Synthesis and biological evaluation of N-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)benzenesulfonamide derivatives as new BET bromodomain inhibitors for anti-hematologic malignancies activities

Article abstract of DOI:10.1007/s11030-016-9707-6

The bromodomain and extra-terminal proteins (BETs), in particular BRD4, has been reported to play important roles in cancer, inflammation, obesity, cardiovascular disease, and neurological disorders. In this paper, a series of benzomorpholinone derivatives were synthesized and biologically evaluated as BETs inhibitors. Detailed structure–activity relationship studies led to the discovery of several new potent compounds, of which 15h and 15i displayed IC ₅₀ values of 2.8 and 4.5 μ M against BRD4 (D1), respectively, and showed good anti-proliferation activities against four hematologic malignancies cell lines at low-micromolar concentrations, including MV4-11, OCI-LY10, Pfeifer, and Su-DHL-6 cells. This chemotype could be further optimized with respect to its potency and drug-like properties in the future.

Full text of DOI:10.1007/s11030-016-9707-6

Mol Divers
DOI 10.1007/s11030-016-9707-6
ORIGINAL ARTICLE
Synthesis and biological evaluation of N-(3-oxo-3,4-dihydro-2H-
benzo[b][1,4]oxazin-7-yl)benzenesulfonamide derivatives as new
BET bromodomain inhibitors for anti-hematologic malignancies
activities
1
2
2
2
2
Li Liu · Yongxia Zhu · Zhihao Liu · Tinghong Ye · Weiqiong Zuo ·
1
2
2
1
Cuiting Peng · Kunjie Xiao · Ningyu Wang · Luoting Yu
Received: 30 August 2016 / Accepted: 31 October 2016
©
Springer International Publishing Switzerland 2016
Abstract The bromodomain and extra-terminal proteins
BETs), in particular BRD4, has been reported to play
Abbreviation
(
important roles in cancer, inflammation, obesity, cardio-
vascular disease, and neurological disorders. In this paper,
a series of benzomorpholinone derivatives were synthe-
sized and biologically evaluated as BETs inhibitors. Detailed
structure–activity relationship studies led to the discovery of
several new potent compounds, of which 15h and 15i dis-
played IC50 values of 2.8 and 4.5 µM against BRD4 (D1),
respectively, and showed good anti-proliferation activities
against four hematologic malignancies cell lines at low-
micromolar concentrations, including MV4-11, OCI-LY10,
Pfeifer, and Su-DHL-6 cells. This chemotype could be fur-
ther optimized with respect to its potency and drug-like
properties in the future.
BRDs
BET
AML
SAR
MTT
Bromodomain-containing proteins
Bromodomain and extra-terminal
Acute myeloid leukemia
Structure–activity relationship
3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-
tetrazolium bromide
Introduction
Bromodomain-containing proteins (BRDs) are a family of
epigenetic protein domains that specifically recognize acety-
lation lysine residues of histone [1]. The bromodomain and
extra-terminal (BET) family belongs to the BRDs family
which consist of four subtypes in mammals: BRD2, BRD3,
BRD4, and BRDT. Each subtype contains two BRD domains
(BD1 and BD2). These BRD domains form deep hydropho-
bic pockets that recognize acetylated lysine residues and an
extra-terminal (ET) domain [2].
Keywords BET inhibitor · BRD4 · Anti-proliferation
activity · Benzomorpholinone · Structure–activity
relationship
Extensive research suggests that the abnormal expression
of BRDs protein plays important roles in a wide variety of
tumor[3,4]. ThetestisspecificisoformBRDThasbeenfound
to be highly expressed in lung cancer [5]. A shRNAs screen
identified BRD4 as an essential protein in acute myeloid
leukemia cells [6]. Genetic knockdown by RNAi or exposure
of cells to BET inhibitors resulted in a significant transcrip-
tional down regulation of c-Myc [7], and the pharmacologic
inhibition of BET family of BRDs by BET inhibitors could
effectively block c-Myc expression in multiple myeloma [8],
Burkitt’s lymphoma [9], and mixed lineage leukemia [10].
In addition, BETs inhibitors have also exhibited therapeutic
potential beyond cancer, such as inflammation [11], obesity
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-016-9707-6) contains supplementary
material, which is available to authorized users.
B Ningyu Wang
scuwny059@163.com
B Luoting Yu
yuluot@scu.edu.cn
1
Department of Pharmaceutical and Bioengineering, School of
Chemical Engineering, Sichuan University, # 24, Section 1,
Ren Min Nan Lu, Chengdu 610000, China
2
State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, West China Medical School, Sichuan
University, # 17, Section 3, Ren Min Nan Lu,
Chengdu 610041, China
[
12], cardiovascular disease [13], and neurological disorders
[14].
1
23

Mol Divers
Fig. 1 BET bromodomains
inhibitors currently reported
Considering the importance of BET bromodomains as
promising targets in the treatment of cancer [10,11], many
pharmaceutical research and development institutions have
focused their efforts on the development of novel inhibitors
of the BET family [15–17]. The main chemical scaffolds of
BET inhibitors are shown in Fig. 1. These inhibitors mainly
interact with the acetyl-lysine binding site of bromodomain.
Even though a number of BET inhibitors have been reported
in recent years, the diversity of the chemical scaffolds of
these leads is limited, and new BET inhibitors with novel
chemotype are worth developing.
Fig. 2 Structure of the racemic mixture 14a and the inhibition curve
against BRD4 (D1). % inhibition at least two independent titrations
series of benzomorpholinone derivatives will be discussed in
this study.
PFI-1(2-methoxy-N-(3-methyl-2-oxo-1,2,3,4-tetrahydro
-
quinazolin-6-yl) -bezenesulfonamide) is a novel lead com-
pound which contains a scaffold of 3,4-dihydro-3-methyl-
(1H)-quinazolinone with an IC50 of 0.1–0.5 µM against
Results and discussion
2
BRD4 (D1) in a biochemical assay [18,19]. As part of our
research program to pursue new scaffolds as BET inhibitors,
we first studied the co-crystallization of PFI-1 with BRD4
Synthesis
Thegeneralsynthesisof N-(3-oxo-3,4-dihydro-2H-benzo[b]
(D1) (PDB: 4E96) and found that the cyclic urea of PFI-1 has
[1,4]oxazin-7-yl) benzenesulfonamide 14a–14m, 15a–15j,
a key interaction with Asn140 of BRD4 (D1) and the urea
carbonyl also makes a water-bridged hydrogen bond inter-
action with the phenolic hydroxyl group of Tyr97 of BRD4
16a–16d, 17a–17e are outlined in Scheme 1. 7-Nitro-
2
H-benzo[b][1,4]oxazin-3(4H)-ones 6–9 were prepared by
cyclization of the α-bromoalkanoic acid ethyl esters 2–5 with
-amino-5-nitrophenol. Reduction of the nitro group of 6–9
(D1) [19]. Then we analyzed the chemical space of PFI-1 and
2
considered to convert quinazolinone to benzomorpholinone
of which the cyclic lactam could mimic the N-methyl urea
to interact with Asn140, while other parts of the structure
of PFI-1 were kept unchanged. A relatively simple com-
pound 14a, (2-methyl-N-(2-methyl-3-oxo-3,4-dihydro-2H-
with iron powder and ammonium chloride at reflux tempera-
ture provides the corresponding anilines 10–13, which were
further reacted with commercially available aryl sulfonyl
chlorides to afford desired final products14a–14m, 15a–15j,
16a–16d, 17a–17e.
benzo[b][1,4]oxazin-7-yl)benzenesulfonamide),
was
designed and synthesized with an IC50 value of 5.4 µM
against BRD4 (D1) (Fig. 2). While this compound was less
potent than PFI-1, we confirmed that this chemotype would
be a new compound class for BET inhibition. The synthesis
and the structure–activity relationships (SAR) of this new
SAR analysis
Our SAR analysis of the benzomorpholinone derivatives was
mainly based on affinity assays results. All of the benzomor-
1
23

Mol Divers
◦
Scheme 1 Synthesis of benzomorpholinone derivatives. Reagents and conditions: i K2CO3, DMF, 90 C, 8–12 h. ii Fe, NH4Cl, CH3CH2OH:H2O =
◦
3
:1, reflux. iii ArSO2Cl, Triethylamine, Tetrahydrofuran, 0 C
pholinones were evaluated using the AlphaScreen assay at a
single concentration of 10 µM (Table 1). Compounds 14b–
against BRD4 (D1) and were better than their para sub-
stituted analogues 15h and 15i. However, compounds 15h
and15i displayed better cytotoxicity activities than 15b, 15c,
15e, and 15f, which showed remarkable inhibitory activities
against the above four human cancer cell lines with IC50 val-
ues ranging from low-micromolar to sub-micromolar values.
The inhibitory activities of the aforementioned com-
pounds against 6 other bromodomains of BETs at the
concentration of 10 µM were evaluated subsequently, as
shown in Table 3. Almost all the compounds exhibited inhi-
bition rates of more than 90% against BRD2 (D1), BRD2
(D2), BRD3 (D1), and BRD4 (D2), while little inhibitory
was observed with respect to BRDT (D1), consistent with
PFI-1 [19], which were also have high selectivity for the
BET family of BRDs.
14g were synthesized to study the influence of different
sulfonyl groups on potency. The replacement of the phenyl
group with propyl 14b, benzyl 14c, 3-naphthalene 14d, 2-
isoxazole 14e , and 4-thiophene 14f did not improve BRD4
inhibitoryactivitywhichsuggeststhatarigidringwithproper
size is crucial for potency. The 8-isoquinoline substituted
analogue 14g demonstrated 94% inhibition at 10 µM against
BRD4 (D1), which is almost equipotent to 14a. However, this
structure was not selected for further optimization due to its
poor water solubility.
Introducing a single substituent on the phenyl ring could in
a large part retain the inhibitory activity against BRD4 (D1),
as illustrated by 14h–14m. The replacement of methyl on
the α-position of the cyclic lactam with an ethyl group could
significantly improve the potency as seen in 15e–15i. The
introduction of a single substituent at the ortho-position of
thephenylringwasfavorablefortheactivity, asdemonstrated
by 15a–15g. Compounds 15b–15g exhibited comparable
potency, suggesting that different electric effect groups on
this position were well tolerated. Electron withdrawing
groups at the para-position of the phenyl ring displayed
decreased BRD4 (D1) activity, such as 15h, 15i , and 15j.
When a single n-propyl group was introduced on the α-
position of the cyclic lactam, the potency of these compounds
Conclusions
In summary, we have successfully synthesized a series of
BET bromodomains inhibitors with potent activities in both
molecular and cellular assays based on a benzenesulfon-
amide scaffold. Our SAR studies revealed that an ethyl
group on the α-position of the lactam could significantly
improve potency against BRD4. Compounds 15h and 15i
displayed IC50 values of 2.8 and 4.5 µM against BRD4 (D1),
respectively, and also showed excellent inhibitory activi-
ties against 4 human cancer cell lines at low-micromolar
to sub-micromolar concentrations, including MV4-11, OCI-
LY10, Pfeifer, and Su-DHL-6 cell lines. Compounds showed
strong inhibition at 10 µM against BRD4 (D1) against sev-
eral human BRDs subtype inhibitory. This work presents
the identification of a new chemotype with BRD4 inhibitory
activity. Further optimization and mechanism studies on this
chemotype are underway.
(
16a–16d) decreased significantly. Lastly, a gem-dimethyl
substituent also on this position (17a–17e) was found to
be unfavorable for potency. These results suggest that alkyl
substituent with two carbon atoms at 2-position of the ben-
zomorpholinone core could be beneficial for BRD4 activity.
BET Bromodomains inhibitory and cytotoxic activity
Compounds which showed inhibition rates of more than 90%
at 10 µM against BRD4 (D1) were selected for IC50 test-
ing against BRD4 (D1) using the AlphaScreen assay and a
cytotoxicity assay against MV4-11, OCI-LY10, Pfeifer, and
Su-DHL-6 cell lines using the MTT assay [20]. These results
are presented in Table 2. Compounds 15b, 15c, 15e, and 15f,
which contain a single substituent at the ortho-position of
the phenyl ring, exhibited low-micromolar inhibitory activity
Materials and methods
General
Chemistry reagents of analytical grade were purchased from
ChangzhengChemical Factory, Chengdu, Sichuan, PR China
1
23

Mol Divers
1
23

Mol Divers
Table 2 Bioactivities of
selected compounds
No.
IC50(µM)
BRD4 (D1) (AlphaScreenTM
)
MV4-11
OCI-LY10
Pfeifer
Su-DHL-6
1
1
1
1
1
1
1
4a
5b
5c
5e
5f
5.4 ± 0.2
2.1 ± 0.1
1.5 ± 0.1
1.7 ± 0.1
2.0 ± 0.1
2.8 ± 0.2
4.5 ± 0.3
0.3 ± 0.1
10.2 ± 0.1
7.5 ± 0.1
9.6 ± 0.2
7.5 ± 0.2
6.3 ± 0.2
3.7 ± 0.2
1.26 ± 0.1
0.78 ± 0.1
9.4 ± 0.2
15.6 ± 0.2
21 ± 0.3
7.82 ± 0.2
>30
24.8 ± 0.3
>30
>30
>30
17.5 ± 0.2
18.8 ± 0.1
2.5 ± 0.1
0.56 ± 0.1
3.1 ± 0.2
>30
>30
>30
>30
5h
5i
3.8 ± 0.1
0.39 ± 0.1
4.4 ± 0.2
7.4 ± 0.2
2.69 ± 0.1
12.3 ± 0.2
PFI-1
IC50 results are an average of at least two independent titrations
Table 3 BET bromodomains
inhibitory profiles of selected
compounds
No.
Inh % (10 µM)
BRD2 (D1)
BRD2 (D2)
BRD3 (D1)
BRD3 (D2)
BRD4 (D2)
BRDT (D1)
1
1
1
1
1
1
1
4a
5b
5c
5e
5f
70 ± 2
99 ± 3
100 ± 2
99 ± 1
98 ± 2
99 ± 1
88 ± 3
93 ± 3
98 ± 1
99 ± 2
97 ± 2
96 ± 3
95 ± 1
81 ± 2
94 ± 1
99 ± 2
99 ± 2
98 ± 3
98 ± 3
95 ± 1
75 ± 2
76 ± 1
99 ± 2
100 ± 2
98 ± 1
98 ± 2
99 ± 1
89 ± 1
79 ± 2
98 ± 1
99 ± 3
95 ± 2
95 ± 3
86 ± 2
54 ± 1
−2 ± 2
2 ± 3
6 ± 3
6 ± 3
7 ± 2
5h
5i
4 ± 3
−1 ± 2
All compounds were tested at a concentration of 10 µM. The percent inhibition is an average of at least two
independent titrations
and were used without further purification. TLC was per-
it is poured onto ice/water (100 mL), and the resulting pre-
cipitate is separated by filtration, washed with water, and
dried. Recrystallization from ethanol afforded the desired
formed on 0.20-mm Silica Gel 60 F254 plates (Qingdao
1
Ocean Chemical Factory, Shandong, China). H-NMR and
1
3
1
C-NMR spectra were recorded on a Bruker Varian Unity
compound 6 as brown solid (0.65 g, 62.5%); H NMR (400
Inova-400 (400/100 MHz) spectrometer using tetramethyl-
silane (TMS) as internal reference chemical. Chemical shifts
MHz, Chloroform-d) δ 8.15 (s, 1H), 7.88–7.79 (m, 2H), 6.83
(d, J = 8.6 Hz, 1H), 4.69 (q, J = 6.9 Hz, 1H), 1.56 (d,
+
(
δ) are quoted in ppm relative to TMS as an internal standard,
J = 6.8 Hz, 3H); MS (ESI), m/z: 208.2 [M+H] .
where (δ) TMS = 0.00 ppm. The multiplicity of the signal
is indicated as s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet, defined as all multipeak signals where overlap or
complex coupling of signals makes definitive descriptions of
peaks difficult. Mass spectra (MS) were measured on a Q-
TOF Premier mass spectrometer (Micromass, Manchester,
UK) utilizing electrospray ionization (ESI).
2-Ethyl-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (7)
The title compound was prepared from ethyl 2-
bromobutanoate according to the procedure for example 6
1
and isolated as brown solid (0.82 g, 73.2%); H NMR (400
MHz, Chloroform-d) δ 8.18 (s, 1H), 7.84 (m, 2H), 6.79 (d,
J = 8.6 Hz, 1H), 4.73 (q, J = 6.9 Hz, 1H), 2.14 (m, 2H),
+
Chemistry
0.94 (d, J = 6.8 Hz, 3H); MS (ESI), m/z: 246.0 [M+ Na] .
2-Methyl-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (6)
7-Nitro-2-propyl-2H-benzo[b][1,4]oxazin-3(4H)-one (8)
2-Amino-5-nitrophenol (5 mmol) and potassium carbonate
The title compound was prepared from ethyl 2-
(
10 mmol) were mixed in anhydrous dimethylformamide (10
bromopentanoate according to the procedure for example 6
and isolated as brown solid (0.79 g, 66.6%); H NMR (400
1
mL). The mixture is stirred at room temperature for 1 h.
Then, ethyl 2-bromopropanoate (5 mmol) is slowly added
to the reaction mixture and the mixture is stirred at reflux
for 8–12 h. After reaction completion (monitored by TLC),
MHz, Chloroform-d) δ 8.15 (s, 1H), 7.88–7.79 (m, 2H), 6.83
(d, J = 8.6 Hz, 1H), 4.69 (m, 1H), 1.63 (m, 2H), 1.39 (m,
+
2H), 0.88 (t, 3H);MS (ESI), m/z: 259.2 [M+Na] .
1
23

Mol Divers
2
,2-Dimethyl-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one
(s, 1H), 6.52 (d, J = 8.2 Hz, 1H), 6.28–6.05 (m, 2H), 4.84
+
(
9)
(s, 2H), 0.95 (d, 6H). MS (ESI); m/z: 215.1 [M+H] .
The title compound was prepared from ethyl 2-bromo-2-
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-
methylpropanoate according to the procedure for example
7-yl)benzenesulfonamide (14a)
1
6
and isolated as brown solid (0.54 g, 48.7%); H NMR (400
MHz, Chloroform-d) δ 8.18 (s, 1H), 7.86–7.74 (m, 2H), 6.79
To a solution of 10 in tetrahydrofuran was added Et N (3.0
eq) followed by a benzenesulfonyl chloride (1.0 eq) at 0 C.
3
◦
(
d, J = 8.6 Hz, 1H), 4.66 (q, 1H), 0.96 (s, 6H);MS (ESI),
+
m/z: 259.1 [M+Na] .
The reaction mixture was allowed to warm up to r.t over a
period of 1 h and reaction overnight. The reaction mixture
was evaporated and dissolved in 10 mL water. 1.0 N HCl was
slowly added to the mixture (pH 4), and the resulting solid
was filtered and rinsed with water. The crude product was
purified on a silica column eluting with petroleum ether/ethyl
7-Amino-2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (10)
To a solution of 6 (3 mmol) in methanol (20 mL) is added
iron powder (15 mmol) followed by an aqueous solution of
ammonium chloride (15 mmol) at room temperature. The
reaction mixture was stirred and heated at reflux for 1 h and
was then allowed to cool to r.t. The solution was filtered
and basified with saturated sodium bicarbonate solution. The
solution was extracted with ethyl acetate (4 × 100 mL). The
combined organic layers were washed with brine, dried over
sodium sulfate, filtered, and the solvent evaporated to give
the desired product 10 and isolated as light yellow solid (0.39
1
acetate (7:1) to get 14a as brown crystals (0.2 g, 62.8%); H
NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 10.11 (s, 1H),
7
(
(
.72 (d, J = 7.9 Hz, 2H), 7.60 (d, J = 6.7 Hz, 1H), 7.55
t, J = 7.2 Hz, 2H), 6.74–6.66 (m, 2H), 6.65 (s, 1H), 4.58
dd, J = 13.4, 6.8 Hz, 1H), 1.35 (d, J = 6.8 Hz, 3H);
1
3
C
NMR (100 MHz, DMSO-d6) δ 166.37 (s), 142.96 (s), 139.28
s), 132.86 (s), 132.63 (s), 129.21 (s), 126.61 (s), 124.33 (s),
15.74 (s), 114.85 (s), 109.24 (s), 72.62 (s), 15.95 (s); MS
(
1
+
1
(ESI), m/z: 341.3 [M+Na] .
g, 73.7%); H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H),
6
.54 (d, J = 8.3 Hz, 1H), 6.23–6.12 (m, 2H), 4.85 (s, 2H),
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)propane-1-sulfonamide (14b)
4.36 (dd, J = 8.0, 4.6 Hz, 1H), 1.37 (d, J = 6.8 Hz, 3H).MS
+
(
ESI), m/z: 201.1 [M+Na] .
To a solution of 10 in tetrahydrofuran was added Et3N (3.0
eq) followed by a benzenesulfonyl chloride (1.0 eq) at 0 C.
7-Amino-2-ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one (11)
◦
The reaction mixture was allowed to warm up to r.t over a
period of 1 h and reaction overnight. The reaction mixture
was evaporated and dissolved in 10 mL water. 1.0 N HCl
was slowly added to the mixture (pH 4), and the resulting
solid was filtered and rinsed with water. The crude product
The title compound was prepared from 7 according to the
procedure for example 10 and isolated as light yellow solid
1
(
(
(
0.56 g, 84.6%); H NMR (400 MHz, DMSO-d6) δ 10.18
s, 1H), 6.54 (d, J = 8.3 Hz, 1H), 6.25–6.10 (m, 2H), 4.84
s, 2H), 4.32 (dd, J = 8.0, 4.6 Hz, 1H), 1.83–1.61 (m, 2H),
was recrystalized from ethanol to obtain desired compound
+
0
.96 (t, J = 7.4 Hz, 3H); MS (ESI), m/z: 215.1 [M+Na] .
1
1
4b as brown crystals (0.16 g, 56.7%); H NMR (400 MHz,
DMSO-d6) δ 10.61 (s, 1H), 9.66 (s, 1H), 6.77–6.83 (m, 3H),
7
-Amino-2-propyl-2H-benzo[b][1,4]oxazin-3(4H)-one (12)
4
3
.53 (m, 1H), 3.12 (m, 2H), 1.56–1.68 (m, 5H), 0.96 (m,
H); C NMR(100 MHz, DMSO-d6) δ 166.42 (s), 143.21
1
3
The title compound was prepared from 8 according to the
procedure for example 10 and isolated as light yellow solid
(s), 133.44 (s), 123.99 (s), 115.92 (s), 114.12 (s), 108.61 (s),
7
2.69 (s), 51.97 (s), 16.75 (s), 16.02 (s), 12.53 (s). MS (ESI),
1
(
(
(
0.57 g, 79.9%); H NMR (400 MHz, DMSO-d6) δ 10.18
s, 1H), 6.54 (d, J = 8.2 Hz, 1H), 6.20–6.12 (m, 2H), 4.85
s, 2H), 4.38 (dd, J = 7.8, 5.1 Hz, 1H), 1.72–1.61 (m, 2H),
+
m/z: 307.1 [M+Na] .
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)-1-phenylmethanesulfonamide (14c)
1
.44 (dp, J = 14.4, 7.0 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H);
+
MS (ESI), m/z: 207.2 [M+Na] .
The title compound was prepared from 10 and phenyl-
methanesulfonyl chloride according to the procedure for
example 14b and isolated as brown crystals (0.16 g, 48.7%);
7
-Amino-2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one
(
13)
1
H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.69 (s,
The title compound was prepared from 9 according to the
procedure for example 10 and isolated as light yellow solid
1H), 7.31 (d, J = 32.7 Hz, 5H), 6.81 (s, 3H), 4.64 (s, 1H),
4.41 (s, 2H), 1.41 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 166.93 (s), 143.68 (s), 134.02 (s), 131.39 (s), 130.01
(0.46 g, 68.3%); 1H NMR (400 MHz, DMSO-d6) δ 10.20
1
23

Mol Divers
(
s), 128.82 (s), 128.64 (s), 124.20 (s), 116.38 (s), 114.17 (s),
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
1
08.75 (s), 73.23 (s), 57.16 (s), 16.55 (s); MS (ESI), m/z:
yl)isoquinoline-8-sulfonamide (14g)
+
355.2 [M+Na] .
The title compound was prepared from 10 and isoquinoline-
8
-sulfonyl chloride according to the procedure for example
1
14a and isolated as brown solid (0.24 g, 64.7%); H NMR
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)naphthalene-2-sulfonamide (14d)
R (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.90 (s, 1H), 9.14
(
s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 8.30 (dd, J = 19.4, 7.4
Hz, 2H), 7.72 (s, 2H), 6.61 (d, J = 6.2 Hz, 3H), 4.49 (d,
The title compound was prepared from 10 and naphthalene-
13
J = 6.1 Hz, 1H), 1.29 (d, J = 6.1 Hz, 3H); C NMR
2
-sulfonyl chloride according to the procedure for example
(100 MHz, DMSO-d6) δ 166.27 (s), 151.45 (s), 142.74 (s),
1
14a and isolated as brown solid (0.19 g, 51.6%); H NMR
1
1
1
3
36.99 (s), 135.02 (s), 134.21 (s), 132.80 (s), 132.08 (s),
28.32 (s), 125.65 (s), 123.91 (s), 122.65 (s), 115.44 (s),
14.30 (s), 108.77 (s), 72.52 (s), 15.88 (s); MS (ESI), m/z:
(
(
400 MHz, DMSO-d6) δ 10.50 (s, 1H), 10.47 (s, 1H), 8.69
d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.16 (d,
J = 7.7 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.76–7.58 (m,
+
92.1 [M+Na] .
3
6
H), 6.61 (dd, J = 21.7, 10.7 Hz, 3H), 4.52 (dd, J = 13.5,
.6 Hz, 1H), 1.30 (d, J = 6.7 Hz, 3H); C NMR (100 MHz,
1
3
2-Methyl-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
DMSO-d6) δ 166.28 (s), 142.91 (s), 134.38 (s), 134.21
[b][1,4]oxazin-7-yl)benzenesulfonamide (14h)
(
(
s), 133.68 (s), 132.49 (s), 129.86 (s), 129.05 (s), 128.09
s), 126.96 (s), 124.46 (s), 124.22 (s), 123.90 (s), 115.72 (s),
The title compound was prepared from 10 and 2-
methylbenzenesulfonyl chloride according to the procedure
1
13.83 (s), 108.22 (s), 72.58 (s), 15.91 (s); MS (ESI), m/z:
+
391.1 [M+Na] .
for example 14a and isolated as brown crystals (0.24 g,
1
7
1
6
3
2.7%); H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H),
0.24 (s, 1H), 7.82 (s, 1H), 7.55 (d, 1H), 7.37 (s, 2H), 6.64–
.70 (d, J = 21.8 Hz, 3H), 4.58 (s, 1H), 2.64(s, 3H), 1.35 (s,
3,5-Dimethyl-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
1
3
H); C NMR (100 MHz, DMSO-d6) δ 166.40 (s), 142.92
[b][1,4]oxazin-7-yl)isoxazole-4-sulfonamide (14e)
(s), 136.68 (s), 131.86 (s), 131.55 (s), 129.21 (s), 126.61 (s),
1
24.33 (s), 115.74 (s), 114.85 (s), 108.40 (s), 72.62 (s), 15.95
The title compound was prepared from 10 and 3,5-
dimethylisoxazole-4-sulfonyl chloride according to the pro-
+
(
s); MS (ESI), m/z: 355.1 [M+Na] .
cedure for example 14a and isolated as brown solid (0.2 g,
1
2-Chloro-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
b][1,4]oxazin-7-yl)benzenesulfonamide (14i)
5
1
4
1
3.2%); H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H),
[
0.20 (s, 1H), 6.80 (d, J = 8.9 Hz, 1H), 6.70–6.64 (m, 2H),
.49 (dd, J = 7.8, 4.4 Hz, 1H), 2.41 (s, 3H), 2.19 (s, 3H),
.68 (d, 3H); 13C NMR (100 MHz, DMSO-d6) δ 173.29
The title compound was prepared from 10 and 2-
chlorobenzenesulfonyl chloride according to the procedure
for example 14a and isolated as brown solid (0.22 g, 58.9%);
(
s), 165.93 (s), 157.27 (s), 142.65 (s), 131.54 (s), 125.02 (s),
1
16.26 (s), 115.80 (s), 115.04 (s), 110.74 (s), 77.13 (s), 23.24
1
+
H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 10.44 (s,
(
s), 15.93 (s), 12.05 (s); MS (ESI), m/z: 360.2 [M+Na] .
1
H), 7.99 (d, J = 7.4 Hz, 1H), 7.63 (d, 2H), 7.51 (t, J = 6.2
Hz, 1H), 6.69 (d, J = 11.2 Hz, 3H), 4.62–4.54 (m, 1H),
1
3
1
.34 (d, J = 6.7 Hz, 3H); C NMR (100 MHz, DMSO-d6)
2,4-Dichloro-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
δ 166.31 (s), 143.00 (s), 134.64 (s), 131.94 (s), 131.55 (s),
[b][1,4]oxazin-7-yl)thiophene-3-sulfonamide (14f)
115.77 (s), 108.40 (s), 72.62 (s), 15.95 (s); MS (ESI), m/z:
+
375.5 [M+Na] .
The title compound was prepared from 10 and 2,4-
dichlorothiophene-3-sulfonyl chloride according to the pro-
2-Bromo-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
cedure for example 14a and isolated as brown solid (0.13 g,
[b][1,4]oxazin-7-yl)benzenesulfonamide (14j)
1
3
1
3.8%); H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H),
0.52 (s, 1H), 7.29 (s, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.72
The title compound was prepared from 10 and 2-
bromobenzenesulfonyl chloride according to the procedure
for example 14a and isolated as brown solid (0.3 g, 76.3%);
(
d, J = 8.0 Hz, 2H), 4.64 (d, J = 6.7 Hz, 1H), 1.38 (d,
13
J = 6.8 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ 166.44
1
(
s), 143.00 (s), 135.46 (s), 131.48 (s), 129.65 (s), 126.77 (s),
H NMR (400 MHz, DMSO-d6) δ 10.50 (d, J = 30.1 Hz,
1
7
26.39 (s), 124.93 (s), 115.88 (s), 115.29 (s), 109.76 (s),
2.68 (s), 15.97 (s); MS (ESI), m/z: 393.1 [M+H] .
2H), 8.03 (s, 1H), 7.83 (s, 1H), 7.54 (s, 2H), 6.70 (s, 3H),
4.59 (s, 1H), 1.35 (s, 3H); C NMR (100 MHz, DMSO-
+
13
1
23

Mol Divers
d6) δ 166.33 (s), 143.00 (s), 138.09 (s), 135.39 (s), 134.58
s), 131.99 (s), 131.73 (s), 128.24 (s), 124.10 (s), 119.15 (s),
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)benzenesulfonamide (15a)
(
1
15.79 (s), 113.88 (s), 108.35 (s), 72.64 (s), 15.95 (s); MS
+
(
ESI), m/z: 419.0 [M+Na] .
To a solution of 11 in tetrahydrofuran was added Et3N (3.0
◦
eq) followed by a benzenesulfonyl chloride (1.5 eq) at 0 C.
The reaction mixture was allowed to warm up to r.t over a
period of 1.5 h and reaction overnight. The reaction mixture
was evaporated and dissolved in 10 mL water. 1.0 N HCl was
slowly added to the mixture (pH 4), and the resulting solid
was filtered and rinsed with water. The crude product was
purified on a silica column to get 15a as brown solid (0.19
4
-Methyl-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
[b][1,4]oxazin-7-yl)benzenesulfonamide (14k)
The title compound was prepared from 10 and 4-
methylbenzenesulfonyl chloride according to the procedure
for example 14a and isolated as light brown solid (0.19 g,
1
g, 57.2%); H NMR (400 MHz, DMSO-d6)δ 10.60 (s, 1H),
1
10.48 (s, 1H), 8.69 (s, 1H), 8.15 (t, 3H), 7.80–7.51 (m, 2H),
5
1
7.7%); H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H),
0.06 (s, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1
1
3
6.58 (d, 2H), 4.38 (s, 1H), 1.64 (m, 2H), 0.89 (t, 3H);
C
NMR (100 MHz, DMSO-d6) δ 165.81 (s), 142.51 (s), 134.34
(s), 129.87 (s), 129.02 (s), 128.06 (s), 126.93 (s), 124.40 (s),
Hz, 2H), 6.75–6.66 (m, 2H), 6.65 (s, 1H), 4.59 (q, J = 6.7
1
3
Hz, 1H), 2.34 (s, 3H), 1.36 (d, J = 6.8 Hz, 3H); C NMR
1
15.59 (s), 113.87 (s), 108.41 (s), 77.01 (s), 23.10 (s), 8.97
(100 MHz, DMSO-d6)δ 166.36 (s), 143.16 (s), 142.95 (s),
+
(
s); MS (ESI), m/z: 333.1 [M+H] .
1
1
36.47 (s), 132.82 (s), 129.63 (s), 126.67 (s), 124.19 (s),
15.74 (s), 114.66 (s), 109.06 (s), 72.62 (s), 20.92 (s), 15.95
+
(
s); MS (ESI), m/z: 355.1 [M+Na] .
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl) -2-methylbenzenesulfonamide (15b)
4
-Chloro-N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo
The title compound was prepared from 11 and 2-
methylbenzenesulfonyl chloride according to the procedure
for example 15a and isolated as brown solid (0.25 g, 73.8%);
[b][1,4]oxazin-7-yl)benzenesulfonamide (14l)
The title compound was prepared from 10 and 4-
chlorobenzenesulfonyl chloride according to the procedure
for example 14a and isolated as brown solid (0.23 g, 64.1%);
1
H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 10.20 (s,
1
H), 7.81 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H),
1
7
.35 (dd, J = 13.4, 7.1 Hz, 2H), 6.73–6.60 (m, 3H), 4.43
H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 10.18 (s,
(
0
dd, J = 7.6, 4.4 Hz, 1H), 2.56 (s, 3H), 1.81–1.59 (m, 2H),
1
6
1
H), 7.70 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H),
1
3
.91 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, DMSO-
.76–6.66 (m, 2H), 6.65 (s, 1H), 4.60 (q, J = 6.8 Hz, 1H),
1
3
d6) δ 166.07 (s), 143.12 (s), 135.98 (s), 132.16 (s), 131.55
s), 129.21 (s), 126.61 (s), 124.33 (s), 115.74 (s), 115.05 (s),
.36 (d, J = 6.7 Hz, 3H); C NMR (100 MHz, DMSO-
(
d6) δ 166.39 (s), 143.00 (s), 138.08 (s), 137.73 (s), 132.25
1
08.40 (s), 72.62 (s), 15.95 (s), 23.10 (s), 8.97 (s); MS (ESI),
(
(
[
s), 129.40 (s), 128.58 (s), 124.62 (s), 115.82 (s), 115.17
s), 109.57 (s), 72.64 (s), 15.95 (s); MS (ESI), m/z: 375.2
M+Na] .
+
m/z: 369.6 [M+Na] .
+
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
N-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)-2 -fluorobenzenesulfonamide (15c)
yl)-4-nitrobenzenesulfonamide (14m)
The title compound was prepared from 11 and 2-
fluorobenzenesulfonyl chloride according to the procedure
for example 15a and isolated as brown solid (0.24 g, 68.6%);
The title compound was prepared from 10 and 4-
nitrobenzenesulfonyl chloride according to the procedure for
1
1
example 14a and isolated brown solid (0.29 g, 82.1%); H
H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 10.41 (s,
NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 10.43 (s, 1H),
1H), 7.77 (t, J = 6.8 Hz, 1H), 7.68 (dd, J = 13.0, 6.3 Hz,
1H), 7.34 (t, J = 7.7 Hz, 1H), 7.1 (t, 1H), 6.73–6.65 (m,
3H), 4.44 (dd, J = 7.7, 4.5 Hz, 1H), 1.82–1.59 (m, 2H),
8
.37 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.7 Hz, 2H), 6.74
(
d, J = 9.0 Hz, 1H), 6.67 (d, 2H), 4.60 (dd, J = 13.6, 6.7
13
13
Hz, 1H), 1.09 (t, J = 7.0 Hz, 3H); C NMR (100 MHz,
0.91 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-
DMSO-d6) δ 166.88 (s), 150.27 (s), 145.27 (s), 143.55 (s),
d6) δ 166.43 (s), 143.18 (s), 133.51 (s), 130.89 (s), 129.50
(s), 128.33 (s), 128.14 (s), 123.67 (s), 115.88 (s), 113.62 (s),
108.19 (s), 72.71 (s), 56.60 (s), 16.04 (s); MS (ESI), m/z:
1
1
3
32.34 (s), 128.73 (s), 125.38 (s), 125.09 (s), 116.38 (s),
16.00 (s), 110.38 (s), 73.15 (s), 16.46 (s); MS (ESI), m/z:
+
+
86.1 [M+Na] .
373.6 [M+Na] .
1
23

Mol Divers
1
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
g, 31.8%); H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H),
yl)-4-methoxy-2-nitrobenzenesulfonamide (15d)
10.48 (s, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 7.0
Hz, 1H), 7.85 (dd, J = 13.6, 7.4 Hz, 2H), 6.74 (d, J = 9.1
Hz, 1H), 6.69 (d, J = 6.8 Hz, 2H), 4.45 (dd, J = 7.6,
4.5 Hz, 1H), 1.81–1.71 (m, 1H), 1.71–1.60 (m, 1H), 0.92 (t,
The title compound was prepared from 11 and 4-methoxy-
2-nitrobenzenesulfonyl chloride according to the procedure
1
3
for example 15a and isolated as brown solid (0.13 g, 31.5%);
J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ 165.82
(s), 142.72 (s), 133.41 (s), 133.30 (s), 132.11 (s), 130.65 (s),
124.18 (s), 115.76 (s), 114.53 (s), 109.06 (s), 77.07 (s), 23.18
1
H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 10.33 (s,
1
H), 7.81 (d, J = 8.9 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H),
+
7
.40–7.23 (m, 1H), 6.72 (m, J = 23.4, 8.3 Hz, 3H), 4.46 (dd,
(s), 8.99 (s); MS (ESI), m/z: 423.3 [M+Na] .
J = 7.6, 4.5 Hz, 1H), 3.87 (s, 3H), 1.73 (ddt, J = 41.9, 14.4,
1
3
7.2 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H); C NMR (100 MHz,
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)-4-methylbenzenesulfonamide (15h)
DMSO-d6) δ 165.88 (s), 162.86 (s), 149.43 (s), 142.67 (s),
1
1
31.71 (d, J = 6.4 Hz), 124.51 (s), 122.24 (s), 117.16 (s),
15.72 (s), 115.19 (s), 109.89 (s), 109.77 (s), 77.10 (s), 56.63
The title compound was prepared from 11 and 4-
methylbenzenesulfonyl chloride according to the procedure
for example 15a and isolated as brown solid (0.2 g, 58.9%);
+
(
s), 23.20 (s), 9.01 (s). MS (ESI), m/z: 430.4 [M+Na] .
1
2-Chloro-N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo
H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 10.01 (s,
[
b][1,4]oxazin-7-yl)benzenesulfonamide (15e)
1H), 7.59 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 6.70
(
d, J = 8.5 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H), 6.64 (s, 1H),
The title compound was prepared from 11 and 2-
chlorobenzenesulfonyl chloride according to the procedure
for example 15a and isolated brown solid (0.16 g, 44.8%); H
NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 10.41 (s, 1H),
8
7
4
4.43 (dd, J = 7.8, 4.5 Hz, 1H), 2.33 (s, 3H), 1.83–1.65(m,
13
2H), 0.92 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-
d6) δ 165.81 (s), 143.15 (s), 142.60 (s), 136.38 (s), 132.82
(s), 129.59 (s), 126.67 (s), 123.97 (s), 115.63 (s), 114.69 (s),
109.22 (s), 77.04 (s), 23.17 (s), 20.92 (s), 9.01 (s); MS (ESI),
1
.05 (t, J = 6.8 Hz, 1H), 7.96 (dd, J = 13.0, 6.3 Hz, 1H),
.57 (t, J = 7.7 Hz, 1H), 7.35 (t, 1H), 6.73–6.65 (m, 3H),
.41 (d, 1H), 1.82–1.63 (m, 2H), 0.88 (t, 3H); 13C NMR (100
+
m/z: 369.5 [M+Na] .
MHz, DMSO-d6) δ 165.76 (s), 142.66 (s), 136.28 (s), 134.62
s), 131.96 (s), 131.78 (s), 131.57 (s), 130.65 (s), 127.68 (s),
4-Chloro-N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo
[b][1,4]oxazin-7-yl)benzenesulfonamide (15i)
(
1
23.95 (s), 115.69 (s), 113.97 (s), 108.53 (s), 77.04 (s), 23.16
+
(
s), 9.00 (s); MS (ESI), m/z: 389.4 [M+Na] .
The title compound was prepared from 11 and 4-
chlorobenzenesulfonyl chloride according to the procedure
for example 15a and isolated as brown solid (0.23 g, 63.2%);
2
-bromo-N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo
1
[b][1,4]oxazin-7-yl)benzenesulfonamide (15f)
H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 10.15 (s,
1
H), 7.69 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H),
The title compound was prepared from 11 and 2-
bromobenzenesulfonyl chloride according to the procedure
for example 15a and isolated as brown solid (0.22 g, 54.7%);
6.73 (d, J = 8.4 Hz, 1H), 6.65 (dd, J = 12.3, 3.9 Hz,
2H), 4.45 (dd, J = 7.8, 4.5 Hz, 1H), 1.84–1.59 (m, 2H),
1
3
0.92 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-
d6) δ 165.99(s), 142.47 (s), 137.96 (s), 137.72 (s), 132.27
(s), 129.34 (s), 128.60 (s), 124.39 (s), 115.73 (s), 115.29 (s),
109.77 (s), 76.85 (s), 23.18 (s), 8.98 (s); MS (ESI), m/z: 389.3
1
H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 10.43 (s,
1
7
3
0
H), 8.02 (dd, J = 7.6, 1.8 Hz, 1H), 7.87–7.77 (m, 1H),
.53 (m, J = 7.4, 5.9 Hz, 2H), 6.69 (m, J = 9.2, 6.1 Hz,
H), 4.44 (dd, J = 7.7, 4.5 Hz, 1H), 1.82–1.59 (m, 2H),
+
[M+Na] .
1
3
.91 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-
d6) δ 165.78 (s), 142.68 (s), 138.05 (s), 135.38 (s), 134.56
s), 132.00 (s), 131.75 (s), 128.20 (s), 123.88 (s), 119.16 (s),
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)-4-nitrobenzenesulfonamide (15j)
(
1
15.69 (s), 113.88 (s), 108.46 (s), 77.05 (s), 23.17 (s), 9.01
+
(
s); MS (ESI), m/z: 433.6 [M+Na] .
The title compound was prepared from 11 and 4-
nitrobenzenesulfonyl chloride according to the procedure for
1
N-(2-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
example 15a and isolated as brown solid (0.27 g, 73.7%); H
yl)-2-(trifluoromethyl)benzenesulfonamide (15g)
NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 10.40 (s, 1H),
8
.37 (d, J = 8.7 Hz, 2H), 7.94 (d, J = 8.7 Hz, 2H), 6.74
The title compound was prepared from 11 and 2-
(d, J = 8.1 Hz, 1H), 6.71–6.62 (m, 2H), 4.45 (dd, J = 7.7,
(trifluoromethyl)benzenesulfonyl chloride according to the
4.5 Hz, 1H), 1.72 (m, J = 43.8, 14.5, 7.3 Hz, 2H), 0.91 (t,
1
3
procedure for example 15a and isolated as brown solid (0.13
J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ 165.85
1
23

Mol Divers
(
(
7
s), 149.78 (s), 144.59 (s), 142.72 (s), 131.72 (s), 128.28
119.17 (s), 115.71 (s), 113.95 (s), 108.52 (s), 75.86 (s), 31.79
(s), 17.56 (s), 13.53 (s); MS (ESI), m/z: 426.9 [M+H] .
+
s), 124.74 (s), 124.60 (s), 115.81 (s), 115.55 (s), 110.06 (s),
+
7.06 (s), 23.18 (s), 8.98 (s); MS (ESI), m/z: 400.2 [M+Na] .
N-(3-oxo-2-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)benzenesulfonamide (16d)
2
-Chloro-N-(3-oxo-2-propyl-3,4-dihydro-2H-benzo
[b][1,4]oxazin-7-yl)benzenesulfonamide (16a)
The title compound was prepared from 12 and benzenesul-
fonyl chloride according to the procedure for example 16a
and isolated as brown solid (0.25 g, 74.7%); H NMR (400
To a solution of 12 in tetrahydrofuran was added Et3N (3.0
eq) followed by a 2-chlorobenzenesulfonyl chloride (1.5 eq)
1
◦
at 0 C. The reaction mixture was allowed to warm up to
MHz, DMSO-d6) δ 10.56 (s, 1H), 10.08 (s, 1H), 7.71 (d,
J = 7.4 Hz, 2H), 7.60 (d, J = 7.1 Hz, 1H), 7.54 (t, J = 7.4
Hz, 2H), 6.74–6.57 (m, 3H), 4.53–4.45 (m, 1H), 1.62 (dd,
J = 20.4, 13.7 Hz, 2H), 1.37 (d, J = 10.1 Hz, 2H), 0.87 (t,
r.t over a period of 2 h and reaction overnight. The reaction
mixture was evaporated and dissolved in 10 mL of water.
1.0 N HCl was slowly added to the mixture (pH 4), and the
resulting solid was filtered and rinsed with water. The crude
13
J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ 165.98
product was purified on a silica column to get 16a as brown
(s), 142.46 (s), 139.24 (s), 132.82 (s), 132.73 (s), 129.16 (s),
1
solid (0.3 g, 81.3%); H NMR (400MHz, DMSO-d6) δ 10.53
1
7
3
26.64 (s), 124.07 (s), 115.65 (s), 114.93 (s), 109.41 (s),
(
(
4
2
s, 1H), 10.42 (s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.67–7.58
5.85 (s), 31.80 (s), 17.58 (s), 13.53 (s); MS (ESI), m/z:
m, 2H), 7.51 (dd, J = 15.6, 7.3 Hz, 1H), 6.72–6.62 (m, 3H),
+
69.1 [M+Na] .
.48 (dd, J = 8.3, 4.2 Hz, 1H), 1.71–1.54 (m, 2H), 1.38 (m,
13
H), 0.87 (t, J = 7.3 Hz, 3H); C NMR (100 MHz, DMSO-
N-(2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo
b][1,4]oxazin-7-yl)-4-methylbenzenesulfonamide (17a)
d6) δ 166.03 (s), 142.54 (s), 138.12 (s), 135.37 (s), 134.58
s), 131.97 (s), 131.83 (s), 128.19 (s), 123.87 (s), 119.17 (s),
[
(
1
15.69 (s), 113.95 (s), 109.52 (s), 75.84 (s), 31.81 (s), 17.60
+
To a solution of 10 in tetrahydrofuran was added Et3N (3.0
(
s), 13.52 (s); MS (ESI), m/z: 403.4 [M+Na] .
eq) followed by a 4-methylbenzenesulfonyl chloride (1.2 eq)
◦
at 0 C. The reaction mixture was allowed to warm up to r.t
4-Chloro-N-(3-oxo-2-propyl-3,4-dihydro-2H-benzo
over a period of 1 h and reaction overnight. The reaction mix-
ture was evaporated and dissolved in 10 mL water. 1.0 N HCl
was slowly added to the mixture (pH 4), and the resulting
solid was filtered and rinsed with water. The crude prod-
uct was purified on a silica column eluting with petroleum
ether/ethyl acetate (7:1) to get the products 14a as brown
[
b][1,4]oxazin-7-yl)benzenesulfonamide (16b)
The title compound was prepared from 12 and 4-
chlorobenzenesulfonyl chloride according to the procedure
for example 16a and isolated as brown solid (0.22 g, 56.9%);
1
H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 10.15
1
crystals (0.19 g, 55.2%); H NMR (400 MHz, DMSO-d6) δ
(
s, 1H), 7.76–7.55 (m, 4H), 6.79–6.57 (m, 3H), 4.50 (dd,
J = 8.4, 4.4 Hz, 1H), 1.80–1.54 (m, 2H), 1.47–1.29 (m,
H), 0.88 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, DMSO-
d6) δ 166.00 (s), 142.48 (s), 137.98 (s), 137.73 (s), 132.28
s), 129.35 (s), 128.61 (s), 124.40 (s), 115.74 (s), 115.30 (s),
1
0.54 (s, 1H), 10.15 (s, 1H), 7.75–7.57 (m, 4H), 6.81–6.54
1
3
1
3
(m, 3H), 2.46 (s, 3H), 1.33 (d, J = 1.5 Hz, 6H); C NMR
2
(100 MHz, DMSO-d6) δ 168.59 (s), 142.37 (s), 138.50 (s),
1
1
38.21 (s), 132.85 (s), 129.82 (s), 129.09 (s), 125.16 (s),
(
1
15.92 (s), 115.74 (s), 110.66 (s), 77.99 (s), 23.70 (s); MS
09.78 (s), 75.86 (s), 31.80 (s), 17.58 (s), 13.52 (s); MS (ESI),
+
+
(ESI), m/z: 347.4 [M+H] .
m/z: 403.3 [M+Na] .
2-Bromo-N-(3-oxo-2-propyl-3,4-dihydro-2H-benzo
N-(2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo
[b][1,4]oxazin-7-yl)benzenesulfonamide (16c)
[b][1,4]oxazin-7-yl)-4-nitrobenzenesulfonamide (17b)
The title compound was prepared from 12 and 2-
bromobenzenesulfonyl chloride according to the procedure
for example 16a and isolated as brown solid (0.34 g, 79.6%);
The title compound was prepared from 13 and 4-
nitrobenzenesulfonyl chloride according to the procedure for
1
example 17a and isolated as brown solid (0.19 g, 49.5%); H
1
H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 10.45 (s,
NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 10.40 (s, 1H),
8.37 (d, J = 8.9 Hz, 2H), 7.94 (d, J = 8.9 Hz, 2H), 6.75 (d,
J = 8.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.2 Hz, 1H), 6.64 (d,
1
H), 8.02 (d, J = 7.1 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.54
(
(
(
dd, J = 14.2, 7.2 Hz, 2H), 6.68 (d, J = 11.4 Hz, 3H), 4.49
1
3
d, J = 3.5 Hz, 1H), 1.61 (dd, J = 20.2, 14.5 Hz, 2H), 1.37
J = 2.1 Hz, 1H), 1.33 (s, 6H); C NMR (100 MHz, DMSO-
d6) δ 168.59 (s), 150.27 (s), 145.09 (s), 142.43 (s), 132.31
(s), 128.76 (s), 125.47 (s), 125.04 (s), 116.01 (s), 110.92 (s),
1
3
d, J = 7.4Hz, 2H), 0.87(t, J = 7.2 Hz, 3H); CNMR(100
MHz, DMSO-d6) δ 165.93 (s), 142.50 (s), 138.01 (s), 135.36
s), 134.54 (s), 132.01 (s), 131.78 (s), 128.17 (s), 123.87 (s),
+
(
78.03 (s), 23.71 (s); MS (ESI), m/z: 400.2 [M+Na] .
1
23

Mol Divers
N-(2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo
mM L-Glutamine. The cell lines were cultured in a humidi-
fied atmosphere with 5% CO2 at 37 C.
◦
[b][1,4]oxazin-7-yl)-4-methoxy-2-nitrobenzenesulfonamide
(
17c)
Cell viability assay
The title compound was prepared from 13 and 4-methoxy-
-nitrobenzenesulfonyl chloride according to the procedure
2
The cell viability treated with the compounds described
above was measured by MTT assay. 100 µl of medium con-
taining various concentrations of compound was added to
96-well micro-titer plates, and then, cells were seeded in
for example 17a and isolated as brown solid (0.27 g, 65.7%);
H NMR (400 MHz, DMSO-d6) δ 10.57 (s,1H), 10.33 (s,
1
1
3
H), 7.81 (s, 1H), 7.56 (s, 1H), 7.32 (s, 1H), 6.69 (s, 3H),
.87 (s, 3H), 1.35 (s, 6H); 13C NMR (100 MHz, DMSO-
4
each well at a density of 5–10 × 10 cells per well. After
d6) δ 165.86 (s), 142.65 (s), 138.00 (s), 137.73 (s), 132.27
s), 129.37 (s), 128.60 (s), 124.42 (s), 115.73 (s), 115.26 (s),
cultured for 72 h, the cells were added 20 µl MTT solution
(
◦
(
5 mg/mL) and incubated for another 1–4 h at 37 C. When
1
09.77 (s), 77.06 (s), 23.18 (s), 9.00 (s); MS (ESI), m/z: 430.6
+
reached the appropriate time, the formazan crystal formed by
living cells was dissolved with 50 µL of the 20% SDS solu-
tion overnight. Then, the optical density was measured using
Spectra MAX M5 microplate spectrophotometer (Molecular
Devices) at 570 nm and the IC50 values were carried out.
[M+Na] .
N-(2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo
b][1,4]oxazin-7-yl)-2-fluorobenzenesulfonamide (17d)
[
The title compound was prepared from 13 and 2-
fluorobenzenesulfonyl chloride according to the procedure
for example 17a and isolated as brown solid (0.1 g, 28.2%);
AlphaScreenTM
1
H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 10.40 (s,
AiphascreenTM assays were performed with minor mod-
1
7
3
H), 7.77 (t, J = 7.2 Hz, 1H), 7.68 (d, J = 5.6 Hz, 1H),
.45–7.37 (m, 1H), 7.34 (t, J = 7.5 Hz, 1H), 6.75–6.61 (m,
H), 1.32 (s, 6H); 1 C NMR (100 MHz, DMSO-d6) δ 168.06
ifications. All reagents were diluted in the recommended
buffer (50 mM HEPES, pH 7.4). BRD4 (D1) protein (Bio-
genie, Canada) was added to microplate followed by non-
biotinylated peptide, solvent, or compound. After incubated
at room temperature for 60 min, the plates were added accep-
tor and donor solution, and then incubated for 60 min at room
temperature under subdued light. When reached the appro-
priate time, the plate was read on a PHERAstar FS plate
reader (BMG Labtech, Germany). The data was fit using
GraphPad Prism 5 to obtain inhibition values using.
3
(
s), 156.82 (s), 141.86 (s), 135.93 (s), 132.14 (s), 130.45 (s),
1
1
3
24.89 (s), 124.40 (s), 117.29 (s), 117.08 (s), 115.38 (s),
14.45 (s), 109.39 (s), 77.48 (s), 23.18 (s); MS (ESI), m/z:
73.3 [M+Na] .
+
N-(2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]
oxazin-7-yl)-2-(trifluoromethyl)benzenesulfonamide (17e)
The title compound was prepared from 13 and 2-
Acknowledgements The authors greatly appreciate the financial sup-
port from the Natural Science Foundation of China (No. 81502919)
and China Postdoctoral Science Foundation (No. 2015M570790, No.
(trifluoromethyl)benzenesulfonyl chloride according to the
procedure for example 17a and isolated as brown solid (0.15
2016T90860).
1
g, 37.2%); H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H),
1
0.45 (s, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.98 (d, J = 7.1
Hz, 1H), 7.84 (td, J = 13.6, 7.7 Hz, 2H), 6.77–6.61 (m, 3H),
.32 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 168.06
1
References
(
(
(
[
s), 141.92 (s), 138.18 (s), 133.40 (s), 133.27 (s), 132.16
s), 130.69 (s), 128.51 (s), 124.44 (s), 115.46 (s), 114.60
s), 109.54 (s), 77.52 (s), 23.19 (s); MS (ESI), m/z: 423.5
1
. Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP,
Barsyte-Lovejoy D, Felletar I, Volkmer R, Muller S, Pawson T,
Gingras AC, Arrowsmith CH, Knapp S (2012) Histone recogni-
tion and large-scale structural analysis of the human Bromodomain
family. Cell 149:214–231. doi:10.1016/j.cell.2012.02.013
+
M+Na] .
2
. Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O,
Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro
S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ,
Schwartz B, Heightman TD, La Thangue N, French CA, Wiest O,
Kung AL, Knapp S, Bradner JE (2010) Selective inhibition of BET
bromodomains. Nature 468:23–30. doi:10.1038/nature09504
. Fu L, Tian M, Li X, Li J, Huang J, Ouyang L, Zhang Y, Liu B
Cell culture
Cell lines MV4-11, OCI-LY10, Pfeifer, and Su-DHL-6 were
purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). All of them were cultured in RPIM
3
1640 or Iscove’s Modified Dulbecco’s Medium supple-
(2015) Inhibition of BET bromodomains as a therapeutic strategy
mented with 10% fetal bovine serum (Gibco, Auckland,
N.Z.), penicillin-streptomycin (Life Technologies), and 4
for cancer drug discovery. Oncotarget 6:5501–5516. doi:10.18632/
oncotarget.3551
1
23

Mol Divers
4
. French CA (2012) Pathogenesis of NUT midline car-
cinoma. Annu Rev Pathol 7:247–265. doi:10.1146/
annurev-pathol-011811-132438
12. Wang F, Liu H, Blanton WP, Belkina A, Lebrasseur NK, Denis GV
(2010) Brd2 disruption in mice causes severe obesity without Type
2 diabetes. Biochem J 425:71–83. doi:10.1042/BJ20090928
13. Sun Y, Huang J, Song K (2015) BET protein inhibition mit-
igates acute myocardial infarction damage in rats via the
TLR4/TRAF6/NF-κB pathway. Exp Therap Med 10:2319–2324.
doi:10.3892/etm.2015.2789
5
. Scanlan MJ, Altorki NK, Gure AO, Williamson B, Jungbluth A,
Chen YT, Old LJ (2000) Expression of cancer-testis antigens
in lung cancer: definition of bromodomain testis-specific gene
(BRDT) as a new CT gene, CT9. Cancer Lett 150:155–164. doi:10.
1
016/S0304-3835(99)00385-7
14. Jung KH, Das A, Chai JC, Kim SH, Morya N, Park KS, Lee YS,
Chai YG (2015a) RNA sequencing reveals distinct mechanisms
underlying BET inhibitor JQ1-mediated modulation of the LPS-
induced activation of BV-2 microglial cells. J Neuroinflammation
12:36–54. doi:10.1186/s12974-015-0260-5
15. Yu L, Wang Z, Zhang Z, Ren X, Lu X, Ding K (2015) Small-
molecule BET inhibitors in clinical and preclinical development
and their therapeutic potential. Curr Top Med Chem 15:776–794.
doi:10.2174/1568026615666150302110135
16. Brand M, Measures AM, Wilson BG, Cortopassi WA, Alexander
R, Höss M, Hewings DS, Rooney TP, Paton RS, Conway SJ (2014)
Small molecule inhibitors of Bromodomain–acetyl-lysine interac-
tions. ACS Chem Biol 10:22–39. doi:10.1021/cb500996u
17. Jung M, Gelato KA, Fernandez-Montalvan A, Siegel S, Haendler B
(2015b)TargetingBETbromodomainsforcancertreatment. Epige-
nomics 7:487–501. doi:10.2217/epi.14.91
18. Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME,
Cook AS, Federov O, Gerstenberger BS, Jones H, Knapp S, Mars-
den B, Nocka K, Owen DR, Philpott M, Picaud S, Primiano MJ,
Ralph MJ, Sciammetta N, Trzupek JD (2012) Identification of a
chemical probe for bromo and extra C-terminal bromodomain inhi-
bition through optimization of a fragment-derived hit. J Med Chem
55:9831–9837. doi:10.1021/jm3010515
6
. Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA,
Magoon D, Qi J, Blatt K, Wunderlich M, Taylor MJ, Johns C,
Chicas A, Mulloy JC, Kogan SC, Brown P, Valent P, Bradner JE,
Lowe SW, Vakoc CR (2011) RNAi screen identifies Brd4 as a
therapeutic target in acute myeloid leukaemia. Nature 478:524–
528. doi:10.1038/nature10334
7
. Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM,
Kastritis E, Gilpatrick T, Paranal RM, Qi J, Chesi M, Schinzel AC,
McKeown MR, Heffernan TP, Vakoc CR, Bergsagel PL, Ghobrial
IM, Richardson PG, Young RA, Hahn WC, Anderson KC, Kung
AL, Bradner JE, Mitsiades CS (2011) BET bromodomain inhibi-
tion as a therapeutic strategy to target c-Myc. Cell 146:904–17.
doi:10.1016/j.cell.2011.08.017
. Chaidos A, Caputo V, Gouvedenou K, Liu B, Marigo I, Chaudhry
MS, Rotolo A, Tough DF, Smithers NN, Bassil AK (2014)
Potent antimyeloma activity of the novel bromodomain inhibitors
I-BET151 and I-BET762. Blood 123:697–705. doi:10.1182/
blood-2013-01-478420
8
9
. Fowler T, Ghatak P, Price DH, Conaway R, Conaway J, Chiang
CM, Bradner JE, Shilatifard A, Roy AL (2014) Regulation of MYC
expression and differential JQ1 sensitivity in cancer cells. PLoS
ONE 9:e87003. doi:10.1371/journal.pone.0087003
1
0. Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff
M, Chan WI, Robson SC, Chung CW, Hopf C, Savitski MM, Huth-
macher C, Gudgin E, Lugo D, Beinke S, Chapman TD, Roberts EJ,
Soden PE, Auger KR, Mirguet O, Doehner K, Delwel R, Burnett
AK, Jeffrey P, Drewes G, Lee K, Huntly BJ, Kouzarides T (2011)
Inhibition of BET recruitment to chromatin as an effective treat-
ment for MLL-fusion leukaemia. Nature 478:529–33. doi:10.1038/
nature10509
19. Picaud S, Da Costa D, Thanasopoulou A, Filippakopoulos P, Fish
PV, Philpott M, Fedorov O, Brennan P, Bunnage ME, Owen
DR, Bradner JE, Taniere P, O’Sullivan B, Muller S, Schwaller
J, Stankovic T, Knapp S (2013) PFI-1, a highly selective protein
interaction inhibitor, targeting BET Bromodomains. Cancer Res
73:3336–3346. doi:10.1158/0008-5472
20. Li J, Wang P, Zhou B, Shi J, Liu J, Li X, Fan L, Zheng Y, Ouyang L
(2016) Development of 4,5-dihydro-benzodiazepinone derivatives
as a new chemical series of BRD4 inhibitors. Eur J Med Chem
121:294–299. doi:10.1016/j.ejmech.2016.05.057
1
1. Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung
CW, Chandwani R, Marazzi I, Wilson P, Coste H, White J, Kir-
ilovsky J, Rice CM, Lora JM, Prinjha RK, Lee K, Tarakhovsky A
(2010) Suppression of inflammation by a synthetic histone mimic.
Nature 468:1119–1123. doi:10.1038/nature09589
1
23