Discovery of a series of dihydroquinoxalin-2(1H)-ones as selective BET inhibitors from a dual PLK1-BRD4 inhibitor

Article abstract of DOI:10.1016/j.ejmech.2017.05.049

Recent years have seen much effort to discover new chemotypes of BRD4 inhibitors. Interestingly, some kinase inhibitors have been demonstrated to be potent bromodomain inhibitors, especially the PLK1 inhibitor BI-2536 and the JAK2 inhibitor TG101209, which can bind to BRD4 with IC₅₀ values of 0.025 μM and 0.13 μM, respectively. Although the concept of dual inhibition is intriguing, selective BRD4 inhibitors are preferred as they may diminish off-target effects and provide more flexibility in anticancer drug combination therapy. Inspired by BI-2536, we designed and prepared a series of dihydroquinoxalin-2(1H)-one derivatives as selective bromodomain inhibitors. We found compound 54 had slightly higher activity than (+)-JQ1 in the fluorescence anisotropy assay and potent antiproliferative cellular activity in the MM.1S cell line. We have successfully solved the cocrystal structure of 52 in complex with BRD4-BD1, providing a solid structural basis for the binding mode of compounds of this series. Compound 54 exhibited high selectivity over most non-BET subfamily members and did not show bioactivity towards the PLK1 kinase at 10 or 1 μM. From in vivo studies, compound 54 demonstrated a good PK profile, and the results from in vivo pharmacological studies clearly showed the efficacy of 54 in the mouse MM.1S xenograft model.

Full text of DOI:10.1016/j.ejmech.2017.05.049

Accepted Manuscript
Discovery of a series of dihydroquinoxalin-2(1H)-ones as selective BET inhibitors
from a dual PLK1-BRD4 inhibitor
Jianping Hu, Yingqing Wang, Yanlian Li, Lin Xu, Danyan Cao, ShanShan Song,
Mohammadali Soleimani Damaneh, Xin Wang, Tao Meng, Yue-Lei Chen, Jingkang
Shen, Zehong Miao, Bing Xiong
PII:
S0223-5234(17)30418-X
10.1016/j.ejmech.2017.05.049
EJMECH 9479
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2017
Revised Date: 19 May 2017
Accepted Date: 23 May 2017
Please cite this article as: J. Hu, Y. Wang, Y. Li, L. Xu, D. Cao, S. Song, M.S. Damaneh, X. Wang, T.
Meng, Y.-L. Chen, J. Shen, Z. Miao, B. Xiong, Discovery of a series of dihydroquinoxalin-2(1H)-ones
as selective BET inhibitors from a dual PLK1-BRD4 inhibitor, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.05.049.
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
1
2
Discovery of a Series of Dihydroquinoxalin-2(1H)-ones as Selective BET
Inhibitors from a Dual PLK1-BRD4 inhibitor
a,c,#
b,#
a,#
b,c
a
b
3
4
5
6
7
8
9
0
1
2
3
Jianping Hu , Yingqing Wang , Yanlian Li , Lin Xu , Danyan Cao , ShanShan Song ,
b,c
a
a
a
a,*
Mohammadali Soleimani Damaneh , Xin Wang , Tao Meng , Yue-Lei Chen , Jingkang Shen ,
b,*
a,*
Zehong Miao , Bing Xiong
a
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
b
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
1
1
1
1
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
201203, China
c
University of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing 100049, China
#
1
1
1
1
1
1
2
2
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
5
6
These authors contributed equally
*
Corresponding authors. Tel: +86 21 50806600 ext. 5412 fax: +86 21 50807088.
Email: (B. X.) bxiong@simm.ac.cn; (Z. M.) zhmiao@simm.ac.cn; (J. S.)
jkshen@simm.ac.cn
Abbreviations
PLK1, Polo-like Kinase 1;
JAK2, Janus kinase 2;
PTMs, post-translational modifications
KAc, acetylated lysine;
BET, Bromodomain and Extra-Terminal;
P-TEFb, positive transcription elongation factor B;
BRD4-BD1, first bromodomain of BRD4;
SAR, structure- activity relationship;

ACCEPTED MANUSCRIPT
1
2
3
FA, fluorescence anisotropy
SSRF, Shanghai Synchrotron Radiation Facility.
4
Abstract
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Recent years have seen much effort to discover new chemotypes of BRD4 inhibitors.
Interestingly, some kinase inhibitors have been demonstrated to be potent
bromodomain inhibitors, especially the PLK1 inhibitor BI-2536 and the JAK2
inhibitor TG101209, which can bind to BRD4 with IC values of 0.025 µM and 0.13
5
0
µM, respectively. Although the concept of dual inhibition is intriguing, selective
BRD4 inhibitors are preferred as they may diminish off-target effects and provide
more flexibility in anticancer drug combination therapy. Inspired by BI-2536, we
designed and prepared a series of dihydroquinoxalin-2(1H)-one derivatives as
selective bromodomain inhibitors. We found compound 54 had slightly higher
activity than (+)-JQ1 in the fluorescence anisotropy assay and potent antiproliferative
cellular activity in the MM.1S cell line. We have successfully solved the cocrystal
structure of 52 in complex with BRD4-BD1, providing a solid structural basis for the
binding mode of compounds of this series. Compound 54 exhibited high selectivity
over most non-BET subfamily members and did not show bioactivity towards the
PLK1 kinase at 10 or 1 µM. From in vivo studies, compound 54 demonstrated a good
PK profile, and the results from in vivo pharmacological studies clearly showed the
efficacy of 54 in the mouse MM.1S xenograft model.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
Keywords: BRD4 inhibitor; kinase; BI-2536; dihydroquinoxalin-2(1H)-one.

ACCEPTED MANUSCRIPT
1
Introduction
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Acetylation of histone lysine residues is one of the most widely studied post-
translational modifications (PTMs) that regulate chromatin structure and gene
,
expression in the cell [1] [2]. Acetylated histones are recognized by “readers”, which
are typically found in chromatin- and transcription-associated proteins that partake in
many protein−protein interactions, facilitating the formation of the protein complexes
,
,
that drive active transcription [3] [4]. So far, three readers (bromodomain [5] [6],
,
double PHD finger [7] [8], and pleckstrin homology domain) [9] were identified to
recognize acetylated lysine (KAc) and among them the bromodomain is the most
thoroughly characterized [10]. Totally there are 61 bromodomains distributed in 46
different proteins encoded in human genome. These bromodomains can be divided
into eight distinct families [11]. Of the 61 human bromodomains known, the bromo
and extra-terminal domain (BET) proteins have recently emerged as druggable targets
for the development of new anticancer agents owing to their roles in the
transcriptional regulation of genes involved in tumor development and survival
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
,
[12] [13]. The BET family is characterized by double bromodomains and consists of
BRD2, BRD3, BRD4, and BRDT [14]. Particularly, given that BRD4 regulate the
transcription of oncogene c-Myc, the inhibition of BRD4 provides an alternative and
indirect strategy to curing disease related to the c-Myc abnormality [15], which is
urgently needed since c-Myc itself is an undruggable transcription factor with no
,
suitable small molecule binding pocket identified yet [16] [17]. In addition, BRD4
inhibition leads to good efficacy in xenograft models representing multiple cancer
,
types [18] [19].
Since the discovery of BRD4 inhibitor (+)-JQ1 (1) [20], some BET bromodomain
inhibitors, including representative molecules I-BET762 (2) [21], PFi-1(3) [22], 4

ACCEPTED MANUSCRIPT
1
2
[23] and RVX-208 (5) [24] (Figure 1), have been investigated as bromodomain
inhibitors [6].
N
N
O
N
N
H
O
N
Cl
I-BET762, 2
3
4
5
Figure 1. Structures of clinical BET bromodomain inhibitors (+)-JQ1 (1), I-BET762
(2), PFi-1 (3), 4, RVX-208 (5) and dual kinase-bromodomain inhibitors BI-2536 (6)
and TG101209 (7).
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Since BRD4 has been shown to be an atypical kinase that phosphorylates Ser2 of
the RNA Pol II carboxy-terminal domain [25], the concept of kinase-bromodomain
dual inhibitors has been explored in recent years. A number of kinase inhibitors have
been identified as bromodomain inhibitors by binding to the KAc binding pocket [26].
Among them, the PLK1 inhibitor BI-2536 (6) and the JAK2 inhibitor TG101209 (7)
[26] (Figure 1) are very potent inhibitors of the BET bromodomains, with IC values
1
1
1
1
1
1
1
1
1
1
5
0
of 0.025 µM and 0.13 µM against BRD4, respectively. On the other side, we hope to
utilize the information of kinase-bromodomain dual inhibitors and obtain more
selective BRD4 inhibitors, as they could offer more flexibility in anticancer drug
combination treatments. Therefore, we designed, synthesized and evaluated a series of
novel dihydroquinoxalin-2(1H)-one derivatives as selective bromodomain inhibitors.
By exploring the structure-activity relationship (SAR) of the new dihydroquinoxalin-

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
2(1H)-one structures, we were able to obtain several potent BRD4 inhibitors in the
fluorescence anisotropy (FA) assay and in the antiproliferation cell based assay.
Comparing with the similar bromodomain inhibitors PFi-1 (3) and 4, we can find that
the scaffold dihydroquinoxalin-2(1H)-one only use the oxygen atom in carbonyl
group to interact with the conserved residue Asn140 of BRD4 and form one hydrogen
bond. While the ring systems of PFi-1 (3) and 4 are flipped, and interact with Asn140
with the amide group to form two hydrogen bonds. Therefore, these chemical similar
inhibitors have very different interaction pattern and distinct binding mode. Finally,
compound 54 was found to be the most potent BRD4 inhibitor with similar activity of
(+)-JQ1 in our FA assay and in the MM.1S cell assay. By solving a cocrystal structure
of BRD4-BD1 complexed with 52, which is very similar to 54, we provided a solid
structure basis for the binding mode of compounds of this series. Compound 54 also
exhibited high selectivity toward most non-BET subfamily members and did not show
bioactivity towards the PLK1 kinase at 10 or 1µM. In in vivo pharmacokinetic
studies, compound 54 demonstrated a good half-life and a sufficient plasma exposure
after oral administration at 10 mg/kg. Additionally, in an in vivo MM.1S cell derived
xenograft model, it demonstrated clear efficacy in inhibiting the tumor growth.
Together, we demonstrated a new direction to obtain selective BRD4 inhibitors
through original dual kinase-bromodomain inhibitors.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
Results and Discussion
Comparison of the binding mode of BI-2536 (6) and our previously-reported 2-
thiazolidinone compound 8 [27] in the BRD4-BD1 binding site showed that both 6
and 8 forms conserved hydrogen bonds with the asparagine residue (N140) that
interacts with the acetylated lysine in histone proteins (Figure 2B, 2C). Further

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
analysis of the crystal structure of 6 bound to BRD4-BD1[28] revealed that the
methylated amide of 6 functions as the ε-N-acetylated lysine group and forms a direct
hydrogen bond with the side chain amide of N140 and a water-mediated hydrogen
bond with Y97, while the methyl group is extended into a hydrophobic subpocket
formed from F83, M132, and C136. These interactions are dominant forces for 6
binding to BRD4-BD1. Additionally, the ethyl group protrudes into a small
hydrophobic subpocket (V87/L92/L94/Y97), and the cyclopentyl moiety and the N-
methylpiperidine point to the solvent, forming no hydrogen bonding with residues
nearby. Analysis of the cocrystal structure of PLK1 bound with 6 (Figure 2A) shows
that one of the pyrimidine nitrogens and the aniline NH form critical hydrogen bonds
with the hinge part of PLK1[29].
1
1
1
1
1
1
1
1
1
1
2
To obtain selective bromodomain inhibitors, one could preserve lactam structure
and remove at least one of nitrogens that made critical hydrogen bonds with hinge
part of PLK1. For example, we used dihydroquinoxalin-2(1H)-one to replace the 7,8-
dihydropteridin-6(5H)-one in BI-2536. Meanwhile, from the superimposed crystal
structures of 6 and 8 with BRD4-BD1, we speculated that substitution at C6 position
of the scaffold may introduce similar interactions with WPF subpocket as compound
4
8 does. Along this line, we explored R substitution in dihydroquinoxalin-2(1H)-one
in order to find a functional group targeting the WPF shelf to enhance its binding
affinity against BRD4.
2
1
A.
B.

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
C.
D.
Figure 2. Analyses of crystal structures of BI-2536 bound to PLK1 and BRD4-BD1.
(A) crystal structure of compound BI-2536 (6) bound to PLK1 kinase domain (PDB
entry: 2RKU); (B) crystal structure of compound BI-2536 bound to BRD4-BD1 (PDB
entry: 4OGI); (C) Superimpose crystal structure of BI-2536 (6) and 8 bound to
BRD4-BD1; (D) Chemical Structures of 8.
Shown in Table 1 are the 1,4-dimethyl-3,4-dihydroquinoxalin-2(1H)-one
2
4
derivatives with various R and R substituents and their BRD4 binding activities. For
4
1
1
1
1
1
1
1
1
R group, 4-methylbenzenesulfonamide substitution (compound 12) showed better
2
potency than other groups (compounds 9-11). For R group, replacement of (S)-Me
with (S)-ethyl reduced the binding activity by almost 2-fold (compare 12 with 13),
while inversion of the stereochemistry of C3 position did not affect the binding
affinity to BRD4 significantly (compare 13 with 14).
2
4
Table 1. Effects of R , R -substituted 1,4-dimethyl-3,4-dihydroquinoxalin-2(1H)-one
a
derivatives on inhibition of BRD4-BD1 by fluorescence anisotropy assay

ACCEPTED MANUSCRIPT
1
2
4
2
4
Cmpd
R
-
R
-
BRD4-BD1
Cmpd
R
R
BRD4-BD1
IC (µM)
IC (µM)
5
0
50
(
+)-JQ1
0.07±0.001
11
1.53±0.22
I-BET762
BI-2536
9
-
-
-
-
0.26±0.01
0.25±0.01
>1.0
12
13
14
0.77±0.14
1.35±0.05
1.47±0.32
1
0
41.89%@1µM
a
2
3
4
5
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE. The fluorescence compound used in the assay was JQ1-FITC (The
synthesis route was provided in our group previous work) [27].
1
3
6
7
8
9
0
1
2
3
Next, we studied the SAR of R and R substitutions and the scaffold
1
modification of dihydroquinoxalin-2(1H)-one. Initially, we explored R group while
preserving the dihydroquinoxalin-2(1H)-one skeleton and N4-cyclopentyl. Exploring
1
1
substitution on the R position with H, Me or Ethyl, we found that R as methyl group
1
1
1
1
had better potency than others (compare 16 with 15 or 17). Secondly, we preserved
1
3
scaffold as dihydroquinoxalin-2(1H)-one and R as Me to explore R group.
Comparison of 16 and 18-24 indicated that better binding activity could be obtained
3
3
when R was cyclopropyl (compound 19), as larger R , including isobutyl, 2-

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
methoxyethyl, 2-morpholinoethyl, (tetrahydrofuran-2-yl)methyl, 2-methoxybenzyl
(compounds 20-24) all reduced the inhibitory activity. Thirdly, we preserved N1-Me
and N4-cyclopropyl to modify scaffold. We changed dihydroquinoxalin-2(1H)-one to
dihydropyrido[2,3-b]pyrazin-2(1H)-one or dihydropyrazino[2,3-b]pyrazin-2(1H)-one
(compare 19 with 25 or 26), and found that 25 had similar potency to 19, while 26
deceased the potency remarkably. Although the scaffold dihydropyrido[2,3-b]pyrazin-
2(1H)-one is very promising, SAR study on this new core is challenging. In current
work, we focus on the scaffold of dihydroquinoxalin-2(1H)-one.
1
3
1
1
Table 2. Effects of R and R substitutions and scaffold modification on dihydroquin-
a
oxalin-2(1H)-one derivatives in the BRD4-BD1 fluorescence anisotropy assay
1
2
BRD4-BD1
1
3
Cmpd
X
Y
R
R
IC (µM)
5
0
(
+)-JQ1
-
-
-
-
-
-
-
-
0.07±0.001
0.26±0.01
I-BET762
BI-2536
-
-
-
-
0.25±0.01
>1.0
1
5
6
C
C
H
1
C
C
Me
0.38±0.04

ACCEPTED MANUSCRIPT
1
1
1
7
8
9
C
C
C
C
0.76±0.21
0.35±0.05
0.25±0.01
C
C
C
Me
Me
Me
20
C
0.73±0.42
2
1
2
C
C
C
C
Me
Me
0.77±0.02
1.03±0.40
2
2
3
4
C
C
C
C
Me
Me
1.04±0.10
>1.0
2
2
5
6
N
N
C
N
Me
Me
0.27±0.02
>1.0
2
a
1
2
The IC in the table was calculated from two independent experimental measurements and
50
expressed as mean ± SE.
3
4
5
6
7
8
Before further exploring the SAR of this series, we need to confirm that the ligand
contacts with the WPF subpocket, and we solved a co-crystal structure of BRD4-BD1
bound with compound 19 (Figure 3A). As expected, the sulfonamide group makes a
turn to enable the 4-methylbenzene go into the WPF subpocket. This showed a
distinct binding conformation as comparing BI-2536 in BRD4-BD1 binding site.

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
Except this remarkable difference, the dihydroquin-oxalin-2(1H)-one scaffold
preserved the essential hydrogen bonding interactions as BI-2536 and located at
almost exact position in the binding site. By comparing the solved structure with PFi-
1 (3) and 4 (PDB entry: 4E96 and 4NYW), we can find that the scaffold
dihydroquinoxalin-2(1H)-one only uses the oxygen atom in carbonyl group to interact
with the conserved residue Asn140 of BRD4 and forms one hydrogen bond. While
the ring systems of PFi-1 (3) and 4 are flipped, and interact with Asn140 with the
amide group to form two hydrogen bonds. Therefore, the comparison indicated that
the binding mode is different from PFi-1 or 4, and represented as an interesting
scaffold for bromodomain inhibitor development.
1
1
1
A.
B.
1
1
1
1
1
2
3
4
5
6
C.
D.
Figure 3. Crystal structures of compounds 19, 35, 52, 53 bound to BRD4-BD1. (A)
superimposed structures of 19 (green, PDB entry: 5XHY) and BI-2536 (yellow)
bound to BRD4-BD1; (B) superimposed structures of 52 (yellow, 5XI2) and 19
(green) bound to BRD4-BD1; (C) superimposed structures of 52 (yellow) and 35

ACCEPTED MANUSCRIPT
1
2
3
(gray, PDB entry: 5XI3) bound to BRD4-BD1; (D) superimposed structures of 52
(yellow) and 53 (blue, PDB entry: 5XI4) bound to BRD4-BD1.
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
To further probe the SAR of R group, we synthesized various sulfonamides,
carbamates and amine groups to attach the core phenyl ring (Table 3). From
4
compounds 27-36, it was found that when R was (S)-1-phenylethan-1-amine (36) it
4
showed better potency than those having R as sulfonamides or carbamates.
Replacement
of
4-methylbenzenesulfonamide
group
with
a
4-
methoxybenzenesulfonamide group reduced the inhibitory activity by almost 2.3-fold
(compare 19 with 27), and replacing the 4-methylbenzenesulfonamide group with a 4-
methylbenzamide group maintained binding affinity (comparing 19 with 31).
Changing the sulfonamides to carbamates reduced potency by almost 3.3-fold
(comparing 19 with 29). Further investigation found that adding an ethyl group to the
nitrogen atom of 4-methylbenzenesulfonamide diminish potency significantly
(compare 19 with 28). In addition, adding an ethyl group to the nitrogen atom of
benzylamine reduced the inhibitory activity by almost 1.7-fold (compare 32 with 33).
These results indicated that adding another alkyl group to the nitrogen atom which
attached to the core phenyl ring was detrimental to potency.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
4
Using commercial available building blocks, we further explored the SAR of R on
the basis of 1-phenylethan-1-amine. As shown in Table 3, we found that compounds
with methyl or ethyl substitution at the methylene of benzylamine (34 or 37) had
better potency that the unsubstituted one (32), larger n-propyl-substituted compound
38 demonstrated marginally reduced potency, and tert-butyl-substituted compound 39
and dimethyl-substituted compound 40 showed significantly decreased potency.

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Cyclizing methyl to the benzene group decreased activity remarkably (comparing 34
with 41).
Next, we turned our attention to the substitutions on benzene of benzylamine.
Comparing 43-46 with 34, we found that Me, F, Cl or OMe substituents at the para
position of benzene improved binding affinity slightly, and the electronic nature of the
substituents on this position was not critical for the binding. As demonstrated by
compounds 42 and 43, the position of the methyl substituent at the benzene ring was
important for the inhibitory activity: the para-methyl substituted compound (43) was
more potent than the ortho-methyl substituted compound (42). Moreover, 2,4-
dimethyl substituted compound 47 had better potency than the 4-methyl substituted
43. These indicated that WPF subpokcet is critical for the binding interaction.
Furthermore, comparing 35 with 36, better potency of (S)-1-phenylethan-1-amine
substituted 36 than (R)-1-phenylethan-1-amine substituted 35 indicated
stereochemistry of the benzylamine is important. We preserved (S)-1-phenylethan-1-
amine and synthesized compounds 48-51. As expected, (S)-compounds are more
potent than their racemic analogues (43, 44, 46 and 47), confirmed the (S)-1-
phenylethan-1-amine was the preferred configuration for binding at WPF shelf.
1
1
1
1
1
1
1
1
1
1
2
2
4
Table 3. Effects of R position in 4-cyclopropyl-1,3-dimethyl-3,4-dihydroquinoxalin
-2(1H)-one derivatives on inhibition of BRD4-BD1 from fluorescence anisotropy
a
assays.
2
2

ACCEPTED MANUSCRIPT
4
4
Cmpd
R
BRD4-BD1
Cmpd
R
BRD4-BD1
IC (µM)
IC (µM)
5
0
50
(+)-JQ1
-
0.07±0.001
38
0.61±0
I-BET762
BI-2536
-
-
0.26±0.01
0.25±0.01
0.25±0.01
39
40
41
20.08%@1µM
19.31%@1µM
43.79%@1µM
1
9
2
2
7
8
0.58±0.07
42
43
0.27±0.02
0.17±0.02
13.16%@1µM
2
3
9
0
0.83±0.05
0.81±0.11
44
45
0.27±0.04
0.21±0.01
3
1
0.24±0.003
46
0.28±0.001
3
3
3
2
3
4
0.46±0.01
0.79±0.05
0.34±0.04
47
48
49
0.12±0.01
0.087±0.01
0.11±0.01
3
5
38.35%@1µM
50
0.23±0.004

ACCEPTED MANUSCRIPT
3
3
6
7
0.18±0.01
51
0.10±0.001
0.41±0.01
a
1
2
3
The IC in the table was calculated from two independent experimental measurements and
50
expressed as mean ± SE.
4
5
6
7
8
9
0
1
2
3
4
Although compounds 48 and 51 had the best activities than others, both
demonstrated as a pair of diastereomers due to the chirality of C3 of scaffold. To
assess the SAR of individual diastereomers, we resolved 48 and 51 by means of
chromatography (supporting information Table S1, Figure S1 and Figure S2) and
obtained compounds 52-55 in Table 4. Surprisingly, we found (3R)-enantiomer had
better potency than (+)-JQ-1, I-BET762 and BI-2536 in FA assays (52 and 54), while
(3S)-enantiomer diminished potency dramatically (53 and 55), implying that the
1
1
1
1
1
2
chirality of R was very important for this type of compounds.
Table 4. Effects of Compounds 36, 43, 47-49 and 51-55 on Inhibition of BRD4-BD1
in FA Assay and Antiproliferation Effects against Cell Lines MM.1S and TY-82
1
5
BRD4-BD1
MM.1S
TY-82
2
5
6
a
a
a
Cmpd
R
-
R
R
-
IC (µM)
IC (µM)
IC (µM)
5
0
50
50
(
+)-JQ1
-
0.07±0.001
0.019±0.009
0.018±0.007

ACCEPTED MANUSCRIPT
I-BET762
BI-2536
-
-
-
-
-
0.26±0.01
0.23±0.027
0.20±0.015
0.001±0.0
b
-
0.25±0.01
N.T.
3
6
3
Me
Me
H
0.18±0.01
0.17±0.02
0.74±0.013
0.38±0.010
0.50±0.071
0.31±0.065
4
Me
Me
4-Me
4
4
4
7
8
9
Me
Me
Me
2,4-dimethyl
4-Me
0.12±0.01
0.087±0.01
0.11±0.01
0.10±0.036
0.081±0.005
0.33±0.019
0.095±0.032
0.058±0.006
0.35±0.026
4-F
5
5
5
1
2
3
Me
2,4-dimethyl
4-Me
0.10±0.001
0.052±0.007
16.98%@1µM
0.10±0.018
0.065±0.016
N.T.
0.23±0.031
0.047±0.016
N.T.
4-Me
5
5
4
5
2,4-dimethyl
0.045±0.002
0.016±0.008
N.T.
0.063±0.020
N.T.
2,4-dimethyl 16.08%@1µM
a
1
2
3
The IC in the table was calculated from two independent experimental measurements and
50
b
expressed as mean ± SE, N.T. represents not tested.
4
5
6
7
8
9
To visualize the stereochemistry effect on the binding activity, we solved three
crystal structures of BRD4-BD1 bound with the representative compounds 35, 52 and
53. From chemical structure point of view, compound 35 contains ((R)-1-
phenylethyl)amino group and a racemic scaffold; compound 52 contains ((S)-1-
phenylethyl)amino group and (R)-4-cyclopropyl-1,3-dimethyl-3,4-dihydroquinoxalin-
2(1H)-one scaffold; while compound 53 differed from 52 only at the scaffold,

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
containing (S)-4-cyclopropyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one. By
comparing the crystal structures of 35 and 52 (Figure 3C), we found that different
chiral phenylethyl-amino groups showed almost no difference in binding
conformation, except for the rotation of cyclopropyl of the scaffold. The notable
difference in BRD4-BD1 around the stereocenter was downward of the residue
TRP81 on WPF shelf, which implies the Van der Waals interactions between 35 and
WPF were attenuated. This is not surprising because this series of BRD4 inhibitors
are mainly exploring the SAR around the WPF shelf and the binding affinity is very
sensitive around this subpocket as demonstrated by several compounds shown in
Table 3. To understand the remarkable difference between compounds 52 and 53, we
compared their cocrystal structures (Figure 3D) and identified the major difference
lies at the dihydroquinoxalin-2(1H)-one scaffold. Binding interactions of 53 with
BRD4-BD1 showed the methyl group stays almost in the plane of scaffold, forcing
the surrounding residues TYR139 slightly shifted. The distance of the methyl carbon
atoms of 53 to the nearest atom of TYR139 is 3.3 Å, which is not in the favorable
range of carbon-carbon interaction. By utilizing Schrödinger program to minimize
compound 53 in water, we also found the methyl group significantly deviated away
from the scaffold plane (see supporting information Figure S4). Both evidences
implied that the ligand 53 must adopted a high-energy conformation to interact with
the binding site of BRD4-BD1. These may account for the diminished binding affinity
of 53 when compared to compound 52.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
Bromodomain Selectivity Screening
Bromodomain is the conserved module in evolution, and they share a common 3D
structure pattern of one short helix (helix Z) and three long helices (helix A−C). The
acetylated lysine can bind to the top of the bromodomains by forming several

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
hydrogen bonds with the conserved residue asparagine (Asn140 in BRD4). As
selectivity is critical for the success of drug discovery, we selected the most potent
BET inhibitor 54 to profile the binding specificity in 32 representative bromodomain
modules by using the DiscoveRx BROMOScan platform at 1 µM concentration. As
showed in Figure 4, it was clearly shown that compound 54 was generally selective
inhibitors for BET subfamily over other non-BET family except its moderate
inhibition on EP300/CREBBP (The detail number was provided as Table S3 in
supporting material).
9
0
1
1
1
1
1
Figure 4. Bromodomain selectivity profile of compound 54. Red, Purple red, Orange,
Purple gray and Gray dots indicate the assay ctrl% range: 0-5%, 5−20%, 20-50%, 50-
70% and 70-100%, respectively, in the DiscoveRX assay at 1 µM concentration. The
ctrl%=(test compound signal – positive control signal)/(negative control signal –
positive control signal) *100%.
1
2
3
4
1
1
5
6
PLK1 Kinase Selectivity Profile

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
We hope to discover a new series of dihydroquinoxalin-2(1H)-one as selective
BRD4 inhibitors by using PLK1-BRD4 dual inhibitor BI-2536 as the starting point.
Thence, on the basis of molecular activity and structural diversity, we chose three
compounds to profile their PLK1 kinase activity, along with BI-2536 and a highly
active ATP competitive kinase inhibitor staurosporine as positive control. From Table
5, we found that staurosporine and BI-2536 showed significant inhibitory effects on
PLK1 kinase at 10 µM or 1 µM. However, compounds 19, 25 and 54 demonstrated
only negligible inhibitory activity for PLK1 kinase at 10 µM or 1 µM as we excepted.
Thus, removing pyrimidine ring nitrogen in BI-2536 as a critical hydrogen acceptor
for kinase hinge binding successfully reduced the inhibitory activity for PLK1 kinase.
1
1
1
Table 5. PLK1 kinase activity of compounds 19, 25 and 54
Cmpd
Inhibition
Inhibition
Cmpd
Inhibition
Inhibition
(%, 10 µM)
(%, 1 µM)
(%, 10 µM)
(%, 1 µM)
Staurosporine
BI-2536
95
100
3
58
101
7
25
54
5
2
7
5
19
1
1
1
2
3
4
Cellular activity
The compounds with better enzymatic activity (IC values ＜0.2 µM) were further
50
1
1
1
1
1
2
5
6
7
8
9
0
assessed in the cellular antiproliferation assay (36, 43, 47-49, 51-52 and 54). The
graphs of cell viability for representative compounds 36, 48, 51, 52, 54, I-BET762
and (+)-JQ-1 were shown in supporting information Figure S5. The results (Table 4)
indicated these compounds had good cellular activities with IC below 1 µM both in
5
0
MM.1S and TY-82 cell lines, and among them compounds 48, 52 and 54 showed
strong proliferation inhibition activity with IC50 below 0.1 µM. Particularly,

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
compound 54 even had similar cellular activities with (+)-JQ1 in MM.1S cell line.
From table 4, we also found BI-2536 showed extremely inhibition on TY-82 cell line,
however, it exhibited moderate binding affinity on BRD4-BD1, so we conjectured its
high cell activity was not only due to its inhibition on BRD4-BD1 but also because of
its inhibition on PLK1 kinase in cell.
Effects on c-Myc Protein and mRNA Expression.
The BRD4 inhibitor (+)-JQ1 induces an antiproliferative effect associated with the
down-regulation of c-Myc transcription. In order to study whether they went through
the BRD4-dependent pathway in the cell, we performed the Western blotting
experiment and quantitative real-time PCR (RT-qPCR) to study the cellular effect
related to c-Myc. On the basis of the protein binding and cellular antiproliferation
assays, we chose 52, 54 and the positive controls (+)-JQ-1, I-BET762, OTX-015, and
BI-2536 to evaluate their effects on c-Myc protein and mRNA expression. As showed
in Figure 5A, both compounds 52 and 54 displayed significant inhibition on the
expression of c-Myc protein both at 0.2 µM and 1 µM. In particular, compound 54
remarkably suppressed the expression of c-Myc protein at 1 µM.
1
1
1
1
1
1
1
1
1
1
2
2
As indicated by RT-qPCR assays in Figure 5B, compounds 52 and 54 strongly
down-regulated the expression of c-myc mRNA in a dose-dependent manner,
showing more than 60% inhibition at 1 µM. Taking the selectivity profiles together,
these data implied that the antiproliferation effects of dihydroquinoxalin-2(1H)-one
series of compounds attributed to the BRD4-dependent pathway.

ACCEPTED MANUSCRIPT
1.2
0.2 µM
1.0 µM
0.8
0.4
0.0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
A.
B.
Figure 5. (A) Inhibition of compounds 52 and 54 on the expression of c-Myc protein.
TY-82 cells were treated with compounds 52 and 54, I-BET762, OTX-015, BI-2536
and (+)-JQ1 (0.2 µM or 1 µM) for 24 h, respectively. Cells were collected and lyzed
for Western blotting. The level of c-Myc protein was detected and GAPDH protein
was chosen as the loading control. The experiments were repeated three times. (B)
Inhibition of compounds 52 and 54 on the expression of c-Myc mRNA. TY-82 cells
were treated as in (A). Then, total RNA was isolated and reverse-transcribed for RT-
qPCR analyses. The data were expressed as mean ± SD, representing the relative
levels of c-myc mRNA from three independent experiments.
1
1
1
1
1
1
1
1
1
1
2
2
In Vivo Study of Compound 54
Based on aforementioned data, compound 54 was selected for further in vivo PK
study. After single oral administration of 10 mg/kg of compound 54 (DMAC/HPMC
formulation) to male ICR (CD-1) mice, the pharmacokinetic parameters of 54 in
plasma samples were summarized in Table 6. The maximum plasma concentration of
54 (869 ng/mL) was observed at 0.33 h after drug administration. At 24 h after
administration, the plasma concentration was below the lower limit of quantity
(LLOQ = 0.3 ng/mL). From Table 6, it showed that compound 54 has a moderate
half-life (2.23 h) and a reasonable plasma exposure of 1689 ng × h/mL, indicating 54
could be advanced for in vivo pharmacological study.

ACCEPTED MANUSCRIPT
1
2
a
Table 6. In Vivo PK Data for Compound 54
Cpd
.
Route of
Dose
Tmax
(h)
Cmax
AUC0-t
AUC0-inf
T1/2
(h)
administration
(mg/kg)
(ng/mL)
(ng·h/mL) (ng·h/mL)
54
p.o.
10
0.33
869
1683
1689
2.23
a
3
4
5
5% dimethylacetamide and 0.5% hydroxypropyl methyl cellulose were used as vehicle.
To evaluate the therapeutic effect of compound 54 in vivo, we established human
6
7
8
9
0
1
2
3
4
MM.1S xenograft model in Balb/c nude mice. Tumor-bearing mice were treated with
54 by intraperitoneal injection (50 mg/kg daily). The result showed that treatment of
compound 54 significantly decreased the growth of xenografts measured by tumor
volume (Figure 6A) but did not cause loss of the body weight (Figure 6B). No animal
death occurred during the experiments. By comparing with the positive control BRD4
inhibitor OTX-015 (oral dosage 50mg/kg daily), 54 and OTX-015 showed similar
inhibition effects about RTV 638% and 603% respectively, although they are
administrated via different route. These data illustrate the therapeutic potential of
compound 54 for tumor treatments.
1
1
1
1
1
1
1
5
6
（A）

ACCEPTED MANUSCRIPT
1
2
（B）
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Figure 6. (A) Relative tumor volume (RTV) of human MM.1S xenografts in Balb/c
nude mice after treatments. **, OTX-015 or compound 54 group versus vehicle
group, p value < 0.01, Formulation: 54: 0.5% Tween80 and 0.5% Methylcellulose
aqueous solution, OTX-015: 5% dimethylacetamide and 0.5% hydroxypropyl methyl
cellulose; (B) Body weight changes of mice.
Chemistry
1
1
1
1
1
1
1
1
1
1
The syntheses of novel dihydroquinoxalin-2(1H)-onederivatives were depicted in
schemes 1-3. In schemes 1, the reaction of commercially available 56 with different
amino acids through aromatic nucleophilic substitutions (S Ar) furnished anilines 57-
N
59, wherein the nitro group directed ortho attack. The dihydroquinoxalin-2(1H)-one
scaffold was next constructed by reductive heterocyclizations of 57-59 into 60-62,
respectively, with tin(II) chloride dihydrate (SnCl ·2H O) and con.HCl. Introduction
2
2
of the methyl group was achieved by deprotonation of the amide NH followed by
quenching with iodomethane to yield 63-65. The 3,6-substituted 1,4-dimethyl-3,4-
dihydroquinoxalin-2(1H)-one derivatives 9-14 were obtained through compounds 63-
65 coupling with different amines or sulfonamides.

ACCEPTED MANUSCRIPT
1
2
Scheme 1 Syntheses of the 3,6-substituted 1,4-dimethyl-3,4-dihydroquinoxalin-
2(1H)-one derivatives 9-14.
3
4
5
6
7
8
2
Reagents and conditions: a) R CH(NH )COOH, K CO , EtOH, reflux, 4 h; b) tin(II) chloride
2
2
3
dihydrate, con.HCl, EtOH, reflux, 5 h; c) NaH, 0 °C, 30 min, iodomethane, rt, 2 h; d) 9 and 10:
different amines, Cs CO , DMF, Pd dba , Xphos, 110 °C, 1h; 11-14: different sulfonamides,
2
3
2
3
K CO , 2-metyhtetrahydrofuran, allylpalladium chloride dimer, tBuXPhos, 85 °C, overnight.
2
3
9
0
1
2
3
4
5
6
7
8
9
0
1
As depicted in Scheme 2, S Ar of different amines to 56 afforded compounds 66-
N
1
1
1
1
1
1
1
1
1
1
2
2
73. Then reduction with Fe powder and NH Cl provided amines 74-81. Compounds
4
82-89 were obtained through two steps, including treatment of 74-81 with 2-
bromopropanoyl bromide to afford corresponding amides, and the ring formation
through intramolecular nucleophilic substitution reaction. For compounds 90-98,
1
introduction of the R group was achieved by deprotonation of the amide NH
followed by quenching with iodomethane or bromoethane. Coupling of 93 and
diverse amines via Buchwald-Hartwig reaction gave compounds 32-51 and 99.
Compounds 15-24 and 27 were obtained through the reaction between intermediates
82 and 90-98 and 4-methylbenzenesulfonamide or 4-methoxybenzenesulfonamide.
Treatment of 93 with tert-butyl carbamate via coupling reaction and subsequent acidic
6
deprotection afforded compound 100. Condensation of 99 with R Cl yielded 28 or 30
in a similar fashion used for producing compound 29 or 31.

ACCEPTED MANUSCRIPT
1
2
Scheme 2. Syntheses of the 1,4,6-substituted 3-methyl-3,4-dihydroquinoxalin-2(1H)-
one derivatives 15-24 and 27-51.
3
4
5
6
7
8
9
0
3
Reagents and conditions: a) R NH , ClCH CH Cl, 80 °C, 12 h; b) Fe, NH Cl, EtOH, 80 °C, 1 h;
2
2
2
4
c) 1. 2-bromopropanoyl bromide, DIPEA, DCM, 0 °C - rt, 2 h; 2. CH CN, DIPEA, 80 °C,
3
overnight; d) iodomethane or bromoethane, NaH, DMF, 0 °C - rt, 2 h; e) 4-
methylbenzenesulfonamide or 4-methoxybenzenesulfonamide, K CO , 2-metyhtetrahydrofuran,
2
3
allylpalladium chloride dimer, tBuXPhos, 85 °C, overnight; f) sodium tert-butoxide, ethylamine
6
7
hydrochloride, toluene, palladium acetate, tBu P, 85 °C, overnight; g) R Cl or R Cl, Et N, DCM,
3
3
1
1
0 °C - rt, overnight; h) 1. tert-butyl carbamate, Cs CO , dioxane, palladium acetate, Xphos, 85 °C,
2 3
1
overnight; 2. TFA, CH Cl , rt, overnight; i) sodium tert-butoxide, toluene, palladium acetate,
2
2
1
1
1
2
3
4
tBu P, 85 °C, overnight.
3
In Scheme 3, treatment of commercially available 101 and 102 with 2-
bromopropanoyl bromide formed amides 103 and 104. The reaction of 103 and 104
with cyclopropylamine gave 105 and 106 through intermolecular nucleophilic
substitution reaction. Compounds 107 and 108 were constructed through
1
1
1
5
6
7

ACCEPTED MANUSCRIPT
1
2
3
4
5
intramolecular S Ar reaction. Deprotonation of the amide NH followed by quenching
N
with iodomethane yielded 109 and 110. Compounds 25 and 26 were obtained through
coupling 109 and 110 with 4-methylbenzenesulfonamide, respectively.
Scheme 3. Synthesis of the dihydropyrido[2,3-b]pyrazin-2(1H)-one or dihydropyra-
zino[2,3-b]pyrazin-2(1H)-one derivative 25 or 26.
6
7
8
9
0
Reagents and conditions: a) 2-bromopropanoyl bromide, Na CO , CH Cl , 0 °C - rt, overnight; b)
2
3
2
2
cyclopropylamine, CH CN, 80 °C, overnight; c) DIPEA, DMF, 150 °C, overnight; d) NaH, 0 °C,
3
30 min, iodomethane, rt, 2 h; e) 4-methylbenzenesulfonamide, K CO , 2-metyhtetrahydrofuran,
2
3
1
1
allylpalladium chloride dimer, tBuXPhos, 85 °C, overnight.
1
2
1
Conclusion
1
1
1
1
1
1
1
3
4
5
6
7
8
9
Inspired by PLK1-BET bromodomain dual inhibitor BI-2536 (6), we rationally
designed, synthesized, and evaluated a series of novel dihydroquinoxalin-2(1H)-one
derivatives as selective bromodomain inhibitors. Through iterative structure-based
designs, we obtained several potent BRD4 inhibitors with IC below 0.1 µM both in
5
0
FA assays and antiproliferation cell based assays. Compound 54 with good potency in
FA and cell assays displayed concentration-dependent inhibition on the expression of
c-Myc protein and c-Myc mRNA, and demonstrated good pharmacokinetic and

ACCEPTED MANUSCRIPT
1
2
3
4
pharmacodynamic properties in mice. Our study demonstrated a practice for
designing selective bromodomain inhibitors starting from the kinase inhibitors.
Experimental Section
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Chemistry
General:
1
H NMR (400 MHz) spectra were recorded by using a Varian Mercury-400 High
Performance Digital FT-NMR spectrometer with tetramethylsilane (TMS) as an
1
3
internal standard. C NMR (126 MHz) spectra were recorded by using a Varian
Mercury-500 High Performance Digital FT-NMR spectrometer. Abbreviations for
peak patterns in NMR spectra: br = broadened, s = singlet, d = doublet, t = triplet, dd
= doublet of doublets and m = multiplet. Low-resolution mass spectra were obtained
with a Finnigan LCQ Deca XP mass spectrometer using a CAPCELL PAK C18
(50mm × 2.0mm, 5 µM) or an Agilent ZORBAX Eclipse XDB C18 (50mm × 2.1m, 5
µM) in positive or negative electrospray mode. High-resolution mass spectra were
recorded by using a Finnigan MAT-95 mass spectrometer. Purity of all compounds
was determined by analytical Gilson high-performance liquid chromatography
(HPLC) using an YMC ODS3 column (50 mm × 4.6 mm, 5 µM). Conditions were as
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
-1
follows: CH CN/H O eluent at 2.5 mLmin flow [containing 0.1% trifluoroacetic acid
3
2
(TFA)] at 35 °C, 8 min, gradient 5% CH CN to 95% CH CN, monitored by UV
3
3
absorption at 214 nm and 254 nm. TLC analysis was carried out with glass precoated
silica gel GF254 plates. TLC spots were visualized under UV light. Flash column
chromatography was performed with a Teledyne ISCO CombiFlash R system. All
f
solvents and reagents were used directly as obtained commercially unless otherwise
noted. All air and moisture sensitive reactions were carried out under an atmosphere

ACCEPTED MANUSCRIPT
1
2
3
4
of dry argon with heat-dried glassware and standard syringe techniques. Melting
points were determined using a SGW X-4 hot stage microscope and are uncorrected.
(spectra data of the synthesized compounds were provided as supporting material)
Synthetic Procedures:
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Compounds 56 and 101-102 were purchased. Other compounds were prepared by one of four
schemes.
Scheme 1. (5-bromo-2-nitrophenyl)-L-alanine (57). To a solution of compound 56 (2 g, 9.09
mmol) and L-alanine (0.81 g, 9.09 mmol) in EtOH (10 mL) was added K CO (1.51 g, 10.91
2
3
mmol) in water (3 mL). The mixture was heated to reflux for 4 hours and monitored by TLC.
Upon completion, EtOH was evaporated. The residue was acidified with 1 N aq. HCl to pH 3 – 4,
diluted with water and extracted with EtOAc (3 × 50 mL). Combined organic layers were washed
with brine, and dried with Na SO to afford compound 57 (2.48 g, 8.58 mmol, 94% yield) as a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
4
1
light yellow solid. H NMR (400 MHz, DMSO-d ) δ 8.41 (d, J = 7.0 Hz, 1H), 8.02 (d, J = 9.1 Hz,
6
1H), 7.24 (s, 1H), 6.90 (d, J = 9.5 Hz, 1H), 4.62 – 4.52 (m, 1H), 1.45 (d, J = 6.9 Hz, 3H).
(S)-6-bromo-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (60). To a solution of compound 57 (2.48
g, 8.58 mmol) in EtOH (20 mL) was mixed with a solution of tin(II) chloride dihydrate (7.74 g,
34.3 mmol) in 10 mL of EtOH and 2.7 mL of con. HCl. The mixture was heated to reflux for 5
hours and monitored through TLC. Upon completion, the reaction mixture was cooled to room
temperature and filtered through Celite. The filtrate was basified with saturated NaHCO solution
3
to pH 6 – 7, diluted with water, and extracted with EtOAc (3 × 50 mL). The combined organic
extracts were washed with brine, dried (Na SO ), and concentrated under reduced pressure.
2
4
Purification by silica gel column chromatography (gradient elution, gradient 0 to 30% EtOAc/60–
90 °C petroleum ether) gave compound 60 (1.80 g, 7.47 mmol, 87% yield) as a light yellow solid.
1
H NMR (400 MHz, CDCl ) δ 9.26 (s, 1H), 6.85 (dd, J = 8.2, 2.0 Hz, 1H), 6.80 (d, J = 1.8 Hz,
3
1H), 6.64 (d, J = 8.2 Hz, 1H), 4.06 – 3.99 (m, 1H), 3.92 (s, 1H), 1.45 (d, J = 6.7 Hz, 3H); LCMS
+
m/z (ESI, positive) found [M + H] 241.05; retention time 2.66 min, > 99% pure.
(S)-6-bromo-1,3,4-trimethyl-3,4-dihydroquinoxalin-2(1H)-one (63). To a solution of compound 60
(1.5 g, 6.22 mmol) in anhydrous DMF (5 mL) was added NaH (1.0 g, 24.88 mmol) portionwise at

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0 °C. The mixture was stirred at 0 °C for 30 min, then iodomethane (1.55 mL, 24.88 mmol) was
added and stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon
completion, the mixture was poured to 100 mL ice water slowly, then the white solid was filtered,
washed with water, dried, and offered the white solid compound 63 (1.37 g, 5.10 mmol, 82%
1
yield). H NMR (400 MHz, CDCl ) δ 6.96 (dd, J = 8.4, 2.1 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 6.74
3
(d, J = 2.1 Hz, 1H), 3.98 (q, J = 6.9 Hz, 1H), 3.34 (s, 3H), 2.84 (s, 3H), 1.12 (d, J = 6.8 Hz, 3H).
(S)-6-((2-methoxyphenyl)amino)-1,3,4-trimethyl-3,4-dihydroquinoxalin-2(1H)-one (9). To
a
solution of compound 63 (220 mg, 0.82 mmol), 2-methoxyaniline (0.14 mL, 1.23 mmol), Cs CO3
2
(534 mg, 1.64 mmol) in anhydrous DMF (30 mL) at room temperature were added Pd dba (23
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
mg, 0.025 mmol) and XPhos (23 mg, 0.049 mmol). The mixture was sealed in a microwave tube
and heated to 110 °C for 1 h. The reaction was monitored by TLC. Upon completion, the reaction
mixture was diluted with water, and extracted with EtOAc (3 × 10 mL). Combined organic layers
were washed with brine, then dried with Na SO . The crude proudct was purified by flash column
2
4
chromatography (gradient elution, gradient 0 to 40% EtOAc/60–90 °C petroleum ether) to give
1
compound 9 (159 mg, 0.51 mmol, 62% yield) as a light white solid. H NMR (400 MHz, CDCl ) δ
3
7.26 – 7.23 (m, 1H), 6.91 – 6.88 (m, 1H), 6.88 – 6.86 (m, 1H), 6.86 – 6.84 (m, 1H), 6.84 – 6.82 (s,
1H), 6.67 (dd, J = 8.4, 2.4 Hz, 1H), 6.46 (d, J = 2.3 Hz, 1H), 3.98 (q, J = 6.8 Hz, 1H), 3.90 (s, 3H),
3.36 (s, 3H), 2.82 (s, 3H), 1.13 (d, J = 6.8 Hz, 3H) (One NH was not seen); LCMS m/z (ESI,
+
positive) found [M + H] 312.20; retention time 3.39 min, > 96% pure.
Following the similar procedures as for compound 9 gave compound 10.
(S)-1,3,4-trimethyl-6-(pyridin-2-ylamino)-3,4-dihydroquinoxalin-2(1H)-one (10). Light yellow
1
solid, 70% yield; H NMR (400 MHz, CDCl ) δ 8.18 (d, J = 4.1 Hz, 1H), 7.48 (t, J = 6.9 Hz, 1H),
3
6.87 (d, J = 8.5 Hz, 1H), 6.85 – 6.80 (m, 2H), 6.73 – 6.69 (m, 1H), 6.66 – 6.62 (m, 2H), 3.99 (q, J
= 6.8 Hz, 1H), 3.37 (s, 3H), 2.84 (s, 3H), 1.14 (d, J = 6.8 Hz, 3H); LCMS m/z (ESI, positive)
+
found [M + H] 283.27; retention time 2.11 min, > 97% pure.
(S)-2-chloro-N-(1,3,4-trimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)benzenesulfonamide
(11). To a solution of compound 63 (150 mg, 0.56 mmol), 2-chlorobenzenesulfonamide (129 mg,
0.67 mmol), K CO (155 mg, 1.12 mmol) in 2-metyhtetrahydrofuran (5 mL) at room temperature
2
3
were added allylpalladium chloride dimer (4 mg, 0.011 mmol) and tBuXPhos (5 mg, 0.011