Development of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives as a novel chemical series of BRD4 inhibitors

Article abstract of DOI:10.1007/s00044-018-2218-5

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histones that regulate gene transcription preferentially at super-enhancer regions. Now BRD4 has been involved into several

Full text of DOI:10.1007/s00044-018-2218-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2218-5
ORIGINAL RESEARCH
Development of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one
derivatives as a novel chemical series of BRD4 inhibitors
Xueting Liu1 Zhenwei Wu2 Jiping Tian2 Xinrui Yuan2 Leilei Zhao2 Pan Chen1 Huibin Zhang2 Jinpei Zhou1
●
●
●
●
●
●
●
Received: 28 November 2017 / Accepted: 6 July 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds
to acetylated histones that regulate gene transcription preferentially at super-enhancer regions. Now BRD4 has been
involved into several types of cancers as a candidate correlated with gene transcription. In this study, we designed and
synthesized 11 novel 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives and evaluated their BRD4 inhibitory activities.
Most of these compounds exhibited moderate BRD4 inhibitory activities in vitro. It is worth noting that compound 14a
showed excellent BRD4(1) and BRD4(2) inhibitory activity with IC50 values of 350 and 290 nm, respectively. Meanwhile,
remarkable anti-proliferative activities toward BRD4-sensitive cancer lines MV4-11 and HL-60 were also observed. In
addition, molecular docking studies was utilized to elaborate the key interactions between compound 14a and BRD4 in
detail. Overall, 7-methylimidazo[1,5-a]pyrazin-8(7H)-one could be employed as a useful scaffold toward more potent BRD4
inhibitors for cancer treatments.
Graphical Abstract
●
●
●
Keywords Gene transcription BRD4 Anticancer Molecular docking
Introduction
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2218-5) contains supplementary
material, which is available to authorized users.
Histone acetylation is a significant component of epigenetic
research which is very important for post-translational
modification (PTM) (Zhao et al. 2010). Bromodomains
(BRDs) are present in a variety of epigenetic regulatory
proteins, including transcriptional activators, histone acet-
yltransferase (HAT), and chromatin (Filippakopoulos et al.
2012). The essence of BRDs is disclosed more and more
thoroughly with the development of BRDs. Although the
proteins of BRDs play a vital function in the treatment of
diseases, but most thorough research at present is the BRD
and extra-terminal (BET) protein. Bromodomain protein 4
(BRD4) is a member of the BET protein family (Shu and
* Huibin Zhang
zhanghb80@cpu.edu.cn
* Jinpei Zhou
jpzhou668@163.com
1
Department of Medicinal Chemistry, China Pharmaceutical
University, 210009 Nanjing, PR China
2
Center of Drug Discovery, Jiangsu Key Laboratory of Drug
Discovery for Metabolic Disease, China Pharmaceutical
University, 210009 Nanjing, PR China

Medicinal Chemistry Research
N
Fig. 1 Structures of unclosed
BRD4 inhibitors
O
N
N
O
Cl
N
O
S
O
N
N
HO
HN
NH
O
N
N
O
N
Cl
O
JQ1
I-BET-151
I-BET726
N
N
N
N
O
H
N
N
N
S
N
H
H
N
N
N
N
F
O
O
O
O
O
O
S
OH
F
N
H
Cl
OTX015
Cl
Abbv-075
I-BET-762
Fig. 2 a Docking conformation of ABBV-075 in BRD4(1) (PDB id: 3P5O). b Docking conformation of 14a in BRD4(1) (PDB id: 3P5O). c
Superimposition docking conformation of ABBV-075 (Yellow) and 14a (Green) in BRD4(1) (PDB id: 3P5O)
Polyak 2017). The BET family of proteins plays a crucial
role in regulating transcriptional programmes (Amorim
et al. 2016; Lin et al. 2016). BET can recruit the positive
transcription elongation factor complex (pTEFb), which is
crucial for transcription elongation via their bromodomains
bounding to acetylated histones (Jang et al. 2005; Mujtaba
et al. 2007; Yang et al. 2005). Meanwhile, gene expression
studies unclosed that BET could moderate key oncogenes
important for cell cycle progression, such as MYC and
E2F1 (Chapuy et al. 2013; Core et al. 2014, 2008; Dawson
et al. 2011; Zhao et al. 2016). Above all, BRD4 bromo-
domain protein has developed to be an interesting drug
target for the treatment of NUT midline carcinoma (NMC),
acute myeloid leukemia (AML), multiple myeloma (MM),
acute lymphoblastic leukemia (ALL), small cell lung car-
cinoma (SCLC), breast cancer, neuroblastoma (NB), and
prostate cancer (Asangani et al. 2014; Ceribelli et al. 2014;
Dawson et al. 2011; Delmore et al. 2011; Lockwood et al.
2012; Mertz et al. 2011; Zuber et al. 2011).
The main body of research in the bromodomain field has
focused on the BET (bromodomain and extra terminal)
family and multiple small molecule inhibitors have been
disclosed, such as JQ1, I-BET 151, I-BET 726, etc. (Fig. 1).
JQ1 was the first unclosed BRD4 inhibitor with IC50 value
as tool compound for a functional role on gene transcription
(Filippakopoulos et al. 2010). However, several BRD4
inhibitors have entered into clinical trials for treating dif-
ferent types of cancers, such as OTX015, GSK762, ABBV-
075, etc. (Fig. 1) (Faivre et al. 2017; Stathis et al. 2016).
ABBV-075 displayed potent antiproliferative activity in
multiple models of resistance to second-generation anti-
androgens (Faivre et al. 2017). However, novel chemical
scaffold for BRD4 inhibitors with excellent ADMET
properties will be necessary for further clinical tests.
Therefore, we designed and synthesized eleven 7-methyli-
midazo[1,5-a]pyrazin-8(7H)-one derivatives via structural
modifications of ABBV-075. Molecular docking studies
were utilized to analyze the interaction with Kac pocket of

Medicinal Chemistry Research
NH2
O
O
O
N
N
NH
N
N
O
a
b
c
N
N
N
N
Br
Br
Br
Br
1
2
3
4
N
O
S
O
O O
N
O
B
O
O
N
Br
F
S
d
e
f
S
R1
R1
O
R1
F
F
F
R1
S
O
O
5a,b
6a,b
7a,b
8a,b
O
O
O
N
O
N
O
N
N
N
N
N
N
N
g
h
i
H
N
H
N
H
N
R2
O
N
R1
N
R1
HN
R1
S
S
S
S
O
O
O
O
O
O
O
9a,b
10a,b
11a-d
Scheme 1 General synthesis of 11a–d. Reagents and conditions: (a)
NaNO2, conc. H2SO4, 0 °C to 45 °C; (b) dimethylsulfate, K2CO3,
acetonitrile, 70 °C; (c) tosylmethyl isocyanide, NaH, THF, 0 °C to rt;
(d) NBS, conc. H2SO4, 50 °C; (e) 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2’-
bi(1,3,2-dioxaborolane), Pd(dppf)Cl2CH2Cl2, KOAc, 1,2-Dimethoxye
thane, 90 °C; (f) 4, Pd(PPh3)4, K2CO3, acetonitrile, toluene, 80 °C; (g)
4-amino-1-Boc-piperidine, DIPEA, DMSO, 110 °C; (h) TFA, CH2Cl2,
0 °C to rt; (i) sulfonyl chloride, 4-methylmorpholine, DMF, rt
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
a
b
c
O
O
O
F
O
O
O
O
HO
R2
R1
R1
R1
R1
S
S
S
S
O
O
O
O
O
O
O
O
8a,b
12a,b
13a,b
14a-c
Scheme 2 General synthesis of 14a–c. Reagents and conditions: (a) methyl 3-hydroxybenzoate, K2CO3, DMF, 85 °C; (b) LiOH, methanol, H2O,
rt; (c) amine, HBTU, DIPEA, DMF, rt
BRD4(1). Carbonyl group of 7-methylimidazo[1,5-a]pyr-
azin-8(7H)-one made a key hydrogen bond with Asn140 at
the bottom of Kac pocket (Fig. 2). Therefore, our deriva-
tives could be as acetylated lysine mimic occupying the
pocket region.
We further assessed the inhibitory potency of our deri-
vatives against BRD4 in vitro. Initial inhibitory activities of
these compounds against BRD4(1) were estimated at a
concentration of 10 μM. Most compounds exhibited mod-
erated BRD4(1) inhibitory activities. The compounds 11d
and 14a–c with relatively higher potency were chosen for
further evaluation of inhibitory activity towards BRD4(1)
and BRD4(2) and antiproliferative activities. We found that
compound showed excellent BRD4(1) and BRD4(2) inhi-
bitory activity with IC50 values of 350 and 290 nm,
respectively. Meanwhile, compound 14a exhibited robust
antiproliferative activity in BRD4-sensitive cancer cells
MV4-11 and HL-60 with IC50 values of 460 and 680 nm
respectively. In addition, compound presented few anti-
proliferative activity in BRD4-independent lines K562.
These findings demonstrated that compound could be a
possible BRD4 inhibitor for cancer treatments.

Medicinal Chemistry Research
Table 1 Structures and BRD4(1) inhibitory activity of compounds 11a-d and 14a-c
Compound
R1
R2
BRD4(1)
(10μM)
BRD4(1)
IC50
BRD4(1) IC50
(μmol/L)
(μmol/L)
b
10a
10b
CH3
CH3CH2
CH3
42%
51%
43%
59%
67%
98%
67%
78%
100%
99%
100%
n.d .
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
11a
11b
CH3
11c
CH3CH2
CH3CH2
CH3
0.897 0.025 0.932 0.021
11d
13a
n.d.
n.d.
n.d.
n.d.
13b
CH3CH2
CH3CH2
CH3
0.350 0.012 0.290 0.013
0.435 0.012 0.490 0.012
0.390 0.012 0.425 0.012
14a
14b
14c
CH3
(+)-JQ1c
ABBV-075c
0.103 0.022 0.098 0.013
0.093 0.034 0.083 0.021
100%
100%
a
The data were expressed as the means SD, representing the relative levels of anti-proliferation from three independent
experiments.
b n.d. = not determined.
c Used as positive control.
aThe data were expressed as the means SD, representing the relative levels of anti-proliferation from three independent experiments.
bn.d. = not determined.
cUsed as positive control.

Medicinal Chemistry Research
Results and discussion
Chemistry
piperidine group. Meanwhile, the inhibition potency also
demonstrated that ethyl was the optimal substituent at R2
region. To analyze the structure–activity relationship,
compound 14a was docked into BRD4(1) protein (PDB id:
3P5O) and overlapped with ABBV-075 via Glide docking
protocol with root mean square deviation (RMSD) value of
0.83 in Maestro 10.2 (Fig. 2).
The general schemes for synthesis of 7-methylimidazo[1,5-
a]pyrazin-8(7H)-one derivatives 11a–d and 14a–c are
summarized in Schemes 1 and 2. H NMR and 13C NMR
1
spectrum, mass spectrometry were executed to verify the
structures. All the derivatives were prepared by column
chromatography over silica gel (200–300 mesh). The purity
was further evaluated by HPLC before being tested in
biological evaluation (purity was >98%).
As shown in Fig. 2, we found that the core 7-methyli-
midazo[1,5-a]pyrazin-8(7H)-one could overlap with 6-
methyl-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one
of
ABBV-075, acted as a KAc mimic. The ketone group of 7-
methylimidazo[1,5-a]pyrazin-8(7H)-one could interact with
Asn140 by a hydrogen bond, which was vital for inhibitory
potency. In addition, methyl group occupied the hydro-
phobic pocket adjacent to Kac region. Compound 11a with
a piperidine group at R2 position exhibited a markedly
reduced inhibitory activity. Therefore, a hydrophobic group
was vital for increasing the lipophilic effects with the
hydrophobic WPF shelf, but the introduction of piperidine
group showed unfavorable steric to decrease the binding
efficiency. Based on above results, we further evaluated
anti-proliferation activities of our target compounds in vitro.
As shown in Scheme 1, the commercial material 5-
bromopyrazin-2-amine (1) reacted with sodium nitrite in
concentrated sulfuric acid to obtain 5-bromopyrazin-2(1H)-
one (2). Then the intermediate 2 was further converted into
5-Bromo-1-methylpyrazin-2(1H)-one (3) by methylation.
Cyclization of intermediate 3 with tosylmethyl isocyanide
prepared 5-Bromo-7-methylimi dazo [1,5-a]pyrazin-8(7H)-
one (4). The pinacol arylboronates (7a, b) were obtained by
bromination of compounds (5a, R1 = Me; 5b, R1 = Et) in
concentrated sulfuric acid, followed by coupling reaction.
Then intermediate 4 reacted with intermediates (7a, b) by
Suzuki coupling reaction to give the key intermediates (8a,
b). The intermediates (8a, b) were further treated with 4-
amino-1-Boc-piperidine, followed by deprotection of Boc
group to get compounds (10a, b). In addition, compounds
(10a, b) were condensed with different sulfonyl chlorides to
prepare the target compounds (11a–d).
In vitro anticancer evaluation against cancer cell
lines
Anti-proliferation activities of compounds 11d and 14a–c
with stronger inhibitory potency against BRD4 were further
evaluated in K564, MV4-11, and HL-60 cells (Table 2). We
found that all compounds showed reasonable anti-
proliferation activities in BRD4-sensitive cells, MV4-11,
and HL-60 cells, while these exhibited few inhibitory
effects in BRD4-independent cell, K564 line. Gratifyingly,
compound 14a presented excellent anti-proliferation activ-
ities in BRD4-sensitive cells, MV4-11 and HL-60 cells with
IC50 of 460 and 680 nm, respectively. Altogether, these
results demonstrated that compound 14a could effectively
As depicted in Scheme 2, the compounds (13a, b) were
synthesized by condensation of intermediates (8a, b) and
methyl 3-hydroxybenzoate, followed by hydrolysis of the
methyl esters. Condensation of compounds (13a, b) with
different amines in the presence of HBTU afforded target
compounds (14a–c).
BRD4 inhibitory activity and SAR study
AlphaScreen assay was performed to evaluate the inhibitory
activities of our target compounds (10a–b, 11a–d, 13a–b,
14a–c) against BRD4. Firstly, we assessed the inhibition
rates of our targets against BRD4(1) at a concentration of
10 μM (Table 1). (+)-JQ1 and ABBV-075 were used as
positive control. Most compounds exhibited moderate
inhibitory activities against BRD4(1). Gratifyingly, com-
pound 11d and 14a–c showed robust inhibitory potency,
and their IC50 values against BRD4(1) and BRD4(2) were
further estimated (Table 1). All compounds exhibited
excellent inhibitory activities against BRD4(1) and BRD4
(2), especially compound 14a presented BRD4(1) and
BRD4(2) inhibitory activity with IC50 values of 350 and
290 nm, respectively. These results indicated that aromatic
moiety at R2 region afforded stronger potency than
Table 2 Anticancer evaluation against cancer cell lines K652, HL-60,
and MV4-11
Compound K562 IC50
HL-60 IC50
MV4-11 IC50
(μM)a
(μM)a
(μM)a
11d
>10
1.864 0.152
0.680 0.143
2.862 0.173
1.944 0.415
0.158 0.037
0.142 0.022
1.243 0.121
0.460 0.114
1.854 0.187
0.785 0.013
0.096 0.021
0.088 0.034
14a
>10
14b
>10
14c
(+)-JQ1b
ABBV-075b >10 μM
>10
9.12 0.08
aThe data were expressed as the means SD, representing the relative
levels of anti-proliferation from three independent experiments.
bUsed as positive control.

Medicinal Chemistry Research
inhibit BRD4 and induce apoptosis in BRD4-sensitive cells,
MV4-11 and HL-60 cells.
5-bromo-1-methylpyrazin-2(1H)-one (3)
Yellow solid (to a solution of 2 (1.51 g, 8.63 mmol) and
K2CO3 (2.39 g, 17.26 mmol) in acetonitrile (30 mL) was
added dimethylsulfate (1.63 g, 12.94 mmol), then stirred at
70 °C for 6 h. After the reaction completed, the mixture was
filtered by a celite pad, and the filtrate was obtained.); H
NMR (300 MHz, CDCl3): δ = 7.97 (s, 1H, Ar–H), 7.32 (s,
1H, Ar-H), 3.52 (s, 3H, –CH3).
Conclusion
1
In this study, we report our structural modification chemical
synthesis and evaluation inhibitory potency of BRD4
in vitro. Eleven novel 7-methylimidazo[1,5-a]pyrazin-8
(7H)-one derivatives were synthesized. Meanwhile, the
inhibitory potency of BRD4 and anti-proliferation activity
were assessed in protein and cell level, respectively. Grat-
ifyingly, compound 14a exhibited robust BRD4(1) and
BRD4(2) inhibitory effect with IC50 of 350 and 290 nm,
respectively. In addition, compound 14a has remarkable
anti-proliferative activities with IC50 of 460 and 680 nm in
BRD4-sensitive cells, MV4-11 and HL-60 cells, respec-
tively. Thus, further research aiming at the potential
mechanisms behind the inhibitory of bromodomian is
requisite when it comes to developing novel BRD4 inhi-
bitors for cancer treatment.
5-bromo-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (4)
Yellow solid (To a solution of 3 (1.60 g, 8.47 mmol) and
tosylmethyl isocyanide (1.99 g, 10.16 mmol) in THF
(30 mL) was added NaH (1.02 g, 35.4 mmol) at 0 °C. After
the reaction completed, the mixture was concentrated under
reduced pressure to get yellow solid without further pur-
1
ification.); H NMR (300 MHz, CDCl3): δ = 8.08 (d, J =
3.3 Hz, 2H, Ar–H), 6.62 (s, 1H, Ar–H), 3.48 (s, 3H, –CH3).
2-bromo-1-fluoro-4-(methylsulfonyl)benzene (6a)
White solid (To a solution of 1-fluoro-4-(methylsulfonyl)
benzene 5a (3 g, 17.22 mmol) in conc. H2SO4 (30 mL) was
added NBS (3.07 g, 17.22 mmol). The reaction mixture was
stirred at 50 °C for 4 h. After the reaction completed, the
mixture was diluted with ice water (100 mL) and the white
precipitate was filtered and washed with water. Then dried
the solid was under reduced pressure to get a white solid.);
1H NMR (300 MHz, DMSO-d6): δ = 8.27 (dd, J = 6.3,
2.2 Hz, 1H, Ar–H), 8.00 (ddd, J = 8.5, 4.5, 2.3 Hz, 1H,
Ar–H), 7.67 (t, J = 8.7 Hz, 1H, Ar–H), 3.30 (s, 3H, –CH3).
Experimental section
Chemistry
All chemical reagents were commercially available and
used without further purification.
1H spectra (DMSO-d6) were measured on Bruker AV-
300 spectrometer at 25 °C and referenced to TMS. Purity of
the target compounds were confirmed by HPLC analysis
(UV detector, wavelength: 272 nm). 1H and 13C NMR
spectra: Bruker ACF-300Q apparatus (300 MHz for 1H
NMR and 75 MHz for 13C NMR), in DMSO-d6 or CDCl3
unless otherwise stated. High-resolution mass spectra were
recorded using an Agilent QTOF 6520 (Beijing, China).
2-bromo-4-(ethylsulfonyl)-1-fluorobenzene (6b)
White solid (To a solution of 1-(ethylsulfonyl)-4-fluor-
obenzene 5b (3.04 g, 16.15 mmol) in conc. H2SO4 (30 mL)
was added NBS (2.87 g, 16.15 mmol). The reaction mixture
was stirred at 50 °C for 4 h. After the reaction completed,
the mixture was diluted with ice water (100 mL) and the
white precipitate was filtered and washed with water. Then
the solid was dried under reduced pressure to get a white
5-bromopyrazin-2(1H)-one (2)
Yellow solid (to a solution of NaNO2 (1.78 g, 25.86 mmol)
in conc., then H2SO4 (15 mL) was added a solution of 1
(3.0 g, 17.24 mmol) in conc. The reaction mixture was
stirred at 45 °C for 2 h. After the reaction completed, the
mixture was diluted with water (200 mL) and extracted with
EtOAc (300 mL). The organic layer was dried over Na2SO4
and concentrated to give Yellow solid.); 1H NMR
(300 MHz, DMSO-d6): δ = 12.24 (s, 1H, –NH–C=O), 8.10
(s, 1H, Ar–H), 7.92 (s, 1H, Ar–H).
1
solid.); H NMR (300 MHz, DMSO-d6): δ = 8.22 (dd, J =
6.4, 2.2 Hz, 1H, Ar–H), 7.96 (ddd, J = 8.6, 4.6, 2.3 Hz, 1H,
Ar–H), 7.68 (t, J = 8.6 Hz, 1H, Ar–H), 3.40 (q, J = 7.4 Hz,
2H, –CH2–), 1.10 (t, J = 7.4 Hz, 3H, –CH3).
2-(2-fluoro-5-(methylsulfonyl)phenyl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (7a)
White solid (A mixture of 6a (3.87 g, 15.29 mmol),
4,4,4ʹ,4ʹ,5,5,5ʹ,5ʹ-octamethyl-2,2ʹ-bi(1,3,2-dioxaborolane)
(4.66 g,
18.35 mmol),
Pd(dppf)Cl2CH2Cl2
(0.34 g,

Medicinal Chemistry Research
0.46 mmol), and KOAc (3 g, 30.58 mmol) in 1,2-dime-
thoxyethane (50 mL) was stirred at 90 °C under nitrogen
atmosphere for 24 h. Then the mixture was diluted with
H2O and EtOAc, and the insoluble material was filtered
through Celite. The organic layer of the filtrate was washed
with water (30 mL) and brine (30 mL), dried over Na2SO4,
and was concentrated under reduced pressure to get a white
solid.); H NMR (300 MHz, DMSO-d6): δ = 8.19–8.05 (m,
2H, Ar–H), 7.52–7.39 (m, 1H, Ar–H), 3.23 (d, J = 3.8 Hz,
3H, –CH3), 1.32 (s, 12H, –C(CH3)2–C(CH3)2–).
Tert-butyl 4-((2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]
pyrazin-5-yl)-4-(methylsulfony l)phenyl)amino)piperidine-1
-carboxylate (9a)
White solid (A mixture of 8a (0.5 g, 1.56 mmol.), 4-amino-
1-Boc-piperidine (0.78 g, 3.89 mmol) and DIPEA (0.6 g,
4.67 mmol) in DMSO (5 mL) was stirred at 110 °C for 24 h.
Then the mixture was poured into water (50 mL) and the
white precipitate was filtered and washed with water. The
solid was then dried under reduced pressure to get a white
1
1
solid.); H NMR (300 MHz, DMSO-d6): δ = 7.83–7.73 (m,
2-(5-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (7b)
2H, Ar–H), 7.68 (d, J = 2.4 Hz, 1H, Ar–H), 7.54 (s, 1H,
Ar–H), 7.08 – 6.97 (m, 2H, Ar–H), 5.88 (d, J = 7.9 Hz, 1H,
–NH–), 3.87 (s, 2H, –CH2–), 3.68 (s, 1H, –CH–), 3.40 (d, J
= 2.4 Hz, 3H, –CH3), 3.12 (d, J = 2.4 Hz, 3H, –CH3), 2.81
(s, 2H, –CH2–), 1.75 (s, 2H, –CH2–), 1.36 (d, J = 2.4 Hz,
9H, –C(CH3)3), 1.25–1.13 (m, 2H, –CH2–).
White solid (A mixture of 6b (4.2 g, 15.72 mmol),
4,4,4ʹ,4ʹ,5,5,5ʹ,5ʹ-octamethyl-2,2ʹ-bi(1,3,2-dioxaborolane)
(4.79 g,
18.87 mmol),
Pd(dppf)Cl2CH2Cl2
(0.35 g,
0.47 mmol) and KOAc (3.09 g, 31.45 mmol) in 1,2-dime-
thoxyethane (50 mL) was stirred at 90 °C under nitrogen
atmosphere for 24 h. Then the mixture was diluted with
H2O and EtOAc, and the insoluble material was filtered
through Celite. The organic layer of the filtrate was washed
with water (30 mL) and brine (30 mL), dried over Na2SO4,
and was concentrated under reduced pressure to get a white
Tert-butyl tert-butyl 4-((4-(ethylsulfonyl)-2-(7-methyl-8-oxo-
7,8-dihy droimi dazo[1,5-a]py razin-5-yl)phenyl)amino)
piperidine-1 -carboxylate (9b)
White solid (A mixture of 8b (0.5 g, 1.49 mmol), 4-amino-
1-Boc-piperidine (0.75 g, 3.73 mmol) and DIPEA (0.58 g,
4.47 mmol) in DMSO (5 mL) was stirred at 110 °C for 24 h.
Then the mixture was poured into water (50 mL) and the
white precipitate was filtered and washed with water. The
solid was then dried under reduced pressure to get a white
solid.)
1
solid.); H NMR (300 MHz, DMSO-d6): δ = 8.14 – 8.02
(m, 2H, Ar–H), 7.48 (t, J = 8.7 Hz, 1H, Ar–H), 3.30 (d, J =
7.3 Hz, 2H, –CH2–), 1.32 (s, 12H, –C(CH3)2–C(CH3)2–),
1.10 (t, J = 7.3 Hz, 3H, –CH3).
5-(2-fluoro-5-(methylsulfonyl)phenyl)-7-methylimidazo[1,5-
a]pyrazin-8(7H)-one (8a)
7-methyl-5-(5-(methylsulfonyl)-2-(piperidin-4-ylamino)
phenyl)imidazo [1,5-a]pyrazin-8(7H)-one (10a)
White solid (A mixture of 4 (1 g, 4.39 mmol), 7a (1.58 g,
5.26 mmol), Pd(PPh3)4 (0.25 g, 0.22 mmol), and K2CO3
(1.21 g, 8.77 mmol) in acetonitrile/toluene (30 mL, 1/5) was
stirred at 80 °C under nitrogen atmosphere for 24 h. Then
the mixture was diluted with CH2Cl2 and then was con-
White solid (This compound was prepared by 9a); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 7.84–7.70 (m,
2H, Ar–H), 7.66 (d, J = 2.4 Hz, 1H, Ar–H), 7.52 (s, 1H,
Ar–H), 7.00 (dd, J = 15.8, 5.9 Hz, 2H, Ar–H), 5.90 (d, J =
7.6 Hz, 1H,–NH–), 3.40 (d, J = 2.9 Hz, 6H, –CH3, –CH3),
3.11 (d, J = 2.9 Hz, 3H, –CH2–, –NH–), 2.88 (d, J =
11.3 Hz, 2H, –CH2–), 1.71 (s, 2H, –CH2–), 1.21 (d, J =
12.6 Hz, 2H, –CH2–). 13C NMR (75 MHz, DMSO-d6): δ =
155.1 (CH, C-6ʹ), 149.7 (C=O, C-6), 135.3 (CH, C-2),
133.1 (CH,C-4ʹ), 131.5 (CH, C-4), 128.6 (C, C-5ʹ), 128.3
(CH, C-8), 127.3 (C, C-5), 126.5 (CH, C-6ʹ), 123.7 (C, C-
1ʹ), 115.7 (C, C-9), 110.3 (CH, C-3ʹ), 62.4 (CH2, Ar-CH2-
N), 50.1 (CH2, –N–CH2–CH2–), 45.1(CH2, –N–
CH2–CH2–), 44.2 (CH3, –S(O2)–CH3), 36.0 (CH3, N-CH3).
MS (ESI, m/z): 402.3 [M+H]+. Anal. calcd. for
C25H27N5O5S2: C, 56.84; H, 5.77; N, 17.44. Found: C,
56.81; H, 5.74; N, 17.40.
1
centrated under reduced pressure to get a white solid.); H
NMR (300 MHz, DMSO-d6): δ = 8.24–8.12 (m, 2H,
Ar–H), 8.06 (dd, J = 3.2, 0.6 Hz, 1H, Ar–H), 7.88 (d, J =
0.6 Hz, 1H, Ar–H), 7.73 (dd, J = 9.7, 8.7 Hz, 1H, Ar–H),
7.26 (s, 1H, Ar–H), 3.42 (s, 3H, –CH3), 3.31 (s, 3H, –CH3).
5-(5-(ethylsulfonyl)-2-fluorophenyl)-7-methylimidazo[1,5-a]
pyrazin-8(7H)-one (8b)
White solid (This compound was prepared by 7b.); 1H
NMR (300 MHz, DMSO-d6): δ = 8.15 (d, J = 5.6 Hz, 2H,
Ar–H), 8.06 (d, J = 2.9 Hz, 1H, Ar–H), 7.88 (s, 1H, Ar–H),
7.74 (t, J = 9.1 Hz, 1H, Ar–H), 7.26 (s, 1H, Ar–H),
3.46–3.35 (m, 5H, –CH2–, –CH3), 1.16 (t, J = 7.3 Hz, 3H,
–CH3).

Medicinal Chemistry Research
5-(5-(ethylsulfonyl)-2-(piperidin-4-ylamino)phenyl)-7-
methylimidazo[1,5-a]pyrazin-8(7H)-one (10b)
(m, 1H, Ar–H), 7.00 (d, J = 7.0 Hz, 2H, Ar–H), 5.91 (d, J
= 7.9 Hz, 1H, –NH–), 3.57 (s, 3H, –CH3), 3.39 (s, 3H,
–CH3), 3.10 (s, 3H, –CH2–, –CH–), 2.42 (d, J = 9.4 Hz, 2H,
–CH2–), 1.87 (t, J = 12.7 Hz, 2H, –CH2–), 1.42 (d, J =
11.4 Hz, 2H, –CH2–); 13C NMR (75 MHz, DMSO-d6): δ =
155.0 (–C=O, C-6), 149.7 (CH, C-2ʹ), 135.3 (C,
–C–SO2–N–), 133.6 (CH, C-2), 132.8 (CH, C-4ʹ), 131.5
(CH, –CH = C–S–), 130.2 (CH, C-4), 129.0 (CH, –C=),
129.0 (C, =C–S–), 128.2 (C, C-5), 126.3 (CH, C-8), 123.4
(CH, C-6ʹ), 122.1 (C, C-5ʹ), 114.0 (C, C-1ʹ), 113.2 (C, C-9),
110.6 (CH, C-3ʹ), 48.0 (CH, –NH–CH–), 45.3 (CH3,
–SO2–CH3), 45.2 (CH2, –SO2–N–CH2–), 34.0 (CH3,
–N–CH3), 30.4 (CH2, –N–CH2–CH2–). MS (ESI, m/z):
548.2 [M+H]+. Anal. calcd. for C23H25N5O5S3: C, 50.44;
H, 4.60; N, 12.79. Found: C, 50.41; H, 4.63; N, 12.76.
White solid (This compound was prepared by 9b); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 7.84–7.70 (m,
2H, Ar–H), 7.54 (d, J = 2.5 Hz, 1H, Ar–H), 7.48 (s, 1H,
Ar–H), 6.88 (dd, J = 13.9, 5.2 Hz, 2H, Ar–H), 5.78 (d, J =
7.1 Hz, 1H, –NH–), 3.25 (d, J = 1.9 Hz, 6H, –CH3, –CH3),
3.28 (q, J = 6.2 Hz, 2H, –CH2–), 2.54 (d, J = 9.2 Hz, 2H,
–CH2–), 1.65 (s, 2H, –CH2–), 1.19 (d, J = 11.3 Hz, 2H,
–CH2–), 1.13 (t, J = 5.3 Hz, 3H, –CH2–). 13C NMR
(75 MHz, DMSO-d6): δ = 154.9 (CH, C-6ʹ), 149.8 (C=O,
C-6), 135.3 (CH, C-2), 133.3 (CH,C-4ʹ), 131.4 (CH, C-4),
128.7 (C, C-5ʹ), 128.5 (CH, C-8), 127.7 (C, C-5), 126.6
(CH, C-6ʹ), 123.7 (C, C-1ʹ), 115.8 (C, C-9), 110.3 (CH, C-
3ʹ), 62.4 (CH2, Ar–CH2–N), 48.3 (CH2, –CH2–CH3), 45.1
(CH2, –N–CH2–CH2–), 40.5(CH2, –N–CH2–CH2–), 36.7
(CH3, N–CH3), 10.4 (CH3, –CH2–CH3). MS (ESI, m/z):
416.3 [M+H]+. Anal. calcd. for C25H27N5O5S2: C, 57.81;
H, 6.06; N, 16.86. Found: C, 57.82; H, 5.98; N, 16.80.
5-(5-(ethylsulfonyl)-2-((1-(phenylsulfonyl)piperidin-4-yl)
amino)phenyl)-7-methylimidazo [1,5-a]pyrazin-8(7H)-one
(11c)
White solid (This compound was prepared by 10b); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 7.82–7.57 (m,
8H, Ar-H), 7.50 (s, 1H, Ar–H), 6.99 (d, J = 11.7 Hz, 2H,
Ar–H), 5.92 (d, J = 7.8 Hz, 1H, –NH–), 3.59 (s, 3H, –CH3),
3.39 (s, 3H, –CH2–, –CH–), 3.21–3.10 (m, 2H, –CH2–),
2.34 (s, 2H, –CH2–), 1.84 (s, 2H, –CH2–), 1.39 (s, 2H,
–CH2–), 1.08 (t, J = 7.3 Hz, 3H, –CH3); 13C NMR
(75 MHz, DMSO-d6): δ = 155.1 (–C=O, C-6), 149.8 (C, C-
2ʹ), 135.5 (C, –N–SO2–C), 133.0 (CH, C-2), 132.2 (CH,
Ar–C), 131.5 (CH, C-4ʹ), 131.0 (CH, C-4), 129.3 (CH,
Ar–C), 128.9 (C, Ar–C), 127.3 (C, C-5), 123.6 (CH, C-8),
123.4 (CH, C-6ʹ), 122.1 (C, C-5ʹ), 114.1 (C, C-1ʹ), 113.2 (C,
C-9), 110.6 (CH, C-3ʹ), 49.7 (CH, –NH–CH–), 48.1 (CH3,
–SO2–CH3), 45.2 (CH2, –NH–CH2–CH2–), 34.0 (CH3,
–N–CH3), 30.4 (CH2, –N–CH2–CH2–), 7.3 (CH3,
-CH2–CH3). MS (ESI, m/z): 556.3 [M+H]+. Anal. calcd.
for C26H29N5O5S2: C, 56.20; H, 5.26; N, 12.60. Found: C,
56.17; H, 5.23; N, 12.64.
7-methyl-5-(5-(methylsulfonyl)-2-((1-(phenylsulfonyl)
piperidin-4yl)amino) phenyl)imida zo[1,5-a]pyrazin-8(7H)-
one (11a)
White solid (This compound was prepared by 10a); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 7.79–7.63 (m,
8H, Ar–H), 7.51 (s, 1H, Ar–H), 7.03–6.95 (m, 2H, Ar–H),
5.91 (d, J = 8.5 Hz, 1H, –NH–), 3.58 (s, 2H, –CH2–),
3.55–3.45 (m, 1H, –CH–), 3.39 (s, 3H, –CH3), 3.10 (s, 3H,
–CH3), 2.34 (d, J = 6.4 Hz, 2H, –CH2–), 1.81 (d, J =
12.2 Hz, 2H, –CH2–), 1.39 (s, 2H, –CH2–); 13C NMR
(75 MHz, DMSO-d6): δ = 155.0 (C=O, C-6), 149.7 (CH,
C-2ʹ), 135.3 (C, –N–SO2–C), 133.1 (CH, C-2), 131.5 (CH,
Ar–C), 130.2 (CH, C-4ʹ), 129.3 (CH, C-4), 128.9 (CH,
Ar–C), 127.3 (CH, Ar–C), 126.2 (C, C-5), 123.3 (CH, C-8),
122.1 (CH, C-6ʹ), 120.4 (C, C-5ʹ), 117.2, (C, C-1ʹ), 113.6
(C, C-9), 110.6 (CH, C-3ʹ), 48.1 (CH, –NH–CH–), 45.1
(CH3, –SO2–CH3), 44.2 (CH2, –NH–CH2– CH2–), 34.0
(CH3, N-CH3), 30.5(CH2, –NH–CH2– CH2–). MS (ESI, m/
z): 542.3 [M+H]+. Anal. calcd. for C25H27N5O5S2: C,
55.44; H, 5.02; N, 12.93. Found: C, 55.41; H, 5.07; N,
12.90.
5-(5-(ethylsulfonyl)-2-((1-(thiophen-2-ylsulfonyl)piperidin-4-
yl)amino)phenyl)-7-methyl imidazo[1,5-a]pyrazin-8(7H)-one
(11d)
White solid (This compound was prepared by 10b); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 8.06 (dd, J =
5.0, 1.3 Hz, 1H, Ar–H), 7.78 (s, 1H, Ar–H), 7.66 (dd, J =
8.8, 2.2 Hz, 1H, Ar–H), 7.63–7.58 (m, 2H, Ar–H), 7.50 (s,
1H, Ar–H), 7.29 (dd, J = 5.0, 3.8 Hz, 1H, Ar–H), 7.01 (t, J
= 4.5 Hz, 2H, Ar–H), 5.92 (d, J = 8.3 Hz, 1H, –NH–), 3.57
(s, 3H, –CH3), 3.39 (s, 3H, –CH2–, –CH–), 3.16 (q, J =
7.2 Hz, 2H, –CH2–), 2.42 (d, J = 9.9 Hz, 2H, –CH2–), 1.87
(t, J = 12.8 Hz, 2H, –CH2–), 1.42 (s, 2H, –CH2–), 1.09 (t, J
= 7.3 Hz, 3H, –CH3); 13C NMR (75 MHz, DMSO-d6): δ =
7-methyl-5-(5-(methylsulfonyl)-2-((1-(thiophen-2-ylsulfonyl)
piperidin-4-yl)amino)phenyl) imidazo[1,5-a]pyrazin-8(7H)-
one (11b)
White solid (This compound was prepared by 10a); m.p. >
200 °C; 1H NMR (300 MHz, DMSO-d6): δ = 8.09–8.03
(m, 1H, Ar–H), 7.78 (s, 1H, Ar–H), 7.71 (dd, J = 8.9,
2.3 Hz, 1H, Ar–H), 7.66 (d, J = 2.2 Hz, 1H, Ar–H),
7.63–7.59 (m, 1H, Ar–H), 7.52 (s, 1H, Ar–H), 7.33 – 7.26

Medicinal Chemistry Research
154.9(–C=O, C-6), 149.8 (C, C-2ʹ), 135.3 (C,
–C–SO2–N–), 133.6 (CH, C-2), 132.8 (CH, C-4ʹ), 132.2
(CH, –CH=C–S–), 131.5 (CH, C-4), 129.0 (CH, –C=),
131.0 (C, =C–S–), 129.2 (C, C-5), 128.2 (CH, C-8), 123.7
(C, C-6ʹ), 122.1 (C, C-5ʹ), 114.1 (C, C-1ʹ), 113.2 (C, C-9),
110.6 (CH, C-3ʹ), 49.7 (CH, –NH–CH–), 48.0 (CH3,
–SO2–CH3), 45.3 (CH2, –SO2–N–CH2–), 34.0 (CH3, N-
CH3), 30.4 (CH2, –N–CH2–CH2–), 7.3 (CH3, –CH2–CH3).
MS (ESI, m/z): 562.3 [M+H]+; Anal. calcd. for
C24H27N5O5S3: C, 51.32; H, 4.85; N, 12.47. Found: C,
51.37; H, 4.81; N, 12.41.
DMSO-d6): δ = 165.1 (C=O, COOH), 155.0 (C=O, C-6),
153.1 (CH, –O–CH=), 149.9 (CH, C-2ʹ), 136.1 (CH, C-4ʹ),
133.6 (CH, C-2), 132.7 (C, C-5ʹ), 130.6 (CH, C-4), 129.4
(CH, =CH-COOH), 128.7 (CH, Ar–C), 127.6 (C, C-5),
126.6 (CH, C-6ʹ), 123.4 (C, C-9), 122.5 (CH, C-8), 120.6
(C, C-1ʹ), 120.6 (CH, Ar–C), 118.7 (CH, Ar–C), 118.3 (CH,
C-3ʹ), 116.9 (CH, Ar–C), 45.3 (CH3, –SO2–CH3), 34.2
(CH3, N-CH3). MS (ESI, m/z): 440.3 [M+H]+. Anal. calcd.
for C25H27N5O5S2: C, 57.40; H, 3.90; N, 9.56. Found: C,
57.32; H, 3.84; N, 9.50.
3-(4-(Ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo
[1,5-a]pyrazin-5-yl)phenoxy) benzoic acid (13b)
Methyl 3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]
pyrazin-5-yl)-4-(methylsulfonyl) phenoxy)benzoate (12a)
White solid (This compound was prepared by 13b); m.p.
162–164 °C; 1H NMR (300 MHz, DMSO-d6): δ = 13.27 (s,
1H, –COOH), 8.23 (s, 1H, Ar–H), 8.08 (d, J = 2.4 Hz, 1H,
Ar–H), 8.00 (dd, J = 8.7, 2.4 Hz, 1H, Ar–H), 7.82 (d, J =
6.0 Hz, 2H, Ar–H), 7.69 (s, 1H, Ar–H), 7.56 (t, J = 7.9 Hz,
1H, Ar–H), 7.45 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 7.20 (s,
1H, Ar–H), 7.10 (d, J = 8.7 Hz, 1H, Ar–H), 3.41 (s, 3H,
–CH3), 3.36 (q, J = 7.3 Hz, 2H, –CH2–), 1.16 (t, J = 7.3 Hz,
3H, –CH3). 13C NMR (75 MHz, DMSO-d6): δ = 165.0
(C=O, COOH), 154.9 (C=O, C-6), 153.1 (CH, –O–CH=),
149.9 (CH, C-2ʹ), 136.2 (CH, C-4ʹ), 133.6 (CH, C-2), 132.6
(C, C-5ʹ), 130.6 (CH, C-4), 129.4 (CH, =CH-COOH),
128.8 (CH, Ar–C), 127.7 (C, C-5), 126.6 (CH, C-6ʹ), 123.3
(C, C-9), 122.5 (CH, C-8), 120.7 (C, C-1ʹ), 120.6 (CH,
Ar–C), 118.4 (CH, Ar–C), 118.2 (CH, C-3ʹ), 116.9 (CH,
Ar–C), 45.3 (CH3, –SO2–CH3), 34.2 (CH3, N-CH3), 7.4
(CH3, –CH2–CH3). MS (ESI, m/z): 454.3 [M+H]+. Anal.
calcd. for C25H27N5O5S2: C, 58.27; H, 4.22; N, 9.27.
Found: C, 58.25; H, 4.20; N, 9.31.
White solid (A mixture of 8a (0.4 g, 1.24 mmol), methyl 3-
hydroxyben zoate (0.28 g, 1.87 mmol) and K2CO3 (0.34 g,
2.49 mmol) in DMF (3 mL) was stirred at 85 °C for 6 h.
Then the mixture was diluted with H2O, extracted with
EtOAc (60 mL), washed with brine (20 mL), dried over
Na2SO4, and concentrated under reduced pressure. The
crude compound was purified by flash chromatography to
1
give a white solid.); H NMR (300 MHz, DMSO-d6): δ =
8.22 (d, J = 2.9 Hz, 1H, Ar–H), 8.15 (d, J = 2.4 Hz, 1H,
Ar–H), 8.06 (dd, J = 8.7, 2.5 Hz, 1H, Ar–H), 7.83 (d, J =
7.7 Hz, 2H, Ar–H), 7.69 (s, 1H, Ar–H), 7.59 (t, J = 7.9 Hz,
1H, Ar–H), 7.46 (d, J = 7.6 Hz, 1H, Ar–H), 7.19 (d, J =
2.9 Hz, 1H, Ar–H), 7.11 (dd, J = 8.7, 2.8 Hz, 1H, Ar–H),
3.85 (d, J = 2.9 Hz, 3H, –CH3), 3.41 (d, J = 2.9 Hz, 3H,
–CH3), 3.28 (d, J = 2.9 Hz, 3H, –CH3).
Methyl 3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-
dihydroimidazo[1,5-a]pyrazin-5-yl)phe noxy)benzoate(12b)
White solid (This compound was prepared by 8b); 1H NMR
(300 MHz, DMSO-d6): δ = 8.23 (s, 1H, Ar-H), 8.08 (d, J =
2.3 Hz, 1H, Ar–H), 8.00 (dd, J = 8.7, 2.4 Hz, 1H, Ar–H),
7.86–7.78 (m, 2H, Ar–H), 7.71 (d, J = 2.1 Hz, 1H, Ar–H),
7.58 (t, J = 7.9 Hz, 1H, Ar–H), 7.48 (dd, J = 7.8, 1.9 Hz,
1H, Ar–H), 7.19 (s, 1H, Ar–H), 7.09 (d, J = 8.7 Hz, 1H,
Ar–H), 3.84 (s, 3H, –CH3), 3.40 (s, 3H, –CH3), 3.36 (s, 2H,
–CH2–), 1.18–1.12 (m, 3H, –CH3).
N-(2-(dimethylamino)ethyl)-3-(4-(ethylsulfonyl)-2-(7-
methyl-8-oxo-7,8-dihydroimidazo [1,5-a]pyrazin-5-yl)
phenoxy)benzamide (14a)
Yellow solid (This compound was prepared by 13b); m.p.
1
164–166 °C; H NMR (300 MHz, DMSO-d6): δ = 8.57 (s,
1H, –NH–C=O), 8.18 (s, 1H, Ar–H), 8.09 (d, J = 2.4 Hz,
1H, Ar–H), 8.02 (dd, J = 8.7, 2.4 Hz, 1H, Ar–H), 7.82 (s,
1H, Ar–H), 7.74 (d, J = 7.8 Hz, 1H, Ar–H), 7.63 (s, 1H,
Ar–H), 7.56 (t, J = 7.9 Hz, 1H, Ar–H), 7.43 (d, J = 9.7 Hz,
1H, Ar–H), 7.21 (s, 1H, Ar–H), 7.07 (d, J = 8.7 Hz, 1H,
Ar–H), 3.49–3.34 (m, 9H, –CH3, –CH2–, –CH2–, –CH2–),
3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-
yl)-4-(methylsulfonyl)phenox y)benzoic acid (13a)
1
White solid (This compound was prepared by 12a); H
2.45 (s, 6H, –N(CH3)2), 1.16 (t, J = 7.3 Hz, 3H, –CH3); 13
C
NMR (300 MHz, DMSO-d6): δ = 13.25 (s, 1H, –COOH),
8.20 (s, 1H, Ar–H), 8.14 (s, 1H, Ar–H), 8.05 (d, J = 8.8 Hz,
1H, Ar–H), 7.81 (s, 2H, Ar–H), 7.66 (s, 1H, Ar–H), 7.55 (t,
J = 7.8 Hz, 1H, Ar–H), 7.42 (d, J = 8.2 Hz, 1H, Ar–H),
7.19 (s, 1H, Ar–H), 7.12 (d, J = 8.6 Hz, 1H, Ar–H), 3.41 (s,
3H, –CH3), 3.28 (s, 3H, –CH3). 13C NMR (75 MHz,
NMR (75 MHz, DMSO-d6): δ = 165.7 (C, –NH–C=O),
155.2 (C=O, C-6), 154.8 (CH, –O–CH=), 154.1 (C, C-2ʹ),
136.9 (CH, –CO–CH=), 133.5 (CH, C-4ʹ), 133.3 (CH, C-
9), 132.8 (CH, C-4), 132.2 (C, C-5ʹ), 130.9 (CH, C-6ʹ),
129.8 (CH, Ar–C), 124.8 (C, C-1), 123.7 (C, C-9), 123.1
(CH, C-8), 122.9 (CH, Ar–C), 121.1 (C, C-1ʹ), 119.6 (CH,

Medicinal Chemistry Research
Ar–C), 117.1 (CH, C-3ʹ), 112.8 (CH, Ar–C), 57.5 (CH2,
–CH2–NH–), 49.9 (CH2, –SO2–CH2–), 44.4 (CH2,
–CH2–NH–), 36.6 (CH3, –NH– (CH3)2), 34.6 (CH3, N-
CH3), 7.6 (CH3, –CH2–CH3); MS (ESI, m/z): 524.3 [M+H]
+; Anal. calcd. for C26H29N5O5S: C, 59.64; H, 5.58; N,
13.38. Found: C, 59.61; H, 5.55; N, 13.41.
(O2)–CH3), 36.3 (CH2, pyrrolidine–N–CH2–), 34.6 (CH3,
N-CH3), 22.9 (CH2, pyrrolidine–CH2–CH2–); MS (ESI, m/
z): 536.3 [M+H]+; Anal. calcd. for C27H29N5O5S: C, 60.55;
H, 5.46; N, 13.08. Found: C, 60.51; H, 5.41; N, 13.11.
Docking studies
N-(2-(dimethylamino)ethyl)-3-(2-(7-methyl-8-oxo-7,8-
dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methylsulfonyl)
phenoxy)benzamide (14b)
All compounds were drawn in ChemDraw, and exported as
sdf style. Ligand preparation protocol of Maestro 10.2 was
utilized to generate all possible tautomers, stereoisomers,
and protonation states of molecules at a simulated pH of
7.4 1.0, then minimization was proceeded at the OPLS
force field (Yang et al. 2016a, b). In our study, BRD4(1)
docking protein was from the crystal complex (PDB id:
3P5O). The Glide protein was created with Glide generation
protocol of Maestro 10.2 and the bond orders were assigned.
Seven molecules of water around the binding site were saved
(i.d.: HOH18, HOH27, HOH33, HOH256, HOH267,
HOH268, HOH358). Hydrogen atoms were added. Finally,
a 10 Å box centered on the geometrical center of the ligand
binding site was created for grid docking. The docking
research was executed with the Glide docking protocol.
After Glide docking completed, only one conformation was
saved for each molecular ligand (Yang et al. 2016a, b).
Yellow solid (This compound was prepared by 13a); m.p.
194-196 °C; 1H NMR (300 MHz, CDCl3): δ = 8.09 (d, J =
2.3 Hz, 1H, –NH–C=O), 8.03–7.97 (m, 2H, Ar–H), 7.75 (s,
1H, Ar–H), 7.59 (d, J = 8.1 Hz, 1H, Ar–H), 7.52 (s, 1H, Ar-
H), 7.42 (t, J = 7.9 Hz, 1H, Ar–H), 7.09 (dd, J = 8.1,
3.4 Hz, 2H, Ar–H), 7.01 (s, 1H, Ar–H), 6.55 (s, 1H, Ar–H),
3.54–3.47 (m, 5H, –CH3, –CH2–), 3.13 (s, 3H, –CH3), 2.52
(t, J = 5.8 Hz, 2H, –CH2–), 2.26 (s, 6H, –N(CH3)2); 13C
NMR (75 MHz, DMSO-d6): δ = 165.6 (C, –NH–C=O),
155.1 (C=O, C-6), 154.9 (CH, –O–CH=), 154.3 (C, C-2ʹ),
136.6 (CH, –CO–CH=), 133.4 (CH, C-4ʹ), 133.3 (CH, C-
9), 132.8 (CH, C-4), 132.3 (C, C-5ʹ), 130.9 (CH, C-6ʹ),
129.9 (CH, Ar-C), 124.9 (C, C-1), 123.5 (C, C-9), 123.2
(CH, C-8), 122.7 (CH, Ar–C), 121.5 (C, C-1ʹ), 119.6 (CH,
Ar–C), 117.5 (CH, C-3ʹ), 112.8 (CH, Ar–C), 57.6 (CH2,
–CH2–NH–), 44.5 (CH2, –CH2–NH–), 44.2 (CH3, –S
(O2)–CH3), 36.7 (CH3, –NH– (CH3)2), 34.6 (CH3, N-CH3);
MS (ESI, m/z): 510.3 [M+H]+; Anal. calcd. for
C25H27N5O5S: C, 58.93; H, 5.34; N, 13.74. Found: C,
58.97; H, 5.31; N, 13.69.
Biological evaluation
Binding effects of target compounds to BRD4(1) and BRD4
(2)
We commissioned Shanghai ChemPartner Co. to proceed
the experiments. BRD4(1) (Active Motif, 44-168aa, Cat.
No. 31380), BRD4(2) (Active Motif, 333-460aa, Cat. No.
31446), (+)-JQ1 (BPS, Cat. No. 27402), ABBV-075 (Lab
preparation). Prepare 1× assay buffer (modified HEPES
buffer). Transfer compounds to assay plates by Echo.
DMSO’s final concentration is 0.1%. Prepare protein solu-
tion in 1× assay buffer (5 nM). Add peptide (H4) in 1×
assay buffer to make the substrate solution. Transfer 5 μL of
protein solution to assay plates or for low control transfer
5 μL of 1× assay buffer, followed with incubation at room
temperature for 15 min. 5 μL of substrate solution was
added into each well, followed with incubation at room
temperature for 60 min. Lastly, 15 μL acceptor and donor
solution was added, followed with incubation for 60 min at
room temperature, subdued light (Yang et al. 2016a, b). The
IC50 values were calculated from the endpoints of EnSpire
with Alpha mode.
3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-
yl)-4-(methylsulfonyl)phenox y)-N-(2-(pyrrolidin-1 -yl)ethyl)
benzamide (14c)
Yellow solid (This compound was prepared by 13a); m.p.
1
187–189 °C; H NMR (300 MHz, DMSO-d6): δ = 8.70 (s,
1H, –NH–C=O), 8.16 (s, 2H, Ar–H), 8.11–8.03 (m, 1H,
Ar–H), 7.82 (s, 1H, Ar–H), 7.74 (d, J = 7.8 Hz, 1H, Ar–H),
7.63–7.52 (m, 2H, Ar–H), 7.45 (d, J = 8.7 Hz, 1H, Ar–H),
7.20 (s, 1H, Ar–H), 7.10 (d, J = 8.8 Hz, 1H, Ar–H), 3.56 (d,
J = 5.3 Hz, 4H, –CH2–, –CH2–), 3.42 (s, 3H, –CH3),
3.32–3.29 (m, 2H, –CH2–), 3.28 (s, 3H, –CH3), 3.05 (s, 2H,
–CH2–), 2.01 (s, 2H, –CH2–), 1.85 (s, 2H, –CH2–); 13C
NMR (75 MHz, DMSO-d6): δ = 165.9 (C, –NH–C=O),
155.2 (C=O, C-6), 154.7 (CH, –O–CH=), 154.4 (C, C-2ʹ),
136.9 (CH, –CO–CH=), 133.5 (CH, C-4ʹ), 133.3 (CH, C-
9), 132.8 (CH, C-4), 132.1 (C, C-5ʹ), 130.9 (CH, C-6ʹ),
129.7 (CH, Ar–C), 124.8 (C, C-1), 123.7 (C, C-9), 123.1
(CH, C-8), 122.9 (CH, Ar–C), 121.5 (C, C-1ʹ), 119.6 (CH,
Ar–C), 117.1 (CH, C-3ʹ), 112.8 (CH, Ar–C), 54.0 (CH2,
–CH2–NH–), 53.7 (CH2, –CH2-NH–), 44.2 (CH3, –S
Cell proliferation assay
HL-60, MV4-11 or K562 cells were seeded in 96-well
plates at a concentration of 1 × 104 cells per well. Cells were

Medicinal Chemistry Research
cultured in 100 μL of IMDM containing 20% fetal bovine
serum. After 12 h, 50 μL of various concentrations of
compounds (triple diluted) were added. The measurement
was executed 72 h after seeding, and 10 μL of cell-counting
kit-8 (CCK-8) reagent was added to each well, followed by
incubation at 37 °C for 4 h. A multi-detection microplate
was utilized to assess the spectrophotometric absorbance of
each well at a wavelength of 450 nm (Yang et al. 2016a, b).
The cell proliferation assay was executed to give the IC50
for the compounds 14a–c and reference compounds repor-
ted in Table 2. GraphPad Prism 5 was used to calculate the
IC50 values of target compounds (Tagde et al. 2016).
factor dependencies by the novel BET inhibitor ABBV-075. Mol
Cancer Res 15(1):35–44
Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP,
Barsytelovejoy D et al. (2012) Histone recognition and large-
scale structural analysis of the human bromodomain family. Cell
149(1):214–231
Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O
et al. (2010) Selective inhibition of BET bromodomains. Nature
468(7327):1067–1073
Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K
(2005) The bromodomain protein Brd4 is a positive regulatory
component of P-TEFb and stimulates RNA polymerase II-
dependent transcription. Mol Cell 19(4):523–534
Lin X, Huang X, Uziel T, Hessler P, Albert DH, Roberts-Rapp LA
et al. (2016) HEXIM1 as a robust pharmacodynamic marker for
monitoring target engagement of BET family bromodomain
inhibitors in tumors and surrogate tissues. Mol Cancer Ther. 16
(2):388–396
Lockwood WW, Zejnullahu K, Bradner JE, Varmus H (2012) Sensi-
tivity of human lung adenocarcinoma cell lines to targeted inhi-
bition of BET epigenetic signaling proteins. Proc Natl Acad Sci
USA 109(47):19408–19413
Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele
DA et al. (2011) Targeting MYC dependence in cancer by inhi-
biting BET bromodomains. Proc Natl Acad Sci USA 108
(40):16669–16674
Acknowledgements This study was supported by the Natural Science
Foundation of Jiangsu Province (No. BK 20141349) and the China
National Key HiTech Innovation Project for the R&D of Novel Drugs
(No. 2013ZX09301303-002).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Mujtaba S, Zeng L, Zhou MM (2007) Structure and acetyl-lysine
recognition of the bromodomain. Oncogene 26(37):5521–5527
Shu S & Polyak K (2017) BET bromodomain proteins as cancer
therapeutic targets. Cold Spring Harb Symp Quant Biol.
Stathis A, Zucca E, Bekradda M, Gomez-Roca C, Delord JP, de La
Motte Rouge T et al. (2016) Clinical response of carcinomas
harboring the BRD4-NUT oncoprotein to the targeted bromo-
References
Amorim S, Stathis A, Gleeson M, Iyengar S, Magarotto V, Leleu X
et al. (2016) Bromodomain inhibitor OTX015 in patients with
lymphoma or multiple myeloma: a dose-escalation, open-label,
pharmacokinetic, phase
e196–e204
1 study. Lancet Haematol 3(4):
domain inhibitor OTX015/MK-8628. Cancer Discov
6
(5):492–500
Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R
et al. (2014) Therapeutic targeting of BET bromodomain proteins
in castration-resistant prostate cancer. Nature 510(7504):278–282
Ceribelli M, Kelly PN, Shaffer AL, Wright GW, Xiao W, Yang Y
et al. (2014) Blockade of oncogenic IkappaB kinase activity in
diffuse large B-cell lymphoma by bromodomain and extra-
terminal domain protein inhibitors. Proc Natl Acad Sci USA 111
(31):11365–11370
Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J et al.
(2013) Discovery and characterization of super-enhancer-
associated dependencies in diffuse large B cell lymphoma. Can-
cer Cell 24(6):777–790
Core, LJ, Martins, AL, Danko, CG, Waters, CT (2014) Analysis of
nascent RNA identifies a unified architecture of initiation regions
at mammalian promoters and enhancers. Nat Genet 46
(12):1311–1320.
Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA sequencing reveals
widespread pausing and divergent initiation at human promoters.
Science 322(5909):1845–1848
Tagde A, Rajabi H, Stroopinsky D, Gali R, Alam M, Bouillez A et al.
(2016) MUC1-C induces DNA methyltransferase 1 and represses
tumor suppressor genes in acute myeloid leukemia. Oncotarget 7
(26):38974–38987
Yang Y, Zhao L, Xu B, Yang L, Zhang J, Zhang H et al. (2016a)
Design, synthesis and biological evaluation of dihydroquinox-
alinone derivatives as BRD4 inhibitors. Bioorg Chem
68:236–244
Yang Y, Zou F, Zhao L, Cheng Y, Zha X, Zhang H et al. (2016b)
Combined pharmacophore models as virtual screening protocol
against BRD4(1) inhibitor. Med Chem Res 25(4):1–11
Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K et al. (2005)
Recruitment of P-TEFb for stimulation of transcriptional elon-
gation by the bromodomain protein Brd4. Mol Cell 19
(4):535–545
Zhao S, Xu W, Jiang W, Yu W, Lin Y, Zhang T et al. (2010) Reg-
ulation of cellular metabolism by protein lysine acetylation.
Science 327(5968):1000–1004
Zhao Y, Liu Q, Acharya P, Stengel KR, Sheng Q, Zhou X et al. (2016)
High-resolution mapping of RNA polymerases identifies
mechanisms of sensitivity and resistance to BET inhibitors in t
(8;21) AML. Cell Rep 16(7):2003–2016
Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA et al.
(2011) RNAi screen identifies Brd4 as a therapeutic target in
acute myeloid leukaemia. Nature 478(7370):524–528
Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff M,
Chan WI et al. (2011) Inhibition of BET recruitment to chromatin
as an effective treatment for MLL-fusion leukaemia. Nature 478
(7370):529–533
Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM et al.
(2011) BET bromodomain inhibition as a therapeutic strategy to
target c-Myc. Cell 146(6):904–917
Faivre EJ, Wilcox D, Lin X, Hessler P, Torrent M, He W et al. (2017)
Exploitation of castration-resistant prostate cancer transcription