Design, synthesis and biological evaluation of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives as BRD4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.008

BRD4 is an attractive target for antitumor due to its important role in regulation of gene transcription. In this paper, we synthesized a series of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives as potent BRD4 inhibitors and evaluated their BRD4 inhibitory activities in vitro and anti-proliferation effects on tumor cells. Gratifyingly, compound 10j exhibited robust potency of BRD4(1) and BRD4(2) inhibition with IC₅₀ values of 130 and 76?nM respectively. Docking studies were performed to explain the structure-activity relationship. Furthermore, compound 10j potently inhibited cell proliferation in BRD4-sensitive cell lines HL-60 and MV4-11 with IC₅₀ value of 0.57 and 0.18?μM respectively. Activity on BRD4-independent K562 cell was weaker than on BRD4-sensitive lines. Overall, these results suggest that compound 10j is a potential BRD4 inhibitor deserving further investigation for cancer treatment.

Full text of DOI:10.1016/j.bmc.2017.03.008

Accepted Manuscript
Design, synthesis and biological evaluation of 7-methylimidazo[1,5-a]pyra-
zin-8(7H)-one derivatives as BRD4 inhibitors
Leilei Zhao, Yifei Yang, Yahui Guo, Lingyun Yang, Jian Zhang, Jinpei Zhou,
Huibin Zhang
PII:
S0968-0896(17)30011-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.008
BMC 13604
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 January 2017
3 March 2017
5 March 2017
Please cite this article as: Zhao, L., Yang, Y., Guo, Y., Yang, L., Zhang, J., Zhou, J., Zhang, H., Design, synthesis
and biological evaluation of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives as BRD4 inhibitors, Bioorganic
&
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.008
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Design, synthesis and biological evaluation of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one
derivatives as BRD4 inhibitors
a
a
b
a
a
b∗
Leilei Zhao , Yifei Yang , Yahui Guo , Lingyun Yang , Jian Zhang , Jinpei Zhou , Huibin
a∗
Zhang
a
Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing 210009, PR China.
b
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China.
Abstract
BRD4 is an attractive target for antitumor due to its important role in regulation of gene
transcription. In this paper, we synthesized a series of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one
derivatives as potent BRD4 inhibitors and evaluated their BRD4 inhibitory activities in vitro and
anti-proliferation effects on tumor cells. Gratifyingly, compound 10j exhibited robust potency of
BRD4(1) and BRD4(2) inhibition with IC50 values of 130 and 76 nM respectively. Docking
studies were performed to explain the structure-activity relationship. Furthermore, compound 10j
potently inhibited cell proliferation in BRD4-sensitive cell lines HL-60 and MV4-11 with IC50
value of 0.57 and 0.18 µM respectively. Activity on BRD4-independent K562 cell was weaker
than on BRD4-sensitive lines. Overall, these results suggest that compound 10j is a potential
BRD4 inhibitor deserving further investigation for cancer treatment.
Keywords: BRD4; Bromodomain; Lysine acetylation; Antitumor
1. Introduction
BRD4 belongs to the bromodomain and extra terminal (BET) family proteins (BRD2, BRD3,
BRD4, and BRDT). BRD4 has a conserved modular architecture including two N-terminal tandem
bromodomain effector modules (BRD4(1) and BRD4(2)), an extra-terminal recruitment domain
(
ET), several conserved motifs (A, B, SEED motifs), and a C-terminal motif (CTM). Each
bromodomain has a conserved architecture, comprising a four-helix bundle (helices αZ, αA, αB,
1
and αC) linked by diverse loop regions (ZA and BC loops). Lysine acetylation is a regulatory
post-translational modification that, in the context of chromatin biology, is associated with open
2
chromatin structure and transcriptional stimulation. BRD4 bromodomains recognize and bind to
acetylated lysine sequences found on histones or other proteins, and then regulate many
downstream cellular processes such as cell cycle, growth, proliferation, and apoptosis. Upon
binding to acetylated histones, BRD4 interacts with the positive transcription elongation factor
3
complex (P-TEFb) complex to influence RNA polymerase II activity. Genome-wide binding and
expression profiling studies show that BRD4 controls both the expression of numerous
protein-coding transcripts and of non-coding enhancer RNAs. For instance, BRD4 play a broad
4
, 5
role in maintaining c-MYC levels, c-MYC is a master regulatory factor of cell proliferation, and
6
pathologic activation of c-MYC plays a central role in cancer pathogenesis. Thus, targeting
BRD4 within the c-MYC transcriptional signaling network can modulate the function of c-MYC
in cancer. As a result, BRD4 is becoming a novel therapeutic candidate target for antitumor drug
7
development.
Recently, a variety of small molecule compounds with potent inhibitory activity against
∗
Corresponding authors. Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
2
10009, PR China Tel: +86-25-83271302; Fax: +86-25-83271480. E-mail: jpzhou668@163.com
∗
Corresponding authors. Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271302; Fax: +86-25-83271480. E-mail:
zhanghb80@cpu.edu.cn (H. Zhang)

8
-14
15
16
17
BRD4 have been reported in the literatures,
such as (+)-JQ1, I-BET762, I-BET151,
1
8
OTX015, and ABBV-075 (Fig. 1). (+)-JQ1 was the first potent BRD4 inhibitor and I-BET762
was obtained from optimization of a hit designed to identify small molecules able to enhance
ApoA1 expression. I-BET762 has entered clinical trials for NUT midline carcinoma and other
cancers. OTX015 has completed Phase I trials for hematologic malignancies and NUT midline
1
9-21
carcinoma, and the results showed clinically meaningful activity at nontoxic doses.
ABBV-075 is in phase I clinical trials for the treatment of patients with solid tumors, acute
myeloid leukemia or multiple myeloma, according to the strong biochemical potency for BRD4
(
ki=15nM). Regardless of these BRD4 inhibitors, none has been approved. New BRD4 inhibitors
with different scaffolds are still necessary to explore their therapeutic potential in different cancers.
Here, 7-methylimidazo[1,5-a]pyrazin-8(7H)-one backbone was utilized to replace the heterocyclic
bicyclic ring of ABBV-075 and a variety of optimal groups were employed for the design of novel
2
2, 23
BRD4 inhibitors (Fig. 2).
In this paper, we report the design, synthesis and evaluation of a
new series of 7-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives (10a-s) as small molecule
BRD4 inhibitors.
Fig. 1. Structures of known BRD4 inhibitors.
Fig. 2. Design of the novel BRD4 inhibitors.
2. Results and discussion
2.1. Chemistry
Target compounds 10a-s were synthesized as outlined in Schemes 1-2. All compounds were
purified by flash chromatography and purity was checked by HPLC before biological evaluation
1
13
(
purity was > 97%). The structures were confirmed by H NMR, C NMR spectrum, and mass
spectrometry.
As depicted in Scheme 1, 5-bromopyrazin-2-amine (1) reacted with sodium nitrite in
concentrated sulfuric acid to give 5-bromopyrazin-2(1H)-one (2).
-Bromo-1-methylpyrazin-2(1H)-one (3) was prepared by methylation of intermediate 2 in the
5
presence of dimethylsulfate. 5-Bromo-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (4) was obtained
by cyclization of intermediate 3 with tosylmethyl isocyanide. Reaction of compounds (5a, R1 =
Me; 5b, R1 = Et) and N-bromosuccinimide in concentrated sulfuric acid afforded intermediates
(
6a, b). The pinacol arylboronates (7a, b) were synthesized by coupling reaction 6a, 6b with

dioxaborolane derivative. By Suzuki coupling reaction of intermediate 4 and 7a, 7b intermediates,
we obtained the key intermediates (8a, b). The target compounds (10a-l) were obtained by
condensation of intermediates (8a, b) with intermediates (9a-g).
N
H
O
O
O
2
N
N
H
N
N
N
N
N
N
N
B
B
B
B
O
N
O
O
O
O
N
O
O
O
N
S
B
S
R
R
B
S
1
1
O
R
1
F
F
F
F
R
1
S
O
O
O
N
R
2
N
N
R
R
3
4
O
H
R
2
R
R
3
4
O
R
1
S
O
O
Scheme 1. General synthesis of 10a-l. Reagents and conditions: (a) NaNO
5℃; (b) dimethylsulfate, K CO
to rt; (d) NBS, conc. H SO , 50℃; (e) 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane),
Pd(dppf)Cl CH Cl , KOAc, 1,2-Dimethoxyethane, 90℃; (f) 4, Pd(PPh , K CO , acetonitrile,
toluene, 80℃; (g) 8a or 8b, K CO , DMF, 80℃.
2
, conc. H
2
SO
4
, 0℃ to
4
2
3
, acetonitrile, 70℃; (c) tosylmethyl isocyanide, NaH, THF, 0℃
2
4
2
2
2
3
)
4
2
3
2
3
In addition, the target compounds (10m-s) were obtained by condensation of compounds
10k, l) with sulfonyl chlorides in the presence of pyridine as shown in Scheme 2.
(
Scheme 2. General synthesis of 10m-s. Reagents and conditions: (a) sulfonyl chloride, pyridine,
CH Cl , rt.
2
2
2.2. BRD4 inhibitory activity and SAR study
BRD4 inhibitory activities of compounds (10a-s) were evaluated by AlphaScreen assay in
vitro. (+)-JQ1 and ABBV-075 were used as positive control. We initially evaluated the inhibition
rates of compounds against BRD4(1) at a concentration of 1 µM, the results were exhibited in
1
Table 1. R was replaced with methyl or ethyl (10a vs 10b, 10k vs 10l, and 10m vs 10n), the
inhibition rate demonstrated that ethyl was the optimal substituent. Furthermore, compounds with
high inhibition rate were selected to investigate their IC50 values against BRD4(1) and BRD4(2).
As showed in Table 2, compounds 10g and 10j exhibited similar activity to (+)-JQ1 and
ABBV-075. To understand the binding mode of this series, we performed docking experiments
with Glide docking in Schrodinger. (+)-JQ1 and 10j were docked into BRD4(1) crystal complex

(
PDB id: 3P5O), and analysis of the docking conformation of (+)-JQ1 and 10j with BRD4(1)
revealed that the ligand-protein interactions were similar for these two molecules (Fig. 3). The key
interactions of (+)-JQ1 and BRD4(1) were shown in Fig. 3(A). The 3-methyl-1,2,4-triazole of
(
+)-JQ1 acted as a KAc mimic, with the methyl group occupying the hydrophobic pocket. One N
formed a water mediated hydrogen bond to a conserved Tyr residue (TRY97), and another N
accepted a hydrogen bond from the conserved Asn residue (ASN140). The key interactions of 10j
and BRD4(1) were shown in Fig. 3(B). The 7-methylimidazo[1,5-a]pyrazin-8(7H)-one acted as a
KAc mimic, with the methyl group occupying the hydrophobic pocket. The O formed a water
mediated hydrogen bond to a conserved Tyr residue (TRY97), and accepted a hydrogen bond from
the conserved Asn residue (ASN140). The 4-chlorophenyl moiety of (+)-JQ1 and the phenylamino
moiety of 10j occupied a hydrophobic part of the WPF shelf. The dimethylthiophene of (+)-JQ1
and the ethylsulfonyl moiety of 10j were directed into the ZA channel.
Table 1. Structures and BRD4(1) inhibitory activity of compounds 10a-s.
a
Compound
0a
0b
0c
0d
0e
0f
R
1
R
2
R
3
R
4
BRD4(1) (1 µM)
98%
1
CH
CH
CH CH
CH
CH
CH
CH CH
CH
CH
CH CH
CH
3
CH
2
2
F
H
H
H
F
F
1
3
F
77%
1
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
98%
1
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
63%
1
3
84%
1
3
67%
1
1
0g
0h
3
2
2
99%
3
74%
1
0i
0j
3
85%
1
3
100%
78%
1
0k
0l
0m
3
NH
2
1
CH
3
CH
CH
2
2
NH
2
89%
1
CH
3
97%
1
0n
0o
0p
0q
CH
3
59%
1
CH
CH
CH
3
CH
CH
CH
2
2
2
94%
1
1
3
92%
3
97%
1
0r
0s
CH
CH
3
66%
1
3
60%
b
(
+)-JQ1
100%
100%
b
ABBV-075
a
b
Inhibition rate mean values at a screening concentration of 1 µM were obtained from three independent experiments.
Used as positive control.

Table 2. BRD4-BD1 and BRD4-BD2 inhibitory effects of compounds with high inhibition rate
Compound
BRD4(1)
IC50 (µmol/L)
0.240±0.021
0.520±0.015
0.200±0.006
0.380±0.011
0.680±0.013
0.130±0.008
0.430±0.019
0.350±0.015
0.130±0.018
0.210±0.022
0.240±0.017
0.390±0.009
0.200±0.012
0.088±0.015
0.074±0.011
BRD4(2)
IC50 (µmol/L)
0.260±0.013
0.580±0.011
0.220±0.022
0.180±0.021
0.320±0.015
0.040±0.024
0.190±0.023
0.180±0.019
0.076±0.015
0.130±0.014
0.170±0.011
0.280±0.007
0.140±0.016
0.087±0.013
0.069±0.015
a
a
1
1
1
1
0a
0b
0c
0e
1
0f
1
1
0g
0h
1
1
0i
0j
1
0m
1
0o
0p
0q
1
1
c
(
+)-JQ1
c
ABBV-075
a
b
c
IC50 values for BRD4(1) and BRD4(2) activities presented are the mean ± SD values of three independent determinations
n.d. = not determined.
Used as positive control.
Fig. 3 (A) Docking conformation of (+)-JQ1 in BRD4(1) (PDB id: 3P5O). (B) Docking
conformation of 10j in BRD4(1) (PDB id: 3P5O). (C) Superimposition docking conformation of
(
+)-JQ1 (Pink) and 10j (Green) in BRD4(1) (PDB id: 3P5O).
2.3. Anticancer evaluation against cancer cell lines in vitro
To investigate the effect of compound 10j on the proliferation of leukemia cells, the reported
sensitive cell lines human promyelocytic leukemia HL-60, acute myeloid leukemia MV4-11 and
BRD4-independent K-562 cells were utilized to validate the cellular proliferation inhibition effects
4
, 24
(
Table 3).
Most of compounds exhibited reasonable anti-proliferative activity. Especially,
compound 10j potently inhibited cell proliferation in cell line HL-60 and MV4-11 with IC50 value
of 0.57 and 0.18 µM respectively. Activity of compound 10j on BRD4-independent K-562 cells
was weaker than on BRD4-sensitive lines. These results indicated that compound 10j could be a
promising lead compound for cancer treatment.

Table 3. Anticancer evaluation against cancer cell lines K562, HL-60 and MV4-11
Compound
K562
IC50 (µM)
>10
HL-60
MV4-11
a
a
a
IC50 (µM)
IC50 (µM)
10a
10b
10c
10e
0.62 ± 0.21
2.86 ± 0.97
0.94 ± 0.41
n.d.
0.41± 0.11
1.85 ± 0.98
0.78 ± 0.31
n.d.
>10
>10
b
n.d
1
0f
>10
>10
>10
>10
>10
>10
n.d.
n.d.
n.d.
1.4 ± 0.48
2.86 ± 0.51
1.53 ± 0.81
6.1 ± 1.91
0.57 ± 0.13
3.36 ± 1.92
n.d.
0.95 ± 0.32
0.17 ± 0.05
1.16 ± 0.74
4.2 ± 1.22
0.18 ± 0.07
1.38 ± 0.94
n.d.
1
1
0g
0h
1
1
0i
0j
1
0m
1
0o
0p
0q
1
n.d.
n.d.
1
n.d.
n.d.
c
(
+)-JQ1
9.12±0.08
0.11 ± 0.03
0.08 ± 0.02
c
ABBV-075
>10µM
0.09 ± 0.02
0.07 ±0.03
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.
Used as positive control.
c
3
. Conclusion
In summary, upon the basis of the previously reported BRD4 inhibitors, nineteen
-methylimidazo[1,5-a]pyrazin-8(7H)-one derivatives were designed and synthesized, and their
7
BRD4-inhibitory activities and anti-proliferative activities were evaluated in vitro. It was found
that compound 10j exhibited robust potency of BRD4(1) and BRD4(2) inhibition with IC50 values
of 130 and 76 nM respectively. The moderated inhibitory difference between BRD4(1) and
BRD4(2) was deserving further investigation for analysis of the potential mechanisms. In addition,
compound 10j potently inhibited proliferation of leukemia cell line HL-60 and MV4-11 with IC50
value of 0.57 and 0.18 µM respectively. These findings demonstrated that compound 10j could be
a potent BRD4 inhibitor and worthy for further investigation.
4. Experimental section
4.1. Chemistry
All chemical reagents were commercially available and treated with standard methods before
use. Solvents were dried and redistilled before use. Column chromatography (CC): silica gel 60
200-300 mesh). Thin-layer chromatography (TLC): silica gel 60F254 plates (250 nm; Qingdao
Ocean Chemical Company, Qingdao,China). m.p.: capillary tube, RY-1 capillary apparatus
Tianjin Optical Instrument Company, Tianjin, China), uncorrected. Purity of the target
(
(
1
13
compounds were determined by HPLC analysis (UV detector, wavelength: 272 nm). H and
C
1
13
NMR spectra: Bruker ACF-300Q apparatus (300 MHz for H NMR and 75 MHz for C NMR), in
DMSO-d6 or CDCl unless otherwise indicated. Mass spectrometry (MS): Hewlett-Packard 1100
LC/MSD spectrometer; elemental analyses: CHNO-Rapid instrument.
.1.1. 5-bromopyrazin-2(1H)-one (2)
2 4
To a solution of NaNO (1.78 g, 25.86 mmol) in conc. H SO (15 mL) was added a solution
3
4
2

2 4
of 1 (3.0 g, 17.24 mmol) in conc. H SO (10 mL) dropwise at 0℃. The reaction mixture was
stirred at 45℃ for 2 h. After cooling to the room temperature, the mixture was poured into ice
water (200 mL) and extracted with EtOAc (300 mL). The organic layer was dried over Na
2
SO
4
1
and concentrated to yield the title compound (1.68 g, 55.69% yield) as a yellow solid. H NMR
(
300 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.10 (s, 1H), 7.92 (s, 1H).
.1.2. 5-bromo-1 -methylpyrazin-2(1H)-one (3)
3
To a solution of 2 (1.51 g, 8.63 mmol) and K CO (2.39 g, 17.26 mmol) in acetonitrile (30
4
2
mL) was added dimethylsulfate (1.63 g, 12.94 mmol). The reaction mixture was stirred at 70℃
for 4 h. After cooling to the room temperature, the mixture was filtered through a celite pad, and
the filtrate was concentrated to afford the title compound (1.36 g, 83.38% yield) as a yellow solid.
1
3
H NMR (300 MHz, CDCl ) δ 7.97 (s, 1H), 7.32 (s, 1H), 3.52 (s, 3H).
4.1.3. 5-bromo-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (4)
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℃. The reaction mixture was stirred at
room temperature for 2 h. The mixture was quenched with methanol and then was concentrated
under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (1.4 g, 72.52% yield) as a yellow solid. H NMR (300 MHz, CDCl
3
) δ 8.08 (d, J =
3.3 Hz, 2H), 6.62 (s, 1H), 3.48 (s, 3H).
4.1.4. 2-bromo-1 -fluoro-4-(methylsulfonyl)benzene (6a)
To a solution of 1-fluoro-4-(methylsulfonyl)benzene 5a (3 g, 17.22 mmol) in conc. H
2 4
SO
(
30 mL) was added NBS (3.07 g, 17.22 mmol). The reaction mixture was stirred at 50℃ for 4 h.
After cooling to the room temperature, the mixture was poured into ice water (100 mL) and the
white precipitate was filtered and washed with water. The solid was then dried under reduced
1
pressure to give the title compound (3.67g, 84.2% yield) as a white solid. H NMR (300 MHz,
DMSO-d6) δ 8.27 (dd, J = 6.3, 2.2 Hz, 1H), 8.00 (ddd, J = 8.5, 4.5, 2.3 Hz, 1H), 7.67 (t, J = 8.7
Hz, 1H), 3.30 (s, 3H).
4.1.5. 2-bromo-4-(ethylsulfonyl)-1 -fluorobenzene (6b)
2 4
To a solution of 1-(ethylsulfonyl)-4-fluorobenzene 5b (3.04 g, 16.15 mmol) in conc. H SO
(
30 mL) was added NBS (2.87 g, 16.15 mmol). The reaction mixture was stirred at 50℃ for 4 h.
After cooling to the room temperature, the mixture was poured into ice water (100 mL) and the
white precipitate was filtered and washed with water. The solid was then dried under reduced
1
pressure to give the title compound (3.31g, 76.72% yield) as a white solid. H NMR (300 MHz,
DMSO-d6) δ 8.22 (dd, J = 6.4, 2.2 Hz, 1H), 7.96 (ddd, J = 8.6, 4.6, 2.3 Hz, 1H), 7.68 (t, J = 8.6
Hz, 1H), 3.40 (q, J = 7.4 Hz, 2H), 1.10 (t, J = 7.4 Hz, 3H).
4
.1.6. 2-(2-fluoro-5-(methylsulfonyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (7a)
mixture of 6a (3.87 g, 15.29
,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (4.66 18.35
CH Cl
50 mL) was stirred at 90℃ under nitrogen atmosphere for 24 h. Then the mixture was diluted
with H O 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 Na SO , and was
A
mmol),
mmol),
4
g,
Pd(dppf)Cl
2
2
2
(0.34 g, 0.46 mmol) and KOAc (3 g, 30.58 mmol) in 1,2-Dimethoxyethane
(
2
2
4
concentrated under reduced pressure. Petroleum ether (30 mL) was added to the crude compound,
the white precipitate was filtered and then dried under reduced pressure to give the title compound
1
(
3.03g, 66.02% yield) as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.19 – 8.05 (m, 2H),

7.52 – 7.39 (m, 1H), 3.23 (d, J = 3.8 Hz, 3H), 1.32 (s, 12H).
4.1.7. 2-(5-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (7b)
A
mixture
,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane)
CH Cl
50 mL) was stirred at 90℃ under nitrogen atmosphere for 24 h. Then the mixture was diluted
with H O 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 Na SO , and was
of
6b
(4.2
g,
15.72
mmol),
4
(4.79
g, 18.87
mmol),
Pd(dppf)Cl
2
2
2
(0.35g, 0.47 mmol) and KOAc (3.09 g, 31.45 mmol) in 1,2-Dimethoxyethane
(
2
2
4
concentrated under reduced pressure. Petroleum ether (30 mL) was added to the crude compound,
the white precipitate was filtered and then dried under reduced pressure to give the title compound
1
(
3.84g, 77.73% yield) as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.14 – 8.02 (m, 2H),
7
.48 (t, J = 8.7 Hz, 1H), 3.30 (q, J = 7.3 Hz, 2H), 1.32 (s, 12H), 1.10 (t, J = 7.3 Hz, 3H).
.1.8. 5-(2-fluoro-5-(methylsulfonyl)phenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (8a)
4
A mixture of 4 (1 g, 4.39 mmol), 7a (1.58 g, 5.26 mmol), Pd(PPh
CO (1.21 g, 8.77 mmol) in acetonitrile/toluene (30 mL, 1/5) was stirred at 80℃ under nitrogen
atmosphere for 24 h. Then the mixture was diluted with CH Cl , and the insoluble material was
3 4
) (0.25g, 0.22 mmol) and
K
2
3
2
2
filtered through Celite. The organic layer was concentrated under reduced pressure. The crude
compound was purified by flash chromatography to give the title compound (1.21 g, 85.88% yield)
1
as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.24 – 8.12 (m, 2H), 8.06 (dd, J = 3.2, 0.6 Hz,
1
3
4
H), 7.88 (d, J = 0.6 Hz, 1H), 7.73 (dd, J = 9.7, 8.7 Hz, 1H), 7.26 (s, 1H), 3.42 (s, 3H), 3.31 (s,
H).
.1.9. 5-(5-(ethylsulfonyl)-2-fluorophenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (8b)
A mixture of 4 (1 g, 4.39 mmol), 7b (1.65 g, 5.26 mmol), Pd(PPh
CO (1.21 g, 8.77 mmol) in acetonitrile/toluene (30 mL, 1/5) was stirred at 80℃ under nitrogen
atmosphere for 24 h. Then the mixture was diluted with CH Cl , and the insoluble material was
3 4
) (0.25g, 0.22 mmol) and
K
2
3
2
2
filtered through celite. The organic layer was concentrated under reduced pressure. The crude
compound was purified by flash chromatography to give the title compound (1.4 g, 95.2% yield)
1
as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.15 (d, J = 5.6 Hz, 2H), 8.06 (d, J = 2.9 Hz,
1
3
4
H), 7.88 (s, 1H), 7.74 (t, J = 9.1 Hz, 1H), 7.26 (s, 1H), 3.46 – 3.35 (m, 5H), 1.16 (t, J = 7.3 Hz,
H).
.1.10. 5-(2-(2,4-difluorophenoxy)-5-(ethylsulfonyl)phenyl)-7-methylimidazo [1,5-a]pyrazin -8
(
7H)-one (10a)
A mixture of 8b (0.05 g, 0.15 mmol), 2,4-difluorophenol (0.03 g, 0.22 mmol) and K
0.04 g, 0.3 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried over Na SO , and
2
CO
3
(
with H
2
2
4
concentrated under reduced pressure. The crude compound was purified by flash chromatography
1
to give the title compound (0.061 g, 91.85% yield) as a white solid. m.p.: 137-139℃; H NMR
(
300 MHz, DMSO-d6) δ 8.09 (s, 2H), 8.00 (d, J = 8.6 Hz, 1H), 7.84 (s, 1H), 7.53 (d, J = 9.9 Hz,
1
3
2
H), 7.24 (s, 2H), 7.06 (d, J = 8.4 Hz, 1H), 3.43 (s, 3H), 3.37 (s, 2H), 1.15 (t, J = 7.3 Hz, 3H); C
NMR (75 MHz, DMSO-d6) δ 159.47, 154.54, 133.10, 132.84, 132.42, 131.93, 129.44, 124.72,
1
22.66, 122.47, 119.92, 114.99, 112.88, 112.57, 112.13, 106.40, 106.05, 105.73, 49.36, 34.15,
+
7.13; ESIMS m/z [M + H] 446.2; Anal. calcd. For C21
H
17
F
2
N
3
O
4
S: C, 56.63; H, 3.85; N, 9.43.
Found: C, 56.67; H, 3.82; N, 9.48.
.1.11. 5-(2-(2,4-difluorophenoxy)-5-(methylsulfonyl)phenyl)-7-methylimidazo[1,5-a]pyrazin-8
4

(
(
7H)-one (10b)
A mixture of 8a (0.11 g, 0.34 mmol), 2,4-difluorophenol (0.09 g, 0.68 mmol) and K
0.09 g, 0.68 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried over Na SO , and
2
CO
3
with H
2
2
4
concentrated under reduced pressure. The crude compound was purified by flash chromatography
1
to give the title compound (0.13 g, 88.02% yield) as a white solid. m.p.: > 200℃; H NMR (300
MHz, DMSO-d6) δ 8.14 (d, J = 2.3 Hz, 1H), 8.08 (s, 1H), 8.03 (dd, J = 8.8, 2.3 Hz, 1H), 7.84 (s,
1
3
1
3
H), 7.60 – 7.49 (m, 2H), 7.22 (d, J = 5.9 Hz, 2H), 7.05 (d, J = 8.7 Hz, 1H), 3.43 (s, 3H), 3.27 (s,
1
3
H); C NMR (75 MHz, DMSO-d6) δ 159.30, 154.46, 135.42, 132.82, 131.77, 131.08, 129.47,
24.73, 124.61, 122.67, 122.49, 119.80, 114.96, 112.89, 112.57, 112.16, 106.39, 106.10, 43.73,
+
4.17; ESIMS m/z [M + H] 432.2; Anal. calcd. For C20
H
15
F
2
N
3
O
4
S: C, 55.68; H, 3.50; N, 9.74.
Found: C, 55.73; H, 3.45; N, 9.69.
.1.12. 5-(5-(ethylsulfonyl)-2-(4-fluorophenoxy)phenyl)-7-methylimidazo[1,5-a]pyrazin-8
7H)-one (10c)
A mixture of 8b (0.13 g, 0.39 mmol), 4-fluorophenol (0.07 g, 0.58 mmol) and K
.78 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted with H
extracted with EtOAc (30 mL), washed with brine (10 mL), dried over Na SO , and concentrated
4
(
2
CO
3
(0.11 g,
O,
0
2
2
4
under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (0.14 g, 84.49% yield) as a white solid. m.p.: > 200℃; H NMR (300 MHz,
DMSO-d6) δ 8.20 (s, 1H), 8.06 (d, J = 2.3 Hz, 1H), 7.99 (dd, J = 8.8, 2.4 Hz, 1H), 7.83 (d, J = 0.6
Hz, 1H), 7.30 (d, J = 6.5 Hz, 4H), 7.19 (s, 1H), 7.03 (d, J = 8.7 Hz, 1H), 3.43 (s, 3H), 3.37 (q, J =
1
3
7
.4 Hz, 2H), 1.16 (t, J = 7.4 Hz, 3H); C NMR (75 MHz, DMSO-d6) δ 160.84, 154.97, 150.12,
1
33.67, 132.98, 132.74, 132.26, 129.88, 123.24, 123.13, 122.85, 120.86, 117.69, 117.38, 116.45,
+
113.02, 49.89, 34.64, 7.68; ESIMS m/z [M + H] 428.2; Anal. calcd. For C21
H18FN
3
O
4
S: C, 59.01;
H, 4.24; N, 9.83. Found: C, 59.05; H, 4.27; N, 9.79.
4.1.13. 5-(2-(3-((4-chlorobenzyl)amino)phenoxy)-5-(methylsulfonyl)phenyl)-7-methylimidazo
[
1,5-a]pyrazin-8(7H)-one (10d)
A mixture of 8a (0.09 g, 0.28 mmol), 3-((4-chlorobenzyl)amino)phenol (0.13 g, 0.56 mmol)
and K
was diluted with H
Na SO , and concentrated under reduced pressure. The crude compound was purified by flash
2
CO
3
(0.12 g, 0.84 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried over
2
4
chromatography to give the title compound (0.13 g, 86.75% yield) as a white solid. m.p.:
1
1
72-174℃; H NMR (300 MHz, DMSO-d6) δ 8.04 (dd, J = 22.5, 5.6 Hz, 3H), 7.83 (s, 1H), 7.35
(
s, 4H), 7.20 – 6.94 (m, 3H), 6.62 – 6.39 (m, 2H), 6.31 (s, 2H), 4.23 (d, J = 4.9 Hz, 2H), 3.41 (s,
1
3
3
H), 3.25 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 160.06, 154.71, 154.45, 150.46, 138.81,
1
34.58, 132.94, 131.48, 131.18, 130.73, 130.42, 129.38, 129.05, 128.21, 122.65, 122.22, 120.32,
+
116.39, 112.67, 109.76, 107.17, 103.74, 45.52, 43.83, 34.12; ESIMS m/z [M + H] 535.2; Anal.
calcd. For C27
.1.14. N-(4-(((3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methylsulfonyl)
phenoxy)phenyl)amino)methyl)phenyl)acetamide (10e)
mixture of 8a (0.1
N-(4-(((3-hydroxyphenyl)amino)methyl)phenyl)acetamide (0.12 g, 0.47 mmol) and K
4 4
H23ClN O S: C, 60.61; H, 4.33; N, 10.47. Found: C, 60.64; H, 4.29; N, 4.37.
4
A
g,
0.31
mmol),
3
CO (0.09 g,
2
0
.62 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted with H
2
O,
extracted with EtOAc (30 mL), washed with brine (10 mL), dried over Na SO , and concentrated
2
4

under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (0.14 g, 80.67% yield) as a white solid. m.p.: 188-190℃; H NMR (300 MHz,
DMSO-d6) δ 9.89 (s, 1H), 8.07 (s, 2H), 7.99 (d, J = 8.9 Hz, 1H), 7.83 (s, 1H), 7.50 (d, J = 8.0 Hz,
2H), 7.23 (d, J = 8.0 Hz, 2H), 7.14 (s, 1H), 7.07 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.46
(
d, J = 8.1 Hz, 2H), 6.29 (d, J = 7.8 Hz, 2H), 4.16 (d, J = 5.5 Hz, 2H), 3.42 (s, 3H), 3.25 (s, 3H),
1
3
2
.02 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 168.10, 160.09, 154.65, 154.37, 150.67, 137.97,
1
34.53, 134.04, 132.92, 131.45, 130.72, 130.33, 129.37, 127.57, 122.66, 122.21, 120.28, 119.02,
+
116.34, 112.68, 109.78, 106.95, 103.70, 45.99, 43.81, 34.11, 23.89; ESIMS m/z [M + H] 558.3;
Anal. calcd. For C29
27 5 5
H N O S: C, 62.46; H, 4.88; N, 12.56. Found: C, 62.40; H, 4.85; N, 12.50.
4.1.15. 5-(2-(3-((4-fluorobenzyl)amino)phenoxy)-5-(methylsulfonyl)phenyl)-7-methylimidazo
[
1,5-a]pyrazin-8(7H)-one (10f)
A mixture of 8a (0.1 g, 0.31 mmol), 3-((4-fluorobenzyl)amino)phenol (0.14 g, 0.62 mmol)
and K
was diluted with H
Na SO , and concentrated under reduced pressure. The crude compound was purified by flash
2
CO
3
(0.13 g, 0.93 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried over
2
4
chromatography to give the title compound (0.15 g, 96.05% yield) as a white solid. m.p.:
1
1
40-142℃; H NMR (300 MHz, DMSO-d6) δ 8.13 – 7.94 (m, 3H), 7.84 (s, 1H), 7.35 (d, J = 5.8
Hz, 2H), 7.19 – 6.96 (m, 5H), 6.57 – 6.39 (m, 2H), 6.31 (d, J = 7.7 Hz, 2H), 4.21 (s, 2H), 3.42 (s,
1
3
3
H), 3.26 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 160.60, 155.20, 151.04, 136.40, 135.05,
1
33.45, 131.98, 131.24, 130.89, 129.88, 129.69, 129.58, 123.17, 122.72, 120.80, 116.87, 115.61,
+
115.34, 110.28, 107.61, 104.20, 90.08, 46.02, 44.32, 34.62; ESIMS m/z [M + H] 519.3; Anal.
calcd. For C27
.1.16. N-(4-(((3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)
phenoxy)phenyl)amino)methyl)phenyl)acetamide (10g)
mixture of 8b (0.11
N-(4-(((3-hydroxyphenyl)amino)methyl)phenyl)acetamide (0.13 g, 0.49 mmol) and K
4 4
H23FN O S: C, 62.54; H, 4.47; N, 10.80. Found: C, 62.58; H, 4.43; N, 10.85.
4
A
g,
0.33
mmol),
3
CO (0.09 g,
2
0
.66 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted with H
2
O,
extracted with EtOAc (30 mL), washed with brine (10 mL), dried over Na SO , and concentrated
2
4
under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (0.15 g, 80% yield) as a white solid. m.p.: 174-176℃; H NMR (300 MHz,
DMSO-d6) δ 9.89 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.94 (dd, J = 8.8, 2.2 Hz, 1H),
7
.82 (s, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.14 (s, 1H), 7.08 (t, J = 8.0 Hz,
H), 7.00 (d, J = 8.8 Hz, 1H), 6.46 (d, J = 7.8 Hz, 2H), 6.30 (d, J = 6.8 Hz, 2H), 4.16 (d, J = 5.8
1
1
3
Hz, 2H), 3.42 (s, 3H), 3.30 (d, J = 7.2 Hz, 2H), 2.02 (s, 3H), 1.15 (t, J = 7.0 Hz, 3H); C NMR
75 MHz, DMSO-d6) δ 168.11, 160.27, 154.60, 154.31, 150.66, 137.97, 134.03, 132.94, 132.14,
(
1
31.58, 130.35, 129.35, 127.69, 127.57, 122.66, 122.20, 120.33, 119.02, 116.28, 112.66, 109.81,
+
106.99, 103.72, 49.46, 45.96, 34.10, 23.89, 7.16; ESIMS m/z [M + H] 572.3; Anal. calcd. For
C
30
H
29
N
5
O
5
S: C, 63.03; H, 5.11; N, 12.25. Found: C, 63.05; H, 5.15; N, 12.21.
4.1.17. 5-(2-(3-((3-methoxybenzyl)amino)phenoxy)-5-(methylsulfonyl)phenyl)-7-methylimidazo
[
1,5-a]pyrazin-8(7H)-one (10h)
A mixture of 8a (0.1 g, 0.31 mmol), 3-((3-methoxybenzyl)amino)phenol (0.11 g, 0.47 mmol)
and K
was diluted with H
Na SO , and concentrated under reduced pressure. The crude compound was purified by flash
2
CO
3
(0.09 g, 0.62 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the mixture
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried over
2
4

chromatography to give the title compound (0.13 g, 78.73% yield) as a white solid. m.p.:
1
1
22-124℃; H NMR (300 MHz, DMSO-d6) δ 8.03 (dd, J = 22.7, 7.5 Hz, 3H), 7.83 (s, 1H), 7.22 (t,
J = 7.9 Hz, 1H), 7.14 – 6.95 (m, 3H), 6.88 (s, 2H), 6.78 (d, J = 7.1 Hz, 1H), 6.46 (d, J = 8.1 Hz,
1
3
2
H), 6.30 (d, J = 8.5 Hz, 2H), 4.20 (d, J = 5.1 Hz, 2H), 3.71 (s, 3H), 3.41 (s, 3H), 3.25 (s, 3H); C
NMR (75 MHz, DMSO-d6) δ 160.59, 155.17, 154.79, 151.19, 141.87, 135.02, 133.42, 131.97,
1
31.23, 130.87, 129.87, 129.83, 123.09, 122.72, 120.79, 119.89, 116.83, 113.41, 113.05, 112.51,
+
110.27, 107.53, 104.19, 97.68, 55.41, 46.78, 44.32, 34.62; ESIMS m/z [M + H] 531.3; Anal.
calcd. For C28
26 4 5
H N O S: C, 63.38; H, 4.94; N, 10.56. Found: C, 63.35; H, 4.91; N, 10.51.
4.1.18. 4-(((3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methylsulfonyl) phe
noxy)phenyl)amino)methyl)benzonitrile (10i)
A mixture of 8a (0.1 g, 0.31 mmol), 4-(((3-hydroxyphenyl)amino)methyl)benzonitrile (0.1 g,
0
.47 mmol) and K
the mixture was diluted with H
dried over Na SO , and concentrated under reduced pressure. The crude compound was purified
2
CO
3
(0.09 g, 0.62 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL),
2
4
by flash chromatography to give the title compound (0.15 g, 91.71% yield) as a white solid. m.p.:
1
1
50-152℃; H NMR (300 MHz, DMSO-d6) δ 8.09 – 7.98 (m, 3H), 7.85 – 7.74 (m, 3H), 7.51 (d, J
=
8.0 Hz, 2H), 7.10 (dd, J = 16.8, 8.4 Hz, 2H), 7.01 (d, J = 8.8 Hz, 1H), 6.66 (s, 1H), 6.44 (d, J =
13
8.1 Hz, 1H), 6.31 (s, 2H), 4.35 (d, J = 5.9 Hz, 2H), 3.42 (s, 3H), 3.26 (s, 3H); C NMR (75 MHz,
DMSO-d6) δ 160.53, 155.25, 154.88, 150.80, 146.54, 135.12, 133.44, 132.73, 131.97, 131.23,
1
31.00, 129.88, 128.51, 123.14, 122.73, 120.83, 119.36, 116.91, 113.15, 110.22, 109.87, 107.88,
+
104.26, 46.33, 44.32, 34.62; ESIMS m/z [M + H] 526.3; Anal. calcd. For C28
H
23
N
5
O
4
S: C, 63.99;
H, 4.41; N, 13.33. Found: C, 63.92; H, 4.45; N, 13.37.
.1.19. 4-(((3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)pheno
4
xy)phenyl)amino)methyl)benzonitrile (10j)
A mixture of 8b (0.1 g, 0.3 mmol), 4-(((3-hydroxyphenyl)amino)methyl)benzonitrile (0.1 g,
0
.45mmol) and K
mixture was diluted with H
over Na SO , and concentrated under reduced pressure. The crude compound was purified by
2
CO
3
(0.08 g, 0.59 mmol) in DMF (2 mL) was stirred at 80℃ for 12 h. Then the
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL), dried
2
4
flash chromatography to give the title compound (0.14 g, 87.01% yield) as a white solid. m.p.:
1
1
40-142℃; H NMR (300 MHz, DMSO-d6) δ 8.06 (d, J = 0.6 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H),
.96 (dd, J = 8.8, 2.4 Hz, 1H), 7.83 (d, J = 0.6 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.2
7
Hz, 2H), 7.15 – 6.98 (m, 3H), 6.66 (t, J = 6.2 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 6.33 (d, J = 7.8 Hz,
1
3
2
H), 4.35 (d, J = 6.0 Hz, 2H), 3.41 (s, 3H), 3.30 (d, J = 7.3 Hz, 2H), 1.15 (t, J = 7.3 Hz, 3H); C
NMR (75 MHz, DMSO-d6) δ 160.70, 155.19, 154.75, 150.79, 146.53, 133.41, 132.72, 132.65,
1
32.09, 131.01, 129.86, 128.49, 128.45, 122.72, 120.83, 119.36, 116.84, 113.13, 110.18, 109.94,
+
109.87, 107.91, 104.27, 49.95, 46.32, 34.60, 7.68; ESIMS m/z [M + H] 540.3; Anal. calcd. For
C
29
H
25
N
5
O
4
S: C, 64.55; H, 4.67; N, 12.98. Found: C, 64.51; H, 4.63; N, 12.94.
.1.20. 5-(2-(3-aminophenoxy)-5-(methylsulfonyl)phenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)
one (10k)
A mixture of 8a (0.3 g, 0.93 mmol), 3-aminophenol (0.2 g, 1.87 mmol) and K
.8 mmol) in DMF (3 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted with H
extracted with EtOAc (50 mL), washed with brine (20 mL), dried over Na SO , and concentrated
4
-
2
CO
3
(0.39 g,
O,
2
2
2
4
under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (0.38 g, 99.16% yield) as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.05 (t,

J = 9.6 Hz, 3H), 7.83 (d, J = 2.2 Hz, 1H), 7.18 (d, J = 2.6 Hz, 1H), 7.05 (dd, J = 9.5, 7.0 Hz, 2H),
.44 (d, J = 8.2 Hz, 1H), 6.28 (dd, J = 9.7, 6.1 Hz, 2H), 5.36 (s, 2H), 3.43 (d, J = 2.5 Hz, 3H), 3.26
d, J = 2.4 Hz, 3H).
.1.21. 5-(2-(3-aminophenoxy)-5-(ethylsulfonyl)phenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)
one (10l)
A mixture of 8b (0.4 g, 1.19 mmol), 3-aminophenol (0.2 g, 1.79 mmol) and K
.39 mmol) in DMF (3 mL) was stirred at 80℃ for 12 h. Then the mixture was diluted with H
extracted with EtOAc (50 mL), washed with brine (20 mL), dried over Na SO , and concentrated
6
(
4
-
2
CO
3
(0.33 g,
O,
2
2
2
4
under reduced pressure. The crude compound was purified by flash chromatography to give the
1
title compound (0.47 g, 92.83% yield) as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.19 –
7
.91 (m, 3H), 7.83 (s, 1H), 7.18 (s, 1H), 7.05 (t, J = 8.4 Hz, 2H), 6.44 (d, J = 7.9 Hz, 1H), 6.28 (d,
J = 12.9 Hz, 2H), 5.35 (s, 2H), 3.43 (s, 3H), 3.33 – 3.23 (m, 2H), 1.15 (t, J = 7.3 Hz, 3H).
.1.22. N-(3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phen
4
oxy)phenyl)-4-methylbenzenesulfonamide (10m)
To a solution of 10l (0.1 g, 0.24 mmol) and 4-methylbenzenesulfonyl chloride (0.07 g, 0.35
mmol) in CH
Then the mixture was diluted with H
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.
2
O, extracted with EtOAc (30 mL), washed with brine (10
2
4
purified by flash chromatography to give the title compound (0.13 g, 95.36% yield) as a white
1
solid. m.p.: 156-158℃; H NMR (300 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.01 (dd, J = 20.6, 8.8 Hz,
3
H), 7.82 (s, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.40 – 7.23 (m, 3H), 7.12 (s, 1H), 6.92 (t, J = 8.0 Hz,
1
3
2H), 6.87 – 6.74 (m, 2H), 3.40 (s, 5H), 2.34 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H); C NMR (75 MHz,
DMSO-d6) δ 159.59, 154.44, 154.14, 143.51, 139.70, 136.23, 132.88, 132.33, 131.64, 131.02,
1
2
9
4
29.76, 129.41, 126.67, 122.62, 122.37, 120.81, 116.52, 115.47, 112.28, 111.24, 49.37, 34.13,
+
0.93, 7.18; ESIMS m/z [M + H] 579.3; Anal. calcd. For C28
.68. Found: C, 58.16; H, 4.49; N, 9.63.
26 4 6 2
H N O S : C, 58.12; H, 4.53; N,
.1.23. 4-methyl-N-(3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methyl sulf
onyl)phenoxy)phenyl)benzenesulfonamide (10n)
To a solution of 10k (0.08 g, 0.19 mmol) and 4-methylbenzenesulfonyl chloride (0.05 g, 0.28
mmol) in CH
Then the mixture was diluted with H
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.
2
O, extracted with EtOAc (30 mL), washed with brine (10
2
4
purified by flash chromatography to give the title compound (0.1 g, 94.41% yield) as a white solid.
1
m.p.: 182-184℃; H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.12 (d, J = 2.1 Hz, 1H), 8.03
(
d, J = 11.1 Hz, 2H), 7.83 (s, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.40 – 7.23 (m, 3H), 7.14 (s, 1H), 6.98
13
–
1
1
5
9
4
6.75 (m, 4H), 3.40 (s, 3H), 3.28 (s, 3H), 2.34 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 159.42,
54.42, 154.16, 143.51, 139.70, 136.16, 135.24, 132.88, 131.67, 131.02, 130.67, 129.77, 129.32,
+
26.67, 122.40, 120.73, 116.50, 115.28, 112.31, 111.22, 43.73, 32.53, 20.94; ESIMS m/z [M + H]
65.2; Anal. calcd. For C27
.87.
24 4 6 2
H N O S : C, 57.44; H, 4.28; N, 9.92. Found: C, 57.41; H, 4.32; N,
.1.24. N-(3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phen
oxy)phenyl)-2-fluorobenzenesulfonamide (10o)
To a solution of 10l (0.1 g, 0.24 mmol) and 2-fluorobenzenesulfonyl chloride (0.07 g, 0.35
2 2
mmol) in CH Cl (2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.

2
Then the mixture was diluted with H O, extracted with EtOAc (30 mL), washed with brine (10
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
4
purified by flash chromatography to give the title compound (0.13 g, 94.71% yield) as a white
1
solid. m.p.: 167-169℃; H NMR (300 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.06 (d, J = 4.2 Hz, 2H),
7
1
3
1
.98 (dd, J = 8.7, 2.2 Hz, 1H), 7.86 – 7.76 (m, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.35 (ddd, J = 20.9,
7.9, 9.3 Hz, 3H), 7.14 (s, 1H), 6.98 – 6.79 (m, 4H), 3.39 (d, J = 9.9 Hz, 5H), 1.17 (t, J = 7.3 Hz,
1
3
H); C NMR (75 MHz, DMSO-d6) δ 159.50, 154.40, 154.25, 139.11, 136.07, 136.07, 133.02,
32.84, 132.33, 131.66, 131.04, 130.39, 129.42, 125.03, 122.63, 122.37, 120.91, 117.48, 117.10,
+
116.81, 116.06, 115.47, 112.24, 110.82, 49.37, 34.11, 7.15; ESIMS m/z [M + H] 583.2; Anal.
calcd. For C27
4 6 2
H23FN O S : C, 55.66; H, 3.98; N, 9.62. Found: C, 55.61; H, 3.95; N, 9.58.
4.1.25. N-(3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phen
oxy)phenyl)thiophene-2-sulfonamide (10p)
To a solution of 10l (0.1 g, 0.24 mmol) and thiophene-2-sulfonyl chloride (0.06 g, 0.35 mmol)
in CH
the mixture was diluted with H
dried over Na SO , and concentrated under reduced pressure. The crude compound was purified
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h. Then
2
O, extracted with EtOAc (30 mL), washed with brine (10 mL),
2
4
by flash chromatography to give the title compound (0.12 g, 89.26% yield) as a white solid. m.p.:
1
1
72-174℃; H NMR (300 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.07 (d, J = 2.7 Hz, 2H), 8.01 (dd, J
=
8.7, 2.3 Hz, 1H), 7.91 (d, J = 5.0 Hz, 1H), 7.83 (s, 1H), 7.53 (d, J = 3.8 Hz, 1H), 7.33 (t, J = 8.1
Hz, 1H), 7.16 – 7.11 (m, 2H), 7.01 – 6.82 (m, 4H), 3.41 (s, 3H), 3.36 (d, J = 7.5 Hz, 2H), 1.16 (t, J
1
3
=
7.4 Hz, 3H); C NMR (75 MHz, DMSO-d6) δ 159.56, 154.44, 154.25, 139.49, 139.39, 133.63,
1
33.01, 132.88, 132.67, 132.33, 131.69, 131.03, 129.42, 127.66, 126.02, 122.65, 122.39, 120.91,
+
116.93, 115.84, 112.27, 111.64, 49.38, 34.13, 7.16; ESIMS m/z [M + H] 571.2; Anal. calcd. For
C
25
H
22
N
4
O
6
S
3
: C, 52.62; H, 3.89; N, 9.82. Found: C, 52.58; H, 3.86; N, 9.87.
4.1.26. N-(3-(4-(ethylsulfonyl)-2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phen
oxy)phenyl)thiophene-2-sulfonamide (10q)
To a solution of 10l (0.1 g, 0.24 mmol) and 4-methoxybenzenesulfonyl chloride (0.07 g, 0.35
mmol) in CH
Then the mixture was diluted with H
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.
2
O, extracted with EtOAc (30 mL), washed with brine (10
2
4
purified by flash chromatography to give the title compound (0.11 g, 78.52% yield) as a white
1
solid. m.p.: 165-167℃; H NMR (300 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.07 (s, 2H), 7.98 (dd, J
=
8.7, 2.3 Hz, 1H), 7.84 (s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.29 (t, J = 8.1 Hz, 1H), 7.15 (s, 1H),
7
7
1
.06 (d, J = 8.9 Hz, 2H), 6.97 – 6.78 (m, 4H), 3.81 (s, 3H), 3.41 (s, 3H), 3.37 (s, 2H), 1.17 (t, J =
1
3
.3 Hz, 3H); C NMR (75 MHz, DMSO-d6) δ 162.54, 159.60, 154.44, 154.14, 139.84, 132.91,
32.88, 132.33, 131.62, 130.99, 130.63, 129.42, 128.89, 122.64, 122.37, 120.83, 116.63, 116.52,
+
115.37, 114.44, 112.29, 111.19, 55.63, 49.40, 34.12, 7.16; ESIMS m/z [M + H] 595.2; Anal. calcd.
For C28
H
26
N
4
O
7
S
2
: C, 56.55; H, 4.41; N, 9.42. Found: C, 56.51; H, 4.46; N, 9.46.
4.1.27. 4-methoxy-N-(3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methyl
sulfonyl)phenoxy)phenyl)benzenesulfonamide (10r)
To a solution of 10k (0.1 g, 0.24 mmol) and 4-methoxybenzenesulfonyl chloride (0.08 g, 0.37
mmol) in CH
Then the mixture was diluted with H
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.
2
O, extracted with EtOAc (30 mL), washed with brine (10
2
4

purified by flash chromatography to give the title compound (0.13 g, 91.9% yield) as a white solid.
1
m.p.: 170-172℃; H NMR (300 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 8.06
(
s, 1H), 8.01 (dd, J = 8.7, 2.4 Hz, 1H), 7.83 (s, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.27 (t, J = 8.1 Hz,
1
3
1
H), 7.14 (s, 1H), 7.05 (d, J = 8.9 Hz, 2H), 6.85 (ddd, J = 16.1, 11.9, 5.1 Hz, 4H), 3.80 (s, 3H),
1
3
.40 (s, 3H), 3.27 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 162.54, 159.42, 154.42, 154.16,
39.83, 135.27, 132.86, 131.68, 130.99, 130.75, 130.61, 129.45, 128.90, 122.63, 122.40, 120.75,
+
116.64, 116.49, 115.35, 114.44, 112.32, 111.16, 55.63, 43.74, 34.14; ESIMS m/z [M + H] 581.2;
Anal. calcd. For C27
24 4 7 2
H N O S : C, 55.85; H, 4.17; N, 9.65. Found: C, 55.81; H, 4.21; N, 9.61.
4.1.27. 3-methyl-N-(3-(2-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(methyl
sulfonyl)phenoxy)phenyl)benzenesulfonamide (10s)
To a solution of 10k (0.1 g, 0.24 mmol) and 3-methylbenzenesulfonyl chloride (0.07 g, 0.37
mmol) in CH
Then the mixture was diluted with H
mL), dried over Na SO , and concentrated under reduced pressure. The crude compound was
2
Cl
2
(2 mL) was added pyridine (1 mL). The reaction mixture was stirred at rt for 2 h.
2
O, extracted with EtOAc (30 mL), washed with brine (10
2
4
purified by flash chromatography to give the title compound (0.13 g, 94.5% yield) as a white solid.
1
m.p.: 156-158℃; H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.13 (d, J = 2.3 Hz, 1H), 8.08 –
7.99 (m, 2H), 7.83 (s, 1H), 7.57 (s, 1H), 7.51 (d, J = 3.7 Hz, 1H), 7.44 (d, J = 5.4 Hz, 2H), 7.29 (t,
J = 8.1 Hz, 1H), 7.16 (s, 1H), 6.88 (ddd, J = 20.8, 9.6, 5.0 Hz, 4H), 3.41 (s, 3H), 3.28 (s, 3H), 2.33
13
(
s, 3H); C NMR (75 MHz, DMSO-d6) δ 159.62, 154.62, 154.16, 139.64, 139.08, 138.97, 135.25,
1
33.74, 132.87, 131.67, 131.56, 131.04, 130.68, 129.43, 129.17, 126.78, 123.79, 122.66, 122.40,
+
120.81, 116.55, 115.48, 112.31, 111.20, 43.72, 34.15, 20.76; ESIMS m/z [M + H] 565.2; Anal.
24 4 6 2
calcd. For C27H N O S : C, 57.44; H, 4.28; N, 9.92. Found: C, 57.40; H, 4.25; N, 9.96.
4.2. Docking studies
All ligand molecules were drawn in ChemDraw 2014, and saved as sdf style. Then ligands
were processed at a simulated pH of 7.4 ± 1.0 to generate all possible tautomers, stereoisomers,
and protonation states and were finally minimized at the OPLS 2005 force field with Ligand
preparation protocol of Maestro 10.2. The crystal complex (PDB id: 3P5O) was selected as the
BRD4(1) docking protein. The protein was opened with Glide generation protocol of Maestro 10.2,
molecules of water around the binding site were saved, and the bond orders were assigned.
Hydrogen atoms were added. Then a 10-Å box centered on the geometrical center of the ligand
binding site was generated for grid calculation. The docking studies were processed with the Glide
docking protocol. After docking finished, only one docking conformation was saved for every
compound.
4.3. Biological evaluation
4.3.1. Binding affinities of target compounds to BRD4(1) and BRD4(2) by AlphaScreen assay.
We commissioned Shanghai ChemPartner Co. to carry out 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 1x assay buffer (modified HEPES
(
Buffer). Transfer compounds to assay plates by Echo. DMSO’s final concentration is 0.1%.
Prepare protein solution in 1x assay buffer (5 nM). Add peptide (H4) in 1x assay buffer to make
the substrate solution. Transfer 5 µL of protein solution to assay plates or for low control transfer 5
µL of 1x assay buffer. Incubate at room temperature for 15 minutes. Add 5 µL of substrate solution

to each well to start reaction. Incubate at room temperature for 60 min. Add 15 µL acceptor and
donor solution, incubate for 60 min at room temperature, subdued light. Read endpoint with
EnSpire with Alpha mode. Fit the data in Excel to obtain inhibition values using equation (1).
Equation (1): Inh % = (Max - Signal) / (Max - Min) × 100.Max signal was obtained from the
action of Enzyme and Substrate. Min signal was obtained from the Substrate only. Fit the data in
GraphPad Prism 5 statistical software to obtain IC50 values using equation (2). Equation (2): Y =
Bottom + (Top - Bottom) / (1 + 10^ ((LogIC50 - X) × Hill Slope)).Y is %inhibition and X is
compound concentration.
4
.3.2. Cell culture and proliferation inhibition assays.
4
HL-60 , MV4-11 or K562 cells were seeded in 96-well plates at a concentration of 1 × 10
cells per well. Cells were grown in 100 µL of IMDM containing 20% fetal bovine serum. After 12
h, 50 µL of which containing various concentrations of compounds (triple diluted) was added. The
measurement was conducted 72 h after seeding, and 10 µL of Cell-counting kit-8(CCK-8) reagent
was added to each well and incubated in 37 °C for 4 h. The spectrophotometric absorbance of each
well was measured by a multi-detection microplate reader at a wavelength of 450 nm. The
inhibition rate was calculated as ((A450 treated - A450 blank) / (A450 control - A450 blank)) ×
100. The IC50 was calculated by GraphPad Prism 5 statistical software.
Acknowledgments
This study was supported by the Natural Science Foundation of Jiangsu Province (No. BK
0141349) and the China National Key HiTech Innovation Project for the R&D of Novel Drugs
No.2013ZX09301303-002).
2
(
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R
2
R
R
OH
3
4
O
O
N
N
N
N
N
N
R
2
R
R
O
F
3
4
R
R
1
1
S
S
O
O
O
O