Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one scaffold for the development of novel chemical inhibitors for Bromodomain and Extraterminal Domain (BET) family

Article abstract of DOI:10.1016/j.bioorg.2019.103044

The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-inhibitory activity with IC₅₀ value of 33 nM and anti-proliferation potency with IC₅₀ value of 110 nM in HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion, these results suggested that compound 28 has merely potential for leukemia treatment.

Full text of DOI:10.1016/j.bioorg.2019.103044

Accepted Manuscript
Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one Scaffold for the De-
velopment of Novel Chemical Inhibitors for Bromodomain and Extraterminal
Domain (BET) Family
Yifei Yang, Pan Chen, Leilei Zhao, Fangqing Zhang, Bing Zhang, Changliang
Xu, Huibin Zhang, Jinpei Zhou
PII:
S0045-2068(19)30699-6
https://doi.org/10.1016/j.bioorg.2019.103044
103044
DOI:
Article Number:
Reference:
YBIOO 103044
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 May 2019
31 May 2019
4 June 2019
Please cite this article as: Y. Yang, P. Chen, L. Zhao, F. Zhang, B. Zhang, C. Xu, H. Zhang, J. Zhou, Exploiting the
7-methylimidazo[1,5-a]pyrazin-8(7H)-one Scaffold for the Development of Novel Chemical Inhibitors for
Bromodomain and Extraterminal Domain (BET) Family, Bioorganic Chemistry (2019), doi:https://doi.org/10.1016/
j.bioorg.2019.103044
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Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one Scaffold for the
Development of Novel Chemical Inhibitors for Bromodomain and Extraterminal
Domain (BET) Family
Yifei Yanga, Pan Chena, Leilei Zhao b, Fangqing Zhang b, Bing Zhang b, Changliang Xuc,, Huibin
Zhangb,, Jinpei Zhoua,
a
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China.
b
Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing 210009, PR China.
c
Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing
University of Chinese Medicine, Nanjing, Jiangsu 210023, PR China.
O
O
O
N
N
N
N
N
N
N
O
O
O
F
O
O
S
S
N
F
F
N
F
O
O
H
H
Compound 17
Compound 28
Fragment 9
BRD4 BD1 IC50=270nM
BRD4 BD2 IC50=310nM
BRD4 BD1 IC50=30nM
BRD4 BD2 IC50=29nM
BRD4 TR-FRET Ki=890nM
BRD4 Engagement EC50=4100nM
Abstract
The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene
expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug
metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported
a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among
them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was
considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-
inhibitory activity with IC50 value of 33 nM and anti-proliferation potency with IC50 value of 110 nM in
HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can
effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion,
these results suggested that compound 28 has merely potential for leukemia treatment.
Keywords: BRD4 inhibitors, Molecular docking, Anticancer, BET family
Introduction
The acetylation of lysine residues were proposed to play crucial roles in post-translational
modifications of histones[1]. Ever since 1992, when the bromodomains comprised of 110-amino acid
were first identified as ‘readers’ of e-N-acetyl-lysine modifications in nucleosomal chromatin, there are
a lot of researches focused on the mechanism of bromodomains in epigenetics [2-4]. The human genome
codes 46 proteins containing 61 BRDs [5], including the bromodomain and extra-terminal (BET) protein
family, which contains four members: BRD2, BRD3, BRD4 and BRDT [6]. These proteins comprise of
conservative tandem bromodomains that recognize acetylated lysine residues on histone tails and recruit
transcriptional factors to promote gene expression [3, 7, 8]. For example, BRD4 recruits positive
transcription elongation factor (P-TEFb) to acetylated chromatin and plays a role in activating the cyclin
dependent kinase (CDK9) subunit, whereby it acts as a vital transcription moderator [9].
BET proteins were confirmed as a promising therapeutic target for competitive inhibitors displacing
bromodomains from chromatin to influence changes in gene expression. Therefore great interests and

Corresponding authors. E-mail addresses: cl.xu81@gmail.com (C. Xu); zhanghb80@163.com (H. Zhang); jpzhou668@163.com (J. Zhou).

attention were attracted for the development of novel BET inhibitors for cancer treatment [10-12].
Several BET inhibitors with different scaffolds have been reported since the first small molecule BRD4
inhibitor JQ1 was disclosed [13]. There are some inhibitors undergoing clinical trials for the treatment
of different diseases, such as OTX015 (Merck & Co. Phase I/II) [14], I-BET762 (GlaxoSmithKline.
Phase II) [15], BI-2536 (Boehringer Ingelheim. Phase II) [16], ABBV-075 (AbbVie . Phase I) [17-19]
(Figure 1). However, the major indications of these inhibitors were hematologic malignancies, such as
multiple myeloma (MM) [20].
N
N
N
N
N
N
O
N
O
O
O
N
N
N
N
S
S
NH
NH
O
N
N
O
N
HN
O
N
OH
N
Cl
Cl
Cl
4
(I-BET151
)
3
(I-BET762
)
1
JQ1
2
(OTX015
(
)
)
N
O
O
O
H
N
N
O
NH2
N
N
O
N
N
N
F
H
O
N
H
N
N
O
S
O
O
N
F
H
Cl
7 (ABBV-075
)
6
BI-2536
)
(
5
CPI-0610
)
(
Figure 1. Structures of known BET bromodomain inhibitors
Recent study also revealed the potency of BET inhibitors in solid tumor, such as pancreatic cancer
[21]. Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers with a median
survival time of 6 months, with less than 5% survival 5 years after diagnosis[22]. PDA surpassed only
by lung cancer, will become the second leading cause of cancer-related deaths in the United States by
2030[23]. According to the important role that BET proteins play in the progression and evolution of
pancreatic cancer, we designed and synthesized a series of imidazolopyridone derivatives containing the
moiety 7-methylimidazo [1, 5-a]pyrazin-8(7H)-one via scaffold hopping as BET inhibitors. Then their
activities in AlphaScreen binding assays and effects on cell viability were evaluated in vitro. Cell cycle
analysis was performed to characterize the molecular mechanisms underlying the cytotoxic effect of
compound 28 in BxPC-3 cell.
Synthesis
Target compounds (15-17, 25-37) were synthesized as outlined in Schemes 1 and 2. All compounds
were purified by chromatography and purity was checked by HPLC before biological evaluation (purity
was >98%). The structures were confirmed by 1H NMR, 13C NMR spectrum, and mass spectra. Some
representative spectrometry was shown in Supporting Information. As depicted in Scheme 1,
commercially available cytosine 8 reacted with bromine in acetic acid to give 6-amino-5-bromo-1H-
pyrimidin-2-one (9). Cyclization of compound 9 with chloroacetaldehyde in dimethyl formamide was
utilized to obtain intermediate 10. The key intermediate 11 was prepared by methylation of intermediate
10 in the presence of dimethylsulfate. Then Suzuki-Miyaura cross coupling reaction was used to get
compound 12, followed by reacting with different phenol in the presence of potassium carbonate. Iron-
catalyzed reduction of the nitro group of 13 in the presence of ammonium chloride followed by
functionalization of the resulting aniline 14 provided facile access to sulfonamides 15-17.

O
O
O
O
N
NH
N
N
N
NH
N
NH
i
iii
iv
ii
N
N
H2N
H2N
Br
Br
Br
R1
R2
9
10
11
15
8
Me
2,6-di-Me-phenyl
2,6-di-Me-phenyl
16
Et
Et
O
O
O
O
17 2,4-di-F-phenyl
N
N
N
N
N
N
N
N
N
N
N
O
N
vi
R1
vii
v
O
F
O
O
R1
O
R1
S
H2N
O2N
R2
N
H
O2N
14a-b
15-17
13a-b
12
Scheme 1. Reagents and conditions: (i) Br2, CH3COOH, 70 °C, 85%; (ii) chloroacetaldehyde, DMF, 100
°C, 65%; (iii) Dimethylsulfate, Cs2CO3, CH3CN, 60 °C, 49%; (iv) Pd(PPh3)4, Na2CO3, toluol
/water/MeOH, 110 °C, 69%; (v) phenol, K2CO3, DMF, 80 °C, 84%; (vi) Fe, NH4Cl, H2O/EtOH, 100 °C,
83%; (vii) RSO2Cl, NEt3, CH2Cl2, 0 °C, then 2N NaOH, 1,4-dioxane, 70 °C, 58-87%.
As shown in Scheme 2, 5-bromopyrazin-2(1H)-one (19) was got via 5-bromopyrazin-2-amine (18)
reacting with sodium nitrite in concentrated sulfuric acid. Methylation reaction was utilized to prepare
5-bromo-1-methylpyrazin-2(1H)-one (20) followed by cyclization with tosylmethyl isocyanide to give
5-bromo-7-methylimidazo[1,5-a]pyrazin-8 (7H)-one (21). The bond between imidazolopyridone and
biaryl ether was formed through Suzuki-Miyaura cross coupling reaction. Then an analogous approach
depicted in Scheme 1 provided access to the target compounds (25-37).
O
O
NH2
O
N
NH
N
iv
iii
N
i
ii
R1
R2
N
N
N
N
N
25
26
27
28
29
30
Me
Et
2,4-di-F-phenyl
3-pyridyl
m-F-phenyl
2,4-di-F-phenyl
3-pyridyl
3,4,5-tri-F-phenyl
Br
Br
Br
18
Br
Et
Et
Me
Me
Me
21
20
19
O
O
O
O
o-F- phenyl
m-F-phenyl
31
32
33
34
35
36
37
N
N
N
N
Me
N
N
N
N
N
N
N
N
2-Me-5-F- phenyl Me
vii
vi
v
2-F-5-Me- phenyl Me
p-isopropylphenyl Me
F
O
O
O
O
S
R1
R1
R1
R2
O
p-Br-phenyl
m-Br-phenyl
Et
Et
O2N
N
H
O2N
H2N
22
23a-k
24a-k
25-37
Scheme 2. Reagents and conditions: (i) NaNO2, conc. H2SO4, 0 °C to 45 °C, 91%; (ii) dimethyl sulfate,
Cs2CO3, CH3CN, 60 °C, 81%; (iii) Tosylmethyl isocyanide, NaH, THF, 0 °C to rt; (iv) Pd(PPh3)4,
Na2CO3, toluol/water/MeOH, 110 °C, 68%; (v) phenol, K2CO3, DMF, 80°C, 95%; (vi) Fe, NH4Cl,
H2O/EtOH, 100 °C, 83%; (vii) RSO2Cl, NEt3, CH2Cl2, 0 °C, then 2 N NaOH, 1,4-dioxane, 70 °C, 58-
87%.
Results and Discussion
2.1 Design strategy
Firstly, to further improve the potency of pyridone-based BET bromodomain inhibitors such as
Fragment 9 [17], imidazole was introduced to afford target compounds (15-17) with 5,6-
dihydroimidazo[1,2-c]pyrimidin scaffold. Biological activities were evaluated using an Amplified
Luminescent Proximity Homogeneous (AlphaScreen) binding assay and three complementary cellular
assays. The AlphaScreen binding assay was used to evaluate the efficacy of compounds for a construct
containing BRD4(BD1) and BRD4(BD2). Growth of cancer cell lines are dependent on BET proteins
[24], the effects of compounds on cancer cell proliferation were measured using the lymphoblast-like
cell line(Raji), promyelocytic cell line (HL-60) and human pancreatic cancer cell line(BxPC-3). However,

potency of BET bromodomain inhibitors with 5,6-dihydroimidazo[1,2-c]pyrimidin scaffold such as 17
was relatively poor (Table 1). To further improve the biochemical efficiency, compound 17 was docked
into BRD4 (BD1) and superimposed with ABBV-075 via Glide docking (PDB id: 3P5O). Docking
studies revealed that although the carbonyl moieties of these cores were situated at an ideal distance away
from the Asn433 NH2 group (2.95 Å), there was not an efficient interaction built between the Asn433
amide carbonyl and moieties of compound 17 (Figure 2). However, 7-methylimidazo[1,5-a]pyrazin-
8(7H)-one-based compounds were designed and evaluated in order to provide a bidentate contact and
improve the binding potency. Compared to compound 17, novel imidazolopyridone of 28 improved both
biochemical and cellular activity by 8-20-fold (Table 1).
Table 1. Biochemical and Cellular Potency of Compounds 15, 16, 17 and 28
O
O
O
O
N
N
N
N
N
N
N
N
N
N
O
N
O
N
O
F
F
O
O
S
O
S
O
S
O
S
O
O
O
O
N
H
F
N
H
N
H
F
N
H
Compound ID
BRD4(BD1) Alpha
Screen IC50(μM)a
BRD4(BD2) Alpha
Screen IC50(μM)
HL-60 Proliferation
IC50 (μM)b
15
16
17
28
0.57±0.022
0.48±0.031
4.3±0.025
3.8±0.021
>10
0.43±0.012
0.39±0.024
3.9±0.019
5.1±0.016
>10
0.27±0.013
0.31±0.011
2.2±0.028
1.8±0.024
5.6
0.033±0.005
0.025±0.009
0.11±0.012
NT
Raji Proliferation
IC50 (μM)b
BxPC-3 Proliferation
IC50 (μM)b
NT
a
BRD4 AlphaScreen IC50 values are reported as the mean ± SD value of three independent
determinations.
b IC50 values presented are the mean derived from three measurements.
Figure 2. Molecular modeling of compound 17 with BRD4(1) (PDB id: 3P5O). (A) Docking
conformation of compound 17 in BRD4(1) (PDB id: 3P5O). (B) Docking conformation of ABBV-075
in BRD4(1) (PDB id: 3P5O). (C) Superimposition docking conformation of compound 17 (Blue) and
ABBV-075 (Green) in BRD4(1) (PDB id: 3P5O).
In order to elaborate the specific interaction between the imidazolopyridone core and BET
bromodomain, we performed docking studies for compound 28 and ABBV-075. Docking studies
indicated that the ligand-protein interactions of two molecules were similar. The 7-methylimidazo[1,5-

a]pyrazin-8(7H)-one acted as a KAc mimic, with the methyl group occupying the small hydrophobic
pocket. The pyridone carbonyl formed a water mediated hydrogen bond with the NH2 of TRY97, and
accepted a hydrogen bond from the conserved Asn residue (ASN140) with an ideal 2.83 Å distance
measured. In addition, the phenyl ether moiety of compound 28 offers valuable interactions by occupying
a hydrophobic part of the WPF shelf and ethylsulfonyl moiety is directed into the ZA channel. The key
hydrogen bond with Asn140 and the additional water mediated interactions would be important for
improving affinity. Gratifyingly, the second imidazolopyridone core exhibited more remarkable binding
effects than the first motif, as evaluated by an improvement in IC50 from 270 to 33 nM (28 vs 17).
Figure 3. Molecular modeling of compound 28 with BRD4(1) (PDB id: 3P5O). (A) Docking
conformation of compound 28 in BRD4(1) (PDB id: 3P5O). (B) Superimposition docking conformation
of compound 28 (Yellow) and Abbv-075 (Green) in BRD4(1) (PDB id: 3P5O).
The 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core exhibited effective and potent inhibitory
activity of BET protein, we evaluated the SAR at other sites of the compounds for further improving
potency (Table 2). Compound 28 with the BRD4(BD1) IC50 of 33 nM was selected as the lead compound.
Different substituents (compound 26-37) were introduced into the phenyl group which was located at the
WPF shelf to further improve the binding affinity. Firstly, pyridines (compound 26) was incorporated
here to reduce lipophilicity and improve the developability profile, though substantial potency was
significantly reduced compared to the phenyl ethers. The mono fluoro substituted compound 27
exhibited moderate improvement. The biochemical activities of compound 36 and 37 with mono bromo
substituent were also limited. These results clearly indicated the importance of the moderate groups
located at WPF shelf for the affinity. In addition, ethyl sulfonamide was preferred over methyl
sulfonamide based upon its modestly improved potency. With these encouraging results in hand, the anti-
proliferation activities were further assessed in several tumor cell lines (Table 2).
Table 2. Biochemical and cellular activities of compound 25-37
ID
R1
R2
BRD4/BD1(
BRD4/
HL60b
IC50(μM)
0.25
7.7
Raji
Inh%(2μM)
40.8
BxPC3
Inh% (2μM)
20.3
IC50nM)a
49
BD2 (IC50nM)
25
26
27
28
29
30
31
32
33
2,4- di-F-phenyl
3-pyridyl
Me
Et
76
73
61
42.1
2.2
m-F-phenyl
Et
33
27
0.2
49.3
46.1
2,4-di-F-phenyl
3-pyridyl
Et
33
25
0.11
6.1
46.23
31.4
43.2
Me
Me
Me
Me
Me
120
90
280
520
NT
NT
110
9.4
3,4,5-tri-F-phenyl
o-F- phenyl
1.5
45.8
7.9
46
0.2
42.9
27.5
m-F-phenyl
52
0.64
0.38
45.2
27.8
2-Me-5-F- phenyl
37
49.9
27.9

34
35
2-F-5-Me- phenyl
p-isopropylphenyl
p-Br-phenyl
Me
Me
Et
74
100
72
NT
84
1.00
0.39
0.27
0.55
0.87
0.12
49.7
27.1
41.3
38.7
47.9
51.2
22.4
30.4
25.8
24.3
31.1
42.7
36
NT
NT
79
37
m-Br-phenyl
Et
52
JQ1
ABBV-
075
87
39
47
a
BRD4 AlphaScreen IC50 values are reported as the mean ± SD value of three independent
determinations.
b IC50 values presented are the mean derived from three measurements.
c The percent of residue via liver microsomes and the mean derived from three measurements.
2.2 Anti-proliferation in cancer cell lines
Furthermore, we assessed the effects of compounds on cancer cell proliferation in the lymphoblast-
like cell line (Raji), promyelocytic cell line (HL-60) and human pancreatic cancer cell line (BxPC-3). As
our previous reports, BET inhibitors show more robust anti-proliferation activities in hematologic
malignancies than solid tumor[25]. In this work, all compounds exhibited preferred effects in HL-60 cell
lines. It is intriguing that compounds 27 and 28 exhibited satisfying anti-proliferation in solid cancer cell,
especially in human pancreatic cancer cell line (BxPC-3). Among them, compound 28 showed anti-
proliferation Inh% of 43.2 in BxPC-3 cell line with 2μM (Table 2). In addition, cell cycle analysis was
performed to characterize the molecular mechanisms underlying the cytotoxic effect of compound 28 in
BxPC-3 cell. BxPC-3 cell was cultured with DMSO control or varied concentration of compound 28 (2,
4, 8 μM) respectively. As shown in Figure 4A, compound 28 treatment resulted in a remarkable increase
in G1 cells at 48 h, which was consistent with previous report[20]. These results indicated that
compounds 27 and 28 were demonstrated promising effects in the therapy of pancreatic cancer.
Figure 4. Cellular effects of compound 28 in BxPC-3 cell lines. (A) Cell cycle analysis was performed
on BxPC-3 cell cultured with DMSO or varied concentration of compound 28 (2, 4, 8 μM) for 48 h. (B)
Data was demonstrated by graph.
2.3 Induce apoptosis via the mitochondrial pathways in pancreatic cancer cells
To further clarify the mechanism of compound 28 triggering apoptosis in pancreatic cancer cells,
we examined the expression pattern of BRD4 and several key proteins in the intrinsic apoptotic pathway.
The results showed that both compound 28 and JQ1 upregulated BRD4 protein, perhaps related to
enrichment occurred in signaling regulation. Compound 28 downregulated c-Myc at 24 hours, consistent
with JQ1. Meanwhile, compound 28 downregulated BcL-xL in BxPc3 pancreatic cancer cell lines to
trigger apoptosis by modulating the intrinsic apoptotic pathway. Early apoptotic events such as caspase
activation (caspase 3 and caspase 9) and PARP cleavage were already apparent at the low concentration
of compound 28 (Figure 5). In addition, the result exhibited that the hexamethylene bisacetamide

inducible protein 1 (HEXIM1), well-characterized PD markers reported by Lin et.al, was significantly
up-regulated [26]. Taken together, these results indicated that compound 28 can effectively trigger
apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway.
Figure 5. Induce apoptosis via the mitochondrial pathways in pancreatic cancer cells. BxPc3 cells were
treated with compound 28 for 24 h, and then the expression of BRD4, c-Myc, PARP, Bcl-xl, caspase 9,
caspase 3 and HEXIM1 were examined by western blot. “+” means 5 μM, “++”means 10 μM, “+++”
means 15 μM.
Conclusion
We have designed and synthesized a series of BET inhibitors with 7-methylimidazo[1,5-a]pyrazin-
8(7H)-one scaffold. Optimization of bidentate contact afforded compounds with high BRD4 potency and
excellent physicochemical properties. Compound 28 exhibited excellent BRD4-inhibitory activity with
IC50 value of 33 nM and anti-proliferation potency with IC50 value of 110 nM in HL-60 cell lines.
Western Blot (WB) indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by
modulating the intrinsic apoptotic pathway. All of these results demonstrated that compound 28 exhibited
excellent preclinical development properties and was progressed into further investigation.
4. Experimental section
4.1 Chemistry. General Methods.
All chemical reagents were commercially purchased and used without further purification. Column
chromatography was performed on silica gel 60 (200-300 mesh). Thin-layer chromatography (TLC):
silica gel 60F254 plates (250 nm; Qingdao Ocean Chemical Company, Qingdao, China) were used to
monitor the reactions. Capillary tubes were used for the measurement of melting points. HPLC analysis
(UV detector, wavelength: 272 nm) was employed to evaluate purity of the target compounds and all
final compounds were purified to ≥95% purity. 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 indicated.
The data of the chemical shifts were showed in δ values (ppm) and the coupling constants (J) in Hz.
Mass spectrometry (MS): Hewlett-Packard 1100 LC/MSD spectrometer; elemental analyses: CHNO-
Rapid instrument.
4-Amino-5-bromopyrimidin-2(1H)-one (9). To a solution of 4-aminopyrimidin-2(1H)-one (2g,
18mmol) in acetic acid (10 mL) was added bromine (3.6 g, 22.5mmol). The reaction mixture was stirred
at 70 °C for 8 h. The reaction mixture was diluted with H2O (30 mL) and the light yellow precipitate was
filtered and washed with water. The solid was then dried under reduced pressure to give the title
compound (2.91g, 84.8% yield). 1H NMR (300 MHz, DMSO-d6) δ 10.86 (s, 1H), 7.75 (s, 1H), 6.85 (s,
1H).
8-Bromoimidazo[1,2-c]pyrimidin-5(6H)-one (10). To a solution of 9 (1 g, 5.26 mmol) in DMF (10

mL) was added 40% chloroacetaldehyde (1.24 g, 6.32 mmol) at rt. The reaction mixture was stirred at
100 °C for 10 h. The reaction mixture was diluted with H2O (20 mL) and the white precipitate was filtered
and washed with water. The solid was then dried under reduced pressure to give the title compound
(0.73g, 64.8% yield). 1H NMR (300 MHz, DMSO-d6) δ 11.92 (s, 1H), 7.89 (d, J = 1.0 Hz, 1H), 7.65 (s,
1H), 7.42 (d, J = 1.0 Hz, 1H).
8-Bromo-6-methylimidazo[1,2-c]pyrimidin-5(6H)-one (11). A solution of 10 (0.25 g, 1.17 mmol)
and K2CO3 (0.32 g, 2.34 mmol) in acetonitrile (20 mL) was stirred at ambient temperature. To this
solution was added dimethylsulfate (0.17 g, 0.14 mmol) and the reaction mixture was stirred at 70 °C for
4 h. After cooling to the room temperature, the mixture was filtered through a celite pad, and the filtrate
was concentrated under reduced pressure. The crude compound was purified by flash chromatography
(silica gel, PE/EA = 10:1~5:1) to afford the title compound (0.13 g, 48.8% yield). 1H NMR (300 MHz,
DMSO-d6) δ 7.92 (d, J = 12.7 Hz, 2H), 7.42 (s, 1H), 3.50 (s, 3H).
8-(2-Fluoro-5-nitrophenyl)-6-methylimidazo[1,2-c]pyrimidin-5(6H)-one (12).
A mixture of
compound 11 (0.3 g, 1.32 mmol), (2-fluoro-5-nitrophenyl)boronic acid (0.495 g, 1.97 mmol), Pd(PPh3)4
(0.152 g, 0.133mmol), and Na2CO3 (0.418 g, 3.95 mmol) in toluol (3 mL), water (3 mL) and EtOH (1
mL) and methanol (1.5 mL) was stirred at 110 °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 was separated and purified by flash chromatography (silica gel, PE/EA = 5:1~2:1) to afford the title
1
compound (0.26 g, 68.57% yield) as a white solid. H NMR (300 MHz, DMSO-d6) δ 8.85 (d, J = 2.9
Hz, 1H), 8.38 (d, J = 3.1 Hz, 1H), 7.96 (s, 2H), 7.69 (d, J = 9.3 Hz, 1H), 7.47 (d, J = 1.4 Hz, 1H), 3.61
(s, 3H).
8-(2-(2,6-Dimethylphenoxy)-5-nitrophenyl)-6-methylimidazo[1,2-c]pyrimidin-5(6H)-one (13a). A
mixture of 12 (0.14 g, 0.49 mmol), 2,6-dimethylphenol (0.12 g, 0.97 mmol) and K2CO3 (0.13 g, 0.97
mmol) in DMF (4 mL) was stirred at 80 °C for 12 h. Then the mixture was diluted with H2O and the
white precipitate was filtered and washed with water. The solid was then dried under reduced pressure
to give the title compound (0.16 g, 84.4% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.63 (d, J = 2.8 Hz,
1H), 8.21 (dd, J = 9.1, 2.9 Hz, 1H), 7.96 – 7.84 (m, 2H), 7.45 (d, J = 1.5 Hz, 1H), 7.25 – 7.10 (m, 3H),
6.55 (d, J = 9.1 Hz, 1H), 3.62 (s, 3H), 2.07 (s, 6H).
8-(5-Amino-2-(2,6-dimethylphenoxy)phenyl)-6-methylimidazo[1,2-c]pyrimidin-5(6H)-one (14a).
To a solution of 13a (0.1 g, 0.26 mmol) and NH4Cl (0.054 g, 1.02 mmol) in 50% EtOH was added Iron
powder (0.057 g, 1.02 mmol) at ambient temperature. Then the mixture was stirred at 100 °C for 2 h.
Then the mixture was diluted with H2O and EtOAc, and the insoluble material was filtered through Celite.
The organic layer was separated and purified by flash chromatography (silica gel, PE/EA = 8:1~2:1) to
afford the title compound (0.077 g, 83.41%). The crude product was further reacted directly for next step
without further purification.
N-(4-(2,6-Dimethylphenoxy)-3-(6-methyl-5-oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl) pheny
l)methanesulfonamide (15). To a solution of compound 14a (0.12 g, 0.332 mmol) and triethylamine (0.13
g, 1.33 mmol) in dichloromethane (6.0 mL) was added dropwise methanesulfonyl chloride (0.13 g, 0.99
mmol) at ice bath. The reaction mixture was stirred for 5 h at ambient temperature. Then the solution
was concentrated under reduced pressure. The residue was diluted with a mixture of 1,4-dioxane (6 mL)
and 2 M aqueous sodium hydroxide (3 mL) and heated for 30min at 70 °C. Then the mixture was diluted
with H2O and EtOAc, brought to pH 7 with 1 M HCl. The organic layer was separated and purified by

flash chromatography (silica gel, PE/EA = 3:1~1:1) to afford the title compound (0.12 g, 81%) as a white
solid. m.p.: 126-128 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.86 (s, 1H), 7.71 (s, 1H), 7.55
(s, 1H), 7.42 (s, 1H), 7.09 (d, J = 8.4 Hz, 4H), 6.33 (d, J = 8.8 Hz, 1H), 3.61 (s, 3H), 3.00 (s, 3H), 2.06
(s, 6H).13C NMR (75 MHz, DMSO) δ 152.43, 150.83, 146.38, 145.12, 133.68, 132.89, 131.85, 131.19,
129.48, 125.99, 125.71, 123.25, 122.27, 113.13, 107.93, 60.21, 40.86, 40.59, 40.31, 40.03, 39.75, 39.47,
39.20, 36.97, 21.20, 16.41, 14.53.MS (ESI, m/z): 439.4[M + H]+; Anal. calcd. for C22H22N4O4S: C, 60.26;
H, 5.06; N, 12.78. Found: C, 60.40; H, 5.11; N, 12.58.
N-(4-(2,6-Dimethylphenoxy)-3-(6-methyl-5-oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl)ph eny
l)ethanesulfonamide (16). Compound 16 was prepared in a similar procedure of compound 15. A white
solid was provided. Yield: 84.9%. m.p.: 130-132 °C; 1H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H), 7.82
(dd, J = 12.3, 2.1 Hz, 2H), 7.56 – 7.43 (m, 2H), 7.26 (s, 1H), 7.08 (s, 3H), 6.82 (dd, J = 8.8, 2.7 Hz, 1H),
6.22 (d, J = 8.8 Hz, 1H), 3.67 (s, 3H), 3.15 (q, J = 7.4 Hz, 2H), 2.03 (s, 6H), 1.41 (t, J = 7.4 Hz, 3H). 13C
NMR (75 MHz, DMSO) δ 185.60, 177.60, 152.24, 150.86, 146.38, 145.12, 133.67, 132.89, 131.84,
131.18, 129.49, 125.66, 122.88, 122.27, 113.15, 113.09, 107.91, 105.33, 90.13, 54.72, 45.47, 40.87,
40.59, 40.32, 40.04, 39.76, 39.48, 39.21, 36.98, 16.42, 8.49. MS (ESI, m/z): 453.3[M + H]+; Anal. calcd.
for C23H24N4O4S: C, 61.05; H, 5.35; N, 12.38. Found: C, 61.18; H, 5.41; N, 12.63.
N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-5-oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl)ph eny
l)ethanesulfonamide (17). Compound 17 was prepared in a similar procedure of compound 15.. A white
solid was provided. Yield: 83.2%; m.p.: 129-131 °C;1H NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.83
(s, 1H), 7.69 (s, 1H), 7.52 (d, J = 2.1 Hz, 1H), 7.37 (d, J = 0.9 Hz, 2H), 7.20 (dd, J = 15.3, 8.8 Hz, 2H),
7.04 (d, J = 7.9 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 3.55 (s, 3H), 3.12 (dd, J = 14.4, 7.1 Hz, 2H), 1.23 (t,
J = 7.2 Hz, 3H).13C NMR (75 MHz, DMSO-d6) δ 199.63, 188.26, 147.35, 145.11, 144.54, 134.25, 133.97,
133.14, 127.32, 124.55, 123.21, 122.76, 122.64, 120.04, 117.18, 112.94, 112.27, 109.68, 106.38, 105.97,
96.93, 78.57, 56.16, 45.61, 44.89, 40.80, 40.60, 40.49, 40.33, 40.22, 40.05, 39.93, 39.77, 39.59, 39.49,
39.34, 37.43, 36.84, 31.43, 19.45, 11.77, 8.45. MS (ESI, m/z): 461.3[M + H]+; Anal. calcd. for
C21H18F2N4O4S: C, 54.78; H, 3.94; N, 12.17. Found: C, 54.18; H, 4.11; N, 12.23.
The synthesis methods of 19-21 would not be shown here because their preparation has been
presented in detail in our previous study [25].
5-Bromopyrazin-2(1H)-one (19). Yellow solid; Yield: 55.7%; 1H NMR (300 MHz, DMSO-d6) δ
12.24 (s, 1H), 8.10 (s, 1H), 7.92 (s, 1H).
5-Bromo-1-methylpyrazin-2(1H)-one (20). Yellow solid; Yield: 83.4%; 1H NMR (300 MHz, CDCl3)
δ 7.97 (s, 1H), 7.32 (s, 1H), 3.52 (s, 3H).
1
5-Bromo-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (21). Yellow solid; Yield: 72.5%; H NMR
(300 MHz, CDCl3) δ 8.08 (d, J = 3.3 Hz, 2H), 6.62 (s, 1H), 3.48 (s, 3H).
5-(2-Fluoro-5-nitrophenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (22). The synthesis method
of 22 was similar to the preparation of 12. White solid; Yield: 68.3%; 1H NMR (300 MHz, DMSO-d6)
δ 8.57 (d, J = 5.7 Hz, 1H), 8.51 (dd, J = 8.6, 3.9 Hz, 1H), 8.15 (s, 1H), 7.91 (s, 1H), 7.75 (t, J = 9.1 Hz,
1H), 7.30 (s, 1H), 3.42 (s, 3H).
5-(2-(2,4-Difluorophenoxy)-5-nitrophenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (23a). The
synthesis method of 23a was similar to the preparation of 13a. White solid; Yield: 95.2%; The crude
product was further reacted directly for next step without further purification.
5-(5-Amino-2-(2,4-difluorophenoxy)phenyl)-7-methylimidazo[1,5-a]pyrazin-8(7H)-one (24a). The
synthesis method of 24a was similar to the preparation of 14a. White solid; Yield: 83.4%;1H NMR (300
MHz, DMSO-d6) δ 7.83 (s, 1H), 7.76 (s, 1H), 7.28 – 7.21 (m, 1H), 7.04 – 6.98 (m, 1H), 6.94 – 6.89 (m,

2H), 6.81 (d, J = 9.5 Hz, 1H), 6.76 – 6.71 (m, 2H), 5.21 (s, 2H), 3.35 (s, 3H).
The synthesis method of 25-37 was similar to the preparation of 15-17.
N-(4-(2,4-Difluorophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)me
thanesulfonamide (25). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.98 (s, 1H),
7.82 (s, 1H), 7.47 – 7.27 (m, 4H), 7.10 (s, 2H), 6.94 (d, J = 8.7 Hz, 1H), 3.40 (s, 3H), 3.05 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ 154.89, 152.40, 139.16, 138.96, 134.57, 133.04, 129.89, 124.66, 124.49,
123.64, 123.50, 123.20, 122.09, 120.77, 117.56, 113.28, 112.72, 112.42, 106.09, 40.85, 40.57, 40.29,
40.02, 39.74, 39.46, 39.19, 34.61. MS (ESI, m/z): 447.0[M+H]+; Anal. calcd. for C20H16F2N4O4S: C,
53.81; H, 3.61; N, 12.55. Found: C, 53.97; H, 3.51; N, 12.42.
N-(3-(7-Methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(pyridin-3-yloxy)phenyl) ethanes
ulfonamide (26). m.p.: 128-130 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.29 (dd, J = 4.6, 1.2
Hz, 1H), 8.24 (d, J = 2.7 Hz, 1H), 8.03 (s, 1H), 7.78 (s, 1H), 7.42 – 7.29 (m, 4H), 7.12 (d, J = 8.5 Hz,
1H), 7.04 (s, 1H), 3.35 (s, 3H), 3.18 (q, J = 7.3 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz,
DMSO-d6) δ 154.81, 153.11, 151.10, 145.23, 140.95, 135.40, 133.13, 129.91, 125.94, 124.94, 123.78,
123.16, 122.39, 122.15, 120.56, 113.17, 45.75, 40.86, 40.58, 40.31, 40.03, 39.75, 39.47, 39.19, 34.58,
8.53. MS (ESI, m/z):426.1[M+H]+; Anal. calcd. for C20H19N5O4S: C, 56.46; H, 4.50; N, 16.46. Found:
C, 56.58; H, 4.91; N, 16.53.
N-(4-(3-Fluorophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)ethan
esulfonamide (27). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.95 (s, 1H), 7.99 (s, 1H), 7.77
(s, 1H), 7.42 – 7.37 (m, 2H), 7.29 (dd, J = 15.2, 8.2 Hz, 1H), 7.17 – 7.11 (m, 1H), 7.00 (s, 1H), 6.93 –
6.85 (m, 1H), 6.84 – 6.71 (m, 2H), 3.34 (s, 3H), 3.18 (q, J = 7.3 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H). 13C
NMR (75 MHz, DMSO-d6) δ 154.81, 150.96, 135.40, 133.12, 131.67, 131.54, 129.87, 123.72, 123.17,
122.55, 122.11, 120.98, 114.58, 113.20, 110.97, 110.69, 106.34, 106.01, 45.76, 40.86, 40.58, 40.30,
40.02, 39.74, 39.47, 39.19, 34.56, 8.52. MS (ESI, m/z):443.0[M+H]+; Anal. calcd. for C21H19FN4O4S: C,
57.01; H, 4.33; N, 12.66. Found: C, 56.84; H, 4.25; N, 12.89.
N-(4-(2,4-Difluorophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)eth
anesulfonamide (28). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.94 (s, 1H),
7.80 (s, 1H), 7.45 – 7.24 (m, 4H), 7.08 (s, 2H), 6.93 (d, J = 8.8 Hz, 1H), 3.40 (s, 3H), 3.15 (q, J = 7.2 Hz,
2H), 1.23 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 154.88, 152.18, 134.61, 132.95, 129.89,
124.38, 124.17, 123.99, 123.63, 123.48, 123.21, 122.06, 120.78, 117.61, 113.26, 112.33, 106.45, 106.09,
105.79, 45.73, 40.87, 40.59, 40.31, 40.03, 39.75, 39.48, 39.20, 34.62, 8.51.MS (ESI, m/z): 461.0[M+H]+;
Anal. calcd. for C21H18F2N4O4S: C, 54.78; H, 3.94; N, 12.17. Found: C, 53.58; H, 3.91; N, 12.13.
N-(3-(7-Methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(pyridin-3-yloxy)phenyl)methan
esulfonamide (29). m.p.: 115-117 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.29 (dd, J = 4.4
Hz, 1.2 Hz,1H), 8.25 (d, J = 2.6 Hz, 1H), 8.04 (s, 1H), 7.78 (s, 1H), 7.44 – 7.29 (m, 4H), 7.13 (d, J = 8.4
Hz, 1H), 7.05 (s, 1H), 3.35 (s, 3H), 3.07 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.80, 153.17, 151.29,
145.18, 140.89, 135.45, 133.08, 129.92, 125.84, 124.87, 124.34, 124.30, 123.21, 122.47, 122.18, 120.66,
113.15, 40.96, 40.68, 40.41, 40.13, 39.92, 39.85, 39.57, 39.29, 34.54. MS (ESI, m/z): 412.0 [M+H]+;
Anal. calcd. for C19H17N5O4S: C, 55.47; H, 4.16; N, 17.02. Found: C, 55.52; H, 4.21; N, 17.38.
N-(3-(7-Methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)-4-(3,4,5-trifluorophenoxy)phenyl)
methanesulfonamide (30). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.06 (s,
1H), 7.80 (s, 1H), 7.42 – 7.36 (m, 2H), 7.18 (d, J = 9.2 Hz, 1H), 7.10 (dd, J = 9.0, 6.1 Hz, 2H), 7.04 (s,
1H), 3.37 (s, 3H), 3.07 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.84, 150.99, 135.62, 133.19, 129.94,
124.13, 123.18, 122.35, 122.19, 120.59, 112.92, 104.67, 104.36, 40.81, 40.53, 40.25, 39.97, 39.81, 39.70,

39.42, 39.14, 34.61. MS (ESI, m/z):487.0[M+Na]+; Anal. calcd. for C20H15F3N4O4S: C, 51.72; H, 3.26;
N, 12.06. Found: C, 52.02; H, 3.18; N, 12.21.
N-(4-(2-Fluorophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)metha
nesulfonamide (31). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.06 (s, 1H),
7.80 (s, 1H), 7.42 – 7.36 (m, 2H), 7.18 (d, J = 9.2 Hz, 1H), 7.10 (dd, J = 9.0, 6.1 Hz, 2H), 7.04 (s, 1H),
3.37 (s, 3H), 3.07 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.87, 152.16, 142.68, 142.52, 134.61,
133.01, 129.88, 125.90, 124.56, 124.46, 123.17, 122.27, 122.08, 121.02, 118.08, 117.67, 117.43, 113.31,
40.81, 40.53, 40.26, 39.98, 39.78, 39.70, 39.42, 39.14, 34.62. MS (ESI, m/z):487.0[M+Na]+; Anal. calcd.
for C20H17F1N4O4S: C, 56.07; H, 4.00; N, 13.08. Found: C, 56.05; H, 4.11; N, 13.12.
N-(4-(3-Fluorophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)metha
1
nesulfonamide (32). m.p. > 220-222 °C; H NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.00 (s, 1H),
7.78 (s, 1H), 7.46 – 7.36 (m, 2H), 7.30 (dd, J = 15.4, 8.0 Hz, 1H), 7.16 (d, 1H), 7.01 (s, 1H), 6.95 – 6.85
(m, 1H), 6.85 – 6.72 (m, 2H), 3.35 (s, 3H), 3.07 (s, 3H).13C NMR (75 MHz, DMSO-d6) δ 154.81, 151.14,
135.45, 133.08, 131.63, 131.50, 129.89, 124.25, 123.22, 122.64, 122.13, 121.10, 114.54, 113.18, 110.91,
110.63, 106.26, 105.93, 40.96, 40.69, 40.41, 40.13, 39.93, 39.85, 39.57, 39.29, 34.52. MS (ESI,
m/z):429.0[M+H]+; Anal. calcd. for C20H17F1N4O4S: C, 56.07; H, 4.00; N, 13.08. Found: C, 55.98; H,
4.13; N, 12.95.
N-(4-(5-Fluoro-2-methylphenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl) phe
nyl)methanesulfonamide (33). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.01
(s, 1H), 7.79 (s, 1H), 7.44 – 7.36 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.11 – 7.03 (m, 2H), 6.86 – 6.76 (m,
1H), 6.73 – 6.66 (m, 1H), 3.35 (s, 3H), 3.07 (s, 3H), 1.91 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ
154.84, 151.18, 135.13, 133.25, 132.56, 132.43, 129.88, 124.37, 124.30, 123.14, 122.23, 121.91, 120.34,
113.25, 110.70, 110.42, 105.41, 105.08, 40.86, 40.58, 40.30, 40.03, 39.83, 39.75, 39.47, 39.19, 34.57,
15.21. MS (ESI, m/z):457.1[M+H]+; Anal. calcd. for C21H19F1N4O4S: C, 51.07; H, 4.33; N, 12.66. Found:
C, 51.08; H, 4.23; N, 12.34.
N-(4-(2-Fluoro-5-methylphenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl) phe
nyl)methanesulfonamide (34). m.p. > 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.95
(s, 1H), 7.79 (s, 1H), 7.42 – 7.33 (m, 2H), 7.20 – 7.10 (m, 1H), 7.07 (s, 1H), 7.02 – 6.90 (m, 3H), 3.38
(s, 3H), 3.06 (s, 3H), 2.19 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.85, 152.27, 142.12, 135.38,
134.59, 132.87, 129.85, 126.38, 126.29, 124.69, 124.53, 123.23, 122.30, 122.03, 120.98, 118.31, 117.06,
116.83, 113.23, 40.97, 40.68, 40.41, 40.13, 39.85, 39.57, 39.29, 34.53, 20.57. MS (ESI, m/z): 443.0
[M+H]+; Anal. calcd. for C21H19F1N4O4S: C, 51.07; H, 4.33; N, 12.66. Found: C, 51.12; H, 4.31; N, 12.54.
N-(4-(4-Isopropylphenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl) met
hanesulfonamide (35). m.p.: 108-110 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 7.99 (s, 1H),
7.78 (s, 1H), 7.36 (d, J = 9.4 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 7.01 (d, J = 5.5 Hz, 2H), 6.87 (d, J = 7.7
Hz, 2H), 3.36 (s, 3H), 3.05 (s, 3H), 2.88 – 2.76 (m, 1H), 1.14 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6) δ 154.83, 154.16, 152.56, 144.43, 134.45, 133.16, 129.76, 128.03, 124.60, 123.12, 121.99,
121.70, 119.63, 119.00, 113.74, 40.76, 40.48, 40.20, 39.93, 39.65, 39.37, 39.09, 34.57, 33.17, 24.39. MS
(ESI, m/z):453.1[M+H]+; Anal. calcd. for C23H24N4O4S: C, 61.05; H, 5.35; N, 12.38. Found: C, 61.07;
H, 5.41; N, 12.61.
N-(4-(4-Bromophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)ethan
esulfonamide (36). m.p. > 220-222 °C;1H NMR (300 MHz, DMSO-d6) δ 9.95 (s, 1H), 7.98 (s, 1H), 7.79
(s, 1H), 7.42 (dd, J = 20.5, 8.1 Hz, 4H), 7.11 (d, J = 9.2 Hz, 1H), 7.00 (s, 1H), 6.89 (d, J = 8.9 Hz, 2H),
3.36 (s, 3H), 3.17 (d, J = 7.3 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H); MS (ESI, m/z): 500.90 [M-H]-. Anal.

calcd. for C21H19BrN4O4S: C, 50.11; H, 3.80; N, 11.13. Found: C, 50.05; H, 3.41; N, 11.34.
N-(4-(3-Bromophenoxy)-3-(7-methyl-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-5-yl)phenyl)ethan
esulfonamide (37). m.p.: 108-110 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.00 (s, 1H), 7.77
(s, 1H), 7.39 (s, 2H), 7.20 (d, J = 29.3 Hz, 4H), 6.97 (d, J = 23.3 Hz, 2H), 3.35 (s, 3H), 3.18 (s, 2H), 1.23
(s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 156.77, 154.30, 150.51, 134.77, 132.67, 131.53, 129.36,
126.56, 123.19, 122.62, 122.03, 121.81, 121.66, 121.08, 120.19, 117.32, 112.72, 45.23, 34.09, 8.03; MS
(ESI, m/z): 500.90 [M-H]-. Anal. calcd. for C21H19BrN4O4S: C, 50.11; H, 3.80; N, 11.13. Found: C, 50.15;
H, 3.79; N, 11.22.
4.2 Docking studies
These docking small molecules were processed with Ligand preparation protocol in Maestro 10.2.
Then all ligands were minimized with OPLS 2005 force field [27]. Glide generation protocol of Maestro
10.2 was performed to process the BRD4 (BD1) protein (PDB id: 3P5O), which was downloaded from
PDB (http://www.rcsb.org/pdb). Seven molecules of water around the Kac-pocket were reserved (id:
HOH18, HOH27, HOH33, HOH256, HOH267, HOH268, HOH358), and the bond orders were assigned
[25]. After that, a glide box using the centroid of the bound ligand and a maximum size of 12 Å was
generated following hydrogen atoms modified. The optimal docking conformations were saved.
4.3. Biological evaluation
4.3.1. AlphaScreen bromodomain binding assay.
The AlphaScreen bromodomain binding assay was performed by Shanghai ChemPartner Co. Firstly
1x assay buffer (modified HEPES Buffer) was prepared for this experiment. Small molecules were
transferred to assay plates with Echo. DMSO’s final concentration is 0.1% following protein solution
prepared in 1x assay buffer (5 nM). Then, to generate the substrate solution, peptide (H4) was added in
1x assay buffer. 5 μL of protein solution or 5 μL of 1x assay buffer was transferred to assay plates. After
that, these assay plates were incubated at room temperature for 15 minutes. To each well 5 μL of substrate
solution was added for reaction. Next, these assay plates were incubated at room temperature for one
hour. In subdued light environment, 15 μL acceptor and donor solution was added and incubated for
another one hour at room temperature. The Alpha mode of EnSpire was performed to show the endpoint.
Finally, the data was imported into Excel to obtain inhibition values using equation (1). Equation (1):
Inh % = (Max - Signal) / (Max - Min) × 100. Meanwhile, max signal was provided from the action of
Enzyme and Substrate and min signal was acquired from the Substrate. GraphPad Prism 5 was used to
get 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 proliferation assay
The anti-proliferation activities of these compounds in cancer cell proliferation lines, lymphoblast-
like cell line(Raji), promyelocytic cell line (HL-60) and human pancreatic cancer cell line(BxPC-3) were
evaluated using MTT colorimetric assay and the procedure and conditions were described in our previous
work[25, 27]. Raji and HL-60 cell line from Key laboratory of SATCM for Empirical Formulae
Evaluation and Achievements Transformation were grown in RPMI 1640 supplemented with 16% fetal
bovine serum (FBS). BxPC-3 cell line from Nanjing Tech University were grown in Dulbecco’s Modifie
d Eagle Medium (DMEM) supplemented with10% FBS. HL-60 DNA content analysis was performed
by flow cytometry (BD, LSR Fortessa TM, San Diego, CA, USA). The percentage of cellsin different
cell cycle phases was determined with modfit LT 2.0 software (BD Biosciences, Pharmingen, San Diego,
CA, USA). Cell cycle analysis was performed following standard protocols.
4.3.3. Western Blot Analysis

Cell lysates were prepared in cell lysis buffer (RIPA Lysis Buffer，Beyotime，P0013B) with
protease inhibitor cocktail (Roche,5892970001). 30μg of total protein was resolved on a 12% SDS
polyacrylamide gel and probed with anti-cMyc (Abcam Ab32072), anti-BRD4 (Cell Signaling #13440),
anti-HEXIM1(Abcam,Ab-25388), anti-PARP (Cell Signaling #9542), anti-Bcl-xl (Abcam Ab-32370),
anti-caspase3 (Cell Signaling # 9665), anti-caspase9 (Cell Signaling # 9508), GAPDH loading control
Cell Signaling#5174. Western blotting was performed following standard protocols.
Acknowledgments
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).
AUTHOR INFORMATION
E-mail: cl.xu81@gmail.com (C. Xu).
E-mail: zhanghb80@163.com (H. Zhang).
E-mail: jpzhou668@163.com (J. Zhou).
ABBREVIATIONS
BET, Bromodomain and Extraterminal Domain; DMPK, drug metabolism and pharmacokinetics; P-
TEFb, positive transcription elongation factor; CDK9, cyclin dependent kinase; PDA, Pancreatic ductal
adenocarcinoma; AlphaScreen, Amplified Luminescent Proximity Homogeneous; HEXIM1,
hexamethylene bisacetamide inducible protein 1; IHC, immunohistochemical; WB, Western Blot
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Novel 7-methylimidazo[1,5-a]pyrazin-8(7H)-one BRD4 inhibitors were designed and synthesized.
·Molecular docking was performed.
·Cell proliferation inhibition was evaluated in the Raji, HL-60 and BxPC-3 cell lines.
·Induce apoptosis via the mitochondrial pathways in BxPC-3 cells.