Design, synthesis, and biological evaluation of novel 4,4-difluoro-1-methyl-N, 6-diphenyl-5, 6-dihydro-4H-pyrimido [4, 5-b] [1, 2, 4] triazolo [4, 3-d] [1, 4] diazepin-8-amine derivatives as potential BRD4 inhibitors

Article abstract of DOI:10.1111/cbdd.13833

Bromodomain-containing protein 4 (BRD4) plays an extremely important physiological role in cancer, and the BRD4 inhibitors can effectively inhibit the proliferation of tumor cells. By taking BI-2536 (PLK1 and BRD4 inhibitor) as the lead compound, sixteen

Full text of DOI:10.1111/cbdd.13833

Received: 13 January 2021
DOI: 10.1111/cbdd.13833
Revised: 10 February 2021
Accepted: 21 February 2021
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R E S E A R C H A R T I C L E
Design, synthesis, and biological evaluation of novel 4,4-difluoro-
1-methyl-N, 6-diphenyl-5, 6-dihydro-4H-pyrimido [4, 5-b] [1, 2, 4]
triazolo [4, 3-d] [1, 4] diazepin-8-amine derivatives as potential
BRD4 inhibitors
Jiuhui Li1
Wenjie Zhang1,2
Qianqian Qiu1
Daoguang Zhou1
Ziying Feng1
Hai Qian1,2
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Zhenzhen Tong1
Wei Shi1
Jiaxin Wei1
Wenlong Huang1,2
Jieming Li3
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1Center of Drug Discovery, State Key
Laboratory of Natural Medicines, China
Pharmaceutical University, Nanjing, China
Abstract
Bromodomain-containing protein 4 (BRD4) plays an extremely important physi-
ological role in cancer, and the BRD4 inhibitors can effectively inhibit the prolif-
eration of tumor cells. By taking BI-2536 (PLK1 and BRD4 inhibitor) as the lead
compound, sixteen novel BRD4 inhibitors with the 4,4-difluoro-1-methyl-N,6-diphe
nyl-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4] diazepine-8-amine
structure were designed and synthetized. Among the target compounds, compound
15h exhibited outstanding inhibition for BRD4-BD1 (IC50 value of 0.42 μM) in the
BRD4-BD1 inhibitory activity assay. Additionally, cell growth inhibition assay dem-
onstrated that compound 15h potently suppressed the proliferation of MV4-11 cells
(IC50 value of 0.51 μM). Besides, compound 15h induced apoptosis and G0/G1 cycle
arrest in MV4-11 leukemia cells effectively, and downregulated the expression of
c-Myc in a dose-dependent manner. In summary, the optimal compound 15h is ex-
pected to become the clinical therapeutic drug for further research.
2Jiangsu Key Laboratory of Drug
Discovery for Metabolic Disease, China
Pharmaceutical University, Nanjing, China
3Henan University of Chinese Medicine,
Zhengzhou, China
Correspondence
Jieming Li, Henan University of Chinese
Medicine, Zhengzhou, China.
Email: jiemingli@hactcm.edu.cn
Hai Qian and Wei Shi, Center of Drug
Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical
University, Nanjing, China.
Emails: qianhai24@163.com;
1520200004@cpu.edu.cn
K E Y W O R D S
Funding information
National Science Foundation of China;
China Postdoctoral Science Foundation
anti-proliferation, anti-tumor, BRD4 inhibitors, cell apoptosis, c-Myc
1
INTRODUCTION
the downstream gene c-Myc, which could increase the occur-
rence of cancers (Yoshida, 2018). Thus, disturbing the inter-
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Bromodomain-containing protein 4 (BRD4), a member of the
best studied bromodomain and extra-terminal domain (BET)
protein family and containing two tandem bromodomains
(BD1 and BD2), regulates gene expression by recruiting tran-
scription factors at specific gene transcription sites and bind-
ing to acetyl lysine (KAc) histones (Fu et al., 2015; Kharenko
et al., 2018). The abnormal expression of BRD4 will activate
action between BRD4 and KAc protein can effectively inhibit
the proliferation of tumor cells and the production of inflam-
matory factors (Chen et al., 2019; Ran et al., 2015; Wang
et al., 2019; Zhou et al., 2019). Due to the extremely import-
ant physiological role, BRD4 has become an important target
in anti-cancer field (Duan et al., 2018; White et al., 2019).
In recent years, several small molecules targeting BRD4
protein have been developed for cancer therapy, such as
(+)-JQ-1 (1, Figure 1), I-BET-762 (2, Figure 1), OTX015
Jiuhui Li and Wenjie Zhang made equal contribution to this work.
Chem Biol Drug Des. 2021;97:1117–1128.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons Ltd.
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(3, Figure 1), and BI-2536 (4, Figure 1; Andrikopoulou
et al., 2020; Ciceri et al., 2014; Huang et al., 2016; Stathis
et al., 2016; Zhao et al., 2013). Wherein, (+)-JQ-1 was the
first reported BRD4 inhibitor, and has been employed widely
to evaluate its therapeutic potential in a great many cancers
(Filippakopoulos et al., 2010). I-BET-762, obtained from op-
timization of a hit which was designed to enhance ApoA1
expression and had entered clinical trials for NUT (nu-
clear protein in testis) midline carcinoma and other cancers
(Delmore et al., 2011; Leal et al., 2017). OTX015 showed
clinically effective activity with nontoxic doses in Phase I
trials for NUT midline carcinoma and hematologic malig-
nancies (Jung et al., 2015; Zhang et al., 2016). BI-2536 was
a dual potent inhibitor of BRD4 and PLK1, which might
provide a new strategy to treat acute myeloid leukemia (Liu
et al., 2017; Steegmaier et al., 2007). Significantly, by explor-
ing the docking assay of (+)-JQ-1 (Figure 2a), I-BET-762
(Figure 2b), and BI-2536 (Figure 2c) with BRD4-BD1, it
was indicated that methyl triazole ring (red, Figure 1) and
methylpiperazine-one ring (blue, Figure 1) could form hydro-
gen bonds with the residues Tyr97 and Asn140 which were
the essential binding sites for the BRD4 inhibitors (Figure 2).
FIGURE 1 Structures of representative
BRD4 inhibitors [Colour figure can be
viewed at wileyonlinelibrary.com]
(a)
(b)
(c)
FIGURE 2 (+)-JQ-1, I-BET-762, and BI-2536 can form hydrogen bonds with the residue Asn140 of KAc recognition region in BRD4: (a)
Eutectic structure of (+)-JQ-1 and BRD4 complex (PDB ID: 3MXF); (b) Eutectic structure of I-BET-762 and BRD4 complex (PDB ID: 3P5O); (c)
Eutectic structure of BI-2536 and BRD4 complex (PDB ID: 4O74) [Colour figure can be viewed at wileyonlinelibrary.com]

LI et aL.
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In this study, to develop the target compounds with high
by HPLC (>95%). Liquid chromatography-mass spectrom-
eter spectra (LC-MS) was carried out on Waters ACQUITY
specificity for BRD4 inhibition, we focused on BI-2536 by
performing extensive structure-activity relationships (SARs)
study. According to the protein-ligand interactions, it was
found that (R)-4-cyclopentyl-3-ethyl-1-methylpiperazine-
2-one group (group I) could form essential hydrogen bonds
with the residues Tyr97 and Asn140 in BRD4 (PDB ID:
4O74; Chen et al., 2015). Furthermore, the more potent com-
pounds could be obtained when group I of BI-2536 (IC50
value of 250 nM) was substituted with 1-cyclopentyl-6,6-difl
uoro-4-methyl-1,4-diazepan-5-one group (group II) of TAK-
960 (IC50 value of 50 nM). In addition, it has been reported
that 3-methyl-7-phenyl-9H-[1,2,4] triazolo[4,3-a] [1,4] diaz-
epine group (group III) of OTX015 (IC50 value of 16 nM)
could form essential hydrogen bonds with the residues Tyr 97
and Asn140 in BRD4, which was important to the inhibition
for BRD4. Therefore, methyl triazole ring could be further
combined with group II of TAK-960 to improve the speci-
ficity for BRD4 inhibition. Above all, we replaced group I
with 9,9-difluoro-3-methyl-7-phenyl-8,9-dihydro-7H-[1,2,4]
triazolo[4,3-d] [1,4] diazepine group and simplified substi-
tuted phenyl to improve the inhibition for BRD4 (Figure 3).
Thus, the novel 4,4-difluoro-1-methyl-N, 6-diphenyl-5,
6-dihydro-4H-pyrimido [4, 5-b] [1, 2, 4] triazolo [4, 3-d] [1,
4] diazepin-8-amine derivatives were designed based on the
SARs of BI-2536.
UPLC-TQD at 25°C in ESI mode. 1H NMR spectrum and 13
C
NMR spectrum were noted on Bruker AV300 (300 MHz).
The unit of coupling constant (J value) is Hz. Chemical shifts
were expressed in ppm (d) relative to tetramethyl silane
(TMS) as internal standard.
2.1.1
General procedure for the synthesis of
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compounds 15a ~ 16h
To a solution of benzotriazole (11.90 g, 99.89 mmol) and
aniline 1 (8.61 ml, 99.89 mmol) in anhydrous ether (100 ml),
37% aqueous formaldehyde was added dropwise (7.95 ml,
99.89 mmol). Then the reaction mixture was stirred at room
temperature for 6 hr. After the reaction was completed, the
mixture was filtered. Then the filtration cake was washed
with cold ether and dried to give 3 as loose white solid
(12.5 g, yield: 56%).
To
a solution of trimethylchlorosilane (4.64 ml,
53.52 mmol) and 300 mesh zinc powder (4.72 g, 71.36 mmol)
under nitrogen in anhydrous THF (80 ml), ethyl difluoro bro-
moacetate (6.96 ml, 53.52 mmol) and compound 3 (8.0 g,
35.68 mmol) were added, respectively, below 0°C. Then the
reaction mixture was stirred below 0°C for 3 hr. When the re-
action was completed, saturated sodium bicarbonate solution
(60 ml) was slowly added to the mixture. Then the mixture
was stirred for 10 min at room temperature and filtered with
diatomite. The filtrate was extracted with ether (60 ml × 3).
The combined organic layers were washed with saturated
saline, dried over anhydrous sodium sulfate, concentrated
under reduced pressure, and purified by silica gel column
chromatography to give 5 as colorless oil (6.7 g, yield: 82%).
To a solution of 2,4-dichloro-5-nitropyrimidine (7.82 g,
40.31 mmol) and anhydrous sodium bicarbonate (12.31 g,
0.15 mol) under nitrogen in anhydrous ethyl acetate
(100 ml), compound 5 (8.4 g, 36.64 mmol) was added
2
EXPERIMENTAL SECTION
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2.1
General chemistry experimental details
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All materials, solvents, and reagents were obtained from
commercial sources. Purifications were implemented by col-
umn chromatography in 200–300 mesh silica gel and moni-
tored by thin-layer chromatography which was performed
on GF/UV 254 plates and visualized by using UV light at
254/365 nm. The purity of the compounds was evaluated
FIGURE 3 The design of the target
compounds [Colour figure can be viewed at
wileyonlinelibrary.com]

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below 0°C. Then the mixture was stirred at room tem-
perature for 16 hr. When the reaction was completed, the
mixture was filtered with diatomite. Then the filtrate was
washed with saturated saline (60 ml), dried over anhydrous
sodium sulfate, concentrated under reduced pressure, and
purified by silica gel column chromatography to give 7 as
yellow solid (7.8 g, yield: 55%).
To a solution of compound 7 (6.5 g, 16.81 mmol) under
nitrogen in acetic acid (50 ml), reduction iron powder (1.41 g,
25.21 mmol) was added. The mixture was stirred at 70°C for
1 hr and then at 100°C for 5 hr. When the reaction was com-
pleted, the mixture was filtered with diatomite. The filtrate
was washed with saturated saline (40 ml), dried over anhy-
drous sodium sulfate, concentrated under reduced pressure,
and purified by silica gel column chromatography to give 9
as yellow solid (3.2 g, yield: 61%).
To a solution of compound 9 (3.8 g, 12.23 mmol) in an-
hydrous tetrahydrofuran (50 ml), Lawesson reagent (2.97 g,
7.34 mmol) was added. The mixture was stirred at 70°C for
6 hr. When the reaction was completed, the mixture was di-
luted with water (20 ml) and extracted with dichloromethane
(40 ml × 3). The combined organic layers were washed with
saturated saline (40 ml), dried over anhydrous sodium sulfate,
concentrated under reduced pressure, and purified by silica
gel column chromatography to give 11 as yellow solid (2.6 g,
yield: 65%).
To a solution of compound 11 (3.4 g, 10.41 mmol) in
methanol and tetrahydrofuran (50 ml, V: V = 1:1), 80% hy-
drazine hydrate (6.51 ml, 0.10 mol) was added. The mixture
was stirred at room temperature for 3 hr, and washed with
saturated saline (40 ml × 2), dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to obtain
intermediate when the reaction was completed. Then, tri-
ethyl orthoacetate (7.67 ml, 41.62 mmol) was added to a
solution of intermediate in toluene (12 ml). Then the mix-
ture was stirred at 110°C for 6 hr. When the reaction was
completed, the mixture was diluted with water (6 ml) and
extracted with ethyl acetate (20 ml × 3). The combined or-
ganic layers were washed with saturated saline, dried over
anhydrous sodium sulfate, and purified by silica gel col-
umn chromatography to give 13 as white solid (2.6 g, yield:
72%).
chromatography to afford target product 15a as white solid
(0.15 g, yield: 65%).
General procedure for the target compounds 15b~16h was
similar to that for compound 15a.
4,4-difluoro-1-methyl-N,6-diphenyl-5,6-dihydro-4H-
pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4] diazepin-8-
amine (15a)
Whitesolid, yield65%. m.p.:156–158°C;1HNMR(300MHz,
DMSO-d6) δ 9.71 (s, 1H), 8.63 (s, 1H), 7.48 (d, J = 7.0 Hz,
2H), 7.37 (d, J = 7.7 Hz, 3H), 7.19 (m, 2H), 6.95 (m, 2H),
6.82 (d, J = 6.7 Hz, 1H), 4.59 (t, J = 12.5 Hz, 2H), 2.63
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 157.03, 153.67,
152.60, 145.32, 139.76, 129.45, 128.04, 125.88, 121.31,
118.24, 113.54, 58.01, 57.61, 57.20, 28.97, 11.90; LC-MS
(ESI) m/z: 406.70 [M + H]+; Anal. calcd. for C21H18F2N7: C,
62.22; H, 4.23; N, 24.18. Found: C, 62.24; H, 4.25; N, 24.22.
4,4-difluoro-N-(4-methoxyphenyl)-1-methyl-6-phenyl-
5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d]
[1,4] diazepin-8-amine (15b)
White solid, yield 68%. m.p.: 177–179°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.59 (s, 1H), 7.32 (m,
7H), 6.54 (s, 2H), 4.58 (t, J = 12.4 Hz, 2H), 3.65 (s, 3H), 2.62
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 157.64, 153.63,
152.69, 145.31, 138.84, 129.62, 127.76, 126.18, 124.76,
119.59, 57.63, 31.10, 11.81; LC-MS (ESI) m/z: 436.07
[M + H]+; Anal. calcd. for C22H20F2N7O: C, 60.68; H, 4.40;
N, 22.52. Found: C, 60.62; H, 4.42; N, 22.54.
N-(4-chlorophenyl)-4,4-difluoro-1-methyl-6-phenyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4]-triazolo[4,3-d] [1,4]
diazepin-8-amine (15c)
White solid, yield 65%. m.p.: 175–177°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.57 (s, 1H), 7.35 (m,
7H), 6.54 (s, 2H), 4.50 (t, J = 12.4 Hz, 2H), 3.64 (s, 3H), 2.66
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 156.69, 153.69,
152.68, 145.31, 138.81, 129.62, 127.77, 126.18, 124.74,
119.59, 57.67, 31.10, 11.88. LC-MS (ESI) m/z: 440.30
[M + H]+; Anal. calcd. for C21H17ClF2N7: C, 57.34; H, 3.67;
N, 22.29. Found: C, 57.40; H, 3.62; N, 22.24.
To a solution of compound 13 (0.2 g, 0.57 mmol), an-
iline (0.059 g, 0.63 mmol) and cesium carbonate (0.37 g,
1.15 mmol) under nitrogen in 1,4-dioxane (40 ml), Tris
(dibenzylacetone) diphenyl (52.52 mg, 0.057 mmol) and 4,5-
bis (diphenylphosphine)—9,9-dimethoxyxanthene (0.37 g,
0.63 mmol) were added, respectively. Then the mixture was
stirred at 110°C for 14 hr. When the reaction was completed,
the mixture was diluted with water (20 ml) and extracted
with ethyl acetate (50 ml × 3). The combined organic lay-
ers were washed with saturated saline (100 ml), dried over
anhydrous sodium sulfate, and purified by silica gel column
4,4-difluoro-N-(4-fluorophenyl)-1-methyl-6-phenyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4]
diazepin-8-amine (15d)
Brown solid, yield 62%. m.p.: 164–166°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.92 (s, 1H), 7.42
(m, 7H), 6.38 (s, 2H), 4.55 (t, J = 12.4 Hz, 2H), 3.60 (s,
3H), 2.64 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 169.98,
167.32, 166.34, 161.02, 141.09, 138.68, 136.22, 131.15,
130.30, 130.24, 129.05, 124.41, 117.73, 116.48, 108.74,
95.93, 55.77, 45.87, 42.05, 34.43, 27.19, 25.86, 25.15,
23.87. LC-MS (ESI) m/z: 424.70 [M + H]+; Anal. calcd.

LI et aL.
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for C21H17F3N7: C, 59.57; H, 3.81; N, 23.16. Found: C,
59.59; H, 3.82; N, 23.14.
C21H17ClF2N8O2: C, 56.01; H, 3.58; N, 24.88. Found: C,
56.06; H, 3.56; N, 24.87.
N-(3,4-dimethoxyphenyl)-4,4-difluoro-1-methyl-6-phenyl-
5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d]
[1,4] diazepin-8-amine (15e)
6-(4-chlorophenyl)-4,4-difluoro-1-methyl-N-phenyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4]
diazepin-8-amine (16a)
White solid, yield 65%. m.p.: 168–170°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.67 (s, 1H), 7.35
(m, 6H), 6.52 (s, 2H), 4.52 (t, J = 12.4 Hz, 2H), 3.67
(s, 3H), 3.62 (s, 3H), 2.64 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) δ 157.43, 153.79, 152.52, 148.41, 145.04,
143.86, 133.36, 129.22, 124.95, 124.19, 111.91, 104.31,
78.43, 55.78, 55.21, 32.87, 31.24, 31.10, 31.04, 28.95,
11.81. LC-MS (ESI) m/z: 466.19 [M + H]+; Anal. calcd.
for C23H22F2N7O2: C, 59.35; H, 4.55; N, 21.06. Found: C,
59.37; H, 4.52; N, 21.04.
White solid, yield 61%. m.p.: 163–165°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.65 (s, 1H), 7.54 (d,
J = 7.0 Hz, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.16 (m, 2H),
6.93 (m, 2H), 6.83 (d, J = 6.7 Hz, 1H), 4.58 (t, J = 12.5 Hz,
2H), 2.64 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 168.59,
168.52, 167.74, 163.53, 159.01, 149.67, 148.71, 149.76,
145.76, 145.63, 145.01, 144.36, 144.31, 143.95, 140.25,
129.47, 129.19, 128.4, 128.28 128.12, 128.07, 124.73,
122.55, 122.51, 121.13, 119.81, 117.47, 54.87, 54.35, 53.90,
11.94. LC-MS (ESI) m/z: 440.02 [M + H]+; Anal. calcd. for
C21H17ClF2N7: C, 57.34; H, 3.67; N, 22.28. Found: C, 57.36;
H, 3.65; N, 22.25.
N-(4-(tert-butyl) phenyl)-4,4-difluoro-1-methyl-6-phenyl-
5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d]
[1,4] diazepin-8-amine (15f)
6-(4-chlorophenyl)-4,4-difluoro-N-(4-methoxyphenyl)-
1-methyl-5,6-dihydro-4H-pyrimido-[4,5-b] [1,2,4]
triazolo[4,3-d] [1,4] diazepin-8-amine (16b)
1
Grayish white solid, yield 65%. m.p.: 145–147°C; H NMR
(300 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.61 (s, 1H), 7.23
(dd, J = 112.6, 37.2 Hz, 8H), 4.71–4.31 (m, 2H), 2.56 (d,
J = 36.3 Hz, 3H), 1.20 (s, 9H). 13C NMR (75 MHz, DMSO-d6)
δ 157.19, 153.67, 152.55, 145.33, 137.08, 129.41, 125.57,
124.57, 118.34, 31.16, 11.89. LC-MS (ESI) m/z: 462.19
[M + H]+; Anal. calcd. for C25H26F2N7: C, 65.06; H, 5.46; N,
21.24. Found: C, 65.07; H, 5.42; N, 21.27.
Whitesolid, yield68%. m.p.:169–171°C;1HNMR(300MHz,
DMSO-d6) δ 9.59 (s, 1H), 8.59 (s, 1H), 7.47 (d, J = 46.9 Hz,
4H), 7.09 (s, 2H), 6.58 (s, 2H), 4.57 (t, J = 12.4 Hz, 2H),
3.67 (s, 3H), 2.61 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
δ 154.59, 154.29, 153.12, 147.90, 144.74, 133.33, 129.84,
113.62, 55.45, 12.34. LC-MS (ESI) m/z: 470.30 [M + H]+;
Anal. calcd. for C22H19ClF2N7O: C, 56.24; H, 3.86; N, 20.87.
Found: C, 56.26; H, 3.82; N, 20.85.
N-(2-chlorophenyl)-4,4-difluoro-1-methyl-6-phenyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4]
diazepin-8-amine (15g)
N,6-bis(4-chlorophenyl)-4,4-difluoro-1-methyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4]-triazolo[4,3-d] [1,4]
diazepin-8-amine (16c)
White solid, yield 65%. m.p.: 173–175°C; 1H NMR
(300 MHz, DMSO-d6) δ 8.63 (d, J = 4.2 Hz, 2H), 7.55–7.21
(m, 7H), 6.94 (m, 2H), 4.59 (t, J = 12.4 Hz, 2H), 2.63 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ 170.14, 167.11, 161.04,
140.97, 138.78, 138.70, 136.24, 131.09, 130.91, 130.28,
129.08, 128.64, 124.49, 123.22, 118.91, 117.16, 116.55,
108.80, 95.83, 55.75, 37.75, 27.22. LC-MS (ESI) m/z: 440.30
[M + H]+; Anal. calcd. for C21H17ClF2N7: C, 57.34; H, 3.67;
N, 22.28. Found: C, 57.36; H, 3.62; N, 22.27.
White solid, yield 75%. m.p.:174–176°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.64 (s, 1H), 7.30
(dd, J = 111.4, 52.8 Hz, 8H), 4.60 (t, J = 11.7 Hz, 2H), 2.56
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 157.21, 154.32,
153.22, 147.92, 144.66, 139.24, 129.98, 128.27, 125.47,
120.32, 58.00, 12.35. LC-MS (ESI) m/z: 474.20 [M + H]+;
Anal. calcd. for C21H16Cl2F2N7: C, 53.18; H, 3.19; N, 20.67.
Found: C, 53.16; H, 3.17; N, 20.70.
4,4-difluoro-1-methyl-N-(4-nitrophenyl)-6-phenyl-5,6-
dihydro-4H-pyrimido[4,5-b] [1,2,4]-triazolo[4,3-d] [1,4]
diazepin-8-amine (15h)
6-(4-chlorophenyl)-4,4-difluoro-N-(4-fluorophenyl)-
1-methyl-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4]
Yellow solid, yield 70%. m.p.: 166–168°C; 1H NMR
(300 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.69 (s, 1H), 8.00–
6.93 (m, 8H), 4.61 (t, J = 12.4 Hz, 2H), 2.63 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ 170.16, 167.10, 167.03, 161.04,
156.31, 140.97, 138.70, 136.27, 135.16, 131.09, 131.02,
130.26, 130.20, 129.06, 124.46, 120.73, 120.62, 117.07,
116.51, 115.34, 115.27, 115.04, 108.82, 95.84, 55.74, 37.65,
27.19. LC-MS (ESI) m/z: 451.30 [M + H]+; Anal. calcd. for
triazolo[4,3-d] [1,4] diazepin-8-amine (16d)
Brown solid, yield 66%. m.p.: 154–156°C; 1H NMR
(300 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.62 (s, 1H), 7.54 (d,
J = 8.2 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.22 (s, 2H), 6.80
(s, 2H), 4.58 (t, J = 12.6 Hz, 2H), 2.61 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) δ 157.26, 154.30, 153.26, 147.92,
144.67, 139.24, 129.92, 128.27, 125.43, 120.34, 58.06,
12.34. LC-MS (ESI) m/z: 458.32 [M + H]+; Anal. calcd. For

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C21H16ClF3N7: C, 55.09; H, 3.30; N, 21.42. Found: C, 55.06;
H, 3.27; N, 21.47.
115.34, 115.27, 115.04, 108.82, 95.84, 55.74, 37.65,
27.19. LC-MS (ESI) m/z: 485.37 [M + H]+; Anal. calcd.
For C21H16ClF2N8O2: C, 52.02; H, 3.12; N, 23.11. Found:
C, 52.06; H, 3.14; N, 23.14.
6-(4-chlorophenyl)-4,4-difluoro-1-methyl-N-
(4-(trifluoromethoxy) phenyl)-5,6-dihydro-4H-
pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4] diazepin-
8-amine (16e)
2.2
Biological assays
|
1
Dark brown solid, yield 64%. m.p.: 158–160°C; H NMR
(300 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.64 (s, 1H), 7.65–
7.49 (m, 2H), 7.41 (d, J = 6.8 Hz, 2H), 7.36–7.17 (m, 2H),
7.03–6.82 (m, 2H), 4.59 (t, J = 11.4 Hz, 2H), 2.62 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ 157.21, 154.36, 153.20,
147.92, 144.68, 142.73, 139.51, 129.97, 121.88, 121.18,
119.99, 57.99, 12.38. LC-MS (ESI) m/z: 524.32 [M + H]+;
Anal. calcd. For C22H16ClF5N7O: C, 50.44; H, 2.89; N,
18.72. Found: C, 50.46; H, 2.85; N, 18.77.
2.2.1
BRD4 inhibition TR-FRET assay
|
BRD4-BD1 TR-FRET Assay Kit (Cayman, 600520) was
used to perform the BRD4 inhibition assay. All assays
were carried out in coning 384 well plates. First, 10 μl of
europium chloride solution of BRD4-BD1 was added to the
384 well plate and the plant was incubated in the dark for
15 min at room temperature. Then, 5 μl of BRD4 ligand/
APC receptor complex was added to each well and the plant
was incubated in the dark at room temperature for 1 hr.
After the incubation, the plate was read on the PerkinElmer
multimode plate reader with TR-FRET technique under
340 nm excitation and 670 nm emission/620 nm emission.
The absorbance between the negative control and the target
compounds was used to calculate the BRD4 inhibition rate,
and IC50 values were calculated from the BRD4 inhibition
rate.
N-(4-(tert-butyl) phenyl)-6-(4-chlorophenyl)-4,4-
difluoro-1-methyl-5,6-dihydro-4H-pyrimido-[4,5-b]
[1,2,4] triazolo[4,3-d] [1,4] diazepin-8-amine (16f)
1
Grayish white solid, yield 65%. m.p.: 162–164°C; H NMR
(300 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.58 (s, 1H), 7.58–
7.32 (m, 4H), 7.15–6.89 (m, 4H), 4.57 (t, J = 11.4 Hz, 2H),
2.61 (s, 3H), 1.22 (s, 9H). 13C NMR (75 MHz, DMSO-d6)
δ 157.00, 153.73, 152.63, 144.29, 143.81, 136.99, 129.41,
124.53, 118.30, 54.83, 33.69, 31.14, 30.64, 11.85. LC-MS
(ESI) m/z: 496.24 [M + H]+; Anal. calcd. For C25H25ClF2N7:
C, 60.54; H, 4.88; N, 19.77. Found: C, 60.56; H, 4.85; N,
19.79.
2.2.2
Cell growth inhibition assay
|
MV4-11 cells were cultured in IMDM modified with 10%
FBS and 1% penicillin-streptomycin in Thermo CO2 incu-
bators at 37°C. Cells were collected in logarithmic growth
phase, and then 100 μl of cell suspension containing 1 × 104
cells was added to each well of 96 well plate. The plate was
incubated in Thermo CO2 incubators at 37°C for 12 hr. After
incubation, 10 μl of compounds with different concentra-
tions were added to each well. After cultured for 72 hr, 5 μl
of CCK-8 was added to each well. The plate was read on
PerkinElmer multimode plate reader at 450 nm and IC50 val-
ues were calculated by GraphPad Prism 7 software.
N-(3-chlorophenyl)-6-(4-chlorophenyl)-4,4-difluoro-
1-methyl-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4]
triazolo[4,3-d] [1,4] diazepin-8-amine (16g)
White solid, yield 69%. m.p.: 166–168°C; 1H NMR
(300 MHz, DMSO-d6) δ 8.67 (d, J = 4.2 Hz, 2H), 7.58–
7.25 (m, 6H), 6.94 (m, 2H), 4.58 (t, J = 12.4 Hz, 2H), 2.64
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 157.07, 156.84,
153.90, 152.66, 143.78, 141.20, 132.95, 130.20, 129.57,
129.37, 126.77, 121.24, 117.49, 116.75, 57.73, 57.32, 56.89,
11.86. LC-MS (ESI) m/z: 474.20 [M + H]+; Anal. calcd.
For C21H16Cl2F2N7: C, 53.18; H, 3.19; N, 20.67. Found: C,
53.16; H, 3.21; N, 20.63.
2.2.3
Apoptosis and cell cycle analysis assay
|
6-(4-chlorophenyl)-4,4-difluoro-1-methyl-N-(4-
nitrophenyl)-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4]
triazolo[4,3-d] [1,4] diazepin-8-amine (16h)
MV4-11 cells were cultured in IMDM modified with 10%
FBS and 1% penicillin-streptomycin in Thermo CO2 incuba-
tors at 37°C. The effects of the target compounds on apopto-
sis and cell cycle were analyzed by flow cytometry. Annexin
V-FITC Apoptosis Detection Kit (KeyGEN BioTECH,
KGA107) and Cell Cycle Detection Kit (KeyGEN BioTECH,
KGA512) were used for cell apoptosis and cell cycle assay,
respectively. Cells were treated with the compound 15h for
24 hr.
White solid, yield 74%. m.p.: 178–180°C; 1H NMR
(300 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.71 (s, 1H), 7.87
(d, J = 8.4 Hz, 2H), 7.62 (d, J = 7.7 Hz, 2H), 7.48–7.27
(m, 4H), 4.62 (t, J = 12.0 Hz, 2H), 2.63 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) δ 170.16, 167.10, 167.03, 161.04,
140.97, 138.70, 136.27, 135.16, 131.09, 131.02, 130.26,
130.20, 129.06, 124.46, 120.73, 120.62, 117.07, 116.51,

LI et aL.
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2.2.4
Western blotting
3
RESULTS AND DISCUSSION
|
|
MV4-11 cells were cultured in IMDM modified with 10%
FBS and 1% penicillin-streptomycin in Thermo CO2 incu-
bators at 37°C. Cells were collected in logarithmic phase
and treated with the compounds with different concen-
trations for another 4 hr. In order to determine the corre-
sponding protein concentration for further, the cells were
put in 1.5 ml centrifuge tube after cracking at 4°C. After
that, 10% separation adhesive and 4% concentration ad-
hesive were prepared, and SDS-polyacrylamide gel was
placed in an electrophoresis tank for sample loading, elec-
trophoresis, and protein transfer. Subsequently, the PVDF
membrane was washed three times with TBST for 10 min
each, immersed in the 5% fat-free milk TBST solution, and
placed in a shaker at room temperature for 2 hr to block
the non-specific protein binding sites. PVDF membrane
was transferred with primary antibody diluent at 4°C over-
night and washed three times with TBST for 10 min each
after the reaction. The secondary antibody was added and
incubated for 2 hr before development. The primary anti-
bodies against c-Myc and β-actin were purchased from Cell
Signaling Technology. BCA Protein Assay Kit was pur-
chased from Beyotime.
3.1
Synthesis of the target compounds
|
General procedure for preparation of the target compounds
was showed in Scheme 1. Initially, 37% formaldehyde was
added to a mixture of aniline (1) and benzotriazole in die-
thyl ether to prepare compound 3, and then zinc powder was
added to a mixture of compound 3, trimethylchlorosilane,
and ethyl difluoroethyl bromide in THF to prepare com-
pound 5. Subsequently, 2,4-dichloro-5-nitropyrimidine was
added to a mixture of compound 5 in ethyl acetate to prepare
compound 7, and then reduction iron powder was added to a
mixture of compound 7 in acetic acid to prepare compound 9.
Lawesson Reagent was added to a mixture of compound 9 in
tetrahydrofuran to prepare compound 11, and then hydrazine
hydrate and triethyl orthoacetate were added to a mixture of
compound 11 in methanol and tetrahydrofuran to prepare the
key intermediate 13. Finally, the target products 15a~15h
were prepared from compound 13 and various substituents
anilines. The synthesis of the target compounds 16a~16h was
similar to that of 15a~15h.
Reagents and conditions: (i) benzotriazole, 37% HCHO,
Et2O, r.t, 6 hr; (ii) trimethylchlorosilane, zinc power (325
mesh), THF, ethyl bromodifluoroacetate, 10°C to r.t, 3–6 hr;
(iii) 2,4-dichloro-5-nitropyrimidine, NaHCO3, EtOAc, r.t,
16 hr; (iv) Fe, AcOH, 70°C, 1 hr, 100°C, 4–5 hr; (v) Lawesson
Reagent, THF, reflux; (vi) hydrazine hydrate, THF/MeOH
(v: v = 1:1), triethyl orthoacetate/toluene (v: v = 2:3), 110°C,
6h; (vii) anilines, Pd2(dba)3, Cs2CO3, xantphos, 1,4-dioxane,
110°C.
2.2.5
Molecular docking calculations
|
We used Schrödinger 2018 for molecular docking calcula-
tions. The crystal structure of BRD4 (PDB ID: 4BJX) was
available from PDB. Proteins were prepared by Protein
Preparation Wizard for adding missing hydrogens, creat-
ing disulfide bonds, and so on. Compounds were prepared
by LigPrep module to generate 3D structures. Receptor Grid
Generation was used to identify the ligand and Schrödinger
XP precision was used to perform the calculation (Bi
et al., 2019). The final result was chosen based on the struc-
ture of the lowest XP score (−3.373).
3.2
BRD4 inhibitory activity assay
|
The inhibitory activities of the target compounds against
BRD4 were tested in vitro by using the time-resolved
fluorescence resonance energy transfer (TR-FRET)
SCHEME 1 General procedure for preparation of the target compounds

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LI et aL.
|
binding assay. As shown in Table 1, the results exhib-
ited that all that target compounds had good inhibition
for BRD4-BD1, especially compound 15h (IC50 value of
0.42 μM) and compound 16h (IC50 value of 0.55 μM).
Based on the results, the SARs were summarized that the
inhibitory activity of the target compounds would be re-
duced when R1 was substituted by chlorine, and the in-
hibitory activity would be increased significantly when
R2 was substituted by electron-withdrawing groups. In
summary, when compared with BI-2536, the optimal
compound 15h demonstrated equivalent inhibitory activ-
ity against BRD4-BD1, which indicated that the 1-methy
l-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4]-triazolo[4,3-d]
[1,4] diazepin-8-amine skeleton could effectively simu-
late the structure of KAc.
3.3
Cell growth inhibition assay
|
Using (+)-JQ-1 and BI-2536 as positive controls, we tested
the anti-proliferative effects of the target compounds on
MV4-11 (biphenotypic B myelomonocytic leukemia) cell
lines. The results demonstrated that all the target compounds
had inhibitory effects on MV4-11 (Table 2). Among these
compounds, compound 15h had outstanding inhibitory effect
on MV4-11 cells (IC50 value of 0.51 μM), which was consist-
ent with the results of the BRD4 inhibitory activity assay.
3.4
Cell apoptosis, cell cycle analysis, and
|
Western blotting assay
Flow cytometry was used to analyze the effect of compound
15h on inducing apoptosis and cell cycle arrest in MV4-11 cell
lines. As showed in Figure 4, compound 15h had equivalent
apoptosis-inducing effect on MV4-11 in a dose-dependent
manner when compared with (+)-JQ-1 and compound 15h
and (+)-JQ-1 could arrest MV4-11 cells at G0/G1 phase in
a dose-dependent manner significantly (Figure 5). It was
reported that BRD4 worked as regulatory factors for c-Myc
protein (Delmore et al., 2011). We performed the western
blotting analysis to research the effects on c-Myc. Compound
15h and (+)-JQ-1 were chosen to evaluate their effects on the
expression of c-Myc. As shown in Figure 6, it was encour-
aging that compound 15h downregulated the expression of
c-Myc in a dose-dependent manner apparently. To sum up,
the compound 15h targeting BRD4 showed excellent inhibi-
tory activities on protein and MV4-11 cell lines in vitro. And
it could be further optimized and modified on the basis of
the structure-activity relationships to obtain BRD4 inhibitors
with higher safety and higher selectivity.
TABLE 1 Structure and inhibitory activity of the target
compounds (15a~16h) against BRD4-BD1
N
N
N
N
F
R2
F
N
H
N
N
R1
Compounds
R1
R2
IC50 (μM)a
15a
15b
15c
15d
15e
15f
15g
15h
16a
16b
16c
16d
16e
16f
16g
16h
H
H
H
H
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
—
H
1.22 0.01**
0.86 0.03*
1.35 0.04***
0.94 0.07*
0.62 0.01
4-OCH3
4-Cl
4-F
3,4-di-OCH3
4-tertiary butyl
2-Cl
4-NO2
H
4-OCH3
4-Cl
4-F
4-OCF3
4-tertiary butyl
3-Cl
4-NO2
—
2.66 0.11***
0.68 0.02*
0.42 0.01
TABLE 2 The anti-proliferative effects of the target compounds
on MV4-11
IC50 (μM)a
Compounds
IC50 (μM)a
1.62 0.10***
1.31 0.08**
2.63 0.01***
1.06 0.04**
1.54 0.02***
4.51 0.02***
1.32 0.13***
0.55 0.01
Compounds
15a
15b
15c
15d
15e
15f
15g
15h
3.09 0.12***
1.15 0.11*
3.56 0.43***
2.74 0.33**
0.76 0.16
16a
16b
16c
16d
16e
16f
16g
16h
BI-2536
4.46 0.62***
3.42 0.25***
4.23 0.46***
2.14 0.73**
3.79 0.85***
8.85 1.12***
3.57 0.26***
0.76 0.22
4.57 0.84***
1.02 0.46*
0.51 0.17
(+)-JQ−1
BI−2536
0.074 0.003***
0.53 0.04
—
—
(+)-JQ-1
0.16 0.05*
0.68 0.06
aThe IC50 value in the table was calculated from the TR-FRET assay. The data
were expressed as the means SD (n = 3).
aThe IC50 value in the table was calculated from the CCK-8 assay. The data were
expressed as the means SD (n = 3).
*p < .05, **p < .01, ***p < .001 versus compound 15h.
*p < .05, **p < .01, ***p < .001 versus compound 15h.

LI et aL.
1125
|
FIGURE 4 Effects of (+)-JQ-1 and compound 15h on apoptosis in MV4-11 cells: (a) Control; (b) (+)-JQ-1(1 μM); (c) (+)-JQ-1 (5 μM); (d)
Compound 15h (1 μM); (e) Compound 15h (5 μM); (f) Cell apoptosis analysis, the values were expressed as the means SD (n = 3). **p < .01,
***p < .001 versus control group [Colour figure can be viewed at wileyonlinelibrary.com]
3.5
Molecular docking study
with the core structure (1-methyl-5,6-dihydro-4H-pyr
imido[4,5-b] [1,2,4]-triazolo[4,3-d] [1,4] diazepin-8-
amine). The results of BRD4 inhibitory activity assay
in vitro showed that the target compounds had good in-
hibitory activities against BRD4-BD1. Among the target
compounds, compound 15h showed excellent BRD4-BD1
inhibitory activity (IC50 value of 0.42 μM), which was
equivalent to that of BI-2536 (IC50 value of 0.53 μM).
Correspondingly, compound 15h had outstanding inhibi-
tory effect on MV4-11 cells in the cell growth inhibition
assay (IC50 value of 0.51 μM). Furthermore, compound
15h had potent apoptosis-inducing effect on MV4-11
cells and could arrest MV-4-11 cells at G0/G1 phase in
a dose-dependent manner significantly. In Western blot-
ting assay, compound 15h down-regulated the expression
of c-Myc apparently. Moreover, based on the docking re-
sults, compound 15h could form key hydrogen bonds with
|
Schrödinger was used to analyze the possible binding mode be-
tween compound 15h and BRD4-BD1 (PDB ID: 4BJX). As ex-
pected, compound 15h (Figure 7a) and (+)-JQ-1 (Figure 2a) had
the equivalent binding mode with BRD4-BD1, they could directly
form key hydrogen bonds with Tyr 97 via a water molecule and
Asn140 in BRD4-BD1. Moreover, the docking results showed that
compound15hformedthepi–pistackingwithTrp81inBRD4-BD1
and the molecular structure of compound 15h had interaction with
the WPF (Trp81, Pro82, Phe83) shelf of BRD4-BD1 (Figure 7b).
4
CONCLUSION
|
In summary, we have taken BI-2536 as the lead com-
pound to design and synthesize the target compounds

1126
LI et aL.
|
FIGURE 5 Effects of (+)-JQ-1 and compound 15h on cell cycle arrest in MV4-11 cells: (a) Control; (b) (+)-JQ-1(1 μM); (c) Compound 15h
(1 μM); (d) (+)-JQ-1 (5 μM); (e) Compound 15h (5 μM); (f) Cell cycle analysis, MV4-11 cells were induced to arrest at G0/G1 phase in a dose-
dependent manner [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 6 Effects of compound 15h on the expression of c-Myc in MV4-11 cells: (a) Representative western blotting band of c-Myc
in MV4-11 cells treated with (+)-JQ-1 and compound 15h; (b) Band intensity of c-Myc expression analysis, the values were expressed
as the means SD (n = 3). *p < .05, **p < .01, ***p < .001 versus control group, ##p < .01, ###p < .001 [Colour figure can be viewed at
wileyonlinelibrary.com]
Tyr97 via a water molecule and Asn140 in BRD4-BD1 di-
rectly, which further indicated that the target compounds
had better binding affinity to BRD4-BD1. Obviously, it
can be confirmed that compound 15h could be an effec-
tive BRD4 inhibitor and be utilized as an optimal com-
pound for further development.

LI et aL.
1127
|
FIGURE 7 Docking results of
compound 15h with BRD4-BD1 (PDB
ID: 4BJX): (a) Compound 15h could
form hydrogen bonds with residue Tyr97
via a water molecule and Asn140, and
form the pi-pi stacking with Trp81 in
KAc recognition region of BRD4-BD1;
(b) The binding site of compound 15h in
BRD4 [Colour figure can be viewed at
wileyonlinelibrary.com]
(a)
(b)
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G., Young, R. A., … Mitsiades, C. S. (2011). BET bromodomain
inhibition as a therapeutic strategy to target c-Myc. Cell, 146(6),
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ACKNOWLEDGMENTS
This study was supported by the National Science Foundation
of China (No. 81872733) and the China Postdoctoral Science
Foundation (No. 2020M681792).
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CONFLICT OF INTEREST
All authors report no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available
in the supplementary material of this article.
ORCID
Hai Qian
https://orcid.org/0000-0002-3827-0992
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