Discovery of 3,5-dimethylisoxazole derivatives as novel, potent inhibitors for bromodomain and extraterminal domain (BET) family

Article abstract of DOI:10.1016/j.bmc.2021.116133

Bromodomain and extra-terminal (BET) is a promising therapeutic target for various hematologic cancers. We used the BRD4 inhibitor compound 13 as a lead compound to develop a variety of compounds, and we introduced diverse groups into the position of the compound 13 orienting toward the ZA channel. A series of compounds (14–23, 38–41, 43, 47–49) bearing triazolopyridazine motif exhibited remarkable BRD4 protein inhibitory activities. Among them, compound 39 inhibited BRD4(BD1) protein with an IC₅₀ of 0.003 μM was superior to lead compound 13. Meanwhile, compound 39 possess activity, IC₅₀ = 2.1 μM, in antiproliferation activity against U266 cancer cells. On the other hand, compound 39 could arrest tumor cells into the G0/G1 phase and induce apoptosis, which was consistent with its results in inhibiting cell proliferation. Biological and biochemical data suggest that BRD4 protein might be a therapeutic target and that compound 39 is an excellent lead compound for further development.

Full text of DOI:10.1016/j.bmc.2021.116133

Bioorg. Med. Chem. 39 (2021) 116133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 3,5-dimethylisoxazole derivatives as novel, potent inhibitors
for bromodomain and extraterminal domain (BET) family
,
,
,
,*
Lincheng Fanga 1, Zhaoxue Hua 1, Yifei Yangc, Pan Chenb, Jinpei Zhoub *, Huibin Zhanga
a Center for 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
c School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of
Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bromodomain and extra-terminal (BET) is a promising therapeutic target for various hematologic cancers. We
used the BRD4 inhibitor compound 13 as a lead compound to develop a variety of compounds, and we intro-
duced diverse groups into the position of the compound 13 orienting toward the ZA channel. A series of com-
pounds (14–23, 38–41, 43, 47–49) bearing triazolopyridazine motif exhibited remarkable BRD4 protein
BET inhibitors
Multiple myeloma (MM)
Molecular docking
Biological mechanism
inhibitory activities. Among them, compound 39 inhibited BRD4(BD1) protein with an IC50 of 0.003
μ
M was
superior to lead compound 13. Meanwhile, compound 39 possess activity, IC50 = 2.1
μM, in antiproliferation
activity against U266 cancer cells. On the other hand, compound 39 could arrest tumor cells into the G0/G1
phase and induce apoptosis, which was consistent with its results in inhibiting cell proliferation. Biological and
biochemical data suggest that BRD4 protein might be a therapeutic target and that compound 39 is an excellent
lead compound for further development.
1. Introduction
showing 95% sequence similarity.11 Although the two have high ho-
mology in the binding pocket, they can specifically recognize different
MM is the second most hematological malignancy characterized by
the uncontrolled proliferation of malignant plasma cells,1–4 which ac-
counts for ~10% of hematological malignancies.5 Despite the contin-
uous development of cancer treatment drugs, MM is still an incurable
disease because of relapse and drug resistance, and the optimal therapy
for advanced-stage multiple myeloma remains poorly defined.6 Thus,
the potent molecules with novel mechanisms are needed to build up the
theoretical basis for MM therapies.
acetylation substrates in H3 and H4 histone tails.12
Some studies have proposed that BRD4 interacts with the N-terminal
regions of histones H3 and H4, and it also plays a crucial role in
recruiting the positive transcription elongation factor b (P-TEFb) to
transcription initiation area promoting transcriptional elongation in a
highly context-dependent manner.13,14 On the other hand, BRD4 can
regulate nuclear factor-κB dependent genes as a response to immune
abnormality.15–17 Furthermore, BRD4 proteins are attracting increasing
attention as a therapeutic target for a variety of human diseases,
including acute myeloid leukemia (AML), MM, NUT midline carcinoma
(NMC), etc.18–20
BET proteins, such as BRD2/3/4 and BRDT, play a major part in
chromatin readers for acetylated lysine residues.7 BRD4 contains two
bromodomains (BD1 and BD2) and an extraterminal (ET) domain, which
is similar to all BET family members, and it also contains four
α
-helices
So far, BRD4 bromodomain inhibitors have been designed to
modulate the function of BRD4 protein, including JQ1, OTX015,
ABBV075 (Fig. 1), some of which have promising therapeutic potential
for disease treatment.18,21 In fact, Several BET inhibitors such as 2
(OTX015) and 3 (I-BET762) have recently entered clinical trials for
(αZ, αA, αB, and αC) that are linked by two divergent loop regions (ZA
and BC loops).8–10 BD1 and BD2 have high homology in amino acid
sequence, achieving >40%. Especially, in the position of the binding
pocket, they only have three crucial residues different from each other,
Abbreviations: BET, Bromodomain and Extraterminal Domain; MM, Multiple myeloma; WB, Western Blot; HEXIM1, Hexamethylene bis-acetamide inducible
protein; MYC, Myelocyto matosis oncogene.
* Corresponding authors.
E-mail addresses: jpzhou668@163.com (J. Zhou), zhanghb80@163.com, zhanghb80@cpu.edu.cn (H. Zhang).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2021.116133
Received 14 January 2021; Received in revised form 18 March 2021; Accepted 23 March 2021
Available online 3 April 2021
0968-0896/© 2021 Published by Elsevier Ltd.

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
cancer therapy. As previously mentioned, we reported that compound
13 containing quinazolinone motif as a BRD4 inhibitor exhibit excellent
biological activities in vitro.22 Here, we set up to introduce different
functional moieties into the position of compound 13 orienting toward
the ZA channel to obtain novel and potent BRD4 inhibitors for the
treatment of MM by exploring the structure–activity relationship (SAR)
more extensively and comprehensively. Additionally, we also have
described the antiproliferation mechanism of compound 39 against
U266 cells.
2.2. Design strategy and SAR study
Protein binding pattern analysis of quinazolinone compound 13 with
BRD4(BD1) protein revealed that the 3,5-Dimethylisoxazole motif as a
Kac mimic extended into the Kac pocket of the BRD4 protein and formed
a hydrogen bond with the key amino acid Asn140, allowing compound
13 to be firmly anchored in the Kac pocket. Simultaneously, the 4-posi-
tion benzene ring of compound 13 extended into the WPF hydrophobic
pocket, which formed by hydrophobic amino acids W81, P82 and F83,
immobilizing the protein by hydrophobic interactions (Figure 2A).
Delightfully, we found that the 3-position amide of compound 13 ori-
ented toward the ZA channel of BRD4 protein, which was exposed to the
solvent region. It meant that diverse active moieties can be introduced
into this position to enhance the physicochemical properties of the lead
compound 13.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds (13–23) as sketched in Scheme 1 and
their structure were confirmed by 1H NMR, 13C NMR spectrum and mass
spectra. Representative spectrometry was displayed in the Supporting
Information. First, 2-Aminobenzophenone as the starting material
reacted with N-Bromosuccinimide, followed by amine reduction with
tert-butyl sulfinate to produce intermediate 9, which was reduced by
sodium borohydride according to reported methods22 and then reacted
with triphosgene to obtain key intermediate 11. Intermediate 12 was
prepared by methylation of compound 11 with iodomethane. The
commercially available 3,5-Dimethylisoxazole pinacol boronate was
used to afford the target compound 13 via Suzuki ꢀ Miyaura coupling
with intermediate 12. Subsequently, lead compound 13 undergoes
nucleophilic substitution with various heterocyclic groups to obtain
target compounds 14–23.
Beginning with the parent molecule compound 13 and SAR study, we
first designed and synthesized compounds 14–23 by introducing
different heterocycles into the 3-position of the lead compound 13 to
explore the effects of the ZA region on the inhibitory activity. Then, most
of these compounds exhibited favorable BRD4 inhibitory activities,
especially the inhibition rates of compounds 14, 15, 17 and 23 were
100% against BRD4(BD1) protein at 1
outstanding activities against BRD4(BD1) with an IC50 of 0.055 and
0.058 M, respectively, that were similar to the activity of the lead
compound 13 (IC50 = 0.053 M). Besides, the introduced heterocyclic
μM. Compounds 14 and 23 had
μ
μ
group extended into the solvent region outside the ZA region by the
docking model of compound 14 to the BRD4 molecule (Figure 2B). We
confirm that the ZA channel has a strong capacity to accommodate
various larger moieties, which can provide a basis for the design of
subsequent BRD4 inhibitors (see Table 1).
Compounds (39–41, 43, 47–49) are synthesized as shown in Scheme
2. The commercially available starting material 3,6-dichloropyridazine
reacted with Semicarbazide, followed by chlorination with POCl3 to
obtain compound 36 and then substituted with piperazine to produce
key intermediate 37. Lead compound 13 was substituted with bromi-
nated alkanes of different lengths to afford intermediates 38a–c. Target
compounds 39–41 were prepared by using intermediates 38a–c and key
intermediate 37 via nucleophilic substitution. Meanwhile, compound 13
also treated with diethylene glycol ditoluenesulfonate, followed by
substitution with compound 37 to give target compound 43. Besides,
Compound 13 could be converted to intermediates 44–46 by reacting
with brominated fatty acids of different lengths. Target products 47–49
were attained through the above methods.
Considered with the above experimental results, we next designed
and synthesized compounds 38–41, 43, 47–49 by introducing a tri-
azolopyridazine structure into this position of the lead compound 13.
We observed that the triazolopyridazine warhead could be able to
extend into the solvent region through the ZA channel, thereby
enhancing the binding ability of small molecules to the protein
(Figure 3A). Meanwhile, most of the compounds exhibited significant
BRD4 (BD1) protein inhibitory activities with an inhibition rate of 100%
against BRD4(BD1) at 0.5 μM in the protein inhibitory activities study,
except for compound 49. Encouragingly, compounds (39–41) with
carbon chains as linkers performed better inhibitory activities than other
N
N
N
N
N
O
O
O
O
N
O
N
N
S
N
S
NH2
NH
O
N
HN
O
N
N
N
OH
N
Cl
Cl
Cl
3 (OTX015
)
4 (I-BET151
)
2
CPI-0610
)
1
JQ1
(
(
)
N
O
N
N
O
H
N
N
O
N
NH
N
N
O
N
N
O
N
H
F
N
N
O
H
O
O
O
S
N
H
F
Cl
6 (ABBV-075
7
BI-2536
)
)
(
5 (I-BET762
)
Fig. 1. Structures of small-molecule BET Bromodomain inhibitors.
2

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
NH2
NH2
NH2
NH2
O
S
H
O
S
c
a
b
O
O
N
N
Br
Br
Br
8
10
9
H
N
O
N
O
N
O
f
e
NH
d
NH
NH
Br
Br
O
N
13
12
11
Compounds
X
Y
Z
R
N
O
Z
Y
O
O
S
N
N
C-Me
N
S
N
C
N
N
C
H
H
H
H
H
F
F
H
F
F
14
15
g
N
R
X
16
17
18
19
20
21
22
23
N
S
O
N
C-Me N
S
C
S
S
N
S
C
C
N
N
N
N
O
14~23
Scheme 1. Reagents and conditions: (a) NBS, DCM, 0˚C; (b) tert-butanesulfinamide, tetraethyl titanate, THF, reflux; (c) NaBH4, THF, H2O, r.t; (d) bis(tri-
chloromethyl) carbonate, THF, r.t; (e) dimethylsulfate; Cs2CO3, acetonitrile, r.t; (f) phenylboronic acids, Pd (PPh3)4, Na2CO3, N2, H2O/EtOH/toluene, 80˚C; (g) 25a, b
or 26 or 28 or 30a, b or 32a, b or 34a,b, Cs2CO3, DMF, r.t.
types of compounds. Among them, compounds 39 and 41 showed the
best activity with an IC50 of 0.003 and 0.0031 M, respectively
2.3.2. Compound 39 induced cell cycle arrest in U266 cells
μ
To validate the effect of compound 39 on the cell cycle distribution of
U266 cells, we used flow cytometry to analyze the cycle distribution of
(Table 2). Especially, compound 39 increased an 8-fold inhibitory of
BRD4(BD1) protein comparing with the lead compound 13. These re-
sults implied that the introduction of the triazolopyridazine group
remarkably enhanced the BRD4 protein inhibitory activity of the lead
compound 13. Therefore, we evaluated the antiproliferative activities of
these compounds in diverse cells.
U266 cells after 48 h treatment with compound 39 at 1 and 2 μM,
respectively. We found that U266 cells could be arrested into the G0/G1
phase after treatment with compound 39 comparing with the control
(Fig. 4), indicating that compound 39 induced U266 cells cycle arresting
into G0/G1 phase.
2.3. Biological evaluation
2.3.3. Compound 39 induced apoptosis in U266 cells
Followed by, to investigate whether compound 39 could cause U266
cell apoptosis, DAPI staining was used to analyze the morphology of
U266 cell nucleus after 48 h treatment with compound 39. The result
showed that U266 nucleus underwent dramatic condensation
comparing with the negative control, indicating that compound 39
induced apoptosis in U266 cells through U266 cell nucleus condensation
(Figure 5A).
2.3.1. Anti-proliferation in cancer cell lines
We next evaluated the antiproliferative potency of compounds
14–23, 39–41, 43, and 47–49 by in vitro human tumor cell line
screening, including MCF10A, CT26, MCF7, HepG2, HCT116 and U266
cells. Together, a majority of compounds possessed excellent anti-
proliferative activity against U266 and nearly no toxicity to normal
breast cells. Most important was that the inhibition rates of compounds
We used flow cytometry to further verified U266 cells apoptosis after
48 h treatment with compound 39. Compound 39 led to significant
U266 cells apoptosis in a concentration-dependent manner comparing
with control (Figure 5B, C). Thus, compound 39 had a significant ability
to induce U266 cell apoptosis which might be the reason for its anti-
proliferative effect.
39–41 were around 50% at 0.1 μM (Table 3), which were consistent
with their potent BRD4 protein activities.
In terms of inhibition of cell proliferation to CT26, HepG2 and U266
cells, compounds 13, 14, 39–41 and 43 exhibited higher anti-
proliferative potency. Therefore, we further sought to the IC50 of these
compounds on CT26, HepG2 and U266 cells (Table 4). It was worth
noting that U266 cells were more sensitive to those compounds
comparing to CT26 and HepG2 cells. The inhibitory activity of com-
3. Conclusion
pounds 39 and 40 on U266 cells (IC50 = 2.11 and 1.99
were similar to that of ABBV-075(IC50 = 2.03 M) but the inhibitory
activity of compounds 39 and 40 (IC50 = 15.77 and 11.77 M, respec-
tively) on CT26 was much better than that of ABBV-075 (IC50 = 48.26
M). Surprisingly, the inhibitory ability of compound 39 on U266 cells
was about 17-fold that of compound 13 (IC50 = 35.35 M).
μ
M, respectively)
In this study, we performed further structural optimization and ac-
tivity evaluation of quinazolinone compound 13, which is previously
reported in our group. The inhibitory activities of compounds 14–23
against BRD4(BD1) protein indicate that compound 13 can accommo-
date large heterocycles at the position orienting toward the ZA channel.
Herein, we introduced a triazolopyridazine structure in this position to
enhance inhibitory potency. The BRD4(BD1) protein assay results
μ
μ
μ
μ
3

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
Cl
Cl
O
Cl
HN
Cl
Cl
N
Cl
N
N
N
a
b
c
N
N
N
N
NH
N
N
N
N
N
N
35
36
37
N
N
N
N
O
N
O
N
O
N
N
Cl
d
NH
N
N
e
N
Br
n
n
O
O
O
N
N
N
39, n=2
40
38a, n=2
38b, n=3
38c, n=4
39~41
13
38
, n=3
41, n=4
N
N
N
O
N
O
N
O
N
N
N
f
Cl
NH
N
OTs
e
N
N
O
O
O
O
N
N
N
O
13
43
42
N
O
N
O
N
O
O
O
n
g
N
h
N
NH
OH
N
n
Cl
N
O
N
O
N
N
N
O
N
N
N
44, n=1
45, n=5
46, n=7
47, n=1
48, n=5
49, n=7
47~49
44~46
13
Scheme 2. Reagents and conditions: (a) semicarbazide hydrochloride, conc. HCl, EtOH, H2O, reflux; (b) POCl3 reflux; (c) piperazine, triethylamine, acetonitrile, r.t;
(d) 1,4-dibromobutane or 1,5-dibromopentane or 1,6-dibromohexane, NaH, DMF, 0˚C to r.t; (e) 37, NaH, DMF, 0˚C to r.t; (f) 1,1′-bis(4-methylbenzenesulfonate),
NaH, DMF, 0˚C to r.t; (g) (1) ethyl bromoacetate or ethyl 6-bromohexanoate or ethyl 8-bromooctanoate, NaH, DMF, 0˚C to r.t; (2) LiOH, MeOH, H2O, r.t; (h) 37, EDCI,
HOBT, triethylamine, DMF, 0˚C to r.t.
Fig. 2. Molecular docking conformation. (A) Conformation of compound 13 (green) in BRD4(BD1) protein (2YEL). (B) Conformation of compound 14 (yellow) in
BRD4(BD1) protein (2YEL). (C) The superposition of compound 13 (green) and 14 (yellow) in BRD4(BD1) protein (2YEL).
exhibit that compound 39 possesses excellent inhibitory activity with an
IC50 of 0.003 M. Biological test results show that compound 39 effec-
tively inhibits the proliferation of U266 myeloma cells with an IC50 of
determined using an RY-1 melting point meter from Tianjin Analytical
Instrument Factory. Infrared spectroscopy (IR) was determined using
potassium bromide for slicing and Nicolet Impact 410 IR tester. Nuclear
magnetic resonance hydrogen (1H NMR) and carbon (13C NMR) spectra
were determined using Bruker AV-300 or Bruker AV-400 magnetic
resonance instruments with tetramethylsilane (TMS) as the internal
standard compound. Mass spectrometry (MS) was determined using an
Agilent 110 liquid combination system. Column chromatography was
performed using 200–300 mesh silica gel from Qingdao Ocean Chemical
Co. All reactions were monitored using high-efficiency thin-layer plates
under 254 nm and 365 nm wavelength UV lamp with either petroleum
ether/ethyl acetate or dichloromethane/methanol systems.
μ
2.1 μM, because it can not only arrest the U266 cells cycle into the G0/
G1 phase, but also has a strong ability to induce cell apoptosis. Due to
the significant in vitro biological activities of compound 39 against
malignant tumors, further molecular binding model and in vivo activity
studies are in progress.
4. Experiment section
4.1. Chemistry
(2-Amino-5-bromophenyl)(phenyl) methanone (8). To the solution of
2-Amino benzophenone (2 g, 10.14 mmol) in dichloromethane (30 mL),
N-bromosuccinimide (1.9 g, 10.65 mmol) was added slowly at 0 ◦C and
stirred in the ice bath for two hours. After completion of the reaction, the
The solvents or reagents used in the chemical experiments were
commercially available analytical or chemically pure products, which
were not treated, unless otherwise specified. The melting point was
4

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
Table 1
Table 2
BRD4(BD1) inhibitory activities of compounds 38–41, 43, and 47–49.
BRD4(BD1) inhibitory activities of compounds 13–23.
Compound No.
Linker
BRD4(BD1)
M(%)
BRD4(BD1) IC50
Compound No.
Het
R
BRD4(BD1)
M (%)
BRD4(BD1) IC50
1
μ
μM
1
μ
μ
Ma
38
39
40
41
43
47
48
49
100
100
100
100
100
100
100
98
0.013
0.003
0.0088
0.0031
0.012
0.0056
0.0092
0.037
13
14
100
100
0.053 ± 0.013
0.055 ± 0.011
H
15
16
17
18
19
20
21
22
23
H
H
H
H
F
100
82
0.105 ± 0.012
NT b
100
89
0.074 ± 0.014
13
100
100
0.053
0.011
NT
JQ1
98
NT
F
74
NT
Table 3
Anti-proliferation activities of 39–41, 43, and 47–49 in cancer cell lines.
H
F
76
NT
Compound No.
Inhibitory% (2
μ
M)
(0.1 μM)
MCF10A CT26 MCF7 HepG2 HCT116
U266
69
NT
13
14
15
16
17
18
19
20
21
22
23
39
40
41
43
47
48
49
JQ1
6.8
12.4
3.7
44.0
30.1
21.2
20.1
28.1
15.5
28.8
16.4
23.5
17.0
24.0
41.0
33.8
20.3
25.1
27.5
25.1
8.8
33.5
34.1
34.0
35.1
35.7
29.1
33.4
27.4
17.5
37.9
26.7
35.5
33.4
33.1
34.7
33.4
33.6
28.7
35.4
32.5
34.7
50.6
29.2
35.2
44.5
26.4
41.1
34.0
31.3
52.8
46.7
39.8
51.9
36.7
28.5
30.7
13.6
22.2
27.8
31.4
30.0
28.3
26.9
26.7
26.0
33.3
28.4
31.8
36.6
61.8
39.0
44.7
18.7
48.9
38.4
14.1
30.3
13.1
26.7
15.1
7.19
20.3
12.7
10.0
12.6
10.4
8.4
F
100
100
0.058 ± 0.015
0.042 ± 0.011
JQ1
4.9
3.3
a
b
The values are presented as means ± SD from three separate determinations.
2.7
NT: no test.
6.5
8.5
4.4
6.8
0.7
26.8
52.3
48.4
45.5
29.5
19.8
31.3
8.5
11.6
9.3
5.3
6.0
10.9
14.7
0.5
6.5
37.9
35.7
Table 4
Fig. 3. Molecular docking conformation. (A) Conformation of compound 39
(orange) in BRD4(BD1) protein (2YEL). (B) The superposition of compound 13
(green) and 39 (orange) in BRD4(BD1) protein (2YEL).
The IC50 of 14, 39–41, and 43 in different cancer cell lines.
Compound No.
IC50
(
μ
M) ± SD a
CT26 HepG2 U266
reaction was quenched by water (40 mL), followed by dichloromethane
(20 mL) solution. The organic phase was separated and dried over
anhydrous sodium sulfate. The crude was purified by column chroma-
tography (petroleum ether/ethyl acetate = 10/1–8/1) to obtain com-
pound 8a (2.4 g, 86% yield) as a light yellow solid.1H NMR (300 MHz,
DMSO‑d6) δ 7.61–7.45 (m, 5H), 7.38 (dd, J = 8.9, 2.4 Hz, 1H), 7.28 (d, J
= 2.4 Hz, 1H), 7.14 (s, 2H), 6.76 (d, J = 8.9 Hz, 1H).
14
21.18 ± 6.31
15.77 ± 4.00
11.77 ± 0.84
7.95 ± 1.64
19.49 ± 3.28
16.73 ± 3.45
14.04 ± 1.01
9.45 ± 0.33
20.98 ± 3.44
18.77 ± 3.28
18.39 ± 1.88
NT b
3.45 ± 0.27
2.11 ± 0.02
1.99 ± 0.01
2.12 ± 0.21
3.05 ± 0.37
35.35 ± 0.22
3.45 ± 0.48
2.03 ± 0.18
39
40
41
43
22.89 ± 4.01
35.98 ± 18.78
28.67 ± 27.84
48.26 ± 9.82
13
JQ1
ABBV-075
a
(E)-N-((2-Amino-5-bromophenyl)(phenyl) methylene)-2-methylpropane-
2-sulfinamide (9). 8 (5 g, 18.11 mmol) and tert-butyl sulfinateamine
(6.58 g, 54.32 mmol) were added to the tetrahydrofuran (60 mL) solution
and stirred for ten minutes at room temperature. Then tetraethyltitanate
(11.47 mL, 54.32 mmol) was added to the above reaction solution, the
temperature was slowly increased to 80 ◦C and the mixture was stirred for
8 h. After cooling the reaction media down to ambient temperature,
abundant tetrahydrofuran solution was evaporated under reduced pres-
sure distillation. The resulting crude product was purified by column
chromatography (petroleum ether/ethyl acetate = 15/1–1/1) to give
Three titrations were performed and data were used to calculate average
IC50 and SD values.
b
NT: no test.
compound 9 (6.1 g, 89% yield) as a light yellow solid.
N-((2-Amino-5-bromophenyl)(phenyl)
methyl)-2-methylpropane-2-
sulfinamide (10). To a stirred solution of compound 9 (1 g, 2.64
mmol) in tetrahydrofuran (20 mL) was then slowly added sodium
borohydride (0.4 g, 10.55 mmol) and the reaction mixture was stirred
for six hours at room temperature. After completion of the reaction,
5

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
Control
1 μM
2 μM
A
PI- A
B
Fig. 4. Compound 39 induced cell cycle arrest in U266 cells. (A, B) U266 cells were stimulated with 1 and 2
μ
M of compound 39 for 48 h, and the cell cycle ratio of
U266 cells was detected by cell cycle staining kit and flow cytometry.
tetrahydrofuran was removed from the reaction solution by reduced
pressure distillation. The residue was purified by column chromatog-
raphy (petroleum ether/ethyl acetate = 11/4–2/1) to afford compound
10 (0.9 g, 90% yield) as a white solid.1H NMR (300 MHz, DMSO) δ 7.36
(d, J = 6.9 Hz, 4H), 7.26 (s, 1H), 7.11 (dd, J = 20.2, 11.6 Hz, 2H),
6.65–6.56 (m, 1H), 5.99 (dd, J = 26.1, 6.2 Hz, 1H), 5.54 (dd, J = 30.2,
6.2 Hz, 1H), 5.25 (s, 2H), 1.13 (d, J = 7.0 Hz, 9H).
(d, J = 8.5 Hz, 1H), 5.53 (s, 1H), 3.19 (s, 3H).
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3,4-dihy-
droquinazolin-2(1H)-one (13). To a solution of 12 (0.3 g, 0.95 mmol) in a
mixture of toluene (3 mL) and ethanol (1 mL) was added, 3,5-dimethy-
lisoxazole-4-boronic acid pinacol ester (0.23 g, 1.0 mmol), 1 mol/L
aqueous sodium carbonate solution (3 mL) and tetraphenylphosphine
palladium (0.109 g, 0.096 mmol) were added in batches. The mixture
was heated to 80˚C and stirred for 12 h under nitrogen. After completion,
the reaction solution was concentrated and purified by column chro-
matography (petroleum ether/ethyl acetate = 5/1–1/1) to obtain
compound 13 (0.21 g, 67% yield) as a white solid in 97% purity. IR (KBr,
6-Bromo-4-phenyl-3,4-dihydroquinazolin-2(1H)-one (11). Bis (tri-
chloromethyl) carbonate (1.17 g, 3.93 mmol) was slowly added to a
solution of compound 10 (1 g, 2.62 mmol) in tetrahydrofuran (15 mL)
and the reaction mixture was stirred for eight hours at ambient tem-
perature. After the reaction was completed, the pH of the reaction so-
lution was adjusted to pH 7.6–10.4 with saturated sodium carbonate
aqueous solution. Then water (20 mL) and ethyl acetate (20 mL) were
added to the above mixture, and the organic phase was separate, the
aqueous phase was extracted twice with ethyl acetate (20 mL). The
organic phase was evaporated and purified by column chromatography
(petroleum ether/ethyl acetate = 5/1–2/1) to obtain compound 11
(0.65 g, 82% yield) as a white solid.
cmꢀ 1
) ν: 3415.94, 1679.16, 1638.13, 1617.58, 1522.13, 1476.05,
1437.20, 1393.99, 1334.82, 1275.37, 1123.55, 721.72, 699.70, 599.58,
568.80, 541.92, 524.80, 489.63; 1H NMR (300 MHz, CDCl3) δ 7.37 (s,
5H), 7.26 (s, 1H), 7.14 (s, 1H), 7.01 (s, 1H), 6.65 (s, 1H), 5.62 (s, 1H),
3.41 (s, 3H), 2.27 (s, 3H), 2.12 (s, 3H). 13C NMR (101 MHz, DMSO) δ
165.22, 158.58, 154.52, 144.71, 138.14, 132.51, 132.03, 131.92,
129.29, 129.17, 129.06, 127.86, 127.60, 126.43, 124.92, 123.39,
115.77, 114.04, 55.92, 40.62, 40.42, 40.21, 40.00, 39.79, 39.58, 39.37,
29.61, 11.73, 10.90.MS (ESI, m/z): 334.0[M+H]+.
6-Bromo-1-methyl-4-phenyl-3,4-dihydroquinazolin-2(1H)-one (12). To
a solution of 11 (1 g, 3.30 mmol) and cesium carbonate (3.22 g, 9.90
mmol) in acetonitrile (15 mL), dimethyl sulfate (0.5 g, 3.96 mmol) was
slowly dripped and the above reaction was stirred for 6 h at room
temperature. Upon completion, the solvent was evaporated and the
residue was isolated by column chromatography (petroleum ether/ethyl
acetate = 5/1–2/1) to give compound 12 (0.8 g, 76% yield) as a white
solid. 1H NMR (300 MHz, DMSO) δ 7.80 (s, 1H), 7.54–7.15 (m, 7H), 6.94
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3-((5-phenyl-1,3,4-
oxadiazol-2-yl) methyl)-3,4-dihydroquinazolin-2(1H)-one (14). To a so-
lution of compound 13 (0.2 g, 0.6 mmol) in N, N-dimethylformamide (6
mL), cesium carbonate (0.59 g, 1.80 mmol) was slowly added and stirred
for 10 min at room temperature. Compound 25a (0.14 g, 0.72 mmol)
was added to the above reaction solution, and the reaction was stirred
for further two hours at room temperature. After the reaction was
6

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
Control
Control
1 μM
1 μM
2 μM
2 μM
8 μM
A
B
8 μM
Annexin V-FITC
40
30
20
10
0
C
**
*
*
M
M
M
ol
ontr
1μ
2μ
8μ
C
Fig. 5. Compound 39 induced apoptosis in U266 cells. (A) U266 cells were stimulated with 1, 2 and 8
μ
M of compound 39 for 48 h, and the nuclear condensation
was detected by DAPI staining, and representative photographs were shown. (B, C) U266 cells were treated with compound 39 at 1, 2 and 8
apoptotic cells were determined by flow cytometry (*p < 0.05, **p < 0.001).
μM for 48 h, and
completed, the above reaction solution was added dropwise to water
(20 mL), then ethyl acetate (20 mL) was added and stirred for 10 min at
room temperature to separate the organic phase. The organic phase was
collected and purified by column chromatography (petroleum ether/
ethyl acetate = 5/1–1/1) to obtain compound 14 (0.18 g, 61% yield) as
in 99% purity. m.p.: 90–92 ◦C. IR (KBr, cmꢀ 1
) ν: 3414.82, 1661.77,
1615.17, 1519.30, 1475.23, 1447.42, 1400.72, 1340.83, 1269.33,
1106.88, 763.00, 695.82, 617.81; 1H NMR (300 MHz, DMSO) δ
7.67–7.51 (m, 4H), 7.36 (t, J = 15.8 Hz, 9H), 7.11 (s, 1H), 5.85 (s, 1H),
5.12 (d, J = 15.1 Hz, 1H), 4.32 (d, J = 17.4 Hz, 1H), 3.35 (d, J = 1.9 Hz,
3H), 2.32 (d, J = 3.2 Hz, 3H), 2.15 (d, J = 3.2 Hz, 3H). 13C NMR (75
MHz, MeOD) δ 163.48, 156.50, 152.48, 139.29, 134.87, 134.83, 126.99,
126.73, 126.54, 126.32, 125.97, 125.40, 125.15, 124.17, 122.29,
121.84, 119.08, 111.79, 60.84, 46.45, 41.18, 27.60, 7.92, 7.17. MS (ESI,
a light yellow solid in 97% purity. m.p.: 136–138 ◦C. IR (KBr, cmꢀ 1
) ν:
3415.62, 1660.20, 1644.05, 1612.81, 1551.70, 1518.37, 1477.56,
1443.76, 1422.74, 1402.98, 1330.44, 1272.67, 1240.61, 1207.66,
1151.59, 1106.89, 1011.42, 830.04, 793.52, 772.73, 736.57, 709.30,
699.81, 689.52, 601.80, 565.95, 526.19, 506.75; 1H NMR (500 MHz,
DMSO) δ 7.88 (d, J = 7.4 Hz, 2H), 7.62 (dt, J = 23.5, 7.4 Hz, 3H), 7.40
(d, J = 7.6 Hz, 2H), 7.36–7.30 (m, 4H), 7.23 (t, J = 7.3 Hz, 1H), 7.16 (d,
J = 8.3 Hz, 1H), 5.93 (s, 1H), 5.19 (d, J = 16.3 Hz, 1H), 4.64 (d, J = 16.3
Hz, 1H), 3.40 (s, 3H), 2.35 (s, 3H), 2.18 (s, 3H). 13C NMR (75 MHz,
DMSO) δ 164.81, 164.18, 163.25, 158.01, 153.30, 141.37, 136.51,
131.91, 129.30, 128.87, 128.76, 127.89, 126.84, 126.43, 125.99,
124.10, 123.63, 123.15, 115.07, 114.00, 61.56, 41.15, 40.35, 40.08,
39.80, 39.52, 39.25, 38.97, 38.70, 30.27, 11.21, 10.33. MS (ESI, m/z):
m/z): 491.3[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-3-((4-methyl-2-phenylthiazol-
5-yl) methyl)-4-phenyl-3,4-dihydroquinazolin-2(1H)-one (16). The prep-
aration method was similar to that of compound 14 and compound 16
(0.14 g, 79% yield) was synthesized as a gray-white solid in 98% purity.
m.p.: 104–106 ◦C. IR (KBr, cmꢀ 1
) ν: 3414.82, 1639.62, 1615.83,
1517.62, 1441.78, 1400.30, 1338.36, 1267.91, 1233.38, 1192.11,
1154.95, 1102.46, 1003.90, 838.17, 699.88, 618.46, 582.16, 522.64; 1H
NMR (500 MHz, DMSO) δ 7.87 (dd, J = 8.7, 5.4 Hz, 2H), 7.36 (d, J = 7.4
Hz, 2H), 7.33–7.23 (m, 7H), 7.07 (d, J = 8.5 Hz, 1H), 5.75 (s, 1H), 5.10
(d, J = 15.7 Hz, 1H), 4.28 (d, J = 15.7 Hz, 1H), 3.28 (s, 3H), 2.31 (s, 3H),
2.24 (s, 3H), 2.14 (s, 3H). 13C NMR (75 MHz, DMSO) δ 164.75, 163.52,
161.36, 157.99, 153.38, 150.97, 141.84, 136.43, 129.69, 128.80,
128.61, 128.18, 128.07, 127.96, 127.82, 126.78, 125.74, 124.06,
492.2[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3-((5-phenyloxazol-
2-yl) methyl)-3,4–dihydroqu inazolin-2(1H)-one (15). The synthesis
method used was similar to that used in the preparation of compound 14
and compound 15 (0.12 g, 74% yield) was given as a grayish-white solid
7

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
123.39, 116.22, 115.93, 115.05, 113.77, 60.80, 41.86, 40.35, 40.06,
[M+H] +
.
39.78, 39.51, 39.23, 38.95, 30.13, 14.87, 11.22, 10.34. MS (ESI, m/z):
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3-((2-phenylthiazol-
4-yl)methyl)-3,4-dihydroqu inazolin-2(1H)-one (21). The synthesis
method used was similar to that used in the preparation of compound 14
and compound 21 (0.79 g, 85% yield) was given as a light yellow solid in
521.3[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3-((5-phenyl-1,3,4-
thiadiazol-2-yl) methyl)-3,4-dihydroquinazolin-2(1H)-one (17). Com-
pound 17 (0.083 g, 89% yield) was obtained as a light yellow solid in
98% purity by similar synthesis procedure used in the preparation of
97% purity. m.p.: 170–172 ◦C. IR (KBr, cmꢀ 1
) ν: 3415.09, 3079.91,
1654.81, 1614.12, 1519.00, 1475.40, 1462.70, 1421.84, 1400.07,
1334.20, 1270.06, 1205.32, 1105.49, 1002.44, 764.46, 733.63, 692.70,
619.11, 518.74; 1H NMR (300 MHz, DMSO) δ 7.87 (d, J = 3.4 Hz, 2H),
7.46 (dd, J = 6.0, 2.9 Hz, 4H), 7.36 (dd, J = 14.2, 6.8 Hz, 5H), 7.26 (d, J
= 7.3 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 5.91 (s, 1H), 5.24 (d, J = 15.4
Hz, 1H), 4.11 (d, J = 15.5 Hz, 1H), 2.32 (s, 3H), 2.16 (s, 3H). 13C NMR
(75 MHz, DMSO) δ 167.20, 164.73, 158.04, 153.56, 153.49, 141.76,
136.89, 132.91, 130.18, 129.11, 128.86, 128.59, 127.73, 126.72,
126.03, 125.90, 124.25, 123.33, 117.00, 115.15, 113.67, 60.88, 45.58,
40.36, 40.07, 39.79, 39.51, 39.23, 38.95, 38.67, 30.17, 11.22, 10.34.
compound 14. m.p.: 238–240 ◦C. IR (KBr, cmꢀ 1
) ν: 3414.33, 1655.48,
1612.21, 1517.71, 1475.19, 1466.37, 1455.32, 1442.19, 1419.93,
1404.05, 1331.82, 1301.69, 1271.04, 1239.18, 1109.93, 978.53,
829.83, 770.50, 753.69, 736.41, 693.67, 641.92, 635.13, 617.51,
562.01, 595.52; 1H NMR (300 MHz, DMSO) δ 7.89 (d, J = 5.5 Hz, 2H),
7.52 (d, J = 6.3 Hz, 3H), 7.31 (dd, J = 17.2, 7.0 Hz, 6H), 7.23 (d, J = 6.2
Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 5.91 (s, 1H), 5.26 (d, J = 15.7 Hz, 1H),
4.61 (d, J = 16.0 Hz, 1H), 2.31 (s, 3H), 2.14 (s, 3H). 13C NMR (75 MHz,
DMSO) δ 166.79, 164.83, 158.29, 153.55, 141.78, 136.54, 132.01,
131.98, 131.51, 131.38, 131.26, 129.38, 128.90, 128.80, 128.76,
128.64, 127.94, 127.53, 126.84, 126.12, 124.07, 123.65, 114.04, 61.79,
45.88, 40.05, 39.77, 39.49, 39.21, 38.94, 30.20, 11.24, 10.36. MS (ESI,
MS (ESI, m/z): 507.2[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-3-((2-(4-fluorophenyl)thiazol-4-yl)
methyl)-1-methyl-4-phenyl-3,4-dihydroquinazolin-2(1H)-one (22). The
synthesis method used was similar to that used in the preparation of
compound 14 and compound 22 (0.66 g, 91% yield) was given as a light
m/z): 508.4[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3-((4-phenylthiazol-
2-yl)methyl)-3,4-dihydroq uinazolin-2(1H)-one (18). The synthesis
method was similar to that of compound 14. Compound 18 (0.091 g,
87% yield) was obtained as a pale yellow solid in 97% purity. m.p.:
yellow solid in 96% purity. m.p.: 166–168 ◦C. IR (KBr, cmꢀ 1
) ν: 3415.70,
3103.02, 2923.64, 1664.50, 1614.13, 1470.96, 1520.90, 1448.72,
1419.85, 1307.80, 1400.61, 1338.31, 1237.51, 1229.08, 1205.29,
1297.86, 1267.51, 1154.15, 1103.56, 1026.74, 1001.17, 837.18,
734.42, 749.03, 694.74, 613.31, 586.04, 566.12, 524.06, 497.79; 1H
NMR (500 MHz, DMSO) δ 7.94 (dd, J = 8.7, 5.4 Hz, 2H), 7.50 (s, 1H),
7.43–7.27 (m, 9H), 7.10 (d, J = 8.4 Hz, 1H), 5.90 (s, 1H), 5.23 (d, J =
15.5 Hz, 1H), 4.14 (d, J = 15.4 Hz, 1H), 3.37 (s, 3H), 2.34 (s, 3H), 2.18
(s, 3H). 13C NMR (75 MHz, DMSO) δ 165.99, 164.73, 161.48, 158.03,
153.55, 153.46, 141.77, 136.87, 129.60, 128.85, 128.60, 128.39,
128.28, 127.72, 126.72, 125.88, 124.24, 123.32, 117.16, 116.27,
115.97, 115.14, 113.68, 60.87, 45.56, 40.33, 40.06, 39.79, 39.51,
94–96 ◦C. IR (KBr, cmꢀ 1
) ν: 3415.44, 1655.21, 1615.26, 1519.05,
1475.27, 1442.70, 1493.61, 1339.18, 1399.45, 1306.83, 1269.39,
1240.19, 1105.94, 827.66, 735.92, 696.64, 617.59, 478.80; 1H NMR
(300 MHz, DMSO) δ 7.99 (s, 1H), 7.88 (d, J = 7.1 Hz, 2H), 7.45–7.25 (m,
10H), 7.13 (d, J = 8.5 Hz, 1H), 5.92 (s, 1H), 5.33 (d, J = 16.1 Hz, 1H),
4.40 (d, J = 16.2 Hz, 1H), 3.38 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). 13
C
NMR (75 MHz, DMSO) δ 167.62, 164.80, 158.03, 153.95, 153.54,
141.47, 136.62, 133.97, 128.89, 128.73, 128.68, 127.95, 127.89,
126.75, 125.99, 125.94, 124.22, 123.60, 115.08, 114.67, 113.93, 61.56,
47.88, 40.05, 39.77, 39.49, 39.21, 38.93, 30.23, 11.23, 10.35. MS (ESI,
39.23, 38.95, 38.68, 30.16, 11.21, 10.33. MS (ESI, m/z): 525.6[M+H] +
.
m/z): 507.3[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-3-((5-(4-fluorophenyl)-1,3,4-oxadiazol-
6-(3,5-Dimethylisoxazol-4-yl)-3-((2-(4-fluorophenyl)-4-methylthiazol-
5-yl) methyl)-1-methyl-4-ph enyl-3,4-dihydroquinazolin-2(1H)-one (19).
The synthesis method used was similar to that used in the preparation of
compound 14 and compound 19 (0.101 g, 83% yield) was given as a
2-yl) methyl)-1-methyl-4-ph enyl-3,4-dihydroquinazolin-2(1H)-one (23).
The synthesis method was similar to that of compound 14. The target
compound 23 (0.78 g, 83% yield) was obtained as a light yellow solid in
98% purity. m.p.: 90–92 ◦C. IR (KBr, cmꢀ 1
) ν: 3415.54, 1638.16,
light yellow solid in 99% purity. m.p.: 98–100 ◦C. IR (KBr, cmꢀ 1
)
ν
:
1616.26, 1521.00, 1498.63, 1475.38, 1400.25, 1341.28, 1269.45,
1238.85, 1157.51, 1107.62, 845.01, 735.91, 614.72; 1H NMR (300
MHz, DMSO) δ 7.91 (dd, J = 8.8, 5.4 Hz, 2H), 7.46–7.26 (m, 9H), 7.13
(d, J = 8.4 Hz, 1H), 5.90 (s, 1H), 5.15 (d, J = 16.3 Hz, 1H), 4.63 (d, J =
16.2 Hz, 1H), 3.37 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). 13C NMR (75 MHz,
MeOD) δ 139.32, 127.27, 127.15, 127.05, 126.78, 126.09, 125.20,
124.34, 124.28, 122.43, 122.17, 114.26, 114.20, 114.15, 113.96,
113.84, 111.90, 61.08, 60.20, 46.44, 39.19, 27.60, 8.00, 7.12. MS (ESI,
3416.13, 2924.92, 1654.99, 1613.46, 1519.11, 1473.93, 1460.42,
1441.76, 1399.91, 1339.14, 1305.31, 1268.24, 1239.47, 1192.13,
1145.85, 1103.16, 1003.60, 826.86, 763.11, 692.01, 524.63; 1H NMR
(300 MHz, DMSO) δ 7.82 (d, J = 3.9 Hz, 2H), 7.47–7.41 (m, 3H),
7.40–7.28 (m, 5H), 7.24 (s, 2H), 7.07 (d, J = 8.4 Hz, 1H), 5.76 (s, 1H),
5.12 (d, J = 15.7 Hz, 1H), 4.28 (d, J = 15.6 Hz, 1H), 3.37 (s, 3H), 2.31 (s,
3H), 2.25 (s, 3H), 2.14 (s, 3H). 13C NMR (75 MHz, DMSO) δ 164.75,
158.07, 153.44, 150.97, 141.84, 136.45, 133.04, 129.96, 129.08,
128.81, 128.62, 128.05, 127.83, 126.78, 125.75, 124.06, 123.38,
115.15, 113.77, 60.78, 41.85, 40.35, 40.07, 39.79, 39.52, 39.24, 38.96,
m/z): 510.6[M+H] +
.
6-Chloro-[1,2,4]triazolo [4,3-b]pyridazin-3(2H)-one (35). To a solu-
tion of 3,6-Dichloropyridazine (5 g, 33.56 mmol) in ethanol (30 mL),
aminourea hydrochloride (7.5 g, 67.13 mmol) was added slowly and
stirred at room temperature for three days. After the reaction was
completed, the reaction mixture was filtered and washed with cold
water. Then the crude was dried to give compound 35 (4.3 g, 75% yield)
as a light yellow solid. 1H NMR (300 MHz, DMSO) δ 7.90 (d, J = 9.8 Hz,
1H), 7.21 (d, J = 9.8 Hz, 1H).
38.67, 30.14, 14.90, 11.22, 10.34. MS (ESI, m/z): 521.3[M+H] +
.
6-(3,5-Dimethylisoxazol-4-yl)-3-((4-(4-fluorophenyl)thiazol-2-yl)
methyl)-1-methyl-4-phenyl-3,4-dihydroquinazolin-2(1H)-one (20). The
synthesis method used was similar to that used in the preparation of
compound 14 and compound 20 (0.77 g, 82% yield) was given as a light
yellow solid in 98% purity. m.p.: 98–100 ◦C. IR (KBr, cmꢀ 1
) ν: 3416.42,
1655.58, 1613.36, 1519.30, 1495.81, 1474.18, 1442.93, 1399.71,
1339.11, 1308.76, 1269.76, 1223.25, 1155.48, 1105.89, 840.50,
749.99, 700.67; 1H NMR (300 MHz, DMSO) δ 8.00–7.88 (m, 3H),
7.40–7.21 (m, 9H), 7.13 (d, J = 8.5 Hz, 1H), 5.91 (s, 1H), 5.30 (d, J =
16.1 Hz, 1H), 4.41 (d, J = 16.0 Hz, 1H), 3.38 (s, 3H), 2.33 (s, 3H), 2.16
(s, 3H). 13C NMR (75 MHz, DMSO) δ 167.77, 164.80, 163.47, 160.22,
158.02, 153.52, 152.87, 141.49, 136.60, 130.57, 128.88, 128.73,
128.04, 127.93, 127.88, 126.74, 125.98, 124.21, 123.59, 115.68,
115.39, 115.07, 114.51, 113.94, 61.56, 47.89, 40.32, 40.05, 39.77,
39.49, 39.21, 38.93, 38.66, 30.22, 11.23, 10.35. MS (ESI, m/z): 525.3
3,6-Dichloro-[1,2,4]triazolo[4,3-b]pyridazine (36). Compound 35
(4.3 g, 33.56 mmol) was dissolved in phosphorus trichloride (15 mL)
and refluxed for 4 h. After the reaction was completed, the reaction was
cooled to room temperature and quenched with ice water (20 mL) and
ethyl acetate (20 mL). The organic phase was collected and purified by
column chromatography (petroleum ether/ethyl acetate = 30/1–10/1)
to obtain compound 36 (3.1 g, 65% yield) as a white solid.
3-Chloro-6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine (37). To
a stirred solution of Piperazine (0.41 g, 4.76 mmol) and triethylamine
(1.47 mL, 10.58 mmol) in acetonitrile (15 mL), compound 36 (1 g, 5.29
8

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
mmol) diluted with acetonitrile (5 mL) was added dropwise, and the
reaction was conducted for ten hours at room temperature. After the
completion of the reaction, the mixture was evaporated and dried to
obtain compound 37 (0.98 g, 77% yield) as a light yellow. 1H NMR (300
MHz, DMSO) δ 9.51 (s, 1H), 8.20 (d, J = 10.2 Hz, 1H), 7.49 (d, J = 10.2
Hz, 1H), 3.85 (s, 4H), 3.23 (s, 4H).
hexyl)-6-(3,5-dimethylisox azol-4-yl)-1-methyl-4-phenyl-3,4-dihydroquinazo
lin-2(1H)-one (41). The synthetic method for compound 41 was similar
tothatofcompound39andcompound41(0.099g, 90%yield)wasgotasa
pale yellow solid in 96% purity. m.p.: 116–118 ◦C. IR (KBr, cmꢀ 1
) ν:
3461.12, 1647.48, 1552.78, 1520.76, 1476.91, 1406.59, 1338.53,
1301.99, 1268.35, 1241.83, 1104.85, 736.75, 511.09; 1H NMR (300 MHz,
DMSO) δ 8.10 (d, J = 10.2 Hz, 1H), 7.46 (d, J = 10.3 Hz, 1H), 7.36 (dd, J =
9.8, 3.1 Hz, 5H), 7.29–7.22 (m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 5.73 (s, 1H),
3.85–3.71 (m, 1H), 3.55 (s, 4H), 3.29 (s, 3H), 2.82 (dd, J = 13.7, 5.7 Hz,
1H), 2.43(s, 4H), 2.36(s, 3H), 2.25(t, J= 7.2Hz, 2H), 2.19(s, 3H), 1.48(d,
J= 44.2Hz, 4H), 1.25(s, 4H). 13CNMR(75MHz, DMSO)δ 164.75, 158.01,
155.50, 153.65, 143.44, 142.45, 137.13, 128.82, 128.50, 127.61, 126.57,
125.68, 124.37, 123.01, 115.33, 113.42, 60.72, 57.52, 52.07, 46.45,
45.10, 40.36, 40.09, 39.82, 39.54, 39.26, 38.98, 38.71, 29.96, 27.28,
26.52, 26.08, 25.92, 11.27, 10.40. MS (ESI, m/z): 654.1[M+H]+.
3-(4-Bromobutyl)-6-(3,5-dimethylisoxazol-4-yl)-1-methyl-4-phenyl-
3,4-dihydroquinazolin-2(1H)-one (38). Compound 13 (0.5 g, 1.50 mmol)
was dissolved in N, N-dimethylformamide (10 mL) and stirred under ice
bath for ten minutes. Then, sodium hydroxide (66 mg, 1.65 mmol)
dissolved in N, N-dimethylformamide (2 mL) was added to the above
reaction solution and stirred under ice bath for further ten minutes.
Then, 1,4-dibromobutane (0.32 g, 1.50 mmol) in N, N-dimethylforma-
mide (1 mL) was slowly added to the above reaction solution and stirred
for eight hours at room temperature. After the reaction was completed,
water (20 mL) and ethyl acetate (20 mL) were added to the above re-
action solution. The organic phase was separated and purified by col-
umn chromatography (petroleum ether/ethyl acetate = 10/1–5/1) to
get compound 38 (0.4 g, 57% yield) colorless oil in 97% purity. 1H NMR
(300 MHz, CDCl3) δ 7.27 (dt, J = 8.6, 6.0 Hz, 5H), 7.10 (dd, J = 8.3, 1.9
Hz, 1H), 7.02–6.88 (m, 2H), 5.46 (s, 1H), 3.93 (dt, J = 13.6, 6.9 Hz, 1H),
3.53–3.31 (m, 5H), 2.93 (dd, J = 13.7, 6.0 Hz, 1H), 2.30 (s, 3H), 2.17 (d,
3-(2-(2-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-
yl)ethoxy)ethyl)-6-(3,5-dimethylisoxazol-4-yl)-1-methyl-4-phenyl-3,4-
dihydroquinazolin-2(1H)-one (43). The synthesis method of compound
43 was the same as that for compound 39 and compound 43 (0.087 g,
78% yield) was obtained as a pale yellow solid in 94% purity. m.p.:
108–110 ◦C. IR (KBr, cmꢀ 1
) ν: 3450.55, 1650.44, 1552.85, 1478.25,
1408.67, 1338.17, 1267.09, 1240.42, 1106.90, 813.55, 736.26, 699.76,
560.52, 514.59; 1H NMR (300 MHz, DMSO) δ 8.08 (d, J = 10.2 Hz, 1H),
7.35 (t, J = 6.2 Hz, 6H), 7.25 (dd, J = 12.2, 6.9 Hz, 2H), 7.04 (d, J = 8.4
Hz, 1H), 5.81 (s, 1H), 3.98 (dd, J = 9.2, 5.1 Hz, 1H), 3.58–3.38 (m, 8H),
3.30 (s, 3H), 3.01–2.91 (m, 1H), 2.46 (s, 6H), 2.31 (s, 3H), 2.14 (s, 3H).
13C NMR (101 MHz, DMSO) δ 165.17, 158.46, 155.92, 154.12, 143.93,
142.72, 137.44, 135.00, 129.39, 129.07, 128.19, 127.07, 126.20,
124.87, 123.64, 115.75, 115.60, 114.05, 68.61, 58.55, 52.61, 46.52,
45.14, 40.61, 40.40, 40.19, 39.98, 39.77, 39.56, 39.36, 30.53, 11.76,
10.90. MS (ESI, m/z): 642.1[M+H]+.
J = 1.6 Hz, 3H), 1.82 (dd, J = 12.8, 5.2 Hz, 2H), 1.78–1.65 (m, 2H). 13
C
NMR (75 MHz, DMSO) δ 164.72, 158.29, 153.55, 151.75, 145.53,
142.25, 129.19, 128.85, 128.54, 128.18, 127.66, 126.59, 125.69,
113.24, 86.33, 60.76, 40.37, 40.10, 39.82, 39.54, 39.26, 38.98, 38.71,
34.75, 34.68, 30.07, 29.99, 29.58, 28.13, 26.12, 11.27, 11.05, 10.40,
10.23. MS (ESI, m/z): 468.3[M+H]+.
3-(4-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)
butyl)-6-(3,5-dimethylisoxa zol-4-yl)-1-methyl-4-phenyl-3,4-dihydroquinazol
in-2(1H)-one (39). To a stirred solution of compound 38 (0.14 g, 0.3
mmol) and 37 (0.085 g, 0.36 mmol) in N, N-dimethylformamide (10 mL),
sodium hydroxide (13.1 mg, 0.33 mmol) in N, N-dimethylformamide (2
mL) wasslowlydroppedat 0◦C. Thereactionmixturewaswarmedtoroom
temperature and stirred for ten hours. After the reaction was completed,
water (20 mL) and ethyl acetate (20 mL) were added to the above reaction
solution and the organic phase was separated. The organic phase was
evaporated and purified by column chromatography (dichloromethane/
methanol = 80/1–20/1) to obtain compound 39 (0.12 g, 64% yield) as a
2-(6-(3,5-Dimethylisoxazol-4-yl)-1-methyl-2-oxo-4-phenyl-1,4-dihy-
droquinazolin-3(2H)-yl)acetic acid (44). Compound 13 (0.15 g, 0.45
mmol) was dissolved in N,N-dimethylformamide (10 mL) and stirred
under ice bath for ten minutes. Then, sodium hydroxide (21.6 mg, 0.54
mmol) dissolved in N, N-dimethylformamide (2 mL) was slowly added to
the above reaction solution and stirred under ice bath for ten minutes.
Then, ethyl bromide acetate (0.11 g, 0.67 mmol) was added to the above
mixture and the reaction was stirred at room temperature for ten hours.
After the reaction was completed, water (20 mL) and ethyl acetate (20
mL) were added to the above reaction solution. The organic phase was
separated and evaporated under reduced pressure distillation. Methanol
(4 mL) and water (2 mL) were added to the residue, and lithium hy-
droxide (21.5 mg, 0.9 mmol) was added and stirred for two hours at
room temperature. After the reaction was completed, the mixture was
concentrated and 1 M of dilute hydrochloric acid was added to adjust the
pH to pH < 5 and the reaction was filtered to give compound 44 (0.15 g,
85% yield) as a grayish-white solid.
gray-white solid in 98% purity. m.p.: 108–110 ◦C. IR (KBr, cmꢀ 1
) ν:
3452.44, 1641.00, 1553.03, 1477.53, 1405.66, 1268.61, 812.61, 736.48,
699.67, 599.29; 1H NMR (300 MHz, DMSO) δ 8.10 (d, J = 10.1 Hz, 1H),
7.45 (d, J = 10.3 Hz, 1H), 7.40–7.30 (m, 5H), 7.26 (d, J = 7.6 Hz, 2H), 7.04
(d, J = 8.3 Hz, 1H), 5.75 (d, J = 5.4 Hz, 1H), 3.88–3.76 (m, 1H), 3.53 (s,
4H), 3.30 (s, 3H), 2.86 (dd, J = 13.3, 6.7 Hz, 1H), 2.42 (s, 3H), 2.36 (s, 3H),
2.28 (s, 2H), 2.19 (s, 3H), 1.63–1.38 (m, 4H), 0.89–0.81 (m, 1H). 13C NMR
(75 MHz, DMSO) δ 164.75, 158.00, 155.51, 153.57, 143.58, 142.49,
137.07, 134.46, 128.82, 128.53, 127.61, 126.59, 125.71, 124.36, 123.06,
115.29, 113.43, 60.76, 57.24, 56.40, 51.98, 46.38, 45.07, 40.39, 40.13,
39.84, 39.57, 39.29, 39.02, 38.75, 29.97, 25.32, 11.26, 10.39. MS (ESI, m/
z): 626.3[M+H]+.
3-(2-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-
2-oxoethyl)-6-(3,5-dime thylisoxazol-4-yl)-1-methyl-4-phenyl-3,4-dihydroqui
nazolin-2(1H)-one (47). To a stirred solution of compound 44 (0.3 g,
0.77 mmol), EDCI (0.2 g, 1.07 mmol) and triethylamine (319 mmL, 2.30
mmol) in N, N-dimethylformamide (10 mL), HOBT (0.12 g, 0.92 mmol)
was added at 0 ◦C and the reaction mixture was stirred in an ice bath for
thirty minutes. Compound 37 (0.2 g, 0.84 mmol) was added to the above
mixture at 0 ◦C and the reaction was stirred at room temperature for ten
hours. After the reaction was completed, the reaction was quenched with
water (20 mL) and ethyl acetate (20 mL) and the organic phase was
collected. The organic phase was evaporated and purified by column
chromatography (dichloromethane/methanol = 60/1–20/1) to obtain
compound 47 (0.23 g, 49% yield) as a white solid in 98% purity. m.p.:
3-(5-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)
pentyl)-6-(3,5-dimethylisox azol-4-yl)-1-methyl-4-phenyl-3,4-dihydroquinazo
lin-2(1H)-one (40). The synthesis method was similar to that of compound
39, and compound 40 was obtained (0.083 g, 85% yield) as a light yellow
solidin97%purity. m.p.:110–112◦C. IR(KBr, cmꢀ 1
)ν:3448.78, 1648.38,
1552.74, 1477.15, 1406.65, 1266.38, 814.20, 736.52, 699.67, 570.38,
512.94; 1H NMR (300 MHz, DMSO) δ 8.08 (d, J = 10.1 Hz, 1H), 7.47–7.17
(m, 8H), 7.02 (d, J = 8.4 Hz, 1H), 5.73 (s, 1H), 3.81 (d, J = 13.1 Hz, 1H),
3.51(s,4H),3.29(s,3H),2.89–2.75(m,1H),2.50(s,2H),2.40(s,3H),2.34
(s, 3H), 2.17 (s, 3H), 1.37 (d, J = 22.8 Hz, 3H), 1.21 (s, 5H). 13C NMR (75
MHz, DMSO) δ 164.67, 158.05, 155.43, 153.67, 143.43, 142.42, 137.06,
128.82, 125.71, 124.35, 115.27, 113.43, 52.03, 45.10, 40.11, 39.84,
39.57, 39.28, 39.01, 29.96, 27.03, 25.72, 24.03, 11.25, 10.38. MS (ESI, m/
z): 640.3[M+H]+.
116–118 ◦C. IR (KBr, cmꢀ 1
) ν: 3453.55, 1647.89, 1553.16, 1520.96,
1477.42, 1407.53, 1237.36, 812.37, 736.54, 700.94, 572.56, 527.59; 1H
NMR (300 MHz, DMSO) δ 8.13 (d, J = 10.2 Hz, 1H), 7.45 (d, J = 10.2 Hz,
1H), 7.40–7.22 (m, 7H), 7.08 (d, J = 8.9 Hz, 1H), 5.68 (s, 1H), 3.62 (d, J =
16.4 Hz, 8H), 3.30 (s, 3H), 2.94 (s, 2H), 2.32 (s, 3H), 2.15 (s, 3H). 13C NMR
3-(6-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)
9

L. Fang et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116133
(75 MHz, DMSO) δ 166.59, 164.72, 158.03, 155.34, 153.33, 143.47,
142.03, 136.98, 134.50, 128.85, 128.56, 127.79, 126.81, 126.21, 124.52,
124.13, 123.21, 115.42, 115.18, 113.58, 61.80, 47.38, 44.90, 44.83,
43.21, 40.75, 40.36, 40.08, 39.80, 39.52, 39.25, 38.97, 38.69, 30.10,
11.23, 10.36. MS (ESI, m/z): 612.1[M+H]+.
4.2.3. Cell cycle analysis
U266 cells (1 × 10 5/2 mL per well) were seeded in six-well plates
and treated with compound 39 at 1 and 2 μM, respectively. After 48 h,
the cells were harvested and fixed in 70% ethanol for 30 min on ice.
After washing with phosphate buffer saline (PBS), the cells were labeled
with propidium iodide (PI, 0.05 mg/mL) in the presence of RNaseA (0.5
mg/mL) and incubated at room temperature in the dark for 30 min. DNA
contents were analyzed using the flow cytometer (MACSQuant® X,
Germany).
3-(6-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-
6-oxohexyl)-6-(3,5-dimeth ylisoxazol-4-yl)-1-methyl-4-phenyl-3,4-dihydroq
uinazolin-2(1H)-one (48). The synthesis procedure was similar to the
method of compound 47 and compound 48 (0.091 g, 88% yield) was given
as a pale yellow solid in 97% purity. m.p.: 102–104 ◦C. IR (KBr, cmꢀ 1
) ν:
3455.08, 2926.62, 1640.23, 1552.73, 1520.13, 1476.33, 1407.77,
1338.47, 1272.09, 1239.31, 1201.10, 1105.03, 817.45, 736.68; 1H NMR
(300MHz, DMSO)δ 8.14(d, J= 9.7Hz, 1H), 7.46(d,J= 10.7Hz,1H), 7.30
(d, J= 23.3Hz,7H),7.02(d,J= 8.3Hz, 1H),5.74(s,1H),3.79(s,1H),3.56
(s, 8H), 3.28 (s, 3H), 2.81 (s, 1H), 2.35 (s, 3H), 2.18 (s, 3H), 1.48 (s, 4H),
1.24 (s, 3H), 0.83 (s, 2H). 13C NMR (75 MHz, DMSO) δ 170.79, 158.05,
155.43, 153.55, 142.45, 137.02, 128.84, 128.50, 127.63, 126.59, 125.69,
124.52, 124.36, 123.01, 115.41, 113.42, 60.69, 46.27, 45.14, 44.91,
43.98, 40.36, 40.28, 40.10, 39.82, 39.54, 39.26, 38.99, 32.01, 29.95,
27.23, 25.93, 24.37, 11.27, 10.41. MS (ESI, m/z): 668.3[M+H]+.
4.2.4. DAPI staining assay
U266 cells were cultured in 12-well culture plates (20000 cells/well)
overnight before treating with compound 39 at 1, 2 and 8 μM for 48 h,
and then washed with PBS. After all, the cell incubated with DAPI for 5
min and washed with PBS. Cells were observed and photographed by a
fluorescent microscope (Olympus IX53).
4.2.5. Annexin V-FITC apoptosis detection assay
After staining with annexin V-FITC and PI using the Annexin V-FITC
apoptosis detection kit (KeyGEN BioTECH, China), cells were detected
by flow cytometry to assess the membrane and nuclear events during
apoptosis. U266 cells (1 × 10 5/2 mL per well) were seeded in six-well
3-(8-(4-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-
8-oxooctyl)-6-(3,5-dimeth ylisoxazol-4-yl)-1-methyl-4-phenyl-3,4-dihydroqui
nazolin-2(1H)-one (49). Compound 49 was synthesized using the same
methods as described for compound 47 and compound 49 (0.087 g, 83%
yield) was obtained as a pale yellow solid in 98% purity. m.p.: 118–120 ◦C.
plates and treated with compound 39 at 1, 2 and 8 μM, respectively.
After 48 h, cells were collected, washed twice with cold PBS, and
resuspended in 500 L of a binding buffer which was then added to 5 μL
μ
IR (KBr, cmꢀ 1
)
ν
: 3460.99, 2927.88, 2854.78, 1642.31, 1553.15, 1520.82,
of annexin V-FITC and incubated at 37 ◦C in the dark for 15 min. Sub-
1476.74, 1407.57, 1338.89, 1271.97, 1239.22, 1104.91, 736.67, 700.29;
1HNMR(300MHz,DMSO)δ 8.16(d,J= 10.2Hz,1H),7.48(d,J= 10.2Hz,
1H), 7.41–7.31 (m, 5H), 7.30–7.23 (m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 5.73
(s, 1H), 3.79(s, 1H), 3.58(s, 8H), 3.28(s, 3H), 2.82(d, J= 5.9Hz, 1H), 2.36
(s, 3H), 2.31 (d, J = 7.8 Hz, 2H), 2.19 (s, 3H), 1.46 (s, 2H), 1.23 (d, J = 2.4
Hz, 8H). 13C NMR (75 MHz, DMSO) δ 170.87, 164.69, 158.01, 155.38,
153.57, 142.45, 137.04, 128.81, 128.50, 127.60, 126.57, 125.68, 124.50,
124.37, 123.01, 115.41, 113.41, 60.74, 46.47, 45.16, 44.96, 43.98, 40.36,
40.09, 39.81, 39.53, 39.25, 38.98, 38.70, 32.15, 29.94, 28.59, 28.48,
27.30, 26.11, 24.54, 11.26, 10.39. MS (ESI, m/z): 696.3[M+H]+.
sequently, the buffer was added to 5
μ
L of PI and incubated at 37 ◦C in
the dark for 5 min. The samples were analyzed by a FACScan flow cy-
tometer (MACSQuant® X, Germany).
4.2.6. Docking study
Molecular docking experiments were used to mimic the way the
compounds interact with the BRD4 protein. The structure of the com-
pound was first sketched into Chemdraw and saved in sdf file format.
The above small molecules were minimized using the MMFF force field
of the Ligand prepare module in the Schrodinger software. Amino acid
modifications and force fields were applied for BRD4 protein using the
Protein prepare module. Glide files for the BRD4(BD1) protein were
generated using the Glide generation module. Finally, small molecule
compounds were docked with the BRD4 protein to produce an optimal
conformation.
4.2. Biological methods
4.2.1. BRD4 protein inhibitory activities
A single or gradient concentration of the active compound to be
tested was performed in a 384-well plate. The concentration of DMSO in
the final solution was ≤0.1%. Then BRD4(BD1) protein solution was
Declaration of Competing Interest
prepared with the 1X buffer. 5
wise to each well, and 5
the blank control. After leaving the plate at room temperature for 15
min, 5 L of substrate solution was added to each well sequentially and
incubated for another 1 h. In the dark environment, 15 L of acceptor
μL of protein solution was added drop-
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
μ
L of buffer was added to the control group as
μ
μ
Acknowledgements
and donor solution were added to each well and incubated for another
hour at room temperature.23 Finally, the absorbance value was
measured using an EnSpire full-wavelength microplate reader, and the
inhibitory activities of compounds were calculated by the formula Inh %
= (maximum signal value-compound signal value)/(maximum signal
value-minimum signal value) × 100.
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) and China Scholarship Council (201907060014).
Appendix A. Supplementary material
4.2.2. Anti-proliferation activities in cancer cell lines
Cryopreserved cells were quickly thawed in a water bath pan (37 ◦C)
and placed in an incubator. After one day, cells were plated at
5000–10,000 cells per well of 96-well plates once sufficient cells were
generated. After plating, shake it slowly and incubated overnight in the
incubator. After cell attachment, a single concentration of compounds of
different gradient concentrations were added to the 96-well plate
described above and incubated in the incubator for another 48 h. MTT
solution was added to each well of the 96-well plate described above and
the absorbance was determined using an enzyme-labeled analyzer.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116133.
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