Design, synthesis and biological evaluation of novel 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as potential BRD4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.05.006

Recently, diverse kinase inhibitors were reported having interaction with BRD4. It provided a strategy for developing a new structural framework for the next-generation BRD4-selective inhibitors. Starting from PLK1 kinase inhibitor BI-2536, we designed 18 compounds by modifying dihydropteridine core. Compound 23 showed potent BRD4 inhibitory activities with IC₅₀ of 79 nM and no inhibitory activities for PLK1. Cell antiproliferation assay was performed and potent inhibitory activity against MV4;11 with IC₅₀ of 1.53 μM. Cell apoptosis and western blotting indicated compound 23 induced apoptosis by down-regulating c-Myc. These novel selective BRD4 inhibitors provided new lead compounds for further drug development.

Full text of DOI:10.1016/j.bmc.2019.05.006

Accepted Manuscript
Design, synthesis and biological evaluation of novel 4,5-dihydro-[1,2,4]triazo-
lo[4,3-f]pteridine derivatives as potential BRD4 inhibitors
Xinzhou Bi, Jieming Li, Jiuhui Li, Wei Shi, Yuxuan Dai, Qifei Li, Wenjie
Zhang, Wenlong Huang, Hai Qian, Cheng Jiang
PII:
S0968-0896(19)30580-2
https://doi.org/10.1016/j.bmc.2019.05.006
BMC 14893
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
10 April 2019
2 May 2019
4 May 2019
Please cite this article as: Bi, X., Li, J., Li, J., Shi, W., Dai, Y., Li, Q., Zhang, W., Huang, W., Qian, H., Jiang, C.,
Design, synthesis and biological evaluation of novel 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as
potential BRD4 inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2
019.05.006
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Design, synthesis and biological evaluation of novel 4,5-
dihydro-[1,2,4]triazolo[4,3-f]pteridine
potential BRD4 inhibitors
derivatives
as
1
1
1
1
1
1
1
Xinzhou Bi , Jieming Li , Jiuhui Li , Wei Shi , Yuxuan Dai , Qifei Li , Wenjie Zhang ,
Wenlong Huang , Hai Qian and Cheng Jiang3,4
1
,2
1,2
1
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
2
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
3
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
4
Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University,
2
4 Tongjiaxiang, Nanjing 210009, PR China
Abstract
Recently, diverse kinase inhibitors were reported having interaction with BRD4. It provided a
strategy for developing a new structural framework for the next-generation BRD4-selective
inhibitors. Starting from PLK1 kinase inhibitor BI-2536, we designed 18 compounds by modifying
dihydropteridine core. Compound 23 showed potent BRD4 inhibitory activities with IC of 79 nM
5
0
and no inhibitory activities for PLK1. Cell antiproliferation assay was performed and potent
inhibitory activity against MV4;11 with IC50 of 1.53 μM. Cell apoptosis and western blotting
indicated compound 23 induced apoptosis by down-regulating c-myc. These novel selective BRD4
inhibitors provided new lead compounds for further drug development.
Keywords
BRD4 inhibitors; anti-tumor; BI-2536; c-myc;

Correspondence to: Hai Qian, Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. E-mail: qianhai24@163.com

Correspondence to: Cheng Jiang, Jiangsu Key Laboratory of Drug Design and Optimization, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. E-mail: jc@cpu.edu.cn

1
. Introduction
BRD4 that contains two tandem bromodomains (BD1) and (BD2) is one member of the
bromodomain and extra-terminal domain (BET) protein family, which regulating gene transcription
by interacting with acetylated lysine residues(KAc) of histones and transcription factors through
[1]
bromodomains. It plays an essential role as an epigenetic “reader” while aberrant activity of BRD4
may have emerged for various diseases including cancers, inflammation, diabetes, acute heart
failure and viral infectious diseases.[2] Hence, BRD4 has been an alternative therapeutic target for
cancer drug research and its generally good druggability is suitable for small molecule inhibitor
development.[3]
The development of first small molecule BRD4 inhibitor (+)-JQ-1(1) was reported by the Dana
Farber Cancer Institute.[4, 5] It achieved effective inhibition through the way that incorporating the
thieno[3,2-f] [1,2,4] triazolo[4,3-a] [1,4] diazepine scaffold (thienotriazolodiazepine) to occupy the
acetylated lysine (KAc) binding site (Figure.1). For this scaffold, the methyltriazole moiety mimics
the acetyl-lysine group of histones. Recently, several kinds of specific small molecular BRD4
inhibitors have been recently developed to interact with acetylated lysine binding site.[2, 6-15] Clinical
candidates OTX015(2) and I-BET762(3) have been developed with scaffold
methyltriazolodiazepines. Meanwhile, multifarious 3,5-dimethylisoxazoles have also been
investigated, as an example of I-BET151(4). PDB data shows that all these core structures form an
essential hydrogen bond with residue ASN140 in BRD4(PDB ID: 4C66 and 3ZYU). Interestingly,
PLK1 kinase inhibitor BI-2536(5) has been reported having an interaction with BRD4 residue
ASN140.[16] This indicate a new strategy to develop new selective BRD4 inhibitors.
Recently, our group developed a series of new scaffold compounds based on the structure of
[5]
BI-2536. Because of the different binding mode of BI-2536 with BRD4 and PLK1 , we modified
triazole to dihydropteridine core to keep interacting with residue ASN140 and TYR97 based on
structure−activity relationships of BI-2536.[17] Due to the modification, the pyrimidine ring may not
be a hydrogen acceptor for binding PLK1. These compounds were designed as selective BRD4
inhibitors. However, these compounds did keep binding activities with BRD4 but no better than BI-
2
536. It provided a strategy for developing a new structural framework for the next-generation
BRD4-selective inhibitors. Further studies should be taken to develop them.

N
N
N
N O
N
N O
N
N
N
S
O
S
NH
N
N
NH
O
N
O
OH
Cl
+)JQ-1,1
Cl
OTX015,2
Cl
(
I-BET762,3
N O
N
O
O
N
N
O
N
N
H
N
N
N
H
O
N
N
HN
O
I-BET151,4
PLK1,BI-2536,5
Figure.1 (+)-JQ-1 and BI-2536 Inhibitors can both form hydrogen bonds with residue ASN140 of BRD4 in KAc
recognition region and form hydrogen bonds with residue Tyr97 through water molecules.
2
. Results and Discussion
2
.1 Synthesis
Synthesis of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives were outlined in Scheme
1
. The target compounds 7-24 were synthesized by 7 steps according to the literature method.
Mannich reaction was used for cyclopentane and glycine methyl ester hydrochloride to afford 1.
Then aromatic nucleophilic substitutions 1 to pyrimidine afforded 2. Fe powder and glacial acetic
acid were used for reduction of nitro group and then for intramolecular nucleophilic substitution

reaction to obtain 3. Then treatment of 3 with Lawesson reagent afforded 4. Materials hydrazine
hydrate and triethyl orthoacetate were obtained to make 6. 6 with substituted aniline via coupling
reaction produced compounds 7-24.
Scheme 1. Synthesis of 5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine
derivatives.
O
O
H
N
O
(i)
O
H
N
+
ClH3N
O
O
NO2
N
N
N
1
(ii)
(iii)
O
Cl
N
Cl
N
N
NO2
Cl
O
N
Cl
N
2
3
N
N
NH2
N
N
N
H
H
N
N
S
N
N
N
N
N
N
N
N
N
(vii)
(iv)
(v)
(vi)
Cl
N
N
Cl
N
N
N
N
Cl
R
H
4
5
6
7-24
Reagents and conditions: (i)sodium triacetoxyborohydride, potassium acetate, ClCH CH Cl, r.t.,
2
2
1
6h; (ii)K CO , acetone, r.t., 12h; (iii)Fe, acetic acid, 80℃, 12h; (iv)Lawesson reagent,
2 3
tetrahydrofuran, 70℃, 6h; (v)70%hydrazine hydrate, tetrahydrofuran, r.t., 10min; (vi)triethyl
orthoacetate, tetrahydrofuran, ethanol, 80℃, 2h; (vii)Cs CO , Pd (dba) , Xantphos, dioxane, 110℃,
2
3
2
3
overnight;
2
.2 BRD4 inhibitory activity assay
To explore inhibition activities of designed compounds to BRD4, these compounds were
evaluated using a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay.
+)-JQ-1 and BI-2536 were used as positive control. We measured the BRD4 inhibition rate of
eighteen compounds. As indicated in Table. 1, all compounds were measured the IC for BRD4
(
5
0
bromodomain 1 except the compound 17. Unfortunately, none of them inhibited BRD4 at sub
micromolar concentration except compound 23 and 24. QSAR research indicated that the group did
have significant connection with activities. The compounds with methoxy group had a relative better
activity compared with other substituents. The compounds with alkyl group all had a low inhibition
rate. This may have relationship with its hydrophobicity. The compounds with tert-butyl group did
not show IC due to its large space. Halogen-substituted compounds all had worse activities and
5
0
were no better than alkyl group. Actually, compounds with electron-rich groups had better activity.
The oxygen or nitrogen atom could have better binding with water molecules around the
Bromodomain protein following the principle of similarity and compatibility (such as 23 and 24).
Amino group and sulfonyl group may expose to solvents water. In general, compounds have groups
of ortho position have better inhibitory activity than groups of meta position.
Table. 1 Structure and inhibitory activity of compounds to BRD4 bromodomain 1 a

N
N
N
N
N
R
N
BRD4
BRD4 BD1
Inhibitory
Activity (IC50,
μM)
BD1Inhibitory
Activity (IC ,
No.
R
No.
R
5
0
μM)
7
8
0.22 ± 0.018
0.16 ± 0.024
16
17
1.43 ± 0.072
>30
N
H
N
H
N
H
O
N
H
F
9
0.18 ± 0.012
18
1.28 ± 0.026
O
O
N
H
N
H
Cl
1
1
1
0
1
2
0.16 ± 0.051
0.22 ± 0.033
0.41 ± 0.043
19
20
21
1.54 ± 0.043
1.43 ± 0.067
0.54 ± 0.091
N
H
N
H
O
O
N
H
Cl
N
H
O2N
N
H
N
H
1
1
1
3
4
5
1.56 ± 0.082
0.38 ± 0.034
22
23
24
N
0.18 ± 0.031
0.079 ± 0.057
N
H
H
NO2
H2N
N
H
N
H
O
S
O
0.86 ± 0.031
0.051 ± 0.018
0.089 ± 0.042
0.066 ± 0.023
N
N
H
H
BI-2536
(+)-JQ-1
a
Each value was reproduced in three experiments.
2
.3 Cell growth inhibition assay
We tested these compounds about their anti-proliferative effects against MV4;11 (biphenotypic

B myelomonocytic leukemia) cell line and HL-60 (acute promyelocytic leukemia) cell line. Clinical
drugs (+)-JQ-1, BI-2536 were used as positive controls. The results of cell growth inhibition assay
induced by synthesized compounds were summarized in Table. 2. Several compounds showed
potency effect against MV4;11 cell lines. Compound 23 and 24 had better effects against MV4;11
than other synthetized compounds while their IC were 1.53μM and 1.92μM. Unfortunately, they
5
0
did not tend good effects like positive controls. All compounds did not have good effects on HL-60
cell line. These compounds tended to be sensitive to MV4;11.
Table. 2 Compounds antiproliferative activity against MV4;11 and HL-60 a
MV4;11
HL-60
antiproliferative
activity (IC50,
μM)
MV4;11
antiproliferative
activity (IC50,
μM)
HL-60
antiproliferative
activity (IC50,
μM)
antiproliferative
activity (IC ,
No.
No.
5
0
μM)
7
8
9
5.53 ± 0.023
4.17 ± 0.046
6.74 ± 0.028
4.84 ± 0.018
6.79 ± 0.014
8.46 ± 0.091
>30
21.45 ± 0.032
19.23 ± 0.046
25.46 ± 0.072
18.72 ± 0.011
26.71 ± 0.041
>30
17
18
19
20
21
22
23
24
>30
>30
>30
>30
>30
>30
1
1
1
1
1
0
1
2
3
4
>30
>30
21.45 ± 0.053
10.69 ± 0.016
1.53 ± 0.043
1.92 ± 0.027
>30
>30
>30
7.16 ± 0.046
7.85 ± 0.038
7.34 ± 0.043
28.97 ± 0.035
(
+)-
1
1
5
6
16.21 ± 0.017
>30
>30
>30
0.57 ± 0.012
0.45 ± 0.008
2.91 ± 0.014
3.42 ± 0.017
JQ-1
BI-
2
536
a
Each value was reproduced in three experiments.
2
.4 Intra-cellular metabolism research
We employed flow cytometry analysis to investigate the ability of compound 23 to induce cell
cycle arrest and apoptosis in the MV4;11 cell lines. As showed in Figure.2(A), compound 23 and
+)-JQ-1 had the effect in inducing apoptosis with the increasing concentration. Compound 23 did
(
not have better effect than (+)-JQ-1. In cell cycle analysis assay, both compound 23 and (+)-JQ-1
induced cell cycle arrest in first gap in dose dependent.
It is reported by Jake E.Delmore that BET bromodomain proteins worked as regulatory factors
for c-Myc.[18] The BRD4 inhibitor (+)-JQ1 induces down-regulation of c-Myc transcription to make
an antiproliferative effect.[4] To further assess our BRD4 selective inhibitors, we performed the
western blotting experiment to study the cellular effect related to c-Myc. We chose compound 23
and the positive controls (+)-JQ-1 to evaluate their effects on levels of c-Myc. As showed in Figure.
2
(C), compound 23 showed significant inhibitory activity for c-Myc protein expression.

Figure. 2 (A) (B) Induction of cell cycle arrest and apoptosis by (+)-JQ-1 and compound 23 in the MV4;11 cell
lines by flow cytometry analysis; (C) Western blotting analysis of c-myc in MV4;11. (+)-JQ-1 and compound 23
were treated with cells. Proteins were probed by specific antibodies;
2
.5 molecular docking study
We use the Schrödinger to analyze the possible binding modes of preferred compounds to
BRD4 BD1. Compound 23 was selected for docking evaluation as representative example and the
predicted binding modes were shown in Figure. 3. As we expected, Compound 23 binds in well-
defined pocket and forms interactions with ASN140 and a second interaction with TYR97, via a
structured water molecule. Meanwhile, it has a second interaction with GLN85, which is the same
as BI-2536 binding modes. It was shown that compound 23 was approach to the ZA channel. It
seems to make sense that compounds with amino group or other hydrophilic groups have better
activity binding with BRD4 because of its hydrophilic property to ZA channel and solvents.

Figure. 3 Docking study for compound 23 with BRD4 BD1 (A) Compound 23 formed hydrogen bonds with residue
ASN140 of BRD4 in KAc recognition region and form hydrogen bonds with residue Tyr97 through water molecules.
(B) Compound 23 had binding site with BRD4.
2
.6 PLK1 Kinase activity assay
At the first beginning, we wanted to develop a selective BRD4 inhibitor through the research
of PLK1-BRD4 dual inhibitor BI-2536. Therefore, we chose preferred compounds 23 and 24 to
profile their PLK1 kinase activities, along with positive control compounds BI-2536. From Table.
3
, as expected, BI-2536 showed a significant inhibitory effect on PLK1 kinase at 1ꢀμM. While
compounds 23 and 24 did not have inhibitory activity on PLK1 kinase even at 30ꢀμM. It was shown
in Figure. 4 that BI-2536 and compound 23 have different binding sites in PLK-1 in molecule
docking. Thus, it’s successful to develop a selective BRD4 inhibitor by modifying triazole to
dihydropteridine core.
Table. 3 PLK1 kinase activity of compounds 23 and 24
No.
PLK1 Inhibition (%, 30μM) PLK1 Inhibition (%, 1μM)
BI-2536
98
5
98
6
2
2
3
4
7
9
Figure. 4 molecule docking study for compound 23 (red) in PLK-1 (PDB ID: 2RKU)
with BI-2536 (green)
3
. Conclusion
In conclusion, we chose the PLK1 kinase inhibitor BI-2536 as the lead compound to explore
the dihydropteridine core structure for binding BRD4. 1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-

f]pteridine structures were offered as novel selective BRD4 inhibitors. BRD4 binding assay showed
that they have inhibitory activities in micromolar concentration. Especially the compound 23 had
antiproliferative activity against MV4;11, which achieved the activity as the same as (+)-JQ-1 but
no better than (+)-JQ-1. Meanwhile, they do not have good effect on HL-60. Synthesized
compounds may be sensitive to MV4;11 only. Apoptosis analysis and western blotting assay
indicated that compound 23 induced cell antiproliferation by down-regulating c-myc, which played
a key role in upregulation of a transcriptional program influencing cell division.[18] However,
compound 23 was given weaker in-vitro potency than (+)-JQ-1, but improved activity in regards to
c-myc expression with (+)-JQ-1. It could be off-targets to other kinases. Anyway, ethyl group of
dihydropteridin most critically enhance BRD4 inhibitory activity[17] and we should do further QSAR
research. Thus, compound 23 could be a potent BRD4 inhibitor and could be utilized as lead
compounds for further development.
4
. Experimental and Section
4
.1 General chemistry experimental details
All materials, reagents and solvents were acquired from commercial sources. They were used
1
13
without further purification unless otherwise indicated. H NMR spectrum and C NMR spectrum
d
were recorded on Bruker AV300 (300MHz). Chemical shifts are expressed as H part per million
(ppm) relative to tetramethyl silane (TMS) as internal standard. Coupling constants (J values) were
carried out in hertz (Hz). Liquid chromatography-mass spectrometer spectra (LC-MS) were
operated on Waters ACQUITY UPLC-TQD in ESI mode. Purifications by column chromatography
were carried out by silica gel (200-300 mesh) and monitored by thin layer chromatography
performed on GF/UV 254 plates and were visualized using UV light at 254 and 365 nm. The purity
of compounds was assessed by HPLC (>95%).
4
.1.1 General procedure for the synthesis of 7-24
To a suspension of glycine methyl ester hydrochloride (5.0 g, 39.8 mmol) in dry
dichloromethane (150 mL) was added potassium acetate (3.9 g, 39.8 mmol) and the reaction mixture
stirred for ten minutes. The reaction mixture was then cooled to 0℃, cyclopentanone (3.5 mL, 39.8
mmol) was added, followed by sodium triacetoxyborohydride (12.7 g, 59.7 mmol) in portions over
a period of ten minutes. Reaction mixture was stirred at room temperature for 16 h after which
saturated sodium bicarbonate solution (60 mL) was added and stirred for another 30 minutes. The
reaction mixture was further basified with aqueous sodium carbonate to pH=~10. The organic layer
was separated and the aqueous layer extracted with dichloromethane (3* 75 mL). The organic layers
were combined, washed with brine, dried over anhydrous sodium sulfate, and evaporated to give
titled compound (yield: 80%).
To a solution of 2, 4-dichloro-5-nitropyrimidine (10.5 g, 54.8 mmol) in acetone (200 mL) was

added methyl N-cyclopentyl-glycine (8.53 g, 54.8 mmol) at -10℃. To the resulting solution was
then added potassium carbonate (9.00 g, 65.1mmol) while maintaining the temperature below 0℃.
Reaction mixture was then warmed to rt and stirred at rt for 16 h. Reaction mixture was filtrated
with diatomite. The organic layer was evaporated. The residue was purified by silica gel flash
chromatography using ethyl acetate-hexane mixture (v: v = 5: 95) as eluent to give target compound
(yield, 60%).
To a solution of methyl N-(2-chloro-5-nitropyrimidin-4-yl)-N-cyclopentyl- glycine (10.25 g,
2.6 mmol) in 100 mL of acetic acid glacial was added iron powder (2.19 g, 39.2 mmol). The
3
reaction mixture was heated at reflux until the completion of the reaction as monitored by TLC. The
reaction was filtered. The filtrate was evaporated to give a residue which was triturated using
isopropyl ether to give target compound (yield: 32%).
To a solution of 2-chloro-8-cyclopentyl-7,8-dihydropteridin-6(5H)-one (320 mg, 1.27 mmol)
in tetrahydrofuran (20.0 ml) was added Lawesson's Reagent (308mg, 0.76mmol) and then stirred at
1
10°C for 3 hours. The reaction mixture was concentrated under reduced pressure. The residue was
purified by silica gel flash chromatography using ethyl acetate-hexane mixture (v: v = 1: 5) as eluent
to give target compound (yield, 88%).
To a solution of 2-chloro-8-cyclopentyl-7,8-dihydropteridine-6(5H)-thione (300mg, 1.12mmol)
in tetrahydrofuran (20.0 ml) was add 80% hydrazine (1.3ml, 11.2mmol) dropwise and then stirred
1
0min. The reaction mixture was concentrated under reduced pressure. The residue redissoluted in
dichloromethane and washed with aqueous sodium hydrogen carbonate, dried over magnesium
sulphate and concentrated.
The residue of crude hydrazide was dissolved in mixture solvent with trimethyl orthoformate
and ethanol (20.0 ml, v: v = 1: 1) and stirred at 80°C for 90 min. The mixture was concentrated
under reduced pressure and purified by flash chromatography on silica gel eluting with methanol-
dichloromethane mixture (v: v = 20: 1) to give 7-chloro-5-cyclopentyl-1-methyl-4,5-dihydro-
[1,2,4]triazolo[4,3-f]pteridine (205 mg, 63 %) as a pale solid.
To a solution of compound 6 (50 mg, 0.17 mmol), aniline (31 μL, 0.34 mmol), Cs CO (83
2
3
mg, 0.26 mmol) in dioxane (10 mL) at room temperature were added Pd (dba) (16 mg, 0.017 mmol)
2
3
and XantPhos (20 mg, 0.034 mmol). The mixture was heated to 110 °C overnight. Then the reaction
mixture was diluted with water, and extracted with dichloromethane (3 × 10 mL). Combined organic
layers were washed with brine, then dried with Na SO . The crude product was purified by flash
2
4
chromatography on silica gel with methanol-dichloromethane mixture to give compound 7 (50 mg,
.14 mmol, 42% yield).
0

Following the similar procedures as for compound 7 gave compound 8-24.
4
.1.1.1 5-cyclopentyl-1-methyl-N-phenyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-amine (7)
1
White solid; yield 43.4%; H NMR (300 MHz, DMSO-d ) δ: 8.29 (s, 1H), 7.60 (d, J = 8 Hz, 2H),
6
7
3
1
6
.35 (t, J = 8 Hz, 2H), 7.20 (s, 1H), 7.07 (t, J = 7 Hz, 1H), 5.23-5.12 (m, 1H), 4.70 (s, 2H), 2.77 (s,
1
3
H), 2.02-1.94 (m, 2H), 1.88-1.80 (m, 2H), 1.76-1.66 (m, 4H); C NMR (75 MHz, DMSO-d ) δ:
6
62.16, 160.56, 160.56, 150.32, 149.34, 138.92, 129.03, 129.03, 122.96, 117.02, 116.63, 90.43,
+
6.13, 56.34, 32.28, 32.28, 25.08, 25.08, 12.62; LC-MS (ESI) m/z: 348.4 [M+H] ; Elemental
Analysis calculated for C H N : C, 65.69; H, 6.09; N, 28.22; Found: C, 65.72; H, 6.11; N, 28.17;
1
9
21
7
4
.1.1.2 5-cyclopentyl-N-(2-methoxyphenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (8)
White solid; yield 35.9%; H NMR (300 MHz, DMSO-d ) δ: 9.52 (s, 1H), 8.43 (s, 1H), 7.48 (s, 1H),
1
6
7
3
1
5
.29-7.26 (m, 1H), 7.21-7.17 (m, 1H), 6.58-6.55 (m, 1H), 5.21-5.17 (m, 1H), 4.66 (s, 2H), 3.73 (s,
1
3
H), 2.68 (s, 3H), 1.87-1.68 (m, 6H), 1.65-1.51 (m, 2H); C NMR (75 MHz, DMSO-d ) δ: 162.71,
6
60.77, 160.77, 150.42, 149.53, 147.04, 132.77, 122.31, 121.36, 113.02, 113.02, 90.43, 66.03, 56.61,
+
5.81, 32.93, 32.93, 24.14, 24.14, 12.22; LC-MS (ESI) m/z: 378.4 [M+H] ; Elemental Analysis
calculated for C H N O: C, 63.64; H, 6.14; N, 25.98; Found: C, 63.58; H, 6.21; N, 25.92;
2
0
23
7
4
.1.1.3 5-cyclopentyl-N-(3-methoxyphenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (9)
White solid; yield 45.9%; H NMR (300 MHz, DMSO-d ) δ: 9.39 (s, 1H), 8.40 (s, 1H), 7.53 (s, 1H),
1
6
7
3
1
5
.28-7.25 (m, 1H), 7.18-7.13 (m, 1H), 6.53-6.50 (m, 1H), 5.17-5.11 (m, 1H), 4.69 (s, 2H), 3.74 (s,
1
3
H), 2.69 (s, 3H), 1.87-1.78 (m, 6H), 1.65-1.53 (m, 2H); C NMR (75 MHz, DMSO-d ) δ: 162.73,
6
60.76, 159.21, 150.42, 149.56, 147.08, 132.72, 122.31, 121.31, 113.02, 113.02, 90.43, 66.08, 56.62,
+
5.81, 32.98, 32.98, 24.13, 24.13, 12.27; LC-MS (ESI) m/z: 378.4 [M+H] ; Elemental Analysis
calculated for: C H N O; C, 63.64; H, 6.14; N, 25.98; Found: C, 63.58; H, 6.21; N, 25.92;
2
0
23
7
4
.1.1.4 5-cyclopentyl-N-(4-methoxyphenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (10)
White solid; yield 41.6%; H NMR (300 MHz, DMSO-d ) δ: 9.20 (s, 1H), 8.37 (s, 1H), 7.62 (d, J =
1
6
9
1
1
2
Hz, 2H), 6.86 (d, J = 9 Hz, 2H), 5.09-5.04 (m, 1H), 4.66 (s, 2H), 3.72 (s, 3H), 2.67 (s, 3H), 1.89-
1
3
.68 (m, 6H), 1.67-1.57 (m, 2H); C NMR (75 MHz, DMSO-d ) δ: 162.23, 160.46, 159.94, 153.34,
6
50.37, 149.32, 131.27, 122.31, 122.31, 113.02, 113.02, 90.43, 66.08, 56.62, 55.81, 32.98, 32.98,
+
4.13, 24.13, 12.27; LC-MS (ESI) m/z: 378.4 [M+H] ; Elemental Analysis calculated for:
C H N O; C, 63.64; H, 6.14; N, 25.98; Found: C, 63.58; H, 6.21; N, 25.92;
2
0
23
7
4
.1.1.5
5-cyclopentyl-N-(3,4-dimethoxyphenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-
f]pteridin-7-amine (11)

1
White solid; yield 40.9%; H NMR (300 MHz, CDCL3) δ: 8.26 (s, 1H), 7.28 (s, 1H), 7.09-7.00 (m,
2
H), 6.87-6.84 (m, 1H), 5.20 (s, 1H), 4.68 (s, 2H), 3.92-3.90 (m, 6H), 2.75 (s, 3H), 1.95 (m, 2H),
1
3
1
.82 (m, 2H), 1.68 (m, 2H); C NMR (75 MHz, CDCL3) δ: 162.24, 160.42, 159.90, 150.34, 149.39,
1
42.10, 131.36, 129.08, 126.54, 123.81, 123.81, 90.83, 66.07, 56.68, 32.92, 24.11, 24.11, 17.81,
+
1
2.27; LC-MS (ESI) m/z: 408.5 [M+H] ; Elemental Analysis calculated for: C H N O ; C, 61.90;
2
1
25
7
2
H, 6.18; N, 24.06; Found: C, 61.78; H, 6.25; N, 24.11;
4
.1.1.6 5-cyclopentyl-1-methyl-N-(o-tolyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-amine (12)
1
White solid; yield 41.6%; H NMR (300 MHz, DMSO-d ) δ: 8.28 (s, 1H), 7.51 (s, 1H), 7.31-7.28
6
(m, 1H), 7.27-7.23 (m, 1H), 7.20-7.16 (m, 1H), 7.01-6.96 (m, 1H), 5.28-5.16 (m, 1H), 4.70 (s, 2H),
1
3
2
.67 (s, 3H), 2.36 (s, 3H),2.05-1.98 (m, 2H), 1.89-1.84 (m, 2H), 1.80-1.74 (m, 4H); C NMR (75
MHz, DMSO-d ) δ: 162.27, 160.46, 159.94, 150.36, 150.36, 142.01, 131.33, 129.04, 126.57, 123.75,
6
1
23.75, 90.84, 66.01, 56.64, 32.96, 24.11, 24.11, 17.83, 12.24; LC-MS (ESI) m/z: 362.5 [M+H]+;
Elemental Analysis calculated for: C H N ; C, 66.46; H, 6.41; N, 27.13; Found: C, 66.52; H, 6.32;
2
0
23
7
N, 27.16;
4
.1.1.7 5-cyclopentyl-1-methyl-N-(m-tolyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-amine (13)
1
White solid; yield 45.3%; H NMR (300 MHz, CDCL3) δ: 8.29 (s, 1H), 7.52 (s, 1H), 7.35-7.33 (m,
1
H), 7.26-7.20 (m, 1H), 7.11 (s, 1H), 6.90 (d, J = 7 Hz, 1H), 5.26-5.15 (m, 1H), 4.70 (s, 2H), 2.76
1
3
(
s, 3H), 2.39 (s, 3H), 2.03-1.95 (m, 2H), 1.87-1.83 (m, 2H), 1.79-1.72 (m, 4H); C NMR (75 MHz,
CDCL3) δ: 162.24, 160.47, 159.98, 150.33, 149.37, 142.33, 139.25, 129.46, 121.04, 119.09, 114.82,
0.84, 66.07, 56.66, 32.93, 32.93, 24.14, 24.14, 21.36, 12.27; LC-MS (ESI) m/z: 362.5 [M+H]+;
Elemental Analysis calculated for: C H N ; C, 66.46; H, 6.41; N, 27.13; Found: C, 66.52; H, 6.32;
9
2
0
23
7
N, 27.16;
4
.1.1.8 5-cyclopentyl-1-methyl-N-(p-tolyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-amine (14)
1
White solid; yield 41.6%; H NMR (300 MHz, CDCL3) δ: 8.28 (s, 1H), 7.48 (d, J = 8 Hz, 2H), 7.17
(
d, J = 8 Hz, 2H), 7.00 (s, 1H), 5.23-5.12 (m, 1H), 4.69 (s, 2H), 2.76 (s, 3H), 2.35 (s, 3H), 2.01-1.94
m, 2H), 1.84-1.78 (m, 2H), 1.75-1.66 (m, 4H); 13C NMR (75 MHz, CDCL3) δ: 162.25, 160.47,
(
1
3
59.93, 150.34, 149.33, 135.97, 131.28, 129.82, 129.82, 120.34, 120.34, 90.87, 66.08, 56.69, 32.95,
+
2.95, 24.18, 24.18, 21.32, 12.21; LC-MS (ESI) m/z: 362.5 [M+H] ; Elemental Analysis calculated
for: C H N ; C, 66.46; H, 6.41; N, 27.13; Found: C, 66.52; H, 6.32; N, 27.16;
2
0
23
7
4
7
.1.1.9 5-cyclopentyl-N-(4-isopropylphenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-
-amine (15)
1
White solid; yield 40.8%; H NMR (300 MHz, DMSO-d ) δ: 9.27 (s, 1H), 8.37 (s, 1H), 7.64 (d, J =
6
8
1
1
Hz, 2H), 7.14 (d, J = 8 Hz, 2H), 5.12-5.04 (m, 1H), 4.66 (s, 2H), 2.93-2.78 (m, 1H), 2.67 (s, 3H),
.86-1.71 (m, 6H), 1.61-1.54 (m, 2H), 1.18 (d, J = 7 Hz, 6H); 13C NMR (75 MHz, DMSO-d ) δ:
6
62.27, 160.44, 159.92, 150.35, 149.35, 138.47, 136.11, 126.96, 126.96, 120.18, 120.18, 90.85,

6
6.04, 56.65, 33.26, 32.96, 32.96, 24.14, 24.14, 23.34, 23.34, 12.21; LC-MS (ESI) m/z: 390.5
+
[
M+H] ; Elemental Analysis calculated for: C H N ; C, 67.84; H, 6.99; N, 25.17; Found: C, 67.79;
2
2
27
7
H, 6.86; N, 25.35;
4
.1.1.10 N-(4-butylphenyl)-5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (16)
White solid; yield 39.4%; H NMR (300 MHz, DMSO-d ) δ: 9.29 (s, 1H), 8.38 (s, 1H), 7.62 (d, J =
1
6
8
1
Hz, 2H), 7.08 (d, J = 8 Hz, 2H), 5.12-5.07 (m, 1H), 4.67 (s, 2H), 2.67 (s, 3H), 1.86-1.72 (m, 6H),
1
3
.61-1.54 (m, 2H), 1.56-1.48 (m, 3H), 1.36-1.24 (m, 3H), 0.89 (t, J = 7 Hz, 3H); C NMR (75 MHz,
DMSO-d ) δ: 162.27, 160.48, 159.96, 150.35, 149.34, 137.54, 136.15, 128.96, 128.96, 120.24,
6
1
20.24, 90.83, 66.04, 56.67, 35.48, 33.44, 32.96, 32.94, 24.17, 24.17, 22.35, 14.11, 12.22; LC-MS
+
(
ESI) m/z: 404.5 [M+H] ; Elemental Analysis calculated for: C H N ; C, 68.46; H, 7.24; N, 24.30;
2
3
29
7
Found: C, 68.46; H, 7.24; N, 24.30;
4
.1.1.11
f]pteridin-7-amine (17)
White solid; yield 42.1%; H NMR (300 MHz, DMSO-d ) δ: 9.30 (s, 1H), 8.38 (s, 1H), 7.64 (d, J =
N-(4-(tert-butyl)phenyl)-5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-
1
6
9
3
1
3
Hz, 2H), 7.27 (d, J = 9 Hz, 2H), 7.27 (t, J = 8 Hz, 1H), 5.13-5.08 (m, 1H), 4.67 (s, 2H), 2.68 (s,
1
3
H), 1.87-1.81 (m, 6H), 1.78-1.76 (m, 2H), 1.27 (s, 9H); C NMR (75 MHz, DMSO-d ) δ: 162.27,
6
60.45, 159.93, 150.31, 149.35, 141.37, 135.88, 128.51, 128.53, 120.04, 120.02, 90.85, 66.04, 56.64,
4.25, 33.23, 32.94, 32.94, 31.34, 31.34, 31.34, 24.17, 24.17, 12.24; LC-MS (ESI) m/z: 404.5
+
[
M+H] ; Elemental Analysis calculated for: C H N ; C, 68.46; H, 7.24; N, 24.30; Found: C, 68.52;
2
3
29
7
H, 7.19; N, 24.29;
4
.1.1.12 5-cyclopentyl-N-(4-fluorophenyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (18)
White solid; yield 38.7%; H NMR (300 MHz, DMSO-d ) δ: 9.43 (s, 1H), 8.38 (s, 1H), 7.62 (d, J =
1
6
8
Hz, 2H), 7.44 (d, J = 8 Hz, 2H), 5.12-5.07 (m, 1H), 4.66 (s, 2H), 2.68 (s, 3H), 1.88-1.75 (m, 6H),
1
.71-1.62 (m, 2H); 13C NMR (75 MHz, DMSO-d ) δ: 162.24, 160.47, 159.93, 150.36, 149.32,
6
1
37.09, 129.87, 129.84, 127.77, 122.12, 122.12, 90.84, 66.07, 56.62, 32.91, 32.91, 24.14, 24.13,
+
1
2.22; LC-MS (ESI) m/z: 366.4 [M+H] ; Elemental Analysis calculated for: C H N F; C, 62.45;
1
9
20
7
H, 5.52; N, 26.83; Found: C, 62.39; H, 5.48; N, 26.77;
4
.1.1.13 N-(4-chlorophenyl)-5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (19)
White solid; yield 46.7%; H NMR (300 MHz, DMSO-d ) δ: 9.55 (s, 1H), 8.40 (s, 1H), 7.78 (d, J =
1
6
9
Hz, 2H), 7.30 (d, J = 9 Hz, 2H), 5.09 (m, 1H), 4.69 (s, 2H), 2.68 (s, 3H), 1.84-1.77 (m, 6H), 1.61
1
3
(
m, 2H); C NMR (75 MHz, DMSO-d ) δ: 162.21, 160.44, 159.93, 150.32, 149.34, 137.09, 129.84,
6
1
29.84, 127.72, 122.13, 122.13, 90.89, 66.04, 56.62, 32.94, 32.93, 24.17, 24.17, 12.22; LC-MS (ESI)

+
m/z: 382.8 [M+H] ; Elemental Analysis calculated for: C H N Cl; C, 59.76; H, 5.28; N, 25.68;
1
9
20
7
Found: C, 59.84; H, 5.19; N, 25.71;
4
.1.1.14 N-(3-chlorophenyl)-5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (20)
White solid; yield 45.8%; H NMR (300 MHz, DMSO-d ) δ: 9.66 (s, 1H), 8.43 (s, 1H), 8.17 (s, 1H),
1
6
7
2
1
3
.50 (d, J = 8 Hz, 1H), 7.27 (t, J = 8 Hz, 1H), 6.95 (d, J = 9 Hz, 1H), 5.13-5.08 (m, 1H), 4.71 (s,
1
3
H), 2.70 (s, 3H), 1.91-1.83 (m, 6H), 1.78-1.63 (m, 2H); C NMR (75 MHz, DMSO-d ) δ: 162.24,
6
60.46, 159.92, 150.34, 149.33, 143.87, 135.12, 130.94, 122.36, 116.72, 115.94, 90.87, 66.05, 56.67,
+
2.92, 32.92, 24.14, 24.14, 12.27; LC-MS (ESI) m/z: 382.8 [M+H] ; Elemental Analysis calculated
for: C H N Cl; C, 59.76; H, 5.28; N, 25.68; Found: C, 59.84; H, 5.19; N, 25.71;
1
9
20
7
4
.1.1.15 5-cyclopentyl-1-methyl-N-(4-nitrophenyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (21)
Yellow solid; yield 48.4%; H NMR (300 MHz, DMSO-d ) δ: 10.22 (s, 1H), 8.49 (s, 1H), 8.20 (d,
1
6
1
3
J = 9 Hz, 2H), 8.02 (d, J = 9 Hz, 2H), 5.18-5.12 (m, 1H), 2.71 (s, 3H), 1.87-1.64 (m, 8H); C NMR
75 MHz, DMSO-d ) δ: 162.25, 160.47, 159.99, 150.34, 149.36, 145.01, 137.97, 124.72, 124.72,
(
6
1
19.24, 119.24, 90.82, 66.04, 56.67, 32.92, 32.92, 24.11, 24.11, 12.23; LC-MS (ESI) m/z: 393.4
+
[
M+H] ; Elemental Analysis calculated for: C H N O ; C, 58.15; H, 5.14; N, 28.55; Found: C,
1
9
20
8
2
5
8.09; H, 5.08; N, 28.64;
4
.1.1.16 5-cyclopentyl-1-methyl-N-(2-nitrophenyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-
amine (22)
Yellow solid; yield 46.3%; H NMR (300 MHz, DMSO-d ) δ: 9.91 (s, 1H), 8.43 (s, 1H), 8.26-8.10
1
6
(m, 1H), 8.10-8.07 (m, 1H), 7.74-7.68 (m, 1H), 7.25-7.19 (m, 1H), 5.01-4.96 (m, 1H),4.71 (s, 2H),
2
1
3
.69 (s, 3H), 2.67 (s, 3H), 1.82-1.57 (m, 8H); 13C NMR (75 MHz, DMSO-d ) δ: 162.24, 160.47,
6
59.98, 150.36, 149.31, 144.65, 137.14, 129.07, 125.98, 119.66, 110.54, 90.86, 66.04, 56.64, 32.97,
+
2.97, 24.17, 24.17, 12.25; LC-MS (ESI) m/z: 393.4 [M+H] ; Elemental Analysis calculated for:
C H N O ; C, 58.15; H, 5.14; N, 28.55; Found: C, 58.09; H, 5.08; N, 28.64;
1
9
20
8
2
4
.1.1.17 N1-(5-cyclopentyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)benzene-1,4-
diamine (23)
White solid; yield 43.7%; H NMR (300 MHz, DMSO-d ) δ: 8.91 (s, 1H), 8.31 (s, 1H), 7.32 (d, J =
1
6
9
1
1
Hz, 2H), 6.51 (d, J = 9 Hz, 2H), 5.07-5.02 (m, 1H), 4.74 (s, 2H), 4.64 (s, 2H), 2.66 (s, 3H), 1.79-
1
3
.75 (m, 8H); C NMR (75 MHz, DMSO-d ) δ: 162.27, 160.45, 159.91, 150.33, 149.34, 138.45,
6
28.93, 118.31, 118.31, 117.12, 117.12, 90.84, 66.05, 56.67, 32.98, 32.98, 24.17, 24.17, 12.27; LC-
+
MS (ESI) m/z: 363.4 [M+H] ; Elemental Analysis calculated for: C H N ; C, 62.96; H, 6.12; N,
1
9
22
8
3
0.92; Found: C, 62.89; H, 6.15; N, 30.96;

4
.1.1.18
f]pteridin-7-amine (24)
White solid; yield 41.1%; H NMR (300 MHz, DMSO-d ) δ: 9.95 (s, 1H), 8.46 (s, 1H), 8.01 (d, J =
5-cyclopentyl-1-methyl-N-(4-(methylsulfonyl)phenyl)-4,5-dihydro-[1,2,4]triazolo[4,3-
1
6
9
Hz, 2H), 7.80 (d, J = 9 Hz, 2H), 5.16-5.11 (m, 1H), 4.72 (s, 2H), 3.12 (s, 3H), 2.70 (s, 3H), 1.90-
1
3
1
.64 (m, 8H); C NMR (75 MHz, DMSO-d ) δ: 162.24, 160.42, 159.97, 150.31, 149.36, 143.88,
6
1
31.01, 129.15, 129.15, 114.56, 114.56, 90.82, 66.01, 56.65, 47.73, 32.94, 32.94, 24.17, 24.17,
+
1
2.27; LC-MS (ESI) m/z: 426.5 [M+H] ; Elemental Analysis calculated for: C H N O S; C, 56.45;
2
0
23
7
2
H, 5.45; N, 23.04; Found: C, 56.39; H, 5.35; N, 23.17;
4
.2 Biological assays
4
.2.1 BRD4 inhibition TR-FRET assay
The BRD4 inhibition assay was performed by using BRD4 bromodomain 1 TR-FRET Assay
Kit (Item No. 600520) obtained from Cayman (Ann Arbor, MI, USA). All assays were carried out
in coning 384 well plates. BRD4 bromodomain 1 Europium Chelate in 10 μL of assay buffer was
added to plate after 5 μL of compounds containing assay buffer. Final solution contained 5% of
DMSO. To this was added BRD4 bromodomain 1 Ligand/APC acceptor mixture to a total volume
of 20 μL. After a 60-minute room temperature incubation plates were read on the PerkinElmer
multimode plate reader with TR-FRET technique. The TR-FRET ratio (670 nm emission/ 620 nm
emission) versus inhibitor concentration on semi-log axes results in a sigmoidal dose-response curve
typical of competitive binding assays. IC was calculated through the data.
5
0
4
.2.2 Cell growth inhibition assays
In all experiments, MV4;11 and HL-60 leukemia cells were used within two months. MV4;11
and HL-60 cells were cultured in IMDM modified with 10% FBS and 1% penicillin-streptomycin
at Thermo CO incubators at 37℃. In this assay, cells were seeded at a density of 10000 cells per
2
well in 100 μL medium in 96 wells cell culture plates. Then compounds with different
concentrations were added 10 μL to per wells. After 72 hours cultured, CCK-8 were added to each
well at 5 μL. The plate was evaluated by PerkinElmer multimode plate reader at 450 nm. IC was
5
0
calculated by the data using GraphPad Prism 7 software.
4
.2.3 Apoptosis and Cell Cycle Analysis assays
In all experiments, MV4;11 leukemia cells were used within two months. MV4;11 cells were
cultured in IMDM modified with 10% FBS and 1% penicillin-streptomycin at Thermo CO2
incubators at 37℃. In this assay, flow cytometry was used to analyze the effects on cell cycle and
apoptosis treated by compounds. Annexin V-FITC Apoptosis Detection Kit (Cat.NO: KGA107) and
Cell Cycle Detection Kit (Cat.NO: KGA512) were obtained from KeyGEN BioTECH. Cells were
treated through the protocol with preferred compound for 24 hours.

4
.2.4 Western blotting
In all experiments, MV4;11 leukemia cells were used within two months. MV4;11 cells were
cultured in IMDM modified with 10% FBS and 1% penicillin-streptomycin at Thermo CO2
incubators at 37℃. BCA Protein Assay Kit was obtained from Beyotime. MV4;11 cells in the
6
logarithmic phase were cultured with concentration of 5 x 10 /mL, under 5% CO , 37℃ in the
2
incubator for 24 hours, then the compounds were added for another 4 h. Then the cells were put in
1
.5 mL centrifuge tube after cracking at 4℃ to determine the corresponding protein concentration
for further. After that, 10% separation adhesive and 4% concentration adhesive were prepared
according to the method reported in previous literature[19], and SDS-polyacrylamide gel was placed
in an electrophoresis tank for sample loading, electrophoresis and protein transfer. Subsequently,
the PVDF membrane was cleaned with TBST for 3 times, each time for 10 min. The PVDF
membrane was immersed in TBST solution of 5% fat-free milk and placed in a shaker at room
temperature to shake slowly for 2 hours to block the non-specific protein binding sites. PVDF
membrane was transferred to a fight with primary antibody diluent at 4 ℃ overnight, after reaction,
the PVDF membrane was washed with TBST for 3 times, each time for 10 min. While the
incubations of secondary antibody were just 2 h before developing. The primary antibodies against
c-myc and β-actin were obtained from Cell Signaling Technology.
4
.2.5 PLK1 inhibition FP assay
In this assay, FP value was calculated by intensities parallel (intparallel, F||) and the
perpendicular fluorescence intensity (Intperpendicular, F⊥ ). This assay was performed on a
SpectraMax MultiMode Microplate Reader (Molecular Devices) using the excitation at 485 nm and
emission filters at 535 nm. FITC-GPMQSpTPLNG-OH was used as fluorescent probe. PLK1 PBD
was added per well at 20 μL. Compounds with different concentrations was added then. The 384-
well black plate was incubated at room temperature for 30 min with gentle shaking prior to FP
values measurements. Competition binding data were analyzed using GraphPad Prism 7 software
(
GraphPad Software, San Diego, CA) and the inhibition constants (IC ) were calculated by
50
nonlinear curve fitting[20]
.
4
.2.6 molecular docking assay
Schrödinger 2017 was obtained for the molecular docking assay. The crystal structure of BRD4
PDB ID: 4O74) was obtained from the Protein Data Bank. Proteins were prepared using Protein
(
Preparation Wizard based on the force field OPLS3 (optimized potentials for liquid simulations).
Protein was prepared under the preprocess that add missing hydrogens, create disulfide bonds, delete
waters beyond 5Ã from het groups and generate het states. Compound 23 was prepared using the
LigPrep module to compute 3D structures based on the force field OPLS3. Receptor Grid
Generation was given to pick to identify the ligand. The calculation was performed based on the
schrödinger XP precision and the structure of lowest-energy was chosen for the final result.

Acknowledgments
This work was supported by National Science Foundation of China (No. 81872733 and No.
8
1573278).
Conflict of Interest
All authors report no conflict of interest.
References:
[1] O.A. Kharenko, R.G. Patel, S.D. Brown, C. Calosing, A. White, D. Lakshminarasimhan, R.K. Suto,
B.C. Duffy, D.B. Kitchen, K.G. McLure, H.C. Hansen, E.H. van der Horst, P.R. Young, Design and
Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors
Targeting a Methionine, Journal of medicinal chemistry, 61 (2018) 8202-8211.
[2] X. Ran, Y. Zhao, L. Liu, L. Bai, C.Y. Yang, B. Zhou, J.L. Meagher, K. Chinnaswamy, J.A.
Stuckey, S. Wang, Structure-Based Design of gamma-Carboline Analogues as Potent and Specific BET
Bromodomain Inhibitors, Journal of medicinal chemistry, 58 (2015) 4927-4939.
[
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