Design, synthesis and biological evaluation of novel pteridinone derivatives as potent dual inhibitors of PLK1 and BRD4

Article abstract of DOI:10.1039/d0nj03477k

To develop novel simultaneous inhibition of PLK1 and BRD4 bromodomain by a single molecule, three series of novel pteridinone derivatives were designed, synthesized and evaluated for their biological activity. Most compounds exhibited moderate to excellent cytotoxic activity against A549, HCT116, PC-3 and MCF-7 cell lines. The most promising compound III4 showed high antiproliferative effects on four cell lines with IC50 values of 1.27 μM, 1.36 μM, 3.85 μM and 4.06 μM, respectively. The enzymatic assay identified III4 as a potent PLK1 and BRD4 dual inhibitor with % inhibition values of 96.6 and 59.1, respectively. Furthermore, to clarify the anticancer mechanism of the target compounds, explorations in the bioactivity were conducted. The results showed that compound III4 obviously inhibited the proliferation of HCT-116 cell lines, induced a great decrease in the mitochondrial membrane potential leading to apoptosis of cancer cells, suppressed the migration of tumor cells, and arrested the S phase of HCT116 cells.

Full text of DOI:10.1039/d0nj03477k

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DOI: 10.1039/D0NJ03477K
Design, synthesis and biological evaluation of novel pteridinone
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derivatives as potent dual inhibitors of PLK1 and BRD4
Yinliang Qia, Le Xua, Zhiwei Lia, Ping Gonga, Tao Hub, Bixi Yinb, Mingze Qina, Yajing
Liua, Yanfang Zhaoa, Yunlei Houa,b
aSchool of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road,
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Shenhe District, Shenyang, 110016, China.
bYangtze River Pharmaceutical Group Co., Ltd, No.1, South Yangtze River Road, Taizhou 225321,
Jiangsu Province, China
Abstract: To develop novel simultaneous inhibition of PLK1 and BRD4 bromodomain
by a single molecule, three series of novel pteridinone derivatives were designed,
synthesized and evaluated for their biological activity. Most compounds exhibited
moderate to excellent cytotoxic activity against A549, HCT116, PC-3 and MCF-7 cell
lines. The most promising compound III4 showed high antiproliferative effects on four
cell lines with IC50 values of 1.27 μM, 1.36 μM, 3.85 μM and 4.06 μM, respectively.
The enzymatic assay identified III4 as a potent PLK1 and BRD4 dual inhibitor with %
inhibition values of 96.6 and 59.1, respectively. Furthermore, to clarify the anticancer
mechanism of target compounds, further explorations in the bioactivity were conducted.
The results showed that compound III4 obviously inhibited proliferation of HCT-116
cell lines, induced a great decrease in mitochondrial membrane potential leading to
apoptosis of cancer cells, suppressed the migration of tumor cells, and arrested S phase
of HCT116 cells.
Key words: dual inhibitors; antitumor; synthesis; pteridinone derivatives
1. Introduction
Cancer remains the second leading disease after cardiovascular of our time owing to
its manifestation through the aberrant regulation of multiple signaling pathways [1].
The effective strategy to inhibit tumor cell proliferation is to inhibit one or more of the
proteins involved in these related signaling pathways. However, single target inhibitors
may function well in vitro, the upregulation of compensatory signaling pathways often
compromises their efficacies in cells [2]. Thus, the dual pharmacology inhibitor may
increase potency for two disease relevant targets/pathways while decreasing toxicity.
Polo-like kinase 1 (PLK1), a serine–threonine kinase, plays a key role in mitosis and
has been demonstrated to be required in centrosome maturation and establishment of a
bipolar spindle [3]. PLK1 was overexpressed in many different tumor types including
lung, colon, prostate, ovary, breast, head and neck squamous cell carcinoma, melanoma,
and overexpression often correlates with poor prognosis [4]. The molecular mechanism
responsible for reciprocal activation between PLK1 and MYC had been identified [5].
Because of its essential role in cell proliferation, there is a high level of interest and an
*Corresponding author. E-mail address: houyunlei901202@163.com(Y.L. Hou). yanfangzhao@126.com (Y.F.
Zhao). lyjpharm@126.com (Y.J. Liu).

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increasing effort to identify and develop small-molecule inhibitors of PLK1. DTOhI:i1s0.l1e03d9/D0NJ03477K
to a number of different compounds to be designed (Fig. 1) which are currently under
clinical investigation [6-7].
Bromodomain-containing protein 4 (BRD4), belonging to the bromodomain and
extra-terminal domain (BET) family of proteins. Inhibition of BRD4 has recently
emerged as an essential transcriptional co-regulator of MYC, and inhibition of the
bromodomain has been shown to be an effective therapeutic approach to target
dysregulated MYC in neuroblastoma [8-10]. Over the past decade, the number of small-
molecule BET inhibitors have expanded dramatically. Some of them (Fig. 1) have been
enrolled into different phases of human clinical trials including RVX-208, I-BET762
and (+)-JQ1. Although, several compounds have progressed to clinical trials for adult
malignancies, the discovery of novel potent inhibitors still attracts many attentions due
to their therapeutic potential for various human diseases [11-12]. Listed below are
structure and the different functions of BRD4 protein.
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3 ((+)-JQ1, BRD4)
2 (Volasertib, PLK1)
Cl
1 (NMS-P937, PLK1)
N
N
O
N
O
HN
N
N
NH
O
HN
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O
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OH
N
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O
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H
N
N
6 (BI2536, PLK1/BRD4)
4 (RVX-208, BRD4)
5 (I-BET762, BRD4)
Fig. 1. Chemical structures of selected PLK1 inhibitors (structures 1-2), BRD4 inhibitors (structures
3-5) and PLK1/BRD4 inhibitor (structure 6) that are currently being evaluated in clinical trials.
Recently, the studies indicated that the mechanism of the PLK1 and BRD4 inhibitors
was associated with the expression of the MYC oncogene, while markedly inhibiting
cancer cell growth and metastasis in hepatocellular carcinoma and neuroblastoma in
vivo. Tontsch-Grunt et al [13]. reported that the BET inhibitor BI 894999 with PLK
inhibitor volasertib exerted synergistic activity of in AML in vitro and in vivo. Liu et al
[14]. discovered that the PLK1 inhibition enhanced the efficacy of BET epigenetic
reader blockade in castration resistant prostate cancer. Together, these preclinical
findings provide evidence for the strong synergistic effect of PLK1 inhibitor and BRD4
inhibitor, warranting future studies in patients with cancer to investigate this paradigm.
In order to achieve this synergistic effect, the single agent combinatorial inhibition of

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DOI: 10.1039/D0NJ03477K
PLK1 and BET bromodomains was an effective process to develop new antineoplastic
drugs. Rationally designed dual PLK1/BRD4 inhibitor carries the promise to deliver
drugs that are more efficacious in cells, as well as overcome the drawbacks associated
with multidrug regimens.
9
Meanwhile, Knapp and Schönbrunn independently discovered that the PLK1
inhibitor BI-2536 (6) is also a potent inhibitor of BRD4 [15-16]. In the binding complex
of BI-2536 with PLK1 and BRD4, the pteridinone moiety (A) occupied the active
domain and retained to exert the key hydrogen bond interactions with Cys 133, Asp
194 and Gln 85, Asn 140 amino acid residues, respectively [17]. However, according
to the cocrystal structure result the 5-methyl and 6-carbonyl groups of pteridinone
moiety did not occupied the ATP cavity, completely. In our previous work, we found
that the incorporation of triazolo or tetrazolo group fit into the pteridinone frameworks
maintained a certain inhibition activity against PLK1 [18]. Therefore, in this work, we
hope to obtain dual inhibitor of PLK1 and BRD4, which persisted the incorporation of
triazolo or tetrazolo group into the pteridinone frameworks (A). On the other hand, the
methoxy group on the aromatic ring moiety (B) was important to maintain kinase
selectivity against PLK1/BRD4, so methoxy group was introduced. Meanwhile, the
‘necessary nitrogen atom’ is prone to be a versatile high-impact design element to
achieve pharmacological profile. In this perspective, the 2-alkoxypyridin-3-yl motif
was attached to B moiety expecting to exert extra interaction between pyridinyl motif
with PLK1 or BRD4 catalytic domain. Furthermore, the hydrophilic “tail” piperidine
(C) motif exposing to the solvent has been investigated extensively in recent years [19],
indicating that modification on the “tail” (C) could be tolerant to maintain efficacies
(Fig. 2).
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DOI: 10.1039/D0NJ03477K
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N
O
6
N
2
2
1
HN
N
N
a
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Selectivities
PK
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O
NH
N
Hydrophilic "tail"
Previous work
BI2536
A
N
N
a
a =
N
N
HN
N
N
O
N
N
N
N
N
N
O
N
R
X
B
Hydrophilic "tail"
C
Target compounds
X = CH or N
This work
Fig. 2. Design strategy for the target compounds.
Meanwhile, compound III2 contained similar structural feature with BI2536
including hydrophilic aniline side chain and pteridinone fragments. In order to better
understand the anti-tumor mechanisms, molecular docking models of III2 were
performed based upon the cocrystal structure of BI2536 with PLK1 and BRD4.
Compound III2 occupied the kinase domain in a similar manner to BI2536. Combining
with the results of docking analysis, PLK1 and BRD4 were very likely to be the
potential drug targets of these pteridinone derivatives.
In this study, we utilized the scaffold of BI-2536 to develop three series of novel
pteridinone derivatives with varied inhibitory activities against PLK1 and BRD4 in
order to perform structure-activity relationship (SAR) studies, as well as understand the
effect of inhibiting both proteins (PLK1 and BRD4) with a single agent in cancer. All
compounds were subsequently assayed for anti-proliferative activities in vitro against
four cancer cell lines A549, PC-3, HCT116 and MCF-7. Based on the anti-proliferative
results, potent compounds were selected for further in vitro enzymatic inhibitory studies.
To further clarify the primary mechanism, III4 was taken forward and examined by in
vitro the migration, apoptosis and cell cycle analysis of HCT116 cells.
2. Chemistry
A series of novel pteridinone derivatives linked with nitrogen heterocycle at N-5,
C-6 position and different aniline at C-2 position were synthesized. The general
synthetic routes of the title compounds were depicted in Scheme 1-3. Starting from

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commercially available 2,4-dichloro-5-nitropyrimidine M1 reactedDOI: 10.1039/D0NJ03477K
with
cyclopentylamine to afford compound M2, which was reduced by using iron powder
and catalytic amounts of concentrated HCl in EtOH/H2O to obtain amide intermediate
M3. The intermediate M4 was prepared from M3 by treatment with ethyl oxalyl
monochloride in acetone. Then carbonyl in compound M4 was chlorinated by thionyl
chloride to afford M5. Intermediate M5 was converted to tetrazole product M8 using
NaN3 as a cyclization reagent in DMF at 0 °C. Subsequently, the intermediate M6 was
available via hydrazinolysis of intermediate M5 with 80% hydrazine monohydrate in
EtOH at 40 °C. The cyclization of the triazole intermediates M7 and M9 on compounds
M6 were performed with trimethyl orthoformate or triethyl orthoformate under 80 °C,
respectively. Side chain anilines M12 were prepared in two steps as shown in Scheme
2. Aromatic nucleophilic substitution of chlorine in 4-chloro-2-methoxy-1-
nitrobenzene or 6-chloro-2-methoxy-3-nitropyridine by secondary amines were readily
achieved in DMF at 40 °C. The resulting nitrobenzenes were reduced to anilines using
Pd/C in EtOH. Subsequently, amination of M7, M8, and M9 by corresponding aryl
amines M12 with p-toluenesulfonic acid as catalyst furnished target compounds I1-I14,
II1-II6 and III1-III14 (Scheme 3).
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H
NO2
Cl
NO2
NH
NH2
NH
N
O
O
N
N
N
N
a
b
c
Cl
N
Cl
N
Cl
N
Cl
N
N
M1
M3
M2
M4
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Cl
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N
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NH2
N
f
d
e
Cl
N
O
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N
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Cl
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O
M5
M6
M7
g
h
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Cl
N
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Cl
N
N
O
O
M9
M8
Scheme 1. General scheme for the synthesis of key intermediates M7, M8 and M9; Reagents and
o
conditions: (a) Cyclopentylamine, NaHCO3, DCM, 25 C, 10 h; (b) Fe powder, HCl (cat.),
EtOH/H2O, reflux, 2 h; (c) i: Ethyl oxalyl monochloride, K2CO3, acetone, 2 h; ii: TEA, EtOH, 100
.
oC, 4 h; (d) SOCl2, DMF (cat.), reflux, 2 h; (e) NH2NH2 H2O, EtOH, 40 oC, 2 h; (f) HC(OEt)3, 80
oC, 2 h; (g) NaN3, DMF, 0 oC, 10 h; (h) CH3HC(OEt)3, 80 oC, 2 h.

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DOI: 10.1039/D0NJ03477K
R1
R1
b
a
H2N
N
R2
O2N
N
R2
O2N
Cl
X
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O
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M10
M11
M12
X = CH, N
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Scheme 2. Preparation of the side chain anilines; Reagents and conditions: (a) Amines R1R2NH,
K2CO3, DMF, 40 °C, 6 h; (b) H2, 10 % Pd/C, EtOH, 50 °C, 5 h.
N
N
N
N
N
HN
N
O
O
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N
R1 R2
I1-I14
N
N
N
N
A
N
N
M12
a
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N
N
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Cl
N
N
O
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N
M7, M8 or M9
R1 R2
II1-II6
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N
R1 R2
III1-III14
Scheme 3. General scheme for the synthesis of target compounds; Reagents and conditions: (a) p-
Toluenesulfonic acid, 1-butanol, 100 oC, 15 h.
3. Results and discussion
3.1. In vitro antiproliferative activity
To evaluate in vitro antitumor activities, all synthesized compounds (I1-I14, II1-II6,
and III1-III14) were investigated against a panel of cancer cell lines, including A549
(human lung adenocarcinoma), HCT116 (human colorectal cancer), PC-3 (human
prostate cancer) and MCF-7 (human breast cancer) cells by the MTT assay. Meanwhile,
JQ1 and BI2536 were served as positive controls. The results were expressed as half-
maximal inhibitory concentration (IC50) values and summarized in Table 1. Most of
pteridinone derivatives I1-I14 showed moderate to significant cytotoxic activities

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against the different cancer cell lines. Several of these compounds have eqDuOivI: a10l.e10n3t9/D0NJ03477K
effects to (+)-JQ1 on one or more cancer cell lines, which suggested that the
combination of 7-aminoaryl-[1,2,4]triazolo[4,3-f]pteridinone framework and
hydrophilic “tail” moiety exhibit potent synergistic antitumor effect. Preliminary SARs
indicated that the introduction of different amino “tail” groups at the C-7 position of
pteridinone moiety had a significant influence on activity. Compounds bearing
substituted piperazine moiety (I1 and I8) displayed excellent anti-tumor activities in the
sigle-digit micromolar range against A549 and HCT116 cells. Notably, one additional
nitrogen atom can be inserted into the position of 7- aminoaryl with maintaining
activities (I8 vs I9). However, compounds I14 containing an unsubstituted piperazine,
compounds I5-I6 containing a morpholine and I12-I13 containing a hydroxyethyl
piperazine respectively led to the diminished potency, which indicated that the presence
of alkyl substituted piperazine was a critical factor in anti-proliferative activities.
Especially, the better promising compound I4 displayed equivalent potency with (+)-
JQ1 against A549, HCT116, PC-3 and MCF-7 cells with IC50 values of 4.12 μM, 6.73
μM, 4.61 μM and 4.22 μM, respectively. Overall, SARs studies identified the 7-amino-
[1,2,4]triazolo[4,3-f]pteridinone moiety bearing piperazine "tails" moieties harboring
cationic nitrogen atoms with favorable potency.
Further studies were performed to examine the effect of shifting the 1,2,3-triazole
motif of I1-I14 series compounds to the tetrazolo series compounds (II1-II6). The results
displayed that most of the tetrazolo series compounds had lower cytotoxicity than 1,2,3-
triazole series. Interestingly, II6 was maintained approximately cytotoxicity in A549,
HCT116, PC-3, MCF-7 cells with IC50 values of 4.87 μM, 2.09 μM, 9.30μM, and 5.40
μM, respectively.
In our previous study, we found that the methyl group acts as an essential factor in
the molecular recognition of endogenous and exogenous substrates by means of
bioreceptors. This can lead to multiple biological effects, such as selectivity among
bioreceptors, increased the binding affinity, and protection against enzyme metabolism.
Accordingly, we next further explored the SAR resulting from a methyl group to the 1
position of 7-amino-[1,2,4]triazolo[4,3-f]pteridinone core on the cytotoxic activity. We
synthesized a series of compounds (III1-III14) containing the methyl group. These
methyl-substituted compounds show superior cytotoxic activities over earlier
compounds against the different cancer cell lines, suggesting that not only the
hydrocarbyl substituted piperazine “tail” but also the methyl-[1,2,4]triazolo moiety
play an important role in cytotoxic activities. Moreover, the examination of SARs
indicated that these analogs had similar SARs as summarized before.
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In conclusion, the most potent compound III4 showed promising cytotoxicity against
A549, HCT116, PC-3 and MCF-7 cell lines with IC50 values of 1.27 μM, 1.36 μM, 3.85
μM and 4.06 μM, respectively. The potency have equivalent effects to (+)-JQ1.
However, the potency was slightly weaker than BI2536. These encouraging results
provided a valuable lead compound III4 and highlighted the potential for further
development of novel pteridinone derivatives as potent antitumor agents.
Table 1.

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Structures and cytotoxicity of compounds (I1-I14, II1-II6, and III1-III14).
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A
N
8
HN
N
N
O
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X
tails
A ring
Compd.
X
“tail”
IC50 a (μM) ± SD b
A549
HCT116
PC-3
MCF-7
6.20 ± 0.09
16.69 ±
0.03
N
N
N
N
I1
I2
CH
N
3.24 ± 0.02
4.57 ± 0.05
3.80 ± 0.06
5.7 ± 0.1
3.52 ± 0.01
14.72 ± 0.05
N
N
N
N
I3
I4
I5
I6
CH
N
>20
4.12 ± 0.04
>20
>20
6.73 ± 0.03
>20
>20
4.61 ± 0.08
>20
>20
4.22 ± 0.08
>20
N
O
O
CH
N
N
>20
>20
>20
>20
10.88 ±
0.02
I7
I8
I9
CH
CH
N
15.78 ± 0.08
6.66 ± 0.07
4.59 ± 0.01
8.48 ± 0.05
3.14 ± 0.08
4.07 ± 0.08
13.07 ± 0.06
4.32 ± 0.01
9.35 ± 0.03
N
NH
N
N
N
N
N
5.86 ± 0.05
11.50 ±
0.06
N
N
17.85 ±
0.04
N
NH
NH
I10
I11
CH
N
17.00 ± 0.05
7.51 ± 0.08
9.60 ± 0.02
3.53 ± 0.05
14.98 ± 0.08
N
10.40 ± 0.05 9.43 ± 0.07
OH
OH
N
N
N
I12
I13
I14
II1
II2
II3
II4
II5
CH
N
>20
>20
>20
>20
>20
>20
>20
>20
N
N
NH
N
CH
CH
CH
N
>20
>20
>20
>20
N
5.06 ± 0.09
>20
10.41 ± 0.03
>20
7.99 ± 0.03
>20
9.31 ± 0.06
>20
N
N
N
N
11.43 ± 0.02
>20
5.26 ± 0.04
>20
16.36 ± 0.06
>20
>20
N
O
N
N
CH
N
>20
N
N
N
O
>20
>20
>20
>20
II6
CH
4.87 ± 0.03
2.09 ± 0.06
9.30 ± 0.05
5.40 ± 0.07
N
NH
N
N
N
III1
III2
CH
N
3.43 ± 0.03
3.76 ± 0.06
4.28 ± 0.08
5.23 ± 0.08
4.46 ± 0.02
6.32 ± 0.03
5.43 ± 0.06
3.06 ± 0.07
13.28 ±
0.07
N
N
N
N
N
N
III3
CH
15.7 ± 0.05
6.84 ± 0.05
>20
N
N
III4
III5
III6
N
CH
N
1.27 ± 0.09
>20
1.36 ± 0.05
>20
3.85 ± 0.03
>20
4.06 ± 0.01
>20
N
N
O
O
>20
>20
>20
>20

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III7
CH
2.37 ± 0.09
3.11 ± 0.02
10.42 ± 0.01 7D.1O7I: 1±0.010.0392/D0NJ03477K
N
NH
5
6
7
8
N
N
N
III8
III9
CH
N
>20
9.42 ± 0.09
2.08 ± 0.07
17.48 ± 0.04
6.02 ± 0.04
>20
6.88 ± 0.05
12.07 ±
0.03
N
3.65 ± 0.05
9
N
NH
NH
III10
III11
CH
N
4.79 ± 0.04
4.22 ± 0.01
3.71 ± 0.07
3.30 ± 0.03
8.25 ± 0.06
8.54 ± 0.09
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13.05 ±
0.03
N
OH
OH
N
N
N
N
III12
III13
CH
N
>20
>20
>20
>20
>20
>20
>20
>20
N
NH
III14
CH
>20
>20
>20
>20
BI2536
JQ1
0.05 ± 0.01
4.58 ± 0.04
1.42 ± 0.04
0.79 ± 0.06
0.46 ± 0.06
3.69 ± 0.05
0.03 ± 0.09
6.88 ± 0.07
a Values are the means of at least three independent experiments. b SD: standard deviation.
3.2. Enzyme activity research
Based on the cellular assays, potent compounds were selected for further in vitro
PLK1 and BRD4 % inhibition at 1 μM. The results were summarized in Fig. 3. Most
of compounds inhibited PLK1 and BRD4 kinases with % inhibition values ranging
from 44.6% to 96.6% and from 12.4% to 59.1%, respectively. In parallel with cellular
results, inhibitors bearing the 1-methyl-1,2,4-triazole at A ring of pteridinone core were
found to more potent than corresponding 1,2,4-triazole or tetrazolo analogues, which
further proved 7-amino-1-methyl-triazolo[4,3-f]pteridin-4(5H)-one a favorable
building block to increase the potency. Even though it showed more selectivity against
PLK1, the inhibitory activity of III4 was still less than BRD4. These results indicated
that three series of novel compounds worth further studying as new potential anticancer
agent for the treatment of human cancers, and the BRD4 inhibitory activities can be
increased by the structural modification.
Fig. 3. Enzymatic activities of the target compounds.
3.3. Preliminary study on mechanism of action
To investigate the molecular mechanisms of action preliminarily, cell apoptosis
analysis of HCT116 cells treated with the optimal compound III4 was performed using
Annexin-V and propidium iodide (PI) double staining by flow cytometry and treating
the HCT-116 cells with 1.0 μM, 3.0 μM and 9.0 μM of III4 for 24 h. As shown in Fig.
4, compound III4 was very effective in the induction of apoptosis in a dose-dependent
manner. Compound III4 proved to induce apoptosis by 68.3 % as compared to 14.6%
of apoptotic cells in the untreated control (Table 2).

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Fig. 4. Compound III4 induced apoptosis in HCT-116 cells. Cells were treated with various
concentrations of III4 for 24 h and then analyzed the Annexin V-FITC/PI staining test by flow
cytometry analysis. Values represent the mean ± S.D, n = 3. P <0.05 versus the control. The
percentage of cells in each part is indicated.
Table 2．
Percent of cell death induced by compound III4 (9.0 μM and 0 μM) on HCT-116 cells.
Apoptosis (%)
Concentrations (III4)
Necrosis (%)
Total
Early
Late
9.0 μM
0 μM
68.3± 0.2
14.6± 0.2
18.4±0.3
6.7±0.3
49.9 ± 0.1
7.9 ± 0.1
1.8 ± 0.2
0.3 ± 0.2
3.4 Migration experiment research
Since migration is an important characteristic for metastatic cancers, the effect of
compound III4 on migration of cancer cells was investigated by the wound healing
assay. As shown in Fig. 5, treatment of HCT116 cells with compound III4 at indicated
concentrations markedly suppressed the wound healing in a concentration-dependent
manner.
Fig. 5. In vitro wound healing assay on HCT116 cells. Phase contrast images were obtained by the
treatment of compounds III4 at indicated concentrations for 0, 24 and 48 h.
3.5 Cell cycle analysis.
The cell cycle analysis was performed to investigate the prevention of proliferation
in HCT116 cells with the most potent compound III4. After treatment of HCT116 cells

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were fixed and stained with PI, the DNA content was analyzed by flow cytometry. The
obtained results were compared with non-treated HCT116 cells, as control. As shown
in Figs. 6 and 7, treatment of HCT116 cells with III4 at 0.3, 1.0, and 9.0 μM
concentrations increased the percentage of S-phase cells from 18.83% (as control group)
to 19.10%, 20.02%, and 37.17%, respectively. These results confirmed that compound
III4 significantly caused S-phase arrest in HCT116 cells.
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Fig. 6. Effect of compound III4 on the cell cycle distribution of HCT116 cells.
Fig. 7. Quantitative analysis of cell cycle distributions; (A) Non-treated cells as control group; (B)
treated with III4 at 0.3 μM; (C) treated with III4 at 1.0 μM; (D) treated with III4 at 9.0 μM.
Fig. 8. The binding models of III4 with PLK1 and BRD4. (A and A’) Predicted binding
conformation for III4 (blue sticks) in the binding site cavity of PLK1 (PDB code: 2RKU) and BRD4
(PDB code: 4O74), (B and B’) 2D diagram of the interaction between III4 and the binding site
cavity of PLK1 and BRD4, (C and C’) III4 overlapping with BI2536 (red sticks).
3.6 Binding Mode of with III4 with PLK1 and BRD4
In order to better understand the binding mode, molecular docking models of III4
was performed based upon the cocrystal structure with BI5236 of PLK1 (PDB code:
2RKU) and BRD4 (PDB code: 4O74), respectively.

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As shown in Fig. 8, III4 occupied the kinase domain in a similar manner toDBOII: 2105.130369/D0NJ03477K
with PLK1 and BRD4, respectively. In the PLK1 kinase domain, III4 was found to form
three hydrogen bonds via the 8-position nitrogen atom, the 7-position NH and the 4-
position oxygen atom of carbonyl of the 7-amino-1-methyl-[1,2,4]triazolo[4,3-
f]pteridinone nuclei with Cys 133 and Cys 67, respectively (Fig. 8A). In the BRD4
kinase domain, III4 was found to form two water-mediated hydrogen bonds with the 2-
position NH, the 7-position NH and the 8-position nitrogen atom; and one hydrogen
bond via the 3-position NH and the 4-position oxygen atom of carbonyl (Fig. 8A’. The
methoxy group on the aromatic ring might be necessary to modulate binding affinity
against the PLK1/BRD4 protein. These results could provide a molecular level
foundation to illustrate that III4 could bind well at the active site of PLK1/BRD4
kinases and it is a potential inhibitor of PLK1/BRD4.
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4. Conclusion
In the present study, three series of novel potent anticancer agents were designed and
synthesized. The results of antiproliferative activities indicated that III4 significantly
exhibits inhibitory activity against A549, HCT116, PC-3 and MCF-7 cell lines with
IC50 values of 1.27 μM, 1.36 μM, 3.85 μM and 4.06 μM, respectively. Combined with
the results of the molecular docking and enzymatic studies, the PLK1 and BRD4 were
very likely to be the drug targets of compound III4. Furthermore, to clarify the
mechanism of the anticancer activity of the pteridinone molecule, the flow cytometry
and wound-healing assays confirmed that III4 induced cell apoptosis. Finally, cell cycle
analysis of III4 by flow cytometry showed cell cycle arrest in S phase, DNA
fragmentation and alteration in mitochondrial membrane potential by compound III4
for the three concentrations tested. In summary, the SARs studies together with the
pharmacological assays on novel pteridinone analogues identified III4 as a promising
anti-cancer agent, which will lead to the promising development of new drugs.
5. Experimental procedures
5.1 Chemistry
Unless otherwise specified, all reagents and solvents were purchased from
commercial sources and used as received. All melting points were obtained on a Mettler
Point MP70 apparatus (Mettler, Toledo, Switzerland) and uncorrected. Mass spectra
(MS) were acquired on an Agilent 1100 LC-MS (Agilent, palo Alto, CA, USA) in ESI
mode. Reactions were monitored on silica plates (F-254) by thin-layer chromatography
(TLC) and visualized under UV light. 1H NMR (600 MHz) spectra were performed on
a Bruker spectrometers (Bruker Bioscience, Billerica, Massachusetts, USA). The
chemical shift (δ) of dimethyl sulfoxide-d6 (DMSO-d6) is based on 2.5ppm at 303 K
and TMS Conducted was used as an internal standard. Column chromatography was
performed using prepacked Qingdao Ocean Chemical Company (Qingdao, Shandong,
China) silica gel (200-300 mesh).
5.1.1 General procedure for preparation of compounds (M2-M9)

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5.1.1.1 2-chloro-N-cyclopentyl-5-nitropyrimidin-4-amine(M2)
2,4-Dichloro-5-nitropyrimidine (M1) (30.0 g, 156 mmol) and NaHCO3 (26.2g, 312
mmol) were dissolved in DCM (200 mL). Cyclopentylamine (15.9 g, 187 mmol) was
dissolved in DCM (300 mL) and then added at 0 ºC. The reaction mixture was stirred
for 10 h at room temperature and then organic was washed by water, and brine. The
combined organic phase was dried over Na2SO4, filtered, and the solvent evaporated to
obtain 30.95g of M2 as a yellow solid, m.p.: 125.2 – 126.6 °C. Yield: 82%; MS (ESI)
m/z: 243.3 [M+H]+.
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5.1.1.2 2-chloro-N4-cyclopentylpyrimidine-4,5-diamine (M3)
To a suspension of compound M2 (30.0 g, 124 mmol) in 300 mL of 95% ethanol was
added HCl (1 mL), and Fe Powder (34.7 g, 620 mmol) in batches. The mixture was
refluxed for 2 h. After completion of the reaction as indicated by TLC, the solution was
then filtered hot through Celite. The filtrate was concentrated in a vacuum to afford
pure product and then purified by silica gel column chromatography to obtain 18.42 g
of M3 as a white solid, m.p.: 128.2 – 130.0 °C. Yield: 70.1%; MS (ESI) m/z: 213.2
[M+H]+ .
5.1.1.3 2-chloro-8-cyclopentyl-5,8-dihydropteridine-6,7-dione (M4)
To a stirred solution of compound M3 (15.0 g, 71 mmol) in acetone (150 mL) was added
K2CO3 (11.6 g, 85 mmol) and ethyl oxalyl monochloride (10.6g, 78 mmol). The
mixture was stirred at room temperature for 2 h. The solution was filtered and the filtrate
was concentrated under reduced pressure. In a pressure flask, the crude ketoester was
dissolved in absolute EtOH (150 mL) and then TEA (8.6 g, 85mmol) was added. The
mixture was heated for an additional 4 h at 100 °C. After indicating the completion of
the reaction by TLC, the solution was filtered to obtain 16.4 g of M4 as a white solid,
m.p.: 210.1 – 212.2 °C. Yield: 86.9%; MS (ESI) m/z: 264.9 [M-H]-.
4.1.1.4 2,6-dichloro-8-cyclopentylpteridin-7(8H)-one (M5)
DMF added to a solution of compound M4 (15.0 g, 56 mmol) in SOCl2 (75 mL), and
the resulting mixture was maintained at this temperature for 2 h. The solvent was
concentrated in vacuum and the reaction mixture was poured into 200 mL
ice water, the resulting precipitate was filtered and dried to obtain 14.8 g of M5 as a
white solid, m.p.: 269.0 – 279.3 °C. Yield: 93.1%; MS (ESI) m/z: 285.26 [M+H]+ .
5.1.1.5 2-chloro-8-cyclopentyl-6-hydrazinylpteridin-7(8H)-one (M6)
A mixture of M5 (14.0 g, 49 mmol) and 80% hydrazine monohydrate (13.7 g, 73.5
mmol) in EtOH (200 mL) was stirred at 40 °C for 2h. After indicating the completion
of the reaction by TLC, a white solid appeared after most solvents were evaporated to
dryness under reduced pressure. The resulting precipitate was filtered off, washed with
water, and dried under vacuum to afford 12.0 g of M6 as a white solid, m.p.: 210.7 –
212.1 °C. Yield: 85.8%; MS (ESI) m/z: 279.0 [M-H]-.
5.1.1.6 7-chloro-5-cyclopentyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (M7)

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To a stirred solution of compound M6 (5.0 g, 17.8 mmol) in trimethyl orthoformate (50
mL). The reaction mixture was stirred at 80 °C for 2h, cooled to room temperature. The
resulting precipitate was filtered off, washed with diethyl ether, and dried to afford 4.7
g of M7 as a white solid, m.p.: 251.8 – 253.1 °C. Yield: 89.9%; MS (ESI) m/z: 313.1
[M+Na]+. 1H NMR (DMSO-d6, 400 MHz) δ 9.90 (s, 1H), 9.49 (s, 1H), 5.76 – 5.60 (m,
1H), 2.21-2.13 (m, 2H), 2.09 – 1.98 (m, 2H), 1.98 – 1.83 (m, 2H), 1.67-1.64 (m, 2H).
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5.1.1.7 7-chloro-5-cyclopentyltetrazolo[1,5-f]pteridin-4(5H)-one (M8)
To a suspension of M5 (5.0 g, 17.5 mmol) and DMF (20 mL) was dropped NaN3 (1.14
g, 17.5 mmol). The mixture was stirred at 0 °C for 10 h. After indicating the completion
of the reaction by TLC, the reaction mixture was cooled to room temperature. Water
was then added, and the solid was filtered and dried under reduced pressure to afford
4. g of M8 as a white solid, m.p.: 185.4 – 186.8 °C. Yield: 83.6%; MS (ESI) m/z: 292.3
[M+H]+. 1H NMR (DMSO-d6, 400 MHz,) δ 9.71 (s, 1H), 5.78 – 5.70 (m, 1H), 2.21 –
2.13 (m, 2H), 2.08 – 2.02 (m, 2H), 2.00 – 1.88 (m, 2H), 1.73 – 1.62 (m, 2H).
5.1.1.8 7-chloro-5-cyclopentyl-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (M
)
9
Compound M6 (5.0 g, 17.8 mmol) was stirred in a solution of triethyl orthoformate (50
ml). The reaction was stirred at 80 ℃ for 2 hours, then cooled to room temperature. The
resulting precipitate was filtered, washed with diethyl ether, and dried to provide 4.6 g
of M9 as a white solid, m.p.: 255.3 – 257.1 °C. Yield: 84.1%; MS (ESI) m/z: 327.2
[M+Na]+.
5.1.2 General Procedure for Preparation of (M11)
In the case of stirring at 40 ℃, substitute amines (1.2 equiv) were added into the DMF
(7 mL/g) mixture of M10 (1 equiv) and K2CO3 (2.0 equiv), and then TLC was detected.
After the mixture is poured into the ice water, the product is precipitated, filtered and
dried to obtain the yellow solid compound M11. After the mixture is poured into the
ice water, the product is precipitated, filtered and dried to obtain the yellow solid
compound M11.
5.1.3 General Procedure for Preparation of (M12)
The nitro compound M11 (1 equiv in a mixture of EtOH-H2O, 95:5, 20 mL) was reduced
with 10% Pd-carbon (5% w/w) The reaction was hydrogenated at 50 ℃ for 5 hours.
The reaction was detected by TLC. After the reaction is completed, the reactant is
pumped and filtered, and the filtrate is vacuum concentrated to the product M12.
5.1.4 General procedure for preparation of compounds (I1-I14,II1-II6 or III1-III14)
Compound M12 (1.1 equiv) and p-toluenesulfonic acid (1 equiv) were added to the
compound to a stirred solution of compound M7, M8, or M9 (1 equiv) in 1-butanol,
placed in a pressure bottle, and heated to 100 ° C for 15 h. The reaction mixture was
quenched with saturated Na2CO3 aqueous solution, then extracted with DCM, washed
with water and dried on anhydrous Na2SO4. The organic layer was concentrated under

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reduced pressure. The product was further purified by fast column chromatography,
and dichloromethane / methanol was used as eluent to obtain I1-I14,II1-II6 or III1-III14
6
7
as a pale yellow solid.
8
9
5.1.4.1 5-cyclopentyl-7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-[1,2,4]
triazolo[4,3-f]pteridin-4(5H)-one(I1)
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Yield: 74.7%; m.p.:242.6-244.4 °C; MS (ESI) m/z:476.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.73 (s, 1H), 7.29 (s, 1H), 6.64 (s, 1H), 6.52 (s,
1H), 5.58 (s, 1H), 3.75 (s, 3H), 3.17 (s, 4H), 2.21 (d, J = 39.5 Hz, 7H), 1.69 (s, 5H),
1.46 (s, 5H).
5.1.4.2 5-cyclopentyl-7-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)-[
1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I2)
Yield: 65.0%; m.p.:263.0-264.9°C; MS (ESI) m/z:477.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.70 (s, 1H), 9.10 (s, 1H), 8.85 (s, 1H), 7.48 (s, 1H), 6.37 (s, 1H), 5.53 (s,
1H), 3.77 (s, 3H), 3.48 (s, 4H), 2.42 (s, 4H), 2.23 (s, 3H), 2.17 (s, 2H), 1.67 (s, 3H),
1.45 (s, 3H).
5.1.4.3 5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)-2-methoxyphenyl)amino)-[1,2
,4]triazolo[4,3-f]pteridin-4(5H)-one(I3)
Yield:74.7 %; m.p.:296.4-298.3 °C; MS (ESI) m/z:530.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.70 (s, 1H), 9.10 (s, 1H), 8.72 (s, 1H), 7.27 (d, J = 8.3 Hz, 1H), 6.63 (d, J
= 2.1 Hz, 1H), 6.50 (dd, J = 8.6, 2.2 Hz, 1H), 5.57 (s, 1H), 3.74 (s, 3H), 3.17 – 3.13 (m,
4H), 2.56 (s, 4H), 2.20 – 2.13 (m, 2H), 1.82 (d, J = 6.1 Hz, 3H), 1.74 – 1.59 (m, 6H),
1.52 (dd, J = 7.1, 5.0 Hz, 3H), 1.45 (d, J = 11.1 Hz, 2H), 1.37 (td, J = 16.3, 8.0 Hz, 3H).
5.1.4.4 5-cyclopentyl-7-((6-(4-cyclopentylpiperazin-1-yl)-2-methoxypyridin-3-yl)amin
o)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I4)
Yield:55.2 %; m.p.:271.5-273.2°C; MS (ESI) m/z:531.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.70 (s, 1H), 9.09 (s, 1H), 8.85 (s, 1H), 7.47 (d, J = 5.3 Hz, 1H), 6.35 (d, J
= 8.4 Hz, 1H), 5.52 (s, 1H), 3.77 (s, 3H), 3.46 (s, 4H), 3.17 (s, 4H), 2.52 (s, 4H), 2.19
– 2.14 (m, 2H), 1.83 – 1.79 (m, 2H), 1.66 – 1.61 (m, 3H), 1.54 – 1.49 (m, 3H), 1.41 –
1.35 (m, 3H).
5.1.4.5 5-cyclopentyl-7-((2-methoxy-4-morpholinophenyl)amino)-[1,2,4]triazolo[4,3-
f]pteridin-4(5H)-one(I5)
Yield: 65.3 %; m.p.: 272.3-274.2 °C; MS (ESI) m/z:463.2[M+H]+; 1H NMR (DMSO-
d6, 600 MHz) δ 9.71 (s, 1H), 9.11 (s, 1H), 8.73 (s, 1H), 7.33 (d, J = 7.3 Hz, 1H), 6.66
(s, 1H), 6.53 (d, J = 7.7 Hz, 1H), 5.59 (s, 1H), 3.76 (s, 7H), 3.13 (s, 4H), 2.18 (d, J =
4.0 Hz, 2H), 1.70 (s, 3H), 1.47 (s, 2H).
5.1.4.6 5-cyclopentyl-7-((2-methoxy-6-morpholinopyridin-3-yl)amino)-[1,2,4]triazolo
[4,3-f]pteridin-4(5H)-one(I6)
Yield: 62.5%; m.p.:276.4-278.1°C; MS (ESI) m/z:464.2[M+H]+; 1H NMR (DMSO-d6,

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600 MHz) δ 9.70 (s, 1H), 9.10 (s, 1H), 8.87 (s, 1H), 7.52 (d, J = 6.1 Hz, 1H), 6D.3O7I: 1(0d.1,03J9/D0NJ03477K
= 8.3 Hz, 1H), 5.54 (s, 1H), 3.78 (s, 3H), 3.74 – 3.70 (m, 4H), 3.45 – 3.41 (m, 4H), 2.17
(s, 2H), 1.67 (s, 3H), 1.45 (s, 3H).
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5.1.4.7 5-cyclopentyl-7-((2-methoxy-4-(3-methylpiperazin-1-yl)phenyl)amino)-[1,2,4]
triazolo[4,3-f]pteridin-4(5H)-one(I7)
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Yield:69.2 %; m.p.: 251.1-253.0°C; MS (ESI) m/z:476.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.72 (s, 1H), 7.28 (d, J = 5.0 Hz, 1H), 6.62 (s,
1H), 6.50 (d, J = 7.7 Hz, 1H), 5.58 (s, 1H), 3.75 (s, 3H), 3.57 – 3.51 (m, 3H), 2.98 (d,
J = 11.3 Hz, 1H), 2.82 (s, 2H), 2.59 – 2.55 (m, 1H), 2.21 (dd, J = 25.6, 14.6 Hz, 3H),
1.69 (s, 3H), 1.46 (s, 3H), 1.05 (d, J = 5.7 Hz, 3H).
5.1.4.8 5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)-2-methoxyphenyl)amino)-[1,2,4]tr
iazolo[4,3-f]pteridin-4(5H)-one(I8)
Yield: 57.2%; m.p.: 254.2-256.3°C; MS (ESI) m/z:490.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.76 – 8.71 (m, 1H), 7.28 (d, J = 5.6 Hz, 1H),
6.64 (s, 1H), 6.51 (d, J = 7.4 Hz, 1H), 5.58 (s, 1H), 3.75 (s, 3H), 3.17 (s, 4H), 2.51 (s,
3H), 2.40 (d, J = 5.8 Hz, 2H), 2.17 (s, 2H), 1.69 (s, 3H), 1.46 (s, 3H), 1.05 (s, 3H).
5.1.4.9 5-cyclopentyl-7-((6-(4-ethylpiperazin-1-yl)-2-methoxypyridin-3-yl)amino)-[1,
2,4]triazolo[4,3-f]pteridin-4(5H)-one(I9)
Yield: 64.2%; m.p.:214.0-216.2 °C; MS (ESI) m/z:491.26[M+H]+; 1H NMR (DMSO-
d6, 600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.86 (s, 1H), 7.48 (s, 1H), 6.37 (s, 1H), 5.53
(s, 1H), 3.78 (s, 3H), 3.50 (d, J = 14.1 Hz, 4H), 2.51 (s, 4H), 2.39 (s, 2H), 2.17 (s, 2H),
1.66 (s, 3H), 1.45 (s, 3H), 1.05 (s, 3H).
5.1.4.10 5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)-2-methoxyphenyl)amino)-[
1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I10)
Yield:68.3 %; m.p.:203.2-206.7 °C; MS (ESI) m/z:490.3.26[M+H]+; 1H NMR (DMSO-
d6, 600 MHz) δ 9.71 (s, 1H), 9.11 (s, 1H), 8.73 (s, 1H), 7.28 (d, J = 8.1 Hz, 1H), 6.64
(d, J = 2.1 Hz, 1H), 6.52 (dd, J = 8.6, 2.3 Hz, 1H), 5.58 (s, 1H), 4.66 (s, 3H), 3.76 (s,
3H), 3.61 (d, J = 11.3 Hz, 3H), 2.97 (s, 3H), 2.20 – 2.14 (m, 3H), 1.69 (s, 3H), 1.46 (s,
3H), 1.09 (d, J = 5.8 Hz, 3H).
5.1.4.11 5-cyclopentyl-7-((6-(3,5-dimethylpiperazin-1-yl)-2-methoxypyridin-3-yl)ami
no)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I11)
Yield: 58.4%; m.p.:205.6-208.3 °C; MS (ESI) m/z:491.3.26[M+H]+;1H NMR (DMSO-
d6, 600 MHz ) δ 9.70 (s, 1H), 9.10 (s, 1H), 8.85 (s, 1H), 7.47 (d, J = 5.1 Hz, 1H), 6.38
(d, J = 8.4 Hz, 1H), 5.54 (s, 1H), 4.16 (d, J = 11.8 Hz, 3H), 3.77 (s, 3H), 2.89 (s, 3H),
2.38 (d, J = 12.5 Hz, 3H), 2.20 – 2.13 (m, 3H), 1.66 (s, 3H), 1.43 (s, 3H), 1.09 (d, J =
5.7 Hz, 3H).
5.1.4.12 5-cyclopentyl-7-((4-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxyphenyl)ami
no)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I12)

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Yield:55.2 %; m.p.: 273.1-275.6°C; MS (ESI) m/z:506.2[M+H]+; 1H NMR (DMSO-d6V,iew Article Online
DOI: 10.1039/D0NJ03477K
600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.73 (s, 1H), 7.28 (d, J = 6.2 Hz, 1H), 6.64 (s,
1H), 6.51 (d, J = 7.8 Hz, 1H), 5.58 (s, 1H), 4.46 (s, 1H), 3.75 (s, 3H), 3.55 (d, J = 4.9
Hz, 2H), 3.16 (s, 4H), 2.58 (s, 4H), 2.46 (d, J = 5.4 Hz, 2H), 2.17 (s, 2H), 1.69 (s, 3H),
1.46 (d, J = 0.9 Hz, 3H).
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5.1.4.13 5-cyclopentyl-7-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-y
l)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(I13)
Yield: 51.0%; m.p.:226.7-229.2°C; MS (ESI) m/z:507.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.86 (s, 1H), 7.47 (d, J = 4.9 Hz, 1H), 6.36 (d, J
= 8.4 Hz, 1H), 5.53 (s, 1H), 4.46 (t, J = 5.1 Hz, 1H), 3.77 (s, 3H), 3.55 (dd, J = 11.3,
5.8 Hz, 2H), 3.47 (s, 4H), 2.52 (s, 4H), 2.44 (t, J = 6.2 Hz, 2H), 2.16 (s, 2H), 1.66 (s,
3H), 1.45 (s, 4H).
5.1.4.14 5-cyclopentyl-7-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)-[1,2,4]triazolo
[4,3-f]pteridin-4(5H)-one(I14)
Yield: 52.9%; m.p.: 264.3-266.8°C; MS (ESI) m/z:462.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.71 (s, 1H), 9.11 (s, 1H), 8.72 (s, 1H), 7.29 (s, 1H), 6.63 (s, 1H), 6.50 (d,
J = 6.8 Hz, 1H), 5.58 (s, 1H), 3.75 (s, 3H), 3.07 (s, 4H), 2.86 (s, 4H), 2.18 (s, 3H), 1.70
(s, 3H), 1.47 (s, 3H).
5.1.4.15 5-cyclopentyl-7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)tetraz
olo[1,5-f]pteridin-4(5H)-one(II1)
Yield: 55.1%; m.p.: 221.2-222.9°C; MS (ESI) m/z:477.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.20 (s, 1H), 9.13 (s, 1H), 7.22 (d, J = 3.9 Hz, 1H), 6.65 (s, 1H), 6.53 (d, J
= 6.8 Hz, 1H), 5.54 (s, 1H), 3.75 (s, 3H), 3.18 (s, 4H), 2.51 (s, 4H), 2.22 (d, J = 40.4
Hz, 5H), 1.70 (s, 3H), 1.46 (s, 3H).
5.1.4.16 5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)-2-methoxyphenyl)amino)te
trazolo[1,5-f]pteridin-4(5H)-one(II2)
Yield: 57.9%; m.p.:248.2-250.1 °C; MS (ESI) m/z:531.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.19 (s, 1H), 9.11 (s, 1H), 7.22 (s, 1H), 6.64 (s, 1H), 6.51 (d, J = 8.2 Hz,
1H), 5.52 (s, 1H), 3.74 (s, 3H), 3.16 (s, 4H), 2.57 (s, 4H), 2.17 (d, J = 13.4 Hz, 2H),
1.82 (d, J = 4.5 Hz, 3H), 1.66 (dd, J = 21.0, 15.5 Hz, 4H), 1.56 – 1.50 (m, 3H), 1.47 –
1.32 (m, 5H).
5.1.4.17 5-cyclopentyl-7-((6-(4-cyclopentylpiperazin-1-yl)-2-methoxypyridin-3-yl)ami
no)tetrazolo[1,5-f]pteridin-4(5H)-one(II3)
Yield: 66.2%; m.p.:219.0-220.8 °C; MS (ESI) m/z:532.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.19 (s, 1H), 9.11 (s, 1H), 7.22 (s, 1H), 6.64 (s, 1H), 6.51 (d, J = 8.2 Hz,
1H), 5.52 (s, 1H), 3.74 (s, 3H), 3.16 (s, 4H), 2.57 (s, 4H), 2.17 (d, J = 13.4 Hz, 2H),
1.82 (d, J = 4.5 Hz, 3H), 1.66 (dd, J = 21.0, 15.5 Hz, 4H), 1.56 – 1.50 (m, 3H), 1.47 –
1.32 (m, 5H).

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5.1.4.18 5-cyclopentyl-7-((2-methoxy-4-morpholinophenyl)amino)tetrazolo[1,5-f]pteVriew Article Online
idin-4(5H)-one(II4)
DOI: 10.1039/D0NJ03477K
Yield: 64.9%; m.p.:240.4-242.2 °C; MS (ESI) m/z:464.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.20 (s, 1H), 9.14 (s, 1H), 7.25 (s, 1H), 6.67 (d, J = 2.2 Hz, 1H), 6.54 (dd,
J = 8.6, 2.2 Hz, 1H), 5.57 (s, 1H), 3.78 – 3.76 (m, 7H), 3.15 – 3.13 (m, 4H), 2.18 (d, J
= 6.8 Hz, 2H), 1.71 (s,3H), 1.46 (s, 3H).
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5.1.4.19 5-cyclopentyl-7-((2-methoxy-6-morpholinopyridin-3-yl)amino)tetrazolo[1,5-
f]pteridin-4(5H)-one(II5)
Yield:45.9 %; m.p.:244.2-246.2 °C; MS (ESI) m/z:465.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.26 (s, 1H), 9.20 (s, 1H), 7.50 (d, J = 3.2 Hz, 1H), 6.39 (d, J = 8.4 Hz,
1H), 5.43 (s, 1H), 3.78 (s, 3H), 3.74 – 3.70 (m, 4H), 3.47 – 3.42 (m, 4H), 2.16 (s, 2H),
1.64 (s, 3H), 1.41 (s, 3H).
5.1.4.20 5-cyclopentyl-7-((2-methoxy-4-(3-methylpiperazin-1-yl)phenyl)amino)tetraz
olo[1,5-f]pteridin-4(5H)-one(II6)
Yield: 53.0%; m.p.:209.2-211.1 °C; MS (ESI) m/z:477.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 9.19 (s, 1H), 9.13 (s, 1H), 7.23 (s, 1H), 6.66 (d, J = 1.9 Hz, 1H), 6.55 –
6.52 (m, 1H), 5.53 (s, 1H), 3.75 (s, 3H), 3.62 (dd, J = 16.9, 12.8 Hz, 3H), 3.09 (d, J =
11.9 Hz, 1H), 2.97 (s, 1H), 2.90 (dd, J = 11.7, 9.4 Hz, 1H), 2.68 (t, J = 10.9 Hz, 1H),
2.38 – 2.34 (m, 1H), 2.18 (s, 2H), 1.69 (s, 3H), 1.45 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H).
5.1.4.21 5-cyclopentyl-7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-met
hyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III1)
Yield:57.2 %; m.p.:261.2-263.1 °C; MS (ESI) m/z:490.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.69 (s, 1H), 7.30 (s, 1H), 6.63 (s, 1H), 6.50 (d, J = 6.5 Hz,
1H), 5.59 (s, 1H), 3.75 (s, 3H), 3.15 (s, 4H), 2.90 (s, 3H), 2.48 (s, 4H), 2.24 (s, 3H),
2.17 (s, 2H), 1.69 (s, 3H), 1.47 (s, 3H).
5.1.4.22 5-cyclopentyl-7-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)-
1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III2)
Yield: 78.1%; m.p.:257.2-258.9 °C; MS (ESI) m/z:491.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.85 (s, 1H), 7.49 (d, J = 1.9 Hz, 1H), 6.37 (d, J = 8.3 Hz,
1H), 5.55 (s, 1H), 3.78 (s, 3H), 3.48 (s, 4H), 2.90 (s, 3H), 2.43 (s, 4H), 2.24 (s, 3H),
2.17 (d, J = 2.0 Hz, 2H), 1.67 (s, 3H), 1.46 (s, 3H).
5.1.4.23 5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)-2-methoxyphenyl)amino)-1
-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III3)
Yield: 55.8%; m.p.:283.8-285.7 °C; MS (ESI) m/z:544.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.69 (s, 1H), 7.28 (d, J = 6.0 Hz, 1H), 6.62 (s, 1H), 6.49 (dd,
J = 8.4, 1.3 Hz, 1H), 5.59 (s, 1H), 3.75 (s, 3H), 3.14 (s, 4H), 2.89 (s, 3H), 2.57 (s, 4H),
2.17 (s, 2H), 1.82 (d, J = 5.6 Hz, 2H), 1.74 – 1.58 (m, 6H), 1.57 – 1.41 (m, 5H), 1.40 –
1.34 (m, 2H).

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DOI: 10.1039/D0NJ03477K
5.1.4.24 5-cyclopentyl-7-((6-(4-cyclopentylpiperazin-1-yl)-2-methoxypyridin-3-yl)ami
no)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III4)
Yield:54.3 %; m.p.: 246.8-248.7°C; MS (ESI) m/z:545.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.83 (s, 1H), 7.48 (d, J = 3.8 Hz, 1H), 6.34 (d, J = 8.4 Hz,
1H), 5.54 (s, 1H), 3.77 (s, 3H), 3.46 (s, 4H), 2.89 (s, 3H), 2.51 (d, J = 3.0 Hz, 4H), 2.16
(d, J = 0.7 Hz, 2H), 1.82 (d, J = 4.8 Hz, 2H), 1.70 – 1.58 (m, 5H), 1.56 – 1.47 (m, 3H),
1.45 (d, J = 5.3 Hz, 1H), 1.39 (dd, J = 12.0, 3.9 Hz, 3H).
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5.1.4.25 5-cyclopentyl-7-((2-methoxy-4-morpholinophenyl)amino)-1-methyl-[1,2,4]tri
azolo[4,3-f]pteridin-4(5H)-one(III5)
Yield: 55.7%; m.p.:284.6-286.3 °C; MS (ESI) m/z:477.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.92 (s, 1H), 8.72 (s, 1H), 7.32 (d, J = 7.9 Hz, 1H), 6.66 (d, J = 2.3 Hz,
1H), 6.52 (dd, J = 8.7, 2.3 Hz, 1H), 5.60 (s, 1H), 3.76 (t, J = 4.6 Hz, 7H), 3.13 – 3.11
(m, 4H), 2.90 (s, 3H), 2.17 (dd, J = 15.5, 8.0, 3.3 Hz, 2H), 1.70 (s, 4H), 1.47 (s, 2H).
5.1.4.26 5-cyclopentyl-7-((2-methoxy-6-morpholinopyridin-3-yl)amino)-1-methyl-[1,
2,4]triazolo[4,3-f]pteridin-4(5H)-one(III6)
Yield: 64.9%; m.p.: 246.6-248.5°C; MS (ESI) m/z:478.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.92 (s, 1H), 8.86 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 6.37 (d, J = 8.4 Hz,
1H), 5.56 (s, 1H), 3.78 (s, 3H), 3.75 – 3.69 (m, 4H), 3.46 – 3.40 (m, 4H), 2.89 (s, 3H),
2.17 (dd, J = 15.2, 8.2 Hz, 3H), 1.67 (s, 3H), 1.46 (s, 3H).
5.1.4.27 5-cyclopentyl-7-((2-methoxy-4-(3-methylpiperazin-1-yl)phenyl)amino)-1-met
hyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III7)
Yield: 63.6%; m.p.:253.5-255.4°C; MS (ESI) m/z:490.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.71 (s, 1H), 7.30 (d, 1H), 6.63 (s, 1H), 6.51 (s, 1H), 5.60 (s,
1H), 3.76 (s, 3H), 3.55 (d, J = 13.4 Hz, 3H), 3.02 (d, J = 9.4 Hz, 1H), 2.92 – 2.89 (m,
3H), 2.87 (s, 2H),2.63 – 2.57 (m, 1H), 2.25 (d, J = 9.6 Hz, 1H), 2.18 (s, 1H), 1.70 (s,
3H), 1.46 (s, 3H), 1.10 – 1.04 (m,3H).
5.1.4.28 5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)-2-methoxyphenyl)amino)-1-meth
yl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III8)
Yield: 69.1%; m.p.:264.3-266.8 °C; MS (ESI) m/z:504.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.92 (s, 1H), 8.72 (s, 1H), 7.29 (d, J = 4.9 Hz, 1H), 6.64 (d, J = 2.1 Hz,
1H), 6.51 (dd, J = 8.6, 2.2 Hz, 1H), 5.59 (s, 1H), 3.51 (s, 3H), 3.16 (s, 4H), 2.90 (s, 3H),
2.53 (s, 4H), 2.40 (dd, J = 13.9, 7.0 Hz, 2H), 2.18 (dd, J = 14.1, 6.9 Hz, 2H), 1.69 (s,
3H), 1.47 (s, 3H), 1.05 (t, J = 7.1 Hz, 3H).
5.1.4.29 5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)-2-methoxyphenyl)amino)-[1,2,4]t
riazolo[4,3-f]pteridin-4(5H)-one(III9)
Yield: 51.9%; m.p.:227.7-231.4 °C; MS (ESI) m/z:505.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.92 (s, 1H), 8.85 (s, 1H), 7.48 (s, 1H), 6.37 (s, 1H), 5.54 (s, 1H), 3.78 (s,
3H), 3.48 (s, 4H), 2.90 (s, 3H), 2.44 (d, J = 68.0 Hz, 6H), 2.17 (s, 2H), 1.66 (s, 3H),
1.45 (s, 3H), 1.05 (s, 3H).

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DOI: 10.1039/D0NJ03477K
5.1.4.30 5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)-2-methoxyphenyl)amino)-1-
methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III10)
Yield:66.9 %; m.p.:259.3-261.8 °C; MS (ESI) m/z:504.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.70 (s, 1H), 7.28 (d, J = 9.1 Hz, 1H), 6.63 (d, J = 2.2 Hz,
1H), 6.50 (dd, J = 8.7, 2.3 Hz, 1H), 5.59 (s, 1H), 3.75 (s, 3H), 3.59 (d, J = 9.6 Hz, 3H),
2.99 – 2.91 (m, 3H), 2.90 (s, 3H), 2.32 – 2.21 (m, 3H), 2.18 (dd, J = 8.7, 5.8 Hz, 3H),
1.68 (s, 3H), 1.46 (s, 3H), 1.07 (d, J = 5.7 Hz, 3H).
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5.1.4.31 5-cyclopentyl-7-((6-(3,5-dimethylpiperazin-1-yl)-2-methoxypyridin-3-yl)ami
no)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III11)
Yield: 53.5%; m.p.:259.3-261.8 °C; MS (ESI) m/z:504.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 2H), 8.84 (s, 2H), 7.47 (s, 2H), 6.37 (d, J = 8.4 Hz, 1H), 5.55 (s,
1H), 4.14 (d, J = 11.3 Hz, 3H), 3.78 (s, 3H), 2.90 (s, 3H), 2.84 (s, 3H), 2.33 (s, 3H),
2.18 (dd, J = 16.5, 9.0 Hz, 3H), 1.66 (s, 3H), 1.46 (s, 3H), 1.07 (d, J = 5.5 Hz, 3H).
5.1.4.32 5-cyclopentyl-7-((4-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxyphenyl)ami
no)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III12)
Yield:56.9 %; m.p.: 273.1-275.6°C; MS (ESI) m/z:520.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.72 (s, 1H), 7.28 (s, 1H), 6.63 (d, J = 2.5 Hz, 1H), 6.50 (dd,
J = 8.7, 2.5 Hz, 1H), 5.32 (s, 1H), 4.46 (s, 1H), 3.75 (s, 3H), 3.55 (d, J = 5.5 Hz, 2H),
3.15 (s, 4H), 2.90 (s, 3H), 2.59 (s, 4H), 2.46 (s, 2H), 2.17 (dd, J = 14.6, 7.2 Hz, 2H),
1.68 (s, 3H), 1.46 (d, J = 6.9 Hz, 3H).
5.1.4.33 5-cyclopentyl-7-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-y
l)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one(III13)
Yield:67.3 %; m.p.: 231.7-234.2°C; MS (ESI) m/z:521.3[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.91 (s, 1H), 8.84 (s, 1H), 7.47 (d, J = 5.5 Hz, 1H), 6.35 (d, J = 8.4 Hz,
1H), 5.54 (s, 1H), 4.44 (t, J = 5.2 Hz, 1H), 3.77 (s, 3H), 3.54 (dd, J = 11.3, 6.0 Hz, 2H),
3.49 – 3.44 (m, 4H), 2.89 (s, 3H), 2.52 (d, J = 4.5 Hz, 4H), 2.43 (t, J = 6.3 Hz, 2H),
2.17 (dd, J = 16.3, 8.9 Hz, 2H), 1.66 (s, 3H), 1.45 (s, 3H).
5.1.4.34 5-cyclopentyl-7-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)-1-methyl-[1,2,
4]triazolo[4,3-f]pteridin-4(5H)-one(III14)
Yield: 54.7%; m.p.: 269.3-271.8°C; MS (ESI) m/z:476.2[M+H]+; 1H NMR (DMSO-d6,
600 MHz) δ 8.92 (s, 1H), 8.71 (s, 1H), 7.29 (d, J = 6.0 Hz, 1H), 6.62 (d, J = 2.4 Hz,
1H), 6.50 (dd, J = 8.6, 2.4 Hz, 1H), 5.60 (s, 1H), 3.76 (s, 3H), 3.07 (d, J = 4.6 Hz, 4H),
2.90 (s, 4H), 2.86 – 2.84 (m, 3H), 2.18 (s, 3H), 1.70 (s, 2H), 1.47 (s, 3H).
5.2 Pharmacology
5.2.1 In vitro antiproliferative assays
MTT assay was performed on A549, HCT116, PC-3 and MCF-7 cells to evaluate
anti-proliferative activity of the target compounds, with BI-2536 as the positive controls.
Cancer cell lines were cultured in minimum essential medium (MEM) supplement with

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10% foetal bovine serum (FBS).
Approximate 5×103 cells, suspended in MEM medium, were plated in a 96-well plate
and incubated in 5% CO2 at 37 °C for 24 h. The compounds at the specified
concentration were added to the culture medium and the cell cultures were incubated
for another 72 h. 10 μL of 5 mg/mL MTT solution were added to each well and
incubated with cells at 37 °C for 4 h. Dissolved the formazan crystals in each well with
100 μL dimethyl sulfoxide, and measured the absorbance at 492 nm (absorbance of
MTT formazan) and 630 nm (reference wave length) with an ELISA reader. The results
were expressed as IC50, which is the average of at least three measured values, and wrer
calculated using Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
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5.2.2 In vitro PLK1and BRD4 enzymatic assays
The in vitro enzymes of the selected compounds were evaluated by the
homogeneous time-resolved fluorescence (HTRF) assay.
Materials and reagents：Enzyme-labeled instrument,384 Plates, PLK1, ATP 10
mM, DTT 1 M, MgCl2 1 M, HTRF KinEASE -STK S1 kit contains STK substrate-1-
biotin, Streptavidin-XL665, STK Antibody-Cryptate, 5x Enzymatic buffer, SEB,
HTRF Detection buffer, Tb-labeled donor, Dye-labeld acceptor, 1xBRD TR-FRET
Assay Buffer, BET Ligand, BRD4(BD1) 3ng/μL.
According to the manufacturer’s instructions (http://www.cisbio.com/kinease),
prepare working solutions (Compounds, TK Substrate-biotin, PLK1, ATP, Sa-XL665
and TK-Antibody-Cryptate). Adding 4 μL of the diluted compounds working solution
to the reaction wells except the control well to which 4 μL of 2.5% DMSO solution was
added. Then add 4 μL of Substrate-STK and ATP working solution to all wells. Adding
2 μL of PLK1enzyme working solution to all reaction wells except negative wells, and
make up the volume with 2 μL of enzyme corresponding to 1 × KB in negative wells.
Seal the plate with a sealing film and mix for 60 minutes at room temperature. Prepare
the test solution 5 minutes before the end of the kinase reaction. After the reaction, add
10 μL of the prepared working solution of Streptavidin-XL665 and TK antibody
europium cryptate to all wells. Seal the plate and mix well. After reaction at room
temperature for 1h, using ENVISION instrument to detect the fluorescence. Add diluted
Tb-labeled donor and dye-labeled acceptor to all wells at 5 μl per well. Add 2 μl of
compound working solution to each well. Add 2 μl 1x BRD TR-FRET Assay Buffer to
each of the negative and positive control wells. Add 5 μL per well of diluted BET
Ligand to the positive control wells and compound wells. Add 5 μl 1x BRD TR-FRET
Assay Buffer to each of the negative control wells. Add diluted BRD4 enzyme to all
wells at 3 μl per well. Reaction at room temperature for 2 hours. Read fluorescence
value at Ex320, Em665/615 nm wavelength.
(Ex320, Em665/615 nm). Emission Ratio(ER)= 665 nm Emission signal/ 615 nm
Emission
signal.
Percent
inhibition
=
(ERpositive―ER
sample)/(ERpositive―ERnegative)*100%
5.2.3 Morphology assays of apoptotic cells

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HCT116 cells were seeded in 6-well plates at a final concentration of 50 0D0O0I:/w10.e10l3l,9/D0NJ03477K
and incubated for 24 hours until the cells adhered. The cells were treated with various
concentrations of compound III4 and cultured for another 48 h, Control cells and treated
cells were collected and washed with PBS, and then resuspended in 100μL 1*binding
buffer incubated in a mixture of 5 μL Annexin V-FTIC and 5μL PI for 10 min at room
temperature in a dark place. Prior to flow cytometry analysis, cells were resuspended
in 400 μL 1 * binding buffer.
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5.2.4 Cell migration assay
Cell migration assay were performed using wound-healing assay. HCT-116 cells
were seeded in six-well plates at a final concentration of 1×106/well and grown to 100%
confluence. Linear wound across the confluent cell layer by a pipette tip. HCT-116 cells
were washed twice with PBS, and then incubated with culture medium containing
DMSO or various concentrations of compound III4. After 0, 24, and 48 hours of
cultivating, the images were taken through a fluorescence microscope (Olympus,
Tokyo, Japan).
5.2.5 Cell cycle assays
The effects of compound III4 on cell cycle progression were verified by using a
standard propidium iodide (PI) staining procedure followed by flow cytometry analysis.
HCT-116 cells were seeded in six-well plates at a final concentration of 25 000/well
and incubated for 24 h. Cells treated with various concentrations of III4 and then
incubated for another 24 h. The cells were harvested and washed twice with ice cold
PBS, then fixed in ice-cold 70% (v/v) ethanol overnight at 4 ℃. The cells were washed
again by PBS, and then the cell DNA was stained with 300 μL PI (Beyotime) for 10
min. Data collection and analysis were carried out by using a flow cytometer.
5.2.6 Molecular docking
Structural alignment was accomplished through the Cresset Forge v10.4.1. The
PLK1 model was built using PDB code: 2RKU as template. The protein coordinate was
downloaded from the Protein Data Bank (http:// www.rcsb.org/pdb/). BI2536 (PDB
code: 2RKU) and BRD4 (PDB code: 4O74) were regarded as reference molecule for
the computation of field points and alignment. The MCS conformation of the molecule
in training set was generated by being aligned on the reference molecule. After the
conformation was obtained, the highest structural arrangement score was recommended
as the best alignment. Molecular field points were computed for the test set of molecule,
together with the molecular field points of the training set structure were revealed to be
the best alignment with the template field points. The analysis of results implemented
on discovery studio visualizer software.
Acknowledgements
The work was financially supported by the Project funded by China Postdoctoral
Science Foundation (2019TQ0215), the Program of Liaoning Revitalization Talents
Program (XLYC1808037), the Doctoral Scientific Research Foundation of Liaoning

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Province (2020-BS-130) and Liaoning XingLiao Talents Program (XLYC1807093).
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References
1 R.L. Siegel, KD Miller, CA: a cancer journal for Clinicians 2016. 2016, 66 (1)，7-
30.
8
9
2 L. Chen, J.L. Yap, M. Yoshioka, ACS Med. Chem. Lett., 2015, 6, 764-769.
3 G Combes, I Alharbi, L G Braga, et al., Playing polo during mitosis: PLK1 takes
the lead, Oncogene., 2017, 36 (34), 4819-4827.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
Z. Zhang, X. Hou, C. Shao, J. Li, J.X. Cheng, S. Kuang, N. Ahmad, T. Ratliff, X.
Liu, Cancer Res, 2014,74 (22),6635-47.
5
D. Xiao, M. Yue, H. Su, P. Ren, J. Jiang, F. Li, Y. Hu, H. Du, H. Liu, G. Qing,
Molecular Cell., 2016, 64 (3), 93–506.
6
7
T.P. Heffron, J. Med. Chem., 2016,59 (22), 10030-10066.
Z. Liu, P. Wang, H. Chen, E.A. Wold, B. Tian, A.R. Brasier, J. Zhou, J. Med.
Chem., 2017,60 (11), 4533-4558.
8
A. Puissant, S.M. Frumm, G. Alexe, C.F. Bassil, J. Qi, Y. Chanthery, E.A. Nekritz,
R. Zeid, W.C. Gustafson, P.Greninger, M.J. Garnett, U. Mcdermott, C.H. Benes,
A.L. Kung, W.A. Weiss, J.E. Bradner, K. Stegmaier, Cancer Discov., 2013,3 (3),
308-323.
9
J. Delmore, G.C. Issa, M.E. Lemieux, P.B. Rahl, J. Shi, H.M. Jacobs, E.
Kastritis, T. Gilpatrick, R.M. Paranal, J. Qi, M. Chesi, A.C. Schinzel, M.R.
Mckeown, T.P. Heffernan, C.R. Vakoc, P.L. Bergsagel, I.M. Ghobrial, P.G.
Richardson, R.A. Young, W.C. Hahn, K.C. Anderson, A.L. Kung, J.E.
Bradner, C.S. Mitsiades, Cell., 2011,146 (6), 904-917.
10
J.A. Mertz, A.R. Conery, B.M. Bryant, P. Sandy, S. Balasubramanian, D.A.
Mele, L. Bergeron, R.J. Sims, Proc. Natl. Acad. Sci., 2011,108 (40), 16669-16674.
11 L. Ouyang, L. Zhang, J. Liu, L. Fu, D. Yao, Y. Zhao, S. Zhang, G. Wang, G.
He, B. Liu, J. Med. Chem., 2017, 60 (24), 9990-10012.
12 M. Zengerle, K. Chan, A. Ciulli, ACS Chem. Biol., 2015,10 (8), 1770-1777.
13 U. Tontschgrunt, D. Rudolph, I. Waizenegger, A. Baum, D. Gerlach, H.
Engelhardt, M. Wurm, F. Savarese, N. Schweifer, N. Kraut, Cancer Lett., 2018,
421, 112-120.
14 F. Mao, J. Li, Q. Luo, R. Wang, Y. Kong, C. Carlock, Z. Liu, B.D. Elzey, X. Liu,
Mol Cancer Ther., 2018, 17 (7), 1554-1565.
15 S.W. Ember, J.Y. Zhu, S.H. Olesen, M.P. Martin, A. Becker, N. Berndt, G.I.
Georg, ACS Chem. Biol., 2014,9 (5), 1160-1171.
16 P. Ciceri, S. Muller, A. Omahony, O. Fedorov, P. Filippakopoulos, J.P. Hunt, E.A.
Lasater, G. Pallares, S. Picaud, C. Wells, S. Martin, L. Wodicka, N.P. Shah, D.K.
Treiber, S. Knapp, Nat. Chem. Biol., 2014,10 (4) , 305-312.
17 S. Liu, H. Yosief, L. Dai, H. Huang, G. Dhawan, X. Zhang, A. Muthengi, J.M.
Roberts, D.L. Buckley, J.A. Perry, L. Wu, J.E. Bradner, J. Qi, W. Zhang, J. Med.
Chem., 2018, 61 (17), 7785-7795.
18 Y. Hou, L. Zhu, Z. Li, Q. Shen, Q. Xu, W. Li, Y. Liu, P. Gong, Eur. J. Med. Chem.,
2019,163, 1690-709.

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19 L. Xiao, X. Yang, M. Zhan, P. Cao, S. Zheng, R. Peng, J. Han, Z. Xie, Z. Tu, C.
Liao, Eur. J. Med. Chem., 2019, 184, 111769.
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