Design, synthesis and biological evaluation of novel pteridinone derivatives possessing a hydrazone moiety as potent PLK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127329

A series of novel pteridinone derivatives possessing a hydrazone moiety were designed, synthesized and evaluated for their biological activity. Most of the synthesized compounds demonstrated moderate to excellent activity against A549, HCT116 and PC-3 cancer cell lines. In particular, compound L₁₉ exhibited the most potent antiproliferative effects on three cell lines with IC₅₀ values of 3.23 μM, 4.36 μM and 8.20 μM, respectively. In kinase assays, the compound L₁₉ also showed potent inhibition activity toward PLK1 with % inhibition values of 75.1. Further mechanism studies revealed that compound L₁₉ significantly inhibited proliferation of HCT-116 cell lines, induced a great decrease in mitochondrial membrane potential resulting in apoptosis of cancer cells, inhibited the migration of tumor cells, and arrested G1 phase of HCT116 cells.

Full text of DOI:10.1016/j.bmcl.2020.127329

Bioorganic&MedicinalChemistryLetters30(2020)127329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel pteridinone derivatives
possessing a hydrazone moiety as potent PLK1 inhibitors
⁎
⁎
Zhiwei Lia, Le Xua, Liangyu Zhua, Yanfang Zhaoa, Tao Hub, Bixi Yinb, Yajing Liua, , Yunlei Houa,b,
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China
b Yangtze River Pharmaceutical Group Pharmaceutical Co., Ltd, 1 South Yangtze River Road Taizhou, Jiangsu 225321, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel pteridinone derivatives possessing a hydrazone moiety were designed, synthesized and eval-
uated for their biological activity. Most of the synthesized compounds demonstrated moderate to excellent ac-
tivity against A549, HCT116 and PC-3 cancer cell lines. In particular, compound L19 exhibited the most potent
antiproliferative effects on three cell lines with IC50 values of 3.23 μM, 4.36 μM and 8.20 μM, respectively. In
kinase assays, the compound L19 also showed potent inhibition activity toward PLK1 with % inhibition values of
75.1. Further mechanism studies revealed that compound L19 significantly inhibited proliferation of HCT-116
cell lines, induced a great decrease in mitochondrial membrane potential resulting in apoptosis of cancer cells,
inhibited the migration of tumor cells, and arrested G1 phase of HCT116 cells.
PLK1
Pteridinone derivatives
Anti-cancer
Cancer, manifesting through abnormal regulation of multiple sig-
naling pathways, remains the second leading disease and one of the
major public health problems.1,2 To overcome cancer, the conventional
strategy was to design and synthesize new antitumor drugs that in-
hibited at least one protein involved in these pathways.3 With the ad-
vancement of scientific and technological methods, the secrets of ma-
lignant proliferation of tumor cells have gradually been discovered,
such as the disorder of cell signaling pathways and uncontrolled cell
proliferation.4 Research on anti-tumor drugs targeting key factors that
regulate the cell cycle has attracted increasing attention from re-
searchers.
have been developed, such as BI-2536, BI-6727 (volasertib), NMS-P937,
GSK461364 and TAK960 have advanced clinical trials and shown en-
couraging anticancer effects in many kinds of tumors10–14 (Fig. 1).
Among these promising anticancer agents, BI-2536 has reached phase II
trials and showed inspiring results.10
The co-crystal structure of the PLK1 catalytic domain in complex
with BI-2536 revealed that BI-2536 aminopyrimidine moiety binded to
hinge region Cys 133 via two hydrogen bonds and pteridinone carbonyl
formed two water-mediated hydrogen bonds with the side chain of Lys
82 and the main chain NH of Asp 194, respectively. Thus the dihy-
dropterinone framework played key roles in the interaction with PLK1
kinase15 (Fig. 2). As a continuation of our previous study of dihy-
droterinone PLK1 kinase inhibitors,16 we modified the framework of BI-
2536 by replacing the 7-position ethyl group with a hydrogen-bond
acceptor carbonyl group in order to form a hydrogen bond with the
protein (Fig. 3).
Polo-like kinases (PLKs), a serine–threonine kinase, have five family
members (PLK1-5) which play a key role in mitosis and have been
proven to be necessary for centrosome maturation and bipolar spindle
formation.5–7 PLK1 is the most widely studied of all PLK family mem-
bers, the molecular mechanism responsible for reciprocal activation
between PLK1 and MYCN (v-myc myelocytomatosis viral related on-
cogene, neuroblastoma derived) have been identified.8 The over-
expression of PLK1 have been found in many different tumor types,
including lung cancer, colon cancer, prostate cancer, ovarian cancer,
breast cancer, head, neck squamous cells cancer and melanoma.9 Due to
its essential role in cell proliferation, PLK1 have been identified as a
broad-spectrum anticancer target.
In addition, hydrazone derivatives showed a broad spectrum of
biological activities in the medical field, such as antimicrobial,17 anti-
tuberculosis18 and antitumor activities.19 Presumably, the bioactivity
based on hydrazone moiety was due to the hydrazone's easy to bind to
molecular targets as a hydrogen bond donor or acceptor. In addition,
the functional group can also give a certain degree of flexibility to the
chemical structure.20,21 Due to its beneficial properties, we are moti-
vated to combine this moiety with the dihydroterinone skeleton to
In recent years, a variety of effective PLK1 small molecule inhibitors
⁎ Corresponding author at: School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
China (Y. Hou).
E-mail addresses: lyjpharm@126.com (Y. Liu), houyunlei901202@163.com (Y. Hou).
https://doi.org/10.1016/j.bmcl.2020.127329
Received 15 April 2020; Received in revised form 3 June 2020; Accepted 4 June 2020
Availableonline08June2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Fig. 1. Structures of PLK1 inhibitors in clinical trials.
Fig. 2. Crystal structure of BI-2536 bound to PLK1 (PDB ID: 2RKU).
Fig. 3. Design strategy for the target compounds.
design novel PLK1 kinase inhibitors (L1–L30) (Fig.3). All compounds
were subsequently assayed for in vitro anti-proliferative activities
against three cancer cell lines A549, HCT116 and PC-3. According to
the antiproliferative results, an effective compound L19 was selected for
further in vitro enzyme inhibition studies. To elucidate the primary
main mechanism, L19 was examined by in vitro the migration, apoptosis
and cell cycle analysis of HCT116 cells.
compounds were described in Scheme 1. Starting from commercially
available 2,4-dichloro-5-nitropyrimidine 1, reacted with cyclopentyla-
mine in DCM to yield intermediate 2, which was further reduced by
using iron powder and catalytic amounts of concentrated HCl in EtOH/
H2O to produce intermediate 3. The intermediate 4 was prepared from
3 by condensed with ethyl oxalyl monochloride in acetone. The com-
pound 4 reacted with methyl iodide to afford N-alkylated product 5.
Subsequently, the intermediate 6 was available via hydrazinolysis of
intermediate 5 with 80% hydrazine monohydrate in EtOH at 40 °C.
A series of novel pteridinone derivatives possessing a hydrazone
moiety were synthesized. The general synthetic routes of the target
2

Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Scheme 1. General scheme for the synthesis
of target compound; Reagents and condi-
tions: (i) Cyclopentylamine, NaHCO3, DCM,
25 °C, 10 h, 85.0%; (ii) Fe powder, HCl
(cat.), EtOH/H2O, reflux, 2 h, 73%; (iii) a:
Ethyl oxalyl monochloride, K2CO3, acetone,
2 h; b: TEA, EtOH, 100 °C, 4 h, 83%; (iv)
CH3I, DCM, rt, 2 h, 97.9%; (v) NH2NH2H2O,
EtOH, 40 °C, 2 h; (vi) appropriate aromatic
aldehyde, EtOH, 80 °C, 5 h.,62.6–83.4%.
Finally, intermediate 6 reacted with appropriate aromatic aldehyde
under standard conditions to obtain the target product L1–L30 in good
or moderate yields.
potential for further development of novel pteridinone derivatives as an
effective antitumor agents.
On the basis of the cellular assays, effective compounds were se-
lected to further in vitro PLK1% inhibition at 1 μM. The results were
shown in Fig. 4. Most of compounds inhibited PLK1 kinases with %
inhibition values ranging from 49.7% to 75.1%. Parallel to the cellular
results, inhibitors bearing electron withdrawing substituent at aromatic
ring were found to more potent than others. Among them, L19 displayed
the best inhibitory activity against PLK1 up to 75.1%. These results
indicated that pteridinone derivatives bearing hydrazone moiety as new
potential anticancer agents for the treatment of human cancers were
worthy of further study, and the PLK1 inhibitory activity may increase
by structural modifications.
To evaluate in vitro antitumor activities, all synthesized compounds
(L1–L30) were investigated against three cancer cell lines including
A549 (human lung adenocarcinoma), HCT116 (human colorectal
cancer) and PC-3 (human prostate cancer) cells by using MTT assay and
BI-2536 was served as the positive control. The antiproliferative results
were expressed as half-maximal inhibitory concentration (IC50) values
and summarized in Table 1. Most of pteridinone derivatives L1–L30
showed moderate to good antiproliferative activities against the dif-
ferent cancer cell lines, which suggested that the combination of 2-
amino-5-methyl-5,8-dihydropteridine-6,7-dione framework containing
hydrazone moiety exhibited potent synergistic antitumor effect. Pre-
liminary SARs indicated that the introduction of different substituted
aromatic rings had a significant influence on activity. Compounds
bearing halogen atoms (F, Cl, and Br) substituted phenyl ring showed
the significant antiproliferative activity, suggesting that the presence of
halogen atoms on phenyl ring was a key factor in the anticancer ac-
tivity. On the other hand, electron-withdrawing compounds displayed
excellent anti-tumor activities in the singular micromolar range against
A549 and HCT116 cells (L22 vs L3). Especially, the most promising
compound L19 exhibited significant potency against A549, HCT116 and
PC-3 cells with IC50 values of 3.23 μM, 4.36 μM and 8.20 μM, respec-
tively. But we also observed that compound L14 shows relatively high
activity even though it contains three electron-donating methyl groups.
Notably, compounds had significantly improved antiproliferative ac-
tivity with inserting another identical substituent into the aromatic ring
(L23 vs L28, L14 vs L16 vs L3). This indicated that incorporating si-
multaneously favorable substitutions could provide additive effects.
The para -methoxy derivative L18 and the corresponding meta-sub-
stituted analog L8 showed similar potency against A549, HCT116 and
PC-3 cells. However, moving the methoxy group to the ortho-position
led to a significant reduction in antiproliferative activity (L17 vs L8, L18).
Decrease in activity may be due to steric hindrance, resulting in the
hydrophobic pocket space collision. These proved that para- and meta-
position were more conducive to increasing activity than ortho-position.
Further studies were performed to examine the effect of shifting the
benzene ring group to the aromatic heterocyclic group (L1 vs L12, L13).
The results indicating that aromatic heterocyclic modification can
preserve anti-proliferative activity.
In order to preliminary study the molecular mechanism of action,
cell apoptosis analysis was performed on the HCT116 cells treating with
1.0 μM, 3.0 μM and 9.0 μM of L19 for 24 h and using Annexin-V and
propidium iodide (PI) double staining by flow cytometry. As shown in
Fig. 5, compound L19 effectively induced apoptosis in a concentration-
dependent manner. Compound L19 proved to induce apoptosis by
38.3% as compared to 18.9% of apoptotic cells in the blank control
(Table 2).
Since migration was an important feature of metastatic cancers, the
effect of compound L19 on migration of HCT116 cells was studied by
the wound-healing assay. As shown in Fig. 6, the drug concentration of
L19 was 0.3 μM and 3 μM, respectively. Compared with the blank con-
trol group, the wound healing rate decreased with the prolongation of
the action time and L19 significantly inhibited the wound healing in a
concentration-dependent manner.
To investigate the effect of optimal compound L19 on the mitotic
cycle of cell, cell cycle analysis of HCT116 cells treated with L19 at
indicated concentrations (0.3, 1.0, 9.0 μM) for 24 h, were fixed and
performed using Annexin-V and propidium iodide (PI) and the DNA
content was analyzed by flow cytometry. The results were compared
with HCT116 cells in non-treated control. As shown in Figs. 7.1 and 7.2,
treatment of HCT116 cells with L19 at 0.3, 1.0, and 9.0 μM concentra-
tions increased the percentage of G1-phase cells from 61.81% (as
control group) to 62.69%, 65.46%, and 67.2%, respectively. These re-
sults certificated that compound L19 markedly caused G1-phase arrest in
HCT116 cells.
In order to verify the rationality of the design and the mode of ac-
tion of these compounds and proteins, we used BI-2536/PLK1 co-crystal
structure (PDB code: 2RKU) as the docking model, and performed
molecular simulation docking of L19 with PLK1 (Fig. 8). In the protein
cavity, compound L19 (purple) and BI-2536 (green) were nearly
Overall, the most potent compound L19 showed promising cyto-
toxicity against A549, HCT116, and PC-3 cell lines with IC50 values of
3.23 μM, 4.36 μM and 8.20 μM, respectively. L19 demonstrated the
3

Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Table 1
Structures and anti-proliferative activities of compound L1–L30
.
a
b
Compound
Aromatic ring
IC50 (μM)
SD
A549
HCT116
> 100
PC-3
L1
L2
L3
> 100
48.20
0.2
> 100
72.52
0.04
> 100
53.59
34.50
0.02
> 100
0.02
L4
L5
6.89
9.41
0.03
0.02
9.81
9.02
0.01
8.42
8.55
0.04
0.02
0.03
L6
L7
84.45
97.78
0.04
86.59
59.30
0.04
26.59
85.15
0.02
0.08
0.01
0.04
0.02
0.02
0.01
L8
27.43
45.75
20.68
L9
40.58
22.92
0.02
0.02
29.04
27.10
0.04
0.04
23.61
17.72
0.05
0.01
L10
L11
39.05
0.05
0.01
26.36
0.02
17.20
0.01
L12
L13
L14
> 100
> 100
12.03
78.74
52.59
7.92
0.08
0.04
72.16
75.63
13.88
0.04
0.08
0.02
0.01
(continued on next page)
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Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Table 1 (continued)
a
b
Compound
Aromatic ring
IC50 (μM)
SD
A549
HCT116
13.80
PC-3
L15
10.99
0.02
0.01
0.04
15.18
0.03
0.02
L16
30.18
0.02
62.93
39.03
L17
> 100
> 100
> 100
L18
L19
> 100
3.23
36.11
4.36
0.05
26.25
8.20
0.02
0.04
0.01
0.05
L20
L21
L22
L23
L24
L25
> 100
6.59
37.24
8.39
0.04
36.30
9.25
0.04
0.02
0.01
0.01
12.30
31.21
12.84
8.78
0.04
15.23
20.73
17.71
5.61
0.01
0.08
0.02
20.32
27.59
11.58
9.26
0.03
0.02
0.02
0.1
0.02
0.01
0.04
0.02
L26
18.27
14.10
0.02
0.02
22.87
9.01
0.01
17.50
21.03
0.01
0.06
L27
0.03
(continued on next page)
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Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Table 1 (continued)
a
b
Compound
Aromatic ring
IC50 (μM)
SD
A549
HCT116
19.77
PC-3
L28
L29
L30
18.31
9.86
0.01
0.01
0.02
13.17
0.04
0.02
0.04
14.03
16.31
12.89
0.05
0.08
6.15
2.03
0.04
7.18
0.46
0.04
BI2536
0.01
0.04
0.01
a
b
Values are the means of at least three independent experiments.
SD: standard deviation.
Fig 4. Enzymatic activities of the target compounds.
Fig 5. Compound L19 induced apoptosis in HCT-116 cells. Cells were treated with various concentrations of L19 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
overlapped, indicating that the modes of binding of the two were ba-
Percent of cell death induced by compound L19 (9.0 μM and 0 μM) on HCT116
sically the same (Fig. 8D). As shown in Fig. 8B, the carbonyls at posi-
tions 6 and 7 of the compound L19 formed three hydrogen bonds (blue)
with His166, Asp194, and Lys83 via water molecules. At the same time,
the bidentate hydrogen bonding of the aminopyrimidine moiety at
position 2 was retained, thereby ensuring its inhibitory activity on
PLK1. The introduction of hydrazone group did change the position of
the aromatic ring and protein (Fig.8D). This may be the reason why the
meta- and para-substituted compound on the benzene ring has good
cells.
Concentrations L19
Apoptosis (%)
Total
Necrosis (%)
Early
Late
9.0 μM
0 μM
38.3
18.9
20.3
7.9
18.0
11.0
0.4
0.2
6

Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Fig. 6. In vitro wound healing assay on HCT116 cells. Phase contrast images were obtained by the treatment of compounds L19 at indicated concentrations for 0, 24
and 48 h.
Fig. 7.1. Cycle distribution of HCT116 cell lines. HCT116 cells were treated with different concentrations of L19 for 24 h (A: control, B: 0.33 μM, C: 1.0 μM and D:
9.0 μM).
activity. On the other hand, in addition to the Pi-cation interactions of
the aromatic ring portion of compound L19 with Arg136 and Leu59, the
fluorine atom in its trifluoromethyl group forms a hydrogen bond with
Arg136, Arg57 and Leu59. This provides unique ideas for follow-up
research.
In this study, we have designed and synthesized a series of novel
pteridinone derivatives possessing a hydrazone moiety based on the
scaffold of PLK1 inhibitor BI-2536. The results of antitumor activity
indicated that L19 obviously exhibited inhibitory activity against A549,
HCT116 and PC-3 cell lines with IC50 values of 3.23 μM, 4.36 μM, and
8.20 μM, respectively. The results of molecular docking and in vitro
enzymatic studies showed that PLK1 maybe a drug target for compound
L
19. Furthermore, to clarify the antiproliferative activity mechanism,
flow cytometry and wound healing tests confirmed that L19 induces
apoptosis. Finally, the cell cycle analysis of L19 by flow cytometry
showed that compound L19 arrested in the G1 phase cell cycle. In
conclusion, SARs research and pharmacological analysis of novel
pteridinone derivatives indicated that L19 as a promising candidate is
worth of further study.
Fig. 7.2. Quantitative analysis of cell cycle distributions; (A) Non-treated cells
as control group; (B) treated with L19 at 0.33 μM; (C) treated with L19 at 1.0 μM;
(D) treated with L19 at 9.0 μM.
7

Z. Li, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127329
Fig. 8. The binding models of L19 with PLK1. (A, B) Predicted binding conformation for L19 (purple sticks) in the binding site cavity of PLK1 (PDB code: 2RKU). (C)
2D diagram of the interaction between L19 and the binding site cavity of PLK1 (D) L19 overlapping with BI-2536 (green sticks).
Declaration of Competing Interest
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127329.
8