Structure-based design and SAR development of novel selective polo-like kinase 1 inhibitors having the tetrahydropteridin scaffold

Article abstract of DOI:10.1016/j.ejmech.2019.111769

Polo-like kinase 1 (Plk1) is a validated target for the treatment of cancer. In this report, by analyzing amino acid residue differences among the ATP-binding pockets of Plk1, Plk2 and Plk3, novel selective Plk1 inhibitors were designed based on BI 2536 a

Full text of DOI:10.1016/j.ejmech.2019.111769

European Journal of Medicinal Chemistry 184 (2019) 111769
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Structure-based design and SAR development of novel selective polo-
like kinase 1 inhibitors having the tetrahydropteridin scaffold
,
b
,
,
b
,
,
, b
Xiao Lv a 1, Xiaoxiao Yang a 1, Mei-Miao Zhan b, Peichang Cao a b, Shihong Zheng a
,
Ruijun Peng b, Jihong Han a b, Zhouling Xie a **, Zhengchao Tu c
,
,
,
b,
, d, ***
Chenzhong Liao a
, b, *
a Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, China
b School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China
c International Cooperative Laboratory of Traditional Chinese Medicine Modernization, Innovative Drug Development of Chinese Ministry of Education,
College of Pharmacy, Jinan University, Guangzhou, 510632, China
d Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Polo-like kinase 1 (Plk1) is a validated target for the treatment of cancer. In this report, by analyzing
amino acid residue differences among the ATP-binding pockets of Plk1, Plk2 and Plk3, novel selective
Plk1 inhibitors were designed based on BI 2536 and BI 6727, two Plk1 inhibitors in clinical studies for
cancer treatments. The Plk1 inhibitors reported herein have more potent inhibition against Plk1 and
better isoform selectivity in the Plk family than these two lead compounds. In addition, by introducing a
hydroxyl group, our compounds have significantly improved solubility and may target specific polar
residues Arg57, Glu69 and Arg134 of Plk1. Moreover, most of our compounds exhibited antitumor ac-
tivities in the nanomolar range against several cancer cell lines in the MTT assay. Through this structure-
based design strategy and SAR study, a few promising selective Plk1 inhibitors having the tetrahy-
dropteridin scaffold, for example, L34, were identified and could be for further anticancer research.
© 2019 Elsevier Masson SAS. All rights reserved.
Received 2 August 2019
Received in revised form
2 October 2019
Accepted 6 October 2019
Available online xxx
Keywords:
Plk1 inhibitor
Isoform selectivity
Structure-based drug design
Structure activity relationship
Anticancer
1. Introduction
box domain (PBD) [9,10], share a high level of structural and
sequential similarity. Whereas, Plk4-5 are structurally the most
The family of Polo-like kinases (Plks) has five members, Plk1-5,
which are serine/threonine kinases of the cell cycle in mammalian
cells and play important roles in cell cycle regulation and are critical
targets for therapeutic invention, mainly in the field of cancer
[1e4]. Plk1 and Plk4 are targets for cancer therapy, and Plk3 is
considered to be a tumor suppressor, whereas, Plk2 has been
debated as a target to treat cancer or a tumor suppressor [5e7]. In
addition, Plk2 is considered as a possible target for Parkinson’s
disease [8].
distinct members of the family [11].
Plk1, a key mitotic regulator, is the most extensively studied and
characterized among Plk1-5 and has been validated as an broad-
spectrum anticancer target [12,13]. Plk1 overexpression is found
in up to 80% of malignancies including breast, non-small cell lung,
colorectal, prostate, pancreatic, papillary thyroid, ovarian, head and
neck and non-Hodgkin’s lymphoma. A few Plk1 inhibitors, such as
BI 2536 [14,15], BI 6727 (volasertib) [16], GSK 461364, GW843682X
[17,18], have advanced into clinical trials and showed encouraging
anticancer effects in many kinds of tumors [2,13,19]. Among these
promising anticancer agents, BI 6727 has reached phase III trials
and showed inspiring results and therefore was awarded break-
through drug status in 2013 and orphan drug status for acute
myeloid leukemia in 2014. Nevertheless, no Plk1 inhibitors have
been licensed by the FDA for cancer treatment.
Plk1-3, possessing two different functionally targeted sites: N-
terminal catalytic domain (NCD) and C-terminal noncatalytic polo-
* Corresponding author. Hefei University of Technology, China.
** Corresponding author. Hefei University of Technology, China.
*** Corresponding author. Jinan University, China.
Drug solubility is a major challenge for drug development.
Especially, anticancer drugs are notorious for their poor solubility
and are hard to design oral dosage forms [20]. BI 2536 and BI 6727
E-mail addresses: zhoulingxie@hfut.edu.cn (Z. Xie), tu_zhengchao@gibh.ac.cn
(Z. Tu), czliao@hfut.edu.cn, chenzhongliao@gmail.com (C. Liao).
1
These authors contributed to this work equally.
https://doi.org/10.1016/j.ejmech.2019.111769
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
have been taken via intravenous drip infusion in the clinical trials
and their solubilities have limited their applications. Therefore,
developing potent selective Plk1 inhibitors with satisfactory solu-
bility is extremely desired in this field.
Plk3 (PDB ID: 4B6L) and noticed there are differences for the resi-
dues around the eOCH3 group (see Fig. 1A) among the ATP-binding
pockets of Plk1-3. Two most noticeable variances are: (1) Tyr161 of
Plk2, in which both of the equivalent ones of Plk1 and Plk3 are
leucine respectively; (2) Arg134 of Plk1, in which both of the cor-
responding residues of Plk2 and Plk3 are serine. We assumed that
an extended polar group from the eOCH3 of BI 2536 may form
hydrogen bonds with part or all of these three polar residues of
Plk1, whereas, it may conflict with Tyr161 of Plk2, and does not
form hydrogen bonds with Ser163 (Plk2) and Ser143 (Plk3) because
of the distances. Hydroxyl is both a hydrogen bond donor and
acceptor, and it has great impact on aqueous solubility of com-
pounds. Therefore, it is reasonable to extend a eOH from the
eOCH3 of BI 2536 and we designed novel Plk1 inhibitors and did
SAR development according to Fig. 2 for the purpose to discover
highly potent selective Plk1 inhibitors with improved solubility.
A supposed compound was docked into the ATP binding site of
Structure-based drug design (SBDD) has played great roles for
drug discovery [21,22]. In our previous work, we have successfully
obtained novel selective Plk2 inhibitors with high potency by
employing a SBDD strategy, mainly by analyzing critical amino acid
residue differences in the binding sites among Plk1, Plk2 and Plk3
[5]. This work reports our similar efforts of identifying more potent
and more soluble selective Plk1 inhibitors than BI 2536 and BI 6727,
two drugs serving for lead compounds herein. Compared with BI
2536, several Plk1 inhibitors with improved solubility, Plk1 inhi-
bition, isoform selectivity, and antiproliferative activity were yiel-
ded through this strategy.
2. Results
€
Plk1 by employing Glide 6.7 in the Schrodinger Suite. The modeling
results verified our assumptions: the extended eOCH2CH2OH
group forms three possible hydrogen bonds with Arg57, Glu69 and
Arg134 (Fig. 1B), whereas, this eOCH2CH2OH group would conflict
with the bulky Tyr161 of Plk2, and only forms one possible
hydrogen bond with Glu78 of Plk3. This result inspired us and we
synthesized many compounds and assayed their potency against
Plk1-3.
2.1. Structure-based design of selective Plk1 inhibitors
It was reported that the eOCH3 group of BI 2536 is an important
specificity determinant against non-Plks by taking advantage of a
small pocket generated by Leu132 in the hinge region of Plk1 [23].
However, we observed that in the close proximity of this eOCH3
group in the ATP binding site of Plk1 (PDB ID: 2RKU [23]), there are
three charged polar residues: Arg57, Glu69, and Arg134. Extending
a polar group from the eOCH3 of BI 2536 to interact with these
three residues, the potency against Plk1 and isoform or kinome
selectivity may be increased. We further aligned kinase-inhibitor
complexes of Plk1 (PDB ID: 2RKU), Plk2 (PDB ID: 4I6F [8]) and
2.2. Biological evaluation
Compounds were assayed for their Plk1-3 inhibitory activities
and isoform selectivity among Plk1-3 using the FRET-based Z0-Lyte
assay system. More details about this assay can be found in the
Supporting Information.
Fig. 1. (A) Differences of the residues among the active sites of Plk1 (green), Plk2
(orange) and Plk3 (grey). BI 2536 binding to Plk1 is shown as green ball and stick
mode. (B) Modeling results indicated that the extended eOCH2CH2OH group could
form three possible hydrogen bonds with Arg57, Glu69 and Arg134 of Plk1. (For
interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
Fig. 2. Design strategy and SAR development of novel Plk1 inhibitors which may
interact with three proximal polar residues, Arg57, Glu69 and Arg134 of Plk1, around
the eOCH3 of BI 2536.

X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
3
Table 1
Enzyme inhibition and isoform selectivity of compounds L1 e L39 against Plk1-3.a.
Compd
R1
R2
R3
R4
X
IC50 (nM) for Plks
Selectivity against Plk1
Plk1
Plk2
3.98
Plk3
6.04
Plk2/Plk1
0.28
Plk3/Plk1
L1
L2
L3
L4
L5
L6
L7
-OCH3
-OCH3
-OCH3
-OCH3
-OH
Me
-H
-H
-H
-H
-H
-H
-H
13.97
0.43
-CH2CH2OH
291.6
1200
52.06
5.89
3474
>10
>10
m
m
M
M
11.73
>8.33
1.46
>33.77
>8.33
57.84
5.16
-CH2OCH3
-H
>10 mM
75.77
7.98
4.72
3011
30.40
18.91
1093
Me
1.35
-CH3
Me
5.75
0.82
3.29
Me
884.6
1796.9
898.4
2.03
1.24
L8
Me
-H
344.7
1252
2.61
3.63
L9
Me
Me
-H
-H
10.17
9.31
36.04
152.0
112.8
452.0
3.54
11.09
48.55
L10
16.32
L11
L12
L13
Me
Me
Me
-H
-H
-H
4.27
9.42
17.29
338.7
230.9
743.9
554.5
150.2
205.2
79.32
24.43
43.02
129.86
15.95
11.87
L14
L15
L16
L17
L18
L19
L20
L21
L22
L23
L24
CH3-
NH2-
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
5.76
131.7
290.5
195.9
314.2
85.11
640.8
626.2
498.0
873.6
385.3
265.1
91.93
162.8
157.4
406.9
142.5
476.7
385.3
440.6
1351
166.1
699.6
22.86
14.02
12.51
14.70
12.41
21.14
15.37
14.25
41.19
21.53
6.974
15.96
7.86
20.72
15.66
21.38
6.86
10.05
19.03
20.78
15.73
9.45
30.31
40.74
34.94
21.21
17.90
38.02
12.61
63.70
9.28
18.40
(continued on next page)

4
X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
Table 1 (continued )
Compd
R1
R2
R3
R4
X
IC50 (nM) for Plks
Selectivity against Plk1
Plk1
Plk2
Plk3
Plk2/Plk1
Plk3/Plk1
L25
L26
L27
L28
L29
L30
L31
L32
L33
L34
L35
L36
L37
L38
L39
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
-H
-H
-H
-F
-F
-F
-F
-F
-F
-F
-F
-F
-F
-F
-F
126.3
>10
m
m
m
M
M
M
>10
m
m
m
M
M
M
>79.18
>79.18
>80.78
>56.50
32.57
16.50
4.39
123.8
177.0
3.58
>10
>10
>10
>10
>80.78
>56.50
34.13
11.90
10.75
13.39
4.70
122.2
61.17
107.2
107.0
33.83
643.0
30.51
167.3
397.3
3180
718.3
868.7
116.6
84.80
43.78
46.93
45.07
436.2
22.14
196.0
366.3
2977
749.7
5.14
9.97
7.99
5.87
7.20
6.26
7.28
88.32
7.84
59.92
5.69
3.89
10.17
20.00
126.8
26.77
52.61
16.45
19.87
25.08
26.83
16.51
19.27
18.32
23.48
28.01
18.51
974.33
Staurosporine
BI 2536
e
e
e
e
e
864.6
7.06
957.2
13.55
>10
20.78
m
M
1.107
1.92
>11.57
2.94
-OCH3
Me
-H
a
Each IC50 is the mean SEM from three experiments.
In our assay, BI 2536, as the positive control compound, received
IC50 values of 7.06, 13.55, and 20.78 nM against Plk1-3 respectively
(see Table 1), which is consistent with the reported values [23],
implying that BI 2536 is a potent but not so selective Plk1 inhibitor.
Another control compound, staurosporine, a natural prototypical
ATP-competitive kinase inhibitor, inhibited Plk1-3 with IC50s of
which the eOCH3 is modified to eOCH2CH2OH. It got its IC50 value
of 4.27 nM against Plk1, ~1.7 folds increment over BI 2536. It
impressed us more with its remarkably improved isoform selec-
tivity to Plk2 (79.32 versus 1.92) and Plk3 (129.86 versus 2.94)
when compared with BI 2536, which confirms our design strategy
is reasonable. Based on L11, we then further modified R2 to different
rings or linear fatty chains and got compounds L12 e L24, among
which, compound L14 demonstrated the best Plk1 inhibition
(IC50 ¼ 5.76 nM) and good isoform selectivity. Compounds L25 e
L27 were yielded by modifying R3 from cyclopentyl to isopropyl
based on compounds L10 e L12, respectively. BI 6727 contains an
isopropyl group, however, this modification led to dramatic drop-
ping of the inhibition against all of Plk1-3 in our study.
Incorporation of fluorine into molecules can modulate their
pharmacokinetics properties, including lipophilicity, electrophi-
licity, metabolic stability, chemical stability, etc., and the strategic
incorporation of fluorine to improve drug potency has become
gradually prevalent in drug discovery [24]. The benzene ring of BI
2653 is sandwiched by Leu59 and Arg136 in the active site of Plk1.
Introducing a fluorine atom into the 2-position of the benzene may
increase the hydrophobic interactions of the designed compounds
864.6 nM, 957.2 nM and more than 10 mM. We initiated our project
by doing a simple SAR by modifying R1, R2, R3 and R4 of the scaffold
(see Table 1). When 1-methylpiperidinyl (R2) of BI 2536 was altered
to cyclopentyl, the activity of compound L1 against Plk1 dropped ~2
folds, and L1 completely lost selectivity. When changing the methyl
group (R4) to eCH2CH2OH (L2), eCH2OCH3 (L3), or just chopping it
(L4), the activities against Plk1 dropped a lot. When altering the
eOCH3 group (R1) to smaller groups such as eOH (L5) or eCH3 (L6),
the inhibition was recovered. Bulky apolar groups (L7 and L8) in
this position led to dramatically dropped inhibitory activities
against all of Plk1-3. This is not surprised because these groups may
conflict with the surrounding residues in the active sites of all Plk1-
3. When R1 is eCH2CH2OH (L9) and eOCH2CH2OH (L10), these two
compounds received improved Plk1 inhibition and isoform selec-
tivity. Compound L11 is the corresponding compound of BI 2536 in

X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
5
Table 2
with Leu59 and Arg136 of Plk1. In addition, the 2-F would form a
possible weak hydrogen bond with the 1-amide attached into this
benzene, and thus configurate a more suitable binding conforma-
tion, which will result in improved potency. Hence, we incorpo-
rated a fluorine in the 2-position of the core, and yielded
compounds L28 e L39. Indeed, through this strategy, the inhibition
against Plk1 was enhanced. For example, L34 got an IC50 value of
3.89 nM against Plk1, in contrast to 21.21 nM of its precursor L22.
Overall the isoform selectivity of L28 e L39 dropped a little bit.
Plk1 is overexpressed in up to 80% of malignancies, and was
identified as a broad-spectrum anticancer target. Therefore, we
investigated the antiproliferative activities of these 39 compounds,
which were tested using six human tumor cell lines: K562 (human
immortalized myelogenous leukemia cell line), MCF-7 (breast
cancer cell line), HuH-7 (hepatocellular carcinoma cells), A549
(human lung cancer cell line), H1975 (human lung carcinoma cell
line), and Hela (human cervical cancer cell line). As the control
compound, BI 2536 received GI50 values of 60.5, 67.0, 28.4, 57.1,
130.0, 34.0 nM against each of them (see Table 2).
Generally, many of our compounds showed good cytotoxic ac-
tivities in the nanomolar range. Among them, compounds L5, L6,
L9, L11, L12, L14, L16, L18, L28, L29, L32, L34 demonstrated
equivalent or even better antiproliferative activities than BI 2536.
The most impressive compound is L34, which showed GI50 values
of 10.6, 28.2, 24.9, 20.2, 107.9, 8.03 nM against K562, MCF-7, HuH-7,
A549, H1975 and Hela, respectively, 1e6 folds improvement over BI
2536 (see Fig. 3). In addition, L34 got GI50 values of 9.47, 6.71,
21.0 nM against other three human cancer cell lines: DU145
(prostate cancer cell line), HT29 (colon adenocarcinoma cell line)
and HL60 (leukemia cell line). The anticancer activities of our
compounds were generally consistent with the IC50 values against
Plk1, verifying the correlation between the in vitro Plk1 inhibitory
activity and cellular cytotoxicity. Incorporation of a fluorine in the
2-position of benzene ring of the core not only improved the in-
hibition against Plk1, but also had good impact on the anti-
proliferative activities. For example, when comparing compound
L22 with L34, it is found that L34 had 17e111 folds improvement to
kill the six cell lines. The replacement of cyclopentyl in the position
R3 by an isopropyl (compounds L25 e L27, L37 e L39) led to seri-
ously worsened antiproliferative activities in our study.
Antiproliferative activities of compounds L1 e L39 against six different human
cancer cell lines.a,b,c,d
Compd
GI50 (nM) to cancer cell lines
K562
MCF-7
HuH-7
A549
H1975
Hela
L1
L2
L3
L4
L5
L6
L7
L8
84.0
4437
>10
809.7
10.7
85.5
3722
>10
744.3
6.25
70.4
4467
6757
429.5
12.2
36.8
322.5
1145
25.5
109.8
71.3
19.9
216.2
56.0
128.6
52.4
81.6
65.9
196.8
NA
64.6
NA
3831
64.2
4820
>10 mM
727.9
9.74
5020
8131
729.4
10.3
mM
m
M
>10
mM
1104
20.2
40.0
NA
23.1
24.7
23.1
20.8
958.4
1983
57.4
91.3
69.6
63.8
65.8
38.1
132.3
46.9
722.6
1802
64.9
98.9
61.5
64.3
140.4
107
251.8
135.1
331.0
162.3
269.0
196.8
153.6
871
207.7
312.0
1818
>10
1273
81.4
572.9
968.1
76.8
71.2
36.0
79.4
138.3
87.6
446.7
84.4
197.1
79.3
321.9
470.0
240.4
540
686.0
282.7
2590
466.4
1504
45.1
50.7
29.2
41.9
122.5
42.0
118.0
25.4
82.3
NA
L9
196.6
157.9
130.9
166.0
170.6
220
1197
361.3
519.2
677.1
676.5
NA
NA
3810
NA
3200
2366
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
L20
L21
L22
L23
L24
L25
L26
L27
L28
L29
L30
L31
L32
L33
L34
L35
L36
L37
L38
L39
BI 2536
98.9
47.4
24.7
143.1
239.1
182.5
187.2
188.3
58.2
1249
6501
772.6
24.6
129.0
152.9
117.1
892.3
187.1
132.8
1117
>10 mM
1748
13.1
33.4
47.14
39.4
28.4
123.1
8.03
NA
876
NA
239.1
1449
mM
>10
m
M
>10
m
M
>10
mM
721.7
66.9
3441
44.0
67.9
171.0
98.3
67.6
3547
501.2
164.9
509.6
252.6
127.5
567.2
107.9
2470
1151
5133
2118
1917
130.0
60.0
43.8
79.3
120.2
86.3
45.2
222.5
130.0
30.6
113.3
140.6
92.4
265.2
10.6
158.4
28.2
349.1
24.9
352.1
20.2
1071
100.1
730.7
540.2
915.1
60.5
907.7
404.7
1546
833.1
931.2
67.0
830.6
231.9
806.7
884.9
1107
28.4
1193
172.2
1717
852.0
1170
57.1
225.1
36.0
434.9
274.5
351.4
34.0
a
BI 2536 was used as the positive control compound.
b
Inhibition of cell growth by the listed compounds was determined by using CCK-
8 assay.
c
2.3. Chemistry
NA: not active up to the highest concentration tested.
Standard error of the GI50 was generally less than 10%.
d
The synthesis of compounds L1 e L6 is illustrated in Scheme 1.
Intermediates dihydropyridinone derivatives were synthesized
according to the method in a previous study of ours [5]. Acylation of
materials 1e3 using a cyclopentylamine afforded intermediates a1
e a3. Nitro reduction of b1 e b3 using iron powder, followed by a
Buchwald-Hartwig reaction, yielded compounds L1 e L6.
The synthesis of compounds L7 e L27 is depicted in Scheme 2.
Commercially available 3-hydroxy-4-nitrobenzoic acid (4) was
converted to intermediates c1 e c14 by an amide reaction. Then,
their nitro groups were reduced to offer intermediates d1 e d14,
which were converted to intermediates e1 e e17 using Buchwald-
Hartwig coupling sequentially. e1 e e17 were further reacted with
various substituted bromine derivatives, respectively, leading to
compounds L7 e L27 by Mitsunobu reaction.
As shown in Scheme 3, compounds L28 e L39 were synthesized
from 3-fluoro-4-nitrobenzonic acid (5), which was reacted with
ethylene glycol to yield intermediate 6, which then was converted
to intermediates f1 e f9 by reacting with several various amides. f1
e f9 were further reduced to intermediates g1 e g9. Compounds
L28
BuchwaldꢀHartwig coupling.
Several of our compounds were calculated for their aqueous
e L39 were obtained from intermediates g1 e g9 by
Fig. 3. Comparison of antiproliferative activities of BI 2536 (blue bars) and L34 (yellow
bars) against nine human cancer cell lines. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)

6
X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
Scheme 1. Synthesis of Compounds L1 e L6. Regents and conditions: I) Amine, HBTU, Et3N, DMF, rt, 12 h; II) AcOH, Fe, 60 ꢁC, 2 h; III) Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 110 ꢁC,
16 h.
Scheme 2. Synthesis of Compounds L7 e L27. Regents and conditions: I) Amine, HBTU, Et3N, DMF, rt, 12 h; II) AcOH, Fe, 60 ꢁC, 2 h; III) Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 110 ꢁC,
16 h; IV) Benzyl bromide, 1-bromo-2-ethyl methyl ether or 2-bromoethanol, K2CO3, acetone, 60 ꢁC, 12 h.
solubility (logS) and permeability (logP) using QikProp 4.4 and MOE
2014. It is clearly demonstrated that compounds containing an
extra eCH2OH group, such as L11, have better aqueous solubility
and lower logP values than the corresponding compounds, such as
BI 2536 (see Table 3).
The calculation was verified by our further measurement of
aqueous solubility of compounds L5, L6, L11, L28, L29, L32, L34, and
BI 2536 by using UVevisible spectrophotometer in PBS buffer (pH
7.4). The UVevis absorption spectra of the various concentration of
four compounds are shown as examples in Fig. 4. BI 2536 has an
aqueous solubility of 0.73 mg/mL. Introducing an extra eCH2OH
(L11) from the methoxyl of BI 2536 remarkably improved the sol-
ubility by ~1.6 fold. Incorporation of a fluorine atom into the 2-
position of L11 dramatically deteriorated the solubility (L28,
0.53 mg/mL). However, L34, the compound having a fluorine atom
and exhibiting the best biological activites both in the kinase and
cell line assays, has an aqueous solubility value of 2.23 mg/mL,
demonstrating our strategy to improve the Plk1 inhibition, selec-
tivity among Plk1-3 and solublitiy is fruitful.
3. Conclusion
Plk1, a serine/threonine protein kinase, is an important regu-
lator of cell cycle progression. Overexpression of Plk1 is often
associated with oncogenesis, therefore, Plk1 is widely recognized
as an oncogene and hence represents an attractive target for cancer

X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
7
Scheme 3. Synthesis of Compounds L28 e L39. Regents and conditions: I) Ethylene glycol, KOH, 80 ꢁC, 8 h; II) Amine, HBTU, Et3N, DMF, rt, 12 h; III) AcOH, Fe, 60 ꢁC, 2 h; IV)
Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 110 ꢁC, 16 h.
Table 3
Predicted and measured aqueous solubility (logSa) and permeability (logPb).
Compd
QikProp 4.4
QPlogS
MOE 2014
logS
Measured solubility (mg/mL)
CIQPlogS
QPlogPo/w
SlogP
logP(o/w)
L5
L6
ꢀ6.35
ꢀ6.90
ꢀ4.87
ꢀ5.15
ꢀ5.73
ꢀ5.90
ꢀ3.53
ꢀ5.36
ꢀ6.00
ꢀ6.31
ꢀ5.02
ꢀ5.37
ꢀ6.28
ꢀ6.08
ꢀ4.17
ꢀ5.13
3.83
4.69
2.93
3.09
3.18
3.64
2.18
3.65
ꢀ5.22
ꢀ5.74
ꢀ4.92
ꢀ5.22
ꢀ5.46
ꢀ5.42
ꢀ4.71
ꢀ5.13
4.10
4.71
2.92
3.06
2.57
3.37
2.21
3.56
3.78
4.39
2.35
2.53
3.14
2.81
1.40
3.04
0.23
<0.07
1.14
0.53
0.18
<0.15
2.23
0.73
L11
L28
L29
L32
L34
BI 2536
a
Predicted aqueous solubility. S in mol$dmꢀ3
Predicted octanol/water partition coefficient.
.
b
intervention. A few Plk1 inhibitors have been entered into clinical
trials, and among them, BI 6727 was awarded breakthrough drug
status, however, no Plk1 inhibitors have been approved for cancer
treatment yet.
To develop more soluble, potent and selective Plk1 inhibitors, in
this report, by taking advantage of residue differences among the
ATP-binding pockets of Plk1, Plk2 and Plk3, and employing a SBDD
strategy and using BI 2536 and BI 6727 as the template compounds,
we designed and synthesized a series of Plk1 inhibitors having the
tetrahydropteridin scaffold, which may target three specific polar
residues Arg57, Glu69 and Arg134 of Plk1.
lity, Plk1 inhibition, isoform selectivity, and antiproliferative ac-
tivity than BI 2536, the most illustrious Plk1 inhibitor. Compound
L34 stood out from the compounds reported herein. It received an
IC50 value of 3.89 nM against Plk1 and good selectivity over Plk2
and Plk3. This compound showed impressive GI50 values of 10.6,
28.2, 24.9, 20.2, 107.9, 8.03, 9.47, 6.71, 21.0 nM against nine cancer
cell lines, K562, MCF-7, HuH-7, A549, H1975, Hela, DU145, HT29,
and HL60 respectively in the antiproliferative activity assay.
Remarkably, the aqueous solubility of this compound had ~3 folds
improvement over BI 2536. All these imply that L34 is a good
candidate for further anticancer research.
Our strategy yielded several Plk1 inhibitors with better solubi-

8
X. Lv et al. / European Journal of Medicinal Chemistry 184 (2019) 111769
Fig. 4. UVevis absorbance. (A)e(D): Absorption spectra of BI 2536, L11, L28 and L34 at various concentrations.
V.R. Pallela, P. Ramkumar, R. Jain, A.K. Aggarwal, E.P. Reddy, Discovery of 2-
Acknowledgments
(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]
pyrimi din-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2
(PLK2), Bioorg. Med. Chem. 24 (2016) 521e544.
This work was financially supported by the National Natural
Science Foundation of China (21672050, 81803352 and 21572003)
and Fundamental Research Funds for the Central Universities
(JZ2018HGBZ0167 and JZ2018HGTA0225). The authors declare no
other conflicts of interest.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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