Identification of highly potent and selective Cdc25 protein phosphatases inhibitors from miniaturization click-chemistry-based combinatorial libraries

Article abstract of DOI:10.1016/j.ejmech.2019.111696

Cell division cycle 25 (Cdc25) protein phosphatases play key roles in the transition between the cell cycle phases and their association with various cancers has been widely proven, which makes them ideal targets for anti-cancer treatment. Though several Cdc25 inhibitors have been developed, most of them displayed low activity and poor subtype selectivity. Therefore, it is extremely important to discover novel small molecule inhibitors with potent activities and significant selectivity for Cdc25 subtypes, not only served as drugs to treat cancer but also to probe its mechanism in transitions. In this study, miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening were used to discover selective Cdc25 inhibitors. The bioassay results showed that compound M2N12 proved to be the most potent Cdc25 inhibitor, which also act as a highly selective Cdc25C inhibitor and was about 9-fold potent than that of NSC 663284. Moreover, M2N12 showed remarkable anti-growth activity against the KB-VIN cell line, equivalent to that of PXL and NSC 663284. An all-atom molecular dynamics (MD) simulation approach was further employed to probe the significant selectivity of M2N12 for Cdc25C relative to its structural homologs Cdc25A and Cdc25B. Overall, above results make M2N12 a promising lead compound for further investigation and structural modification.

Full text of DOI:10.1016/j.ejmech.2019.111696

Journal Pre-proof
Identification of highly potent and selective Cdc25 protein phosphatases inhibitors
from miniaturization click-chemistry-based combinatorial libraries
Lanlan Jing, Gaochan Wu, Xia Hao, Fisayo A. Olotu, Dongwei Kang, Chin Ho Chen,
Kuo-Hsiung Lee, Mahmoud E.S. Soliman, Xinyong Liu, Yuning Song, Peng Zhan
PII:
S0223-5234(19)30848-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111696
EJMECH 111696
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 July 2019
Revised Date: 3 September 2019
Accepted Date: 11 September 2019
Please cite this article as: L. Jing, G. Wu, X. Hao, F.A. Olotu, D. Kang, C.H. Chen, K.-H. Lee,
M.E.S. Soliman, X. Liu, Y. Song, P. Zhan, Identification of highly potent and selective Cdc25 protein
phosphatases inhibitors from miniaturization click-chemistry-based combinatorial libraries, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111696.
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Identification of highly potent and selective Cdc25 protein
phosphatases inhibitors from miniaturization click-chemistry-based
combinatorial libraries
Lanlan Jinga, Gaochan Wua, Xia Haoa, Fisayo A. Olotub, Dongwei Kanga, Chin Ho
Chenc, Kuo-Hsiung Lee d,e, Mahmoud E. S. Solimanb*, Xinyong Liua,*, Yuning Songf,*,
Peng Zhana,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012 Ji'nan, Shandong, PR China
b Molecular Bio-computation and Drug Design Laboratory, School of Health Sciences,
University of KwaZulu-Natal, Westville Campus, Durban 4001, South Africa.
cDuke University Medical Center, Box 2926, Surgical Oncology Research Facility,
Durham, NC 27710, USA
dNatural Products Research Laboratories, Eshelman School of Pharmacy, University
of North Carolina, Chapel Hill, North Carolina 27599, United States.
eChinese Medicine Research and Development Center, China Medical University and
Hospital, Taichung 40402, Taiwan.
fDepartment of Clinical Pharmacy, Qilu Hospital of Shandong University, 250012,
Ji’nan, China.
*Corresponding authors. E-mail address: Soliman@ukzn.ac.za (Soliman M.);
xinyongl@sdu.edu.cn
(Liu
X.Y.);
syncat827@163.com
(Song
Y.N.);
zhanpeng1982@sdu.edu.cn (Zhan P.)
1

Abstract
Cell division cycle 25 (Cdc25) protein phosphatases play key roles in the
transition between the cell cycle phases and their association with various cancers has
been widely proven, which makes them ideal targets for anti-cancer treatment.
Though several Cdc25 inhibitors have been developed, most of them displayed low
activity and poor subtype selectivity. Therefore, it is extremely important to discover
novel small molecule inhibitors with potent activities and significant selectivity for
Cdc25 subtypes, not only served as drugs to treat cancer but also to probe its
mechanism in transitions. In this study, miniaturized parallel click chemistry synthesis
via CuAAC reaction followed by in situ biological screening were used to discover
selective Cdc25 inhibitors. The bioassay results showed that compound M2N12
proved to be the most potent Cdc25 inhibitor, which also act as a highly selective
Cdc25C inhibitor and was about 9-fold potent than that of NSC 663284. Moreover,
M2N12 showed remarkable anti-growth activity against the KB-VIN cell line,
equivalent to that of PXL and NSC 663284. An all-atom molecular dynamics (MD)
simulation approach was further employed to probe the significant selectivity of
M2N12 for Cdc25C relative to its structural homologs Cdc25A and Cdc25B. Overall,
above results make M2N12 a promising lead compound for further investigation and
structural modification.
Keywords
Cdc25 inhibitors; anticancer agents; subtype selectivity; click chemistry.
1. Introduction
Although there are different types of cancers, with each cancer type having
diverse causes and various clinical symptoms, one of the most common features they
all share is a disordered cell cycle. Therefore, a major strategy in anti-cancer treatment
is to target molecules which regulate the cell cycle. The Cdc25 phosphatases are
highly conserved dual specificity phosphatases that play important roles in cell cycle
2

under normal conditions and in response to DNA damage [1]. They could activate
cyclin-dependent kinases (CDKs) through the dephosphorylation of pThr14 and
pTyr15 [2]. Cyclin-dependent kinases, the central regulators of the eukaryotic cell
cycle, could phosphorylate and activate downstream players like retinoblastoma (Rb)
protein to promote cell cycle progression [3]. Several studies have shown that the
Cdc25 phosphatases are overexpressed in a wide variety of cancers [4]. Therefore,
Cdc25 phosphatases have been demonstrated as promising targets for the discovery of
novel small molecule antitumor drugs [5-7].
Cdc25 phosphatases are present in all eukaryotes except plants [8]. There are
three subtypes of Cdc25 phosphatases including Cdc25A, Cdc25B and Cdc25C.
Cdc25A is thought to be regulators of G1/S and G2/M transition, and it could
dephosphorylate and activate the CDK2/cyclin A and CDK2/cyclin E complexes
which is necessary for mitosis [9]. Cdc25B and Cdc25C are regulators of G2/M
transition through their ability to dephosphorylate and activate Cdk1/cyclin A,
Cdck2/cyclin A and Cdk1/cyclin B [1]. Cdc25 phosphatases also play a vital role in
the checkpoint control in response to DNA damage induced by ionizing radiation (IR)
or ultraviolet light (UV). Especially, Cdc25C is a crucial downstream effector of
Chk1 response on account of its phosphorylation on Ser216 which can generate
14-3-3-mediated separation of the phosphatases away from their substrates and a
consequential balance of Cdk1/cyclin B within the phosphorylated and inhibited state
[10]. Previous research about FPD/AML (familial platelet disorder/acute myeloid
leukemia) has illustrated that mutated Cdc25C could disrupt the G2/M checkpoint and
promote malignant transformation [11]. It is also found that Cdc25C and
phospho-Cdc25C (Ser216) play a key role in the development and progression of
vulvar carcinomas [12]. Moreover, overexpression of Cdc25C and an alternatively
spliced variant were found in prostate cancer, but the role of Cdc25C and its spliced
variant is not clear [13]. Taken together, these results could present Cdc25C as a
novel target for the treatment of cancer while potent and selective Cdc25C inhibitors
could help disrupt the pathogenesis of these cancers.
Recently, many new Cdc25 phosphatase inhibitors were derived from a
3

para-quinonoid structure using VK3 as a lead compound. Besides, small molecule
Cdc25 phosphatase inhibitors derived from natural products have also served as
research hotspots [14-17]. Currently, although Cdc25 phosphatase inhibitors have
been developed, only few of them demonstrate inhibitory activities in vivo [18]. And
most of the Cdc25 phosphatase inhibitors demonstrated with low activity and poor
selectivity. Moreover, small molecule inhibitors with subtype selectivity can not only
serve as probe tools to study the mechanism of the targets, but also have a broad
application prospects in clinical drug development [19]. Above all, it is necessary to
discover novel Cdc25 phosphatase inhibitors with potent activity and significant
subtype selectivity. Previous research indicated that the privileged quinone structure
plays an important role to maintain the compounds inhibitory activity against Cdc25
phosphatase. Thus, with the aim to discover promising Cdc25 phosphatase inhibitors
with potent inhibitory activity and high subtype selectivity, we chose the
representative quinone inhibitors NSC 663284, NSC 668394 and Cpd5 as lead
compounds. For the catalytic domains of Cdc25 lack any apparent substrate
recognition site and their active site face is surprisingly flat, these lead to difficulties
in rational drug design according to structure-activity relationship [20]. Therefore, it
is necessary to introduce structural diversity of substituents to modify quinone
scaffold.
In order to rapidly generate a large number of diverse molecules, we employed
copper(I)-catalyzed azide–alkyne (3+2) dipolar cycloaddition (CuAAC) reaction to
yield 1,4-disubstituted-1,2,3-triazoles. The reaction is one of the extant click
reactions which is the most versatile, enabling exclusive formation of
1,4-disubstituted-1,2,3-triazoles in high yields under mild reaction conditions. Due to
its reliability, specificity, and biocompatibility, the 1,2,3-triazole structural motif is
regarded as a safe bioequivalent surrogate (nonclassical bioisostere) widely
employed in drug design [21]. Besides, previous research demonstrated that “click
chemistry” coupled with miniaturization synthesis and in-situ screening is a rapid,
reliable and efficient tool to discover novel compounds with different biological
activities [22]. Therefore, in this study, we devised a method of parallel click
4

chemistry synthesis via CuAAC reaction followed by in situ biological screening. In
this method, we introduced the alkyne functional group onto to the quinone skeletons
and yielded the privileged alkynyl fragments M1 and M2 (Fig. 1) and obtained a
variety of azide substituents. Then each reaction was carried out by incubating an
azide substituent with alkynyl fragment in 96-wall plates yielded 96-member
quinone derivatives library, and the reaction mixtures were used to test the inhibitory
activity against Cdc25s directly without purification. Further in vitro investigations
and molecular dynamics (MD) simulation led to the identification of compound
M2N12 as a promising lead compound for further investigation.
Figure 1. Discovery of Cdc25 phosphatase inhibitors based on click chemistry and in situ
screening strategy.
2. Results and discussion
2.1. Building and characterizing the libraries by click chemistry[23, 24]
5

The goal of this work was the synthesis of large libraries of quinone derivatives
to identify a suitable lead compound for the search of novel selective Cdc25C
inhibitors. Therefore, based on the detailed analysis of the structure-activity
relationship (SAR) of substituted quinone compounds demonstrated as Cdc25s
inhibitors, we reasoned that generating broad unbiased structural diversity might be
more powerful than incremental optimization for developing improved Cdc25s
inhibitors. Therefore, we retained the quinone scaffold, and focused on the
modification of side chain. At first, we designed two terminal alkynes M1 and M2
and a total of 48 substituents bearing azido groups (Fig. 2). Two terminal alkynes
were synthesized from commercially available starting materials (Scheme 1, Scheme
2). Briefly, menadione was reacted with prop-2-yn-1-amine (A1) to yield compound
M1, and compound M2 was prepared from 6,7-dichloroquinoline-5,8-dione (A2) by
nucleophilic substitution reaction. Among the 48 substituents, N33, N37-39
substituents were obtained from commercial sources, and the other substituents were
synthesized according to the existing methods. The synthesis of azide substituents is
shown in Scheme 3, Scheme 4, Scheme 5 and Scheme 6. N1-N22, N40 and N48
were obtained directly from the halogen-bearing substituents by nucleophilic
substitution with NaN3. The substituted phenylamine (A23-A27), morpholine (A34),
pyrrolidine (A35) or cyclopropanamine (A36) were converted to the intermediates
B23-B27 and B34-B36 by acylation, then the intermediates were reacted with NaN3
to provide the corresponding azide substituents N23-N27 and N34-N36. Further
oxidation of N22 in the presence of 3-chloroperbenzoic acid generated compounds
N41 and N42. Substituted aromatic boronic acid B28-B32 and B43-B47 were treated
with NaN3 by the catalytic CuSO4·5H2O to afford the corresponding azide
substituents N28-N32 and N43-N47, respectly. Finally, with azide substituents and the
privileged alkynyl fragments in hand, we performed click reactions by using the
optimized parallel synthesis condition in the presence of CuSO4·5H2O, sodium
ascorbate
and
1-(1-benzyltriazol-4-yl)-N,N-bis[(1-benzyltriazol-4-yl)methyl]methanamine (TBTA)
in DMSO and yield 96 quinone derivatives. The newly synthesized compounds were
6

characterized by MS and NMR spectra.
Scheme 1. Synthesis of M1. Reagents and conditions. (i): EtOH, r.t..
Scheme 2. Synthesis of M2. Reagents and conditions. (i): EtOH, r.t..
7

Scheme 3. Synthesis of azide substituents N1-N22, N40 and N48. Reagents and conditions. (i):
NaN3, DMF, r.t. or 70°C.
Scheme 4. Synthesis of azide substituents N23-27 and N34-N36. Reagents and conditions. (i):
Et3N, THF, 0°C; (ii): NaN3, DMF, r.t.
Scheme 5. Synthesis of N41 and N42. Reagents and conditions. (i): 3-Chloroperbenzoic acid,
CH2Cl2, 0 , 1h; (ii): 3-chloroperbenzoic acid, CH2Cl2, 15h at r.t..
.
Scheme 6. Synthesis of N28-32 and N43-N47. Reagents and Conditions. (i): NaN3, CuSO4 5H2O,
CH3OH.
8

N3
N3
N3
N3
N3
N3
N3
N3
N3
NO2
Cl
CN
Cl
NO2
N3
CN
CN
N3
N3
N3
N10
N3
N12
NO2
Cl
N9
N11
N5
N4
N7
N1
N3
N8
N6
N2
N3
O
O
N3
NH2
S
N3
O
N3
S
NH2
O
N3
N3
O
N17
N20
N16
N18
O
N19
N13
N15
N14
O
O
O
COOH
N3
N3
N3
N3
NH
NH
NH
NH
F
O
N3
O
S
N3
N
N3
N
N3
H
O
O
S O
Br
CF3
N21
N3
N24
OH
O
N26
N23
N25
N27
N22
N28
CH3
CH3
H3C
O
O
O
Cl
O
O
O
N
HN
N3
O
N3
N
O
O
N3
N
NH
N3
N3
COOH
N3
O
N3
O
O
N3
N30
N34
N29
N31
N35
N32
N36
H2N
N37
N33
N3
N3
O
OH
O
S
O
S
H3C
CH3
N3
N3
N3
O
O
N3
N3
O
O
O
N
CH3
N43
N3
N45
N3
N46
N44
N41
N42
N40
N38
N39
N3
O
B
N3
HO
OH
N47
N48
Figure 2. Structures of diverse azide substituents.
2.2. Generation of the combinatorial library
A 96-member quinone derivatives library was constructed in in microtiter plates
and in millimolar scale. Refer to our previous established reaction conditions by
CuAAC click chemistry (Table 1), we selected CuSO4·5H2O as a catalyst and
performed the click chemical reaction according to the molar ratio (azide : alkyne :
TBTA : CuSO4·5H2O : sodium ascorbate = 1.4 : 1.0 : 0.2 : 5.0 : 0.2). The detailed
reaction conditions were shown in Table1.
Table 1. The detailed reaction conditions
9

Reagents
Azide-unit
Alkyne-unit
TBTA
Concentration
35 mM DMSO
25 mM DMSO
10 mM DMSO
4 mMMilliQ
Volume
20 µL
20 µL
10 µL
25 µL
25 µL
Final Concentration
7 mM
5 mM
20 mol%
20 mol%
CuSO 5H O
・
4
2
Sodium Ascorbate
20 mM MilliQ
100 mol%
The prepared azide substituents in DMSO solution (35 mM, 20 µL), alkyne
fragments in DMSO solution (25 mM, 20 µL),
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) in DMSO solution (10
mM, 10 µL), CuSO4·5H2O aqueous solution (4 mM, 25 µL) and sodium ascorbate
aqueous solution (20 mM, 25 µL) were added in sequence to a 96-well microtiter
plate, and the reaction mixtures were stirred on the constant temperature oscillation
incubator at room temperature for 24 h. The completion of the synthetic process was
checked by thin-layer chromatography (TLC) that was performed on glass-backed
silica gel plates, and spots were visualized with iodine vapors and UV light (λ = 254,
365 nm). Then the reaction mixtures were diluted with 150 µL DMSO and the final
concentration of target compounds in each well was 2 mM. Finally, we successfully
obtained 96 quinone derivatives. The reaction mixtures could be used to perform the
enzymatic assays against Cdc25s directly.
2.3. In situ screening against the Cdc25 (A, B and C)
In order to discover the potent Cdc25 inhibitors, all compounds in the library
were tested in enzymatic assays against the Cdc25A, B and C. An inhibitor
concentration of 10 µM was chosen which could permit the rapid identification of
novel Cdc25 phosphatases inhibitors. The biological results are presented as
percentages of phosphatase activity inhibition. NSC 663284 was selected as reference
drug. The percentages of inhibition of all of the compounds of sublibraries M1NX to
Cdc25A, Cdc25B and Cdc25C were lower than NSC 663284, indicating that their
inhibitory activities were weaker than NSC 663284.
10

Figure 3. Cdc25 inhibitory activity in the presence of 10 µM M2NX.
Next, we tested the biological activities of sublibraries M2NX (Fig. 3). It is
noteworthy that the inhibition percentages of compound M2N1 (57.18%), M2N2
(69.14%), M2N3 (52.21%), M2N12 (74.36%), M2N30 (59.39%), M2N36 (55.30%),
M2N37 (54.37%), M2N41 (53.27%), M2N42 (52.90%), M2N47 (53.15%) and
M2N48 (56.02%) were higher than NSC 663284 (48.40%) against Cdc25A,
indicating that their inhibitory activities were more potent than NSC 663284. In terms
of Cdc25B, the M2NX compounds exhibited slight reduced or equivalent inhibition
percentages comparable to NSC 663284. Notably, almost all of the M2NX
compounds showed significantly improved Cdc25C inhibitory activity with an
exception of M2N10, M2N43 and M2N46.
On the whole, after in-depth analyzing the inhibitory activity of sublibraries
M2NX against Cdc25A, B and C, three hits emerged from screen. Crude compounds
M2N1, M2N2 and M2N12 exhibited prominent potency against those three Cdc25
subtypes. Therefore, for M2N1, M2N2 and M2N12 used in the primary were crude
compounds, we decided to synthesize these three compounds（Scheme 7）and further
determine their enzyme inhibitory activity and in vitro anti-tumor activity.
11

O
Cl
CN
N3
N
N
N
H
CN
N
N
O
O
N
N
N
M2N1
Cl
N1
O
CN
Cl
i
N
H
N
N
H
N3
O
O
O
M2
N
CN
N2
M2N2
Cl
H3C
N3
N12
N
N
H
N
N
O
N
M2N12
CH3
.
Scheme 7. Synthesis of M2N1, M2N2, M2N12. Reagents and conditions. (i) CuSO4 5H2O，VcNa，
THF/H2O.
2.4. Biological evaluation
2.4.1. Enzyme inhibition assay
The newly synthesized compounds M2N1, M2N2 and M2N12 were
subsequently tested for their ability to inhibit the Cdc25A, B and C respectively
xianshi under different concentration gradients. Thus, the IC50 values were determined
by using assay conditions previously described for in situ screening. As shown in
Table 2, all the tested compounds showed excellent inhibitory activities toward Cdc25
(A, B and C) with IC50 values ranging from 0.09 to 2.79 µM, being superior to or
comparable to that of the reference drug NSC 663284 (IC50 = 0.76-1.36 µM). Among
these derivatives, compound M2N12 was proved to be the most potent Cdc25 (A, B
and C) inhibitors. In case of the Cdc25A and Cdc25B, M2N12 (IC50(Cdc25A) = 0.53 µM,
IC50(Cdc25B) = 1.39 µM) displayed slightly improved or equipotent inhibitory activity
compared to NSC 663284 (IC50(Cdc25A) = 1.24 µM, IC50(Cdc25B) = 1.36 µM). Notably,
M2N12 displayed the most promising activity against Cdc25C with IC50 value of 0.09
µM, which was about 9-fold more potent than NSC 663284 (IC50(Cdc25C) = 0.76 µM).
Meanwhile, the inhibitory activity of M2N12 against Cdc25C was 6- and 15-fold
12

greater than against Cdc25A and Cdc25B respectively, indicating that M2N12 could
act as a highly selective Cdc25C inhibitor.
Table 2. The enzyme activity of M2N1, M2N2 and M2N12a
IC50 (µM)b
Compounds
Cdc25A
1.20±0.50
0.54±0.10
0.53±0.03
1.24±0.66
Cdc25B
2.79±0.87
1.00±0.61
1.39±0.95
1.36±0.97
Cdc25C
0.42±0.07
0.21±0.01
0.09±0.01
0.76±0.15
M2N1
M2N2
M2N12
NSC 663284
aAll values are micromolar concentrations and are the mean ± S.E.M. of three or more independent determinations
bIC50 values were evaluate by in vitro fluorimetric assay and were determined by GraphPad Prism software.
2.4.2. In vitro anti-growth activity
The target compounds M2N1, M2N2 and M2N12 were evaluated for their in
vitro cytotoxic activity against five tumor cell lines and a normal cell line including
A-549 (lung carcinoma), MDA-MB-231 (breast carcinoma), KB (nasopharyngeal
carcinoma), KB-VIN (MDR KB subline), MCF-7 (breast adenocarcinoma) and HBE
(normal human bronchial epithelial cell) by using the sulforhodamine B colorimetric
assay. And the paclitaxel (PXL) and NSC 663284 were selected as the positive
control. The biological results were shown in Table 3.
As depicted in Table 3, all the tested compounds displayed prominent in vitro
inhibitory activity against the five tumor cell lines with IC50 values ranging from 3.92
to 11.62 µM. Though the compounds showed equipotent or increased cytotoxic effects
toward the normal HBE cells compared to PXL and NCS663284, comparisons of the
IC50 values between tumor line A549 cells and HBE cells indicated that M2N12 are
more cytotoxic to tumor cells than normal cells, exhibiting some selectivity to cancer
cells. In summary, those novel compounds exhibited equipotent or slightly impaired
inhibitory potency against the tested tumor cell lines compared to PXL and NSC
663284. Especially, the most potent compound M2N12 (IC50 = 6.810 µM) showed
remarkable anti-growth activity against the KB-VIN cell line, equivalent to that of
13

PXL (IC50 = 3.146 µM) and NSC 663284 (IC50 = 4.87 µM). This inspiring result
indicated that compound M2N12 will be a promising lead compound for further
optimization.
Table 3. The anti-growth activity of M2N1, M2N2 and M2N12a
IC50
Cells
PXL
(nM)
NSC663284
M2N1
M2N2
M2N12
(µM)
(µM)
(µM)
(µM)
7.52±0.55
9.74±1.24
2.76±0.42
4.97±0.32
4.89±0.52
4.87±0.18
1.0±0.076
3.31±0.23
4.50±0.17
4.86±0.02
5.13±0.26
9.02±0.39
4.70±0.21
2.18±0.42
4.92±0.13
4. 90±0.16
5.01±0.28
11.62±0.18
4.84±0.28
6.43±0.33
3.92±0.15
4.63±0.47
5.05±0.41
6.81±0.23
4.71±0.33
6.00±0.18
A549
MDA-MB-231
KB
6.72±0.34
3145.83±514.05
12.06±1.33
36497±127
KB-VIN
MCF-7
HBE
aAll values are micromolar concentrations and are the mean ± S.E.M. of three or more independent determinations
2.5. X-ray diffraction assay
It is worth noting that in the nucleophilic substitution process of the starting
material A-2 to afford the alkyne-bearing key intermediate M2, there involves
regioselectivity issue, namely the selective substitution of the 6-position or 7-position
chlorine atom of A-2. The substitution sites for propargylamine substituents in the
intermediate M2 was identified by X-ray diffraction (XRD), a powerful and effective
non-destructive technique for characterizing crystalline materials. The spectral data of
the intermediate M2 was obtained by X-ray diffraction, and subsequently analyzed by
OLEX software as depicted in Fig. 4. The X-ray diffraction experiment results
confirmed that the propargylamine group replaced 7-position chlorine atom of A-2,
which is consistent with the reported results [25].
14

Figure 4. ORTEP drawing of compound M2
2.6. Molecular dynamics simulation studies
To investigate the dynamics of selective and high-affinity binding of M2N12
towards Cdc25C as against its structurally similar homologs; Cdc25A and Cdc25B, an
all-atom MD simulation method was employed. We performed time-scale
investigations into the binding site dynamics of the three proteins (Cdc25A, Cdc25B
and Cdc25C) given that they are highly similar structural homologs [20, 26].
Particularly we determined the differential binding modes of M2N12 within the
protein binding sites across the MD simulation period which lasted for 100ns.
2.6.1 Analyses of Cdc25A, B and C active site architecture and corresponding
M2N12 stability
Considering the widely reported effects of active site attributes on drug binding
and affinity [27-29], we determined the compactness and solvent accessibility of
M2N12-binding sites across the three proteins. This served as the first step in
providing a rational explanation for reasons why M2N12 has significant selectivity
for Cdc25C. To perform this analysis, we selected residues that correspond to the
M2N12-binding site region, particularly those within 5Å distance as shown in Fig. 5.
Figure 5. Binding site sequences for Cdc25A, Cdc25B and Cdc25C that constitutive of M2N12
docked region. These residues include those defined in previous studies [30-32] and herein not
arranged sequentially but randomly based on their possible involvements in M2N12 binding.
A striking observation was the dynamic nature of Cdc25C catalytic site region
relative to Cdc25A and B as monitored in Fig. 6. Unique to this Cdc25C site was the
stable transient motions of constituent residues which could be more favorable to the
15

binding of M2N12. On the contrary, while the active site of Cdc25A was highly
unstable (unfavorable for ligand binding), the uniform but lower Ca motion peak
demonstrated by constituent Cdc25B active pocket residues could restrain in-depth
hydrophobic M2N12 binding.
Figure 6. Binding site dynamics of M2N12-bound Cdc25A, Cdc25B and Cdc25C over 100ns MD
simulation period. Above: RMSD and SASA plots comparing stability and Ca motions of
constituent site residues Below: Ca RMSD and RoG plots monitoring the motions of M2N12
within the target proteins. Cdc25A, Cdc25B and Cdc25C systems are depicted in yellow, cyan and
brown colorations respectively.
Probing solvent accessibility using SASA analytical method further clarified our
predictions. Presumably, while binding site residues (side chains) were highly
exposed to solvent space in Cdc25A, constituent Cdc25B pocket residues remained
overly buried, which could in turn prevent optimal M2N12 binding, motion and
stabilization. On the other hand, a moderate increase in surface-exposure at the
binding site of Cdc25C could allow optimal M2N12 motion towards crucial site
residues necessary for high-affinity interactions. Estimations of per-residue
fluctuations for constituent binding site residues across the three proteins revealed
16

high → low → moderate side chain flexibility in Cdc25A → Cdc25B →
Cdc25C. (Fig. 7). Mean values for the respective estimations are presented in Table 4.
Figure 7. Superposed-3D representation and RMSF plot showing the per-residue fluctuations of
M2N12 interacting residues within and outside the active site region (blue inset). Yellow, cyan and
brown colorations depict Cdc25A, Cdc25B and CDC25C systems.
Table 4. Estimated average values indicating the degree of active sites and ligand motions in
target proteins
Complexes
Active site
RMSD (Å)
4.2±0.8
Active site
SASA (Å2)
1198.4±0.5
463.1±0.2
622.4±0.4
Active site
RMSF (Å)
1.7±0.2
M2N12
RMSD (Å)
2.3±0.7
M2N12
RoG (Å)
4.8±0.5
4.6±0.5
3.6±0.1
M2N12-Cdc25A
M2N12-Cdc25B
M2N12-Cdc25C
1.5±0.1
1.2±0.1
2.7±0.7
1.9±0.6
1.6±0.1
1.2±0.3
To gain further insights into the dynamical motions of individual active site
residues, we calculated the per-residue RMSF of these residues across the binding
sites of Cdc25A, Cdc25B and Cdc25C. Results revealed relatively higher fluctuations
among active site residues in Cdc25A, with Arg566 having a RMSF value of 5.66Å
while corresponding Met531 and Met435 in Cdc25B and Cdc25C have RMSF values
of 1.14Å and 3.30Å respectively. This suggests high → low → moderate active
site motions in Cdc25A → Cdc25B → Cdc25C. As presented in Table 5, the least
fluctuated residues at the Cdc25A active site were His429 (1.00Å) while Gly511
(0.53Å) and Phe375 (0.54Å) had the least motions in Cdc25B and Cdc25C
respectively. This further reveal differential active motions across the three proteins
17

which could in turn influence the binding and stability of M2N12.
Table 5. Per-residue RMSF estimations of active site residues in Cdc25A, Cdc25B and Cdc25C.
Cdc25A
Cdc25B
Cdc25C
Active site
Active site
Active site
RMSF (Å)
RMSF (Å)
RMSF (Å)
residues
Arg385
Tyr386
Pro387
Tyr388
Glu389
His429
Cys430
Glu431
Phe432
Ser433
Gly467
Gly468
Tyr469
Lys470
Glu471
Tyr485
Arg486
Pro487
Met488
His489
His490
residues
Cys426
Arg472
Tyr428
Pro429
Tyr430
Ile470
residues
Cys330
Arg331
Tyr332
Pro333
Tyr334
Val374
Phe375
His376
Cys377
Glu378
Leu412
Lys413
Gly414
Gly415
Tyr416
Gln430
Ser431
Tyr432
Cys433
Pro434
Met435
1.22
2.00
1.48
2.22
1.82
1.00
1.09
1.98
2.28
1.44
1.32
1.05
1.15
1.67
1.44
1.59
2.22
2.59
4.61
4.70
5.66
0.65
0.57
0.90
0.75
0.74
0.67
0.56
0.89
0.72
0.80
0.58
1.02
0.59
0.53
0.57
1.23
1.24
0.87
0.83
0.81
1.14
1.37
1.12
1.61
1.49
1.91
0.59
0.54
1.33
0.98
0.82
0.68
1.00
0.90
0.76
0.80
1.36
1.04
0.89
1.74
1.68
3.30
Phe471
His472
Cys473
Glu474
Ile508
Lys509
Gly510
Gly511
Tyr512
Gln526
Asp527
Tyr528
Arg529
Pro530
Met531
Moreover, we measured the systemic motions of M2N12 across the binding sites
of Cdc25A, B and C over the 100ns simulation run. We observed that amongst the trio,
M2N12 was more stable at the binding site of Cdc25C compared to the highly
unstable motions exhibited in Cdc25A and B. This can be related to the differential
site attributes of the three proteins as earlier indicated, and could suggest that the
stable nature of Cdc25C binding site favored M2N12 stabilization.
Motions of M2N12 at the binding sites of the respective proteins over the
18

simulation period was monitored using the Ca RMSD and RoG metrics as presented
in Fig. 6.
The ligand Ca RoG plot reflected a highly unstable motion of M2N12 which
occurred from peaks as low as 3.2Å to peaks as high as 5.45Å in both Cdc25 A and B.
On the other hand, variations in M2N12 motion was about 0.75Å in Cdc25C
indicative of its stability.
2.6.2. Unprecedented binding modes of M2N12 at Cdc25A, B and C binding sites
We examined the differential binding patterns and orientations of M2N12 at
different simulation trajectories to further understand the effects of the protein binding
site architecture. This approach was important to gain insights into the structural
stability of M2N12 at the respective binding sites of Cdc25A, Cdc25Band Cdc25C
since deviations from the starting structure could depict systematic alterations binding
modes and dynamics.
To obtain a clear representation of the distinct binding modes, we obtained
snapshots of the protein-ligand complexes at 1000 - 100000 frames which
corresponds to 1-100 ns. 2D conformations of M2N12 were then prepared at specific
time-frames and their binding modes over the various time-frames were presented as
shown in Table 6. Inter-molecular interactions elicited by M2N12 at its various
binding modes in Cdc25A, B and C are further shown in Fig. 10 below.
Table 6. Predicted binding modes of M2N12 within the active sites of Cdc25A/B/C over 100ns
MD simulation. Numbering represents definite time-frames at which the snapshots were taken; 1
= 1000 frames (1ns), 2 = 20000 frames (20ns), 3 = 40000 frames (40ns), 4 = 60000 frames, 5 =
80000 frames (80ns), 6 = 100000 frames (100ns).
19

As shown, M2N12 demonstrated unstable or irregular binding modes at the
active sites of Cdc25A and B indicative of its degree of instability compared to its
stable orientations at the active site of Cdc25C. The binding modes/orientations of
M2N12 appeared to be uniform from 1-60ns while a re-orientation occurred at 80ns
and lasted until the end of the simulation.
This could imply that while the compact and stable active site attribute of
Cdc25C held M2N12 in position until it exerted its inhibitory functionality, the highly
flexible and unstable attributes of Cdc25A/B active sites disfavored the stable binding
of M2N12.
20

Figure 8: PCA plot for M2N12 showing its dynamics and motions across Cdc25A, B and C active
sites over 100ns MD simulation period.
To further corroborate this observation, we estimated principal components of
M2N12 across the 100ns MD simulation period to obtain insights into its relative
motion and dynamics when bound to Cdc25A, B and C. This is graphically depicted
in Fig. 8, and as shown, M2N12 exhibited highly scattered or dispersed motions at the
active sites of Cdc25A and Cdc25B which could correspond to their unstable or
irregular binding modes as estimated in Table 6. Relatively, M2N12 exhibited highly
stable motions at the active site of Cdc25C as shown in Fig. 8.
Furthermore, Fig. 9 shows the initial and final positioning of M2N12 at the
active sites of Cdc25A/B/C over 100ns simulation period. This was to examine
systemic deviations of the M2N12 along the resulting trajectories of the three
complexes.
21

Figure 9. Positional motions of M2N12 in Cdc25A (yellow), Cdc25B (cyan) and Cdc25C (brown)
to capture systemic deviations from the starting complexes (center). Time-frames at which the
snapshots were taken were also indicated.
As shown, M2N12 gradually moved out of the active sites of Cdc25A and B,
probably due to their high instability as previously determined (Fig. 6, 8). On the
contrary, M2N12 remained deeply buried at the catalytic site core of Cdc25C which
demonstrated distinct compactness that favored its stabilization as earlier reported.
The ability of M2N12 to maintain high-affinity interactions and distinct stability at
the active site of Cdc25C relative to Cdc25A and B could possibly explain why it
demonstrated a higher inhibition (IC50) as experimentally reported. The patterns of
M2N12 motion in Cdc25A/B was highly deviated from the starting (pre-MD)
structures where it was bound (docked) to active site regions similar to Cdc25C.
22

2.6.3 Systemic involvements of Cdc25A, B and C site residues in M2N12 binding,
stabilization and affinity
Furthermore, we examined the consequential effects of the variations in active
site conformations on M2N12 binding among the various proteins. We employed the
MM/GBSA method to estimate total delta binding energies (∆G) of M2N12 when
bound to Cdc25A/B/C. Energies were averaged across two phases; the initial (1-50ns)
and final (60-100ns) periods to monitor the time-scale interactions of M2N12. Our
findings are presented in Table 7 and Fig. 10.
Time-scale inter-molecular interactions mediated by M2N12 (Fig. 10)
corroborates binding modes earlier reported in Table 6.
As shown, averages of resulting energies across the simulation period indicate
that M2N12 exhibited favorable and high-affinity binding in Cdc25C compared to
Cdc25A and B. van der Waals (∆EvdW) and electrostatic (∆Eele) interactions are most
favorable in M2N12-Cdc25C complex. These were in addition to a highly
unfavorable polar desolvation energy (∆EGB) which reflect the hydrophobic (stable
active site) binding of M2N12. Likewise, a more negative (favorable) non-polar
energy (∆ESA) further corroborates the hydrophobic effects of Cdc25C active site
towards ligand binding and stabilization. Total delta energy (∆Gbind) is most favorable
in M2N12-Cdc25C complex compared to Cdc25A and B-M2N12 complexes.
Furthermore, we examined the nature and types of interactions mediated by
M2N12 at the respective active sites of Cdc25A, B and C. Findings are presented in
Fig. 9.
Table 7. Delta binding energy analyses of M2N12 complexes
M2N12- M2N12-
M2N12-
Cdc25C
M2N12-
Cdc25A
M2N12- M2N12-C
Complexes
Cdc25A
Cdc25B
Cdc25B
dc25C
∆Evdw
(kcal/mol)
∆Eele
-7.2±0.4
-5.5±0.3
-15.0±0.2
-4.4±0.4
-1.4±0.3
-5.4±0.2
-14.0±0.1
-2.0±0.2 -3.7±0.4
-5.3±0.1
23
-3.7±0.2
-6.2±0.1

(kcal/mol)
∆EGB
5.3±0.3 6.2±0.4
10.2±0.1
3.4±0.4
6.2±0.2
10.4±0.1
(kcal/mol)
∆ESA
-0.8±0.04 -0.7±0.03 -1.5±0.01
-0.45±0. -0.7±0.02 -1.4±0.01
03
(kcal/mol)
∆Ggas
(kcal/mol)
∆Gsol
-9.2±0.5 -9.2±0.6
4.5±0.3 5.5±0.4
-4.8±0.3 -3.7±0.2
-20.3±0.2
8.7±0.1
-5.8±0.6
3.0±0.4
-2.8±0.3
-9.2±0.3
5.5±0.2
-3.6±0.1
-20.2±0.2
9.0±0.2
(kcal/mol)
∆Gbind
-11.6±0.1
-11.2±0.1
(kcal/mol)
∆Eele = electrostatic energy; ∆EvdW = van der Waals energy; ∆Gbind = total binding energy; ∆Gsol
= solvation energy ∆Ggas = gas phase energy; ∆EGB = polar desolvation energy; ∆ESA non-polar
solvation energy
As shown, there were distortions in the primary catalytic site binding of M2N12
in Cdc25A and Cdc25B. The ‘presumed’ unstable site architectures of these proteins
were relatively less favorable for the binding and stabilization of M2N12. In Cdc25A,
while the ligand was initially bound at the catalytic site via strong hydrogen, van der
Waals and aromatic interactions (similar to Cdc25C), interactions with residues
distant from the core catalytic region; Tyr386 and Tyr388 were observed in
subsequent trajectories. A similar occurrence was observed in Cdc25B as evidenced
by subsequent interactions with non-catalytic site residues, which altogether
corroborates findings earlier mentioned. However, the ‘closed’ compact and stable
nature of Cdc25C active site held M2N12 in position until the end of the simulation.
24

Figure 10. M2N12 interacting residues in Cdc25A, Cdc25B and Cdc25C across 100ns MD
simulation.
This stable binding was characterized by the steady occurrence of strong
hydrogen and aromatic interactions as shown. This altogether explains its stable
motion (Fig. 6, 8) and highly ordered binding modes (Table 6). An important
interaction was the pi-pi stacking mediated by Tyr416 with the saturated
methyl-benzene ring of M2N12. This interaction, however, similar to Tyr485 in
Cdc25A which occurred at the initial stage of M2N12 binding but was lost
subsequently since it was unstable. Also, it can be seen that a higher number of
non-polar residues contributed to M2N12 binding and stabilization at the hydrophobic
core of Cdc25c compared to Cdc25A and B.
2.7. In silico prediction of physicochemical properties
By the classical definition for drug evaluation, a potential drug candidate should
25

own favorable druglikeness features. Therefore, in order to further guide subsequent
structural optimization and preliminary drug-like properties evaluation, some
significant physicochemical properties of representative compounds M2N1, M2N2
and M2N12 were predicted comprehensively by using free online molinspiration
software (http://www.molinspiration.com/), including molecular weight (MW),
hydrogen bond acceptors (nON), hydrogen bond donors (nOHNH), rotatable bonds
(nrotb), topological polar surface area (TPSA), miLogp, natoms and molecular
volume (MV). As shown in Table 7, all the tested compounds were accurately accord
with the “Lipinski's rule of five”. The results suggested that those three compounds
have achieved a well balance between moderate efficacy and desirable
physicochemical properties.
Table 7. Physicochemical properties of representative compounds
MW
(Da)
TPSA
(A2)
MV
(m3/mol)
Compound
nON nOHNH nrotb
miLogP nViol natoms
Accepted range
M2N1
<500
<10
8
<5
1
≤10
<140
113.57
113.57
89.78
<5
-
-
404.82
404.82
393.83
5
5
5
1.17
1.20
1.91
0
0
0
29
29
28
330.70
330.70
330.40
M2N2
8
1
M2N12
7
1
a
Using free on-line software (http://www.molinspiration.com/); MW = molecular weight; nON = no. of hydrogen bond acceptors;
nOHNH = no. of hydrogen bond donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area; miLogP = molinspiration
predicted LogP; nViol = number of violations; natoms = no. of atoms; MV = molar volume.
3. Conclusion
For the Cdc25s were key phosphatases in cell cycle and overexpressed in various
types of cancer, Cdc25 inhibitors would be a good target for cancer treatment. In this
work, a total of 96 quinone derivatives were synthesized by microscale click
chemistry reaction and tested for in vitro biological activities by in situ screening, and
interestingly, all the tested compounds showed excellent inhibitory activities toward
Cdc25 (A, B and C). Particularly, M2N12, a promising lead compound, with potent
activity and significant Cdc25C selectivity was discovered and also exhibited
remarkable suppression on KB-VIN cell growth. In addition, the molecular dynamics
study revealed that the unique architecture of the Cdc25C catalytic site favored
26

optimal binding and stabilization of M2N12 compared to Cdc25A and B. Cdc25C
exhibited a highly stable and compact catalytic site which in turn enhanced the deep
hydrophobic pocket binding of M2N12.
In summary, these data revealed that M2N12 is regarded as a novel and
promising Cdc25C inhibitor for further investigation and can be used as a small
molecule probe to explore the specific mechanism of Cdc25C.
4. Experimental section
4.1. Chemistry
1H NMR and 13C NMR spectra were obtained via a Bruker Avance-400 NMR
spectrometer in Cdcl3 or DMSO using TMS as internal reference. Chemical shifts
were expressed in δ units (ppm) and J values were presented in hertz (Hz). All
melting points of the compounds were determined on a micro melting point apparatus
and were uncorrected. Related mass spectra dates were determined by a LC
Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel
GF254 for TLC and spots were visualized by irradiation with UV light (λ = 254 nm
and 365nm). Meanwhile, flash column chromatography was performed on column
packed with Silica Gel 60 (200-300 mesh). Rotary evaporator was used to concentrate
the reaction solutions under reduced pressure condition. Solvents were of reagent
grade and purified with standard methods when necessary.
4.1.1. 2-methyl-3-(prop-2-yn-1-ylamino) naphthalene-1,4-dione (M1)
Menadione（0.581 mmol, 1.0 eq）and prop-2-yn-1-amine (1.16 mmol, 2.0 equiv)
were mixed in anhydrous ethanol and stirred at room temperature for 10-12h. The
solvent was removed under reduced pressure and then water (20mL) was added,
extracted with ethyl acetate (3×30 mL). The combined organic layer was washed with
saturated salt water, dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to yield the crude product, which was purified by flash column
chromatography to afford compound M1 as russet solid with the yield of 82%.1H
NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 7.6 Hz, 2H), 7.87 – 7.66 (m, 2H), 6.97 (t, J
27

= 6.8 Hz, 1H), 4.29 (dd, J = 6.9, 2.4 Hz, 2H), 3.28 (d, J = 2.4 Hz, 1H), 2.16 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 182.63, 182.40, 146.48, 134.89, 132.87,
130.71, 126.11, 113.76, 82.48, 75.02, 34.36, 10.64. ESI-MS: m/z 223.88 (M - H)-,
C14H11NO2 (225.08).
4.1.2. 6-chloro-7-(prop-2-yn-1-ylamino) quinoline-5,8-dione (M2)
6,7-dichloroquinoline-5,8-dione (0.42 mmol, 1.0 equiv), prop-2-yn-1-amine
(0.84 mmol, 2.0 equiv) and K2CO3 (0.42 mmol, 1.0 equiv) were mixed in anhydrous
ethanol and stirred at room temperature for 10-12h. The solvent was removed under
reduced pressure and then water (20mL) was added, extracted with ethyl acetate
(3×30 mL). The combined organic layer was washed with saturated salt water, dried
over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to yield the
crude product, which was purified by flash column chromatography to afford
compound M2 as russet solid with the yield of 52%.1H NMR (400 MHz, DMSO-d6) δ
8.93 (dd, J = 4.6, 1.6 Hz, 1H), 8.34 (dd, J = 7.9, 1.6 Hz, 1H), 7.97 – 7.73 (m, 2H),
13
4.48 (dd, J = 6.7, 2.4 Hz, 2H), 3.28 (s, 1H, CH). C NMR (100 MHz, DMSO-d6) δ
178.55, 175.66, 153.60, 146.72, 146.04, 134.26, 129.33, 128.89, 81.66, 75.08, 34.33.
ESI-MS: m/z 244.93 (M - H)-, C12H7ClN2O2 (246.02).
4.1.3. General procedure for the synthesis of azide substituents (N1-N22, N40 and
N48)
B1-B22, B40 or B48 (1.0 mmol, 1.0equiv) were dissolved in 5 mL
N,N-dimethylformamide (DMF), and sodium azide was added to the above solution.
The reaction system was stirred at room temperature or 70
for 24h. Then water
(30mL) was added, extracted with ethyl acetate (3×10 mL). The organic phase was
combined, dried over anhydrous Na2SO4, filtered, and concentrated to yield the crude
azide substituents N1-N22, N40 and N48, which were used for the next step without
further purification.
2-cyanobenzyl azide (N1)
28