Discovery of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives as novel tubulin polymerization inhibitors for treatment of cancer

Article abstract of DOI:10.1016/j.ejmech.2019.111728

A new series of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives were designed, synthesized and demonstrated to act as tubulin polymerization inhibitors. These new derivatives showed significant antitumor activities, among which

Full text of DOI:10.1016/j.ejmech.2019.111728

Journal Pre-proof
Discovery of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one
derivatives as novel tubulin polymerization inhibitors for treatment of cancer
Qiangsheng Zhang, Xi Hu, Guoquan Wan, Jia Wang, Lu Li, Xiuli Wu, Zhihao Liu,
Luoting Yu
PII:
S0223-5234(19)30880-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111728
EJMECH 111728
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2019
Revised Date: 20 September 2019
Accepted Date: 21 September 2019
Please cite this article as: Q. Zhang, X. Hu, G. Wan, J. Wang, L. Li, X. Wu, Z. Liu, L. Yu, Discovery of 3-
(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives as novel tubulin polymerization
inhibitors for treatment of cancer, European Journal of Medicinal Chemistry (2019), doi: https://
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Discovery of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one
derivatives as novel tubulin polymerization inhibitors for treatment of cancer
a1
a1
a
a
a
a
Qiangsheng Zhang , Xi Hu , Guoquan Wan , Jia Wang , Lu Li , Xiuli Wu , Zhihao
a
*
a
*
Liu , Luoting Yu
a
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University, and Collaborative Innovation Center for
Biotherapy, 17#3rd Section, Ren Min South Road, Chengdu 610041, China.
1
These authors contribute equally to this work.
*
Corresponding authors. Address: Lab of Medicinal Chemistry, State Key Laboratory
of Biotherapy and Cancer Center, West China Hospital, Sichuan University and
Collaborative Innovation Center for Biotherapy, 17#3rd Section, Ren Min South Road,
Chengdu 610041, China. Phone: +86-28-85503817; Fax: +86-28-85164060. E-mail
address: yuluot@scu.edu.cn (L.-T. Yu) & liuzhihao@scu.edu.cn (Z.-H. Liu)
Abstract:
A new series of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one
derivatives were designed, synthesized and demonstrated to act as tubulin
polymerization inhibitors. These new derivatives showed significant antitumor
activities, among which SKLB0533 demonstrated to be the most potent compound,
with IC values ranging from 44.5 and 135.5 nM against seven colorectal carcinoma
5
0
(CRC) cell lines. Remarkably, SKLB0533 exhibited no activity against other potential

targets, such as 420 kinases and EZH2. Besides, SKLB0533 inhibited tubulin
polymerization, arrested the cell cycle at the G2/M phase and induced apoptosis in
CRC cells. Furthermore, SKLB0533 suppressed tumour growth in the HCT116
xenograft model without inducing notable major organ-related toxicity, suggesting
that SKLB0533 could be used as a promising lead compound for the development of
new antitumor agents.
Keywords: Microtubule, Tubulin polymerization inhibitors, Anti-cancer agents,
Colorectal carcinoma
1
. Introduction
Morbidity and mortality due to cancer are growing rapidly worldwide, and
cancer has become a serious public health problem. Estimates from the of 2018 global
cancer report indicate that CRC is the third most common causes of cancer death
worldwide in both males and females [1]. More than 1.5 million men and women
living in the United States are estimated to have a previous colorectal cancer
diagnosis, and 145,600 new cases will be diagnosed in 2019[2]. Moreover, an
increasing number of young people are dying from CRC[3].
Microtubules (MTs) are dynamic filamentous cytoskeletal polymers of tubulin,
which involved in numerous essential cellular functions, such as cell growth, division,
motility and intracellular trafficking[4-6]. Disruption of microtubule dynamics
increases the number of cells in metaphase arrest and mitotic catastrophe, which
closely associated with the progression of cancer, including CRC [7, 8]. Due to the

important role of microtubules, especially in mitosis, they have become an important
target for the design of new anticancer agents[9-11].
Today, microtubule-targeted drugs span a wide structural heterogeneity and
contain several potent natural and synthetic compounds, such as Paclitaxel[12],
Epothilone D[13], Laulimalide[14], Vincristine[15], Rhizoxin[16] and Colchicine[17].
Recently, a variety of colchicine binding site tubulin inhibitors containing a
trimethoxyphenyl (TMP) moiety were designed for the treatment of cancer, such as
Combretastatin A-4 (CA-4)[18], Ombrabulin[19] and Compound 8c4[20] (Fig. 1).
Fig. 1 Representative microtubule stabilizing and destabilizing agents.
Despite the advances have been undoubtedly made in microtubule-targeted
agents, existing agents suffer from some significant drawbacks[21], such as limited
effectiveness which can lead to treatment failure, drug resistance and adverse

toxicities can be dose-limiting, indicating a narrow therapeutic window [22].
Collectively, these shortcomings have encouraged medicinal chemists to develop
novel antimitotic agents for cancer therapy[23-25].
Among the reported microtubule inhibitors, compounds owning concise
double-ring core structure attracted our interest, such as MPC-6827[26], Compound
3
0·HCl[27], Myoseverin[28] and E2GG[29]. By investigating these compounds, we
found that such microtubule inhibitors were mainly composed of three parts (Fig. 2):
the core structure (yellow box), which serves as skeletal support; the aromatic ring
(blue box), which may participate in a π-π stacking with the amino acid residues
around the binding site[30]; and the linker (purple box),which connects the core
structure to the aromatic ring.
Based on the above findings, we chose purine as the core structure, aminomethyl
as the linker firstly. Then, we replaced the aromatic ring with pyridin-2(1H)-one using
a bioelectronic isosteric strategy. From the co-crystal structure of Colchicine bound to
the tubulin (PDB code: 4O2B), we could see that trimethoxyphenyl motif surrounded
by residues Cysβ241, Leuβ248, Asnβ249, Alaβ250, Aspβ251 and Lysβ254. So
compared to the phenyl ring, pyridin-2(1H)-one retained aromaticity and contained a
hydrogen bond acceptor and hydrogen bond donor, which may increase its hydrogen
bonding
-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives were
designed.
interactions[31].
Herein,
a
series
of
3

O
O
N
N
N
N
HCl
N
N
core structure
linker
Compound 30•HCl
MPC-6827
HN
O
O
O
HN
N
N
N
R2
N
aromatic ring
HN
N
HN
H
R1
N
N
N
N
N
tagert compounds
HN
N
HN
N
O
O
Myoseverin
E2GG
Fig. 2 Rational design of new tubulin-targeted compounds.
2
2
. Results and discussion
.1. Chemistry
3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one
derivatives
were synthesized by a two-four step procedure summarized in Scheme 1 Compounds
a-3d were synthesized from commercially available 2,6-dichloro-1H-purine in two
3
steps: (1) 2,6-dichloro-1H-purine was reacted with halogenated alkane under basic
conditions to give compounds 2a-2c[32]. Compound 2d was obtained by protecting
the amino group with tetrahydropiran (THP) under p-toluenesulfonic acid (p-TsOH).
(2) Compounds 3a-3d were synthesized in high yields by reacting compounds 2a-2d
with 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one[33]. Compounds 4a1-4d8 were
obtained using the Suzuki-Miyaura reaction by coupling them to corresponding boric
acid or borate derivatives in a 1,1'-bis(diphenylphosphino) ferrocene catalytic system
in the presence of sodium carbonate (Na CO ) as the base in dioxane/water at 100 °C
2
3
for 4 h. Finally, compounds 4a7, 4b7, 4c7 and 4d1-4d8 were stirred at room

temperature overnight with trifluoroacetic acid (TFA) to remove the THP or
tert-butyloxycarbonyl (Boc) protecting group and obtain the target compounds 5a-5k.
The
structures
of
3
-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives were
1
13
confirmed by NMR ( H & C) and HRMS spectral analyses. The purity of all the
newly synthesized compounds was more than 98% as analysed by HPLC.
Scheme 1 Synthesis of the target compounds. (a) Halogenated alkane, K CO ,
2
3
anhydrous DMSO,15 ℃ , overnight. (b) 3,4-Dihydro-2H-pyran, p-TsOH, DCM, r.t.,8 h.
c) 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one, EtOH, 80 ℃ , 6 h. (d) Boric acid
or borate derivatives, PdCl (dppf)· CH Cl , Na CO , dioxane/water, 100 ℃, 4 h. (e)
(
2
2
2
2
3
TFA, DCM, r.t., overnight.
.2. Structure -activity relationships
All the 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one
2

derivatives were evaluated to the antiproliferative activity against three cancer cell
lines HepG2 (hepatocellular carcinoma cells), HCT116 (colorectal carcinoma cells)
and A549 (lung cancer cells) using the MTT assay in terms of half maximal inhibitory
concentration (IC ) values. For comparison, Myoseverin and CA-4 were selected as
5
0
the positive references (Tab. 1).
Generally, most of the synthesized compounds inhibited the growth of the three
cancer cells lines, with IC values less than 5 µM. Approximately one-third of tested
50
compounds were superior to Myoseverin with IC values less than 1 µM.
5
0
The 2-Pr,3-pentyl or cyclopentyl substituents on the N9-position of the purine
derivatives (3a-3d, 4a1-4a7, 4b1-4b7 and 4c1-4c7) provided increased
antiproliferative activity. The antiproliferative activity was significantly reduced if the
2
-Pr,3-pentyl or cyclopentyl substituents were absent at position 1 (as in 5a-5k
compared to 3a-3d, 4a1-4a7, 4b1-4b7 and 4c1-4c7) or if the substituent at position 1
was a tetrahydropiran instead of 2-Pr,3-pentyl or cyclopentyl (compare 4d1-4d8
versus 3a-3d, 4a1-4a7, 4b1-4b7 and 4c1-4c7). These results suggested that suitable
alkyl substituents at the N9-position may be advantageous.
Comparison of compounds 3a-3d with other compounds revealed that a 2-chloro
substitution on the purine ring led to a remarkable improvement in antiproliferative
activity. The antiproliferative activity of the synthesized compounds with different
substituents at position 2 increased in the following order: 3a-3d (chloro) > 4a1, 4b1,
4
c1 (4-(trifluoromethoxy)phenyl) > 4a2-4a4, 4b2-4b4, 4c2-4c4 (4-morpholinophenyl,
-morpholinophenyl, 4-(morpholinomethyl)phenyl) > 4a5-4a7, 4b5-4b7, 4c5-4c7
3

(
4-(4-methylpiperazin-1-yl)phenyl,
6-(4-methylpiperazin-1-yl)pyridin-3-yl,
-(4-Boc-piperazin-1-yl)pyridin-3-yl). Compared 3c (SKLB0533) with compound 3a,
b, 3d and 3e, we hypothesize that different substituents at the N9 position may affect
6
3
the interaction of C6 chlorine and surrounding residues. SKLB0533 could inhibit the
polymerization of tubulin more effectively. By comparing the antiproliferative activity
of a C2-position hydrogen substitution, chlorine substitution and benzene ring
substitution, we can speculate that the suitable size substituent group at the
C2-position may be beneficial.
In this study, we did not examine the effect of different pyridin-2(1H)-one
substitutions on antiproliferative activity, which will be the focus of our next stage of
work.
Notably, SKLB0533 exhibited the most potent antiproliferative activity among
the synthesized compounds, with IC50 values ranging between 0.06 and 0.10 µM.
SKLB0533 was more active against the three tested cancer cell lines than Myoseverin
(3.06 to >10 µM). Meanwhile SKLB0533 was 10- to 20-fold less active than CA-4
against the HpeG2 and A549 cell lines, which was superior to CA-4 against the
HCT116 cell lines in our test.

Tab. 1 Antiproliferative activities of the target compounds.
IC /µM
IC /µM
5
0
50
Cpd.
Cpd.
HepG2
0.15±0.01
0.20±0.06
HCT116
1.86±033
1.33±0.26
A549
HepG2
7.63±0.84
1.62±0.00
HCT116
>10
A549
5.17±0.06
>10
3
3
a
1.13±0.05
1.51±0.06
4c6
4c7
b
1.01±0.00
3
c
（
SKLB 0.08±0.01
0.06±0.00
0.10±0.01
4d1
5.22±0.08
5.83±0.06
9.46±0.04
0
533）
3
d
0.41±0.01
/
2.71±0.13
1.05±0.23
0.55±0.08
1.03±0.08
7.42±0.54
8.01±0.36
7.93±0.75
8.99±0.03
4.14±0.36
0.78±0.02
>10
1.93±0.27
>10
4d2
4d3
4d4
4d5
4d6
4d7
4d8
5a
>10
>10
>10
>10
>10
3
e
>10
4a1
4a2
4a3
4a4
4a5
4a6
4a7
2.47±0.07
1.11±0.07
>10
9.31±0.01
4.30±0.28
>10
6.67±0.19
>10
8.27±0.27
>10
>10
>10
>10
>10
>10
0.93±0.02
5.81±0.14
>10
>10
>10
9.05±0.00
9.07±0.04
1.19±0.05
3.94±0.03
0.85±0.08
1.08±0.01
6.91±0.19
5.66±0.31
6.84±1.34
>10
>10
4.72±0.12
>10
>10
>10
2.88±0.22
5.72±0.00
1.71±0.10
1.29±0.09
6.89±0.06
4.74±0.01
5.19±0.63
>10
>10
>10
0.49±0.01
5.38±0.30
5.90±0.05
>10
5b
6.13±0.02
>10
4b1
4b2
4b3
4b4
4b5
4b6
4b7
1.42±0.13
1.54±0.030
2.22±0.16
1.82±0.01
1.99±0.09
0.82±0.10
0.52±0.13
6.21±0.41
0.98±0.05
5c
5d
>10
>10
5e
>10
2.99±0.01
>10
6.13±0.12
5.09±0.02
>10
5f
>10
5g
>10
0.65±0.23
0.88±0.07
7.55±0.78
0.52±0.04
5h
>10
8.21±0.11
>10
5i
9.83±0.87
>10
>10
>10
4
c1
c2
5j
>10
>10
4
>10
5k
6.02±0.07
2.65±0.10
>10
Myosev-
erin
4
c3
0.89±0.020 5.32±0.19
2.65±0.19
3.06±0.31
3±2
7.82±0.17
212±0
>10
7±0
CA-4
4
c4
c5
1.93±0.07
2.59±0.30
2.46±0.20
1.37±0.18
>10
(nM)
4
6.21±0.01

Data are expressed as the mean±SEM for at least 2 independent experiments.
2
.3. Effect on tubulin polymerization
Since the vital role of tubulin-microtubule system in the maintenance of cellular
morphology and basic cellular functions, we examined the effect of SKLB0533 on the
organization of the microtubule network in living cells. Based on the obtained IC50
values against the three cancer cell lines, SKLB0533 was evaluated for cell-based
immunofluorescence staining assays at 50 nM. As shown in Fig. 3A, cells without
drug treatment (control group) maintained a well-organized spindle and microtubule
network. However, after cells were treated with SKLB0533 for 18 h, the microtubule
network became disorganized and condensed around the nucleus, similar to the
disruption produced by CA-4 at the same concentration. These results confirm that
SKLB0533 is able to disrupt the microtubule network at low concentrations in
HCT116 and SW620 cells.
An in vitro tubulin polymerization assay was used to prove the impact of
SKLB0533 on the tubulin polymerization dynamics, CA-4 was used as a positive
control and paclitaxel was utilized as a negative control. As illustrated in Fig. 3B,
paclitaxel immediately and obviously increased the absorbance, indicating that this
compound enhanced tubulin polymerization. By contrast, CA-4 and SKLB0533
decreased the absorbance, indicating that tubulin polymerization was inhibited.
Meanwhile, low concentration of SKLB0533 (0.05 µM) could inhibit microtubule
polymerization. The microtubule inhibition was enhanced with the increased
concentration of SKLB0355, which showed strong inhibition at 30 µM. These results

clearly indicate that SKLB0533 significantly inhibits the polymerization of tubulin in
vitro.
Fig. 3 Effects of SKLB0533 on tubulin polymerization in vitro. (A) Effect of 50 nM
SKLB0533 on the organization of the microtubule network. HCT116 and SW620
cells were treated with 50 nM SKLB0533 for 18 h. Control was treated with 0.1%
DMSO. Tubulin labelled with a monoclonal β-tubulin antibody (green) and nuclei
labelled with DAPI (blue) were observed under a confocal fluorescence microscope
(scale bar = 10 µm). (B) Polymerization of tubulin at 37 °C in the presence of

paclitaxel (3 µM), CA-4 (1.25 µM), and SKLB0533 (0.05, 1.25, 6 and 30 µM).
2
.4. Molecular modeling
To investigate the possible binding mode for this series of compounds, the most
potent compound SKLB0533 was docked into the tubulin-Colchicine complex (PDB
code: 4O2B). As shown in Fig. 4, SKLB0533 was bound in the same location as the
Colchicine, with the purine-aminomethyl-pyridone located towards the β-subunit and
surrounded by residues Cysβ241, Leuβ248, Asnβ249, Alaβ250, Aspβ251, Lysβ254,
Leuβ255, Asnβ258, Metβ259, Thrβ314, Val β315, Alaβ316, Thrβ353 and Alaβ354.
There was hydrogen bond between the ketone of pyridin-2(1H)-one fragment and the
NH of Aspβ251.And the N7 of purine ring formed a hydrogen interaction with the
NH of Aspβ258. Besides the hydrogen interactions, there were some hydrophobic
interactions with the surrounding residues.

Fig. 4 Predicted binding mode of SKLB0533 (yellow stick) with tubulin (PDB code:
O2B) and overlapping with Colchicine (blue stick). Hydrogen bonds are shown by
green dashed lines.
4
2
.5. Inhibition of other targets
Considering that the 2,6,9-trisubstituted purine fragment in SKLB0533 was often
reported in kinase inhibitors, such as CDK1[34],CDK2[35], Src[36], AMPK[37] etc.
Therefore, we tested the activity of SKLB0533 against a panel of 420 kinases by
Eurofins Kinase Profiler. Here a single concentration (1 µM) of SKLB0533 was used.
The results showed that SKLB0533 had no activity against any of the kinases from
the
panel
(Fig.
5,
Supplementary
Tab.
S1).
Furthermore,
the

4
,6-dimethylpyridin-2(1H)-one fragment in SKLB0533 was often reported in
enhancer of zeste homologue 2 (EZH2) inhibitors[38, 39], we selected four
structurally diverse compounds to test EZH2 inhibition activity. Compound 3b, 4b2,
5
a and SKLB0533 did not exhibit significant inhibition of EZH2 at 1 µM or even at
0 µM (Supplementary Tab. S2).
1
Fig. 5 Heat map of the inhibition rate against activity of 420 kinds of kinases.
These results indicate that SKLB0533 specifically inhibits of microtubule
polymerization, and exhibits no activity against other potential targets, such as the
4
20 tested kinases and EZH2. These results suggest that SKLB0533 may have few
toxic side effects since its off-target effects are minimal.
2
.6. In vitro antiproliferative activities
We chose a panel of cancer cell lines of different histotypes to investigate the
antiproliferative effect of SKLB0533. The results demonstrated that SKLB0533
obviously inhibited all the tested cell lines after 72 h of incubation, with an optimal
inhibitory effect on the CRC cell lines at IC values between 44.5 and 135.5 nM (Tab.
5
0
3
). Thus, we decided to study the effect of SKLB0533 on CRC cells.

Tab. 3 Effects of SKLB0533 on cell viability.
Cell lines
HCT116
SW620
Cell type
IC (nM)
50
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Breast cancer
57.5±4.60
60.0±1.41
45.5±2.48
CT26
SW480
64.5±3.18
135.5±6.72
61.0±8.49
71.0±3.54
200.5±2.48
80.0±12.00
286.0±9.19
98.0±8.00
DLD-1
HCT15
HT29
MDA-MB-231
HepG2
Hepatocellular carcinoma
Hepatocellular carcinoma
Lung cancer
PLC/PRF/5
A549
Data are expressed as the mean±SEM for at least 2 independent experiments.
To evaluate the anticancer activity of SKLB0533 on CRC cells, the HCT116 and
SW620 cell lines were exposed to graded concentrations of SKLB0533 for either 24 h,
4
8 h or 72 h, and caused a marked decrease in viability (Fig. 6A and 6B). These
results demonstrate that SKLB0533 inhibits the proliferation of HCT116 and SW620
cells in a time and concentration dependent manner. Colony formation assays were
performed to further validate the anti-proliferating effect of SKLB0533. Notably,
colonies in treatment group were fewer and smaller than those in vehicle group (Fig.
6
C), and when the concentration reached 50 nM, almost no colony formation was
observed in SW620 cell lines.

Fig. 6 SKLB0533 suppressed proliferation of HCT116 and SW620 cells. (A)(B)
Tumour cells were treated with different concentrations of SKLB0533 for 24 h, 48 h
or 72 h. MTT assays were performed to measure cell proliferation. (C) Effect of
SKLB0533 on cell colony formation after two weeks of treatment.
2
.7. Cell cycle arrest
Both microtubule stabilizers and destabilizers alter the tubulin-microtubule
equilibrium causing mitotic arrest at the G2/M phase and ultimately apoptotic cell
death. A flow cytometry analysis was performed to examine the effect of SKLB0533
on HCT116 and SW620 cell cycle progression (Fig. 7A). Treatment of SKLB0533
resulted in the gradual accumulation of cells in the G2/M phase of the cell cycle, and
this accumulation occurred in a concentration-dependent manner, whereas the control

cells were mainly in the G1 phase. Thus, the cell cycle distribution indicated that
SKLB0533 could arrest HCT116 and SW620 cells in the G2/M phase.
To gain insight into the molecular mechanism underlying cell cycle arrest,
several cell cycle-related proteins were detected by western blot analysis. As shown in
Fig. 7B, after treatment with SKLB0533 for 24 h, the expression of CyclinB1 and
CDK1 were both upregulated. Furthermore, the level of cdc25c, which catalyses the
dephosphorylation of pCDK1(Y15), was also upregulated. Then we observed a
decrease in the level of pCDK1(Y15). These results suggest that high levels of
CyclinB1-CDK1 complex induce the cell cycle to remain in the stage of G2/M
transformation.
Fig. 7 SKLB0533 induced G2/M arrest in HCT116 and SW620 cells. (A) HCT116
and SW620 cell lines were treated with increasing doses of SKLB0533 (0, 25, 50 and

7
5 nM) for 24 h and were stained with 50 mg/mL propidium iodide (PI). (B) Effects
of SKLB0533 on the expression of G2/M phase related regulator proteins. HCT116
and SW620 cell lines were treated with SKLB0533 for 24 h at the indicated
concentrations (0, 25, 50, 100 and 150 nM).
2
.8. Cell apoptosis analysis
Mitotic arrest of tumour cells by tubulin-directed agents is generally associated
with cellular apoptosis. To investigate the effects of SKLB0533 inducing apoptosis,
AnnexinV/PI staining was performed to further determine whether SKLB0533 had a
pro-apoptotic effect on CRC cell lines. As shown in Fig. 8A, after exposure of
HCT116 cells to SKLB0533 for 48 h, both early apoptotic cells (Q3) and late
apoptotic cells (Q2) remarkably increased from 14.3% to 54.9% as the SKLB0533
concentration increased from 25 nM to100 nM. Meanwhile, only a few apoptotic cells
were detected in the control group. We also observed similar results in SW620 cells.
These data suggested that SKLB0533 induces apoptosis of HCT116 and SW620 cells
in a concentration-dependent manner.
Meanwhile, we also detected changes in several key proteins in the apoptosis
pathway. As shown in Fig. 8B, caspase9, which is involved in the
mitochondrial-mediated intrinsic apoptosis pathway, was activated after 48 h of
SKLB0533 treatment, as we observed that the level of cleaved caspase9 increased in a
dose-dependent manner. Additionally, the main executor of apoptosis, caspase3, was
then cleaved and activated. The significant increase in the expression of cleaved

PARP further indicated that caspase3 was activated after 48 h of SKLB0533 treatment.
We also detected the expression of some Bcl-2 family proteins, which were involved
in regulating apoptosis. These results showed that the level of Bcl-2 decreased, while
the level of Bax increased, in both cell lines.
Fig. 8 SKLB0533 induced apoptosis of HCT116 and SW620 cells. (A) HCT116 and
SW620 cells were treated with SKLB0533 for 48 h and apoptosis was detected by
flow cytometry after Annexin V/PI staining. (B) Effects of SKLB0533 on the
expression of apoptosis related regulator proteins. The levels of Bcl-2, Bax, cleaved
caspase9, cleaved caspase3 and cleaved PARP were determined via western blot
analysis.
2
.9. In vivo antitumor efficacy in human xenografts of SKLB0533
To evaluate the in vivo therapeutic efficacy of SKLB0533, HCT116 colon cancer

cells were subcutaneously engrafted into nude mice to establish a xenograft model.
Capecitabine, a first-line treatment for colorectal cancer, was selected as the positive
reference. SKLB0533 was orally administered at 40 or 80 mg/kg once daily over a
3
period of 4 weeks, at which point the tumour size reached approximately 100 mm .
As shown in Fig.9A, 9B and 9D, oral administration of 40 and 80 mg/kg SKLB0533
once daily for 4 weeks showed TGI values of 39.9% and 71.6%, respectively, on day
2
8. However, under the same condition, the TGI from the group treated with 540
mg/kg capecitabine was only 61.6%. We also observed a significant reduction in
tumour weight in Fig. 9C. These results indicate that the SKLB0533 potently and
effectively inhibits colorectal tumour growth in xenograft models.
2
.10. Preliminary safety profile of SKLB0533
To determine the potential toxicity of SKLB0533, we observed changes in body
weight and determined the routine parameters and biochemical analysis of blood. As
illustrated in Fig. 10A and 10B, no significant changes were observed in body weight,
and the haematological and serum biochemical values were comparable with those of
the vehicle group after 4 weeks SKLB0533 treatment. Moreover, microscopic
examination of the heart, liver, spleen, lung and kidneys further showed that
SKLB0533 (80 mg/kg) treatment did not cause obvious toxicity in mice (Fig. 10C).

Fig. 9 Effects of SKLB0533 on the growth of HCT116 xenografts in BALB/c mice.
BALB/c mice bearing HCT116 (n = 7) were orally treated with the vehicle control or
SKLB0533 once every day for 4 weeks. (A) Images of excised tumours from each
group. (B) Effects of SKLB0533 on tumour growth in a mouse xenograft. (C) Weight
of tumours from sacrificed mice. (D) Change in tumour volume at day 28. Data are
presented as the mean ± SD of 7 mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 10 Preliminary safety evaluation of SKLB0533 in BALB/c mice. (A) The
differences in body weight between three treatment groups (SKLB0533 40 mg/kg,
SKLB0533 80 mg/kg and Capecitabine) and the vehicle group were not significant.
Data are expressed as the mean±SD (n=7). (B) SKLB0533 did not cause significant
changes in routine blood analysis. The parameter units are as follows: WBC (white
9
12
blood cell) and PLT (platelet),10 /L; RBC (red blood cell), 10 /L; ALB (albumin),
GLB (globulin) and TP (total protein), g/L; ALT (alanine transarninase) and AST
(aspartate aminotransferase), U/L; UA (uric acid), µM; CHO (cholesterol) and GLU
(glucose), mM. A/G, ALB/ GLB. (C) Paraformaldehyde fixed organs (the heart, liver,
spleen, lungs and kidneys) were processed for paraffin embedding and then stained
with hematoxylin and eosin. The images shown are representatives from each group.
Scale bars represent 50 µm.

3
. Conclusion
In this study, we designed, synthesized and evaluated a series of novel
-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives as
3
potential inhibitors of tubulin polymerization using bioelectronic isosteric strategy.
SKLB0533 displayed the most potent activity against a wide variety of cancer cell
lines, with an optimal inhibitory effect on CRC cell lines. The immunofluorescence
analysis and tubulin polymerization assay results indicated that SKLB0533 could
effectively inhibit tubulin polymerization. Furthermore, this compound exhibited no
activity against 420 different kinases and EZH2, indicating considerable selectivity. In
mechanism, SKLB0533 could disturb the dynamics of tubulin, arrest the cell cycle in
the G2/M phase and induce apoptosis in HCT116 and SW620 cells. Moreover,
SKLB0533 was demonstrated significant antitumor e℃ cacy in the HCT116-xenograft
mode without overt toxicities. In conclusion, these results highlighted the SKLB0533
as a promising anti-tubulin agent for the treatment of CRC.
4
. Experimental section
4
.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers
and used without further purification. Melting point (mp) was measured on hot-stage
1
13
microscope (Shanghai Precision Scientific Instruments Co., Ltd, X-4). The H and C
NMR spectra were recorded on a Bruker Avance 400 spectrometer at 25 ℃ using
DMSO℃ d , CD OD or CDCl as the solvent. Chemical shifts (δ) are reported in ppm
6
3
3

relative to Me Si (internal standard), coupling constants (J) are reported in hertz, and
4
peak multiplicity are reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br s (broad singlet). High resolution mass analysis was performed on a
Waters Q-TOF Premier mass spectrometer with electron spray ionization (ESI). Thin
layer chromatography (TLC) was performed on 0.20 mm silica gel F-254 plates
(Qingdao Haiyang Chemical, China). Visualization of TLC was accomplished with
UV light and/or aqueous potassium permanganate or I in a silica gel. Column
2
chromatography was performed using silica gel 60 of 300-400 mesh (Qingdao
Haiyang Chemical, China).
4
.1.1.
,6-dichloro-9-substituted-9H-purine derivatives (2a-2c).
,6-dichloro-9-isopropyl-9H-purine (2a). 2,6-dichloro-9H-purine (1.0 g, 5.29
mmol), and potassium carbonate (K CO ) (2.193 g, 15.87 mmol) were dissolved in 20
The
representative
procedure
for
the
preparation
of
2
2
2
3
ml anhydrous DMSO. Iodopropane (2.64 mL, 26.45 mmol) was added dropwise at
5 ℃ . The reaction mixture was stirred at 15 ℃ overnight. Upon completion of the
1
reaction, the reaction solution was poured into ice water, and a white solid
precipitated. The solution was filtered and dried under a vacuum to give 1.1 g of light
1
yellow solid. Yield: 90.02%. H NMR (400 MHz, DMSO-d ) δ 8.86 (s, 1H), 4.83 (p, J
6
=
6.8 Hz, 1H), 1.56 (d, J = 6.8 Hz, 6H).
1
2
,6-dichloro-9-(pentan-3-yl)-9H-purine (2b). White solid, yield: 81.75%.
H

NMR (400 MHz, DMSO-d ) δ 8.85 (s, 1H), 4.38 (tt, J = 8.8, 5.6 Hz, 1H), 2.10 -1.85
6
(m, 4H), 0.72 (t, J = 7.4 Hz, 6H).
1
2
,6-dichloro-9-cyclopentyl-9H-purine (2c). White solid, yield: 58.82%. H NMR (400
MHz, DMSO-d ) δ 8.82 (s, 1H), 4.93 (p, J = 7.4 Hz, 1H), 2.20 (dq, J = 12.7, 6.7 Hz,
6
2
H), 2.02 (dq, J = 13.6, 7.1 Hz, 2H), 1.89 (m, 2H), 1.77 – 1.64 (m, 2H).
4
.1.2.
The
representative
procedure
for
the
preparation
of
3
-(((2-chloro-9-substituted-9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-
one derivatives (3a-3c).
3-(((2-chloro-9-isopropyl-9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1
H)-one (3a). 2,6-dichloro-9-isopropyl-9H-purine (2a) (540 mg, 2.34 mmol),
3
-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (427 mg, 2.81 mmol ，obtained
following the reference procedure [40]), and triethylamine (1.626 mL, 11.7 mmol)
were added to ethanol (10 mL), and the reaction solution was reacted under reflux at
8
0 ° C for 6 h. A white solid precipitated during the reaction. After the reaction was
completed, the suspension obtained was filtered and washed with cold ethanol (10 mL)
to afford a solid product. The crude product was purified by silica gel column
chromatography to afford target compounds as white solid with yield 50.52%, mp
1
1
5
3
41-143 °C. H NMR (400 MHz, DMSO-d ) δ 11.52 (s, 1H), 8.24 (s, 1H), 7.73 (t, J =
6
.7 Hz, 1H), 5.87 (s, 1H), 4.66 (m, J = 6.7 Hz, 1H), 4.45 (d, J = 5.3 Hz, 2H), 2.28 (s,
1
3
H), 2.11 (s, 3H), 1.49 (d, J = 6.8 Hz, 6H). C NMR (101 MHz, Chloroform-d) δ

1
65.35, 155.06, 154.13, 150.85, 149.71, 143.23, 137.16, 121.96, 119.10, 109.66,
6.77, 37.00, 22.80 (2C), 19.70, 18.87. HRMS (ESI): calcd. for C H ClN O
4
16
19
6
+
[
M+Na] : 369.1207, found: 369.1204.
3
-(((2-chloro-9-(pentan-3-yl)-9H-purin-6-yl)amino)methyl)-4,6
1
dimethylpyridin-2(1H)-one (3b). White solid, yield: 64.23%, mp 224-225 °C. H
NMR (400 MHz, DMSO-d ) δ 11.52 (s, 1H), 8.21 (s, 1H), 7.75 (t, J = 5.7 Hz, 1H),
6
5
.88 (s, 1H), 4.44 (d, J = 5.2 Hz, 2H), 4.19 (m, 1H), 2.29 (s, 3H), 2.11 (s, 3H), 1.90
13
(
ddt, J = 18.1, 14.1, 7.1 Hz, 4H), 0.69 (t, J = 7.3 Hz, 6H). C NMR (101 MHz,
Chloroform-d) δ 165.34, 155.02, 154.15, 150.82, 143.17, 138.22, 122.06, 109.66,
5
8.83, 36.96, 27.78 (2C), 19.72, 18.89, 10.49 (2C). HRMS (ESI): calcd. for
+
C H ClN O [M+Na] : 397.1520, found: 397.1525.
18
23
6
3
-(((2-chloro-9-cyclopentyl-9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)
1
-
one (3c). White solid, yield: 82.76%, mp 220-221 °C. H NMR (400 MHz, DMSO-d )
6
δ 11.52 (s, 1H), 8.21 (s, 1H), 7.73 (t, J = 7.9 Hz,1H), 5.87 (s, 1H), 4.82 – 4.71 (m, 1H),
13
4
.45 (d, J = 5.3 Hz, 2H), 2.28 (s, 3H), 2.11 (m, 5H), 1.89 (m, 2H), 1.68 (m, 2H). C
NMR (101 MHz, Chloroform-d) δ 164.87, 154.88, 154.16, 151.31, 150.05, 143.01,
1
37.78, 121.93, 118.68, 109.82, 55.70, 36.75, 32.85 (2C), 23.75 (2C), 19.65, 18.64.
+
HRMS (ESI): calcd. for C H ClN O [M+Na] : 395.1363, found: 395.1362.
18
21
6
4
.1.3. The representative procedure for the preparation of 3-(((2-substituted-9-

substituted -9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives
4a1-4a7;4b1-4b7;4c1-4c7).
-(((9-isopropyl-2-(4-(trifluoromethoxy)phenyl)-9H-purin-6-yl)amino)methyl)-4
6 dimethylpyridin-2(1H)-one (4a1). To a stirred solution of 3a (140 mg, 0.40 mmol)
(
3
,
in a dioxane–water mixture (16 mL/4 mL), (4-(trifluoromethoxy)phenyl) boronic acid
pinacol ester (172.8 mg, 0.60 mmol) was added, followed by the addition of Na CO
2
3
(169.6 mg, 1.60 mmol). The solution was purged with argon for 15 min and then
PdCl (dppf)· CH Cl (29.2 mg, 0.04 mmol) was added and the solution was again
2
2
2
purged with argon for an additional 10 min. The reaction mixture was stirred at 100 ℃
for 5 h. After completion (monitored by TLC), the reaction mixture was diluted with
water and extracted with 10% MeOH/DCM. The combined organic layers were dried
over anhydrous sodium sulphate, filtered and concentrated under reduced pressure.
The crude compound was purified by column chromatography eluting with
MeOH/DCM to afford the desired compound 4a1 as a white solid. Yeild:53.40%, mp
1
2
8
64-266 °C. H NMR (400 MHz, DMSO-d ) δ 11.55 (s, 1H), 8.55 (d, J = 8.4 Hz, 2H),
6
.25 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 5.7 Hz, 1H), 5.85 (s, 1H), 4.83 (m, J
=
6.8 Hz, 1H), 4.71 (d, J = 5.3 Hz, 2H), 2.26 (s, 3H), 2.11 (s, 3H), 1.58 (d, J = 6.7 Hz,
1
3
6
1
1
H). C NMR (101 MHz, Chloroform-d) δ 165.29, 157.42, 154.43, 150.25, 142.95,
37.54, 129.67 (2C), 122.78, 120.35 (2C), 109.44, 46.90, 29.70, 22.76 (2C), 19.79,
+
8.91. HRMS (ESI): calcd. for C H F N O [M+H] : 473.1913, found: 473.1914.
2
3
23
3
6
2
3
-(((9-isopropyl-2-(4-morpholinophenyl)-9H-purin-6-yl)amino)methyl)-4,6 dimethyl-

1
pyridin-2(1H)-one (4a2). Brown solid, yield: 68.06%, mp 190-192 °C. H NMR (400
MHz, Chloroform-d) δ 11.87 (s, 1H), 8.44 (d, J = 8.5 Hz, 2H), 7.71 (s, 1H), 6.97 (d, J
=
8.9 Hz, 2H), 6.67 (t, J = 5.7 Hz 1H), 5.86 (s, 1H), 4.96 (d, J = 5.3 Hz, 2H), 4.88 (m,
J = 6.8 Hz, 1H), 3.89 (t, J = 5.9, 3.7 Hz, 4H), 3.30 – 3.20 (t, 4H), 2.41 (s, 3H), 2.30 (s,
1
3
3
1
1
H), 1.61 (d, J = 6.8 Hz, 6H). C NMR (101 MHz, Chloroform-d) δ 164.80, 158.93,
54.05, 152.15, 150.97, 149.88, 142.73, 137.02, 130.65, 129.27 (2C), 122.83, 118.16,
14.65 (2C), 109.74, 66.77 (2C), 48.78 (2C), 46.75, 35.97, 22.59 (2C), 19.73, 18.49.
+
HRMS (ESI): calcd. for C H N O [M+H] : 474.2617, found: 474.2617.
26
31
7
2
3
-(((9-isopropyl-2-(3-morpholinophenyl)-9H-purin-6-yl)amino)methyl)-4,6-dimethyl-
1
pyridin-2(1H)-one (4a3). Off-white solid, yield: 57.59%, mp 146-148 °C. H NMR
(
400 MHz, DMSO-d ) δ 11.55 (s, 1H), 8.22 (s, 1H), 8.06 (s, 1H), 7.91 (d, J = 7.7 Hz,
6
1
H), 7.33 (t, J = 7.9 Hz, 1H), 7.15 (t, J = 5.7 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 5.86 (s,
H), 4.83 (m, J = 6.8 Hz, 1H), 4.73 (d, J = 5.3 Hz, 2H), 3.79 (t, J = 4.7 Hz, 4H), 3.19
1
1
3
(
t, J = 4.8 Hz, 4H), 2.30 (s, 3H), 2.11 (s, 3H), 1.57 (d, J = 6.7 Hz, 6H). C NMR (101
MHz, Chloroform-d) δ 165.31, 158.76, 154.36, 151.35, 142.88, 140.26, 137.32,
1
28.89, 123.01, 120.40, 117.15, 115.65, 109.33, 67.08 (2C), 49.73 (2C), 46.66, 22.84
+
(
2C), 19.89, 18.95. HRMS (ESI): calcd. for C H N O [M+H] : 474.2617, found:
2
6
31
7
2
4
74.2613.
3
-(((9-isopropyl-2-(4-(morpholinomethyl)phenyl)-9H-purin-6-yl)amino)methyl)-4,6-d
imethylpyridin-2(1H)-one (4a4). Light yellow solid, yield: 21.68%, mp