Design, synthesis and biological evaluation of a novel tubulin inhibitor SKLB0565 targeting the colchicine binding site

Article abstract of DOI:10.1016/j.bioorg.2020.103695

A series of 3-(((9H-purin-6-yl) amino) methyl) pyridin-2(1H)-one derivatives were designed, synthesized and confirmed as tubulin polymerization inhibitors. All compounds were evaluated for their anti-proliferative activities on three colorectal carcinoma (CRC) cell lines. Among these compounds, SKLB0565 displayed noteworthy potency against eight CRC cell lines with IC₅₀ values ranging from 0.012 μM and 0.081 μM. Besides, SKLB0565 inhibited tubulin polymerization, caused G2/M phase cell cycle arrest, depolarized mitochondria and induced cell apoptosis in CRC cells. Furthermore, SKLB0565 suppressed cell migration and disrupted the capillary tube formation of human umbilical vein endothelial cells (HUVECs). Our data clarified that SKLB0565 is a promising anti-tubulin agent for CRC therapy which is worthy of further evaluation.

Full text of DOI:10.1016/j.bioorg.2020.103695

Bioorganic Chemistry 97 (2020) 103695
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of a novel tubulin inhibitor
SKLB0565 targeting the colchicine binding site
1
1
⁎
Xi Hu , Lu Li , Qiangsheng Zhang, Qianqian Wang, Zhanzhan Feng, Ying Xu, Yong Xia ,
⁎
Luoting Yu
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
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of 3-(((9H-purin-6-yl) amino) methyl) pyridin-2(1H)-one derivatives were designed, synthesized and
confirmed as tubulin polymerization inhibitors. All compounds were evaluated for their anti-proliferative ac-
tivities on three colorectal carcinoma (CRC) cell lines. Among these compounds, SKLB0565 displayed note-
worthy potency against eight CRC cell lines with IC50 values ranging from 0.012 μM and 0.081 μM. Besides,
SKLB0565 inhibited tubulin polymerization, caused G2/M phase cell cycle arrest, depolarized mitochondria and
induced cell apoptosis in CRC cells. Furthermore, SKLB0565 suppressed cell migration and disrupted the ca-
pillary tube formation of human umbilical vein endothelial cells (HUVECs). Our data clarified that SKLB0565 is a
promising anti-tubulin agent for CRC therapy which is worthy of further evaluation.
Tubulin polymerization inhibitors
Colchicine binding site
Anti-proliferative
Colorectal carcinoma
1
. Introduction
and BNC105 [18]. We designed and synthesized a series of 3-(((9H-purin-
6
-yl)amino)methyl-4,6-dimethylpyridin-2(1H)-one derivatives using a
Microtubules, which are widely expressed in eukaryotic cells, are
bioelectronic isosteric strategy in our previous study. Biological evalua-
tion indicated that this class of compounds were antimitotic agents and
exerted their anticancer action through inhibition of tubulin poly-
merization. In particular, one of the compounds SKLB0533 showed strong
anti-proliferative effects in CRC cells. Furthermore, SKLB0533 showed
excellent target selectivity and exhibited no inhibitory activities against
other potential targets, including 420 kinases and EZH2 [19]. All of these
indicated that this series of compounds deserve further study to obtain
more potent compounds, as well as to explore their structure activity
relationships (SARs) more perfectly.
tubular polymers formed by polymerization of α- and β-tubulin with
molecular weights of about 55 kDa [1,2].They are main components of
the cytoskeleton and play pivotal roles in numerous cellular activities,
such as maintaining cell morphology and transporting intracellular
material, especially during cell division and proliferation [3–5]. Mi-
crotubules maintain the balance of dynamic polymerization and depo-
lymerization in normal cells. However, the irreversible destruction of
microtubule dynamic polymerization balance is often closely related to
the progression of cancer [6]. Therefore, microtubules have been con-
sidered to be a promising target for cancer chemotherapy [7,8].
Lots of tubulin inhibitors have been successfully used in the clinic and
many more are under preclinical and clinic evaluation, such as paclitaxel
In this study, we focus on the effects of different pyridine-2(1H)-one
substitutions on their anti-proliferative activities and aim to find more
potent compounds targeting the colchicine binding site to complement
our previous research. We designed and synthesized a series of 3-(((9H-
purin-6-yl) amino) methyl) pyridin-2(1H)-one derivatives, analyzed the
SARs of different pyridine-2(1H)-one substitutions and evaluated their
anti-CRC activities of this class of compounds (Fig. 1B).
[
9], colchicine [10], combretastatin A-4 (CA-4) [11,12] and SAMRT [13]
(
Fig. 1A). Based on the analysis of the chemical structure of reported
microtubule inhibitors, our research interest focuses on novel microtubule
inhibitors containing concise double-ring core structure, such as MPC-
6
827 [14], Compound 30·HCl [15], Compound 60 [16], Myoseverin [17]
⁎
Corresponding authors at: 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.
E-mail addresses: scuxiayong@163.com (Y. Xia), yuluot@scu.edu.cn (L. Yu).
1
These authors contribute equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103695
Received 28 January 2020; Received in revised form 21 February 2020; Accepted 22 February 2020
Available online 24 February 2020
0
045-2068/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(
http://creativecommons.org/licenses/BY-NC-ND/4.0/).

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 1. (A) Chemical structures of some tubulin polymerization inhibitors containing double-ring core structure. (B) Further study of 2, 6, 9-trisubstituted purine
derivatives in this study.
2
. Results and discussion
group with tetrahydropiran (THP) under p-toluenesulfonic acid (p-
TsOH). (2) Compounds 3a1–3d3 were synthesized in high yields by
reacting compounds 2a–2d with 3-(aminomethyl)-4-ethyl-6-methyl-
pyridin-2(1H)-one, 3-(aminomethyl)-4,6-diethylpyridin-2(1H)-one and
4-(aminomethyl)-1-methyl-5,6,7,8-tetrahydroisoquinolin-3(2H)-one
[20]. The chlorine in the C2‐position of the intermediates was sub-
stituted with a methylamino group on treatment with aqueous methy-
lamine in a sealed tube to give corresponding compounds 4a1–4b4 in
excellent yields.
2.1. Chemistry
Scheme 1 illustrated the reaction routes employed for synthesis of
the 3-(((9H-purin-6-yl) amino) methyl) pyridin-2(1H)-one derivatives.
Compounds 3a1–3d3 were synthesized from commercially available 2,
6
-dichloro-1H-purine in two steps: (1) 2, 6-dichloro-1H-purine was re-
acted with halogenated alkane under basic conditions to give com-
pounds 2a–2c. Compound 2d was obtained by protecting the amino
2

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Scheme 1. Synthesis of target compounds. (a) Halogenated alkane, K
2
CO
3
, anhydrous DMSO, 15 °C, overnight. (b) 3,4-Dihydro-2H-pyran, p-TsOH, DCM, r.t.,8h. (c)
3
3
-(aminomethyl)-4-ethyl-6-methylpyridin-2(1H)-one, 3-(aminomethyl)-4,6-diethylpyridin-2(1H)-one or 4-(aminomethyl)-1-methyl-5,6,7,8-tetrahydroisoquinolin-
(2H)-one, EtOH, 80 °C, 6 h. (d) Aqueous methylamine, sealed tube, 110 °C, 5 h.
2
.2. Structure-activity relationships
3d1–3d3). In our previous studies, the steric effect of substituents on
the C2-position of purine ring was crucial to the anti-proliferative ac-
tivities [21]. To exclude the possibilities that the anti-proliferative ef-
fects were caused by the departure of chlorine atom, the chlorine atom
at 2-position of purine in 3c2 was replaced with aminomethyl sub-
stitution to obtain 4b3, which maintained the anti-proliferative ac-
tivity, suggesting that 3c2 might not exert anti-proliferative efficacy
through the departure of chlorine atom.
To better understand the SARs of the newly synthesized compounds,
the IC50 values were obtained by MTT cytotoxicity assay. According to
previous studies, SKLB0533 exhibited notable anti-proliferative effects
on CRC cells. Therefore, we continued to evaluate the anti-proliferative
activities of these compounds against three CRC cells lines CT26, SW20
and HCT116, CA-4 and SKLB0533 served as the positive references
(
Table 1).
Then we focused on the effects of different pyridin-2(1H)-one sub-
stitutions at the 9-position of purine ring on anti-proliferative activities.
As shown in Table 1, the anti-proliferative activities of the synthesized
compounds with different pyridin-2(1H)-one substitutions increased in
the following order: compounds 3a2, 3b2, 3c2, 3d2, 4a3 and 4b3 (4,6-
diethylpyridin-2(1H)-one) > compounds 3a1, 3b1, 3c1, 3d1, 4a2 and
In general, when the N9-position of purine ring was substituted by
2
-Pr, 3-pentyl, cyclopentyl or THP, all of the compounds exhibited good
anti-proliferative activities. Incorporation of a cyclopentyl group at 9-
position of purine ring (3c1–3c3), demonstrated improved cellular
activities compared to 3-pentyl and THP substituents (3b1–3b3,
4
3
b2 (4-ethyl-6-methylpyridin-2(1H)-one) > compounds 3a3, 3b3,
c3, 3d3 and 4b4 (1-methyl-5,6,7,8-tetrahydroisoquinolin-3(2H)-one).
Table 1
When comparing compound 3c3 with SKLB0533, compound 4a3 with
compound 4a1 and compound 4b3 with compound 4b1, the data in-
dicated that the 4,6-diethylpyridin-2(1H)-one substituent might in-
teract more strongly with the amino acids surrounding the binding
pocket, which was confirmed in subsequent molecular modeling stu-
dies.
a
Anti-proliferative activities of the compounds against three CRC cell lines.
IC50 values (µM)b
Cpd.
CT26
SW620
HCT116
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
a1
0.101 ± 0.011
0.022 ± 0.011
> 5
0.072 ± 0.001
0.009 ± 0.002
1.350 ± 0.054
0.409 ± 0.058
0.244 ± 0.001
1.908 ± 0.164
0.031 ± 0.004
0.040 ± 0.011
0.312 ± 0.010
0.097 ± 0.018
0.043 ± 0.007
0.958 ± 0.026
0.176 ± 0.053
0.103 ± 0.006
0.317 ± 0.037
0.214 ± 0.017
0.120 ± 0.011
0.078 ± 0.011
1.133 ± 0.057
0.062 ± 0.001
0.029 ± 0.006
< 0.002
Most of the synthesized compounds inhibited the growth of the
tested CRC cell lines, with IC50 values less than 1 μM. Notably, com-
pound 3c2 (SKLB0565) exhibited the most potent anti-proliferative
activity among all compounds, with IC50 values ranging between 0.021
and 0.057 µM. Moreover, SKLB0565 was more active against the tested
CRC cell lines than SKLB0533 in our study. Therefore, SKLB0565 was
selected for further pharmacodynamic study.
a2
0.058 ± 0.003
0.038 ± 0.002
0.077 ± 0.003
0.104 ± 0.007
3.232 ± 0.022
0.510 ± 0.390
0.021 ± 0.003
0.839 ± 0.021
2.761 ± 0.197
0.039 ± 0.000
2.250 ± 0.243
0.142 ± 0.013
0.099 ± 0.018
0.479 ± 0.088
0.138 ± 0.029
0.043 ± 0.003
0.019 ± 0.007
1.329 ± 0.077
0.072 ± 0.003
0.128 ± 0.005
a3
b1
0.023 ± 0.003
0.021 ± 0.006
0.086 ± 0.021
0.049 ± 0.002
0.057 ± 0.007
1.255 ± 0.193
0.088 ± 0.007
0.064 ± 0.019
4.772 ± 0.158
2.790 ± 0.72
0.619 ± 0.162
> 5
b2
b3
c1
c2 (SKLB0565)
c3
d1
d2
d3
a1
a2
a3
b1
b2
b3
b4
2.3. Effects of SKLB0565 on tubulin polymerization and microtubule
The effects of SKLB0565 on tubulin polymerization were de-
termined by a fluorescence-based tubulin polymerization kit in cell free
condition, and paclitaxel and CA-4 were used as positive controls. As
shown in Fig. 2A, paclitaxel exhibited significant promotion of tubulin
polymerization, while compound SKLB0565, which was similar to CA-
1.498 ± 0.023
0.616 ± 0.115
0.192 ± 0.064
> 5
SKLB0533
CA-4
0.052 ± 0.012
0.114 ± 0.013
4
, displayed a concentration-dependent inhibition of tubulin poly-
merization. These results indicated that SKLB0565 was able to effec-
tively inhibit the polymerization of tubulin in vitro.
a
b
Cell lines were treated with different concentrations of compounds for 72 h.
IC50 values are indicated as the mean ± SEM of at least two independent
Since inhibition of tubulin polymerization would cause damage of
microtubule networks and cytoskeleton [22], we subsequently
experiments.
3

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 2. Effects of SKLB0565 on tubulin poly-
merization (A) and microtubule networks (B) in
vitro. (A) 2 mg/mL purified tubulin protein in re-
action buffer was incubated at 37 °C in the presence
of 0.1% DMSO, SKLB0565 (0.05, 1.25, 6.0, 30 μM),
Paclitaxel (3 μM) or CA-4 (1.25 μM).
Polymerizations were followed by an increase in
fluorescence excitation at 355 nm and fluorescence
emission at 460 nm over a 60 min period at 37 °C.
(
B) Both cells were treated with 0.1% DMSO,
SKLB0565 (50 nM) or CA-4 (50 nM) for 18 h.
Microtubule networks were visualized with an anti-
β-tubulin-FITC antibody (green), and the cell nu-
cleus were visualized with DAPI (blue). (For inter-
pretation of the references to colour in this figure
legend, the reader is referred to the web version of
this article.)
performed the immunofluorescence assays to investigate the effects of
compound SKLB0565 on microtubule networks in HCT116 and SW620
cells. As shown in Fig. 2B, the microtubule networks in the cells ex-
hibited a normal filamentous arrangement without drug treatment.
However, when treated with SKLB0565 or CA-4 at the concentration of
the CRC cells after 72 h of treatment, and the IC50 values were all below
100 nM. Therefore, we chose HCT116 and SW620 to further study the
efficacy of SKLB0565 against CRC cells.
As shown in Fig. 4A, after treatment with different concentrations of
SKLB0565 for 24, 48 or 72 h, the viabilities of both HCT116 and SW620
cells were inhibited in different degrees, indicating that SKLB0565
suppressed cell proliferation in a time- and concentration-dependent
manner. Colony formation assay was also used to assess the sensitivity
of CRC cells to SKLB0565 over a relatively long exposure time
5
0 nM for 18 h, the microtubule organization in the cytosol was dis-
rupted and shrank to the nucleus. These results indicated that
SKLB0565 could cause the disintegration of intracellular microtubule
networks, which would potentially lead to cell cycle disorder [23].
(
14 days). As shown in Fig. 4B, both the size and number of clones in
HCT116 and SW620 cells were decreased at low concentration of
SKLB0565.
2.4. Molecular docking
The binding mode and type of interaction between SKLB0565 and
tubulin (PDB code: 4O2B) were investigated by molecular docking. As
shown in Fig. 3A, SKLB0565 could well bind to colchicine binding site
of tubulin, and the binding mode was similar to that of colchicine.
There were hydrogen bonds between the ketone of pyridin-2(1H)-one
fragment and the NH of Aspβ251, the N7 of purine ring and the NH of
Asnβ258. The NH of pyridin-2(1H)-one fragment of SKLB0565 could
form a hydrogen interaction with the carbonyl of Aspβ251, but
SKLB0533 did not form the hydrogen interaction (Fig. 3B). We specu-
lated that the 4, 6-diethyl group of pyridin-2(1H)-one caused a slight
change in the spatial conformation of pyridine, which could better
adapt to the binding pocket. Besides the hydrogen interactions, there
were some hydrophobic interactions with the surrounding residues.
2
.6. SKLB0565 treatment caused G2/M cell cycle arrest of CRC cells
Since most microtubule polymerization inhibitors could disrupt cell
mitosis and exert the G2/M arrest on cell cycle [24,25], the effects of
SKLB0565 on cell cycle progression in HCT116 and SW620 cells were
examined by flow cytometry (FCM) analysis after propidium iodide (PI)
staining. As shown in Fig. 5A, the proportion of cells in G2/M phase
increased with the increase of SKLB0565, suggesting that SKLB0565
could cause G2/M phase cell cycle arrest in a dose-dependent manner.
The alterations of protein expression involved in G2/M phase reg-
ulations were detected to elucidate how SKLB0565 caused cell cycle
arrest. As indicated in Fig. 5B, SKLB0565 increased the expression of
CyclinB1 and CDK1, and decreased that of pCDK1 (Y15). Meanwhile,
the expression of cdc25c, which catalyzes the dephosphorylation of
pCDK1 (Y15), was upregulated. Our data indicated that SKLB0565
caused the increase activities of CyclinB1-CDK1 complex in CRC cells
and might lead to cell cycle arrest through this mechanism.
2.5. In vitro anti-proliferative activities of SKLB0565
The growth inhibitory activities of SKLB0565 were further eval-
uated by MTT assay against some other CRC cell lines. The data in
Table 2 showed that SKLB0565 dramatically inhibited the viabilities of
4

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 3. Predicted binding mode of SKLB0565 (yellow stick) with tubulin (PDB code: 4O2B) and overlapping with colchicine (blue stick) (A) and SKLB0533 (carmine
stick) (B). Hydrogen bonds are shown as green dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
treatment with SKLB0565 for 48 h, indicating the activation of caspase-
Table 2
Effects of SKLB0565 on CRC cell viabilities .
9. This phenomenon indicated that the mitochondria-mediated intrinsic
apoptotic pathway might be involved in SKLB0565 induced apoptosis.
The disruption of ΔΨm is an important event in the mitochondrial
apoptosis pathway [29,30]. Then, a mitochondria-specific and voltage-
dependent dye Rhodamine 123 (Rh123) was used to detect the ΔΨm
changes in both cell lines. The results in Fig. 7B showed that ΔΨm
decreased after treatment with the increasing concentration of
SKLB0565, indicating that SKLB0565 induced apoptosis probably via
the intrinsic mitochondrial apoptosis pathway. Furthermore, the ex-
pression of PARP, a downstream cleavage substrate of caspase-3, and
the expression of Bcl-2 and Bax, two Bcl-2 family proteins, which are
involved in the regulation of mitochondrial membrane permeability
[31,32], were also detected (Fig. 7A). After exposure to SKLB0565 for
48 h, the level of cleaved PARP increased significantly. Additionally,
the expression of Bcl-2 was down-regulated and that of Bax was up-
regulated after SKLB0565 treatment for 48 h. All of these results sug-
gested that the endogenous mitochondrial mediated apoptosis pathway
was involved in SKLB0565 induced apoptosis.
a
IC50 (μM)b
Cell lines
Cell type
HCT116
SW620
SW480
SW48
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
Colorectal carcinoma
0.012 ± 0.003
0.028 ± 0.001
0.081 ± 0.004
0.015 ± 0.003
0.074 ± 0.008
0.029 ± 0.001
0.029 ± 0.001
0.052 ± 0.001
DLD-1
HCT15
HT29
CT26
a
b
Cell lines were treated with different concentrations of SKLB0565 for 72 h.
IC50 values are indicated as the mean ± SEM of at least two independent
experiments.
2.7. SKLB0565 treatment caused apoptosis of CRC cells
To analyze the mode of cell death induced by SKLB0565, FCM
analysis was performed after FITC-Annexin-V/PI staining. PI, could
enter dead cells and intercalate in DNA, and Annexin-V, could bind to
phosphatidyl serine of valgus at the initial stage of apoptosis [26]. It
can be observed from Fig. 6A that the total percentages of early and late
apoptotic cells were significantly elevated with the increasing con-
centration of SKLB0565. The data showed that 100 nM of SKLB0565
caused apoptosis rates of 43.27% and 31.23% after 48 h treatment in
HCT116 and SW620 cells, respectively. Whereas the apoptosis rates of
untreated cells were only 3.39% and 8.60%. The morphological
changes of SW620 cells’ nuclei after SKLB0565 treatment were ana-
lyzed through Hoechst 33342 staining [27]. As seen in Fig. 6B, the
apoptotic cells showed bright blue fluorescent and condensed nuclei,
which were only seen in the SKLB0565 treated cells. These results
showed that SKLB0565 significantly induced CRC cell apoptosis in a
dose-dependent manner.
2.9. In vitro anti-vascular activity of SKLB0565
HUVECs were used to investigate the anti-vascular activity of
SKLB0565. Because microtubule networks control some cell migration
events [5], the inhibition of SKLB0565 on HUVECs migration was de-
tected by wound healing assay. As we can see in Fig. 8A, the scratch in
the SKLB0565 treated cell cultures healed slower than that in the un-
treated cell culture.
Colchicine site binding agents could block the vasculogenesis within
solid tumors and a number of them have been in anticancer clinical
investigations [33]. Thus, we carried out capillary tube formation assay
to evaluate the anti-vascular activity of SKLB0565. HUVECs could form
capillary-like tubes with multicentric junctions after being seeded on
matrigel. As shown in Fig. 8B, after exposure to SKLB0565 at doses of
50 and 100 nM for 6 h, the capillary-like tubes were damaged in dif-
ferent levels, suggesting that SKLB0565 effectively inhibited the tube
formation of HUVECs. These results confirmed that SKLB0565 was able
to inhibit vasculogenesis in vitro.
2.8. Effects of SKLB0565 on the mitochondrial membrane potential (ΔΨm)
In order to gain insight into the underlying mechanism of cell
apoptosis induced by SKLB0565, several apoptosis-related proteins
were detected by western blot. As shown in Fig. 7A, the expression of
cleaved caspase-9, which plays an important role in mitochondrial-
mediated intrinsic apoptosis pathway [28,29], was up-regulated after
3. Conclusions
In the present study, we have designed, synthesized and evaluated
the anti-CRC efficacies of a series of novel 3-(((9H-purin-6-yl) amino)
5

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 4. Anti-proliferative activities of SKLB0565
on HCT116 and SW620 cells measured by MTT
and colony formation assays. (A) The growth in-
hibitory activities of SKLB0565 against HCT116
and SW620 cells at different time points. (B)
Representative colony formation images of
HCT116 and SW620 cells after exposure to
SKLB0565 for 14 days.
methyl) pyridin-2(1H)-one derivatives based on the previously reported
SKLB0533. Anti-proliferative screening of these newly synthesized
compounds showed that the representative compound SKLB0565 ex-
hibited noteworthy nanomolar potency against a series of CRC cell
lines. It showed inhibitory activity on tubulin polymerization and the
ability to bind to colchicine site. Further mechanism studies illustrated
that SKLB0565 caused abnormal cellular microtubules and G2/M phase
cell cycle arrest, depolarized mitochondria, and induced cell apoptosis
in CRC cell lines. Additionally, wound healing and capillary tube for-
mation assays showed that SKLB0565 was a potent vascular disrupting
agent. Taken together, SKLB0565 showed impressing anti-CRC activ-
ities in vitro and is a potential anti-tubulin agent for CRC treatment.
4.1.1. The representative procedure for the preparation of 2,6-dichloro-9-
substituted-9H-purine derivatives (2a–2c)
2,6-dichloro-9-isopropyl-9H-purine (2a). 2,6-dichloro-9H-purine
(1.0 g, 5.29 mmol), and potassium carbonate (K
2
CO ) (2.193 g,
3
15.87 mmol) were dissolved in 20 mL anhydrous DMSO. Iodopropane
(2.64 mL, 26.45 mmol) was added dropwise at 15 °C. The reaction
mixture was stirred at 15 °C overnight. Upon completion of the reaction,
the reaction solution was poured into ice water, and a white solid pre-
cipitated. The solution was filtered and dried under a vacuum to give
1
1.1 g of light yellow solid. Yield: 90.02%. H NMR (400 MHz, DMSO‑d
δ 8.86 (s, 1H), 4.83 (p, J = 6.8 Hz, 1H), 1.56 (d, J = 6.8 Hz, 6H).
6
)
2,6-dichloro-9-(pentan-3-yl)-9H-purine (2b). White solid, yield:
1
8
5
1.75%. H NMR (400 MHz, DMSO‑d
6
) δ 8.85 (s, 1H), 4.38 (tt, J = 8.8,
.6 Hz, 1H), 2.10–1.85 (m, 4H), 0.72 (t, J = 7.4 Hz, 6H).
4
. Experimental
2,6-dichloro-9-cyclopentyl-9H-purine (2c). White solid, yield:
1
5
8.82%. H NMR (400 MHz, DMSO‑d
6
) δ 8.82 (s, 1H), 4.93 (p,
4.1. Chemistry
J = 7.4 Hz, 1H), 2.20 (dq, J = 12.7, 6.7 Hz, 2H), 2.02 (dq, J = 13.6,
7
.1 Hz, 2H), 1.89 (m, 2H), 1.77–1.64 (m, 2H).
Unless otherwise noted, all materials were obtained from commer-
cial suppliers and used without further purification. Melting point (mp)
was measured on hot-stage microscope (Shanghai Precision Scientific
4.1.2. Synthesis of 2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
(2d)
1
13
Instruments Co., Ltd, X-4). The H and C NMR spectra were recorded
on a Bruker Avance 400 spectrometer at 25 °C using DMSO‑d
or CDCl as the solvent. Chemical shifts (δ) are reported in ppm relative
to Me Si (internal standard), coupling constants (J) are reported in
hertz, and peak multiplicity are reported as s (singlet), d (doublet), t
6
, CD
3
OD
2,6-dichloro-9H-purine (4.62 g, 24.03 mmol), 3,4-dihydropyran
(4.38 g, 48.06 mmol) and p-toluenesulfonic acid (0.27 g, 1.44 mmol)
were added to a 250 mL round bottom flask. DCM was used as the
solvent, and the solution was reacted at room temperature for 8 h. After
the reaction was completed, the reaction solution was washed with
water. The organic phase was collected and dried under a vacuum to
give crude the product. The crude product was then purified by silica
3
4
(
triplet), q (quartet), m (multiplet), or br s (broad singlet). High re-
solution mass analysis was performed on a Waters Q-TOF Premier mass
spectrometer with electron spray ionization (ESI). Thin layer chroma-
tography (TLC) was performed on 0.20 mm silica gel F-254 plates
gel column chromatography to afford 6.5 g of the target compounds as
1
(
Qingdao Haiyang Chemical, China). Visualization of TLC was accom-
white solid with yield 99.01%. H NMR (400 MHz, DMSO‑d
6
) δ 8.95 (s,
plished with UV light and/or aqueous potassium permanganate or I
2
in
1H), 5.74 (dd, J = 10.8, 2.3 Hz, 1H), 4.10–3.93 (m, 1H), 3.78–3.66 (m,
1H), 2.31–2.19 (m, 1H), 2.07–1.88 (m, 2H), 1.82–1.71 (m, 1H), 1.60 (p,
J = 5.9, 4.9 Hz, 2H).
a silica gel. Column chromatography was performed using silica gel 60
of 300–400 mesh (Qingdao Haiyang Chemical, China).
6

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 5. SKLB0565 caused G2/M phase cell cycle arrest in HCT116 and SW620 cells. (A) Cells were treated with increasing doses of SKLB0565 for 24 h and then
analyzed by FCM. (B) Quantified histograms display the effects of SKLB0565 on HCT116 and SW620 cell cycle progression. Data are expressed as means ± SD for 3
independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle control group by t-test. (C) After treatment with indicated con-
centrations of SKLB0565 for 24 h, the cells were harvested and lysed for detection of several key cell cycle regulating proteins.
4
.1.3. The representative procedure for the preparation of 3-(((2-chloro-9-
substituted-9H-purin-6-yl)amino)methyl)-pyridin-2(1H)-one derivatives
3a1–3d3)
-(((2-chloro-9-isopropyl-9H-purin-6-yl)amino)methyl)-4-ethyl-6-
methylpyridin -2(1H)-one (3a1). 2,6-dichloro-9-isopropyl-9H-purine
2a) (300.00 mg, 1.30 mmol), 3-(aminomethyl)-4-ethyl-6-methylpyr-
1H), 5.92 (s, 1H), 4.71–4.62 (m, 1H), 4.49 (d, J = 5.2 Hz, 2H), 2.63 (q,
J = 7.3 Hz, 2H), 2.13 (s, 3H), 1.49 (d, J = 6.8 Hz, 6H), 1.11 (t,
1
3
(
J = 7.6 Hz, 3H). C NMR (101 MHz, DMSO‑d ) δ 164.01, 155.01,
6
3
153.13, 149.73, 143.84, 139.90, 121.03, 118.96, 106.36, 47.22, 36.63,
25.90, 22.55 (2C), 18.78, 14.90. HRMS (ESI): calcd. for C17
H
21ClN NaO
6
+
(
[M+Na] : 383.1363 found: 383.1355.
idin-2(1H)-one (258.95 mg, 1.56 mmol, obtained following the re-
ference procedure [21]), and triethylamine (0.90 mL, 6.50 mmol) were
added to ethanol (10 mL), and the reaction solution was reacted under
reflux at 80° C for 5 h. After completion (monitored by TLC), the re-
action mixture was removed under vacuum and extracted with DCM.
The organic layers were dried over anhydrous sodium sulphate, filtered
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography to afford target com-
3-(((2-chloro-9-isopropyl-9H-purin-6-yl)amino)methyl)-4,6-diethyl-
pyridin-2(1H)-one (3a1). White solid, yield: 87.32%, mp 187–189 °C.
1
H NMR (400 MHz, DMSO‑d ) δ 11.55 (s, 1H), 8.24 (s, 1H), 7.69 (t,
6
J = 7.9 Hz, 1H), 5.94 (d, J = 4.4 Hz, 1H), 4.71–4.62 (m, 1H), 4.49 (d,
J = 5.5 Hz, 2H), 2.64 (q, J = 7.7 Hz, 2H), 2.43 (q, J = 7.7 Hz, 2H),
1
3
1.48 (t, J = 6.9 Hz, 6H), 1.12 (t, J = 6.8, 6.1 Hz, 6H). C NMR
(101 MHz, Chloroform-d) δ 165.72, 156.34, 154.99, 154.11, 149.70,
149.27, 137.16, 121.27, 119.11, 106.18, 46.75, 36.56, 26.35, 26.20,
1
pounds as white solid with yield 91.23%, mp 215–217 °C. H NMR
22.79 (2C), 14.81, 12.69. HRMS (ESI): calcd. for C18
H23ClN NaO [M
6
+
(
400 MHz, DMSO‑d
6
) δ 11.56 (s, 1H), 8.24 (s, 1H), 7.69 (t, J = 5.5 Hz,
+Na] : 397.1520 found: 397.1513.
7

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 6. SKLB0565 induced apoptosis in HCT116 and SW620 cells. (A) The cells were treated with increasing doses of SKLB0565 for 48 h and analyzed by FCM after
Annexin V/PI staining. (B) Quantified histograms display the proportions of apoptotic cells. Data are expressed as means ± SD for 3 independent experiments,
*
p < 0.05, compared with the vehicle control group by t-test. (C) After treatment with indicated concentrations of SKLB0565 for 48 h, the cells were stained with
Hoechst 33422 (10 μg/mL) and visualized by fluorescence microscopy.
4
-(((2-chloro-9-isopropyl-9H-purin-6-yl)amino)methyl)-1-methyl-
(101 MHz, Chloroform-d) δ 165.74, 156.38, 154.91, 154.14, 150.49,
5
9
8
,6,7,8-tetrahydro isoquinolin-3(2H)-one (3a3). White solid, yield:
143.77, 138.24, 121.10, 118.89, 107.98, 58.81, 36.48, 27.76 (2C),
1
3.10%, mp 284–286 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.54 (s, 1H),
26.22, 18.95, 14.75, 10.48 (2C). HRMS (ESI): calcd. for C19
H
25ClN NaO
6
+
.23 (s, 1H), 7.79 (t, J = 5.2 Hz, 1H), 4.76–4.53 (m, 1H), 4.46 (d,
[M+Na] : 411.1676 found: 411.1673.
J = 5.1 Hz, 2H), 2.87 (s, 2H), 2.38 (s, 2H), 2.10 (s, 3H), 1.64 (t,
3-(((2-chloro-9-(pentan-3-yl)-9H-purin-6-yl)amino)methyl)-4,6-die-
1
3
J = 5.2 Hz, 5H), 1.49 (d, J = 6.8 Hz, 6H). C NMR (101 MHz,
thylpyridin-2(1H)-one (3b2). White solid, yield: 86.58%, mp
1
DMSO‑d
6
) δ 162.16, 155.14, 153.09, 150.26, 149.71, 141.05, 139.82,
146–148 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.55 (s, 1H), 8.21 (s, 1H),
1
1
20.97, 118.96, 112.27, 47.21, 36.83, 27.02, 24.66, 22.55, 22.44,
7.73 (t, J = 5.5 Hz, 1H), 5.94 (s, 1H), 4.49 (d, J = 5.2 Hz, 2H),
+
6.44. HRMS (ESI): calcd. for C19
H23ClN
6
NaO [M+Na] : 409.1520
4.24–4.14(m, 1H), 2.65 (q, J = 7.5 Hz, 2H), 2.43 (q, J = 7.5 Hz, 2H),
1
3
found: 405.1519.
2.00–1.75 (m, 4H), 1.19–1.07 (m, 6H), 0.69 (t, J = 7.3 Hz, 6H).
C
3
-(((2-chloro-9-(pentan-3-yl)-9H-purin-6-yl)amino)methyl)-4-ethyl-
NMR (101 MHz, DMSO‑d ) δ 164.11, 155.12, 154.93, 153.20, 150.50,
6
6
1
7
4
-methylpyridin-2(1H)-one (3b1). White solid, yield: 89.38%, mp
149.33, 140.93, 121.32, 118.80, 104.84, 59.30, 36.67, 27.34 (2C),
1
97–199 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.56 (s, 1H), 8.21 (s, 1H),
26.02, 25.84, 14.96, 13.46, 10.91 (2C). HRMS (ESI): calcd. for
+
.72 (t, J = 5.5 Hz, 1H), 6.04–5.75 (m, 1H), 4.49 (d, J = 5.0 Hz, 2H),
C
20
H27ClN
6
NaO [M+Na] : 425.1833 found: 425.1828.
.25–4.09 (m, 1H), 2.64 (q, J = 7.6 Hz, 2H), 2.13 (s, 3H), 1.99–1.70
4-(((2-chloro-9-(pentan-3-yl)-9H-purin-6-yl)amino)methyl)-1-me-
1
3
(
m, 4H), 1.12 (t, J = 7.6 Hz, 3H), 0.69 (t, J = 7.3 Hz, 6H). C NMR
thyl-5,6,7,8-tetra hydroisoquinolin-3(2H)-one (3b3). White solid, yield:
8

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Fig. 7. Effects of SKLB0565 on the mi-
tochondrial membrane potential. (A) After
treatment with indicated concentrations of
SKLB0565 for 48 h, the cells were harvested
and lysed for detection of some apoptosis-
related proteins. (B) The cells were treated
with SKLB0565 for 48 h and ΔΨm was
analyzed by FCM after Rh123 staining.
1
3
J = 7.8 Hz, 3H). C NMR (101 MHz, DMSO‑d
6
) δ 164.03, 155.00,
1
3
53.17, 150.11, 143.85, 140.33, 121.00, 119.01, 106.36, 55.93, 36.63,
2.46 (2C), 25.90, 23.93 (2C), 18.78, 14.89. HRMS (ESI): calcd. for
+
C
19
H
23ClN
6
NaO [M+Na] : 409.1520 found: 409.1511.
3
-(((2-chloro-9-cyclopentyl-9H-purin-6-yl)amino)methyl)-4,6-die-
thylpyridin-2(1H)-one (3c2). White solid, yield: 93.32%, mp
1
2
21–223 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.56 (s, 1H), 8.21 (s, 1H),
7
.71 (t, J = 5.5 Hz, 1H), 5.94 (s, 1H), 4.81–4.72 (m, 1H), 4.50 (d,
J = 5.3 Hz, 2H), 2.65 (q, J = 7.7 Hz, 2H), 2.43 (q, J = 7.5 Hz, 2H),
2
2
.18–2.08 (m, 2H), 1.98–1.88 (m, J = 14.4, 7.5 Hz, 1H), 1.88–1.79 (m,
13
H), 1.74–1.61 (m, 2H), 1.13 (t, J = 6.8, 6.0 Hz, 6H). C NMR
(
101 MHz, Chloroform-d) δ 165.65, 156.43, 154.96, 154.15, 150.18,
1
2
49.21, 137.70, 121.32, 119.03, 106.24, 55.60, 36.56, 32.93, 26.35,
6.21, 23.80, 14.81, 12.69. HRMS (ESI): calcd. for C20
H
25ClN NaO [M
6
Fig. 8. Effects of SKLB0565 on HUVECs migration and tube formation. (A)
Scratches of the cells were created with 200 μL pipette and images were cap-
tured after treatment with indicated concentrations of SKLB0565 for 24 h. (B)
Images depicting the formation of capillary-like tubes of HUVECs after treat-
ment with indicated concentrations of SKLB0565 for 6 h.
+
+
Na] : 423.1676 found: 423.1676.
4
-(((2-chloro-9-cyclopentyl-9H-purin-6-yl)amino)methyl)-1-methyl-
5
,6,7,8-tetra hydroisoquinolin-3(2H)-one (3c3). White solid, yield:
1
91.96%, mp 178–180 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.52 (s, 1H),
8
.20 (s, 1H), 7.80 (t, J = 5.5 Hz, 1H), 4.80–4.70(m, 1H), 4.46 (d,
J = 4.3 Hz, 2H), 2.87 (s, 2H), 2.38 (s, 2H), 2.20–2.11 (m, 2H), 2.10 (s,
1
8
8
4
3.79%, mp 252–254 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.54 (s, 1H),
3H), 1.97–1.86 (m, 2H), 1.856–1.80(m, 1H), 1.74–1.66 (m, 2H), 1.64
1
3
.20 (s, 1H), 7.80 (t, J = 5.5 Hz, 1H), 4.46 (d, J = 5.2 Hz, 2H),
(s, 4H). C NMR (101 MHz, Chloroform-d) δ 163.61, 155.10, 154.11,
.24–4.15 (m, 1H), 2.89 (s, 2H), 2.37 (s, 2H), 2.11 (s, 3H), 1.97–1.78
151.32, 150.16, 141.17, 137.71, 121.05, 119.10, 114.40, 55.58, 36.73,
1
3
(
m, 4H), 1.65 (s, 4H), 0.69 (t, J = 7.3 Hz, 6H). C NMR (101 MHz,
32.93 (2C), 27.28, 24.95, 23.81 (2C), 22.44, 22.30, 16.82. HRMS (ESI):
+
Chloroform-d) δ 163.69, 155.04, 154.11, 151.28, 150.45, 141.28,
calcd. for C21
H
25ClN
6
NaO [M+Na] : 435.1676 found: 435.1671.
1
2
38.25, 121.05, 118.95, 114.43, 58.78, 36.69, 27.27 (2C), 24.96,
3-(((2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)
2.44, 22.31, 16.78, 10.49 (2C). HRMS (ESI): calcd. for C21
H
27ClN
6
NaO
methyl)-4-ethyl-6-methylpyridin-2(1H)-one (3d1). White solid, yield:
+
1
[
M+Na] : 437.1833 found: 437.1828.
88.43%, mp 204–206 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.56 (s, 1H),
3
-(((2-chloro-9-cyclopentyl-9H-purin-6-yl)amino)methyl)-4-ethyl-
8.34 (s, 1H), 7.82 (t, J = 7.9 Hz, 1H), 5.92 (d, J = 1.0 Hz, 1H),
5.65–5.43 (m, 1H), 4.49 (d, J = 5.3 Hz, 2H), 4.02–3.95 (m, 1H),
3.75–3.62 (m, 1H), 2.60 (q, J = 7.6 Hz, 1H), 2.25–2.15 (m, 1H), 2.13
(s, 3H), 1.93 (m, 2H), 1.73 (m, 1H), 1.56 (m, 2H), 1.10 (t, J = 7.6 Hz,
6
1
7
-methylpyridin-2(1H)-one (3c1). White solid, yield: 82.52%, mp
1
91–193 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.55 (s, 1H), 8.21 (s, 1H),
.71 (t, J = 5.5 Hz,1H), 5.92 (s, 1H), 4.86–4.69 (m, 1H), 4.49 (d,
1
3
J = 4.3 Hz, 2H), 2.62 (q, J = 7.3 Hz, 2H), 2.20–2.05 (m, 5H),
3H). C NMR (101 MHz, Chloroform-d) δ 164.71, 157.03, 154.64,
1
.99–1.89 (m, 2H), 1.88–1.80 (m, 2H), 1.77–1.62 (m, 2H), 1.11 (t,
154.45, 149.00, 143.21, 137.60, 120.94, 118.08, 108.10, 81.50, 68.62,
9

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
3
6.16, 31.80, 26.06, 24.68, 22.56, 18.50, 14.43. HRMS (ESI): calcd. for
C
19
H
28
N
7
O [M+H]+: 370.2355 found: 370.2352.
+
C
19
H
23ClN
6
NaO
2
[M+Na] : 425.1469 found: 425.1463.
3-(((9-cyclopentyl-2-(methylamino)-9H-purin-6-yl)amino)methyl)-
3
-(((2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)
4,6-dimethyl pyridin-2(1H)-one (4b1). White solid, yield: 90.12%, mp
1
methyl)-4,6-diethylpyridin-2(1H)-one (3d2). White solid, yield:
184–186 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.51 (s, 1H), 7.72 (s, 1H),
1
8
5.68%, mp 226–228 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.57 (s, 1H),
6.62 (t, J = 3.8 Hz ,1H), 6.24 (q, J = 4.6 Hz, 1H), 5.85 (s, 1H), 4.65 (p,
J = 7.5 Hz, 1H), 4.50 (d, J = 5.3 Hz,2H), 2.80 (d, J = 4.8 Hz, 3H),
8
.34 (s, 1H), 7.84 (t, J = 7.9 Hz, 1H), 5.94 (s, 1H), 5.56 (d,
1
3
J = 10.8 Hz, 1H), 4.50 (d, J = 5.4 Hz, 2H), 4.02–3.95 (m, 1H),
2.26 (s, 3H), 2.10 (s, 3H), 2.08–1.61 (m, 8H). C NMR (101 MHz,
3
2
.75–3.61 (m, 1H), 2.64 (q, J = 7.6 Hz, 2H), 2.43 (q, J = 7.5 Hz, 2H),
DMSO‑d ) δ 163.96, 160.16, 154.76, 151.52, 148.74, 143.09, 136.17,
6
.26–2.13 (m, 1H), 1.93 (m, 2H), 1.73 (m, 1H), 1.56 (m, 2H), 1.13 (t,
123.04, 114.19, 108.02, 55.14, 36.36, 32.29 (2C), 28.82, 24.11 (2C),
1
3
+
J = 7.6 Hz, 6H). C NMR (101 MHz, DMSO‑d
6
) δ 164.08, 155.25,
19.53, 18.64. HRMS (ESI): calcd. for C19
H
26
N
7
O [M+H] : 368.2199
1
8
54.98, 153.71, 149.64, 149.35, 139.91, 121.17, 118.58, 104.85,
found: 368.2196.
1.40, 68.14, 36.69, 30.45, 26.01, 25.84, 24.94, 22.77, 14.92, 13.45.
3-(((9-cyclopentyl-2-(methylamino)-9H-purin-6-yl)amino)methyl)-
+
HRMS (ESI): calcd. for C20
H
6
25ClN NaO
2
[M+Na] : 439.1626 found:
4-ethyl-6-methylpyridin-2(1H)-one (4b2). White solid, yield: 87.82%,
1
4
39.1624.
mp 156–158 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.53 (s, 1H), 7.72 (s,
4
-(((2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)
1H), 6.62 (t, J = 3.8 Hz ,1H), 6.25 (q, J = 5.2 Hz, 1H), 5.89 (s, 1H),
4.65 (p, J = 7.4 Hz, 1H), 4.53 (d, J = 5.3 Hz,2H), 2.80 (d, J = 4.8 Hz,
3H), 2.63 (q, J = 7.6 Hz, 2H), 2.12 (s, 3H), 2.10–1.59 (m, 8H), 1.08 (t,
methyl)-1-methyl-5,6,7,8-tetrahydroisoquinolin-3(2H)-one
(3d3).
1
White solid, yield: 89.02%, mp 175–177 °C. H NMR (400 MHz,
1
3
DMSO‑d
6
) δ 11.54 (s, 1H), 8.33 (s, 1H), 7.93 (t, J = 7.9 Hz, 1H), 5.55
J = 7.5 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 165.76, 160.30,
(
d, J = 11.2 Hz, 1H), 4.46 (d, J = 5.2 Hz, 2H), 4.02–3.95 (m, 1H),
155.83, 154.69, 151.35, 143.61, 134.70, 121.98, 114.54, 107.82,
3
3
.75–3.57 (m, 1H), 2.85 (s, 2H), 2.37 (s, 2H), 2.18 (td, J = 11.6,
55.13, 35.77, 32.63 (2C), 28.86, 26.35, 24.04 (2C), 18.91, 14.73. HRMS
+
.9 Hz, 1H), 2.10 (s, 3H), 1.93 (m, 1H), 1.75 (m, 1H), 1.64 (s, 4H), 1.56
(ESI): calcd. for C20
H
28
N
7
O [M+H] : 382.2355 found: 382.2351
1
3
(
m, J = 4.8, 4.3 Hz, 2H). C NMR (101 MHz, Chloroform-d) δ 163.55,
3-(((9-cyclopentyl-2-(methylamino)-9H-purin-6-yl)amino)methyl)-
1
1
2
4
55.06, 154.45, 151.45, 149.20, 141.36, 137.54, 120.81, 118.64,
4,6-diethylpyridin-2(1H)-one (4b3). White solid, yield: 91.27%, mp
1
14.49, 81.45, 77.26, 68.72, 36.74, 32.12, 27.26, 24.90, 22.76, 22.40,
194–196 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.53 (s, 1H), 7.72 (s, 1H),
2.26, 16.77. HRMS (ESI): calcd. for C21
H
6
25ClN NaO
2
[M+Na]+
:
6.63 (t, J = 3.8 Hz ,1H), 6.25 (q, J = 4.9 Hz, 1H), 5.91 (s, 1H), 4.64 (p,
J = 7.6 Hz, 1H), 4.54 (d, J = 5.3 Hz,2H), 2.80 (d, J = 4.7 Hz, 3H),
51.1626 found: 451.1618.
2
.65 (q, J = 7.5 Hz, 2H), 2.49 (t, J = 1.9 Hz, 3H), 2.41 (q, J = 7.6 Hz,
13
4
.1.4. The representative procedure for the preparation of 3-(((2-
2H), 2.17–1.55 (m, 8H), 1.12 (t, J = 7.5 Hz, 3H). C NMR (101 MHz,
methylamino-9-substituted-9H-purin-6-yl)amino)methyl)-pyridin-2(1H)-
DMSO‑d ) δ 164.36, 160.15, 154.60, 154.39, 151.58, 149.00, 136.20,
6
one derivatives (4a1–4b4)
122.61, 114.15, 104.93, 55.14, 35.82, 32.29 (2C), 28.77, 26.20, 25.82,
+
3
-(((9-isopropyl-2-(methylamino)-9H-purin-6-yl)amino)methyl)-
24.10 (2C), 15.19, 13.49. HRMS (ESI): calcd. for C21
H
30
7
N O [M+H]
:
4
,6-dimethyl pyridin-2(1H)-one (4a1). A mixture of 3-(((2-chloro-9-
396.2512 found: 396.2509.
isopropyl-9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-
one (190.00 mg, 0.55 mmol) and aqueous methylamine (30%, 10 mL)
was stirred in a sealed 35 mL sealed tube at 110 °C for 5 h. A white solid
precipitated during the reaction. After the reaction was completed, the
suspension obtained was filtered and washed with water (10 mL) to
afford a solid product. The crude product was purified by silica gel
4-(((9-cyclopentyl-2-(methylamino)-9H-purin-6-yl)amino)methyl)-
1-methyl-5,6,7,8-tetrahydroisoquinolin-3(2H)-one (4b4). White solid,
1
yield: 89.16%, mp 150–152 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.53
(s, 1H), 7.72 (s, 1H), 6.69 (t, J = 3.8 Hz ,1H), 6.25 (q, J = 5.7 Hz, 1H),
4.65 (p, J = 7.4 Hz, 1H), 4.51 (d, J = 5.3 Hz, 2H), 2.89–2.74 (m, 4H),
2.50 (p, J = 1.9 Hz, 8H), 2.10 (s, 3H), 2.00–1.77 (m, 4H), 1.63 (d,
1
3
column chromatography to afford target compounds as white solid with
J = 0.8 Hz, 3H). C NMR (101 MHz, DMSO‑d ) δ 162.42, 160.12,
6
1
yield 90.52%, mp 206–208 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.51
154.73, 149.41, 140.72, 136.17, 122.29, 114.16, 112.34, 55.15, 35.94,
32.28 (2C), 28.80, 27.15, 24.63, 24.10 (2C), 22.56, 22.42, 16.40. HRMS
(
s, 1H), 7.75 (s, 1H), 6.62 (t, J = 3.8 Hz ,1H), 6.25 (q, J = 5.5 Hz, 1H),
5
2
.85 (s, 1H), 4.53 (dd, J = 13.2, 6.5 Hz, 3H), 2.80 (d, J = 4.8 Hz, 3H),
(ESI): calcd. for C22
H
30
N
7
O [M+H]+: 408.2512 found: 408.2513.
1
3
.26 (s, 3H), 2.10 (s, 3H), 1.45 (d, J = 6.8 Hz, 6H). C NMR (101 MHz,
) δ 163.95, 160.17, 154.75, 148.71, 145.66, 143.09, 135.55,
23.07, 114.14, 108.01, 49.07, 46.03, 28.82, 22.54 (2C), 19.53, 18.64.
DMSO‑d
6
4.2. Molecular modelling
1
+
HRMS (ESI): calcd. for
42.2040.
C
17
H
24
7
N O
[M+H]
:
342.2042 found:
The 3D structure of the tubulin was downloaded from the PDB
(http://www.rcsb.org/, PDB code 402B). The protein was prepared
with discovery studio 3.1. Molecule SKLB0565 was built with
ChemBio3D and optimized at molecular mechanical level. The structure
of colchicine was directly extracted from the crystal structure composed
of tubulin and colchicine. Then SKLB0565 was docked to the binding
3
4
-ethyl-3-(((9-isopropyl-2-(methylamino)-9H-purin-6-yl)amino)me-
thyl)-6-methylpyridin-2(1H)-one (4a2). White solid, yield: 88.98%, mp
1
1
6
66–168 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.55 (s, 1H), 7.75 (s, 1H),
.62 (t, J = 3.8 Hz ,1H), 6.25 (q, J = 5.5 Hz, 1H), 5.90 (s, 1H), 4.53 (p,
J = 6.7 Hz, 3H), 2.81 (d, J = 4.7 Hz, 3H), 2.63 (q, J = 7.6 Hz, 2H),
site of tubulin by employing a protein-ligand docking program
1
3
2
.12 (s, 3H), 1.45 (d, J = 6.7 Hz, 6H), 1.09 (t, J = 7.5 Hz, 3H).
C
GOLD5.2. Scoring function GOLDSCORE was used for exhaustive
searching, solid body optimizing, and interaction scoring.
NMR (101 MHz, DMSO‑d
6
) δ 164.29, 160.16, 154.60, 154.27, 151.18,
1
2
3
43.52, 135.57, 122.30, 114.11, 106.44, 55.37, 46.03, 28.77, 26.08,
2.54 (2C), 18.76, 15.15. HRMS (ESI): calcd. for C18
56.2199 found: 356.2200.
H
26
N
7
O [M+H]+
:
4.3. In vitro tubulin polymerization assay
4
,6-diethyl-3-(((9-isopropyl-2-(methylamino)-9H-purin-6-yl)amino)
The effects of different concentrations of SKLB0565 (0.05, 1.25, 6.0,
30 μM) on tubulin polymerization were performed using a fluorescence-
based tubulin polymerization kit (BK011P, Cytoskeleton, USA) ac-
cording to the manufacturer’s protocol. Paclitaxel (3 μM) and CA-4
(1.25 μM) served as reference compounds and DMSO served as a ve-
hicle control. When the compounds (5 μL) in 96-well plate were in-
cubated to 37 °C, the corresponding 50 μL of tubulin reaction mix which
contain 2 mg/mL porcine brain tubulin was added to each well. The
increase in fluorescence was immediately monitored by excitation at
methyl)pyridin-2(1H)-one (4a3). White solid, yield: 91.30%, mp
1
1
6
76–178 °C. H NMR (400 MHz, DMSO‑d
6
) δ 11.54 (s, 1H), 7.76 (s, 1H),
.63 (t, J = 3.8 Hz ,1H), 6.26 (q, J = 5.4 Hz, 1H), 5.91 (s, 1H), 4.53 (p,
J = 6.7 Hz, 3H), 2.81 (d, J = 4.8 Hz, 3H), 2.65 (q, J = 7.6 Hz, 2H),
2
7
1
2
.42 (q, J = 7.6 Hz, 2H), 1.45 (d, J = 6.8 Hz, 6H), 1.11 (dt, J = 12.7,
13
.6 Hz, 6H). C NMR (101 MHz, DMSO‑d ) δ 164.38, 160.17, 154.60,
6
54.40, 151.15, 148.99, 135.56, 122.63, 114.11, 104.94, 46.04, 35.90,
8.76, 26.20, 25.82, 22.52 (2C), 15.18, 13.48. HRMS (ESI): calcd. for
10

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
3
55 nm and emission at 460 nm in a multimode reader over a 60 min
4.10. Immunofluorescence (IF) staining
period at 37 °C.
The microtubule organization was observed after immuno-
fluorescence staining of the cells [25]. After treated with the respective
compound for 18 h, the cells were fixed in 4% paraformaldehyde for
4.4. Cell lines and cell culture
5
min, washed with PBS and permeabilized with 0.1% Triton X-100 in
All the cell lines used in our study were purchased from the
PBS for 10 min at room temperature (RT). After incubating with the
blocking agent for 45 min at RT, the cells were incubated with anti-β-
tubulin monoclonal antibody (10094-1-AP, Proteintech, China) over-
night at 4 °C. Then the cells were washed three times by PBS following
staining by FITC conjugated secondary antibody and labeling of nuclei
by DAPI. The cells were finally visualized using a confocal microscope.
American Type Culture Collection (ATCC). Cells were cultured in RPMI-
1
640 containing 10% fetal serum (FBS) and 1% antibiotics (Penicillin
and streptomycin) with 5% CO at 37 °C.
2
4.5. MTT cell proliferation assay
Cell growth was monitored by absorbance using the MTT (3-(4, 5-
4
.11. Protein extraction and western blotting analysis
dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) assay
3
[
34]. Cells were seeded at the density of 2–5 × 10 cells per well in 96-
The cells were harvested and homogenized with RIPA buffer con-
well cell plates with 100 μL complete medium containing 10% FBS for
taining cocktail and phosphatase inhibitors for western blot analysis.
Equal amounts of proteins were separated by SDS-PAGE gel and
transferred onto nitrocellulose (NC) filter membranes. After blocking in
2
4 h. The cells were incubated with different doses of the test com-
pounds for 24, 48 or 72 h. Then, 20 μL of MTT (5 mg/mL) was added
and incubated for another 2.5 h at 37 °C. The formazan was dissolved in
5
% dried milk in TBS/T, the membranes were incubated with primary
1
5
50 μL DMSO and the absorbance value of each well was measured at
70 nm after the crystal of purple formazan was dissolved. The IC50
antibodies overnight at 4 °C. After incubation with appropriate horse-
radish peroxidase (HRP)-conjugated secondary antibodies, the expres-
sion of proteins was detected by enhanced chemiluminescence (ECL).
were calculated using Graphpad Prism 8 software.
4.6. Colony formation assay
4.12. Wound healing assay
Cells were seeded at low density (600–800 cells per well) in stan-
The wound healing assay was conducted as described previously
with some modification [38]. HUVEC cells were overspread in 6-well
plates. Scratches were made in confluent monolayers using 200 μL
pipette tip. Then, the wounds were washed twice with sterile PBS to
remove nonadherent cell debris. Serum-free medium containing dif-
ferent concentrations of the SKLB0565 (0, 50, 100 nM) were added to
the petri dishes. Images were taken by an OLYMPUS inverted digital
camera after 24 h incubation.
dard 6-well plates and exposed to different concentrations of
SKLB0565. The medium was refreshed every three days. After treat-
ment for 14 days, the cells were fixed with 4% paraformaldehyde for
5
min, and then stained with 0.5% crystal violet for another 10 min.
PBS was used to rinse for 2–3 times at last.
4
.7. Cell cycle and apoptosis analysis by FCM
4
.13. Capillary tube formation assay
For the cell cycle analysis, after treatment with SKLB0565 for 24 h,
the cells were harvested and fixed with 75% cold ethanol for at least 2 h
and stained with a solution containing PI for 10 min in the dark. Then
the cells were analyzed by FCM. The data were analyzed using the
FlowJo software.
EC Matrigel matrix thawed at 4 °C was used to coat each well of a 96-
well culture plate and allowed to polymerize for an hour at 37 °C. Then,
4
5
0 μL of HUVECs (2 × 10 cells per well) were seeded on matrigel-
coated well with increasing concentrations of SKLB0565. HUVECs were
incubated for 6 h to allow formation of tube-like structures at 37 °C. The
images were taken by an OLYMPUS inverted digital camera.
For the apoptosis assays, the Annexin V- FITC apoptosis detection
kit was used as described previously [35]. Cells treated with SKLB0565
for 48 h were harvested and analyzed by FCM using the apoptosis de-
tection kit according to the manufacturer’s instructions. The data were
analyzed with FlowJo software.
4
.14. Statistical analysis
All quantitative results were expressed as means ± SD in 3 in-
4
.8. Mitochondrial membrane potential (ΔΨm) assay
dependent experiments. Statistically significant differences were ob-
tained using Student’s t-test. Differences were considered to be statis-
tically significant with a P value less than 0.05.
The ΔΨm of cancer cells was determined after Rh123 staining as
described previous [36]. After incubation with different concentrations
of SKLB0565 for 48 h, the cells were collected and incubated with
Rh123 (5 μg/mL) in the dark at 37 °C for 30 min. Then the fluorescence
emitted from Rh123 was detected by FCM. The data were analyzed
using the FlowJo software.
Declaration of Competing Interest
The authors declare no conflict of interest about this article.
Acknowledgements
4.9. Morphological analysis after Hoechst staining
We are grateful for Shuhui Xu and Lihua Zhou of State Key
Laboratory of Biotherapy (Sichuan University) for NMR measurements
and Professor Lijuan Chen's team of State Key Laboratory of Biotherapy
(Sichuan University) for HRMS measurements.
Morphological changes associated with apoptosis in SW620 cells
were detected by Hoechst 33342 (Sigma, USA) staining as described
5
previously [37]. Cells were seeded at 1 × 10 cell per well in 6-well
plates and incubated for 24 h. Then different concentrations of
SKLB0565 were added and incubated for another 48 h. Finally, the cells
were stained with a solution containing Hoechst 33342 (5 μg/mL) ac-
cording to the manufacturer’s instructions. The stained cells were ob-
served and taken photos under an inverted fluorescence microscopy.
Funding
This work was supported by the National S&T Major Special Project
on Major New Drug Innovations of China (2018ZX09201018), the
11

X. Hu, et al.
Bioorganic Chemistry 97 (2020) 103695
Sichuan Provincial Science and Technology Program for Key Research
and Development, China (No. 2018SZ0007), and the Fundamental
Research Funds for the Central Universities, China (2017SCU12046, the
Postdoctoral Foundation of Sichuan University).
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