Synthesis and bioevaluation of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amines as tubulin polymerization inhibitors with anti-angiogenic effects

Article abstract of DOI:10.1016/j.bmc.2020.115985

A new series of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine derivatives as tubulin polymerization inhibitors were synthesized, and evaluated for the anti-proliferative activities. A structure–activity relationship study revealed that the free amino moiety of 1H-pyrazolo[3,4-b]pyridin-3-amine played an essential role in the anti-proliferative activities. Especially, compound 15c displayed the strongest anti-proliferation against MCF-7 cells with IC₅₀ value of 0.067 ± 0.003 μM, and high selectivity over the normal human embryonic lung WI-38 cells with IC₅₀ value of 23.41 ± 1.53 μM. Further mechanistic studies revealed that 15c showed strong anti-tubulin polymerization activity, changed the morphology of tubulin, and arrested the cell cycle at the G2/M transition in MCF-7 cells. Molecular docking analysis suggested that 15c well occupied the colchicine-binding pocket of tubulin. Additionally, 15c demonstrated anti-angiogenic activities with blocking the migration, invasion and tube formation, disrupting the newly formed tube, and regulating both MMP-9 and TIMP-1 in HUVEC cells. In summary, our results highlight that compound 15c is a potential antitumor compound that are worthy of further development.

Full text of DOI:10.1016/j.bmc.2020.115985

Bioorg. Med. Chem. 31 (2021) 115985
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioevaluation of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b]pyridin-3-amines as tubulin polymerization inhibitors with
anti-angiogenic effects
Shu-Yi Hao1, Zhi-Yuan Qi1, Shuai Wang, Xing-Rong Wang, Shi-Wu Chen*
School of Pharmacy, Lanzhou University, Lanzhou 730000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine derivatives as tubulin poly-
merization inhibitors were synthesized, and evaluated for the anti-proliferative activities. A structure–activity
relationship study revealed that the free amino moiety of 1H-pyrazolo[3,4-b]pyridin-3-amine played an essential
role in the anti-proliferative activities. Especially, compound 15c displayed the strongest anti-proliferation
Aniogenesis
Tubulin polymerization
Inhibitors
Pyrazolo[3,4-b]pyridine
Antitumor
against MCF-7 cells with IC50 value of 0.067 ± 0.003
onic lung WI-38 cells with IC50 value of 23.41 ± 1.53
μ
M, and high selectivity over the normal human embry-
μ
M. Further mechanistic studies revealed that 15c showed
strong anti-tubulin polymerization activity, changed the morphology of tubulin, and arrested the cell cycle at the
G2/M transition in MCF-7 cells. Molecular docking analysis suggested that 15c well occupied the colchicine-
binding pocket of tubulin. Additionally, 15c demonstrated anti-angiogenic activities with blocking the migra-
tion, invasion and tube formation, disrupting the newly formed tube, and regulating both MMP-9 and TIMP-1 in
HUVEC cells. In summary, our results highlight that compound 15c is a potential antitumor compound that are
worthy of further development.
1. Introduction
identification of new compounds that may be more potent or more se-
lective in targeting tissues or tumors.6
Microtubules are a key component of the cytoskeleton and are pre-
sent in almost all eukaryotic cells except for a few types such as human
Combretastatin-A4 (CA-4, 1, Fig. 1) is a natural cis-stilbene product
isolated from the bark of the African willow tree, Combretum caffrum,
and it is a well-known molecule that strongly inhibits tubulin poly-
merization by binding to the colchicine-binding site, causing rapid
vascular shutdown and cell death in tumor cells.7 However, the poor
water solubility and bioavailability of CA-4 have hindered its develop-
ment into an antitumor drug.8 To overcome these shortcomings, its
water-soluble ester, CA-4P, was developed as a tumor vascular-targeting
agent and entered into clinical trials for the treatment of platinum-
resistant ovarian cancer.9 Unfortunately, the study was terminated
owing to a lack of efficacy in improving progression-free survival,
combined with an unfavorable objective response rate.10
red blood cells.1 They consist of globular proteins composed of
α- and
β-tubulin heterodimers, and they play key roles in cellular processes
such as organellar movement, intracellular transport, the formation and
maintenance of cell shape, and cell division.2 On the other hand, the
inhibition of tubulin assembly into microtubules, or, inversely, the
depolymerization of microtubules, leads to the arrest of cell division and
eventually to apoptosis.3 This makes microtubule-targeting agents
(MTAs) are an effective and attractive molecular target for cancer che-
motherapeutics.4 Current MTAs include the colchicine, vinca alkaloids,
and taxanes sites with the first two binding to the so-called colchicine-
and vinca-binding sites and acting as tubulin polymerization destabil-
izers, whereas the latter bind to the taxanes-binding site and act as
polymerization stabilizers.5 These MTAs can interfere with the micro-
tubule equilibrium process, and the success of tubulin polymerization
inhibitors as anticancer agents has stimulated significant interest in the
Due to its important pharmacological characteristics and simple
structure, a wide variety of CA-4 analogs have been developed and
evaluated in structure-activity (SAR) studies.11–16 These studies showed
that the 3,4,5-trimethoxyphenyl (TMP) moiety (A ring) is an important
pharmacodynamic point for tubulin binding, and the oxygen atom of its
* Corresponding author.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
1
These authors made equal contributions to this work.
https://doi.org/10.1016/j.bmc.2020.115985
Received 11 November 2020; Received in revised form 21 December 2020; Accepted 24 December 2020
Available online 31 December 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
4-methoxy group interacts with the Cys241 residue at the colchicine-
binding site.17,18 While the B ring can be replaced with a various het-
erocycle, such as benzofuran, benzothiophene, and indole, among
others, the resulting compounds retain their tubulin polymerization
inhibitory activity.5,11 Thus the modification of the B ring has become
the focus of research, and some derivatives with superior antitumor
activites were found. For example, isoazaerianin (3), in which the cis-
stilbene was substituted with methylamino, displays anti-cancer activ-
ities with nanomolar IC50 cytotoxicity against a panel of cancer cells and
inhibits tubulin assembly at a micromolar level.19 Further, compound 4
with benzofuran showed good proliferation inhibitory activity against
the HeLa, A549, HT-29, MCF-7, and HL-60 cell lines, with IC50 values in
the range of 13–24 nM.20 In addition, the Tung group synthesized
compound 5 with a 1H-indazole and found that its IC50 toward KB cells
was 20 nM.21
Compounds 14a,b were further reacted with corresponding haloalkane
under NaH in DMF to obtain the target compounds 15a,b, respectively.
Simultanously, reaction of compounds 14e,f with various haloalkane,
and further deprotection yielded the target compounds 15c–l. All target
derivatives were fully characterized by 1H NMR, 13C NMR, and HRMS
spectroscopic techniques, and their purities were more than 95% based
on HPLC analysis. The spectral data were in full agreement with the
expected structures (see Experimental Section and Supporting
Information).
2.2. Biological evaluation
2.2.1. Anti-proliferative activities of target compounds 14a–d, 15a–l
Target compounds 14a–d and 15a–l were evaluated for the anti-
proliferative activity against five human cancer cell lines (HCT116,
colon cancer; A549, non-small-cell lung cancer; HeLa, human epithelial
cervical cancer; MCF-7, human breast cancer; and HepG2, hepatic car-
cinoma) and the WI-38 cell line (normal human embryonic lung cells)
using the MTT assay with CA-4 as the reference drug.24 The IC50 values
of the compounds are summarized in Table 1.
Hybrid molecules have the potential to enhance efficacy, overcome
drug resistance, and reduce toxicity, and several hybrid molecules have
been subjected to clinical evaluations for the treatment of various dis-
eases.22 We previously described a series of 1-(benzofuran-3-yl)-4-
(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole derivatives as tubulin poly-
merization inhibitors with marked cytotoxicity.23 As part of our ongoing
search for novel tubulin polymerization inhibitors based on CA-4, we
designed and synthesized a series of N-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[3,4-b]pyridin-3-amine derivatives that are hybrids of com-
pounds 4 and 5 and further modified using bioisosteric replacement of
the benzene moiety with pyridine (Fig. 2). Herein, we report the syn-
thesis and structure-activity relationship of a set of novel target com-
pounds 14–15. Furthermore, we present the biological activities of a
representative compound 15c, including their effects on in vitro tubulin
polymerization, intracellular microtubule networks, cell cycle transi-
tion, as well as anti-angiogenic effects in HUVEC cells. In addition,
molecular modeling analysis of the colchicine-binding site of tubulin
was performed with 15c.
Compounds 14a–d and 15a,b, in which the amino of 1H-pyrazolo
[3,4-b]pyridine at N1 was substituted with various R1, generally showed
poor proliferation inhibitory activity against the five human cancer cell
lines. Encouragingly, compounds 15c–l with a free amino group at N1 of
the 1H-pyrazolo[3,4-b]pyridine had dramatically improved anti-
proliferative activities. In addition, the introduction of CH2CH2F at the
bridge connection improved this anti-proliferative activity (15 g vs
15d), while the activities of 15 h, in which the amino of 1H-pyrazolo
[3,4-b]pyridine was substituted with CH2CHF2, were superior compared
with those of unsubstituted 15d, and weaker than those of 15g. In
particular, compounds 15c and 15g showed not only superior anti-
tumor activities but also low toxicities, as the IC50 of compound 15c
against MCF-7 cells was 0.067 ± 0.003
μM and 23.41 ± 1.53 μM against
WI-38 cells, while the IC50 of 15g against HeLa cells was 0.012 ± 0.009
2. Results and discussion
μ
M and 16.48 ± 1.60 μM against WI-38 cells. These SAR studies showed
that derivatives with an unsubstituted 1H-pyrazolopyridine ring dis-
played better activity profiles, and when the NH at the connection was
substituted with a small group, this activity improved.
2.1. Chemistry
The synthetic route to the target compounds is outlined in Scheme 1.
Initially, the substitution of 2-chloro-6-methoxypyridine as the starting
material with N-bromosuccinimide (NBS) in the presence of benzoyl
peroxide (BPO) yielded 3-bromo-2-chloro-6-methoxypyridine (9), and
the Bouveault reaction of 9 provided 2-chloro-6-methoxynicotinylalde-
hyde (10), which reacted with hydrazine hydrate under refluxing con-
2.2.2. In vitro tubulin polymerization inhibition activities of 15c and 15 g
Mechanistic researches have revealed that the potent anticancer
activity of CA-4 is based on its inhibition of tubulin polymerization.8 To
elucidate whether 15c and 15g also target the tubulin-microtubule
system, the in vitro tubulin polymerization inhibitory activities of 15c
and 15g were evaluated using the method previously described.23 As
shown in Fig. 3, after tubulin was incubated with 15c and 15g at various
concentrations, the absorbance values decreased compared with the
control. These results indicated that 15c and 15g both inhibited tubulin
ditions
produced
6-methoxy-1H-pyrazolo[3,4-b]pyridine
(11).
Subsequent bromination of 11 at the 3-position with NBS generated 3-
bromo-6-methoxy-1H-pyrazolo[3,4-b]pyridine (12), and substitution of
the latter with different haloalkanes in the presence of NaH in dime-
thylformamide (DMF) afforded N1-substituted pyridopyrazole de-
rivatives 13a–e, while protection with 3,4-dihydro-2H-pyran in the
presence of p-toluenesulfonic acid in tetrahydrofuran (THF) yielded 13f.
Next, Buchwald-Hartwig coupling of 13a–f with 3,4,5-trimethoxyani-
line catalyzed by Pd(OAc)2 provided the compounds 14a–f.
polymerization in vitro and that this inhibition occurred in
concentration-dependent manner, with IC50 values of 14.0 ± 3.3 M and
55.7 ± 2.0 M respectively. Moreover, 15c showed stronger inhibition
of tubulin polymerization than that of 15g, although weaker than CA-4
(1.8 ± 0.1 M). Considering compound 15c possessed more potent anti-
a
μ
μ
μ
Fig. 1. Structures of CA-4 (1), colchicine (2) and isoazaerianin (3).
2

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
Fig. 2. Design of target compounds.
Scheme 1. Reagents and conditions: (i) NBS, BPO, CH3CN, 90 ◦C, 3 h; (ii) DMF, n-BuLi, dry THF, ꢀ 78 ◦C, 1.5 h; rt, 6 h; (iii) N2H4⋅H2O, H2O, reflux, 5 h; (iv) NBS,
CH2Cl2, ꢀ 30 ◦C, 30 min; (v) haloalkane (for 13a–e), NaH, DMF, rt, 24 h; or p-TsOH, 3,4-dihydro-2H-pyran (for 13f), THF, 80 ◦C, 3 h; (vi) 3,4,5-trimethoxyaniline,
xantphos, Pd(OAc)2, Cs2CO3, dioxane, reflux, 3 h; (vii) 14a and MeI (for 15a), or 14b and cyclopentylchloride (for 15b), NaH, DMF, rt, 24 h; (viii) 14e,f, haloalkane,
NaH, DMF, rt, 24 h; then HCl/EtOH, rt, 2–12 h (for 15c–l).
tubulin polymerization and less toxicity against WI-38 normal cells than
those of 15g, Therefore, compound 15c was chosen to further study of
its biological activity and action mechanisms.
affinity with β2-tubulin. In addition, the TMP ring can go deep into the
binding pocket composed of Ala250, Leu242, Cys241, Val238, and
Ile378. Among these residues, the mercapto group in the Cys241 is very
close to the 4′-OMe of 15c, which shows a certain interaction force
(Fig. 4C). From Fig. 4B, it can be found that the vincristine binding site
2.2.3. Docking study
CA-4 has good anti-tubulin polymerization activity because it can
bind to the colchicine-binding site, thereby inhibiting the polymeriza-
tion of tubulin.15 As our above experiments showed, compound 15c
showed good anti-tubulin polymerization activity. In order to identity
that 15c inhibits microtubule polymerization by binding to colchicine
of 15c exists on α2-tubulin and is adjacent to GDP end in β1-tubulin.
However, 15c does not penetrate into the binding pocket of vincristine,
and fails to form hydrogen bonds and hydrophobic interactions with key
amino acid residues in the pocket, such as Asn329, Pro175, Val177,
Ser178, Pro325 and Val353 (Fig. 4D). Therefore, the docking simula-
tions indicated that compound 15c, like CA-4, is a tubulin destabilizer
that binds to the colchicine site.
¨
site instead of vincristine, we used Schrodinger software for molecular
docking simulations at the colchicine and vincristine sites of tubulin
(PDB code: 1Z2B).17,25 As shown in Fig. 4A, the TMP ring of 15c is
embedded in the β2-tubulin end, and the pyrazolopyridine ring is
located toward the GTP end. Furthermore, 15c has almost the same
spatial conformation with CA-4, and the N1H of pyrazolopyridine forms
stable hydrogen bonding with Thr179, which further enhances the
2.2.4. Effect of 15c on microtubule organization
Since tubulin plays a vital role in maintaining cell shape and basic
cell function, we used immunofluorescence to investigate whether 15c
could disrupt microtubule dynamics in living cells (MCF-7 cells). As
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S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
shown in Fig. 5, in the control group, the microtubule network showed
normal arrangement and organization. However, after the addition of
15c, the spindle formation showed obvious abnormalities and breakage.
When the concentration of compound 15c was 10 nM, the microtubules
gradually began to shrink around the nucleus. When the concentration
increased to 100 nM, the microtubule spindle showed clear shrinkage
around the center of the cell, forming a clump. The morphological
microtubule changes indicated that compound 15c dramatically dis-
rupted the microtubule organization in a concentration-dependent
manner, and such changes may eventually lead to cell cycle disruption.
Table 1
Anti-proliferative activities of compounds 14a–d and 15a–l.
Compds
IC50
(μ
M) a,b
HCT-
116
A549
HeLa
MCF-7
HepG2
WI-38
14a
14b
14c
14d
15a
15b
15c
15d
15e
15f
>50
>50
>50
6.07 ±
0.43
>50
6.24 ±
0.13
2.43 ±
0.81
>50
6.28 ±
1.03
22.35 ±
1.18
>50
73.83 ±
3.71
12.00 ±
4.49
31.86 ±
3.34
12.85 ±
2.58
13.24 ±
2.69
20.04 ±
0.57
4.32 ±
0.67
>50
>50
22.50 ±
3.01
>50
38.84 ±
7.46
12.56 ±
4.48
2.2.5. Effect of 15c on cell cycle
Since most tubulin destabilizers can disrupt regulated cell cycle
distribution, we chose flow cytometry to detect whether 15c was able to
block the cell cycle.26 As shown in Fig. 6A, after the addition of 5 nM and
10 nM 15c, the G2/M population increased from 16.81% (control) to
63.50% and 80.73%, respectively. The results indicate that compounds
15c can block cancer cells in the G2/M phase. However, compound 15c
did not induce apoptosis of MCF-7 cells like others CA-4 derivatives as
tubulin polymerization inhibitors (Fig. S1).
>50
>50
>50
>50
0.59 ±
0.26
>50
>50
2.52 ±
0.32
22.80 ±
2.94
>50
>50
138.43
± 6.95
23.41
1.53
0.26
6.00
0.21
0.067
0.003
0.81 ±
0.15
4.79
0.02
2.24
0.04
1.56
16.67 ±
7.25
31.17 ±
7.49
0.70 ±
0.13
9.64 ±
3.79
8.03 ±
1.25
5.97 ±
1.38
>50
0.73 ±
0.02
1.37 ±
0.78
6.26 ±
0.88
2.99 ±
1.76
To further validate these findings, we used western blot analysis to
detect the effect of compounds 15c on the cyclins Cdc25c, Cdc2, and
Cyclin B1 at the protein level. It is known that the activation of Cdc2
kinase is controlled by Cyclin B1 binding and that Cdc25c phosphory-
lation can promote the mitosis of eukaryotic cells, leading to cell divi-
sion.27 As shown in Fig. 6B, compared with the control group, the
protein levels of Cdc25c, Cdc2, and Cyclin B1 were significantly reduced
following treatment with 15c at 10 nM, 50 nM and 100 nM. From
densitometry analysis of these western blots (Fig. 6C), it can be seen that
the expression of these proteins showed a concentration-dependent
decrease with increasing compound concentration. The results suggest
that compound 15c arrest MCF-7 cells in the G2/M transition by
reducing the levels of Cdc25c, Cdc2, and Cyclin B1.
>50
>50
21.17 ±
2.02
>50
24.46 ±
2.26
6.04 ±
0.76
15 g
15 h
15i
0.12
0.20
0.012
0.009
0.17 ±
0.02
0.102
0.012
21.73 ±
3.28
5.79
16.48
1.60
0.03
0.08
0.56
0.47 ±
0.14
2.42 ±
0.52
18.75 ±
2.34
1.69 ±
0.26
2.18 ±
0.55
20.56 ±
2.54
0.18 ±
0.04
2.35 ±
0.93
5.46 ±
1.49
1.29 ±
0.11
15j
3.66 ±
0.88
31.77 ±
9.04
1.77 ±
0.37
1.72 ±
0.69
>50
30.85 ±
2.47
15 k
15 l
CA-4
26.23 ±
2.11
22.71 ±
8.38
28.43 ±
4.67
>50
>50
10.41 ±
6.03
3.37 ±
0.31
>50
3.19 ±
1.04
10.35 ±
1.97
7.38 ±
0.72
1.67 ±
0.01
0.029 ±
0.005
0.058 ±
0.005 ±
0.002
0.034 ±
0.008
0.28 ±
0.13
31.57 ±
6.06
0.004
a
IC50 values are presented as the means ± SD of triplicate experiments.
2.2.6. Effect of 15c on VEGF-A in MCF-7 cells
b
Drug treatment for 72 h.
Vascular endothelial growth factor (VEGF) can be secreted by tumor
cells and subsequently promote HUVECs growth in Ischemic area of
Fig. 3. Tubulin polymerization inhibitory activities of 15c, 15g and CA-4. Purified tubulin protein at 10
(A), 15g (B) and CA-4 (C) at the indicated concentrations; (D) Chemical structures of 15c and 15g.
μ
M was incubated at 37 ◦C in the absence or presence of 15c
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S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
Fig. 4. Molecular docking of 15c at different binding site of the tubulin (PDB code: 1Z2B). (A) Surface representation of 15c at colchicine binding site. (B) Surface
representation of 15c at vincristine binding site. (C) The interaction of 15c (yellow sticks) with surrounding amino acid residues (pink sticks) of β2-tubulin compared
with CA-4 (green). (D) The interaction of 15c (yellow sticks) with surrounding amino acid residues (pink sticks) of α2-tubulin compared with vincristine (purple).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
tissue, leading to “starvation” of the tumor.
2.2.7. Effects of 15c on HUVEC cells migration and invasion
The migration and invasion of HUVEC plays an important role in the
early stage of tumor angiogenesis.29 The IC50 value of 15c against
HUVEC cells was 23.5 ± 1.80 μM in MTT method, thus we chose HUVEC
cell cultures to evaluate the ability of compound 15c (from 0.1 to 1 μM)
to inhibit HUVEC cell migration and invasion.30 As shown in Fig. 8A,
after 24 h the control group showed complete coverage of the scratches
due to the migration of the HUVEC cells. In contrast, treatment with 15c
at any of the tested concentrations prevented the migration of the
HUVEC cells. Compared with the 100% migration rate of the control
group at 24 h, the cell migration rates following treatment with com-
pound 15c at 0.1 μM, 0.5 μM, and 1 μM were 26.4%, 26.6%, and 22.4%,
respectively (Fig. 8B). From these results, it can be concluded that
compound 15c can inhibit the migration of HUVEC cells. Similarly, the
results from the transwell chamber revealed that 15c could significantly
suppress the invasion ability of HUVECs in a dose-dependent manner
(Fig. 8C and 8D). The above results are consistent with previous findings
that CA-4 has an inhibitory effect on the angiogenesis.31
Matrix metalloproteinases (MMPs), in particularly MMP-9, play
critical roles in remodeling the extracellular matrix (ECM) by digesting
various ECM proteins in the basement membrane.32 Through their
MMP-inhibitory activities, tissue inhibitors of metalloproteinases
(TIMPs) such as TIMP-1 are capable of inhibiting tumor invasion and
metastasis in experimental cancer models.33 Next, we used western
blotting to measure expression levels of MMP-9 and TIMP-1 in HUVEC
cells exposed to various concentrations of 15c for 24 h.32 Western
blotting revealed that decreased levels of MMP-9, and increased levels of
TIMP-1 in HUVEC cells following exposure to 15c (Fig. 9). These find-
ings suggest that 15c presumably inhibit migration and invasion of
HUVEC cells by down-regulation the activity of MMP-9 and up-
regulation TIMP-1.
Fig. 5. Effect of 15c on microtubule organization of MCF-7 cells. MCF-7 cells
were plated in confocal dishes and incubated with 15c at indicated concen-
trations for 6 h. The images were observed and photographed with a laser
scanning confocal microscope. Scale bar: 100 μm.
tumor tissues to draw adequate nutrition, and the VEGF-A is considered
to be one of the important regulators of angiogenesis.28 Therefore,
reducing the expression of VEGF-A in tumor tissues plays a vital role in
tumor angiogenesis. Thus, we tested the effects of 15c on VEGF-A in
MCF-7 cells, and found a apposite decrease in VEGF-A expression in the
group with high-concentration 15c treatment (Fig. 7). The result
revealed that 15c has a significant effect on angiogenesis in tumor
Fig. 6. Effect of compound 15c on cell cycle. (A) MCF-7 cells were treated with 0, 5 and 10 nM 15c for 24 h, and detected by flow cytometry. (B) MCF-7 cells were
treated with 0, 10, 50 and 100 nM 15c for 24 h, and the level of Cdc25c, Cdc2 and Cyclin B1 were tested by west blotting. (C) The histogram of 15c on cycle-related
proteins Cdc25c, Cdc2 and Cyclin B1 in average of two independent experiments.
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S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
Fig. 7. Effect of compound 15c on VEGF-A in MCF-7 cells. (A) MCF-7 cells were treated with 15c at various concentrations for 24 h. (B) quantitative analysis of the
VEGF-A protein expression, result are showed as mean ± SD of three independent experiments.
Fig. 8. Effects of 15c on migration and invasion in HUVEC cells. (A) HUVEC cells treated with 15c at indicated concentrations for 12 and 24 h, and the number of
relative closure of scratches in wound healing assay; (C) HUVEC cells were treated with 15c at indicated concentrations for 24 h, and the number of invaded cells in
transwell invasion assay. The statistical histogram of the effects of 15c on migration (B) and invasion (D) of HUVEC cells. ***P < 0.001. Data are presented as mean
± SD of three independent experiments.
2.2.8. Effect of 15c on tube formation of HUVEC cells
lumen-like structure.34 We, therefore, evaluated the anti-vascular ac-
tivity of compound 15c based on HUVEC tubule formation (Fig. 10). The
control group formed an extensive network lumen-like structure, and
In the late stage of angiogenesis, endothelial cells have completed the
steps of proliferation and migration and are arranged to form a long
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S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
Fig. 9. The effects of 15c on expression of MMP-9 and TIMP-1 in HUVECs. (A) Analysis of expression of MMP-9 and TIMP-1 in HUVECs exposed to 0, 0.05, 0.1, 0.5
μ
M and 1 μM 15c for 24 h by western blotting. (B) Quantitative level of proteins in HUVECs after 15c treatment. All data were expressed as the mean ± SD of three
independent experiments.
the small tubes were tightly connected. When 15c was added at a con-
site rather than vincristine site. By reducing the expression of cyclin Cdc
2, Cdc25c and Cyclin B1, 15c arrested tumor cells in the G2/M phase. In
addition, 15c played an anti-angiogenic role by blocking tubule for-
mation and inhibiting migration and invasion of HUVEC cells. Studies
have shown that compound 15c as an effective tubulin polymerization
inhibitor is worthy of subsequent in vivo pharmacokinetic experiments
and evaluation of in vivo antitumor experiments.
centration of 0.1 μM, tubule formation was inhibited, and as the con-
centration increased, the inhibitory effect on tubule formation became
more obvious. When the concentration of 15c was 1 μM, the formation
of HUVEC cell tubules was almost completely inhibited. These results
showed that compound 15c can inhibit HUVEC cell tubule formation in
a dose-dependent manner, thereby effectively inhibiting angiogenesis.
2.2.9. Effect of 15c on disruption of newly formed vascular structures of
HUVEC cells
4. Experimental protocols
Since microtubule disruption is a major aspect of the antivascular
activity of CA-4 and its prodrug CA-4-phosphate,35 so we investigated
whether 15c displayed vascular targeting effects in vitro models by
disrupting the formed tube-like structures. In vitro, HUVECs seeded on a
permissive thick layer of the reconstituted basement membrane,
matrigel rapidly aligned and formed a network of cords, which is
reminiscent of newly formed vessels after 6 h. The addition of 15c (0,
4.1. Chemistry
All starting materials and regents were purchased commercially and
used without further purified, unless otherwise stated. All reactions
were monitored by thin layer chromatograph (TLC) on silica gel GF254
(0.25 mm thick). Column chromatography (CC) was performed on Silica
Gel 60 (230–400 mesh, Qingdao Ocean Chemical Ltd., China). HPLC
analysis using a UltiMate300 DAD HPLC system equipped with a PU-
2089 Plus quaternary gradient pump and a UV-2075 Plus UV–vis de-
tector, using an Alltech Kromasil C18 column with dimensions of 250
0.1, 0.5 or 1 μM) to these newly formed tube-like structures, after in-
cubation for 6 h, photographs were taken. As showed in Fig. 11, 15c
disrupts the integrity of the network in a dose-dependent manner. Taken
together, these data indicated that 15c displayed not only tubule
formation-inhibiting activity, but also vascular-disrupting activity.
mm × 4.6 mm and 5
μ
m particle size. 1H NMR and 13C NMR spectrum
were recorded on an Agilent NMR inova 600 spectrometer with TMS as
an internal standard, all chemical shift values were reported as ppm.
Mass spectra were recorded on an Esquire 6000 (ESI-ION TRAP) spec-
trometer or LTQ-Orbitrap-ETD (Thermo Scientific, USA) with ESI source
as ionization, respectively.
3. Conclusion
In summary, we designed and synthesized a series of novel pyr-
azolopyridine tubulin polymerization inhibitors using hybrid approach.
After preliminary biological activity evaluation and mechanism
research, we found compound 15c displayed better anti-proliferation on
tumor cells and weak toxicity to normal cells WI-38. Furthermore, 15c
showed strong anti-tubulin polymerization activity, and changed the
intracellular tubulin morphology, and formed similar combination
mode to CA-4 and better hydrophobic effect at the colchicine site,
indicating that it can inhibit microtubules by binding to the colchicine
4.1.1. 3-Bromo-2-chloro-6-methoxypyridine (9)
To a solution of 2-chloro-6-methoxypyridine (8) (30.00 g, 209.0
mmol) and dibenzoyl peroxide (BPO) (2.53 g, 10.4 mmol) in acetonitrile
(120 mL), N-bromosuccinimide (55.79 g, 313.4 mmol) was slowly added
in small portions at room temperature followed by further refluxing for
3 h. After the reaction was completed, the solution was cooled and
concentrated under reduced pressure. The residue was dissolved with
Fig. 10. Effects of 15c on the formation of HUVEC cell tubules. HUVEC cells were seeded with 15c at concentration of 0, 0.1, 0.5 and 1
plates, and the representative images of preformed capillary-like tubules were observed under a microscope (×100).
μM for 6 h on matrigel coated
8

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
Fig. 11. Disruption of 15c on newly formed vascular structures in vitro. Representative images of control vehicle or 15c (0.1, 0.5 and 1
like tubular network (×100).
μM) treated HUVEC capillary-
H2O (50 mL), and the aqueous phase was extracted with ethyl acetate (3
× 50 mL). The combined organic extracts were dried over anhydrous
Na2SO4, filtered, and the solvent was evaporated in vacuo. The crude
product was purified by column chromatography (PE: EA = 100:1 ~
50:1) to provide 9 as white solid. Yield: 43.85 g (95%). 1H NMR (600
MHz, CDCl3) δ 7.73 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 3.93 (s,
3H).
Subsequently, haloalkane (9.9 mmol) in anhydrous DMF (5 mL) was
added dropwise. The cooling bath was removed and stirring was
continued for 24 h at room temperature. The solvent was evaporated in
vacuo and the residue was dissolved in ethyl acetate (30 mL). The so-
lution was washed with a saturated solution of Na2CO3, dried over
anhydrous Na2SO4, filtered, and the solvent was evaporated in vacuo.
The residue was purified by column chromatography to obtain 13a–e.
4.1.2. 2-Chloro-6-methoxynicotinic aldehyde (10)
4.1.5.1. 3-Bromo-6-methoxy-1-methyl-1H-pyrazolo[3,4-b]pyridine
To a pre-cooled (ꢀ 78 ◦C) solution of 9 (10.00 g, 45.3 mmol) in
anhydrous THF (50 mL), a solution of n-butyl lithium (2.5 M, 24.0 mL)
was slowly dropped in efficient agitation under atmosphere of argon,
and the reaction mixture was allowed to stir for another 1.5 h. Subse-
quently, N,N-dimethylformamide (7.0 mL, 90.0 mmol) was added por-
tionwise and the resulting mixture was allowed to slowly warm up to
room temperature and stirred for 6 h. Next, H2O (20 mL) was added and
the mixture was extracted with ethyl acetate (3 × 30 mL). The combined
organic extracts were dried over Na2SO4, filtered, and the solvent was
evaporated in vacuo. The crude product was purified by column chro-
matography (PE: EA = 80:1 ~ 50:1) to give 10 as a light yellow solid.
Yield: 4.89 g (63%). 1H NMR (600 MHz, CDCl3) δ 10.30 (s, 1H), 8.10 (d,
J = 8.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.04 (s, 3H).
(13a). White solid; yield: 64%; 1H NMR (600 MHz, CDCl3) δ 7.72 (d, J
= 9.0 Hz, 1H), 6.61 (d, J = 9.0 Hz, 1H), 4.02 (s, 3H), 4.01 (s, 3H); 13
C
NMR (150 MHz, CDCl3) δ 164.9, 149.6, 131.2, 118.9, 110.7, 107.8, 53.8,
33.8; HRMS (ESI) m/z calculated for C8H9BrN3O [M+H]+ 241.9924,
found 241.9916.
4.1.5.2. 3-Bromo-1-cyclopentyl-6-methoxy-1H-pyrazolo[3,4-b]pyridine
(13b). White solid; yield: 58%; 1H NMR (600 MHz, CDCl3) δ 7.70 (d, J
= 8.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 5.26–5.20 (m, 1H), 4.01 (s, 3H),
2.17–2.13 (m, 4H), 2.01–1.96 (m, 2H), 1.73–1.69 (m, 2H); 13C NMR
(150 MHz, CDCl3) δ 164.5, 149.1, 131.2, 118.5, 110.9, 107.7, 58.1, 53.8,
32.1 (2C), 24.7 (2C); HRMS (ESI) m/z calculated for C12H15BrN3O
[M+H]+ 296.0393, found 296.0387.
4.1.3. 6-Methoxy-1H-pyrazolo[3,4-b]pyridine (11)
4.1.5.3. 3-Bromo-1-isopropyl-6-methoxy-1H-pyrazolo[3,4-b]pyridine
(13c). Light yellow solid; yield: 62%; 1H NMR (600 MHz, CDCl3) δ 7.71
(d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 5.13–5.08 (m, 1H), 4.01 (s,
3H), 1.57 (s, 3H), 1.56 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 164.5,
148.6, 131.2, 118.6, 110.8, 107.7, 53.8, 49.2, 21.9 (2C); HRMS (ESI) m/
z calculated for C10H13BrN3O [M+H]+ 270.0237, found 270.0230.
A solution of 10 (4.50 g, 26.3 mmol) and hydrazine hydrate (20 mL)
in H2O (10 mL) was stirred for 5 h at 130 ◦C. Next, the resulting mixture
was cooled to room temperature, and extracted with ethyl acetate (3 ×
10 mL). The combined organic extract was washed with saturated
NaHCO3 solution (15 mL), and dried over Na2SO4, filtered, and the
solvent was evaporated in vacuo. The crude product was purified by
column chromatography (PE: EA = 50:1 ~ 30:1) to give 11 as a white
4.1.5.4. 3-Bromo-6-methoxy-1-phenyl-1H-pyrazolo[3,4-b]pyridine
(13d). Light yellow solid; yield: 57%; 1H NMR (600 MHz, CDCl3) δ 8.26
(d, J = 7.2 Hz, 2H), 7.82 (d, J = 9.0 Hz, 1H), 7.53–7.52 (m, 1H),
7.51–7.49 (m, 2H), 6.74 (d, J = 9.0 Hz, 1H), 4.07 (s, 3H); 13C NMR (150
MHz, CDCl3) δ 164.0, 147.9, 138.1, 131.3, 130.6, 129.9, 127.9, 127.8,
124.9, 119.5, 111.4, 107.5, 53.1; HRMS (ESI) m/z calculated for
C13H11BrN3O [M+H]+ 304.0080, found 304.0081.
1
solid. Yield: 2.30 g (58%). H NMR (600 MHz, CDCl3) δ 7.93 (s, 1H),
7.90 (d, J = 9.0 Hz, 1H), 6.63 (d, J = 9.0 Hz, 1H), 4.02 (s, 3H); MS (ESI)
m/z 150.1 for [M+H]+.
4.1.4. 3-Bromo-6-methoxy-1H-pyrazolo[3,4-b]pyridine (12)
To a pre-cooled (ꢀ 30 ◦C) solution of 11 (2.0 g, 13.4 mmol) in
dichloromethane (20 mL), N-bromosuccinimide (2.38 g, 13.4 mmol)
was slowly added in portions followed by further stirred for 30 min. The
resulting mixture was warmed to room temperature, and added H2O (10
mL). The aqueous phase was extracted with dichloromethane (2 × 10
mL), the combined organic phases were dried over anhydrous magne-
sium sulphate, filtered, and the solvent was evaporated in vacuo. The
crude product was purified by column chromatography (PE: EA = 100:1
~ 30:1) to get 12 light yellow solid. Yield: 1.56 g (51%); 1H NMR (600
MHz, CDCl3) δ 7.78 (d, J = 9.0 Hz, 1H), 6.68 (d, J = 9.0 Hz, 1H), 4.02 (s,
3H).
4.1.5.5. 1-Benzyl-3-bromo-6-methoxy-1H-pyrazolo[3,4-b]pyridine
(13e). Light yellow solid; yield: 66%; 1H NMR (600 MHz, CDCl3) δ 7.71
(d, J = 9.0 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.31–7.29 (m, 2H),
7.27–7.25 (m, 1H), 6.62 (d, J = 8.4 Hz, 1H), 5.54 (s, 2H), 4.02 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 165.0, 149.4, 136.7, 131.3, 128.6 (2C),
128.2(2C), 127.8, 119.6, 110.8, 108.0, 53.9, 51.0; HRMS (ESI) m/z
calculated for C14H12BrN3NaO [M+Na]+ 340.0056, found 340.0050.
4.1.6. 3-Bromo-6-methoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo
[3,4-b]pyridine(13f)
4.1.5. General procedures for preparation of compounds 13a–e
A solution of 12 (1.50 g, 6.6 mmol) and sodium hydride (0.24 g, 9.9
mmol) in anhydrous DMF (5 mL) was stirred for 30 min at 0 ◦C.
To a solution of 12 (1.50 g, 6.6 mmol) and p-toluenesulfonic acid
(0.23 g, 1.32 mmol) in anhydrous tetrahydrofuran (10 mL), 3,4-dihydro-
2H-pyran (1.8 mL, 19.8 mmol) was slowly added under the atmosphere
9

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
of argon. The mixture was heated to reflux and stirring was continued
for 3 h. The solvent was evaporated in vacuo and the residue, saturated
NaHCO3 solution (20 mL) was added, and the mixture was extracted
with dichloromethane (3 × 20 mL). The combined organic extracts were
dried over anhydrous Na2SO4, filtered, and the solvent was evaporated
in vacuo. The residue was purified by column chromatography (PE: EA
= 70:1 ~ 50:1) to obtain 13f as white solid. Yield: 2.1 g (80%); 1H NMR
(600 MHz, CDCl3) δ 7.73 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H),
5.94 (dd, J = 10.2, 2.4 Hz, 1H), 4.14–4.13 (m, 1H), 4.04 (s, 3H),
3.79–3.75 (m, 1H), 2.64–2.60 (m, 1H), 2.16–2.14 (m, 1H), 1.97–21.94
(m, 1H), 1.81–1.76 (m, 2H), 1.63–1.61 (m, 1H); 13C NMR (150 MHz,
CDCl3) δ 165.1, 149.6, 131.4, 121.5, 111.2, 108.5, 82.3, 67.9, 54.0,
29.4, 25.0, 23.0; HRMS (ESI) m/z calculated for C12H15BrN3O2 [M+H]+
334.0162, found 334.0159.
130.9, 128.8 (2C), 124.0 (2C), 119.0 (2C), 105.4 (2C), 104.0, 94.8 (2C),
61.0, 55.9 (2C), 53.8; MS (ESI) m/z 407.2 for [M+H]+; HRMS (ESI) m/z
calculated for C22H23N4O4 [M+H]+ 407.1719, found 407.1728.
4.1.7.5. 1-Benzyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b]pyridin- 3-amine (14e). Yellow solid; yield: 61%; 1H NMR (600
MHz, CDCl3) δ 7.69 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.30 (t,
J = 7.2 Hz, 2H), 6.56 (s, 2H), 6.48 (d, J = 8.4 Hz, 1H), 6.19 (brs, 1H),
5.48 (s, 2H), 4.03 (s, 3H), 3.80 (s, 3H), 3.76 (s, 6H); 13C NMR (150 MHz,
CDCl3) δ 164.8 (2C), 153.6 (2C), 149.5, 138.5, 137.4 (2C), 131.3 (2C),
128.5 (2C), 128.2, 127.6, 105.2 (2C), 94.8 (2C), 61.0, 55.9, 53.7, 50.0,
29.7; MS (ESI) m/z 421.2 for [M+H]+.
4.1.7.6. 6-Methoxy-1-(tetrahydro-2H-pyran-2-yl)-N-(3,4,5-trimethox-
yphenyl)-1H- pyrazolo[3,4-b]pyridin-3-amine (14f). White solid; yield:
72%; 1H NMR (600 MHz, CDCl3) δ 7.66 (d, J = 8.4 Hz, 1H), 6.57 (s, 2H),
6.48 (d, J = 8.4 Hz, 1H), 6.23 (s, 1H), 5.93 (dd, J = 8.4, 2.4 Hz, 1H), 4.15
(d, J = 9.0 Hz, 1H), 4.03 (s, 3H), 3.82–3.78 (m, 10H), 2.61–2.59 (m, 1H),
2.17–1.2.15 (m, 1H), 2.02–1.99 (m, 1H), 1.87–1.75 (m, 2H), 1.64–1.62
(m, 1H); 13C NMR (150 MHz, CDCl3) δ 164.6, 153.6, 149.9, 143.9,
138.4, 132.5, 131.4, 105.6 (2C), 102.7, 95.1 (2C), 81.7, 68.0, 61.0, 55.9
(2C), 53.6, 29.4, 25.2, 23.2; MS (ESI) m/z 415.2 for [M+H]+.
4.1.7. General procedures for preparation of compounds 14a–f
To a solution of 4,5-bisdiphenylphosphine-9,9-dimethylxanthene
and Pd(OAc)2 in dried 1,4-dioxane, a solution of 3,4,5-trimethoxyani-
line in 1,4-dioxane was added, and the resulting mixture was heated
under reflux for 5 min in the atmosphere of argon. Subsequently,
respective of intermediate 13a–f and cesium carbonate were added in
sequence, and the resulting mixture was heated under reflux for 3 h.
Next, the solvent was evaporated in vacuo, and the residue was purified
by column chromatography to obtain the target compounds 14a–d and
the intermediates 14e,f.
4.1.8. General procedures for preparation of compounds 15a,b
To a pre-cooled (0 ◦C) solution of 14a,b (0.8 mmol) in DMF (5 mL),
sodium hydride (1.0 mmol) was slowly add, and the resulting mixture
was stirred for 30 min at 0 ◦C. Subsequently, haloalkane (1.0 mmol) in
anhydrous DMF (5 mL) was added dropwise, and the cooling bath was
removed and stirring was continued for 24 h at room temperature. The
solvent was evaporated in vacuo and H2O (10 mL) was added. Next, the
mixture was extracted with ethyl acetate (3 × 10 mL), and the combined
organic extracts were dried over anhydrous Na2SO4, filtered, and the
solvent was evaporated in vacuo. The residue was purified by column
chromatography to give the corresponding product.
4.1.7.1. 6-Methoxy-1-methyl-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (14a). White solid; yield: 64%; m.p.: 199–201
◦C; HPLC purity: 96.44% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 6.109
min); 1H NMR (600 MHz, CDCl3) δ 7.68 (d, J = 8.4 Hz, 1H), 6.53 (s, 2H),
6.45 (d, J = 8.4 Hz, 1H), 6.11 (brs, 1H), 4.02 (s, 3H), 3.94 (s, 3H), 3.82
(s, 9H); 13C NMR (150 MHz, CDCl3) δ 164.5, 153.6, 149.5, 142.7, 138.8,
131.3 (2C), 104.7 (2C), 101.8, 94.6 (2C), 61.0, 55.9 (2C), 53.5, 33.1; MS
(ESI) m/z 345.2 for [M+H]+; HRMS (ESI) m/z calculated for
C
17H21N4O4 [M+H]+ 345.1563, found 345.1568.
4.1.8.1. 6-Methoxy-N,1-dimethyl-N-(3,4,5-trimethoxyphenyl)-1H-pyr-
azolo[3,4-b] pyridin-3-amine (15a). White solid; yield: 51%; m.p.:
124–126 ◦C; HPLC purity: 96.31% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 10.268 min); 1H NMR (600 MHz, CDCl3) δ 7.02 (d, J = 8.4 Hz, 1H),
6.40 (s, 2H), 6.25 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.86 (s,
3H), 3.75 (s, 6H), 3.48 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 164.1,
153.6 (2C), 150.0, 147.6, 143.8, 134.4, 133.1 (2C), 104.0, 101.8, 100.2
(2C), 61.0, 56.1 (2C), 53.4, 40.3, 33.0; MS (ESI) m/z 359.2 for [M+H]+;
HRMS (ESI) m/z calculated for C18H23N4O4 [M+H]+ 359.1719, found
359.1726.
4.1.7.2. 1-Cyclopentyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyr-
azolo[3,4-b] pyridin-3-amine (14b). White solid; yield: 75%; m.p.:
203–205 ◦C; HPLC purity: 97.23% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 11.367 min); 1H NMR (600 MHz, CDCl3) δ 7.69 (d, J = 8.4 Hz, 1H),
6.66 (s, 2H), 6.45 (d, J = 8.4 Hz, 1H), 6.17 (brs, 1H), 5.25 (brs, 1H), 4.01
(s, 3H), 3.83 (s, 9H), 2.18–2.12 (m, 4H), 2.02–1.97 (m, 2H), 1.76–1.72
(m, 2H); 13C NMR (150 MHz, CDCl3) δ 164.4, 153.5, 148.9, 142.5,
138.3, 131.7 (2C), 104.8 (2C), 101.5, 94.7 (2C), 61.0, 57.4, 55.6 (2C),
53.6, 31.8 (2C), 24.5 (2C); MS (ESI) m/z 399.2 for [M+H]+; HRMS (ESI)
m/z calculated for C21H27N4O4 [M+H]+ 399.2032, found 399.2039.
4.1.8.2. N,1-Dicyclopentyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[3,4-b] pyridin-3-amine (15b). White solid; yield: 49%; m.p.:
111–113 ◦C; HPLC purity: 97.23% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 11.506 min); 1H NMR (600 MHz, CDCl3) δ 6.71 (d, J = 8.4 Hz, 1H),
6.32 (s, 2H), 6.19 (d, J = 8.4 Hz, 1H), 5.21–5.18 (m, 1H), 4.53–4.50 (m,
1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.75 (s, 6H), 2.15–2.11 (m, 6H),
2.02–1.18 (m, 4H) 1.71–1.62 (m, 6H); 13C NMR (150 MHz, CDCl3) δ
163.6, 153.3 (2C), 149.3, 146.4 (2C), 135.1, 132.7 (2C), 104.1, 103.3
(2C), 61.3, 61.0, 57.0, 56.1 (2C), 53.4, 31.8 (2C), 29.9 (2C), 24.8 (2C),
23.5 (2C); MS (ESI) m/z 467.3 for [M+1]+; HRMS (ESI) m/z calculated
for C26H35N4O4 [M+H]+ 467.2658, found 467.2668.
4.1.7.3. 1-Isopropyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (14c). Yellow solid; yield: 66%; m.p.: 172–174
◦C; HPLC purity: 96.30% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 8.101
min); 1H NMR (600 MHz, CDCl3) δ 7.69 (d, J = 8.4 Hz, 1H), 6.63 (s, 2H),
6.46 (s, 1H), 6.13 (brs, 1H), 5.08 (brs, 1H), 4.01 (s, 3H), 3.83 (s, 9H),
1.55 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ 164.1, 153.4 (2C),
151.7, 149.2, 139.0, 136.6, 133.4, 106.4 (2C), 104.4, 102.4, 61.0, 56.2
(2C), 53.7, 49.8, 20.6 (2C); MS (ESI) m/z 373.2 for [M + H]+; HRMS
(ESI) m/z calculated for C19H25N4O4 [M+H]+ 373.1876, found
373.1882.
4.1.7.4. 6-Methoxy-1-phenyl-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (14d). Yellow solid; yield: 68%; m.p.: 189–191
◦C; HPLC purity: 95.03% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 8.854
min); 1H NMR (600 MHz, CDCl3) δ 8.35 (d, J = 9.0 Hz, 2H), 7.80 (d, J =
8.4 Hz, 1H), 7.48 (t, J = 8.4 Hz, 2H), 7.21 (t, J = 8.4 Hz, 1H), 6.85 (s,
4.1.9. General procedures for preparation of compounds 15c-l
The pre-cooled solution of 14f (0.8 mmol) in DMF (5 mL) (0 ◦C) was
treated with various haloalkane by similar procedure as description to
synthesize 14a,b. Then the obtained intermediate (0.6 mmol) was
directly dissolved in ethanol (5 mL), and added concentrated hydro-
chloric acid (3 mL) at room temperature, and stirring was continued at
room temperature for 24 h. The solvent was evaporated in vacuo and the
2H), 6.58 (d, J = 8.4 Hz, 1H), 4.07 (s, 3H), 3.88 (s, 6H), 3.84 (s, 3H); 13
C
NMR (150 MHz, CDCl3) δ 164.5, 153.4, 148.6, 144.3, 140.0, 137.8,
10

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
residue was dissolved in ethyl acetate (10 mL) and H2O (5 mL). The
organic layer was washed with a saturated solution of Na2CO3, dried
over anhydrous Na2SO4, filtered, and the solvent of evaporated in vacuo.
Next, the residue was purified by column chromatography to provide the
corresponding target compounds 15c–l.
1H), 6.51 (s, 2H), 6.30–6.28 (m, 2H), 4.21–4.15 (m, 2H), 3.99 (s, 3H),
3.89 (s, 3H), 3.77 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 164.7, 153.8
(2C), 151.8, 148.2, 141.9, 136.0, 114.0 (t, J = 241.5 Hz, 1C), 105.2,
102.8 (2C), 101.1, 65.5, 61.1, 56.2 (2C), 53.8; MS (ESI) m/z 395.2 for
[M+H]+; HRMS (ESI) m/z calculated for C18H21F2N4O4 [M+H]+
395.1531, found 395.1537.
4.1.9.1. 6-Methoxy-N-methyl-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (15c). White solid; yield: 64%; m.p.: 159–161
◦C; HPLC purity: 95.59% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 5.182
min); 1H NMR (600 MHz, CDCl3) δ 7.03 (d, J = 8.4 Hz, 1H), 6.44 (s, 2H),
6.31 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.87 (s, 3H), 3.77 (s, 6H), 3.50 (s,
3H); 13C NMR (150 MHz, CDCl3) δ 164.0, 153.6 (2C), 150.3, 142.8,
132.1 (2C), 129.2, 108.0, 106.9, 104.2 (2C), 61.0, 56.3 (2C), 53.6, 33.6;
MS (ESI) m/z 345.2 for [M+H]+; HRMS (ESI) m/z calculated for
4.1.9.7. N-(cyclopropylmethyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)-
1H-pyrazolo [3,4-b]pyridin-3-amine (15i). Light yellow solid; yield:
55%; m.p.: 118–120 ◦C; HPLC purity: 96.39% (MeOH: H2O = 85:15, 1.0
mL/min, tR = 5.908 min); 1H NMR (600 MHz, CDCl3) δ 6.88 (d, J = 8.4
Hz, 1H), 6.49 (s, 2H), 6.27 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.89 (s, 3H),
3.77 (s, 6H), 3.74 (d, J = 6.6 Hz, 2H), 1.29–1.25 (m, 1H), 0.49 (d, J =
6.6 Hz, 2H), 0.24 (d, J = 6.6 Hz, 2H); 13C NMR (150 MHz, CDCl3) δ
164.5, 153.6 (2C), 151.8, 149.2, 142.4, 133.5, 104.7 (2C), 102.8 (2C),
61.1, 57.1, 56.2 (2C), 53.7, 9.9, 3.9 (2C); MS (ESI) m/z 385.2 for
[M+H]+; HRMS (ESI) m/z calculated for C20H25N4O4 [M+H]+
385.1876, found 385.1882.
C
17H21N4O4 [M+H]+ 345.1563, found 345.1570.
4.1.9.2. N-Ethyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (15d). Yellow solid; yield: 65%; m.p.: 137–139
◦C; HPLC purity: 96.88% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 5.476
min); 1H NMR (600 MHz, CDCl3) δ 6.91 (d, J = 8.4 Hz, 1H), 6.43 (s, 2H),
6.28 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.96 (q, J = 7.2 Hz, 2H), 3.88 (s,
3H), 3.77 (s, 6H), 1.32 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
164.4, 153.6 (2C), 151.7, 148.7, 142.0, 133.5, 104.6 (2C), 102.0 (2C),
61.0, 56.1 (2C), 53.8, 47.1, 12.9; MS (ESI) m/z 359.2 for [M+H]+;
HRMS (ESI) m/z calculated for C18H23N4O4 [M+H]+ 359.1719, found
359.1727.
4.1.9.8. 6-Methoxy-N-(2-methoxyethyl)-N-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo [3,4-b]pyridine-3-amine (15j). White solid; yield: 52%; m.p.:
127–129 ◦C; HPLC purity: 97.55% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 5.170 min); 1H NMR (600 MHz, CDCl3) δ 6.95 (d, J = 8.4 Hz, 1H), 6.55
(s, 2H), 6.29 (d, J = 8.4 Hz, 1H), 4.09 (t, J = 4.8 Hz, 2H), 4.01 (s, 3H),
3.88 (s, 3H), 3.76 (s, 9H), 3.72 (t, J = 4.8 Hz, 2H); 13C NMR (150 MHz,
CDCl3) δ 164.4, 153.5, 151.7, 148.4, 142.5, 133.6, 130.9, 128.8, 104.6,
102.0, 101.8, 65.5, 61.0, 58.9, 56.0 (2C), 53.8, 52.2; MS (ESI) m/z 389.2
for [M+H]+; HRMS (ESI) m/z calculated for C19H25N4O5 [M+H]+
389.1825, found 389.1832.
4.1.9.3. 6-Methoxy-N-propyl-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b]pyridin- 3-amine (15e). Yellow solid; yield: 55%; m.p.: 140–142
◦C; HPLC purity: 96.99% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 5.952
min); 1H NMR (600 MHz, CDCl3) δ 6.93 (d, J = 8.4 Hz, 1H), 6.43 (s, 2H),
6.29 (d, J = 8.4 Hz, 1H), 4.02 (s, 3H), 3.88 (s, 3H), 3.84 (t, J = 6.6 Hz,
2H), 3.76 (s, 6H), 1.81–1.79 (m, 2H), 1.00 (t, J = 6.6 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 164.4, 153.6, 151.7, 149.0, 142.5, 135.0, 133.5,
130.9, 128.8, 104.6, 101.9 (2C), 61.2, 56.1 (2C), 54.5, 53.7, 20.9, 11.5;
MS (ESI) m/z 373.2 for [M+H]+; HRMS (ESI) m/z calculated for
4.1.9.9. N-Cyclopentyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyr-
azolo[3,4-b] pyridin-3-amine (15k). White solid; yield: 56%; m.p.:
145–148 ◦C; HPLC purity: 95.27% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 6.598 min); 1H NMR (600 MHz, CDCl3) δ 6.49 (d, J = 9.0 Hz, 1H), 6.45
(s, 2H), 6.22 (d, J = 9.0 Hz, 1H), 4.61–4.60 (m, 1H), 4.00 (s, 3H), 3.93 (s,
3H), 3.80 (s, 6H), 2.09–2.08 (m, 2H), 1.65–1.62 (m, 4H) 1.60–1.57 (m,
2H); 13C NMR (150 MHz, CDCl3) δ 164.4, 153.4 (2C), 151.7, 150.0,
140.0, 133.3, 130.8, 128.8, 106.4, 104.7, 102.5, 61.1, 61.0, 56.2 (2C),
53.6, 29.7 (2C), 23.1 (2C); MS (ESI) m/z 399.3 for [M+H]+; HRMS (ESI)
m/z calculated for C21H27N4O4 [M+H]+ 399.2032, found 399.2038.
C
19H25N4O4 [M+H]+ 373.1876, found 373.1883.
4.1.9.4. N-Isopropyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (15f). Light yellow solid; yield: 60%; m.p.:
110–112 ◦C; HPLC purity: 96.20% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 5.650 min); 1H NMR (600 MHz, CDCl3) δ 6.48 (d, J = 9.0 Hz, 1H), 6.43
(s, 2H), 6.19 (d, J = 9.0 Hz, 1H), 4.68–4.66 (m, 1H), 3.95 (s, 3H), 3.91 (s,
3H), 3.78 (s, 6H), 1.29 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ
164.3, 153.4 (2C), 151.7, 149.4, 138.9, 133.4, 106.5 (2C), 104.5, 61.1,
56.2 (2C), 53.7, 49.7, 20.6 (2C); MS (ESI) m/z 373.2 for [M+H]+; HRMS
(ESI) m/z calculated for C19H25N4O4 [M+H]+ 373.1876, found
373.1882.
4.1.9.10. N-Benzyl-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[3,4-b] pyridin-3-amine (15l). White solid; yield: 58%; m.p.: 167–169
◦C; HPLC purity: 97.43% (MeOH: H2O = 85:15, 1.0 mL/min, tR = 6.255
min); 1H NMR (600 MHz, CDCl3) δ 7.38 (d, J = 6.6 Hz, 1H), 7.31–7.28
(m, 3H), 7.23 (d, J = 6.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.35 (s, 2H),
6.33 (d, J = 8.4 Hz, 1H), 5.16 (s, 2H), 3.98 (s, 3H), 3.84 (s, 3H), 3.66 (s,
6H); 13C NMR (150 MHz, CDCl3) δ 164.5, 153.5 (2C), 151.7, 148.6,
142.5, 139.1, 133.4, 128.4 (2C), 127.5 (2C), 126.9 (2C), 105.0 (2C),
102.0, 100.7 (2C), 61.0, 56.2, 56.0, 53.8; MS (ESI) m/z 385.2 for
[M+H]+; HRMS (ESI) m/z calculated for C23H25N4O4 [M+H]+
421.1876, found 421.1883.
4.1.9.5. N-(2-fluoroethyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[3,4-b] pyridin-3-amine (15g). White solid; yield: 63%; m.p.:
147–149 ◦C; HPLC purity: 95.79% (MeOH: H2O = 85:15, 1.0 mL/min, tR
= 5.233 min); 1H NMR (600 MHz, CDCl3) δ 6.89 (d, J = 8.4 Hz, 1H), 6.52
(s, 2H), 6.28 (d, J = 8.4 Hz, 1H), 4.79 (t, J = 4.8 Hz, 1H), 4.71 (t, J = 4.8
Hz, 1H), 4.21 (t, J = 4.8 Hz, 1H), 4.16 (t, J = 4.8 Hz, 1H), 3.97 (s, 3H),
3.88 (s, 3H), 3.76 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 164.6, 153.7,
151.9, 148.3, 142.3, 135.6, 133.5, 130.9, 128.8, 104.9, 102.6, 101.6,
80.9, 65.5, 61.1, 56.1 (2C), 53.1; MS (ESI) m/z 377.3 for [M+H]+;
HRMS (ESI) m/z calculated for C18H22FN4O4 [M+H]+ 377.1625, found
377.1632.
4.2. Biology
4.2.1. MTT assay
Cells grown in the logarithmic phase were seeded into 96-well plates
(5 × 103 cells/well) for 24 h, and then exposed to different concentra-
tions of the test compounds for 72 h. After attached cells were incubated
with 5 mg/mL MTT (Sigma, USA) for another 4 h, the suspension was
discarded, and subsequently the dark blue crystals (formazan) were
solubilized in dimethyl sulfoxide (DMSO). The absorbance of the solu-
tion at 490 nm was measured using a multifunction microplate reader
(Molecular Devices, Flex Station 3), and each experiment was performed
at least in triplicate. IC50 values, which represent the drug
4.1.9.6. N-(2,2-Difluoroethyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)-
1H-pyrazolo [3,4-b]pyridine-3-amine (15h). White solid; yield: 56%; m.
◦
p.: 147–149 C; HPLC purity: 98.03% (MeOH: H2O = 85:15, 1.0 mL/
min, tR = 5.302 min); 1H NMR (600 MHz, CDCl3) δ 6.84 (d, J = 8.4 Hz,
11

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
concentrations required to cause 50% cancer cell growth inhibition,
were used to express the cytotoxic effects of each compound and were
calculated with GraphPad Prism Software version 5.02 (GraphPad Inc.,
La Jolla, CA, USA).
incubated overnight with specific primary antibodies (CST, USA) at 4 ◦C.
After three washes in TBST, the membranes were incubated with the
appropriate HRP-conjugated secondary antibodies at room temperature
for 2 h. The blots were developed with enhanced chemiluminescence
(Pierce, Rockford, Illinois, USA) and were detected by an LAS4000
imager (GE Healthcare, Waukesha,Wisconsin, USA).
4.2.2. In vitro tubulin polymerization assay
Pig brain microtubule protein was isolated by three cycles of
temperature-dependent assembly/disassembly according to Shelanski et
al in 100 mM PIPES (pH 6.5), 1 mM MgSO4, 2 mM EGTA, 1 mM GTP and
1 mM 2-mercaptoethanol. In the first cycle of polymerization, glycerol
and phenylmethylsulfonyl fluoride were added to 4 M and 0.2 mM,
respectively. Homogeneous tubulin was prepared from microtubule
protein by phosphocellulose (P11) chromatography. The purified pro-
teins were stored in aliquots at ꢀ 70 ◦C. Tubulin protein was mixed with
different concentrations of compound in PEM buffer (100 mM PIPES, 1
mM MgCl2, and 1 mM EGTA) containing 1 mM GTP and 5% glycerol.
Microtubule polymerization was monitored at 37 ◦C by light scattering
at 340 nm using a SPECTRA MAX 190 (Molecular Device) spectropho-
tometer. The plateau absorbance values were used for calculations.
4.2.6. Wound healing assay
MCF-7 cells (5 × 105 cells/well) were grown in petridishes for 24 h.
Scratches were made in confluent monolayers using 200 μL pipette tip.
Then, wounds were washed twice with PBS to remove non-adherent cell
debris. The media containing different concentrations of the compound
15c were added to the petridishes. Cells which migrated across the
wound area were photographed under the phase contrast microscope
(Nikon) after 0, 12 and 24 h treatment. The number of cells migrated in
to the wound area was counted manually.
4.2.7. Transwell invasion assay
The Transwell (12 μm, Corning Incorporated) was pre-coated with
70
harvested and resuspended in serum-free medium containing 0, 0.05,
μ
L Matrigel for 5 h at 37 ◦C to achieve solidification. MCF-7 cells were
4.2.3. Immunofluorescence microscopy
In a 10 mm confocal culture dish, 3 × 104 cells were grown for 24 h
and then incubated in the presence or absence of compound 15c at the
indicated concentrations for another 6 h. After being washed with
phosphate-buffered solution (PBS) and fixed in 4% prewarmed (37 ◦C)
paraformaldehyde for 15 min, the cells were permeabilized with 0.5%
Triton X-100 for 15 min and blocked for 30 min in 10% goat serum.
Then, the cells were incubated with mouse anti-tubulin antibody (CST,
0.1, 0.5 and 1 μM of compound 15c and added into the upper wells of
the Transwell chamber at the density of 5 × 105 cells/mL. While 600
μL
of ECM containing 10% FBS was added into the lower chambers which
had been coated with 70
μ
L of Matrigel (1:8 dilution in serum-free
◦
medium, Corning/BD Biosciences). After 24 h of incubation at 37 C,
the invasion cells were fixed with methanol and stained with 0.1%
crystal violet for 30 min, respectively. Then, the chambers were washed
with PBS and left to dry. Images were photographed using an inverted
fluorescence microscope (Zeiss, VERT1, USA) and counted by Image J
software for three independent fields randomly.
◦
USA) at 4 C overnight, washed with PBS three times, and incubated
with goat antimouse IgG/Alexa-Fluor 488 antibody (Invitrogen, USA)
for 1 h. The samples were immediately visualized on a Zeiss LSM 570
laser scanning confocal microscope (Carl Zeiss, Germany) after the
nuclei were stained with DAPI (Solarbio, Chin) in the dark at room
temperature for 10 min.
4.2.8. Tubule formation assay
The Matrigel was slowly driven into the pre-cooled 96-well plate
with a pre-cooled pipette tip. Wrap the 96-well plate with plastic wrap
and put it in a 4 ◦C refrigerator. After the matrigel is covered with the
bottom of the well and stabilized, place the 96-well plate in a 37 ◦C cell
incubator and polymerize for 30 min. The digested HUVEC cells were
resuspended in ECM medium containing 20% FBS, and HUVEC cells
were seeded in 96-well plates at 5 × 104 cells per well using a cell
counting plate. Then add 15c of different concentration for 6 h. After 6
h, observe the formation of cell tubules and take pictures under an
inverted microscope.
4.2.4. Cell cycle analysis
Cells were seeded in 6-well plates (3 × 105 cells/well), incubated in
the presence or absence of compound 15c at the indicated concentra-
tions for 24 h, harvested by centrifugation, and then fixed in ice-cold
70% ethanol overnight. After the ethanol was removed the next day,
the cells were resuspended in ice-cold PBS, treated with RNase A
(Keygen Biotech, China) at 37 ◦C for 30 min, and then incubated with
the DNA staining solution propidium iodide (PI, Keygen Biotech, China)
at 4 ◦C for 30 min. Approximately 10 000 events were detected by flow
cytometry (Beckman Coulter, Epics XL) at 488 nm.
To examine neovessel disruption, the formed capillary-like struc-
tures were exposed to serum-free culture DMEM medium with either
vehicle or 15c (0.1, 0.5 and 1 μM). After incubation for 6 h, photographs
4.2.5. Western blot analysis
were taken. The number of left tubes was quantified and the disruption
of neovasculature networks was expressed as [1-(tubestreated/tubesve-
hicle)] × 100%.
MCF-7 or HUVEC cells (5.0 × 105 cells/dish) were incubated with or
without 15c at various concentrations for 6 h. After incubation, the cells
were collected by centrifugation and washed twice with phosphate-
buffered saline chilled to 0 ◦C. Then, the cells were homogenized in
RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% (w/
v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 1
mM EDTA (Beyotime, China). The lysates were incubated on ice for 30
min, intermittently vortexed every 5 min, and centrifuged at 12,500 g
for 15 min to harvest the supernatants. Next, the protein concentrations
were determined by a BCA Protein Assay Kit (Thermo Fisher Scientific,
Rockford, Illinois, USA). The protein extracts were reconstituted in
loading buffer containing 62 mM Tris-HCl, 2% SDS, 10% glycerol, and
5% β-mercaptoethanol (Beyotime, China), and the mixture was boiled at
100 ◦C for 10 min. An equal amount of the proteins (50 mg) was sepa-
rated by 8–12% sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis (SDS-PAGE) and transferred to nitrocellulose membranes
(Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Then,
the membranes were blocked with 5% nonfat dried milk in TBS con-
taining 1% Tween-20 for 90 min at room temperature and were
4.2.9. Molecular docking
In this study, the tubulin structure was downloaded from the Protein
Data Bank (PDB code 1SA0). Using Schrodinger software prepared the
protein by removing of Mg2+, water molecules and colchicine. Then the
ligand 15c was minimized, and the docking procedure was performed by
employing docking program in schrodinger software, and the structural
image was obtained using PyMOL software.
4.3. Statistical analysis
All data in this study were obtained from three independent exper-
iments and then expressed as the means ± SD. Statistically significant
differences were assessed via SPSS 18.0 software (SPSS, IL, USA). A
value of p < 0.05 was considered statistically significant.
12

S.-Y. Hao et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115985
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cancer. Bioorg Med Chem. 2017;25:2226–2233.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
17 Sharma S, Poliks B, Chiauzzi C, et al. Characterization of the colchicine binding site
on avian tubulin isotype βVI. Biochemistry. 2010;49:2932–2942.
18 Bai R, Covell DG, Pei XF, et al. Mapping the binding site of colchicinoids on β-tubulin
2-chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with
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Acknowledgments
This work was financially supported by the National Natural Sci-
ences Foundations of China (21672093).
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115985.
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