3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors: Rational design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.12.053

A series of novel 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines as tubulin polymerization inhibitors targeting the colchicine site were designed via ring tethering strategy, which was supported by conformational analysis. The general, chemically unstable and rot

Full text of DOI:10.1016/j.ejmech.2018.12.053

Accepted Manuscript
3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors:
Rational design, synthesis and biological evaluation
Min'an Zhai, Shiyuan Liu, Meiqi Gao, Long Wang, Jun Sun, Jianan Du, Qi Guan, Kai
Bao, Daiying Zuo, Yingliang Wu, Weige Zhang
PII:
S0223-5234(18)31096-1
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.053
EJMECH 10987
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 September 2018
Revised Date: 10 December 2018
Accepted Date: 21 December 2018
Please cite this article as: Min'. Zhai, S. Liu, M. Gao, L. Wang, J. Sun, J. Du, Q. Guan, K. Bao, D. Zuo,
Y. Wu, W. Zhang, 3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors:
Rational design, synthesis and biological evaluation, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2018.12.053.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization
inhibitors: Rational design, synthesis and biological evaluation
Min’an Zhaia, Shiyuan Liua, Meiqi Gaob, Long Wanga, Jun Sund, Jianan Dub, Qi Guana*, Kai Baoc, Daiying Zuob,
Yingliang Wub*, Weige Zhanga*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China
b
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, China
c
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, China
d
Clinical Pharmacology Laboratory, Henan Province People’s Hospital, 7 Weiwu Road, Jinshui District,
Zhengzhou 450003, China
Corresponding author
E-mail: zhangweige2000@sina.com (W. Zhang), yingliang_1016@163.com (Y. Wu), guanqi@syphu.edu.cn (Q.
Guan).

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Abstract
A series of novel 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines as tubulin polymerization inhibitors
targeting the colchicine site were designed via ring tethering strategy, which was supported by
conformational analysis. The general, chemically unstable and rotational linker, carbanyl group,
was locked by 1H-pyrazolo[3,4-b]pyridine to avoid carbonyl reduction and restrict the instability
of molecular conformation caused by the rotation of the carbon-carbon single bond beside
carbonyl group. All of target compounds were synthesized and evaluated for their antiproliferative
activities against three human cancer lines (SGC-7901, A549 and HeLa) by MTT assay. Most of
these compounds showed prominent in vitro potency and the most potent compound in this
scaffold 13d (SGC-7901: IC50 = 13 nM) could significantly inhibit tubulin polymerization and
strongly disrupt cytoskeleton. The results of molecular modeling study revealed that 13d interacts
with tubulin by binding to the colchicine site.
Keywords: Ring tethering; Conformational analysis; Pyrazolo[3,4-b]pyridine; Colchicine site

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1. Introduction
Microtubules are vital components of filamentous cytoskeleton consisted of α,β-tubulin
heterodimers in eukaryotic cells.[1] It is known that microtubules play important roles in driving
many cellular physiological processes such as cell signaling, intracellular transport, intracellular
macromolecular assemblies and cell division.[2] The critical characteristic of microtubule is that it
is always in a dynamic equilibrium of polymerization and depolymerization, and this is the
foundation of tubulin polymerization inhibitors’ functionality.[3] The widely known tubulin
polymerization inhibitors such as colchicine, CA-4 and SMART (Fig. 1) could be used for
damaging or killing various types of tumor cells through breaking the equilibrium by binding to
colchicine site in tubulin.[4]
As SMART analogues, compounds 4-6 (Fig. 1) were designed according to bioisosterism and
evaluated antiproliferative activity previously, with the IC50 values of 52-500 nM, 25-39
nM, >3000 nM, respectively.[5] The loss of compound 6’s activity impels us to explore the
fundamental causes. Compared the chemical structures of compound 6 with 4, 5 and SMART,
they have a similar molecular skeleton containing three conjugated six-membered aromatic rings
(A-, B-, and C-ring, respectively) with a ketone linker between A- and B-ring.[6] Generally,
structural modifications on B-ring using bioisosterism can be tolerated in maintaining original
bioactivity.[7] It is known that this antitubulin effect stems from the interactions between ligands
(tubulin polymerization inhibitors) and receptor (α,β-tubulin) with appropriate binding mode.
Therefore the difference of binding mode between 6 with the other compounds with α,β-tubulin
may be the key point to explain the loss of activity of 6. Unfortunately, co-crystal of SMART or
its analogues with α,β-tubulin heterodimer complex has not been obtained up to now. Note that
this class of compounds has two vastly different and relatively stable conformations, “bent” and
“straight” conformation respectively (Fig. 2). Previously we reported that the “bent” conformation
of SMART analogues may be critical for maintaining antitubulin activity based on the results of
density functional theory (DFT).[8] Inspired by this, we speculated that the difference of the
preferred conformations of compounds 4-6 may be the reason that compound 6’s low activity. To
verify our speculation, DFT calculation was performed to compare the energy of two different
conformations of the above compounds. As the results shown in Table 1, Compounds 3, 4 and 5
may adopt stable “bent” conformation to interact with α,β-tubulin, which appears to be necessary

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for potent biological activity. On the other hand, compound 6 has an entirely different and stable
“straight” conformation and could not effectively interact with tubulin. Given the above analysis,
“bent” conformation of molecule may be the key factor for the design of antitubulin inhibitors.
The pharmacokinetic behaviour of carbonyl group on SMART analogues is unstable because
it can be easily reduced to hydroxyl in vivo, resulting in loss of bioactivities.[9] Ring tethering
strategy is an efficient means in drug design, which can be used for enhancing molecular stability
through protecting sensitive group and restricting bioactive conformation. For example, CA-4 was
often modified by the introduction of a five-membered aromatic heterocycle to lock the unstable
cis carbon-carbon double bond.[10-14]
1H-pyrazolo[3,4-b]pyridine is a special and significant fused aromatic heterocyclic ring,
which has been used as a key pharmacophore in drug design. For example, BAY 41-2272 is a
stimulator of soluble guanylate cyclase (sGC), which contains the 1H-pyrazolo[3,4-b]pyridine
fragment;[15] 6-Aryl pyrazolo[3,4-b]pyridine was also reported as inhibitor of glycogen synthase
kinase-3 (GSK-3) in 2003.[16]
Based on the above findings, a stable fragment 1H-pyrazolo[3,4-b]pyridine was chosen to
replace the carbonyl linkage and B-ring through rational ring tethering strategy to restrict
bioactive conformation and stabilize carbonyl group. Herein,
a
series of
3,5-diaryl-1H-pyrazolo[3,4-b]pyridines (12a-m, 13a-h, and 13l-m) were designed and synthesized
as antitubulin agents (Fig. 3). The preliminary tests of bioactivity in vitro, including
antiproliferative activity, tubulin polymerization, immunofluorescence staining, cell cycle analysis,
and cytotoxicity test were performed to explore the preliminarily structure-activity relationship
and illuminate the pharmacologic mechanism. Additionally, molecular modeling was carried out
to investigate the possible binding mode of target compounds.

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Fig. 1. Structures of colchicine, CA-4, SMART and analogues 4-6.
Fig. 2. Two conformations of compounds 3-6 (the blue represent six-membered aromatic ring).
Table 1. The relationships between conformation and bioactivity.
Compound
Ebent (a.u.)a
Estraight (a.u.)
Ebent-Estraight (kJ/mol)b
IC50 (nM)c
SMART, 3
-1488.05297426
-1151.24384014
-1167.28482948
-1183.31988660
-1488.04806398
-1151.24359019
-1167.27773895
-1183.32631705
-12.89
-0.66
21-71 (6 cell lines)
52-500 (6 cell lines)
25-39 (6 cell lines)
>3000 (6 cell lines)
4
5
6
-18.62
+16.88
a 1 a.u. = 2625.50 kJ/mol; b Negtive value represents “bent” conformation is preferred conformation; c cited from
literature [4].
Fig. 3. Structure-based rational design via ring tethering strategy.
2. Results and discussion
2.1. Chemistry
The synthetic route for the target compounds (12a-m, 13a-h, and 13l-m) is outlined in Scheme 1.
The key intermediate 3-(substituted phenyl)-1H-pyrazol-5-amine (compound 10) was prepared in
two steps. Briefly, the oxidative condensation of substituted benzaldehyde was carried out with
acetonitrile to produce 3-oxo-3-(substituted phenyl)propanenitrile (compound 9),[17] followed by
cyclization of compound 9 with 80% hydrazine hydrate in absolute ethanol.[18] Subsequently, the

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target compounds 12a-m were obtained by treating compound 10 with corresponding
indole-3-carboxaldehyde derivatives using direct one-step synthetic method.[19] Finally, the
amino-compounds 12a-h were converted to the target compounds 13a-h and 13l-m via reductive
deamination.[20]
Scheme 1. Synthesis of the target compounds. Reagents and condition: (a) CuCl2, KOH, CH3CN, DMA, O2
(balloon), rt, 24 h; (b) N2H4 H2O (80%), TsOH, EtOH, reflux, 2 h; (c) AlCl3, MeOH, 80oC, 4-8 h; (d) Isoamyl
.
nitrite, THF, rt, 2-6 h.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activities and cytotoxicity assay
All the newly synthesized 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines were screened against
SGC-7901 (human gastric carcinoma cell line), A549 (human lung carcinoma cell line) and HeLa
(Human cervical carcinoma cell line) by standard MTT assay to evaluate their antiproliferative
activities in terms of half maximal inhibitory concentration (IC50) values. By comparison, SMART
was selected as the positive reference (Table 2). Obviously, the strategy of introducing
fused-heterocyclic moiety is effective and almost all of the tested compounds exhibited moderate
to potent antiproliferative activity except for compounds 12l-m and 13l-m (IC50 > 30 µM), which
have no 3,4,5-trimethoxyphenyl group. It can be seen that 3,4,5-trimethoxyphenyl group is a
necessary pharmacophore for retaining bioactivities, which is consistent with previous reports
about CA-4, SMART and its analogues.[3] Compounds 12a-h containing amino group at the
C2-position of C-ring showed the IC50 values of 0.081 to 10.25 µM. The antiproliferative activity

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was reduced when the amino group of compounds 12d-f was methylated. A clear growth of IC50
value was observed when the amino group of compounds 12a, 12b and 12d was removed, and
compound 13d exhibited most potent antiproliferative activity against SGC-7901 cell with IC50
value of 13 nM, which was superior to SMART. The above analysis suggested that bulky
substituents on C2-position of C-ring may be adverse. Compounds 12e-f and 13e-f possessing
fluorine or chlorine atom at C4-position of C-ring were observed to be more active against HeLa
and SGC-7901 cell lines in comparison with compounds 12g and 13g, which containing bromine
atom at C4-position of C-ring. One possible reason for this phenomenon was that the bromine
atom has lower electronegativity and bigger atom size. Meanwhile, one of most distinct
characteristics of these compounds was that they exhibited selective antiproliferative activities on
different tumor cell lines. For example, compounds 12b, 12d-f, 13b and 13h showed strong and
selective inhibition to the proliferation of Hela and SGC-7901 cell lines (IC50 value 0.013-0.62
µM) while A549 cell line was not sensitive to these compounds (IC50 > 1.31 µM).
Table 2. Antiproliferative activities of target compounds.
(IC50 ± SD, µM)a
Compounds
SGC-7901
4.37 ± 0.18
0.62 ± 0.05
9.51 ± 0.87
0.23 ± 0.021
0.40 ± 0.017
0.58 ± 0.024
5.52 ± 0.16
3.73 ± 0.14
6.31 ± 0.39
3.76 ± 0.21
8.48 ± 0.76
>30
A549
HeLa
2.55 ± 0.11
0.38 ± 0.02
4.93 ± 0.77
0.16 ± 0.011
0.081 ± 0.008
0.36 ± 0.017
2.61 ± 0.12
1.44 ± 0.49
3.84 ± 0.15
1.67 ± 0.37
6.49 ± 0.70
>30
1.42 ± 0.09
4.43 ± 0.24
10.25 ± 1.02
7.83 ± 0.45
1.31 ± 0.10
3.24 ± 0.18
8.47 ± 0.23
5.36 ± 0.24
9.25 ± 0.44
4.27 ± 0.20
14.02 ± 1.03
>30
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
>30
>30
>30
12m

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0.037 ± 0.004
0.051 ± 0.006
1.91 ± 0.21
0.65 ± 0.043
>30
0.057 ±0.007
0.086 ± 0.012
0.46 ± 0.025
0.045 ± 0.008
0.89 ± 0.12
13a
13b
13c
13d
13e
13f
7.66 ± 0.48
0.082 ± 0.016
>30
0.013 ± 0.005
2.31 ± 0.17
4.82 ± 0.96
14.61 ± 1.24
17.43 ± 1.10
0.87 ± 0.15
20.15 ± 1.78
10.86 ± 1.97
13g
13h
13l
>30
>30
0.13 ± 0.010
0.28 ± 0.016
>30
>30
>30
>30
>30
13m
SMART (3 b
)
0.018 ± 0.006
0.029 ± 0.009
0.024 ± 0.011
a Data from three independent experiments; b Used as positive control.
Next, cytotoxicity test was carried out by using normal fibroblasts L929 cell line to assess the
cytotoxicity of the most potent compound 13d against non-tumor cells, and SMART was utilized
as positive control. As shown in Table 3, the data demonstrated that the cytotoxicity of compound
13d (IC50 value 37.5 µM) was obviously weaker than SMART (IC50 value 25.8 µM).
Table 3. Cytotoxicity test of compound 13d and SMART against L929 cells.
Compound
(IC50 ± SD, µM)
37.5 ± 2.4
13d
SMART
25.8 ± 2.0
2.2.2. Tubulin polymerization in vitro
In order to shed light on the biological mechanism of this series of compounds, the most potent
compound 13d was selected to examine the antitubulin activity, meanwhile SMART was used as
positive control and taxol, the first compound known to interact with and stabilize tubulin , was
utilized as negative control. As shown in Fig. 4, 13d significantly exhibited antitubulin activity
which was very close to SMART. Additionally, compound 13d inhibited tubulin polymerization
in a dose-dependent manner. These data indicated that this series of compounds most likely
targeted at tubulin.

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Fig. 4. Effects of compound 13d (12.5-100 µM), paclitaxel (5 µM), and SMART (10 µM) on tubulin
polymerization in vitro. The assay was performed using a fluorescence-based tubulin polymerization assay kit
(Cytoskeleton-Cat. #BK011P). Absorbance was monitored at 37oC every minute for 80 min.
2.2.3. Immunofluorescence staining of tubulin
To further investigate the impacts of 13d on intracellular microtubules, SGC-7901 cell was
utilized for immunofluorescence assay to directly observe the changes in morphology of
microtubules and SMART was utilized as reference. As shown in Fig. 5, the microtubule network
(green) in normal SGC-7901 cells was well-assembled and evenly distributed in cytoplasm.
Treatment of SGC-7901 cells with 36 nM (2-fold IC50) of SMART and 26 nM (2-fold IC50) of
compound 13d for 24 h, respectively, led to profound changes of the microscopic shape and
structure. The changes were outstandingly shown in two interrelated aspects: one is that the
cytoskeleton was destroyed and cell morphology became round; another one is the presence of
multinucleated cells. This phenomenon means that compound 13d may be a colchicine site binder,
similar to SMART.

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Fig. 5. Effects of SMART (36 nM) and 13d (26 nM) on the morphology of SGC-7901 cells by
immunofluorescence assay. Microtubule and unassembled tubulin were stained green; nuclei were stained blue.
2.2.4. Cell cycle arrest
It was known that tubulin polymerization inhibitors could trigger the alteration of cell cycle
parameters resulting in a preferential G2/M blockade. Therefore, cell cycle analysis assay was
performed to investigate the effects of the most potent compound 13d on the cell cycle (Fig. 6).
SGC-7901 cells were incubated with SMART (36 nM, Fig. 6A) or 13d (26 nM, Fig. 6B), and the
proportion of tested cells at different cell cycle phases was analyzed by flow cytometry after 0, 12,
24, 36, 48, and 72 h of treatment, respectively. As the results depicted in Fig. 6A, SMART
induced a sharply decrease of G1 cell population, and an increase of G2/M cell population.
Meanwhile, massive multinucleated cells were observed, which was consistent with the
immunofluorescence assay. As shown in Fig. 6B, the G2/M cell population increased significantly
from 24.47% to 59.62% after treatment with 26 nM of 13d for 12 h. Thus, the cell cycle
distribution indicated that compound 13d could arrest SGC-7901 cell in G2/M phase.

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Fig. 6. The effects of SMART (6A) and 13d (6B) on the cell cycle distributions.
2.3. Molecular modeling
For the aim to research the potential binding mode of the target compounds, the most potent
compound 13d was docked into the tubulin-CA-4 complex (PDB code: 5LYJ) by following the
procedure of our previous report.[8] The docking-derived superimposition of 13d (green), 4
(purple), and 5 (yellow) was illustrated in the Fig. 7A. 13d showed a binding pose similar to
compounds 4 and 5, and the molecular skeletons of these three could be well superimposed. As
the 2D diagram of 13d-tubulin interactions shown in Fig. 7B, the oxygen atom of methoxyl group
at C4-position of A-ring which located in β-tubulin deeply could establish a hydrogen bond with
β-Cys241 (2.17
Å). Meanwhile, the methyl group at C4-position of C-ring which extended toward

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α-tubulin could form alkyl hydrophobic interaction with α-Val181 (3.57
Å). The docking results
suggested that 13d may exhibit its biological activities by binding to colchicine site.
Fig. 7. (A) Possible binding modes of compound 4, 5 and 13d in colchicine site (purple-4; yellow-5; green-13d);
(B) Compound 13d-tubulin interactions (green dotted line represents hydrogen bond, purple dotted line represents
alkyl hydrophobic interaction). All of the nonpolar hydrogen atoms were removed for the purpose of clarity.
3. Conclusion
A new series of 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines was designed rationally through the
strategy of ring tethering which was supported by the conformational analysis. All of the designed
compounds 12a-12m, 13a-13h, and 13l-13m were synthesized and characterized. MTT assay was
used to evaluate the antiproliferative activity preliminarily against three human tumor cells
(SGC-7901, A549, and HeLa) and most of the target compounds showed potent inhibitory activity
at sub-micromolar to nanomolar level. The most potent compound 13d was selected to illuminate
the biological mechanism through tubulin polymerization assay, immunofluorescence staining,
and cell cycle analysis. Results indicated that 13d could inhibit tubulin assembly effectively in
vitro, destroy the skeleton of intracellular microtubule significantly, and induce cell cycle arrest in
G2/M phase. Furthermore, molecular modeling studies showed that 13d could bind to colchicine
site strongly. More importantly, this successful instance of molecular design based on
conformation revealed that “bent” conformation is a critical factor in the rational design of
SMART analogues. In addition, we believe that some chain or ring-like polar fragments which are
beneficial to maintain the “bent” conformation of SMART analogues can probably be introduced
on B-ring to adjust the physicochemical properties so as to meet the requirements of druggability.
It will be our main objective which should be researched continuously and the correlational study
will be duly published.

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4. Experimental section
4.1. Chemistry
All of reagents and solvents were purchased from chemical company. TLC analysis was used
for determining the extent of reactions under UV light (wavelength: 365 nm and 254 nm). 1H (600
MHz) and 13C NMR (150 MHz) spectra were tested on a Bruker AVANCE 600 by using
DMSO-d6 or CDCl3 as solvent (0.03% TMS as internal reference) at room temperature. Melting
point was measured (uncorrected) on hot-stage microscope (Beijing Taike, X-4). Mass spectra
were performed on an Agilent 1100-sl mass spectrometer with electrospray ionization (ESI)
source from Agilent Co.Ltd. High resolution mass determinations (HRMS) for target compounds
were obtained on an Agilent 6530 accurate-mass Q-TOF LC-MS system.
4.1.1. Synthesis of intermediates 9a-c
The mixture of 8 (10 mmol), CH3CN (100 mmol), CuCl2 (0.3 mmol) and KOH (30 mmol) in
N,N-dimethylacetamide (DMA) was stirred vigorously under O2 atmosphere at 25oC for 24 h.
When the reaction was completed, the volatile solvent was removed under reduced pressure and
the unsolvable impurities were excluded by filter. 200 mL water was added into the filtrate, then
the pH was adjusted to 7 by slowly adding 20% hydrochloric acid and massive amounts of solid
precipitated out. Finally, the solid was collected by filtration and washed three times with water to
furnish intermediates 9a-c as white solid with yield ranged from 72% to 76%.
4.1.1.1. 3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (9a)
1
White solid, yield 75%, H NMR (600 MHz, CDCl3): δ 3.93 (6H, s), 3.95 (3H, s), 4.08 (2H, s),
7.15 (2H, s). MS (ESI): [M+H]+ = 236.1.
4.1.1.2. 3-oxo-3-(3,4-dimethoxyphenyl)propanenitrile (9b)
1
White solid, yield 76%, H NMR (600 MHz, CDCl3): δ 3.95 (s, 3H), 3.98 (s, 3H), 4.04 (s, 2H),
6.92 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.51 (s, 1H). MS (ESI): [M+H]+ = 206.0.
4.1.1.3. 3-oxo-3-(4-methoxyphenyl)propanenitrile (9c)
White solid, yield 72%, 1H NMR (600 MHz, CDCl3): δ 3.90 (s, 3H), 4.03 (s, 2H), 6.98 (d, J = 8.9
Hz, 2H), 7.90 (d, J = 8.9 Hz, 2H). MS (ESI): [M+H]+ = 176.1
4.1.2. Synthesis of key intermediates 10a-c
To a solution of 9 (5 mmol) and p-toluenesulfonic acid (TsOH, 0.1 mmol) in ethanol was
added 80% hydrazine hydrate dropwise at 78oC. On completion of the reaction (TLC check), the

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solvent was removed under reduced pressure and the crude product was purified by silica gel
column chromatography to afford intermediates 10a-c as white solid in excellent yields.
4.1.2.1. 3-(3,4,5-Trimethoxyphenyl)-1H-pyrazol-5-amine (10a)
1
White solid, yield 93%, H NMR (600 MHz, DMSO-d6): δ 3.66 (s, 3H), 3.81 (s, 6H), 4.75 (brs,
2H), 5.78 (s, 1H), 6.95 (s, 2H), 11.70 (brs, 1H). MS (ESI): [M+H]+ = 250.1, [M+Na]+ = 272.1.
4.1.2.2. 3-(3,4-Dimethoxyphenyl)-1H-pyrazol-5-amine (10b)
1
White solid, yield 93%, H NMR (600 MHz, DMSO-d6): δ 3.76 (s, 3H), 3.79 (s, 3H), 4.71 (brs,
2H), 5.71 (s, 1H), 6.94 (d, J = 8.3 Hz, 1H), 7.16 (dd, J = 8.3, 1.9 Hz, 1H), 7.24 (d, J = 1.9 Hz, 1H),
11.63 (brs, 1H). MS (ESI): [M+H]+ = 220.1.
4.1.2.3. 3-(3-Methoxyphenyl)-1H-pyrazol-5-amine (10c)
1
White solid, yield 95%, H NMR (600 MHz, DMSO-d6): δ 3.76 (s, 3H), 4.70 (brs, 2H), 5.68 (s,
1H), 6.94 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 11.68 (brs, 1H). MS (ESI): [M+H]+ =
190.1.
4.1.3. Synthesis of target compounds 12a-m
To a solution of 10 (0.5 mmol) and 1H-indole-3-carbaldehyde derivatives 11 (0.5 mmol) in 2
mL anhydrous methanol, AlCl3 (0.05 mmol) was added then the mixture was stirred for 4-8 h in
15 mL heavy-wall pressure vessel at 70oC. When the completion of reaction (TLC check), the
mixture was naturally cooled to room temperature, and the resulting solid was filtered. The crude
product was purified by silica gel column chromatography or recrystallization in ethyl acetate to
afford target compounds 12a-m as white solid with yield ranged from 56% to 75%.
4.1.3.1. 2-(3-(3,4,5-Trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12a)
1
White solid, yield 78%, mp 214-216oC, H NMR (600 MHz, DMSO-d6): δ 3.72 (s, 3H), 3.88 (s,
6H), 5.04 (s, 2H), 6.66-6.69 (m, 1H), 6.79 (d, J = 8.1 Hz, 1H), 7.07-7.10 (m, 1H), 7.12 (d, J = 7.4
Hz, 1H), 7.25 (s, 2H), 8.48 (s, 1H), 8.56 (s, 1H), 13.81 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ
56.5 (2C), 60.6, 104.6 (2C), 112.5, 115.9, 117.3, 123.2, 129.0, 129.3, 129.4, 130.0, 131.3, 138.1,
143.4, 146.5, 150.3, 152.4, 153.8 (2C). HRMS (ESI): calcd. for C21H20N4NaO3 [M+Na]+ 399.1428,
found 399.1432.
4.1.3.2. 4-Methyl-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12b)
1
White solid, yield 64%, mp 192-193oC, H NMR (600 MHz, DMSO-d6): δ 2.21 (s, 3H), 3.73 (s,
3H), 3.88 (s, 6H), 4.95 (s, 2H), 6.73 (d, J = 8.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.96 (s, 1H),

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7.24 (s, 2H), 8.48 (s, 1H), 8.56 (s, 1H), 13.81 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 20.5,
56.5 (2C), 60.6, 104.7 (2C), 112.4, 116.3, 123.4, 126.0, 129.3, 129.4, 129.5, 129.9, 131.7, 138.1,
143.4, 143.6, 150.3, 152.4, 153.8 (2C). HRMS (ESI): calcd. for C22H22N4NaO3 [M+Na]+ 413.1584,
found 413.1589.
4.1.3.3. 4-Methoxy-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12c)
1
White solid, yield 75%, mp 208-210oC, H NMR (600 MHz, DMSO-d6): δ 3.69 (s, 3H), 3.73 (s,
3H), 3.89 (s, 6H), 4.63 (s, 2H), 6.74-6.79 (m, 3H), 7.25 (s, 2H), 8.53 (d, J = 1.4 Hz, 1H), 8.62 (d, J
= 1.4 Hz, 1H), 13.82 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 55.8, 56.5 (2C), 60.6, 104.6 (2C),
112.4, 115.2, 116.3, 117.3, 124.2, 129.3, 129.4, 130.1, 138.1, 140.1, 143.5, 150.3, 151.9, 152.4,
153.8 (2C). HRMS (ESI): calcd. for C22H23N4O4 [M+H]+ 407.1714, found 407.1717;
C22H22N4NaO4 [M+Na]+ 429.1533, found 429.1537.
4.1.3.4. 5-Methyl-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12d)
1
White solid, yield 63%, mp 180-182oC, H NMR (600 MHz, DMSO-d6): δ 2.22 (s, 3H), 3.73 (s,
3H), 3.88 (s, 6H), 4.96 (s, 2H), 6.50 (d, J = 7.5 Hz, 1H), 6.62 (s, 1H), 7.01 (d, J = 7.5 Hz, 1H),
7.24 (s, 2H), 8.44 (d, J = 1.6 Hz, 1H), 8.54 (d, J = 1.6 Hz, 1H), 13.79 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 21.4, 56.4 (2C), 60.6, 104.6 (2C), 112.5, 116.4, 118.3, 120.6, 129.3, 129.4, 129.9,
131.2, 138.0, 138.1, 143.3, 146.2, 150.3, 152.4, 153.8 (2C). HRMS (ESI): calcd. for
C22H22N4NaO3 [M+Na]+ 413.1584, found 413.1588.
4.1.3.5. 5-Fluoro-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12e)
1
White solid, yield 56%, mp 223-224oC, H NMR (600 MHz, DMSO-d6): δ 3.73 (s, 3H), 3.88 (s,
6H), 5.38 (s, 2H), 6.43-6.46 (m, 1H), 6.57 (d, J = 11.1 Hz, 1H), 7.12-7.14 (m, 1H), 7.25 (s, 2H),
8.45 (s, 1H), 8.52 (s, 1H), 13.82 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C), 60.6,
101.5 (d, J = 23.9 Hz), 103.4 (d, J = 21.6 Hz), 104.6 (2C), 112.5, 119.6, 128.4, 129.3, 130.3, 132.8
(d, J = 10.3 Hz), 138.1, 143.4, 148.5 (d, J = 11.7 Hz), 150.3, 152.5, 153.8 (2C), 164.1 (d, J =
238.8 Hz). HRMS (ESI): calcd. for C21H20FN4O3 [M+H]+ 395.1514, found 395.1525;
C21H19FN4NaO3 [M+Na]+ 417.1333, found 417.1351.
4.1.3.6. 5-Chloro-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12f)
1
White solid, yield 69%, mp 227-229oC, H NMR (600 MHz, DMSO-d6): δ 3.73 (s, 3H), 3.88 (s,
6H), 5.39 (s, 2H), 6.66 (d, J = 7.7 Hz, 1H), 6.84 (s, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.24 (s, 2H),
8.47 (s, 1H), 8.53 (s, 1H), 13.84 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C), 60.6,

ACCEPTED MANUSCRIPT
104.6 (2C), 112.5, 114.7, 116.5, 122.0, 128.2, 129.2, 130.2, 132.8, 133.4, 138.1, 143.5, 148.2,
150.1, 152.5, 153.8 (2C). HRMS (ESI): calcd. for C21H20ClN4O3 [M+H]+ 411.1218, found
411.1217; C21H19ClN4NaO3 [M+Na]+ 433.1038, found 433.1056.
4.1.3.7. 5-Bromo-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12g)
1
White solid, yield 61%, mp 132-134oC, H NMR (600 MHz, DMSO-d6): δ 3.72 (s, 3H), 3.88 (s,
6H), 5.37 (s, 2H), 6.79 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 8.0
Hz, 1H), 7.24 (s, 2H), 8.46 (d, J = 1.9 Hz, 1H), 8.52 (d, J = 1.9 Hz, 1H), 13.83 (s, 1H); 13C NMR
(150 MHz, DMSO-d6): δ 56.5 (2C), 60.6, 104.6 (2C), 112.4, 117.6, 119.3, 122.0, 128.2, 129.2,
129.8, 130.2, 133.1, 138.1, 143.5, 148.4, 150.1, 152.5, 153.8 (2C). HRMS (ESI): calcd. for
C21H20BrN4O3 [M+H]+ 455.0713, found 455.0723.
4.1.3.8. 5-Methoxy-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12h)
1
White solid, yield 65%, mp 202-204oC, H NMR (600 MHz, DMSO-d6): δ 3.72 (s, 3H), 3.73 (s,
3H), 3.88 (s, 6H), 5.08 (s, 2H), 6.28 (dd, J = 8.6 Hz, J = 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz, 1H),
7.04 (d, J = 8.6 Hz, 1H), 7.24 (s, 2H), 8.42 (d, J = 1.9 Hz , 1H), 8.53 (d, J = 1.9 Hz, 1H), 13.77 (s,
1H); 13C NMR (150 MHz, DMSO-d6): δ 55.2, 56.4 (2C), 60.6, 100.9, 103.4, 104.6 (2C), 112.5,
116.4, 129.2, 129.4, 129.8, 132.1, 138.1, 143.3, 147.6, 150.4, 152.3, 153.8 (2C), 160.4. HRMS
(ESI): calcd. for C22H22N4NaO4 [M+Na]+ 429.1533, found 429.1557.
4.1.3.9. N,5-dimethyl-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)aniline (12i)
White solid, yield 75%, mp 232-234oC, 1H NMR (600 MHz, DMSO-d6): δ 2.30 (s, 3H), 2.65 (d, J
= 5.0 Hz, 3H), 3.72 (s, 3H), 3.88 (s, 6H), 5.18 (q, J = 5.0 Hz, 2H), 6.47 (s, 1H), 6.52 (d, J = 7.4 Hz,
1H), 7.00 (d, J = 7.4 Hz, 1H), 7.23 (s, 2H), 8.40 (d, J = 1.9 Hz, 1H), 8.48 (d, J = 1.9 Hz, 1H),
13.80 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 21.9, 30.6, 56.4 (2C), 60.6, 104.6 (2C), 111.0,
112.5, 117.3, 121.4, 129.0, 129.3, 130.1, 130.4, 131.0, 138.1, 143.4, 147.4, 150.6, 152.5, 153.8
(2C). HRMS (ESI): calcd. for C23H25N4O3 [M+H]+ 405.1921, found 405.1929; C23H24N4NaO3
[M+Na]+ 427.1741, found 427.1748.
4.1.3.10.
5-Fluoro-N-methyl-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)
aniline (12j)
1
White solid, yield 66%, mp 260-262oC, H NMR (600 MHz, DMSO-d6): δ 2.65 (d, J = 4.8 Hz,
3H), 3.72 (s, 3H), 3.88 (s, 6H), 5.55 (q, J = 4.8 Hz, 1H), 6.40-6.42 (m, 1H), 6.45-6.48 (m, 1H),
7.10-7.13 (m, 1H), 7.24 (s, 2H), 8.41 (d, J = 1.7 Hz, 1H), 8.47 (d, J = 1.7 Hz, 1H), 13.83 (s, 1H);

ACCEPTED MANUSCRIPT
13C NMR (150 MHz, DMSO-d6): δ 30.5, 56.4 (2C), 60.6, 97.0 (d, J = 26.0 Hz), 102.1 (d, J = 21.6
Hz), 104.7 (2C), 112.6, 120.3 (d, J = 1.7 Hz), 128.1, 129.2, 130.8, 132.4 (d, J = 10.4 Hz), 138.1,
143.5, 149.6 (d, J = 11.4 Hz), 150.6, 152.6, 153.8 (2C), 164.0 (d, J = 241.3 Hz). HRMS (ESI):
calcd. for C22H21FN4NaO3 [M+Na]+ 431.1490, found 431.1502.
4.1.3.11.
5-Chloro-N-methyl-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)
aniline (12k)
1
Pale yellow solid, yield 72%, mp 269-271oC, H NMR (600 MHz, DMSO-d6): δ 2.65 (d, J = 4.7
Hz, 3H), 3.72 (s, 3H), 3.88 (s, 6H), 5.56 (q, J = 4.7 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.70 (dd, J =
7.9 Hz, J = 1.8 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 7.23 (s, 2H), 8.42 (d, J = 1.7 Hz, 1H), 8.48 (d, J
= 1.7 Hz, 1H), 13.84 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 30.4, 56.4 (2C), 60.6, 104.7 (2C),
109.6, 112.5, 115.7, 122.9, 127.8, 129.2, 130.8, 132.5, 134.3, 138.1, 143.6, 149.0, 150.4, 152.7,
153.8 (2C). HRMS (ESI): calcd. for C22H22ClN4O3 [M+H]+ 425.1375, found 425.1384.
4.1.3.12. 2-(3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)-5-methylaniline (12l)
1
White solid, yield 68%, mp 239-241oC, H NMR (600 MHz, DMSO-d6): δ 2.22 (s, 3H), 3.82 (s,
3H), 3.86 (s, 3H), 4.91 (s, 2H), 6.50 (dd, J = 7.5 Hz, J = 0.9 Hz, 1H), 6.62 (s, 1H), 6.99 (d, J = 7.5
Hz, 1H), 7.09 (d, J = 8.0 Hz, 2H), 7.56-7.57 (m, 2H), 8.42 (d, J = 1.9 Hz , 1H), 8.52 (d, J = 1.9 Hz,
1H), 13.70 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 21.4, 55.9, 56.0, 110.3, 112.4, 112.5, 116.3,
118.3, 119.8, 120.7, 126.5, 129.3, 130.0, 131.2, 138.1, 143.2, 146.2, 149.4, 149.5, 150.2, 152.4.
HRMS (ESI): calcd. for C21H20N4NaO2 [M+Na]+ 383.1478, found 383.1498.
4.1.3.13. 2-(3-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)-5-methylaniline (12m)
1
White solid, yield 65%, mp 236-238oC, H NMR (600 MHz, DMSO-d6): δ 2.23 (s, 3H), 3.82 (s,
3H), 4.89 (s, 2H), 6.50 (dd, J = 7.6 Hz, J = 0.7 Hz, 1H), 6.62 (s, 1H), 6.99 (d, J = 7.6 Hz, 1H),
7.08 (d, J = 8.7 Hz, 2H), 7.97 (d, J = 8.7 Hz, 2H), 8.40 (d, J = 1.8 Hz , 1H), 8.52 (d, J = 1.8 Hz,
1H), 13.69 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 21.4, 55.6, 112.4, 114.9 (2C), 116.2, 118.2,
120.8, 126.4, 128.4 (2C), 129.3, 130.0, 131.2, 138.1, 143.1, 146.2, 150.1, 152.4, 159.7. HRMS
(ESI): calcd. for C20H18N4NaO [M+Na]+ 353.1373, found 353.1397.
4.1.4. Synthesis of target compounds 13a-h
The mixture of 12 (0.2 mmol) and isoamyl nitrite (0.3 mmol) in THF was stirred for 2-6 h at
room temperature. The solvent was removed under reduced pressure and the residue was purified

ACCEPTED MANUSCRIPT
by silica gel column chromatography to afford target compounds 13a-h as white solid with yield
ranged from 64% to 83%.
4.1.4.1. 5-Phenyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13a)
1
White solid, yield 72%, mp 172-174oC, H NMR (600 MHz, DMSO-d6): δ 3.79 (s, 3H), 3.97 (s,
6H), 7.32 (s, 2H), 7.46-7.48 (m, 1H), 7.56-7.59 (m, 2H), 7.89 (d, J = 7.4 Hz, 2H), 8.70 (d, J = 1.8
Hz ,1H), 8.93 (d, J = 1.8 Hz 1H), 13.93 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C),
60.6, 104.7 (2C), 112.6, 127.8 (2C), 127.9, 128.4, 129.2, 129.6 (2C), 130.3, 138.2, 138.5, 143.7,
148.8, 152.9, 153.8 (2C). HRMS (ESI): calcd. for C21H19N3NaO3 [M+Na]+ 384.1319, found
384.1325.
4.1.4.2. 5-(m-Tolyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13b)
1
White solid, yield 67%, mp 158-160oC, H NMR (600 MHz, DMSO-d6): δ 2.41 (s, 3H), 3.74 (s,
3H), 3.91 (s, 6H), 7.23 (d, J = 7.4 Hz, 1H), 7.26 (s, 2H), 7.39-7.42 (m, 1H), 7.61 (d, J = 7.8 Hz,
1H), 7.65 (s, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.86 (d, J = 2.0 Hz, 1H), 13.87 (s, 1H); 13C NMR (150
MHz, DMSO-d6): δ 21.6, 56.5 (2C), 60.6, 104.8 (2C), 112.6, 124.9, 128.2, 128.5, 128.6, 129.2,
129.4, 130.4, 138.2, 138.4, 138.8, 143.7, 148.9, 152.8, 153.8 (2C). HRMS (ESI): calcd. for
C22H21N3NaO3 [M+Na]+ 398.1475, found 398.1481.
4.1.4.3. 5-(3-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13c)
1
White solid, yield 75%, mp 173-175oC, H NMR (600 MHz, DMSO-d6): δ 3.74 (s, 3H), 3.85 (s,
3H), 3.92 (s, 6H), 6.97-6.99 (m, 1H), 7.28 (s, 2H), 7.39-7.44 (m, 3H), 8.66 (d, J = 1.7 Hz, 1H),
8.89 (d, J = 1.7 Hz, 1H), 13.87 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 55.7, 56.5 (2C), 60.6,
104.7 (2C), 112.5, 113.2, 113.8, 120.1, 128.6, 129.2, 130.2, 130.6, 138.2, 140.0, 143.8, 148.9,
152.9, 153.8 (2C), 160.3. HRMS (ESI): calcd. for C22H22N3O4 [M+H]+ 392.1605, found 392.1624.
4.1.4.4. 5-(p-Tolyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13d)
1
White solid, yield 83%, mp 183-185oC, H NMR (600 MHz, DMSO-d6): δ 2.37 (s, 3H), 3.73 (s,
3H), 3.91 (s, 6H), 7.26 (s, 2H), 7.32 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 8.60 (d, J = 2.0
Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H), 13.84 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 22.6, 56.5
(2C), 60.6, 104.7 (2C), 112.6, 127.7 (2C), 128.0, 129.2, 130.2 (2C), 130.3, 135.6, 137.3, 138.2,
143.6, 148.7, 152.8, 153.8 (2C). HRMS (ESI): calcd. for C22H22N3O3 [M+H]+ 376.1656, found
376.1655; C22H21N3NaO3 [M+Na]+ 398.1475, found 398.1476.
4.1.4.5. 5-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13e)

ACCEPTED MANUSCRIPT
1
White solid, yield 75%, mp 218-219oC, H NMR (600 MHz, DMSO-d6): δ 3.74 (s, 3H), 3.91 (s,
6H), 7.27 (s, 2H), 7.34-7.37 (m, 2H), 7.88-7.90 (m, 2H), 8.64 (d, J = 1.5 Hz, 1H), 8.86 (d, J = 1.5
Hz, 1H), 13.87 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C), 60.6, 104.8 (2C), 112.5,
116.3 (2C, d, J = 21.3 Hz), 128.4, 129.3, 129.9 (2C, d, J = 8.0 Hz), 130.1, 135.0 (d, J = 2.6 Hz),
138.2, 143.8, 148.8, 152.8, 153.8 (2C), 163.2 (d, J = 245.6 Hz). HRMS (ESI): calcd. for
C21H19FN3O3 [M+H]+ 380.1405, found 380.1406; C21H18FN3NaO3 [M+Na]+ 402.1224, found
402.1229.
4.1.4.6. 5-(4-Chlorophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13f)
1
White solid, yield 64%, mp 220-221oC, H NMR (600 MHz, DMSO-d6): δ 3.74 (s, 3H), 3.91 (s,
6H), 7.27 (s, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.5 Hz, 2H), 8.67 (d, J = 2.0 Hz, 1H), 8.88
(d, J = 2.0 Hz, 1H), 13.90 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C), 60.6, 104.8 (2C),
112.5, 128.6, 129.0, 129.1, 129.4 (2C), 129.6 (2C), 132.9, 137.4, 138.2, 143.8, 148.7, 152.9, 153.8
(2C). HRMS (ESI): calcd. for C21H19ClN3O3 [M+H]+ 396.1109, found 396.1105;
C21H18ClN3NaO3 [M+Na]+ 418.0929, found 418.0926.
4.1.4.7. 5-(4-Bromophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13g)
1
White solid, yield 71%, mp 250-252oC, H NMR (600 MHz, DMSO-d6): δ 3.74 (s, 3H), 3.91 (s,
6H), 7.27 (s, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H), 8.67 (d, J = 2.0 Hz, 1H), 8.87
(d, J = 2.0 Hz, 1H), 13.90 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 56.5 (2C), 60.6, 104.8 (2C),
112.5, 121.5, 129.1, 130.0 (2C), 130.1, 132.4 (2C), 137.7, 138.2, 143.9, 148.7, 152.9, 153.8 (2C).
HRMS (ESI): calcd. for C21H18BrN3NaO3 [M+Na]+ 462.0424, found 462.0435.
4.1.4.8. 5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (13h)
1
White solid, yield 70%, mp 199-201oC, H NMR (600 MHz, DMSO-d6): δ 3.73 (s, 3H), 3.82 (s,
3H), 3.91 (s, 6H), 7.08 (d, J = 8.7 Hz, 2H), 7.26 (s, 2H), 7.77 (d, J = 8.7 Hz, 2H), 8.57 (d, J = 1.3
Hz, 1H), 8.84 (d, J = 1.3 Hz, 1H), 13.82 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 55.7, 56.5
(2C), 60.6, 104.6 (2C), 112.6, 115.0, 127.6, 129.0 (2C), 129.2, 130.1, 130.8, 138.1, 143.5, 148.6,
152.6, 153.8 (2C), 159.4. HRMS (ESI): calcd. for C22H22N3O4 [M+H]+ 392.1605, found 392.1625;
C22H21N3NaO4 [M+Na]+ 414.1424, found 414.1447.
4.1.4.9. 3-(3,4-dimethoxyphenyl)-5-(p-tolyl)-1H-pyrazolo[3,4-b]pyridine (13l)
1
White solid, yield 53%, mp 229-231oC, H NMR (600 MHz, DMSO-d6): δ 2.38 (s, 3H), 3.84 (s,
3H), 3.88 (s, 3H), 7.11 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.8 Hz, 2H), 7.57 (s, 1H), 7.66 (d, J = 7.6

ACCEPTED MANUSCRIPT
Hz, 1H), 7.73 (d, J = 7.8 Hz, 2H), 8.61 (s , 1H), 8.85 (s, 1H), 13.75 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 21.2, 56.0, 56.1, 110.3, 112.5, 120.0, 126.4, 127.6 (3C), 127.9, 130.1 (3C), 135.6,
137.2, 143.5, 148.6, 149.5, 149.6, 152.8. HRMS (ESI): calcd. for C21H20N3O2 [M+H]+ 346.1550,
found 346.1559.
4.1.4.10. 3-(4-methoxyphenyl)-5-(p-tolyl)-1H-pyrazolo[3,4-b]pyridine (13m)
1
White solid, yield 50%, mp 197-199oC, H NMR (600 MHz, DMSO-d6): δ 2.38 (s, 3H), 3.84 (s,
3H), 7.11 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.74 (d, J = 7.9 Hz, 2H), 8.05 (d, J = 8.8
Hz, 2H), 8.63 (d, J = 2.0 Hz, 1H), 8.84 (d, J = 2.0 Hz, 1H), 13.74 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 21.2, 55.7, 112.4, 114.9 (2C), 126.2, 127.6 (2C), 127.9, 128.5 (2C), 130.0, 130.1
(2C), 135.5, 137.2, 143.4, 148.5, 152.8, 159.7. HRMS (ESI): calcd. for C20H18N3O [M+H]+
316.1444, found 316.1458; C20H17N3NaO [M+Na]+ 338.1264, found 338.1278.
4.2. Biology experiments
4.2.1. Antiproliferation assay in vitro
The antiproliferative activities in vitro of SMART and all the target compounds (12a-m, 13a-h
and 13l-m) were tested by MTT assay followed the procedures as previous report.[21]
4.2.2. Tubulin polymerization in vitro
The tubulin polymerization assay was performed by using the commercial tubulin polymerization
assay kit (Cytoskeleton-Cat. #BK011P) referred to the protocol of manufacturer.
4.2.3. Immunofluorescence staining of tubulin
Immunofluorescence staining studies of SMART and compound 13d were investigated using the
reported method.[22]
4.2.4. Cell cycle arrest
Cell cycle analysis assay was followed the procedure of relevant report.[22]
4.3. Computational assay
4.3.1. DFT computation
Structural optimization of compounds 3-6 were performed at B3LYP/6-31G(d) level of Gaussian
09 software.[23] The results of optimization showed the energy of two conformations for any
molecule, and the unit a.u. of original energy value was converted into standard unit kJ·mol-1 (1
a.u. = 2625.50 kJ·mol-1). The results were listed in Table 1.
4.3.2. Molecular modeling

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The molecular modeling studies were performed by using Accelrys Discovery Studio 3.0. The
X-ray crystal structures of tubulin in complex with CA-4 (PDB: 5LYJ) was downloaded from the
RCSB Protein Data Bank (http://www.rcsb.org/structure/5LYJ). The ligand 4, 5, and 13d were
energy-minimised with the CHARMm force-field and docked into colchicine binding site using
CDOCKER protocol. The pictures in Figure 6 were edited with Discovery Studio 4.5 Visualizer.
Acknowledgements
We gratefully acknowledge the National Natural Science Foundation of China (81673293 and
81602969), Project funded by China Postdoctoral Science Foundation (215746), Plan for
Development of Young Scholars of Shenyang Pharmaceutical University, National Natural
Science Foundation of Liaoning (201602687), Doctoral Research Funding of Liaoning Province
(201601144), Education Fund Item of Liaoning Province (201610163L12), and Liaoning Province
Undergraduate Training Program for Innovation (201610163019, 201710163000202 and
201710163000094) for the generous financial support. This work was also supported by the
Scientific and Technological Project of Henan Province (172102310102), Program for Innovative
Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team
in University.
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Highlights
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Conformations of SMART analogues were analyzed based on DFT calculation.
Target compounds were design rationally via ring tethering strategy.
Most of the target compounds showed significant antiproliferative activities.
Compound 13d inhibited tubulin polymerization and caused cells arrest in G2/M phase.
The binding mode of 13d was determined by docking studies.