Synthesis and biological evaluation of 1-(benzofuran-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103392

The key functions of microtubules and the mitotic spindle in cell division make them attractive targets for cancer therapy. In this study, a series of 1-(benzofuran-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole derivatives was synthesized, and their

Full text of DOI:10.1016/j.bioorg.2019.103392

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of 1-(benzofuran-3-yl)-4-(3,4,5-
trimethoxyphenyl)-1H-1,2,3-triazole derivatives as tubulin polymerization
inhibitors
⁎
Zhi-Yuan Qia,1, Shu-Yi Haoa,1, Heng-Zhi Tiana, Hong-Li Biana, Ling Huib, Shi-Wu Chena,
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
a 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:
The key functions of microtubules and the mitotic spindle in cell division make them attractive targets for cancer
therapy. In this study, a series of 1-(benzofuran-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole derivatives
was synthesized, and their antiproliferative activities against HCT116, HeLa, HepG2, and A549 cells were
evaluated. 6-Methoxy-N-phenyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)benzofuran-2-carboxamide
(17g) exhibited the strongest antiproliferative activities, with IC50 values ranging from 0.57 to 5.7 μM.
Mechanistic studies showed that 17g inhibited tubulin polymerization, leading to the disruption of mitotic
spindle formation, cell cycle arrest in the G2/M phase, and apoptosis of A549 cells. A docking study indicated
that 17g was a good molecular fit at the colchicine binding site of tubulin. These results showed that 17g is a
potential anticancer compound that is worthy of further development as a tubulin polymerization inhibitor.
Microtubule
Tubulin polymerization
Benzo[b]furan
1,2,3-Triazole
Antitumor
1. Introduction
shortcomings, its water-soluble ester, CA-4P (3), was prepared and
entered clinical trials for the treatment of platinum-resistant ovarian
Microtubules are long, hollow tubular structures that predominantly
consist of α,β-tubulin dimers [1]. They are involved in many structural
functions, including cell division, signaling, motility, cell maintenance,
and intracellular vesicle transport, and have become attractive targets
for novel antimitotic drugs for cancer therapy [2]. The binding sites of
microtubule-targeting drugs include the colchicine, vinca, and taxane
sites. In general, compounds that bind to the colchicine and vinca sites
are tubulin polymerization inhibitors, whereas those that bind to the
taxane site are polymerization stabilizers [3,4]. Inhibitors that bind to
the colchicine site have drawn the most attention, because these com-
pounds have the potential to overcome ABC-transporter-mediated drug
resistance and vascular disrupting properties [5–7].
cancer. Unfortunately, that study was terminated in 2017 owing to a
lack of efficacy for improving progression-free survival (PFS), combined
with an unfavorable objective response rate (ORR) [11].
Structure–activity relationship studies of CA-4 derivatives have in-
dicated that the 3,4,5-trimethoxybenzene (A ring) is critical for bioac-
tivity [12–16] and that the 3-hydroxy-4-methoxybenzene (B ring)
might be replaced with various heterocycles [17,18]. Furthermore,
there is evidence to suggest that maintaining a cis-configuration be-
tween the A and B rings is important for optimal bioactivity [17,18].
Flynn and coworkers reported that BNC105 (4), which contains a benzo
[b]furan as the B ring, exhibited excellent antiproliferative activity
against MCF-7 cells (IC50 = 0.002 μM) and tubulin polymerization in-
hibitory activity (IC50 = 3.0 μM) [19]. Furthermore, its water-soluble
phosphate, BNC105P (5), exhibited potent effects on mesothelioma,
and ovarian and renal cancers, in clinical trials [20]. Odlo et al. also
reported that the vinyl group in CA-4 can be substituted with 1,2,3-
triazolyl to maintain the cis-configuration of the A and B rings. The
resulting compound (6) exhibited strong antiproliferative activity
against K562 cells, with an IC50 value of 3.20 μM [21].
Colchicine (1, Fig. 1) was the first drug known to function as a tu-
bulin polymerization inhibitor [8]. However, the therapeutic value of
its anticancer activity is restricted by its toxicity and low therapeutic
index [9]. Combretastatin A-4 (CA-4, 2), another inhibitor that binds at
the colchicine site, was first isolated from the bark of Combretum caf-
frum, and displays potent cytotoxicity against a wide range of cancer
cell lines. However, its poor water solubility and bioavailability hin-
dered its development into an antitumor drug [10]. To overcome these
Triazoles are five-membered rings with numerous applications as
⁎ 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.bioorg.2019.103392
Received 22 June 2019; Received in revised form 4 September 2019; Accepted 21 October 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Zhi-YuanQi,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103392

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hydrolyzed to give 13a,b, and the resulting carboxylic acids were es-
terified with isopropanol using 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDC·HCl) and N,N-dimethylaminopyridine
(DMAP) catalysis to give 14b and 15b [27]. Amidation of 13a,b with
various amines using EDC·HCl and 1-hydroxybenzyltriazole hydrate
(HOBt) catalysis yielded 14c–i and 15c–i [28]. Finally, the 18 target
compounds were synthesized by click reactions between 12a, 14b–i or
12b, 15b–i, and 9 [21,29].
2.2. Biological profiling
Fig. 1. Chemical structures of colchicine (1), CA-4 (2) and CA-4P (3).
2.2.1. Antiproliferative activities of compounds 16a-i, 17a-i
Novel compounds 16a–i and 17a–i were evaluated for anti-
proliferative activity against four human cancer cell lines (HCT116
colon cancer, HepG2 hepatic carcinoma, HeLa human epithelial cer-
vical cancer, and A549 non-small-cell lung cancer) using the MTT assay
with CA-4 as a reference drug [30]. The IC50 values of the compounds
(concentrations corresponding to 50% inhibition of cell growth) are
summarized in Table 1.
linkers in medicinal chemistry [22,23]. To discover novel inhibitors
based on CA-4 with strong antitumor activities, we designed a series of
1-(benzofuran-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole deri-
vatives that are hybrids of compounds 4 and 6. These hybrid com-
pounds contain both benzo[b]furan and 1,2,3-triazolyl moieties
(Fig. 2). Herein, we report the synthesis and antiproliferative activities
of a set of novel target compounds. Furthermore, we report the biolo-
gical activity of representative compound 17g, including its effect on in
vitro tubulin polymerization, intracellular microtubule networks, cell
cycle arrest, cell apoptosis, and the mitochondrial membrane potential
(MMP) of A549 cells. Furthermore, molecular modeling studies of the
colchicine binding site of tubulin were performed with 17g.
Compounds 16a–i were unsubstituted at the 6-position, whereas
their analogues 17a–i were the corresponding 6-methoxy derivatives.
This allowed us to investigate the effect of the 6-methoxy group on
antiproliferative activity. As shown in Table 1, the IC50 values of 17a–i
were lower than those of the corresponding unsubstituted compounds
16a–i (for example, 17a vs. 16a, 17b vs. 16b), indicating that the 6-
methoxy group was beneficial for antiproliferative activity. Compounds
16a,b and 17a,b, possessing either ethoxycarbonyl or iso-
propoxycarbonyl groups at the 2-position, showed poor anti-
proliferative activities, while 2-amido-substituted compounds 16c–i
and 17c–i showed higher activities. Among them, 6-methoxy-N-phenyl-
3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole-1-benzofuran-2-car-
boxamide (17g) exhibited the highest activity, with IC50 values against
2. Results and discussion
2.1. Chemistry
The synthetic routes to the target compounds are outlined in
Schemes 1 and 2. Initially, we synthesized intermediate 5-ethynyl-
1,2,3-trimethoxybenzene (9) from 3,4,5-trimethoxybenzaldehyde (7) as
the starting material using the Corey–Fuchs reaction [24]. Specifically,
5-(2,2-dibromovinyl)-1,2,3-trimethoxybenzene (8) was obtained from
the reaction of 7 with PPh3 and CBr4. Compound 8 was then treated
with n-BuLi at − 78 °C to yield alkyne derivative 9 [25].
HCT-116, HeLa, HepG2, and A549 cells of 0.87
0.79, 0.73
0.67,
5.74 1.21 and, 0.57 0.31 μM, respectively. Furthermore, the
compounds substituted with cyclopropylamine, piperidine, and mor-
pholine showed lower cytotoxic activities than those substituted with
aniline (16h,i vs. 16g, 17h,i vs. 17g). Furthermore, compounds 16c,
16g and 17g were also evaluated for their toxicity toward normal
human liver cells HL-7702, the results evaluated that 16g and 17g
showed lower toxicity than that of CA-4 (Table 1). Considered com-
pound 17g showed the most potent inhibitory activities against tumor
cells and less toxicity toward normal cells, following compound 17g
Tandem one-pot cyclization of 2-hydroxy benzonitrile derivatives
10a,b with ethyl bromoacetate and K2CO3 in DMF afforded 2-ethox-
ycarbonyl-3-amino benzo[b]furan derivatives 11a,b [17]. Next, the
azidation of 11a,b was accomplished in the presence of NaNO2 and
NaN3 in acetic acid to obtain 12a,b [26]. The ester group of 12a,b was
Fig. 2. Design of target compounds 16a-i and 17a-i.
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Scheme 1. Reagents and conditions: (i) CBr4, PPh3, DCM, 0 °C, 2 h; (ii) n-BuLi, dry THF, –78 °C, 5 h.
was selected to further study its action mechanism in A549 cells.
the control group exhibited a normal arrangement, with slim and fi-
brous microtubules wrapped around the cell nucleus. However, when
the cells were treated with 17g at concentrations of 1.25 and 2.50 μM,
the microtubule network tissue was destroyed and shrank to the nu-
cleus. When the compound concentration was increased to 5.00 μM, the
spindle formation adopted distinct abnormalities and was heavily dis-
rupted. Furthermore, the microtubule spindles shrank around the
center of the cells, and a number of dotted disorder formations were
observed. This effect was analogous to that of CA-4 at 5.00 μM.
Therefore, 17g disrupted the microtubule organization in A549 cells,
which could potentially lead to cell cycle disorder [4].
2.2.2. In vitro tubulin polymerization inhibition activity of compound 17g
Mechanistic research has shown that the potent anticancer activity
of CA-4 is based on its inhibition of tubulin polymerization [10]. To
identify the target of compound 17g, we measured the in vitro tubulin
polymerization inhibition activity of 17g using the method originally
described by Shelanski et al. [31]. As shown in Fig. 3A and 3B, the
absorbance intensity of purified and unpolymerized tubulin increased
with time, indicating that tubulin polymerization had occurred. After
tubulin was incubated with 17g at various concentrations (ranging
from 0.6 to 20.0 μ M), the change in absorbance intensity was reduced
compared with the control (Fig. 3A). This indicated that 17g inhibited
tubulin polymerization in a dose-dependent manner. The IC50 value of
2.2.4. Effect of 17g on the A549 cell cycle in the G2/M phase
Microtubules play an important role in the movement of chromo-
somes during cell division. As most tubulin destabilizing agents disrupt
the regulated cell cycle distribution [33], flow cytometry analysis was
performed to determine the effect of 17g on the cell cycle. As shown in
Fig. 5A and 5B, when A549 cells were treated with 17g at 0.5 μM for
24 h, 60% of the cells were arrested in the G2/M phase. This effect was
intensified when the concentration of 17g was increased to 2.5 μM,
with the percentage of cells at the G2/M phase increasing to 63%. For
CA-4, the proportion of cells in the G2/M phase increased from 6% in
the control to 69% at 2.5 μM. These results verified that 17g induced
cell cycle arrest in the G2/M phase.
polymerization inhibition was determined as 4.1
0.1 μM (Fig. 3A),
which was only slightly higher than that of CA-4 (1.0
0.1 μM,
Fig. 3B). Therefore, compound 17g was a potent inhibitor of tubulin
polymerization.
2.2.3. Effect of 17g on microtubule organization in A549 cells
The tubulin–microtubule system plays a vital role in the main-
tenance of cell shape and basic cellular functions. The inhibition of
tubulin polymerization leads to the destruction of microtubule tissue
and the cytoskeleton [2]. An immunofluorescence assay was performed
to determine whether 17g disrupted the microtubule organization in
living A549 cells [32]. As shown in Fig. 4, the microtubule networks in
Furthermore, cyclin B1, Cdc25C, and Cdc2 are known to be key
regulators in cell cycle progression [34,35]. Therefore, the effect of 17g
Scheme 2. Reagents and conditions: (i)
ethyl bromoacetate, K2CO3, DMF, 60 °C
for 8 h, then 140 °C for 8 h; (ii) NaNO2,
NaN3, 10:1 acetic acid/H2O, 0 °C, 2.5 h;
(iii) 6 N NaOH, THF and H2O, rt, 2d; (iv)
isopropanol (for 14b and 15b), EDC·HCl,
DMAP, 24 h; or various amines (for 14c-
i and 15c-i), EDC·HCl, HOBt, DCM, rt,
overnight; (v) 9, CuSO4·5H2O, sodium
ascorbate, 1:1 t-BuOH/H2O, rt, over-
night.
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Table 1
Antiproliferative activities of compounds 16a–i and 17a–i.
Compd
R1
R2
IC50 (μM)a,b
HCT-116
> 100
HeLa
HepG2
A549
HL-7702
16a
16b
16c
16d
16e
16f
16g
16h
16i
H
H
H
H
H
H
H
H
H
OCH2CH3
> 100
59.30
6.84
30.97
58.00
2.92
6.24
9.48
> 100
> 100
9.29
–
–
OCH(CH3)2
41.71
16.43
> 100
8.94
4.19
5.15
0.34
0.22
3.66
18.34
–
6.04
1.26
> 100
> 100
> 100
6.07
0.38
43.37
24.99
1.83
7.55
7.71
36.82
51.12
8.34
5.43
9.75
22.52
94.74
0.83
2.65
8.93
0.34
–
8.59
5.47
0.31
–
1.02
0.52
2.53
36.87
73.52
> 100
17.17
> 100
> 100
65.23
64.85
5.42
2.98
73.99
> 100
7.86
–
–
17a
17b
17c
17d
17e
17f
17g
17h
17i
OMe
OMe
OMe
OMe
OCH2CH3
36.64
40.21
52.46
> 100
7.30
4.23
8.74
> 100
29.48
2.84
11.57
14.79
6.29
3.59
5.02
> 100
50.41
–
–
–
–
OCH(CH3)2
0.67
0.95
7.48
1.26
7.88
3.17
2.93
> 100
> 100
78.38
> 100
OMe
OMe
OMe
OMe
OMe
–
1.27
0.38
7.66
20.26
41.65
5.74
6.83
9.05
6.01
1.21
3.21
4.65
0.02
18.95
> 100
0.57
–
5.56
1.76
0.79
7.56
0.86
0.03
29.99
0.73
5.59
–
0.87
47.79
80.08
0.08
0.67
0.31
16.90
6.96
34.33
4.60
> 100
49.75
0.17
38.79
37.85
0.04
–
11.74
0.01
10.92
0.15
–
CA-4
–
0.01
16.60
4.53
a
b
IC50 values are presented as the means
Drug treatment for 72 h.
SD of triplicate experiments.
on the expression of these proteins was investigated by Western blot
analysis. As shown in Fig. 5C, after A549 cells were treated with 17g at
concentrations of 0.125, 0.25, and 0.5 μM for 12 h, the expression of
cyclin B1, Cdc25C, and Cdc2 proteins decreased in a dose-dependent
manner. These results indicated that 17g can block A549 cells at the
G2/M phase, analogous to CA-4, by regulating the expression of cycle-
related proteins.
2.2.5. Effect of 17g on A549 cells apoptosis
Mitotic arrest of tumor cells by tubulin destabilizing agents is gen-
erally associated with cellular apoptosis [36,37]. Therefore, the apop-
totic effect of 17g on A549 cells was measured, using CA-4 as a re-
ference compound. As shown in Fig. 6A, after A549 cells were treated
with 17g at concentrations of 0.1 and 2.5 μM for 12 h, the percentage of
apoptotic cells was determined to be 5.68% and 8.72%, respectively.
These percentages were greater than that under the control conditions,
which afforded a 3.34% apoptosis rate, indicating that 17g induced
A549 cell apoptosis. However, the 8.72% apoptosis rate caused by 17g
at 2.5 μM was significantly lower than that of CA-4 at an equivalent
concentration (30.71%). This was consistent with the lower anti-
proliferative activities of 17g compared with those of CA-4.
Bcl-2 and Bcl-xl are important antiapoptotic proteins, while Bax is
an important proapoptosis protein [38]. Therefore, the effect of 17g on
the expression of Bcl-2, Bcl-xl, and Bax proteins was investigated. As
shown in Fig. 6B and 6C, when A549 cells were treated with 17g at
different concentrations (0.125, 0.25, and 0.5 μM) for 6 h, the
Fig. 3. Tubulin polymerization inhibitory activity of 17g (A) and CA-4 (B).
Purified tubulin protein at 10 μM was incubated at 37 °C in the absence or
presence of 17g and CA-4 at the indicated concentrations.
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Fig. 4. Effect of compound 17g on organization of the cellular microtubule network in A549 cells. A549 cells were plated in confocal dishes and incubated with 17g
or CA-4 at the indicated concentrations for 6 h, followed by direct microscopy, with representative immunofluorescence shown. Scale bar: 10 μm.
expression level of antiapoptotic proteins (Bcl-2, Bcl-xl) was down-
regulated significantly. However, 17g had no significant effect on the
expression level of proapoptosis protein Bax. These results demon-
strated that 17g, similar to CA-4, can downregulate the expression of
antiapoptotic proteins, leading to cell apoptosis.
9B. As expected, 17g occupied the colchicine binding site at the in-
terface of α, β-tubulin, and was mostly confined in a deep pocket in β-
tubulin opposite a GTP molecule bound in a pocket in α -tubulin
(Fig. 8A). Specifically, the 3,4,5-trimethoxybenzene ring (A ring) of 17g
was buried deep within β-tubulin, while its benzo[b]furan ring (B ring)
was facing α -tubulin, and the compound was tightly wrapped by the
surrounding amino acid residues (Fig. 8B). The A ring penetrated into
the hydrophobic pocket composed of Cys241, Val328, Tyr202, Leu255,
and Ala354, and was involved in a hydrophobic interaction with
Cys241. The benzo[b]furan sat deep in a pocket composed of Lys254,
Val74, Gln11, Tyr224, and Asn101, and the 2-N-phenyl amide was
positioned close to Ser178 and Val181. Altogether, compound 17g
exhibited efficient binding to the colchicine site of tubulin, which might
support its inhibitory effect against tubulin.
2.2.6. Effect of 17g on the mitochondrial membrane potential (MMP)
Previous studies have indicated that mitochondria play a vital role
in the progression of apoptosis [39]. Decreased MMP (ΔΨm) has been
implicated as an early event in apoptotic cells. Therefore, we measured
the effect of 17g on MMP changes in A549 cells. As shown in Fig. 7,
after A549 cells were exposed to 17g at concentrations of 0.1, 0.5, and
2.5 μM, the number of monomer forms (green fluorescence) gradually
increased compared with the control group, while the number of ag-
gregate forms (red fluorescence) gradually decreased. This change in-
dicated that 17g decreased the MMP in a dose-dependent manner.
Furthermore, the results indicated that 17g was able to induce MMP
collapse and mitochondrial dysfunction, and downregulate the ex-
pression of antiapoptotic proteins, leading to A549 cell apoptosis.
3. Conclusion
In summary, we synthesized a series of novel 1-(benzofuran-3-yl)-
1H-1,2,3-triazole derivatives as tubulin polymerization inhibitors.
Compound 17g showed the most potent antiproliferative activity and
less toxicity among this series. Mechanistic studies indicated that 17g
inhibited tubulin polymerization, strongly disrupted microtubule or-
ganization in A549 cells, blocked the cell cycle in the G2/M phase by
affecting cycle-related proteins, and led to apoptosis by downregulating
2.2.7. Molecular docking
To investigate the possible binding mode of 17g, a molecular
docking study was performed at the colchicine site of tubulin (PDB
code: 1SA0) [40]. The resulting binding mode is shown in Fig. 8A and
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Fig. 5. (A) Effect of 17g on cell cycle arrest in A549 cells. A549 cells were treated with compound 17g and CA-4 at the indicated concentrations for 24 h. (B)
Quantitative analysis of the percentage of cells in each cell cycle phase. (C) Expression of proteins cyclin B1, Cdc25c, and Cdc2 analyzed by Western blot.
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Fig. 6. (A) Effect of 17g on A549 cell apoptosis. A549 cells were treated with compound 17g and CA-4 at the indicated concentrations for 12 h. (B) Expression of
proteins Bcl-2 and Bcl-xl analyzed by Western blot. (C) Expression of protein Bax analyzed by Western blot.
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Fig. 7. Effect of 17g on the MMP. A549 cells were treated with DMSO (0.01%) or 17g at the indicated concentrations (0.1, 0.5 and 2.5 μM) for 6 h, followed by
incubation with fluorescence probe JC-1 for 30 min. The cells were then analyzed by fluorescence microscopy. Scale bar: 10 μm.
tubulin polymerization inhibitor 17g is a potential cancer treatment,
warranting its further investigation and development.
4. Experiment
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). 1H
NMR and 13C NMR spectra were recorded with Agilent NMR inova 600
or Bruker AM 400 spectrometers with TMS as an internal standard, all
chemical shift values were reported as ppm. Mass spectra were recorded
on a Bruker Dalton APEXII49e and Esquire 6000 (ESI-ION TRAP)
spectrometer with ESI source as ionization, respectively.
4.1.1. 5-(2,2-Dibromovinyl)-1,2,3-trimethoxybenzene (8)
To a solution of CBr4 (10 g, 15.3 mmol) in dry dichloromethane
(50 mL) was slowly added PPh3 (16 g, 30.6 mmol). The reaction mix-
ture was stirred at room temperature for 1 h. Then added 7 (4.0 g,
10.2 mmol) at 0 °C and stirred for another 2 h. After the reaction was
completed, the solid was filtered off, and the solution was concentrated
under reduced pressure to give crude product. The crude product was
purified by column chromatography (50:1 petroleum ether/ethyl
acetate) to give 5-(2,2-dibromovinyl)-1,2,3-trimethoxybenzene
8
(3.7 g, yield 52%) as a yellow liquid. 1H NMR (600 MHz, CDCl3) δ 7.41
(s, 1H), 6.80 (s, 2H), 3.87 (s, 3H), 3.86 (s, 6H); MS (ESI) m/z 353.0 for
[M+H]+
.
Fig. 8. Molecular docking of 17g at the colchicine binding site of the α,β-tu-
bulin interface. (A) Surface representation of 17g. (B) 17g (green sticks) and
surrounding amino acid residues (gray sticks) of tubulin.
4.1.2. 5-Ethynyl-1,2,3-trimethoxybenzene (9)
antiapoptotic proteins and decreasing the MMP of A549 cells. A
docking study showed that 17g was a good molecular fit at the col-
chicine binding site. Collectively, these results highlight that novel
To a solution of 8 (3.7 g, 10.5 mmol) in dry THF (50 mL) was slowly
added n-BuLi (12.5 mL, 31.5 mmol, 2.5 M in hexane) with a syringe at
−78 °C under nitrogen protection. Stirring the mixture at −78 °C for
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3 h and continue to react for 2 h at room temperature. After the reaction
was finished, the mixture was quenched with saturated aqueous NH4Cl,
and the aqueous phase was extracted with Et2O for three times. The
combined organic extracts were dried over MgSO4, filtered, and con-
centrated under reduced pressure. Then the crude product was purified
by column chromatography (100:1–60:1 petroleum ether/ethyl
acetate) to give 9 (1.1 g, yield 55%) as a yellow solid. 1H NMR
(600 MHz, CDCl3) δ 6.73 (s, 2H), 3.85 (s, 3H), 3.83 (s, 6H), 3.03 (s, 1H);
4.1.6. General procedures for preparation of compounds 14b and 15b
To a solution of 13a (0.25 g, 1.23 mmol) in DCM (20 mL) was added
EDC·HCl (0.35 g, 1.89 mmol), DMAP (0.03 g, 0.25 mmol) and iso-
propanol (1.0 mL). The reaction mixture was stirred at room tempera-
ture for 24 h. Then the mixture was added H2O (50 mL) and extracted
with DCM. The combined organic extracts were dried over Na2SO4,
filtered, and concentrated under reduced pressure. Then the crude
product was purified by column chromatography (50:1–20:1 petroleum
ether/ethyl acetate) to give 14b. A similar procedure was operated to
provide pure compound 15b.
MS (ESI) m/z 193.1 for [M+H]+
.
4.1.3. General procedures for preparation of compounds 11a,b
To a solution of 2-hydroxybenzonitrile (8.0 g, 67.2 mmol) in DMF
was added ethyl bromoacetate (13.5 g, 80.7 mmol) and K2CO3 (18.5 g,
134.4 mmol). The mixture was stirred at 60 °C for 8 h, then heated to
140 °C and stirred for 8 h. Then the mixture was added water (500 mL)
and extracted with ethyl acetate. The combined organic extracts were
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography (10:1–6:1
petroleum ether/ethyl acetate) to give 11a. A similar procedure was
operated to provide pure 11b.
4.1.6.1. Isopropyl 3-azidobenzofuran-2-carboxylate (14b). Yield: 70%;
yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.73 (d, J = 8.4 Hz, 1H),
7.54 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.33 (t, J = 7.8 Hz,
1H), 5.39–5.37 (m, 1H), 1.45 (d, J = 6.0 Hz, 6H).
4.1.6.2. Isopropyl 3-azido-6-methoxybenzofuran-2-carboxylate (15b). Yield:
76%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.57 (d, J = 9.0 Hz, 1H),
6.99 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 5.36–5.35 (m, 1H), 3.86 (s, 3H), 1.44
(d, J = 9.6 Hz, 6H).
4.1.7. General procedures for preparation of compounds 14c-i, 15c-i
To a solution of 13a (0.24 g, 1.18 mmol) in DCM (20 mL) was added
EDC·HCl (0.29 g, 1.54 mmol), HOBt (0.21 g, 1.54 mmol) and propyla-
mine (1.0 mL). The reaction mixture was stirred at room temperature
for overnight. After the reaction was finished, the mixture was added
H2O (50 mL) and extracted with DCM. The combined organic extracts
were dried over Na2SO4, filtered, and concentrated under reduced
pressure. Then the crude product was purified by column chromato-
graphy (50:1–20:1 petroleum ether/ethyl acetate) to give 14c. Similar
procedures were operated to provide pure compounds 14d-i, 15c-i.
4.1.3.1. Ethyl 3-aminobenzofuran-2-carboxylate (11a). Yield 73%;
yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.56 (d, J = 7.8 Hz, 1H),
7.46 (d, J = 3.0 Hz, 2H), 7.25 (s, 2H), 4.45 (q, J = 7.2 Hz, 2H), 1.44 (t,
J = 7.2 Hz, 3H); MS (ESI) m/z 206.1 for [M+H]+
.
4.1.3.2. Ethyl 3-amino-6-methoxybenzofuran-2-carboxylate (11b). Yield:
64%; yellow solid; 1H NMR (600 MHz, DMSO- d6) δ 7.78 (d, J = 8.4 Hz,
1H), 7.05 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 6.6 Hz, J = 1.8 Hz, 1H),
6.29 (brs, 2H), 4.24 (q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 1.28 (t,
J = 7.2 Hz, 3H). MS (ESI) m/z 236.1 for [M+H]+
.
4.1.7.1. 3-Azido-N-propylbenzofuran-2-carboxamide (14c). Yield: 79%;
yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.73 (d, J = 8.4 Hz, 1H),
7.49–7.45 (m, 2H), 7.32 (t, J = 7.8 Hz, 1H), 6.65 (brs, 1H), 3.46 (q,
J = 6.6 Hz, 2H), 1.69–1.66 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H).
4.1.4. General procedures for preparation of compounds 12a,b
To a solution of 11a (8.9 g, 43.4 mmol) in acetic acid (120 mL) and
H2O (12 mL) was added NaNO2 (7.5 g, 108.5 mmol) and NaN3 (7.0 g,
108.5 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2.5 h.
After the reaction was finished, the mixture was added aqueous
NaHCO3 (100 mL) and extracted with ethyl acetate. The combined or-
ganic extracts were dried over Na2SO4, filtered, and concentrated under
reduced pressure. Then the crude product was purified by column
chromatography (20:1–10:1 petroleum ether/ethyl acetate) to give
12a. A similar procedure was operated to provide pure 12b.
4.1.7.2. 3-Azido-N-cyclopropylbenzofuran-2-carboxamide (14d). Yield:
42%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz,
1H), 7.47 (d, J = 3.6 Hz, 2H), 7.33–7.30 (m, 1H), 6.71 (brs, 1H),
2.95–2.92 (m, 1H), 0.91 (q, J = 6.0 Hz, 2H), 0.70–0.67 (m, 2H).
4.1.7.3. 3-Azido-N-cyclopentylbenzofuran-2-carboxamide (14e). Yield:
40%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.72 (d, J = 7.8 Hz,
1H), 7.49–7.45 (m, 2H), 7.31 (t, J = 7.8 Hz, 1H), 6.56 (brs, 1H),
4.45–4.42 (m, 1H), 2.10 (t, J = 6.0 Hz, 2H), 1.80–1.76 (m, 2H),
1.7–1.68 (m, 2H), 1.60–1.55 (m, 2H).
4.1.4.1. Ethyl 3-azidobenzofuran-2-carboxylate (12a). Yield: 47%;
yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 1H),
7.55–7.49 (m, 2H), 7.32 (t, J = 7.2 Hz, 1H), 4.50 (q, J = 7.2 Hz, 2H),
1.46 (t, J = 7.2 Hz, 3H).
4.1.7.4. 3-Azido-N-cyclohexylbenzofuran-2-carboxamide
(14f). Yield:
84%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz,
1H), 7.49–7.45 (m, 2H), 7.33–7.30 (m, 1H), 6.56 (d, J = 7.8 Hz, 1H),
4.03–4.02 (m, 1H), 2.05 (dd, J = 9.0, 3.6 Hz, 2H), 1.77 (dt, J = 13.8,
3.6 Hz, 2H), 1.67 (dd, J = 9.6, 4.2 Hz, 1H), 1.47–1.41 (m, 2H),
1.35–1.22 (m, 4H).
4.1.4.2. Ethyl 3-azido-6-methoxybenzofuran-2-carboxylate (12b). Yield:
43%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.57 (d, J = 9.0 Hz,
1H), 6.98 (d, J = 1.8 Hz, 1H), 6.93 (dd, J = 7.2, 1.8 Hz, 1H), 4.47 (q,
J = 7.2 Hz, 2H), 3.86 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).
4.1.5. General procedures for preparation of compounds 13a,b
To a solution of 12a (4.7 g, 20 mmol) in THF (30 mL) and H2O
(1 mL) was added NaOH (4.9 g, 120 mmol). The reaction mixture was
stirred at room temperature for 2 days. Then the reaction solution was
acidified to pH = 1 by the dropwise addition of 3 M HCl and extracted
with DCM. The combined organic extracts were dried over Na2SO4,
filtered, and concentrated under reduced pressure to give the product 3-
azidobenzofuran-2-carboxylic acid (13a). A similar procedure was op-
erated to provide 3-azido-6-methoxybenzofuran-2-carboxylic acid 13b.
These two compounds were directly used in next reaction without
further purification.
4.1.7.5. 3-Azido-N-phenylbenzofuran-2-carboxamide (14 g). Yield: 80%;
yellow solid; 1H NMR (600 MHz, CDCl3) δ 8.36 (brs, 1H), 7.78 (d,
J = 7.8 Hz, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.56–7.50 (m, 2H), 7.41–7.35
(m, 3H), 7.17 (t, J = 7.8 Hz, 1H).
4.1.7.6. (3-Azidobenzofuran-2-yl)(piperidin-1-yl)methanone
(14 h). Yield: 76%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.68 (d,
J = 7.8 Hz, 1H), 7.45–7.39 (m, 2H), 7.27 (t, J = 7.2 Hz, 1H), 3.68 (s,
2H), 3.64 (s, 2H), 1.70–1.65 (m, 6H).
4.1.7.7. (3-Azidobenzofuran-2-yl)(piperidin-1-yl)methanone
9

Z.-Y. Qi, et al.
BioorganicChemistryxxx(xxxx)xxxx
(MeOH:H2O = 80:20, 0.5 mL/min, tR = 8.71 min).
(14i). Yield: 76%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.72 (d,
J = 7.8 Hz, 1H), 7.47–7.45 (m, 2H), 7.32 (t, J = 6.0 Hz, 1H), 3.80 (brs,
8H).
4.1.8.2. Isopropyl 3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)
benzofuran-2-carboxylate (16b). Yield: 65%; yellow solid; m.p.:
171–173 °C; 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.04 (d,
J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H),
7.38 (t, J = 8.0 Hz, 1H), 7.12 (s, 2H), 5.27 (p, J = 4.0 Hz, 1H), 3.90 (s,
6H), 3.84 (s, 3H), 1.33 (d, J = 4.0 Hz, 6H); 13C NMR (150 MHz, CDCl3)
δ 158.1, 153.8 (2C), 153.5, 138.6, 135.2, 129.2, 125.7, 124.9 (2C),
123.1 (2C), 122.8, 112.4 (2C), 103.3 (2C), 70.3, 60.9, 56.3 (2C), 21.8
(2C); MS (ESI) m/z 438.2 for [M+1]+; HRMS (ESI) 438.1668 for [M
+H]+ (calcd 438.1660 for C23H24N3O6); HPLC purity 98.7% (MeOH:
H2O = 75:25, 0.8 mL/min, tR = 7.96 min).
4.1.7.8. 3-Azido-6-methoxy-N-propylbenzofuran-2-carboxamide
(15c). Yield: 68%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.58 (d,
J = 8.4 Hz, 1H), 6.96 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.56 (brs, 1H),
3.87 (s, 3H), 3.44 (q, J = 6.6 Hz, 2H), 1.68–1.64 (m, 2H), 1.00 (t,
J = 7.2 Hz, 3H).
4.1.7.9. 3-Azido-N-cyclopropyl-6-methoxybenzofuran-2-carboxamide
(15d). Yield: 74%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.57 (d,
J = 9.6 Hz, 1H), 6.94–6.92 (m, 2H), 6.63 (brs, 1H), 3.86 (s, 3H),
2.92–2.90 (m, 1H), 0.90–0.86 (m, 2H), 0.66 (s, 2H).
4.1.8.3. N-propyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)
benzofuran-2-carboxamide (16c). Yield: 70%; yellow solid; m.p.:
167–169 °C; 1H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.32 (d,
J = 8.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 2H), 7.45 (dt, J = 8.2, 4.1 Hz,
1H), 7.21 (s, 2H), 6.99 (s, 1H), 3.97 (s, 6H), 3.91 (s, 3H), 3.49 (q,
J = 6.8 Hz, 2H), 1.77–1.67 (m, 2H), 1.04 (t, J = 8.0 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 158.1, 153.7 (2C), 152.5, 146.8, 138.5, 136.0,
128.6, 125.8, 124.9, 124.1 (2C), 123.0 (2C), 111.7, 103.6 (2C), 60.9,
56.4 (2C), 41.3, 22.8, 11.4; MS (ESI) m/z 437.1 for [M+1]+; HRMS
(ESI) 437.1832 for [M+H]+ (calcd 437.1819 for C23H25N4O5); HPLC
purity 98.5% (MeOH:H2O = 35:65, 0.8 mL/min, tR = 4.21 min).
4.1.7.10. 3-Azido-N-cyclopentyl-6-methoxybenzofuran-2-carboxamide
(15e). Yield: 68%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.71 (d,
J = 3.6 Hz, 1H), 6.95 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.48 (d,
J = 6.0 Hz, 1H), 4.43 (q, J = 7.2 Hz, 1H), 3.86 (s, 3H), 2.10 (q,
J = 6.0 Hz, 2H), 1.76–1.70 (m, 2H), 1.69–1.66 (m, 2H), 1.55–1.51
(m, 2H).
4.1.7.11. 3-Azido-N-cyclohexyl-6-methoxybenzofuran-2-carboxamide
(15f). Yield; 69%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.57 (d,
J = 8.4 Hz, 1H), 6.96 (s, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.42 (d,
J = 6.6 Hz, 1H), 4.02–4.00 (m, 1H), 3.87 (s, 3H), 2.04–2.02 (m, 2H),
1.78–1.71 (m, 2H), 1.67–1.64 (m, 1H), 1.46–1.41 (m, 2H), 1.33–1.25
(m, 3H).
4.1.8.4. N-cyclopropyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-carboxamide (16d). Yield: 65%; yellow solid; m.p.:
175–176 °C; 1H NMR (600 MHz, CDCl3) δ 9.30 (s, 1H), 8.34 (d,
J = 8.4 Hz, 1H), 7.57–7.53 (m, 2H), 7.44 (t, J = 7.2 Hz, 1H), 7.22 (s,
2H), 7.05 (s, 1H), 3.95 (s, 6H), 3.91 (s, 3H), 2.95 (d, J = 3.0 Hz, 1H),
0.97 (d, J = 6.6 Hz, 2H), 0.75 (s, 2H); 13C NMR (150 MHz, CDCl3) δ
159.5, 153.7 (2C), 152.4, 146.7, 138.6, 135.5, 128.7, 125.8, 124.8,
124.2, 123.2, 123.0, 122.9, 111.6, 103.5, 103.4, 60.8, 56.3 (2C), 29.6,
22.6, 6.7; MS (ESI) m/z 435.2 for [M+1]+; HRMS (ESI) 435.1671 for
[M+H]+ (calcd 435.1663 for C23H23N4O5); HPLC purity > 99%
(MeOH: H2O = 35:65, 0.8 mL/min, tR = 4.38 min).
4.1.7.12. 3-Azido-6-methoxy-N-phenylbenzofuran-2-carboxamide
(15 g). Yield: 66%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 8.27
(brs, 1H), 7.69 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 9.6 Hz, 1H), 7.38 (d,
J = 7.8 Hz, 2H), 7.17 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.97 (d,
J = 8.4 Hz, 1H), 3,89 (s, 3H).
4.1.7.13. (3-Azido-6-methoxybenzofuran-2-yl)(piperidin-1-yl)methanone
(15 h). Yield: 68%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.56 (d,
J = 9.0 Hz, 1H), 6.94–6.91 (m, 2H), 3.86 (s, 3H), 3.67 (brs, 4H),
1.71–1.67 (m, 6H).
4.1.8.5. N-cyclopentyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-carboxamide (16e). Yield: 75%; yellow solid; m.p.:
178–180 °C; 1H NMR (600 MHz, CDCl3) δ 9.25 (s, 1H), 8.30 (d,
J = 7.8 Hz, 1H), 7.57 (s, 2H), 7.43 (t, J = 6.0 Hz, 1H), 7.21 (s, 2H),
6.93 (d, J = 7.2 Hz, 1H), 4.44 (q, J = 7.2 Hz, 1H), 3.98 (s, 6H), 3.91 (s,
3H), 2.14 (q, J = 6.0 Hz, 2H), 1.81 (s, 2H), 1.72–1.70 (m, 2H),
1.64–1.59 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 157.7, 153.7 (2C),
152.4, 146.8, 138.6, 136.1, 128.6, 125.9, 124.9, 124.1 (2C), 123.0
(2C), 111.7, 103.6 (2C), 60.9, 56.4 (2C), 51.4, 33.2 (2C), 23.8 (2C); MS
(ESI) m/z 463.2 for [M+1]+; HRMS (ESI) 463.1982 for [M+H]+
4.1.7.14. (3-Azido-6-methoxybenzofuran-2-yl)(morpholino)methanone
(15i). Yield: 68%; yellow solid; 1H NMR (600 MHz, CDCl3) δ 7.58 (d,
J = 4.2 Hz, 1H), 6.94–6.93 (m, 2H), 3.88 (s, 3H), 3.79 (brs, 8H).
4.1.8. General procedures for preparation of compounds 16a-i, 17a-i
To a solution of 9 (0.24 g, 1.18 mmol) in t-BuOH (10 mL) and H2O
(10 mL) was added CuSO4·5H2O (0.29 g, 1.54 mmol), sodium ascorbate
(0.21 g, 1.54 mmol) and 12a (0.13 g, 0.58 mmol). The reaction mixture
was stirred at room temperature for overnight. Then the mixture was
added 3 M HCl (100 mL) and extracted with CHCl3. The combined or-
ganic extracts were dried over Na2SO4, filtered, and concentrated under
reduced pressure. Then the crude product was purified by column
chromatography (10:1–2:1 petroleum ether/ethyl acetate) to give 16a.
Similar procedures were operated to provide pure compounds 16b-i,
17a-i.
(calcd
463.1976
for
C25H27N4O6);
HPLC
purity
97.2%
(MeOH:H2O = 45:55, 0.8 mL/min, tR = 4.10 min).
4.1.8.6. N-cyclohexyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-carboxamide (16f). Yield: 63%; yellow solid; m.p.:
176–178 °C; 1H NMR (400 MHz, CDCl3) δ 9.16 (s, 1H), 8.23 (d,
J = 8.0 Hz, 1H), 7.53–7.44 (m, 2H), 7.37 (td, J = 8.2, 6.9, 4.2 Hz,
1H), 7.14 (s, 2H), 6.75 (d, J = 8.0 Hz, 1H), 3.93 (s, 1H), 3.90 (s, 6H),
3.83 (s, 3H), 2.02–1.97 (m, 2H), 1.64–1.60 (m, 3H), 1.43–1.27 (m, 3H);
13C NMR (150 MHz, CDCl3) δ 157.2, 153.7 (2C), 152.4, 146.8, 138.5,
136.1 128.6, 125.9, 124.9, 124.1, 123.1 (2C), 123.0, 111.7, 103.5 (2C),
60.9, 56.4 (2C), 48.6, 33.1 (2C), 25.4 (2C), 24.8; MS (ESI) m/z 477.2 for
[M+1]+; HRMS (ESI) 477.2142 for [M+H]+ (calcd 477.2132 for
4.1.8.1. Ethyl3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)
benzofuran-2-carboxylate (16a). Yield: 79%; yellow solid; m.p.:
168–170 °C; 1H NMR (600 MHz, CDCl3) δ 8.78 (s, 1H), 8.14 (d,
J = 8.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 8.4 Hz, 1H),
7.44 (t, J = 7.8 Hz, 1H), 7.19 (s, 2H), 4.48 (q, J = 7.2 Hz, 2H), 3.97 (s,
6H), 3.91 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ 158.6, 153.8 (2C), 153.5, 147.0, 138.7, 134.5, 129.4, 125.6, 125.5,
124.9, 123.3, 122.8, 122.3, 112.4, 103.5 (2C), 62.2, 60.9, 56.3 (2C),
14.1; MS (ESI) m/z 424.2 for [M+1]+; HRMS (ESI) 424.1511 for [M
+H]+ (calcd 424.1503 for C22H22N3O6); HPLC purity > 99%
C
26H29N4O5); HPLC purity > 99% (MeOH:H2O = 50:50, 0.8 mL/min,
tR = 4.20 min).
4.1.8.7. N-phenyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)
benzofuran-2-carboxamide (16 g). Yield: 60%; yellow solid; m.p.:
10

Z.-Y. Qi, et al.
BioorganicChemistryxxx(xxxx)xxxx
175–177 °C; 1H NMR (600 MHz, CDCl3) δ 9.18 (s, 1H), 8.71 (s, 1H),
8.33 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.64–7.62 (m, 2H),
7.49 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 7.23 (s, 1H), 7.21 (s,
2H) 3.98 (s, 6H), 3.91 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 156.0,
153.7 (2C), 152.6, 147.1, 136.7, 135.7, 129.3 (2C), 129.2, 125.7,
125.5, 125.2, 124.2, 124.1, 123.1, 122.9, 120.6 (2C), 112.0 (2C), 103.5
(2C), 61.0, 56.4 (2C); MS (ESI) m/z 471.1 for [M+1]+; HRMS (ESI)
471.1676 for [M+H]+ (calcd 471.1663 for C26H23N4O5); HPLC
purity > 99% (MeOH:H2O = 60:40, 0.8 mL/min, tR = 4.09 min).
(ESI) m/z 467.2 for [M+1]+; HRMS (ESI) 467.1936 for [M+H]+
(calcd
467.1925
for
C24H28N4O6);
HPLC
purity > 99%
(MeOH:H2O = 20:80, 0.8 mL/min, tR = 4.25 min).
4.1.8.13. N-Cyclopropyl-6-methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazol-1-yl)benzofuran-2-carboxamide (17d). Yield: 65%; yellow
solid; m.p.: 174–176 °C; 1H NMR (400 MHz, CDCl3) δ 9.31 (s, 1H), 8.15
(q, 4.0 Hz, 1H), 7.14 (s, 2H), 6.96 (dt, J = 8.9, 2.3 Hz, 1H), 6.92–6.86
(m, 2H), 3.90 (d, J = 1.5 Hz, 6H), 3.81 (s, 3H), 3.80 (s, 3H), 2.91–2.81
(m, 1H), 0.87 (q, J = 4.0 Hz, 2H), 0.69–0.62 (m, 2H); 13C NMR
(150 MHz, CDCl3) δ 161.3, 159.7, 153.8, 153.6, 146.7, 138.3, 134.3,
125.9, 124.9, 123.6, 122.9, 116.0, 114.6, 103.3 (2C), 95.2 (2C), 60.9,
56.3 (2C), 55.8, 22.6, 6.8 (2C); MS (ESI) m/z 465.2 for [M+1]+; HRMS
(ESI) 465.1779 for [M+H]+ (calcd 465.1769 for C24H25N4O6); HPLC
purity > 99% (MeOH:H2O = 50:50, 0.8 mL/min, tR = 4.16 min).
4.1.8.8. Piperidin-1-yl(3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-yl)methanone (16 h). Yield: 61%; yellow solid; m.p.:
182–184 °C; 1H NMR (600 MHz, CDCl3) δ 8.67 (s, 1H), 8.07 (d,
J = 8.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H),
7.43 (t, J = 7.8 Hz, 1H), 7.17 (s, 2H), 3.97 (s, 6H), 3.90 (s, 3H), 3.73 (s,
2H), 3.43 (s, 2H), 1.69 (s, 4H), 1.59 (s, 2H); 13C NMR (600 MHz, CDCl3)
δ 158.7, 153.7 (2C), 152.8, 147.4, 139.3, 138.4, 127.6, 125.7, 124.8,
122.5, 122.0, 121.7, 121.0, 112.0, 103.2 (2C), 61.0, 56.3 (2C), 48.2,
43.7, 26.5, 25.5, 24.3; MS (ESI) m/z 463.2 for [M+1]+; HRMS (ESI)
463.1989 for [M+H]+ (calcd 463.1976 for C25H27N4O5); HPLC
purity > 99% (MeOH:H2O = 40:60, 0.8 mL/min, tR = 4.41 min).
4.1.8.14. N-Cyclopentyl-6-methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazol-1-yl)benzofuran-2-carboxamide (17e). Yield: 70%; yellow
solid; m.p.: 171–173 °C; 1H NMR (600 MHz, CDCl3) δ 9.33 (s, 1H), 8.22
(d, J = 9.0 Hz, 1H), 7.21 (s, 2H), 7.20 (s, 2H), 7.06 (dd, J = 7.2, 1.8 Hz,
1H), 7.03 (d, J = 2.4 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 4.44–4.42 (m,
1H), 3.97 (s, 6H), 3.91 (s, 1H), 3.90 (s, 1H),2.13 (dd, J = 7.2, 6.0 Hz,
2H), 1.82–1.79 (m, 2H), 1.72–1.70 (m, 2H), 1.62–1.57 (m, 2H); 13C
NMR (150 MHz, CDCl3) δ 161.1, 157.8, 153.8, 153.6, 146.7, 138.3,
134.9, 125.9, 124.7, 123.3, 122.9. 116.2, 114.5, 103.4 (2C), 95.3 (2C),
60.9, 56.3 (2C), 55.8, 51.3, 33.2 (2C), 23.8 (2C); MS (ESI) m/z 493.2 for
[M+1]+; HRMS (ESI) 493.2094 for [M+H]+ (calcd 493.2080 for
4.1.8.9. Morpholino(3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)
benzofuran-2-yl)methanone (16i). Yield: 76%; yellow solid; m.p.:
178–180 °C; 1H NMR (600 MHz, CDCl3) δ 8.72 (s, 1H), 8.06 (d,
J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.2 Hz, 1H),
7.44 (t, J = 7.2 Hz, 1H), 7.18 (s, 2H), 3.97 (s, 6H), 3.91 (s, 3H), 3.80 (s,
4H), 3.70 (s, 2H), 3.60 (s, 2H); 13C NMR (600 MHz, CDCl3) δ 158.8,
153.7 (2C), 152.7, 147.4, 138.4, 138.2, 128.0, 125.5, 125.0, 122.3
(2C), 122.1, 121.8, 112.0, 103.2 (2C), 66.7, 66.5, 60.9, 56.3 (2C), 47.4,
43.0; MS (ESI) m/z 465.2 for [M+1]+; HRMS (ESI) 465.1777 for [M
+H]+ (calcd 465.1769 for C24H25N4O6); HPLC purity > 99% (MeOH:
H2O = 50:50, 0.8 mL/min, tR = 4.18 min).
C
26H29N4O6); HPLC purity > 99% (MeOH:H2O = 50:50, 0.8 mL/min,
tR = 4.35 min).
4.1.8.15. N-Cyclohexyl-6-methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazol-1-yl)benzofuran-2-carboxamide (17f). Yield: 67%; yellow
solid; m.p.: 177–179 °C; 1H NMR (600 MHz, CDCl3) δ 9.33(s, 1H), 8.21
(d, J = 9.0 Hz, 1H), 7.20 (s, 2H), 7.05 (dd, J = 7.2, 1.8 Hz, 1H), 7.03 (d,
J = 1.8 Hz, 1H) 6.72 (d, J = 8.4 Hz, 1H), 4.02–3.98 (m, 1H), 3.97 (s,
6H), 3.91 (s, 3H), 3.90 (s, 3H), 2.06 (dd, J = 9.0, 3.0 Hz, 2H), 1.80 (td,
J = 6.6, 3.6 Hz, 2H), 1.68 (dd, J = 6.0, 3.6 Hz, 1H), 1.49–1.42 (m, 2H),
1.38–1.31 (m, 2H), 1.27–1.25 (m, 1H); 13C NMR (150 MHz, CDCl3) δ
161.2, 157.4, 153.8, 153.7, 146.8, 138.6, 135.0, 126.0, 124.8, 123.5,
123.0, 116.3, 114.6, 103.6 (2C), 95.4 (2C), 61.0, 56.4 (2C), 55.8, 48.5,
33.2 (2C), 25.5, 24.9 (2C); MS (ESI) m/z 507.2 for [M+1]+; HRMS
(ESI) 507.2253 for [M+H]+ (calcd 507.2238 for C27H31N4O6); HPLC
purity > 99% (MeOH:H2O = 50:50, 0.8 mL/min, tR = 4.20 min).
4.1.8.10. Ethyl
6-methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzofuran-2-carboxylate (17a). Yield: 61%; yellow solid;
m.p.: 169–171 °C; 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 1H), 8.06 (d,
J = 8.8 Hz, 1H), 7.19 (s, 2H), 7.12–7.03 (m, 2H), 4.47 (q, J = 8.0 Hz,
2H), 3.97 (s, 6H), 3.91 (s, 3H), 3.90 (s, 3H), 1.44 (t, J = 8.0 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 161.9, 158.7, 155.1, 153.8 (2C), 147.0,
138.6, 133.2, 126.1, 125.7, 124.1, 122.3, 115.9, 115.5, 103.4 (2C),
95.4, 62.0, 60.9, 56.4 (2C), 55.8, 14.2; MS (ESI) m/z 454.2 for [M
+1]+
;
HRMS (ESI) 454.1623 for [M+H]+ (calcd 454.1609 for
C
23H24N3O7); HPLC purity > 99% (MeOH:H2O = 50:50, 0.8 mL/min,
tR = 4.23 min).
4.1.8.16. 6-Methoxy-N-phenyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzofuran-2-carboxamide (17g). Yield: 58%; yellow solid;
m.p.: 184–176 °C; 1H NMR (600 MHz, CDCl3) δ 9.19 (s, 1H), 8.68 (s,
1H), 8.15 (d, J = 8.4 Hz, 1H), 7.61–7.59 (m, 2H), 7.51–7.49 (m, 2H),
7.18 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 3.96 (s, 6H), 3.90 (s,
3H), 3.89 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 161.6, 156.1, 154.0,
153.7, 146.9, 138.6, 136.9, 134.5, 129.2 (2C), 125.7, 125.2, 124.7,
124.3, 122.8, 120.5 (2C), 116.1, 114.9, 103.6 (2C), 95.4 (2C), 60.9,
56.4, 55.8; MS (ESI) m/z 501.2 for [M+1]+; HRMS (ESI) 501.1781 for
[M+H]+ (calcd 501.1769 for C27H25N4O6); HPLC purity > 99%
(MeOH:H2O = 60:40, 0.8 mL/min, tR = 4.28 min).
4.1.8.11. Isopropyl 6-methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzofuran-2-carboxylate (17b). Yield: 61%: yellow solid;
m.p.: 171–173 °C; 1H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.94 (d,
J = 8.0 Hz, 1H), 7.08 (s, 2H), 7.02 (d, J = 4.0 Hz, 1H), 6.98 (q,
J = 4.0 Hz, 1H), 5.26 (p, J = 8.0 Hz, 1H), 3.90 (s, 6H), 3.84 (s, 6H),
1.33 (d, J = 8.0 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ 161.8, 158.2,
155.1, 153.8 (2C), 138.7, 133.8, 125.7, 123.8 (2C), 116.1, 115.4 (2C),
103.4 (2C), 95.5 (2C), 70.0, 60.9, 56.3 (2C), 55.8, 21.8 (2C); MS (ESI)
m/z 468.2 for [M+1]+; HRMS (ESI) 468.1779 for [M+H]+ (calcd
468.1765 for C24H26N3O7); HPLC purity > 99% (MeOH:H2O = 35:65,
0.8 mL/min, tR = 4.53 min).
4.1.8.17. (6-Methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-yl)(piperidin-1-yl)methanone (17 h). Yield: 62%; yellow
4.1.8.12. 6-Methoxy-N-propyl-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzofuran-2-carboxamide (17c). Yield: 63%; yellow solid;
m.p.: 169–171 °C; 1H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 8.22 (d,
J = 8.0 Hz, 1H), 7.20 (s, 2H), 7.12–6.96 (m, 2H), 6.88 (s, 1H), 3.97 (s,
6H), 3.91 (s, 3H), 3.90 (s, 3H), 3.47 (q, J = 8.0 Hz, 2H), 1.70 (s, 2H),
1.04 (t, J = 8.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 161.2, 158.2,
153.8, 153.6, 146.7, 134.9, 125.9, 124.6, 123.3, 122.9, 116.2, 114.5
(2C), 103.5 (2C), 95.4 (2C), 60.9, 56.3 (2C), 55.8, 40.1, 22.8, 11.3; MS
solid; m.p.: 179–181 °C; 1H NMR (400 MHz, CDCl3)
δ 8.66 (d,
J = 4.0 Hz, 1H), 7.90 (q, J = 4.0 Hz, 1H), 7.10 (s, 2H), 7.00–6.97 (m,
2H), 3.90 (s, 6H), 3.85 (s, 3H), 3.83 (s, 3H), 3.65 (s, 2H), 3.39 (s, 2H),
1.62 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 160.5, 159.0, 154.1, 153.8
(2C), 147.3, 138.6, 137.9, 125.8, 122.7, 121.6, 115.8, 114.4, 103.4
(2C), 95.8, 61.0, 56.4 (2C), 55.8, 48.2, 43.8, 26.5, 25.6, 24.4 (2C); MS
(ESI) m/z 493.2 for [M+1]+; HRMS (ESI) 493.2093 for [M+H]+
(calcd
493.2082
for
C26H29N4O6);
HPLC
purity > 99%
11

Z.-Y. Qi, et al.
BioorganicChemistryxxx(xxxx)xxxx
(MeOH:H2O = 60:40, 0.8 mL/min, tR = 4.22 min).
(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.
4.1.8.18. (6-Methoxy-3-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-
yl)benzofuran-2-yl)(piperidin-1-yl)methanone (17i). Yield: 57%; yellow
solid; m.p.: 183–185 °C; 1H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H), 7.90
(q, J = 4.0 Hz, 1H), 7.11 (s, 2H), 7.03–6.95 (m, 2H), 3.90 (s, 6H), 3.85
(s, 3H), 3.83 (s, 3H), 3.75–3.55 (m, 8H); 13C NMR (150 MHz, CDCl3) δ
160.9, 159.1, 154.1, 153.8 (2C), 147.3, 138.6, 136.8, 125.7, 122.9,
122.0, 115.7, 114.7, 103.5 (2C), 95.7 (2C), 66.8 (2C), 61.0, 56.4 (2C),
55.9, 47.5, 43.1; MS (ESI) m/z 495.2 for [M+1]+; HRMS (ESI)
495.1888 for [M+H]+ (calcd 495.1874 for C25H27N4O7); HPLC
purity 96.1% (MeOH:H2O = 30:70, 0.8 mL/min, tR = 4.08 min).
4.2.5. Western blot analysis
A549 cells (5.0 × 105 cells/dish) were incubated with or without
17g 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, in-
termittently 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 separated by 8–12% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose mem-
branes (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
Then, the membranes were blocked with 5% nonfat dried milk in TBS
containing 1% Tween-20 for 90 min at room temperature and were
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 tem-
perature for 2 h. The blots were developed with enhanced chemilumi-
nescence (Pierce, Rockford, Illinois, USA) and were detected by an
LAS4000 imager (GE Healthcare, Waukesha, Wisconsin, USA).
4.2. Biology
4.2.1. Antiproliferation assays
Cells grown in the logarithmic phase were seeded into 96-well
plates (5 × 103 cells/well) for 24 h, and then exposed to different
concentrations of the test compounds for 72 h. After attached cells were
incubated with 5 mg/mL MTT (Sigma, USA) for another 4 h, the sus-
pension was discarded, and subsequently the dark blue crystals (for-
mazan) were solubilized in dimethyl sulfoxide (DMSO). The absorbance
of the solution at 490 nm was measured using a multifunction micro-
plate reader (Molecular Devices, Flex Station 3), and each experiment
was performed at least in triplicate. IC50 values, which represent the
drug concentrations required to cause 50% cancer cell growth inhibi-
tion, 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).
4.2.2. In vitro tubulin polymerization assay
Pig brain microtubule protein was isolated by three cycles of tem-
perature-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) spectro-
photometer. The plateau absorbance values were used for calculations.
4.2.6. Apoptosis analysis
A549 cells were seeded in 6-well plates (3 × 105 cells/well), in-
cubated in the presence or absence of compound 17g at the indicated
concentrations for 12 h. After incubation, cells were harvested and in-
cubated with 5 μL of Annexin-V/FITC (Keygen Biotech, China) in
binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH
7.4) at room temperature for 15 min. PI solution was then added to the
medium for another 10 min incubation. Almost 10 000 events were
collected for each sample and analyzed by flow cytometry (Beckman
Coulter, Epics XL).
4.2.7. Mitochondrial membrane potential assay
A lipophilic cationic dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-
benzimidazolcarbocyanine (JC-1, Beyotime, China), was used to
monitor the level of MMP in the cells. At normal state, the MMP is high
and JC-1 appears as aggregates, indicated by red fluorescence. When
apoptosis occurs, the MMP is reduced and JC-1 appears as monomers,
indicated by green fluorescence. Two methods including flow cyto-
metry and fluorescence microscopy were used to detect the MMP. For
the fluorescence microscopy detection, A549 cells were plated in 6-well
plates (3 × 105 cells/well), incubated for 24 h, and treated with 17g at
the indicated concentrations for another 6 h. Then, the cells were
stained with 2 μM JC-1 at 37 °C for 30 min and washed with PBS, and
then the cell nuclei were stained with DAPI (Solarbio, China) for 10 min
in the dark. The cell images were immediately visualized on a Zeiss LSM
570 laser scanning confocal microscope (Carl Zeiss, Germany).
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 17g 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,
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. Molecular docking
4.2.4. Cell cycle analysis
In this study, the tubulin structure was downloaded from the Protein
Data Bank (PDB code 1SA0). Using Schrodinger software to prepare the
protein by removing of Mg2+, water molecules and colchicine. Then the
ligand 17g was minimized. The docking procedure was performed by
employing docking program in schrodinger software, and the structural
image was obtained using PyMOL software.
A549 cells were seeded in 6-well plates (3 × 105 cells/well), in-
cubated in the presence or absence of compound 17g at the indicated
concentrations 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
12

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BioorganicChemistryxxx(xxxx)xxxx
Acknowledgements
Marcotullio, A. Gulino, B. Ricci, A. Soriani, A. Santoni, M. Caraglia, S. Porto, E. Da
Pozzo, C. Martini, A. Brancale, L. Marinelli, E. Novellino, S. Vultaggio, M. Varasi,
C. Mercurio, C. Bigogno, G. Dondio, E. Hamel, P. Lavia, R. Silvestri, New indole
tubulin assembly inhibitors cause stable arrest of mitotic progression, enhanced
stimulation of natural killer cell cytotoxic activity, and repression of hedgehog-
dependent cancer, J. Med. Chem. 58 (2015) 5789–5807.
This work was financially supported by the National Natural
Sciences Foundations of China (21672093).
[19] B.L. Flynn, G.S. Gill, D.W. Grobelny, J.H. Chaplin, D. Paul, A.F. Leske,
T.C. Lavranos, D.K. Chalmers, S.A. Charman, E. Kostewicz, D.M. Shackleford,
J. Morizzi, E. Hamel, M.K. Jung, G. Kremmidiotis, Discovery of 7-hydroxy-6-
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Declaration of Competing Interest
The authors declare that they have no competing interests.
Appendix A. Supplementary material
[20] G. Kremmidiotis, A. Leske, J. Simpson, E. Doolin, J. Iglesias, Identification of
plasma biomarker concentration changes resulting from the administration of the
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