Synthesis and biological evaluation of thiabendazole derivatives as anti-angiogenesis and vascular disrupting agents

Article abstract of DOI:10.1016/j.bmc.2015.03.085

Abstract Thiabendazole, already approved by FDA for oral use as an anti-fungal and anti-helminthic drug since 1967, has recently been repurposed as a vascular disrupting agent. By optimization of the structure of the lead compound, we successfully identified compound TBZ-19 and the new derivative is over 100-fold more potent than the lead compound against the growth of four different cell lines (A549, HCT-116, HepG2 and HUVECs). The most potent two candidates TBZ-07 and TBZ-19, exhibiting moderate inhibitory cell proliferation activity, were also verified as anti-angiogenesis and vascular disrupting agents. Therefore, TBZ-07 and TBZ-19 would be promising candidates with vasculature targeting activity and merit further development.

Full text of DOI:10.1016/j.bmc.2015.03.085

Accepted Manuscript
Synthesis and Biological Evaluation of Thiabendazole Derivatives as Anti-an-
giogenesis and Vascular Disrupting Agents
Chao Zhang, Bo Zhong, Simin Yang, Liangkun Pan, Siwang Yu, Zhongjun Li,
Shuchun Li, Bin Su, Xiangbao Meng
PII:
S0968-0896(15)00292-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.03.085
BMC 12211
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
5 February 2015
30 March 2015
31 March 2015
Please cite this article as: Zhang, C., Zhong, B., Yang, S., Pan, L., Yu, S., Li, Z., Li, S., Su, B., Meng, X., Synthesis
and Biological Evaluation of Thiabendazole Derivatives as Anti-angiogenesis and Vascular Disrupting Agents,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.03.085
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Synthesis and Biological Evaluation of
Thiabendazole Derivatives as Anti-angiogenesis and
Vascular Disrupting Agents
Chao Zhang, Bo Zhong, Simin Yang, Liangkun Pan, Siwang Yu, Zhongjun Li, Shuchun Li, Bin Su, and Xiangbao Meng
1

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis and Biological Evaluation of Thiabendazole Derivatives as Anti-
angiogenesis and Vascular Disrupting Agents
Chao Zhanga, Bo Zhongb, Simin Yang a, Liangkun Pana, Siwang Yua, Zhongjun Lia, Shuchun Lia, Bin Sub,
and Xiangbao Menga*
aState Key Laboratory of Natural and Biomimetic Drugs; Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing
100191, China
bDepartment of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH, 44115, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Thiabendazole, already approved by FDA for oral use as an anti-fungal and anti-helminthic drug
since 1967, has recently been repurposed as a vascular disrupting agent. By optimization of the
structure of the lead compound, we successfully identified compound TBZ-19 and the new
derivative is over 100-fold more potent than the lead compound against the growth of four
different cell lines (A549, HCT-116, HepG2 and HUVECs). The most potent two candidates
TBZ-07 and TBZ-19, exhibiting moderate inhibitory cell proliferation activity, were also
verified as anti-angiogenesis and vascular disrupting agents. Therefore, TBZ-07 and TBZ-19
would be promising candidates with vasculature targeting activity and merit further
development.
Received in revised form
Accepted
Available online
Keywords:
thiabendazole
anti-cancer
vascular targeting
tubulin
2015 Elsevier Ltd. All rights reserved
anti-angiogenesis
that is likely to be susceptible to their activity, and the schedule
of treatment. Both approaches are proved to be successful in
defense of cancer as there are several molecules which were
validated to be effective both in preclinical and clinical trials.
Among them, bevazizumab (AvastinTM) has been proved as an
1. Introduction
2
As early as 1980s, Denekamp first proposed the concept that
vascular targeting agents (VTAs) would be promising for the
treatment of cancer.1 Researchers observed that solid tumors
degenerate as tumor blood vessels were naturally blocked and
hypothesized that physically occlusion of blood vessels in tumor
issues by means of VTAs to result in tumor regression. This
antiangiogenic VTA, which is
a recombinant humanized
monoclonal antibody that binds to vascular endothelial cell
growth factor (VEGF) and blocks VEGF interaction with its
corresponding receptors.6,7 While biologics as VDAs is costly
and proved to be unpractical, the small molecule approach has
increased significantly over the past decade and today includes
approximately a dozen compounds that are in human clinical
trials. Many well-described antimitotic agents are derived from
natural sources. Colchicine and Vinca alkaloids, the first tubulin
binding agents discovered, cause destabilization of microtubules
and, as a consequence, induce the cell to undergo apoptosis.8 On
the other hand paclitaxel, a standard antitumor agent originally
extracted from Taxus brevifolia, binds to a different site within
tubulin leading to the stabilization of microtubules.9 Also, a large
number of synthetic or semi-synthetic small molecules has been
proved to be powerful microtubule targeting agents, including
CA-4P10, BNC 10511, isoCA-412, Isoerianin13, azaisoerianin14,
combreatabenzodiazepines15, etc. Therefore, inhibition of
microtubule function using tubulin targeting agents is a validated
approach to anticancer therapy and merits further development to
improve bioavailability, solubility and to overcome emerging
drug resistance in clinic .10,11, 16,17,18
hypothesis was later proved to be effective. VTAs are usually
anti-angiogenic agents
2-5
divided into two separate categories,
(AIAs), which halt the formation of new blood vessels, and
vascular disrupting agents (VDAs) which mainly target
endothelial cells and pericytes of the already established tumor
vasculature, resulting tumor ischemia and necrosis. In addition,
VDAs fall into two general classes referred to as ligand-directed
VDAs and small molecule VDAs.
advantage of antibodies, polypeptides or growth factors which
selectively bind with endothelial cells of tumor issues and
contribute to the necrosis of tumor. Rather than specifically
localizing to the tumor endothelium, these agents take advantage
of the pathophysiological differences between normal and tumor
endothelium to induce selective occlusion of tumor vessels.
While the latter, mainly consists of tubulin targeting agents,
could either inhibit the polymerization of tubulin or stabilized the
formation of microtubule. It is important to emphasize that there
is a very clear distinction between these two approaches based on
key three aspects: the physiologic target, the extension of disease
2
The former type takes
*Corresponding Author: Tel.: 86-10-82801714;
E-mail addresses: xbmeng@bjmu.edu.cn
2

2. Results and discussion
2.1 Synthesis
The synthesis of twenty-four compounds (see Table 1) based
on thiabenzole core was accomplished by condensations of o-
phenyldiamine derivatives with thiazole-4-aldehyde, pydrine-2-
aldehyde or tert-butyloxycarbonyl (4-formylthiazol-5-yl)
carbamate catalysed by sodium pyrosulfite in DMF at 120ºC,
respectively, which is described in Scheme 1. Among these
aldehydes, the synthesis of tert-butyloxycarbonyl (4-
formylthiazol-5-yl) carbamate would be relative complicated, the
synthetic route18 is described in Scheme 2. Synthesis of TBZ-19
was based on the former building block, tert-butyloxycarbonyl
(4-formylthiazol-5-yl) carbamate. It was further condensed with
o-phenyldiamine,
cyclopropylisothiocyanate to afford TBZ-19and the synthetic
deprotected,
reacted
with
route was also described in Scheme 2.
Figure 1. Structure of antimitotic agents with vascular-disrupting
activities.
Review of recent published literature reveals that many anti-
helmintic drug on market have been verified to be anti-mitotic
agents. Among them, albendazole and mebendazole are currently
under development for the treatment of cancer. Albendazole is
well tolerated, relatively safe and has evidence of anti-tumor
effects in patients with advanced cancer.19 While mebendazole is
a potent antitumor agents both in vitro and in vivo due to its
property of inducing mitotic arrest and apoptosis by
depolymerizing tubulin.20 Thiabendazole (TBZ;4-(1H-1,3-
benzodiazol-2-yl)-1,3-thiazole), already approved by the U.S.
Food and Drug Administration (FDA) for systemic oral use in
humans (as an anti-fungal and anti-helminthic treatment), has
recently been verified to be vascular disrupting agent and thus as
a potential complementary therapeutic for use in combination
with current anti-angiogenic therapies.21 However, the IC50 value
of TBZ was as high as over 250 µM to disrupt vascular, which
seems limit its future application as an anti-cancer agent.
Scheme 1. Reagents and conditions: (a) Na2S2O5, DMF, 120ºC,
51-85%; (b) NaH, DMF, r.t. 68-92%.
Table 1. Structures of TBZ derivatives
No.
R1
H
R2
H
R3
H
R4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R5
H
R6
4-thiazole
4-thiazole
4-thiazole
4-thiazole
4-thiazole
4-thiazole
4-thiazole
TBZ
H
Me
H
H
H
TBZ-01
TBZ-02
TBZ-03
TBZ-04
TBZ-05
TBZ-06a
TBZ-07
TBZ-08
TBZ-09
TBZ-10
TBZ-11
TBZ-12
TBZ-13
TBZ-14
TBZ-15
TBZ-16
TBZ-17
TBZ-18
TBZ-19
TBZ-20
Me
H
H
H
Me
H
Me
Cl
H
H
H
H
H
F
H
H
H
OMe
H
H
H
Bz
H
4-thiazole
4-thiazole
H
H
H
H
H
H
H
H
H
NO2
H
H
4-thiazole
4-thiazole
4-thiazole
4-thiazole
4-thiazole
2-Py
H
Me
Bn
pMB
mNB
H
H
H
H
H
Figure 2. Structures of representative benzimidazoles with anti-
helmintic activity.
H
H
Me
H
H
Considering the unique selectivity and effectiveness of VTAs, we
aimed to develop a new class of anticancer drugs bearing dual
biological activity- anti-angiogenesis and vascular disruption,
which is hypothesized to be synergistic for the treatment of
cancer both in period of initiation and progression. Herein, we
report the design, synthesis, and biological evaluation of
thiabendazole derivatives as anticancer agents with vascular
disrupting and anti-angiogenesis activity.
H
H
4-thiazole
4-thiazole
4-thiazole
4-thiazole
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
benzene
H
H
Bn
3

In addition, electron withdrawing group such as Cl or CF3 at 6
position showed moderate inhibitory activity, whereas with
benzoyl substitution the compound exhibited the best activity. 5,
6 fused benzene group derivative TBZ-08 also showed moderate
inhibitory activity against HUVECs at 100 µM. 3, 4, 5-
trimethoxy benzyl substitution at 1 position showed better
activity compared with parent compound TBZ. Among all
compounds, TBZ-19 bearing cyclopropanylthiourea substitution
at 5 position of thiazole showed the best inhibitory activity in
these assays.
H
H
H
H
H
H
H
H
H
TBZ-21
TBZ-22
2-Py
2-Py
a. tautomer was determined by NMR spectroscopy analysis
b. Bn= benzyl; pMB= p-methoxyl benzyl; m-NB= m-nitro benzyl; 2-py= 2-
pyridyl; Bz= benzoyl.
It was encouraging that compound TBZ-07 and TBZ-19
displayed significant improvement in potency both at low and
high concentration when compared with the parental compound
TBZ. Furthermore, we determined the IC50 values of these two
compounds against HUVECs, HCT116, HepG2, and A549 cell
lines and the data was shown in Table 3.
Table 3. IC50 (µM) values of selected compounds toward four
cancer cell linesa
Scheme 2. Reagents and conditions: (a) NaNO2, H3PO4, H2O,
51%; (b) Na2S2O4, aqueous NaHCO3, CH2Cl2, 80%; (c) HCOOH,
(CH3CO)2O, N2, 0 ºC, 93%; (d) Lawesson's regent, toluene,
reflux, 41%; (e) (Boc)2O, DMAP, 0 ºC, anhydrous CH2Cl2, 24%;
(f) DIBAL-H, -78ºC, Ar, anhydrous CH2Cl2, 62%; (g) o-
phenylenediamine, Na2S2O5, DMF, 120ºC, 94%; (h) 10% TFA,
DCM, 99%; (i) cyclopropylisothiocyanate, pyrdine, reflux, 99%.
Compd. HUVEC
>100
HCT116
HepG2
A549
>100
>100
>100
TBZ
TBZ-07 9.22 5.42 2.46 1.15 7.58 0.43 7.70 1.53
1.23 0.61 0.43 0.08 0.79 0.12 1.02 0.17
TBZ-19
a MTT assay was used for evaluation, and values were expressed as mean IC50 of
the triplicate experiments.
2.2 Biology
2.2.2
Anti-angiogenesis Activity Assays
To further investigate the anti-cancer activity of TBZ-07 and
TBZ-19, both compounds were determined to interfere with the
angiogenesis process. One of the most widely used in vitro
assays to model the reorganization stage of angiogenesis is the
tube formation assay. This assay measures the ability of
endothelial cells, plated at subconfluent densities with the
appropriate extracellular matrix support, to form capillary-like
structures.19,20 Therefore, HUVEC tube formation had been used
first as a model to test in vitro angiogenesis, as vascular
endothelial tube network formed in vitro has many similarities
with capillary vessels formed in vivo. Molecules significantly
disturb the tube formation process are promising to be anti-
angiogenic agents. If the process being disturbed, partially
dissected tube remains or even totally blocked tubes could be
observed. While the HUVECs were plated, elongated and robust
tube-like structures were well established after incubation for 1 h
in the negative control group; but treatment of HUVECs with
compounds TBZ-07 and TBZ-19 at the concentration of 0.03, 0.1
0.33, 1.00, 3.00, 10.0 µM inhibited the formation of tubular
structures in different ranks. Encouragingly, TBZ-07 and TBZ-19
could significantly inhibit the tube formation process of
HUVECs at 0.33 µM, while positive control sunitinib partially
inhibited its formation at 0.033 µM and completely blocked it at
0.33 µM (Fig. 3).
2.2.1
Cell viability assays
We firstly evaluated anti-proliferative activity of the newly
synthesized compounds in vitro. These compounds were tested
with human umbilical vein endothelial cells (HUVECs).
Inhibitory data of compounds TBZ-01 to TBZ-24 at different
concentrations (10 µM and 100 µM) to HUVECs are listed in
Table 2.
Table 2. Inhibition rate(%) of TBZ derivatives against HUVECsa
Compd.
TBZ
10µM
0
100µM
11
56
59
0
TBZ-01
TBZ-02
TBZ-03
TBZ-04
TBZ-05
TBZ-06
TBZ-07
TBZ-08
TBZ-09
TBZ-10
TBZ-11
TBZ-12
TBZ-13
TBZ-14
TBZ-15
TBZ-16
TBZ-17
TBZ-18
TBZ-19
TBZ-20
TBZ-21
TBZ-22
TBZ-23
TBZ-24
3
8
0
7
53
28
19
58
59
30
3
2
3
50
2
7
0
4
46
41
21
10
45
25
57
51
59
38
8
0
2
7
compounds
Con.
9
10
5
µM
TBZ-07
TBZ-19
sunitinib
2
63
7
0
9
8
0
8
0
7
0
0.03
aall assays were run in triplicate
Based on the above anti-proliferative data, a preliminary SAR
was summarized. Compounds with methyl group at 6 or 7
position showed better potency at 100 µM compared with 10 µM.
4

3.00
10.0
0.10
0.33
1.00
3.00
10.0
Figure 4. Rat aortic ring assay
2.2.3 Tubulin Intensity Assays
We then move on to examine the effects of these two compounds
on microtubule polymerization. We began by assessing the
integrity of the microtubule network by immunofluorescence
microscopy, using an antibody against α-tubulin. Tubulin
intensity assays were carried on to examine the ability of TBZ-07
and TBZ-19 to interfere with microtuble assembly. As shown in
Fig. 5, the DMSO-treated control cells displayed a well –
developed array of microtubules radiating from the juxtanuclear
microtubule organizing center. In contrast, both TBZ-07 can
TBZ-19 can inhibit the formation of tubulin at 10 µM as they
both exhibited a tubulin staining pattern that was diffuse and
disorganized. In addition, TBZ at 250µM and vinblastine at 50
nM showed similar effect on microtubule organization of A549
cell line.
Figure 3. Tube formation assay of HUVEC cells
Considering that rat thoracic aorta rings (TARs) model is more
close to in vivo condition compared with HUVEC tube formation
model in view of multi-steps involved in angiogenesis: sprouting,
proliferation, migration and differentiation, TARs assay was then
employed to evaluate the antiangiogenic activity of
compounds.21,22 In this assay, aortic rings cultured in collagen gel
give rise to microvascular networks composed of branching
endothelial channels. Compounds with anti-angiogenic activity
could significantly inhibit the formation and number of branches
in the aortic rings. According to the experiment, TBZ-07 and
TBZ-19 significantly inhibited the outgrowth of microvessels at 3
to 10 µM in a concentration-dependent manner, while positive
control sunitinib demonstrated almost complete inhibition of
angiogenesis at 0.33 µM (Fig.4).
TBZ-07 10 μM
Control
compounds
Con.
µM
TBZ-07
TBZ-19
sunitinib
0
TBZ 250 μM
TBZ-19 10 μM
0.03
0.10
0.33
1.00
Vincristine 50 nM
Figure 5. Effect of selective compounds on tubulin intensity in
A549 cells.
2.2.4 Tubulin polymerization assays
5

using commercial silica gel HSGF254 plates. Column
chromatography was performed on Silica Gel 60(E. Merck, 230-
400 mesh). Melting points were measured on an X-5 micro-
melting point apparatus and were uncorrected. The 1H (400
MHz) and 13C NMR (100.6 MHz) spectra were recorded with
BrukerAM-400 spectrometer in CDCl3 and DMSO-d6 solution.
Chemical shifts were referenced with tetramethylsilane (TMS).
The HR-ESI-MS data were measured on a Bruker Apex IV
FTMS.
Tubulin can be affected in two different manners. Taxanes and
epothilones stabilize tubulin polymerization, whereas vinca
alkaloids, halichondrins, and colchicine inhibit tubulin
polymerization.23-26 In order to have a better understanding of the
binding manner, tubulin inhibitory activity of TBZ-07 and TBZ-
19 was determined with tubulin polymerization assay by
monitoring the absorptions at 340 nm. In this assay, we observed
here that TBZ-07 and TBZ-19 can act as microtubule
depolymerizing agents time-dependently at 5 µM concentration.
As illustrated in Fig.6, the curve of TBZ-07 and TBZ-19 under
the curve of DMSO demonstrated that these two compounds
acted in a similar mechanism with Nacodazole, a well-known
tubulin inhibitor used as a positive control for the assay, to inhibit
tubulin polymerization.27 While Taxol at 10 µM which appears
above the control line means its microtubule stabilization effect.
Therefore, this study indicates that TBZ-07 and TBZ-19 bind at
the colchicine site on tubulin. To sum up, the above results
indicate that these compounds play anti-angiogenic and vascular-
disrupting role through inhibition of the proliferative and tube
formation of HUVECs, and microtubule depolymerization during
mitosis, respectively.
4.1.2. General procedure for the synthesis of TBZ compounds
(TBZ-1-9, 14-17, 23-24).
A solution of substituted o-phenyldiamine (1.0eq), thiazole-4-
aldehyde or pydrine-2-aldehyde (1.0eq) with sodium pyrosulfite
in DMF was stirred at 120 ºC overnight. On completion of the
reaction monitored by TLC, the solvent was evaporated and the
residue was purified by silica gel chromatography by
DCM/MeOH system to afford the final product. If necessary, the
crude product could be recrystallized in DCM or dichloroethane
to afford pure sample.
4.1.3. General procedure for the synthesis of TBZ compounds
(TBZ-10, 11, 12, 13, 18, 20).
To a solution of TBZ (1.0eq) (for TBZ-10,11,12,13,18) or 2-
phenyl-1H-benzo[d]imidazole (1.0eq) (for TBZ-20) with sodium
hydride (1.2eq) in DMF for 15 minutes, then corresponding
iodomethane, benzyl bromide, 4-methoxybenzylchloride, 3-
nitrobenzyl bromide, 5-(chloromethyl)-1,2,3-trimethoxybenzene
(1.0eq) was added slowly at room temperature. On completion of
the reaction monitored by TLC, the solvent was evaporated and
the residue was purified by silica gel chromatography by
DCM/MeOH system to afford the final product.
0.35
0.30
0.25
0.20
0.15
4.1.4. Procedure for the synthesis of TBZ-20
0.10
A solution of TBZ (1.0eq), 2-bromo-pyridine (2.0eq), Cs2CO3
(2.0eq), CuI (1.0eq), 1,3-di(pyridin-2-yl)propane-1,3-dione
(1.0eq) in DMF was stirred at room temperature under Ar for 30
minutes, then the solution was heated to 110ºC for 48 hours. The
precipitate was removed off by celite, the filtrate was diluted
with EtOAc for 3 times, washed by brine, dried over MgSO4，
purified by silica gel chromatography to afford final product
TBZ-20.
DMSO
0.05
TBZ-07(5µM)
TBZ-19(5µM)
Nocodazole(3µM)
Taxol (10uM)
0.00
0
4
8
12
16
Time (min)
Figure 6. Tubulin polymerization in the presence of TBZ-07,
TBZ-19, Nocodazole, and Taoxl
4.1.5. Procedure for the synthesis of TBZ-21
Following the former mentioned procedure for the synthesis of
TBZ compounds, TBZ-21 was synthesized by the general
procedure, the Boc group was deprotected by 5% TFA in DCM,
the solvent was evaporated off and the remaining was neutralized
by aqueous NaHCO3, extracted by DCM, the organic layer was
combined and dried over MgSO4. DCM was evaporated to afford
TBZ-21 without further purification and used directly in the next
step.
3. Conclusion
In conclusion, we successfully synthesized 24 derivatives of lead
compound TBZ and optimized the anti-angiogenic and tumor
vasculature disruption effect. Among all the synthesized
compounds, TBZ-07 and TBZ-19 showed superiority compared
to the parental TBZ. In particularly, TBZ-19 was proved to be the
most potent one, with nearly 100-fold improvement of potency
against the growth of HUVECs, A549, HCT116, and HepG2 cell
lines. Biological evaluations also confirm their mechanism of
anti-angiogenic and tumor vasculature disruption effect.
Consequently, compound TBZ-07 and TBZ-19 would be
promising anticancer agents and merit further investigation.
4.1.6. 4-(1H-benzo[d]imidazol-2-yl) thiazole (TBZ). White solid;
yield 85%; 1H-NMR (400 MHz, DMSO-d6) δ 9.32 (d, J = 1.9 Hz,
1H), 8.45 (d, J = 1.9 Hz, 1H), 7.61 – 7.54 (m, 2H), 7.22 – 7.14 (m,
2H).
4.1.7. 4-(5-methyl-1H-benzo[d]imidazole-2-yl) thiazole (TBZ-
01). Brown solid; yield 58%; 1H-NMR (400 MHz, DMSO-
d6+5%CF3COOH) δ 9.51 (d, J = 1.4 Hz, 1H), 8.93 (d, J = 1.4 Hz,
1H), 7.73 (d, J = 8.4 Hz, 1H), 7.63 (s, 1H), 7.43 (d, J = 8.4 Hz,
1H). note : the signal of methyl group was buried in DMSO.
4. Experiment Section
4.1 Chemistry
4.1.1. General
4.1.8. 4-(4-methyl-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
02). Brown solid; yield 63%; 1H-NMR (400 MHz, DMSO-
d6+5%CF3COOH) δ 9.48 (d, J = 1.8 Hz, 1H), 8.98 (d, J = 1.9 Hz,
Unless otherwise noted, all reagents were obtained from
commercial suppliersand used without further purification.
Reactions were monitored by thin-layer chromatography (TLC)
6

1H), 7.60 (d, J = 8.2 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.35 (d, J
= 7.4 Hz, 1H), 2.64 (s, 3H).
4.1.19.4-(1-(3-nitrobenzyl)-1H-benzo[d]imidazol-2-yl) thiazole
(TBZ-13). Light brown solid; yield 88%; H-NMR (400 MHz,
1
CDCl3) δ 8.87 (d, J = 2.0 Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.16
(s, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.45 –
7.38 (m, 2H), 7.35 – 7.27 (m, 3H), 6.16 (s, 2H).; 13C-NMR (101
MHz, CDCl3) δ 153.26, 148.45, 146.63, 143.06, 139.37, 135.62,
132.78, 129.75, 123.68, 123.24, 122.65, 122.04, 121.70, 120.05,
110.01, 77.38, 77.06, 76.74, 47.94. HR-EI-MS ：Calcd for
C17H13N4O2S [M+H]+: 337.07537; found: 337.07515.
4.1.9. 4-(5, 6-dimethyl-1H-benzo[d]imidazol-2-yl) thiazole
(TBZ-03). Brown solid; yield 80%; 1H-NMR (400 MHz, DMSO-
d6+5%CF3COOH) δ 9.48 (dd, J = 7.7, 1.8 Hz, 1H), 8.86 (d, J =
1.8 Hz, 1H), 7.57 (s, 2H), 2.39 (s, 6H).
4.1.10. 4-(6-chloro-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
04). Grey solid；yield 62%；1H-NMR (400 MHz, DMSO-d6) δ
13.18 (s, 1H), 9.34 (s, 1H), 8.49 (d, J = 1.4 Hz, 1H), 7.58 (s, 2H),
7.23 (dd, J = 8.5, 1.6 Hz, 1H).
4.1.20.5-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (TBZ-
14). Brown solid; yield 62%; 1H-NMR(400 MHz, DMSO-d6-
5%TFA, ppm): 2,51 (3H, s), 7,42 (1H, d, J=8.2 Hz), 7,63 (1H, s),
7,72-7,77 (2H,m), 8,22 (1H, td, J=7.9, 1.6 Hz), 8,41 (1H, d, J=7.9
Hz), 8,91 (1H, d, 4.1 Hz).
4.1.11. 4-(6-fluoro-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
05). Brown solid; yield 67%; H-NMR (400 MHz, DMSO-d6-
1
5%TFA) δ 9.48 (d, J = 1.8 Hz, 1H), 8.90 (d, J = 1.8 Hz, 1H), 7.84
(dd, J = 9.0, 4.5 Hz, 1H), 7.66 (dd, J = 8.5, 2.4 Hz, 1H), 7.43 (td,
J = 9.4, 2.5 Hz, 1H).
4.1.21.
Methyl
2-(thiazol-4-yl)-1H-benzo[d]imidazole-5-
carboxylate (TBZ-15). Pale yellow solid; m.p. 110-111ºC; yield
62%; 1H-NMR (400 MHz, DMSO) δ 13.36 (s, 1H), 9.37 (s, 1H),
8.56 (s, 1H), 8.20 (s, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.67 (s, 1H),
3.88 (s, 3H).; 13C-NMR (101 MHz, DMSO) δ 155.88, 155.39,
4.1.12.4-(6-methoxy-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
1
06). Brown solid; yield 65%; H-NMR (400 MHz, DMSO-d6-
5%TFA) δ 9.48 (d, J = 1.8 Hz, 1H), 8.86 (d, J = 1.8 Hz, 1H), 7.71
147.23, 118.48, 111.84, 55.41.
; HR-EI-MS: Calcd for
(d, J = 9.0 Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.18 (dd, J = 9.0, 2.4
C12H10N3O2S [M+H]+: 260.04882, found: 260.04898.
Hz, 1H), 3.87 (s, 3H).； 13C-NMR (101 MHz, DMSO-d6) δ
167.18, 156.33, 150.26, 149.52, 147.79, 146.91, 143.87, 138.43,
134.56, 124.26, 124.13, 123.91, 123.41, 121.43, 121.07, 120.95,
119.05, 114.05, 112.27, 52.42.; HR-EI-MS: Calcd for
C11H10N3OS[M+H]+: 232.05391; found: 232.053892.
4.1.22. Methyl 2-(thiazol-4-yl)-1H-benzo[d]imidazole-7-
carboxylate (TBZ-16). Light brown solid ; m.p. 141-143 ºC;
yield 65%; 1H-NMR (400 MHz, CDCl3) δ 11.05 (s, 1H), 8.91 (d,
J = 1.5 Hz, 1H), 8.31 (d, J = 1.5 Hz, 1H), 7.97 (dd, J = 19.3, 7.8
Hz, 2H), 7.34 (t, J = 7.8 Hz, 1H), 4.03 (s, 3H).; 13C-NMR (101
MHz, DMSO) δ 165.72, 155.64, 148.32, 146.04, 144.71, 133.33,
124.66, 124.42, 121.82, 121.05, 113.92, 52.15.; HR-EI-MS:
calcd for C12H10N3O2S[M+H]+: 260.04882, found: 260.04905.
4.1.13.Phenyl(2-(thiazol-4-yl)-1H-benzo[d]imidazol-5-
yl)methanone (TBZ-07). Yellow solid; yield 51%; 1H-NMR (400
MHz, CDCl3) δ 8.92 (s, 1H), 8.35 (s, 1H), 8.14 (s, 1H), 7.84 (d, J
= 7.5 Hz, 3H), 7.72 (s, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.49 (t, J =
7.5 Hz, 2H).
4.1.23.4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) thiazole
1
(TBZ-17). Light brown solid; m.p. 153-154 ºC; yield 55%; H-
4.1.14. 4-(1H-naphtho[2,3-d]imidazol-2-yl) thiazole (TBZ-08).
Off-white solid; yield 65%; 1H-NMR (400 MHz, DMSO-d6-
5%TFA) δ 9.49 (d, J = 1.2 Hz, 1H), 9.05 (d, J = 1.4 Hz, 1H), 8.30
(s, 2H), 8.13 (dd, J = 6.3, 3.3 Hz, 2H), 7.52 (dd, J = 6.5, 3.2 Hz,
2H).; 13C-NMR (101 MHz, DMSO-d6-5%TFA) δ 159.0, 158.6,
158.2, 157.8, 146.9, 139.3, 131.2, 130.7, 128.8, 127.9, 125.6,
116.4, 113.5, 110.; HR-EI-MS: Calcd for C14H10N3S[M+H]+:
252.05899, found: 252.05902.
NMR (400 MHz, CDCl3) δ 8.92 (d, J = 1.9 Hz, 1H), 8.36 (d, J =
1.9 Hz, 1H), 7.95 (s, 1H), 7.63 (d, J = 51.3 Hz, 1H), 7.54 (d, J =
8.2 Hz, 1H). ; 13C-NMR (101 MHz, DMSO) δ 208.47, 156.39,
149.93, 146.78, 129.69, 126.85, 124.15, 123.45, 123.14, 121.40,
119.35; HR-EI-MS：Calcd for C11H7F3N3S [M+H]+: 270.03073;
found: 270.03044.
4.1.24.4-(1-(3, 4, 5-trimethoxybenzyl)-1H-benzo[d]imidazol-2-yl)
thiazole (TBZ-18). Pale yellow solid; m.p. 147-149 ºC; yield
4.1.15. 4-(6-nitro-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-09).
Yellow solid; yield 62%; 1H-NMR (400 MHz, DMSO-d6-
5%TFA) δ 9.43 (s, 1H), 8.79 (s, 1H), 8.52 (s, 1H), 8.24 (d, J =
8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H).
1
68%; H-NMR (400 MHz, CDCl3) δ 8.92 (d, J = 2.1 Hz, 1H),
8.34 (d, J = 2.1 Hz, 1H), 7.87 – 7.75 (m, 1H), 7.36 (dd, J = 6.8,
1.2 Hz, 1H), 7.33 – 7.24 (m, 2H), 6.41 (s, 2H), 5.98 (s, 2H), 3.78
(s, 3H), 3.69 (s, 6H).; 13C-NMR (101 MHz, CDCl3) δ 153.53,
153.17, 136.05, 132.77, 123.46, 123.03, 121.58, 119.89, 110.75,
4.1.16. 4-(1-methyl-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
10). White solid; yield 90%; 1H-NMR (400 MHz, CDCl3) δ 8.93
(s, 1H), 8.31 (s, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.42 (d, J = 7.1 Hz,
1H), 7.36 – 7.28 (m, 2H), 4.23 (s, 3H).
104.06, 60.91, 56.14, 48.77. HR-EI-MS ： Calcd for
C20H20N3O3S [M+H]+: 382.12199; found: 382.12128.
4.1.25.1-(4-(1H-benzo[d]imidazol-2-yl)
thiazol-5-yl)-3-
4.1.17. 4-(1-benzyl-1H-benzo[d]imidazol-2-yl) thiazole (TBZ-
11). White solid; yield 87%; 1H-NMR (400 MHz, CDCl3) δ 8.87
(d, J = 2.0 Hz, 1H), 8.32 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 7.8 Hz,
1H), 7.34 – 7.28 (m, 2H), 7.26 – 7.21 (m, 4H), 7.15 (d, J = 6.6 Hz,
2H), 6.08 (s, 2H).
cyclopropylthiourea (TBZ-19). Light yellow solid; m.p. 244-246
ºC; yield 83%; 1H-NMR (400 MHz, DMSO-d6) δ 13.31 (s, 1H),
13.12 (s, 1H), 9.34 (s, 1H), 8.64 (s, 1H), 7.68 – 7.46 (m, 2H),
7.24 (s, 2H), 2.91 (d, J = 16.8 Hz, 1H), 1.17 (s, 2H), 0.79 (s, 2H).;
13C-NMR (101 MHz, DMSO-d6) δ 178.13, 148.69, 145.31,
142.38, 139.62, 133.19, 128.63, 122.73, 121.99, 117.86, 111.71,
24.63, 7.54. ; HR-EI-MS ：Calcd for C14H14N5S2 [M+H]+:
316.06851; found: 316.06893.
4.1.18.4-(1-(4-methoxybenzyl)-1H-benzo[d]imidazol-2-yl)
thiazole (TBZ-12). White solid; m.p. 150-152ºC; yield 82%;1H-
NMR (400 MHz, CDCl3) δ 8.86 (d, J = 2.1 Hz, 1H), 8.30 (d, J =
2.1 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.34 – 7.20 (m, 3H), 7.10
(d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.7 Hz, 2H), 5.97 (s, 2H), 3.70 (s,
3H).; 13C-NMR (101 MHz, CDCl3) δ 158.98, 152.98, 147.89,
146.86, 143.08, 135.96, 129.16, 128.12, 123.22, 122.77, 121.32,
119.74, 114.05, 110.71, 77.43, 77.11, 76.79, 55.21, 47.98.; HR-
4.1.26. 1-benzyl-2-phenyl-1H-benzo[d]imidazole (TBZ-20).
White solid; yield 92%; 1H-NMR (400 MHz, CDCl3) δ 7.87 (d, J
= 8.0 Hz, 1H), 7.72 – 7.65 (m, 2H), 7.48 – 7.41 (m, 3H), 7.35 –
7.26 (m, 4H), 7.25 – 7.19 (m, 2H), 7.13 – 7.06 (m, 2H), 5.44 (s,
2H).
EI-MS ： Calcd for C18H16N3OS[M+H]+: 322.10086; found:
322.10142.
4.1.27. 4-(1H-benzo[d]imidazol-2-yl) thiazol-5-amine (TBZ-21).
Light yellow solid; yield 99%; H-NMR (400 MHz, DMSO) δ
1
7

12.47 (s, 1H), 8.22 (s, 1H), 7.55 (s, 1H), 7.41 (s, 3H), 7.20 – 7.07
(m, 2H). ; HR-EI-MS：Calcd for C10H9N4S [M+H]+: 217.05424;
found 217.05412.
3, every 10 s/min 3 to min 5, every 30 s/min 5 to min10, and
every 60 s/min 10 to min 17).
4.2.5. Tubulin intensity assay
4.1.28. 4-(1-(pyridin-2-yl)-1H-benzo[d]imidazol-2-yl) thiazole
1
(TBZ-22). Brown solid; 177-178 ºC; yield 73%; H-NMR (400
A549 cells were treatment for 24h in the presence of 10µM,
25µM of TBZ-19 and TBZ-07, 50nM vinblastine, 250µM TBZ,
and stained for α-tubulin(red) with TRITC and nucleus(green)
with Hoechst 33258, images were taken by Operetta(20×).
MHz, CDCl3) δ 8.64 – 8.59 (m, 2H), 8.09 (d, J = 2.0 Hz, 1H),
7.86 (ddd, J = 13.1, 6.7, 2.7 Hz, 2H), 7.42 – 7.36 (m, 2H), 7.36 –
7.27 (m, 3H). ; 13C-NMR (101 MHz, DMSO) δ 154.52, 149.81,
149.16, 146.55, 146.12, 142.35, 138.93, 135.67, 123.77, 123.75,
123.11, 122.42, 121.77, 119.38, 111.09. ; HR-EI-MS：Calcd for
C15H11N4S [M+H]+: 279.06989; found: 279.06971 .
Acknowledgement
We gratefully acknowledge the National Natural Science
Foundation of China (NSFC No. 81273370) for generous
financial support.
4.1.29. 4-(1H-imidazo[4,5-c]pyridin-2-yl) thiazole (TBZ-23).
White solid; yield 62%; 1H-NMR (400 MHz, DMSO-d6) δ 9.39 (t,
J = 9.8 Hz, 1H), 8.93 (s, 1H), 8.60 (t, J = 7.9 Hz, 1H), 8.32 (d, J =
5.5 Hz, 1H), 7.56 (s, 1H).
References and Notes
4.1.30. 4-(1H-imidazo[4,5-b]pyridin-2-yl) thiazole (TBZ-24).
Purple solid; yield 55%; 1H-NMR (400 MHz, DMSO-d6-5%TFA)
δ 9.40 (s, 1H), 8.84 (s, 1H), 8.63 (d, J = 5.3 Hz, 1H), 8.45 (d, J =
7.9 Hz, 1H), 7.75 – 7.37 (m, 1H).
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9