Synthesis and biological evaluation of 2-amino-1-thiazolyl imidazoles as orally active anticancer agents

Article abstract of DOI:10.1007/s10637-010-9547-7

Designed from a high throughput screened hit compound, novel 2-amino-1-thiazolyl imidazoles were synthesized and demonstrated cytotoxicity against human cancer cells. 1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)- 1Himidazol- 2-amine (compound 2), a 2-amino-1-thiazolyl imidazole, inhibited tubulin polymerization, interacted with the colchicine-binding sites of tubulins, and caused cell cycle arrest at the G₂/M phase in human gastric cancer cells. Disruption of the microtubule structure in cancer cells by compound 2 was also observed. Compound 2 concentration-dependently inhibited the proliferation of cancer cells in histocultured human gastric and colorectal tumors. Given orally, compound 2 prolonged the lifespans of leukemia mice intraperitoneally inoculated with the murine P388 leukemic cells. We report 2-amino-1-thiazolyl imidazoles as a novel class of orally active microtubuledestabilizing anticancer agents. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s10637-010-9547-7

Invest New Drugs (2012) 30:164–175
DOI 10.1007/s10637-010-9547-7
PRECLINICAL STUDIES
Synthesis and biological evaluation of 2-amino-1-thiazolyl
imidazoles as orally active anticancer agents
Wen-Tai Li & Der-Ren Hwang & Jen-Shin Song & Ching-Ping Chen & Tung-Wei Chen &
Chi-Hung Lin & Jiunn-Jye Chuu & Tzu-Wen Lien & Tsu-An Hsu & Chen-Lung Huang &
Huan-Yi Tseng & Chu-Chung Lin & Heng-Liang Lin & Chung-Ming Chang &
Yu-Sheng Chao & Chiung-Tong Chen
Received: 14 July 2010 /Accepted: 21 September 2010 /Published online: 2 October 2010
#
Springer Science+Business Media, LLC 2010
Summary Designed from a high throughput screened hit
compound, novel 2-amino-1-thiazolyl imidazoles were
synthesized and demonstrated cytotoxicity against human
cancer cells. 1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)-1H-
imidazol-2-amine (compound 2), a 2-amino-1-thiazolyl
imidazole, inhibited tubulin polymerization, interacted with
the colchicine-binding sites of tubulins, and caused cell
cycle arrest at the G2/M phase in human gastric cancer
cells. Disruption of the microtubule structure in cancer cells
by compound 2 was also observed. Compound 2
concentration-dependently inhibited the proliferation of
cancer cells in histocultured human gastric and colorectal
tumors. Given orally, compound 2 prolonged the lifespans
of leukemia mice intraperitoneally inoculated with the
murine P388 leukemic cells. We report 2-amino-1-thiazolyl
imidazoles as a novel class of orally active microtubule-
destabilizing anticancer agents.
Introduction
Microtubules, the important cytoskeletal filaments in cells,
are assembled by polymerized tubulins whose normal
arrangement is essential for cell replication functions.
Microtubule is one of the validated molecular targets
through which anticancer agents are in action [1, 2]. The
tubulin binding agents interact with the tubulins on the
binding sites and cause aberrations in the tubulin assembly
kinetics and thus altered stabilities of the microtubule
structure. A number of microtubule-targeted tubulin bind-
ing agents are clinically effective drugs in cancer treatment
[3–6]. Paclitaxel, docetaxel, and epothilones bind to the
taxane-binding site and stabilize microtubules [7, 8]. Vinca
alkaloids vincristine, vinblastine, and a third-generation
Vinca alkaloid vinorelbine bind to the Vinca domain, and
inhibit tubulin polymerization or interfering with microtu-
bule disassembly disrupts several cellular functions [4, 9].
Moreover, CA-4 and BPR0L075 are the colchicine-site
binders that inhibit microtubule polymerization [5, 10–12].
All these above-mentioned tubulin interacting drugs and
investigational agents are available only in injectable
dosage forms. Compared to parenteral dosing routes, the
oral dosing route is better in its patient compliance, ease of
use, and low cost [13, 14]. Searching for orally active
anticancer agents including tubulin binders [3] has therefore
been a major focus of research in the discovery of
anticancer drugs.
.
.
.
Keywords Microtubule Cell cycle arrest Apoptosis
.
.
Cytotoxicity Orally active cancer therapeutics Leukemia
:
:
:
:
:
W.-T. Li D.-R. Hwang J.-S. Song C.-P. Chen T.-W. Chen
:
:
:
:
:
J.-J. Chuu T.-W. Lien T.-A. Hsu C.-L. Huang H.-Y. Tseng
C.-C. Lin H.-L. Lin C.-M. Chang Y.-S. Chao
:
:
:
:
C.-T. Chen (*)
Institute of Biotechnology and Pharmaceutical Research,
National Health Research Institutes,
35 Keyan Road, Zhunan, Miaoli 35053, Taiwan
e-mail: ctchen@nhri.org.tw
High-throughput screening (HTS) platform is a powerful
tool for indentifying hit compounds with potential biolog-
ical activities [15, 16]. By using cytotoxicity as the primary
screening system, we had identified a cytotoxic HTS hit
compound, 2-((1E)-(1-(1-phenylethylideneamino)-4-phe-
nyl-1H-imidazol-2-ylimino)methyl)-4,6-dibromophenol.
:
T.-W. Chen C.-H. Lin
Institute of Biophotonics, National Yang Ming University,
Shih-Pai, Taipei 11221, Taiwan

Invest New Drugs (2012) 30:164–175
165
The hit compound consists of a core amino imidazole
structure. It has been demonstrated that compounds
containing an amino imidazole structure possess anticancer
activities [17, 18]. Further modified from the chemical
skeleton of this hit compound, novel core chemical
scaffolds containing amino imidazole have been designed
and a number of the structural derivatives have been
synthesized and evaluated their anticancer activities, in-
cluding a novel class of orally active anticancer agents
2-amino-1-arylidenamino imidazoles previously reported
[19]. These 2-amino-1-arylidenamino compounds consist
of an (E)-exocyclic imine with acceptable chemical stability
for exerting proper oral bioavailability and thus the in vivo
activities [19]. Nonetheless, imine moiety could be metab-
olized by liver microsomal enzymes in vivo [20] and the
imine metabolic mechanism has been utilized to design
prodrugs [21]. Heteroaromatic rings have been commonly
incorporated as bioisosteres into new chemical entities
which often show greater pharmacological activities [22, 23],
and metabolic stabilities. [24]. Pietrancosta et al reported
imino-tetrahydro-benzothiazole consisted of an imine in
cyclic form is more potent in p53 inhibition than its open
form [22]. It is therefore interested to know whether a
compound designed by replacing the free secondary imine of
the 2-amino-1-arylidenamino imidazoles by a more stable
heterocyclic aromatic ring such as thiazole ring retains its
biological activities (Chart 1).
guanidine 1 in 87% yield [25]. 2-Bromo-1-(thiophen-2-yl)
ethanone was synthesized from the bromination of 1-
(thiophen-2-yl)ethanone according to the previously
reported procedure [26]. Subsequently, cyclization reaction
of benzyl guanidine 1 and 2-bromo-1-(thiophen-2-yl)etha-
none was conducted by refluxing the reaction mixtures at
80°C to afford 2-amino-1-thiazolyl imidazole 2 in 62%
yield [27].
All reactions were conducted in dried glassware in an
oven at 120°C overnight and cooled in a desiccator over
anhydrous CaSO4 or silica gel. All reagents were used as
received from the commercial suppliers unless otherwise
stated. Tetrahydrofuran was distilled from sodium/benzo-
phenone ketyl under a nitrogen atmosphere. Other solvents,
including acetone, dichloromethane, and ethanol, were
distilled over CaH2 under nitrogen. The proton NMR
spectra were obtained on a Varian Mer-Vx-300 (300 MHz)
spectrometer. Chloroform-d and DMSO-d6 of spectrograde
were used as solvents. All NMR chemical shifts were
reported as δ values in parts per million (ppm), and coupling
constants (J) were given in hertz (Hz). The splitting pattern
abbreviations are as follows: s, singlet; d, doublet; t, triplet;
q, quartet; br, broad; m, unresolved multiplet due to the field
strength of the instrument; dd, doublet of doublet; dt, doublet
of triplet; and ddd, doublet of doublet of doublet. Mass
spectra were carried out on a Hewlett-Packard (Model 1100
MSD) mass spectrometer. Purification was performed by
using preparative separations in gravity column chromatog-
raphy (Merck silica gel 60, particle size of 230–400 mesh).
Analytical TLC was carried out on precoated plates
(Merck silica gel 60, F254). Compounds analyzed on the
TLC plates were visualized by using UV light, I2 vapor, or
2.5% phosphomolybdic acid in ethanol with heating. The
purity of the synthetic compound was measured by using
HPLC with the detection wavelength at 254 nm and
determined using area peaks of the chromatograms. UV
spectra (λ, nm) were determined on an Agilent 1024-element
diode array.
In the present study, we designed and synthesized
imidazole compounds possessing the thiazolyl ring, i.e.,
2-amino-1-thiazolyl imidazoles. The biological activities of
these novel anticancer 2-amino-1-thiazolyl imidazoles were
described.
Experimental
Chemical synthesis and characteristics The general syn-
thesis method of 2-amino-1-thiazolyl imidazoles is shown
in Chart 2. 2-Bromoacetophenone and amidinothiourea
mixed in ethanol were condensed to provide benzyl
Cancer cell growth inhibition assay As previously reported
[28], human gastric cancer NUGC-3 cells were seeded at
4,500 cells/100 μL/well in 96-well plates and incubated for
24 h at 37°C in a 5% CO2 incubator. Compounds were
dissolved in DMSO purchased from Sigma (St. Louis, MO,
USA) and diluted for treating cells in culture medium
containing DMSO at the final concentration of ≤0.3%.
Cancer cells were treated with testing compounds of various
concentrations in duplicates per concentration in culture
medium (200 μL/well) and incubated at 37°C in a CO2
incubator for 72 h. Actinomycin D of 10 nM and 0.3%
DMSO vehicle were used as the positive and negative
controls, respectively. A colorimetric assay using the 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetra-
Design
H
N
N
S
N
N
NH2
NH2
N
N
S
S
2
Chart 1

166
Invest New Drugs (2012) 30:164–175
Chart 2
zolium (MTS) and phenazinemethosulfate (PMS) system
was used to determine the cytotoxic activity of the testing
compounds. MTS and PMS were purchased from Promega
(Madison, WI, USA) and Sigma Inc. (St. Louis, MO, USA),
respectively. Optical density (OD) values at 490 nm were
measured with a 1420-multilabel counter VICTOR® from
Wallac (Turku, Finland). IC50, the concentration that
inhibited 50% of the cancer cell growth activity, was
estimated.
and recorded at optimal time intervals for 30 min. The area
under the curve normalized to that of vehicle control was
calculated and used to estimate the IC50 that inhibits 50% of
the tubulin polymerization activity by using a modified
sigmoidal model with SigmaPlot® from SPSS Inc. (Chicago,
IL, USA).
Colchicine binding assay Binding of [3H]-colchicine ([ring C,
methoxy-3H]-colchicine, from PerkinElmer, Waltham, MA,
USA) to bovine brain microtubule associated protein-rich
tubulins (Cytoskeleton Inc., Denver, CO, USA) was measured
by column centrifugation as described with modifications
[19, 30]. Solution mixtures of the heterodimeric tubulins and
[3H]-colchicine (0.1 Ci/mmol) both at 2.5 μM in the presence
of the compound or cold colchicine were incubated in the
polymerization buffer at room temperature for 1 h. 50 μL of
the solutions were centrifuged through Sephadex G-50
column and the eluates were analyzed for radioactivity in
counts per min (cpm) by scintillation counting. The percent-
age of the compound bound was calculated using the
equation of ðcpm with compound ꢀ cpm without tubulinÞ=
ðcpm withDMSO ꢀ cpm without tubulinÞ ꢁ 100%:
Cell cycle analysis by flow cytometry Human gastric cancer
NUGC-3 cells seeded at 1×106 cells/10-cm dish were
incubated with the compound at 37°C for 24 h. The cell
pellets were collected, permeabilized with a buffer: 0.5%
Triton X-100, 0.2 μg/ml Na2EDTA2H2O, and 1% bovine
serum albumin (BSA) in PBS, and placed on ice for
15 min. The mixture was then combined with pre-cooled
methanol and centrifuged at 300×g for 5 min. The nuclear
DNAs were stained with a sodium citrate (3.4 mM) solution
containing propidium iodide (50 μg/ml) and RNase A
(5 Kunits/ml) in the dark at 4°C for 30 min. The data of
fluorescence distribution were collected, analyzed for DNA
contents, and displayed as single histograms for FL2-H by
using a FACSCalibur flow cytometer (Becton Dickinson,
Bedford, MA, USA) [11, 29].
Immunostaining of tubulins in cancer cells Human gastric
cancer NUGC-3 cells were treated with the compound of
1 μM for 16 h, washed with phosphate-buffered solution
(PBS) and fixed with 4% formaldehyde at room tempera-
ture for 10 min. After rinsed with PBS, the fixed cells were
blocked with 5 mg/mL BSA at room temperature for
30 min followed by an incubation with 1:200 dilution of
monoclonal anti-β-tubulin (mouse IgG1 isotype, Cat. No.
T42026) from Sigma Inc. (St. Louis, MO, USA) at 4°C
overnight. The stained tubulins were then visualized in
green fluorescence by a reaction with 1:300 dilution of the
goat anti-mouse IgG-FITC conjugates (Cat. No. F0257,
Sigma) at room temperature for 1 h. The cells were then
stained for DNA with Hoechst 33258 (1 μg/mL) in 0.1%
Tubulin polymerization assay Microtubule turbidity assay
was conducted with modifications based on a previous
report [19]. Bovine brain microtubule-associated protein-
rich heterodimeric tubulins from Cytoskeleton Inc. (Denver,
CO, USA) were dissolved in 100 μL polymerization buffer
containing PIPES (100 mM, pH=6.9), MgCl2 (2 mM),
GTP (1 mM), and DMSO (2%, v/v). The mixtures were
placed into 96-well microtiter plates in the presence of 2 of
various concentrations. The increase in absorbance at
340 nm was measured in a microplate reader PowerWave
X from Bio-Tek Instruments (Winooski, VT, USA) at 37°C

Invest New Drugs (2012) 30:164–175
167
Triton X-100 PBS at room temperature for 5 min. The
cellular images were visualized and photos taken by using a
fluorescence confocal laser-scanning microscope (Leica
TCS SP2, Heidelberg, Germany) [19].
initiated on 1 day after the cell inoculation. Mice of 7 to
8 per treatment group were included in the treated and
vehicle control groups. Compound 2 was dissolved in
DMSO and then dispersed to 20 mg/ml in the dosing
vehicle 2% carboxymethyl cellulose (Sigma, St. Louis,
MO, USA) with the final concentration of DMSO less than
0.5% for oral administration. Vinblastine (Sigma, St. Louis,
MO, USA) at 1 mg/ml was dissolved in the dosing vehicle
mixture of DMSO/Cremophor EL/saline (5%/20%/75%:
v/v/v) for intravenous administration. Another group of mice
orally treated with the oral dosing vehicle was included as
the negative control. The P388 cells-inoculated leukemic
animals were monitored for survival twice daily. The time
period that 50% of the leukemic mice still survive was
defined as the median survival time and used to calculate a
percentage (normalized to the median survival time of the
control group) of increase in the lifespan of the treated
animals.
Ex vivo histocultures of human tumors Modified from a
previous report [31], histocultures of human gastric MKN-
45 and colorectal SW480 tumors were established. Cancer
cells of 1×106/mouse were inoculated subcutaneously to
the flank of nude mice. The subcutaneously growing
tumors were harvested at a size of approximately 1 cm3,
dissected into pieces of 1 to 2 mm in diameter, and placed
on presoaked gelatin gels of 1 cm3 at 5–6 tumor pieces per
gel in 6-well culture plates containing culture medium of
MEM:DMEM (1:1), 2 mML-glutamine, 10% fetal bovine
serum (FBS), 1 mM sodium pyruvate, 0.1 mM non-
essential amino acids, and 40 μg/mL gentamicin (pH=
7.4) at 7 mL per well. The histocultures were kept in a
humidified atmosphere containing 95% air and 5% CO2 at
37°C in a CO2 incubator from Binder GmbH (Tuttlingen,
Germany). After a 3-day incubation, the culture media were
replaced with freshly prepared culture media containing the
compound of various concentrations ranging from 0.0001
to 10 μg/mL. The histocultures were treated for 96 h
followed by an incubation with 40 μM 5-bromo-2-
deoxyuridine (BrdU) for 48 h, washed 3 times with PBS,
fixed in 10% neutralized formalin, dehydrated in ethanol
solutions, and then embedded in paraffin for tissue
sectioning. Tumor tissue sections of 5-cm thick were
prepared and immunostained with BrdU antibody (M744)
for the DNA-incorporated BrdU in the proliferating tumor
cells. Proliferating tumor cells were visualized in brown
nuclei, whereas non-proliferating cells were hematoxylin
counterstained for nuclei (blue). Labeling index (LI) is
defined as the number of the BrdU-labeled (brown) tumor
cells divided by the number of total (brown and blue) tumor
cells and used as an index for the proliferation ability of the
histocultured tumor cells. The concentration IC50 that
inhibits 50% proliferation activity of the histocultured
tumor cell was estimated using the software SigmaPlot®
from (SPSS Inc., Chicago, IL, USA).
Statistical analysis Data were statistically analyzed by
ANOVA followed by Student-Newman-Keuls test and the
log rank test was used to analyze a difference in the lifespan
between groups using the SPSS® software (SPSS Inc.,
Chicago, IL, USA). A p value of <0.05 between groups was
considered statistically significant.
Results and discussion
All the presented synthetic compounds 2 and 3–16 listed in
Chart 3 showed a purity of >95%. Their chemical
properties were individually described as followings.
R1
N
S
R2
R3
N
N
NH2
3: R1= phenyl,
4: R1= phenyl,
5: R1= 2-pyridinyl, R2 = H,
6: R1= 2-thiophenyl, R2 = H,
7: R1= phenyl,
8: R1= 3-thiophenyl, R2 = H,
9: R1= 3-thiophenyl, R2 = H,
10: R1= phenyl,
11: R1= 2-pyridyl,
12: R1= phenyl,
13: R1= 2-pyridyl,
14: R1= methyl,
15: R1= phenyl,
16: R1= phenyl,
R2 = H,
R2 = H,
R3= phenyl
R3= 2-furanyl
R3= phenyl
R3= phenyl
R3= 3-thiophenyl
R3= phenyl
R3= 3-thiophenyl
R3= 2-thiazolyl
R3= 2-thiophenyl
R3= 2-pyridyl
R3= 2-pyridyl
R3= 2-thiophenyl
In vivo leukemia mouse study The in vivo anticancer
activity of the compound was evaluated by a murine
leukemia mouse model as previously described [19, 28].
Inbred male DBA/2 J mice of 6 weeks old (The National
Laboratory Animals Center, Taipei, Taiwan) were intrave-
nously inoculated with the murine leukemic P388 cells
(Japanese Collection of Research Bioresources, Osaka,
Japan) at 1×106 cells per mouse. P388 cells were
propagated in RPMI1640 medium supplemented with the
MEM-non-essential amino acids, 50 μM 2-mercaptoethanol
and 10% FBS. Treatments with the compounds were
R2 = H,
R2 = H,
R2 = H,
R2 = H,
R2 = H,
R2 = H,
R2 = phenyl, R3= phenyl
R2 = phenyl, R3= H
Chart 3

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Invest New Drugs (2012) 30:164–175
1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-
amine (2) A mixture of amidinothiourea (2.0 g, 16.9 mmol),
2-bromoacetophenone (3.4 g, 16.9 mmol) was heated at
reflux in ethanol (12 mL) for 4 h. The mixture was cooled
to room temperature and removed half of the solvent. The
precipitate was filtered, washed with acetone, and dried to
give N-(4-phenyl-1,3-thiazol-2-yl)guanidine (3.2 g, 87%).
A solution of N-(4-phenyl-1,3-thiazol-2-yl)guanidine
(3.2 g, 14.7 mmol) and 2-bromo-1-(2-thienyl)-1-ethanone
(1.5 g, 7.35 mmol) in ethanol (5 mL) was heated to 80°C
for 2 h. The mixture was left at room temperature for
additional 10 h, filtered, washed with hot water and recrystal-
lized from ethanol to give 2 (1.48 g, 62%). 1H NMR
(CDCl3): δ 7.85 (d, J=6.9 Hz, 2H), 7.48-7.38 (m, 3H), 7.33
(d, J=3.3 Hz, 1H), 7.25-7.20 (m, 2H), 7.02-7.03 (m, 2H),
6.36 (br, 2H). 13C NMR (CDCl3): δ 157.4, 154.0, 142.5,
138.3, 131.5, 131.2, 128.8, 128.7, 127.7, 126.5, 125.8,
125.4, 118.2, 112.8. ESMS m/z: 325.0 (MH+).
1-(4-Phenylthiazol-2-yl)-4-(thiophen-3-yl)-1H-imidazol-2-
amine (7) H NMR (CDCl3): δ 7.97 (d, J=7.2 Hz, 2H),
1
7.91 (s, 1H), 7.62-7.54 (m, 3H), 7.51-7.45 (m, 3H), 7.39
(t, J=7.2 Hz, 1H), 7.00 (s, 2H). 13C NMR (CDCl3): δ
158.3, 155.0, 143.5, 139.3, 137.2, 133.5, 131.6, 126.8,
126.1, 125.5, 124.3, 123.4, 116.3, 110.7. ESMS m/z: 325.6
(M+1)+.
4-Phenyl-1-(4-(thiophen-3-yl)thiazol-2-yl)-1H-imidazol-2-
1
amine (8) H NMR (CDCl3): δ 8.13 (t, J=2.1 Hz, 1H),
7.80 (d, J=7.2 Hz, 2H), 7.74 (s, 1H), 7.70 (s, 1H), 7.65 (d,
J=2.1 Hz, 2H), 7.37 (t, J=7.2 Hz, 2H), 7.23 (t, J=7.2 Hz,
1H), 7.02 (s, 2H). 13C NMR (CDCl3): δ 157.5, 149.5,
144.9, 142.4, 137.4, 129.8, 128.5, 128.3, 127.6, 126.2,
124.7, 124.1, 113.2, 106.4. ESMS m/z: 325.8 (M+1)+.
4-(Thiophen-3-yl)-1-(4-(thiophen-3-yl)thiazol-2-yl)-1H-
1
imidazol-2-amine (9) H NMR (CDCl3): δ 8.12 (t, J=
1.8 Hz, 1H), 7.72 (s, 1H), 7.65-7.60 (m, 3H), 7.57-7.54 (m
2H), 7.49 (d, J=4.8 Hz, 1H), 7.02 (s, 2H). 13C NMR
(CDCl3): δ 158.7, 143.1, 142.3, 142.1, 139.8, 136.7, 128.9,
128.7, 128.3, 128.3, 128.1, 128.0, 113.1, 106.4. ESMS m/z:
331.1 (M+1)+.
4-(Phenyl)-1-(4-phenylthiazol-2-yl)-1H-imidazol-2-amine
(3) H NMR (CDCl3): δ 7.98 (d, J=7.2 Hz, 2H), 7.93
1
(s, 1H), 7.81 (d, J=7.2 Hz, 2H), 7.74 (s, 1H), 7.48 (t, J=
7.2 Hz, 2H), 7.41-7.34 (m, 3H), 7.24 (t, J=7.2 Hz, 1H),
7.01 (s, 2H). 13C NMR (CDCl3): δ 157.4, 154.0, 149.4,
138.7, 134.3, 129.4, 128.7, 128.8, 128.7, 128.4, 125.1,
125.0, 115.8, 110.3. ESMS m/z: 319.6 (M+1)+, 331.5
(M+23)+.
1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-
1
amine (10) H NMR (CDCl3): δ 8.01-7.98 (m, 4H), 7.85
(d, J=3.3 Hz, 1H), 7.74 (s, 1H), 7.65 (d, J=3.3 Hz, 1H),
7.48 (t, J=7.2 Hz, 2H), 7.39 (t, J=7.2 Hz, 1H), 7.17 (br, 2H).
13C NMR (CDCl3): δ 158.2, 155.0, 141.5, 138.3, 137.2,
131.6, 131.3, 128.8, 128.5, 127.4, 126.3, 125.5, 117.3,
110.8. ESMS m/z: 325.5 (M+1)+.
4-(Furan-2-yl)-1-(4-phenylthiazol-2-yl)-1H-imidazol-2-
1
amine (4) H NMR (CDCl3): δ 7.98 (d, J=7.5 Hz, 2H),
7.93 (s, 1H), 7.66 (s, 1H), 7.48 (t, J=7.5 Hz, 2H), 7.41-7.35
(m, 2H), 7.03 (s, 2H), 6.54 (s, 2H). 13C NMR (CDCl3): δ
157.7, 156.0, 142.9, 139.3, 137.1, 132.5, 131.7, 128.9,
127.5, 126.3, 125.4, 123.4, 116.2, 111.8. ESMS m/z: 309.5
(M+1)+, 331.4 (M+23)+.
1-(4-(Pyridin-2-yl)thiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-
1
2-amine (11) H NMR (CDCl3): δ 8.64 (d, J=4.5 Hz, 1H),
8.16 (d, J=7.5 Hz, 1H), 8.09 (s, 1H), 7.92 (dt, J=7.5,
1.5 Hz, 1H), 7.63 (s, 1H), 7.42-7.37 (m, 3H), 7.09-7.07
(m, 3H). 13C NMR (CDCl3): δ 156.2, 149.5, 149.3, 148.4,
147.8, 139.4, 137.4, 137.1, 128.5, 127.1, 125.4, 124.3,
121.8, 119.6, 110.5. ESMS m/z: 326.1 (M+1)+.
4-Phenyl-1-(4-(pyridin-2-yl)thiazol-2-yl)-1H-imidazol-2-
1
amine (5) H NMR (CDCl3): δ 8.65-8.63 (m, 1H), 8.15
(d, J=7.8 Hz, 1H), 8.10 (s, 1H), 7.92 (dt, J=7.8, 1.8 Hz, 1H),
7.82 (d, J=7.2 Hz, 2H), 7.75 (s, 1H), 7.42-7.34 (m, 3H), 7.24
(t, J=7.2 Hz, 1H), 7.01 (s, 2H). 13C NMR (CDCl3): δ 158.3,
155.1, 150.8, 149.2, 146.4, 138.2, 134.2, 134.1, 129.4, 128.6,
126.7, 125.3, 121.7, 112.5, 111.0. ESMS m/z: 320.5 (M+1)+,
342.6 (M+23)+.
1-(4-Phenylthiazol-2-yl)-4-(pyridin-2-yl)-1H-imidazol-2-
1
amine (12) H NMR (CDCl3): δ 7.85 (d, J=6.9 Hz, 2H),
7.48-7.32 (m, 6H), 7.26 (s, 1H), 7.19 (s, 1H), 6.75 (s, 1H),
5.97 (br, 2H). 13C NMR (CDCl3): δ 157.3, 154.1, 149.5,
149.3, 148.5, 137.2, 135.8, 134.2, 128.7, 128.5, 126.2,
124.3, 122.6, 119.2, 109.8. ESMS m/z: 320.5 (M+1)+.
4-Phenyl-1-(4-(thiophen-2-yl)thiazol-2-yl)-1H-imidazol-2-
1
amine (6) H NMR (CDCl3): δ 7.80 (d, J=7.2 Hz, 2H),
7.74 (s, 1H), 7.72 (s, 1H), 7.66 (dd, J=3.6, 1.2 Hz, 1H),
7.58 (dd, J=5.1, 1.2 Hz, 1H), 7.37 (t, J=7.2 Hz, 2H), 7.24
(t, J=7.2 Hz, 1H), 7.15 (dd, J=5.1, 3.6 Hz, 1H), 7.00 (br,
2H). 13C NMR (CDCl3): δ 157.6, 149.3, 148.2, 137.6,
137.3, 128.3, 128.7, 128.4, 127.9, 126.1, 125.4, 122.3,
119.6, 110.4. ESMS m/z: 325.1 (M+1)+.
4-(Pyridin-2-yl)-1-(4-(pyridin-2-yl)thiazol-2-yl)-1H-imida-
zol-2-amine (13) H NMR (CDCl3): δ 8.64 (d, J=4.5 Hz,
1H), 8.53 (d, J=4.5 Hz, 1H), 8.17 (d, J=7.8 Hz, 1H), 8.13
(s, 1H), 7.93 (dt, J=7.8, 1.8 Hz, 1H), 7.85-7.77 (m, 2H),
7.69 (s, 1H), 7.42-7.37 (m, 1H), 7.27-7.23 (m, 1H), 7.05
(s, 2H). 13C NMR (CDCl3): δ 157.2, 156.2, 155.4, 148.6,
1

Invest New Drugs (2012) 30:164–175
169
148.4, 144.6, 142.8, 139.5, 138.2, 137.2, 124.5, 124.2,
123.6, 123.4, 111.0, 107.8. ESMS m/z: 321.6 (M+1)+
343.5 (M+23).+
ring, compounds 2, 3, 4, 7, 10, and 11 with a substituent of
2-thiophenyl, phenyl, 2-furanyl, 2-thiazolyl, and 2-pyridyl
at the 4-position of 2-amino imidazole, respectively,
exhibited a cytotoxic IC50 value of 0.05, 0.55, 0.23, 0.39,
and 0.33 μM against NUGC-3 cells. 2-Thiophenyl substi-
tution at the 4-position of 2-amino imidazole rendered the
best potency among them and, at the same position, phenyl
substitution showed the least potency. On the other hand,
compounds 3, 5, 6 and 8, with a phenyl substituent on the
4-position of the 2-amino imidazoles, showed IC50 values
of 0.55, 0.28, 0.26 and 0.10 μM when the 4-position of
thiazolyl ring was substituted with a phenyl, 2-pyridinyl, 2-
thiophenyl, and 3-thiophenyl group, respectively. It appears
that a substitution with a 5-membered aromatic ring is
better than a 6-membered aromatic ring on the 4-position of
either the thiazolyl ring or 2-amino imidazole ring.
However, compound 9, with a 3-thiophenyl substituent on
both the 4-positions of the thiazolyl ring and 2-amino
imidazole ring, did not show an improved cytotoxicity
more potent than those of compounds 2–8 and 10–11. With
dual substitutions of two 6-membered aromatic rings,
compounds 3, 5, 12 and 13 showed similar potencies to
those with a 6-membered and a 5-membered aromatic rings.
A methyl substituent on the thiazolyl ring resulted in a
significant decrease of cytotoxicity as compound 14
showed, indicating the necessity of a 5- or 6-membered
aromatic ring for potent cytotoxicity. Furthermore, a phenyl
substitution at the 5-position of 2-amino imidazole com-
pletely abolished the cytotoxic activities of the 2-amino-1-
thiazolyl imidazoles against NUGC-3 cells as demonstrated
by compounds 15 and 16. A potent active 2-amino-1-
thiazolyl imidazole, compound 2, was further examined for
subcellular and molecular mechanisms mediating the
anticancer activity and in vivo antitumor activity.
1-(4-Methylthiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-
1
amine (14) H NMR (CDCl3): δ 7.50 (s, 1H), 7.39-7.35 (m,
2H), 7.08-7.03 (m, 2H), 6.99 (br, 2H), 2.35 (s, 3H). 13C NMR
(CDCl3): δ 157.4, 154.0, 142.5, 138.3, 137.1, 131.5, 131.2,
125.8, 118.2, 112.8. 17.1. ESMS m/z: 263.8 (M+1)+, 285.1
(M+23).+
4,5-Diphenyl-1-(4-phenylthiazol-2-yl)-1H-imidazol-2-amine
1
(15) H NMR (CDCl3): δ 7.90 (d, J=7.2 Hz, 2H), 7.85
(s, 1H), 7.49-7.34 (m, 10H), 7.23-7.12 (m, 3H), 6.84 (s, 2H).
13C NMR (CDCl3): δ 154.7, 149.5, 139.2, 137.5, 136.2,
133.5, 129.3, 129.2, 128.7, 128.6, 127.9, 127.8, 127.3,
127.1, 126.9, 124.4, 123.2, 110.4. ESMS m/z: 395.6
(M+1)+.
5-Phenyl-1-(4-phenylthiazol-2-yl)-1H-imidazol-2-amine
1
(16) H NMR (CDCl3): δ 8.02 (s, 1H), 7.96 (d, J=7.2 Hz,
2H), 7.47 (t, J=7.2 Hz, 2H), 7.38 (t, J=7.2 Hz, 1H), 7.31
(d, J=7.2 Hz, 2H), 7.27-7.20 (m, 3H), 6.81 (s, 1H), 6.47
(br, 2H). 13C NMR (CDCl3): 155.6, 148.5, 139.6, 138.5,
135.2, 129.3, 128.5, 127.5, 127.2, 127.1, 126.8, 123.4,
121.8, 109.4. ESMS m/z: 319.6 (M+1)+.
Cytotoxicity and structure activity relationships The above-
described newly synthesized 2-amino-1-thiazolyl imida-
zoles were evaluated for cytotoxic activity against the
human gastric cancer NUGC-3 cells using the colorimetric
MTS/PMS assay system. The IC50s of compounds 2–16
against NUGC-3 cells were determined and summarized in
Table 1. Structure-activity relationships for the substitutions
on the thiazolyl and amino imidazole rings of the 2-amino-
1-thiazolyl imidazoles were evaluated. With the same
substituent of a phenyl group at the 4-position of thiazolyl
Cell cycle arrest at G2/M phase By using the flow
cytometry analysis, we investigated the effects of com-
pound 2 on cancer cell cycle progression. Distribution of
the cell populations treated with compound 2 in different
phases of the cell cycle was determined. After treated with
compound 2 of various concentrations for 24 h, human
gastric cancer NUGC-3 cells were arrested at the G2/M
phase in a concentration-dependent manner as shown in
Fig. 1a–f. As the compound 2 concentration was increased,
the S-phase population was decreased while the G2/M-
phase population was increased. Notably, a significantly
large group of the cancer cells that contain polyploidy
DNAs was induced by compound 2 treatments of 1 and
10 μM as shown in Fig. 1e and f, respectively. These
results indicated that compound 2 causes cancer cell cycle
arrest at the G2/M phase and induces polyploidy in the
cancer cells, which together lead to inhibition of the
proliferation of the human cancer cells.
Table 1 Anticancer activity of 2-amino-1-thiazolyl imidazoles against
human gastric cancer NUGC-3 cellsa
Compound
IC50
Compound
IC50
0.39
2
3
4
5
6
7
8
9
0.05
0.55
0.23
0.28
0.26
0.07
0.10
0.38
10
11
12
13
14
15
16
0.34
0.33
0.85
5.65
>10
>10
a IC50 values expressed in μM are the means of triplicates from at least
three experiments

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Invest New Drugs (2012) 30:164–175
Fig. 1 Compound 2 caused cell
cycle arrest in human cancer
cells. Proliferating human gas-
tric cancer NUGC-3 cells were
arrested at the G2/M phase by
treatments of compound 2 in a
concentration-dependent man-
ner. a: vehicle control; b–f:
0.001, 0.01, 0.1, 1, and 10 μM,
respectively. g: The populations
of the NUGC-3 cells in G2/M
and those with polyploidy were
increased by compound 2
Inhibition of tubulin polymerization Tubulin turbidity assay
was performed to explore whether the kinetics of microtu-
bule assembly is a potential target via which compound 2
exerted cytotoxicity against human cancer cells. As shown
in Fig. 2, compound 2 concentration-dependently inhibited
the tubulin polymerization as observed by the decreased
OD350 values (turbidity) of the polymerizing microtubule
mixtures containing compound 2. A maximum inhibition
on the tubulin polymerization in the presence of 10 μM
compound 2 was observed. An IC50 value was estimated at
7.8 μM.
tubulins at 25 μM. It was less potent than cold colchicine
on interfering with the colchicine binding to tubulins. A
concentration-dependent inhibition on the [3H]-colchicine
binding kinetics in the presence of colchicine (0.25, 2.5,
and 25 nM) was observed and therefore demonstrated the
specificity and quality of the assay system. Therefore,
compound 2 causes disruption of microtubule assembly via
interfering with the colchicine binding sites on tubulin
which leads to cancer cell cycle arrest at the G2/M phase
and thus cancer cell proliferation.
Disruption of microtubule structure in human cancer
cells The microtubule structures of human gastric cancer
NUGC-3 cells were visualized by immunostained FITC
Interference on colchicine binding to tubulins As illustrated
in Fig. 3, compound 2 inhibited colchicine binding to

Invest New Drugs (2012) 30:164–175
171
DNA-incorporated BrdU (brown) and hematoxylin-
counterstained for nuclei (blue) of tumor cells were shown
in Fig. 5. Compound 2 caused a concentration-dependent
reduction in the number of the BrdU-labeled proliferating
human gastric MKN-45 tumor cells as shown in Fig. 5a–c.
A similar phenomenon was also observed in the compound
2-treated human colorectal SW-480 tumors (Fig. 5d–f). The
BrdU LI of each compound 2-treated tumor specimens was
calculated and the data obtained from the human gastric
MKN-45 and colorectal SW-480 tumors were plotted in
Fig. 6a and b, respectively. The IC50 concentrations that
reduce 50% of the LI (i.e., the tumor cell proliferation
activity) in the two histocultured human tumor types for
compound 2 were 12 and 17 μM, respectively. Therefore,
compound 2 demonstrated activities against human solid
tumors and was further investigated for in vivo oral
antitumor activity by using the murine leukemia P388
cells-inoculated mouse model.
Fig. 2 Concentration-dependent inhibition of tubulin polymerization
by compound 2. Black circle: vehicle control; White circle: 0.02 μM;
Delta: 0.2 μM; White square: 2 μM; and Diamond: 20 μM
green fluorescence. Figure 4 showed the representative
photos of a vehicle-treated control (left panel) and
compound-treated (right panel) NUGC-3 cells. Compared
to that in the vehicle-treated, the microtubule structure was
disarrayed and a diffused and thus not as concentrated
green fluorescent tubulin immuno-reactivity was observed
in the compound-treated NUGC-3 cells. It was also noted
that the cell number was less in the compound-treated
group and the nuclei of some of the compound-treated cells
exhibited irregular shapes. Microtubule is one of the
molecular targets for compound 2 actions and has been
demonstrated by not only the results of inhibiting the
tubulin polymerization but also the changes in the mor-
phology of microtubules.
In vivo oral activity on lifespan prolongation of leukemic
mice Adult male DBA/2 J mice orally gavaged with the
vehicle survived for 6 to 7 days after the i.v. inoculation of
murine leukemic P388 cancer cells. As shown in Fig. 7, the
leukemia mice orally administered with compound 2 at
200 mg/kg/day for 4 consecutive days survived longer than
the vehicle-treated mice. A positive control mouse group
treated with colchicine-targeting vinblastine (5 mg/kg, i.v.
once) was included to demonstrate the validity of the
testing system. Compound 2 showed oral activity and
significantly prolonged (75% longer compared to that of the
vehicle control group) the lifespan of the leukemia mice as
shown in Table 2.
The present study described the synthesis of a class of
novel anticancer 2-amino-1-thiazolyl imidazoles and dem-
onstrated the in vitro, ex vivo, and in vivo anticancer
activities of a representative compound of the class.
Controlled regulation of microtubule assembly kinetics is
critical for cell division and thus tumor growth. The
microtubule assembly system is a well-validated molecular
system of drug target from which a number of clinically
used anticancer drugs have been discovered. By interacting
with the colchicine-binding sites on tubulins, 2-amino-1-
thiazolyl imidazoles cause microtubule disruption resulting
in arrest of the cell cycle progression and thus DNA
polyploidy leading to cytotoxicity against cancer cells. We
have also shown that compound 2 is in vivo orally active in
prolonging the lifespan of the leukemia mice. The oral
activity suggests that compound 2 is not only orally
absorbable but also distributes efficiently to the tumor
tissues at effective concentrations in order to achieve the
observed anticancer activity in the animals. To further
explore the in vivo oral activity spectrum against human
tumors of different organ types in the preclinical tumor
Ex vivo antitumor activity against human tumors Histocul-
tured human gastric MKN-45 and colorectal SW-480
tumors were treated with compound 2 for 96 h and the
representative tumor tissue sections immunostained for
Fig. 3 Inhibition of [3H]-colchicine binding to tubulins by compound
2. The percentage of tubulin-bound [3H]-colchicine was reduced in the
presence of colchicine (0.25, 2.5, and 25 μM) or compound 2 (2.5 and
25 μM). *: p<0.05; **: p<0.05 vs. colchicine of the same
concentration

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Invest New Drugs (2012) 30:164–175
Fig. 4 Morphological aberra-
tions in the microtubule ar-
rangement caused by compound
2 in cancer cells. Human gastric
cancer NUGC-3 cells were trea-
ted with compound 2 of 1 μM
for 16 h followed by DNA
staining using Hoechst 33258
(blue). Tubulins were immunos-
tained with primary mouse anti-
tubulin IgGs and visualized by
FITC-conjugated secondary goat
anti-mouse IgGs (green). Disor-
ganized green fluorescent mi-
crotubule structures indicated by
arrowheads in the compound
2-treated cancer cells were
shown. Scale bars=50 μm
Fig. 5 Compound 2 inhibited
the tumor cell proliferation in ex
vivo histocultured human
tumors. Tissue sections of the
human tumor xenografts, treated
with compound 2 for 96 h, were
immunostained for DNA-
incorporated BrdU (in brown
nuclei) and hematoxylin-
counterstained for the nuclei
(in blue) of the histocultured
tumor cells. Representative
tissue sections of human gastric
MKN-45 tumors treated with
vehicle (a), 10 (b), and 1 μg/mL
(c) of compound 2; and human
colorectal SW-480 tumors trea-
ted with vehicle (d), 10 (e), and
1 μg/mL (f) of compound 2
were shown, respectively

Invest New Drugs (2012) 30:164–175
173
Fig. 7 Orally administered compound 2 prolonged lifespan of
leukemia mice. Murine leukemia P388 cells were i.v. inoculated at
1×106 cells/mouse through the tail vein into DBA/2 mice. At 24 h
after the inoculation, compound 2 at 200 mg/kg/day (White circle) and
vehicle (Black circle), were p.o. given once daily for 4 consecutive
days. A group of mice treated with vinblastine of 5 mg/kg, i.v. once
(White square) was included as a reference control of the experimental
system. Orally gavaged compound 2 significantly increased the
lifespan of the leukemia mice
also to be determined and studies such as single-dose/
repeated-dose pharmacokinetics, toxicology/toxicokinetics,
and safety pharmacology in animals are also to be
performed. These are the necessary information all together
for the decision making and preparations for drug develop-
ment toward further clinical studies in patients. Neverthe-
less, we have discovered and reported here a novel class of
orally active microtubule-destabilizing 2-amino-1-thiazolyl
imidazoles, which can be further investigated and devel-
oped into chemotherapeutics against cancerous diseases.
Fig. 6 Concentration-dependent activities of compound 2 against the
growth of human tumors. Histocultured human gastric MKN45
tumors (a) and colorectal SW480 tumors (b) were treated with
compound 2 for 96 h followed by BrdU-immunostaining for
proliferating cancer cells. The labeling index (LI) and IC50 values
were estimated as indicated (n=19–30)
models (eg. subcutaneously or orthotopically xenografted
growing tumors models) in animals is warranted.
Neutropenia and peripheral neuropathy [3, 32, 33] are
the two most frequently observed dose-limiting toxicities in
patients associated with the currently available microtubule
targeting drugs. It is important to further investigate if this
novel class of compounds causes neutropenia and/or
neuropathy in animals before further development for
clinical studies [34]. Multidrug resistance (MDR), mediated
such as by over-expression of P-glycoproteins, significantly
limits the clinical efficacies of the microtubule-targeting
drugs [35, 36]. There are newly discovered synthetic small
microtubule-targeting agents whose activities were found
less or not affected by the MDR mechanisms [3, 37].
Furthermore, the demonstrated oral anticancer activity of
compound 2, again, implies a satisfactory penetration
ability through the P-glycoprotein-expressing epithelial cell
layers of the intestinal lumens into the systemic blood
circulation and thus an acceptable oral bioavailability of the
compound [38, 39]. Whether 2-amino-1-thiazolyl imida-
zoles can overcome the MDR mechanism and are active
against taxanes-resistant cancer cells are to be further
examined. The tissue distribution after oral absorption is
Conclusions
We reported here the syntheses and anticancer activities of
2-amino-1-thiazolyl imidazoles. From which, selective
compounds showed potent cytotoxic activities against
human cancer cells. An example compound 1-(4-phenyl-
thiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-amine dem-
onstrated in vivo oral antitumor activity on prolonging the
lifespan of the leukemia mice. The possible underlying
molecular mechanisms of its anticancer action are the
Table 2 Lifespan prolongation in leukemia mice by compound 2
Treatment
Prolonged lifespan
(% of vehicle control)
Compound 2, 200 mg/kg (p.o. ×4)
75%*
83%*
Vinblastine, 5 mg/kg (i.v. ×1)
* p<0.05 vs. vehicle control

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Invest New Drugs (2012) 30:164–175
interferences on the colchicine binding sites of tubulins and
on the kinetics of microtubule assembly, which then causes
cell cycle arrest at the G2/M phase, inhibition of tumor cell
proliferation, and induction of tumor cell apoptosis. The 2-
amino-1-thiazolyl imidazoles may, therefore, be explored
further in pre-clinical and clinical developments as antican-
cer chemotherapeutics.
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Acknowledgements This study was supported by Grants BP-090-
PP01, BP-090-CF03 and BP-091-CF03 of The National Health
Research Institutes, Zhunan, Miaoli, Taiwan, R.O.C.
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