Synthesis and biological activities of 2-amino-1-arylidenamino imidazoles as orally active anticancer agents

Article abstract of DOI:10.1021/jm901501s

2-Amino-l-arylidenaminoimidazoles, a novel class of orally (po) active microtubule-destabilizing anticancer agents, were synthesized. The compounds were designed from a hit compound identified in a drug discovery platform by using cancer cell-based high throughput screening assay. Selective synthesized compounds exerted cell cytotoxicity against human cancer cells. The underlying mechanisms for the anticancer activity were demonstrated as interacting with the tubulins and inhibiting microtubule assembly, leading to proliferation inhibition and apoptosis induction in the human tumor cells. Furthermore, two compounds showed in vivo anticancer activities in both po and intravenously (iv) administered routes and prolonged the life spans of murine leukemic P388 cells-inoculated mice. These new po active antimitotic anticancer agents are to be further examined in preclinical studies and developed for clinical uses.

Full text of DOI:10.1021/jm901501s

J. Med. Chem. 2010, 53, 2409–2417 2409
DOI: 10.1021/jm901501s
Synthesis and Biological Activities of 2-Amino-1-arylidenamino Imidazoles as Orally Active Anticancer
Agents
Wen-Tai Li,† Der-Ren Hwang,† Jen-Shin Song,† Ching-Ping Chen,† Jiunn-Jye Chuu,† Chih-Bo Hu,† Heng-Liang Lin,†
Chen-Lung Huang,† Chiung-Yi Huang,† Huan-Yi Tseng,† Chu-Chung Lin,† Tung-Wei Chen,†,‡ Chi-Hung Lin,‡
Hsin-Sheng Wang,† Chien-Chang Shen,† Chung-Ming Chang,† Yu-Sheng Chao,† and Chiung-Tong Chen*,†
†Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli 35053, Taiwan,
ROC, and ‡Institute of Biophotonics, National Yang Ming University, Shih-Pai, Taipei 11221, Taiwan, ROC
Received October 9, 2009
2-Amino-1-arylidenaminoimidazoles, a novel class of orally (po) active microtubule-destabilizing
anticancer agents, were synthesized. The compounds were designed from a hit compound identified
in a drug discovery platform by using cancer cell-based high throughput screening assay. Selective
synthesized compounds exerted cell cytotoxicity against human cancer cells. The underlying mechan-
isms for the anticancer activity were demonstrated as interacting with the tubulins and inhibiting
microtubule assembly, leading to proliferation inhibition and apoptosis induction in the human tumor
cells. Furthermore, two compounds showed in vivo anticancer activities in both po and intravenously
(iv) administered routes and prolonged the life spans of murine leukemic P388 cells-inoculated mice.
These new po active antimitotic anticancer agents are to be further examined in preclinical studies and
developed for clinical uses.
Introduction
that they were biologically active inhibiting the polymeriza-
tion of tubulins.
The high-throughput screening (HTSa) platform has been
utilized in the discovery of drugs for many years.1-3 To
identify active compounds (i.e., hits) by using HTS against a
library of a large amount of compounds using cell-based
colorimetric assays is a now one of the routine activities in
drug discovery.4-7 There are currently marketed drugs and
drug candidates in the developmental stages that were suc-
cessfully discovered by using the HTS platform.7 We had
identified several potential HTS hit compounds by using a
colorimetric human cancer cell-based cytotoxicity HTS assay
against in-house synthesized and commercially available
compounds. 2-((1E)-(1-(1-Phenylethylideneamino)-4-phen-
yl-1H-imidazol-2-ylimino)methyl)-4,6-dibromophenol is one
of the identified hit compounds and contains aminoimidazole
as the chemical skeleton. 4-Amino-5-imidazole derivatives
have been found to possess potent anticancer activities,8-10
while some bis[2-chloroethyl]aminoimidazole derivatives
were DNA binding agents and topoisomerase II inhibitors.11
Naamidine A, a 2-aminoimidazole alkaloid,12,13 was a potent
antagonist of the epidermal growth factor receptor whose
downstream signaling pathways, when overexpressed, leads
to cancer cell growth and tumorigenesis. We designed and
synthesized 2-amino-1-arylidenaminoimidazoles and found
Microtubule has been a proven molecular target via which
anticancer drugs have been demonstrated with efficacies and
currently used in patients.14-16 There are clinically used
antimitotic microtubule-destabilizers, vinca alkaloids vincris-
tine, vinblastine, and a third-generation vinca alkaloid vinor-
elbine.17 Inhibiting tubulin polymerization or interfering with
microtubule disassembly disrupts several cellular functions,
including cell motility and mitosis. The colchicine-site binders
that inhibit microtubule polymerization are another class of
promising compounds in discovery and development, includ-
ing combretastatin A-4 phosphate.18-20 New tubulin target-
ing agents are currently in preclinical and clinical develop-
ment, among which very few are po active.20
Here we described the syntheses and biological functions of
novel po active anticancer agents, 2-amino-1-arylidenaminoi-
midazoles designed by modifying the core structure of a high-
throughput screening hit identified by a human cancer cell-
based cytotoxicity assay.
Chemistry
The general method for the synthesis of 2-amino-1-arylide-
naminoimidazoles is shown in Scheme 1. Starting with ami-
noguanidine, the free amine was obtained by adding hydro-
chloride to liberate carbon dioxide, coupled with 4-chloro-
benzaldehyde to generate guanylhydrazone (1) in a good yield
of 91%. Ring closure reaction was accomplished by the
treatment of 2-bromoacetophenone with 1 to afford 1-(4-
chlorobenzylideneamino)-4-phenyl-1H-imidazol-2-amine (2)
in a yield of 77%.21-24 The synthetic procedures illustrated in
Scheme 1 were then carried out to give the desired 2-amino-1-
arylidenaminoimidazoles (2-23) listed in Chart 1.
*To whom correspondence should be addressed. Phone: 886-37-246-
166, extension 35711. Fax: 886-37-586-456. E-mail: ctchen@nhri.org.tw.
a Abbreviations: HTS, high-throughput screening; NOE, nuclear
Overhauser effect; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PMS, phenazine
methosulfate; DMSO, dimethyl sulfoxide; FITC, fluorescein isothio-
cyanate; BrdU, 5-bromo-2-deoxyuridine; LI, 5-bromo-2-deoxyuridine
labeling index; OD, optical density.
r
2010 American Chemical Society
Published on Web 02/19/2010
pubs.acs.org/jmc

2410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Li et al.
The stereochemistry of the 2-amino-1-arylidenaminoimi-
dazoles were characterized by nuclear overhauser effect
(NOE) experiments. (E)-Configuration of the exocyclic
CdN bond of the 2-amino-1-arylidenaminoimidazoles was
the only one isomer found. There was a strong NOE effect
between the two protons of the exocyclic CHadN and
imidazole CHb of 2, and this result was also consistent with
the previous report.25 The chemical stability of the 2-amino-1-
arylidenaminoimidazoles was also examined by the treatment
of 2 in hydrochloride (pH 2) solution (CH3CN/H2O = 1:1)
with shaking for 12 h. Although Schiff base hydrolysis
happens under acidic conditions, the stability test indicated
that there were no imine hydrolysis products found. The
chemical stability indicated that 2 may also be stable in the
acidic environment in the stomach when po administered.
The chemical structures of these 2-amino-1-arylidenami-
noimidazoles are novel for targeting tubulins. Previously
reported tubulin-targeting compounds contain a core struc-
ture with an imidazole26,27 or benzimidazole26 different from
the presently reported one. The core structures of these
imidazole-containing compounds reported in the present
study are different from those of the literature-reported
tubulin-interacting compounds that contain an imidazole.
active 2-amino-1-arylidenaminoimidazoles were then further
examined for possible molecular and cellular mechanisms of
anticancer activities. Interactions with tubulins affected the
microtubule assembly kinetics, induction of apoptosis as
visualized by DNA fragmentation analysis in the human
gastric cancer NUGC-3 cells, and inhibition of the prolifera-
tion of human tumors in the ex vivo histoculture system.29
Furthermore, the in vivo antitumor activities were also ex-
plored by using the murine leukemic P388 cells-inoculated
mouse model.30 Compounds 2, 3, and 5 showed in vitro
anticancer activities and were further evaluated for biological
activities in the biological functional studies.
Results and Discussion
In Vitro Anticancer Activity against Human Gastric Cancer
Cells. The IC50 concentrations of 2-23 against human
gastric cancer NUGC-3 cells were determined and summari-
zed in Table 1. Structure-activity relationships between the
substitutions of 2-amino-1-arylidenaminoimidazole have
been evaluated. Compounds 6, 7, and 13 with the arylidene
substituents of phenyl, 3-methyl-2-thiophenyl, and 4-quino-
lyl, respectively, exhibited cytotoxic IC50 values of 0.84, 0.91,
and >10 μM against NUGC-3 cancer cells. Among them,
phenyl substitution was superior to the others. In addition,
the substituent on the phenyl ring of arylidene indicated that
4-fluorophenyl (3) has comparable activity to 4-chlorophe-
nyl (2) with IC50 values of 0.05 and 0.06 μM, respectively,
whereas a 4-bromophenyl (4) led to a decrease in the activity.
Compound 12, with 2-chlorophenyl of arylidene, resulted in
the apparent decrease of cytotoxicity compared to the 4-
chlorophenyl (2). Therefore, substitution at the 4-position of
Biology
All the synthesized 2-amino-1-arylidenaminoimidazoles
(2-23) were initially evaluated for cytotoxicity in the human
gastric cancer NUGC-3 cells using a colorimetric 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium (MTS) and phenazine methosulfate
(PMS) assay system28,29 by measuring the residual cancer cell
activity after treatment with the test compounds. Selective
Table 1. Anticancer Activity of 2-Amino-1-arylidenaminoimidazoles
against Human Gastric Cancer NUGC-3 Cellsa
Scheme 1a
compd
IC50
compd
IC50
compd
18
19
IC50
2
3
4
5
6
7
8
9
0.06
0.05
0.58
0.23
0.84
0.91
5.77
>10
10
11
12
13
14
15
16
17
0.74
5.12
3.97
>10
>10
5.20
3.28
0.05
0.31
0.06
0.86
0.08
>10
0.51
0.01
20
21
22
23
colchicine
a Reagents and conditions: (a) 4-chlorobenzaldehyde, reflux, 10 min;
a IC50 values expressed in μM as the mean values of triplicate wells
from at least three experiments.
(b) 2-bromoacetophenone, NaOH, EtOH, 70 °C, 4 h.
Chart 1. Selective 2-Amino-1-arylidenaminoimidazoles

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2411
Figure 1. Apoptotic DNA fragmentation induction was observed
in human gastric NUGC-3 cancer cells treated with 2, 5, and
colchicine at 100 nM for 24 h.
the phenyl ring seems to play a critical role for the cytotoxic
activity. A similar phenomenon was also observed with substi-
tuents of 3,4-dichlorophenyl (10) and 2,3-dichlorophenyl (11)
on arylidene and showed IC50 values of 0.74 and 5.12 μM,
respectively. Interestingly, 4-chloro-2-nitrophenyl of arylidene
(9) showed no activity against the NUGC-3 cancer cells. The
structure-activity relationship with the comparisons of 17 and
21 versus 18 and 23 revealed that 2-thiophenyl and 3-thiophenyl
at the 4-position of the imidazole ring exhibited the most potent
cytotoxic activities against the NUGC-3 cancer cells. Com-
pound 5, with a 3-pyridyl group at the 4-position of imidazole,
revealed a similar activity to 18 containing a 2-pyridyl group. In
addition, 19, with substitution of 4-fluorophenyl of arylidene
and 3-pyridyl group at the 4-position of imidazole, exhibited a
potent cytotoxic IC50 of 0.06 μM. Substitution on the 5-
position of imidazole caused loss of activity, similar to that
shown by compound 22.
DNA Fragmentation Induction in Human Gastric Cancer
Cells. Human gastric NUGC-3 cancer cells were treated with
2-amino-1-arylidenaminoimidazoles for 24 h, and the induc-
tion of apoptosis was demonstrated by DNA fragmentation
analysis. As shown in Figure 1, compounds 2 and 5 at 100
nM induced DNA fragmentation in the cancer cells. Apop-
tosis induction may be a mechanism by which these 2-amino-
1-arylidenaminoimidazoles killed the cancer cells. A dimeth-
yl sulfoxide (DMSO) vehicle negative control and colchicine
as positive control were included.
Figure 2. Inhibition on tubulin polymerization by 2 (A), 5, (B), and
colchicine (C) at the following concentrations (μM): (b) 10, (O) 1,
(1) 0.1, (4) 0.01, (9) 0.001, (0) vehicle control.
Table 2. Inhibition of Tubulin Polymerization by 2, 5, and Colchicine
IC50 (μM)a
compd
2
5
2.65 ( 0.41
0.27 ( 0.03
0.29 ( 0.05
colchicine
a IC50 expressed as the mean ( SD.
Inhibition of Tubulin Polymerization. Tubulin polymeriza-
tion assays were performed to explore whether microtubule
is the potential target through which the 2-amino-1-arylide-
naminoimidazoles exerted anticancer activities. With the
inclusion of the reference colchicine, 2 and 5 interacted
with and inhibited the microtubule assembly, as shown in
Figure 2. The estimated IC50 values are summarized in Table 2.
As previously reported for antimicrotubule agents,31,32 the
potencies for inhibiting the microtubule assembly were not
necessarily correlated to those for inhibiting the cancer cell
proliferation. The inhibition on the tubulin polymerization
and colchicine binding kinetics provide evidence at the
molecular level for the interference on microtubule assembly,
which is one of the proven molecular mechanisms of antic-
ancer action and for the compounds presented in the study.
Furthermore, the calculated partition coefficients (calcu-
lated log P) of compounds 2 and 5 are 4.152 and 2.655,
respectively. The difference in the hydrophorbic character-
istics is likely to be attributed, in part, to the different
potencies between the molecular and cellular systems.
Inhibition of Colchicine Binding to Tubulins. Both com-
pounds 2 and 5 concentration-dependently interfered with
Figure 3. [3H]Colchicine binding to tubulins was concentration-
dependently inhibited by treatments of 2 and 5.
[3H]colchicine binding to tubulins as illustrated in Figure 3.
Compounds 2 and 5 at 25 μM potently interfered with the
colchicine binding to tubulins and showed activity compar-
able to that caused by cold colchicine of the same concentra-
tion. A concentration-dependent inhibition on the colchicine

2412 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Li et al.
on the targeted microtubules were not only demonstrated
by the inhibitions on the tubulin polymerization and colchi-
cine binding but also visualized by fluorescence imaging
observations of the microtubules.
Ex Vivo Antitumor Activity against Human Tumors. His-
tocultured human gastric MKN-45 and colorectal SW-480
tumors were treated with 2 and 3 for 96 h. The representative
tumor tissue sections immunostained with DNA-incorpo-
rated 5-bromo-2-deoxyuridine (BrdU) brown and hematox-
ylin-counterstained blue nuclei of the tumor cells are shown
in Figure 5. As in Figure 5A-D, 2 caused a concentration-
dependent reduction in the number of the BrdU-labeled
human gastric MKN-45 tumor cells. A similar phenomenon
was also observed in the human colorectal SW-480 tumors
treated with 2 as shown in Figure 5E-G. Compound 3 also
demonstrated antiproliferative activities against both gastric
and colorectal tumors because a small number of residual
BrdU-labeled tumor cells were observed at the highest con-
centration (10 μg/mL) treated. It was, however, noted in
Figure 5H that the nuclei of the residual BrdU-labeled
gastric tumor cells after 3 treatments were mainly fragmen-
ted or presented in debris, indicating that the cells were
undergoing apoptosis. The BrdU labeling index (LI) of each
treated tumor specimens was counted and the calculated
data for the treatments with 2 and 3 in the human gastric
MKN-45 tumors were plotted in Figure 6A and Figure 6B,
respectively. The IC50 concentrations that reduced 50% of
the LI (i.e., the tumor cell proliferation activity) in the
histocultured human gastric tumors for 2 and 3 were 13
and 34 μM, respectively.
In Vivo Activity on Prolongation of Leukemic Mouse
Cancer Survival. Female DBA/2J mice of a vehicle control
group consistently survived for 6-7 days after single iv
inoculation of the murine leukemic P388 cancer cells. A
positive quality control group using doxorubicin (10 mg/
kg, once, iv) was also included in each experiment to
demonstrate a consistent effect (approximately 110% in-
crease in life span) of doxorubicin in prolonging the survival
period in the leukemic DBA/2J mice and thus the proper
response sensitivity of the testing system. Compound 2
exhibited both po and iv dose-dependent activities in in-
creasing the life spans of the cancer cells-bearing animals as
shown in Figure 7A without evident loss of body weight
(data not shown). A significant dose-dependent relationship
(p < 0.05, ANOVA) was observed. The oral doses of 2 at 25,
100, and 200 (mg/kg)/day for 9 consecutive days increased
the life spans by 14%, 71%, and 114%, respectively. On the
other hand, iv doses of 2 at 2.5 and 7.5 (mg/kg)/day for 4
consecutive days increased the life spans by 17% and 100%,
respectively. Compound 3 (200 (mg/kg)/day ꢀ 9 days, po)
exhibited a 110% prolongation of the survival of leukemic
animals as shown in Figure 7B.
Figure 4. Aberration of microtubule arrangements in the human
gastric cancer NUGC-3 cells treated by 2 at 1 μM for 16 h. DNA was
stained by Hoechst 33258 (blue), and tubulins were visualized by
FITC (green). Disorganized green fluorescent microtubule struc-
tures and slightly fragmented nucleus DNAs indicated by arrow-
heads in the 2-treated cancer cells were observed.
binding kinetics was observed and thus demonstrated the
specificity of interactions on the colchicine binding sites and
the quality of the assay system.
Interference on the Microtubule Assembly in Human Gas-
tric Cancer Cells. Treated by 2 at 1 μM for 16 h, NUGC-3
gastric cancer cells were immunostained for microtubules as
visualized by fluorescein isothiocyanate (FITC), in green
shown in Figure 4. A DMSO vehicle control was included.
Compared to the well-arranged microtubule structures in the
untreated cancer cells, the disarranged microtubule struc-
tures were observed as diffused green fluorescent tubulin-
immunoreactive activities in those 2-treated NUGC-3 cells.
It was also noted that the blue fluorescence-stained DNA of
the treated cancer cells were fragmented, as also observed in
Figure 1 from the DNA fragmentation assay. The cells then
underwent apoptotic processes and eventually died, as
shown in the cytotoxicity assay. Therefore, the molecular
interaction effects of 2-amino-1-arylidenaminoimidazoles
Conclusions
Cancer has been the leading cause of death worldwide.
Chemotherapy remains one of the major treatment options
available for cancer, and most of the currently used anticancer
drugs are administered to patients via a parenteral infusion or
bolus injection. Clinical complications with the parenteral
administrations have been documented. Extra care is needed
because of inappropriate patient compliances, and extra cost
associated with hospitalization is necessary. Efforts in search-
ing for po active anticancer agents have been extensive,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2413
Figure 5. Inhibition activities of 2 and 3 against on the tumor cell proliferation in ex vivo histocultured human gastric MKN-45 and colorectal
SW-480 tumors. Representative tumor tissue sections with immunostained DNA-incorporated BrdU (in brown nuclei and/or fragmented
apoptotic bodies) and hematoxylin-counterstained (in blue nuclei) tumor cells were shown as in MKN-45 tumors treated with vehicle (A) and
with 2 at 0.01 (B), 1 (C), and 10 (D) μg/mL; in SW-480 tumors treated with vehicle (E) and with 2 at 1 (F) and 10 (G) μg/mL; in MKN-45 tumors
with 3 at 10 μg/mL (H); and in SW-480 tumors treated with 3 at 10 μg/mL (I).
Figure 6. Concentration-dependent anticancer activities of 2 and 3
against ex vivo histocultured human gastric MKN-45 tumors. The
labeling indices (LIs) were calculated from the histocultured gastric
MKN-45 tumors treated with 2 (A) and 3 (B) for 96 h, and the IC50
values were estimated as indicated.
including the anticancer drug category of tubulin binding
agents from which only injectable drugs are available such
as taxanes and vinca alkaloids. We report here the synthesis
Figure 7. Dose-dependent prolongation in the survival of P388 cells-
inoculated leukemia DBA/2J mice po gavaged or iv administered with 2
and biological functions of 2-amino-1-arylidenaminoimida-
zoles as po active anticancer agents. Selective compounds had
(A) and 3 (B). (A) (1) 200 (po ꢀ 9), (O) 200 (po ꢀ 4), (4) 100 (po ꢀ 9),
(9) 25 (po ꢀ 9), (0) 7.5 (iv ꢀ 4), ([) 2.5 (iv ꢀ 4) mg/kg, (b) vehicle con-
shown potent effects in the interference on the colchicine
binding to tubulins, inhibition of tumor cell proliferation,
trol. A significant oral dose-dependent relationship (p < 0.05, AN-
OVA) was observed. (B) (O) 200 mg/kg (po ꢀ 4), (b) vehicle control.

2414 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Li et al.
and induction of human cancer cell apoptosis. Given po or iv,
the compounds demonstrated in vivo anticancer activities and
prolonged the survival of leukemia mice. The 2-amino-1-
arylidenaminoimidazoles may therefore be further developed
as anticancer therapeutics in patients.
Visualizations of DNA and Tubulins in Cancer Cells. Human
gastric cancer NUGC-3 cells were treated with 1 μM test com-
pound for 16 h. The cells were washed with phosphate-buffered
solution (PBS) and fixed with 4% formaldehyde at room tempera-
ture for 10 min. After being rinsed with PBS, the fixed cells were
then blocked with 5 mg/mL bovine serum albumin at room
temperature for 30 min followed by an incubation with 1:200
dilution of monoclonal anti-β-tubulin (mouse IgG1 isotype, cata-
log no. T42026, Sigma, St. Louis, MO) at 4 °C overnight. The
antitubulin IgG1-recognized tubulins were then visualized as green
fluorescence by a reaction with 1:300 dilution of the goat anti-
mouse IgG FITC (catalog no. F0257, Sigma) at room temperature
for 1 h. The cells were then incubated for DNA staining in the
Hoechst 33258 (1 μg/mL) in 0.1% 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).
Ex Vivo Histocultures of Xenografted Human Tumors. As
reported previously,29 histocultures of human gastric MKN-45
and colorectal SW480 tumors were established. The tumor cells
of 1 ꢀ 106 were inoculated subcutaneously to the nude mice and
monitored for tumor growth twice weekly. The subcutaneously
growing tumor xenografts at a size of approximately 1 cm3 in
nude mice were then harvested and dissected into pieces of 1-2
mm in diameter. Gelatin gels cut into pieces of 1 cm3 were
presoaked overnight in culture medium in six-well culture
plates. Five to six of the tumor pieces were placed on each
gelatin gel and cultured in medium consisting of MEM/DMEM
(1:1), 2 mM L-glutamine, 10% FBS, 1 mM sodium pyruvate, 0.1
mM nonessential 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 histoculture media were replaced with
freshly prepared culture media containing the test compound at
various concentrations ranging from 0.0001 to 10 μg/mL. The
histocultures were further incubated for 96 h in a CO2 incubator.
After the compound treatments, the histocultures were incu-
bated with 40 μM 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 thickness were prepared and immunos-
tained with BrdU antibody (M744) for the DNA-incorporated
BrdU in the proliferating tumor cells. Proliferating tumor cells
were visualized in brown nuclei, whereas nonproliferating cells
were counterstained in blue nuclei with Mayer’s hematoxylin
from Shandon (Pittsburgh, PA). LI was 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.
Experimental Section
In Vitro Growth Inhibition Study. As previously reported,30
human gastric cancer NUGC-3 cells were seeded at a density of
4500 cells in 100 μL per well in 96-well flat-bottom plates and
incubated for 24 h at 37 °C in a 5% CO2 incubator. Test
compounds were dissolved in DMSO purchased from Sigma (St.
Louis, MO), further diluted with the culture media to obtain an
optimal range of concentrations for treatments in duplicate per
concentration (200 μL/well), and incubated at 37 °C in a CO2
incubator for 72 h. All treatment media contained the final DMSO
concentration of e0.3%. Actinomycin D of 10 nM and 0.3%
DMSO were used as the positive and vehicle controls, respectively.
A colorimetric assay using the MTS/PMS system was used to
determine the cytotoxic activity of the test compounds. Both MTS
and PMS were purchased from Promega Corp. (Madison, WI).
The optical density (OD) values at 490 nm were measured with a
1420-multilabel counter VICTOR from Wallac (Turku, Finland).
The IC50, the concentration that inhibited 50% of the cancer cell
growth activity, was then determined.
DNA Fragmentation Analysis. Oligonucleosomal fragments
of the genomic DNA induced by treatments with the com-
pounds in human gastric cancer NUGC-3 cells were isolated
and analyzed by agarose gel electrophoresis according to pre-
viously described procedures30 with modification. In brief, 2 ꢀ
106 cancer cells were incubated with the test compound for 24 h.
Both floating and adherent cells were collected at the end of
incubation, washed once with PBS, and resuspended in the lysis
buffer. After incubation at 50 °C overnight, the samples were
treated with RNase A at 37 °C for 1 h. The DNA was extracted
with phenol/chloroform/isoamyl alcohol (25:24:1), and the
supernatant was collected, separated on a 2% agarose gel, and
stained with ethidium bromide. Colchicine was used as a
positive control.
Tubulin Polymerization Assay. Microtubule turbidity assay
was conducted according to a previously reported procedure
with modifications.33 In brief, bovine brain microtubule-asso-
ciated protein-rich heterodimeric tubulins from Cytoskeleton
Inc. (Denver, CO) were dissolved in a 100 μL of polymerization
buffer containing 100 mM PIPES (pH 6.9), 2 mM MgCl2, 1 mM
GTP, and 2% (v/v) DMSO and placed in 96-well microtiter
plates in the presence of 2 or 5. The increase in absorbance at 340
nm was measured in a microplate reader PowerWave X from
Bio-Tek Instruments (Winooski, VT) at 37 °C and recorded at
optimal time points for 30 min. The area under the curve
normalized to that of vehicle control was used to estimate an
IC50 that inhibited tubulin polymerization by 50% using non-
linear regression with SigmaPlot from SPSS Inc. (Chicago, IL).
Colchicine was served as a reference control.
In Vivo Cancer Survival Study. The in vivo anticancer activi-
ties of the compounds were evaluated by a murine leukemic
mouse model.30 Inbred female DBA/2J mice of 5 weeks old were
purchased from The National Laboratory Animals Center
(Taipei, Taiwan). Murine leukemic P388 cells purchased from
the Japanese Collection of Research Bioresources (Osaka,
Japan) were cultured and propagated in RPMI 1640 medium
supplemented with the MEM-nonessential amino acids, 50 μM
2-mercaptoethanol, and 10% fetal bovine serum. All mice were
iv inoculated with 1 ꢀ 106 P388 cells per mouse. The treatments
were initiated in one day after the cell inoculation. Seven to eight
mice per treatment group were used in the treated and vehicle
control groups. Test compounds were dissolved in DMSO and
then diluted with the dosing vehicle 0.5% carboxymethyl cellu-
lose with a final concentration of DMSO of less than 0.5%. The
compounds were po gavaged to the mice (0.1 mL per mouse).
Mice of the negative control group were treated with the dosing
vehicle only. The P388 cell-inoculated leukemic animals were
monitored twice daily for survival, and the survival fractions of
the treatment groups were recoded. The time period for which
Colchicine Binding Assay. Binding of [3H]colchicine ([ring C,
methoxy-3H]colchicine, from PerkinElmer, Waltham, MA) to
bovine brain microtubule associated protein-rich tubulins
(Cytoskeleton Inc., Denver, CO) was measured by column
centrifugation as previously described with modifications.33,34
Solution mixtures of the heterodimeric tubulins and [3H]col-
chicine (0.1 Ci/mmol), both at 2.5 μM in the presence of the
compound or cold colchicine, were incubated in the polymeri-
zation buffer at room temperature for 1 h. An amount of 50 μL
of the solutions was centrifuged through a Sephadex G-50
column, and the eluates were analyzed for radioactivity in
counts per min (cpm) by scintillation counting. The percentage
of the compound bound was calculated using the equation [(cpm
with compound) - (cpm without tubulin)]/[(cpmwith DMSO) -
(cpm without tubulin)] ꢀ 100.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2415
50% of the leukemic mice survived was defined as the median
survival time and used to calculate the percentage (normalized
to the median survival time of the control group) of increase in
the life span of the treated animals. The percentage of increase in
life span was then used as an index for treatment response.
Chemistry. All commercially available materials were used
without further purification unless otherwise stated. For anhy-
drous reactions, glassware was dried overnight in an oven at
120 °C and cooled in a desiccator over anhydrous CaSO4 or
silica gel. Diethyl ether and tetrahydrofuran were obtained by
distillation from the sodium ketyl of benzophenone under
nitrogen. Other solvents, including chloroform, dichloro-
methane, ethyl acetate, and methanol, were distilled over
CaH2 under nitrogen. Melting points were obtained with a
Yanaco (MP-500D) melting point apparatus. Infrared spectra
were recorded on a Perkin-Elmer (Spectrum RX1) spectro-
photometer. 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 are reported as δ values in parts per
million (ppm), and coupling constants (J) are 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; 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 chromatography (Merck silica gel 60, particle
size of 230-400 mesh). Analytical TLC was carried out on
precoated plates (Merck silica gel 60, F254). Compounds ana-
lyzed 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 compounds was checked by HPLC
with the detection wavelength of 254 nm. The purity of indivi-
dual compounds was determined from the area peaks in the
chromatogram of the sample solution. UV spectra (λ, nm) were
determined on an Agilent 1024-element diode array. All the
presented compounds showed a purity of >95%.
1-(4-Bromobenzylideneamino)-4-phenyl-1H-imidazol-2-amine
(4). 1H NMR (CDCl3): δ 8.53 (s, 1H), 7.96 (s, 1H), 7.84 (d, J =
8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 4H), 7.34 (t, J = 7.5 Hz, 2H),
7.19 (t, J = 7.2 Hz, 1H), 6.25 (br, 2H). 13C NMR (CDCl3): δ
157.0, 140.5, 135.4, 133.9, 132.8, 131.8, 130.4, 129.3, 128.8,
127.5, 125.4, 119.0. ESMS m/z: 341.5 (M þ 1)þ.
1-(4-Chlorobenzylideneamino)-4-(pyridin-3-yl)-1H-imidazol-
2-amine (5). 1H NMR (CDCl3): δ 8.89 (s, 1H), 8.55 (s, 1H), 8.38
(d, J = 5.1 Hz, 1H), 8.10 (s, 1H), 7.99-7.93 (m, 3H), 7.57 (d, J =
7.5 Hz, 2H), 7.37 (t, J = 5.1 Hz, 1H), 6.34 (br, 2H). 13C NMR
(CDCl3): δ 162.0, 155.1, 151.0, 148.5, 137.6, 135.0, 132.1, 131.1,
130.9, 129.6, 126.0, 122.0, 117.0. ESMS m/z: 298.5 (M þ 1)þ.
1-(Benzylideneamino)-4-phenyl-1H-imidazol-2-amine (6). 1H
NMR (CDCl3): δ 8.56 (s, 1H), 8.00 (s, 1H), 7.94 -7.90 (m,
3H), 7.70 (d, J = 7.2 Hz, 2H), 7.52-7.47 (m, 3H), 7.34 (t, J = 7.5
Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 6.19 (s, 2H). 13C NMR
(CDCl3): δ 155.4, 140.5, 136.0, 134.7, 131.6, 131.1, 130.6, 129.3,
128.9, 128.8, 127.5, 118.4. ESMS m/z: 263.6 (M þ 1)þ.
1-((3-Methylthiophen-2-yl)methyleneamino)-4-phenyl-1H-imi-
dazol-2-amine (7). 1H NMR (CDCl3): δ 8.89 (s, 1H), 8.55 (s, 1H),
8.38 (d, J = 5.1 Hz, 1H), 8.10 (s, 1H), 7.99-7.93 (m, 3H), 7.57
(d, J = 7.5 Hz, 2H), 7.37 (t, J = 5.1 Hz, 1H), 6.34 (br, 2H). 13
C
NMR (CDCl3): δ 163.7, 140.5, 139.5, 136.0, 133.1, 128.7, 126.8,
126.3, 124.9, 124.5, 123.2, 121.0, 11.7. ESMS m/z: 283.5 (M þ
1)þ, 305.6 (M þ 23)þ.
1-(4-(Trifluoromethyl)benzylideneamino)-4-phenyl-1H-imida-
zol-2-amine (8). 1H NMR (CDCl3): δ 8.64 (s, 1H), 8.13 (d, J =
8.1 Hz, 2H), 8.00 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.70 (d, J =
7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H),
6.32 (s, 2H). 13C NMR (CDCl3): δ 159.3, 138.5, 136.1, 135.2,
132.9, 131.1, 129.5, 129.3, 127.8, 125.5, 123.3, 122.2, 119.0.
ESMS m/z: 331.5 (M þ 1)þ.
1-(4-Chloro-2-nitrobenzylideneamino)-4-phenyl-1H-imidazol-
2-amine (9). 1H NMR (CDCl3): δ 8.76 (s, 1H), 8.48 (d, J = 8.4
Hz, 1H), 8.24 (d, J = 1.8 Hz, 1H), 8.19 (s, 1H), 7.95 (dd, J = 8.4,
1.8 Hz, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H),
7.28 (t, J = 7.2 Hz, 1H), 7.16 (br, 2H). 13C NMR (CDCl3): δ
160.1, 154.3, 143.5, 138.5, 134.0, 133.1, 131.1, 129.5, 126.3,
125.8, 123.5, 122.4, 121.1, 118.0. ESMS m/z: 342.5 (M þ 1)þ.
1-(3,4-Dichlorobenzylideneamino)-4-phenyl-1H-imidazol-2-
amine (10). 1H NMR (CDCl3): δ 8.52 (s, 1H), 8.28 (d, J = 1.5
Hz, 1H), 7.91 (s, 1H), 7.82 (dd, J = 8.4, 1.5 Hz, 1H), 7.76 (d, J =
8.4 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H),
7.19 (t, J = 7.5 Hz, 1H), 6.39 (s, 2H). 13C NMR (CDCl3): δ
157.8.0, 142.5, 137.0, 136.7, 133.5, 131.3, 130.1, 129.7, 128.4,
127.8, 126.7, 124.5, 121.0, 21.3. ESMS m/z: 331.1 (M þ 1)þ.
1-(2,3-Dichlorobenzylideneamino)-4-phenyl-1H-imidazol-2-
amine (11). 1H NMR (CDCl3): δ 8.95 (s, 1H), 8.70 (s, 1H), 8.47
-8.42 (m, 3H), 7.85 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.8 Hz,
2H), 7.57-7.49 (m, 3H), 7.42 (t, J = 7.8 Hz, 1H). 13C NMR
(CDCl3): δ 161.2, 142.5, 136.5, 135.8, 134.5, 131.1, 130.6, 128.9,
127.3, 126.8, 124.7, 122.4, 121.5, 118.0. ESMS m/z: 331.5 (M þ
1)þ.
General Procedure for the Synthesis of 2-23. 1-(4-Chlo-
robenzylideneamino)-4-phenyl-1H-imidazol-2-amine (2). Hydro-
chloric acid (20%, 4 mL) and 4-chlorobenzaldehyde (2.0 g, 15.8
mmol) were mixed together, and this mixture was slowly added
to a solution of aminoguanidine bicarbonate (2.2 g, 15.8 mmol)
in water (12 mL). After liberation of carbon dioxide, the mixture
was heated to reflux and then allowed to cool to room tempera-
ture. A solution of 40% aqueous potassium hydroxide (7 mL)
was added, and the mixture was heated at reflux for an addi-
tional 10 min. The resulting solution was filtered, washed with
water until the wash water was at pH 7, dried, and recrystallized
from ethanol to give (4-chlorobenzylideneamino)guanidine 1
(2.6 g, 91%) as a yellow solid.
Compound 1 (0.74 g, 4.06 mmol) was added to a solution of 2-
bromoacetophenone (0.40 g, 2.03 mmol) in ethanol (10 mL),
and the mixture was heated to 70 °C for 4 h. Aqueous sodium
hydroxide solution was added dropwise. A yellow precipitate
was formed in the reaction mixture which was cooled to room
temperature for an additional 10 h. The precipitates were then
filtered, washed with hot water, and recrystallized from ethanol
to give 2 (0.44 g, 77%) as a yellow solid. 1H NMR (CDCl3): δ
8.56 (s, 1H), 8.01-7.96 (m, 3H), 7.69 (d, J = 7.2 Hz, 2H),
7.37-7.30 (m, 4H), 7.18 (t, J = 7.2 Hz, 1H), 6.20 (s, 2H). 13C
NMR (CDCl3): δ 169.2, 157.0, 140.5, 136.0, 133.1, 130.8, 129.4,
129.3, 128.8, 127.5, 122.0, 115.6. ESMS m/z: 281.5 (MHþ).
1-(4-Fluorobenzylideneamino)-4-phenyl-1H-imidazol-2-amine
(3). 1H NMR (CDCl3): δ 8.55 (s, 1H), 7.97-7.92 (m, 3H), 7.69
(d, J = 6.0 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 6.0 Hz,
2H), 7.19 (t, J = 6.0 Hz, 1H), 6.25 (s, 2H). 13C NMR (CDCl3): δ
161.0, 142.5, 138.6, 137.0, 133.1, 131.9, 131.6, 129.3, 129.0,
127.8, 126.7, 125.5, 122.0. ESMS m/z: 297.5 (M þ 1)þ.
1-(2-Chlorobenzylideneamino)-4-phenyl-1H-imidazol-2-amine
(12). 1H NMR (CDCl3): δ 8.65 (s, 1H), 8.34 (d, J = 7.2 Hz, 1H),
8.16 (s, 1H), 7.76 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.5 Hz, 1H),
7.51-7.41 (m, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.19 (t, J = 7.8 Hz,
1H), 6.31 (s, 2H). 13C NMR (CDCl3): δ 154.0, 147.5, 137.6,
135.0, 134.5, 133.9, 132.3, 130.8, 129.3, 127.0, 124.8, 121.5,
119.0. ESMS m/z: 297.6 (M þ 1)þ, 319.5 (M þ 1)þ.
1-((Quinolin-4-yl)methyleneamino)-4-phenyl-1H-imidazol-2-
1
amine (13). H NMR (CDCl3): δ 9.24 (s, 1H), 9.02-8.98 (m,
2H), 8.81 (d, J = 8.4 Hz, 1H), 8.39 (t, J = 3.9 Hz, 1H), 8.30 (d,
J = 4.5 Hz, 1H), 8.19 (d, J = 4.5 Hz, 1H), 8.10 (dd, J = 8.4, 3.9
Hz, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.22
(t, J = 7.5 Hz, 1H). 13C NMR (CDCl3): δ 161.0, 153.1, 144.0,
139.5, 137.4, 134.0, 131.1, 128.9, 127.4, 127.3, 125.0, 124.8,
123.5, 122.0 121.0, 115.0. ESMS m/z: 314.4 (M þ 1)þ.
1-(4-Chlorobenzylideneamino)-4-biphenyl-1H-imidazol-2-amine
1
(14). H NMR (CDCl3): δ 8.57 (s, 1H), 8.04 (s, 1H), 7.95 (d,

2416 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Li et al.
J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.71-7.65 (m, 4H),
7.57 (d, J = 8.4 Hz, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.5
Hz, 1H), 6.29 (s, 2H). 13C NMR (CDCl3): δ 157.9, 142.5, 137.9,
137.6, 136.5, 136.1, 132.0, 131.9, 130.6, 129.3, 129.0, 128.4,
127.0, 125.9, 122.7, 118.0. ESMS m/z: 273.5 (M þ 1)þ.
1-(4-Chlorobenzylideneamino)-4-(2-methoxyphenyl)-1H-imi-
dazol-2-amine (15). 1H NMR (CDCl3): δ 8.60 (s, 1H), 8.01-7.98
(m, 3H), 7.86 (s, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.22-7.16 (m,
1H), 7.05-6.94 (m, 2H), 6.19 (s, 2H), 3.95 (s, 3H). 13C NMR
(CDCl3): δ 161.2, 144.5, 137.6, 135.0, 133.9, 131.6, 127.8, 126.0,
125.5, 124.0, 122.6, 108.0, 52.2. ESMS m/z: 327.6 (M þ 1)þ.
1-(4-Chlorobenzylideneamino)-4-(5-chlorothiophen-2-yl)-1H-
imidazol-2-amine (16). 1H NMR (CDCl3): δ 8.51 (s, 1H), 7.94 (d,
J = 7.8 Hz, 2H), 7.86 (s, 1H), 7.57 (d, J = 7.8 Hz, 2H), 7.05 (s,
2H), 6.39 (s, 2H). 13C NMR (CDCl3): δ 158.4, 147.8, 139.6,
137.0, 134.9, 133.6, 131.0, 128.5, 126.2, 125.1, 123.1, 119.0.
ESMS m/z: 337.4 (M þ 1)þ.
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1-(4-Chlorobenzylideneamino)-4-(thiophen-3-yl)-1H-imida-
zol-2-amine (17). 1H NMR (CDCl3): δ 8.49 (s, 1H), 7.94 (d, J =
8.4 Hz, 2H), 7.79 (s, 1H), 7.58-7.51 (m, 4H), 7.32 (d, J = 4.8 Hz,
1H), 6.24 (s, 2H). 13C NMR (CDCl3): δ 159.4, 143.3, 142.5,
137.6, 134.6, 132.0, 130.9, 127.0, 124.7, 122.3, 120.0, 118.6.
ESMS m/z: 303.5 (M þ 1)þ.
€
€
1-(4-Chlorobenzylideneamino)-4-(pyridin-2-yl)-1H-imidazol-
2-amine (18). 1H NMR (CDCl3): δ 8.73 (s, 1H), 8.48 (d, J = 4.8
Hz, 1H), 8.09 (s, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.79-7.15 (m,
2H), 7.56 (d, J = 8.4 Hz, 2H), 7.20-7.15 (m, 1H), 6.28 (br, 2H).
13C NMR (CDCl3): δ 158.4, 156.4, 150.3, 146.5, 139.2, 137.6,
137.0, 132.9, 131.6, 127.0, 126.2, 124.0, 119.8. ESMS m/z: 298.3
(M þ 1)þ.
1-(4-Fluorobenzylideneamino)-4-(pyridin-3-yl)-1H-imidazol-
2-amine (19). 1H NMR (CDCl3): δ 8.96 (d, J = 1.8 Hz, 1H), 8.48
(dd, J = 4.8, 1.8 Hz, 1H), 8.20 (s, 1H), 8.05 (dt, J = 8.1, 1.8 Hz,
1H), 7.81 (dd, J = 8.7, 5.4 Hz, 2H), 7.47 (s, 1H), 7.31 (ddd, J =
8.1, 4.8, 1.8 Hz, 1H), 7.18 (t, J = 8.7 Hz, 2H), 4.95 (br, 2H). 13
C
NMR (CDCl3): δ 166.2, 159.0, 149.5, 148.3, 144.5, 137.0, 133.1,
132.1, 131.8, 129.4, 124.0, 122.0, 117.6. ESMS m/z: 282.3 (M þ
1)þ.
1-(2,3-Dihydronaphthalen-4(1H)-ylideneamino)-4-phenyl-1H-
imidazol-2-amine (20). 1H NMR (CDCl3): δ 8.31 (d, J = 7.8 Hz,
1H), 7.68 (d, J = 7.2 Hz, 2H), 7.45-7.38 (m, 2H), 7.32-7.25 (m,
4H), 7.12 (t, J = 7.2 Hz, 1H), 5.72 (s, 2H), 2.93 (t, J = 6.3 Hz,
2H), 2.85 (t, J = 6.0 Hz, 2H), 1.92-1.83 (m, 2H). 13C NMR
(CDCl3): δ 164.4, 140.5, 139.7, 136.4, 133.1, 131.2, 129.3, 129.1,
128.8, 128.6, 128.3, 127.5, 126.2, 122.1, 31.6, 26.5. ESMS m/z:
303.3 (M þ 1)þ, 325.1 (M þ 23)þ.
1-(4-Chlorobenzylideneamino)-4-(thiophen-2-yl)-1H-imidazol-2-
amine (21). 1H NMR (CDCl3): δ 8.52 (s, 1H), 7.93 (d, J = 8.4
Hz, 2H), 7.80 (s, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 4.2
Hz, 1H), 7.21 (d, J = 4.2 Hz, 1H), 7.04 (t, J = 4.2 Hz, 1H), 6.31
(br, 2H). 13C NMR (CDCl3): δ 160.4, 145.3, 138.7, 137.2, 132.9,
131.6, 130.2, 128.9, 128.6, 124.5, 121.3, 118.0. ESMS m/z: 303.4
(M þ 1)þ.
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(22). 1H NMR (CDCl3): δ 7.90 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H),
7.49-7.34 (m, 9H), 7.20-7.06 (m, 3H), 6.11 (br, 2H). 13C NMR
(CDCl3): δ 158.4, 137.6, 136.5, 132.4, 132.9, 131.6, 131.5, 129.4,
128.3, 127.0, 126.8, 125.5, 124.5, 122.5, 121.0. ESMS m/z: 373.5
(M þ 1)þ.
1-(4-Chlorobenzylideneamino)-4-(thiazol-2-yl)-1H-imidazol-
2-amine (23). 1H NMR (CDCl3): δ 8.72 (s, 1H), 8.08 (s, 1H), 7.96
(d, J = 8.1 Hz, 2H), 7.79 (d, J = 3.3 Hz, 1H), 7.59-7.56 (m, 3H),
6.45 (br, 2H). 13C NMR (CDCl3): δ 162.2, 157.4, 145.9, 137.6,
135.3, 132.9, 130.4, 127.0, 121.3, 120.3, 116.6. ESMS m/z: 304.5
(M þ 1)þ.
Acknowledgment. This study was supported by Grants
BP-090-PP01, BP-090-CF03, and BP-091-CF03 of The Na-
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