Synthesis and antitumor activity of 1-mesityl-3-(2-naphthoylmethano)-1H-imidazolium bromide

Article abstract of DOI:10.1016/j.bmcl.2010.01.163

An imidazolium salt, 1-mesityl-3-(2-naphthoylmethano)-1H-imidazolium bromide (MNIB), has been investigated for its antitumor properties. In vitro studies demonstrate that MNIB is active against K562, SMMC-7721, EJ, AGZY, HEP-2, A549, HepG2, and Raji tumor

Full text of DOI:10.1016/j.bmcl.2010.01.163

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1844–1847
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activity of 1-mesityl-3-(2-naphthoylmethano)-1H-im-
idazolium bromide
a,
Xianghui Zeng a, , Xiaodong Yang a, , Yanli Zhang b, , Chen Qing b, , Hongbin Zhang
*
*
a Key Laboratory of Medicinal Chemistry for Natural Resource (Yunnan University), Ministry of Education, School of Chemical Science and Technology,
Yunnan University, Kunming 650091, PR China
b The Yunnan Key Laboratory of Pharmacology for Natural Products Research, Kunming Medical College, Kunming 650031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An imidazolium salt, 1-mesityl-3-(2-naphthoylmethano)-1H-imidazolium bromide (MNIB), has been
investigated for its antitumor properties. In vitro studies demonstrate that MNIB is active against K562,
SMMC-7721, EJ, AGZY, HEP-2, A549, HepG2, and Raji tumor cells, and can induce the G1 phase cell cycle
arrest and apoptosis in K562 cells. Moreover, administration of MNIB significantly inhibited tumor growth
in human non-small lung tumor (A549) xenografts.
Received 19 November 2009
Revised 27 January 2010
Accepted 30 January 2010
Available online 6 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
MNIB
Apoptosis
Cell cycle
Antitumor activity
Imidazolium salts have attracted considerable interests for their
versatile properties in chemistry as well as pharmacology. A number
of imidazolium salts have been reported in recent years, including
imidazolium alkaloids (lepidiline B) isolated from natural source,1
with interesting biological activities such as antimicrobial and anti-
fungal (l,3-dialky imidazolium chlorides),2 antitumor (l,3-dialky
imidazoliumiodides),3 antimuscarinic (1,3-disubstituted imidazoli-
um halides),4 thromboxane synthetase inhibition (1,3-disubstituted
imidazolium halides),5 antiinflammatory (enol betaines of phenacyl
halides),6 antiarrhythmic (1,3-disubstituted imidazolium halides),7
and plasmid DNA cleavage (monometallic cyclen complexes con-
taining 1,3-disubstituted imidazolium bromides group).8 In our pre-
vious paper, we reported the synthesis of a number of phenacyl
imidazolium salts and the preliminary results for its cytotoxic eval-
uation towards a number of tumor cell lines.9 We found that, for the
first time, the synthetic phenaxyl imidazolium salts are potent anti-
tumor agents. As part of our ongoing research efforts on the design
anddevelopmentof imidazolering-basedcancertherapeuticagents,
we report herein the in vitro cytotoxic properties of 1-mesityl-3-
(naphthoylmethano)-1H-imidazolium bromide (MNIB) against tu-
mor cells. We also present our findings that MNIB elongates the G1
phase of the cell cycle leading to apoptosis, and exhibits significant
in vivo cytotoxic properties.
A modified procedure for the synthesis of 1-arylimidazoles was
reported by our group.10 Following the method, we obtained the
desired 1-mesityl-1H-imidazole (5, 43% yield). Then, MNIB (1)
was prepared by treatment of compound 5 with 2-bromo-1-(naph-
thalen-3-yl)ethanone (6) in toluene and heated to reflux for 12 h
(92%, Scheme 1).11
In order to investigate whether the synthesized compound inhi-
bit cancer cell proliferation, K562 (human chronic myelogenous
leukemia cell), SMMC-7721 (human hepatocellular carcinoma
cell), EJ (human bladder tumor cell), Hep-2 (human laryngeal car-
cinoma cell), A549 (human lung tumor cell), human hepatocellular
liver carcinoma (HepG2), and Raji (human Burkitt’s lymphoma)
were treated at different concentrations from 0.1 to 100 lg/mL
for 48 h.12 The cytotoxic properties of MNIB on the cell lines were
evaluated by MTT assay, and the results are summarized in Table 1.
Growth of human tumor cells was strongly inhibited by the com-
pound MNIB with IC50 value of 0.3–5.0 lg/mL (Table 1).
The cell inhibition data (Table 1) clearly indicate that the new
imidazolium bromide agent MNIB inhibited the growth of different
types of tumor cells. Among the seven tested cell lines, k562, Raji,
SMMC-7721, EJ, Hep-2, and HepG2 exhibited more sensitivity to
MNIB (IC50 values, 0.3–2.2 lg/mL) than A549 (5.0 lg/mL). Particu-
larly, Raji and Hep-2 cell lines were ꢀeightfold more sensitive to
MNIB than A549. This compound demonstrated excellent antipro-
liferative activities on various human tumor cell lines from a di-
verse set of target organs, including leukemia and solid tumors
(bladder, laryngeal, lung, and liver cancer cell lines). These results
suggested that MNIB had promising antitumor activity against a
broad spectrum of human tumors.
* Corresponding authors. Tel.: +86 871 5031119; fax: +86 871 5035538 (H.Z.);
tel.: +86 871 5332985; fax: +86 871 5316884 (C.Q.).
E-mail addresses: qingchenhh@yeah.net (C. Qing), zhanghb@ynu.edu.cn,
zhanghbyd@gmail.com (H. Zhang).
These authors contributed equally to this research.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.163

X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1844–1847
1845
H
H
O
H
H
O
O
a
b
N
NH2
+
N
N
two steps 43%
2
3
4
5
5
O
O
4
1'
4'
Br
c
N
N +
N
N
3
1
2
92%
Br
6
5
1
Scheme 1. Synthesis of MNIB (1). (a) Reagents and conditions: CH3OH, rt; (b) HCHO, NH4Cl or NH4Ac, 85% H3PO4; (c) PhCH3, reflux.
Table 1
g/mL)]a
(lg/mL)
To detect the role of MNIB in conferring sensitivity to apoptosis,
K562 cells were exposed to increasing concentrations of MNIB.13
The degree of apoptotic cell death was quantified with Annexin V-
FITC/PI double-labeled cytometry. As shown in Figure 1, there were
little binding of Annexin V-FITC in untreated K562 cells. However,
Cytotoxic activity data for compound MNIB [IC50 (l
Compound
MNIB
IC50
EJ
1.2
K562
1.7
SMMC-7721
1.8
Hep-2
0.3
A549
5.0
HepG2
2.2
Rajib
0.7
after treatment of cells with MNIB at 2, 10 lg/mL doses for 24 h,
a
Cytotoxicity as IC50 values for each cell line, the concentration of compound
the apoptosis rate was 8.1 1.1%, and 15.5 0.8%, respectively,
which were statistically different from the control (1.9 0.3%).
The results of cell cycle analysis on synchronized K562 cells by
flow cytometry are summarized in Table 2.14 The data demonstrate
that the percent of cells in the G1 phase was significantly higher for
that caused 50% reduction in absorbance at 570 nm relative to untreated cells using
the MTT assay.
b
Human chronic myelogenous leukemia (K562), human hepatocellular carci-
noma (SMMC-7721), human bladder tumor (EJ), human laryngeal carcinoma (Hep-
2), human lung tumor (A549), human hepatocellular liver carcinoma (HepG2), and
human Burkitt’s lymphoma (Raji).
B
A
18
16
14
12
10
8
**
**
6
4
2
0
0
2
10
MNIB (µg/mL)
Figure 1. MNIB caused significant apoptotic death of K562 cells. (A) Cells were treated with 2, 10 lg/mL MNIB for 24 h. The induction of apoptosis was determined by
Annexin V-FITC/PI double-staining assay. The figures were representative of three separate experiments. (B) The degree of apoptotic cell death was quantified. Data
**
represented the mean S.D. of at least three independent experiments ( P <0.01).

1846
X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1844–1847
Table 2
crease, whereas tumor volumes in MNIB treatment groups obtained
Flow cytometry analysis of compound MNIB in K562 cellsa
5.3-fold (4 mg kgÀ1 doseÀ1) and 3.1-fold (8 mg kgÀ1 doseÀ1) in-
creases, respectively. A 2.5-fold increasein the tumor volume was ob-
tained in the group treated with 1 mg/kg cisplatin. Acute toxicity in
mice was not found when MNIB was administered in the range 10–
80 mg/kg.
Cell cycle phase
% K562 cells
Control With MNIB = 2 g/mL With MNIB = 10 lg/mL
l
G1
S
G2/M
22
62
16
40
45
16
56
26
19
At the current time, to the best of our knowledge, there are
no chemotherapeutic agents available that can effectively control
the growth and metastasis of human non-small cell lung tumor
cells. In this context, new agents that decrease the rate of prolif-
eration either directly or by increasing the rate of programmed
cell death, allowing for improved treatment of human non-small
cell lung tumor, are clearly needed. Our investigation on imi-
dazolium bromide agent MNIB has demonstrated its excellent
efficacy in inhibiting the growth of K562, SMMC-7721, EJ, AGZY,
HEP-2, A549, HepG2, and Raji tumor cells. The remarkable effi-
cacy of MNIB in suppressing the growth of various tumor cells
is of particular significance for its potential use in the treatment
of a wide range of cancers. It is also important to note that MNIB
has demonstrated unique selectivity in elongating (or arresting)
the G1 phase of the cell cycle. With this cell cycle specificity,
MNIB can be potentially used in combination with other chemo-
therapeutic agents for a greater combined efficacy. Therefore, the
potential clinical applications of the new chemotherapeutic
agent MNIB in the treatment of various cancers are promising.
Further studies on the in vivo activity of MNIB, its pharmacoki-
netics, and its schedule dependency in animals are currently
underway.
a
Data represent percentage of cells in a particular cell cycle phase of the cell
cycle after 24 h of treatment with MNIB. Data are representative of three inde-
pendent experiments.
cell samples that were incubated with MNIB than for the control set,
and the proportion of G1 phase cells treated with MNIB increased
gradually in a dose-dependent manner. At the same time, the frac-
tion of cells in S phase decreased accordingly, while the proportion
of G2/M phase cells had no significant change. Moreover, many of
the cells went through apoptosis when MNIB reached 2 lg/mL. This
resultsuggeststhatMNIB mayinduceapoptosisviaelongationof the
G1 phase in the cell cycle.
This result is significant because disruption or malfunction of
cell cycle control within the G1 phase has been recognized as
the most important biochemical phenomenon for tumor progres-
sion and tumorigenesis. The ability of certain small molecules to
control cell cycle machinery within the G1 phase has provided
exciting new opportunities with hopes of developing new types
of drugs efficacious against refractory cancers (e.g., non-small
cell lung cancer).
We have also examined the in vivo antitumor activity of MNIB,
usingA549xenograftmodels.15 Ininvivoexperimentsoftumor-bear-
ing mice, average tumor size was about 100 mm3 at the initiation of
treatment (day 1). MNIB was administrated ip once every 2 days for
19 days. Mice weight and tumor volume were recorded every 2 days
until animals were sacrificed at 19 days. Tumor volume (mm3) was
measured with calipers and calculated as (W2 Â L)/2, where W,
width; and L, length. The tumor volume at day n was expressed as rel-
ative tumor volume (RTV) according to the following formula:
RTV = TVn/TV1, where TVn is the tumor volume at day n and TV1 is
the tumor volume at day 1. The tumor volumes were significantly
inhibited (P <0.05–0.001) from days 12 to 19 in the groups treated
with MNIB (4 and 8 mg kgÀ1 doseÀ1). As shown in Figure 2, from days
1 to19, the tumorvolumes inthecontrol groupachieved a 7.8-foldin-
Acknowledgments
This work was supported by National Basic Research Program of
China (973 Program 2009CB522300), the Natural Science Founda-
tion of Yunnan Province (2007B0006Z), the Foundation of the Chi-
nese Ministry of Education for Fostering Important Scientific
Program(706052), and YunnanEducationalCommission(08Y0011).
References and notes
1. Cui, B.; Zheng, B. L.; He, K.; Zheng, Q. Y. J. Nat. Prod. 2003, 66, 1101.
2. (a) Vik, A.; Hedner, E.; Charnock, C.; Tangen, L. W.; Samuelsen, Ø.; Larsson, R.;
Bohlinb, L.; Gundersen, L. L. Bioorg. Med. Chem. 2007, 15, 4016; (b)
Demberelnyamba, D.; Kim, K. S.; Choi, S.; Park, S. Y.; Lee, H.; Kim, C. J.; Yoo, I.
12
control
DDP 1 mg/kg
MNIB 8 mg/kg
MNIB 4 mg/kg
10
8
6
4
2
0
5
8
12
15
19
Treatment days
Figure 2. Antitumoric effects of compound MNIB in nude mice. MNIB antitumor potency in human non-small lung tumor A549 xenograft model. Treatment was initiated
when average tumors reached a mean group size of 100 mm3. MNIB was administered (ip) every 2 days for 19 days (n = 8 per group).

X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1844–1847
1847
D. Bioorg. Med. Chem. 2004, 12, 853; (c) Pernak, J.; Skrzypzzak, A.; Kucharski, S.;
Krysinski, J. Arch. Pharm. 1984, 317, 430.
56.29, 20.99, 17.27. ESI-MS: m/e 356 [M+1ÀBr]+ (85), 355 [MÀBr]+ (100). HR-
ESI-MS: m/z calcd for C24H23BrN2O 434.0994, found 434.0992.
3. (a) Fortuna, C. G.; Barresi, V.; Berellini, G.; Musumarra, G. Bioorg. Med. Chem.
2008, 16, 4150; (b) Ballistreri, F. P.; Barresi, V.; Benedetti, P.; Caltabiano, G.;
Fortuna, C. G.; Longo, M. L.; Musumarraa, G. Bioorg. Med. Chem. 2004, 12, 1689.
4. Miyachi, H.; Kiyota, H.; Segawa, M. Bioorg. Med. Chem. Lett. 1999, 9, 3003.
5. Iizuka, K.; Kamijo, T.; Yamamoto, R.; Harada, H. U.S. Patent 4, 461,905, 1984.
6. Haugwitz, R. D.; Narayanan, V. L. U.S. Patent 3,852,301, 1974.
7. Lis, R.; Morgan, T. K., Jr.; DeVita, R. J.; Davey, D. D.; Lumman, W. C., Jr.; Wohl, R.
A.; Diamond, J.; Wong, S. S.; Sullivan, M. E. J. Med. Chem. 1987, 30, 696.
8. Li, Q. L.; Huang, J.; Wang, Q.; Jiang, N.; Xia, C. Q.; Lin, H. H.; Wu, J.; Yu, X. Q.
Bioorg. Med. Chem. 2006, 14, 4151.
12. The cytotoxicity assay was in seven kinds of cells line (K562, Hep-2, HepG2,
HL60, EJ, Raji, and SMMC-7721). Cells were cultured at 37 °C under
a
humidified atmosphere of 5% CO2 in RPMI 1640 medium supplemented with
10% fetal serum and dispersed in replicate 96-well plates. Compounds were
then added. After 48 h exposure to the compounds, cells viability were
determined by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (MTT) cytotoxicity assay by measuring the absorbance at 570 nm
with a microplate spectrophotometer. Each test was performed in triplicate.
13. Evaluation of apoptosis. According to the method described in Annexin V-FITC
detection kit, cells (1 Â 106/mL) treated with or without varying
9. Yang, X.-D.; Zeng, X.-H.; Zhang, Y.-L.; Qing, C.; Song, W.-J.; Li, L.; Zhang, H.-B.
Bioorg. Med. Chem. Lett. 2009, 19, 1892.
10. Liu, J. P.; Chen, J. B.; Zhao, J. F.; Zhao, Y. H.; Li, L.; Zhang, H. B. Synthesis 2003,
2661.
concentrations of MNIB (2, 10
of binding buffer. Afterwards, cells were stained with 5
solution and 10 L PI solution for 15 min at room temperature in the dark.
Then the samples were diluted with 300 L of binding buffer and analyzed by
l
g/mL) for 24 h were resuspended in 100
lL
l
L Annexin V-FITC
l
l
11. Typical procedure for the synthesis of compound MNIB.
flow cytometry using the FACScalibur (Becton–Dickinson, San Jose, CA).
14. Cell cycle analysis. To analyze the DNA content by flow cytometry, cells were
trypsinized from the culture flask. After centrifugation at 300g for 5 min at
room temperature, the supernatant was removed. The cells were then washed
twice with PBS solution and fixed with 3 mL of ice-cold 70% EtOH overnight.
Fixed cells were harvested by centrifugation at 300g for 3 min at room
temperature and washed twice with PBS containing 1% FBS. Collected cells
(A) Synthesis of 1-(2,4,6-trimethylphenyl)-1H-imidazole (5) [4]: 2,4,6-
trimethylaniline (1.35 g, 10 mmol) in MeOH (10 mL) was treated with 30%
glyoxal (1.62 mL, 10 mmol) for 16 h at room temperature. A yellowish mixture
was formed. NH4Cl (1.07 g, 20 mmol) was added followed by 37% aqueous
formaldehyde (1.6 mL). The mixture was diluted with MeOH (40 mL) and the
resulting mixture was refluxed for 1 h. H3PO4 (1.4 mL, 85%) was added over a
period of 10 min. The resulting mixture was then stirred at reflux for a further
8 h. The reaction was monitored by TLC. After removal of the solvent, the dark
residue was poured onto ice (30 g) and neutralized with 40% KOH aqueous
solution until the pH 9. The resulting mixture was extracted with Et2O
(5 Â 30 mL). The organic phases were combined and washed with H2O, brine
and dried (NaSO4). The solvent was removed and the residue was
chromatographed on silica gel to afford compound 5 (43% yield).
were resuspended in PBS (100
l
L/L Â 105 cells) and treated with 100
lg/mL of
RNase A at 37 °C for 30 min. Propidium iodide was then added to a final
concentration of 50
lg/mL for DNA staining and 20,000 fixed cells were
analyzed on FACScalibur (Becton–Dickinson, San Jose, CA). Cell cycle
a
distribution was analyzed using the Modifit’s program (Becton–Dickinson).
15. For the evaluation of in vivo antitumor activity, A549 human non-small lung
tumor cells (2 Â 107 cells/mL) were implanted subcutaneously into the right
flank of nude mice on day 0. Compounds were dissolved in 0.5% CMC-Na and
were intraperitoneously administered at a concentration of 4 or 8 mg/kg every
2 days for 19 days. Cisplatin (DDP) was used as a reference compound and its
dosage was 1 mg/kg. Test substances were administrated in a volume of 0.2 mL
per 20 g body weight of animals. Tumor volume (mm3) was measured with
calipers and calculated as (W2 Â L)/2, where W, width; and L, length. The
tumor volume at day n was expressed as RTV according to the following
formula: RTV = TVn/TV1, where TVn is the tumor volume at day n and TV1 is
the tumor volume at day 1. Mice weight and tumor volume were recorded
until animals were sacrificed at 19 days. Animal experiments were performed
under the permission according to institutional guidelines.
(B) Synthesis of compound MNIB (1): compound 5 (374 mg, 2 mmol) in toluene
(10 mL) was treated with 2-bromo-1-(naphthalen-3-yl)ethanone (6, 592 mg,
2.4 mmol) and heated to reflux for 12 h. A white precipitate was formed and
collected by filtration, washed with toluene (3 Â 10 mL), then dried to give the
compound MNIB (1, 799 mg, 92% yield). MNIB (1): white powder, mp 294–
296 °C. IR (KBr) 3418, 3156, 3119, 2954, 2916, 2838, 1693, 1600, 1571, 1512,
1462, 1422, 1364, 1244, 1214, 1177, 1113, 1065, 1015, 983, 861, 839, 812, 745,
670 cmÀ1 1H NMR (300 MHz, DMSO-d6) d 9.42 (s, 1H, Himidazole-2), 8.86 (s, 1H,
.
Himidazole-5), 8.23–8.03 (m, 6H, PhH and Himidazole-4), 7.79–7.69 (m, 2H, PhH),
7.19 (s, 2H, PhH), 6.29 (s, 2H, PhCOCH2), 2.36 (s, 3H, CH3), 2.11 (s, 6H, 2 Â CH3).
13C NMR (75 MHz, DMSO-d6) d 191.44, 140.71, 139.40, 135.95, 134.68, 132.43,
131.54, 131.28, 131.07, 129.68, 129.21, 128.26, 127.77, 125.12, 123.87, 123.59,