Cytotoxicity of substituted benzimidazolyl curcumin mimics against multi-drug resistance cancer cell

Full text of DOI:10.5012/bkcs.2013.34.4.1272

1272 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 4
http://dx.doi.org/10.5012/bkcs.2013.34.4.1272
Notes
Cytotoxicity of Substituted Benzimidazolyl Curcumin Mimics Against Multi-Drug
Resistance Cancer Cell
a
Young Woo Eom, Sangtae Oh, Ho Bum Woo, Jungyeob Ham, Chan Mug Ahn, and Seokjoon Lee
†,a
‡
§
‡,*
#,*
Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea
†
Department of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, Korea
‡
Department of Basic Science, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea
*
E-mail: ahn0707@yonsei.ac.kr
§
Marine Chemomics Laboratory, Natural Medicine Center, Korea Institute of Science and Technology,
Gangneung 210-340, Korea
#
Department of Pharmacology, Kwandong University College of Medicine, Gangneung 210-701, Korea
*
E-mail: sjlee@kwandong.ac.kr
Received December 26, 2012, Accepted January 21, 2013
Key Words : Curcumin, Benzimidazole, Cytotoxicity, Multi-drug resistance, Curcumin mimics
10
library (6) against various cancer cells. Because the benzi-
Curcumin (diferuloyl methane, 1), a major constituent of
the root of Curcuma longa L., has useful biological pro-
midazole molecules exhibit a variety of biological properties
1
2
perties, which are antiinflammatory, antioxidant, antiviral,
3
11
12
such as anticancer, antiviral, and anti-hypertensive activity,
13
4 5
chemopreventive, anti-infective activity, and wound-heal-
we can expect their novel cytotoxic properties come from
benzimidazolyl groups attached to the feruloyl structure.
To date, the most efficient tool to treat cancer, viral infec-
tion, and so on is chemotherapy, but the occurrence of drug
resistance due to clinically used small molecular drugs has
become a significant obstacle in cancer and antiviral treat-
6
ing properties. We have previously reported that various
alkyl amide and aryl amide linked curcumin mimics (2) have
7
an angiogenesis inhibition effect and curcumin derivatives
(3) possessing diverse sulfonyl amide-linked functionalities
exhibit a vasodilatation effect on the basilar artery of white
+
8
14
ment. In particular, because multidrug resistance (MDR) of
rabbit induced by high K ion. In addition, we discovered
that substituted triazolyl curcumin derivatives (4) synthe-
sized from Cu(I)-catalyzed Huisgen 1,3-cycloaddition ex-
hibit moderate to strong inhibitory activity against the
osteoclastogenesis induced by the receptor activator of NF-
cancer cells caused by prolonged administration of a certain
drug can result in resistance toward multiple drugs, the cure
rate and survivability of cancer patients by chemotherapy is
critically reduced. Therefore, it is very urgent to study the
recurrence of MDR and discover novel anticancer agents
inhibiting MDR cells. Recently, curcumin and its related
9
κB ligand (RANKL), which means that curcumin mimics
with triazole groups will be potential drug candidates for
treating osteoporosis. Based on the screening results for all
curcumin mimics in our in-house library, their diverse bio-
logical properties mainly depend on the additional functional
groups attached to the feruloyl structure and they generally
exhibit mild cytotoxicity. However, very recently, we dis-
covered that introduction of benzimidazole groups (5) to the
feruloyl scaffold through aldol condensation greatly improv-
ed the cytotoxicity of newly synthetic curcumin mimics
natural products were reported to modulate the human
15-17
MDR1 gene expression.
We also previously reported
that amide-linked curcumin derivatives (2) showed a potent
MDR reversal activity by inhibiting drug efflux function of
18,19
P-glycoprotein (P-gp).
When we are considering the our
previous studies on the cytotoxicity against cancer cells and
inhibitory effect on drug efflux function of P-gp of curcumin
mimics library, we can anticipate the recent curcumin library
Figure 1. Structures of curcumin and synthetic curcumin mimic derivatives.
a
These authors contributed equally to this work.

Notes
Bull. Korean Chem. Soc. 2013, Vol. 34, No. 4 1273
Scheme 1. Reagents and conditions: (a) Glycolic acid (3 eq), 4 N HCl, reflux, 6 h; (b) Iodomethane (3 eq), tetrabutylammonium bromide
o
o
(0.2 eq), potassium hydroxide (1.1 eq), tetrahydrofuran, 0 C; (c) Dess-Martin periodinane (1.1 eq), methylene chloride, 0 C, 6 h; (d)
o
Tribromoborane (2 eq), methylene chloride, −78 C; (e) 11 or 12 (each 1 eq), 40% KOH, Ethanol, ice bath, 10 h.
o
23
C. The curcumin mimic library like 13 and its constitu-
(6) possessing benzimidazole functionalities may inhibit the
MDR cancer cell. In order to prove our postulation, we
compared the cytotoxicity of curcumin derivatives (6) against
multidrug resistant ovarian cancer cell lines with over-
expressed P-gp and its origin ovcar-8 without P-gp and will
report the result in this paper.
tional isomeric curcumin derivatives such as 14 were obtain-
ed from the aldol reaction of 11 and 12 with synthetic
benzimidazolyl 2-carbaldehyde compounds (10a-10g), re-
8
spectively. The instrumental data of curcumin library con-
1 13
firmed through H NMR and C NMR spectroscopy were
10
We synthesized benzimidazolyl curcumin mimic library
(5) shown in Scheme 1. The reaction of variously substituted
1,2-phenylenediamine (7a-7g) with glycolic acid (3 eq) in
4 N hydrochloric acid produced substituted benzimidazolyl
already reported in our previous report.
To evaluate the cytotoxicity of novel curcumin mimics
with structurally diverse benzimidazol moieties against MDR
cancer cells, we conducted a cytotoxicity assay by employ-
20
methanol (8a-8g) after reflux for 6 h. The benzimidazolyl
24
ing the MTT colorimetric method against NCI/ADR-RES
methanols 8a-8g were reacted with iodomethane (3 eq) in
the presence of tetrabutylammonium bromide (TBAB, 0.2
eq) and potassium hydroxide (KOH, 1.1 eq) in tetrahydro-
cell with over-expressed P-gp and OVCAR-8 without P-
25
gp. The cytotoxicity of benzimidazolyl curcumin mimic
library (13a-13h and 14a-14h) against non-MDR cancer
cells OVCAR-8 shows quite strong to moderate potency,
namely is much stronger than their mother compound, cur-
cumin. It means that the addition of the benzimidazole group
to feruloyl structure dramatically increases the inhibitory
potency on cancer cells by about 5-340 times with the IC50
of curcumin (1), indicating that we can design novel drug
candidates from the adequate addition of diverse functional
o
furan (THF) at 0 C to afford 1-methylbenzimidazolyl
21
compounds (9a-9g), which were subsequently oxidized
with the Dess-Martin periodinane in methylene chloride at 0
o
C to produce substituted benzimidazolyl 2-carbaldehyde
22
(10a-10g). 5-Hydroxy benzimidazolyl 2-carbaldehyde
(10h) was also obtained from the demethylation of 10e using
tribromoborane (BBr3, 2 eq) in methylene chloride at −78
Table 1. Inhibitory concentration of curcumin mimic library against various cancer cell lines
a
a
Cancer cells (IC50 µM)
Cancer cells (IC50 µM)
b
b
Entry
RF
Entry
RF
NIH/ADR-RES
OVCAR-8
NIH/ADR-RES
OVCAR-8
a
b
c
d
e
f
21.3
9.6
5.1
5.6
0.7
50.7
6.5
6.3
6.2
5.8
4.2
1.7
33.1
1.6
5.4
3.8
4.7
3.2
a
b
c
d
e
f
23.7
9.8
5.4
5.7
4.4
1.7
6.1
6.6
5.2
4.5
2.0
1.4
23.2
79.5
35.0
24.0
29.2
18.4
443.7
45.2
30.8
28.5
117.6
8.2
72.2
6.8
13
14
5.9
6.4
g
h
g
h
59.9
5.7
curcumin
854.5
138.5
238.5
0.001
3.6
Paclitaxel
70.0
0.005
14000
vincristine
138500
a
b
IC50 was calculated from nonlinear regression by Graphpad Prism software. Resistance factor (RF) to each drug was calculated as the ratio of the IC50
values of NIH/ADR cells to that of OVCAR-8 cells

1274 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 4
Notes
2. Sharma, O. P. Biochem. Pharmacol. 1976, 25, 1811.
groups to feruloyl structure. In order to clarify the anti-MDR
cancer cell activity of our curcumin library, we test the same
screening using MDR ovarian cancer cell (NCI/ADR-RES).
As shown in Table 1, the inhibitory effect on MDR ovarian
cancer cell is weaker than in case of test on OVCAR-8.
3. Li, C. J.; Zhang, L. J.; Dezube, B. J.; Crumpacker, C. S.; Pardee,
A. B. Proc. Natl. Acad. Sci. 1993, 90, 1839.
4. Kawamori, T.; Lubet, R.; Steele, V. E.; Kelloff, G. J.; Kaskey, R.
B.; Rao, C. V.; Reddy, B. S. Cancer Res. 1999, 59, 597.
5. Brouet, I.; Ohshima, H. Biochem. Biophys. Res. Commun. 1995,
206, 533.
26
However, when considering the resistance factor (RF)
6. Sidhu, G. S.; Mani, H.; Gaddipati, J. P.; Singh, A. K.; Seth, P.;
Banaudha, K. K.; Patnaik, G. K.; Maheshwari, R. K. Wound Rep.
Reg. 1999, 7, 362.
which calculate as the ratio of the IC50 values of NIH/ADR
cells to that of OVCAR-8 cells, benzimidazolyl curcumin
mimic library (13a-13h and 14a-14h) shows a small RF
value, which means that the difference of the inhibitory
potency between MDR ovarian cancer cell (NCI/ADR-RES)
and non-MDR ovarian cancer cell (OVCAR-8) is narrow
and they may be good candidates for treating cancer cells
with MDR. Most important anticancer drugs such as pacli-
taxel and vincristine exhibit 140,000 and 138,500 values,
respectively. In particular, 13b and 14b have a strong cyto-
toxic effect on both each ovarian cancer cells and low RF
value (each 1.7) and 14h also shows a strong inhibitory
effect and 1.4 of RF value. In case of 13c, its inhibition
effect is quite strong (IC50 = 23.2 μM on NCI/ADR-RES but
0.7 μM on OVCAR-8) but relatively high RF value (33.1).
Thus it could not differentiate MDR cancer cell from non-
MDR cell. Based on the screening results of Table 1, because
curcumin have a weak anticancer activity but low RF value,
it is possible to discover novel anticancer drug candidates by
structurally modifying curcumin mimics that have an inhibitory
properties against cancer cells with multi-drug resistance.
When considering that our previous amide-linked curcumin
7. Woo, H. B.; Shin, W.-S.; Lee, S.; Ahn, C. M. Bioorg. Med. Chem.
Lett. 2005, 15, 3782.
8. Ahn, C. M.; Park, B.-G.; Woo, H. B.; Ham, J.; Lee, S. Bioorg.
Med. Chem. Lett. 2009, 19, 1481.
9. Park, S.-K.; Oh, S.; Shin, H. K.; Kim, S. H.; Ham, J.; Song, J.-S.;
Lee, S. Bioorg. Med. Chem. Lett. 2011, 21, 3573.
10. Woo, H. B.; Eom, Y. W.; Park, K.-S.; Ham, J.; Ahn, C. M.; Lee, S.
Bioorg. Med. Chem. Lett. 2012, 22, 933.
11. Nofal, Z. M.; Soliman, E. A.; El-Karim, S. S. A.; El-Zahar, M. I.;
Srou, A. M.; Sethumadhavan, S.; Maher, T. J. Acta. Pol. Pharm.-
Drug Res. 2011, 68, 519.
12. Porcari, A. R.; Devivar, R. V.; Kucera, L. S.; Drach, J. C.;
Townsend, L. B. J. Med. Chem. 1998, 41, 1252.
13. Kumar, J. R.; Jawahar, J. L.; Pathak, D. P. E-J. Chem. 2006, 3,
278.
14. Aouali, N.; Eddabra, L.; Macadré, J.; Morjani, H. Crit. Rev. Oncol./
Hematol. 2005, 56, 61.
15. Chearwae, W.; Anuchapreeda, S.; Nandigama, K.; Ambudkar, S.
V.; Limtrakul, P. Biochem. Pharmacol. 2004, 68, 2043.
16. Anuchapreeda, S.; Leechanachai, P.; Smith, M. M.; Ambudkar, S.
V.; Limtrakul, P. Biochem. Pharmacol. 2002, 64, 573.
17. Romiti, N.; Tongiani, R.; Cervelli, F.; Chieli, E. Life Sci. 1998, 62,
2349.
18. Kim, Y. K.; Song, Y. J.; Seo, D. W.; Kang, D. W.; Lee, H. Y.;
Rhee, D. K.; Han, J. W.; Ahn, C. M.; Lee, S.; Kim, S. N. Biochem.
Biophys. Res. Commun. 2007, 355, 136.
mimics (2) inhibit drug efflux function of P-gp and increase
18,19
accumulation of anticancer drugs in the cancer cells,
it is
19. Um, Y.; Cho, S.; Woo, H. B.; Kim, Y. K.; Kim, H.; Ham, J.; Kim,
S. N.; Ahn, C. M.; Lee, S. Bioorg. Med. Chem. 2008, 16, 3608.
20. Orjales, A.; Alonso-Cires, L.; López-Tudanca, P.; Tapia, I.;
Mosquera, R.; Labeaga, L. Eur. J. Med. Chem. 1999, 34, 415.
21. Roggen, H.; Charnock, C.; Gundersen, L.-L. Tetrahedron 2009,
65, 5199.
possible to postulate that feruloyl group contribute to decrease
the drug efflux function of P-gp and benzimidazole group
increase the potency of cytotoxicity.
In conclusion, a novel curcumin mimics library (13a-13h
and 14a-14h) through the aldol reaction of (E)-4-(4-hydroxy-
3-methoxyphenyl)but-3-en-2-one (11) or (E)-4-(3-hydroxy-
4-methoxyphenyl)but-3-en-2-one (12) with diversely sub-
stituted benzimidazolyl-2-cabaldehyde (10a-10h) have been
synthesized with the purpose of discovering novel anti-
cancer drug candidates that inhibit a proliferation both MDR
cancer cells and non-MDR cancer cells at the same time. On
the basis of the MTT assay against the cancer cells on NCI/
ADR-RES and OVCAR-8, we confirmed that our novel
curcumin mimics inhibit the growth of MDR cancer cells
more strongly than our previous in-house curcumin mimic
libraries and curcumin. Among the tested derivatives, 13b,
14b, and 14h are the strongest candidates inhibiting multi-
drug resistance cancer cells.
22. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
23. Matarrese, M.; Soloviev, D. V.; Moresco, R. M.; Ferri, V.; Simonelli,
P.; Magni, F.; Colombo, F.; Todde, S.; Carpinelli, A.; Fazio, F.;
Kienle, M. G. Bioorg. Chem. 1998, 26, 91.
24. OVCAR-8 and NCI/ADR-RES cells overexpressing P-gp were
obtained from National Cancer Institute (Frederick, MD). These
cells were maintained in RPMI 1640 culture medium containing
10% FBS and antibiotics (100 U/mL penicillin G and 100 µg/mL
streptomycin). All experiments were carried out 24 h after cells
were seeded. Chemical compounds were dissolved in DMSO and
diluted with DMEM medium to final concentrations of 1 nM to
100 µM. OVCAR-8 and NCI/ADR-RES cells were incubated
with chemical compounds for 48 h and then the MTT assay was
performed. A 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium
bromide (MTT) reduction was determined to examine the effect
of chemical compounds on cell viability. Briefly, following the
treatment of cells with different concentrations of chemical compound
(final concentration, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, and
100 µM) for 48 h, cells were incubated with MTT solution (0.5
Acknowledgments. This work was supported by a
research grant from Yonsei University Wonju College of
Medicine (YUWCM 2012-58). The authors also thank for
the technical support from Natural Medicine Center at Korea
Institute of Science and Technology.
o
mg/mL) for 3 h at 37 C. The violet precipitate (formazan) in MTT-
treated cells was dissolved with 100 µL of DMSO and then ana-
lyzed at a wavelength of 595 nm with a microplate reader (Beckman
Coulter, Brea, CA). Each assay was performed in triplicate.
25. Qiang, F.; Lee, B.-J.; Ha, I.; Kang, K. W.; Woo, E.-R.; Han, H.-K.
Eur. J. Pharm. Sci. 2010, 41, 226.
References and Notes
26. Lu, J.-J.; Cai, Y.-J.; Ding, J. Mol. Cell Biochem. 2012, 360, 253.
1. Srimal, R. C.; Dhawan, B. N. J. Pharm. Pharmacol. 1973, 25, 447.