Cytotoxicity against KB and NCI-H187 cell lines of modified flavonoids from Kaempferia parviflora

Article abstract of DOI:10.1016/j.bmcl.2010.03.054

Flavones 1-4 isolated from Kaempferia parviflora were used for structural modification. Sixteen flavonoid derivatives, including four new derivatives, were synthesized and evaluated for cytotoxicity against KB and NCI-H187 cell lines. Flavanones 2a-4a demonstrated higher cytotoxic activity than the parent compounds. Cytotoxicity against KB cell line of oxime 1c was about 7 times higher than the ellipticine standard. Interestingly, oximes 1c and 2c exhibited highly potent cytotoxicity against NCI-H187 cell line with IC₅₀ values of 0.014 and 0.23 μM, respectively. Oximes 4c and 5c showed strong cytotoxicity against NCI-H187 cell line with IC₅₀ values of 4.04 and 2.32 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2010.03.054

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2821–2823
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cytotoxicity against KB and NCI-H187 cell lines of modified flavonoids
from Kaempferia parviflora
*
Chavi Yenjai , Suchana Wanich
Natural Products Research Unit, Center for Innovation in Chemistry, Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Flavones 1–4 isolated from Kaempferia parviflora were used for structural modification. Sixteen flavonoid
derivatives, including four new derivatives, were synthesized and evaluated for cytotoxicity against KB
and NCI-H187 cell lines. Flavanones 2a–4a demonstrated higher cytotoxic activity than the parent com-
pounds. Cytotoxicity against KB cell line of oxime 1c was about 7 times higher than the ellipticine stan-
dard. Interestingly, oximes 1c and 2c exhibited highly potent cytotoxicity against NCI-H187 cell line with
Received 24 November 2009
Revised 6 March 2010
Accepted 11 March 2010
Available online 15 March 2010
IC50 values of 0.014 and 0.23
H187 cell line with IC50 values of 4.04 and 2.32
l
M, respectively. Oximes 4c and 5c showed strong cytotoxicity against NCI-
M, respectively.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
l
Kaempferia parviflora
Flavanone oximes
Cytotoxicity
KB cells
NCI-H187 cells
Kaempferia parviflora belongs to the family of Zingiberaceae and
is known in Thai as kra-chai-dam. The rhizomes of this plant have
been used for treatment of gastrointestinal disorders,1 as health-
promoting herbs and as anti-inflammatory agents.2 In recent years,
there have been many reports of biological activities of this plant,
for example, antibacterial, antiplasmodial,3 anti-peptic ulcer,1 anti-
viral protease effects,4 anti-allergic activity5 as well as modulators
of multidrug resistance in cancer cells.6 In addition, it was found
that this plant has no testosterone-like effect on reproduction in
male rats.7 In our previous study, we reported the quantitation of
11 flavonoids in K. parviflora by gas chromatography. The major
components in this plant are 5,7-dimethoxyflavone, 5,7,40-trimeth-
oxyflavone, 5-hydroxy-3,7-dimethoxyflavone, and 5-hydroxy-7-
methoxyflavone.8 It was reported that 5,7-dimethoxyflavone
showed cancer chemopreventive properties in a variety of human
cell lines while 5,7-dihydroxyflavone, an unmethylated analog, had
potential chemopreventive properties but very poor bioavailabil-
ity.9,10 Trifluoromethyl derivatives of 5,7-dimethoxyflavone were
synthesized and evaluated for their inhibitory effect on the cell cy-
cle of U2OS cells in vitro and it was found that 4-trifluoromethyl
derivative possessed strong cytotoxicity.11 It has been reported
that 6-amino-5,7-dihydroxyflavone demonstrates potent and spe-
drugs from natural products, we therefore planned to modify the
chemical structure of flavonoids from K. parviflora and also
screened for cytotoxic activity of the derivatives of those flavo-
noids. We recently reported that aminoflavonoid derivatives from
this plant demonstrated strong cytotoxicity against the KB cell
line.13 Moreover, flavanone oximes exhibited cytotoxicity against
HepG2 and T47D cell lines.14 We report herein the simple synthetic
methods and active cytotoxic structures of modified flavonoids.
Catalytic hydrogenation of flavones 1–4 was performed under
hydrogen gas and using palladium on charcoal as a catalyst to fur-
nish flavanones 1a–4a as the major products (Scheme 1). Flava-
none 2a was obtained as
a single diastereomer as cis
configuration which was confirmed by the correlation of protons
at C2 and C3 in the NOESY experiment (Fig. 1). The 1H NMR spec-
trum of 2a showed resonance signals at d 5.34 (1H, d, J = 2 Hz) and
d 3.73 (1H, d, J = 2 Hz) which were assigned to H-2 and H-3, respec-
tively. Dihydrochalcones 1b, 3b, and 4b were detected as minor
products which were obtained from benzylic cleavage of flavanon-
es 1a, 3a, and 4a, respectively (Scheme 1). However, there was no
product from benzylic cleavage of 2a in the same condition. This
may be due to the methoxy group at C3 position retarding the ben-
zylic cleavage.
The demethylation reaction of flavanone 3a was carried out
using HBr in AcOH to obtain the corresponding 5,7-dihydroxyfla-
vanone 5a in 48% yield (Scheme 2). After treatment of 1a–5a with
hydroxylamine hydrochloride in the presence of KOH, oximes 1c–
5c were obtained in high yield (Scheme 3). The 13C NMR spectra of
oxime derivatives showed upfield shift signals of carbonyl carbons
(d 189.2–195.7) to the oxime carbons (d 148.8–157.2). The IR
cific rat intestinal
a
-glucosidase inhibitory activity.12 Due to the
continuing need for new cancer chemopreventive agents with no-
vel structures and as part of our research program on anti-tumor
* Corresponding author. Tel.: +66 43 202222 41x12243; fax: +66 43 202373.
E-mail address: chayen@kku.ac.th (C. Yenjai).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.054

2822
C. Yenjai, S. Wanich / Bioorg. Med. Chem. Lett. 20 (2010) 2821–2823
R4
+
R4
R4
R3
O
O
1a
2a
3a
4a
R3
O
O
R3
OH
O
H2, Pd/C
EtOAc
R1
R1
R1
R2
R2
R2
1 R1=R4=H, R2=OH, R3=OCH3
1b
_
2 R1=R3=OCH3, R2=OH, R =H
4
3 R1=R4=H, R2=R3=OCH3
4 R1=H, R2=R3=R4=OCH3
3b
4b
Scheme 1. Reagents and conditions: H2, Pd/C, EtOAc, rt, 24 h, 1a (57%), 1b (27%), 2a (66%), 3a (60%), 3b (17%), 4a (48%), 4b (18%).
(GFP) based assay.16 Ellipticine was included as a reference
H3CO
O
H3CO
O
substance.
The cytotoxicity results are shown in Table 1. Compound 2a
showed cytotoxicity against KB and NCI-H187 with IC50 values of
30.37 and 98.23 M, respectively. Flavanones 3a and 4a demon-
strated moderate cytotoxicity against NCI-H187 cells with IC50 val-
ues of 33.06 and 25.10 M, respectively, while showing weak
cytotoxicity against KB cells with IC50 values of 39.85 and
78.80 M, respectively. However, these three compounds exhib-
H
H
OCH3
OCH3
H
H
H
OH O
HO OH
l
2a
( )
( ) 2d
l
Figure 1. NOESY experiments of 2a and 2d.
l
ited more cytotoxicity than the parent flavones. These results sug-
gest that the flavanone skeleton is essential for cytotoxicity. In
contrast, flavanone 1a did not show cytotoxicity against any cell
lines. Unmethylated flavanone 5a showed weak cytotoxicity
H3CO
O
O
HO
O
HBr
AcOH
reflux
CH3O
OH O
3a
5a
(IC50 ꢁ 95–100
lM) in comparison with the parent compound (3a).
Dihydrochalcones 1b, 3b, and 4b showed no cytotoxicity
Scheme 2. Reagents and conditions: HBr, AcOH, reflux, 24 h, 48%.
against any cell lines, except 1b which exhibited strong cytotoxic-
ity against NCI-H187 cells with an IC50 value of 9.70 lM. This may
be due to the structure of this compound containing two phenolic
groups.
R4
R3
O
N
NH2OH
KOH
On attempting to find active substances, other functional
groups were produced and we anticipate obtaining useful results.
From the cytotoxicity of 2a–4a, we found that the flavanone struc-
ture showed potent cytotoxicity and therefore we decided to
change the carbonyl group to oxime and investigate the activity
of a series of oxime derivatives. Fortunately, 1c showed strong
cytotoxic activity against KB cell line with an IC50 value of
1a-5a
R1
OH
R2
1c-5c
Scheme 3. Reagents and conditions: NH2OHꢂHCl, 20% KOH, EtOH, reflux, 1–3 h, 1c
(96%), 2c (80%), 3c (89%), 4c (82%), 5c (98%).
0.26
improvement in potency against NCI-H187 cells by showing IC50
values of 0.014 and 0.23 M, respectively, which is higher than
the ellipticine standard (IC50 = 2.77 M) by nearly 200- and 12-
lM. Interestingly, oximes 1c and 2c showed dramatic
spectra showed characteristic absorption bands of OH stretching
between mmax of 3445–3345 cmꢀ1
.
l
l
Flavanones 2a–4a were treated with NaBH4 at room tempera-
ture to afford a single diastereomer of flavanols 2d–4d in moderate
to high yield (Scheme 4). The NOESY experiment of 2d showed rel-
atively cis orientation of protons at C2, C3, and C4 (Fig. 1). This
means that the insertion of the hydride ion occurred on the less
hindered side. The 13C NMR spectra of alcohol derivatives showed
signals of C4 upfield shifted from carbonyl carbons (d 189.2–195.7)
to oxycarbons (d 63.3–67.0). In addition, the 1H NMR spectra
showed signals of H-4 at d 5.24–5.27. In total, sixteen flavonoid
derivatives were synthesized and four of them (2a, 2c, 2d, and
4c) were new derivatives.
fold. In addition, oximes 4c (IC50 = 4.04
lM) and 5c
Table 1
Cytotoxicity of modified flavonoidsa
Compound
Cytotoxicity, IC50
NCI-H187
(lM)
KB
Vero cells
1–4
1a
2a
Inactiveb
Inactiveb
30.37
Inactiveb
Inactiveb
98.23
Inactiveb
—
—
3a
39.85
33.06
—
4a
5a
1b
3b
4b
1c
2c
3c
4c
5c
2d
3d
78.80
99.16
25.10
95.53
9.70
—
—
—
—
Cytotoxicity assays against human epidermoid carcinoma of
oral cavity (KB) and human small cell lung cancer (NCI-H187) cell
lines were performed employing Resazurin Microplate Assay
(REMA).15 Cytotoxicity assay against African green monkey kidney
cell line (Vero cells) was evaluated using Green Fluorescent Protein
Inactiveb
Inactiveb
Inactiveb
0.26
Inactiveb
Inactiveb
0.014
—
2.67
41.20
—
23.91
0.23
53.19
4.04
2.32
Inactiveb
43.15
R4
Inactiveb
Inactiveb
—
R3
O
NaBH4
CH3OH
84.90
56.79
2a-4a
95.56
R1
Inactiveb
Inactiveb
2.77
—
—
2.29
R2
92.14
OH
4d
Inactiveb
1.79
2d-4d
Ellipticine
a
Data shown are replicate experiments.
Inactive at >100 lM.
Scheme 4. Reagents and conditions: NaBH4, CH3OH, rt, 1 h, 2d (95%), 3d (92%), 4d
(76%).
b

C. Yenjai, S. Wanich / Bioorg. Med. Chem. Lett. 20 (2010) 2821–2823
2823
(IC50 = 2.32 lM) showed strong cytotoxicity against NCI-H187 cell
Acknowledgments
line. The results show convincingly that the oxime group at posi-
tion 4 of flavanone plays an important role in cytotoxicity. In con-
trast, 3c showed weak cytotoxicity against NCI-H187 cell line and
was inactive against KB cells. As in the previous report, this com-
pound showed weak cytotoxicity against HepG2 and T47D cell
lines.14 These results suggest that the hydroxyl group at C5 posi-
tion is crucial for cytotoxicity. In case of 5c, it showed stronger tox-
We thank the National Research Council of Thailand for finan-
cial support. Thanks are also due to the Bioassay Laboratory of
the National Center for Genetic Engineering and Biotechnology,
Pathumthani, Thailand, for biological activity assays. The Center
for Innovation in Chemistry (PERCH-CIC), Commission on Higher
Education, Ministry of Education is gratefully acknowledged.
icity against KB and NCI-H187 cell lines (IC50 = 84.90 and 2.32 lM,
respectively) than the corresponding methylated derivative 3c.
This derivative also showed good activity against HepG2 and
T47D cell lines.14 These results confirm that the hydroxyl group
is favorable to the activity. In addition, it is interesting to note that
the structure of compounds 1c and 2c are 5-hydroxy-7-methoxy-
flavanone oxime derivatives. These substances are likely to be
useful as lead compounds for the development of a novel class of
cytotoxic agents. However, cytotoxicity against normal cells
should be concerned. Therefore, cytotoxicity of these four com-
pounds (1c, 2c, 4c, and 5c) was evaluated against Vero cells. As
shown in Table 1, 4c and 5c exhibited no cytotoxicity against this
cell lines. Compound 1c showed an IC50 value of 2.67 lM against
Vero cells which is about 10- and 190-fold lower toxicity than
KB and NCI-H187 cell lines, respectively. In case of 2c, it demon-
strated an IC50 value of 41.20 lM against Vero cells which is about
180-fold lower toxicity than NCI-H187 cell line. All results show
convincingly that these substances are likely to be useful as lead
compounds for the development of a novel class of cytotoxic
agents, especially against lung cancer. Finally, flavanols 2d–4d
showed less potent cytotoxicity against KB and NCI-H187 cell lines
in comparison with the parents 2a–4a. These results confirm that
the 4-carbonyl groups or carbonyl analogs of flavanone are crucial
for the activity.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.054.
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In summary, we prepared a series of modified flavonoids under
simple reactions and evaluated their cytotoxicity. We found that
oxime derivatives, especially 1c and 2c possess stronger cytotoxic-
ity than the ellipticine standard and may be promising leads for the
development of cytotoxic agents. These results encourage the syn-
thesis of flavonoid analogs for improving cytotoxic activity.