Chemical modification and anticancer effect of prenylated flavanones from Taiwanese propolis

Article abstract of DOI:10.1080/14786419.2010.535146

Our previous studies demonstrated that eight prenylated flavanones (1-8), isolated from Taiwanese propolis, were capable of a broad spectrum of biological activities. Among them, nymphaeol A (3), nymphaeol B (4) and nymphaeol C (7), abundant in Taiwanese

Full text of DOI:10.1080/14786419.2010.535146

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Chemical modification and anticancer
effect of prenylated flavanones from
Taiwanese propolis
Chia-Nan Chen a , Che-Jen Hsiao b , Shoei-Sheng Lee b , Jih-Hwa
Guh b , Po-Cheng Chiang b , Chih-Chiang Huang c & Wei-Jan Huang
c
a NatureWise Biotech & Medicals Corporation, Taipei 115, Taiwan
b School of Pharmacy, College of Medicine, National Taiwan
University, Taipei 100, Taiwan
c Graduate Institute of Pharmacognosy, Taipei Medical University,
Taipei 110, Taiwan
Version of record first published: 27 Jul 2011
To cite this article: Chia-Nan Chen, Che-Jen Hsiao, Shoei-Sheng Lee, Jih-Hwa Guh, Po-Cheng
Chiang, Chih-Chiang Huang & Wei-Jan Huang (2012): Chemical modification and anticancer effect
of prenylated flavanones from Taiwanese propolis, Natural Product Research: Formerly Natural
Product Letters, 26:2, 116-124
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Natural Product Research
Vol. 26, No. 2, January 2012, 116–124
Chemical modification and anticancer effect of prenylated flavanones
from Taiwanese propolis
Chia-Nan Chena, Che-Jen Hsiaob, Shoei-Sheng Leeb, Jih-Hwa Guhb,
b
Po-Cheng Chiang , Chih-Chiang Huang and Wei-Jan Huang
c
c
*
aNatureWise Biotech & Medicals Corporation, Taipei 115, Taiwan;
bSchool of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan;
cGraduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
(Received 9 November 2009; final version received 17 September 2010)
Our previous studies demonstrated that eight prenylated flavanones (1–8),
isolated from Taiwanese propolis, were capable of a broad spectrum of
biological activities. Among them, nymphaeol A (3), nymphaeol B (4) and
nymphaeol C (7), abundant in Taiwanese propolis, exhibited cytotoxicity
against cancer cell lines. It therefore seemed interesting to improve their
activity via a semi-synthetic strategy. In this study, 12 novel prenylated
flavanones were synthesised in our laboratory and their activities were
assessed for two human prostate cancer cell lines, PC-3 and DU-145, and a
human hepatoma cell line, Hep-3B. Of these compounds, 10c, 11 and 12
showed more potent cytotoxicity against the PC-3 cell line than 5-Fu.
Using cytometric analysis followed by double staining with annexin
V-FITC and propidium iodide, it was observed that these compounds
induced apoptosis as well. This suggests that prenylated flavanones 10c,
11 and 12 may have anticancer potential for further development.
Keywords: prenylated flavanones; cytotoxicity; PC-3; DU-145; Hep-3B;
apoptosis
1. Introduction
Flavanones, widely distributed in the plant kingdom, are found in about 4000
naturally occurring compounds (Rusak, Herwig, & Jutta, 2002), exhibiting a large
number of biological effects including anti-inflammatory, antiviral and anticancer
properties (Harborne & Williams, 2000). Among various flavanones, prenylated
flavanones, characterised by C5 isoprenoid units, attracted interest for their
association with several biological targets such as oestrogen receptors (ERs)
(Milligan et al., 1999), heat shock proteins (HSP-90) (Hadden, Galam, Matts, &
Blagg, 2007), cyclooxygenases (COXs) (Barret, Gordon,
& Evans, 1985),
P-glycoproteins (PgPs) (Tchamo et al., 2000) and phosphodiesterases (PDEs) (Shin
et al., 2002). Prenylated flavanones were reported to inhibit NO production by
interfering with inducible nitric oxide synthase (iNOS) expression immediately
without the inhibition of iNOS after lipopolysaccharide (LPS)/interferon (IFN)-ꢀ
*Corresponding author. Email: wjhuang@tmu.edu.tw
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2010.535146
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Natural Product Research
117
macrophage stimulation (Cerqueira et al., 2008). Pedro et al. reported that a
prenylated flavanone, artelastin, demonstrated cytotoxicity against 52 human
tumour cell lines ranging from 0.8 to 20.8 mM and showed a dual function as a
disrupter of microtubule and inhibitor of DNA replication in cultured human breast
cancer cell line MCF-7 (Pedro et al., 2005). In addition, it was reported that 8-
prenylated naringenin exhibited strong oestrogenic activity and rapid absorption
after oral administration in contrast to the poor bioavailability of general flavanones
(Rad et al., 2006). This suggests that the prenyl groups on aromatic rings may
increase the lipophilicity and confer a strong affinity to biological membranes
(Barron, Balland, Possety, Ravanel, & Desfougeres, 1996).
We previously isolated and structurally characterised three novel prenylated
flavanones: propolins A (1), B (2) and E (5) (Chen, Wu, Shy, & Lin, 2003; Chen,
Weng, Wu, & Lin, 2004; Chen, Wu, & Lin, 2004) together with five known ones:
nymphaeol A (3), nymphaeol B (4), nymphaeol C (7) (Yakushijin, Shibayama,
Murata, & Furukawa, 1980), isonymphaeol B (6) (Kumazawa et al., 2004) and
30-geranyl-naringenin (8) (Jayasinghe, Rupasinghe, Hara, & Fujimoto, 2006;
Kumazawa et al., 2007) from Taiwanese propolis (Figure 1). Most of them exhibited
a broad spectrum of biological activities such as anticancer, antioxidant and
antimicrobial functions (Chen et al., 2003, Chen, Weng, Wu & Lin, 2004, 2008;
Chen, Wu, & Lin, 2007; Huang et al., 2007; Weng et al., 2007). Of these compounds,
Figure 1. Structures of the prenylated flavanones (1–8) isolated from Taiwanese propolis.

118
C.-N. Chen et al.
nymphaeol A (3), nymphaeol B (4) and nymphaeol C (7), abundant in Taiwanese
propolis, showed activity against human melanoma cells. So, it seemed interesting to
improve their cytotoxicity via a semi-synthetic strategy. The study of the cytotoxic
effect of these flavanone derivatives was carried out using three human cancer cell
lines of PC-3, DU-145 and Hep-3B. Here we show that compounds 10c, 11 and 12
exhibited higher cytotoxicity against PC-3 cancer cells than 5-Fu and induced
apoptosis, observed through cell cycle progression, followed by double staining with
annexin V-FITC and propidium iodide (PI). The following section describes the
outcome of these efforts.
2. Results and discussion
Our previous result indicated that the hydration of the terminal olefin on a prenyl
side chain of prenylated flavanones can increase their cytotoxicity against melano-
cyte (Chen, Weng, Wu & Lin, 2004; Chen, Wu & Lin, 2004). Hydration of the
terminal olefin either on a prenyl or geranyl group was achieved as described in
Scheme 1. Tetramethylation of nymphaeol A (3), nymphaeol B (4) and nymphaeol C
(7) in the presence of dimethyl sulphate and potassium carbonate under a general
condition afforded 9a–c, respectively. Treatment of 9b–c with 49% H2SO4(aq) using
THF as a co-solvent gave the corresponding monohydrated geranyl products 10b–c.
However, under the similar condition, 9a afforded the dihydrated geranyl product
10a. The different reactivity of the geranyl group at C-6 of 9a might be attributed to
its steric hindrance, which is less than that of 10b–c. Direct hydration of nymphaeol
A (3) and B (4) also yielded 10d–e, respectively. However, hydration of nymphaeol C
(7) under conditions similar to those described above had failed. In addition, a
selective trimethylation of nymphaeol C (7) at C-7, C-30 and C-40 according to the
reported method (Chu, Wu, & Lee, 2004) gave the expected product 9d (Phommart,
P. Sutthivaiyakit, Chimnoi, Ruchirawat, & S. Sutthivaiyakit, 2005) and when
followed by acidic hydration afforded 10f. Increased nymphaeol C (7) lipophilicity
was achieved as described in Scheme 2. The conversion of two tertiary alcohols of
10c into carbonate in the treatment of ethyl chloroformate and triethylamine had
1
failed, while leading to the C-ring opened product chalcone 11. The H-NMR of 11
showed the characteristic chemical shift at ꢁ 8.11 (1H, d, J ¼ 15.4 Hz) and 7.76 (1H,
d, J ¼ 15.4 Hz), which supports this structure. The HRESIMS spectrum of 11
showed a quasi-molecular ion (MþH)þ at m/z 585.3412, consistent with that
calculated for C34H49O8. Atmospheric hydrogenation of 10c using palladium on
carbon as a catalyst produced saturated product 12. It is noted that 12 has a Rf
similar to that of 10c. The HREIMS spectrum of 12 showed a molecular ion (M)þ at
m/z 586.3506, consistent with the expected formula C34H50O8. Tetra-acetylation of
nymphaeol B (4) and C (7) with acetic anhydride and pyridine provided 13a
(Yakushijin et al., 1980) and 13b, respectively. Investigation of the effect of the
prenyl side chain on flavanone skeletons was conducted as described in Scheme 2.
Ozonolysis of 9c and subsequent treatment of thiourea split the olefins into the
dialdehyde 14 (Huang, Chen, & Lee, 2003). The 1H-NMR of 14 showed two
additional signals at ꢁ 9.73 (1H, s) and 9.63 (1H, s) compared to 9c, indicating the
presence of aldehyde. The HREIMS spectrum of 14 showed a molecular ion (M)þ at
m/z 428.1469, consistent with the expected formula C23H24O8. Sodium chlorite

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119
OH
OMe
OMe
R''
OH
R''
OMe
R''
OMe
HO
R'
O
MeO
R'
O
MeO
R'
O
a
b
OH
O
OMe O
OMe O
3: R„=R2; R„„=H
4: R„=H; R„„=R2
7: R„=R1; R„„=R2
9a: R„=R2; R„„=H
9b: R„=H; R„„=R2
9c: R„=R1; R„„=R2
10a: R„=R5; R„„=H
10b: R„=H; R„„=R4
10c: R„=R3; R„„=R4
OH
OH
R''
OH
R''
O
OH
HO
R'
O
HO
R'
b
OH
O
OH
O
10d: R„=R5; R„„=H
10e: R„=H; R„„=R4
3: R„=R2; R„„=H
4: R„=H; R„„=R2
OH
OMe
R''
OMe
R''
O
OH
OMe
R''
OMe
HO
R'
MeO
R'
O
MeO
R'
O
c
b
OH
O
OH
O
OH
O
9d: R„=R1; R„„=R2
10f: R„=R3; R„„=R4
7
OH
R1=
R4=
R2=
R3=
OH
OH
OH
R5=
Reagents and conditions: (a) K2CO3, DMS, acetone, Δ, 24 h (b) H2SO4-THF, rt; (c) K2CO3, DMS, acetone, Δ, 6h;
Scheme 1. Syntheses of prenylated flavanones 10a-f.
oxidation (Andrus, Asgari, & Sclafani, 1997) of 14 gave the diacid 15. The HREIMS
spectrum of 15 showed a molecular ion (M)þ at m/z 460.1382, consistent with the
expected formula C23H24O10. The physical data for all compounds are provided as
supplementary material (available online only).
The cytotoxic effect of flavanones was assessed against two human prostate
cancer cell lines, PC-3 and DU-145, and a human hepatoma cell line, Hep-3B, using
5-Fu as a control compound (Supplementary Table S1 – online only). It indicated
that most compounds were not capable of selective cytotoxicity against these three
cancer cells. Of these compounds, compounds 10c, 11 and 12 showed higher
cytotoxic effect on PC-3 than 5-Fu. Compared to 7, compound 10c displayed
stronger potency, suggesting that this two-step chemical modification made a good
contribution to activity, whereas it showed a marginal effect on compounds 10a–b.
Compared to 12, compound 10c showed selective cytotoxicity towards PC-3, which
might be attributable to the importance of C20–C30 double bond on the geranyl side
chain. The stronger cytotoxicity of compound 10c compared to 10b might be

120
C.-N. Chen et al.
OMe
OMe
OMe
HO
HO
HO
HO
HO
HO
OMe
OMe
OMe
MeO
O
MeO
O
a
MeO
OH
b
OMe O
OMe O
OMe O
12
11
10c
OAc
OH
OAc
OH
AcO
R
O
HO
R
O
O
c
OAc O
OH
13a R=H
13b R=R1
4 R=H
7 R=R1
R1=
O
O
H
O
O
OH
OMe
OMe
OMe
OMe
OMe
OMe
MeO
O
MeO
O
MeO
O
e
d
OMe O
OMe O
OMe O
9c
H
OH
15
14
Reagents and conditions: (a) ClCO2Et, Et3N, toluene,
Δ; (b) H2, Pd-C, rt; (c) (Ac)2O, pyr, rt; (d)1)O3, CH2Cl2-
MeOH, -40oC, 2)(NH2)2CS, 0oC~rt; (e) NaClO2, NaH2PO4, rt;
Scheme 2. Syntheses of prenylated flavanones 11–12, 13a–b, 15.
attributed to the presence of a prenyl group at C6 position. The decrease in activity
of compound 10e compared to 10b indicated the importance of methoxy groups in
the hydrated nymphaeol B (4). The weaker activity of compounds 13a–b suggested
that acetyl groups showed a marginal effect on nymphaeol B (4) and C (7).
In order to investigate the effect of compounds 10c, 11 and 12 and the reference
compound 5-Fu on cell cycle progression, a flow cytometric analysis was performed
against PC-3 cells at time periods 12 and 24 h, respectively (Supplementary Figure S1
– online only). With a treatment at 30 mM, compounds 10c, 11 and 12 increased the
sub-G1 population by 10-, 3- and 7-fold, respectively, at 12 h. Furthermore, after
incubation for 24 h, compounds 10c, 11 and 12 significantly increased the sub-G1
population by 17-, 4-, and 8-fold, respectively. However, 5-Fu had only a slight effect
on cell cycle progression at each time period. In order to investigate whether
compounds 10c, 11 and 12 could induce cell death related to apoptosis, a
cytofluorometric analysis using annexin V-FITC and PI double staining was carried
out at time periods of 12 and 24 h. Annexin V (þ)/PI (ꢀ) and annexin V (þ)/PI (þ)
showed that cells were at the early and the late stages of apoptosis, respectively
(Supplementary Figure S2 – online only). At a concentration of 30 mM, compounds
10c, 11 and 12 increased the population of annexin V (þ)/PI (ꢀ) compared to

Natural Product Research
121
annexin V (þ)/PI (þ) by 27.48%, 40.32% and 16.32% compared to 6.84%, 4.56%
and 9.18%, respectively, at 12 h. In addition, treatment with compounds 10c, 11 and
12 for 24 h, resulted in an increased population of annexin V (þ)/PI (ꢀ) compared to
annexin V (þ)/PI (þ) by 50.73%, 57.72% and 50.49% compared to 30.26%, 19.96%
and 20.14%, respectively. This suggested that compounds 10c, 11 and 12 may induce
apoptosis in a time-dependent manner. In conclusion, this study reveals the
cytotoxicity of prenylated flavanones derived from three isolated Taiwanese propolis
towards several cancer cell lines and further investigates their effect on cancer cell
deaths, which may be useful for therapeutic applications. Further experiments to
study the mechanisms are being undertaken in this laboratory.
3. Experimental
3.1. General
The physical and spectral data of prepared compounds were obtained from the
following instruments: Fisher–Johns melting point apparatus (uncorrected); JEOL
JMX-HX110 (HREIMS and HRFABMS), JMS-SX102A (EIMS and FABMS) and
Finnigan MAT 95S (HRESIMS and ESIMS) mass spectrometers; and Bruker
AV-400 and AV-500 spectrometers using solvent peak as reference standard.
3.2. Nymphaeol A (3), B (4) and C (7)
Further information about nymphaeol A (3), B (4) and C (7), such as propolis origin,
extraction and isolation is supplied as supplementary material (available online
only).
3.3. General procedure for preparation of 9a–c
To the mixtures of prenylated flavanones (3, 4 or 7, 0.31 mmol), K2CO3
(431 mg, 3.1 mmol) and acetone (15 mL), Me2SO4 (0.25 mL, 2.48 mmol) was added
and the resulting final solution was heated and refluxed under N2 for 24 h. After
removal of the organic solvent, the residue was dissolved in CH2Cl2 (50 mL) and
washed with distilled H2O (50 mL ꢁ 3). The organic layer was dried over Na2SO4 and
evaporated under reduced pressure. The residue obtained was purified by a silica gel
column (EtOAc: n-hexane ¼ 1:6) to give 9a–c.
3.4. General procedure for preparation of (10a–f)
To a solution of (9a–d or 3 or 4, 0.17 mmol) in THF (6 mL), 49% H2SO4 (4 mL) was
added drop-wise in an ice bath. After complete addition, the reaction mixture was
stirred at room temperature (RT) for 8 h and then diluted with H2O. The aqueous
layer was extracted with CH2Cl2 (50 mL ꢁ 3). The combined organic layers were
dried over Na2SO4 and evaporated under reduced pressure to give a residue, which
was purified by a silica gel column (EtOAc: n-hexane ¼ 1:6ꢂ1:1) to give the pure
oil 10a–f.

122
C.-N. Chen et al.
3.5. 30,40,7-O-Trimethylnymphaeol C (9d)
To the mixture of 7 (300 mg, 0.61 mmol), K2CO3 (252 mg, 1.83 mmol) and acetone
(20 mL), Me2SO4 (0.20 mL, 2.1 mmol) was added and the resulting solution was
heated to reflux under N2 for 6 h. After removal of acetone, the residue was dissolved
in CH2Cl2 (50 mL) and washed with distilled H2O (50 mL ꢁ 3). The organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The residue was purified
by a silica gel column (EtOAc: n-hexane ¼ 1:6) to give 9d (120 mg, yield: 37%).
3.6. 3-(3-Hydroxy-3-methylbutyl)-20-(7-hydroxy-3,7-dimethyloct-3-enyl)-
6-hydroxy- 30,40,2,4-tetramethoxychalcone (11)
To a solution of 9c (100 mg, 0.17 mmol) in toluene (10 mL), ethyl chloroformate
(0.4 mL) and pyridine (0.1 mL) was added and the resulting solution was heated to
120ꢃC under N2 for 48 h. After removal of the solvent, the residue was purified by a
silica gel column (EtOAc: n-hexane ¼ 1:1) to afford 11 (75 mg, yield: 75%).
3.7. 6-(3-Hydroxy-3-methylbutyl)-20-(7-hydroxy-3,7-dimethyloctyl)-30,40,5,7-tetra
methoxyflavanone (12)
To the mixture of 9c (100 mg, 0.17 mmol) and EtOH (10 mL), Pd-C (30 mg) was
added, and the resulting solution was stirred at RT under atmospheric H2 overnight.
The reaction mixture was filtered through celite and concentrated under reduced
pressure. The residue was purified by a silica gel column (EtOAc: n-hexane ¼ 1:6) to
give pure oil 12 (80 mg, yield: 79%).
3.8. General procedure for the preparation of 13a–b
To a solution of 7 or 4 (0.61 mmol) in pyridine (4 mL), acetic anhydride (2 mL) was
added and the reaction mixture was stirred at RT for 6 h. The reaction mixture was
diluted with EtOAc (25 mL) and washed with 0.1 N HCl (10 mL ꢁ 3). The organic
layer was dried over Na2SO4 and evaporated under reduced pressure. The residue
was purified by a silica gel column (EtOAc: n-hexane ¼ 1:5) to yield pure 13a–b.
3.9. 20,6-Di-(2-oxo-ethyl)-30,40,5,7-tetramethoxyflavanone (14)
The mixture of 9c (5.00 g, 9.12 mmol), CH2Cl2 (100 mL) and MeOH (100 mL) was
cooled to ꢀ20ꢃC and then treated with ozone until all of the starting material was
exhausted. After addition of (NH2)2CS (1.04 g, 13.68 mmol) to the above solution,
the reaction mixture was warmed to RT for 30 min, washed with 1% NaHCO3
(50 mL ꢁ 3) and dried over Na2SO4. The organic solvents were removed under
reduced pressure to give the residue, which was subjected to a silica gel column
(EtOAc: n-hexane ¼ 1:1) to yield pure 14 (2.93 g, yield: 74%).

Natural Product Research
3.10. 20,6-Di-(2-carboxylethyl)-30,40,5,7-tetramethoxyflavanone (15)
123
Compound 14 (2.31 g, 4.40 mmol) was dissolved in t-BuOH (15 mL), and then
2-methyl-2-butene (3 mL) and 11 mL of an aqueous solution of NaClO2 (3.78 g,
44 mmol) and NaH2PO4-H2O (1.52 g, 10.93 mmol) were added drop-wise over a
period of 20 min. The reaction mixture was stirred at RT overnight. The t-BuOH was
removed under reduced pressure and then the residue was partitioned between
CH2Cl2 (50 mL) and water (30 mL ꢁ 3). The CH2Cl2 layer was dried over Na2SO4
and evaporated under reduced pressure to give a residue, which was purified by a
silica gel column (EtOAc: n-hexane ¼ 1:1) to give 15 (1.64 g, yield: 81%).
Supplementary material
Physical data for all compounds and further experimental details regarding
nymphaeol A (3), B (4) and C (7) are available online, alongside Supplementary
Table S1 and Supplementary Figures S1–S2.
Acknowledgements
We gratefully acknowledge the financial support from the Ministry of Economics and the
National Science Council, Taiwan, Republic of China under grants IZ-950295 and
NSC97-2320-B-038-010-MY3, respectively.
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