Cytotoxicity against cholangiocarcinoma and HepG2 cell lines of lignan derivatives from Hernandia nymphaeifolia

Article abstract of DOI:10.1007/s00044-018-2214-9

Twelve lignan derivatives were synthesized from deoxypodophyllotoxin isolated from Hernandia nymphaeifolia. Cytotoxicity evaluation against cholangiocarcinoma, KKU-100, and HepG2 cell lines showed that compounds 3, 9, 10, and 13 exhibited stronger cytotoxicity than the starting material, 1, with IC₅₀ ranging from 0.42 to 2.01 μM. Compound 10 displayed interesting activity by showing IC₅₀ values of 0.75 and 0.46 μM against KKU-100 and HepG2 cell lines, respectively. From these observation, 10 seems to be useful as a lead compound for the development of anticancer agents.

Full text of DOI:10.1007/s00044-018-2214-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2214-9
ORIGINAL RESEARCH
Cytotoxicity against cholangiocarcinoma and HepG2 cell lines of
lignan derivatives from Hernandia nymphaeifolia
Jittra Suthiwong1 Jaroon Wandee2 Siripit Pitchuanchom3 Punchapat Sojikul4 Veerapol Kukongviriyapan2
Chavi Yenjai1
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Received: 6 April 2018 / Accepted: 28 June 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Twelve lignan derivatives were synthesized from deoxypodophyllotoxin isolated from Hernandia nymphaeifolia.
Cytotoxicity evaluation against cholangiocarcinoma, KKU-100, and HepG2 cell lines showed that compounds 3, 9, 10,
and 13 exhibited stronger cytotoxicity than the starting material, 1, with IC50 ranging from 0.42 to 2.01 μM. Compound 10
displayed interesting activity by showing IC50 values of 0.75 and 0.46 μM against KKU-100 and HepG2 cell lines,
respectively. From these observation, 10 seems to be useful as a lead compound for the development of anticancer agents.
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Keywords Hernandia nymphaeifolia Deoxypodophyllotoxin KKU-100 HepG2
Introduction
P388 lymphocytic leukemia and human cancer, KB16,
A549, and HT-29 cell lines (Chen et al. 1997). Many lig-
nans from medicinal plants have shown anti-oxidant, anti-
estrogenic, anti-mitotic, and anti-viral activities (Pettit et al.
2004). Because of this pharmacological information, we are
interested in the structural modification of a major lignan,
deoxypodophyllotoxin, from the seeds of H. nymphaeifolia
for cancer therapy, especially for cholangiocarcinoma.
Deoxypodophyllotoxin has shown cytotoxicity against
several cancer cell lines such as KB16, A549, HT-29,
Colo205, k562, LNCaP, and PC-3 (Bogucki and Charlton
1995; Wickramaratne et al. 1995; Lim et al. 1999; Jiang
et al. 2007). In addition, this compound inhibits 12-O-
tetradecanoylphorbol 13-acetate-induced ornithine dec-
arboxylase in cultured mouse epidermal cells (Chang et al.
2000).
Hernandia nymphaeifolia is a coastal tree that grows
throughout the tropical and subtropical areas. It is found in
the southern part of Thailand and called Pho Kra Ding. Its
seed is used as a cathartic (Kan 1970). This plant mainly
contains lignans and aporphine alkaloids (Chen et al. 1996;
Chao et al. 2002; Suthiwong et al. 2018). Several com-
pounds from this plant have shown antiplatelet aggregation
and cytotoxicity (Chen et al. 2000). It has been reported that
some compounds exhibit anticancer activity against murine
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2214-9) contains supplementary
material, which is available to authorized users.
In Thailand, cholangiocarcinoma is the cancer of most
interest, due to its being one of the major health problems,
especially in the northeastern area (Sripa and Pairojkul
2008). It is believed that infection by the liver fluke,
Opisthorchis viverrini, is the cause of the disease (McGlynn
et al. 2006). In this country, this cancer has been found in
men more than women because of the high prevalence of
liver fluke infections. There is no effective chemotherapy
treatment with advanced cholangiocarcinoma patients
(Sampson et al. 1997). Surgical resection is the best treat-
ment and is a potentially curative therapy for this cancer.
Thus, effective therapeutic agents from natural sources are
still needed. It has been reported that KKU-100 was the
* Chavi Yenjai
chayen@kku.ac.th
1
Department of Chemistry, Faculty of Science, Natural Products
Research Unit, The Center of Excellence for Innovation in
Chemistry, Khon Kaen University, Khon Kaen 40002, Thailand
2
Department of Pharmacology, Faculty of Medicine, Khon Kaen
University, Khon Kaen 40002, Thailand
3
Department of Chemistry, Faculty of Science, Natural Products
Research Unit, The Center of Excellence for Innovation in
Chemistry, Mahasarakham University, Mahasarakham 44150,
Thailand
4
Department of Biotechnology, Faculty of Science, Mahidol
University, Bangkok 10400, Thailand

Medicinal Chemistry Research
Scheme 1 Derivatives of deoxypodophyllotoxin
least sensitive cell line which showed the highest IC50 value
of 25.21 μM to ellipticine, thus this cell line was selected for
the study (Songsiang et al. 2010).
derivatives 4, 5, and 6 were synthesized by treatment of 2
with benzoyl chloride, methanesulfonyl chloride and
valeroyl chloride, respectively, in the presence of Et3N
(Scheme 1). Compounds 3–6 showed the coupling constant
of the cis configuration as J7′/8′ = 5.7–5.9 Hz. After treat-
ment of 1 with LAH in THF at −10 °C, deox-
ypicropodophyllotoxin (7) was obtained with 66% yield,
and no reduced product was obtained. This result indicates
that LAH acts as a base and abstracted acidic proton at the
alpha position leads to the epimerization of the starting
material. The 1H NMR spectrum displayed a double of
doublet signal of H-8′ at δ 3.34 (J = 9.6, 3.0 Hz) where 1
showed a multiplet signal at δ 2.73. In addition, compound
7 showed a doublet signal of H-7′ at δ 4.37 (J = 3.0 Hz)
where 1 exhibited at δ 4.60. Further treatment of 1 with
LAH at room temperature for 68 h, interestingly, carboxylic
acid derivative 8 (22%) and epimerized product 7 (67%)
were observed. The 13C NMR spectrum of 8 displayed
signals at δ 31.4 (C-7) and δ 63.8 (C-9), where 1 showed at
δ 33.3 (C-7) and δ 72.2 (C-9). Bromination of 1 with NBS
in the presence of aqueous acetonitrile at room temperature
yields monobromolignan 9 (39%) and dibromolignan 10
(24%) (Scheme 2). The effect of high electron density at C-
2′ (and C-6′) in 1 led to bromination at the C-2′ position.
The 13C NMR data of 9 at C-2′ (C-Br) showed a signal at δC
114.6 where 1 showed at δC 108.5 and the proton signal of
H-6′ changed from δH 6.34 to δH 6.12 (Feliciano et al.
1993). It was found that the addition of the second bromine
atom at C-2 in 10 may be due to steric hindrance at C-6′.
The HMBC spectrum of dibromolignan 10 showed
Results and discussion
Chemistry
Deoxypodophyllotoxin (1), a lignan derivative, was isolated
using a chromatographic method from the EtOAc extract of
the seed of H. nymphaeifolia with a good yield (Suthiwong
et al. 2018). In our cytotoxicity screening, this compound
showed cytotoxicity against cholangiocarcinoma (KKU-
M100) and HepG2 cell lines, with IC50 values of 25.78 μM
and 8.10 μM, respectively. To explore the structure-
cytotoxicity relationship, a series of deoxypodophyllotoxin
derivatives have been synthesized and evaluated for cyto-
toxicity to find novel compounds for cholangiocarcinoma
and HepG2 treatment. Twelve lignan derivatives were
successfully synthesized using simple organic reactions.
Treatment of 1 with conc. sulfuric acid in methanol at room
temperature yields methyl ester 2 (48%) (Scheme 1). The
stereochemistry at C-8′ is still the same as the starting
material by showing a coupling constant of cis configura-
tion, J7′/8′ = 5.7 Hz, while trans configuration showed J7′/8′
= 6.4 Hz. (Gordaliza et al. 1997). The 1H NMR spectrum of
2 showed a singlet signal at δ 3.53, indicating the presence
of a methyl ester group. Alcohol 2 was further treated with
acetic anhydride in pyridine affording 3 (98%). Lignan

Medicinal Chemistry Research
Scheme 2 Bromolignan
derivatives of
deoxypodophyllotoxin
Table 1 Cytotoxicity of all compounds (IC50, μM)*
correlations between H-7 (δ7a 3.31 and δ7b 2.56) and C-2 (δ
103.2). The reaction of 7 with NBS in the same condition
gave monobromolignan 11 (28%), dibromolignan 12 (20%)
and tribromolignan 13 (11%). The 13C NMR signal of 11 at
C-2′ changed from δC 105.3 to δC 110.9 where the signal of
H-6′ changed from δH 6.33 to δH 6.31. In the case of 7, the
bromination of the second bromine atom occurring at C-6′
may be due to this compound being less steric than 10. The
spectroscopic data of dibromolignan 12 exhibited the con-
taining of bromine atoms at C-2′ and C-6′ positions, which
were different from 10. The 1H and 13C NMR of 12 showed
signals at δH/δC 6.69/108.7 (H-2) and δH/δC 6.06/106.5 (H-
5), which correlated with C-3 (δ 146.1) and C-4 (δ 146.9) in
the HMBC experiment. In the case of tribromolignan 13, it
showed only one aromatic proton at δH/δC 6.04/105.8 of the
H-5 position. Correlations between H-5 and C-3 (δ 144.8),
C-4 (δ 146.6), C-1 (δ 127.3) and C7′ (δ 45.1) were observed
in the HMBC spectrum. In this spectrum, the correlations
between H-7 and C-2 (δ 103.0), C-6 (δ 131.7) were also
observed in the HMBC spectrum.
Compound
KKU-100
HepG2
1
25.78 1.78
1.63 0.79
2.01 0.74
14.96 7.85
0.84 0.47
4.47 1.94
9.04 5.70
34.58 10.80
1.84 0.54
0.75 0.52
4.82 2.09
22.6 203
1.01 0.39
25.21 0.20
8.10 2.00
6.73 1.37
1.94 1.12
3.55 1.44
16.12 4.32
41.39 10.49
1.50 0.53
14.65 7.20
1.90 0.77
0.46 0.19
5.86 1.88
14.92 7.37
0.42 0.29
2
3
4
5
6
7
8
9
10
11
12
13
Ellipticine
Cisplatin
2.2 0.70
*Data shown are from triplicate experiments
Biological activity
was 14.96 μM), which is about 5–30 fold stronger than the
starting material. The results show convincingly that
polarity may play an important role in cytotoxicity. Among
the ester derivatives, 3 exhibited strong cytotoxicity against
both cells with IC50 values of 2.01 and 1.94 μM, respec-
tively. Compound 7 displayed cytotoxicity with IC50 values
of 9.04 and 1.5 μM, respectively, which were 2.8 and 5.4
fold stronger than the starting material. These results sug-
gest that the stereochemistry which led to the molecular
form may affect the activity. Comparing between com-
pounds 2 and 8, carboxylic acid, which is more polar than
methyl ester, exhibited weaker cytotoxicity (IC50 = 34.58
and 14.65 μM). These results confirm that the polarity of a
compound may play an important role in activity. In cases
of bromide derivatives, they showed strong cytotoxicity,
Natural lignan from H. nymphaeifolia, deox-
ypodophyllotoxin, was used as the starting material and 12
derivatives were synthesized. All compounds were eval-
uated for cytotoxicity against cholangiocarcinoma cells
(KKU-M100, poorly-differentiated adenocarcinoma) and
HepG2 cell lines (Tusskorn et al. 2013). KKU-100 cell is a
poorly differentiated adenocarcinoma and is the least sen-
sitive among cholangiocarcinoma cells. Most lignan deri-
vatives displayed stronger cytotoxicity against two cell
lines, KKU-100 and HepG2 cells, except compounds 8 and
12 (Table 1). In cases of KKU-100 cells, methyl ester
derivatives (2–6) showed cytotoxicity with IC50 values
ranging from 0.84 to 4.47 μM, except compound 4 (IC50

Medicinal Chemistry Research
with the exception of compound 12, against KKU-100 and
HepG2 cell lines, with IC50 values ranging from 0.42 to
5.86 μM. Comparing between compounds 9 and 10, the
presence of a bromine atom at the C-2 position increased
cytotoxicity. In cases of compounds 12 and 13, the bromine
atom at the C-2 position also improved the cytotoxicity
against the two cell lines. Compound 13 exhibited the
activity with IC50 values of 1.01 and 0.42 μM to KKU-100
and HepG2 cells, respectively. These results confirm that
the bromine atom at the C-2 position can enhance the
cytotoxicity. Among all derivatives, 10 is the most active
compound, showing IC50 values of 0.75 and 0.46 μM
against KKU-100 and HepG2 cell lines, respectively.
Extraction and isolation of deoxypodophyllotoxin
(1)
Air-dried seed (300 g) of H. nymphaeifolia were ground and
successively extracted at room temperature with EtOAc
(3 × 1.5 L), and MeOH (3 × 1.5 L). After filtration and
evaporation, the crude EtOAc (79 g) and MeOH (23 g)
extracts were obtained. The crude EtOAc extract was
separated by silica gel flash column chromatography (FCC)
and eluted with a gradient system of hexane and EtOAc to
obtain 4 fractions, F1-F4. Fraction F1 (30 g) was identified
as the natural oil. Fraction F2 was purified by silica gel
FCC, and 5% EtOAc-hexane was used as an eluent to
obtain two subfractions, F2.1 and F2.2. Further purification
of subfraction F2.2 by CC and elution with 100% CH2Cl2
afforded 1 (321 mg, 0.11%) which was used as the starting
material.
Conclusion
Lignan derivatives were synthesized by using deox-
ypodophyllotoxin as the starting material. All compounds
were evaluated for cytotoxicity against cholangiocarcinoma
cells, KKU-100, and hepatoma carcinoma cells, HepG2.
Compounds 3, 9, 10, and 13 exhibited cytotoxicity against
the two cell lines. It is believed that polarity of these
compounds may play an important role in cytotoxicity.
Among bromide derivatives (9–13), it was found that the
bromine atom at the C-2 position was favorable for cyto-
toxicity. Compound 10 displayed highly potent activity
against the two cell lines.
Deoxypodophyllotoxin (1)
White solid; mp 166–168 °C, [α]23 −54° (c 0.1 CHCl3);
D
FT-IR (film) νmax cm−1: 2923, 1763, 1588, 1504, 1479,
1459, 1421, 1377, 1336, 1270, 1219, 1121, 1033, 997, 929;
1H NMR (400 MHz, CDCl3) δ 6.66 (1H, s, H-2), 6.52 (1H,
s, H-5), 6.34 (2H, br s, H-2′,6′), 5.95 (1H, s, –OCH2O–),
5.92 (1H, s, –OCH2O–), 4.60 (1H, br s, H-7′), 4.45 (1H, dd,
J = 8.3, 5.4 Hz, H-9), 3.92 (1H, t, J = 9.0 Hz, H-9), 3.80
(3H, s, 4′–OCH3), 3.75 (6H, s, 3′,5′–OCH3), 3.07 (1H, br d,
J = 11.3 Hz, H-7a), 2.73 (3H, m, H-7b, 8, 8′); 13C NMR
(100 MHz, CDCl3) δ 175.0 (C-9′), 152.7 (C-3′, 5′), 147.2
(C-3), 146.9 (C-4), 137.3 (C-4′), 136.4 (C-1′), 130.8 (C-6),
128.4 (C-1), 110.6 (C-5), 108.6 (C-2), 108.5 (C-2′, 6′),
101.3 (–OCH2O–), 72.2 (C-9), 60.9 (4′-OCH3), 56.4 (3′, 5′-
OCH3), 47.6 (C-8′), 43.9 (C-7′), 33.3 (C-7), 32.9 (C-8);
HRESI-MS m/z 421.1277 [M + Na]+ (calcd. for C22H22O7
+Na, 421.1263).
Experimental part
General experimental procedures
All melting points were determined on a SANYO Gallen-
kamp (UK) melting point apparatus. Optical rotations were
identified using a JASCO P–1020 digital polarimeter. UV
spectra were recorded using an Agilent 8453 UV-Visible
spectrophotometer (Germany). IR spectra were taken as thin
films using a Perkin Elmer Spectrum One FT-IR spectro-
Preparation of lignan derivatives
Delactonization
1
photometer (UK) H NMR spectra were determined with a
Varian Mercury plus spectrometer (UK) operating at
400 MHz (1H NMR) and at 100 MHz (13C NMR). Mass
spectra were recorded on a Micromass Q-TOF 2 hybrid
quadrupole time-of-flight (Q-TOF) mass spectrometer with
a Z-spray ES source (Micromass, UK). Silica gel 60
(100–200 mesh, Merck) was employed for column chro-
matography. Preparative TLC was carried out using silica
gel 60 GF254. TLC was examined on silica gel 60 F254
(Merck) precoated aluminum sheets. Spot on TLC were
visualized under UV light and by spraying with acidic
anisaldehyde solution followed by heating. All solvents
were distilled throughout the separation process.
A solution of compound 1 (172.4 mg, 0.4337 mmol) in
MeOH (3 mL) was treated with conc. H2SO4 (0.2 mL) at
100 °C for about 24 h. Water was added and the mixture
was extracted with EtOAc (2×50 mL). The organic layer
was combined, washed with water, brine and dried with
anhydrous Na2SO4. Evaporation of solvent gave a crude oil,
which was purified by PLC (50 % EtOAc:hexane) to give a
semi-solid of compound 2 (89.1 mg, 48%).
Methyl deoxypodophyllate (2) White solid; mp 154–157 °
24
C, ½αŠ −95° (c 0.1 CHCl3); FT-IR (film) νmax cm−1: 3498,
D
2936, 1734, 1588, 1503, 1483, 1459, 1419, 1328, 1224,

Medicinal Chemistry Research
1123, 1035, 1005, 925, 862, 749; 1H NMR (400 MHz,
CDCl3) δ 6.58 (1H, s, H-2), 6.34 (1H, s, H-5), 6.09 (2H, s,
H-2′,6′), 5.83 (2H, br s, –OCH2O–), 4.32 (1H, d, J =
5.7 Hz, H-7′), 3.74 (3H, s, 4′-OMe), 3.70 (6H, s, 3′ and 5′-
OMe), 3.64 (1H, dd, J = 10.9, 3.8 Hz, H-9a), 3.54 (1H, dd
overlap, J = 10.9, 5.4 Hz, H-9), 3.53 (3H, s, 9′-OMe),
3.06–2.89 (2H, m, H-8′, 7a), 2.65 (1H, dd, J = 17.1,
10.8 Hz, H-7b), 2.35 (1H, m, H-8); 13C NMR (100 MHz,
CDCl3) δ 173.9 (C-9′), 152.7 (C-3′,5′), 146.6 (C-4), 146.2
(C-3), 138.0 (C-4′), 137.1 (C-1′), 130.0 (C-6), 128.5 (C-1),
109.2 (C-5), 108.0 (C-2), 106.8 (C-2′ and 6′), 100.8
(-OCH2O-), 65.4 (C-9), 60.8 (4′-OMe), 56.1 (3′ and 5′-
OMe), 51.5 (9′-OMe), 48.3 (C-8′), 47.8 (C-7′), 32.9 (C-8),
32.1 (C-7); HRESI-MS m/z 453.1521 [M + Na]+ (calcd. for
C23H26O8+Na, 453.1525).
30 min. The entire reaction mixture was poured into cold
water and extracted with EtOAc (2×20 mL). The organic
layer were combined, washed with water, saturated NaCl,
dried over anhydrous Na2SO4 and evaporated to give a
crude oil, which was purified by PLC (20 % EtOAc:hexane)
to give 4 (17.4 mg, 96%).
The reaction of 2 with MsCl or valerory chloride was
examined in the same procedure as described above and
then 5 (92%) or 6 (96%) were obtained, respectively.
Methyl benzyloxydeoxypodophyllate (4)
White solid; mp 171–173 °C; ½αŠ25 −86° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2934, 1718, 1587, 1503, 1483, 1454,
1
1271, 1222, 1123, 1036, 1005, 938, 749, 710; H NMR
(400 MHz, CDCl3) δ 8.01 (2H, d, J = 7.9 Hz, H-3″), 7.56
(1H, t, J = 7.6 Hz, H-5″), 7.44 (2H, t, J = 7.6 Hz, H-4″),
6.63 (1H, s, H-2), 6.43 (1H, s, H-5), 6.16 (2H, s, H-2′ and
6′), 5.90 (1H, s, –OCH2O–), 5.88 (1H, s, –OCH2O–), 4.48
(1H, dd, J = 11.1, 3.1 Hz, H-9a), 4.41 (1H, d, J = 5.7 Hz,
H-7′), 4.39 (1H, m overlap, H-9b) 3.81 (3H, s, 4′-OMe),
3.76 (6H, s, 3′,6′-OMe), 3.55 (3H, s, 9′-OMe), 3.14 (1H,
dd, J = 11.5, 5.7 Hz, H-8′), 3.09 (1H, dd, J = 16.9, 5.8 Hz,
H-7a) 2.84 (1H, dd, J = 16.9, 10.8 Hz, H-7b), 2.77–2.66
(1H, m, H-8); 13C NMR (100 MHz, CDCl3) δ 172.6 (C-9′),
166.6 (C-1″), 152.9 (C-3′,5′), 146.9 (C-4), 146.5 (C-3),
137.8 (C-4′), 137.4 (C-1′), 133.2 (C-5″), 130.2 (C-6), 130.0
(C-2″), 129.7 (C-3″), 128.6 (C-4″), 128.1 (C-1), 109.4 (C-
5), 108.0 (C-2), 106.9 (C-2′ and 6′), 101.0 (−OCH2O−),
67.1 (C-9), 61.0 (4′-OMe), 56.3 (C-3′ and 5′-OMe), 51.6
(9′-OMe), 47.9 (C-8′), 47.8 (C-7′), 32.1 (C-8), 30.4 (C-7);
HRESI-MS m/z 557.1789 [M+Na]+ (calcd. for C30H30O9
+Na, 557.1788).
Acetylation
To a solution of compound 2 (15.3 mg, 0.0355 mmol) in
pyridine (1 mL) was added dropwise Ac2O (excess) at 0 °C,
and the reaction mixture was stirred at room temperature for
2 h. The entire reaction mixture was poured into the cold
water and extracted with EtOAc (2×20 mL). The organic
layers were combined, washed with water, saturated NaCl,
dried over anhydrous Na2SO4 and evaporated. After
recrystallization, compound 3 was obtained as a colorless
solid (16.5 mg, 98%).
25
D
Methyl acetoxydeoxypodophyllate (3) Viscous oil; ½αŠ
−75° (c 0.1 CHCl3); FT-IR (film) νmax cm−1: 2930, 1738,
1588, 1503, 1484, 1460, 1230, 1127, 1037; 1H NMR
(400 MHz, CDCl3) δ 6.62 (1H, s, H-2), 6.40 (1H, s, H-5),
6.12 (2H, s, H-2′ and 6′), 5.89 (1H, s, –OCH2O–), 5.88 (1H,
s, –OCH2O–), 4.37 (1H, d, J = 5.8 Hz, H-7′), 4.21 (1H, dd,
J = 11.0, 3.2 Hz, H-9a), 4.13 (1H, dd, J = 11.0, 5.8 Hz, H-
9b), 3.79 (3H, s, 4′-OMe), 3.74 (6H, s, 3′ and 6′-OMe),
3.54 (3H, s, 9′-OMe), 3.00 (1H, dd, J = 11.0, 5.8 Hz, H-8′),
2.98 (1H, dd, J = 16.9, 5.8 Hz, H-7a) 2.70 (1H, dd, J =
16.9, 10.8 Hz, H-7b), 2.62–2.50 (1H, m, H-8), 2.05 (3H, s,
H-2″); 13C NMR (100 MHz, CDCl3) δ 172.6 (C-9′), 171.2
(C-1″), 152.9 (C-3′ and 5′), 146.8 (C-4), 146.4 (C-3), 137.8
(C-4′), 137.3 (C-1′), 130.0 (C-6), 128.0 (C-1), 109.4 (C-5),
107.9 (C-2), 106.9 (C-2′ and 6′), 101.0 (-OCH2O-), 66.8
(C-9), 60.9 (4′-OMe), 56.3 (3′ and 5′-OMe), 51.5 (9′-OMe),
47.73 (C-7′), 47.69 (C-8′), 31.9 (C-7), 30.1 (C-8), 21.0 (C-
2″); HRESI-MS m/z 495.1628 [M + Na]+ (calcd. for
C25H28O9+Na, 495.1631).
Methyl mesyloxydeoxypodophyllate (5)
White solid; mp 175–177 °C; ½αŠ25 −62° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2935, 1733, 1588, 1503, 1483, 1460,
1
1420, 1352, 1331, 1223, 1172, 1124, 1035, 926, 752; H
NMR (400 MHz, CDCl3) δ 6.63 (1H, s, H-2), 6.39 (1H, s,
H-4), 6.10 (2H, s, H-2′ and 6′), 5.90 (2H, s, −OCH2O−),
4.42 (1H, d, J = 5.9 Hz, H-7′), 4.40–4.32 (2H, m, H-9),
3.80 (3H, s, 4′-OMe), 3.74 (6H, s, 3′ and 5′-OMe), 3.61
(3H, s, 9′-OMe), 3.10–3.00 (2H, m, H-7a and 8′), 2.98 (3H,
s, SO2Me), 2.85 (1H, dd, J = 17.1, 11.2 Hz, H-7b),
2.68–2.56 (1H, m, H-8); 13C NMR (100 MHz, CDCl3) δ
172.3 (C-9′), 153.0 (C-3′ and 5′), 147.0 (C-4), 146.6 (C-3),
137.4 (C-1′ and 4′), 129.7 (C-6), 127.5 (C-1), 109.3 (C-5),
107.9 (C-2), 106.9 (C-2′ and 6′), 101.1(−OCH2O−), 72.2
(C-9), 61.0 (4′-OMe), 56.3 (3′ and 5′-OMe), 51.7 (9′-OMe),
47.6 (C-8′), 46.8 (C-7′), 37.2 (SO2Me), 31.6 (C-8), 30.4 (C-
7); HRESI-MS m/z 531.1283 [M + Na]+ (calcd. for
C24H28O10+Na, 531.1301).
General procedure to prepare 4–6
To a solution of compound 2 (14.6 mg, 0.034 mmol) in
Et3N (1 mL) was added dropwise BzCl (excess) at 0 °C and
the reaction mixture was stirred at room temperature for

Medicinal Chemistry Research
Methyl valeroyloxydeoxypodophyllate (6)
(C-7′), 33.2 (C-8), 32.2 (C-7); HRESI-MS m/z 421.1261
[M + Na]+ (calcd. for C22H22O7+Na, 421.1263).
White solid; mp 168–169 °C; ½αŠ25 −52° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2954, 1732, 1587, 1503, 1483, 1459,
1419, 1390, 1330, 1222, 1162, 1124, 1035, 1007, 937, 752;
1H NMR (400 MHz, CDCl3) δ 6.62 (1H, s, H-2), 6.40 (1H,
s, H-5), 6.12 (2H, s, H-2′ and 6′), 5.89 (1H, s, –OCH2O–),
5.88 (1H, s, –OCH2O–), 4.37 (1H, d, J = 5.7 Hz, H-7′),
4.20 (1H, dd, J = 11.2, 3.0 Hz, H-9a), 4.15 (1H, dd, J =
11.0, 5.6 Hz, H-9b), 3.80 (3H, s, 4′-OMe), 3.74 (6H, s, 3′
and 5′-OMe), 3.54 (3H, s, 9′-OMe), 3.05–2.95 (2H, m, H-
7a and 8′), 2.71 (1H, dd, J = 16.9, 10.8 Hz, H-7b),
2.61–2.51 (1H, m, H-8), 2.31 (2H, t, J = 7.6 Hz, H-2″), 1.59
(2H, m, H-3″), 1.33 (2H, m, H-4″), 0.91 (3H, t, J = 7.3 Hz,
H-5″); 13C NMR (100 MHz, CDCl3) δ 173.9 (C-1″), 172.6
(C-9′), 152.9 (C-3′,5′), 146.8 (C-4), 146.4 (C-3), 137.8 (C-
4′), 137.3 (C-1′), 130.0 (C-6), 128.1 (C-1), 109.4 (C-5),
108.0 (C-2), 106.9 (C-2′ and 6′), 101.0 (−OCH2O−), 66.4
(C-9), 61.0 (4′-OMe), 56.3 (3′ and 5′-OMe), 51.5 (9′-OMe),
47.7 (C-8′), 47.7 (C-7′), 34.1 (C-2″), 32.0 (C-8), 30.2 (C-7),
27.2 (C-3″), 22.4 (C-4″), 13.8 (C-5″); HRESI-MS m/z
537.2101 [M + Na]+ (calcd. for C28H34O9+Na, 537.2101).
Hydrolysation of 1
A solution of compound 1 (89.7 mg, 0.2251 mmol) in THF
at −10 °C was treated with LAH (4.5 mmol, 1 M solution in
THF) under argon. The reaction mixture was stirred at
−10 °C for 2 h. and then warmed to room temperature until
the disappearance of the starting material (68 h) which was
determined by TLC. The reaction mixture was quenched
with 10% HCl followed by extraction of the aqueous layer
with EtOAc (2×20 mL). The combined organic layers were
washed with water and brine, dried with Na2SO4, and
concentrated in vacuo. Chromatography on silica gel with
EtOAc-hexanes (2:3) as the eluent afforded products 8
(20.5 mg, 22%) and 7 (60.3 mg, 67%).
Deoxypicropodophyllic acid (8)
White solid; mp 162–165 °C; ½αŠ25+21° (c 0.1 CH3OH);
D
FT-IR (film) νmax cm−1: 3402, 2918, 1762, 1589, 1504,
1
1482, 1461, 1424, 1328, 1228, 1128, 1007, 982; H NMR
Epimerization
(400 MHz, Methanol-d4) δ 6.62 (1H, s, H-2), 6.38 (2H, s,
H-2′ and 6′), 6.30 (1H, s, H-5), 5.86 (1H, s, –OCH2O–),
5.85 (1H, s, –OCH2O–), 4.36 (1H, d, J = 6.1 Hz, H-7′),
3.75 (6H, s, 3′ and 5′-OMe), 3.73 (3H, s, 4′-OMe), 3.70
(1H, dd, J = 10.9, 5.9 Hz, H-9a), 3.55 (1H, dd, J = 10.9,
8.3 Hz, H-9b), 3.06 (1H, dd, J = 6.1, 3.5 Hz, H-8′), 2.95
(1H, dd, J = 16.6, 5.5 Hz, H-7a), 2.84 (1H, dd, J = 16.6,
7.8 Hz, H-7b), 2.40–2.31 (1H, m, H-8); 13C NMR
(100 MHz, CD3OD) δ 176.9 (C-9′), 154.3 (C-3′ and 5′),
147.8 (C-4), 147.5 (C-3), 143.4 (C-1′), 137.7 (C-4′), 131.0
(C-6), 129.9 (C-1), 110.4 (C-5), 109.3 (C-2), 107.7 (C-2′
and 6′), 102.0 (-OCH2O-), 63.8 (C-9), 61.1 (4′-OMe), 56.6
(3′ and 5′-OMe), 50.6 (C-8′), 47.5 (C-7′), 37.0 (C-8), 31.4
(C-7); HRESI-MS m/z 439.1372 [M + Na]+ (calcd. for
C22H24O7+Na, 439.1369).
A solution of compound 1 (46.2 mg, 0.1159 mmol) in THF
at −10 °C was treated with LAH (4.5 mmol, 1 M solution in
THF) under argon. The reaction mixture was stirred at
−10 °C for 2 h. The reaction mixture was quenched with
10% HCl followed by extraction of the aqueous layer with
EtOAc (2×20 mL). The combined organic layers were
washed with water and brine, dried with Na2SO4, and
concentrated in vacuo. Chromatographic separation on
silica gel with EtOAc-hexane (2:3) as the eluent afforded
the epimerized product 7 (30.5 mg, 66%).
Deoxypicropodophyllotoxin (7)
White solid; mp 159–161 °C, ½αŠ24 +61° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2924, 1761, 1589, 1505, 1478, 1459,
Addition with N-Bromosuccinamide
1
1423, 1325, 1245, 1180, 1121, 1033, 1005, 926, 791; H
NMR (400 MHz, CDCl3) δ 6.66 (1H, s, H-2), 6.58 (1H, s,
H-5), 6.33 (2H, s, H-2′, 6′), 5.95 (1H, br s, –OCH2O–), 5.92
(1H, br s, –OCH2O–), 4.45 (1H, dd, J = 9.2, 7.4 Hz, H-9a),
4.37 (1H, d, J = 3.0 Hz, H-7′), 3.97 (1H, dd, J = 9.2,
3.2 Hz, H-9b), 3.82 (3H, s, 4′-OMe), 3.78 (6H, s, 5′ and 3′-
OMe), 3.34 (1H, dd, J = 9.6, 3.0 Hz, H-8′), 3.06–2.96 (1H,
m, H-8), 2.86 (1H, dd, J = 15.4, 6.4 Hz, H-7a), 2.48 (1H,
dd, J = 15.4, 5.5 Hz, H-7b); 13C NMR (100 MHz, CDCl3) δ
178.5 (C-9′), 153.5 (C-3′,5′), 147.0 (C-3), 146.9 (C-4),
138.4 (C-1′), 130.6 (C-6), 128.4 (C-1), 110.0 (C-5), 108.9
(C-2), 105.3 (C-2′), 105.2 (C-6′), 101.1 (−OCH2O−), 72.9
(C-9), 61.0 (4′-OMe), 56.4 (3′,5′-OMe), 46.5 (C-8′), 45.5
To a solution of 1 (33 mg, 0.0828 mmol) in EtOAc (2 mL)
was added dropwise the solution of NBS (2.2 eq.) in
MeCN: H2O (2 mL) at 0 °C and this was stirred at 0 °C for
1 h and kept stirring at room temperature for 1 h. The entire
reaction mixture was poured into cold water and extracted
with EtOAc (2×20 mL). The organic layers were combined,
washed with water, saturated NaCl, dried over anhydrous
Na2SO4 and evaporated to give solid products. After pur-
ification by PLC, lignan derivatives 9 (15.5 mg, 39%) and
10 (10.9 mg, 24%) were obtained.
The reaction of 7 (29.9 mg) with NBS was examined in
the same procedure as described above and then 11

Medicinal Chemistry Research
(10.2 mg, 28%), 12 (8.4 mg, 20%) and 13 (11.3 mg, 24%)
were obtained.
= 3.8 Hz, H-7′), 4.43 (1H, dd, J = 9.2, 6.3 Hz, H-9a), 4.08
(1H, dd, J = 9.2, 2.0 Hz, H-9b), 3.92 (3H, s, 5′-OMe), 3.89
(3H, s, 4′-OMe), 3.70 (3H, s, 3′-OMe), 3.35 (1H, dd, J =
8.8, 3.8 Hz, H-8′), 2.97 (1H, dd, J = 15.4, 6.5 Hz, H-7a),
2.92–2.82 (1H, m, H-8), 2.60 (1H, dd, J = 15.4, 7.8 Hz, H-
7b); 13C NMR (100 MHz, CDCl3) δ 177.7 (C-9′), 152.7 (C-
3′), 151.6 (C-5′), 147.0 (C-4), 146.9 (C-3), 142.3 (C-4′),
137.1 (C-1′), 129.8 (C-6), 128.4 (C-1), 110.9 (C-2′), 110.1
(C-6′), 109.2 (C-5), 108.5 (C-2), 101.1 (–OCH2O–), 72.8
(C-9), 61.20 (5′-OMe), 61.16 (4′-OMe), 56.3 (3′-OMe),
45.1 (C-8′), 45.0 (C-7′), 33.3 (C-8), 32.2 (C-7); HRESI-MS
m/z 499.0376, 501.0353 [M + Na]+ (calcd. for C22H21BrO7
+Na, 499.0368, 501.0348).
2′-Bromodeoxypodophyllotoxin (9)
White solid; mp 221–223 °C; ½αŠ25 −93° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2927, 1779, 1563, 1480, 1389, 1334,
1313, 1283, 1224, 1197, 1164, 1103, 1037, 999, 944, 928,
872, 754; 1H NMR (400 MHz, CDCl3) δ 6.63 (1H, s, H-2),
6.38 (1H, s, H-5), 6.12 (1H, br s, H-6′), 5.90 (2H, s,
–OCH2O–), 5.28 (1H, d, J = 5.9 Hz, H-7′), 4.51 (1H, dd, J
= 8.6, 6.7 Hz, H-9a), 3.93 (1H, dd, J = 10.2, 8.6 Hz, H-9b),
3.91 (3H, s, 3′-OMe), 3.86 (3H, s, 4′-OMe), 3.63 (3H, s, 5′-
OMe), 3.08 (1H, dd, J = 15.5, 4.7 Hz, H-7a), 3.03–2.89
(1H, m, H-8), 2.83–2.73 (2H, m, H-7b and 8′); 13C NMR
(100 MHz, CDCl3) δ 173.4 (C-9′), 152.3 (C-5′), 151.0 (C-
3′), 147.1 (C-4), 147.0 (C-3), 142.7 (C-4′), 136.6 (C-1′),
131.3 (C-6), 127.6 (C-1), 114.6 (C-2′), 110.8 (C-6′), 110.4
(C-5), 108.5 (C-2), 101.3 (–OCH2O–), 71.7 (C-9), 61.2 (4′-
OMe), 61.1 (3′-OMe), 56.5 (5′-OMe), 46.8 (C-8′), 41.5 (C-
7′), 34.4 (C-8), 32.9 (C-7); HRESI-MS m/z 499.0385,
501.0365 [M + Na]+ (calcd. for C22H21BrO7+Na,
499.0368, 501.0348).
2′,6′-Dibromodeoxypicropodophyllotoxin (12)
White solid; mp 220–223 °C ½αŠ24+23° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2925, 1772, 1503, 1481, 1464, 1407,
1388, 1334, 1242, 1215, 1159, 1126, 1088, 1038, 1009,
1
929, 837, 754; H NMR (400 MHz, CDCl3) δ 6.69 (1H, s,
H-2), 6.06 (1H, s, H-5), 5.91 (1H, br s, –OCH2O–), 5.87
(1H, br s, –OCH2O–), 5.16 (1H, d, J = 6.4 Hz, H-7′), 4.51
(1H, dd, J = 9.3, 5.6 Hz, H-9a), 4.26 (1H, d, J = 9.3 Hz, H-
9b), 3.98 (3H, s, 4′-OMe), 3.96 (3H, s, 5′-OMe), 3.89 (3H,
s, 3′-OMe), 3.43 (1H, t, J = 7.3 Hz, H-8′), 2.90–2.80 (2H,
m, H-7a, 8), 2.62 (1H, t, J = 14.5 Hz, H-7b); 13C NMR
(100 MHz, CDCl3) δ 178.6 (C-9′), 151.6 (C-3′), 151.1 (C-
5′), 146.9 (C-4), 146.9 (C-4′), 146.1 (C-3), 136.3 (C-1′),
129.8 (C-6), 128.2 (C-1), 119.5 (C-2′), 112.7 (C-6′), 108.7
(C-2), 106.5 (C-5), 101.0 (–OCH2O–), 71.9 (C-9), 61.4 (4′-
OMe), 61.2 (5′-OMe), 61.2 (3′-OMe), 46.0 (C-8′), 44.6 (C-
7′), 35.9 (C-8), 32.8 (C-7); HRESI-MS m/z 576.9452,
578.9453, 580.9416 [M + Na]+ (calcd. for C22H20Br2O7
+Na, 576.9473, 578.9453, 580.9433).
2,2′-Dibromodeoxypodophyllotoxin (10)
White solid; mp 225–227 °C; ½αŠ25 −84° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2936, 1778, 1563, 1465, 1390, 1334,
1313, 1223, 1198, 1167, 1104, 1040, 1000, 932, 837, 752;
1H NMR (400 MHz, CDCl3) δ 6.39 (1H, s, H-5), 6.10 (1H,
br s, H-6′), 6.01 (1H, s, –OCH2O–), 5.99 (1H, s,
–OCH2O–), 5.29 (1H, br d, J = 6.4 Hz, H-7′), 4.56 (1H, dd,
J = 8.5, 6.8 Hz, H-9a), 3.98 (1H, dd, J = 10.5, 8.5 Hz, H-
9b), 3.91 (3H, s, 5′-OMe), 3.86 (3H, s, 4′-OMe), 3.66 (3H,
s, 3′-OMe), 3.31 (1H, dd, J = 16.4, 5.0 Hz, H-7a),
3.00–2.86 (1H, m, H-8), 2.75 (1H, dd, J = 14.3, 6.4 Hz, H-
8′), 2.56 (1H, dd, J = 16.4, 11.6 Hz, H-7b); 13C NMR
(100 MHz, CDCl3) δ 173.0 (C-9′), 152.4 (C-3′), 151.2 (C-
5′), 146.9 (C-4), 145.8 (C-3), 142.9 (C-4′), 135.9 (C-1′),
133.5 (C-6), 127.2 (C-1), 144.7 (C-2′), 110.9 (C-6′), 109.8
(C-5), 103.2 (C-2), 101.7 (–OCH2O–), 71.7(C-9), 61.2 (4′-
OMe), 61.1 (5′-OMe), 56.7 (3′-OMe), 46.4 (C-8′), 42.0 (C-
7′), 34.3 (C-8), 32.5 (C-7); HRESI-MS m/z 576.9457,
578.9458, 580.9421 [M + Na]+ (calcd. for C22H2Br2O7
+Na, 576.9473, 578.9453, 580.9433).
2,2′,6′-Tribromodeoxypicropodophyllotoxin (13)
White solid; mp 233–235 °C ½αŠ24+53° (c 0.1 CHCl3); FT-
D
IR (film) νmax cm−1: 2924, 1774, 1607, 1499, 1465, 1406,
1388, 1316, 1238, 1160, 1086, 1041, 1009, 930, 837, 754;
1H NMR (400 MHz, CDCl3) δ 6.04 (1H, s, H-5), 5.99 (1H,
br s, –OCH2O–), 5.97 (1H, br s, –OCH2O–), 5.16 (1H, d, J
= 6.5 Hz,, H-7′), 4.54 (1H, dd, J = 9.4, 5.9 Hz, H-9a), 4.32
(1H, d, J = 9.4 Hz, H-9b), 3.98 (3H, s, 4′-OMe), 3.96 (3H,
s, 3′-OMe), 3.89 (3H, s, 5′-OMe), 3.46–3.37 (2H, m, H-7a,
8′), 2.87–2.78 (1H, m, H-8), 2.42 (1H, dd, J = 15.1,
13.0 Hz, H-7b); 13C NMR (100 MHz, CDCl3) δ 178.2 (C-
9′), 151.6 (C-3′), 151.1 (C-5′), 147.0 (C-4′), 146.6 (C-4),
144.8 (C-3), 135.8 (C-1′), 131.7 (C-6), 127.3 (C-1), 119.5
(C-2′), 114.8 (C-6′), 105.8 (C-5), 103.0 (C-2), 101.4
(-OCH2O-), 71.9 (C-9), 61.4 (4′-OMe), 61.3 (3′-OMe), 61.2
(5′-OMe), 45.9 (C-8′), 45.1 (C-7′), 35.5 (C-8), 30.6 (C-7);
HRESI-MS m/z 654.8581, 656.8575, 658.8549, 660.8506
2′-Bromodeoxypicropodophyllotoxin (11)
Viscous oil; ½αŠ24+34° (c 0.1 CHCl3); FT-IR (film) νmax cm
D
−1: 2928, 1768, 1566, 1481, 1390, 1328, 1256, 1161, 1104,
1
1036, 933, 801, 756; H NMR (400 MHz, CDCl3) δ 6.67
(1H, s, H-2), 6.40 (1H, s, H-5), 6.31 (1H, s, H-6′), 5.93 (1H,
br s, –OCH2O–), 5.90 (1H, br s, –OCH2O–), 4.65 (1H, d, J

Medicinal Chemistry Research
[M + Na]+ (calcd. for C22H19Br3O7+Na, 654.8518,
656.8558, 658.8538, 660.8518).
References
Bogucki DE, Charlton JL (1995) An asymmetric synthesis of
(-)-deoxypodophyllotoxin. J Org Chem 60:588–593
Chang LC, Song LL, Park EJ, Luyengi L, Lee KJ, Farnsworth NR,
Pezzuto JM, Kinghorn AD (2000) Bioactive constituents of Thuja
occidentalis. J Nat Prod 63:1235–1238
Cell culture
The human cholangiocarcinoma (CCA) cell lines; KKU-
M100 cells were routinely cultured in Ham’s F12, sup-
plemented with 10% fetal bovine serum, 12.5 mM N-2-
hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES,
pH 7.3), 100 U/ml penicillin G and 100 μg/ml gentamicin.
Human hepatoma cell line, HepG2 cells, from the Amer-
ican Type Culture Collection (ATCC HB 8065), were
grown in Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% fetal bovine serum (1%), MEM
nonessential amino acids (Gibco), 12.5 mM HEPES, pH
7.3, 100 U/ml penicillin and 100 μg/ml gentamicin. Cul-
tured cells were maintained at 37 °C in a humidified
atmosphere of 5% CO2. The cells were subcultured every
2–3 days before cultured cell confluence using 0.25%
trypsin–EDTA, and medium was changed after an over-
night incubation.
Chao YY, Su W, Jan CR, Ko YC, Chen JJ, Cheng JS, Liu CP, Lo YK,
Chou KJ, Lee KC, Chen WC, Chen IS (2002) Novel action of
lignans isolated from Hernandia nymphaeifolia on Ca2+ signaling
in human neutrophils. Arch Toxicol 75:695–702
Chen IS, Chen JJ, Duh CY, Tsai IL (1997) Cytotoxic lignans from
Formosan Hernandia nymphaeifolia. Phytochemistry 45:991–996
Chen JJ, Chang YL, Teng CM, Chen IS (2000) Anti-platelet aggre-
gation alkaloids and lignans from Hernandia nymphaeifolia.
Planta Med 66:251–256
Chen JJ, Tsai IL, Chen IS (1996) New oxoaporphine alkaloids from
Hernandia nymphaeifolia. J Nat Prod 59:156–158
Feliciano AS, Medarde M, Lamamie de Clairaca RP, López JL, Puebla
P, Gravalos DG, Lázaro PR, Garcia de Quesada T (1993)
Synthesis and biological activity of bromolignans and cyclo-
lignans. Arch Pharm 326:421–426
Gordaliza M, Castro A, Miguel del Corral J, López-Vázquez L, Garcia
PA, Feliciano AS, Garcia-Grávalos D, Broughton H (1997)
Preparation and cytotoxicity of podophyllotoxin derivatives
lacking the lactone ring. Tetrahedron 53:15743–15760
Jiang RW, Zhou JR, Hon PM, Li SL, Zhou Y, Li LL, Ye WC, Xu HX,
Shaw PC, But PPH (2007) Lignans from Dysosma wersipellis
with inhibitory effects on prostate cancer cell lines. J Nat Prod
70:283–286
Cytotoxicity assay
KKU-M100 and HepG2 cells were seeded onto 96-well
plates at a density of 7.5 × 103 and 1.5 × 104 cells/well,
respectively. After an overnight incubation, cultured media
were changed to serum-free media. Test compounds were
dissolved in DMSO and diluted with medium to various
concentrations before use. The compounds were added into
cultured cells and incubated for 24 h. The cytotoxicity was
assessed by the sulphorhodamine B (SRB) assay as pre-
viously described (Tusskorn et al. 2013). In brief, cultured
cells were fixed with 15% trichloroacetic acid and stained
with 0.4% SRB. The protein-bound dye was solubilized
with 10 mM Tris-base solution for determination of the
absorbance at 540 nm with a microplate reader. The cyto-
toxicity was calculated as percent absorbance of controls.
The IC50 value was calculated by a non-linear curve-fitting
program.
Kan WS (1970) Manual of Medicinal Plants in Taiwan. Vol. 1.
National Research Insititute of Chinese Medicine, Taiwan, p
178–179
Lim YH, Leem MJ, Shin DH, Chang HB, Hong SW, Moon EY, Lee
DK, Yoon SJ, Woo WS (1999) Cytotoxic constituents from the
roots of Anthriscus sylvestris. Arch Pharm Res 22:208–212
McGlynn KA, Tarone RE, El-Serag HB (2006) A comparison of
trends in the incidence of hepatocellular carcinoma and intrahe-
patic cholangiocarcinoma in the United States. Cancer Epidemiol
Biomark Prev 15:1198–1203
Pettit GR, Meng Y, Gearing RP, Herald DL, Pettit RK, Doubek DL,
Chapuis JC, Tackett LP (2004) Antineoplastic Agents. 522.
Hernandia peltata (Malaysia) and Hernandia nymphaeifolia
(Republic of Maldives). J Nat Prod 67:214–220
Sampson LK, Vickers SM, Ying W, Phillips JO (1997) Tamoxifen-
mediated growth inhibition of human cholangiocarcinoma. Can-
cer Res 57:1743–1749
Songsiang U, Pitchuanchom S, Boonyarat C, Hahnvajanawong C,
Yenjai C (2010) Cytotoxicity against cholangiocarcinoma cell
lines of zerumbone derivatives. Eur J Med Chem45:3794–3802
Sripa B, Pairojkul C (2008) Cholangiocarcinoma: lessons from Thai-
land. Curr Opin Gastroenterol 24:349–356
Suthiwong J, Boonloh K, Kukongviriyapan V, Yenjai C (2018) Cyto-
toxicity against cholangiocarcinoma and HepG2 cell lines of lig-
nans from Hernandia nymphaeifolia. Nat Prod Commun 13:61–63
Tusskorn O, Prawan A, Senggunprai L, Kukongviriyapan U,
Kukongviriyapan V (2013) Phenethyl isothiocyanate induces
apoptosis of cholangiocarcinoma cells through interruption of
glutathione and mitochondrial pathway. Naunyn Schmiede Arch
Pharmacol 386:1009–1016
Acknowledgements The Post-Doctoral Training Program from
Research Affairs and Graduate School, Khon Kaen University for
Jittra Suthiwong is acknowledged (Grant no. 590012). This work was
supported by the Thailand Research Fund (RSA5980022), Khon Kaen
University and The Center of Excellence for Innovation in Chemistry
(PERCH-CIC), Office of the Higher Education Commission, Ministry
of Education.
Compliance with ethical standards
Wickramaratne DB, Mar W, Chai H, Castlllo J, Farnsworth NR,
Soejarto DD, Cordell GA, Pezzuto IM, Kinghorn AD (1995)
Cytotoxic constituents of Bursera permollis. Planta Med
61:80–81
Conflict of interest The authors declare that they have no conflict of
interest.