Structure-activity relationships and the cytotoxic effects of novel diterpenoid alkaloid derivatives against A549 human lung carcinoma cells

Article abstract of DOI:10.1007/s11418-010-0452-3

The cytotoxicity of three alkaloids from the roots of Aconitum yesoense var. macroyesoense as well as 36 semi-synthetic C₂₀-diterpenoid atisine-type alkaloid derivatives against A549 human lung carcinoma cells was examined. Ten acylated alkaloid derivatives, pseudokobusine 11-veratroate (9), 11-anisoate (12), 6,11-dianisoate (14), 11-p-nitrobenzoate (18), 11,15-di-p-nitrobenzoate (22), 11-cinnamate (25) and 11-m- trifluoromethylbenzoate (27), and kobusine 11-p-trifluoromethylbenzoate (35), 11-m-trifluoromethylbenzoate (36) and 11,15-di-p-nitrobenzoate (39), exhibited cytotoxic activity, and 11,15-dianisoylpseudokobusine (16) was found to be the most potent cytotoxic agent. Their IC₅₀ values against A549 cells ranged from 1.72 to 5.44 μM. In the occurrence of cytotoxic effects of atisine-type alkaloids, replacement by an acyl group at both C-11 and C-15 resulted in the enhancement of activity of the parent alkaloids compared to that from having hydroxy groups at this position, and the presence of a hydroxy group at the C-6 position was required for the cytotoxic effects. These acylated alkaloid derivatives inhibit cell growth through G1 arrest.

Full text of DOI:10.1007/s11418-010-0452-3

J Nat Med (2011) 65:43–49
DOI 10.1007/s11418-010-0452-3
ORIGINAL PAPER
Structure–activity relationships and the cytotoxic effects of novel
diterpenoid alkaloid derivatives against A549 human lung
carcinoma cells
•
Koji Wada Masaharu Hazawa Kenji Takahashi
•
•
•
Takao Mori Norio Kawahara Ikuo Kashiwakura
•
Received: 28 April 2010 / Accepted: 5 July 2010 / Published online: 14 August 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract The cytotoxicity of three alkaloids from the
roots of Aconitum yesoense var. macroyesoense as well as
36 semi-synthetic C20-diterpenoid atisine-type alkaloid
derivatives against A549 human lung carcinoma cells was
examined. Ten acylated alkaloid derivatives, pseudokobu-
sine 11-veratroate (9), 11-anisoate (12), 6,11-dianisoate
(14), 11-p-nitrobenzoate (18), 11,15-di-p-nitrobenzoate
(22), 11-cinnamate (25) and 11-m-trifluoromethylbenzoate
(27), and kobusine 11-p-trifluoromethylbenzoate (35), 11-m-
trifluoromethylbenzoate (36) and 11,15-di-p-nitrobenzoate
(39), exhibited cytotoxic activity, and 11,15-diani-
soylpseudokobusine (16) was found to be the most potent
cytotoxic agent. Their IC50 values against A549 cells ran-
ged from 1.72 to 5.44 lM. In the occurrence of cytotoxic
effects of atisine-type alkaloids, replacement by an acyl
group at both C-11 and C-15 resulted in the enhancement
of activity of the parent alkaloids compared to that from
having hydroxy groups at this position, and the presence of
a hydroxy group at the C-6 position was required for the
cytotoxic effects. These acylated alkaloid derivatives
inhibit cell growth through G1 arrest.
Keywords Diterpenoid alkaloids Á Pseudokobusine Á
Kobusine Á Cytotoxic agents Á A549 human
lung carcinoma cells Á Structure–activity relationship
Introduction
Diterpenoid alkaloids are classified according to their
chemical structure as C19-norditerpenoid alkaloids, which
consist of an aconitine or a lycoctonine skeleton, and C20-
diterpenoid alkaloids, consisting of an atisine or a veat-
chine skeleton. A large number of diterpenoid alkaloids
have been isolated from various species of Aconitum and
Delphinium (Ranunculaceae) [1, 2]. The pharmacological
properties of C19-norditerpenoid alkaloids, including
aconitine, mesaconitine, hypaconitine and jesaconitine,
have been studied extensively and reviewed [1, 2]. Acon-
itine and mesaconitine are representative toxins that exhibit
activity both centrally and peripherally, with predominant
effects on the cardiovascular and respiratory systems, by
preventing the normal closing of sodium channels [3, 4]. In
contrast, there is little information regarding the pharma-
cological properties of C20-diterpenoid alkaloids and their
chemically transformed products. Kobusine (1) and
pseudokobusine (2), the major alkaloid constituents of
Aconitum yesoense var. macroyesoense, and certain semi-
synthetic derivatives of diterpenoid alkaloids have been
shown by using a Doppler-type laser blood flow meter to
significantly increase cutaneous blood flow in the hind feet
of anaesthetized mice [5–7].
K. Wada (&)
School of Pharmacy, Hokkaido Pharmaceutical University,
7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan
e-mail: kowada@hokuyakudai.ac.jp
M. Hazawa Á K. Takahashi Á I. Kashiwakura
The majority of drugs used in cancer chemotherapy can
be divided into alkylating agents, anti-metabolites, antibi-
otics, plant alkaloids, topoisomerase inhibitors, monoclonal
antibodies and other antitumor agents [8–16]. However,
little information on the cytotoxic properties of Aconitum
Graduate School of Health Sciences, Hirosaki University,
66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
T. Mori Á N. Kawahara
Research Center, North Japan Chemical, Inc., Eniwa RBP 308,
1-1, 3-Meguminokita, Eniwa, Hokkaido 061-1374, Japan
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J Nat Med (2011) 65:43–49
alkaloids has been reported, despite their intense toxicities.
Two reports on the effects of C19-norditerpenoid alkaloids
on cancer cells have appeared in recent years. 8-O-Azeloyl-
14-benzoylaconine, an aconitine-type C19-norditerpenoid
alkaloid, exhibited anti-proliferative activity [17] and the
cytotoxic effects of various C19-norditerpenoid alkaloids
against tumor cell lines have been reported [18]. Our pre-
vious study demonstrated the effects of various naturally
occurring and semi-synthetic diterpenoid alkaloids on the
growth of the A172 human malignant glioma cell line [19].
The results of previous studies showed that seven acylated
alkaloid derivatives, 12-acetylluciculine, pseudokobusine
11-veratroate (9), 11-anisoate (12), 11-p-nitrobenzoate
(18), 11-cinnamate (25) and 11-m-trifluoromethylbenzoate
(27), and 11-(m-trifluoromethylbenzoyl)kobusine (36), had
significant cytotoxic effects on the growth of A172 cells.
Esterification of the hydroxyl group at C-11 may, thus,
contribute to the enhancement of activity of the parent
alkaloids more than that of the OH group at C-11.
Cytotoxic properties and radiation-sensitizing effects of
various types of novel derivatives prepared from Aconitum
alkaloids have also been investigated [20]. 11-Anisoyl-
pseudokobusine (12) and 11-m-trifluoromethylbenzoyl
pseudokobusine (36) showed significant suppressive effects
against the non-Hodgkin’s lymphoma Raji cell line [21].
11-m-Trifluoromethylbenzoylpseudokobusine (36) clearly
inhibited the phosphorylation of extracellular signal-regu-
lated kinase, induced enhanced phosphoinositide 3-kinase
phosphorylation and led to the subsequent accumulation of
G1 and/or sub-G1 phase in Raji cells. In addition, sup-
pressive effects of 11-anisoylpseudokobusine (12) and
11-m-trifluoromethylbenzoylpseudokobusine (36) on the
growth of human CD34? hematopoietic stem/progenitor
cells were observed.
impact (EI) mass spectra were measured on Hitachi
M-2000 and JEOL JMS-700 spectrometers. IR spectra
were recorded with an IR spectrophotometer, Perkin-Elmer
Spectrum 100. All products reported showed 1H-NMR
spectra and mass spectra in agreement with the assigned
structures. Reactions were carried out under an inert
atmosphere of dry nitrogen or argon, unless otherwise
described. Standard syringe techniques were used for
transferring dry solvents. Reaction courses and product
mixtures were monitored routinely by TLC on silica
gel (precoated Merck F254 plates) and visualized with
Dragendorff reagent. Chromatography was performed
using silica gel and the indicated solvent system. All other
chemicals used were of analytical grade.
Alkaloids
The diterpenoid alkaloids kobusine (1), pseudokobusine (2)
and 15-veratroylpseudokobusine (10) were used after
extraction from the roots of A. yesoense var. macroyesoense,
followed by purification and identification by methods
described previously [22, 23]. Thirty acyl deriva-
tives, N-benzyl-N,6-seco-6-dehydropseudokobusine (3) [19],
N,15-dibenzyl-N,6-seco-6-dehydropseudokobusine (4) [19],
6-benzoylpseudokobusine (5) [23], 6,11-dibenzoylpseudo-
kobusine (6) [23], 15-benzoyl-6,11-di-p-nitrobenzoylpseu-
dokobusine (7) [19], 6-veratroylpseudokobusine (8) [7],
11-veratroylpseudokobusine (9) [7], 6-anisoylpseudokobu-
sine (11) [7], 11-anisoylpseudokobusine (12) [7], 15-anis-
oylpseudokobusine (13) [7], 6,11-dianisoylpseudokobusine
(14) [7], 6,15-dianisoylpseudokobusine (15) [7], 11,15-
dianisoylpseudokobusine (16) [7], 6-p-nitrobenzoylpseu-
dokobusine (17) [23], 11-p-nitrobenzoylpseudokobusine
(18) [7], 15-p-nitrobenzoylpseudokobusine (19) [24],
6,11-di-p-nitrobenzoylpseudokobusine (20) [24], 6,15-di-p-
nitrobenzoylpseudokobusine (21) [24], 11,15-di-p-nitro-
benzoylpseudokobusine (22) [7], 6,11,15-tri-p-nitrobenzoyl
pseudokobusine (23) [24], 6-cinnamoylpseudokobusine
(24) [6], 11-cinnamoylpseudokobusine (25) [6], 6-(m-tri-
fluoromethylbenzoyl)pseudokobusine (26) [19], 11-(m-tri-
fluoromethylbenzoyl)pseudokobusine (27) [19], 11-benzoyl
kobusine (30) [6], 11-anisoylkobusine (31) [7], 11-vera-
troylkobusine (32) [7], dihydrokobusine (33) [7], 11-cinna
moylkobusine (34) [6] and 11-(m-trifluoromethylben-
zoyl)kobusine (36) [19], were prepared by methods
described previously. Six semi-synthetic derivatives,
6-(p-trifluoromethylbenzoyl)pseudokobusine (28), 11-(p-tri-
fluoromethylbenzoyl)pseudokobusine (29), 11-(p-trifluoro-
methylbenzoyl)kobusine (35), 11-p-nitrobenzoylkobusine
(37), 15-p-nitrobenzoylkobusine (38) and 11,15-di-p-nitro-
benzoylkobusine (39), were prepared from kobusine (1) and
pseudokobusine (2). These semi-synthetic alkaloids were
synthesized at controlled reaction times and temperatures.
In the present study, the effects of various semi-syn-
thetic novel C20-diterpenoid alkaloids on the growth of the
A549 human lung cancer cell line were examined. Twenty
novel derivatives were prepared from natural compounds.
In order to carry out structure–activity relationship studies
of the anti-proliferative effect against A549 cells, three
natural and 36 semi-synthetic diterpenoid alkaloids were
tested.
Materials and methods
General experimental procedures
Melting points were determined on a Yanagimoto micro
melting point apparatus and are uncorrected. 1H-NMR
spectra in CDCl3 were recorded on JEOL GX-270 and
AL-400 spectrometers using tetramethylsilane as an inter-
nal standard. Chemical shifts are given in ppm. Electron
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J Nat Med (2011) 65:43–49
45
EIMS m/z: 485 (M?), 312, 296, 173. HREIMS m/z:
485.2157 (calcd. for C28H30F3NO3: 485.5376).
Synthesis of 6-(28) and 11-(p-trifluoromethylbenzoyl)
pseudokobusine (29)
A solution of 2 (0.111 g, 0.34 mmol) and p-trifluoro-
methylbenzoyl chloride (0.1 ml, 0.67 mmol) in pyridine
(2 ml) was stirred for 30 min at ambient temperature. After
adding water, the reaction mixture was extracted with
chloroform after the addition of aqueous NH4OH. The
organic layer was washed with aqueous saturated NaHCO3
and brine, and then dried over anhydrous MgSO4. The
solvent was evaporated under reduced pressure and the
resulting residue was purified by silica gel column chro-
matography eluting with 1% methanol–NH4OH–saturated
CHCl3 to give 28 (19 mg, 11%), 29 (34 mg, 20%) and
2 (14 mg). 6-(p-Trifluoromethylbenzoyl)pseudokobusine
(28): amorphous. 1H-NMR (CDCl3, 400 MHz) d: 0.98 (3H,
s, H-18), 3.96 (1H, s, H-15), 4.08 (1H, d, J = 4.8 Hz,
H-11), 5.14 and 5.24 (each 1H, s, H-17), 7.71 (2H, d,
J = 8.0 Hz, H–Ar), 8.16 (2H, d, J = 8.0 Hz, H–Ar). IR
(ATR) cm-1: 3316, 1726, 1562, 1322, 1259, 1163, 896.
EIMS m/z: 501 (M?), 429, 329, 190, 173. HREIMS m/z:
501.2130 (calcd. for C28H30F3NO4: 501.2127). 11-(p-Tri-
fluoromethylbenzoyl)pseudokobusine (29): colorless crys-
tals (acetone–hexane), mp 293°C (dec.). 1H-NMR (CDCl3,
400 MHz) d: 1.18 (3H, s, H-18), 4.00 (1H, s, H-15), 5.06
and 5.23 (each 1H, s, H-17), 5.32 (1H, d, J = 4.8 Hz,
H-11), 7.64 (1H, d, J = 8.3 Hz, H–Ar), 8.00 (1H, d,
J = 8.3 Hz, H–Ar). IR (ATR) cm-1: 3291, 1717, 1557,
1323, 1267, 1166, 901. EIMS m/z: 501 (M?), 328, 173.
HREIMS m/z: 501.2125 (calcd. for C28H30F3NO4:
501.2127).
Synthesis of 11-(37), 15- (38) and 11,15-di-p-
nitrobenzoylkobusine (39)
A solution of 1 (0.106 g, 0.34 mmol) and p-nitrobenzoyl
chloride (0.252 mg, 1.36 mmol) in pyridine (2 ml) was
stirred for 2 h at 0°C (ice bath). After adding water, the
reaction mixture was extracted with chloroform after the
addition of aqueous NH4OH. The organic layer was
washed with aqueous saturated NaHCO3 and brine, and
then dried over anhydrous MgSO4. The solvent was
evaporated under reduced pressure, and the resulting resi-
due was purified by silica gel column chromatography
eluting with 0.5% methanol–NH4OH–saturated CHCl3 to
give 37 (55 mg, 35%), 38 (14 mg, 9%), 39 (52 mg, 25%)
and 1 (4 mg). 11-p-Nitrobenzoylkobusine (37): amor-
1
phous. H-NMR (CDCl3, 270 MHz) d: 0.99 (3H, s, H-18),
4.05 (1H, d, J = 5.6 Hz, H-15), 5.08 and 5.25 (each 1H, s,
H-17), 5.42 (1H, d, J = 4.9 Hz, H-11), 8.13 (2H, d,
J = 8.5 Hz, H–Ar), 8.28 (2H, d, J = 8.5 Hz, H–Ar). IR
(ATR) cm-1: 3377, 1717, 1605, 1525, 1279, 1103, 900.
EIMS m/z: 462 (M?), 433, 312, 295. HREIMS m/z:
462.2179 (calcd. for C27H30N2O5: 462.2153). 15-p-Nitro-
benzoylkobusine (38): colorless crystals (acetone–hexane),
1
mp 214–216°C. H-NMR (CDCl3, 270 MHz) d: 0.96 (3H,
s, H-18), 4.14 (1H, d, J = 4.6 Hz, H-11), 5.25 and 5.39
(each 1H, s, H-17), 5.73 (1H, s, H-15), 8.21 (2H, d,
J = 8.9 Hz, H–Ar), 8.30 (2H, d, J = 8.9 Hz, H–Ar). IR
(ATR) cm-1: 3055, 1710, 1606, 1524, 1269, 1104, 901.
EIMS m/z: 462 (M?), 432, 312, 296. HREIMS m/z:
462.2131 (calcd. for C27H30N2O5: 462.2153). 11,15-p-
Nitrobenzoylkobusine (39): colorless crystals (acetone–
Synthesis of 11-(p-trifluoromethylbenzoyl)kobusine
(35)
1
hexane), mp 232–233°C. H-NMR (CDCl3, 270 MHz) d:
A solution of 1 (0.041 g, 0.13 mmol) and p-trifluoro-
methylbenzoyl chloride (0.04 ml, 0.26 mmol) in pyridine
(1 ml) was stirred for 1.5 h at 0°C (ice bath). After adding
water, the reaction mixture was extracted with chloroform
after the addition of aqueous NH4OH. The organic layer
was washed with aqueous saturated NaHCO3 and brine,
and then dried over anhydrous Na2SO4. The solvent was
evaporated under reduced pressure, and the resulting resi-
due was purified by silica gel column chromatography
eluting with 2% methanol–NH4OH–saturated CHCl3 to
give 35 (16 mg, 26%). 11-(p-Trifluoromethylbenzoyl)ko-
busine (35): white crystals (acetone–hexane), mp
0.97 (3H, s, H-18), 5.19 and 5.41 (each 1H, s, H-17), 5.51
(1H, d, J = 4.6 Hz, H-11), 5.81 (1H, s, H-15), 8.02 (2H, d,
J = 8.9 Hz, H–Ar), 8.05 (4H, d, J = 8.9 Hz, H–Ar), 8.07
(2H, d, J = 8.3 Hz, H–Ar). IR (ATR) cm-1: 3073, 1711,
1606, 1524, 1260, 1102, 902. EIMS m/z: 611 (M?), 581,
461, 432. HREIMS m/z: 611.2265 (calcd. for C34H33N3O8:
611.2265).
Inhibition of growth of the human lung cancer cell line
A549
All test alkaloids were dissolved in dimethyl sulfoxide
(DMSO) at 1 or 5 mg/ml immediately before use and
diluted in the medium before addition to the cells. Cells
were cultured in a DMEM medium supplemented with
10% heat-inactivated fetal bovine serum and antibiotics
[penicillin (100 UI/ml) and streptomycin (100 UI/ml)].
1
213–216°C. H-NMR (CDCl3, 400 MHz) d: 0.98 (3H, s,
H-18), 4.03 (1H, s, H-15), 5.08 and 5.25 (each 1H, s,
H-17), 5.41 (1H, d, J = 4.8 Hz, H-11), 7.69 (2H, d,
J = 8.3 Hz, H–Ar), 8.07 (2H, d, J = 8.3 Hz, H–Ar). IR
(ATR) cm-1: 3361, 1718, 1555, 1323, 1275, 1164, 895.
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J Nat Med (2011) 65:43–49
To determine the effects of alkaloids on cell growth,
exponentially growing A549 cells (4 9 103 cells/well)
were seeded in 24-well plates (Falcon, Becton–Dickinson
Biosciences, Franklin Lakes, NJ, USA) with 500 ml of
medium, and each alkaloid (final concentration: 1 or 5 lg/ml)
was added to each plate 24 h later. The total cell numbers
were determined after 3 days using a particle counter
(model ZTM series, Coulter Electronics, Hialeah, FL,
USA). The results are expressed as inhibition values in
comparison to untreated controls and as IC50 values (con-
centration causing 50% inhibition relative to untreated
controls). All experiments were repeated at least three
times. Excluding the possible anti-proliferative effects of
DMSO, the maximum concentration of DMSO (0.5%) was
used in control culture and did not affect the growth of
A549 cells. Pemetrexed (Eli Lily and Company, India-
napolis, IN, USA) was used as the reference control.
Alkaloids 1 and 2 contain two and three hydroxy groups,
respectively, in the common basic structure of the atisine
skeleton, being devoid of any other substituents. In the
molecules of 1 and 2 (Fig. 1), semi-synthetic derivatives
were tested for suppressive effects at 1 lg/ml additions and
IC50 values against the growth of A549 cells were examined
(Table 1). N-Benzyl-N,6-seco-6-dehydropseudokobusine
(3) and N,15-dibenzyl-N,6-seco-6-dehydropseudokobusine
(4) were inactive. Among the benzoyl derivatives (5–7) of 2,
6-benzoylpseudokobusine (5) and 15-benzoyl-6,11-di-p-
nitrobenzoylpseudokobusine (7) were inactive. 6,11-
Dibenzoylpseudokobusine (6) had a weak cytotoxic effect,
which was altered by an aryl substituent at C-11 or by a
hydroxy group at C-15. Among the veratroyl derivatives (8–
10) of 2, 6-veratroylpseudokobusine (8) was inactive.
15-Veratroylpseudokobusine (10) displayed little cytotoxic
effect. In contrast, 11-veratroylpseudokobusine (9) had a
significant cytotoxic effect. Therefore, the suppressive
effects were elicited by the presence of an acyl substituent at
C-11. Among the anisoyl (11–16) and p-nitrobenzoyl
(17–23) derivatives of 2, 6-anisoylpseudokobusine (11), 6,15-
dianisoylpseudokobusine (15), 6,15-di-p-nitrobenzoylpseu-
dokobusine (21) and 6,11,15-tri-p-nitrobenzoylpseudoko-
busine (23) were inactive. 6-p-Nitrobenzoylpseudokobusine
(17) and 15-p-nitrobenzoylpseudokobusine (19) displayed
little cytotoxic effect, and 15-anisoylpseudokobusine (13)
and 6,11-di-p-nitrobenzoylpseudokobusine (20) showed
only weak cytotoxic effects. 11-Anisoylpseudokobusine
(12), 6,11-dianisoylpseudokobusine (14), 11,15-diani-
soylpseudokobusine (16), 11-p-nitrobenzoylpseudokobu-
sine (18) and 11,15-di-p-nitrobenzoylpseudokobusine (22)
had significant cytotoxic effects. Accordingly, the cytotoxic
effects of 6-substrates (11, 17) were weaker than those of
6,11-disubstrates (14, 20), and 11-substrates (12, 18) had
more potent cytotoxic effects than those of 6,11-disubstrates
(14, 20). In fact, 11-acyl derivatives (25, 27) exhibited more
potent cytotoxic effects than those of 6-substrates (24, 26),
but p-trifluoromethylbenzoyl derivatives (28, 29) were
inactive. In addition 11,15-dianisoylpseudokobusine (16)
and 11,15-di-p-nitrobenzoylpseudokobusine (22) were
found to be about 1.3-fold and 2-fold more potent than
11-anisoylpseudokobusine (12) and 11-p-nitrobenzoylpseu-
dokobusine (18), respectively. Substitution of the hydroxy
group at C-11 of pseudokobusine had variable effects.
Benzoate (6) and p-trifluoromethylbenzoate (29) were in
active. Veratroate (9, IC50 = 4.07 lM), p-nitrobenzoate
(18, IC50 = 5.08 lM), cinnamate (25, IC50 = 4.24 lM)
and m-trifluoromethylbenzoate (27, IC50 = 4.67 lM) showed
significant cytotoxic effects. p-Trifluoromethylbenzoate (29)
had little effect at 5 lg/ml, whereas the effect of m-tri-
fluoromethylbenzoate (27) was more potent than that of 29.
As to the effects of the substitution position by these
benzoyl groups, m-position gave good result. Anisoate
Cell cycle analysis by flow cytometry
A549 cells were treated with each compound at doses of its
IC50 and double IC50 values and incubated for 24 h. The
harvested cells were treated with PBS containing 0.1%
Triton X-100 (Wako) and were stained with propidium
iodide (50 lg/ml, Sigma). Analysis of cell cycle distribu-
tion was performed using a flow cytometer (Beckman–
Coulter, Cell Lab QuantaTM SC MPL, Fullerton, CA,
USA).
Statistical analysis
The data are expressed as the mean SD of 3 cultures of a
group, and the significance of differences between the
control and experimental groups were determined using
either Student’s t test or Mann–Whitney’s U-test, depend-
ing on the data distribution. Statistical analysis was per-
formed using the Excel 2003 software package (Microsoft,
Redmond, WA, USA) with the add-in software Statcel 2
(OMS, Saitama, Japan).
Results and discussion
Aconitum diterpenoid alkaloids and their novel derivatives
were examined for the suppressive effects on the growth of
the A549 human lung cancer cell line [20]. C19-nordit-
erpenoid aconitine-type alkaloids (five alkaloids) and
lycoctonine-type alkaloids (seven alkaloids) were found to
be inactive. Among the seven C20-diterpenoid veatchine-
type alkaloids tested, 12-acetylluciculine and 12-benzoyl-
luciculine showed slight inhibitory activities against
growth.
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J Nat Med (2011) 65:43–49
47
(12, IC50 = 2.20 lM) was found to be about 2-fold more
potent than these substrates. Consequently, in the occur-
rence of cytotoxic effects of atisine-type alkaloids,
replacement by an acyl group at C-11 resulted in the
enhancement of activity of the parent alkaloids more than
when a hydroxy group was present at this position, and the
presence of a hydroxy group at the C-6 position was required
for the cytotoxic effects. Furthermore, replacement by an
acyl group at both C-11 and C-15 [e.g., 11,15-diani-
soylpseudokobusine (16) and 11,15-di-p-nitrobenzoylpseu-
dokobusine (22)] was required for the enhancement of the
cytotoxic effect of 11-substrates (12, 18).
Table 1 Cytotoxic effects of atisine-type C20-diterpenoid alkaloids
against A549 cell lines
Compound
1 lg/mla
5 lg/mla
IC50 (lM)
Control
1.00
1.00
–
1
1.07 0.16
1.12 0.16
1.01 0.03
0.98 0.02
1.02 0.01
0.88 0.06*
0.96 0.06
0.98 0.06
0.65 0.06*
0.94 0.01
1.02 0.01
0.65 0.08*
0.89 0.03
0.66 0.04*
1.04 0.14
0.54 0.14*
0.93 0.04
0.75 0.08*
0.93 0.05
0.86 0.11
0.98 0.11
0.74 0.14*
1.05 0.06
0.95 0.04
0.59 0.10*
1.05 0.06
0.69 0.09*
1.03 0.05
1.09 0.19
1.06 0.19
0.98 0.14
1.04 0.25
1.09 0.06
0.93 0.19
0.72 0.11*
0.64 0.06*
0.91 0.19
0.92 0.23
0.75 0.22
0.14 0.05*
1.00 0.05
0.93 0.04
ND
ND
2
ND
3
ND
4
ND
ND
5
ND
ND
6
ND
ND
7
ND
ND
8
0.86 0.03
ND
ND
Similarly, the suppressive effects of kobusine deriva-
tives (30–39) at 1 lg/ml additions and IC50 values against
the growth of A549 cells were examined (Table 1).
11-Benzoylkobusine (30), 11-veratroylkobusine (32) and
dihydrokobusine (33) were inactive. 11-Anisoylkobusine
(31) and 11-cinnamoylkobusine (34) displayed little cyto-
toxic effects. 11-(p-Trifluoromethylbenzoyl)kobusine (35)
and 11-(m-trifluoromethylbenzoyl)kobusine (36) had sig-
nificant cytotoxic effects. Among the p-nitrobenzoyl
derivatives (37–39) of 1, 11-p-nitrobenzoylkobusine (37)
and 15-p-nitrobenzoylkobusine (38) had little cytotoxic
effects, and the effect of 37 was slightly more potent than
that of 38. However, 11,15-di-p-nitrobenzoylkobusine (39)
9
4.07 0.00
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Pemetrexed
ND
1.05 0.04
ND
ND
2.20 0.11
ND
0.56 0.04*
0.15 0.04*
0.25 0.06*
0.03 0.01*
0.83 0.03
ND
3.68 0.30
ND
1.72 0.03
ND
5.08 0.15
ND
0.47 0.04*
0.11 0.08*
0.42 0.10*
0.03 0.01*
0.91 0.05
0.90 0.02
ND
ND
ND
2.66 0.21
ND
13
12
17
R1O 11
20
HO
ND
16
14
1
4.24 0.00
ND
9
6
15
2
3
OR2
10
OH
8
N
N
ND
7
5
4
ND
4.67 0.08
ND
19
18
33
1.17 0.31
0.72 0.07*
0.64 0.11*
0.35 0.04*
0.50 0.06*
1.04 0.05
0.44 0.11*
0.21 0.04*
ND
R1
R2
1
30
31
H
H
H
H
Bz
As
Ac = COCH3
As = COC6H4OCH3 (p)
Bz = COC6H5
ND
HO
ND
32 Vr
H
H
H
H
H
NB
Cm = COCH=CHC6H5
MB = COC6H4CF3 (m)
NB = COC6H4NO2 (p)
PB = COC6H4CF3 (p)
Vr = COC6H3(OCH3)2 (3,4)
11.42 0.71
ND
34
35
36
37
38
Cm
PB
MB
NB
H
OR
C6H5H2C
N
ND
O
ND
39 NB NB
3: R = H
4: R = CH2C6H5
5.44 0.41
3.75 0.14
ND
R2O
0.37 0.10*
0.89 0.04*
0.27 0.24*
0.13 0.04*
OR3
N
ND
3.02 0.47
0.48 0.07
OR1
R1
R2
R3
R1
R2
R3
R1
R2
R3
2
5
6
7
8
H
H
H
H
H
13
14
15
16
17
18
19
20
21
H
As
H
As
H
NB
H
As
H
As
As
H
H
NB
H
22
23
24
25
26
27
28
29
NB NB
NB NB
H
H
H
ND not determined
a
Each value is the mean SD of three determinations
Bz
Bz
NB
Vr
H
H
As
H
As
As
H
NB
H
H
NB
NB
NB
Cm
H
MB
H
Bz
NB Bz
H
Vr
H
H
H
H
H
H
H
H
Cm
H
* p \ 0.05, indicating significant difference from the control value
H
H
Vr
H
H
9
MB
H
10
11
12
PB
H
H
As
NB
H
PB
had a significant cytotoxic effect and showed an IC50 value
against A549 cells of 3.02 0.47 lM. The cytotoxic
effects of kobusine derivatives were weak compared with
NB
Fig. 1 Structure of C20-diterpenoid alkaloids and their derivatives
123

48
J Nat Med (2011) 65:43–49
Control
Compound 16
the cytotoxic effects of pseudokobusine derivatives, except
trifluoromethylbenzoate (35, 36). Hence, the presence of a
hydroxy group at the C-6 position enhanced the suppres-
sive effects against the growth of A549 cells.
Compound 14
Compound 35
80
60
40
20
0
In a test at 5 lg/ml additions against the growth of A549
cells, 26 alkaloids (1, 2, 8, 11, 13–17, 19–24, 28–35, 37–
39) were examined. Pseudokobusine (2) and alkaloids 8,
17, 23, 24 and 38 displayed weak cytotoxic effects. Alka-
loids 13, 19, 21, 29, 30, 32 and 34 had significant cytotoxic
effects and were found to be about 1.4- to 2.4-fold more
potent than 1 lg/ml addition. In contrast, alkaloids 14, 15,
20, 31, 35, 37 and 39 had significantly more potent cyto-
toxic effects and were found to be about 3- to 8-fold more
potent than 1 lg/ml addition. Alkaloids 16 and 22 showed
the strongest cytotoxic activities against A549 cells.
24 hr
48 hr
Time
Fig. 3 The effects of compounds 14, 16 and 35 on the G1 phase
population of A549 cells. A549 cells following treatment with vehicle
alone and each compound at doses of double IC50 values for 24 and
48 h
The cell cycle distribution of A549 cells at 24 and 48 h
after treatment was analyzed by a fluorescence cell ana-
lyzer. Compounds 14, 16 and 35 showed remarkable
enhancement at the G1 phase for up to double IC50 dose at
24 h (Fig. 2), and these compounds increased the G1 phase
population of A549 cells in a time-dependent manner
(Fig. 3). These results suggest that cytotoxic derivatives
can disturb G1 to S phase entrance [25–27]. It is well
established that cell cycle progression is highly dependent
on cyclins, Cdks (cyclin-dependent kinases) and Cdk
inhibitors [28]. Raf-MEK-Erk signaling and/or PTEN-
PI3K-AKT signaling contributes to G1 to S transition via
subsequent cyclin production and the inhibition of MEK
and PI3K activity induced complete G1 phase arrest [29].
Moreover, in a previous study, a C-11 acyl derivative
showed an inhibitory effect on the growth of A549 cells
without enhancement of apoptosis or DNA damage [20].
Thus, it appears that these diterpenoid alkaloid derivatives
do not induce genotoxic stress and inhibit cell growth
through G1 arrest.
The results of this study suggest that C-6 and C-15
hydroxyl groups in pseudokobusine are necessary for a
cytotoxic effect. Esterification of the hydroxyl group at
C-11 may, thus, contribute to the enhancement of activity
of the parent alkaloids more than that of the OH group at
C-11. Furthermore, replacement by an acyl group at C-15
in 11-substrates, such as 11,15-dianisoylpseudokobusine
(16), 11,15-di-p-nitrobenzoylpseudokobusine (22) and
11,15-di-p-nitrobenzoylkobusine (39), was involved in the
activation of the cytotoxic effect. These three compounds
incorporated all of the favorable modifications identified to
date. They possess a novel structure and show remarkable
IC50 value in the sub-micromolar range. Substitution of the
hydroxyl group had variable effects. Benzoyl and benzyl
substitutions were inactive. Cinnamoyl, p-nitrobenzoyl,
m-trifluoromethylbenzoyl and veratroyl substitutions were
effective. Anisoate was found to be about 2-fold more
potent than these substrates. Current studies are focused on
the use of semi-synthetic analogues of diterpenoid alka-
loids to further probe the mechanisms of the cytotoxic
effect on the growth of the A549 human lung cancer cell
line. The present results suggested that novel alkaloid
derivatives affect the metabolism of tumor cells as a part of
the anti-proliferative activities. The suppressive effects of
these alkaloids on the growth of human CD34? hemato-
poietic stem/progenitor cells will be examined in the
future.
Control
200
G1: 63.6%
1000
Compound 14
Compound 35
Compound 16
200
200
200
G1: 62.5%
G1: 66.7%
G1: 65.1%
IC50
1000
1000
1000
200
200
200
G1: 73.3%
G1: 79.7%
G1: 74.7%
2×IC50
1000
1000
1000
Fig. 2 The effects of compounds 14, 16 and 35 on the cell cycle
distribution of A549 cells. A549 cells treated with vehicle (DMSO)
alone and each compound at doses of its IC50 and double IC50 values
for 24 h were fixed, and the cell cycle distribution was then analyzed
by flow cytometry. Representative cytograms are shown
123

J Nat Med (2011) 65:43–49
49
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