A new abietene diterpene and other constituents from Kaempferia angustifolia Rosc

Article abstract of DOI:10.3390/molecules16043018

A new abietene diterpene, kaempfolienol (5S,6S,7S,9S,10S,11R,13S-abiet- 8(14)-enepenta-6,7,9,11,13-ol, 1), was isolated from a rhizome extract of Kaempferia angustifolia Rosc. along with the known compounds crotepoxide, boesenboxide, zeylenol, 2′-hydroxy-

Full text of DOI:10.3390/molecules16043018

Molecules 2011, 16, 3018-3028; doi:10.3390/molecules16043018
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Abietene Diterpene and Other Constituents from
Kaempferia angustifolia Rosc.
Sook Wah Tang 1, Mohd Aspollah Sukari 1,*, Mawardi Rahmani 1, Nordin Hj. Lajis 1 and
Abdul Manaf Ali 2
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
2
Universiti Sultan Zainal Abidin, Kampus Kota, Jalan Sultan Mahmud, 20400 Kuala
Terengganu, Malaysia
* Author to whom correspondence should be addressed; E-Mail: aspollah@science.upm.edu.my;
Tel.: +603-8946 6797.
Received: 16 March 2011; in revised form: 1 April 2011 / Accepted: 2 April 2011 /
Published: 7 April 2011
Abstract: A new abietene diterpene, kaempfolienol (5S,6S,7S,9S,10S,11R,13S-abiet-8(14)-
enepenta-6,7,9,11,13-ol, 1), was isolated from a rhizome extract of Kaempferia
angustifolia Rosc. along with the known compounds crotepoxide, boesenboxide, zeylenol,
2′-hydroxy-4,4′,6′-trimethoxychalcone, (24S)-24-methyl-5α-lanosta-9(11),25-dien-3β-ol,
β-sitosterol and β-sitosterol-3-O-β-D-glucopyranoside. The structures of all compounds
were elucidated on the basis of mass spectroscopic and NMR data. Zeylenol (2), the major
constituent of the plant, was derivatized into diacetate, triacetate and epoxide derivatives
through standard organic reactions. The cytotoxic activity of compounds 1, 2 and the
zeylenol derivatives was evaluated against the HL-60, MCF-7, HT-29 and HeLa cell lines.
Keywords: Kaempferia angustifolia; abietene diterpene; kaempfolienol; zeylenol;
cytotoxic
1. Introduction
Kaempferia angustifolia, which is also known as Kunci pepet, Kunci menir or Kunci kunot can be
found growing wild in forests of Western and Central Java of Indonesia and in parts of Thailand. The

Molecules 2011, 16
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pleasant-smelling plant is usually used as a remedy for colds, stomach ache and dysentery, while its
rhizomes are used for coughs and as a masticatory [1]. Our group has previously reported on the
chemical constituents [1], essential oil characterization [2] and the larvicidal activity of Kaempferia
angustifolia against Aedes aegypti larvae [3]. Besides oxygenated cyclohexane derivatives [4],
pimarane diterpenes [5], phenylpropanoids [6] and flavonoids [7] have also been isolated from other
Kaempferia species. This paper reports our recent isolation of a new abietene diterpene, kaempfolienol (1),
along with chalcone, triterpene, glycoside and other cyclohexane derivatives from the rhizome of
Kaempferia angustifolia and the cytotoxic properties of selected compounds. Several derivatives of the
major component zeylenol were also prepared to evaluate the potential enhancement of its
cytotoxic activity.
2. Results and Discussion
Phytochemical investigation on the rhizomes of Kaempferia angustifolia Rosc. from Indonesia has
resulted in the isolation of new abietene diterpene kaempfolienol and seven other known compounds.
The structures of crotepoxide [1], boesenboxide [1], 2′-hydroxy-4,4′,6′-trimethoxychalcone [1],
(+)-zeylenol [4], (24S)-24-methyl-5α-lanosta-9(11),25-dien-3β-ol [8], β-sitosterol [9] and β-sitosterol-
3-O-β-D-glucopyranoside [10] were elucidated based on comparison of their physical and spectral data
with reported literature values.
Compound 1, kaempfolienol (Figure 1), was isolated from the chloroform extract of Kaempferia
angustifolia rhizome as colourless needle-shaped crystals with a melting point of 278-280 °C. It has an
optical rotation value of [α]D = + 1.8 ° (c 0.13, MeOH). A sodium adduct molecular ion at m/z
377.2746 (calcd. 377.2304) [M+Na]+ corresponding to the molecular formula C20H34O5Na was
observed in the HRESIMS spectrum, requiring four unsaturation equivalents. The absorptions at 3,438
cm-1 and 1,018 cm-1 in the IR spectrum were attributed to hydroxyl group and C-O, respectively,
whereas a weak absorption band at 1,628 cm-1 was due to olefinic C=C stretching. These data
indicated that compound 1 is an unsaturated alcohol derivative. The 1H-NMR spectrum was integrated
for 29 protons. The presence of five methyl signals at δ 1.27, 1.21, 1.02, 0.96 and 0.93, of which the
latter two, due to an isopropyl group, resonated as a doublet with J = 7.0 Hz, indicating that compound 1
is an abietane-type diterpene.
Figure 1. Structures of compounds.
(2) R1 = R2 = R3 = H
(3) R1 = H, R2 = R3 = Ac
(4) R1 = R2 = R3 = Ac
(1)
Ph’/Ph’’ = C6H5

Molecules 2011, 16
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Figure 1. Cont.
7
''
HO
OCOPh
HO
OH
2
6
5
3
'
OCOPh
4
O
(5)
In addition, an olefinic proton at δ 5.78 (s) and three methine protons at δ 4.35 (dd, J = 11.3, 4.9 Hz),
4.18 (t, J = 2.8 Hz) and δ 3.89 (d, J = 2.8 Hz) in the lower field region were also observed. The proton
at δ 4.18 showed a cross peak with the signal at δ 3.89 in the COSY spectrum, revealing that they were
linked to adjacent carbons. The occurrence of an isopropyl group was also supported by the COSY
correlations between two methyls at δ 0.96 (H-16) and 0.93 (H-17) with the methine proton (H-15) at δ
1.71 (m) (Figure 2).
Figure 2. COSY and HMBC correlations of compound 1.
From the 13C-NMR spectrum, 19 carbon resonances which corresponded to six methine, four
methylene, five methyl and four quartenary carbons from DEPT analysis were observed. One of the
carbon signals at δ 45.0 was possibly due to overlapping of two carbon resonances since the intensity
of the peak was about one-fold higher than the rest and the assignments remained to be clarified. A
pair of olefinic carbons was observed at δ 139.9 (q) and 135.8 (methine), while crowded signals in the
high-field region δ 15-50 were typical for a terpene skeletons.
The HMQC spectrum showed carbons and protons connected by one bond whilst HMBC
correlations determined two and three-bond carbon-proton couplings (Figure 2). Two quartenary
methyls at δ 1.27 (H-18) and 1.02 (H-19) were correlated to δ 35.0 (C-4) in the HMBC spectrum; the
former signal also showed cross peak with methylene carbon at δ 45.0 (C-3), while the latter exhibited
a 3J correlation with the methine carbon at δ 44.3 (C-5).
Furthermore, H-5 (δ 1.96) displayed a correlation to another quartenary methyl (C-20: δ 21.2), and
its protons (H-20: δ 1.21) were in turn correlated to the δ 37.1 methylene carbon (C-1), oxygenated

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quartenary carbon δ 78.0 (C-9) and the signal at δ 45.0 in the HMBC spectrum. Therefore, the carbon
resonance at δ 45.0 was assigned to the quartenary carbon C-10 in addition to C-3. Correlations
between H-5 and δ 4.18 (H-6) and between H-6 and δ 3.89 (H-7) were demonstrated in the COSY
spectrum, thus revealing the connectivities of C5-C7.
Methyl protons (H-16 and H-17) and the methine proton H-15 from the isopropyl fragment were
correlated to the oxygenated quartenary carbon at δ 73.3 (C-13). H-15 also demonstrated long-range
correlations to the methylene carbon δ 37.6 (C-12) and the olefinic carbon δ 135.8 (C-14). Again, the
elucidation of structure was substantiated by COSY correlations between the olefinic proton H-14 at δ
5.78 and H-12 at δ 1.61 (via a w-coupling) which also displayed a cross peak with the oxygenated
methine proton H-11 (δ 4.35). The connectivities shown from the evidences mentioned above revealed the
double-bond at C-8 (δ 139.9) and C-14 (δ 135.8) and the placement of isopropyl group at C-13.
Additional HMBC correlations of H-11/C-10, H-14/C-7 (δ 81.2) and H-1 (δ 1.54)/C-9 (78.0)
established the linkage between three cyclic rings. Proton signals for hydroxyl groups were not observed,
but the placement of hydroxyl groups at C-6, C-7, C-9, C-11 and C-13 was reasonable since the
respective carbons resonated in a more deshielded region (δ 65 – 81) and conform to the type of carbons
in DEPT spectrum. Consequently, compound 1 was identified as abiet-8(14)-enepenta-6,7,9,11,13-ol and
named kaempfolienol. The NMR spectral data of this compound are summarized in Table 1.
Table 1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectral data of kaempfolienol (1)
(CD3OD).
1H-NMR δ
(multiplicity,
J in Hz)
HMBC
13C -NMR
Position
COSY
2J
3J
(δ)
1
2
3
1.55 (m)
-
-
-
37.1
20.0
45.0
C-2
C-9
-
1.76 (dd, 13.1, 3.4)
1.46 (dt, 13.1, 3.4)
1.66 (m)
-
-
1.25 (br d, 3.4)
1.33 (br d, 11.3)
-
C-1
-
4
5
-
35.0
44.3
-
1.96 (d, 2.8)
H-6
C-6, C-4,
C-10
C-9, C-20,
C-18, C-3
C-10, C-8
C-5, C-9,
C-14
6
7
4.18 (t, 2.8)
3.89 (d, 2.8)
H-5, H-7
H-6
73.1
81.2
C-7
C-6, C-8
8
-
-
-
139.9
78.0
45.0
65.4
37.6
-
-
9
-
-
-
-
10
11
12
-
-
-
4.35 (dd, 4.9, 11.3)
1.61 (dd, 11.3, 1.5)
1.63 (dd, 4.9, 1.5)
H-12
H-11
C-12
C-10
C-13, C-11
C-14, C-9,
C-15

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Table 1. Cont.
13
14
-
-
73.3
-
-
5.78 (s)
H-12
135.8
C-13
C-7, C-9,
C-12, C-15
C-12, C-14
C-13, C-17
C-13, C-16
C-3, C-19
C-18, C-5,
C-3
15
16
17
18
19
1.71 (m)
H-16, H-17
39.3
16.9
17.6
25.0
34.3
C-13, C-16
C-15
0.96 (d, 7.0)
0.93 (d, 7.0)
1.27 (s)
H-15
H-15
C-15
-
-
C-4
1.02 (s)
C-4
20
1.21 (s)
-
21.2
C-10
C-9, C-1
The less clear stereochemical aspects of 1, such as the ambiguity around the stereochemistry of
chiral centres, led us to perform an X-ray diffraction analysis of the compound. The single crystal
XRD data has been deposited with the Cambridge Crystallographic Data Centre (CCDC) [11]. The
structure of 1 was thus confirmed as 5S,6S,7S,9S,10S,11R,13S-abiet-8(14)-enepenta-6,7,9,11,13-ol,
and the compound was named as kaempfolienol. Abietane diterpenes are rarely described from
Kaempferia species and the Zingiberaceae family. To date, the only mention found is isolation of
abieta-8,11,13-trien-11-ol from Kaempferia atrovirens reported in a dissertation [12] and hence these
findings contribute to the chemotaxonomic significance.
2.1. Derivatives of Zeylenol
Zeylenol (2), an oxygenated cyclohexene derivative, was isolated as the major compound from the
chloroform extract of the plant. The synthesis of zeylenol derivatives has been reported previously [13]
but some of the 13C-NMR spectral data have never been reported and their cytotoxic properties are yet
to be studied.
More efficient synthesis methods were implemented for the structural-modification of zeylenol (2) to
give zeylenol diacetate (3) and zeylenol triacetate (4) in high yield. A higher yield (99.3%) for conversion
of zeylenol (2) to derivative 3 was accomplished by using same reagents as in the literature [13], albeit
with shorter reaction duration (2 hours) as compared to the overnight stirring used in the reference. The
use of acetic anhydride under base (pyridine)-catalyzed conditions at reflux led to the formation of
single product 4 (77.5% of yield), whereas a mixture of 3 (33%) and 4 (31%) was obtained by
employing 4-dimethylaminopyridine and stirring at RT overnight [13].
The formation of diacetate derivative 3 (Figure 1) could be evidenced from two singlet peaks at δ
1
2.09 (2-OCOCH3) and 2.12 (6-OCOCH3) in the H-NMR spectrum. In addition, the signals for
acetoxyl methyls [δ 20.9 (2-OCOCH3) and 20.8 (6-OCOCH3)] and the corresponding carbonyl carbons
(δ 170.3 and 169.7) could also be observed in the 13C-NMR spectrum.
The 1H-NMR spectrum of zeylenol triacetate (4, Figure 1) is similar to that of zeylenol (2), except
for the presence of three extra singlet peaks at δ 1.96, 2.03 and 2.13 which can be assigned to the
acetoxy methyls 2-OCOCH3, 6-OCOCH3 and 1-OCOCH3, respectively. The oxymethine protons H-2,
H-3 and H-6 appeared more deshieded, whereas the inequivalent methylene protons of H-7 resonated
at lower field upon acetylation compared to the starting material 2. Besides, the formation of triacetoxy

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derivative could be evidenced from 13C-NMR shifts by having three methyl signals at δ 20.7
(2-OCOCH3), 20.8 (6-OCOCH3) and 21.6 (1-OCOCH3) as well as three carbonyl groups at δ 169.2
(1-OCOCH3), 169.5 (6-OCOCH3) and 169.9 (2-OCOCH3).
As for the elucidation of zeylenol epoxide (5, Figure 1), the signals at δH 5.81 (δC 127.8) and δH
5.94 (δC 131.3) corresponding to the olefinic group of zeylenol (2), were no longer present. Instead,
two doublet of doublet peaks were observed at the higher field region at δ 3.79 (H-4) and 3.67 (H-5) in
the 1H-NMR spectrum. In the 13C-NMR spectrum, two additional methine carbon resonances at δ 57.0
(C-4) and 54.8 (C-5) were observed. Furthermore, 3J correlations between H-4 and C-2 as well as H-5
versus C-1 in HMBC spectrum proved the assignments of C-4 and C-5. The 13C-NMR spectral data of
compounds 3 and 5 are reported for the first time in this paper.
2.2. Cytotoxic Activity of Compounds
The cytotoxic activities of compounds 1-5 were evaluated against HL-60 (human promyelocytic
leukemia), MCF-7 (human breast cancer), HT-29 (human colon cancer) and HeLa (human cervical cancer)
cell lines and the results are summarized in Table 2. Kaempfolienol (1) demonstrated moderate inhibition
against HL-60 and MCF-7 cell lines with IC50 values 24.22 ± 0.30 µg/mL and 23.50 ± 1.70 µg/mL,
respectively. Zeylenol (2) was selectively active towards inhibition of leukemic cancer cell (HL-60)
with IC50 value of 11.65 ± 0.52 µg/mL. Acetylation of the hydroxyl groups of zeylenol (2) resulted in
the elimination of its antileukemic activity for compounds 3 and 4 while weaker activities were also
observed for the epoxide derivative (5) (IC50 20.91 ± 1.28 µg/mL). As for zeylenol diacetate (3), the
anticancer property against HT-29 cell line (IC50 6.73 ± 0.68 µg/mL) was better than its starting
material [zeylenol (2)]. To the best of our knowledge, this is the first report of the cytotoxic activities
of (+)-zeylenol (2) and its derivatives (3-5).
Table 2. Cytotoxic activity of compounds against HL-60, MCF-7, HT-29 and HeLa cells.
IC50(μg/mL)
Compounds
HL-60
MCF-7
HT-29
HeLa
1
24.22 ± 0.30
23.50 ± 1.70
-
-
-
-
-
-
2
11.65 ± 0.52
-
-
-
-
-
3
-
6.73 ± 0.68
4
-
-
5
20.91 ± 1.28
NT
Positive control
Goniothalamin
5-Fluorouracil
Tamoxifen
1.23 ± 0.20
4.20 ± 0.30
3.12 ± 0.20
1.10 ± 0.21
Notes: HL-60 (Human Promyelocytic Leukemia), MCF-7 (Human Breast Cancer), HT-29 (Human
Colon Cancer), HeLa (Human Cervical Leukemia).
- : not active, IC50 > 30 μg/mL; NT: not tested.
* Results are expressed as IC50 values (μg/mL) ± Standard deviation of three experiments
performed in triplicate.

Molecules 2011, 16
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3. Experimental
3.1. General
Melting points were determined using a Barnstead Electrothermal IA 9100 series melting point
equipment and were uncorrected. Optical rotations were measured on a JASCO P-2000 series
polarimeter. UV spectra were obtained by using Shimadzu 1650 PC spectrophotometer. IR spectra were
recorded using a Perkin Elmer FTIR spectrophotometer model Spectrum BX. The HRESIMS mass
spectra were obtained from a Bruker Daltonics micrOTOF-Q ESI-Qq-TOF mass spectrometer. 1H-NMR
13
and C-NMR spectra were recorded with a JEOL FT NMR spectrometer (500 MHz and 125 MHz for
compound 1 and 400 MHz/100 MHz for other compounds) using TMS as internal standard. Silica gel
(70-230 mesh, 230-400 mesh, Merck) was used for column chromatography. TLC was performed on
silica gel plates (Merck DC-Alufolien 60 F254) and the spots were visualized under UV (254 nm, 366 nm)
followed by spraying with 10% aqueous H2SO4 and further heating on a hot plate.
3.2. Plant Material
Rhizomes of Kaempferia angustifolia Rosc. were collected from Yogyakarta, Indonesia in 2001 and
identified by Sugeng Riyanto from Gadjah Mada University, Indonesia. A voucher specimen was
deposited in the herbarium of the institution.
3.3. Extraction and Isolation
Rhizomes of Kaempferia angustifolia Rosc. were air-dried and ground into powder form. The plant
sample (765 g) was extracted with methanol three times for three days each to give crude methanol
extract which was then partitioned with hexane, water with methanol (10%) and chloroform (1 L each)
to give hexane-soluble fraction (10.60 g) and chloroform-soluble fraction (42.50 g). The aqueous with
methanol fraction was further discarded. The hexane extract (8.00 g) was subjected to vacuum column
chromatography and eluted with mixtures of hexane, ethyl acetate and methanol to give 85 fractions
(200 mL each) which were then combined according to their similar TLC profiles. Upon purification,
(24S)-24-methyl-5α-lanosta-9(11),25-dien-3β-ol (40 mg), β-sitosterol (35 mg), 2′-hydroxy-4,4′,6′-
trimethoxychalcone (7 mg), crotepoxide (10 mg) and β-sitosterol-3-O-β-D-glucopyranoside (8 mg)
were obtained. Part of the chloroform extract (40.00 g) was fractionated in the similar manner to
obtain 100 fractions (200 mL each) which were combined into major fractions based on their TLC
profiles. 2′-Hydroxy-4,4′,6′-trimethoxychalcone (10 mg), boesenboxide (16 mg), crotepoxide (0.79 g),
zeylenol (0.97 g) and kaempfolienol (abiet-8(14)-enepenta-6,7,9,11,13-ol, 1, 20 mg) were obtained.
3.4. Structural Modification of Zeylenol
Zeylenol (2, 0.10 g; 2.604 × 10-4 mole) was subjected to acetylation using acetic anhydride (1.0 mL;
1.06 × 10-2 mole) and pyridine (2 mL) with stirring for 2 hours at room temperature. The reaction was
monitored by using thin-layer chromatography. Upon completion, the reaction crude product was
poured into iced-water (10 mL) and extracted with dichloromethane (2 × 10 mL). The organic layer
was then washed with 20% hydrochloric acid and further dried by adding anhydrous sodium sulphate.

Molecules 2011, 16
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The crude product (0.17 g) was obtained after filtering off the drying agent and evaporating the solvent
under reduced-pressure. Purification of the product using silica gel 9385 column chromatography was
carried out. Eluent with a solvent system of hexane-ethyl acetate (70:30 → 60:40) gave zeylenol
diacetate (3, 0.12 g; 99.3% yield).
Acetic anhydride (3.0 mL; 3.17 × 10-2 mole) was added to zeylenol (2, 0.10 g; 2.604 × 10-4 mole) in
pyridine (4.0 mL) and the mixture refluxed for six hours. The reaction product was worked-up
similarly to 3 to give crude product (0.20 g). The yellowish solid was then chromatographed on a silica
gel column to yield a pale yellow oil which solidified at room temperature and was recrystallized with
methanol to afford colourless needle-shaped crystals of zeylenol triacetate (4, 0.11 g; 77.5% yield).
Zeylenol (2, 0.05 g; 1.30 × 10-4 mole) was reacted with meta-chloroperbenzoic acid (MCPBA) (25 mg;
1.45 × 10-4 mole) in dichloromethane (10.0 mL) by reluxing for 24 hours. The solvent was evaporated
from the reaction product under reduced-pressure and the crude mixture (94 mg) was purified by
silica-gel column chromatography. Elution with hexane-ethyl acetate (70:30) afforded zeylenol
epoxide (5, 12 mg; 23.0% yield).
3.5. Spectral Data
Kaempfolienol (1). Colourless needle-shaped crystals. m.p. 278-280 °C. [α]D = + 1.8 ° (c 0.13,
MeOH). UV (EtOH) λmax nm (log ε): 273 (2.70), 202 (4.06). IR (cm-1, KBr disc) νmax : 3,438, 2,934,
1
13
2,884, 1,628, 1,436, 1,384, 1,342, 1,018. For H- and C-NMR spectra, see Table 1. HR-ESIMS m/z
(theoretical mass): 377.2746 ([M+Na]+, 377.2304), 355.3210 ([M+H]+, 355.2484).
Zeylenol (2). White amorphous solid. m.p. 108-110 °C (Lit. [4]: 125-126 °C). [α]D = + 123.4 ° (c 0.05,
CHCl3) [Lit.: [α]D = + 121.6 ° (c 0.05, CHCl3)] [4]. UV (CHCl3) λmax nm (log ε): 275 (3.35), 241
(4.18). IR (cm-1, KBr disc) νmax : 3,470, 3,062, 2,912, 1,692 (C=O), 1,602, 1,586, 1,452, 1,374, 1,122,
1,072, 712. 1H-NMR (400 MHz, CD3OD): δ 8.03 (2H, d, J = 7.4 Hz, H-2″/6″), 7.99 (2H, d, J = 7.4 Hz,
H-2′/6′), 7.56 (2H, m, H-4′/4″), 7.42 (4H, m, H-3′/5′, H-3″/5″), 5.94 (1H, dd, J = 10.1, 4.6 Hz, H-5),
5.81 (1H, dd, J = 10.1, 2.4 Hz, H-4), 5.69 (1H, dd, J = 6.4, 2.4 Hz, H-3), 4.68 (1H, d, J = 11.9 Hz, H-7a),
4.59 (1H, d, J = 11.9 Hz, H-7a), 4.34 (1H, br d, J = 4.6 Hz, H-6), 4.22 (1H, d, J = 6.4 Hz, H-2).
13C-NMR (100 MHz, CD3OD): δ 168.3 (7-OCO), 167.9 (3-OCO), 134.3 (C-4″), 134.1 (C-4′), 131.5
(C-1″), 131.4 (C-1′), 131.3 (C-5), 130.7 (C-2″/6″), 130.6 (C-2′/6′), 129.5 (C-3″/5″), 129.4 (C-3′/5′),
127.8 (C-4), 76.6 (C-1), 75.5 (C-3), 71.0 (C-2), 70.0 (C-6), 68.1 (C-7). HR-ESIMS m/z (theoretical
mass): 407.1655 ([M+Na]+, 407.1107), 385.1801 ([M+H]+, 385.1287).
Zeylenol diacetate (3). Colourless oil. [α]D = + 24.8 ° (c 0.14, CHCl3) [Lit. [13]: [α]D = + 17 ° (c 1.0,
CHCl3)]. UV (CHCl3) λmax nm (log ε): 282 (3.60), 275 (3.67), 240 (4.52). IR (cm-1, NaCl disc) νmax
:
1
3,464, 2,962, 1,728, 1,602, 1,448, 1,372, 1,264, 1,110, 712. H-NMR (400 MHz, CDCl3): δ 7.99 (4H,
m, H-2′/6′, H-2″/6″), 7.56 (2H, m, H-4′/4″), 7.42 (4H, m, H-3’/5’, H-3″/5″), 6.01 (1H, dd, J = 10.1, 2.8
Hz, H-4), 5.95 (1H, ddd, J = 10.1, 4.6, 1.8 Hz, H-5), 5.88 (1H, ddd, J = 7.3, 2.8, 1.8 Hz, H-3), 5.71
(1H, d, J = 7.3 Hz, H-2), 5.51 (1H, d, J = 4.6 Hz, H-6), 4.65 (1H, d, J = 12.0 Hz, H-7a), 4.46 (1H, d,
J = 12.0 Hz, H-7b), 3.16 (1H, s, 1-OH), 2.12 (3H, s, 6-OCOCH3), 2.09 (3H, s, 2-OCOCH3). 13C-NMR
(100 MHz, CDCl3): δ 170.3 (2-OCO), 169.7 (6-OCO), 166.7 (7-OCO), 165.9 (3-OCO), 133.5 (C-4″),

Molecules 2011, 16
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133.3 (C-4′), 129.2 (C-5), 125.8 (C-4), 129.7 (C-2′/6′, C-2″/6″), 129.3 (C-1″), 129.0 (C-1′), 128.5
(C-3″/5″), 128.4 (C-3′/5′), 74.1 (C-1), 71.7 (C-2), 70.9 (C-3), 70.3 (C-6), 65.9 (C-7), 20.9
(2-OCOCH3), 20.8 (6-OCOCH3). HR-CIMS m/z (theoretical mass): 469.1497 ([M+H]+, required
469.1499).
Zeylenol triacetate (4). Colourless needle-shaped crystals. m.p. 150-151 °C (Lit. [13]: 158-159 °C).
[α]D = + 26.7 ° (c 0.30, CHCl3) [Lit. [13]: [α]D = + 24 ° (c 0.6, CHCl3)]. UV (CHCl3) λmax nm (log ε):
283 (3.24), 275 (3.35), 242 (4.14). IR (cm-1, KBr disc) νmax: 3,062, 2,986, 2,954, 1,752, 1,726, 1,604,
1,454, 1,374, 1,104, 712. 1H-NMR (400 MHz, CDCl3): δ 8.03 (4H, m, H-2′/6′, H-2″/6″), 7.57 (2H, m,
H-4′/4″), 7.47 (2H, d, J = 8.3 Hz, H-3″/5″), 7.43 (2H, d, J = 8.3 Hz, H-3′/5′), 6.19 (1H, br d, J = 4.6
Hz, H-6), 6.06 (1H, dd, J = 10.1, 2.8 Hz, H-4), 5.99 (1H, d, J = 8.3 Hz, H-2), 5.96 (1H, ddd, J = 10.1,
4.6, 1.8 Hz, H-5), 5.89 (1H, ddd, J = 8.3, 2.8, 1.8 Hz, H-3), 5.09 (1H, d, J = 12.0, H-7a), 4.82 (1H, d,
13
J = 12.0, H-7b), 2.13 (3H, s, 1-OCOCH3), 2.03 (3H, s, 6-OCOCH3), 1.96 (3H, s, 2-OCOCH3). C-
NMR (100 MHz, CDCl3): δ 169.9 (2-OCOCH3), 169.5 (6-OCOCH3), 169.2 (1-OCOCH3), 166.0
(7-OCO), 165.5 (3-OCO), 133.4 (C-4″), 133.3 (C-4′), 130.0 (C-5), 129.8 (C-2″/6″), 129.6 (C-2′/6′),
129.4 (C-1″), 129.2 (C-1′), 128.5 (C-3′/5′, C-3″/5″), 125.3 (C-4), 81.4 (C-1), 70.9 (C-3), 70.7 (C-2),
67.9 (C-6), 63.1 (C-7), 21.6 (1-OCOCH3), 20.8 (6-OCOCH3), 20.7 (2-OCOCH3). HR-CIMS m/z (%
intensity): 511.1607 ([M+H]+, required 511.1604).
Zeylenol epoxide (5). Colourless needle-shaped crystals. m.p. 164-166 °C (Lit. [13]: 173-175 °C).
[α]D = + 61.4 ° (c 0.08, CHCl3) [Lit. [13]: [α]D = + 68 ° (c 0.3, CHCl3)]. UV (CHCl3) λmax nm (log ε):
283 (3.14), 276 (3.23), 242 (4.05). IR (cm-1, KBr disc) νmax : 3,476, 2,924, 1,696, 1,600, 1,450, 1,279,
1,069, 709. 1H-NMR (400 MHz, CDCl3): δ 8.10 (2H, d, J = 8.3 Hz, H-2′/6′), 8.04 (2H, d, J = 8.3 Hz,
H-2″/6″), 7.59 (2H, d, J = 8.3 Hz, H-4′/4″), 7.44 (4H, t, J = 8.3 Hz, H-3′/5′, H-3″/5″), 5.71 (1H, dd,
J = 8.2, 1.8 Hz, H-3), 4.84 (1H, d, J = 11.9 Hz, H-7a), 4.58 (1H, d, J = 11.9 Hz, H-7b), 4.16 (1H, d, J = 4.6 Hz,
H-6), 4.02 (1H, d, J = 8.2 Hz, H-2), 3.79 (1H, dd, J = 3.7, 1.8 Hz, H-4), 3.67 (1H, dd, J = 3.7, 4.6 Hz,
13
H-5), 3.39 (1H, br s, OH), 2.96 (1H, br s, OH), 2.61 (1H, d, J = 8.2 Hz, 6-OH). C-NMR (100 MHz,
CDCl3): δ 167.6 (7-OCO), 167.1 (3-OCO), 133.5 (C-4′/4″), 130.0 (C-2′/6′), 129.8 (C-2″/6″), 129.4 (C-1″),
129.3 (C-1′), 128.5 (C-3′/5′, C-3″/5″), 77.1 (C-1), 74.1 (C-3), 68.9 (C-2), 68.1 (C-6), 66.6 (C-7), 57.0
(C-4), 54.8 (C-5). HRFAB-MS m/z (theoretical mass): 401.1242 ([M+H]+, required 401.1236).
3.6. Cytotoxicity Assays
Compounds were assayed for cytotoxic test against HL-60, MCF-7, HT-29 and HeLa cell lines
using the MTT method [6]. Results were expressed as IC50 (μg/mL) ± standard deviation from three
separate experiments, each performed in triplicate.
4. Conclusions
From the rhizome extract of Kaempferia angustifolia Rosc., a new abietene diterpene identified as
5S,6S,7S,9S,10S,11R,13S-abiet-8(14)-enepenta-6,7,9,11,13-ol to which the trivial name kaempfolienol
has been assigned was isolated. Cyclohexane derivatives, chalcone, triterpenes and glycoside were

Molecules 2011, 16
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also obtained from the plant. Several derivatives of zeylenol were synthesized and the cytotoxic
properties against some selected cancer cell lines of these compounds were studied.
Acknowledgements
We would like to acknowledge the financial support from the Ministry of Science, Technology and
Innovation (MOSTI) for e-Science fund provided and Universiti Putra Malaysia for a Graduate
Research Fellowship scholarship provided to one of the authors (S.W. Tang). We also thank Mohd.
Ambar Yarmo (UKM) for HRESIMS spectra, Mohd. Azlan Nafiah (UPSI) for NMR spectra,
Mohamed Ibrahim Mohamed Tahir, Sidik Silong and Kwong Huey Chong of Chemistry Department
UPM for the assistance regarding single crystal XRD analysis.
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Sample Availability: Contact the corresponding author.
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