Article

pubs.acs.org/jnp

Terpenoids from the Root Bark of Pterolobium macropterum

Jittra Suthiwong, Siripit Pitchuanchom, Wareeporn Wattanawongdon, Chariya Hahnvajanawong,
and Chavi Yenjai*,

Natural Products Research Unit, Center of Excellence for Innovation in Chemistry, Department of Chemistry, Faculty of Science,
and Department of Microbiology, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen
University, Khon Kaen 40002, Thailand

Laboratory of Natural Products, Faculty of Science and Center of Excellence for Innovation in Chemistry, Lampang Rajabhat
University, Lampang 52100, Thailand
S Supporting Information
*

ABSTRACT: Five new compounds, including pteroloterins AC (1, 3, and 4), 1-acetoxytaepeenin C (2), and 8ahydroxycadinenal (5), and 11 known compounds were isolated from the root bark of Pterolobium macropterum. All compounds
were evaluated for cytotoxicity against the cholangiocarcinoma cell lines. Compound 9 showed weak cytotoxicity against the
KKU-M139 cell line with an IC50 value of 23.24 0.18 M and showed no activity against normal cells.

observed that crude extracts (hexanes, EtOAc, and MeOH) of

the root bark of P. macropterum exhibited cytotoxicity with IC50
values ranging between 9 and 45 g/mL. Thus, investigating
the root bark of P. macropterum resulted in the isolation of four
new cassane diterpenoids (14) and a new cadinane derivative
(5). All of the pure compounds were evaluated for cytotoxicity
against the CCA, KKU-M136, KKU-M159, and KKU-M216
cell lines.

holangiocarcinoma (CCA) is one of the major health

problems that aects northeast Thailand. Liver uke
infection and hepatolithiasis have been identied as the
etiological factors that inuence the regional frequency of
CCA. The liver uke, Opisthorchis viverrini, is endemic to
northeast Thailand and is closely related to the high incidence
of bile duct cancer.1 The infection of O. viverrini occurs
primarily in the liver, extrahepatic bile ducts, and gall bladder.
The only potentially curative treatment for CCA is a surgery
that is appropriate in less than 50% of cases.2 In patients with
irresectable and metastatic CCA, chemotherapy has been used
to control the disease and improve the patients survival rates.
However, a relatively poor response rate of CCA to
chemotherapy has been demonstrated in clinical studies.3
Therefore, a search for new and eective therapeutic agents is
still needed. This has led to screening chemical constituents
from Thai medicinal plants for cytotoxicity against cholangiocarcinoma cell lines.46
Pterolobium macropterum, which belongs to the family
Leguminosae, is a medicinal plant that is found throughout
China, Bhutan, India, Indonesia, and Southeast Asia. In
Thailand, this plant has been used to treat toothache and
fever and to promote wound healing.7 In northeast Thailand,
the plant is known as nam kra jai. In a study on the isolation
of cytotoxic agents against CCA from natural sources, it was
2014 American Chemical Society and
American Society of Pharmacognosy

RESULTS AND DISCUSSION

Crude hexanes and EtOAc extracts from the root bark of P.
macropterum were separated by column chromatography and
preparative TLC, leading to ve new compounds, pteroloterins
AC (1, 3, and 4), 1-acetoxytaepeenin C (2), and 8ahydroxycadinenal (5). In addition, 11 known compounds,
including taepeenin A (6),8 taepeenin C (7),8 epi-benthaminin
2 (8),9 nortaepeenin A (9),8 neocaesalpin I methyl ester (10),10
neocaesalpin H methyl ester (11),10 neocaesalpin H (12),10 4norcadin-5-en-4-one (13),11 7-hydroxy-4-norcadin-5-en-4-one
(14),11 cyperotundone (15),12 and n-hexacosyl ferulate (16),13
were isolated (Figure 1). The known compounds were
Received: June 11, 2014
Published: October 22, 2014
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Figure 1. Structures of compounds 116.

proton at 7.32 (H-11) and C-8, C-10, C-12, and C-13 were
observed in the HMBC experiment, along with correlations
between H-16 ( 7.52) and C-12 and C-13 and between H-15
( 6.72) and C-12. In the HMBC spectrum, two methyl ester
protons at 3.77 and 3.75 correlated with carbons at 172.4
and 173.6, respectively, conrming the presence of two ester
groups. The 1H1H COSY spectrum displayed correlations in
the aliphatic region, representing H-1/H-2/H-3 and H-5/H-6/
H-7. The methine proton at 2.42 (H-5) correlated with C-4,
C-6, C-10, C-18, and C-19. The 1H and 13C NMR data were
similar to those of taepeenin A (6),8 except the methyl group at
C-4 in 6 was replaced by a methyl ester group in 1. The relative
conguration of 1 was determined based on the coupling
constant between H-5 and H-6 (J = 11.6 Hz), which indicated
a diaxial orientation of these two protons. The NOESY
spectrum showed a cross-peak between Me-20 and CO2Me-19,
which indicated that these groups were cofacial. Thus, the
structure of compound 1, named pteroloterin A, was
established as shown.
Compound 2 was obtained as colorless needles (MeOH). A
molecular formula of C23H28O6 was determined from the 13C
NMR data and its quasi-molecular ion peak at m/z 423.1784
[M + Na]+ in the HRESIMS spectra, corresponding to 10
indices of hydrogen deciency. In the IR spectrum, the
absorption bands at 3447 and 1731 cm1 indicated the

identied by spectroscopic and spectrometric data, including

those obtained from 1D and 2D NMR, IR, and MS, and by
comparison with literature values.
Compound 1 was obtained as colorless needles (MeOH). A
molecular formula of C22H26O5 was determined from the 13C
NMR data and its quasi-molecular ion peak at m/z 393.1664
[M + Na]+ in the HRESIMS spectra, corresponding to 10
indices of hydrogen deciency. The carbonyl group showed an
absorption band at 1726 cm1 in the IR spectrum. The 13C
NMR and DEPT spectra showed 22 carbon signals, including
two methyl, two methyl ester, ve methylene, four methine
(one aliphatic and three aromatic), seven quaternary (two
aliphatic and ve aromatic), and two carbonyl carbons. The 1H
NMR data showed two singlets at 7.52 and 6.72, which were
assigned as H-16 and H-15, respectively (Table 1). These
protons were connected to carbons at 144.5 and 105.1 in the
HMQC spectrum, which suggested a furan moiety. The singlet
at 7.32 was assigned as H-11 and correlated to a carbon at
105.6 in the HMQC experiment. The 13C NMR data displayed
ve other aromatic carbons at 128.1 (C-8), 145.3 (C-9),
153.7 (C-12), 125.8 (C-13), and 128.4 (C-14) (Table 1). In the
HMBC spectrum, the methyl protons at 2.36 (Me-17)
correlated with C-8 and C-13, and correlations between the
methyl protons at 1.13 (Me-20) and C-1, C-5, C-9, and C-10
were observed (Figure 2). Correlations between the aromatic
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Table 1. 1H and 13C NMR Data (400 MHz, CDCl3) for Compounds 13
1
position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
OCH3-18
OCH3-19
OCOCH3
OCOCH3

C, type
39.8
20.0
34.2
57.8
47.0
23.8
29.3
128.1
145.3
38.5
105.6
153.7
125.8
128.4
105.1
144.5
16.1
173.6
172.4
23.8
52.8
52.0

CH2
CH2
CH2
C
CH
CH2
CH2
C
C
C
CH
C
C
C
CH
CH
CH3
C
C
CH3
CH3
CH3

2
H (J in Hz)

1.50, 2.32, m
1.75, 2.07, m
1.47, 2.49, m
2.42, br d (11.6)
1.94, 2.11, m
2.75, 2.90, m

7.32, s

6.72, br s
7.52, br s
2.36, s

1.13, s
3.75, s
3.77, s

C, type
77.1
24.7
40.2
53.1
48.1
68.5
39.2
123.9
145.1
37.7
105.5
154.0
126.8
128.9
105.1
144.7
16.3
176.8
13.4
27.6
52.6

CH
CH2
CH2
C
CH
CH
CH2
C
C
C
CH
C
C
C
CH
CH
CH3
C
CH3
CH3
CH3

21.2 CH3
170.2 C

3
H (J in Hz)

5.19, dd (10.8, 5.6)

1.74, 1.99, m
2.28, 2.31, m
2.28, br s
4.21, br d (5.6)
2.91, d (17.2); 3.08, dd (17.2, 5.6)

7.35, s

6.73, br d (2.0)
7.54, br d (2.0)
2.36, s
1.68, s
1.68, s
3.69, s

C, type
38.8
19.1
34.6
57.5
50.0
25.4
30.6
36.8
51.8
37.2
22.9
152.2
119.0
142.5
106.5
141.6
104.2
173.6
172.5
13.5
52.8
52.0

CH2
CH2
CH2
C
CH
CH2
CH2
CH
CH
C
CH2
C
C
C
CH
CH
CH2
C
C
CH3
CH3
CH3

H (J in Hz)
1.50, 1.75, m
1.39, 2.40, m
2.02,
1.10,
2.31,
2.21,
1.51,

br d (12.0)
1.79, m
2.41, m
br t (11.8)
m

2.74, dd (16.8, 5.6); 2.47, dd (16.8, 10.8)

6.44, br d (1.6)
7.23, br s
4.87/5.07, br s

0.79, s
3.70, s
3.75, s

2.03, s

(br d, J = 5.6 Hz) was assigned as H-6 and attached to the

carbon at 68.5 in the HMQC spectrum. This proton
displayed correlations to H-5 and H-7 in the 1H1H COSY
spectrum. In the HMBC spectrum, correlations between H-7
and C-5, C-8, and C-9 were observed. The coupling constant
between H-1 and H-2 was 10.8 Hz (diaxial orientation), and it
was 5.6 Hz between H-1 and H-2 (axialequatorial
orientation), indicating that the acetoxy substituent at C-1
was in the -equatorial orientation. The orientations of H-5 and
H-6 were deduced from the small coupling constants of H-5 (
2.28, br s), indicating that H-5 and H-6 were oriented in -axial
and -equatorial positions, respectively. Thus, the structure of
compound 2, named 1-acetoxytaepeenin C, was established as
shown.
Compound 3 was obtained as an amorphous powder. A
molecular formula of C22H28O5 was determined from the 13C
NMR data and its quasi-molecular ion peak at m/z 395.1888
[M + Na]+ in the HRESIMS spectra, corresponding to nine
indices of hydrogen deciency. The 13C NMR and DEPT
spectra showed 22 carbon signals, including a methyl, two
methyl ester, seven methylene (six aliphatic and one olenic),
ve methine (three aliphatic and two olenic), ve quaternary
(two aliphatic, one olenic, and two aromatic), and two
carbonyl carbons (Table 1). The 1H and 13C NMR data
showed the presence of a furan moiety, as observed for
compounds 1 and 2. In the HMQC spectrum, two protons at
H 4.87 and 5.07 correlated with the same carbon at C 104.2,
indicating the presence of an exocyclic double bond. These
protons displayed correlations with C-8 and C-13 in the
HMBC spectrum (Figure 2). The methylene protons (H-11)
gave rise to two doublets of doublets at 2.74 (dd, J = 16.8, 5.6
Hz) and 2.47 (dd, J = 16.8, 10.8 Hz). These protons displayed
correlations with C-8, C-9, and C-13. Signals at H/C 0.79/13.5
were assigned to the Me-20 group and correlated with C-1, C-5,

Figure 2. HMBC correlations of compounds 15.

presence of a hydroxy group and a carbonyl group, respectively.

The 13C NMR and DEPT spectra showed 23 carbon signals,
including four methyl, one methyl ester, three methylene, six
methine (three aliphatic and three aromatic), seven quaternary
(two aliphatic and ve aromatic), and two carbonyl carbons.
The 1H NMR data showed similar signals of aromatic protons
to those of compound 1, which indicated a benzofuran moiety.
The doublet of doublets at 5.19 (J = 10.8, 5.6 Hz) assigned to
H-1 correlated with the carbon at 77.1 in the HMQC
experiment (Table 1). The methyl group Me-20 (H/C 1.68/
27.6) showed correlations with C-1, C-5, C-9, and C-10 in the
HMBC experiment (Figure 2). The methyl group at H/C 2.03/
21.2 correlated with the carbonyl at 170.2, indicating the
presence of an acetyl group. The methyl protons, Me-19, and
CO2Me-18 correlated with the ester carbonyl at 176.8 in the
HMBC spectrum. In addition, the correlation of H-19 with C-3
and C-4 was observed. In the 1H NMR data, the signal at 4.21
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C-9, and C-10. Two singlets at 3.75 and 3.70 were assigned to
two groups of methyl ester protons, and these protons
correlated with carbons at 172.5 and 173.6, respectively. In
the HMBC spectrum, correlations between H-5 (C 50.0) and
C-4, C-6, C-7, C-10, C-18, C-19, and C-20 were observed. The
relative conguration of this compound is the same as 1, which
showed trans ring fusion and an -axial orientation of H-5 (
2.02, br d, J = 12.0 Hz). The -axial orientation of H-8 was
determined from the large coupling constant (J = 11.8 Hz), and
the NOESY spectrum showed a cross-peak between H-8 and
Me-20, which indicated that these groups were cofacial. The orientation of H-9 was also determined from a cross-peak with
H-5 in the NOESY experiment. Thus, the structure of 3, named
pteroloterin B, was determined as shown.
Compound 4 was obtained as an amorphous powder. It was
assigned a molecular formula of C19H28O3, based on its 13C
NMR data and quasi-molecular ion peak at m/z 327.1930 [M +
Na]+, corresponding to six indices of hydrogen deciency. The
IR spectrum displayed the absorption band of the conjugated
carbonyl group at 1667 cm1. The 13C NMR and DEPT spectra
exhibited 19 carbon signals, including three methyl, one methyl
ester, six methylene, four methine (three aliphatic and one
olenic), three quaternary (one olenic and two aliphatic), and
two carbonyl carbons. The 1H NMR displayed a singlet at
5.83, which was assigned as H-13 and correlated with the
carbon at 126.8 in the HMQC spectrum (Table 2). This
proton showed correlations to C-8 and C-15 in the HMBC
experiment (Figure 2). The methylene protons at 2.42 (dd, J
= 15.6, 3.2 Hz, H-11a) and 2.07 (t, J = 15.6 Hz, H-11b)

correlated with C-8 and C-12 in the HMBC experiment. The

correlations of the methyl protons at 1.89 (Me-15) to C-8
and C-14 were observed in the HMBC spectrum. Two methyl
groups resonating at 1.19 and 0.93 were assigned as Me-17
and Me-18, respectively, and showed correlations to carbons at
16.7 and 14.8, respectively. Correlations between Me-17 and
C-3, C-5, and C-16 were observed in the HMBC spectrum. The
Me-18 protons showed correlations with C-1, C-5, C-9, and C10. The singlet at 3.65 correlated with the carbonyl carbon at
179.1 in the HMBC experiment, indicating the presence of a
methyl ester group. The large coupling constant (br d, J = 12.4
Hz) of H-5 indicated the -axial orientation of this proton. The
NOESY experiment showed a correlation of the Me-18 with H8, which conrmed the cofacial orientation of these protons.
Thus, the structure of 4, named pteroloterin C, was determined
as shown.
Compound 5 was obtained as a colorless oil with []23D
121 (c 0.001, CHCl3). A molecular formula of C15H24O2 was
established based on the 13C NMR and HRESIMS data,
corresponding to four indices of hydrogen deciency. The
absorption bands of the hydroxy group at 3418 cm1 and the
carbonyl group at 1691 cm1 were observed in the IR spectrum.
This compound contained 15 carbons, including three methyl,
four methylene, ve methine (four aliphatic and one olenic), a
carbonyl, an oxygenated, and an olenic carbon. The 1H NMR
data showed a singlet at 9.48, which was assigned as a formyl
proton. It also showed an olenic proton at 6.78 that
correlated with a carbon at 148.2 (C-1) (Table 2). The 13C
NMR signal at 144.0 was assigned as the C-2 olenic carbon.
The 1H1H COSY spectrum showed correlations between H3/H-4/H-4a and H-5/H-6/H-7/H-8. Correlations of H-13
with C-2 and C-3 were observed in the HMBC spectrum
(Figure 2). In this spectrum, the correlations of H-1 with C-3,
C-13, C-4a, and C-8a conrmed the unsaturated cyclic system.
The 1H NMR data displayed three methyl doublets at 1.06 (J
= 6.8 Hz), 0.97 (J = 7.2 Hz), and 0.93 (J = 6.4 Hz), which were
assigned as Me-11, Me-10, and Me-12, respectively. The
HMBC spectrum exhibited correlations of H-10 and H-11 with
C-8 and C-9. Correlations between H-9 and C-7, C-8, and C-8a
were also observed. The COSY spectrum showed correlations
between the methine proton (H-9) and Me-10 and Me-11,
which conrmed the presence of an isopropyl group at C-8.
The 13C NMR data showed a signal at 73.0, which was
assigned as an oxygenated carbon (C-8a). The HMBC
spectrum displayed the correlations of H-12 with C-5, C-6,
and C-4a. The coupling constants and data from the NOESY
experiment were used to elucidate the relative conguration of
5. The large coupling constant (J = 12.8 Hz) of H-8 is
characteristic of a diaxial relationship with H-7 and indicates the
equatorial orientation of the C-8 isopropyl group. The axial
orientation of H-4a was conrmed by the correlation between
H-4a and H-8 in the NOESY experiment. Thus, the structure of
5, named 8a-hydroxycadinenal, was established as shown.
The cytotoxicity against human CCA, KKU-M156, KKUM213, and KKU-M139 cell lines as well as normal cells (Vero
cells) was tested using the modied Skehan method.14
Compound 9 showed weak cytotoxicity against the KKUM139 and KKU-M213 cell lines, with IC50 values of 23.24
0.18 and 34.83 0.18 M, respectively (Table 3). In addition,
this compound was inactive against normal cells. Compounds
1, 4, 5, and 1013 exhibited cytotoxicity against the KKUM213 cell line, with IC50 values ranging from 43 to 88 M.
Compounds 6 and 1113 exhibited cytotoxicity against KKU-

Table 2. 1H and 13C NMR Data (400 MHz, CDCl3) for

Compounds 4 and 5
4
position

C, type

5
H (J in Hz)

1.03, 1.73, m
1.59, m
1.56, m

C, type
148.2 CH
144.0 C
17.8 CH2

1
2
3

37.5 CH2
18.0 CH2
36.9 CH2

47.5 C

4a
5
6

49.1 CH
24.4 CH2

1.78, br d (12.4)
1.27, 1.56, m

50.0 CH
31.1 CH
34.9 CH2

7
8

30.1 CH2
40.0 CH

1.17, 2.19, m
2.28, m

25.1 CH2
53.1 CH

8a
9
10

53.3 CH
36.4 C

1.63 m

73.0 C
25.8 CH
20.5 CH3

11

37.5 CH2

2.07, t (15.6); 2.42,

dd (15.6, 3.2)

25.0 CH3

12

200.4 C

13
14
15
16
17
18
OCH3-16

126.8
165.2
22.0
179.1
16.7
14.8
51.9

CH
C
CH3
C
CH3
CH3
CH3

18.6 CH2

20.3 CH3
5.83, s

194.9 CH

H (J in
Hz)
6.78, s
1.97,
2.31, m
1.89,
2.04, m
1.29, m
0.89, m
1.04,
1.68, m
1.64, m
1.39, br d
(12.8)
2.29, m
0.97, d
(7.2)
1.06, d
(6.8)
0.93, d
(6.4)
9.48, s

1.89, s
1.19, s
0.93, s
3.65, s
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Table 3. Cytotoxicity of Isolated Compounds (M)a

compound

KKU-M156

KKU-M213

KKU-M139

Vero cells

crude hexanes
crude EtOAc
crude MeOH
1
2
4
5
6
7
9
10
11
12
13
14
15
ellipticine

45.15 1.76b
8.94 0.05b
38.70 0.76b
83.6 2.3
115.36 1.79
inactivec
inactivec
80.08 1.44
112.72 0.44
147.69 0.73
inactivec
61.25 0.47
82.94 0.40
88.75 7.66
138.58 2.02
inactivec
37.92 6.74

16.10 0.23b
27.69 0.70b
9.16 0.48b
44.16 0.67
98.23 1.49
66.78 0.09
88.77 0.97
144.23 9.93
105.28 0.93
34.83 0.18
47.58 0.58
43.95 0.47
68.85 5.37
87.34 10.81
inactivec
138.28 0.96
6.58 1.74

27.58 0.6b
15.09 0.51b
10.29 0.002b
80.20 0.94
155.04 1.87
196.80 1.47
inactivec
70.61 1.10
121.51 1.17
23.24 0.18
inactivec
76.28 11.42
77.26 10.39
70.81 8.68
148.16 4.86
inactivec
18.27 1.14

69.18 3.72b
75.56 0.26b
133.67 0.88b
65.68 6.8
inactivec
50.48 1.28
inactivec
57.56 3.00
inactivec
inactivec
94.41 4.62
59.18 2.87
97.09 4.85
153.89 2.96
153.52 5.13
inactivec
15.51 2.11

Data shown are from triplicate experiments. bg/mL. cInactive at >200 M.

M156, with IC50 values ranging from 61 to 88 M. Compounds

1, 6, and 1113 displayed cytotoxicity against KKU-M139,
with IC50 values ranging from 70 to 80 M. Comparison
between 13 and 14 suggested that the C-7 hydroxy group may
be detrimental to cytotoxicity.
In conclusion, four new cassanes (14) and a new cadinane
(5) were isolated from the root bark of P. macropterum.
Cytotoxicity against three CCA cell lines and normal cells were
evaluated. Cytotoxicity results showed that 9 had the highest
cytotoxicity against the KKU-M139 cell line, with an IC50 value
of 23.24 0.18 M.

EtOAc, and MeOH. On the basis of their TLC characteristics, the

fractions that contained the same major compounds were combined to
give ve fractions, HF1 to HF5. Fraction HF2 was puried by silica gel
FCC, and 10% EtOAchexanes was used as an eluent to give two
subfractions, HF2.1 and HF2.2. Further purication of these two
subfractions by preparative thin layer chromatography (PLC) and
developing with 1% acetonehexanes aorded 8 (7.4 mg, 0.0004%)
and 6 (15.4 mg, 0.0008%), respectively. Fraction HF3 was puried by
silica gel FCC and eluted with an isocratic system of 50% CH2Cl2
hexanes to give three subfractions, HF3.1HF3.3. Subfractions HF3.1
and HF3.2 were further puried by PLC using 50% CH2Cl2hexanes
and 10% EtOAchexanes as eluting solvents to give 3 (3.5 mg,
0.0002%) and 13 (10.7 mg, 0.00005%), respectively. The purication
of subfraction HF3.3 by silica gel FCC (50% CH2Cl2hexanes) gave
three subfractions, HF3.3.1HF3.3.3. Further purication of HF3.3.1 with
PLC (100% CH2Cl2) aorded 15 (13.6 mg, 0.0007%). Purications of
HF3.3.2 and HF3.3.3 were carried out on PLC using 10% EtOAc
hexanes as the eluting solvent to give 9 (33.7 mg, 0.0017%) and 10
(13.8 mg, 0.0007%), respectively. Fraction HF4 was puried by silica
gel column chromatography and eluted with a gradient of 20%
EtOAchexanes to give three subfractions, HF4.1HF4.3. Subfraction
HF4.1 was puried by Sephadex LH-20 column chromatography and
eluted with 100% MeOH to aord 1 (13.2 mg, 0.0007%). Subfraction
HF4.2 was puried using PLC, and 30% acetonehexanes was used as
developing solvent to give 5 (5.3 mg, 0.0003%). Subfraction HF4.3 was
puried by Sephadex LH-20 column chromatography (100% MeOH)
followed by PLC (100% CH2Cl2) to yield 4 (9.5 mg, 0.0005%).
The crude EtOAc extract was subjected to silica gel FCC and eluted
with a gradient system of hexanes, EtOAc, and MeOH to aord ve
subfractions, EF1EF5. Subfraction EF2 was subjected to silica gel
FCC and eluted with a gradient of 20% EtOAchexanes to give three
subfractions, EF2.1EF2.3. Subfraction EF2.2 was puried by PLC (10%
acetonehexanes) to yield 13 (10.7 mg, 0.0005%). Further
purication of EF2.3 by silica gel FCC and elution with a gradient of
50% CH2Cl2hexanes gave 11 (9.8 mg, 0.0005%) and 16 (9.6 mg,
0.0005%). Fraction EF3 was puried by silica gel FCC (20% EtOAc
hexanes as eluent) to give subfractions EF3.1 and EF3.2. Further
purication of EF3.1 with PLC (100% CH2Cl2) yielded 7 (18.3 mg,
0.0009%). Purication of EF3.2 by reversed-phase column chromatography and elution with 20% H2OMeOH aorded 2 (7.3 mg,
0.0004%). Fraction EF4 was puried by silica gel FCC and eluted with
30% EtOAchexanes to give two subfractions, EF4.1 and EF4.2.
Subfraction EF4.2 was subjected to gel ltration over Sephadex LH-20
(MeOH) to aord two subfractions, EF4.2.1 and EF4.2.2. After
purication of subfraction EF4.2.1 with PLC (20% EtOAchexanes),
14 (11.4 mg, 0.0006%) was obtained. Subfraction EF4.2.2 was

EXPERIMENTAL SECTION

General Experimental Procedures. A SANYO Gallenkamp

(UK) melting point apparatus was used to determine melting points. A
JASCO DIP-1000 digital polarimeter was used to identify the optical
rotation. An Agilent 8453 UVvisible spectrophotometer (Germany)
was used to record the UV spectra. IR spectra were recorded as thin
lms using a PerkinElmer Spectrum One FT-IR spectrophotometer
(UK). The NMR spectra were recorded on a Varian Mercury plus
spectrometer (UK) operating at 400 MHz (1H) and at 100 MHz
(13C). The solvent residual peak was used for chemical shift
referencing (H 3.31, C 49.0 for methanol-d4 and H 7.26, C 77.2
for CDCl3). Mass spectra were obtained on a Micromass Q-TOF 2
hybrid quadrupole time-of-ight (Q-TOF) mass spectrometer with a
Z-spray ES source (Micromass, UK). Column chromatography (CC)
was carried out using silica gel 60 (100200 mesh, Merck). TLC was
performed on silica gel 60 F254 (Merck) precoated aluminum sheets.
The compounds were visualized under UV light and by spraying with
acidic anisaldehyde solution followed by heating. Gel ltration was
carried out over Sephadex LH-20 (Pharmacia) suspended in MeOH.
Distilled solvents were used throughout the separation process.
Plant Material. The roots of Pterolobium macropterum were
collected from Khon Kaen Province, Thailand, in August 2012. The
plant was identied by Prof. Dr. Pranom Chantaranothai, Faculty of
Science, Khon Kaen University, where a voucher specimen
(KKU032012) is deposited.
Extraction and Isolation. Air-dried roots (2.0 kg) of P.
macropterum were ground and successively extracted at room
temperature with hexanes (3 12 L), EtOAc (3 5 L), and
MeOH (3 5 L). After the evaporation of solvents, the crude hexanes
(29 g), EtOAc (48 g), and MeOH (125 g) extracts were obtained. The
crude hexanes extract was separated by silica gel ash column
chromatography (FCC) and eluted with a gradient system of hexanes,
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dx.doi.org/10.1021/np500476h | J. Nat. Prod. 2014, 77, 24322437

Journal of Natural Products

Article

recrystallized with EtOAchexanes (1:1) and gave 12 (14.2 mg,

0.0007%) as a colorless solid.
Cytotoxicity Assay. Cells ((12) 104 cells/well) were seeded in
96-well plates at 37 C. After 24 h incubation, cells were treated with
0.1% DMSO (as solvent-control cells) and the compounds by adding
10 L/well of each concentration in triplicate to obtain a nal
concentration of 0.02520 g/well at 37 C. After incubation for 1 h
(starting cells) and 72 h, cell growth was determined using the
sulforhodamine B (SRB) assay.14 The percentage of cell viability was
calculated as [(OD treated cells on day 3 OD starting cells)/(OD
control on day 3 OD starting cells)] 100. The 50% growth
inhibitory concentrations (IC50) of the compounds on the CCA cell
lines were calculated from the doseresponse curves. The IC50 values
were calculated through computation using the CalcuSyn software.
Pteroloterin A (1): colorless needles (MeOH); mp 148150 C;
[]23D +51 (c 0.001, CHCl3); UV (CHCl3) max (log ) 255 (4.10)
nm; IR (neat) max 2951, 1726, 1435, 1247, 1141, 1016, 765 cm1; 1H
NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table
1; HRESIMS m/z 393.1664 [M + Na]+ (calcd for C22H26O5Na,
393.1678).
1-Acetoxytaepeenin C (2): colorless needles (MeOH); mp 179
183 C; []22D 60 (c 0.001, CHCl3); UV (MeOH) max (log ) 208
(4.33), 252 (3.92) nm; IR (neat) max 3447, 2924, 2854, 1731, 1668,
1245, 1140, 1032, 766 cm1; 1H NMR (400 MHz) and 13C NMR
(100 MHz) data (CDCl3), see Table 1; HRESIMS m/z 423.1784 [M
+ Na]+ (calcd for C23H28O6Na, 423.1784).
Pteroloterin B (3): colorless, amorphous powder; []22D +24 (c
0.001, CHCl3); UV (CHCl3) max (log ) 253 (3.71) nm; IR (neat)
max 2951, 1724, 1436, 1249, 1225, 1141, 754 cm1; 1H NMR (400
MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 1;
HRESIMS m/z 395.1888 [M + Na]+ (calcd for C22H28O5Na,
395.1834).
Pteroloterin C (4): colorless, amorphous powder; []23D 32 (c
0.001, CHCl3); UV (CHCl3) max (log ) 244 (3.89) nm; IR (neat)
max 2932, 1723, 1667, 1437, 1380, 1247, 1177, 1142 cm1; 1H NMR
(400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 2;
HRESIMS m/z 327.1930 [M + Na]+ (calcd for C19H28O3Na,
327.1936).
8a-Hydroxycadinenal (5): colorless oil; []23D 121 (c 0.001,
CHCl3); UV (CHCl3) max (log ) 243 (3.66) nm; IR (neat) max
3418, 2925, 1691, 1455, 1373, 1242, 1003 cm1; 1H NMR (400 MHz)
and 13C NMR (100 MHz) data (CDCl3), see Table 2; HRESIMS m/z
237.1834 [M + H]+ (calcd for C15H24O2Na, 237.1855).

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ASSOCIATED CONTENT

S Supporting Information
*

H and 13C NMR spectra for 15 are available free of charge

via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Tel: +66-4320-2222-41, ext 12243. Fax: +66-4320-2373. Email: chayen@kku.ac.th (C. Yenjai).
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We thank the Royal Golden Jubilee Scholarship (PHD/0020/
2556) and the National Research University Project of
Thailand through the Advanced Functional Materials Cluster
of Khon Kaen University for nancial support. The Center of
Excellence for Innovation in Chemistry (PERCH-CIC), Oce
of the Higher Education Commission, Ministry of Education, is
gratefully acknowledged.
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