Phytochemistry 122 (2016) 286–293

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Cassane diterpenes from the seed kernels of Caesalpinia sappan

Hai Xuan Nguyen a, Nhan Trung Nguyen a, Phu Hoang Dang a, Phuoc Thi Ho a, Mai Thanh Thi Nguyen a,⇑,
Mao Van Can b, Dya Fita Dibwe c, Jun-ya Ueda c, Suresh Awale c,⇑
a
Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, Viet Nam
b
Vietnam Military Medical University, Ha Noi, Viet Nam
c
Division of Natural Drug Discovery, Department of Translational Research, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Eight structurally diverse cassane diterpenes named tomocins A–H were isolated from the seed kernels of
Received 23 June 2015 Vietnamese Caesalpinia sappan Linn. Their structures were determined by extensive NMR and CD spectro-
Received in revised form 18 December 2015 scopic analysis. Among the isolated compounds, tomocin A, phanginin A, F, and H exhibited mild prefer-
Accepted 31 December 2015
ential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived condition
Available online 5 January 2016
without causing toxicity in normal nutrient-rich conditions.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Caesalpinia sappan
Caesalpiniaceae
Cassane diterpene
Antiausterity agent
PANC-1

1. Introduction 2014). As a part of an ongoing screening to discover unique anti-

Caesalpinia sappan Linn. (Caesalpiniaceae) is a tree commonly
distributed in Vietnam, Myanmar, Thailand, India and other South-
east Asian countries. In Ayurveda, the heartwood is often pre-
scribed for balancing ‘‘Pitta dosha” (a type of body constitution),
which controls digestion, metabolism and energy production,
including burning sensation, ulcers, diarrhea and diabetes
(Badami et al., 2004). In Vietnam, the tree is known as ‘‘To Moc”,
and the decoction of its heartwood is used to treat rheumatism
and inflammatory diseases and as an emmenagogue agent
(Nguyen, 1999). The heartwood contains brazilins, dibenzoxocins,
homoisoflavonoids, and chalcones as the major bioactive con-
stituents (Zhao et al., 2013; Lai et al., 2011; Kim et al., 2012;
Nguyen et al., 2005), which possessed xanthine oxidase inhibitory
(Nguyen et al., 2004, 2005), antioxidant (Badami et al., 2003), anti-
inflammatory (Cuong et al., 2012), antidiabetic (Moon et al., 1990),
hepatoprotective (Srilakshmi et al., 2010) and vasorelaxation (Xie
et al., 2000) activities.
The seed kernel of this plant is a rich source of cassane diter-
penes. Several cassane diterpenes having either antibacterial activ-
ity or cytotoxicity have been reported (Wu et al., 2014; Yodsaoue
et al., 2008; Kinoshita et al., 2005; Zhang et al., 2013; Ma et al.,
⇑ Corresponding authors.
E-mail addresses: nttmai@hcmus.edu.vn (M.T.T. Nguyen),
u-toyama.ac.jp (S. Awale).
austerity strategy based anticancer agents (Ueda et al., 2014;
Nguyen et al., 2014, 2013; Awale et al., 2012a,b; Awale et al.,
2006a,b, 2008), the CH2Cl2 extract of the seed kernel of C. sappan
showed preferential cytotoxicity in nutrient-deprived medium
(NDM) against PANC-1 human pancreatic cancer cells. Phytochem-
ical investigation of this extract yielded twelve compounds, includ-
ing eight structurally diverse cassane diterpenes named tomocins
A–H (1–8, Fig. 1). Reported herein are the structures of these com-
pounds, together with their preferential cytotoxic activity against
the PANC-1 human pancreatic cancer cell line.
2. Results and discussion
Tomocin A (1) was isolated as a colorless amorphous solid. Its
molecular formula was determined by HR-ESI-MS to be C22H28O8.
Its IR spectrum showed the absorptions of hydroxyl (3429 cm 1),
ester carbonyl (1740 cm 1), a,b-unsaturated ester carbonyl
(1745 cm 1), and aldehyde carbonyl (1705 cm 1) groups. The 1H
NMR spectrum (Table 1) displayed signals due to an aldehyde (dH
9.86, H-20), an olefinic proton (dH 5.74, H-15), two O-methyls (dH
3.72, 18-OCH3; 3.64, 19-OCH3) and a secondary methyl (dH 1.18,
H-17) group. The 13C NMR spectra, in combination with DEPT anal-
ysis, showed the presence of an aldehyde (dC 207.7), three ester
carbonyls (dC 174.0, 173.0, 172.6), a pair of olefinic carbons (dC
suresh@inm. 174.6, 114.4), an acetal carbon (dC 106.7), six methylenes (dC
39.6, 35.2, 33.0, 31.6, 25.6, 20.9), four methines (dC 50.7, 45.2,

http://dx.doi.org/10.1016/j.phytochem.2015.12.018
0031-9422/Ó 2016 Elsevier Ltd. All rights reserved.
 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293 287

O O
O 16 O O
HO HO
15 O
20 12 R1 R2
11 13
1 R2 H 20
20 H OCH3
O H
2
H
14
17 H H
10 8 19 O
3 4 6 7 H
H H
H 3COOC R1 H 3COOC
18 19 H
2 R1 = OH, R 2 = H H 3COOC
1R1 =COOCH 3, R2 =CHO 3 R1 = OCH3, R 2 = H
13 R1 = R 2= CH3 4 R1 = H, R 2 = OCH3 6
5 R1 = H, R 2 = OC 2H 5

O
O O O O
R3 R3
H O R1 R2 O
R2 H H H CHO H
O
H H H H

H H H H
H 3COOC H 3COOC H 3COOC H 3COOC COOCH3
R1
9 R1 = H, R 2 = OH, R 3 = H 11 12
7 R1 = CH3, R 2 = H, R 3 = H
8 R1 = CHO, R 2 = OCH3, R 3 = OH 10 R1 = OH, R 2 = H, R 3 = OH

Fig. 1. Chemical structures of compounds 1–8.

Table 1
1
H and 13C NMR spectroscopic data of tomocins A–D (1–4).

Position 1a 2b 3a 4a
dH dC dH dC dH dC dH dC
1 1.08, m 39.6 1.23, m 37.3 1.32, m 39.9 1.00, td (13.5, 5.7) 34.1
2.45, m 2.02, m 2.09, m 2.44, dd (13.0, 5.1)
2 1.61, m 20.9 1.59, m 20.8 1.63, m 22.1 1.57, m 22.1
2.26, m 2.24, m 2.32, m 2.33, m
3 1.67, m 35.2 1.91, m 35.6 1.94, m 36.9 1.86, m 37.2
2.34, d (12.7) 1.96, m 2.04, m 1.97, m
4 59.8 45.5 46.7 47.0
5 2.15, dd (12.9, 2.0) 50.7 1.55, m 44.9 1.63, m 46.6 1.75, m 48.2
6 1.66, m 25.6 1.37, m 29.0 1.28, m 24.9 1.30, m 24.2
2.49, m 1.62, m 2.05, m 1.82, m
7 1.02, dd (13.1, 3.7) 31.6 1.22, m 23.7 1.22, m 30.2 1.50, m 30.7
2.46, m 2.11, m 2.11, m 1.67, m
8 1.96, m 43.2 1.61, m 41.3 1.61, m 43.1 1.69, m 42.6
9 1.84, m 45.2 2.29, m 41.4 2.21, m 42.8 1.53, m 43.8
10 52.0 38.4 39.9 40.6
11 1.08, m 33.0 1.62, m 37.8 1.62, m 38.4 1.48, m 41.1
2.45, m 2.56, d (10.5) 2.54, dd (12.0, 2.5) 2.52, m
12 106.7 106.3 107.5 107.7
13 174.6 174.0 176.0 175.5
14 3.03, m 38.0 2.89, m 37.2 2.94, m 38.4 3.00, m 38.0
15 5.74, s 114.4 5.66, s 113.2 5.74, s 113.8 5.72, s 113.8
16 173.0 171.9 173.3 173.4
17 1.18, d (7.3) 12.5 1.13, d (7.0) 11.7 1.14, d (7.3) 12.2 1.15, d (7.3) 13.1
18 174.0 175.5 176.9 177.0
19 172.6 3.67, m 61.3 3.64, m 62.3 3.85, dd (12.1, 0.9) 67.1
4.33, dd (11.5, 1.5) 4.13, dd (11.5, 2.0) 4.17, dd (12.1, 2.4)
20 9.86, s 207.7 4.83, s 96.6 4.30, s 104.9 4.83, s 102.7
18-OCH3 3.72, s 53.2 3.67, s 51.6 3.68, s 52.1 3.66, s 52.2
19-OCH3 3.64, s 52.4
20-OCH3 3.23, s 54.6 3.35, s 56.1
a
Measured in CD3OD.
b
Measured in CDCl3.

43.2, 38.0) a secondary methyl (dC 12.5) and two quaternary car-
bons (dC 59.8, 52.0), respectively. Excluding the signals for the
two O-methyls, compound 1 has, a total of 20 carbons in its main
framework suggesting a diterpene. Furthermore, these assign-
ments were quite close to the cassane diterpenes possessing an
a,b-unsaturated c-lactone moiety, as reported from Caesalpinia
crista (Kinoshita et al., 2005), Caesalpinia minax (Ma et al., 2012),
Bowdichia nitida (Matsuno et al., 2008), and Myrospermum frutes-
cens (Mendoza et al., 2003). The 1H–1H COSY and HSQC spectra
showed partial connectivity (bold line) of C-1–C-2–C-3, C-5–C-6–
C-7–C-8–C-9–C-11, and C-8–C-14–C-17, which were connected
further on the basis of long-range HMBC correlations (Fig. 2). The
288 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293

O O
16 O
O O
HO 12 HO O
HO
11 OH
13
20
OCH3
1 CHO O
14 1 O
8 17 10
19 19
3 7
5
O O
H 3C C18 19 C CH3 C CH3
O O O 18 O C CH3
O O

1 2 3; 4

O O
O
O O O O
HO O 11
HO
OCH3 O O
OCH 2CH3 H 3CO
20
O
O
10

H 3C H 3C
C CH3 O C CH3 O C
O HC O
C CH3 O O O
O O
6 7 8
5

Fig. 2. Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) observed for 1–8.

O O O
O
H
16
HO H H 3CO H H OCH3H
O H H O O O
H HH O O
OMe CHO O H
15
H H H H H H
O H H H 20 15 H
H H 11
8
H
20 15
H 11 H H
19 H 11
8 19
H
9 OH 14 H 8 OH
9 OH H
9 OH 14 H
Me H 3
6
3 6 Me
MeO 2C H Me Me MeO 2C H
H
17
MeO 2C H MeO 2C H H
H H

1 2 3 4

O O O
H OCH3 O OCH3
OCH 2CH 3 O O 15 H H O O 15 H OH O H
H H H H 20
H H
H
H 19 HH H
20
O O H
H 20
HH
H
11
H Me H CHO H H
H 8 H H
19 H H
9 14
H 14
OH
H 3
6
OH
5 Me Me Me
H MeO 2C H MeO 2C H MeO 2C H
Me H H H
MeO 2C H H
6 8
7
5

Fig. 3. Key NOESY correlations (solid arrows) observed for 1–8.

HMBC correlations from the aldehyde proton at dH 9.86 to dC 39.6 (Fig. 3) H-20/-OCH3 (C-19), H-20/H-8, H-20/H-11b and H-5/H-9,
(C-1), dC 50.7 (C-5), dC 45.2 (C-9) indicated the location of aldehyde H-9/H3-17 suggested that rings A and B were trans-fused and
group at C-20. The HMBC correlations from dH 3.72 to an ester car- adopted a chair conformation with H-20, H-8 in b-axial and H-5,
bonyl at dC 174.0 (C-18) and dH 3.64 to an ester carbonyl at dC 172.6 as well as H-9 and H3-17 in a-axial orientation. This was further
(C-19), and from dH 2.15 (H-5) to C-18 and C-19, suggested the supported by a large trans-diaxial coupling constant of H-3a
location of the two O-methyl groups at C-18 and C-19, respectively. (J = 12.7 Hz), H-5 (J = 12.9 Hz), H-7a (J = 13.1 Hz). The NOESY cor-
The HMBC correlations of protons H2-1, H-5, H-8 and H-9 estab- relations between H-14/H-15, H-14/H-8 suggested a b-equatorial
lished rings A and B while HMBC correlations of protons H-11, orientation of H-14, and that the ring C adopts a boat conforma-
H-14, H3-17, and H-15 furnished the construction of ring C tion. Thus, the relative configuration at C-5, C-8 C-9, C-12, and C-
attached to an a,b-unsaturated c-lactone moiety (ring D) at C-12 14 were established. The CD of the a,b-unsaturated c-lactone rings
and C-13 (Fig. 2), leading to a complete determination of structure with a chiral c-carbon showed Cotton effects in the region of 200–
1. The relative configuration was assigned on the basis of NOESY 235 nm corresponding to the p ? p⁄ transition and 235–270 nm cor-
correlations and coupling constant data. The NOESY correlations responding to the n ? p⁄ transition (Uchida and Kuriyama, 1974;
 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293
Gawronski et al., 1996; Beecham, 1972; Lee et al., 2008). As a n ?
p⁄ Cotton effect may be easily influenced by external asymmetry,
the p ? p⁄ Cotton effect is decisive in order to assign the absolute
configuration of the a,b-unsaturated c-lactone ring (Chen et al.,
2014). Compound 1 showed a negative p ? p⁄ Cotton effect at
219 nm (Fig. 4) indicating the configuration of C-12 as R. So, the
absolute configuration of tomocin A was established to as
5R,8S,9S,10S,12R,14R (1).
Tomocin B (2) was isolated as a colorless amorphous solid and
its molecular formula C21H28O7 was determined by HR-ESI-MS.
The 1H and 13C NMR (Table 1) spectroscopic data closely resembled
those of 1 and showed the presence of signals characteristic of an
a,b-unsaturated butenolide ring (dC-12 106.3, dC-13 174.0, dC-15
113.2, dC-16 171.9), and a secondary methyl (dH-17 1.13, dC-17
11.7) group. However, an obvious difference was observed due to
a disappearance of the signal of the aldehyde group at C-20 (dH
9.86, dC 207.7) and one of the methyl ester groups in 1. Compound
2 instead showed the presence of an oxymethylene (dH 4.33, 3.67,
dC 61.3) and a hemiacetal group (dH 4.83, dC 96.6). These two sig-
nals were assigned to be for C-19 and C-20 based on long-range
HMBC correlations (Fig. 2). Further, the correlations between the
oxymethylene and the acetal group indicated the presence of an
acetal linkage between C-19 and C-20. The relative configuration
of 2 was determined by NOESY and the coupling constant data
was the same as in 1 for rings A, B and C. As for the bridged ring,
NOESY correlations (Fig. 3) between H-20/H-6b, H-20/H-8 and
H-20/H-11b suggested a b orientation for H-20. Compound 2 showed
a negative p ? p⁄ Cotton effect at 225 nm (Fig. 4), indicating an R
absolute configuration at C-12 (Uchida and Kuriyama, 1974;
Gawronski et al., 1996). Accordingly, the structure of tomocin B
was established as 4R,5R,8S,9S,10S,12R,20S (2).
Tomocin C (3) and D (4) were both isolated as colorless amor-
phous solids and they possessed the same molecular formula
C22H30O7 as determined by HR-ESI-MS. The 1H and 13C NMR spec-
troscopic data (Table 1) were similar to each other and tomocin B
(2), except for the presence of a signal due to an O-methyl group in
both 3 and 4. The location of this O-methyl group was determined
to be at C-20 based on HMBC (Fig. 2) correlations between the
O-methyl protons [dH 3.68 (3), dH 3.66 (4)] and C-20 [dC 104.9
(3), dC 102.7 (4)]. Therefore, 3 and 4 must have a same overall
structure with a difference in configuration at C-20. In tomocin C
(3), the NOESY correlations (Fig. 3) between H-20/H-6b, H-20/
H-8 and H-20/H-11b suggested a b orientation of H-20. While, in
tomocin D (4), the O-methyl group showed NOESY correlations
(Fig. 3) to H-6b, H-8 and H-11b suggesting the b orientation of
O-methyl group i.e. H-20 in 4 is a oriented. Both 3 and 4 displayed
Fig. 4. Circular dichroism (CD) spectra of 1–8 in EtOH.
289
negative Cotton effects corresponding to p ? p⁄ transitions (Fig. 4)
indicating the absolute configuration at C-12 as R in both com-
pounds (Uchida and Kuriyama, 1974; Gawronski et al., 1996).
The 1H and 13C NMR spectroscopic data of tomocin E (5) were
also similar to tomocin D (4) with the only difference being due
to the presence of an O-ethyl signal in 5 instead of an O-methyl
group in 4 as determined by 1H and 13C NMR spectroscopic analy-
sis (Table 2). The presence of the O-ethyl group at C-20 was estab-
lished based on HMBC data analysis (Fig. 2). As in 4, the O-ethyl
group showed NOESY correlations (Fig. 3) with H-6b, H-8, and
H-11b, suggesting the b orientation of ethoxy group substituent.
The CD spectrum of 5 also showed negative p ? p⁄ Cotton effect
(Fig. 4) suggesting absolute chirality at C-12 as R (Uchida and
Kuriyama, 1974; Gawronski et al., 1996). So, the structure of 5
was concluded as 4R,5R,8S,9S,10S,12R,20R (5). Laboratory solvents
such as CH2Cl2, in general, contain a trace amount of EtOH (0.3–1%)
as a stabilizer. Therefore, although, ethanol has not been used dur-
ing extraction and throughout the isolation process, possibility 5 to
be an artifact cannot be ignored.
Tomocin F (6) was isolated as colorless amorphous solid. Its
molecular formula was assigned to be C22H28O6 from the HR-ESI-
MS. Its 1H and 13C NMR data (Table 2) were those of ring C and
D of neocaesalpin V (Ma et al., 2012) and those of rings A and B
in tomocin D (4). In the HMBC spectrum, tomocin F (6) showed
the presence of an acetal linkage between C-19 and C-20 and sim-
ilar substituents at C-18 and C-19 (Fig. 2) as in tomocin D (4). Fur-
thermore, in the NOESY spectrum (Fig. 3), the correlations between
the O-methyl group at C-20 and H-6b, H-8 and H-11b indicated
their proximity as in tomocin D (4). Tomocin F (6) displayed a neg-
ative Cotton effect at 254 nm (Fig. 3). Based on biogenetic consid-
eration of isolated related analogs, the absolute chirality is
assigned as 5R,8S,9R,10S,14R. Accordingly, the structural formula
of tomocin F (6) was established as shown.
Tomocin G (7) was isolated as a colorless amorphous solid hav-
ing the molecular formula C21H28O5. As in tomocins B–F (2–6), the
1
H and 13C NMR spectroscopic data (Table 2) showed signals due to
the presence of an a,b-unsaturated c-lactone moiety. An obvious
difference was observed, however, due to presence of a tertiary
methyl group (dH 0.97, H-19; dC 15.8, C-19), and the presence of
two oxymethine protons (dH 4.02, H-11; dH 5.04, H-12) when com-
pared to 2–5. The connectivity between C-1–C-2–C-3, C-5–C-6–C-
7–C-8–C-9–C-11 and C-8–C-14–C-17 was established by 1H–1H
COSY, HSQC and long-range correlations in the HMBC spectrum
(Fig. 2). The oxymethine proton at dH 5.04 (H-12) gave HMBC cor-
relations to C-16 (dC 173.3), C-15 (dC 114.6) and C-13 (dC 173.4) of
the a,b-unsaturated butenolide ring as well as to the oxymethine
290 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293

Table 2
1
H and 13C NMR spectroscopic data of tomocins E–H (5–8).

Position 5a 6b 7b 8c
dH dC dH dC dH dC dH dC
1 1.65, m 37.4 1.67, m 36.4 1.07, m 35.6 1.16, m 34.5
1.93, m 1.93, m 1.85, m 1.56, m
2 1.57, m 22.1 1.66, m 21.1 1.62, m 18.2 1.63, m 18.9
2.34, m 2.37, m 1.71, m 1.58, m
3 1.28, m 39.2 1.27, m 37.9 1.63, m 36.8 1.49, m 28.8
2.25, m 2.27, m 1.80, m 2.10, m
4 50.9 49.8 47.3 59.9
5 1.70, m 45.4 1.77, dd (12.5, 3.5) 44.1 2.11, dd (11.0, 2.0) 46.7 1.51, m 46.6
6 1.29, m 31.1 1.30, m 29.5 1.29, m 23.9 1.47, m 28.8
1.57, m 1.55, m 2.14, m
7 1.94, m 25.1 1.99, m 23.9 1.32, m 28.8 1.05, m 27.3
2.14, m 2.12, m 1.64, m
8 1.58, m 43.5 1.86, m 39.6 1.99, m 34.7 2.66, m 31.0
9 1.58, m 42.6 2.06, dd (11.6, 2.7) 44.1 1.43, m 49.5 1.51, m 46.1
10 36.4 49.8 45.9 47.3
11 1.62, m 39.2 5.84, s 109.6 4.02, dd (5.5, 3.5) 80.2 4.03, d (7.0) 81.0
2.50, dd (12.0, 2.5)
12 107.4 150.5 5.04, dd (3.5, 2.0) 85.8 104.7
13 175.3 161.5 173.4 172.7
14 2.99, m 38.3 2.86, m 34.0 2.90, m 33.8 2.75, m 32.1
15 5.74, s 114.1 5.76, s 110.6 5.78, s 114.6 6.05, s 116.7
16 173.2 170.0 173.3 170.2
17 1.15, d (7.0) 12.6 1.05, d (7.3) 13.9 1.15, d (7.0) 12.5 1.01, d (7.0) 11.4
18 176.4 174.6 178.2 173.0
19 3.42, m 63.2 3.96, dd (11.5, 2.7) 62.1 0.97, s 15.8 9.71, s 199.3
3.99, dd (11.5, 3.0) 3.36, dd (11.5, 1.2)
20 4.85, s 102.3 4.77, s 102.6 3.90, s 69.7 4.44, s 107.7
18-OCH3 3.68, s 52.1 3.69, s 51.7 3.68, s 51.9 3.68, s 52.3
20-OCH3 3.32, s 55.6 3.22, s 54.4
20-OCH2CH3 3.45, m 64.7
3.72, m
20-OCH2CH3 1.15, t (7.0) 15.5

a
Measured in CD3OD.
b
Measured in CDCl3.
c
Measured in DMSO-d6.

carbon C-11 (dC 80.2) and C-9 (dC 49.5) (Fig. 2). On the other hand,
the oxymethine proton at dH 4.02 (H-11) gave HMBC correlations
to C-20 (dC 69.7) and C-9. This evidence suggested the presence
of an ether linkage between C-20 and C-11 in 7. The tertiary methyl
group at dH 0.97 was assigned as C-19 based on its HMBC correla-
tions to C-4 (dC 47.3), C-18 (dC 178.2) and C-3 (dC 36.8) (Fig. 2).
Therefore the structure of tomocin H (7) was established, and its
relative configuration was determined based on NOESY correla-
tions (Fig. 3). The NOESY between H-19/H-20 indicated that they
are b-axial oriented while the NOESY between H-9/H-17 indicated
that they are a-axial oriented, and where rings A/B adopted a chair
conformation. Contrary to tomocin A–F (1–6), tomocin G (7) dis-
played a positive p ? p⁄ Cotton effect at 234 nm (Fig. 4) indicating
a S absolute configuration at C-12 (Uchida and Kuriyama, 1974;
Gawronski et al., 1996). The structure of 7 was thus concluded as
4R,5R,8S,9S,10S,10R,11R,12S tomocin H.
Tomocin H (8) was isolated as a colorless amorphous solid. The
IR absorptions at 3430, 1740, 1743, and 1708 cm 1 indicated the
presence of hydroxyl, ester carbonyl, c-lactone, and aldehyde car-
bonyl groups, respectively. Its HR-ESI-MS gave a molecular formula
of C22H28O9. The 1H NMR (Table 2) of tomocin H (8) displayed sig-
nals due to an aldehyde (dH 9.71), two O-methyl groups (dH 3.68, dH
3.22), together with a secondary methyl, five methylene, and seven
methine moieties, including an olefinic methine (dH 6.05, H-15)
and two oxymethines (dH 4.03, H-11; dH 4.44, H-20). The 13C
NMR spectroscopic data (Table 2) showed a total of twenty-two
carbons including those for two O-methyl groups, an a,b-unsatu-
rated c-lactone moiety, an acetal (dC 104.7, C-12) and hemiacetal
(dC 107.7; C-20) carbons. As in tomocin G (7), 8 also showed similar
partial connectivity between C-1–C-2–C-3, C-5–C-6–C-7–C-8–C-9–
C-11 and C-8–C-14–C-17 as established by 1H–1H COSY and HSQC
spectra (Fig. 2). In the HMBC spectrum, the aldehyde proton at dH
9.71 (H-20) gave correlations with C-4, C-3 and C-18 suggesting
its location to be at C-19. Similarly, one of the O-methyl group also
gave HMBC correlations to C-18 suggesting the methyl ester group
at C-18. The HMBC correlations from H-15 to the acetal carbon at
dC 104.7 (C-12), 170.2 (C-16), 172.7(C-13) indicated the presence
of an a,b-butenolide ring D system with a hydroxyl group at C-
12 as in 1–5. As in 7, the acetal linkage between C-20 and C-11
was established by HMBC correlations from the oxymethine proton
at d 4.03 (H-11) and vice versa. The remaining O-methyl group at d
3.22 was assigned to be located at C-20 based on the HMBC corre-
lations between d 3.22 to dC 107.7 (C-20) and vice versa. Therefore,
the structure of tomocin H (8) was established. In the NOESY spec-
trum (Fig. 3), correlations between H-19/H-20 indicated a b-axial
orientation of the aldehyde group while the NOESY between H-9/
H-17 indicated that they were a-axial oriented, and that rings
A/B adopted a chair conformation. Similarly, a NOESY correlation
between a methoxyl at C-20 and H-8 was observed suggesting
their proximity. As in tomocin G (7), tomocin H (8) also displayed
a positive p ? p⁄ Cotton effect at 224 nm (Fig. 4) indicating a S
absolute configuration at C-12 (Uchida and Kuriyama, 1974;
Gawronski et al., 1996). Accordingly, the absolute
stereochemistry of structure of 8 was concluded as
4R,5R,8S,9S,10R,10R,11R,12S,20S.
The five known compounds phanginin A (9), phanginin F (10)
and phanginin H (11), phanginin K (12) and neocasalpinin H (13)
were identified by comparison of their spectroscopic data with
 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293
Fig. 5. Preferential cytotoxicity and morphological change induced by tomocin A (1) treatment for 24 h against PANC-1 cells.
those published in literature (Yodsaoue et al., 2008; Kinoshita
et al., 2005).
The isolated compounds were tested for their cytotoxic activity
against a human pancreatic PANC-1 cancer cell line in normal
nutrient-rich medium (DMEM) and nutrient-deprived medium
(NDM), using an antiausterity strategy (Awale et al., 2006a,b).
Compounds possessing preferential cytotoxicity in NDM without
toxicity in DMEM are considered as antiausterity agents. The pref-
erential cytotoxicity is represented as PC50 value, the concentration
at which 50% of the cells were preferentially killed in NDM without
toxicity at nutrient-rich medium. Among the test compounds, 1
(PC50, 58 lM), 9 (PC50, 67 lM), 10 (PC50, 51 lM) and 11 (PC50,
75 lM) exhibited mild preferential cytotoxicity against PANC-1
cell line. Arctigenin, a positive control (Awale et al., 2006) in this
study displayed the most potent preferential cytotoxicity (PC50,
0.8 lM). Further, ethidium bromide and acridine orange (EB/AO)
staining assay was performed to see the morphological change of
PANC-1 cells induced by tomocin A (1) (Shakya et al., 2014). AO
is a cell permeable dye and emits bright green fluorescence in live
cells. EB is permeable in dead cells and gives red fluorescence. As
shown in Fig. 5, cells treated with tomocin A (1) induced round
morphological alterations indicative of dying cells (weak green flu-
orescence) and increase in dead cell populations (red fluorescence)
of PANC-1 cells in a concentration-dependent manner.
3. Conclusion
In the present study, twelve secondary metabolites including
eight new structurally diverse cassane diterpenes were isolated
from the seed kernels of the Vietnamese medicinal plant C. sappan.
Their structures were characterized by extensive NMR and CD
spectroscopic analysis. Among the new compounds, tomocin A
(1), phanginin A, F, and H (9, 10 and 11) exhibited mild preferential
cytotoxicity against PANC-1 human pancreatic cancer cells under
nutrition-deprived condition without causing toxicity in normal
nutrient-rich conditions. It should be noted that human pancreatic
cancer cells have remarkable tolerance to nutrition starvation and
are resistant to conventional anticancer drugs in clinical use such
as paclitaxel, gemcitabine, 5-FU with PC50 > 200 lM (Ueda et al.,
2014). In this regard, tomocin A and its analogs may be a structural
template worth exploiting for antiausterity drug development.
291
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a JASCO DIP-140 digital
polarimeter. CD and UV measurements were carried out simulta-
neously on a JASCO J-805 spectropolarimeter. IR spectra were mea-
sured with a Shimadzu IR-408 spectrophotometer in CHCl3
solutions. NMR spectra were taken on a Bruker Avance III 500
(Bruker Biospin) with TMS as an internal standard, and chemical
shifts are expressed in d values. HR-ESI-MS measurements were
carried out on a Bruker microTOF-QII spectrometer. Column
chromatography (CC) was performed with BW-820MH Si gel (Fuji
Silisia, Kasagai, Japan). Analytical and preparative TLC was carried
out on precoated silica gel 60F254 and RP-18F254 plates (Merck, 0.25
or 0.50 mm thickness).
4.2. Plant material
The seed kernels of C. sappan were collected at the Seven-
Mountain area, An Giang province, Vietnam in February 2011.
The plant was identified by Ms. Hoang Viet (Faculty of Biology,
University of Science, Vietnam National University-Ho Chi Minh
City). A voucher specimen (AN-2215) is deposited at the Depart-
ment of Analytical Chemistry, Faculty of Chemistry, University of
Science, Vietnam National University-Ho Chi Minh City.
4.3. Extraction and isolation
Air-dried seed kernels of C. sappan L. (3.2 kg) were extracted
with CH2Cl2 (20 L, 3 h  3, under conditions of reflux), and the
combined solvent were evaporated under reduced pressure to give
a CH2Cl2 extract (200 g). The latter was subjected to silica gel CC
(120  12 cm) with an EtOAc–hexane gradient system to give 6
fractions (fr.1: 10% EtOAc eluate, 40.3 g; fr.2: 20% EtOAc, 10.5 g;
fr.3: 30% EtOAc, 15.6 g; fr.4: 40% EtOAc, 20.5 g; fr.5: 50% EtOAc,
45.3 g; fr.6: 100% EtOAc, 50.3 g), respectively.
Fraction 1 was separated by silica gel CC with EtOAc–hexane
(0–50% EtOAc) to afford four subfractions (fr.1-1, 14.6 g; fr.1-2,
5.8 g; fr.1-3, 8.2 g; fr.1-4, 10.8 g). Subfraction 1-2 was applied to
reversed-phase preparative TLC with MeOH:H2O (1:1) to give 1
292 H.X. Nguyen et al. / Phytochemistry 122 (2016) 286–293
(22.4 mg) and 13 (10.1 mg). Fraction 2 was subjected to silica gel
CC, eluted with EtOAc–hexane (0–30% EtOAc) to afford three sub-
fractions (fr.2-1, 3.8 g; fr.2-2, 450 mg; fr.2-3, 3.7 g). Subfraction 2-1
was recrystallized with EtOAc–hexane (1:1) to give 9 (500 mg),
while subfraction 2-2 was purified further on silica gel with 1%
MeOH in CHCl3 as eluant, followed by preparative TLC with
MeOH–CHCl3 (2:98, v/v) to give 2 (6.8 mg) and 3 (7.7 mg). Fraction
3 was purified further on silica gel with EtOAc–hexane (0–20%) to
afford four subfractions (fr.3-1, 2.5 g; fr.3-2, 1.8 g; fr.3-3, 4.0 g; fr.3-
4, 4.5 g). Subfraction 3-1 was subjected to further silica gel CC, with
EtOAc–hexane (10:90) as eluent, followed by preparative TLC with
EtOAc–hexane (20:80) to give 5 (7.0 mg). Subfraction 3-2 was sep-
arated by preparative TLC with MeOH–CHCl3 (1:99) to give 10
(7.0 mg). Subfraction 3-3 was applied to a preparative TLC plate
eluted with MeOH–CHCl3 (1:99), followed by preparative TLC with
acetone–hexane (40:60) to give 6 (5.0 mg) and 7 (10.5 mg). Frac-
tion 4 was subjected to silica gel CC, eluted with MeOH–CHCl3
(0–10%) to afford three subfractions (fr.4-1, 2.7 g; fr.4-2, 4.5 g;
fr.4-3, 5.5 g). Subfraction 4-1 was subjected to additional CC with
a MeOH–CHCl3 (1:99) and then followed by preparative TLC with
MeOH–CHCl3 (2:98) to give 8 (9.0 mg). Subfraction 4-2 was puri-
fied using normal-phase preparative TLC in an acetone–hexane
(1:10) solvent system to give 4 (20 mg). Fraction 5 was applied
to a silica gel CC with MeOH–CHCl3 (0–10%) gradient system, and
then followed by reversed-phase preparative TLC with MeOH–
H2O (1:1) to give 11 (5.0 mg) and 12 (7.7 mg).
4.3.1. Tomocin A (1)
Colorless amorphous solid; [a]25
D 95 (c 0.01, EtOH); CD (c
2.38  10 4 M, EtOH) [h]204 + 3096, [h]219 13882; UV kmax
220 nm (EtOH); IR mmax (CHCl3) 3429, 2930, 1745, 1740, 1705,
755 cm 1; for 1H and 13C NMR (CD3OD) spectroscopic data, see
Table 1; HR-ESI-MS m/z 443.1672 [calcd for C22H28O8Na (M+Na)+,
443.1682].
4.3.2. Tomocin B (2)
Colorless amorphous solid; [a]25
D 83 (c 0.01, EtOH); CD (c
2.55  10 4 M, EtOH) [h]205 + 29920, [h]225 36738; UV kmax
220 nm (EtOH); IR mmax (CHCl3) 3395, 1745, 1743, 752 cm 1; for
1
H and 13C NMR (CDCl3) spectroscopic data, see Table 1; HR-ESI-
MS m/z 415.1734 [calcd for C21H28O7Na (M+Na)+, 415.1733].
4.3.3. Tomocin C (3)
Colorless amorphous solid; [a]25
D 112 (c 0.01, EtOH); CD (c
2.46  10 4 M, EtOH) [h]204 + 18236, [h]226 38620; UV kmax
220 nm (EtOH); IR mmax (CHCl3) 3404, 1745, 1743, 754 cm 1; for
1
H and 13C NMR (CD3OD) spectroscopic data, see Table 1; HR-ESI-
MS m/z 429.1884 [calcd for C22H30O7Na (M+Na)+, 429.1889].
4.3.4. Tomocin D (4)
Colorless amorphous solid; [a]25
D 265 (c 0.01, EtOH); CD (c
2.00  10 4 M, EtOH) [h]204 + 6040, [h]224 13537; UV kmax
218 nm (EtOH); IR mmax (CHCl3) 3403, 1745, 1743, 757 cm 1; for
1
H and 13C NMR (CD3OD) spectroscopic data, see Table 1; HR-ESI-
MS m/z 429.1887 [calcd for C22H30O7Na (M+Na)+, 429.1889].
4.3.5. Tomocin E (5)
Colorless amorphous solid; [a]25
D + 42 (c 0.01, EtOH); CD (c
2.38  10 4 M, EtOH) [h]204 + 21418, [h]224 28072; UV kmax
220 nm (EtOH); IR mmax (CHCl3) 3440, 1745, 1743, 760 cm 1; for
1 2012. Phenolic compounds from Caesalpinia sappan heartwood and their anti-
H and 13C NMR (CD3OD) spectroscopic data, see Table 2; HR-ESI-
MS m/z 443.2041 [calcd for C23H32O7Na (M+Na)+, 443.2046].
4.3.6. Tomocin F (6)
Colorless amorphous solid; [a]25 D + 59 (c 0.01, EtOH); CD (c
2.00  10 4 M, EtOH) [h]204 + 12361, [h]258 35933, [h]268 38088,
[h]276 34177; UV kmax 276 nm (EtOH); IR mmax (CHCl3) 1743,
1740, 754 cm 1; for 1H and 13C NMR (CDCl3) spectroscopic data,
see Table 2; HR-ESI-MS m/z 389.1965 [calcd for C22H29O6 (M+H)+,
389.1964].
4.3.7. Tomocin G (7)
Colorless amorphous solid; [a]25
D + 423 (c 0.01, EtOH); CD (c
2.77  10 4 M, EtOH) [h]207 10097, [h]234 + 58619; UV kmax
234 nm (EtOH); IR mmax (CHCl3) 1747, 1740, 757 cm 1; for 1H and
13
C NMR (CDCl3) spectroscopic data, see Table 2; HR-ESI-MS m/z
383.1818 [calcd for C21H28O5Na (M+Na)+, 383.1834].
4.3.8. Tomocin H (8)
Colorless amorphous solid; [a]25
D + 248 (c 0.01, EtOH); CD (c
2.38  10 4 M, EtOH) [h]205 7652, [h]224 + 20995; UV kmax
220 nm (EtOH); IR mmax (CHCl3) 3430, 2930, 1743, 1740, 1708,
755 cm 1; For 1H and 13C NMR (DMSO-d6) spectroscopic data, see
Table 2; HR-ESI-MS m/z 421.1855 [calcd for C22H29O8 (M+H)+,
421.1862].
Acknowledgements
This work was supported by a grant from Toyama Support Cen-
ter for Young Principal Investigators in Advanced Life Sciences, and
a Grant in Aid for Scientific Research (No. 24510314) from the
Japan Society for the Promotion of Science (JSPS), Japan to SA,
and a grant B2011-18-04TD from Vietnam National University –
Ho Chi Minh City (VNU-HCM) to MTTM.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2015.
12.018.
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