Phytochemistry Letters 4 (2011) 147–150

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Clerodane diterpenoids and a trisubstituted furan from Croton oblongifolius

Khanitha Pudhom a, Damrong Sommit b,*
a
Research Center for Bioorganic Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b
Department of Chemistry, Faculty of Science, Mahanakorn University of Technology, Bangkok 10530, Thailand

A R T I C L E I N F O A B S T R A C T

Article history: A new clerodane diterpene, 3,4,15,16-diepoxy-cleroda-13(16),14-diene-12,17-olide (1), and a new

Received 25 November 2010 trisubstituted furan, 3(30 -methoxy-50 -phenylfuran-20 -yl)propan-1-ol (2), were isolated from the bark of
Received in revised form 9 February 2011 Croton oblongifolius, together with six known compounds (3–8). The structures of these compounds were
Accepted 17 February 2011
elucidated by analysis of NMR spectroscopic and mass spectrometric data. Compounds 4–7 showed
Available online 5 March 2011
broad cytotoxicity on all cancer cell lines tested.
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Clerodane
Diterpenoid
Trisubstituted furan
Croton oblongifolius
Cytotoxicity

1. Introduction 2. Results and discussion

Plants belonging to the genus Croton (Euphorbiaceae) are well- 3,4,15,16-Diepoxy-cleroda-13(16),14-diene-12,17-olide (1), iso-
known for producing a variety of diterpenoids including pimarane, lated as colorless crystals, possessed the molecular formula
kaurane, labdane, cembrane, cleisthantane, and clerodane diter- C20H26O4 as established by HR-ESI-MS ion at m/z 353.1728
penoids, with a wide range of biological activities (Block et al., [M + Na]+ (calcd 353.1729), implying eight degrees of unsaturation.
2004; Cai et al., 1993; Garcia et al., 2006; Kuo et al., 2007; The 1H NMR spectrum (Table 1) displayed the typical signals of three
McChesney and Silveira, 1989; Pudhom et al., 2007). Particularly, tertiary methyls at dH 1.00, 1.12 and 1.19, and of a b-substituted
previous investigation on one of the plants in this genus, Croton furan ring at dH 6.39, 7.40 and 7.42. The 13C NMR (Table 1) and
oblongifolius Roxb., has led to the isolation of an array of heteronuclear single quantum coherence (HSQC) data revealed the
structurally diverse diterpenes with cytotoxicity on cancer cell presence of 20 nonequivalent carbons including one ester carbonyl,
lines (Roengsumran et al., 1999, 2001, 2002, 2004; Sommit et al., three tertiary methyls, five methylenes, seven methines (two
2003; Youngsa-ad et al., 2007). We recently also identified four oxygenated and three olefinic), and four quaternary carbons (one
furanocembranoids from the stem bark of C. oblongifolius collected olefinic). Since a carbonyl group and a furan ring of 1 accounted for
from Loei province. In the present study, we describe the isolation four out of eight units of unsaturation, the remaining four units
and characterization of a new clerodane diterpene (1) and a indicated a tetracyclic core system. The existence of a d-lactone ring
trisubstituted furan (2), together with six known diterpenoids (3– was corroborated by the strong heteronuclear multiple bond
8), from the hexane extract of the bark of C. oblongifolius collected correlations (HMBC) from H-12 (dH 5.50, dd, J = 7.2, 11.2 Hz) and
from Prachuabkhirikhan Province, as well as their cytotoxicity on H-8 (dH 2.14, m) to the ester carbonyl (C-17) at dC 172.3 (Fig. 2).
five human cancer cell lines. The structures of the known Additionally, the presence of an oxirane ring located between C-3 (dC
compounds were determined by comparison of their spectroscopic 61.8) and C-4 (dC 66.0) was confirmed by HMBC cross peaks of Me-
data with those in the literature (Bohlmann et al., 1985; 18/C-3, Me-18/C-4, and Me-19/C-4 (Fig. 2). The above NMR data
Harinantenaina et al., 2006; Heymann et al., 1994; Ngamrojana- strongly suggested that 1 was a clerodane-type diterpenoid. The
vanich et al., 2003; Rojatkar and Nagasampagi, 1994). NMR data of 1 were closely related to those of ravidin A (Qin and Li,
2004), except for the presence of a methylene group at C-6 instead of
a ketone carbonyl in ravidin A. This was clearly verified by the
homonuclear proton-proton spin system of CH2-6–CH2-7–CH-8
fragment, observed in the 1H–1H correlation spectroscopy (COSY)
* Corresponding author. Tel.: +66 2 988 3655; fax: +66 2 988 3655.
spectrum. Moreover, the connectivity of this fragment to C-5, C-9
E-mail address: [email protected] (D. Sommit). and C-17 was established by HMBC correlations of Me-19/C-6,

1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.02.004
148 [()TD$FIG]
K. Pudhom, D. Sommit / Phytochemistry Letters 4 (2011) 147–150

Table 1
1 13
NMR spectroscopic data (400 MHz for H and 100 MHz for C, CDCl3) for
compounds 1 and 2.

Position 1 2

dH, mult. (J in Hz) dC, mult dH, mult. (J in Hz) dC, mult
1 1.29, dd (4.0, 10.4) 15.5, CH2 4.35, t (6.8) 64.3, CH2
2 1.70, m 28.2, CH2 2.11, q (6.8, 14.8) 30.5, CH2
2.18, m
3 2.93, br s 61.8, CH 2.72, t (7.6) 32.5, CH2
4 66.0, qC
5 37.5, qC
6 1.49, dd (5.2, 12.8) 36.0, CH2
1.84, dt (2.4, 12.8)
7 1.73, dd (2.4, 12.8) 19.2, CH2 Fig. 2. 1H–1H COSY and key HMBC correlations for compounds 1 and 2.
2.01, m
8 2.14, m 51.1, CH
9 36.7, qC
10 0.94, dd (2.8, 10.4) 54.1, CH Me-20/C-8, and H-8/C-17. The single-crystal X-ray diffraction
11 1.57, d (11.2) 44.0, CH2 analysis of 1 (Fig. 3) confirmed its planar structure and allowed
2.30, dd (7.2, 14.8) the determination of its relative configuration.
12 5.50, dd (7.2, 11.2) 71.9, CH
3(30 -Methoxy-50 -phenylfuran-20 -yl)propan-1-ol (2) was
13 126.0, qC
14 6.39, br s 108.5, CH obtained as a colorless gum and its molecular formula was
15 7.40, br s 143.8, CH determined as C14H16O3 by HR-ESI-MS (m/z 255.0994 [M + Na]+,
16 7.42, br s 139.3, CH calcd 255.0997), implying seven degrees of unsaturation. The 1H
17 172.3, qC NMR spectrum (Table 1) showed the signals of a methoxy group at
18 1.19, s 19.5, CH3
19 1.12, s 17.1, CH3
dH 3.86, and a monosubstituted benzene ring at dH 7.45 (2H, t,
20 1.00, s 14.7, CH3 J = 6.8 Hz), 7.57 (1H, t, J = 6.8 Hz), and 8.05 (2H, d, J = 6.8 Hz).
10 Another characteristic signal at dH 6.43 (s) accounting for a furanyl
20 132.3, qC ring was suggestive of the existence of an additional aromatic
30 146.9, qC
moiety in the molecule. Analysis of the 13C NMR spectrum and its
40 6.43, br s 104.9, CH
50 166.6, qC
2D NMR data (1H–1H COSY, HSQC, and HMBC) revealed the
100 130.3, qC presence of three aromatic carbon resonances for a benzene ring,
200 , 600 8.05, d (6.8) 129.5, CH four aromatic carbons for a trisubstituted furan ring (dC 104.9 CH,
300 , 500 7.45, t (6.8) 128.4, CH 132.3 qC, 146.9 qC, and 166.6 qC), and a methoxy group (dH 3.86
400 7.57, t (6.8) 133.0, qC
and dC 56.2). The existence of an n-propanol (-CH2-CH2-CH2-OH)
3-OMe 3.86, s 56.2, CH3
fragment was deduced by 1H–1H COSY correlation (Fig. 2). On the
basis of HMBC data, the cross-peak observed from H2-3 to C-20 and
from the methoxy protons to C-30 allowed the n-propyl chain and
methoxy group to be connected to C-20 and C-30 of the furan ring,
respectively. Therefore, the structure of 2 was established to be a
2,3,5-trisubstituted furan as shown in Fig. 1.

[()TD$FIG]

Fig. 1. Structures of the isolated compounds 1–8.

[()TD$FIG] K. Pudhom, D. Sommit / Phytochemistry Letters 4 (2011) 147–150 149

spectrometer at 400 MHz for 1H NMR and at 100 MHz for 13C NMR
using TMS (tetramethylsilane) as the internal standard. HRESIMS
spectra were obtained with a Bruker micrOTOF.

3.2. Plant material

The bark of C. oblongifolius was collected from Prachuabkhiri-

khan Province, Thailand, in April 2010. Plant materials were
identified by Royal Forest Department, Bangkok, Thailand. A
voucher specimen (BKF 084729) was deposited at the Forest
Herbarium, Royal Forest Department, Bangkok, Thailand.

3.3. Extraction and isolation

Air-dried and powdered bark of C. oblongifolius (1.5 kg) was

extracted with MeOH (5 L  3, each for two days) at room
temperature. Extracts were pooled and the solvent was removed
under reduced pressure. The combined MeOH crude extract was
then suspended in H2O (500 mL) and partitioned with hexane
(500 mL  3) and EtOAc (500 mL  3) to afford the hexane (35.2 g)
and EtOAc (43.5 g) crude extracts. The hexane extract was
chromatographed on a silica gel column eluted with a gradient
of acetone–hexane (from 1:15 to 1:1) to yield ten fractions (I–X).
Fraction I was subjected to column chromatography over silica gel
eluting with acetone–hexane (1:19) to give 4 (25.0 mg) and 6
(53.2 mg). Fraction III was chromatographed on a silica gel column
using acetone–hexane (1:19) to afford 8 (8.5 mg). Fraction V was
further purified by a silica gel column (acetone–hexane, 1:4) to
Fig. 3. ORTEP diagram for compound 1.
yield 3 (22.4 mg), whereas purification of fraction VII by flash
column chromatography, eluted with acetone–hexane (1:4), gave
Compounds 1–7 were tested in vitro for cytotoxic activity, using 1 (62.2 mg). Fraction VIII was subjected to column chromatogra-
the MTT colorimetric method (Carmichael et al., 1987; Twenty- phy over silica gel with a mixture of MeOH–CH2Cl2 (1:98) to yield 5
mann and Luscombe, 1987), against the five human cancer cell (8.5 mg). Compounds 2 (12.1 mg) and 7 (5.6 mg) were obtained
lines, HEP-G2, SW-620, CHAGO, KATO-3 and BT-474 in tissue from fraction IX after separation by flash column chromatography
culture (Table 2). Compounds 4–7 showed broad cytotoxicity on eluting with MeOH–CH2Cl2 (1:98).
cancer cell lines tested, while compound 3 displayed only weak
activity on KATO-3 cell lines with IC50 value of 9.45 mg/mL. 3.4. 3,4,15,16-Diepoxy-cleroda-13(16),14-diene-12,17-olide (1)
Unfortunately, compounds 1 and 2 did not exhibit any detectable
cytotoxicity against any of the five cell lines tested at the initial Colorless crystals: mp 132–134 8C; [a]D25 + 12.0 (c 0.1, MeOH);
screening dose of 1 mg/mL. Interestingly, the only difference UV (MeOH) lmax (log e) 282 (2.47) nm; IR (KBr) nmax 3152, 2960,
between the inactive compound 1 and other clerodane diterpenes 1726, 1514, 1436, 1226, 1152, 1082 and 1008 cm1; 1H and 13C
isolated is the presence of the lactonization of C-17 to C-12 in 1. NMR (CDCl3) see Table 1; HR-ESI-MS m/z 353.1729 [M + Na]+
Therefore, this implied that the flexible side chain at C-12 might (calcd for C20H26O4Na, 353.1728).
play an important role for their cytotoxicity.
3.5. X-ray crystallographic analysis of 3,4,15,16-diepoxy-cleroda-
3. Experimental 13(16),14-diene-12,17-olide (1)

3.1. General experimental procedures Crystal data: colorless crystal (MeOH); C20H26O4, Mr = 330.41,
monoclinic, P212121, a = 10.677(2) Å, b = 7.5368(10) Å,
Melting points were measured with a Fisher-Johns melting c = 11.614(2) Å, Z = 2 and V = 852.7(3) Å3, Mo Ka radiation,
point apparatus. Optical rotations were measured on a PerkinEl- l = 0.71073 Å. The intensity data were collected at 293 K to a
mer 341 polarimeter. UV spectra were recorded on a CARY 50 maximum 2u value of 56.568. Of the 5126 reflections collected,
Probe UV–vis spectrophotometer. IR spectra were recorded on a 3071 were unique (Rint = 0.0226). The crystal structure was solved
PerkinElmer Model 1760X Fourier Transform Infrared Spectro- by direct methods and using the SHELXS97 program (Sheldrick,
photometer. NMR spectra were recorded on a Bruker AV400 1997a,b). Refinements were made by full-matrix least squares on
all F2 data using SHELXL97 to final R values [I > 2((I)] of
R1 = 0.0651, wR2 ¼ 0:1817 and Goodness of fit on F2 = 1.035. All
Table 2
Cytotoxic data for compounds 3—7.
non-hydrogen atoms were anisotropically refined. All hydrogen
atoms were added at calculated positions and refined using a rigid
Compound IC50 (mg/mL)/cell line model. Crystallographic data for 1 have been deposited with the
BT-474 KATO-3 CHAGO SW-620 Hep-G2 Cambridge Crystallographic Data Center (deposition number CCDC
3 >10 9.45 >10 >10 >10 785563). Copies of the data can be obtained, free of charge, via
4 6.18 7.22 9.18 4.81 8.23 www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_re-
5 4.77 6.71 7.83 5.20 7.59 [email protected], or by contacting The Cambridge Crystallo-
6 3.26 6.78 6.67 4.44 6.37
graphic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK:
7 4.07 3.56 6.02 4.16 6.58
fax: +44 1223 336033.
150 K. Pudhom, D. Sommit / Phytochemistry Letters 4 (2011) 147–150

3.6. 3(30 -Methoxy-50 -phenylfuran-20 -yl)propan-1-ol (2) Cai, Y., Chen, Z.P., Phillipson, J.D., 1993. Clerodane diterpenoids from Croton lechleri.
Phytochemistry 34, 265–268.
Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, B., 1987. Evaluation
Colorless gum: UV (MeOH) lmax (log e) 272 (3.26) nm; IR (KBr) of a tetrazolium-based semiautomated colorimetric assay: assessment of che-
nmax 3439, 3101, 2939, 1717, 1608, 1517, 1456, 1273, and mosensitivity testing. Cancer Res. 47, 936–942.
Garcia, A., Ramirez-Apan, T., Cogordan, J.A., Delgado, G., 2006. Absolute configura-
1113 cm1; 1H and 13C NMR (CDCl3) see Table 1; HR-ESI-MS m/ tion assignments by experimental and theoretical approaches of ent-labdane-
z 255.0994 [M + Na]+ (calcd for C14H16O3Na, 255.0997). and cis-ent-clerodane-type diterpenes isolated from Croton gabellus. Can. J.
Chem. 84, 1593–1602.
Harinantenaina, L., Takahara, Y., Nishizawa, T., Kohchi, C., Soma, G.-I., Asakawa,
3.7. Cytotoxicity assay Y., 2006. Chemical constituents of Malagasy liverworts, part V: prenyl
bibenzyls and clerodane diterpenoids with nitric oxide inhibitory activity
All stock cultures were grown in T-25 flasks. Freshly trypsinized from Radula appressa and Thysananthus spathulistipus. Chem. Pharm. Bull. 54,
1046–1049.
cell suspensions were seeded in 96-well microtiter plates at
Heymann, H., Tezuka, Y., Kikuchi, T., Supriyatna, S., 1994. Constituents of Sindora
densities of 5000 cells per well with compounds added from sumatrana MIQ, III. New trans-clerodane diterpenoids from the dried pods.
DMSO-diluted stock. After three days in culture, attached cells Chem. Pharm. Bull. 42, 1202–1207.
were stained with MTT (3-[4,5-dimethylthiazol-2-yl-2,5-diphe- Kuo, P.C., Shen, Y.C., Yang, M.L., Wang, S.H., Thang, T.D., Dung, W.X., Chiang, P.C., Lee,
K.H., Lee, E.J., Wu, T.S., 2007. Crotonkinins A and B and related diterpenoids from
nyltetrazolium] bromide). The absorbency at 540 nm was mea- Croton tonkinensis as anti-inflammatory and antitumor agents. J. Nat. Prod. 70,
sured using a microplate reader after solubilizing the bound dye. 1906–1909.
The mean IC50 was the concentration of agent that inhibited cell McChesney, J.D., Silveira, E.R., 1989. 12-Hydroxyhardwickic acid and sonderianial,
neo-clerodane from Croton sonderianus. Phytochemistry 28, 3411–3414.
growth by 50% under the experimental conditions and was the Ngamrojanavanich, N., Tonsiengsom, S., Lertpratchya, P., Roengsumran, S., Puthong,
average from at least six independent determinations that were S., Petsom, A., 2003. Diterpenoids from the stem barks of Croton robustus. Arch.
reproducible and statistically significant. The following human Pharm. Res. 26, 898–901.
Pudhom, K., Vilaivan, T., Ngamrojanavanich, N., Dechangvipart, S., Sommit, D.,
tumor cell lines were used in the assay: human breast ductol Petsom, A., Roengsumran, S., 2007. Furanocembranoids from the stem bark
carcinoma ATCC No. HTB 20 (BT474), undifferentiated lung of Croton oblongifolius. J. Nat. Prod. 70, 659–661.
carcinoma (CHAGO), liver hepatoblastoma (Hep-G2), gastric Qin, H.-L., Li, Z.-H., 2004. Clerodane-type diterpenoids from Nannaglottis ravida.
Phytochemistry 65, 2533–2537.
carcinoma ATCC No. HTB 103 (KATO-3), and colon adeno Roengsumran, S., Singtothong, P., Pudhom, K., Ngamrojanavanich, N., Petsom, A.,
carcinoma ATCC No. CCL 227 (SW-620). All cell lines were obtained Chaichantipyuth, C., 1999. Neocrotocembranal from Croton oblongifolius. J. Nat.
from the Institute of Biotechnology and Genetic Engineering, Prod. 62, 1163–1164.
Roengsumran, S., Petsom, A., Kuptiyanuwat, N., Vilaivan, T., Ngamrojanavanich, N.,
Chulalongkorn University and were cultured in RPMI-1640
Chaichantipyuth, C., Puthong, S., 2001. Cytotoxic labdane diterpenoids from
supplemented with 25 mM HEPES, 0.25% (w/v) sodium bicarbon- Croton oblongifolius. Phytochemistry 56, 103–107.
ate, 5% (v/v) fetal bovine serum, and 100 mg/mL kanamycin. Roengsumran, S., Musikul, K., Petsom, A., Vilaivan, T., Sangvanich, P., Pornpakakul,
S., Puthong, S., Chaichantipyuth, C., Jaiboon, N., Chaichit, N., 2002. Croblongi-
folin, a new anticancer clerodane from Croton oblongifolius. Planta Med. 68,
Acknowledgments 274–277.
Roengsumran, S., Pornpakakul, S., Muangsin, N., Sangvanich, P., Nhujak, T., Sing-
This work was supported by Ratchadaphisek Somphot Endow- tothong, P., Chaichit, N., Puthong, S., Petsom, A., 2004. New halimane diterpe-
noids from Croton oblongifolius. Planta Med. 70, 87–89.
ment Fund (AG001B), and the Thai Government Stimulus Package Rojatkar, S.R., Nagasampagi, B.A., 1994. Diterpenes from Cipadessa fruticosa. Phyto-
(TKK2555) under the Project for Establishment of Comprehensive chemistry 37, 505–507.
Center for Innovative Food, Health Products and Agriculture. We Sheldrick, G.M., 1997a. SHELXS-97 Program for Crystal Structure Refinement.
University of Göttingen, Germany.
also thank the Center for Petroleum, Petrochemicals, and Advanced Sheldrick, G.M., 1997b. SHELXS-97 Program for Crystal Structure Solution. Univer-
Materials, Chulalongkorn University for partial support. sity of Göttingen, Germany.
Sommit, D., Petsom, A., Ishikawa, T., Roengsumran, S., 2003. Cytotoxic activity of
References natural labdanes and their semi-synthetic modified derivatives from Croton
oblongifolius. Planta Med. 69, 167–170.
Twentymann, P.R., Luscombe, M.A., 1987. Study of some variables in a tetrazolium
Block, S., Baccelli, C., Tinant, B., Meervelt, L.C., Rozenberg, R., Jiwan, H.J., Llabres, G.,
dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 56,
Pauw-Gillet, D.M., Quetib-Leclercq, J., 2004. Diterpenes from the leaves of
279–285.
Croton zambesicus. Phytochemistry 65, 1165–1171.
Youngsa-ad, W., Ngamrojanavanich, N., Mahidol, C., Ruchirawat, S., Prawat, H.,
Bohlmann, F., Banerjee, S., Jakupovic, J., Grenz, M., Misra, L.N., Schmeda-Hirsch-
Kittakoop, P., 2007. Diterpenoids from the roots of Croton oblongifolius. Planta
mann, G., King, R.M., Robinson, H., 1985. Clerodane and labdane diterpenoids
Med. 73, 1494–11494.
from Baccharis species. Phytochemistry 24, 511–515.