(20R,23E)-eupha-8,23-diene-3β,25-diol from Tripetalum cymosum

Article abstract of DOI:10.1016/S0031-9422(98)00565-2

A new euphane triterpenoid, (20R,23E)-eupha-8,23-diene-3β,25-diol, was isolated from the leaves of Tripetalum cymosum and its structure was determined by spectral analysis and chemical correlation with euphol. Caryophyllene oxide, friedelin, euphol and putranjivic acid were also obtained from the leaves whilst the bark afforded euphol, glutin-5-en-3β- ol, butyrospermol and dammara-20,24-dien-3β-ol.

Full text of DOI:10.1016/S0031-9422(98)00565-2

PERGAMON
Phytochemistry 50 (1999) 849±857
(20R,23E)-Eupha-8,23-diene-3b,25-diol from Tripetalum cymosum
Yuan-Wah Leong, Leslie J. Harrison *
Department of Chemistry, National University of Singapore, 10, Kent Ridge Crescent, Singapore, 119260, Singapore
Received 24 March 1998
Abstract
A new euphane triterpenoid, (20R,23E)-eupha-8,23-diene-3b,25-diol, was isolated from the leaves of Tripetalum cymosum and
its structure was determined by spectral analysis and chemical correlation with euphol. Caryophyllene oxide, friedelin, euphol
and putranjivic acid were also obtained from the leaves whilst the bark a€orded euphol, glutin-5-en-3b-ol, butyrospermol and
dammara-20,24-dien-3b-ol. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Tripetalum cymosum; Guttiferae; Triterpenoids; Euphane; (20R,23E)-eupha-8,23-diene-3b,25-diol
1. Introduction
Members of the Guttiferae are well known sources
of aromatic metabolites such as xanthones (Bennett, &
Lee, 1989; Peres, & Nagem, 1977) and coumarins
(McKee, Fuller, Covington, Cardellina et al., 1996).
Whilst many such compounds have been reported
from large genera (>100 species) such as Garcinia
(Harrison, Leong, Leong, Sia, Sim et al., 1994),
Calophyllum (Iinuma, Ito, Tosa, Tanaka et al., 1997),
Clusia (Henry, Jacobs, Carrington, McLean,
&
Reynolds, 1996) and Hypericum (Ishiguro, Nagareya,
Suitani, & Fukumoto, 1997), the Guttiferae also con-
tains a number of small genera, e.g., Archytaea
lack the aromatic compounds which are typical of the
Guttiferae.
(Kubitzki, Lins Mesquita,
&
Gottlieb, 1978),
Pentadesma (Gunasekera, Sivapalan, Sultanbawa, &
Ollis, 1977) and Ploiarium (Bennett, Lee, Lowrey,
1990; Bennett, Harrison, Sia, Sim, & Connolly, 1992;
Bennett, Harrison, Lim, Sim et al., 1991), which con-
tain fewer than 10 species each (Willis, 1973). One
such genus is Tripetalum, the sole member of which, T.
cymosum K. Schum., is native to Papua New Guinea
where it is cultivated by the natives who use the juice
from the fruits to stain their teeth black (Usher, 1984).
There are no previous reports concerning the constitu-
ents of this plant. We have now studied the hexane
extracts of both bark and leaves and found them to
2. Results and discussion
Chromatographic separation of the hexane extract
of the bark gave the known triterpenoids glutin-5-en-
3b-ol (1) (Kitajima, Arai, & Tanaka, 1994; Carvalho,
& Seita, 1993; Mahato, Das, & Sahu, 1981), euphol (2)
(De Pascual Teresa, Urones, Marcos, Basabe et al.,
1987; Gewali, Hattori, Tezuka, Kikuchi, & Namba,
1990), butyrospermol (eupha-7,24-dien-3b-ol) (3) (De
Pascual Teresa et al., 1987) and dammara-20,24-dien-
3b-ol (4) (Mills, & Werner, 1955) which were ident-
i®ed by comparison of their physical properties with
literature values. As euphol is dicult to distinguish
from its C-20 epimer tirucallol, its identity was con-
* Author to whom correspondence should be addressed.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00565-2

850
Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
®rmed by conversion to the acetate (5) (De Pascual
Teresa et al., 1987). The 13C NMR spectrum of
dammara-20,24-dien-3b-ol (see Table 1) has not
previously been reported. The present assignments
were made by comparison with those of dam-
marenediol II (6) (Tori, Matsuda, Sono, & Asakawa,
1988).
Table 1
13C NMR Shifts for Dammara-20,24-dien-3b-ol
(4) and Dammarenediol II (6) [21].
Carbon
4a
6b
Euphol has previously been isolated from Garcinia
myrtifolia (Guttiferae) (Mills & Werner, 1955) and sev-
eral Euphorbia species (Euphorbiaceae) (De Pascual
Teresa, Urones, Marcos, Basabe et al., 1987; Gewali,
Hattori, Tezuka, Kikuchi, & Namba, 1990). Its 13C
NMR signals were recently completely assigned by 2D
NMR (Gewali et al., 1990; (Spino, Lal, Sotheeswaran
& Aalbersberg, 1995) and some of the previous assign-
ments (De Pascual Teresa et al., 1987) were revised.
Glutin-5-en-3b-ol has been previously isolated from
Mammea acuminata (Guttiferae) (Bandaranayake,
Karunanayake, Sotheeswaran, & Sultanbawa, 1980) as
well as from several Euphorbia species (Fisher, &
Seiler, 1961; Starratt, 1966) and Securinega tinctoria
(Euphorbiaceae) (Carvalho, & Seita, 1993). Plants
from a variety of families, e.g., Gramineae (Akihisa,
Yamamoto, Tamura, Kimura, et al., 1992), Moraceae
(Kitajima, Arai, & Tanaka, 1994), Scrophulariaceae
(Mahato, Das, & Sahu, 1981), Celastraceae (Gonzalez,
Ferro, & Ravelo, 1987) and Verbenaceae (Lin, Kuo, &
Che, 1989) are also sources of this compound.
Butyrospermol (3) has been isolated from a number of
sources, one being the herbaceous shrub Euphorbia
broteri (Euphorbiaceae) (De Pascual Teresa, 1987).
Dammara-20,24-dien-3b-ol (4), which was ®rst found
in the dammar resins from trees of the
Dipterocarpaceae family (Mills, & Werner, 1955), was
also isolated from Vismia guaramirangae (Camele,
1
39.1
39.0
2
27.1
79.0
39.0
56.0
18.3
35.5
40.5
51.0
37.3
21.4
27.5
45.3
49.5
31.4
25.0
47.9
15.7
16.0
152.8
107.5
39.1
28.9
124.5
131.4
25.7
17.7
28.0
15.4
16.2
27.4
78.9
39.0
55.9
18.3
35.3
40.4
50.7
37.1
21.6
27.6
42.3
50.3
31.2
24.9
49.9
15.5
16.2
75.4
25.4
40.5
22.6
124.8
131.5
25.7
17.7
28.0
15.4
16.5
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
b
In CDCl3. 125 MHz, 100 MHz.

Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
851
Table 2
13C NMR Shifts for Friedelin (8) [26], Putranjivic Acid (9) and
Methyl Putranjivate (10).
Carbon
8a
9b
10b
1
22.3
21.2
21.4
2
41.5
213.2
58.2
42.1
41.3
18.2
53.1
37.4
59.4
35.6
30.5
39.7
38.3
32.4
36.0
30.0
42.8
35.3
28.1
32.7
39.2
6.8
41.6
179.5
151.0
42.1
37.2
18.0
53.1
38.4
58.3
35.26
30.3
39.7
38.7
32.3
36.1
30.0
42.9
35.34
28.2
32.9
39.3
111.0
18.0
18.2
18.8
20.2
31.9
32.1
35.0
±
41.6
174.3
151.0
42.1
37.3
18.0
53.1
38.4
58.4
35.3
30.3
39.7
38.7
32.3
36.1
30.0
42.9
35.2
28.2
32.8
39.3
110.8
18.0
18.2
18.8
20.2
31.8
32.1
35.0
51.4
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
14.6
17.9
20.2
18.6
32.1
35.0
31.8
±
Delle Monache, Delle Monache, Marini Bettolo, &
Alves De Lima, 1982) and V. martiana (Guttiferae)
(Nagem, & Faria, 1990).
The leaf extract was also subjected to extensive chro-
matographic separation to give the known compounds
caryophyllene oxide (7) (Heymann, Tezuka, Kikuchi,
& Supriyatna, 1994), friedelin (8), euphol (1) (De
Pascual Teresa et al., 1987; Gewali et al., 1990) and
putranjivic acid (9) (Chopra, Jain, & Seshadri, 1969;
Aoyagi, Tsuyuki, & Takahashi, 1973) which were
identi®ed by comparison of their physical properties
with those in the literature or, in the case of friedelin,
by comparison with an authentic sample. The 13C
NMR data for putranjivic acid and its methyl ester
(10) (Chopra, Jain, & Seshadri, 1969; Aoyagi, Tsuyuki,
& Takahashi, 1973) (see Table 2) are given here for
the ®rst time, the assignments being made by compari-
son with those of friedelin.
Caryophyllene oxide (7) has been found in the essen-
tial oils of a number of plants belonging to a variety
of families, e.g., Myrtaceae (Zhen, Kennery, & Lam,
1992), Dipterocarpaceae (Gupta,
&
Dev, 1971),
Zingiberaceae (Damodaran, & Dev, 1968), Labiatae
(Maurer, & Hauser, 1983). Recently, it was isolated

852
Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
Table 3
13C NMR Shifts for Compounds 2, 11, 14 [41], 15 [18] and 17.
Carbon
2a
11a
14b
35.6
15c
31.7
17a
35.6
1
35.3
35.3
2
28.0
79.0
38.9
51.0
19.0
27.7
134.0
133.6
37.3
21.5
30.9
44.1
50.0
29.8
28.2
49.7
15.6
20.2
35.9
18.9
35.4
24.8
125.2
130.8
17.7
25.8
28.1
15.5
24.5
27.9
79.0
38.9
51.0
18.9
27.7
134.0
133.5
37.3
21.5
31.0
44.2
50.0
29.8
27.9
49.6
15.8
20.2
36.2
19.1
38.2
125.7
139.3
70.8
29.88
29.93
28.1
15.5
24.5
27.8
79.0
38.9
50.4
18.2
26.5
134.4
134.4
37.0
21.0
30.9
44.4
49.8
30.8
28.2
50.4
15.7
19.1
36.2
18.6
36.3
24.9
125.2
130.9
25.7
17.6
27.9
15.4
24.2
26.9
80.8
39.5
47.3
20.9
28.1
47.8
20.2
26.1
25.8
35.6
45.4
48.9
32.9
26.6
52.1
18.0
29.7
36.5
18.3
39.1
125.7
139.5
70.7
29.96
30.05
19.3
15.2
25.5
27.9
79.0
38.9
50.4
18.3
26.5
134.5
134.4
37.1
21.0
30.93
44.5
49.9
30.89
28.2
50.2
15.8
19.2
36.7
18.7
39.1
125.6
139.4
70.8
29.9
30.0
28.0
15.4
24.3
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
In CDCl3. 125 MHz, 75 MHz, 50.3 MHz.
b
c
from the roots and rhizomes of Valeriana fauriei
(Valerianaceae) (Nishiya, Kimura, Takeya, & Itokawa,
1992) and the pods of Sindora sumatrana
(Leguminosae) (Heymann 1994). Putranjivic acid (9)
and its methyl ester (10) were ®rst isolated from the
leaves of Putranjiva roxburghii (Euphorbiaceae)
(Chopra, Jain & Seshadri, 1969).
In addition to the known compounds, the most
polar compound from the leaves, (20R,23E)-eupha-
8,23-diene-3b,25-diol (11), was obtained as a gum,
C30H50O2 (m/z 442.3806), [a]D +17.0. Its IR spectrum
showed a characteristic hydroxyl absorption band
1
(3480 cm 1). The H NMR and 13C NMR spectra (see
Table 3) exhibited resonances for a disubstituted
double bond [dH 5.58 (2H, br s, H-23 and H-24); dC
139.3 and 125.7 (each d, C-23 and C-24)], a fully sub-
stituted double bond [dC 134.0 and 133.5 (each s, C-8
and C-9)], a secondary alcohol methine [dH 3.23 (1H,
dd, J= 4.5 and 11.7, H-3a; dC 79.0 (d, C-3)], a tertiary

Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
853
corresponding carbons of (23E)-3b-acetoxycycloart-23-
en-25-ol (15) (see Table 3) from Euphorbia broteri (De
Pascual Teresa et al., 1987). The compound was there-
fore (20R,23E)-eupha-8,23-diene-3b,25-diol (11) or
its 20-epimer (20S,23E)-tirucalla-8,23-diene-3b,25-diol
(16).
In order to determine the C-20 con®guration, 11
was prepared by photo-oxygenation of euphol (2).
This reaction is well-known and has been previously
carried out for the preparation of (23E)-lanosta-8,23-
diene-3b,25-diol (17) from lanosterol acetate (Nagano,
Poyser, Cheng, Luu et al., 1977). The physical proper-
ties of synthetic 11 were identical to those of the natu-
ral product and established its identity as (20R,23E)-
eupha-8,23-diene-3b,25-diol. The other product of the
photo-oxygenation was one of the two 24-hydroxy iso-
mers of eupha-8,25-diene-3b,24x-diol (18). The con-
®guration at C-24, however, was not assigned. The 13C
assignments for the nuclei of compounds 11 and 18
were made by comparison with those of euphol (2)
while the shifts for the side chains of both 11 and 18
were assigned by comparison with the corresponding
carbons of compound 15 (see Table 3).
The photo-oxygenation of lanosterol (13) was
repeated since the 13C NMR shifts for (23E)-lanosta-
8,23-diene-3b,25-diol (17) were not available in the lit-
erature and they are useful for purposes of compari-
son. In addition to 17, an inseparable mixture of the
24R- and 24S-isomers of 19 was also obtained.
Oxidation of this mixture with MnO2 gave 3b-hydro-
xylanosta-8,25-dien-24-one (20). The 13C NMR shifts
of 17 and 20 are reported below (see Experimental
Section 3).
alcohol carbon [dC 70.8 (s, C-25)], one secondary [dH
0.82 (3H, d, J=6.0 Hz, H3-21)] and seven tertiary
methyl groups [dH 1.31 (6H, s, H3-26 and H3-27), 1.00,
0.95, 0.88, 0.80 and 0.78 (each 3H, s, CH3)], in ad-
dition to nine methylene carbons, three methine car-
bons and four quaternary carbons. The molecular
formula indicated the presence of six units of unsatura-
tion and since there are only two ole®nic groups, the
compound must be tetracarbocyclic.
The compound formed a monoacetate (12) [nmax
1
1
1733 cm
2.05 (3H, s, CH3CO)] upon treatment with acetic
(C.O), 1246 and 1026 cm
(C±O); dH
1
anhydride/pyridine and a ketone 13 [nmax 1708 cm
(C.O)] upon oxidation with pyridinium chlorochro-
mate. The similarity between the A ring carbon shifts
of 11 and those of euphol (2) and lanosterol (14) (see
Table 3) indicated that C-3 is b-hydroxylated.
Comparison of the 13C NMR shifts of the ring car-
bons of compound 10 with those of lanosterol (14)
(see Table 3) revealed di€erences in the chemical shifts
of C-6, C-7, C-9, C-15, C-17 and C-19 (DdC 0.7, 1.2,
0.9, 1.0, 0.8 and 1.1). On the other hand, the nuclear
carbon shifts for compounds 2 and 11 were virtually
identical. This suggested that 11 had a euphane/tirucal-
lane nucleus with a D8,9 double bond. The other
double bond was hence located in the side chain. The
overlapping signals at dH 5.58, attributed to two
ole®nic protons, did not o€er much information as to
3. Experimental
1
the nature of the double bond. However, when the H
Mps: uncorr. [a]D: CHCl3. UV: EtOH. IR: CCl4.
NMR: 500 MHz (1H) and 125 MHz (13C) in CDCl3
relative to TMS at d=0.00. 13C multiplicities were
determined using the DEPT pulse sequence. CC: silica
gel (Baker, 40 mm) or C18 (Bakerbond, 40 mm). GPC:
Sephadex LH-20 (CHCl3±MeOH, 1:1). HPLC: silica
gel (Partisil, 5 mm, 4.6 Â 250 mm) or C18 (Whatman
ODS2, 5 mm, 4.6 Â 250 mm); RI detection.
NMR spectrum was recorded in C6D6, the signal chan-
ged to an AB quartet with one member further
coupled to a neighbouring methylene [dH 5.67 (1H,
ddd, J= 5.9, 7.4 and 15.6 Hz, H-23), and 5.61 (1H, d,
J= 15.6 Hz, H-24)], establishing the presence of a
trans-double bond in the side chain. The deshielded
nature (dH 1.31) of the C-25 methyls (H3-26 and H3-
27) indicated that they must be attached to the fully
substituted oxygenated carbon (dC 70.8, C-25) which,
in turn, must carry the tertiary hydroxyl group. The
structure of the side chain is therefore [±CH2±
CH.CH±C(CH3)2OH] which is a fairly common side
chain in fungal lanostanes (Vrkoc, Budesinsky, &
Dolejs, 1976) and is also known from higher plants
(De Pascual Teresa, 1987). The 13C NMR shifts of the
side chain were virtually identical to the shifts of the
The leaves and bark of Tripetalum cymosum were
obtained from the Forest Research Institute, Lae,
Papua New Guinea (voucher no. 8866). A specimen
(TC1) is retained in the Dept. of Chemistry, NUS. The
samples were air dried and ground. The ground ma-
terials (1 kg bark, 280 g leaves) were then subjected to
exhaustive extraction using hot hexane and concen-
trated in vacuo to a€ord the crude leaf extract (4.8 g)
and the crude bark extract (1.8 g).

854
Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
3.1. Bark extract
H-3a), 2.05 (3H, s, CH3CO), 1.69, 1.61 (each 3H, s,
H3-26, H3-27), 0.98, 0.884 (each 3H, s, CH3), 0.876
(6H, s, 2Â CH3), 0.86 (3H, d, J= 6.2 Hz, H3-21), 0.75
(3H, s, CH3); 13C NMR (300 MHz): d 171.0 (s), 134.0
(s), 133.7 (s), 130.9 (s), 125.2 (d), 81.0 (d), 51.1 (d),
50.1 (s), 49.7 (d), 44.2 (s), 37.9 (s), 37.2 (s), 35.9 (d),
35.4 (t), 35.0 (t), 30.9 (t), 29.8 (t), 28.1 (t), 28.0 (q),
27.6 (t), 25.7 (q), 24.8 (t), 24.5 (q), 24.3 (t), 21.6 (t),
21.3 (q), 20.2 (q), 18.92 (q), 18.86 (t), 17.7 (q), 16.6 (q),
15.6 (q).
CC of the extract (silica gel, 3±5% EtOAc±hexane
gradient) a€orded two frs. HPLC [silica gel, 6.25%
EtOAc±hexane followed by C18, Me2CO±H2O±AcOH
(88:11:1)] of fr 1 (708 mg) a€orded glutin-5-en-3b-ol
(1) (10.8 mg), euphol (2) (367 mg) and eupha-7,24-
dien-3b-ol (butyrospermol) (3) (20 mg). Fr 2 (263 mg)
was also subjected to HPLC [silica gel, 6.25% EtOAc±
hexane, followed by C18, Me2CO±H2O±AcOH
(88:11:1)] to give 2 (147 mg), 3 (30 mg) and dammara-
20,24-dien-3b-ol (4) (4.6 mg).
3.5. Eupha-7,24-dien-3b-ol (Butyrospermol) (3)
3.2. Glutin-5-en-3b-ol (1)
Gum (lit. 108±1108C (De Pascual Teresa, et al.,
1987); [a]D 4.5 (c 0.70) (lit. 12.0 [18]). FT±IR nmax
cm 1: 3448 (OH), 1458 and 1376 [ gem-dimethyl]. EI±
MS m/z (rel. int.): 426 [M] + (26), 411 [M-CH3] +
(100), 393 (48). HREI±MS: m/z 426.3835 (C30H50O
Colourless solid, mp. 207±2088 (CH3OH-CHCl3) (lit.
209±2108C (Mahato, Das, & Sahu, 1981); [a]D + 52.5
(c 0.32) (lit. +61.0 [17]). FT±IR nmax cm 1: 3375
(OH), 1448 and 1373 ( gem-dimethyl). EI±MS m/z (rel.
int.): 426 [M] + (15), 408 [M-H2O] + (23), 274
[C20H34] + (100), 259 [C20H34-CH3] + (78), 205 (50),
152 [C10H16O] + (13), 137 (52), 134 [C10H14] + (82),
121 (51), 119 (49), 109 (53), 95 (63), 81 (53), 69 (62),
55 (74); HREI±MS: m/z 426.3859 (C30H50O requires
1
requires m/z 426.3862); H and 13C NMR spectra iden-
tical to lit. values (De Pascual Teresa, 1987).
3.6. Dammara-20,24-dien-3b-ol (4)
Colourless solid, mp. 133±1348C (CH3OH) (lit. 136±
1388C (Mills, & Werner, 1955); [a]D +28.5 (c 0.40) (lit.
+47 [20]). FT±IR nmax cm 1: 3500 (OH), 1637 (C.C),
1452 and 1376 ( gem-dimethyl). EI±MS m/z (rel. int.):
426 [M] + (18), 357 (5); HREI±MS: m/z 426.3851
(C30H50O requires m/z 426.3862). 1H NMR: d 5.13
(1H, t sept, J= 1.4 and 7.0 Hz, H-24), 4.74 (1H, br s,
H-21), 4.71 (1H, br s, J= 1.5 Hz, H-21), 3.20 (1H, dd,
J =4.9 and 11.3 Hz, H-3a), 1.69, 1.62 (each 3H, br s,
H3-26 and H3-27), 0.980, 0.977, 0.87, 0.85, 0.78 (each
3H, s, CH3), 0.74 (1H, dd, J=2.3 and 12.0 Hz, H-5).
13C NMR: see Table 1. Di€erence NOE: H3-28 [H-3a
(4.8), H-5], H3-30 [H3-19 (0.8)], H3-19 [H3-30 (2.2), H3-
29 (1.1)], H3-29 [H3-28 (0.6), H3-19 (1.7)].
1
m/z 426.3862). H NMR and 13C NMR spectra were
identical to those in the literature (Kitajima, Arai, &
Tanaka, 1994; Carvalho, & Seita, 1993).
3.3. Eupha-8,24-dien-3b-ol (Euphol) (2)
Colourless needles, mp. 114±1158C (CH3OH) (lit.
115±1168C (De Pascual Teresa, 1987); [a]D +30.0 (c
0.30), +29.4 (c 4.40, C6H6) (lit. +31.0, CHCl3 [18]).
FT±IR nmax cm 1: 3481 (OH), 1670, 1454 and 1374
( gem-dimethyl), 1024 (C±O). EI±MS m/z (rel. int.):
426 [M] + (21), 135 (15), 133 (15), 121 (20), 119 (16),
109 (37), 107 (20), 105 (18), 95 (28), 81 (27), 79 (12),
69 (100), 67 (18), 55 (46), 43 (29), 41 (59); HREI±MS:
m/z 426.3860 (C30H50O requires m/z 426.3862). 1H
NMR: see Table 9; 13C NMR: see Table 3.
3.7. Leaf extract
CC on silica gel (acetone±hexane gradient) gave six-
teen frs. GPC of fr 1 (398 mg) followed by recrystalli-
sation (CHCl3±hexane) gave friedelin (8) (22 mg)
which was identi®ed by comparison with an authentic
sample. CC (silica gel, 4% EtOAc±hexane) and HPLC
(silica gel, 2% EtOAc±hexane) of the concentrated
mother liquors (114 mg) a€orded caryophyllene oxide
(7) (10.5 mg). Fr 2 (735 mg) was subjected to GPC,
CC (silica gel, 7% acetone±hexane) and HPLC (silica
gel, 1% acetone±hexane) to give euphol (2) (323 mg).
Fr 3 contained mainly 2 and was not further investi-
gated. Fr 4 contained mainly putranjivic acid (9). Fr 5
(242 mg) was subjected to GPC and CC (C18, 85%
acetone±H2O), followed by HPLC (C18, 90% acetone±
H2O) to a€ord putranjivic acid (9) (30 mg). Fr 6
(293 mg) was not investigated as TLC of this fraction
3.4. Euphol acetate
The alcohol 1 (10 mg) was acetylated with Ac2O±
pyridine. After the usual workup, CC of the residue
(silica gel, 1% EtOAc±hexane) yielded the monoace-
tate (5) (9 mg) as a colourless solid, mp. 107±1098
(CH3OH) (lit. 107±1098C (De Pascual Teresa, 1987).
[a]D + 37.2 (c 0.91) (lit. +38.5 [18]). IR nmax cm
1724 (C.O), 1443 and 1361 ( gem-dimethyl), 1242 and
1019 (C±O). EI±MS m/z (rel. int.): 468 [M] + (18), 453
[M-CH3] + (57), 408 [M±AcOH] + (9), 393 [M±CH3±
AcOH]+ (58), 109 (41), 95 (36), 69 (100), 55 (49).
HREI±MS: m/z 468.3939 (C32H52O2 requires m/z
468.3967); 1H NMR (300 MHz): d 5.10 (1H, br t,
J= 7.0 Hz, H-24), 4.50 (1H, dd, J= 4.7 and 11.7 Hz,
1
:

Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
855
revealed no distinct spots. Frs 7 and 8 (534 mg) were
combined, and upon GPC, CC (twice, C18, 76%
acetone±water, followed by silica gel, CH2Cl2) and
®nal puri®cation by HPLC (silica gel, 12% acetone±
hexane) a€orded (20R,23E)-eupha-8,23-diene-3b,25-
diol (11) (18 mg).
409 [M±H2O±CH3] + (82), 391 [M±2H2O±CH3] +
(42), 109 (100); HREI±MS: m/z 442.3806 (C30H50O2
requires m/z 442.3811). H NMR: d 5.58 (2H, br s, H-
1
23 and H-24), 3.23 (1H, dd, J =4.5 and 11.7 Hz, H-
3a), 2.34 (1H, br d, J =12.7 Hz), 1.31 (6H, s, H3-26
and H3-27), 1.00, 0.95, 0.88 (each 3H, s, CH3), 0.82
(3H, d, J= 6.0 Hz, H3-21), 0.80, 0.78 (each 3H, s,
1
3.8. (6R,7R)-Caryophyllene oxide (7)
CH3). H NMR (C6D6): d 5.67 (1H, ddd, J=5.9, 7.4
and 15.6 Hz, H-23), 5.61 (1H, d, J= 15.6 Hz, H-24),
3.07 (1H, m, H-3a), 2.43 (1H, br d, J= 8.1 Hz), 1.25
(6H, s, H3-26 and H3-27), 1.04, 0.96, 0.93 (each 3H, s,
CH3), 0.93 (3H, d, J=6.1 Hz, H3-21), 0.87, 0.85 (each
3H, s, CH3). 13C NMR: see Table 3.
Colourless solid, mp. 57±588 (CH3OH) (lit. 61±628C
(Nishiya et al., 1992); [a]D 63.1 (c 0.70) (lit. 66.7
(Nishiya et al., 1992). FT±IR nmax cm 1: 2932, 1630,
1454, 1383, 864. HREI±MS: m/z 220.1834 (C15H24O
1
requires m/z 220.1827); H and 13C NMR spectra iden-
tical to literature values (Heymann et al., 1994).
3.12. Acetylation of 11
3.9. Putranjivic acid (9)
The diol (11) (7.5 mg) was acetylated with Ac2O±
pyridine. The crude product was puri®ed by HPLC
(silica gel, 7% acetone±hexane) to give the monoace-
tate (12) (4 mg) as a colou`rless solid, mp. 112±1138
(CHCl3). IR nmax cm 1: 1733 (ester C.O), 1456 and
1372 ( gem-dimethyl), 1246 and 1026 (C±O). EI±MS
m/z (rel. int.): 484 [M] + (25), 466 (23), 452 (84), 370
(89); HREI±MS: m/z 484.3930 (C32H52O3 requires m/z
Colourless needles, mp. 178±1808C (CH3OH) (lit.
173.0±173.58C (Aoyagi, Tsuyuki, & Takahashi, 1973);
[a]D 4.6 (c 0.51) (lit.
12 (Aoyagi, Tsuyuki, &
Takahashi, 1973). FT±IR nmax cm 1: 3283±2614 (car-
boxylic OH), 1708 (C.O). EI±MS m/z (rel. int.): 442
[M] + (6), 427 (10), 205 (66); HREI±MS: m/z 442.3814
(C30H50O2 requires m/z 442.3811). 1H NMR: d 5.62
(1H, dd, J =10.7 and 17.4 Hz, H-4), 4.93 (1H, d,
J= 10.7 Hz, H-23E), 4.91 (1H, d, J=17.4 Hz, H-
23Z), 2.33 (2H, m, H2-2), 1.18 (3H, s, H3-5), 1.02 (3H,
s, CH3), 1.00 (6H, s, 2Â CH3), 0.95 (3H, s, CH3), 0.89
(3H, s, CH3). 13C NMR: see Table 2.
1
484.3916). H NMR: d 5.58 (2H, m, H-23 and H-24),
4.50 (1H, dd, J =4.6 and 11.8 Hz, H-3a), 2.34 (1H, br
d, J=12.6 Hz), 2.05 (3H, s, CH3CO), 1.31 (6H, s, H3-
26 and H3-27), 0.98, 0.884 (each 3H, s, CH3), 0.876
(6H, s, 2Â CH3), 0.82 (3H, d, J= 5.9 Hz, H3-21), 0.77
(3H, s, CH3).
3.10. Methylation of 9
3.13. Oxidation of 11
The acid (9 mg) was methylated with CH2N2. CC of
the residue (silica gel, 1% EtOAc±hexane) a€orded
methyl putranjivate (10) (6.0 mg) as a colourless solid,
mp. 135±1378 (CH3OH) (lit. 136.0±136.58C (Aoyagi,
Tsuyuki, & Takahashi, 1973); [a]D 5.5 (c 0.58) (lit.
8.3 (Aoyagi, Tsuyuki, & Takahashi, 1973). FT±IR
nmax cm 1: 1740 (C.O), 1241 and 1049 (C±O). EI±MS
m/z (rel. int.): 456 [M] + (9), 441 [M-CH3] + (14), 331
(8), 301 (9), 273 (23), 250 (19), 223 (52), 218 (57), 205
(85), 95 (100). HREI±MS: m/z 456.3988 (C31H52O2
requires m/z 456.3967). 1H NMR: d 5.61 (1H, d,
J= 10.8 and 17.4 Hz, H-4), 4.92 (1H, d, J =10.7 Hz,
H-23E), 4.90 (1H, d, J =17.4 Hz, H-23Z), 3.63 (3H, s,
OCH3), 2.29 (2H, m, H2-2), 1.17 (3H, s, H3-5), 1.02
(3H, s, CH3), 0.99 (6H, s, 2 ÂCH3), 0.98 (3H, s, CH3),
0.94 (3H, s, CH3), 0.88 (3H, s, CH3). 13C NMR: see
Table 2.
The alcohol 11 (11.5 mg) was oxidised with PCC in
CH2Cl2. The crude product was puri®ed by ¯ash chro-
matography (silica gel, 7% acetone±hexane) to give
the ketone 13 (2 mg) as a colourless solid, mp. 132±
1338 (CHCl3). IR nmax cm 1: 1708 (C.O), 1459 and
1376 ( gem-dimethyl). EI±MS m/z (rel. int.): 440 [M] +
(11), 407 (70), 325 (97); HREI±MS: m/z 440.3640
(C30H48O2 requires m/z 440.3654). 1H NMR: d 5.59
(2H, m, H-23 and H-24), 2.56 (1H, ddd, J=7.5, 10.0
and 15.9 Hz, H-2a), 2.47 (1H, ddd, J= 3.9, 7.7 and
15.9 Hz, H-2b), 2.35 (1H, br d, J =12.4 Hz), 1.32 (6H,
s, H3-26 and H3-27), 1.10, 1.062, 1.057, 0.90 (each 3H,
s, CH3), 0.83 (3H, d, J =6.0 Hz, H3-21), 0.78 (3H, s,
CH3).
3.14. Photo-oxygenation of euphol (2)
A mixture of euphol (260 mg) and methylene blue
(1.5 mg) in EtoH (30 ml) was irradiated with a 500 W
lamp. The reaction mixture was stirred rapidly to e€ect
the introduction of oxygen and its temperature was
maintained at 258. Upon consumption of the euphol,
excess NaBH4 was added and the reaction mixture was
3.11. (20R,23E)-Eupha-8,23-diene-3b,25-diol (11)
Gum. [a]D +17.0 (c 0.45). IR nmax cm 1: 3380
(OH), 1455 and 1374 ( gem-dimethyl), 1025 (C±O). EI±
MS m/z (rel. int.): 442 [M] + (4), 424 [M-H2O]+ (32),

856
Y. Leong, L.J. Harrison / Phytochemistry 50 (1999) 849±857
stirred for 15 mins. Water was then added to decom-
pose unreacted NaBH4 and the EtOH was distilled o€.
The crude product was partitioned between CH2Cl2
and water. The organic layer was dried with Na2SO4,
concentrated and ¯ash chromatographed (silica gel,
8% acetone±hexane) to give (20R)-eupha-8,25-diene-
3b,24x-diol (18) (17 mg) and (20R,23E)-eupha-8,23-
diene-3b,25-diol (11) (31 mg), identical to the natural
product.
3.18. Mixture of 24-epimers of lanosta-8,25-diene-3b,24-
diol (19)
1H NMR: d 4.92 (2H, quintet of d, J =0.9, 5.7 Hz,
2 Â H-26), 4.83 (2H, m, 2Â H-26), 4.01 (2H, t, 2Â H-
24), 3.23 (2H, dd, J= 4.5 and 11.6 Hz, 2 Â H-3a), 1.72
(6H, br s, 2 Â H3-27), 1.00, 0.98 (each 6H, s, 2Â CH3),
0.91 (6H, d, J= 6.3 Hz, 2 Â H3-21), 0.87, 0.81, 0.69
(each 6H, s, 6 Â CH3). 13C NMR: d 147.8 (s), 147.5 (s),
134.5 (s), 134.4 (s), 111.4 (t), 110.9 (t), 79.0 (d), 76.8
(d), 76.3 (d), 50.4 (d), 50.3 (d), 49.8 (s), 44.5 (s), 38.9
(s), 37.1 (s), 36.3 (d), 35.6 (t), 31.95 (t), 31.92 (s), 31.7
(t), 31.5 (t), 31.0 (t), 30.9 (t), 28.2 (t), 28.1 (t), 28.0 (q),
27.9 (t), 26.5 (t), 24.3 (q), 21.0 (t), 19.2 (q), 18.7 (q),
18.3 (t), 17.6 (q), 17.2 (q), 15.8 (q), 15.4 (q).
3.15. (20R)-Eupha-8,25-diene-3b,24x-diol (18)
Gum. [a]D + 19.1 (c 1.70). IR nmax cm 1: 3389
(OH), 1465 and 1374 ( gem-dimethyl), 1025 (C±O). EI±
MS m/z (rel. int.): 442 [M] + (25), 427 (34), 409 (100),
391 (42), 175 (27), 173 (24), 149 (27), 133 (36); HREI±
MS: m/z 442.3801 (C30H50O2 requires m/z 442.3811).
1H NMR: d 4.92, 4.83 (each 1H, br s, H2-26), 4.01
(1H, t, J =6.1 Hz, H-24), 3.23 (1H, dd, J=4.5 and
11.7 Hz, H-3a), 1.72 (3H, br s, H3-27), 0.99, 0.94, 0.86
(each 3H, s, CH3), 0.85 (3H, d, J= 6.2 Hz, H3-21),
0.79, 0.75 (each 3H, s, CH3); 13C NMR: d 147.6 (s),
134.0 (s), 133.5 (s), 111.1 (t), 79.0 (d), 76.6 (d), 51.0
(d), 50.0 (s), 49.7 (d), 44.1 (s), 38.9 (s), 37.3 (s), 36.0
(d), 35.3 (t), 31.6 (t), 31.1 (t), 30.9 (t), 29.8 (t), 28.0 (q),
28.0 (t), 27.9 (t), 27.7 (t), 24.5 (q), 21.5 (t), 20.1 (q),
19.0 (q), 18.9 (t), 17.4 (q), 15.7 (q), 15.5 (q).
3.19. b-Hydroxylanosta-8,25-dien-24-one (20)
The mixture of 24-epimers of 19 (8 mg) was dis-
solved in C6H6. Activated MnO2 in C6H6 was added
to the soln and the reaction mixture was stirred and
monitored by TLC. MnO2 was removed by ®ltration
through Celite and the crude product obtained was
puri®ed by HPLC (DIOL, 10% EtOAc±hexane) to
a€ord 20 (4.1 mg) as a colourless solid, mp. 152±1538
(CHCl3). IR nmax cm 1: 1682 (C.O), 1453 and 1372
( gem-dimethyl). EI±MS m/z (rel. int.): 440 [M] + (16),
425 (53), 407 (70); HREI±MS: m/z 440.3648
(C30H48O2 requires m/z 440.3654). 1H NMR: d 5.95
(1H, br s, H-26), 5.75 (1H, br s, H-26), 3.23 (1H, dd,
J =4.6 and 11.6 Hz, H-3a), 2.72 (1H, ddd, J =5.3,
10.3 and 15.8 Hz, H-23), 2.61 (1H, ddd, J=5.9, 10.0
and 15.8 Hz, H-23), 1.87 (3H, br s, H3-27), 0.91 (3H,
d, J =6.3 Hz, H3-21), 1.00, 0.98, 0.88, 0.81 and 0.70
(each 3H, s, CH3). 13C NMR: 202.9 (s), 144.6 (s),
134.5 (s), 134.4 (s), 124.2 (t), 79.0 (d), 50.4 (2 Â d), 44.6
(s), 38.9 (s), 37.1 (s), 36.3 (d), 35.6 (t), 34.7 (t), 31.1 (t),
31.0 (t), 30.8 (t), 28.2 (t), 28.0 (q), 27.9 (t), 26.5 (t),
24.3 (q), 21.0 (t), 19.2 (q), 18.6 (q), 18.3 (t), 17.8 (q),
15.8 (q), 15.4 (q).
3.16. Photo-oxygenation of lanosterol (13)
Commercially obtained lanosterol (250 mg) (which
contained dihydrolanosterol as impurity) was used.
The procedure and the workup are as described above.
The crude product was ¯ash chromatographed (silica
gel, 15% EtOAc±hexane) to give unreacted dihydrola-
nosterol (44.1 mg), (23E)-lanosta-8,23-diene-3b,25-diol
(17) (25.4 mg) and a mixture of the 24-epimers of
lanosta-8,25-diene-3b,24-diol (19) (25.5 mg).
3.17. (23E)-Lanosta-8,23-diene-3b,25-diol (17)
Acknowledgements
Colourless plates, mp. 197±1988C (CHCl3), (lit.
192.5±193.58C (Nagano et al., 1977); [a]D +46.5 (c
1.40). IR nmax cm 1: 3450 (OH), 1454 and 1372 ( gem-
dimethyl). EI±MS m/z (rel. int.): 442 [M] + (3), 424
[M±H2O]+ (12), 409 [M±H2O±CH3] + (35), 391 [M±
2H2O±CH3] + (27), 109 (100); HREI±MS: m/z
We thank the National University of Singapore for
®nancial support and for the award of a postgraduate
scholarship (to Y.-W.L.).
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