Cytotoxic and anti-HIV-1 constituents of Gardenia obtusifolia and their modified compounds

Article abstract of DOI:10.1016/S0040-4020(02)00999-7

5α-Cycloart-24-ene-3,23-dione (1), 5α-cycloart-24-ene-3,16,23-trione (2) and methyl 3,4-seco-cycloart-4(28),24-diene-29-hydroxy-23-oxo-3-oate (3), together with five known flavones 5,7,4′-trihydroxy-3,8-dimethoxyflavone (4), 5,7,4′-trihydroxy-3,8,3′-tri-methoxyflavone (5), 5,7,4′-trihydroxy-3,6,8-trimethoxyflavone (6), 5,4′-dihydroxy-3,6,7,8-tetramethoxyflavone (7) and 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (8) have been isolated from the leaves and twigs of Gardenia obtusifolia. The structures were assigned on the basis of spectroscopic methods. Compounds 3-8 and some of the modified compounds showed significant cytotoxic activities in several mammalian cell lines, especially 8 and its diacetate 21 which exhibited potent cytotoxicities (compound 8: P-388 0.05μg/mL, KB 0.09μg/mL, BCA-1 0.63μg/mL, Lu-1 0.09μg/mL, ASK 0.70μg/mL; its diacetate: P-388 0.27μg/mL, KB 0.06μg/mL, BCA-1 0.53μg/mL, Lu-1 0.49μg/mL). It was also found that 5, 8 and 21 showed antimitotic acitivity in the ASK assay. Compounds 2, 4, 6, 7 and some of the modified compounds displayed interesting anti-HIV activity in the syncytium assay, but were inactive or exhibited weak activity in the HIV-1 RT assay; while compound 3 was found to be active in the HIV-1 RT assay (99.9 % inhibition at 200μg/mL), but cytotoxic in the syncytium assay.

Full text of DOI:10.1016/S0040-4020(02)00999-7

Tetrahedron 58 (2002) 8073–8086
Cytotoxic and anti-HIV-1 constituents of Gardenia obtusifolia and
their modified compounds
a
a
a
Patoomratana Tuchinda,a Wilart Pompimon, Vichai Reutrakul, Manat Pohmakotr,
Chalobon Yoosook,b Natedao Kongyai,b Samaisukh Sophasan,c Kulawee Sujarit,c
Suchart E. Upathumd and Thawatchai Santisuke
,
*
aDepartment of Chemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
bDepartment of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
cDepartment of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
dDepartment of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
eThe Forest Herbarium, Royal Forest Department, Phaholyothin Road, Bangkok 10900, Thailand
Received 30 May 2002; revised 19 July 2002; accepted 8 August 2002
Abstract—5a-Cycloart-24-ene-3,23-dione (1), 5a-cycloart-24-ene-3,16,23-trione (2) and methyl 3,4-seco-cycloart-4(28),24-diene-29-
hydroxy-23-oxo-3-oate (3), together with five known flavones 5,7,40-0trihydroxy-3,8-dimethoxyflavone (4), 5,7,40-trihydroxy-3,8,30-tri-
methoxy0flavone (5), 5,7,40-trihydroxy-3,6,8-trimethoxyflavone (6), 5,4 -dihydroxy-3,6,7,8-tetramethoxyflavone (7) and 5,30-dihydroxy-
3,6,7,8,4 -pentamethoxyflavone (8) have been isolated from the leaves and twigs of Gardenia obtusifolia. The structures were assigned on the
basis of spectroscopic methods. Compounds 3–8 and some of the modified compounds showed significant cytotoxic activities in several
mammalian cell lines, especially 8 and its diacetate 21 which exhibited potent cytotoxicities (compound 8: P-388 0.05 mg/mL, KB
0.09 mg/mL, BCA-1 0.63 mg/mL, Lu-1 0.09 mg/mL, ASK 0.70 mg/mL; its diacetate: P-388 0.27 mg/mL, KB 0.06 mg/mL, BCA-1
0.53 mg/mL, Lu-1 0.49 mg/mL). It was also found that 5, 8 and 21 showed antimitotic acitivity in the ASK assay. Compounds 2, 4, 6, 7 and
some of the modified compounds displayed interesting anti-HIV activity in the syncytium assay, but were inactive or exhibited weak activity
in the HIV-1 RT assay; while compound 3 was found to be active in the HIV-1 RT assay (99.9 % inhibition at 200 mg/mL), but cytotoxic in
the syncytium assay. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
has resulted in the isolation of four ring-A seco-cycloartane
triterpenes. In this work, the chloroform fraction of
Gardenia obtusifolia Roxb. was studied and led to the
isolation of 5a-cycloart-24-ene-3,23-dione (1), 5a-cycloart-
24-ene-3,16,23-trione (2), methyl 3,4-seco-cycloart-
4(28),24-diene-29-hydroxy-23-oxo-3-oate (3); along with
five known flavones 5,7,40-trihydroxy-3,8-dimethoxy-
flavone (4), 5,7,40-trihydroxy-3,8,30-trimethoxyflavone (5),
5,7,40-trihydroxy-3,6,8-trimethoxyflavone (6), 5,40-dihy-
droxy-3,6,7,8-tetramethoxyflavone (7) and 5,30-dihydroxy-
3,6,7,8,40-pentamethoxyflavone (8) (Fig. 1). Compound 1
has been isolated previously from the stem bark of
Monocyclanthus vignei,14 the bud exudates of Fijian
Gardenia species15 and the leaves of Guarea trichilioides,16
while 2 and 3 are new compounds. For further structure–
activity studies, the new cycloartane derivatives 9–15 and the
known flavone derivatives 16–22 (Fig. 1) wereprepared from
the isolated compounds. The structures of all compounds were
elucidated on the basis of spectroscopic methods. We herein
describe the isolation, the modification and the determination
of the structures, including their cytotoxic and anti-HIV
activities. There have been no reports either on phytochem-
istry or biological activity of G. obtusifolia prior to our work.
More than 80 species in the genus Gardenia (Rubiaceae) are
widely distributed among the tropical forests in various
parts of the world. Fifteen species of Gardenia were
reported in Thailand.1 Several species of Gardenia have
been recorded to be used ethnomedically in various
countries primarily for abortifacient2 and contraceptive2–4
purposes. Some species are used as a febrifuge,5 for the
treatment of headaches6 and as a larvicides.7 Extracts of
various Gardenia species showing anti-implantation and
abortifacient effects,8 as well as antiulcer,9 antibacterial,10
analgesic,11 diuretic,11 hypertensive11 and larvicidal
activity12 have been previously reported. As part of our
ongoing project on the discovery of new anti-cancer and
anti-HIV agents from plants, several species of Gardenia
have been collected from various parts of Thailand. Our
previous work on Gardenia coronaria and G. sootepensis13
Keywords: cycloartane-3-one; ring-A seco-cycloartane; 3-methoxyflavone;
cytotoxic activity; anti-HIV activity.
*
Corresponding author. Tel.: þ66-2-2015159; fax: þ66-2-2477050;
e-mail: scptc@mahidol.ac.th
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00999-7

8074
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Figure 1.
2. Results and discussion
methyl at C-21. The fragmentation ions in the mass
spectrum of 1 at m/z 438 [Mþ], 340, 125, 98, 83 (base
peak) and 55 were useful in obtaining the structure of 1.15
The presence of the carbonyl group and the unsaturation in
the side chain was confirmed as evidenced by the fragment
ions at m/z 125 for C8H13Oþ, 83 for C5H7Oþ and 55 for
C4Hþ7 . The ions at m/z 340 and m/z 98 were the results of a
McLafferty rearrangement involving the carbonyl group and
the g-hydrogen in the side chain. Compound 1 was finally
proved to be 5a-cycloart-24-ene-3,23-dione by direct
Compound 1 exhibited [M]þ peak at m/z 438 in the EIMS
corresponding to a molecular formula C30H46O2. Its IR
(CHCl3) spectrum showed the absorption bands at nmax
1698 cm21 (for 6-membered ring ketone), 1683 cm21 (for
conjugated ketone), and 1616 cm21 (for CvC). The H
1
NMR spectrum (Table 1) of 1 displayed a characteristic pair
of doublets for cyclopropane methylene protons17–24 at d
0.58 and 0.79 (1H each, d, J¼4.3 Hz), as well as the three
singlet signals for the four methyl groups of the cyclo-
artanone-fused moiety at d 0.91 (3H), 1.05 (6H) and 1.10
(3H), which suggested that 1 was a normal cycloartanone-
type triterpenoid. Additionally, the presence of a terminal
dimethylvinyl group connected to a carbonyl group in the
side chain was characterized by the low-field shifts of the
signal of an olefinic proton at d 6.06 (m), and also the signals
of two geminal vinylmethyls at d 2.15 and 1.87 (d each,
J¼1.1 Hz), respectively. Apart from the singlets of four
tertiary methyls, a doublet of a secondary methyl group was
observed at d 0.89 (J¼6.3 Hz), which was referred to the
1
comparison of its H and 13C NMR (Table 1) spectral data
with those reported in the literature.14–16 The results from
2D NMR experiments supported the assignments of protons
and carbons in the structure (see Section 5 and Table 2).
The molecular formula of compound 2 was determined as
C30H44O3 by its EIMS which showed the molecular ion
peak at m/z 452 [M]þ. Compound 2 is clearly related to
compound 1, except that 2 has one extra carbonyl group in
the structure. The IR (CHCl3) spectrum of 2 exhibited three
carbonyl stretching bands at 1727, 1700 and 1682 cm21

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Table 1. 300 MHz 1H- and 75 MHz 13C NMR data of cycloartanes 1–3 (CDCl3)
8075
a
dH
b
dC
Position
1
2
3
1
2
3
1
2
(a) 1.85 (obsc.)
(b) 1.53 (ddd, 14, 6.5, 2.6)
(a) 2.71 (ddd, 14, 14, 6.4)
(b) 2.30 (ddd, 14, 4.3, 2.6)
(a) 1.89 (obsc.)
(b) 1.56 (obsc.)
(a) 2.72 (ddd, 13.9, 13.9, 6.3)
(b) 2.32 (obsc.)
–
–
1.75 (dd, 12.2, 4.3)
(a) 1.62 (obsc.)
(b) 0.97 (dddd, 12.6, 12.6, 12.6, 2.6)
(a) 1.37 (obsc.)
(b) 1.21 (obsc.)
1.69 (dd, 12.3, 4.6)
–
–
(a) 2.16 (obsc.)
(b) 1.28 (obsc.)
1.85 (obsc.)
(a) 2.16 (obsc.)
(b) 1.37 (obsc.)
(a) 2.50 (obsc.)
(b) 2.28 (ddd, 15.5, 11, 4.5)
–
–
2.53 (obsc)
(a) 1.67 (obsc)
(b) 1.03 (obsc.)
(a) 1.30 (obsc.)
(b) 1.06 (obsc.)
1.56 (obsc.)
–
33.41
37.44
33.13
37.30
28.90
31.55
3
4
5
6
–
216.50
50.21
48.42
21.47
215.99
50.15
48.30
21.27
174.50
152.38
42.07
–
1.71 (dd, 12.5, 4.6)
(a) 1.56 (obsc.)
(b) 0.95 (dddd, 12.5, 12.5, 12.5, 2.4)
(a) 1.39 (obsc.)
(b) 1.14 (obsc.)
1.60 (obsc)
–
28.85
7
25.83
26.18
25.21
8
9
10
11
47.82
21.01
25.98
26.66
47.28
20.37
26.45
26.20
47.85
21.72
27.37
26.85
–
–
(a) 2.02 (obsc.)
(b) 1.20 (obsc.)
1.64 (obsc)
–
–
1.33 (obsc.)
(a) 2.12 (obsc.)
(b) 1.23 (obsc.)
1.66 (obsc.)
–
–
1.32 (obsc.)
12
13
14
15
32.69
45.45
48.87
35.51
31.25
42.07
45.29
50.90
32.84
45.15
48.96
35.58
–
–
(a) 2.07 (d, 18.5)
(b) 2.01 (d, 18.5)
–
16
(a) 1.90 (obsc.)
(b) 1.31 (obsc.)
1.66 (obsc.)
1.05 (s)
(a) 0.79 (br d, 4.3, endo)
(b) 0.58 (d, 4.3, exo)
2.04 (obsc.)
0.89 (d, 6.3)
(a) 2.51 (dd, 14.4, 2.3)
(b) 2.12 (obsc.)
–
6.06 (m)
–
2.15 (d, 1.1)
1.87 (d, 1.1)
1.05 (s)
(a) 1.87 (obsc.)
(b) 1.30 (obsc.)
1.63 (obsc.)
1.01 (obsc.)
(a) 0.73 (br d, 4.4, endo)
(b) 0.48 (d, 4.4, exo)
2.04 (obsc.)
0.88 (d, 6.1)
(a) 2.51 (obsc.)
(b) 2.13 (obsc.)
–
28.34
219.26
28.24
17
18
19
2.30 (obsc.)
1.20 (s)
52.53
18.12
29.53
60.87
18.95
30.04
52.44
18.19
30.20
(a) 0.86 (br d, 4.4, endo)
(b) 0.65 (d, 4.4, exo)
2.33 (obsc.)
0.99 (d, 5.7)
(a) 3.20 (m)
(b) 2.34 (obsc.)
–
6.11 (m)
–
2.14 (d, 1.0)
1.88 (d, 1.3)
1.06 (s)
20
21
22
33.39
19.29
51.72
27.40
20.21
49.98
33.39
19.28
51.67
23
24
25
26
27
28
201.52
124.40
154.61
20.65
27.65
22.17
200.71
124.40
154.27
20.72
27.60
22.15
201.59
124.32
154.67
20.62
27.63
110.37
6.06 (m)
–
2.14 (d, 1.3)
1.89 (d, 1.1)
(a) 5.11 (br dd, 2.9, 1.5)
(b) 5.08 (br s)
4.14 (br s)
29
30
3-OMe
1.10 (s)
0.91 (s)
–
1.11 (s)
1.10 (s)
–
20.75
19.37
–
20.64
19.70
–
64.62
19.31
51.50
0.94 (s)
3.64 (s)
a
Chemical shift given in ppm using TMS as internal reference; multiplicities and coupling constants (Hz) are given in parentheses; obsc.¼obscured signal.
Chemical shift given in ppm; CDCl3 signal at dC 77.00 as reference.
b
corresponding to the CvO of 5-membered ring ketone,
Consequently, the structure of compound 2 was established
as 5a-cycloart-24-ene-3,16,23-trione.
CvO of 6-membered ring ketone and CvO of conjugated
ketone, respectively. In its 1H NMR spectrum, the
characteristic pair of doublets at d 0.65 and 0.86
(J¼4.4 Hz) in combination with the four singlets at d
1.06, 1.10, 1.11 and 1.20 are compatible with those obtained
for a cycloartane triterpene bearing a carbonyl group at C-3.
The low-field shifts of the signals of H-15a, H-15b, H-17, H-
20, H-21, H-22a and H-22b (d 2.07, 2.01, 2.30, 2.33, 0.99,
3.20 and 2.34, respectively), comparable with the data of 1,
supported the proximity of these protons to the C-16
carbonyl group (see Table 1). Compound 2 was suggested to
have the same side chain as in 1. The fragment ions in the
mass spectrum at m/z 354, 125, 98, 83 (base peak) and 55
were in accordance with those occurred in compound 1. The
similarity of the side chain as in 1 was confirmed by the low-
field signals of the vinyl proton at d 6.11 (m) together with
the two geminal vinylmethyls at d 2.14 (d, J¼1.0 Hz) and
1.88 (d, J¼1.3 Hz). On the basis of 2D NMR spectral
analyses (see Section 5 and Table 2), the chemical shifts in
the 1H and 13C NMR spectra (Table 1) were assigned.
Compound 3 was obtained as a colorless oil. The IR
(CHCl3) spectrum of 3 exhibited the absorption bands at
1681 cm21 (conjugated CvO stretching) and 1615 cm21
(CvC stretching) which suggested that 3 had a conjugated
ketone in the structure. The EIMS of 3 showed the [M]þ at
m/z 484, corresponding to a molecular formula of C31H48O4.
The presence of the ion peaks at m/z 386, 125, 98, 83 (base
peak) and 55 supported the same side chain as in 1 and 2.
Compound 3 was characterized to contain an ester group by
the observed CvO stretching band at 1729 cm21. The 13C
NMR spectral data at dC 201.59 and 174.50 confirmed the
presence of the conjugated carbonyl and the ester groups,
1
respectively. The H NMR data (Table 1) of compound 3
showed signals of C-19 methylene in the cyclopropane ring
of a cycloartane triterpene (d 0.48 and 0.73, d, J¼4.4 Hz),
along with the signals of two tertiary methyls (d 0.94 and
1.01, s), one secondary methyl (d 0.88, d, J¼6.1 Hz) and
two geminal vinylmethyls connected to a carbonyl group (d

8076
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Table 2. Observed HMBC correlations in compounds 1–3
ester signals at dH 3.64 (s, COOCH3) and dC 51.50
(COOCH3). In general, the 13C NMR data correlated
closely with our caronalolic acid previously isolated from
G. coronaria,13 which differed from compound 3 only in the
side chain and the methyl ester group at C-3. By complete
analyses of these 2D NMR spectra (see Section 5 and Table
2) in combination with the 1H and 13C NMR data (Table 1),
compound 3 was identified as methyl 3,4-seco-cycloart-
4(28),24-diene-29-hydroxy-23-oxo-3-oate (3).
C
1
1 correlated H
2 correlated H
3 correlated H
2a, 2b, 5, 19a,
19b
2a, 2b, 5, 19a,
19b
1a, 1b
1b, 2a, 2b, 28,
29
2a (w), 5, 28,
29
1b, 6a, 6b, 7a,
19a, 19b, 28, 29
2b, 5, 19a, 19b
2
3
1a, 1b
1a, 1b
1a, 1b, 2a, 2b,
5, 28, 29
2a, 5, 28, 29
1a, 1b, 2a, 2b,
3-OMe
5, 28a, 28b, 29
4
5
1a, 1b, 6a, 6b,
7a, 7b, 19b, 28,
29
5, 8
5, 8
1b, 6a, 7a, 7b,
19a, 19b, 28a, 28b,
29
5, 7b, 8
6b, 8
In order to gain more information about the structure–
activity relationship, the isolated cycloartanes 1 and 2 were
modified as shown in Scheme 1. It was found that the side
chain was easily hydrogenated (H2, 10% Pd/C in MeOH)
and gave the corresponding 9 and 10 in 95 and 96% yields,
respectively. Compounds 9 and 10 were clearly proved to be
the dihydro-derivatives of 1 and 2 as evidenced by their
molecular ions at m/z 440 (for C30H48O2) and 454 (for
C30H46O3), respectively, in the EIMS. The disappearance of
the olefinic proton signal of H-24 in the 1H NMR spectrum
(Table 3) of each compound indicated that compounds 1 and
2 were completely hydrogenated. The correlations observed
in the COSY (see Section 5) and HMQC spectra, in
combination with the HMBC (Table 5) spectral data made
the assignments of the chemical shifts in the 13C NMR
spectra of 9 and 10 (Table 4) possible.
6
7
8
5, 7a, 7b
5, 6a, 6b, 8
1a (w), 6a, 7a, 11b, 7b, 11b, 15a, 15b, 6a, 6b, 7a, 7b,
15, 19a, 19b, 30
1b, 5, 7a, 7b,
8, 11a, 11b, 19a,
19b
19a, 19b, 30
1a, 5, 7a, 7b,
8, 11a, 11b, 12,
19a, 19b
15, 19a, 19b, 30
1a, 1b, 6a, 6b,
8, 12, 19a, 19b
9
10
1a, 1b, 2a, 2b,
5, 6a, 6b, 8,
19a, 19b
1a, 1b, 2a, 2b,
5, 6a, 8, 19a,
19b, 28 (w), 29
(w)
1a, 1b, 2a, 2b,
6a, 8, 19a, 19b
11
12
8, 12, 19a, 19b
11a, 11b, 18
8, 12
11a, 11b, 15, 17,
18
12, 19a, 19b
11a, 11b, 17, 18
13
11b, 12, 15, 16a,
17, 18, 30
8, 12, 15a, 15b,
18, 30
8, 11a, 12, 15,
16a, 16b, 17, 18,
30
14
15
8, 12, 16a, 16b,
17, 30
8, 16a, 16b, 17,
30
15
12, 15, 18, 22a
(w), 22b (w)
12, 17
1a, 1b, 5, 8,
11a, 11b
17, 21, 22a, 22b
7a, 12, 15a, 15b,
17, 18, 30
8, 17, 30
8, 12, 15, 16b,
17, 18, 30
16b, 30
Since there have been few natural seco-cycloartanes
reported and these compounds exhibited interesting bio-
logical activities, hence the oxidative transformation of ring
A of the isolated cycloartanes was undertaken. The Baeyer–
Villiger oxidation of cycloartanone 1 with MCPBA
(1.1 equiv.) in CH2Cl2 at room temperature for 7 h afforded
the heptanolide 11 (36% yield) and the heptanolide–
epoxides 13A, 13B (ratio 1:1.2) as an inseparable mixture
of diastereomers (3% yield), along with the recovered
starting material (24% yield). However, the oxidation
reaction was completed by using 2.2 equiv. of MCPBA.
Under these conditions, the heptanolide 11 and the
inseparable diastereomers 13A, 13B (ratio 1:1.2) were
isolated in 39 and 24% yields, respectively. Similarly, the
Baeyer–Villiger oxidation of cycloartanone 2 with
1.1 equiv. of MCPBA gave the heptanolide 12, the
inseparable diastereomers of heptanolide–epoxides 14A,
14B (ratio 1:1.3) and the recovered starting material in 46, 8
and 25% yields, respectively. The complete oxidation of 2
could also be achieved by treatment with 2.2 equiv. of
MCPBA under the same conditions, and led to the formation
of the heptanolide 12 (44% yield) and the inseparable
diastereomers of heptanolide–epoxides 14A, 14B (ratio
1:1.3, 24% yield). The heptanolides 11 and 12 were
determined to have one oxygen more than 1 and 2,
respectively, by their EIMS at m/z 454 [M]þ for 11
(C30H46O3) and m/z 468 [M]þ for 12 (C30H44O4). The IR
(CHCl3) spectra of both compounds showed the absorption
band corresponding to the CvO stretching of 7-membered
ring lactone at 1702 cm21, while the CvO of 5-membered
16
17
15a, 15b, 17
12, 18, 21, 22a,
22b
15, 17
12, 15, 18, 22a,
22b
12, 17
1a, 5, 8, 11a,
11b
18
19
12, 17
1a, 5, 8, 11a,
11b
17, 21, 22a, 22b
20
16b, 17, 21, 22a,
22b
21
22
23
17, 22a, 22b
21
20 (w), 22a, 22b,
17, 20, 22a, 22b
17, 20, 21
22a, 22b, 24, 26
(w), 27 (w)
26, 27
26, 27
24, 27
24, 26
5, 29
17, 22a, 22b
21
22a, 22b, 24, 27
(w)
26, 27
24, 26, 27
24, 27
24, 26
24
26, 27
26, 27
24, 27
24, 26
5, 29
5, 28
8, 15
–
24
25
26
27
28
29
30
3-OMe
5, 29
5, 28
8, 15a, 15b
–
5, 28a, 28b
8, 15, 16b
2b
w¼weak correlation.
2.14, d, J¼1.3 Hz and 1.89, d, J¼1.1 Hz). Furthermore, a
broad doublet of doublets (d 5.11, J¼2.9, 1.5 Hz) and a
broad singlet (d 5.08) of an sp2 methylene of H-28 signified
a terminal double bond which exhibited allylic coupling
with the two-proton broad singlet of H-29 at d 4.14.
Comparable with the analogous signals in compounds 1 and
2, the pair of doublets corresponding to H-19a and H-19b of
compound 3 appeared shifted downfield. All of these data
suggested a change in ring-B substitution pattern, which led
to the proposal for 3 of a 3,4-seco-cycloartane structure with
a primary alcohol group at C-29. Information from the 13C
and DEPT-spectra indicated that one of the methylene
carbons resonated at d 64.62, providing a strong support for
the primary alcohol unit. The presence of a carbomethoxy
ester group in the structure was confirmed by the methyl
1
ring ketone in 12 absorbed at 1727 cm21. The H and 13C
NMR spectral data of 11 and 12 were quite similar to those
of 1 and 2 with some differences observed in ring-A. With
respect to the analogous signals observed in 1 and 2, the
downfield shifts of the C-4 gem-dimethyl signals (H-28: dH
1.40 for 11 and 1.42 for 12; H-29: dH 1.46 for 11 and 1.47

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
8077
Scheme 1.
for 12) and of the C-28 signals in the 13C NMR spectra at dC
31.01 for 11 and dC 30.91 for 12 were due to the g-effect of
the C-5–C-6 bond and the C-3 carbonyl in the ring-A
lactone. Other connectivities were confirmed by 2D NMR
correlation experiments (see Section 5 and Table 5).
Cycloartanes with 7-membered lactone in ring-A have
been reported from natural sources, such as kadsudilactone
isolated from Kadsura coccinea22 and root of Buxus
papillosa.25 The diastereomers A and B of heptanolide–
epoxides in 13 or 14 could not be separated from each other
by chromatography; they were characterized as a mixture of
diastereomers. Although the structure of each diastereomer
of 13 possesses two carbonyl groups, the IR spectrum
(CHCl3) of the mixture shows only one CvO stretching
band at 1703 cm21. In this case, the CvO absorptions of
both 7-membered lactone and side-chain ketone appeared at
the same position. The IR spectrum (CHCl3) of 14A, 14B
mixture exhibited one extra CvO absorption band at
1725 cm21, which was assigned to the absorption of 5-
membered ring ketone, while the CvO absorptions of 7-
membered lactone and the side-chain ketone were also
observed as a strong band at 1706 cm21. The ratios of the
diastereomers 13A/13B and 14A/14B were determined
from the integration of the separated singlet signals of H-24
in the 1H NMR spectra. The chemical shifts of other
singlet of three protons at d 3.66 and the carbon signal at d
51.58 were referred to the methyl signals of the ester group.
Information from the mass spectrum, which showed the
fragment ion at m/z 59 (C3H7Oþ), confirmed the presence of
the isopropanol group in the structure of 15. All of the
1
signals in the H and 13C NMR spectra were completely
assigned by analyses of the 2D NMR spectral data (see
Section 5 and Table 5).
The relative stereochemistry of the isolated compounds 1–3
and their modified products 9–15 were determined by
NOESY NMR experiments. The results are in agreement
with the ROE correlations of nigranoic acid observed in the
ROESY spectrum.18 The NOESY correlations observed are
as shown in Fig. 2.
Compounds 4–8 are 3-methoxyflavone derivatives. The
structures were identified by direct comparison of their
melting points and spectral data to the values recorded in the
literature.26–33 The acetylated and methylated derivatives
16–22 were prepared by using Ac2O/4-dimethylamino-
pyridine (DMAP) at room temperature and Me2SO4/K2CO3
in acetone at reflux, respectively.
1
obscured signals in the H NMR spectra of both mixtures
were deduced from the correlation positions observed in the
2D-spectra (see Section 5 and Table 6).
3. Bioassay evaluations
Pure compounds 1–22 were evaluated for cytotoxic effects
against a panel of cultured mammalian cell lines.34 The
results including their antimitotic activities are as shown in
Table 7. Moderate to high potency of cytotoxicities were
found in compounds 3–8, 12, 20 and 21, while compounds
5, 8 and 21 exhibited antimitotic effects in the ASK assay. It
should be noted that compound 8 and its diacetate 21
The heptanolide 12 was used as an example for the synthesis
of seco-cycloartane. The seco-cycloartane 15 was obtained
in 80% yield by the cleavage of the heptanolide 12 with
1.1 equiv. of NaOMe in MeOH at room temperature for 6 h.
The structure of 15 was confirmed by its spectral data. The

8078
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Table 3. 300 MHz 1H NMR data of compounds 9–12 and 15 (CDCl3)
a
Position
dH
9
10
(a) 1.89 (obsc.)
(b) 1.56 (obsc.)
(a) 2.72 (ddd, 13.8, 13.8, 6.5)
(b) 2.34 (obsc.)
11
12
15
1
2
(a) 1.86 (obsc.)
(b) 1.55 (obsc.)
(a) 2.71 (ddd, 13.9, 13.9, 6.5)
(b) 2.31 (obsc.)
(a) 1.80 (obsc.)
(b) 1.52 (obsc.)
(a) 2.71 (obsc.)
(b) 2.66 (obsc.)
–
(a) 1.85 (obsc.)
(b) 1.57 (obsc.)
(a) 2.73 (obsc.)
(b) 2.69 (obsc.)
–
(a) 2.71 (obsc.)
(b) 1.40 (obsc.)
(a) 2.67 (obsc.)
(b) 2.25 (obsc.)
–
3
4
5
6
–
–
–
–
–
–
–
1.72 (dd, 12.3, 4.4)
(a) 1.56 (obsc.)
(b) 0.96 (obsc.)
(a) 1.39 (obsc.)
(b) 1.14 (obsc.)
1.61 (obsc.)
–
1.76 (obsc.)
(a) 1.62 (obsc.)
(b) 0.98 (obsc.)
(a) 1.35 (obsc.)
(b) 1.20 (obsc.)
1.70 (obsc.)
–
2.04 (obsc.)
(a) 1.81 (obsc.)
(b) 0.67 (obsc.)
(a) 1.32 (obsc.)
(b) 1.08 (obsc.)
1.43 (obsc.)
–
2.12 (obsc.)
(a) 1.86 (obsc.)
(b) 0.71 (obsc.)
(a) 1.32 (obsc.)
(b) 1.18 (obsc.)
1.53 (obsc.)
–
1.92 (obsc.)
(a) 1.76 (m)
(b) 0.72 (obsc.)
(a) 1.26 (obsc.)
(b) 1.10 (obsc.)
1.43 (obsc.)
–
7
8
9
10
11
–
–
–
–
–
(a) 2.04 (obsc.)
(b) 1.18 (obsc.)
1.65 (obsc.)
–
–
1.34 (obsc.)
(a) 2.17 (obsc.)
(b) 1.26 (obsc.)
1.86 (obsc.)
–
(a) 2.06 (obsc.)
(b) 1.09 (obsc.)
1.69 (obsc.)
–
–
1.34 (obsc.)
(a) 2.21 (obsc.)
(b) 1.22 (obsc.)
1.87 (obsc.)
–
–
2.04 (br.s)
(a) 2.24 (obsc.)
(b) 1.30 (obsc.)
1.85 (obsc.)
–
–
2.03 (br.s)
12
13
14
15
–
(a) 2.07 (d, 18.4)
(b) 2.00 (d, 18.4)
–
16
(a) 1.88 (obsc.)
(b) 1.31 (obsc.)
1.64 (obsc.)
1.03 (s)
(a) 0.80 (d, 4.1)
(b) 0.59 (d, 4.1)
2.05 (obsc.)
0.88 (d, 6.2)
(a) 2.44 (dd, 15.7, 2.2)
(b) 2.14 (obsc.)
–
2.26 (obsc.)
2.15 (obsc.)
0.92 (d, 6.7)
0.91 (d, 6.5)
1.04 (s)
(a) 1.88 (obsc.)
(b)1.31 (obsc.)
1.64 (obsc.)
1.03 (s)
(a) 0.66 (obsc.)
(b) 0.61 (d, 4.9)
2.03 (obsc.)
0.89 (d, 6.2)
(a) 2.51 (dd, 14.7, 2.3)
(b) 2.12 (obsc.)
–
6.06 (m)
–
2.16 (d, 1.0)
1.89 (d, 1.1)
1.40 (s)
–
–
17
18
19
2.27 (obsc.)
1.18 (s)
2.32 (obsc.)
1.17 (s)
2.30 (obsc.)
1.16 (s)
(a) 0.86 (d, 4.4)
(b) 0.66 (d, 4.4)
2.35 (obsc.)
0.97 (d, 5.9)
(a) 3.15 (m)
(b) 2.38 (obsc.)
–
2.30 (obsc.)
2.15 (obsc.)
0.92 (d, 6.5)
0.91 (d, 6.6)
1.06 (s)
(a) 0.73 (d, 4.6)
(b) 0.69 (d, 4.6)
2.34 (obsc.)
0.98 (d, 5.7)
(a) 3.22 (m)
(b) 2.35 (obsc.)
–
6.11 (m)
–
2.14 (d, 1.1)
1.88 (d, 1.2)
1.42 (s)
(a) 0.75 (d, 4.8)
(b) 0.61 (d, 4.8)
2.32 (obsc.)
0.97 (d, 5.8)
(a) 3.22 (m)
(b) 2.31 (obsc.)
–
6.11 (m)
–
2.14 (d, 1.1)
1.88 (d, 1.1)
1.25 (s)
20
21
22
23
24
25
26
27
28
29
30
3-OMe
1.10 (s)
0.91 (s)
–
1.11 (s)
1.09 (s)
–
1.46 (s)
0.92 (s)
–
1.47 (s)
1.12 (s)
–
1.23 (s)
1.12 (s)
3.66 (s)
a
Chemical shift given in ppm using TMS as internal reference; multiplicities and coupling constants (Hz) are given in parentheses.
showed high potent cytotoxicity comparable to other
compounds in Table 7.
flavonol methyl ethers have been reported in several species
of plants, compounds possessing significant cytotoxic and
anti-HIV activities are particularly rare. To the best of our
knowledge, the structure modification of the isolated
cycloartanes to obtain better biological activities has not
been attempted. Our work represents an example of an
investigation of both chemical and biological aspects.
All of the isolated and modified compounds were also tested
employing HIV-1 reverse transcriptase (RT),35 and a
syncytium assay36 using DTat/RevMC99 virus and 1A2 cell
line system37 (see Table 8). It was found that only 3 showed
potent inhibitory activity (99.9% inhibition at 200 mg/mL)
against HIV-1 RT (fagaronine chloride was used as a
positive control35). Some of the compounds were active in
the DTat/RevMC99 syncytium assay, using AZT as a
reference.38 However, compounds 3, 5, 8, 12, 17, 21 and
22 were found to be very toxic to the cell lines used in this
assay. It is noteworthy that compound 10, which was
modified from the isolated 2, gave the best therapeutic index
(TI.32.1; EC50,3.9 mg/mL; IC50.125 mg/mL) compar-
able with other compounds.
5. Experimental
5.1. General procedures
Mps: uncorr.; UV: EtOH or MeOH; IR: CHCl3 or KBr.
NMR spectra were recorded on a Bruker DPX 300 in CDCl3
using TMS as an internal standard, otherwise stated; CC was
carried out on silica 60, 70–230 mesh.
5.2. Plant material
4. Conclusion
The leaves and twigs of G. obtusifolia Roxb. (Rubiaceae)
were collected from Chiang Mai Province of Thailand in
March, 1996, and identified by one of us (T. S.). A voucher
In conclusion, it is interesting to note that although the
occurrence of cycloartane triterpenes and highly oxygenated

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
8079
Table 4. 75 MHz 13C NMR data of compounds 9–12 and 15 (CDCl3)
ene-3,23-dione (1) (180.1 mg) and 5a-cycloart-24-ene-
3,16,23-trione (2) (4.11 g). Another compound was isolated
as a white semi-solid and identified as methyl 3,4-seco-
cycloart-4(28),24-diene-29-hydroxy-23-oxo-3-oate (3)
(53.8 mg). Elution with 15–20% CH3COCH3-n-hexane
led to the separation of a fraction (36.3 g). Repeated CC
(silica gel, CH3COCH3-n-hexane gradient) afforded two
flavones which were recrystallized with EtOH and identified
as 5,7,40-trihydroxy-3,8-dimethoxyflavone (4) (2.52 g) and
5,7,40-trihydroxy-3,8,30-trimethoxyflavone (5) (1.22 g).
Elution with 20–30% CH3COCH3-n-hexane yielded a
semi-solid mixture (29.3 g) of three compounds. Further
separation by CC (silica gel, CH3COCH3/n-hexane gradi-
ent) followed by recrystallization from EtOH resulted in the
isolation of 5,7,40-trihydroxy-3,6,8-trimethoxyflavone (6)
(1.30 g), 5,40-dihydroxy-3,6,7,8-tetramethoxyflavone (7)
(1.02 g) and 5,30-dihydroxy-3,6,7,8,40-pentamethoxyflavone
(8) (59.4 mg), respectively. Elution with 30–50% CH3-
COCH3-n-hexane yielded a gum (95.5 g). This fraction was
further purified by CC (silica gel, CH3COCH3-n-hexane
gradient) twice, followed by recrystallization from EtOH to
give 7 (2.98 g) and 8 (572.4 mg).
a
dC
Position
9
10
11
12
15
1
2
3
4
5
6
7
8
33.36
37.41
216.58
50.17
48.36
21.42
25.78
47.76
20.94
25.91
26.59
32.63
45.37
48.82
35.42
28.26
52.25
18.08
29.48
32.72
19.36
50.67
211.30
52.57
24.49
22.51
22.63
22.12
20.71
19.23
–
33.11
37.27
215.98
50.12
48.25
21.23
26.14
47.24
20.31
26.39
26.14
31.25
42.03
45.19
50.87
219.35
60.65
18.89
30.00
26.81
20.39
49.03
210.51
52.24
24.60
22.52
22.62
22.11
20.70
19.64
–
30.17
35.09
175.46
87.23
49.83
26.05
25.38
48.74
23.01
27.26
27.27
32.77
45.03
48.88
35.75
28.38
52.56
18.39
29.61
33.31
19.35
51.65
201.46
124.41
154.66
20.65
27.64
31.01
23.11
19.42
–
29.79
34.87
175.20
86.94
49.61
25.58
25.64
47.92
22.19
27.68
26.77
31.30
41.97
44.74
51.00
219.08
60.73
19.05
29.86
27.32
20.02
49.88
200.63
124.28
154.32
20.57
27.55
30.91
22.98
19.75
–
29.90
32.05
174.91
76.04
45.10
25.06
26.00
47.91
21.87
27.18
26.20
31.66
42.21
44.86
51.39
219.53
60.93
19.22
31.38
27.48
20.03
50.05
200.78
124.38
154.26
20.63
27.60
31.75
26.16
19.92
51.58
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-OMe
5.3.1. 5a-Cycloart-24-ene-3,23-dione (1). Colorless needles
from MeOH–CHCl3, mp 135.8–136.08C (Lit.14 137–
1388C). [a] ¼þ7.29 (c 0.85, CHCl3) [Lit.14 [a]D21¼þ7
26
589
(c 1.14)]. UV (EtOH) lmax nm (log 1): 235 (4.76). IR
(CHCl3) nmax: 1698 (CvO stretching of 6-membered ring
ketone), 1683 (CvO stretching of conjugated ketone), 1616
1
(CvC), 1447, 1382, 1235, 1113, 1038, 973 cm21. H and
13C NMR data: see Table 1. COSY correlations H/H: 1a/1b,
2a, 2b, 19a; 1b/1a, 2a, 2b; 2a/1a, 1b, 2b; 2b/1a, 1b, 2a; 5/6a,
6b, 19a; 6a/5, 7a, 7b; 6b/5, 7a; 7a/6a, 6b, 8; 7b/6a; 8/7a, 19b,
30; 11a/11b, 12; 11b/11a, 12; 12/11a, 11b, 18; 15/16a, 16b,
30; 16a/15, 16b; 16b/15, 16a; 17/16b, 18, 20; 18/12, 17, 30;
19a/1a, 5, 19b; 19b/5, 8, 19a; 20/17, 21, 22a, 22b; 21/17, 20,
22a, 22b; 22a/20, 21, 22b; 22b/20, 21, 22a; 24/26, 27; 26/24,
27; 27/24, 26. HMBC correlations: see Table 2. EIMS
(70 eV) m/z (%): 438 [M]þ (3), 423 (1), 355 (1), 340 (13),
325 (3), 313 (3), 298 (1), 285 (1), 271 (1), 255 (1), 243 (1),
231 (1), 217 (2), 202 (5), 187 (5), 175 (5), 161 (5), 147 (21),
125 (56), 98 (42), 91 (13), 83 (100), 67 (12), 55 (42). Anal.
calcd for C30H46O2: C, 82.14; H, 10.57. Found: C, 81.93; H,
10.41.
a
Chemical shift given in ppm; CDCl3 signal at dC 77.00 as reference.
specimen (BKF no. 092419) of G. obtusifolia has been
deposited at the Forest Herbarium, Royal Forest Depart-
ment, Bangkok, Thailand.
5.3. Extraction and isolation
The air-dried and finely powdered leaves and twigs of G.
obtusifolia (7.9 kg) were successively extracted with
methanol (75 L£21 days£5 times) at room temperature,
followed by filtration. The filtrates were combined and
evaporated to dryness under vacuum to afford a crude
MeOH extract (1093 g). The methanol extract was
suspended in water (1 L) and partitioned with CHCl3
(5£2.5 L), EtOAc (3£2.5 L) and n-BuOH (2£2 L), respec-
tively. Removal of solvents from each fraction yielded the
CHCl3 fraction (496 g), EtOAc fraction (31 g), n-BuOH
fraction (196 g) and H2O fraction (360 g), respectively.
5.3.2. 5a-Cycloart-24-ene-3,16,23-trione (2). Colorless
26
589
needles from EtOAc, mp 173.0–173.88C. [a] ¼292.7
(c 0.85; CHCl3). UV (EtOH) lmax nm (log 1): 236 (4.19). IR
(CHCl3) nmax: 1727 (CvO of 5-membered ring ketone),
1700 (CvO stretching of 6-membered ring ketone), 1682
(CvO stretching of conjugated ketone), 1618 (CvC),
1448, 1385, 1243, 1114, 1037, 974 cm21. 1H and 13C NMR
data: see Table 1. COSY correlations H/H: 1a/1b, 2a, 2b,
19a; 1b/1a, 2a, 2b; 2a/1a, 1b, 2b, 29; 2b/1a, 1b, 2a, 28; 5/6a,
6b, 19a, 19b; 6a/5, 6b, 7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b;
7b/6a, 6b, 7a, 8; 8/7a, 7b, 15, 19a, 19b, 30; 11a/11b, 12, 19a,
19b; 11b/11a, 12, 19b; 12/11a, 11b, 18; 15a/15b, 17, 30;
15b/15a, 17, 30; 17/15a, 15b, 18; 18/12, 17; 19a/1a, 5, 8,
11a, 19b; 19b/1a, 5, 8, 11a, 11b, 19a; 20/21, 22a, 22b; 21/20,
22a, 22b; 22a/20, 21, 22b; 22b/20, 21, 22a; 24/26, 27; 26/24,
27; 27/24, 26; 28/2b, 5, 29; 29/2a, 28; 30/8, 15a, 15b.
HMBC correlations: see Table 2. EIMS (70 eV) m/z (%):
452 [M]þ (1), 437 (16), 381 (8), 355 (2), 354 (,1), 339 (1),
The CHCl3 extract (496 g) was subjected to a coarse
separation on a silica gel column (1.8 kg), eluting with
various proportions of CH3COCH3-n-hexane, followed by
increasing amount of MeOH in CH3COCH3 and finally with
MeOH. Fractions (500 mL each) were collected and
combined on the basis of TLC behavior. Elution with 15%
CH3COCH3-n-hexane yielded a fraction (115 g), which
consisted of three compounds. Repeated column chromato-
graphy of this fraction (silica gel, CH3COCH3-n-hexane
gradient) resulted in the isolation of three compounds.
Further purification of the two isolated compounds by
recrystallization gave colorless needles of 5a-cycloart-24-

8080
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Table 5. Observed HMBC correlations in compounds 9–12 and 15 (in CDCl3)
C
dH
9
10
11
12
15
1
2
3
2a, 2b, 5, 19a, 19b
1a, 1b
1a, 1b, 2a, 2b, 5, 28,
29
2b, 5, 28, 29
1a, 1b, 6a, 6b, 7a, 7b,
19a, 19b, 28, 29
5, 7a, 7b, 8
2a, 2b, 5, 19a, 19b
1a, 1b
1a, 1b, 2a, 2b, 5, 28,
29
2b, 5, 28, 29
1a, 1b, 6a, 6b, 7a, 7b,
19a, 19b, 28, 29
5, 7a, 7b, 8
2a, 2b, 5, 19a, 19b
1a, 1b
1a, 1b, 2a, 2b, 28, 29
2a, 2b, 5, 19a, 19b
1a, 1b
1a, 1b, 2a, 2b, 28 (w)
2a, 2b, 5, 19a, 19b
1a, 1b, 19a
1a, 1b, 2a, 2b, 3-OMe
4
5
5, 28, 29
5, 6b (w), 28, 29
5, 6b, 28, 29
19a, 19b
1a, 1b, 6a, 7b, 19a, 19b, 28, 29 1a, 1b, 6a, 7a, 7b, 19a,
19b, 28, 29
5, 8
5, 8
6a (w), 7a, 7b, 11b, 19a,
19b, 30
7b, 8, 11a, 11b, 12, 19a,
19b
1a, 1b, 2a, 2b, 8, 11a,
19a, 19b
8, 11b, 12, 19a, 19b
11a, 11b, 18
11b, 12, 16a, 16b, 17, 18,
30
6
7
8
5, 7a, 7b, 8
5, 6a, 6b, 8
5, 8
5, 8
5, 6a, 6b, 8
5, 6a, 6b, 8
6a, 6b, 7a, 7b, 11a, 11b,
15, 19a, 19b, 30
5, 7a, 7b, 8, 11a, 11b,
19a, 19b
1a, 1b, 2a, 2b, 5, 6a,
6b, 8, 11a, 11b, 19a, 19b
8, 12, 19a, 19b
11a, 11b, 17, 18
8, 11a, 11b, 12, 15, 16a,
16b, 18, 30
6a, 6b, 7a, 7b, 11a, 11b,
15a, 15b, 19a, 19b, 30
1b (w), 5, 7a, 7b, 8,
11a, 11b, 12, 19a, 19b
1a, 1b, 2a, 2b, 5, 6a,
6b, 8, 11a, 11b, 19a, 19b
8, 12, 19a, 19b
6a (w), 7a, 7b, 15, 19a, 6a, 7a, 11b, 15, 19a, 19b,
19b, 30
1a, 1b, 7a, 7b, 8, 11a,
11b, 12, 19a, 19b
1a, 1b, 2a, 2b, 5, 6a,
6b, 8, 11a
8, 12, 19a, 19b
11a, 11b, 18
8, 12, 15, 18, 30
30
9
1a, 1b, 7a, 7b, 8, 11a,
11b, 12, 19a, 19b
1a, 1b, 2a, 2b, 5, 6a,
19a, 19b
8, 12, 19a, 19b
11a, 11b, 18
8, 12, 15, 18, 30
10
11
12
13
11a, 11b, 15a, 15b, 17, 18
8, 12, 15a, 15b, 18, 30
14
8, 12, 16a, 16b, 17, 30
8, 11a, 11b, 12, 15a, 15b,
17, 18, 30
8, 17 (w)
15a, 15b, 17, 20
12, 15a, 15b, 18, 20, 21,
22a, 22b
7a, 7b, 8, 12, 17, 18,
30
16b, 30
15
12, 18, 21
7b, 8 (w), 12, 15, 17,
8, 12, 15, 17, 18, 30
18, 30
8, 30
15, 17
15
16
17
8, 16a, 16b, 30
15, 17, 21
12, 15, 16a, 16b, 18, 21,
30 (w)
12, 17
1a, 1b, 5, 8, 11a, 11b
16a, 16b, 17, 21, 22a, 22b
20, 22a, 22b
21
22a, 22b, 24, 25
8, 30
15, 17, 20
12, 15, 18, 20, 21, 22a, 12, 15, 18, 21, 22a, 22b
22b
12, 17
1a, 1b, 5, 8, 11a, 11b
17, 21, 22a, 22b
17, 20, 22a, 22b
17, 20, 21
22a, 22b, 24, 26 (w), 27 22a, 22b, 26 (w), 27 (w)
(w)
18
19
20
21
22
23
12, 17
12, 17
12, 17
1a, 1b, 5, 8, 11a, 11b
17, 21, 22a, 22b
17, 20, 22a, 22b
20, 21, 24
1a, 5, 8, 11a, 11b
21, 22a, 22b
20, 22a, 22b
21
1b, 5, 11a, 11b
17, 21, 22a
20, 22a
20, 21, 22
21, 22a, 22b, 24, 25
22a, 22b, 24
24
25
26
27
28
29
30
3-OMe
22a, 22b, 26, 27
24, 26, 27
24, 25, 27
24, 25, 26
5, 29
5, 28
15
–
25, 26, 27
24, 26, 27
24, 25, 27
24, 25, 26
5, 29
2a, 2b, 5, 28
8, 15a, 15b
–
22b, 26, 27
26, 27
24, 27
24, 26
29
5, 28
8, 15
–
26, 27
26, 27
24, 27
24, 26
5, 29
5, 28
8, 15
–
22b (w), 26, 27
26, 27
27
26
29
5, 28
8, 15
–
w¼weak correlation.
313 (1), 299 (1), 269 (1), 233 (1), 203 (1), 185 (2), 177 (2),
161 (2), 147 (3), 135 (5), 125 (15), 98 (24), 91 (15), 83
(100), 69 (21), 55 (62). Anal. calcd for C30H44O3: C, 79.60;
H, 9.80. Found: C, 79.49; H, 9.61.
339 (3), 313 (2), 299 (3), 287 (4), 271 (2), 260 (4), 247 (5),
231 (5), 219 (5), 187 (10), 173 (12), 159 (12), 147 (38), 125
(34), 121 (38), 105 (28), 98 (13), 91 (32), 83 (100), 67 (20),
55 (55), 41 (28). HRFABMS calcd for C31H49O4 [MþH]þ
485.3618, found 485.3621.
5.3.3. Methyl 3,4-seco-cycloart-4(28),24-diene-29-
hydroxy-23-oxo-3-oate (3). Colorless semi-solid. [a]
31
589
¼
5.3.4. 5,7,40-Trihydroxy-3,8-dimethoxyflavone (4). Yellow
needles from EtOH, mp 256.9–257.48C after darkening at
245.1–245.68C (Lit.26 2598C after darkening at 2488C,
Lit.27 242–2448C and 254–2568C). IR (KBr) nmax: 3424
(O–H stretching), 3227 (O–H stretching), 1656 (CvO
stretching of conjugated ketone), 1610, 1555, 1504, 1436,
1365, 1287, 1230, 1172, 1013, 930 cm21. Anal. calcd for
C17H14O7: C, 61.82; H, 4.27. Found: C, 62.04; H, 4.27. The
triacetate 16 crystallized from EtOH as colorless needles,
mp 164.9–165.68C. (Lit.27 167–1688C).
þ58.7 (c 0.8; CHCl3). UV (EtOH) lmax nm (log 1): 237
(4.16). IR (CHCl3) nmax: 3483 (O–H stretching of alcohol),
1729 (CvO stretching of ester), 1681 (CvO stretching of
conjugated ketone), 1615 (CvC), 1438, 1379, 1358, 1282,
1228, 1170, 1041, 904 cm21. 1H and 13C NMR: see Table 1.
COSY correlations H/H: 1a/1b, 2a, 2b; 1b/1a, 2a, 2b; 2a/2b,
1a, 1b; 2b/1a, 1b, 2a; 5/6a, 6b, 19a, 19b; 6a/5, 6b, 7a, 7b;
6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b, 8; 7b/6a, 6b, 7a, 8; 8/7a, 7b,
19a, 19b, 30; 11a/11b, 12, 19a, 19b; 11b/11a, 12, 19b;
12/11a, 11b, 18; 15/16a, 16b; 16a/15, 16b, 17; 16b/15, 16a;
17/16a, 16b; 18/12, 17; 19a/1a, 5, 8, 11a, 19b; 19b/5, 8, 11a,
11b, 19a; 20/21, 22a, 22b; 21/20, 22a, 22b; 22a/20, 21, 22b;
22b/20, 21, 22a; 24/26, 27; 26/24, 27; 27/24, 26; 28a/28b,
29; 28b/28a, 29; 29/28a, 28b; 30/8, 15. HMBC correlations:
see Table 2. EIMS (70 eV) m/z (%): 484 [M]þ (1), 469 (9),
453 (8), 438 (5), 411 (1), 397 (3), 386 (7), 371 (12), 353 (7),
5.3.5. 5,7,40-Trihydroxy-3,8,30-trimethoxyflavone (5).
Yellow needles from EtOH; mp 215.4–216.98C (Lit.26
217–2188C, Lit.27 215–2178C, Lit.28 216–2188C). IR
(KBr) nmax: 3367 (O–H stretching), 1651 (CvO stretching
of conjugated ketone), 1595, 1505, 1450, 1380, 1320, 1300,
1209, 1163, 1021, 983, 936 cm21. The triacetate 17

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Table 6. Observed HMBC correlations in compounds 13A, 13B and 14A, 14B
13A, 13B
Correlated H
8081
14A, 14B
Correlated H
C
C
1
2
3
4
2, 5, 19a, 19b
1a, 1b
1a, 1b, 2, 28
1
2
3
4
5
2, 5, 19a, 19b
1a, 1b
1a, 1b, 2, 28, 29
5, 28, 29
1a, 1b, 6a, 6b, 7a, 7b, 19a, 19b, 28, 29
5, 28, 29
5 (A)
5 (B)
6
1a, 6a, 28, 29
1a, 6a, 28, 29
5, 7b, 8 (A), 8 (B)
5, 6a, 6b
6
7
5, 7a, 7b, 8
5, 6a, 6b, 8
7
8 (A)
8 (B)
9
10
11
6a, 7b, 19a, 19b
6a, 7b, 19a, 19b
5, 7b, 11b, 19a, 19b
2, 5, 6a, 11a, 19a, 19b
12, 19a, 19b
8 (A)
8 (B)
9
10
11
6a, 6b, 7a, 7b, 11b, 15, 19a, 19b, 30 (A)
6a, 6b, 7a, 7b, 11b, 15, 19a, 19b, 30 (B)
5, 7a, 7b, 8, 11a, 11b, 12, 19a, 19b
1a, 1b, 2, 5, 6a, 6b, 8, 11a, 19a, 19b
8, 12, 19a, 19b
12 (A)
12 (B)
13 (A)
13 (B)
14 (A)
14 (B)
15 (A)
15 (B)
16 (A)
16 (B)
17 (A)
17 (B)
18 (A)
18 (B)
19
20 (A)
20 (B)
21 (A)
21 (B)
22 (A)
22 (B)
23 (A)
23 (B)
24 (A)
24 (B)
25 (A)
25 (B)
26 (A)
26 (B)
27
11a, 11b, 18 (A)
11a, 11b, 18 (B)
11b, 16a, 17 (A), 18 (A), 30
11b, 16a, 17 (B), 18 (B), 30
17 (A), 18 (A)
17 (B), 18 (B)
16a, 16b, 30
16a, 16b, 30
15, 17 (A)
15, 17 (B)
12
11a, 11b, 17 (A), 17 (B), 18
13
14
8, 12, 15, 18, 30 (A), 30 (B)
7a, 12, 15, 17 (A), 17 (B), 18, 30 (A), 30 (B)
15 (A)
15 (B)
16 (A)
16 (B)
17 (A)
17 (B)
18 (A)
18 (B)
19
8, 12, 17 (A), 30 (A)
8, 12, 17 (B), 30 (B)
15, 17 (A)
15, 17 (B)
18 (A), 21 (A), 22a, 22b, 30
18 (B), 21 (B), 22a, 22b, 30
12
12, 15, 18, 20, 21 (A), 22a (A), 22b (A)
12, 15, 18, 20, 21 (B), 22a (B), 22b (B)
12, 17 (A)
12, 17 (B)
1a, 1b, 5, 8, 11a, 11b
17 (A), 17 (B), 21 (A), 21 (B), 22a (A), 22a (B), 22b (A), 22b (B)
12
1a, 1b, 5, 8 (A), 8 (B), 11a, 11b
21 (A), 22a, 22b
21 (B), 22a, 22b
22a, 22b
22a, 22b
21 (A)
21 (B)
22a, 22b, 24 (A)
22a, 22b, 24 (B)
22b, 26 (A), 27
22b, 26 (B), 27
24 (A), 26 (A), 27
24 (B), 26 (B), 27
24 (A), 27
20
21
17 (A), 17 (B), 22a (A), 22a (B), 22b (A), 22 (B)
22 (A)
22 (B)
23 (A)
23 (B)
24
17 (A), 20, 21 (A)
17 (B), 20, 21 (B)
22a (A), 22b (A), 24 (A)
22a (B), 22b (B), 24 (B)
26 (A), 26 (B), 27
25 (A)
25 (B)
26
24 (A), 26 (A), 27
24 (B), 26 (B), 27
24 (A), 24 (B), 27
24 (B), 27
26 (A), 26 (B)
27 (A)
27 (B)
28
24 (A), 26 (A)
24 (B), 26 (B)
5, 29
28
5, 29
5, 28
5, 28
8 (A), 8 (B), 15
29 (A)
29 (B)
30
29
5, 28
30 (A)
30 (B)
8, 15
8, 15
crystallized from EtOH as colorless plates, mp 138.6–
139.48C (Lit.39 mp 138–1398C). HRFABMS calcd for
C24H23O11 [MþH]þ 487.1233, found 487.1244.
5.3.7. 5,40-Dihydroxy-3,6,7,8-tetramethoxyflavone (7).
Yellow needles from EtOH, mp 226.2–226.68C (Lit.29 mp
225–2268C). UV (MeOH) lmax nm (log 1): 283 (4.70), 343
(4.73). IR (KBr) nmax: 3222 (O–H stretching), 1648 (CvO
stretching of conjugated ketone), 1612, 1575, 1547, 1509,
1481, 1371, 1288, 1207, 1178, 1046, 1004, 920 cm21. Anal.
calcd for C19H18O8: C, 60.96; H, 4.85. Found: C, 61.21; H,
4.99. The diacetate 20 crystallized from EtOH as pale
yellow needles, mp 128.0–129.58C. The methylated
product 19 crystallized from EtOH as pale yellow needles,
mp 130.1–131.58C (Lit.40,41 130–1318C, Lit.42 131–
1328C).
5.3.6. 5,7,40-Trihydroxy-3,6,8-trimethoxyflavone (6).
Yellow needles from EtOH, mp 244.1–245.68C (Lit.26
244–2458C). UV (MeOH) lmax nm (log 1): 280 (3.20), 332
(3.17). IR (KBr) nmax: 3424 (O–H stretching), 3227 (O–H
stretching), 1656 (CvO stretching of conjugated ketone),
1609, 1555, 1504, 1436, 1365, 1287, 1230, 1172, 1012,
930 cm21. The triacetate 18 crystallized from EtOH as pale
yellow needles, mp 158.1–158.98C (Lit.40 159–1608C).
HRFABMS calcd for C24H23O11 [MþH]þ 487.1233, found
487.1258. The methylated product 19 crystallized from
EtOH as pale yellow needles, mp 130.1–131.58C (Lit.40,41
130–1318C, Lit.42 131–1328C).
5.3.8. 5,30-Dihydroxy-3,6,7,8,40-pentamethoxyflavone (8).
Yellow needles from EtOH, mp 171.6–172.28C (Lit.31
1708C, Lit.32 169–1708C, Lit.33 176–1778C). IR (CHCl3)

8082
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
Figure 2. NOSEY correlations of the isolated 1–3 and the modified 9–15.
30
589
n
max: 3546 (O–H stretching), 1647 (CvO stretching of
151.18C. [a] ¼þ64.0 (c 0.05, CHCl3). IR (CHCl3) nmax
:
conjugated ketone), 1596, 1561, 1512, 1481, 1463, 1376,
1275, 1249, 1135, 1052, 1006, 981 cm21. Anal. calcd for
C20H20O9: C, 59.41; H, 4.99. Found: C, 59.61; H, 5.12. The
diacetate 21 crystallized from EtOH as pale yellow needles,
mp 165.8–166.38C. The methylated product 22 crystallized
from EtOH as pale yellow needles, mp 130.5–131.88C
(Lit.43 131–1328C).
1702 (CvO of 6-membered ring ketone and CvO of the
side-chain ketone), 1467, 1383, 1366, 1213, 1113, 1008,
916 cm21. 1H and 13C NMR data see Tables 3 and 4. COSY
correlations H/H: 1a/1b, 2a, 2b, 19a; 1b/1a, 2a, 2b, 19b;
2a/1a, 1b, 2a, 29; 2b/1a, 1b, 2a, 28; 5/6a, 6b, 19a, 19b; 6a/5,
6b, 7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b; 7b/6a, 6b, 7a;
8/6b, 7a, 7b; 11a/11b, 12, 19a, 19b; 11b/11a, 12, 19b;
12/11a, 11b; 15/16a, 16b; 16a/15, 16b, 17; 16b/15, 16a, 17;
17/16a, 16b, 18, 20; 18/12, 17; 19a/1a, 5, 11a, 19b; 19b/1b,
5, 11a, 11b, 19a; 20/17, 21, 22a, 22b; 21/20, 22a; 22a/20, 21,
22b, 24; 22b/20, 22a, 24; 24/22a, 25, 26, 27; 25/24, 26, 27;
26/24, 25; 27/24, 25; 28/29; 29/28; 30/8, 15. HMBC
correlations: see Table 5. EIMS (70 eV) m/z (%): 440 [M]þ
(11), 425 (6), 355 (8), 340 (66), 325 (21), 313 (8), 302 (20),
271 (6), 255 (6), 243 (6), 221 (9), 217 (12), 202 (27), 187
(23), 175 (27), 161 (28), 147 (99), 135 (40), 121 (99), 107
5.4. Structure modification
5.4.1. Preparation of 5a-cycloart-24,25-dihydro-3,23-
dione (9). Compound 1 (30.5 mg, 0.097 mmol) in MeOH
(4 mL) was hydrogenated in the presence of 10% palladium
on carbon catalyst (0.7 mg). The catalyst was filtered, and
the filtrate after evaporation and recrystallization from
EtOH gave 9 (29.1 mg, 95%) as colorless plates, mp 149.5–

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
8083
Table 7. Cytotoxic and antimitotic activities (ASK assay) of the isolated 1–
8 and the modified compounds 9–22
(6 mL) was hydrogenated in the presence of 10% palladium
on carbon catalyst (5.9 mg). After work-up and recrystalli-
zation from EtOH, 10 (243.8 mg, 96%) was obtained as
Compound
Cytotoxicity
Cell line
ASK assay
30
colorless needles, mp 128.1–129.18C. [a] ¼258.18 (c
589
0.06, CHCl3). IR (CHCl3) nmax: 1727 (CvO of 5-membered
ring ketone), 1703 (CvO of 6-membered ring ketone and
CvO of saturated ketone in the side chain), 1466, 1417,
P-388 KB Col-2 BCA-1 Lu-1 ASK
1386, 1367, 1245, 1167, 1145, 1114, 976 cm21. 1H and 13
C
1
2
3
4
5
6
7
8
.20 .20 .20
.20 .20 .20
.20
.20
10.9
9.4
2.7
1.2
.20 .20
.20 .20
2
2
2
2
þ
2
2
þ
2
2
2
2
2
2
2
2
2
2
2
2
þ
2
NMR data: see Tables 3 and 4. COSY correlations H/H:
1a/1b, 2a, 2b, 19a; 1b/1a, 2a, 2b; 2a/1a, 1b, 2b; 2b/1a, 1b, 2a;
5/6a, 6b; 6a/5, 6b, 7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b;
7b/6a, 6b, 7a, 8; 8/7a, 7b; 11a/11b, 12; 11b/11a, 12; 12/11a,
11b; 15a/15b, 30; 15b/15a, 30; 17/18, 20, 21; 18/12, 17;
19a/1a, 19b; 19b/19a; 20/17, 21, 22a, 22b; 21/17, 20, 22a,
22b; 22a/21, 22b; 22b/20, 22a; 24/25, 26, 27; 25/24, 26, 27;
26/24, 25, 27; 27/24, 25, 26; 28/5, 29; 29/28; 30/15a, 15b,
18. HMBC correlations: see Table 5. EIMS (70 eV) m/z
(%): 454 [M]þ (2), 439 (100), 421 (4), 355 (3), 339 (4), 313
(4), 301 (8), 185 (3), 147 (3), 135 (4), 119 (3), 109 (3), 105
(5), 95 (3), 93 (4), 91 (5), 85 (8), 79 (4), 67 (4), 57 (12), 41
(8). HRFABMS calcd for C30H47O3 [MþH]þ 455.3513,
found 455.3515.
3.3
2.4
3.1
16.4 9.1
.20 .20
14.8 15.7
5.8
.20 16.3
13.1 5.9
.20 17.1
13.0 3.9
10.9
13.8 .20 .20
2.7 19.9 .20
0.05 0.09 8.8
13.8 .20 .20
8.8 .20 .20
.20 12.6 .20
4.8
0.63
.20
.20
.20
13.3
15.5
.20
.20
11.7
.20
15.7
.20
2.7
0.09 0.70
.20 .20
.20 .20
16.6 .20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1.8
7.3
8.7 4.2
.20 10.8
5.9
11.2
19.3 .20
.20 .20
.20 .20 .20
14.0 11.6 9.4
5.3 .20 .20
18.3 .20 .20
17.5 14.1 14.1
.20 16.8 .20
10.4 14.8 .20
0.27 0.06 13.0
18.1 7.00 .20
9.1
.20
16.9 13.1
.20 .20
.20 14.7
.20 .20
11.4 11.1
0.49 2.36
.20 .20
5.4.3. Reaction of 5a-cycloart-24-ene-3,23-dione (1) with
2.2 equiv. of MCPBA. To a stirred solution of 1 (100 mg,
0.2283 mmol) in anhydrous CH2Cl2 (6 mL) was sequen-
tially added MCPBA (85%, 102 mg, 0.5023 mmol,
2.2 equiv.) and anhydrous NaHCO3 (57.5 mg, 0.6844 mmol,
3 equiv.). The reaction mixture was left stirring at room
temperature for 7 h, then diluted with CH2Cl2 (15 mL),
washed with a 1:1 mixture of 5% Na2S2O3 and 5% NaHCO3
(3£20 mL) to remove excess MCPBA, and finally with H2O
(3£20 mL). The organic layer was dried with anhydrous
Na2SO4, filtered and evaporated to dryness to afford a white
solid (115.2 mg). Purification by preparative thin-layer
chromatography on silica plates (EtOAc/hexane, 1:4)
0.53
.20
Cytotoxic assay: ED50#5 mg/mL is considered active; P-388: murine
lymphocytic leukemia, KB: human nasopharyngeal carcinoma, Col-2:
human colon cancer, BCA-1: human breast cancer, Lu-1: human lung
cancer, ASK: rat glioma; þ¼active; 2¼inactive.
(56), 105 (51), 95 (45), 93 (41), 85 (57), 79 (29), 67 (35), 57
(100). HRFABMS calcd for C30H49O2 [MþH]þ 441.3720,
found 441.3716.
5.4.2. Preparation of 5a-cycloart-24,25-dihydro-3,16,23-
trione (10). Compound 2 (252.5 mg, 0.56 mmol) in MeOH
Table 8. Anti-HIV activities of the isolated 1–8 and the modified 9–22 by syncytium and HIV-1 RT assays
Compound
Anti-syncytium (MC99þ1A2)
Anti-HIV-1 RT
% Inhibition
IC50 (mg/mL)
EC50 (mg/mL)
TI (IC50/EC50
)
Activity
Activity
1
2
3
4
5
6
7
8
215
.250
,3.9
14.0
,3.9
41.8
77.0
,7.8
64.1
.125
18.0
,7.8
20.5
.125
12.3
16.4
,3.9
16.8
.125
6.7
–
–
–
–
Ia
Aa
T
A
T
A
Aa
T
A
Aa
A
12.0
7.0
99.9
35.2
19.1
25.2
9.4
15.0
19.3
22.0
35.8
12.9
2.6
7.8
13.9
8.3
0
I
I
A
W
I
I
I
I
W
I
W
I
I
I
I
I
I
I
I
146.2
–
,3.9
–
,3.9
,3.9
–
17.6
,3.9
6.2
–
5.1
–
,3.9
,3.9
–
,3.9
–
,3.9
–
.1.7
.3.6
.10.7
.19.5
–
9
3.6
.32.1
2.9
–
4.0
–
10
11
12
13
14
15
16
17
18
19
20
21
22
T
Aa
Ia
Aa
Aa
T
Aa
Ia
Aa
T
.3.1
.4.2
.4.3
.1.7
–
–
6.0
0
0
0
0.8
I
I
I
,3.9
,3.9
–
–
–
T
Syncytium assay: A¼active in the assay for inhibition of syncytium formation by MC99-infected cells, Aa¼active in the assay for reduction of syncytium
formation by MC99 virus, I¼inactive in the syncytium inhibition assay, Ia¼inactive in the reduction assay, T¼toxic; Radioisotopic RT assay: A¼very active
(.70% inhibition), M¼moderately active (.50–70% inhibition), W¼weakly active (30–50% inhibition), I¼inactive (,30% inhibition).

8084
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
afforded heptanolide 11 (40.4 mg, 39%) and a 1:1.2
diastereomeric mixture of 13A and 13B (25.5 mg, 24%).
30; 18 of B/12, 17 of B, 30; 19a/1a, 5, 19b; 19b/5, 8 of A and
B, 19a; 20/17 of A and B, 21 of A and B, 22a, 22b; 21 of
A/20, 22a of A; 21 of B/20, 22a of B, 22a/20, 21 of A and B;
24 of A/26 of A, 27; 24 of B/26 of B, 27; 26 of A/24 of A,
27; 26 of B/24 of B, 27; 27/24 of A and B, 26 of A and B;
28/29; 29/5, 28; 30/8 of A and B, 15. HMBC correlations
see Table 6. EIMS (70 eV) m/z (%): 470 [M]þ (2), 455 (5),
430 (4), 397 (4), 383 (8), 365 (15), 356 (39), 341 (41), 329
(11), 327 (18), 313 (19), 283 (16), 269 (15), 253 (12), 241
(18), 233 (26), 213 (27), 201 (31), 189 (32), 187 (42), 175
(58), 173 (60), 161 (72), 147 (100), 141 (9), 133 (75), 121
(80), 107 (70), 99 (8), 93 (76), 91 (75), 79 (59), 67 (29), 55
(20). HRFABMS calcd for C30H46O4Na [MþNa]þ
493.3282, found 493.3259.
The heptanolide 11 was recrystallized from EtOH to give
30
589
colorless needles, mp 134.8–135.48C. [a] ¼þ18.3 (c
0.07, CHCl3). UV (EtOH) lmax nm (log 1): 234 (4.19). IR
(CHCl3) nmax: 1702 (CvO of 7-membered lactone), 1685
(CvO of conjugated ketone), 1616 (CvC), 1446, 1389,
1
1294, 1225, 1193, 1113, 1103, 1035, 1012, 980 cm21. H
and 13C NMR data see Tables 3 and 4. COSY correlations
[dH/dH (H/H)]: 1a/1b, 2a, 2b, 19a, 19b; 1b/1a, 2a, 2b; 2a/1a,
1b, 2b; 2b/1a, 1b, 2a; 5/6a, 6b, 19a, 19b, 28, 29; 6a/5, 6b, 7a,
7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b, 8; 7b/6a, 6b, 7a, 8; 8/7a,
7b, 30, 19b; 11a/11b, 12, 19a, 19b; 11b/11a, 12, 19a, 19b;
12/11a, 11b, 18; 15/16a, 16b, 30; 16a/16b, 17; 16b/15, 16a,
17; 17/16a, 16b, 21; 18/12, 17; 19a/1a, 5, 11a, 11b, 19b;
19b/1a, 5, 8, 11a, 11b, 19a; 20/17, 21, 22a, 22b; 21/20, 22a,
22b; 22a/20, 21, 22b; 22b/20, 21, 22a; 24/26, 27; 26/24, 27;
27/24, 26; 28/29; 29/5, 28; 30/8. HMBC correlations: see
Table 5. EIMS (70 eV) m/z (%): 454 [M]þ (2), 439 (4), 421
(1), 371 (1), 356 (9), 341 (11), 327 (2), 313 (3), 298 (2), 283
(3), 257 (1), 233 (3), 219 (2), 201 (3), 187 (4), 175 (5), 161
(6), 147 (19), 133 (10), 125 (16), 121 (22), 105 (15), 91 (20),
83 (100), 79 (19), 67 (14), 55 (47), 41 (22). HRFABMS
calcd for C30H47O3 [MþH]þ 455.3513, found 455.3527.
5.4.4. Reaction of 5a-cycloart-24-ene-3,16,23-trione (2)
with 2.2 equiv. of MCPBA. According to the procedure
described in Section 5.4.3, compound
2 (100 mg,
0.2212 mmol) in anhydrous CH2Cl2 (6 mL) was reacted
with MCPBA (85%, 98.8 mg, 0.4867 mmol, 2.2 equiv.) and
anhydrous NaHCO3 (55.8 mg, 0.6637 mmol, 3 equiv.).
After left stirring at room temperature for 7 h and usual
work-up, a white solid (108.9 mg) was obtained. Purifi-
cation by preparative thin-layer chromatography on silica
plates (EtOAc/CH2Cl2/hexane, 3:1:6 as eluent) afforded
heptanolide 12 (45.9 mg, 44%) and a 1:1.3 diastereomeric
mixture of heptanolide–epoxides 14A/14B (25.7 mg, 24%).
The mixture of 13A and 13B was obtained as a colorless
solid, mp 129.1–130.78C. [a] ¼þ25.5 (c 0.06, CHCl3).
30
589
IR (CHCl3) nmax: 1703 (CvO of 7-membered lactone and
CvO of the side-chain ketone), 1447, 1459, 1390, 1294,
The heptanolide 12 was recrystallized from EtOH to give
30
589
colorless needles, mp 117.8–119.08C. [a] ¼248.6 (c
1
1193, 1114, 1030, 1012, 979 cm21. H NMR (300 MHz)
0.07, CHCl3). UV (EtOH) lmax nm (log 1): 231 (4.19). IR
(CHCl3) nmax 1727 (CvO of 5-membered ring ketone),
1702 (CvO of 7-membered lactone), 1687 (CvO of
conjugated ketone), 1618 (CvC), 1542, 1447, 1389, 1293,
dH: 3.41 (s, H-24 of A), 3.34 (s, H-24 of B), 2.69 (obsc., H-
2), 2.55 (obsc., H-22a) 2.34 (obsc., H-22b), 2.10 (obsc., H-
11a), 2.09 (obsc., H-5), 2.08 (obsc., H-20), 1.93 (m, H-16a),
1.81 (obsc., H-1a), 1.80 (obsc., H-6a), 1.69 (obsc., H-17 of
A), 1.67 (obsc., H-12), 1.65 (obsc., H-17 of B), 1.52 (m., H-
1b), 1.46 (s, H-29), 1.45 (obsc., H-8 of A), 1.44 (s, H-26 of
A), 1.43 (obsc., H-8 of B), 1.43 (s, H-26 of B), 1.40 (s, H-
28), 1.36 (obsc., H-15), 1.35 (obsc., H-16b), 1.30 (obsc., H-
7a), 1.27 (s, H-27), 1.10 (obsc., H-11b), 1.09 (obsc., H-7b),
1.02 (s, H-18 of B), 1.01 (s, H-18 of A), 0.93 (s, H-30), 0.91
(d, J¼6.5 Hz, H-21 of B), 0.90 (d, J¼6.5 Hz, H-21 of A),
0.69 (obsc., H-6b), 0.68 (d, J¼5.0 Hz, H-19a), 0.62 (d,
J¼5.0 Hz, H-19b). 13C NMR (75 MHz) dC: 206.89 (C-23 of
B), 206.70 (C-23 of A), 175.43 (C-3), 87.19 (C-4), 65.73 (C-
24 of A), 65.61 (C-24 of B), 61.03 (C-25 of A), 60.96 (C-25
of B), 52.32 (C-17 of B), 52.21 (C-17 of A), 49.76 (C-5 of
A), 49.75 (C-5 of B), 48.89 (C-14 of B), 48.88 (C-14 of A),
48.67 (C-8 of B), 48.65 (C-8 of A), 48.14 (C-22 of B), 48.09
(C-22 of A), 45.00 (C-13 of A), 44.99 (C-13 of B), 35.65 (C-
15 of A), 35.64 (C-15 of B), 35.05 (C-2), 32.85 (C-20 of A),
32.72 (C-12 of A), 32.70 (C-12 of B), 32.44 (C-20 of B),
30.97 (C-28), 30.13 (C-1), 29.56 (C-19), 28.41 (C-16 of A),
28.34 (C-16 of B), 27.24 (C-10), 27.16 (C-11), 26.00 (C-6),
25.32 (C-7), 24.76 (C-26 of B), 24.74 (C-26 of A), 23.08 (C-
29 of B), 23.07 (C-29 of A), 22.91 (C-9), 19.47 (C-30),
19.39 (C-21 of A), 19.35 (C-21 of B), 18.51 (C-27), 18.36
(C-18 of A), 18.34 (C-18 of B). COSY correlations H/H:
1a/1b, 2, 19a; 1b/1a, 2; 2/1a, 1b; 5/6a, 6b, 19a, 19b, 29; 6a/5,
7b; 6b/5, 7a; 7a/6b, 8 of A and B; 7b/6a, 8 of A and B; 8 of A
and B/7a, 7b, 19b; 11a/11b, 12; 11b/11a, 12; 12/11a, 11b;
15/16a, 16b, 30; 16a/15, 16b, 17 of A and B; 16b/15, 16a, 17
of A and B; 17 of A and B/16a, 16b, 20; 18 of A/12, 17 of A,
1
1225, 1116, 1101, 980 cm21. H and 13C NMR data: see
Tables 3 and 4. COSY correlations H/H: 1a/1b, 2a, 2b, 19a;
1b/1a, 2a, 2b, 19b; 2a/1a, 1b, 2b; 2b/1a, 1b, 2a; 5/6a, 6b, 29;
6a/5, 6b, 7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b, 8; 7b/6a, 6b,
7a, 8; 8/7a, 7b; 11a/11b, 12, 19a; 11b/11a, 12; 12/11a, 11b;
15/17, 30; 17/15, 18, 21; 18/12, 17, 30; 19a/1a, 5, 11a, 11b,
19b; 19b/1a, 5, 8, 11b, 19a; 20/17, 21, 22a, 22b; 21/17, 20,
22a, 22b; 22a/20, 21, 22b; 22b/20, 21, 22a; 24/26, 27; 26/24,
27; 27/24, 26; 28/29; 29/5, 28; 30/8, 15. HMBC corre-
lations: see Table 5. EIMS (70 eV) m/z (%): 468 [M]þ (8),
453 (63), 397 (19), 371 (8), 355 (8), 339 (10), 234 (6), 135
(10), 125 (23), 105 (13), 98 (16), 91 (17), 83 (100), 69 (17),
55 (41), 41 (17). HRFABMS calcd for C30H45O4 [MþH]þ
469.3306, found 469.3313.
The mixture of 14A and 14B was obtained as a colorless
31
589
solid, mp 200.0–201.58C. [a] ¼þ10 (c 0.06, CHCl3). IR
(CHCl3) nmax 1725 (CvO of 5-membered ring ketone),
1706 (CvO of 7-membered lactone and CvO of the side-
chain ketone), 1459, 1447, 1389, 1293, 1245, 1187, 1115,
1101, 1033, 980 cm21. 1H NMR (300 MHz) dH: 3.49 (s, H-
24 of A), 3.44 (s, H-24 of B), 3.34 (dd, J¼17.3, 2.5 Hz, H-
22a of A), 3.29 (dd, J¼17.3, 2.9 Hz, H-22a of B), 2.72 (m,
H-2), 2.52 (dd, J¼17.3, 11.2 Hz, H-22b of A), 2.50 (dd,
J¼17.3, 11.6 Hz, H-22b of B), 2.38 (m, H-20), 2.29 (d,
J¼9.1 Hz, H-17 of B), 2.27 (d, J¼9.1 Hz, H-17 of A), 2.23
(m, H-11a), 2.12 (dd, J¼12.7, 5.3 Hz, H-5), 2.04 (br s,
H-15), 1.88 (obsc., H-12), 1.85 (obsc., H-6a), 1.84
(obsc., H-1a), 1.56 (obsc., H-1b), 1.54 (obsc., H-8), 1.47 (s,

P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
8085
H-29), 1.45 (s, H-26 of A), 1.44 (s, H-26 of B), 1.42 (s, H-
28), 1.30 (obsc., H-7a), 1.29 (s, H-27), 1.24 (obsc., H-11b),
1.20 (obsc., H-7b), 1.16 (s, H-18), 1.12 (s, H-30 of B), 1.11
(s, H-30 of A), 1.00 (d, J¼6.2 Hz, H-21 of B), 0.99 (d,
J¼6.2 Hz, H-21 of A), 0.73 (d, J¼4.9 Hz, H-19a), 0.71
(obsc., H-6b), 0.69 (d, J¼4.9 Hz, H-19b). 13C NMR
(75 MHz) dC: 219.10 (C-16 of A), 219.04 (C-16 of B),
205.78 (C-23 of B), 205.58 (C-23 of A), 175.15 (C-3), 86.92
(C-4), 65.57 (C-24), 60.96 (C-25 of B), 60.89 (C-25 of A),
60.54 (C-17 of B), 60.49 (C-17 of A), 50.98 (C-15 of A),
50.95 (C-15 of B), 49.62 (C-5), 47.96 (C-8 of B), 47.94 (C-8
of A), 46.83 (C-22 of B), 46.80 (C-22 of A), 44.72 (C-14),
42.02 (C-13), 34.92 (C-2), 31.41 (C-12), 30.94 (C-28),
29.84 (C-1 and C-19), 27.70 (C-10), 26.76 (C-11), 26.67 (C-
20), 25.67 (C-6), 25.60 (C-7), 24.71 (C-26), 23.03 (C-29),
22.19 (C-9), 20.33 (C-21), 19.77 (C-30 of B), 19.75 (C-30 of
A), 19.02 (C-18 of A), 18.97 (C-18 of B), 18.40 (C-27 of B),
18.16 (C-27 of A). COSY correlations H/H: 1a/1b, 2, 5, 19a;
1b/1a, 2, 19a, 19b; 2/1a, 1b; 5/1a, 6a, 6b, 19b, 29; 6a/5, 6b,
7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b, 8; 7b/6a, 6b, 7a, 8;
8/7a, 7b, 15; 11a/11b, 12, 19a; 11b/11a, 12; 12/11a, 11b, 18;
15/8, 17 of A and B, 30; 17 of A/15, 18, 20, 21 of A; 17 of
B/15, 18, 20, 21 of B; 18/12, 17 of A and B; 19a/1a, 1b, 5, 8,
11a, 11b, 19b; 19b/1a, 1b, 5, 8, 11b, 19a; 20/17 of A and B,
21 of A and B, 22a of A and B, 22b of A and B; 21 of A/20,
22a of A, 22b of A; 21 of B/20, 22a of B, 22b of B; 22a of
A/20, 21 of A, 22b of A; 22a of B/20, 21 of B, 22b of B; 22b
of A/20, 21 of A, 22a of A; 22b of B/20, 21 of B, 22a of B;
24 of A/26 of A, 27; 24 of B/26 of B, 27; 26 of A/24 of A,
27; 26 of B/24 of B, 27; 27/24 of A and B, 26 of A and B;
28/5, 29; 29/5, 28; 30 of A and B/8, 15. HMBC correlations:
see Table 6. EIMS (70 eV) m/z (%): 484 [M]þ (8), 469 (9),
451 (6), 426 (2), 413 (11), 397 (100), 355 (6), 339 (25), 311
(3), 287 (7), 259 (5), 243 (4), 229 (8), 203 (5), 191 (11), 178
(24), 161 (10), 139 (15), 119 (21), 105 (21), 91 (26), 83 (33),
69 (55), 55 (37), 43 (63). HRFABMS calcd for C30H44O5Na
[MþNa]þ 507.3075, found 507.3072.
21/17, 22a, 22b; 22a/20, 21, 22b; 22b/20, 21, 22a; 24/26, 27;
26/24, 27; 27/24, 26; 28/29; 29/5, 28; 30/12, 15. HMBC
correlations: see Table 5. EIMS (70 eV) m/z (%): 501
[MþH]þ (3), 482 (5), 467 (16), 453 (45), 427 (51), 422 (8),
397 (19), 371 (27), 355 (8), 339 (14), 299 (6), 271 (3), 233
(8), 177 (11), 147 (8), 125 (26), 91 (17), 83 (100), 73 (24),
59 (42), 43 (16). HRFABMS calcd for C31H49O5 [MþH]þ
501.3567, found 501.3603.
Acknowledgements
We wish to acknowledge the financial support from the
National Research Council of Thailand (NRCT). We
also thank the Higher Education Development Project:
Postgraduate Education and Research Program in Chem-
istry and the Thailand Research Fund (TRF) through a
Senior Scholar Award to V. R. for the partial support. W. P.
is grateful to the Institutional Strengthening Program,
Faculty of Science, Mahidol University, for providing a
research scholarship.
References
1. Smitinand, T. Thai Plant Names; Pra Cha Chon Co., Ltd:
Bangkok, 2001; Revised edition by The Forest Herbarium,
Royal Forest Department, pp 248–249.
2. Woo, W. S.; Lee, E. B.; Shin, K. H.; Kang, S. S.; Chi, H. J.
Korean J. Pharmacogn. 1981, 12, 153–170.
3. Mbela, T. K. M.; Shabani, M.; Dieyi, S.; Cimanga, K.; Moswa,
L. Fitoterapia 1992, 63, 179–181.
4. Jain, S. P. Int. J. Crude Drug Res. 1989, 27, 29–32.
5. El-Hamidi, A. Planta Med. 1970, 18, 278–280.
6. Croft, K. D.; Tu’Ipulotu, P. S. Pac. J. Nat. Sci. 1980, 1, 45–47.
7. Nayar, S. L. Bull. Natl Inst. Sci. India 1955, 4, 137–145.
8. Lu, R. F.; Aeng, Y. R.; Wang, W. C. Sheng Chih Yu Bi Yun
1981, 12, 16–18.
5.4.6. Methanolysis of heptanolide 12. A solution of
heptanolide 12 (50 mg, 0.1068 mmol) in dry methanol
(2 mL) was added dropwise to a stirred mixture of NaOMe
(6.34 mg, 0.1177 mmol, 1.1 equiv.) in dry MeOH (6 mL) at
08C. The reaction mixture was left stirring at room
temperature for 6 h and then acidified with 2N HCl until
the pH,6.5–7.0. After dilution with water (50 mL) and
extraction with CH2Cl2 (3£20 mL), the combined organic
layer was washed with water (3£50 mL), dried over
anhydrous Na2SO4, filtered and evaporated to dryness to
give a white semi-solid (48.2 mg). Purification by prepara-
tive thin-layer chromatography on silica plates, eluting with
9. Takase, H.; Imanishi, K.; Miura, O.; Yumioka, E.; Watanabe,
H. Jpn. J. Pharmacol. 1989, 49, 301–308.
10. Laurens, A.; Mboup, S.; Tignokpa, M.; Sylla, O.; Masquelier,
J. Pharmazie 1985, 40, 482–485.
11. Hussain, M. M.; Sokomba, E. N.; Shok, M. Int. J. Pharmacogn.
1991, 29, 94–100.
12. Manson, D. J. Malaria Inst. India 1939, 2, 85–93.
13. Silva, G. L.; Gil, R. R.; Cui, B.; Chai, H.; Santisuk, T.; Srisook,
E.; Reutrakul, V.; Tuchinda, P.; Sophasan, S.; Sujarit, S.;
Upatham, S.; Lynn, S. M.; Farthing, J. E.; Yang, S.-L.; Lewis,
J. A.; O’Neill, M. J.; Farnsworth, N. R.; Cordell, G. A.;
Pezzuto, J. M.; Kinghorn, A. D. Tetrahedron 1997, 53,
529–538.
EtOAc–hexane (1:4) gave the hydroxy-ester 15 (42.6 mg,
80% yield) as a semi-solid. [a] ¼259.5 (c 0.39, CHCl3).
30
589
UV (EtOH) lmax nm (log 1): 235 (5.57). IR (CHCl3) nmax
:
3492 (O–H stretching), 1727 (CvO of ester and 5-
membered ring ketone), 1684 (CvO of conjugated ketone),
1618 (CvC), 1447, 1387, 1292, 1244, 1187, 1173, 1116,
1101, 1053, 1035, 979 cm21. 1H and 13C NMR see Tables 3
and 4. COSY correlations H/H: 1a/1b, 2a, 2b; 1b/1a, 2a, 2b;
2a/1a, 1b, 2b; 2b/1a, 1b, 2a; 5/6a, 6b, 19a, 19b, 28; 6a/5, 6b,
7a, 7b; 6b/5, 6a, 7a, 7b; 7a/6a, 6b, 7b, 8; 7b/6a, 6b, 7a, 8;
8/7a, 7b, 15; 11a/11b, 12, 19a, 19b; 11b/11a, 12, 19b;
12/11a, 11b, 18; 15/8, 17, 30; 17/15, 18, 21; 18/12, 17;
19a/1b, 5, 11a, 19b; 19b/1b, 5, 11a, 19a; 20/21, 22a, 22b;
14. Achenbach, H.; Frey, D. Phytochemistry 1992, 31,
4263–4274.
15. Davies, N. W.; Miller, J. M.; Naidu, R.; Sotheeswaran, S.
Phytochemistry 1992, 31, 159–162.
16. Furlan, M.; Roque, N. F.; Filho, W. W. Phytochemistry 1993,
32, 1519–1522.
17. Kikuchi, M.; Yoshikoshi, A. Chem. Lett. 1972, 725–728.
18. Sun, H.-D.; Qui, S.-X.; Lin, L.-Z.; Wang, Z.-Y.; Lin, Z.-W.;
Pengsuparp, T.; Pezzuto, J. M.; Fong, H. H. S.; Cordell, G. A.;
Farnsworth, N. R. J. Nat. Prod. 1996, 59, 525–527.

8086
P. Tuchinda et al. / Tetrahedron 58 (2002) 8073–8086
19. Liu, J.-S.; Huang, M.-F.; Ayer, W. A.; Bigam, G. Tetrahedron
Lett. 1983, 24, 2355–2358.
33. Shi, Q.; Chen, K.; Li, L.; Chang, J.-J.; Autry, C.; Kozuka, M.;
Konoshima, T.; Estes, J. R.; Lin, C. M.; Hamel, E.; McPhail,
A. T.; McPhail, D. R.; Lee, K.-H. J. Nat. Prod. 1995, 58,
475–482.
20. Raldugin, V. A.; Kukina, T. P.; Yaroshenko, N. I.; Pentegora,
V. A. Khim. Prir. Soedin. 1987, 2, 306–307.
21. Chen, Y.; Lin, Z.; Zhang, H.; Sun, H. Phytochemistry 1990,
29, 3358–3359.
34. Likhitwitayawuid, K.; Angerhofer, C. K.; Cordell, G. A.;
Pezzuto, J. M.; Ruangrungsi, N. J. Nat. Prod. 1993, 56, 30–38.
35. Tan, G. T.; Pezzuto, J. M.; Kinghorn, A. D.; Hughes, S. H.
J. Nat. Prod. 1991, 54, 143–154.
22. Tan, R.; Xue, H.; Li, L.-N. Planta Med. 1991, 57, 87–88.
23. Chen, G.-F.; Li, Z.-L.; Pan, D.-J.; Tang, C.-M.; He, X.; Xu, G.-
Y.; Chen, K.; Lee, K.-H. J. Nat. Prod. 1993, 56, 1114–1122.
24. Cabrera, G. M.; Gallo, M.; Seldes, A. M. J. Nat. Prod. 1996,
59, 343–347.
36. Nara, P. L.; Hatch, W. C.; Dunlop, N. M.; Roeby, W. G.;
Arthur, L. O.; Gonda, M. A.; Fischinger, P. J. AIDS Res. Hum.
Retrovir. 1987, 3, 283–302.
25. Atta-ur-Rahman; Nasir, H.; Asif, E.; Ali, S. S.; Iqbal, Z.;
Choudhary, M. I.; Clady, J. Tetrahedron 1992, 48,
3577–3584.
37. Kiser, R.; Makovsky, S.; Terpening, S. J.; Liang, N.; Clayton,
D. A. J. Virol. Methods 1996, 58, 99–109.
38. Tuchinda, P.; Pohmakotr, M.; Reutrakul, V.; Thanyachareon,
W.; Sophasan, S.; Yoosook, C.; Santisuk, T.; Pezzuto, J. M.
Planta Med. 2001, 67, 572–575.
26. Roitman, J. N.; James, L. F. Phytochemistry 1985, 24, 835–848.
27. Ghisalberti, E. L.; Jefferies, P. R.; Stacey, C. I. Aust. J. Chem.
1967, 20, 1049–1053.
39. Horie, T.; Tsukayama, M.; Kawamura, Y.; Seno, M. J. Org.
Chem. 1987, 52, 4702–4709.
28. Wang, Y.; Hamburger, M.; Gueho, J.; Hostettmann, K.
Phytochemistry 1989, 28, 2323–2327.
40. Herz, W.; Anderson, G. D.; Wagner, H.; Maurer, G.; Maurer,
I.; Flores, G.; Farkas, L. Tetrahedron 1975, 31, 1577–1581.
41. Herz, W.; Anderson, G. D.; Wagner, H.; Maurer, G.; Flores,
G.; Farkas, L. Tetrahedron Lett. 1973, 28, 2571–2572.
42. Rashid, M. A.; Armstrong, J. A.; Gray, A. I.; Waterman, P. G.
Phytochemistry 1992, 31, 1265–1269.
29. Gupta, S. R.; Seshadri, T. R.; Sood, G. R. Phytochemistry
1973, 12, 2539–2540.
30. El-Ansari, M. A.; Barron, D.; Abdalla, M. F.; Saleh, N. A. M.;
´ ´
Le Quere, J. L. Phytochemistry 1991, 30, 1169–1173.
31. Lichius, J. J.; Thoison, O.; Montagnac, A.; Pais, M.; Gueritte-
Voegelein, F.; Sevenet, T.; Cosson, J.-P.; Hadi, A. H. A. J. Nat.
Prod. 1994, 57, 1012–1016.
43. Martino, V. S.; Ferraro, G. E.; Coussio, J. D. Phytochemistry
1976, 15, 1086–1087.
32. Wollenweber, E.; Stern, S.; Roitman, J. N.; Yatskievych, G.
Phytochemistry 1989, 28, 303–305.