Triterpenes from natural and transformed roots of Plocama pendula

Article abstract of DOI:10.1021/np0680026

Plocama pendula root cultures transformed by Agrobacterium rhizogenes were established. From these cultures two new pentacyclic triterpenes, 3-epi-pomolic acid 3α-acetate (3α-acetoxy-19α-hydroxyursa-12-en-28-oic acid) (1) and baloic acid (3α-acetoxy-19α-h

Full text of DOI:10.1021/np0680026

1092
J. Nat. Prod. 2006, 69, 1092-1094
Triterpenes from Natural and Transformed Roots of Plocama pendula
Braulio M. Fraga,* Carmen E. D´ıaz, and Nayra Quintana
Instituto de Productos Naturales y Agrobiolog´ıa, CSIC, AVenida Astrof´ısico F. Sa´nchez 3, 38206 La Laguna, Tenerife, Spain
ReceiVed February 3, 2006
Plocama pendula root cultures transformed by Agrobacterium rhizogenes were established. From these cultures two
new pentacyclic triterpenes, 3-epi-pomolic acid 3R-acetate (3R-acetoxy-19R-hydroxyursa-12-en-28-oic acid) (1) and
baloic acid (3R-acetoxy-19R-hydroxyursa-12,20(30)-dien-28-oic acid) (9), were isolated. The former was also obtained
from P. pendula natural roots, together with another two new pentacyclic triterpenes, 3-epi-pomolic acid (3R,19R-
dihydroxyursa-12-en-28-oic acid) (3) and 19R-hydroxyoleanonic acid (19R-hydroxy-3-oxo-olean-12-en-28-oic acid) (12).
Table 1. 13C NMR Data of 1, 4, 8, 9, and 12
Plocama pendula Ait. (Rubiaceae) is an endemic plant of the
Canary Islands. As with other species of this family, it is
carbon
1
4
8
9
12
characterized phytochemically by its content of anthraquinones.1,2
In this paper we describe the isolation of two new triterpenes, 3-epi-
pomolic acid 3R-acetate (1) and baloic acid (9), from transformed
root cultures by Agrobacterium rhizogenes and another two new
compounds, 3-epi-pomolic acid (3) and 19R-hydroxyoleanonic acid
(12), from the natural roots of P. pendula.
1
2
3
4
5
6
7
8
33.4
22.6
78.2
39.9
49.9
18.1
32.4
36.4
46.9
36.8
23.5
129.4
137.7
41.1
28.1
25.3
47.6
52.8
73.0
41.0
25.9
37.3
27.4
21.8
15.0
16.9
24.6
183.2
27.4
16.1
32.8
25.1
76.1
40.0
48.8
18.3
32.6
37.0
46.9
37.2
23.5
129.3
137.9
41.1
28.1
25.9
47.8
53.2
73.1
41.1
25.4
37.4
27.4
22.2
14.9
16.6
24.6
178.3
28.2
16.1
38.1
23.6
80.9
37.7
55.2
18.2
32.6
39.9
47.1
36.9
23.5
129.3
137.9
41.0
28.2
25.3
47.8
52.8
73.1
41.0
25.9
37.1
27.4
16.6
15.3
16.9
24.4
183.6
28.0
16.1
33.6
22.7
78.2
39.7
50.1
18.1
32.9
36.5
47.0
37.0
23.4
129.8
137.8
41.5
27.8
25.5
47.7
54.1
72.5
152.3
27.4
35.8
27.8
21.9
15.2
17.1
23.7
181.6
29.9
107.3
38.9
34.2
217.4
47.5
55.4
19.7
32.1
39.6
47.2
37.0
23.8
124.9
142.7
41.4
27.9
27.5
45.3
43.6
81.6
34.6
28.1
32.5
26.3
21.4
14.7
17.0
24.4
183.0
28.1
24.9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
and C-5, due to the γ-gauche effect.6 The 13C NMR spectrum (Table
1), which was assigned unambiguously using 2D NMR data (COSY,
HSQC, and HMBC), was in accordance with this structure. This
was also confirmed chemically. Methylation of 1 with diazomethane
afforded the acetate methyl ester 2. Hydrolysis of this led to the
3R-alcohol, 4, which was oxidized with Jones reagent to afford
the 3-oxo derivative, 6. This was identical with the methyl ester of
pomonic acid.7 Accordingly, the structure of compound 1 was
determined as 3-epi-pomolic acid 3R-acetate (3R-acetoxy-19R-
hydroxyursa-12-en-28-oic acid).
The HRMS of 1 showed a molecular ion at m/z 514.3655,
corresponding to the molecular formula, C32H50O5, and the loss of
acetic acid at m/z 454. The 1H NMR spectrum showed the signals
of six angular methyls, the C-30 secondary methyl at δ 0.96 (d, J
) 7 Hz), the methyl of an acetate group, the H-18 singlet at δ
2.55, the H-3 singlet at 4.63, geminal to the acetoxy group, and
the H-12 triplet at δ 5.37 (J ) 3.5 Hz). The resonances of H-18,
the C-29 methyl (δ 1.24), geminal to a hydroxyl group, and the
C-30 methyl were characteristic of pomolic acid (7) derivatives,3,4
which possess an R-alcoholic group at C-19. The location of the
acetoxy group at C-3 was determined considering the correlation
of the two methyls at C-4 (H-23 and H-24) with C-3 in the HMBC
spectrum. The R-axial orientation of this acetoxy group was
established by the form of the signal of its geminal proton observed
at δ 4.63 (br s)5 and the high-field shift of the resonances of C-1
A second new triterpene obtained from the hairy roots of P.
pendula was named baloic acid (9). The HRMS showed the
1
molecular formula, C32H48O5. Its H NMR spectrum showed six
angular methyls, an acetate group, a proton geminal to an axial
acetoxy group (δ 4.64, br s), an exocyclic methylene (δ 4.79, s
and 5.03, s), and a vinylic proton (δ 5.44, t). This spectroscopic
behavior was similar to that of 1, except for an exocyclic double
bond instead of a secondary methyl group. The 13C NMR spectrum
(Table 1) showed the resonance of the carbons bearing oxygens,
C-3, C-19, and C-28, at δ 78.2, 72.5, and 181.6, respectively, while
the vinylic carbons C-12, C-13, C-20, and C-30 appeared at δ 129.8,
* To whom correspondence should be addressed. Tel: 34-922251728.
Fax: 34-922260135. E-mail: bmfraga@ipna.csic.es.
10.1021/np0680026 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/30/2006

Notes
Journal of Natural Products, 2006, Vol. 69, No. 7 1093
System Gold 125P and a Beckman ultrasphere column (Si 1 × 25 cm,
5 µm) was used. Conformations of minimum energy were determined
by computational methods employing the Hyperchem 7.1 program of
Hypercube.
Plant Material. The plant P. pendula, commonly known as “balo”,
was collected in January 2004, at the coast of Ch´ıo, and the seeds in
July 2001, at the coast of Barranco Hondo (Tenerife Island). Both were
identified by Prof. P. Pe´rez de Paz, Department of Plant Biology,
Botanical Section, University of La Laguna, Tenerife.
137.8, 152.3, and 107.3, respectively. The structure was confirmed
by 2D NMR spectroscopy. Thus, in the HMBC spectrum, correla-
tions were observed of H-18 with C-12, C-13, C-14, C-16, C-17,
C-19, C-22, C-28, and C-29, of H-23 and H-24 with C-3, C-4, and
C-5, of H-25 with C-1, C-5, C-9, and C-10, of H-26 with C-7,
C-9, and C-14, of H-27 with C-14, of H-29 with C-18, C-19, and
C-20, and of H-30 with C-19 and C-21. Thus, the structure of baloic
acid was determined as 3R-acetoxy-19R-hydroxyursa-12,20(30)-
dien-28-oic acid (9). The corresponding alcohol 11 is unknown,
but a 24-hydroxy derivative of this, coussaric acid, has been
obtained from another plant in the Rubiaceae, Coussarea breV-
icaulis.8
Sterile plantlets of P. pendula were obtained by seed germination
in aseptic conditions. Agrobacterium rhizogenes ATCC-15834 was
directly inoculated by a needle to the stem of the plantlets, cultured on
agar medium containing 30 g L-1 sucrose and half-strength Murashige
and Skoog salts.16 Roots appeared at the inoculation site after 4 weeks.
The induced hairy roots were excised and cultured on hormone-free
1/2 Gamborg B5 solid medium17 supplemented with 30 g L-1 sucrose
and 0.5 mg mL-1 ampicillin to eliminate the bacteria. The axenic hairy
roots thus obtained were subcultured in the dark at 25 °C on the same
solid medium without antibiotics every 25-30 days.
DNA Extraction and Analysis. Total genomic DNA was extracted
from transformed root tissue and from the untransformed root plants
using a GenElute plant genomic DNA miniprep kit (Sigma). Plasmid
DNA from A. rhizogenes strain ATCC-15834 was used as a positive
control. Polymerase chain reaction was performed using REDExtract-
N-Amp plant PCR kit (Sigma) to detect the insertion of TL-DNA and
TR-DNA of A. rhizogenes ATCC 15834 in the transformed roots. The
oligonucleotide primers for TL-DNA were 5′-ATGGATCCCAAAT-
TGCTATTCCTTCCA-3′ and 5′-TTAGGCTTCTTTCTTCAGG TTTA-
3′, which amplify a segment complementary to the 5′ coding sequence
of rol B to the 3′ coding sequence of rol C on the TL-DNA region, and
5′-CGGAAATTGTGGCTCGTTGTGGAC-3′ and 5′-AATCGTTCA-
GAGAGCGTCCGAAGTT-3′, which amplify the ags gene of the TR-
DNA region. PCR amplification was performed in a DNA thermal
cycler (Applied Biosystems 2700, Foster City, CA) under the following
conditions: initial denaturation at 94 °C for 5 min, followed by 35
cycles of 94 °C for 1 min, annealing at 55 °C for 1 min, with a final
extension at 72 °C for 10 min. The PCR reaction mixture was
electrophoresed on a 1.2% agarose gel using Tris-acetate-EDTA buffer
and visualized by ethidium bromide staining under ultraviolet light at
260 nm.
Culture Methods. The hairy roots were cultivated in the dark at 25
°C in 250 mL Erlenmeyer flasks containing 100 mL of hormone-free
1/2 Gamborg B5 liquid medium supplemented with 30 g L-1 sucrose
and shaken on a rotary shaker at 80 rpm. After 4 weeks, the hairy
roots (581 g) were harvested and separated from the culture medium
by filtration through filter paper under vacuum.
Extraction and Isolation. The hairy roots were freeze-dried (45.3
g), powdered, and extracted with ethanol in a Soxhlet. The ethanolic
extract was evaporated to dryness under reduced pressure, affording a
syrupy extract (11.7 g). Then this extract was separated into six fractions
by vacuum liquid chromatography (VLC) using an n-hexane-EtOAc
gradient. All fractions were further chromatographed on a silica gel
column and/or by preparative normal-phase HPLC on an Inertsil Prep-
sil (Gasukuro Kogyo) 25 × 2 i.d. column and Ultrasphere Si (Beckman)
25 × 1 i.d. to give in order of polarity a mixture of campesterol,
â-sitosterol, and stigmasterol (380 mg), 3-epi-pomolic acid 3R-acetate
(1) (8 mg), baloic acid (9) (6 mg), the acetates of oleanolic and ursolic
acids (8 mg), and ursolic and oleanolic acids (40 mg).
Known sterols such as campesterol, stigmasterol, and â-sitosterol,
and triterpenes such as oleanolic and ursolic acids, and the acetates
of these acids, were also isolated from the hairy roots of P. pendula.
From the natural roots of P. pendula another two new triterpenes
were obtained and characterized as 3-epi-pomolic acid (3) and 19R-
hydroxyoleanonic acid (12), respectively. Compound 3 was obtained
1
in a very small amount and identified only by considering its H
NMR spectroscopic and MS data. The former were very similar to
that of the corresponding alcohol 1, but showing now in 3 the H-3â
signal at δ 3.41, while this was δ 4.63 in 1. The resonances of
H-16, H-18, and the methyl groups could also be assigned by
comparison with those observed for 1. Thus, the structure 3R,16R-
dihydroxyursa-12-en-28-oic acid (3R-epi-pomolic acid) (3) was
given to this new triterpene. A compound analogous to 3, but
hydroxylated at C-24 and named barbinervic acid, has been isolated
from Clethra barbinerVis9 and PetiVeria alliacea.10
The structure of the final new triterpene was determined as 12.
1
The HREIMS showed the molecular formula, C30H46O4. The H
NMR spectrum displayed characteristic signals of seven angular
methyl groups, a proton geminal to an oxygen function (δ 3.35, br
s), and an olefinic hydrogen (δ 5.46, t). The 13C NMR spectrum
showed signals of seven methyls, nine methylenes, three methines,
an oxygenated methine (δ 81.6), six quaternary carbons, a double
bond (δ 124.9 and 142.7, C-12 and C-13), an oxo group (δ 217.4),
and a carbonyl of an acid group (δ 183.0). Consequently, compound
12 must be a triterpene acid of the oleanane type containing an
oxo group and a secondary alcohol. Correlations of H-23 and H-24
with the oxo group were observed in the HMBC spectrum, which
indicated that this function was at C-3, while correlations of H-29
and H-30 with C-19 indicated that the secondary alcohol was at
C-19. An R-configuration was assigned to this group considering
the pattern of resonance of H-19, a broad singlet. Other cross-peaks
displayed were those of H-18 (δ 3.11) with C-12 and C-13. The
assignment of the 13C NMR spectrum is given in Table 1.
Consequently, the structure of this new compound was determined
as 19R-hydroxy-3-oxo-olean-12-en-28-oic acid (19R-hydroxy-
oleanonic acid) (12). The corresponding C-3â acetate, rubiprasin
C, has been isolated from Rubia cordifolia.11
The known triterpenoids and sterols isolated from the hairy roots
were also obtained from the natural roots. Moreover, from the latter,
a mixture of the sterols campest-4-en-3-one, â-sitost-4-en-3-one,
and stigmast-4-en-3-one, and the triterpenes 3â-hydroxyolean-11,-
13(18)-dien-28-oic acid (13),12,13 pomolic acid 3â-acetate14 (8), and
pomonic acid (5),3,15 were also obtained.
The dry crust from roots of P. pendula (1.8 kg) was finely cut and
extracted with ethanol. In this way, a 120 g extract was obtained, which
was chromatographed using the same methods employed for the extract
of the hairy roots, giving a mixture of campest-4-en-3-one, â-sitost-
4-en-3-one, and stigmast-4-en-3-one (16 mg), campesterol, â-sitosterol,
and stigmasterol (650 mg), pomonic acid (5) (2 mg), 3-epi-pomolic
acid 3R-acetate (1) (2 mg), pomolic acid 3â-acetate (8) (10 mg), a
mixture of the acetates of oleanolic and ursolic acid (120 mg), oleanolic
acid and ursolic acid (42 mg), 3â-hydroxyolean-11,12(13)-dien-28-oic
acid (13) (1 mg), 3-epi-pomolic acid (3) (0.8 mg), and 19R-hydroxy-
oleanonic acid (12) (3 mg).
3-epi-Pomolic acid 3r-acetate (1): [R]D +31.7 (c 0.06, CHCl3);
1H NMR (500 MHz, CDCl3) δ 0.74 (3H, s, H-26), 0.85 (3H, s, H-23),
0.88 (3H, s, H-24), 0.94 (3H, s, H-25), 0.96 (3H, d, J ) 7 Hz, H-30),
1.06 (1H, br d, J ) 14 Hz, H-15â), 1.21 (3H, s, H-29), 1.31 (3H, s,
H-27), 2.07 (3H, s, -OAc), 2.54 (1H, m, H-16), 2.55 (1H, s, H-18),
Experimental Section
General Experimental Procedures. Melting points were determined
with a Reichert Thermovar apparatus and are uncorrected. IR spectra
1
were recorded using a Perkin-Elmer 1600 FT spectrometer. H NMR
spectra were recorded in CDCl3 solution at 500.13 MHz with a Bruker
AMX2-500 spectrometer. 13C NMR spectra were run in CDCl3 at 50.32
and 125.13 MHz with a Bruker AC-200 or a Bruker AMX2-500
spectrometer, respectively. Chemical shifts are given in ppm (δ). Mass
spectra and HRMS were taken at 70 eV in a Micromass Autospec
spectrometer. Dry column chromatography was performed on Merck
silica gel 0.02-0.063 mm. Semipreparative HPLC with a Beckman

1094 Journal of Natural Products, 2006, Vol. 69, No. 7
Notes
4.63 (1H, br s, H-3), 5.37 (1H, t, J ) 3.5 Hz, H-12); EIMS m/z 514
[M]+ (1), 496 (1), 468 (17), 454 (12), 396 (14), 336 (4), 264 (8), 248
(54), 246 (23), 218 (16), 203 (56), 202 (25), 201 (34), 200 (20), 190
(100), 189 (52), 187 (28), 185 (22), 175 (46), 173 (23), 171 (14), 161
(17), 159 (18), 157 (13), 149 (19), 146 (83), 133 (43); HREIMS m/z
514.3655 (calcd for C32H50O5, 514.3658).
1.90 (1H, m, H-11), 2.07 (3H, s, -OAc), 2.24 (1H, br d, J ) 13 Hz,
H-21), 2.48 (1H, td, J ) 13 and 5 Hz, H-16), 2.71 (1H, td, J ) 13 and
6 Hz, H-21), 2.91 (1H, s, H-18), 3.62 (3H, s), 4.64 (1H, t, J ) 2.5 Hz,
H-3), 4.79 and 5.03 (each 1H, s, H-30), 5.44 (1H, t, J ) 3.5 Hz, H-12);
EIMS m/z 526 [M]+ (8), 508 (12), 466 (61), 451 (7), 424 (8), 276
(18), 263 (17), 244 (12), 217 (30), 203 (26), 190 (100), 187 (29), 179
(38), 175 (32), 173 (47); HREIMS m/z 526.3623 (calcd for C33H50O5,
528.3658).
3r-Acetate methyl ester of 3-epi-pomolic acid (2): 1H NMR (500
MHz, CDCl3) δ 0.71 (3H, s, H-26), 0.87 (3H, s, H-23), 0.91 (3H, s,
H-24), 0.95 (3H, s, H-25), 0.97 (3H, d, J ) 7 Hz, H-30), 1.23 (3H, s,
H-29), 1.33 (3H, s, H-27), 2.09 (3H, s, -OAc), 2.54 (1H, m, H-16),
2.62 (1H, s, H-18), 3.62 (3H, s), 4.65 (1H, br s, H-3), 5.39 (1H, t, J )
3.5 Hz, H-12); EIMS m/z 528 [M]+ (2), 468 (38), 453 (7), 260 (12),
218 (16), 201 (34), 190 (60), 189 (28), 179 (100), 175 (25), 146 (57);
HREIMS m/z 528.3801 (calcd for C33H52O5, 528.3814).
1
19r-Hydroxyoleanonic acid (12): H NMR (500 MHz, CDCl3) δ
0.80 (3H, s, H-26), 0.98 (3H, s, H-29), 1.00 (3H, s, H-30), 1.06 (3H,
s, H-24), 1.07 (3H, s, H-25), 1.10 (3H, s, H-23), 1.29 (3H, s, H-27),
3.11 (1H, br s, H-18), 3.35 (1H, br s, H-19), 5.46 (1H, t, J ) 3.5 Hz,
H-12); EIMS m/z 470 [M]+ (13), 455 (3), 452 (7), 437 (4), 424 (48),
352 (23), 264 (73), 246 (87), 231 (42), 205 (40), 201 (100), 146 (87);
HREIMS m/z 470.3406 (calcd for C30H46O4, 470.3396).
3-epi-Pomolic acid (3): 1H NMR (500 MHz, CDCl3) δ 0.75 (3H, s,
H-26), 0.84 (3H, s, H-24), 0.92 (3H, s, H-25), 0.96 (3H, s, H-23), 0.96
(3H, d, J ) 7 Hz, H-30), 1.06 (1H, td, J ) 14 and 5 Hz, H-15â), 1.22
(3H, s, H-29), 1.28 (3H, s, H-27), 2.54 (1H, m, H-16), 2.55 (1H, s,
H-18), 3.41 (1H, t, J ) 2.8 Hz, H-3), 5.38 (1H, t, J ) 3.5 Hz, H-12);
EIMS m/z 472 [M]+ (3), 454 (12), 439 (7), 426 (13), 264 (32), 246
(53), 231 (29), 218 (20), 207 (28), 201 (74), 190 (100), 175 (51) 146
(93); HREIMS m/z 472.3571 (calcd for C30H48O4, 472.3553).
Hydrolysis of 2. A solution of the acetate methyl ester 2 (4 mg) in
methanol (1 mL) was treated with 5% methanolic KOH (3 mL) at room
temperature for 96 h. The usual workup afforded 3-epi-pomolic acid
methyl ester (4): 1H NMR (500 MHz, CDCl3) δ 0.68 (3H, s, H-26),
0.84 (3H, s, H-24), 0.92 (3H, s, H-25), 0.94 (3H, d, J ) 7 Hz; H-30),
0.95 (3H, s, H-23), 1.24 (3H, s, H-29), 1.28 (3H, s, H-27), 2.51 (1H,
m, H-16), 2.60 (1H, s, H-18), 3.41 (1H, t, J ) 2.8 Hz, H-3), 3.60 (3H,
s, -OMe), 5.4 (1H, t, J ) 3.6 Hz, H-12); EIMS m/z 486 [M]+ (3), 468
(21), 450 (19), 426 (12), 408 (14), 391 (13), 390 (4), 351 (24), 333
(25), 260 (20), 247 (15), 219 (20), 207 (26), 201 (57), 190 (74), 179
(100), 175 (42), 146 (57); HREIMS m/z 486.3708 (calcd for C31H50O4,
486.3709).
3â-Hydroxyolean-11,13(18)-dien-28-oic acid (13): 1H NMR (500
MHz, CDCl3) δ 0.78, 0.79, 0.80, 0.91, 0.95, 0.98 and 0.99 (each 3H,
s), 1.68 (1H, d, J ) 14 Hz, H-19), 2.54 (1H, dd, J ) 14 and 2 Hz,
H-19), 3.24 (1H, dd, J ) 11.5 and 5 Hz; H-3), 5.66 (1H, br d, J )
10.5 Hz, H-12) and 6.43 (1H, dd, J ) 10.5 and 3 Hz, H-11); EIMS
m/z 454 [M]+ (77), 436 (8), 421 (7), 409 (27), 391 (7), 300 (13), 255
(29), 234 (25), 207 (38), 203 (48), 201 (23), 189 (100), 187 (42), 175
(34), 173 (28), 163 (29), 147 (25), 145 (31); HREIMS m/z 454.3433
(calcd for C30H50O3, 454.3447).
Acknowledgment. This work has been supported by the Ministerio
de Educacio´n y Ciencia, Spain (BQU2002-00765). N.Q. thanks the
Spanish Research Council (CSIC) and the European Social Foundation
for an I3P fellowship.
References and Notes
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(2) Gonza´lez, A. G.; Cardona, R. J.; Lo´pez-Dorta, H.; Medina J. M.;
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(3) Cheng, D. L.; Cao, X. P. Phytochemistry 1992, 31, 1317-1320.
(4) Kakuno, T.; Yoshikawa, K.; Arihara, S. Phytochemistry 1992, 31,
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(5) Chen, T. K.; Ales, D. C.; Baenziger, N. C.; Wiemer, D. F. J. Org.
Chem. 1983, 48, 3525-3531.
(6) Chavez, M. I.; Julian, A.; Delgado, G. Magn. Reson. Chem. 2003,
41, 143-144.
Oxidation of 4. A solution of the alcohol 4 (3.1 mg) in acetone (2
mL) was treated with Jones reagent (0.25 mL) at 0 °C for 1 h. The
excess chromic acid was destroyed with 2-propanol, after which the
solvent was partially evaporated and the residue diluted with water
and extracted with CHCl3. Usual workup and methylation of the residue
with diazomethane afforded pomonic acid methyl ester (6). This
compound was identical with that obtained by methylation of pomonic
acid (5) with diazomethane.
1
Pomolic acid 3â-acetate (8): H NMR (500 MHz, CDCl3) δ 0.73
(7) Misra, L.; Laatsch, H. Z. Naturforsch. 2000, 55b, 768-770.
(8) Su, B. N.; Kang, Y. H.; Pinos, R. E.; Santarsiero, B. D.; Mesecar,
A. D.; Soejarto, D. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A.
D. Phytochemistry 2003, 64, 293-302.
(3H, s, H-26), 0.85 (3H, s, H-24), 0.88 (3H, s, H-23), 0.94 (3H, s,
H-25), 0.95 (3H, d, J ) 6.5 Hz, H-30), 1.21 (3H, s, H-29), 1.25 (3H,
s, H-27), 2.04 (3H, s), 2.53 (1H, s, H-18), 4.50 (1H, dd, J ) 10 and 6
Hz, H-3), 5.34 (1H, t, J ) 3.6 Hz, H-12); EIMS m/z 514 [M]+ (3), 468
(31), 454 (24), 439 (9), 396 (10), 246 (25), 218 (22), 203 (16), 201
(27), 190 (80), 187 (16), 185 (13), 175 (22), 173 (16), 165 (12), 159
(15), 146 (100), 133 (26); HREIMS m/z 514.3620 (calcd for C32H50O5,
514.3658).
(9) Takani, M.; Kubota, K.; Nozawa, M.; Ushiki, T.; Takahashi, K. Chem.
Pharm. Bull. 1977, 25, 981-985.
(10) Delle Monache, F.; Menichini, F.; Suarez, L. E. C. Gazz. Chim. Ital.
1996, 126, 275-278.
(11) Itokawa, H.; Qiao, Y. F.; Takeya, K.; Iitaka, Y. Chem. Pharm. Bull.
1989, 37, 1670-1672.
Baloic acid (9): 1H NMR (500 MHz, CDCl3) δ 0.77 (3H, s, H-26),
0.86 (3H, s, H-23), 0.89 (3H, s, H-24), 0.94 (3H, s, H-25), 1.26 (3H,
s, H-27), 1.41 (3H, s, H-29), 2.08 (3H, s, -OAc), 2.24 (1H, ddd, J )
13, 6 and 2 Hz, H-21), 2.48 (1H, td, J ) 13 and 5 Hz, H-16), 2.71
(1H, td, J ) 13 and 6 Hz, H-21), 2.85 (1H, s, H-18), 4.64 (1H, br s,
H-3), 4.79 and 5.03 (each 1H, s, H-30), 5.44 (1H, t, J ) 3.5 Hz, H-12);
EIMS m/z 434 [M - AcOH - H2O]+ (3), 393 (14), 322 (100), 250
(18), 210 (22), 149 (15).
(12) Ikuta, A.; Kamiya, K.; Satake, T.; Saiki, Y. Phytochemistry 1995,
38, 1203-1207.
(13) Xu, X.; Zhang, Y.; Yang, J.; Zhu, Z. Zhongguo Zhongyao Zazhi 1998,
23, 733-734.
(14) Valcic, S.; Wa¨chter, G. A.; Montenegro, G.; Timmermann, B. N. Z.
Naturforsch. 1997, 52c, 264-266.
(15) Brieskorn, C. H.; Wunderer, H. Chem. Ber. 1967, 100, 1252-1265.
(16) Murashige, T.; Skoog, F. Physiol. Plant. 1962, 15, 473-479.
(17) Gamborg, O. L.; Miller, R. A.; Ojima, K. Exp. Cell Res. 1968, 50,
151-158.
Methyl ester of baloic acid (10): [R]D +12 (c 0.1, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 0.74 (3H, s, H-26), 0.85 (3H, s, H-23), 0.89 (3H,
s, H-24), 0.94 (3H, s, H-25), 1.25 (3H, s, H-27), 1.41 (3H, s, H-29),
NP0680026