Triterpenes from Garcia parviflora. Cytotoxic evaluation of natural and semisynthetic friedelanes

Article abstract of DOI:10.1021/np100440p

Three new friedelane-type triterpenes, 1,2-dehydro-2,3-secofriedelan-3-oic acid (1), 1β-hydroxyfriedelin (2), and 3β-hydroxyfriedelan-23-oic acid (3), and the known compounds friedelin-3,4-lactone (4), acetyl aleuritolic acid (5), 4-hydroxy-5-propionyl-1,3-di-O-methylpyrogallol, elemicin, and (-)-syringaresinol were isolated from the leaves of Garcia parviflora. The structures of 1-3 were elucidated by spectroscopic methods, including 1D and 2D NMR, HREIMS, X-ray, and CD analysis. Some derivatives of 2 (6-14) were prepared via oxidation, reduction, and esterification. The natural triterpenes and the semisynthetic friedelane derivatives were tested for cytotoxic activity against human cancer cell lines U251, PC-3, K562, HCT-15, MCF-7, and SKLU-1. Compound 5 was cytotoxic against U251 cells.

Full text of DOI:10.1021/np100440p

J. Nat. Prod. 2010, 73, 1839–1845
1839
Triterpenes from Garcia parWiflora. Cytotoxic Evaluation of Natural and Semisynthetic
Friedelanes
Blanca M. Reyes, Mar´ıa Teresa Ram´ırez-Apan, Rube´n A. Toscano, and Guillermo Delgado*
Instituto de Qu´ımica, UniVersidad Nacional Auto´noma de Me´xico, Ciudad UniVersitaria, Circuito Exterior, Coyoaca´n 04510, Me´xico D. F
ReceiVed July 12, 2010
Three new friedelane-type triterpenes, 1,2-dehydro-2,3-secofriedelan-3-oic acid (1), 1ꢀ-hydroxyfriedelin (2), and 3ꢀ-
hydroxyfriedelan-23-oic acid (3), and the known compounds friedelin-3,4-lactone (4), acetyl aleuritolic acid (5), 4-hydroxy-
5-propionyl-1,3-di-O-methylpyrogallol, elemicin, and (s)-syringaresinol were isolated from the leaves of Garcia
parViflora. The structures of 1-3 were elucidated by spectroscopic methods, including 1D and 2D NMR, HREIMS,
X-ray, and CD analysis. Some derivatives of 2 (6-14) were prepared via oxidation, reduction, and esterification. The
natural triterpenes and the semisynthetic friedelane derivatives were tested for cytotoxic activity against human cancer
cell lines U251, PC-3, K562, HCT-15, MCF-7, and SKLU-1. Compound 5 was cytotoxic against U251 cells.
Members of the plant family Euphorbiaceae have provided
various bioactive metabolites,1 including pro-inflammatory,2 anti-
inflammatory,3 antiviral,4 and cytotoxic5 compounds, represented
by cytotoxic triterpenes,6,7 diterpenes,8 and sesquiterpenes.7,9
Garcia parViflora Lundell (Euphorbiaceae) is a tree endemic to
Mexico,10 and its chemical constituents have not been investigated
previously. Three new friedelanes (1-3) have been isolated and
characterized from this material, together with some known
compounds. Previous publications11,12 have reported the preparation
of several compounds by modifying the A ring of friedelane
triterpenes, which afforded derivatives with cytotoxic activity.
Therefore, we prepared some selected derivatives of 2 (6-14) via
oxidation, reduction, and esterification. The natural triterpenes and
the semisynthetic friedelane derivatives were tested for cytotoxicity
against breast (MCF-7), leukemia (K562), central nervous system
(U251, Glia), prostate (PC-3), colon (HCT-15), and lung (SKLU-
1) cancer cell lines.
extract led to the isolation and characterization of the new triterpene
1,2-dehydro-2,3-secofriedelan-3-oic acid (1) and the known com-
pounds friedelin-3,4-lactone (4),14 acetyl aleuritolic acid (5),15,16
and ꢀ-sitosterol. The new natural products 1ꢀ-hydroxyfriedelin (2)
and 3ꢀ-hydroxyfriedelan-23-oic acid (3) and the known compounds
4-hydroxy-5-propionyl-1,3-di-O-methylpyrogallol17 and elemicin18,19
were isolated from the ethyl acetate extract. The methanol extract
yielded the known compounds (s)-syringaresinol20,21 and ꢀ-sito-
steryl-ꢀ-D-glucopyranoside.
Compound 1 was obtained as a white solid. Its molecular formula
was established as C30H50O2 from the HRFABMS (m/z 443.3886
[M + H]+), indicating six degrees of unsaturation. The IR spectrum
showed absorptions indicating OH (3375 cm-1) and carbonyl (1693
cm-1) groups. Analysis of the 13C NMR (Table 1) spectrum, with
the aid of DEPT and HSQC experiments, revealed the presence of
30 signals involving eight methyl (δC 34.9, 32.1, 31.8, 20.8, 20.3,
18.9, 18.7, and 11.7), nine sp3 methylene (δC 39.3, 36.6, 36.0, 35.3,
34.3, 32.8, 32.3, 30.3, and 17.7), one sp2 methylene (δC 118.7),
four sp3 methine (δC 60.6, 52.4, 47.8, and 42.8) with one sp2 (δC
135.2), and seven quaternary carbons including the carbonyl group
of a carboxylic acid (δC 179.5, 39.7, 38.6, 38.5, 37.0, 30.0, and
1
28.2). The H NMR spectrum (Table 1) showed seven tertiary
methyl singlets and one secondary methyl doublet; these data were
in agreement with the structure of a friedelane skeleton, ascribing
the signal at δH 1.09 (J ) 7.0 Hz) to H-23. The signals at δH 5.74
(ddd, J ) 10.0, 10.5, 17.0 Hz), 5.13 (dd, J ) 10.0, 2.5 Hz), and
4.98 (dd, J ) 17.0, 2.5 Hz) indicated a terminal double bond,
attributed to H-1, H-2a, and H-2b, respectively, in agreement with
the assignments from the HSQC spectrum for the signals at δC 135.2
(C-1) and 118.7 (C-2). The olefinic hydrogens exhibited HMBC
correlations with a methine carbon at δC 60.6 (C-10) and its
corresponding hydrogen exhibited a resonance at δH 1.72 (d, J )
10.5 Hz, H-10R), confirming that the vinylic residue was at C-10.
The 1H NMR spectrum revealed a quartet at δH 2.48 (J ) 7.0 Hz),
which corresponded to the hydrogen attached to C-4 (δC 47.8), and
this signal showed HMBC correlations with the quaternary carbons
at δC 179.5 (C-3) and 38.6 (C-5), with a methylene carbon at δC
34.3 (C-6), with a methine carbon at δC 60.6 (C-10), and with the
methyl carbons at δC 11.7 (C-23) and 20.8 (C-24). The H-23 methyl
protons (δH 1.09) showed HBMC correlations with the carboxylic
acid carbon C-3 (δC 179.5) and with the quaternary carbon C-5
(δC 38.6). These correlations confirmed the location of the car-
boxylic group, and the additional 1H and 13C NMR signals (Table
1) were similar to those of friedelin. Thus, 1 was 1,2-dehydro-2,3-
secofriedelan-3-oic acid, and the configuration at C-4 was proposed
as R considering analogy with other naturally occurring friedelanes.
Results and Discussion
The hexane, ethyl acetate, and methanol extracts obtained from
the aerial parts of G. parViflora were evaluated in a cytotoxicity
assay using the sulforhodamine B colorimetric method.13 In this
assay, the hexane and ethyl acetate extracts inhibited growth of
cells in the U251, PC3, K562, SKLU-1, and MCF-7 cancer cell
lines (between 60% and 100% at 50 µg/mL), which encouraged us
to investigate their bioactive principles. Fractionation of the hexane
* Corresponding author. Tel: (+52-55)-5622-4446. Fax: (+52-55)-5616-
2217. E-mail: delgado@servidor.unam.mx.
10.1021/np100440p  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/19/2010

1840 Journal of Natural Products, 2010, Vol. 73, No. 11
Reyes et al.
Table 1. 1Ha and 13Cb NMR Spectroscopy Data for 1-3 (δ in ppm)
1
2
3
position
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
2a
2b
3
4
5
6a
6b
7
8
135.2, CH
118.7, CH2
5.74, ddd (17.0, 10.5, 10.0)
5.13, dd (10.0, 2.5)
4.98, dd (17.0, 2.5)
71.4, CH
52.7, CH2
4.84, br s
2.66, dd (14.0, 4.5)
2.38, dd (14.0, 2.5)
16.0, CH2
33.5, CH2
1.50c
1.94, m
1.50c
179.5, qC
47.8, CH
38.6, qC
34.3, CH2
211.3, qC
59.1, CH
43.9, qC
34.3, CH2
68.2, CH
59.5, CH
38.5, qC
42.9, CH2
4.15, ddd (2.5,2.5,2.5)
2.20, d (2.5)
2.48, q (7.0)
2.30, q (6.5)
1.64, ddd (13.5, 3.5, 3.0)
1.78, ddd (13.5, 3.5, 3.5)
1.76, br d (12.5)
1.37c
1.53c
1.47c
1.20c
17.7, CH2
52.4, CH
37.0, qC
60.6, CH
36.6, CH2
18.8, CH2
53.9, CH
38.6, qC
61.6, CH
35.7, CH2
18.0, CH2
53.7, CH
37.9, qC
61.0, CH
36.0, CH2
1.45c
1.33c
1.36c
9
10
11a
11b
12a
12b
13
14
15a
15b
16a
16b
17
18
19a
19b
20
21a
21b
22a
22b
23
24
25
26
27
28
29
30
1.72, d (10.5)
1.19c
1.14, m
1.47, br s
1.64, ddd (13., 3.0, 3.0)
0.98, br s
1.45c
30.3, CH2
1.28, m
30.4, CH2
1.39, m
31.0, CH2
1.34, m
39.7, qC
38.5, qC
32.3, CH2
39.8, qC
38.5, qC
32.7, CH2
40.2, qC
38.8, qC
32.8, CH2
1.50c
1.30c
1.55c
1.36c
1.55c
1.29, m
1.52, m
1.31, m
1.57c
36.0, CH2
36.2, CH2
36.5, CH2
1.36c
30.0. qC
42.8, CH
35.3, CH2
30.1. qC
43.0, CH
35.5, CH2
30.5. qC
43.4, CH
35.8, CH2
1.53c
1.36c
1.19c
1.58, dd (11.0, 6.0)
1.57, dd (11.0, 6.0)
1.38c
1.22, dd (14.0, 6.0)
28.2, qC
32.8, CH2
28.2, qC
33.0, CH2
28.5, qC
33.3, CH2
1.46c
1.50c
1.50c
1.25c
1.28, m
39.3, CH2
1.49, m
39.3, CH2
1.50, m
39.7, CH2
1.49c
0.93c
0.94c
11.7, CH3
20.8, CH3
18.9, CH3
20.3, CH3
18.7, CH3
32.1, CH3
31.8, CH3
34.9, CH3
1.09, d (7.0)
1.11, s
0.95, s
0.995, s
1.02, s
1.17, s
0.990, s
0.94, s
6.9, CH3
0.94, d (6.5)
1.08, s
1.31, s
1.04, s
1.05, s
1.19, s
1.01, s
0.96, s
177.8, qC
18.40, CH3
18.43, CH3
20.4, CH3
18.9, CH3
32.2, CH3
32.4, CH3
35.2, CH3
17.4, CH3
19.2, CH3
20.2, CH3
18.7, CH3
32.2, CH3
31.8, CH3
34.9, CH3
1.19, s
0.89, s
1.01, s
1.04, s
1.19, s
1.00, s
0.95, s
a Recorded in CDCl3 (1, 2 [50 °C]), CDCl3-CD3OD (1:1) (3) at 500 MHz. b Recorded in CDCl3 (1, 2 [50 °C]), CDCl3-CD3OD (1:1) (3), at 125
MHz (1, 3) and 75 MHz (2). c Overlapping signals.
Compound 2 was isolated as colorless needles. Its IR spectrum
exhibited bands for OH (3370 cm-1) and carbonyl (1703 cm-1
similar to those reported for friedelane trierpenes. The structure of
2 was confirmed by X-ray diffraction analysis of crystals grown
from ethyl acetate (Figure 1). Its absolute configuration was
determined as depicted in 2 from a circular dichroism spectrum
that showed a negative Cotton effect at 295 nm (∆ε -50.41) due
to the n f π* transition of the carbonyl group.22 This configuration
is characteristic for naturally occurring friedelanes.
)
groups. The molecular formula was established as C30H50O2 from
the HRFABMS ion at m/z 443.3884 [M + H]+, indicating six
degrees of unsaturation. The 13C NMR spectrum (Table 1) had 30
resonances, and the DEPT spectra showed the presence of eight
methyl, 10 methylene, five methine including one carbinol (δC 71.4),
and seven quaternary carbons with one carbonyl group at δC 211.3.
Compound 3 was obtained as white crystals. Its molecular
formula, C30H50O3 (HRFABMS, m/z 459.3841 [M + H]+), indicated
six degrees of unsaturation. IR absorptions at 3508 and 1717 cm-1
and the 13C signals at δC 68.2 and 177.8 established the presence
of OH and carbonyl groups. The 13C NMR and DEPT spectra
exhibited 30 signals involving seven methyl, 11 methylene, five
1
The H NMR spectrum (Table 1) showed eight methyl signals,
seven singlets (δH 1.31, 1.19, 1.08, 1.05, 1.04, 1.01, and 0.96), and
a doublet at δH 0.94 (J ) 6.5 Hz), similar to those described for 1.
This last signal was ascribed to H-23 since it showed a HSQC
correlation with the shielded methyl carbon resonance at δC 6.9
and HMBC correlations with a methine carbon at δC 59.1 (C-4)
and with the quaternary carbons at δC 43.9 (C-5) and 211.3 (C-3),
establishing a ketone group at C-3. A secondary OH group was
1
methine, and seven quaternary carbons. The H NMR spectrum
showed six tertiary methyl singlets, of which two were overlapping
methyls (H3-24 and H3-28). The signal at δH 4.15 (ddd, J ) 2.5,
2.5, 2.5 Hz, H-3R) placed a secondary OH at C-3 in 3, supported
by an oxygenated carbon at δC 68.2 in the 13C NMR spectrum that
correlated with H-1 (δH 1.50) in the HMBC spectrum. The doublet
at δH 2.20 (J ) 2.5 Hz) was assigned to the hydrogen attached to
C-4 (δC 59.5), since it showed HMBC correlations with the
carboxylic acid group at C-23 (δC 177.8), with C-5 (δC 38.5), and
with C-24 (δC 18.4), and a COSY correlation with H-3R. The
coupling constant between H-3 and H-4 (2.5 Hz) established the
ꢀ-orientation of the OH and carboxylic groups, and this was
confirmed by NOESY interactions of H-4R with H-3R and H-10R.
1
evident in the H NMR spectrum by a broad signal located at δH
4.84, which corresponded to the carbinol methine proton H-1. The
existence of this group was supported by the signal of an oxygenated
carbon at δC 71.4 in the 13C NMR spectrum. Two doublets of
doublets [δH 2.66 (J ) 14.0, 4.5 Hz) and 2.38 (J ) 14.0, 2.5 Hz)]
were assigned to methylene protons H-2a and H-2b, respectively,
attached to C-2 (δC 52.7). The quartet signal at δH 2.30 (J ) 6.5
Hz) corresponded to the axial proton H-4R. The OH group was
assigned a ꢀ-orientation from the NOESY experiments, which
exhibited interactions of equatorial proton H-1R with H-10R, H-8R,
and H-2a and H-2b. The additional 1H and 13C NMR signals were

Triterpenes from Garcia parViflora
Journal of Natural Products, 2010, Vol. 73, No. 11 1841
Scheme 1. Friedelane Derivativesa
Figure 1. ORTEP drawing of 2 with atom labeling.
1
The additional H and 13C NMR (Table 1) signals were similar to
those reported for friedelane trierpenes, verifying structure 3.
Friedelanes functionalized at ring A possess a variety of bio-
logical activities,23 and we considered that compound 2 offered
the possibility of various structural modifications. Therefore, some
derivatives were prepared in order to explore the cytotoxic potential
of the resulting semisynthetic friedelane derivatives. Reduction of
2 with NaBH4 in THF-H2O24 under reflux afforded 1ꢀ,3ꢀ-
dihydroxyfriedelane (6) in good yield, which was subjected to
acetylation with Ac2O-pyridine, yielding the monoacetyl (7) and
diacetyl (8) derivatives. Oxidation of 2 by Jones’s reagent25 at room
temperature produced the previously reported 1,3-diketone 9 in
moderate yield.26,27 Reaction of 2 with hydroxylamine chloride28
afforded oxime 10, which was treated with p-TsCl,28 producing
a Reagents: (a) NaBH4; (b) Ac2O, Py; (c) Jones reagent; (d) NH2OH; (e):
p-TsCl, Py; (f) m-CPBA.
SX 102 A mass spectrometers, respectively. Analytical TLC was carried
out on precoated silica gel 60 F254 sheets (Merck). Column chroma-
tography (CC) was performed using silica gel (230-400). All solvents
were dried and distilled before use.
Plant Material. Aerial parts of G. parViflora were collected in
Zacatal, Veracruz, Me´xico, during April 2003, and identified by
Francisco Ramos Marchena. A voucher specimen was deposited at the
National Herbarium (MEXU, voucher 1104455), Instituto de Biolog´ıa,
Universidad Nacional Auto´noma de Me´xico.
29
the lactam 11. Treatment of 2 with m-CPBA in CHCl3 under
reflux afforded 12-14. Compound 13 has been reported previ-
ously.30 The structures of the compounds obtained by these
transformations (Scheme 1) were determined by conventional
spectroscopic analyses (Tables 2 and 3).
Extraction and Isolation. The air-dried plant material (leaves and
branches, 5.7 kg) was powdered and extracted at room temperature
consecutively with n-hexane (24 L × 3), ethyl acetate (24 L × 3), and
methanol (24 L × 2), and the extracts were concentrated to dryness in
vacuo to obtain 27.7, 57.4, and 54.0 g of residues, respectively. The
hexane extract was chromatographed on a silica gel (90 g) column with
a gradient solvent system of hexane-EtOAc. The fractions (100 mL
each) were monitored by TLC and pooled into seven main fractions.
Fraction 3 (38.0 mg) was separated by preparative TLC using
benzene-EtOAc (9:1) to yield 1,2-dehydro-2,3-secofriedelan-3-oic acid
(1, 25 mg) and acetyl aleuritolic acid (5, 8 mg). Fraction 4 was subjected
to CC using mixtures of n-hexane and EtOAc of increasing polarity.
Five subfractions were collected. Subfraction 3 (87 mg) was purified
by preparative chromatography using benzene-EtOAc (98:2) to obtain
compound 1 (50 mg), friedelin-3,4-lactone (4, 6 mg), and ꢀ-sitosterol
(19 mg). The ethyl acetate extract was suspended in acetone, filtered,
and washed with a mixture of hexane-ethyl acetate (1:1), yielding 1ꢀ-
hydroxyfriedelin (2, 2.0 g). The filtrate was evaporated to dryness, and
this residue (55.0 g) was chromatographed on a silica gel column eluted
with mixtures of n-hexane, n-hexane-EtOAc, and n-EtOAc-MeOH
of increasing polarity to afford 11 main fractions. Fraction 2 (1.48 g)
was fractionated by silica gel CC eluted with n-hexane-EtOAc mixtures
to give eight subfractions. (8.1-8.8). Subfraction 8.6 (82 mg) was
purified by preparative TLC using toluene-EtOAc (7:3) to afford 63
mg of elemicin. Fraction 6 (3.7 g) was subjected to silica gel CC and
eluted with a gradient of n-hexane-EtOAc to give 13 subfractions
(13a-13m). Subfraction 13g (0.97 g) was subjected to CC over silica
gel using an n-hexane-EtOAc mixture of increasing polarity to yield
seven fractions. Fraction 13g-4 (46 mg) was purified by preparative
The cytotoxicity of compounds 1-14 was tested against six
human tumor cell lines. Compound 5 was cytotoxic against the
U251 cell line, with IC50 8.4 µM. Compounds 1, 4, and 6 exhibited
weak activity in the same cell line, with IC50 36.8, 17.1, and 22.8
µM, respectively, when compared to the positive control adriamycin
(IC50 0.3 µM). Compound 14 exhibited weak cytotoxicity against
the HCT-15 cell line (IC50 41.8 µM compared with adriamycin (IC50
0.23 µM). Friedelin-3,4-lactone (4) and acetyl aleuritolic acid (5)
were more cytotoxic than 1,2-dehydro-2,3-secofriedelan-3-oic acid
(1). All other compounds were noncytotoxic against the tested cell
lines.
Experimental Section
General Experimental Procedures. Melting points were determined
on a Fisher Johns apparatus and are uncorrected. Optical rotations were
measured in CHCl3 on a Perkin-Elmer 341 polarimeter using a sodium
lamp at the wavelength 589 nm. UV data were measured on a Shimadzu
UV-160 spectrophotometer. The CD spectrum was measured with a
JASCO J-720 spectrometer. IR spectra were recorded on a Nicolet
Magna FT-IR 750 spectrometer. 1H (500 MHz),13C (125.0 MHz), and
2D NMR spectra (in CDCl3, DMSO, and CD3OD) were obtained on a
Varian Inova 500, and 13C NMR (75.0 MHz) were obtained on a Varian
Unity 300 spectrometer. Chemical shifts were expressed in parts per
million (δ) relative to TMS as internal standard. EI and HRFAB mass
spectra were measured on JEOL JMS-AX 505 HA and JEOL JMX-

1842 Journal of Natural Products, 2010, Vol. 73, No. 11
Reyes et al.

Triterpenes from Garcia parViflora
Journal of Natural Products, 2010, Vol. 73, No. 11 1843
Table 3. 13C NMR Data of Compoundsa 6-8, 10-12, and 14
(δ in ppm)
(27), 90 (33), 77 (20), 57 (50), 18 (57); HRFABMS m/z [M + H]+
443.3884 (calcd for C30H51O2, 443.3889).
3ꢀ-Hydroxyfriedelan-23-oic acid (3): white crystals (from CH3Cl3:
MeOH), mp 237-240 °C; [R]25D +37.7 (c 0.13 CHCl3); IR (KBr) νmax
3508, 2934, 2867, 1717, 1453, 1385, 1235, 1188, 1002, 687 cm-1; 1H
and 13C NMR, see Table 1; FABMS m/z 459 [M + H]+ (2), 441 (3),
391 (4), 307 (11), 289 (13), 165 (22), 154 (80), 136 (72), 107 (40), 77
(100), 63 (56), 39 (62), 27 (32), 14 (8); HRFABMS m/z [M + H]+
459.3841 (calcd for C30H51O3, 459.3838).
Preparation of 1ꢀ,3ꢀ-Dihydroxyfriedelane (6). 1ꢀ-Hydroxyfriede-
lin (2) (50.0 mg, 0.11 mmol) was suspended in THF-H2O (3:0.1 mL),
and NaBH4 (9.4 mg, 0.25 mmol) was added. The resulting mixture
was stirred under reflux and monitored by TLC (hexane-EtOAc, 7:3).
After completion of the reaction (3 h), distilled water (3 mL) was added
to the reaction mixture, and it was then stirred for an additional 5 min
at rt. The mixture was extracted with CH2Cl2 (3 × 10 mL) and dried
over anhydrous Na2SO4. The crude material was purified by CC (silica
gel, hexane-EtOAc, 8:2), affording compound 6 (47.8 mg, 95.2%
yield).
position
6
7
8
10
11
12
14
1
2
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
68.4
41.9
74.1
49.5
38.1
44.4
17.8
53.8
39.3
61.8
35.5
30.5
39.7
38.4
32.4
36.1
30.0
42.8
35.3
28.2
32.8
39.3
12.0
18.4
19.4
20.0
18.7
32.1
31.8
35.0
67.2
39.8
74.8
48.3
37.6
44.4
18.0
53.8
38.2
61.4
35.3
30.4
39.7
38.4
32.4
36.0
30.0
42.8
35.3
28.2
32.8
39.3
11.6
18.2
19.2
20.0
18.7
32.1
31.8
35.0
68.1
36.1
68.1
37.6
64.7
45.0
65.5 150.7
43.7 127.4
72.8 166.6 177.4 172.6 198.3
48.0
37.8
43.7
17.9
53.9
37.8
60.4
35.3
30.3
39.6
38.4
32.4
36.0
30.0
42.8
35.3
28.1
32.8
39.3
11.5
17.2
18.8
20.1
18.6
32.1
31.8
34.9
51.2
42.5
42.6
18.1
52.9
38.5
60.7
34.5
29.6
38.9
37.6
31.7
35.3
29.3
42.1
34.6
27.5
32.2
38.5
8.1
60.3
41.2
41.4
19.2
53.9
40.0
67.2
36.0
31.2
39.9
39.2
33.0
36.6
30.6
43.5
35.9
28.6
33.4
39.8
15.7
15.8
19.4
20.4
19.0
32.4
32.1
35.2
85.2
41.7
40.4
18.4
53.4
39.3
66.2
35.6
30.5
39.5
38.6
32.6
36.1
30.0
42.9
35.4
28.2
32.9
39.3
15.2
16.2
19.2
20.0
18.6
32.1
31.8
34.9
78.4
45.5
32.7
17.3
51.6
36.3
52.9
34.9
30.2
39.7
38.2
32.1
35.9
30.0
42.8
35.4
28.1
32.7
39.2
16.4
17.2
20.3
19.9
18.7
32.1
31.7
35.0
1ꢀ,3ꢀ-Dihydroxyfriedelane (6): white needles (CHCl3-MeOH), mp
217-219 °C; [R]25 +7.0 (c 0.1, CHCl3); IR (KBr) νmax 3428, 2937,
D
2867, 1459, 1385, 1145, 1055, 976, 848, 578 cm-1; 1H and 13C NMR,
see Tables 2 and 3, respectively; EIMS m/z 444 [M]+ (7), 429 (21),
291 (22), 260 (27), 220 (18), 205 (35), 191 (33), 163 (28), 137 (38),
123 (68), 95 (100), 83 (95), 69 (95), 55 (82), 43 (83), 18 (77);
HRFABMS m/z 444.3973 [M]+ (calcd for C30H51O2, 444.3967).
Acetylation of 1ꢀ,3ꢀ-Dihydroxyfriedelane (6). Compound 6 (30.5
mg) was treated with Ac2O (1.5 mL) in pyridine (1.0 mL) at room
temperature for 72 h. The formation of products was monitored by
TLC using n-hexane-CH2Cl2-EtOAc (8:1:1). The reaction mixture
was worked up as usual, and the residue was subjected to CC over
silica gel. Elution with an n-hexane-CH2Cl2-EtOAc solvent system
afforded 7 (12.5 mg, 41%), 8 (3.6 mg, 9.9%), and recovered starting
material (6, 10.7 mg, 35.1%).
16.2
18.3
19.4
18.1
31.5
31.3
34.4
acetate-1
CdO
CH3
acetate-3
CdO
CH3
170.0
21.6
3ꢀ-Acetoxy-1ꢀ-hydroxyfriedelane (7): colorless needles (CH2Cl2),
mp 213-215 °C; [R]25D -5.45 (c 0.11, CHCl3); IR (CHCl3) νmax 2934,
1
2865, 1731, 1462, 1378, 1251, 1230, 1139, 1019, 978 cm-1; H and
170.2 170.5
21.3 21.2
13C NMR, see Tables 2 and 3; EIMS m/z 486 [M]+ (3), 471 (10), 426
(9), 333 (8), 273 (14), 260 (18), 229 (15), 218 (31), 205 (21), 191
(26), 149 (27), 121 (60), 109 (73), 95 (100), 69 (82), 55 (62), 43 (45),
41 (27), 29 (6); HRFABMS m/z 486.4065 [M]+ (calcd for C32H54O3,
486.4073).
a Recorded in CDCl3 (6-8, 12, 14), CDCl3-DMSO (3:1) (10),
CDCl3-MeOH (1:1) (11) at 125 MHz (6-8, 10-12) and 75 MHz (14).
1ꢀ,3ꢀ-Diacetoxyfriedelane (8): colorless needles (CH2Cl2), mp
219-221 °C; [R]25D +9.2 (c 0.13, CHCl3); IR (CHCl3) νmax 2939, 2867,
1726, 1462, 1371, 1268, 1242, 1140, 1022, 977 cm-1; 1H and 13C NMR,
see Tables 2 and 3; EIMS m/z 528 [M]+ (6), 468 (4), 426 (9), 408 (8),
393 (9), 376 (11), 273 (17), 260 (26), 205 (48), 149 (40), 137 (44),
123 (90), 109 (97), 95 (100), 81 (75), 69 (98), 55 (52), 43 (68);
HRFABMS m/z 528.4186 [M]+ (calcd for C34H56O4, 528.4179).
Preparation of Friedelane-1,3-dione (9). 1ꢀ-Hydroxyfriedelin (2)
(14.0 mg, 0.03 mmol) was suspended in THF (5 mL). Jones reagent
was added dropwise until the orange color remained. The reaction
mixture was stirred (30 min) at room temperature, and then it was
quenched with MeOH, producing a blue-green solution. The solvents
were eliminated under vacuum, and water was added. The product was
extracted with CHCl3 (3 × 3 mL), washed with aqueous NaHCO3 and
brine, and dried over anhydrous Na2SO4. The product was purified by
CC (silica gel, hexane-EtOAc, 8:2) to afford 1,3-diketofriedelane (9)
(9.0 mg, 64% yield) and recovered 1ꢀ-hydroxyfriedelin (2) (2.0 mg,
14.3% yield). Compound 9 was identified by direct comparison with
TLC using n-hexane-EtOAc (6:4) to yield 36.8 mg of 4-hydroxy-5-
propionyl-1,3-di-O-methylpyrogallol. Subfraction 13i (0.38 g) was
suspended in a mixture of hexane-CH2Cl2 (1:1), the suspension was
filtered, and the solids were washed with hexane-CH2Cl2 (1:1) and
crystallized from CHCl3-MeOH to obtain 7.8 mg of compound 3. The
methanol extract of G. parViflora (54 g) was chromatographed on a
silica gel column with mixtures of n-hexane-EtOAc and EtOAc-MeOH
of increasing polarity, yielding 15 main fractions. The 13th fraction
(152 mg) was purified by preparative chromatography using
n-hexane-EtOAc-MeOH (40:55:5) to afford 43 mg of a yellow oil,
which was further purified by analytical TLC using CH2Cl2-
EtOAc-MeOH (80:15:5) to yield (-)-syringaresinol (21 mg). The last
fraction from chromatography of the MeOH extract yielded ꢀ-sitosterol-
ꢀ-D-glucopyranoside (10 mg).
1,2-Dehydro-2,3-secofriedelan-3-oic acid (1): white solid (from
CHCl3); mp 205-208 °C; [R]25 +16.8 (c 0.095, CHCl3); IR (KBr)
D
ν
max 3375, 2928, 2860, 1693, 1463, 1388, 1231, 1079, 1007, 919, 674
1
cm-1; H and 13C NMR, see Table 1; EIMS m/z 442 [M]+ (31), 427
(32), 245 (39), 205 (96), 191 (55), 163 (49), 137 (53), 109 (79), 95
(100), 69 (81), 57 (61), 43 (40), 29 (7); HRFABMS m/z [M + H]+
443.3886 (calcd for C30H51O2, 443.3889).
data reported in the literature,26,27 [R]25 -5.0 (c 0.12, CHCl3), lit.27
D
[R]25 -4.2.
D
Preparation of 1ꢀ-Hydroxyfriedelin-3-oxime (10). Compound 2
(100 mg, 0.22 mmol) and hydroxylamine hydrochloride (39.3 mg) were
dissolved in a mixture of anhydrous pyridine (1.5 mL) and absolute
ethanol (1.5 mL), and the reaction mixture was refluxed for 30 min
and cooled to rt.28 Then, a solution of HCl (10%, 2 mL) was added,
obtaining a precipitate, which was filtered. The solid was washed with
EtOAc to obtain 10 (98.9 mg, 95.6%).
1ꢀ-Hydroxyfriedelin (2): colorless needles (from EtOAc) relatively
insoluble in solvents such as acetone, ethyl acetate, THF, and CH2Cl2.
This limited solubility could be explained by two intermolecular
hydrogen bonds (identified by X-ray analysis) between the carbonyl
and OH groups that enhance the crystalline stability.31 Mp >300 °C;
[R]25D -38.0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 243 (2.42) nm;
CD (CHCl3) λmax ∆ε295 -50.41; IR (KBr) νmax 3370, 2938, 2865, 1704,
1454, 1384, 1235, 1174, 1121, 1024, 917, 651 cm-1; 1H and 13C NMR,
see Table 1 (the spectra were recorded at 50 °C due to the low solubility
of 2 in CDCl3 at rt); EIMS m/z 442 [M]+ (5), 424 (4), 344 (100), 289
(7), 258 (41), 230 (14), 205 (11), 193 (77), 184 (22), 135 (39), 129
1ꢀ-Hydroxyfriedelin-3-oxime (10): colorless crystals, mp 242-244
°C; [R]25 +18.4 (c 0.10, CHCl3); IR (KBr) νmax 3611, 3289, 2933,
D
1
2867, 1674, 1460, 1385, 936 cm-1; H and 13C NMR, see Tables 2
and 3; EIMS m/z 457 [M]+ (17), 439 (33), 422 (100), 408 (19), 205
(96), 191 (21), 166 (38), 138 (44), 123 (68), 109 (76), 95 (96), 69

1844 Journal of Natural Products, 2010, Vol. 73, No. 11
Reyes et al.
(79), 55 (50), 41 (22), 18 (35); HRFABMS m/z [M + H]+ 458.3997
(calcd for C30H52O2N, 458.3998).
automatic diffractometer with a CCD area detector at 173(2) K using
graphite- monochromated Mo KR radiation (λ ) 0.71073 Å). The
structure was solved by direct methods and refined by full-matrix least-
squares on F2 using the program SHELXS-97.32 The crystal data are
summarized as follows: empirical formula C30H50O2; formula weight
442.70 amu; crystal color and habit, colorless prism, orthorhombic,
crystal size 0.268 × 0.056 × 0.038 mm, space group P212121, Z ) 4,
a ) 6.230(3) Å, b ) 14.029(8) Å, c ) 29.261(16) Å, V ) 2557(2) Å3;
Dcalcd ) 1.150 Mg/m3; F(000) ) 984, µ ) 0.069 mm-1; 18 537 collected
reflections (2.01° e θ e 25.44°), -7 e h e 7, -16 e k e 16, 35 e
l e 35; 2723 independent reflections (Rint ) 0.0877); goodness-of-fit
on F2 is 1.020, final R indices for I > 2σ(I), R1 ) 0.0877, wR2 ) 0.1724,
R indices for all data R1 ) 0.1560, wR2 ) 0.2023; refining 301
parameters and no restraints; the largest difference peak and hole was
0.289 and -0.266 e Å-3; completeness to θ (25.44°) 99.2%, maximum
transmission 0.9999, minimum transmission 0.9811.
Preparation of 1ꢀ-Hydroxyfriedelin-3,4-lactam (11). Compound
10 (42.0 mg, 0.09 mmol) and p-toluenesulfonyl chloride (14.0 mg, 0.07
mmol) were dissolved in pyridine (1 mL), and the mixture was refluxed
for 5 h. Then, the reaction mixture was cooled and diluted with water
(3 mL), and the product was extracted with CHCl3 (3 × 5 mL). The
organic phases were combined, washed with HCl (10%) and brine, and
dried over anhydrous Na2SO4. Concentration of the organic phase
afforded 11 (39.4 mg, 93.8%).
1ꢀ-Hydroxyfriedelin-3,4-lactam (11): white powder, mp 262-264
°C; [R]25 -13.3 (c 0.10, CHCl3); IR (KBr) νmax 3422, 3257, 3938,
D
1
2868, 1667, 1452, 1192 cm-1; H and 13C NMR, see Tables 2 and 3;
EIMS m/z 457 [M]+ (25), 439 (8), 424 (32), 218 (21), 205 (48), 191
(36), 163 (22), 137 (29), 123 (39), 109 (48), 95 (56), 81 (47), 69 (45),
55 (31), 44 (100), 28 (11); HRFABMS m/z [M + H]+ 458.4000 (calcd
for C30H52O2N, 458.3998).
Acknowledgment. We thank CONACyT (Me´xico) and Programa
de Maestr´ıa y Doctorado en Ciencias Qu´ımicas (UNAM) for financial
support. We also thank E. M. Mart´ınez (Instituto de Biolog´ıa, UNAM),
A. Cano, M. I. Cha´vez, R. Patin˜o, L. Velasco, and J. Pe´rez Flores
(Instituto de Qu´ımica, UNAM) for technical assistance.
Reaction of 1ꢀ-Hydroxyfriedelin (2) with m-CPBA. Compound
2 (30 mg, 0.06 mmol) was dissolved in CHCl3 (15 mL), and then
m-CPBA29 (29.3 mg, 0.16 mmol) was added. The mixture was
refluxed for 6 h. The reaction mixture was diluted with CHCl3, and
the solution was washed successively with 5% Na2SO3(aq), saturated
NaHCO3, and brine and dried with Na2SO4. The solution was
evaporated in vacuo, and the residue was separated by silica gel
CC eluted with mixtures of n-hexane and EtOAc, to afford
compounds 12 (4.2 mg, 13.5%), 13 (4.8 mg, 16.6%), and 14 (3.7
mg). Compound 13 was identified by direct comparison with data
reported in the literature.12,30
1
Supporting Information Available: H and 13C spectra of com-
pounds 1-3, 6-8, 10-12, and 14, CD curve, and X-ray crystal-
lographic data for 2 are provided. This information is available free of
charge via the Internet at http://pubs.acs.org.
1ꢀ-Hydroxyfriedelin-3,4-lactone (12): amorphous solid (CHCl3);
[R]25D +17.1 (c 0.10, CHCl3); IR (KBr) νmax 3364, 2939, 2867, 1720,
1447, 1384, 1306, 1265, 1183, 1076 cm-1; 1H and 13C NMR, see Tables
2 and 3; EIMS m/z 458 [M]+ (1), 173 (12), 83 (100), 55 (35), 43 (7),
18 (5); HRFABMS m/z 459.3832 [M + H]+ (calcd for C30H51O3,
459.3838).
References and Notes
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Triterpenes from Garcia parViflora
Journal of Natural Products, 2010, Vol. 73, No. 11 1845
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Centre (CCDC No. 766372). Copies of the data can be obtained, free
of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
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(31) Crystallographic data for the structure of compound 2 reported in this
paper have been deposited with the Cambridge Crystallographic Data
NP100440P