New cytotoxic bis 5-alkylresorcinol derivatives from the leaves of Oncostemon bojerianum from the Madagascar rainforest

Article abstract of DOI:10.1021/np0201568

Bioassay-directed fractionation of a CH₂Cl₂-MeOH extract of the leaves of Oncostemon bojerianum resulted in the isolation of eight new 5-alkylresorcinols, named oncostemonols A-H (1-8), and two known derivatives, (8′Z)-1,3-dihydroxy-5-[16′-(3′′, 5′′-dihydroxyphenyl)-8′-hexadecen-1′-yl] benzene (9) and (8′Z)-1,3-dihydroxy-5-[14′-(3′′, 5′′-dihydroxyphenyl)-8′-tetradecen-1′-yl] benzene (10). The structures of the new compounds 1-8 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic interpretation and chemical derivatization. All the compounds exhibited cytotoxic activity against the A2780 ovarian cancer cell line.

Full text of DOI:10.1021/np0201568

J . Nat. Prod. 2002, 65, 1627-1632
1627
New Cytotoxic Bis 5-Alk ylr esor cin ol Der iva tives fr om th e Lea ves of On costem on
bojer ia n u m fr om th e Ma d a ga sca r Ra in for est1
†
†
‡
§
V. S. Prakash Chaturvedula, J ennifer K. Schilling, J ames S. Miller, Rabodo Andriantsiferana,
§
,†
Vincent E. Rasamison, and David G. I. Kingston*
Department of Chemistry, M/ C 0212, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,
Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, and Centre National d’Application et des
Recherches Pharmaceutiques, B.P. 702, Antananarivo 101, Madagascar
Received April 8, 2002
2 2
Bioassay-directed fractionation of a CH Cl -MeOH extract of the leaves of Oncostemon bojerianum
resulted in the isolation of eight new 5-alkylresorcinols, named oncostemonols A-H (1-8), and two known
derivatives, (8′Z)-1,3-dihydroxy-5-[16′-(3′′,5′′-dihydroxyphenyl)-8′-hexadecen-1′-yl]benzene (9) and (8′Z)-
1
,3-dihydroxy-5-[14′-(3′′,5′′-dihydroxyphenyl)-8′-tetradecen-1′-yl]benzene (10). The structures of the new
compounds 1-8 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic interpretation
and chemical derivatization. All the compounds exhibited cytotoxic activity against the A2780 ovarian
cancer cell line.
As a part of our continuing research on the isolation of
bioactive compounds from the Suriname and Madagascar
rainforests2 as a part of the mission of the Suriname-
Madagascar International Cooperative Biodiversity Group
(
ICBG), we obtained a sample of the CH
extract of the leaves of Oncostemon bojerianum A. DC
Myrsinaceae) from a collection made in Madagascar.
2 2
Cl -MeOH
(
Oncostemon A. J uss. is a member of the Myrsinaceae, a
family of about 33 genera and 850 mostly tropical species.
One of the largest genera in the family, Oncostemon, is
endemic to Madagascar, the Comores, and the Mascarenes.
Although about 100 species have been described, the genus
is tremendously complex and in serious need of taxonomic
revision, as it has not been treated since the Flore de
3
Madagascar, and undoubtedly many species remain un-
described. The present species was identified as O. boje-
rianum A. DC by the collectors, but the name must be
accepted with some reservation pending further taxonomic
study.
The extract of O. bojerianum was selected for bioassay-
guided fractionation on the basis of both its cytotoxicity,
with an IC50 15.7 µg/mL against the A2780 ovarian cancer
cell line, and the absence of any reported phytochemistry
of this genus. Many 5-alkylresorcinols have been reported
4
-7
from the Myrsinaceae and from other plant sources,
but
none from Oncostemon. The crude extract after extensive
chromatography followed by reversed-phase HPLC fur-
nished the eight new bioactive 5-alkylresorcinols, oncos-
temonols A-H (1-8), in addition to the two known
5
-alkylresorcinols 9 and 10.
oncostemonols A-H (1-8), in addition to the two known
-alkylresorcinols 9 and 10. The structures of the two
known compounds were identified as (8′Z)-1,3-dihydroxy-
-[16′-(3′′,5′′-dihydroxyphenyl)-8′-hexadecen-1′-yl]ben-
5
Resu lts a n d Discu ssion
5
Initial liquid-liquid partition of the crude extract indi-
cated that the bioactivity was concentrated in the CHCl -
3
zene (9) and (8′Z)-1,3-dihydroxy-5-[14′-(3′′,5′′-dihydroxy-
phenyl)-8′-tetradecen-1′-yl]benzene (10), by comparison of
soluble portion of a CHCl -aqueous MeOH partition.
3
8
their spectral data with values reported in the literature.
Chromatography of this fraction on a Sephadex LH-20
column followed by column chromatography over reversed-
phase (LRP-2) silica gel and then finally by reversed-phase
HPLC furnished the eight new bioactive 5-alkylresorcinols,
Oncostemonol A (1) was isolated as a colorless viscous
44 6
liquid whose molecular formula was established as C32H O
from HRFABMS, 13C NMR, and DEPT spectral data.
Compound 1 gave a green coloration with methanolic
*
To whom inquiries should be addressed. Tel: (540) 231-6570. Fax: (540)
FeCl
3
, indicating the presence of a phenolic hydroxyl group,
2
31-7702. E-mail: dkingston@vt.edu.
and this was supported by the observation of an absorption
at 3450 cm in its IR spectrum. The IR spectrum also
showed the presence of a carbonyl group at 1735 cm , and
†
Virginia Polytechnic Institute and State University.
Missouri Botanical Garden.
Centre National d’Application et des Recherches Pharmaceutiques.
-
1
‡
§
-1
1
0.1021/np0201568 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/11/2002

1
628 J ournal of Natural Products, 2002, Vol. 65, No. 11
Chaturvedula et al.
Ta ble 1. 1H NMR Data for Compounds 1-8 and 11a (acetone-d6, 500 MHz)
position
1
2
3
4
5
6
7
8
11
2
4
6
1
2
3
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
4
6
1
6.36 t 2.1
6.37 d 2.1
6.55 d 2.1
2.49 t 7.5
1.53 m
1.30 m
1.53 m
2.04 m
5.34 t 4.6
5.34 t 4.6
2.04 m
1.53 m
1.30 m
1.30 m
1.30 m
1.53 m
2.49 t 7.5
6.36 t 2.1
6.39 d 2.0
6.56 d 2.1
2.48 t 7.6
1.54 m
1.30 m
1.54 m
2.04 m
5.33 t 4.5
5.33 t 4.5
2.04 m
1.54 m
1.30 m
1.30 m
1.30 m
1.54 m
2.48 t 7.6
6.35 t 1.9
6.28 d 2.1
6.17 d 2.1
2.52 t 7.6
1.58 m
1.31 m
1.58 m
2.03 m
5.33 t 4.5
5.33 t 4.5
2.03 m
1.58 m
1.31 m
1.31 m
1.31 m
1.58 m
2.52 t 7.6
6.24 t 2.1
6.28 d 2.1
6.21 d 2.0
2.49 t 7.5
1.57 m
1.29 m
1.57 m
2.04 m
5.34 t 4.5
5.34 t 4.5
2.04 m
1.57 m
1.29 m
1.29 m
1.29 m
1.57 m
2.49 t 7.5
6.27 t 2.0
6.29 d 2.0
6.19 d 2.0
2.51 t 7.6
1.56 m
1.29 m
1.56 m
2.03 m
5.33 t 4.6
5.33 t 4.6
2.03 m
1.56 m
1.29 m
1.29 m
1.29 m
1.56 m
2.51 t 7.5
6.40 t 2.0
6.41 d 2.1
6.56 d 2.0
2.54 t 7.5
1.59 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.59 m
2.54 t 7.5
6.19 t 2.1
6.18 d 2.2
6.18 d 2.2
2.42 t 7.5
1.54 m
1.29 m
1.29 m
1.29 m
1.54 m
6.18 t 2.0
6.17 d 2.1
6.17 d 2.1
2.42 t 7.5
1.54 m
1.30 m
1.30 m
1.30 m
1.54 m
6.76 t 2.1
6.84 d 2.1
6.84 d 2.1
2.60 t 7.5
1.59 m
1.32 m
1.59 m
2.04 m
5.34 t 4.6
5.34 t 4.6
2.04 m
1.59 m
1.32 m
1.32 m
1.32 m
1.59 m
2.60 t 7.5
′
′
′-5′
′
′
′
′
0′
1′
2′
3′
4′
5′
6′
7′
8′
9′
0′
2.03 m
2.03 m
5.34 t 4.6
5.34 t 4.6
2.03 m
1.54 m
1.29 m
1.29 m
1.29 m
1.29 m
1.29 m
5.33 t 4.5
5.33 t 4.5
2.03 m
1.54 m
1.30 m
1.30 m
1.30 m
1.54 m
2.42 t 7.5
1.54 m
2.42 t 7.5
6.18 d 2.2
6.19 t 2.1
6.18 d 2.2
8.01
′′
′′
′′
6.55 d 2.1
6.36 t 2.1
6.37 d 2.1
6.17 d 2.1
6.18 t 2.2
6.17 d 2.1
6.17 d 2.1
6.35 t 1.9
6.28 d 2.1
6.17 d 2.1
6.15 t 2.2
6.17 d 2.1
6.32 d 2.1
6.26 t 2.2
6.32 d 2.1
6.56 d 2.0
6.40 t 2.0
6.41 d 2.1
6.17 d 2.1
6.18 t 2.0
6.17 d 2.1
8.02
6.84 d 2.1
6.76 t 2.1
6.84 d 2.1
,3′′,3,5′′-OH
-OCOCH3
-OCH3
2.19 s
2.18 s
2.19 s
2.18 s
3.77 s
3.75 s
3.75 s
a
Assignments made on the basis of COSY and HMQC spectral data and comparison with the literature values.8
F igu r e 1. HMBC correlations for oncostemonol A (1).
F igu r e 2. Key mass spectroscopic fragmentations for oncostemonol
A (1).
this was confirmed as an acetoxyl group from its signals
in the 1H (δ 2.19, s) and C (δ 168.7 and 20.2) NMR
spectra, respectively. The fragment ions observed at m/z
13
further. The stereochemistry at the double bond between
C-8′/C-9′ of 1 was assigned as Z, as in the case of 9, on the
1
3
4
64 and 404 in the EIMS indicated the successive loss of
basis of the close similarity of coupling constants and
C
1
two acetic acid molecules from the molecular ion. The H
NMR values. Acetylation of 1 furnished the new tetraac-
etate (11), whose molecular formula was confirmed as
C H O by HRFABMS; the identical compound was also
36 48 8
prepared by acetylation of 9. Thus, the structure of oncos-
temonol A (1) was assigned as (8′Z)-1-acetoxy-3-hydroxy-
5-[16′-(3′′-acetoxy-5′′-hydroxyphenyl)-8′-hexadecen-1′-yl]-
benzene.
NMR spectrum contained two doublets at δ 6.37 (2H, J )
2
.1 Hz) and 6.55 (2H, J ) 2.1 Hz), three triplets at δ 6.36
(
4
(
2H, J ) 2.1 Hz), 2.49 (4H, J ) 7.5 Hz), and 5.34 (2H, J )
.6 Hz), two multiplets centered at δ 2.04 (4H) and 1.53
8H), and a broad singlet at δ 1.30 (12H). This spectrum
was almost identical to that of (8′Z)-1,3-dihydroxy-5-[16′-
8
(3′′,5′′-dihydroxyphenyl)-8′-hexadecen-1′-yl]benzene (9), ex-
Oncostemonol B (2) was also obtained as a colorless
cept for differences in the chemical shift values of the
protons in the aromatic region.
42 5
liquid, and its molecular formula was assigned as C30H O
by HRFABMS. From the molecular formula it was observed
that compound 2 contained one acetate unit less than 1.
Its IR spectrum also showed the presence of hydroxyl (3425
The absence of any terminal methyl group or carboxylic
acid, coupled with the observation that some of the signals
in the 13C NMR spectrum of 1 integrated for more than
one carbon, indicated that it was a symmetrical 5-alkyl-
resorcinol. Since the mass spectrum of 1 had its molecular
-1
-1
cm ) and ester carbonyl (1745 cm ) groups, as for 1. The
1
H NMR spectrum of 2 was almost identical to that of
oncostemonol A (1), except for the presence of three new
aromatic proton signals at δ 6.17 (2H, J ) 2.1 Hz) and 6.18
(1H, J ) 2.2 Hz) and a reduction of the integration of the
three signals at δ 6.56, 6.36, and 6.39 from two protons to
a single proton each. This indicated that the two aromatic
rings in 2 were not identical. Similarly the singlet at δ 2.18
corresponded to three protons as compared with six in 1,
indicating the presence of only one acetate group in 2.
These data confirm the assignment of 2 as the monoacetate
derivative of 9; this was supported by an ion at m/z 422
formed by the loss of acetic acid from the molecular ion. A
ion at m/z 524, which is 84 mass units higher than that of
1
9
, and since the singlet at δ 2.19 in its H NMR spectrum
integrated for six protons, the structure 1 was assigned as
1
the diacetate derivative of 9. The H NMR signals of 1 at
δ 6.36, 6.37, and 6.55, each corresponding to two protons,
confirmed that the two aromatic rings in 1 are identical
and, hence, that the two acetate groups are located at C-1
and C-3′. This conclusion is supported by the HMBC
correlations shown in Figure 1.
The 13C NMR values for all the carbons of 1 were
assigned on the basis of HMQC and HMBC spectral data
and were in good agreement with the assigned structure
1
close comparison of the H NMR values of 2 with those of
1 and 9 confirmed the structure completely. As final
(
2
Table 2). The mass fragments observed at m/z 359, 289,
35, and 165 in its EIMS (Figure 2) supported the structure
confirmation, acetylation of 2 with Ac
nished the tetraacetate 11. As in the case of 1 and 9, the
2
O-pyridine fur-

Cytotoxic Bis 5-Alkylresorcinol Derivatives
J ournal of Natural Products, 2002, Vol. 65, No. 11 1629
Ta ble 2. 13C NMR Data for Compounds 1-8 and 11a (acetone-d6, 125 MHz)
carbon
1
2
3
4
5
6
7
8
11
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
152.0
112.4
158.4
106.7
145.1
112.5
35.6
31.7
29.0
29.2
29.4
29.5
26.9
129.9
129.9
26.9
29.0
29.2
29.4
29.5
152.1
112.5
158.5
106.5
145.1
112.7
35.6
31.8
29.0
29.2
29.3
29.4
27.0
129.8
129.8
27.0
29.0
29.2
29.3
29.4
158.5
98.7
161.0
105.3
145.1
107.9
35.9
31.3
28.5
28.7
29.1
29.4
26.9
129.8
129.8
26.9
28.5
28.7
29.1
29.4
31.2
35.8
158.5
98.5
158.5
98.6
158.4
112.7
152.1
106.6
145.0
112.4
35.7
158.4
100.1
158.4
106.9
145.1
106.9
35.8
158.4
100.2
158.4
106.9
145.1
106.9
35.8
151.4
113.2
151.4
118.9
145.2
118.9
35.3
31.0
28.9
29.0
29.1
29.2
27.0
129.9
129.7
27.0
28.9
29.0
29.1
29.2
c
c
c
c
161.0
105.4
145.1
107.7
36.0
161.0
105.4
145.1
107.8
36.1
′
′
′
′
′
′
′
′
31.3
31.3
31.8
31.3
31.7
d
d
d
d
d
d
d
d
c
c
c
c
c
b
29.1c
28.7c
28.7c
c
c
c
c
28.7
28.c
29.0c
29.0c
29.2
c
28.9
c
28.9
c
c
c
29.3c
29.1c
29.0c
29.1
29.2
29.4c
26.9
29.3c
27.0
29.4
c
29.2
c
29.2
c
29.6c
29.3c
29.4c
c
29.5c
29.5c
d
d
129.8
129.8
26.9
129.8
129.8
27.0
29.6
29.6c
27.0
d
27.1
d
′
0′
1′
2′
3′
4′
5′
6′
7′
8′
9′
0′
′′
′′
′′
′′
′′
′′
29.6c
129.8e
129.7e
129.8e
129.9e
d
d
d
d
d
d
d
d
c
c
c
c
c
b
c
c
c
c
c
28.7
28.c
29.1
29.0c
29.1c
29.4c
31.3
29.0c
29.2c
29.3c
31.3
29.2c
29.3c
29.4c
31.8
26.9
d
27.0
d
29.5c
29.2c
29.1c
28.9c
28.9c
29.0c
29.2c
29.4c
31.6
31.6
35.6
31.8
35.8
27.0
35.3
36.0
36.0
35.7
c
28.7
28.9c
31.2
35.8
35.8
145.1
112.5
152.0
112.4
158.4
106.7
168.7
20.2
145.1
106.8
158.5
100.0
158.5
106.8
168.8
20.3
145.1
107.9
158.5
98.7
161.0
105.3
145.1
106.9
158.5
100.2
158.5
106.9
145.1
98.7
161.0
97.0
161.0
105.4
145.0
112.4
158.4
112.7
152.1
106.6
168.8
20.2
145.1
106.9
158.4
100.1
158.4
106.9
145.1
106.9
158.4
100.2
158.4
106.9
145.2
118.9
151.4
113.2
151.4
118.9
168.6
20.2
OCOCH3
OCOCH3
OCH3
54.5
54.6
54.6
a
Assignments made on the basis of HMQC and HMBC and comparison with the literature data.8 b Values having the same superscripts
in the respective columns are interchangeable.
stereochemistry between the double bond at C-8′/C-9′ was
assigned as Z on the basis of the almost identical coupling
constants and 1 C NMR values of the signals for the C-8′/
C-9′ protons and carbons in the three compounds. On the
basis of the above spectral data, the structure of oncoste-
monol B (2) was established as 8(′Z)-1-acetoxy-3-hydroxy-
spectrum of 4 was similar to that of oncostemonol C (3),
except for minor differences in the chemical shifts of the
protons in the aromatic region and the lack of a signal for
one of the methoxy groups of 3. Thus instead of the three
proton signals at δ 6.17 (2H, J ) 2.1 Hz), 6.28 (2H, J ) 2.1
Hz), and 6.35 (2H, J ) 1.9 Hz) of oncostemonol C (3),
compound 4 showed three doublets at δ 6.17 (2H, J ) 2.1
Hz), 6.21 (1H, J ) 2.0 Hz), and 6.28 (1H, J ) 2.1 Hz) and
two triplets at δ 6.15 (1H, J ) 2.2 Hz) and 6.24 (1H, J )
2.1 Hz), indicating that compound 4 is not a symmetrical
structure like 3. The molecular formula of 4 differed from
3
5
-[16′-(3′′,5′′-dihydroxyphenyl)-8′-hexadecen-1′-yl]ben-
zene.
Oncostemonol C (3) was obtained as a colorless liquid
and was determined to have the molecular formula C30
by HRFABMS. Its IR spectrum showed the presence of a
44 4
H O
-
1
hydroxyl group (3430 cm ) and the absence of an ester
carbonyl group. Its H NMR spectrum was very similar to
2
that of 3 by one CH group, confirming that it is a
1
monomethyl ether derivative of 9. This conclusion was
supported by the observation of a fragment ion at m/z 422
in the EIMS, corresponding to the loss of only one molecule
of methanol from the molecular ion. Methylation of 4 with
that of 1, except for the absence of the acetate singlet at δ
1
2
.19. Instead, the H NMR spectrum showed the presence
of a six-proton singlet at δ 3.77, indicating that the two
acetate groups in 1 had been replaced by two methoxyl
groups in 3. This was supported by the presence of a carbon
signal at δ 54.5 and by the observation of fragment ions at
m/z 436 and 404 in its EIMS, formed by the successive loss
of two molecules of methanol from the molecular ion. The
2 2
CH N yielded compound 12, identical in all respects with
that prepared from 3. On the basis of the above spectral
data, the structure of oncostemonol D (4) was established
as (8′Z)-1-hydroxy-3-methoxy-5-[16′-(3′′,5′′-dihydroxyphen-
yl)-8′-hexadecen-1′-yl]benzene.
1
3
C NMR values for all the carbons were assigned on the
Oncostemonol E (5) was isolated as a colorless oil, with
a molecular formula of C H O (HRFABMS). A compari-
basis of HMQC and HMBC spectra and were in good
agreement with the structure (Table 2). Methylation of 3
with diazomethane furnished a product whose spectral data
3
1
46
4
1
son of the H NMR data of 5 with that of 3 and 4 indicated
the similar nature of the three compounds, with the major
difference being that the singlet at δ 3.75 corresponded to
nine protons compared with three and six protons in
oncostemonols D (4) and C (3), respectively. This indicated
that compound 5 is the trimethyl ether of 9, and this was
supported by fragments at m/z 450, 418, and 386 observed
in the EIMS, corresponding to the successive loss of three
molecules of methanol from the molecular ion. Methylation
1
13
(
H, C NMR and MS) matched those of (8′Z)-1,3-dimethoxy-
-[16′-(3′′,5′′-methoxyphenyl)-8′-hexadecen-1′-yl]benzene
5
8
(12), confirming the structure. Oncostemonol C (3) was
thus assigned as (8′Z)-1-hydroxy-3-methoxy-5-[16′-(3′′-hy-
droxy-5′′-methoxyphenyl)-8′-hexadecen-1′-yl]benzene.
The molecular formula of oncostemonol D (4) was
1
determined to be C29
H
42
O
4
by HRFABMS. The H NMR

1
630 J ournal of Natural Products, 2002, Vol. 65, No. 11
Chaturvedula et al.
F igu r e 3. Key mass spectroscopic fragmentations for oncostemonol
F (7).
F igu r e 4. Key mass spectroscopic fragmentations for oncostemonol
G (8).
Ta ble 3. Cytotoxicities of Compounds 1-10 to A2780 Cellsa
of 5 with diazomethane furnished a product identical to
1
2 in all spectral characteristics. The structure of oncos-
compound
IC50, µg/mL
temonol E (5) was thus assigned as (8′Z)-1-hydroxy-3-
methoxy-5-[16′-(3′′,5′′-dimethoxyphenyl)-8′-hexadecen-1′-
yl]benzene.
1
2
3
4
5
6
7
8
9.4, 10.2, 11.0
10.2, 11.2
9.8, 10.6, 11.4
10.0, 11.2
9.8, 10.6
9.5, 10.3
10.2, 11.4
9.4, 11.2
The molecular formula of oncostemonol F (6) was de-
1
duced as C32
H
46
O
6
by HRFABMS. The H NMR data of 6
were almost identical to those of 1 (Table 1), except for the
absence of signals for the double bond between C-8′ and
C-9′. Thus the absence of a two-proton triplet at about δ
9
10
10.2, 11.0
9.8, 10.6, 11.2
5
1
.35, together with the observation of a broad singlet at δ
.30 for 16 protons, indicated the saturated nature of the
a
The concentration of compound that inhibited 50% (IC50) of
alkyl chain. This information, together with the mass
spectrum which indicated a molecular weight two mass
units greater than that of 1, confirmed the structure of
the growth of the A2780 mammalian cell line according to the
procedure described.1
,10
oncostemonol F as dihydro-oncostemonol A, or 6. The 13
structure of oncostemonol H (8) was thus assigned as
(10′Z)-1,3-dihydroxy-5-[18′-(3′′,5′′-dihydroxyphenyl)-10′-oc-
tadecen-1′-yl]benzene.
C
NMR values of 6 were assigned on the basis of HMQC and
HMBC correlations and were in good agreement with the
structure. Thus, oncostemonol F (6) was assigned as
The isolated compounds were tested for cytotoxicity
against A2780 ovarian cancer cells. As shown in Table 3,
all the isolated compounds (1-10) were found to be weakly
cytotoxic, with IC50 values ranging between 9.4 and 11.4
µg/mL (Table 3). The fact that all the compounds isolated
had similar activities suggests that the activity is mainly
dependent on the basic skeleton of the 5-alkylresorcinols,
rather than on any specific substituents thereon. It should
also be noted that the compounds isolated represent only
a fraction of the bioactivity of the starting extract. The
bioactivity in this extract did however decline on storage,
suggesting that the bioactive constituents may be unstable.
This is consistent with the phenolic nature of the bioactive
constituents, and it is likely that the compounds oxidized
during the isolation process. It is also noteworthy that
Hecht and his collaborators have found that 5-alkylresor-
cinols function as DNA-cleaving agents,9 and it seems
possible that these dialkylresorcinols act in the same way.
1
-acetoxy-3-hydroxy-5-[16′-(3′′-acetoxy-5′′-hydroxyphenyl)-
hexadecan-1′-yl]benzene.
Oncostemonol G (7) was isolated as a viscous liquid, and
its molecular formula was established as C32
48 4
H O by
1
13
HRFABMS. The H and C NMR data (Tables 1 and 2)
were very similar to those of 9, except for the integration
of the broad singlet at δ 1.29, which corresponded to 20
protons compared with 12 protons in 9. This suggested the
presence of four additional methylene groups between the
two aromatic rings of 9. Previous studies indicated that
the location of the double bond in similar compounds could
be established on the basis of mass spectroscopic fragmen-
6
4
tation patterns as an alternative to ozonolysis or oxidative
5
degradation studies. Since compound 7 was isolated in
small quantity, ozonolysis or oxidative degradation experi-
ments could not carried out, and hence the location of the
double bond was determined on the basis of its mass
spectroscopic fragmentation as shown in Figure 3. The
fragments at m/z 373, 372, 275, 274, 221, 220, 123, and
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. IR and UV spectra
were measured on MIDAC M-series FTIR and Shimadzu UV-
1
22 observed in the EIMS (Figure 3) indicated the sym-
metrical nature of the molecule, confirming the presence
of the double bond at C-10′/C-11′. The stereochemistry at
the double bond between C-10′/C-11′ of 1 was assigned as
Z on the basis of its almost identical coupling constants
and 13C NMR values with those of 9. On the basis of these
spectral data, the structure of oncostemonol G (7) was
established as (10′Z)-1,3-dihydroxy-5-[20′-(3′′,5′′-dihydroxy-
phenyl)-10′-dodecen-1′-yl]benzene.
1
201 spectrophotometers, respectively. NMR spectra were
obtained on a J EOL Eclipse 500 spectrometer. Mass spectra
were obtained on a J EOL HX-110 instrument. The chemical
shifts are given in δ (ppm) with TMS as internal reference
and coupling constants in Hz. Sephadex LH-20 and reversed-
phase Si gel (LRP-2, 200 µm) were used for column chroma-
tography. Reversed-phase HPLC was performed on a Shimad-
zu LC-10AT instrument with an ODS C18 column.
The molecular formula of oncostemonol H (8) was
Cytotoxicity Bioa ssa ys. The A2780 ovarian cancer cell
line assay was carried out by the reported methods.
1
10
deduced as C30
H
44
O
4
by HRFABMS. The H NMR spectrum
of 8 was almost identical to that of 7, except that the signal
at δ 1.30 corresponded to 16 protons, indicating that
compound 8 had two fewer methylene units than 7. The
location of the double bond in 8 was also established as
being between C-10′/C-11′ on the basis of its EI mass
spectroscopic fragmentation pattern (Figure 4), with key
fragment ions observed at m/z 345, 344, 275, 274, 247, 246,
P la n t Ma ter ia l. The leaves of Oncostemon bojerianum A.
DC. (Myrsinaceae) were collected by St e´ phan Rakotonan-
drasana, S. Randrianasolo, C. Birkinshaw, and P. Antilahi-
mena. The plants were collected in Madagascar in the province
of Toamasina, near Antanandava, 2 km north of the village of
Ankosy, just outside the Parc National de Zahamena in dense
humid forest at 1000 m in elevation. Voucher specimens have
been deposited at the herbaria in Antananarivo (TEF and
CNRP), Paris (P), and the Missouri Botanical Garden, St.
Louis (MO).
2
21, 193, 123, and 122. Comparison with the spectra of 7
and 9 indicated a Z stereochemistry at C-10′/C-11′, and the

Cytotoxic Bis 5-Alkylresorcinol Derivatives
J ournal of Natural Products, 2002, Vol. 65, No. 11 1631
-
1 1
Extr a ct P r ep a r a tion . The plant samples were dried,
3440, 2950, 1645, 1615, 1445, 1121, 1054, 745 cm ; H NMR,
see Table 1; 13C NMR, see Table 2; EIMS m/z (rel int) 454 [M ]
(9), 437 (4), 422 (11), 405 (13), 331 (38), 221 (18), 207 (14), 193
+
ground, and extracted with CH
dried extract MG 003.
2 2
Cl -MeOH (1:1) to give the
Extr a ction a n d Isola tion . The crude extract (0.75 g) was
suspended in aqueous MeOH (MeOH-H O, 9:1, 200 mL) and
extracted with n-hexane (3 × 200 mL). The aqueous layer was
then diluted to 60% MeOH (v/v) with H O and extracted with
CHCl
(3 × 200 mL). The aqueous layer was concentrated, and
the residue obtained was suspended in H O (25 mL) and
extract was
(11), 149 (23), 137 (100), 123 (72); HRFABMS m/z 455.3156
+
2
[M + H] (calcd for C29
H
43
O
4
, 455.3161).
Meth yla tion of On costem on ol D (4). Methylation of
oncostemonol D (4, 0.8 mg) as previously described gave a
2
1
3
product (0.6 mg) that was identical ( H NMR and MS) with
2
12.
extracted with n-BuOH (3 × 25 mL). The CHCl
3
On costem on ol E (5): colorless liquid; UV (MeOH) λmax 279
found to be the most cytotoxic and was fractionated over
Sephadex LH-20 using n-hexane-EtOAc (100:0 to 0:100) and
nm (ꢀ 13 600); IR ν
max
3450, 2953, 1635, 1451, 1116, 1050, 742
cm-1
1
H NMR, see Table 1; 13C NMR, see Table 2; EIMS m/z
(rel int) 482 [M ] (16), 465 (7), 450 (15), 418 (24), 386 (17),
331 (27), 221 (14), 151 (72), 136 (41), 123 (100); HRFABMS
m/z 483.3462 [M + H] (calcd for C H O , 483.3475).
;
then MeOH-H
2
O (100:0 to 40:60) to furnish eight fractions
+
(
A-H), of which fractions A-D were found to be the most
active. Fraction A on column chromatography over LRP-2 Si
gel using MeOH-H O (70:30) followed by reversed-phase
HPLC with the mobile phase CH CN-H O (80:20) yielded the
new 5-alkylresorcinol 6 (1.4 mg). Fraction B on reversed-phase
preparative TLC (MeOH-H O, 70:30) followed by reversed-
phase HPLC with the mobile phase CH CN-H O (70:30)
yielded the new 5-alkylresorcinol 1 (1.8 mg). Fraction C on
O (80:
0) followed by reversed-phase HPLC with mobile phase CH
+
3
1
47
4
2
Meth yla tion of On costem on ol E (5). Methylation of
oncostemonol C (5, 0.8 mg) as described previously gave a
3
2
1
product (0.6 mg) that was identical ( H NMR and MS) with
2
1
2.
On costem on ol F (6): colorless liquid; UV (MeOH) λmax 275
nm (ꢀ 13 250); IR νmax 3450, 2960, 1738, 1640, 1615, 1445, 1125,
3
2
column chromatography over LRP-2 using MeOH-H
2
-1
1
13
1
050, 1015, 750 cm ; H NMR, see Table 1; C NMR, see
2
3
-
+
Table 2; EIMS m/z (rel int) 526 [M ] (5), 508 (6), 466 (17), 403
48), 402 (28), 297 (6), 250 (21), 235 (13), 221 (17), 193 (14),
2
CN-H O (75:25) furnished the four new 5-alkylresorcinols 2
(
1
(1.7 mg), 3 (1.5 mg), 4 (1.2 mg), and 5 (1.1 mg). Fraction D on
+
35 (32), 123 (35), 59 (100); HRFABMS m/z 526.3301 M (calcd
reversed-phase preparative TLC (MeOH-H
2
O, 85:15) followed
CN-H
90:10) yielded the two new 5-alkylresorcinols 7 (0.9 mg) and
for C32 , 526.3293).
46 6
H O
by reversed-phase HPLC with the mobile phase CH
3
2
O
On costem on ol G (7): colorless liquid; UV (MeOH) λmax 291
nm (ꢀ 13 900); IR νmax 3390, 2945, 1640, 1450, 1125, 1055, 742
(
8
(0.7 mg) as well as the two known compounds 9 (4.2 mg)
-1
1
13
cm ; H NMR, see Table 1; C NMR, see Table 2; EIMS m/z
(
2
and 10 (2.3 mg). The two known compounds 9 and 10 were
+
rel int) 496 [M ] (15), 479 (4), 461 (15), 373 (18), 372 (16),
89 (26), 288 (42), 275 (28), 274 (18), 235 (21), 221 (15), 220
21), 207 (31), 206 (24), 137 (14), 123 (46), 122 (100); HR-
identified by comparison of their spectral data with literature
8
values.
(
On costem on ol A [(8′Z)-1-a cetoxy-3-h yd r oxy-5-[16′-(3′′-
a c e t o x y -5′′-h y d r o x y p h e n y l)-8′-h e x a d e c e n -1′-y l]b e n -
zen e] (1): colorless liquid; UV (MeOH) λmax 276 nm (ꢀ 14 600);
IR νmax 3450, 2960, 1735, 1645, 1615, 1453, 1128, 1065, 753
+
FABMS m/z 497.3618 [M + H] (calcd for C32
49 4
H O , 497.3631).
On costem on ol H (8): colorless liquid; UV (MeOH) λmax 286
nm (ꢀ 14 300); IR νmax 3410, 2950, 1635, 1435, 1105, 1055, 750
-1
1
13
cm ; H NMR, see Table 1; C NMR, see Table 2; EIMS m/z
(
(
-
1
1
13
cm ; H NMR, see Table 1; C NMR, see Table 2; EIMS m/z
(
+
rel int) 468 [M ] (11), 450 (6), 435 (10), 420 (8), 359 (15), 358
23), 345 (34), 344 (17), 275 (19), 274 (21), 247 (18), 246 (15),
+
rel int) 524 [M ] (6), 507 (4), 464 (12), 404 (21), 359 (18), 289
(
23), 235 (10), 205 (4), 165 (43), 164 (28), 123 (12), 61 (100);
2
35 (17), 221 (28), 207 (19), 193 (14), 137 (23), 123 (72), 122
+
HRFABMS m/z 525.3213 [M + H] (calcd for C32
H
45
O
6
,
+
(
4
45 4
100); HRFABMS m/z 469.3321 [M + H] (calcd for C30H O ,
5
0
25.3216).
Acetyla tion of On costem on ol A (1). Oncostemonol A (1,
.8 mg) was treated with Ac O-Py (1:1, 0.4 mL) at room
69.3318).
Acetyla tion of 9. Acetylation of 9 (1.5 mg) as described
2
1
above gave a product (1.2 mg) that was identical (TLC and H
temperature for 4 h. Usual workup gave product 11 (0.7 mg):
NMR) with 11.
1
13
H NMR, see Table 1; C NMR, see Table 2; EIMS m/z (rel
+
int) 608 [M ] (12), 548 (16), 488 (15), 291 (13), 277 (25), 207
Ack n ow led gm en t. This work was supported by Interna-
tional Cooperative Biodiversity Grant No. U01 TW/CA-00313
from the Fogarty Center, National Institutes of Health, and
this support is gratefully acknowledged. Field work essential
to this study was conducted under a collaborative agreement
between the Missouri Botanical Garden (MBG), the Parc
Botanique et Zoologique de Tsimbazaza, the United States
National Cancer Institute, and the Centre National d’Appli-
cations et des Recherches Pharmaceutiques (CNARP). We
gratefully acknowledge courtesies extended by the Government
of Madagascar (Minist e` re des Eaux et For eˆ ts). We thank
St e´ phan Rakotonandrasana, S. Randrianasolo, Chris Birkin-
shaw (MBG), and P. Antilahimena for plant collection, and
Adam Bradley (MBG) for bibliographic and database help. We
also thank Mr. Bill Bebout (VPISU) for obtaining the mass
spectra.
(
6
17), 205 (28), 165 (23), 123 (22), 61 (100); HRFABMS m/z
09.3439 [M + H]+ (calcd for C36
, 609.3427).
On cost em on ol B [8(′Z)-1-a cet oxy-3-h yd r oxy-5-[16′-
3′′,5′′-d ih yd r oxyp h en yl)-8′-h exa d ecen -1′-yl]ben zen e] (2):
colorless liquid; UV (MeOH) λmax 272 nm (ꢀ 13 980); IR νmax
49 8
H O
(
425, 2960, 1745, 1655, 1625, 1450, 1135, 1060, 750 cm ; 1H
-
1
3
1
3
NMR, see Table 1; C NMR, see Table 2; EIMS m/z (rel int)
4
2
+
82 [M ] (8), 465 (12), 422 (15), 359 (24), 289 (26), 235 (21),
05 (6), 165 (48), 123 (17), 61 (100); HRFABMS m/z 483.3097
+
[
M + H] (calcd for C30
H
43
O
5
, 483.3110).
Acetyla tion of On costem on ol B (2). Acetylation of on-
costemonol B (2, 0.6 mg) as previously described and usual
workup gave a product (0.5 mg) that was identical (TLC and
1
H NMR) with 11.
On costem on ol C [(8′Z)-1-h yd r oxy-3-m eth oxy-5-[16′-(3′′-
h yd r oxy-5′′-m e t h oxyp h e n yl)-8′-h e xa d e ce n -1′-yl]b e n -
zen e] (3): colorless liquid; UV (MeOH) λmax 268 nm (ꢀ 14 100);
IR νmax 3430, 2965, 1642, 1450, 1120, 1065, 751 cm ; H NMR,
see Table 1; C NMR, see Table 2; EIMS m/z (rel int) 468 [M ]
(
(
[
-1 1
Refer en ces a n d Notes
1
3
+
(
1) Biodiversity Conservation and Drug Discovery in Madagascar, Part
. For Part 4, see: Chaturvedula, V. S. P.; Schilling, J . K.; Miller, J .
12), 450 (4), 437 (3), 436(21), 404 (17), 331 (46), 221 (21), 207
5
16), 193 (16), 149 (36), 137 (100); HRFABMS m/z 469.3315
S.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I. J . Nat.
Prod. 2002, 65, 1222-1224.
2) Yang, S.-W.; Zhou, B,-N.; Malone, S.; Werkhoven, M. C. M.; van der
Troon, F.; Wisse, J . H.; Kingston, D. G. I. J . Nat. Prod. 1999, 62,
+
M + H] (calcd for C30
45 4
H O , 469.3318).
(
Meth yla tion of On costem on ol C (3). Oncostemonol C (3,
.8 mg) was treated with excess ethereal CH . Evaporation
0
2 2
N
1
173-1174.
and purification by preparative TLC gave a product (0.6 mg)
that was identical ( H, C NMR and MS) with compound 12.
On cost em on ol D [(8′Z)-1-h yd r oxy-3-m et h oxy-5-[16′-
(3) Perrier de la B aˆ thie, H. Myrsinac e´ es. In Flore de Madagascar et des
Comores; Humbert, H. Ed.; Firmin-Didot: Paris, 1953.
1
13
(
4) Deng, J ,-Z.; Starck, S. R.; Hecht, S. M. J . Nat. Prod. 1999, 62, 477-
4
80.
(
3′′,5′′-d ih yd r oxyp h en yl)-8′-h exa d ecen -1′-yl]ben zen e] (4):
(5) Fukuyama, Y.; Kiryama, Y.; Kodama, M.; Iwaki, H.; Hosazawa, S.;
colorless liquid; UV (MeOH) λmax 281 nm (ꢀ 11 650); IR νmax
Shinji, M. K. Chem. Pharm. Bull. 1995, 43, 1391-1394.

1
632 J ournal of Natural Products, 2002, Vol. 65, No. 11
Chaturvedula et al.
(
6) Suzuki, Y.; Esumi, Y.; Yamaguchi, I. Phytochemistry 1999, 52, 281-
(10) McBrien, K. D.; Bery, R. L.; Lewes, S. E.; Nedderman, K. M.;
Bursuker, I.; Huang, S.; Klehr, S. E.; Leet, J . E. J . Antibiot. 1995,
48, 1446-1452.
2
89.
(7) Tyman, J . H. P. Chem. Soc. Rev. 1979, 25, 499-537.
(8) Furstner, A.; Seidel, G. J . Org. Chem. 1997, 62, 2332-2336.
(9) Scannell, R. T.; Barr, J . R.; Murty, V. S.; Reddy, K. S.; Hecht, S. M.
J . Am. Chem. Soc. 1988, 110, 3650-3651.
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