Isolation and synthesis of two antiproliferative calamenene-type sesquiterpenoids from Sterculia tavia from the Madagascar Rain Forest

Article abstract of DOI:10.1016/j.bmc.2012.10.012

Investigation of the endemic Madagascan plant Sterculia taiva Baill. (Malvaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of two new bioactive calamenene-type sesquiterpenoids, named tavinin A (2) and

Full text of DOI:10.1016/j.bmc.2012.10.012

Bioorganic & Medicinal Chemistry 20 (2012) 6940–6944
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isolation and synthesis of two antiproliferative calamenene-type
sesquiterpenoids from Sterculia tavia from the Madagascar Rain Forest q
Yumin Dai a, Liva Harinantenaina a, Peggy J. Brodie a, Martin W. Callmander b, Sennen Randrianasolo b,
Etienne Rakotobe c, Vincent E. Rasamison c, David G. I. Kingston a,
⇑
a Department of Chemistry and the Virginia Tech Center for Drug Discovery, M/C 0212, Virginia Tech, Blacksburg, VA 24061, United States
b Missouri Botanical Garden, B.P 3391, Antananarivo 101, Madagascar
c Centre National d’Application des Recherches Pharmaceutiques, B.P 702, Antananarivo 101, Madagascar
a r t i c l e i n f o
a b s t r a c t
Article history:
Investigation of the endemic Madagascan plant Sterculia taiva Baill. (Malvaceae) for antiproliferative
activity against the A2780 ovarian cancer cell line led to the isolation of two new bioactive calamen-
ene-type sesquiterpenoids, named tavinin A (2) and epi-tavinin A (3) together with the known sesquit-
erpenoid mansonone G (1). The structures of the two new compounds were elucidated based on
analysis of their 1D and 2D NMR spectra and mass spectrometric data, and were confirmed by de novo
synthesis. The three isolated sesquiterpenoids (1–3) had modest antiproliferative activities against the
Received 16 August 2012
Revised 8 October 2012
Accepted 15 October 2012
Available online 23 October 2012
Keywords:
A2780 ovarian cancer cell line, with IC50 values of 10.2, 5.5 and 6.7
lM, respectively.
Calamenene-type sesquiterpenoid
Antiproliferative activity
Sterculia tavia
Ó 2012 Elsevier Ltd. All rights reserved.
Synthesis
Nuclear magnetic resonance
1. Introduction
2. Results and discussion
Discovering antiproliferative natural products from both tropi-
cal dry forests and rainforests of Madagascar has been one of the
objectives of our group’s involvement for more than 15 years in
the International Cooperative Biodiversity Group (ICBG) program.1
As a part of this research, an EtOH extract from the bark of Sterculia
tavia Baill. (Malvaceae) from the rain forest of northern Madagascar
was found to exhibit antiproliferative activity against the A2780
The EtOH extract of the bark part of the S. tavia was subjected to
liquid–liquid partitioning to give an active dichloromethane frac-
tion with an IC50 value of 9.6 lg/mL. Bioassay-guided separation,
including LH-20 size-exclusion, normal-phase silica gel chroma-
tography, and C-18 reverse-phase HPLC, was used to obtain the
known cadinane-type sesquiterpenoid mansonone (1), and the
two new calamenene-type sesquiterpenoids designated tavinin A
(2) and epi-tavinin A (3) as the major antiproliferative constituents
of the extract. Herein, we report the structural elucidation and syn-
thesis of the two new compounds.
human ovarian cancer cell line, with an IC50 value of 14 lg/mL.
The Malvaceae family is a family of flowering plants containing over
200 genera with close to 2300 species.2 Sterculia, one of the largest
genera of this family, is a rich source of alkaloids, saponins and fla-
vonoid glycosides, which are well-known for their broad range of
bioactivity, including antimicrobial, antifungal, insecticidal, cyto-
toxic, antioxidant, and anti-inflammatory activities among others.3
Although the genus Sterculia has been well investigated, the present
species is endemic to Madagascar and has not been explored for its
phytochemical composition and biological activity. The EtOH ex-
tract of S. tavia was thus selected for bioassay-guided fractionation
to isolate its antiproliferative components.
Compound 1 was identified as the cadinane-type sesquiterpe-
noid mansonone G (6-hydroxy-5-isopropyl-3,8-dimethyl-naphtha-
lene-1,2-dione) by comparison of its chemical and spectroscopic
data with those reported in the literature.4
Tavinin A (2), ½a D21
ꢀ
+15° (c 1.2, MeOH), was isolated as a light
yellow oil. Its positive ion HRESIMS revealed quasi-molecular ion
peaks at m/z 293.1399 [M+H]+ and 315.1194 [M+Na]+, correspond-
ing to a molecular formula of C16H20O5, with seven degrees of
unsaturation. Based on the similarity of the NMR spectral data of
2 and those of compound 1, compound 2 was identified as a ses-
quiterpenoid with a similar carbon skeleton. The six aromatic 13C
NMR resonances: d 162.9, 145.3, 127.8, 123.1, 116.7 and 107.4,
as well as the two singlet signals (Table 1) observed in the aromatic
region of its 1H NMR spectrum (d 7.64 and 7.37), revealed the
q
Biodiversity Conservation and Drug Discovery in Madagascar, Part 53. For Part
52, see Ref. 1b.
⇑
Corresponding author. Tel.: +1 540 231 6570; fax: +1 540 231 3255.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.012

Y. Dai et al. / Bioorg. Med. Chem. 20 (2012) 6940–6944
6941
Table 1
hydrogen-bonded carbonyl groups in the structure. Since the ben-
zene rings and two carbonyl groups contribute four and two de-
grees of unsaturation, respectively, the remaining one was
assigned to a ring. The above data indicated that the basic skeleton
of 2 was that of an oxygenated calamenene-type sesquiterpenoid.6
The methyl proton signal located at d 2.27 and the methoxy proton
at d 4.04 (both singlets) were assignable to a C-6 methyl carbon
and to the carbon of a C-7 methoxyl group of the benzene ring. This
assignment was supported by HMBC correlations (Fig. 1a) between
H-15 (d 2.27) and C-7 (d 162.9) and the C-5 methine carbon (d
127.8), and between H-16 (d 4.04) and C-7. An oxygen-bearing
quaternary carbon (d 88.9, C-4) was determined to be attached to
C-10 of the benzene ring (d 123.1) due the presence of a 3J HMBC
crosspeak between the methine proton at H-5 and C-4. The 1H
NMR spectrum exhibited resonances at d 0.67 (d, J = 6.7 Hz, H-
12) and d 0.88 (d, J = 6.7 Hz, H-13) for the two methyl groups of
an isopropyl group, and these showed COSY correlations with a
methine proton at d 2.52 (sept, J = 6.8 Hz, H-11). The 3J HMBC cor-
relation between the protons of one of the doublet methyl groups
(H-12/H-13) and the carbon at d 88.9 allowed us to determine the
connection of the isopropyl unit to be at C-4. Moreover, the 2J
HMBC correlation observed between the protons at d 1.62 (H-14)
and C-1, the 3J HMBC crosspeaks between H-14 and C-9 (d 145.3)
and between H-14 and one carbonyl carbon at d 204.0 (C-2), as
well as the 3J HMBC correlations between the septet methine pro-
ton (H-11) and the carbon at d 196.7 (C-3), allowed us to determine
the location of a second oxygen-bearing quaternary carbon (d 73.8)
and the two carbonyl carbons to be at C-1, C-2 and C-3, respec-
tively. The relative configuration of 2 (Fig. 1a) as well as the assign-
ment of the aromatic methyl and methoxy groups to C-6 and C-7,
respectively, rather than the reverse, was deduced by the interpre-
tation of the results of the 1D NOE-difference experiment. When
the H-14 protons (d 1.62) were irradiated, only the signal at d
7.37 (H-8) was amplified (1.23%). Similarly, irradiation of H-11 (d
2.52) only enhanced the aromatic signals at d 7.64 (H-5) by 3.18%.
NMR spectroscopic data for 2 and 3 in acetone-d6 (600 MHz)
14
O
OH
OH
O
O
O
O
9
O
7
1
4
16
O
OH
O
10
15
5
13
HO
HO
11
12
1
2
3
Position
2
3
dH (J in Hz)
dCa, type
dH (J in Hz)
dCa, type
1
2
3
4
5
6
7
8
—
—
—
—
73.8, C
204.0, C
196.7, C
88.9, C
127.8, CH
116.7, C
162.9, C
107.4, CH
145.3, C
—
—
—
—
7.68 s
—
—
7.33 s
74.7, C
208.2, C
200.7, C
91.2, C
7.64 s
128.1, CH
116.9, C
163.2, C
108.0, C
144.8, C
122.8, C
35.6, CH
15.6, CH3
16.2, CH3
27.4, CH3
15.1, CH3
55.5, CH3
—
—
7.37 s
9
—
—
—
—
10
11
12
13
14
15
16
123.1, C
2.52 sept
36.1, CH
15.4, CH3
15.9, CH3
28.1, CH3
15.1, CH3
55.5, CH3
2.35 sept
0.67 d (J = 6.7 Hz)
0.88 d (J = 6.7 Hz)
1.62 s
0.81 d (J = 6.7 Hz)
0.90 d (J = 6.7 Hz)
1.86 s
2.27 s
4.05 s
2.27 s
4.04 s
The 13C NMR spectral data were generated from gHSQC and gHMBC spectra.
a
Table 2
Antiproliferative activity of compounds 1–3 and synthetic intermediates 7–12 against
A2780 ovarian cancer cells
Compound
IC50 (lM)
1
2
10.2 0.9
5.5 0.9
5.9 0.7
6.7 0.3
6.3 0.4
>50
rac-2
3
rac-3
7
8
9
10
11
The second compound isolated, epi-tavinin A (3), ½a D21
ꢀ
+8° (c 1.2,
MeOH) had the same molecular formula (C16H20O5) as 2 by inter-
pretation of its HRESIMS data. The NMR spectroscopic data of com-
pound 3 were very similar to those of 2 except for the 1H chemical
shifts of H-11, H-12, H-13 and H-14. Compounds 2 and 3 were thus
assigned as a pair of diastereomers differing in one of their two ste-
reogenic centers (C-1 and C-4). The relative configuration of 3 was
determined in the same manner as that of 2. Irradiation of H-14 in
a 1D NOE-difference experiment enhanced the signals of both H-8
and H-11 by 1.32% and 1.35%, respectively, indicating a syn rela-
tionship between the methyl and isopropyl groups in 3. Based on
these observations, the relative configurations of C-1 and C-4 of
compound 2 were assigned as R⁄ and R⁄ while those of C-1 and
C-4 of 3 were assigned as S⁄ and R⁄.
>50
33.9 1.5
4.0 0.5
22.1 3.4
35.5 2.7
0.028 0.003
12
Paclitaxel
presence of a tetrasubstituted benzene ring with two protons lo-
cated in the para-position.5 The presence of two deshielded carbon
signals at d 204.0 and 196.7, as well as the compound’s IR
absorption (
m
max = 1659 cmꢁ1), indicated the presence of two
H
H
H
H
H
H
OH
OH
H
H
HMBC
O
O
O
O
O
O
H
H
H
H
H
H
COSY
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HO
HO
NOESY
H
H
H
H
H
H
H
H
Figure 1. (a) HMBC, COSY, and NOESY correlations of 2; (b) HMBC, COSY and NOESY correlations of 3.

6942
Y. Dai et al. / Bioorg. Med. Chem. 20 (2012) 6940–6944
The structures of compounds 2 and 3 were confirmed by syn-
products 2 and 3 showed weak inhibition of the A2780 ovarian
cancer cells, with IC50 values of 5.5 and 6.7 M, respectively. The
synthetic compounds rac-2 and rac-3 had identical values within
experimental error, indicating that bioactivity is not dependent
on stereochemistry in this series. Among the synthetic intermedi-
thesis (Scheme 1). The synthesis initially followed McCormick’s
method for the synthesis of lacinilene C methyl ether (10) from
2-methylanisole.7 The literature procedure was modified by run-
ning the Grignard reaction to convert the known ketoester 4 to
5-isopropyl-5-(4-methoxy-3-methylphenyl)-dihydrofuran-2-one
(6) at low temperature, which increased the yield of 6 from 20% to
42% and of the reduction byproduct 5 from 23% to 30%. Dihydrof-
uranone 6 was then converted to cycloalkene 7, diol 8, and ketone
9 as previously described,7 except that conversion of 8 to 9 was
carried out using the sulfur trioxide pyridine complex instead of
the Swern oxidation originally used.7 Oxidation of the carbon–
carbon double bond of compound 10 to triol 13 could not be
accomplished directly with osmium tetroxide in the presence of
N-methylmorpholine N-oxide because of the electron deficient
l
ates, only lacinilene C methyl ether (10, IC50 value of 4.0 lM) was
of comparable potency to the natural products; the other calamen-
ene-type sesquiterpenoid intermediates were less active or inac-
tive. The cytotoxic activity of compound 10 may be due to the
presence of an
a,b-unsaturated ketone function in the molecule;
such groups are electrophilic and are able to bind to receptors,
leading to facile reactions with protein thiols, and ultimately to
induction of apoptosis.10
4. Experimental section
nature of the ab-unsaturated double bond. The ketone 10 was thus
reduced to alcohol 118 by Luche reduction9 in a yield of 90.3%. The
product appeared to consist of a single compound as judged by
HPLC and by the presence of only one singlet for H-2 in its 1H
NMR spectrum, presumably because the orientation of the hydride
ion attack was governed by complexation of the borohydride with
the adjacent C-1 hydroxyl group. Oxidation of 11 with osmium
tetroxide in the presence of N-methylmorpholine N-oxide afforded
tetraol 12 as a mixture of stereoisomers. Without separation of this
mixture, the tetraols 12 were further oxidized by the sulfur triox-
ide pyridine complex to give a mixture of four diones as two pairs
of enantiomers in the relatively low yield of 43.2%. The diones were
separated by HPLC to afford synthetic racemic 2 and synthetic
racemic 3. The structures of the natural products isolated were
confirmed by comparison of their 1H NMR, UV spectrometric and
HRESIMS data with those of their synthetic counterparts.
4.1. General experimental procedures
Optical rotations were recorded on a JASCO P-2000 polarimeter.
IR spectroscopic data were measured on a MIDAC M-series FTIR
spectrophotometer. NMR spectra were recorded in acetone-d6 or
CDCl3 on Bruker Avance 500 or Bruker Avance 600 spectrometers.
The chemical shifts are given in d (ppm), and coupling constants (J)
are reported in Hz. Mass spectra were obtained on an Agilent 6220
LC–TOF–MS in the positive ion mode.
4.2. Antiproliferative bioassays
Antiproliferative activities were obtained at Virginia Tech
against the drug-sensitive A2780 human ovarian cancer cell line11
as previously described.1c
3. Bioassay data
4.3. Plant material
The isolated tavinin A (2) and epi-tavinin A (3), the synthetic
compounds rac-2 and rac-3 as well as the intermediates involved
in the synthetic route were tested for antiproliferative activity
against the A2780 ovarian cancer. As listed in Table 2, both natural
A sample of the bark parts of Sterculia tavia Baill. was collected
in October 2005. The sample was collected from a tree 24 m high,
with yellowish seeds and light brown fruits. The collection was
made in the rainforest 8 km from Ankijabe, in the Daraina region
O
COEt
O
O
O
O
O
O
O
O
a
b
O
O
+
O
O
three steps
O
4
5
6
7
HO
HO
HO
HO
HO
HO
O
O
O
O
c
d
e
f
9
8
11
10
X
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
g
+
HO
O
HO
12
rac-2
rac-3
13
Scheme 1. Synthesis of compound 2 and 3. Reagents and conditions: (a) isopropylmagnesium chloride, THF, ꢁ15 °C, 1 h, 42.1%; (b) 2% Osmium teroxide, N-Methyl-
morpholine-N-oxide (NMO), dichloromethane, rt, 48 h, 75.4%; (c) sulfur trioxide–pyridine complex, dichloromethane, 0 °C, 2 h, 91.9%; (d) 2,3-dichloro-5,6-dicyanobenzo-
quinone (DDQ, benzene), reflux, 24 h, 58%; (e) NaBH4, CeCl3, 7H2O, rt, 5 min, 90.3%; (f) 2% Osmium tetroxide, N-methyl-morpholine-N-oxide (NMO), dichloromethane, rt,
48 h; (g) sulfur trioxide–pyridine complex, dichloromethane, 0 °C, 2 h, 13% (rac-2) 10% (rac-3).

Y. Dai et al. / Bioorg. Med. Chem. 20 (2012) 6940–6944
6943
of Madagascar, at coordinates 13°1601200S 049°3604000E and eleva-
tion 617 m. Voucher specimens have been deposited at the Parc
Botanique and Zoologique de Tsimbazaza (TAN), at the Centre Na-
tional d’Application des Recherches Pharmaceutiques in Antanan-
arivo, Madagascar (CNARP), the Missouri Botanical Garden in St.
Louis, Missouri (MO), and the Muséum National d’Histoire Natu-
relle in Paris, France (P), voucher Sennen Randrianasolo et al. 533.
were washed with saturated NaCl solution, dried over Na2SO4
and concentrated to afford 468 mg brown oil, which was refluxed
together with 500 mg KOH in 100 mL 95% EtOH for 4 h. The EtOH
was removed from the mixture under reduced pressure, 100 mL of
water was added, and the resulting mixture was extracted three
times with 50 mL Et2O. The aqueous portion was acidified to pH
1 with HCl and stirred at rt for 2 h. The acidified solution was ex-
tracted three times by another 50 mL of Et2O, and the ethereal po-
tion was washed with saturated Na2CO3 and NaCl solution,
respectively, dried by Na2SO4 and concentrated under reduced
pressure to afford 313 mg of a yellow oil. Chromatographic purifi-
cation of the crude lactone (50 g of silica gel; eluted with EtOAc/
hexane, 3:7) afforded 167 mg of compound 6 (Rf 0.56, silica gel
TLC, EtOAc/hexane, 3:7) and 140 mg of compound 5 (Rf 0.40).
The NMR and mass spectrometric data were consistent with the
data reported.7
4.4. Extraction and isolation
Dried bark parts of S. tavia (252 g) were ground in a hammer
mill, then extracted with EtOH by percolation for 24 h at room
temperature to give the crude extract MG 3532 (5.2 g), of which
1.42 g was shipped to Virginia Tech for bioassay-guided isolation.
A 1.4 g sample of MG 3532 (IC50 14 lg/mL) was suspended in aque-
ous MeOH (MeOH/H2O 9:1, 100 mL), and extracted with hexane
(3 ꢂ 100 mL portions). The aqueous layer was then diluted to
60% MeOH (v/v) with H2O and extracted with dichloromethane
(DCM) (3 ꢂ 150 mL portions). The hexane fraction was evaporated
4.8. 1,2,3,4-Tetrahydro-1,2-dihydroxy-4-isopropyl-7-methoxy-
1,6-dimethylnaphthalenes (8)
in vacuo to leave 497.5 mg of material with IC50 = 15
residue from the DCM fraction (213.5 mg) had an IC50 of 9.6
l
g/mL. The
g/
mL, and the remaining aqueous MeOH fraction had an IC50
>20 g/mL. LH-20 size exclusion open column chromatography
l
This compound was prepared from 4-hydroxy-4-(4-methoxy-3-
methylphenyl)-5-methyl-hexanoic acid lactone (6) in four steps as
previously described; its spectroscopic data matched the literature
data.7
l
(DCM/MeOH = 1:1) of the DCM fraction was used to obtain six frac-
tions, of which the two most active fractions F3 (67.7 mg) and F4
(51.4 mg) had IC50 values of 5.9 and 4.8
tion F4 was then applied to a silica gel column with elution by hex-
ane/EtOAc, 7:3 to give seventeen fractions, of which fraction 4
l
g/mL, respectively. Frac-
4.9. l-Hydroxy-4-isopropyl-7-methoxy-1,6-dimethyl-2-
naphthalenone (10)
(2.3 mg) was the most active (IC50 3.7
l
g/mL), and yielded com-
Diol mixture 8 (50 mg) was dissolved in 50 mL of CH2Cl2 and
the solution cooled to 0 °C. Sulfur trioxide pyridine complex
(150.6 mg) was added to the solution during a 5 min period. After
stirring for 2 h, 50 mL of saturated Na2CO3 solution was added, and
the layers were separated. The aqueous layer was extracted with
50 mL of CH2Cl2 three times, and the combined organic layers were
washed with saturated NaCl solution, dried over Na2SO4, and con-
centrated under reduced pressure to afford 45.3 mg of ketone 9 as
a yellow oil. The crude ketone and 56.6 mg dichlorodicyanobenzo-
quinone were stirred in 50 mL of benzene for 24 h at room temper-
ature, after which the solvent was evaporated under reduced
pressure. The resulting oil was purified by column chromatography
(50 g of silica gel; eluted with EtOAc/hexane, 2:3) to give 28.9 mg
(58%) of compound 10 (Rf 0.40). The NMR and mass spectrometric
data were consistent with the reported data.7
pound 1 (1.5 mg, IC50 2.5 g/mL) on chromatography on C-18 HPLC
l
with elution by 85% MeOH in water, with the retention time of
14.9 min. Fraction F3 was also applied to a silica gel column with
elution by hexane/EtOAC = 4:1 to give six fractions, of which frac-
tion 5 (26.2 mg) was the most active (IC50 2.7
lg/mL). Compounds
2 (0.32 mg, IC50 1.7 g/mL) and 3 (0.28 mg, IC50 1.9
l
lg/mL), with
retention times of 14.7 and 15.2 min, respectively, were obtained
by using C-18 HPLC eluted by 70% MeOH in water to purify fraction
5.
4.5. (1R,4R)-1,4-dihydroxy-1-isopropyl-6-methoxy-4,7-
dimethylnaphthalene-2,3(1H,4H)-dione (2, Tavinin A)
Light yellow oil; ½a D21
ꢀ
+15 (c 1.2, MeOH); UV (MeOH) kmax (e)
207 (3.20), 232 (2.35), 280 (1.62); IR mmax cmꢁ1: 3418, 2926,
1659, 1463, 1052 cmꢁ1
.
1H NMR (600 MHz, acetone-d6), and 13C
4.10. 4-Isopropyl-7-methoxy-1,6-dimethyl-1,2-
dihydronaphthalene-1,2-diol (11)
NMR (125 MHz, acetone-d6), see Table 1; HRESIMS m/z 315.1194
[M+Na]+ (calcd for C16H20NaO5, 315.1208).
Compound 10 (26.0 mg) was dissolved in 25 mL of 0.4 mM
methanolic CeCl3ꢃ7H2O at room temperature, and 1 equiv of NaBH4
(3.8 mg) was added to the solution. After stirring for 5 min, the
mixture was dried under reduced pressure, redissolved in 25 mL
of water and extracted three times with 25 mL of dichloromethane.
The organic portion was washed with saturated NaCl solution,
dried over Na2SO4 and concentrated under reduced pressure. The
resulting light yellow oil was purified by column chromatography
(50 g of silica gel; eluted with EtOAc/hexane, 2:3) to afford 23.7 mg
(90%) of diol 11. The product appeared to consist of a single com-
pound as judged by HPLC and by the presence of only one singlet
for H-2 in its 1H NMR spectrum, presumably because the orienta-
tion of the hydride ion attack was governed by complications of
the borohydride with the adjacent C-1 hydroxyl group. m/z
262.1658; 1H NMR (CDCl3) d 7.12 (H-8, s, 1), 7.06 (H-5, s, 1), 5.56
(H-3, br s, 1), 4.48 (H-2, br s, 1), 3.86 (H-16, s, 3), 2.87 (H-11, sept,
1, J = 6.65), 2.19 (H-15, s, 3), 1.30 (H-14, s, 3), 1.19 (H-12, d, 3,
J = 6.68), 1.06 (H-13, d, 3, J = 6.68); 13C NMR (CDCl3) d 157.5 (C-7,
C), 142.6 (C-4, C), 142.2 (C-9, C), 126.2 (C-5, CH), 125.2 (C-10, C),
4.6. (1R,4S)-1,4-dihydroxy-1-isopropyl-6-methoxy-4,7-
dimethylnaphthalene-2,3(1H,4H)-dione (3, Epi-Tavinin A)
Light yellow oil; ½a D21
ꢀ
+8 (c 1.2, MeOH); UV (MeOH) kmax (e) 207
(3.20), 232 (2.35), 280 (1.62); IR mmax cmꢁ1: 3432, 2932, 1671,
1459, 1078 cmꢁ1 1H NMR (600 MHz, acetone-d6), and 13C NMR
.
(125 MHz, acetone-d6), see Table 1; HRESIMS m/z 315.1201
[M+Na]+ (calcd for C16H20NaO5, 315.1208).
4.7. Synthesis of 4-hydroxy-4-(4-methoxy-3-methylphenyl)-5-
methyl-hexanoic acid lactone (6)
Ketoester 412 (500 mg) was dissolved in 50 mL of THF, and the
solution cooled to ꢁ15 °C. Isopropyl magnesium chloride (1.5 mL
of a 2.0 M solution in THF) was added during a 15 min period. After
stirring for 2 h, the mixture was dried under reduced pressure, and
50 mL of saturated NH4Cl solution was added. The aqueous solu-
tion was extracted with 50 mL Et2O three times, and the extracts

6944
Y. Dai et al. / Bioorg. Med. Chem. 20 (2012) 6940–6944
125.2 (C-6, C), 121.7 (C-3, CH), 105.7 (C-8, CH), 77.1 (C-2, CH), 77.0
(C-1, C), 55.7 (C-16, CH3), 28.2 (C-11, CH); 22.7 (C-14, CH3), 21.6 (C-
12, CH3), 20.8 (C-13, CH3), 16.4 (C-15, CH3).
work essential for this project was conducted under a collaborative
agreement between the Missouri Botanical Garden and the Parc
Botanique et Zoologique de Tsimbazaza and a multilateral agree-
ment between the ICBG partners, including the Centre National
d’Applications des Recherches Pharmaceutiques. We gratefully
acknowledge courtesies extended by the Government of Madagas-
car (Ministère des Eaux et Forêts).
4.11. 1,4-Dihydroxy-1-isopropyl-6-methoxy-4,7-
dimethylnaphthalene-2,3-diones rac-2 and rac-3
Diol 11 (22.0 mg) was dissolved in 6 mL of acetone and 2.5 mL
of water and the solution cooled to ꢁ25 °C. N-methylmorpholine
N-oxide (0.05 mL of a 50% solution in water) and 0.1 mL of 2% os-
mium tetroxide aqueous solution were then added under nitrogen.
The mixture was allowed to warm to room temperature and stirred
for an additional 48 h, and then 50 mg of Na2S2O4 was added. The
acetone was removed under reduced pressure, the pH was ad-
justed to 2 by HCl, and the solution was extracted with 20 mL
dichloromethane three times. The combined extracts were washed
with saturated NaCl solution, dried by Na2SO4 and concentrated
under reduced pressure to afford product 12 as a light brown oil
The crude product was oxidized directly with sulfur trioxide pyri-
dine complex as described in the synthesis of compound 10 above.
The resulting 6.6 mg of light brown oil was applied to C-18 HPLC
and eluted by 68% MeOH in water to afford 3.2 mg of light yellow
oil (rac-2) and 2.5 mg of light yellow oil (rac-3), at retention times
of 19.5 and 20.7 min, respectively. The NMR and mass spectromet-
ric data of both compounds were identical to those of the corre-
sponding natural products.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.012.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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This project was supported by the Fogarty International Center,
the National Cancer Institute, the National Science Foundation, the
National Heart, Lung and Blood Institute, the National Institute of
Mental Health, the Office of Dietary Supplements, and the Office
of the Director of NIH, under Cooperative Agreement U01
TW000313 with the International Cooperative Biodiversity Groups.
This project was also supported by the National Research Initiative
of the Cooperative State Research, Education and Extension
Service, USDA, Grant #2008-35621-04732. These supports are
gratefully acknowledged. This work was also supported by the Na-
tional Science Foundation under Grant no. CHE-0619382 for pur-
chase of the Bruker Avance 500 NMR spectrometer and Grant no.
CHE-0722638 for the purchase of the Agilent 6220 mass spectrom-
eter. We thank Mr. Bill Bebout for obtaining the mass spectra and
Dr. Hugo Azurmendi for assistance with the NMR spectra. Field
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