Sesquiterpenes from Blumea balsamifera

Article abstract of DOI:10.1021/np100646n

Five new guaiane sesquiterpenes, blumeaenes E1 (1), E2 (2), K (3), L (4), and M (5), and one new eudesmane sesquiterpene, samboginone (6), along with three known compounds, cryptomeridiol, 3,3′,5,7-tetrahydroxy-4′- methoxyflavanone, and austroinulin, were isolated from the leaves of the Philippine medicinal herb sambong, Blumea balsamifera. The absolute configuration of the new guaiane core was determined as 1S,7S,9S,10R by employing the modified Mosher's method. In the structure of 1, the absolute configuration of the epoxyangelic acid moiety was identified as 2S,3S using (R)-PGME as a chiral anisotropic auxiliary.

Full text of DOI:10.1021/np100646n

NOTE
pubs.acs.org/jnp
Sesquiterpenes from Blumea balsamifera
Osamu Shirota,*,‡ Jennifer M. Oribello,§ Setsuko Sekita,‡ and Motoyoshi Satake¥
Division of Pharmacognosy and Phytochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501,
Japan
S Supporting Information
b
ABSTRACT: Five new guaiane sesquiterpenes, blumeaenes E1 (1), E2
(2), K (3), L (4), and M (5), and one new eudesmane sesquiterpene,
samboginone (6), along with three known compounds, cryptomeridiol,
3,30,5,7-tetrahydroxy-40-methoxyflavanone, and austroinulin, were iso-
lated from the leaves of the Philippine medicinal herb sambong, Blumea
balsamifera. The absolute configuration of the new guaiane core was
determined as 1S,7S,9S,10R by employing the modified Mosher’s method.
In the structure of 1, the absolute configuration of the epoxyangelic acid moiety was identified as 2S,3S using (R)-PGME as a chiral
anisotropic auxiliary.
he Philippines archipelago, with some 8000 higher plant
T
species, is one of the eight most plant-rich areas in Asia.1 The
vegetative pattern of the Philippine area suggests that most of
these plants have considerable and distinct medicinal values.
Further, some species are being screened for their pharmacolo-
gical importance. However, only a few are recognized in the
standard herbal pharmacopoeia of the Philippines.
Sambong (Blumeae Balsamiferae Folium) is one of the most
commonly used medicinal herbs in the Philippines and is the
fresh or dried leaf of Blumea balsamifera (L.) DC (Asteraceae).
The plant is an erect, suffrutescent, pubescent shrub with strong
aromatic properties that grows up to 3 m high on roadsides and in
fields, lowlands, and mountainous regions in India, Myanmar,
South China, Taiwan, Thailand, Malaysia, Indonesia, and the
Philippines. It has several common names, such as sambong (in
Tagalog), lakdabulan (in Bikol), subsusob (in Ilokano), and
blumea camphor (in English) and is traditionally used for
arthritis, rheumatism, and chest pain; the crushed leaves mixed
with coconut oil are applied on the affected area. A decoction of
the leaves is ingested as a tea for cough and gas pain in adults and
children older than seven. Crushed leaves are mixed with oil and
applied on the abdomen of children to relieve gas pain. Doctors
in the Philippines now routinely prescribe sambong for the
dissolution of kidney stones. Sambong is also known as a diuretic
and is used in cases of hypertension and mild to moderate
congestive heart failure. Previous chemical investigations of this
plant led to the isolation of several sesquiterpenoids and
flavonoids.2-14
Compounds 1 and 2 were obtained as amorphous solids. Their
identical molecular formula, C20H30O6, was established by
HRFABMS data of m/z 349.1995 [M þ H - H2O]þ. The 1H
NMR spectra of both compounds (Table 1) showed similar
chemical shifts and coupling constants; three methyl doublets,
We investigated the chemical constituents of the Philippine
sambong to be used as reference compounds for TLC analysis of
monographs of Philippine herbal drugs. Chromatography of a
leaf extract of B. balsamifera afforded six new sesquiterpenes (1-
6) along with three known compounds, cryptomeridiol,15
3,30,5,7-tetrahydroxy-40-methoxyflavanone,2 and austroinulin.16
Special Issue: Special Issue in Honor of Koji Nakanishi
Received: September 13, 2010
Published: February 14, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
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olefinic carbon at δC 137.1 (C-5). The same HMBC correlations
were obtained in 2. Thus, 1,9,10-trihydroxyguaian-4-en-6-one was
deduced as a basic core of 1 and 2. The relative configuration of
theguaianeskeletonwasdetermined bymeansof NOESY analysis
as shown in Figure 2. The relative configuration of 1 and 2 was the
same. The remaining unit c was found to be a glycidic acid moiety,
which could be derived from an angelic acid or a tiglic acid,
connected to the guaiane skeleton with an ester linkage at C-9
based on HMBC analysis. This glycidic acid moiety was derived
from an angelic acid in both 1 and 2, as a NOESY correlation was
observed between a methine proton at δH 3.08/3.09 (H-30) and
singlet methyl protons at δH 1.38/1.38 (H3-40) of 1 and 2.
Therefore, the structural difference between 1 and 2 should be
the configuration of the glycidic acid moiety. To determine the
configuration of the glycidic acid and the guaiane core, the
following strategy was employed. Angelic acid was oxidized to
obtain glycidic acid (epoxyangelic acid) as an enantiomeric pair.
This glycidic acid was attached to (R)-(-)-phenylglycine methyl
ester (PGME),17 which acted as a chiral anisotropic auxiliary, and
products were separated by ODS HPLC to obtain each diaster-
eomer in pure form. NMR analysis of these two diastereomers
revealed that the first-eluting isomer on the ODS HPLC should
have a 2R,3R-configuration concerning the epoxy ring, whereas
theslow-elutingisomer should have a 2S,3S-configurationbecause
an anisotropic effect was observed on the H-4 methyl group (δH
1.10) of the 2R,3R-isomer (the first-eluting isomer), and corre-
sponding NOESY correlations for each stereoisomer were also
observed as shown in Figure 3. Compound 1 was hydrolyzed to
obtain the glycidic acid (epoxyangelic acid) and the guaiane core.
Although NMR measurements of the (R)-PGME derivative of
the glycidic acid from 1 failed because of the limited amount of
the derivative and the water solubility of the glycidic acid, the
retention time on the ODS HPLC of the (R)-PGME derivative
was identical to that of the slow-eluting isomer of the reference
compound. Thus the configuration of the glycidic acid moiety of 1
was confirmed as 20S,30S. In contrast, the remaining guaiane core
was successfully subjected to the modified Mosher's method to
establish the configuration as 1S,7S,9S,10R (Figure 4).18 There-
fore, diastereomer 2 should have a (2R,3R)-epoxyangelic acid
moiety. After our investigation was completed, we found that
Chen et al. recently reported the isolation and structural determi-
nation of 10 sesquiterpenoid esters, blumeaenes A-J, from the
same plant material.14 We realized that blumeaene E, one of their
isolated compounds, had a similar relative configuration to that of
1 and 2; however, the absolute configuration of both the
sesquiterpene core and the glycidic acid moiety was not deter-
mined. Therefore, compounds 1 and 2 were named blumeaenes
E1 and E2, respectively.
Figure 1. COSY correlations of 1 and 2.
Figure 2. Principal HMBC and NOESY correlations of 1 and 2.
three methyl singlets, one oxymethine, two D2O exchangeable
signals, and nine methine and/or methylene signals between 1.4
and 3.2 ppm were observed. Thedifferences between 1 and 2 were
restricted to the chemical shifts of two D2O exchangeable signals
and one methylene proton at δH 1.77 (ddd, J = 1.4, 3.3, 14.2 Hz)
for 1 and δH 1.86 (ddd, J = 1.3, 3.2, 14.2 Hz) for 2. The 13C NMR
spectra of the two compounds (Table 1) were similar, consisting
of one ketone carbonyl, one ester carbonyl, one set of tetrasub-
stituted double bonds, two oxygen-attached quaternary carbons,
one oxymethine, and six methyl carbons, along with the remaining
three methylene, three methine, and one quaternary carbon in
each compound. The analysis of the H-H COSY spectra of both
compounds revealed the same three units, a-c, as shown in
Figure 1. Construction of the gross structure for 1 and 2 was
Compound 3 was obtained as an amorphous solid, which had
a molecular formula of C20H32O7 according to HRMS. The 1H
NMR spectrum of 3 (Table 1) showed a similar spectroscopic
pattern to that of 1 and 2, except for the low-field shift of H-30
(δH 3.08/3.09 for 1 and 2; δH 4.36 for 3) and the appearance of
one additional D2O exchangeable proton at δH 3.39. A low-field
shift of C-20 (δC 60.2/59.8 for 1 and 2; δC 77.2 for 3) was
observed in the 13C NMR spectrum. Therefore, cleavage of the
epoxy rings in 1 and 2 was assumed for 3 from these spectro-
scopic features and was in good agreement with its molecular
formula. Chemical shift assignments of 3 were performed
by a combination of HSQC and HMBC spectroscopic analyses,
and the similarity of the relative configuration of the guaiane
core to those of 1 and 2 was confirmed by NOESY correlations.
1
performed by HMBC analysis (Figure 2). In the H NMR
spectrum of 1, a methyl singlet at δH 2.10 (H3-15) of unit a
showed HMBC correlations with two olefinic carbons at δC 137.1
(C-5) and 160.6 (C-4) and one ketone carbonyl carbon at δC
201.6 (C-6). This carbonyl carbon correlated with one methine
proton at δH 2.35 (H-11) of unit b, thus confirming the connec-
tion between units a and b through an R,β-unsaturated carbonyl
group. Similarly, the methyl singlet at δH 1.15 (H3-14) of unit b
had an HMBC correlation with an oxygen-substituted quaternary
carbon at δC 90.4 (C-1). This quaternary carbon correlated with
one set of methylene protons at δH 1.37/2.71 (H2-2) of unit a.
This set of methylene protons also gave correlations with the
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Figure 3. Absolute configuration of (2R,3R)- and (2S,3S)-epoxyangelic acid (R)-PGME conjugates.
Figure 4. Absolute configuration of the guaiane core of 1 by means of
the modified Mosher’s method.
Figure 5. Principal HMBC and NOESY correlations of 6.
protons at δH 1.22 had long-range correlations with C-9 (δC
70.8), a carbonyl carbon at δC 217.5, a quaternary carbon at
δC 52.2, and an olefinic carbon at δC 138.4; methylene protons
H2-2 (δH 2.38/2.79) and H2-3 (δH 2.38/2.58) showed correla-
tions with the carbonyl carbon. These long-range correlations
revealed that the methyl group was attached to the quaternary
carbon next to C-9, and the carbonyl carbon was located between
the quaternary carbon and C-2. Thus, a eudesmane core,
9-hydroxyeudesman-4-ene-1,6-dione, was found as the skeleton
of 6, as shown in Figure 5. The relative configuration including
the β-oriented methyl group at C-10 was established by NOESY
analysis; thus the structure of 6 was proposed for samboginone.
Thus, the structure of 3 for blumeaene K was proposed as
shown.
Compound 4, an amorphous solid, had a molecular formula of
C17H26O5. The 1H NMR spectra of 4 (Table 1) showed the same
spectroscopic pattern of the guaiane core as those of 1-3 and an
acetyl methyl proton signal instead of angelic acid derivative
signals. Therefore, it was assumed that 4 was the acetyl derivative
of the guaiane core, which we prefer to call the blumeaene core.
Thus, the structure of 4 was proposed for blumeaene L. 2D NMR
spectroscopic analyses fully agreed with the structure.
Compound 5, an amorphous solid having a molecular formula
1
of C20H30O6, showed similar patterns in both the H and 13C
NMR spectra to those of 1 and 2 including a glycidic acid moiety.
However, chemical shift differences were observed especially at
H-14 (δH 3.08/3.09 for 1 and 2; δH 4.36 for 5), H-8β (δH 1.51/
1.50 for 1 and 2; δH 2.51 for 5), and H-9 (δH 5.56/5.52 for 1 and
2; δH 5.00 for 5). NOESY analysis of 5 revealed that the
blumeaene core was epimerized at C-9 because a NOESY
correlation was observed between H-9 and H-14. This spectro-
scopic analysis also confirmed epoxyangelic acid as a glycidic acid
moiety, which showed a NOESY correlation between H-30 and
H-40, connected at C-9 of the blumeaene core via an ester linkage.
Therefore, the structure of 5 was assigned as blumeaene M.
Compound 6, an amorphous solid, had a molecular formula of
C15H22O3. The 1H and 13C NMR spectroscopic patterns were
somewhat different from those of the other compounds, and it
showed no acid moiety esterified to the sesquiterpene core. The
COSY spectrum revealed similar connectivity with units a and b
of the blumeaene core. In the HMBC spectrum, the methyl
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a JASCO P-1030 polarimeter. UV, CD, and IR spectra
were obtained with a JASCO V-560 UV/vis spectrophotometer, a
JASCO J-820 spectropolarimeter, and a JASCO FT/IR-6300 spectro-
meter with an ATR option, respectively. 1D and 2D 1H and 13C NMR
spectra were recorded on a Varian Unity Plus 400 spectrometer at 300 K
by using Varian standard pulse sequences. Phase-sensitive NOESY
experiments were conducted with a mixing time of 800 ms. A 3.57 ms
(140 Hz) delay was used to optimize one-bond coupling in the HSQC
spectra and suppress them in the HMBC spectra, and the evolution delay
for long-range couplings in the HMBC spectra was set at 62.5 ms (8 Hz).
FABMS and HRFABMS spectra were obtained on a JEOL AX-505H
spectrometer. ESITOF and HRESITOFMS spectra were obtained on a
Q-Tof micro mass spectrometer (Micromass/Waters). Silica gel open-
column chromatography was performed on silica gel 60 (Merck). MPLC
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was performed with a prepacked glass column (Ultra Pack: 26 mm i.d. ꢀ
300 mm; Yamazen Corp., Kyoto, Japan) packed with 40 μm of silica gel.
HPLC was performed with an Inertsil PREP-ODS column (5 mm i.d. ꢀ
250 mm for analysis, 20 mm i.d. ꢀ 250 mm for preparative; GL Science
Inc. Tokyo, Japan) packed with 10 μm of ODS. TLC was conducted on
precoated silica gel 60 F254 (Merck) and/or RP-18 F254s (Merck), and
the spots were detected by heating after spraying with vanillin-sulfuric
acid reagent.
Plant Material. Air-dried B. balsamifera (L.) DC (Asteraceae)
leaves were obtained in August 2000 from the University of the
Philippines Los Banos, Laguna. Plant identification was confirmed by
Dr. Wilfredo Vendivil of the Herbarium in the Department of Botany,
the National Museum of the Philippines. Voucher specimens (#2000.8-
SBF) were deposited at the Division of Pharmacognosy and Phyto-
chemistry, National Institute of Health Sciences, Japan, and Food-Drug
Regulation Office, Bureau of Food and Drugs, Department of Health,
Philippines.
Blumeaene L (4):. amorphous solid; [R]23 -18 (c 0.05,
D
MeOH); UV (MeCN) λmax (log ε) 246 (3.91) nm; CD (MeCN) λmax
(Δε) 330 (-0.8), 285 (-0.2), 252 (-2.8), 202 (þ1.5) nm; IR (ATR)
3428, 2929, 1720, 1666, 1606, 1462, 1431, 1370, 1242, 1107, 1038, 977,
877, 837, 669, 617 cm-1; 1H NMR (CDCl3, 400 MHz) see Table 1; 13
C
NMR (CDCl3, 100 MHz) see Table 2; ESITOFMS m/z 333.2 (100, [M
þ Na]þ), 282.3 (41), 233.2 (78), 215.1 (32), 191.1 (33); HRESI-
TOFMS m/z 333.1690 (calcd for C17H26O5Na, 333.1678).
Blumeaene M (5):. amorphous solid; [R]23 þ48 (c 0.01,
D
MeOH); UV (MeCN) λmax (log ε) 247 (3.90) nm; CD (MeCN) λmax
(Δε) 319 (-0.4), 296 (-0.2), 279 (-0.4), 247 (þ3.1), 204 (-3.2) nm;
IR (ATR) 3445, 2960, 1727, 1667, 1612, 1445, 1372, 1264, 1112, 877,
760, 668 cm-1; 1H NMR (CDCl3, 400 MHz) see Table 1; 13C NMR
(CDCl3, 100 MHz) see Table 2; ESITOFMS m/z 389.2 (100, [M þ
Na]þ), 331.2 (17), 233.2 (61), 215.1 (45), 191.1 (27); HRESITOFMS
m/z 389.1944 (calcd for C20H30O6Na, 389.1940).
Samboginone (6):. amorphous solid; [R]23 -13 (c 0.114,
D
MeOH); UV (MeCN) λmax (log ε) 237 (3.78) nm; CD (MeCN) λmax
(Δε): 306 (-0.8), 242 (þ4.9), 202 (-1.6) nm; IR (ATR) 3428, 2959,
1694, 1445, 1371, 1263, 1236, 1161, 1067, 1007, 917, 803, 759, 669, 541
cm-1; 1H NMR (CDCl3, 400 MHz) see Table 1; 13C NMR (CDCl3,
100 MHz) see Table 2; ESITOFMS m/z 233.2 (100, [M - H2O þ
H]þ), 215.2 (52); HRESITOFMS m/z 233.1539 (calcd for C15H21O2,
233.1542).
Preparation of Epoxyangelic Acids and Their (R)-PGME
Derivatives. To an MeOH solution (60 mL) of angelic acid methyl
ester (3.5 g; 30 mmol) was added 35 mL of 1 mol/L LiOH, and the
mixture stirred overnight. The resulting solution was concentrated,
acidified with 1 mol/L H2SO4, saturated with NH4Cl, and then extracted
with DCM five times. The organic layer was washed with brine, dried
over Na2SO4, and concentrated to obtain angelic acid as a colorless,
crystalline solid (2.15 g; 71.5%): 1H NMR (CDCl3, 400 MHz) δ 1.92
(3H, dq, J = 1.6, 1.6 Hz), 2.05 (3H, dq, J = 1.6, 7.2 Hz), 6.23 (1H, qq, J =
1.6, 7.2 Hz); 13C NMR (CDCl3, 100 MHz) δ 16.1 (q), 20.4 (q), 127.2
(s), 141.2 (d), 173.9 (s).
Extraction and Isolation. B. balsamifera leaves (24.3 g) were
powdered and extracted with MeOH (3 ꢀ ca. 100 mL) at 40-50 ꢀC,
and the MeOH solution was evaporated in vacuo at 37 ꢀC to yield a
MeOH extract (3.23 g). The extract was chromatographed over a silica
gel open column eluted with n-hexane/EtOAc (9:1 to 0:1) and then
EtOAc/MeOH (1:1 to 0:1) as stepwise gradient solvent systems to
yield 10 fractions. Fraction 6 (199 mg) was separated by silica gel
MPLC eluted with n-hexane/EtOAc (6:4, 1:1, and 4:6). Fraction 7 (209
mg) was separated by silica gel MPLC eluted with n-hexane/EtOAc (4:6
and 3:7). The MPLC-derived fractions were further purified by
ODS HPLC with aqueous MeCN as the eluent to yield six new
compounds: blumeaene E1 (1; 17.3 mg, 0.071% from dried leaves),
blumeaene E2 (2; 2.1 mg, 0.009%), blumeaene K (3; 1.2 mg, 0.005%),
and blumeaene L (4; 0.6 mg, 0.002%) from Fr. 7 and blumeaene
M (5; 0.8 mg, 0.003%) and samboginone (6; 6.4 mg, 0.026%) from Fr. 6.
The known compounds, cryptomeridiol (7.2 mg, 0.030%),15 3,30,5,7-
tetrahydroxy-40-methoxyflavanone (20.2 mg, 0.083%),2 and austroinu-
lin (5.2 mg; 0.021%),16 were also isolated from Frs. 9 and 10. All
of these extraction and isolation experiments were performed in
2000-2001.
To a mixture of angelic acid (573 mg; 5.7 mmol) and Na2WO4 H2O
3
(190 mg; 0.57 mmol) was added 6 mL of 1 mol/L KOH. The resulting
aqueous solution was acidified to pH 5-6 with 1 mol/L H2SO4, and
then 0.6 mL of 10 mol/L H2O2 was added and the mixture stirred for 30
min. The reaction mixture was acidified to pH 2-3 with 1 mol/L
H2SO4, saturated with NH4Cl, and then extracted with DCM five times.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated to obtain epoxyangelic acid as a colorless oil (515.4 mg;
77.8%): 1H NMR (CDCl3, 400 MHz) δ 1.40 (3H, d, J = 5.6 Hz), 1.61
(3H, s), 3.15 (1H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) δ 13.5
(q), 18.6 (q), 59.7 (s), 60.6 (d), 174.8 (s).
Blumeaene E1 (1):. amorphous solid; [R]22 -41 (c 0.09,
D
MeOH);14 UV (MeCN) λmax (log ε) 247 (3.92) nm; CD (MeCN)
λmax (Δε) 330 (-0.9), 289 (-0.2), 253 (-3.1), 203 (þ1.8) nm; IR
(ATR) 3466, 2959, 1728, 1673, 1607, 1447, 1369, 1266, 1161, 1110,
1082, 972, 876, 839, 764, 576 cm-1; 1H NMR (CDCl3, 400 MHz) see
Table 1; 13C NMR (CDCl3, 100 MHz) see Table 2; FABMS m/z 389.2
(16, [M þ Na]þ), 349.2 (21, [M þ H - H2O]þ), 307.2 (17), 233.2
(57), 191.2 (27), 154.1 (100), 136.1 (71); HRFABMS m/z 349.1995
(calcd for C20H29O5, 349.2015).
Blumeaene E2 (2):. amorphous solid; [R]22 -41 (c 0.11,
To a mixture of epoxyangelic acid (43 mg; 0.37 mmol), (R)-(-)-
D
MeOH);14 UV (MeCN) λmax (log ε) 247 (3.90) nm; CD (MeCN)
λmax (Δε) 331 (-1.0), 283 (-0.2), 251 (-2.8), 200 (þ1.1) nm; IR
(ATR) 3389, 2957, 1747, 1660, 1609, 1468, 1432, 1385, 1266, 1160,
1110, 1081, 973, 876, 841, 825, 758, 648, 575 cm-1; 1H NMR (CDCl3,
400 MHz) see Table 1; 13C NMR (CDCl3, 100 MHz) see Table 2;
FABMS m/z 389.2 (3, [M þ Na]þ), 349.2 (8, [M þ H - H2O]þ),
307.2 (20), 233.2 (17), 191.2 (7), 154.1 (100), 136.1 (66); HRFABMS
m/z 349.2008 (calcd for C20H29O5, 349.2015).
PGME HCl (85 mg; 0.41 mmol), 1-hydroxybenzotriazole (65 mg; 0.42
3
mmol), and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro-
phosphate (218 mg; 0.42 mmol) in 1 mL of DMF was added 135 μL of N-
methylmorpholine (1.22 mmol), and the mixture stirred overnight. The
resulting mixture was diluted with EtOAc and washed with 1 mol/L HCl,
saturated NaHCO3, and brine sequentially. The organic layer was con-
centratedandappliedtopreparative HPLCpurification to obtain one set of
pure diastereomers of epoxyangelic acid (R)-PGME derivative (the first
eluted peak: 27.5 mg, 28.2%; the second eluted peak: 27.1 mg, 27.8%).
(R,R)-Epoxyangelic acid (R)-PGME derivative (the first
eluted peak):. amorphous powder; 1H NMR (CDCl3, 400 MHz) δ
1.10 (3H, d, J = 5.5 Hz), 1.56 (3H, s), 3.05 (1H, q, J = 5.5 Hz), 3.74 (3H,
s), 5.57 (1H, d, J = 7.7 Hz), 7.22 (1H, br s, J = 7.7 Hz), 7.31-7.36 (5H,
m); 13C NMR (CDCl3, 100 MHz) δ 13.4 (q), 18.8 (q), 52.8 (q), 56.0
(d), 61.4 (d), 61.8 (s), 127.4 (d ꢀ 2), 128.7 (d), 129.0 (d ꢀ 2), 136.3 (s),
169.2 (s), 170.8 (s); ESITOFMS m/z 286.1 (100, [M þ Na]þ);
HRESITOFMS m/z 286.1051 (calcd for C14H17NO4Na, 286.1055).
Blumeaene K (3):. amorphous solid; [R]23 -20 (c 0.10,
D
MeOH); UV (MeCN) λmax (log ε) 246 (3.91) nm; CD (MeCN) λmax
(Δε) 330 (-0.9), 285 (-0.2), 252 (-2.8), 202 (þ1.2) nm; IR (ATR)
3396, 2958, 1727, 1660, 1611, 1443, 1371, 1267, 1161, 1109, 1080, 973,
877, 842, 759, 648 cm-1; 1H NMR (CDCl3, 400 MHz) see Table 1; 13
C
NMR (CDCl3, 100 MHz) see Table 2; FABMS m/z 425.2 (4, [M þ
Na]þ), 385.2 (6, [M - H2O þ H]þ), 307.2 (17), 233.2 (21), 154.1
(100), 136.1 (70); HRFABMS m/z 385.1792 (calcd for C20H32O7,
385.1804).
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Table 2. 13C NMR Spectroscopic Data (100 MHz, CDCl3) for 1-6
1
2
3
4
5
6
position
δH, mult.
δH, mult.
δH, mult.
δH, mult.
δH, mult.
δH, mult.
1
90.4, C
36.5, CH2
36.8, CH2
160.6, C
137.1, C
201.6, C
51.6, CH
27.9, CH2
79.4, CH
78.6, C
90.4, C
36.6, CH2
36.8, CH2
160.8, C
137.0, C
201.4, C
51.6, CH
27.7, CH2
79.8, CH
78.5, C
90.1, C
36.2, CH2
36.9, CH2
162.8, C
137.2, C
201.3, C
51.3, CH
27.7, CH2
81.2, CH
78.5, C
90.1, C
89.7, C
38.1, CH2
37.4, CH2
160.5, C
138.9, C
204.1, C
56.3, CH
26.0, CH2
82.2, CH
78.8, C
52.2, C
2
36.6, CH2
36.9, CH2
160.7, C
35.3, CH2
33.1, CH2
136.6, C
3
4
5
137.0, C
138.4, C
6
201.8, C
204.2, C
7
51.6, CH
27.7, CH2
78.7, CH
78.5, C
53.8, CH
28.0, CH2
70.8, CH
217.5, C
8
9
10
11
12
13
14
15
10
20
30
40
50
26.7, CH
17.9, CH3
20.7, CH3
15.8, CH3
17.8, CH3
170.0, C
60.2, C
26.8, CH
18.0, CH3
20.8, CH3
16.0, CH3
17.8, CH3
170.0, C
59.8, C
26.8, CH
18.1, CH3
20.8, CH3
16.9, CH3
17.8, CH3
174.2, C
77.2, C
26.8, CH
17.9, CH3
20.8, CH3
16.1, CH3
17.8, CH3
171.3, C
29.1, CH
18.6, CH3
20.8, CH3
19.7, CH3
17.9, CH3
170.0, C
59.9, C
25.6, CH
17.9, CH3
20.7, CH3
17.8, CH3
20.6, CH3
21.3, CH3
60.0, CH
13.8, CH3
19.3, CH3
60.2, CH
13.7, CH3
19.3, CH3
62.7, CH
23.1, CH3
18.0, CH3
60.1, CH
13.8, CH3
19.2, CH3
(S,S)-Epoxyangelic acid (R)-PGME derivative (the second
eluted peak):. amorphous, oily solid; 1H NMR (CDCl3, 400 MHz) δ
1.47 (3H, d, J = 5.6 Hz), 1.53 (3H, s), 3.12 (1H, q, J = 5.6 Hz), 3.72 (3H,
s), 5.54 (1H, d, J = 7.7 Hz), 7.11 (1H, br s, J = 7.7 Hz), 7.32-7.42 (5H,
m); 13C NMR (CDCl3, 100 MHz) δ 13.4 (q), 18.7 (q), 52.7 (q), 56.1
(d), 61.8 (d), 61.9 (s), 127.4 (d ꢀ 2), 128.8 (d), 129.2 (d ꢀ 2), 135.6 (s),
169.2 (s), 170.9 (s); ESITOFMS m/z 286.1 (100, [M þ Na]þ);
HRESITOFMS m/z 286.1056 (calcd for C14H17NO4Na, 286.1055).
Hydrolysis of Blumeaene E1 (1). To a solution of blumeaene
E1 (ca. 1.0 mg; 2.7 μmol) in MeOH was added 0.5 mol/L LiOH (10 μL;
5 μmol), and the mixture stirred for 2 h at rt. The reaction mixture was
diluted with H2O and extracted with DCM three times and with n-
hexane one time, and the combined organic extract was concentrated to
obtain the blumeaene core. The remaining aqueous phase was acidified
with 1 mol/L HCl and extracted with DCM three times and with EtOAc
one time, and then the combined organic extract was concentrated to
obtain epoxyangelic acid.
(R)- and (S)-MTPA Esterification of Blumeaene Core. To a
mixture of the blumeaene core (<ca. 3 μmol), (R)-MTPA (ca. 2 mg; 8.5
μmol), EDC (ca. 2 mg; 10 μmol), and DMAP (ca. 0.1 mg; 0.8 μmol) was
added CH2Cl2 (0.5 mL), and the mixture stirred overnight at rt. The
reaction mixture was concentrated and applied to preparative TLC to
obtain the (R)-MTPA ester of the blumeaene core (ca. 1.5 mg; 3 μmol).
The (S)-MTPA ester of the blumeaene core was prepared in a similar
manner.
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of all new com-
b
pounds (1-6). This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ81-87-894-5111. Fax: þ81-87-894-0181. E-mail: shirota@
kph.bunri-u.ac.jp.
Present Addresses
‡Laboratory of Pharmacognosy and Natural Products Chemistry,
Kagawa School of Pharmaceutical Sciences, Tokushima Bunri
University, 1314-1 Shido, Sanuki-City, Kagawa 769-2193,
Japan.
§Food-Drug Regulation Office, Bureau of Food and Drugs,
Department of Health, Civic Drive, Filinvest Corporate City,
Alabang, Muntinlupa City, Manila 1770, Philippines.
¥Center of Environmental Science for Human Life, Ochanomizu
University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
’ ACKNOWLEDGMENT
Preparation of the (R)-PGME Amide of Epoxyangelic Acid
from Blumeaene E1. To a mixture of epoxyangelic acid (<ca. 3
This work was supported in part by the Japan International
Cooperation Agency (JICA) for a training program of the
Philippine pharmacopoeia project.
μmol) from the hydrolysis of 1, (R)-(-)-PGME HCl (13 mg; 64
3
μmol), HOBt (8 mg; 50 μmol), and PyBOP (20 mg; 50 μmol) in 0.5 mL
of DMF was added 8 μL of NMM (75 μmol), and the mixture
stirred overnight. The resulting mixture was diluted with EtOAc
and sequentially washed with 1 mol/L HCl, saturated NaHCO3, and
brine. The organic layer was concentrated and applied to an analytical
HPLC to identify the diastereomer of epoxyangelic acid (R)-PGME
amides.
’ DEDICATION
Dedicated to Dr. Koji Nakanishi of Columbia University for his
pioneering work on bioactive natural products.
475
dx.doi.org/10.1021/np100646n |J. Nat. Prod. 2011, 74, 470–476

Journal of Natural Products
NOTE
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