γ-lactones and ent-eudesmane sesquiterpenes from the endophytic fungus Eutypella sp. BCC 13199

Article abstract of DOI:10.1021/np900316x

Two new γ-lactones, eutypellins A (1) and B (2), and two ent-eudesmane sesquiterpenes, ent-4(15)-eudesmen-11-ol-1-one (3) and ent-4(15)-eudesmen-1α,11-diol (4), together with three known pimarane diterpenes, diaporthein B, scopararane A, and libertellenon

Full text of DOI:10.1021/np900316x

1720
J. Nat. Prod. 2009, 72, 1720–1722
γ-Lactones and ent-Eudesmane Sesquiterpenes from the Endophytic Fungus Eutypella sp.
BCC 13199
Masahiko Isaka,*,† Somporn Palasarn,† Sanisa Lapanun,† Rungtiwa Chanthaket,† Nattawut Boonyuen,† and
Saisamorn Lumyong‡
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand, and Department of Biology, Faculty of Science, Chiang Mai UniVersity,
Chiang Mai 50200, Thailand
ReceiVed May 28, 2009
Two new γ-lactones, eutypellins A (1) and B (2), and two ent-eudesmane sesquiterpenes, ent-4(15)-eudesmen-
11-ol-1-one (3) and ent-4(15)-eudesmen-1R,11-diol (4), together with three known pimarane diterpenes, diaporthein
B, scopararane A, and libertellenone C, were isolated from the endophytic fungus Eutypella sp. BCC 13199. The
structures of these compounds were elucidated by interpretation of spectroscopic data. The absolute configuration
of 4 was confirmed by application of the modified Mosher’s method. Eutypellin A (1) and sesquiterpene 3 exhibited
weak cytotoxic activities.
Endophytic fungi have been the source of a wide range of novel
bioactive secondary metabolites.1-3 As part of our research program
on the utilization of fungal sources in Thailand, we have investigated
the endophyte Eutypella sp. BCC 13199, as an extract of this strain
showed cytotoxic activity against human small-cell lung cancer cells
(NCI-H187) with an IC50 value of 7.1 µg/mL. Scale-up fermentation
and chemical studies led to the isolation of the new γ-lactones
eutypellins A (1) and B (2), the ent-eudesmane sesquiterpenes ent-
4(15)-eudesmen-11-ol-1-one (3) and ent-4(15)-eudesmen-1R,11-
diol (4), and three known pimarane diterpenes, diaporthein B,4
scopararane A,5 and libertellenone C.6 Details of the isolation,
structure elucidation, and biological activities of the new compounds
are presented here.
Eutypellin A (1) was isolated as a yellow, amorphous solid. The
molecular formula of 1 was determined to be C11H12O5 by
HRESIMS in conjunction with the 1H and 13C NMR spectroscopic
data. The IR spectrum showed broad and intense carbonyl absorp-
1
tion bands at νmax 1749 and 1678 cm-1. The H and 13C NMR,
DEPT, and HMQC spectroscopic data revealed that this compound
The molecular formula of eutypellin B (2) was determined by
contained an aldehyde (δH 9.70, d, J ) 7.8 Hz; δC 194.9), an ester
HRESIMS as C11H14O5. The signals in its 1H and 13C NMR spectra
closely matched those of 1 except for the difference that the formyl
(δC 170.7), a quaternary sp2 carbon (δC 137.6), five olefinic
methines, two oxymethines (δC 71.0 and 85.9), and an oxymeth-
group (C-1) in 1 was replaced with a hydroxymethyl (δH 4.13, 2H,
ylene (δC 60.8) group. The COSY data indicated the connections
dd, J ) 4.3, 1.5 Hz; δC 61.4). The location of the hydroxymethyl
from C-1 to C-4 and from C-6 to C-10. The E-configuration of
group (H-1) was confirmed by the COSY correlations, indicating
C-2/C-3 and C-8/C-9 olefinic bonds was evident from the respective
1
a vicinal H-1H coupling with H-2 and an allylic coupling with
J-values of 15.3 and 15.5 Hz. The R-alkylidene-γ-lactone moiety
H-3 (δH 6.70, ddt, J ) 15.2, 11.6, 1.5 Hz). H-6 resonated as a
was established by the HMBC correlations from H-4 and H-7 to
the carbonyl carbon at δH 169.3 (C-11), from H-3 and H-6 to C-5,
and from H-4 to C-6. An intense NOESY correlation between H-3
and H-6 indicated the 4E-configuration; therefore, compound 1
possesses the 2E,4E,8E triene geometry. To address the relative
configuration at C-6 and C-7, the γ-lactone conformations for two
possible diastereomers, cis- and trans-H-6/H-7, were examined
using molecular model as well as computational energy minimiza-
broad singlet with narrow peak width, which demonstrated the same
relative configuration (trans-H-6/H-7) as 1. Detailed interpretation
of the NMR spectroscopic data confirmed that eutypellin B (2) is
the C-1 alcohol derivative of 1.
To determine the absolute configuration, we examined the
application of the modified Mosher’s method.7,8 The reaction of 2
with p-bromobenzoyl chloride in pyridine gave the bis-p-bromoben-
zoate 5. However, the reaction of 5 with (S)- or (R)-MTPACl in
tion (MM2 and MOPAC). The cis-H-6/H-7 isomer adopted a stable
pyridine or DMAP/CH2Cl2, in all cases, did not provide the expected
conformation with the dihedral angle of H-C(6)-C(7)-H close
to 0°, whereas the trans-H-6/H-7 isomer had a dihedral angle within
6-O-MTPA ester derivative but gave only polymeric products.
Attempts at crystallization of 5 and other mono- and bis-acylated
the range 90-120°. Therefore, the latter configuration (trans) was
derivatives of 2 for X-ray crystallographic analysis were also
more consistent with the observed small J-value (2.3 Hz) between
unsuccessful; therefore, the absolute configuration of the eutypellins
H-6 and H-7.
remains unassigned.
The molecular formula of sesquiterpene 3 was determined by
* Corresponding author. Tel: +66-25646700, ext 3554. Fax: +66-
HRESIMS as C15H26O2. The IR absorption at νmax 1702 cm-1 and
25646707. E-mail: isaka@biotec.or.th.
† National Center for Genetic Engineering and Biotechnology.
‡ Chiang Mai University.
a
13C NMR resonance at δC 215.0 indicated the presence of an
aliphatic ketone functionality. Detailed analyses of the NMR
10.1021/np900316x CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/09/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 9 1721
plugs and inoculated into 3 × 1 L Erlenmeyer flasks containing 250
mL of potato dextrose broth (PDB; potato starch 4.0 g, dextrose 20.0 g,
per liter). After incubation at 25 °C for 3 days on a rotary shaker (200
rpm), 600 mL of the primary culture was transferred into a 10 L
bioreactor containing 5.4 L of malt extract broth (MEB; malt extract
6.0 g, yeast extract 1.2 g, maltose 1.8 g, dextrose 6.0 g, per liter), and
final fermentation was carried out at 25 °C for 12 days under agitation
at 200 rpm and aeration at 0.5 vvm. The cultures were filtered to
separate broth (filtrate) and mycelia (residue). Culture broth was
extracted with EtOAc (2 × 6 L), and the combined organic phase was
concentrated to obtain a brown gum (broth extract; 3.46 g). The MeOH
extract from mycelia (191 mg) did not contain any unique compounds.
The broth extract was subjected to column chromatography (CC) on
silica gel (5.0 × 15 cm, MeOH/CH2Cl2, step gradient elution from 0:100
to 100:0) to obtain five pooled fractions: fraction 1 (630 mg), 2 (1.40 g),
3 (732 mg), 4 (534 mg), and 5 (170 mg). Fraction 1 was triturated in
MeOH (2 mL) and filtered. The residual solid (40 mg) was subjected
to preparative HPLC using a reversed-phase column (LiChroCART
250-10, 10 × 250 mm, 10 µm; mobile phase MeCN/H2O, 65:35, flow
rate 4 mL/min) to furnish 5 (16 mg, tR 5 min) and 6 (5 mg, tR 9 min).
The filtrate from trituration was chromatographed on a Sephadex LH-
20 column in MeOH to obtain four fractions: fraction 1-1 (300 mg),
1-2 (160 mg), 1-3 (10 mg), and 1-4 (56 mg). Fraction 1-1 was separated
by preparative HPLC (NovaPak HR C18, 40 × 100 mm, 10 µm; mobile
phase MeOH/H2O, 60:40, flow rate 15 mL/min) to collect fractions
for tR 5 min (fraction 1-1-1, 51 mg) and tR 9 min (fraction 1-1-2, 200
mg). Fraction 1-1-1 was further purified by CC on silica gel (2.0 × 15
cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 100:0) to afford
4 (37 mg). Fraction 1-1-2 was also purified by CC on silica gel to
furnish 3 (169 mg). Fractions 1-2 and 1-3 were combined and
fractionated by preparative HPLC to obtain diaporthein B (51 mg) and
scopararane A (14 mg). Fractions 2 (1.40 g) and 3 (732 mg), from the
first silica gel CC, were combined and further fractionated by CC on
silica gel. The major fraction (853 mg) was subjected to preparative
HPLC (NovaPak HR C18, 40 × 100 mm, 10 µm; mobile phase MeOH/
H2O, 60:40, flow rate 15 mL/min) to obtain 1 (36 mg; tR 6 min) and
a fraction (tR 8.5 min, 6 mg) mainly composed of libertellenone C.
Libertellenone C was further purified by short CC on silica gel (MeOH/
CH2Cl2) to a colorless solid (3.1 mg). Fractions 4 and 5 were combined
and triturated in MeOH (2 mL). The filtrate was concentrated to leave
2 (610 mg).
Figure 1. ∆δ values [δS - δR] of the MTPA esters 6a and 6b.
spectroscopic data (1H-1H COSY, HMQC, and HMBC) revealed
the eudesmene skeleton with an exomethylene (C-4/C-15), a ketone
(C-1), and a tertiary alcohol (C-11) functionality. Compound 4,
C15H26O2 (HRESIMS), was a C-1 alcohol analogue of 3. The vicinal
J-values of 11.6 and 4.6 Hz for H-1 (δH 3.41, dd, J ) 11.6, 4.6
Hz) with H2-2 indicated an axial orientation of H-1. LiBH4 reduction
of 3 gave an exclusive product whose MS and 1H NMR data were
identical to those of 4.9-12 The observed levorotation for 3 ([R]27
D
-87, c 0.05, CHCl3) and 4 ([R]28 -48, c 0.725, CHCl3) was
D
opposite of those of the known eudesmane sesquiterpenes. Thus,
compound 4 is the enantiomer of 4(15)-eudesmen-1ꢀ,11-diol, which
was previously isolated from higher plants Pterocarpus marsupium
([R]31D +56.4, c 1.5, CHCl3),9 Cryptomeria japonica ([R]30D +56,
c 1.5, CHCl3),10 and Cymbopogon proximus.11 Compound 3 is the
enantiomer of the semisynthetic compound previously obtained by
Jones’ oxidation of 4(15)-eudesmen-1ꢀ,11-diol9 or as one of the
acid degradation products ([R]D +110.0, c 0.05, CHCl3) of hinesol.13
The absolute configuration of 4, isolated from BCC 13199, was
further confirmed by application of the modified Mosher’s method.7,8
The ∆δ values [δS - δR] of the MTPA esters 6a and 6b indicated
the 1S-configuration (Figure 1).
Eutypellin A (1): brown, amorphous solid; [R]25 +31 (c 0.60,
Compounds 1-4 were tested for their cytotoxic activities against
three cancer cell lines, NCI-H187, MCF-7 (human breast cancer),
and KB (human oral carcinoma), and noncancerous Vero cells
(African green monkey kidney fibroblasts) (Table 2). Eutypellin A
(1) exhibited weak cytotoxicity, whereas its alcohol analogue, 2,
was almost inactive. ent-Eudesmanone 3 also showed weak
cytotoxicity, but the corresponding alcohol derivative 4 was inactive
to all cell lines. Compounds 1-4 were also subjected to our
antimalarial (Plasmodium falciparum K1), antituberculosis (My-
cobacterium tuberculosis H37Ra), and antifungal (Candida albicans
and Magnaporthe grisea) assays; however, they were all inactive.
D
MeOH); UV (MeOH) λmax (log ε) 272 (4.55), 329 sh (3.79) nm; IR
1
(KBr) νmax 3423, 1749, 1678, 1651, 1193, 1099, 1043, 976 cm-1; H
NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6),
Table 1; HRMS (ESI-TOF) m/z 247.0586 [M + Na]+ (calcd for
C11H12O5Na 247.0582).
Eutypellin B (2): pale brown, amorphous solid; [R]24D +14 (c 0.60,
MeOH); UV (MeOH) λmax (log ε) 269 (4.63) nm; IR (KBr) νmax 3424,
1746, 1649, 1195, 1091, 1046, 977 cm-1; 1H NMR (500 MHz, DMSO-
d6) and 13C NMR (125 MHz, DMSO-d6), Table 1; HRMS (ESI-TOF)
m/z 249.0734 [M + Na]+ (calcd for C11H14O5Na, 249.0739).
ent-4(15)-Eudesmen-11-ol-1-one (3): colorless solid; [R]27 -87
D
(c 0.05, CHCl3); IR (KBr) νmax 3343, 2948, 1702 cm-1; 1H NMR (500
MHz, CDCl3) δ 5.01 (1H, br d, J ) 1.3 Hz, Ha-15), 4.79 (1H, br d, J
) 1.3 Hz, Hb-15), 2.70 (1H, m, Ha-2), 2.61 (1H, m, Ha-3), 2.44 (1H,
m, Hb-3), 2.38 (1H, m, Hb-2), 2.14 (1H, br d, J ) 11.1 Hz, H-5), 1.83
(2H, m, Ha-6 and Ha-9), 1.77 (1H, m, Ha-8), 1.59 (1H, dt, J ) 4.0,
13.7 Hz, Hb-9), 1.37 (1H, m, Hb-6), 1.33 (1H, m, H-7), 1.25 (1H, m,
Experimental Section
General Experimental Procedures. Melting points were measured
with an Electrothermal IA9100 digital melting point apparatus. Optical
rotations were measured with a JASCO P-1030 digital polarimeter. UV
spectra were recorded on an Analytikjena SPEKOL 1200 spectropho-
tometer. FTIR spectra were taken on a Perkin-Elmer 2000 spectrometer.
NMR spectra were recorded on Bruker DRX400 and AV500D
spectrometers using the signals of the residual solvent protons and the
solvent carbons as internal references (δH 2.50/δC 40.0 for DMSO-d6,
and δH 7.26/δC 77.0 for CDCl3). ESITOF mass spectra were measured
with Micromass LCT and Bruker micrOTOF mass spectrometers.
Fungal Material. The fungus used in this study was isolated from
Earth Ginger Etlingera littoralis in Doi Suthep-Pui National Park,
Chiang Mai Province, Thailand, and it was deposited in the BIOTEC
Culture Collection (BCC) on February 12, 2003, as BCC 13199. This
fungus was identified as a Eutypella sp. (order Xylariales, family
Diatrypaseae) on the basis of the sequence data of the 18S rDNA and
ITS genes by one of the authors (N.B.).
Hb-8), 1.23 (3H, s, H-12), 1.22 (3H, s, H-13), 0.99 (3H, s, H-14); 13
C
NMR (125 MHz, CDCl3) δ 215.0 (C, C-1), 146.6 (C, C-4), 109.1 (CH2,
C-15), 72.6 (C, C-11), 48.5 (CH, C-7), 48.4 (C, C-10), 47.9 (CH, C-5),
38.1 (CH2, C-2), 34.6 (CH2, C-3), 32.1 (CH2, C-9), 27.6 (CH3, C-12),
27.0 (CH3, C-13), 24.6 (CH2, C-6), 22.0 (CH2, C-8), 16.6 (CH3, C-14);
HRMS (ESI-TOF) m/z 259.1671 [M + Na]+ (calcd for C15H24O2Na,
259.1674).
ent-4(15)-Eudesmen-1r,11-diol (4): colorless solid; [R]28D -48 (c
1
0.725, CHCl3); IR (KBr) νmax 3357, 2936, 1016, 888 cm-1; H NMR
(500 MHz, CDCl3) δ 4.76 (1H, d, J ) 1.3 Hz, Ha-15), 4.51 (1H, d, J
) 1.3 Hz, Hb-15), 3.41 (1H, dd, J ) 11.6, 4.6 Hz, H-1), 2.30 (1H,
ddd, J ) 13.6, 4.9, 2.2 Hz, Ha-3), 2.10 (1H, dt, J ) 5.2, 13.6 Hz,
Hb-3), 1.96 (1H, ddd, J ) 12.3, 3.4, 2.8 Hz, Ha-9), 1.81 (1H, m, Ha-
2), 1.55 (1H, dq, J ) 5.0, 12.2 Hz, Hb-2), 1.70 (1H, m, H-5), 1.68
(2H, m, Ha-6 and Ha-8), 1.34 (1H, tt, J ) 12.0, 3.2 Hz, H-7), 1.23
(1H, m, Hb-8), 1.21 (1H, m, Hb-6), 1.20 (6H, s, H-12 and H-13), 1.16
Fermentation and Isolation. Eutypella sp. BCC 13199 was
maintained on potato dextrose agar at 25 °C. The agar was cut into

1722 Journal of Natural Products, 2009, Vol. 72, No. 9
Notes
Table 1. NMR Spectroscopic Data (DMSO-d6) for Eutypellins A (1) and B (2)
eutypellin A (1)
eutypellin B (2)
position
δC, mult.
δH, mult. (J in Hz)
HMBC
δC, mult
δH, mult. (J in Hz)
HMBC
2, 3
1, 4
1, 2, 4, 5
2, 5, 6, 11
1
2
3
4
5
6
194.9, CH
138.9, CH
145.2, CH
136.0, CH
137.6, qC
71.0, CH
9.70, d (7.8)
2
61.4, CH2
147.8, CH
124.0, CH
140.2, CH
127.4, qC
70.8, CH
4.13, dd (4.3, 1.5)
6.66, dd (15.3, 7.8)
7.76, dd (15.3, 11.7)
7.37, dd (11.7, 1.8)
1, 4
1, 5
2, 6, 11
6.49, dt (15.2, 4.3)
6.70, ddt (15.2, 11.6, 1.5)
7.16, dd (11.6, 1.1)
4.86, m
5
4.62, br s
4, 5, 8, 11
6-OH
6.29, br d (6.4)
not obsd
7
8
9
10
11
85.9, CH
125.1, CH
135.8, CH
60.8, CH2
169.3, qC
4.81, ddd (6.9, 2.3, 0.7)
5.71, ddt (15.5, 6.9, 1.8)
5.92, ddt (15.5, 0.7, 4.2)
3.98, m
6, 11
6, 7, 9, 10
85.8, CH
125.8, CH
134.6, CH
60.9, CH2
170.6, qC
4.73, m
6, 9, 11
7, 9, 10
10
5.64, ddt (15.5, 6.6, 1.7)
5.83, ddt (15.5, 1.0, 4.4)
3.94, d (4.4)
8, 9
8, 9
(1H, m, Ha-2), 1.86 (1H, m, Ha-9), 1.74-1.60 (3H, m, H-5, Ha-6 and
Ha-8), 1.60 (1H, m, Hb-2), 1.36 (1H, m, H-7), 1.30-1.22 (2H, m, Hb-6
and Hb-8), 1.22 (1H, m, Hb-9), 1.21 (6H, s, H-12 and H-13), 0.72
(3H, s, H-14); HRMS (ESITOF) m/z 477.2226 [M + Na]+ (calcd for
C25H33O4F3Na, 477.2223).
Table 2. Cytotoxic Activities of Compounds 1-4 (IC50, µM)
compound
NCI-H187
MCF-7
KB
Vero
1
2
3
4
12
206
11
>210
0.25
3.6
84
>221
20
38
144
32
>210
0.28
2.5
88
>221
32
1
(R)-MTPA Ester 6b. colorless gum; H NMR (400 MHz, CDCl3)
>210
1.5
>210
δ 7.53-7.38 (5H, m, phenyl), 4.90 (1H, dd, J ) 11.8, 4.6 Hz, H-1),
4.81 (1H, br s, Ha-15), 4.68 (1H, br s, 11-OH), 4.57 (1H, br s, Hb-3),
3.55 (3H, br s, OCH3), 2.38 (1H, m, Ha-3), 2.20 (1H, m, Hb-3), 2.03
(1H, m, Ha-2), 1.85 (1H, m, Ha-9), 1.75 (1H, m, Hb-2), 1.72 (1H, m,
H-5), 1.62-1.55 (2H, m, Ha-6 and Ha-8), 1.33 (1H, m, H-7), 1.28-1.18
(2H, m, Hb-6 and Hb-8), 1.20 (6H, s, H-12 and H-13), 1.16 (1H, m,
Hb-9), 0.72 (3H, s, H-14); HRMS (ESITOF) m/z 477.2202 [M + Na]+
(calcd for C25H33O4F3Na, 477.2223).
Biological Assays. The cytotoxic activities against KB cells (oral
human epidermoid carcinoma), MCF-7 cells (human breast cancer),
and NCI-H187 cells (human small-cell lung cancer) were performed
using the resazurin microplate assay.14 Cytotoxicity to Vero cells
(African green monkey kidney fibroblasts) was evaluated using the
green fluorescent protein microplate assay (GFPMA).15
b
doxorubicina
ellipticinea
-
b
-
5.5
a Reference compounds for the cytotoxicity assays. b Not tested.
(1H, m, Hb-9), 0.67 (3H, s, H-14); 13C NMR (125 MHz, CDCl3) δ
148.9 (C, C-4), 106.8 (CH2, C-15), 79.3 (CH, C-1), 72.8 (C, C-11),
49.0 (CH, C-7), 47.5 (CH, C-5), 40.2 (C, C-10), 37.0 (CH2, C-9), 34.2
(CH2, C-3), 31.5 (CH2, C-2), 27.2 (CH3, C-12), 27.1 (CH3, C-13), 24.5
(CH2, C-6), 22.2 (CH2, C-8), 10.2 (CH3, C-14); HRMS (ESI-TOF) m/z
261.1823 [M + Na]+ (calcd for C15H26O2Na, 261.1830).
Preparation of the Bis-p-Bromobenzoate Derivative 5. Compound
2 (56 mg) was treated with p-bromobenzoyl chloride (170 mg) in
pyridine (1.0 mL) at room temperature for 16 h. The mixture was diluted
with EtOAc and washed with H2O and 1 M NaHCO3, and the organic
layer was concentrated in vacuo. The residue was subjected to
preparative HPLC (MeCN/H2O, 75:25) to furnish the bis-p-bromoben-
zoate 5 (60 mg) along with a mono-p-bromobenzoate derivative (60
mg).
Supporting Information Available: NMR spectra of compounds
1-4. This material is available free of charge via the Internet at http://
pubs.acs.org.
Compound 5: colorless solid; 1H NMR (500 MHz, CDCl3) δ 7.91
(2H, d, J ) 8.3 Hz, p-bromobenzoyl), 7.90 (2H, d, J ) 8.3 Hz,
p-bromobenzoyl), 7.61 (2H, d, J ) 8.3 Hz, p-bromobenzoyl), 7.59 (2H,
d, J ) 8.3 Hz, p-bromobenzoyl), 7.31 (1H, d, J ) 11.7 Hz, H-4), 6.81
(1H, dd, J ) 15.1, 11.7 Hz, H-3), 6.42 (1H, dt, J ) 15.1, 5.5 Hz, H-2),
6.05 (1H, dt, J ) 15.5, 5.2 Hz, H-9), 5.85 (1H, dd, J ) 15.5, 5.3 Hz,
H-8), 4.98 (2H, d, J ) 5.5 Hz, H-1), 4.87-4.81 (4H, m, H-6, H-7, and
H-10), 2.48 (1H, d, J ) 6.9 Hz, 6-OH); MS (ESITOF) m/z 612.94,
614.94, and 616.94 [M + Na]+.
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LiBH4 Reduction of 3. To a solution of 3 (4.0 mg) in THF (0.3
mL), cooled on an ice-water bath, was added LiBH4 (7 mg), and the
mixture was stirred for 1.5 h. Water was added and the mixture extracted
with EtOAc. The organic layer was concentrated under reduced pressure
to leave a colorless solid, which was purified by a short silica gel column
1
(3.3 mg). The ESIMS and H NMR spectroscopic data were identical
to those of the isolated compound 4.
Preparation of the MTPA Esters 6a and 6b. Compound 4 (1.0
mg) was treated with (-)-(R)-MTPACl (20 mg) in pyridine (0.2 mL)
at room temperature for 14 h. The mixture was diluted with EtOAc
and washed with H2O and 1 M NaHCO3, and the organic layer was
concentrated in vacuo. The residue was purified by a short silica gel
column (MeOH/CH2Cl2) to obtain the (S)-MTPA ester 5a (1.1 mg).
Similarly, (R)-MTPA ester 5b was prepared from 4 and (+)-(S)-
MTPACl.
(12) Anglea, T. A.; Pinder, A. R. Tetrahedron 1987, 43, 5537–5543.
(13) Itokawa, H.; Nakanishi, H.; Mihashi, S. Chem. Pharm. Bull. 1983,
31, 1991–1997.
(14) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000,
267, 5421–5426.
(15) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob.
Agents Chemother. 2003, 47, 3682–3687.
1
(S)-MTPA Ester 6a: colorless gum; H NMR (400 MHz, CDCl3)
δ 7.52-7.39 (5H, m, phenyl), 4.86 (1H, dd, J ) 11.7, 4.6 Hz, H-1),
4.80 (1H, br s, Ha-15), 4.68 (1H, br s, 11-OH), 4.57 (1H, br s, Hb-3),
3.51 (3H, br s, OCH3), 2.34 (1H, m, Ha-3), 2.19 (1H, m, Hb-3), 1.98
NP900316X