Ring B aromatic norpimarane glucoside from a Xylaria sp.

Article abstract of DOI:10.1021/np100873t

A novel 20-norpimarane glucoside, xylopimarane (1), together with the known sphaeropsidin C (2) and clonostachydiol (3), was isolated from the fungus Xylaria sp. BCC 4297. Compound 1 exhibited cytotoxicity to cancer cell lines KB, MCF-7, and NCI-H187 with respective IC₅₀ values of 1.0, 13, and 65 μM.

Full text of DOI:10.1021/np100873t

NOTE
pubs.acs.org/jnp
Ring B Aromatic Norpimarane Glucoside from a Xylaria sp.
Masahiko Isaka,* Arunrat Yangchum, Patchanee Auncharoen, Kitlada Srichomthong, and
Prasert Srikitikulchai
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand
S Supporting Information
b
ABSTRACT: A novel 20-norpimarane glucoside, xylopimarane (1), together with the known
sphaeropsidin C (2) and clonostachydiol (3), was isolated from the fungus Xylaria sp. BCC 4297.
Compound 1 exhibited cytotoxicity to cancer cell lines KB, MCF-7, and NCI-H187 with
respective IC50 values of 1.0, 13, and 65 μM.
ungi belonging to the genus Xylaria have been the source of
bioactive compounds such as xyloketals and xyloallenolide
3426 and 1636 cm-1, which suggested the presence of OH
1
F
groups and an aromatic chromophore. The H and 13C NMR,
from Xylaria sp. (No. 2508),1,2 multiplolides from X. multiplex
BCC 1111,3 xylactam from X. euglossa,4 integric acid from a
Xylaria sp.,5 xylarenals from X. persicaria,6 and eremophilanolides
from Xylaria sp. BCC 21097.7 As part of our research program on
the utilization of fungal sources in Thailand, we investigated the
Xylaria sp. strain BCC 4297, as an extract of this fungus showed
moderate cytotoxic activity against several cancer cell lines. Scale-
up fermentation and chemical studies resulted in the isolation of
a novel 20-norpimarane glucoside, xylopimarane (1), along with
the known sphaeropsidin C (2)8 and clonostachydiol (3).9,10
DEPT135, and HMQC spectroscopic data demonstrated that 1
was composed of a sugar unit (C6) and aglycone (C19). The
pyranose ring (C-10-C-60) was addressed by COSY data. The
coupling constants observed for H-30 (t, J = 9.3 Hz) and H-400 (t, J =
9.4 Hz) indicated that methine protons H-20, H-30, H-40, and H-50 all
occupied axial positions. The small coupling constant (J = 3.8 Hz) of
H-10/H-20 revealed an equatorial orientation of the anomeric proton
H-10. Therefore, the sugar unit was assigned as R-glucopyranose. The
aglycone (C19) contained six aromatic quaternary carbons, a vinyl
group (δC 147.6/δH 6.25; δC 110.2/δH 5.02 and 4.96), a hydro-
xylated methine (δC 68.1, δH 4.49, d, J = 4.7 Hz; OH, δH 4.27, d, J =
4.7 Hz), two quaternary carbons, five methylenes, and three methyl
groups. The planar structure of the aglycone was deduced from
COSY and HMBC (Table 1) correlations. Two methyl groups at δH
1.42 (δC 27.9, CH3-18) and 1.38 (δC 27.2, CH3-19) showed HMBC
correlations to each other and also exhibited correlations to a
quaternary carbon at δC 34.1 (C-4), an aromatic quaternary carbon
at δC 131.7 (C-5), and a methylene carbon at δC 42.0 (C-3), which
indicated the connections of these methyl groups to the same
quaternary carbon (C-4). HMBC data also demonstrated that the
quaternary carbon at δC 38.6 (C-13) was connected to a vinyl group
(C-15/C-16), a methyl group at δC 19.8 (δH 0.79, CH3-17), a
hydroxylated methine (CH-14), and a methylene carbon at δC 25.8
(CH2-12). The presence of a fully substituted benzene (ring B) was
indicated by HMBC correlations to the aromatic carbons: from H2-1,
H2-2, and HR-11 (δH 2.60) to C-10 (δC 132.6), from H2-1, H2-3,
H3-18, and H3-19 to C-5 (δC 131.7), from H2-1, H2-11, and Hβ-12
(δH 1.50) to C-9 (δC 123.2), from H2-11, H-14, and 14-OH to C-8
(δC 129.6), and from H-14 to C-7 (δC 143.2). The quaternary
carbon at δC 147.4, which lacked a HMBC correlation, was assigned
The molecular formula of 1 was determined by HRESIMS as
C25H36O8. The IR spectrum exhibited absorption bands at νmax
Received: December 1, 2010
Published: January 12, 2011
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np100873t J. Nat. Prod. 2011, 74, 300–302
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
Table 1. NMR Spectroscopic Data for 1 in Acetone-d6
transformation occurred during fermentation of BCC 4297. Thus,
compound 4, produced by oxidation of sphaeropsidin C (2), is the
possible biosynthetic precursor of 1. Glucoside formation should be
the last biosynthetic step.
(500 MHz for 1H, and 125 MHz for 13C)
position
δC, mult.
δH, mult. (J in Hz)
HMBC
2, 3, 5, 10
1
27.8, CH2 2.48-2.46,m
19.4, CH2 1.74-1.69,m
42.0, CH2 1.60-1.57,m
34.1, qC
2
1, 4, 10
3
1, 2, 4, 5, 18, 19
4
5
131.7, qC
6
147.4, qC
7
143.2, qC
8
129.6, qC
9
123.2, qC
10
11
132.6, qC
23.2, CH2 R 2.60, dd (17.3, 6.8)
β 2.43, m
8, 9, 10, 12, 13
8, 9, 10, 12
Xylopimarane (1) exhibited cytotoxic activity against the human
cancer cell lines KB (oral carcinoma), MCF-7 (breast cancer), and
NCI-H187 (small-cell lung cancer), with IC50 values of 1.0, 13, and
65 μM, respectively. It also showed cytotoxicity (IC50 41 μM) to
nonmalignant Vero cells (African green monkey kidney fibroblasts).
12
25.8, CH2 R 2.21, m
β 1.50, dd (12.9, 7.0)
38.6, qC
11, 13, 14, 17
9, 11, 13, 14, 15, 17
13
14
15
16
68.1, CH
4.49, d (4.7)
7, 8, 9, 12, 13, 15, 17
12, 13, 14, 17
13, 15
147.6, CH
6.25, dd (17.7, 10.9)
’ EXPERIMENTAL SECTION
110.2, CH2 5.02, dd (17.7, 1.4)
4.96, dd (10.9, 1.4)
19.8, CH3 0.79, s
27.9, CH3 1.42, s
27.2, CH3 1.38, s
9.47, br s
General Experimental Procedures. Melting points were mea-
sured with an Electrothermal IA9100 digital melting point apparatus.
Optical rotations were measured with a JASCO P-1030 digital polarimeter.
UV spectra were recorded on a GBC Cintra 404 spectrophotometer. FTIR
spectra were taken on a Bruker VECTOR 22 spectrometer. NMR spectra
were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF
mass spectra were measured with a Bruker micrOTOF mass spectrometer.
Fungal Material. The fungus used in this study was isolated from
an unidentified dead wood in Hala Bala Wildlife Sanctuary, Narathiwat
Province, Thailand, and it was deposited in the BIOTEC Culture Collection
on February 24, 2000, as BCC 4297. On the basis of the characteristics of
fruiting bodies and morphology, this fungus was assigned to the genus
Xylaria (family Xylariaceae) by one of the authors (P.S.).
Fermentation and Isolation. The fungus BCC 4297 was main-
tained on potato dextrose agar at 25 °C. The agar was cut into small plugs
and inoculated into 2 ꢀ 250 mL Erlenmeyer flasks containing 25 mL of
potato dextrose broth (PDB; potato starch 4.0 g/L, dextrose 20.0 g/L). After
incubation at 25 °C for 8 days on a rotary shaker (200 rpm), each primary
culture was transferred into a 1 L Erlenmeyer flask containing 250 mL of the
same liquid medium (PDB) and incubated at 25 °C for 8 days on a rotary
shaker (200 rpm). These secondary cultures were pooled, each 25 mL
portion was transferred into 20 ꢀ 1 L Erlenmeyer flasks containing 250 mL
of malt extract broth (MEB; malt extract 6.0 g/L, yeast extract 1.2 g/L,
maltose 1.8 g/L, dextrose 6.0 g/L), and final fermentation was carried out at
25 °C for 83 days under static conditions. The culture was filtered to separate
the residue (mycelia) and the filtrate (broth). The broth was extracted with
EtOAc (3 ꢀ 5 L), and the combined organic layer was concentrated to
obtain a brown gum (834 mg; extract A). The mycelium was macerated in
MeOH (1 L, rt, 2 days) and filtered. H2O (150 mL) and hexanes (1 L) were
added to the filtrate, and the layers were separated. The aqueous MeOH
phase was partially evaporated, and the residue was extracted with EtOAc
(2 ꢀ 600 mL). The combined EtOAc solution was washed with H2O
(100 mL) and concentrated under reduced pressure to obtain a brown gum
(183 mg, extract B). Extract A was subjected to Sephadex LH-20 column
chromatography (3.0 ꢀ 50 cm, MeOH) to obtain six pooled fractions,
A1-A6. Fraction A3 (545 mg) was chromatographed on silica gel (3.0 ꢀ
15 cm, MeOH/CH2Cl2, step gradient elution from 2:98 to 10:90) to obtain
11 fractions, A3-1-A3-11. Fraction A3-4 (58 mg) was purified by pre-
parative HPLC using a reversed-phase column (SunFire Prep C18 OBD,
19 ꢀ 150 mm, 5 μm; mobile phase MeCN/H2O, gradient from 30:70 to
13, 15
17
12, 13, 14, 15
3, 4, 5, 19
18a
19a
6-OH
14-OH
10
20
30
40
50
3, 4, 5, 18
4.20, d (4.7)
8, 13, 14
7, 30, 50
30
20, 40
30, 50, 60
40
103.6, CH
72.2, CH
73.3, CH
70.2, CH
75.6, CH
5.01, d (3.8)
3.75, m
3.91, t (9.3)
3.45, t (9.4)
4.13, m
60
61.4, CH2 3.98, br d (11.6); 3.74, m 50
a The assignment of CH3-18 and CH3-19 can be interchanged.
to C-6 to constitute a benzene ring. The chemical shifts of C-6 and
C-7 suggested that these carbons were attached with an oxygen atom.
Therefore, the planar structure of the aglycone was assigned as 20-
norpimara-5,7,9-triene-6,7,14-triol, wherein either 6-OH or 7-OH
should form a glucoside. HMBC correlation from the anomeric
proton (H-10) to C-7 indicated the location of the glucose unit. The
co-occurrence of 1 with the known sphaeropsidine C (2) and the
plausible biosynthetic relation as described below strongly suggested
that these compounds should have the same sense of C-13 absolute
configuration. Intense NOESY correlations of H-14 and H3-17 and
the absence of the cross-peak for H-14 and H-15 indicated the β-face
orientation of H-14. Assignment of the protons of two methylene
groups (H2-11 and H2-12) was addressed on the basis of the NOESY
correlations from H3-17 to Hβ-11 (δH 2.43) and Hβ-12 (δH 1.50).
Conformation of ring C was uncertain from the available NMR data.
Finally, the D-glucose unit was confirmed by acid hydrolysis of 1.
To our knowledge, this is the first report of a natural ring B
aromatic 20-norpimarane skeleton. In 1972, Ellestad and co-workers
reported the isolation of LL-S491β (4) and LL-S491γ (7β-hydroxy
analogue of 4) from the fungus Aspergillus chevalieri.11,12 The paper
also describes that ethanolic hydrochloric acid treatment of 4
gave decarboxylation/aromatization products 5a and 5b.11 Since
5a is identical to the aglycone of 1, we assume that the same
301
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Journal of Natural Products
NOTE
80:20, flow rate 10 mL/min) to furnish 2 (7.7 mg). Fraction A3-6 (91 mg)
was also further fractionated by preparative HPLC (MeCN/H2O, gradient
from 20:80 to 40:60) to afford 3 (20.0 mg). Fraction A4 (97 mg) was sub-
jected to silica gel column chromatography (2.0 ꢀ 15 cm, MeOH/CH2Cl2,
step gradient elution from 2:98 to 11:89) to give 1 (13.2 mg). Extract B was
chromatographed on Sephadex LH-20 (2.0 ꢀ 60 cm, MeOH) and pre-
parative HPLC (MeCN/H2O) to furnish 3 (1.1 mg) and 1 (6.0 mg).
(9) Grabley, S.; Hammann, P.; Thiericke, R.; Wink, J. J. Antibiot.
1993, 46, 343–345.
(10) Rao, A. V. R.; Murthy, V. S.; Sharma, G. V. M. Tetrahedron Lett.
1995, 36, 143–146.
(11) Ellestad, G. A.; Kunstmann, M. P.; Mirando, P.; Morton, G. O.
J. Am. Chem. Soc. 1972, 94, 6206–6208.
(12) The same compound (4) was later isolated from the fungus
Sphaeropsis sapinea f. sp. cupressi and named sphaeropsidin A: Evidente,
A.; Sparapano, L.; Motta, A.; Giordano, F.; Fierro, O.; Frisullo, F.
Phytochemistry 1996, 42, 1541–1546.
(13) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem.
2000, 267, 5421–5426.
Xylopimarane (1): colorless solid; mp 174-175 °C; [R]26 þ75
D
(c 0.11, MeOH); UV (MeOH) λmax (log ε) 207 (4.66), 228 sh (3.95),
288 (3.44) nm; IR (KBr) νmax 3420, 2928, 1636, 1429, 1029, 1008 cm-1
;
1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6)
data, see Table 1; HRMS (ESI-TOF) m/z 487.2301 [M þ Na]þ (calcd for
C25H36O8Na, 487.2302).
(14) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Anti-
microb. Agents Chemother. 2003, 47, 3682–3687.
Hydrolysis of 1. Compound 1 (1.9 mg) was hydrolyzed in 5%
aqueous hydrochloric acid (0.5 mL) at 90 °C for 2 h. The mixture was
washed with Et2O (2 ꢀ 1 mL), and the aqueous layer was concentrated
under reduced pressure to obtain D-glucose (0.7 mg, 1H NMR in D2O/
CD3OD); [R]27D þ85 (c 0.035, H2O)).
Biological Assays. Anticancer activities against KB cells (oral
human epidermoid carcinoma), MCF-7 cells (human breast cancer),
and NCI-H187 cells (human small-cell lung cancer) were evaluated
using the resazurin microplate assay.13 The IC50 values of the standard
compound doxorubicin hydrochloride were 0.27 μM for KB cells,
4.9 μM for MCF-7 cells, and 0.13 μM for NCI-H187 cells. Cytotoxicity
to Vero cells (African green monkey kidney fibroblasts) were performed
using the green fluorescent protein microplate assay (GFPMA).14
Ellipticine was used as the standard of cytotoxicity (IC50 7.3 μM).
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of 1. This material
b
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ66-25646700, ext 3554. Fax: þ66-25646707. E-mail: isaka@
biotec.or.th.
’ ACKNOWLEDGMENT
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology
(BIOTEC), is gratefully acknowledged.
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