Neuritogenic cerebrosides from an edible Chinese mushroom. Part 2: Structures of two additional termitomycesphins and activity enhancement of an inactive cerebroside by hydroxylation

Article abstract of DOI:10.1016/S0968-0896(01)00125-0

Termitomycesphins E and F, novel cerebrosides that are hydroxylated around the middle of the long-chain base (LCB), have been isolated from the edible Chinese mushroom Termitomyces albuminosus (Berk.) Heim. ('Jizong' in Chinese) together with termitomycesphins A-D, and shown to induce neuronal differentiation in rat PC12 cells. Their stereostructures have been determined based on their chemical derivatization and spectroscopic analysis. The major cerebroside obtained from the same mushroom was not hydroxylated around the middle of the LCB and was inactive against PC12 cells, suggesting the importance of the extra hydroxyl group on LCB. The Di- and tetrahydroxylation of this inactive cerebroside resulted in the enhancement of its neuritogenic activity.

Full text of DOI:10.1016/S0968-0896(01)00125-0

Bioorganic & Medicinal Chemistry 9 (2001) 2171–2177
Neuritogenic Cerebrosides from an Edible Chinese Mushroom.
Part 2y: Structures of Two Additional Termitomycesphins and
Activity Enhancement of an Inactive Cerebroside by
Hydroxylation
Jianhua Qi, Makoto Ojika* and Youji Sakagami
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received 16 February 2001; accepted 14 April 2001
Abstract—Termitomycesphins E and F, novel cerebrosides that are hydroxylated around the middle of the long-chain base (LCB),
have been isolated from the edible Chinese mushroom Termitomyces albuminosus (Berk.) Heim. (‘Jizong’ in Chinese) together with
termitomycesphins A–D, and shown to induce neuronal differentiation in rat PC12 cells. Their stereostructures have been deter-
mined based on their chemical derivatization and spectroscopic analysis. The major cerebroside obtained from the same mushroom
was not hydroxylated around the middle of the LCB and was inactive against PC12 cells, suggesting the importance of the extra
hydroxyl group on LCB. The Di- and tetrahydroxylation of this inactive cerebroside resulted in the enhancement of its neuritogenic
activity. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
termitomycesphins A–D (1–4).1 These cerebrosides all
possessed a branch allylic alcohol system around the
middle of the long-chain base (LCB). A further investi-
gation of this mushroom resulted in the finding of two
additional derivatives, termitomycesphins E (5) and F
(6), accompanied by a known cerebroside 7 (Fig. 1).
Furthermore, as part of the structure–activity relation-
ship studies, the inactive cerebroside 7 was converted to
the di- and tetrahydroxylated derivatives (12ab, 13ab,
and 13cd) (Fig. 3), which turned out to become neur-
itogenic. In this paper, we describe the isolation and
structural determination of two new termitomycesphins
5 and 6, the conversion of the inactive cerebroside 7 into
the active derivatives, and the evaluation of the neur-
itogenic activity of these natural and artificial cerebro-
sides.
The nerve growth factor (NGF) is currently the best
characterized neurotrophic factor essential for the sur-
vival and functioning of nerve cells.2,3 Findings suggest
that NGF can be effective for the treatment of dementia
and cerebral paralysis.4À7 Therefore, NGF was con-
sidered as a good candidate for the treatment of neuro-
degenerative diseases. However, it is very difficult to use
NGF as a drug, because it is too large in size to cross
the blood–brain barrier. Low-molecular-weight com-
pounds exhibiting NGF-like activity could lead to pro-
mising therapeutic drugs to treat neurodegenerative
diseases. The PC12 cell line derived from rat pheochro-
mocytoma cells is well-known to express neuronal
properties (elongation of neuronal processes) against
NGF and is a useful model system for such studies.8
A previous search for such neuritogenic compounds
from the Chinese mushroom Termitomyces albuminosus
(Berk.) Heim. (‘Jizong’ in Chinese) was performed in
our laboratory using the PC12 cell line system. This
resulted in the isolation of four unusual cerebrosides,
Results and Discussion
The isolation of termitomycesphins A–D (1–4) was
described in detail in a previous paper.1 A further study
of the other fractions from the last HPLC separation
resulted in the isolation of termitomycesphins E (5)
(0.00036%, base on dry mushroom) and
(0.00057%). The examination of a less polar fraction
than that of the termitomycesphins led to the isolation
F (6)
*Corresponding author. Tel.: +81-52-789-4117; fax: +81-52-789-
4118; e-mail: ojika@agr.nagoya-u.ac.jp
yFor Part 1, See ref 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00125-0

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J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
of a known cerebroside 79,10 (0.042%) as a major cere-
broside component.
glucose. Since the carbon signals assigned to C4, C5,
and C7–C10are split into two similar chemical shifts in
the ratio of ca. 1:1, we presumed that 6 is an inseparable
mixture of C8 or/and C9 epimers. Two carbon-chain
lengths of the long-chain base (LCB) and fatty acid
moieties of 6 were determined by the FAB-MS fragment
analysis. It was reported that cerebrosides gave the
characteristic fragment ions of (LCB+H)+ and
(LCB+HÀH2O)+ in the positive mode FAB-MS
measurement.11 The FAB-MS spectrum of 6 displays
the marked fragment ions at m/z 312 and 294 that could
be due to (LCB+H)+ and (LCB+HÀH2O)+, respec-
tively. An additional fragment peak at m/z 276
(LCB+HÀ2H2O)+ also arises from a further dehydra-
tion of the LCB moiety, revealing that 6 possesses the
same carbon-chain length of LCB as that of 2 and,
consequently, the same fatty acid moiety as 2 taking the
molecular formula into consideration. The absolute ste-
reochemistry of 6 except for the C8 and C9 positions
was determined to be the same as that of 2, because
the hydrogenation product 8 (Fig. 3) from 6 showed
an identical optical rotation [a]2D5+9.3 (c 0.14,
MeOH)], FAB-MS, and 1H NMR data to those for
the hydrogenation product from 21 [a]D25 +10( c 0.19,
MeOH)].
Termitomycesphin F (6) has the molecular formula of
C43H83NO10, which was determined by HRESI-MS
measurement [m/z 774.6087 (M+H)+, Á+0.8 mmu]. The
IR and NMR spectra of 6 indicated the presence of a
fatty acid (large signal at dH 1.24), an amide (1648 and
1537 cmÀ1), and a sugar moiety (complex NMR signals
at dH 3.89–4.90) that are similar to those of the known
termitomycesphins (1–4), indicating that 6 is a deriva-
tive of the termitomycesphins. The notable difference in
the NMR spectra between 2 and 6 is that the signals due
to the exomethylene (C9–C19) in 2 is replaced by a sec-
ondary methyl group (dH19 1.06 and 1.08, dH9 1.62 and
1.72; dC19 14.4 and 15.9, dC9 39.3 and 39.8) in 6. The
other NMR data are similar to those for 2 (Table 1). An
analysis of the HMQC and DQFCOSY spectra allowed
us to draw the following partial structures: b-glucoside
moiety, C1–C10, C20–C40, and two terminal methyls
connected to methylenes. In an HMBC spectrum, H2,
N–H, and H20 are correlated to the carbonyl carbon at
d 175.6, revealing the connectivity of the fatty acyl and
LCB moieties. The correlations of H1/anomeric C100
and anomeric H100/C1 indicate the location of the b-
Table 1. 1H (600 MHz) and 13C (150MHz) NMR data for termito-
a
mycesphins E (5) and F (6) in pyridine-d5
Position
1H (ppm) (J in Hz)
13C (ppm)
70.1 t
LCB
1a
1b
2
4.22 dd (9.3, 3.5)
4.68 dd (9.3, 4.5)
4.80m
54.6 d
3
4
5
6a
6b
7a
7b
8
9
101.30
11-17
18
19
2-NH
4.77 m
72.3 d
6.05 dd (15.2, 2.8)
6.01 dd (15.2, 4.6)
2.35, 2.37 mb
2.55, 2.56 mb
1.66, 1.71 mb
1.72, 1.80m
3.68, 3.77 mb
1.62, 1.72 mb
131.5, 131.6 db
132.9, 133.1 db
29.8 t
b
34.0, 34.8 t
b
73.7, 74.6 db
39.3, 39.8 db
32.6, 34.0t
22.8–32.0t
14.2 q
15.9, 14.4 qb
b
b
, 1.34 m
c
1.24 m
0.85 t (6.8)d
1.08, 1.06 d (7.4)b
8.35 d (8.5)
Acyl
10
175.6 s
72.4 d
35.5 t
20
4.56 m
2.00 m
2.20m
1.68 m
1.80m
30a
30b
40a
25.9 t
40b
50–150 (or 170)
1.24 m
0.84 t (6.8)d
22.8–32.0t
14.2 q
c
160 (or 180)
S0u0 gar
100
4.90d (7.6)
4.01 dd (7.6, 7.1)
4.19 m
4.20m
3.89 m
105.5 d
75.0 d
78.4 d
71.5 d
78.4 d
62.6 t
200
3
400
500
600a
600b
4.34 dd (11.7, 5.2)
4.49 br d (11.7)
aBoth data for 5 and 6 are superimposable.
b1:1 ratio due to C-8 and/or C-9 epimers.
cd=22.8, 27.8, 27.9, 29.5, 29.7, 29.8, 30.4 and 32.0.
dInterchangeable.
Figure 1. Structures of termitomycesphins A–F (1–6) and the known
cerebroside 7.9,10

J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
2173
The molecular formula of termitomycesphin E (5),
C41H79NO10, was determined to be less than that of 6
by C2H4 based on the HRESI-MS measurement [m/z
746.5764 (M+H)+, Á +1.8 mmu]. The 1H and 13C
NMR spectra of 5 are superimposable on those of 6
(Table 1), indicating that 5 is a homologue of 6 posses-
sing a shorter carbon chain. The positive FAB-MS
spectra of both 6 and 5 displayed the same fragment
ions at m/z 312 (LCB+H)+, 294 (LCB+HÀH2O)+,
and 276 (LCB+HÀ2H2O)+, revealing the presence of
the same LCB moiety in 5 and 6. The absolute stereo-
chemistry of 5 could be the same as that of 6 because of
the superimposable NMR data and a similar optical
rotation [5: [a]D24 +2.8 (c 0.20, MeOH); 6: [a]2D4+2.0( c
0.15, MeOH)].
of 14 mM. Such a tendency was also observed for termi-
tomycesphins A–D (1–4); 1 and 3 were more active than
2 and 4.1 On the other hand, the known cerebroside 7
showed almost no neuritogenic activity. These findings
suggest that not only the fatty acyl-chain length but also
the polarity of the molecules (the presence of the extra
hydroxyl group around the middle of the LCB) play
important roles in the neuritogenic activity of these cer-
ebrosides (Fig. 3).
To obtain supporting evidence for the importance of the
extra hydroxyl group around the middle of the LCB of
the termitomycesphins, we tried to introduce hydroxyl
groups into the LCB of the major inactive cerebroside 7.
Dihydroxylation of 7 with microencapsulated osmium
tetraoxide (MC OsO4) and N-methylmorpholine N-
oxide (NMO) gave the 8,9-dihydro-8,9-dihydroxyl deri-
vative 12ab in 44% yield. The number and positions of
the introduced hydroxyl groups were determined from
the FAB-MS data [m/z 784 (M+Na)+] and the follow-
ing newly observed 1H NMR signals assigned by a
DQFCOSY spectrum (Table 3): 3.79 (m, H-8), 1.41 (s,
H-19), and 5.71 (brs, 8-OH). The split peaks of C4–C5
(dC 133.0/132.9 and 131.7/131.6, respectively) observed
in 13C NMR spectrum of 12ab indicated that 12ab was a
1:1 mixture of diastereomers. The hydroxylation using
an excess amount of MC OsO4 gave the 4,5,8,9-tetra-
hydro-4,5,8,9-tetrahydroxy derivatives, 13ab and 13cd,
in 66 and 20% yields, respectively, both of which turned
out to be a 1:1 diastereomeric mixture from the HPLC
The gross structure of the known cerebroside 7 was
determined by a comparison of the NMR and FAB-MS
data with the reported ones.9,10 The degradation of 7
was carried out to determine the stereochemistry and
the two carbon chain lengths (Fig. 2). The hydrogena-
tion of 7 gave the tetrahydro derivative, which, upon
methanolysis, gave methyl d-glucopyranoside (2:1 mix-
ture of a and b-anomers), the fatty acid methyl ester 9,
and free LCB 10. The configuration of the methyl glu-
coside was determined to be the d form by a compar-
ison of the optical rotation [[a]2D5 +74 (c 0.027, MeOH)]
with the reported one.12 The R configuration of the
methyl ester 9 was determined by the comparison of the
optical rotation [[a]2D5 À5 (c 0.11, CHCl3)].13 The long-
chain base 10 was converted to the tribenzoyl derivative
1
analysis and H NMR spectra. The introduction of the
1
11. The CD spectrum and partial H NMR data (Table
four hydroxyl groups in 13ab and 13cd was revealed by
the FAB-MS data [m/z 818 (M+Na)+] and the newly
observed 1H NMR signals due to three oxymethines
and a branch methyl (Table 3): 4.65/4.67 (1H, m, H-5),
4.19 (1H, m, H-4), 3.85/3.89 (1H, m, H-8), 1.39/1.40
(3H, s, H-19) for 13ab; 4.36/4.39 (1H, m, H-5), 4.04
(1H, m, H-4), 3.82/3.86 (1H, m, H-8), 1.41/1.42 (3H, s,
H-19) for 13cd. The difference between 13ab and 13cd
can be attributed to the difference in the configuration
at the C4–C5 stereogenic centers, because the differences
in the NMR chemical shifts are mainly observed around
H4 and H5. To determine the absolute stereochemistry
at C4–C5 of 13ab, it was converted to an acetonide and
then finally to a tetraacetate triacetonide 14 (Fig. 4).
2) of 11 agreed with those reported for the benzoate of a
(2S,3R)-LCB,14 revealing the 2S,3R stereochemistry of
11. The structure and absolute stereochemistry of 7 was
thus determined to be the same as the reported ones as
shown in Figure 1.9,10
The neuritogenic activities of 5, 6, and 7 were evaluated
using the PC12 cell line system. Figure 5 shows the time
course of the neuronal differentiation of the PC 12 cells
induced by these compounds. Termitomycesphin E (5)
possessing a C16-fatty acyl moiety showed a much
higher activity than termitomycesphin F (6) possessing a
longer (C18) fatty acyl moiety at the same concentration
Figure 2. Degradation scheme of 7.

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J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
The relative stereochemistry of C3–C5 in 14 was deter-
mined to be anti–syn on the basis of the coupling constants
and NOE correlations as shown in Figure 4, suggesting
that 14 adopted a twisted boat conformation.15 Thus, the
absolute configuration of C4–C5 of 13ab was determined
to be 4R,5S, and C4–C5 of 13cd was 4S,5R.
(5 ng/mL) rather than that of termitomycesphin E (5)
(gradual increase). We presume that a peptidic com-
pound, NGF, and highly hydroxylated lipids such as
12ab and 13a–d are relatively susceptible to metabolic
degradation in the cell-culture conditions.
The neuritogenic activity of 12ab and 13a–d (Fig. 5)
indicated that hydroxylation markedly endowed 7 with
the activity (25% maximum neuronal differentiation at
a concentration of 2.5 mM), though these showed cyto-
toxicity at a concentration higher than 2.5 mM. The
times and concentrations required for reaching the
maximum activity of 12ab and 13a–d are much shorter
and lower than those of 5, respectively. Interestingly,
the time course of their activities (rapid increase and
then gradual lowering) was quite similar to that of NGF
Experimental
General procedures
Preparative HPLC was performed using JASCO PU-
980pumps (Japan). Optical rotations were measured on
a JASCO DIP-370digital polarimeter. IR spectra were
recorded on a JASCO FT/IR-7000S. UV spectra were
recorded on a JASCO Ubest-50UV/VIS spectrometer.
CD spectra were recorded with a JASCO J-720spec-
trometer. FAB-MS spectra were recorded on a JEOL
DX-705L (Japan) mass spectrometer, using m-nitro-
benzyl alcohol as the matrix in the positive mode. High-
resolution FAB-MS spectra were recorded on a JEOL
Mstation JMS-700 mass spectrometer, using a mixture of
the glycerol/thioglycerol/m-nitrobenzyl alcohol (3:1:1)
+NaI as the matrix and PEG 600 or PEG 1000 as the
calibration standard in the positive mode. HRESI-MS
spectra were recorded on a MDS SCIEX (Canada)
QSTAR mass spectrometer using a mixture of CsI and
the sex pheromone inhibitor iPD1 (MW 829) (Bachem,
Switzerland) as the calibration standards in the positive
mode. NMR spectra were recorded on a Bruker AMX-
600 or a Bruker ARX-400 spectrometer. The NMR
chemical shifts in d (ppm) were referenced to the solvent
peak of dC 39.7 for DMSO-d6, dH 7.26 for CDCl3 (resi-
dual CHCl3), and dH 7.19 and dC 123.4 for pyridine-d5.
Isolation
The extraction and purification procedures were descri-
bed in detail in a previous paper.1 The BuOH fraction
from the EtOH extract of the mushroom T. albuminosus
was chromatographed on ODS to give four fractions.
The HPLC purification of the third fraction yielded ter-
mitomycesphin E (5) (15.7 mg, 0.00036%, tR=120min)
and termitomycesphin
F (6) (24.7 mg, 0.00057%,
tR=205 min) as well as 1–4.1 A portion (100 mg) of the
fourth fraction (8.9 g), which was eluted with MeOH,
was purified by HPLC [Develosil ODS-10( f 20ꢀ250
mm; Nomura Chemical, Japan), solvent: MeOH/H2O
(98:2), flow rate: 8 mL/min, two injections, detection at
205 nm] to yield the known cerebroside 7 (20.4 mg,
0.042% calcd yield, tR=36 min).
Figure 3. Structures of cerebroside derivatives.
Termitomycesphin E (5): a colorless powder; [a]2D4 +2.8
(c 0.20, MeOH); IR (KBr) 3375, 2923, 2853, 1648, 1537,
1468, and 1079 cmÀ1; HRESI-MS m/z 746.5764, calcd
for C41H80NO10 (M+H) 746.5782; 1H and 13C NMR in
Table 1.
Table 2. Selected 1H NMR data (CDCl3, 400 MHz) and CD data
(MeOH) for tribenzoate 11
1H NMR
CD
Termitomycesphin F (6): a colorless powder; [a]2D4 +2.0
(c 0.15, MeOH); IR (KBr) 3375, 2921, 2853, 1648, 1538,
1468, and 1079 cmÀ1; HRESI-MS m/z 774.6087, calcd
JNHÀH2 JH2ÀH3 dNH dH3
dH2
7.09 5.38 4.88 4.61/4.67
dH1
l (ÁE)
7.9
3.9
238
223
(À7.8) (+5.8)
1
for C43H84NO10 (M+H) 774.6095; H and 13C NMR
Coupling constants J are in Hz, chemical shifts d are in ppm, and wave
length l in nm.
were superimposable on those of 5 (Table 1).

J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
2175
Figure 4. NMR analysis of tetraacetate triacetonide derivative 14 obtained from 13ab to determine the absolute configuration of C4–C5 of 13ab.
Partial chemical shifts (in ppm) are in italics.
was stirred under a hydrogen atmosphere at room tem-
perature for 4 h. The reaction mixture was filtered, and
the filtrate was concentrated to give 5.2 mg of tetra-
hydro derivative 8: [a]2D5+9.3 (c 0.14, MeOH), FAB-MS
1
m/z 798 (M+Na)+, H NMR data were the same as
that from 2.1
Degradation of 7
The known cerebroside 7 (5.4 mg) was hydrogenated
under the above conditions, and the crude product was
subjected to acidic methanolysis in the same conditions
as those for 2 and 4,1 giving methyl a- and b-d-gluco-
pyranoside (0.4 mg) [a]2D5 +74 (c 0.027, MeOH)], LCB
10, and fatty acid methyl ester 9 (1.4 mg): colorless
powder, [a]D24 À5 (c 0.11, CHCl3) [lit.13 [a]2D5 À3.6
(CHCl3)], FAB-MS m/z 287 (M+H)+. LCB 10 from
the EtOAc fraction was benzoylated,1,14 and the pro-
duct was purified by preparative TLC (silica gel, EtOAc/
hexane=1:9, developed twice, Rf=0.18) to give (2S,3R)-
2-benzoylamido-1,3-dibenzoyloxy-9-methyloctadecane
(11) (0.7 mg) as a colorless powder: HRFAB-MS m/z
1
628.4061, calcd for C40H54NO5 (M+H) 628.4002. H
NMR (400 MHz, CDCl3) d 8.02 (2H, m), 7.95 (2H, m),
7.77 (2H, m), 7.35–7.58 (9H, m), 7.08 (1H, d,
J=8.6 Hz), 5.38 (1H, m), 4.88 (1H, m), 4.63 (2H, m),
1.05–1.95 (27H, m, methylenes and one methine), 0.87
Figure 5. Time course of neuronal differentiation induced in PC12
cells by termitomycesphins E and F (5 and 6) and other cerebroside
(3H, t, J=6.8 Hz), and 0.80 (3H, d, J=6.5 Hz). The
concentration of 11 was calculated from an e value of
derivatives in comparison with nerve growth factor (NGF) as a posi-
32000 at 228 nm.14
tive control. The percentages of the cells with longer processes than the
diameter of the cell body were plotted as neuronal differentiation.
Hydroxylation of 7 to 12ab and 13a–d
Known cerebroside 7: a colorless powder; [a]2D5 +4.5 (c
0.46, MeOH); IR (KBr) 3371, 2921, 2853, 1643, 1536,
A mixture of 7 (9.5 mg), MC OsO4 (4.0mg, Wako,
Japan), and NMO (10.0 mg) in H2O/acetone/aceto-
nitrile (1:1:1, 60 mL) was stirred for 48 h at 50 ꢁC. The
catalyst was separated by filtration and washed with
MeOH. The combined filtrates were concentrated to
dryness, which was purified by preparative TLC (silica
gel, CHCl3/MeOH=4:1, developed twice, Rf=0.42) to
afford 12ab (4.2 mg, 44%): colorless powder, [a]2D3+5.5
(c 0.24, MeOH), IR (KBr) 3376, 2923, 2853, 1642, 1542,
1467, and 1080 cmÀ1; HRFAB-MS m/z 784.5500, calcd
for C41H79NO11Na (M+Na) 784.5550. 1H NMR in
Table 3; 13C NMR (pyridine-d5, 100 MHz) d 175.6,
133.0/132.9 (1:1), 131.7/131.6 (1:1), 105.5, 78.4, 78.3,
77.3/77.2 (1:1), 75.0, 74.4, 72.4, 72.3, 71.5, 70.0, 62.6,
1468, and 1081 cmÀ1; FAB-MS m/z 750(M+Na) + 13C
;
NMR (DMSO+2% D2O): d 173.91, 135.08, 131.26,
131.05, 123.61, 103.56, 77.00, 76.63 (76.62, CH–OD),
73.49 (73.46), 71.20 (71.17), 70.81 (70.71), 70.25 (70.20),
68.86, 61.33 (61.21), 53.04 (53.00, CH–ND–), 39.90,
34.53, 32.23, 31.36, 29.10, 28.98, 28.74, 27.49, 27.38,
24.61, 22.14, 15.83, and 13.95.
Hydrogenation of 6
To a solution of termitomycesphin F (6, 5.3 mg) in
EtOH (1 mL) were added PtO2 (1.0mg) and the mixture

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J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
Table 3. 1H NMR data of dihydroxylated and tetrahydroxylated cerebrosides (600 MHz, in pyridine-d5)
12ab
13ab (13a/13b)a
13cd (13c/13d)a
LCB
1a
1b
2
3
4.24 dd (10.9, 3.1)
4.66 m
4.78 m
4.78 m
6.11 m
4.51 m
4.67 m
5.31 m
4.68 m
4.19 m
4.20 m
4.77 m
4.88 m
4.41 m
4.04 m
4
5
6a
6b
7a
7b
8
10–17
18
19
6.06 m
2.39 m
2.70m
1.78 m
1.90m
3.79 m
1.25 br s, 1.63–1.90m
0.86 t (6.7)b
1.41 s
4.65/4.67 m
2.29/2.18 m
2.54/2.61 m
1.89/2.04 m
2.29/2.14 m
3.85/3.89 m
1.23–1.30m, 1.58–2.62 m
0.85 t (7.0)b
1.39/1.40s
4.36/4.39 m
2.05/2.19 m
2.54/2.38 m
1.85/2.05 m
2.33/2.13 m
3.82/3.86 m
1.20–1.32 m, 1.62–2.55 m
0.86 t (6.9)b
1.41/1.42 s
8.63 d (8.8)
NH
Acyl
20
8.33 d (8.2)
8.51/8.52 d (8.6)
4.56 m
2.00 m
2.20m
1.63–1.90m
1.63–1.90m
1.25 br s
0.85 t (5.7)b
4.51 m
1.88 m
2.13 m
1.62 m
1.71 m
4.55 m
1.93 m
2.15 m
1.67 m
1.74 m
30a
30b
40a
40b
50–150
160
S0u0 gar
100
1.23–1.30m
0.86 t (7.0)b
1.20–1.32 m
0.87 t (6.9)b
4.89 d (7.9)
4.00 dd (8.0, 7.7)
4.17 m
4.19 m
3.89 m
4.91/4.92 d (7.9)
3.95 dd (7.9, 7.5)
4.16 m
4.15 m
3.82 m
4.86 d (7.8)
3.98 m
4.15 m
4.15 m
3.88 m
200
3
400
500
600a
600b
4.34 dd (11.4, 4.3)
4.49 br d (11.4)
4.30m
4.43 d (11.5)
4.32 m
4.49 d (12.5)
aA 1:1 mixture of diastereomers. Measured at 310K.
bInterchangeable.
54.6, 39.2, 35.5, 32.0, 31.8/31.7 (1:1), 30.9, 30.4/30.3
(1:1), 30.0, 29.9, 29.5, 25.9, 24.0, 22.8, 22.4, and 14.2.
mixture was extracted three times with ether (2.5 mL).
The ether layers were combined, concentrated, and the
residue was purified by preparative TLC (silica gel,
acetone/hexane=4:6, Rf=0.3) to afford a mixture of
triacetonides (0.7 mg). The second trial using 13ab
(2.0mg), acetone (1.5 mL), 2,2-dimethoxypropane
A large excess of MC OsO4 (12.0mg) was used to oxi-
dize 10.0 mg of 7. The mixture was stirred for 24 h at
50 ꢁC and then filtered. The filtrate was evaporated and
the residue was dissolved in MeOH and passed through
a TOYOPAK ODS-M cartridge (TOSOH, Japan),
which was then washed with MeOH. The filtrate was
concentrated and subjected to preparative HPLC
[Develosil ODS-10( f 20ꢀ250mm), solvent: CH 3CN/
H2O/MeOH (75:15:10), flow rate: 8 mL/min] to give two
mixtures of tetrahydroxylated derivatives 13ab (6.6 mg,
66%, tR=50–66 min) and 13cd (2.0mg, 20%, tR=68–
73 min). 13ab: colorless powder, [a]2D3 À1.2 (c 0.41,
MeOH), IR (KBr) 3398, 2926, 2853, 1624, 1541, 1467,
and 1084 cmÀ1; HRFAB-MS m/z 818.5565. calcd for
C41H81NO13Na (M+Na) 818.5605. 1H NMR in Table 3.
13cd: colorless powder, [a]2D3 +12 (c 0.094, MeOH), IR
(KBr): 3384, 2922, 2852, 1647, 1541, 1467, and
ꢁ
(1.5 mL), and PPTS (1.0mg) at 0 C for 1 h then room
temperature for 4 h afforded a mixture of triacetonides
(0.7 mg). The combined mixtures (1.4 mg) of triaceto-
nides were subjected to HPLC [Develosil ODS-HG-5 (f
10ꢀ250mm), solvent: MeOH/H 2O (93:7), flow rate:
2.5 mL/min] and four peaks were collected (tR=94, 98,
101 and 116 min). The first fraction (0.3 mg,
tR=94 min), FAB-MS m/z 938 (M+Na)+, was acetyl-
ated with a mixture of dried pyridine (0.5 mL) and ace-
tic anhydride (0.5 mL) at room temperature for 5 h to
afford 14 (0.5 mg); HRFAB-MS m/z 1106.6895, calcd
for C58H101NO17Na (M+Na) 1106.6967. 1H NMR
(CDCl3, 600 MHz) d 6.30(1H, d, J=8.9 Hz, NH), 5.14
(1H, m, H-20), 5.12 (1H, m, H-3’’), 4.99 (1H, dd, J=3.8,
2.2 Hz, H-4), 4.89 (1H, dd, J=9.3, 8.0Hz, H-2’’), 4.50
(1H, d, J=7.8 mz, H-1’’), 4.19 (1H, m, H-2), 3.96
(1H, m, H-1b), 3.93 (1H, m, H-600b), 3.88 (1H, m, H-5),
3.82 (1H, dd, J=7.5, 6.4 Hz, H-3), 3.72 (1H, m, H-600a),
3.69 (1H, m, H-400), 3.67 (1H, m, H-1a), 3.34 (1H, m,
H-500), 2.18, 2.12, 2.04 and 2.03 (12H, s, 4 methyls from
–COCH3), 1.66 (2H, m, H-7), 1.65 (1H, m, H-6b), 1.45
(1H, m, H-6a), 1.46, 1.40, 1.38, 1.36 (3H each, s each, 4
methyls from acetonide), 1.30(6H, s, 2 methyls from
acetonide), 1.04–2.23 (40H, m, methylenes), 1.88 (1H,
1079 cmÀ1
; HRFAB-MS m/z 818.5565, calcd for
C41H81NO13Na (M+Na) 818.5605. 1H NMR in Table 3.
Conversion of 13ab to 14
A solution of 13ab (2.0mg) in a mixture of acetone
(0.5 mL), 2,2-dimethoxypropane (0.5 mL), and pyr-
idinium p-toluene sulfonate (PPTS) (1.0mg) was stirred
at room temperature for 2 h. The reaction was quenched
by adding saturated NaHCO3 solution (2.5 mL). The

J. Qi et al. / Bioorg. Med. Chem. 9 (2001) 2171–2177
2177
m, H-30b), 1.72 (1H, m, H-30a), and 0.86 (3H, t,
J=6.8 Hz).
4. Fischer, W.; Wictorin, K.; Bjorklund, A.; Williams, L. R.;
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Bioassay method
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Biochemistry 1999, 38, 7294.
Acknowledgements
This work was supported by a grant, Research for the
Future Program, from JSPS.
11. Sawabe, A.; Morita, M.; Okamoto, T.; Ouchi, S. Biol.
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