Structure of TMC-69, a new antitumor antibiotic from Chrysosporium sp. TC 1068

Article abstract of DOI:10.1016/S0040-4020(01)00009-6

A new antitumor antibiotic, TMC-69, possessing inhibitory activity against cdc25A phosphatase, has been isolated from the fermentation broth of a mitosporic fungus, Chrysosporium sp. TC 1068. This antibiotic was labile in solid state as well as in solution, and thus was converted to the more stable derivatives, diacetyl TMC-69 and hexahydro TMC-69, to elucidate the structure and to evaluate the biological activities. The planar structure of TMC-69 was determined, through the analysis of various NMR experiments of diacetyl TMC-69 and ESI-MS data of TMC-69, to be [5Z(E,E)]-1,4-dihydroxy-5-phenyl-3-[tetrahydro-3-methyl-5- (6-methyl-2,4-octadienylidene)-2H-pyran-2-yl]-2(1H)-pyridinone. Furthermore, the absolute configuration of tetrahydropyranyl moiety was determined by degradation studies of TMC-69, followed by the application of modified Mosher's method.

Full text of DOI:10.1016/S0040-4020(01)00009-6

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 1731±1735
Structure of TMC-69, a new antitumor antibiotic from
Chrysosporium sp. TC 1068
Jun Kohno,p Naomitsu Hirano, Kazutoshi Sugawara, Maki Nishio, Tomiki Hashiyama,
Noriyuki Nakanishi and Saburo Komatsubara
Discovery Research Laboratory, Tanabe Seiyaku Co. Ltd., 2-2-50, Kawagishi, Toda-shi, Saitama 335-8505, Japan
Received 2 November 2000; accepted 27 December 2000
AbstractÐA new antitumor antibiotic, TMC-69, possessing inhibitory activity against cdc25A phosphatase, has been isolated from the
fermentation broth of a mitosporic fungus, Chrysosporium sp. TC 1068. This antibiotic was labile in solid state as well as in solution, and thus
was converted to the more stable derivatives, diacetyl TMC-69 and hexahydro TMC-69, to elucidate the structure and to evaluate the
biological activities. The planar structure of TMC-69 was determined, through the analysis of various NMR experiments of diacetyl TMC-69
and ESI-MS data of TMC-69, to be [5Z(E,E)]-1,4-dihydroxy-5-phenyl-3-[tetrahydro-3-methyl-5-(6-methyl-2,4-octadienylidene)-2H-pyran-
2-yl]-2(1H)-pyridinone. Furthermore, the absolute con®guration of tetrahydropyranyl moiety was determined by degradation studies of
TMC-69, followed by the application of modi®ed Mosher's method. q 2001 Elsevier Science Ltd. All rights reserved.
In the course of our screening program for new antitumor
substances, a new antitumor antibiotic, TMC-69 (1) was
isolated from the fermentation broth of a mitosporic fungus,
Chrysosporium sp. TC 1068. TMC-69 (1) was a labile
compound, and the more stable derivatives, diacetyl
TMC-69 (2) and hexahydro TMC-69 (3) were prepared to
elucidate the structures and to evaluate the biological
activities. TMC-69 (1) and the derivatives 2 and 3 showed
cytotoxic activities in vitro against various tumor cell lines,
and 3 induced signi®cant prolongation of survival time of
mice transplanted with B16 melanoma as well as P388
leukemia. Moreover, hexahydro TMC-69 (3) was found to
have inhibitory activity against cdc25A phosphatase. In this
paper, we report the isolation, structure elucidation and
cytotoxicity in vitro of 1±3. The taxonomy, fermentation,
and detailed evaluation of biological activities will be
reported elsewhere.1
1. Results
1.1. Isolation
The fermentation broth (6.3 L) of Chrysosporium sp. TC
1068 was extracted with 1-butanol. The extract was puri®ed
by Sephadex LH-20 column chromatography and prepara-
tive HPLC to afford 43.3 mg of TMC-69 (1).
1.2. Structure determination
TMC-69 (1) was obtained as colorless powder, which was
completely decomposed after two weeks at 08C under air. In
the organic solvent, e.g. DMSO, methanol, acetone, ethyl
acetate, chloroform and benzene, up to 50% of 1 was
decomposed within two days. TMC-69 (1) showed charac-
teristic UV absorption maxima at 262, 275 and 287 nm,
suggesting the presence of a triene group. The IR spectrum
of 1 indicated the presence of a hydroxyl (3230 cm21) and
an amide (1640 and 1550 cm21) group.
Diacetyl TMC-69 (2), obtained by acetylation of 1 with
acetic anhydride and pyridine, was stable in methanol at
room temperature for three days, even though about 50%
of 2 was decomposed in chloroform or DMSO.
Hexahydro TMC-69 (3), obtained by catalytic hydrogena-
tion of 1 with palladium charcoal, was stable in the organic
solvents described above at room temperature for more than
two weeks. The UV spectrum of 3 lacked the characteristic
absorption, attributed to triene group, observed in 1.
Keywords: TMC-69; antitumor antibiotic; Chrysosporium; 2(1H)-pyridin-
one.
Corresponding author. Tel.: 181-48-433-2732; fax: 181-48-433-2734;
e-mail: jun-k@tanabe.co.jp
p
1
The H and 13C NMR spectra in CDCl3 of 1 are shown in
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00009-6

1732
J. Kohno et al. / Tetrahedron 57 (2001) 1731±1735
Table 1. 13C and 1H NMR data for 1±3
Position
1a
2b
3a
13C
1H
13C
1H
13C
1H
2
3
4
5
6
7
8
9
157.0 sc
108.6 s
160.8 s
113.6 s
129.9 d
80.8 d
157.3 s
124.4 s
157.1 s
117.6 s
134.9 d
79.4 d
37.1 d
43.0 t
157.0 s
109.3 s
160.9 s
113.5 s
129.9 d
81.7 d
31.0 d
36.1 t
7.69 (1H, s)
4.88 (1H, d, 9.8)d
2.08 (1H, m)
7.87 (1H, s)
7.67 (1H, s)
4.68 (1H, d, 10.5)
2.08 (1H, m)
4.62 (1H, d, 9.9)
2.40 (1H, m)
2.45 (1H, m)e
2.15 (1H, m)f
38.3 d
41.7 t
2.41 (1H, brd, 12)e
2.23 (1H, t, 12)f
1.77 (1H, brd, 12)e
1.53 (1H, m)f
10
11
133.4 sg
67.3 t
137.1 s
67.3 t
34.0 d
72.5 t
1.63 (1H, m)
4.88 (1H, d, 13.0)e
4.01 (1H, d, 13.0)f
5.91 (1H, d, 9.8)
6.21±6.23 (1H, m)
6.21±6.23 (1H, m)
6.05 (1H, dd, 8.9, 15.0)
5.62 (1H, dd, 7.8, 15.0)
2.11 (1H, m)
1.33 (2H, m)
0.85 (3H, t, 7.4)
0.88 (3H, d, 6.4)
1.00 (3H, d, 6.7)
4.77 (1H, d, 12.8)e
3.83 (1H, d, 12.8)f
5.94 (1H, brd, 11.3)
6.46 (1H, dd, 11.3, 14.2)
6.22 (1H, dd, 10.6, 14.2)
6.13 (1H, ddd, 0.9, 10.6, 14.8)
5.60 (1H, dd, 7.9, 14.8)
2.10 (1H, m)
1.33 (2H, m)
0.85 (3H, t, 7.4)
0.80 (3H, d, 6.5)
0.98 (3H, d, 6.9)
3.96 (1H, brd, 11.6)e
3.72 (1H, dd, 11.6, 2.5)f
1.52 (2H, m)
12
13
14
15
16
17
18
19
20
21
10
20,60
30,50
40
CH3CO
CH3CO
CH3CO
CH3CO
OH
OH
125.6 dg
124.3 dg
134.2 d
128.6 dg
141.9 d
38.6 d
29.7 t
11.7 q
17.6 q
125.1 d
126.3 d
134.0 d
130.1 d
141.2 d
39.4 d
30.4 t
12.0 q
17.8 q
20.4 q
134.2 s
129.7 d
129.3 d
128.7 d
17.8 q
20.5 q
167.4 s
167.3 s
30.9 t
30.0 t
27.1 t
27.8 t
36.9 t
1.25±1.35 (9H, m)
34.4 d
29.5 t
1.11 (2H, m)
11.4 q
17.9 q
19.2 q
133.2 s
129.2 d
128.4 d
127.7 d
0.85 (3H, t, 7.3)
0.80 (3H, d, 6.6)
0.83 (3H, d, 6.3)
20.0 q
133.0 sg
122.9 d
128.4 d
127.7 dg
7.43 (2H, m)
7.39 (2H, m)
7.33 (1H, m)
7.46 (2H, m)
7.41 (2H, m)
7.35 (1H, m)
7.33±7.42 (5H, m)
2.35 (3H, s)
1.90 (3H, s)
9.22 (1H, brs)
N.d.
9.51 (1H, brs)
N.d.
a
Measured in CDCl3.
Measured in acetone d6.
Multiplicity.
Proton number, multiplicity and coupling constants in Hz.
Equatorial protons.
Axial protons.
b
c
d
e
f
g
May be exchangeable.
Table 1. Owing to unstability of 1, poorly resolved 2D-
NMR spectra of 1 were obtained in DMSO-d6, acetone-d6
and benzene-d6 as well as in CDCl3. The more stable
derivative 2, however, showed well-resolved 2D-NMR
spectra in acetone-d6. The structure of 1 was thus derived
from the analysis of various NMR experiments of 2.
of 1 by C4H4O2 unit. The 1H and 13C NMR spectra of 2 were
similar to those of 1, except for the additional signals of two
methyls (1H: d 2.35 and 1.90; 13C: d 17.8 and 20.5) and two
carbonyls (13C: d 167.4 and 167.3). These results indicated
that the structure of 2 was diacetyl derivative of 1. The 13C
NMR spectrum of 2 displayed 30 signals composed of 3
methyls, 3 methylenes, 3 methines, 17 sp2 carbons as well
as two acetyl groups. In the DQF-COSY experiment, the
cross peaks, observed unambiguously, indicated the
presence of a 6-methyl-2,4-octadienylidene moiety (C-12±
C-19 and C-21) as shown in Fig. 1.
1.3. Diacetyl TMC-69 (2) and TMC-69 (1)
The molecular formulae of 1 and 2 were elucidated to be
C26H31NO4 and C30H35NO6 from their HRFAB-MS [m/z 1:
found 444.2173 (M1Na)1, calcd 444.2151; 2: found
528.2373 (M1Na)1, calcd 528.2362] and 1H and 13C
NMR data. The molecular formula of 2 differed from that
The structure of a tetrahydro-3-methyl-2H-pyranyl moiety was
deduced from a proton network of ±H7±H8(H20)± H9±,
Figure 1. 2D-NMR experiments of 2.

J. Kohno et al. / Tetrahedron 57 (2001) 1731±1735
1733
Figure 2. Key NOE correlations and 1H±1H coupling constants of 3.
and HMBC correlations from H-11 (d 4.77 and 3.83) to C-7
(d 79.4), C-9 (d 43.0) and C-10 (d 137.1). The HMBC
correlations from H-11 to C-10 (d 137.1) and C-12 (d
125.1), and from H-12 to C-9 (d 43.0) indicated that the
octadienylidene moiety was connected with the pyranyl
moiety at C-10. The presence of a mono substituted benzene
relative con®guration at C-10 was deduced from the 1H±1H
3
coupling (3JH-10,H-11axˆ2.5 Hz and JH-10,H-11eqˆ1 Hz) and
NOE (H-8/H-12, H-10/H-9ax, and H-10/H-11ax) correla-
tions as shown in Fig. 2.
1.5. Absolute structure
1
ring was also indicated from the characteristic 13C and H
NMR signals, and HMQC and HMBC correlations. The
structure of 1,4-diacetoxy-2(1H)-pyridinone moiety was
made up by the remaining ®ve sp2 carbons (13C: d 157.3,
157.1, 134.9, 124.4 and 117.6), three oxygens, one nitrogen
and two acetyl groups. The structure was con®rmed by
HMBC correlations from H-7 to C-3 (d 124.4), C-2 (d
157.3) and C-4 (d 157.1), and from H-6 (d 7.87) to C-2
and C-4, leading the plane structure of 2.
The absolute con®guration of the tetrahydropyranyl moiety
was determined by degradation studies of 1, followed by the
application of modi®ed Mosher's method.2 TMC-69 (1) was
methylated with trimethylsilyldiazomethane to afford
dimethyl TMC-69 (4). Ozonolysis of 4 gave a 10-oxotetra-
hydropyranyl derivative 5, which was reduced with sodium
borohydride to yield a 10-hydroxytetrahydropyranyl deriva-
tive 6. The hydroxyl group at C-10 of 6 was determined to
be located at equatorial position based on the large
vicinal coupling constants, 3JH-10,H-9axˆ12 Hz and
3JH-10,H-11axˆ10.3 Hz. Finally, the 10-hydroxyl group of 6
was converted to (S)-(2)- and (R)-(1)-a-methoxy-a-
(tri¯uoromethyl)-phenylacetyl (MTPA) esters to give 7
and 8, respectively.
The relative stereochemistry at C-7 and C-8 was determined
1
to be trans on the basis of the H±1H coupling constant,
3JH-7,H-8ˆ9.9 Hz, and NOE correlation, CH3-20/H-7. The
con®guration of the double bond at C-10 was assigned as
Z form from NOE correlations, H-9eq/H-12, and H-11eq/
H-13. The con®guration of the two double bonds at
C-13 and C-15 was determined to be E form, based on
Distribution of the positive and negative d values of the
MTPA esters was in agreement with 10S stereochemistry
(Fig. 3). From the above result along with the relative
stereochemistry of the tetrahydropyranyl moiety, the
absolute stereochemistry of 1, 2 and 3 was established to
be 7R, 8R and 7R, 8R and 7R, 8R, 10R, respectively.
3
the large coupling constants, JH-13,H-14ˆ14.2 Hz and
3JH-15,H-16ˆ14.8 Hz, and NOE correlations H-12/H-14,
H-13/H-15, H-14/H-16, and H-15/H-17 (Fig. 1). The gross
structure of 2 derived from the above results, demonstrated
that the structure of 1 was [5Z(E,E)]-1,4-dihydroxy-5-phenyl-
3-[tetrahydro-3-methyl-5-(6-methyl-2,4-octadienylidene)-2H-
pyran-2-yl]-2(1H)-pyridinone.
TMC-69 (1) was thus determined to be [2R-trans,5Z(E,E)]-
1,4-dihydroxy-5-phenyl-3-[tetrahydro-3-methyl-5-(6-methyl-
2,4-octadienylidene)-2H-pyran-2-yl]-2(1H)-pyridinone. The
stereochemistry at C-17 has not yet been established.
1.4. Hexahydro TMC-69 (3)
The molecular formula of 3 (C26H37NO4, HRFAB-MS: m/z
found 444.2173 (M1Na)1, calcd 444.2151) with the
absence of the triene absorption in the UV spectrum
suggested that 3 was the hexahydro derivative of 1. In the
13C NMR spectrum, one methine carbon C-10 (d 34.0) and
®ve methylene carbons C-12±C-16 (d 30.9, 30.0, 27.1, 27.8
and 36.9) in 3 were observed in place of the corresponding
ole®nic carbons in 1. The structure of 3 was thus determined
to be a C-10 and C-12±C-16-hexahydro derivative of 1. The
Several fungal metabolites, possessing phenyl-2(1H)-
pyridinone skeleton, namely tenellin and bassianin as
yellow pigments,3,4 ilicicolin H as an antifungal antibiotic,5
sambutoxin as a mycotoxin,6 etc. have been reported.
Among them, a tetrahydropyranyl moiety is only found in
sambutoxin. Furthermore, 2(1H)-pyridinone skeleton with
the tetrahydropyranyl moiety is seen in an antifungal
antibiotic, funiculosin.7 Both have the same substitution,
1
Figure 3. H chemical shift differences (Ddˆd72d8) between 7 and 8.

1734
J. Kohno et al. / Tetrahedron 57 (2001) 1731±1735
5-methyl-6-(1,3,5-trimethylhept-1-enyl)-, on the tetra-
hydropyranyl moiety. None of the known metabolites,
however, shows the same substitution pattern on the tetra-
hydropyranyl moiety of TMC-69. Thus, TMC-69 is classi-
®ed to be a new type of the 2(1H)-pyridinone antibiotic.
time: 33 min). This preparative HPLC was repeated 55
times for the remaining crude solid, and combined active
eluates were concentrated and extracted with 1-butanol. The
butanol extract was washed with water, concentrated and
lyophilized to afford a pure TMC-69 (1, 43.3 mg) as color-
less amorphous powder; UV (MeOH) lmax 287 (4.63), 275
(4.76), 262 (4.69) and 245sh (4.54) nm (log e); IR (KBr)
nmax 3230, 2960, 2925, 2875, 1640, 1580, 1550, 1455,
1220, 1050, 990, 780 and 700 cm21; FAB-MS m/z 422
(M1H)1, 444 (M1Na)1; ESI-MS m/z 422 (M1H)1, 334,
308, 282, 256, 216, 179 and 161. HRFAB-MS m/z 444.2173
(M1Na)1, (calcd for C26H31NO4Na 444.2151); The 1H and
13C NMR data are given in Table 1.
1.6. Biological activity
TMC-69 (1), and the derivatives 2 and 3 showed cyto-
toxicity against several tumor cell lines, human colon
carcinoma HCT-116 (IC50 (mM); 1: 6.8, 2: 6.0, 3: 0.8),
B16 mouse melanoma (IC50 (mM); 1: 6.6, 2: 7.6, 3: 1.9)
and murine lymphoid neoplasm P388D1 (IC50 (mM); 1:
6.6, 2: 6.8, 3: 0.9). These results indicated that the free
N-hydroxyl group and the triene moiety were not essential
for cytotoxicity. TMC-69 derivatives 3, exhibited signi®-
cant in vivo antitumor activity in B16 melanoma or P388
leukemia bearing mice.1 Moreover, 3 inhibited cdc25A
phosphatase activity.1 Although several related compounds
having the 2(1H)-pyridinone skeleton have been reported,
only TMC-69 derivatives showed promising antitumor
activity as far as we know. Structure±activity relationships
based on the synthetic study of TMC-69 will be reported in a
near future.
2.1.2. Diacetyl TMC-69 (2). Compound 1 (70 mg) and
acetic anhydride (1.8 mL) in dry pyridine (7.0 mL) were
stirred at room temperature for 30 min. The reaction
mixture was diluted with ethyl acetate (100 mL), washed
with water, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The resulting crude syrup was puri-
®ed by preparative HPLC (column: YMC D-ODS-5B,
30 i.d.£250 mm; elution: 75% aqueous acetonitrile; ¯ow
rate: 25 mL min21; detection: UV at 254 nm; retention
time: 55.3 min) followed by Sephadex LH-20 column chro-
matography with acetone to afford 18.8 mg of 2 as colorless
amorphous powder; UV (75% aqueous acetonitrile) lmax
284, 274, 265 nm; IR (KBr) nmax 3430, 2960, 2925, 2875,
2. Experimental
1810, 1775, 1670, 1540, 1365, 1160, 990 and 700 cm21
;
FAB-MS m/z 506 (M1H)1, 528 (M1Na)1; HRFAB-MS
m/z 528.2373 (M1Na)1, (calcd for C30H35NO6Na
528.2362); The 1H and 13C NMR data are given in Table 1.
2.1. General methods
Optical rotations were determined using the sodium D line
on a Horiba model SEPA-200 high sensitive polarimeter.
UV spectrum of 1 and 3 were measured on a Shimadzu
model UV-2200A spectrophotometer. UV spectrum of 2
was obtained on Hewlett±Packard HP-1100 diode array
detector. IR spectra were recorded on a JASCO model
100 infrared spectrophotometer. The samples were prepared
and mounted as KBr micropellets. FAB-MS spectra were
obtained on a JEOL JMS HX-100 spectrometer and ESI-MS
spectra were obtained using MStation 700 tandem type mass
2.1.3. Hexahydro TMC-69 (3). Compound 1 (100.5 mg) in
methanol (2.0 mL) was reduced with hydrogen over 50%
wet-10% palladium charcoal (20.0 mg) for 1 h. The reaction
mixture was ®ltered and concentrated under reduced pres-
sure. The resulting syrup was puri®ed by preparative HPLC
(column: YMC D-ODS-5-B, 30 i.d.£250 mm; elution: 85%
aqueous acetonitrile; ¯ow rate: 25 mL min21; detection: UV
at 254 nm; retention time: 55.3 min) followed by Sephadex
LH-20 column chromatography with CH2Cl2/MeOH (1:1)
to give 6.5 mg of 3. Colorless, amorphous powder;
1
spectrometer. H and 13C NMR spectra were recorded on a
JEOL GSX-400 (400 MHz) and Varian Jemini 2000
(300 MHz) NMR spectrometer at 308C. The chemical shifts
are given in ppm (d) relative to tetramethylsilane as an
internal standard. Wakogel C-200 (Wako Pure Chemical
Industries Ltd.) and Chromatorex DM1020 (Fuji Silysia
Chemical Ltd.) were used for silica gel and amino silica
gel column chromatography, respectively.
25
[a]D ˆ1143.48(c 0.480, MeOH); UV (MeOH) lmax 300
(3.65), 241 (4.38), 210 (4.36) nm (log e); IR (KBr) nmax
3180, 2960, 2925, 2855, 1640, 1580, 1560, 1500, 1455,
1380, 1220, 1050, 980, 870, 775 and 700 cm21; FAB-MS
m/z 428 (M1H)1, 450 (M1Na)1; HRESI-MS m/z 426.2621
(M2H)2, (calcd for C26H36NO4 426.2644); The 1H and 13
C
NMR data are given in Table 1.
2.1.1. Isolation of TMC-69 (1). The fermentation broth
(6.3 L) of Chrysosporium sp. TC 1068 was extracted with
1-butanol (2.7 L). The solvent layer (1.7 L) was separated
and concentrated in vacuo to dryness. The residue was
extracted twice with acetone (200 mL) to give a crude
syrup (3.85 g). This syrup was applied onto a column of
Sephadex LH-20 (40 i.d.£640 mm) and eluted with
CH2Cl2/MeOH (1:1). The appropriate fractions, determined
by TLC analysis, were combined and evaporated under
reduced pressure to yield a crude solid (568 mg). A portion
of this solid (10 mg) was further puri®ed by preparative
HPLC (column: YMC D-ODS-5-B, 30£250 mm; eluent:
75% CH3CN±10 mM phosphate buffer (pH 3.5); ¯ow
rate: 25 mL min21; detection: UV at 254 nm; retention
2.1.4. Methylation of 1. To a solution of 1 (655 mg) in
CH2Cl2 (100 mL) and MeOH (20 mL) was added excess
diazomethane in diethyl ether. The mixture was stirred at
08C for 30 min and then at room temperature for 3 h. After
removing the solvent, the residue was puri®ed on amino
silica gel column with EtOAc/n-hexane (1:1), followed by
silica gel column with EtOAc/n-hexane (1:4, 1:2, 1:1; step-
wise) to yield dimethyl TMC-69 (4, 120 mg, 17% yield).
1
ESI-MS m/z 450 (M1H)1; H NMR (CDCl3, 300 MHz) d
7.50 (H-6, 1H, s), 7.33±7.45 (Ph, 5H, m), 6.30 (H-13, 1H,
dd, 14, 11.2), 6.15 (H-14, 1H, dd, 14, 10.3), 6.05 (H-15, 1H,
14.7, 10.3), 5.87 (H-12, 1H, d, 11.2), 5.58 (H-16, 1H, dd,
14.7, 7.7), 4.85 (H-11eq, 1H, brd, 13), 4.68 (H-7, 1H, d,

J. Kohno et al. / Tetrahedron 57 (2001) 1731±1735
1735
1
10.1), 4.10 (N±OCH3, 3H, s), 4.00 (H-11ax, 1H, d, 13), 3.35
(OCH3, 3H, s), 2.46 (H-9eq, 1H, dd, 14, 4.2), 2.70 (H-8, 1H,
m), 2.0±2.2 (H-9ax and H-17, 2H, m), 1.32 (H-18, 2H, m),
1.01 (H-21, 3H, d, 6.8), 0.85 (H-19, 3H, t, 7.3), 0.82 (H-20,
3H, d, 6.6).
C29H31NO7F3 562.2053); H NMR (CDCl3, 400 MHz) d
7.503 (H-6, 1H, s), 7.4±7.5 (Ph£2, 10H, m), 5.234 (H-10,
1H, m), 4.514 (H-7, 1H, d, 11), 4.145 (H-11eq, 1H, ddd,
10.5, 5.0, 2.0), 4.098 (N±OCH3, 3H, s), 3.557 (OCH3, 3H,
s), 3.362 (OCH3, 3H, s), 3.348 (H-11ax, 1H, t, 10.5), 2.833
(H-8, 1H, m), 2.370 (H-9eq, 1H, m), 1.468 (H-9ax, q, 12),
0.843 (H-20, 3H, d, 6.6).
2.1.5. Preparation of 6 from 4. Dry O3 was bubbled into
the solution of 4 (80 mg, 0.18 mmol) in MeOH (5 mL) at
2788C, until the starting material was disappeared on TLC
(SiO2, EtOAc/n-hexane (1:1)). To the reaction mixture,
Me2S was added (200 mL, 2.7 mmol) and then the mixture
was stirred at room temperature for 1 h. To the resulting
mixture, NaBH4 was added (27 mg, 0.72 mmol) and then
the mixture was stirred at 08C for 30 min. After adding
acetone (1 mL) to the reaction mixture, water was added
and the mixture was extracted with CH2Cl2. The organic
phase was washed with water, saturated NaCl, and dried
over anhydrous Na2SO4. The solvent was concentrated in
vacuo and the residual oil was puri®ed on preparative TLC
(SiO2, 6% MeOH±CHCl3) to yield a colorless oil of 6
2.2. In vitro cytotoxic activity
The cells used for assay were cultured in the following
medium; HCT-116: complete McCoy's 5A supplemented
with 10% fetal bovine serum, B16: complete D-MEM
supplemented with 10% fetal bovine serum, P388D1:
complete RPMI-1640 supplemented with 5% fetal bovine
serum.
In vitro cytotoxic activity was tested in 96-well microtiter
plates of which well containing 1£104 each cell lines in
135 mL medium. The test samples were dissolved in 10%
DMSO. The serially diluted DMSO solution (15 mL) was
added to each well of plates. After addition, the cells were
incubated at 378C for 72 h in a humidi®ed 5% CO2
atmosphere. In vitro cytotoxic activity was evaluated by
the microculture tetrazolium (MTT) assay method for
each cell and by the colorimetrical determination method
at 540 nm.
1
(9 mg, 22% yield). ESI-MS m/z 346 (M1H)1; H NMR
(CDCl3, 300 MHz) d 7.51 (H-6, 1H, s), 7.35±7.45 (Ph,
5H, m), 4.46 (H-7, 1H, d, 10), 4.15 (H-11eq, 1H, ddd,
10.3, 5, 2), 4.10 (N±OCH3, 3H, s), 3.95 (H-10, 1H, m),
3.36 (OCH3, 3H, s), 3.24 (H-11ax, 1H, t, 10.3), 2.75 (H-8,
1H, m), 2.24 (H-9eq, 1H, m), 1.25 (H-9ax, 1H, q, 12), 0.82
(H-20, 3H, d, 6.8).
2.1.6. (S)-(2)-MTPA ester of 6. To a stirred solution of 6
(2.3 mg, 0.0067 mmol) in dry CH2Cl2 (0.5 mL), (R)-(2)- a-
methoxy-a-tri¯uoromethylphenylacetic chloride (MTPACl;
12 mg, 0.05 mmol), triethylamine (12 mg, 0.1 mmol) and
4-dimethylaminopyridine (DMAP; catalytic amount) was
added and the mixture was stirred at room temperature for
7 h. The reaction mixture was directly puri®ed on prepara-
tive TLC (SiO2, 5% MeOH±CHCl3) to yield 2 mg of
(S)-(2)-MTPA ester, 7 (53% yield). FAB-MS m/z 562
(M1H)1; HRFAB-MS m/z 562.2050 (M1H)1, (calcd for
Acknowledgements
We wish to thank Ms Naoko Fukui for NMR measurements,
and Dr Noriko Ohashi and Ms Sonoko Shiina for mass
measurements.
References
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1
C29H31NO7F3 562.2053); H NMR (CDCl3, 400 MHz) d
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2.1.7. (R)-(1)-MTPA ester of 6. To a stirred solution of 6
(3.0 mg, 0.0087 mmol) in dry CH2Cl2 (0.5 mL), (S)-(1)-
MTPACl (12 mg, 0.05 mmol), triethylamine (12 mg,
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