Zygosporin D and two new cytochalasins produced by the fungus Metarrhizium anisopliae

Article abstract of DOI:10.1021/np990331t

Zygosporin D (3) and two new cytochalasins (4 and 5) were isolated from the culture filtrate of the fungus Metarrhizium anisopliae and characterized on the basis of their spectral data and chemical conversions. The new cytochalasins, 4 and 5, were determi

Full text of DOI:10.1021/np990331t

132
J . Nat. Prod. 2000, 63, 132-135
Zygosp or in D a n d Tw o New Cytoch a la sin s P r od u ced by th e F u n gu s
Meta r r h iziu m a n isoplia e
Yuzo Fujii,† Hiroko Tani,† Masakatsu Ichinoe,‡ and Hiromitsu Nakajima*,†
Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, J apan, and
Laboratory of Food Microbiology, Tokyo Kasei University, Itabashi-ku, Tokyo 173-0003, J apan
Received J uly 9, 1999
Zygosporin D (3) and two new cytochalasins (4 and 5) were isolated from the culture filtrate of the fungus
Metarrhizium anisopliae and characterized on the basis of their spectral data and chemical conversions.
The new cytochalasins, 4 and 5, were determined to be deacetylcytochalasin C and (6R,16S,18R,21R)-
18,21-dihydroxy-16,18-dimethyl-10-phenyl[11]cytochalasa-13(E),19(E)-diene-1,7,17-trione, respectively. Of
these three cytochalasins, only zygosporin D is an effective inhibitor of shoot elongation of rice seedlings.
During our screening-based search for natural plant
growth retardants among the metabolites produced by soil
fungi,1 we found that the fungus, Metarrhizium anisopliae
(Metschn.) Sorok., produced growth retardant(s) to rice
seedlings. M. anisopliae, one of the most important insect
pathogens, has a high degree of specialization and occurs
almost solely in two families of the Coleoptera, the Ela-
teridae and Curculionidae.2 Production of cytochalasins C
(1) and D (2)3 and of insecticidal cyclodepsipeptides, the
destruxins,4 by M. anisopliae has been reported. The
culture filtrate from our fungus contained three cytochal-
asins, one of which showed plant growth-retardant activity
and was identified as zygosporin D (3). The second,
cytochalasin 4, is the first isolation as a natural product,
but it has been reported as a chemical conversion product
of cytochalasin C (1). The third, 5, was determined to be a
new type of cytochalasin. These last two cytochalasins, 4
and 5, had no retardant activity.
of cytochalasin C (1). Deacetylcytochalasin C is reported
to have been obtained only by mild alkaline hydrolysis of
cytochalasin C (1).3 This is the first report of deacetylcy-
tochalasin C as a natural product.
The structure of 5 was elucidated by comparing its NMR
data with those of 4 and by chemical conversion. The
molecular formula of 5 is the same as that of 3 and 4. These
compounds, therefore, have the same unsaturation. There
were two resonances in the 13C NMR spectrum of 5
assignable to ketonic carbonyl carbons (δ 210.4 and 214.2),
whereas in that of 4 there was only one such resonance (δ
210.0). Four resonances assignable to olefinic carbons (δ
126.9, 127.2, 133.3, and 136.8) were present in the 13C
NMR spectrum of 5, whereas 4 had six such resonances (δ
126.2, 126.4, 131.0, 131.7, 132.6, and 138.1). Instead of the
two additional olefinic carbon signals present in the 13C
NMR spectrum of 4, in that of 5 there were two signals
1
due to sp3 methine carbons (δ 35.6 and 45.8). The H NMR
Cytochalasins 3-5 were isolated from the culture filtrate
of the fungus in respective yields of 1, 11, and 0.4 mg/L.
On the basis of the HREIMS and 13C NMR data, they have
the same molecular formula, C28H35NO5. The NMR data
for 3 suggested that it was zygosporin D, the deacetyl
derivative of cytochalasin D (2). Spectral data reported in
the original paper,5 however, were not sufficient to identify
it unambiguously as zygosporin D; therefore, chemical
conversion was attempted. Compound 3 was acetylated by
the usual method, affording 6, which was identical in all
respects to the compound obtained by acetylation of cy-
tochalasin D (2).
spectrum of 5 had four methyl signals (one singlet and
three doublets); in that of 4 there were also four methyl
1
signals (three singlets and one doublet). The H-1H COSY
spectrum of 4 indicated a coupling system in which the
proton that resonated at δ 2.40 (H-8) was coupled with two
protons at δ 3.78 (H-7) and 5.89 (H-13). In the 1H-1H
COSY spectrum of 5 the proton that resonated at δ 3.79
(H-8) appeared to be coupled only with the proton at δ 5.66
(H-13). These NMR data indicate that 5 has a structure in
which the double bond at C-5 in 4 is hydrogenated to a
single bond and the hydroxy group at C-7 in 4 is oxidized
to a ketone group. HMBC data support this conclusion.
The structure of cytochalasin 4 was clarified by compar-
ing its NMR data with the data for 3 and by the chemical
conversion. Two resonances (δ 114.0 and 148.1) due to the
exocyclic double bond that are present in the 13C NMR
spectrum of 3 were missing; instead, the carbon signals (δ
126.2 and 132.6) due to a tetrasubstituted double bond
Chemical conversion of cytochalasin D (2) and 5 con-
firmed this and established the stereochemistry of 5.
Catalytic hydrogenation of 2 afforded two products, 8 and
9, which were diastereomers due to the configuration of
C-6. NOE experiments were conducted to establish the
stereochemistry of C-6. In those for 8, irradiation of H-11
caused NOE enhancement of the signals due to H-3, H-5,
and H-6, and irradiation of H-12 caused NOE enhancement
of the signals due to H-5, H-6, and H-8. In those for 9,
irradiation of H-11 caused NOE enhancement of the signals
due to H-3 and H-5, and irradiation of H-12 caused NOE
enhancement of the signals due to H-3, H-6, and H-7. These
results indicate that the absolute stereochemistry of C-6
in 8 is R and in 9 is S. Compound 8 was oxidized by
pyridinium chlorochromate (PCC) yielding 10 which was
identical in all respects to the compound obtained by the
acetylation of 5. The structure of 5, therefore, was estab-
1
were present in 4. Furthermore, in the H NMR spectrum
of 4, the signal (δ 1.54) due to the C-11 methyl was a
singlet, not a doublet. These findings suggested that 4 is
an isomer of 3, a deacetyl derivative of cytochalasin C (1).
Chemical conversion was performed to confirm this. Com-
pound 4 was acetylated, giving 7, which was identical in
all respects to the compound obtained by the acetylation
* To whom correspondence should be addressed. Tel. and Fax: +81-857-
31-5362. E-mail: nakajima@muses.tottori-u.ac.jp.
† Tottori University.
‡ Tokyo Kasei University.
10.1021/np990331t CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/19/1999

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 1 133
its morphological features.2 It has been maintained on potato-
dextrose agar.
lished as (6R,16S,18R,21R)-18,21-dihydroxy-16,18-dimethyl-
10-phenyl[11]cytochalasa-13(E),19(E)-diene-1,7,17-trione.
To date only the protophomin from Phoma exigua has been
reported as a 7-keto cytochalasin, in which the ketone
function is in the cyclohexene ring.6,7 The cytochalasin
reported here is a new type, in which the 7-ketone function
is in the cyclohexane ring.
F er m en ta tion a n d Extr a ction . The fungus was grown
without shaking at 24 °C for 14 days in the dark in 500-mL
conical flasks (50) containing liquid medium (200 mL/flask)
composed of glucose (30 g/L), peptone (3 g/L), the extract from
50 g/L of malt and H2O. Metabolites were extracted from the
culture filtrate with EtOAc (3 × 10 L) after adjusting its pH
to 2.0 with HCl. The EtOAc extracts were dried over Na2SO4,
concentrated, washed with 1M NaHCO3 (2 × 0.5 volume), then
dried over Na2SO4, and concentrated to dryness.
P u r ifica tion . The residue (0.58 g) was purified by Si gel
column chromatography (Daisogel IR-60, 11 × 135 mm).
Elution was done with 650 mL (130 mL × 5) each of 10, 20,
and 40% acetone in n-hexane. The first fraction, eluted with
40% acetone in n-hexane (195 mg), was recrystallized from
EtOAc, yielding colorless needles of 4 (102 mg). The cytochal-
asins in the mother liquor were purified by Sephadex LH-20
column chromatography (20 × 400 mm, MeOH). The column
was developed with MeOH, and 5-mL portions of the eluate
were collected. Fractions 18-20 were combined and evaporated
to dryness. The cytochalasins in the residue (35 mg) were
purified by HPLC (70% MeOH, 1.0 mL/min), giving 3 as a
white powder (12.3 mg, tR 18.7 min), colorless needles of 4 (11.0
mg, tR 21.8 min), and 5 as a white powder (4.3 mg, tR 23.8
min).
Zygosp or in D (3): [R]25D +20.6° (c 0.25, EtOH) and -15.5°
(c 0.58, dioxane); IR (film) νmax 3374 (OH, NH), 1700 (ketone
CO), 1682 (amide CO) cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.32
(2H, m, H-3′, -5′), 7.26 (1H, m, H-4′), 7.12 (2H, m, H-2′, -6′),
6.21 (1H, dd, J ) 16.2, 2.4 Hz, H-20), 5.65 (1H, dd, J ) 15.9,
10.2 Hz, H-13), 5.53 (1H, s, NH), 5.41 (1H, dd, J ) 16.2, 2.6
Hz, H-19), 5.31 (1H, s, H-12), 5.27 (1H, ddd, J ) 15.9, 10.8,
5.4 Hz, H-14), 5.11 (1H, s, H-12), 4.05 (1H, dd, J ) 2.6, 2.4
Hz, H-21), 3.79 (1H, d, J ) 10.7 Hz, H-7), 3.29 (1H, ddd, J )
8.3, 4.3, 3.8 Hz, H-3), 2.9-2.8 (1H, m, H-5), 2.88 (1H, dd, J )
13.6, 3.5 Hz, H-10), 2.83 (1H, dd, J ) 10.7, 10.2 Hz, H-8), 2.72
(1H, ddq, J ) 11.0, 1.6, 6.5 Hz, H-16), 2.57 (1H, m, H-4), 2.55
(1H, m, H-10), 2.48 (1H, ddd, J ) 13.0, 11.0, 10.8 Hz, H-15),
2.00 (1H, ddd, J ) 13.0, 5.4, 1.6 Hz, H-15), 1.55 (3H, s, H-23),
1.19 (3H, d, J ) 6.8 Hz, H-22), 1.11 (3H, d, J ) 6.8 Hz, H-11);
13C NMR (CDCl3, 100 MHz) δ 210.2 (s, C-17), 175.0 (s, C-1),
148.1 (s, C-6), 137.3 (s, C-1′), 137.1 (d, C-20), 133.7 (d, C-19),
130.9 (d, C-14), 129.2 (d, C-2′, -6′), 128.9 (d, C-3′, -5′), 127.1
(d, C-4′), 127.0 (d, C-13), 114.0 (t, C-12), 77.7 (s, C-18), 76.4
(d, C-21), 69.7 (d, C-7), 54.2 (s, C-9), 53.5 (d, C-3), 50.0 (d, C-4),
45.6 (d, C-8), 45.3 (t, C-10), 42.3 (d, C-16), 37.7 (t, C-15), 32.9
(d, C-5), 24.2 (q, C-23), 19.4 (q, C-22), 13.9 (q, C-11); EIMS
m/z 465 [M]+ (5), 437 (15), 419 (13), 365 (36), 338 (100), 254
In a microdrop bioassay of rice seedlings (Oryza sative
L. cv. Yamahikari), the administration of 1 nmol/plant of
3 (zygosporin D) reduced the second leaf sheath length of
treated plants to 41% of that of the water control. It is
noteworthy that 3 had this effect without doing damage to
the plant cells. The same amount of 4 (deacetylcytochalasin
C) and 5 produced, respectively, 83 and 94% growth in
terms of the second leaf sheath length of treated plants
compared to the control.
(58), 120 (18), 91 (53); HREIMS m/z 465.2500 (calcd for C28H35
-
NO5, 465.2515).
Acetylcytoch a la sin D (6). Compound 3 (3.2 mg) was
treated overnight with 0.5 mL of acetic anhydride and 0.5 mL
of pyridine. The products were purified by HPLC (85% MeOH,
1.0 mL/min), giving 6 (2.6 mg, tR 16.1 min) as a white powder.
Com p ou n d 6: [R]25 +30.2° (c 1.00, EtOH); IR (film) νmax
D
3451 (OH, NH), 1736 (acetyl CO), 1699 (ketone CO and amide
CO), 1229 cm-1 1H NMR (CDCl3, 400 MHz) δ 7.4-7.1 (5H,
;
m, H-2′-6′), 6.09 (1H, dd, J ) 15.6, 2.7 Hz, H-20), 5.66 (1H,
dd, J ) 2.7, 2.4 Hz, H-21), 5.60 (1H, s, NH), 5.57 (1H, dd, J )
15.9, 9.8 Hz, H-13), 5.24 (1H, s, H-12), 5.19 (1H, d, J ) 10.2
Hz, H-7), 5.18 (1H, m, H-14), 5.11 (1H, dd, J ) 15.6, 2.4 Hz,
H-19), 5.03 (1H, s, H-12), 3.23 (1H, m, H-3), 3.07 (1H, dd, J )
10.2, 9.8 Hz, H-8), 2.83-2.66 (4H, m, H-5, -10, -16), 2.44 (1H,
ddd, J ) 13.2, 11.3, 10.2 Hz, H-15), 2.28 (3H, s, 21-OAc), 2.15
(1H, m, H-4), 1.93 (1H, m, H-15), 1.92 (3H, s, 7-OAc), 1.49 (3H,
s, H-23), 1.17 (3H, d, J ) 6.8 Hz, H-22), 0.91 (3H, d, J ) 6.6
Hz, H-11); EIMS m/z 549 [M]+ (14), 521 (76), 458 (67), 401
(59), 358 (61), 310 (66), 254 (100), 120 (40), 91 (93).
Cytochalasin D (2) (5 mg, Sigma, from Zygosporium man-
sonii) was subjected to the procedure used for 3, giving 6 (4.6
mg).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. IR spectra were
recorded on a J ASCO FT-IR-5300 spectrometer. Optical
rotation was measured with a Horiba SEPA-200 high sensitive
polarimeter. EIMS were obtained with a J EOL AX505HA
spectrometer (direct probe, 70 eV). NMR spectra were mea-
sured with a J EOL Lambda 400 or a J EOL GX 270 NMR
spectrometer. Chemical shifts were referenced against CDCl3
(δH 7.26, δC 77.20) and DMSO-d6 (δC 39.60). Preparative HPLC
was performed on a Cosmosil 5C18-AR column (Nacalai Tesque,
10 × 250 mm), monitoring at 220 nm.
F u n ga l Ma ter ia l. A fungus (A-173) isolated from a soil
sample collected in Tottori Prefecture, J apan, was identified
as Metarrhizium anisopliae (Metchn.) Sorok. 1883, based on
Dea cetylcytoch a la sin C (4): [R]25D +27.6° (c 0.25, EtOH);
IR (film) νmax 3434 (OH, NH), 1692 (ketone CO and amide CO),
1644 (CdC) cm-1; 1H NMR (CDCl3, 270 MHz) δ 7.37-7.11 (5H,

134 J ournal of Natural Products, 2000, Vol. 63, No. 1
Notes
m, H-2′-6′), 6.15 (1H, dd, J ) 15.4, 2.4 Hz, H-20), 5.89 (1H,
dd, J ) 16.2, 10.3 Hz, H-13), 5.64 (1H, s, NH), 5.46 (1H, dd, J
) 15.4, 2.4 Hz, H-19), 5.26 (1H, ddd, J ) 16.2, 10.3, 5.4 Hz,
H-14), 4.51 (1H, m, H-21), 3.78 (1H, br d, J ) 9.7 Hz, H-7),
3.35 (1H, m, H-3), 3.00 (1H, br s, H-4), 2.96 (1H, dd, J ) 13.5,
9.6 Hz, H-10), 2.81 (1H, dd, J ) 13.5, 5.7 Hz, H-10), 2.72 (1H,
m, H-16), 2.52 (1H, ddd, J ) 12.8, 11.0, 10.3 Hz, H-15), 2.40
(1H, dd, J ) 10.3, 9.7 Hz, H-8), 2.00 (1H, br dd, J ) 12.8, 5.4
Hz, H-15), 1.69 (3H, s, H-12), 1.55 (3H, s, H-23), 1.54 (3H, s,
H-11), 1.20 (3H, d, J ) 7.0 Hz, H-22); 13C NMR (DMSO-d6,
67.8 MHz) δ 210.0 (s, C-17), 176.0 (s, C-1), 138.1 (d, C-20),
138.1 (s, C-1′), 132.6 (s, C-6), 131.7 (d, C-19), 131.0 (d, C-14),
129.6 (d, C-2′, -6′), 128.4 (d, C-3′, -5′), 126.5 (d, C-4′), 126.4 (d,
C-13), 126.2 (s, C-5), 77.6 (s, C-18), 73.2 (d, C-21), 68.2 (d, C-7),
59.6 (d, C-3), 54.1 (s, C-9), 48.5 (d, C-4), 48.0 (d, C-8), 44.5 (t,
C-10), 41.5 (d, C-16), 38.1 (t, C-15), 24.9 (q, C-23), 19.5 (q, C-22),
16.6 (q, C-12), 14.3 (q, C-11); EIMS m/z 465 [M]+ (12), 437 (18),
419 (20), 365 (60), 338 (100), 320 (87), 256 (70), 120 (52), 91
(45); HREIMS m/z 465.2522 (calcd for C28H35NO5, 465.2515).
Acetylcytoch a la sin C (7). Compound 4 (2.0 mg) was
acetylated by the procedure used for 3. The products were
purified by HPLC (85% MeOH, 1.0 mL/min) giving 7 (1.8 mg,
tR 17.8 min) as colorless needles.
The reaction mixture was filtered and evaporated in vacuo.
The products were separated by HPLC (65% MeOH, 0.8 mL/
min) giving compounds 8 (4.0 mg, tR 49.9 min) and 9 (3.7 mg,
tR 48.3 min).
Com p ou n d 8: [R]25 +13.0° (c 0.20, EtOH); IR (film) νmax
D
3362 (OH, NH), 1744 (acetyl CO), 1692 (ketone CO and amide
CO), 1028 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.36-7.12 (5H,
m, H-2′-6′), 6.13 (1H, dd, J ) 15.9, 2.7 Hz, H-20), 5.65 (1H,
dd, J ) 15.7, 10.3 Hz, H-13), 5.62 (1H, dd, J ) 2.7, 2.2 Hz,
H-21), 5.52 (1H, s, NH), 5.27 (1H, ddd, J ) 15.7, 11.1, 5.4 Hz,
H-14), 5.13 (1H, dd, J ) 15.9, 2.2 Hz, H-19), 4.64 (1H, s, OH),
3.55-3.45 (2H, m, H-3, 7), 2.91 (1H, dd, J ) 13.5, 4.1 Hz,
H-10), 2.73 (1H, m, H-16), 2.65 (1H, dd, J ) 13.5, 9.5 Hz, H-10),
2.63 (1H, dd, J ) 10.3, 9.7 Hz, H-8), 2.49 (1H, ddd, J ) 12.4,
11.8, 11.1 Hz, H-15), 2.24 (3H, s, 21-OAc), 2.06 (1H, dd, J )
4.3, 3.8 Hz, H-4), 2.00 (1H, br dd, J ) 12.4, 5.4 Hz, H-15),
1.77-1.63 (2H, m, H-5, 6), 1.50 (3H, s, H-23), 1.19 (3H, d, J )
7.0 Hz, H-22), 0.93 (3H, d, J ) 6.2 Hz, H-12), 0.88 (3H, d, J )
6.2 Hz, H-11); 13C NMR (CDCl3, 100 MHz) δ 210.3, 174.4,
169.6, 137.5, 133.9, 132.7, 131.1, 129.03, 128.96, 127.4, 127.1,
77.7, 77.2, 68.5, 53.8, 53.7, 50.9, 46.1, 45.4, 42.4, 37.7, 34.7,
33.3, 24.2, 20.8, 19.4, 16.1, 12.6; EIMS m/z 509 [M]+ (4), 481
(39), 449 (16), 422 (25), 421 (44), 379 (20), 378 (43), 360 (24),
340 (67), 330 (38), 323 (33), 322 (30), 312 (29), 256 (30), 254
(29), 120 (37), 91 (100).
Com p ou n d 7: [R]25 +56.0° (c 0.25, EtOH); IR (film) νmax
D
3362 (OH, NH), 1742 (acetyl CO), 1698 (ketone CO and amide
CO), 1231, 1028 cm-1; H NMR (CDCl3, 270 MHz) δ 7.4-7.1
Com p ou n d 9: [R]25 -22.0° (c 0.10, EtOH); IR (film) νmax
1
D
(5H, m, H-2′-6′), 6.00 (1H, dd, J ) 15.1, 2.2 Hz, H-20), 5.92
(1H, dd, J ) 2.2, 2.2 Hz, H-21), 5.76 (1H, dd, J ) 15.7, 10.8
Hz, H-13), 5.60 (1H, s, NH), 5.30 (1H, br d, J ) 7.6 Hz, H-7),
5.16 (1H, m, H-14), 5.12 (1H, dd, J ) 15.1, 2.2 Hz, H-19), 4.66
(1H, s, 18-OH), 3.33 (1H, t, J ) 7.6 Hz, H-3), 2.97 (2H, d, J )
7.6 Hz, H-10), 2.78-2.38 (4H, m, H-4, 8, 15, 16), 2.28 (3H, s,
21-OAc), 1.94 (3H, s, 7-OAc), 1.49 (3H, s, H-23), 1.49 (3H, br
s, H-11 or -12), 1.44 (3H, br s, H-11 or -12), 1.17 (3H, d, J )
7.6 Hz, H-22); EIMS m/z 549 [M]+ (11), 521 (60), 489 (78), 458
(84), 429 (42), 401 (54), 358 (55), 338 (62), 320 (100), 310 (80),
240 (64), 228 (63), 120 (100), 91 (76).
3387 (OH, NH), 1738 (acetyl CO), 1684 (ketone CO and amide
CO), 1018 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.34-7.09 (5H,
m, H-2′-6′), 6.13 (1H, dd, J ) 15.9, 2.7 Hz, H-20), 5.67 (1H,
dd, J ) 15.6, 9.8 Hz, H-13), 5.60 (1H, dd, J ) 2.7, 2.4 Hz, H-21),
5.56 (1H, s, NH), 5.30 (1H, ddd, J ) 15.6, 10.7, 5.1 Hz, H-14),
5.12 (1H, dd, J ) 15.9, 2.4 Hz, H-19), 4.64 (1H, s, OH), 3.61
(1H, m, H-3), 3.07 (1H, dd, J ) 11.2, 5.6 Hz, H-7), 2.82 (1H,
dd, J ) 13.4, 4.9 Hz, H-10), 2.76-2.66 (2H, m, H-8, -16), 2.61
(1H, dd, J ) 13.4, 9.3 Hz, H-10), 2.50 (1H, ddd, J ) 12.7, 11.2,
11.0 Hz, H-15), 2.22 (3H, s, 21-OAc), 2.16 (1H, m, H-5), 2.04-
1.96 (2H, m, H-4, -15), 1.84 (1H, s, OH), 1.72 (1H, m, H-6),
1.50 (3H, s, H-23), 1.18 (3H, d, J ) 6.8 Hz, H-22), 1.01 (3H, d,
J ) 7.6 Hz, H-12), 0.87 (3H, d, J ) 7.3 Hz, H-11); 13C NMR
(CDCl3, 100 MHz) δ 210.3, 174.5, 169.6, 137.3, 134.8, 132.8,
130.2, 129.03, 128.97, 127.4, 127.1, 77.6, 77.4, 74.0, 54.4, 53.9,
50.5, 47.0, 46.2, 42.4, 39.6, 37.8, 30.2, 24.2, 20.8, 19.4, 16.1,
15.1; EIMS m/z 509 [M]+ (5), 481 (39), 449 (16), 422 (29), 421
(44), 406 (22), 390 (21), 378 (42), 360 (21), 341 (32), 340 (96),
330 (42), 322 (25), 312 (24), 272 (21), 256 (24), 254 (25), 120
(35), 91 (100).
Cytochalasin C (1) (5 mg, Sigma, from Metarrhizium
anisopliae) was subjected to the procedure used for 3, giving
7 (3.3 mg).
Com p ou n d 5: [R]25 -29.5° (c 0.2, EtOH); IR (film) νmax
D
3387 (OH, NH), 1705 (ketone CO), 1695 (ketone and amide
CO), 1032 cm-1 1H NMR (CDCl3, 400 MHz) δ 7.32 (2H, m,
;
H-3′, -5′), 7.26 (1H, m, H-4′), 7.13 (2H, m, H-2′, -6′), 6.20 (1H,
dd, J ) 15.9, 2.4 Hz, H-20) 5.69 (1H, br s, NH), 5.66 (1H, ddd,
J ) 15.6, 9.5, 1.5 Hz, H-13), 5.40 (1H, dd, J ) 15.9, 2.2 Hz,
H-19), 5.05 (1H, ddd, J ) 15.6, 10.8, 4.6 Hz, H-14), 4.08 (1H,
br s, H-21), 3.79 (1H, d, J ) 9.5 Hz, H-8), 3.58 (1H, ddd, J )
9.0, 4.2, 3.4 Hz, H-3), 2.99 (1H, dd, J ) 13.4, 3.4 Hz, H-10),
2.70 (1H, ddq, J ) 11.2, 1.5, 6.8 Hz, H-16), 2.61 (1H, dd, J )
4.9, 4.2 Hz, H-4), 2.57 (1H, dd, J ) 13.4, 9.0 Hz, H-10), 2.48
(1H, ddd, J ) 13.0, 11.2, 10.8 Hz, H-15), 2.32 (1H, m, H-5),
2.03-1.95 (2H, m, H-6, -15), 1.52 (3H, s, H-23), 1.17 (3H, d, J
) 6.8 Hz, H-22), 1.14 (3H, d, J ) 7.1 Hz, H-12), 1.08 (3H, d, J
) 7.1 Hz, H-11); 13C NMR (CDCl3, 100 MHz) δ 214.2 (s, C-7),
210.4 (s, C-17), 173.8 (s, C-1), 136.8 (d, C-20), 136.7 (s, C-1′),
133.3 (d, C-14), 129.3 (d, C-2′, -6′), 129.1 (d, C-3′, -5′), 127.3
(d, C-4′), 127.2 (d, C-19), 126.9 (d, C-13), 77.6 (s, C-18), 76.5
(d, C-21), 55.6 (s, C-9), 53.2 (d, C-3), 51.0 (d, C-4), 50.4 (d, C-8),
45.8 (d, C-6), 45.8 (t, C-10), 42.3 (d, C-16), 38.1 (t, C-15), 35.6
(d, C-5), 24.2 (q, C-23), 19.3 (q, C-22), 15.9 (q, C-11 or -12),
15.8 (q, C-11 or -12); long-range correlations in the HMBC
spectrum (optimized for J C,H of 7 Hz) C-1 (H-8), C-3 (NH, H-10),
C-4 (H-11), C-5 (H-11, H-12), C-6 (H-11, H-12), C-7 (H-6, H-8,
H-12), C-8 (H-21), C-9 (NH, H-8, H-21), C-10 (H-2′, H-6′), C-11
(H-6), C-12 (H-6), C-13 (H-8, H-15), C-14 (H-8, H-15), C-15 (H-
13, H-22), C-16 (H-22), C-17 (H-15, H-22, H-23), C-18 (H-19,
H-20, H-23), C-19 (H-20, H-21, H-23), C-20 (H-19), C-21 (H-
19, H-20), C-23 (H-19), C-1′ (H-10), C-2′, -6′ (H-10); EIMS m/z
465 [M]+ (12), 447 (14), 437 (37), 419 (20), 374 (37), 365 (60),
338 (100), 254(33), 190 (18), 120 (20), 91 (48); HREIMS m/z
465.2500 (calcd for C28H35NO5, 465.2515).
Com p ou n d 10. Compound 8 (3.3 mg) in CH2Cl2 (2 mL) was
added dropwise to the solution of PCC (8.7 mg) in CH2Cl2 (0.21
mL), and the whole was stirred for 1 h. Diethyl ether (2 mL)
and anhydrous Mg2SO4 (5 g) then were added to the reaction
mixture, which was stirred for 10 min. After filtering the
mixture, the filtrate was concentrated in vacuo and purified
by HPLC (65% MeOH, 0.8 mL/min), giving compound 10 (2.1
mg, tR 51.0 min).
Com p ou n d 10: [R]25 -43.9° (c 0.5, EtOH); IR (film) νmax
D
3431 (OH, NH), 1741 (acetyl CO), 1711 (ketone CO), 1700
(amide CO, sh), 1225 cm-1; 1H NMR (CDCl3, 270 MHz) δ 7.36-
7.11 (5H, m, H-2′-6′), 6.13 (1H, dd, J ) 15.6, 2.7 Hz, H-20),
5.72 (1H, dd, J ) 15.4, 9.7 Hz, H-13), 5.68 (1H, dd, J ) 2.7,
2.4 Hz, H-21), 5.52 (1H, s, NH), 5.15 (1H, dd, J ) 15.6, 2.4
Hz, H-19), 5.11 (1H, ddd, J ) 15.4, 11.0, 4.8 Hz, H-14), 4.62
(1H, br s, OH), 3.71 (1H, d, J ) 9.7 Hz, H-8), 3.53 (1H, m,
H-3), 2.92 (1H, dd, J ) 13.4, 4.1 Hz, H-10), 2.78-2.61 (3H, m,
H-4, -10, -16), 2.53 (1H, ddd, J ) 12.7, 11.2, 11.0 Hz, H-15),
2.30 (3H, s, 21-OAc), 2.10-1.95 (3H, m, H-5, -6, -15), 1.78 (1H,
s, OH), 1.49 (3H, s, H-23), 1.19 (3H, d, J ) 6.8 Hz, H-22), 1.11
(3H, d, J ) 7.0 Hz, H-12), 0.97 (3H, d, J ) 6.5 Hz, H-11); EIMS
m/z 507 [M]+ (3), 447 (21), 419 (25), 404 (39), 376 (42), 338
(100), 328 (20), 282 (22), 254 (26), 190 (43), 120 (20), 91 (44).
Compound 5 (1.4 mg) was acetylated by the procedure used
for 3, giving 10 (1.5 mg).
Micr od r op Bioa ssa y. Seeds of rice (Oryza sativa L. cv.
Yamahikari) were soaked in H2O for 48 h at 28° under
fluorescent light. Sets of six germinated seeds were planted
on 1% agar medium in 30-mL beakers and incubated for 48 h
Com p ou n d s 8 a n d 9. Cytochalasin D (2) (10 mg, Sigma,
from Zygosporium mansonii) in EtOAc (3 mL) containing PtO2
(2 mg) was stirred under H2 at room temperature for 30 min.

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 1 135
Refer en ces a n d Notes
under continuous light. A 1-µL sample containing 50% aqueous
acetone was placed as a drop between the shoot and first leaf
of a seedling. After incubation for 72 h under continuous light,
the length of the second leaf sheath of the seedling was
measured.
(1) Fujii, Y.; Fukuda, A.; Hamasaki, T.; Ichimoto, I.; Nakajima, H.
Phytochemistry 1995, 40, 1443-1446.
(2) Domsch, K. H.; Gams, W.; Anderson, T.-H. Compendium of Soil Fungi;
Academic Press: London, 1980; pp 413-415.
(3) Aldridge, D. C.; Turner, W. B. J . Chem. Soc. (C) 1969, 923-928.
(4) Suzuki, A.; Tamura, S. Agric. Biol. Chem. 1972, 36, 896-898.
(5) Minato, H.; Katayama, T. J . Chem. Soc. (C) 1970, 45-47.
(6) Binder, M.; Tamm, C. Helv. Chim. Acta 1973, 56, 2387-2396.
(7) Natori, S.; Yahara, I. In Mycotoxins and Phytoalexins; Sharma, R.
P., Salunkhe, D. K., Eds.; CRC Press: Boca Raton, FL, 1991; pp
291-336.
Ack n ow led gm en t. We are grateful to Dr. Yukihiro Sug-
imoto, Arid Land Research Center, of this university for his
assistance in measuring NMR. This work was supported in
part by a Grant-in-Aid for Scientific Research to H.N. from
the Ministry of Education, Science and Culture of J apan (no.
09660118).
NP990331T