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Isolation of aspirochlorine (=antibiotic A30641) possessing a novel dithiodiketopiperazine structure from Aspergillus Flavus
Tetrahedron Letters,Vo1.23,No.20,pp Printed in Great Britain
ISOLATION
(=ANTIBIOTIC A306411 POSSESSING A
0~ AsPIROCHLORINE
NOVEL DITHIODIKETOPIPERAZINE
Kanzo Sakata,*
Hajime
The Institute
A very unusual
CgH12N205S2C1, gillus
produced
flavus, which
of Agriculture,
been isolated
aspirochlorine
filtrate
has been identified
Penicillium
structure
active
(sh., 5600); Ai!loMe
Japan.
University,
together
phytopathogenic flavus. 2)
as canadensolide
(~=0.33, MeOH);
AlET
fungi,
Phytophthora
from culture
CllH14
broth of
principle
nm (E) 250 (sh., 4800),
360 (M+, C12HgN205S2C1), 5, 296 (M-S 2, C12HgN205S2C1),
spp. have
The less polar compound,
The polar active
nm (E) 263 (sh., 7600), 313 (8000); vmax
by Asper-
A 30641 from -~ A. tamarii.
which was isolated
NRRL 8101. 4)
to aspiroclorine,
with canadensolide
with the antibiotic
of Aspergillus
canadense 3) and -~ A. tamarii
(1): [a];' t66.7"
Takahashi
Wako-shi,
(1) has been assigned
substance
principles 1) against
from the culture
04, [a];' -1B2", fungi,
active
FLAVUS
Kyoto 606, Japan.
has been found to be identical
Two bilogically
and Nobutaka
Research,
Kyoto Prefectural
dithiodiketopiperazine as a biologically
FROM ASPERGILLUS
Akira Sakurai
and Chemical
Sakyo-ku, Sumnary:
STRUCTURE
Masago,#
of Physical
# Faculty
004o-4039/82/202095-04$03.00/O 01982 Pergamon Press Ltd.
2095-2098,1982
named
298 (6300), 305
(CHC13) 1726, 1622, 1604 cm-'; m/l
265, 241, 221, 210, 209, 182, 181; G(CDC13)
3.96 (3H, s), 4.89 (lH, d, 1.2), 5.15 (lH, d, 4.6), 5.97 (lH, br.), 6.78 (lH, s), 7.14 (lH, d, 1.2), 7.30 (lH, br.); was concluded 30641 isolated However, signals tertiary
[8]&
by comparison
+17,000,
[8]24g -23,000,
of their spectroscopic
from -A. tamarii
NRRL 8101,4'6)
bridgehead
have been reported8)
carbon
assigned
(C-8).
7)
13
except
for a signal
C NMR spectra
of Agricultural 422, Japan.
the structure
by the structure
carbon
A
6) All the
2.
(6 102.6) which must be ascribed
of a few epidithiodiketopiperazine
Chemistry,
2095
with the antibiotic
2. had been assigned.
could not be explained
and the signal of the particular
* Present address: Department versity, 836 Ohya, Shizuoka
[8]280.5 f0, [8]303 +ll,OOO,
data to be identical
to which
the 13C NMR data of 1 we obtained
(Fig.) were reasonably
[8]26g +2900,
is observed
to the
compounds
in a region 6 74-78.
Faculty of Agriculture,
Shizuoka
Uni-
The
2096
H
0
1: R=H
_
3: R = AC
154.1 165.0 7
165.7
200
180
160
140
120
100
Fig. C-13 NMR Spectrum difference
of the chemical
This fact together has
Coloration
Acetylation
which
suggests In
that the structure
of j_ indicated
conditions
the presence
anhydride
product
of desulfurized
the presence
of lwere
hydroxyl
methine
structure;C$
(X=0 or N).
spectroscopic chlorine.
data described
The following
(6 5.15, d) shifted
carbon signal Considering
signal
downfield
on 1 support
derivative
shift (15
(3): m/z -- 444
Amino acid analysis aromatic
of the
amino acid,
(6 3.96) and two olefinic
to a methine
proton
to 6 6.21 changing
(6 102.6) should be assigned biosynthesis
in
in 1.
is coupled
above allow us to propose
reactions
must be revised.
The bathochromic
and an unidentified
a methoxyl
Another
The tertiary
linkage.
9)
group in 1.
structure
(8 6.78, s and 7.14, d, 1.2), one of which
acetylation.
of the i-0 linkage.
principle
gave an N,&-diacetyl
1 gave glycine
observed
0
(1725 cm-') for the amide carbonyl
2.37, 2.72 (3H each s).
signals
proton signal
by the effect
for theactive
of a disulfide
of a diketopiperazine
the 'H NMR spectrum
20
with the ions of -m/z 256 (M-S2) and 64 (S2)")
and pyridine
380 (M-S2); G(CDC13)
40
MHz, CDC13, PND).
absorption
-2 proposed
showed a phenolic
with acetic
(M+, C16H13N207S2Cl), acid hydrolysis
high carbonyl
with a AgN03 reagent lo) together
the mass spectrum nm) in alkaline
of l(ZZ.53
60
shift is too big to be rationalized
with the extremely
led us to conclude
80
the structure.
(8 4.89, d, 1.2).
into singlet
on
to a carbon with a partial
of a diketopiperazine a quite unusual
proton
compound
structure
and all the
1 for
aSpirO-
2097
NaOMe/MeOH
6 or _7 _
Treatment methyl
of 1 with ethereal
(5) derivatives.
diazomethane
Q: main product;
in MeOH yielded
an Tj,c-dimethyl (4) and an -O,O-di-
m/l 388 (M+, C14H,3N205S2Cl),
6 (COC13) 3.12, 3.92, 3.97 (3H each, s), 4.92
(lH, d, 1.2), 5.00, 6.72 (1H each, s), 7.16 (lH, d,
5; minor
1.2); A:tiH nm 259 (sh.), 297, 305 (sh.).
324 (M-S2, C14H13N205Cl);
product,
less polar; IIJ/~388 (M+), 324 (M-S2);
6 (CDC13) 3.92, 3.93, 3.96 (3H each, s), 4.69 (lH, d, 1.2), 5.26, 6.63 (1H each, s), 7.17 (lH, d, The three Q-methyl
1.2).
4 support implies
signals
and the lower chemical
the enol ether structure
that no change occurred
One-pot two isomers
reaction (5 and 7).
The CD spectrum
5.
of 1 with Me1 in pyridine
librium mixture
(6
ed by participation
by SBH reduction
and the carbonyl
absorption
of the disulfide
of the lone pair electons which
12) gave
371 (M-SMe); 6 (CDC13) 2.11,
1.85, 2.43, 3.18, 3.88, 3.90 (3H each,
linkage.
: 1 i 1 : 1.3) in MeOH at r.t. overnight.
to form an ionic intermediate
in MeOH
(lH, s), 5.10 (lH, d, 1.2), 6.55 (lH, s), 7.22 (lH, d,
(lH, br. s), 6.61 (lH, s), 7.23 (lH, d, 1.2).
opening with methylation
to that of 1, which
of j_ on this methylation.
387 (M-OMe),
371 (M-SMe); G(CDC13)
and 2.19 in 6 and 1.85 and 2.43 in 1 show reductive
at r.t. followed
6: m/z 418 (M+, C16HlgN205S2Cl),
7: m/g 418 (M+), 387 (M-OMe),
s), 4.71 (lH, s), 4.77
of 4 is very similar
in the frame work of the structure
2.19, 3.10. 3.87, 3.94 (3H each, s), 4.87 1.2).
shift of H-11 (6 5.26) of 5 than that of
results
on the nitrogen
in liberation
Two z-methyl
(1691 cm-')')
carrying
reachesto
isomerization
a S-methyl
of an anomer at C-8.
(6 2.11
of the mixture
Each compound
This facile
signals
an equi-
is explain-
group on it
2098
Reaction
of the mixture
(M+, C17H23N206S2C1), The bathochromic hydroxyl
in 5.
compounds
(23 nm) in alkaline
The nucleophilic surprising
naturally.
with a similar
conditions
attack of methoxide
that a compound
partial
FT-NMR measurements. measurements
adduct
(8):
nm 230 (sh.), 295; hi!$oMe
strongly
suggests
IJ/~ 450 318 nm.
the presence
of a phenolic
anion on C-8 produced 8.
13)
as thiosulfenic
are, to our knowledge,
known synthetic
structure.
of the structure
of lwill
be published
We thank Prof. S. Abe of Kyoto Prefectural
discussions.
a methanol
with such a labile functionarity
of the fungus and Prof. T. Oishi of the Institute helpful
i:iF
E-(Thiosulfenyl)phthalimides
Further comfirmation Acknowledgements.
in MeOH yielded
403 (M-SMe), 371 (M-MeOH-SMe);
shift
It is rather amide occurrs
6 and 1 with NaOMe
We are greatful
of Physical
to Drs. S. Takeuchi
We are also indebted
elsewhere.
University
for the identification
and Chemical
Research
(IPCR) for
and Y. Kohno of IPCR for their help for
to Mr. Y. Esumi of IPCR for high resolution
and to Miss H. Tanaka of IPCR for her assistance
for the fermentation
mass
of the fungus.
References
1)
Aspirochlorine (1) has different activity from that of canadensolide. 1 shows not only growth inhibition of mycelium but also enormous numbers of spherical swellings on the hyphae of P&&hthora cinnamomi (A mating type). Isolation and the biological activity of lwill be pub+--lia-* 1s e ln detail els &where.
2)
This fungus was isolated from the air and identified Agriculture, Kyoto Prefectural University, Sakyo-ku,
by Prof. Shigeo Abe of Faculty Kyoto 606, Japan.
of
Wright, P.W. Brian, S.M. Clarke and S.A. Hutchinson, 3) N.J. McCorkinkale, J.L.C. 727 (1968); M. Kato, R. Tanaka and A. Yoshikoshi, Chem. Comn., 1561 (1971).
Tet. Lett.,
4) D.H. Berg, R.P. Massing,
22, 394 (1976).
5) The formulae
M.M. Hoehn,
L.D. Boeck and R.L. Hamill,
of the ions in this paper were
6) D.H. Berg, R.L. Hamill (1976); idem., ibid.,
confirmed
J. Antibiot.,
by high resolution
mass spectrometry.
and M.M. Hoehn, GA., 86, 418029 (1977): U. S. Patent, 87671s (1977): U. S.-patent, 4,001,086 (1977).
3,991,052
gj,
7) An approximate calculation of the chemical shifts of aromatic carbons was made based on the assumption that the substituents at C-l and C-6 are equivalent to methoxyl and ethyl groups, respectively, using a table in the text: E. Breitmair and W. Voelter, "Methods and Application in Organic Chemistry, Vol. 5,C-13 NMR Spectroscopy Monographs in Modern Chemistry," Verlag Chemie Weinheim, New York, 1978, p. 213. 8) J.W. Ronaldson, Aust. J. Chem., ?S, 2307 (1976); D. Brewer, A.G. McInnes, D.G. Smith, A. Taylor and J.A Walter, J. Chem. Sot., Perkin I, 1248 (1978); G. Deffeux, M.A. Baute, R. Baute and M.J. Filleau, J. Antibiot., 31, 1102 (1978). 9) K.H. Michel, M.O. Channey, N.D. Jones, M.M. Hoehn and R. Nagarajan, S. Safe and A. Taylor, J. Chem. Sot. (C), 1189 (1971). 10) K.C. Murdock,
J. Antibiot.,
J. Med. Chem., iz, 827 (1974).
11) H. Budzikiewicz, C. Djerassi and D.H. Williams, "Structure Elucidation Mass Spectrometry," Holden Day, San Francisco, 1964, Vol. 2, p. 249. 12) W.D. Jamison, 13) A.B. Sulivan
02, 57 (1974);
R. Rahman and A. Taylor, and K. Boetany,
J. Chem. Sot. (C), 1564 (1969).
Int. J. Sulfur
(Received in Japan 11 January 1982)
Chem., A, 1, 207 (1971).
of Natural
Products
by
ISOLATION
(=ANTIBIOTIC A306411 POSSESSING A
0~ AsPIROCHLORINE
NOVEL DITHIODIKETOPIPERAZINE
Kanzo Sakata,*
Hajime
The Institute
A very unusual
CgH12N205S2C1, gillus
produced
flavus, which
of Agriculture,
been isolated
aspirochlorine
filtrate
has been identified
Penicillium
structure
active
(sh., 5600); Ai!loMe
Japan.
University,
together
phytopathogenic flavus. 2)
as canadensolide
(~=0.33, MeOH);
AlET
fungi,
Phytophthora
from culture
CllH14
broth of
principle
nm (E) 250 (sh., 4800),
360 (M+, C12HgN205S2C1), 5, 296 (M-S 2, C12HgN205S2C1),
spp. have
The less polar compound,
The polar active
nm (E) 263 (sh., 7600), 313 (8000); vmax
by Asper-
A 30641 from -~ A. tamarii.
which was isolated
NRRL 8101. 4)
to aspiroclorine,
with canadensolide
with the antibiotic
of Aspergillus
canadense 3) and -~ A. tamarii
(1): [a];' t66.7"
Takahashi
Wako-shi,
(1) has been assigned
substance
principles 1) against
from the culture
04, [a];' -1B2", fungi,
active
FLAVUS
Kyoto 606, Japan.
has been found to be identical
Two bilogically
and Nobutaka
Research,
Kyoto Prefectural
dithiodiketopiperazine as a biologically
FROM ASPERGILLUS
Akira Sakurai
and Chemical
Sakyo-ku, Sumnary:
STRUCTURE
Masago,#
of Physical
# Faculty
004o-4039/82/202095-04$03.00/O 01982 Pergamon Press Ltd.
2095-2098,1982
named
298 (6300), 305
(CHC13) 1726, 1622, 1604 cm-'; m/l
265, 241, 221, 210, 209, 182, 181; G(CDC13)
3.96 (3H, s), 4.89 (lH, d, 1.2), 5.15 (lH, d, 4.6), 5.97 (lH, br.), 6.78 (lH, s), 7.14 (lH, d, 1.2), 7.30 (lH, br.); was concluded 30641 isolated However, signals tertiary
[8]&
by comparison
+17,000,
[8]24g -23,000,
of their spectroscopic
from -A. tamarii
NRRL 8101,4'6)
bridgehead
have been reported8)
carbon
assigned
(C-8).
7)
13
except
for a signal
C NMR spectra
of Agricultural 422, Japan.
the structure
by the structure
carbon
A
6) All the
2.
(6 102.6) which must be ascribed
of a few epidithiodiketopiperazine
Chemistry,
2095
with the antibiotic
2. had been assigned.
could not be explained
and the signal of the particular
* Present address: Department versity, 836 Ohya, Shizuoka
[8]280.5 f0, [8]303 +ll,OOO,
data to be identical
to which
the 13C NMR data of 1 we obtained
(Fig.) were reasonably
[8]26g +2900,
is observed
to the
compounds
in a region 6 74-78.
Faculty of Agriculture,
Shizuoka
Uni-
The
2096
H
0
1: R=H
_
3: R = AC
154.1 165.0 7
165.7
200
180
160
140
120
100
Fig. C-13 NMR Spectrum difference
of the chemical
This fact together has
Coloration
Acetylation
which
suggests In
that the structure
of j_ indicated
conditions
the presence
anhydride
product
of desulfurized
the presence
of lwere
hydroxyl
methine
structure;C$
(X=0 or N).
spectroscopic chlorine.
data described
The following
(6 5.15, d) shifted
carbon signal Considering
signal
downfield
on 1 support
derivative
shift (15
(3): m/z -- 444
Amino acid analysis aromatic
of the
amino acid,
(6 3.96) and two olefinic
to a methine
proton
to 6 6.21 changing
(6 102.6) should be assigned biosynthesis
in
in 1.
is coupled
above allow us to propose
reactions
must be revised.
The bathochromic
and an unidentified
a methoxyl
Another
The tertiary
linkage.
9)
group in 1.
structure
(8 6.78, s and 7.14, d, 1.2), one of which
acetylation.
of the i-0 linkage.
principle
gave an N,&-diacetyl
1 gave glycine
observed
0
(1725 cm-') for the amide carbonyl
2.37, 2.72 (3H each s).
signals
proton signal
by the effect
for theactive
of a disulfide
of a diketopiperazine
the 'H NMR spectrum
20
with the ions of -m/z 256 (M-S2) and 64 (S2)")
and pyridine
380 (M-S2); G(CDC13)
40
MHz, CDC13, PND).
absorption
-2 proposed
showed a phenolic
with acetic
(M+, C16H13N207S2Cl), acid hydrolysis
high carbonyl
with a AgN03 reagent lo) together
the mass spectrum nm) in alkaline
of l(ZZ.53
60
shift is too big to be rationalized
with the extremely
led us to conclude
80
the structure.
(8 4.89, d, 1.2).
into singlet
on
to a carbon with a partial
of a diketopiperazine a quite unusual
proton
compound
structure
and all the
1 for
aSpirO-
2097
NaOMe/MeOH
6 or _7 _
Treatment methyl
of 1 with ethereal
(5) derivatives.
diazomethane
Q: main product;
in MeOH yielded
an Tj,c-dimethyl (4) and an -O,O-di-
m/l 388 (M+, C14H,3N205S2Cl),
6 (COC13) 3.12, 3.92, 3.97 (3H each, s), 4.92
(lH, d, 1.2), 5.00, 6.72 (1H each, s), 7.16 (lH, d,
5; minor
1.2); A:tiH nm 259 (sh.), 297, 305 (sh.).
324 (M-S2, C14H13N205Cl);
product,
less polar; IIJ/~388 (M+), 324 (M-S2);
6 (CDC13) 3.92, 3.93, 3.96 (3H each, s), 4.69 (lH, d, 1.2), 5.26, 6.63 (1H each, s), 7.17 (lH, d, The three Q-methyl
1.2).
4 support implies
signals
and the lower chemical
the enol ether structure
that no change occurred
One-pot two isomers
reaction (5 and 7).
The CD spectrum
5.
of 1 with Me1 in pyridine
librium mixture
(6
ed by participation
by SBH reduction
and the carbonyl
absorption
of the disulfide
of the lone pair electons which
12) gave
371 (M-SMe); 6 (CDC13) 2.11,
1.85, 2.43, 3.18, 3.88, 3.90 (3H each,
linkage.
: 1 i 1 : 1.3) in MeOH at r.t. overnight.
to form an ionic intermediate
in MeOH
(lH, s), 5.10 (lH, d, 1.2), 6.55 (lH, s), 7.22 (lH, d,
(lH, br. s), 6.61 (lH, s), 7.23 (lH, d, 1.2).
opening with methylation
to that of 1, which
of j_ on this methylation.
387 (M-OMe),
371 (M-SMe); G(CDC13)
and 2.19 in 6 and 1.85 and 2.43 in 1 show reductive
at r.t. followed
6: m/z 418 (M+, C16HlgN205S2Cl),
7: m/g 418 (M+), 387 (M-OMe),
s), 4.71 (lH, s), 4.77
of 4 is very similar
in the frame work of the structure
2.19, 3.10. 3.87, 3.94 (3H each, s), 4.87 1.2).
shift of H-11 (6 5.26) of 5 than that of
results
on the nitrogen
in liberation
Two z-methyl
(1691 cm-')')
carrying
reachesto
isomerization
a S-methyl
of an anomer at C-8.
(6 2.11
of the mixture
Each compound
This facile
signals
an equi-
is explain-
group on it
2098
Reaction
of the mixture
(M+, C17H23N206S2C1), The bathochromic hydroxyl
in 5.
compounds
(23 nm) in alkaline
The nucleophilic surprising
naturally.
with a similar
conditions
attack of methoxide
that a compound
partial
FT-NMR measurements. measurements
adduct
(8):
nm 230 (sh.), 295; hi!$oMe
strongly
suggests
IJ/~ 450 318 nm.
the presence
of a phenolic
anion on C-8 produced 8.
13)
as thiosulfenic
are, to our knowledge,
known synthetic
structure.
of the structure
of lwill
be published
We thank Prof. S. Abe of Kyoto Prefectural
discussions.
a methanol
with such a labile functionarity
of the fungus and Prof. T. Oishi of the Institute helpful
i:iF
E-(Thiosulfenyl)phthalimides
Further comfirmation Acknowledgements.
in MeOH yielded
403 (M-SMe), 371 (M-MeOH-SMe);
shift
It is rather amide occurrs
6 and 1 with NaOMe
We are greatful
of Physical
to Drs. S. Takeuchi
We are also indebted
elsewhere.
University
for the identification
and Chemical
Research
(IPCR) for
and Y. Kohno of IPCR for their help for
to Mr. Y. Esumi of IPCR for high resolution
and to Miss H. Tanaka of IPCR for her assistance
for the fermentation
mass
of the fungus.
References
1)
Aspirochlorine (1) has different activity from that of canadensolide. 1 shows not only growth inhibition of mycelium but also enormous numbers of spherical swellings on the hyphae of P&&hthora cinnamomi (A mating type). Isolation and the biological activity of lwill be pub+--lia-* 1s e ln detail els &where.
2)
This fungus was isolated from the air and identified Agriculture, Kyoto Prefectural University, Sakyo-ku,
by Prof. Shigeo Abe of Faculty Kyoto 606, Japan.
of
Wright, P.W. Brian, S.M. Clarke and S.A. Hutchinson, 3) N.J. McCorkinkale, J.L.C. 727 (1968); M. Kato, R. Tanaka and A. Yoshikoshi, Chem. Comn., 1561 (1971).
Tet. Lett.,
4) D.H. Berg, R.P. Massing,
22, 394 (1976).
5) The formulae
M.M. Hoehn,
L.D. Boeck and R.L. Hamill,
of the ions in this paper were
6) D.H. Berg, R.L. Hamill (1976); idem., ibid.,
confirmed
J. Antibiot.,
by high resolution
mass spectrometry.
and M.M. Hoehn, GA., 86, 418029 (1977): U. S. Patent, 87671s (1977): U. S.-patent, 4,001,086 (1977).
3,991,052
gj,
7) An approximate calculation of the chemical shifts of aromatic carbons was made based on the assumption that the substituents at C-l and C-6 are equivalent to methoxyl and ethyl groups, respectively, using a table in the text: E. Breitmair and W. Voelter, "Methods and Application in Organic Chemistry, Vol. 5,C-13 NMR Spectroscopy Monographs in Modern Chemistry," Verlag Chemie Weinheim, New York, 1978, p. 213. 8) J.W. Ronaldson, Aust. J. Chem., ?S, 2307 (1976); D. Brewer, A.G. McInnes, D.G. Smith, A. Taylor and J.A Walter, J. Chem. Sot., Perkin I, 1248 (1978); G. Deffeux, M.A. Baute, R. Baute and M.J. Filleau, J. Antibiot., 31, 1102 (1978). 9) K.H. Michel, M.O. Channey, N.D. Jones, M.M. Hoehn and R. Nagarajan, S. Safe and A. Taylor, J. Chem. Sot. (C), 1189 (1971). 10) K.C. Murdock,
J. Antibiot.,
J. Med. Chem., iz, 827 (1974).
11) H. Budzikiewicz, C. Djerassi and D.H. Williams, "Structure Elucidation Mass Spectrometry," Holden Day, San Francisco, 1964, Vol. 2, p. 249. 12) W.D. Jamison, 13) A.B. Sulivan
02, 57 (1974);
R. Rahman and A. Taylor, and K. Boetany,
J. Chem. Sot. (C), 1564 (1969).
Int. J. Sulfur
(Received in Japan 11 January 1982)
Chem., A, 1, 207 (1971).
of Natural
Products
by