An Epitetrathiodioxopiperazinewith 3S ,6S Configurationfrom
Hyalodendron sp.
GEORGEM. STRUNZ,MASATOSHI
KAKUSHIMA,
A N D MERLYN
A. STILLWELL
Cnnndintl Forestry Service, P.O. Box 4000, Fredericton, N e w Br~ttlswickE3B 5G4
Received July 24, 1974
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GEORGE
M. STRUNZ,
MASATOSHI
KAKUSHIMA,
and MERLYNA. STILLWELL.
Can. J. Chem.
53,295 (1975).
(3S,6S)3-Benzyl-6-hydroxymethyl-1,4dimethyl-3,6-epitetrathiopiperazine-2,5-dione
has been
isolated from cultures of Hynlodendron sp. The same compound is produced when hyalodendrin is heated with methanol - hydrochloric acid.
A. STILLWELL.
Can. J. Chem. 53,
KAKUSHIMA
et MERLYN
GEORGE
M. STRUNZ,MASATOSHI
295 ( 1975).
La ( 3 s ,6S) 3-Benzyl-6-hydroxymtthyl-1,4dimtthyl-3,6-epitetrathiopiperazine-2,5-dione
aCt6
isolt de cultures d'une espkce d'Hynlodendron. Le m&me compost est produit en chauffant
I'hyalodendrine avec une solution de methanol et d'acide chlorhydrique.
The fungitoxic epidithiodioxopiperazine hyalodendrin, 1, and the corresponding di(methy1thio) ether, 2, both with 3S,6S configuration have
recently been isolated from culture filtrates of
Hyalodendron sp. (1-3). Metabolites 1and 2, and
the trisulfide 3, of undetermined absolute configuration, were obtained from cultures of an unidentified fungus (NRRL 3888) (4). The metabolite 1, with R configuration at positions 3 and 6,
and compounds 2 and 4, evidently belonging to
the same stereochemical series, are produced by
Penicillium turbatum (5). Gliovictin, a metabolite
of Helminthosporium victoriae also has structure 2
(6) and is likewise enantiomeric (3R,6R) with the
corresponding Hyalodendron product.
We record here some observations made
during a study of the 3S,6S tetrasulfide 4,
isolated from fermentations of Hyalodendron
sp. This compound, Cl,H16N,0,S,, was present
in chloroform extracts of the filtered culture
medium in amounts which ranged up to 3% of
the yield of pure hyalodendrin. It was slightly
more polar than the latter, and could be separated and purified by preparative-layer chromatography, followed by crystallization. Comparison of the spectral characteristics of 4 (see
Experimental section) with those of its co-metabolites 1 and 2 leave no doubt as to its constitution. Comparison of the circular dichroism curve
of the Hyalodendron tetrasulfide (see Experimental section) with that published for the
Penicillium metabolite (5) establishes that these
compounds are enantiomeric at positions 3 and
6. A detailed discussion of the geometry of the
tetrasulfide system of 4 is deferred at this stage,
although it is noted that the synthetic compound
N,N1- dimethyl - 3,6 - epitetrathiopiperazine - 2,5dione and sporidesmin G have been shown by
X-ray analysis to be structurally similar with
respect to the epitetrathiodioxopiperazine moiety
(7, 8).
A tetrasulfide, identical in all respects (including c.d. spectrum) with the Hyalodendron
tetrasulfide, was obtained in modest yield when
hyalodendrin was refluxed in methanolic hydrochloric acid until no starting material remained
(t.1.c.). By-products from this transformation,
depleted in sulfur, resisted preliminary purifica-
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296
CAN. J. CHEM. VOL. 53, 1975
tion attempts and have not been investigated
further at this stage. Tetrasulfide formation was
also effected in low yield by shaking a solution of
hyalodendrin in the culture medium (3) (initial
p H 3.5, methanol co-solvent) for a period comparable with the fermentation time. This result
raises the possibility that the tetrasulfide isolated
could be an artifact, formed from hyalodendrin
by a similar acid-catalyzed mechanism. Although such a possibility cannot be excluded at
present, the fact that the P. turbatum tetrasulfide
is produced in a substantially different medium
(5), and that sporidesmin G appears to be a true
metabolite' seems t o attest the 'natural' origin
of at least part of the Hyalodendron tetrasulfide.
Refluxing hyalodendrin in aqueous methanol
solution without addition of hydrochloric acid
also led to the formation of a tetrasulfide. This
transformation occurred more slowly than that
in acid solution and the yield of tetrasulfide obtained was substantially higher. The product
formed under these conditions was racemic.
Conversion of epidithiodioxopiperazines to
the corresponding tetrasulfides (with retention
of configuration) has previously been effected by
the action of dihydrogen disulfide (9, 10). In
bioassays, the 3S,6S tetrasulfide 4 was in general
comparable with hyalodendrin (in some cases
slightly less effective) in its ability t o inhibit the
growth of selected test fungi.l
Experimental
See refs. 1 and 2 for description of instrumentation, etc.
Isolation of 'Na/ural' Tetrasulfide 4
The production and isolation of hyalodendrin and
bisdethiodi(methy1thio) hyalodendrin have been detailed
elsewhere (1-3). In a typical isolation sequence, material
extracted with chloroform from 37 1 of filtered culture
medium which had supported growth of Hyalodendron
sp. for 12 days was chromatographed on a column of
silica gel (500 g Kieselgel, 100-200 mesh, Gebr. Herrmann, Koln). The eluates were monitored by t.1.c.
Elution with benzene-chloroform (65 :35) afforded
initially 2 g of hyalodendrin. In the later fractions, the
emergence of a second compound, with slightly lower
R, became evident (t.1.c.). Fractions containing the new
compound were combined and rechromatographed on a
preparative-layer plate of silica gel (benzene-acetone,
9: 1, R,
0.5). This separation provided 78 mg of crude
tetrasulfide, which gave 60 mg of crystalline material
from benzene-cyclohexane. Recrystallization from benzene-cyclohexane afforded colorless crystals, m.p. 133139" (pulverized crystals, 125-132"); c.d. (c 2.81 x lo-"
-
'Dr. A. Taylor. Personal communication.
2M. A. Stillwell. Unpublished data.
M in MeOH, 1 mm cell) h nm (AE): 370(0), 328(+0.9),
3 16(0), 297(- 4.4), 287(0), 263(+ 13.6), 225(inflection
12.1), 230(0), and < 220 (- ve max) ; v,,,(KBr) inter
alia-3400, 1666, 1648, 1375, 1060, 742, 735, 701, and
657 cm-'; n.m.r. (CDCI,, 220 MHz) 6 3.03 (3H, s), 3.07
15 Hz), broadened base), 3.83
(3H, s) 3.25 (2H, d, J
(lH,d,J-12H~),4.02(1H,d,J-15H~),4.20(1H,d,
J
12 Hz), 7.09-7.27 (5H, m). T h e signal for the
hydroxyl proton (broad absorption around 6 3.25) disappeared on deuterium exchange. Mass spectrum inter
alia, m/e 260 (base peak) ( M - S4)+, 242 ( M - S4 H 2 0 ) + , 169 ( M - S 4 - C7H7)+,and 64.
Anal. Calcd. for C14H16N2S403:C, 43.27; H, 4.15; N,
7.21; S, 33.01, Found: C , 43.42; H , 4.11; N, 7.20; S,
33.02.
Acid Treatment of Hyalodendrin
Methanol - Hydrochloric Acid
A solution of hyalodendrin (582 mg, 1.79 mmol) in
methanol (30 ml) containing 1 N aqueous hydrochloric
acid (3.0 ml) was heated under reflux for 24 h, when no
starting material remained. (Longer reaction times, up to'
16 days, did not appear t o have much effect on either the
yield or c.d. properties of the tetrasulfide product.) After
addition of saturated sodium chloride solution, the mixture was extracted thoroughly with chloroform. The
extracts were washed with saturated sodium bicarbonate
solution, dried (MgS04), and evaporated in vacuo. The
tetrasulfide 4 was separated and purified by repeated
chromatography o n preparative-layer plates of silica gel
(benzene-acetone, 94:6). Crystallization from benzenecyclohexane gave 100 mg (0.257 mmol) of colorless
crystals of 4, m.p. 132-136" (pulverized crystals, 125132"); mixture melting point with 'natural' 4 : 125-132";
c.d., i.r. (KBr), n.m.r. (CDCI,, 220 MHz), and mass
spectra identical with 'natural' 4 (vide supra). An analytical sample was prepared by recrystallizations from benzene: colorless prisms, m.p. 135-138".
Anal. Calcd. for Cl4Hl6N2S4O3: C, 43.27; H, 4.15;
N, 7.21; S, 33.01. Found: C, 43.30; H, 4.16; N, 7.28; S,
32.99.
Culture Medium, pH 3.5 ( 3 )
Because of the low solubility of hyalodendrin in water,
methanol was used a s co-solvent. An aliquot (I ml) of a
solution of hyalodendrin (2 g) in methanol (40 ml) was
added to each of 40 1-1 Erlenmeyer flasks, eachcontaining
500 ml of synthetic culture medium a t p H 3.5 (3). T h e
flasks were stoppered with foam-plastic plugs (as used in
fermentation) and shaken on a gyratory shaker at 22"
for 14 days (final p H 4). Unchanged hyalodendrin
and tetrasulfide 4 were recovered from the chloroform
extracts by chromatography as described above (isolation
of 'natural' tetrasulfide 4). Recrystallization from benzene-cyclohexane afforded 37 mg of 4 a s almost colorless
crystals, m.p. 133-142"; c.d. (c 2.75 x lo-, M in MeOH,
1 m m cell) h nm (AE): 370 (O), 330 (+0.9), 318 (O), 298
(-4.2), 288 (O), 264 (+ 12.8), 254 (inflection 10.4), 231
(O), and < 220 (- ve max); i.r. (KBr), and mass spectra
identical with those of 'natural' 4.
+
-
-
-
+
Formation of Racemic Tetrasulfide 4
A solution of hyalodendrin (1.0 g, 3.08 mmol) in
methanol (40 ml) a n d water (4 ml) was heated under
reflux for 18 days, when no unchanged starting material
remained (t.1.c.). A saturated aqueous solution of sodium
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STRUNZ E T AL.: PIPERAZINE-2,s-DIONE
chloride was added and the mixture was extracted with
chloroform. Material from the chloroform extracts was
chromatographed on preparative layer plates of silica gel
(benzene-acetone, 9: I), and the product corresponding
in R, with the tetrasulfide 4 was collected. Recrystallization from benzene afforded 410 mg (1.05 mmol, 68%
theoretical) of tetrasulfide as almost colorless crystals,
m.p. 155-166"; c.d. (c 2.92 x 10-3 M in MeOH, 1 mm
cell) 600-250 nm, AE = 0 ; n.m.r. (CDCi3, 220 MHz)
and mass spectra identical with those of 'natural' tetrasulfide 4. The main peaks in the i.r. (KBr) spectrum were
essentially as quoted for 'natural' 4: minor differences
were detectable in the fingerprint region. An analytical
sample was prepared by recrystallizations from benzenechloroform, m.p. 159-166".
Anal. Calcd. for CI4Hl6NZS4O3:C, 43.27; H , 4.15;
N, 7.21; S, 33.01; 0, 12.35. Found: C, 43.26; H, 4.20;
N, 7.22; S, 32.97; 0, 12.28.
Mol. Wt. Calcd. for C14H16NZS403:388.6. Found
(osmometer, CHCl solvent): 399.
NOTEADDED IN PROOF:An additional correlation of the
Hyalodendron tetra- and disulfide has been effected by
sodium borohydride reduction of the former, followed by
oxidation with iodine-iodide solution (cf: ref. 11). This
treatment yielded a product, identical in all respects,
including c.d. spectrum, with hyalodendrin.
The enantiometric relationship of bisdethiodi(methy1thio)hyalodendrin and gliovictin was confirmed by
297
direct comparison of the former with a sample of the
latter, kindly provided by Professor Arigoni.
We thank Mr. J. Hoyle, University of Alberta for the
circular dichroism measurements and Mrs. M. Austria,
University of New Brunswick for the mass spectra.
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M. A. STILLWELL,
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and C. J. HEISSNER.
(1973).
M. K A K U S H I M A , ~ ~ ~
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M. A. STILLWELL.
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L. P. MAGASI,
andG. M. STRUNZ.
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