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A novel sterol peroxide from the sea anenome Metridium senile
261
3305 A NOVEL
STEROL
PEROXIDE
John
A.
FROM THE SENILE
Findlay
and
SEA
Ashok
ANENOME
METRIDIUM
D. Patil
D e p a r t m e n t of C h e m i s t r y , U n i v e r s i t y of New B r u n s w i c k Fredericton, New B r u n s w i c k , C a n a d a E3B 6E2 Received 2-14-85. ABSTRACT Nine 5 ~ , 8 ~ - e p i d i o x y A 6 and H6,9(II) sterols were i d e n t i f i e d i n c l u d i n g the h i t h e r t o u n k n o w n ( 2 2 E ) - 5 , 8 epidioxy-5~,8~-stigmasta-6,9(ll),22-trien-3B-ol 9 from the sea a n e n o m e M e t r i d i u m senile based on 200 MHz 1Hnmr and mass s p e c t r a l data.
5~,8~-Epidioxy organisms have
now
report
were
chloroform
investigators
chopped
and
near
reversed
(p-porasil,
followed
(95:5)
providing
sterols
2 (6),
3 (6,7),
8 (6),
MHz
iHnmr
and
comparison
was of
spectra
which and
with
a new
encountered.
The
novel
September1984
(i-8).
We
the
high
compound
with
epidioxy (Linnaeus)
eight
4 (6),
were
resolution
gel
purification HPLC
known
deduced
mass
and
on
employing epidioxy
6 (7),
7
from
200
spectra
(HRMS)
data.
resolution
established
S - J e ~ o
silica
5 (6,7),
epidioxy-trien-ol high
to
i0 m i c r o n s ) the
New
methanol
by r e p e a t e d
structures
literature
In a d d i t i o n ,
the
marine
Andrews,
subjected
C18,
methanol/water
and
from
senile
St.
extracted
portion
on TLC
(4,6),
of
Metridium
collected
chromatography
1 (4),
isolated
constitution
from
soluble
phase
of
been
findings.
organisms,
Brunswick,
the
derived our
have
groups
examined
fraction
The
the
several
recently
sterol and
by
sterols
the
x x)~
formulated
mass
spectrum
molecular
as 9 (HRMS)
formula
Volume 44, Number3
262
~
T
~, ~ o
x ~
R
4
2
R --
"
'
-
f:l --
~
7
~ 1
3
R -"
7
0
R
-
R
HO
7 g
R--
"
--.(~(
T'm
C29H4403 intense type
(m/z ion
440.3285,
m/z
accounted
for
by
M-O 2 f r a g m e n t , (M-CH3), provide iHnmr
422.3184
(d,
gives
0.87
(6H,
21CH3), and
J = 7.2
methyl
a triplet
multiplet
this
sidechain
sterol
is
are
the
ions
at m/z
Hz)
high Hz,
and
at ~ 0.82
due
to the for
Thus,
we
the
9.
the
The C6H
J = 8.5
displays (3H,
and
and
C7H while
(J = 5.5 doublets
Hz, at 6
J = 7.2 Hz,
29CH3).
C3H
the
200 MHz
Hz),
(18CH3) , 1.09
(J = 6 . 1 H z ,
from
is
425.3050
at ~ 5.44
0.91
251.1719
(M-O2-H20-CH3)
for
(d,
region
26/27CH3),
typical
(4,6).
6.25
doublet
field
1.01 is
signals
an
Diels-Alder
sidechain
formulation
shows
displayed
at m/z
for
singlets
5.17
ion
support
at ~ 4.02
and
to retro
375.3041
at ~
5.24
and
and
to a double
The
and
prominent
J = 8.5
J = 1.3 Hz).
due
intense
of water
(CDCI3)
rise
263
(M-H20),
additional
at 6 6.51 CIIH
loss
An
x ~
440.3280)
(M-02)
(6).
while
spectrum
calcd.
408.3395
fragmentation
~ o
(19CH3) ,
A broad
multiplets
trans
~22
conclude
that
at
protons this
in
new
(22E)-5,8-epidioxy-5~,8~-stigmasta-6,9(ll),22-
trien-3B-ol,
9.
EXPERIMENTAL H i g h r e s o l u t i o n mass s p e c t r a w e r e r e c o r d e d on an A.E.I. M S - 5 0 m a s s s p e c t r o m e t e r c o u p l e d to a DS 50 c o m p u t e r ( U n i v e r s i t y of A l b e r t a ) . iHnmr s p e c t r a were r e c o r d e d in CDCI 3 s o l u t i o n on a V a r i a n ~ L 2 0 0 i n s t r u m e n t . H P L C was c a r r i e d out on a W a t e r s s y s t e m e m p l o y i n g 440 a b s o r b a n c e and R401 r e t r a c t i v e i n d e x d e t e c t o r s and using r e v e r s e d p h a s e ( ~ - p o r a s i l C-18) c o l u m n (30 cm x 3.9 mm). Infrared s p e c t r a w e r e r e c o r d e d on a P e r k i n - E l m e r 598 s p e c t r o p h o t o m e t e r and o p t i c a l r o t a t i o n s were m e a s u r e d in c h l o r o f o r m s o l u t i o n u s i n g a P e r k i n - E l m e r 241 p o l a r i m e t e r .
264
S
.x- ~ , ~ o
EXTRACTION
x x~ m
AND ISOLATION
Sea anenomes Metridium senile (wet wt 1.98 kg) were collected near St. Andrews (New Brunswick). The specimens were chopped and extracted in a Soxhlet apparatus with methanol. Removal of methanol in vacuo gave a pale brown solid (23 g). The crude extract was dissolved in water (350 mL) and extracted with chloroform (3 x 400 mL). The chloroform soluble extract (14.5 g) was chromatographed over a column of silica gel G (300 g) using chloroform and chloroform-methanol (95:5) mixture for elution. Early fractions containing lipids and fats (5.2 g) were not pursued. Subsequent fractions yielded long chain fatty acids (1.3 g) and conventional sterols (2.22 g) and were followed by the epidioxy sterol mixture (0.123 g). The latter fraction was further purified by preparative TLC on silica gel using 6% methanol in chloroform. The TLC-purified epidioxy sterol mixture (68 mg) was subjected to reverse- phase HPLC separation employing methanol. Further purification using 5% water in methanol as mobile phase resulted in the isolation of the following compounds in order of elution: 1 (12 mg), 3 (7 mg), 2 (3 mg), 9 (5 mg), 7 (4 mg), 8 (2 mg), 5 (8 mg), 6 (9 mg), and 4 (5 mg). (22E)-5,8-Epidioxy-5~,8~-stigmasta-6,9(ll),22-trien-3B -oi 9: An oil (5 m !:~ I~0(CHCI3)3370, 2950, 1460, 1370, 1310, 1085, 950 cm -~', [~jN = +1.7 (c = 0.16, CHCI3) ; HRMS m/z 440.3285(0.5, M + C29H4403, Calcd. 440.3280), 425.3050(4.3), 422.3184(8.2), 408.3395(35.8), 375.3041 (11.5), 251.1797(25.1), 211.1482(13.7), 297.1324(15.7).
ACKNOWLEDGEMENTS The assistance of Dianne Fournier in the initial phase of this work is acknowledged and we thank Barry Hill for collection and identification of organisms and the Natural Sciences and Engineering Research Council for financial assistance. REFERENCES i. 2. 3. 4. 5.
Sheikh, Y. M. and Djerassi, C., TETRAHEDRON, 30, 4095 (1974). Fattorusso, E., Mango, S., Santacrose, C., and Sica, D., GAZZ °. CHIM. ITAL., 104, 409 (1974). Malorni, A., Minale, L., and Riccio, R., NOUV. J. CHIM., ~, 351 (1978). Stonard, R. J., Petrovich, J. C., and Anderson, R. J., STEROIDS, 36, 81 (1980). Guyot, M. and Durgeat, M., TETRAHEDRON LETT., 1391
(1981),
T
6. 7. 8.
~, ~ o
x
:Din
Gunatilaka, A. A. L., Gopichand, Y., Schmitz, F. J., and Djerassi, C., J. ORG. CHEM., 46, 3860 (1981). Sugnaux, F. R. and Djerassi, C., J. CHROMATOG., 251, 189 (1982). Finamore, E., Minale, L., and Ali Mohammed, M., REND. ACCAD. SCI. FIS. MAT. NAPLES, 50, 81 (1983).
265
3305 A NOVEL
STEROL
PEROXIDE
John
A.
FROM THE SENILE
Findlay
and
SEA
Ashok
ANENOME
METRIDIUM
D. Patil
D e p a r t m e n t of C h e m i s t r y , U n i v e r s i t y of New B r u n s w i c k Fredericton, New B r u n s w i c k , C a n a d a E3B 6E2 Received 2-14-85. ABSTRACT Nine 5 ~ , 8 ~ - e p i d i o x y A 6 and H6,9(II) sterols were i d e n t i f i e d i n c l u d i n g the h i t h e r t o u n k n o w n ( 2 2 E ) - 5 , 8 epidioxy-5~,8~-stigmasta-6,9(ll),22-trien-3B-ol 9 from the sea a n e n o m e M e t r i d i u m senile based on 200 MHz 1Hnmr and mass s p e c t r a l data.
5~,8~-Epidioxy organisms have
now
report
were
chloroform
investigators
chopped
and
near
reversed
(p-porasil,
followed
(95:5)
providing
sterols
2 (6),
3 (6,7),
8 (6),
MHz
iHnmr
and
comparison
was of
spectra
which and
with
a new
encountered.
The
novel
September1984
(i-8).
We
the
high
compound
with
epidioxy (Linnaeus)
eight
4 (6),
were
resolution
gel
purification HPLC
known
deduced
mass
and
on
employing epidioxy
6 (7),
7
from
200
spectra
(HRMS)
data.
resolution
established
S - J e ~ o
silica
5 (6,7),
epidioxy-trien-ol high
to
i0 m i c r o n s ) the
New
methanol
by r e p e a t e d
structures
literature
In a d d i t i o n ,
the
marine
Andrews,
subjected
C18,
methanol/water
and
from
senile
St.
extracted
portion
on TLC
(4,6),
of
Metridium
collected
chromatography
1 (4),
isolated
constitution
from
soluble
phase
of
been
findings.
organisms,
Brunswick,
the
derived our
have
groups
examined
fraction
The
the
several
recently
sterol and
by
sterols
the
x x)~
formulated
mass
spectrum
molecular
as 9 (HRMS)
formula
Volume 44, Number3
262
~
T
~, ~ o
x ~
R
4
2
R --
"
'
-
f:l --
~
7
~ 1
3
R -"
7
0
R
-
R
HO
7 g
R--
"
--.(~(
T'm
C29H4403 intense type
(m/z ion
440.3285,
m/z
accounted
for
by
M-O 2 f r a g m e n t , (M-CH3), provide iHnmr
422.3184
(d,
gives
0.87
(6H,
21CH3), and
J = 7.2
methyl
a triplet
multiplet
this
sidechain
sterol
is
are
the
ions
at m/z
Hz)
high Hz,
and
at ~ 0.82
due
to the for
Thus,
we
the
9.
the
The C6H
J = 8.5
displays (3H,
and
and
C7H while
(J = 5.5 doublets
Hz, at 6
J = 7.2 Hz,
29CH3).
C3H
the
200 MHz
Hz),
(18CH3) , 1.09
(J = 6 . 1 H z ,
from
is
425.3050
at ~ 5.44
0.91
251.1719
(M-O2-H20-CH3)
for
(d,
region
26/27CH3),
typical
(4,6).
6.25
doublet
field
1.01 is
signals
an
Diels-Alder
sidechain
formulation
shows
displayed
at m/z
for
singlets
5.17
ion
support
at ~ 4.02
and
to retro
375.3041
at ~
5.24
and
and
to a double
The
and
prominent
J = 8.5
J = 1.3 Hz).
due
intense
of water
(CDCI3)
rise
263
(M-H20),
additional
at 6 6.51 CIIH
loss
An
x ~
440.3280)
(M-02)
(6).
while
spectrum
calcd.
408.3395
fragmentation
~ o
(19CH3) ,
A broad
multiplets
trans
~22
conclude
that
at
protons this
in
new
(22E)-5,8-epidioxy-5~,8~-stigmasta-6,9(ll),22-
trien-3B-ol,
9.
EXPERIMENTAL H i g h r e s o l u t i o n mass s p e c t r a w e r e r e c o r d e d on an A.E.I. M S - 5 0 m a s s s p e c t r o m e t e r c o u p l e d to a DS 50 c o m p u t e r ( U n i v e r s i t y of A l b e r t a ) . iHnmr s p e c t r a were r e c o r d e d in CDCI 3 s o l u t i o n on a V a r i a n ~ L 2 0 0 i n s t r u m e n t . H P L C was c a r r i e d out on a W a t e r s s y s t e m e m p l o y i n g 440 a b s o r b a n c e and R401 r e t r a c t i v e i n d e x d e t e c t o r s and using r e v e r s e d p h a s e ( ~ - p o r a s i l C-18) c o l u m n (30 cm x 3.9 mm). Infrared s p e c t r a w e r e r e c o r d e d on a P e r k i n - E l m e r 598 s p e c t r o p h o t o m e t e r and o p t i c a l r o t a t i o n s were m e a s u r e d in c h l o r o f o r m s o l u t i o n u s i n g a P e r k i n - E l m e r 241 p o l a r i m e t e r .
264
S
.x- ~ , ~ o
EXTRACTION
x x~ m
AND ISOLATION
Sea anenomes Metridium senile (wet wt 1.98 kg) were collected near St. Andrews (New Brunswick). The specimens were chopped and extracted in a Soxhlet apparatus with methanol. Removal of methanol in vacuo gave a pale brown solid (23 g). The crude extract was dissolved in water (350 mL) and extracted with chloroform (3 x 400 mL). The chloroform soluble extract (14.5 g) was chromatographed over a column of silica gel G (300 g) using chloroform and chloroform-methanol (95:5) mixture for elution. Early fractions containing lipids and fats (5.2 g) were not pursued. Subsequent fractions yielded long chain fatty acids (1.3 g) and conventional sterols (2.22 g) and were followed by the epidioxy sterol mixture (0.123 g). The latter fraction was further purified by preparative TLC on silica gel using 6% methanol in chloroform. The TLC-purified epidioxy sterol mixture (68 mg) was subjected to reverse- phase HPLC separation employing methanol. Further purification using 5% water in methanol as mobile phase resulted in the isolation of the following compounds in order of elution: 1 (12 mg), 3 (7 mg), 2 (3 mg), 9 (5 mg), 7 (4 mg), 8 (2 mg), 5 (8 mg), 6 (9 mg), and 4 (5 mg). (22E)-5,8-Epidioxy-5~,8~-stigmasta-6,9(ll),22-trien-3B -oi 9: An oil (5 m !:~ I~0(CHCI3)3370, 2950, 1460, 1370, 1310, 1085, 950 cm -~', [~jN = +1.7 (c = 0.16, CHCI3) ; HRMS m/z 440.3285(0.5, M + C29H4403, Calcd. 440.3280), 425.3050(4.3), 422.3184(8.2), 408.3395(35.8), 375.3041 (11.5), 251.1797(25.1), 211.1482(13.7), 297.1324(15.7).
ACKNOWLEDGEMENTS The assistance of Dianne Fournier in the initial phase of this work is acknowledged and we thank Barry Hill for collection and identification of organisms and the Natural Sciences and Engineering Research Council for financial assistance. REFERENCES i. 2. 3. 4. 5.
Sheikh, Y. M. and Djerassi, C., TETRAHEDRON, 30, 4095 (1974). Fattorusso, E., Mango, S., Santacrose, C., and Sica, D., GAZZ °. CHIM. ITAL., 104, 409 (1974). Malorni, A., Minale, L., and Riccio, R., NOUV. J. CHIM., ~, 351 (1978). Stonard, R. J., Petrovich, J. C., and Anderson, R. J., STEROIDS, 36, 81 (1980). Guyot, M. and Durgeat, M., TETRAHEDRON LETT., 1391
(1981),
T
6. 7. 8.
~, ~ o
x
:Din
Gunatilaka, A. A. L., Gopichand, Y., Schmitz, F. J., and Djerassi, C., J. ORG. CHEM., 46, 3860 (1981). Sugnaux, F. R. and Djerassi, C., J. CHROMATOG., 251, 189 (1982). Finamore, E., Minale, L., and Ali Mohammed, M., REND. ACCAD. SCI. FIS. MAT. NAPLES, 50, 81 (1983).
265