Triterpenoid constituents of Euphorbia cyparissias


Starratt, A.N.

Phytochemistry 5(6): 1341-1344

1966


The non-saponifiable fraction from the light petroleum extract of Euphorbia cyparissias has been investigated and shown to contain euphol, 24-methylenecycloartanol, cycloart-23-en-3β,25-diol,β-amyrin, glut-5(6)-en-3-one, glut-5(6)-en-3α-ol, glut-5(6)-en-3β-ol, and β-sitosterol. This is the first recorded instance of glut-5(6)-en-3α-ol occurring in nature.

Phytochemistry,
1966,
VoL
5,
pp.
1341
to
1344.
Pergamon
Press
Ltd.
Printed
in
England
SHORT
COMMUNICATION
TRITERPENOID
CONSTITUENTS
OF
EUPHORBIA
CYPARISSIAS
A.
N.
STARRATT
Research
Institute,
Canada
Department
of
Agriculture,
London,
Ontario,
Canada
(Received
30
March
1966)
Abstract—The
non-saponifiable
fraction
from
the
light
petroleum
extract
of
Euphorbia
cyparissias
has
been
investigated
and
shown
to
contain
euphol,
24-methylenecycloartanol,
cycloart-23-en-3/1,25-diol,f1-amyrin,
glut-5(6)-en-3-one,
glut-5(6)-en-3a-ol,
glut-5(6)-en-3/1-ol,
and
fl-sitosterol.
This
is
the
first
recorded
instance
of
glut-5(6)-en-3a-ol
occurring
in
nature.
IN
CONNEXION
with
other
studies
concerning
cypress
spurge
(Euphorbia
cyparissias),
a
noxious
weed
found
in
eastern
Canada,
we
have
undertaken
a
reinvestigation
of
the
chemical
con-
stituents
of
this
plant.
The
results
of
an
earlier
study'
were
suggestive
of
the
presence
of
triterpenes
known
to
occur
in
other
Euphorbia
species.
Chemotaxonomic
work
on
the
Euphorbiaceae
has
recently
been
reviewed
2
and
an
analytical
method
presented
2,
3
for
the
rapid
identification
of
the
tetracyclic
triterpenes
in
the
latex
of
these
plants.
The
method
employed
in
the
present
work
was
designed
to
permit
the
preparative
separa-
tion
and
identification
of
both
the
tetracyclic
and
pentacyclic
triterpenes
present
in
E.
cyparissias.
However,
the
unsaponifiable
portion
of
a
light
petroleum
extract
of
the
plant
proved
to
be
a
mixture
of
considerable
complexity,
and
success
in
the
resolution
was
achieved
only
by
repeated
chromatography
in
combination
with
acetylation
procedures.
By
these
means,
it
was
possible
to
isolate
and
identify
unambiguously
the
following
compounds
:
euphol,
24-methylenecycloartanol,
cycloart-23-en-313,25-dio1,13-amyrin,
glut-5(6)-en-3-one,
glut-5(6)-en-3(z-ol,
glut-5(6)-en-3/3-ol,
and
fl-sitosterol.
These
compounds
or
derivatives
thereof
were
identified
by
comparison
with
the
relevant
authentic
specimens.
However,
neither
the
24-methylenecycloartanol
nor
the
derived
acetate
could
be
freed
from
a
con-
taminant
which
lowered
the
melting
point
but
other
physical
characteristics
(see
Experi-
mental)
leave
no
doubt
as
to
their
identity.
In
addition,
two
other
crystalline
substances
were
obtained
but
in
quantities
insufficient
to
permit
complete
purification
and
identification.
Although
glut-5(6)-en-3/3-ol
has
been
isolated
previously
4
from
a
natural
source
and
more
recently
5
from
an
Euphorbia
species
(E.
royleana),
there
appears
to
be
no
prior
report
of
the
natural
occurrence
of
glut-5(6)-en-3a-ol.
This
latter
alcohol
has
previously
been
prepared
6
by
lithium
aluminium
hydride
reduction
of
the
corresponding
ketone.
In
the
E.
HUPPERT,
H.
SWIATKOWSKI
and
J.
ZELLNER,
Monatsh.
48,
491
(1927).
2
G.
PoNstNur
and
G.
OmussoN,
Phytochem.
4,
799
(1965).
3
G.
PoNstrsisr
and
G.
OtaussoN,
Phytochem.
4,
813
(1965).
4
F.
G.
FISCHER
and
N.
&u.
nt,
Ann.
them.
644,
162
(1961).
5
P.
SENGUPTA
and
S.
GHOSH,
J.
Ind.
Chem.
Soc.
42,
543
(1965).
6
S.
CHAPON
and
S.
DAVID,
Bull.
Soc.
Chim.
France
333
(1953);
J.
M.
BEATON,
F.
S.
SPRING
and
R.
STEVENSON,
J.
Chem.
Soc.
2616
(1955).
85
1341
1342
A.
N.
STARRATT
present
work
it
was
found
that
sodium
borohydride
reduction
of
glut-5(6)-en-3-one
followed
by
acetylation
gave,
as
the
main
product,
glut-5(6)-en-3a-y1
acetate
together
with
a
small
amount
of
the
epimer,
glut-5(6)-en-3fl-y1
acetate,
identical
with
the
major
pentacyclic
triter-
penoid
consistuent
of
E.
cyparissias.
This
is
the
second
report
of
the
natural
occurrence
of
cycloart-23-en-3/3.25-dio1
7
which
probably
arises
biogenetically
by
oxidation
of
cycloartenol.
Although
not
detected
by
us,
cycloartenol
is
a
common
Euphorbia
constituent
2
and
also
would
be
expected
to
give
rise
to
24-methylenecycloartanol.
The
wide
variety
of
isoprenoid
compounds,
which
have
been
isolated,
demonstrates
the
presence,
in
this
plant,
of
enzymes
capable
of
effecting
cyclization
of
squalene
in
two
different
conformations
leading
to
both
tetracyclic
and
pentacyclic
systems,
and
of
causing
a
number
of
subsequent
modifications.
The
pentacyclic
triterpenes
present
in
the
Euphorbia
are
undoubtedly
as
significant
taxonomically
as
the
tetracyclic
triterpenes
being
investigated
in
this
regard
by
the
French
workers.
2
EXPERIMENTAL
Melting
points
were
uncorrected
and
determined
on
a
Klifler
hot
stage.
Alumina
was
Woelm
neutral
standardized
according
to
Brockmann.
Silica
gel
(Kieselgel)
was
used
for
thin-layer
chromatography
(TLC).
All
specific
rotations
were
determined
in
chloroform.
Petrol
refers
to
light
petroleum;
fractions
of
b.p.
35-60'
used
for
extraction
and
chromato-
graphy
and
fraction
of
b.p.
60-80'
used
for
recrystallization.
Extraction
and
Isolation
of
the
Non-saponifiable
Fraction
Dried
ground
spurge
(410
g)
was
exhaustively
extracted
with
petrol
in
a
Soxhlet
apparatus.
On
standing
the
petrol
extract
deposited
a
product
which
showed
no
triterpenoid
charac-
teristics.
This
substance,
m.p.
77-78
,
[o]
r
,
+
0',
is
almost
certainly
the
previously
reported'
ceryl
alcohol.
The
clear
filtrate
was
concentrated
to
afford
material
(17.1
g)
which
was
refluxed
(15
min)
in
tetrahydrofuran
(200
ml)
with
10%
aq.
KOH
(25
ml).
The
non-saponi-
fiable
fraction
(13.7
g)
was
isolated
from
the
alkaline
solution
with
ether.
Separation
and
Ideniffication
of
Triterpenes
A
partial
fractionation
of
the
non-saponifiable
portion
(13.7
g)
was
achieved
by
chromato-
graphy
on
alumina
(Grade
HI;
180
g).
Successive
elution
with
petrol-benzene
mixtures,
benzene,
and
ether
afforded
the
following
fractions:
(a)
ketone
(1.7
g),
(b)
alcohol
(2.3
g),
(c)
alcohol
(1.8
g),
(d)
alcohol
(0.6
g),
(e)
crystalline
alcohol
(0.7
g),
and
(
f
)
alcohol
(2.3
g).
A
large
fraction
consisting
apparently
of
a
mixture
of
hydrocarbons
(TLC,
i.r.)
w
as
removed
from
the
column
before
the
ketone
fraction.
Glut-5(6)-en-3-one.
Rechromatography
of
fraction
(a)
on
alumina
(Grade
I;
150
g)
and
elution
with
benzene-ether
(9:1)
yielded
a
crystalline
substance
(60
mg),
m.p.
237-240
,
[x]p
+
27
'
(c,
0.57),
Vmax
1700
cm
-1
(in
CHC1
3
)
identified
as
glut-5-en-3-one.
Reduction
with
lithium
aluminium
hydride
gave
an
alcohol,
m.p.
198-201',
which
upon
acetylation
afforded
a
compound,
m.p.
233-236
,
identical
(TLC,
mixed
m.p.,
and
i.r.)
with
authentic
glut-5(6)-
en-30c-y1
acetate.
6
Fractions
(b),
(c),
and
(d)
were
acetylated
since
attempts
to
separate
them
further
proved
unsuccessful.
Ceryl
acetate,
formed
from
ceryl
alcohol
described
above,
could
be
readily
7
C.
DJERASSI
and
R.
MCCRINDLE,
J.
Chem.
Soc.
4034
(1962).
Triterpenoid
constituents
of
Euphorbia
cyparissias
1343
separated
as
it
was
hydrolyzed
by
standing
for
2
hr
on
the
alumina
column
(Grade
I)
whereas
the
triterpenoid
acetates
were
unchanged.
Careful
chromatography
of
fraction
(b)
on
alumina
(Grade
I;
200
g)
with
benzene
con-
taining
increasing
amounts
of
ether
afforded,
in
order
of
elution,
the
following
triterpenoid
acetates,
pure
after
recrystallization:
euphyl
acetate
(283
mg),
P-amyrin
acetate
(trace),
glut-5(6)-en-3x-y1
acetate
(6
mg),
and
glut-5(6)-en-3/3-y1
acetate
(215
mg).
Similarly,
fraction
(c)
afforded
the
following
acetates:
P-amyrin
acetate
(32
mg),
24-methylenecycloartanyl
acetate
(552
mg),
and
glut-5(6)-en-30c-y1
acetate
(10
mg).
Fraction
(d)
was
chromatographed
over
alumina
(Grade
III;
64
g)
and
elution
with
petrol
gave
fractions
which
were
recrystallized
to
afford
the
following:
P-amyrin
acetate
(27
mg),
24-methylenecycloartanyl
acetate
(195
mg),
and
an
unknown
acetate
(3
mg;
m.p.
150-154°).
The
identity
of
these
acetates
was
demons-
trated
as
described
below.
Euphyl
acetate.
Recrystallization
from
chloroform—acetonitrile
yielded
needles,
m.p.
106-107°,
kb,
+
40°
(c,
1.17),
which
upon
hydrolysis
gave
material,
m.p.
112-114°
(ether—
methanol),
[a]p
+
34°
(c,
145).
Treatment
with
benzoyl
chloride—pyridine
gave
a
benzoate
recrystallized
from
ether—alcohol
to
afford
euphyl
benzoate,
m.p.
135-137°,
identified
by
direct
comparison
(TLC,
i.r.,
mixed
m.p.)
with
an
authentic
specimen.
fi-Amyrin
acetate.
Recrystallization
from
ether—petrol
gave
material,
m.p.
239-241°,
undepressed
upon
admixture
with
authentic
p-amyrin
acetate.
Comparison
of
i.r.
spectra
further
confirmed
the
identity.
Glut-5(6)-en-lx-y1
acetate.
Recrystallization
from
chloroform—methanol
gave
needles,
m.p.
236-237°,
identical
(TLC,
i.r.,
mixed
m.p.)
with
authentic
glut-5(6)-en-3a-y1
acetate.
Ghat-5(6)-en-313-yl
acetate.
Recrystallization
from
ether—petrol
afforded
crystals,
m.p.
190-191°,
undepressed
upon
admixture
with
glut-5(6)-en-3p-y1
acetate
prepared
as
described
below.
Glut-5(6)-en-3-one
(60
mg)
in
tetrahydrofuran
(6
ml)
and
alcohol
(2
ml)
was
treated
with
excess
sodium
borohydride
for
20
min
at
room
temperature.
The
product,
shown
by
thin-
layer
chromatography
to
be
a
mixture
of
alcohols,
was
acetylated
(acetic
anhydride—pyridine)
and
chromatographed
carefully
on
alumina
(Grade
I;
10
g).
Benzene—ether
(19:1)
eluted
a
substance
(56
mg)
shown
to
be
glut-5(6)-en-3a-y1
acetate
described
above.
Further
elution
with
the
same
solvent
gave
a
chromatographically
(TLC)
pure
fraction
(3
mg)
recrystallized
from
ether—petrol
to
afford
glut-5(6)-en-3P-y1
acetate,
m.p.
184-187°.
24-Methylenecycloartanyl
acetate.
Recrystallization
from
ether—acetone
gave
material,
[a]D+
56°
(c,
1.37),
v.
1640,
890
cm
-1
(in
CHC1
3
),
softening
in
the
range
80-100°
and
melting
after
resetting
at
107-109°.
On
some
occasions
crystalline
material,
m.p.
111-113°,
could
be
obtained
by
recrystallization
from
chloroform—alcohol.
The
NMR
spectrum,*
showing
bands
at
T
9.14,
9.08,
9.03
for
the
methyl
groups
and
T
9.71, 9.64,
9.46,
9.37
multiplet
attributed
to
a
cyclopropane
grouping,
was
very
similar
to
that
reported
2
for
24-methylenecycloartanyl
acetate.
No
melting
point
depression
was
observed
upon
ad-
mixture
with
authentic
24-methylenecycloartanyl
acetate
Hydrolysis
gave
material,
m.p.
109-113°,
[oc]
D
+
44°
(c,
0.67)
(Found:
C,
84-55;
H,
11.53.
Calc.
for
C
33
H
32
0
:
C,
84.48;
H,
11.89
%).
Admixture
with
authentic
24-methylenecyclo-
artanol
(m.p.
121-122°)
showed
no
melting
point
depression
although
the
melting
point
of
this
substance
was
lower.
Sublimation
in
vacuo
did
not
raise
the
melting
point.
)9-Sitosterol.
Fraction
(e)
on
recrystallization
from
chloroform—methanol
gave
/3-
sitosterol,
m.p.
and
mixed
m.p.
137-138°.
Benzoylation
yielded
p-sitosterol
benzoate,
m.p.
*
Determined
in
CDC1
3
solution
using
a
Varian
A-60
spectrometer
with
TMS
as
an
internal
standard.
1344
A.
N.
STARRATT
and
mixed
m.p.
144-146'
(acetone-methanol).
Further
proof
of
the
identity
was
obtained
from
comparison
of
i.r.
spectra
and
TLC
behavior.
Cycloart-23-ene-313,25-diol.
Fraction
(f)
was
chromatographed
on
alumina
(Grade
III;
117
g)
and
elution
with
benzene-ether
(9:1)
gave
a
fraction
recrystallized
from
chloroform-
petrol
to
afford
an
alcohol
(40
mg),
m.p.
198-201',
[oc]
D
+
40'
(c,
0.56)
(monoacetate,
m.p.
147-149
-
),
exhibiting
no
melting
point
depression
upon
admixture
with
cycloart-23-ene-
3/3,25-dio1.
7
Eluted
slightly
earlier
than
this
compound
was
another
substance,
recrystallized
from
ether-alcohol
as
plates,
m.p.
149-163',
of
insufficient
quantity
to
permit
further
purification
and
characterization.
Acknowledgements—The
technical
assistance
of
Mrs.
M.
Stevens
is
gratefully
acknowledged.
Identification
of
13-sitosterol
was
carried
out
by
Mr.
M.
Ktenas.
The
author
is
grateful
to
Dr.
P.
Harris
for
collecting
the
spurge
and
to
Drs.
C.
Djerassi,
B.
0.
Lindgren,
P.
de
Mayo,
M.
Shimizu,
and
R.
Stesenson
for
prodding
reference
specimens.