THE VERATRINE ALKALOIDS A review of the literature on earlier investigations of the alkaloid ...

12 Pages·2003·0.83 MB·English

THE VERATRINE ALKALOIDS
XXV. THE ALKALOIDS OF VERATRUM VIRIDE
BY WALTER A. JACOBS AND LYMAN C. CRAIG
(From the Laboratories of The Rockefeller Institute for Medical Research, New York)
(Received for publication, July 16, 1945)
A review of the literature on earlier investigations of the alkaloid content
of American hellebore or T’eratrum viride Aiton has been given by Seiferle,
Johns, and Richardson (1). In the earlier work the occurrence of only
jervine, pseudojervine, and rubijervine had been described. In their study
they were able to isolate, in addition to these alkaloids, germine and its es-
ter, protoveratridine. Our own more recent studies of the alkaloids of this
plant have now added to the list and have also necessitated reinterpreta-
tions of earlier data. In a previous paper (2) we have reported on one phase
of this study concerned with the isolation of pseudojervine and the new
alkaloid veratrosine, which were characterized as glucosides of jervine and
veratramine respectively.1 These substances were found in the alcoholic
extract of the plant material which followed preliminary extraction of the
latter with benzene. A study of alkaloids contained in the benzene extract
is the subject of the present report. The yield of partly crystalline crude
basic material obtained after preliminary purification varied with different
commercial sampleso f American hellebore, and any data given as to yields
can be regarded only as approximations. About 11 gm. were extracted
from each kilo of mat’erial. A preliminary separation of the bases was
effected essentially as employed by Saito (3) in his study of Veratrum grandi-
florum Loes. fil. By conversion to the sulfates a large sparingly soluble
sulfate fraction was first obtained which after reconversion to the free bases
was then transformed in alcoholic solution to the hydrochlorides. The
sparingly soluble salt which crystallized was the hydrochloride of jervine,
the principal alkaloidal constituent.
From the more soluble hydrochloride fraction a crystalline’base was iso-
lated which agreed very closely in properties with those recorded by Saito
for veratramine. However, the analytical results obtained with our
alkaloid were in closer agreement with the formulation C&II,,OzN than
with that of C&H8sOzNd erived by him. As a check on the identity of our
substance, it was hydrogenated to a dihydro derivative with properties
essentially as given by Saito for dihydroveratramine. Our analytical data
again were more consistent with a formulation, C27H4102N. Saito’s so
called “diacetyl” derivative was also prepared. His analytical results as
well as our own, however, are more satisfactory for a triacetylveratramine,
555
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556 VERATRINE ALKALOIDS. xxv
C&50$. The latter on saponification with alkali yielded the substance
described by him as a “low melting veratramine,” but which we have since
found to be the neutral W-acetylveratramine, C2J1410JN.
Veratramine, therefore, is an unsaturated secondary base which possesses
two acylatable hydroxyl groups. Since the presence of only one double
bond was directly shown by hydrogenation, and since it is a secondary and
-
220 240 260 280 300 320 340 360 380 4Kl 420
Amm,L
FIG. 1. Absorption spectrum curves. Curve 1, veratramine; Curve 2, dihydro-
veratramine; Curve 3, the new alkaloid; Curve 4, jervine.
presumably steroid base, there remained three points of unsaturation to be
explained.
Information on this point was derived from the ultraviolet absorption
spectrum of the alkaloid. Curve 1, Fig. 1, was obtained in ethanol solution
with the Beckman quartz spectrophotometer. The similarity of the curve
to that recorded in the literature for neoergosterol (4) and for trianhydro-
strophanthidin (5), as contrasted to those recorded for simple conjugated
double bonds, leaves little doubt that the three points of unsatnration are
W. A. JACOBS AND L. C. CRAIG 557
contained in a benzene ring system in veratramine. If this is the case, and
the alkaloid has the formula C&,H3902N, then some modification of the
regular sterol skeleton must be present,, since in a regular sterol with 27
carbon atoms an angular methyl group would be inconsistent with the
benzenoid character of the ring to which it is attached. In veratramine the
heterocyclic port.ion could not give the ultraviolet absorpt.ion spectrum
noted because of the secondary character of the nitrogen atom.
Since veratramine forms the glucoside, veratrosine, presumably analogous
to pseudojervine, it is probable that there is present the usual hydroxyl
group on carbon atom 3 and since no phenolic hydroxyl can be detected,
Ring A should not be benzenoid. In work with jervine to be described on a
later occasion, it will be shown that this alkaloid reacts in a way consistent
with a hydroxyl group on carbon atom 3 and a A5 double bond, since it can
be converted through a ketone, presumably A4-jervone, to an allojervine
which gives the color test of a 3-hydroxy-A4 derivative. Jn the case of
veratramine, although it was changed readily to amorphous material with
aluminum tertiary butylate, no evidence could be obtained that this was of
the usual 3-0x0-A4 character. It appears probable, as a tentative interpre-
tation, that Ring B rather than Ring C is the site of the benzenoid structure
and that, in consequence, the usual steroid angular carbon atom 18 attached
to carbon atom 10 has been shifted in veratramine to carbon atom 1, as has
been assumed to occur in trianhydrostrophanthidin (6).
Although the veratrine alkaloids are unquestionably steroid, attempts to
relate any one of them directly to the sterols by dehydrogenation to Diels’
hydrocarbon have in each case given a different result. Rubijervine is
precipitated by digifonin and gives the transformations of cholesterol (7)
in regard to a 3(P)-hydroxy-A5 structure, but no Diels’ hydrocarbon was
detected upon dehydrogenation. Instead, a lower melting isomeric hydro-
carbon (8) was obtained which was shown to possess a phenanthrene ring
system by ultraviolet absorption spectrum study. Its properties and those
of its trinitrobenzene derivative approximated closely the values recorded
in the literature for 1’-methyl-1,2-cyclopentenophenanthrene (9). The
possibility must also be considered, among others, that this hydrocarbon
can prove to be 5-methyl-1,2-cyclopentenophenanthrene. The formulation
of the latter would be consistent with a methyl group on carbon atom 1. It
is now our intention to determine the identity of this hydrocarbon by
comparison with synthetic material. Although this substance is a high
temperature product, if it should prove to be I’-methyl-l ,Zcyclopenteno-
phenanthrene, its formation might raise the question as to whether carbon
atom 15 in rubijervine is the site of a methyl group instead of carbon atom
13.
In the case of dihydroveratramine, the absorption curve, Curve 2, Fig. 1,
558 VERATRINE ALKALOIDS. XXV
has been found to resemble quite closely that of veratramine, and therefore
indicates not only retention of the benzenoid structure but that the double
bond of veratramine which can be hydrogenated is not conjugated with the
benzene ring. If the base contains the usual angular methyl group on
carbon atom 13, this double bond would be restricted to Ring D or the sterol
side chain which carries the nitrogen atom.
Contrary to jervine, veratramine was found to be relatively resistant to
acid and was recovered unchanged when submitted to the action of methyl
alcoholic HCI. As previously described, veratrosine yields veratramine and
d-glucose on hydrolysis.
An appreciable fraction of alkaloidal material remained in solution as
more soluble salts after precipitation of the above sparingly soluble sulfate
fraction. A partial separation of the bases recovered from this fraction
was effected by chromatographing through aluminum oxide. The major
portion was gradually eluted from the column with benzene which contained
2.5 per cent of methanol. The crystalline substance which emerged with
amorphous material in the early fractions proved to be identical with the
isorubijervine first obtained from Veratrmm album. This overlapped into
later fractions which contained rubijervine. The separation of rubij ervine
from isorubijervine and a relatively large amount of amorphous material
was effected by taking advantage of the sparing solubility in moist chloro-
form of rubijervine which contained water of crystallization.
After the elution of rubijervine, at first only amorphous material was ob-
tained, but fractions were then encountered from which it was possible to
isolate a new crystalline alkaloid in very small amount. 12 kilos of plant
material yielded about 0.5 gm. This alkaloid contained solvent and, after
preliminary softening, melted up to about 170-175”, then crystallized again
and remelted at 272-274”. The analytical data suggested a provisional
formula, C27H4104N (or C&H3904N). This was supported by analyses of a
neutral N-acetyl derivative, C29H430~N, and of a nitroso derivative,
C2,H4,,05N2. The formation of these derivatives showed the secondary
basic character of the alkaloid which is thus the third secondary alkaloidal
base to be isolated in succession t.o jervine and veratramine (and their glu-
cosides). The N-acetyl derivative was obtained by saponification of the
uncrystallized product of the direct acetylation of the alkaloid. The num-
ber of OH groups involved in this intermediate acetyl derivative was not
determined. The alkaloid did not react with hydroxylamine.
The ultraviolet absorption spectrum, Curve 3, Fig. 1, obtained with the
alkaloid indicates the presence of conjugated double bonds and resembles
the curve obtained on a previous occasion with jervine (lo), except that a
second but lower broad band with a maximum at 360 rnp has been found.
Since the earlier observations with jervine had not been extended so far,
W. A. JACOBS Ai’iD L. C. CRAIG 559
the latter has now been reexamined and has been found to exhibit a similar
second broad band with a maximum at 360 rnp, Curve 4, Fig. 1. Although
certain implications might be drawn from these observations, their inter-
pretation will be left to a later occasion in connection with other data which
will be available.
There was evidence of the presence of other crystalline alkaloids in frac-
tions from which the new alkaloid was obtained but in amounts too small to
make profitable their study at the moment. Further elution of the column
with benzene which contained more methanol continued to yield fractions
of amorphous material from which small amounts of germine were even-
tually obtained. Germine was more readily isolated, although in small
amount, by continued extraction with chloroform of the aqueous phase
which remained after the preliminary removal of the more easily extracted
major alkaloid fraction discussed above. The identity of this germine was
confirmed by the preparation of the acetonyl derivative (11).
A relatively large amorphous fraction of alkaloid material still remained
which will require future study.
EXPERIMENTAL
Ground roots and rhizomes of commercial Veratrum viride were extracted
essentially as previously described (2) in portions of 2 kilos with benzene
and dilute ammonia. The combined benzene extracts from 6 kilos were
concentrated under reduced pressure and brought to 4 liters in benzene.
This was extracted repeatedly with portions of a total of 5 liters of 5 per
cent aqueous acetic acid. The free bases were reprecipitated from the
aqueous ext,ract with excess 25 per cent NaOH and then reextracted with
benzene. A small persistent precipitate which gradually separated at the
interface and retarded separation was discarded and the benzene solution
after washing with water was dried over sodium sulfate. The remaining
alkaline aqueous phase by further repeated extractions with chloroform
yielded the germine to be described below. The benzene solution on con-
centration to dryness gave a partly crystalline residue of approximately 65
gm. of a crude alkaloid mixture. The solution of this material in 950 cc.
of 5 per cent acetic acid was treated with 135 cc. of saturated ammonium
sulfate solution. The copious rather gelatinous precipitate was warmed,
which caused partial formation of minute microcrystalline aggregates. It
was collected with 5 per cent ammonium sulfate solution. The precipitate
was resuspended in fresh wash solution and recentrifuged several times to
insure proper washing of the crude sulfate. The soluble sulfate fraction
contained in the mother liquors was worked up as given below. The sulfate
was then resuspended in water and treated with excess NaOH solution.
The mixture was shaken thoroughly with chloroform to redissolve the lib-
560 VERATRINE ALKALOIDS. XXV
erated bases. The extract was separated by centrifugation and after
washing with water was cleared with sodium sulfate and concentrated to
small bulk. After addition of absolute ethanol, all residual chloroform was
boiled off. To the mixture (250 cc.), which had partly crystallized, HCl
(sp. gr. 1.19) was carefully added in slight excess. Immediate deposition of
jervine hydrochloride occurred. After cooling and collection with absolute
ethanol, 32 gm. were obtained. Partial concentration of the mother liquor
under reduced pressure yielded a small additional fraction. If carried too
far, this material was contaminated with the salt of veratramine.
When the hydrochloride was dissolved in hot, somewhat diluted ethanol
and decomposed with ammonia, the base separated as needles which melted
at 240-241” (uncorrected).
[a]: = -146” (c = 0.99 in absolute ethanol)
Cn~HBsOsN. Calculated, C 76.18, H 9.24; found, C 76.24, H 9.22
Verutramine-The mother liquor from crude jervine hydrochloride was
treated with excess ammonia and carefully diluted to incipient turbidity.
Veratramine gradually separated as a copious deposit which increased on
further dilution. The alkaloid was collected with 50 per cent ethanol and
additional amounts were obtained from the mother liquor. The yield was
7to8gm.
On recrystallization from 95 per cent ethanol, it separated as delicate
needles which melted at 204-207” after preliminary sintering and contained
water of crystallization; the air-dried material showed no ethoxyl content.
[oL]: = -69” (c = 0.98 for the anhydrous substance in methanol)
For analysis, the substance was dried at 110” and 0.2 mm.
C&H3902N. Calculated. C 79.16, H 9.60
Found. (a) “ 78.88, ‘I 9.57
@I I‘ 78.69, “ 9.73
Saito reported a rotation of [c,]:’ = -70” (c = 1.065 in methanol) ap-
parently for the undried material.
Attempts to raise the melting point to 209.5-210.5” as reported by Saito
were unsuccessful by simple recrystallization. This was accomplished,
however, as follows, which suggestst hat small persistent amounts of con-
taminating jervine may have been removed in the process.
0.2 gm. of the product was dissolved with chilling in 4 cc. of methanol
saturated at 0” with HCI. The solution was allowed to come to room tem-
perature in a sealed tube. After 1.5 hours the base was recovered by dilu-
tion, addition of alkali, and extraction with chloroform. It crystallized
from dilute ethanol as delicate needles and after recrystallization melted at
W. A. JACOBS AND L. C. CRAIG 561
209-210.5”. There was no appreciable depression when mixed with vera-
tramine.
[(Y]: = -68” (c = 0.88 for the anhydrous substance in methanol)
For analysis, the substance was dried at 110’ and 0.2 mm.
Found, C 79.12, H 9.65
As a check on its identity, it was acetylated and the product was found to
be indistinguishable in properties and melting point from the triacetyl
derivative described below.
Dihyclroveratramine-O.2 gm. of veratramine was hydrogenated with 50
mg. of platinum oxide catalyst in acetic acid solution, as described by Saito.
The base recovered from the reaction was obtained as leaflets from dilute
ethanol which melted at 198-200” after slight preliminary sintering. Saito
reported 197-198”.
For analysis, it was dried at 110” and 0.2 mm.
C&TH~~O~N. Calculated, C 78.77, H 10.05; found, C 78.75, H 10.10
Triucetylveratrumine-This was prepared, essentially as described by
Saito, by boiling in acetic anhydride but for only 30 minutes. The residue
obtained on concentration crystallized directly from dilute ethanol. On
recrystallization from 95 per cent ethanol, it formed needles which at about
120-130” showed a change in appearance, presumably due to loss of solvent,
and then melted at 204-206” after preliminary sintering. Saito reported
205-206.5” for his ‘Ldiacetylveratramine.‘J
For analysis, it was dried at 110’ and 0.2 mm.
CSaHa60aN. Calculated. C 73.97, H 8.47
Found. (a) ‘( 74.00, “ 8.25
@I “ 74.09, “ 8.47
Acetylveratramine-80 mg. of the triacetyl derivative were refluxed for 2
hours in 2 cc. of 2 N NaOH in absolute ethanol. On dilution to incipient
turbidity, it gradually crystallized. When recrystallized from dilute
ethanol, it formed almost rectangular leaflets which melted at 177-180”
after slight preliminary softening. Saito reported 179-180”. It did not
dissolve in dilute acids.
For analysis it was dried at 110” and 0.2 mm.
C~QH~~O~N. Calculated, C 77.11, H 9.16; found, C 77.07, H 9.21
The combined mother liquor and washings of the crude insoluble sulfate
fraction were made alkaline and repeatedly extracted with chloroform.
562 VERATRINE ALKALOIDS. XXV
The washed extract was cleared with sodium sulfate and concentrated. A
resinous residue of mixed bases was obtained which varied in different ex-
periments from 16 to about 25 gm. for 6 kilos of plant material.
32.5 gm. of mixed bases were dissolved in 200 cc. of benzene and passed
through a column of 650 gm. of active alumina. Attempted elution with
twelve 100 cc. portions of benzene was ineffective and so 2.5 per cent
of methanol in benzene was used. After a liter had passed through, dis-
solved material began to emerge from the column and was collected in 100
cc. portions. The amounts obtained in each fraction after removal of sol-
vent are recorded in Table I. Data obtained beyond Fraction 32, how-
TABLE I
Chromatogram of Bases from Soluble Sulfate Fraction
Fraction No. Weight &ted Fraction No. Weight &ted
I I
2.5% methanol 5% methanol
gm. m.
1 0.37 17 0.05
2 1.9 18 0.05
3 3.05 19 0.05
4 4.1 20 0.05
5 3.6 21 0.05
6 3.0 22 0.06
7 2.02 23 0.08
8 1.35 24 0.10
9 1.05 25 0.10
10 0.71 26 0.10
11 0.35 27 0.15
12 0.27 28 0.19
13 0.17 29 0.18
14 0.15 30 0.14
15 0.09 31 0.10
16 0.05 32 0.09
ever, in which 10 per cent methanol was employed, are not recorded.
About 70 per cent of the original weight was recovered in Fractions 1
through 15.
Each fraction up to Fraction 16 was dissolved in a small volume of chloro-
form and treated with a few drops of water to furnish water of crystalliza-
tion for rubijervine. The latter crystallized only on seeding in small
amount in Fraction 5, then in gradually increasing relative amounts up to
Fraction 15, and again diminished. In each casei t was collected with moist
chloroform. The combined yield was 1.5 gm. On recrystallization from 95
per cent ethanol, it crystallized as needlesw hich melted at 242-244’.
W. .J. JACOBS AND L. C. CRAIG 563
[a],” = +20.5” (c = 0.97 in absolute ethanol)
For analysis, it was dried at 110” and 0.2 mm.
CUHGO~N. Calculated, C 78.38, H 10.49; found, C 78.38, H 10.61
Fractions 2 to 5 (after filtration from rubijervine) were concentrated to
remove chloroform, and the residue was dissolved in ethanol. On careful
dilution, isorubijervine crystallized copiously as needles and was collected
with 70 per cent ethanol. 2.3 gm. were obtained, which melted at 216”.
The rubijervine mother liquors of Fractions 6 to 15 were combined and,
after removal of chloroform, yielded slowly an additional 1.2 gm. of crude
isorubijervine.
On recrystallization from dilute ethanol, isorubijervine was obtained
which contained solvent and melted at 213-215” after preliminary sintering.
For analysis, it was dried at 110” and 0.2 mm.
Found, C 78.42, H 10.57
In other cases, the relative and absolute yields of the two alkaloids varied
considerably and appeared to depend somewhat upon striking optimum
manipulative conditions for their separation from the larger amount of still
uncharacterized alkaloid material.
The Alkaloid C’z~H4104N (or C2iH3904N)-AS Fraction 1G emerged from
the column, the eluting solvent was changed to 5 per cent methanol in ben-
zene. Fractions 20 to 32 were found to contain a new alkaloid which crys-
tallized from a methanol solution of the residues obtained after removal of
the eluting solvent. Crystallization was induced by seeding with material
obtained in preliminary work. The yield was 0.48 gm. In another ex-
periment, 44 gm. of chromatographed alkaloid mixture (from 12 kilos of
plant material) yielded 0.5 gm. of the new alkaloid.
On recrystallization from methanol, it formed six-sided platelets, some
sufficiently long and narrow to constitute needles. After preliminary sin-
tering above 130”, it gradually softened to a melt with effervescence at
about 170-175”, crystallized again on further heating, and then melted at
272-274”. It dissolved readily in chloroform, acetone, appreciably in ben-
zene, rather sparingly in methanol or ethanol, and very sparingly in ether.
[a]: = -78” (c = 0.63 in methanol)
It contained solvent and for analysis was dried at 110’ and 0.2 mm.
C~TH~~O~N. Calculated. C 73.03, H 9.32
CL,HagOgN. “ “ 73.42, “ 8.91
Found. (a) “ 73.41, “ 9.30
(b) “ 73.10, “ 9.42
(cl “ 72.93, “ 9.24
564 VERATRINE ALKALOIDS. XXV
The new alkaloid did not yield a sparingly soluble sulfate. The hydro-
chloride crystallized in the presence of excess dilute HCl.
The N-Acetyl Derivative-The alkaloid was boiled for a minute with acetic
anhydride. After decomposition with water, the mixture was directly
extracted with chloroform. The latter after concentration left a resin of
the polyacetyl derivative which did not crystallize. It was dissolved in a
small volume of methanol, treated with excess alkali, and boiled a ‘few
minutes to saponify the ester groups. The diluted mixture was acidified
with HzS04 and all neutral material was extracted with chloroform. The
latter on concentration yielded a resin which finally crystallized from a
small volume of dilute methanol. It formed flat micro prisms or short
broad needles which contained solvent and melted at 168-170”.
For analysis, it was dried at 100” and 0.2 mm.
CZQHQO~N. Calculated, C 71.70, H 8.93; found, C 71.41, H 9.07
The Nitroso Derivative-50 mg. of the alkaloid were dissolved in 2 cc. of
water and 0.2 cc. of acetic acid. Upon addition of sodium nitrite solution,
it soon became a mass of crystals. After standing, the mixture was ex-
tracted with chloroform and the extract was shaken with dilute H&SO+ then
with water, and dried. This yielded on concentration material which crys-
tallized from dilute ethanol as lustrous, microscopic, six-sided platelets, and
melted at 263-265”.
It contained solvent and for analysis was dried at 110’ and 0.2 mm.
CLTH~~OEN~. Calculated. C 68.60, H 8.53
Found. “ 68.91, “ 8.48
Germ&e-The above alkaline aqueous phase which remained after re-
extraction of the crude alkaloid mixture with benzene was subsequently
extracted twelve times with chloroform. The combined extracts after
drying and concentration yielded a residue of 1.8 gm. When dissolved in
methanol and seeded with germine, crystallization occurred. 0.23 gm. of
alkaloid was obtained, although continued extraction of the aqueous phase
yielded additional amounts of the alkaloid. On recrystallization from
methanol, it formed small prisms which melted gradually at 163-168” to a
paste filled with bubbles.
[a]: = +5” (c = 0.97 in 95% ethanol)
For analysis, it was dried at 110” and 0.2 mm.
C&HpaOsN. Calculated, C 63.61, H 8.51; found, C 63.52, H 8.70
For further characterization, the acetonyl derivative was prepared as
previously described (11) for acetonylgermine through the hydrochloride,