THE ACONITE ALKALOIDS

XVII. FURTHER STUDIES WITH HETISINE

BY WALTER A. JACOBS AND CHARLES F. HUEBNER

(From, the Laboratories of The Rockefeller Institute for Medical Research, Netit York)

(Received for publication, May 14, 1947)

In the course of the isolation of the alkaloids from the roots of Aconitum heterophyllum obtained in a more recent commercial shipment fromIndia, it has been found that the base hetisine occurs in larger amounts than originally suspected. About, 1 gm. per kilo has now been isolated.

The procedure used for the separation of the alkaloid fractions was es- sentially as previously described (1). But following the extraction of the strongly basic alkaloid fraction with benzene a continued extraction of the remaining aqueous phase with chloroform has now yielded a fraction which contains most of the hetisine and which could be readily isolated as the hydrochloride. To complete the extraction it was found necessary to continue the process for a number of days. The isolation of this material has made possible a more extensive study of the alkaloid which became especially desirable because of its relatively simple formulation of CzoH,703N. Further analytical studies with the base and its derivatives have confirmed this formulation.

At first the attempt was made which was but partly successful to carry its degradation through the steps of exhaustive methylation. Methyl- hetisinium iodide was readily prepared, which was in turn converted to the methine base, des-methylhetisine. The latter and its hydrochloride were crystalline. The base with methyl iodide yielded a crystalline methyl- des-methylhetisinium iodide from which a chloride was also prepared. When, however, the attempt was made at the next stage of degradation to pyrolyze the ammonium hydroxide, methanol was split off instead of water with the production again of des-methylhetisine. This result was accompanied by the production of considerable unidentified amorphous material. How- ever, the attempt to repeat the methylation of this amorphous fraction, followed by pyrolysis of its ammonium hydroxide, resulted again inalmost quantitative recovery of basic material. The experiment was also unsuc- cessful at this stage for an Emde reduction to the methochloride.

As a modification of the procedure, des-methylhetisine hydrochloride was hydrogenated. At this point, instead of a tetrahydro derivative to be expected because of the original reactive double bond present in hetisine as well as that of the new methine bond, only 1 mole of II, was absorbed with the production of a dihydro-des-methylhetisine hydrochloride. The free

189

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190 ACONITE ALKALOIDS. XVII

base C2&03N could not be crystallized. It seems probable, from the experience with dihydrohetisine discussed below, that t,he new mcthine bond must be the one hydrogenated and that in some way the original hetisine double bond has lost it#s reactivity. The free base yielded an ap- parent mixture of possibly isomeric crystalline derivatives with methyl iodide, each of which analyzed as a dihydro-des-methylhetisinium methiodide. Here again pyrolysis of the hydroxide resulted in loss of methanol with reformation of dihydro-des-methylhetisine.

Of interest was the behavior of dihydro-des-methylhetisine on oxidation with permanganate. It was smoothly converted to a base, C21H2903, which readily crystallized and proved to be different from and apparently isomeric with des-methylhetinine. It is hoped that we can return to its study at a later time.

An attempt was made to apply the Hofmann degradation to dihydro- hetisine as a starting point. However, a difficulty was at once encountered in the failure to obtain crystalline substances. At t’his point it may be mentioned that dihydrohetisine, previously described only as the hydro- chloride, has since been obtained as the crystalline base. Methyldihydro- hetisinium iodide was obtained only as a resin. Nevertheless, the free hydroxide was pyrolyzed to yield des-methyldihydrohetisine. The resinous base gradually crystallized on standing, but no satisfactory method for its recrystallization was found. On hydrogenation as the HCl salt, it ab- sorbed 1 mole of HZ to form an amorphous dihydro-des-methyldihydro- hetisine. Since the original double bond of hetisine had been disposed of, it appears that the new methine double bond must have been the point, of hydrogenation. The hydrogenated base gave in turn a resinous methi- odide. However, on pyrolysis of the ammonium base, it lost methanol, as shown by the N-methyl content of the sublimed material.

A preliminary study of the dehydrogenation of hetisine with selenium has also been made. A tarry reaction product was obtained which could be separated into neutral and basic fractions. From the neutral fraction, by chromatographing through A&O3 and sublimation, it was possible to isolate several hydrocarbon fractions. One of these proved of significance, since it yielded, although in very small amount, a fraction which was finally crystallized as the trinitrobenzene compound. The hydrocarbon re- covered from the latter was found to be pimanthrene, as shown by analysis, absorption spectra (Fig. l), and by comparison of its properties and those of the trinitrobenzene compound with a sample of pimanthrene already isolated from staphisine (2). As in the case of the latter, the possibility of a partly hydrogenated diterpenoid structure is therefore suggested in the make-up of hetisine.

The major part of the hydrocarbon fraction from hetisine was a non-

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IV. A. JACOBS AND C. F. HUEBNER 191

fluorescent material with a comparatively high hydrogen content. It could be separated from the fluorescent phenanthrene hydrocarbons with A&OS. The molecular weight and analytical data indicated C&O, or a system of six rings, as the formulation of the material. Since the fraction was a liquid and available in too small amount for fractional distillation, there could be no assurance of its homogeneity. The notable fact is, however, that a fraction of such comparatively high molecular weight and high hydrogen content is produced. -4lthough t,his might suggest a pos-

4.6

3.8

2200

FIG. 1. 0, pimanthrene from hetisine; @, pimanthrene from staphisine

sible double molecule for hetisine of C40H640&2, such a hydrocarbon could result from the condensation of smaller fragments during the dehydrogena- tion. This was shown by the determination of the molecular weight of hetisine. Because of its sparing solubility in camphor as originally re- ported, the Barger method has since been used which verified the simpler molecule of CzoHz703N.

At this point it may be reported that the failure of hetisine to consume significant amounts of lead tekaacetate or sodium periodate are against the presence in it of reactive vicinal hydroxyl groups.

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192 ACONITE ALKALOIDS. XVII

The basic fraction from the dehydrogenation was similarly separated with alumina into a number of fractions and at least partly purified by recrystallization of the picrates.

Three fractions were obtained, one which on analysis gave C~~HUN.- CGH307N3, a second either as C~~H~~N.CJ&O~N~ or C~~HX,N*C~H~OYN~, and a third as C19H260N. CcHs07N3. The first and second fractions may be identical. These substances were produced in very small amounts and, since the free bases themselves could not be crystallized, any attempt to interpret their significance would be premature.

3.8

3.4

3.0

2.6

IL, 2.2

0” 4 1.8

1.4

1.0

0.6

I

0.2 2000 2200 2400 284x 2rm 3330 31

FIG. 2. 0, hetisine in ethanol; O, hetisine in ethanol with excess HCl

The ultraviolet absorption spectra of hetisine and its hydrochloride taken in alcoholic solution (Fig. 2) were found to show much less difference than in the case of atisine and heteratisine. The absorption as seen in Fig. 2 is mostly end-absorption of an unspecific type which does not permit of obvious conclusions.

Since the nitrogen atom of hetisine is tertiary and apparently common to two rings, the additional three rings of the perhydrophenanthrene portion would require a minimum of five rings for the alkaloid. Although no crystalline 0-acyl derivatives of the latter have been obtained, the active hydrogen determination has indicated the presence of three OH groups (1).

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W. A. JACOBS AND C. F. HUEBNER 193

One double bond has been shown by hydrogenation. From the formula- tion CzoH2703N for the base, there remain therefore two undetermined points of unsaturation. Whether these consist of resistant double bonds or rings remains a problem.

EXPERIMENTAL

The rhizomes of Aconitum heterophyllum used in the following studies were obtained from an Indian source through S. B. Penick and Company.

The procedure for the extraction of the ground material was essentially as previously described, with a few changes. Each 8 kilo lot was extracted for a day vuith 24 liters of 70 per cent alcohol and then by three succeeding daily treatments with 16 liters of solvent. After concentration of the ex- tract to a thin aqueous syrup and acidification with 10 per cent H$Oa, the mixture was extracted four times with a total of 2 liters of chloroform, which in turn was washed several times with small portions of water. The combined aqueous fractions were carefully neutralized with Na3C03 solu- tion and extracted with three 1 liter portions of benzene. This benzene extract of the weaker bases was washed several times with minimal amounts of water and kept separate. The combined aqueous phase was then emulsified with 1500 cc. of benzene and rapidly made strongly alkaline with 200 cc. of 25 per cent NaOH and well shaken. The extraction was repeated six times with 500 cc. portions of benzene. After washing with several small portions of water the dried extract was concentrated in vacua to a resin of bases which were in turn converted as previously described into the HCI salts in absolute alcoholic solution. Successive fractions of crystalline salts were obtained which totaled about 40 gm. from each 8 kilos and consisted mainly of atisine hydrochloride, although the final fractions yielded small amounts of heteratisine and hctisine hydrochloride.

Following the above extraction with benzene, the alkaline aqueous phase (about 4 liters) was extracted consecutively in several funnels six times with a total of about 1500 cc. of chloroform. The washed and dried ex- tract yielded a resinous residue which was converted in alcoholic solution to the HCl salt. The latter crystallized copiously and after collection weighed about 4.3 gm. At this point the amount varied in different ex- periments, the variation being possibly due to differences in the extraction conditions. Such differences, however, were as a rule compensated for by the amount obtained in the succeeding continuous extraction.

[(Y]: = $12.5” (C = 1.01 in 05% ethanol) CzoH,03N.HC1. Calculated. C 65.63, H 7.72

Found. (a) “ 65.48, “ 7.61 (6) “ 65.10, “ 7.42

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194 ACONITE ALKALOIDS. XVII

Under the microscope the substance changed above 320” and gradually lost its double refraction at 328-335”.

For comparison, a sample of hetisine hydrochloride was prepared from the hetisine previously described (1). Under t,he microscope it. began to change above 320” and gradually lost. its double refraction at 330-340”.

[al: = +12.7” (c = 1.06 in 95% ethanol) Found, C 65.80, I-T 7.72

At this stage it was found advisable to continue the extraction process in a continuous liquid extractor for 5 or 6 days. After each day the boiling chloroform solution of extracted material was replaced by fresh solvent, since an apparent condensation of chloroform with substances in the al- kaline aqueous phase caused formation of obscure resinous productswhich made purification of the alkaloid more difficult. The basic material re- covered from the combined chloroform extracts was converted in alcoholic solution to the HCI salt. 3.8 gm. more of hetisine hydrochloride were thus obtained.

Found, (a) C 65.45, H 7.72; (b) C 65.23, H 7.45

The total amount of crude hetisine hydrochloride recovered from ap- proximately 119 kilos of ground atis root was about 112 gm.

The base prepared from the hydrochloride separated gradually from di- lute alcohol as glistening stout prisms which contained solvent and ef- fervesced at 145”. The melting point varied with the conditions of crystallization. For analysis it was dried at 100” and 0.2 mm.

CzaHz,03N. Calculated, C 72.90, I-1 8.27; found, C 72.90, H 8.03

Hetisine was found to sublime almost completely at 0.1 mm. from a bath at 190-210’. If the material used contained a solvent, a small amount flashed over even at 145-160’.

Found, C 72.89, H 8.23

A solution of 41.2 mg. of hetisine in 1 cc. of ethanol was found in equi- librium with a solution of 42.0 mg. of isoatisine (mol. wt. 343.3) in 1 cc. of ethanol, as determined by the Barger method.

CZOHZ~OZN. Calculated, mol. wt., 329.3; found, 336

Dihydrohetisine-Instead of the procedure originally reported, the con- ditions used by Suginome and Shimanouti (3) for the hydrogenation of kobusine were repeated here in an unsuccessful effort to carry the hydro- genation beyond the dihydro stage.

0.1 gm. of alkaloid dissolved in acetic acid was hydrogenated with 50 mg. of platinum oxide catalyst,. After the prompt absorption of approxi-

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W. A. JACOBS AND C. F. HUEBNER 195

mately 1 mole beyond the catalyst requirement, there was no significant further absorption, even after addition of more catalyst. After 1 week the process was discontinued; the filt,rate from the catalyst was concen- trated, the base was liberated in aqueous solution with alkali, and then ex- tracted with chloroform. A resin was obtained on concentration which was dissolved in a small volume of methanol. After addition of water the solution was boiled down to 3 cc. to remove methanol. On seeding, di- hydrohetisine crystallized slowly and incompletely as pointed microprisms which contained solvent. Under the microscope it softened to a resin at 136-139” which gradually crystallized again on further heating. The ap- parently anhydrous material then partly softened, especially above 220”, and melted at 250-255”.

For analysis, the substance was dried at 100” and 0.2 mm.

C2~H2~03N. Calculated, C 72.45, H 8.82; found, C 72.27, H 8.77

Methylhetisinium Iodide-Q.3 gm. of hetisine was heated with 1 cc. of methyl iodide in a sealed tube at 100” for 2 hours. The partly crystalline mass was boiled down to remove the excess reagent and treated with methanol. After concentration to smaller volume and addition of ether, the reaction product crystallized as needles or rods. 0.19 gm. was col- lected in a first fraction. Under the microscope the substance, after pre- liminary sintering above 300”, did not lose its double fraction till 320-325”.

C21H,003NI. Calculated. C 53.48, H 6.42, I 26.94 Found. $‘ 53.28, “ 6.26, “ 26.94

lrhe mother liquor on further addition of ether gave a second fraction of 0.12 gm.

Found, C 53.22, H 6.28, I 26.32

Des-methylhetisine-2.5 gm. of the methiodide were dissolved in a small volume of methanol and decomposed with a slight excess of a suspension in methanol of freshly prepared Ag,O. The filtrate was concentrated in the sublimation apparatus under reduced pressure and the residual resinous mass was gradually heated to 100” under t’he water pump as long as bubbling of the resin (due to loss of solvent and perha.ps water) continued. The pressure was reduced with an oil pump to 0.3 to 0.4 mm. and the heating continued with progressive temperature elevation to about 160” in 20 minutss. Although some condensation on the condenser became apparent above 335”, possibly due to continued II20 cleavage, the sub- limation became especially profuse and rapid from 170-180”. In the course of an additional 20 minutes the bath was raised to 200” and the opera- tion then interrupted. The practically colorless resinous sublimate was

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196 ACONITE ALKALOIDS. XVII

washed off with methanol and again freed from solvent. The yield was 1.55 gm. This material gradually crystallized on long standing, but the base was preferably prepared from the hydrochloride described below by addition of excess of ammonia to its aqueous solution. The base separated as sparingly soluble flat needles which showed a micro melting point of 122-124’ after slight preliminary softening.

For analysis it was dried at 100” and 0.2 mm.

Cz,HzpO~N. Calculated, C 73.42, H 8.52; found, C 73.30, H 8.55

The hydrochloride crystallized from methanol-ether as needles which melted under the microscope at 303-305” after softening above 297”.

CZ~HMO~NCI. Calculated. C 66.37, H 7.96, Cl 9.34 Found. “ 66.20, “ 7.70, “ 9.24

Methyl-des-methylhetisinium Iodide-l.55 gm. of the previous des base were heated with 8 cc. of methyl iodide for 3 hours. The initial clear solution deposited a resin which crystallized as arborescent masses of prisms. After removal of the excess reagent, the mass was crystallized from chilled methanol as colorless prisms. The first two fractions (a and b) amounted to 1 gm.

C22H3203NI. Calculated. C 54.41,H 6.65, I 26.16, N(CHa) 6.18 Found. (a) “ 54.42, “ 6.55

“ (b) “ 54.56, “ 6.49, N(CHa) 6.10, 5.90 “ “ 54.79, “ 6.68, “ 25.78

After recrystallization from methanol, it sintered under the microscope above 237” and gradually softened to a melt at 246-250”.

When the attempt was made to recover material in the original mother liquor by the addition of ether, the obvious peroxide content of the latter caused liberation of iodine with crystallization of further fractions, but these were found to be high in iodine, apparently due to the addition of the latter to a double bond. This material was not further studied.

Chloride-The chloride was obtained from the iodide in methanol with AgCl. It crystallized from methanol-ether as flat needles which gradually melted under the microscope at 285-290”.

CzsHJ203NC1. Calculated. C 67.05, H 8.19, Cl 9.01 Found. “ 67.04, “ 7.95, “ 8.80

The attempt was made to carry the methiodide through the next stage of degradation. 0.15 gm. of methiodide was converted with Ag,O in methanol to the hydroxide. After removal of solvent, the resinous residue was slowly heated in a sublimation apparatus at 0.2 to 0.3 mm. The bath during 20 minutes gradually reached 250-255” and was kept at this point 15 minutes more. The sublimate was washed off with methanol and after

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W. A. JACOBS AND C. F. HUEBNER 197

removal of the solvent weighed 0.11 gm. In methanol it yielded a hydro- chloride (46 mg.) which separated as delicate needles on addition of ether. Analysis, however, showed the loss of a CH3 group and reconversion to des-methylhetisine hydrochloride.

CzlHaoOaNC1. Calculated. C 66.37, H 7.96, N(CHs) 3.95 Found. (a) “ 66.77, “ 7.93 “ 4.44

(b) “ 66.25, “ 7.78, “ 4.30

Since again in a second experiment, but with a larger amount, only a fraction of the sublimed base crystallized as the hydrochloride (0.2 gm. from 0.53 gm. of sublimed base), the attempt was made to carry the dissolved’ material through the final step of degradation if still a dimethyl derivative. After removal of Cl- in methanol with AgzO, during which an odor of trimethylamine became apparent, the dissolved base was freed from solvent and sublimed as usual. 0.33 gm. of resinous sublimate was obtained which was found to be still entirely basic. The material could not be crystallized.

Dihydro-des-methylhetisine-1.42 gm. of des-N-methylhet,isine hydro- chloride were hydrogenated in methanol with 0.1 gm. of PtOz catalyst. After 3 hours the reaction appeared complete when 87 cc. or 1 mole of HZ had been absorbed by t,he substance.

The product separated in a volume brought to 5 cc. as prismatic needles. 0.83 gm. was collected at 0” with methanol. Under the microscope it began to change above 300” but especially at 308-310”, and then disap- peared by apparent sublimation from the hot stage at 315-318”. This general behavior, the point of which varied somewhat with different samples or fractions, was not altered by recrystallization from methanol.

CZIH320~NCI. Calculated, C 66.02, H 8.45; found, C 66.10, H 8.25

Additional fractions were obtained by addition of ether to the mother liquor.

The free base could not be obtained crystalline. An attempt to increase the absorption of H, in the presence of an excess

of HCI in methanol solution did not change the result.

Found, C 65.80, H 8.41

Dihydro-des-methylhetisine Methiodide-The resinous base obtained from 0.22 gm. of the above salt was heated with 2 cc. of methyl iodide at 100” for 6 hours. An appreciable crystalline deposit formed which was sepa- rated from the solvent. The crystals on recrystallization from methanol yielded 95 mg. of compact prisms or thin almost square platelets which melted under the microscope at 249-251” after slight preliminary softening.

Cz2HJ,03NI. Calculated. C 54.19, H 7.03, N(CH3) 6.16 Found. “ 54.47, “ 7.14, “ 6.50

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198 ACONITE ALKALOIDS. XVII

The above methyl iodide mother liquor concentration yielded an ap- preciable resinous residue which crystallized from 1 cc. of methanol at 0” as flat needles or platelets. This fraction (46 mg.) sintered gradually above 263” and after some progressive softening melted incompletely from 273-280”, but crystals persisted till 295”.

Found, C 53.80, H 6.77, N(CHJ 6.98

The attempt to carry this methiodide through the next stage of degrada- tion was as follows: 73 mg. of methiodide (first fraction) after decomposi- tion with Ag,O in methanol were freed from solvent and then sublimed at 0.2 mm. After heating to 215’ the process was interrupted. The resin recovered from the condenser weighed 53 mg. After conversion to the HCI salt, 22 mg. were obtained in a first fraction from methanol-ether. This material under the microscope gradually sublimed with decomposition at 296-300”.

ChHa,03NC1. Calculated. C 66.02, H 8.45, N(CH1) 3.93 Found. ” 65.93, “ 8.42, “ 4.33

Oxidation of Dihydro-des-methylhetisine-0.1 gm. of the hydrochloride of this base in methanol was decomposed with a slight excess of silver oxide. After concentration of the clear filtrate all attempts to crystallize the free base were futile. The resin was dissolved in 10 cc. of acetone and 0.1 cc. of acetic acid. On addition of small amounts of powdered potassium permanganate, a gradual but definite reaction occurred, but then reached a point where the reagent was very slowly used up and the amount added had totaled about 25 to 30 mg. The filtrate was concentrated to remove the solvent and, after addition of water, the base was liberated with alkali and extracted with chloroform. The latter yielded on concentration 80 mg. of a colorless resin which gradually crystallized. The base crystallized from a chilled concentrated methanol solution as well formed long needles or flat prisms. It melted under the microscope at 139-141” after slight preliminary sintering.

C21H2803N. Calculated. C 73.42, H 8.52 Found. (a)” 73.20, “ 8.24

(b) If 73.34, “ 8.22

Dehydrogenation of Iietisine-The resinous base obtained from 6gm. of hetisine hydrochloride by decomposition with NaOH and extraction with chloroform was mixed with 15 gm. of selenium and heated in a slow stream of COZ for 2.5 hours at 340”. The amount of substance which distilled was inappreciable and was therefore discarded. The reaction mass was thoroughly extracted with hot benzene. This extract was shaken first with 10 per cent HCI and then with 5 per cent NaOH. The acid extract

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W. A. JACOBS AND C. F. HUEBNER 199

was made alkaline and extracted with ether. 0.75 gm. of a crude basic fraction was thus obtained (Fraction B). The Na0I-I extract was found to contain no appreciable phenolic or acidic products. The benzene solu- tion which remained and contained the neutral fraction yielded 2.25 gm. of a brown tar on evaporation (Fraction N). Fraction B (0.75 gm.) of crude bases was dissolved in benzene and passed through a 12 mm. X 10 cm. column of Al203 (E. Merck and Company). On development with benzene three zones were noted, a lower zone which was distinguished by a blue fluorescence in ultraviolet light, an intermediate yellow zone, and an upper dark brown zone. The three zones were successively eluted with benzene. Fraction 1 amounted to 35 mg., Fraction 2 was 130 mg., and Fraction 3 was 235 mg. None of these could be crystallized directly.

On addition of an equal amount of picric acid in ethanol to Fraction 1 it crystallized copiously. After two recrystallizations from ethanol, 15 mg. of small yellow platelets were obtained; micro m.p. 225-230” with decomposition.

C~H~TN.C&O~N~. Calculated, C 60.48, II 4.23; found, C 60.44, H 4.39

Fraction 2 (130 mg.) was rechromatographed on Al,03 from petroleum ether solution. By development with 40 per cent benzene in petroleum ether, a lower blue fluorescing band, followed in the ultraviolet light, was eluted from the column. 30 mg. of an oil resulted, from which a crystalline picrate was readily prepared. On recrystallization from acetone, 28 mg. of yellow-orange platelets were obtained; micro m.p., 235-245” with decomposition.

C,&~N.C~H~O~NN~. Calculated. C 60.48, H 4.23 C~sH~sN.C&O~Nz. “ “ 60.73, “ 3.84

Found. “ 60.67, “ 3.99

A mixture of this picrate with the picrate prepared from Fraction 1 melted at 225-235” with decomposition.

Fraction 3 yielded in similar manner a partially crystalline picrate which after two recrystallizations from acetone gave 10 mg. of a light yellow powder, micro m.p. 320-325”.

C19H260N.C H 0 N 6 3 7 3. Calculated. C 58.54, H 5.52, N 10.93 ChH~~ON.C~H~O~N3. (‘ “ 58.31, “ 5.89 ‘I 10.88

Found. “ 58.46, “ 5.65, “ 10.51

The neutral fraction (2.25 gm.) was chromatographed from benzene on a 12 mm. x 10 cm. column of A&OS. It was developed with benzene till the lower, blue fluorescing band (in ultraviolet light) was removed. 0.42 gm. of a mobile yellow oil was obtained in this fraction. A second yellow zone was then eluted with benzene to yield 0.32 gm. of a resin. The

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200 ACONITE ALKALOIDS. XVII

uppermost dark brown zone was finally eluted with 1 per cent methanol in benzene and yielded 1.36 gm. of a brown tar. These last two fractions were not further studied. The first fraction was rechromatographed on alumina from petroleum ether and then developed with a mixture of 10 per cent benzene in petroleum ether. The eluate, collected before the fluorescent zone had begun to emerge, yielded 180 mg. of a coloriess oil. The latter was distilled twice in a sublimation apparatus at 130’ and 0.1 mm.

C,&. Calculated. C 86.87, H 13.13, mol. wt. 446.45 Cd&a. “ “ 86.47, “ 13.53, “ “ 448.47

Found. “ 86.70, “ 13.40, “ “ 445, 450

The micro boiling point determined at 760 mm. indicated it to be above 260”, and at 26 mm. at about 185”. ni’r = 1.4805.

The succeeding blue fluorescent zone, on elution with 30 per cent benzene in petroleum ether, yielded 20 mg. of an oil. On addition of an equal weight of trinitrobenzene in acetone to this fraction and concentration to dryness, a residue was obtained which crystallized from ethanol as yellow needles, micro m.p. 156-159”.

C1JTll.C&1306N3. Calculated. C 62.98, H 4.09 C,JL~~C&O~N3. “ “ 63.71, “ 4.42

Found. “ 63.78, “ 4.48

By chromatographing this substance through A1203 from benzene, the hydrocarbon was separated from the trinitrobenzene. The oil obtained crystallized from ethanol and after recrystallization from ethanol, 4mg. of plates were obtained, micro m.p. 77-80”.

Cd,,. Calculated, C 93.15, H 6.85; found, C 93.10, H 6.89

The melting point of a mixture of pimanthrene obtained by the dehydro- genation of staphisine (m.p. 80-84’) with this hydrocarbon was 80-84”. The trinitrobenzene derivative again prepared from the crystalline C&HI4 hydrocarbon formed yellow needles from ethanol which melted at 166- 168”. The melting point of the trinitrobenzene derivative prepared from staphisine-pimanthrene was 166-168” and the melting point of a mixture of the two derivatives showed no depression. A mixture of the pimanthrene derivative with the above crude trinitrobenzene derivative having a melting point of 156-159” melted at 162-168”. It is most probable that this substance was an impure pimanthrene compound, as also indicated by the analysis. The melting point of the recrystallized complex of pimanthrene and trinitrobenzene, determined in a capillary, was 161.5-162”. Ruzicka and Sternbach (4) have reported the melting point as 158-160.5”.

All analytical work was performed by Mr. D. Rigakos.

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W. A. JACOBS AND C. F. HUEBNER 201

SUMMARY

An improved method for the isolation of hetisine has been found which involves the continuous extraction of a strongly alkaline aqueous solution with chloroform after preliminary extraction of the other alkaloids of the crude mixture with benzene. Degradation of hetisine by exhaustive methylation proceeded one step to des-methylhetisine but the latter was regenerated by pyrolysis of its quaternary ammonium hydroxide. Des- methylhetisine was hydrogenated to a dihydro derivative whose quaternary ammonium hydroxide also lost methanol to regenerate the original dihydro derivative. The Hofmann degradation applied to dihydrohetisine was also unsuccessful. In this case no crystalline intermediates could be isolated. Selenium dehydrogenation of hetisine yielded the hydrocarbon, piman- threne, and very small amounts of three basic substances, C!~~H~‘IN, C!1gH1bN or C18H17N, and C1BH260N. The latter were isolated as picrates.

BIBLIOGRAPHY

1. Jacobs, W. A., and Craig, L. C., J. Biol. Chem., 143, 605 (1942). 2. Craig, L. C., and Jacobs, W. A., J. Biol. Chem., 162, 645 (1944). 3. Suginome, H., and Shimanouti, F., Ann. Chem., 646, 220 (1940). 4. Ruzicka, L., and Sternbach, L., Helv. chim. acta, 23, 124 (1940).

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Walter A. Jacobs and Charles F. HuebnerFURTHER STUDIES WITH HETISINE

THE ACONITE ALKALOIDS: XVII.

1947, 170:189-201.J. Biol. Chem. 

  http://www.jbc.org/content/170/1/189.citation

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