TWO PRXDICTED HYDRATES OF KITRIC ACID. 443

XXVII.-Contributions to OUT Knowledge of the Aconite Alkaloi'ds. Part IV. On Isaconitine. (Napelline.)

Bp WYNPHAX R. DUNSTAN and E. F. HARRISON, Demonstrator in the Research Laboratory of the Pharmaceutical Society.

IN a, previous paper (Part 11, Trans., 1892,61,385), a new alkalo'id was briefly described, which had been found, together with aconitine, in the rcots of true Aconitum Napellus. This alkalo'id it was suggested should be provisionally called nupellbze, a name long ago applied to a supposed alkaloid of this plant, which has since been proved to be a mixture of several substances.

YOL. LXXII. 21

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444 DUNSTAN AKD HARRISON ON ISACONITINE.

The impure napelline then obtained was descl*ibed as an uncrys- tallisable alkaloid, resinous when dry, and as we had not succeeded in crystallising' any of its salts, no sure criterion of purity was available. Since then, however, several of the salts of the new alkalo'id have been crystallised, and during t,he past year we hare been occupied in the very tedious work of preparing sufficient pure material to enable us to examine the chemical properties of this important constituent of Acon i tum Napellus. By degrees, we have worked out a satisfactory process for separating napelline from aconitine, and also from another amorphous alkalo'id closely resembling it, which has been provisionally named homonapelline. The process is as follows.

Separation of Napelline (Isaconitine) from the Total Alkaloi'ds of A. Napellus.

The total alkaloids obtained from the root in the manner described in Part IX (Zoc. cit.) are dissolved in cold dilute hpdrobromic acid (5 per cent,.), excess of acid being carefully avoided. The clear solution is then precipitated with a slight excess of dilute ammonia, and the liquid shaken several times wit.h ether; the ethereal solution is washed with a small quantity of water, and then distilled. The residue, which consists chiefly of nconitine, is dissolved in just sufficient dilute hydrobromic acid, and the exactly neutral solut'ion is allowed to evaporate in a desiccator over calcium chloride, when crystals of acorbitine hydrobromide separate. The mother liquor contains some homonapelline and napelline.

The aqueous solution which has been extracted with ether is now extracted with chloroform until nothing further is removed. The solution in chloroform is washed with a little water and distilled. The residue, consisting largely of napelline, is dissolved in a sufficiency of dilute hydrochloric acid ( 5 per cent.), and the liquid, after being exactly neut ralised with dilute ammonia if necessary, is evaporated 011 the water bath. If the liquid is stirred occasionally, a crust of crystals separates after a time from the hot solution. These consist of napelline hydrochloride, and when some quantity bas formed, it is filtered off and washed with a little cold water, these washings being added to the original liquid. The evaporation and stirring are then repeated, and more crystals obtained, which are dealt with as before. By repeating the operation, as many as nine fractions of crystals may be separated. It has been found tha t by crystallising out from the hot liquid the napelline hydrochloride is obtained purer than by the usual process of evaporating to a small ~ o l u t n e and allowing the hot liquid to cool. The different fractions

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DUNSTBN AND HARRISON ON ISBOONITIXE. 445

are then mixed together and recrystallised in the same way until they melt ab 268" (corr.).

The mother liquors contain the hydrochlorides of homoisaconitine (homonapelline) and aconine, but some quantity of aconine still remains dissolved in the liquid which has been extracted with chloro- form.

Composition and Prop2rtie.v of Isaconitiite (Napelline) and its Salts. Piire napelline may be produced from the pure hydrochloride by

adding dilute ammonia in slight excess to a cold aqueous solution, when the base is precipitated in white flocks. Potash and soda act similarly, but the use of these alkalis is to be avoided, as the alkalo'id is readily bydrolysed by contact with them. Tbe slightly alkaline liquid is then extracted several times whh ether, and if the solution in ether is washed with water, dried with calcium chloride, and the ether distilled off, the base remains as a colourless, tramparent varnish, which has so far resisted all attempts to crystallise it. When quite dry, it is very friable, and is readily powdered. It melts indefiniteIy near 125".

Napelline is slightly soluble in water, furnishing a bitter solution, which, however, does not produce the tingling sensation on the tongue which is so characteristic of aconitine. The aqueous sollition is alka- line to litmus. The base is readily soluble in alcohol or chloroform, and also in ether. The alcoholic solution is dextrorotatory, but less so than that of aconitine. The determination of the specific rotation in alcoholic solution furnished the following results :-

a[15"] = 42" 3' ; Z = 2 dm. ; c = 7.86,

whence [a]= = +448. 100 x 0.705 2 x 786

-_____-

A solution of the base in chloroform is only feebly dextrorotatory. When burned in a current of oxygen in the usual manner,

the alkaloid furnished the following data. Two specimens were anal y se d.

C = 61.11 ; H = 6.96. 11. 0,2299 ,, 0.5145 CO, ,, 0.145 H,O. c' = 61-03; H = 7.15.

Cs3H,8Ol2 requires C = 61.20; H = 6.95 per cent.

The composition of napelline is, therefore, identical with that of aconitine, bo t8h alkalojids being represented by the formula C,H,,NO,,. The alternative name of isaconitine may t,hus be used with ad- vantage for this alkaloyd, since i t is isomeric with aconitine, and the provisionally suggested name of napelline may be abaiidlmed.

2 1 2

I. 0.2379 gave 0.5311 CO, and 0.2486 H,O.

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34(i DUNSTAN AXD HARRISON OX ISACOSITIXE.

By dissolution of the base in a sufficient quantity of the correspond- ing acid, diluted with water, the hydrochloride, hydrobromide, hydr i - odide, and vzitrate have been obtained in a crystalline state. These dissolre readily in water and alcoh ol forming very bitter solutions.

The hydrochloride, C&,,NO,,,HCI, crptailises in needles which are soluble in water and alcohol. The aqueous solution is intensely bitter, but does ao t produce the slightpst tingling sensation. This affords a most delicate test for the presence of aconitine salt, a small drop of the very dilute solution of isaconitine hydrochloride being placed on the tip of the outotretched tongue and allowed to remain for about a minute. It is then washed off with water; after the lapse of a few minutes, a distinct tingling sensation will be ex- perienced if the salt was contaminated with even a very minute quantity of aconitine hydrochloride. We have found this to be a far more delicate test for minute quantities of aconitine than any chemical reaction, and in preparing the material used in this enquiry, the recry stallisation of the isaconitine hydrochloride was contiiiued until it saturated solution of the salt produced no tingling when tested in the manner above described. The complete removal of these minute traces o€ acoiiitine is frequently a very long operation.

The crystals of the salt deposited from aqueous solution and air- dried were analysed with the following results :-

I. 03543 lost 0.01 H,O at loo", and gave 0.0721 AgCI.

11. 0.2919 lost 04088 HzO at loo", and gave 0.0621 AgC1.

H,O =

H,O = 2-82 ; C1 = 5.03 per cent.

3.01 ; C1 = 5.26 per cent. 111. 0.3755 gave 0.0794 AgC1. C1 = 5.24 per cent,.

C,H4,N0,2,HC1,H20 requires H,O = 2.56 ; C1 = 5.06 per cent.

When crystallised from a hot solution, as previously described in connection wi th the isolation of isaconitine, small crystals are obtained. Large crystals, often well defined, may be produced by the slow evaporation of an aqueous solution, or by the cautious addition of ether to a sohition of the salt in alcohol. When pure, the crystals, dried at loo", melt at 268" (corr.).

On some occasions what is apparently an isomeric form of tbe salt has been obtained melting at 217" (corr.).

Like the corresponding aconitine compound, an aqueous solution of the salt is laevorotatory. The specific rotation has been calculated from the following data:-

a[15'] = 1'9'; I = 4 dm.; c = 1,

whenoe [a]= = -28.74'. 100 x 1.15" 1 x 4

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DUXSTAX AND HARRISON ON ISACONITINB. 447

The specific rotation of aconitine hydrobromide (Parts I and 31) is [aID = -30*47", from which it would appear that amnitine and isaconitine salts are about equally lsevorotatory.

The hydrobromide, C33H,,N0,,,HBr, cry stallises in needles melting at 282" (corr.), and closely resembles hhs hydrochloride.

The hydriodidp, CsH45N012,HI, forms granular crystals, like those of aconitine hydriodide. Like this salt, it is sparingly soluble in water; it is, therefore, precipitated when potassium iodide is added to a, solution of isaconitine hydrochloride. The crystals melt at 246" (corr.). An aqueous solution of this salt is lsevorotatory, [ a ] ~ = -2694".

React ion of Isaconnitine with Auric Chloride.

Anrie chloride reacts in a remarkable manner with isaconitine hydrochloride. When aqueous solutions of these salts are mixed, a bulky yellow precipitate is thrown down, which in its appearance re- calls the precipitate produced under the same conditious with aconitine hydrochloride. When this precipitate, dried over calcium chloride, is dissolved in alcohol, and the solution slowly evaporated in a. desiccator, almost colourless crystals separate. These melt with decomposition at 204". They are more readily obtained by adding light petroleum to the strong alcoholic solution, and allowing the mixture to stand. The original precipitate has been dissolved in various liquids, always with the same result, that on evaporation the nearly colondess crystals are deposited, entirely differing from the yeIlow crystals of aconi tine aiirochIoride which are produced under similar conditiolas.

On analysis, this compound furnished the following data :-

I. 0-2033 gave 0.0436 Au and 0.0645 AgCl. 11. 0.1533 ,, 0.0334Au 7 7 0.0480 AgC1.

Found. Calculated for <------ -

I . 11. CBH,NO,, H CL,AUClp C,H~~(AUCI,) NOI:. An . .. . . 21.44 21-79 19-87 2 1646 CI.. . . . . 7-88 7-74 14.40 7.77

It t h u s appears that this salt, which is colourless when quite pure, is not an aurochloride, but an arirochloro-deriwatciwe of isaconitine, in which one atom of hydrogen in the alkalo'id is Ireplaced by the gro-crp AuCl,. The formation of this compound is represented, by the equa- tion :-

C33H4,NOl,,HCI + AuCl, = C,H4,(AuCl,)NOl2 + 2HC1. ( I saconitine (Aurochlor-

hydra4doride.) imconitine.)

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443 DUNSTAN AND HARRISON ON IXACONITINE.

The only alkalojidal compound of this type which has been obtained, so far, is the aurochlorocaffeine, of which an account was given to the Society by one of u s a short time ago (t,his vol., p. 195).

Amochlorisaconitine differs, however, from aurochlorocaffe~ne in tlie important respect that it is not, converted by the action of hydro- gen chloride into the aurochloride, CB,H,,NO,,,HCl,AuC1,. Possibly, t h e yellow, amorphous precipitate obtained by the interaction of aqueous solutions of the two salts is the nurochloride which loses two molecular proportions of hydrochloric acid when it is dissolved.

The formation of this interesting derivative constitutes a trust- worthy method of identifying isaconitine, and of distinguishing it from aconitine, since this alkalo'id furnishes, under the same condi- ticns, a stable aurochloride, which apparently shows no tendency to pass into an aurochloro-derivative.

H@-Olpis of Isamnithe.

Isaconitine undergoes hydrolysis when it is heated for many hours w i t h water in a closed tube. Potash and soda effect the hydrolysis of t h e alkaloid most easily, and, indeed, act even in the cold. So f a r as we have been able to compare the behaviour of the two alkalojds, isaconitine appears to be more easily hydrolysed by alkalis than aconitiiie. Ammonia acts least rapidly, and in dilute solutions very slowly indeed.

W e have made a number of experiments wihh pure isaconitine in order to ascertain the nature of the hydrolytic products and the pro- portions in which they are formed.

By heating the base with water at 130" for 48 hours, some darken- i ~ g occurred, and i t was found that hydrolysis was nearly complete. The resulting solution, being acid, was made alkaline with ammonia a n d extracted with ether, which removed only traces of unaltered isacoiiitine. Ib mas now acidified with hydrogen chloride, and again extracted with ether, which removed a solid acid, slight'ly soluble in cold water, After crystallisation, ih i s acid melted at l d l " , and exhi- bited all the properties of bensoac acid. No alcohol or other acid could be detected.

The amount of benzoic acid formed from the weight of alkalo'id taken corresponded with 18.5 per cent.

The acid liynid, which had been extracted with ether, was now made alkaline with ammonia, and extracted with chloroform. T hia removed a base which was left as a coloured varnish, on the evapora- tion of the chloroform, and could not be crystallised. It dissolved readily in alcohol and in xater, but was nearly insoluble in ether. A further quantity of this substance was obtained by neutralisiug the

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DUNSTAN AND HARRISON ON ISACONXTINE. 449

acid liquid, evapoi*ating to dryness, and extracting the residue with alcohol.

Other experiments were made in order to ascertain the nature of this base, which exhibited a very marked resemblance to aconine, the hydrolytic product of aconitine, but considerable difficulty was experi- enced in purifying it from the coloured products which were formed along with i t during hydrolysis. Axany hydrolyses were made in aqueous, alkaline, and acid solutions, necessarily with small quantities of materials, and owing to the difficulty of purifying the product, the pure base was not obtained in sufficient quantity for complete examination. These experiments proved coi~clnsively, however, that benzoic acid was the constant and only acid product of hydroljsis, and that it is formed in the same proportion as when aconitine is hydrolysed, namely, 18.5 per cent. It was thus rendered highly probable that the basic product of hydrolysis was nconilae, or a com- pound isomeric with it.

To settle this question, we prepared a, fresh quantity of several grams of pure isaconitine from the total alkaloid of A. Napellus, a long and tedious operation. The hydrolybis was effected by adding considerable excess of aqueous soda to a strong aqueous solution of isaconitine hydrochloride. The liquid wits warmed on the water-bath for two hours, and then allowed to stand for about 24 hours a t tho ordinary temperature, after which hydroiysis was complete, ether extracting virtually nothing from the solution.

The alkaline solution was now acidified with nydrochloric acid, and the beneoic acid extracted with ether. The acid liquid was then neutralised, evaporated to dryness, and the residue extracted with alcohol. The residue left on evaporating the alcohol was extracted with chloroform, and the salt left on distilling off the chloroform dis- solved in water. By slow evaporation, the characteristic crystals of aconine hydrochloride separated. These were drained on a tile and recrystaIlised by the cautious addition of ether to an alcoholic soh- tion. This salt exactly corresponded in its properties with the crys- talline sconine hydrochloride obtained from aconitine and described in Part 111. It was there shown that the salt is hvorotatory, [a],, = -7.71". The salt derived from isaconitine was found to be lavo- rotatory to the same extent. The following results were obtained with an aqueous solution :-

a[15O] = 0.3706 ; I = 2 dm.; c = 2.398 (anhydrous salt),

whence

Like aconine hydrochloride, the Ralt began to melt at 176", and decomposed with effervescence at about 191". It h w been shown that

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450 DUNSTAN AND HARRISON ON ISSCONITINE.

aconine hydrochloride crystallises with 2H20, one being lost at lOC;", and the other a t 120". The amount of water was therefore deter- mined in the salt derived from isaconitine, and as a further con- firmation of its composition, the chlorine was estimated. The follow- ing results were obtained :-

I . 0.2488 lost 0.0066 H,O at 100". 11. 0'2044 ,, 0.0056 HzO at loo", 0.0059 more H20 at 120", and

111. 0.1791 lost 0.0046 HzO at loo", 0.0055 more H,O at 120°, and gave 0.0496 AgCl.

gave 0.0432 AgC1.

Found. r--L-- 7 Calculated fo: I. 11. 111. CzcHdlNO11 ,HC1,2 €320.

Water a t 100" . . . 2.65 2-74 2.56 per cent. 2.94 per cent. Water a t 120" . . . - 5-63 5-63 ,, 5.88 ,, Chlorine . . . . . . . . - 6.0 3-96 7, 5.76 ,, On combustion, the following results were obtained :- 0.1282 air-dried salt gave C = 50.58. H = 7-60 per cent.

C26H41N01,,HC1,2H20 requires C = 50.69 ; H = 7-47 per cent.

The salt is thus proved to have the same composition as aconine hydrochloride, with which i t appears to be identical in every respect.

From the pure hydrochloride the base was regenerated as a nearly colourless varnish. Like aconine, it dissolved in water, forming .z powerfully alkaline solution, capable of expelling ammonia from its salts. The solution also acted as a powerful reducing agent. We have most carefully compared the propeyties of the aconine from aconitine with that derived from isaconitine, and have been unable to detect any difference between them.

Isaconitine, then, not only has the same composition as aconitine, but it also furnishes on hydrolysis the same proportions of benzoic acid and aconine. The decomposition of both alkaloids is therefore represented by the same equation :-

C3JLiNOit + €320 = C7H602 + c?aJ%iNO11.

We hope shortly to be able to throw some light on the nature of the relationship which subsists between these two alkalo'ids ; from the dif- ference in their reactions with auric chloride, it is clear that the isomerism is more deep-Reated than that eubsisting between atropine and byoscysmine. I n fact it is certain that there is a distinct structural difference in the constitution of the two substances, and not merely a stereochemical difference. I t is probable that this difference resides in the atoms or groups which are attached to, o r in the immediate neighbourhood of, the nitrogen atom.

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DUNSTAN bND HARRISON Oh’ ISACONITINE, 451

On the other hand, the asymmetlric carbon atoms, or atoms which confer on aconitine its rotatory power, would appear to be also pre- sent, in the molecule of isaconitine, since the salts of both alkaloids are optically active in the same direction, and nearly to the same extent.

That the constitutional connection of aconitine and isaconitine is very close, and the structuml difference between them is but small, i s evident from the identity of the hydrolytic products, which, more- over, are formed in the same proportion.

.It should be remarked that isaconitine differs both in composition and properties from the amorphous bases described by previous workers as existing in A. Napellus. It may be safely inferred from the great difficulty we have experienced in isolating isaconitine, and from the circumstance that special methods of purification have been found to be necessary, that these ‘‘ amorphous bases ” were not single substances bat mixtures.

Although isaconitine bears certain resemblances to the amorphou8 picraconitine once, but only once, obtained by T. B. Groves €rom a collection of roots supposed to be those of A. Napellus, i t is evidently an entirely different substance. Picraconitine wm found by Alder Wright to contain 62.91 per cent. of carbon and 7-88 per cent, of hydrogen, its composition being expressed by the formula C,,H,NO,,.

The substance called “ napelline ” by Hubschmann was undoubtedly a mixture.

Jnrgens (Inaug. Dissert., Dorpat, 1884) obtained from the root of il. napellus an amorphous base which contained 67.74 per cent. of carbon and 8.4 per cent. of hydrogen. No proof was given of its homogeneity .

Physiological Action of Isoacvnitine. Professor Cash, of Aberdeeo, has kindly undertaken to investigat.e

the physiological action of the pure alkslojids which we are obtaining from A. Nu,peZlus. Our knowledge of the physiological action of aconitine and the aconite alkaloids is at present no less confused than that of their chemistry.

Professor Cash’s investigation is not yet completed, but some inter- esting preliminary results have been obtained by a comparison of the physiological action of aconitine and isaconitine. The behariour of the two alkaloids, although in some respects similar, appears to be generally distinct. Noreover, whilst aconitine is a most violent poison, even in excessively minute doses, isaconitine is only toxic to small animals when exhibited in comparatively large doses. With guinea pigs, it was found that whilst the lethal quantity (per kiln. of the animals’ weight) of aconitine, administered as a solution of

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452 KTPPIX'O : ACTION OF PHOSPHORIC ANHYDRIDE

the hydrochloride by hypodermic injection, was only 0*000064 gram (0*001 grain), that needed of isaconitine under the same con- ditions was between 0.0194 and 0.0259 gram (0.3-0.4 grain). I n the case of frogs, it was observed tha t an average animal, weighing abont 25 grams, was killed by 0.000032 gram (0.0005 grain) of aconitine, whereas 0.00953 gram (0.07 grain) of isaconitine was needed to pro- duce a lethal result.

Research Laboratoi-y of the Yharmnceuticul Socieiy, Lorzdon.

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