THE PICROLONATES OF CERTAIN ALKALOIDS. BY W. H. WARREN AND R. S. WEISS. (From the Chemical Laboratory of the Medical Department of Washington University, St. Louis, Missouri.) (Received for publication, June q, 1907.) (Plates III-VIII.) Our attention was called to the use of picrolonic acid in pre- cipitating alkaloids and we have studied its behavior with some of the more important representatives of this group. Like picric acid picrolonic acid precipitates many basic nitrogen compounds and, since the latter has given better results in other instances, we thought it advisable to determine the relative value of these reagents as alkaloidal precipitants. They are widely different as far as ease of obtaining them is concerned. Picric acid is cheap and quickly prepared, whereas picrolonic acid is not listed by manufacturers and its preparation is rather difficult. Knorr’ gave the name “p&olonic acid” to I-p-nitro-phenyl- J:methyl-4-nitro-5-pyrazolon, CH,---C=N I \N--C,H,-NO,. / h-0,--c =c \ OH which he preferred to picric acid in studying 1,2-ethanol-amine and certain morpholine derivatives, because of the greater insolu- bility of their picrolonates and the ease with which these could be recrystallized and identified. Recently Steude12 has pointed out the value of this reagent inisolating and identifying the two hexone bases, arginin and histidin. 1 Ber. d. dew&h. chenz. Gesellsch., xxx, p. 909, and xxxii, pp. 732 and 736. 2 Zeitschr. f. physiol. Ckem., xxxvii, p. 219. 327 by guest on May 10, 2018 http://www.jbc.org/ Downloaded from
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THE PICROLONATES OF CERTAIN ALKALOIDS.
BY W. H. WARREN AND R. S. WEISS.
(From the Chemical Laboratory of the Medical Department of Washington University, St. Louis, Missouri.)
(Received for publication, June q, 1907.)
(Plates III-VIII.)
Our attention was called to the use of picrolonic acid in pre- cipitating alkaloids and we have studied its behavior with some of the more important representatives of this group. Like picric acid picrolonic acid precipitates many basic nitrogen compounds and, since the latter has given better results in other instances, we thought it advisable to determine the relative value of these reagents as alkaloidal precipitants. They are widely different as far as ease of obtaining them is concerned. Picric acid is cheap and quickly prepared, whereas picrolonic acid is not listed by manufacturers and its preparation is rather difficult.
Knorr’ gave the name “p&olonic acid” to I-p-nitro-phenyl- J:methyl-4-nitro-5-pyrazolon,
CH,---C=N
I \N--C,H,-NO,. /
h-0,--c =c \ OH
which he preferred to picric acid in studying 1,2-ethanol-amine and certain morpholine derivatives, because of the greater insolu- bility of their picrolonates and the ease with which these could be recrystallized and identified. Recently Steude12 has pointed out the value of this reagent inisolating and identifying the two hexone bases, arginin and histidin.
1 Ber. d. dew&h. chenz. Gesellsch., xxx, p. 909, and xxxii, pp. 732 and 736. 2 Zeitschr. f. physiol. Ckem., xxxvii, p. 219.
Methyl-phenyl-pyrazolon, prepared from acetacetic ester and phenylhydrazine, according to Knorr,‘was converted into picro- Ionic acid by Bertram’s method as described by Steudel.2 In the hope of improving the yield we tried several modifications of this method but had indifferent success. Finally we employed the method of Braur2 which gave a result so much better than any we had obtained up to that time that we can recommend it for the preparation of picrolonic acid, As this method is some- what inaccessible. we have described our manner of carrying it
out.
Six hundred cc. of 90 per cent nitric acid (sp. gr., r.49), prepared by diluting 540 cc. of strongest fuming nitric acid (sp. gr., 1.52 = 99.7 per cent) with 60 cc. of water, were kept cold with ice-water in a wide-mouth bottle. Two hundred grams of methyl-phenyl-pyrazolon were added gram at a time, the mixture being mechanically stirred all the while and the temperature being kept below 15’ C. Each addition of substance caused a vigorous reaction attended with escape of red fumes. Halfway in the process, a reddish-yellow, crystalline product began to appear. At the end, the mixture was stirred for thirty minutes and then the crystal- line mass was collected upon a small porcelain disc covered with asbes- tos, freed from acid by suction and washed successively with dilute nitric acid and with water. As this crude product gradually undergoes decom- position, it is well to proceed at once with the next step in the process.
Fifteen hundred cc. of 38 per cent acetic acid, about six times the weight of the crude product, were mechanically stirred in a beaker on the water- bath. The crude product finely ground was added and the temperature was not allowed to exceed 60’ C. This substance, which is heavy and granular, tends to settle to the bottom of the beaker. At low tempera- tures it remains unaltered but gradually changes as the temperature rises. The change is rapid from 50’ to 60’. A mass of light-yellow, flocculent crystals replaces the reddish-yellow compound and fills the liquid. Sa- ponification is complete in I 5 to 40 minutes. The product may be exam- ined occasionally under the microscope, if necessary, and the reaction is complete when only needles of picrolonic acid are to be seen.
The crude picrolonic acid, freed from acid on a Btichner filter, was washed with water and then ground in a mortar with I 5 o grams of crystal- lized sodium carbonate. Carbon dioxide was given off and the yellow sodium salt was formed. This was pressed out and crystallized from a
1 Ber. d. datsch. them. Gesellsch., xvi, p. 2597 2 Zeitschr. f . physiol. Chem., xliv, p. r 57. 3 Inaugural Dissertation, Jena. 1899.
mixture of three volumes of strong alcohol and one volume of water. Sodium picrolonate was then decomposed with concentrated hydro- chloric acid. The acid was warmed in a beaker on the water-bath, stirred as before and the sodium salt was added in small portions. Picrolonic acid separated as a fine, mealy powder. It was thrown on a porcelain disc covered with asbestos, freed from acid by suction and washed with water to remove acid and sodium chloride. We obtained 166 grams of picrolonic acid by this method from 200 grams of methyl-phenyl-pyra- zolon. If further purification is desired, picrolonic acid may be crystal- lized from strong alcohol from which it separates in fine yellow needles which melt at 116.5~ C.
USE OF PICROLONIC ACID.
A saturated alcoholic solution of picrolonic acid is the most convenient form in which to use this reagent for ordinary pur- poses. Such a solution may be employed to precipitate alka- loids from water, alcohol or from other solvents miscible with alcohol. Although picrolonic acid itself is only slightly soluble in water, sufficient alcohol is present to prevent the reagent from being precipitated when added in excess to aqueous solu- tions.
In some cases it may be an advantage, after extracting an alkaloid from an aqueous, alkaline solution with such solvents as ether, chloroform, benzol, acetic ether and amyl alcohol, to substitute for the alcoholic reagent a saturated solution of picro- ionic acid in the particular solvent used to extract the alkaloid. In isolating morphine, for example, the alkaline solution may be extracted either with hot chloroform or with hot amyl alcohol and this extract may be tested directly with a solution of picrolonic acid in the corresponding solvent. The following table shows the relative solubility of picrolonic acid in the solvents men- tioned above :
SOLUBILITIES OF PICROLONIC ACID AT ORDINARY TEMPERATURE.
To recover an alkaloid from its picrolonate, it is necessary only to warm the latter with dilute sulphuric acid which dis- charges the bright yellow color of the picrolonate, causing the alkaloid to pass into solution and precipitating pale-yellow picrolonic acid. By extracting with acetic ether, in which picro- ionic acid is especially soluble, the aqueous solution of the alka- loid is left colorless. This has suggested a method of purifying alkaloids after their extraction in the usual way from animal tissues. An alkaloid like strychnine, whose picrolonate is very insoluble, may be precipitated from aqueous solution and thus separated from other substances which interfere with its purifi- cation. The precipitated picrolonate may be collected on a filter, washed with water and then warmed with dilute sulphuric acid. After extracting the free picrolonic acid as before, a very pure solution of the alkaloid will remain.
COMPARISON OF PICROLONIC ACID AND PICRIC ACID.
Although picric acid may be used either in aqueous or in alcoholic solution, we were limited in the case of picrolonic acid to alcohol because of the slight solubility of this substance in water. The two reagents were prepared by saturating 90 per cent alcohol with these acids. Such a solution of picric acid con- tains at ordinary temperature 0.0596 gram in I cc. and the cor- responding solution of picrolonic acid contains 0.0209 gram in I cc. There is no precipitation of either reagent upon adding it to 5 cc. of water volume for volume, or in smaller quantity. A precipitate, therefore, in an actual test is due to alkaloid and not to dilution of the reagent with water.
The solution of each alkaloid was prepared either by dissolv- ing one of its salts in water, or by dissolving the alkaloid itself in water with the aid of dilute acetic acid. We began in each case with a I per cent solution, aliquot volumes of which were diluted with water until a point was reached where the test failed. Time is a factor to be considered in deciding whether a test is positive or negative, especially when the solutions are dilute. The precipitate may not be immediate but it will some- times appear upon standing. We adopted fifteen minutes as an arbitrary limit and called the test negative when there was no
precipitate within that time. Frequently there was a turbidity instead of a distinct precipitate but this sometimes became crystalline upon standing. We decided to accept as positive those tests where a turbidity gave such a result. The relative delicacy of the two reagents toward the alkaloids studied appears in the following table:
DELICACY OF REAGENTS AS ALKALOIDAL PRECIPITANTS.
PICRW ACID.
Remarks.
PIcRoLONIc Acm. --- I I..-----’
/ .Positire i I m15min. Remarks. !I
-- Coniine.. . . I-250
/ Turbidity with- 11 l-800 / Ver;5 s~lig”,; in out precipitate 11 I
Nicotine.. . / l-200001 Slight crystalline / precipitate II
l-20000; Slight crystalline / precipitate
Strychnine.. . 1 l-40000i Very slight in / l-750001 Very slight in I 15 min. I I 15 min.
Brucine.. . l-20000/ Positive in 12 ’ ! min. II
1-10000~ Very slight in 15 min.
Morphine.. . ’ l-400 ’ Very slight in / l-500 15 min. 1
/ Positive in 30
I / min. in l-1000
Codeine. . . . l-1000 j Very slight -I
Atropine.. . . . l-1000 / Turbidity which
i ; / becomes crystal- / line
becomes crystal- line
Quinine. . l-.50000/ Turbiditv with- I out pregipitate
l-50000 Turbidity with-
I- out precipitate
Hydrastine. . l-10000/ Turbidity with- / l-10000 Turbidity which out precipitate becomes crystal-
line -- - - ~-
Our results show considerable variation in the limit of dilution within which alkaloids can be detected with these reagents. Strychnine, which can be detected by picrolonic acid in 1-75000,
affords the most delicate test, whereas coniine can barely be detected by picric acid in 1-250. Yet the difference between these reagents is not as marked as we had expected when we began our experiments. Upon the whole picrolonic acid is the
more sensitive, especially toward coniine, strychnine and mor- phine. On the other hand, picric acid is the more sensitive toward brucine and codeine. They appear about equally sensi- tive toward nicotine, quinine, atropine and hydrastine. As a matter of fact picric acid is a better reagent for nicotine, because the precipitate is distinctly crystalline and quite characteristic in I-zoooo. This reagent would seem to be well-adapted for detecting this particular alkaloid.
The recognition of these alkaloidal picrolonates and picrates by means of the microscope depends entirely upon the method of preparing these compounds. It is difficult, if not impossible, to distinguish most of these substances when they have been precipitated from aqueous solution. We should hardly expect it to be otherwise. We observed a difference in those cases where there was a turbidity which became crystalline upon standing. The crystalline form was more distinct. For this reason we have always redissolved the substance, preferably in alcohol, and allowed crystallization to take place slowly. This applies more particularly to our later work where we had occasion to examine crystals carefully either to describe them or to take photo-micrographs. Since our results with alkaloidal picrates are for the most part like those of Popoff,’ who has made an extensive study of picric acid as an alkaloidal precip- itant, we have described and photographed only a few of the more important of these compounds.
PROPERTIES OF ALKALOIDAL PICROLONATES AND ANALYTICAL
RESULTS.
In preparing alkaloidal picrolonates for analysis and for observ- ing their properties, we have obtained the best results by adding an excess of alcoholic reagent to a hot solution of the free alka- loid in as small a quantity of alcohol as possible. Some picrolo- nates appear immediately; others crystallize as the solution cools and yet others require long standing or concentration of the solution. Before being analyzed, these compounds were recrystal- lized at least once and dried at 100-1 IO’ C., or by standing in desiccator over sulphuric acid.
1 Le Laborattire d.e Toxicologic: Brouardel et Ogier, pp. 203-220, 1S9r.
Coniine picrolonate was prepared by adding a slight excess of reagent to Merck’s pure coniine dissolved in a small quantit> of alcohol. Since the precipitate, which appeared immediately, did not show the characteristic crystalline form of this substance, it was recrystallized once from alcohol somewhat diluted with water. Coniine picrolonate forms large rhombohedrons when it crystallizes slowly from alcohol, but when crystallization is rapid it appears as short, thick, irregular crystals with pointed ends and these are often in rosettes (Fig. I). These crystals are quite different from those of the corresponding nicotine com- pound and serve to distinguish these two liquid alkaloids. Co- niine picrolonate is light-yellow, it dissolves readily with heat either in water or in alcohol and melts at 195.5’ at which point it becomes dark owing to decomposition. Upon analysis it gave the following result :
o. 2252 gram of substance gave 35.5 cc. of nitrogen gas at 21” and 748 mm.
Calculated for CdbsN~0~: Found:
Iv................... 17.9 per cent. 17.7 per cent.
Nicotine Picrolonate-C,,H,,N,.C,,H,N,O,.
As our available alkaloid showed signs of change due probably to long standing, we prepared the dioxalate and recrystallized this compound until it melted at 110” C. Nicotine picrolonate was precipitated by adding an excess of reagent to an aqueous solution of the dioxalate. The precipitate was crystallized from alcohol somewhat diluted with water. This compound forms long, thin, prismatic needles with square ends which are often grouped in fan-shaped clusters (Fig. a). It is light-yellow and melts at 213~ previously subliming somewhat and turning dark. Upon analysis it gave the following result:
o.2015 gram of substance gave 35.6 cc. of nitrogen gas at 19~ and 747 mm.
Calculated for CzdhN~0.s: Found :
TV................... 19.72 per cent. 19.96 per cent.
Nicotine, though a diacid base, appears to combine with but one molecule of picrolonic acid. On the other hand it combines with two molecules of picric acid. This compound has been
described by Pinner and Wolfenstein’ and, as stated elsewhere, we have found it preferable to the picrolonate. Aqueous picric acid precipitates nicotine picrate as small yellow needles which melt at 218~ and dissolve with difficulty. Crystallized from hot alcohol it appears in short, hexagonal prisms often terminated by hexagonal pyramids (Fig. 3). Some forms resemble quartz crystals.
Strychnim Picrolonate-C,,H,,N,O,.C,,H,N,O,.
The picrolonates of the two nux vomica alkaloids, strychnine and brucine, melt with difficulty, or not at all, and differ mainly in crystalline form. Strychnine picrolonate was prepared by adding the reagent in excess to a hot alcoholic solution of the alkaloid. The precipitate consists of crystalline leaflets but, when crystallized slowly from alcohol, it appears in rectangular prisms, more or less irregular in form, which are sometimes arranged in star-shaped clusters (Fig. 4). This compound is light-yellow and very insoluble in water or in alcohol of any strength. When heated it begins to darken at 2~6~ and the color increases as the temperature rises, making it difficult to deter- mine the precise melting-point which seemed to be about 275’.
Upon analysis it gave the following result:
o. 2016 gram of substance gave 24.9 cc. of nitrogen gas at 20“ and 747 mm.
Calculated for ‘A&3&: Found:
N . . . . . . . . . . . . . 14.04 per cent. 13.9 per cent.
In comparing strychnine picrolonate with the picrate, we found the latter compound mentioned by Guaresch? who refers to Gerock’s use of the picrates of strychnine and brucine in separating these alkaloids but does not give their properties. Strychnine picrate, prepared by adding an excess of alcoholic reagent to a hot alcoholic solution of the alkaloid, separates from solution in leaflets. Redissolved in alcohol and allowed to crystallize slowly, it appears in short, slightly thickened trape- zoidal plates, the non-parallel ends of which are usually irregular
1 Ber. d. deutsch. them. Gesellsch., xxiv, p. 66.
2 Einftihrung in das Stud&n der Alkaloide, p. 505, 1896
in outline (Fig. 5). It undergoes decomposition when heated and up to 300~ does not melt.
Brucine PicroZona2e-C,,H,,N,O,.C,,H,N,O,.
Brucine picrolonate was prepared by adding the reagent in excess to an alcoholic solution of the alkaloid. The precipitate. which is almost immediate, consists of cubical crystals man) of which look like rhombohedrons (Fig. 6). This compound is very insoluble in water or in alcohol of any strength. When heated it begins to darken at 234’ and finally melts at 256’. Upon analysis it gave the following result:
o. 2011 gram of substance gave 22.8 cc. of nitrogen gas at zoo and 753 mm.
Calculated for CasHsrNoO,,: Found :
N . . . . . . . . . . . . . . . . . . . 12.7 per cent. 12.8 per cent.
In comparing brucine picrolonate with the picrate, we noticed a peculiarity in the behavior of the latter. An alcoholic solution of brucine was added to an alcoholic solution of picric acid. There was an immediate yellow crystalline precipitate and, when this was allowed to stand without being filtered, an orange-red compound also appeared gradually. Upon heating the solution to boiling, the yellow substance was dissolved and, as the solution cooled, only the orange-red compound reappeared. This latter substance crystallizes in cubical crystals and in elongated, rect- angular prisms terminated by low pyramids (Fig. 7). When heated up to 31oO, neither the yellow nor the orange-red com- pound melts though both undergo decomposition. At 213’ the yellow compound becomes orange-red. Judged by the follow- ing analyses, there is not a wide difference in their composition:
Yellow Compound-C,,H,,N,O,.C,H,(NO,),OH.
o. 2007 gram of substance gave 19.8 cc. of nitrogen gas at 18’ and 739 mm.
Calculated for ‘&J-LoNJO~I: Found :
N . . . . . . . . . . . . 11.23 per cent. 11.08 per cent.
Orange-red Compound-C,,H,,N,O,.C,H,(NO,),OH.
o. 2016 gram of substance gave 20 cc. of nitrogen gas at 19~ and 75 I mm.
Calculated for ‘SnHzsNdh, : Found :
N . . . . . . . . . . . 11.23 per cent. 11.32 per cent.
This compound was prepared by adding the reagent in excess to an alcoholic solution of the alkaloid and evaporating the solution until crystallization began. Morphine picrolonate forms broad, flattened needles which resemble knife-blades and are sometimes arranged in fan-shaped clusters (Fig. 8). It is quite readily soluble in alcohol. When heated it melts at 186.5’ and turns dark-brown near its melting-point. Upon analysis it gave the following result:
o. 2024 gram of substance gave 22 cc. of nitrogen gas at 16~ and 749 mm.
Calculated for ‘.XbNrDs: Found:
N................... 12.7 per cent. 12.5 per cent.
Codeine Picrolonate-C,,H,,NO,.C,,H,N,O,.
This compound was prepared by adding the reagent in excess to an alcoholic solution of the alkaloid. When the solution is very concentrated and crystallization is rapid, codeine picro- lonate appears in short, thick crystals which form rosettes; but when it crystallizes slowly from hot alcohol it forms character- istic crystals looking like truncated pyramids (Fig. 9). It is deep- yellow and melts at 219’, turning orange-red just before melting and becoming dark as it melts. Upon analysis it gave the following result :
o. 2054 gram of substance gave 22.4 cc. of nitrogen gas at 20~ and 750 mm.
Crdculated for Cz&&L’s: Found:
N . . . . . . 12.4 per cent. 12.3 per cent.
Atropilze Picrolonate-C,,H,,NO,.C,,H,N,O,.
This compound was prepared by adding the reagent in excess to an alcoholic solution of the alkaloid. The yellow precipitate was recrystallized from alcohol in which atropine picrolonate is readily soluble. It appears as short, pointed crystals which are grouped in thick, spherical masses (Fig. IO). It melts at 194' and turns orange-red. Upon analysis it gave the following result :
o .2009 gram of substance gave 2 2.4 cc. of nitrogen gas at 20' and 7 5 2 mm.
Calculated for ‘&Ha~Ns0~: Found:
N...........,....... 12.66 per cent. 12.63 per cent.
This compound was prepared by adding a hot alcoholic solu- tion of quinine to a hot alcoholic solution of picrolonic acid con- taining the reagent in the proportion of two molecules to one molecule of the alkaloid. The picrolonate crystallized slowly and seemed rather soluble in alcohol, but in recrystallizing it from hot alcohol we found that a much larger volume of solvent was required for complete solution. This compound appears in hair-like needles which form spherical masses or sheaf-like clusters (Fig. I I). It begins to darken at 223O and melts at 22~~. Upon analysis it gave the following result:
o. 2007 gram of substance gave 28.8 cc. of nitrogen gas at 19' and 750 mm.
Caloulated for ‘&hoN1o01z: Found:
TV................... 16.4 per cent. 16.28 per cent.
Hydrastine Picrolonate-C,,H,,NO,. C,,H,N,O,.
This compound was prepared by adding the reagent in excess to a hot alcoholic solution of the alkaloid. There was an imme- diate crystalline precipitate which was recrystallized from hot alcohol somewhat diluted with water. Hydrastine picrolonate is light-yellow and melts at 220~. It appears as long, flat, pris- matic needles with square or pointed ends and these are some- times in spherical clusters (Fig. 12). Upon analysis it gave the following result :
o. 2013 gram of substance gave 19.7 cc. of nitrogen gas at 21' and 747 mm.
N . . . . . . . . . . . . . . . .
Calculated for ‘&&~WII:
10.81 per cent.
Found:
10.95 per cent.
Other Alkaloids and Picrolonic Acid.
In addition to the preceding nine alkaloids, which form definite crystalline compounds with picrolonic acid, we have also studied the action of this reagent upon cocaine, aconitine and caffeine. We did not succeed in obtaining as satisfactory results with these three alkaloids. Hager’ found that a saturated aqueous solution of picric acid would not precipitate caffeine and there was some doubt about its giving a precipitate with aconitine.
Pure cocaine hydrochloride, dissolved in water and treated with alcoholic picrolonic acid, gave only a thick resinous pre- cipitate which did not show the least sign of becoming crystal- line after long standing, or when dissolved in alcohol in which it is readily soluble. The same result was obtained when the alkaloid itself was dissolved in alcohol and treated with the reagent. Consequently we did not make any further exami- nation of this substance.
We had a hope that aconitine, for which the number of well- defined tests is limited, might give a characteristic result with picrolonic acid. When pure crystallized aconitine was dis- solved in alcohol and treated with picrolonic acid, there was not an immediate precipitate. As the solution evaporated, crystal- lization began but it was evident that these crystals were not homogeneous. Some of these were yellow and others were white. A result quite similar to this was given also by caffeine. We did not, therefore, continue the examination of the picrolo- nates of these alkaloids.
In conclusion we desire to acknowledge our obligations to Prof. H. Steudel who directed our attention to the subject con- sidered in these pages and very kindly supplied us with a portion of the picrolonic acid required for the work.
