THE VERATRINE ALKALOIDS

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1 THE VERATRINE ALKALOIDS XIX. ON PROTOVERATRINE AND ITS ALKAMINE, PROTOVERINE BY WALTER A. JACOBS AND LYMAN C. CRAIG (From the Laboratories of The Rockefeller Znstitute for Medical Research, New York) (Received for publication, May 19, 1943) Protoveratrine is a sparingly soluble alkaloid which comprises an appreciable fraction of the crystallizable alkaloids of T/era&urn album. It was first isolated by Salzberger (I.), who ascribed to it the formulation C32H510nN. This formulation has since been revised to CMHMOMN by Poethke (2), who succeeded in isolating from it on alkaline hydrolysis acetic acid, methylethylacetic acid, and methylethylglycolic acid. A supposed alkamine, protooerine, for which a corresponding formulation, C28H46010N, was assumed, was obtained with difficulty and only in amorphous form and no crystalline derivatives were prepared from it. Protoveratrine was thus concluded to be the triacyl ester of the alkamine, protoverine. In our previous communication on the dehydrogenation of protoveratrine itself (3), we have described the production of 2-ethyld-met,hylpyridine, which had already been shown to be a characteristic basic dehydrogenation product from cevine (4) and jervine (5), and which more recently has been found to be a product also from rubijervine (G), germine (7), and from the potato alkamine, In addition, the formulations of jervine, rubijervine (and isorubijervine), and germine have been revised to C$TH3903N, C&H4302N, and C~,HQO~N, respectively, so that, like cevine, &H4308N, and solanidine, CUH~~ON, they are all CW 1 Since the appearance of our paper (8) on the correlation of the veratrine alkaloids with the potato alkaloids, the recent article of Prelog and Szpilfogel (9) has reached us. These workers have also shown that the base which results on dehydrogenation of solanidine is 2-ethyl-5-methylpyridine. They have confirmed our earlier conclusions as to the identity of this base by its comparison with a base obtained by synthesis. They have adopted the same structural hexacyclic arrangement of the ring system for solanidine which we have already clearly expressed in the case of the veratrine alkaloids (IO) with the exception of the possibility that Ring B can be 5- membered. Our discussion contained also the definite suggestion of a resemblance to the picture to be found in the case of the potato alkamine, in which, however, Ring B must be B-membered as in t-he sterols, since Diels hydrocarbon had been obtained from it on dehydrogenation. At that time, it was already our intention to test this suggested analogy by a similar study of the solanum alkamines, the results of which we have since published. WC shall continue to regard the solanum alkamines as a subject of our studies, since they are members of the general group to which the veratrine alkaloids also belong. 271

2 272 VERATRINE ALKALOIDS. XIX derivatives. In view of the apparent discrepancy in the case of protoveratrine, we have returned to a study of this alkaloid. It has now been found possible to isolate the alkamine, protoverine, in crystalline form by continuous extraction of the saponification mixture with chloroform. As will be presented further on, preliminary saturation of the diluted mixture with COZ was found necessary to reduce to a minimum isomerization to an isoprotoverine. Protoverine readily crystallized on concentration of the chloroform extract with chloroform of crystallization. This permitted its sharp separation from isoprotoverine, which remained in the mother liquor. Protoverine also crystallized from methanol and from water with solvent of crystallization. Analyses of the anhydrous substance have agreed with the formulation C2,H4309N and not with C&H45010N, as derived by Poethke. This formulation has been supported by the results obtained with a number of its derivatives. Attempts to hydrogenate protoverine with platinum oxide catalyst as the free base, or as the hydrochloride in methanol, were unsuccessful. However, on reduction with sodium in butanol, a dihydro base, G,HJE,O~N, was obtained. The same dihydro base was obtained also from isoprotoverine described below. Whether it is to be regarded as a dihydroprotoverine or dihydroisoprotoverine has not as yet been determined. Although the hydrochloride of protoverine could not be made to crystallize, the alkamine readily formed a crystalline acetonylprotoverine hydrochloride, which in turn yielded the crystalline acetonylprotoverine, C3,,H470~N. The formation of this derivative parallels that of acetonylgermine (7) and indicates that two of the hydroxyl groups in each of these alkamines are situated on 2 vicinal carbon atoms, or on carbon atoms removed by an intervening carbon atom. It is probable that these positions will be found to be identical in both of these alkamines. The chloroform mother liquor of the above protoverine-chloroform compound after removal of solvent yielded material which crystallized from methanol as needles contaminated with prisms of protoverine, which were separated by fractionation. The needles were found to consist of isoprotoverine, G~H4309N. This substance was shown to be the result of the isomerizing action of alkali on protoverine, since the latter, when treated under certain conditions with alkali, was partly converted into isoprotoverine. If too strong alkali or too high a temperature was employed, the alkamine was further altered with the formation of a deep yellow solution, from which very little crystalline material could be isolated. Contrary to protovcrine, isoprotoverine could be hydrogenated to a dihydroisoprotoverine, C&H*E,O~N, which was distinct from the product of the sodium reduction described above. This isomerism is probably due to the different position occupied by the double bond in each of these bases. As will be presented in a subsequent paper, a parallel picture is to

3 W. A. JACOBS AND L. C. CRAIG 273 be found in the relationship between germine and isogermine. Although, as in the case of protoverine, isoprotoverine was found to react with acetone, no crystalline product could be obtained from the reaction mixture. The formation of dihydro derivatives from protoverine, together with other observations, points definitely to the presence of a double bond in this alkamine. Like cevine, germine, and rubijervine, protoverine behaves as a tertiary base. The results of the active H determinations were in close agreement with the presence of nine hydroxyl groups in the alkamine. In support of this, acetonylprotoverine gave approximately 7 moles of methane as required by the presence of seven free OH groups. This conclusion was further supported by the result of the Zerewitinoff determination on the alkaloid protoveratrine itself. This approximated the requirements of seven free OH groups. Although the alkaloid must be a triacyl ester of protoverine, the extra OH group is contained in the oc-hydroxymethylethylacetyl group. Since it contains nine hydroxy1 groups and one double bond, protoverine, &Hd309N, like cevine, germine, and solanidine, must therefore be a hexacyclic tertiary sterol (or modified sterol) base.,4s the triacyl ester of protoverine with acetic, methylethylacetic, and hydroxymethylethylacetic acids, the formulation of protoveratrine, therefore, must be C39Ha013N. All of our attempts to obtain proper analytical results with the alkaloid after repeated recrystallization, however, have not been satisfying. It is possible that retention of solvent may have contributed to this result. Somewhat better analytical results were obtained with the alkaloid which had been recovered after exposure to the conditions for the production of an acetonyl derivative, such as that used in the case of its alkamine, protoverine. The failure to form such an acetonyl derivative shows that one or both of the hydroxyl groups responsible for this function in the alkamine must be covered by acylation in the alkaloid. It is probable that one of the acylated OH groups must be situated on carbon atom 3 of the sterol or modified sterol skeleton. This point will be discussed further in a subsequent paper. EXPERIMENTAL Proloveratrine-For the isolation of this alkaloid from the crude benzene extract of mixed alkaloids, we have followed essentially the procedure outlined in our previous paper (3). The analytical results from the material obtained by a repetition of our previous recrystallization procedure have been essentially duplicated. Again the substance separated on addition of ammonia to the solution of the acetate in alcohol as small four-sided platelets which decomposed at 275 (uncorrected)2 after pre- 2 The uncorrcctcd melting points were taken in the usual manner, but not corrected for stem exposure. The others are corrected micro melting points.

4 274 VERATRINE ALKALOIDS. XIX liminary darkening and sintering. The decomposition point depended upon the rate of heating. When the substance was dried both at 120 (a) and 140 (b) at 2 mm., no appreciable loss could be noted. C&JI,OI~N. Calculated. C 62.25, H 8.18 Found. (a) 61.48, 7.95 (b) 61.76, 7.72 This material was recrystallized several times by addition of ether to the chloroform solution. No retention of solvent could be detected on drying. Found, C 61.28, H 8.11 Similar results were obtained when recrystallization was accomplished by addition of 95 per cent alcohol to the solution in chloroform, followed by boiling off the chloroform. The substance separated as six-sided platelets and prisms, which decomposed at 275 (uncorrected) after preliminary discoloration. Found, C 61.48, H 7.98; C 61.56, H mg. of substance gave cc. of CH, (25, mm.); at 95 there was no change. Found, H 0.99;, calculated for 7H, Protoveratrine was then treated with acetone and HCl under conditions which caused the production of an acetonyl derivative from protoverine as follows: Although the alkaloid itself was recovered, the analytical data approached more nearly the theoretical values. 0.1 gm. of protoveratrine was suspended in 2 cc. of acetone and continuously treated with HCI (1.19) until acid to Congo red and solution was complete. The mixture could not be made to crystallize, even on addition of ether. After dilution with water, the mixture was promptly made alkaline and extracted with ether. The latter on concentration gave the original alkaloid. Found, C 61.91, H 8.02 In another experiment, the acidified acetone solution of the alkaloid was allowed to stand for 20 hours. It was treated directly with Na2C03 solution, then diluted, and extracted with chloroform. The extracted material crystallized from ether as six-sided platelets. Found, C 61.84, H 8.08 In an experiment in which the mixture stood for 3 days, similar results were obtained. The substance recrystallized from chloroform-ether decomposed at (uncorrected) after preliminary darkening. Found, C 61.87, 61.55, H 7.68, 8.16

5 W. A. JACOBS AND L. C. CRAIG 275 Protoveriae-3.8 gm. of protoveratrine were treated with 26 cc. of a solution of 4 gm. of NaOH in 100 cc. of methanol. Gentle warming caused rapid solution and the odor of fatty acid esters quickly became apparent. After about 7 minutes warming, the weakly colored solution was cooled, made just acid with dilute HzS04, and then made faintly alkaline to phenolphthalein with KazC03 solution. The mixture after dilution to about 100 cc. was placed in a special continuous extractor for extraction wit.h chloroform. The condensed chloroform, which fell in a continuous spray through the aqueous mixture to a lower chloroform layer, continuously passed through an overflow into the flask of boiling chloroform below. After about 20 hours, the extraction was interrupted. The flask contained a copious suspension of delicate needles. The collected material amounted to 1.8 gm. For recrystallization,. the chloroform compound was dissolved in a necessarily large volume of methanol and concentrated to 20 cc. The base separated as small, glistening, sparingly soluble prisms. The yield was 1.28 gm. It softened gradually to a slowly effervescing resin at , after preliminary sintering. The melting point, however, varied with different preparations, which was due, apparently, to a varying solvent content. [a]: = -12 (c = 0.96 in pyridine) For analysis, the substance was dried at 120 and 2 mm. CziH4,0&.2CH,0H. Calculated, CHaOH 10.87; found, 9.92 CzJLQoN. C 61.67, I Found. (a) 61.45, 8.30 (b) 62.06, 8.32 A sample of the substance was dissolved by heating in a small volume of diluted methanol and, after addition of sufficient water, the methanol was boiled off. The base gradually crystallized as prismatic needles, which slowly softened to a melt at (uncorrected). The aqueous solution foamed readily. For analysis, the substance was dried at 120 and 2 mm. C~TH~~O~N.H~O. Calculated, Hz0 3.31; found, 3.22 Anhydrous Substance-Found, C 61.37, H mg. of substance gave 1.58 cc. of CH, (26, mm.); at 95 there was no change. Found, II 1.69; calculated for 9H, When the chloroform mother liquor of the above chloroform compound of protoverine was concentrated in vacua to dryness, the resinous residue weighed 1.24 gm. When dissolved in a small volume of methanol, it readily crystallized on rubbing as minute platelets mixed with prisms and occasionally needles. After collection with methanol, 0.63 gm. was

6 276 VERATRINE ALKALOIDS. XIX obtained. On treatment with chloroform, this was found to yield an additional amount of the chloroform compound of protoverine. The mother liquor of this contained a small amount of isoprotoverine. If the diluted saponification mixture was not first made weakly alkaline, as described above, before the continuous extraction with chloroform, a larger proportion of isoprotoverine was obtained as described below under isoprotoverine. Acetonylprotoverine-80 mg. of protoverine were suspended in 1. cc. of methanol and then brought into solution with sufficient HCl (1.19) to render the mixture distinctly acid to Congo red. On cautious addition of acetone t o the initial precipitation point, the precipitate gradually redissolved, and it soon became possible to continue the addition of acetone without the formation of the amorphous material. On being rubbed, the hydrochloride of the acetonyl derivative crystallized in excellent yield. On recryst allization from methanol-acetone, it separated as microscopic aggregates of short, flat needles which melted with decomposition at (uncorrected) after preliminary discoloration and sintering. For analysis, the salt was dried at 110 and 2 mm. C&480!XCl. Calculated, C 59.82, H 8.04; found, C 59.62, H 8.10 The free base was readily obtained by addition of an excess of Xa2C03 solution to the solution of the salt in 50 per cent methanol. For recrystallization, a relatively large volume of methanol was required and, when concentrated to smaller volume, acetonylprotoverine separated as microscopic flat needles or elongated platelets. After initial sintering and coloring above 235, it gradually melted to a dark mass at (uncorrected). C~ H~TOJ. Calculated, C 63.67, II 8.38; found, C 63.58, H mg. of substance gave 1.40 cc. of CH, (26, mm.); at 95 there was no change. Found, H 1.15; calculated for 7H, Reduction 01 Protoverine (Dihydroprotoverine?)-2 gm. of sodium were added t,o a boiling solution of 0.5 gm. of protoverine in 20 cc. of butanol, and the mixture was at once vigorously shaken. The reaction mixture colored when air gained access to it. After completion, the chilled mixture was treated with water and saturated with COS. The butanol was removed under reduced pressure, and the diluted mixture was cont,inuously extracted with chloroform overnight. The accumulated chloroform extract contained crystalline material. After concentration to small volume, the crystals were collected with chloroform. The yield was 0.16 gm. For recrystallization, the product was dissolved in a necessarily large volume of methanol. After concentration to 10 cc., it separated without solvent

7 W. A. JACOBS AND L. C. CRAIG 277 as micro platelets which, although mostly irregular, showed a tendency to be triangular. Under the microscope, it did not exhibit a real melting point. It began to show decomposition above 300, which gradually increased in rate, especially at [LY]; = -54 (c = 0.50 in pyridinc) C,J1,60sW. Calculated. C 61.44, H 8.60 Found. (a) 61.34, 61.52, 8.61 Isoprotoverine-In a number of experiments in which the precaution was not taken to neutralize the saponification mixture before extraction, a larger proportion of this alkamine resulted. The mother liquors of the chloroform compound of protoverine, which had accumulated from the saponification of 15 gm. of protoveratrine, were concentrated to remove chloroform. The residue was dissolved in a small volume of methanol and then diluted with water and, after addition of Na2C03 solution, the mixture was reextracted in a continuous extractor with chloroform for 20 hours. The chloroform extract on concentration yielded a slowly crystallizing fraction of needles, which proved to be isoprotoverine. The filtrate was concentrated and the residual chloroform was boiled off after addition of methanol. From a volume of about 20 cc. of methanol, a copious separation of needles occurred. This fraction of the isoalkamine, together with what crystallized directly from the above concentrated chloroform solution, amounted to 1.06 gm. The methanol mother liquor on further concentration formed a thick paste of a mixture of needles of isoprotoverine and rosettes of stouter crystals of protoverine. Isoprotoverine separated on recrystallization from methanol as needles or thin, short prisms, which were practically without solvent. It began to color above 240, then sintered and darkened on further heating, and effervesced at 264 (uncorrected). [a]; = -42 (c = 0.99 in pyridine) C27H,309N. Calculated. C 61.67, H 8.25 Found. (a) 61.54, 8.34 I (b) 61.70, 8.37 Attempts to obtain a crystalline hydrochloride were unsuccessful. The same substance was obtained directly from protoverine as follows: 0.2 gm. of protoverine was dissolved by warming in a mixture of 1 cc. of methanol and 3 cc. of HzO. 1 cc. of N NaOH was added and the solution was warmed to 50 for 2 hours. After a few minutes, a turbidity developed which gradually increased to a deposit. The solution was treated with 5 cc. of methanol and diluted to about 80 cc. The mixture was extracted with chloroform for 18 hours in a continuous extractor. The addition

8 278 VERATRINE ALKALOIDS. XIX of methanol helped to reduce the tendency to form an emulsion during the extraction. The chloroform extract on concentration to a few cc. yielded a paste of needles which were collected with chloroform. 67 mg. were obtained. This consisted of unchanged protoverine. The mother liquor was concentrated to remove solvent and again dissolved in a little chloroform. il small second fraction was collected. Finally, a third small crop was obtained. The final mother liquor was freed from chloroform by boiling down with methanol. The residue weighed 0.11 gm., representing roughly 50 per cent of transformed starting material. The solution in a small volume of methanol crystallized first as a mixture of pointed micro platelets, followed by a mass of delicate needles. The collected material weighed 53 mg. The homogeneity of the substance remained in question, since it did not dissolve completely in chloroform. By rapid fractionation from methanol, it was possible to remove first a rapidly crystallizing, small fraction of protoverine. This was followed by a fraction of needles of isoprotoverine. The substance so obtained sintered above 200, then gradually softened and darkened on further heating. The mass then melted with effervescence at 254 (uncorrected). [CL], = -37 (c = 0.70 in pyridine) For analysis, it was dried at 110 and 2 mm., since it was found to contain solvent. CzyH4309N. Calculated, C 61.67, H 8.25; found, C 61.63, H 8.25 As a check on identity, this substance was found to yield the following dihydro derivative. Dihyclroisoprotoverine-0.1 gm. of isoprotoverine (which had been obtained as a by-product on saponification of the alkaloid) was hydrogenated in methanol with 50 mg. of platinum oxide catalyst. The absorption was roughly 1 mole in excess of that required by the catalyst. During the hydrogenation, the substance which gradually dissolved was replaced by a suspension of the sparingly soluble product. A relatively large volume of a mixture of methanol and chloroform was required to redissolve the substance for separation from the catalyst. The filtrate, after concentration to remove the chloroform, yielded the substance as microscopic, short, flat needles or narrow platelets which contained solvent. In the micro melting point apparatus, it showed a slow decomposition at after preliminary softening and discoloration. When treated under the microscope with a drop of water, it first dissolved and then crystallized again as delicate needles, apparently due to interchange of solvent. [a], = -49 (c = 0.67 in pyridine)

9 W. A. JACOBS AND L. C. CRAIG 279 For analysis, the substance was dried at 120 and 2 mm. CzTH,,O&.BCH,OH. Calculated, CH,OH 10.82; found, Anhydrous Substance-Cz,HajO&. Calculated. C 61.44, I Found. (a) 61.29, , I 8.50 Isoprotoverine, which was obtained by isomerization of protoverine, yielded on hydrogenation a substance indistinguishable in properties from the above. Anhydrous Subslance-Found, C 61.44, H All microanalyses and active H determinations were performed by Mr. D. Rigakos of this laboratory. BIBLIOGRAPHY 1. Salzbcrger, G., Arch. Pharm., 228, 462 (1890). 2. Poethke, W., Arch. Phar,m., 276, 357, 571 (1937). 3. Craig, L. C., and Jacobs, W. A., J. Biol. Chem., 143, 427 (1942). 4. Jacobs, W. A., and Craig, L. C., J. Biol. Chem., 120, 447 (1937); 124, 659 (1938). 5. Jacobs, W. A., Craig, L. C., and Lavin, G. I., J. Biol. Chem., 141, 51 (1941). 6. Jacobs, 1%. A., and Craig, L. C., J. Biol. Chem., 148, 41 (1943). 7. Craig, L. C., and Jacobs, W. A., J. Biol. Chem., 148, 57 (1943). 8. Craig, L. C., and Jacobs, W. -I., Science, 97, 122 (1943). 9. Prelog, V., and Szpilfogel, S., Helv. chim. acta, 25, 1306 (1942). 10. Craig, L. C., and Jacobs, W. A., J. Biol. Chem., 141, 253 (1941).

10 THE VERATRINE ALKALOIDS: XIX. ON PROTOVERATRINE AND ITS ALKAMINE, PROTOVERINE Walter A. Jacobs and Lyman C. Craig J. Biol. Chem. 1943, 149: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1

THE OXIDATION OF ESTRONE BY HYDROGEN PEROXIDE

THE OXIDATION OF ESTRONE BY HYDROGEN PEROXIDE BY W. W. WESTERFELD* (Prom the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, and the Department of Biological