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Investigation of the alkaloids of Punica granatum L
.4RCHIVES
OF BIOCHEMISTRY
Investigation
AND
BIOPHYSICS
of the Alkaloids
69,
27-32 (1957)
of Punica granatum L.’
J. P. Wibaut and U. Hollstein Iirom the Laboratory
for Organic Chemistry, Rrnsterdam, Holland
Xunicipa~l
Cinioersity
01’
Received December 14, 1956
The alkaloid pelletierine CsH150N has been mentioned in the chemical, pharmaceutical, and medical literature for about 80 years. Pelletierinr is said to occur in the bark of Punica granatum L. together with pseudopelletierine, C&H170N; isopelletierine, C$H,,ON; and methylisopelletierine, C9H170N. It is thought that pelletierine has anthelmintic properties and that the anthelmintic activity of the mixture of alkaloids from Pzsnica granatum is mainly (or at least to a considerahlc extent) due to the presence of pelletierine in these mixtures. On the strength of various reactions, Hess (l), in 1917--l& ascribed the structure of 3-(2’-piperidyl)propanal to pelletierine. although the degradation evidence as given by him seems to support this conclusion, there may be an unsuspected weak link in his chain of argument. Several attempts during the period 1940-3933 to synthesize the 3-(2’ pyridyl)propionaldehyde were unsuccessful (2-4). Though the diethylacetal of this aldehyde has been synthesized, it has not, been possible to obtain the aldehyde itself. Although this aldehyde is no doubt formed primarily through hydrolysis of the acetal, only resinous substances are obtained. Ot,her synthetic approaches also failed. Therefore, in recent years t,he existence of pelletierine has been questioned. Galinovsky and Vogl (5) prepared t’he N-bcnzoyl derit-abive from synthetic isopelletierine, l-(2’-piperidyl)propanone. On the other hand, from a commercial sample of “pelletierine hydrobromide,” in whirh according to their investigation, the hydrohromides of some alkaloids from Punica granatzcm were present, they prepared N-henzoylisopelle1 Compare WIBAUT, J. P., AND HOLLSTEIN? tenschap. Ser. B 69, 426 (1956). 27
I;., Pror.
Koninkl.
Ned. Akad.
We-
28
WIBAUT
AND HOLLSTEIN
tierine; it appeared from the melting point and mixed melting point that this preparation was identical with the synthetic product. On the strength of these as well as other observations, Galinovsky and Hiillinger (6) hold the view that the so-called pelletierine does not exist, but that the alkaloid described as such is in reality isopelletierine. We have made similar observations; a sample of so-called “nn-pelletierine hydrobromide,” which had been isolated from the bark of Pun& grunatum by Wellcome Research Laboratories (England), was found to be identical with the hydrobromide of synthetically prepared nn-isopelletierine (4). Obviously, these observations do not warrant a definite answer to the question as to whether a substance having the structural formula of 3-(2’-piperidyl)propanal occurs in Punica granatum. Therefore we started a new investigation using modern analytical methods (7). In a previous communication (8) on this subject we described the separation of the alkaloids of Punica granatum L. by means of chromatography on paper. We examined three samples of bark of different origin. All the three samples contain, in addition to pseudopelletierine, isopelletierine, and methylisopelletierine, other basic components, which give a color reaction with Dragendorff’s reagent. We have improved the chromatographic separation. The results obtained with three samples of bark of different origin are given in this communication. RESULTS
AND
DISCUSSION
Table I first of all gives the Rf values of some still unknown bases, which for the time being are indicated by A0 , A1, AZ, Aa, and Ad, TABLE A0
Rf
A1
I A2
At
A4
A-total -------
Me-
%;:
thyl-
0.35
0.48
Isop.
isop.
0.03
0.07
0.11
0.17
0.21
%
%
%
%
%
%
%
2 9 10 5
86 83 78 79 82
3 3 4 4
0.55
-p---p_I_p %
Extract Extract Extract Extract Extract bined
1 2 3, neutral 3, alkaline 3, com-
11 9 11 15 9
2
5 2 2 3
%
ALKALOIDS
OF
PUNICA
GRANATUM
29
and the Rf values of pseudopelletierine, methylisopelletierine, and isopelletierine. The numbers 1, 2, and 3, respectively, in Table I, relate to: 1. bark of unknown origin, obtained from England; 2. bark of Italian origin; and S. bark of Spanish origin. The percentages of pseudo-, methyliso-, and isopelletierine have been calculated from calibration curves representing the relation between the size of the spots and t’he quantity of synthetic alkaloid deposited. The percentages of the unknown bases A have been calculated from a fictitious calibration curve, which we obtained by averaging the three well known calibration curves. Relative errors of about 50 % should be taken into account for percentages under 10 %. In paper chromatography of bark 1 the technique had not yet been developed to such an extent as to permit separation of substances A into their components, so that we can only give A4otal. The alkaline and neutral extraction, respectively, of bark 3 depends on whether the bark has been subjected to an alkaline pretreatment (with Ca(OH)z). The combined extraction has been described in our previous communication (8). By adapting t,he circular preparative paper chromatography according to Giri (9) to our separation problem we succeeded in isolating the bases A, , Az , and A4 as picrolonates from the combined extract of bark 3. The A,, and A8 concentrations were so low that isolation was not possible. On the assumption that the unknown bases contain only one nitrogen atom per molecule, we find from the analyses of the picrolonates the following simplest formulas for the bases: A,: CgHli02N; AZ: C,oH,,02N; A4:C7H90N. These formulas show that, in comparison with A, , Az contains an additional CHZ group, so that the bases Al and A2 might be considered as homologs; the low hydrogen content of A4 points toward an aromatic character. The principal conclusion, however, is that apart from isopelletierine, the bark does not contain an isomeric compound of the composition CsH160N. The slight quantities of Aa and A, , which cannot be isolated, can be left out of consideration. Besides, in quite a different way could we demonstrate that the various bark extracts examined do not contain a substance of the structure 3-(2’-piperidyl)propanal. In spite of the instability of this aldehyde, former investigations by Galinovsky and co-workers (3) showed that this substance is formed as an intermediary during the last’ step of the synthesis before resinification occurs. This conclusion could be confirmed by
30
WIBAUT
AND
HOLLSTEIN
experiments in which the 3-(2’-piperidyl)propanal-2,4dinitrophenylhydrazone hydrochloride (m.p. 191-193”) was formed from the acetal after acid hydrolysis. The presence in the hydrolysis product of an aldehyde group could be demonstrated with Schiff’s reagent and by means of a-though less specific-reaction with ammoniacal silver solution. The 3-(2’-piperidyl)propanal was prepared in dilute solution in three ways, which have previously been tested for a synthesis, viz. by acid hydrolysis of the acetal (2), by dehydrogenation of 4-(2’-piperidyl)butane-1,2-diol (4), and by reduction of 3-ketooctahydropyrrocoline. From the close agreement of Rf values of these three reaction products in each of 11 different paper chromatographic systems, we concluded that these products are identical. These spots, which we therefore ascribe to 3-(2’piperidyl)propanal, do not occur in the bark extracts of the samples under investigation. We now consider it very improbable that the aldehyde 5-(Y-piperidyl)propanal occurs in nature. The fact that the resinification of 3-(2’-piperidyl)propanal is actually based on autocondensation, as presumed by former investigators, has now been proved in our opinion by the infrared spectra of the hydrolysis product of 3-(2’-piperidyl)propanal acetal in carbon tetrachloride and of the hydrobromide of this hydrolysis product in a potassium bromide disk. Using carbon tetrachloride as solvent, we observed bands at 1682 cm.-’ and at 1643 cm.-‘; for the potassium bromide disk the bands were found at 1677 cm.-’ and at 1632 cm.-‘. We ascribe these bands to an (Y, p-unsaturated aldehyde and to a carbon-carbon double bond. The resinification is therefore based on a polyaldol condensation, followed by dehydration. Galinovsky’s supposition that the pelletierine described in the literature is identical with isopelletierine, would now seem to us to be correct. The melting points of the hydrochloride, the hydrobromide, and the picrate of isopelletierine closely approximate the values stated in the literature for the hydrochloride, hydrobromide, and the picrate of pelletierine. The same applies to the melting point of the N-benzoyl compound of isopelletierine compared with the literature value for the N-benzoyl compound of pelletierine. We have prepared the oxime of nn-isopelletierine (m.p. 100-104”) and its picrate (m.p. 175-176”). The melting points of these compounds do
ALKALOIDS OF PUNICA GRANATUM
31
not differ much from the values stated by Hess for the melting points of the pelletierine oxime and its picrate, respectively. In connection with indications in the literature that pelletierine shows anthehnintic activity, it is interesting to find out to what extent 3-(2’piperidyl)propanal possesses this property in comparison with #-pelletierine, isopelletierine, and methylisopelletierine. This investigation has been made by Dr. J. van Noordwijk at the Pharmacotherapeutical Laboratory of the University of Amsterdam.2 For this investigation dilute solutions of the hydrochlorides of $-pelletierine, nn-isopelletierine, and nn-methylisopelletierine were used; these substances have been synthesized at our laboratory. The pharmacological activity of these substances has been investigated on the liver fluke according to the method of Chance and Mansour (10). Isopelletierine hydrochloride is still active at a concentration of l/16,000 to l/32,000, while the hydrochloride of S-(d’-piperidyl)propanal is not active at a concentration of l/1000. Methylisopelletierine and +pelletierine (as hydrochlorides) are still active in a solution of l/4000 to l/8000. SUMMARY
Of the Punica granatum alkaloids, the isopelletierine shows the greatest activity (with respect to the liver fluke), methylisopelletierine and #-pelletierine being less active. It follows therefore that the anthehnintic activity of extracts from Punica granutum is chiefly due to isopelletierine. This is in agreement with the conception of Galinowsky and ourselves, that the pelletierine mentioned in the literature is identical with isopelletierine. REFERENCES 1. HESS, K., AND EICHEL, A., Ber. 66, 1192 (1917). 2. BEETS, M. G. J., AND WIBAUT, J. P., Rec. trav. chim. 60, 905 (1941); SPIELMAN. M. A., SWADESH, S., AND MORTENSON, C. W., J. Org. Chem. 6, 780 (1941). 3. GALINOVSKY, F., VOQL, O., AND WEISER, R., Monatsh. 83, 114 (1952). 4. WIBAUT, J. P., AND HIRSCHEL, M. I., Rec. trav. chim. 76, 225 (1966). 5. GALINOVSKY, F., AND VOQL, O., Monatsh. 83, 1055 (1952). 6. GALINOVSKY, F., AND H~LLINQER, R., Monatsh. 86, 1012 (1954). 7. WIBAUT, J. P., BEYERMAN, H. C., AND ENTHOVEN, P. H., Rec. trav. chim. 73, 102 (1954). 2 We are indebted to Prof. Dr. B. Mendel, director of this laboratory, who placed the necessary equipment at our disposal and to Dr. J. van Noordwijk, who permitted us to mention the results obtained by him in this communication.
32
WIBAUT
AND
HOLLSTEIK
8. WIBAUT, J. P., BEYERMAN, H. C., HOLLSTEIN, U., IMULLER, Y. M. F., AND GREUELL, E., Proc. Konink. Ned. Akad. Wetenschap. Ser. B 63, 56 (1955). 9. GIN, K. V., J. Indian Inst. Sci. 37, 1 (1955). 10. CHANCE, M. R. A., AND MANSOUR, T. E., Brit. J. Pharmacol. 4, 7 (1949); NOORDWIJK, J. v., AND HOLLSTEIN, U., Acta Physiol. et Pharmacol. Neerl. 6, 212 (1956).
OF BIOCHEMISTRY
Investigation
AND
BIOPHYSICS
of the Alkaloids
69,
27-32 (1957)
of Punica granatum L.’
J. P. Wibaut and U. Hollstein Iirom the Laboratory
for Organic Chemistry, Rrnsterdam, Holland
Xunicipa~l
Cinioersity
01’
Received December 14, 1956
The alkaloid pelletierine CsH150N has been mentioned in the chemical, pharmaceutical, and medical literature for about 80 years. Pelletierinr is said to occur in the bark of Punica granatum L. together with pseudopelletierine, C&H170N; isopelletierine, C$H,,ON; and methylisopelletierine, C9H170N. It is thought that pelletierine has anthelmintic properties and that the anthelmintic activity of the mixture of alkaloids from Pzsnica granatum is mainly (or at least to a considerahlc extent) due to the presence of pelletierine in these mixtures. On the strength of various reactions, Hess (l), in 1917--l& ascribed the structure of 3-(2’-piperidyl)propanal to pelletierine. although the degradation evidence as given by him seems to support this conclusion, there may be an unsuspected weak link in his chain of argument. Several attempts during the period 1940-3933 to synthesize the 3-(2’ pyridyl)propionaldehyde were unsuccessful (2-4). Though the diethylacetal of this aldehyde has been synthesized, it has not, been possible to obtain the aldehyde itself. Although this aldehyde is no doubt formed primarily through hydrolysis of the acetal, only resinous substances are obtained. Ot,her synthetic approaches also failed. Therefore, in recent years t,he existence of pelletierine has been questioned. Galinovsky and Vogl (5) prepared t’he N-bcnzoyl derit-abive from synthetic isopelletierine, l-(2’-piperidyl)propanone. On the other hand, from a commercial sample of “pelletierine hydrobromide,” in whirh according to their investigation, the hydrohromides of some alkaloids from Punica granatzcm were present, they prepared N-henzoylisopelle1 Compare WIBAUT, J. P., AND HOLLSTEIN? tenschap. Ser. B 69, 426 (1956). 27
I;., Pror.
Koninkl.
Ned. Akad.
We-
28
WIBAUT
AND HOLLSTEIN
tierine; it appeared from the melting point and mixed melting point that this preparation was identical with the synthetic product. On the strength of these as well as other observations, Galinovsky and Hiillinger (6) hold the view that the so-called pelletierine does not exist, but that the alkaloid described as such is in reality isopelletierine. We have made similar observations; a sample of so-called “nn-pelletierine hydrobromide,” which had been isolated from the bark of Pun& grunatum by Wellcome Research Laboratories (England), was found to be identical with the hydrobromide of synthetically prepared nn-isopelletierine (4). Obviously, these observations do not warrant a definite answer to the question as to whether a substance having the structural formula of 3-(2’-piperidyl)propanal occurs in Punica granatum. Therefore we started a new investigation using modern analytical methods (7). In a previous communication (8) on this subject we described the separation of the alkaloids of Punica granatum L. by means of chromatography on paper. We examined three samples of bark of different origin. All the three samples contain, in addition to pseudopelletierine, isopelletierine, and methylisopelletierine, other basic components, which give a color reaction with Dragendorff’s reagent. We have improved the chromatographic separation. The results obtained with three samples of bark of different origin are given in this communication. RESULTS
AND
DISCUSSION
Table I first of all gives the Rf values of some still unknown bases, which for the time being are indicated by A0 , A1, AZ, Aa, and Ad, TABLE A0
Rf
A1
I A2
At
A4
A-total -------
Me-
%;:
thyl-
0.35
0.48
Isop.
isop.
0.03
0.07
0.11
0.17
0.21
%
%
%
%
%
%
%
2 9 10 5
86 83 78 79 82
3 3 4 4
0.55
-p---p_I_p %
Extract Extract Extract Extract Extract bined
1 2 3, neutral 3, alkaline 3, com-
11 9 11 15 9
2
5 2 2 3
%
ALKALOIDS
OF
PUNICA
GRANATUM
29
and the Rf values of pseudopelletierine, methylisopelletierine, and isopelletierine. The numbers 1, 2, and 3, respectively, in Table I, relate to: 1. bark of unknown origin, obtained from England; 2. bark of Italian origin; and S. bark of Spanish origin. The percentages of pseudo-, methyliso-, and isopelletierine have been calculated from calibration curves representing the relation between the size of the spots and t’he quantity of synthetic alkaloid deposited. The percentages of the unknown bases A have been calculated from a fictitious calibration curve, which we obtained by averaging the three well known calibration curves. Relative errors of about 50 % should be taken into account for percentages under 10 %. In paper chromatography of bark 1 the technique had not yet been developed to such an extent as to permit separation of substances A into their components, so that we can only give A4otal. The alkaline and neutral extraction, respectively, of bark 3 depends on whether the bark has been subjected to an alkaline pretreatment (with Ca(OH)z). The combined extraction has been described in our previous communication (8). By adapting t,he circular preparative paper chromatography according to Giri (9) to our separation problem we succeeded in isolating the bases A, , Az , and A4 as picrolonates from the combined extract of bark 3. The A,, and A8 concentrations were so low that isolation was not possible. On the assumption that the unknown bases contain only one nitrogen atom per molecule, we find from the analyses of the picrolonates the following simplest formulas for the bases: A,: CgHli02N; AZ: C,oH,,02N; A4:C7H90N. These formulas show that, in comparison with A, , Az contains an additional CHZ group, so that the bases Al and A2 might be considered as homologs; the low hydrogen content of A4 points toward an aromatic character. The principal conclusion, however, is that apart from isopelletierine, the bark does not contain an isomeric compound of the composition CsH160N. The slight quantities of Aa and A, , which cannot be isolated, can be left out of consideration. Besides, in quite a different way could we demonstrate that the various bark extracts examined do not contain a substance of the structure 3-(2’-piperidyl)propanal. In spite of the instability of this aldehyde, former investigations by Galinovsky and co-workers (3) showed that this substance is formed as an intermediary during the last’ step of the synthesis before resinification occurs. This conclusion could be confirmed by
30
WIBAUT
AND
HOLLSTEIN
experiments in which the 3-(2’-piperidyl)propanal-2,4dinitrophenylhydrazone hydrochloride (m.p. 191-193”) was formed from the acetal after acid hydrolysis. The presence in the hydrolysis product of an aldehyde group could be demonstrated with Schiff’s reagent and by means of a-though less specific-reaction with ammoniacal silver solution. The 3-(2’-piperidyl)propanal was prepared in dilute solution in three ways, which have previously been tested for a synthesis, viz. by acid hydrolysis of the acetal (2), by dehydrogenation of 4-(2’-piperidyl)butane-1,2-diol (4), and by reduction of 3-ketooctahydropyrrocoline. From the close agreement of Rf values of these three reaction products in each of 11 different paper chromatographic systems, we concluded that these products are identical. These spots, which we therefore ascribe to 3-(2’piperidyl)propanal, do not occur in the bark extracts of the samples under investigation. We now consider it very improbable that the aldehyde 5-(Y-piperidyl)propanal occurs in nature. The fact that the resinification of 3-(2’-piperidyl)propanal is actually based on autocondensation, as presumed by former investigators, has now been proved in our opinion by the infrared spectra of the hydrolysis product of 3-(2’-piperidyl)propanal acetal in carbon tetrachloride and of the hydrobromide of this hydrolysis product in a potassium bromide disk. Using carbon tetrachloride as solvent, we observed bands at 1682 cm.-’ and at 1643 cm.-‘; for the potassium bromide disk the bands were found at 1677 cm.-’ and at 1632 cm.-‘. We ascribe these bands to an (Y, p-unsaturated aldehyde and to a carbon-carbon double bond. The resinification is therefore based on a polyaldol condensation, followed by dehydration. Galinovsky’s supposition that the pelletierine described in the literature is identical with isopelletierine, would now seem to us to be correct. The melting points of the hydrochloride, the hydrobromide, and the picrate of isopelletierine closely approximate the values stated in the literature for the hydrochloride, hydrobromide, and the picrate of pelletierine. The same applies to the melting point of the N-benzoyl compound of isopelletierine compared with the literature value for the N-benzoyl compound of pelletierine. We have prepared the oxime of nn-isopelletierine (m.p. 100-104”) and its picrate (m.p. 175-176”). The melting points of these compounds do
ALKALOIDS OF PUNICA GRANATUM
31
not differ much from the values stated by Hess for the melting points of the pelletierine oxime and its picrate, respectively. In connection with indications in the literature that pelletierine shows anthehnintic activity, it is interesting to find out to what extent 3-(2’piperidyl)propanal possesses this property in comparison with #-pelletierine, isopelletierine, and methylisopelletierine. This investigation has been made by Dr. J. van Noordwijk at the Pharmacotherapeutical Laboratory of the University of Amsterdam.2 For this investigation dilute solutions of the hydrochlorides of $-pelletierine, nn-isopelletierine, and nn-methylisopelletierine were used; these substances have been synthesized at our laboratory. The pharmacological activity of these substances has been investigated on the liver fluke according to the method of Chance and Mansour (10). Isopelletierine hydrochloride is still active at a concentration of l/16,000 to l/32,000, while the hydrochloride of S-(d’-piperidyl)propanal is not active at a concentration of l/1000. Methylisopelletierine and +pelletierine (as hydrochlorides) are still active in a solution of l/4000 to l/8000. SUMMARY
Of the Punica granatum alkaloids, the isopelletierine shows the greatest activity (with respect to the liver fluke), methylisopelletierine and #-pelletierine being less active. It follows therefore that the anthehnintic activity of extracts from Punica granutum is chiefly due to isopelletierine. This is in agreement with the conception of Galinowsky and ourselves, that the pelletierine mentioned in the literature is identical with isopelletierine. REFERENCES 1. HESS, K., AND EICHEL, A., Ber. 66, 1192 (1917). 2. BEETS, M. G. J., AND WIBAUT, J. P., Rec. trav. chim. 60, 905 (1941); SPIELMAN. M. A., SWADESH, S., AND MORTENSON, C. W., J. Org. Chem. 6, 780 (1941). 3. GALINOVSKY, F., VOQL, O., AND WEISER, R., Monatsh. 83, 114 (1952). 4. WIBAUT, J. P., AND HIRSCHEL, M. I., Rec. trav. chim. 76, 225 (1966). 5. GALINOVSKY, F., AND VOQL, O., Monatsh. 83, 1055 (1952). 6. GALINOVSKY, F., AND H~LLINQER, R., Monatsh. 86, 1012 (1954). 7. WIBAUT, J. P., BEYERMAN, H. C., AND ENTHOVEN, P. H., Rec. trav. chim. 73, 102 (1954). 2 We are indebted to Prof. Dr. B. Mendel, director of this laboratory, who placed the necessary equipment at our disposal and to Dr. J. van Noordwijk, who permitted us to mention the results obtained by him in this communication.
32
WIBAUT
AND
HOLLSTEIK
8. WIBAUT, J. P., BEYERMAN, H. C., HOLLSTEIN, U., IMULLER, Y. M. F., AND GREUELL, E., Proc. Konink. Ned. Akad. Wetenschap. Ser. B 63, 56 (1955). 9. GIN, K. V., J. Indian Inst. Sci. 37, 1 (1955). 10. CHANCE, M. R. A., AND MANSOUR, T. E., Brit. J. Pharmacol. 4, 7 (1949); NOORDWIJK, J. v., AND HOLLSTEIN, U., Acta Physiol. et Pharmacol. Neerl. 6, 212 (1956).