Pilocarpine and Isopilocarpine

The presence of alkaloids in jaborandi was first reported by M. Byasson (73) and the principal alkaloid, pilocarpine, was isolated independently by A. W. Gerrarci (74-77) and E. Hardy (78) both of whom characterized it by the preparation of several crystalline salts. A. Petit and M. Polonovski (50) showed the presence of a second alkaloid, isomeric with pilocarpine although pilocarpine is easily converted into the new base under a variety of conditions (see Section 5), they maintained that it also occurs in the leaves and is not purely an artifact formed during the isolation processes. Jowett (28) named this base isopilo- 

No detailed directions for the isolation of the jaborandi alkaloids on the laboratory scale appear to have been published, but F. Chemnitius 

An improvement in the extraction procedure (90) involves adding an acidic reagent to the leaves and extracting with benzene to remove fatty materials. The leaves are then rendered alkaline and the alkaloids are extracted with alcohol or benzene and purified through their salts. Other procedures have been outlined (78, 91). 

The separation of isopilocarpine from pilocarpine is often very difficult since mixtures containing between 50% and 66% of isopilocarpine yield nitrates that on recrystallization give products of constant melting 

Pilocarpine and isopilocarpine are usually obtained as colorless, viscous oils, but both have been crystallized as low-melting, hygroscopic solids (39, 50). Pilocarpine is triboluminescent (93). It is readily soluble in water, alcohol, and chloroform (28, 78, 50), fairly soluble in benzene (50), and almost insoluble in ether or light petroleum (28). Isopilocarpine is very similar in its solubility (28, 50). Pilocarpine base, in alcoholic solution, shows an absorption maximum of low intensity at 2630 A (94) in pilocarpine salicylate the bands of salicylic acid dominate the spectrum. The absorption in the ultraviolet of solutions of the nitrates of the alkaloids, recorded by Jowett (33), appear to be due solely to the nitrate ion. The infrared absorption spectra of pilocarpine and its hydrochloride have been examined (95). 

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Constitvtion of Pilocarpine and isoPilocarpine. Though numerous interesting observations have been made on these two alkaloids by M. and M. Polonovski, knowledge of their constitution is due principally to the work of Jowett and of Pinner. ... 

Roylance showed that pilocarpidine on -methylation yielded two products, pilocarpine and neopilocarpine (see below), thus confirming Hamack s suggestion that pilocarpidine is the imino-base corresponding to pilocarpine, Spath and Kunz showed that pilocarpidine, on treatment with alcoholic sodium ethoxide, is converted into isopilocarpidine (nitrate, m.p. 109-111°), which, on quaternary methylation, yields isopilocarpine metho-salts (methopicrate, m.p. 119-120°). The confirmation of these observations by the synthesis of pilocarpidine and isopilocarpidine and their conversion into pilocarpine and isopilocarpine has been described already. 

Pilosine, CjgHjgOgNj, obtained by Pyman (and almost simultaneously by L ger and Roques, who named it carpidine) from mother liquors remaining after the isolation of pilocarpine and isopilocarpine from the total alkaloids of P. mi.crophyllus, crystallises from alcohol in large colourless plates, m.p. 187°, [a]n + 39-9° (EtOH), lasvorotatory in alkaline solution. The salts do not crystallise readily the sulphate,. H SO, forms clusters of plates, m.p. 194-5°, fajD + 21° (HgO) the acid tartrate, B. H2C4HiOg, has m.p. 135-6°, [a]n + 24-2°, and the aurichloride, B. HAuCb, m.p. 143-4°. 

Fig. 7.13. HO -Catalyzed ring opening of pilocarpine (7.76) and isopilocarpine (7.77) to pilocarpic acid and isopilocarpic acid, respectively, and proton-catalyzed lactonization of the two acids to the respective lactone. Note that pilocarpine and isopilocarpine interconvert by a base-catalyzed reaction of epimerization (Reaction a).

Pilocarpine (P), a drug used in treating glaucoma, can potentially contain its epimer, isopilocarpine (I) as an impurity. In a study it was not possible to completely separate pilocarpine and isopilocarpine by variation of the pH of the running buffer. The optimal pH for separation should be 6.9 where both compounds are ca 50% ionised but even at this pH separation was incomplete.- ... 

Separation of pilocarpine and isopilocarpine by inclusion of P-cyclodextrin, the running buffer. (A) With addition of cyclodextrin. (B) Without addition of cyclodextrin. 

In 1900 Jowett (38) suggested that the differences between pilocarpine and isopilocarpine had their origin in the steric structures. This was in contrast to the opinion of Pinner et al. who assumed the differences to be caused by N-methylation at different sites in the imidazole ring (39). Lan-genbeck in 1924 assumed on the basis of chemical reactions, such as the formation of quaternary salts and ozonolysis, that the differences were of a steric nature (40,41). Preobrazhenski et al. in 1936 (42) supposed on the basis of different stability and optical rotation that pilocarpine and isopilocarpine should possess the cis and trans configuration, respectively. An identical conclusion was drawn by Zavyalov (43) in 1952, on the basis of... 

A stereoselective synthesis of (+)-pilocarpine (7) starting from L-histidine (2) has been worked out by Noordam et al. (88 - 90). Use was made of the S configuration of the amino acid, which is the same as that of C-3 of the lactone ring in both (+)-pilocarpine and (+)-isopilocarpine. Furthermore, regioselective N-alkylation reactions of the imidazole nucleus of histidine had been developed by Beyerman et al. (29,91). Schemes 3 and 4 depict the different ways of the regioselective alkylations. For the synthesis of pilocarpine, the N7I-methylation has been performed via Nb-protection with the 4-nitrobenzenesulfonyl group, instead of the benzoyl group (29). 

Metapilocarpine was first reported by Pinner he obtained it by heating pilocarpine hydrochloride at 225-235°C (118). Polonovski proposed a betaine structure (58) for the compound (119). On reinvestigation, metapilocarpine was shown to be a racemic mixture of isopilocarpine (120). The structure was proved by spectral analysis and by GLC comparison with authentic isopilocarpine. The pharmacological activity of the racemic product was compared to that of (+)-pilocarpine and (+)-isopilocarpine (120). 

Pilocarpine was gas chromatographed by Brochmann-Hanssen and Baerheim Svendsen1 on a packed column of 1.15 SE-30 at 175°C. Brochmann-Hanssen and Fontan chromatographed pilocarpine on packed columns of various polarity, e.g. XE-60, EGSS-V, Hl-EFF 8 B, as well as NGS and PVP + NGS. Massingill and Hodgkins used packed columns of various polarities and they separated pilocarpine and isopilocarpine on II JXR and 0.5 % Epon 1001 Resin with retention times of 9.08 min and 8.75 min, respectively, on JXR, and 11.50 min and 11.67 min, respectively, on Epon 1001 Resin. 

Bundgaard and Hansen separated pilocarpine and its degradation products on a silica gel column using methanol - 2 M phosphoric acid - water (3 5 92) containing 33S sodium sulfate as mobile phase (Fig.14.2). The poor separation of pilocarpine and isopilocarpine at a column temperature of 20-25° C was improved by increasing the temperature to 40° C. 

Examples of using cyclodextrin modifiers for enhancement of separation has been shown by Baeyens, et al.[44] and Yeo, et al. [45]. The first group demonstrated the separation of pilocarpine and isopilocarpine in ophthalmic preparations, while the later demonstrated the separation of plant growth hormones. 

Both pilocarpine and isopilocarpine behave as monoacidic bases, and a large number of crystalline salts of the two alkaloids have been described (see Section VI). By conductimetric (96) and potentiometric (97) titration, the first dissociation constant of pilocarpine was found to be 1 X 10 and 1.07 X 10" respectively that of isopilocarpine is 0.68 X 10 (97). By the indicator method, J. M. Kolthoff (98) estimates the first and second dissociation constants of pilocarpine to be 7 X 10" and... 

The minor alkaloid pilosine was isolated irom Pilocarpus microphyllus by F. L. Pyman (46) and independently by E. Leger and F. Roques (116, 117) who named it carpiline. The crude alkaloid was obtained by fractional precipitation of the bases remaining in the mother liquor after the removal of pilocarpine and isopilocarpine, and it was purified by crystallization from 90% alcohol. The yield corresponded to a content of only 0.007 % of the leaves, and it was established that no other alkaloid was present in the leaves in quantity greater than 0.003% (46). 

Pilosinine is a monoacidic base, and is optically active, the rotatory power falling rapidly in aqueous solution. It gives no color with diazo-benzenesulfonic acid and does not immediately decolorize permanganate. In its physical and chemical properties it closely resembles pilocarpine and isopilocarpine, and accordingly Pyman (46) proposed structures I and II for pilosine and pilosinine respectively, the alkaline degradation of pilosine being attributed to a reversed aldol condensation of a known type (177). On this basis, anhydropilosine has structure III. As pilosine... 

Tertiary aliphatic amines are rather difficult to detect, but they can be detected at 254 run after quatemization with p-NBBr [66]. Pilocarpine and isopilocarpine were derivatized in a sealed ampoule for 24 h at 40 °C, then separated by ion-pair HPLC. Tertiary amines can also be determined by post-chromatographic derivatization. Kudoh et al. described a method [67] involving derivatization with 1% dtric acid in acetic anhydride at 120 C, separation on a chemically bonded silica gel Nucleosil 5N(CH3)2, column, and detection at 550 nm. 

L. D. Dunn, B. S. Scott and E. D. Dorsey, Analysis of Pilocarpine and Isopilocarpine in Ophthalmic Solutions by Normal-Phase High-Performance Liquid Chromatography, J. Pharm. Sci. 70 446 (1981). 

Isopilocarpine coexists with pilocarpine in nature in aqueous solutions, pilocarpine can hydrolyze to both pilocarpinic and isopilocarpinic acids. The latter, in acidic pH, can recyclize to isopilocarpine (Scheme 25.2). The contrary does not happen, because the isopilocarpine hydrolysis yields only isopilocarpinic acid [25]. This change in the spatial structure decreases the binding affinity of isopilocarpine to approximately one-tenth to that of pilocarpine in bovine muscarinic cholinergic receptors [71]. 

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