Alkaloids Derived from Histidine

2.3 Alkaloids Derived from Histidine


The amino acid L-histidine, containing the heterocyclic imidazole ring, is considered to be the right precursor of alkaloids that essentially comprise of this ring-system.
A good number of Pilocarpus species, belonging to family Rutaceae, found to contain plethora of alkaloids with an imidazole ring, namely: pilocarpine, isopilocarpine, and pilosene. It has been observed that the alkaloids in these species invariably reside in the leaves. Pilocarpine constitutes 0.5-1.0% of the dried leaf material. Isopilocarpine appears to vary significantly within a range from 5 to 7.5% of the total alkaloids. Further, the alkaloids are located mostly in the upper epidermal leaves of the cells of the leaves, and also in the cells of the mesophyll bordering upon the lower epidermis.
The three major alkaloids derived from histidine shall be described in the sections that follows.

2.3.1 Pilocarpine

Synonyms Ocusert Pilo.
Biological Source Pilocarpine is obtained from the leaves of closely related plants of the genus Pilocarpus, belonging to the natural order Rutaceae. However, the genus comprised of a variety of species commonly known by various names, such as: Pilocarpus jaborandi (Pernambuco Jaborandi), (Pilocarpus pennatifolius (Paraguay Jaborandi); Pilocarpus microphyllus (Maranham Jaborandi); Pilocarpus selloanus (Rio Jaborandi); Pilocarpus trachylophus (Ceara Jaborandi); Pilocarpus spicatus (Aracati Jaborandi); Pilocarpus heterophyllus (Barqui Simento Jaborandi); and Pilocarpus racemosus (Guadeloupe).
It is worthwhile to mention here that P. microphyllus is the major commercial source of this drug.
Chemical Structure

(3S-cis)-3-Ethyldihydro-4-[1-methyl-1H-imidazol-5-yl) methyl]-2(3H)-furanone (C11H16N2O2).
Pilocarpine is a monoacidic tertiary base comprising of a lactone ring and an imidazole nucleus. It is the lactone of pilocarpic acid, an acid with a glyoxaline nucleus, as given below:


lactone of pilocarpic acid, an acid with a glyoxaline nucleus
Isolation The finely powdered leaves of Jaborandi is first extracted with ethanol (95% v/v) containing 1% HCl. The ethanol is distilled off under vacuo and the residue is taken up with a little water and neutralized carefully by the addition of dilute ammonia. The resins separating out are filtered off and the filtrate is concentrated to a small volume. The resulting concentrated filtrate is alkalified with an excess of ammonia and the liberated alkaloids are shaken out with at least three successive portions of chloroform. The chloroform is removed from the combined extract under vacuo.
The residue is dissolved in a minimum volume of distilled water and neutralized with dilute HNO3 (6N). The mixture of nitrates of pilocarpine and isopilocarpine crystallizes out upon cooling; which may be further separated by fractional crystallization from ethanol.
Characteristic Features
1. It is found as oil or crystals having mp 34°C.
2. It boils at bp5 260°C with partial conversion to its isomer isopilocarpine.
3. Its specific rotation is [ α]D18  +106o (C = 2) and dissociation constant pK1 (20°C) 7.15; and pK2 (20°C) 12.57.
4. It is soluble in water, alcohol, chloroform, sparingly soluble in ether and benzene; and practically insoluble in petroleum ether.
5. It exhibits an absorption maximum at 263 nm.
6. It behaves as a monoacidic base.
7. It usually gives distinct precipitates with a number of reagents, such as: Wagner’s Reagent, Mayer’s Reagent, Hager’s Reagent, silicotungstic acid, phosphomolybdic acid, gold and platinic halides.
Note: Some of these precipitates do help in the identification of pilocarpine.
8. Cessation of Lactone-Ring: The lactone ring is opened-up (undergoes cessation) by treatment with strong alkalies like NaOH, KOH, which ultimately form salts with the formation of pilocarpic acid as given below:

Cessation of Lactone-Ring: salts with the formation of pilocarpic acid
Note: The cessation of the lactone-ring absolutely destroys the physiological activity of pilocarpine; and the lactone-ring is not affected by either ammonia or alkali carbonates.
9. KMnO4-Oxidation: KMnO4 oxidation destroys the imidazole ring in pilocarpine and yields ammonia, methyl amine, pilopic acid, homopilopic acids plus other products.

Kmno4-oxidation yields ammonia, methyl amine, pilopic acid, homopilopic acids
Isomerism Pilocarpine and isopilocarpine are stereoisomers, that essentially exhibit the stereochemical difference in the lactone moiety of the molecule as shown below:

Isomerism Pilocarpine and isopilocarpine are stereoisomers
However, the above observation is based on the experimental evidence, which specifically depicts that the isomerism of the above two alkaloids still persists, even when the imidazole moiety undergoes destruction under mild experimental conditions.
Identification Tests
1. Helch’s Violet-Colour Test: Pilocarpine readily forms a violet coloured compound when a solution of either the base or its salt is first treated with hydrogen peroxide (H2O2) and then with potassium dichromate (K2Cr2O7) in the presence of few drops of dilute sulphuric acid (Helch, 1902).
Note:
(i) The violet-coloured compound (i.e., pilocarpine perchromate) is soluble in chloroform and benzene. It was further characterized as pilocarpine perchromate by Biedebach (1933).
(ii) Shupe successfully employed the Helch’s reaction to determine pilocarpine quantitatively by the colourimetric assay.
2. Ekkert’s Colour Tests: Add to 1 ml of 1% (w/v) solution of pilocarpine hydrochloride (C11H16N2O2.HCl) 1 ml of sodium nitroprusside solution (2% w/v) and 1 ml of NaOH solution (1N). Allow the reaction mixture to stand for 6-8 minutes and then acidify with dilute HCl when a wine or red colour develops.
(Note: Isopilocarpine hydrochloride also gives a similar colour test.)
Further, when a few drops of 0.1 N sodium thiosulphate solution are added to the wine or red colour solution, it changes to distinct green colouration.
Note: Elvidge (1947) put forward a method for the assay of the total alkaloids of Pilocarpus leaves based on the Ekkert’s colour test.
Uses
1. Pilocarpine possesses miotic and diaphoretic actions.
2. Pilocarpine nitrate is used extensively as an ophthalmic drug having cholinergic action.
3. It is also employed to reduce the intra-ocular pressure in glaucoma patients.

2.3.2 Isopilocarpine

Synonym β-Pilocarpine.
Biological Source It is same as stated under Section 2.3.1 on pilocarpine.
Chemical Structure

Isopilocarpine
Isolation It has been discussed under Section 2.3.1 above
Characteristic Features
1. It is a hygroscopic oily liquid or prisms.
2. It has the following physical parameters: bp10 261°C; [α]18D +50 C° (C = 2); pK1 (18°C) 7.17.
3. It is miscible with water and alcohol; very soluble in chloroform; less soluble in ether and benzene; and almost insoluble in petroleum-ether.
Identification Tests Its derivatives have the specific physical parameters, namely:
1. Isopilocarpine hydrochloride hemihydrate (C11H16N2O2 . HCl.½H2O): It is obtained as scales from ethanol having mp 127°C; when anhydrous mp 161°C; [α]D18 +39o (C = 5). It is soluble 0.27 part water and 2.1 parts ethanol.
2. Isopilocarpine nitrate (C11H16N2O.HNO3): It occurs as prisms from water, scales from ethanol, having mp 159°C; [α]D18  +39o C (C = 2). It is soluble in 8.4 parts of water and in 350 parts of absolute ethanol.
Uses
1. It is used as an antiglaucoma agent
2. It is also employed as miotic.

2.3.3 Isopilosine

Synonyms Carpiline; Carpidine; Pilosine (this compound was originally called pilosine i.e., the cis-isomer of isopilosine.
Biological Source It is obtained from the dried leaflets of Pilocarpus microphyllus (Rutaceae), which has the total alkaloidal content (0.5-1.%) that consists principally pilocarpine along with small portion of isopilosine, pilosine and related structures.
Chemical Structure

Isopilosine
[3S-[3α (S*), 4β]]-Dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-5-yl) methyl]-2 (3H)-furanone. (C16H18N2O3).
Isolation It is isolated from the leaves of P. microphyllus Stapf. (Rutaceae) by adopting standard procedures.*
Characteristic Features
1. It is obtained as needles from ethanol mp 182-182.5°C.
2. Its specific rotation [α ]D20  + 839o ethanol; uvmax (ethanol) : 210 nm (log € 4.10).
Biosynthesis of Imidazole Alkaloids L-Histidine, an amino acid contains an imidazole ring; and is, therefore, the most probable precursor of alkaloids containing this ring system. The imidazole alkaloids usually found in Jaborandi leaves (P. microphyllus and P. jaborandi; Rutaceae) are most likely derived from histidine; however, sufficient experimental data are lacking. Interestingly, the additional carbon atoms may originate from acetate or supposedly from threonine in the case of pilocarpine, whereas pilosine incorporates a phenylpropane C6H3 unit as shown below.

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