Alkaloids Derived from Tryptophan

2.8 Alkaloids Derived from Tryptophan

L-Tryptophan is a neutral heterocyclic amino acid containing essentially an indole ring system. It has been observed that it serves as a precursor for a wide spectrum of indole alkaloids. However, there exists an ample concrete evidence that major rearrangement reaction may convert the predominant indole-ring system into a quinoline-ring system thereby enhancing further the overall ability of tryptophan to act broadly as an alkaloid precursor.

The various alkaloids derived from tryptophan are conveniently classified into the following categories, namely: (i) Simple Indole Alkaloids; (ii) Simple b-Carboline Alkaloids; (iii) Terpenoid Indole Alkaloids; (iv) Quinoline Alkaloids; (v) Pyrroloindole Alkaloids; (vi) Ergot Alkaloids.

These aforesaid categories of alkaloids shall be discussed separately with typical important examples followed by the possible biosynthetic pathways, wherever necessary.

2.8.1 Simple Indole Alkaloids

L-Tryptophan (i.e., a-aminoindole-3-propanoic acid) on decarboxylation yields tryptamine. The Nmethyl and N, N-dimethyl derivatives of the latter are broadly distributed in the plant kingdom as serotonin—a simple hydroxylated derivative. Sequential biotransformation viz., decarboxylation,

N-methylation and hydroxylation gives rise to the formation of psilocin; whereas, phosphorylation of the OH group in psilocin yields psilocybin.

The three alkaloids, namely: serotonin, psilocin and psilocybin shall be discussed in the sections that follow:

A. Serotonin

Synonyms 5-Hydroxytryptamine; 5-HT; Enteramine; Thrombocytin; Thrombotonin;

Biological Sources The root bark of Gossypium hirsutum L. (Malvaceae) (American Unplanted

Cotton) contains serotonin.

Chemical Structure

3-(2-Aminoethyl)-1H-indol-5-ol; (C₁₀H₁₂N₂O).

Identification Tests

1. Serotonin Hydrochloride (C₁₀H₁₂N₂O.HCl) It is obtained as hygroscopic crystals, sensitive to light having mp 167-168°C. It is water soluble and the aqueous solutions are found to be stable at pH 2-6.4.

2. Serotonin complex with Creatinine Sulphate Monohydrate (C₁₄H₂₁ N₅O₆S.H₂O)

(Antemovis) It is obtained as plates which decomposes at 215°C. Its uvmax (water at pH 3.5): is 275 nm (ε 15,000). It has two dissociation constants pK₁' = 4.9 and pK₂' = 9.8. The pH of a 0.01 molar aqueous solution is 3.6. It is found to be soluble in glacial acetic acid; very sparingly soluble in methanol and ethanol (95%); and insoluble in absolute ethanol, acetone, pyridine, ethyl acetate, chloroform, benzene and ether.

Uses

1. It is a potent vasoconstrictor.

2. It is also a neurotransmitter in the CNS and is important in sleep-walking-cycles.

B. Psilocin

Synonyms Psilocyn.

Biological Sources It is obtained from the sacred mushroom of Mexico known as Teonanacatl. It is also found in the fruiting bodies of Psilocybe maxicana Heim, (Agaricaceae).

Chemical Structure

3-[2-(Dimethylamino) ethyl]-1H-indol-4-ol; (C₁₂H₁₆N₂O);

Isolation It has been successfully isolated in trace amounts from the fruiting bodies of Psilocybe mexicana*.

Characteristic Features

1. It is obtained as plates from methanol having mp 173–176°C.

2. It is an amphoteric substance.

3. It is unstable in solution, more precisely in an alkaline solution.

4. It is very slightly soluble in water.

5. Its uv_max: 222, 260, 267, 283, 293 nm (log ε 4.6, 3.7, 3.8, 3.7, 3.6).

Uses It is a hallucinogenic substance

Note It is a controlled substance listed in the U.S. Code of Federal Regulations, Title 21 Part 1308, 11 (1995).

C. Psilocybin

Synonym Indocybin;

Biological Sources These are same as mentioned in psilocin ‘B’ above.

Chemical Structure

3-[2-(Dimethylamino) ethyl]-1H-indol-4-ol dihydrogen phosphate ester; (C₁₂H₁₇N₂O₄P).

Isolation The method of isolation of psilocybin is the same as stated under psilocin.

Characteristic Features

1. Psilocybin is obtained as crystals from boiling water having mp 220-228°C; and from boiling methanol mp 185-195°C.

2. It has uv_max (methanol): 220, 267, 290 nm (log ε 4.6, 3.8, 3.6).

3. The pH of a saturated solution in 50% aqueous ethanol is 5.2.

4. Solubility Profile: It is soluble in 20 parts of boiling water, 120-parts of boiling methanol; sparingly soluble in ethanol; and practically insoluble in chloroform, benzene.

Uses It is a hallucinogenic substance and exerts its action at a dose level of 6-20 mg.

Biosynthesis of Serotonin, Psilocin and Psilocybin The different steps involved in the biosynthesis of serotonin, psilocin and psilocybin may be summarized as stated below:

1. L-Tryptophan upon oxidation gives rise to the corresponding hydroxylated derivative known as 5-hydroxyl-L-tryptophan, which further undergoes decarboxylation to yield serotonin also termed as 5-hydroxytryptamine (or 5-HT).

2. L-Tryptophan undergoes decarboxylation to yield the tryptamine, which affords N-Methylation and N,N-dimethylation in the presence of S-adenosylmethionine (SAM). The resulting dimethyl derivative upon oxidation gives rise to the product psilocin another hydroxylated derivative.

3. Phosphorylation of the hydroxyl function in psilocin affords psilocybin.

4. Interestingly, both psilocin and psilocybin are solely responsible for attributing the hallucinogenic properties of the so-called ‘magic mushrooms’, that include species of Psilocybe, Panaeolus and the like.

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* Hofmann et al., Experientia, 14, 107 (1958); Heim et al., Helv. Chim. Acta, 42, 1557 (1959).

2.8.2 Simple β-Carboline Alkaloids

The alkaloids based on the β-carboline ring system obviously suggest the formation of a new sixmembered heterocyclic ring employing the ethylamine side-chain present in tryptamine exactly in the same manner to the evolution of tetrahydroisoquinoline alkaloids (see Section 2.7.2). The exact mechanism whereby the above rearrangement is accomplished may be explained by virtue of the fact that C-2 of the indole nucleus is nucleophilic due to the adjacent nitrogen atom. Therefore, C-2 can conveniently participate in a Mannich/Pictet-Spengler type reaction, thereby enabling it to attack a Schiff base produced from tryptamine and either an aldehyde (or keto acid) as given below:

It has been observed that relatively simpler structures make use of keto-acids, such as: harman, harmaline, harmine and elaeagnine. These alkaloids shall be treated individually in the sections that follow.

Interestingly, the comparatively complex carbolines, for instance: the terpenoid indole alkaloids e.g., ajmaline are usually generated by the help of a pathway that specifically utilize an aldehyde, such as: secologanin. This particular section shall be dealt with separately under Section 7.2.8.3.

A. Harman

Synonyms Aribine; Loturine; Passiflorin; 2-Methyl-b-carboline; 3-Methyl-4-carboline;

Biological Sources It is obtained from the bark fruit of Passiflora incarnata L. (Passifloraceae) (May pop, Passion flower); seed of Peganum harmala L. (Rutaceae) (Harmel, Syrian Rue, African Rue), bark of Sickingia rubra (Mart.) K. Schum. (Arariba rubra Mart.), (Rubiaceae); and bark of Symplocus racemosa Roxb. (Symplocaceae).

Chemical Structure

1-Methyl-9H-pyrido [3, 4, b] indole; (C₁₂ H₁₀ N₂).

Isolation Poindexter and Carpenter* isolated this alkaloid from the cigarette smoke.

Characteristic Features

1. It is obtained as orthorhombic crystals from heptane and cyclohexane having mp 237-238°C.

2. It has a bitter taste.

3. It exhibits distinct bright blue fluorescence in uv light.

4. It pKa’s are 7.37 and 144.6.

5. It has uvmax (methanol): 234, 287, 347 nm (log ε 4.57, 4.21, 3.66).

6. It is practically insoluble in water and freely soluble in dilute acids.

Identification Test Harman Hydrochloride (C12H₁₀N₂.HCl) It is obtained as rosettes of needles from a mixture of ethanol + 20% HCl in water which sublimes at 120-130°C.

Uses It is a narcotic hallucinogen.

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* Poindexter and Corpenter, Chem. & Ind. (London), 1962, 176.

B. Harmaline

Synonyms Harmidine; Harmalol Methyl Ether; O-Methyl-harmalol; 3, 4-Dihydroharmine;

Biological Sources It is obtained from the seeds of Peganum harmala L. (Zugophyllaceae); and Banisteria cappi Spruce (Malpighiaceae). It is also obtained from the fruit of Passiflora incarnata L. (Passifloraceae) (Passionflower, Maypop).

Chemical Structure

4, 9-Dihydro-7-methoxy-1-methyl-3H-pyridol [3, 4,-β] indole; (C₁₃H₁₄N₂O).

Characteristic Features

1. It is obtained as orthorhombic bipyramidal prisms, or tablets from methanol; and as rhombic octahedra from ethanol having the same mp 229-231°C.

2. Its solutions give a blue fluorescence.

3. Its dissociation constant pK_a 4.2.

4. It has uv_max (methanol): 218, 260, 376 nm (log ε 4.27, 3.90 and 4.02)

5. It is found to be slightly soluble in water, ethanol, ether; and very soluble in dilute acids and hot ethanol.

Identification Tests Harmaline forms definite derivatives as shown below:

1. Harmaline Hydrochloride Dihydrate (C₁₃H₁₄N₂O.HCl.2H₂O): It is obtained as slender, yellow needles that are found to be moderately soluble in ethanol and water.

2. N-Acetylharmaline: It is obtained as needles having mp 204-205°C.

Uses

1. It is recognized as a narcotic hallucinogen.

2. It is used as a CNS-stimulant.

C. Harmine

Synonyms Telepathine; Leucoharmine; Yageine; Banisterine;

Biological Sources It is obtained from the seeds of Peganum harmala L. (Zygophyllaceae); Banisteria caapi Spruce. (Malpighiaceae); and Banisteriopsis inebrians Morton. (Malpighiaceae). It is also obtained from the fruit of Passiflora incarnata L. (Passifloraceae).

Chemical Structure

7-Methoxy-1-methyl-9H-pyrido [3, 4-β] indole; (C₁₃H₁₂N₂O).

Isolation Harmin may be isolated from the seeds of Peganum harmala L. (Zygophyllaceae) by the method suggested by Reinhard et al.

Characteristic Features

1. It is obtained as slender, orthorhombic prisms from methanol having mp 261°C (decomposition).

2. It sublimes and has pKa value of 7.70.

3. It as uvmax (methanol): 241, 301, 336 nm (log ε 4.61, 4.21, 3.69).

4. It is found to be slightly soluble in water, ethanol, ether and chloroform.

Identification Tests

Harmine Hydrochloride Dihydrate (C₁₃H₁₂N₂O.HCl.2H₂O) It is obtained as crystals having mp 262°C (decomposition), but when anhydrous mp 321°C (decomposition), but when anhydrous mp 321°C. The aqueous solution exhibits a distinct blue fluorescence. It is found to be soluble in 40 parts of water and freely soluble in hot water.

Uses It finds its usage as a CNS-stimulant and also as a narcotic hallucinogen.

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* Rainhard et al. Phytochemistry, 7, 503, (1968).

D. Elaeagnine

Biological Source It is obtained from the bark of Elaeagnus angustifolia Linn., (Synonyms: E. hortensis Bieb.) (Elaeagnaceae).

Chemical Structure

Biosynthesis of Elaeagnine, Harman, Harmaline and Harmine The various steps involved in the biosynthesis of the above mentioned four alkaloids are briefly summarized as under:

1. Tryptamine and acetyl carboxylic acid (i.e., keto acid) undergoes a Mannich-like reaction to yield a β-carboline carboxylic acid, which on oxidative decarboxylation gives rise to 1-methyl β-carboline.

2. The resulting product on subsequent reduction gives rise to the alkaloid elaeagnine.

3. The 1-methyl β-carboline upon mild oxidation yields the alkaloids harman with the elimination of a mole of water from the 6-membered heterocyclic nucleus.

4. The 1-methyl β-carboline upon hydroxylation followed by methylation produces harmaline.

5. Harmaline on further oxidation generates harmine by the loss of a mole of water from the 6-membered pyridine ring at C-3 and C-4 positions.

The these steps are sequentially arranged in the following course of reactions:

2.8.3 Terpenoid Indole Alkaloids

Terpenoid indole alkaloids is perhaps one of the major groups of alkaloids in the plant kingdom which comprise of more than 3000 recognized alkaloids till date Interestingly, they are found to be confined to eight different natural orders (i.e., families), of which the Apocynaceae, the Loganiaceae, and the Rubiaceae are predominantly the best known sources.

However, it is pertinent to mention here that practically in all the structure a tryptamine residue is strategically located in the molecule; while the remaining fragment is invariably recognized as a C9 or C10 residue.

The wisdom, relentless efforts and meticulous in-depth studies carried out by numerous groups of researchers dealing with plant substances across the globe ultimately led to three main structural variants entirely based on their good judgement and understanding namely:

(a) Coryanthe Type e.g., ajmalicine and akuammicine,

(b) Aspidosperma Type e.g., tabersonine, and

(c) Iboga Type e.g., catharanthine.

It has since been established beyond any reasonable doubt that the C9 or C10 component present in the aforesaid three types of structural variants i.e., Carynanthe, Aspidosperma and Iboga groups was definitely of the terpenoid origin. Besides, it was also confirmed that the secoridoid secologanin was duly proclaimed to be the terpenoid derivative, which perhaps must have initially combined with the tryptamine residue of the molecule. From these scientific and logical evidences one may safely infer that the three above mentioned groups of alkaloids might be not only related but also rationalized in terms of rearrangements taking place exclusively in the terpenoid portion of the various structural variants as shown in the pathway given below.

Salient Features The salient features of the above pathway are as follows:

1. Secologanin (a secoridoid and a terpenoid derivative) is formed through geraniol via loganin, which essentially contains the 10C-framework a typical characteristic feature of the Coryanthe moiety.

2. The resulting Coryanthe C-skeleton undergoes subsequent rearrangements to give rise to Aspidosperma and Iboga groups.

3. This intra-molecular rearrangement may be represented by detachment of a 3C-unit, which is subsequently reunited to the remaining C7 fragment in one of the two different manners as shown in the pathway.

4. Interestingly, where C9 terpenoid units are complied with, the alkaloids usually, seem to have lost a C-atom marked in the circle, which exactly corresponds to the carboxylate function of secologanin molecule. Therefore, its ultimate elimination by way of hydrolysis/decarboxylation is now understood without any reasonable doubt.

5. Thus, the Coryanthe type of C-skeleton yields ajmalicine and akuammicine.

6. The Aspidosperma type of C-skeleton yields tabersonine and vindoline.

7. The Iboga type of C-skeleton gives rise to catharanthine.

A few typical examples of terpenoid indole alkaloids, namely: Ajmalicine (Raubasine); Akuammicine; Vindoline; and Catharanthine shall be discussed below:

A. Ajmalicine

Synonyms Raubasine; Circolene; Hydrosarpan; Lamuran; Isoarteril;

Biological Sources It is obtained from the plants of Catharanthus lanceus Pichon (Boj.) (Apocynaceae) (Lanceleaf Periwinkle); Catharanthus roseus (L.) G. Don (Apocynaceae) (Periwinkle, Madagascar or Cape Periwinkle, Old Maid]; leaves of Mitragyna speciosa Korth. (Rubiaceae) (Katum, Kutum, Krantum); Rauvolfia scrpentina (L.) Benth. (Apocynaceae) (Rauvolfia, Chandra, Sarpaganda); and bark of Corynanthe johimbe K. Schum., (Rubiaceae).

Chemical Structure

(19α)-16, 17-Didehydro-19-methyl-oxayohimban-16-carboxylic acid methyl ester; (C₂₁H₂₄N₂O₃).

Isolation Ajmalicine may be isolated either from the bark of Corynanthe johimbe by the method suggested by Heinemann*, or from the roots of Rauwolfia serpentina by the procedure adopted by Hofmann.**

Characteristic Features

1. It is obtained as prisms from methanol which decompose at 257°C.

2. It has specific optical rotation [α]20D – 60° (C = 0.5 in chloroform); [α]²⁰_D – 45° (C = 0.5 in pyridine); and [α]²⁰_D – 39° (C = 0.25 in methanol).

3. It exhibits uvmax (methanol): 227, 292 nm (log e 4.61, 3.79).

Identification Tests

1. Ajmalicine Hydrochloride (C₂₁H₂₄N₂O₃.HCl): It is obtained as leaflets from ethanol having mp 290°C (decomposed); [α]²⁰_D – 17° (C = 0.5 in methanol); and is sparingly soluble in water or dilute HCl.

2. Ajmalicine Hydrobromide (C₂₁H₂₄N₂O₃.HBr): It is obtained as diamond-shaped plates from methanol having mp 295-296°C.

Uses

1. It is mostly used as antihypertensive and anti-ischemic agent (both ceretral and peripheral).

2. It has a broad application in the relief of obstruction of normal cerebral blood flow.

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* Heinemann, H., Ber. 67, 15 (1934).

** Hofmann, A, Helv. Chim. Acta. 37, 849, (1954).

B. Akuammicine

Biological Source It is obtained from the plant substance of Catharanthus roseus (L.) G. Don (Apocyanaceae) (Periwinkle, Madagascar or Cape Periwinkle, Old Maid); and also from the seeds of Picralima klaineana, Pierre, belonging to the natural order (Apocyanaceae).

Chemical Structure

2, 16, 19-20-Tetradehydrocuran-17-oic acid methyl ster; (C₂₀H₂₂N₂O₂).

Characteristic Features

1. It is obtained as plates from a mixture of ethanol and water having mp 182°C.

2. Its physical parameters are: [α]¹⁶_D – 745° (C = 0.994 in ethanol); pK_a 7.45; and uv_max (ethanol): 227, 330 and 330 nm (log ε 4.09, 4.07, 4.24).

Identification Tests It forms the following derivatives:

1. Akuaminicine Hydrochloride Dihydrate (C₂₀H₂₂N₂O₂.HCl.2H₂O): It is obtained as leaflets from ethanol or water having mp 171°C; and has [α]²¹_D – 610° (C = 1.430 in ethanol).

2. Akuaminicine Perchlorate Monohydrate (C₂₀H₂₂N₂O₂.HClO₄.H₂O): It is obtained as needles from a mixture of ethanol and water having mp 134-136°C.

3. Akuammicine Hydroiodide Monohydrate (C₂₀H₂₂N₂O₂.HI,H₂O): It is obtained as square plates from water having mp 128°C.

4. Akuammicine Methiodide: It is obtained as crystals from water with mp 252°C.

5. Akuammicine Nitrate: It is obtained as needles from hot water having mp 182.5°C.

Uses The drug exhibits a slight digitalis-like reaction; and is, therefore, believed to act as a heart poison.

C. Vindoline

Biological Sources It is obtained from the plant Catharanthus roseus (L.) G. Don (Apocynaceae) (Periwinkle, Madagascar or Cape Periwinkle; Old Maid). It is found to be the major alkaloid from the leaves of Vinca rosea Linn. (Apocynaceae).

Chemical Structure

(2β, 3β, 4β, 5α,12β, 19α)-4-(Acetyloxy)-6, 7-didehydro-3-hydroxy-16-methoxy-1 methylaspidospermidine-3-carboxylic acid methyl ester; (C₂₅H₃₂N₂O₆).

Isolation It is isolated from the leaves of Vinca rosea by the method suggested by Gorman et al.*

Characteristic Features

1. Vinodoline is obtained in two forms: first, as needles from a mixture of acetone and petroleum ether having mp 164-165°C; and secondly, as prisms having mp 174-175°C.

2. It has [α]²⁰_D - 18° (chloroform) and dissociation constant pKa 5.5 in 66% DMF.

3. It has uv_max (ethanol): 212, 250, 304 nm (log ε 4.49, 3.74, 3.57).

Identification Tests It gives specific derivatives as.

1. Vindoline Hydrochloride (C₂₅H₃₂N₂O₆.HCl): It is obtained as crystals from acetone having mp 161-164°C.

2. Demethoxy Vindoline (C₂₄H₃₀N₂O₅) (Vindorosine, Vindolidine): It is obtained as needles from benzene and petroleum ether having mp 167°C. It has [α]¹⁶_D -31° (Chloroform); and uv_max (methanol): 250, 302 nm (log ε 3.98, 3.52).

D. Catharanthine

Biological Sources It is obtained in the plant of Catharanthus lanceus Pichon (Boj.) (Apocynaceae) (Lanceleaf Periwinkle); and Catharanthus roseus (L.) G. Don (Apocyanaceae) (Periwinkle, Madagascar or Cape Periwinkle, Old Maid). It is also found in Vinca rosea Linn. (Apocynaceae).

Chemical Structure

3, 4-Didehydroibogamine-18-carboxylic acid methyl ester; (C₂₁H₂₄N₂O₂).

Isolation It may be isolated from Vinca rosea Linn by the method recommended by Gorman et al.**

Characteristic Feature

1. Its crystals obtained from methanol has mp 126-128°C.

2. It has uvmax (ethanol): 226, 284, 292 nm (log ε 4.56, 3.92, 3.88).

3. It has specific optical rotation [α]²⁷_D + 29.8° (CHCl₃); and dissociation constant pKa′ 6.8.

Uses

1. Its pharmacological action resembles to that of R. serpentina.

2. It also shows beneficial growth inhibition effects in certain human tumors.

3. It is used as a diuretic.

Biosynthesis of Ajmalicine, Vindoline and Catharanthine The various steps involved in the biosynthesis of ajmalicine, vindoline and catharanthine are summarized below:

1. Condensation of secologanin with tryptamine in a Mannich-type reaction gives rise to the tetrahydro-b-carboline system and generates strictosidine.

2. The structural variations involved in converting the Coryanthe type skeleton into the corresponding Aspidosperma and Iboga types are evidently quite complex and are given in the pathway as under.

3. Preakuammicine is obtained from strictosidine via the enol-form of dehydrogeissoschizine.

4. Preakuammicine undergoes intramolecular rearrangement to produce stemmadenine, which subsequently gives rise to a hypothetical intermediate.

5. The hypothetical intermediate may be redrawn which undergoes Diel's-Alder type reaction to produce catharanthine.

6. Dehydrogeissoschizine yields ajmalicine.

7. The hypothetical intermediate gives rise to vindoline via tabersonine.

It is pertinent to mention here that the sequence of alkaloid formation has been proved initially by noting carefully which alkaloids become labelled as a feeding experiment progresses, but more recently it has been confirmed by suitable enzymatic experimental studies.

It is important to mention here that there exists a plethora of structural variants of terpenoid indole alkaloids which may be exemplified with the help of the following specific examples of certain potent alkaloids, namely:

(i) Yohimbine: It is a carboxyclic variant related to ajmalicine and appears to arise from dehydrogeissoschizine by an elaborated mechanism.

(ii) Reserpine: It is a trimethoxybenzoyl ester of yohimbine-like alkaloid. It has an additional-OCH₃ moiety at C.-11 of the indole nucleus.

(iii) Rescinnamine: It is a trimethoxycinnamoyl ester of yohimbine-like alkaloid. It also contains an additional methoxyl substituent on the indole-system at C-11.

(iv) Vinblastine: The nucleophilie vindoline, C-5 of the indole nucleous is being activated adequately by the OMe at C-6, besides the N-atom of the indole moiety. The resulting adduct is subsequently reduced in the dihydropyridinium ring by the NADH-dependent 1, 4-addition, giving the substrate for hydroxylation. Its ultimate reduction gives rise to vinblastine.

(v) Vincristine: It is the oxidized product of vinblastine whereby the inherent N-formyl group on the indoline fragment is transformed.

(vi) Strychnine: The loss of one C from a preakuammicine-like structure via hydrolysis/decarboxylation followed by an addition of the additional two C-atoms by means of aldolcondensation with the formyl moiety, complexed as a hemiacetal in the well-known Wieland-Gumlich aldehyde. The ultimate formation of strychnine from its hemiacetal is by virtue of the formation of both ether and amide linkages.

The above mentioned six structural variants of the terpenoid indole alkaloids shall now be discussed individually in the sections that follow.

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* Gorman et al. J. Am. Pharm. Assoc. 48, 256, (1959).

** M. Gorman et al., J. Arn. Pharm. Assoc. Sci. Ed. 48, 256 (1959).

A. Yohimbine

Synonyms Quebrachine; Corynine; Aphrodine;

Biological Sources It is found in the root bark of Alchornea floribunda Muell. Arg. (Euphorbiaceae) (Niando); plant* of Catharanthus lanceus Pichon (Boj.) (Apocyraceae) (Lanceleaf Periwinkle); bark of Pausinystalia johimbe (K. Schum.) (Rubiaceae) (Yohimbe); root of Rauvolfia serpentina (L.) Benth. (Apocynaceae) (Rauvolfia, Chandra, Sarpaganda); and plant of Rauvolfia tetraphylla L. (Apocynaceae) (Pinque-Pinque).

Chemical Structure

(16a, 17a)-17-Hydroxyyohimban-16-carboxylic acid methyl ester; (C21H26N2O3).

Characteristic Features

1. It is obtained as orthorhombic needles from dilute alcohol having mp 234°C.

2. Its specific optical rotations are: [α]²⁰_D + 50.9° to + 62.2° (ethanol); [α]²⁰_D + 108° (pyridine); and [α]²⁰₅₄₆ + 129° (C = 0.5 in pyridine).

3. It has uvmax (methanol): 226, 280, 291 nm (log ε 4.56, 3.88, 3.80).

4. It is freely soluble in ethanol, chloroform, hot benzene; moderately soluble in ether; and sparingly soluble in water.

Identification Tests

Yohimbine Hydrochloride (C₂₁H₂₆N₂O₃.HCl) (Aphrodyne, Yocon, Yohimex, Yohydrol): It is obtained as orthorhombic plates or prisms from ethanol which decompose at 302°C. Its specific optical rotation [α]²²_D + 105° (water). It is found to be soluble in nearly 120 ml water, 400 ml ethanol, and the aqueous solution is almost neutral.

Uses

1. It is an aderenergic blocking agent, which has been used extensively in angina pectoris and arteriosclerosis.

2. It has been used successfully for the treatment of impotency in patients with vascular or diabetic problems.

3. It is invariably employed as a pharmaiological probe for the study of α2-adrenoreceptor.

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* Emboden reported that this plant contains upto 5% yohimbin.

B. Reserpine

Synonyms Crystoserpine; Eskaserp; Rau-sed; Reserpoid; Rivasin; Serfin; Sandril; Sedaraupin; Serpasil; Serpine; Serpasol; Serpiloid.

Biological Sources It is obtained from the plant Catharanthus roseus (L.) G. Don (Apocynaceae) (Periwinkle, Madagascar or Cape Periwinkle, Old Maid); root of Rauvolfia serpentina (L.) Benth (Apocynaceae) (Rauvolfia, Chandra, Sarpaganda); root of Rauvolfia tetraphylla L. (Apocynaceae) (Pinque-Pinque); and from the plant of Vinca minor L. (Apocynaceae) (Periwinkle).

Chemical Structure

(3β, 16β, 17α, 18β, 20α)-11, 17-Dimethoxy-18-[(3, 4, 5-trimethoxy benzoyl)oxy] yohimban-16-carboxylic acid methyl ester; (C₃₃H₄₀N₂O₉);

Isolation Reserpine may be isolated by adopting the following steps in a sequential manner:

1. The powdered and sieved roots are allowed to swell in a NaHCO3 solution (10% w/v) for a period of 10-12 hours. The resulting solution is extracted with benzene, until the extracts give a weak positive reaction with HgI₂.

2. The combined benzene extracts are concentrated and ether is added to the benzene solution. The resulting mixture is extracted with dilute HCl. The combined acidic solution is washed with ether, filtered and extracted with chloroform in a successive manner.

Note: The chloroform will specifically extract the weakly basic alkaloids, such as: Reserpine and Rescinnamine.

3. The combined chloroformic extract is washed subsequently with 10% (w/v) sodium carbonate solution and followed by water so as to get rid of any free acids present. The resulting extract is finally evaporated to dryness under vacuo.

4. The residue is dissolved in anhydrous methanol and seeded with a pure crystal of reserpine and allowed to cool gradually when reserpine will crystallize out.

5. However, rescinnamine, deserpidine and other minor weakly basic alkaloids could be obtained from the mother liquor conveniently.

6. The mother liquor is evaporated to dryness, and the residue is dissolved in the minimum quantity of benzene and subjected to column chromatography over a column packed with acid-washed alumina. The alkaloids are eluted in the different fractions by making use of benzene, chloroform, methanol (10%) in a sequential manner.

Characteristic Features

1. It is obtained as long prisms from dilute acetone which get decomposed at 264-265°C; (decomposes at 277-277.5°C in an evac-tube).

2. Its specific optical rotations are: [α]²³_D - 118° (CHCl3); [α]²⁶_D - 164° (C = 0.96 in pyridine; [α]²⁶_D - 168° (C = 0.624 in DMF).

3. It has uvmax (CHCl3): 216, 267, 295 nm (61700, 17000, 10200).

4. Reserpine is weakly basic in nature, pKa 6.6.

5. It is found to be freely soluble in chloroform (~ 1g/6 ml), glacial acetic acid, methylene chloride; soluble in benzene, ethyl acetate; slightly soluble in acetone, methanol, ethanol (1g/1800 ml), ether, in aqueous solutions of citric and acetic acids; and very sparingly soluble in water.

Identification Tests

1. Most solutions of reserpine upon standing acquire a distanct yellow colouration and a marked and pronounced fluorescence; especially after the addition of an acid or upon exposure to light.

2. Reserpine Hydrochloride Hydrate (C₃₃H₄₀N₂O₉.HCl.H₂O): It is obtained as crystals which decompose at 224°C.

Uses

1. It is a hypotensive drug which exhibits strong hypotensive and sedative activity.

2. It is also employed to alleviate mild anxiety conditions i.e., the drug shows a mild tranquillizing effect.

C. Rescinnamine

Synonyms Reserpinine; Anaprel; Apoterin S; Cartric; Cinnaloid; Moderil;

Biological Sources It is obtained from the roots of Rauvolfia serpentina (L.) Benth. (Apocynaceae)

(Rauvolfia, Chandra, Sarpaganda).

Chemical Structure

3, 4 5-Trimethoxy-cinnamic acid ester of methyl reserpate; (C₃₅H₄₂N₂O₉).

Isolation Rescinnamine may be isolated from step (5) onwards as described under Morphine.

Characteristic Features

1. It is obtained as fine needles from benzene having mp 238-239°C (under vacuum).

2. Its specific optical rotation is [α]²⁴_D - 97° (C = 1 in chloroform).

3. It has uvmax (methanol): 228, 302 nm (log ε 4.79, 4.48).

4. Solubility Profile: It is moderately soluble in methanol, benzene, chloroform and other organic solvents; and practically insoluble in water.

Uses It is mostly used as an antihypertensive.

D. Vinblastine

Synonyms Vincaleukoblastine; VLB; 29060-LE;

Biological Source It is obtained from Vinca rosea Lin.. (Apocynaceae).

Chemical Structure

Isolation It may be isolated from Vinca rosea Linn., either by the method suggested by Noble et al*. or by Gorman et al.,**

Characteristic Features

1. It is obtained as solvated needles from methanol having mp 211-216°C.

2. Its specific optical rotation [α]²⁶_D + 42° (chloroform).

3. It has uvmax (ethanol): 214, 259 nm (log ε4.73, 4.21).

4. It is soluble in alcohols, chloroform, acetone, ethyl acetate and is practically insoluble in water and petroleum ether.

Identification Tests It forms derivatives as given below:

1. Vinblastine Sulphate (C₄₆H₅₈N₄O₉.H₂SO₄) (Exal, Vebe, Velban): It is obtained as crystals mp 284-285°C. Its physical parameters are: [α]²⁶_D – 28° (C = 1.01 in methanol); pKa1 5.4; pK_a 27.4. It has uv_max (methanol): 212, 262, 284, 292 nm (log € 4.75, 4.28, 4.22, 4.18). One part is soluble in 10 parts of water, 50 parts of chloroform; very slightly soluble in ethanol; and practically insoluble in ether.

2. Vinblastine Dihydrochloride Dihydrate (C₄₆H₅₈N₄O₉.2HCl.2H₂O): It is obtained as crystals that decompose at 244-246°C.

Uses

1. The alkaloid is used for the treatment of a wide variety of neoplasms.

2. It is also recommended for generated Hodgkin’s disease, lymphocytic lymphoma, hystiocytic hymphoma, mycosis fungoides, advanced testicular carcinoma, Kaposi's sarcoma, and choriocarcinoma and lastly the breast cancer unresponsive to other therapies.

3. It is effective as a single entity, however, it is normally given along with other neoplastic agents in combination therapy for the increased therapeutic effect without any noticeable additive toxicity.

4. It arrests mitosis at the metaphase.

5. It is found to be effective in the acute leukemia of children.

-------------------------------------------------

* Noble et al. Ann. N.Y. Acad. Soc. 76, Art 3, 892-894 (1958)

** Gorman et al. J. Am. Chem. Soc., 81, 4745, 4754, (1959).

E. Vincristine

Synonyms Leurocristine; VCR; LCR.

Biological Sources It is also obtained from Vinca rosea Lin., (Catharanthus roseus G. Don) belonging to the natural order Apocynaceae.

Chemical Structure Please see the chemical structure under Vinblastine. It may also be named as: 22-Oxovincaleukoblastine.

Isolation Vincristine may be isolated from Vinca rosea Linn., by the method suggested by Svoboda.*

Characteristic Features

1. It is obtained as blades from methanol having mp 218-220°C.

2. Its specific optical rotation [α]²⁵_D + 17°; [α]²⁵_D + 26.2° (ethylene chloride); pKa: 5.0, 7.4 in 33% DMF.

3. It has uv_max (ethanol): 220, 255, 296 nm (log am 4.65, 4.21, 4.18).

Identification Tests

Vincristine Sulphate (C46H56N4O10.H2SO4) (Vincrex, Oncovin, Vincosid, Kyocrystine): Its crystals are obtained from ethanol and is found to be unstable.

Uses

1. Vincristine sulphate is recommended for the treatment of acute lymphocytic leukemia, and in combination therapy in Hodgkin's disease, lymphosarcoma, reticulum cell sarcoma, neuroblastoma, Wilm's tumour and rhabdomyosarcoma.

Note: Viucristine sulphate being highly unstable; therefore, its refregerated storage in sealed ampules is absolutely essential.

2. It is broadly used as an antineoplastic agent.

---------------------------------

* Svobada, Lyoydia, 24, 173 (1961)

F. Strychnine

Biological Sources It is abundantly found in the seeds of Strychnos Nux Vomica L. (Loganiaceae) (Nux Vomica, Strychnine); beans of Strychnos ignatti Berg. (Loganiaceae); roots of S. cinnamomifolia Thw.; seeds, bark and wood of S. colubrina Linn.; and plant of S. malaccensis Benth. (Syn: S. gautheriana Pierre).

Chemical Structure

Strychnidine-10-one; (C₂₁H₂₂N₂O₂)

Salient Features

1. Strychnine contains two N-atoms even then it happens to be a mono-acidic base.

2. Strychnine readily forms a variety of salts, such as: nitrate, N6-oxide, phosphate and sulphate. Interestingly, the N-atom which is specifically involved in the salt formation is the one that is located farthest from the aromatic benzene ring.

3. The second N-atom is strategically positioned as an amide nitrogen; and, therefore, it does not exhibit any basic characteristics.

Isolation Strychnine may be isolated from the seeds of S. nux vomica by adopting the following steps sequentially:

1. The seeds of nux vomica are dried, ground and sieved which are mixed with an adequate quantum of pure slaked lime and made into a paste by adding a requisite amount of water. The wet mass thus obtained is dried at 100°C and extracted with hot chloroform in a continuous extractor till the extraction is completed.

2. The alkaloids are subsequently removed from the chloroform solution by shaking with successive portions of dilute sulphuric acid (2N). The combined acid extracts are filtered to get rid of any foreign particles or residue.

3. To the resulting acidic filtrate added an excess of ammonia to precipitate the alkaloids (strychnine + brucine).

4. The precipitate is extracted with ethanol (25% v/v) several times which exclusively solubilizes brucine, and ultimately leaves strychnine as an insoluble residue.

5. The residue containing strychnine is filtered off and is finally purified by repeated recrystallization from ethanol.

Characteristic Features

1. It is obtained as brilliant, colourless cubes from a mixture of chloroform and ether having mp 275-285°C, and d¹⁸ 1.359.

2. Its specific optical rotation [α]¹⁸_D-104.3° (C = 0.254 in ethanol); [α]²⁵_D-13° (C = 0.4 in chloroform).

3. Its dissociation constant pK_a (25°) 8.26.

4. It has uvmax (95% ethanol); 2550, 2800, 2900 Å (E1% 1cm 377, 130, 101).

5. Solubility Profile: 1g dissolves in 182 ml ethanol, 6.5 ml chloroform, 150 ml benzene, 250 ml methanol, 83 ml pyridine; and very slightly soluble in water and ether.

6. A solution of strychnine containing 1 part in 700,000 parts of water gives a distinct bitter taste.

Identification Tests Strychnine may be identified either by specific colour tests or by specific derivatives:

(a) Colour Tests

1. Sulphuric Acid-Dichromate Test: Strychnine (5-10 mg) when dissolved in a few drops of concentrated sulphuric acid and stirred with a crystal of pure potassium dichromate [K₂Cr₂O₇] it gives an instant reddish-violet to purple colouration.

Note: Strychnine derivatives will also give this test except strychnine nitrate.

2. Mandelin’s Reagent Test: Strychnine or its corresponding salt when treated with Mandelin’s Reagent* it gives rise to a violet to blue colouration.

3. Ammonium Vanadate (V) Test: Strychnine or its salt when treated with a saturated solution of ammonium vanadate, it produces a violet to blue colouration.

4. Nitric Acid Test: Strychnine on being treated with a trace of HNO3 (conc.) yields an instant yellow colouration.

Note: A similar test with Brucine gives an intense orange-red colouration. It may be used to differentiate between strychnine and brucine.

(b) Strychnine Derivatives: The various important strychnine derivatives are as given under:

1. Strychnine Nitrate (C₂₁H₂₃N₃O₅): It is obtained as colourless, odourless needles or while crystalline powder 1g dissolves in 42 ml water, 10 ml boiling water, 150 ml ethanol, 80 ml ethanol at 60°C, 105 ml chloroform, 50 ml glycerol; and insoluble in ether. It shows a pH ~ 5.7.

2. Strychnine N⁶– Oxide (C₂₁H₂₂N₂O₃): It is obtained as monoclinic prisms from water which decompose at 207°C. It has pK value 5.17. It is found to be freely soluble in ethanol, glacial acetic acid, chloroform; fairly soluble in water; sparingly soluble in benzene; and practically insoluble in ether and petroleum ether.

3. Strychnine Phosphate (C₂₁H₂₅N₂O₆P): It is usually obtained as its dihydrate salt (2O₆P.2H₂O) which is colourless or while crystals or white powder. 1g dissolves in slowly in ~ 30 ml water, more soluble in hot water, and slightly soluble in ethanol. The aqueous solution is acidic to litmus.

4. Strychnine Sulphate (C₄₂H₄₆N₄O₈S): It normally crystallizes as pentahydrate [2C₂₁H₂₂N₂O₂.H₂SO₄.5H₂O]. It is colourless, odourless, very bitter crystals or white crystalline powder. It effloresces in dry air and loses all its water of crystallization at 100°C. It shows mp when anhydrous ~ 200°C with decomposition. 1g dissolves in 35 ml water, 7 ml boiling water, 81 ml ethanol, 26 ml ethanol at 60°C, 220 ml chloroform, 6 ml glycerol, and insoluble in ether. A 1 : 100 solution shows pH 5.5.

5. Strychnine Gluconate Pentahydrate (C₂₇H₃₄N₂O₉.5H₂O): Its crystals darken above 80°C. It is soluble in 2 parts water ~ 40 parts ethanol. The aqueous solution is found to be neutral.

6. Strychnine Glycerophosphate Hexahydrate (C₄₅H₅₃N₄O₁₀P.6H₂O): 1g dissolves in ~ 350 ml water, ~ 310 ml ethanol; slightly soluble in chloroform; and very slightly soluble in ether.

7. Strychnine Hydrochloride Dihydrate (C₂₁H₂₃ClN₂O₂.2H₂O): It is obtained as trimetric prisms which are efflorescent in nature. 1g dissolves in ~ 40 ml water, ~ 80 ml ethanol, and insoluble in ether. The pH of a 0.01 M solution is 5.4.

Uses

1. Strychinine is extremely interesting pharmacologically and is regarded as a valuable tool in both physiologic and neuroanatomic research.

2. It is extremely toxic, and functioning as a central stimulant.

3. It causes excitation of all parts of the central nervous system and blocks inhibitory spinal inpulses at the post synaptic level. This may lead to an exaggeration in reflexes ultimately leading to tonic convulsions.

4. The drug is rarely used in modern medical practice but is utilized as a vermin killer i.e., animal or insect killer.

5. It is used chiefly in poison baits for rodents.

Biosynthesis of Yohimbine, Reserpine, Rescinnamine, Vinblastine, Vincristine and Strychnine Dehydogeissoschizine (keto-form) undergoes isomerization by means of the nucleophilic attack on to carbonyl through a conjugated system, which subsequently forms an onium ion that upon reduction produces yohimbine as shown below:

Reserpine and deserpidine are essentially the trimethoxybenzoyl esters of yohimbine-type alkaloids; whereas, rescinnamine is a trimethoxycinnamoyl ester. Interestingly, both reserpine and rescinnamine contain an additional methoxyl moiety present strategically on the indole ring system at C-11, which is accomplished by virtue of hydroxylation and methylation at a late stage along the pathway. A predominant and characteristic feature of these alkaloids is that they exhibit the opposite stereochemistry at C-3 to yohimbine and strictosidine as depicted below:

The biosynthetic pathway leading to vinblastine and vincristine is supposedly involve the following vital steps:

1. An oxidative reaction on catharanthine, catalysed by an enzyme peroxidase, thereby producing a peroxide that aptly loses the peroxide as a leaving group, ultimately breaking a carbon-carbon covalent bond as shown in the diagram given below.

2. The intermediate electrophilic ion is attacked on to the conjugated iminium system by the vindoline, whereby C-5 of the indole nucleus being appropriately activated by the –OCH₃ moiety located at C-6, and also by the N-atom present in the indole ring.

3. The resulting adduct is subsequently reduced in the dihydropyridinium ring by NADH*-dependent 1, 4-addition thereby giving rise to the substrate for hydroxylation.

4. Ultimately, reduction of the above resulting product generates vinblastine.

5. The oxidized product from vinblastine, with its N-formyl moiety rather than N-methyl on the vindoline fragment, may finally yield vincristine.

The biosynthetic pathway leading to strychnine essentially comprise of the following steps, namely:

1. Preakuammicine loses one C-atom via hydrolysis followed by decarboxylation.

2. Addition of the two extra C-atoms is accomplished by means of Aldol-condensation reaction with acetyl-CoA, whereby it yields the Wieland-Gumlich aldehyde as a complexed hemiacetal form.

3. The subsequent construction of ether and amide linkages gives rise to the formation of stryctinine from the above hemiacetal as shown below.

--------------------------------------------------

* NADH = Nicotinamide adenine dinucleotide (reduced form).

2.8.4 Quinoline Alkaloids

A good number of very prominent and remarkable examples of the ‘quinoline-alkaloids’ derived from tryphphan are nothing but the modifications of the terpenoid indole alkaloids commonly found in the genus Cinchona belonging to the natural order Rubiaceae.

Interestingly, more than twenty alkaloids have been isolated and characterized from the bark of Cinchona calisaya and Cinchona ledgeriana, very commonly known across the globe as the Yellow Cinchona; besides the other equally well-known species Cinchona succirubra, popularly known in trade as the Red Cinchona. However, the four long prized and most popular quinoline alkaloids known for their antimalarial activities are namely: quinine, cinchonine, quinidine, and cinchonidine. These alkaloids shall now be described individually in the sections that follow. It is worthwhile to state here that these structures are not only unique but also remarkable wherein the indole nucleus is replaced by a quinoline system through an intramolecular rearrangement as given below:

A. Quinine

Biological Sources The cinchona species (Rubiaceae) specifically contains quinine in the bark upto 16% (mostly 6-10%) in a variety of its species, namely: Cinchona calisaya Wedd.; C. ledgeriana Moens ex Trimen; C. officinalis Linn. f.; C. robusta How.; and C. succirubra Pavon ex Klotzsch. The representative samples of dried cinchona, cinchona bark or peruvian bark is found to contain nearly 0.4 to 4% quinine.

Chemical Structure

(8a, 9R)-6′-Methoxycinchonan-9 ol; (C20H24N2O2).

Isolation of Quinine, Cinchonine, Cinchonidine and Quinidine The isolation of all the four important quinoline alkaloid, such as: quine, cinchonine; cinchonidine and quinidine may be accomplished by adopting the following steps carefully and sequentially.

Step 1: The cinchona bark is dried, powdered, sieved and treated with calcium oxide (slaked lime), NaOH solution (10% w/v) and water and kept as such for 6-8 hours.

Step II: The resulting mixture is treated with benzene in sufficient quantity and refluxed for 12-16 hours. The mixture is then filtered while it is hot.

Step III: The hot filtrate is extracted successively with 6N. sulphuric acid. The mixture of alkaloidal bisulphate is heated upto 90°C and maintained at this temperature upto 20-30 minutes.

Step IV: The resulting solution is cooled to room temperature and made alkaline by the addition of solid pure sodium carbonate till a pH 6.5 is attained.

Step V: The alkaloidal sulphate solution thus obtained is treated with sufficient quantity of activated charcoal powder (1g per 1L), boil, shake vigorously and filter.

Step VI: Cool the hot filtrate slowly in a refrigerator (2-10°C) overnight and again filter. Collect the residue and the filtrate separately.

Step VII: The residue (or precipitate) of quinine sulphate is boiled with water and made alkaline by adding cautiously solid sodium carbonate. The resulting precipitate is that of quinine.

Step VIII: The filtrate obtained from step-VI comprises of cinchonine, cinchonidine and quinidine; which is treated with NaOH solution (10% w/v) very carefully to render it just alkaline. It is successively extracted with adequate quantity of ether. The lower (aqueous layer) and the upper(ethereal layer) are collected separately.

Step IX: The aqueous layer contains cinchonine. It is evaporated to dryness in a Rotary Film Evaporator, extracted with absolute ethanol, decolourized with activated charcoal powder and allow it to crystallize slowly in a refrigerator (2-10°C) overnight. The crystals of cinchonine are obtained.

Step X: The ethereal layer obtained in step-VIII contains quinidine and cinchonidine. It is extracted with dilute HCl (2N) several times till a drop of the extract on evaporation does not give a positive test for alkaloids. Neutralize the combined acidic extract by adding solid sodium potassium tartrate carefully. Filter the resulting mixture and collect the precipitate and the filtrate separately.

Step XI: The precipitate of cinchonidine tartrate is treated with dilute HCl carefully. The resulting solution of alkaloid hydrochloride is made alkaline by the addition of dilute ammonium hydroxide when cinchonidine is obtained as a precipitate.

Step XII: The filtrate obtained from Step-X contains quinidine tartrate which is treated with solid potassium iodide powder carefully till the whole of quinidine gets precipitated as quinidine hydroiodide salt. It is filtered and the solid residue is finally treated with dilute NH₄OH to obtain the precipitate of quinidine.

Characteristic Features

1. It is obtained as triboluminescent, orthorhombic needles from absolute ethanol having mp 177° (with some decomposition).

2. It sublimes in high vacuum at 170-180°C.

3. Its specific optical rotations are: [α]¹⁵_D - 169° (C = 2 in 97% ethanol); [α]¹⁷_D - 117° (C = 1.5 in chloroform); [α]¹⁵_D - 285° (C = 0.4 M in 0.1 N H₂SO₄).

4. Its dissociation constants are: pK₁ (18°) 5.07; and pK₂ 9.7.

5. The pH of its saturated solution in 8.8.

6. It gives a distinct and characteristic blue fluorescence which is especially strong in dilute sulphuric acid.

7. Solubility Profile: 1 g dissolves in 1900 ml water; 760 ml boiling water; 0.8 ml ethanol; 80 ml benzene; 18 ml benzene at 50°; 1.2 ml chloroform; 250 ml by ether; 20 ml glycerol; 1900 ml of 10% ammonia water; and almost insoluble in petroleum ether.

Identification Tests Quinine may be identified either by a series of Colour Tests or by the formation of several known derivatives having characteristic features; and these shall be discussed separately as under:

(a) Colour Tests: These are, namely

1. Oxygenated Acids: Oxygenated acids, such as: sulphuric acid or acetic acid gives a strong blue fluorescence with quinine. This test is very sensitive even in extremely dilute solutions.

------------------------------------------------

* Herpathile The iodo sulphate of quinine (or sulphate of iodo-quinine) is nown as Herpathitie after the name of its

discover [Formula: B₄ . 3H₂SO₄ . 2HI . I4 + 3H₂O]

Note: Halogen quinine compounds and hydrochloride salts of quinine do not give

fluorescence in solution.

2. Herpathite Test: To a boiling mixture of quinine (0.3g) in 7.5 ml glacial acetic acid, 3 ml ethanol (90% v/v) and 5 drops of concentrated H2SO4, add 3.5 ml of I2 solution (1% w/v) in ethanol, crystals of iodosulphate of quinine or Herpathite* separates out on cooling. The crystals thus obtained exhibit metallic lustre, appears dark in reflected light and alive-green in transmitted light.

3. Thalleioquin Test: When a few drops of bromine water are added to 2 or 3 ml of a weakly acidic solution of quinine salt, followed by the addition of 0.5-1.0 ml of strong ammonia solution, it produces a distinct characteristic emerald green colouration. It is an extremely sensitive colour test which may detect quinine even upto a strength as low as 0.005% (w/v). The end coloured product is known as thalleioquin for which the exact chemical composition is not yet known.

Note: (a) This test is given by quinidine and also by other Remijia alkaloids e.g., cupreine.

(b) Both cinchonine and cinchonidine do not respond to the Thalleioquin Test.

4. Erythroquinine Test (or Rosequin Test): Dissolve a few mg of quinine in dilute acetic acid, add to it a few drops of bromine water (freshly prepared), followed by a drop of a 10% (w/v) solution of potassium ferrocyanide [K₄Fe(CN)₆]. Now, the addition of a drop of concentrated NH₄OH solution gives rise to a red colouration instantly. If shaken quickly with 1-2 ml of chloroform, the red colouration is taken up by the lower chloroform-layer.

(b) Derivatives/Salts of Quinine: These are as follows:

1. Quinine Trihydrate: It is obtained as a microcrystalline powder having mp 57°C. It effloresces and loses one mol of water in air, two moles of water over H2SO4, and becomes anhydrous at 125°C.

2. Quinine Bisulphate Heptahydrate (C₂₀H₂₄N₂O₂.H₂SO₄.7H₂O) [Synonyms: Quinbisan, Dentojel, Biquinate): It is obtained as very bitter crystals or crystalline powder. It effloresces on exposure to air and darkens on exposure to light. 1 g dissolves in 9 ml water, 0.7 ml boiling water, 23 ml ethanol, 0.7 ml ethanol at 60°C, 625 ml chloroform, 2500 ml ether, 15 ml glycerol and having a pH 3.5.

3. Quinine Dihydrochloride (C₂₀H₂₄N₂O­.2HCl) (Synonyms: Quinine dichloride; Acid quinine hydrochloride; Quinine bimuriate): It is obtained as a powder or crystals having a very bitter taste. 1g dissolves in about 0.6 ml water, 12 ml ethanol; slightly soluble in chloroform; and very slightly soluble in ether. The aqueous solutions are found to be strongly acidic to litmus paper (pH about 2.6).

4. Quinine Hydrochloride Dihydrate (C₂₀H₂₄N₂O₂.HCl.2H₂O): It is obtained as silky needles having a bitter taste. It effloresces on exposure to warm air. It does not lose all its water below 120°C. 1 g dissolves in 16 ml water, in 0.5 ml boiling water, 1.0 ml ethanol, 7.0 ml glycerol, 1 ml chloroform, and in 350 ml ether. A 1% (w/v) aqueous solution shows a pH 6.0-7.0.

5. Quinine Sulphate Dihydrate [(C₂₀H₂₄N₂O₂)₂.H₂SO₄.2H₂O] (Synonyms: Quinamm; Quinsan; Quine, Quinate): It is obtained as dull needles or rods, making a light and readily compressible mass. It loses its water of crystallization at about 110 °C. It becomes brownish on exposure to light. Optical rotation [α]¹⁵_D - 220° (5% solution in about 0.5 N . HCl). 1g dissolves in 810 ml water, 32 ml boiling water, 120 ml ethanol, 10 ml ethanol at 78°C; slightly soluble in ether and chloroform, but freely soluble in a mixture of 2 vols. chloroform and 1 vol. absolute ethanol. Its aqueous solutions are neutral to litmus. The pH of a saturated solution in 6.2.

Uses

1. It is frequently employed as a flavour in carbonated beverages.

2. It is used as an antimalarial agent.

3. It is also employed as a skeletal muscle relaxant.

4. It has been used to treat hemorrhoids and varicose veins.

5. Quinine is also used as a oxytocic agent.

6. Quinine is supposed to be prophylactic for flu.

Biosynthesis of Quinine A survey of literature reveals that the intrinsic details of the biosynthetic pathways are lacking; however, an assumed biogenetic process essentially involving the followingsteps:

1. L-Tryptophan and secologanin yields strictosidine, which upon hydrolysis and decarboxylation produces coryantheal.

2. Coryantheal undergoes intramolecular changes, first-by cleavage of C-N bond (via iminium), and secondly-by formation of an altogether new C-N bond (again via iminium). This gives rise to an intermediate.

3. The resulting intermediate undergoes further intramolecular changes to yield cinchoninone having a quinoline nucleus.

4. Cinchoninone in the presence of NADPH* reduces the carbony function and generates quinine:

-------------------------------------

* NADPH = Nicotinamide adenine dinucleotide phosphate (reduced form).

B. Cinchonine

Biological Sources It occurs in most varieties of cinchona bark as mentioned under quinine (section ‘A’). Besides, cinchonine especially occurs in the bark of Cinchona micrantha R & P. belonging to the natural order Rubiaceae.

Chemical Structure

(9S) - Cinchonan-9-ol; (C₁₉H₂₂N₂O)

Isolation The detailed method of isolation has been given under quinine (section ‘A’). Besides, Rabe* has put forward another method of isolation of cinchonine.

Characteristic Features

1. Cinchonine is obtained as needles from ethanol or ether having mp 265°C.

2. It begins to sublime at 220°C.

3. Its specific optical rotation is [α]_D + 229° (in ethanol).

4. Solubility Profile: 1g dissolves in 60 ml ethanol, 25 ml boiling ethanol, 110 ml chloroform, 500 ml ether; and practically insoluble in water.

5. It has two distinct dissociation constants: pK₁ 5.85 and pK₂ 9.92.

Identification Tests Cinchonine may be identified by forming its specific derivatives, namely:

1. Cinchonine Hydrochloride Dihydrate (C₁₉H₂₂N₂O.HCl.2H₂O): It is obtained as fine crystals. The mp of its anhydrous salt is 215 °C with decomposition. 1g dissolves in 20 ml water, 3.5 ml boiling water 1.5 ml alcohol, 20 ml chloroform; and slightly soluble in ether. The aqueous solution is almost neutral.

2. Cinchonine Dihydrochloride (C₉H₂₂N₂O.2HCl): It is usually obtained as white or faintly yellow crystals or crystalline powder. It is found to be freely soluble in water or ethanol.

3. Cinchonine Sulphate Dihydrate [(C₁₉H₂₂N₂O)2.H₂SO₄.2H₂O]: It is commonly obtained as lustrous extremely bitter crystals. Its anhydrous salt has mp 198°C. 1g dissolves in 65 ml water, 30 ml hot water, 12.5 ml ethanol, 7 ml hot ethanol, 47 ml chloroform; and slightly soluble in ether. The aqueous solution is practically neutral.

4. Epicinchonine [Synonyms (9R)-Cinchonan-9-ol]: It has mp 83°C; and [α]²²_D + 120.3° (C =0.806 in ethanol).

Uses

1.      It is used as an antimalarial agent.

2. It is employed as a tonic in waters, bitters and liqueurs.

3. It is broadly used for febrifuge, schizonticide, stomachic, amebiasis, dysentry, flu, fever, and as

a mild stimulant of gastric mucosa.

-----------------------------

* Rabe, Ber. 41, 63 (1908)

C. Quinidine

Synonyms Conquinine; Pitayine; b-Quinine;

Biological Source Quinidine is obtained from the various species of Cinchona as described under quinine (section ‘A’). It is reported to be present in cinchona barks ranging between 0.25-3.0%.

Chemical Structure It is the dextrorotatory stereoisonter of quinine

(9S)-6′-Methoxycinchonan-9-ol; (C₂₀H₂₄N₂O₂).

Isolation Quinidine may be isolated from the cinchona bark by the method stated under quinine (section ‘A’).

Characteristic Features

1. Quinidine is obtained as triboluminescent crystals having mp 174-175°C after drying of the solvated crystals.

2. Its specific optical rotations are: [α]¹⁵_D + 230° (C = 1.8 in chloroform); [α]¹⁷_D + 258° (ethanol); and [α]¹⁷_D + 322° (C = 1.6 in 2m HCl).

3. It has two dissociation constants, namely: pK₁ (20°) 5.4; and pK₂ 10.0.

4. It gives a distinct and characteristic blue fluorescence in dilute sulphuric acid (2N).

5. The uv absorption spectrum is identical with that of quinine.

6. Solubility Profile: 1 g gets dissolved in 2000 ml cold water, 800 ml boiling water, 36 ml ethanol, 56 ml ether, 1.6 ml chloroform; very soluble in methanol; and practically insoluble in petroleum ether.

Identification Tests The various derivatives of quinidine have specific characteristic features as enumerated below:

1. Quinidine Sulphate Dihydrate [(C₂₀H₂₄N₂O₂)₂.H₂SO₄.2H₂O] (Synonyms Quinidex; Quinicardine; Quinora; Extentabs; Cin-Quin): It is mostly obtained as white, very bitter, odorless, fine crystals which is frequently cohering in masses. It does not lose all of its water of crystallization below 120°C. It has been found to darken on exposure to light. It has [α]²⁵_D ~ + 212° (in 95% ethanol); and ~ + 260° (in dilute HCl). The pH of a 1% (w/v) solution between 6.0-6.8. Its pKa values are : 4.2 and 8.8. 1 g dissolves in 90 ml water, 15 ml boiling water, 10 ml ethanol, 3 ml methanol, 12 ml chloroform; and insoluble in ether and benzene.

Note: Quinidine sulphate dihydrate is the salt of an alkaloid obtained either from various species of Cinchona and their hybrids, or from Cuprea bark, obtained from Remijia pedunculata and Remijia purdieana belonging to the natural order Rubiaceae.

2. Quinidine Gluconate (C₂₆H₃₆N₂O₉) (Synonyms Quinaglute; Duraquin; Gluconic acid quinidine salt): It is obtained as crystals having mp 175-176.5°C; and soluble in 9 parts of water and 60 parts of ethanol.

3. Quinidine Polygalacturonate (C₂₀H₂₄N₂O₂.C₆H₁₀O₇.H₂O) [Synonyms Galactoquin; Cardioquin; Naticardina): It is obtained as an amorphous powder mp 180°C (decomposes). The anhydrous substance is found to be insoluble in methanol, ethanol, chloroform, ether, acetone, dioxane; and soluble in 40% methanol or ethanol: 12%; in water at 25°C: ~ 2%.

4. Quindine Hemipentahydrate: It is obtained as prisms from dilute ethanol, mp ~ 168°C, and loses 1/2 H₂O on exposure to air.

5. Quinidine Hydrogen Sulphate Tetrahydrate (C₂₀H₂₄N₂O₂.H₂SO₄.4H₂O) (Synonyms Kiditard; Kinichron; Kinidin Durules; Quiniduran; Chinidin - Duriles; Quinidine Bisulphate): It is obtained as rods which is soluble in 8 parts of water and emitting a distinct blue fluorescence.

6. Neutral Hydroiodide of Quinidine (C₂₀H₂₄N₂O₂.HI): It is obtained as a crystalline powder when KI is added to a neutral aqueous solution of a quinidine salt. It is very sparingly soluble in water (1 part in 1250 parts at 15°C). It is found to be much less soluble than that of the other cinchona alkaloids.

Quinidine also gives a specific colour test as given below:

Ferrocyanide Test for Quinidine A small quantum (10-15 mg) of a quinidine salt is mixed thoroughly with 0.5-1.0 ml of freshly prepared bromine water in an evaporating dish. The contents are transferred carefully into a test tube with the help of 1 ml of distilled water. To this is added 1 ml of chloroform, contents shaken and then allowed to stay for a few minutes. A few drops of a 10% (w/v) solution of potassium ferrocyanide [K₄ Fe(CN)₆] and 3 ml of a 5N. NaOH solution are added with continuous shaking. The chloroform layer attains a red colour.

Note: Quinine or its salt under identical treatment gives a negative test, and hence it may be used to distinguish between quinidine and quinine.

Uses

1. It is used as an antiarrhythmic agent (Class 1A)*.

2. It finds its applications as an antimalarial drug.

3. It is most commonly employed to treat various cardiac arrhythmias, namely: atrial flutter, AV junctional and ventricular contractions, atrial and ventricular tachycardia, atrial fibrillation, and premature atrial condition.

-----------------------------------------------

* Class 1A Antiarrhythmic Agent When the antiarrhythmic mechanisms is accomplished through membrane

stabilization.

D. Cinchonidine

Synonyms Cinchovatine; α-Quinidine;

Biological Sources It is obtained in most varieties of the cinchona bark as described under quinine (section ‘A’). It is, however, observed to be present especially in the bark of Cinchona pubescens Vahl. (C. succirubra Pav.) and Cinchona pitayensis Wedd., (Rubiaceae).

Chemical Structure

(8a, 9R)-Cinchonan-9-ol; (C₁₉H₂₂N₂O).

Isolation Cinchonidine can be conveniently isolated from the bark of various species of cinchona as described explicitely under quinine (section ‘A’). However, it may also be isolated by the method suggested by Leers.*

Characteristic Features

1. It is obtained as orthorhombic prisms or plates from ethanol having mp 210°C.

2. It has specific optical rotation [α]²⁰_D - 109.2° (in ethanol).

3. Solubility Profile: It is found to be freely soluble in chloroform and ethanol; moderately soluble in ether; and practically insoluble in water.

4. It has two dissociation constants: pK₁ 5.80 and pK₂ 10.03.

Identification Tests Cinchonidine may be identified by preparing its specific derivatives that possess characteristic features, such as:

1. Cinchonidine Dihydrochloride (C₁₉H₂₂H₂O.2HCl): It is obtained as white or slightly yellow crystals or powder. It is freely soluble in ethanol and water.

2. Cinchonidine Hydrochloride Dihydrate (C₁₉H₂₂N₂O.HCl.2H₂O): It is obtained as a crystalline powder. It losses all of its water of crystallization at 120°C. It has [α]²⁰_D - 117.5° (in water). It is soluble in 25 parts of cold water, more soluble in boiling water; soluble in chloroform and ethanol; and slightly soluble in ether. The aqueous solution is almost neutral in nature.

3. Cinchonidine Sulphate Trihydrate [(C₁₉H₂₂N₂O)₂.H₂SO₄.3H₂O]: It is obtained as silky, acicular crystals which effloresce on being exposed to air and get darkened in light. The mp of anhydrous salt is nearly 240°C with decomposition. 1g dissolves in 70 ml water, 20 ml hot water, 90 ml ethanol, 40 ml hot ethanol, 620 ml chloroform; practically insoluble in ether. The aqueous solution is more or less neutral.

4. Epicinchonidine [Synonyms: (8α, 9S)-Cinchonan-9-ol)]: It has mp 104°C; and [α]²⁰_D + 63°(C = 0.804 in ethanol).

Uses It is mostly used as an antimalarial agent.

Totaquine Totaquine is nothing but a mixture of the total alkaloids of the well-known cinchona bark. It is invariably exploited as a ‘cheap substitute’ for quinine in an unethecal practice in trade. It is found to contain not less than 7% and not more than 12% of quinine units anhydrous form; and not more than 80% of the total anhydrous crystallizable cinchona alkaloids.

The following table summarizes the characteristic features and specific tests for the four major cinchona alkaloids, namely: Quinine, Quinidine, Cinchonine and Cinchonidine.

Differences Among Four Major Cinchona Alkaloids

Biosynthesis of Cinchonine, Quinidine and Cinchonidine The various sequential steps involved in the biosynthesis of Cinchonine, Quinidine and Cinchonidine are stated as under:

1. Strictosidine is obtained by the interaction of L-tryptophan and secologanin as already shown in the Biosynthesis of Quinine.

2. Strietosidine undergoes a molecular rearrangement to form an aldehyde which upon hydrolysis and decarboxylation yields coryantheal.

3. Coryantheal generates cinchoninone by virtue of two transformations; first: an intermediate formed due to the cleavage of C-N bond (via iminium) then formation of a new C-N bond (again via iminium); and secondly: cleavage of the indole C-N bond. The resulting product loses a molecule of water to yield cinchonionone.

4. Cinchoninone undergoes epimerization at C-8 via enol to form the stereoisomer, which upon interaction with NADPH gives rise to chnchonine and quindine respectively.

5. Cinchonione with direct interaction with NADPH gives rise to cinchonidine and quinine respectively.

The outline of the biosynthesis elaborated above from (1) through (5) may be summarized as depicted below:

---------------------------

** Leers, Ann., 82, 147 (1952)

2.8.5 Pyrroloindole Alkaloids

The indole nucleus has two C-atoms in the heterocyclic portion, viz., C-2 and C-3. Interestingly, both C-2 and C-3 may be regarded as nucleophilic in character. However, it has been established beyond any reasonable doubt that the reactions essentially involving C-2 appear to be the most common in alkaloid biosynthesis.

It is, however, pertinent to mention here that the nucleophic character of C-3 has been duly exploited thereby generating the almost rare pyrroloindole nucleus as given below:

Physostigmine is a typical example of this specific category of alkaloid which shall now be discussed in details as under:

Physostigmine

Synonyms Eserine; Cogmine;

Biological Sources It is obtained from the seeds of Physostigma venenosum Balf. (Fabaceae) (Calabar Bean, Ordeal Bean) yielding not less than 0.15% of the total alkaloids of physostigma.

Chemical Structure

(3aS-cis)-1, 2, 3, 3α, 8, 8α-Hexahydro-1, 3α, 8-trimethylpyrrolol [2, 3-b] indol-5-ol methylcarbamate (ester); (C₁₅H₂₁N₃O₂).

Isolation Physostigmine may be isolated by adopting the following two steps, namely:

Step I: The seeds are dried, powdered, sieved and extracted by continuous percolation with hot ethanol (95%) and the solvent is subsequently removed by distillation under vacuo. Water is added to the residue and the floating fatty layer is separated The lower aqueous layer is subjected to alkalinization with sodium carbonate and the liberated alkaloid is then extracted with ether successively.

Step II: The combined ethereal extract is then concentrated to a small volume and washed with 5% (w/v) sulphuric acid repeatedly unless and until the washings give a positive acidic reaction to litmus paper. To this aqueous acidic solution (containing the alkaloids as sulphates) is added an excess of a saturated solution of sodium salicylate when the physostigmine salicylate separates out as a crystalline product. The physostigmine may be recovered from the resulting salt by treating it with sodium carbonate followed by an immediate extraction with ether successively. The ether is evaporated in a Rotary Thin-Film Evaporator and the desired physostigmine is collected as prisms or clusters.

-----------------------------------------------------

* Schwyzer, Die Fabrikation Pharmazeutischer and Chemisch-Technischer Produkte (Berlin, 1931) p 338.

However, physostigmine may also be isolated by the methods described by Schwyzer* and Cheminitius.*

Characteristic Features

1. It is obtained as orthorhombic sphenoidal prisms or clusters of leaflets from ether or benzene having mp 105-106°C. It is also available as an unstable, low melting form mp 86-87°C.

2. Its specific optical rotations are: [α]¹⁷_D - 76° (C = 1.3 in chloroform); and [α]²⁵_D - 120° (benzene).

3. It has two dissociation constants: pKa₁ 6.12, and pKa₂ 12.24.

4. Solubility Profile: It is slightly soluble in water; soluble in ethanol, benzene, chloroform and oils.

Identification Tests Physostigmine may be identified either by specific colour tests or by preparing their derivatives as stated below:

(a) Colour Tests: These are as follows:

1. Physostigmine or its salts, a few mg, when warmed with 1 ml of strong ammonia solution it gives rise to a yellowish-red colouration. On further evaporation to dryness on a steam-bath, a bluish residue (eserine blue) is obtained that is soluble in ethanol forming a blue solution.

2. Both solid and solutions of physostigmine eventually turn red on being exposed to heat, light and air; and also on contact with traces of metals. This colour change indicates hydrolysis to eseroline and oxidation to rubreserine.

3. Physostigmine gives an instant blue colouration when treated with potassium ferricyanide [K₃Fe(CN)₆] and a few drops of FeCl₃ solution (1% w/v).

4. Physostigmine produces a deep-yellow colouration on being heated with 0.5-1 ml of KOH solution (1% w/v).

Note: (a) This is a very sensitive test and can detect it upto 10 mcg level.

(b) Under controlled experimental parameters the intensity of the yellow colour produced may be measured spectrophotometrically at 470 nm and can serve as an assay method.

5. When a small quantity of physostigmine is heated in a porcelain basin on a steam both with a drop or two of fuming HNO₃, a yellow solution is obtained. The resulting solution on evaporation to dryness forms a green residue due to the formation of chloreserine, which is readily soluble in ethanol to give a green solution.

6. Physostigmine when treated with a solution of phosphomolybdic acid and ammonium meta vanadate in H₂SO₄ it gives rise to an emerald green colour.

(b) Derivatives: Major derivatives of physostigmine are:

1. Physostigmine Salicylate (C₂₂H₂₇N₃O₅) (Antilirium): It is obtained as acicular crystals having mp 185-187°C. It has uvmax (methanol: 239, 252, 303 nm (log ε 4.09, 4.04, 3.78). 1g dissolves in 75 ml water at 25°C. The pH 0.5% (w/v) aqueous solution is 5.8. It is soluble in 16 ml ethanol; 5 ml of boiling ethanol; 6 ml of chloroform; and 250 ml of ether.

2. Physostigmine Sulphate [(C₁₅H₂₁N₃O₂)₂.H₂SO₄]: It is mostly obtained as deliquescent scales having mp 140°C (after drying at 100°C). 1g dissolves in 0.4 ml ethanol, 4 ml water, 1200 ml

ether. The pH of 0.05 M aqueous solution is 4.7. The solutions of the sulphate salt are more

prone to change colour than those of the corresponding salt of the salicylate.

3. Physostigmine Sulphite [(C₁₅H₂₁N₃O₂)₂.H₂SO₃]: The white powder is found to be freely soluble in ethanol and water. The aqueous solution is observed to remain colourless for a long duration.

Uses

1. It possesses a cholinergic (anticholinesterase) and miotic activities.

2. It was used earlier to treat myasthenia gravis; but now it is more frequently used for the eye.

3. It is employed as an antidote for reversing CNS and cardiovascular (viz., arrythmia and tachycardia) effects of excessive dosages with tricyclic antidepressants.

4. It helps in the contraction of the ciliary muscle of eye, and a decrease in the intraocular pressure produced by an increased out-flow of the aqueous humor.

5. Physostigmine is employed frequently in ophthalmology to treat glaucoma.

Biosynthesis of Physostigmine The various steps involved in the biosynthesis of physostigmine are as follows.

1. Tryptamine undergoes C-methylation at C-3 of the indole nucleus due to its nucleophilic character.

2. Formation of the ‘third pyrrole’ ring takes place by virtue of the nucleophilic attack of the primary amine function on to the iminium ion.

3. Further substitution on the phenyl ring leads to the formation of physostigmine.

The above three steps are summarized as given below:

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* Chemnitius, J. Prabt. Chem. 116, 59 (1927).

2.8.6 Ergot Alkaloids

Ergot is a fungal disease very commonly and widely observed on a good number of wild as well as cultivated grasses, and is produced by different species of claviceps. This particular disease is usually characterized by the formation of hard and seedlike ‘ergots’ in place of the normal seeds. However, these specific structures are frequently termed as sclerotia, which represent the ‘resting stage’ of the fungus.

The generic name, ‘claviceps’, usually refers to the club-like nature of the sclerotium*, whereas purpurea signifies its purple colour. As these sclerotia are elongated and somewhat pointed in shape and appearance, hence the common name of spurred rye has been assigned to the drug.

Medicinal ergot is the dried sclerotium of the fungus Claviceps purpurea (Fries) belonging to the natural order Clavicipitaceae developed in the ovary of rye, Secale cereale (Germinae/Poaceae).

There are certain other species of Claviceps which have been found to produce ergots in the ovaries of other member of Graminae and Cyperaceae.

In fact, there exist four main categories of ergot alkaloids which may be distinguished, namely:

(a) clavine alkaloids, (b) lysergic acids, (c) lysergic acid amides, and (d) ergot peptide alkaloids.

There are, in fact, ten ergot peptide alkaloids which are: ergotamine, ergosine, ergocristine, ergocryptine, ergocornine, ergotaminine, ergosinine, ergocristinine, ergocryptinine, and ergocorninine; however, the last five alkaloids being isomers of the first five. The aforesaid alkaloids are beautifully typified by a structure comprising of four components, viz., lysergic acid, dimethylpyruvic acid, proline and phenylalanine strategically joined together in amide linkages as depicted below:

Interestingly, the poisonous properties of ergots in grain, specifically rye, for animal as well as human consumption, purposefully and unknowingly, have long been recognized. The dreadful causative agents are collectively termed as the ‘ergot alkaloids’, containing essentially an indole nucleus. These, group of alkaloids are also referred to as ‘ergolines’.

The three important and typical members of the ergot alkaloids (ergolines), namely: ergonovine, ergotamine and lysergamde (ergine) shall be discussed individually in the sections that follows:

------------------------------------------------

* Sclerotium A hardened mass formed by the growth of certain fungi. THe sclerotium formed by ergot on rye is of

medical importance due to its toxicity.

A. Ergonovine

Synonyms Ergometrine; Ergobasine; Ergotocine; Ergostetrine; Ergotrate; Ergoklinine; Syntometrine.

Biological Sources It is obtained from the seeds of Ipomea violaceae Linn. (Ipomea tricolor Cav.) belonging to family Convolvulaceae (Morning glory, Tlitliltzen, Ololiuqui); and also from the dried seeds of Rivea corymposa Hall. F. (Convolvulaceae) (Snakeplant).

The percentage of Ergometrine and Ergine present in the Rivea and Ipomea species are as given below:

Chemical Structure

[8β (s)]-9, 10-Diadehydro-N-(2-hydroxy-1-methylethyl)-6-methylergoline-8-carboxamide; (C₁₉H₂₃N₃O₂).

Isolation The following steps may be followed stepwise:

1. The seeds are dried, powdered, sieved and finally defatted with n-hexane in a Soxhlet apparatus.

2. The defatted mare is extracted with hot dilute sulphuric acid (6N) successively. The acid extract is then treated with on excess of barium sulphate, and the barium is removed with CO₂ and subsequent filtration.

3. The resulting filtrate is then concentrated by evaporation under reduced pressure.

4. The concentrated solution is taken up in ethanol, made alkaline with NH4OH and subjected to extraction with chloroform successively.

5. The resulting chloroform extract is further extracted with dilute H₂SO₄ (6N). The acidic solution is made alkaline with ammonia and saturated with NaCl and then extracted with ether several times.

6. The solvent is removed from the ether extract under vacuo leaving the alkaloidal residue.

7. Ergonovine may be recrystallized from acetone.

It may also be prepared from D-lysergic acid and L (+)-2-amino-1-propanol by the method of Stoll and Hofmann.*

Characteristic Features

1. Ergonovine is obtained as tetrahedral crystals from ethyl acetate, and as fine needles from benzene. It tends to form solvated crystals having mp 162°C.

2. It has specific optical rotation [α]²⁰_D + 90° (in water).

3. Its dissociation constant is pKa 6.8.

4. It is found to be freely soluble in lower alcohols, acetone and ethyl acetate; more soluble in water than the other principal alkaloids of ergot; and slightly soluble in chloroform.

Identification Tests As per se the ergot alkaloids may be identified either by general precipitation and colour reactions or by preparing their derivatives as stated below:

(a) Precipitation Reactions

(i) The ergot alkaloids are readily precipitated by the alkaloidal reagents. However, Mayers reagent is regarded to be the most sensitive test whereby on opalescence in dilutions of 1 ppm can be obtained.

(ii) Iodine solution in KI also gives an instant precipitate with very dilute solutions of ergot alkaloids.

(b) Colour Tests: The most vital colour tests are given as under:

(i) Keller's Test: To a solution of the alkaloid in glacial acetic acid add a few mg of solid FeCl₃ and then add 1-2 ml of concentrated sulphuric acid along the side of the tube. The appearance of an intense blue colouration is accomplished at the junction of the two layers.

(ii) Van Urk Test: When a solution containing an ergot alkaloid is mixed with Van Urk Reagent**, it gives rise to a characteristic deep blue colouration.

Note: (a) Van Urk Reagent may also be used in spraying developed paper chromatograms of the ergot alkaloids, and for this purpose 10% (v/v) HCl is used instead of H₂SO₄.

(b) The spectrophotometric assay for total ergot alkaloids is also based on the blue colour given with Van Urk Reagent.

(iii) Glyoxylic Acid Reagent Test: Ergot alkaloids gives a blue colouration with the addition of Glyoxylic acid reagent and a few drops of concentrated H₂SO₄.

(iv) Fluorescence Test: The aqueous solution of the salts of ergot alkaloids produce a distinct blue fluorescence.

(c) Derivatives of Ergonovine: The various derivatives of ergonovine are as follows:

(i) Ergonovine Maleate (Ergometrine Maleate) (C₁₉H₂₃N₂O₂.C₄H₄O₄) [Synonyms Cornocentin; Ermetrine; Ergotrate Maleate]: It is obtained as crystals that decompose at 167°C. It has specific optical rotation [α]²⁵_D + 48° to + 57°. 1g dissolves in 36 ml water and 120 ml ethanol. It is almost insoluble in chloroform and ether.

(ii) Methylergonovine Maleate (C₂₀H₂₅N₂O₂.C₄H₄O₄): It is a semisynthetic homologne of ergonovine and prepared from lysergic acid and 2-aminobutanol. It is obtained as a white to pinkish-tan microcrystalline powder.

(iii) Ergonovine Tartrate Hydrate (Ergometrine Tartrate Hydrate) [(C₁₉H₂₃N₃O₂)₂. C₄H₆O₆.H₂O] (Basergin, Neofermergen): It is obtained as crystals that are slightly soluble in water.

Uses

1. Ergonovine is used as an oxytocic.

2. Ergonovine maleate also acts as an oxytocic and produces much faster stimulation of the uterine muscles as compared to other ergot alkaloids.

3. Methylergonovine meleate is observed to act as an oxytocic whose actions are slightly more active and longer acting than ergonovine.

-----------------------------------------------

* Stoll, A., and Hofmann, A., Helv. Chim Acta, 26, 944 (1943).

** Van Urk Reagent Mix togetehr 0.125g of para-dimethylamino. benzaldehyde; 0.1 ml of FeCl3 soln. (5% w/v),

and 15% (v/v) H2SO4 to make 100 ml.

B. Ergotamine

Biological Source It is obtained from the seeds of Claviceps purpurea (Fr.) Tul. (Hypocreales) (Ergot).

Chemical Structure The chemical structure of ergotamine has been given in Section 7.2.8.6.

Isolation The method of Stoll* may be adopted as stated below:

1. The powdered dried ergot is first defatted with n-bexane or petroleum ether (40-60°)

2. The marc consisting of the defatted powdered ergot is thoroughly mixed with aluminium sulphate and water so as to fix the alkaloids by converting them into the double salts.

3. The resulting alkaloidal double salts are subjected to continuous extraction with hot benzene that removes the alkaloid exclusively on one hand; and the unwanted substances e.g., ergot oil, soluble acid, neutral substances like-phytosterol, colouring matter and organic acids on the other.

4. The benzene is removed under vacuo and the residue thus obtained is stirred for several hours with a large volume of benzene and subsequently made alkaline by passing NH₃ gas.

5. The resulting solution is filtered and the benzene extract is concentrated under vacuo to approximately 1/50th of the original volume, whereupon ergotamine crystallizes out.

6. An additional quantity of ergotamine may also be crystallized from the mother liquour by treatment with petroleum ether.

7. Ergotamine may be further purified by crystallization from aqueous acetone.

Characteristic Features

1. It is obtained as elongated prisms from benzene that get decomposed at 212-214°C.

2. It usually becomes totally solvent-free only after prolonged heating in a high vacuum.

3. It is found to be highly hygroscopic in nature; and darkens and decomposes on exposure to air, heat and light.

4. It has specific optical rotation [α]²⁰_D - 160° (chloroform).

5. It is soluble in 70 parts methanol, 150 parts acetone, 300 parts ethanol; freely soluble in chloroform, pyridine, glacial acetic acid; moderately soluble in ethyl acetate; slightly soluble in benzene; and practically insoluble in petroleum ether and water.

Identification Tests The precipitation reactions and the colour tests are the same as described under ergonovine. However, the specific derivatives of ergotamine are as stated below:

1. Ergotamine Tartrate [(C₃₃H₃₅N₅O₅)₂.C₄H₆O₆] (Ergomar; Ergate; Ergotartrat; Ergostat; Exmigra; Fermergin; Lingraine; Gynergen; Lingran): It is normally obtained as solvated crystals e.g., the dimethanolate; also occurs as heavy rhombic plates from methanol having mp 203°C (decomposes). It has specific optical rotation [α]²⁵_D – 125° to – 155° (C = 0.4 in chloroform). One gram dissolves in either 500 ml of ethanol or water.

2. Ergotamine Hydrochloride [C₃₃H₃₅N₅O₅.HCl]: It is obtained as rectangular plates from 90% (v/v) ethanol which get decomposed at 212°C. It is found to be soluble in water-ethanol mixtures; and sparingly in water or ethanol alone.

3. Dihydroergotamine Mesylate (C₃₃H₃₇N₅O₅.CH₃SO₃H) (Agit;1 Dihydro-ergotamine methane sulphonate; Angionorm; DET MS; Dergotamine; D.H.E. 45; Diergo; Dihydergot; Dirgotarl; Endophleban; Ergomimet; Ikaran; Migranal; Morena; Ergont; Ergotonin; Orstanorm; Tonopres; Verladyn; Seglor): It is obtained as large prisms from 95% (v/v) ethanol having mp 230-235°C; and moderately soluble in water.

Note: (a) It is the salt of a semisynthetic alkaloid prepared from ergotamine by hydrogenation of the ∆⁹ double bond in the lysergic acid nucleus.

(b) It is mostly used in the treatment of migraine because it is found to be better in efficacy and more tolerated than the parent alkaloid.

Uses

1. It is employed as a potent antimigraine drug.

2. Ergotamine possesses oxytocic properties, but it is not employed for that effect.

3. Ergotamine tartrate is used invariably to prevent or abort vascular headaches, including migraine and cluster headaches. The mechanism of action is perhaps due to direct vasoconstriction of the dilated carotid artery bed with concomitant lowering in the amplitude of pulsations.

4. Ergotamine tartrate is also an antagonist of the serotonin activity.

5. Ergotamine tartrate is frequently used along with caffeine for the management and control of migraine headache. Both serve as cerebral vasoconstrictors; while the latter is considered to increase the action of the former.

6. Methylergonovine maleate is an oxytocic reported to be longer acting and more active than ergonovine.

------------------------------------

* Stoll, Helv. Chim. Acta 28, 1283, (1945)

C. Ergine

Synonyms Lysergamide; Lysergic acid amide;

Biological Sources It is obtained from the immature seeds of Argyreja nervosa (Burm.) Bojer (Convolvulaceae) (Wood Rose, Silver Morning Glory); Beeds of Ipomea Violaceae L. (Convolulaceae) (Tlitliltzen, Ololiuqui); seeds of Rivea corymbosa Hall. F. (Convolvulaceae) (Snakeplant); and also from the seeds of Ipomea tricolor Cav (Convolvulaceae).

Chemical Structure

9, 10-Didehydro-6-methylergoline-8β-carboxamide; (C₁₆H₁₇N₃O).

Isolation It is isolated from the seeds of Rivea corymbosa (L.) and from Ipomea tricolor Cav. By the method of Hofmann and Tscherter.*

Characteristic Features

1. It is obtained as prisms from methanol which get decomposed at 242°C.

2. It has a specific optical rotation of [α]²⁰₅₄₆₁ + 15° (C = 0.5 in pyridine).

Identification Tests The precipitation reactions and the colour tests are the same as described under ergonovine (Section A).

Ergine may also be identified by forming its derivative as stated below:

Ergine Methane Sulphonate (C₁₆H₁₇N₃O.CH₃SO₃H) It is obtained as prisms from a mixture of methanol and acetone that get decomposed at 232°C.

Uses It has a pronounced depressant action.

Note: It is a controlled substance listed in the U.S. Code of Federal Regulations. Title 21 Part 1308, 13 (1995).

Biosynthesis of Ergotamine The various steps involved in the biosynthesis of ergotamine are as enumerated below:

1. Three amino acids, viz., L-alanine, L-phenylalanine, and L-proline in the presence of ATP and enzyme SH; or D-(+)-lysergic acid in the presence of ATP and enzyme SH undergo two steps: first-activation via AMP esters, and secondly-attachment to the respective enzymes, thereby giving rise to an intermediate. It is worthwhile to observe that the enzyme is comprised of two subunits that essentially bind the substrates as indicated in the biosynthetic pathway given below.

2. The comparatively more complex structures comprising of the peptide fragments, such as: ergotamine are eventually formed by sequential addition of amino acid residues to the thioesterbound lysergic acid, yielding a linear lysergyl-tripeptide covalently attached to the enzyme complex.

3. The resulting complex undergoes lactam formation followed by release from enzyme. In other words, the cyclized tripetide residue is rationalized instantly by the formation of a lactam (amide) that releases ultimately the product from the enzyme.

4. This resulting product first affords hydroxylation then followed by generation of a hemeketallike linkage to give rise to the formation of ergotamine.

All these aforesaid steps (1) through (4) have been duly depicted in the following biosynthetic pathway.

Peptide Alkaloids in Ergot Interestingly, it has been observed critically that three amino acids, namely: alamine, phenylatine and proline, actually from the basis for the various structures which are encountered in the domain of the ‘ergot alkaloids’. Therefore, these known and established structures may be subdivided into three major groups which are: ergotamine group, ergoxine group, and ergotoxine group.

The various alkaloids having the peptide linkages found in ‘ergot’ are depicted as under.

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* Hofmann and Tscherter, Experientia, 16, 414 (1964).
Source:Pharmacognosy And Pharmacobiotechnology By Ashutosh Kar