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]15D - 169° (C = 2 in 97% ethanol); [α]17D - 117° (C = 1.5 in chloroform); [α]15D - 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.
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* 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]15D - 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:
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* 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]22D + 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.
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* 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]15D + 230° (C = 1.8 in chloroform); [α]17D + 258° (ethanol); and [α]17D + 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]25D ~ + 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.
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* 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]20D - 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]20D - 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]20D + 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:
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** Leers, Ann., 82, 147 (1952)
