METABOLIC PRODUCTS OF THE CINCHONA ALKALOIDS IN HUMAN URINE*

BY BERNARD B. BRODIE,? JOHN E. BAER,$ AND LYMAN C. CRAIG

(From the Research Service, Third (New York University) Medical Division, Goldwater Memorial Hospital, and the Department of Biochemistry, New York University

College of Medicine, New York, New York, the National Heart Institute, National Institutes of Health, Bethesda, Maryland, and The Rockefeller

Institute for Medical Research, New York, New York)

(Received for publication, August 14, 1950)

In spite of the wide use of quinine in the treatment of malaria, and of quinidine in the therapy of cardiac arrhythmias, little information exists concerning the metabolic fate of the cinchona alkaloids administered to man. As early as 1869, Kerner (1) isolated from the urine of subjects receiving quinine a substance thought to be quinetine, a derivative of quinine in which the vinyl side chain of the quinuclidine nucleus has been oxidized to a carboxyl group. Halberkann (2) suggested that the com- pound was not a metabolite of quinine but had been formed by oxidation during the analytical procedure. Nierenstein (3) also claimed to have isolated quinetine from the urine of a single subject receiving quinine. In addition this investigator isolated from the urine of subjects with blackwater fever a product that exhibited marked hemolytic properties for red blood cells and that appeared to account for the symptoms of the disease (4). This substance, hemoquinic acid, was characterized as 6- methoxyquinoline-4-ketocarboxylic acid. Lipkin (5) obtained a product by incubation of quinine with minced sheep liver which, on the basis of its chemical properties, he considered quinetine.

Kelsey, Geiling, Oldham, and Dearborn (6) isolated a different type of metabolic product when quinine was incubated with rabbit liver. This derivative was a phenol whose structure was characterized by Mead and Koepfli (7) as a carbostyril in which the quinoline nucleus had been oxi- dized in position 2.

*Part of the work described in this paper was done under a contract, recom- mended by the Committee on Medical Research, between the Office of Scientific Research and Development and New York University. A portion of the work was presented at the meetings of the Federation of American Societies for Experimental Biology, March, 1946 (Federation Proc., 6, 168 (1946)) and was also discussed in a I-Iarvey Lecture by Dr. James A. Shannon, October 25, 1945.

t Present address, National Heart Institute, National Institutes of Health, Bethesda, Maryland.

$ Present address, Carleton College, Northfield, Minnesota.

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568 CINCHONA ALKALOIDS

Recent studies on the excretion of the cinchona alkaloids have shown that quinine, quinidine, cinchonine, and cinchonidine are extensively me- tabolized in man, only a small fraction of the ingested drugs being excreted unchanged in the urine (8, 9). In the development of analytical methods for the estimation of the drugs in biological fluids, the existence of meta- bolic products was observed, and some of their solubility characteristics were noted (10). This observation led to the present work, which is concerned with the isolation and the elucidation of the structure of certain metabolic products of quinine, quinidine, cinchonine, and cinchonidine.

Procedures

Experience has shown that, as a rule, basic organic compounds are metabolized in the body to substances which are more water-soluble than the parent compounds and, therefore, have lower partition ratios in organic solvent-water systems. Their differential partition ratios have been exploited to fractionate the cinchona alkaloids and the various meta- bolic transformation products that are excreted in urine. First, the parent drug was extracted from an aqueous phase with the relatively non-polar solvent, heptane, which left behind all or a considerable fraction of the more water-soluble metabolic products. Following this, alkaloidal ma- terial was extracted from the aqueous phase with solvents of progressively increasing polarity, whereby a rough fractionation of the metabolic prod- ucts was achieved. The extracted material in the various solvent phases was usually returned to an aqueous acid phase preparatory to counter- current distribution. The organic bases in such a polar solvent as isoamyl alcohol can be returned quantitatively to an aqueous phase by decreasing the polarity of the solvent by the addition of heptane.

Further fractionation of the metabolites was effected by counter-current distribution, by means of a separatory funnel technique involving eight transfers (11). In general, isoamyl alcohol, containing various amounts of heptane, and an aqueous buffer (1 M phosphate or 0.6 M borate or a mix- ture of both) were used as the two immiscible phases. Both phases were previously saturated one with the other. For the most effective distribu- tion of a substance it should be distributed about equally between the two phases. This is commonly achieved by adjusting the pH of the aqueous phase. It was desirable to work at a low pH so as to permit the use of high concentrations of the organic bases under study. The partition ratio was adjusted, therefore, by varying the polarity of the organic solvent phase, usually by suitable adjustment of the proportion of isoamyl alcohol to heptane. After distribution, the partition ratio of the material in each separatory funnel was determined by measuring the total concen- tration of cinchona alkaloids in each phase, as described below. The

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B. B. BRODIE, J. E. BAER, AND L. C. CRAIG 569

contents of separatory funnels with similar partition ratios in a given run were then pooled. The aqueous phase of the pooled sample was alkalized with ammonium hydroxide and the total cinchona material transferred to the organic phase by shaking. Further purification involved a transfer to a small acid phase, followed by further counter-current distribution, or by the evaporation of the solvent to dryness and recrystallization from appropriate solvents.

Spectrophotometric measurements of the alkaloidal material were made in dilute sulfuric acid by means of the Beckman quartz spectrophotometer. Aqueous phases were analyzed by measurements of an aliquot which had been diluted with 0.1 N sulfuric acid; organic phases after extraction of an aliquot into 0.1 N sulfuric acid. The initial assumption made was that the absorption spectra of the cinchona metabolites and the parent com- pounds are not far different, an approximation subsequently shown to be valid and very useful. The measurements for the quinine and quinidine products were made therefore at 350 rnp, while those for cinchonine and cinchonidine were made at 315 mp.

The basic dissociation constants of some of the metabolites were meas- ured and are expressed as the pH of the half neutralized base (pKJ.

The melting points were determined on the micro hot stage melting point apparatus unless otherwise stated.

EXPERIMENTAL

Cinchonine

15 liters of urine from subjects who had received cinchonine orally were made ammoniacal and extracted, first with ether, then with isoamyl alcohol. The ether-soluble material was returned to a small volume of dilute acid, and the acid phase was made ammoniacal and extracted several times with heptane. The cinchona material remaining in the ammoniacal aqueous phase was then extracted with ether.

The heptane phase was shaken with 2.5 N NaOH, which served to remove a small amount of material which had amphoteric properties. The alkaline phase was neutralized with acid, then ammonium hydroxide was added, and the amphoteric material (weakly acid and basic) extracted into ether. This ether extract was added to the main ether fraction.

Heptane Fraction (0.2 Gm.)-A counter-current distribution of the ma- terial between buffer at pH 5.8 and 10 per cent isoamyl alcohol-heptane yielded two fractions. One had the properties of cinchonine; its partition ratio in the above two-phase system and its absorption curve in 0.1 N

H&Oh (Fig. 1) were almost identical with those of cinchonine. The other fraction, which was highly fluorescent in 0.1 N HzS04, was distributed in

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570 CINCHONA ALKALOIDS

the system heptane-buffer at pH 11.5. Crystallization of the material in the middle funnels from chloroform-acetone yielded needles and leaflets, melting at 174-179”; reported melting point for quinine 174.5-175” (12).

The cinchonine administered to the subjects was found, on the basis of its fluorescence and its absorption at 360 rnp, to contain quinine as an impurity to the extent of about 10 per cent. It was presumed, therefore, that the quinine found in the urine originated from the quinine present as’ an impurity in the ingested cinchonine.

Ether Fraction (S Gm.)-Three successive counter-current distributions between buffer at pH 4.1 and isoamyl alcohol were carried out. The

I 300

WAVE LENGTH MjU

FIG. 1. The absorption spectra of cinchonine (broken line), cinchonine carbostyril (solid line), and oxidized cinchonine carbostyril (dotted line). The substances were dissolved in 0.1 N H&JO4 at a concentration of 10 y per ml. Cell thickness, 1 cm.

major fraction was taken into a small volume of acid, and ammonium hydroxide was added, yielding a white precipitate; m.p. 268-270”. Re- crystallization of a small amount of material from methanol yielded rhombic crystals; m.p. 271-273”. The basic dissociation constants were found to be as follows: pK,‘, 8.34 (in 28 per cent ethanol); pK/, 3.11 (in water). The solubility of the compound in alkali was suggestive of a phenolic compound.

Analysis-C,eHzzOzNz. Calculated. C 73.51, H 7.14 Found. (‘ 73.44, “ 7.12

The analysis indicates that the compound has 1 more oxygen atom than cinchonine. The absorption spectrum in 0.1 N HzS04 was distinctly

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B. B. BRODIE, J. E. BAER, AND L. C. CRAIG 571

different from that of cinchonine (Fig. 1). Thus it appears that the additional oxygen is in the quinoline ring, since its presence in the qui- nuclidine moiety presumably would result in a negligible alteration of the absorption curve. A sample of the compound was submitted to Dr. J. B. Koepfli of the California Institute of Technology, who characterized it as the carbostyril, 2-hydroxycinchonine (2’-hydroxy-3-vinylruban-9-ol).1

Isoamyl Alcohol Fraction (2.6 ti.)-A counter-current distribution of this material in the system isoamyl alcohol-buffer at pH 4.7 showed two components. The component favoring the alcohol phase was found to be residual carbostyril analogue which had not been completely extracted by ether. The principal component was purified by a counter-current distribution in isoamyl alcohol-buffer at pH 6.3. Material in the middle funnels was recrystallized from chloroform-petroleum ether. The sub- stance was further purified by a counter-current distribution in the system 30 per cent butanol-cyclohexane-buffer at pH 6.6. The material in funnels 2 to 6 was recrystallized from methanol-acetone and yielded rosettes of needles; m.p. 278-279”.

Analyszs-CLsHarOaN2. Calculated. C 69.96, H 6.80 Found. “ 69.50, “ 7.07

The compound appears to be a derivative of cinchonine containing 2 additional oxygen atoms. The absorption curve of the material in 0.1 N

H&S04 was found to be almost identical with that of the carbostyril ana- logue described above (Fig. 1). Therefore, the compound appears to be cinchonine carbostyril with an additional oxygen atom located in the quinuclidine moiety where its presence would not appreciably influence the absorption curve. The basic dissociation constants were pK,’ 7.65, pK,” 2.76 (both in water). A monopicrate prepared in acetone and re- crystallized from the same solvent melted at 283-286”.

Analysis-ClpH220aNz. C~H~O7N~. Calculated. C 54.04, H 4.54 Found. “ 54.24, “ 4.32

Cinchonidine

22 liters of urine from patients who had received cinchonidine orally were adjusted to pH 10 and extracted twice with isoamyl alcohol. The alcohol extract contained about 6.5 gm. of cinchona material, estimated spectrophotometrically. The alkaloidal material was extracted into a small volume of acid, and the acid was adjusted to a pH of 10 and ex-

1 Koepfli, J. B., unpublished work.

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572 CINCHONA ALKALOIDS

tracted, successively, four times with heptane, seven times with ether, and twice with isoamyl alcohol. The heptane phase was extracted with 2.5 N

NaOH, which served to remove a small amount of material which had amphoteric properties. The alkaline phase was adjusted to pH 10 and the amphoteric material extracted into ether. This ether extract was added to the main ether fraction.

Heptane Fraction (0.7 Gm.)-A counter-current distribution in the sys- tem 18 per cent isoamyl alcohol-heptane-buffer at pH 6.0 was carried out. The material in the middle funnels was taken into 0.1 N sulfuric acid. The absorption spectrum of the material was almost identical with that of a known sample of cinchonidine (Fig. 2). Further evidence that the com-

I I I 1 I I I I

I

380

WAVE LENGTH M/l

FIG. 2. The absorption spectra of cinchonidine (broken line), cinchonidine carbo- styril (solid line), and oxidized cinchonidine carbostyril (dotted line). The sub- stances were dissolved in 0.1 N H&04 at a concentration of 10 y per ml. Cell thick- ness 1 cm.

pound was cinchonidine was obtained by comparing its partition ratios in ethylene dichloride-water at various pH levels with those of authentic cinchonidine; these were found to be almost identical.

Ether Fraction (3.0 Gm.)-An aliquot of this fraction was subjected to two counter-current distributions in the system 60 per cent isoamyl alcohol-heptane-buff er at pH 5.9. Funnels 3 to 7 contained 520 mg. of product, which was precipitated from a small volume of acid with am- monia to yield 190 mg. of white crystals; m.p. 222-229’. A distribution in buffer at pH 6.88 and chloroform indicated that the substance was of high purity. Material from the middle funnels of this distribution was recrystallized from methanol-mater. The compound almost completely

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B. B. BRODIE, J. E. BAER, AND L. C. CRAIG 573

melted at 160” and resolidified to melt at 220”, a few well formed square- ended needles persisting to 227”.

Analysis-ClsHg20zNe. Calculated. C 73.51, H 7.14 Found. “ 73.52, “ 7.18

The analysis indicates that the compound contains 1 more oxygen atom than cinchonidine. The absorption spectrum of the compound (Fig. 2) was found to be almost identical with that of the carbostyril metabolite of cinchonine. The compound was soluble in alkali as well as acid, sug- gestive of a phenolic basic compound. It would appear, therefore, that this compound is the carbostyril of cinchonidine, 2-hydroxycinchonidine.

Isoamyl Alcohol Fraction (0.6 Gm.)-This fraction was colored by uri- nary pigments. Four counter-current distributions in isoamyl alcohol- buffer at pH 5.9 were required to remove all coloration from the major component. Material from Funnels 3 to 7 was recrystallized slowly from chloroform-petroleum ether to yield a substance which lost its double refraction at 227” and melted at 230’; the melting point was not sharp. It was not possible to prepare a crystalline gold salt with chloroauric acid.

analysis-CleHzzOsNz. Calculated. C 69.90, H 6.86 Found. I‘ 68.32, “ 6.56

The analytical data indicate that the compound is still impure. The absorption curve (Fig. 2) was almost identical with that of the carbostyril, 2-hydroxycinchonidine. The solubility properties and oxygen content would suggest that the chief component is the’carbostyril of cinchonidine further oxidized in the quinuclidine ring, an isomer of the more highly oxidized cinchonine carbostyril.

Quinine

48 liters of urine of subjects who had received quinine orally were ad- justed to pH 10 and exhaustively extracted with ether. The alkaloidal material was returned to an aqueous acid which, after the addition of ammonia, was successively extracted several times with heptane, then with chloroform, and finally with isoamyl alcohol.

Heptane Fraction (3.3 &.)-A small aliquot was distributed in 30 per cent isoamyl alcohol-heptane buffer at pH 4.95. The material was found to be essentially pure and to have the same partition ratio in this system

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574 CINCHONA ALKALOIDS

as quinine. Its absorption curve was almost identical with that of au- thentic quinine (Fig. 3). The partition coefficients of the substance and authentic quinine between ethylene dichloride and water at various pH values were compared and found to be almost the same. In view of the above data, the substance was considered to be unchanged quinine.

Chloroform Fraction-The chloroform extract was shaken with alkali to remove phenolic material. In the case of cinchonine and cinchonidine the only organic-soluble metabolic products excreted into the urine were phenolic. However, with quinine, a considerable chloroform-soluble, non- phenolic fraction (3.4 gm.) was apparent. This material represented the

WAVE LENGTH M,LL

FIG. 3. The absorption spectra of quinine (solid line) and monohydroxy (dotted line) and dihydroxy (broken line) non-phenolic metabolites of quinine. The sub- stances were dissolved in 0.1 N HzSOc at a concentration of 10 y per ml. Cell thick- ness, 1 cm.

bulk of isolated metabolic products of quinine. A 900 mg. aliquot was subjected to three counter-current distributions in 40 per cent isoamyl alcohol-heptane-buffer at pH 6.0. Funnels 3 to 7 of the third distribution contained 360 mg. of material, which was recrystallized from benzene; it formed rosettes melting at 147-149” (capillary). After recrystallization of a small sample from methanol-acetone, the melting point was 145-148’ with previous sintering. The basic dissociation constants were pK,’ 7.24, pK,” 4.12 (in water). The basic dissociation constants of quinine were measured under comparable conditions and found to be pK,’ 8.32, pK,” 4.25.

Analysis-CzoH240aNz. Calculated. C 70.50, H 7.11 Found. “ 70.38, “ 6.77

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B. B. BRODIE, J. E. BAER, AND L. C. CRAIG 575

Analysis of the compound indicates that it contains 1 more oxygen atom than quinine. The absorption curve in 0.1 N HzS04 was almost the same as that of quinine (Fig. 3), indicating that oxidation had occurred in the quinuclidine ring.

The 2.5 N NaOH solution, which contained the chloroform-soluble phenolic fraction (1.2 gm.), was neutralized with acid, ammonium hy- droxide was added, and the alkaloidal material extracted into isoamyl alcohol. Distribution of this fraction in 85 per cent isoamyl alcohol- heptane-buffer at pH 5.0 demonstrated considerable impurity. In ad- dition, it was observed that a pink color appeared, becoming more intense overnight. The impurities, including the color, were removed by the extraction of an aqueous phase of pH 4.8 with chloroform. An additional counter-current distribution of the water-soluble material yielded 300 mg. of material which had the same melting point (248’), absorption spec- trum, fluorescence characteristics, and partition coefficient (in the above system) as the product obtained by the action in vitro of rabbit liver on quinine described by Kelsey et al. (6), which has been identified by Mead and Koepfli (7) as the carbostyril analogue of quinine.

Isoumyl Alcohol Fraction (3.0 &.)-This material was subjected to several counter-current distributions between buffer at pH 8.3 and 50 per cent isoamyl alcohol-heptane. Several hundred mg. of material were obtained, which was recrystallized from chloroform; m.p. 110-114”. Re- crystallization of a small amount from methanol-acetone yielded well formed stout needles or columns and rosettes. When heated rapidly, the substance partially melted at 110”. When heated slowly, the crystals lost double refraction at 128-131” and set to a viscous liquid which became quite fluid at 135”. No crystalline picrate or hydrochloride could be obtained. The basic dissociation constants were found to be pK,’ 7.93, pKio” 4.00 (in water).

AnaZysis-C~oH~~0~Nz. Calculated. C 67.39, H 6.79, N 7.86 Found. “ 66.84, “ 7.19, ‘I 8.03

The analytical data indicate that the compound possesses 2 oxygen atoms more than quinine. The absorption spectrum in 0.1 N H,SO, (Fig. 3) was almost identical with that of quinine, suggesting that both oxygens were in the quinuclidine ring.

The isoamyl alcohol fraction also contained other substances in small amounts. Some of these probably represent metabolites previously de- scribed. One product, more water-soluble than the other metabolites so far described, and presumably a still more oxygenated derivative, was present in quantities too small for further investigation.

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576 CINCHONA ALKALOIDS

The urine of subjects who had received quinidine orally was exhaus- tively extracted with ether. The alkaloidal material was fractionated as described for quinine.

Heptane Fraction (2.7 &.)-A counter-current distribution of this frac- tion in 30 per cent isoamyl alcohol-heptane-buffer at pH 5.0 indicated one main component. Material from the middle funnels was brought into a small volume of acid and precipitated by the addition of ammonium hydroxide. The resulting solid melted at 1733174”; the melting point was not depressed by the admixture of authentic quinidine. The absorption spectrum of the material in 0.1 N HzS04 was almost identical with that o

WAVE LENGTH M,&

FIG. 4. The absorption spectra of quinidine (solid line), quinidine carbostyril (broken line), and a monohydroxy non-phenolic metabolite of quinidine (dotted line). The substances were dissolved in’ 0.1 N H&S04 at a concentration of 10 y per ml. Cell thickness, 1 cm.

authentic quinidine (Fig. 3). The fraction was presumed to be unchanged quinidine.

Chloroform Fraction, Non-Phenolic (0.5 Gm.)-This fraction was purified by two counter-current distributions in 60 per cent isoamyl alcohol- heptane-buffer at pH 5.0. The material in the middle funnels was re- distributed in chloroform-buffer at pH 6.2. Recrystallization from ace- tone of the material in Funnel 4 yielded crystals melting at 210-212”.

AnaZysis-C~,,H~~0~N~. Calculated. C 70.56, H 7.11 Found. “ 70.54, “ 7.13

The analysis indicates that this compound contains 1 more oxygen than quinidine. Since the absorption spectrum in 0.1 N H&O, was identical

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B. B. BRODIE, J. E. BAER, AND L. c. CRAIG 577

with that of unchanged quinidine (Fig. 4), the additional oxygen atom is presumably attached to the quinuclidine portion of the molecule.

Chloroform Fraction, Phenolic (0.3 Gm.)-This material was distributed between 54 per cent isoamyl alcohol-heptane-buffer at pH 4.95. Two fractions were found. The major fraction was subjected to another counter-current distribution with the above two-phase system. The ma- terial in the middle funnels was recrystallized from chloroform petroleum ether; m.p. 245-260”. An additional distribution yielded 10 mg. of ma- terial; when recrystallized from methanol-water, 5.4 mg. of well formed crystals were obtained. The compound did not have a sharp melting point; 235-260”.

Analysis-Cz,,HzaOOaNz. Calculated. C 70.56, H 7.11 Found. “ 70.60, “ 7.47

The analysis and solubility characteristics of the compound suggest that it is the quinidine carbostyril, 2-hydroxyquinidine. This was confirmed by the absorption spectrum curve which proved to coincide with the quinine carbostyril curve (Fig. 4).

The minor phenolic fraction, which was present in only small quantities, was found to be more water-soluble than quinidine carbostyril. The ab- sorption curve was found to be similar to that of the carbostyril. It is likely that this compound has both the carbostyril group and the qui- nuclidine oxygen of the chloroform-soluble non-phenolic compound.

Isoamyl Alcohol Fraction-This fraction contained a mixture of com- ponents, some of which may have been residual quantities of previously removed substances. One fraction, m.p. 120-124’, which was not in- vestigated further, appeared to have been a further oxygenated derivative.

DISCUSSION

All four carbostyrils derived by oxidation of the quinoline ring at po- sition 2 have been isolated and characterized. The acidic character of the aromatic hydroxyl group explains the removal of these compounds from an organic phase when it is shaken with alkali. The absorption spectra of the carbostyrils differ from those of the parent compounds, as would be anticipated from a consideration of the resonance effect of the -NH-C=0 tautomer on the structure.

No evidence was found of a compound in which only the quinuclidine ring of cinchonine or cinchonidine was oxidized. On the other hand, both quinine and quinidine are oxidized to such compounds, Indeed, it ap-

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578 CINCHONA ALKALOIDS

pears that the quinine molecule can be metabolized to a compound with 2 additional oxygen atoms in the quinuclidine ring.

Another difference between the demethoxy alkaloids (cinchonine and cinchonidine) and quinine and quinidine is that the carbostyrils of the former are further oxidized, the 2nd oxygen appearing in the quinuclidine nucleus.

The introduction of oxygen into the quinuclidine nucleus of quinine to form a non-phenolic derivative lowers the first basic dissociation constant from pK 8.32 to 7.24. Such a large decrease in basicity can be explained by a transformation to an cY-hydroxyamine structure. It is reasonable to assume that all the cinchona derivatives with additional oxygen in the quinuclidine moiety have had the oxygen inserted as a hydroxyl group adjacent to the tertiary nitrogen.

I II III FIG. 5. Proposed scheme for the route of metabolism of cinchonine and cinchoni-

dine in the human. (I) Cinchonine or cinchonidine; (II) cinchonine carbostyril or cinchonidine carbostyril; (III) oxidized cinchonine carbostyril or oxidized cinchon- idine carbostyril.

The differences among these metabolic products suggest that the de- methoxy alkaloids are metabolized through a somewhat different route than are quinine and quinidine. The route of metabolism proposed for cinchonine and cinchonidine in the human involves two serial steps (Fig. 5); the first of these is the addition of oxygen to the quinoline nucleus with the formation of a carbostyril, the second, an addition of oxygen to the quinuclidine nucleus of the carbostyril, probably at the ring carbon ad- jacent to the nitrogen.

The route of metabolism proposed for quinine and quinidine appears to involve two parallel pathways (Fig. 6). The first pathway is the forma- tion of a carbostyril by the addition of oxygen to the quinoline nucleus; the second is the addition of oxygen to the quinuclidine nucleus to form a monohydroxy non-phenolic derivative. In the case of quinine, at least, this latter compound appears to be oxidized further in the quinuclidine nucleus to form a dihydroxy derivative.

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B. B. BRODIE, J. E. BAER, AND L. C. CRAIG 579

Only in the case of cinchonine have quantitative estimations been made of the total amount of parent compound and its metabolic products ex- creted in the urine after single oral doses of the parent drug. The results indicate that about 80 per cent of orally administered cinchonine is re- coverable in the urine as cinchonine and its derived products, about 4 per cent as unchanged cinchonine, 55 per cent as the 2-hydroxy derivative, and 22 per cent as the further oxidized product (13). That the second metabolic product is derived in the body from the carbostyril was shown by experiments in which the latter compound was administered orally (13).

I”

\

II m

0

cp CH:CH2

CHOH ‘,:

CH30 ’ ’

6, \ N’ OH m

FIG. 6. Proposed scheme for the route of metabolism of quinine and quinidine in the human. (I) Quinine or quinidine; (II) monohydroxy non-phenolic metabolite of quinine or quinidine; (III) dihydroxy non-phenolic metabolite of quinine or quinidine; (IV) quinine carbostyril or quinidine carbostyril.

2-Hydroxycinchonine has been found by Earle, Welch, and Shannon (14) to possess about one-fifth the antimalarial activity of its parent compound. These authors concluded that the 2-hydroxy compound plays a negligible rale in the antimalarial effect achieved when cinchonine is administered.

Although similar determinations have not been made for the other cinchona alkaloids, a rough indication can be given of the relative amounts of unchanged alkaloid and transformation products. In the case of cin- chonidine, the carbostyril was the principal product; unchanged cin- chonidine and the dihydroxy metabolite were present in about equal amounts, the amount of each being about one-fifth that of the carbostyril. The principal quinine product was not the carbostyril but the non- phenolic monohydroxy metabolite. It was present in about the same

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580 CINCHONA ALKALOIDS

amount as unchanged quinine and in 3 times the amount of the carbo- styril. In the case of quinidine also, the non-phenolic monohydroxy derivative was the main metabolite. However, the latter was present in smaller quantities than the unchanged quinidine. The more water-soluble metabolites of both quinine and quinidine were also present in significant amounts.

The enzyme in rabbit liver that appears to be involved in the oxidation of the cinchona alkaloids and other quinolines to their corresponding carbostyrils has been isolated by Knox (15). This enzyme, which has been prepared in about 5 per cent purity, is associated with the flavo- protein, liver aldehyde oxidase. It is unlikely that this enzyme is also responsible for the oxidation that occurs in the quinuclidine nucleus.

SUMMARY

Metabolic products of each of the four principal cinchona alkaloids have been isolated from human urine. The separation of the products from each other and from the parent drug was based on differences in their partition ratios in different systems.

2-Hydroxycinchonine, 2-hydroxycinchonidine, 2-hydroxyquinidine, and 2-hydroxyquinine were all isolated in crystalline form. The assignment of carbostyril structures is supported by evidence from chemical analyses, absorption spectra, basic dissociation constants, and solubility properties.

Cinchonine and cinchonidine are also converted to dihydroxy com- pounds; the second hydroxyl group appears in the quinuclidine ring. The absence of other metabolic products suggests a mechanism of stepwise oxidation of these two alkaloids.

Quinine and quinidine both add 1 oxygen atom in the quinuclidine ring to form non-phenolic isomers of the carbostyrils. The concomitant de- crease in basicity indicates that the hydroxyl group is located on a carbon adjacent to the quinuclidine nitrogen atom. A dihydroxy derivative of quinine was also found which appears to have both hydroxyl groups in the quinuclidine ring. Other metabolic products of quinine and quinidine were present in small amounts, but were not identified.

BIBLIOGRAPHY

1. Kerner, C., Arch. gee. Physiol., 2, 200 (1869). 2. Halberkann, J., Biochem. Z., 96, 24 (1919). 3. Nierenstein, M., J. Roy. Army Med. Corps, 32, 215 (1918). 4. Nierenstein, M., J. Roy. Army Med. Corps, 32, 218 (1918). 5. Lipkin, J., Ann. Trop. Med., 13, 149 (1919). 6. Kelsey, F. E., Geiling, E. M. K., Oldham, F. K., and Dearborn, E. H., J. Phar-

macol. and Exp. Therap., 80, 391 (1944). 7. Mead, J., and Koepfli, J. B., J. Biol. Chem., 164, 507 (1944).

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CraigBernard B. Brodie, John E. Baer and Lyman C.

URINECINCHONA ALKALOIDS IN HUMAN

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