METABOLIC PRODUCTS OF THE CINCHONA ALKALOIDS IN HUMAN URINE* (Received for ...

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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.
567
This is an Open Access article under the CC BY license.
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
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
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
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.
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
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
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
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.
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 substancews ere dissolvedi n’ 0.1 N H&S04a t 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