The Metabolism of Pyrazolo(3,4-Ã-/)pyrimidines
by the Rat't
PHILIPFEIGELSON
ANDJACKD. DAVIDSON^
(Departments of Biochemistry and Medicine, Collegeof Physicians and Surgeons, Columbia University, New York, N.Y.)
The recent development of a new series of carcinostatic purine analogs, the pyrazolo(3,4-d)pyrimidines (5), has led to their study as substrates
and inhibitors of certain purine oxidation systems
in vitro (1,4). In the course of these studies, it was
observed that purified xanthine oxidase incubated
in vitro with 4-aminopyrazolo(3,4-d)pyrimidine
(pyrazoloadenine) resulted in its oxidation to 4amino-6-hydroxypyrazolo(3,4-d)pyrimidine
(pyrazoloisoguanine) (2). It seemed of interest to as
certain whether this in vitro phenomenon repre
sents an in vivo catabolic pathway of this drug.
Knowledge of the catabolism of carcinostatic
agents is both of intrinsic interest and of possible
importance in gaining insight into tissue specifici
ties and the development of resistance toward
such compounds by tumors.
MATERIALS AND METHODS
Sprague-Dawley strain stock rats (300-400 gm.)
were maintained on Purina chow and water ad
libitum. A homogenized 400 mg. per cent saline
suspension of pyrazoloadenine1 was administered
intraperitoneally, each animal receiving a single
dose of 0.5 ml/100 gm body weight. Control rats
received comparable doses of saline. The animals
were immediately transferred to metabolism cages;
food was withheld, but water was allowed ad
libitum. Urines were collected under toluene for
24 hours. Each urine sample was alkalinized with
concentrated ammonium hydroxide, and the re
sultant precipitate was removed by centrifugation; silver nitrate was then added in excess. The
silver-pyrazolopyrimidine precipitate was permit
ted to flocculate overnight at 4°C. The precipitate
* This study was supported by U.S.P.H.S. grants numbers
C 2046 and C 2332.
t This material was presented in part at the 48th Annual
Meeting of the American Association for Cancer Research,
Chicago, April 12-14, 1957.
ÃŽ
Present address: National Cancer Institute, Bethesda 14,
Maryland.
1The pyrazolo(3,4-d)pyrimidines were kindly supplied by
Dr. Roland K. Robins, Arizona State College, Tempe, Arizona.
was then collected by centrifugation, washed by
suspension in dilute ammonium hydroxide, and
finally recovered by centrifugation. Further re
moval of contaminants was achieved by solution
of the silver-pyrazolopyrimidine precipitate in
excess 0.3 N hydrochloric acid with gentle warm
ing. The silver chloride was removed by centrifu
gation. Following alkalinization with ammonia,
the pyrazolopyrimidines were reprecipitated as
silver salts, washed, and recovered as previously
described. The silver-pyrazolopyrimidines were
again decomposed with excess hydrochloric acid,
the silver chloride was removed, and the solution
of pyrazolopyrimidines was taken to dryness un
der a jet of dry air at room temperature. These
isolated urinary pyrazolopyrimidines were dis
solved in a small volume of water and applied as
spots to sheets of Whatman No. 1 filter paper for
descending Chromatographie separation by use of
the indicated solvent systems. Spots of chemically
synthesized pyrazolo(3,4-¿)pyrimidines were em
ployed as reference compounds (5). The positions
of the chromatographed compounds were located
by a 254-m/i ultraviolet light source. At this wave
length pyrazolopyrimidines either absorbed ultra
violet light or fluoresced.
Typical results of one such experiment are pre
sented in Table 1. It is evident that animals given
pyrazoloadenine excrete the following pyrazolo
pyrimidines: pyrazoloadenine; 4-amino-6-hydroxypyrazolo(3,4-d)pyrimidine (pyrazoloisoguanine) ;
4-hydroxypyrazolo(3,4-<i)pyrimidine (pyrazolohypoxanthine) ; 4,6-dihydroxypyrazolo(3,4-d)pyrimidine (pyrazoloxanthine). The pyrazoloxanthine
and pyrazoloisoguanine analogs are excited by ul
traviolet light and fluoresce, while the other com
pounds quench. There is an as yet unidentified
compound (compound X), which appears in the
urine of these rats, which does not appear in that
of control animals and which has the following
Ry's:0.27 in the Hanes-Isherwood system (3),
0.15 in the Price system,2 0.86 in a modified Wyatt
system (2). These experiments indicated that the
'K. K. Tsuboi and T. D. Price, unpublished data.
Received for publication September 26, 1957.
226
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FEIGELSONANDDAVIDSON—PyrazolopyrimidineMetabolism
rat possesses the ability to both oxidize and oxidatively deaminate administered pyrazoloadenine
and suggested the following as a possible metabolic
sequence :
-NH3
pyrazoloadenine
> pyrazolohypoxanthine
sults are shown in Table 2. These data provide
evidence that pyrazolohypoxanthine is and pyr
azoloisoguanine is not a precursor of pyrazoloxan
thine. The rat, therefore, cannot oxidatively de
aminate pyrazoloisoguanine but can oxidize pyr
azolohypoxanthine to pyrazoloxanthine. In all
cases some of the administered compound was
excreted unchanged in the urine. Both pyrazolo
adenine and pyrazolohypoxanthine seem to be
metabolic precursors of compound "X."
Pyrazolopyrimidine metabolism in the rat,
therefore, may be summarized as depicted in
Chart 1.
+IÕ2O
[0]
[0]
pyrazoloisoguanine
-NH3
227
1
»pyrazoloxanthine
+IÕ20
To ascertain whether the pyrazoloxanthine
arose from pyrazolohypoxanthine by oxidation or
from pyrazoloisoguanine by oxidative deamination, replicate adult rats were injected intraperitoneally with doses of 20 mg/kg of one of the fol
lowing compounds suspended in saline: pyrazolo
hypoxanthine, pyrazoloisoguanine, and pyrazolo
adenine. All urines were collected, and the pyrazolopyrimidines were isolated and chromatographically identified as previously described. The re
DISCUSSION
The xanthine oxidase-catalyzed in vitro oxida
tion of pyrazoloadenine to pyrazoloisoguanine has
been demonstrated to proceed in an analogous
fashion in the intact rat. Pyrazoloisoguanine is ap
parently not metabolized to pyrazoloxanthine.
Using an as yet unidentified enzyme system, the
rat oxidatively deaminates the pyrazoloadenine to
TABLE1
RF OF PYRAZOLO(3,4-d)PYRIMIDINES
AND URINARY PYRAZOLOPYRIMIDINE EXCRETION
FOLLOWING
ADMINISTRATION
OFPYRAZOLOADENINE
TOTHERAT
RF IN SOLVENT SYSTEMS
Urinary pyrazolopyrimidines
n-PROPANOL:NHiOH:HiO(S)
.69
n-Butanol : NH<HCOi :HiO
.51
ljopropanol:HCI (Z)
.70
.35
.09(F1)
.15
.59
.43 (Fl)*
.34 (Fl)
.27
.62(F1)
.56 (Fl)
.86
Reference compounds:
Pyrazoloadenine
.55
Pyrazolophypoxanthine
.56
.36
Pyrazoloxanthine
.48(F1)
.07(F1)
Pyrazoloisoguanine
.34(F1)
.07(F1)
* RF values followed by (Fl) indicate that these spots fluoresce under light of 254
other values represent spots which absorb light at this wave length.
.70
.65
.58(F1)
.55 (Fl)
wave length. All
TABLE 2
CHROMATOGRAPHIC
IDENTIFICATION
OFURINARY
CATABOLITES
FOLLOWING
THE
ADMINISTRATION
OFVARIOUS
PYRAZOLO(3,4-d)pYRiMiDiNES
TOTHERAT
RF IN SOLVENT SYSTEMS
Urinary pyrazolopyrimidines
from rats given:
Saline
Pyrazoloadenine
Pyrazolohypoxanthine
Pyrazoloisoguanine
Reference compounds:
Pyrazoloadenine
Pyrazolohypoxanthine
Pyrazoloxanthine
Pyrazoloisoguanine
n-Butanol : NH.HCOi :H.O
n-Propanol :NH4ÛH: H.O (3)
.07 (Fl)*; .13; .37 ;.56
.29 (Fl); .41 (Fl); .53; .63
.07(F1); .13; .35
.32; .41 (Fl); .53
.09(F1)
.30(F1)
.54
.36
.07(F1)
.07(F1)
.62
.51
.41 (Fl)
.27(F1)
UBINÕBT
CATABOLITO
None
Pyrazoloadenine; pyrazolohypo
xanthine; pyrazoloisoguanine;
pyrazoloxanthine; compound X
Pyrazolohypoxanthine; pyrazolo
xanthine; compound X
Pyrazoloisoguanine
* See footnote *, Table 1.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
228
Cancer Research
pyrazolohypoxanthine, which is then further me
tabolized to pyrazoloxanthine. This latter reaction
can be catalyzed by xanthine oxidase (4).3
It is of interest to note that xanthine oxidase
converts in vivo and in vitro the relatively weak in
hibitor of itself, pyrazoloadenine, with a K¡of
Vol. 18, February,
1958
underlie the sensitivity or resistance of various tis
sues and tumors towards these compounds remains
yet to be investigated.
SUMMARY
The metabolism of various pyrazolo(3,4-d)pyrimidines by the adult rat has been studied. As has
been previously shown with purified xanthine oxi
dase in vitro, pyrazoloadenine is oxidized in vivo to
pyrazoloisoguanine. The rat metabolizes pyrazolo(3,4-d)pyrimidines as follows:
-NH3
pyrazoloadenine
[0]
xanthine
oxidase
pyrazoloisoguanine
+H20
pyrazolohypoxanthine
[0]
xanthine
oxidase
pyrazoloxanthine
REFERENCES
Pyrazololsoguanine
Pyrazoloxanthine
CHART1.—The metabolism of pyrazolo(3,4-<i)pyrimidines
in the rat.
10~4 M, to pyrazoloisoguanine with a K¡of IO"6M
(2). This, then, is an example of an enzyme's
furthering its own inhibition. Whether this phe
nomenon is of pharmacological significance re
mains speculative. Whether variations in the catabolism of these drugs by the tissues concerned
8P. Feigebon, unpublished data.
1. FEIOELSON,P., and DAVIDSON,
3. D. Studies on the Inhibi
tion of Xanthine Oxidase by 8-Azaguanine and Pyrazolopyrimidines, Proc. Am. Âssoc. Cancer Research, 2:108,
1956.
2. FEIOELSON,P.; DAVIDSON,
3. D.; and ROBINS,R. K. Pyrazolopyrimidines as Inhibitors and Substrates of Xanthine
Oxidase. 5. Biol. Chem., 226:993-1000, 1957.
8. HANES,C. S., and ISHERWOOD,
F. A. Separation of Phos
phoric Esters on the Filter Paper Chromatogram. Nature,
164:1107-12, 1949.
4. LORZ,D. C., and HTTCHINGS,
G. H. Specificity of Xanthine
Oxidase. Abst. Am. Chem. Soc., 30c, 1956.
5. ROBINS,R. K. Potential Furine Antagonists. I. Synthesis of
Some 4,6-Substituted Pyrazolo(3,4-d)pyrimidines, J. Am.
Chem. Soc., 78:784-90, 1956.
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The Metabolism of Pyrazolo(3,4-d)pyrimidines by the Rat
Philip Feigelson and Jack D. Davidson
Cancer Res 1958;18:226-228.
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