1139
SOTES
tetrazole (1) resists metabolic degradation by rat, dog, and man. When 14C-labeled 1 wa5 administered intravenously to rats approximately 65% of the radioactivity was recovered in urine arid approximately 207' in feces witliiii tlic first 24 hr (Table I). Subsequent excreta 'r.IBLE
1
.~I>MIiXISTK.\TIO.V O F " C - ~ - ( : ~ - P ~ R I D ~ ~ L ) T E T R . ~ Z o L E TO THE
It.IT
Species
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Itat, #1 $2 I ) o y pl
60 iv 200 iv 30 oi,al (gelutiii wphtde) 30 iv
$2
.ISD IJOG
% of adrnin radioactivity r e d , ---C-24 1ir after dose--Urine Feces
74 60 6s
18 24
87
contained a small or insignificant quantity of radioactivity. Papergrain chromatographic assays of urine or fecal extracts followed by radioactive strip scanning showed only one single radioactive peak with R f identical with the control 5-(3-pyridyl)tetrazole (1). Similar
as unchanged drug after administration t o rats.: Although the heterocyclic portion of 2 offers no convenient "handle" for conversion, the saturated ring would appear to be a suitable substrate for oxidation. Approximately 30% of cyclohexane administered to rabbits is recovered unchanged, but the major portioii, approximately BO%, is metabolized aia hydroxylatioii.fi Williams5 discusses the possibility that 2 is excreted a i a labile conjugate but reverts to 2 under the acid conditions of urine. E'ollowing the administration of 1 t o rats or dogs no attempt was made to alter the pH of urine, and aliquots of urine were subjected to paper chromatography shortly after collection. All papergrams after scanning showed a single peak of radioactivity with a mobility consistent with 1; no evidence for the presence of a r:diouctive conjugate \\;is detcctcd. Until additioiial data 011 the met ;ibolisni of tet r u o l c become available, it will riot be possible to generalize from the two examples discussed that the presence of a tetrazole group in a drug confers characteristic< rendering the entire molecule unsuitable for metabolic transformations. Experimental Section
"C
1
2
results were obtained following the oral and intravenous administration of 1 to dogs. To further establish the identity of recovered radioactivity a portion of dog urine was subjected to ion-exchange chromatography. The eluted radioactivity was diluted with unlabeled 1 and recrystallized to constant specific activity. Paper chromatography of the recrystallized material indicated homogeneity of label and mass. 111 human studies the administration of unlabeled 1 in doses ranging from 8 to 25 mg/kg, resulted in the 24-hr urinary recovery of 30-95yo of the administered dose a.: unchanged 1 . 2 The observed metabolic stability of 1 appears to be unique among 3-substituted pyridines. 1:ollowing the administration of I4C-3acetylpyridine to rats, 25% of the dose is recovered in urine as 3-acetyl-6-pyridone and 0.3% as S-methyl-3acetyl-6-pyridone. With a dose as high as 1000 mg/kg, no unchanged 3-acetylpyridine could be found in rat u r i ~ i e . ~The major portion of I4C-labeled nicotinic acid administered to the rat and dog is recovered methylated as the S-methyl quaternary compound, l-niethylnicotiiianiide.4 Thus, in the example of :+substituted pyridines, the presence of a tetrazole substituent prevents metabolic alterations of the pyridine moiety. 1,S-Pentamethylenetetrazole (2) is another tetrazole derivative known t o be metabolically stable. Approximately 75Y0 of the dose of 2 is recovered in the urine ( 2 ) 'I'ticse experiment3 wcrr conducted in rollahoration with Dr-. 8 . Uilgore and Y. Del'elice of oiir Clinical I'harmacology Depart,ment. ( 3 ) V. Neulioff and T. Harris, Arch. E x p t l . I'athol. Phurmokol., 2 4 9 , 11 (106-1). ( 4 ) It. T. \\'iliiartis, "l)ctosiratir,n Alrclianisin," 2nd ell, John \ \ i l c y ani1 Sun., Jnc., N c n \ ork, N. \.., 1930, 1, X I .
Synthesis of %-Labeled 5 4 3-Pyridy1)tetrazole ( 1 ). Nicotinonitrile-7-14C.-Nicotino~iitrile was synthesized from nicotinamide by the met,hod of Teague arid Short.' A mixture of nicotinamide7-14C, 21.2 mg ( 2 xncuries, New England Nuclear Corp.), and 1.198 g of unlabeled nicotinamide was used in the synthe 309 mg (49% of 3-cyanopyridine). 5-(3-Pyridyl)tetrazole (1 ).-To 309 mg of l4C-laheled 3-cyanopyridine was added 198 mg of unlabeled 3-cyariopyridirie, 592 mg of NaN3, 487 mg of KH4C1, 11.5 mg of LiC1, and 9.2 ml of dry DMF. The reaction mixture was stirred for 16 hr at 128125O, allowed to cool, and the precipitate was recovered by ceritrifugation. The crude product was recrystallized once from 30 ml of hot water to yield 465 mg (46Vc) of 1, specific activity 1.13 pcuries/mg, mp 235-236" dec, lit.1 238" dec. Charles River CU rats weighing approximately 180 g a i l t l mongrel dogs, approximately 10 kg, were \wed. The driig with administ'ered iiitravenously as an aqueous solutiuii iii the tail vein of rats or in the femoral artery of dogs. Radioactivity in feces was recovered by EtOA4cesll,ac.tioti of a water-fecal slurry acidified with 3 -V HC1 t o pH 3.4. ITriiie radioactivity was recovered by chromatography over a columil of Amberlite 111-120 resin which had been pi,evioiisly sliuried in 1 S HCl and washed (H&) until iieutral. Essentially all of the radioactivity applied t.o the column was recovered by elution with 1 S "?OH. The fractions contairiirig radioactivity were combined and lyophilized, cold 5-(3-pyridyl)tetraaole was added, and the mistnre was recrystallized to constant specific activity. When subjected to paper chromatography this material showed a single radioactive peak with Rf of 1. Paper chromatography was carried out in BuOH-diosarie",OH or BuOH-AcOH-H20. Papergrams were examined for radioactivity in an Atomic Accessories, Model KSC-18O continuous papergram strip scanner. All radioactive samples were measired in a Suclear Chicago Xodel 723 liquid scint illatiori spectrometer. Quenching corrections were made by the ititeriial standard procedure employing 14C-toluene. .411 samples wei'e assayed in a scintillator consisting of 0 . 3 2 2,.Fdipheiiylosazole (PPO) arid 0 . 0 1 p-bis[2-(5-phenyloxazolyl)) ~~ benzene (POPOP) in a mixture of 30% absolute ethanol and 70% toluene.
Acknowledgment.-We wish to thank Mrs. C. Fordham and Illr. P. Chernowsky for their capable :mist anw. ( 5 ) Reference 4, 617. ( f j j Iteferewu 1, p 11.;. ( i j 1'. C. 'l'e;lguc and \ \ . .\. bliurt, V7y. byii., 33, 5 2 (lYj3)
