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Accession number 208 r0f'?. CO"WOP L0VTcl'f.r ~=~-j L;.; ~~I.., GV*aCrTsbora COMPARISON OF OXOLAMINE CITRATE AND PMO METABOLISM: Theoretical Explanation of the Carcinogenicity of Oxolamine Citrate and Non-Carcinogenicity of PMO 'Submittied by: Dr. P. D~. Schickedantz Report number: Date: May 30, 1973 Summary or Abstract: From a search of the literature on 3-phenyl-5-(2-diethyl- aminoethyl)-1,2,4-oxadiazole (oxolamine) and related compounds, it has been concluded that the bladder carcinogenesis of oxolamine in rats is related to the diethylaminoethyl group and not the 3-phenyl-1,2,4-oxadiazoyl moiety. Diethylamine, a urinary metabolite of oxolamine, may possibly be nitrosated in vivo to N-nitrosodiethylamine, a potent carcinogen~. N- Hydroxylated aminoethyloxadiazoles and an epoxy-substituted oxadiazole are other possible sources of carcinogenicity which also are not structurally possible fromiPMO1. PDS:njw ~ . ~. Xc: Dr. H. J. Minnemeyer ~ Dr. R. S. Marmor ~ _ C+ Library - ~

COMPARISON OF OXOLAMINE CITRATE AND PMO METABOLISM: Theoretical Explanation of the Carcinogenicity of~ Oxolamine Citrate and Non-Carcinogenicity of PMO The effects of relatively large doses of oxolamine citrate, I, (50-1000mg/kg) have been variously described ranging from causing severe bladder irritation in mice, rats and dogs (Silvestrini, et. al., 1963) to being defi- nitely carcinogenic in the bladders of rats and dogs (Barron, 1963). 0 CH N J--CH2CHZN 2 5 "-C2H5 •CI T RATE I, Oxol'amine citrate = 3-phenyl-5-(2-diethylaminoethyl)- ' 1,2,4-oxadiazole, citrate salt Dogs whose ureters were surgically re-routed to avoid passing urine to the bladder did not develop the irritation when given oxolamine citrate (Silvestrini, et. al., 1963b). Hence, the inflammatory effects appear to be caused by urinary metabolites. Also, the inflammatory effect seems to be associated with the in vivo formation of diethylamine. Oxol- amine free base, as well as other 5-(2-aminoethyl)-1,2,4- O oxidiazoles, are cleaved exceptionally easily into secondary 0 c,D amines and 5-vinyloxadiazoles by mild heating in aqueous base CA (Catanese, et. al., 1963). However, of the compounds studied, V7 only those which also produced diethylamine, or other secondary amines, in the urine were bladder irritants. A separate study (Silvestrini, et. al., 1963a) showed that indeed

-2- diethylamine caused bladder irritation when administered intravenously in doses of 10 mg/kg, orally at 300 mg/kg or when solutions as dilute as 0.001 per cent were surgically instilled directly into the bladder. A number of other amines behaved similarly. Other toxicity studies have shown that diethylamine causes degenerative damage, including necrosis, in liver and kidney (Grice, et. al., 1971). Experimental evidence (Catanese, et. al., 1963) makes it seem unlikely that the bladder irritation is associated with other oxolamine metabolites. For example, 3-phenyl- 5-vinyl-1,2,4-oxadiazole, 3-phenyl-5-ethyl-1,2,4-oxadiazole, as well as,:.3-phenyl-5-methyl-1,2,4-oxadiazole (PMO) were not bladder irritants when administered orally to rats in doses of 1000 mg/kg. However, a suspected metabolite, 3-phenyl-5-(2-hydroxyethyl)-1,2,4-oxadiazole, (Silvestrini, et. al., 1964) has not been synthesized and hence has not been tested. Aside from the irritative effect of diethylamine, there is a strong possibility that the carcinogenic effect of oxolamine is due to diethylamine. Sen et. al. (1970) found that diethylnitrosamine, a well known carcinogen of liver, bladder and esophagus (Weisburger and Weisburger, 1966), is formed when diethylamine and sodium nitrite are incubated with gastric juices. This nitrosation was shown to occur in vivo in cats and rabbits. Wolf and Wasserman - (1972) point out that the possible hazardous formation of carcinogenic nitrosamines from dietary nitrites and

drugs containing secondary amine groups is still in need of more study. In order to rationalize the peculiar lability of the 5-(2-diethylaminoethyl)-1,2,4-oxadiazoles, the following are proposed as possible non-enzymatic pathways: (1) ~ elimination, reminiscent of the reverse Michael reaction (Cram and Hammond, 1959), equation 1. 0 I H J--CH2CH2N\CC25 H-~ (equation 1) R HCN2N\R (2) Elimiraation via an allyl carbonium which is stablized by resonance with the oxadiazole ring (R. Marmor, personal comm.)-equation 2. .eR HZCH2N\ R \ Hp --4 R + HN `R CH=CH2 H-C H2 ---- (equation 2)

Several metabolic pathways are available for degrada- tion of oxolamine which are not possible with PMO. Some of the possible intermediates contain functional groups, N-hydroxy and epoxide, which have been strongly implicated with carcinogenic activity (Miller and Miller, 1967; Badger, 1962; Daly, et. al., 1972). Silvestrini, et. al. (1964) believe that oxolamine metabolism involves elimination of diethylamine to form 3-phenyl-5-vinyl-1,2,4-oxadiazole which can then add water to form~3-phenyl-5-(2-hydroxyethyl)- 1,2,4-oxadiazole. The current theory would be that the alcohol is formed via the epoxide, equation 3. ~0 C2H5 -HN(CZHS)2 i N~\25 -)J&_CHZCH2NCH N 0 NiJ--CH=CH2. 0 NJ-CH CH2 --3 (equation 3) •

-5- N-hydroxylation may take place as follows, equation 4: J--CH2CH2N\ C2H5 enmymatic dealkylation N 5;-'I-CHZCHZN\ C2H5 (equ.ation 4) -0 , H N;~'-J-CH2CHZN\ H C2 5 . N 0 II pH N 0 ."C2H 5 N 0 N J-CH2CHZN 'o'0H ~H

6 Since PMO has no dialkylaminoethyl group, there is no possibility for formation of the same irritating,or carcin- ogenic intermediates such as were possible with oxolamine. PMO and'oxolamine have, of course, the 3-phenyl-1,2,4- oxadiazoyl group in common. The main argument implicating oxolamine as a carcinogen is its uniquely labile 2-diethyl- aminoethyl group. The lack of any carcinogenic effect attributable to PMO is confirmed by the results of the chronic toxicity studies in laboratory animals performed at Hazleton Labora- tories. These results are reported in Section~VII C of this report.

REFERENCES Badger, G. M., "The Chemical Basis of Carcinogenic Activity," C. C. Thomas, Publ., Springfield, Ill., 1962, p. 54. Barron, C. N., "Observations on the Chronic Toxicity of 3-Phenyl-5p-Diethylaminoethyl-1,2,4-Oxadiazole in the Rat and Dog," Exptl. Mol. Pathol. Suppl. 2, 1-27 (1963). Catanese, B., Palazzo, G., Pozzatti, C., and Silvestrini, B., "Toxicologic Studies on 1,2,4-Oxadiazole Derivatives: Relationship between Chemical Structure and Bladder Irritation," Exptl. Mol. Pathol. Suppl. 2, 28-40 (1963). Cram, D. J. and Hammond, G. S., "Organic Chemistry," McGraw Hill, New York, N. Y., 1959, p. 399. Daly, J. W., Jerina, D. M., Wilkop, B., "Arene Oxides and the NIH Shift: The Metabolism, Toxicity and Carcinogenicity of Aromatic Compounds,"' Experimentia, 28, 1129-1264 (1972). Grice, H. C., Barth, M. L., Cornish, H. H., Foster, G. V., and Gray, R. H.., "Correlation between Serum Enzymes, Isoenzyme Patterns and Histological Detectable Organ Damage," Food Cosmet. Toxicol. 9(6), 847-855 (1971). Miller, J. A. and Miller, E. C., "Activation of Carcinogenic Aromatic Amines and Amides by N-hydroxylation In~Vivo," in "Carcinogenisis: A Broad Critique," Williams and Wilkins, Baltimore, Md., 1967, pp. 397-420. Sen, L. P., Smith, D. C., and Schwinghamer, L. "Formation of'N-Nitrosamines from Secondary Amines and Nitrite in Human and!Animal Gastric Juice," Food Cosmet. Toxicol. 7(4), 301-7 (1969; C. A. 72, 1316f (197U': - Silvestrini, B., Catanese, B., Garau, A., and Pozzatti, C., °Toxicity Studies on 3-Phenyl-5)B-Diethylaminoethyl-1,2,4- Oxadiazole: Vesical Irritating Activity of Diethylamine and Other Simple Amines," Exptl. Mol. Pathol. Suppl. 2, ~ 41-49 (1963a). - © O Silvestrini, B., Bignami, A., Garau, A., and Pozzatti, C., ~ 'Toxicity Studies of 3-Phenyl-5)3-Diethylaminoethyl-1,2,4 Oxadiazole in Mice, Rats and'Dogs." Exptl. Mol. Pathol. ~ i 2 50-64 (1963b) Su . , pp . Silvestrini, B., Catanese, B., Corsi, G., and Ridolfi, P. *The Urinary Metabolites of 5-(2-Diethylaminoethyl)-3- Phenyl-1,2,4-Oxadiazole," J. Pharm. Pharmacol. 16, 38-42 (1964).

Wolff, I. A. and Wasserman, A. E., "Nitrates, Nitrites and!Nitrosamines,"' Science, 177, 15-19 (July 7, 1972). Weisberber, J. H., and Weisburger, E. K.,"Chemicals as Causes of Cancer,"Chem. Enq. News, 124-141 (Feb. 7, 1966).