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And the Failure to Detect N-Acetylation of 2-Aminofluorene in the Dog*

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And the Failure to Detect N-Acetylation of 2-Aminofluorene in the Dog* The N- and Ring-Hydroxylation of 2-Acetylaminofluorene and the Failure To Detect N-Acetylation of 2-Aminofluorene in the Dog* LIONEL A. P0IRIER, JAMES A. MILLER, AND ELIZABETH C. MILLER (McArdle Memorial Latoratoryfor Cancer Research, Medical School, University of Wiscon.rin, Madison, Wisconthi) SUMMARY N-Hydroxy-2-acetylaminofluorene (N-hydroxy-AAF), in conjugated form, was identified as a urinary metabolite of 2-acetylaminofluorene (AM') in male mongrel dogs. This metabolite was isolated and characterized in crystalline form. 7-Hydnoxy AM', in conjugated form, and AM? were also found in the urine of dogs fed AAF; no 1-, 3-, or 5-hydroxy-AAF was detected. The ingestion of N-hydroxy-AAF led to the urinary excretion of the same metabolites; however, none of these acetylated metabo lites was detected in the urine of dogs fed 2-aminofluorene, N-hydroxy-2-aminofluorene, 1-hydroxy-AAF, or 3-hydroxy-AAF. Dietary supplementation with calcium pantoth enate and riboflavin and an attempt to induce acetylase activity by feeding 2-amino fluonene for several days did not lead to the urinary excretion of any recognizable acetylated urinary metabolites of 2-aminofluorene. Furthermore, under similar condi tions the specific activities of the acetylated urinary metabolites of 2-(acetyl-1'-C'4) aminofluonene fed in mixtures with unlabeled 2-aminofluorene were not appreciably different from the specific activity of the ingested acetyl-labeled AAF. In a dog fed a single dose of AAF-9-C'4 63 pen cent of the C'4 was excreted in the feces, and 19 per cent of the C'4 was found in the urine during the next 5 days. Approximately 3 per cent of an oral dose of AAF was found as 7-hydnoxy-AAF in the feces collected during the 1st day. No N-hydroxy amides were detected in the urine of dogs after ingestion of the fol lowing amides: acetanilide and its p-vinyl, p-fluoro, and p-ethoxy derivatives; trans 4-acetylaminostilbene; 2-propionylaminofluonene; and 2-n-butyrylaminofluorene. Ad ministration of 2-acetylaminonaphthalene to a dog led to the urinary excretion of very small amounts of this amide and its N-hydroxy metabolite. The synthesis of N-hydroxy-2-aminofluorene, a new compound, is described. The versatile carcinogenic activity of 2-acetyl 26) . Other aromatic amides and amines such as aminofluorene (AM') has been demonstrated by 2-aminonaphthalene (5, 16), 4,4'-diaminobiphenyl the variety of species and tissues in which this (benzidine) (30, 33'), 4-aminobiphenyl (11, 33) compound can induce malignant tumors (34) . In and 4-acetylaminobiphenyl (18) also exhibit car the dog AAF has been found to be a carcinogen cinogenic activity in the urinary bladder of the for the urinary bladder (26) and the liver (3, 18, dog. Likewise several of these carcinogens—viz., a This investigation was supported by a research training 2-aminonaphthalene, 4,4'-diaminobiphenyl, and grant, CRTY-5002, and by Grant C355 of the National Cancer 4-aminobiphenyl—pnoduce tumors of the urinary Institute, United States Public Health Service; by a grant bladder in humans following industrial exposure from the Jane Coffin Childs Memorial Fund for Medical Re (7, 14, 21, 32). Thus, studies on the metabolism of search; and by the Alexander and Margaret Stewart Trust Fund. We are grateful to Mrs. Wai Won Moy for valuable carcinogenic aromatic amines and amides in the technical assistance. 1 The article by Walpole et at. (33) contains further data on Received for publication January 21, 1963. the carcinogenicity of benzidine in the dogs of Spitz et at. (SO). 790 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. P0IRIER ci al.—Metabolism of AAF in the Dog 791 dog may provide clues to the mechanism by which butyrylaminofluorene (173°—175°),@trans4-acetyl these compounds induce bladder tumors in both aminostilbene (231°—232°)(4),5p-vinylacetanilide species.2 (140°—141°) (29), and p-fluoroacetanilide (151°— In the rat the carcinogenic action of AAF (22) 152°) (20). The following compounds were com and of 4-acetylaminobiphenyl (25) appears to pro mercial products: acetanilide (115°—116°)(Baker ceed through the N-hydroxylation of the parent and Adamson, N. F.), p-ethoxyacetanilide (134°— amide. The dog is known to N-hydroxylate 4-ace 136°) (Matheson, Coleman, and Bell), 2-acetyl tylaminobiphenyl (2.5), and both the dog (6, 31) aminonaphthalene (131°—132°)(Aldrich). and the human (31) N-hydroxylate 2-aminonaph N-Hydroxy-2-aminofluorene is a new com thalene. This report presents data on : (a) the pound and was prepared in this laboratony@ from N- and ning-hydroxylation of AAF and related 2-nitrofluorene by an adaptation of the general compounds in the dog and (b) our failure to dem procedure of Willstätter and Kubli (40). One gram onstrate the N-acetylation of 2-aminofluorene in of pure 2-nitrofluonene (m.p., 157°—158°C.)(Al this species. drich) was dissolved in 800 ml. of 95 per cent ethanol with heat. The solution was placed in a MATERIALS AND METHODS large ice bath and was saturated with NH3 at 10°— Compounds.—Except where noted the melting 15°C.; this required about 45 mm. The solution points (°C.) are uncorrected. The respective melt was then saturated with H2S. This step also re ing points and references to the syntheses of the quired about 45 mm. and was followed by the ap compounds prepared in this laboratory were as pearance of crystals of NH4SH. The solution was follows : AAF (192°—194°)(24),2-(acetyl-1'-C'4)- allowed to stand in the ice bath for 20 hours, by aminofluorene (192°—193°)(28),2-aminofluorene which time the ice had melted and the solution was (126°—127°) (19), N-hydroxy-AAF (145°—147°) nearly at room temperature. Two volumes of ethyl (22),@ 2-propionylaminofluorene (203°—205°),@2-n- ether were added, and the precipitate of NH4SH 2 Nomenclature in this paper (Chemical Abstracts nomen was removed by gravity filtration through coarse clature): paper. The filtrate was placed in a large pear 2-acetylaminofluorene (N-2-fluorenylacetamide) 2-propionylaminofluorene (N-2-fluorenylpropionamide) shaped sepanatony funnel fitted with an air-driven 2-n-butyrylaminofiuorene (N-2-fluorenyl-n-butyra stirrer and a tube for the introduction of N2. mide) Under air-free conditions the solution was extract 4-acetylaminobiphenyl (4'-phenylacetanilide) ed repeatedly with 50-ml. volumes of air-free 2-acetylaminaphthalene (N-2-naphthylacetamide) water (held in another sepanatory funnel under N2) 4-acetylaminostilbene (4'-styrylacetanilide) N-hydyoxy-2-acetylamino (N-hydroxy-N-2-fluorenyl until the ether-ethanol layer was only a very light fluorene acetamide) yellow in color and the aqueous extracts were x-hydroxy-2-acetylamino (N-(x-hydroxy)-2-fluorenyl colorless. Strict avoidance of 02 is important at fluorene acetamide) this point, since the dissolved hydroxylamine is 2-aminofluorene (2-fluorenamine) readily oxidized to azoxyfluorene. The ether-etha 4-aminobiphenyl (4-biphenylamine) 2-aminonaphthalene (2-naphthylamine) nol layer was then transferred anaerobically to a 4,4'-diaminobiphenyl (same) distillation apparatus and concentrated in vacuo p-x-acetanilide (4'-x-acetanilide) in a water bath at 40°C. When the volume of the 2-nitrofluorene (same) solution reached about 100 ml., the hydroxylamine 3 A modified procedure was employed in our more recent preparations of N-hydroxy-AAF. A solution of pure 2-nitro started to crystallize. The concentration was con fluorene (m.p., 157°—158°C.)(Aldrich) in ethyl acetate was tinued until the solution was filled with crystals. reductively acetylated as previously described (22). The hy The suspension of crystals was then quickly fil drolysis with ammonium hydroxide was carried out at room temperature for 4 hour with vigorous mixing of the two liquid tered in air through hard paper in a small suction phases. After removal of the ethyl acetate in vacuo the residue funnel. The crystals were washed with cold water, was dissolved in ethyl ether and extracted twice with dilute and the funnel and contents were dried in vacuo sodium hydroxide solution. The combined alkaline extract was extracted repeatedly with ethyl ether until the ether extracts over concentrated sulfuric acid in the dark. The were only slightly yellow.The alkaline extract was then acidi 4 The propionyl and n-butyryl derivatives of 2-aminofluo fled in an ice bath to pH 3. At this pH the precipitate of N- rene were prepared by the complete reductive acetylation of hydroxy-AAF was readily filtered by suction on paper sup 2-nitrofluorene with the corresponding anhydride in a manner ported by glass cloth. The precipitate was washed with water analogous to that employed in the preparation of N-hydroxy and dried in vacuoover sulfuric acid. At this stage many of AAF (22). These amides have been described previously the preparations have melted in the desired range (145°- (12, 27). 147°C.); however, other presumably identical preparations have required crystallization from benzene to attain this melt 5 This compound was generously supplied by Dr. R. A. An ing poinL All the products have given titrations of 98—100per dersen of this laboratory. cent theory upon reduction with titanium trichloride (15). 6 J@ A. Miller and G. S. Brenner. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. 7992 Cancer Research Vol. 923, June 1963 light yellow microcrystalline mass separated readi ded in about 75 gm. of ground horsemeat (K-9, ly from the paper, and it was relatively stable to K-9 Canning Co., Madison, Wis.). Such feedings oxygen as long as it was kept in the dank in the were repeated 3 and 6 hours after the initial inges refrigerator.
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