The Conversion of Noncarcinogenic Aromatic Amides to ... · mide-a-'@C was cleaved in vivo from the arylhydroxamic acid, as shown by the isolation of N-benzoyl-a-â€šC-glycine

(CANCER RESEARCH 27 Part 1, 1443-1455, August 1967J

The Conversion of Noncarcinogenic Aromatic Amides to Carcinogenic

Aryihydroxamic Acids by Synthetic N-Hydroxylatio&

H. R. GUTMANN, S. B. GALITSKI,AND W. A. FOLEYLaboratory for Cancer Research, Veterans Administration Hospital, and the Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota .55417

hydroxylation to N-arylhydroxamic acids such as N-OH-AAF(7, 27), and that these hydroxamic acids are proximate agents in

carcinogenesis by aromatic amides (26, 28). This concept is basedlargely, though not entirely, on the observation that a hydroxamic acid, such as N-OH-AAF, induces a greater number oftumors in a susceptible species, such as the rat, than does thecorresponding aromatic amide. The difficulty inherent in a cornparison of the carcinogenicity of two compounds, both of whichgive rise to neoplasia, is that the size of the sample (i.e., thenumber of animals) which can be employed in such comparativeexperiments is usually not large enough for statistical evaluation.The differences in tumor incidence must therefore be striking ifdefinitive conclusions are to be drawn. In the present work adifferent approach has been taken to subject the concept of theessential role of N-hydroxylation in carcinogenesis by aromaticamides to a rigorous test. It seemed that the necessity of N-hydroxylation for the initiation of neoplasia could be demonstrated if it were possible to transform a noncarcinogenic aromatic amide into an active species by synthetic N-hydroxylation.Preliminary experiments indicated that the inactive pamidofluorenol, 7-OH-AAF, which is a major metabolite ofN-2-fluorenylacetamide in the rat (3), was converted into theactive carcinogen, N-OH-7-OH-AAF, by synthetic N-hydroxylation (12). This idea has been further explored by comparing thecarcinogenicity of BAF, an aromatic amide which is only mildly,if at all, carcinogenic (36), with that of the corresponding newarylhydroxamic acid, N-OH-BAF. It was anticipated that theuse of N-OH-BAF would answer the question whether N-2-

2 The Chemical Abstracts nomenclature for the N-arylhydroxa

mic acids and one related derivative is as follows: N-OH-AM', N-hydroxy-N-2-fluorenylacetamide; N-fluoren-2-yl-acetohydroxamic acid; N-OH-BAF, N-hydroxy -N -2- fluorenylbenzamide;N-fluoren-2-yl-benzohydroxamic acid; N-OH-7-OH-AAF, N-hydroxy-N-(7-hydroxy-2-fluorenyl)acetamide ; N-7-hydroxyfluoren2 - yl - acetohydroxamic acid; N - benzoylphenylhydroxylamine;N-phenylbenzohydroxamic acid; N-benzoyloxy-2-fluorenylbenzamide ; N-fluoren-2-yl-N-benzoyloxybenzamide. The nomericlature employed in the text for the other compounds correspondsto that of Chemical Abstracts. The following abbreviations are alsoused: 7-OH-AAF, N-(7-hydroxy-2-fluorenyl)acetamide; BAF,N-2-fluorenylbenzamide; 1-OH-BAF, N-(1-hydroxy-2-fluorenyl)benzamide; BA, benzanilide; N-OH-BA, N-benzoylphenylhydroxylamine; 3-OH-BAF, N-(3-hydroxy-2-fluorenyl)benzamide; N-OH-BAF-a-'4C, N-hydroxy-N-2-fluorenylbenzamidea-'4C; BAF-a-'4C, N-2-fluorenylbenzamide-a-'4C; MeOH, methanol; TLC, thin-layer chromatography.

SUMMARY

Two essentially noncarcinogenic aromatic amides, N-2-fiuorenylbenzamide and N-(7-hydroxy-2-fluorenyl)acetamide,were converted, by synthetic N-hydroxylation, to the highlycarcinogenic arythydroxamic acids, N-hydroxy-2-fluorenylbenzamide and N-hydroxy-(7-hydroxy-2-fluorenyl)acetamide,respectively. N-Hydroxy-2-fiuorenylbenzamide when administered intraperitoneally gave a 100% tumor incidence in thoseyoung female rats which were alive 8 weeks after the administration of the compound. The tumor incidence in male rats was 75%.The majority of the tumors (8/11) in female rats and all of thetumors (6/6) in male rats were rapidly growing and highly invasive intraperitoneal sarcomas. In addition, female rats developedmammary adenocarcinomas. N-Benzoylphenylhydroxylamine,an analog of N-hydroxy-2-fiuorenylbenzamide in which thefluorenyl moiety was replaced by the phenyl group, was notcarcinogenic. These results suggest that metabolic N-hydroxylation may be a necessary, but not sufficient, condition for carcinogenesis by aromatic amides.

Metabolic studies carried out in the female rat with the use ofN-hydroxy-2-fiuorenylbenzamide and of N-2-fluorenylbenzamide labeled with 14(Jin the a-carbon atom of the benzoyl groupindicated that the benzoyl group of N-hydroxy-2-fluorenylbenzamide-a-'@C was cleaved in vivo from the arylhydroxamic acid, asshown by the isolation of N-benzoyl-a-â€•C-glycine and of benzoica-'@C acid from the urine. The data imply that N-2-fluorenylhydroxylamine, potentially a proximate agent in carcinogenesisby N-2-fiuorenylacetamide and N-hydroxy-2-fiuorenylacetamide,is a product of the metabolism of N-hydroxy-2-fluorenylbenzamide. Examination of rat urine or bile after the intraperitonealadministration of N-2-fiuorenylbenzamide-a-'@C disclosed onlynegligible amounts of N-hydroxy-2-fluorenylbenzamide. Theseexperiments favor the view that the lack of carcinogenicity ofcertain aromatic amides for the rat may be explained by the inability of this species to N-hydroxylate the amide to the corresponding hydroxamic acid at a significant rate.

INTRODUCTION

Recent evidence indicates that carcinogenic aromatic amides,such as N-2-fiuorenylacetamide, are converted by metabolic N-

1 This investigation was supported by USPHS Research Grant

CA-02571 from the National Cancer Institute.Received January 12, 1967; accepted April 18, 1967.

1443AUGUST 1967

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H. R. Gutmann, S. B. Galitski, and W. A . Foley

fluorenylhydroxylamine, a metabolite of N-OH-AAF in vitro(10, 18, 19), is a proximate agent in carcinogenesis by arylhydroxamic acids derived from N-2-fiuorenylacetamide (10, 37). Sincethe arnide linkage in BAF is relatively stable to cleavage in vivoand in vitro (13, 30), it was expected that the bond which linksthe benzoyl grOUp to the nitrogen in N-OH-BAF would also beresistant to metabolic attack. If this were the case and if N-OHBAF were carcinogenic, N-2-fluorenylhydroxylamine wouldpresumably be eliminated as an obligatory intermediate forcarcinogenesis by N-OH-BAF and, by inference, by other arylhydroxamic acids as well.

MATERIALS AND METHODS

Preparation of Unlabeled Compounds

BAF, rn.p. 223â€”224Â°C,X@H 303 rnj.@(â‚¬,25,400) (1, 13);1-OH-BAF, m.p. 227_230cC (11),3 BA, m.p. 165Â°C(35); N-OHBA, ml). 122â€”123Â°C(2) ; 7-OH-AAF, m.p. 232â€”233Â°C(4);N-OH-AAF, rn.p. 149â€”151Â°C(26) ; and N-benzoylglycine, m.p.194Â°C, x@:@'@225 m@ (e, 11,300) (16), were prepared by thepublished procedures. Ethyl N-benzoylglycinate was synthesizedby a modification of the published method (15, 34). Benzoic acid,rn.p. 122Â°C, x@:@ 225 rnj@ (e, 10,900), was purchased fromMallinckrodt Chemical Works, St. Louis, Mo.

N-OH-BAF

Method A. To a solution of 2-nitrofluorene, m.p. 158â€”160Â°C(22), (2.5 gm, 10 mrnoles) in ethyl acetate (125 ml) were addedbenzoyl chloride (9.5 ml, 80 mmoles), 10% palladium on carboncatalyst (0.40 gm) (29), and triethylamine (0.20 ml). The mixturewas hydrogenated at room temperature and at atmosphericpressure until 27 rnmoles of hydrogen had been taken up. Afterseparation of the catalyst, ammonium hydroxide (80 ml, 50%,v/v) was added, and the mixture was heated under a refiux for 1hour with mechanical stirring. The organic phase was extracted10 times with 50 ml of 1 N sodium hydroxide and 8 times with 25ml of 2 N sodium hydroxide, and the combined alkaline extractswere acidified with concentrated hydrochloric acid. The precipitate was recrystallized from ethanol-water to give 1.1 gm ofcrude N-OH-BAF, rn.p. 160â€”165Â°C,36% yield. The material wasdissolved in the minimum amount of hot methanol, and to thissolution was added cupric acetate monohydrate (0.80 gm) inmethanol (120 ml). The yellow precipitate was collected afterstanding overnight, washed twice with ether and acetone, andsuspended in 95% ethanol (100 ml). Hydrogen suffide followedby nitrogen was passed through the suspension for 15 and 30minutes, respectively. The cupric sulfide was coagulated with0.5 N hydrochloric acid (2 ml) and separated by centrifugation.

3 These authors reported a m.p. of 234â€”236Â°C for N-(1-hydroxy

2-fluorenyl)benzamide. The purity of the present preparation,m.p. 227-230Â°C,was checked by thin-layer chromatography andby the elemental analysis. The compound migrated as a singlecomponent when it was chromatographed on Silica Gel GF254with ether:acetone (4:6) or petroleum-ether:acetone (7:3) assolvents. The RF values were 0.80 and 0.51, respectively.

Calculated for C20H15O2N: C, 79.71; H, 5.02; N, 4.65

Found:

The supernatant liquid was evaporated at reduced pressure, andthe residue was recrystallized twice from ethanol-water to giveN-OH-BAF, m.p. 173â€”175Â°C,X@@â€•303 m@z(e, 21,000) withshoulders at 295 and 283 mgi;@ 3,160 cm' (â€”OHâ€”) 1,610cm' (C =0) . Descending chromatography of the compound onWhatrnan paper No. 1 or 3MM with cyclohexane :t-butyl alcohol:acetic acid :water (18 :2 : 1 : 1) as the mobile phase gave a singlespot (RF 0.74 and 0.81, respectively) when the chromatogramwas sprayed with the Folin-Ciocalteu (9, 38) reagent. The cornpound was also detected as a fluorescence-quenching spot underultraviolet light (2537 A). N-OH-BAF when subjected to thinlayer chromatography (TLC) on Silica Gel GF254 with methanol:glacial acetic acid (96 :4) or with dioxane :water (9 : 1) as solventsgave a single, fluorescence-quenching spot (RF = 0.74 and 0.72,respectively).

Calculated for C20H,5NO2: C, 79.71; H, 5.02; N, 4.65

Found: C, 79.44; H, 4.88; N, 4.77

Method B. To a solution of N-2-fiuorenylhydroxylamine (33)(1.3 gm, 6.6 mmoles)in a pyridine:benzenemixture (30 ml:60ml) were added benzoyl chloride (1.2 ml, 10.4 mmoles) and a fewcrystals of hydroquinone. After the mixture had been stirred for1 hour, sufficient water was added to effect separation of thebenzene, which was washed with water, then extracted 4 timeswith 50 ml of 0.5 N sodium hydroxide. The extract was acidifiedwith concentrated hydrochloric acid, and the precipitate was recrystallized twice from ethanol-water to give 1.04 gm of N-OHBAF, m.p. 187â€”190Â°C(with decomposition), 50% yield. Theinfrared and ultraviolet spectra, as well as the chromatographicproperties of the compound, were identical with those of N-OHBAF prepared by Method A.4

N-Benzoyloxy-2-fiuorenylbenzamide. To N-OH-BAF (0.15gm, 0.50 mmole), prepared by the above Method B and dissolved in pyridine (5 ml), was added benzoylchloride (0.20 ml,1.7 mmoles) , and the solution was heated on a steam bath for1 hour. After it had stood at room temperature overnight, thesolution was poured into ice water and the mixture was extractedwith two 50-mi portions of ethyl acetate. The ethyl acetate waswashed with water, twice with 5% sodium bicarbonate, and againwith water. The ethyl acetate was then dried over anhydroussodium sulfate, and the solvent was evaporated. The residue wasrecrystallized from 95% ethanol to give 0. 11 gm of N-benzoyloxy2-fiuorenylbenzamide, m.p. 181â€”182.5Â°C,52% yield;@271 mj.@(e, 24,400) with a shoulder at 304 mjz; v@t@1730 cm@(Oâ€”C=O). TLC of the compound on Silica Gel GF@4 withchloroform :methanol (97 :3) as a solvent showed a single, fluorescence-quenching spot, RF = 0.74.

Calculated for C27H1903N: C, 79.98; H, 4.72; N, 3.46

Found: C, 79.70; H, 4.59; N, 3.49

4 The melting points of different preparations of N-OH-BAF

and of N-OH-7-OH-AAF varied by as much as 10-15Â°C.However,the ultraviolet and infrared spectra as well as the chromatographicproperties of the various products were the same as those of theanalytically pure samples. Because of the variability of the melting points the purity of individual preparations was judged by thereproducible spectral and chromatographic properties. TheN-OH-BAF employed in the carcinogenicity tests was preparedby Method B.C, 80.05; H, 5.15; N, 4.47

1444 CANCER RESEARCH VOL. 27



Conversion of Noncarcinogenic Aromatic Amides

3-OH-BAF. To a solution of 2-amino-3-fluorenol (obtainedfrom Aldrich Chemical Co., Milwaukee, Wis.) (0.10 gm, 0.5mmole) in pyridine (5 ml) was added benzoyl chloride (0.13ml, 1.0 mmole), and the solution was stirred at room temperaturefor 30 minutes. The mixture wa.s poured into water (30 ml), andthe precipitate was redissolved in ethyl acetate. Ammoniumhydroxide (20 ml, 50%, v/v) was added, and the mixture washeated under a refiux for 1 hour with stirring. The organic phasewas extracted twice with 0.5 N sodium hydroxide, and 0. 11 gm3-OH-BAF, m.p. 255â€”258Â°C,73% yield, was precipitated byacidifying the combined extracts with concentrated hydrochloricacid. The compound gave a single fluorescence-quenching spot onTLC (Silica Gel GFn4) with ether :acetone (7 :3) (RF = 0.80)or petroleum-ether :acetone (7 :3) (RF = 0.48) as solvents,x@:@327m@s(e,22,000);@ 1650cm' (C=O),1550cm@(â€”NH-â€”,amide II).

Calculated for C20H15N02: C, 79.71 ; H, 5.02; N, 4.65

Found:

of the compound on Silica Gel GF254 with methanol as a solventalso showed a single, fluorescence-quenching spot, RF = 0.60.

The copper chelate of N-OH-7-OH-AAF was prepared by dissolving 35 mg of N-OH-7-OH-AAF in the minimum amount ofmethanol and by adding dropwise a solution of cupric acetatemonohydrate (0.20 gm) in methanol (30 ml). The green l)recipitate was washed with ether, 95% ethanol, and ether to give 32mg of the chelate, m.p. > 300Â°C,78 % yield.

Calculated for C30H24O6N2Cu: C, 62.96; H, 4.29; N, 4.89

Found: C, 62.10; 11, 4.01; N, 4.59

Decomposition of the chelate with hydrogen sulfide asdescribed above and recrystallization of the resulting productfrom ethanol-water gave N-OH-7-OH-AAF, rn.p. 213â€”215Â°C.

Ethyl N-Benzoylglycinate. N-Benzoylglycine (0.50 grn,2.8 mmoles) was dissolved in hot 95% ethanol (10 ml). Concentrated sulfuric acid (0.15 ml, 3.2 mmoles) was added, and thesolution was heated under a reflux for 10 hours. The reactionmixture was diluted with cold distilled water (15 ml) , and theproduct which crystallized on standing overnight at 4Â°C wascollected and washed with water and 10% sodium bicarbonate.Ethyl N-benzoylglycinate, m.p. 59Â°C(15), was obtained iii 35%yield (0.20 gm) ;@ 225 mj.@ (e, 10,600) ;@ 3300cm@ (Nâ€”H), 1748 cm@ (Oâ€”C=O), 1641 cm' (C=O).

Preparation of Labeled Compounds. N-OH-BAF-a-'4Cwas prepared essentially according to Method B described abovefor the unlabeled material. N-2-Fluorenylhydroxylamine (33),(0.14 gm, 0.71 mmole), in a mixture (20 ml) of pyridine :benzene(1 :3) was treated with benzoyl-a-'4C chloride (obtained fromNuclear Research Chemicals, Inc., Orlando, Fla.) (0.75 mmole,specific radioactivity = 1.34 mc/mmole) to give 78 rng of crudeN-OH-BAF-a-14C, m.p. 187â€”190Â°C,37 % yield. The ultravioletspectrum of the labeled product exhibited the usual absorptionmaximum at 303 m@and, in addition, a peak at 250 m@due to anunknown contaminant. The compound was purified by dissolvingit in ethyl acetate and by stirring the solution with ammoniumhydroxide (50%, v/v) at room temperature for 30 minutes. Theethyl acetate was extracted with several portions of 0.5 N sodiumhydroxide and N-OH-BAF-a-'4C, m.p. 184â€”186Â°C(specific radioactivity = 2.63 x 10@dpm/mmole), was precipitated by acidifying the combined extracts with concentrated hydrochloric acid.The ultraviolet absorption spectrum of the purified labeled compound was identical with that of authentic N-OH-BAF. BAF-aâ€˜@Cwas prepared by benzoylating N-fluorenamine, m.p. 129â€”131Â°C(0.14 gm, 0.75 mmole), in pyridine (5 ml) with benzoyl-aâ€˜@Cchloride (obtained from Nuclear Research Chemicals, Inc.,Orlando, Fla.) (0.75 mmole, specific radioactivity = 1.34 mc/mmole). After heating on the steam bath for 15 minutes, thereaction mixture was poured into cold water (10 ml) and sufficientbenzene was added to form two phases. The benzene layer waswashed with 5% sodium bicarbonate and with several portions ofdistilled water. The saturation of the benzene with water precipitated BAF-a-14C, m.p. 225Â°C.The compound was combined withmaterial, m.p. 225Â°C,obtained by evaporating the benzene, andwas recrystallized from 95% ethanol. There was obtained 0.13gm of BAF-a-'4C, 63% yield, with a specific radioactivity of1.57 x 10@dpm/mmole. The ultraviolet absorption spectrum ofthe labeled compound was identical with that of authentic BAF.

Animals, Diets, and Maintenance of Animals. The rats usedin these studies were purchased from the Holtzman Co., Madison,

C, 80.00; H, 5.28; N, 4.79

N-OII-7-OH-AAF. 2-Hydroxy-7-nitrofluorene, m.p. 251â€”253Â°C (obtained from Aldrich Chemical Co., Milwaukee, Wis.),(0.91 gm, 4.0 mmoles) was dissolved in warm ethyl acetate (80 ml).After insoluble material had been removed by filtration, freshlydistilled acetic anhydride (4.0 ml, 42 mmoles) and triethylamine(0.4 ml) were added. The mixture was hydrogenated at roomtemperature and at atmospheric pressure with 10% palladium oncarbon (29) as a catalyst until the theoretical amount of hydrogenhad been taken up. After the catalyst had been separated, ammonium hydroxide (80 ml, 50%, v/v) was added to the filtrateand the mixture was heated under a reflux for 15 minutes withmechanical stirring. The organic solvent was evaporated and theresidue was triturated with a small amount (25 ml) of cold ethylacetate. The material insoluble in the ethyl acetate was recrystallized from ethanol-water (with charcoal) to give 72 mg of lighttan N-OH-7-OH-AAF, m.p. 213â€”215Â°C(with decomposition);x@s@ ethanol 294 m@z (e, 24,800) with a shoulder at 314 m@i;

3250 cm@ (0â€”H), 1620 cm' (C=O). Additional material(0.11 gm) with the same melting point and with the identicalinfrared spectrum was obtained by acidifying the ammoniumhydroxide solution with concentrated hydrochloric acid. In subsequent preparations the procedure was simplified by stirring thehydrogenated reaction mixture with an equal volume of ammonium hydroxide (50%, v/v) at room temperature for 1 hour.Under these conditions N-OH-7-OH-AAF was extracted quantitatively into the aqueous phase and was obtained in yields ranging from 25 to 33% by acidifying the aqueous phase with concentrated hydrochloric acid and by recrystallizing the precipitatefrom ethanol-water (with charcoal).

Calculated for C15H13O3N: C, 70.57; H, 5.13; N, 5.49

Found: C, 70.52; H, 5.28; N, 5.54

Descending chromatography of the compound on WhatmanNo. 1 paper with 8ec-butyl alcohol:3% ammonium hydroxide(38), or with cyclohexane :t-butyl alcohol: glacial acetic acid:water (18 :2: 1 : 1) as solvents gave a single spot (RF = 0.89 and0.21, respectively) which was detected by spraying the papereither with the Folin-Ciocalteu reagent (9, 38) or with 1%dimethylaminobenzaldehyde in 1 N hydrochloric acid (7). TLC

AUGUST 1967 1445



H. R. Gutmann, S. B. Galitski, and W. A. Foley

Wisconsin. The female rats used in the carcinogenicity tests hadinitial weights of 45â€”60grn, while the female rats employed in themetabolic experiments weighed from 75 to 100 gm. Since youngmale rats (50â€”60gm) were very susceptible to the toxic action ofN-OH-BAF and rarely survived 8 weeks, the male rats selectedfor the carcinogenicity tests weighed from 125 to 140 gm. In oneexperiment 3 female rats were maintained on the standard dietcontaining 0.038% BAF for 7 weeks prior to administration ofthe BAF-a-â€•C. The final weights of these rats ranged from 170 to185 gui. The female rats with fistulas of the bile duct weighed275 gm.

For the carcinogenicity tests the rats were caged individuallyand maintained on the semisynthetic 20% caseine diet usedroutinely in this laboratory (21) with the exception of the animalswhich received N-OH-AAF and N-OH-7-AAF. These rats werefed the grain diet employed by Miller et at. during tests of thecarcinogenicity of N-OH-AAF (26) in order to permit a directcomparison of the activity of N-OH-7-OH-AAF with thecarcinogenicity of N-OH-AAF. Food and water were allowedad libitum and the animals were weighted daily. After the ad

ministration of the compounds had been completed, the animalswere weighed once weekly. Autopsies were performed on allanimals immediately after death or at the termination of theexperiments. The tumors arid certain other tissues (lungs, liver,and kidneys) were fixed in buffered formalin and sectioned. Inmetabolic studies designed to examine the urine for the presenceof certain metabolites of N-OH-BAF-a-'4C and of BAF@a@HC,the animals were set up iii stainless steel metabolism cages whichpermitted the separate collection of urine and feces. The urinewas collected in containers packed in Dry Ice. At the end of thecollection period, the cages were rinsed with distilled water andthe wash liquid was added to the urine. The urine samples werestored at â€”15Â°C.The feces were dried in open containers overcalcium chloride prior to extraction as described below. Food andwater were allowed ad libitum.

In the experiments in which the bile was collected after theintrapentoneal administration of BAF-a-'4C, a polyethylenetubing (PE-50, o.d. = 0.023 inch) was inserted into the bile ductunder light ether anesthesia and under semisterile conditions(25). The rats were placed into restraining cages, and the bile wascollected in containers cooled in ice. Water and a solution of 5%sodium bicarbonate in saline were allowed immediately followingthe operation. Food was withheld for 24 hours and was allowedad libitzim thereafter. The diet was supplemented every 3rd day

by 3 drops each of Aquasol A (5000 TJ.S.P. units of vitamin A)and of Aquasol E (5 IU of vitamin E) (obtained from U. S.,Vitamin and Pharmaceutical Corp., Arling-Funk LaboratoriesDivision, New York, N.Y.).

Administration of Compounds. The compounds whichwere tested for their carcinogemcity were injected intraperitoneally 3 times weekly for 4 weeks at a level of 4.5 mg/100gm of body weight. The compounds were dispersed in 0.9%sodium chloride containing 1.75% gum acacia (1.0 mg of cornpound/0.1 ml of vehicle) by homogenizing in a Potter-Elvejhemtype glass homogenizer prior to each injection (26). The 14C-labeled compounds were dispersed by homogenization in 0.5 mlof the above vehicle and injected intraperitoneally through adisposable needle (gauge No. 23) . Appropriate corrections weremade for losses of radioactive material in the homogenizer, thesyringe, and the needle.

Radioactivity Measurements. All radioactivity measurements were made by liquid scintillation spectrometry in 22-milow-potassium vials. The radioactivity of the â€œC-labeled cornpounds was assayed in scintillator Solution B (10 ml) (20). Theethanol-soluble fecal radioactivity was determined in scintillatorSolution B on aliquots of a solution (25 ml) obtained by continuous extraction of the powdered feces with 95% ethanol for 72hours. The total radioactivity of the urine or of the bile wasdetermined on aliquots (usually 0.5 ml) in scintillator SolutionA (15 ml) (20). All samples were counted in duplicate with anerror of 5% or less and were corrected for quenching by thechannel ratio procedure.

Fractionation of Urine and Bile. Prior to the fractionationof the metabolites of N-OH-BAF-a-'4C and of BAF-a-â€•C according to the scheme shown in Chart 1, O-glucuronides and 0-sulfatesin the urine were cleaved by fl-glucuronidase and Taka-diastase(7, 17, 26, 33, 38). The urine was pooled, adjusted to pH 6 with

glacial acetic acid, and made up to 200â€”250ml with deionizedwater. Fifty-mi portions were placed into 125-mi Erlenmeyerflasks. To each flask were added sodium acetate (5 ml, 1 at),chloroform (0.75 ml), fl-glucuronidase5 (50 mg), and Takadiastase(obtainedfrom Parke,Davis andCo.,Ann Arbor,Mich.)(50 mg), and the open flasks were shaken in a water bath at 37Â°Cfor 18 hours. At the end of the incubation the contents of theflasks were combined and the total radioactivity of duplicatealiquots (usually 0.5 ml) was determined. The carrier compoundsdissolved in methanol were added with vigorous shaking, and themixture was acidified with concentrated hydrochloric acid. Theexpected metabolites of N-OH-BAF-a-'4C and of BAF-a-1@Cwere then separated by solvent extraction as shown in Chart 2.Thc cleavage of conjugates in the bile and the fractionation ofthe hydrolysate were carried out essentially in the same mannerexcept that the Taka-diastase was omitted and the hydrolysis ofO-glucuronides was carried out at nil 6.8. This pH was chosenbecause the 1)11optimum of bacterial f3-glucuronidase appears tobe near neutrality (23). The bile (approximately 50 ml adjustedto pH 6.8 with 20% acetic acid) was diluted to 200 ml with 0.1M phosphate buffer, pH 6.8, and the mixture was divided into 50-

ml portions. Chloroform (0.75 ml) and $-glucuronidase (50 mg)were added to each 50-mi portion.

Purification of Urinary Metabolites. BAF@a@HC, N-benzoyl-a-'@C-glycine, and benzoic-a-'kJ acid, obtained from theappropriate urinary fraction by solvent evaporation, were furtherpurified by TLC. The residue was dissolved in methanol (5â€”10ml), and aliquots (0.10â€”0.20ml) were chromatographed on SilicaGel GFn4 (0.25-mm thickness). The following solvents were used:(A) methanol, (B) methanol: water (9 : 1), (C) methanol: chioroform (9 : 1), (D) n-butyl alcohol :acetic acid :water (8 : 1 : 1), (E)benzene :dioxane :acetic acid (90 :25 :4), (F) methanol :petroleum-ether (9 : 1), (G) petroleum-ether :acetone (7 :3), (H) chloro

5 Two preparations of @-glucuronidase, Lot@ 125B-6430 (bac

terial @-glucuronidase, Type II) and Lot * 16B-6200 (bacterialfl-glucuronidase, Type I) were obtained from Sigma ChemicalCompany, St. Louis, Missouri. Determination of the specificactivities of these preparations at pH 6.8 and 37Â°Cwith phenolphthalein glucuronidate as substrate and in the presence of chloroform according to the directions of the manufacturer gave valuesof 96,000 Fishman units/gm and of 74,100 Fishman units/gm,respectively.





Urine or Bile1) incubated with $-glucuronidase and/or takadiastase at 37tand pH 6.1 (urine) or 6.8 (bile)2) total radioactivity measured3) carrier compounds (N-benzoylglycine, benzoic acid, BAF, N-OH-BAY) added4) acidified (pH 2-4) with concentrated hydrochloric acid

5) extractedwith EtOAc

EtOAcextractedwith 57.NaRCO3

4EtOAc(F-l)

contains(1) BAF, purifi@ by TLC(2) N-OH-BAF-O- C, purified

a)by paper chromatographyfollowedby conversionto Benzoyloxy-BAF

b)throughthe Cu chelatefollowedby conyÃ§rsionto BenzoyloxyBAF-a-@'@C

form:ethyl acetate :acetone (6 :3 : 1). N-Benzoylglycine had RFvalues of 0.50â€”0.64, 0.60â€”0.70, 0.46â€”0.59, and 0.59â€”0.61 inSolvents (A), (B), (C), and (D). Ethyl N-benzoylglycinate hadR@ values of 0.75-0.78 and 0.52â€”0.59in Solvents (C) and (E).The RF values for benzoic acid in Solvents (B), (C), (E), and(F) were 0.67â€”0.70, 0.63â€”0.70, 0.59â€”0.66, and 0.66â€”0.69. BAFgave RF values of 0.74-0.79, 0.51â€”0.53,and 0.68â€”0.70in Solvents(A), (G), and (H). After development of the chromatograms, thearea of the gel containing the compound was scraped from theplate and the compound was eluted with methanol (2 x 2.0 ml).The identity of the eluted compound was checked routinely bycomparing its ultraviolet absorption spectrum with that of anauthentic sample. The radioactivity of the solution (or of a suitable aliquot) was measured in scintillator Solution B. The specificradioactivities calculated from the radioactivity measurementsand from the ultraviolet absorbance at the wave length of maxima! extinction were expressed as dpm,mmole and are the averageof triplicate determinations. The isolated compounds were considered radiochernically pure when the specific radioactivityremained unchanged after rechromatography of the compound inat least one different solvent. The purity of N-benzoyl-a-'4C-glycine was also demonstrated by the single peaks in theradioactivity profile exhibited by the labeled amino acid and itsethyl ester upon TLC (Chart 2). The ester was obtained byheating F-2 (Chart 1) in absolute ethanol (5 ml) and concentrated sulfuric acid (0.07 ml) under a reflux for 10 hours. Ethyl N-benozyl-a-1'C-glycinate was isolated by dilution of the reactionmixture with water and by subsequent extraction with ethyl acetate. Prior to TLC the ethyl acetate was dried over anhydroussodium sulfate. The purity of N-benzoyl-a-'4C-glycine shown

Aqueous Phase,discarded

4EtOAc(F-2)

contains(1) benzoic-ft@4@ acid, purified by TLC(2) N-benzoyl-@- 4C-glycine, purified

by TLC and bj@conversion to EthylN-benzoyl-U- 4C-glycinate

above was confirmed by the virtual identity of the specific radioactivities of the two compounds (2.18 x 10@and 2.02 x 10@dpm/mmole).

TLC was unsatisfactory for the purification of N-OH-BAF-a14C, and the following method was adopted for the isolation of thelabeled hydroxamic acid. Fraction F-i was shaken with 0.5 Nsodium hydroxide which removed urinary or biliary pigmentswithout extracting appreciable amounts of N-OH-BAF. The ethylacetate was washed with water and evaporated, and the residue,dissolved in methanol, was streaked onto Whatman No. 3MMpaper ( 48 x 56 cm). The widthof theband was 1.0â€”1.5cm, and theamount of compound applied was limited to 3â€”4mg in order toavoid spreading of the band during chromatography. The chromatogram was developed by the descending technic with theupper phase of cyclohexane : t-butyl alcohol :acetic acid :water(18 :2 : 1 : 1). Prior to development the paper was equilibratedwith both phases of the solvent system for 12 to 18 hours. N-OHBAF-a-14C was detected by the brown color which the compoundassumed during chromatography and/or by the fluorescencequenching property of N-OH-BAF-a-'4C on exposure of thechromatogram to ultraviolet light. The band, RF = 0.81â€”0.83,was cut out, and the N-OH-BAF-a-'4C was eluted from the stripby descending chromatography with ethyl acetate. After solventevaporation the residue, dissolved in benzene (10 ml), was benzoylated with benzoyl chloride (0.05 ml) and pyridine (0.01 ml)by heating the mixture under a reflux for 3 hours. The reactionmixture was washed with water, 5 % sodium bicarbonate, andwater, and the benzene was evaporated. The residue, dissolved inacetone, was chromatographed on Silica Gel GFn4 (1-mm thickness) with chloroform :methanol (97 :3) as a solvent. The N-

NaHCO3acidified (pH 2) with conc. HC1

extracted with EtOAc

EtOAcextractedwith 0.5N NaOH

4Aqueous Phase,discarded

3O.SN NaOH

CHART 1. Scheme for the extraction of labeled metabolites from rat urine or bile after the intraperitoneal administration of N-hy

droxy-2-fluorenylbenzamide-a-14C or of N-2-fluorenylbenzamide-ci-'4C. BAF, N-2-fluorenylbenzamide; N-OH-BAF, N-hydroxy-2-fluorenylbenzam.ide; EtOAc, ethyl acetate; TLC, thin-layer chromatography.

AUGUST 1967 1447



H. R. Gulmann, S. B. Galitski, and W. A . Foley

aromatic amides, BAF and BA, were either very slightly active(BAF) or inactive (BA). The o-amidofluorenols, 1-OH-BAF and3-OH-BAF, as well as the extended p-amidofluorenol, 7-OHAAF, likewise displayed no carcinogenic activity. The o-amidofluorenols were tested because the o-hydroxyamines which mightarise from them metabolically have been considered to play a rolein the mechanism of action of carcinogenic aromatic amides (6).The lack of carcinogenicity of 7-OH-AAF which had been established previously by oral feeding (4, 36) was confirmed in thepresent experiments by another route of administration. Incontrast to the above compounds, the arylhydroxamic acidscorresponding to BAF and 7-OH-AAF were highly carcinogenic.Thus, N-OH-BAY gave a tumor incidence of 100% and of 75%in the female and male rat, respectively. Similarly, N-OH-7-OHAAF and N-OH-AAF induced neoplasms in 50% and 92% of therats, resl)ectively. The high tumor incidence in the young femalerat after the intraperitoneal administration of N-OH-AAF was insubstantial agreement with the incidence reported by Milleret al. (26) in adult male and female rats as well as in immature

female rats with this hydroxamic acid. The lack of carcinogenicityof N-benzoylphenylhydroxylamine, an arylhydroxamic acid inwhich the fluorene moiety of N-OH-BAF was replaced by thephenyl group, contrasted strikingly with the high carcinogenicactivity of N-OH-BAF. This observation suggested that the sizeof the aryl moiety is a major factor in determining the carcinogenicity of an arylhydroxamic acid. The majority of the tumors(8/1 1 in female rats and 6/6 in male rats) induced by N-OHBAF were fast-growing intraperitoneal sarcomas. The tumorssurrounded and compressed the hollow viscera and coated thesurface of the liver and spleen. The tumor mass and the bowelpresented a firm, irregular, partly cystic structure which wasadherent to the posterior peritoneal surface. Ascites was cornmonly present, and hydronephrosis due to ureteral obstructionwas observed in two animals (Fig. 1). Histologically, the tumorswere characterized by their pleomorphism. A dominant featurewas the presence of eosinophilic fiber bundles in an irregulararrangement. The nuclei were generally elongated with bluntedends. A few cells showed a vacuolated or â€œfoamyâ€•cytoplasm.Areas of tumor necrosis were common (Figs. 2, 3). There washistologic evidence of invasion of the intestinal mucosa, thediaphragm, the body wall, the myometrium, the lymph nodes,and the vascular spaces. In one instance, distant metastases tothe lungs were noted. Because of the variability of the histologicpattern seen, it was felt that these tumors were best classified aspoorly differentiated sarcomas. The gross and histologic appearance of the tumors was similar to that of the sarcomas obtainedby Miller et at. (26) in male and female adult rats after the intraperitoneal administration of N-OH-AAF. In addition to thesesarcomas, several female animals (3/8) developed adenocarcinomas of the breast. These tumors (3/11) were similar inhistologic appearance to the spontaneously occurring mammarytumors of the rat breast (5). The malignant potential of thesetumors has been reported, and the factors influencing theirincidence have been reviewed (31). One of the rats treated withBAF also had one mammary adenocarcinoma. However, none ofthe controls which were given the vehicle developed either intraperitoneal sarcomas or mammary adenocarcinomas. These datashowed clearly that the intraperitoneal sarcomas were attributable to the administration of N-OH-BAF. In addition, the corn

SW

280

2.40

200E0.

120

80

40

CHART 2. Radioactivity profiles of N-benzoyl-a-'4C-glycine in

Fraction F-2 of rat urine and of ethyl N-benzoyl-a-'4C-glycine derived from the ethyl N-benzoyl-a-'4C-glycinate. The urine of arat that had received N-hydroxy-2-fluorenylbenzamide-a-'4C (2.3mg; 1.76 X 10@dpm) intraperitoneally was fractionated accordingto the scheme of Chart 1. Aliquots of F-2 and of the ethyl acetatesolution, obtained by esterifyiiig F-2 as described in the text,were chromatographed on Silica Gel GF254 (0.25-mm thickness)with Solvents (A) and (E), respectively. Following thin-layerchromatography, the gel was scraped from the plate at 0.5-cm intervals and the radioactivity of the gel, suspended in scintillatorSolution B, was measured. The radioactivity of every 2nd sectionis plotted against the distance from the origin. The R, values ofthe labeled compounds were identical with those of authenticsamples run concurrently.

benzoyloxy-2-fluorenylbenzamide-a-'4C, RF = 0.7 1â€”0.73,wasextracted from the gel with acetone (3 x 50 ml), and the residueremaining after solvent evaporation was dissolved in methanol(10 ml). The ultraviolet absorption spectrum of an aliquot wassuperimposable on that of authentic N-benzoyloxy-2-fluorenylbenzamide. The specific radioactivity of the compound wascalculated from the radioactivity of the solution and from theextinction of N-benzoyloxy-2-fluorenylbenzamide at 271 [email protected] alternative method used in the isolation of N-OH-BAF-a-â€•Cfrom bile, the hydroxamic acid was separated as the chelate. Theresidue in Fraction F-i which remained after evaporation of theethyl acetate was dissolved in methanol (10 ml), and 3 ml of asolution of cupric acetate monohydrate (0.2 gm/30 ml methanol)were added. After standing at 0Â°Cfor 12 hours the chelate waswashed with cold ether (2 x 5 ml), suspended in 95% ethanol,and decomposed with hydrogen sulfide (8, 17, 33). Cupric sulfidewas removed by filtering the reaction mixture through a layer ofCelite. The residue remaining after solvent evaporation wastaken up in methanol and the radioactivity of the solution as wellas its ultraviolet absorption spectrum, which was indistinguishable from that of authentic N-OH-BAF, were determined. Thehydroxamic acid was then benzoylated, and the specific radioactivity of the resulting N-benzoyloxy-2-fluorenylbenzamide-a-â€˜SCwas obtained as described above.

RESULTS

Carcinogenicity Tests. The data on the carcinogenicity ofthe compounds investigated are summarized in Table 1. The

2. 4 (, 8 10 12 14 2 4 (, 8 10 12 14

Distanoe@ from Origin, cm

CANCER RESEARCH VOL. 271448




TABLE 1

The Carcinogenic'ities of Aromatic Amides and of the Corresponding Arylhydroxamic Acidsafter Intraperitoneal Injection to the Rat

Duration ofexperiments

(weeks)

20

343420

20

2420

2033343434353228

No. of rats survivingAverage

amount ofcompound

administered(mmole/rat)

0.200.310.150.210.160.200.370.330.240.230.23

%turnormci

dence@'

000080

100750000050

92

No. ofrats used

121210

1012101710

109

1010101212

At 8weeksAt

20weeksAt

nation ofexperi

ment1212121212999810981212121098800800998988101091010910109121212121110

Sex

FFFMFMFMFFFFFFF

Compound administered

None : Control 1None: Control 2None : Control 3None : Control 4BAP'

N-OH-BAF

1-OH-BAF3-OH-BAF

BAN-OH-BA

7-OH-AAFN-OH-7-OH-AAFN-OH-AAF

No. oftumorbearing

ratsÂ°

00001 11108 [hId6 [61000006 l10]@

11 [26]'

aThenumberinbracketsisthetotalnumberoftumors.b Calculated on the basis of the numbers of animals surviving 8 weeks.

C BAF; N-2-fluorenylbenzamide ; N-OH-BAF, N-hydroxy-N-2-fluorenylbenzamide ; 1-OH-BAF,

N-(1-hydroxy-2-fluorenyl)benzamide ; 3-OH-BAF, N-(3-hydroxy-2-fluorenyl)benzamide ; BA, benzanilide; N-OH-BA, N-benzoylphenylhydroxylamine; 7-OH-AAF, N-(7-hydroxy-2-fluorenyl)acetamide;N-OH-7-OH-AAF, N-hydroxy-N-(7-hydroxy-2-fluorenyl)acetamide ; N-OH-AAF, N-hydroxy-N-2-fluorenylacetamide.

d Three of the tumors were adenocarcinomas of the breast.

I All of the tumors were adenocarcinomas of the breast.

pound induced mammary adenocarcinomas at a comparativelylow frequency. In contrast, the administration of N-OH-7-OHAAF and of N-OH-AAF led exclusively to the formation ofmammary tumors. From a consideration of the tumor incidenceand of the amounts of compound required for tumor induction inthe present experiments, it would appear that N-OH-BAF andN-OH-AM' are about equally carcinogenic.

It seems noteworthy that, in agreement with the data alreadypublished (26), N-OH-AAF when injected intraperitoneally intothe young female rat yielded exclusively mammary tumors, whileN-OH-BAF at a comparable dose level produced tumors preferentially at or near the site of application. Although intraperitoneal sarcomas have been elicited in adult male and female ratsby the administration of N-OH-AAF (26), the quantities ofN-OH-AM' injected were about 4â€”5times greater than theamounts of N-OH-BAF administered in the present experiments.The available evidence suggests that N-OH-BAF, in contrast toN-OH-AAF, is primarily a locally acting carcinogen, and further

studies on this point are in progress.In addition to its carcinogenicity, N-OH-BAF exhibited

marked toxicity. Thus, 9/17 female rats died within 8 weeksafter the initiation of the experiments with no evidence of tumors(Table 1). Similarly, 12/12 young male rats with an averageinitial weight of 70 gm which received the compound subcutaneously in a separate series (4.5 mg/100 gui of body weight) 3times weekly for one month died within 4 weeks after completion

of the injections with no overt signs of tumors. In addition tothese acute toxic effects, N-OH-BAF had a profound, long-termeffect on the growth of the rat, as measured by the gain in bodyweight (Chart 3). The compound depressed the rate of growthafter the first injection, and the difference in weight gain from thecontrols persisted after completion of the injections and throughout the life-span of the rats. N-OH-AAF decreased the growth ofthe rat in a similar manner and to approximately the same extent.In contrast, N-OH-7-OH-AAF exerted a less pronounced effecton growth (15% depression at 3 months) than either N-OH-BAFor N-OH-AAF, and the noncarcinogenic N-OH-BA had no effect.These data suggested an inverse relation between the carcinogenic activity and the growth inhibition due to arylhydroxamicacids. Inhibitory effects of carcinogens on growth are well knownand have been recorded previously (14, 39, 40). It is not clear,however, whether carcinogenic activity and growth retardationare causally related or coincidental.

Metabolic Experiments. In order to rationalize the highcarcinogenicity of N-OH-BAF and the almost complete lack ofcarcinogenicity of BAF, the elimination of the two compounds inthe urine, feces, and bile as well as several specific metabolicreactions were studied by means of tracer technics with the use ofN-OH-BAF and of BAF labeled with â€˜4Cin the carbonyl carbonof the benzoyl group.

A comparison of the radioactivity recovered from the urineand the feces after the administration of the two radioactive

1449AUGUST 1967



20 40 (,O 80 100 120


zoo

160

120

80Eâ€˜@24OC

_; 0@ 200

.4-

_; 120

200

1 (,0

120

80

40

00

..

L

-. -I-.

-@ @E@ --

HoursCHART 4. The fraction of the administered radioactivity ex

creted in the urine after intraperitoneal administration of N-hydroxy-2-fiuorenylbenzamide-a-'@C (Experiment 1: 1.28 mg,1.11 X 10@dpm; Experiment 2: 2.30 mg, 1.76 X 10@dpm) and ofN-2-fluorenylbenzamide-a-'4C (Experiment 3: 4.64 mg, 2.55 X10@dpm; Experiment 4: 5.16 mg, 2.84 X 10@dpm) to the female rat.

Time,hours

CHART 5. The fraction of the administered radioactivity ex

creted in the bile after intraperitoneal administration of N-2-fluorenylbenzamide-a-14C (Experiment 1: 5.1 mg, 2.73 X 10@dpm;Experiment 2: 8.2 mg, 4.42 X 10@dpm) to the female rat.

the excreta were purified by TLC to constant specific radioactivity, the principal criterion of the radiochemical purity of theisolated products, as shown in Tables 2 and 3. The data obtainedin these experiments and summarized in Table 4 indicated that5% of N-OH-BAF-a-'@C was debenzoylated to yield N-benzoyl

a-â€•C-glycine and benzoic-a-â€•C acid, whereas only 1% of BAF-a14Cwas metabolized in this fashion. When the sum of the urinary

Time (da9s)

CHART 3. The average weight gain of female rats that received

the test compounds listed in Table 3. Each point represents theaverage weight gain of all rats alive at that time. Average weightgain = average weight at time t â€”average initial weight. BA,benzanilide; N-OH-BA, N-benzoylphenylhydroxylamine; BAF,N-2-fluoronylbenzamide; 1-OH-BAF, N-(1-hydroxy-2-fluorenyl)benzamide; 3-OH-BAF, N-(3-hydroxy-2-fluorenyl)benzamide; N-OH-BAF, N-hydroxy-N-2-fluorenylbenzamide; N-OH-AAF, N-hydroxy-N-2-fiuorenylacetamide; 7-OH-AAF, N-(7-hydroxy-2-fluorenyl)acetamide; N-OH-7-OH-AAF, N-hydroxy-N-(7-hydroxy2-fluorenyl)acetamide.

compounds revealed marked differences in the distribution of theexcreted â€˜IC.The major share (80â€”90%)of the total radioactivityexcreted after the intraperitoneal injection of N-OH-BAF-a-'4Cappeared in the urine within 48 hours (Table 4, Chart 4). Incontrast, only 30% of the total radioactivity excreted within 48hours after the administration of BAF-ce-'4C was recovered fromthe urine, and 70% was found in the ethanolic extract of the feces(Table 4, Chart 4). These patterns suggested the bile as a majorroute for the excretion of the radioactivity derived from BAF-aâ€˜IC,but not from N-OH-BAF-a-'4C. The elimination of BAF-aâ€˜@C(or metabolites thereof) by way of the bile was subsequentlysubstantiated in two experiments in which 23 and 11%(average = 17 %) of the administered radioactivity were recovered from the bile 96 hours after the injection of the labeledcompound (Chart 5).

The specific metabolic reactions studied by radioactive tracermethods in the female rat (debenzoylation of N-OH-BAF and ofBAF, reduction of N-OH-BAF and N-hydroxylation of BAF)are outlined in Chart 6. The carrier compounds isolated from

24.0

â€˜@).4-

4-

eI.Lj

>-â€˜3 12.0

@8.

â€˜@ 4.


1z 243648&072 849&



Ex .penm1@rit

@.0.g@mo1es

ofcom

poundadminis

tered5Compound

isolatedAmount ofcarrierused(smoles)Specific

radioactivity of isolated compound(dpm/@@mo1e)CMetabolite

excreted@(@imoIes)After

TLC-1

2.18 X 10@

1.62X l0@After

TLC-2

2.18 X 10@

1.47X 10@After

TLC-3

2.19 X 10@

5.70X 102In

theurine

0.348In

thefeces

0.002'16.70N-BenzoylglycineN-2-Fluorenylbenzamide412

10.024.22N-Benzoylglycine

Benzoic acidN-2-Fluorenylbenz

amide30.1

44 .217.61.50

X 10@2 .58 X [email protected] [email protected]

X 10@

1.97 X [email protected] [email protected]

X 10@

1.99 X [email protected] [email protected]

0.0350.026

w

0 0@__â€¢@s__@ 1411 _____ 1411

@ w c@115â€”C--oH+ (J@â€”@''j NH2

CHART 6. Metabolic reactions of N-hydroxy-N-2-fluorenyl

benzamide and of N-2-fluorenylbenzamide investigated by radioactivetracertechnics.

N-benzoyl-a-'4C glycine and benzoic-a-'4C acid was expressed interms of the total urinary â€œC,the ratio of these metabolites tothe total [email protected] was 1.6-fold greater after the administration of N-OH-BAF-a-'4C than after the injection of BAF-a-'4C.The difference in the deacylation of the two compounds becomeseven more striking when account is taken of the fact that themolar quantities of N-OH-BAF-a-'4C administered were onlyone-third of the molar amounts of BAF-ce-'4C injected. Since onlytrace amounts of BAF@a@UC,corresponding to 0.60 and 0.03%of the administered N-OH-BAF-a-'4C, were isolated from theurine and feces, respectively (Table 2), the reduction of N-OHBAF to BAF in vivo appeared to be negligible. The formation of

aN-OH-BAF-a-'4C,N-hydroxy-2-fluorenylbenzamide-a-'4C; TLC, thin-layerchromatography.b The specific activity of N-OH-BAF-a-â€•C was 2.62 X 10Â° dpm/Mmole.

C Each value is the average of triplicate determinations.

d gsmolesof metabolite excreted =specific radioactivity of isolated compound X @.@molescarrier added

specific radioactivity of administered compounda l)etermined after isolation of the carrier compound from an ethanolic extract of the feces as de

scribed under Materials and Methods.


TABLE 2

The Excretion of Metabolites in the Urine and Feces after Intraperiloneal Administrationof N-OH-J3AF-a-@C' to the Female Rat

N-benzoylglycine and of benzoic acid after the administration ofN-OH-BAF was therefore the result of the debenzoylation ofN-OH-BAF and did riot involve BAF as an intermediate. Itwould thus appear that the bond which links the acyl moiety tothe nitrogen in N-OH-BAF is metabolically not as stable as theamide linkage of BAF. The data imply that N-2-fluorenylhydroxylamine, a metabolite of N-OH-AAF (10, 19), is also a productof the metabolism of N-OH-BAF in the rat.

Examination of the urine of female rats failed to disclose thelresence of significant quantities of N-OH-BAF-a-'4C after singledoses or after repeated injections of BAF-a-'4C. In the latterexperiment, the inverse isotopic dilution @a.scarried out on thepooled urines of 3 rats which had been fed the standard diet contaming 0.039% BAF for 7 weeks. This dietary regime was instituted because the urinary excretion of N-OH-AAF by the ratreaches a maximum after the ingestion of N-2-fluorenylacetamidefor several weeks (7).

Because of a recent report that the bile contains as much as16â€”20% of a single dose of N-2-fluorenylacetamide as the 0-glucuronide cf N-OH-AAF (42), the elimination of N-OH-BAF-aâ€˜4Cin the bile after single doses of l3AF-a@4C was likewise investigated. However, only trace quantities of N-OH-BAF-a-'4C (0.06and 0.08% of the administered BAF-a-â€•C) were obtained fromthe bile in two experiments. These data support the view thatN-hydroxylation of BAF is not a major metabolic reaction in therat.

DISCUSSION

The conversion, by synthetic N-hydroxylation, of 2 essentiallynoncarcinogenic aromatic amides, 7-OH-AAF and BAF, to highlyactive arylhydroxamic acids gives strong support to the view,previously expressed by other workers in this field (10, 26, 28,37), that metabolic N-hydroxylation is a critical reaction in theimtiation of neol)lasia by aronratic arnides and that arylbydrox

AUGUST 1967 1451

Ic6h5Lc_NK_cH2._c_oh

I



.ExperimentNo.5moles

ofcompound

administaredCompound

isolatedAmountof

carrierused

(smoles)Specific

radioactivity of isolated compound(dpm/iimole)eMetabolite

excretedd(ismoles)After

TLC-1After TLC-2AfterTLC-31

2

316.3

18.1

80.0iV-Benzoylglycine

Benzoicacid

N-BenzoylglycineBenzoic acidN-Hydroxy-2-fluorenylbenz

amide

N-Hydroxy-2-fluorenylbenzamide24.5

33.4

11.014.86.95

16.69.90

X [email protected] 10@

7.90 X [email protected] X 10@

2.70 X 1021.03

X 10@

2.69X 10@

8.00 X [email protected] X [email protected]

X 10@

2.70X 10@

8.20 X [email protected] X [email protected]

0.058

0.0560.0290.0003'

0.0031

aBAF-a-'4C, N-2-fluorenylbenzamide-a-'4C;TLC, thin-layer chromatography.b The specific radioactivity of BAF-a-'4C was 1.57 X 106 dpm/@smole.

C Each value is the average of triplicate determinations.

d @smoles of metabolite excreted =

specific radioactivity of isolated compound X j@molescarrier added

specific radioactivity of administered compound6 In this experiment Fraction F-i (Chart 1) was chromatographed on paper as described under Mate

rials and Methods. The band containing the N-OH-BAF (RF = 0.82) was eluted, and the radioactivityof the eluate (3.95 X 10@dpm) was determined. The @molesof N-OH-BAF in the eluate were then calculated by dividing the radioactivity of the eluate by the specific radioactivity of the administered cornpound.

I Determined on the pooled urines of 3 rats prefed with 0.039% BAF in the diet for 7 weeks. Each rat

received 3.8 mg of BAF-a-14C in 2 injections, 24 hours apart, and the urine was collected for 96 hours.

ExperimentNo.Compound administerediimoles administered%

of administeredradioactivity excreted%

of administeredcompound metabolizedtoeIn

urineIn fecesN-Benzoyla@14C@g1ycineBenzoica-'@CacidN-OH-BAFa-â€•C1

23

45N-OH-BAF-a-'4C

N-OH-BAF-a-'4CBAF-a-'4C

BAF@a@i4CBAF-a-'4C4.22

6.7016.3

18.180.036.2â€•

25.1'10.2â€•

6.&'94f@d

2.14'@34d

13.7â€•4.1

5.21.0

0.310.840.35

0.16<0.005<0.005

(dpm/@imole carrier isolated X @@molecarrier added X 10@dpm administered


amic acids are proximate agents in carcinogenesis by the former as important for carcinogenesis as is N-hydroxylation. This viewcompounds. However, the lack of carcinogenicity of N-OH-BA is supported by the observation that N-hydroxy-1-fluorenylsuggests that N-hydroxylation alone is not a sufficient condition acetamide, a recently synthesized position isomer of N-hydroxyfor carcinogenesis. It would appear that the structural features of 2-fluorenylacetamide, was not carcinogenic when injected intrathe aromatic portion of the molecule (size and configuration) are peritoneally into the immature female rat at a level of 4.5 mgI

TABLE 3

The Excretion of Urinary Metabolites after Intraperitoneal Administration of BAF-a-â€•C'to the Female Rat

TABLE 4

The Fraction of the Administered Radioactivity Excreted and the Urinary Metabolites Isolated after theIntraperitoneal Administration of N-Hydroxy-@-fluorenylbenzamide-a-'4C (N-OH-BAF-a-â€•C)Â°

or of N-5-Fluorenylbenzamide-a-14C (BAF-a-â€•C)5

a Specific radioactivity = 2.63 X 10@dpm/pmole.SSpecific radioactivity = 1.57 X 10 dpm/,&mole.C Percent of administered compound metabolized =

d Collected for 96 hours.

e Collectedfor@51 hours.





100 gm of body weight 3 times weekly for 1 month (H. R. Gutmann and Y. Yost, unpublished experiments).

The debenzoylation of N-OH-BAF on the scale observed wasunexpected because of the resistance of the amide bond of BAFto metabolic attack referred to above. The appearance of urinaryN-benzoylglycine and of benzoic acid may be due to the hydrolytic cleavage of the benzoyl group from the hydroxamic acid.However, the instability of the bond which joins the benzoylgroup in N-OH-BAF to the remainder of the molecule and theresulting excretion of N-benzoylglycine and of benzoic acid mayalso be accounted for by the disproportionation of N-OH-BAF to2-nitrosofluorene and benzaldehyde. The aldehyde may, in turn,be oxidized to benzoic acid (41). Since N-hydroxy-N-phenylbenzenesulfonamide is decomposed quantitatively to nitrosobenzene and benzene sulfinic acid in alkaline media and at roomtemperature (32), the disproportionation of N-OH-BAF in a likemanner would seem to be a distinct possibility. Further studiesare needed to show which of the two mechanisms is operative inproducing N-benzoylglycine and benzoic acid from N-OH-BAFin vivo.

The failure of N-OH-BAF to undergo reduction to BAF in therat was likewise unexpected since the reduction of N-OH-AAF toN-2-fluorenylacetamide by this species is a major metabolicreaction in vivo and in vitro (24, 27). It is possible that N-OHBAF may not be a suitable substrate for the reductase, becausethe bulky benzoyl group may sterically hinder enzymatic attack.Further studies are desirable to settle the problem of whether therat (or tissues thereof) is capable of reducing N-OH-BAF to BAF.

The inability to detect N-OH-BAF-a-'4C in the urine or bileafter single or repeated doses of BAF-a-1@C argues for the viewthat the rat does not N-hydroxylate this noncarcinogenic aromatic amide. It should be noted that the experimental conditionsand the dietary regime employed in the present study were verysimilar to those which permitted the detection and isolation ofN-OH-AAF from rat or rabbit urine after the enzymatic hydrolysis of the 0-glucuronide (7, 17, 26, 27, 38). Since molecularmodels gave no evidence that the ether linkage of the 0-glucuronide of N-OH-BAF is sterically hindered (C. C. Irving, personalcommunication), there is noreasontoassumethat the @-glucuronidase preparation employed which was highly active towardphenolphthalein glucuronidate would have failed to hydrolyze

the 0-glucuronide of N-OH-BAF. On the basis of the availableevidence the argument appears persuasive that aromatic amides

which cannot be transformed by the rat to aryihydroxamic acids

by metabolic N-hydroxylation are also not carcinogenic for this

species.6Since N-2-fluorenylhydroxylamine is a probable metabolite of

N-OH-BAF, the present experiments provide no answer to the

question whether N-OH-BAF is carcinogenic per se or whether itsactivity is mediated through N-2-fiuorenylhydroxylamine or

through other as yet unknown metabolites. The solution of thisproblem depends on the availability of N-acyl- or N-aryl-N

6Attempts to detect N-OH-7-OH-AAF, by paper chromatography, in rat urine after single or repeated intraperitoneal injections of 7-OH-AAF or after oral ingestion of this compound for 9weeks were likewise unsuccessful (S. F. Chang and H. R. Gutmann, unpublished experiments).

fluorenylhydroxylamines which are not cleaved to N-2-fiuorenylhydroxylamine. Experiments along these lines are in progress.

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FIG. 1. Gross photograph of a partially dissected rat which had received N-hydroxy-N-2-fluorenylbenzamide by intraperitoneal in

jectiotis. Tumor niass encases intestine an I covers peritoneal surface laterally.Fi;. 2. Sarcoma from rat treated with .V-hvdroxy-.V-2-fluorenylhenzamide as described in the text. Small intestinal mucosa above.

Note fibrous appenratice of cells. X 100.F'IG. 3. Same tumor as show,i in Fig. 2. Note cvtoplasmic vacuolization, tumor giant cells, arid nuclear and cytoplasmic pleomor

phism. X 400.




Conversion of Noncorcinogenic Aromatic Amides

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1967;27:1443-1455. Cancer Res H. R. Gutmann, S. B. Galitski and W. A. Foley -Hydroxylation

NCarcinogenic Arylhydroxamic Acids by Synthetic The Conversion of Noncarcinogenic Aromatic Amides to

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The Conversion of Noncarcinogenic Aromatic Amides to ... · mide-a-'@C was cleaved in vivo from the arylhydroxamic acid, as shown by the isolation of N-benzoyl-a-â€šC-glycine