Novrmher, 1963
ACETYLHYDRAZINE AS A
hfET.4BOLIC IKTERMEDIATE
1.727
ethanol filtrate was treated with 2,4-dinitrophenylhydrazineand a hydrazone was obtained. It was recrystallized from acetone and had m.p. 306-307" dec. Reported for benzyl methyl ketone 2,4-dinitrophenylhydrazone, m.p. 1560.17 (c) In Aqueous Buffered Solution.-A solution of 2.0 g. (0.012 mole) of amphetamine hydrochloride in 500 ml. of 0.0625 M potassium dihydrogen phosphate was adjusted to pH 7.0 by the addition of potassium hydroxide solution, 1.0 g. (0.0048 mole) of dehydroascorbir acid-methanol complex in 20 ml. of buffer was added, and the pH was again brought up to 7.0. The solution was then kept a t room temperature for 36 hr., during which time it turned dark amber. Then 25 ml. of concd. sulfuric acid was added and the solution was distilled into a saturated aqueous hydrochloric acid solution of 2,4-dinitrophenglhydrazine. No hydrazone dzrivative could be isolated. (17) S. M. McElvain, "The Characterization of Organic Compounds," The Macmillan Co., New York, N. T., 1953, p. 236.
Acetylhydrazine as an Intermediate i n the Metabolism of Aroylhydrazines',2 LESI'OXB. TURSBULL, A L L ~s. S Y.4RD, and HERBERT MCKENXIS, *JR. DppaTtmpnt of Phnmiacoloqy, Medzcal College of Virginia, Richmond, Vu.
Received May 3, 1969 The oral administration of benzhydrazide or 1-acetyl-2-benzoylhydrazine leads in the rabbit to the urinary excretion of 1,2-diacetylhydrazine. l-Acetyl-C14-2benzoylhydrazine, prepared from benzhydrazide and acetic anhydride-l-CI4, in the rat similarly led to the urinary excretion of 1,2-diacetylhydrazine, which had a specific activity of approximately SOYo of the administered compound. 1,2-Diacetylhydrazine-C14itself was converted only slightly (0.1-0.3%) to respiratory carbon dioxide. Acetylhydrazine in the dog was converted in part t o hydrazine. These data are consistent with previous experiments on isoniazid and its metabolite acetylisoniazid. They suggest that one of the routes for the metabolism of certain aroylhydrazines in man, the rabbit, and the rat is: aroylhydrazine -* 1-acetyl-2-aroylhydrazine -+ acetylhydrazine + 1,2-diacetylhydrazine.
Previous s t ~ d i e swere ~ , ~ designed t o test a hypothetical routes in which the metabolism of isonicotinic acid hydrazide leads t o the (1) Preeented in part a t the meeting of the American Society for Pharmacology and Experimental Therapeutics, April 14-18, 1958, Philadelphia, Penna. (2) .4ided by a research grant (RG-5337)from the National Institutes of Health, U. S. Public Health Service. (3) A. S.Yard and H. McKennis, Jr.. Federation P r o c . , 17, 421 (1958). (4) .4. S.Yard and €1. McKennis, Jr., J. N e d . Pharm. Chem., 6, 196 (1962). (5) H.McKennis, Jr., 4 . 8. Yard, J. H. Weatherby, and J. A. Hagy, J . Pharmacol. E r p l l . Therap.. 1116, 109 (1959).
1328
L. B. TURKBULL, A. S. YARD,A N D HERBERTM c K s u s ~ s,JR. ,
Ynl. 5
formation of 1,2-diacetylhydrazine by may of t,he intermediaks 1acetyl-2-isonicotinylhydrazine6 and acetylhydrazine. This led to thv demonstration that the anesthetized dog after administration of acetylisoniazid excreted acetylhydrazine in the urine. It was shown5 also in the dog, by means of the iodate reaction,7 that after t,he administration of acetylhydrazine or hydrazine under comparable conditions t'he urine was devoid or essentially devoid of I ,"diacet,ylhydrazine. Following a single oral dose of 1-acetyl-2-isonicotinylhydrazine to the human, l12-diacetylhydrazine was isolated from tht. urine. The foregoing and related experimental data support the vicn. t'hat acet,ylhydrazine participates in some species as an intermediatcl in t,he metabolism of isoniazid and tend to support the general q u a tions for the metabolism of aroylhydrazines: R,COSHSH2+- RC( )XHSHCOCH3 -+ CH&ONHNH, + CH3CONHNHCOCH3. The present study provides new data on the metabolism of benzhydrazide, acetylbenzhydrazide (l-acetyl-2-henzoylhydrazine), and acetylhydrazine in the dog, rat, and rabbit'. I t was of part,icular int'erest to study benzhydrazide as an analog of isoniazid since the metabolism of benzhydrazide is uncomplicat,ed by the possibilities of amine oxide and of quaternary ammonium compound formation. which may contribut'e significantly to the metabolism of isoniazid by may of methyl acceptance at the pyridine nitrogens or by way of phosphonucleotide f ~ r m a t ~ i o lo n . ~An , inquiry into aspects of the metabolism of benzhydrazide also assumed particular importance since El Masri, Smith, and Williams1' concluded from their studies in the rabbit t'hat the major route in the metabolism of benzhydrazide, p-chlorobenzhydrazide and p-methylbenzhydrazide could be represented by RCsH4CONHNH2 n'H2XH2 RC6H4COpH RCeH4COKHCH2C02H, where R = H, C1 or CH3. Following administ,rat,ion of benzhydrazide to the rabbit, these investigators found hgdrazine (isolat,ed by conversion to benzalazine) in the urine to the extent of 5% of t'he administered dose. In our present series with Sew Zealand White female rabbits, intraperitoneal administration of benzhydrazide (102 mg./kg.) g a w rise to urinary excret,ion of 1,2-diacylhydrazine as indicated by the -+
+
-+
(6) H. R. Hughes, ibid., 109, 444 (1953). (7) H. McRennis, J r . , J. H. Weatherby, and E. P. Dellis, Anal. Chem., 30, 499 (1958). (8) H. McKennis, Jr., A . R. Yard, and I?. V. Pahnelas, Am. Reo. Tubsrc. PuZmonarU Diseases, 7 5 , 966 (1956). (9) I,. .J. Zatinan, N. 0. Kaplan, S. P. Colowick, and h l . A i . Ciotti, .J. Am. Chem. SOC..7 5 , 3293 (1953). (IO) D. 5. Goldmnn, ibid., 7 6 , 2841 (1954). (11) A. 31. El lRIasri, J . N. Smith, a n d R. T. Wiliianis, R i o r h e m . J . , 6 8 , 5x7 (1SSSj.
Novcmhcr, 1902
ACETYLHYDRAZINE AS A METABOLIC INTERMEDIATE 1399
1830
r,. B. TURFBULL, A. S. YARD,
.INI) HERBERT MCKESXIS,?heme in which acetylhydrazine and 1-acetyl-2-aroylhydrnzines arc' intermediates in the metabolism of aroylhydrazines, 1-acetyl-C 14henzoylhydrazine was prepared by reaction of benzoylhydrazine and acetic. anhydride-l-C14 in pyridine. The radioactive l-acetyl-2henzoylhydrazine (total dose of 135 mg.) was given to three male albino rath. The pooled urine from these animals at 2 1 hours i t a < processed by methods similar t o those previously employed,j arid 1,2-diacet~-lhydrazine-C~~ with a specific activity caorreiponding t o 8156 of the parent compound was obtained. In the present btudies, it \\-as also shown (Table I) that C1) of the acetyl group in labeled 1-acetyl-2-benzoylhydrazine is metabolized to cli402.From this and previous studies it appears that the major metabolism of the labeled carbon of 1-ac~ety1-Ci~--2-benzo~~lhydrazinc t o C1?OZoccurs prior to the formation of 1,2-diacetyIhydrazine. Thc studies in which acetylhydrazine, but not 1,2-diacetylhydrazine, I! as +own6 to produce fatty livers in rabbits in a manner comparable t o hydrazine s e n e also to suggest a relative stability of 1,"diacetylhydrazine to hydrolysis in zivo. Such stability was again indicated in the present investigation by studies on the metabolism of I,?diacetylhydrazine-C" to C1402in the rat. 1,2-Diacetylhydrazine-C was conveniently synthesized by treating hydrazine with acetic anhydride-l-C1* in pyridine. The radioactivity of 1,2-diacetglhydrazine-C1* following oral administration to the rat was excreted in the urine to the extent of 85-907; during :i (12)
h S Yard and 1% XIcIiennia TI
J I'ha*rnncof LzptZ Thernp. 114, 3 9 1 (193.5).
Xovember, 1962
ACETYLHYDRAZINE AS A METABOLIC INTERMEDIATE
1331
48-hour period (Table 11). The combined recovery of radioactivity from urine, feces, and expired air averaged 95.0% of the dose. Only 0.6-1.3% of the administered radioactivity was found in the form of respiratory carbon dioxide. The excretion of radioactivity as carbon dioxide indicated that in the rat hydrolysis and resynthesis of 1,2diacetylhydrazine probably would not be sufficient to account for the observed metabolic dilution of the CI4 label in the sequence going from l-acetyl-2-benzoylhydrazine to 1,2-diacetylhydrazine. Of the two remaining metabolic steps which would make acetate available for oxidation to carbon dioxide, hydrolysis of acetylbenzhydrazide to benzhydrazide and acetate and hydrolysis of acetylhydrazine to hydrazine and acetate, only the latter was investigated in the dog, taking advantage of its inability to acetylate acetylhydrazine and excrete 1,2-diacetylhydrazine t h e r e f r ~ m . ~A male mongrel dog under pentobarbital anesthesia received an intravenous injection of acetylhydrazine fumarate. The combined urine at 2 hours after administration of the compound contained hydrazine which was isolated as benzalazine upon addition of benzaldehyde. All of the foregoing data are consistent with the concept that aroylhydrazines can be metabolized in certain species, viz., man, rat, and rabbit, b y way of a metabolic route involving the intermediates l-aroyl-2-acetylhydrazine, acetylhydrazine, and finally the relatively stable 1,2-diacetylhydrazine. The hydrolysis of acetylhydrazine, as indicated in experiments in the dog, suggests acetylhydrazine may be in part the immediate precursor of the hydrazine which has been observed by some investigators in studies on the metabolism of aroylhydrazines. Acknowledgments.-The authors wish to thank Mrs. Patricia Ladd and Mr. Robert Leavelle for technical assistance. Experimental Paper Chromatograms.-All paper chromatograms were prepared by the descending method a t ambient temperature using Whatman No. 1 paper. The solvent system was ammonia-ethanol-butanol5 and acidic p-dimethylaminobenzaldehyde was used5 to disclose the hydrazine compounds. Metabolism of I-Acetyl-2-benzoylhydrazineby the Rabbit.-The urine of 4 Ncw Zealand White female rabbits (1.25-1.70 kg.) that had received a total of 774 mg. of I-acetyl-2-benzoylhydrazine (133.6 mg./kg.) intraperitoneally was collected up t o 30 hr. after administration of the compound. After filtration, the pooled urine was passed through Dowex 50 ( H + ) and Dowex 3 (OH-) as previously described.5 The combined effluent and water wash was treated with decolorizing carbon and then concentrated almost to dryness under diminished pressure. ,4 solution of the residue in 20 ml. of ethanol was placed on six sheets of Whatman
So. 1 paper (18 l / 4 " X 22 l/2''j and chroinatogritphed. The areas correspondirig5 in /o dryness under diminished pressure. The rcsitlue was recrystallized from acetonc. 'I'hc:
November. 1962
ACETYLHYDRAZINE AS A METABOLIC INTERMEDIATE 1333
colorless crystals, m.p. 137.5-139', were collected and washed with cold acetone. The yield of dried product was 58.3 mg. (70%). Metabolism of C14-Hydrazides.-Msle albino Wistar-strain rats were housed in glass metabolism cages. All animals were allowed food and water ad Zzbitum. Respiratory carbon dioxide was collected in bubble towers containing aqueous potassium hydroxide (10% w./w.). Radioactivity of aliquote was determined on infinitely thick samples of barium carbonate following addition of barium chlcride. Feces were separated from urine by collection on a screen. Urine and feces were removed from the cages daily. Feces were dried overnight a t 100' and pulverized to a uniform powder for counting a t infinite thickness against standards prepared by adding l-acetyl-C14-2-benzoylhydrazine to control feces. Urine samples were dried on talc a t 100" and similarly counted a t infinite thiclrness. Tables I and I1 summarize the data which were obtained. Isolation of 1,2-Diacetylhydrazine and 1-Acetyl-2-benzoylhydrazine from Urine.-Urine samples were filtered through Celite and then concentrated to dryness under diminished pressure. The residue was triturated with absolute ethanol. The ethanolic solution was filtered and concentrated to dryness under diminished pressure. The residue was dissolved in water and then placed upon a column of Dowex 50 (H+). The effluent and water wash from the column were combined and placed upon a column of Dowex 21K (OH-). The latter was eluted with 1 J f acetic acid. The effluent and the acetic acid eluate were then combined and processed for metabolites. The combined urines from rats (no. 2 and no. 3 ) obtained during the first 24-hr. period after administration of radioactive acetylbenzhydrazide were concentrated to dryness. The ethanol-soluble components of the residue were dissolved in water and plared upon Dowex 21K (OH-). The column was eluted with 1 Af acetic acid. The residue from evaporation of the solvent (99';; of the radioactivity which had been placed on the column) waa triturated with methanol. The methanol-soluble Components were chromatographed on large sheets of Whatman S o . 1 paper. The radioactive area, corresponding in R, value to 1,2diacetylhydrazine, which was located by radioautographs, was cut from the sheets and extracted with methanol in a Soxhlet extractor. The residue (18 mg.) from evaporation of the solvent was diluted with 62 mg. of non-isotopic. 1,%diacetylhydrazine and dissoh ed in acetone-benzene. The solution upon rooling and evaporation depositrd 38.8 mg. of 1,2-diacetylhydrazine (3.47 X IO4 v p.m./mg. a t infinite thinness). The melting point was unchanged on further recrystallization and the specific activity (4.10 and 3.84 X lo4 c.p m./mg.) was constant within experimental error. The final product weighed 20.6 mg. The radioactive zone6 in the paper chromatograms which corresponded in Ki value to 1-acetyl-2-benzoylhydrazine was located and then extracted with methanol as described above. An aqueous solution of the solids obtained from evaporation of the methanol was processed on Dowex 21K (OH-) as described above. The acetic acid eluate was concentrated to dryness. Nun-isotopic carrier (62 mg ) was added. The mixture yielded upon repeated recrystallization from acctcinv 1-acetyl-2-benzoylhydrazine,m.p. 172-173", 36.8 mg., 3.06 x l o 4 c.p.m /mg at infinite thinness. Further recrystallization t o give 25.1 mg., 2.86 X lo4 c.p.ni. and finally 16.0 mg., 2.94 X IO4 c.p.m. a t infinite thinness, did not appreciably affect the specific activity. Specific Activity of Urinary 1,2-Diacetylhydrazine Arising from an Oral Dose of 1-A~etyl-C~~-2-benzoylhydrazine.-Three male albino rats (393, 395, and 462 g.)
were given oral doses of acetylbpiizhydrazide (45 nig., 5.43 x 107 c*.p.iii./innide a t infinite thinness). The pooled in-ine a t 21 hr. was neutralized with sodium hydroxide and, after filtration through Celit,e, placed upon ii column of T)ower 50 (H+). The effluent and water wash which cwntained 14.2 istered radioactivity were conwntr:tted t o d r p e s s under dim The residue was triturated with hot absoiutt. ethanol. .In aqueous solution of the solids obtained from evaporat.ion of the et,hanol was placed on Dowex 21X (OH-). The acetic acid eluate r a s evaporated to dryness and chromatographed on paper as in prrceding experiment>sto give radioactive zones corrrsponding in Rr value to diacetylhydrazine and acetylhenzhydrazide. The diacetylhydrazine zone was extracted with met'hanol. An aqueous solution of the solids from evaporation of the methanol was reprocessed on 1)ow?); 2lK (OH-). The residue from the acrtic arid eliiate uf the rolunin cwntained 12 mg. of impure tliacetylhydrazine. Trit>urationwith acet'one servrtl to rcniuw :I small amount of non-radioactive solids. By concc,ntrat,ion of the solvent, crvstals of diacetylhydrazine were obtained. -After twu re(.rystallizutioris the product. m.p. 138-139", weighed 2.0 m y . Aliquot,s in ethanol were platrd at infinite thinness (xi aluminuni planchrts. Thc trverage sperific. :ictivity for three sani mmole. The specific act.ivitj~ T virtually unchanged lizat,ioris t,o give a final prodii( 0.3M I n g . ~ 111.1). . 13% x 107 c.p.m./mniolc. Determination of Metabolic 1,2-Diacetylhydrazine by Isotopic Dilution with 1,2-Dia~etyl-C'~-hydrazine.-Tothe poolcd 24-hr. urine from 3 male albino rate (385,400, and 500 g.1 nhich had each rrreivrd orally 50 nig. of acetylbenzhvdrazidc in :3 ml. of water was added 7 mg. of c~iac.et,ylli;\.drnzine(4.5i x lofic.p.m./nig. I . .\ pure samplr of diac.etyllivdraziiic \VM isolated I,- tlie priic~eduresdesrrihcd * of the samplt~!m.p. 1 :38-13!1 :liter ri.crvstallizati(,ii abovo. The s l 1 ~ 3 i cart,ivity from acetone was 1.S.1 x 100 r.pm./iiig., represent,ing an m t i m of 17.2 inp. [ i f diacetylhydrazine rquivalent, on :I molar basis t o 13.2 of thc administcrrtl acety1benahydr:izi~e.
