251
DEMETHYLATIOX STUDIES.
Xarch 1968
spot appeared light green in color. Thin layer chromatography was carried out on silica GF (hLerck A. G.) plates employing one of five solvent systems for development: (1) CsHs-EteXH (19: 1 v/v), ( 2 ) EtOAc, (3) EtOH-EtOAc (1: 1 v/v), (4) XeOHEtOBc ( 7 :3 v/v), and ( 5 ) C6H6-AcOH (Y: 1 v/v). Demethylation in Vitro.-Livers were removed from 300-g male rats that had been sacrificed by decapitation. The livers were immediately homogenized in 4 vol of 0.1 J I phosphate huffer (pH 7.4) in a Potter-Elvehjem homogenizer at 0" using a Teflon pestle. About 1.5 mill of grinding time was required to produce a preparation of maximum activity. The homogenate was then centrifuged at 15,000g for 30 miii and the supernatant fraction containing the soluble plus microsomal fraction was removed by decantation. Livers were removed fiom 300-g male rats pretreated with phenobarbital (40 mg/kg) daily for the 4 days immediately prior t o the day of sacrificing aiid processed to the 15,OOOg supernatant fraction as above. For the in vilro demethylation experiments a mixture of 1 In1 of siipernataiit fraction (200 mg of liver) toget>herwith 300 pmol of phosphate biiffer (pH 7.4), 0.3 pmol of T P N + , 11 pmol of glucose 6-phosphate, 43 pmol of semicarbazide, 30 pmol of l1gCl2, aiid 50 pmol of riicot.inamide in H,O (2 ml) was added t o 5 pmol of sribstrate. The cofactor concentrations q-ere chosen as those that gave maximum formaldehyde production from biityiiamine.12 The reaction mixtures were then incubated iii air with shaking at 37" for 1 hr. The reactions were terminated by the addition of 4 ml of 10% ZriCl, arid the formaldehyde formed was determiiied by the method of Cochin atid A ~ e 1 r o d . l ~ Inhibition experiments were carried out in a manner similar to that described above except, that various quantities of DPEA ranging from 5 X J I were added to the reaction mixture. The substrate employed in these experiments was N,N-dimethyl3,3,7-trimethyladamaiitanecarboxamide(11). Demethylation in Vivo.-The rates of in viz'o demethylation were determined by followiiig the rates of 14C02expiration after iiitraperitoiieal administratioil of the I4C-labeled dimethyl- arid acid. monomethylamides of 3,.i,i-trimethyladamantanecarboxyIic For these experiment*, 1.50-g male rats were [[sed at a dose of 40 mg/kg in polyethylene glycol solution. I n order t o determine the rate of expiratioii of 14C02,a radioreapirometer similar to that developed and described by Tulbert l 4 , I 5 was employed. In urtr iiistrrimerit the rat cage and the ionization chamber had a volume of .NO ml. X flow rate of 500 ml/min of air was employed. I n the iiihibit ion stitdies DPEA (2,4-dichIoro-6-phenylpheiioxyethylamine hydrochloride) was given intraperitoneally (50 pmol/kg) 10 min before admiiiistratioii of the amide. 111 the illduction st,udies, male rats (150 g ) were given a single dose of phenobarbital (40 mg/kg) daily for 4 days immediately prior to the admiiiistratioii of the amide. Urinary Excretion Studies.-The i n ciz'o fate of I1 and its Nmethy1-l4C analog (111) was studied in 200-g male Pardiie\!-istar rat,s. After dosing with 40 mg of radiolabeled amide/kg ip, the aiiimali were kept in staiiilesh steel metabolism cages. Urine samples were collected for 24 hr and the radioactive content was determiiied by liquid scintillatioIi coiuiting. 111 order to investigate t,he uature of the urinary metabolites, the 24-hr urine samples were first extracted with CHZCL, acidified t o pII 5.5 with AcOH, and incubated for 18 hr with 1.0 ml of Gliisiilase wliition (mixture of p-glucrironidase and sulfatase, Etido Prodrrcts, Inc.)/100 ml of urine. The liberated metabolites preheiit were theii extracted with CH2CI, and studied by chromatographic method>.
Resul ts As ail initial step in the investigation, a comparative study of the demethylation of I and 11 and their monomethyl analogs was carried out in the in vivo rat liver microsomal system. The demethylation of a typical lipid-soluble tertiary amide, d-propoxyphene, which is (12) R . 1,;. ~ 1 r A I a l i o na n d N . R . I,:art
