DEMETHYLATION STUDIES. VI
1Iarch 1969
207
Demethylation Studies. VI. The Inhibition of Hepatic Rilicrosomal Oxygenation by 2,4-Dichloro(6-pheny1phenoxy)ethylamine'and Related Compounds ROBERT E. R I C k I A H O S , JACK ~ ~ L L ES~ ,L X I A NW. C U L P , W I L L I A M R. GIBSON, WARRENAI. MILLER,AND FREDERICK J. NARSHALL The Lilly Research Laboratories, Indianapolis, Indiana 46206 Received August 19, 1968
A series of o-phenylphenoxyethylamines has been studied as inhibitors of microsomal drug oxidation. Optimum activity resided in primary amines having an o-chloro substituent and unbranched ethylene chain. 2,4-Dichloro(6-pheny1phenoxy)ethylamine (1, DPEA) was shown to be a particularly effective inhibitor of the dealkylation of a variety of drugs. I n the intact rat, 1 inhibited the demethylation of butynamine and imipramine but not of propoxyphene or p-bromoanisole. Compound 1 prolonged hexobarbital and ethinamate sleeping time in mice and in dog, I he I,csiilt.iiig d N t a were ~ ~ ~ ( ~ rcont d e iiiuoiisly ti 011 a riii~difiecl r,(;cds ~ < J l t h ~~riiillipoin~ up st rip c:harl recorder. 'Tht,ev i t (wis WC'I'L' rcwiidcd, total CO, (respiratory rate), total I C , and bpccific activity. The total amouiit of 14C02expired was readillobtained by integration of the area under t,he 14C curve. While the CO, arid 14C curves are frequently irregidar due to changes of respiratory rate the specific activity was usually a smooth ciirvc'. The labeled substrates chosen for this study produced peak specsific activities about 15 rnin following intraperitoneal injection. The peak specific activity attained reflects the amount of enzyme ~ i ~ ~ s e i Fir i i . Iliat, r e : i ~ i i it lit. t i e i ~ ~ ~ ~i i :i i specific w act ivity h:il w i ~ citrcl i :I.< the iiieityiiw t ~ in f ciit~itihibi1ioii ('Fable \'I). . . ... -. ..-- (12) 195Iil.
U. AI. ' r u l l ~ e r l ,A I . l i i r k , i ~ i i d1:.
U a k i . r , .L,n. ,/, /'h.q,\iu/,, 185, 2(i!J
I N H r n I m n OF THE
Substrate
Propoxyphene But,ynamine p-(p-Chlorophenyl)dimethylpropylnmir~~ Aminopyriiie Ethylmorphiiie Imipramine Amitriptyline Acetylmethadol l>inmylmet~hglamine ?;,?l’-l>imethyldiphenylet hylami I I P 1-1Iethosy11,zpht~haleiir .kiiisoiiitde llriht ,xyhii I:tnioxmie
TABLE V XICROSOM.AL DEALKYLATION O F T-ARIUCS SUBSTRATES Rate, mwmul/ g min
Reaction
N-Demethylation N-Demethylation X-Demethylation N-Demethylatiori K-Demethy lation N-Demethylation N-Demethylation N-Demethylation N-Demethylation N-Demethylation 0-Demethglation 0-Ilemethylrttion 0-1)rmr t hyla t i o i i
manner (see above). Each liver was done separately and the results were expressed as nanomoles metabolized per milligram of microsomal protein during the 15-min incubation. Both the in vivo and the i n vitro results are summarized in Table VIII. Each result is the average of results from six rats.
1f7 122 68 137 338 102 118 34 $18 314 1.i 22 100
OF
,-
% inhibition by
-Compound 10-6 M
5 X 10-6 .If
44 96 91 63 66 62 37 66 87
13 81 70 83 7 3.i
13 35 41 41 11 44 1ti
79 49 f2 80
7
-Compound 7 10-6 . ‘tf 5 X lO-aM
8
10
84
93
72
81 64 .iB
48 3i 8 0
7 84 54 34 35 :Hi
18 0 17 94 74 62 56 80
TABLE VI INFLUENCE OF PREDOSING TIMEO N THE INHIBITION in J7iv0 BUTYNAMINE DEMETHYL~TIONQ BY COMPOUND 1 -of
Time of predosing, min
Results and Discussion
-1
R a t e of demethylation butynaminc+N-methyl-CIIndividual v a h e s Mean (pCi/mol of (rCi/mol COz)
42.0,62.5,72.5 59.0 7.4,9.5,12.7 9.9 8.5,12.7,16.5 12.6 10.6,12.1,15.9 12.9 5.3,8.8,9.9 8.0 16.5,41 .O, 73 . O 43.5 predosed with 15.9 mg/kg (50 fimol/kg) of 1 by
Control (noinhihitor)
I n the structure-activity studies three test substrates were used. They were butynamine, a hypotensive amine, meperidine, and 2-butylaminomethyl8-methoxy-1 ,&benzodioxane (methoxybutamoxane) The S-demethylation of butynamine5 was chosen as an example of a readily inhibited reaction while the x‘demethylation of meperidine represents a case in which inhibition is more difficult. Nethoxybutamoxane, which like its ethoxy analog’ is metabolized by 0dealkylation, was chosen as a typical 0-demethylation substrate. The first parameter to be investigated was the influence of ring substitution. The results are shown in Table I. The most obvious result is that the o-chloro substituent is necessary. Compounds 3 and 4, which are without the o-chloro group, are essentially without activity. The p-chloro substituent appears to have a small positive effect in the o-phenyl series but is without effect in the o-benzyl series. It is clear that the ophenyl group in 1 can be replaced by o-benzyl ( 5 ) without loss of activity. The effect of alterations in the nature of the carbon bridge connecting oxygen and nitrogen was next investigated (Table 11). Lengthening the ethylene chain by one more C resulted in some loss of activity as did branching on the chain. I n this regard the negative effect of a branching (10) was substantially greater than with /3 branching (9). The corresponding amide (11) was without activity. Table I11 summarized the data on the effect of substitution on S . The diethyl analog of 1 (13) was substantially less active than the primary amine. Substitution of one ethyl group (12) had less effect. The basis for the effect of S substitution is unknolvn. However, steric factors may be involved, since replacement of the diethylamino group (13) by the less hindered piperidino (14) or morpholino groups (15) had a smaller negative effect. I n virw of diffieultics in thc preparation of analogs of 1 i n \rhich t h c osygcri :itom hnrl bcmi rrplnctd hy
.
10
100 250 500
1000 Rats were the intraperitoneal route. Bt the specified postdose times butynamine-N-methy1-l4C was administered intraperitoneally a t a dose of 14 mg/kg ( f 5 pmol/kg). The radiocarbon dose wm 1.36 pCi/l5O-g rat. * The specific activity of respired 14C02at, 15 min after administration of radiobutynamine.
other atoms, this question was investigated with the less active /3-naphthyloxyethylamine series. The results (Table IV) show that the substituion of S for 0 leads to a substantial increase in activity. Replacement by CH2 had no effect while replacement by ?; essentially destroyed the activity. Based on these studies, 1, was chosen for further studies. I n Table V its in vitro activity against a variety of substrates compared with that of 7 is presented. The degree of inhibition produced by 1 and by 7 against various substrates proved to be very similar with the notable exception of imipramine and acetylmethadol, both of which were much more readily inhibited by 1 than by 7. This similarity of response of 1 and 7 to various substrates suggests that they act by the same mechanism. However, Schenkman, et aZ.,13 have studied the spectral changes resulting from interaction of these inhibitors with hepatic microsomal cytochrome and found that 7 gives a type I interaction while 1 gives a type I1 change. Thuq there appears to be a difference in the way these two compounds interact with microsomes (cf. also ref 2b). The inhibitory activity of 1 in in vivo systems was next investigated. I n earlier studies 1 was shown t o be effective :IS an inhibitor of the in i:iiio demethylntion
of the hypotensive amine, butynamine, in rats a t doses as low as 2 mg/kg.5 I n the present study a higher dose (15.9 mg/kg, 50 ,umol/kg) was chosen in order to estimate the duration of action of 1 in rats. The results are shown in Table TI. I'nder these conditions 1 produced a dramatic reduction in the rate of demethylation of butyriamine that persisted for at least 500 min. ,4t 1000 min demethylation rates appeared to have returned to normal. Inhibition of the i n viuo demethylation of other suh
