Journal o f Medicinal Chemistry J
@ Copyright 1971 b y the American Chemical Societu
VOLUME14, NUMBER9
SEPTEMBER 1971
Influence of Hydrophobic Character on the Relative Rate of Oxidation of Drugs by Rat Liver Microsomes YVONNEC. MARTIN* Chemica,l Pharmacology Department, Abbott Laboratories, North Chicago, Illinois 60064 AND
CORWINHANSCH
Department of Chemistry, Pomona College, Cluremont, California 91711 Received November 12, 19YO The relative Michaelis constant (Km)of a series of 14 drugs for the mixed-fufiction oxidases of rat liver microsomes increases with an increase in the octanol-water partition coefficient of the drug. For ionized drugs a correction for ionization improved the results. I n a series of dimethylanilines there was no correlation of K , with u or u+.
It was thus of interest to extend such studies t o include a wider variety of molecules and to expand the reactions studied to several of the oxidations catalyzed by the NADPH-dependeht, mixed-functioa oxidases of liver microsomes.
The relative rate of oxidative metabolism of drugs has long been considered to be related to the relative lipophilic character of the molecule. Although the correlation was not analyzed statistically, Gaudette and Brodie presented evidence that the relative rates of demethylation of a series of a variety of drugs was related to their CHCL-pH 7.4 buffer partition coefficients.' Maze1 and Henderson have reported similar studies with the opposite conclusion: in a series of 14 compounds there was no relationship between the extent of N-demethylation and CHCL-pH 7.4 buffer partition coefficient.2 I n neither of these studies was a distinction made between structure-activity relationships in substrate binding to the enzymes and in the maximal velocity. I n another study, Hansch and coworkers3 showed that the relative rates of demethylation of a series of aliphatic tertiary amines originally studied by McMahon was indeed related to the partition coefficient and that the data could be expressed by eq l.3 I n this
Experimental Section Enzyme Activity.-Adult male rats were pretreated with 100 mg/kg of phenobarbital ip daily for 5 days. After 18-hr starvation the liver microsomes were isolated' and stored frozen a t -20' until use. The rate of oxidation of the drugs by liver microsomes (0.5 mg of protein) was studied in pH 7.4 phosphate buffer a t 37" by following the rate of disappearance of 10-3 M NADPH spectrophotometrically.' This system was chosen because it is not necessary to add other commonly used cofactors such as nicotinamide or MgCll and because the system is relatively free from extraneous protein. The advantages of such a system are evident from the reports that nicotinamide6 and protein' can interfere with the reaction. Only those substrates for which the rate of NADPH consumption was double that of the endogenous rate were considered. The Km'sand Vmax'swere calcd from the data with a FORTRAN program which incorporates the statistical treatment of Wi1kinson.s Partition Coefficients.-The partition coefficients in Table I marked by an asterisk are experimental values. The others are calcd values. The derivs of PhNMep were calcd by adding r x from the aniline8 series to log P for PhSMez. Log P for N,Ndimethyl-@-naphthylamine was calcd by adding T ~ (of ~0.18 ~ from the benzene system to log P of naphthalene (3.37). Log P for codeine was calcd from log P (0.76) for morphine. To this was added: T = log Panreole- log P p h e n o l = 2.11 - 1.46 =
log BR = 0.470 log P - 0.268 (pKa - 9.5) - 1.305 n = 18 r = 0.890 s = 0.222
(1) equation n is the number of points used in the regression analysis, r is the multiple correlation coefficient, and s is the standard deviation. The biological response, BR, is the relative rate of i n vitro demethylation by rat microsomes, P is the octanolHzO partition coefficient, and Ka is the ionization constant. Both the partition coefficient and the pK, were statistically significant predictors of the relative rate of demethylation.
(4) L. Ernster, P. Siekevitz, and G. E . Palade, J. Cell. Biol., 16, 541 (1962). ( 5 ) Y.C.Martin, Biochem. Pharmacol., 16,2041 (1967). (6) J. B . Schenkman a n d R. W. Estabrook, Fed. Proc., Fed. Amer. Soc. E z p . Biol., 26, 729 (1967). (7) J. R. Fouts, Tozicol. A p p l . Pharmacol., 16, 48 (1970). ( 8 ) G.N . Wilklnson, Biochem. J.,80, 324 (1961). (9) T. Fujita, J. Iwasa, dnd C. Hansch, J. Amer. Chem. Soc., 86, 5175 (1964).
(1) L. E . Gaudette a n d B. B. Brodie, Biochem. Pharmacol., 2 , 89 (1959). (2) P.Maze1 and J. Henderson, rbid., 14, 92 (1965). (3) C.Hansch, A. R. Steward, and J. Iwasa, J. Med. Chem., 8,868 (1965).
777
~
)
~
778 Journul of
Medicinal Chemistry, 1071, Vol. 14, S o . 9
RIICH.IELIS COSSTIST A N D V,,,
FOR
XIARTIN ASD HAKSCH
T A B L EI V.LRIOUSSUBSTRATES OF KAT MICROSOMAL NADPH OXIDASE Frnax
h.0.
Compti
Log P
Log fraction not ionized
pK,
nmoles/ min per mg of protein
- Log K m , M Obsd Calcdd
7--
Log Vma,
-1)imethyl-p-iiapht hglamiiie 3 . 33 5.63 3,38 172 2.236 ta-Chloro-.\~,S-dimethylaiiiline 3.29 4.94 19 109 2.037 t,i-hIethyl-S,,\'-dime thylanilirie 2 81 4.73 4.84 220 2.342 p-~\lethgI-S,~~-dimethylaIiiliiie 2.80 4 70 4.84 177 2.230 1 Pentobarbital 2.07* 7.680.19 4 48 4 . 13 185 2,267 (i Hexobarbital 1.49* 7 9Oi1 0.13 4 22 3.76 311 2.493 7 .\',S-L)imet hglaiiiliiie 2.31* 4 19 4.47 195 2.290 S Codeine 1.41 7 .9,ib 0.66 3.36 3.31 376 2.575 9 p-Amiiio-S,S-dimethylariilirie 1 0s 3 87 3.56 151 2.179 10 nz-A~nirio-.\:,~\'-dimethylaniline 1.08 3 . Rt5 3.56 136 2.134 11 Ephedrine 0.93* 9 21c 1.81 1 97 2.11 569 2.755 12 Barbital O.G.i* 7.46O 0.-3 2 77 3.07 161 2.207 I3 Physostigmine 1 Oi* 6.12" 0 2 94 3 . 3 251 2.400 I4 Caffeine -0.07* 2.86 2.71 298 2.474 Me& Index, 8th ed, Paul G. Stecher, Ed., llerck arid Co., Rahway, X. J., 1968. fl AI. E. Krahl, ,f. P h y s . C h i . , 44, 449 (1940). 1,. G . Silleii aiid A . E. Martell, Cheni. Soc. Sptc. Publ., 17 (1964). d From eq 4, Table II. 1 2 3 4
c
Log 1' for PhCH2NlIel was calcd from log P (l.
