Journal ofMedicina1 Chemistry @ Cop~,rwhl 1965 b y the American Chemical Society
YOLUME6, XCMBER 4
JULY9, 1963
Demethylation Studies. IV. The in citro and in civo Cleavage of Alkyl- and Arylalkyl-p-nitrophenyl Ethers ROBERT E. ALC~\IAHON, HILKLVW. CULP,JACK 1 1 1 ~ ~ASD 5 , FREDERICK ,J. N.UISHALL Lzlly Research Laborcdories, Indzalmpolzs 6 , Indiana
Received January
4,1963
Tlie oxidative 0-dealkylation of a series of alkyl and arylalkyl p-nitroplien;\-l ethers by rat liver microsomes has been studied. For saturated alkyl groups t,he rat,e tends t,o decrease u-ith increasing bulk of t,he group. Certain electron donating groups such as ethylene, cyano, and phenyl when at,tached to the reactive methylene increase the rate of reaction. Ring subst,itution of any type in the benzyl group of p-nitrophenyl benzyl ether tends to reduce the rate. This is thought t o be simply a steric effect. I n c Z ' L . ~ dealkylation rates correlated well with i n citro results.
Hiiggiiii, .Jeiiscii, aiid Clei.elaiidl studied the zn &o iuetaholism of p-nitroanisole aiid p-iiitropheiietole in the rat aiid foliiid them to be extensively cleaved to p-iiiti*opIiciiol. 17sing.tissue hoinogeiiates, they found that both I i i er aiid kidiiey could rarry out this cleavage. In a study of the zn rtztro coiiversioii of rodeiiie t o inorpliiiic i u e l i m l ? f o w d the enzymatic activity responsible resided i i i IIVCI. iiii(8rosoines and required SXDI'H aiid oxygeii. rl'lie methyl group appeared as formaldehyde. Thus the reactioii was shown to he oiie of the sevrral oxidatii c imctioiis that are catalyzed h y mammaliaii I i L er micarosomal eiizyinei aiid which possess a reqiiireinciit for SA4DI'H aiid .ixelrod4 also studied zn 1 z t ~ o0-dealkylation of a variety of riiig suhstituted aiiiwl~saiid plieiietoles by the mi(-rosomal systeni. Ailtliou~li the c.lea\.age of a wide \.ariety of arylalkyl ctheis ha\-e heeii reported,j iioiie have beeii in\ estigated i l l \\-hicsli the alkyl group was other thaii methyl or ctliyl. In the present study the aryl group was held cnoiistaiit (p-iiitioplieiiyl) and tlie iiatm.c>of tlie alkyl grotip 1-ai.ied o i w a wide range.
Experimental Materials.-Substituted p-nitrophenyl cthers needed for this study 1vei-e prepared by st,andard organic synthesis procedures. Paper chromatography was used t o ehox t h a t each ether \vas free of p-nitrophenol. The preparation of C" labeled p-nitrophenyl benzyl ether will serve to illustrate tlie method used. A mixture of 330 pmoles of sodium p-nitrophenoxide and 200 pnioles of benayl-l-Cl4 chloride in 3 ml. of absolute ethanol was refluxed 16 hr. and then evaporated t o dryness. The product (1) C. Huggins, E. F'. Jensen, a n d A . S. Cleveland, I'roc. So?. h'xp. Bioi. .Ired., 68, 477 (1948). ( 2 ) J . Axelrod, J . Pham"ol. Ezptl. Therap., 115, 239 (19.55). (3) B. Brodie, J. .kxelrod, J. R. Cooper, L. Gaudette, B. N. L a U u , C. .\Iitoma, a n d S. Udenfriend, Science, 121, 603 (1955). ( 4 ) J. Axelrod, Biochem. J . , 6 3 , fi34 (19%). ( 5 ) R. T. TYilliarns, "Detoxication AIeciianisnis," Jolin !Gley a n d Sons, h'ew I'ork, N. Y., 1959. 1). 321.
was taken up in etlier and unreacted phenol renioved by estrsction with alkali. The ether solution was reduced to dryness m d the product recrystallized from metli\-lryc.lohexane. The prnduct weighed 20 nig. (43':t) and melted at 103-105" (reporteds 106'). Paper chromatography was used t o demonstrate that no unreacted p-nitrophenol or benzyl-1-C" chloride remained in t h e product. Methods. Microsomee.-The livers of iiornwl m:de rats were q u i i ~ k removed. l~~ folio .[-in de ,:.p:taL'cin. nnd p':t(.ed in ice ro!d 0.1 .Ii phoq)hnte bu"er. pH 7.4. ?he livers were weighed, minced, and put i n t o a (*old Potter-E1veh:eni homogenizer. Phosphate buffer ivas added in the rat,io of 1:I and the livers were homogenized with :I Teflon pestle for 1-1.5 min. The pooled honiogenates ',rere caentrifuged a t 20,000/ for 30 niin. in :t Spinco LIiidel I, rentiifuge ;it 0". The resulting siip-rniitmt n - ~ sthen recentrifuged at S0,OOO:i for 30 niin. and an aliquot of the clear supernatant, !ree of visible fat, was removed and saved. The remainder of the supernatant was decanted and discarded. The niic rosonies wverr resrispended b>- honiogenizing tlie niicrosome pcllet with suitable volumes of huffer and S0.000:/ supernat:tnt t o \-ield :i final si ispension containing in each 1111. tlie microsomes froin 200 iiig. of liver and 80,0007 supernatant f r o m 40 ing. nf liver I n T'ilro Dealkjrlaticns.--Var:.ing amounts of substrate dissolved in 0.2 ml. of polyethj-lene glycol ( P E G 400) were pl;tcaed in 20 nil. beakers and to this 11-as added 1 nil. of mic*rosome suspension, 50 pnioles of nicotinamide? 50 pmoles of niagnesiuni vhloride, 10 pnioles of glucose-6-phosphate, 0.5 pniole of S,ZDP, and sufficient, buffer ( p H 7.4) t o bring t,he final volume t o 3 nil. I n the runs involving inhibitors the inhibitor was added with the substrate. Incubations were carried out in a shaker a t 37" in an ntmosphere of oxygen for 30 min. The presence of oxygen completely inhibited the activity of the microsomal reductase' and p-nitrophenol carried through the incubation could be recovered quantit,atively. The incubation was stopped by t,he addition of 1 nil. of 10yctrichloroacetic acid and the precipitated protein renioved by centrifugat,ion. After adding 1 nil. of 0.T5 .I' S a O H t o 3 nil. of supernatant, unreacted subst,rate was removed by extraction with 5 nil. of methylene chloride. Removal of unreacted substrate was necessary since its presence interfered with the p-nitrophenol reading. p-Sitrophenol was determined spectropliotometrically at 400 mp. That the product was indeed p-nitroT
(6) C . l i i i r u p f , .4nn.. 234, 123 (1881). (7) J . R. Fouts a n d B. B. Brodie. J . Phaimacol. E z p l l . T h e r a p . . 119, 197 (19,771.
343
July, 1963
DEMETHYLATIOS STUDIES.I Y
The apparent discrepancy shown by the methyl compound most probably relates to the appreciable water solubility of this ether. The microsomal fraction is the major lipid bearing subfraction of the liver cell, and in the incubation mixture one has in effect a two-phase system, L e . , an aqueous phase and a lipid phase (microsomes). S-Dealkylation ~ t u d i e s * ~ have - l j led to the belief that only those substrates which can penetrate into the lipid phase will interact with enzyme. If this speculation were correct it mould mean that a true measure of K,, would be that concentration in the microsome fraction which results in one-half maximum rate. I n a well defined group of water insoluble lipid soluble compounds such as those under discussion, the apparent K , values measured, though iiot the true values, would relate to eaeh other in a proportional manner. This would not however be true in the case of a water soluble compound such as the p-nitropheiiyl methyl ether, and the K , value found would be deceptively high. When the niaxiniuni rates (Ir,,,, Table 11) are compared, it is seen that the methyl ether iiow falls iii line and is the most artire, with the others showing decreased reactivity as the size of the alkyl groups inrreases. The inverse relationship between rate and the size of the alkyl group inay result siiiiply from a decreasing chemical reactivity with iiicreasiiig bulk. It might also be merely a matter of iiicreasiiig steric hind raiice to react ion, The effect of incorporating various fuiictioiial groups iiito the alkyl group was investigated next. The inwrporation of a double bond iiito the three-carbon side chains (9) resulted iii a considerable increase in activity in dealkylation compared to the corresponding saturated ether (3). If, as is likely,16 the dealkylation reaction involves electrophilic attack upon the methylene carbon, then any electron doiior group in ail adjacent position would be expected to increase the rate of rea(-tion. The same considerations also explain the increased rate shown by tlie cyanomethyl ether (8). =Is would be expected p-iiitroplieiioxyacetic acid (11) is i1iactii.e. I t should be completely ionized at pH 7.4 and iiot available for distribution into the microsomes. The reason for the lack of activity of the p-piperidinoethyl ether (12) is not clear. , h o t h e r electron doiior group which, n.hen attached to the active iiiethyleiie carbon, would be expected to lead t o increased reactivity is the phenyl group. This is the case. Indeed, p-iiitrophenyl benzyl ether (12) is tlie most active substrate a t low concentrations. Unfortunately this interesting ether exhibited substrate inhibition of dealkylation rate even at relatively low c.oiireiitratiou. -1scan be seen from the data presented in I’ig. 1 the rate reached a maximum a t a conceatratioii of less than 3 X JI. Actually substrate inhibition was observed with nearly all of the substituted benzyl ethers studied and it n-as not possible to study enzyme binding and ,,,,T’ in this important series. It was thought that a study of the effect of ring substitution upon rate would be instructive in attempts to understand the niechanism of the reaction. However, (13) L. Gaudette and B. B. Brodie. Biockem. Pkarmacol., 2 , 89 (1959). (1-1) R . E. l I c h l a h o n , J . M e d . Pharm. Chem., 4, 67 ( 1 9 6 1 ) . f1.5) R . E. l l c h l a h o n a n d N. R. Easton, ihid.. 4, 437 (1961). 116) ,J. R. Gillette a n d J. J. Kamiii, J . Pkaimacol. Ezptl. Therap., 130, 262 (1Q60,.
015,
0
2
I
SUBSTRATE
3
CONCK,XIO~lM/
Fig 1.-The effect of substrate concentration upon the rate of cleavage of p-nitrophenyl benzyl ether. Incubation conditions are described under methods.
it was found that without exception all of the riiig substituted benzyl ethers were cleaved a t a slower rate than the parent compound. I;or example, both the p iiitro group, which should decrease the electronegativity of the active methylene group, and the p-inethyl group, which should increase it, lead to substrates which are essentially inactive. A n examination of the o-halo derivatives yields a clue to the nature of the effect of substituents. It mas found that in going from benzyl to o-fluorobenzyl (13) to o-chlorobenzyl (14) to o-bromobenzyl (15) the rate decreased in that order, i.e., the rate decreases with increasing bulk of the substituent. The methyl group which is similar in size to bromine yields an o-methyl derivative (16) which differs little in rate from the o-bromo compound. Substitution in the para position seems to be even more detrimeiital. The bulky p-nitro group (20) and p-methyl group (19) yield derivatives which are without activity as substrates. The smaller chloro substituent has a greater depressant effect in the para position than in the ortho position. Thus the uiisubstituted beiizeiie ring seems to be the optimal configuration for this series of ethers. Ally substitutioii o n the ring reduces substrate activity possibly through steric interference with the approach of the ring to the enzyme surfare. The phenethyl ether (21) in which the beiizeiie ring is too far removed from the active methylene to affect its electronegativity is predictably much less active than the benzyl ether. p-Xitrodiphenyl ether is also a substrate for this enzyme thus confirming the carlier i n ziuo observations of Huggiiis, et al.‘ The effect of various inhibitors upon the cleavage of p-nitrophenetole was also studied (Table 111). DPEA (2,4-dichloro-6-phenylphenoxyethylamine)which is a caompetitive inhibitor of S-demethylationli is an effective inhibitor although relatively high concentrations are required. Cupric ion also inhibits effectively. It is possible that copper ion inhibits by blocking YADPH oxidase thus depressing the rate of formation of ‘active hydroxyl.”* Cyanide, dipyridyl and iodoacetate did iiot inhibit at the concentrations used. I n Vivo.-The cleaTage of this interesting series of ethers was also studied in the whole animal. Since (17) R. E. M c l l a h o n and J. Mills, J . M e d . Pkarm. Ckem., 4, 211 (1961). (18) J. R. Gillette, B. B. Brodie, and B. S . L a D u , J . Pkarmacol. Ezptl. Therap., 119, 532 (1967).
OH
1
