Interactions of nitrogen heterocycles with cytochrome P-450 and monooxygenase activity

Chem.-Biol. Interactions, 50 (1984) 267--275 Elsevier Scientific Publishers Ireland Ltd.

267

INTERACTIONS OF NITROGEN HETEROCYCLES WITH CYTOCHROME P-450 AND MONOOXYGENASE ACTIVITY

M. MURRAY* and C.F. WILKINSON**

Department of Entomology, Cornell University, ithaca, N Y 14853 (U.S.A.) (Received December 19th, 1983) (Revision received March 23rd, 1984) (Accepted March 26th, 1984)

SUMMARY

Three groups of isomeric nitrogen heterocycles, phenylpyridines, phenylimidazoles and pyridylimidazoles were studied in relation to the effect of steric factors on type II binding to cytochrome P-450 and inhibition of aryl hydrocarbon (benzo[a] pyrene) hydroxylase (AHH) activity in hepatic microsomes from phenobarbital(PB)- and ~-naphthoflavone(~NF)-induced rats. Type II binding affinity was lower (higher Ks) in compounds with substituents on the carbon adjacent to the nitrogen undergoing ligand interaction than in those where steric hindrance near the nitrogen was minimal. Binding affinities of the compounds as measured by their Ks values, were quite similar in both PB- and ~NF-induced microsomes. In PB-induced microsomes, type II binding affinity was generally reflected by the ability of the compounds to inhibit AHH activity. In contrast, most of the compounds evaluated were inactive as AHH inhibitors in ~NF-induced microsomes.

Key words: Nitrogen heterocycles -- Cytochrome P-450 -- Aryl hydrocarbon hydroxylase -- Inhibition INTRODUC~ON

Several groups of nitrogen heterocycles are recognised as effective in vitro and in vivo inhibitors of a variety of monooxygenase reactions catalyzed by the microsomal mixed-function oxidase (MFO) system of mammals and other species. The most potent compounds yet reported are the 1- and 4(5)-substi-

*Present address: Department of Medicine, University of Sydney, Sydney, Australia. **To whom correspondence should be sent. Abbreviations: AHH, aryl hydrocarbon (benzo[a]pyrene) hydroxylase; ~NF, ~-naphthoflavone; MFO, mixed-function oxidase; PB, phenobarbital. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

268 tuted imidazoles [1--4], although benzimidazoles [5--7] and several pyridine derivatives [ 8--10] are also effective MFO inhibitors. The inhibitory potency of most of the nitrogen heterocycles is considered to result from direct ligand interaction between the ring nitrogen and the heme moiety of cytochrome P-450 [3]. This view is supported by correlations between MFO inhibition and spectral (type II) dissociation constants (Ks} [3,4,7,11--13] and by the observation that steric hindrance around the nitrogen atom in substituted imidazoles [13], oxazoles and thiazoles [14] leads to dramatic decreases in type II binding and inhibitory potency. Recent studies in this laboratory utilizing the technique of equilibrium dialysis have clearly established that 1 [ [ 4-SH-phenyl] imidazole exhibits considerable selectivity in its ability to bind to cytochrome P-450 [15], the affinity of 1-phenylimidazole for the 'PB-type' cytochrome P-450 being approx. 40-fold greater than that for the '3-methylcholanthrene (3MC)- or ~NF-type' cytochrome P-448. These data were reflected by the potent inhibitory action of 1-phenylimidazole towards AHH in PB-induced microsomes and by its failure to inhibit AHH activity in microsomes from rats induced with 3MC or~NF [15]. Preliminary studies in this laboratory indicated that this apparent selectivity of 1-phenylimidazole was not reflected in its type II spectral interactions since both binding affinity and maximum spectral change (4 absorbance 430--395 nm) were approximately the same in microsomes from both PBand ~NF-induced rats; this suggested that type II spectral parameters may not constitute good indicators of the inhibitory potency of nitrogen heterocycles. The present study was undertaken to further evaluate the relationships between type II spectral interactions and MFO (AHH) inhibition and to investigate relationships between these parameters and the steric and structural character of three groups of nitrogen heterocycles, the phenylpyridines, phenylimidazoles and pyridylimidazoles. All studies were conducted in microsomes from rats induced with PB or flNF. M ATER I A L S AND METHODS

Chemicals The isomeric phenylpyridines (I--III) and 4(5)-phenylimidazole (VI) were purchased from Aldrich Chemical Co. {Milwaukee, WI), 1-phenylimidazole (IV) from Transworld Chemicals, In. (Washifigton, DC) and 2-phenylimidazole (V) from ICN-K and K Laboratories (Plainview, NY). All were purified by recrystallization or vacuum distillation prior to use. The pyridylimidazoles (VII--IX) were synthesized according to the method of ChisweU et al. [16]. The reaction mixture of glyoxal, pyridine carboxaldehyde (Aldrich Chemical Co.) and ammonia solution, was exhaustively extracted with diethyl ether and the concentrated ether extract applied to an alumina column packed in ether. Elution with ether yielded the desired compounds that were purified by recrystallization. 2-(2'-Pyridyl)-imidazole {VII) m.p. 135--136°C (literature m.p. 134--135°C [16] was recrystaUized from

269 ethyl acetate, 2-(3'pyridyl)imidazole m.p. 206--209°C (literature m.p. 206-207°C [ 17] ) from ethyl acetate, and 2-(4'-pyridyl)-imidazole m.p. 208--211°C (literature 206--208°C [17] ) from ethyl acetate/ethanol. Biochemicals were purchased from Boehringer Mannheim (Indianapolis, IN) and all other solvents and chemicals were of analytical reagent grade.

Animals Male Sprague--Dawley-derived rats (200--250 g) were purchased from Blue Spruce Farms, Altamont, NY. PB was administered i.p. in normal saline at a dose of 100 mg/kg once daily for 3 days and ~NF was similarly administered in corn oil at a dose of 40 mg/kg. Animals were killed by decapitation 24 h following the last treatment with the inducer. Microsomal prepara tion Hepatic microsomal fractions were prepared as previously described [ 18]. Protein was determined by the m e t h o d of Lowry et al. [19] using bovine serum albumin as a standard. Enzyme assays AHH activity was determined by a modification of the direct spectrofluorometric assay of Yang and Kicha [20] using an Aminco SPF-125 spectrofluorometer. Incubations contained 0.2 mg of microsomal protein and 5 nmol of benzo[a]pyrene in 0.1 M potassium phosphate buffer (pH 7.4; final volume 2.0 ml). NADPH (50 nmol) was used to initiate the reaction which was followed by the change in fluorescence intensity (excitation ~, = 387 nm; emission k = 407 nm) with time. Inhibitors were added to the reaction mixtures in 50/~l ethanol and ethanol alone (50ul) was added to the corresponding control. Is0 (M)-values were obtained from plots of percent inhibition versus log inhibitor concentration each plot being constructed from the mean percent inhibition observed at four or five different inhibitor concentrations in 2--4 separate determinations. Control AHH activities (mean +S.D.) were 1.90 + 0.18 (N = 8) and 9.69 + 0.63 (N = 10) nmol benzo[a]pyrene metabolized/mg protein/min, respectively, in microsomes from PBand ~NF-induced rats. Cytochrome P-450 was measured by the m e t h o d of Omura and Sato [21] using an Aminco-Chance DW-2 spectrophotometer and employing an extinction coefficient of 91 mM -1 cm -~ for the ferrocytochrome P-450-carbonyl spectral complex. Optical difference spectroscopy Difference spectra were measured at 37°C in an Aminco-Chance DW-2 spectrophotometer using 1 cm cuvettes containing 1-ml aliquots of microsomal suspensions ( 1 mg microsomai protein per ml) in potassium phosphate buffer (0.1 M, pH 7.4). Test compounds were added to the sample cuvette in appropriate volumes (5--20 ~1) of ethanol and the type II difference spectra were recorded between 380 and 500 nm; appropriate volumes of ethanol

. 2-Phenyl 3-Phenyl 4-Phenyl 1-Phenyl 2-Phenyl 4 (5)-Phenyl 2'-Pyridyl 3~-Pyridyl 4'-Pyridyl

310 4.2 6.1 1.5 1300 2.6 2600 210 19

3.10 2.33 4.03 4.25 2.40 5.40 8.64 1.62 2.89

x x x x x x x × ×

10 -2 10 -2 10 -2 10 -2 10 -: 10 .2 10 3 10 -2 10 -5

23 21 18 7.3 430 12 > 1000 > 1000 480

b

130 2.7 11 3.5 2100 4.0 210 85 48

Ks

AHH/Is0 c

Kas

AAbmax

~NF-induced

(VII- IX)

PB-induced

(IY-Yl)

Substitution

3

4'

aSpectral dissociation c o n s t a n t (uM) for t y p e II optical d i f f e r e n c e s p e c t r u m . Maximal spectral change ~ a b s o r b a n c e u n i t s ( 4 3 0 - - 3 9 5 n m ) / n m o l c y t o c h r o m e P-450. cI~,, (uM) t o w a r d s A H H activity.

I II III IV V VI VII VIII IX

Compound

(I-III)

H

2.70 4.93 5.63 4.8 3.23 3.93 2.86 2.20 3.31

× x x x x x x x ×

10 -2 10 -2 10 -2 10 -2 10 -2 10 -2 10 -3 10 2 10 -2

L~Amax

I N T E R A C T I O N OF I S O M E R I C P H E N Y L P Y R I D I N E S , P H E N Y L I M I D A Z O L E S A N D 2 - P Y R I D Y L I M I D A Z O L E S WITH C Y T O C H R O M E ( S ) P-450 A N D A H H A C T I V I T Y IN H E P A T I C M I C R O S O M E S F R O M PB- A N D ~ N F - I N D U C E D R A T S

TABLE I

>1000 39 320 >1000 >1000 >1000 >1000 >1000 >1000

AHH/Iso

b~

271 were added to the reference cuvette. Spectral dissociation constants (Ks) and maximal spectral change values (AAmax) were obtained from double reciprocal plots of ligand concentration versus A absorbance between 430 nm (maximum} and 395 nm (minimum). At least four different concentrations of the test ligand were used for each determination and 2--4 separate determinations were made for each compound. RESULTS AND DISCUSSION Table I shows cytochrome P-450 binding characteristics (Ks and AAmax) and inhibitory potency towards AHH activity, of nine nitrogen heterocycles in hepatic microsomes from PB- and ~NF-induced rats. Although all of the compounds evaluated exhibited type II optical difference spectra and were thus able to undergo ligand interaction with the ferricytochrome(s) P-450 in both 2B-- and ~NF-induced microsomes, the Ksvalues for different compounds varied by almost three orders of magnitude. As indicated by the K~values, the relative order of the affinities of the compounds within each of the three isomeric series were the same in PB- and /3NF-induced microsomes and, indeed, the actual Ks-values were quite similar in both types of microsomes with the exception of the 10-fold difference with 2-(2'-pyridyl)imidazole (VII). In agreement with previous reports [13,14], Ks-values within each series of compounds appear to reflect steric factors relating to the accessibility of the nitrogen atom involved in ligand interaction. Thus, in the case of the phenylimidazoles (IV--VI), the 1-, 2and 4(5)-isomers exhibit K~values of 1.5, 1300 and 2.6 uM, respectively, in PB-induced microsomes (3.5, 2100 and 4.0/aM in ~NF-induced microsomes) and clearly reflect the steric hindrance associated with substitution on carbon-2 adjacent to the nitrogen. A similar effect undoubtedly accounts for the relatively low affinities of compounds I and VII that also contain substituents adjacent to the nitrogen. In the case of the 2-pyridylimidazoles (VII-IX) it is likely that ligand interaction with cytochrome P-450 occurs through the pyridine nitrogen rather than through that on the imidazole ring since all these compounds are 2-substituted imidazoles in which imidazole nitrogen binding should be minimal as in compound V [13]. In summary, therefore, it appears that the affinity of the nitrogen heterocycles (I--IX) for cytochrome P-450 in both PB-~and ~NF-induced microsomes depends to a large extent on the st~ri~ accessibility of a nitrogen atom on either the pyridine (I--II, VII--IX) or imidazole (IV--VI) rings. It is possible that the uniformly lower affinities (higher Ks-values) of the 2-pyridylimidazoles (VII--IX) compared with those of the phenylpyridines (I--III) reflect the somewhat lower hydrophobic character of the former since lipophilicity is also considered to be an important factor in type II binding affinity [4]. The apparent relationship between steric character and binding affinity of the nitrogen heterocycles used in this study does not appear to hold true with respect to the extent of binding of the different compounds as measured by AAmax 430--395 nm. Indeed, with the exception of the very low level of

272 binding observed with VII, AAmax-Values are relatively constant and show no obvious or consistent trends associated with either chemical structure or with the type of microsomes employed. It can be concluded that hepatic microsomes from both PB- and ~NF-induced rats do not differ greatly in their ability to bind different nitrogen heterocycles, at least as measured by type II spectral interactions. AHH activity in microsomes from PB-induced rats was susceptible to inhibition by several of the nitrogen heterocycles, the most potent being 1and 4(5)-phenylimidazole (IV and VI) and the three isomeric phenylpyridines (I--III) having Is0-values between 7.3 and 23 pM. The remaining compounds were much less effective inhibitors, In general, the most effective AHH inhibitors were those compounds with the highest type II binding affinities, although a good correlation between Ks and I50 was apparent only with the phenylimidazoles (IV--VI). All of the phenylpyridines (I--III) were equally effective AHH inhibitors in PB-induced microsomes in spite of the fact that the Ks of the 2-phenyl isomer (I) was approx. 75- and 50-fold higher than the 3- and 4-phenylpyridines, respectively. None of the pyridylimidazoles were good inhibitors although 2-(4'-pyridyl)imidazole (IX) had a reasonably high binding affinity {Ks 19 pM). There is no evidence of any relationship between AAmax-Vaiues of the nitrogen heterocycles and their inhibitory potency towards AHH activity. In marked contrast to the situation in PB-induced microsomes, none of the compounds were effective inhibitors of AHH activity in j3NF-induced microsomes with the exception of 3-phenylpyridine (II) that with an I50 of 39 /~M could be the most potent monoheterocyclic inhibitor yet described for 13NF/AHH activity. Although this compound also exhibited the highest affinity towards ~NF-induced microsomes (Ks = 2.7 pM), this was not much higher than those measured with 1- and 4(5)-phenylimidazoles that showed no inhibition of AHH activity at concentrations of up to 100/~M. Clearly, in the case of microsomes from ~NF-induced rats there is little or no relationship between binding affinity and inhibitory activity towards AHH. Indeed, it is of considerable interest to note that as described earlier [15], many of the nitrogen heterocycles that inhibit AHH activity in PB-induced microsomes cause a significant stimulation of AHH activity in/3NF-induced microsomes (Table II). An adequate explanation of this observation has not yet been proposed. The results of the present study clearly indicate that no uniform relationship exists between the type II spectral binding characteristics of nitrogen heterocycles and their inhibitory activity towards MFO activity; this is particularly true of MFO activity (e.g. AHH) associated with/3NF-induced microsomes where the major isozyme is considered to be cytochrome P-450c (cytochrome P-448) [22]. Cytochrome P-450 c differs from other isozymes in spectral, structural and catalytic properties [23,24]. In particular, cytochrome P-450c is able to catalyze the oxygenation of sterically hindered positions of relatively large polycyclic molecules, suggesting that it has a somewhat larger active center than other forms of cytochrome P-450 [25,26].

273 T A B L E II E N H A N C E M E N T O F A H H A C T I V I T Y BY N I T R O G E N H E T E R O C Y C L E S IN MICROSOMES FROM 5NF-INDUCED RATS

Compound

Conc. ( . M )

A H H activity a (X ± S.D.; N)

activity

None (control) l-Phenylirnidazole(IV) 2-Phenylimidazole (V)

0 100 10 100 1000 10 100 1000 10 100 500 1000

9.69 12.31 10.24 10.39 9.48 10.95 9.72 9.96 11.29 10.43 8.99 7.35

100 127 b 106 107 98 113 b 100 103 117 b 108 93 76 b

2-(2'-pyridyl)Imidazole (VII)

1-(3'-pyridyl)Imidazole (VIII)

± ± ± ± ± ± + + ± ± ± ±

0.63; 0.69; 0.16; 0.61; 1.47; 0.67; 0.54; 0.62; 0.81; 0.77; 0.34; 0.07;

10 14 3 3 3 3 3 3 3 3 3 3

% Control

aAHH activity data are expressed in nmol benzo[a ] pyrene m e t a b o l i z e d / m i l l i g r a m p r o t e i n / rain and are means ± S.D. of N determinations. bSignificantly different f r o m c o n t r o l activity at the 0.05 confidence level as measured by the unpaired S t u d e n t ' s t-test (2-tai!ed).

The ability of the eUipticines [26,27] and ~NF [28] to selectively inhibit reactions catalyzed by cytochrome P-450c may result, in part, from the relatively large size of these compounds; steric bulk has also been proposed to account for why clotrimazole [ 1-(o-chlorophenyldiphenylmethyl)imidazole] [29] and n o t 1-phenylimidazole is an effective inhibitor of cytochrome P-450c-mediated AHH activity [ 15]. The ability of 1-phenylimidazole (IV) and other nitrogen heterocycles to exhibit type II spectral interactions with cytochrome P-450c in ~NF-induced microsomes and yet cause.no inhibition of AHH activity (Table I) indicates that factors other than molecular size are involved. A recent report by Phillipson et al. [ 30] provides evidence that cytochrome P-450c may possess two type I substrate binding sites with different substrate specificities, one of which is different from that of other isozymes. A similar explanation could also account for the fact that although cytochrome P-450c forms type II (455 nm) spectral complexes with methylenedioxyphenyl (e.g. isosafrole) metabolites, such complexes are n o t inhibitory towards AHH activity [31]. On the other hand, both type II and type III spectral complexes are thought to involve direct binding to the heme moiety of cytochrome P-450, and it is n o t at all clear why such complexes would n o t prove inhibitory to all monooxygenase reactions. ACKNOWLEDGEMENT

The work was supported by grant ES 01902 from the National Institutes of Health.

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