Chem. Res. Toxicol. 1989, 2, 57-59
57
Isotopic Sensitivity in the Microsomal Oxidation of the Dihydropyridine Calcium Entry Blocker Nifedipine J. L. Born* and W. M. H a d l e y The Toxicology Program, College of Pharmacy, The University of New Mexico, Albuquerque, New Mexico 87131 Received November 4, 1988
T h e primary deuterium isotope effect on V , for the microsomal oxidation of the dihydropyridine calcium entry blocker nifedipine [4-(2-nitrophenyl)-2,6-dimethyl-3,5-bis(methoxycarbonyl)-1,4-dihydropyridine] has been measured. The magnitude of the kinetic isotope effect, 6.7, suggests that the rate-limiting step in the mechanism of microsomal oxidation of nifedipine involves the loss of a hydrogen atom rather than nitrogen oxidation. Thus the microsomal oxidation of nifedipine is mechanistically different from that of other 1,4-dihydropyridines.
The dihydropyridine calcium entry blocker nifedipine [4-(2-nitrophenyl)-2,6-dimethyl-3,5-bis(methoxycarbonyl)-l,4-dihydropyridine] undergoes rapid first-pass metabolism producing metabolites that are inactive as antihypertensive agents (1). The oxidative metabolism of nifedipine proceeds via a two-electron oxidation catalyzed by P-450NF (2, 3). Both 4-aryl- and 4-alkyl-2,6-dimethyl-3,5-bis(alkoxycarbonyl)-l,4-dihydropyridines are rapidly oxidized by cytochrome P-450; however, 4-alkyl1,Cdihydropyridines are suicide substrates for cytochrome P-450 ( 4 ) ,while 4-aryl-substituted 1,4-dihydropyridines have not been implicated in the destruction of cytochrome P-450. The oxidation of 4-ethyl-2,6-dimethyl-3,5-bis(ethoxycarbonyl)-l,4-dihydropyridine (1) and 4-phenyl-2,6-dimethyl-3,5-bis(methoxycarbonyl)-1,4-dihydropyridine (2) are thought to proceed via a one-electron oxidation at the dihydropyridine nitrogen to produce a radical cation which undergoes a second, one-electron oxidation to produce a pyridine metabolite ( 4 , 5 ) . The second one-electron oxidation of 1 occurs via the loss of the ethyl group from position 4 of the radical cation to produce a pyridine metabolite ( 4 ) . The cytochrome P-450 mediated oxidation of 4-aryl-1,4-dihydropyridines results in 4-arylpyridine metabolites via the loss of a hydrogen from position 4 of the dihydropyridine ring rather than the loss of an aryl radical. The lack of an isotopically sensitive step in the oxidation of 2 ( 5 ) suggests that the cytochrome P-450 mediated oxidation of 2 proceeds, as shown in path A of Figure 1, via the formation of the radical cation 3. The radical cation 3 loses a proton followed by redistribution of an electron to produce the benzylic radical 4, which undergoes a second one-electron oxidation to give a pyridine metabolite (3,5). This mechanism is consistent with the mechanism of oxidation of 4-alkyl-1,4-dihydropyridines, in that it involves nitrogen oxidation to give a radical cation, and is similar to the mechanism for N-dealkylation of amines in which a proton loss is an integral part of the mechanism ( 6 ) . An alternative mechanism, which would explain the products of the oxidation of 4aryl-1,4-dihydropyridines, is shown in path B of Figure 1. This mechanism involves the abstraction of hydrogen from position 4 of the dihydropyridine ring to produce the benzylic radical 3, which foliowing a second one-electron
* Address correspondence to this author.
oxidation produces a pyridine metabolite. The mechanisms of cytochrome P-450 mediated aliphatic hydroxylations, N-dealkylations, aromatic hydroxylations, and 0-dealkylations have been investigated via the use of kinetic isotope effects involving either deuterium- or tritium-labeled substrates. Kinetic isotope studies have provided evidence that the microsomal (or purified cytochrome P-450) dealkylation of amines is not isotopically sensitive, thus implicating the formation of a radical cation as a rate-limiting step (6, 7, 9). Similar experiments have shown that aliphatic hydroxylations and 0-dealkylation reactions mediated by cytochrome P-450 are isotopically sensitive, thus implicating hydrogen abstraction as an important component of the rate-limiting step (6-8, 10, 11). The object of this study was to employ kinetic isotope experiments in the examination of the mechanism of microsomal oxidation of nifedipine.
Materials and Methods Nifedipine was prepared via the Hantzsch reaction (12). [42H]Nifedipine was prepared in a similar manner by using [a2H]-2-nitrobenzaldehyde, which was synthesized via the reduction of 2-nitrobenzoyl chloride with LiA12H(O-tBu)3according to the procedure described for the production of 2-nitrobenzaldehyde (13). The mass spectrum of [4-2H]nifedipinewas consistent with the incorporation of a deuterium a t position 4 of the dihydropyridine ring with a parent peak a t mle 347, a p + 1peak at 348, and a peak a t m/e 225 representing the loss of a nitrophenyl radical. Conversely, nifedipine has a parent peak at 346, a p + 1 peak a t 347, and a peak a t 224 resulting from the loss of the nitrophenyl radical. The ring-oxidized metabolite of nifedipine, 4-(2-nitrophenyl)-3,5-bis(carboxymethyl)-2,6-dimethylpyridine, and the internal standard 4-(2-nitrophenyl)-3,5-bis(carboxyethyl)-2,6-dimethylpyridinewere prepared by the oxidation of the corresponding dihydropyridines (14). Rat liver microsomes (male Sprague-Dawley rats, 150-185 g each) were prepared as previously reported (15). Protein concentration was measured (16), and the concentration of cytochrome P-450was determined as described by Omura and Sat0 (17). Incubation mixtures contained nifedipine or [2H]nifedipine (12, 6, 3, 2.25, and 1.5 fimol), MgC12 (50 pmol), glucose 6-phosphate (30 fimol), glucose-6-phosphate dehydrogenase ( 2 units), microsomal protein (5mg), and pH 7.4 Tris buffer (0.5 M, p H 7.4) to give a total volume of 5 mL. The samples were preincubated (5 min) under a constant flow of oxygen a t 37 OC with shaking (100 oscillations/min) in a Dubnoff shaking incubator. The reactions were initiated by the addition of substrate in 100 fiL of ethanol.
0893-228~/89/2702-0057$01.50/0 0 1989 American Chemical Society
58 Chem. Res. Toxicol., Vol. 2, No. 1, 1989
Born and Hadley
0
m
t
P
H
Nifedipine R =
4 4
H
I
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V
0
10
20
30
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40
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Figure 2. Lineweaver-Burk representations of the oxidation of and [4-2H]nifedipine( 0 )by rat liver microsomes. nifedipine (0) The concentration of nifedipine was measured by HPLC as indicated under Materials and Methods; the values are i the standard deviation with n = 4.
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