Diclofenac and its derivatives as tools for studying human cytochromes P450 active sites: Particular efficiency and regioselectivity of P4502Cs

A comparison of the oxidations of diclofenac with microsomes of yeasts expr essing various human liver cytochromes P450 showed that P450 2C9 regioselec tively led to 4'-hydroxy diclofenac (4'-OHD) whereas P450 3A4 only led to 5 -hydroxy diclofenac (5-OHD). P450 2C19, 2C18, and 2C8 led to the simultaneo us formation of 4'-OHD and 5-OHD (respective molar ratios of 1.3, 0.37, and 0.17), and P450 1Al, 1A2, 2D6, and 2E1 failed to give any detectable hydro xylated metabolite under identical conditions. P450 2C9 was found to be muc h more efficient for diclofenac hydroxylation than all the other P450s test ed (k(cat)/K-M of 1.6 min(-1) mu M-1 instead of 0.025 for the second more a ctive P450), mainly because of markedly lower K-M values (15 +/- 8 instead of values between 170 and 630 mu M). Oxidation of diclofenac with chemical model systems of cytochrome P450 based on iron porphyrin catalysts exclusiv ely led to the quinone imine derived from two-electron oxidation of 5-OHD, in an almost quantitative yield. Two derivatives of diclofenac lacking its COO- function were then synthesized; their oxidation by recombinant human P 450 2Cs always led to a major product coming from their 5-hydroxylation. Su bstrate 2, which derives from reduction of the COO- function of diclofenac to the CH2OH function, was studied in more detail. All the P450s tested (1A 1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, and 3A4) almost exclusively led to its 5 -hydroxylation. P450s of the 2C subfamily were found to be the mast efficie nt catalysts for this reaction, with k(cat)/K-M values between 0.2 and 1.6 min(-1) mu M-1. Oxidation of 2 with an iron porphyrin based chemical model of cytochrome P450 also led to a product derived from the oxidation of 2 at position 5. These results show that oxidation of diclofenac and its deriva tive 2, either with chemical model systems of cytochrome P450 or with recom binant human P450s, generally occurs at position 5. This position, para to the NH group on the more electron-rich aromatic ring of diclofenac derivati ves, is thus, as expected, the privileged site of reaction of electrophilic , oxidant species. The most spectacular exception to this chemoselective 5- oxidation of diclofenac derivatives was found for oxidation of diclofenac i tself with P450 2C9 (and P450 2C19 and 2C18 to a lesser extent), which only led to 4'-OHD. A likely explanation for this result is a strict positionin g of diclofenac in the P450 2C9 active site, via its COO- function, to comp letely orientate its hydroxylation toward position 4', which is not chemica lly preferred. P450 2C19, 2C18, and 2C8 would not lead to such a strict pos itioning as they give mixtures of 4'-OHD and 5-OHD. The above results show that diclofenac derivatives are interesting tools to compare the active sit e topologies of human P450 2Cs.