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The active site topology, substrate specificity, and biological roles of the human cytochrome P450 CYP2J2, which is mainly expressed in the cardiovascular system, are poorly known even though recent data suggest that it could be a novel biomarker and potential target for therapy of human cancer. This paper reports a first series of high-affinity, selective CYP2J2 inhibitors that are related to terfenadine, with Ki values as low as 160 nM, that should be useful tools to determine the biological roles of CYP2J2.
In the human genome, 57 genes have been found to code for cytochromes P450 (CYPs) that are involved in the oxidative metabolism of endogenous compounds and xenobiotics.1 The main CYPs implicated in drug metabolism, such as CYP3A4, CYP2C9 or CYP2D6, and those responsible for the biosynthesis of steroid hormones have been extensively studied,1 and several X-ray structures of human CYPs have been recently published.2 Much less is known about recently discovered human CYPs such as CYP2J2.1,3 This cytochrome seems to be primarily expressed in heart3; it has also been found in kidney, liver, lung,4 and the gastrointestinal tract.5 Its biological role is presently unclear, even though it has been found to catalyze the oxidation of a few drugs such as ebastine in the intestine.6,7 Moreover, recombinant CYP2J2 catalyzes the epoxidation of arachidonic acid to four cis-epoxyeicosatrienoic acids (EETs), with regio- and stereo-selectivities that match those of the EETs isolated from heart tissue.3 Some EET-derived metabolites play important roles in regulation of vascular tone8 and in a host of processes related to cancer cell behavior, angiogenesis, and tumor pathogenesis.9 Very recent data suggest that CYP2J2 promotes the neoplastic phenotype of carcinoma cells and may represent a novel biomarker and potential target for therapy of human cancers.10 However, little data are presently available on the active site topology and substrate specificity of CYP2J2.1 This communication reports the design and synthesis of a first series of high-affinity inhibitors of human CYP2J2. These inhibitors should be useful tools to determine the biological roles of this cytochrome.
Hydroxylation of the drug ebastine by recombinant CYP2J27 expressed in baculovirus-infected Sf9 insect cells3 was used as an assay to find CYP2J2 inhibitors. During a first screening for such inhibitors, compound 1, derived from the drug terfenadine by oxidation of its benzylic alcohol function, was found to inhibit CYP2J211 with an IC50 value of 0.7 ± 0.1 μM. This value was much lower than the IC50 found for terfenadine itself (8.1 ± 0.4 μM) (Table 1), that was previously described as an inhibitor of CYP2J2.12 Then, compound 1, called terfenadone in the following, was used as a starting point for the design of high-affinity inhibitors of CYP2J2. Ebastine,7 terfenadine,13 and compound 1 are all hydroxylated by CYP2J2 at a site that is weakly reactive from a chemical standpoint, a CH3 of the t-butyl group (Scheme 1). This regioselectivity in favor of the least reactive part of these substrates implies their strict positioning in the CYP2J2 active site in order to maintain the t-butyl group in close proximity of the heme iron for transfer of an oxygen atom from O2. Therefore, a series of compounds derived from 1 by replacement of its t-butyl group with various R groups of different size and polarity was synthesized (Scheme 2). This included compounds bearing functions previously known to lead to suicide inactivation of cytochromes P450 after in situ oxidation.14 This is the case for the terminal double bond of compound 5, since terminal alkenes act as mechanism-based inhibitors of cytochromes P450 after N-alkylation of the heme by an intermediate derived from P450-catalyzed oxidation of the substrate double bond.14 The choice of the CHF2 and benzodioxole functions of compounds 12 and 13 was also made on the basis of literature data on suicide substrates of cytochrome P450.14 In situ hydroxylation of the C–H bond of CHF2 groups leads to an electrophilic intermediate able to acylate the P450 protein, whereas inactivation of cytochrome P450 by benzodioxole derivatives is due to the formation of an iron–carbene bond after oxidation of the benzodioxole CH2 group.14 The structure of compounds 5, 12, and 13 has been chosen in order that CYP2J2-catalyzed oxidations occur at the site leading to inactivating metabolites, assuming that hydroxylation of 5, 12, and 13 should occur on the homobenzylic position as the hydroxylation of compound 1 and terfenadine.
The general synthetic route used for the preparation of terfenadone derivatives (Scheme 2) with R = (CH2)nCH3 (with n = 0–3), (CH2)nOH and (CH2)nOCOCH3 (with n = 2 or 3), Br, CH2CHF2, OCH2O–, and OCH3 involved an acylation of the benzenic starting compound with 4-chlorobutanoic acid chloride in the presence of FeCl3, AlCl3 or SnCl4 and reaction of the corresponding product with α,α-diphenyl-4-piperidinomethanol. In the case of 7 and 8, the starting compounds were the acetates of 2-phenylethanol and 3-phenylpropanol, respectively. Deprotection of the alcohol function was done as the last step of the synthesis. Compound 5 was obtained from reaction of 11 with allyltributyltin, in the presence of tetrakis(triphenylphosphine)palladium(0).
The structures of all the terfenadone derivatives listed in Table 1 were completely established from their 1H NMR and mass spectra; 1H NMR spectroscopy analysis in the presence of an internal standard showed that all these compounds were more than 95% pure.
Table 1 compares the IC50 values measured for the inhibition of ebastine hydroxylation catalyzed by recombinant CYP2J2. It shows that most of the synthesized terfenadone derivatives are good CYP2J2 inhibitors with IC50 values at the low μM range. Compounds 4 and 5 had the highest affinity with an IC50 value of 0.4 μM. In fact, increasing the chain length from R = methyl to R = propyl results in a gradual decrease of the IC50 value, whereas a further increase of the chain length (R = butyl) leads to a loss of affinity. Introduction of a polar function in the R substituent generally leads to a decrease in the affinity of the inhibitors. Compounds such as 10 and 13 in which oxygen atoms have been introduced at benzylic positions exhibit IC50 values one order of magnitude greater than those observed for compounds bearing an alkyl chain (R = Et or Pr, 3 or 4 for instance). Compounds such as 7, 8, 9, and 12 in which an OH, OAc or F substituent have been introduced in the R-chain farther from the phenyl ring exhibit intermediate IC50 values, around 2 μM. Thus, the best inhibitors (in terms of IC50 value) were compounds 4 and 5. Preliminary experiments showed that compound 4 is a competitive inhibitor of CYP2J2-catalyzed hydroxylation of ebastine with a Ki of 160 ± 30 nM and also a competitive substrate of CYP2J2. Compound 5 seems to be a time-dependent inhibitor, as expected for a compound bearing a terminal double bond.14 Interestingly, compounds 12 and 13 involving a CHF2 and benzodioxole function, respectively, also led to time-dependent inhibitory effects that suggest a mechanism-based type of inhibition.
Table 2 compares the inhibitory effects of the best inhibitors found for CYP2J2, compounds 4 and 5, toward the other main human cytochromes P450 that are present in the cardiovascular system, CYP2C8, CYP2C9, CYP2B6, and CYP3A4.21 The data clearly show that compounds 4 and 5 are selective inhibitors of CYP2J2, as they are nearly inactive toward CYP2C8 and their IC50 values for CYP2C9, CYP2B6, and CYP3A4 are 1–3 orders of magnitude higher than those observed for CYP2J2.
In conclusion, the aforementioned results have led to the first selective, high-affinity inhibitors of CYP2J2, compounds 4 and 5, that exhibit IC50 values around 400 nM. Compound 4 is a competitive inhibitor characterized by a Ki of 160 nM, a value that is remarkably low for a human cytochrome P450 inhibitor.14 Additional studies are underway to determine the type of inhibition exhibited by compounds 5, 12, and 13, and to use these new inhibitors as tools to study the biological roles of CYP2J2 in vitro and in vivo. In light of the recent findings that CYP2J2 promotes the neoplastic phenotype of carcinoma cells, these compounds are also currently being investigated as potential anti-cancer therapeutics.