Single Heteroatom Substitutions in the Efavirenz Oxazinone Ring Impact Metabolism by CYP2B6

Abstract

Previously, we observed that the oxazinone ring is important for CYP2B6 activity toward efavirenz ((4S)-6-Chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one), a CYP2B6 substrate used to treat HIV. Here, to further understand the structural characteristics of efavirenz that render it a CYP2B6 substrate, we test the importance of each heteroatom of the oxazinone ring. We assembled a panel of five analogues: 6-Chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-2-methyl-4-(trifluoromethyl)-2H-3,1-benzoxazine (1), (4S)-6-Chloro-4-[(1E)-2-cyclopropylethenyl]-3,4-dihydro-4-(trifluoromethyl)-,2(1H)-quinazolinone (2), (4S)-6-Chloro-4-(2-cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (3), 6-Chloro-4-(cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinolinone (4), and 6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-4H-benzo[d][1,3]dioxin-2-one (5). Metabolism of 1–5 was investigated using human liver microsomes, individual P450s, and mass spectrometry or UV absorbance detection. Steady-state analysis of CYP2B6 metabolism of 1–5 showed KM values ranging from 0.3 to 3.9 fold different than observed for efavirenz (KM of 3.6 ± 1.7 μM). The lowest KM values approximating 1 μM, were observed for metabolism of 1, while the largest KM, 14 ± 6.4 μM, was found for 4. Our work reveals that analogues with heteroatom changes in the oxazinone ring are still CYP2B6 substrates, though the changes KM suggest altered substrate binding.

Crystal Structure of CYP2B6 in Complex with an Efavirenz Analog

“…Interestingly, the chlorine of the efavirenz analog 2-desoxo-2-methyl (abbreviated as I for convenience) forms a π bond with F108 in a similar fashion to that illustrated in our recent study using monoterpenes [ 21 ]. The orientation of the methyl group towards the heme-iron corroborates the putative metabolic transformation at the oxazinone ring shown more recently by Cox and Bumpus [ 20 ]. The trifluoro group was located near the I101 and F115 side chains, whereas the cyclopropyl group was placed between the side chains of F206 and T302.…”

“…To extend this study to halogenated drugs that are major substrates of CYP2B6, we determined the crystal structure of CYP2B6 in complex with an analog of the important anti-retroviral drug efavirenz. The 2-desoxo-2-methyl analog contains a methyl group in place of the carbonyl oxygen in efavirenz [ 20 ]. The crystal structure revealed how CYP2B6 accommodates this important drug analog in the active site near the heme ( Figure 1 ).…”

“…A recent study investigated the chemical features of efavirenz that make crucial contacts in the active site of this enzyme [ 19 ]. The results further demonstrated the importance of the oxazinone ring and the ability of the CYP2B family of enzymes to metabolize certain efavirenz analogs predominantly compared to any other families of CYPs [ 20 ]. In this study, we report a crystal structure of CYP2B6 in complex with an analog of efavirenz.…”

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

“…CYP2B6 is pharmacologically and clinically relevant, and several CYP2B6 substrates are chiral, with varying degrees of enantioselective metabolism, and enantioselectivity may vary among different CYP2B6 variants (Wang et al, 2018). As a prototypic CYP2B6 substrate, Sefavirenz and analogs have been used to inform on the structure, activity, and catalytic mechanism of wild-type CYP2B6 (Bumpus and Hollenberg, 2008;Cox and Bumpus, 2014;Cox and Bumpus, 2016;Shah et al, 2018) and variants such as CYP2B6.4 (Bumpus et al, 2005). These compounds, together with molecular modeling, have provided insights into the active site configuration of CYP2B6.…”

The Reaction of o‐Aminoacetophenone N‐Tosylhydrazone and CO2 toward 1,4‐Dihydro‐2H‐3,1‐benzoxazin‐2‐ones

“…1,4-Dihydro-2H-3,1-benzoxazin-2-one shows abundant potential biological activities such as progesterone receptor antagonist, [10] HIV-1 reverse transcriptase inhibitor (Scheme 1). [11] Typical synthetical methods includes the catalytic chlorocyclization of 2vinylphenylcarbamates, [12] the selenium-catalyzed oxidative carbonylation of 2-aminobenzyl alcohol, [13] the copper(I)/N-heterocyclic carbene-catalyzed addition of terminal alkynes to trifluoromethyl ketones, [14] and the intramolecular conversion of N,N-bis (2-picolyl)ureas to cyclic carbamates. [15] To the best of our knowledge, example on the application of CO 2 as a C1 source to access such frameworks has never been reported so far.…”