The and configurations seem to be more preferable for the association to CYP3A4, as the conformers tend to ligate with the unfavorable reverse side-group orientation. Finally, for the first time, this study demonstrated that less structurally complex inhibitors that are more potent than ritonavir could be rationally developed. (iii) the relationship between the R1/R2 configuration and preferential binding to CYP3A4 is complex and depends on the side-group functionality/interplay and backbone spacing; and (iv) three inhibitors, 5a-b and 7d, were superior to ritonavir (IC50 of 0.055C0.085 M vs. 0.130 M, respectively). configuration if the hydroxyl group is removed. Based on our previous studies on the interaction of CYP3A4 with ritonavir and its analogues [9C12], we developed a pharmacophore model for a CYP3A4-specific inhibitor [13] and utilized a build-from-scratch approach to elucidate the relative importance of each pharmacophoric determinant. Three groups of inhibitors (series I-III) with different backbones and side-groups attached to the pyridine ring, serving as the heme-ligating moiety, MK-2894 have been already characterized [14C16]. These studies demonstrated that the binding and inhibitory strength of ritonavir-like compounds depends on the backbone length and composition, spacing between the functional groups, H-bonding to the active site Ser119, hydrophobic interactions mediated by the R1/R2 side-groups and, to a lesser degree, their stereo configuration. The current study was designed to test one of the earlier predictions that an increase in the R2 hydrophobicity could improve the inhibitory strength [15, 16]. Eight (series IV) analogues were developed by modifying the R2 functionality in two different scaffolds used for synthesis of 8f, the most potent series II inhibitor [15], and 4e-h, the high-affinity subgroup from series III [16] (Figure 1). Here we report that, indeed, compounds with the larger, more hydrophobic naphthalene ring at R2 position tend to bind tighter and inhibit CYP3A4 more potently MK-2894 than their phenyl- or indole-containing counterparts. The backbone spacing and side-group configuration were other factors that strongly influenced the inhibitory strength MK-2894 and preferential binding to CYP3A4. Most importantly, for the first time, this study identified three compounds that were chemically simpler than ritonavir but inhibited CYP3A4 twice as stronger. MATERIALS AND METHODS Chemistry General Methods C All reactions were performed with commercially available reagents (Aldrich, Thermo-Fisher, Alfa Aesar, Acros, Oakwood, FZD10 Millipore) without further purification. Anhydrous solvents were acquired through a solvent purification system (Inert MK-2894 PureSolv and JC Meyer systems) or purified according to standard procedures. 1H NMR spectra were recorded on Bruker DRX 400 MHz, Bruker DRX 500 MHz, or Bruker Avance 600 MHz spectrometer and processed using TopSpin 3.5 software. LRMS and HRMS data were obtained via ESI LC-TOF on a Waters (Micromass) LCT Premier spectrometer (Waters), with PEG as the calibrant for HRMS. Optical rotation was recorded on a Rudolph Autopol III Automatic Polarimeter at room temperature in methanol. TLC was performed using EMD Millipore silica gel 60 F254 aluminum plates. Separation by column chromatography was conducted using Fisher silica MK-2894 gel 60 (230C400 mesh). Purity of final products was verified by 1H NMR with TMS as a standard and by UPLC-MS (Waters Acquity UPLC H-class QDA with a FTN) with a C18 column (2.1 x 50 mm; Acquity UPLC BEH C18; 1.7 m particles). All investigated compounds were 95% pure as determined by UPLC-MS. High resolution mass spectrometry data, NMR spectra, and UPLC-MS chromatograms are included in the Supplementary Material. Synthesis of analogues Typical procedure for amino alcohols from amino acids (compounds 1a-e)[17, 18] C To a flask containing DL-1-Naphthylalanine (1a) (1.0 g, 4.6 mmol), TMSCl (1.51 g, 14 mmol, 3 eq) was added, followed by anhydrous methanol (10 mL). The reaction was allowed to stir at room temperature overnight. Upon completion, the volatiles were removed affording 2a as a white powder (0.87 g, 94%), which was used without further purification. 1H NMR (400 MHz, CDCl3T) 8.05 (d, = 7.0 Hz, 1H), 7.88 (d, = 9.1.