A novel series of RORγ inhibitors was identified starting with the HTS hit 1. After SAR investigation based on a prospective consideration of two drug-likeness metrics, ligand efficiency (LE) and fraction of sp 3 carbon atoms (Fsp 3 ), significant improvement of metabolic stability as well as reduction of CYP inhibition was observed, which finally led to discovery of a selective and orally efficacious RORγ inhibitor 3z.KEYWORDS: Th17, immunological diseases, nuclear receptor, RORγ, ligand efficiency (LE), fraction of sp 3 carbon atoms (Fsp 3 )T wo decades after the discovery of Th1 and Th2 cells, a third subset of T helper cells called Th17 cells was identified and has drawn considerable attention since it was suggested to play a central role in the pathogenesis of various autoimmune diseases such as psoriasis and rheumatoid arthritis. 1,2 Among several regulatory pathways in which Th17 development and function are involved, the one regulated by the nuclear receptor RORγ appears to be crucial for controlling the differentiation and function. 3 Given its validity as an emerging drug target for treatment of immunological diseases, many research groups have made significant efforts in the discovery of RORγ modulators in recent years. 4−19 Since starting our RORγ inhibitor program in 2003, we discovered several structurally diverse hits after a HTS campaign. 20 From these hits we selected compound 1 as the first hit-to-lead series for optimization. In addition to being reasonably potent against RORγ (hLUC EC 50 = 1.7 μM, FRET EC 50 = 0.85 μM), compound 1 also demonstrated >20-fold selectivity over five nuclear receptors (hRORα, hFXR, hRXRα, hPR, and hPPARγ) and was structurally unique in comparison to other nuclear receptor modulators. 16−18 However, this compound has several drawbacks. For example, the microsomal stability in liver microsomes is poor with only 18% remaining at 10 min in human liver microsomes. It also has a modest time-dependent human CYP3A4 inhibition (IC 50 = 4 μM) probably due to some reactive metabolites formed by the oxidation of 1. The ligand efficiency is only 0.25, far below the literature consensus value (0.30) for a drug-like molecule. 21 The concept of ligand efficiency (LE) was first introduced by Kuntz 22 and is widely accepted as a reliable index of drug-like qualities. 23 Improvement of LE inevitably results in lower molecular weight and higher potency. We reasoned that a strategy of increasing LE and lowering the lipophilicity should therefore significantly improve the drug-like properties of compound 1. In addition, compound 1 is a rather flat molecule with a fraction of saturated carbons (Fsp 3 ) of 0.24. Fsp 3 is a newer index representing drug-likeness. 24 Lovering et al. pointed out that a decrease of Fsp 3 value would result in an increased incidence of CYP inhibition. 25 The desired Fsp 3 value is over 0.47 according to the literature. 24 Thus, we considered that improvement of the poor Fsp 3 value of compound 1 would be a rational way to overcome the CYP inhibi...

Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor ␣ (RXR␣) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at Ϫ7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them ␣-half-sites. Introduction of a mutation into either an ␣ or ␤ half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXR␣ heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with ␣ and ␤ half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.

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-CYP3A4 is an important drug-metabolizing enzyme induced by various compounds causing drug-drug interactions. However, the molecular mechanism of CYP3A4 induction is not completely understood. CYP3A4 induction is caused by pregnane X receptor (PXR) through binding to some PXR binding elements. These elements comprise an everted repeat separated by six nucleotides in the promoter region and distal nuclear receptor binding element 1 (dNR-1) as well as the essential distal nuclear receptor binding element for CYP3A4 induction (eNR3A4) in the enhancer region of the CYP3A4 gene. Recently, we found that polycyclic aromatic hydrocarbons including anthracene induce CYP3A4 in HepG2 cells with a different induction profile from that of rifampicin (RF), a typical PXR ligand. When a CYP3A4 reporter plasmid in which the eNR3A4 DNA fragment binds directly to the CYP3A4 promoter (-362 bases) was evaluated in a reporter assay, dibenz [a,h]anthracene (DBA) induced reporter activity, while RF did not. To be induced reporter activity by RF, more 14 nucleotides 5′ upstream of the eNR3A4 (rifampicin eNR3A4: reNR3A4) DNA fragment were required. However, eNR3A4 and reNR3A4 did not respond to recombinant PXR without dNR-1. These results suggest that eNR3A4 and reNR3A4 are necessary for CYP3A4 induction by DBA and RF, respectively, and that dNR-1 is indispensable for full induction through PXR.