Gratifyingly compound also demonstrated improved physicochem

Gratifyingly, compound also demonstrated improved physicochemical properties with significant improvement in aqueous solubility. Compound was sufficiently potent to determine its pharmacokinetic (PK) and pharmacodynamics (PD); data is summarized in . shows ADME characteristics for compound and as a result of reduced hydrophobicity. Indeed, while the introduction of 4-pyran in the place of CF3-enhances the cell potency, and maintained its selectivity. A key issue at this stage was poor oral exposure, low bioavailability and reduced passive permeability with increased plasma clearance. This presented us with conundrum in that although the pyran ring was the most efficient replacement for the aryl ring in terms of hydrophobicity/potency (LogP = 0.95), it suffered from poor permeability and low oral exposure. Compound had been shown to be a substrate for P-glycoprotein (P-gp) in vitro (), but this did not appear to impact its oral absorption. With the decrease in Ppass of compound and increase in efflux ratio, we suspected that oral Dihydro-β-erythroidine hydrobromide receptor of compound might be impacted by P-gp mediated efflux. Therefore, to confirm oral absorption impacted by increased P-gp efflux, we examined oral exposure of compound in P-gp and breast cancer resistance protein (Bcrp) KO mice. Compound had 5-fold greater plasma exposure in P-gp KO mice compared to normal mice at the same dose (). Bcrp alone and together with P-gp was not a factor, which was consistent with in vitro results showing that compound is not a Bcrp substrate. Both passive permeability and P-gp substrate recognition appeared to change with relative constancy of hydrogen bond (HB) count and topological polar surface area (TPSA). So, based upon a negative relationship between Ppass and total HB strength and a positive relationship between P-gp efflux ratio and total HB strength (, ). We focused on reducing total HB strength especially with regard to imidazole substitutions. So, we decided to revisit the fluorinated alkyl substituent that could maintain the potency with improved permeability. We reasoned that if we could extend the alkyl chain farther (from simple CF3 substitution ) while reaching into the enzyme p-loop pocket deep enough, then that should maintain its activity similar to the aryl () or cyclic ring () system. In addition, such a fluorinated alkyl group with reduced polarity and hydrogen bond accepting strength should improve passive permeability and decrease the P-gp effect. To validate our design hypothesis, we synthesized trifluoropropyl substituted compound as shown in . In vivo PK parameters obtained in rat after intravenous administration at 10 mg/kg and oral dosing at 5 mg/kg are highlighted in . Replacing the fluorinated aryl ring and hydrophilic 4-pyranyl in with an appropriate fluorinated alkyl led to compound displaying improved permeability and oral bioavailability in rat as expected. PK attributes were also favorable in mouse and dog (data not shown in manuscript preparation). In addition, compound demonstrated enhanced cell potency and ideal physicochemical properties. Based on these encouraging results compound was tested in vivo for efficacy in a 4 weeks U87MG rat xenograft model. Oral doses were chosen to be 25, 50 mg/kg once or 25 mg/kg twice, daily as crystalline suspensions in Hydroxyethyl cellulose (HEC). Tumor volume was measured relative to controls animal treated with HEC vehicle only. Compound induced dose-dependent growth inhibition and was well tolerated with no body-weight loss observed at all doses and dose regimens tested (). The synthesis of final compounds – is outlined in , , . The synthesis of enantiomerically pure ()- and ()-4-chloro-5-R-6,8-dihydro-5-pyrido[2,3-]pyrimidin-7-one started from commercially available propanedioic acid dimethyl ester subjected for Michael addition with methyl crotonate (()-methyl but-2-enoate) followed by an in situ ring formation to give . Compound was prepared by chlorination of the hydroxyl group with POCl in the presence of base such as ,-diethylaniline. The racemic compound was obtained by treating excess amount of aqueous ammonium hydroxide. In order to improve the throughput of the chiral resolution was further protected as -Boc derivative by treating with Boc anhydride in the presence of DMAP to provide . The chiral lactam was prepared by chiral chromatography and subsequent removal of the protecting group under acidic condition furnished the final compound with good optical purity.