Yohei Kosugi

Synthesis of a Novel Series of Tricyclic Dihydrofuran Derivatives: Discovery of 8,9-Dihydrofuro[3,2-c]pyrazolo[1,5-a]pyridines as Melatonin Receptor (MT1/MT2) Ligands

Novel tricyclic dihydrofuran derivatives were designed, synthesized, and evaluated as melatonin receptor (MT(1)/MT(2)) ligands based on the previously reported 1,6-dihydro-2H-indeno[5,4-b]furan 1a. By screening the central tricyclic cores, we identified 8,9-dihydrofuro[3,2-c]pyrazolo[1,5-a]pyridine as a potent scaffold with a high ligand-lipophilicity efficiency (LLE) value. Subsequent optimization of the side chains led to identification of the potent MT(1)/MT(2) agonist 4d (MT(1), K(i) = 0.062 nM; MT(2), K(i) = 0.420 nM) with good oral absorption and blood-brain barrier (BBB) penetration in rats. The oral administration of compound 4d exhibited a sleep-promoting action in freely moving cats at 0.1 mg/kg.

Evaluation of cytochrome P450-mediated drug–drug interactions based on the strategies recommended by regulatory authorities

Herein, we aimed to evaluate the recently proposed risk assessment strategies of a cytochrome P450 (CYP) mediated drug–drug interaction (DDI) according to the European Medicines Evaluation Agency (EMEA) draft guideline, and discuss the differences between this guideline and the Food and Drug Administration (FDA) draft guidance. A retrospective study on reported 35 clinical DDI cases revealed that the EMEA assessment successfully predicts moderate-to-strong DDIs, i.e. drugs that cause more than 2-fold increase in the area under the curve in the presence and absence of CYP inhibitor (AUCi/AUC); however, EMEA tends to overlook weak DDIs with AUCi/AUC ≤ 2 to > 1.25. For CYP3A4 inhibitors, even clinically insignificant DDIs were overemphasized if the intestinal DDI is considered. The differences between unbound fraction in plasma and microsomes account for the discrepancies in DDI risk assessment results between EMEA and FDA assessments. Comparing two assessment results for CYP2D6 and CYP2C9 inhibitors, the FDA assessment suggested potential DDI risks for sulphinpyrazone and amitriptyline, while the EMEA assessment indicated no potential risk for these drugs. Through a retrospective study, we showed practical differences in the DDI assessment strategies of EMEA and FDA and suggested improvements in their current strategies.

Design and Synthesis of Potent Inhibitor of Apoptosis (IAP) Proteins Antagonists Bearing an Octahydropyrrolo[1,2-a]pyrazine Scaffold as a Novel Proline Mimetic.

To develop novel inhibitor of apoptosis (IAP) proteins antagonists, we designed a bicyclic octahydropyrrolo[1,2-a]pyrazine scaffold as a novel proline bioisostere. This design was based on the X-ray co-crystal structure of four N-terminal amino acid residues (AVPI) of the second mitochondria-derived activator of caspase (Smac) with the X-chromosome-linked IAP (XIAP) protein. Lead optimization of this scaffold to improve oral absorption yielded compound 45, which showed potent cellular IAP1 (cIAP1 IC(50): 1.3 nM) and XIAP (IC(50): 200 nM) inhibitory activity, in addition to potent tumor growth inhibitory activity (GI(50): 1.8 nM) in MDA-MB-231 breast cancer cells. X-ray crystallographic analysis of compound 45 bound to XIAP and to cIAP1 was achieved, revealing the various key interactions that contribute to the higher cIAPI affinity of compound 45 over XIAP. Because of its potent IAP inhibitory activities, compound 45 (T-3256336) caused tumor regression in a MDA-MB-231 tumor xenograft model (T/C: -53% at 30 mg/kg).

Design, Synthesis, and Biological Evaluation of a Series of Piperazine Ureas as Fatty Acid Amide Hydrolase Inhibitors

A series of piperazine ureas were designed, synthesized, and evaluated for their potential as novel orally efficacious fatty acid amide hydrolase (FAAH) inhibitors for the treatment of neuropathic and inflammatory pain. We carried out an optimization study of compound 5 to improve its in vitro FAAH inhibitory activity, and identified the 2-pyrimidinylpiperazine derivative 21d with potent inhibitory activity, favorable DMPK profile and brain permeability. Compound 21d showed robust and dose-dependent analgesic efficacy in animal models of both neuropathic and inflammatory pain.

Antitumor Activity and Pharmacodynamic Biomarkers of a Novel and Orally Available Small-Molecule Antagonist of Inhibitor of Apoptosis Proteins

Inhibitor of apoptosis proteins (IAP), which are key regulators of apoptosis, are inhibited by second mitochondria-derived activator of caspase (SMAC). Small-molecule IAP antagonists have recently been reported as novel therapeutic treatments for cancer. In this study, we showed that the octahydro-pyrrolo[1,2-a]pyrazine derivative, T-3256336, is a novel and orally available small-molecule IAP antagonist. T-3256336 selectively binds to and antagonizes protein interactions involving cellular IAP-1 (cIAP-1), cIAP-2, and X-linked IAP (XIAP). T-3256336 induced the rapid proteasomal degradation of cIAP-1 and activated TNF-α–dependent extrinsic apoptosis signaling in cultured cells. In a MDA-MB-231-Luc breast cancer xenograft model, T-3256336 induced cIAP-1 degradation, TNF-α production, and caspase activation in tumors, which resulted in strong antitumor activities. T-3256336 induced increases in the plasma levels of TNF-α and fragmented cytokeratin-18, which correlated with the antitumor potency in MDA-MB-231-Luc xenograft models. This study provided further insights into biomarkers of IAP antagonists. Furthermore, our data provided evidence that T-3256336 is a promising new anticancer drug worthy of further evaluation and development. Mol Cancer Ther; 12(2); 230–40. ©2012 AACR.

Design, synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells.

We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC50 23 nM and cellular IAP [cIAP]: IC50 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI50 2.8 nM) with high permeability and low potential of MDR1 substrate.

Evaluation of Acid Tolerance of Drugs Using Rats and Dogs Controlled for Gastric Acid Secretion

We attempted to establish animal models to evaluate the effects of drug degradation in the stomach on oral bioavailability. In addition, we assessed the utilization of animal studies in determining the need for enteric-coated formulations. In order to control the gastric pH in rats and dogs, appropriate dosing conditions were investigated using pentagastrin and rabeprazole, which stimulate and inhibit gastric acid secretion. Using animals controlled for gastric acid secretion, the area under curve (AUC) ratios (AUC with rabeprazole/AUC with pentagastrin) of all compounds unstable under acidic conditions were evaluated. The AUC ratios of omeprazole and erythromycin, which are administered orally to humans, as enteric-coated tablets, were greater than 1.9 in the rats and dogs controlled for gastric acid secretion. On the contrary, the AUC ratios of clarithromycin, azithromycin, and etoposide (commercially available as a standard immediate-release form) were less than 1.3 each. In conclusion, in vivo models using rats and dogs were optimized to evaluate the effects of gastric acid on the oral bioavailability of drugs, and demonstrated that in vivo models can lead to a better understanding of the oral bioavailability, with respect to the formulation development.

Species differences and substrate specificity of CYP3A heteroactivation by efavirenz

Abstract 1. The purpose of this study was to clarify species differences in the heteroactivation of CYP3A substrates by efavirenz, which is known from clinical studies to activate midazolam 1′-hydroxylation, and to assess the feasibility of an animal model. 2. In monkey and human liver microsomes, efavirenz activated CYP3A-mediated midazolam 1′-hydroxylation, but had no effect in rat liver microsomes. The activating effect of efavirenz was also observed with recombinant human CYP3A4 and CYP3A5. Midazolam 4-hydroxylation, testosterone 6β-hydroxylation and the oxidation of nifedipine were not activated by efavirenz in any of the microsomes. 3. In an in vivo study using monkeys, the AUC ratio of midazolam/1′-hydroxymidazolam was reduced from 0.85 to 0.30 by efavirenz treatment, which was comparable to that obtained in clinical studies. However, the AUC changes of midazolam caused by efavirenz were smaller than those observed in clinical results, therefore the effect of efavirenz on monkeys was not completely consistent with that seen in humans. 4. In conclusion, this is the first report that efavirenz specifically activates midazolam 1′-hydroxylation only in monkey and human liver microsomes, revealing marked species differences and high substrate specificity in the heteroactivation. A further study is required to clarify whether this in vitro result reflects the in vivo situation.

Risk assessment of drug–drug interactions using hepatocytes suspended in serum during the drug discovery process

Abstract 1. This study optimized the reported approach for the prediction of drug–drug interactions (DDIs) using hepatocytes suspended in serum (HHSS) and provided a practical usage of HHSS in the early and late phases of drug discovery. 2. First, the IC50 was determined using HHSS and evaluated as a qualitative index for DDI risks in the early phase. A retrospective study on clinical DDI cases revealed that inhibitors with IC50 < 100 μmol/L caused clinical DDIs while those with IC50 > 100 μmol/L showed weak or no potential for DDIs. Meanwhile, a pragmatic cutoff value could not be determined using previously reported Ki values of recombinant human cytochrome P450s. 3. Second, for a more substantial DDI risk assessment in the later phase, quantitative predictions of clinical DDI based on a static model were attempted by optimizing the most appropriate inhibitor concentration ([I]). The use of hepatic input plasma concentrations as a surrogate for [I] achieved the most successful predictions of the magnitude of increase in the AUC (within a 2-fold range of the observed values for 93.8% of inhibitors). 4. Through this study, we proposed the practical application of HHSS for an effective workflow to explore and profile candidates with less DDI liability.

Discovery of 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole, a novel CRF1 receptor antagonist

Compound 1 exhibits potent binding inhibition activity against a corticotropin-releasing factor 1 (CRF1) receptor (IC50=9.5nM) and in vitro antagonistic activity (IC50=88nM) but is rapidly metabolized by human hepatic microsomes (182μL/min/mg). Here we identified metabolically stable compounds with potent CRF binding inhibitory activity. Structure-activity relationship (SAR) studies considering in vitro metabolic stability revealed that 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole 24d was more stable in human microsomes (87μL/min/mg) than compound 1. Compound 24d demonstrated potent CRF binding inhibitory activity (IC50=4.1nM), in vitro antagonistic activity (IC50=44nM), and slow dissociation from the CRF1 receptor. Orally administered compound 24d (6-24μmol/kg) showed ex vivo CRF1 receptor binding in the rat pituitary, olfactory bulb, and frontal cortex and suppressed stress-induced adrenocorticotropic hormone (ACTH) secretion. In this report, we discuss SAR studies on the metabolic stability as well as CRF binding inhibitory activity of the benzimidazole series as CRF1 receptor antagonists and the pharmacological profiles of compound 24d.