Kazunobu Aoyama

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).

Discovery of the First Potent and Orally Available Agonist of the Orphan G-Protein-Coupled Receptor 52

G-protein-coupled receptor 52 (GPR52) is an orphan Gs-coupled G-protein-coupled receptor. GPR52 inhibits dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation, and therefore, GPR52 agonists may have potential as a novel class of antipsychotics. A series of GPR52 agonists with a bicyclic core was designed to fix the conformation of the phenethyl ether moiety of compounds 2a and 2b. 3-[2-(3-Chloro-5-fluorobenzyl)-1-benzothiophen-7-yl]-N-(2-methoxyethyl)benzamide 7m showed potent activity (pEC50 = 7.53 ± 0.08) and good pharmacokinetic properties. Compound 7m significantly suppressed methamphetamine-induced hyperactivity in mice after oral administration of 3 mg/kg without disturbance of motor function.

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.

In vitro and in vivo antitumor activities of T-3764518, a novel and orally available small molecule stearoyl-CoA desaturase 1 inhibitor

Abstract Most cancer cells are characterized by elevated lipid biosynthesis. The rapid proliferation of cancer cells requires de novo synthesis of fatty acids. Stearoyl‐CoA desaturase‐1 (SCD1), a key enzyme for lipogenesis, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. Therefore, it has been studied as a candidate target for cancer therapy. In this study, we demonstrate the pharmacological properties of T‐3764518, a novel and orally available small molecule inhibitor of SCD1. T‐3764518 inhibited stearoyl‐CoA desaturase‐catalyzed conversion of stearoyl‐CoA to oleoyl‐CoA in colorectal cancer HCT‐116 cells and their growth. Further, it slowed tumor growth in an HCT‐116 and a mesothelioma MSTO‐211H mouse xenograft model. Comprehensive lipidomic analyses revealed that T‐3764518 increases the membrane ratio of saturated: unsaturated fatty acids in various lipid species such as phosphatidylcholines and diacylglycerols in both cultured cells and HCT‐116 xenografts. Treatment‐associated lipidomic changes were followed by activated endoplasmic reticulum (ER) stress responses such as increased immunoglobulin heavy chain‐binding protein expression in HCT‐116 cells. These T‐3764518‐induced changes led to an increase in cleaved poly (ADP‐ribose) polymerase 1 (PARP1), a marker of apoptosis. Additionally, bovine serum albumin conjugated with oleic acid, an SCD1 product, prevented cell growth inhibition and ER stress responses by T‐3764518, indicating that these outcomes were not attributable to off‐target effects. These results indicate that T‐3764518 is a promising new anticancer drug candidate.

Design, synthesis, and pharmacological evaluation of 4-azolyl-benzamide derivatives as novel GPR52 agonists

G protein-coupled receptor 52 (GPR52) agonists are expected to improve the symptoms of psychiatric disorders. During exploration for a novel class of GPR52 agonists with good pharmacokinetic profiles, we synthesized 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methylbenzamide (4u; half maximal effective concentration (EC50)=75nM, maximal response (Emax)=122%) starting from a high-throughput screening hit 3 (EC50=470nM, Emax=56%). The structural features of a reported GPR52 agonist were applied to 3, led to design 4-azolylbenzamides as novel GPR52 agonists. A structure-activity relationship study of 4-azolylbenzamide resulted in the design of the 1,2,4-triazole derivative 4u, which demonstrated excellent bioavailability in rats (F=53.8%). Oral administration of 4u (10mg/kg) significantly suppressed methamphetamine-induced hyperlocomotion in mice. Thus, 4u is a promising lead compound for drug discovery research of GPR52 agonists.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.

Design and synthesis of 1-(1-benzothiophen-7-yl)-1 H -pyrazole, a novel series of G protein-coupled receptor 52 (GPR52) agonists

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC50 = 21 nM, Emax = 103%, logD = 2.21, Solubility at pH 6.8 = 21 μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.