Design and synthesis of 6-methylpyridin-2-one derivatives as novel and potent GluN2A positive allosteric modulators for the treatment of cognitive impairment.

N-Methyl-D-aspartate receptors (NMDARs) are key regulators of synaptic plasticity in the central nervous system. Potentiation of NMDARs containing GluN2A subunit has been recently recognized as a promising therapeutic approach for neurological disorders. We identified a novel series of GluN2A positive allosteric modulator (PAM) with a pyridin-2-one scaffold. Initial lead compound 1 was discovered through in silico-based screening of virtual ligands with various monocyclic scaffolds. GluN2A PAM activity was increased by introduction of a methyl group at the 6-position of the pyridin-2-one ring and a cyano group in the side chain. Modification of the aromatic ring led to the identification of potent and brain-penetrant 6-methylpyridin-2-one 17 with a negligible binding activity for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Oral administration of 17 significantly enhanced rat hippocampal long-term potentiation (LTP). Thus, 17 would be a useful in vivo pharmacological tool to investigate complex NMDAR functions for the discovery of therapeutics toward diseases associated with NMDAR dysfunction.

Discovery of Pyrazolo[1,5-a]pyrazin-4-ones as Potent and Brain Penetrant GluN2A-Selective Positive Allosteric Modulators Reducing AMPA Receptor Binding Activity.

N-Methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family and play a crucial role in learning and memory by regulating synaptic plasticity. Activation of NMDARs containing GluN2A, one of the NMDAR subunits, has recently attracted attention as a promising therapeutic approach for neuropsychiatric diseases such as schizophrenia, depression, and epilepsy. In the present study, we developed potent and brain-penetrable GluN2A-selective positive allosteric modulators. Lead compound 2b was generated by scaffold hopping of hit compound 1, identified from the internal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-focused compound library through a high-throughput screening campaign. Subsequent optimization of the lead compound, including a structure-based drug design approach, resulted in the identification of a potent GluN2A PAM (R)-9, which possessed high selectivity against both subtypes of AMPAR and NMDAR. Furthermore, (R)-9 significantly enhanced long-term potentiation in the rat hippocampus 24 h after oral administration, indicating that this molecule is a potentially useful in vivo pharmacological tool for treating psychiatric diseases.

Discovery of Potent, Selective, and Brain-Penetrant 1 H-Pyrazol-5-yl-1 H-pyrrolo[2,3- b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors.

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is predominantly expressed in the brain and implicated in neuronal development and cognition. However, the detailed function of ALK in the central nervous system (CNS) is still unclear. To elucidate the role of ALK in the CNS, it was necessary to discover a potent, selective, and brain-penetrant ALK inhibitor. Scaffold hopping and lead optimization of N-(2,4-difluorobenzyl)-3-(1 H-pyrazol-5-yl)imidazo[1,2- b]pyridazin-6-amine 1 guided by a cocrystal structure of compound 1 bound to ALK resulted in the identification of (6-(1-(5-fluoropyridin-2-yl)ethoxy)-1-(5-methyl-1 H-pyrazol-3-yl)-1 H-pyrrolo[2,3- b]pyridin-3-yl)((2 S)-2-methylmorpholin-4-yl)methanone 13 as a highly potent, selective, and brain-penetrable compound. Intraperitoneal administration of compound 13 significantly decreased the phosphorylated-ALK (p-ALK) levels in the hippocampus and prefrontal cortex in the mouse brain. These results suggest that compound 13 could serve as a useful chemical probe to elucidate the mechanism of ALK-mediated brain functions and the therapeutic potential of ALK inhibition.

Development of two fluorine-18 labeled PET radioligands targeting PDE10A and in vivo PET evaluation in nonhuman primates.

INTRODUCTION: Phosphodiesterase 10A (PDE10A) is a member of the PDE enzyme family that degrades cyclic adenosine and guanosine monophosphates (cAMP and cGMP). Based on the successful development of [11C]T-773 as PDE10A positron emission tomography (PET) radioligand, in this study our aim was to develop and evaluate fluorine-18 analogs of [11C]T-773. METHODS: [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were synthesized from the same precursor used for 11C-labeling of T-773 in a two-step approach via 18F-fluoromethylation and 18F-fluoroethylation, respectively, using corresponding deuterated synthons. A total of 12 PET measurements were performed in seven non-human primates. First, baseline PET measurements were performed using High Resolution Research Tomograph system with both [18F]FM-T-773-d2 and [18F]FE-T-773-d4; the uptake in whole brain and separate brain regions, as well as the specific binding and tissue ratio between putamen and cerebellum, was examined. Second, baseline and pretreatment PET measurements using MP-10 as the blocker were performed for [18F]FM-T-773-d2 including arterial blood sampling with radiometabolite analysis in four NHPs. RESULTS: Both [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were successfully radiolabeled with an average molar activity of 293 ± 114 GBq/µmol (n=8) for [18F]FM-T-773-d2 and 209 ± 26 GBq/µmol (n=4) for [18F]FE-T-773-d4, and a radiochemical yield of 10% (EOB, n=12, range 3%-16%). Both radioligands displayed high brain uptake (~5.5% of injected radioactivity for [18F]FM-T-773-d2 and ~3.5% for [18F]FE-T-773-d4 at the peak) and a fast washout. Specific binding reached maximum within 30 min for [18F]FM-T-773-d2 and after approximately 45 min for [18F]FE-T-773-d4. [18F]FM-T-773-d2 data fitted well with kinetic compartment models. BPND values obtained indirectly through compartment models were correlated well with those obtained by SRTM. BPND calculated with SRTM was 1.0-1.7 in the putamen. The occupancy with 1.8 mg/kg of MP-10 was approximately 60%. CONCLUSIONS: [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were developed as fluorine-18 PET radioligands for PDE10A, with the [18F]FM-T-773-d2 being the more promising PET radioligand warranting further evaluation.

Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A inhibitors interacting with Tyr683 in the PDE10A selectivity pocket.

Utilizing structure-based drug design techniques, we designed and synthesized phosphodiesterase 10A (PDE10A) inhibitors based on pyridazin-4(1H)-one. These compounds can interact with Tyr683 in the PDE10A selectivity pocket. Pyridazin-4(1H)-one derivative 1 was linked with a benzimidazole group through an alkyl spacer to interact with the OH of Tyr683 and fill the PDE10A selectivity pocket. After optimizing the linker length, we identified 1-(cyclopropylmethyl)-5-[3-(1-methyl-1H-benzimidazol-2-yl)propoxy]-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (16f) as having highly potent PDE10A inhibitory activity (IC50=0.76nM) and perfect selectivity against other PDEs (>13,000-fold, IC50=>10,000nM). The crystal structure of 16f bound to PDE10A revealed that the benzimidazole moiety was located deep within the PDE10A selectivity pocket and interacted with Tyr683. Additionally, a bidentate interaction existed between the 5-alkoxypyridazin-4(1H)-one moiety and the conserved Gln716 present in all PDEs.

Development of a series of novel carbon-11 labeled PDE10A inhibitors.

Phosphodiesterase 10A (PDE10A) is a member of the PDE family of enzymes that degrades cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our aim was to label a series of structurally related PDE10A inhibitors with carbon-11 and evaluate them as potential positron emission tomography (PET) radioligands for PDE10A using nonhuman primates. The series consisted of seven compounds based on the 3-(1H-pyrazol-5-yl)pyridazin-4(1H)-one backbone. These compounds were selected from the initial larger library based on a number of parameters such as affinity, selectivity for hPDE10A in in vitro tests, lipophilicity, and on the results of multidrug resistance protein 1 (MDR1)-LLCPK1 and the parallel artificial membrane permeability assays. Seven radioligands (KIT-1, 3, 5, 6, 7, 9, and 12) were radiolabeled with carbon-11 employing O-methylation on the hydroxyl moiety using [(11)C]methyl triflate. In vivo examination of each radioligand was performed using PET in rhesus monkeys; analysis of radiometabolites in plasma also was conducted using HPLC. All seven radioligands were labeled with high (>90%) incorporation of [(11)C]methyl triflate into their appropriate precursors and with high specific radioactivity. Carbon-11 labeled KIT-5 and KIT-6 showed high accumulation in the striatum, consistent with the known anatomical distribution of PDE10A in brain, accompanied by fast washout and high specific binding ratio. In particular [(11)C]KIT-6, named [(11)C]T-773, is a promising PET tool for further examination of PDE10A in human brain.

Characterization of the binding properties of T-773 as a PET radioligand for phosphodiesterase 10A.

INTRODUCTION: Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP and is selectively expressed in striatal medium spiny neurons. Recent studies have suggested that PDE10A inhibition is a novel approach for the treatment of disorders such as schizophrenia and Huntington's disease. A positron emission tomography (PET) occupancy study can provide useful information for the development of PDE10A inhibitors. We discovered T-773 as a candidate PET radioligand for PDE10A and investigated its properties by in vitro autoradiography and a PET study in a monkey. METHODS: Profiling of T-773 as a PET radioligand for PDE10A was conducted by in vitro enzyme inhibitory assay, in vitro autoradiography, and PET study in a monkey. RESULTS: T-773 showed a high binding affinity and selectivity for human recombinant PDE10A2 in vitro; the IC50 value in an enzyme inhibitory assay was 0.77nmol/L, and selectivity over other PDEs was more than 2500-fold. In autoradiography studies using mouse, rat, monkey, or human brain sections, radiolabeled T-773 selectively accumulated in the striatum. This selective accumulation was not observed in the brain sections of Pde10a-KO mice. The binding of [(3)H]T-773 to PDE10A in rat brain sections was competitively inhibited by MP-10, a selective PDE10A inhibitor. In rat brain sections, [(3)H]T-773 bound to a single high affinity site of PDE10A with Kd values of 12.2±2.2 and 4.7±1.2nmol/L in the caudate-putamen and nucleus accumbens, respectively. In a monkey PET study, [(11)C]T-773 showed good brain penetration and striatum-selective accumulation. CONCLUSION: These results suggest that [(11)C]T-773 is a potential PET radioligand for PDE10A.

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists.

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.

Design, synthesis, and structure-activity relationships of dihydrofuran-2-one and dihydropyrrol-2-one derivatives as novel benzoxazin-3-one-based mineralocorticoid receptor antagonists.

Dihydrofuran-2-one and dihydropyrrol-2-one derivatives were identified as novel, potent and selective mineralocorticoid receptor (MR) antagonists by the structure-based drug design approach utilizing the crystal structure of MR/compound complex. Introduction of lipophilic substituents directed toward the unfilled spaces of the MR and identification of a new scaffold, dihydropyrrol-2-one ring, led to potent in vitro activity. Among the synthesized compounds, dihydropyrrol-2-one 11i showed an excellent in vitro activity (MR binding IC50=43nM) and high selectivity over closely related steroid receptors such as the androgen receptor (AR), progesterone receptor (PR) and glucocorticoid receptor (GR) (>200-fold for AR and PR, 100-fold for GR).

Identification of benzoxazin-3-one derivatives as novel, potent, and selective nonsteroidal mineralocorticoid receptor antagonists.

Mineralocorticoid receptor (MR) blockade has come into focus as a promising approach for the treatment of cardiovascular diseases such as hypertension and congestive heart failure. In order to identify a novel class of nonsteroidal MR antagonists that exhibit significant potency and good selectivity over other steroidal hormone receptors, we designed a novel series of benzoxazin-3-one derivatives and synthesized them from 6-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-benzoxazin-3(4H)-one (1a), high-throughput screening (HTS) hit compound. Our design was based on a crystal structure of an MR/compound complex and a docking model. In the course of lead generation from 1a, a 1,2-diaryl framework was characterized as a key structure with high binding affinity. On the basis of scaffold hopping and optimization studies, benzoxazin-3-one derivatives possessing 1-phenyl-3-trifluoromethylpyrazol-5-yl moiety at the 6-position were identified as a novel series of potent and selective MR antagonists. Among these compounds, 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n) showed highly potent activity and good selectivity and also exhibited a significant antihypertensive effect in deoxycorticosterone acetate-salt hypertensive rats. On the basis of these results, compound 14n was progressed for further pharmacological evaluation.

Acid-free, aminoborane-mediated Ugi-type reaction leading to general utilization of secondary amines.

A variety of secondary amines have become utilized in the Ugi reaction by using aminoborane 1 as an iminium ion generator. Aldehydes, secondary amines, and isocyanides are coupled in the presence of 1 at room temperature, giving the corresponding alpha-amino amides in good yields. The nonacidic reaction conditions are beneficial for unique chemoselectivity, where the aldimine functionality is left intact in the present Ugi-type reaction.