An Approach to Discovering Novel Muscarinic M1 Receptor Positive Allosteric Modulators with Potent Cognitive Improvement and Minimized Gastrointestinal Dysfunction.

Activation of muscarinic M1 receptor (M1R) is a promising approach for improving cognitive impairment in Alzheimer's disease. However, an M1R-selective positive allosteric modulator (PAM), benzyl quinolone carboxylic acid (BQCA), at 30 mg/kg, induced diarrhea in wild-type mice, but not in M1R knockout mice. Moreover, BQCA (0.1-1000 nM) augmented electric field stimulation (EFS)-induced ileum contraction in an in vitro Magnus assay. Thus, we decided to establish a drug-screening strategy to discover novel M1 PAMs producing potent cognitive improvement with minimized gastrointestinal (GI) dysfunction. We assessed PAM parameters of various M1 PAMs with ≥100-fold selectivity over other muscarinic receptor subtypes by using in vitro binding and functional analysis. Evaluation of these M1 PAMs in the Magnus assay revealed a significant correlation between percentage of ileum contractions at 1 µM and their α-value, a PAM parameter associated with the binding cooperativity between acetylcholine and M1 PAM. M1 PAMs with lower α-value showed lower impact on EFS-induced ileum contraction. Next, we characterized in vivo profiles of two M1 PAMs: compound A (log α = 1.18) and compound B (log α = 3.30). Compound A, at 30 mg/kg, significantly improved scopolamine-induced cognitive deficits without prominent signs of diarrhea at up to 1000 mg/kg in mice. In contrast, compound B, at 10 mg/kg, showed both significant improvement of scopolamine-induced cognitive deficits and severe diarrhea. Thus, fine adjustment of the α-values could be a key to discovering M1 PAMs yielding potent cognitive improvement with a lower risk of GI effects.

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists.

A new class of corticotropin releasing factor 1 (CRF1) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a-e were designed as CRF1 receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF1 receptor antagonist. The synthesized tricyclic derivatives 2a-e showed CRF1 receptor binding activity with IC50 values of less than 400 nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF1 receptor binding activity (IC50 = 7.1 nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF1 receptor binding activity (IC50 = 58 nM) with good oral bioavailability (F = 68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [125I]-CRF binding in the frontal cortex (5 and 10 mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10 mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF1 receptors in the brain and exhibits the potential to be further examined for clinical studies.

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.

Discovery of a 7-arylaminobenzimidazole series as novel CRF1 receptor antagonists.

A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing factor 1 (CRF1) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of 1, predicted to occupy a large lipophilic pocket of a CRF1 receptor, with an aryl group. During the course of this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhibitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog 16g, showed potent CRF binding inhibitory activity against a human CRF1 receptor and human CRF1 receptor antagonistic activity (IC50=27nM, 56nM, respectively). This compound exhibited ex vivo (125)I-Tyr(0) ((125)I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland at 20mg/kg after oral administration. In this report, we discuss the structure-activity-relationship of these 7-arylamino-1H-benzimidazoles and their synthetic method.

Discovery of pyrrolo[2,3-d]pyrimidin-4-ones as corticotropin-releasing factor 1 receptor antagonists with a carbonyl-based hydrogen bonding acceptor.

A new class of pyrrolo[2,3-d]pyrimidin-4-one corticotropin-releasing factor 1 (CRF(1)) receptor antagonists has been designed and synthesized. In general, reported CRF(1) receptor antagonists possess a sp(2)-nitrogen atom as hydrogen bonding acceptor (HBA) on their core scaffolds. We proposed to use a carbonyl group of pyrrolo[2,3-d]pyrimidin-4-one derivatives as a replacement for the sp(2)-nitrogen atom as HBA in classical CRF(1) receptor antagonists. As a result, several pyrrolo[2,3-d]pyrimidin-4-one derivatives showed CRF(1) receptor binding affinity with IC(50) values in the submicromolar range. Ex vivo (125)I-sauvagine binding studies showed that 2-(dipropylamino)-3,7-dimethyl-5-(2,4,6-trimethylphenyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (16b) (30 mg/kg, p.o.) was able to penetrate into the brain and inhibit radioligand binding to CRF(1) receptors (frontal cortex, olfactory bulb, and pituitary) in mice. We identified pyrrolo[2,3-d]pyrimidin-4-one derivatives as the first CRF(1) antagonists with a carbonyl-based HBA.

T-495, a novel low cooperative M1 receptor positive allosteric modulator, improves memory deficits associated with cholinergic dysfunction and is characterized by low gastrointestinal side effect risk.

M1 muscarinic acetylcholine receptor (M1 R) activation can be a new therapeutic approach for the treatment of cognitive deficits associated with cholinergic hypofunction. However, M1 R activation causes gastrointestinal (GI) side effects in animals. We previously found that an M1 R positive allosteric modulator (PAM) with lower cooperativity (α-value) has a limited impact on ileum contraction and can produce a wider margin between cognitive improvement and GI side effects. In fact, TAK-071, a novel M1 R PAM with low cooperativity (α-value of 199), improved scopolamine-induced cognitive deficits with a wider margin against GI side effects than a high cooperative M1 R PAM, T-662 (α-value of 1786), in rats. Here, we describe the pharmacological characteristics of a novel low cooperative M1 R PAM T-495 (α-value of 170), using the clinically tested higher cooperative M1 R PAM MK-7622 (α-value of 511) as a control. In rats, T-495 caused diarrhea at a 100-fold higher dose than that required for the improvement of scopolamine-induced memory deficits. Contrastingly, MK-7622 showed memory improvement and induction of diarrhea at an equal dose. Combination of T-495, but not of MK-7622, and donepezil at each sub-effective dose improved scopolamine-induced memory deficits. Additionally, in mice with reduced acetylcholine levels in the forebrain via overexpression of A53T α-synuclein (ie, a mouse model of dementia with Lewy bodies and Parkinson's disease with dementia), T-495, like donepezil, reversed the memory deficits in the contextual fear conditioning test and Y-maze task. Thus, low cooperative M1 R PAMs are promising agents for the treatment of memory deficits associated with cholinergic dysfunction.

TAK-071, a novel M1 positive allosteric modulator with low cooperativity, improves cognitive function in rodents with few cholinergic side effects.

The muscarinic M1 receptor (M1R) is a promising target for treating cognitive impairment associated with cholinergic deficits in disorders such as Alzheimer's disease and schizophrenia. We previously reported that cooperativity (α-value) was key to lowering the risk of diarrhea by M1R positive allosteric modulators (M1 PAMs). Based on this, we discovered a low α-value M1 PAM, TAK-071 (α-value: 199), and characterized TAK-071 using T-662 as a reference M1 PAM with high α-value of 1786. Both TAK-071 and T-662 were potent and highly selective M1 PAMs, with inflection points of 2.7 and 0.62 nM, respectively. However, T-662 but not TAK-071 augmented isolated ileum motility. TAK-071 and T-662 increased hippocampal inositol monophosphate production through M1R activation and improved scopolamine-induced cognitive deficits in rats at 0.3 and 0.1 mg/kg, respectively. TAK-071 and T-662 also induced diarrhea at 10 and 0.1 mg/kg, respectively, in rats. Thus, taking into consideration the fourfold lower brain penetration ratio of T-662, TAK-071 had a wider margin between cognitive improvement and diarrhea induction than T-662. Activation of M1R increases neural excitability via membrane depolarization, reduced afterhyperpolarization, and generation of afterdepolarization in prefrontal cortical pyramidal neurons. T-662 induced all three processes, whereas TAK-071 selectively induced afterdepolarization. Combining sub-effective doses of TAK-071, but not T-662, with an acetylcholinesterase inhibitor, significantly ameliorated scopolamine-induced cognitive deficits in rats. TAK-071 may therefore provide therapeutic opportunities for cognitive dysfunction related to cholinergic deficits or reduced M1R expression, while minimizing peripheral cholinergic side effects.

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats.

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF1 receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF1 receptor binding affinities (15 and 10nM) and functional activities in reporter gene assay (15 and 9.5nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10mg/kg inhibited [125I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [125I]-CRF binding, indicating that only Compound A inhibited central [125I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF1 receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 µM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 µmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.