Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe

Abstract

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M 1 PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M 1 PAM with an in vitro profile comparable to the prototypical M 1 PAM, BQCA, but with an improved brain to plasma ratio.The muscarinic acetylcholine receptors (mAChRs) are members of the family A G-proteincoupled receptors (GPCRs) and include five subtypes denoted M 1 -M 5 . All five of the mAChRs are known to play critical roles in multiple basic physiological processes and represent attractive therapeutic targets for a number of peripheral and CNS pathologies.1 -3 Within the mAChRs, a major challenge has been a lack of subtype selective ligands to study the specific contribution of discrete mAChRs in various disease states.3 , 4 To address this limitation, we have focused on targeting allosteric sites on mAChRs as a means to develop subtype selective small molecules, both allosteric agonists and positive allosteric modulators (PAMs).5 -9 Moreover, the emerging phenomenon of ligand-biased signaling requires the development of diverse chemical scaffolds of M 1 ligands to successfully dissect of the roles of M 1 activation through multiple, discrete ligand-biased signaling pathways.10 , 11As members of the Molecular Libraries Production Center Network (MLPCN),12 we performed a real-time cell-based calcium-mobilization assay employing a rat M 1 /CHO cell Correspondence to: Michael R. Wood. † these authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Perusal of the HTS data, which also yielded the non-selective hit 5, identified a second weak M 1 PAM hit 7, VU0108370, with an EC 50 of ∼13 μM. Confirmation of 7 from fresh powder and counter-screening against M 2 -M 5 increased our enthusiasm for this highly M 1 mAChR selective hit (Fig. 2); however, the CRC did not plateau, suggesting the M 1 EC 50 was actually >13 μM. Despite the weak potency, the confirmation of a novel M 1 PAM scaffold with high M 1 selectivity initiated a lead optimization campaign to improve M 1 potency while maintaining the high M 2 -M 5 selectivity. NIH Public AccessOur initial optimization strategy is outlined in Figure 3, and as SAR with allosteric ligands is often shallow, we employed an iterative parallel synthesis approach, along with targeted syntheses for structures encompassing more speculative modifications. Attempted mod...

Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs

“…54,55 Here, commercial 4,6-difluoroindole 18 is a linchpin to access diversely functionalized derivatives 21 , 24 , and 27 . Analogues 21 are readily prepared in four steps by first deprotonation of 18 and alkylation with the desired benzyl bromide to provide 19 .…”

“…22,23,40–44,54 To ensure sufficient potentiation of M 1 was present in the aforementioned rat AHL studies (dosed at 30 mg/kg), we evaluated 16 and 28a in the novel object recognition paradigm in rats (Figure 9). Here, at doses of 1, 3, and 10 mg/kg, 16 did not enhance cognitive performance in this task ( p = 0.0529), but did trend toward significance.…”

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

“…Achieving high-quality small-molecule probes required significant medicinal chemistry optimization of the initial screening hits, leading to the identification of an M1-selective PAM (ML169; Fig. 3) (Bridges et al , 2010; Reid et al , 2011; Tarr et al , 2012), an M4-selective PAM (ML253; Fig. 3) (Le et al , 2013; Niswender et al , 2010) and an M5-selective PAM (ML326; Fig.…”

Further optimization of the M1 PAM VU0453595: Discovery of novel heterobicyclic core motifs with improved CNS penetration

“…27 Subsequent optimization efforts identfied ‘molecular switches’ that gave rise to a series of highly selective M 5 PAMs, 30-32 as well as ML137 ( 2 ), a highly selective M 1 PAM by virtue of the fluorophenyl pyrazole moiety. 28 Carbonyl deletion provided lactam 3 , and surveying regioisomeric lactams afforded VU0451725 ( 4 ), with improved DMPK properties over 2 and 3 . 33 Finally, an aza-scan identified the the 6,7-dihydro-5 H -pyrrolo[3,4- b ]pyridine-5-one core of VU0453595 ( 5 ), which proved a useful in vitro and in vivo tool, demonstrating efficacy in rodent models of pharmacologically-induced NMDA hypofunction.…”