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Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284

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Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284 1521-0111/90/3/177–187$25.00 http://dx.doi.org/10.1124/mol.116.104737 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:177–187, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284 Deborah L. Smith, Jennifer E. Davoren, Jeremy R. Edgerton, John T. Lazzaro, Che-Wah Lee, Sarah Neal, Lei Zhang, and Sarah Grimwood Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut Received April 14, 2016; accepted June 30, 2016 Downloaded from ABSTRACT 3 3 Selective activation of the M1 muscarinic acetylcholine recep- of ACh required to inhibit [ H]N-methylscopolamine ([ H]NMS) tor (mAChR) via a positive allosteric modulator (PAM) is a new binding to M1, left-shifting the ACh Ki approximately 19-fold at approach for the treatment of the cognitive impairments asso- 10 mM. Saturation analysis of a human M1 mAChR stable cell line 3 molpharm.aspetjournals.org ciated with schizophrenia and Alzheimer’s disease. Herein, showed that [ H]PT-1284 bound to M1 mAChR in the presence of we describe the characterization of an M1 PAM radioligand, 1mMAChwithKd, 4.23 nM, and saturable binding capacity (Bmax), 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)- 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one [3H]PT-1284 binding in a concentration-responsive manner, whereas 3 ([ H]PT-1284), as a tool for characterizing the M1 allosteric M1 allosteric and orthosteric agonists showed weak affinity (.30 mM). 2 binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2- A strong positive correlation (R 5 0.86) was found to exist Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9- between affinity values generated for nineteen M1 PAMs in the 3 dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 (1)) was [ H]PT-1284 binding assay and the EC50 values of these ligands shown to potentiate acetylcholine (ACh) in an M1 fluorometric in a FLIPR functional potentiation assay. These data indicate that imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and there is a strong positive correlation between M1 PAM binding carbachol in a hippocampal slice electrophysiology assay affinity and functional activity, and that [3H]PT-1284 can serve as at ASPET Journals on September 26, 2021 (EC50, 165 nM). PT-1284 (1) also reduced the concentration a tool for pharmacological investigation of M1 mAChR PAMs. Introduction hippocampus, striatum, and cerebral cortex (Wall et al., 1991; Levey 1996; Porter et al., 2002) and activation of these Muscarinic acetylcholine receptors (mAChRs) are mem- receptors in regions known for memory and cognitive func- bers of the superfamily of G protein-coupled receptors (GPCR). tion are expected to bestow a procognitive effect (Caulfield Five mammalian subtypes have been identified and are re- et al., 1983; Hagan et al., 1987; Messer et al., 1990). ferred to as M –M , with distribution in the central nervous 1 5 The orthosteric agonist xanomeline achieved proof of con- system as well as the periphery. mAChR subtypes M1,M3,and cept in human clinical trials for improvements in positive, M5 couple to Gq/11 proteins, which results in IP3 production and negative, and cognitive symptoms associated with schizophre- subsequent calcium mobilization, whereas subtypes M2 and M4 nia (Shekhar et al., 2008), as well as improvements in cognitive couple to Gi/o proteins, thereby inhibiting adenylyl cyclase function associated with Alzheimer’s disease (Bodick et al., activity (Bonner et al., 1987; Caulfield 1993; Caulfield and 1997). Although reported as an M1/M4 mAChR-preferring Birdsall, 1998). The M1 subtype is highly expressed in the agonist, xanomeline caused peripherally mediated adverse effectssuchasnausea,vomiting, salivation, and gastroin- dx.doi.org/10.1124/mol.116.104737. testinal distress, which led to a high dropout rate in these ABBREVIATIONS: ACh, acetylcholine; Bmax, saturable binding capacity; BQCA, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, benzyl quinolone carboxylic acid; CHO, Chinese hamster ovary; CNS, central nervous system; compound 9, 8-((1S,2S)-2-hydroxycyclohexyl)- 5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; compound 10, (2-((1S,2S)-2-hydroxycyclohexyl)-5-((6-methylpyridin- 3-yl)methyl)-1,2-dihydro-3H-benzo[e]isoindol-3-one; compound 12, 5-((6-chloropyridin-3-yl)methyl)-8-((1S,2S)-2-hydroxycyclohexyl)-8,9-dihydro- 7H-pyrrolo[3,4-hour]quinolin-7-one; compound 13,8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(tributylstannyl)pyridin-3-yl)methyl)-8,9-dihydro-7H- pyrrolo[3,4-hour]quinolin-7-one; DMF, dimethylformamide; FBS, fetal bovine serum; FLIPR, fluorometric imaging plate reader; Fu,b, fraction 3 unbound in brain; GPCR, G protein-coupled receptors; hM1, human M1; HPLC, high-performance liquid chromatography; [ H]PT-1284, 8-((1S,2S)- 2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; mAChR, muscarinic acetylcholine recep- tor; MPO, multiparameter optimization; NMS, N-methylscopolamine; 1H NMR, proton nuclear magnetic resonance; PAM, positive allosteric modulator; PET, positron emission tomography; PQCA, 1-{[4-cyano-4-(2-pyridinyl)-1-piperidinyl]methyl}-4-oxo-4H-quinolizine-3-carboxylic acid; PT-1284 (1), 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; 13C NMR, carbon-13 nuclear magnetic resonance. 177 178 Smith et al. clinical trials. Alleviating these adverse effects and preserving Pfizer animal care facilities that supported this work are fully biologic efficacy by developing subtype-selective orthosteric accredited by the Association for Assessment and Accreditation of agonists has proven to be challenging (Messer, 2002) owing to Laboratory Animal Care (AAALAC) International. complete homology of the muscarinic receptors at the orthos- teric acetylcholine (ACh) site (Kruse et al., 2013). PT-1284 (1) Synthesis A new approach for achieving subtype selectivity is the All solvents and reagents were obtained from commercial sources development of allosteric modulators that bind at sites distinct and were used as received. All reactions were monitored by thin layer from the orthosteric binding site (Kruse et al., 2013). Unlike the chromatography (TLC plates F254; Merck) or ultra high-performance orthosteric binding site, allosteric binding sites display hetero- liquid chromatography–mass spectrometry analysis (Waters Acquity, geneity across mAChR subtypes, providing opportunities for ESCi Multi-Mode Ionization Source). Proton nuclear magnetic reso- achieving desired subtype selectivity (Ma et al., 2009; Kruse nance (1H NMR) and carbon-13 nuclear magnetic resonance (13C et al., 2013). Therefore, by targeting allosteric binding sites, NMR) spectra were obtained using deuterated solvent on a Varian selective positive allosteric modulators (PAMs) could be iden- 400 MHz instrument (LabX, Midland, ON, Canada). All 1HNMR tified to enhance both the affinity and intrinsic activity of the shifts are reported in d units (ppm) relative to the signals for chloroform (7.27 ppm) and methanol (3.31 ppm). All 13C shifts are endogenous agonist, ACh, at the M1 mAChR without activation reported in d units (ppm) relative to the signals for chloroform (77.0 of other mAChR subtypes. This profile could potentially lead to 1 ppm) and methanol (49.1 ppm) with H-decoupled observation. All Downloaded from therapeutics with improved safety and toleration profiles, coupling constants (J values) are reported in hertz. NMR abbrevia- compared with nonselective mAChR activators. In 2009, tions are as follows: br, broadened; s, singlet; d, doublet; t, triplet; q, BQCA, a highly selective M1 PAM, was disclosed by Merck quartet; p, pentuplet; m, multiplet; dd, doublet of doublets; ddd, (Kenilworth, NJ; Ma et al., 2009). As a follow-on to BQCA, the doublet of doublet of doublets. second generation M1 PAM PQCA (Kuduk et al., 2011) was Compound 12. 6-Chloropyridin-3-yl)methyl)zinc(II) chloride shown to improve cognition in animal models designed to (12 ml, 6.0 mmol, 0.5 M in tetrahydrofuran) was added to a solution measure recognition memory, spatial working memory, and of compound 11 (1.8 g, 5.0 mmol), which was prepared according to molpharm.aspetjournals.org executive function (Uslaner et al., 2013; Lange et al., 2015; Puri previously reported procedures (Kuduk et al., 2012), with palladium et al., 2015; Vardigan et al., 2015). tris-tert-butylphosphine (128 mg, 0.25 mmol). The resulting mixture Availability of a potent and selective radioligand that binds was stirred at 60°C under a nitrogen atmosphere for 18 hours. The mixture was cooled to room temperature and concentrated under to the M allosteric site will facilitate the understanding of M 1 1 reduced pressure. The crude material was purified by flash chroma- PAM pharmacology and the characterization of M1 selective tography through silica using 5% methanol in dichloromethane to PAMs (Trankle et al., 1996). Herein, we describe our efforts in afford a brown solid, which was washed with methanol (10 ml) to 1 the identification and in vitro pharmacology profiling of an afford compound 12 as a white solid.
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