Design and Synthesis of Î³- and Î´-Lactam M1 Positive Allosteric

Article pubs.acs.org/jmc

Design and Synthesis of γ- and δ‑Lactam M1 Positive Allosteric Modulators (PAMs): Convulsion and Cholinergic Toxicity of an M1‑Selective PAM with Weak Agonist Activity Jennifer E. Davoren,*,† Michelle Garnsey,∥ Betty Pettersen,⊥ Michael A. Brodney,† Jeremy R. Edgerton,‡ Jean-Philippe Fortin,‡ Sarah Grimwood,‡ Anthony R. Harris,∥ Stephen Jenkinson,# Terry Kenakin,∇ John T. Lazzaro,∥ Che-Wah Lee,∥ Susan M. Lotarski,‡ Lisa Nottebaum,# Steven V. O’Neil,∥ Michael Popiolek,‡ Simeon Ramsey,§ Stefanus J. Steyn,† Catherine A. Thorn,‡ Lei Zhang,† and Damien Webb∥ †

Medicine Design, ‡Internal Medicine Research Unit, and §Inflammation and Immunology Research Unit, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, United States ∥ Medicine Design and ⊥Drug Safety Research and Development, Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States # Drug Safety Research and Development, Pfizer Worldwide Research and Development, La Jolla, California 92121, United States ∇ Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, United States S Supporting Information *

ABSTRACT: Recent data demonstrated that activation of the muscarinic M1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M2 and M3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M1-selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M1 is sufficient to elicit cholinergic AEs.

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INTRODUCTION The muscarinic acetylcholine receptors (mAChR) M1−M5 are a family of five G protein-coupled receptors that have long been attractive targets for drug development.1 In two separate phase II clinical trials for Alzheimer’s disease (AD) and schizophrenia, the M1,M4-preferring muscarinic agonist xanomeline demonstrated significant pro-cognitive and antipsychotic effects in both patient populations.2 Though the clinical data was compelling, further development of xanomeline was halted due to high patient drop-out rates that were attributed to classic cholinergic side effects such as sweating, salivation, and © 2017 American Chemical Society

gastrointestinal (GI) and cardiovascular (CV) adverse events (AEs). Guided by the tissue distribution, potentiating effects of the M1 receptor on synaptic plasticity,3 and learning deficits observed in M1 knockout mice,4 researchers have postulated that the positive effects of cholinergic drugs on cognition likely involve M1 receptor activation (Figure 1).5 M1 is predominantly expressed in the brain, with the highest mRNA expression Received: April 20, 2017 Published: June 9, 2017 6649

DOI: 10.1021/acs.jmedchem.7b00597 J. Med. Chem. 2017, 60, 6649−6663

Journal of Medicinal Chemistry

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Figure 1. Comparison of M1 mRNA expression levels in reads per kilobase per million mapped reads (rpkm) in the CNS vs peripheral organs from human cadaver tissue. Data were mined from the Genotype−Tissue Expression (GTEx) Project.11 CNS tissues are highlighted in the red box. A fully labeled version of this figure is available in the Supporting Information.

Figure 2. Representative examples of M1 PAMs.

subtypes.9 This concept is currently being tested by a recent clinical entry from Heptares.10 In 2009, Merck disclosed the discovery of an M1-selective positive allosteric modulator (PAM), BQCA (2)12 (Figure 2), that binds to an allosteric pocket in the M1 receptor. This pocket is believed to be distinct and separate from the orthosteric site.9 Optimization of 2 provided an improved tool compound, PQCA (3),13 which demonstrated efficacy in nonhuman primate (NHP) studies focused on cognition and learning.14 Further optimization led to the discovery of a vast array of other chemotypes15,16 including tricyclic lactam 417 and MK-7622 (5).18 The latter was advanced into a phase II clinical

being found in the cortex and hippocampus, brain regions which are intimately involved in cognitive processing. M1 has low mRNA expression levels outside the CNS, with the exception of the prostate6 and salivary glands7 (Figure 18). Combined, this data supports a rationale that selective activation of the M1 receptor could enhance cognition and memory without the GI side effects seen with nonselective agents. Development of a selective M1 agonist, however, has proved to be challenging, mainly due to difficulties in achieving subtype selectivity; this is a consequence of the orthosteric binding site being highly conserved across the five mAChR 6650



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Figure 3. (A) M1 functional dose−response curves in the presence (PAM mode, blue diamond) and absence (agonist mode, red circle) of an EC20 level of ACh in a FLIPR assay for compounds 5−7. (B) Functional concentration−response curves for compounds 5−7 in the presence (PAM mode, blue diamond) and absence (agonist mode, red circle) of an EC20 level of carbachol (CCh) in a hippocampal slice electrophysiology assay.

trial (May 2013)19 as an adjunct therapy for patients with AD. For reasons not disclosed at the time of this publication, the study was terminated (April 2016). This impressive body of work has garnered significant attention from the medicinal chemistry community. Our own efforts in this area have yielded multiple chemical series exemplified by azaindole PF-06764427 (6)20 and pyridine PF06767832 (7).21 Others working in this area at the time of our program included Vanderbilt University22 (822b), Takeda23 (924), Asceneuron,25 Monash University,26 and Roche.27 Our recent disclosure on compound 721 included a comprehensive discussion on its pharmacology, selectivity, preclinical efficacy, and safety threshold. Despite being devoid of M2 and M3 activity up to 10 μM (>180-fold selectivity in vitro), exposure to this compound resulted in CV and GI side effects similar to those of nonselective muscarinic agents plus M1 mediated convulsion,28 thus contradicting the original hypothesis that cholinergic effects are exclusively driven by M2/ M3. A similar conclusion was independently drawn by researchers at Bristol-Myers Squibb using structurally distinct M1-selective PAM compounds.29 Both studies used ligands whose pharmacology can be classified as PAM-agonist (examples of PAM-agonist pharmacology for compounds 5− 7 are shown in Figure 3) and were therefore unable to demonstrate whether it was direct activation of M1 (allosteric agonism) or PAM potentiation of the endogenous agonist acetylcholine (ACh) that led to cholinergic AEs and convulsion. These studies leave open the possibility that an M1 PAM with less agonist activity could potentially have a larger therapeutic index (TI) between efficacy, cholinergic AEs, and convulsion. Herein, we report on the design, synthesis, and optimization of a series of novel bicyclic lactams (11, 12) that culminated in the discovery of the CNS-penetrant M1-selective lactam PAM, 1 (PF-06827443). Compound 1 has reduced allosteric agonist activity relative to compounds such as 5−7 and was advanced

into safety toxicology studies in rat and dog. In rats, compound 1 showed no clear signs of GI AEs but was dose-limited by convulsion. In dogs, we observed clear signs of GI AEs and convulsion, which resulted in narrow therapeutic indexes (TI’s) comparable to those of PAM-agonist compound 7.21 Taken together with previous reports,21,29 these results strongly suggest that the observed cholinergic side effects are not chemotype-specific, are primarily driven by the positive allosteric modulation of the endogenous ligand ACh, and do not require direct allosteric agonism at the M1 receptor.

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RESULTS AND DISCUSSION

Structure activity relationship (SAR) data from our previous work on M1 PAMs such as azaindole 620 and pyridine 721,30 demonstrated that an intramolecular hydrogen bond (IMHB) enforces a conformation of the amide carbonyl that can lead to an interaction with Gln177. We also demonstrated that a heterocycle at the para-position of the benzyl tail group significantly increased potency by occupying an aromatic, tyrosine-rich pocket. The 3-substituted 1-methyl-1H-pyrazole featured in 6 emerged early as an optimal heterocycle for potency and CNS penetration but was found to be rapidly demethylated by hepatocytes to form the CNS-restricted Ndesmethyl bioactive metabolite 10. In the pyridine series, the 4substituted thiazole afforded improved potency without such metabolic concerns. In the work reported herein, we explored the viability of replacing the aforementioned IMHB with a covalent constraint in the form of γ- and δ-lactam, to enforce the bioactive conformation (Figures 4 and 5). As with our pyridine series, methyl substitution at the 5-position of the fused bicyclic system was retained to ensure appropriate benzyl tail group orientation. The γ- and δ-lactams 11 and 12 were synthesized from the corresponding benzolactams 13 and 14 via Pd-catalyzed crosscoupling reactions (Scheme 1). The lactams were derived from benzolactones 15 and 16, which themselves could be prepared 6651



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Scheme 2. Synthetic Route for Synthesis of the γ- and δLactam Series from Common Starting Material 17a

Figure 4. Design of lactam M1 allosteric modulators utilizing SAR data from azaindole 6 and pyridine 7.

Reagents and conditions: (a) CH2Br2, Pd(OAc)2, K2HPO4, 140 °C, 32 h, 59%; (b) (1) SOCl2, BnEt3N+Cl−, BF3•OEt2, 90 °C, 38 h, (2) iPr2NEt, MeOH, CH2Cl2, −10 °C, 10 min, 69%; (c) K2CO3, EtOH, 20 °C, 6 h then 100 °C, 20 h, 64%; (d) Br(CH2)2Br, Pd(OAc)2, K2HPO4, 120 °C, 16 h, 33%;32 (e) (1) DMF, SOCl2, 72 °C, 18 h, (2) 19, iPr2NEt, CH2Cl2, 0 °C, 1 h, (3) KOt-Bu, THF, 0 °C to rt, 3 h, 50% (3 steps); (f) B2Pin2, KOAc, Pd(dppf)Cl2, dioxane, 80 to 100 °C, 65− 77% (g) ArylCH2Cl, Pd(PPh3)4, 3.0 M Cs2CO3, 1,4-dioxane, 80 °C, 55%. a

Figure 5. Overlay of compounds 7 (yellow) and 11b (gray).

from the 3-bromo-4-methylbenzoic acid (17) by means of a Pd(II)-catalyzed C−H alkylation/lactonization reaction originally reported by the Yu research group.31 Palladium-mediated C−H activation of 17 in the presence of the dielectrophile dibromomethane or 1,2-dibromoethane directly afforded the γ- and δ-lactones 15 and 16, respectively, in low to moderate yield (Scheme 2). Conversion of γ-lactone 15 to γ-lactam 13 was achieved via a chloroalkyl ester intermediate 18, itself obtained upon treatment of 15 with thionyl chloride followed by methanol. The desired γ-lactam 13 was obtained by the reaction of 18 with amino alcohol 1921 under basic conditions. The δ-lactam 14 was synthesized using a similar pathway that commenced with treatment of δ-lactone

16 with neat thionyl chloride. The intermediate chloroalkyl acid chloride was treated with amino alcohol 19 followed by potassium tert-butoxide to give the desired lactam 14. Bromolactams 13 and 14 were subjected to Pd-mediated borylation to give the penultimate compounds 20 and 21. This short synthetic sequence allowed us to rapidly prepare gram quantities of pinacol boronates 20 and 21 which were subjected to a variety of Suzuki reactions (representative conditions shown in Scheme 2; other conditions are described

Scheme 1. Retrosynthetic Analysis of the γ- and δ-Lactam Series 11 and 12 from Common Starting Material 17

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Table 1. SAR of the Lactam Series

a Compound EC50 values were measured in a potentiation assay format, with a calcium mobilization readout, on a FLIPR384 fluorometric plate reader (FLIPR) in the presence of an EC20 concentration of ACh. bEC50 values represent the geometric mean of at least three experiments. c Lipophilic efficiency (LipE)34 defined as M1 PAM pEC50-ceLogD.35 dIntrinsic clearance in human liver microsomes. eRatio of permeability, measured as a rate in 10−6 cm/s in and out (BA/AB) of a Madin−Darby canine kidney (MDCK) epithelial cell line transfected with the MDR1 gene encoding the P-gp efflux transporter (MDCKI-MDR1, acquired from the NIH).36 fCompound 1 is built upon scaffold 11.

1). For example, compound 11b (M1 PAM EC50 = 24 nM), was approximately 2-fold more potent than its pyridine counterpart 7 (EC50 = 60 nM). In general, δ-lactams (12a− d) were 2- to 3-fold more potent than their γ-lactam counterparts (1 and 11a−g). However, the concomitant increase in lipophilicity, as a consequence of the larger lactam ring, resulted in no meaningful gains in lipophilic efficiency (LipE) and gave rise to higher human liver microsomal (HLM) clearance. Consistent with SAR from the azaindole and pyridine series, the substituents at the para-position of the benzyl tail had profound effects on potency and MDR efflux ratio, with the 4oxazole analogues (1 and 12d) exhibiting the lowest MDR BA/ AB values (11 and 2.7, respectively).33 Further heteroaryl variations led to either elevated HLM clearance (pyrazole 11e) or exceptionally high MDR efflux activity (imidazoles 11f and

in the Supporting Information) to couple with various benzyl halides in which to probe the SAR at this vector. The efficient C−H functionalization-driven synthesis described above allowed us to concurrently prepare and evaluate γ- and δ-lactam analogues. On the basis of the good SAR alignment of the lactam series with 6 and 7, we elected to pursue a focused effort based on the established SAR from the azaindole20 and pyridine21 series. For the head group moiety we employed (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol (19), because it had been demonstrated to provide improved lipophilic efficiency (LipE) 34 and microsomal stability compared to its cyclohexyl counterpart.21 For the benzyl tail group, para-substituted heteroaryls were employed to balance potency and in vitro ADME properties. Gratifyingly, the previously established SAR translated to the lactam series and afforded a number of γ- and δ-lactam M1 PAMs (1, 11a−g, and 12a−d) with excellent potencies (Table 6653



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activity (>10 μM) at any additional targets, with the exception of PDE11A4 (IC50 = 5.06 μM). In an hM1 recombinant cell system using a FLIPR (Ca2+) readout, compound 1 exhibited weak allosteric agonist activity (Figure 6A, N = 19) relative to other compounds synthesized as part of this program (for example, compounds 5−737). This profile was desirable as we and others22b,29 sought to explore whether reduced agonism could offer a larger TI between efficacy and cholinergic side effects/convulsion. Unfortunately, we were unable to identify any compounds that were completely devoid of allosteric agonist activity (pure-PAM agents) in our recombinant FLIPR assay system in order to test this hypothesis. As noted above, compound 1 showed weak agonist activity in the recombinant M1 FLIPR assay. However, in a rat hippocampal slice electrophysiology assay measuring spontaneous action potentials in CA1 pyramidal neurons,38 no effect on CA1 spike rates was observed in agonist mode, while in PAM mode the expected concentration-dependent response was seen (Figure 6B). The PAM response was antagonized by the addition of the reportedly M1-selective muscarinic antagonist N-[3-oxo-3-[4-(4-pyridinyl)-1-piperazinyl]propyl]2,1,3-benzothiadiazole-4-sulfonamide (VU0255035, 22).39 The lack of observed agonist activity in this preparation may be due to the fact that the top concentration tested in this assay was lower than that used in the FLIPR assay (3 μM versus 10 μM, respectively) and/or that it may be attributable to differences in receptor expression and coupling in native tissue compared to our M1-overexpressing cell lines.40 In any case, in both the FLIPR and hippocampal slice preparation readout, compound 1 exhibited reduced allosteric agonist activity relative to compounds 5−7. We previously demonstrated that a guinea pig ileum (GPI) longitudinal muscle myenteric plexus (LMMP) tissue bath preparation is responsive to selective M1 allosteric agonist and PAM activation. The acetylcholinesterase inhibitor donepezil, which enhances the endogenous ACh levels in this preparation was used in order to measure the PAM activity of the compounds. Indeed, donepezil alone produces a concentrationdependent contraction in this tissue assay. For the measurement of agonist activity, the compound is added in the absence of donepezil. However, due to the basal release of ACh in this preparation,41 even a pure-PAM agent would show activity in

11g). Compounds 1 and 12d provided the best overall profiles and were advanced into further pharmacological profiling. A key differentiation between compounds 1 and 12d was revealed in a high-throughput time-dependent inhibition (TDI) assay examining their ability to inactivate CYP3A4: compound 12d was approximately 6-fold more potent than compound 1. This finding, in conjunction with the lower intrinsic HLM clearance of compound 1, prompted us to select 1 for extensive profiling of pharmacokinetic (PK), in vitro and in vivo pharmacology, and safety characteristics. In mouse, rat and dog, compound 1 exhibited brain permeability (Cb,u/Cp,u) of 0.2−0.3 (Table 2). In vivo PK studies in both rats and dogs demonstrated that 1 has low IV clearance, a long half-life and excellent oral-bioavailability. Table 2. Mouse, Rat, and Dog PK for Compound 1 B/Pa Cb,u /Cp,ub,c Fp,u,d T1/2 (h)e (oral) Cl (mL/min/kg)f (IV) F%

mouse

rat

dog

0.1 0.3 0.013 n.d. n.d. n.d.

0.2 0.2 0.035 6.7 2.4g 91%h

0.7 0.3 0.059 7.3 1.7i 65%j

a

AUC ratio of the total drug level in brain (B) to the total drug level in plasma (P). bAUC ratio of the unbound drug concentration level in brain (Cb,u) to the unbound drug concentration level in plasma (Cp,u). c Rat fraction (Fu,b) unbound in the brain = 0.03. dFraction unbound in the plasma. eHalf-life following single-dose oral administration. f Observed plasma clearance following a single IV bolus. gDosed as a solution: 0.5 mg/kg in 20% hydroxypropyl-β-cyclodextrin (HPBCD). h Dosed as a suspension: 2 mg/kg in 0.5% methylcellulose. iDosed as a solution: 0.1 mg/kg in 20% HPBCD. jDosed as a suspension: 0.5 mg/ kg in 0.5% methylcellulose; n.d. (not determined).

In a recombinant panel of functional human mAChR assays, compound 1 had no appreciable M2−M5 agonist or PAM activity when tested at concentrations up to 10 μM (>200-fold selectivity for M1). Compound 1 was also evaluated in a panel of assays that included GPCRs (32 targets), ion channels (12 targets), nuclear receptors (3 targets), amine transporters (5 targets), phosphodiesterases (PDE; 11 targets), and a panel of enzyme and kinase targets (13 targets); 1 had no measurable

Figure 6. (A) Functional dose−response curves for compound 1 in the presence (PAM mode) and absence (Agonist mode) of an EC20 of ACh in the M1 FLIPR assay (agonist EC50 ≥ 5 μM; PAM EC50 = 47 nM). (B) Functional concentration−response curves for 1 in the presence (PAM mode) and absence (Agonist mode) of an EC20 level of carbachol (CCh) in the hippocampal slice firing rate assay. (C) Functional concentration− response curves for 1 in the absence (agonist mode, EC50 = 92 nM) and presence (PAM mode, EC50 = 46 nM) of an EC20 level of donepezil relative to the maximal effect of CCh (300 nM) in the guinea pig ileum tissue bath preparation. 6654



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Figure 7. Representative M1 ACh shift data using (A) FLIPR and (B) β-arrestin in the presence of fixed concentrations of compound 1. (C) Binding and functional parameters for compound 1 from the functional allosteric model: aintrinsic efficacy of ACh; bintrinsic efficacy of 1; and cfunctional cooperativity of 1 with ACh.

Figure 8. Compound 1 (A) In vivo C57BL/6 mouse striatal IP1 increase versus free brain exposure of compound 1. Data points are from individual mice. (B) C57BL/6 mouse amphetamine-stimulated locomotor activity (aLMA) was reduced by compound 1 at doses of 0.32, 1, and 3.2 mg/kg. (C) Rats given scopolamine (0.32 mg/kg) had to swim farther to find the hidden platform in the Morris water maze, and compound 1 significantly reversed this impairment at 0.32 and 1 mg/kg. (D) Summary of exposure data from the aLMA and scopolamine deficit MWM studies. a Concentration of unbound compound in plasma (nM); bconcentration of unbound compound in brain (nM); clisted compound concentrations were measured in the plasma and brain from a satellite study (N = 2/dose) at T = 1 h using the same dosing solution (solution in 10/90 Cremophor/water) and route of administration (sc) as the aLMA study; dcompound concentrations measured in plasma only at T = 1.25 h from a satellite study (N = 2/dose) using the same dosing solution (solution in 20% hydroxypropyl β-cyclodextrin (HPBCD) and route of administration (sc) from the MWM study; eCb,u was estimated from Cp,u using the AUC-derived Cb,u/Cp,u of 0.2.

(200 nM) enhanced the activity of compound 1, revealing its PAM activity (Figure 6C). To evaluate the functional cooperativity of compound 1 with ACh in FLIPR and β-arrestin assays,42 the τA values for ACh

the agonist read of this assay. That hypothesis is in line with what is seen for compound 1: under basal conditions (low basal ACh levels), compound 1 produced a moderate concentrationdependent increase in tone, while the addition of donepezil 6655



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Table 3. Single-Dose Dose-Escalation Study in Rats: Summary of Clinical Signs and Exposure Following Oral Administration of Compound 1 treatment exposures Cmax

AUC (0−24 h)

dosea mg/kg

sexb

Cp,u (nM)

Cp,u (nM·h)

treatment findingse

1 5 15 45

M/F M/F M/F M/F

73 313 1507 2312−3827d

1048 4927 20609 n.d.

none none soft feces (4/6 rats, 1−1.5 HPDc) convulsions (5/6 rats, at 1−6 HPD)

a

Compound 1 was dosed orally in a 0.5% methylcellulose suspension at 1 and 5 mg/kg and in 1.25% w/v hydroxypropyl cellulose SL, 0.05% docusate sodium in purified water nanosuspension at 15 and 45 mg/kg. bN = (3/sex)/dose. cHPD = hours postdose. dPlasma drug levels associated with convulsions. TI based on Ceff of 16.6 nM or 398 nM·h.; n.d. = not determined. eAll times listed as HPD are approximate.

Table 4. Single-Dose Dose-Escalation Study in Dogs: Summary of Clinical Signs and Exposures Following Oral Administration of Compound 1 treatment exposures Cmax

AUC (0−24 h)

sexa

Cp,u (nM)

Cp,u (nM·h)

treatment findingsd

b

5

M/F

446

3850

5c 15b

M/F F

303 1067

3370 NA

Male: transient salivation (1, 3, and 4 HPD), emesis (4 bouts: 1−4 HPD), ataxia (2 HPD), circling behavior (4.3 HPD) and asymptomatic at 6 HPD. Found dead the following day (cause of death not identified). Female: signs limited to soft stools (1.5 HPD) Watery stools (2/2 dogs, 0.5−1.3 HPD), emesis (M, 1 bout, 1.75 HPD) Convulsion 1.2 HPD proceeded by ataxia at 0.8 HPD, salivation, increased activity, and vocalization at 1 HPD.

dose mg/kg

a

Compound 1 was dosed orally as 1/sex/dose at 5 mg/kg in both formulations and 1 female at 15 mg/kg. bCompound 1 was dosed orally as a spray-dried dispersion (SDD) (23% drug loading with 0.5% methylcellulose and 100 mM citrate buffer). cCompound 1 was dosed orally as a nanosuspension (hydroxypropyl cellulose SL, 0.05% docusate sodium in purified water). dAll times listed as HPD are approximate.

Table 5. M1 Pharmacology and Safety Summary for Compounds 1 and 5−7 M1 in vitro pharmacology and safety data (nM) agonist

PAM

binding

2-fold IP1 accumulation

rat Cmax convulsion

dog Cmax convulsion

CNS Cmax TIb

cmpd

EC50

%c

EC50

Kid

Cb,ua

Cb,ue

Cp,u

Cb,ue

Cp,u

rat

dog

1 5 6 7

>5,000 >2,000 >1,000 >4,000

23 62 38 34

47 63 55 60

16 8 40 19

22 4 32 14

440 130f n.d. n.d.

2,310 550g n.d. n.d.

320 n.d. n.d. 290i

1,070 n.d. n.d. 950

20 33 >14h >29j

15 n/a n/a 20

a

Cb,u exposures that provide a 2-fold increase in IP1 accumulation in striatal brain tissue46 were derived as described. Compound 1 was estimated by interpolation of the data presented in Figure 8A, compound 5 corresponds to a measured 2.0-fold (±0.6) increase using data from a published study46 and one additional unpublished study [see Supporting Information for this data set], compound 6 corresponds to a measured 2.1-fold (±0.4) increase of striatal IP1,46 and compound 7 corresponds to a measured 2.0-fold (±0.5) increase of striatal IP1.21 bTI was calculated by dividing speciesrelevant Cb,u associated with convulsion by the IP1 Cb,u. cAgonist % max is defined as the mean average agonist response at 13 μM. dMeasured by inhibition of the M1 PAM radioligand [3H]PT-1284.58 eCb,u was calculated from measured Cp,u using the species-appropriate measured AUC Cb,u/ Cp,u. fPfizer generated data: AUC Cb,u/Cp,u rat = 0.24. gData generated at Pfizer in a rat single-dose dose-escalation study format, convulsion in 1/6 rats approximately 4 HPD; peak plasma exposure for that animal was achieved at 2 HPD. hData generated at Pfizer in a rat single-dose doseescalation study format, calculated TI is based on a no-convulsion highest exposure achieved study Cp,u = 2,900 nM (Cb,u = 370 nM from a Cb,u/Cp,u = 0.1520). iAUC dog Cb,u/Cp,u = 0.30.21 jBased on a no-convulsion highest exposure in rat Cp,u = 1,200 nM21 (Cb,u = 410 nM from a measured rat Cb,u/ Cp,u = 0.34).

were fitted to the control concentration−response curves (Figure 7A and B) according to the Black/Leff operational model.43,44 The changes due to the addition of compound 1 in the affinity of the receptor for ACh are represented by the α values, while changes in efficacy are represented by β values (Figure 7C). The αβ/KB product is a single parameter estimate of the potentiation of each signaling pathway. The KB values represent the affinity of compound 1 for the receptor in the absence of cobinding ligand and cannot be used to estimate receptor occupancies.45 Differences in αβ/KB values indicate induced bias in the natural signaling to ACh. In this case, the binding of compound 1 produces a minor 1.4-fold bias toward

β-arrestin in vitro;38 the translation and relevance of this pharmacology is not known. Compound 1 was evaluated in three in vivo studies that we have been previously shown21 to be sensitive to M1 activation (Figure 8A−C). Accumulation of brain IP1, which is a downstream product of Gq G-protein signaling was assessed. Mice were dosed with compound 1 or vehicle, and both the striatal IP1 level and the free brain concentration (Cb,u) of compound 1 were measured. Compound 1 robustly increased striatal IP1 levels in mice in a manner that correlated to the brain concentration of the drug (R2 = 0.78) (Figure 8A). The exposure−response relationship is similar to what we previously 6656



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reported21,46 and underscores the ability of an M1 PAM to potentiate ACh under endogenous tone. Next, compound 1 was tested to see if it could reduce hyperactive behavior in mice stimulated by D-amphetamine in a pharmacodynamic assay sensitive to M1 PAM activation.12,21 Compound 1 significantly reduced amphetamine-stimulated locomotor activity (aLMA) in mice at all doses administered (Figure 8B), with the lowest dose (0.32 mg/kg) leading to a free brain concentration of 3.8 nM in a satellite group of mice (Figure 8D). To assess compound 1’s effects on cognition, we subjected it to the rat Morris water maze (MWM) assay, which assesses a compound’s ability to reverse spatial learning and memory deficits induced by scopolamine47 (Figure 8C). All doses of compound 1 significantly attenuated the disruptive effects of scopolamine on swim distance in the MWM model (see Supporting Information for additional end points). The middle dose of 1.0 mg/kg (Cb,u = 13 nM, Figure 8D) produced the most robust reversal of scopolamine, similar to donepezil, across days 3−5.48 Compound 1’s cholinergic AE profile and convulsive threshold was determined in rats (Table 3) and dogs (Table 4) using a single-dose dose-escalation (DE) study model. In rat, the no-observed-adverse-effect level (NOAEL) was 15 mg/kg (Cmax Cp,u = 1.5 μM) in this study. At this exposure compound 1 was well tolerated: soft feces, noted in 4 of the 6 rats shortly after dosing, did not persist past 90 min. At 45 mg/kg (Cmax Cp,u = 2.3−3.8 μM), 5 of the 6 rats convulsed over a span of 1 to 6 h postdose (HPD).49 The estimated rat Cb,u at convulsion was approximately 460 to 760 nM50 (Table 5). Because compound 1 had low thermodynamic solubility,51 we simultaneously evaluated a nanomilled suspension52 and a spray-dried dispersion (SDD)53 formulation in a dog safety study (Table 4). Compared with rats, similar exposures in dogs were poorly tolerated. Using the SDD formulation, at 5 mg/kg (Cmax Cp,u = 446 nM) one animal’s clinical signs were mild and limited to soft stool at 1.5 HPD. The second animal administered the same dose/formulation had cholinergic signs that consisted of soft/watery stools, emesis, and salivation. In addition, this animal displayed ataxia and circling behavior. All symptoms resolved by 6 HPD; however, the next morning the animal was found dead. Although the cause of death was not apparent at necropsy, it is our hypothesis that death was caused by convulsion after a period of latency.28a,54 At the 5 mg/kg dose using the nanomilled formulation, lower exposures were achieved (Cmax Cp,u = 303 nM), and clinical signs were limited in both animals to watery stools and emesis, which resolved by 2 HPD. The definitive convulsive threshold for this compound was achieved at 15 mg/kg (Cmax Cp,u = 1.1 μM) using the SDD formulation. Normally, the combined rat and dog nonclinical safety data would be used to cap initial clinical studies at 1/10th the Cmax value of the NOAEL of the species most sensitive to convulsion.55 In this case, because we believed the death at the 5 mg/kg nanomilled formulation dose was likely treatmentrelated, we would need to establish a clean NOAEL with no overlapping PK. To achieve this goal, we would need to target 1/3 of the exposure at the tainted dose (approximately 150 nM), which would make the 1/10th NOAEL Cmax Cp,u = 15 nM, directly within the range in which we observed efficacy in preclinical pharmacodynamic models. To put this safety data into context, a TI can be calculated as the ratio of the exposure associated with convulsion obtained from toxicology studies divided by the exposure that is

predicted to be efficacious in treating the disease (Ceff). Determining a preclinical Ceff based on a cognition end point that translates to humans is challenging. Development of assays that translate to the clinic with sufficient throughput for preclinical research programs is a continuing quest.56 In this program, we utilized the ex vivo IP1 accumulation assay described in Figure 8A to compare compounds in vivo. For example, brain concentrations (Cb,u) corresponding to an approximate 2-fold accumulation57 of IP1 for compounds 1 and 5−7 are reported in Table 5. It is noteworthy that by this measure compound 5 was more potent than its FLIPR EC50 suggested. This result is in line with its more potent binding Ki as measured by displacement of [3H]PT-1284.58 By contrast, azaindole 6 demonstrated the least in vivo ACh cooperativity, while compounds 1 and 7 were roughly similar. The Cmax concentration of M1 PAM in the brain (Cb,u) associated with convulsion can be estimated from the Cmax plasma levels (Cp,u) using a measured species appropriate AUCderived Cb,u/Cp,u. Dividing the Cb,u associated with convulsion by the IP1 Cb,u provides a TI against convulsion using CNSrelevant concentrations (Table 5, rat, compounds 1 and 5; dog, compounds 1 and 7). Data from compounds not dosed high enough for convulsion in a toxicology study are also reported as they may be of interest to researchers (Table 5, rat, compounds 6 and 7). Of this cohort, compounds 1 and 5 have the least and most allosteric agonist activity, respectively, and caused convulsion in rats at comparable TI’s. Compounds 1 and 7 were both dosed to convulsion in dogs, and again, TI’s were similar for both compounds, with compound 7 appearing to have a slightly larger TI in both species.

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CONCLUSION Using an efficient C−H activation approach for synthesis and SAR knowledge from our previous work, we discovered a number of lactams that proved to be potent and selective PAMs of the M1 receptor with clean ancillary pharmacology. Optimization of the heterocycle at the 4-position of the benzyl group provided the series lead, compound 1, which was selected for its optimal balance of PAM potency, physical chemical properties, and P-gp efflux liability. Compound 1 has excellent oral bioavailability and low clearance, but modest CNS penetration in rats and dogs. Compound 1 exhibited excellent allosteric cooperativity with ACh both in vitro and in vivo, where a robust potentiation response in the striatum (>20-fold) was observed when measuring IP1 accumulation in a wild-type mouse under endogenous levels of ACh; additionally, robust in vivo activity in the aLMA and MWM assays was observed at low free-brain (Cb,u) concentrations. Compound 1 also exhibited reduced allosteric agonist activity in our FLIPR and electrophysiology assays, relative to other M1 PAMs such as compounds 5−7. This profile made compound 1 suitable to test whether direct activation (allosteric agonism), rather than allosteric potentiation, was responsible for the cholinergic AEs and convulsion seen with M1 PAMs in preclinical safety studies. Studies in rats and dogs to evaluate safety and cholinergic AE in vivo were performed on compound 1. While we observed almost no findings, aside from transient loose stool in a rat study at exposures as high as 1.5 μM (Cp,u), at the next dose 4/ 6 animals convulsed at an average Cmax Cp,u of 2.3 μM. At similar exposures, dogs proved to be a more sensitive species. At exposures of 310 nM (Cp,u), we observed loose stool and emesis. At 1.1 μM (Cp,u), dogs displayed ataxia and convulsion. Calculated estimates of Cb,u in both studies demonstrate that 6657



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2-((3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-6-(4(oxazol-4-yl)benzyl)isoindolin-1-one (1). A 3.0 M aq solution of Cs2CO3 (0.27 mL, 0.81 mmol) was added to a mixture of 4-(4(chloromethyl)phenyl)oxazole hydrochloride (74 mg, 0.32 mmol) and 20 (101 mg, 0.27 mmol) in anhydrous 1,4-dioxane (5.4 mL) at rt. Pd(PPh3)4 (55.6 mg, 0.027 mmol) was added, and the mixture was heated to 80 °C for 2 h, whereupon it was cooled to rt, quenched with water, and extracted with EtOAc (3 × 50 mL). The combined organic extracts were dried with MgSO4, filtered, then concentrated in vacuo to give a yellow oil. The crude material was purified by silica gel chromatography (eluent: CH2Cl2/MeOH 100:1 to 95:5) to afford a solid, which was crystallized from EtOAc/MeOH to afford 1 as an offwhite crystalline solid (see Supporting Information for PXRD) (60 mg, 55%): 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 1.0 Hz, 1H), 8.44 (d, J = 0.8 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H), 7.38 (s, 1H), 7.21 (d, J = 8.0 Hz, 2H), 5.07 (d, J = 5.7 Hz, 1H), 4.42 (d, J = 2.7 Hz, 2H), 4.08 (s, 2H), 4.05−3.96 (m, 1H), 3.91−3.82 (m, 2H), 3.74−3.63 (m, 1H), 3.43−3.34 (m, 1H), 3.07 (t, J = 10.5 Hz, 1H), 2.32 (s, 3H), 1.82 (dq, J = 4.7, 12.5 Hz, 1H), 1.69−1.61 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 168.13, 153.0, 140.8, 140.6, 140.4, 139.6, 139.4, 135.3, 131.4, 129.6, 129.1, 125.9, 125.2, 123.9, 72.0, 66.7, 66.5, 55.3, 46.2, 38.7, 30.2, 20.3; LC-MS m/z [M + H]+ calcd for C24H24N2O4, 405.17; found, 405.5; melting point, 205 °C. 2-((3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl)-6-methyl-7-(4(oxazol-4-yl)benzyl)-3,4-dihydroisoquinolin-1(2H)-one (12d). A 3.0 M aq solution of Cs2CO3 (1.29 mL, 3.87 mmol) was added to a mixture of 4-(4-(chloromethyl)phenyl)oxazole hydrochloride (297 mg, 1.29 mmol) and 21 (500 mg, 1.29 mmol) in anhydrous THF (7.2 mL) at rt. Pd(t-Bu3P)2 (267.2 mg, 0.258 mmol) was added, and the mixture was heated to 75 °C for 3 h whereupon it was cooled to rt, quenched with water, and extracted with CH2Cl2. The combined organic extracts were dried with MgSO4, filtered, then concentrated in vacuo to give a yellow oil. The residue was purified by silica gel chromatography (eluent: heptanes/EtOAc 1:0 to 0:1) to give the product (374 mg) as a yellow solid. A portion of this material (364 mg) was dissolved in hot EtOAc (20 mL) and MeOH (1.5 mL), then slowly cooled to rt. After 18 h, the suspension was cooled to 0 °C and filtered to give 120 mg of a white solid primarily composed of 12d. A portion of this material (102 mg) was combined with 30 mg of another lot prepared in a similar manner and recrystallized from a mixture of hot EtOAc and MeOH to provide 60.6 mg of 12d as a white solid: 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.8 Hz, 2H), 7.88 (s, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 6.99 (s, 1H), 4.75 (ddd, J = 12.2, 10.5, 4.3 Hz, 1H), 4.14 (dd, J = 11.4, 5.1 Hz, 1H), 4.06−3.90 (m, 3H), 3.72 (td, J = 10.2, 5.1 Hz, 1H), 3.45− 3.62 (m, 3H), 3.19−3.28 (m, 1H), 2.95 (m, 2H), 2.24 (s, 3H), 1.90 (td, J = 12.4, 4.9 Hz, 2H), 1.78−1.72 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 166.6, 151.3, 141.4, 140.3, 140.1, 137.7, 136.2, 133.5, 129.9, 129.0, 128.9, 128.6, 127.1, 125.7, 72.4, 67.6, 67.0, 56.8, 40.6, 39.2, 28.9, 27.9, 19.8; LC-MS m/z [M + H]+ calcd for C25H26N2O4, 419.19; found, 419.5. 6-Bromo-2-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methylisoindolin-1-one (13). A mixture of 18 (4.1 g, 14.77 mmol), 19 (1.73 g, 14.8 mmol), and K2CO3 (6.12 g, 44.3 mmol) in EtOH (50 mL) was stirred at 20 °C for 6 h. After 6 h, the temperature was increased to 100 °C, and the mixture stirred at this temperature for 20 h. The mixture was then filtered and the filtrate concentrated in vacuo. The resulting residue was purified by silica gel chromatography (eluent: CH2Cl2/MeOH 100:1 to 9:1) to afford 13 as a white solid (3.1 g, 64%): 1H NMR (400 MHz, CD3OD) δ 7.91 (s, 1H), 7.54 (s, 1H), 4.50 (s, 2H), 4.20−4.11 (m, 1H), 4.05−3.93 (m, 2H), 3.85 (dt, J = 5.0, 10.0 Hz, 1H), 3.51 (dt, J = 2.0, 12.0 Hz, 1H), 3.21 (t, J = 10.5 Hz, 1H), 2.51 (s, 3H), 2.04−1.91 (m, 1H), 1.87−1.78 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 168.7, 141.6, 140.4, 131.6, 127.1, 124.8, 124.5, 72.1, 68.2, 66.8, 55.8, 46.1, 30.1, 23.6; LC-MS m/z [M + H]+ calcd for C14H16BrNO3, 327.19; found, 327.9. 7-Bromo-2-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-6-methyl-3,4-dihydroisoquinolin-1(2H)-one (14). Anhydrous DMF (0.51 mL, 6.60 mmol) was added to a mixture of 16 (1.59 g, 6.60 mmol) in thionyl chloride (19.2 mL, 264 mmol). The reaction mixture was

convulsion occurred at concentrations in the brain well below where we would expect allosteric agonist activity (440 nM in rats and 330 nM in dogs), providing supporting evidence that positive allosteric modulation of ACh, not allosteric agonism, was responsible for the observed convulsions. Likewise, in dogs, the peripheral exposures at which we saw signs of GI AE were below concentrations where we would expect allosteric agonism with this compound. Holistically, these study results and convulsive thresholds are similar to those previously reported for compound 7, suggesting that these AEs are not compound- or chemotypespecific. Of greatest concern is the unexpected overnight death of a dog in our 5 mg/kg dose group, long after all signs of cholinergic toxicity had resolved. While an isolated incident is difficult to analyze and it is inconclusive whether this was treatment-related, we did observe similar delayed convulsion and death with compounds 5 and 7. In consideration of this unexpected death, compound 1 had no TI relative to efficacious doses in our preclinical behavior assays. Further efforts targeting the M1 receptor with a PAM should acknowledge all of the safety risks presented by such ligands. The GPI assay may be of use as an early phenotypic screen for GI AEs, but it is ultimately the risk of convulsion that is of greatest concern with this mechanism. Researchers pursuing the M1 PAM mechanism must take appropriate precautions to ensure the safety of compounds going into the clinic.

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EXPERIMENTAL SECTION

All procedures performed on animals in this study were in accordance with established guidelines and regulations, and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. Pfizer animal care facilities that supported this work are fully accredited by AAALAC International. Chemistry General Information. All solvents and reagents were obtained from commercial sources and were used as received. All reactions were monitored by TLC (TLC plates F254, Merck) or UPLC-MS analysis (Waters Acquity, ESCI ±, APCI ±). Melting points were obtained with a Thomas−Hoover melting point apparatus and are uncorrected. Mass spectrometry data obtained using a Waters SQ MS (single quad) tune: ESI-3.5 kV capillary/APCI (in ESCI mode)-0.3 μA corona pin, 30 V cone, source 150 °C, desolvation 475 °C, and desolvation gas N2 400L/h. 1H NMR spectra were obtained using deuterated solvent on a Varian 400 MHz instrument. All 1H NMR shifts are reported in δ units (ppm) relative to the signals for chloroform (7.27 ppm), DMSO (2.50 ppm), and MeOH (3.31 ppm). All coupling constants (J values) are reported in hertz (Hz). NMR abbreviations are as follows: br, broadened; s, singlet; d, doublet; t, triplet; q, quartet; p, pentuplet; m, multiplet; dd, doublet of doublets; and ddd, doublet of doublet of doublets. HPLC purity analysis of the final test compounds was carried out using one of three methods. Method A: UPLC/UV. WuXi AppTec, Shanghai, China. Column: Agilent Xtimate C18, 5 × 30 mm, 3 μm; UV purity detected at 220 nm; mobile phase A = 0.1% TFA in H2O; mobile phase B = 0.1% TFA in CH3CN. Gradient: 1% B to 100% B in 5.0 min. Flow rate: 1.2 mL/ min. Method B: UPLC/UV WuXi AppTec, Shanghai, China. Column: XBridge C18, 2.1 × 50 mm, 5 μm; UV purity detected at 220 nm; mobile phase A = 0.0375% TFA in H2O; mobile phase B = 0.01875% TFA in CH3CN. Gradient: 1% B to 5% B in 0.6 min, 5% B to 100% B in 4.4 min, 100% B to 1% B for 0.3 min, and held at 1% B for 0.4 min. Flow rate: 0.8 mL/min. Method C: column, Waters Atlantis C18, 4.6 × 50 mm, 5 μm; UV purity detected at 215 nm; mobile phase A, 0.05% TFA in H2O (v/v); mobile phase B, 0.05% TFA in CH3CN (v/ v); gradient, 5% B linear to 95% B in 4.0 min, held at 95% B to 5.0 min. Flow rate: 2 mL/min. All final compounds were determined to have a purity of >95% by one of the aforementioned methods unless stated otherwise. 6658



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heated in a sealed tube to 72 °C for 18 h, then concentrated in vacuo to give approximately 1.95 g of the chloroalkyl acid chloride, which was used directly without purification: 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.25 (s, 1H), 3.72 (t, J = 6.7 Hz, 2H), 3.29 (t, J = 6.7 Hz, 2H), 2.48 (s, 3H). N,N-Diisopropylethylamine (3.44 mL, 19.9 mmol) was added to a mixture of the chloroalkyl acid chloride (1.95 g) in CH2Cl2 (33 mL) at 0 °C. After 5 min, 19 (926 mg, 7.91 mmol) was added. After 1 h at 0 °C, the reaction was quenched with water (200 mL), and the aq layer was extracted with EtOAc (2 × 400 mL). The combined organic extracts were dried with MgSO4, filtered, then concentrated in vacuo to give approximately 2.48 g of chloroalkyl amide, which was used in the next step without purification: 1H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.19 (s, 1H), 6.00 (d, J = 6.2 Hz, 1H), 4.08 (dd, J = 4.7, 11.7 Hz, 1H), 4.04−3.94 (m, 2H), 3.84−3.78 (m, 2H), 3.68 (d, J = 4.7 Hz, 1H), 3.59 (tt, J = 4.6, 9.5 Hz, 1H), 3.47 (dt, J = 11.9, 2.3 Hz, 1H), 3.28−3.13 (m, 3H), 2.43 (s, 3H), 2.09−2.01 (m, 1H), 1.75−1.63 (m, 1H); LC-MS m/z [M + H]+ calcd for C15H19BrClNO3, 376.02; found, 378.3. Potassium tert-butoxide (1.49 g, 13.2 mmol) was added to a mixture of chloroalkyl amide (2.48 g) in anhydrous THF (44 mL) at 0 °C. The reaction mixture was slowly warmed to rt over 3 h, then quenched with water (50 mL). The aq phase was extracted with CH2Cl2 (2 × 400 mL), and the combined organic layers were dried with MgSO4, filtered, and concentrated in vacuo to give a white solid that was purified by silica gel chromatography (eluent: heptanes/EtOAc 4:1 to 0:1) to give 14 as a white solid (1.11 g, 50% isolated yield over 3 steps): 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.03 (s, 1H), 4.72 (ddd, J = 4.4, 10.3, 12.1 Hz, 1H), 4.16 (dd, J = 5.1, 11.0 Hz, 1H), 4.01 (dd, J = 4.8, 11.4 Hz, 1H), 3.82−3.70 (m, 1H), 3.70−3.40 (m, 3H), 3.27 (dd, J = 10.5, 10.5 Hz, 1H), 3.11 (d, J = 6.8 Hz, 1H), 3.06−2.76 (m, 2H), 2.36 (s, 3H), 1.87 (dq, J = 4.8, 12.4 Hz, 1H), 1.82−1.68 (m, 1H); LC-MS m/z [M + H]+ calcd for C15H18BrNO3, 340.05; found, 340.3. 6-Bromo-5-methyl-2-benzofuran-1(3H)-one (15). A mixture of 3bromo-4-methylbenzoic acid (17) (10 g, 47 mmol), K2HPO4·3H2O (13.4 g, 140 mmol), and Pd(OAc)2 (10.4 g, 46.5 mmol) in CH2Br2 (155 mL) was heated to 140 °C in a Parr reactor for 2 days. After cooling to rt, the resulting black mixture was filtered through diatomaceous earth, and the filter cake was rinsed with EtOAc. The filtrate was concentrated in vacuo to afford the crude product as an orange solid. Trituration with EtOH and collection of the resulting solid by filtration afforded 15 as a pale brown solid (6.25 g, 59%): 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.38 (s, 1H), 5.24 (s, 2H), 2.55 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.6, 145.7, 144.8, 129.4, 125.7, 125.3, 124.0, 69.2, 24.0. 7-Bromo-6-methylisochroman-1-one (16). A mixture of 3-bromo4-methylbenzoic acid (17) (10 g, 46.5 mmol), Pd(OAc)2 (5.22 g, 23.3 mmol), and K2HPO4 (24.3 g, 140 mmol) in BrCH2CH2Br (155 mL) was stirred overnight at 120 °C. The mixture was concentrated in vacuo to furnish a black tar, which was then dissolved in EtOAc (500 mL) and filtered. The filtrate was concentrated in vacuo and purified by silica gel chromatography (eluent: heptanes/EtOAc 1:0 to 0:1) to give 16 as a tan solid (3.73 g, 33%): 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.15 (s, 1H), 4.59−4.43 (m, 2H), 2.97 (t, J = 6.1 Hz, 2H), 2.44 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.9, 144.3, 138.4, 134.0, 129.4, 124.5, 123.9, 67.2, 27.3, 23.3; LC-MS m/z [M + H]+ calcd for C10H9BrO2, 240.98; found, 243.2. Methyl 5-Bromo-2-(chloromethyl)-4-methylbenzoate (18). To a mixture of 15 (15 g, 63 mmol) in thionyl chloride (300 mL) was added BF3·Et2O (534 mg, 3.77 mmol) followed by benzyltriethylammonium chloride (858 mg, 3.77 mmol). The resulting mixture was heated at 90 °C for 16 h. TLC analysis (petroleum ether/ethyl acetate 3:1) showed that some starting material remained; additional BF3· Et2O (534 mg, 3.77 mmol) and benzyltriethylammonium chloride (858 mg, 3.77 mmol) were added and the mixture stirred at 90 °C for 22 h. The mixture was then concentrated in vacuo and the resulting residue dissolved in dry CH2Cl2 (300 mL). The solution was cooled in an ice−EtOH bath for 5 min, and dry MeOH (100 mL) was added dropwise. This mixture was basified to pH 8 by addition of N,Ndiisopropylethylamine, whereupon the mixture was concentrated, and EtOAc (300 mL) was added. The resulting white precipitate was

removed by filtration and the filtrate concentrated in vacuo. Purification by silica gel chromatography (eluent: petroleum ether/ EtOAc 30/1 to 20/1) afforded 18 as a pale yellow solid (12.1 g, 69%): 1 H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.41 (s, 1H), 4.99 (s, 2H), 3.93 (s, 3H), 2.46 (s, 3H). 2-((3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-6(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one (20). 13 (800 mg, 2.45 mmol), bis(pinacolato)diboron (818 mg, 3.19 mmol), KOAc (313 mg, 3.19 mmol), and Pd(dppf)Cl2·CH2Cl2 (204 mg, 0.245 mmol) were mixed with 1,4-dioxane (20 mL) in a vial. The vial was sealed and heated to 80 °C. After 2 h, the temperature was reduced to 60 °C and stirring was continued for 16 h. The mixture was then allowed to cool to rt, filtered through diatomaceous earth, and concentrated in vacuo. The resulting crude material was purified by silica gel chromatography (eluent: heptane/EtOAc 1:0 to 0:1) to afford 20 as a brown oil (700 mg, 77%): 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.19 (s, 1H), 4.56−4.43 (m, 1H), 4.43−4.22 (m, 2H), 4.21−4.13 (m, 1H), 4.02 (d, J = 10.1 Hz, 1H), 3.82 (br s, 1H), 3.59− 3.48 (m, 1H), 3.30 (t, J = 9.6 Hz, 1H), 2.56 (s, 3H), 2.45 (br s, 1H), 1.90 (br s, 2H), 1.36 (s, 12H); LC-MS m/z [M + H]+ calcd for C20H28BNO5, 374.26; found, 374.5. 2-((3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl)-6-methyl-7(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroisoquinolin-1(2H)-one (21). A suspension of 14 (1.33 g, 4.62 mmol), B2Pin2 (2.37 g, 9.23 mmol), Pd(dppf)Cl2·CH2Cl2 (192 mg, 0.23 mmol), and KOAc (1.36 g, 13.8 mmol) in anhydrous 1,4-dioxane (25.6 mL) was heated in a pressure tube to 100 °C for 18 h. The reaction was cooled to rt and filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo to give a brown oil. The crude material was purified by silica gel chromatography (eluent: heptanes/EtOAc 7:3 to 0:1) to give 21 as a gray solid (1.16 g, 65%): 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 6.98 (s, 1H), 4.75 (ddd, J = 4.7, 10.3, 12.3 Hz, 1H), 4.15 (dd, J = 5.9, 11.3 Hz, 1H), 4.02 (dd, J = 4.7, 11.3 Hz, 1H), 3.77−3.66 (m, 1H), 3.60−3.43 (m, 3H), 3.25 (dd, J = 10.0, 11.1 Hz, 1H), 3.04−2.86 (m, 2H), 2.71 (d, J = 6.6 Hz, 1H), 2.55 (s, 3H), 1.91 (dq, J = 4.7, 12.4 Hz, 1H), 1.80−1.70 (m, 1H), 1.34 (s, 12H); 13C NMR (100 MHz, CDCl3) δ 166.8, 149.2, 140.2, 136.2, 128.2, 126.0, 83.6, 72.4, 67.8, 67.0, 56.7, 40.4, 29.0, 28.5, 24.94, 24.90, 22.3; LC-MS m/z [M + H]+ calcd for C21H30BNO5, 388.22; found, 388.5.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00597. Supplementary figures and data from functional assays; PXRD and thermodynamic solubility for compound 1 (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail jennifer.e.davoren@pfizer.com. ORCID

Jennifer E. Davoren: 0000-0002-7312-0666 Michael Popiolek: 0000-0001-7372-1982 Notes

The authors declare no competing financial interest. Compound 1 is commercially available from Sigma−Aldrich (catalog # PZ0359).

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ACKNOWLEDGMENTS We would like to acknowledge Simon Xi for mining the GTex data base and producing figures on human mRNA expression levels for M1−M5, Lois Chenard and our Wuxi team for 6659



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Alzheimer’s disease and schizophrenia. Neuropsychiatr. Dis. Treat 2014, 10, 183−191. (6) Witte, L. P. W.; Chapple, C. R.; de la Rosette, J. J. M. C. H.; Michel, M. C. Cholinergic innervation and muscarinic receptors in the human prostate. Eur. Urol. 2008, 54, 326−334. (7) Ryberg, A. T.; Warfvinge, G.; Axelsson, L.; Soukup, O.; Götrick, B.; Tobin, G. Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man. Arch. Oral Biol. 2008, 53, 66−74. (8) Unabridged figures of full-body M1-M5 mRNA expression can be found in the Supporting Information of this article. (9) Kruse, A. C.; Ring, A. M.; Manglik, A.; Hu, J.; Hu, K.; Eitel, K.; Hubner, H.; Pardon, E.; Valant, C.; Sexton, P. M.; Christopoulos, A.; Felder, C. C.; Gmeiner, P.; Steyaert, J.; Weis, W. I.; Garcia, K. C.; Wess, J.; Kobilka, B. K. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 2013, 504, 101−106. (10) Heptares. https://www.heptares.com/news/261/74/HeptaresAnnounces-Positive-Results-from-Phase-1b-Clinical-Trial-withHTL9936-a-First-In-Class-Selective-Muscarinic-M1-Receptor-Agonistfor-Improving-Cognition-in-Dementia-and-Schizophrenia.html (accessed Apr 18, 2017). (11) Lonsdale, J.; Thomas, J.; Salvatore, M.; Phillips, R.; Lo, E.; Shad, S.; Hasz, R.; Walters, G.; Garcia, F.; Young, N.; Foster, B.; Moser, M.; Karasik, E.; Gillard, B.; Ramsey, K.; Sullivan, S.; Bridge, J.; Magazine, H.; Syron, J.; Fleming, J.; Siminoff, L.; Traino, H.; Mosavel, M.; Barker, L.; Jewell, S.; Rohrer, D.; Maxim, D.; Filkins, D.; Harbach, P.; Cortadillo, E.; Berghuis, B.; Turner, L.; Hudson, E.; Feenstra, K.; Sobin, L.; Robb, J.; Branton, P.; Korzeniewski, G.; Shive, C.; Tabor, D.; Qi, L.; Groch, K.; Nampally, S.; Buia, S.; Zimmerman, A.; Smith, A.; Burges, R.; Robinson, K.; Valentino, K.; Bradbury, D.; Cosentino, M.; Diaz-Mayoral, N.; Kennedy, M.; Engel, T.; Williams, P.; Erickson, K.; Ardlie, K.; Winckler, W.; Getz, G.; DeLuca, D.; MacArthur, D.; Kellis, M.; Thomson, A.; Young, T.; Gelfand, E.; Donovan, M.; Meng, Y.; Grant, G.; Mash, D.; Marcus, Y.; Basile, M.; Liu, J.; Zhu, J.; Tu, Z.; Cox, N.; Nicolae, D.; Gamazon, E.; Im, H.; Konkashbaev, A.; Pritchard, J.; Stevens, M.; Flutre, T.; Wen, X.; Dermitzakis, E. T.; Lappalainen, T.; Guigo, R.; Monlong, J.; Sammeth, M.; Koller, D.; Battle, A.; Mostafavi, S.; McCarthy, M.; Rivas, M.; Maller, J.; Rusyn, I.; Nobel, A.; Wright, F.; Shabalin, A.; Feolo, M.; Sharopova, N.; Sturcke, A.; Paschal, J.; Anderson, J. M.; Wilder, E. L.; Derr, L. K.; Green, E. D.; Struewing, J. P.; Temple, G.; Volpi, S.; Boyer, J. T.; Thomson, E. J.; Guyer, M. S.; Ng, C.; Abdallah, A.; Colantuoni, D.; Insel, T. R.; Koester, S. E.; Little, A. R.; Bender, P. K.; Lehner, T.; Yao, Y.; Compton, C. C.; Vaught, J. B.; Sawyer, S.; Lockhart, N. C.; Demchok, J.; Moore, H. M. The Genotype-Tissue Expression (GTEx) project. Nat. Genet. 2013, 45, 580−585. (12) Ma, L.; Seager, M. A.; Wittmann, M.; Jacobson, M.; Bickel, D.; Burno, M.; Jones, K.; Graufelds, V. K.; Xu, G.; Pearson, M.; McCampbell, A.; Gaspar, R.; Shughrue, P.; Danziger, A.; Regan, C.; Flick, R.; Pascarella, D.; Garson, S.; Doran, S.; Kreatsoulas, C.; Veng, L.; Lindsley, C. W.; Shipe, W.; Kuduk, S.; Sur, C.; Kinney, G.; Seabrook, G. R.; Ray, W. J. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 15950−15955. (13) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Pitts, D. R.; Greshock, T. J.; Ma, L.; Wittmann, M.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.; Ray, W. J. Discovery of a selective allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold. J. Med. Chem. 2011, 54, 4773−4780. (14) (a) Uslaner, J. M.; Eddins, D.; Puri, V.; Cannon, C. E.; Sutcliffe, J.; Chew, C. S.; Pearson, M.; Vivian, J. A.; Chang, R. K.; Ray, W. J.; Kuduk, S. D.; Wittmann, M. The muscarinic M1 receptor positive allosteric modulator PQCA improves cognitive measures in rat, cynomolgus macaque, and rhesus macaque. Psychopharmacology 2013, 225, 21−30. (b) Lange, H. S.; Cannon, C. E.; Drott, J. T.; Kuduk, S. D.; Uslaner, J. M. The M1 muscarinic positive allosteric modulator PQCA improves performance on translatable tests of memory and attention in rhesus monkeys. J. Pharmacol. Exp. Ther. 2015, 355, 442− 450.

technical assistance in running libraries, Deborah Smith for running radioligand binding assays, Feng Shao and Bruce Rogers for helpful discussions, Rebecca O’Connor and HD Biosciences for screening assistance, Gregory Kauffman, Romelia Salomon-Ferrer, Joy Yang, and Xinjun Hou for modeling help, Clinton Bourbonais and Tim McNally for help with locomotor assays and PK submission, Yuxia Mao and Sheri Shamblin for formulation and thermodynamic solubility measurement assistance, Neal Sach for reaction optimization assistance and Katherine Brighty for editing.

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ABBREVIATIONS USED B 2 p in 2 , bis(pinacolato )diboron; B 2 p i n 2 , 1, 1′-bis(diphenylphosphino)ferrocene (dppf); Pd 2 (dba) 3 , tris(dibenzylideneacetone)dipalladium(0); HPD, hours post dose; ACh, acetylcholine; PAM, positive allosteric modulator; AE, adverse event; GI, gastrointestinal; CV, cardiovascular; PXRD, powder X-ray diffraction; GPI, guinea pig ileum; LMMP, longitudinal muscle myenteric plexus; mAChR, muscarinic acetylcholine receptor; CNS, central nervous system; NHP, nonhuman primate; AUC, area under the curve; AD, Alzheimer’s disease; MDR, multidrug resistance protein; HLM, human liver microsome; LipE, lipophilic efficiency; PK, pharmacokinetics; MWM, Morris water maze; aLMA, amphetamine-stimulated locomotor activity; B/P, brain/plasma; Fb,u, fraction unbound in brain; Fp,u, fraction unbound in plasma; Cb,u, concentration unbound in brain and; Cp,u, concentration unbound in plasma

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REFERENCES

(1) Kruse, A. C.; Kobilka, B. K.; Gautam, D.; Sexton, P. M.; Christopoulos, A.; Wess, J. Muscarinic acetylcholine receptors: novel opportunities for drug development. Nat. Rev. Drug Discovery 2014, 13, 549−560. (2) (a) Messer, W. S., Jr. The utility of muscarinic agonists in the treatment of Alzheimer’s disease. J. Mol. Neurosci. 2002, 19, 187−193. (b) Lieberman, J. A.; Javitch, J. A.; Moore, H. Cholinergic agonists as novel treatments for schizophrenia: the promise of rational drug development for psychiatry. Am. J. Psychiatry 2008, 165, 931−936. (c) Bodick, N. C.; Offen, W. W.; Levey, A. I.; Cutler, N. R.; Gauthier, S. G.; Satlin, A.; Shannon, H. E.; Tollefson, G. D.; Rasmussen, K.; Bymaster, F. P.; Hurley, D. J.; Potter, W. Z.; Paul, S. M. Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease. Arch. Neurol. 1997, 54, 465−473. (d) Shekhar, A.; Potter, W. Z.; Lightfoot, J.; Lienemann, J.; Dubé, S.; Mallinckrodt, C.; Bymaster, F. P.; McKinzie, D. L.; Felder, C. C. Selective muscarinic receptor agonist xanomeline as a novel treatment approach for schizophrenia. Am. J. Psychiatry 2008, 165, 1033−1039. (3) (a) Buchanan, K. A.; Petrovic, M. M.; Chamberlain, S. E. L.; Marrion, N. V.; Mellor, J. R. Facilitation of long-term potentiation by muscarinic M1 receptors is mediated by inhibition of SK channels. Neuron 2010, 68, 948−963. (b) Seol, G. H.; Ziburkus, J.; Huang, S.; Song, L.; Kim, I. T.; Takamiya, K.; Huganir, R. L.; Lee, H. K.; Kirkwood, A. Neuromodulators control the polarity of spike-timingdependent synaptic plasticity. Neuron 2007, 55, 919−929. (c) Shinoe, T.; Matsui, M.; Taketo, M. M.; Manabe, T. Modulation of synaptic plasticity by physiological activation of M1 muscarinic acetylcholine receptors in the mouse hippocampus. J. Neurosci. 2005, 25, 11194− 11200. (4) Anagnostaras, S. G.; Murphy, G. G.; Hamilton, S. E.; Mitchell, S. L.; Rahnama, N. P.; Nathanson, N. M.; Silva, A. J. Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice. Nat. Neurosci. 2002, 6, 51−58. (5) Foster, D. J.; Choi, D. L.; Conn, P. J.; Rook, J. M. Activation of M1 and M4 muscarinic receptors as potential treatments for 6660



Article

(15) (a) Kuduk, S. D.; Beshore, D. C. SAR studies on carboxylic acid series M1 selective positive allosteric modulators (PAMs). Curr. Top. Med. Chem. 2014, 14, 1738−1754. (b) Kuduk, S. D.; Chang, R. K.; Greshock, T. J.; Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T. Identification of amides as carboxylic acid surrogates for quinolizidinone-based M1 positive allosteric modulators. ACS Med. Chem. Lett. 2012, 3, 1070−1074. (c) Kuduk, S. D.; Di Marco, C. N.; Saffold, J. R.; Ray, W. J.; Ma, L.; Wittmann, M.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.; Beshore, D. C. Identification of a methoxynaphthalene scaffold as a core replacement in quinolizidinone amide M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2014, 24, 1417−1420. (d) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T. Quinolizidinone carboxylic acids as CNS penetrant, selective M1 allosteric muscarinic receptor modulators. ACS Med. Chem. Lett. 2010, 1, 263−267. (e) Kuduk, S. D.; Di Marco, C. N.; Cofre, V.; Pitts, D. R.; Ray, W. J.; Ma, L.; Wittmann, M.; Veng, L.; Seager, M. A.; Koeplinger, K.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T. N-Heterocyclic derived M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2010, 20, 1334−1337. (f) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T. Quinolizidinone carboxylic acid selective M1 allosteric modulators: SAR in the piperidine series. Bioorg. Med. Chem. Lett. 2011, 21, 1710−1715. (g) Kuduk, S. D.; Di Marco, C. N.; Cofre, V.; Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T. Fused heterocyclic M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2011, 21, 2769−2772. (16) (a) Kuduk, S. D.; Di Marco, C. N.; Yang, Z.-Q. N-Linked Quinolineamide M1 Receptor Positive Allosteric Modulators. WO 2012158474, May 11, 2012. (b) Kuduk, S. D.; Di Marco, C. N.; Greshock, T. J. N-Methyl-tetrahydroquinoline M1 Receptor Positive Allosteric Modulators. WO 2012158473, May 11, 2012. (c) Kuduk, S. D.; Chang, R. K.; Greshock, T. J. Dihydrobenzoquinazolinone M1 Receptor Positive Allosteric Modulators. WO 2012047702, Sep 29, 2011. (d) Kuduk, S. D.; Beshore, D. C.; Yang, Z.-Q.; Shu, Y.; Pitts, D. Preparation of Benzoisoindolylmethyllactam Derivatives and Analogs for Use as M1 Receptor Positive Allosteric Modulators. WO 2012158475, May 11, 2012. (e) Kuduk, S. D.; Beshore, D. C.; Yang, Z.; Shu, Y. Fused Isoindolone M1 Receptor Positive Allosteric Modulators and Their Preparation and Use for the Treatment of M1Mediated Diseases. WO 2012003147, Jun 27, 2011. (f) Kuduk, S. D.; Skudlarek, J. W. Preparation of Heterocyclic Fused Phenanthrolinones as M1 Receptor Positive Allosteric Modulators for the Treatment of Diseases Mediated by the M1 Receptor. WO 2011159553, Jun 10, 2011. (g) Kuduk, S. D.; Schlegel, K.-A.; Yang, Z.-Q. Quinoline-amide Derivatives as M1 Receptor Positive Allosteric Modulators and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO 2011084368, Dec 13, 2010. (h) Kuduk, S. D.; Gilbert, K. F. Preparation of Tetrahydroquinolinylamide Derivatives for Use as M1 Receptor Positive Allosteric Modulators. WO 2011159554, Jun 10, 2011. (i) Kuduk, S. D.; Chang, R. K.; Greshock, T. J. Benzoquinazolinone Derivatives as M1 Receptor Positive Allosteric Modulators and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO 2011025851, Aug 26, 2010. (j) Kuduk, S. D.; Chang, R. K.; Greshock, T. J. Quinolizine-3carboxamide Derivatives as M1 Receptor Positive Allosteric Modulators and Their Preparation and for the Treatment of Diseases. WO 2011062853, Nov 15, 2010 (k) Kuduk, S. D.; Chang, R. K.; Greshock, T. J. Pyridoquinazolinone as M1 Receptor Positive Allosteric Modulators and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO 2011075371, Dec 09, 2010. (l) Kuduk, S. D.; Beshore, D. C.; Di Marco, C. N.; Greshock, T. J. Aminobenzoquinazolinone M1 Receptor Positive Allosteric Modulators. WO 2011084371, Dec 13, 2010. (m) Beshore, D. C.; Kuduk, S. D.; Dipardo, R. M. Pyrazoloquinolinone Derivatives as M1 Receptor Positive Allosteric Modulators and

Their Preparation and Use for the Treatment of M1 ReceptorMediated Diseases. WO 2011049731, Oct 01, 2010. (n) Beshore, D. C.; Kuduk, S. D. Heterocyclic-Fused Pyrazolo[4,3-c]pyridin-3-one M1 Receptor Positive Allosteric Modulators. WO 2011041143, Sep 20, 2010. (o) Beshore, D. C.; Kuduk, S. D. Heterocyclic Quinolizine Derived M1 Receptor Positive Allosteric Modulators. WO 2011137049, Apr 25, 2011. (p) Beshore, D. C.; Di Marco, C. N.; Kuduk, S. D. Naphthalene Carboxamide Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2011149801, May 23, 2011. (q) Kuduk, S. D.; Di Marco, C. N. Preparation of 1-Benzyl-4-oxo-1,4dihydroquinoline-3-carboxylates as M1 Receptor Positive Allosteric Modulators. WO 2010019391, Feb 18, 2010. (r) Kuduk, S. D.; Di Marco, C. N. Preparation of Quinolizidinone M1 Receptor Positive Allosteric Modulators. WO 2010042347, Sep 29, 2009. (s) Kuduk, S. D.; Di Marco, C. N. Preparation of Fused Heterocyclic M1 Receptor Positive Allosteric Modulators. WO 2010047990, Oct 13, 2009. (t) Beshore, D. C.; Kuduk, S. D. Pyrazolo[4,3-c]cinnolin-3-one Derivatives as M1 Receptor Positive Allosteric Modulators and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO 2010096338, Feb 12, 2010. (u) Beshore, D. C.; Kuduk, S. D. Preparation of Fused Pyrazolopyridazines as Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2010123716, Apr 13, 2010. (v) Kuduk, S. D.; Di Marco, C. N.; Shipe, W. D. Preparation of Oxodihydroquinoline Derivatives for Use as M1 Receptor Positive Allosteric Modulators. WO 2009134668, Apr 23, 2009. (w) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N. Quinolizidinone M1 Receptor Positive Allosteric Modulators. WO 2009102574, Feb 03, 2009. (x) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N. Preparation of Quinolizidinone M1 Receptor Positive Allosteric Modulators. WO 2009117283, Mar 11, 2009. (y) Kuduk, S. D.; Chang, R. K. Synthesis of Quinolizidinone M1 Receptor Positive Allosteric Modulators. WO 2009051715, Oct 15, 2008. (z) Kuduk, S. D.; Chang, R. K. Preparation of Heterocyclylmethyl Oxoquinolizinecarboxylates as Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2009094279, Jan 15, 2009. (aa) Beshore, D. C.; Dipardo, R. M.; Kuduk, S. D. Preparation of Fused Pyridone M1 Receptor Positive Allosteric Modulators. WO 2009102588, Feb 04, 2009. (ab) Lindsley, C.; Shipe, W. D.; Yang, F.; Bunda, J. L. Preparation of Benzyl-Substituted Quinolone M1 Receptor Positive Allosteric Modulators. WO 2008002621, Jun 27, 2007. (ac) Shipe, W. D.; Lindsley, C.; Hallett, D. Quinolone Derivatives, Processes for Preparing Them, Pharmaceutical Compositions Containing Them, and Their Use as M1 Receptor Positive Allosteric Modulators. WO 2007067489, Dec 04, 2006. (ad) Shipe, W. D.; Lindsley, C.; Hallett, D. Preparation of Quinolone M1 Receptor Positive Allosteric Modulators. WO 2007100366, Dec 04, 2006. (17) Yang, Z. Q.; Shu, Y.; Ma, L.; Wittmann, M.; Ray, W. J.; Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.; Kuduk, S. D. Discovery of naphthyl-fused 5-membered lactams as a new class of M1 positive allosteric modulators. ACS Med. Chem. Lett. 2014, 5, 604−608. (18) (a) Kuduk, S. D.; Beshore, D. C.; Di Marco, C. N.; Greshock, T. J. Benzoquinazolinone Derivatives as M1 Receptor Positive Allosteric Modulators and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO 2010059773, May 27, 2010. (b) Chen, Q.; Scott, J.; Krska, S. W.; Baxter, C.; Stewart, G. W.; Tan, L.; Gibb, A.; Maligres, P. E. Process for making M1 receptor positive allosteric modulators. WO2017058691, Apr 6, 2017. (c) Shultz, C. S.; Maguire, C. K.; Xu, Y. Crystal Forms of a M1 Receptor Positive Allosteric Modulator. WO 2017058689, Apr 6, 2017. (19) ClinicalTrials.gov Identifier: NCT01852110 (accessed May 19, 2017). (20) Davoren, J. E.; O’Neil, S. V.; Anderson, D. P.; Brodney, M. A.; Chenard, L.; Dlugolenski, K.; Edgerton, J. R.; Green, M.; Garnsey, M.; Grimwood, S.; Harris, A. R.; Kauffman, G. W.; LaChapelle, E.; Lazzaro, J. T.; Lee, C. W.; Lotarski, S. M.; Nason, D. M.; Obach, R. S.; Reinhart, V.; Salomon-Ferrer, R.; Steyn, S. J.; Webb, D.; Yan, J.; Zhang, L. Design and optimization of selective azaindole amide M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2016, 26, 650− 655. 6661



Article

(21) Davoren, J. E.; Lee, C. W.; Garnsey, M.; Brodney, M. A.; Cordes, J.; Dlugolenski, K.; Edgerton, J. R.; Harris, A. R.; Helal, C. J.; Jenkinson, S.; Kauffman, G. W.; Kenakin, T. P.; Lazzaro, J. T.; Lotarski, S. M.; Mao, Y.; Nason, D. M.; Northcott, C.; Nottebaum, L.; O’Neil, S. V.; Pettersen, B.; Popiolek, M.; Reinhart, V.; SalomonFerrer, R.; Steyn, S. J.; Webb, D.; Zhang, L.; Grimwood, S. Discovery of the potent and selective M1 PAM-agonist N-[(3R,4S)-3hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4-yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of efficacy and cholinergic side effects. J. Med. Chem. 2016, 59, 6313−6328. (22) (a) Bender, A. M.; Conn, P. J.; Lindsley, C. W.; Emmitte, K. A.; Han, C.; Engers, J. L. Preparation of Substituted Imidazopyridine and Triazolopyridine Analogs as Positive Allosteric Modulators of Muscarinic Acetylcholine Receptor M1. WO 2016172547, Oct 27, 2016. (b) Rook, J. M.; Abe, M.; Cho, H. P.; Nance, K. D.; Luscombe, V. B.; Adams, J. J.; Dickerson, J. W.; Remke, D. H.; Garcia-Barrantes, P. M.; Engers, D. W.; Engers, J. L.; Chang, S.; Foster, J. J.; Blobaum, A. L.; Niswender, C. M.; Jones, C. K.; Conn, P. J.; Lindsley, C. W. Diverse effects on M1 signaling and adverse effect liability within a series of M1 ago-PAMs. ACS Chem. Neurosci. 2017, 8, 866−883. (c) Bridges, T. M.; Kennedy, J. P.; Noetzel, M. J.; Breininger, M. L.; Gentry, P. R.; Conn, P. J.; Lindsley, C. W. Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part II: Development of a potent and highly selective M1 PAM. Bioorg. Med. Chem. Lett. 2010, 20, 1972−1975. (d) Han, C.; Chatterjee, A.; Noetzel, M. J.; Panarese, J. D.; Smith, E.; Chase, P.; Hodder, P.; Niswender, C.; Conn, P. J.; Lindsley, C. W.; Stauffer, S. R. Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1:2,5-Dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators. Bioorg. Med. Chem. Lett. 2015, 25, 384−388. (e) Melancon, B. J.; Poslusney, M. S.; Gentry, P. R.; Tarr, J. C.; Sheffler, D. J.; Mattmann, M. E.; Bridges, T. M.; Utley, T. J.; Daniels, J. S.; Niswender, C. M.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. Isatin replacements applied to the highly selective, muscarinic M1 PAM ML137: Continued optimization of an MLPCN probe molecule. Bioorg. Med. Chem. Lett. 2013, 23, 412−416. (f) Panarese, J. D.; Cho, H. P.; Adams, J. J.; Nance, K. D.; Garcia-Barrantes, P. M.; Chang, S.; Morrison, R. D.; Blobaum, A. L.; Niswender, C. M.; Stauffer, S. R.; Conn, P. J.; Lindsley, C. W. Further optimization of the M1 PAM VU0453595: Discovery of novel heterobicyclic core motifs with improved CNS penetration. Bioorg. Med. Chem. Lett. 2016, 26, 3822− 3825. (g) Poslusney, M. S.; Melancon, B. J.; Gentry, P. R.; Sheffler, D. J.; Bridges, T. M.; Utley, T. J.; Daniels, J. S.; Niswender, C. M.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. Spirocyclic replacements for the isatin in the highly selective, muscarinic M1 PAM ML137: The continued optimization of an MLPCN probe molecule. Bioorg. Med. Chem. Lett. 2013, 23, 1860−1864. (h) Reid, P. R.; Bridges, T. M.; Sheffler, D. J.; Cho, H. P.; Lewis, L. M.; Days, E.; Daniels, J. S.; Jones, C. K.; Niswender, C. M.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe. Bioorg. Med. Chem. Lett. 2011, 21, 2697−2701. (i) Lindsley, C. W.; Conn, P. J.; Wood, M. R.; Hopkins, C. R.; Melancon, B. J.; Poslusney, M. S.; Engers, D. W. Substituted 2-(4-Heterocyclylbenzyl)isoindolin-1-one Analogs as Positive Allosteric Modulators of the Muscarinic Acetylcholine Receptor M1. WO 2013063549, Oct 28, 2012. (j) Lindsley, C. W.; Conn, P. J.; Wood, M. R.; Hopkins, C. R.; Melancon, B. J.; Poslusney, M. S. Preparation of Substituted Benzylspiroindolin-2-one Derivatives as Positive Allosteric Modulators of the mAChR M1 Receptor. WO 2013071201, Nov 10, 2012. (k) Lindsley, C. W.; Conn, P. J.; Wood, M. R.; Melancon, B. J.; Poslusney, M. S.; Tarr, J. C. Substituted Pyrazol-benzyl Analogues as Positive Allosteric Modulators of mAChR M1 Receptors. WO 2013106795, Jan 12, 2013. (l) Lindsley, C. W.; Conn, P. J.; Wood, M. R.; Melancon, B. J.; Poslusney, M. S. Preparation of Substituted 1-Benzylindolin-2-one Analogs as Positive Allosteric Modulators of Muscarinic Acetylcholine M1 Receptors for Treating Psychiatric and Neurological Disorders. US 20140194471, Jul

10, 2014. (m) Lindsley, C. W.; Conn, P. J.; Stauffer, S. R.; Panarese, J. D. Substituted 1-Benzylquinoxalin-2(1H)-one Analogs as Positive Allosteric Modulators of Muscarinic Acetylcholine Receptor M1. WO 2014179226, Apr 28, 2014. (n) Lindsley, C. W.; Conn, P. J.; Stauffer, S. R.; Panarese, J. D. Quinazolinone and Pyridopyrimidinone Derivatives as Positive Allosteric Modulators of Muscarinic Acetylcholine Receptor M1 and Their Preparation and Use for the Treatment of Neurological and Psychiatric Disorders. WO 2014179237, Apr 28, 2014. (o) Lindsley, C. W.; Conn, P. J.; Stauffer, S. R.; Panarese, J. D. Preparation of Benzyldihydrobenzooxazinecarboxamide Derivatives for Use as Positive Allosteric Modulators of Muscarinic Acetylcholine Receptor M1. WO 2015080904, Nov 18, 2014. (p) Lindsley, C. W.; Conn, P. J.; Wood, M. R.; Tarr, J. C.; Bridges, T. M. Indole Compounds as Positive Allosteric Modulators of the Muscarinic Receptor. WO 2011163280, Jun 21, 2011. (23) (a) Ogino, M.; Kimura, E.; Suzuki, S.; Ashizawa, T.; Imaeda, T.; Fujimori, I.; Arai, R. Preparation of Nitrogen-Containing Heterocyclic Compound as Muscarinic M1 Receptor Positive Allosteric Modulators for Treatment of Nervous System Disease. WO 2015190564, Jun 11, 2015. (b) Yamada, M.; Suzuki, S.; Sugimoto, T.; Nakamura, M.; Sakamoto, H.; Kamata, M. Isoindolin-1-one Derivatives as Cholinergic Muscarinic M1 Receptor Positive Allosteric Modulators and Their Preparation and Use for the Treatment of Diseases. WO 2015163485 Apr 22, 2015. (c) Sugimoto, T.; Suzuki, S.; Sakamoto, H.; Yamada, M.; Nakamura, M.; Kamata, M.; Shimokawa, K.; Ogino, M.; Kimura, E.; Murakami, M.; Yonemori, J.; Kojima, T. Preparation of 2,3-dihydro4H-1,3-benzoxazin-4-one Derivatives as Modulators of Cholinergic Muscarinic M1 Receptor. WO 2016208775, Dec 29, 2016. (24) Sugimoto, T.; Shimokawa, K.; Kojima, T.; Sakamoto, H.; Fujimori, I.; Nakamura, M.; Yamada, M.; Murakami, M.; Kamata, M.; Suzuki, S. Nitrogen-Containing Heterocyclic Compounds and Their Use for Pharmaceuticals and Cholinergic Muscarinic M1 Receptor Positive Allosteric Modulation. WO 2015174534, Nov 19, 2015. (25) Swinnen, D.; Montagne, C.; Pomel, V.; Quattropani, A.; Molette, J.; Gerber, P. Tetraaza-cyclopenta[a]indenyl Derivatives as M1 Receptor Positive Allosteric Modulators Useful in the Treatment of Diseases Mediated by the Muscarinic M1 Mediator and Their Preparation. WO 2013091773, Nov 30, 2012. (26) (a) Mistry, S. N.; Lim, H.; Jörg, M.; Capuano, B.; Christopoulos, A.; Lane, J. R.; Scammells, P. J. Novel fused arylpyrimidinone based allosteric modulators of the M1 muscarinic acetylcholine receptor. ACS Chem. Neurosci. 2016, 7, 647−661. (b) Mistry, S. N.; Valant, C.; Sexton, P. M.; Capuano, B.; Christopoulos, A.; Scammells, P. J. Synthesis and pharmacological profiling of analogues of benzyl quinolone carboxylic acid (BQCA) as allosteric modulators of the M1 muscarinic receptor. J. Med. Chem. 2013, 56, 5151−5172. (c) Mistry, S. N.; Jörg, M.; Lim, H.; Vinh, N. B.; Sexton, P. M.; Capuano, B.; Christopoulos, A.; Lane, J. R.; Scammells, P. J. 4Phenylpyridin-2-one derivatives: A novel class of positive allosteric modulator of the M1 muscarinic acetylcholine receptor. J. Med. Chem. 2016, 59, 388−409. (27) (a) Ballard, T. M.; Flohr, A.; Groebke Zbinden, K.; Pinard, E.; Ryckmans, T.; Schaffhauser, H. Preparation of Pyrrolopyridine and Pyrazolopyridine Derivatives as Muscarinic M1 Receptor Modulators. WO 2015028483, Aug 08, 2014. (b) Ballard, T. M.; Groebke Zbinden, K.; Pinard, E.; Ryckmans, T.; Schaffhauser, H. Preparation of Indole and Indazole Derivatives as Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2015044072, Sep 22, 2014. (c) Groebke Zbinden, K.; Pinard, E.; Ryckmans, T. Preparation of Fluoro-naphthyl Derivatives as Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2015110370, Jul 30, 2015. (28) (a) Cruickshank, J. W.; Brudzynski, S. M.; McLachlan, R. S. Involvement of M1 muscarinic receptors in the initiation of cholinergically induced epileptic seizures in the rat brain. Brain Res. 1994, 643, 125−129. (b) Hamilton, S. E.; Loose, M. D.; Qi, M.; Levey, A. I.; Hille, B.; McKnight, G. S.; Idzerda, R. L.; Nathanson, N. M. Disruption of the M1 receptor gene ablates muscarinic receptordependent M current regulation and seizure activity in mice. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 13311−13316. (c) Bymaster, F. P.; 6662



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for β-arrestin, compound 1 has 50% of the intrinsic efficacy of ACh. These fractions are assay-dependent. (45) This is because for PAMs, the effective receptor occupancy is determined by KB divided by the alpha-factor. (46) Popiolek, M.; Nguyen, D. P.; Reinhart, V.; Edgerton, J. R.; Harms, J.; Lotarski, S. M.; Steyn, S. J.; Davoren, J. E.; Grimwood, S. Inositol phosphate accumulation in vivo provides a measure of muscarinic M1 receptor activation. Biochemistry 2016, 55, 7073−7085. (47) (a) Buresova, O.; Bolhuis, J. J.; Bures, J. Differential effects of cholinergic blockade on performance of rats in the water tank navigation task and in a radial water maze. Behav. Neurosci. 1986, 100, 476−482. (b) Morris, R. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 1984, 11, 47− 60. (48) The lowest dose of 0.32 mg/kg (Cb,u = 3.2 nM) was statistically significant on days 4 and 5, whereas the high dose was similar, but with statistical significance observed only on day 4. It is unclear why this Ushaped curve was observed with compound 1. Previously, we reported on compound 7 in scopolamine-disrupted MWM, and we did not observe this profile, and effects were dose-dependent. (49) A true Cmax was not measured at this dose, but the convulsions were associated with the highest measured plasma drug level for each animal. (50) Estimated using the measured rat Cb,u/Cp,u = 0.2. (51) Fessif thermodynamic solubility 0.0397 mg/mL at pH 6.3; for additional solubility measurements and PXRD of measured lot, please see the Supporting Information. (52) Patel, V. R.; Agrawal, Y. K. Nanosuspension: An approach to enhance solubility of drugs. J. Adv. Pharm. Technol. Res. 2011, 2, 81− 87. (53) Friesen, D. T.; Shanker, R.; Crew, M.; Smithey, D. T.; Curatolo, W. J.; Nightingale, J. A. Hydroxypropyl methylcellulose acetate succinate-based spray-dried dispersions: An overview. Mol. Pharmaceutics 2008, 5, 1003−1019. (54) Curia, G.; Longo, D.; Biagini, G.; Jones, R. S. G.; Avoli, M. The pilocarpine model of temporal lobe epilepsy. J. Neurosci. Methods 2008, 172, 143−157. (55) NOAELhttp://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/ucm292340.pdf (accessed Jan 15, 2017). (56) (a) Pratt, J.; Winchester, C.; Dawson, N.; Morris, B. Advancing schizophrenia drug discovery: Optimizing rodent models to bridge the translational gap. Nat. Rev. Drug Discovery 2012, 11, 560−579. (b) Hånell, A.; Marklund, N. Structured evaluation of rodent behavioral tests used in drug discovery research. Front. Behav. Neurosci. 2014, 8, 252. (57) The 2-fold value was derived from a PK/PD model of compounds with known clinical benefit. This research will be published in an independent manuscript. (58) Smith, D. L.; Davoren, J. E.; Edgerton, J. R.; Lazzaro, J. T.; Lee, C. W.; Neal, S.; Zhang, L.; Grimwood, S. Characterization of a novel M1 muscarinic acetylcholine receptor positive allosteric modulator radioligand, [3H]PT-1284. Mol. Pharmacol. 2016, 90, 177−187.

Carter, P. A.; Yamada, M.; Gomeza, J.; Wess, J.; Hamilton, S. E.; Nathanson, N. M.; McKinzie, D. L.; Felder, C. C. Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpineinduced seizure activity. Eur. J. Neurosci. 2003, 17, 1403−1410. (29) Alt, A.; Pendri, A.; Bertekap, R. L., Jr.; Li, G.; Benitex, Y.; Nophsker, M.; Rockwell, K. L.; Burford, N. T.; Sum, C. S.; Chen, J.; Herbst, J. J.; Ferrante, M.; Hendricson, A.; Cvijic, M. E.; Westphal, R. S.; O’Connell, J.; Banks, M.; Zhang, L.; Gentles, R. G.; Jenkins, S.; Loy, J.; Macor, J. E. Evidence for classical cholinergic toxicity associated with selective activation of M1 muscarinic receptors. J. Pharmacol. Exp. Ther. 2016, 356, 293−304. (30) Brodney, M. A.; Davoren, J. E.; Garnsey, M. R.; Zhang, L.; O’Neil, S. V. Pyridine Derivatives as Muscarinic M1 Receptor Positive Allosteric Modulators. WO 2016009297 Jul 02, 2015. (31) Zhang, Y. H.; Shi, B. F.; Yu, J. Q. Palladium(II)-catalyzed ortho alkylation of benzoic acids with alkyl halides. Angew. Chem., Int. Ed. 2009, 48, 6097−6100. (32) Because of the heterogeneity of the reaction mixture, the yields were highly variable in this step. (33) It is worth mentioning that during the course of our work on the M1 receptor, our organization replaced our legacy MDR cell line with a new line purchased from the NIH; therefore, the MDR ER values published in this article are not directly comparable to our previous reports on M1 PAM chemical matter. (34) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Rapid assessment of a novel series of selective CB(2) agonists using parallel synthesis protocols: A Lipophilic Efficiency (LipE) analysis. Bioorg. Med. Chem. Lett. 2009, 19, 4406−4409. (35) Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F. ElogD(oct): a tool for lipophilicity determination in drug discovery. 2. Basic and neutral compounds. J. Med. Chem. 2001, 44, 2490−2497. (36) Park, M. S.; Okochi, H.; Benet, L. Z. Is ciprofloxacin a substrate of P-glycoprotein? Arch. Drug Inf. 2011, 4, 1−9. (37) Composite M1 agonist and PAM curves for all compounds in Table 1 can be viewed in the Supporting Information for comparison. (38) (a) Dasari, S.; Gulledge, A. T. M1 and M4 receptors modulate hippocampal pyramidal neurons. J. Neurophysiol. 2011, 105, 779−792. (b) Langmead, C. J.; Austin, N. E.; Branch, C. L.; Brown, J. T.; Buchanan, K. A.; Davies, C. H.; Forbes, I. T.; Fry, V. A. H.; Hagan, J. J.; Herdon, H. J.; Jones, G. A.; Jeggo, R.; Kew, J. N.; Mazzali, A.; Melarange, R.; Patel, N.; Pardoe, J.; Randall, A. D.; Roberts, C.; Roopun, A.; Starr, K. R.; Teriakidis, A.; Wood, M. D.; Whittington, M.; Wu, Z.; Watson, J. Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28−1. Br. J. Pharmacol. 2008, 154, 1104−1115. (39) Weaver, C. D.; Sheffler, D. J.; Lewis, L. M.; Bridges, T. M.; Williams, R.; Nalywajko, N. T.; Kennedy, J. P.; Mulder, M. M.; Jadhav, S.; Aldrich, L. A.; Jones, C. K.; Marlo, J. E.; Niswender, C. M.; Mock, M. M.; Zheng, F.; Conn, P. J.; Lindsley, C. W. Discovery and development of a potent and highly selective small molecule muscarinic acetylcholine receptor subtype I (mAChR 1 or M1) antagonist in vitro and in vivo probe. Curr. Top. Med. Chem. 2009, 9, 1217−1226. (40) Indeed, differences in the potency of the muscarinic agonist carbachol were noted between these two preparations (129 nM for FLIPR versus 329 nM for the slice preparation). (41) Gustafsson, L.; Hedqvist, P.; Fredholm, B. B.; Lundgren, G. Inhibition of acetylcholine release in guinea pig ileum by adenosine. Acta Physiol. Scand. 1978, 104, 469−478. (42) Luttrell, L. M.; Lefkowitz, R. J. The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals. J. Cell Sci. 2002, 115, 455−465. (43) Black, J. W.; Leff, P. Operational models of pharmacological agonism. Proc. R. Soc. London, Ser. B 1983, 220, 141−162. (44) The τB values represent the intrinsic efficacy of compound 1, and these are expressed as a fraction of the τA values in the assay; i.e., for Ca++ FLIPR, compound 1 has 35% of the efficacy of ACh, while 6663


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