Probing Structural Requirements of Positive Allosteric Modulators of

Sep 27, 2013 - ABSTRACT: The M4 mAChR is implicated in several CNS disorders and ... focused library of putative M4 PAMs derived from VU0152100 and ...
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Probing Structural Requirements of Positive Allosteric Modulators of the M4 Muscarinic Receptor Tracey Huynh,† Celine Valant,‡ Ian T. Crosby,†,§ Patrick M. Sexton,‡ Arthur Christopoulos,*,‡ and Ben Capuano*,† †

Medicinal Chemistry and ‡Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381-399 Royal Parade, Parkville VIC 3052, Australia S Supporting Information *

ABSTRACT: The M4 mAChR is implicated in several CNS disorders and possesses an allosteric binding site for which ligands modulating the affinity and/or efficacy of ACh may be exploited for selective receptor targeting. We report the synthesis of a focused library of putative M4 PAMs derived from VU0152100 and VU10005. These compounds investigate the pharmacological effects of previously identified methoxy and fluoro substituents, providing useful estimates of affinity (KB), cooperativity (αβ), and direct agonist properties (τB).

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INTRODUCTION Muscarinic acetylcholine receptors (mAChR) are part of the G protein-coupled receptor (GPCR) superfamily and, to date, five mAChR subtypes have been identified (M1−M5). Each of these subtypes is distributed throughout the periphery and central nervous system (CNS). The M1 and M4 subtypes have been of particular interest for the treatment of a range of CNS disorders, as they are abundantly expressed in the forebrain, including the striatum, cerebral cortex, and hippocampus,1,2 and have been implicated in neuropsychiatric disorders such as Alzheimer’s disease and schizophrenia. Importantly, xanomeline (Figure 1), an M1/M4-preferring orthosteric agonist, was

propyl-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxamide (LY2033298, 2) (Figure 1) as a robust positive allosteric modulator (PAM) of the endogenous ligand, ACh, at the M4 mAChR. Further mechanistic studies8 demonstrated that 2 enhances both the affinity and the efficacy of ACh, suggesting multiple modes by which selectivity can be attained via an allosteric mechanism to potentially limit side effects. Shirey et al.9 identified a bicyclic scaffold, VU10010 (Figure 1), similar to that of 2, which produced positive allosteric modulation of agonist function at the M4 mAChR. Further chemical optimization produced additional promising M4 mAChR PAMs, namely VU0152100 (1a) and VU10005 (1i) (Figure 1).9,10 To date, the bulk of studies focusing on target- and mechanism-based chemical optimization of M4 mAChR PAMs have relied on modulator titration curves, whereby a fixed concentration (e.g., EC20) of orthosteric agonist is tested against increasing concentrations of the prospective allosteric ligand to determine modulator potency.10 Although undoubtedly useful for high throughput screening and optimization, a limitation of this approach is that it may often yield apparently “flat” structure−activity relationship (SAR) profiles if a single potency parameter is used as the sole determinant of compound activity. This is because, operationally, allosteric modulator activity is characterized by minimally four pharmacological parameters, namely the affinity of the allosteric ligand for the free receptor (KB), the cooperativity factors that define the magnitude and direction of the allosteric ligand’s effect on the orthosteric ligand’s affinity (α) and/or downstream efficacy (β), and the intrinsic agonist efficacy of the allosteric ligand (τ B ). 11,12 In an interaction paradigm exemplified by the titration curve experimental design, the potency of the modulator and its apparent maximal effect are amalgams of these four parameters. Changes in one of the parameters as a consequence of chemical modification may be

Figure 1. The M1/M4-preferring orthosteric agonist xanomeline, the M4 PAM from Eli Lilly (2), and M4 PAMs containing the thienopyridine scaffold developed by Vanderbilt University (VU).

introduced into clinical trials and exhibited dose-dependent improvements in cognitive deficits and positive symptoms (e.g., hallucinations, delusions) associated with schizophrenia.3 Despite these promising clinical benefits, xanomeline showed unfavorable side effects as a result of nonselective binding to other mAChR subtypes in the periphery. This is due to the high amino acid sequence conservation of the orthosteric site of each of the mAChR subtypes, a notorious limitation to the discovery of highly subtype-selective ligands. An alternative avenue to attaining greater selectivity in such instances is to target topographically distinct allosteric sites, which have been identified for all of the mAChR subtypes.4−6 A major breakthrough in this regard was the study by Chan et al.,7 which reported the discovery of 3-amino-5-chloro-N-cyclo© 2013 American Chemical Society

Received: July 11, 2013 Published: September 27, 2013 8196

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Brief Article

offset by changes in another of the parameters in the opposite direction, hence potentially accounting for an apparent flat SAR based on potency alone. At a minimum, we have shown that delineation of three operational characteristics of allostery i.e., modulator affinity (KB), overall cooperativity (αβ), and (any) direct allosteric agonism (τB), can be routinely obtained from a more comprehensive functional concentration−response curve analysis13,14 and propose that this can yield a more “enriched” SAR for describing GPCR allostery. As a proof of concept, we have selected 1a and 1i as model lead compounds to define the aforementioned operational parameters of allosterism for the M4 mAChR and report the chemical synthesis of a focused library of compounds incorporating modifications to the substitution pattern of the arylmethyl motif of the VU lead compounds. Specifically, we aim to investigate the electronic and positional effects of previously identified substituents, namely the methoxy and fluoro groups15 on the three operational parameters of GPCR allostery described above. In addition to providing the first quantification of these properties for such compounds, we reveal a correlation between the degree of direct allosteric agonism and magnitude of the PAM effect, highlighting how such enriched SAR can provide new insights into the mode of action of GPCR modulators.

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Scheme 1. Chemical Synthesis of the Target Thienopyridines (1a−q) from the Key Intermediate Core (5) and the Substituted N-Benzyl Chloroacetamides (8a− q)a

RESULTS AND DISCUSSION

Chemistry. The general synthesis for the target thieno[2,3b]pyridine structures (1) is illustrated in Scheme 1. Synthesis of the key pyridinethione core (4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile, 5)16 was furnished by a condensation reaction between acetylacetone (3) and 2-cyanothioacetamide (4) and isolated in good yield. The chloroacetamide derivatives 8a−q were synthesized in good to excellent yield in accordance with literature,17 whereby chloroacetyl chloride (6) was reacted with the appropriately substituted benzylamine (7a−q). The synthetic route for the final muscarinic M4 PAM analogues was initially derived from the procedure described by Kennedy et al.,15 which combined 5 and 8 in the presence of triethylamine and DMF at room temperature. This procedure unexpectedly led to the synthesis of the monocyclic intermediates 9a−c,e,g,h,k,l,p rather than the desired bicyclic thienopyridine compounds (1) (Scheme 1). The use of triethylamine was insufficiently basic to form the bicyclic core (1) as suggested.15 The Thorpe−Ziegler cyclization of 9a− c,e,g,h,k,l,p was carried out using potassium hydroxide in DMF at room temperature, and within 20 min, furnished the target bicyclic compounds 1a−c,e,g,h,k,l,p in good yields (Scheme 1). The alternative synthesis of the bicyclic compounds 1d,f,i,j,m−o,q was effected following the procedure described by Shirey et al.9 (Scheme 1 (v)), whereby the chloroacetamide (rather than the described bromoacetamide) was reacted with 5 in the presence of aqueous potassium hydroxide in DMF at elevated temperature. This was followed by the conversion of the primary aromatic amine of the target compounds to their respective monohydrochloride salts for biological evaluation using ethereal hydrogen chloride; all target compounds afforded bright-yellow solids in quantitative yield. Biological Activity. To assess the biological activity of both the monocyclic (9a−c,e,g,h,k,l,p) and bicyclic (1a−q) series of compounds, cell-based functional assays of M4 receptormediated ERK1/2 phosphorylation assays were performed

a

Reagents and conditions: (i) CH3OH, rt, 2 h, reflux, 2 h, then 6 M HCl (aq), 61%; (ii) Et3N, DCM, 0 °C to rt, 45−99%; (iii) Et3N, DMF, rt, 30 min, 65−94%; (iv) KOH (aq), DMF, rt, 15−20 min, (46−92%); (v) KOH (aq), DMF, rt, 24 h followed by 50 °C, 15 h, (35−78%).

using intact FlpIn-CHO cells stably transfected with the human muscarinic M4 receptor. Time-course assays were initially performed to ascertain the optimal incubation time for maximum ERK1/2 phosphorylation for each compound. Subsequently, concentration− response curves were established at this time point to determine the potency and maximal effect each compound as a potential direct agonist of the M4 mAChR. Finally, ACh interaction studies were performed to identify prospective M4 PAMs from both series 9a−c,e,g,h,k,l,p and 1a−q and to quantify their activity; an example is shown for 1n in Figure 2A. Although five of the 17 compounds synthesized have been previously reported (two in the literature9,10 (1a and 1i) and three (1g and 1j−k) as part of the Vanderbilt University Specialized Chemistry Center for Accelerated Probe Development database (Vanderbilt University Medical Center), a detailed pharmacological assessment of the allosteric profiles of these ligands has not been performed to date. We thus applied an operational model of allosterism to estimate the negative logarithm of the binding affinity of the ligands at the allosteric site (pKB), their respective intrinsic efficacies (τB) as direct allosteric agonists, and the composite cooperativity (αβ) as a global measure of the allosteric ligand’s effect on ACh affinity and efficacy.8 8197

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The novel monocyclic series 9a−c,e,g,h,k,l,p did not display any significant allosteric modulatory effects. In contrast, interaction studies revealed varying degrees of activity for the bicyclic series (1a−q). The binding affinity, functional cooperativity, and intrinsic efficacy estimates for all test compounds (1a−q) on the aforementioned assay are tabulated in Table 1. For comparison, we also determined empirical EC50 and Emax (EM′) parameters for the effect of each compound on an EC20 concentration of ACh, thus mimicking the more typical titration curve experimental design (Table 1). Modification of the methoxy substitution pattern from the 4position of the literature compound 1a (KB = 1.14 μM) to the 3-position (1b) resulted in a significant (p < 0.05) reduction in affinity by an order of magnitude (KB = 11.4 μM) but, interestingly, the cooperativity with ACh showed a trend toward improvement. In contrast, a comparison of the EC50 values from the more standard titration curve data indicated no significant effect on modulator potency (Table 1). This example illustrates how the same structural modification can have opposing effects on different properties underlying allosteric modulator actions (i.e., affinity vs cooperativity) that can thus contribute to the appearance of a “flat” SAR in titration curve mode. In contrast, the 2-methoxy analogue (1c), along with the di- and trimethoxy substituted compounds 1d−f, demonstrated no apparent activity at the M4 mAChR. To ascertain whether this reflected lack of interaction at the receptor, or neutral modulation with ACh, these compounds were also tested in combination with known PAMs of the M4 mAChR (see Supporting Information). The results of these experiments showed that 1c−f and 1q did not compete for the allosteric site. The literature compound 1a retained the best allosteric profile within the methoxy-substituted series (1a−e).

Figure 2. (A) Effect of increasing concentrations of 1n on AChmediated ERK1/2 phosphorylation at the M4 mAChR stably expressed in CHO FlpIn cells. (B) Correlation analysis of efficacy (τB) and cooperativity (αβ) parameter estimates for bicyclic series 1a−q.

Table 1. Binding Affinity (KB), Functional Cooperativity (αβ), and Intrinsic Efficacy (τB) Estimates of Test Compounds on M4 mAChR-Mediated ERK1/2 Phosphorylation in the Presence of ACha

compd

R

1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q

4-OCH3 3-OCH3 2-OCH3 3,4-(OCH3)2 3,5-(OCH3)2 3,4,5-(OCH3)3 2-F 3-F 4-F 2,3-F2 2,5-F2 2,4,5-F3 2,3,5-F3 3-F-4-OCH3 2-F-4-OCH3 2,3-F2-4-OCH3 2,5-F2-4-OCH3

pEC50 (EC50, μM)

EM′ (%)

6.89 ± 0.23 6.25 ± 0.17 na na na na 6.90 ± 0.16 6.51 ± 0.12 6.66 ± 0.27 6.98 ± 0.17 6.88 ± 0.25 6.24 ± 0.41 6.85 ± 0.12 7.00 ± 0.11 6.46 ± 0.34 6.62 ± 0.23 na

85 ± 92 ± na na na na 92 ± 98 ± 98 ± 91 ± 85 ± 83 ± 85 ± 95 ± 56 ± 89 ± na

(0.13) (0.56)

(0.13) (0.31) (0.22) (0.11) (0.13) (0.58) (0.14) (0.10) (0.35) (0.24)

4 5

3 3 5 3 3 7 2 2 5 5

pKB (KB, μM) 5.95 ± 0.16 4.95 ± 0.18 na na na na 5.93 ± 0.11 5.21 ± 0.17 5.60 ± 0.15 5.76 ± 0.13 5.77 ± 0.11 5.63 ± 0.21 5.89 ± 0.11 5.59 ± 0.11 5.74 ± 0.21 5.73 ± 0.16 na

(1.14) (11.4)

(1.17) (6.24) (2.52) (1.72) (1.69) (2.32) (1.29) (2.58) (1.82) (1.88)

log αβ (αβ) 0.68 ± 0.22 1.37 ± 0.18 na na na na 1.10 ± 0.15 1.63 ± 0.21 1.12 ± 0.20 1.15 ± 0.19 0.76 ± 0.14 0.70 ± 0.16 0.81 ± 0.12 1.53 ± 0.14 0.49 ± 0.07 0.83 ± 0.22 na

(4.77) (23.4)

(12.4) (42.2) (13.2) (14.3) (5.70) (4.96) (6.41) (34.0) (3.11) (6.69)

log τB (τB) 0.22 ± 0.06 (1.68) 0.34 ± 0.10 (2.18) na na na na 0.36 ± 0.06 (2.30) 0.76 ± 0.13 (5.71) 0.44 ± 0.09 (2.77) 0.45 ± 0.08 (2.80) 0.15 ± 0.05 (1.43) −0.27 ± 0.10 (0.54) 0.03 ± 0.04 (1.08) 0.48 ± 0.07 (3.04) −1.12 ± 0.17 (0.08) 0.41 ± 0.09 (2.59) na

Also shown are empirical potency (EC50) and Emax (EM′) for the effects of each compound on a single (EC20) concentration of ACh. Values represent the mean SEM from at least three experiments performed in duplicate. na: not active.

a

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Journal of Medicinal Chemistry Examination of the biological activity of the fluoro substituted compounds 1g,h,j−m revealed no substantial changes in modulator affinity for the free receptor compared to the 4-fluoro substituted literature compound 1i (KB = 2.52 μM). The 3-fluoro substituted analogue (1h) demonstrated the most promising profile, characterized by an approximate 3-fold improvement in functional cooperativity with ACh compared to 1i, although this was not statistically significant. Of note are 1g and 1j (the 2-fluoro and 2,3-difluoro substituted analogues, respectively), which exhibited binding, functional cooperativity, and intrinsic efficacy profiles commensurate with that of 1i. The analogue brandishing the 2,5-difluoro substitution pattern, 1k, previously identified by Kennedy et al. as displaying significant activity in their ACh “fold-shift” assay,15 exhibited a diminished profile to that of 1i when incorporated into the simplified scaffold. The trifluoro-substituted analogues 1l and 1m offered no advantage over their monofluoro-substituted counterparts 1g−i. Compounds incorporating both fluoro and methoxy substituents (1n−q) exhibited mixed profiles for affinity, functional cooperativity, and intrinsic efficacy compared to the fluoro-substituted analogues (1g−j) and methoxy-substituted analogues (1a−b). 1n (3-fluoro-4-methoxy analogue), which furnishes the desired positional and electronic features of 1a (4-methoxy analogue) and 1h (3-fluoro analogue) described previously, exhibited the best modulatory and intrinsic agonism properties within this subset. Modifying the position of the fluorine substituent from the 3-position to the 2-position as exemplified in 1o (2-fluoro-4-methoxy analogue) afforded a significant (p < 0.05) reduction in functional cooperativity (log αβ = 0.49 ± 0.07; αβ = 3.11) and virtually abolished intrinsic efficacy (τB = 0.08) compared to 1n (log αβ = 1.53 ± 0.14; αβ = 34.0; τB = 3.04). Subsequent incorporation of an additional fluorine substituent at the 3-postion, as denoted by 1p (2,3-difluoro-4methoxy analogue), restored the intrinsic efficacy (τB = 2.59) to a level comparable to 1n (τB = 3.04). Surprisingly, the 2,5difluoro-4-methoxy analogue 1q failed to display any appreciable binding affinity, cooperativity, or intrinsic efficacy, highlighting the shallow SAR often associated with libraries of allosteric modulators.5 Finally, the ability to resolve the SAR around these compounds in terms of separate allosteric model parameters yielded additional insights into their mechanism of action. Specifically, as shown in Figure 2B, we found a significant correlation (slope = 1.00 ± 0.28) between the cooperativity and efficacy estimates, suggesting that the mechanism of positive modulation involves, at least in part, the ability of these molecules to preferentially stabilize an active state of the M4 mAChR. To ascertain whether this stabilization was manifested at the level of orthosteric ligand affinity, signaling efficacy, or both, we investigated the effects of representative compounds of series 1 (1a, 1h−j, 1n, and 1p) on the equilibrium binding of [3H]NMS (see Supporting Information). No significant difference was identified between the values of log αβ derived from analysis the cell-based functional data, and log α derived from interaction binding studies with [3H]NMS, suggesting that the observed cooperativity of these ligands (1a, 1h−j, 1n, and 1p) is primarily manifested as modulation of the ACh affinity, rather than efficacy.

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CONCLUSION

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EXPERIMENTAL SECTION

Brief Article

A focused library of compounds derived from the previously described VU series of M4 PAMs were synthesized and characterized, probing the ring substitution patterns of incorporating fluorine and methoxy substituents and combinations thereof. The target compounds were pharmacologically profiled in an ERK1/2 phosphorylation assay using intact FlpIn-CHO cells stably transfected with the human muscarinic M4 receptor to elucidate their binding affinity to the allosteric site (pKB), intrinsic efficacies (τB), and cooperativities (αβ) of the orthosteric ligand ACh. The novel monocyclic series 9a− c,e,g,h,k,l,p exhibited no allosteric modulatory effects. It is noteworthy that all synthesized target ligands that demonstrated positive allosteric modulation of the M4 mAChR also exhibited intrinsic agonism (τB) at the allosteric site in their own right, a pharmacological feature also observed for the M4 PAM (2). The compounds that exhibited the most promising affinity, cooperativity, and intrinsic efficacy profiles compared to the Vanderbilt University reference compounds 1a and 1i included the 3-fluoro (1h), the 3-fluoro-4-methoxy (1n), and the 2,3-difluoro-4-methoxy (1p) analogues. A more detailed synthetic and pharmacological investigation of this unique scaffold is warranted to further probe the structural requirements of the allosteric site of the muscarinic M4 receptor. This information will enhance the robustness and reliability of such SAR studies and significantly contribute to future M4 receptorbased allosteric drug discovery programs

General Procedure for Synthesis of Target Thienopyridines 1a−c,e,g,h,k,l,p from Monocyclic Precursors. The monocyclic compounds 9a−c,e,g,h,k,l,p (1 equiv) was dissolved in dry DMF (3−4 mL), and 1 M aqueous KOH solution (1 equiv) was added while stirring. The reaction mixture was stirred at room temperature, and the reaction was complete after 15−20 min. The product was partitioned between DCM (30 mL) and H2O (30 mL). Saturated brine solution was added to dissipate the emulsion. The aqueous layer was extracted with DCM (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The products were dissolved in EtOAc and converted to the hydrochloride salt with ethereal hydrogen chloride. The solvent was removed by rotary evaporation, and the products were suspended in Et2O and filtered, all of which yielded bright-yellow solids in quantitative yields. 3-Amino-N-(3-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide Hydrochloride (1b). Bright-yellow solid (39 mg, 46%); mp 257−259 °C dec. 1H NMR (DMSO-d6) δ 2.60 (s, 3H), 2.80 (s, 3H), 3.73 (s, 3H), 4.39 (d, J = 6 Hz, 2H), 6.06 (br s, 2H), 6.73−6.83 (m, 1H), 6.87−6.90 (m, 2H), 7.18 (s, 1H), 7.24 (app t, J = 7.5 Hz, 1H), 8.42 (app t, J = 6 Hz, 1H) ppm. 13C NMR (DMSO-d6) δ 19.9 (CH3), 22.4 (CH3), 42.1 (CH2), 54.9 (CH3), 97.9 (C), 111.8 (CH), 113.0 (CH), 119.3 (CH), 122.2 (CH), 124.2 (C), 129.2 (CH), 141.4 (C), 147.0 (C), 147.4 (C), 155.8 (C), 157.0 (C), 159.1 (C), 164.8 (C). HRMS (ESI+): exact mass calcd for C18H20N3O2S 342.1271, found 342.1268. HPLC (λ = 254 nm) tR (gradient) = 7.5 min, 98.8% purity. General Procedure for the Synthesis of the Target Thienopyridines 1d,f,i,j,m−o,q from Pyridinethione (5). 5 (1 equiv) and a substituted N-benzyl chloroacetamides 8d,f,i,j,m−o,q (1.5 equiv) were dissolved in DMF (5 mL) to which solid KOH (5 equiv) dissolved in H2O (1 mL) was added. The mixture was stirred at room temperature for 24 h and then heated to 50 °C for 15 h. The reaction mixture was partitioned between EtOAc (30 mL) and H2O (30 mL). The organic layer was removed, and the aqueous layer was further extracted with EtOAc (2 × 20 mL). The organic layers were combined, washed with brine (20 mL), dried over anhydrous Na2SO4, then filtered and concentrated in vacuo. The products were dissolved 8199

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in EtOAc and converted to the hydrochloride salt with ethereal hydrogen chloride. The solvent was removed by rotary evaporation, and the products were suspended in Et2O and filtered, all of which yielded bright-yellow solids in quantitative yields. 3-Amino-N-(3-fluoro-4-methoxybenzyl)-4,6-dimethylthi-eno[2,3b]pyridine-2-carboxamide Hydrochloride (1n). Bright-yellow solid (176 mg, 50%); mp 235−236 °C dec. 1H NMR (DMSO-d6) δ 2.55 (s, 3H), 2.75 (s, 3H), 3.81 (s, 3H), 4.33 (d, J = 6 Hz, 2H), 4.51 (br s, 3H), 7.08−7.14 (m, 4H), 8.33 (t, J = 6 Hz, 1H). 13C NMR (DMSOd6) δ 19.9 (CH3), 22.9 (CH3), 41.4 (CH2), 56.0 (CH3), 97.6 (C), 113.7 (d, 4JCF = 2 Hz, CH), 114.9 (d, 2JCF = 18 Hz, CH), 122.1 (CH), 123.5 (d, 3JCF = 3.5 Hz, CH), 123.9 (C), 132.9 (d, 3JCF = 5.5 Hz, C), 145.8 (d, 2JCF = 10.5 Hz, C), 146.2 (C), 147.6 (C), 151.2 (d, 1JCF = 243.5 Hz, C), 156.7 (C), 157.6 (C), 165.0 (C). HRMS (ESI+): exact mass calcd for C18H19FN3O2S 360.1177, found 360.1181. HPLC (λ = 254 nm) tR (gradient) = 11.9 min, 99.5% purity.

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(5) Melancon, B. J.; Hopkins, C. R.; Wood, M. R.; Emmitte, K. A.; Niswender, C. M.; Christopoulos, A.; Conn, P. J.; Lindsley, C. W. Allosteric Modulation of Seven Transmembrane Spanning Receptors: Theory, Practice, and Opportunities for Central Nervous System Drug Discovery. J. Med. Chem. 2011, 55, 1445−1464. (6) Holzgrabe, U.; Mohr, K. Allosteric modulators of ligand binding to muscarinic acetylcholine receptors. Drug Discovery Today 1998, 3, 214−222. (7) Chan, W. Y.; McKinzie, D. L.; Bose, S.; Mitchell, S. N.; Witkin, J. M.; Thompson, R. C.; Christopoulos, A.; Lazareno, S.; Birdsall, N. J. M.; Bymaster, F. P.; Felder, C. C. Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 10978−10983. (8) Leach, K.; Loiacono, R. E.; Felder, C. C.; McKinzie, D. L.; Mogg, A.; Shaw, D. B.; Sexton, P. M.; Christopoulos, A. Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties. Neuropsychopharmacology 2009, 35, 855−869. (9) Shirey, J. K.; Xiang, Z.; Orton, D.; Brady, A. E.; Johnson, K. A.; Williams, R.; Ayala, J. E.; Rodriguez, A. L.; Wess, J.; Weaver, D.; Niswender, C. M.; Conn, P. J. An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission. Nature Chem. Biol. 2008, 4, 42−50. (10) Brady, A. E.; Jones, C. K.; Bridges, T. M.; Kennedy, J. P.; Thompson, A. D.; Heiman, J. U.; Breininger, M. L.; Gentry, P. R.; Yin, H.; Jadhav, S. B.; Shirey, J. K.; Conn, P. J.; Lindsley, C. W. Centrally Active Allosteric Potentiators of the M4Muscarinic Acetylcholine Receptor Reverse Amphetamine-Induced Hyperlocomotor Activity in Rats. J. Pharmacol. Exp. Ther. 2008, 327, 941−953. (11) Leach, K.; Sexton, P. M.; Christopoulos, A. Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology. Trends Pharmacol. Sci. 2007, 28, 382−389. (12) Canals, M.; Lane, J. R.; Wen, A.; Scammells, P. J.; Sexton, P. M.; Christopoulos, A. A Monod−Wyman−Changeux Mechanism Can Explain G Protein-Coupled Receptor (GPCR) Allosteric Modulation. J. Biol. Chem. 2012, 287, 650−659. (13) Aurelio, L.; Valant, C.; Flynn, B. L.; Sexton, P. M.; Christopoulos, A.; Scammells, P. J. Allosteric Modulators of the Adenosine A1 Receptor: Synthesis and Pharmacological Evaluation of 4-Substituted 2-Amino-3-benzoylthiophenes. J. Med. Chem. 2009, 52, 4543−4547. (14) 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. (15) Kennedy, J. P.; Bridges, T. M.; Gentry, P. R.; Brogan, J. T.; Kane, A. S.; Jones, C. K.; Brady, A. E.; Shirey, J. K.; Conn, P. J.; Lindsley, C. W. Synthesis and Structure−Activity Relationships of Allosteric Potentiators of the M4 Muscarinic Acetylcholine Receptor. ChemMedChem 2009, 4, 1600−1607. (16) Eyduran, F.; Ozyurek, C.; Dilek, N.; Ocak Iskeleli, N.; Sendil, K. 4,6-Dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile. Acta Crystallogr., Sect. E: Struct. Rep. Online 2007, 63, o2415−o2417. (17) Ferraccioli, R.; Forni, A. Selective Synthesis of Isoquinolin-3-one Derivatives Combining Pd-Catalysed Aromatic Alkylation/Vinylation with Addition Reactions: The Beneficial Effect of Water. Eur. J. Org. Chem. 2009, 2009, 3161−3166.

ASSOCIATED CONTENT

* Supporting Information S

Full details of synthesis, characterization, and pharmacological evaluations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For A.C.: E-mail, [email protected]. *For B.C.: phone, +61 3 9903 9556; E-mail, ben.capuano@ monash.edu . Present Address §

I.T.C.: E-mail: [email protected].

Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Many thanks to Dr. Jason Dang (Monash University) for helping obtain NMR spectral data, HRMS, and HPLC chromatograms. This work was supported by Program Grant 519461 of the National Health and Medical Research Council (NHMRC) of Australia. A.C. and P.M.S. are Principal Research Fellows of the NHMRC. T.H. is a recipient of the Australian Postgraduate Award (APA).

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ABBREVIATIONS USED mAChR, muscarinic acetylcholine receptor; GPCR, G proteincoupled receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; CHO, Chinese hamster ovary

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REFERENCES

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