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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity

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Author Information

Received: ; Last Update: March 14, 2013.

This probe (ML173, CID 45142486) can be used both in vitro and in vivo to study the role of selective M4 receptor activation. This probe possesses excellent selectivity versus M1, M2, M3 and M5, as well as a large panel of G-protein-coupled receptors (GPCRs), ion channels, and transporters. Moreover, this probe displays an order of magnitude greater potency at the human M4 receptor versus the rat M4 receptor (human M4 EC50 = 95 nM) surpassing our initial M4 positive allosteric modulator (PAM) probe, and now affects a large fold shift at both the human (60x) and rat (44x) receptors. While close analogs are active in rodent preclinical antipsychotic behavioral models, the greatly improved human potency may warrant testing in antipsychotic behavioral models with non-human primates. ML173 possess improved metabolic stability over the earlier M4 PAM probe (CID 864492) making it a potentially better in vivo tool.

Assigned Assay Grant #: MH077607-1

Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver

Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley

Assay Submitter & Institution: Colleen M. Niswender, Vanderbilt University

PubChem Summary Bioassay Identifier (AID): 2616

Probe Structure & Characteristics

3-amino-N-(2,3-difluorobenzyl)-4-methyl-6-(pyridin-4-ylmethoxy)thieno[2,3-b]pyridine-2-carboxamide, MW = 440.5, logP = 3.1, tPSA = 90.1 Å2

ML173.

ML173

CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]§
CID 45142486/ML173rM4
hM4
2440
95
[SID 92392533, AID 2616]
M1, M2, M3, M5> 30 μM [SID 92392533, AID 1932, AID 1930, AID 1929, AID 1928]> 100ACh Fold-Shift (44-fold) [SID 92392533, AID 449769]

Recommendations for Scientific Use of the Probe

This probe (ML173, CID 45142486) can be used both in vitro and in vivo to study the role of selective M4 receptor activation. This probe possesses excellent selectivity versus M1, M2, M3 and M5, as well as a large panel of GPCRs, ion channels and transporters. Moreover, this probe displays an order of magnitude greater potency at the human M4 receptor versus the rat M4 receptor (human M4 EC50 = 95 nM) surpassing our initial M4 PAM probe, and now affects a large fold shift at both the human (60x) and rat (44x) receptors. While close analogs are active in rodent preclinical antipsychotic behavioral models, the greatly improved human potency may warrant testing in antipsychotic behavioral models with non-human primates. ML173 possess improved metabolic stability over the earlier M4 PAM probe (CID 864492) making it a potentially better in vivo tool.

1. Introduction

Specific AIM: To identify small molecule positive allosteric modulators and/or allosteric agonists of the M4 muscarinic acetylcholine receptor that are cell permeable, possess low- to submicromolar potency and show greater than 10-fold selectivity over the other mAChRs (M1, M2, M3 and M5) employing a functional HTS approach.

Significance: To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1–M5) and play important roles in mediating the actions of acetylcholine (ACh) in the peripheral and central nervous systems.1 Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer’s disease, and schizophrenia.24 In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M2 and M3 mAChRs.5,6 Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. Clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent.79 In follow-up studies in rats, xanomeline displayed an antipsychotic-like profile comparable to clozapine.10 However, a long standing question concerns whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia.5,6,11 This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neuro-transmission and that loss of M4 function results in a state of dopamine hyperfunction.12 These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression,13 suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients. However, multiple studies suggest that M1 may also play an important role in the antipsychotic effects of mAChR agonists and that the relative contributions of M1 and M4 to the antipsychotic efficacy of xanomeline or antipsychotic-like effects of this compound in animal models are not known. Unfortunately, highly selective centrally penetrant activators of either M1 or M4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. Only recently did we develop a highly selective M1 allosteric agonist probe (CID 25010775) and two highly selective M1 PAM probes (CID 44251556 and CID 44475955) along with our initial M4 PAM probe (CID 864492) to study the role of selective M1 and/or M4 activation in vitro and in vivo. Additionally, selective M5 activation can now be studied using our most recent, highly selective M5 PAM probe (CID 42633508).

Rationale: In recent years, major advances have been made in the discovery of highly selective agonists of other GPCRs that act at an allosteric site rather than the orthosteric site,14 as well as positive allosteric modulators (PAMs).15,16 By screening for compounds that act at an allosteric site on the receptor, it is anticipated that compounds that can selectively activate the M4 receptor versus the other mAChR subtypes may be identified. In conjunction with our MLPCN M1 allosteric agonist probe (CID 25010775), development of a potent and selective M4 allosteric agonist or PAM will enable the biomedical community to dissect the pharmacology of xanomeline and determine the pharmacology and therapeutic potential of selective M1 and M4 activation.

2. Materials and Methods

2.1. Assays

PubChem Primary Assay Description: Chinese hamster ovary (CHO K1) cells stably expressing rat (r)M1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to their recommendations. CHO cells stably expressing human (h) M2, hM3, and hM5 were generously provided by A. Levey (Emory University, Atlanta, GA); rM4 cDNA provided by T. I. Bonner (National Institutes of Health, Bethesda, MD) was used to stably transfect CHO-K1 cells purchased from the ATCC using Lipofectamine 2000. To make stable hM2 and rM4 cell lines for use in calcium mobilization assays, cell lines were cotransfected with a chimeric G protein (Gqi5) using Lipofectamine 2000. rM2, hM3, and hM5 cells were grown in Ham’s F-12 medium containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax I, 20 mM HEPES, and 50 μg/mL G418 sulfate. hM2-Gqi5 cells were grown in the same medium supplemented with 500 μg/mL hygromycin B. Stable rM4 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax I, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 20 mM HEPES, and 400 μg/mL G418 sulfate; rM4-Gqi5 cells were grown in the same medium supplemented with 500 μg/mL hygromycin B. CHO cells stably expressing rM1, hM3, or hM5 were plated at a seeding density of 50,000 cells/100 μL/well. CHO cells stably coexpressing hM2/Gqi5 and rM4/Gqi5 were plated at a seeding density of 60,000 cells/100 μL/well. For calcium mobilization, cells were incubated in antibiotic-free medium overnight at 37 °C/5% CO2 and assayed the next day.

Calcium Mobilization Assay: Cells were loaded with calcium indicator dye [2 μM Fluo-4 acetoxymethyl ester (50 μL/well) prepared as a stock in DMSO and mixed in a 1:1 ratio with 10% Pluronic acid F-127 in assay buffer (1xHanks’ balanced salt solution supplemented with 20 mM HEPES and 2.5 mM probenecid, pH 7.4)] for 45 min at 37 °C. Dye was removed and replaced with the appropriate volume of assay buffer. All compounds were serially diluted in assay buffer for a final 2x stock in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine (EC20 concentration or full dose-response curve) was prepared at a 10x stock solution in assay buffer before addition to assay plates. Calcium mobilization was measured at 25 °C using a FLEXstation II (Molecular Devices, Sunnyvale, CA). Cells were preincubated with test compound (or vehicle) for 1.5 min before the addition of the agonist, acetylcholine. Cells were then stimulated for 50 s with a submaximal concentration (EC20) or a full dose-response curve of acetylcholine. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine.

List of PubChem bioassay identifiers generated for this screening project

AID 625, AID 643, AID 1921, AID 1923, AID 1928, AID 1929, AID 1930, AID 1932, AID 1938, AID 1939, AID 2616, AID 449765, AID 449767, AID 449769, AID 449770

2.2. Probe Chemical Characterization

Synthetic procedure (large scale) and spectral data for ML173 (CID 45142486)

Image ml173fu2

Probe compound ML173 (CID 45142486) was prepared according to the above scheme and provided the following characterization data: LCMS (>98%) m/z = 441 [M+H+] (2.03 min retention, 214 nm). 1H NMR (400MHz, DMSO-d6): δ = 8.92 (d, J = 6.8 Hz, 2H), 8.30 (t, J = 5.6 Hz, 1H), 8.09 (d, J = 6.4 Hz, 2H), 7.32 (m, 1H), 7.16 (m, 2H), 6.85 (s, 1H), 5.75 (s, 2H), 4.46 (d, J = 5.6 Hz, 2H), 2.74 (s, 3H). 13C (100MHz, DMSO-d6): δ = 165.2, 162.1, 157.8, 157.0, 150.8, 148.8, 148.3, 148.1, 146.4, 146.2, 141.4, 129.3 (d, J = 11.0 Hz), 124.5, 124.3, 120.9, 115.6 (d, J = 17.0 Hz), 109.5, 65.3, 35.7, 19.9. HRMS calculated for C22H19N4O2F2S [M + H] 441.1197, found 441.1196.

Solubility. Solubility in PBS (at pH = 7.4) was determined to be less than 0.03 μM. However, given the engineered presence of a pyridine nitrogen it was not surprising to find improved solubility under alternate conditions; as its HCl salt, ML173 shows good solubility in these acceptable vehicles (>10 mg/mL in 20% β-cyclodextrin, PEG400/H2O or pH 3 saline) and >100 μM in DMSO.

Stability. Stability (at room temperature = 23 °C) for ML173 in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) is shown in the table below. After 48 hours, the percent of parent compound remaining was 117%, indicating that this probe molecule was stable to the assay conditions, but that assay variability over the course of the experiment ranged from a low of 85% (at 2 hours) to a high of 117% (at 48 hours).

Percent Remaining (%)
Compound0 Min15 Min30 Min1 Hour2 Hour24 Hour48 Hour
ML173100102101958590117

Reactivity. As assessed through a glutathione (GSH) trapping experiment in phosphate buffered saline (with a substrate concentration of typically 5–50 μM and a GSH concentration of 5 mM, at t = 60 minutes) ML173 was found to not form any detectable GSH adducts.22

Compounds added to the SMR collection (MLS#s): 002919691 (ML173, CID 45142486, 500 mg), 002919689, 002919690, 002919692, 002919693, 002919694.

2.3. Probe Preparation

3-amino-N-(2,3-difluorobenzyl)-4-methyl-6-oxo-6,7-dihydrothieno[2,3-b]pyridine-2-carboxamide (3): To a flask containing 2-mercapto-4-methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile 1 (6.0 mmol) was added DMF (30 mL) followed by TEA (6.6 mmol), and then the mixture was cooled to 0 °C with an ice bath. A solution of 2-chloro-N-(2,3-difluorobenzyl)acetamide 2 (6.0 mmol) in DMF (30 mL) was slowly added to the reaction via addition funnel over 30 minutes and then allowed to return to ambient temperature overnight. The reaction was concentrated then partitioned between DCM (100 mL) and H2O (100 mL), separated, and the aqueous layer washed twice with DCM (100 mL). The organic layer was dried with solid MgSO4, filtered, and concentrated to a tan solid which was washed with EtOAc (3× 30 mL) to afford pure 3-amino-N-(2,3-difluorobenzyl)-4-methyl-6-oxo-6,7-dihydrothieno[2,3-b]pyridine-2-carboxamide 3 as a light brown solid (50%).

3-amino-N-(2,3-difluorobenzyl)-4-methyl-6-(pyridin-4-ylmethoxy)thieno[2,3-b]pyridine-2-carboxamide dihydrochloride (ML173, CID 45142486): To a 20 mL microwave vial containing 3-amino-N-(2,3-difluorobenzyl)-4-methyl-6-oxo-6,7-dihydrothieno[2,3-b]-pyridine-2-carboxamide 3 (2.3 mmol), potassium iodide (2.3 mmol), and cesium carbonate (4.6 mmol) was added DMF (10 mL). To this was added 4-(chloromethyl)pyridine hydrochloride 4 (2.3 mmol) and the reaction was stirred until bubbling ceased. The vessel was capped and the reaction mixture was heated to 160 °C for 30 min. After cooling to ambient temperature, the reaction was concentrated, and then partitioned between DCM (100 mL) and H2O (100 mL), separated, and the aqueous layer washed twice with DCM (100 mL). The organic layer was dried with solid MgSO4, filtered, and concentrated to a dark brown solid. This material was crystallized from a mixture of MeOH:DCM:Hexanes with 4N HCl:Dioxanes (5 mL) to afford solid ML173 (CID 45142486) as a light brown solid (60%).

3. Results

Center Summary of Screen: This screen was performed in the pilot phase, the MLSCN, when the MLSMR compound collection at Vanderbilt only contained 12,364 compounds. From the primary M4 screen of 12,364 compounds, 69 putative M4 activators were identified with an average Z′ score of 0.67±0.093. The confirmation screen (singles at 10 μM) produced 25 active compounds. After a selectivity screen versus M1, only 4 compounds (Figure 1) appeared to be M4 selective. Chemistry was pursued around these four screening hits 5–8, and 48 analogs were synthesized (25 analogs of 5–7 and 23 analogs of 8); unfortunately, none of these confirmed as selective M4 agonist or PAMs.

Figure 1. M4 HTS hits.

Figure 1

M4 HTS hits.

The project was shelved until a preliminary report from Eli Lilly (personal communication leading to ML108) showed that a new compound, LY2033298, was a robust, but weak, M4 PAM that is highly selective for human M4 (Figure 2). However, to study selective M4 activation in a preclinical, academic setting, we, and the biomedical community at large, require a tool which is potent and selective for rat M4. Therefore, we initiated a cheminformatics and database mining effort based on the LY2033298 scaffold, to attempt to deliver a useful M4 PAM probe for the MLPCN Network that would possess appropriate potency (rM4 EC50 < 500 nM), and be free from patent restrictions.16 A substructure search for commercially available compounds in the ChemBridge Corporation’s chemical database containing a core similar to LY2033298 delivered a novel lead (CID 714286, not shown) and related analogs, which were free of intellectual property barriers. CID 714286 was a potent PAM of rat M4 (EC50 = 400 nM) which induced a 47-fold leftward shift in the ACh Concentration Response Curve (CRC). Moreover, CID 714286 was highly selective for rat M4 (EC50 > 50 μM for rM1, hM2, hM3 and hM5), binds at an allosteric site on the M4 receptor, increases affinity for ACh and increases coupling to G proteins.17 However, CID 714286 possessed a high log P (4.6) and was virtually insoluble in anything except DMSO.17 Subsequent optimization of this lead (CID 714286) to our first highly selective rM4 Probe Molecule (CID 864492, rM4 EC50 = 380 nM, Figure 2) was the subject of a previous probe report and has been published elsewhere.18 Although an excellent probe molecule, compound CID 864492 did display some less than optimal characteristics that we sought to address. Paramount among these was the poor metabolic stability of CID 864492, such that after 90 minutes in the presence of human or rat liver microsomes (in vitro) less than 10% of the parent remained. This instability likely resulted in the suboptimal rodent PK for CID 864492, which were adequate but not ideal for in vivo studies.18

Figure 2. LY2033298 and cheminformatics and database mining approach which lead to the first M4 probe compound (CID 864492).

Figure 2

LY2033298 and cheminformatics and database mining approach which lead to the first M4 probe compound (CID 864492).

Probe Chemical Lead Optimization Strategy: Numerous modifications to the thieno [2,3-b]pyridine scaffold, including substitution of the primary amine, deletion or extension at the 4-methyl position and variation of the pyridine to a pyrazine or benzene ring, were all previously found to compromise activity regardless of amide side chain substituent.18 Metabolite identification experiments indicated that hydroxylation of the 6-methyl group on the pyridine ring was the major oxidative metabolite. Thus, the replacement of this 6-methyl group with ether analogs at this position, to remove the possibility of benzylic oxidation, was explored following the general route of Scheme 1. Having tentatively designated the p-methoxy-benamide moiety of CID 864492 as a favored substituent based on its in vitro functional activity at rat M4, we first held this side chain constant (10a, R1 = p-methoxybenzylamino) while exploring alternate ethers (R2, 12a). Accordingly, the pyridone core 919 was cyclized with α-chloro-p-methoxybenzamide (10a) to give the key intermediate 11a. Alkylation of 11a with various alkyl chlorides, followed by mass-directed preparative HPLC purification, gave the analogs 12a appearing in Table 1.

Scheme 1. Synthesis of Analogues 8a and 9.

Scheme 1

Synthesis of Analogues 8a and 9.

Table 1. Structure and SAR for Analogues 12a.

Table 1

Structure and SAR for Analogues 12a.

About half of these analogs possessed EC50 values over 10 μM with potentiation effects emerging only at the 10 and 30 μM concentrations from the full CRCs, however encouraging values were seen for some promising, novel structures. Particularly, picolyl analogues CID 44626302, 44626303, and 44626304 each exhibited an EC50 value of ~2 μM. A full CRC for CID 44626302 in the presence of a fixed ACh EC20 is presented in Figure 3a. CID 44626302 elicited a robust potentiation of M4 activation, elevating the submaximal ACh response to over 130% of the maximum response induced by a high concentration of ACh alone (Figure 3b). Looking ahead to in vivo studies, the structure of CID 44626302 was particularly attractive as the presence of a basic amine would allow for an HCl salt to confer greater aqueous solubility for vehicle formulation. Based on these potency data, the six compounds that exhibited EC50 values below 10 μM were examined for their ability to shift a full ACh CRC to the left when applied at a fixed 30 μM concentration in a similar functional Ca2+ assay with rM4/Gqi5-expressing cells (i.e. fold-shift assay). In the case of other allosteric potentiators of GPCRs, compound potency often fails to correlate tightly with fold-shift magnitude. For example, a potentiator with high potency but low efficacy can exhibit next to no fold-shift effect, and conversely one with low potency but high efficacy can induce a substantial fold-shift. Hence, evaluation of fold-shift for novel potentiators having upper single-digit micromolar potencies can sometimes uncover helpful SAR that would have otherwise been missed.

Figure 3. Calcium mobilization assays in rat M4/Gqi5-expressing CHO cells.

Figure 3

Calcium mobilization assays in rat M4/Gqi5-expressing CHO cells. a) Concentration response curve for CID 44626302 in the presence of a fixed submaximal (~EC20) concentration of ACh (PAM EC50 = 2.0 μM). b) Concentration response curve for ACh in (more...)

As shown in Table 1, purely alkyl ethers (CID 45142474, 45142475 and 45142477) failed to produce measurable potentiation. The same lack of effect was seen with the tertiary amine analogues CID 45142469, 45142470 and 45142473. However, the ether linked morpholino CID 45142471 and pyrrolidine CID 45142472 analogues demonstrated strong leftward fold-shifts (37× and 25×, respectively). Interestingly, movement of the nitrogen from the 2-position or 3-position of the picolyl ethers CID 44626304 and CID 44626303 toward the 4-position of CID 44626302 progressively increased the fold-shift from 5× to 9× and ultimately 50× (Figure 3b showing CID 44626302 only), respectively. However, despite retention of robust potentiation properties in terms of fold-shift for analogues CID 45142471, CID 45142472 and particularly CID 44626302, the potency of these analogues was moderately diminished relative to the parent compound CID 864492 (EC50 = 380 nM). Furthermore, the SAR for this library underlines the aforementioned importance of considering both fold-shift and potency when evaluating allosteric potentiators. Although each of the three picolyl ether analogues had ~2 μM EC50 values, their leftward-shift effects on the ACh CRC revealed dramatic differences in potentiation efficacy. For the next library iteration, we postulated that with the picolyl or ethyl morpholine ether moieties on the left-hand side of the molecule, the p-methoxybenzyl of the right-hand side might no longer be favored for M4 potency. Therefore, we opted to rescan the amide with select R3 groups (Scheme 1, compound 13), while sampling each of the three picolyl ether modifications, the morpholino ether, and the dimethylpropylamine ether. The morpholino and 4-picolyl were clear choices based on their degree of fold-shift, but the 2-picolyl and 3-picolyl were also included to be comprehensive. The dimethylpropylamine ether was chosen to provide for the possibility that a different amide side chain may rescue the activity of CID 45142469 (i.e. a matrix-like approach to broaden SAR).

This second generation library began with the cyclization between pyridone 9 and ethyl chloroacetate (10b, Scheme 1) to produce thienopyridone ethyl ester 11b. To obtain the alkyl ethers 12b, compound 11b was alkylated with the five selected side chains from our previously mentioned library (R2). These scaffolds were saponified and immediately coupled with amines to produce analogs 13 (Table 2).

Table 2. Structure and SAR for Analogues 13.

Table 2

Structure and SAR for Analogues 13.

All these compounds possessed an EC50 value below 10 μM except for derivative CID 45142485, the difluorobenzyl-substituted 2-picolyl analogue. Similar to earlier libraries, the fold-shift magnitude did not track closely with potency, as shown, for example, with CID 45142483. This tert-butyl-substituted morpholine analogue had near 9 μM potency but caused a robust 62-fold shift of the ACh CRC. Furthermore, the two dimethylpropyl analogues CID 45142477 and 45142478 displayed approximately 3 μM potency yet produced only a moderate ACh fold-shift. Interestingly, this dimethylpropylamine moiety at R2 conferred poor potency (>10 μM) in its parent compound CID 45142469 that possessed the p-methoxybenzyl amide, but this second library discovered side chains at R3 that rescued activity for this left-hand side modification. In general, difluorinated benzylic substitutions at R3 were favored, providing analogues with EC50 values in the 2–5 μM range at rat M4 and broad fold-shift values. The 4-picolyl moieties of R2 with the 2,3-difluoro and 2,5-difluoro substitutions at R3 of compounds CID 45142486 and CID 45142487 proved most desired when seeking a balance of both potency and potentiation efficacy, consistent with previous SAR. However, the morpholines at R2 (CIDs 45142479, 45142480, 45142489, 45142482, and 45142483) with bare alkyl and mono-oxygenated side chains at R3 possessed strong fold-shift effects despite moderately weaker potency compared to CID 45142486 and CID 45142487. Figure 4 presents the CRC for elevation of an ACh ~EC20 and fold-shift on a full ACh CRC for analogue CID 45142486. Interestingly, this 2,3-difluorobenzyl substituted analogue did not elevate the maximal response of ACh at the top of the CRC (Figure 4b), which contrasts with 4-methoxybenzyl analogue CID 44626302 (Figure 3b). Despite generation of a multidimensional library of analogues varying both sides of the lead scaffold, the approximately 400 nm potency (rat M4) of the first generation compound CID 864492 could not be maintained despite retention of strong potentiation activity in terms of ACh CRC fold-shift (e.g. >50x). Indeed, compounds CID 44626302, 45142480 and 45142487 each caused substantial leftward shift of ACh CRCs when applied at 30 μM, but were approximately an order of magnitude less potent than the first generation compounds at the rat M4 receptor. These SAR suggest the presence of a possible ~2 μM potency floor for this chemotype with 6-position ether modifications, as variation of the amide side chain failed to provide congers with EC50 values below this level.

Figure 4. Calcium mobilization assays in rat M4/Gqi5-expressing CHO cells.

Figure 4

Calcium mobilization assays in rat M4/Gqi5-expressing CHO cells. a) Concentration response curve for CID 45142486 in the presence of a fixed submaximal (~EC20) concentration of ACh (PAM EC50 = 2.4 μM). b) Concentration response curve for ACh in (more...)

In parallel, we evaluated the microsomal stability of CIDs 44626302, 45142483, 45142486 and 45142487 in both rat and human microsomes. Replacement of the metabolically labile 6-methyl group with the ether linkage did indeed improve metabolic stability for all four analogues CIDs 44626302, 45142483, 45142486 (ML173) and 45142487 (>90% parent remaining after 90 min) as compared to CID 864492 (<10% parent remaining after 90 min). Moreover, incorporation of the basic amine moieties in CIDs 44626302, 45142483, 45142486 and 45142487 also improved solubility providing either homogeneous solutions or uniform microsuspensions, as the HCl salts at 10 mg/mL, across a panel of pharmaceutically acceptable vehicles (β-cyclodextrin, PEG400/H2O, etc.) relative to CID 864492, which was only soluble in 10% Tween80. In fact, CID 45142486 (ML173) afforded a homogeneous solution at 15 mg/mL in pH 3 saline.

Despite micromolar potency at rat M4, we evaluated CIDs 44626302, 45142486 (ML173) and 45142487 in our standard reversal of amphetamine-induced hyperlocomotion in vivo model, since a long-standing question in the PAM field has centered on whether EC50 or fold-shift is more relevant to provide in vivo efficacy.16 As reported previously, CID 864492 (rM4 EC50 = 390 nM, fold-shift of 70x) were efficacious in this model. Interestingly, both CID 44626302 and CID 45142487 produced modest decreases in amphetamine-induced hyper locomotion while CID 45142486 (ML173) had no effect over the time course tested (Figure 5). These findings suggest that the diminished potency of these new compounds may have translated to reduced in vivo efficacy relative to CID 864492.

Figure 5. Reversal of amphetamine-induced hyper locomotion rat behavioral study.

Figure 5

Reversal of amphetamine-induced hyper locomotion rat behavioral study. Pretreatment with vehicle (10% Tween 80 i.p., n = 9, dark circle) or a 56.6 mg/kg dose of either CID 44626302 (dark triangle), CID 45142486 (light triangle), or CID 45142487 (dark (more...)

Primarily, our efforts reported here were aimed at exploring SAR at rat M4 and optimizing this series for beneficial DMPK and vehicle formulation properties for in vivo rodent behavioral studies. While stability and physiochemical properties were improved, potency at rat M4 was diminished to a point where in vivo efficacy was reduced and, in the case of CID 45142486 (ML173), in vivo efficacy was lost. However, rat and human mAChRs do diverge and species differences have been noted for other mAChR PAMs. Therefore, we opted to evaluate representative compounds CIDs 44626302, 45142483, 45142486 (ML173) and 45142487 in analogous functional cell-based Ca2+ assays using cells expressing the human M4 receptor (and promiscuous Gq15 for Ca2+ mobilization readout). To this end, these four compounds were submitted to Millipore Corp. (St. Charles, USA) and assayed by their GPCR Profiler Service, which provided potency and ACh CRC fold-shift values with the human M4 receptor. Remarkably, each compound possessed EC50 values approximately in the 100–200 nM range at human M4 (Figure 6a), more than an order of magnitude greater potency than at the rat M4 receptor. Each compound also elicited large leftward shifts of the control ACh CRC in human M4 cells (Figure 6b) similar to their respective fold-shifts at rat M4. In contrast, the first M4 PAM Probe CID 864492 and about 20 other first generation analogues, displayed near equivalent EC50 values at rat and human M4, suggesting the basic residues in these newer analogues contact divergent residues in human M4. While receptor expression levels in the two cell lines is not known, ACh EC50 values in the two cell lines are equivalent (rat M4 ACh, EC50 = 154 nM; human M4 ACh, EC50 = 100 nM), and all first generation analogues lacking basic residues were also equipotent. These human M4 data exemplify the differences that may exist between species in terms of compound potency, efficacy, and other pharmacological parameters, despite relatively high structural similarity between rat and human mAChRs.

Figure 6. Calcium mobilization assays in human M4/Gq15-expressing CHO cells.

Figure 6

Calcium mobilization assays in human M4/Gq15-expressing CHO cells. a) Concentration response curve for CID 44626302 (square, EC50 = 100 nM), CID 45142483 (up triangle, EC50 = 192 nM), CID 45142486 (ML173) (down triangle, EC50 = 95 nM), and CID 45142487 (more...)

In addition, CIDs 44626302, 45142483, 45142486 and 45142487 and related second generation analogues remained highly M4 selective at both human and rat mAChR cell lines (Figure 7). Whereas a 30 μM concentration of CIDs 44626302, 45142483, 45142486 (ML173) and 45142487 afforded large leftward shifts (44–63x) of the ACh CRCs of M4, these same concentrations of compound had no effect on the ACh CRCs of M1, M2, M3 or M5 (data shown is for rat mAChRs).

Figure 7. Calcium mobilization assays in a) M1, b) M2/Gqi5, c) M3, and M5 expressing CHO cells.

Figure 7

Calcium mobilization assays in a) M1, b) M2/Gqi5, c) M3, and M5 expressing CHO cells. Concentration response curves for ACh in the absence (square) or presence of 30 μM CID 44626302, CID 45142483, CID 45142486, or CID 45142487, which display no (more...)

The calculated physical properties appearing in Table 3 for the initial M4 probe molecule (CID 864492) and this current probe (CID 45142486, ML173) were generated using TRIPOS software. Also included in Table 3 are the averages from the MDDR database of compounds both entering Phase I and launched drugs. These numbers indicate that both probes are within the average values for Phase I compounds, except when predicting the number of hydrogen bond donors and calculated solubilities. However, for these two calculated values there are extenuating circumstances that warrant further comment. In the case of hydrogen bond donors, the amide carbonyl may be internally masking one of the hydrogen bond donors on the primary amine through an intramolecular hydrogen bond. Fewer hydrogen bond donors tends to increase the probability of CNS exposure and it has already been shown that the initial probe (CID 864492) and close analogs of the current probe were efficacious in reversing amphetamine-induced hyperlocomotor activity in rats (Figure 5), implying some level of CNS exposure. Next, with respect to LogS, although the calculations predict CID 864492 to be more soluble than the current probe CID 45142486, we know from experience that this is not the case, when comparing hydrochloride salts. The pyridine in CID 45142486 (ML173) was deliberately, and moreover successfully, introduced to improve solubility and points to the importance of not solely relying on neutral forms to judge/predict solubility.

Table 3. Calculated Property Comparison with MDDR Compounds.

Table 3

Calculated Property Comparison with MDDR Compounds.

To more fully characterize this novel M4 PAM probe molecule, CID 45142486 was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to not significantly interact with 64 out of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM).20 CID 45142486 (ML173) did have activity at the following 4 targets (human targets at 10 μM): Adenosine A3 (65%), NET (55%), DAT (59%), and Cysteinyl CysLT1 (70%). However it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% activities.” Thus, CID 45142486 (ML173) is highly selective and can be used to dissect the role of hM4in vitro and potentially in vivo with non-human primates.

In summary, a lead optimization campaign around the initial M4 PAM probe CID 864492 provided novel analogues with improved metabolic stability and physiochemical properties, but diminished efficacy at rat M4 (EC50 values ~2 μM) while retaining comparable fold-shift (14 – 67x) of the ACh CRC. Moreover, though weak at rat M4, several close analogues displayed modest in vivo efficacy in reversing amphetamine-induced hyper locomotion, a classic preclinical antipsychotic model. Surprisingly, we noted significant species differences within this new series of M4 PAMs, where analogues such as CID 45142486 (ML173) displayed an order of magnitude greater potency at human M4 (EC50 = 95 nM) than at the rat M4 receptor (EC50 = 2.4 μM) with comparable fold-shifts (human, 60x; rat, 44x) and high M4 mAChR subtype selectivity.

3.1. Summary of Screening Results

Image ml173fu25

3.2. Dose Response Curves for Probe

See Figures 4, 6 and 7 (vide supra).

3.3. Scaffold/Moiety Chemical Liabilities

No chemical liabilities for the probe molecule have been identified at the present time.

3.4. SAR Table

Table 4SAR Analysis for rat M4 Positive Allosteric Modulators

Image ml173fu26.jpg
EntryCIDSIDVU Number*XYZRat M4 EC50 (μM) **Rat M4 ACh fold-shift ***
14514246992392513VU0152117Sdimethylaminon-propyl4-methyoxybenzyl> 101x
24514247092392514VU0152118Sdimethylaminoethyl4-methyoxybenzyl> 10-
34514247192392515VU0152119Smorpholinoethyl4-methyoxybenzyl6.537x
44514247292392516VU0152120Spyrrolidin-1-ylethyl4-methyoxybenzyl8.225x
54514247392392517VU0152121S1-methylpiperidin-3-ylmethylene4-methyoxybenzyl> 10-
64514247492392518VU0152122S-n-propyl4-methyoxybenzyl>10-
74514247592392519VU0152123S-i-propyl4-methyoxybenzyl>10-
84514247692392523VU0152133S-n-pentyl4-methyoxybenzyl>10-
94462630292392520VU0152129Spyridin-4-ylmethylene4-methyoxybenzyl2.050x
104462630392392521VU0152130Spyridin-3-ylmethylene4-methyoxybenzyl2.29x
114462630492392522VU0152131Spyridin-2-ylmethylene4-methyoxybenzyl1.85x
124514247792392524VU0359439Sdimethylaminon-propyloxetan-3-yl2.587x
134514247892392525VU0359451Sdimethylaminon-propyl2,5-difluorobenzyl2.7514x
144514247992392526VU0359453Smorpholinoethyloxetan-3-yl7.4240x
154514248092392527VU0359454Smorpholinoethyl3-methoxypropyl6.2567x
164514248992392537VU0359455Smorpholinoethylphenyl5.8151x
174514248292392529VU0359459Smorpholinoethyli-propyl7.1536x
184514248392392530VU0359460Smorpholinoethylt-butyl8.7362x
194514248492392531VU0359479Spyridin-2-ylmethylene2,3-difluorobenzyl5.4028x
204514248592392532VU0359480Spyridin-2-ylmethylene2,5-difluorobenzyl> 1041x
214514248692392533VU0359508Spyridin-4-ylmethylene2,3-difluorobenzyl2.4444x
224514248792392534VU0359509Spyridin-4-ylmethylene2,5-difluorobenzyl3.7864x
*

S = synthesized, P = purchased.

**

Data represent the mean values from at least 3 experiments with similar results [AID 2616].

***

Leftward shift of an ACh CRC in the presence of 30 μM compound relative to ACh CRC control [AID 449769].

3.5. Cellular Activity

This series of positive allosteric modulators displayed functional activity (Ca+2 mobilzation) in CHO cells stably expressing the human and rat M4 receptors [AID 2616 and 449769]. Although not demonstrated with the probe molecule (ML173) close analogs did show modest decreases in amphetamine-induced hyper locomotion in rats, implying at least some level of CNS exposure was obtained, while not showing any signs of overt toxicity or general sedation.

3.6. Profiling Assays

The probe molecule (ML173) and 3 close analogs were profiled in functional cell-based Ca+2 mobilization assays across the five muscarinic subtypes at Millipore Corp. (St. Charles, USA) and were found to elicit measureable potentiation at only the M4 receptor subtype. Furthermore, ML173 was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to not significantly interact with 64 out of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM).20 CID 45142486 (ML173) did have activity at the following 4 targets (human targets at 10 μM): Adenosine A3 (65%), NET (55%), DAT (59%), and Cysteinyl CysLT1 (70%). However it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% activities.”

4. Discussion

4.1. Comparison to existing art and how the new probe is an improvement

The first selective M4 PAM disclosed was LY2033298 (see Figure 2). Similar to ML173, this compound showed significantly reduced potency between the rat and human receptors. Although low nanomolar potency was observed at the human M4 receptor, this decrease in potency at the rodent receptor necessitated the co-administration of a submaximal dose of oxotremorine (a non-selective muscarinic agonist) to observe the in vivo M4 potentiating effects of LY2033298 in a condition avoidance response rodent model.21 Additionally, when the same experiment was conducted in M4 Knock Out (KO) mice the actions of LY2033298 were significantly attenuated but not abolished as might be expected for a highly selective M4 PAM. Still, LY2033298 served as the inspiration, along with cheminformatics and database mining efforts, to produce our first M4 PAM probe (ML108). This compound possessed a rM4 EC50 = 380 nM, and more importantly showed efficacy in a different antipsychotic rat model (reversal of amphetamine-induced hyperlocomotor activity) without needing to be co-dosed with a non-selective muscarinc agonist. The major shortcomings associated with ML108 were metabolic stability and solubility. Relative to our initial M4 PAM probe (ML108) this second generation probe (ML173) possesses improved metabolic stability and physiochemical properties, but diminished efficacy at rat M4 (EC50 values ~2 μM) while retaining a comparable fold-shift (44x) of the ACh CRC. Though weakly efficacious at rat M4, several close analogues displayed modest in vivo efficacy in reversing amphetamine-induced hyper locomotion, a classic preclinical antipsychotic model. We noted significant species differences within this new series of M4 PAMs, where analogues such as CID 45142486 (ML173) displayed an order of magnitude greater potency at human M4 (EC50 = 95 nM, surpassing our initial M4 probe) than at the rat M4 receptor (EC50 = 2.4 μM) with comparable fold-shifts (human, 60x; rat, 44x) and high M4 mAChR subtype selectivity. Lastly, this probe (ML173), along with our initial probe (ML108), is not encumbered with patent restrictions and rests firmly in the public domain.

4.2. Mechanism of Action Studies

It is believed that this probe is functioning by positive allosteric modulation of the muscarinic acetylcholine M4 receptor given its lack of activity on the other muscarinic receptor subtypes (M1–3,5), its dependence on the presence of ACh to elicit functional activity and its nearly clean profile in the Ricerca lead profiling screen.

4.3. Planned Future Studies

At the present time no further studies are planned with this probe molecule. However, given its excellent selectivity profile it will continue to be used as a reference standard for in vitro experiments exploring hM4 receptor activation and downstream signaling. Ultimately, if the opportunity arises to study selective M4 receptor activation in non-human primates this probe will be among our top candidates for initial testing.

5. References

1.
Wess J. Crit, Rev Neurobiol. 1996;10:69–99. [PubMed: 8853955]
2.
Bymaster FP, Felder C, Ahmed S, McKinzie D. Curr Drug Targets. 2002;1:163–181. [PubMed: 12769625]
3.
Messer WS Jr. Curr Top Med Chem. 2002;2:353–358. [PubMed: 11966459]
4.
Raedler TJ, Bymaster FP, Tandon R, Copolov D, Dean B. Mol Psychiatry. 2007;12:232–246. [PubMed: 17146471]
5.
Bymaster FP, Carter PA, Yamada M, Gomeza J, Wess J, Hamilton SE, Nathanson NM, McKinzie DL, Felder CC. Eur J Neurosci. 2003;17:1403–1410. [PubMed: 12713643]
6.
Bymaster FP, McKinzie DL, Felder CC, Wess J. Neurochem Res. 2003;28:437–442. [PubMed: 12675128]
7.
Bodick NC, Offen WW, Levey AI, Cutler NR, Gauthier SG, Satlin A, Shannon HE, Tollefson GD, Rasmussen K, Bymaster FP, et al. Arch Neurol. 1997;54:465–473. [PubMed: 9109749]
8.
Shekhar A, Potter WZ, Lienemann J, Sunblad K, Lightfoot J, Herrera J, Unverzagt F, Bymaster FP, Felder C. 40th Annual Meeting of American College of Neuropsychopharmacology; 2005 Dec 11–15; Waikoloa, HI. Nashville, TN: American College of Neuropsychopharmacology; 2001.
9.
Shekhar A, Potter WZ, Lightfoot J, Lienemann J, Dubé S, Mallinckrodt C, Bymaster FP, McKinzie DL, Felder CC. Am J Psychiatry. 2008;165:1033–1039. [PubMed: 18593778]
10.
Stanhope KJ, Mirza NR, Bickerdike MJ, Bright JL, Harrington NR, Hesselink MB, Kennett GA, Lightowler S, Sheardown MJ, Syed R, et al. J Pharmacol Exp Ther. 2001;299:782–792. [PubMed: 11602695]
11.
Felder CC, Porter AC, Skillman TL, Zhang L, Bymaster FP, Nathanson NM, Hamilton SE, Gomeza J, Wess J, McKinzie DL. Life Sci. 2001;68:2605–2613. [PubMed: 11392633]
12.
Tzavara ET, Bymaster FP, Davis RJ, Wade MR, Perry KW, Wess J, McKinzie DL, Felder C, Nomikos GG. FASEB J. 2004;18:1410–1412. [PubMed: 15231726]
13.
Scarr E, Sundram S, Keriakous D, Dean B. Biol Psychiatry. 2007;61:1161–1170. [PubMed: 17239354]
14.
Marino MJ, Williams DL Jr, O’Brien JA, Valenti O, McDonald TP, Clements MK, Wang R, DiLella AG, Hess JF, Kinney GG, et al. Proc Natl Acad Sci U S A. 2003;100:13668–13673. [PMC free article: PMC263871] [PubMed: 14593202]
15.
Conn PJ, Jones CK, Lindsley CW. Trends in Pharm Sci. 2009;30:148–159. [PMC free article: PMC2907736] [PubMed: 19201489]
16.
Conn PJ, Christopolous A, Lindsley CW. Nat Rev Drug Discov. 2009;8:41–54. [PMC free article: PMC2907734] [PubMed: 19116626]
17.
Shirey JK, Xiang Z, Orton D, Brady AE, Johnson KA, Williams R, Ayala JE, Rodriguez AL, Wess J, Weaver D, et al. Nat Chem Biol. 2008;4:42–50. [PubMed: 18059262]
18.
Brady A, Jones CK, Bridges TM, Kennedy PJ, Thompson AD, Breininger ML, Gentry PR, Yin H, Jadhav SB, Shirey J, Conn PJ, Lindsley CW. J Pharm & Exp Ther. 2008;327:941–953. [PMC free article: PMC2745822] [PubMed: 18772318]
19.
Litvinov VPS, Yu A, Promonenkov VK, Rodinovskaya LA, Shestopalov AM. Seriya Khimicheskaya. 1984;8:1869–1870.
20.
For information on the Ricerca (formerly MDS Pharma) Lead Profiling Screen see: https:​//pharmacology​.ricerca.com/Catalog/
21.
Leach K, Loiacono RE, Felder CC, McKinzie DL, Mogg A, Shaw DB, Sexton PM, Christopolous A. Neuropsychopharmacology. 2010;35:855–869. [PMC free article: PMC3055367] [PubMed: 19940843]
22.
Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www​.absorption.com/site

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