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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 and development of the a highly selective M1 Positive Allosteric Modulator (PAM)

,1 ,2 ,3 ,4 and 5.

Received: ; Last Update: October 20, 2010.

The specific aim of this project is to identify, via a functional high-throughput screening (HTS) approach, small molecule positive allosteric modulators (PAMs) and/or allosteric agonists of the M1 muscarinic acetylcholine receptor (mAChR) that are cell permeable, possess submicromolar potency, and show greater than 10-fold selectivity over the other mAChRs (M2-M5). The identified small molecular probe ML137 (CID-44251556) can be used for in vitro molecular pharmacology and electrophysiology experiments to study the receptor trafficking profile and role of selective M1 receptor activation by this unique M1 PAM chemotype. Probes developed from these efforts will greatly advance the current state of the art by aiding in the understanding of M1's role in cell-based physiology, and may extend the clinical understanding of psychotic and cognitive symptoms associated with neurodegenerative disorders, such as Alzheimer's Disease and schizophrenia.

Assigned Assay Grant #: MH077606-1

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: P. Jeffrey Conn, Vanderbilt University

PubChem Summary Bioassay Identifier (AID): AID-2543

Probe Structure & Characteristics

1-(2-fluoro-4-(1-methyl-1H-pyrazol-4-yl)benzyl)indoline-2,3-dione

MW = 335.1, logP = 3.0, tPSA = 55

Image ml137fu1
CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti- target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 44251556
ML137
M1830 [8528605, 2425]M2, M3, M4, M5

MDS Pharma
> 30 μM [8528605, 2430, 2428, 2438, 2433]>30ACh Fold-shift (> 3-fold)

[8528605, 2434]

Recommendations for the scientific use of this probe

This probe (CID 44251556) can be used for in vitro molecular pharmacology and electrophysiology experiments to study the receptor trafficking profile and role of selective M1 receptor activation by this unique M1 PAM chemotype. This probe possesses high selectivity versus M2–M5, as well as a large panel of GPCRs, ion channels and transporters. CID 44251556 is moderately centrally penetrant (B/P ratio is 0.22), and while in vivo studies are possible, it has not been optimized for CNS penetration. CID 44251556 also displays reasonable solubility in acceptable vehicles (>5 mg/mL) in 20% β-cyclodextrin and >100 μM in DMSO.

Specific AIM

To identify small molecule positive allosteric modulators (PAMs) and/or allosteric agonists of the M1 muscarinic acetylcholine receptor that are cell permeable, possess submicromolar potency and show greater than 10-fold selectivity over the other mAChRs (M2–M5) employing a functional HTS approach. Out of this effort aimed at M1, which afforded a highly selective M1 antagonist (CID 24768606) and a highly selective M1 allosteric agonist (CID 25010775), we also identified and optimized the first M5 ligand, an M5 PAM (CID 42633508). From the same pan-Gq M1, M3, M5 PAM lead that afforded the M5 PAM, we have now been able to develop a highly selective M1 PAM with submicromolar EC50. Another MLSCN screening effort identified a highly selective M4 PAM (CID 864492); thus, two MLSCN/MLPCN screens have provided a toolkit of highly selective mAChR ligands available from the MLPCN to study individual mAChR function both in vitro and in vivo.

Significance

The five cloned muscarinic acetylcholine receptor subtypes (mAChR1-5 or M1–M5) are known to play highly important and diverse roles in many basic physiological processes. (1–3) Correspondingly, muscarinic agonists and antagonists targeting one or more subtypes have been used preclinically and clinically for research and treatment of a wide range of pathologies. (3,4) Based on the high sequence homology of the mAChRs across subtypes, and particularly within the orthosteric acetylcholine (ACh) binding site, discovery of truly subtype-selective compounds has proven historically difficult. Due to the scarcity of selective compounds, a detailed understanding of the precise roles of each subtype in neurobiology and in various central nervous system (CNS) disorders has thus remained elusive. (3,4) In numerous Phase II and III clinical trials, pan-mAChR agonists were shown to improve cognitive performance in AD patients, but the GI-and/or cardiovascular side effects, resulting from activation of peripheral mAChRs, were deemed intolerable and the trials were discontinued. (5,6) Importantly, several pan-mAChR agonists demonstrated a decline in the concentration of Aβ42 in the cerebral spinal fluid of AD patients, suggesting that mAChR activation has the potential to be disease modifying as well as providing palliative cognitive therapy. (7) More recent studies in 3xTg-AD mice further support a disease modifying role for mAChR activation, and several Ph III trials demonstrated that mAChR activation lowered Aβ42 in patients. (8) Interestingly, the M1/M4 preferring xanomeline, in addition to improving cognitive performance, had robust therapeutic effects on the psychotic symptoms and behavioral disturbances associated with AD and recently published clinical trial data indicates efficacy in schizophrenic patients. (9,10) Probes developed from these efforts will greatly advance the current state of the art by aiding in the understanding of M1’s role in cell-based physiology and may extend the clinical understanding of psychotic and cognitive symptoms associated with neurodegenerative disorders like Alzheimer’s Disease and schizophrenia.

Rationale

In recent years, major advances have been made in the discovery of highly selective agonists of other G protein-coupled receptors (GPCRs) that act at an allosteric site rather than the orthosteric binding site. (11,12) By screening for compounds that act at an allosteric site on the receptor, it is anticipated that compounds that selectively activate M1 versus the other muscarinic subtypes may be identified. (13–18) While allosteric M1 agonists have been identified, AC-42 and TBPB, they both suffer from undesirable ancillary pharmacology, poor physicochemical properties, poor pharmacokinetics and/or limited CNS exposure. (19,20) Thus, to truly enable the biomedical community to dissect the relative contributions of selective M1 activation in preclinical models of AD and schizophrenia and to understand the role of M1 in the pronounced efficacy of the M1/M4 preferring xanomeline, improved M1 probes are required. Recently, a number of novel highly subtype-selective allosteric ligands for M1 and M4 have emerged from functional cell-based screening efforts – several are MLPCN probes along with the prototypical M1 PAM, BQCA. (13–18,20–22) Although considerable interest was initially generated around BQCA, this level of interest appears to have waned and may point to a fatal flaw in its general chemotype (See Figure 2). Our initial report on the discovery of CID 3008304, a pan Gq M1, M3, M5 PAM, also described three other series of weak M1 PAMs, and identified that different M1 PAM chemotypes displayed different modes of activity on downstream receptor signaling/trafficking despite similar profiles in Ca2+ assays. (13) Thus, all allosteric M1 activation is not equivalent, and additional tool compounds representing diverse chemotypes are required to truly dissect and study M1 function in the CNS. Based on our ability to develop an M5 selective PAM from a pan Gq M1, M3, M5 PAM,(21,22) we initiated an effort to optimize CID 3008304 for M1 PAM activity in an attempt to add a unique chemotype to our tool kit of selective M1 activators. (23)

Figure 2. M5 PAM, CID 42633508, and M1 PAM, BQCA.

Figure 2

M5 PAM, CID 42633508, and M1 PAM, BQCA.

Assay Implementation and Screening

PubChem Bioassay Name

Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Positive Allosteric Modulator (PAM)

List of PubChem bioassay identifiers generated for this screening project (AIDs)

AID-2543, AID-2438, AID-2434, AID-2425, AID-2430, AID-2428, AID-2433

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. hM2, 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 ratM4/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.

Summary of Screen

This screen was performed in the pilot phase, the MLSCN, when the MLSMR compound collection at Vanderbilt only contained 65K compounds. From the primary M1 screen of 65K compounds, ~12 putative M1 PAMs were identified with an average Z’ score of 0.70+0.09. The confirmation screen (singles at 10 μM) produced two lead compounds, one of which is still in chemical lead optimization. The other, CID 3008304, represented a unique, and never before seen, pharmacological profile (Figure 1) in that it was a PAM of all the Gq-coupled mAChRs (M1 EC50 = 6.1 μM, M3 EC50 = 6.4 μM and M5 EC50 = 4.1 μM), but devoid of activity at the Gi/o-coupled M2 and M4.13 This led us to predict it would be possible to dial-in or dial-out different mAChR sub-types and potentially develop an M1 selective PAM, an M3 selective PAM and/or an M5 selective PAM from this non-selective lead through chemical optimization. This effort delivered the first M5 ligand, an M5 PAM (CID 42633508), and key SAR was established that ‘dialed out’ activity (Figure 2). (21,22) Efforts now focused on ‘dial in’ M1 PAM activity while abolishing M3 and M5 PAM activity to provide an M1 PAM (23) distinct from the BQCA chemotype. (14,15)

Figure 1. CRCa at M1–M5 for HTS lead CID 3008304.

Figure 1

CRCa at M1–M5 for HTS lead CID 3008304. CID 3008304 is a pan Gq-M1, M3, M5 PAM.

Probe Chemical Lead Optimization Strategy

Our initial optimization strategy is outlined in Figure 3, and as SAR with allosteric ligands is often shallow, we employed an iterative parallel synthesis approach. From our M5 Probe work where we counter-screened on M1, we quickly learned that most substitutions on the isatin ring led to pan mAChR activation profiles with various degrees of potency, efficacy, and subtype-selectivity. Thus, our first libraries employed an unsubstituted isatin core and surveyed diversity on the southern benzyl moiety. (23)

Figure 3. Initial optimization strategy for CID 3008304, a pan Gq M1, M3, M5 PAM.

Figure 3

Initial optimization strategy for CID 3008304, a pan Gq M1, M3, M5 PAM.

Libraries were prepared according to Scheme 1, wherein commercial indoline-2,3-dione 1 was alkylated with phenethyl bromides to provide analogs 2, or benzyl bromides/heteroarylmethyl halides to deliver analogs 3 (Figure 4). In parallel, CID 3008304 was coupled to diverse aryl/heteroaryl boronic acids employing an microwave-assisted protocol, to produce a 10-member Suzuki library to explore the effect of introduction of biaryl and heterobiaryl motifs replacing the bromine of CID 3008304, and providing analogs 4 (Figure 5). As previously documented for allosteric ligands, SAR was shallow, with all analogs 2 (such as CIDs 44602488, 44602487) and 3 (such as CIDs 2048702 and 10946670) devoid of M1 PAM activity (M1 EC50 >10 μM). Analogs 4 did not fare much better, with functionalized biaryls (such as CIDs 44602480 and 44602486) and heterobiaryls (such as CIDs 44602481 and 44602485) also devoid of M1 PAM activity (M1 EC50 >10 μM). However, one compound stood out (Figure 6), an N-Me pyrazole congener CID 44251554, that displayed an M1 EC50 of 2.3 μM, and good selectivity versus M3 and M5. Moreover, CID 44251554 afforded an ~5-fold leftward shift of the M1 ACh CRC at 10 μM, and a larger ~14-fold shift at 30 μM, with ~30% intrinsic allosteric agonism. (23) Intriguingly, the 5-OCF3 congener of CID 44251554 is an equipotent M5-preferring PAM, highlighting the aforementioned ‘molecular switch’ to engender M5 preference. However, it was exciting to see that we could develop an M1-preferring PAM from our initial pan Gq M1, M3, M5 PAM lead, CID 3008304, but it did not meet probe criteria in terms of potency or our desired level of mAChR selectivity.

Scheme 1. Synthesis of analogs of CID 3008304.

Scheme 1

Synthesis of analogs of CID 3008304.

Figure 4. Representative analogs 2 and 3 that were inactive (EC50 >10 μM) as M1 PAMs.

Figure 4

Representative analogs 2 and 3 that were inactive (EC50 >10 μM) as M1 PAMs.

Figure 5. Representative analogs 4 that were inactive (EC50 >10 M) as M1 PAMs.

Figure 5

Representative analogs 4 that were inactive (EC50 >10 M) as M1 PAMs.

Figure 6. CID 44251554, an M1-prefering PAM with an EC50 of 2.3 μM.

Figure 6

CID 44251554, an M1-prefering PAM with an EC50 of 2.3 μM.

As SAR for this series proved to be incredibly shallow, we next incorporated subtle changes, in the form of fluorine atoms, to the CID 44251554 scaffold. Following the synthetic routes outlined in Scheme 1, analogs with fluorine on both the isatin scaffold and the benzyl ring were readily prepared and evaluated for their ability to potentiate an EC20 of ACh at M1. This effort proved more productive (Table 1) with five of the analogs 5a–e displaying potentiation of M1, and two analogs provided M1 EC50s below 1 μM. Fluorine substitution was well tolerated on both the isatin core (4,7-difluoro or 7-fluoro) and on the benzyl ring (2-fluoro and 2,6-difluoro). The addition of a single fluorine atom to the 2-position of the benzyl ring delivered CID 44251556, with an M1 EC50 of 830 nM (65% ACh Max) – comparable to BQCA (M1 EC50 = 845 nM), but without the carboxylic acid moiety, which is presumed to be responsible for BQCA’s poor CNS exposure. (23)

Table 1. Structures and activities of analogs 5.

Table 1

Structures and activities of analogs 5.

This single change afforded a three-fold increase in potency over CID 44251554. A 2,6-difluorobenzyl congener CID 44251555 provides equivalent M1 potency with a slightly diminished ACh Max (60%) to CID 44251556. As fluorine content increased CIDs 44251557, 44251558 and 44251559 (fluoro-substitution on both the isatin core and benzyl ring) provided comparable M1 potency, but lower ACh Max (40–55%). CID 44251556 was studied further (Figure 7). (23) Gratifyingly, CID 44251556 was found to be a highly selective M1 PAM, with minimal/no activation of M2–M5 up to 30 μM (Figure 7A–B). However, in M1 ACh CRC fold-shift experiments, CID 44251556 as well as the di-fluoro congener CID 44251555 displayed only a subtle effect, increasing the potency of ACh by only 3x and 2x respectively, at 30 μM (Figure 7C). The smaller fold-shift appears to correlate with the lower overall ACh Max for this series. Lack of correlation between PAM potency and fold-shift is commonly observed within series of mAChR allosteric modulators and underscores the importance of determining both parameters when establishing SAR. Nonetheless, CID 44251556 represents the second known chemotype to provide potent and selective M1 positive allosteric modulation. (23)

Figure 7. A) CRCs for CID 44251555 at M1–M5; B) CRCs for CID 44251556 at M1–M5; C) Fold-Shift for CID 44251555 and CID 44251556 at 30 μM.

Figure 7

A) CRCs for CID 44251555 at M1–M5; B) CRCs for CID 44251556 at M1–M5; C) Fold-Shift for CID 44251555 and CID 44251556 at 30 μM.

In addition to high mAChR selectivity, CID 44251556 was tested at MDS Pharma’s Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to only bind to hERG (72%@10μM). (24) Thus, CID 44251556 is highly selective and can be used to dissect the role of M1 in vitro. To determine if CID 44251556 could be employed as an in vivo probe, we evaluated rat pharmacokinetics and brain exposure after i.p. dosing. Unfortunately, CID 44251556 was a rapidly cleared compound in rat, with a short half-life (< 1h) and low brain exposure (B/P = 0.2). Thus, CID 44251556 should best be employed as part of in vitro pharmacology and electrophysiology experiments gauged at examining receptor trafficking phenomenon. In silico properties of CID 44251556 were also calculated using TRIPOS software and compared to the MDDR database of compounds both entering Phase I and launched drugs (Table 2), and found to have an overall favorable profile.

Table 2. Calculated Property Comparison with MDDR Compounds.

Table 2

Calculated Property Comparison with MDDR Compounds.

Thus, optimization of a pan Gq mAChR M1, M3, M5 PAM (CID 3008304), which previously led to the discovery of the first selective M5 PAM (CID 42633508), provided CID 44251556, a highly selective and potent M1 PAM. CID 44251556 possesses comparable potency to BQCA and represents only the second known chemotype to provide highly selective M1 potentiation. Efforts to develop an M3 PAM from this chemotype have thus far proven unsuccessful; however, the ability to dial in or out M1 and M5 PAM activity within a single scaffold is unprecedented. Further in vitro and in vivo characterization of CID 44251556 is in progress in the assay submitter’s lab, and data will be reported in due course.

Synthetic procedure and spectral data for CID 44251556

Image ml137fu7

CID 44251556, 1-(2-fluoro-4-(1-methyl-1H-pyrazol-4-yl)benzyl)indoline-2,3-dione [ML137]

To a glass vial containing acetonitrile (1 mL) was added isatin (50.0 mg, 0.340 mmol), potassium carbonate (94.0 mg, 0.680 mmol), potassium iodide (6.0 mg, 0.034 mmol), and 4-bromo-2-fluorobenzyl bromide (110 mg, 0.409 mmol). The solution was stirred overnight at room temperature and then partitioned between CH2Cl2 and H2O. The organics were then passed through a disposable phase separator column (Isolute) and concentrated to afford the crude intermediate. Purification by HPLC (acetonitrile/H2O with 0.1% TFA) afforded the pure intermediate (42.0 mg, 0.126 mmol, 37% yield, LCMS = > 98%). This intermediate (42.0 mg, 0.126 mmol) was then dissolved in tetrahydrofuran (1 mL) in a glass vial to which 1-methylpyrazole-4-boronic acid (40.0 mg, 0.189 mmol), cesium carbonate (approximately 250 μL of 1 M solution in H2O, 0.25 mmol), and tetrakis(PPh3)Pd(0) (15 mg, 0.013 mmol) was added. This solution was microwave irradiated at 120 °C for 20 minutes and then passed through a disposable celite column (Isolute) using CH2Cl2 (excess). The organics were concentrated and the crude product was purified by mass-directed preparative HPLC (acetonitrile/H2O with 0.1% TFA) to afford the probe compound (12 mg, 0.035 mmol, 27%). LCMS = > 98% 214 nm, RT = 2.89 min, m/z = 336 (m+1). 1H NMR (400 MHz, CDCl3) 7.74 (s, 1H), 7.61 (m, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.20 (m, 2H), 7.11 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.97 (s, 2H), 3.96 (s, 3H). HRMS found 336.1145; calc’d for C19H14FN3O2, 336.1148, m+1).

MLS#s

002699061 (Probe, 500 mg), 002699059, 002699058, 002699057, 002699056, 002699060

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For information on the MDS Pharma Lead Profiling Screen see: www​.mdsps.com

APPENDIX I. Solubility, Stability and Reactivity data as determined by Absorption Systems

Solubility

Solubility in PBS (at pH = 7.4) for ML137 was 0.75 μM.

Stability

Stability (at room temperature = 23 °C) for ML137 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 121%, but the assay variability over the course of the experiment ranged from a low of 100% (at 0 minutes) to a high of 121% (at 48 hours).

Percent Remaining (%)
Compound0 Min15 Min30 Min1 Hour2 Hour24 Hour48 Hour
ML137100117110112117113121

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) ML137 was found to not form any detectable GSH adducts.*

APPENDIX II. Liquid Chromatography-Mass Spectrometry (LCMS) and Nuclear Magnetic Resonance (NMR) as prepared by Vanderbilt Specialized Chemistry Center

Image ml137fu8
Image ml137fu9
Image ml137fu10
Image ml137fu11
Image ml137fu12
Image ml137fu13
Image ml137fu14
Image ml137fu15

Footnotes

*

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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