This probe (ML172, CID 44602489), which possesses improved muscarinic selectivity over our previous M₅ positive allosteric modulator (PAM) probe (ML129, CID 42633508), can be used for in vitro molecular pharmacology and electrophysiology experiments to study the role of selective M₅ receptor activation. This probe possesses unprecedented selectivity versus M₁, M₂, M₃ and M₄. The probe (ML172, CID 44602489) is not readily CNS penetrant, and so would need to be administered i.c.v. to study the role of central M₅ activation in vivo.

Probe Structure & Characteristics

1-(4-phenoxybenzyl)-5-(trifluoromethoxy)indoline-2,3-dione, MW = 413.4, logP = 5.8, tPSA = 107.0 Ų

ML172

Recommendations for Scientific Use of the Probe

This probe (ML172, CID 44602489), which possesses improved muscarinic selectivity over our previous M₅ PAM probe (ML 129, CID 42633508), can be used for in vitro molecular pharmacology and electrophysiology experiments to study the role of selective M₅ receptor activation. This probe possesses unprecedented selectivity versus M₁, M₂, M₃ and M₄. The probe (ML172, CID 44602489) is not readily CNS penetrant, and so would need to be administered i.c.v. to study the role of central M₅ activation in vivo.

1. Introduction

Specific AIM: To identify small molecule positive allosteric modulators (PAMs) and/or allosteric agonists of the M₅ muscarinic acetylcholine receptor that are cell permeable, possess low- to sub-micromolar potency and show greater than 10-fold selectivity over the other mAChRs (M₁, M₂, M₃ and M₄) employing a functional HTS approach. Out of this effort primarily aimed at M₁, which afforded a highly selective M₁ allosteric agonist (CID 25010775) and two highly selective M₁ PAM probes (CID 44251556, and CID 44475955), we also identified and optimized the first selective M₅ PAM (CID 42633508), and now report the next-generation M₅ PAM described herein. Another MLSCN screening effort identified a highly selective M₄ PAM (CID 864492) and a subsequent highly selective M₄ PAM endowed with improved human potency (ML 173, CID 45142486); thus, a toolkit containing highly selective, allosteric mAChR ligands are available from the MLPCN to study individual mAChR function.

Significance: The five cloned muscarinic acetylcholine receptor subtypes (mAChR1-5 or M_1–5) 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 challenging.^3,4 A number of novel highly subtype-selective allosteric ligands for M₁ and M₄ have emerged from functional cell-based screening efforts.^5,6 Only recently have the first M₅ selective ligands been disclosed from these laboratories.¹³ Relative to the other mAChRs, little is known about M₅, which is expressed at very low levels in the CNS and peripheral tissues.^2–4

Rationale: Data from studies using mAChR5 knock-out (M₅-KO) mice suggest that M₅ is the sole mediator of ACh-induced vasodilation in the cerebral vasculature and thereby may have therapeutic relevance for cerebrovascular diseases or acute ischemic stroke.^7,8 M₅-KO mice have also been found to exhibit deficits in long-term potentiation (LTP) at the hippocampal mossy fiber-CA3 synapse and show deficits in hippocampal-dependent behavioral cognitive tests.⁸ In light of these and related findings, activation of M₅ has been suggested as a potential target for treatment of Alzheimer’s disease, perhaps in combination with M₁ activation.⁹ Consistent with the putative post-synaptic localization of M₅ in the ventral tegmental area (VTA), other M₅-KO data suggest this subtype plays an important role in regulation of mesolimbic dopamine transmission.^3,9 Indeed, M₅-KO mice exhibit decreased reward responses to morphine, decreased self-administration of cocaine, and less pronounced drug withdrawal symptoms, suggesting that M₅ antagonists or negative allosteric modulators may have therapeutic value in the treatment of illicit drug addiction.^9–11 Further pharmacological exploration of these and related hypotheses greatly depends on the discovery of novel M₅-preferring or selective small molecule tools.

2. Materials and Methods

2.2. Probe Chemical Characterization

Synthetic procedure (large scale) and spectral data for ML172 (CID 44602489)

Probe compound ML172 (CID 44602489) was prepared according to the above scheme and provided the following characterization data: LCMS (>98%) m/z = 414 [M+H⁺] (1.65 min retention, 214 nm),¹H NMR (>95%) (400 MHz, CDCl₃) δ =7.52 (s, 1H), 7.36 (m, 6H), 7.15 (t, J = 7.2 Hz, 1H), 7.10 (t, J = 5.2 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.93 (s, 2H). ¹³C-NMR (100MHz, d₆ - DMSO) δ 182.2, 157.8, 157.6, 156.4, 145.3, 130.9, 129.8, 129.0, 128.3, 123.7, 121.6, 120.4, 119.2, 119.0, 118.5, 118.1, 111.9, 43.7, HRMS (Q-TOF): m/z calc for C₂₂H₁₅NO₄F₃ [M + H]: 414.0953, found 414.0959.

Solubility. Solubility in PBS (at pH = 7.4) was determined to be less than 0.10 μM although alternative formulations could potentially improve this number. For example, ML172 shows good solubility in DMSO (>100 μM), but this is not a universally acceptable vehicle.

Stability. Stability (at room temperature = 23 °C) for ML172 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 134%, but the assay variability over the course of the experiment ranged from a low of 84% (at 24 hours) to a high of 167% (at 30 minutes). It is important to point out that ML172 showed very poor LCMS sensitivity and exhibited high levels of non-specific binding. Given these caveats, it is difficult to draw any firm conclusions with respect to ML172’s stability from this experiment.

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	Percent Remaining (%)
Compound	0 Min	15 Min	30 Min	1 Hour	2 Hour	24 Hour	48 Hour
ML172	100	164	167	154	114	84	134

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) ML172 was found to not form any detectable GSH adducts.²¹

Compounds added to the SMR collection (MLS#s): 003108019 (ML172, CID 44602489, 500 mg), 003108020, 003108021, 003108022, 003108023, 003108024.

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 65K compounds. From the primary M₁ screen of 65K compounds, ~12 putative M₁ PAMs were identified giving an average Z’ score of 0.70±0.09. The confirmation screen (singles at 10 μM) produced two lead compounds, one of which was recently developed into a structurally novel M₁ PAM (CID 44475955). The other, CID 3008304, represented a unique, and never before seen, pharmacological profile (Figure 1) in that it was a PAM of all the G_q-coupled mAChRs (M₁ EC₅₀ = 6.1 μM, M₃ EC₅₀ = 6.4 μM and M₅ EC₅₀ = 4.1 μM), but devoid of activity at the G_i/o-coupled M₂ and M₄.¹² 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.^13a As predicted, CID 3008304 served as the common starting point for the development of the two highly selective probe molecules shown in Figure 2: the M₁ PAM (CID 44251556) and the M₅ PAM (CID 42633508, EC₅₀ = 1.1 μM, 91% ACh Max). Although very selective for M₅, CID 42633508 did retain a detectable amount of activity at the M₁ and M₃ receptors at concentrations of 30 μM and above. Therefore we chose to explore the possibility of further optimizing CID 42633508 as described next.

Figure 1

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

Figure 2

Two MLPCN probe molecules derived from CID 3008304.

Probe Optimization Strategy: For the continued optimization of CID 42633508, we chose the strategy depicted in Figure 3, and as SAR with allosteric ligands is often shallow, we employed an iterative parallel synthesis approach. From the development of our initial M₅ probe, we were confident that the 5-OCF₃ group was essential for M₅-preferring activity and selectivity, so this moiety was universally retained. Libraries were prepared according to Scheme 1, wherein the commercially available indoline-2,3-dione 1 was alkylated with 4-bromobenzylbromide to deliver intermediate 2. An eleven-membered Suzuki library was then prepared to explore the biaryl and heterobiaryl replacements for the methyl ether of CID 42633508, thus providing analogs 3. Concomitantly, 1 was alkylated with functionalized phenethyl bromides 4 to explore the effect of chain homologation present in analogs 5. Compounds from these libraries were then triaged by a single point (10 μM) screen for their ability to potentiate an EC₂₀ of ACh in M₅-CHO cells with those results appearing in Figure 4.

Figure 3

Optimization strategy for CID 42633508, a highly M₅-preferring PAM.

Scheme 1

Reagents and conditions. (a) p-bromobenzylbromide, K₂CO₃, KI, CAN, rt, 16 h (99%); (b) R-B(OH)₂, Pd(t-Bu₃P)₂, Cs₂CO₃, THF:H₂O, mw, 120 °C, 20 min (10–90%); (c) K₂CO₃, KI, CAN, rt, 16 h (50–90%)

Figure 4

Ca2+ mobilization screen with M₅-CHO cells using 10 μM test compound in the presence of a submaximal concentration of Ach (~EC₂₀) to triage libraries 3 and 5 for PAM activity. Data represent means ± SEM from 3 independent determinations (more...)

In general, chain homologation in analogs 5 did not produce improved profiles relative to the benzyl analogs 3. Although M₅ PAM efficacy was maintained it came at the cost of right-shifted EC₅₀ values. For example, CID 45281805, the direct phenethyl analog of CID 42633508 possessed an M₅ EC₅₀ of 4.9 and an 80% ACh maximum response (data not shown). Biaryl and heterobiaryl analogs 3 proved far more productive, affording a number of M₅ PAMs with high selectivity versus M₁ (M₁ EC₅₀s >30 μM) and low micromolar M₅ EC₅₀s (Table 1). The remaining 5 analogs of 3 displayed M5 EC₅₀s > 10 μM. A wide variety of R- groups could be tolerated, both five- (CIDs 45281797 and 45281803) and six-membered heterocycles (CIDs 45281798 and 45281801) provided active compounds, as did bare phenyl (CID 45281794) and substituted phenyl (CID 45281796). Potency was virtually identical for all of the active analogs 3 (M₅ EC₅₀s 2.7 – 4.8 μM) with similar ACh Max values (70–85%). Shallow SAR was again noted for these allosteric ligands, with compounds either being active in the micromolar potency range or inactive as M₅ PAMs.

Table 1

Structures and activities of analogs 3.

The two most potent analogs from Table 1 (CIDs 45281794 and 45281797) were selected for additional follow-up. Figure 5 depicts only the G_q mAChR (M₁, M₃ and M₅) concentration response curves (CRCs) for CID 45281794 and CID 45281797. Note that CID 45281794 possesses improved M₅ selectivity versus our initial M₅ probe CID 42633508, with almost negligible activation of M₃ at 30 μM (Figure 5A). Both analogs CID 45281794 and CID 45281797 elicit significant leftward shifts (> 50-fold) of the ACh CRC at M5 (Figure 5C), as compared to the 14-fold shift of our initial M₅ probe CID 42633508. As seen with the M₁ PAM BQCA^14–16 and other ago-potentiators for class C GPCRs.^17–19 Figure 5C indicates moderate intrinsic allosteric agonism at 30 μM for both analogs at the M₅ receptor.

Figure 5

Concentration-response curves in Ca²⁺ mobilization assays with M₁-,M_2Gqi5-, M₃, M_4/Gqi5-, and M₅-CHO cells for (A) CID 45281794 (M₅ EC₅₀ = 2.7 μM) and (B) CID 45281797 (M5 EC50 = 2.8 μM). (C) Ach concentration-response fold-shift Ca2+ (more...)

Encouraged by the potency and mAChR selectivity of the phenyl-containing CID 45281794 and the ether-containing CID 42633508 (Figure 2), a hydrid analog was prepared (Scheme 2) that possessed a biphenyl ether moiety, CID 44602489 (ML 172), and displayed an M₅ EC₅₀ of 1.9 μM with a 75% ACh Max. More Importantly, CID 44602489 (ML 172) was completely selective versus M_1–4, affording no elevation of an ACh EC₂₀ at M_1–4 at concentrations as high as 30 μM (Figure 6). Notably, CID 44602489 (ML 172) represents the most selective M₅ PAM described to date; however, unlike CID 45281794 and CID 45281797, this hybrid analog only afforded a ~5-fold shift of the M₅ ACh CRC at 30 μM and did not display intrinsic allosteric agonism (graph not shown). While subsequent compounds were prepared in an effort to improve this fold shift, potency and fold shift do not always track in the same direction, and in this case our attempts were unfruitful. Therefore CID 44602489 (ML172), the most highly M₅ muscarinic selective PAM known to date, was chosen as our next-generation M₅ probe molecule.

Scheme 2

Reagents and conditions: (a) K₂CO₃, KI, CAN, mw, 160 °C, 10 min (68%).

Figure 6

Concentration-response curves in Ca²⁺ mobilization assays with M₁-,M_2Gqi5-, M₃, M_4/Gqi5-, and M₅-CHO cells for CID 44602489 (M₅ EC₅₀ = 1.9 μM). Data represent means ± SEM from 3 independent determinations

Subsequent follow up demonstrated that many of these analogs displayed moderate to poor PK in rats with limited brain exposure (AUC_Brain/AUC_Plasma ~ 0.25), presumably due to the bis-carbonyl of the isatin moiety. However, these are important tools to study M₅ function in cells, in electrophysiology and by i.c.v. injection. We did not examine the brain exposure when a DMSO-containing vehicle was employed, but using a DMSO-containing vehicle has been shown to improve brain levels.^17–18

The calculated physical properties appearing in Table 2 for the in initial M₅ probe molecule (CID 42633508) and this current probe ML172 (CID 44602489) were generated using TRIPOS software. Also included in Table 2 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 and launched compounds with respect to MW, hydrogen bond donors/acceptors and number of rotatable bonds. However, both molecules have relatively high cLogP values, which may contribute to their moderate to poor PK, and is also supported by the calculated low LogS (Solubility) values of −4.94 and −6.67 (CID 42633508 and CID 44602489, respectively). Similarly, the greater than 100 square angstroms of polar surface area for each molecule, although not necessarily high enough to preclude entry into the CNS, might be contributing to their less than ideal CNS exposure.

Table 2

Calculated Property Comparison with MDDR Compounds.

To more fully characterize this novel M₅ PAM probe molecule, ML172 (CID 44602489) was tested at MDS Pharma’s (now Ricerca’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 36 out of the 68 assays conducted (no inhibition > 50% at 10 μM).²⁰ ML172 (CID 44602489) did have activity at the following 32 targets (human targets at 10 μM, unless stated as rat): Adenosine A₁ (50%), Adenosine A₃ (97%), rat Adrenergic α_1A (79%), Adrenergic α_1D (66%), Adrenergic α_2A (100%), Adrenergic β₁ (89%), NET (93%), Bradykinin B₁ (52%), rat Calcium Channel L-Type, Dihydropyridine (74%), CB₁ (109%), D₁ (99%), D_2S (85%), D₃ (79%), DAT (90%), rat GABA transporter (56%), H₁ (99%), H₂ (101%), rat Imidazoline, central (50%), Leukotriene, Cysteinyl CysLT₁ (83%), MT₁ (60%), Muscarinic M₁ (98%), Muscarinic M₂ (96%), Neuropeptide Y Y₁ (83%), KOP (98%), MOP (99%), PAF (58%), hERG (81%), EP₄ (66%), 5-HT_1A (69%), 5-HT_2B (90%), SERT (80%) and rat Sodium Channel, site 2 (97%). 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.” This is best illustrated by the fact that we see no functional activity for ML172 (CID 44602489) at either the M₁ or M₂ receptor at concentrations as high as 30 μM (Figure 6), while the lead profiling screen indicates 98% and 96% inhibition of radio ligand binding at M₁ and M₂, respectively, at just 10 μM.

In summary, ML172 (CID 44602489) represents a marked improvement over our initial M₅ probe with respect to muscarinic selectivity, however ancillary pharmacology, at the binding level, appears to have suffered. CNS penetration is poor, using DMSO-free vehicles, possibly restricting ML172 (CID 44602489) to use as an in vitro or i.c.v. probe.

3.1. Summary of Screening Results

3.4. SAR Table

Table 3SAR Analysis for M₁ Positive Allosteric Modulators

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Entry	CID	SID	VU Number	*	X	Y	Z	M1 PAM EC50 (μM)**	M1 ACh fold-shift***
1	45281794	92845376	VU0365114-1	S	methylene	phenyl	-	2.7	> 50
2	45281795	92845377	VU0365115-1	S	methylene	3,5-dichlorophenyl	-	~ 10	ND
3	45281796	92845378	VU0365116-1	S	methylene	phenyl	2-methoxy	3.9	ND
4	45281797	92845379	VU0365117-1	S	methylene	thiophen-3-yl	-	2.8	> 50
5	45281798	92845380	VU0365118-1	S	methylene	pyridin-3-yl	6-chloro	4.8	ND
6	45281799	92845381	VU0365119-1	S	methylene	phenyl	4-chloro	~ 10	ND
7	45281800	92845382	VU0365120-1	S	methylene	2,5-difluorophenyl	-	~ 10	ND
8	45281801	92845383	VU0365121-1	S	methylene	pyridin-3-yl	6-fluoro	3.6	ND
9	45281802	92845384	VU0365122-1	S	methylene	phenyl	4-methoxy	~ 10	ND
10	45281803	92845385	VU0365123-1	S	methylene	1H-pyrazol-4-yl	1-methyl	3.3	ND
11	45281804	92845386	VU0365138-1	S	methylene	phenyl	4-propoxy	> 10	ND
12	45281805	92845387	VU0365124-1	S	ethyl	4-methoxy	-	4.9	ND
13	45281806	92845388	VU0365125-1	S	ethyl	4-bromo	-	~10	ND
14	44602489	87352032	VU0400265-1	S	methylene	O	phenyl	1.9	5

*

S = synthesized, P = purchased.

**

Data represent the mean values from at least 3 experiments with similar results [AID 2416].

***

Leftward shift of an ACh CRC in the presence of 30 μM compound relative to ACh CRC control [AID 2186].

4. Discussion

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For information on the Ricerca (formerly MDS Pharma) Lead Profiling Screen see: https:​//pharmacology​.ricerca.com/Catalog/

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Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www​.absorption.com/site