Development of a Highly Selective, Orally Bioavailable and CNS Penetrant M₁ Agonist Derived from the MLPCN Probe ML071

Abstract

Herein we report the discovery and SAR of a novel series of M₁ agonists based on the MLPCN probe, ML071. From this, VU0364572 emerged as a potent, orally bioavailable and CNS penetrant M₁ agonist with high selectivity, clean ancillary pharmacology and enantiospecific activity.

The muscarinic acetylcholine receptors, mAChRs or M₁–M₅, are members of the class A G protein-coupled receptors (GPCRs) that mediate a broad range of actions of the neurotransmitter acetylcholine (ACh) in the central nervous system and other tissues.^1–3 Previous attempts to develop compounds that are highly selective for M₁ have failed because of the high conservation of the orthosteric ACh binding site and difficulty in developing truly specific compounds, not only for M₁ (versus M₂–M₅) but also other biogenic amine receptors (BARs).^4–6 Despite these major shortcomings, multiple orthosteric M₁ ‘preferring’ agonists have provided proof of concept in Phase II and III clinical trials for both Alzheimer’s disease (AD) and schizophrenia, generating great enthusiasm for selective M₁ activation.^7–14

Current efforts are focused on the selective activation of M₁ by targeting less conserved allosteric sites, and this approach is proving highly successful for multiple GPCRs.^4,5,15,16 Allosteric and/or bi-topic M₁ partial agonists first appeared, such as 1–4, with improved mAChR selectivity in many instances, but a general lack of selectivity versus BARs (D₂, 5HT_2c, 5HT_2B, opiate, etc.) and poor DMPK properties precluded their utility as tools to probe selective M₁ activation in vivo (Fig. 1).^6,17–20 Recently, numerous additional M₁ full and partial agonists have been reported with improved properties and efficacy in cognition models.^21–23 Positive allosteric modulators (PAMs) from three structural classes, 5–7, have also been reported, with exquisite selectivity for M₁ versus M₂–M₅ and BARs, allowing limited in vivo proof of concept studies to be conducted.^{24–26,28,29,31,32} For all of these PAMs, both DMPK properties and CNS penetration are barely adequate for in vivo studies; ^{24–26,28,29,31,32} however, two scaffolds variants of the BQCA PAM scaffold have overcome the DMPK issues and afforded PAMs with good properties and CNS exposure.^27,30

Figure 1.

Prototypical ‘M₁-preferring’ allosteric (bi-topic) agonists 1–4, and M₁ PAM series 5–7.

Based on the need for additional M₁ tools, we recently performed a functional M₁ HTS of the 65,000 compound MLPCN library using a high-expressing M₁ rat CHO cell line.^33,34 From this effort, we identified M₁ agonists, antagonists and PAMs, which were quickly optimized to afford the M₁ selective antagonist ML012 (VU0255035),³³ M₁ PAMs ML137 (VU0366369)³¹ and ML169 (VU0405562)³² as well as the M₁ agonist ML071 (VU0357017).³⁴ ML071 (8) proved to be a highly selective M₁ partial agonist (EC₅₀ = 200 nM, 81% ACh Max, >30 µM versus M₂–M₅) with clean ancillary pharmacology (no activity greater than 50% in a 10 µM radioligand binding panel of 68 GPCRs, ion channels and transporters, including the BARs) and favorable DMPK properties and CNS penetration (Fig. 2).³¹ ML071 demonstrated that selective M₁ activation potentiated NMDA receptor currents, provided a significant increase in soluble APP (sAPPα) in cell culture, and dose-dependently reversed scopolamine-induced disruption of contextual fear conditioning responses.³⁴ However, ML071 also displayed functional D₂ antagonism (IC₅₀ = 4.5 µM), which we hoped to eliminate through chemical optimization. As previously detailed, SAR for ML071 was shallow, with few modifications tolerated.³⁴ In this Letter, we describe the introduction of cyclic constraints to impart improved potency, efficacy and DMPK properties (Fig. 2).

Figure 2.

Prior SAR for ML071 and proposal to introduce cyclic constraints.

A number of cyclic constraints ((R)- and (S)-3-aminopyrroldines (9–12), piperazine (13), [2.2.1] (14) and [3.3.0] (15) congeners) were synthesized (Fig. 3A) and evaluated, affording less than 30% M₁ activation at 10 µM (Fig. 3B). However, the (R)-aminopiperidine constraint 17 (VU0364572) afforded full activation of M₁, while the (S)-enantiomer 16 was inactive, providing the first reported example of enantioselective M₁ activation. VU0364572 proved to be highly selective for M₁ (Fig. 3C) with an EC₅₀ of 110 nM and ~95% ACh Max.

Figure 3.

A) Structures of cyclic constrained analogs of 8. B) 10 µM M₁ single point screen of cyclic constraint variants of ML071. C) Full CRCs at M₁–M₅ for VU0364572 (17).

This finding led us to synthesize libraries of analogs around both 16 and 17 wherein we varied the amide moiety; interestingly, all (S)-enantiomers 16 were inactive (M₁ EC₅₀s >10 µM). Analogs 17 were synthesized according to Scheme 1. As shown in Table 1, SAR was shallow, as with ML071,³⁴ with only 12 of 36 analogs possessing M₁ potencies below 5 µM; moreover, 17 proved to be the best compound within this series in terms of M₁ potency, ACh Max and mAChR selectivity. Efforts now focused on full characterization of VU0364572, 17.

Scheme 1.

Reagents: (a) (R)-3-amino-3-N-Boc-piperidine, NaBH(OAc)₃, DCE, 95%; (b) HCl, dioxane, rt, 96%; (c) RCOCl, DIEA, DCM, rt, 65–95%.

Table 1.

Structures and activities of M₁ agonist analogs 20a–k.

Cmpd	R	M1 EC50 (µM)a	ACh Max (%)a	M2–M5 EC50s (µM)a
8		0.2	81	>30
17		0.11	96	>30
20a		0.06	77	>10
20b		0.13	91	>10
20c		0.14	92	>10
20d		0.25	91	>10
20e		0.31	84	>10
20f
20g		0.89	41	>10
20h		0.69	44	>10
20i		1.3	61	>10
20j		0.96	63	>10
20k		2.6	57	>10
		5.0	35	>10

^aAverage of at least three determinations in our rat HTS high-expressing CHO cell line

We then evaluated VU0364572 (17) in a radioligand binding panel of 68 GPCRs, ion channels and transporters (including all the BARs),³⁵ and no significant activities were reported (no inhibition >30% @10 µM). Importantly, 17 showed minimal activity at hERG (22%@10 µM), despite possessing a classical hERG pharmacophore. Furthermore, based on the previously observed weak functional D₂ antagonism of ML071 (D₂ IC₅₀ = 4.5 µM), we evaluated 17, and were delighted to find no functional activity at the D₂ receptor (Fig. 4). Based on this, VU0364572 is a highly selective M₁ agonist with exceptionally clean ancillary pharmacology, and a valuable small molecule tool to dissect the role of direct and selective M₁ activation.^1–20

Figure 4.

A) CRC for ML071 for D₂ antagonism, affording an IC₅₀ of 4.5 µM. B) CRC for VU0364572 for D₂ antagonism, affording an IC₅₀ > 30 µM.

Further in vitro characterization followed, evaluating the ability of 17 to shift APP processing towards the non-amyloidogenic pathway. Employing our standard model^20,22,28,29 in TREx293-hM₁ cells, carbachol (CCh) (10 µM) affords a significant increase in soluble APP (sAPPα), whereas an identical concentration of 17 provides a more robust increase (~3-fold) in sAPPα. Based on this data, selective activation of M₁, via 17, may have a disease modifying role in AD.

Activation of NMDA receptor currents by M₁ is postulated to play a critical role in the cholinergic regulation of cognitive function and circuitry that underlie the efficacy of mAChR agonists in schizophrenia (the NMDA receptor hypofunction hypothesis of schizophrenia)³⁶ and AD. As shown in Figure 5, VU0364572 (17), was found to potentiate NMDA receptor currents in hippocampal CA1 pyramidal cells, further validating selective M₁ activation as a means to promote synaptic plasticity.

Figure 5.

VU0364572 potentiates NMDA receptor currents in hippocampal CA1 pyramidal cells. A) Representative whole cell traces of NMDA-evoked currents and B) time course of normalized amplitude of NMDAR currents before, during and after application of 30 µM VU0364572 (17). Notably, even after washout, the potentiation persists for 10 min. Error bars represent mean ± SEM for five independent determinations.

The in vitro data was tremendously exciting, prompting the evaluation of 17 in our tier 1 DMPK battery. 17 displayed low plasma protein binding for both human (f_u = 5.8%) and rat f_u = 14.9%) and had a clean CYP profile (3A4, 2C9, 1A2 and 2D6; IC₅₀ >25 µM). Intrinsic clearance experiments (rat CL_INT = 23.4 mL/min/kg and human CL_INT = 11.2 mL/min/kg) suggested 17 would be a low to moderate clearance compound, and rat PK confirmed a good in vitro/in vivo correlation. A standard rat IV(1 mg/kg)/PO (10 mg/kg) study found 17 to possess a CL of 14.7 mL/min/kg, with a V_ss of 0.98 L/kg and a t_1/2 of 46 min. Importantly, 17 was orally bioavailable with a %F of 37 (AUC_IV = 1.1 µg*hr/mL, AUC_PO = 4.2 µg*hr/mL). In parallel, we performed an oral plasma:brain level (PBL) study with 17 in male Sprague-Dawley rats (Table 2). At a dose of 10 mg/kg with a 90 minute endpoint, 17 achieved an average Brain_AUC/Plasma_AUC of 1.35, providing excellent CNS exposure.

Table 2.

10 mg/kg Oral Plasma:Brain Level Study with 17.

Dose (mg/Kg)	Animal	Plasma (µM)	Brain (µM)	Brain:Plasma
10	1	1.45	2.23	1.54
10	2	1.62	1.89	1.17

As it became time to profile 17 on human M₁–M₅, our research team had moved away from the high-expressing HTS lines and developed both rat and human M₁–M₅ with expression more closely resembling native expression levels. In these new cell lines, both 17 and ML071 experienced an ~10-fold right shift in potency (EC₅₀s of 1.3 µM and 2.3 µM, respectively) with slightly lower ACh Max, yet still remained highly selective (M₂–M₅ EC₅₀s >30 µM). As M₁ expression levels vary amongst neuronal tissues and brain regions, compounds such as 17 may behave as full agonists in one population and as weak, partial agonists in another. The ramifications of this, and further in depth in vitro and in vivo pharmacological studies are in progress and will be reported shortly.³⁷

In summary, the chemical lead optimization of the MLPCN M₁ agonist probe ML071 (VU0357017), led to the discovery of VU0364572 (17), an M₁ agonist with high selectivity (versus M₂–M₅ as well as the BARs), exceptional PK and brain exposure, robust effects on APP processing and potentiation of NMDA receptor currents and enantiospecific M₁ activation. Further in depth in vitro and in vivo pharmacological studies with VU0364572 (17) are in progress and will be reported shortly.