Synthesis and SAR of N-(4-(4-alklylpiperazin-1-yl)phenyl)benzamides as muscarinic acetylcholine receptor subtype 1 (M₁) anatgonists

Abstract

This Letter describes the synthesis and SAR, developed through an iterative analog library approach, of a novel series of selective M₁ mAChR antagonists, based on an N-(4-(4-alkylpiperazin-1-yl)phenyl)benzamide scaffold for the potential treatment of Parkinson's disease, dystonia and other movement disorders. Compounds in this series possess M₁ antagonist IC₅₀s in the 350 nM to >10 μM range with varying degrees of functional selectivity versus M₂-M₅.

There are five subtypes of muscarinic acetylcholine receptors (mAChR1-5 or M₁-M₅), members of the G Protein-Coupled Receptor (GPCR) family A, that mediate the metabotropic actions of the neurotransmitter acetylcholine.^1,2 M₁, M₃ and M₅ activate phospholipase C and calcium mobilization through G_q whereas M₂ and M₄ block the action of adenylyl cyclase through G_i/o.^1,2 The cholinergic system, mediated by mAChRs, plays a critical role in a wide variety of CNS and peripheral functions including memory and attention mechanisms, motor control, nociception, regulation of sleep wake cycles, cardiovascular function, renal and gastrointestinal function to mention only a few.^1-4 As a result, agents that can selectively modulate the activity of mAChRs have the potential for therapeutic use in multiple peripheral and central pathological states. Due to high sequence conservation within the orthosteric binding site of the five mAChR subtypes, it has been historically difficult to develop mAChR subtype-selective ligands.^1-5 Based on brain expression and cellular localization, data from mAChR knock-out mice and clinical trials with muscarinic agents, the M₁ subtype is an attractive molecular target for the treatment of CNS disorders. M₁ has been implicated in the pathologies of Alzheimer's disease (AD), Parkinson's disease (PD) and dystonia due to its role in cognition and motor control.⁶

The majority of reported muscarinic antagonists are unselective, such as a scopolamine, 1.⁷ Recently, pirenzapine, 2 has emerged as a relatively selective M₁ receptor antagonist (20- to 50-fold versus M₂-M₅) and there are numerous reports of moderately selective M₃ antagonists (20- to 50-fold versus M₂) such as 3.⁸ Interestingly, the most selective M₁ antagonist, MT7, 4, the 65 amino acid peptide, (>1,000-fold versus M₂-M₅) was derived from venom extracts of the green mamba snake (Fig. 1).⁹ From an M1 functional screen within the MLSCN, we identified M₁ antagonists such as 5 (M₁ IC₅₀ of 441 nM and with >340-fold selectivity versus M₄, but modest selectivity versus M₂, M₃ and M₅ (7.9-fold, 7-fold, and 2.4-fold, respectively)) and 6 (M₁ IC₅₀ of 5.0 μM and with >30-fold selective versus M₂-M₅).^10-12 Based on the M₁ selectivity of 6, attractive physiochemical properties (MW < 350, clogP 3.6) and the fact that it was the only benzamide-containing analog in the series, we initiated a library synthesis effort¹³ to develop SAR around 6.

Figure 1.

Structures of representative mAChR antagonists.

As shown in Scheme 1, the first round of library synthesis focused on benzamide analogs of 6. Commercially available 3-chloro-(4-(4-ethylpierazin-1yl)aniline 7 was acylated under standard conditions employing polymer-supported reagents and scavengers¹³ to afford a 24-member library of analogs 8, along with resynthesized 6. All analogs were then purified by mass-guided HPLC to analytical purity.¹⁴ To effectively screen small libraries of potential mAChR ligands, we have adopted a strategy to triage compounds in single-point screens (at 10 μM) at M₁, M₃ and M₅ – the G_q-coupled mAChRs – to identify active and selective compounds prior to running full concentration-response curves (CRCs).¹⁵ Figure 2 shows the 10 μM single-point screens for the first 25-member library of benzamide analogs 8.

Scheme 1.

Library synthesis of first generation analogs 8. All library compounds were purified by mass-guided HPLC to >98% purity.¹⁴

Figure 2.

Single-point EC₈₀ plus 10 μM compound triage screen at M₁, M₃ and M₅ to select compounds for full CRCs.

As Shown in Table 1, re-synthesized 6 displayed comparable potency and mAChR selectivity to the original sample (M₁ IC₅₀ = 3.2 μM, IC₅₀ ≫10 μM for M₂-M₅). Functionalized benzamide analogs 8 possessed a wide range of M₁ potency and mAChR selectivity, and we initially evaluated anlaogs 8 against M₁, M₃ and M₅. Substitution in the 2-position, 8a (2-Cl) and 8b (2-OMe) possessed submicromolar M₁ IC₅₀s (960 nM and 820 nM, respectively), but also showed low micromolar activity at M₃ and M₅. A pentafluorophenyl congener 8e (Fig. 3A) proved to be a submicromolar antagonist of both M₁ and M₅ (IC₅₀s of 350 nM and 830 nM, respectively). Substitution at the 4-position, as with the 4-OMe derivative 8f, was comparable to the original 6. Interestingly, a 2,5-bisCF₃ analog 8h had an M₁ IC₅₀ of 490 nM, with ∼ 9-fold functional selectivity versus M₃ and M₅ (Fig. 3B). Intrigued by this potent and selective M₁ antagonist, we screend against M₂ and M₄ as well, but found that 8h possessed only 3- to 4-fold selectivity versus the G_i/o-coupled mAChRs (Table 1). 8i, a 3,5-bisCF₃ analog possessed a unique profile as a dual M₁/M₄ antagonist (IC₅₀s of 2.6 μM and 3.7 μM, respectively), with little effect on an ACh EC₈₀ at 10 μM on M₂, M₃ or M₅. Finally, a 3,4-difluoro 8j derivative was also comparable to the original 6. While this library afforded interesting results, further optimization was required.

Table 1.

Structures and mAChR activities of analogues 8.

Cmpd	Ar	M1 IC50 (μM)a	M2 IC50 (μM)a	M3 IC50 (μM)a	M4 IC50 (μM)a	M5 IC50 (μM)a
6		3.2	>10	>10	>10	>10
8a		0.96	ND	8.2	ND	2.3
8b		0.82	ND	5.6	ND	1.3
8c		2.9	6.9	>10	3.7	>10
8d		2.1	ND	>10	ND	3.5
8e		0.35	ND	3.7	ND	0.83
8f		3.2	ND	>10	ND	>10
8g		2.9	>10	4.3	3.7	4.1
8h		0.49	2.7	4.2	1.5	4.1
8i		2.6	>10	>10	3.7	>10
8j		4.7	>10	>10	>10	>10

^aIC₅₀s are an average of three independent experiments using mAChR (CHO) cell lines. ND = not determined.

Figure 3.

CRCs for M₁, M₃ and M₅ for (A) compound 8e (M₁ IC₅₀ = 350 nM) and (B) compound 8h (M₁ IC₅₀ = 490 nM), showing ∼9-fold functional selectivity versus M₃ and M₅.

Having surveyed the amide moiety while maintaining the N-ethyl piperazine, we next generated two-dimensional libraries wherein the nature of the alkyl group was varied (9-12) while also surveying diverse benzamides to generate analogs 9a-f, 10 a-f, 11a-f and 12a-f (Scheme 2).

Scheme 2.

Library synthesis of second generation analogs 9a-f, 10a-f, 11a-f and 12a-f. All library compounds were purified by mass-guided HPLC to >98% purity.¹⁴

Application of the same strategy to triage compounds in single-point screens (at 10 μM) at M₁, M₃ and M₅ to identify active and selective compounds prior to running full (CRCs) was employed, but >75% of these new analogs possessed no M₁ antagonist activity. The SAR for this series was incredibly shallow, with only an N-propyl congener with the 3,5-dicholrobenzamide moiety 11i displaying reasonable activity (M₁ IC₅₀ = 3.7 μM, IC₅₀ >10 μM for M₃ and M₅), and all other analogs possessing M₁ IC₅₀s in the 6-9 μM range.

In summary, a two-dimensional parallel synthesis library campaign was performed around 6, an M₁ antagonist identified in a functional HTS screen. SAR for this series was shallow, but we were able to improve the M₁ antagonist activity of 6 into the 350 to 500 nM range with analogs 8, while maintaining good mAChR selectivity. Interestingly, 8i is the first reported dual M₁/M₄-preferring antagonist, which compliments the prototypical M₁/M₄-preferring agonist xanomeline. Other chemical series from our M₁ functional screen are currently under chemical optimization, and further refinements will be reported in due course.