Discovery of VU6077564: A Selective M₁ Antagonist That Promotes Neurite Outgrowth in Adult Sensory Neurons

Abstract

Previously, we reported the potent and highly selective M₁ antagonists VU0415248 and VU0452865; however, these compounds were limited by high predicted hepatic clearance in rat. This work reports the development of cyclobutylsulfonamide-based analogs of VU0415248 and VU0452865. From this exercise, we identified VU6077564, a peripherally restricted and selective M₁ antagonist. Furthermore, we demonstrate the ability of VU6077564 to promote neurite outgrowth in adult rat dorsal root ganglia sensory neurons, highlighting the potential of selective M₁ antagonists for the treatment of peripheral neuropathy.

Introduction

Acetylcholine (ACh), the primary neurotransmitter and neuromodulator of the cholinergic system, exerts its effects through two major classes of receptors: muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs). These receptors are distributed throughout both the central nervous system (CNS) and the peripheral nervous system (PNS), where they regulate essential functions such as memory, learning, attention, arousal, and involuntary muscle movement. The muscarinic receptors are a family of five (M₁–M₅) G protein-coupled receptors (GPCRs) belonging to the α-branch of class A. The M₁, M₃, and M₅ subtypes preferentially couple to G_q/11, resulting in stimulation of phospholipase C (PLC) and intracellular calcium mobilization. The M₂ and M₄ subtypes primarily couple to G_i/o proteins, which primarily inhibit cAMP production from ATP by inhibiting adenylate cyclase (AC).

Of the mAChRs, M₁ accounts for 60% of mAChRs expressed in the CNS, primarily in the cerebral cortex, hippocampus, and striatum, emphasizing its vital role in synaptic plasticity, learning, and memory. , Antagonism of M₁ has shown potential for the treatment of conditions and diseases such as multiple sclerosis, Parkinson’s disease, and Fragile X syndrome. − Beyond the CNS, M₁ has also emerged as a therapeutic target for maladies of the PNS. We have previously demonstrated that M₁ antagonists enhance neurite outgrowth and may represent potential therapeutic agents for peripheral neuropathy by promoting axonal repair across a variety of neuropathic animal models. Notably, in cultured adult dorsal root ganglia (DRG) sensory neurons the pan-muscarinic antagonists hexamethonium and mecamylamine did not exert any detectable effect in elevating neurite outgrowth. However, M₁ selective antagonists pirenzepine, muscarinic toxin 7 (MT7), and ML012 (VU0255035) (Figure ) demonstrated significant activation of AMP-activated protein kinase (AMPK) and augmentation of mitochondrial function to drive elevated neurite outgrowth. The translational significance of M₁ antagonists was recently highlighted in phase 2 clinical trials in patients with type 2 diabetes and exhibiting mild-moderate sensory neuropathy. For 5–6 months, daily topical treatment with oxybutynin (a potent, nonselective M₁ antagonist that is available over-the-counter) or pirenzepine (a selective M₁ antagonist with limited penetration into the CNS) elevated nerve fiber levels in the skin as well as reversed clinical signs of pain and improved quality of life (as assessed using the Norfolk scale). , Future development of oxybutynin for therapy in diseases of the PNS is discouraged due to its lack of selectivity for M₁ and propensity to cross the blood-brain barrier. Consequently, M₁ antagonists with improved selectivity and enhanced PNS distribution compared with the CNS are needed to further investigate the therapeutic potential of M₁ blockade in treating peripheral neuropathy.

1.

Pan-muscarinic antagonists (A) and selective M₁ antagonists (B).

The ubiquity of mAChRs throughout biological compartments and their highly conserved orthosteric binding pocket pose a significant challenge in the development of highly selective therapeutic agents. Notably, most known muscarinic antagonists, such as scopolamine, oxybutynin, and diphenhydramine, are nonselective (Figure ). Moreover, moderately selective M₁ receptor antagonists, such as pirenzepine, have limited utility in treating CNS disorders due to poor blood-brain barrier penetration. However, pirenzepine shows promise in the treatment of neuropathic diseases of the PNS. , Conversely, S-(−)-ET 126 (Figure ), an analog of scopolamine, is a brain-penetrant compound with moderate selectivity for M₁ over M₂ and M₃. However, its affinity for M₄ and M₅ remains unclear. MT7, a 65-amino-acid peptide with over 1000-fold selectivity for M₁ over M_2–5, was derived from the venom of the green mamba snake. The peptidic nature of MT7, unfortunately, makes it an unlikely drug candidate.

Our lab has been at the forefront of developing selective M₁ antagonists for over a decade, starting with the Molecular Libraries Screening Center Network (MLPCN) development program, supported by the NIH Molecular Libraries Roadmap. This program led to the discovery of the highly selective M₁ antagonist, ML012 (1), which exhibited impressive selectivity for M₁, ranging from 45- to 159-fold over M_2–5 (Figure ). In a rodent model, ML012 ameliorated pilocarpine-induced seizures, a condition caused by the stimulation of M₁ mAChR subtype receptor. Moreover, ML012 mitigated pilocarpine-induced seizures without adversely affecting memory, highlighting the superior potential of selective M₁ antagonists over pan-muscarinic antagonists, like scopolamine, for therapeutic development. Encouraged by these results, we initiated a hit-to-lead optimization campaign using an iterative parallel synthesis approach, resulting in the development of the isoquinoline sulfonamide VU0415248 (2), an M₁ antagonist with improved selectivity over M_2–5. Further optimization led to the discovery of the more potent M₁ antagonist VU0452865 (3) without affecting M₁ selectivity. Unfortunately, VU0415248 and VU0452865 exhibited high predicted rat hepatic clearance (CL_hep) values of 68.6 mL/min/kg and 49.5 mL/min/kg, respectively. Subsequent efforts led by Consortium Pharmaceuticals (formerly PIPELINE Therapeutics) to explore this chemical space resulted in the development of PIPE-359 and later PIPE-307 (structure undisclosed), which was in Phase 2 clinical trials for patients with relapsing-remitting multiple sclerosis (RRMS). − While this compound was tolerated and showed an acceptable safety profile at all doses tested, it did not demonstrate any significant change to visual function when binocular 2.5% low contrast letter acuity was assessed. Herein, we provide an update on our efforts to develop a viable in vivo tool as part of our ongoing pursuit in the discovery and development of M₁ antagonists. Modifications were made to the western aryl, core, and eastern aza-heterocycle regions of the VU0452865 scaffold, resulting in the development of potent and selective M₁ antagonists (Figure ).

2.

Timeline for the development of selective M₁ antagonists.

3.

SAR studies strategy to improve VU0452865.

Results and Discussion

Previously, our work toward the development of VU0452865 (3) featured a 4-pyridyl group in the eastern region of our scaffolds. Concerned over the presence of a nucleophilic 4-dimethylaminopyridine (DMAP)-like moiety in our scaffold, we synthesized the 2-pyridyl analog 6 (Figure ). While a 5-fold loss in potency was observed with this change (hM₁ IC₅₀ = 110 nM vs hM₁ IC₅₀ = 530 nM, respectively), both compounds display similar in vitro properties. Based on these results, we further explored structure–activity relationships (SAR) around 6.

The synthesis of compounds 6, 10, and 11 began with the coupling of corresponding commercially available carboxylic acids (7) with the commercially available 2-(piperidin-4-yl) pyridine (8) (Scheme A). Following a Boc-deprotection with TFA in DCM, azetidines 9 could undergo sulfonamide formation with isoquinoline-5-sulfonyl chloride to afford analogs 6, 10, and 11. For the synthesis of cyclobutyl analogs 15 and 16 (Scheme B), isoquinoline-5-sulfonyl chloride hydrochloride (12) was first treated with sodium sulfite and sodium bicarbonate to generate the corresponding sulfinate salt. The crude sulfinate salt was then reacted with cis or trans bromocyclobutanes 13 to give the corresponding sulfones. The ester moieties were then saponified to afford carboxylic acids 14, which were then coupled with piperazine 8 under standard HATU conditions to furnish analogs 15 and 16.

1. Synthesis of M₁ Antagonist Analogs 6, 10, 11, 15, 16, 20–24, and 28 .

^a Reagents and conditions: (a) HATU, DIPEA, DMF, 17 h; (b) TFA, DCM, 2 h, quant. over two steps; (c) sulfonyl chloride, DIPEA, DCM, 2 h, 22–70%; (d) Na₂SO₃, NaHCO₃ H₂O, DMSO, microwave irradiated at 120 °C, 1 h, then DMF, alkyl-bromide, Cs₂CO₃, 17 h; (e) NaOH, DMF, 4 h, 26–32% over three steps; (f) HATU, DIPEA, DMF, 2 h, 26–53%; (g) DIPEA, DCM, 17 h, 99% (h) NaOH, 1,4-dioxane, 17 h, 99%; (i) aza-heterocycle, HATU, DIPEA, DMF, 2 h, 34–95%; (j) HATU, DIPEA, DMF, 17 h, 36%; (k) TFA, DCM, 4 h, quantitative; (l) aryl sulfonyl chloride, DIPEA, DCM, 1–4 h, 20–98%; for 28a, perfluorophenyl quinoline-4-sulfonate, DIPEA, DCE, 50 °C, 1 h, 17%.

For the synthesis of cyclobutylsulfonamide (20–24, and 28), we deployed iterative parallel synthesis approaches starting from commercially available amines 18 (Scheme C) or carboxylic acid 25 (Scheme D). Briefly, aryl sulfonyl chloride 17 was reacted with 18 followed by saponification to obtain the carboxylic acid 19. The carboxylic acid was then coupled with various substituted aza-heterocycles to obtain the final compounds 20–24. Alternatively, 25 was subjected to HATU amide coupling conditions with aza-heterocycle 26, followed by subsequent Boc-deprotection with TFA to obtain the TFA salt 27. Finally, intermediate 27 was subjected to sulfonation conditions with the corresponding aryl sulfonyl chlorides to give analogs 28.

With final compounds in hand, analogs were screened against human M₁ (hM₁) in a calcium mobilization assay to determine their potency. Attempts to functionalize the azetidine ring were unsuccessful as both fluorination (10) and methylation (11) resulted in >3-fold reduction in potency (hM₁ IC₅₀ = 2,440 nM and 1,850 nM, respectively) (Table ). Next, we explored the effect of introducing cis–trans geometric isomerism. Unfortunately, attempts to replace the azetidine moiety with cyclobutane (compounds 15 and 16) or cyclobutylamine (compounds 20 and 21) groups resulted in weak M₁ antagonist activity (hM₁ IC₅₀ s > 10 μM).

1. Structure and M₁ Antagonist Activity of 6 and Its Analogs .

^aCalcium mobilization assays with hM₁-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 1–3 independent experiments in triplicate.

Assessment of historical SAR showed that VU0415248 (2) contains a quinoline-5-sulfonamide rather than the isoquinoline-5-sulfonamide found in VU0452865 (3). Based on these results, we synthesized quinoline-5-sulfonamide analogs of 22a and 22b (Figure ). While compound 22a displayed weak M₁ antagonism (hM₁ IC₅₀ > 10 μM), compound 22b displayed >3-fold improvement in potency for M₁ (hM₁ IC₅₀ = 138 nM) in comparison to VU0415248 (2) and VU0452865 (3). Additionally, compound 22b exhibited excellent selectivity for M₁ over M_2–5 (hM₂ IC₅₀ = 7.7 μM, hM_3–5 IC₅₀ > 10 μM). However, 22b was found to have high predicted hepatic clearance in rat (rCL_hep = 63.0 mL/min/kg).

4.

Impact of quinoline-5-sulfonamides on hM₁ potency and in vitro clearance.

To better understand the major metabolic liabilities associated with 22b to help direct SAR, a soft spot analysis was conducted in rat S9 fractions. While 59% of 22b remained unchanged after incubation, a total of five metabolites were identified (Figure ). Metabolite M467b, arising from the oxidation of the quinoline moiety was the most abundant (17%), followed by metabolite M515 (10%). Overall, the results indicated that oxidation of the 5-quinoline and 2-pyridylpiperazine moieties represented the primary source of metabolic liability, drawing our focus to these two regions of the molecule.

5.

Soft spot analysis of 22b in rat S9 fractions.

Based on the results of the soft spot analysis of 22b, we first attempted to functionalize or replace the piperazine ring with other aza-heterocycles to block metabolism of this moiety (Table ). Octa-deuteration of the piperazine ring (23a) resulted in a 6-fold loss in potency for M₁ (hM₁ IC₅₀ = 820 nM). Methylation of the piperazine ring (analogs 23b–e) was not tolerated, resulting in >8-fold loss in potency (hM₁ IC₅₀ s > 1.0 μM). While replacing the piperazine moiety with a piperidine (23f) also resulted in >20-fold loss in potency (hM₁ IC₅₀ = 3.2 μM), the piperazine isostere 3,7-diazabicyclo[3.3.0]­octane analog 23g showed excellent potency for M₁ (hM₁ IC₅₀ = 70 nM). However, like 22b, the predicted rat hepatic clearance for 23g was high (rCL_hep = 63.0 mL/min/kg). The 3,9-diazabicyclo[4.2.1]­nonane analog 23h was equipotent (hM₁ IC₅₀ = 150 nM) to 22b but also suffered from high predicted rat hepatic clearance (rCL_hep = 68.0 mL/min/kg). Additionally, 23h was screened against other mAChR subtypes, and the results (hM₂ IC₅₀ = 890 nM, hM₄ IC₅₀ > 10 μM) indicated that while a bridged ring system can serve as a bioisostere for the piperazine ring, it reduces selectivity against M₂ in this scaffold.

2. Structure and Activity of M₁ Antagonist Analogs (23a–h) with Replacement and Substitution of the Piperazine Core .

^aCalcium mobilization assays with hM₁-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 1–3 independent experiments in triplicate.

As functionalization and replacement of the piperazine ring did not afford any analogs with improved microsomal stability, we next focused our attention on exploring SAR around the 2-pyridine ring (Table ). While the 3-pyridine analog 24a displayed acceptable potency for M₁ (hM₁ IC₅₀ = 96 nM), its predicted hepatic clearance in rat was also high (rCL_hep = 57.0 mL/min/kg). Substitution on the 2-pyridine at either the 5- or 6-positions (24b-24e) was well tolerated, generating compounds with IC₅₀ s ≤ 105 nM. However, rat microsomal clearance remained high for all compounds in question (rCL_hep s ≥ 56.0 mL/min/kg). In this series, 24c stood out to us for its excellent potency for M₁ (hM₁ IC₅₀ = 5.8 nM). Additional substitution on the 24c pyridine ring system was explored as a method for improving metabolic stability. Methylation at either the 3- or 4- positions (24f and 24g) resulted in >50-fold loss in potency (hM₁ IC₅₀ s = 300 nM and 721 nM, respectively) with no improvement to rat hepatic clearance (rCL_hep s = 64.0 mL/min/kg and 60 mL/min/kg, respectively). Gratifyingly, methylation at the 6-position (24h) not only retained excellent potency (hM₁ IC₅₀ = 11 nM) but also modestly improved predicted rat hepatic clearance (rCL_hep = 41.0 mL/min/kg). Replacement of the 24h methyl group with a trifluoromethyl group (24i) maintained good potency (hM₁ IC₅₀ = 20 nM), while incorporation of an additional methyl group (24j) to 24h led to a 30-fold loss in potency (hM₁ IC₅₀ = 331 nM). Both transformations led to compounds with high predicted hepatic clearances in rat (rCL_hep s ≥ 59.0 mL/min/kg). Bicyclic ring systems mimicking the electronics of 24h were also explored. The 1,5-naphthyridine 24k showed weak potency for M₁ (hM₁ IC₅₀ > 10 μM), whereas the 1,8-naphthyridine 24l retained excellent potency for M₁ (hM₁ IC₅₀ = 10 nM). The predicted hepatic clearance in rat for 24l remained high (rCL_Hep = 59.0 mL/min/kg).

3. Structure and Activities of Selected M₁ Antagonist Analogs (24a–l) with Changes to the Eastern Aryl Terminus .

^aCalcium mobilization assays with hM₁-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 1–3 independent experiments in triplicate.

Encouraged by its excellent potency and improved predicated hepatic clearance in rat, we explored optimization of the 24h western aryl ring (Table ). Replacement of the 5-quinoline ring with either a 4-quinoline (28a) or other bicyclic ring systems (28b–d) was well tolerated with hM₁ IC₅₀ s < 150 nM; however, high predicted hepatic clearance in rat persisted (rCL_hep s ≥ 51.0 mL/min/kg). Substitution at the 8-position (28e) of the 5-quinoline ring of 24h with a methyl group led to >170-fold loss in potency (hM₁ IC₅₀ = 1.9 μM). On the other hand, fluorination at this same position (28f) proved more promising, generating a highly potent compound (hM₁ IC₅₀ = 71 nM) while displaying similar rat predicted hepatic clearance to 24h (rCL_hep = 43.0 mL/min/kg).

4. Structure and Activities of Selected M₁ Antagonist Analogs (28a–f), Exploing the Western Aryl Terminus of Compound 24h .

^aCalcium mobilization assays with hM₁-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 1–3 independent experiments in triplicate.

Intrigued by the results with 28f, we revisited a methyl scan of the piperazine core, (Table ). The synthesis of compounds 31a–d is outlined in Scheme , using the iterative parallel synthesis approaches described earlier. Methylation of the piperazine led to >5-fold loss in potency for 31a–c (hM₁ IC₅₀s = 390 nM–1,130 nM). However, this loss in potency was offset by a modest improvement in in vitro rat clearance (rCL_hep s = 34.0–38.0 mL/min/kg). Compound 31d was an outlier, showing ∼2-fold improvement in potency for M₁ (hM₁ IC₅₀ = 31 nM) compared to 28f while maintaining moderate predicted hepatic clearance in rat (rCL_hep = 40 mL/min/kg).

5. Structure and Activities of M₁ Antagonist Analogs (31a–d) with Methyl Substitution of the Piperazine Core .

^aCalcium mobilization assays in hM₁-CHO cells were performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 2–3 independent experiments in duplicate or triplicate.

2. Synthesis of M₁ Antagonist Analogs 31a–d .

^a Reagents and conditions: (a) DIPEA, DCM, rt, 17 h, 64%; (b) 1:1 TFA/H₂O, 100 °C, 1 h, 99%; (c) aryl piperazine, HATU, DIPEA, DMF, rt, 2 h, 35–47%.

Compounds with hM₁ IC₅₀ s < 100 nM and predicted rat hepatic clearance values of ≤ 40 mL/min/kg were progressed for additional studies, narrowing our focus to compounds 24h, 28f, and 31d. As 22b showed weak activity toward hM₂ (hM₂ IC₅₀ = 7.7 μM), we evaluated these three compounds against hM₂ (Table ). While compounds 28f and 31d showed excellent selectivity for hM₁ versus hM₂ (>140-fold selectivity), compound 24h was only 12-fold selective for hM₁ versus hM₂. Based on these data, compounds 28f and 31d were advanced for further in vitro and in vivo studies (Table ).

6. Counter Screening of M₁ Antagonists 24h, 28f, and 31d against hM₂ .

	VU6073552 (24h)	VU6074545 (28f)	VU6077564 (31d)
hM1 IC50 (nM)	11	71	31
hM2 IC50 (nM)	132	>10,000	>10,000
hM2/hM1	12	∼141	∼323

^aCalcium mobilization assays in hM₁-CHO and hM_2/Gqi5-CHO cells were performed in the presence of an EC₈₀ fixed concentration of acetylcholine, n = 2–3 independent experiments in duplicate or triplicate.

7. Additional In Vitro Pharmacology Characterization, In Vitro DMPK, and Rat In Vivo PK Profile of Analogs 28f and 31d .

	VU6074545 (28f)	VU6077564 (31d)
MW	508.6	522.6
xLogP	1.78	2.28
tPSA (Å)	119.3	119.3
hM1 IC50 (nM)	71	31
rM1 IC50 (nM)	50.9	39.5
hM1 KB (nM)	25	30
rM1 KB (nM)	22	12
hM2–5 (μM)	>10	>10
Rat in vitro PK parameters
rCLint (mL/min/kg)	114	94
rCLhep (mL/min/kg)	43.4	40.1
f u,plasma (rat)	0.020	0.019
f u,brain (rat)	0.036	0.031
MDCKII-MDR1 P-gp ER	48.0	46.6
Rat in vivo PK parameters
CLp (mL/min/kg)	17.6	71.7
V ss (L/kg)	0.32	27.6
Elim. t 1/2 (h)	0.33	35.2
K p	0.03	0.08
K p,uu	0.05	0.13

^a f _u = Fraction unbound; equilibrium dialysis assay; brain = rat brain homogenates.

^bMale Sprague–Dawley rats (n = 2); IV PK: 0.2 mg/kg, vehicle = 12% ethanol, 48% PEG400, 40% DMSO (0.5 mL/kg).

^c K _p = total brain to total plasma ratio.

^d K _p,uu = unbound brain (brain f _u × total brain) to unbound plasma (plasma f _u × total plasma) ratio.

Evaluation of 28f and 31d against the hM_3–5 subtypes revealed excellent selectivity profiles, with IC₅₀ s > 10 μM (Table ). Compounds 28f and 31d were then tested in progressive fold-shift (PFS) experiments with Schild analysis to investigate their pharmacological activity at M₁ in greater depth by determining their mechanism of action and affinity (K _B) for the M₁ receptor. These experiments demonstrated that 28f and 31d are reversible competitive antagonists (i.e., surmountable antagonism) and exhibit potent K _B values for human M₁ (hK_B = 25 nM and 30 nM, respectively) (Figure S1). Additionally, 28f and 31d were also tested at rat M₁ to assist in correlating data from rat models (rK_B = 22 nM and 12 nM, respectively) (Figure S2). Both compounds also showed modest free fraction in rat plasma (f _u,plasma ∼ 0.02) and rat brain homogenates (f _u,brain ∼ 0.031–0.036). When evaluated for rat in vivo pharmacokinetics (PK), 28f displayed low clearance (CL_p = 17.6 mL/min/kg), a short elimination half-life (t _1/2 ∼ 20 min), and low volume of distribution (V _ss = 0.32 L/kg). Compound 28f also displayed low CNS penetration (rat brain:plasma K _p = 0.03) and low CNS distribution of unbound drug (K _p,uu = 0.05), which is in agreement with the high efflux ratio (ER = 48.0) obtained from in vitro studies utilizing MDCKII-MDR1 transfected cells.

Compound 31d was found to have an in vitro-in vivo correlation (IVIVC) disconnect, displaying high in vivo clearance in rat (CL_p = 71.7 mL/min/kg) with a long elimination half-life of 35.2 h and high volume of distribution (27.6 L/kg). Compound 31d also demonstrated low CNS penetration and distribution of unbound drug (K _p = 0.08 and K _p,uu = 0.13), which is aligned with the high efflux ratio observed in vitro (ER = 46.6). The relatively high topological polar surface area (tPSA) of 119.3 Å for 28f and 31d may contribute to the limited brain penetration observed with both compounds.

While 31d displayed high in vivo clearance in rat, we were still encouraged by its excellent potency and selectivity for M₁. We explored the neuroprotective potential of 31d in neuropathic disease by studying its ability to elevate neurite outgrowth in cultured adult sensory neurons. We have previously shown that M₁ antagonists can prevent or reverse key features of peripheral neuropathy in rodent models of diabetes, including loss of sensory nerve terminals, thermal hypoalgesia, and slowed nerve conduction. For this experiment various concentrations of 31d were applied to adult rat DRG sensory neurons under defined conditions. This dissociated neuron culture system exhibits robust neurite outgrowth upon plating and the process of outgrowth and branching of axons is homologous to axonal sprouting that occurs in target tissues during innervation. Furthermore, this in vitro model permits the study of mechanisms of drug-induced axonal repair in response to disease states such as diabetes and chemotherapy treatment that trigger a distal dying-back of axons in the peripheral tissues. , Sensory neurons were treated for 42 h with a range of 31d concentrations in the presence of a low dose cocktail of neurotrophic factors (to provide optimal conditions for survival and growth and mimic the milieu in vivo) (Figure ). A medium dose cocktail of neurotrophic factors was added in a separate group and acted as an internal control to judge the level of response between independent experiments. An approximate 2-fold maximal induction of neurite outgrowth was observed at 1000 nM of drug compared with low dose growth factor treatment. A statistically significant 1.7-fold elevation of total neurite outgrowth was induced by 100 nM and 300 nM of 31d. The ED₅₀ for elevation of neurite outgrowth by 31d was determined as 55 nM (Figure S3), which aligns well with the rat functional potency and binding affinity (rat IC₅₀ = 39.5 nM and rat K _B = 12 nM). There was no effect on the survival of sensory neurons across the full range of concentrations of 31d.

6.

Effect of increasing doses of VU6077564 (31d) on neurite outgrowth of adult rat sensory neurons. Dissociated DRG sensory neurons were cultured for 42 h under defined conditions in the presence of a low dose cocktail of growth factors (LDGF). Neurons were exposed to a range of concentrations of VU6077564 (VU) and a separate group were treated with a medium dose cocktail of growth factors (MDGF) as an internal control. Neurons were fixed, stained for neuron-specific peripherin and total neurite outgrowth assessed. Fluorescent images from cultures are shown in (A) MDGF, (B) 0 nM drug (LDGF alone), (C) 100 nM VU6077564 and (D) 1000 nM VU6077564. Bar = 100 μm. In (E) is a bar chart showing data for total neurite outgrowth. Values are means ± SEM, n = 6 replicate cultures. Data analyzed by one-way ANOVA with Tukey’s post hoc test.* P < 0.05, ** P < 0.01, *** P < 0.001, # significantly different at P < 0.01 vs all groups except 1000 nM drug.

Conclusion

Overall, we identify VU6077564 (31d) as a lead compound within a new series of potent and highly selective cyclobutylsulfonamide-based M₁ antagonists. VU6077564 displays high in vivo clearance in rat and is peripherally restricted (K _p = 0.08, K _p,uu = 0.13, ER = 46.6). Herein, we demonstrate VU6077564 promotes neurite outgrowth in cultured adult DRG sensory neurons, further supporting the therapeutic potential of peripherally restricted and selective M₁ antagonists for therapy in axonal dying-back diseases of the PNS, such as diabetic neuropathy, chemotherapy-induced peripheral neuropathy, and HIV-neuropathy. Moreover, valuable lessons have been gleaned from this work that inform strategies to improve in vivo clearance, the details of which will be reported in due course.

Methods

General Information

All chemicals were purchased from commercial vendors and used without further purification. All NMR spectra were recorded on a 400 MHz AMX Bruker NMR spectrometer. ¹H and ¹³C chemical shifts are reported in δ values in ppm downfield with the deuterated solvent as the internal standard. Low resolution mass spectra were obtained on an Agilent 6120/6150 or Waters QDa (Performance) SQ MS with ESI source. High resolution mass spectra were obtained on an Agilent 6540 UHD Q-TOF with ESI source. Normal phase column chromatography was performed on a Teledyne ISCO CombiFlash Rf+ system. For compounds that were purified on a Gilson preparative reversed-phase HPLC, the system comprised of a 333 aqueous pump with solvent selection valve, 334 organic pump, GX 271 or GX-281 liquid hander, two column switching valves, and a 155 UV detector. Solvents for extraction, washing and chromatography were HPLC grade. All final compounds were found to be >95% pure by HPLC-MS analysis.

Synthesis. 2-Methyl-6-(piperazin-1-yl)­nicotinonitrile; 2,2,2-trifluoroacetate (26)

Step 1: To a solution of 6-chloro-2-methylnicotinonitrile (60 mg, 0.39 mmol) in MeCN (2.6 mL) was added tert-butyl piperazine-1-carboxylate (110 mg, 0.59 mmol) and DIPEA (0.137 mL, 0.79 mmol). After stirring at 80 °C for 17 h, the reaction mixture was cooled to rt, diluted with water, and extracted with DCM (3×). The combined organics were passed through a phase separator and concentrated. Purification via normal-phase column chromatography on silica gel (0–50% EtOAc/hexanes) afforded tert-butyl 4-(5-cyano-6-methylpyridin-2-yl)­piperaine-1-carboxylate as a white solid (110 mg, 93% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.66 (d, J = 9.0 Hz, 1H), 6.68 (d, J = 9.0 Hz, 1H), 3.75–3.67 (m, 4H), 3.55–3.48 (m, 4H), 2.52 (s, 3H), 1.48 (s, 9H). HRMS (Q-TOF, ES+): Calculated for C₁₆H₂₂N₄O₂ (M + H)⁺, 303.1816; Observed, 303.1822. Step 2: To a solution of tert-butyl 4-(5-cyano-6-methylpyridin-2-yl)­piperaine-1-carboxylate (100 mg, 0.33 mmol) in DCM (2 mL) was added TFA (0.215 mL, 2.81 mmol). After stirring for 17 h, the mixture was concentrated in vacuo and carried forward without further purification in quantitative yield. ¹H NMR (400 MHz, D₂O) δ 7.89 (d, J = 9.1 Hz, 1H), 6.89 (d, J = 9.1 Hz, 1H), 3.99 (dd, J = 5.4, 5.3 Hz, 4H), 3.39 (dd, J = 5.4, 5.3 Hz, 4H), 2.61 (s, 3H). HRMS (Q-TOF, ES+): Calculated for C₁₁H₁₄N₄ (M + H)⁺, 203.1291; Observed, 203.1296.

6-(4-((1s,3s)-3-Aminocyclobutane-1-carbonyl)­piperazin-1-yl)-2-methylnicotinonitrile 2,2,2-trifluoroacetate (27 )

Step 1: To a solution of 26 (107 mg, 0.33 mmol) in DMF (1.4 mL) was added cis-3-(tert-butyoxycarbonylamino)­cyclobutanecarboxylic acid (60 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (0.146 mL, 0.84 mmol). After stirring a rt for 17 h, the reaction mixture was syringe filtered and purified using RP-HPLC (20–60% ACN/0.5% aqueous NH₄OH) to give tert-butyl ((1s,3s)-3-(4-(5-cyano-6-methylpyridin-2-yl)­piperazine-1-carbonyl)­cyclobutyl)­carbamate as a white solid (40 mg, 36% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.68 (d, J = 8.9 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 4.06–3.94 (m, 1H), 3.77–3.63 (m, 6H), 3.61–3.55 (m, 2H), 3.12–3.04 (m, 1H), 2.56–2.47 (m, 5H), 2.17–2.06 (m, 2H), 1.43 (s, 9H). HRMS: (Q-TOF, ES+): Calculated for C₂₁H₂₉N₅O₃ (M+H)⁺, 400.2343; Observed, 400.2351. Step 2: To a solution of tert-butyl ((1s,3s)-3-(4-(5-cyano-6-methylpyridin-2-yl)­piperazine-1-carbonyl)­cyclobutyl)­carbamate (37 mg, 0.092 mmol) in DCM (0.5 mL) was added TFA (0.06 mL, 0.7835 mmol). After stirring at rt for 3 h, the mixture was concentrated in vacuo and carried forward without further purification in quantitative yield. ¹H NMR (400 MHz, D₂O) δ 8.02 (d, J = 9.5 Hz, 1H), 7.13 (d, J = 9.6 Hz, 1H), 3.93–3.73 (m, 9H), 3.45–3.31 (m, 1H), 2.73 (s, 3H), 2.70–2.61 (m, 2H), 2.44–2.32 (m, 2H). HRMS: (Q-TOF, ES+): Calculated for C₁₆H₂₁N₅O (M + H)⁺, 300.1819; Observed, 300.1827.

N-((1s,3s)-3-(4-(5-Cyano-6-methylpyridin-2-yl)­piperazine-1-carbonyl)­cyclobutyl)-8-fluoroquinoline-5-sulfonamide (28f)

To a solution of 27 (38 mg, 0.092 mmol) in DCM (0.5 mL) was added 8-fluoroquinoline-5-sulfonyl chloride (25 mg, 0.10 mmol) and TEA (0.04 mL, 0.58 mmol). After stirring at rt for 1.5 h, the reaction mixture was diluted with water and extracted with DCM (3×). The combined organics were passed through a phase separator and concentrated. Purification via RP-HPLC (20–60% ACN/0.5% aqueous NH₄OH) afforded an off-white solid (28 mg, 60% yield). ¹H NMR (400 MHz, (CD₃)₂CO) δ 9.15 (ddd, J = 8.8, 1.6 Hz, J _HF = 1.7 Hz, 1H), 9.07 (dd, J = 4.1, 1.6 Hz, 1H), 8.32 (dd, J = 8.3 Hz, J _HF = 5.0 Hz, 1H), 7.79 (dd, J = 8.8, 4.1 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.67 (dd, J = 8.3 Hz, J _HF = 9.9 Hz, 1H), 7.32 (d, J = 9.1 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 3.86–3.73 (m, 1H), 3.69–3.59 (m, 4H), 3.58–3.53 (m, 2H), 3.49–3.42 (m, 2H), 2.96 (tt, J = 9.8, 7.9 Hz, 2H), 2.48 (s, 3H), 2.26–2.13 (m, 1H), 2.03–1.93 (m, 2H). ¹³C NMR (101 MHz, (CD₃)₂CO) δ 172.11, 161.92, 161.87 (d, J _CF = 263.3 Hz), 159.84, 152.32 (d, J _CF = 2.1 Hz), 141.74, 139.73 (d, J _CF = 11.7 Hz), 134.26 (d, J _CF = 2.5 Hz), 134.09 (d, J _CF = 4.9 Hz), 131.04 (d, J _CF = 9.5 Hz), 126.79 (d, J _CF = 2.5 Hz), 124.58, 119.19, 113.08 (d, J _CF = 20.1 Hz), 104.73, 96.39, 45.22, 45.13, 44.87, 41.97, 34.79, 34.76, 23.84. HRMS (Q-TOF, ES+): Calculated for C₂₅H₂₅FN₆O₃S (M + H)⁺, 509.1766; Observed, 509.1772.

cis-3-[(8-Fluoroquinolin-5-yl)­sulfonylamino]­cyclobutane-1-carboxylic acid (30)

Step 1: To a solution of cis-methyl 3-aminocyclobutanecarboxylate hydrochloride (371 mg, 2.24 mmol) and DIPEA (1.8 mL, 10.18 mmol) in DCM (10 mL) in an ice bath was added 8-fluoroquinoline-5-sulfonyl chloride (500 mg, 2.04 mmol). The resulting reaction mixture was allowed to warm to rt and stirred for 17 h. The reaction was then quenched with saturated aqueous NaHCO₃ solution and extracted with DCM (2×). The combined organic layers were concentrated and purified by using normal-phase column chromatography on silica gel (10–100% EtOAc/hexanes) to provide cis-methyl 3-[(8-fluoroquinolin-5-yl)­sulfonylamino]­cyclobutane-1-carboxylate (440 mg, 64% yield) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.18 (ddd, J = 8.8, 1.6 Hz, J _HF = 1.6 Hz, 1H), 9.03 (dd, J = 4.3, 1.6 Hz, 1H), 8.31 (dd, J = 8.3 Hz, J _HF = 5.0 Hz, 1H), 7.82 (dd, J = 8.9, 4.3 Hz, 1H), 7.63 (dd, J = 8.3 Hz, J _HF = 9.9 Hz, 1H), 3.76–3.61 (m, 1H), 3.57 (s, 3H), 2.67 (tt, J = 10.0, 7.9 Hz, 1H), 2.22–2.14 (m, 2H), 1.96–1.86 (m, 2H). HRMS: (Q-TOF, ES+): Calculated for C₁₅H₁₅FN₂O₄S (M+H)⁺, 339.0809; Observed, 339.0814. Step 2: To cis-methyl 3-[(8-fluoroquinolin-5-yl)­sulfonylamino]­cyclobutane-1-carboxylate (170 mg, 0.5 mmol) was added water (1 mL) and TFA (1 mL, 13.06 mmol), and the mixture was stirred at 100 °C for 2 h. The solvent was removed in vacuo to afford a white powder that was carried forward without further purification 162 mg¹H NMR (400 MHz, CD₃OD) δ 9.18 (ddd, J = 8.8, 1.6 Hz, J _HF = 1.6 Hz, 1H), 9.03 (dd, J = 4.2, 1.5 Hz, 1H), 8.31 (dd, J = 8.3 Hz, J _HF = 4.9 Hz, 1H), 7.82 (dd, J = 8.9, 4.2 Hz, 1H), 7.63 (dd, J = 8.3 Hz, J _HF = 9.9 Hz, 1H), 3.68 (tt, J = 9.2, 7.5 Hz, 1H), 2.62 (tt, J = 10.0, 7.9 Hz, 1H), 2.32–2.06 (m, 2H), 1.97–1.84 (m, 2H). HRMS: (Q-TOF, ES+): Calculated for C₁₄H₁₃FN₂O₄S, (M + H)⁺, 325.0653; Observed, 325.0656.

(S)-2-Methyl-6-(3-methylpiperazin-1-yl)­nicotinonitrile; (2,2,2-trifluoroacetate)

Tert-butyl (2S)-2-methyl-1-piperazinecarboxylate (74 mg, 0.37 mmol) was dissolved in MeCN (1 mL). Cesium carbonate (71 mg, 0.73 mmol) and 6-fluoro-2-methylnicotinonitrile (50 mg, 0.37 mmol) were added sequentially, and the reaction mixture was refluxed for 17 h. The reaction mixture was diluted with water followed by extraction with DCM (2×). The combined organic layers were passed through a phase separator and concentrated. To the concentrated residue was added DCM (0.5 mL) and TFA (0.113 mL, 1.47 mmol). After stirring at rt for 2 h, solvents were removed in vacuo, and the sample carried forward without further purification as a brown solid (120.9 mg). ¹H NMR (400 MHz, CD₃OD) δ 7.76 (d, J = 8.9 Hz, 1H), 6.82 (d, J = 8.9 Hz, 1H), 4.66–4.55 (m, 2H), 3.48 (dt, J = 12.6, 2.8 Hz, 1H), 3.44–3.35 (m, 1H), 3.34–3.25 (m, 1H), 3.18 (td, J = 12.2, 3.3 Hz, 1H), 3.07 (dd, J = 14.4, 10.6 Hz, 1H), 2.55 (s, 3H), 1.39 (d, J = 6.6 Hz, 3H). HRMS (Q-TOF, ES+): Calculated for C₁₂H₁₆N₄ (M + H)⁺, 217.1448; Observed, 217.1448.

N-((1R,3s)-3-((S)-4-(5-Cyano-6-methylpyridin-2-yl)-2-methylpiperazine-1-carbonyl)­cyclobutyl)-8-fluoroquinoline-5-sulfonamide (31d)

To a mixture of intermediate 30 (20 mg, 0.062 mmol), (S)-2-methyl-6-(3-methylpiperazin-1-yl)­nicotinonitrile; (2,2,2-trifluoroacetate) (20 mg, 0.062 mmol), and HATU (28 mg, 0.074 mmol) in DMF (1 mL) was added DIPEA (0.035 mL, 0.25 mmol). After stirring at rt for 2 h, the mixture was syringe filtered and purified using RP-HPLC (15–70% ACN/0.5% aqueous NH₄OH) to give a white solid (15 mg, 47% yield). ¹H NMR (400 MHz, CDCl₃) (mixture of rotamers) δ 9.13–9.02 (m, 2H), 8.28 (dd, J = 8.3 Hz, J _HF = 4.9 Hz, 1H), 7.63 (dd, J = 8.7, 4.2 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.46 (dd, J = 8.3 Hz, J _HF = 9.4 Hz, 1H), 6.40 (d, J = 8.9 Hz, 1H), 6.20 (d, J = 9.6 Hz, 0.5H), 6.11 (d, J = 9.4 Hz, 0.5H), 4.84–4.72 (m, 0.5H), 4.48–4.34 (m, 1H), 4.24 (d, J = 13.4 Hz, 0.5H), 4.14 (d, J = 12.1 Hz, 0.5H), 4.06–3.90 (m, 1H), 3.79 (h, J = 8.2 Hz, 1H), 3.55–3.31 (m, 1H), 3.31–3.22 (m, 1H), 3.16–2.93 (m, 1.5H), 2.86 (dt, J = 17.7, 8.4 Hz, 1H), 2.57 (s, 3H), 2.39–2.26 (m, 2H), 2.19–2.00 (m, 2H), 1.13 (dd, J = 20.2, 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) (mixture of rotamers) δ 172.2 (d, J _CF = 39.6 Hz), 161.8, 161.4 (d, J _CF = 265.5 Hz), 158.8, 151.4, 141.2, 138.9 (d, J _CF = 12.0 Hz), 133.5, 132.1 (d, J _CF = 3.3 Hz), 130.3 (d, J _CF = 9.5 Hz), 126.1 (d, J _CF = 2.8 Hz), 123.8, 118.7, 112.2 (d, J _CF = 20.1 Hz), 103.2 (d, J _CF = 7.1 Hz), 96.3, 49.5, 48.3, 48.0, 46.1, 44.9, 44.7, 44.4, 40.4, 36.8, 34.8, 34.5, 34.4, 34.2, 30.7, 30.2, 23.7, 17.6, 16.0. HRMS (Q-TOF, ES+): Calculated for C₂₆H₂₇FN₅O₃S (M + Na)⁺, 545.1742; Observed, 545.1741

Molecular Pharmacology

Calcium Mobilization Assays

Compound-evoked decrease to an EC₈₀ concentration of acetylcholine (ACh) in intracellular calcium were measured using Chinese hamster ovary (CHO) cells stably expressing human or rat muscarinic receptors (M₁–M₅; M₂ and M₄ cells were coexpressed with chimeric G_qi5). The stable cells were cultured in F12 medium containing 10% fetal bovine serum, 20 mM HEPES, 100 units/mL antibiotics/antimycotic, 0.5 mg/mL G418, and 0.2 mg/mL hygromycin (M₂ and M₄ G_qi5 coexpressing cells only). All reagents used were from Life Technologies (Carlsbad, CA) unless otherwise noted. Briefly, the day before the assay, cells (15,000 cells/20 μL/well) were plated in black-walled, clear-bottomed, 384 well plates (Greiner Bio-One, Monroe, NC) in the culture medium without G418 and hygromycin, and then incubated overnight at 37 °C in the presence of 5% CO₂. The next day, calcium assay buffer (Hank’s balanced salt solution (HBSS), 20 mM HEPES, 2.5 mM Probenecid, 4.16 mM sodium bicarbonate (Sigma-Aldrich, St. Louis, MO)) was prepared to dilute compounds, agonists, and Fluo-4-acetomethoxyester (Fluo-4-AM), fluorescent calcium indicator dye. Compounds were serially diluted 1:3 or 1:5 into 10-point concentration response curves in DMSO using the Bravo Liquid Handler (Agilent, Santa Clara, CA), transferred to a 384 well daughter plates using an Echo acoustic liquid handler (Beckman Coulter, Indianapolis, Indiana), and diluted in assay buffer to a 2× final concentration. The agonist plates were prepared using acetylcholine (ACh, Sigma-Aldrich, St. Louis, MO) concentrations for the EC₂₀, EC₈₀, and EC_Max responses by diluting in assay buffer to a 5× final concentration. The 2× dye solution (2.3 μM) was prepared by mixing a 2.3 mM Fluo-4-AM stock in DMSO with 10% (w/v) pluronic acid F-127 in a 1:1 ratio in assay buffer. Using a microplate washer (BioTek, Winooski, VT), cells were washed with assay buffer 3 times to remove medium. After the final wash, 20 μL of assay buffer remained in the cell plates. Immediately, 20 μL of the 2× dye solution (final 1.15 μM) was added to each well of the cell plate using a Multidrop Combi dispenser (Thermo Fisher, Waltham, MA). After cells were incubated with the dye solutions for 45 min at 37 °C in the presence of 5% CO₂, the dye solutions were removed and replaced with assay buffer using a microplate washer, leaving 20 μL of assay buffer in the cell plate, and the cell plate allowed to incubate for 10 min at 37 °C. The compound, agonist, and cell plates were placed inside the Functional Drug Screening System 7000 (FDSS7000, Hamamatsu, Japan) to measure the calcium flux. After establishment of a fluorescence baseline for 2–3 s (2–3 images at 1 Hz; excitation, 480 ± 20 nm; emission, 540 ± 30 nm), 20 μL (2×) of test compound or vehicle was added to the cells, and the response was measured. 140 s later, 10 μL (5X) of an EC₂₀ concentration of ACh or vehicle was added to the cells, and the response of the cells was measured. Approximately 125 s later, an EC_80 or EC_Maxconcentration of ACh was added. Calcium fluorescence was recorded as fold over basal fluorescence and raw data were normalized to the maximal response to ACh. Compound-evoked decreases in calcium response in the presence of ACh EC₈₀ agonist were determined as inhibition activity, and potency (IC₅₀) and maximum inhibition responses (% ACh_Min) of compounds were determined using a four-parameter logistical equation using GraphPad Prism (La Jolla, CA) or the Dotmatics software platform (Woburn, MA)

$$y=\mathrm{bottom}+\frac{\mathrm{top}-\mathrm{bottom}}{1+{10}^{(\mathrm{logEC}50-A)\mathrm{Hillslope}}}$$

where A is the molar concentration of the compound; bottom and top denote the lower and upper plateaus of the concentration–response curve; HillSlope is the Hill coefficient that describes the steepness of the curve; and EC₅₀ is the molar concentration of compound required to generate a response halfway between the top and bottom.

Glossary

Abbreviations

AC

Adenylate cyclase

ACh

acetylcholine

AMPK

AMP-activated protein kinase

CLhep

hepatic clearance

CNS

central nervous system

DMAP

4-dimethylaminopyridine

DRG

dorsal root ganglia

DMPK

drug metabolism and pharmacokinetics

ER

efflux ratio

GPCRs

G protein-coupled receptors

hM1

muscarinic acetylcholine receptor subtype 1, human

hM2

muscarinic acetylcholine receptor subtype 2, human

K_B

binding affinity

mAChR

muscarinic acetylcholine receptor

nAChR

nicotinic acetylcholine receptor

MDGF

medium dose cocktail of growth factors

MLPCN

Molecular Libraries Screening Center Network

PK

pharmacokinetic

PNS

peripheral nervous system

rM1

muscarinic acetylcholine receptor subtype 1, rat

RRMS

relapsing-remitting multiple sclerosis

SAR

structure–activity relationship

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.6c00367.

• General methods for the synthesis and characterization of key compounds, experimental details for calcium mobilization assays, in vitro and in vivo DMPK protocols, soft spot analysis protocols, and adult rat DRG sensory neuron culture protocols (PDF)

T.I.B., E.S.C., and C.J.D. performed synthetic chemistry. T.I.B. and E.S.C. performed compound characterization. J.C.W., L.P., A.L.R., and H.P.C performed and analyzed molecular pharmacology. D.R.S. and P.F. designed, performed, and analyzed the cultured DRG neuron studies. S.C., S.S., S.K., I.Z.I., K.J.W., and O.B. performed and analyzed DMPK experiments. A.T.G. and C.K.J. performed in vivo pharmacology experiments. C.C.P. conducted the soft spot analysis. E.S.C., D.W.E., H.P.C., C.M.N., C.K.J., P.F., and C.W.L. oversaw experimental design. T.I.B. and E.S.C. drafted the manuscript with input from all authors.

P.F. is a cofounder and shareholder in WinSanTor Inc., which is performing human drug discovery work and clinical trials in the area of antimuscarinic drugs.

The authors declare no competing financial interest.