Explorations of Agonist Selectivity for the α9* nAChR with Novel Substituted Carbamoyl/Amido/Heteroaryl Dialkylpiperazinium Salts and Their Therapeutic Implications in Pain and Inflammation

Abstract

There is an urgent need for nonopioid treatments for chronic and neuropathic pain to provide effective alternatives amid the escalating opioid crisis. This study introduces novel compounds targeting the α9 nicotinic acetylcholine receptor (nAChR) subunit, which is crucial for pain regulation, inflammation, and inner ear functions. Specifically, it identifies novel substituted carbamoyl/amido/heteroaryl dialkylpiperazinium iodides as potent agonists selective for human α9 and α9α10 over α7 nAChRs, particularly compounds 3f, 3h, and 3j. Compound 3h (GAT2711) demonstrated a 230 nM potency as a full agonist at α9 nAChRs, being 340-fold selective over α7. Compound 3c was 10-fold selective for α9α10 over α9 nAChR. Compounds 2, 3f, and 3h inhibited ATP-induced interleukin-1β release in THP-1 cells. The analgesic activity of 3h was fully retained in α7 knockout mice, suggesting that analgesic effects were potentially mediated through α9* nAChRs. Our findings provide a blueprint for developing α9*-specific therapeutics for pain.

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) are cation-selective, ligand-gated ion channels (LGICs) that mediate an exceptionally diverse array of physiologic processes, including fast neurotransmission in the peripheral nervous systems (at the skeletal neuromuscular junction and in the autonomic nervous system) and modulation of the synaptic function in the central nervous system, as well as immunomodulatory functions in peripheral tissues.^1,2

Functional nAChRs as LGIC result from the assembly of either five identical or different subunits, giving rise to homomeric or heteromeric pentamers, respectively.³ Neuronal nAChRs are formed from among nine identified α (α2 to α10) and three β (β2 to β4) subunits.⁴ It has been proposed that the nAChR function can be modulated to treat various nervous system disorders, such as Alzheimer’s disease, schizophrenia, depression, attention deficit hyperactivity disorder (ADHD), and tobacco addiction,^5,6 as well as chronic pain,⁷ Parkinson’s disease,⁸ and hearing disorders.⁹ Much of the work regarding nAChRs as therapeutic targets has focused on the subtypes expressed at high levels in the brain, heteromeric receptors containing α4 and β2 subunits, and homomeric α7-containing receptors. However, the identification of nAChR expression in a variety of immune cells has provided evidence for a cholinergic anti-inflammatory system (CAS)^10,11 that modulates inflammatory disease and neuropathic pain. This discovery has promoted a new direction for the development of pain therapeutics. In addition to their canonical ionotropic functions, noncanonical, flux-independent, and exclusively metabotropic functions have been proposed for nAChRs and other ligand-gated ion channels. The function of nAChRs in the CAS may rely more on metabotropic than ionotropic signaling¹²⁻¹⁴ and the receptor subunits most strongly implicated as targets are α7 and the less-well-understood α9 and α9α10 receptor subunits,^15,16 which until recently have only been associated with auditory function.^17,18 The α9 subunits are known to combine with α10 subunits to form α9α10 nAChRs with kinetic properties slightly different from homomeric α9 nAChRs, with a likely (α9)₂(α10)₃ stoichiometry.¹⁹

The heteromeric nAChRs on hair cells contain α9 and α10 subunits and have several distinguishing characteristics.²⁰ The α9α10 receptor features antagonism by nicotine, which, as noted above, typically activates nAChRs, and potent block by strychnine and bicuculline, which are also antagonists of glycine and GABA receptors, respectively.²¹ The α9α10 nAChRs are among the most calcium-permeable LGICs known,²² although their endogenous ion channel activity has only been recorded in cochlear and vestibular hair cells. However, expression of α9α10 has been described in dorsal root ganglion neurons,^23,24 lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary gland.²⁵ This widespread distribution of α9* nAChRs may be associated with diverse physiological roles for these receptors in neuronal, sensory, metabolic, and immune tissues.¹⁸ The α9 and α10 subunits share homology with other nAChRs, yet are structurally and pharmacologically distinct, having the lowest degree of sequence similarity with other nAChRs, making them a promising target for developing selective drugs. In addition to potentially modulating CAS, compounds that target α9 and α9α10 may be useful for treating various hearing disorders, such as noise-induced hearing loss or the debilitating disorders, vertigo, or tinnitus.¹⁸

We have recently shown that compounds previously identified as silent agonists of α7, with potential metabotropic activity, could, based on specific structural epitopes, function as potent α9 agonists or antagonists.²⁶ We confirmed that one of the α9 agonists was an effective inhibitor of the ATP-induced maturation and release of the pro-inflammatory cytokine interleukin (IL)-1β in a cell-based assay.²⁷ In the present study, we extend our evaluation of potential agonists of α9 and α9α10 receptors, relative to their activity for α7 receptors. We began the study with two reference compounds (Figure 1) that we recently reported as α9 agonists: 4-(4-carbamoylphenyl)-1,1-diethylpiperazin-1-ium iodide (1, p-CONH2 diEPP) and 1,1-diethyl-4-(4-((6-methylpyridin-2-yl)-carbamoyl)phenyl)piperazin-1-ium iodide, (2, APA-diEPP), which is a derivative of the simpler amide 1. 1 was a full α9 agonist with 66-fold greater potency for α9 than for α7. The carboxamide derivative 2 was also a full α9 agonist with 10-fold greater potency for α9 than for α7. Thus, α9 tolerated the addition of the relatively bigger picolyl group to the amide; in our prior work, we discussed an extended binding pocket (shown in Figure 1A) on the (−) face of the α9 subunit interface.²⁶

Figure 1.

(A) α9 homopentamer binding site in the extracellular domain with a zoomed-in view of the orthosteric binding site with compound 1 bound at the (+)α9(−)α9 interface. The extended binding pocket is shown by the area under the dotted square. Structures of previously characterized phenylpiperaziniums 1 and 2 used as reference compounds for this study. (B) APA-diEPP (2) showing enhanced α9 selectivity over α7 applications on cells expressing α7 and α9 with the average normalized responses. (C) Structural variation explored in the present study.

Building on the success of compound 2 as an α9 agonist, we aimed to create new compounds selectively targeting α9* receptors, while assessing the design constraints for this class. We synthesized a focused compound library around compounds 1 and 2 (Figure 2). We explored how altering the amide’s hydrogen bonding ability (3a–l, Figure 2), its bioisosteric replacement with heterocylces (3j–l, Figure 2), and the nature of substituents on amide, i.e., alkyl or pyridyl and phenyl amides (3a–f, 3g–i, Figure 2) could influence activity for α9 vs α9α10 or α7 nAChR receptors. Given that we were varying the size of the group on the amide nitrogen, we also wished to determine if smaller groups on the ammonium nitrogen might have compensatory, synergistic, or antagonistic effects when a large amide group was part of the molecule. We also aimed to reverse the amide linkage in the potent analogs to create reverse amide analogs (Figure 2), thus examining the effect of altering the distance of the hydrogen bond donor to the binding site residues. Our core hypothesis posits that the phenylpiperazine scaffold can be successfully optimized for α9-containing receptor (α9*) selectivity, and such probes could offer new insights into nAChR’s roles in auditory, analgesic, and anti-inflammatory processes. To address these questions, we therefore synthesized the new set of compounds (Figure 2).

Figure 2.

Structures of novel phenylpiperaziniums synthesized in this study with their systematic variations from the precursor molecules. All the compounds are in the iodide form of the ammonium salt. The dotted arrows point toward the new modified molecules with respect to its precursor 3a–3l.

2. Results and Discussion

2.1. Chemistry

The general approach used for the synthesis of N,N-dialkyl-4-(substituted aryl/alkyl)carbamoyl)phenyl)piperazinium iodides (3a–f) and the reverse amide analogs (3g), (3h,i), and 4-(4-(heteroaryl)phenyl)-N,N-dialkyl piperazinium iodides (3j–l) is depicted in Schemes 1–4. Amidation of p-bromo ethylbenzoate 4 (Scheme 1) formed 4-bromo-N-(aryl)benzamides (6a–e) by acyl substitution with amines (5a–e); these reactions proceeded in excellent 90–95% yield at room temperature. This transition-metal-free amidation was carried out using alkyl or arylamines (2.0 equiv), lithium hexamethyldisilazide (LiHMDS) 3.0 equiv as a base, and tetrahydrofuran as a solvent at ambient conditions in the presence of argon. It is noteworthy that the reactions proceeded rapidly and in high yields. The second step was the key step in the synthesis, i.e., the C–N cross-coupling reaction for N-arylation of ethylpiperazine (7a) or methylpiperazine (7b) utilizing 4-bromo-N-(aryl)benzamides (6a–e), leading to the formation of 4-(4-ethylpiperazin-1-yl)-N-alkyl/aryl benzamides (8a–e). As previously described²⁶ Buchwald–Hartwig C–N cross-coupling reactions were superior to Ullman chemistry,²⁸ when the coupling targets included amide functionality. Initially, we used palladium diacetate (10 mol %) and (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl) ligand (BINAP) (20 mol %) in toluene. The reaction failed to complete, even after 4 days, leading to exceptionally low yields. We also employed tetrakis(triphenylphosphine)palladium(0) [palladium tetrakis] (10 mol %) instead of Pd(OAc)_2, using sodium tertiary butoxide (2 equiv) as a base in toluene; however, it ended up with a number of impurities that could not be separated even by column chromatography. Finally, the reaction utilizing 8–10 mol % of tris(dibenzylideneacetone)dipalladium⁰ (Pd₂(dba)₃) in cesium carbonate (2.0 equiv) as a base and BINAP (10–20 mol %) in 1–2 mL of dioxane afforded a good yield (60–78%) of the coupled product after purification. Once obtained, the 4-(4-ethylpiperazin-1-yl)-N-alkyl/aryl benzamides (7a–e) were then converted into the quaternary ammonium salts by alkylation with ethyl iodide or methyl iodide in tetrahydrofuran and then purified by precipitation to afford the N,N-dialkyl-4-(substituted aryl/alkyl) carbamoyl)phenyl)piperazinium iodides (3a–f).

Scheme 1. Synthesis of 1,1-Dialkyl-4-(substituted aryl/alkyl)carbamoyl)phenyl)piperazinium Iodides (3a–f).

Reagents and conditions: (a) Lithium bis(trimethylsilyl)amide (LiHMDS) [1.0 M in THF] (3.0 equiv), r.t, 24 h, under Ar; (b) Tris(dibenzylideneacetone)dipalladium(0) (8–10 mol %), cesium carbonate (2.0 equiv), (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl) BINAP (20 mol %), 7a; N-ethylpiperazine or 7b; N-methylpiperazine (4 equiv), dioxane, 8 h, 90 °C. (c) 9a; iodoethane or 9b; iodomethane, THF, 25 °C, 2–24 h. Details provided in Experimental Section and Supporting Information.

Scheme 4. Scheme for the Synthesis of 4-(4-(Heteroaryl)phenyl)-1,1-diethylpiperazinium Iodides(3j–l).

Reagents and conditions: (a) [Pd₂(dba)₃] (10 mol %, cesium carbonate (2.0 equiv), BINAP (30 mol %), N-ethylpiperazine, dioxane (2 mL), 48 h, 120 °C. (b) Iodoethane, THF, 25 °C, 48 h. Details are provided in the Experimental Section and Supporting Information.

In Scheme 2, for making reverse amide analog 3g, 4-bromoaniline (10) was made to react with methyl 6-methylpicolinate (11) in the presence of lithium hexamethyldisilazide (LiHMDS) to yield N-(4-bromophenyl)-6-methylpicolinamide (12), which was further reacted under Buchwald–Hartwig amination to yield N-(4-(4-ethylpiperazin-1-yl)phenyl)-6-methylpicolinamide (13). However, reverse amide analog in this case ended up with lower yield (38%). The coupled compound was further treated with iodoethane to yield 1,1-diethyl-4-(4-(6-methylpicolinamido)phenyl)piperazin-1-ium iodide (3g).

Scheme 2. Scheme for the Synthesis of 1,1-Diethyl-4-(4-(6-methylpicolinamido)phenyl)piperazin-1-ium Iodide (3g).

Reagents and conditions: (a) Lithium bis(trimethylsilyl)amide LiHMDS [1.0 M in THF] (3.0 equiv), r.t, 24 h, under Ar; (b) [Pd₂(dba)₃] (8 mol %), cesium carbonate (2.0 equiv), BINAP (20 mol %), 7a; N-ethylpiperazine (4 equiv), THF, 8 h, 98 °C. (c) 9a; iodoethane, THF, 25 °C, 36 h. Details are provided in the Experimental Section and Supporting Information.

Owing to the lower yield of reverse amide in Scheme 2, we changed the synthetic route for 3h and 3i (Scheme 3). We started with 4-nitroiodobenzene. We first coupled 4-nitroiodobenzene (14) with methyl piperazine 7b using Buchwald–Hartwig amination to yield 1-methyl-4-(4-nitrophenyl)piperazine (15) in 82% yield, followed by the reduction of nitro functionality using Raney Ni and hydrogen gas in methanol/tetrahyrofuran leading to the formation of 4-(4-methylpiperazin-1-yl)aniline (16) in 91% yield. 16 was then utilized for the aminolysis of esters 11 and 17 using LiHMDS in THF yielding amides 18 and 19, which were then alkylated using iodoethane to yield the desired piperazinium reverse amide salts 3h and 3i.

Scheme 3. Scheme for the Synthesis of 4-(4-Aryl/Alkylamidophenyl)-1-ethyl-1-methylpiperazin-1-ium Iodides (3h, 3i).

Reagents and conditions: (a) [Pd₂(dba)₃] (4 mol %), cesium carbonate (1.0 equiv), BINAP (10 mol %), 7b; N-methylpiperazine (4 equiv), THF, 3h, 98 °C. (b) Raney Ni/H₂, Methanol/THF (c) LiHMDS [1.0 M in THF] (3.0 equiv), r.t., 24 h, under Ar; (d) 9a; iodoethane, THF, 25 °C, 36 h. Details are provided in Experimental Section and Supporting Information.

For making bioisosteres of carboxamide moiety, a procedure similar to Scheme 1 (Scheme 4) was adopted for synthesis of N,N-diethyl-4-heteroarylphenyl)piperazin-1-ium iodides (3j–l), utilizing N-(4-heteroarylphenyl)-N′-ethylpiperazines (21a–c) with some modifications as discussed in the Experimental Section. The formation of the final products was confirmed by spectroscopic techniques. The formation of compounds 3a–l was confirmed through ¹H NMR spectroscopy. A triplet for six protons and a quartet of four protons corresponding to 2 methyl groups and 2 methylene groups, respectively, confirmed the desired transformation into N,N-diethyl salts for the compounds 3a–l.

2.2. Electrophysiology

Xenopus oocytes were injected with RNAs to produce functional nAChR, which were characterized by two-electrode voltage-clamp measurements. Figures 3–6 present concentration–response curves (CRCs) for the new compounds tested with human α9 and α9α10 vs α7 receptors; Table 1 presents the values for I_max and EC₅₀ with these receptors for new [(1,1-dialkyl-4-(substituted aryl/alkyl)carbamoyl) phenyl)piperazinium iodides (3a–f)] with α9-containing and α7 nAChR; Table 2 shows the corresponding values of reverse amides (3g–i), while Table 3 tabulates the corresponding values of aromatic carboxamide isosteres (3j–l) in comparison to carboxamide bearing diEPP scaffold 1. and Table 5 summarizes inhibition data for the compounds with heteromeric nAChR.

Figure 3.

Concentration–response curves for compounds 1 and 3a–3d with α7, α9, and α9α10 nAChR expressed in Xenopus laevis oocytes. The data for reference compound 1 (taken from Papke et al. 2022²⁶). The test compound applications were alternated with control applications of 60 μM ACh to confirm the stability of the control ACh responses. Experiment responses were measured relative to the preceding ACh control responses and then normalized relative to the ratio of the ACh controls to the ACh maximum (0.63) as determined in previous experiments.^26,30 Each point is the average (n ≥ 5 ± SEM). Experimental data were fit to the Hill curve as described in the Experimental Section.

Figure 6.

On the left, compound 3h docking within the α9(+)α9(−) interface. Compound 3h (depicted in green) is represented in stick mode, revealing its preferred conformation within the orthosteric binding site on the extracellular domain. α9(+) is illustrated in pink cartoon and α9(−) in cyan. On the right, a zoomed-in view presents a 3D ligand interaction diagram, with ligand interactions denoted by dashed lines. π-Cation interactions are depicted in magenta, salt bridge interactions in red, and hydrogen bond interactions in green.

Table 1. I_max and EC₅₀ Values for Reference and New Agonists (1,1-Dialkyl-4-(substituted aryl/alkyl)carbamoyl)phenyl)piperazinium Iodides (3a–f)) with α9-Containing and α7 nAChR.

^aI_max values are presented relative to ACh (I_max = 1).

^bEC₅₀ values are in units of μM. See Experimental Section for details. *Data on 2 are taken from Papke et al. 2022.

Table 2. I_max and EC₅₀ Values of Normal Amide 3f vs Reverse Amide Analogs (3g–i) on α9-Containing and α7 nAChR.

^aI_max values are presented relative to ACh (I_max = 1).

^bEC₅₀ values are in units of μM. See Experimental Section for details.

Table 3. I_max and EC₅₀ Values of Compound 1 vs Isosteres Containing Heterocyclic Analogs (3j–i) with α9-Containing and α7 nAChR.

^aI_max values are presented relative to ACh (I_max = 1).

^bEC₅₀ values are in units of μM. See Experimental Section for details.

Table 5. Effects of 3 μM Co-Application on the Control ACh (30 μM for α4β2 and α1β1εδ and 100 μM for α3β4 Receptors) Responses^a.

compound	α3β4	α4β2	α1β1εδ
3a	0.849 ± 0.034	0.919 ± 0.022	0.541 ± 0.048
3b	0.650 ± 0.039	0.946 ± 0.032	0.709 ± 0.086
3c	0.789 ± 0.057	0.697 ± 0.027	0.506 ± 0.082
3d	1.039 ± 0.015	0.807 ± 0.048	0.555 ± 0.060
3e	0.838 ± 0.037	0.912 ± 0.038	0.709 ± 0.091
3f	0.849 ± 0.018	0.858 ± 0.045	0.413 ± 0.070
3g	0.415 ± 0.054	1.066 ± 0.099	0.380 ± 0.070
3h	0.659 ± 0.056	1.047 ± 0.064	0.558 ± 0.026
3i	0.798 ± 0.047	1.191 ± 0.07	0.409 ± 0.050
3j	0.507 ± 0.032	0.823 ± 0.043	0.353 ± 0.071
3k	0.736 ± 0.034	0.913 ± 0.026	0.590 ± 0.062
3l	0.387 ± 0.057	0.709 ± 0.086	0.691 ± 0.094

^aThese data are the normalized responses to coapplications of the test compounds with ACh to the initial control responses to ACh alone They therefore have no units (current/current).

Electrophysiology results (Tables 1–3) show that almost all the new compounds except 3e and 3l are full agonist of α9 nAChRs with varying degrees of potency.

2.2.1. Structure–Activity Relationships

The pCN-diEPP compound was recently shown to be a full agonist with an EC₅₀ value of 0.368 ± 0.10 μM at homomeric α9 nAChRs and also a full agonist at heteromeric α9α10 nAChR with an EC₅₀ of 2.57 ± 0.57 μM with an I_max of 0.94 ± 0.06 that of ACh.²⁹ It behaves as a partial agonist at α7 nAChR. However, this compound was only 7-fold selective for homomeric α9 nAChRs over heteromeric α9α10 nAChR. The p-CONH₂ diEPP, 1, behaves as a potent agonist (EC₅₀ = 1.15 μM; I_max = 0.80 ± 0.03) (Table 1) at α9 nAChR. It behaves as a full agonist at α9α10 nAChR but with 11-fold reduced potency. It behaves as a weak partial agonist at α7 nAChR (EC₅₀ = 76 μM; I_max = 0.34 ± 0.03) (Table 1). The p-CONH₂ diEPP, 1, was selected as a lead for the current investigation over pCN-diEPP as it was more selective (11-fold) for homomeric α9 nAChRs, and it provides additional opportunities for further functionalization at the amide function to fine-tune the activity and selectivity at these three receptor subtypes. The significance of amide function was clear in our first analog compound 3a bearing the pyrrolidinyl ring with both hydrogen bond donors removed. It behaved as a partial agonist (I_max = 0.69 ± 0.09) with significantly reduced potency (EC₅₀ = 45 μM) at α9 nAChR (Table 1). The compound also exhibited reduced potency and efficacy at heteromeric α9α10 nAChR (EC₅₀ = 15 μM; I_max = 0.56 ± 0.04) with negligible activity at homomeric α7 nAChR (Table 1 and Figure 3). Compound 3b, retaining one of the H-bond donors and bearing an n-propyl substitution, significantly brought back the activity at both α9 nAChR and α9α10 nAChR. It behaved as a full agonist with an EC₅₀ value of 9 ± 6 μM and I_max = 1.13 ± 0.15 (Table 1) and a partial agonist at α9α10 nAChR with an EC₅₀ value of 11 ± 6 μM, indicating that keeping at least one NH for such pharmacophores may be important for α9* and α7 efficacy. Compound 3b bearing the lipophilic N-propyl group and exhibiting higher efficacy at α9 nAChR hinted toward the possibility of having a lipophilic secondary pocket at α9* nAChR that could be probed further with the lipophilic aliphatic group or alicyclic/aromatic rings. The APA-diEPP, 2 (Table 1; taken from Papke et al., 2022), bearing a 2-picolinyl ring, though α9-selective over α7, exhibited a significant boost in potency and efficacy across all three nAChR subtypes studied relative to p-CONH-diEPP (1). It was an efficacious and potent full agonist at homomeric α9 nAChR (EC₅₀ = 0.66 ± 0.05 μM) with I_max of 1.06 ± 0.02 and at heteromeric α9α10 nAChR (EC₅₀ = 0.98 ± 0.09 μM with I_max of 0.9 ± 0.02). It also exhibited significantly improved potency and efficacy at α7 nAChR (EC₅₀ = 6.4 ± 1.4 μM and I_max of 0.42 ± 0.02) as compared to compounds 1, 3a, and 3b; therefore, we shifted our attention and preference to aromatic analogs.

Figures 3 and 4 present data for the CRCs for compounds 3a–d and 2 and 3e–3i, respectively, with the values for I_max and EC₅₀ presented in Table 1. From a qualitative perspective, it is evident that, with the exception of compound 3e, all members of the alkyl/aryl carbamoyl series (3b–f) exhibit enhanced efficacy, potency, and selectivity for the α9 and α9α10 receptors when compared to the α7 receptor. Notably, compound 3f, which serves as an analog of 2, functions as a full agonist for α9 and α9α10 receptors, while displaying only weak partial agonist activity on the α7 receptor. Remarkably, it demonstrates about 12-fold selectivity for α9 and α9α10 receptors over α7, with EC₅₀ values of 510 nM (I_max of 1.19 ± 0.03) and 480 nM (I_max of 0.92 ± 0.04, for α9 and α9α10 receptors, respectively.

Figure 4.

Concentration–response curves for compounds 2 and 3e–3i with α9 and α9α10 vs α7 nAChR. The test compound applications were alternated with control applications of 60 μM ACh to confirm the stability of the control ACh responses. Experiment responses were measured relative to the preceding ACh control responses and then normalized relative to the ratio of the ACh controls to the ACh maximum (0.63), as determined in previous experiments.^26,30 Each point is the average (n ≥ 5 ± SEM). Experimental data were fit to the Hill curve as described in the Experimental Section.

2.2.2. Nature of the Arylamide

Compound 3c was employed to assess the importance of the methyl group on the pyridine ring, while compound 3d retained the methyl group but eliminated the pyridyl nitrogen, thereby providing insights into the significance of a pyridine ring compared to a benzene ring. Analysis of CRCs for compound 3c (Figure 3) has unveiled a noteworthy trend. Specifically, with regards to 3c, a potent full agonist for α9 and α9α10 nAChRs, a distinct shift toward higher potency for α9α10 over α9 nAChRs, has been observed. Among the carbamoyl series 3a–f, all bearing a pyridyl substitution on the carboxamide moiety, compound 3c emerges as the most selective for α9α10 nAChR. Notably, 3c exhibits a 15-fold decrease in potency on α9 nAChR compared to a 22-fold decrease on α7 nAChR (Table 1 and Figure 3). This observation suggests the possibility that its binding affinity to the agonist binding sites at α9α10 interfaces is stronger than its interaction with homomeric α9 nAChR. Consequently, it raises the intriguing possibility of pharmacological discrimination between α9 and α9α10 receptors through variations in the size and substituents of the arylamide aryl ring. Compound 3d, a structural analog of compound 2 in which the pyridine ring is substituted with a phenyl ring, acts as a full agonist for both α9 and α9α10 receptors, yet remains a potent partial agonist for α7 nAChR. Intriguingly, compound 3d demonstrates increased potency for α7 when compared to its homologues in the 3a–f series, thereby decreasing selectivity for α9* nAChR. This suggests that the presence of pyridyl nitrogen is pivotal for selectivity toward α9* nAChR over α7 nAChR. Additionally, the data imply that hydrophobic electron-rich aromatic rings as in 3d may enhance the agonistic binding affinity for the α7 receptor.

Focusing on the relative potencies on α9 homomeric receptor, compound 3f, an N-ethyl, N-methyl analog of compound 2, emerged as the most potent full agonist among 3a–3f. It displayed an EC₅₀ value of 0.51 ± 0.07 μM and a maximum response (I_max) of 1.19 ± 0.03, as tabulated in Table 1. A remarkable ∼51-fold increase in potency compared to acetylcholine (ACh) and a 2.25-fold increase relative to compound 1 on the α9 nAChR were observed. Conversely, removing the 6-methyl group on the pyridyl ring of compound 2 substantially attenuated the potency of compound 3c, resulting in a 27-fold decrease, as indicated by its EC₅₀ of 18.36 ± 36.2 μM (see Table 1 and Figure 3). Interestingly, retaining the methyl group while replacing the pyridyl ring with a phenyl ring (compound 3d) restored receptor activation and potency, yielding an EC₅₀ value of 1.25 ± 0.14 μM. This potency was either comparable to or slightly less than that of p-CONH2-diEPP (compound 1). However, the absence of the pyridyl nitrogen in 3d resulted in a potency reduced to half that of compound 2 (APA-diEPP).

Regarding potency on a heteromeric α9α10 receptor, from 3a–3f, compound 3f again emerged as the most potent agonist (EC₅₀ = 0.48 ± 0.09 μM) to its activity on the α9 homomer. This compound exhibited full agonism and was 27-fold and 62-fold more potent than compounds 1 and ACh, respectively (refer to Table 1 and Figure 3). In contrast to its behavior with the α9 homomer, compound 3d—characterized by an electron-rich phenyl ring—demonstrates enhanced potency on the α9α10 heteromer, registering an EC₅₀ value of 0.61 ± 0.08 μM and an I_max of 1.12 ± 0.03 (as delineated in Table 1 and Figure 3).

All analogs from 3a–f exhibited characteristics of either partial or weak partial agonism toward the α7 nAChR. Of particular note, compound 3d, distinguished by its meta-tolyl ring, demonstrated the highest partial agonistic activity toward α7 nAChR (I_max of 0.6 ± 0.02) and the highest potency with an EC₅₀ value of 1.34 ± 0.1 μM. It, however, rendered it the least selective across the three nAChR subtypes under study (Table 1 and Figures 3 and 4).

2.2.3. Effects of Steric Bulk on the Ammonium Group

Compound 3f diverges from its parent compound 2 by the substitution of an ethyl group with a methyl group at the ammonium nitrogen, thus decreasing steric bulk in this region. This alteration yielded a modest increase in potency toward the α9 nAChR subtype (0.51 ± 0.07 μM vs 0.66 ± 0.05 μM) and approximately doubled the potency against the α9α10 nAChR subtype (0.48 ± 0.09 μM vs 0.98 ± 0.09 μM). Efficacy-wise, compound 3f functioned as a full agonist for α9 and was nearly as efficacious for α9α10, with I_max values of 1.19 ± 0.03 and 0.92 ± 0.04, respectively. Notably, this enhancement in potency did not extend to the smaller dimethyl analog, compound 3e. For this variant, potencies toward the α9 homomeric receptor subtype declined approximately 10-fold and about 4-fold toward the α9α10 heteromeric receptor, even though 3e maintained its full agonistic activity for α9α10 but acted as a partial agonist for α9 nAChR.

Interestingly, the α7 subtype exhibited a surprising indifference to these structural modifications at the ammonium moiety. The I_max and EC50 values for α7 were largely invariant, falling within the ranges of 0.40–0.57 and 4.3–6.4 μM, respectively, across compounds 2, 3f, and 3e. This result suggests that substitution at the ammonium nitrogen may be a useful way to tune for α9α10 selectivity. However, it should be noted that the relative insensitivity of the α7 responses to steric bulk in this domain was somewhat unexpected since an earlier study with simple piperidinium compounds³¹ showed a strong effect of increased size of groups on the ammonium, reducing efficacy for α7 channel activation. It was reported that diM-piperidinium (an analog of 3e) was essentially a full agonist of α7, while diE-piperidinium (an analog of 3d) was an α7-silent agonist, giving responses only when coapplied with the α7-positive allosteric modulator (PAM) PNU-120596).³¹ Although the core pharmacophore for all these compounds is the amine on the piperidinium, the addition of bulk to the amine in the form of two ethyl groups converted both diE-Pip and diEPP to silent agonists. However, our data indicate that specific substitutions on the phenyl group of the diEPP compounds can have a big impact on α7 efficacy, suggesting that these distant groups may reposition the amine in the binding pocket in such a way as to restore efficacy.

To assess the functional impact of normal amide versus reverse amide linkages on the activity of compound 3f toward the α9* receptor, we synthesized three more analogs 3g–3i (Table 2). These analogs serve as structural mimics of the lead compound 3f. Our objective was to evaluate whether these structural changes would yield different hydrogen-bonding interactions, thereby influencing potency, efficacy, and selectivity. Specifically, the compounds 3g and 3h are the diethyl phenyl piperazinyl (diEPP) and methyl ethyl phenyl piperazinyl (MEPP) analog of 3f. Additionally, compound 3i was designed to further probe the influence of reverse amide linkage by substituting the 6-methyl pyridyl carboxamide group of 3f with an acetamido moiety tethered to the central phenyl ring. Our findings indicate that compounds 3g–3i functioned as α9-selective full agonists, with higher selectivity over α7 receptors. Notably, compounds 3g and 3i displayed robust partial agonism for α9α10 (Figure 4 and Table 2). When the picolinyl group in compound 3h was substituted with a methyl group, as seen in compound 3i, there is a marked shift in receptor selectivity favoring α9* over α7 nAChR. Additionally, the agonist activity of the compound transitioned from full to partial agonism on α9* with reduced potency (Figure 4 and Table 2). These observations suggest that smaller alkyl groups, such as methyl, are potentially not optimal for the α9* binding pocket, given the greater spatial availability in the extended binding region. Among the reverse amide analogs, compound 3h emerged as the most potent α9 full agonist, exhibiting a potency of 230 nM (340-fold selective for α9 over α7 nAChR)—remarkably, the highest across all compound series. This represents a 113-fold increase in potency relative to ACh. Importantly, reverse amide 3h is twice as potent as its normal amide counterpart, 3f.

2.2.4. Amide Group Isosteres

To investigate the impact of aromatic isosteres as replacements for carboxamide, the biological activity of compound 1 was juxtaposed with isosteric analogs 3j–3l—namely, oxazole, pyrazole, and pyrrole (Figure 2). These isosteres were chosen for their potential to confer differential binding patterns upon interacting with the receptor, as compared to the parent p-CONH₂ -diEPP (compound 1). Figure 5 and Table 3 detail the CRCs and tabulated parameters for these compounds, benchmarked against reference compound 1.

Figure 5.

Concentration–response curves for reference compound 1 (upper left) and compounds 3j–l with α7, α9, and α9α10 nAChR. The test compound applications were alternated with control applications of 60 μM ACh to confirm the stability of the control ACh responses. Experiment responses were measured relative to the preceding ACh control responses and then normalized relative to the ratio of the ACh controls to the ACh maximum (0.63) as determined in previous experiments.^26,30 Each point is the average (n ≥ 5 ± SEM). Experimental data were fit to the Hill curve, as described in the Experimental Section.

Upon analysis of Figure 5, it became evident that oxazole 3j and pyrazole 3k displayed full agonism toward α9 receptors, although pyrazole 3k was a partial agonist toward α9α10. The compounds exhibited low-to-moderate micromolar potencies for α7 receptors. Remarkably, 3j showed submicromolar potency for α9-containing receptors, achieving an I_max value of 1.29 ± 0.13 and emerging as the second most potent analog in the series, with a potency of 380 nM. Interestingly, both 3j and 3k were very weak partial agonists for α7 receptors. A comparative analysis revealed a 10-fold decrease in potency for 3k as compared to 3j against α7 receptors (EC₅₀ = 18 ± 0.60 μM), emphasizing their higher selectivity toward α9 and α9α10 receptors. When the nitrogen count was reduced to a single atom in the pyrrole derivative 3l, a significant attenuation in α9* receptor activation occurred. Compound 3l emerged as a very weak partial agonist with reduced potency (Table 3 and Figure 5).

2.3. Molecular Docking

To explore the binding mode of the ligands in the α9 receptor and their interactions with nearby residues, we conducted molecular docking studies of compounds 3a–l. We utilized our recently reported model for α9 for docking studies.²⁶ Within these studies, we observed some similarities and difference in binding modes, we also observed some interesting ligand interactions with some particular residues that are unique to the α9 receptor and absent in α7 such as D121, T119, and R113. Of significant importance is T119, considering its presence in α9 and absence in α10 receptor, where it is replaced by R119. While all molecules were subjected to docking, compound 3h, the most potent a9 analog, was selected to illustrate its likely binding mode, as shown in Figure 6.

The compounds 3a–k displayed favorable glide eModel values ranging from −56.9 to −40.18 kcal/mol and good glide scores from −14.96 to −7.93 except for 3l with lowest score (−4.96) that is in good correlation with experimental results. Molecular docking of ligands with α9 homomer reveals a common binding feature that all ligands share, which is that the cationic piperazine ring of the studied compounds is enclosed by an electron-rich aromatic cage formed by residues W151, Y192, and Y99 in the (+)-subunit under the C-Loop showing π–cation interactions as shown by compound 3h in Figure 6 (such a type of cation–pi interactions at the orthosteric site was also observed in cryoEM structures of α7 agonists by Noviello et al.³² and Zhao et al.)³³

Present in α9 and α10 but not α7, the D121 carboxylate on the (−) face of the orthosteric binding site also appeared to have salt bridge interactions for all the ligands with the piperazinyl quaternary piperazinium ion. Notably, the aromatic/aliphatic head groups stretch out of the aromatic cage under the C-Loop, in the proximity of the positively charged R113 and inserting into a polar hydrophobic pocket formed by V111, L117, and T119 in the (−) α9 subunit. The π-cation interaction between the π electrons of either phenyl, pyridyl, or heterocyclic rings of ligands and one of the guanidinium amino groups of R113 on the (−) face (Figure 6) varies that may lead to slight differences in agonist activity. The π-cation distances in the top poses for compounds 3c–h were 3.1 ± 1.2 Å. It should be noted that the angle defining these putative π-cation interactions was not ideal; however, the flexibility of the R113 side chain and the vagaries of docking lead us to at least consider this proposal viable because of the close distance. Pyrrolidinyl in compound 3a (a partial agonist) was ineffective to form such an interaction thereby prefers to have weaker alkyl–alkyl interactions with a tilted conformation Compound 3b (less potent but efficacious agonist) bearing n-propyl moiety, although fits well into the pocket makes weak alkyl–alkyl interactions with nearby L117 and alkyl part of R113 show loss of effective π-cation interaction. A good correlation has also been observed with the observed activity of heterocycles 3j–l in the α9 receptor. Oxazole-bearing analog (3j) is 3-fold more potent than p-CONH₂, where amide hydrogens are replaced by aromatic ring depicting that electron-rich aromatic ring is important for enhancing π-cation interactions.

Both normal amide and reverse amide showed a similar interaction except with T119. When 3f was compared with 3h, it was found that 3f did not show any appreciable hydrogen bond interaction with T119 in α9; however, its reverse amide analog 3h showed strong H-bonding interactions with T119 at 1.9 Å (Figure 6).

These investigations revealed some putative binding modes of ligands with certain residues unique to the α9 receptor not α7. Our findings enable us to provide insights into why aromatic head groups, in compounds 3c–3i, demonstrate superior efficacy when compared to aliphatic counterparts. Additionally, our predictions effectively account for the better activity of reverse amide compounds over normal amide.

2.3.1. Silent Agonism of Newly Synthesized Compounds at α7 nAChR

Our initial investigations into α9* nicotinic receptor agonists and antagonists²⁶ commenced with a previously identified group of compounds postulated as silent agonists for the α7 nicotinic subtype.²⁸ These molecules exhibited minimal activation of the α7 receptor when administered alone, yet they could engender a PAM-sensitive desensitized state, which has been hypothesized to possess metabotropic functions within cholinergic anti-inflammatory system (CAS).¹³ In this context, our experimental compounds, which were characterized by their minimal efficacy toward α7 receptor activation in solitary application, were assessed for their capacity to potentiate receptor activity upon simultaneous administration with the α7 PAM PNU-120596. This was benchmarked against the response elicited by coapplying 30 μM acetylcholine with 10 μM PNU-120596, as delineated in Table 4. Notably, the conjoint application of ACh and PNU-120596 yielded peak current responses that surpassed those elicited by ACh alone by a factor of 21.6 ± 4.7.

Table 4. Responses to the Drugs at 30 μM Co-applied with 10 μM PNU-120596, Normalizes to the Responses of 30 μM ACh Co-applied with 10 μM PNU-120596.

compound	aα7 Imax (drug alone)	PNU ± 0.03
ACh	1.00 ± 0.04	1 ± 0.33
1	0.34 ± 0.01	0.18 ± 0.03
2a	0.42 ± 0.02	0.17 ± 0.01
3a	0.06 ± 0.11	0.01 ± 0.00
3b	0.38 ± 0.09	0.19 ± 0.01
3c	0.46 ± 0.08	0.12 ± 0.01
3d	0.60 ± 0.02	0.17 ± 0.01
3e	0.57 ± 0.03	0.16 ± 0.03
3f	0.40 ± 0.02	0.13 ± 0.01
3g	0.14 ± 0.004	31.84 ± 7.95
3h	0.41 ± 0.025	110.51 ± 27.53
3i	0.00	0.15 ± 0.09
3j	0.12 ± 0.01	0.05 ± 0.00
3k	0.088 ± 0.001	0.17 ± 0.02
3l	0.041 ± 0.002	0.08 ± 0.00

^aTaken from Table 1.

The test compounds, when combined with PNU-120596, produced α7 responses that were modest and directly correlated with their inherent α7 activity when applied alone. However, compounds 3k and 3l, despite their nominal α7 efficacy in isolation, demonstrated amplified responses upon coapplication with PNU-120596—approximately double of the ACh control values—positioning them near the threshold for classification as silent agonists.²⁸ In the case of compound 3k, this enhanced α7 activity may potentially augment α9* agonism within CAS, whereas for compound 3l, the likelihood of such an interaction appears diminished. Intriguingly, the reverse amide derivatives, compounds 3g and 3h, demonstrated potent silent agonist activity at the α7 receptor, yielding a remarkable enhancement in responses—227-fold and 270-fold, respectively—when coadministered with PNU-120596.

2.3.2. Initial Profiling of Novel Compounds against Heteromeric Nicotinic Receptors

We evaluated the test compounds for their ability to activate or inhibit other nAChR subtypes. Agonist activity was evaluated by applying the compounds at 100 or 30 μM to human α4β2 and α3β4 as well as to mouse muscle α1β1εδ nAChR expressed in Xenopus oocytes. In no case did the compounds evoke a response as large as 1% of the respective ACh controls (30 μM for α4β2 and α1β1εδ and 100 μM for α3β4 receptors), i.e., basically below our limit of detection (data not shown). To evaluate inhibitory activity on the same receptor subtypes, compounds were coapplied with 3 μM of the test compounds, 3 μM being a concentration in the range of the potency for α9 activity. The inhibitory activity was generally low (Table 5).

2.4. Cholinergic Control of ATP-Mediated Interleukin-1β Release by Monocytic THP-1 Cells

Effect of Select α9* nAChR Agonists on ATP-Mediated IL-1β Release by Monocytic THP-1 Cells

We previously demonstrated that stimulation of nAChRs containing α7 and/or α9/α10 subunits potently inhibits the ATP-induced inflammasome-dependent cleavage and release of the pro-inflammatory cytokine IL-1β by monocytic cells.^34,35 In the light of the potent agonistic effects of compounds 2, 3f, and 3h at conventional ionotropic α9* nAChRs shown previously²⁶ and in the present study, we hypothesized that these compounds could down-regulate the ATP-mediated release of IL-1β by human monocytic THP-1 cells (Figure 7). These cells were primed with lipopolysaccharide (LPS; 1 μg/mL) for 5 h and stimulated for another 40 min with the P2 × 7 receptor agonist (2′(3′)-O-(4-benzoyl–benzoyl) ATP (BzATP, 100 μM) in the presence or the absence of ACh (10 μM) and different concentrations of compounds 2, 3f, or 3h (Figure 8). IL-1β concentrations were measured in cell culture supernatants by ELISA. Untreated cells (IL-1β = 1 pg/mL) and cells primed with LPS (IL-1β = 7 pg/mL, n = 24) released low amounts of IL-1β, while priming of cells with LPS followed by stimulation with BzATP resulted in elevated IL-1β levels (IL-1β = 78 pg/mL, n = 24; Figure 8). In line with our hypothesis, compounds 2, 3f, and 3h significantly and dose-dependently inhibited (IC₅₀ values 2 = 14 μM; 3f = 9 μM; 3h = 0.5 μM) the BzATP-induced release of IL-1β, similar to ACh,^29,34,35 which was included as a positive control (Figure 8A–C). Compounds 3f and 3h seemed to be more effective compared to compound 2 (Figure 8A–C). In the absence of BzATP, neither ACh nor compounds 2, 3f, or 3h induced the release of IL-1β by THP-1 cells that were primed with LPS (Figure 8C). In none of the experimental settings, cell death was increased, as measured by the lactate dehydrogenase activity in cell culture supernatants (data not shown).

Figure 7.

Suggested mechanism of the cholinergic control of ATP-induced release of interleukin-1β (IL-1β) by noncanonical nicotinic receptors (NCNRs). Injury causes the release of cytoplasmic danger-associated molecular patterns (DAMPs) like extracellular ATP, and frequently, microbiota, a source of pathogen-associated molecular patterns (PAMPs), get access to the damaged tissue. In mononuclear phagocytes, DAMPs and PAMPs can bind to pattern recognition receptors such as toll-like receptors (TLR) and induce the synthesis of the inactive pro-IL-1β, the cytoplasmic precursor of IL-1β. Extracellular ATP is sensed by the P2 × 7 receptor (P2 × 7R), resulting in NLRP3 inflammasome assembly, caspase-1 activation, and maturation and release of IL-1β. Activation of NCNRs containing subunits α7, α9, and/or α10 by classical nicotinic ligands and novel α9* selective compounds (2, 3f, and 3h) down-regulates the response of the ATP-sensitive P2 × 7R and, consequently, the maturation and release of IL-1β.

Figure 8.

Impact of compounds 2, 3f, and 3h on the ATP-mediated release of interleukin (IL)-1β by human monocytic THP-1 cells. THP-1 cells were primed with lipopolysaccharide (LPS; 1 μg/mL, 5 h). Thereafter, the P2 × 7 receptor agonist BzATP ((2′/3′-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate, tri(triethylammonium) salt) was added for another 40 min to trigger IL-1β release, which was measured by ELISA. The BzATP (100 μM)-induced release of IL-1β was investigated in the presence and absence of acetylcholine (ACh; 10 μM) and compounds 2, 3f, or 3h. The concentration of IL-1β released in response to BzATP was calculated by subtracting the IL-1 β concentrations measured in supernatants of cells treated with LPS alone. In each experiment, the IL-1β concentrations obtained after stimulation with BzATP + solvent were set to 100%, and all other values were calculated accordingly. Data are presented as individual data points, bar represents median, and whiskers encompass the 25th to 75th percentile. *p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone. The Friedman test was followed by the Wilcoxon signed-rank test.

These in vitro experiments suggest that compounds 2, 3f, and 3h hold therapeutic promise for alleviating inflammation resulting from cellular damage of diverse origins, as well as for pain management. Additionally, nAChR stimulation in mononuclear phagocytes activates a broad array of anti-inflammatory signaling pathways.³⁶ The nAChR-mediated control of ATP signaling is only one of them. Of these, the control of ATP signaling via α9* nAChRs appears most pertinent in the modulation of ATP-induced IL-1β release, although further experimental validation is required to substantiate this mechanistic inference.

We further tested two compounds on freshly isolated human peripheral blood mononuclear cells (PBMCs; Figure 9). Human PBMCs are much more sensitive compared to THP-1 cells. Monocytic THP-1 cells and primary human PBMCs differed in the response toward compounds 2 and 3f. While high concentrations (10–100 μM) of both compounds were needed in monocytic THP-1 cells to efficiently inhibit the ATP-mediated release of IL-1β, 100 fM were sufficient for similar effects in PBMCs (Figures 8 and 9). High concentrations of 2 and 3f were, however, ineffective in PBMCs (Figure 9). We can only speculate on possible explanations for these phenomena and much more research is needed to test them. At present, the exact subunit composition of functional nAChRs at the surface of these cells is unknown. It may be that PBMCs express nAChR subunits or subunit combinations at which high concentrations of 2 and 3f exert antagonistic functions that offset agonist functions at another subunit. The strikingly high sensitivity of PBMCs toward compounds 2 and 3f may be influenced for instance by endogenous prototoxins that have been shown to modulate nAChR sensitivity and function.³⁷ Further, we have to consider that PBMCs, although enriched for monocytes by adherence selection, are composed of a mixture of mononuclear leukocytes that might interfere in their reaction toward cholinergic compounds. Anyhow, in contrast to a cell line such as monocytic THP-1 cells, human PBMCs are much better predictors for the effect of compounds in vivo. Hence, compounds 2 and 3f deserve further in-depth analyses to test if they are suited as anti-inflammatory therapeutics.

Figure 9.

Impact of the compounds 2 and 3f on the ATP-mediated release of interleukin (IL)-1β by primary human peripheral blood mononuclear cells (PBMCs). Human PBMCs were isolated from freshly collected blood samples of healthy human volunteers (male and female). Cells were primed with lipopolysaccharide (LPS; 5 ng/mL, 20 min) during the isolation process. After 3 h of culture, adherent cells were enriched by adherence selection. Thereafter, the P2 × 7 receptor agonist BzATP ((2’/3′-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate, tri(triethylammonium) salt) was added for another 30 min to trigger IL-1β release, which was measured by ELISA. The BzATP (100 μM)-induced release of IL-1β was investigated in the presence and absence of the compounds 2, 3f, or acetylcholine (ACh; 10 μM). In each experiment, the IL-1β concentrations obtained after stimulation with BzATP + solvent were set to 100%, and all other values were calculated accordingly. Data are presented as individual data points, bar represents median, and whiskers encompass the 25th to 75th percentile. *p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone. The Friedman test was followed by the Wilcoxon signed-rank test.

2.5. Aqueous Solubility, Plasma Protein Binding, Metabolism, Plasma Stability, and Permeability Studies

Compounds 3h and 3f exhibited high aqueous solubility (>1000 μM; Table S4) and were found to be stable in pooled murine plasma (Figure 10). In vitro metabolic stability studies of both compounds were performed in pooled human liver microsomes and rat hepatocytes. Both compounds showed low clearance, and extrapolated hepatic extraction ratios are <0.12 in both microsomal- and hepatocyte-based assays (Table S4). Plasma protein binding of 3h and 3f was 54 and 32%, respectively (Table S4).

Figure 10.

Stability of 3h, 3f, and positive control (procaine) in pooled mouse plasma (n = 3, mean ± SD).

2.5.1. Caco-2 Permeability

The P_app values across the human colorectal adenocarcinoma cell (Caco-2) monolayer for 3h and 3f are shown in Table 6. The P_app(A→B) values for propranolol and atenolol were found to be 20.0 ± 1.6 × 10^–6 and 2.9 ± 0.3 × 10^–6 cm/s, respectively, validating the integrity of the Caco-2 cell monolayer throughout the experiment. The P_app(A→B) and P_app(B→A) values for digoxin were found to be 5.4 ± 0.4 × 10^–6 and 11.9 ± 1.3 × 10^–6 cm/s, respectively, with an efflux ratio of 2.2, validating the functioning of p-gp of the Caco-2 monolayer. Both the compounds were found to have low permeability at all three tested pH conditions, and there is no significant difference in permeabilities with the change in pH of the apical compartment.

Table 6. Permeability Across the Caco-2 Monolayer (n = 3, Mean ± SD).

compound	pH condition	permeability (Papp) (×10–6 cm/s)		efflux ratio	permeability class (high/low)	transporter mediated
		A → B	B → A
3f	5.0	1.9 ± 0.4	1.9 ± 1.2	0.96	low	no
	6.8	2.2 ± 1.3	2.6 ± 1.0	1.18	low	no
	7.4	2.2 ± 0.2	2.9 ± 0.5	1.36	low	no
3h	5.0	2.5 ± 0.3	2.9 ± 0.3	1.19	low	no
	6.8	2.8 ± 0.1	3.0 ± 0.6	1.08	low	no
	7.4	2.4 ± 0.2	3.1 ± 0.2	1.28	low	no

Due to the presence of a quaternary ammonium group, both the compounds were found to have low permeability (1.9–2.8 × 10^–6 cm/s) (Table 6) across the Caco-2 monolayer at all three tested pH conditions (pH, 5, 6.8, and 7.4), and there is no significant difference in permeabilities with the change in pH of the apical compartment. The efflux ratios below 1.4 suggest that there was no to minimal efflux of compounds through the intestine. Based on preliminary in vitro ADME and pharmacodynamic studies, it is evident that both compounds may have limited extravascular distribution, but systemic exposure may be adequate to achieve peripheral therapeutic activity to study nAChRs ligands.

2.5.2. Stability in Murine Plasma

The plasma stability was performed using plasma from BALB/c mice. Half-life of procaine in plasma was found to be less than 5 min, but both 3h and 3f were stable (>90%) up to 2 h incubation in murine plasma at 37 °C (Figure 10).

2.6. In Vivo Studies in Models for Pain

2.6.1. Compound 3h (GAT2711) Fully Attenuates Inflammatory Pain in an α7 nAChR-Independent Manner

Animals treated with complete Freund́s adjuvant (CFA) showed greatly reduced mechanical pain thresholds compared to vehicle-treated controls (P = 0.001, Supporting Information Tables S1 and S2). Mechanical thresholds were elevated by treatments with 3h (Figure 11 and Supporting Information, Tables S1). Note that these data were not well fit to normal distributions, especially under control conditions where the majority of responses were the same high threshold value of 3.63 g. Therefore, the data in Figure 11 are the mean ± SD scores for each condition. Statistical analysis was conducted using a Kruskal–Wallis test followed by Mann–Whitney rank sum test at each time point following 3h treatment. There were significant effects for the dose of 10 mg/kg body weight (bw) tested at the 1 and 3 h time points (P = 0.002 and 0.002; Table S11c,d), for 2 and 10 mg/kg bw doses at the 6 h (P = 0.01 and 0.002, Table 1e) and the 24 h time points (P = 0.024 and 0.045, Table S11f), and for 10 mg/kg dose at the 5 days time point (P = 0.002, Table S11h) with no significant effects at the 72 h time point. In sham-treated mice, 10 mg/kg 3h did not alter von Frey responses. In addition, 3h significantly reduced paw edema (F(2,21) = 13.65; P = 0.0002, Student’s t-test; Figure 11B), with mice treated with 10 mg/kg dose differing from the vehicle group (P < 0.0001). At that dose of 10 mg/kg, 3h did not significantly alter locomotor activity of mice compared to vehicle-treated animals (P = 0.4717) (Figure 11C), as assessed by Student’s t-test.

Figure 11.

Effects of systemic administration of GAT2711 (3h) in the CFA-induced chronic inflammatory pain model. (A) Antiallodynic effects after intraperitoneal administration of various doses of GAT2711 (2 and 10 mg/kg body weight, i.p.) in mice. The mechanical paw withdrawal thresholds were determined 3 days after intraplantar injection of CFA (100%) at 1, 3, 6, 24, and 72 h and 5 days after the drug administration (n = 7–8/group, 50% male and 50% female). GAT2711 fully reversed the mechanical hypersensitivity in a dose-related manner. (B) Systemic GAT2711 (2 and 10 mg/kg body weight) reduced paw edema at 6 h after administration. ΔPaw diameter = pretreatment paw diameter-post treatment (*** = P < 0.0001). (C) Intraperitoneal administration of GAT2711 (10 mg/kg body weight) did not alter the locomotor activity of mice 6 h after treatment. (D) Antiallodynic effects of systemic GAT2711 in the CFA model was not lost in the α7 KO mice. Antiallodynic effects after intraperitoneal administration of GAT2711 (10 mg/kg body weight) in WT and in α7 KO mice. The mechanical paw withdrawal thresholds were determined 3 days after intraplantar injection of CFA (100%) at 6 h after the drug administration. Plotted are the mean ± standard deviation under each condition. P values (A,B,D) as determined by the Kruskal–Wallis followed by Mann–Whitney rank sum test (Table S1a–h) or (C) Student’s t-test are indicated in the figure.

In a separate experiment (Figure 11D), we tested CFA-treated α7 WT and KO mice with 3h (10 mg/kg bw) or vehicle and evaluated their mechanical hypersensitivity 3 days after CFA. Our experiments indicated that the antinociceptive effect of 3h was independent of α7 nAChRs since the effects of 10 mg/kg bw 3h were essentially the same in both WT and α7 nAChR KO animals (Kruskal–Wallis test followed by Mann–Whitney rank sum test; Table S1).

2.6.2. Compound 3f (1-HA-84) Attenuates CFA-Induced Inflammatory Pain

Since compound 3f was moderately less potent than 3h in activating α9 nicotinic subtypes, we tested it at the higher dose of 20 mg/kg bw in the CFA model. As observed in Figure 12A, 3f reduced CFA-induced mechanical hypersensitivity at the 1, 3, 6, 24, and 72 h time points after injection (Figure 12A and Table S2; Kruskal–Wallis test followed by Mann–Whitney rank sum test). In sham-treated mice, 20 mg/kg bw 3f did not alter von Frey responses (P ≥ 0.05). As with Figure 12A, data presented are the mean ± SD scores for each condition. In addition, 3f significantly reduced paw edema (t = 2.240, df = 12; P = 0.0448, Figure 12B), with mice treated with 20 mg/kg dose bw differing from the vehicle group. At that dose of 20 mg/kg, 3f did not significantly alter locomotor activity of mice compared to vehicle-treated animals (P = 0.2047) (Figure 12C) as assessed by Student’s t-test.

Figure 12.

Effects of systemic administration of 3f in the CFA-induced chronic inflammatory pain model. (A) Antiallodynic effects after intraperitoneal administration of 3f (20 mg/kg body weight, i.p.) in mice. The mechanical paw withdrawal thresholds were determined 3 days after intraplantar injection of CFA (100%) at 1, 3, 6, 24, and 72 h after the drug administration (n = 7–8/group, 50% male and 50% female). Compound 3f fully reversed the mechanical hypersensitivity at the dose tested. (B) Systemic 3f (20 mg/kg body weight) partially reduced paw edema at 6 h after administration (*P = 0.0448). ΔPaw diameter = pretreatment paw diameter-post treatment. (C) Intraperitoneal administration of 3f (20 mg/kg body weight) did not alter the locomotor activity of mice 6 h after treatment. Plotted are the mean ± standard deviation under each condition. P values (A,B) as determined by the Kruskal–Wallis followed by Mann–Whitney rank sum test (Table S2) or (C) Student’s t-test are indicated in the figure.

3. Conclusions

In summary, in the present study, we have advanced the understanding of small molecules as full and selective agonists for α9 and α9α10 nAChRs, a domain previously marked by a scarcity of detailed reports. Our approach involved refining the N-phenylpiperazine scaffold, known for its α7 silent agonism, to unearth agonists with significant selectivity and efficacy for α9 and α9α10 over α7 nAChRs. Building on our prior work,²⁶ we confirm that variations at the para position of the core phenyl piperazine distinctly influence activity profiles across different nAChRs. Our findings also underscore the vital role of steric bulk alterations at the core of piperazine’s ammonium nitrogen in modulating the differential receptor affinity between α9 and α9α10 nAChR. This insight opens new avenues for further targeted investigations around this scaffold.

These findings are instrumental in guiding the design of novel compounds targeting α9-containing subtypes. By pinpointing specific structural epitopes that modulate activity, we expand the repertoire of pharmacological agents for probing the functions of α9 and α9α10 nAChRs in pain, inflammation, and other diseases.

While there is compelling evidence for both α7 and α9* receptors being potential targets in CAS,^10,13,14 the details of how these receptors should be targeted remain unclear. Our study sheds light on this complexity. Interestingly, compound 3h (GAT2711; 340-fold selective for α9 over α7 nAChR), akin to desensitizing α7 agonists, may offer a novel approach to managing inflammatory or neuropathic pain. The retention of full analgesic activity of 3h in α7 nAChR KO animals strongly implicates the possibility of α9* nAChR-dependent mechanism.

The optimal strategy for targeting α9* in inflammatory pain is yet to be fully understood. Although α9 antagonists, like conotoxins, suggest antagonist-based targeting,^15,38,39 their precise mechanism remains ambiguous. While α9-mediated CAS activity is associated with receptor activation and/or desensitization,⁴⁰ the studies with α9 conopeptide antagonists^{16,38,39,41,42} suggested that α9 CAS activity would arise from α9 inhibition in vivo.¹⁴ However, this was inconsistent with the effect of the conopeptide in the cell-based assays, which suggested that the α9 nAChR agonism was the basis for the CAS effects. Our cell-based assays indicate that both unconventional α9 agonists and partial agonists²⁷ related to our study compounds can mediate α9*-dependent anti-inflammatory activity. In conclusion, the diverse profiles of our study compounds, exemplified by 3h and 3i, represent a significant leap in developing precision tools for differentiating the roles of α7 and α9 receptors in cholinergic anti-inflammatory pathways.

In this study, a noteworthy observation pertains to the extended duration of action exhibited by the novel agonists investigated. In contrast to previously²⁹ reported α9 nAChR agonists such as pCN-diEPP, which demonstrated efficacy in ameliorating CFA-induced pain like behaviors for a period ranging from 3 to 6 h, the novel compounds 3h and 3f showed a significantly prolonged effective duration of approximately 72 h. This extended analgesic effect is less likely attributable to a potential depot effect linked to their quaternary ammonium groups, as this is a common feature shared among these compounds. Our ongoing research aims to elucidate the underlying mechanisms responsible for this prolonged action, with findings to be detailed in forthcoming publications. Furthermore, the activation of α9* nAChR receptors by these small-molecule agonists not only facilitates longer-lasting analgesia but also does so without compromising motor coordination. This characteristic positions these compounds as promising candidates for supplanting addictive opioid analgesics, potentially heralding a new era in pain management.

4. Experimental Section

4.1. General Procedures

Reagents for chemical synthesis were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). All were of reagent quality or were purified before use. Organic solvents were of analytical grade or were purified by standard procedures. Reactions were monitored on EMD Millipore 0.25 mm silica gel thin layer chromatography (TLC) plates (with fluorescence UV indicator F254) using the solvent system specified in the corresponding experimental protocol. TLC plates were visualized under UV illumination at 254 nm. Column chromatography was performed with silica gel 60 (230–400 mesh). Melting points were obtained on a MFB-595010 M Gallenkamp apparatus equipped with a digital thermometer and are uncorrected. Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker spectrometer (400 and 600 MHz for ¹H and 101 and 151 MHz for ¹³C) using CDCl₃ or Methanol-d₄ and DMSO-d₆ as solvent. Chemical shifts (δ scale) are given in parts per million (ppm) relative to the peak of residual solvent. Processing of the spectra was performed using MestReNova 14.1.1. ¹H and ¹³C NMR and mass spectra for new compounds are provided in the Supporting Information section. Reactions were carried out in flame-dried glassware under argon when anhydrous conditions were required. In those cases, anhydrous solvents were used in the reactions. Compound purity was more than 96% as determined by HPLC and ¹H NMR analyses (see Supporting Information). The syntheses of compounds 1 and 2 have been described.^26,28

4.1.1. Synthesis of 4-Bromo-N-alkyl/arylbenzamides (6a–d)

4.1.1.1. (4-Bromophenyl)(pyrrolidin-1-yl)methanone (6a)

An oven-dried round-bottom flask was flushed with Ar and charged with pyrrolidine (5a) (1.5 mL, 18.24 mmol, 2.0 equiv). Lithium bis(trimethylsilyl)amide (LiHMDS) 1.0 M in THF, 3.0 equiv (27.3 mL, 27.4 mmol), was added with vigorous stirring at room temperature, followed by dropwise addition of ethyl 4-bromobenzoate (4) (neat, 1.46 mL, 9.12 mmol 1.0 equiv), and the reaction mixture was stirred at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with aqueous ammonium chloride (1.0 M, 10 mL) and then diluted with dichloromethane (30 mL), and the resulting the organic layer was washed with water (1 × 30 mL) and brine (1 × 30 mL), then dried, and concentrated and dried under vacuum. 2.10 g, 8.22 mmol of (4-bromophenyl)(pyrrolidin-1-yl)methanone (6a) was obtained as a brown oil. Yield: 90%; TLC R_f = 0.23 in n-hexane: ethyl acetate (6:4). ¹H NMR (400 MHz, Chloroform-d) δ 7.37 (dd, J = 2.1, 8.5 Hz, 2H), 7.31–7.17 (m, 2H), 3.45 (dt, J = 3.7, 7.2 Hz, 2H), 3.25 (dt, J = 3.8, 7.6 Hz, 2H), 1.75 (m, 4H). ¹³C NMR (101 MHz, Chloroform-d): δ 168.15, 135.55, 131.05, 128.50, 123.70, 49.22, 45.94, 26.02, 24.01. LCMS (ESI) calcd for C₁₁H₁₂BrNO, [M + H] 254.01, Found. 254.02; HRMS (ESI) exact mass calcd for C₁₁H₁₂BrNO [M + H]⁺, 254.0175; found, 254.0188.

4.1.1.2. 4-Bromo-N-propylbenzamide (6b)

This compound was prepared from ethyl 4-bromobenzoate (4) (1.46 mL, 9.12 mmol 1.0 equiv) and n-propylamine 5b (1.5 mL, 18 mmol, 2.0 equiv) using the procedure described for 6a to afford the desired product 6b as a shiny white solid (2.11 g, 8.72 mmol, 96%). mp 78.1–79.5 °C, TLC R_f = 0.33 in n-hexane: ethyl acetate (6:4). ¹H NMR (400 MHz, Chloroform-d): δ 7.69–7.60 (m, 2H), 7.58–7.50 (m, 2H), 6.44 (s, 1H), 3.39 (ddd, J = 5.9, 7.2, 8.0 Hz, 2H), 1.63 (h, J = 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 166.60, 133.60, 131.64, 128.46, 125.85, 41.81, 22.80, 11.38. LCMS-(ESI) calcd C₁₀H₁₂BrNO [M + H], 242.02; found, 242.01.

4.1.1.3. 4-Bromo-N-(pyridin-2-yl)benzamide (6c)

This compound was prepared from ethyl 4-bromobenzoate (4) (1.46 mL, 9.12 mmol 1.0 equiv) and 2-aminopyridine 5c (1.71g, 18.2 mmol, 2.0 equiv) using the procedure described for 6a to afford the desired product 6c as a light beige solid (2.39 g, 8.62 mmol, 95% yield). mp 123.2–125.7 °C, TLC R_f = 0.26 in n-hexane: ethyl acetate (7:3). ¹H NMR (600 MHz, Chloroform-d): δ 8.88 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 8.22 (t, J = 4.1 Hz, 1H), 7.82–7.74 (m, 3H), 7.62 (d, J = 8.1 Hz, 2H), 7.07 (dd, J = 4.9, 7.3 Hz, 1H). ¹³C NMR (151 MHz, Chloroform-d): δ 164.83, 151.39, 147.86, 138.54, 133.11, 132.05, 128.83, 127.05, 120.10, 114.28. LCMS (ESI) calcd C₁₂H₉BrN₂O [M + H], 277.00; found, 277.03.

4.1.1.4. 4-Bromo-N-(m-tolyl)benzamide (6d)

This compound was prepared from ethyl 4-bromobenzoate (4) (1.46 mL, 9.12 mmol 1.0 equid) and m-toluidine 5d (1.97 mL, 18.2 mmol, 2.0 equiv) using the procedure described for 6a to afford the desired product 6d as a shiny beige solid (2.52 g, 8.87 mmol, 95% yield) mp 133.1–134.6 °C, TLC R_f = 0.35 in n-hexane: ethyl acetate (7:3). ¹H NMR (400 MHz, Chloroform-d): δ 8.05 (s, 1H), 7.73–7.65 (m, 2H), 7.54 (dd, J = 1.6, 8.5 Hz, 2H), 7.46 (d, J = 1.9 Hz, 1H), 7.40 (dd, J = 2.1, 8.0 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.97 (m, 1H), 2.33 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 164.93, 138.98, 137.53, 133.76, 131.85, 128.85, 128.63, 126.41, 125.60, 121.07, 117.51, 21.44. LCMS (ESI) calcd C₁₄H₁₂BrNO [M + H], 290.02; found, 290.7.

4.1.1.5. 4-Bromo-N-(6-methylpyridin-2-yl)benzamide (6e)

This compound was prepared from ethyl 4-bromobenzoate (4) (1.46 mL, 9.12 mmol 1.0 equiv) and 6-methylpyridin-2-amine 5e (1.97g, 18.2 mmol, 2.0 equiv) using the procedure described for 6a to afford the desired product 6e as a pale white solid (2.43 g, 8.35 mmol, 92% yield), m.p: 73.7–74.3 °C, TLC R_f = 0.31 in n-hexane: ethyl acetate (6:4). ¹H NMR (600 MHz, Methanol-d₄): δ 8.02 (dd, J = 4.6, 8.5 Hz, 1H), 7.88 (ddd, J = 1.7, 6.4, 10.3 Hz, 2H), 7.75–7.65 (m, 3H), 7.07–7.01 (m, 1H), 2.49 (s, 3H, CH₃). ¹³C NMR (151 MHz, Methanol-d₄): δ 157.01, 150.89, 138.51, 133.24, 131.55, 129.11, 126.38, 119.33, 111.68, 111.66, 22.59. LCMS (ESI) calcd C₁₃H₁₁BrN₂O [M + H], 291.01; found, 291.0.

4.1.2. Synthesis of 4-(4-Ethylpiperazin-1-yl)-N-alkyl/aryl Benzamides (8a–e)

4.1.2.1. (4-(4-Ethylpiperazin-1-yl)phenyl)(pyrrolidin-1-yl)methanone (8a)

In a round-bottom flask with a continuous supply of nitrogen, (4-bromophenyl)(pyrrolidin-1-yl)methanone (6a) (0.89g, 3.52 mmol), Pd₂(dba)₃ (0.32 g, 0.352 mmol (10 mol %)), (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ligand (BINAP) (0.438g, 0.704 mmol, 20 mol %), 1-ethylpiperazine (7a) (1.78 mL, 14.08 mmol), and Cs₂CO₃ (2.94 g, 7.04 mmol) were dissolved in 1 mL of dioxane and 3 mL of dry THF and stirred at 90 °C for 8 h. The reaction mixture was evaporated, and the residue was dissolved in dichloromethane and then filtered over Celite. The filtrate was extracted with water (30 mL) to remove excess of ethyl piperazine. The dichloromethane (DCM) layer was concentrated, and the product was purified by column chromatography on silica gel, eluting with ethyl acetate. The fractions containing the desired product were combined, evaporated, and dried. Compound 8a was obtained as a pale brown oil (0.65 g, 2.3 mmol, yield: 64% yield). TLC R_f = 0.24 in 95% ethyl acetate in n-hexane. ¹H NMR (400 MHz, Chloroform-d): δ 7.48 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 3.61 (t, J = 7.0 Hz, 2H), 3.50 (t, J = 6.4 Hz, 2H), 3.32–3.20 (m, 4H), 2.58 (m, 4H), 2.46 (q, J = 7.2 Hz, 2H), 2.00–1.77 (m, 4H), 1.12 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 169.55, 152.17, 128.87, 127.14, 114.21, 52.59, 52.29, 49.75, 48.16, 46.27, 26.49, 24.39, 11.95. HRMS (ESI) exact mass calcd for C₁₇H₂₅N₃O [M + H]⁺, 288.2070; found, 288.2079 and [M + Na]⁺, 310.1890; found, 310.1884.

4.1.2.2. 4-(4-Ethylpiperazin-1-yl)-N-propylbenzamide (8b)

It was prepared from 6b (0.852 g, 3.52 mmol) and N-ethyl piperazine (1.78 mL, 14.1 mmol) in 1 mL of dioxane and 3 mL of toluene using the procedure and conditions described for 8a to afford the desired product 8b as a pale white solid (0.90 g, 3.27 mmol, 92% yield). mp 126.4–128.8 °C TLC R_f = 0.28 in 95:5 ethyl acetate/n-hexane. ¹H NMR (400 MHz, Chloroform-d): δ 7.67 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.02 (s, 1H), 3.39 (m, 2H), 3.34–3.27 (m, 4H), 2.64–2.56 (m, 4H), 2.48 (q, J = 7.2 Hz, 2H), 1.68–1.54 (m, 2H), 1.13 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 167.35, 153.42, 128.40, 124.85, 114.50, 52.77, 52.56, 48.12, 41.82, 23.27, 12.15, 11.68. HRMS (ESI) exact mass calcd for C₁₆H₂₅N₃O [M + H]⁺, 276.2070; found, 276.2078 and [M + Na]⁺, 298.1890; found, 298.1887.

4.1.2.3. 4-(4-Ethylpiperazin-1-yl)-N-(pyridin-2-yl)benzamide (8c)

It was prepared from 6c (0.975 g, 3.52 mmol) and N-ethyl piperazine (1.78 mL, 14.1 mmol) in 4 mL of dioxane and toluene (1:3) using the procedure and conditions described for 8a to afford the desired product 8c as a pale white solid (0.73 g, 2.4 mmol, 67% yield). mp 124.3–126 °C TLC R_f = 0.28 in 95% ethyl acetate in n-hexane. ¹H NMR (400 MHz, Chloroform-d): δ 8.72 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 4.9 Hz, 1H), 7.91–7.76 (m, 2H), 7.71 (td, J = 1.8, 7.9 Hz, 1H), 7.00 (dd, J = 5.0, 7.3 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 3.34 (t, J = 5.1 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.46 (q, J = 7.2 Hz, 2H), 1.12 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 165.26, 153.65, 151.94, 147.70, 138.28, 128.80, 123.25, 119.38, 114.06, 113.94.43, 52.26, 47.46, 11.90. HRMS (ESI) exact mass calcd for C₁₈H₂₂N₄O [M + H]⁺, 311.1866; found, 311.1880 and [M + Na]⁺, 333.168; found, 333.1691.

4.1.2.4. 4-(4-Ethylpiperazin-1-yl)-N-(m-tolyl)benzamide (8d)

This compound was prepared from 6d (1.02 g, 3.52 mmol) and N-ethyl piperazine (1.78 mL, 14.1 mmol) in 1 mL of dioxane and 3 mL of toluene using the procedure and conditions described for 8a to afford the desired product 8d as a pale-yellow solid (0.92 g, 2.8 mmol, 81% yield). mp 154–156.6 °C TLC R_f = 0.32 in ethyl acetate: methanol (9:1). ¹H NMR (400 MHz, Chloroform-d): δ 7.82–7.76 (m, 2H), 7.74 (s, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 2.2, 7.9 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.97–6.89 (m, 3H), 3.38–3.31 (m, 4H), 2.61 (t, J = 5.1 Hz, 4H), 2.49 (d, J = 7.2 Hz, 2H), 2.36 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 165.22, 153.48, 138.88, 138.23, 128.79, 128.48, 124.85, 124.32, 120.67, 117.07, 114.17, 52.53, 52.33, 47.72, 21.52, 11.98.

4.1.2.5. 4-(4-Methylpiperazin-1-yl)-N-(6-methylpyridin-2-yl)benzamide (8e)

This compound was prepared from 6e (1.02 g, 3.52 mmol) and N-methyl piperazine (1.56 mL, 14.1 mmol) in 3 mL of dioxane and toluene (1:2) using the procedure and conditions described for 8a to afford the desired product 8e as an off-white solid (0.89 g, 2.9 mmol, 82% yield). m.p: 128.3–131.1 °C TLC R_f = 0.29 in ethyl acetate/methanol (9:1). ¹H NMR (400 MHz, Methanol-d₄): δ 8.04 (d, J = 8.3 Hz, 1H), 7.95–7.86 (m, 2H), 7.72 (t, J = 7.9 Hz, 1H), 7.05 (dd, J = 8.1, 10.4 Hz, 3H), 3.44–3.36 (m, 4H), 2.67–2.59 (m, 4H), 2.50 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, Methanol-d₄): δ 168.00, 158.34, 155.37, 152.77, 140.08, 130.31, 124.61, 120.39, 115.38, 113.01, 55.85, 48.36, 46.22, 24.02.

4.1.3. Synthesis of 1,1-Dialkyl-4-(4-alkyl/arylcarbamoyl)phenyl)piperazin-1-ium Iodides (3a–f)

4.1.3.1. 1,1-Diethyl-4-(4-(pyrrolidine-1-carbonyl)phenyl)piperazin-1-ium Iodide (3a)

In a sealed vial, the coupled compound 8a (0.22 g, 0.76 mmol, 1 equiv) was dissolved in 0.5 mL of dry THF, and iodoethane (0.62 mL, 7.7 mmol, 10 equiv) was added; the resulting mixture was stirred at 25 °C for 24 h until complete consumption of the starting material [TLC in ethyl acetate: methanol (7:3)]. Upon completion of the reaction, hexane was added to the reaction mixture to remove excess iodoethane. The hexane solution was pipetted from the mixture leaving behind a residue that was dissolved in DCM (2 mL). Addition of pentane (8 mL) led to the formation of a precipitate that was allowed to settle, and the resulting supernatant was carefully removed by pipetting. Solvent was further removed from the resulting solid under high vacuum to afford 3a (0.20 g, 0.45 mmol, 59%) as a pale yellow hygroscopic solid that was stored under argon. TLC R_f = 0.33 in ethyl acetate: methanol (7:3). ¹H NMR (400 MHz, Chloroform-d): δ 7.46 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 3.60 (m, 16H), 1.97–1.91 (m, 2H), 1.90–1.84 (m, 2H), 1.39 (t, J = 7.1 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d): δ 169.11, 149.87, 129.31, 128.83, 115.31, 77.32, 77.00, 76.68, 57.48, 53.71, 49.74, 46.33, 42.28, 26.45, 24.40, 7.75. HRMS (ESI) exact mass calcd for C₁₉H₃₀N₃O⁺ [M]⁺, 316.2389; found, 316.2393.

4.1.3.2. 1,1-Diethyl-4-(4-(propylcarbamoyl)phenyl)piperazin-1-ium Iodide (3b)

This compound was prepared from 8b (0.35 g, 1.27 mmol) and iodoethane (1.02 mL, 12.1 mmol) using the procedure and conditions described for 3a to afford the desired product 3b. The product was precipitated from ethyl acetate and obtained as a white solid (0.390 g, 0.904 mmol, 71% yield). mp 231–233.8 °C TLC R_f = 0.29 in ethyl acetate/methanol (7:3). ¹H NMR (400 MHz, Methanol-d₄): δ 7.83–7.74 (m, 2H), 7.11–7.02 (m, 2H), 3.65 (s, 8H), 3.56 (q, J = 7.3 Hz, 4H), 1.62 (h, J = 7.3 Hz, 2H), 1.37 (t, J = 7.3 Hz, 6H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, Methanol-d₄): δ 169.83, 153.22, 129.85, 127.17, 115.97, 58.50, 54.24, 42.89, 42.78, 23.91, 11.86, 7.41. HRMS (ESI) exact mass calcd for C₁₈H₃₀N₃O⁺ [M]⁺, 304.2383; found, 304.2398.

4.1.3.3. 1,1-Diethyl-4-(4-(pyridin-2-ylcarbamoyl)phenyl)piperazin-1-ium Iodide (3c)

This compound was prepared from 8c (0.25 g, 0.80 mmol) and iodoethane (0.65 mL, 8.05 mmol) using the procedure and conditions described for 3a to afford the desired product 3c. The product was obtained as a pink-white solid after precipitation from ethyl acetate (0.27 g, 0.57 mmol, 72% yield). mp 226.1–227.9 °C, TLC R_f = 0.23 in ethyl acetate/methanol (7:3). ¹H NMR (400 MHz, Methanol-d₄): δ 8.39–8.32 (m, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.02–7.93 (m, 2H), 7.84 (m, 1H), 7.21–7.12 (m, 3H), 3.72 (d, J = 5.7 Hz, 4H), 3.70–3.63 (m, 4H), 3.59 (q, J = 7.2 Hz, 4H), 1.40 (t, J = 7.3 Hz, 6H). ¹³C NMR (101 MHz, Methanol-d₄): δ 167.67, 153.51, 153.12, 148.84, 139.42, 130.20, 126.02, 120.91, 116.05, 115.57, 58.14, 53.97, 42.34, 7.13. HRMS (ESI) exact mass calcd for C₂₀H₂₇N₄O⁺ [M]⁺, 339.2179; found, 339.2180.

4.1.3.4. 1,1-Diethyl-4-(4-(m-tolylcarbamoyl)phenyl)piperazin-1-ium Iodide (3d)

This compound was prepared from 8d (0.35 g, 1.08 mmol) and iodoethane (0.87 mL, 11 mmol) using the procedure and conditions described for 3a to afford the product as a beige-white solid after precipitation from ethyl acetate (0.38 g, 0.79 mmol, 73% yield). mp 201.1–203.3 °C, TLC R_f = 0.25 in ethyl acetate/methanol (7:3). ¹H NMR (600 MHz, DMSO-d₆): δ 9.90 (s, 1H), 7.96–7.91 (m, 2H), 7.60 (d, J = 1.8 Hz, 1H), 7.58–7.54 (m, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.13–7.08 (m, 2H), 6.89 (d, J = 7.5 Hz, 1H), 3.65 (t, J = 5.2 Hz, 4H), 3.57 (t, J = 5.2 Hz, 4H), 3.50 (q, J = 7.2 Hz, 4H), 2.30 (s, 3H), 1.23 (t, J = 7.2 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆): δ 164.70, 151.59, 139.31, 137.61, 129.04, 128.35, 124.92, 123.99, 120.83, 117.47, 113.87, 56.16, 51.88, 40.58, 40.15, 21.23, 6.76. HRMS (ESI) exact mass calcd for C₂₂H₃₀N₃O⁺ [M]⁺, 352.2383; found, 352.2400.

4.1.3.5. 1,1-Dimethyl-4-(4-((6-methylpyridin-2-yl)carbamoyl)phenyl)piperazin-1-ium Iodide (3e)

This product was prepared from 8e (0.35 g, 1.1 mmol) and iodomethane (0.70 mL, 11 mmol) using the procedure and conditions described for 3a with a reaction time of 2 h to afford the product as a pale yellow solid after precipitation from ethyl acetate (0.40 g, 0.88 mmol, 78% yield). m.p: 135.5–137.7 °C, TLC R_f = 0.27 in ethyl acetate: methanol (7:3). ¹H NMR (400 MHz, Methanol-d₄): δ 8.02 (d, J = 8.2 Hz, 1H), 7.98–7.89 (m, 2H), 7.72 (t, J = 7.9 Hz, 1H), 7.19–7.11 (m, 2H), 7.04 (d, J = 7.5 Hz, 1H), 3.72 (dd, J = 6.0, 17.3 Hz, 8H), 3.34 (s, 6H), 2.49 (s, 3H). ¹³C NMR (101 MHz, Methanol-d₄): δ 167.73, 158.41, 153.76, 152.63, 140.13, 131.81, 130.43, 126.49, 120.57, 116.14, 113.08, 62.50, 52.00, 43.25, 24.03. HRMS (ESI) exact mass calcd for C₁₉H₂₅N₄O⁺ [M]⁺, 325.2023; found, 352.2037.

4.1.3.6. 1-Ethyl-1-methyl-4-(4-((6-methylpyridin-2-yl)carbamoyl)phenyl)piperazin-1-ium Iodide (3f)

This compound was prepared from 8e (0.27 g, 0.87 mmol) and iodoethane (0.70 mL, 8.7 mmol) using the procedure and conditions described for 3a to afford the product 3f as a pink-white solid after precipitation from ethyl acetate (0.29 g, 0.62 mmol, 72% yield). mp 207.2–209.4 °C TLC R_f = 0.26 in ethyl acetate/methanol (7:3). ¹H NMR (400 MHz, DMSO-d₆): δ 10.42 (s, 1H), 8.07–7.96 (m, 3H), 7.70 (t, J = 7.9 Hz, 1H), 7.13–7.05 (m, 2H), 7.00 (d, J = 7.4 Hz, 1H), 3.80–3.48 (m, 10H), 3.12 (s, 3H), 2.45 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Methanol-d₄): δ 167.65, 158.33, 153.71, 152.56, 140.03, 130.33, 126.29, 120.48, 115.99, 112.99, 60.97, 60.45, 46.67, 42.92, 23.98, 7.79. HRMS (ESI) exact mass calcd for C₂₀H₂₇N₄O⁺ [M]⁺, 339.2179; found, 339.2193.

4.1.4. Synthesis of N-(4-Bromophenyl)-6-methylpicolinamide (12)

An oven-dried round-bottom flask was flushed with Ar and charged with 4-bromoaniline (10) (1.81g, 12 mmol, 2 equiv). Lithium bis(trimethylsilyl)amide (LiHMDS) 1.0 M in THF, 3.0 equiv (18 mL, 18.05 mmol), was added with vigorous stirring at room temperature, followed by dropwise addition of methyl 6-methylpicolinate (11) (neat, 0.88 mL, 6.0 mmol 1.0 equiv), and the reaction mixture was stirred at room temperature. After completion of the reaction (monitored by TLC and LC–MS), the reaction mixture was quenched with aqueous ammonium chloride (1.0 M, 10 mL) and then diluted with dichloromethane (40 mL), and the resulting the organic layer was washed with water (1 × 40 mL), brine (1 × 40 mL) and then concentrated. The product was purified by column chromatography using n-hexane: Ethyl acetate yielding 1.4 g, 4.8 mmol of N-(4-bromophenyl)-6-methylpicolinamide (12). Yield: 80.2%; TLC R_f = 0.19 in ethyl acetate: methanol (9:1). ¹H NMR (500 MHz, Chloroform-d): δ 10.06 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.70–7.63 (m, 2H), 7.48–7.41 (m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 2.59 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 162.23, 157.25, 148.76, 137.83, 136.96, 131.97, 126.41, 121.23, 119.51, 116.70, 24.28.

4.1.5. Synthesis of N-(4-(4-Ethylpiperazin-1-yl)phenyl)-6-methylpicolinamide (13)

In a sealed vial purged with nitrogen, N-(4-bromophenyl)-6-methylpicolinamide (12) (0.7 g, 2.4 mmol), Pd₂(dba)₃ (0.18 g, 0.19 mmol (8 mol %)), (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ligand (BINAP) (0.29g, 0.48 mmol, 20 mol %), 1-ethylpiperazine (1.2 mL, 9.6 mmol), and Cs₂CO₃ (1.56 g, 4.8 mmol) were dissolved in 3 mL THF and stirred at 98 °C for 10 h. The reaction mixture was evaporated, and the residue was dissolved in dichloromethane, filtered over Celite, concentrated, and purified by column chromatography on silica gel, eluting with 90% ethyl acetate in hexane. The fractions containing the desired product were combined, evaporated, and dried under high vacuum. Compound 13 was obtained as a pale yellow solid (0.3 g, 0.92 mmol, yield: 38%). TLC R_f = 0.22 (5% methanol in ethyl acetate). ¹H NMR (500 MHz, Chloroform-d): δ 9.94 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.71–7.65 (m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 6.99–6.93 (m, 2H), 3.29–3.11 (m, 4H), 2.62 (s, 3H), 2.58 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 161.83, 157.03, 149.33, 148.13, 137.68, 130.41, 125.93, 120.85, 119.31, 116.60, 55.11, 49.49, 46.14, 24.27, 7.21.

4.1.6. Synthesis of 1,1-Diethyl-4-(4-(6-methylpicolinamido)phenyl)piperazin-1-ium Iodide (3g)

In a sealed vial, the coupled compound 13 (0.2 g, 0.61 mmol, 1 equiv) was dissolved in 0.5 mL dry THF, and iodoethane (0.50 mL, 6.16 mmol, 10 equiv) was added; the resulting mixture was stirred at 25 °C for 48 h until complete consumption of the starting material [TLC in ethyl acetate/methanol (7:3)], LC–MS. Upon completion of the reaction, hexane was added to the reaction mixture to remove excess iodoethane. The hexane solution was pipetted out from the mixture leaving behind a residue that was dissolved in DCM, rotavaped, and recrystallized with ethyl acetate. A pure solid was dried under high vacuum to afford 3g (0.22 g, 0.45 mmol, 74%) as a light-yellow solid. TLC R_f = 0.11 in ethyl acetate: methanol (7:3). ¹H NMR (500 MHz, Methanol-d₄): δ 7.99 (d, J = 7.7 Hz, 1H), 7.88 (td, J = 1.4, 7.7 Hz, 1H), 7.75–7.69 (m, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.12–7.05 (m, 2H), 3.64 (t, J = 5.1 Hz, 4H), 3.55 (q, J = 7.0 Hz, 8H), 2.65 (s, 3H), 1.37 (t, J = 7.3 Hz, 6H). ¹³C NMR (126 MHz, Methanol-d₄).: δ 164.46, 159.21, 150.42, 147.91, 139.22, 132.75, 127.61, 122.79, 120.39, 118.03, 58.72, 54.17, 44.22, 24.21, 7.30.

4.1.7. Synthesis of 1-Methyl-4-(4-nitrophenyl)piperazine (15)

In a sealed vial purged with nitrogen, 4-nitroiodobenzene (1.5 g, 6.02 mmol), Pd₂(dba)₃ [0.22 g, 0.24 mmol (4 mol %)], (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ligand (BINAP) (0.37g, 0.60 mmol, 10 mol %), 1-methylpiperazine (2 mL, 18.7 mmol), and Cs₂CO₃ (1.96 g, 6.02 mmol) were dissolved in 3 mL THF and stirred at 98 °C for 3h. The reaction mixture was evaporated, and the residue was dissolved in dichloromethane, filtered over Celite, concentrated, and purified by column chromatography on silica gel, eluting with ethyl acetate. The fractions containing the desired product were combined, evaporated, and dried under high vacuum. Compound 15 was obtained as an orange solid (1.1 g, 2.3 mmol, yield: 82.5%). TLC R_f = 0.76 in 5% methanol in ethyl acetate. ¹H NMR (500 MHz, Chloroform-d): δ 8.11 (d, J = 9.3 Hz, 2H), 6.82 (d, J = 9.4 Hz, 2H), 3.49–3.40 (m, 4H), 2.58–2.48 (m, 4H), 2.35 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 154.83, 138.43, 132.12, 128.54, 125.93, 112.66, 54.53, 46.99, 46.07.

4.1.8. Synthesis of 4-(4-Methylpiperazin-1-yl)aniline (16)

Raney Nickel (0.02 g, 8 mol %) was added in a mixture of 1-methyl-4-(4-nitrophenyl)piperazine (15) (1 g, 4.52 mmol) in methanol/THF 8 mL (1:1) in a three neck round-bottom flask equipped with a supply of hydrogen gas contained in a balloon. The reaction is allowed to stir, and hydrogen gas is released at a slow rate. The reaction mixture is allowed to stir for 2 h at room temperature. After the completion of reaction, the reaction mixture is diluted with dichloromethane passed through Celite and concentrated, recrystallized with DCM: Hexane, and dried under high vacuum. Compound 16 was obtained as a brown solid (0.79 g, 4.13 mmol, yield: 91.3% yield). TLC R_f = 0.25 in 2% methanol in ethyl acetate. ¹H NMR (500 MHz, Chloroform-d): δ 6.83–6.77 (m, 2H), 6.66–6.60 (m, 2H), 3.33 (d, J = 74.1 Hz, 2H), 3.11–3.00 (m, 4H), 2.62–2.50 (m, 4H), 2.33 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 144.48, 140.11, 118.54, 116.19, 55.34, 50.86, 46.16.

4.1.9. Synthesis of N-(4-(4-Methylpiperazin-1-yl)phenyl)-6-methylpicolinamide (18)

An oven-dried round-bottom flask was flushed with argon and charged with 4-(4-methylpiperazin-1-yl)aniline (16) (0.75g, 3.92 mmol, 1.1 equiv). Lithium bis(trimethylsilyl)amide (LiHMDS) 1.0 M in THF, 3.0 equiv (10.5 mL, 10.5 mmol), was added with vigorous stirring at room temperature, followed by dropwise addition of methyl 6-methylpicolinate (11) (neat, 0.51 mL, 3.52 mmol 1.0 equiv), and the reaction mixture was stirred at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with aqueous ammonium chloride (1.0 M, 10 mL) and then diluted with dichloromethane (30 mL), and the resulting the organic layer was washed with water (1 × 40 mL) and brine (1 × 40 mL), then dried, and concentrated. Crude was purified by reverse phase column chromatography, and the product was eluted at 100% water containing 0.1% formic acid. The fractions containing product were neutralized with sodium bicarbonate, followed by extraction again with DCM. 0.9 g, 2.89 mmol of N-(4-(4-methylpiperazin-1-yl)phenyl)-6-methylpicolinamide (18). Yield: 82.1%; TLC R_f = 0.19 in ethyl acetate/methanol (9:1). ¹H NMR (500 MHz, Chloroform-d): δ 9.94 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.71–7.65 (m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 6.99–6.93 (m, 2H), 3.29–3.11 (m, 4H), 2.62 (s, 3H), 2.58 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 161.83, 157.03, 149.33, 148.13, 137.68, 130.41, 125.93, 120.85, 119.31, 116.60, 55.11, 49.49, 46.14, 24.27.

4.1.10. Synthesis of N-(4-(4-Methylpiperazin-1-yl)phenyl)acetamide (19)

An oven-dried round-bottom flask was flushed with argon and charged with 4-(4-methylpiperazin-1-yl)aniline (16) (0.70g, 3.65 mmol, 1.1 equiv). Lithium bis(trimethylsilyl)amide (LiHMDS) 1.0 M in THF, 2.0 equiv (6.57 mL, 6.5 mmol), was added with vigorous stirring at room temperature, followed by dropwise addition of ethyl acetate (neat, 0.32 mL, 3.29 mmol 1.0 equiv), and the reaction mixture was stirred at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with aqueous ammonium chloride (1.0 M, 10 mL) and then diluted with dichloromethane (30 mL), and the resulting the organic layer was washed with water (1 × 40 mL) and brine (1 × 40 mL), then dried, and concentrated. Crude was purified by reverse phase column chromatography, and the product was eluted at 100% water containing 0.1% formic acid. The fractions containing the product were neutralized with sodium bicarbonate, followed by extraction again with DCM. 0.61 g, 2.61 mmol of N-(4-(4-methylpiperazin-1-yl)phenyl)acetamide (19). Yield: 79.4%; TLC R_f = 0.16 in ethyl acetate/methanol (9:1). ¹H NMR (500 MHz, Chloroform-d): δ 7.35 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 7.9 Hz, 2H), 3.15 (s, 4H), 2.59–2.54 (m, 5H), 2.34 (s, 3H), 2.12 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ 168.20, 148.29, 130.39, 121.52, 116.63, 55.02, 49.44, 46.05, 24.37.

4.1.11. Synthesis of 1-Ethyl-1-methyl-4-(4-(6-methylpicolinamido)phenyl)piperazin-1-ium Iodide (3h)

In a sealed vial, the coupled compound 18 (0.25 g, 0.80 mmol, 1 equiv) was dissolved in 0.5 mL dry THF, and iodoethane (0.64 mL, 8.05 mmol, 10 equiv) was added; the resulting mixture was stirred at 25 °C for 48 h until complete consumption of the starting material [TLC in ethyl acetate/methanol (7:3)]. Upon completion of the reaction, hexane was added to the reaction mixture to remove excess iodoethane. The hexane solution was pipetted out from the mixture, leaving behind a residue that was dissolved in DCM, rotavaped, and recrystallized with ethyl acetate. A pure solid was dried under high vacuum to afford 3h (0.33 g, 0.70 mmol, 87.8%) as a light-yellow solid. TLC R_f = 0.2 in ethyl acetate/methanol (7:3). ¹H NMR (500 MHz, Methanol-d₄): δ 7.98 (d, J = 7.7 Hz, 1H), 7.87 (t, J = 7.7 Hz, 1H), 7.77–7.66 (m, 2H), 7.46 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 8.7 Hz, 2H), 3.71–3.39 (m, 10H), 3.18 (s, 3H), 2.64 (s, 3H), 1.44 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, Methanol-d₄): δ 164.42, 159.18, 150.42, 147.89, 139.19, 132.79, 127.58, 122.77, 120.37, 118.16, 61.04, 60.83 46.65, 44.57, 24.21, 7.73.

4.1.12. Synthesis of 4-(4-Acetamidophenyl)-1-ethyl-1-methylpiperazin-1-ium Iodide (3i)

In a sealed vial, N-(4-(4-methylpiperazin-1-yl)phenyl)acetamide (19) (0.25 g, 1.07 mmol, 1 equiv) was dissolved in 0.5 mL of dry THF, and iodoethane (0.86 mL, 10.7 mmol, 10 equiv) was added; the resulting mixture was stirred at 25 °C for 24 h until complete consumption of the starting material [TLC in ethyl acetate: methanol (7:3) and LC–MS analysis]. Upon completion of the reaction, hexane was added to the reaction mixture to remove excess iodoethane. The hexane solution was pipetted out from the mixture leaving behind a residue that was dissolved in DCM, rotavaped, and recrystallized with ethyl acetate/hexane (9:1). A pure solid was dried under high vacuum to afford 3i (0.28 g, 0.71 mmol, 67.0%) as a light-yellow solid. TLC R_f = 0.17 in ethyl acetate/methanol (7:3). ¹H NMR (500 MHz, Methanol-d₄): δ 7.46 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 3.59 (qd, J = 6.2, 12.2, 12.9 Hz, 10H), 3.16 (s, 3H), 2.09 (s, 3H), 1.42 (d, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, Methanol-d₄): δ 170.02, 146.13, 132.29, 121.16, 116.70, 59.43, 53.42, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 45.20, 43.27, 22.23, 6.28.

4.1.13. Synthesis of N-(4-Heteroarylphenyl)-N′-ethylpiperazines (21a–c)

4.1.13.1. Synthesis of 2-(4-(4-Ethylpiperazin-1-yl)phenyl)oxazole (21a)

In a sealed vial purged with nitrogen, 2-(4-iodophenyl)oxazole (20a) (0.85g, 3.1 mmol, 1 equiv), Pd₂(dba)₃ (0.28 g, 0.31 mmol (10 mol %)), (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ligand (BINAP) (0.586 g, 0.94 mmol, 30 mol %), 1-ethylpiperazine (1.6 mL, 2.5 mmol, 4 equiv), and Cs₂CO₃ (2.04 g, 6.27 mmol, 2 equiv) were dissolved in dioxane (2 mL) and toluene (2 mL) and stirred at 120 °C for 48 h. The reaction mixture was evaporated, and the residue was dissolved in dichloromethane and then filtered over Celite. The filtrate was extracted with water (30 mL) to remove excess ethyl piperazine. The DCM layer was concentrated, and the product was purified by column chromatography on silica gel, eluting with ethyl acetate. The fractions containing the desired product were combined, evaporated, and dried under high vacuum. 21a (0.50 g, 1.9 mmol, 62%) was obtained as a pale white solid. mp 120.2–122.8 °C TLC R_f = 0.28 in ethyl acetate/methanol (9:1). ¹H NMR (400 MHz, Chloroform-d): δ 7.96–7.86 (m, 2H), 7.63 (s, 1H), 7.16 (s, 1H), 6.98–6.90 (m, 2H), 3.38–3.29 (m, 4H), 2.63 (t, J = 5.0 Hz, 4H), 2.50 (q, J = 7.2 Hz, 2H), 1.15 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 162.57, 152.55, 137.81, 128.25, 127.76, 118.38, 115.06, 52.75, 52.54, 48.13, 12.08. HRMS (ESI) exact mass calcd for C₁₅H₁₉N₃O [M + H]⁺, 258.1601; found, 258.1606.

4.1.13.2. 1-(4-(1H-Pyrazol-1-yl)phenyl)-4-ethylpiperazine (21b)

This compound was prepared from 1-(4-iodophenyl)-1H-pyrazole 20b (0.70 g, 2.6 mmol) and ethyl piperazine (1.3 mL, 10 mmol) using the procedure and conditions described for 21a to afford the desired product 21b (0.38 g, 1.5 mmol, 57% yield) as a pale yellow solid. mp 116.8–119.1 °C TLC R_f = 0.27 in ethyl acetate/methanol (9:1). ¹H NMR (400 MHz, Chloroform-d): δ 7.80 (d, J = 2.4 Hz, 1H), 7.66 (d, J = 1.8 Hz, 1H), 7.54 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 6.40 (s, 1H), 3.31–3.14 (m, 4H), 2.63 (dd, J = 3.9, 6.3 Hz, 4H), 2.48 (q, J = 7.2 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Methanol-d₄): δ 151.63, 141.51, 134.89, 129.08, 121.91, 117.77, 108.21, 53.82, 53.42, 49.87, 11.88. HRMS (ESI) exact mass calcd for C₁₅H₂₀N₄ [M + H]⁺, 257.1761; found, 257.1771.

4.1.13.3. 1-(4-(1H-Pyrrol-1-yl)phenyl)-4-ethylpiperazine (21c)

This compound was prepared from 1-(4-iodophenyl)-1H-pyrrole 20c (0.70 g, 2.6 mmol) and ethyl piperazine (1.32 mL, 10.4 mmol) using the procedure and conditions described for 21a to afford the desired product 21c (0.35 g, 1.4 mmol, 53% yield) as a pale white solid. mp 115.6–117.2 °C TLC R_f = 0.30 in ethyl acetate/methanol (9:1). ¹H NMR (400 MHz, Methanol-d₄): δ 7.32 (d, J = 9.0 Hz, 2H), 7.07–6.99 (m, 4H), 6.22 (t, J = 2.2 Hz, 2H), 3.25–3.16 (m, 4H), 2.70–2.60 (m, 4H), 2.51 (q, J = 7.2 Hz, 2H), 1.15 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d): δ 143.34, 130.51, 121.82, 119.57, 116.80, 109.66, 52.72, 52.35, 49.25, 12.08. HRMS (ESI) exact mass calcd for C₁₆H₂₁N₃ [M + H]⁺, 256.1808; found, 256.1815.

4.1.14. Synthesis of 1,1-Diethyl-4-heteroarylphenyl)piperazin-1-ium Iodides (3j–l)

4.1.14.1. Synthesis of 1,1-Diethyl-4-(4-(oxazol-2-yl)phenyl)piperazin-1-ium Iodide (3j)

In a sealed vial, the coupled compound 21a (0.30 g, 1.2 mmol) was dissolved in dry THF (0.5 mL); iodoethane (0.93 mL, 11.65 mmol) was added; and the resulting mixture was stirred at 25 °C for 2 days until complete consumption of the starting material (TLC in ethyl acetate: methanol (7:3). Upon completion, hexane was added to the reaction mixture, followed by filtration. 3j was obtained as a pale white solid after precipitation from ethyl acetate (0.312 g, 0.755 mmol, 65%; mp 222.2–224 °C TLC R_f = 0.29 in ethyl acetate/methanol (7:3) ¹H NMR (500 MHz, Methanol-d₄): δ 7.97–7.91 (m, 3H), 7.26–7.23 (m, 1H), 7.19–7.12 (m, 2H), 3.68 (s, 8H), 3.59 (q, J = 7.1 Hz, 4H), 1.39 (t, J = 7.2 Hz, 6H). ¹³C NMR (101 MHz, Methanol-d₄): δ 163.77, 152.62, 140.23, 128.79, 128.72, 120.14, 116.73, 58.51, 54.30, 42.92, 7.52. HRMS (ESI) exact mass calcd for C₁₇H₂₄N₃O⁺ [M]⁺, 286.1914; found, 286.1926.

4.1.14.2. 4-(4-(1H-pyrazol-1-yl)phenyl)-1,1-diethylpiperazin-1-ium Iodide (3k)

This compound was prepared from 21b (0.25 g, 0.98 mmol) and iodoethane (0.78 mL, 9.8 mmol) using the procedure and conditions described for 3j to afford the product as a light beige solid after precipitation from ethyl acetate (0.193 g, 0.468 mmol, 48% yield). m.p: 217.4–219.1 °C, TLC R_f = 0.27 in ethyl acetate: methanol (7:3). ¹H NMR (400 MHz, Methanol-d₄): δ 8.11 (d, J = 2.5 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.16 (d, J = 9.1 Hz, 2H), 6.50 (s, 1H), 3.71–3.50 (m, 12H), 1.38 (t, J = 7.3 Hz, 6H). ¹³C NMR (101 MHz, Methanol-d₄): δ 151.63, 141.51, 134.89, 129.08, 121.91, 117.77, 108.21, 58.51, 54.30, 42.92, 7.52. HRMS (ESI) exact mass calcd for C₁₇H₂₅N₄⁺ [M]⁺, 285.2074; found, 285.2084.

4.1.14.3. 4-(4-(1H-Pyrrol-1-yl)phenyl)-1,1-diethylpiperazin-1-ium Iodide (3l)

This compound was prepared from 21c (0.25 g, 0.98 mmol) and iodoethane (0.8 mL, 10 mmol) using the procedure and conditions described for 3j to afford the product as a light pink solid after precipitation from ethyl acetate (0.187 g, 0.455 mmol, 46% yield). mp 218.3–220 °C, TLC R_f = 0.31 in ethyl acetate: Methanol (7:3). ¹H NMR (400 MHz, Methanol-d₄): δ 7.40 (d, J = 8.9 Hz, 2H), 7.12 (d, J = 9.0 Hz, 2H), 7.08 (t, J = 2.2 Hz, 2H), 6.24 (t, J = 2.2 Hz, 2H), 3.72–3.48 (m, 12H), 1.37 (t, J = 7.3 Hz, 6H). ¹³C NMR (101 MHz, Methanol-d₄): δ 161.49, 148.79, 122.27, 120.15, 118.71, 111.07, 58.78, 54.25, 44.28, 7.36. HRMS (ESI) exact mass calcd for C₁₈H₂₆N₃⁺ [M]⁺, 284.2121; found, 284.2124.

4.2. Molecular Biology

Plasmid DNAs encoding the human α7 and heteromeric nAChR were obtained from Jon Lindstrom (University of Pennsylvania, Philadelphia, PA). Mouse muscle subunit clones were obtained from Jim Boulter (Salk Institute, La Jolla CA) and Paul Gardner (Dartmouth, Hanover NH). The human resistance-to-cholinesterase 3 (RIC3) clone was obtained from Millet Treinin (Hebrew University, Jerusalem, Israel) and RNA coinjected with α7 to improve the level and speed of receptor expression without affecting their pharmacological properties.⁴³ Plasmid DNA encoding the human α10 nAChR was obtained from J. Michael McIntosh. Plasmid DNA encoding the human α9 nAChR and the human receptor-associated protein of the synapse (RAPSYN) with codon optimization for expression in Xenopus laevis were obtained from Katrin Richter. RAPSYN RNA was coinjected with the α9 and α10 to improve expression.³⁴ After linearization and purification of the plasmid DNAs, RNAs were prepared using the mMessage mMachine in the vitro RNA transcription kit (Ambion, Austin, TX). Frogs were maintained in the Animal Care Service facility of the University of Florida, and all procedures were approved by the University of Florida Institutional Animal Care and Use Committee (approval number 202002669). In brief, the animals were first anesthetized for 15–20 min in 1.5 L frog tank water containing 1 g of MS-222 buffered with sodium bicarbonate. Oocytes were obtained surgically from mature female X. laevis (Nasco, Ft. Atkinson WI, USA) and treated with 1.4 mg/mL type 1 collagenase (Worthington Biochemicals, Freehold NJ, USA) for 2–4 h at room temperature in Ca²⁺-free Barth’s solution (88 mM NaCl, 1 mM KCl, 2.38 mM NaHCO₃, 0.82 mM MgSO₄, 15 mM HEPES, and 12 mg/L tetracycline, pH 7.6) to remove the ovarian tissue and the follicular layers. Stage V oocytes were injected with 4–6 ng CHRNA7 RNA and 2–3 ng RIC3 RNA (2:1 ratio) in 50 nL water, or with 12 ng CHRNA9 RNA and 3 ng RAPSN RNA, or along with 12 ng CHRNA10 RNA in 50 nL water. Oocytes were maintained in Barth’s solution containing 0.32 mM Ca(NO₃)₂ and 0.41 mM CaCl₂, and recordings were carried out 2–20 days after injection.

4.3. Electrophysiology

Two-electrode voltage-clamp experiments were conducted using OpusXpress 6000A (Molecular Devices, Union City CA, USA).⁴⁴ Both the voltage and current electrodes were filled with 3 M KCl. Oocytes were voltage-clamped at −60 mV at room temperature. The oocytes were perfused with Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, 1 μM atropine, pH 7.2) at 2 mL/min. To evaluate the effects of experimental compounds, responses were compared to control ACh-evoked responses, defined as the average of two initial applications of 60 μM ACh made before test applications. Drug applications were 12 s in duration followed by 181 s washout periods.

The responses were calculated as both peak-current amplitudes and net charge, as previously described.⁴⁵ Data were collected at 50 Hz, filtered at 20 Hz, and analyzed by Clampfit (Molecular Devices) and Excel (Microsoft, Redmond, WA, United States). Data are expressed as means ± SEM from at least five oocytes for each experiment and plotted with Kaleidagraph 4.5.2 (Abelbeck Software, Reading, PA, United States). Each episode of data acquisition was a total of 210 s and included an initial 30 s period used to define the baseline for the drug-evoked responses. After 30 s, drugs were applied, and the following 120 s was defined as the drug response period for analysis. Data reported for α7 are net charge, while peak currents are used for α9 and α9/α10 responses since these receptors do not show the same concentration-dependent desensitization that invalidates peak currents as measurements of α7 concentration-dependent responses.²⁷ The values for the curve fits were generated using the Levenberg–Marquardt algorithm to obtain the best Chi-Square fit to the Hill equation using the Kaleidagraph 4.5.2 plotting program. The errors in the tables are the calculated standard errors of the fit parameters based on the goodness of fit.

4.4. Molecular Docking Studies

All docking studies were performed using glide docking by Schrodinger ¨ (Maestro version 2023-2).^46,47 We utilized our recently reported homology model (template PDB: 7KOQ) for α9 for docking studies.²⁶ The protein was prepared, optimized, and minimized and the receptor grid was generated.⁴⁸ Ligand_out files of ligands 3a–3l were prepared using the LigPrep, at the OPLS2005 force field and target pH (7.0 ± 2.0) protonation state.⁴⁹ The grid employed was sufficiently large as to encompass the orthosteric site of the receptor; grid was set to a cube of 20 × 20 × 20 Å. Using the Glide docking module of Schrodinger-Maestro 13.2, XP flexible ligand docking was performed within partial charge cutoff and the van der Waals scaling factor was selected to be 0.15 and 0.80, respectively, for ligand atoms.⁵⁰ Finally, the conformations with the most favorable free energy of binding were selected for analyzing the interactions between the receptor and ligands. PyMOL version 2.5.2⁵¹ and Chimera 1.6 software⁵² were used for 3D molecular graphics, structural alignments, and visualizations.

4.5. Caco-2 Permeability Assay

Caco-2 permeability was assessed using a similar procedure reported by Kamble et al.⁵³ Briefly, Caco-2 cells at a passage of 33 were seeded on 24-well Transwell cell culture inserts (Corning Incorporated, Corning, NY, USA) with 0.4 μm pore size and surface area of 0.33 cm² and used for study after 21–25 days postseeding. Prior to the experiment, DMEM was replaced with warm HBSS buffer (pH 7.4). The integrity of the Caco-2 monolayer was verified by measuring the transepithelial electrical resistance (TEER) value across the monolayer using Millicell, ERS meter (Millipore, Bedford, MA), and the wells with a TEER value over 250 Ω cm² were used for permeability assessment. The pH-dependent permeability of 3h and 3f was assessed at pH 5.0, 6.8, and 7.4 at a concentration of 5 μg/mL. The pH of the basolateral compartment was kept at pH 7.4 for all experiments, and all experiments were conducted in triplicate. Propranolol (5 μg/mL) and atenolol (10 μg/mL) were used as high and low permeability markers, while digoxin (10 μg/mL) was used as a p-glycoprotein (p-gp) substrate to validate the assay. The apical (A) to basolateral (B) transport experiments at different pH across the Caco-2 monolayer were conducted by adding 0.1 mL of compound solution in the apical compartment of the insets and 0.6 mL blank HBSS buffer (pH = 7.4) in the basolateral compartment. The basolateral (B) to apical (A) transport experiments across the Caco-2 monolayer were conducted by adding 0.1 mL of blank HBSS buffer in the apical compartment of the insets and 0.6 mL compound solution in the basolateral compartment. Aliquots (25 μL) were collected from the basolateral and apical compartments at 0 and 2 h, and 100 μL of acetonitrile containing phenacetin as internal standard was added and vortex-mixed for 2 min, followed by filtration using 0.45 μm polytetrafluoroethylene (PTFE) filter plates by centrifugation for 5 min at 2000 rpm (1500g). The filtrates were injected into UPLC–MS/MS for quantitative analysis. Permeability marker (propranolol and atenolol) samples were also processed in a similar manner and analyzed by UPLC–MS/MS.

4.5.1. Data Analysis

The apparent permeability coefficient (P_app) was calculated as

where [drug]_acceptor is the concentration of the drug in the acceptor compartment, and [drug]_{initial, donor} is the initial concentration of the drug in the donor compartment; area is the surface area of the membrane (0.33 cm²); time is the total transport time in seconds; V_A is the volume (in mL) in the acceptor well. The efflux ratio was calculated using the following equation

4.5.2. Mice Plasma Stability

To know the stability of the compounds in the mice plasma, the in vitro stability study was conducted using pooled BALB/c mice plasma, purchased from commercial source (BioIVT). The blank mice plasma (250 μL) was preincubated in a shaking incubator for 10 min prior to spiking the compound at 37 °C. The compounds were spiked at a concentration of 1 μM and incubated again in a shaking incubator. Procaine was used as a positive control. A 25 μL of the plasma was sampled at 0, 5, 10, 15, 20, 30, 60, 90 and 120 min. Plasma proteins were precipitated by adding 100 μL of acetonitrile containing phenacetin as internal standard and vortex-mixed for 2 min, followed by filtration using 0.45 μm PTFE filter plates by centrifugation for 5 min at 2000 rpm. The filtrate was injected to UPLC–MS/MS for quantitative analysis.

4.5.3. Quantification of 3h and 3f

Both 3h and 3f were quantified using a Waters Acquity Class-I UPLC coupled with a Xevo TQ-S Micro triple quadrupole mass spectrometer (Milford, MA, USA). Chromatographic separation was achieved on a Waters Acquity BEH C18 column (1.7 μm, 2.1 × 50 mm). The mobile phases used were 0.1% formic acid in water (A) and acetonitrile (B) at a 0.35 mL/min flow rate and with the following gradient: initial condition as mobile phase A at 95% held for 0.4 min, linearly decreased to 20% reaching 1.6 min, and maintained until 2.4 min, then sharply decreased back to the initial conditions by 2.5 min, and maintained until 3.0 min for re-equilibration. The column and autosampler temperatures were kept at 50 and 4 °C, respectively. The mass spectrometer was operated in positive ion mode, and detection of the ions was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions of m/z 339.3 precursor ion [M + H]⁺ to the m/z 86.0 product ion for both 3h and 3f, m/z 180.12 precursor ion [M + H]⁺ to the m/z 110.03 product ion for internal standard (phenacetin). MassLynx 4.2 was used for data acquisition and TargetLynx for data analysis. The ion spray voltage was set at 2000 V, the desolvation temperature was 400 °C, the desolvation gas flow was 900 L/h, and the cone gas flow was 40 L/h.

4.6. Cell Culture Experiments on Monocytic THP-1 Cells and Freshly Isolated Human PBMCs

The monocytic THP-1 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). THP-1 cells were cultured under 5% CO₂ atmosphere at 37 °C in RPMI 1640 medium (Capricorn, Ebsdorfergrund, Germany, Cat# RPMI-A) supplemented with 10% fetal bovine serum (FBS) from Capricorn (Cat# FBS-16A). For the experiments, the cells were spun down (500 g, 8 min, 19 °C) and resuspended in FBS-free RPMI 1640 medium. Thereafter, 0.5 × 10⁶ cells/0.5 mL and per well were seeded in 48-well plates (Greiner Bio-One, Frickenhausen, Germany) as described previously.²⁷ The cells were primed with LPS (1 μg/mL; E. coli O26:B6, Merck, Darmstadt, Germany, Cat# L2654) for 5 h under 5% CO2 atm at 37 °C. Thereafter, the P2 × 7 receptor agonist BzATP (100 μM; Jena Bioscience, Jena, Germany, Cat# NU-1620-5) was added for 40 min in the absence or presence of the compounds 2, 3f, and 3h or the cholinergic agonist ACh (10 μM; Merck, Cat# A6625). After the treatment, cells were spun down (500 g, 8 min, 4 °C) to collect the cell-free supernatants, which were stored at −20 °C for later measurements of IL-1β and lactate dehydrogenase (LDH) activity.

Human PBMCs were isolated from blood samples obtained from healthy (self-reported) female and male nonsmoking adult volunteers. The study was approved by the ethics committee of the medical faculty Giessen, Germany (no. 90/18) and performed in accordance with the Helsinki Declaration. PBMC isolation was performed using Leucosep gradients (Greiner Bio-One, Cat# 227288), as described previously.²⁷ To prime the cells with LPS, 5 ng/mL LPS was added to blood samples before the gradient centrifugation. After the isolation process, 0.5 × 10⁶ cells/0.5 mL and per well were seeded in 48-well plates (Greiner Bio-One) in Monocyte Attachment Medium (PromoCell, Heidelberg, Germany, Cat# C-28051) for 3 h. Nonadherent cells were removed, and cell culture medium was replaced by fresh RPMI 1640 medium (Sigma-Aldrich, Cat# R8758). Stimulation with BzATP in the presence or absence of the compounds 2, 3f, or ACh was done as described for THP-1 cells.

To measure IL-1β concentrations in the cell-free supernatants, the human IL-1 beta/IL-1F2 DuoSet enzyme-linked immunosorbent assay (ELISA) from R&D Systems (Cat# DY201) was used according to the supplier’s instructions. In parallel, LDH activity was measured to test for cell viability at the end of the cell culture experiments, using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, United States; Cat# G1780) according to the supplier’s instructions. The viability of the cells was not impaired in any condition tested (data not shown).

Results obtained in the BzATP-induced IL-1β release experiments were analyzed using SPSS (Version 27, IBM, Armonk, NY, United States). The data were analyzed first by the Friedman test followed by the Wilcoxon signed-rank test. Data were visualized using Inkscape version 0.48.5 r10040 (Free and Open Source Software licensed under the GPL). The number (n) of individual experiments is indicated in the figures and refers to independent experiments, which were performed on different days with different cell passages.

4.6.1. Measurements of Inflammatory Pain

4.6.1.1. Animals

Experiments were conducted using adult (10–15 weeks) male and female C57BL/6J mice from Jackson Laboratory (Bar Harbor ME, USA): α7 WT and KO mice on a C57BL/6J background. Mice null for the α7 subunit along with their WT littermates were initially procured from Jackson Laboratory and later bred in an approved animal care facility at Virginia Commonwealth University. The breeding scheme involved crossing heterozygous mice and backcrossing progeny for at least 12 to 15 generations, to control for irregularities that might occur crossing solely mutant animals, to generate both mutant and WT animals. Then mice were weaned at 21 days of age and subsequently housed in groups of two to five with Teklad corn cob bedding (#7097, Envigo Teklad, Madison WI, USA). Initially, they were maintained in a temperature- and humidity-controlled vivarium space (21 ± 3 °C, 55 ± 10%) on a 12 h light/dark cycle (lights on at 7:00 AM) with free access to food (Teklad LM-485 mouse sterilized diet, Harlan Laboratories Inc., Indianapolis IN, USA) and water until needed. Then mice were retrieved from the vivarium and housed (4–5 mice per cage) for the duration of the study in a temperature- and humidity-controlled out-of-vivarium space on the same light/dark cycle. They were given ad libitum food and water. All experiments were performed during the light cycle. This study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University (approval #AM10142) and carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All experimental animals were included in further behavioral testing and none of them showed behavioral disturbances unrelated to the pain induction procedure.

4.6.1.2. Induction of Inflammatory Pain by Complete Freund’s Adjuvant

We explored the effects of 3h and 3f in the CFA test, composed of inactivated and dried Mycobacterium tuberculosis and adjuvant, a widely used model of persistent inflammatory pain.⁵⁴ CFA was purchased from Sigma-Aldrich (St. Louis MO, USA). The CFA model is based on hypersensitivity, paw swelling, and nuclear factor-κB—mediated transcription of tumor necrosis factor α involved in the formation of the principal mediators of inflammation.⁵⁵ Mice were injected intraplantarly with 20 μL of CFA (50%, diluted in mineral oil; Sigma-Aldrich). Mechanical sensitivity (see the measurement of the von Frey test) was measured before and 3 days after CFA injection. Compound 3h [2 and 10 mg/kg body weight (bw)] and 3f (20 mg/kg bw), dissolved in a mixture of 1:1:18 [1 volume ethanol/1 volume Emulphor-620 (Rhone-Poulenc, Inc., Princeton NJ, USA)/18 volumes distilled water] or vehicle, was injected intraperitoneally (i.p.) on day 3 after CFA injection, and mice were tested for mechanical sensitivity at different time points (1, 3, 6, 24, and 72 h and 5 days for compound 3h and 1, 3, 6, 24, and 72 h for compound 3f after drug injection.

4.6.1.3. Evaluation of Mechanical Sensitivity

Mechanical sensitivity thresholds were determined according to the method of Chaplan et al.⁵⁶ and as adapted in Bagdas et al., 2015.⁵⁷ A series of calibrated von Frey filaments (Stoelting, Wood Dale IL, USA) with logarithmically incremental stiffness ranging from 2.83 to 5.07 expressed as diameter sensitivity (ds) log 10 of 10 x force (in milligrams) were applied to the paw with a modified up–down method.⁵⁸ The mechanical threshold was expressed as log 10 of 10× force (in mg), indicating the force of the von Frey hair to which the animal reacted (paw withdrawn, licking, or shaking). All behavioral testing on animals was performed in a blinded manner.

The data obtained were discrete values that were not normally distributed and, hence, not suitable for parametric analysis. The data were therefore first evaluated with the nonparametric Kruskal–Wallis test, a one-way analysis of variance by ranks. A significant Kruskal–Wallis test indicated that at least one sample stochastically dominated the other samples, in this case, the baseline data for all groups. For analyzing the specific sample pairs at the different time points for stochastic dominance, as a second step the groups were tested pairwise with the Mann–Whitney rank sum test. The P values calculated by the Mann–Whitney rank sum test are provided in the Tables 1a–h or shown in the Figures 11 and 12.

4.6.1.4. Locomotor Activity

Mice were placed into individual Omnitech photocell activity cages (28 × 16.5 cm) (Columbus OH, USA) 6 h after administration of either vehicle or compound 3h (10 mg/kg bw, i.p.). Interruptions of the photocell beams (two banks of eight cells each) were then recorded for the next 60 min. Data are expressed as number of photocell interruptions.

Glossary

Abbreviations

ACh

acetylcholine

BINAP

2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

CAS

cholinergic anti-inflammatory system

PAM

positive allosteric modulator

nAChR

nicotinic acetylcholine receptors

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02429.

• ¹H NMR, ¹³C NMR spectra, HRMS, and the HPLC chromatograms of synthesized precursors and final compounds for biological assays (PDF)

• Molecular formula strings (CSV)

• Docking model of compound 3f at ECD at the α9-α9 interface (PDB)

• Docking model of compound 3h at ECD at the α9-α9 interface (PDB)

Author Contributions

H.A., N.A.H., R.L.P., and G.A.T. contributed to the conception and design of the study and conduction of experiments and majorly contributed to manuscript writing and reviewing. N.A.H., G.A.T., and R.L.P. supervised the entire project. H.A. conducted the synthesis of all compounds, their purification, and characterization—¹HNMR, ¹³CNMR, LC–MS, and HRMS—and was responsible for making Supporting Information and writing of manuscript and preparation of figures, tables, schemes, and so forth. H.A. and N.A.H. carried out computational studies and writeup and prepared figures. C.S. and R.L.P. conducted electrophysiology experiments, analyzed data, prepared figures, and wrote the manuscript. K.R. and V.G. were responsible for the cell-based assays and data collection for the experiments on monocytic THP-1 cells and human PBMCs and performed related experimental writeup. S.M.H., K.C., and M.I.D. conducted in vivo studies including anti-inflammatory activity mechanical hypersensitivity and locomotor activity. S.R.R.K. and A.S. conducted permeability and plasma stability studies in murine plasma. All authors reviewed the final version of the manuscript before submission.

The authors declare no competing financial interest.