Subnanomolar Affinity and Selective Antagonism at α7 Nicotinic Receptor by Combined Modifications of 2-Triethylammonium Ethyl Ether of 4-Stilbenol (MG624)

Abstract

Modifications of the cationic head and the ethylene linker of 2-(triethylammonium)ethyl ether of 4-stilbenol (MG624) have been proved to produce selective α9*-nAChR antagonism devoid of any effect on the α7-subtype. Here, single structural changes at the styryl portion of MG624 lead to prevailing α7-nAChR antagonism without abolishing α9*-nAChR antagonism. Nevertheless, rigidification of the styryl into an aromatic bicycle, better if including a H-bond donor NH, such as 5-indolyl (31), resulted in higher and more selective α7-nAChR affinity. Hybridization of this modification with the constraint of the 2-triethylammoniumethyloxy portion into (R)-N,N-dimethyl-3-pyrrolidiniumoxy substructure, previously reported as the best modification for the α7-nAChR affinity of MG624 (2), was a winning strategy. The resulting hybrid 33 had a subnanomolar α7-nAChR affinity and was a potent and selective α7-nAChR antagonist, producing at the α7-, but not at the α9*-nAChR, a profound loss of subsequent ACh function.

Introduction

The triethylammonium ethyl ether of 4-stilbenol (1a, MG624) has returned to the fore in very recent years after being reported in the 1950s as a ganglioplegic agent with very weak antimuscarinic activity and no activity on the neuromuscular junction^1,2 and first characterized in 1998 as an antagonist of the homopentameric α7 nicotinic acetylcholine receptors (nAChRs) with moderate and high selectivity, respectively, over the β4- and the β2-containing nAChRs.³ An expanded knowledge of biochemistry, molecular pharmacology, and physiology of nAChR subtypes, along with a number of structure–activity relationship (SAR) studies, has allowed a fuller understanding of MG624′s pharmacological profile and its multifaceted potential as a therapeutic hit.⁴⁻⁶ Starting from the proven ability of nicotine to promote growth and metastasis of lung tumors by acting on α7- and α9α10-nAChRs, we have initially demonstrated that 1a blocks these proproliferative effects on adenocarcinoma cells expressing such nAChRs.⁴ We have enlarged the investigation to glioblastoma and to analogues of 1a with elongated O–N alkylene linker, further confirming the antitumor activity and finding that it is greatly advantaged by ethylene bridge lengthening, which generally corresponds to the increasing potency of α7- and α9α10-nAChR antagonism.⁵

A deeper pharmacological and functional characterization of 1a and its two analogues with tetramethylene and octamethylene O–N linker (1b and 1c, respectively; Chart 1) led us to conclude that, at the α7-nAChRs, they behave as a very weak partial agonist (1a), a silent agonist (1b), and a full antagonist (1c) and that their antiproliferative and cytotoxic effects are not only due to the action on nAChRs.⁶ Other non-nicotinic intracellular mechanisms are involved, such as the reduction of the production of mitochondrial and glycolytic adenosine triphosphate (ATP),^5,6 and further studies are needed to understand whether they are independent or cooperative with nicotinic antagonism. Leaving aside the multiple and incompletely defined mechanisms underlying antiproliferative effects (which are therefore hard to interpret), we returned to the electrophysiological assessment of α7- and α9α10-nAChR subtypes and a systematic SAR study. We have very recently reported a series of analogues of 1a modified at the ammonium head or at the two-carbon O–N linker.⁷ Some of these modifications, detrimental to the α7-nAChR affinity, such as the inclusion of the linker in six-membered nitrogen heterocycles (1e, 1f, and 1g; Chart 1) or oversized increase or decrease of the ammonium head volume (1d and 1h, respectively; Chart 1), led to selective antagonists of human α9α10-nAChR, devoid of any antagonist activity at the α7-nAChR and showing partial agonism at high supramicromolar concentrations. As noted in our recent publication, their selective α9α10-nAChR antagonist activity appeared to consist of opening and rapidly engaging the channel and then blocking it in an open but nonconducting state. These observations are compatible with an open-channel block mechanism,⁷ although we emphasize that a definitive demonstration of such a mechanism would require extensive further testing (e.g., competition and voltage-dependence experiments). Among these selective α9α10-nAChR antagonists, the cyclohexyldimethylammonium analogue 1d (Chart 1) stands out for having no α7-nAChR agonist or antagonist effect and very low affinity for the ganglionic α3β4 nicotinic subtype, thus proposing itself as an invaluable tool to define the therapeutic potential of the α9α10-nAChR antagonism.⁷

Chart 1. 1a (MG624) and its Analogues Modified at the Ammonium Ethyl Residue.

As a second part of the SAR investigation on 1a, we considered modifications at its stilbene scaffold, more specifically at the styryl portion, which represents the distal part of such scaffold and whose modifications were expected to be highly influential, as evidenced by the present results, on the interaction with the α7- and α9α10-nAChR subtypes. Here, we report the synthesis and the biological evaluation of compounds 3–33 (Chart 2), in which (a) the styryl residue of 1a is totally or partially abolished (3–5), made linear (7), derigidified (6), or further rigidified (8 and 9) also with phenyl bioisosteric replacement (30–32), decorated at phenyl (12–22) or benzo-condensed (10 and 11), or modified at the vinylene portion by the introduction of heteroatoms and cyclization (23–29) and (b) the two most productive modifications of 1a in terms of the α7-nAChR affinity of this series and of the previous one,⁷ respectively, represented by the indolyl analogue 31 and the stilbenoxypyrrolidine 2 (Chart 2), are combined to give hybrid 33. The biological evaluation was performed similarly to that for previously reported analogues of 1a modified at the ammonium ethyl residue.⁷ First, an extensive determination of the nAChR subtype binding affinities was performed, followed by the functional screening of a large selection of compounds for α7- and α9α10-nAChR antagonisms and then more detailed tests on a few best hits to further study the mechanism of the antagonist activity at the two receptor subtypes.

Chart 2. 1a (MG624), its Analogues Modified at the Styryl Portion (3–32), its Previously Reported Analogue Modified at the Ammonium Ethyl Residue (2), and its Analogue Modified at Both the Substructures (33).

Results

Chemistry

Compounds 3–5, 10–20, and 23 were synthesized from phenol (compound 3), 4-phenylphenol (compound 5), hydroquinone (compound 23), and p-hydroxybenzaldehyde (compounds 4 and 10–20) according to Scheme 1.

Scheme 1. Reagents and Conditions.

(a) 2-Chloro-N,N-diethylethylamine hydrochloride, K₂CO₃, KI, acetone or methyl ethyl ketone, reflux; (b) iodoethane in 1,2-dichloroethane, rt for 3, 23; dichloromethane (DCM), reflux for 4, 5; neat, reflux for 17; EtOH, 70 °C for 10, 11; tetrahydrofuran (THF), reflux for 12–14, 16, 18, 67; (c) 1,2-dibromoethane, K₂CO₃, KI, methyl ethyl ketone, reflux; (d) NaI, acetone, reflux; (e) diethylamine, toluene, 60 °C; (f) benzyl bromide, K₂CO₃, acetone, reflux; (g) acetic anhydride, pyridine, rt; (h) methyltriphenylphosphonium bromide, K₂CO₃, THF, reflux; (i) 5 M NaOH, THF, 0 °C; (j) appropriate aryl iodide, Pd(OAc)₂, triethylamine, CH₃CN, reflux; (k) 1.25 M HCl in MeOH, reflux; (l) triethylamine, toluene, reflux; (m) 1-naphthylmethyltriphenylphosphonium chloride, sodium, EtOH, 10 °C to rt; and (n) 2-naphthylmethyltriphenylphosphonium bromide, sodium, EtOH, 10 °C to rt.

Phenol was O-alkylated with diethylaminoethyl chloride to give 34, which was quaternarized to 3 with ethyl iodide.

To obtain the p-vinylphenyl ether 4, p-hydroxybenzaldehyde was acetylated (40), submitted to Wittig olefination with methylenetriphenylphosphorane (41), desacetylated (42), O-alkylated with 1,2-dibromoethane (43), converted into the iodoethyl ether 44, and then reacted with diethylamine to give 45, which was quaternarized with ethyl iodide (4). Starting from 4-phenylphenol, these last four steps (O-bromoethylation, bromine/iodine exchange, diethylamine reaction, quaternarization) led to 5.

Intermediate 42 was also used to synthesize compounds 12–20. The three positional isomers 12–14 were prepared from 42 by coupling with 2-bromo-, 3-bromo-, and 4-bromoiodobenzene, respectively, followed by etherification of phenol with diethylaminoethyl chloride and quaternarization with iodoethane. By the same steps, but using 4-trifluoromethyl-, 3-methoxy-, and 4-methoxyiodobenzene, respectively, we synthesized compounds 16–18. The synthesis of 15 started from 3-trifluoromethyliodobenzene, which was coupled with 42. The resulting intermediate 58 was O-bromoethylated with 1,2-dibromoethane (60), converted into the 2-iodoethyl analogue 62, and then reacted with triethylamine to give 15. The two positional isomers 19 and 20 were prepared by coupling 42 with MEM-protected 3-iodophenol yielding 59 and with 4-iodophenol yielding 65. For the synthesis of 19, the subsequent steps were O-bromoethylation (61), bromine/iodine exchange (63), MEM deprotection (64), and reaction with triethylamine. For the synthesis of 20, the O-MEM intermediate 65 was reacted with diethylaminoethyl chloride (66), quaternarized with iodoethane (67), and MEM-deprotected.

The olefination of p-hydroxybenzaldehyde with 1-naphthylmethylenetriphenylphosphorane yielded intermediate 68, which was treated with diethylaminoethyl chloride to give the tertiary amine 69 and then converted into 10 by treatment with ethyl iodide. The olefination of p-hydroxybenzaldehyde with 2-naphthylmethylenetriphenylphosphorane provided intermediate 70 and its cis isomer, which were separated by chromatography. Successive etherification of 70 with diethylaminoethyl chloride and quaternarization with iodoethane gave 11.

Compound 23 was obtained from hydroquinone by etherification of one hydroxyl with benzyl bromide (38) and of the other with diethylaminoethyl chloride (39), followed by quaternarization of the tertiary amine 39 with iodoethane.

Compounds 6, 7, and 22 were synthesized from trans-4-stilbenol, 4-(2-phenylethynyl)phenol, and resveratrol, respectively, according to Scheme 2. Stilbenol was hydrogenated to 4-(2-phenylethyl)phenol (72), O-alkylated with diethylaminoethyl chloride (73), and quaternarized with iodoethane to give 6. 4-(2-Phenylethynyl)phenol was O-alkylated with 1,2-dibromoethane (74) and, after bromine/iodine exchange (75), converted to 7 by reaction with triethylamine. Resveratrol was chloroethylated at the 4′-hydroxyl with 1-bromo-2-chloroethane (76) and, after chlorine/iodine exchange (77), converted to 22 by reaction with triethylamine.

Scheme 2. Reagents and Conditions.

(a) H₂, Pd/C, MeOH, rt; (b) 2-chloro-N,N-diethylethylamine hydrochloride, K₂CO₃, KI, methyl ethyl ketone, reflux; (c) iodoethane, toluene, 90 °C; (d) 1,2-dibromoethane, K₂CO₃, KI, methyl ethyl ketone, reflux; (e) NaI, acetone, reflux; (f) triethylamine, toluene, rt for 7, reflux for 22; and (g) 1-bromo-2-chloroethane, K₂CO₃, N,N-dimethylformamide (DMF), 60 °C.

Scheme 3 shows the syntheses of compounds 8, 9, 25–27, and 30–33. 4-Hydroxyphenyl boronic acid was coupled with 2-bromo- and 1-bromonaphthalene and the resulting intermediates, 78 and 80, respectively, were O-alkylated with diethylaminoethyl chloride (79 and 81) and quaternarized to 8 and 9, respectively, with ethyl iodide. By the same reaction sequence, we synthesized the final compounds 30 and 32 from 4-hydroxyphenyl boronic acid using 6-bromoindole and 5-bromobenzofuran respectively. For the synthesis of 31, 4-hydroxyphenyl boronic was coupled with N-tosyl-5-bromoindole and the resulting intermediate tosyl amide 96 was hydrolyzed to 97, O-alkylated with diethylaminoethyl chloride (98), and converted to 31 with iodoethane. Intermediate 96 was coupled, by the Mitsunobu reaction, with (R)-N-boc-3-hydroxypyrrolidine to give 99. Subsequent reduction with LiAlH₄ provided the N-methyl pyrrolidine 100, which was converted to 33 by treatment with iodomethane. The preparation of 25 and 26 was accomplished from 4-benzamidophenol (88) and 4-hydroxybenzanilide (84), respectively, through the same sequence of reactions: O-chloroethylation (89 and 85), chlorine/iodine exchange (90 and 86), reaction with diethylamine (91 and 87), and quaternarization with iodoethane (25 and 26). 4-Benzamidophenol (88) was prepared from 4-aminophenol, while 4-hydroxybenzanilide (84) was prepared from p-salicylic acid by acetylation (82), conversion into p-hydroxybenzoyl chloride, and reaction with aniline (83) and desacetylation. For the synthesis of 27, phenol was coupled with benzenediazonium salt, generated in situ from aniline, and the obtained compound 92 was reacted with diethylaminoethyl chloride (93) and quaternarized to 27 with iodoethane.

Scheme 3. Reagents and Conditions.

(a) Appropriate aryl bromide, Pd(PPh₃)₄, tetrabutyl ammonium bromide (TBAB), EtOH/2 M_aq Na₂CO₃, 1,2-dimethoxyethane or EtOH/toluene, reflux; (b) 2-chloro-N,N-diethylethylamine hydrochloride, K₂CO₃, KI, methyl ethyl ketone, reflux; (c) iodoethane: neat, reflux for 8, 9, 25, 26; DCM, rt for 27; THF, reflux for 30–32; (d) Ac₂O, H₂SO₄, 80 °C; (e) 1° step: ClCOCOCl, DCM, DMF, rt; 2° step: aniline, DCM, rt; (f) 1 M NaOH, MeOH, rt; (g) 1-chloro-2-bromoethane, Cs₂CO₃, DMF, 60 °C; (h) NaI, acetone, reflux; (i) diethylamine, reflux; (j) benzoic anhydride, sodium octyl sulfate, H₂O, CH₃CN, rt; (k) 1° step: aniline, NaNO₂, 37% HCl, H₂O, 0 °C to rt; 2° step: NaHCO₃, rt; (l) KOH, MeOH, reflux; (m) tert-butyl (S)-3-hydroxypyrrolidine-1-carboxylate, PPh₃, diisopropyl azodicarboxylate (DIAD), THF, −10 °C to reflux; (n) LiAlH₄, THF, −10 °C to reflux; and (o) iodomethane, THF, 40 °C.

Scheme 4 shows the syntheses of compounds 24, 28, and 29. 4-Hydroxybenzaldehyde was etherified with diethylaminoethyl chloride to intermediate 103, reduced to benzyl alcohol 104, transformed into phenyl ether 105 by the Mitsunobu reaction with phenol, and quaternarized to 24 with ethyl iodide. Intermediate 103 was also used to synthesize the benzoxazole nucleus of 28 and the benzimidazole nucleus of 29. The addition–elimination reaction of 103 with o-aminophenol provided the Schiff base 106, which was transformed into 107 by oxidative cyclization and then quaternarized to 28 with ethyl iodide. The benzimidazole intermediate 108 was directly obtained from 103 by reaction with 2-aminoaniline in the presence of lead tetraacetate. Final quaternarization with ethyl iodide provided 29.

Scheme 4. Reagents and Conditions.

(a) 2-Chloro-N,N-diethylethylamine hydrochloride, K₂CO₃, KI, methyl ethyl ketone, reflux; (b) NaBH₄, MeOH, rt; (c) phenol, PPh₃, DEAD, THF 0 °C to rt; (d) iodoethane, rt; (e) o-aminophenol, EtOH, reflux; (f) Pb(OAc)₄, EtOH, reflux; (g) o-phenylenediamine, Pb(OAc)₄, EtOH, reflux; and (h) iodoethane, 1,2-dichloroethane, rt.

Biology

Binding Studies

The binding affinities (K_i) of all of the compounds were determined by competition binding experiments on the α7 human subtype, transiently expressed in the SH-SY5Y neuroblastoma cells,⁵ and the results are shown in Table 1. With the exception of a few compounds that had a modest affinity for α7-nAChR, competitive binding affinity was also assessed at the human α3β4-nAChR subtype stably transfected in SH-EP1 cells⁸ and only select compounds were also tested on the human α4β2-nAChR subtype stably transfected in HEK 293 cells (a generous gift from Dr. Jon Lindstrom⁹).

Table 1. Affinity (Ki in μM) of Compounds for the Human α7, α3β4, and α4β2-nAChR Subtypes.

	α7-nAChR [125I]-αBgtx Ki (μM)	α3β4-nAChR [3H]-Epi Ki (μM)	α4β2-nAChR [3H]-Epi Ki (μM)		α7-nAChR [125I]-αBgtx Ki (μM)	α3β4-nAChR [3H]-Epi Ki (μM)	α4β2-nAChR [3H]-Epi Ki (μM)
1a	0.104 (0.55–0.202)	0.433 (0.227–0.823)	5.7 (3–10.6)	18	0.353 (0.146–0.853)	0.187 (0.090–0.388)	nd
2	0.023 (0.09–0.055)	2.700 (1.800–4.200)	9.3 (2.9–30)	19	0.573 (3.79–0.866)	0.998 (0.714–1.280)	10.1 (5.5–18.5)
3	27 (17.6–44.3)	10 (6.2–19)	19 (10–37)	20	0.342 (225–0.520)	0.873 (0.484–1.575)	9.82 (5.8–16.6)
4	0.285 (0.110–0.379)	1.070 (0.618–1.800)	24 (6.5–91)	21	0.189 (0.096–0.393)	0.676 (0.406–1.125)	1.2 (0.49–3.3)
5	0.129 (0.048–0.349)	0.440 (0.247–0.784)	11.8 (7–37)	22	0.242 (0.139–0.422)	0.793 (0.579–1.084)	7.24 (4.2–12.4)
6	1.646 (0.643–4.208)	1.249 (0.590–2.643)	nd	23	1.010 (0.380–2.670)	4.700 (3.400–6.400)	7.5 (10–37)
7	0.664 (0.416–1.061)	0.912 (0.693–1.200)	19.4 (3.5–105)	24	2.600 (1.020–6.800)	nd	nd
8	0.081 (0.046–0.145)	0.458 (0.191–1.000)	1.8 (0.69–4.7)	25	0.862 (0.522–1.400)	1.070 (0.208–5.500)	4.5 (0.77–27)
9	3.107 (1.532–6.299)	0.097 (0.418–2.263)	nd	26	0.250 (0.110–0.623)	0.113 (0.026–0.488)	23 (3.9–100)
10	1.347 (0.488–3.713)	0.501 (0.255–0.985)	nd	27	0.526 (0.310–0.805)	nd	nd
11	1.004 (0.425–2.374)	0.425 (0.192–0.985)	nd	28	0.166 (0.071–0.391)	0.653 (0.316–1.346)	nd
12	0.525 (0.293–0.940)	nd	nd	29	0.0336 (0.016–0.072)	0.345 (0.152–0.781)	nd
13	1.036 (0.539–1.990)	nd	nd	30	0.184 (0.091–0.374)	0.174 (0.067–0.453)	nd
14	0.723 (0.421–1.240)	nd	nd	31	0.0187 (0.0086–0.0402)	0.177 (0.078–0.403)	nd
15	1.525 (0.987–2.358)	1.411 (0.926–2.150)	21.8 (17–28)	32	0.450 (0.198–1.022)	0.343 (0.155–0.759)	nd
16	1.334 (0.734–2.423)	0.874 (0.597–1.280)	10.4 (4–26.7)	33	0.00082 (0.00065–0.00123)	0.365 (0.274–0.485)	5.1 (3.1–8.4)
17	0.621 (0.415–0.921)	nd	nd

Heterologously expressed human receptors were used. α4β2 and α3β4-nAChR subtypes were expressed in HEK 293 or SH-EP1 cells, respectively; human α7-nAChR was expressed in SH-SY5Y human neuroblastoma cells. Binding was determined using as ligand [³H]epibatidine for α4β2- and α3β4-nAChR subtypes and [¹²⁵I] α-bungarotoxin for the α7-subtype. Saturation and competition binding data were evaluated by one-site competitive binding curve-fitting procedures using GraphPad Prism version 6 (GraphPad Software, CA). In the saturation binding assay, the maximum specific binding (B_max) and the equilibrium binding constant (K_d) values were calculated using one-site—specific binding with the Hill slope—model. Inhibition constants (K_i) were obtained by fitting three independent competition binding experiments, each performed in duplicate for each compound on each subtype and were estimated by reference to the K_d of the radioligand, obtained in separate saturation binding experiments, according to the Cheng–Prusoff equation and expressed in micromolar. The numbers in parentheses of K_i values represent the confidence interval of the value.

We found that, among the compounds modified by structural simplification or rigidification of the styryl residue (3–9), only compounds 5 and 8 had an α7-nAChR K_i value close to that of the parent compound 1a (K_i = 104 nM) and maintained a modest α7- vs α3β4-nAChR and a high α7- vs α4β2-nAChR selectivity.

The second set of analogues of 1a, those decorated at the distal phenyl by substituents or an additional condensed benzene (compounds 10–22), showed both a lower affinity for α7-nAChR and reduced selectivity over α3β4-nAChR (where measured).

The same outcome was seen across the third set of analogues (compounds 23–27), those with an ether, an amide, or a diazo linker in place of vinylene. However, replacement of the vinylene linker with an imino linker locked into oxazole or imidazole condensed with the distal phenyl (compounds 28 and 29) led, in the case of benzimidazole 29, to an improved α7-nAChR affinity (K_i = 33.6 nM) and α7- vs α3β4-nAChR selectivity (10.3 ratio) compared to 1a (K_i = 104 nM; α7- vs α3β4-nAChR selectivity = 4.2). Among the last set of compounds (30–32), formally derived from 8 by replacement of 2-naphthyl with 5- or 6-indolyl or 5-benzoxazolyl, analogous results were obtained for indole 31 (18.7 nM K_i and 9.5 ratio).

Finally, we determined for compound 33, a hybrid between compounds 31 and 2, very high α7-nAChR affinity (0.82 nM K_i), and α7- vs α3β4- and α4β2-nAChR selectivities (445 and 6200 ratios, respectively). Compound 2 is a previously reported analogue of 1a,⁷ modified at the O–N linker and endowed with high α7-nAChR affinity (23 nM K_i) and α7- vs α3β4- and α4β2-nAChR selectivities (117 and 404 ratios, respectively).

The binding affinities of the compounds for the α3β4- and α7-nAChR subtypes were generally similar or moderately different (≤10-fold ratio), except for the above-mentioned compounds 2 and 33 that had >400-fold preference for α7- over α3β4-nAChR and for compound 9, which had ≈30-fold higher affinity for the α3β4- than for the α7-nAChR. Approximately half of the compounds were also tested for their affinity for the α4β2-nAChR, and we determined that all had low affinity (K_i > 1.2 μM) including compounds 1a, 2, 5, 8, and 33 that we have determined to have high α7-nAChR affinity.

In Vitro Functional Activity on α7 and α9α10-nAChR Subtypes

Compound 1a was earlier shown to be an antagonist of chicken α7-nAChR expressed in Xenopus laevis oocytes (IC₅₀ = 109 nM) and, more recently, at human α7 and α9α10-nAChR expressed in X. laevis oocytes (IC₅₀ = 41 and 10 nM, at the respective subtypes).^3,5

Of the compounds reported in this manuscript, nine (6, 25–31, and 33) were chosen for further testing in functional assays. The selection was driven by the significantly higher α7-nAChR binding potency and selectivity for α7- over α3β4-nAChR binding shown by 29 and 31 in comparison with 1a, suggesting further rigidification and introduction of a weakly acidic NH in a suitable position as critical modifications of the styryl moiety of 1a. Therefore, in vitro functional tests were extended also to benzamides 25 and 26, benzoxazole 28, and indole 30, in which one or both the above modifications at the styryl moiety are featured. Compound 33 was selected on the basis of its greatly improved (subnanomolar) α7-nAChR affinity and better α7- over α3β4-nAChR selectivity compared to any of the other compounds considered in this study, while the diazo derivative 27 and the phenylethyl analogue 6 were included for the significance of their respective linker modifications. Antagonism of currents activated by 1 mM ACh was determined using X. laevis oocytes that expressed human α7- or α9α10-nAChR. Test compounds were coapplied during agonist stimulation. The approach and apparatus were similar to those earlier published for α7-nAChR.¹⁰ However, in this case, α9α10-nAChR was also tested (from oocytes injected with α9 to α10 cRNAs at a 9:1 ratio). As noted in our recent publication,⁷ the injection of α9-nAChR cRNA alone produces very little function. In contrast, the injection of our chosen ratio of α9 and α10 cRNA (9:1) produced the most function. Use of this α9:α10 cRNA injection ratio will likely produce functional α9α10-nAChR incorporating subunits in two different stoichiometries: (α9)₂(α10)₃ and (α9)₃(α10)₂.¹¹ The just noted increase in function following coinjection of the α10 subunit (compared to that if the α9 subunit cRNA is injected alone) further reassures us that the α9-only-nAChR function will be either minimal or absent under the 9:1 α9:α10 cRNA coinjection condition that we use in this and our previous manuscript. We chose to use the same experimental approaches for the present study to allow comparisons to be made to our recently published data.⁷ Functional responses of α7-nAChR were assessed using both the measurement of peak currents and net charge gated (area under curve or AUC) to determine whether the rapid-desensitizing property of this subtype at high agonist concentrations might alter the IC₅₀ values obtained.¹² The concentration response curves thus obtained are shown in Figure 1, and the IC₅₀ values calculated in each case are summarized in Table 2. Also given in Table 2 are IC₅₀ values for the lead compound (1a) and for 2 (for comparison since, together with 31, it is the parent compound of 33). As may be seen, in this experiment, under the conditions applied here, IC₅₀ values calculated using either peak current or AUC measurements of the α7-nAChR function were extremely similar. Despite this, it is worth mentioning that the exceptionally rapid kinetics of the α7-nAChR function at high agonist concentrations raise a concern that the coapplication of antagonists may result in inhibition being measured when drug application is incomplete. For this reason, later parts of this study examined the effects of applying the test compounds by themselves, rather than in a coapplication format.

Figure 1.

Inhibition concentration response profiles of test compounds at α7- or α9α10-nAChR subtypes. mRNA encoding human α7-nAChR subunit was coinjected in X. laevis oocytes along with mRNA for NACHO (in enhanced expression of α7-nAChR; ● or ⧫). Separate batches of oocytes were injected at a 9:1 ratio with mRNA encoding human α9- and α10-nAChR subunits, respectively (open circles). In both cases, the function was tested 1 week after injection, employing two-electrode voltage clamp electrophysiology. Initial stimulations were ACh-only (1 mM, 1 s stimulation, 60 s wash between stimulations, five repeats). These initial stimulations were used to confirm that agonist-alone responses were stabilized, and to provide a positive control, before test compounds were applied. Test compounds were coapplied with ACh stimulations (same 1 mM ACh concentration, 1 s application time, and 60 s wash between applications, as was used for the initial ACh-only stimulations). Concentrations of test compounds were increased from the lowest shown to a maximum of 100 μM in half-log steps. For α7-nAChR, responses were measured in two different ways (as peak currents (●) or as area under the curve (⧫). In all cases, responses when test compounds were coapplied were normalized to the mean of the magnitude of the final two positive control responses that preceded the introduction of the test compound. Each point is the mean ± standard error of mean (S.E.M.) of five to six responses, with each response being collected from an individual oocyte. Error bars are included for all points but are not visible where the size of the point exceeds that of the corresponding error bars. Even coapplication of compound 6 at 100 μM produced no inhibition of the α7-nAChR function; the resulting data have been omitted to increase clarity.

Table 2. Inhibition Potency (IC₅₀) of Test Compounds at α7- and α9α10-nAChR Determined from Concentration Response Curves Illustrated in Figure 1.

	α7 IC50 μM (peak current)	α7 IC50 μM (AUC)	α9α10 IC50 μM
1a	1.99 (1.78–2.24)	2.08 (1.10–3.93)	6.68 (5.62–7.76)
2	1.49 (1.23–1.78)	2.17 (1.32–3.58)	36.5 (17.0–77.6)
6	NA	NA	9.12 (7.76–10.72)
25	25.7 (21.9–30.2)	26.9 (20.4–35.5)	115 (41.7–316)
26	>100	>100	75.9 (61.7–93.3)
27	11.2 (9.77–12.9)	5.54 (4.27–7.18)	15.8 (11.7–21.4)
28	1.78 (1.45–2.29)	2.25 (1.78–2.85)	72.4 (31.6–207.0)
29	182 (129–257)	197 (112–347)	202 (144–288)
30	1.91 (1.48–2.45)	1.95 (1.63–2.34)	6.46 (5.49–7.49)
31	2.01 (1.70–2.34)	1.86 (1.22–2.81)	8.13 (6.31–13.2)
33	1.07 (0.89–1.29)	1.12 (0.62–2.03)	15.9 (11.8–21.4)

The summary of the test compound antagonist potency (IC₅₀ values) is derived using the concentration response curves illustrated in Figure 1. Details of the protocols used are given in the Experimental Section and Figure 1 (legend). Please note that IC₅₀ values at α7-nAChR were calculated using both peak current and area under the curve (AUC) approaches. Both approaches yielded similar values for all compounds tested. Confidence intervals (95% values) are provided in parentheses, which represent the 95% confidence interval of the mean value. “NA”, not applicable (i.e., agonist-induced function was not inhibited by the coapplication of the test compound even at 100 μM).

Except for 6, which had no effect at the α7-subtype, all of the tested compounds were able to inhibit ACh activity at both the subtypes: 27 and 29 with almost identical potency at α7 and α9α10-nAChR, 26 with selectivity toward the α9α10-nAChR, and the remaining 1a, 2, 25, 28, 30, 31, and 33 with higher potency at the α7-nAChR subtype. As shown in Figure 1, some compounds were not able to produce complete inhibition of the nAChR function. In some cases, inhibitory concentration response curves reached a plateau of incomplete antagonism. In others, the maximum test compound concentration of 100 μM was insufficient to produce complete inhibition. However, complete or nearly complete inhibition was observed at both α7- and α9α10-nAChR subtypes for 27, 30, 31, and 33. Among these four compounds, 33, the most potent α7-nAChR antagonist of the whole series (1.07 μM IC₅₀), showed the highest α7- vs α9α10-nAChR selectivity. Importantly, none of the compounds produced biphasic inhibition of the α9α10-nAChR function. This indicates that in no case do any of the test compounds discriminate between the alternate α9α10-nAChR stoichiometries described in the prior paragraph as likely to be present under the experimental conditions used in this study.

We emphasize here that while sequential applications of test compounds at progressively higher concentrations are common practice, it could result in compounding of effects (in this case, antagonism) produced by previous applications. For this reason, four compounds of special interest were selected to examine their potential intrinsic agonist affinity at α7- and α9α10-nAChR and the ability to affect function induced by a subsequent application of an ACh control response. These were compound 6, which exerted no inhibition of α7-nAChR but essentially full inhibition of α9α10-nAChR responses, and compounds 2, 28, and 33, all exhibiting the highest and most selective α7-nAChR antagonist activity (∼1 μM IC₅₀, 15–40-fold selectivity over α9α10-nAChR IC₅₀). As for the preceding experiment, repeated applications of ACh (1 mM) were used to ensure the stability of functional responses and define an agonist positive control response. Subsequently, each compound of interest was applied at a single concentration of 100 μM (no ACh present) to oocytes expressing α9α10-nAChR or, excluding 6, to oocytes expressing α7-nAChR. The 100 μM concentration was chosen since it matches the final concentration of the test compounds when they were coapplied with ACh in Figure 1, allowing outcomes to be compared directly. The application of a single concentration, in the absence of ACh, addresses the concern stated at the beginning of this paragraph that sequential applications of the test compounds could result in compounding of their effects.

Application of an individual 100 μM pulse of any of compounds 2, 6, 28, or 33 resulted in the partial agonism of α9α10-nAChRs (efficacy of 5–55% of ACh control when responses were measured in terms of the peak current). Interestingly, as previously reported for some analogues of 1a modified at the ammonium ethyl portion,⁷ all of the α9α10-nAChR functional responses induced by the test compounds were shorter-lasting than those evoked by the application of the ACh control. When responses were considered in terms of AUC, this resulted in efficacy being reduced to 0.2–3% of ACh control responses. Similar outcomes were found at α7-nAChR for compounds 2, 28, and 33 (15–50% efficacy compared to ACh control). When the intrinsic activity was assessed in terms of AUC, it was reduced somewhat (to 10–22% of the ACh control). This suggests that responses produced at α7-nAChR by the test compound were also somewhat truncated compared to those induced by ACh, albeit to a lesser extent than was seen at α9α10-nAChR. However, compound 6, which did not affect ligand binding at the α7-subtype also, did not have an intrinsic activity at α7-nAChRs (Figure 2). Of interest, compound 1a has also been reported recently to be an α7-nAChR partial agonist (response to a 100 μM application noted to be ≈40% of the 200 μM ACh control stimulation).⁶

Figure 2.

Partial agonism of human α7- or α9α10-nAChRs by compounds 2, 6, 28, or 33 (applied alone). Compounds 2, 6, 28, and 33 were selected (please see the test for criteria) to determine whether they were able to activate human α7- or α9α10-nAChR (intrinsic activity). Two-electrode voltage clamp protocols were similar to those used in Figure 1, including the use of an initial train of ACh (1 mM) control pulses to ensure the stability of responses and collect positive control data for a full agonist. After a further 1 min wash period, compounds of interest were applied for 1 s at 100 μM (the same as the highest concentration applied in Figure 1; in this case, test compounds were applied alone instead of coapplied with ACh). In this case, responses at both α7- and α9α10-nAChR were quantified in terms of both peak currents and AUC. For each individual oocyte, and for each method of quantification, responses when test compounds were coapplied were normalized to the mean of the magnitude of the final two positive control responses that preceded the introduction of the test compound. Each bar represents the mean response collected from three individual oocytes, with error bars representing the S.E.M. Points represent responses from individual oocytes.

Further, at α9α10-nAChR, rebound currents were observed, subsequent to the recovery of the short-duration currents produced in response to the test compound application. These rebound currents lasted longer than initial currents evoked by the test compounds or even preceding the ACh control responses. This phenomenon is illustrated in example traces, shown in Supporting Information Data (pages 20–26). In contrast, at α7-nAChR, only compound 33 evoked a rebound current (example traces are also provided in the same section of Supporting Information Data). Figure 3A illustrates the size of the poststimulation rebound currents evoked by 2, 6, 28, and 33 at α9α10-nAChRs and of 2, 28, and 33 at α7-nAChR. In each case, responses are normalized to the size of control responses previously evoked by ACh positive control responses. Responses are again presented in terms of both peak currents and AUC. As may be seen, peak currents attained during these rebound currents following test compound application varied between 25 and 40% of those produced by ACh control stimulations. However, when AUC was considered, rebound currents varied between 27 and 100% of control. This reflects the effects of the relatively slow onset and recovery of the rebound currents when compared to the initial responses to test compound application.

Figure 3.

Illustration of rebound current magnitudes and residual ACh activity following the application of compounds of interest. (A) Magnitudes of rebound currents appearing after the cessation of test compound application were recorded from α7- and α9α10-nAChRs. As for Figure 2, responses at both α7- and α9α10-nAChR were quantified in terms of both peak currents and AUC. (B) Residual activation evoked by a final ACh control application (1 mM, 1 s) applied 1 min after stimulation with each compound of interest. In this case, too, responses at both nAChR subtypes were measured as both peak current and AUC and, in each case, normalized to the ACh control responses that preceded the application of the test compound. Each bar represents the mean response collected from three to seven individual oocytes, with error bars representing the S.E.M. Points represent responses from individual oocytes.

Responses were also measured for a final ACh (1 mM, 1 s) control stimulation, applied 1 min after test compound application to each oocyte. These final ACh control applications produced a response that was reduced (in some cases, much reduced) in amplitude (whether in terms of peak current or AUC) than the initial ACh control applications. In Figure 3B, we illustrate the residual activities induced by these concluding ACh (1 mM) applications, subsequent to the application of compounds 2, 6, 28, and 33 to α9α10-nAChRs or compounds 2, 28, and 33 to α7-nAChRs. As illustrated in Figure 3B, 6 (which has no intrinsic efficacy at α7-nAChR) did, however, significantly block subsequent ACh-induced function at α9α10-nAChRs.

Moving to α7-nAChR responses, compound 33 (which has the highest α7-nAChR affinity and antagonist potency) was the only one in the series to produce an α7-nAChR rebound current (Figure 3A). This α7-nAChR rebound current induced by 33 was small in terms of peak amplitude, increased slowly, and was very slow to return to baseline. As a result of these slow response kinetics, the intrinsic activity was significantly higher when assessed as AUC than in terms of peak current (57 vs 7%, respectively). Notably, no distinct peak of function was induced by a subsequent control application of ACh; the block of subsequent ACh-induced α7-nAChR activity by compound 33 was thus essentially complete (Figure 3B).

We wished to examine if there was a correlation between the recovery of the rebound currents induced by test compounds at α9α10-nAChR and suppression of the final ACh control stimulation that follows the test compound application. These were calculated, respectively, as “recovery of rebound current” (the percentage by which the rebound current had returned to the prior baseline 1 min following application of the test compound; normalized for each individual oocyte to the peak amplitude of the rebound current over baseline) and “residual ACh-induced current” (i.e., the final ACh control stimulation applied 1 min following the application of the test compound, normalized for each oocyte as a percentage of the amplitude of the mean of the ACh control applications applied before the test compound was applied). Please refer to the Supporting Information Data (page 20) for an illustration of these terms. In our prior publication, we speculated that slow and incomplete recovery of the rebound current preceding the application of the final ACh control pulse could substantially suppress the functional response to the subsequent and final ACh control application.⁷ In Figure 4, we plot “residual ACh-induced current” (y-axis) against “recovery of rebound current” (x-axis) for the previously published compounds 1d, 1e, 1f, 1g, and 2 along with the new compounds 6, 28, and 33. Please note that, in this figure, only current amplitude data could be used since our previous publication assessed only peak response, and not AUC, values. As can be seen, there is a strong correlation between incomplete recovery of rebound current when the final ACh control pulse is delivered and increased inhibition of ACh-induced currents at α9α10-nAChR. Compounds 2, 28, and 33 showed an almost complete recovery and the highest residual ACh-induced currents, whereas compounds 1d, 1e, and 1f showed a largely incomplete recovery and the lowest residual ACh-induced currents. This confirms what we had speculated in our prior publication,⁷ reinforcing the suggestion that the longer the duration of the rebound current (i.e., the slower the disassociation of the compound of interest from the α9α10-nAChR), the greater the suppression of subsequent ACh-induced activity is.

Figure 4.

Relationship between residual ACh function after the application of test compounds to α9α10-nAChR and the extent to which the rebound currents that they induce are able to recover before the final ACh application is made (see the text for how these values were calculated). On the Y-axis, the mean amplitude ± S.E.M. of the residual ACh-induced current as % of the previously established ACh control amplitude; on the X-axis, the mean ± S.E.M. recovery of the rebound current as % of the peak rebound current.

Discussion

We began by determining the α7-nAChR binding affinity and selectivity over α3β4-nAChR. Regardless of their electronic effects, all of the accomplished modifications of 1a (K_i value at α7-nAChR = 104 nM) imply an increase of the steric bulk of the distal phenyl group (compounds 10–22) resulting in significantly lower α7-nAChR affinities. These bulk-increasing modifications also lowered, and sometimes reversed, α7- vs α3β4-nAChR selectivity. Such outcomes indicate that the extension of the styryl residue of 1a is a critical issue. Otherwise, within the set of compounds 3–9 (which abolished the distal phenyl or simply varied its positioning), the 2-naphthyl analogue 8 showed a profile of α7-nAChR affinities and α7- over α3β4-nAChR selectivities very similar to that of 1a. This suggests that the coplanarity of vinylene and phenyl is a requisite of the active conformer of 1a. Among the compounds modified at the vinylene linker (compounds 23–27), moderate α7-nAChR affinities are shown only by 26 and 27. Notably, these two compounds, having an amide and diazo linker, respectively, maintain the original styryl rigidity (unlike 23 and 24, which have a flexible methyleneoxy linker). Further, unlike the other benzamide 25, compounds 26 and 27 are superimposable to 1a and to its intramolecular cyclized 2-naphthyl analogue 8, respectively. These SARs are supported by the subsequent five compounds (28–32), which are all isosteres of 8, thus rigidified analogues of 1a, in which the 2-naphthyl of 8 is replaced by a heteroaromatic bicycle without extension with respect to the original styryl moiety but with additional interaction potential due to the presence of heteroatoms. Three of them (28, 30, and 32) show moderate α7-nAChR affinity and the other two, 29 and 31, show high α7-nAChR affinity (33.6 and 18.7 nM K_i, respectively). Compared to 1a and 8, benzimidazole 29 and indole 31 have not only significantly higher α7-nAChR affinity but also increased α7- vs α3β4-nAChR selectivity. In both, a critical role is played by NH, as indicated by the loss of α7-nAChR affinity resulting from its replacement with O (cf. 29 with 28 and 31 with 32) or its repositioning (cf. 31 with 30). Consistently with all of these observations, a great step forward is achieved by combining the two best modifications of 1a, in terms of α7-nAChR affinity and selectivity, at the stilbene scaffold and at the 2-ammonium ethyl portion, respectively: the replacement of the stilbene scaffold with 4-(5-indolyl)phenyl (compound 31, 18.7 nM K_i) and the previously reported constraint of the 2-ammoniumethyloxy portion into (R)-3-pyrrolidiniumoxy substructure (compound 2, 23 nM K_i). The effects of these two modifications are synergic and the resulting hybrid 33 displays subnanomolar α7-nAChR affinity and very high α7- vs α3β4- and α4β2-nAChR selectivities. In Chart 3, all of the above structure–affinity relationships are summarized and visualized reproducing, for clarity, the same subdivision of the styryl modifications as in Chart 2 and representing the productive and the unproductive ones compared to 1a in green and red, respectively.

Chart 3. 1a Analogues with Productive (in Green) and Unproductive (in Red) Modifications in Terms of α7-nAChR Affinity and α7- vs α3β4-nAChR Selectivity (Both Reported for the Ref (1)a and for the Improving Modifications).

To further elucidate the structural determinants for so high an increase in affinity at the α7-nAChR subtype, the molecular docking of 1a and 33 at the orthosteric binding pocket of the α7α7 dimer extracted and refined from the recently reported cryo-EM structure 7EKP was performed.¹³ As shown in Figure 5, both 33 and 1a assume a similar binding pose at the α7α7 subunit interface, superimposable with EVP-6124, the ligand complexed with the receptor in the original cryo-EM (not shown). In detail, the permanently charged quaternary ammonium head of both 33 and 1a is accommodated within the aromatic box formed by Tyr-115, Trp-171, Tyr-217, and Tyr-210, with which they establish π–cation interactions. Comparison between the shorter and more rigid N–O linker of 33 and the longer and flexible linker of 1a highlights an important difference in how far the aromatic moiety protrudes into the binding pocket: whereas the indole ring of 33 is still embedded within it, the distal aromatic ring of 1a extends out. As illustrated from the binding site analysis, both the O-linked styryl portion of 1a and the biphenyl portion of 33 are sandwiched in a lipophilic and narrow area (in yellow) between Leu-141 and Gln-79 at the top and Trp-77 and Ser-58 at the bottom. Instead, the terminal pyrrole group of (R)-33 is positioned at the hydrophilic entrance of the binding pocket, where H-bond donors are strongly preferred due to the presence of multiple H-bond acceptors on the target (such as the side chain of Ser-56 or the carbonyl of Glu-184). The additional H-bond network, together with a better fit in the binding pocket, is compatible with the 120 times increase of affinity from 1a to 33.

Figure 5.

Proposed binding mode of 33 (pink) and 1a (green) at the α7-nAChR orthosteric binding site (PDB ID: 7EKP). The receptor backbone is represented by sky-blue cartoons, and individual residues defining the binding site are colored in gray. π–cation interactions are shown as dashed green lines, while hydrogen bonds are shown as yellow dashed lines. The inner surface of the binding pocket is depicted in gray, lipophilic areas are in yellow, and hydrophilic areas where the H-bond donor is favored are in blue. Superimposition of the docking poses of 33 and 1a reveals the critical H-bonding between the indole moiety of 33 and the distal hydrophilic area of the α7α7 binding site, plausibly responsible for 120 times higher affinity.

Also here, as for the previously reported analogues of 1a modified at the ammonium ethyl residue,⁷in vitro functional activity at the α7 and α9α10-nAChRs was determined for a selection of analogues, 9 among the 31 initially tested for binding affinities. As explained above, the selection was centered on benzimidazole 29 and indoles 31 and 33, having the best α7-nAChR profiles; some of their strictest analogues (25, 26, 28, and 30) and, for the representativeness of the vinylene modification, compounds 6 and 27 were then recruited. According to such criteria, as in the previously reported selection of 12 1a analogues modified at the ammonium ethyl residue,⁷ compounds with modest or moderate α7-nAChR affinity (see 6, 25, 26, and 27) were tested for in vitro functional activity as well as compounds with good or high α7-nAChR affinity (28, 29, 30, 31, and 33). It is therefore significant that we obtained, applying selection criteria including a wide range of α7-nAChR affinities in both cases, divergent results for the two series of compounds (those made here vs in the preceding study⁷). Indeed, among the previously published 1a analogues (those modified at the ethyl ammonium head), we found only compounds unable to produce 100% inhibition of the ACh-induced function at the α7-nAChR or even completely devoid of α7-nAChR antagonism, but all antagonizing ACh activity at α9α10-nAChR. In contrast, among the present 1a analogues modified at the stilbene scaffold in the current study, only one compound, the 4-(2-phenylethyl)phenyl analogue 6, was devoid of α7-nAChR antagonism; all of the other compounds inhibited ACh-induced function at both α7- and α9α10-nAChR subtypes and, in the case of the three indolyl analogues 30, 31, and 33, produced 100% inhibition. Notably, compound 6 is, among the selected nine compounds, the one with the poorest α7-nAChR affinity, which could be imputed to the loss of that beneficial coplanarity suggested by the comparison of 8 with 1a. Overall, the modifications at the ethyl ammonium portion of 1a seem effective in impairing the interaction with the α7-nAChR, while α7- vs α9α10-nAChR selective antagonism can be achieved only by modifying both the stilbene scaffold and the ethyl ammonium head, as demonstrated by hybrid 33, endowed with subnanomolar α7-nAChR affinity, 100% inhibition of ACh-induced function at the α7-nAChR, and good antagonist selectivity for α7- over α9α10-nAChR.

Conclusions

If one considers the results obtained with the present modifications and those previously reported of 1a, one can immediately see that we have found in our prior publication several 1a analogues producing antagonism through a mechanism that we speculated was compatible with the open-channel block at α9α10-nAChR while being completely devoid of α7-nAChR antagonism. In contrast, the present study identified no 1a analogue with the opposite profile (i.e., block of α7-nAChR without α9α10-nAChR antagonism). Against this trend, all of the compounds that behave as antagonists at both the receptor subtypes are more potent at α7- than at α9α10-nAChR, except 26 in the present series. However, a marked α7- vs α9α10-nAChR-selective antagonism remains elusive. Only 33 shows a significantly selective antagonism at the α7-nAChR together with 100% inhibition of ACh-induced function at both the receptor subtypes. As depicted in Figure 3, it is the only one of the tested compounds that produces a profound loss of subsequent ACh-induced function at the α7-nAChR subtype (Figure 3B) and the only one that also produces a measurable rebound current at this same subtype (Figure 3A). These features of 33 at α7-nAChR are similar to those that we have described for multiple structurally related (but α9α10-nAChR-selective) antagonists and previously noted to be compatible with an open-channel blocker mechanism. However, as noted in the Introduction section, further experimentation is required to draw a firm conclusion as to the precise mechanism by which these compounds exert antagonism.

Overall, these results show that making the α7- and α9α10-nAChR antagonist 1a ineffective on one of the two subtypes or highly subtype-selective is not equally simple in both directions. Single modifications, such as the increase of the ammonium head bulkiness or rigidification of the ethylene linker (1d–1g),⁷ but also simple saturation of the vinylene bridge (6) are sufficient to profoundly or completely impair the effects at the α7-nAChR while maintaining the inhibition of ACh function at the α9α10-nAChR. On the other hand, we have not found single modifications of 1a resulting in the exact opposite behavior. However, we were able to obtain a complete loss of residual ACh-induced function at the α7-nAChR while leaving almost unaltered the residual ACh-induced function at the other subtype by making modifications at both the portions of 1a, the stilbene and the ethyl ammonium head. These modifications leading to 33 were suggested by the high α7-nAChR affinities of compounds 2 and 31, modified at the ethyl ammonium head and at the stilbene, respectively.

We can thus note that, with regard to 1a modifications, the α9α10-nAChR shows a wider tolerance for structural modifications than the α7-nAChR and this may account for the fact that differentiating α9α10-nAChR antagonism from α7-nAChR antagonism, using 1a as a starting hit, is less difficult than the reverse outcome, for which a finer modulation of the molecular features of the hit is required.

There is a great interest in the physiological roles of α7- and α9α10-nAChR and their druggability for the development of optimized therapeutics.¹⁴ To this end, the production of ligands that can reliably discriminate functional effects mediated by α7- or α9α10-nAChR is absolutely critical. The identification of 33 and 1d as selective antagonists, at one or the other receptor subtype, having strictly related structures and the same potential mechanism of action, provides a valuable pair of tools and a great aid to future work that will rationally generate new, even-more selective agents.

Experimental Section

Chemistry

All chemicals and solvents were used as received from commercial sources or prepared, as described in the literature. Flash chromatography purifications were performed using KP-Sil 32–63 μm 60 Å cartridges. Thin-layer chromatography (TLC) analyses were carried out on alumina sheets precoated with silica gel 60 F254 and visualized with UV light. The content of saturated aqueous solution of ammonia in eluent mixtures is given as v/v percentage. R_f values are given for guidance. ¹H NMR spectra were recorded at 600, 400, 300, or 200 MHz, while ¹³C NMR spectra were recorded at 150, 100, or 75 MHz using FT-NMR spectrometers. Chemical shifts are reported in ppm relative to residual solvent (CHCl₃, MeOH, or DMSO) as the internal standard. Melting points were determined by a Buchi Melting Point B-540 apparatus. Optical rotations were determined using a Jasco P-1010 polarimeter. Liquid chromatography–mass spectrometry (LC–MS) analysis was performed using an Agilent 1200 series solvent delivery system equipped with an autoinjector coupled to a PDA and an Agilent 6400 series triple quadrupole electrospray ionization detector. Gradients of 5% aqueous MeCN + 0.1% HCO₂H (solvent A), and 95% aqueous MeCN + 0.05% HCO₂H (solvent B) were employed. Purity was measured by analytical high-performance liquid chromatography (HPLC) on an UltiMate HPLC system (Thermo Scientific) consisting of an LPG-3400A pump (1 mL/min), a WPS-3000SL autosampler, and a DAD-3000D diode array detector using a Gemini-NX C18 column (4.6 mm × 250 mm, 3 μm, 110 Å); gradient elution 0–100% B (MeCN/H₂O/TFA, 90:10:0.1) in solvent A (H₂O/TFA, 100:0.1) over 20 min. Data were analyzed using Chromeleon Software v. 6.80. Purity is ≥ 95%, and retention times (R_t) are reported.

Method A

Under a nitrogen atmosphere, a suspension of the appropriate phenol (10 mmol, 1 equiv), K₂CO₃ (2.0–4 equiv), and KI (0.1 equiv) in the specified solvent (15 mL) was vigorously stirred at reflux temperature for 30 min. The appropriate alkylating agent (1.2–4.2 equiv) was added portionwise or dropwise, and the resulting mixture was refluxed overnight unless specified otherwise. The reaction mixture was cooled to room temperature, and the solid was removed by filtration. The filtrate was concentrated under vacuum, and the crude was purified as specified. The desired products 34, 35, 39, 43, 52–57, 60, 61, 66, 69, 71, 73, 74, 76, 79, 81, 93, 95, 98, 102, and 103 were obtained as oils or solids in variable yields (23–100%).

Method B

The appropriate tertiary amine (2.59 mmol, 1 equiv) was dissolved in the specified solvent (5 mL), and iodoethane (1.2–50 equiv) was added dropwise. The reaction mixture was vigorously stirred at the specified temperature for 1–24 h. The reaction was worked up and purified as specified. The desired compounds 3–6, 8–14, 16–18, 23–32, and 67 were obtained as solids in variable yields (20–100%).

Method C

All of the solvents used were previously degassed. Under an inert atmosphere, the specified aryl bromide (1.3 mmol, 1 equiv) was dissolved in either 1,2-dimethoxyethane or a mixture toluene/EtOH 1:1 (5 mL). Upon the addition of a solution of Pd(PPh₃)₄ (0.35 equiv) in the same solvent (2 mL), the reaction mixture was stirred for 20 min. Afterward, a mixture of EtOH (2 mL)/2 M_aq Na₂CO₃ (4 mL) was added dropwise. When specified, TBAB (0.05 equiv) was also added. A solution of the appropriate boronic acid (1.1 equiv) in 1,2-dimethoxyethane (5 mL) was added dropwise, and the reaction mixture was refluxed overnight. Upon evaporation of the solvent under reduced pressure, the residue was diluted in DCM and filtered through a silica pad, and the solvent was evaporated under reduced pressure. The crude was purified as specified, providing compounds 78, 80, 94, 96, and 101 as oils or solids in moderate to high yields (42–92%).

Method D

The appropriate alkyl halide (2.20 mmol, 1 equiv) was dissolved in a saturated solution of NaI in acetone (10 mL), and the reaction mixture was stirred at reflux temperature overnight. A 10% aqueous solution of Na2S2O5 (20 mL) was added, and the mixture was stirred for 1 h at room temperature. After evaporation of acetone under reduced pressure, the resulting aqueous suspension was extracted with diethyl ether twice. The organic layers were combined and washed with water and then brine. The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The desired products 36, 44, 62, 63, 75, 77, 86, and 90 were obtained as oils or solids in high yields (72–97%).

Method E

Unless specified otherwise, a solution of the appropriate alkyl iodide (1.35 mmol, 1 equiv) and diethylamine (50 equiv) in toluene (10 mL) was heated at 60 °C for 3–4 h. Upon cooling to room temperature, the mixture was washed with water three times. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified as specified, providing the desired compounds 37, 45, 87, and 91 as oils or solids in high yields (84–100%).

Method F

Under an inert atmosphere, a mixture of the appropriate aryl iodide (2.14 mmol, 1 equiv), Pd(OAc)₂ (0.1 equiv), and anhydrous triethylamine (2.1 equiv) in CH₃CN (5 mL) was stirred at room temperature for 30 min and then 4-vinylphenol 42 (1.1–1.5 equiv) was added. The resulting mixture was stirred at reflux temperature overnight. Upon cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was diluted with cold 10% aqueous HCl solution (10 mL) and extracted with EtOAc three times. The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified as specified, providing the desired compounds 46–51, 58, 59, and 65 as oils or solids in low to modest yields (21–66%).

Synthesis of N,N,N-Triethyl-2-phenoxyethan-1-aminium Iodide (3)

Obtained from N,N-diethyl-2-phenoxyethan-1-amine 34 (500 mg, 2.59 mmol, 1 equiv) and iodoethane (8 equiv) in 1,2-dichloroethane (5 mL), according to Method B, overnight at room temperature. Upon rotary evaporation of the volatiles, the residue was dissolved in MeOH and diluted with diethyl ether. The suspension was filtered, and the solid was washed with diethyl ether. Trituration with diisopropyl ether/2-propanol provided the desired product 3 as a dark solid in a 48% yield. Mp = 79–83 °C. R_t (LC-MS) = 2.867 min. LC-MS (ESI): m/z calcd for C₁₄H₂₄NO [M]⁺ = 222.19, found 222.2. R_t (HPLC) = 9.28 min. ¹H NMR (300 MHz, chloroform-d) δ 7.27 (t, J = 7.7 Hz, 2H), 7.03–6.86 (m, 3H), 4.50–4.40 (m, 2H), 4.01–3.87 (m, 2H), 3.55 (q, J = 7.2 Hz, 6H), 1.42 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, chloroform-d) δ 157.0, 129.9, 122.2, 114.6, 62.0, 57.0, 54.8, 8.6.

Synthesis of N,N,N-Triethyl-2-(4-vinylphenoxy)ethan-1-aminium Iodide (4)

Obtained from N,N-diethyl-2-(4-vinylphenoxy)ethan-1-amine 45 (150 mg, 0.68 mmol, 1 equiv) and iodoethane (1.2 equiv) in DCM (2 mL) according to Method B, at reflux temperature for 2 h. Upon cooling, the suspension was filtered and the solid was recrystallized from diethyl ether, providing the desired product as a pale-yellow solid in a 43% yield. Mp = 100.1 °C. R_t (LC-MS) = 3.158 min. LC-MS (ESI): m/z calcd for C₂₄H₃₀NO [M]⁺ = 248.20, found 248.2. R_t (HPLC) = 11.01 min. ¹H NMR (300 MHz, chloroform-d) δ 7.33 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.62 (dd, J = 17.6, 10.9 Hz, 1H), 5.60 (d, J = 17.6 Hz, 1H), 5.14 (d, J = 10.9 Hz, 1H), 4.49 (t, J = 4.6 Hz, 2H), 4.03–3.92 (m, 2H), 3.57 (q, J = 7.2 Hz, 6H), 1.44 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, chloroform-d) δ 156.8, 135.9, 132.0, 127.8, 114.8, 112.7, 62.2, 57.1, 54.9, 8.7.

Synthesis of 2-([1,1′-Biphenyl]-4-yloxy)-N,N,N-triethylethan-1-aminium Iodide (5)

Obtained from 2-([1,1′-biphenyl]-4-yloxy)-N,N-diethylethan-1-amine 37 (680 mg, 2.52 mmol, 1 equiv) and iodoethane (4 equiv) in DCM (7 mL) according to Method B, at reflux temperature for 2 h. Upon cooling, the suspension was filtered and the solid was recrystallized from diethyl ether, providing the desired compound 5 as an off-white solid in an 80% yield. Mp = 167.4 °C. R_t (LC-MS) = 3.608 min. LC-MS (ESI): m/z calcd for C₂₀H₂₈NO [M]⁺ = 298.22, found 298.3. R_t (HPLC) = 12.63 min. ¹H NMR (300 MHz, chloroform-d) δ 7.56–7.48 (m, 4H), 7.44–7.36 (m, 2H), 7.34–7.27 (m, 1H), 7.07–6.97 (m, 2H), 4.55 (t, J = 4.5 Hz, 2H), 4.09–4.01 (m, 2H), 3.60 (q, J = 7.2 Hz, 6H), 1.47 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, chloroform-d) δ 156.6, 140.3, 135.4, 128.9, 128.6, 127.2, 126.9, 115.1, 62.4, 57.2, 55.0, 8.7.

Synthesis of N,N,N-Triethyl-2-(4-phenethylphenoxy)ethan-1-aminium Iodide (6)

Obtained from 73 (150 mg, 0.504 mmol) and iodoethane (4 equiv) in toluene (6 mL) at 90 °C, according to Method B. After 48 h, diethyl ether was added to the mixture and the solid was collected by filtration to give 6 as an off-white solid (96 mg, 42%). Mp = 122–123 °C. R_t (LC-MS) = 3.756 min. LC-MS (ESI): m/z calcd for C₂₂H₃₂NO⁺ [M]⁺ = 326.25, found 326.3. R_t (HPLC) = 13.56 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.26–7.18 (m, 2H), 7.17–7.07 (m, 5H), 6.89 (d, J = 8.6 Hz, 2H), 4.44–4.35 (m, 2H), 3.78–3.70 (m, 2H), 3.48 (q, J = 7.2 Hz, 6H), 2.86 (s, 4H), 1.37 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 157.1, 142.9, 136.5, 130.8, 129.6, 129.2, 126.8, 115.4, 62.6, 57.2, 54.9, 39.2, 38.1, 7.9.

Synthesis of N,N,N-Triethyl-2-(4-(phenylethynyl)phenoxy)ethan-1-aminium Iodide (7)

A solution of 1-(2-iodoethoxy)-4-(phenylethynyl)benzene 75 (125 mg, 0.36 mmol, 1 equiv) in toluene (3 mL) and triethylamine (3 mL) was stirred at room temperature overnight. The resulting suspension was filtered, and the solid was washed with EtOAc, providing the desired product 7 as an off-white solid in a 37% yield. Mp = 162.2 °C dec. R_t (LC-MS) = 3.787 min. LC-MS (ESI): m/z calcd for C₂₂H₂₈NO⁺ [M]⁺ = 322.22, found 322.2. R_t (HPLC) = 13.69 min. ¹H NMR (600 MHz, chloroform-d) δ 7.52–7.46 (m, 4H), 7.36–7.30 (m, 3H), 6.94 (d, J = 8.8 Hz, 2H), 4.56 (t, J = 4.7 Hz, 2H), 4.12–4.05 (m, 2H), 3.60 (q, J = 7.2 Hz, 6H), 1.51–1.44 (m, 9H). ¹³C NMR (150 MHz, chloroform-d) δ 156.9, 133.5, 131.6, 128.5, 128.3, 123.4, 117.4, 114.8, 89.0, 88.9, 62.4, 57.2, 55.0, 8.7.

Synthesis of N,N,N-Triethyl-2-(4-(naphthalen-2-yl)phenoxy)ethan-1-aminium Iodide (8)

Obtained from N,N-diethyl-2-(4-(naphthalen-2-yl)phenoxy)ethan-1-amine 79 (33 mg, 0.1 mmol, 1 equiv) according to Method B, using ethyl iodide as a solvent (2 mL) for 30 min at reflux temperature. Upon cooling, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered. The solid was washed with DCM, providing the desired product 8 as a white solid in a 76% yield. Mp = 242 °C. R_t (LC-MS) = 3.864 min. LC-MS (ESI): m/z calcd for C₂₄H₃₀NO [M]⁺ = 348.23, found 348.3. R_t (HPLC) = 14.11 min. ¹H NMR (300 MHz, methanol-d₄) δ 8.04 (d, J = 1.9 Hz, 1H), 7.96–7.83 (m, 3H), 7.79–7.71 (m, 3H), 7.55–7.40 (m, 2H), 7.14 (d, J = 8.8 Hz, 2H), 4.56–4.47 (m, 2H), 3.84–3.76 (m, 2H), 3.51 (q, J = 7.2 Hz, 6H), 1.40 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 158.6, 139.0, 136.1, 134.0, 129.6, 129.5, 129.1, 128.6, 127.4, 126.9, 126.1, 126.0, 124.4, 116.1, 62.7, 57.2, 55.0, 7.9.

Synthesis of N,N,N-Triethyl-2-(4-(naphthalen-1-yl)phenoxy)ethan-1-aminium Iodide (9)

Obtained from N,N-diethyl-2-(4-(naphthalen-1-yl)phenoxy)ethan-1-amine 81 (70 mg, 0.22 mmol, 1 equiv) according to Method B, using iodoethane as a solvent (2 mL), at reflux temperature, for 30 min. Upon cooling, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered. The solid was washed with DCM, providing the desired product 9 as a pale-yellow solid in a 54% yield. Mp = 178 °C. R_t (LC-MS) = 3.792 min. LC-MS (ESI): m/z calcd for C₂₄H₃₀NO [M]⁺ = 348.23, found 348.3. R_t (HPLC) = 13.97 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.96–7.78 (m, 3H), 7.57–7.35 (m, 6H), 7.16 (d, J = 8.9 Hz, 2H), 4.59–4.49 (m, 2H), 3.87–3.77 (m, 2H), 3.53 (q, J = 7.2 Hz, 6H), 1.41 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 158.3, 140.9, 135.7, 135.4, 133.0, 132.4, 129.4, 128.6, 127.9, 127.0, 126.8, 126.7, 126.4, 115.6, 62.8, 57.2, 55.0, 8.0.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(2-(naphthalen-1-yl)vinyl)phenoxy)ethan-1-aminium Iodide (10)

Obtained from compound 69 (1.00 g, 2.89 mmol) and iodoethane (10 equiv) in EtOH according to Method B at 70 °C for 18 h. The mixture was concentrated under vacuum and purified by flash chromatography (DCM/MeOH 95:5) to give 10 as a pale-yellow solid in a 48% yield. Mp = 164.5–166.5 °C (crystallized from EtOH/MeOH 8:2). R_t (LC-MS) = 3.951 min. LC-MS (ESI): m/z calcd for C₂₆H₃₂NO [M]⁺ = 374.25, found 374.2. R_t (HPLC) = 14.66 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 8.45–8.36 (m, 1H), 8.01–7.91 (m, 2H), 7.86 (dd, J = 7.7, 2.6 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.69–7.48 (m, 3H), 7.26 (d, J = 16.1 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 4.45 (t, J = 4.7 Hz, 2H), 3.70 (t, J = 4.7 Hz, 2H), 3.40 (q, J = 7.0 Hz, 6H), 1.25 (t, J = 7.0 Hz, 9H). ¹³C NMR (100 MHz, dimethylsulfoxide-d₆) δ 157.1, 134.4, 133.4, 130.8, 130.7, 130.7, 128.4, 128.2, 127.5, 126.1, 125.9, 125.8, 123.8, 123.0, 122.8, 114.8, 61.1, 55.2 52.9, 7.3.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(2-(naphthalen-2-yl)vinyl)phenoxy)ethan-1-aminium Iodide (11)

Obtained from compound 71 (1.00 g, 2.89 mmol) and iodoethane (10 equiv) in EtOH according to Method B at 70 °C for 14 h. The mixture was concentrated under vacuum and purified by flash chromatography (DCM/MeOH 95:5). The product was crystallized from MeOH affording 11 as a white solid in a 24% yield. Mp = 223–227 °C. R_t (LC-MS) = 4.080 min. LC-MS (ESI): m/z calcd for C₂₆H₃₂NO [M]⁺ = 374.25, found 374.2. R_t (HPLC) = 14.77 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 8.01–7.95 (m, 1H), 7.94–7.81 (m, 4H), 7.70–7.60 (m, 2H), 7.55–7.43 (m, 2H), 7.39 (d, J = 16.5 Hz, 1H), 7.30 (d, J = 16.5 Hz, 1H), 7.10–6.99 (m, 2H), 4.44 (t, J = 4.8 Hz, 2H), 3.70 (t, J = 4.8 Hz, 2H), 3.40 (q, J = 7.1 Hz, 6H), 1.25 (t, J = 7.1 Hz, 9H). ¹³C NMR (100 MHz, dimethylsulfoxide-d₆) δ 157.1, 134.9, 133.3, 132.4, 130.6, 128.4, 128.1, 127.8, 127.7, 127.5, 126.6, 126.4, 125.9, 125.8, 123.5, 114.9, 61.1, 55.2, 52.9, 7.3.

Synthesis of (E)-2-(4-(2-Bromostyryl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (12)

Obtained from (E)-2-(4-(2-bromostyryl)phenoxy)-N,N-diethylethan-1-amine 52 (21 mg, 0.56 mmol, 1 equiv) and iodoethane (10 equiv) in THF (5 mL), according to Method B, at reflux temperature, overnight. Upon cooling at room temperature, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, providing the desired product 12 as a pale-yellow solid in a 100% yield. Mp = 133.3 °C. R_t (LC-MS) = 3.926 min. LC-MS (ESI): m/z calcd for C₂₂H₂₉BrNO [M]⁺ = 402.14, 404.14, found 402.1, 404.1. R_t (HPLC) = 14.53 min. ¹H NMR (300 MHz, chloroform-d) δ 7.62 (dd, J = 7.8, 1.6 Hz, 1H), 7.55 (dd, J = 8.1, 1.2 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.35–7.25 (m, 2H), 7.14–7.03 (m, 1H), 6.99–6.91 (m, 3H), 4.57–4.49 (m, 2H), 4.05–3.96 (m, 2H), 3.57 (q, J = 7.2 Hz, 6H), 1.45 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, chloroform-d) δ 157.0, 137.1, 133.1, 131.3, 130.5, 128.8, 128.5, 127.7, 126.7, 126.2, 124.1, 115.0, 62.3, 57.1, 54.9, 8.7.

Synthesis of (E)-2-(4-(3-Bromostyryl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (13)

Obtained from (E)-2-(4-(3-bromostyryl)phenoxy)-N,N-diethylethan-1-amine 53 (290 mg, 0.78 mmol, 1 equiv) and iodoethane (10 equiv) in THF (5 mL), according to Method B at reflux temperature, overnight. Upon cooling at room temperature, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, providing the desired product 13 as a white solid in a 100% yield. Mp = 133.3 °C. R_t (LC-MS) = 3.985 min. LC-MS (ESI): m/z calcd for C₂₂H₂₉BrNO [M]⁺ = 402.14, 404.14, found 402.1, 404.1. R_t (HPLC) = 14.60 min. ¹H NMR (300 MHz, chloroform-d) δ 7.59 (t, J = 1.8 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.39–7.30 (m, 2H), 7.18 (t, J = 7.8 Hz, 1H), 7.04–6.90 (m, 3H), 6.86 (d, J = 16.3 Hz, 1H), 4.57–4.46 (m, 2H), 4.02–3.94 (m, 2H), 3.56 (q, J = 7.2 Hz, 6H), 1.44 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, chloroform-d) δ 156.9, 139.6, 131.1, 130.3, 129.9, 129.2, 129.1, 128.3, 125.9, 125.1, 122.9, 115.0, 62.3, 57.0, 54.9, 8.6.

Synthesis of (E)-2-(4-(4-Bromostyryl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (14)

Obtained from (E)-2-(4-(4-bromostyryl)phenoxy)-N,N-diethylethan-1-amine 54 (315 mg, 0.84 mmol, 1 equiv) and iodoethane (2 equiv) in THF (5 mL), according to Method B at reflux temperature, overnight. Upon cooling at room temperature, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, providing the desired product 14 as a white solid in a 79% yield. Mp = 244.4 °C. R_t (LC-MS) = 3.950 min. LC-MS (ESI): m/z calcd for C₂₂H₂₉BrNO [M]⁺ = 402.14, 404.14, found 402.1, 404.1. R_t (HPLC) = 14.68 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 7.60 (d, J = 8.8 Hz, 2H), 7.55–7.53 (m, 4H), 7.27 (d, J = 16.5 Hz, 1H), 7.12 (d, J = 16.5 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 4.42 (t, J = 4.7 Hz, 2H), 3.69 (t, J = 4.7 Hz, 2H), 3.39 (q, J = 7.1 Hz, 6H), 1.23 (t, J = 7.1 Hz, 9H). ¹³C NMR (75 MHz, dimethylsulfoxide-d₆) δ 157.2, 136.6, 131.6, 130.3, 128.8, 128.2, 128.0, 125.3, 120.1, 114.9, 61.1, 55.1, 52.9, 7.4.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(3-(trifluoromethyl)styryl)phenoxy)ethan-1-aminium Iodide (15)

Obtained from 62 (47 mg, 0.112 mmol, 1 equiv) and triethylamine in toluene (1:1 v/v, 5 mL) at reflux for 16 h. Upon cooling to room temperature, diethyl ether was added and the solid was collected by filtration to give 15 as an off-white solid in a 32% yield. Mp = 178.6 °C. R_t (LC-MS) = 3.963 min. LC-MS (ESI): m/z calcd for C₂₃H₂₉F₃NO [M]⁺ = 392.22, found 392.2. R_t (HPLC) = 14.72 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.85–7.75 (m, 2H), 7.64–7.55 (m, 2H), 7.55–7.46 (m, 2H), 7.26 (d, J = 16.4 Hz, 1H), 7.14 (d, J = 16.4 Hz, 1H), 7.08–6.98 (m, 2H), 4.48 (t, J = 4.6 Hz, 2H), 3.83–3.74 (m, 2H), 3.50 (q, J = 7.2 Hz, 6H), 1.38 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 158.9, 140.2, 132.3, 132.1 (q, J = 33.7 Hz), 131.0, 130.6 (q, J = 1.3 Hz), 130.5, 129.3, 126.6, 124.6 (q, J = 3.9 Hz), 123.9 (q, J = 3.8 Hz), 115.9, 62.7, 57.1, 55.0, 7.9.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(4-(trifluoromethyl)styryl)phenoxy)ethan-1-aminium Iodide (16)

Obtained from (E)-N,N-diethyl-2-(4-(4-(trifluoromethyl)styryl)phenoxy)ethan-1-amine 55 (60 mg, 0.17 mmol, 1 equiv) and iodoethane (10 equiv) in THF (2 mL), according to Method B, at reflux temperature overnight. Upon cooling to room temperature, the reaction mixture was diluted with diethyl ether, and the resulting suspension was filtered, affording the desired compound 16 as a white solid in a 55% yield. Mp = 243.8 °C. R_t (LC-MS) = 3.999 min. LC-MS (ESI): m/z calcd for C₂₃H₂₉F₃NO [M]⁺ = 392.22, found 392.3. R_t (HPLC) = 14.70 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 7.79 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 16.5 Hz, 1H), 7.24 (d, J = 16.5 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 4.43 (t, J = 4.8 Hz, 2H), 3.69 (t, J = 4.8 Hz, 2H), 3.39 (q, J = 7.1 Hz, 6H), 1.24 (t, J = 7.1 Hz, 9H). ¹³C NMR (75 MHz, dimethylsulfoxide-d₆) δ 157.5, 141.5, 130.8, 130.0, 128.3, 127.1 (q, J = 31.6 Hz), 126.7, 125.6 (q, J = 3.7 Hz), 125.0, 120.8 (q, J = 272.0 Hz), 115.0, 61.1, 55.1, 52.9, 7.3.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(3-methoxystyryl)phenoxy)ethan-1-aminium Iodide (17)

Obtained from (E)-N,N-diethyl-2-(4-(3-methoxystyryl)phenoxy)ethan-1-amine 56 (20 mg, 0.02 mmol, 1 equiv) according to Method B, using iodoethane as a solvent (2 mL), at reflux temperature overnight. Upon cooling at room temperature, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, affording the desired product 17 as a white solid in a 100% yield. Mp = 186 °C. R_t (LC-MS) = 3.736 min. LC-MS (ESI): m/z calcd for C₂₃H₃₂NO₂ [M]⁺ = 354.24, found 354.3. R_t (HPLC) = 13.62 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.55 (d, J = 8.8 Hz, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.17–7.04 (m, 4H), 7.00 (d, J = 8.8 Hz, 2H), 6.81 (ddd, J = 8.0, 2.5, 0.9 Hz, 1H), 4.52–4.40 (m, 2H), 3.82 (s, 3H), 3.80–3.74 (m, 2H), 3.49 (q, J = 7.2 Hz, 6H), 1.38 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 161.5, 158.5, 140.4, 132.8, 130.6, 129.1, 129.0, 128.3, 120.0, 115.9, 114.0, 112.6, 62.6, 57.1, 55.7, 54.9, 7.9.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(4-methoxystyryl)phenoxy)ethan-1-aminium Iodide (18)

Obtained from (E)-N,N-diethyl-2-(4-(4-methoxystyryl)phenoxy)ethan-1-amine 57 (100 mg, 0.31 mmol, 1 equiv) and iodoethane (10 equiv) in THF (5 mL), according to Method B, at reflux temperature, overnight. The reaction mixture was concentrated under vacuum, the residue was diluted with diethyl ether, and the resulting suspension was filtered, affording the desired compound 18 as a white solid in a 98% yield. Mp = 222.1 °C. R_t (LC-MS) = 3.723 min. LC-MS (ESI): m/z calcd for C₂₃H₃₂NO₂ [M]⁺ = 354.24, found 354.3. R_t (HPLC) = 13.58 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.50 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.02–6.97 (m, 4H), 6.90 (d, J = 8.8 Hz, 2H), 4.45 (t, J = 4.5 Hz, 2H), 3.80 (s, 3H), 3.79–3.74 (m, 2H), 3.49 (q, J = 7.2 Hz, 6H), 1.38 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 160.7, 158.2, 133.3, 131.7, 128.63, 128.55, 128.0, 126.7, 115.8, 115.1, 62.6, 57.1, 55.7, 54.9, 7.9.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(3-hydroxystyryl)phenoxy)ethan-1-aminium Iodide (19)

A solution of (E)-3-hydroxy-4′-(2-iodoethyloxy)stilbene 64 (85 mg, 0.23 mmol, 1 equiv) was dissolved in 3 mL of triethylamine and 3 mL of toluene and stirred at reflux temperature for 5 h. Upon cooling at room temperature, the suspension was filtered and washed with CH₃CN obtaining the desired product 19 as a pale brown solid in a 23% yield. Mp = 196.3 °C. R_t (LC-MS) = 3.326 min. LC-MS (ESI): m/z calcd for C₂₂H₃₀NO₂ [M]⁺ = 340.23, found 340.2. R_t (HPLC) = 11.97 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.52 (d, J = 8.7 Hz, 2H), 7.15 (t, J = 7.8 Hz, 1H), 7.10–6.92 (m, 6H), 6.67 (dd, J = 7.8, 1.9 Hz, 1H), 4.50–4.39 (m, 2H), 3.80–3.71 (m, 2H), 3.48 (q, J = 7.2 Hz, 6H), 1.37 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 158.8, 158.5, 140.4, 132.8, 130.6, 128.9, 128.8, 128.4, 119.1, 115.8, 115.5, 113.7, 62.6, 57.1, 54.9, 7.9.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(4-hydroxystyryl)phenoxy)ethan-1-aminium Iodide (20)

A mixture of 67 (32 mg, 0.058 mmol) and an excess of a 1.25 M HCl solution in MeOH was stirred at reflux overnight. Upon cooling to room temperature, the resulting mixture was concentrated under vacuum to give 20 as a yellow solid in a 100% yield. Mp = 139.4–139.9 °C. R_t (LC-MS) = 3.301 min. LC-MS (ESI): m/z calcd for C₂₂H₃₀NO₂ [M]⁺ = 340.23, found 340.3. R_t (HPLC) = 11.80 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.54–7.43 (m, 2H), 7.41–7.32 (m, 2H), 7.04–6.86 (m, 4H), 6.82–6.71 (m, 2H), 4.44 (t, J = 4.6 Hz, 2H), 3.80–3.71 (m, 2H), 3.48 (q, J = 7.2 Hz, 6H), 1.38 (t, J = 7.2 Hz, 9H).¹³C NMR (75 MHz, methanol-d₄) δ 158.3, 158.0, 133.4, 130.6, 128.7, 128.5, 128.3, 125.9, 116.5, 115.8, 62.6, 57.1, 54.9, 7.9.

Synthesis of (E)-2-(4-(3,5-Dihydroxystyryl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (22)

A solution of (E)-5-(4-(2-iodoethoxy)styryl)benzene-1,3-diol 77 (290 mg, 0.76 mmol, 1 equiv) was dissolved in 5 mL of triethylamine and 5 mL of toluene and stirred at reflux temperature for 5 h. Upon cooling at room temperature, the suspension was filtered and washed with CH₃CN and EtOH providing the desired product 22 as a pale brown solid in a 12% yield. Mp = 236.5 °C. R_t (LC-MS) = 3.014 min. LC-MS (ESI): m/z calcd for C₂₂H₃₀NO₃ [M]⁺ = 356.22, found 356.3. R_t (HPLC) = 10.52 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.49 (d, J = 8.8 Hz, 2H), 7.06–6.95 (m, 3H), 6.88 (d, J = 16.3 Hz, 1H), 6.47 (d, J = 2.1 Hz, 2H), 6.19 (t, J = 2.1 Hz, 1H), 4.49–4.40 (m, 2H), 3.78–3.71 (m, 2H), 3.47 (q, J = 7.2 Hz, 6H), 1.37 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 159.7, 158.4, 140.9, 132.8, 128.9, 128.6, 127.3, 115.8, 105.9, 102.9, 62.6, 57.1, 54.9, 7.9.

Synthesis of 2-(4-(Benzyloxy)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (23)

Obtained from 2-(4-(benzyloxy)phenoxy)-N,N-diethylethan-1-amine 39 (280 mg, 0.94 mmol, 1 equiv) and iodoethane (4 equiv) in 1,2-dichloroethane (3 mL), according to Method B, at room temperature, overnight. The reaction mixture was concentrated under reduced pressure, the residue was triturated in diisopropyl ether/2-propanol, and then filtered, providing the desired compound 23 as a white solid in an 84% yield. Mp = 172.4–173.9 °C. R_t (LC-MS) = 3.515 min. LC-MS (ESI): m/z calcd for C₂₁H₃₀NO₂ [M]⁺ = 328.23, found 328.3. R_t (HPLC) = 12.73 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.47–7.25 (m, 5H), 7.00–6.90 (m, 4H), 5.03 (s, 2H), 4.40–4.33 (m, 2H), 3.76–3.69 (m, 2H), 3.47 (q, J = 7.2 Hz, 6H), 1.36 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 155.2, 153.2, 138.8, 129.5, 128.8, 128.6, 117.1, 116.7, 71.6, 63.2, 57.3, 55.0, 8.0.

Synthesis of N,N,N-Triethyl-2-(4-(phenoxymethyl)phenoxy)ethan-1-aminium Iodide (24)

Obtained from N,N-diethyl-2-(4-(phenoxymethyl)phenoxy)ethan-1-amine 105 (100 mg, 0.33 mmol, 1 equiv) according to Method B, using iodoethane as a solvent (2 mL), at room temperature, overnight. The reaction mixture was diluted with diethyl ether, and the resulting suspension was filtered, providing the desired compound 24 as a white solid in a 50% yield. Mp = 153 °C. R_t (LC-MS) = 3.629 min. LC-MS (ESI): m/z calcd for C₂₁H₃₀NO₂ [M]⁺ = 328.23, found 328.3. R_t (HPLC) = 12.69 min. ¹H NMR (400 MHz, methanol-d₄) δ 7.44–7.38 (m, 2H), 7.30–7.21 (m, 2H), 7.09–6.99 (m, 2H), 6.99–6.94 (m, 2H), 6.92 (td, J = 7.2, 1.1 Hz, 1H), 5.01 (s, 2H), 4.45 (t, J = 4.8 Hz, 2H), 3.79–3.73 (m, 2H), 3.48 (q, J = 7.2 Hz, 6H), 1.37 (t, J = 7.2 Hz, 9H). ¹³C NMR (100 MHz, methanol-d₄) δ 160.2, 158.6, 132.2, 130.44, 130.43, 121.9, 116.0, 115.7, 70.5, 62.7, 57.2, 55.0, 8.0.

Synthesis of 2-(4-Benzamidophenoxy)-N,N,N-triethylethan-1-aminium Iodide (25)

Obtained from N-(4-(2-(diethylamino)ethoxy)phenyl)benzamide 91 (100 mg, 0.31 mmol, 1 equiv) according to Method B, using iodoethane (1 mL) as a solvent, at reflux temperature for 5 h. Upon cooling, the reaction mixture was diluted in DCM and the resulting suspension was filtered. The solid was washed repeatedly with DCM, providing the desired compound 25 as a white solid in a 46% yield. Mp = 202 °C. R_t (LC-MS) = 3.082 min. LC-MS (ESI): m/z calcd for C₂₁H₂₉N₂O₂ [M]⁺ = 341.22, found 341.2. R_t (HPLC) = 10.69 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 10.16 (s, 1H), 7.95 (d, J = 6.7 Hz, 2H), 7.72 (d, J = 9.0 Hz, 2H), 7.63–7.47 (m, 3H), 7.00 (d, J = 9.0 Hz, 2H), 4.39 (t, J = 4.8 Hz, 2H), 3.68 (t, J = 4.8 Hz, 2H), 3.38 (q, J = 7.1 Hz, 6H), 1.24 (t, J = 7.1 Hz, 9H). ¹³C NMR (75 MHz, dimethylsulfoxide-d₆) δ 165.1, 153.6, 134.9, 133.0, 131.5, 128.4, 127.5, 121.9, 114.6, 61.2, 55.2, 52.9, 7.3.

Synthesis of N,N,N-Triethyl-2-(4-(phenylcarbamoyl)phenoxy)ethan-1-aminium Iodide (26)

Obtained from 4-(2-(diethylamino)ethoxy)-N-phenylbenzamide 87 (100 mg, 0.31 mmol, 1 equiv) according to Method B, using iodoethane (1 mL) as a solvent, at reflux temperature for 5 h. Upon cooling, the reaction mixture was diluted in DCM and the resulting suspension was filtered. The solid was washed repeatedly with DCM, providing the desired compound 26 as an off-white solid in a 46% yield. Mp = 192 °C. R_t (LC-MS) = 3.108 min. LC-MS (ESI): m/z calcd for C₂₁H₂₉N₂O₂ [M]⁺ = 341.22, found 341.3. R_t (HPLC) = 10.89 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 10.09 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.40–7.26 (m, 2H), 7.20–7.05 (m, 3H), 4.50 (t, J = 4.8 Hz, 2H), 3.72 (t, J = 4.8 Hz, 2H), 3.40 (q, J = 7.2 Hz, 6H), 1.25 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, dimethylsulfoxide-d₆) δ 164.6, 160.0, 139.2, 129.6, 128.5, 127.7, 123.5, 120.4, 114.2, 61.3, 55.1, 52.9, 7.3.

Synthesis of (E)-N,N,N-Triethyl-2-(4-(phenyldiazenyl)phenoxy)ethan-1-aminium Iodide (27)

Obtained from 93 (218 mg, 733 mmol) and iodoethane (4 equiv) in DCM (2.5 mL) according to Method B at room temperature for 16 h. The reaction mixture was diluted with diethyl ether (5 mL). The suspension was stirred for 15 min, and then the solid was isolated by filtration. The solid was redissolved in the smallest amount of EtOH, diethyl ether was added, and the formed solid was isolated by filtration to give 27 as a yellow solid in a 32% yield. Mp = 152.2–155.0 °C. R_t (LC-MS) = 3.634 min. LC-MS (ESI): m/z calcd for C₂₀H₂₈N₃O [M]⁺ = 326.22, found 326.2. R_t (HPLC) = 12.89 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.99–7.91 (m, 2H), 7.91–7.82 (m, 2H), 7.59–7.42 (m, 3H), 7.23–7.14 (m, 2H), 4.56 (t, J = 4.6 Hz, 2H), 3.86–3.77 (m, 2H), 3.50 (q, J = 7.3 Hz, 6H), 1.39 (t, J = 7.3 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 161.3, 154.0, 148.9, 131.9, 130.2, 125.8, 123.6, 116.2, 63.0, 57.1, 55.0, 8.0.

Synthesis of 2-(4-(Benzo[d]oxazol-2-yl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (28)

A solution of 2-(4-(benzo[d]oxazol-2-yl)phenoxy)-N,N-diethylethan-1-amine hydrochloride 107 (130 mg, 37 mmol, 1 equiv) in Na₂CO₃ 1 M (5 mL) was extracted with EtOAc (3 × 5 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The resulting tertiary amine was reacted according to Method B, using iodoethane as a solvent (2 mL), at room temperature, overnight. The reaction mixture was diluted with diethyl ether, and the resulting suspension was filtered, providing the desired compound 28 as a white solid in a 90% yield. Mp = 213–218 °C (dec). R_t (LC-MS) = 3.386 min. LC-MS (ESI): m/z calcd for C₂₁H₂₇N₂O₂ [M]⁺ = 339.21, found 339.2. R_t (HPLC) = 12.29 min. ¹H NMR (300 MHz, chloroform-d) δ 8.19 (d, J = 9.0 Hz, 2H), 7.74–7.69 (m, 1H), 7.57–7.52 (m, 1H), 7.35–7.30 (m, 2H), 7.10 (d, J = 9.0 Hz, 2H), 4.69–4.61 (m, 2H), 4.17–4.10 (m, 2H), 3.62 (q, J = 7.2 Hz, 6H), 1.50 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 163.0, 160.5, 150.5, 141.4, 129.2, 125.0, 124.6, 120.1, 118.9, 115.0, 110.3, 61.5, 55.6, 53.6, 6.6.

Synthesis of 2-(4-(1H-Benzo[d]imidazol-2-yl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (29)

Obtained from 2-(4-(1H-benzo[d]imidazol-2-yl)phenoxy)-N,N-diethylethan-1-amine 108 (100 mg, 0.32 mmol, 1 equiv) and iodoethane (8 equiv) in 1,2-dichloroethane, according to Method B, at room temperature, overnight. The volatiles were removed under reduced pressure, and the residue was diluted with diethyl ether. The resulting suspension was filtered, and the desired product 29 was obtained as an off-white solid in a 50% yield. Mp = 176.2–180.1 °C. R_t (LC-MS) = 2.525 min. LC-MS (ESI): m/z calcd for C₂₁H₂₈N₃O [M]⁺ = 338.22, found 338.2. R_t (HPLC) = 8.24 min. ¹H NMR (300 MHz, methanol-d₄) δ 8.09 (d, J = 8.9 Hz, 2H), 7.62 (dd, J = 6.1, 3.2 Hz, 2H), 7.30 (dd, J = 6.1, 3.2 Hz, 2H), 7.22 (d, J = 8.9 Hz, 2H), 4.61–4.49 (m, 2H), 3.86–3.77 (m, 2H), 3.51 (q, J = 7.2 Hz, 6H), 1.39 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 161.0, 152.7, 139.1, 129.8, 124.4, 123.5, 116.5, 115.5, 63.0, 57.1, 55.0, 8.0.

Synthesis of 2-(4-(1H-Indol-6-yl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (30)

Obtained from 2-(4-(1H-indol-6-yl)phenoxy)-N,N-diethylethan-1-amine 95 (19 mg, 0.06 mmol, 1 equiv) and iodoethane (50 equiv) in THF (5 mL) according to Method B at reflux temperature overnight. Upon cooling, the reaction mixture was diluted with diisopropyl ether, and the resulting suspension was filtered, providing the desired compound 30 as an off-white solid in a 56% yield. Mp = 206.3–207.4 °C. R_t (LC-MS) = 3.464 min. LC-MS (ESI): m/z calcd for C₂₂H₂₉N₂O [M]⁺ = 337.23, found 337.3. R_t (HPLC) = 12.51 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.63 (d, J = 8.9 Hz, 2H), 7.60–7.53 (m, 2H), 7.28–7.21 (m, 2H), 7.07 (d, J = 8.9 Hz, 2H), 6.44 (dd, J = 3.1, 0.9 Hz, 1H), 4.52–4.42 (m, 2H), 3.82–3.72 (m, 2H), 3.49 (q, J = 7.2 Hz, 6H), 1.38 (t, J = 7.2 Hz, 9H). ¹³C NMR (150 MHz, methanol-d₄) δ 157.8, 138.3, 137.9, 135.2, 129.3, 128.7, 126.3, 121.5, 119.5, 115.9, 110.1, 102.2, 62.7, 57.2, 55.0, 7.9.

Synthesis of 2-(4-(1H-Indol-5-yl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (31)

Obtained from 98 (40 mg, 0.13 mmol, 1 equiv) and iodoethane (50 equiv) in THF (5 mL) according to Method B at reflux temperature overnight. Upon cooling, the reaction mixture was diluted with diisopropyl ether, and the resulting suspension was filtered, providing 31 as an off-white solid in a 20% yield. Mp = 203 °C dec. R_t (LC-MS) = 3.414 min. LC-MS (ESI): m/z calcd for C₂₂H₂₉N₂O [M]⁺ = 337.23, found 337.2. R_t (HPLC) = 12.32 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.72 (dd, J = 1.8, 0.7 Hz, 1H), 7.65–7.56 (m, 2H), 7.43 (dt, J = 8.4, 0.8 Hz, 1H), 7.33 (dd, J = 8.5, 1.8 Hz, 1H), 7.25 (d, J = 3.1 Hz, 1H), 7.09–7.02 (m, 2H), 6.48 (dd, J = 3.1, 0.9 Hz, 1H), 4.50–4.44 (m, 2H), 3.82–3.67 (m, 2H), 3.50 (q, J = 7.2 Hz, 6H), 1.39 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 157.6, 138.4, 133.1, 130.1, 129.3, 127.3, 126.3, 121.7, 119.2, 115.9, 112.4, 102.7, 62.8, 57.5, 55.2, 7.9.

Synthesis of 2-(4-(Benzofuran-5-yl)phenoxy)-N,N,N-triethylethan-1-aminium Iodide (32)

A solution of 2-(4-(benzofuran-5-yl)phenoxy)-N,N-diethylethan-1-amine hydrochloride (48 mg, 0.14 mmol, 1 equiv) in DCM was washed with a solution of 1 M NaOH and with brine. The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated under vacuum to afford the corresponding free base. The resulting residue was reacted with iodoethane (50 equiv) in THF (5 mL) according to Method B at reflux temperature overnight. Upon cooling, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, providing the desired compound 32 as an off-white solid in a 22% yield. Mp = 253 °C. R_t (LC-MS) = 3.731 min. LC-MS (ESI): m/z calcd for C₂₂H₂₈NO₂ [M]⁺ = 338.21, found 338.2. R_t (HPLC) = 13.07 min. ¹H NMR (300 MHz, methanol-d₄) δ 7.80–7.78 (m, 1H), 7.77 (dd, J = 2.2, 0.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.56–7.47 (m, 2H), 7.09 (d, J = 8.9 Hz, 2H), 6.88 (dd, J = 2.2, 1.0 Hz, 1H), 4.53–4.45 (m, 2H), 3.82–3.76 (m, 2H), 3.50 (q, J = 7.2 Hz, 6H), 1.39 (t, J = 7.2 Hz, 9H). ¹³C NMR (75 MHz, methanol-d₄) δ 158.2, 155.8, 147.1, 137.1, 136.8, 129.6, 129.5, 124.6, 120.2, 116.0, 112.3, 107.8, 62.7, 57.2, 55.0, 8.0.

Synthesis of (R)-3-(4-(1H-Indol-5-yl)phenoxy)-1,1-dimethylpyrrolidin-1-ium Iodide (33)

Compound 100 (65 mg, 0.22 mmol) was dissolved in THF (5 mL). Iodomethane (277 μL, 4.45 mmol) was added, and the reaction mixture was added at 40 °C for 16 h. Upon cooling to room temperature, diethyl ether was added and the solid was isolated by vacuum filtration, washed with diethyl ether, and dried to give 33 as a white solid in a 93% yield. Mp = 226.1–228.7 °C. [α]_D²⁵ = −9.86 (c 0.5, dimethylsulfoxide). R_t (LC-MS) = 3.205 min. LC-MS (ESI): m/z calcd for C₂₀H₂₃N₂O [M]⁺ = 307.18, found 307.2. R_t (HPLC) = 11.51 min. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 11.11 (s, 1H), 7.75 (dd, J = 1.8, 0.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.45 (dt, J = 8.4, 0.8 Hz, 1H), 7.39–7.31 (m, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.46 (ddd, J = 3.0, 1.9, 0.8 Hz, 1H), 5.31 (d, J = 7.6 Hz, 1H), 3.93 (dd, J = 13.2, 5.9 Hz, 1H), 3.87–3.75 (m, 2H), 3.70–3.56 (m, 1H), 3.27 (s, 3H), 3.22 (s, 3H), 2.92–2.74 (m, 1H), 2.39–2.24 (m, 1H). ¹³C NMR (75 MHz, dimethylsulfoxide-d₆) δ 154.9, 135.5, 135.2, 130.8, 128.2, 127.9, 126.0, 120.1, 117.6, 115.9, 111.7, 101.4, 74.9, 69.3, 64.1, 52.6, 52.4, 30.1.

Synthesis of N,N-Diethyl-2-phenoxyethan-1-amine (34)

Obtained from phenol (1.00 g, 10.63 mmol), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and 2-(diethylamino)ethyl chloride hydrochloride (1.5 equiv) in acetone (15 mL) according to Method A. The crude was diluted with diethyl ether and washed three times with a 1 M NaOH solution. The organic phase was dried over anhydrous Na₂SO₄ and filtered. The filtrate was evaporated under vacuum, providing the desired product 34 as a colorless oil in a 75% yield. R_f = 0.6 (DCM/MeOH 95:5 + 0.5% NH_3(aq20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.34–7.24 (m, 2H), 7.03–6.85 (m, 3H), 4.06 (t, J = 6.4 Hz, 2H), 2.89 (t, J = 6.4 Hz, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.08 (t, J = 7.2 Hz, 6H).

Synthesis of 4-(2-Bromoethoxy)-1,1′-biphenyl (35)

Obtained from 4-phenylphenol (2.00 g, 11.75 mmol, 1 equiv), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and 1,2-dibromoethane (4.2 equiv), according to Method A, at reflux temperature for 48 h. The residue was suspended in chloroform (100 mL) and washed with an aqueous solution of 10% NaOH (2 × 20 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 95:5) providing the desired product 35 as a white powder in a 46% yield. R_f = 0.57 (cyclohexane/EtOAc 9:1) mp = 112 °C (coherent with the literature¹⁵). ¹H NMR (300 MHz, chloroform-d) δ 7.56 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.40–7.45 (m, 2H), 7.30–7.38 (m, 1H), 6.92 (d, J = 8.1 Hz, 2H), 4.30 (t, J = 6.3 Hz, 2H) 3.65 (t, J = 6.3 Hz, 2H).

Synthesis of 4-(2-Iodoethoxy)-1,1′-biphenyl (36)

Obtained from 4-(2-bromoethoxy)-1,1′-biphenyl 35 (1.50 g, 5.44 mmol, 1 equiv) according to Method D, providing the desired compound 36 as a white solid in a 92% yield. R_f = 0.51 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d) δ 7.56 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.40–7.45 (m, 2H), 7.30–7.38 (m, 1H), 6.92 (d, J = 8.1 Hz, 2H), 4.30 (t, J = 6.3 Hz, 2H) 3.45 (t, J = 6.3 Hz, 2H).

Synthesis of 2-([1,1′-Biphenyl]-4-yloxy)-N,N-diethylethan-1-amine (37)

Obtained from 4-(2-iodoethoxy)-1,1′-biphenyl 36 (900 mg, 2.79 mmol, 1 equiv) according to Method E at 60 °C for 4 h. The residue was purified by silica gel flash column chromatography (DCM/MeOH 98:2 + 0.5% NH_3(aq20%)) providing the desired compound 37 as a pale-yellow oil in an 84% yield. R_f = 0.42 (DCM/MeOH 98:2 + 0.5% NH_3(aq20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.56 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.40–7.45 (m, 2H), 7.30–7.38 (m, 1H), 6.92 (d, J = 8.1 Hz, 2H), 4.12 (t, J = 6.3 Hz, 2H), 2.93 (t, J = 6.3 Hz, 2H), 2.69 (q, J = 7.1 Hz, 4H), 1.09 (t, J = 7.1 Hz, 6H).

Synthesis of 4-(Benzyloxy)phenol (38)

Under a nitrogen atmosphere, a suspension of hydroquinone (1.00 g, 9.08 mmol, 1 equiv) and K₂CO₃ (0.62 g, 4.54 mmol, 0.5 equiv) in acetone (10 mL) was vigorously stirred at reflux temperature for 30 min. A solution of benzyl bromide (0.78 g, 4.54 mmol) in acetone (0.5 mL) was added dropwise, and the resulting mixture was refluxed overnight. The reaction mixture was cooled to room temperature, and the solid was removed by filtration. The filtrate was concentrated under vacuum, and the residue was diluted with EtOAc and washed with water. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the filtrate was evaporated under vacuum. The crude was purified by silica gel flash chromatography (cyclohexane/EtOAc 85:15), providing the desired product 38 as a white solid in a 52% yield. R_f = 0.45 (cyclohexane/EtOAc 8:2). Mp = 123 °C (coherent with the literature¹⁶). ¹H NMR (300 MHz, chloroform-d) δ 7.47–7.29 (m, 5H), 6.86 (d, J = 9.0 Hz, 2H), 6.76 (d, J = 9.0 Hz, 2H), 5.01 (s, 2H).

Synthesis of 2-(4-(Benzyloxy)phenoxy)-N,N-diethylethan-1-amine (39)

Obtained from 4-(benzyloxy)phenol 38 (250 mg, 1.25 mmol, 1 equiv), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.5 equiv) in acetone according to Method A. The crude was diluted with diethyl ether and washed three times with a 1 M NaOH solution. The organic phase was dried over anhydrous Na₂SO₄ and filtered. The filtrate was evaporated under vacuum, providing the desired product 39 as a colorless oil in a 75% yield. R_f = 0.58 (DCM/MeOH 95:5 + 0.5% NH_3(20% aq)). ¹H NMR (300 MHz, chloroform-d) δ 7.52–7.26 (m, 5H), 6.98–6.70 (m, 4H), 5.01 (s, 2H), 4.02 (t, J = 6.3 Hz, 2H), 2.87 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.08 (t, J = 7.2 Hz, 6H).

Synthesis of 4-Formylphenyl Acetate (40)

A solution of 4-hydroxybenzaldehyde (3.00 g, 24.6 mmol, 1 equiv) in pyridine (20 mL) was stirred at 0 °C for 30 min. Upon the dropwise addition of acetic anhydride (3.5 mL, 37 mmol, 1.5 equiv) for 30 min, the reaction mixture was warmed to room temperature and stirred until TLC showed full conversion. Afterward, the pH was adjusted to 7 by the dropwise addition of 1 M HCl (10 mL), and the product was extracted in diethyl ether. The combined organic phases were washed with 1 M HCl and 1 M NaOH and then dried over anhydrous Na₂SO₄, filtered under reduced pressure, and evaporated, providing the desired product 40 as a pale-yellow oil in an 80% yield. R_f = 0.43 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d) δ 9.9 (s, 1H) 7.8 (d, J = 8.5 Hz, 2H) 7.30 (d, J = 8.5 Hz, 2H) 2.30 (s, 3H).

Synthesis of 4-Vinylphenyl Acetate (41)

Under an inert atmosphere, methyltriphenylphosphonium bromide (7.16 g, 20.04 mmol, 1 equiv) was added portionwise to a suspension of 4-formylphenyl acetate 40 (2.74 g, 16.7 mmol, 1.2 equiv) and K₂CO₃ (2.76 g, 20.04 mmol, 1.2 equiv) in anhydrous THF (35 mL). The reaction mixture was refluxed for 6 h and then concentrated under reduced pressure. The residue was diluted with diethyl ether and washed with water. The water layer was re-extracted with diethyl ether, and the combined organic phases were dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 95:5), providing the desired compound 41 as a colorless oil in a 62% yield. R_f = 0.57 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d) δ 7.42 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 6.71 (dd, J = 17.6, 10.9 Hz, 1H), 5.71 (dd, J = 17.6, 0.7 Hz, 1H), 5.25 (dd, J = 10.9, 0.7 Hz, 1H), 2.30 (s, 3H).

Synthesis of 4-Vinylphenol (42)

A solution of 4-vinylphenyl acetate 41 (3.00 g, 18.5 mmol, 1 equiv) in THF (30 mL) was cooled to 0 °C. A solution of 5 M NaOH (9 mL, 46.25 mmol, 2.5 equiv) was added dropwise for 5 min, and the reaction mixture was stirred at the same temperature for 4 h. The mixture was quenched for 15 min by the dropwise addition of cold 1.5 M HCl (30 mL) and then further diluted with 60 mL of cold water. The aqueous phase was extracted four times with diethyl ether, and the combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated by a rotary evaporator at 25 °C. The residue was taken in absolute EtOH (30 mL) and evaporated again at 25 °C, providing the desired compound 42 as a solid in a 100% yield. The compound was stored as an ethanolic solution at 0 °C to avoid polymerization. R_f = 0.45 (cyclohexane/EtOAc 8:2). Mp = 72–74 °C (coherent with the literature¹⁷). ¹H NMR (300 MHz, chloroform-d) δ 7.30 (d, J = 8.5 Hz, 2H) 6.80 (d, J = 8.5 Hz, 2H) 6.70 (dd, J = 10.9, 17.6 Hz, 1H), 5.60 (d, J = 17.6 Hz, 1H) 5.10 (d, J = 10.9 Hz, 1H) 4.70 (s, OH, exchange with D₂O).

Synthesis of 1-(2-Bromoethoxy)-4-vinylbenzene (43)

Obtained from 4-vinylphenol 42 (1.15 g, 9.6 mmol, 1 equiv), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and dibromoethane (4.2 equiv) in anhydrous methyl ethyl ketone (20 mL), according to Method A, at reflux temperature for 48 h. The residue was resuspended in chloroform (100 mL) and washed with an aqueous solution of 10% NaOH. The organic layer was dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 95:5) providing the desired product 43 as a pale-yellow oil in a 55% yield. R_f = 0.63 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d) δ 7.39–7.30 (m, 2H), 6.92–6.82 (m, 2H), 6.66 (dd, J = 17.6, 10.9 Hz, 1H), 5.62 (d, J = 17.6 Hz, 1H), 5.14 (d, J = 10.9 Hz, 1H), 4.30 (t, J = 6.3 Hz, 2H), 3.63 (t, J = 6.3 Hz, 2H).

Synthesis of 1-(2-Iodoethoxy)-4-vinylbenzene (44)

Obtained from 1-(2-bromoethoxy)-4-vinylbenzene 43 (500 mg, 2.20 mmol, 1 equiv) according to Method D, providing the desired intermediate 44 as a pale-yellow oil in an 80% yield. R_f = 0.58 (cyclohexane/EtOAc 95:5). ¹H NMR (300 MHz, chloroform-d) δ 7.35 (d, J = 8.9 Hz, 2H), 6.86 (d, J = 8.9 Hz, 2H), 6.66 (dd, J = 17.6, 10.9 Hz, 1H), 5.62 (d, J = 17.6 Hz, 1H), 5.14 (d, J = 10.9 Hz, 1H), 4.36–4.18 (m, 2H), 3.50–3.32 (m, 2H).

Synthesis of N,N-Diethyl-2-(4-vinylphenoxy)ethan-1-amine (45)

Obtained from 1-(2-iodoethoxy)-4-vinylbenzene 44 (370 mg, 1.35 mmol, 1 equiv) according to Method E, at 60 °C for 4 h, as a pale-yellow oil, in a 98% yield. R_f = 0.75 (DCM/MeOH 95:5 + 0.5% NH_3(aq. 20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.33 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.65 (dd, J = 17.6, 10.9 Hz, 1H), 5.60 (d, J = 17.6 Hz, 1H), 5.12 (d, J = 10.9 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 2.90 (t, J = 6.3 Hz, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.09 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-4-(2-Bromostyryl)phenol (46)

Obtained from 1-bromo-2-iodobenzene (500 mg, 1.77 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (from cyclohexane/EtOAc 9:1 to 7:3), providing the desired product 46 as a white solid in a 31% yield. R_f = 0.32 (cyclohexane/EtOAc 9:1). Mp = 115.3 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.64 (dd, J = 7.7, 1.6 Hz, 1H), 7.57 (dd, J = 7.9, 1.3 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.35–7.26 (m, 2H), 7.09 (ddd, J = 7.9, 7.3, 1.6 Hz, 1H), 6.98 (d, J = 16.2 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 5.02 (s, 1H).

Synthesis of (E)-4-(3-Bromostyryl)phenol (47)

Obtained from 1-bromo-3-iodobenzene (500 mg, 1.77 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (from cyclohexane/EtOAc 9:1 to 7:3), providing the desired product 47 as a pale-yellow solid in a 53% yield. R_f = 0.27 (cyclohexane/EtOAc 9:1). Mp = 133.3 °C (coherent with the literature¹⁸). ¹H NMR (300 MHz, chloroform-d) δ 7.63 (t, J = 1.8 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.38–7.32 (m, 2H), 7.20 (t, J = 7.9 Hz, 1H), 7.04 (d, J = 16.3 Hz, 1H), 6.91–6.80 (m, 3H), 5.00 (s, 1H, exchanges with D₂O).

Synthesis of (E)-4-(4-Bromostyryl)phenol (48)

Obtained from 1-bromo-4-iodobenzene (500 mg, 1.77 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (from cyclohexane/EtOAc 9:1 to 7:3), providing the desired product 48 as a white solid in a 66% yield. R_f = 0.29 (cyclohexane/EtOAc 9:1). Mp = 191.7 °C (coherent with the literature¹⁹). ¹H NMR (300 MHz, chloroform-d) δ 7.48–7.30 (m, 6H), 7.03 (d, J = 16.4 Hz, 1H), 6.93–6.79 (m, 3H), 4.98 (s, 1H, exchanges with D₂O).

Synthesis of (E)-4-(4-(Trifluoromethyl)styryl)phenol (49)

Obtained from 4-iodobenzotrifluoride (300 mg, 1.1 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (gradient from cyclohexane/EtOAc 9:1 to 7:3), providing the desired compound 49 as a white solid in a 54% yield. R_f = 0.84 (cyclohexane/EtOAc 8:2 + 1% triethylamine). Mp = 158.3–161.0 °C (coherent with the literature²⁰). ¹H NMR (300 MHz, chloroform-d) δ 7.62–7.54 (m, 4H), 7.44 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 16.3 Hz, 1H), 6.97 (d, J = 16.3 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 4.88 (broad s, 1H, exchanges with D₂O).

Synthesis of (E)-4-(3-Methoxystyryl)phenol (50)

Obtained from 1-iodo-3-methoxybenzene (200 mg, 0.86 mmol, 1 equiv) and 4-vinylphenol 42 (1.5 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (DCM), providing product 50 as a beige solid in a 35% yield. R_f = 0.36 (cyclohexane/EtOAc 8:2). Mp = 117.6 °C (coherent with the literature²¹). ¹H NMR (300 MHz, methanol-d₄) δ 7.39 (d, J = 8.6 Hz, 2H), 7.22 (t, J = 7.9 Hz, 1H), 7.11–7.00 (m, 3H), 6.93 (d, J = 16.3 Hz, 1H), 6.81–6.73 (m, 3H), 3.81 (s, 3H).

Synthesis of (E)-4-(4-Methoxystyryl)phenol (51)

Obtained from 1-iodo-4-methoxybenzene (500 mg, 2.14 mmol, 1 equiv) and 4-vinylphenol 42 (1.5 equiv) according to Method F. The crude was purified by crystallization from MeOH, providing product 51 as a white solid in a 21% yield. R_f = 0.39 (cyclohexane/EtOAc 8:2). Mp = 136–138 °C (coherent with the literature²²). ¹H NMR (300 MHz, methanol-d₄) δ 7.42 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 6.94–6.86 (m, 4H), 6.76 (d, J = 8.8 Hz, 2H), 3.80 (s, 3H).

Synthesis of (E)-2-(4-(2-Bromostyryl)phenoxy)-N,N-diethylethan-1-amine (52)

Obtained from (E)-4-(2-bromostyryl)phenol 46 (140 mg, 0.51 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.5 equiv) in methyl ethyl ketone (10 mL) according to Method A. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 52 as a yellow oil in a 100% yield. R_f = 0.24 (cyclohexane/EtOAc 9:1 + 1% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.64 (dd, J = 7.9, 1.7 Hz, 1H), 7.57 (dd, J = 8.0, 1.3 Hz, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.36–7.26 (m, 2H), 7.09 (ddd, J = 8.0, 7.3, 1.7 Hz, 1H), 6.98 (d, J = 16.2 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 4.08 (t, J = 6.3 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-2-(4-(3-Bromostyryl)phenoxy)-N,N-diethylethan-1-amine (53)

Obtained from (E)-4-(3-bromostyryl)phenol 47 (240 mg, 0.87 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.5 equiv) in methyl ethyl ketone (10 mL), according to Method A. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 53 as a yellow oil in an 89% yield. R_f = 0.24 (cyclohexane/EtOAc 9:1 + 1% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.63 (t, J = 1.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.40–7.32 (m, 2H), 7.20 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 16.3 Hz, 1H), 6.94–6.83 (m, 3H), 4.08 (t, J = 6.3 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-2-(4-(4-Bromostyryl)phenoxy)-N,N-diethylethan-1-amine (54)

Obtained from (E)-4-(4-bromostyryl)phenol 48 (300 mg, 1.09 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (10 mL), according to Method A. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 5 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 54 as a yellow oil in an 80% yield. R_f = 0.24 (cyclohexane/EtOAc 9:1 + 1% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.48–7.40 (m, 4H), 7.34 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 16.3 Hz, 1H), 6.95–6.83 (m, 3H), 4.07 (t, J = 6.3 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-N,N-Diethyl-2-(4-(4-(trifluoromethyl)styryl)phenoxy)ethan-1-amine (55)

Obtained from (E)-4-(4-(trifluoromethyl)styryl)phenol 49 (150 mg, 0.57 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL), according to Method A. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 20 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 55 as a white solid in a 31% yield. R_f = 0.41 (DCM/EtOAc 9:1 + 1% triethylamine). Mp = 112.8 °C. ¹H NMR (300 MHz, methanol-d₄) δ 7.68 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 16.4 Hz, 1H), 7.08 (d, J = 16.4 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 4.15 (t, J = 5.6 Hz, 2H), 3.02 (t, J = 5.6 Hz, 2H), 2.78 (q, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H).

Synthesis of (E)-N,N-Diethyl-2-(4-(3-methoxystyryl)phenoxy)ethan-1-amine (56)

Obtained from (E)-4-(3-methoxystyryl)phenol 50 (60 mg, 0.27 mmol) K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL), according to Method A. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 56 as a yellow oil in a 23% yield. R_f = 0.31 (DCM/EtOAc 9:1 + 1% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.44 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.9 Hz, 1H), 7.13–6.95 (m, 4H), 6.90 (d, J = 8.8 Hz, 2H), 6.80 (dd, J = 7.9, 2.0 Hz, 1H), 4.10 (t, J = 6.2 Hz, 2H), 3.84 (s, 3H), 2.93 (t, J = 6.2 Hz, 2H), 2.69 (q, J = 7.1 Hz, 4H), 1.09 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-N,N-Diethyl-2-(4-(4-methoxystyryl)phenoxy)ethan-1-amine (57)

Obtained from (E)-4-(4-methoxystyryl)phenol 51 (100 mg, 0.44 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL) according to Method A. The residue was diluted with EtOAc, washed with water and then extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH until pH 12 and then extracted with EtOAc (3 × 15 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure providing the desired compound 57 as a white solid in an 84% yield. R_f = 0.32 (DCM/EtOAc 9:1 + 1% triethylamine). Mp = 137.6 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.45–7.38 (m, 4H), 6.94–6.86 (m, 6H), 4.18 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H), 3.02 (t, J = 6.0 Hz, 2H), 2.79 (q, J = 7.0 Hz, 4H), 1.17 (t, J = 7.0 Hz, 6H).

Synthesis of (E)-4-(3-(Trifluoromethyl)styryl)phenol (58)

Obtained from 3-iodobenzotrifluoride (1.0 g, 3.68 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F at reflux temperature overnight. The crude was purified by silica gel flash column chromatography (gradient from cyclohexane/EtOAc 9:1 to 7:3), providing the desired compound 58 as a pale-yellow solid in a 44% yield. R_f = 0.41 (cyclohexane/EtOAc 8:2). Mp = 119.1–123.0 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.75–7.69 (m, 1H), 7.67–7.60 (m, 1H), 7.51–7.39 (m, 4H), 7.11 (d, J = 16.3 Hz, 1H), 6.97 (d, J = 16.3 Hz, 1H), 6.90–6.80 (m, 2H), 4.80 (s, 1H).

Synthesis of (E)-4-(3-((2-Methoxyethoxy)methoxy)styryl)phenol (59)

Obtained from 1-iodo-3-((2-methoxyethoxy)methoxy)benzene 109 (870 mg, 2.82 mmol, 1 equiv) and 4-vinylphenol 42 (1.2 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (gradient from cyclohexane/EtOAc 9:1 to 7:3), providing the desired product 59 as a yellow oil in a 65% yield. R_f = 0.25 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d) δ 7.39 (d, J = 8.8 Hz, 2H), 7.30–7.17 (m, 2H), 7.18–7.09 (m, 2H), 7.04 (d, J = 16.5 Hz, 1H), 6.91 (d, J = 16.5 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 5.32 (s, 2H), 5.27 (s, 1H, exchanges with D₂O), 3.90–3.84 (m, 2H), 3.63–3.57 (m, 2H), 3.41 (s, 3H).

Synthesis of (E)-1-(4-(2-Bromoethoxy)styryl)-3-(trifluoromethyl)benzene (60)

Obtained from 58 (40 mg, 0.15 mmol, 1 equiv), K₂CO₃ (3 equiv), KI (0.1 equiv), and 1,2-dibromoethane (30 μL, 0.315 mmol, 2.1 equiv) in methyl ethyl ketone (2 mL), according to Method A. The residue was purified by flash chromatography (cyclohexane/EtOAc gradient from 9:1 to 8:2) affording 60 as a colorless oil in an 89% yield. R_f = 0.37 (cyclohexane/EtOAc 8:2). ¹H NMR (300 MHz, chloroform-d) δ 7.76–7.70 (m, 1H), 7.67–7.61 (m, 1H), 7.52–7.41 (m, 4H), 7.12 (d, J = 16.3 Hz, 1H), 6.99 (d, J = 16.3 Hz, 1H), 6.96–6.88 (m, 2H), 4.32 (t, J = 6.3 Hz, 2H), 3.66 (t, J = 6.3 Hz, 2H).

Synthesis of (E)-1-(4-(2-Bromoethoxy)styryl)-3-((2-methoxyethoxy)methoxy)benzene (61)

Obtained from (E)-4-(3-((2-methoxyethoxy)methoxy)styryl)phenol 59 (430 mg, 1.43 mmol, 1 equiv), K₂CO₃ (1.5 equiv), KI (0.1 equiv), and 1,2-dibromoethane (2.1 equiv) in methyl ethyl ketone (15 mL) according to Method A. The crude was purified by silica gel flash column chromatography (gradient from cyclohexane/EtOAc 9:1 to 7:3), providing the desired intermediate 61 as a yellow oil in a 26% yield. R_f = 0.39 (cyclohexane/EtOAc 8:2). ¹H NMR (300 MHz, chloroform-d) δ 7.45 (d, J = 8.7 Hz, 2H), 7.29–7.23 (m, 1H), 7.21–7.11 (m, 2H), 7.05 (d, J = 16.4 Hz, 1H), 6.99–6.87 (m, 4H), 5.31 (s, 2H), 4.32 (t, J = 6.3 Hz, 2H), 3.89–3.79 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 3.62–3.51 (m, 2H), 3.39 (s, 3H).

Synthesis of (E)-1-(4-(2-Iodoethoxy)styryl)-3-(trifluoromethyl)benzene (62)

Obtained from 60 (50 mg, 0.135 mmol, 1 equiv) according to Method D, providing the desired compound 62 as a colorless oil in an 83% yield. R_f = 0.40 (cyclohexane/EtOAc 8:2). ¹H NMR (300 MHz, chloroform-d) δ 7.75–7.70 (m, 1H), 7.69–7.56 (m, 1H), 7.50–7.44 (m, 4H), 7.12 (d, J = 16.4 Hz, 1H), 6.99 (d, J = 16.4 Hz, 1H), 6.95–6.86 (m, 2H), 4.33–4.23 (m, 2H), 3.48–3.39 (m, 2H).

Synthesis of (E)-1-(4-(2-Iodoethoxy)styryl)-3-((2-methoxyethoxy)methoxy)benzene (63)

Obtained from (E)-1-(4-(2-bromoethoxy)styryl)-3-((2-methoxyethoxy)methoxy)benzene 61 (150 mg, 0.37 mmol, 1 equiv) according to Method D, providing the desired compound 63 as a yellow oil in a 77% yield. R_f = 0.39 (cyclohexane/EtOAc 8:2). ¹H NMR (300 MHz, chloroform-d) δ 7.45 (d, J = 8.7 Hz, 2H), 7.29–7.26 (m, 1H), 7.21–7.09 (m, 2H), 7.05 (d, J = 16.2 Hz, 1H), 6.99–6.84 (m, 4H), 5.31 (s, 2H), 4.27 (dd, J = 7.4, 6.4 Hz, 2H), 3.87–3.82 (m, 2H), 3.61–3.55 (m, 2H), 3.43 (dd, J = 7.4, 6.4 Hz, 2H), 3.39 (s, 3H).

Synthesis of (E)-3-(4-(2-Iodoethoxy)styryl)phenol (64)

A mixture of (E)-3-(2-methoxyethoxymethyloxy)-4′-(2-iodoethyloxy)stilbene 63 (129 mg, 0.28 mmol, 1 equiv) and an excess of methanolic solution of 1.25 M HCl was stirred at 65 °C overnight. Upon cooling at room temperature, the resulting mixture was concentrated under reduced pressure, providing the desired product 64 as a pale-pink solid in an 82% yield. R_f = 0.29 (cyclohexane/EtOAc 8:2). Mp = 113.4 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.44 (d, J = 8.7 Hz, 2H), 7.25–7.18 (m, 1H), 7.09–6.99 (m, 2H), 6.99–6.87 (m, 4H), 6.72 (dd, J = 8.1, 2.4 Hz, 1H), 4.30–4.24 (m, 2H), 3.47–3.39 (m, 2H).

Synthesis of (E)-4-(4-((2-Methoxyethoxy)methoxy)styryl)phenol (65)

Obtained from 1-iodo-4-((2-methoxyethoxy)methoxy)benzene²³ (750 mg, 2.43 mmol, 1 equiv) and 4-vinylphenol 42 (1.1 equiv) according to Method F. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc gradient from 9:1 to 7:3), providing the desired product 65 as a white solid in a 65% yield. R_f = 0.4 (DCM/EtOAc 9:1). Mp = 113.4–114.1 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.45–7.33 (m, 4H), 7.07–6.98 (m, 2H), 6.92 (s, 2H), 6.86–6.77 (m, 2H), 5.28 (s, 2H), 4.98 (s, 1H), 3.89–3.79 (m, 2H), 3.62–3.53 (m, 2H), 3.39 (s, 3H).

Synthesis of (E)-N,N-Diethyl-2-(4-(4-((2-methoxyethoxy)methoxy)styryl)phenoxy)ethan-1-amine (66)

Obtained from 65 (200 mg, 0.67 mmol, 1 equiv), K₂CO₃ (3.0 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methylethylketone (25 mL) according to Method A. The residue was diluted with water and extracted with EtOAc (3 × 15 mL). The organic phases were combined and extracted with 1 M HCl. The aqueous phase was basified with 1 M NaOH and extracted with EtOAc (3 × 30 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give 66 as a yellow oil in a 20% yield. R_f = 0.2 (DCM/EtOAc 9:1 + 1% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.43 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.93 (s, 2H), 6.88 (d, J = 8.8 Hz, 2H), 5.29 (s, 2H), 4.58–4.51 (m, 2H), 3.86–3.81 (m, 2H), 3.59–3.54 (m, 2H), 3.51–3.41 (m, 2H), 3.38 (s, 3H), 3.26 (q, J = 7.4 Hz, 4H), 1.47 (t, J = 7.4 Hz, 6H).

Synthesis of (E)-N,N,N-Triethyl-2-(4-(4-((2-methoxyethoxy)methoxy)styryl)phenoxy)ethan-1-aminium Iodide (67)

Obtained from 66 (50 mg, 0.13 mmol) and iodoethane (9.6 equiv) in THF (2 mL) according to Method B at reflux for 16 h. Upon cooling to room temperature, the reaction mixture was diluted with diethyl ether and the resulting suspension was filtered, affording 67 as a light-yellow solid in a 46% yield. Mp = 130.4–130.8 °C. ¹H NMR (300 MHz, methanol-d₄): δ 7.51 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.05–6.96 (m, 6H), 5.27 (s, 2H), 4.50–4.39 (m, 2H), 3.83–3.78 (m, 2H), 3.81–3.72 (m, 2H), 3.58–3.54 (m, 2H), 3.48 (q, J = 7.2 Hz, 6H), 3.33 (s, 3H), 1.37 (t, J = 7.2 Hz, 9H).

Synthesis of (E)-4-(2-(Naphthalen-1-yl)vinyl)phenol (68)

1-Naphthylmethyltriphenylphosphonium chloride (28.5 g, 64.9 mmol) was added to a solution of sodium (2.85 g, 124 mmol) in EtOH (100 mL) at T < 10 °C. A solution of 4-hydroxybenzaldehyde (7.93 g, 64.9 mmol) in EtOH (50 mL) was added to the previous mixture, and the reaction mixture was stirred at room temperature for 48 h. The mixture was concentrated under reduced pressure. The residue was taken in diethyl ether and 3 M HCl, and the phases were separated. The organic phase was washed with water, dried over anhydrous Na₂SO₄, filtered, and evaporated affording the crude product. Flash chromatography (cyclohexane/EtOAc from 90:10 to 85:15—second eluted isomer) and subsequent crystallization from cyclohexane/CHCl₃ afforded compound 68 in a 53% yield. Mp = 135–138 °C. R_f = 0.3 (cyclohexane/EtOAc 8:2). ¹H NMR (200 MHz, chloroform-d) δ 8.28–8.20 (m, 1H), 7.95–7.70 (m, 4H), 7.65–7.45 (m, 5H), 7.11 (d, J = 16.0 Hz, 1H), 7.00–6.80 (m, 2H), 5.15 (s, 1H).

Synthesis of (E)-N,N-Diethyl-2-(4-(2-(naphthalen-1-yl)vinyl)phenoxy)ethan-1-amine (69)

Obtained from 68 (2.00 g, 8.12 mmol), 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv), and K₂CO₃ (2.24 g, 16.2 mmol) in acetone (30 mL) according to Method A at reflux for 4 h. Upon filtration, the filtrate was taken in diethyl ether and 2 M NaOH. The organic phase was separated and washed again with 2 M NaOH, water, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give 69 as a yellow oil in an 80% yield. R_f = 0.8 (DCM/MeOH 98:2 + 1.0% NH_3(aq20%)). ¹H NMR (200 MHz, chloroform-d) δ 8.30–8.20 (m, 1H), 7.90–7.70 (m, 4H), 7.60–7.40 (m, 5H), 7.11 (d, J = 16.0 Hz, 1H), 7.06–6.86 (m, 2H), 4.19–3.99 (m, 2H), 2.99–2.88 (m, 2H), 2.80–2.55 (m, 4H), 1.22–0.99 (m, 6H).

Synthesis of (E)-4-(2-(Naphthalen-2-yl)vinyl)phenol (70)

2-Naphthylmethyltriphenylphosphonium bromide (36.77 g, 76 mmol) was added to a solution of sodium (3.49 g, 152 mmol) in EtOH (100 mL) at T < 10 °C. A solution of 4-hydroxybenzaldehyde (9.28 g, 76 mmol) in EtOH (50 mL) was added to the previous mixture, and the reaction mixture was stirred at room temperature for 72 h. The mixture was concentrated under reduced pressure. The residue was taken in diethyl ether and 3 M HCl, and the phases were separated. The organic phase was washed with water, dried over anhydrous Na₂SO₄, filtered, and evaporated affording the crude product. The crude product was suspended in diethyl ether, and the mixture was vigorously stirred for 30 min. The solid was isolated by filtration and purified by flash chromatography (cyclohexane/EtOAc 8:2) to give compound 70 in a 31% yield. Mp = 208–211 °C (coherent with the literature²⁰). R_f = 0.55 (cyclohexane/EtOAc 1:1). ¹H NMR (200 MHz, dimethylsulfoxide-d₆) δ 9.68 (s, 1H), 8.05–7.80 (m, 5H), 7.60–7.45 (m, 4H), 7.37 (d, J = 16.0 Hz, 1H), 7.22 (d, J = 16.0 Hz, 1H), 6.95–6.74 (m, 2H).

Synthesis of (E)-N,N-Diethyl-2-(4-(2-(naphthalen-2-yl)vinyl)phenoxy)ethan-1-amine (71)

Obtained from 70 (2.00 g, 8.12 mmol), 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv), and K₂CO₃ (2.24 g, 16.2 mmol) in acetone (30 mL) according to Method A at reflux for 4 h. Upon filtration, the residue was purified by flash column chromatography (DCM/MeOH 95:5) to give 71 as a yellow oil in a 53% yield. R_f = 0.75 (DCM/MeOH 98:2 + 1.0% NH_3(aq20%)). ¹H NMR (200 MHz, chloroform-d) δ 7.90–7.70 (m, 5H), 7.55–7.40 (m, 4H), 7.22 (d, J = 16.0 Hz, 1H), 7.12 (d, J = 16.0 Hz, 1H), 7.02–6.84 (m, 2H), 4.18–4.00 (m, 2H), 2.99–2.79 (m, 2H), 2.80–2.52 (m, 4H), 1.18–1.00 (m, 6H).

Synthesis of 4-Phenethylphenol (72)

Pd/C (0.60 g) was added to a solution of 4-hydroxystilbene (1.00 g, 5.09 mmol) in MeOH (50 mL), and the reaction mixture was stirred under a hydrogen atmosphere for 24 h. The reaction mixture was filtered through a short layer of celite, and the volatiles were evaporated under vacuum to give 72 as a white solid (1.00 g, 100%). Mp = 97.2–98.2 °C (coherent with the literature²⁴). R_f = 0.5 (cyclohexane/EtOAc 7:3). ¹H NMR (300 MHz, chloroform-d) δ 7.36–7.11 (m, 5H), 7.09–6.98 (m, 2H), 6.80–6.68 (m, 2H), 2.98–2.74 (m, 4H).

Synthesis of N,N-Diethyl-2-(4-phenethylphenoxy)ethan-1-amine (73)

Obtained from compound 72 (0.5 g, 2.52 mmol, 1 equiv), K₂CO₃ (4.0 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (20 mL), according to Method A, at reflux temperature for 24 h. The residue was taken in 1 M HCl (100 mL) and washed with diethyl ether (50 mL). The water phase was basified with 3 M NaOH and extracted with EtOAc (3 × 50 mL). The combined organic phase was washed with brine, dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. Purification by silica gel flash column chromatography (DCM/MeOH 95:5 + 0.5% NH_3(aq20%)) afforded 73 as a colorless oil (0.62 g, 82%). R_f = 0.4 (DCM/MeOH 95:5 + 0.5% NH_3(aq20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.32–7.24 (m, 2H), 7.23–7.14 (m, 3H), 7.11–7.04 (m, 2H), 6.86–6.78 (m, 2H), 4.22–4.04 (m, 2H), 3.07–2.93 (m, 2H), 2.92–2.84 (m, 4H), 2.84–2.67 (m, 4H), 1.39–0.92 (m, 6H).

Synthesis of 1-(2-Bromoethoxy)-4-(phenylethynyl)benzene (74)

Obtained from 4-(phenylethynyl)phenol (280 mg, 1.44 mmol, 1 equiv), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and 1,2-dibromoethane (4.2 equiv) in methyl ethyl ketone (5 mL) according to Method A. The crude was purified by silica gel flash column chromatography (gradient from cyclohexane to cyclohexane/EtOAc 9:1), providing the desired product 74 as a white solid in a 66% yield. R_f = 0.56 (cyclohexane/EtOAc 9:1). Mp = 88.7 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.54–7.44 (m, 4H), 7.36–7.29 (m, 3H), 6.89 (d, J = 8.9 Hz, 2H), 4.31 (t, J = 6.3 Hz, 2H), 3.65 (t, J = 6.3 Hz, 2H).

Synthesis of 1-(2-Iodoethoxy)-4-(phenylethynyl)benzene (75)

Obtained from 1-(2-bromoethoxy)-4-(phenylethynyl)benzene 74 (118 mg, 0.39 mmol, 1 equiv) according to Method D, providing the desired compound 75 as a white solid in a 92% yield. R_f = 0.67 (cyclohexane/EtOAc 9:1). Mp = 108.3 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.56–7.41 (m, 4H), 7.39–7.28 (m, 3H), 6.88 (d, J = 9.0 Hz, 2H), 4.33–4.21 (m, 2H), 3.50–3.35 (m, 2H).

Synthesis of (E)-5-(4-(2-Chloroethoxy)styryl)benzene-1,3-diol (76)

Obtained from resveratrol (446 mg, 1.95 mmol, 1 equiv), K₂CO₃ (1.1 equiv), and 1-bromo-2-chloroethane (1.5 equiv) in DMF (2 mL) according to Method A, at 60 °C, overnight. The reaction mixture was concentrated under reduced pressure, diluted with EtOAc, and washed with 1 M HCl. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified by silica gel flash chromatography providing the desired compound 76 as a white solid in a 40% yield. R_f = 0.2 (DCM/EtOAc). Mp = 161 °C (coherent with the literature⁵). ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 9.17 (s, 2H, exchanges with D₂O), 7.49 (d, J = 8.8 Hz, 2H), 7.04–6.81 (m, 4H), 6.37 (d, J = 2.1 Hz, 2H), 6.10 (t, J = 2.1 Hz, 1H), 4.28–4.20 (m, 2H), 3.95–3.87 (m, 2H).

Synthesis of (E)-5-(4-(2-Iodoethoxy)styryl)benzene-1,3-diol (77)

Obtained from (E)-5-(4-(2-chloroethoxy)styryl)benzene-1,3-diol 76 (230 mg, 0.79 mmol, 1 equiv), according to Method D, providing the desired compound 77 as a pale-yellow solid in a 97% yield. R_f = 0.2 (DCM/EtOAc). Mp = 138 °C. ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 9.23 (s, 2H, exchanges with D₂O), 7.51 (d, J = 8.2 Hz, 2H), 6.98–6.89 (m, 4H), 6.39 (d, J = 2.2 Hz, 2H), 6.12 (t, J = 2.2 Hz, 1H), 4.27 (t, J = 6.3 Hz, 2H), 3.52 (t, J = 6.3 Hz, 2H).

Synthesis of 4-(Naphthalen-2-yl)phenol (78)

Obtained from 2-bromonapthalene (273 mg, 1.24 mmol, 1 equiv), p-hydroxyphenyl boronic acid (1.1 equiv), Pd(PPh₃)₄ (0.35 equiv), and TBAB (0.05 equiv) in 1,2-dimethoxyethane, according to Method C. The crude was purified by silica gel flash column chromatography (toluene/EtOAc 95:5), providing the desired compound 78 as a white solid in a 92% yield. R_f = 0.48 (cyclohexane/EtOAc 9:1). Mp = 167 °C (coherent with the literature²⁵). ¹H NMR (300 MHz, chloroform-d) δ 8.01–7.96 (m, 1H), 7.92–7.82 (m, 3H), 7.71 (dd, J = 8.6, 1.9 Hz, 1H), 7.62 (d, J = 8.7 Hz, 2H), 7.53–7.43 (m, 2H), 6.96 (d, J = 8.7 Hz, 2H), 4.86 (s, 1H).

Synthesis of N,N-Diethyl-2-(4-(naphthalen-2-yl)phenoxy)ethan-1-amine (79)

Obtained from 4-(naphthalen-2-yl)phenol 78 (210 mg, 0.95 mmol, 1 equiv), K₂CO₃ (2.5 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (10 mL) according to Method A. The crude was diluted with diethyl ether and washed three times with a 1 M NaOH solution. The organic phase was dried over anhydrous Na₂SO₄ and filtered. The filtrate was evaporated under vacuum, providing the desired product 79 as a white solid in a 27% yield. R_f = 0.29 (DCM/MeOH 95:5 + 0.5% NH_3(20% aq)). Mp = 51 °C. ¹H NMR (300 MHz, chloroform-d) δ 8.01–7.97 (m, 1H), 7.92–7.83 (m, 3H), 7.72 (dd, J = 8.5, 1.9 Hz, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.55–7.42 (m, 2H), 7.03 (d, J = 8.9 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 2.93 (t, J = 6.4 Hz, 2H), 2.68 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H).

Synthesis of 4-(Naphthalen-1-yl)phenol (80)

Obtained from 1-bromonapthalene (158 mg, 0.76 mmol, 1 equiv), p-hydroxyphenyl boronic acid (1.1 equiv), Pd(PPh₃)₄ (0.35 equiv), and TBAB (0.05 equiv) in 1,2-dimethoxyethane according to Method C. The crude was purified by silica gel flash column chromatography (toluene/EtOAc 95:5), providing the desired product 80 as a white solid in a 50% yield. R_f = 0.48 (cyclohexane/EtOAc 7:3). Mp = 91 °C (coherent with the literature²⁶). ¹H NMR (300 MHz, chloroform-d) δ 8.00–7.87 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H), 7.58–7.34 (m, 6H), 6.99 (d, J = 8.8 Hz, 2H), 5.22 (s, 1H, exchanges with D₂O).

Synthesis of N,N-Diethyl-2-(4-(naphthalen-1-yl)phenoxy)ethan-1-amine (81)

Obtained from 4-(naphthalen-1-yl)phenol 80 (84 mg, 0.38 mmol, 1 equiv), K₂CO₃ (4 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL), according to Method A, at reflux temperature, overnight. The residue was diluted in diethyl ether and washed with water. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 7:3 + 0.5% triethylamine), providing the desired compound 81 as a pale-yellow oil in a 46% yield. R_f = 0.20 (cyclohexane/EtOAc 7:3 + 0.5% triethylamine). ¹H NMR (300 MHz, chloroform-d) δ 7.95–7.78 (m, 3H), 7.54–7.36 (m, 6H), 7.03 (d, J = 8.6 Hz, 2H), 4.16 (t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.5 Hz, 2H), 2.70 (q, J = 7.0 Hz, 4H), 1.12 (t, J = 7.0 Hz, 6H).

Synthesis of 4-Acetoxybenzoic Acid (82)

A solution of 4-hydroxybenzoic acid (1.00 g, 7.24 mmol, 1 equiv) in acetic anhydride (4 mL, 36.28 mmol, 5 equiv) and 96% of sulfuric acid (0.1 mL) was heated at 80 °C for 2 h. After cooling to 0 °C, the mixture was slowly diluted with water and precipitation of a white solid occurred. The suspension was filtered, and the solid was washed with water three times, affording the desired compound 82 as a white solid in a 97% yield. R_f = 0.47 (cyclohexane/EtOAc 9:1). Mp = 199–201 °C (coherent with the literature²⁷). ¹H NMR (300 MHz, chloroform-d) δ 8.15 (d, J = 7.6 Hz, 2H), 7.32–7.10 (m, 2H), 2.47–2.15 (s, 3H).

Synthesis of 4-(Phenylcarbamoyl)phenyl Acetate (83)

The reaction was performed under inert conditions. A catalytic amount of DMF (5 drops) was added dropwise to a suspension of 4-acetoxybenzoic acid 82 (350 mg, 1.94 mmol, 1 equiv) in DCM (15 mL). The mixture was cooled to 0 °C, and oxalyl chloride (493 mg, 3.88 mmol, 2 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure to obtain a crude containing the activated acyl chloride, which was directly dissolved in DCM (10 mL) under a nitrogen atmosphere. The mixture was cooled to 0 °C, and a solution of aniline (542 mg, 5.82 mmol, 3 equiv) in DCM (3 mL) was added dropwise to the mixture. The resulting suspension was stirred for 1 h at room temperature and then washed with a 1 M HCl aqueous solution. The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified through silica gel flash column chromatography (toluene/EtOAc 9:1), affording the desired compound 83 as a white solid in a 96% yield. R_f = 0.67 (toluene/EtOAc 1:1). Mp = 168 °C (coherent with the literature²⁸). ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 10.25 (s, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.41–7.24 (m, 4H), 7.10 (t, J = 7.5 Hz, 1H), 2.31 (s, 3H).

Synthesis of 4-Hydroxy-N-phenylbenzamide (84)

An aqueous solution of 1 M NaOH (3 mL) was added to a suspension of 4-(phenylcarbamoyl)phenyl acetate 83 (520 mg, 2.04 mmol, 1 equiv) in MeOH (5 mL). The resulting solution was stirred at room temperature for 30 min. The reaction mixture was diluted with water (5 mL), the pH was adjusted to 6 by the dropwise addition of 1 M HCl, and the product was extracted with EtOAc. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, affording the desired compound 84 as a white powder in a 100% yield. R_f = 0.47 (cyclohexane/EtOAc 1:1). Mp = 203 °C (coherent with the literature²⁹). ¹H NMR (300 MHz, dimethylsulsoxide-d₆) δ 10.07 (s, 1H), 9.96 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.75 (dd, J = 8.6, 1.2 Hz, 2H), 7.33 (t, J = 8.6 Hz, 2H), 7.06 (tt, J = 7.2, 1.2 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H).

Synthesis of 4-(2-Chloroethoxy)-N-phenylbenzamide (85)

A suspension of 4-hydroxy-N-phenylbenzamide 84 (420 mg, 1.97 mmol, 1 equiv) and Cs₂CO₃ (1.2 equiv) in DMF (10 mL) was stirred at room temperature for 30 min. 1-Chloro-2-bromoethane (1.5 equiv) was added dropwise, and the resulting mixture was stirred at 60 °C overnight. The suspension was diluted with diethyl ether, washed with 1 M NaOH, and the organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing the desired compound 85 as a pale-yellow powder in a 39% yield. R_f = 0.30 (cyclohexane/EtOAc 1:1). Mp = 174 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.85 (d, J = 8.7 Hz, 2H), 7.74 (s, 1H), 7.63 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.14 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 4.30 (t, J = 5.8 Hz, 2H), 3.85 (t, J = 5.8 Hz, 2H).

Synthesis of 4-(2-Iodoethoxy)-N-phenylbenzamide (86)

Obtained according to Method D from 4-(2-chloroethoxy)-N-phenylbenzamide 85 (220 mg, 0.80 mmol, 1 equiv), providing the desired intermediate 86 as a white powder in an 84% yield. R_f = 0.35 (cyclohexane/EtOAc 1:1). Mp = 172 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.85 (d, J = 8.7 Hz, 2H), 7.71 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.37 (m, 2H), 7.14 (t, J = 7.0 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 4.32 (t, J = 6.7 Hz, 2H), 3.45 (t, J = 6.7 Hz, 2H).

Synthesis of 4-(2-(Diethylamino)ethoxy)-N-phenylbenzamide (87)

Obtained from 4-(2-iodoethoxy)-N-phenylbenzamide 86 (100 mg, 0.27 mmol, 1 equiv), according to Method E, using diethylamine as a solvent (3 mL), at reflux temperature for 3 h. The reaction mixture was concentrated under reduced pressure, and the residue was diluted in diethyl ether. The organic phase was extracted with 1 M HCl, the pH of the aqueous phase was adjusted to 9 with NH_3(aq 30%), and the product was re-extracted with EtOAc. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing the desired compound 87 as a pale brown oil in a 100% yield. R_f = 0.60 (DCM/MeOH 9:1 + 0.5% NH_3(aq30%)). ¹H NMR (300 MHz, chloroform-d) δ 7.84 (d, J = 8.8 Hz, 2H), 7.77 (s, 1H), 7.63 (dd, J = 7.5, 1.2 Hz, 2H), 7.42–7.29 (m, 2H), 7.14 (tt, J = 7.1, 1.2 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H), 2.99 (t, J = 6.0 Hz, 2H), 2.75 (q, J = 7.1 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H).

Synthesis of N-(4-Hydroxyphenyl)benzamide (88)

A suspension of 4-aminophenol (546 mg, 5 mmol, 1 equiv) and sodium octyl sulfate (0.02 equiv) was warmed under stirring in water (20 mL) until a clear solution was obtained. Afterward, a solution of benzoic anhydride (1 equiv) in CH₃CN (5 mL) was added dropwise and the reaction mixture was stirred at room temperature for 15 min. The solution was concentrated under vacuum, and the resulting brown suspension was filtered. The crude was triturated in chloroform and filtered, providing the desired compound 88 as an off-white solid in a 99% yield. R_f = 0.47 (cyclohexane/EtOAc 1:1). Mp = 216 °C (coherent with the literature³⁰). ¹H NMR (300 MHz, dimethylsulfoxide-d₆) δ 9.98 (s, exchanges with D₂O, 1H), 9.21 (s, NH, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.61–7.41 (m, 5H), 6.72 (d, J = 8.8 Hz, 2H).

Synthesis of N-(4-(2-Chloroethoxy)phenyl)benzamide (89)

A suspension of N-(4-hydroxyphenyl)benzamide 88 (860 mg, 4 mmol, 1 equiv) and Cs₂CO₃ (1.2 equiv) in DMF (20 mL) was stirred at room temperature for 30 min. 1-Chloro-2-bromoethane (1.5 equiv) was added dropwise, and the resulting mixture was stirred at 60 °C overnight. The suspension was diluted with diethyl ether, washed with 1 M NaOH, and the organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing the desired compound 89 as a pale-yellow solid in a 19% yield. R_f = 0.30 (cyclohexane/EtOAc 7:3). Mp = 177 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.92–7.79 (m, 2H), 7.71 (s, 1H), 7.59–7.45 (m, 5H), 6.94 (d, J = 9.0 Hz, 2H), 4.24 (t, J = 5.9 Hz, 2H), 3.82 (t, J = 5.9 Hz, 2H).

Synthesis of N-(4-(2-Iodoethoxy)phenyl)benzamide (90)

Obtained according to Method D from N-(4-(2-chloroethoxy)phenyl)benzamide 89 (230 mg, 0.83 mmol, 1 equiv), providing the desired compound 90 as a white powder in a 72% yield. R_f = 0.35 (cyclohexane/EtOAc 7:3). Mp = 173 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.86 (d, J = 8.3 Hz, 2H), 7.70 (s, 1H), 7.60–7.43 (m, 5H), 6.92 (d, J = 8.3 Hz, 2H), 4.26 (t, J = 7.3 Hz, 2H), 3.42 (t, J = 7.3 Hz, 2H).

Synthesis of N-(4-(2-(Diethylamino)ethoxy)phenyl)benzamide (91)

Obtained from N-(4-(2-iodoethoxy)phenyl)benzamide 90 (100 mg, 0.27 mmol, 1 equiv), according to Method E, using diethylamine as a solvent (3 mL), at reflux temperature for 3 h. The reaction mixture was concentrated under reduced pressure, and the residue was diluted in diethyl ether. The organic phase was extracted with 1 M HCl, the pH of the aqueous phase was adjusted to 9 with NH_3(aq30%), and the product was extracted again with EtOAc. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing the desired compound 91 as a pale brown oil in a 100% yield. R_f = 0.60 (DCM/MeOH 9:1 + 0.5% NH_3(30%aq)). ¹H NMR (300 MHz, chloroform-d) δ 7.86 (d, J = 8.9 Hz, 2H), 7.71 (s, 1H), 7.57–7.44 (m, 5H), 6.92 (d, J = 8.9 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 2.94 (t, J = 6.1 Hz, 2H), 2.71 (q, J = 7.1 Hz, 4H), 1.12 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-4-(Phenyldiazenyl)phenol (92)

Aniline (490 μL, 5.37 mmol) was dissolved in H₂O (2.5 mL) and 37% HCl (1.3 mL, 16.11 mmol) at 0 °C. A solution of NaNO₂ (370 mg, 5.37 mmol) in H₂O (2.5 mL) was added dropwise at the same temperature and stirred for 15 min. A solution of phenol (505 mg, 5.37 mmol) in EtOH (2 mL) was added, and the resulting mixture was stirred for 1 h at room temperature. A saturated solution of NaHCO₃ was added up to pH 7, and stirring was continued for 30 min. The formed solid was isolated by vacuum filtration, washed with water, and dried to give 92 as a brown solid in a 92% yield. Mp = 149 °C dec (coherent with the literature³¹). R_f = 0.65 (cyclohexane/EtOAc 7:3). ¹H NMR (300 MHz, chloroform-d) δ 7.92–7.83 (m, 4H), 7.56–7.40 (m, 3H), 7.00–6.91 (m, 2H).

Synthesis of (E)-N,N-Diethyl-2-(4-(phenyldiazenyl)phenoxy)ethan-1-amine (93)

Obtained from 92 (0.84 g, 4.24 mmol), K₂CO₃ (4 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (30 mL), according to Method A, at reflux temperature, overnight. The residue was obtained by filtration, and evaporation was taken in 1 M HCl (30 mL) and washed with diethyl ether (2 × 20 mL). NaOH (2 M) was added to the water phase up to pH 13, and then extraction with EtOAc (3 × 30 mL) was performed. The combined organic layer was washed with brine (30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated. Purification by silica gel flash chromatography (DCM/MeOH gradient from 0 to 30% MeOH) afforded 93 as a dark red oil in a 68% yield. R_f = 0.80 (DCM/MeOH + 1% NH_3(30%aq)). ¹H NMR (300 MHz, chloroform-d) δ 7.98–7.82 (m, 4H), 7.55–7.39 (m, 3H), 7.06–6.97 (m, 2H), 4.16 (t, J = 6.2 Hz, 2H), 2.94 (t, J = 6.2 Hz, 2H), 2.69 (q, J = 7.2 Hz, 4H), 1.11 (t, J = 7.2 Hz, 6H).

Synthesis of 4-(1H-Indol-6-yl)phenol (94)

Obtained from 6-bromoindole (300 mg, 0.15 mmol, 1 equiv), p-hydroxyphenyl boronic acid (2 equiv), and Pd(PPh₃)₄ (0.35 equiv) in EtOH/toluene 1:1 (5 mL) according to Method C, at reflux temperature overnight. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 7:3), followed by recrystallization from diisopropyl ether, providing the desired product 94 as a pink solid in a 42% yield. R_f = 0.26 (cyclohexane/EtOAc 7:3). Mp = 94 °C. ¹H NMR (300 MHz, chloroform-d) δ 8.20 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.57–7.49 (m, 3H), 7.33 (dd, J = 8.2, 1.7 Hz, 1H), 7.27–7.19 (m, 1H), 6.91 (d, J = 8.6 Hz, 2H), 6.57 (ddd, J = 3.2, 2.0, 1.0 Hz, 1H), 4.76 (s, 1H).

Synthesis of 2-(4-(1H-Indol-6-yl)phenoxy)-N,N-diethylethan-1-amine (95)

Obtained from 4-(1H-indol-6-yl)phenol 94 (52 mg, 0.25 mmol, 1 equiv), K₂CO₃ (4 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL), according to METHOD A, at reflux temperature, overnight. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The pH of the water phase was adjusted to 9 with 1 M NaOH and further extracted with EtOAc (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing the desired compound 95 as a red oil in a 25% yield. ¹H NMR (300 MHz, chloroform-d) δ 8.30 (s, 1H), 7.67 (dt, J = 8.2, 0.8 Hz, 1H), 7.60–7.49 (m, 3H), 7.34 (dd, J = 8.2, 1.6 Hz, 1H), 7.22 (dd, J = 3.1, 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 6.56 (ddd, J = 3.1, 2.0, 0.8 Hz, 1H), 4.14 (t, J = 6.2 Hz, 2H), 2.96 (t, J = 6.2 Hz, 2H), 2.72 (q, J = 7.2 Hz, 4H), 1.12 (t, J = 7.2 Hz, 6H).

Synthesis of 4-(1-Tosyl-1H-indol-5-yl)phenol (96)

Obtained from 5-bromo-1-tosyl-1H-indole (316 mg, 0.90 mmol, 1 equiv), p-hydroxyphenyl boronic acid (2 equiv), Pd(PPh₃)₄ (0.08 equiv), and TBAB in EtOH/toluene 1:3 (5 mL) according to Method C, at reflux temperature for 5 h. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 8:2), affording 96 as a pale-yellow oil in a 69% yield. R_f = 0.4 (cyclohexane/EtOAc 7:3). ¹H NMR (300 MHz, chloroform-d) δ 8.01 (dt, J = 8.6, 0.8 Hz, 1H), 7.83–7.74 (m, 2H), 7.65 (dd, J = 1.9, 0.7 Hz, 1H), 7.57 (d, J = 3.7 Hz, 1H), 7.53–7.37 (m, 3H), 7.25–7.19 (m, 2H), 6.94–6.84 (m, 2H), 6.68 (dd, J = 3.7, 0.8 Hz, 1H), 5.09 (broad s, 1H), 2.34 (s, 3H).

Synthesis of 4-(1H-Indol-5-yl)phenol (97)

To a solution of 96 (135 mg, 0.317 mmol) in MeOH (15 mL), KOH (108 mg, 1.92 mmol) was added. The resulting mixture was stirred at reflux for 3 h. KOH (190 mg, 3.39 mmol) was added, and stirring was continued at the same temperature for 3 h. The solvent was evaporated, and the residue was taken in EtOAc and washed with 1 M HCl. The organic phase was washed with 1 M NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered, and the solvent was concentrated under vacuum. Purification by silica gel flash chromatography (cyclohexane/EtOAc gradient from 8:2 to 1:1) provided 97 as an orange oil in a 90% yield. R_f = 0.2 (cyclohexane/EtOAc 7:3). ¹H NMR (300 MHz, chloroform-d) δ 8.17 (s, 1H), 7.80 (dt, J = 1.6, 0.9 Hz, 1H), 7.57–7.49 (m, 2H), 7.47–7.35 (m, 2H), 7.23 (dd, J = 3.2, 2.4 Hz, 1H), 6.95–6.88 (m, 2H), 6.60 (ddd, J = 3.1, 2.0, 0.8 Hz, 1H), 4.99 (s, 1H).

Synthesis of 2-(4-(1H-Indol-5-yl)phenoxy)-N,N-diethylethan-1-amine (98)

Obtained from 97 (128 mg, 0.25 mmol, 1 equiv), K₂CO₃ (4.1 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (5 mL), according to Method A, at reflux temperature, overnight. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The pH of the water phase was adjusted to 9 with 1 M NaOH, and the product was extracted with EtOAc (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure, providing 98 as a yellow oil in a 26% yield. ¹H NMR (300 MHz, chloroform-d) δ 8.24 (s, 1H), 7.80 (dt, J = 1.6, 0.8 Hz, 1H), 7.62–7.51 (m, 2H), 7.49–7.36 (m, 2H), 7.25–7.23 (m, 1H), 7.02–6.92 (m, 2H), 6.59 (ddd, J = 3.1, 2.0, 0.8 Hz, 1H), 4.25 (t, J = 5.9 Hz, 2H), 3.09 (t, J = 5.9 Hz, 2H), 2.87 (q, J = 7.3 Hz, 4H), 1.22 (t, J = 7.3 Hz, 6H).

Synthesis of tert-Butyl (R)-3-(4-(1-Tosyl-1H-indol-5-yl)phenoxy)pyrrolidine-1-carboxylate (99)

Under a nitrogen atmosphere, 96 (513 mg, 1.41 mmol) and tert-butyl (S)-3-hydroxypyrrolidine-1-carboxylate (264 mg, 1.41 mmol) were dissolved in anhydrous THF (5 mL). A solution of triphenylphosphine (444 mg, 1.69 mmol) was added, and the resulting mixture was cooled at −10 °C. A solution of DIAD (332 μL, 1.69 mmol) in anhydrous THF (10 mL) was added dropwise. At the end of addition, the mixture was stirred at reflux overnight. The solvent was evaporated, and the residue was taken in diethyl ether (200 mL), and washed with water and brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was concentrated under reduced pressure. Purification by two-column chromatography (first: DCM/EtOAc gradient from 0 to 10% EtOAc; second: toluene/EtOAc 9:1) provided 99 as an off-white solid in a 33% yield. ¹H NMR (300 MHz, chloroform-d) δ 8.01 (dt, J = 8.7, 0.8 Hz, 1H), 7.82–7.75 (m, 2H), 7.69–7.63 (m, 1H), 7.57 (d, J = 3.6 Hz, 1H), 7.54–7.44 (m, 2H), 7.25–7.14 (m, 3H), 6.97–6.88 (m, 2H), 6.68 (dd, J = 3.7, 0.8 Hz, 1H), 4.97–4.85 (m, 1H), 3.71–3.46 (m, 4H), 2.35 (s, 3H), 2.28–2.03 (m, 2H), 1.47 (s, 9H).

Synthesis of (R)-5-(4-((1-Methylpyrrolidin-3-yl)oxy)phenyl)-1H-indole (100)

Under a nitrogen atmosphere, a solution of 99 (140 mg, 0.267 mmol) in anhydrous THF (5 mL) was added dropwise to a suspension of LiAlH₄ (60 mg, 1.58 mmol) at −10 °C. At the end of addition, the reaction mixture was refluxed for 5 h. Upon cooling to 0 °C, water was added and the insoluble mixture was removed by filtration on celite. The solvent was evaporated, the residue was taken in EtOAc, and the organic layer was washed with a saturated solution of Na₂CO₃ and brine. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. Purification by flash column chromatography (DCM/MeOH gradient from 0 to 30% MeOH) provided 100 as an off-white solid in an 86% yield. Mp = 146.0–148.4 °C. ¹H NMR (300 MHz, chloroform-d) δ 8.17 (s, 1H), 7.80 (dt, J = 1.6, 0.8 Hz, 1H), 7.59–7.50 (m, 2H), 7.48–7.37 (m, 2H), 7.25–7.22 (m, 1H), 7.00–6.86 (m, 2H), 6.59 (ddd, J = 3.1, 2.0, 0.8 Hz, 1H), 4.93–4.82 (m, 1H), 2.90–2.81 (m, 3H), 2.56–2.44 (m, 1H), 2.42 (s, 3H), 2.39–2.29 (m, 1H), 2.05 (dddd, J = 13.7, 8.1, 6.0, 2.5 Hz, 1H).

Synthesis of 4-(Benzofuran-5-yl)phenol (101)

Obtained from 5-bromobenzofurane (200 mg, 1.02 mmol, 1 equiv) and p-hydroxyphenyl boronic acid (1.1 equiv) according to Method C. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 9:1), providing the desired product 101 as a white solid in an 80% yield. R_f = 0.2 (cyclohexane/EtOAc 9:1). Mp = 194 °C. ¹H NMR (300 MHz, chloroform-d) δ 7.73 (dd, J = 1.9, 0.8 Hz, 1H), 7.64 (d, J = 2.2 Hz, 1H), 7.56–7.41 (m, 4H), 6.92 (d, J = 8.8 Hz, 2H), 6.80 (dd, J = 2.2, 0.8 Hz, 1H), 4.70 (broad s, 1H).

Synthesis of 2-(4-(Benzofuran-5-yl)phenoxy)-N,N-diethylethan-1-amine Hydrochloride (102)

Obtained from 4-(benzofuran-5-yl)phenol 101 (170 mg, 0.81 mmol, 1 equiv), K₂CO₃ (4 equiv), KI (0.1 equiv), and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv), in methyl ethyl ketone (5 mL), according to Method A, at reflux temperature, overnight. The residue was diluted with EtOAc, washed with water, and then extracted with 1 M HCl. The pH of the water phase was adjusted to pH 9 with 1 M NaOH and further extracted with EtOAc (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The residue was taken in diethyl ether (3 mL) and warmed until complete dissolution. A solution of 2 M HCl in diethyl ether (0.5 mL) was added dropwise, and the resulting suspension was filtered, providing the desired compound 102 as a white solid in a 65% yield. Mp = 213 °C. ¹H NMR (300 MHz, methanol-d₄) δ 7.81–7.74 (m, 2H), 7.65–7.57 (m, 2H), 7.57–7.47 (m, 2H), 7.16–7.07 (m, 2H), 6.88 (s, 1H), 4.48–4.36 (m, 2H), 3.70–3.58 (m, 2H), 3.45–3.35 (m, 4H), 1.40 (t, J = 7.4 Hz, 6H).

Synthesis of 4-(2-(Diethylamino)ethoxy)benzaldehyde (103)

Obtained from p-hydroxybenzaldehyde (2.00 g, 16.38 mmol, 1 equiv), K₂CO₃ (2.5–4 equiv), and KI (0.1 equiv) and 2-chloro-N,N-diethylethylamine hydrochloride (1.2 equiv) in methyl ethyl ketone (100 mL), according to Method A, at reflux temperature, overnight. The residue was dissolved in HCl and washed with diethyl ether. The aqueous phase was basified to pH 10 with 1 M NaOH and extracted with EtOAc (3 × 10 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was concentrated under reduced pressure. The crude was purified by silica gel flash column chromatography (DCM/MeOH 9:1 + 1% NH_3(aq30%)), providing the desired compound 103 as a colorless oil in a 68% yield. R_f = 0.36 (diisopropyl ether/2-propanol 85:15 + 1% NH_3(aq 30%)). ¹H NMR (300 MHz, chloroform-d) δ 9.88 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 2.90 (t, J = 6.2 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 1.07 (t, J = 7.1 Hz, 6H).

Synthesis of (4-(2-(Diethylamino)ethoxy)phenyl)methanol (104)

A solution of 4-(2-(diethylamino)ethoxy)benzaldehyde 103 (1.92 g, 8.68 mmol, 1 equiv) in MeOH (20 mL) was treated with NaBH₄ (2 equiv) and stirred at room temperature for 3 h. Afterward, the solvent was evaporated under reduced pressure, and the residue was diluted with water (10 mL) and extracted with EtOAc (3 × 20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (gradient from DCM to DCM/MeOH 9:1 + 1%NH_3(aq20%)), providing the desired product 104 as a colorless oil in a 45% yield. R_f = 0.67 (DCM/MeOH 9:1 + 1%NH_3(aq20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.27 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 4.61 (s, 2H), 4.05 (t, J = 6.3 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.08 (t, J = 7.2 Hz, 6H).

Synthesis of N,N-Diethyl-2-(4-(phenoxymethyl)phenoxy)ethan-1-amine (105)

Under an inert atmosphere, a solution of (4-(2-(diethylamino)ethoxy)phenyl)methanol 104 (0.77 g, 3.45 mmol, 1 equiv), PPh₃ (1.2 equiv), and phenol (1 equiv) in THF (12 mL) was cooled to 0 °C and DEAD (1.2 equiv) was added dropwise. The reaction mixture was stirred at room temperature overnight. The volatiles were evaporated under reduced pressure, and the residue was purified by silica gel flash column chromatography (gradient from DCM to DCM/MeOH 9:1 + 1.5% NH_3(aq20%)), providing the desired compound 105 as an off-white solid in a 20% yield. Mp = 54.0–57.8 °C. R_f = 0.6 (DCM/MeOH + 1% NH_3(aq20%)). ¹H NMR (300 MHz, chloroform-d) δ 7.35 (d, J = 8.8 Hz, 2H), 7.35–7.26 (m, 2H), 7.03–6.93 (m, 3H), 6.92 (d, J = 8.8 Hz, 2H), 4.98 (s, 2H), 4.07 (t, J = 6.3 Hz, 2H), 2.90 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.09 (t, J = 7.1 Hz, 6H).

Synthesis of (E)-2-((4-(2-(Diethylamino)ethoxy)benzylidene)amino)phenol (106)

To a refluxed solution of 4-(2-(diethylamino)ethoxy)benzaldehyde 103 (700 mg, 3.16 mmol, 1 equiv) in EtOH, o-aminophenol (1 equiv) was added. The reaction mixture was stirred at reflux temperature for 1 h. The solvent was evaporated under reduced pressure, providing the desired compound 106 as a brown oil, which was used in the next step without further purification. R_f = 0.6 (diisopropyl ether/2-propanol 85:15 + 1% NH_3(aq 20%)). ¹H NMR (300 MHz, chloroform-d) δ 8.62 (s, 1H), 7.91–7.83 (m, 2H), 7.31–7.24 (m, 1H), 7.17 (ddd, J = 8.1, 7.3, 1.5 Hz, 1H), 7.05–6.96 (m, 3H), 6.89 (ddd, J = 8.0, 7.4, 1.4 Hz, 1H), 4.15 (t, J = 6.2 Hz, 2H), 2.93 (t, J = 6.2 Hz, 2H), 2.69 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H).

Synthesis of 2-(4-(Benzo[d]oxazol-2-yl)phenoxy)-N,N-diethylethan-1-amine Hydrochloride (107)

A solution of (E)-2-((4-(2-(diethylamino)ethoxy)benzylidene)amino)phenol 106 (987 mg, 3.16 mmol) and Pb(OAc)₄ (1.5 equiv) in EtOH was refluxed for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in MeOH (2 mL) and cooled to 0 °C. Under vigorous stirring, a methanolic solution of 4 M HCl (0.2 mL) was added dropwise. After 30 min, the reaction mixture was diluted with diethyl ether, and the resulting suspension was filtered, affording the desired product 107 as a white solid in a 30% yield. Mp = 192–193 °C. ¹H NMR (300 MHz, methanol-d₄) δ 8.23 (d, J = 9.0 Hz, 2H), 7.73–7.68 (m, 1H), 7.68–7.63 (m, 1H), 7.42–7.37 (m, 2H), 7.23 (d, J = 9.0 Hz, 2H), 4.54–4.44 (m, 2H), 3.72–3.63 (m, 2H), 3.38 (q, J = 7.3 Hz, 4H), 1.40 (t, J = 7.3 Hz, 6H).

Synthesis of 2-(4-(1H-Benzo[d]imidazol-2-yl)phenoxy)-N,N-diethylethan-1-amine (108)

A solution of 4-(2-(diethylamino)ethoxy)benzaldehyde 103 (610 mg, 2.76 mmol, 1 equiv) in EtOH (3 mL) was heated to reflux temperature, and o-phenylenediamine (300 mg, 2.76 mmol, 1 equiv) and Pb(OAc)₄ were added. The reaction mixture was stirred at reflux temperature overnight. Upon cooling, the volatiles were removed under reduced pressure, and the residue was purified by silica gel flash column chromatography (EtOAc/2-propanol gradient from 0 to 5% 2-propanol + 3% NH_3(aq20%)). Product 108 was obtained as a light brown solid in a 44% yield. R_f = 0.15 (diisopropyl ether/2-propanol 85:15 + 1% NH_3(aq20%)). Mp = 189.3–192.1 °C (coherent with the literature³²). ¹H NMR (300 MHz, chloroform-d) δ 7.98 (d, J = 8.8 Hz, 2H), 7.67–7.57 (m, 2H), 7.25–7.21 (m, 2H), 6.94 (d, J = 8.8 Hz, 2H), 4.16 (t, J = 5.9 Hz, 2H), 2.99 (t, J = 5.9 Hz, 2H), 2.77 (q, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H).

Synthesis of 1-Iodo-3-(2-methoxyethoxymethyloxy)benzene (109)

Under a nitrogen atmosphere at 0 °C, MEMCl (0.44 mL, 3.82 mmol) was added dropwise to a solution of 3-iodophenol (600 mg, 2.73 mmol) and DIPEA (0.84 mL, 4.84 mmol) in DCM (4 mL). Upon stirring at 35 °C for 5 h, the mixture was quenched with a saturated NH₄Cl solution at 0 °C. The aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with 1 M HCl, saturated NaHCO₃ solution, and brine, and the organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under vacuum obtaining 109 as a pale-yellow oil in a 96% yield. R_f = 0.5 (cyclohexane/EtOAc 9:1). ¹H NMR (300 MHz, chloroform-d): δ 7.45–7.39 (m, 1H), 7.36–7.29 (m, 1H), 7.05–6.94 (m, 2H), 5.24 (s, 2H), 3.86–3.75 (m, 2H), 3.63–3.49 (m, 2H), 3.38 (s, 3H).

Biological Assays

All methods are the same as those used in our recent publication.⁷ We provide brief outlines of these approaches next.

Binding Affinity to α7, α3β4, and α4β2 Nicotinic Receptors

For (±)-[³H]epibatidine (specific activity of 56–60 Ci/mmol; Perkin Elmer, Boston, MA), saturation binding studies were carried out on membrane homogenates. These were prepared from either SH-EP1 cells stably transfected with α3- and β4-nAChR subunit cDNAs⁸ or HEK 293 cells stably transfected with the α4 and β2 cDNAs (generous gift of Dr. Jon Lindstrom).⁹

For saturation experiments, the membrane homogenate aliquots were incubated overnight at 4 °C with 0.01–5 nM concentrations of (±)-[³H]epibatidine. Nonspecific binding was determined in parallel by adding 100 nM unlabeled epibatidine (Sigma-Aldrich) to the incubation solutions, as described previously.³³ At the end of the incubation, the samples were filtered on a GFC filter soaked in 0.5% polyethylenimine and washed with 10 mL of ice-cold phosphate-buffered saline (PBS) and the filters were counted in a β counter.

For [¹²⁵I]-αBungarotoxin ([¹²⁵I]αBgtx) (specific activity 200–213 Ci/mmol, Perkin Elmer, Boston, MA), saturation binding studies were carried out on a membrane homogenate prepared from SH-SY5Y cells transfected with human α7 cDNA, as described previously.⁵ Aliquots of the membrane homogenates were incubated overnight with 0.1–10.0 nM concentrations of [¹²⁵I]Bgtx at rt. Nonspecific binding was determined in parallel by including in the assay mixture 1 μM of unlabeled αBgtx (Sigma-Aldrich). After incubation, the samples were filtered as described for (±)-[³H]epibatidine binding.

For competition studies, the inhibition of [³H]epibatidine and [¹²⁵I] αBgtx binding was measured by incubating the membranes transfected with the appropriate subtype with increasing concentrations of the compounds (1 nM to 1 mM) 5 min followed by overnight incubation at 4 °C, with 0.1 nM of [³H]epibatidine for the α4β2 subtype or 0.25 nM of [³H]epibatidine for the α3β4 subtype or at rt with 2–3 nM of [¹²⁵I]αBgtx in the case of the α7-subtype. At the end of the incubation time, the samples were processed as described for the saturation studies.

[³H]epibatidine binding was determined by liquid scintillation counting in a β counter, and [¹²⁵I] αBgtx binding was determined by direct counting in a γ counter. Saturation binding data were evaluated by one-site competitive binding curve-fitting procedures using GraphPad Prism version 6 (GraphPad Software, CA). In the saturation binding assay, the maximum specific binding (B_max) and the equilibrium binding constant (K_d) values were calculated using one-site—specific binding with the Hill slope—model. K_i values were obtained by fitting three independent competition binding experiments, each performed in duplicate for each compound on each subtype. Inhibition constants (K_i) were estimated by reference to the K_d of the radioligand, according to the Cheng–Prusoff equation and are expressed as nM values.

Two-Electrode Voltage Clamp (TEVC) Recording of α7- and α9α10-nAChR Functions

For functional pharmacology studies, two-electrode voltage clamp recordings were performed, using human nAChR subunits heterologously expressed in X. laevis oocytes. Approaches were closely related to those previously detailed.¹⁰ Briefly, X. laevis oocytes were purchased from Ecocyte Bioscience US (Austin, TX), and the incubation temperature was 13 °C. Harvesting of oocytes from X. laevis by EcoCyte follows the guidelines of the National Institute of Health’s Office of Laboratory Animal Welfare and was authorized under IACUC number #1019-1 (valid through December 2022). Injections of nAChR subunit mRNA were made using glass micropipettes (outer diameter ≈40 μm, resistance 2–6 MΩ), and mRNA was injected in a total volume of 40 nL. For α7-nAChR, 1.25 ng of α7-nAChR subunit mRNA was injected per oocyte along with 0.125 ng of NACHO mRNA to improve functional expression.³⁴ For α9α10-nAChR, a total of 10 ng of nAChR subunit mRNA was injected using α9 to α10 cRNAs in a 9:1 ratio by mass.

TEVC recordings were made in oocyte saline solution (82.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1.8 mM CaCl₂·₂H₂O, and 1 mM MgCl₂·₆H₂O, pH 7.4) and were performed at room temperature (20 °C). One week after injection, oocytes were voltage-clamped (−70 mV; Axoclamp 900A amplifier, Molecular Devices, Sunnyvale, CA). Recordings were sampled at 10 kHz (low-pass Bessel filter, 40 Hz; high-pass filter, DC) and saved to disk (Clampex v10.2; Molecular Devices). To ensure the quality of recordings, oocytes with leak currents (I_leak) > 50 nA were discarded without being recorded. In all cases, initial control stimulations (ACh, 1 mM, applied for 1 s) were performed, with a 60 s washout (no drug) between control stimulations (total of five stimulations). This allowed us to define a 100% response control and to ascertain that run-down or desensitization was not occurring due to repeated ACh stimulation.

For antagonist concentration response curves, test compounds were applied simultaneously with 1 mM ACh, starting with the lowest concentration of the test compound and increasing in half-log steps to a maximum concentration of 100 μM. The standard 1 min spacing between stimulation was maintained. Data for each oocyte were normalized by expressing the peak function in the presence of test compounds as % of the control function (the mean peak function measured across the initial control stimulations was defined as 100% for each oocyte). IC₅₀ values were calculated from these normalized nAChR-mediated currents through nonlinear least-squares curve fitting (GraphPad Prism 5.0; GraphPad Software, Inc., La Jolla, CA).

The intrinsic agonist efficacy of test compounds was measured by applying them (alone at 100 μM, 1 s application time, no ACh coapplication) 1 min following the last initial control stimulation. The peak function following the addition of the test compound was normalized for each oocyte in the same way just described for antagonist concentration curves. The same normalization was applied to the peak of any rebound current observed during the 60 s washout period following the application of the test compound and to the peak function induced by a final control application of ACh (1 mM, 1 s application time).

Computational Modeling

Compounds 1a and 33 were drawn with the two-dimensional (2D) sketch editor of Maestro and prepared for docking using Ligprep, with default settings. The dimeric α7α7 interface containing EVP-6124 was extracted from the cryo-EM of the full-length structure of the human α7-nAChR (7EKP) and prepared with the Protein Preparation Wizard according to default settings. Compound 33 was docked using the Induced Fit Protocol of Schrodinger,³⁵ selecting the current ligand (EVP-6124) as the docking centroid, Glide XP redocking, and a scaling factor of 1.0, to avoid excessive deformation of the binding site. The best-scoring pose according to the IFD score and the XP GScore also respected the best-known conserved ligand−α7-nAChR interaction, by placing the positively charged nitrogen within the aromatic box and was therefore selected. Compound 1a was docked using Glide XP docking with default settings, with a grid centered on ligand 33, and the best-scored pose according to the XP GScore was selected. The binding site analysis was performed using Sitemap, centered on 33 and default settings.

Glossary

Abbreviations Used

B_max

maximum specific binding

cryo-EM

cryogenic electron microscopy

DIAD

diisopropyl azodicarboxylate

eq

equivalent

K_d

equilibrium binding constant

K_i

inhibition constant

nAChR

nicotinic acetylcholine receptor

R_t

retention time

S.E.M.

standard error of mean

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01256.

• ¹H NMR and ¹³C NMR of the final compounds; HPLC traces of key final compounds (6, 28, and 33); example traces of two-electrode voltage clamp recordings (PDF)

• Molecular formula strings (CSV)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was supported by the Università degli Studi of Milan and by the National Institutes of Health awards R01 DA043567 and R01 DA042749 to P.W.

The authors declare no competing financial interest.