Abstract
The homomeric α7 nicotinic receptor (α7 nAChR) is widely expressed in the human brain that could be activated to suppress neuroinflammation, oxidative stress and neuropathic pain. Consequently, a number of α7 nAChR agonists have entered clinical trials as anti-Alzheimer's or anti-psychotic therapies. However, high-resolution crystal structure of the full-length α7 receptor is thus far unavailable. Since acetylcholine-binding protein (AChBP) from Lymnaea stagnalis is most closely related to the α-subunit of nAChRs, it has been used as a template for the N-terminal domain of α-subunit of nAChR to study the molecular recognition process of nAChR-ligand interactions, and to identify ligands with potential nAChR-like activities.
Here we report the discovery and optimization of novel acetylcholine-binding protein ligands through screening, structure-activity relationships and structure-based design. We manually screened in-house CNS-biased compound library in vitro and identified compound 1, a piperidine derivative, as an initial hit with moderate binding affinity against AChBP (17.2% inhibition at 100 nmol/L). During the 1st round of optimization, with compound 2 (21.5% inhibition at 100 nmol/L) as the starting point, 13 piperidine derivatives with different aryl substitutions were synthesized and assayed in vitro. No apparent correlation was demonstrated between the binding affinities and the steric or electrostatic effects of aryl substitutions for most compounds, but compound 14 showed a higher affinity (Ki=105.6 nmol/L) than nicotine (Ki=777 nmol/L). During the 2nd round of optimization, we performed molecular modeling of the putative complex of compound 14 with AChBP, and compared it with the epibatidine-AChBP complex. The results suggested that a different piperidinyl substitution might confer a better fit for epibatidine as the reference compound. Thus, compound 15 was designed and identified as a highly affinitive acetylcholine-binding protein ligand. In this study, through two rounds of optimization, compound 15 (Ki=2.8 nmol/L) has been identified as a novel, piperidine-based acetylcholine-binding protein ligand with a high affinity.
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Introduction
Acetylcholine is an important neurotransmitter in the central nervous system (CNS). Dysfunctional acetylcholine signaling is associatedwith significant pathological conditions (eg, Alzheimer's disease1, Parkinson's disease2,3,4,5 and schizophrenia6,7,8). Its cholinergic effects are mediated through either muscarinic (mAChR) or nicotinic (nAChR) receptors. Cumulative evidence suggests that the homomeric α7 receptor, one subtype of the most widely expressed nicotinic receptor in the human brain, could be activated to suppress neuroinflammation9,10, oxidative stress9,11 and neuropathic pain12 in animal models. Moreover, improvement in the triad of positive symptoms, negative symptoms and cognitive dysfunction in schizophrenia is shown to be mediated by the α7 receptor in both preclinical and clinical studies13,14,15. Several α7 nAChR agonists (eg, tropisetron16,17, GTS-2118,19, TC-5619 (bradanicline)20,21,22, EVP-612423, ABT-12624, AQW05125, Figure 1) have entered clinical trials as anti-Alzheimer's or anti-psychotic therapies. FORUM Pharmaceuticals Inc recently completed a phase-III clinical trial of EVP-6124 as an adjunctive pro-cognitive treatment in schizophrenia subjects during chronic, stable, atypical antipsychotic therapy (ClinicalTrials.gov Identifier: NCT01716975). Additional α7 nAChR agonists are expected to move into clinical trials to address the unmet medical needs of patients suffering from psychotic disorders. Unfortunately, similar to other ligand-gated ion channels (LGICs) such as 5-HT3 and GABAA, there is no available high-resolution crystal structure of the α7 receptor. However, some evidence-based hypotheses26,27,28 and theoretical models29,30,31 have been developed.
Acetylcholine-binding protein (AChBP) is a soluble protein separated from Lymnaea stagnalis32, which is most closely related to the α-subunit of nAChRs. Nearly all residues conserved within the nAChR family are present in AChBP, including those relevant to ligand binding. AChBP also binds to known nAChR agonists and competitive antagonists, such as acetylcholine, nicotine, d-tubocurarine and α-bungarotoxin33. Therefore, AChBP could be utilized as a template for the N-terminal domain of an α-subunit of nAChR to study the molecular recognition process of nAChR-ligand interactions34,35,36,37 and to identify ligands with potential nAChR-like activities38,39,40.
Based on the previously described AChBP-based binding assay41, our in-house CNS-biased compound library was manually screened and compound 1, a piperidine derivative, was identified as an initial hit with moderate binding affinity against AChBP (17.2% inhibition at 100 nmol/L). Since the pyridine substructure is considered to be a privileged substructure for typical nAChR agonists (such as nicotine and epibatidine) and methoxypyridineis also common in nAChR ligands42,43,44, the naphthalene substructure of compound 1 was changed to a pyridine ring in compound 2, which marginally increased binding to AChBP (21.5% inhibition at 100 nmol/L). Moreover, with compound 2 as the starting point of this study, we conducted structure-activity relationship (SAR) analysis, and structure-based optimization, which eventually yielded a low-nanomolar affinity compound (compound 15) against AChBP, as shown in Scheme 1.
Materials and methods
Chemistry
Chemicals were purchased from Sigma Aldrich (Sigma-Aldrich Shanghai Trading Co Ltd, Shanghai, China), AcrosOrganics (Fisher Scientific Worldwide (Shanghai) Co, Ltd, Shanghai, China), Energy Chemical (Sun Chemical Technology (Shanghai) Co, Ltd, Shanghai, China) and Shanghai Chemical Reagent Company (Shanghai, China) and were used directly without further purification. All compounds were purified by silica gel thin-layer chromatography (>95%), and their structures were determined with nuclear magnetic resonance spectra and low-resolution mass spectra. 1H spectra were recorded in DMSO-d6 or CDCl3 on Varian Mercury-300 or Varian Mercury-400 instruments. Mass spectra were generated with electric ionization (ESI) produced by an HP5973N analytical mass spectrometer.
General procedure for the preparation of 2a and 15a
A solution of commercially available substituted piperidine derivative (12.7 mmol) in anhydrous DCM (100 mL) was cooled to 0 °C. Tert-butyldicarbonate (2.6 g, 12.1 mmol) in DCM (20 mL) was added drop-wise into solution. After being stirred for 10 min, the reaction mixture was allowed to adjust to room temperature and stirred overnight. After dilution with water, the crude product was extracted with DCM (100 mL×2). The combined organic extracts were washed with hydrochloric acid (0.5 mol/L, 50 mL), and brine (100 mL), dried over Na2SO4, filtered, and concentrated to yield compounds 2a and 15a. The crude product was used without further purification.
Detailed analytical information can be found in the supporting information.
General procedure for the preparation of 2b–15b
A solution of commercially available lithium diisopropylamide (2 mol/L, 21.7 mmol) in anhydrous THF (100 mL) was cooled to −78 °C. Compound 2a (12.1 mmol) in anhydrous THF (20 mL) was added drop-wise into the solution. After being stirred for 10 min, the reaction mixture was allowed to adjust to −30 °C and stirred for 30 min. Various substituted benzyl bromides (24.2 mmol) in anhydrous THF (20 mL) were added drop-wise into the solution, which was cooled to −78 °C again. After being stirred for 2 h at −78 °C, the solution was adjusted to room temperature and stirred overnight. The reaction mixture was quenched by the addition of saturated ammonium chloride solution (30 mL) at 0 °C. After dilution with water, the crude product was extracted with ethyl acetate (200 mL×2). Combined organic extracts were washed with water (100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography over silica using petroleum ether/ethyl acetate (15/1) to yield compounds 2b–15b.
Detailed analytical information can be found in the supporting information.
General procedure for the preparation of 2c–15c
A solution of compounds 2b–15b (1.39 mmol) in anhydrous THF (50 mL) was cooled to 0 °C. Lithium aluminum hydride (1 mol/L, 1.39 mmol) in anhydrous THF was added drop-wise into the solution. After being stirred at 0 °C for 1 h, the reaction mixture was quenched by the addition of saturated ammonium chloride solution (10 mL) at 0 °C. After dilution with water, the crude product was extracted with ethyl acetate (100 mL×2). The combined organic extracts were washed with water (100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated to yield compounds 2c–15c. The crude product was used without further purification.
Detailed analytical information can be found in the supporting information.
General procedure for the preparation of 2d–15d
A solution of compounds 2c–15c (0.69 mmol) in anhydrous DMF (8 mL) was cooled to 0 °C. Sodium hydroxide (3.45 mmol) was added to the solution and stirred for 15 min at room temperature. Then, 3-fluoropyridine (3.45 mmol) was added and the reaction mixture was heated to 75 °C for 10 h. The reaction mixture was quenched by the addition of saturated ammonium chloride solution (30 mL) at 0 °C. After dilution with water, the crude product was extracted with ethyl acetate (30 mL×2). The combined organic extracts were washed with water (10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography over silica using petroleum ether/ethyl acetate (5/1) to yield compounds 2d–15d.
Detailed analytical information can be found in the supporting information.
General procedure for the preparation of 2–15
Compounds 2d–15d (0.24 mmol) were dissolved in DCM (3 mL) along with TFA (1 mL), and the reaction mixture was stirred at room temperature for 1 h and then concentrated. The crude product was dissolved in DCM (50 mL) and basified with sodium hydroxide solution (20%, 2 mL). Organic extracts were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography over silica using DCM/methanol (10/1) to yield compounds 2–15.
3-[(4-benzylpiperidin-4-yl)methoxy]pyridine (2)
Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 9.80–9.00 (brs, 1H), 8.33–8.30 (brs, 1H), 8.22–8.19 (brs, 1H), 7.24–7.14 (m, 5H), 7.02–6.98 (m, 2H), 3.67 (s, 2H), 3.40–3.20 (m, 4H), 2.86 (s, 2H), 1.94–1.88 (m, 4H). ESI-MS (m/z): 283 [M+H]+. HRMS (m/z): calculated for C18H22N2O [M+H]+, 283.1810; observed, 283.1812.
3-{[4-(2-chlorobenzyl)piperidin-4-yl]methoxy}pyridine (3)
Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 8.23–8.21 (m, 1H), 8.20–8.16 (m, 1H), 7.28–7.17 (m, 1H), 7.16–7.06 (m, 5H), 3.80 (s, 2H), 3.00–2.90 (m, 2H), 2.84–2.80 (m, 2H), 2.35–2.25 (brs, 1H), 1.78–1.59 (m, 4H). ESI-MS (m/z): 317 [M+H]+. HRMS (m/z): calculated for C18H21ClN2O [M+H]+, 317.1421; observed, 317.1415.
3-{{4-[2-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine (4)
Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 8.30–8.19 (s, 2H), 7.68–7.62 (m, 1H), 7.48–7.41 (m, 1H), 7.40–7.35 (m, 1H), 7.35–7.25 (m, 1H), 7.18–7.10 (m, 1H), 3.98–3.92 (m, 2H), 3.38–3.34 (m, 2H), 3.12–3.09 (m, 2H), 2.01–2.00 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C19H21F3N2O [M+H]+, 351.1684; observed, 351.1669.
3-{[4-(2-methylbenzyl)piperidin-4-yl]methoxy}pyridine (5)
Pale yellow oil 98%. 1H NMR (400 MHz, CD3OD) δ 8.29–8.28 (m, 1H), 8.19–8.17 (m, 1H), 7.50–7.46 (m, 1H), 7.43–7.39 (m, 1H), 7.18–7.08 (m, 4H), 3.98 (s, 2H), 3.28–3.14 (m, 4H), 2.97 (s, 2H), 2.32 (s, 3H), 2.01–1.94 (m, 2H), 1.92–1.82 (m, 2H). ESI-MS (m/z): 297 [M+H]+. HRMS (m/z): calculated for C19H24N2O [M+H]+, 297.1967; observed, 297.1962.
3-{[4-(3-chlorobenzyl)piperidin-4-yl]methoxy}pyridine (6)
Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 8.36–8.25 (m, 1H), 7.95–7.75 (m, 2H), 7.07–6.97 (m, 2H), 6.92-6.79 (m, 2H), 3.90–3.75 (m, 2H), 3.40–3.20 (m, 5H), 2.84–2.65 (m, 2H), 1.95–1.65 (m, 4H). ESI-MS (m/z): 317 [M+H]+. HRMS (m/z): calculated for C18H21ClN2O [M+H]+, 317.1421; observed, 317.1417.
3-{{4-[3-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine (7)
Pale yellow oil 95%. 1H NMR (600 MHz, CDCl3) δ 8.35–8.33 (m, 1H), 8.26–8.24 (m, 1H), 7.47–7.43 (m, 1H), 7.35-7.31 (m, 2H), 7.27–7.22 (m, 2H), 7.20–7.16 (m, 1H), 4.91–4.88 (brs, 1H), 3.68 (s, 2H), 3.18–3.12 (m, 2H), 3.05–3.00 (m, 2H), 2.96 (s, 2H), 1.78–1.73 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C19H21F3N2O [M+H]+, 351.1684; observed, 351.1674.
3-{[4-(3-methylbenzyl)piperidin-4-yl]methoxy}pyridine (8)
Pale yellow oil 99%. 1H NMR (600 MHz, CDCl3) δ 8.36–8.35 (m, 1H), 8.24–8.22 (m, 1H), 7.25–7.19 (m, 2H), 7.12–7.09 (m, 1H), 7.01–6.99 (m, 1H), 6.87–6.85 (m, 2H), 4.46–4.45 (brs, 1H), 3.70 (s, 2H), 3.16–3.11 (m, 2H), 3.06–3.01 (m, 2H), 2.85 (s, 2H), 2.21 (s, 3H), 1.76–1.72 (m, 4H). ESI-MS (m/z): 297 [M+H]+. HRMS (m/z): calculated for C19H24N2O [M+H]+, 297.1967; observed, 297.1957.
3-{[4-(4-chlorobenzyl)piperidin-4-yl]methoxy}pyridine (9)
Pale yellow oil 95%. 1H NMR (600 MHz, CD3OD) δ 8.72–8.21 (m, 1H), 8.11–8.05 (m, 1H), 7.10–7.08 (m, 1H), 7.06–6.81 (m, 5H), 3.52 (s, 2H), 3.31–3.25 (m, 2H), 3.21–3.12 (m, 2H), 2.70 (s, 2H), 1.91–1.78 (m, 4H). ESI-MS (m/z): 317 [M+H]+. HRMS (m/z): calculated for C18H21ClN2O [M+H]+, 317.1421; observed, 317.1407.
3-{{4-[4-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine (10)
Pale yellow oil 95%. 1H NMR (400 MHz, CD3OD) δ 8.32–8.31 (m, 1H), 8.20–8.18 (m, 1H), 7.56–7.53 (m, 2H), 7.51–7.47 (m, 1H), 7.43–7.39 (m, 1H), 7.37–7.34 (m, 2H), 3.86 (s, 2H), 3.33–3.28 (m, 2H), 3.24–3.16 (m, 2H), 3.05 (s, 2H), 1.94–1.80 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C19H21F3N2O [M+H]+, 351.1684; observed, 351.1669.
3-{[4-(4-methylbenzyl)piperidin-4-yl]methoxy)}pyridine (11)
Pale yellow oil 96%. 1H NMR (400 MHz, CD3OD) δ 8.32–8.31 (m, 1H), 8.20–8.17 (m, 1H), 7.51–7.47 (m, 1H), 7.43–7.39 (m, 1H), 7.06–7.00 (m, 4H), 3.83 (s, 2H), 3.42–3.36 (m, 2H), 3.34–3.26 (m, 2H), 2.91 (s, 2H), 2.26 (s, 3H), 1.96–1.84 (m, 4H). ESI-MS (m/z): 297 [M+H]+. HRMS (m/z): calculated for C19H24N2O [M+H]+, 297.1967; observed, 297.1966.
3-{{4-[(1,1'-biphenyl)-4-ylmethyl]piperidin-4-yl}methoxy}pyridine (12)
Pale yellow oil 99%. 1H NMR (400 MHz, CD3OD) δ 8.33–8.31 (m, 1H), 8.19–8.17 (m, 1H), 7.56–7.53 (m, 2H), 7.49–7.45 (m, 3H), 7.42–7.36 (m, 3H), 7.32–7.27 (m, 1H), 7.20–7.18 (m, 2H), 3.85 (s, 2H), 3.40–3.34 (m, 2H), 3.29–3.22 (m, 2H), 2.96 (s, 2H), 1.96–1.84 (m, 4H). ESI-MS (m/z): 359 [M+H]+. HRMS (m/z): calculated for C24H26N2O [M+H]+, 359.2123; observed, 359.2102.
3-{[4-(2,3-dichlorobenzyl)piperidin-4-yl]methoxy}pyridine (13)
Pale yellow oil 96%. 1H NMR (400 MHz, CD3OD) δ 8.20–8.15 (m, 2H), 7.43–7.37 (m, 3H), 7.24–7.15 (m, 2H), 4.00 (s, 2H), 3.15 (s, 2H), 3.11–3.04 (m, 2H), 3.02–2.95 (m, 2H), 1.88–1.75 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C18H20Cl2N2O [M+H]+, 351.1031; observed, 351.1008.
3-{[4-(3,4-dichlorobenzyl)piperidin-4-yl]methoxy}pyridine (14)
Pale yellow oil 98%. 1H NMR (400 MHz, CD3OD) δ 8.31–8.29 (m, 1H), 8.19–8.17 (m, 1H), 7.42–7.37 (m, 3H), 7.26–7.25 (m, 1H), 7.07–7.03 (m, 1H), 3.81 (s, 2H), 3.22–3.15 (m, 2H), 3.12–3.04 (m, 2H), 2.89 (s, 2H), 1.84–1.70 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C18H20Cl2N2O [M+H]+, 351.1031; observed, 351.1021.
3-{[3-(3,4-dichlorobenzyl)piperidin-3-yl]methoxy}pyridine (15)
Pale yellow oil 96%. 1H NMR (400 MHz, CDCl3) δ 8.36–8.34 (m, 1H), 8.25–8.22 (m, 1H), 7.28–7.19 (m, 4H), 6.94–6.90 (m, 1H), 3.82–3.78 (m, 1H), 3.72–3.68 (m, 1H), 2.90–2.82 (m, 3H), 2.78–2.69 (m, 3H), 2.36–2.22 (brs, 1H), 1.70–1.54 (m, 3H), 1.50–1.42 (m, 1H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for C18H20Cl2N2O [M+H] +, 351.1031; observed, 351.1023.
AChBP-based [3H]-epibatidine binding assay
The inhibition of 3H]-epibatidine binding with Ls-AChBP was conducted according to previously reported methods41. In brief, Ls-AChBP was diluted in PBS-Tris binding buffer (final concentration of 1.4 mmol/L KH2PO4, 4.3 mmol/L Na2HPO4, 137 mmol/L NaCl, 2.7 mmol/L KCl, 20 mmol/L Trizma base, 4% DMSO, 0.05% Tween 20, pH 7.4) to obtain a quantity of 1.3 ng per well. AChBP was incubated with 10−4–10−11mol/L ligands in the presence of approximately 1.5 nmol/L 3H]-epibatidine. Displacement of 3H]-epibatidine was recorded to represent binding to AChBP. Five or six log-dilution concentrations of representative compounds were tested in duplicate. The IC50 (concentration of the compound that produces 50% inhibition of binding) was determined by least squares nonlinear regression using GraphPad Prism software. Nicotine served as the positive control. The results of the binding assay revealed that the affinity of nicotine was similar to a previous report (Ki=777.7 nmol/L in our article vs Ki=478.0 nmol/L in reported article).
Molecular modeling
All molecular models were implemented based on the molecular modeling packages in Schrodinger Maestro 10.1. The structures of epibatidine, compound 14, (S)-15, and (R)-15 were built and optimized. The crystal complex of AChBP in complex with epibatidine was acquired from the RCSB protein data bank (PDB ID: 2BYQ) and appropriately treated by the protein preparation wizard module. The Glide function, available in the packages, was employed for molecular docking. The procedures were as follows: (1) ligand preparation (the Ligprep module was used to create several conformations of the ligand), (2) receptor grid generation, and (3) ligand docking. For the receptor grid generation procedure, the residues Tyr 55, Tyr 188, Ser 189 and Tyr 195 were chosen as the 'Rotatable Groups' because the exact conformations of these residues were flexible within several reported structures.
To validate the docking methodology, epibatidine was chosen as the test ligand and the RMSD value was calculated using the core pattern comparison. The RMSD value (1.33–2.5) confirmed the reliability of the docking method. Finally, compound 14 was docked to AChBP to provide putative docked models.
The hydrogen bond, cation-π interactions, π–π interactions, as well as the measurements of the distances of certain atoms, were elucidated by the software Pymol 1.5.0.3. The 2D diagram of the putative binding mode was produced by Schrodinger Maestro 10.1.
Results
Chemistry
The piperidine derivatives were prepared in a five-step procedure as demonstrated in Scheme 2. The addition of a Boc group to a commercially available ethyl piperidine-4-carboxylate yielded compound 2a. The Boc-protect compound 2a reacted with various substituted benzyl bromide in the presence of LDA to afford the corresponding nucleophilic substitution products (compound 2b–14b) in 45%–58% yields. The alcohol compounds 2c–14c were synthesized via reduction using a LiAlH4 solution at 0 °C. The reaction of compounds 2c–14c with 3-fluoropyridine and subsequent deprotection of the Boc group by TFA furnished the target compounds 2–14.
The synthesis of compound 15 is described in Scheme 3 utilizing a procedure similar to that used for compounds 2–14.
Biological evaluation
The binding data of the synthesized compounds are shown in Table 1. Compared with nicotine, most of the compounds displayed slightly higher binding affinities for AChBP at a test concentration of 100 nmol/L. Compound 14 displayed a 7-fold higher binding affinity when compared to nicotine.
Molecular docking
To elucidate the binding mode of compound 14, the molecular docking strategy was utilized using the epibatidine-bound Ls-AChBP (PDB: 2BYQ). As shown in Figure 2, compound 14 was completely buried within the protein at the interface between the principal side of the interface and the complementary side.
Each subunit consisted of an N-terminal α-helix, two short α3 helices, and a 10-stranded β-sandwich core. On the principal side, the nitrogen atom, which served as the protonation center in the piperidine ring of compound 14 at physiological pH, resided within hydrogen-bonding distance of the backbone carbonyl atom of Tyr 93. Moreover, three cation-π interactions were predicted between the protonated nitrogen atom and the phenyl groups of Tyr 188 and Tyr 195 as well as the indole group of Trp 147. The 3,4-dichloro aryl formed a T-shaped π-π interaction with Trp 147. On the complementary side, the 3,4-dichloro aryl entered into a hydrophobic sub-pocket formed by Ile 106, Phe 117, Ala 107, Met 116, and Val 108. Alternatively, the pyridine ring occupied another pocket comprising Ser 189, Tyr 55, Cys 190, and Gln 57. The pyridine ring also shared a π–π interaction with the Cys 190-Cys 191 ring.
Discussion
With the purpose of finding novel AChBP ligands, our in-house CNS-biased compound library was screened manually in vitro, and compound 1, a piperidine derivative, was chosen as the starting hit for this study. With the substructure of naphthalene changed to the privileged pyridine for compound 1, we used compound 2 as the prototype compound for further optimization. A series of compounds with different aryl substitutions were designed and synthesized. Most compounds demonstrated somewhat higher binding affinities against AChBP than compound 2 and nicotine at 100 nmol/L. Para- or meta-substitutedaryl substitution was associated with marginal increase of affinities compared with ortho-substituted aryl substitution. No definite correlation between binding affinities and the steric or electrostatic effects of substituted aryl substitutions was observed. During this 1st optimization, the affinity was optimized for compound 14, which was 7-fold higher than that of nicotine.
Molecular docking was utilized to predict the binding mode of compound 14 for further structure optimization. Epibatidine was reported to have the highest binding affinity with AChBP in natural products comprising an aromatic ring and an endocyclic secondary amino group45. These two groups were positioned at an optimal distance and an optimal relative spatial orientation to provide ideal interactions with the residues at the binding site36,46. Minimal changes in the chemical structure of these compounds have been found to exert profound effects on their pharmacological properties42. As shown in Figure 3, the overlapped conformation of epibatidine versus compound 14 suggested that the protonation center located in the piperidine ring of compound 14 at physiological pH was within the hydrogen-bonding distance of the backbone carbonyl atom of Tyr 93. In contrast, epibatidine formed a hydrogen-bond between the potentially protonated nitrogen atom of the aliphatic ring and the backbone carbonyl of Tyr 147.
The distance between the alpha-carbon (Cα) and the nitrogen atom in aliphatic ring of compound 14 with the nitrogen atom in aliphatic ring of epibatidine was calculated. The results suggested that the Cα-N distance (1.08 Å) was shorter than N–N distance (2.24 Å). It should be noted that, if this overlapped conformation is true for epibatidine and compound 14 in complex with AChBP, a change of 4-piperidinyl substitution to 3-piperidinyl substitution might fit compound 14 to epibatidine better and might contribute to significantly increased AChBP binding affinities. Thus, compound 15 with the expected 3-piperidinyl substitution was designed and synthesized (Scheme 3) during the 2nd round of optimization.Follow-up bio-assays suggested that compound 15 was a very potent AChBP ligand and was two orders of magnitude more potent than compound 14 (Table 1).
To explain the dramatically increased affinity of compound 15, molecular docking was utilized. Since compound 15 had a chiral center, the binding modes of (S)-15 and (R)-15 were predicted separately. As shown in Figure 4, the protonated nitrogen atom of (S)-15 shared two hydrogen bonds with Tyr 93 and Trp 147 and two cation-π interactions with Trp 147 and Tyr 188. At the same time, the protonated nitrogen atom of (R)-15 only had one hydrogen-bond with Trp 147, but it also shared three cation-π interactions, two with Trp 148 and one with Tyr 195. The two aromatic rings of each enantiomer entered the same hydrophobic pockets as compound 14. As the protonated nitrogen of epibatidine also shared a hydrogen-bond with Trp 147, it is possible that Trp 147 was a key residue for ligand-receptor interactions.
Enhancing interactions with Trp 147 might drastically increase the affinity of the compounds. Furthermore, as the aromatic rings entered the same pocket, changing the position of the protonated nitrogen atom altered the distance and the orientation between the protonation center and the aromatic rings. Thus, changing the position of the protonated nitrogen atom may also improve the affinity of this compound.
In conclusion, pyridine-based compound 1 was identified from our in-house CNS-biased compound library by manual in vitro screening. With introduction of the privileged pyridine, compound 2 (21.5% inhibition at 100 nmol/L) was selected as the prototype compound for further optimization. Within the 1st of optimization, compound 14 displayed the highest binding affinity (Ki=105.6 nmol/L). Using the molecular docking results of compound 14 with Ls-AChBP, a 2nd of optimization was implemented with a structure-based strategy, which led to the discovery of the highly affinitive compound 15 (Ki=2.8 nmol/L) as a potent, novel, piperidine-based acetylcholine-binding protein ligand. The binding mode of compound 15 was then predicted to explain the drastic increase in binding affinity.
Author contribution
Li-ming SHAO, Lu ZHOU, and Wei LI conceived the research; Jian SHEN screened the in-house library based the biological assay which was set up by Lu ZHOU; Jian SHEN and Xi-cheng YANG designed and synthesized the target compounds, as well as biological assays under the supervision of Li-ming SHAO and Wei LI; Ming-cheng YU, Xun-jie ZHANG, Guan-xin PAN, Yu-rong YAN, and Si-chen WANG participated in synthetic works and biological assays; Xi-cheng YANG completed the works of molecular modeling; Yong-hui WANG, Qiong XIE, Lin-qian YU, Li XIAO, Hui-jiao SUN, and Hao CHEN were involved in data analysis and interpretation; Jian Shen and Xi-cheng Yang drafted the manuscript, which was revised and refined by Wei LI, Lu ZHOU, and Li-ming SHAO. All authors approved the final edition of manuscript.
Structural formulae of α7 nAChR agonists under clinical development.
A putative binding mode of compound 14 in complex with Ls-AChBP. The hydrogen-bond (red dash), the cation-π interactions (brown dash) and the π–π interactions (blue dash) were shown. (A) The 3D view of the putative binding mode of compound 14 (colored by yellow). The principal side chain (colored by blue) and the complementary side (colored by green) were shown. (B) The 2D view of the putative binding mode of compound 14.
Comparison of the binding mode of compound 14 (colored by blue) with that of epibatidine (colored by yellow). The hydrogen bonds (red dash) as well as the distance (black dash) between the alpha-carbon (Cα) and the nitrogen atom in aliphatic ring of compound 14 with the nitrogen atom in aliphatic ring of epibatidine were shown.
A putative binding mode of compound (S)-15 and (R)-15 in complex with Ls-AChBP. The hydrogen-bond (red dash), the cation-π interactions (brown dash) and the π–π interactions (blue dash) were shown. (A) The 3D view of the putative binding mode of compound (S)-15 (colored by yellow). The principal side chain (colored by blue) and the complementary side (colored by green) were shown. (B) The 2D view of the putative binding mode of compound (S)-15. (C) The 3D view of the putative binding mode of compound (R)-15 (colored by yellow). The principal side chain (colored by blue) and the complementary side (colored by green) were shown. (D) The 2D view of the putative binding mode of compound (R)-15.
The discovery path of compound 15 as a potent AChBP ligand.
Synthesis of the piperidine derivatives. Reagents and conditions: a) Boc2O, DCM, RT, overnight (98%); b) substituted benzyl bromide, LDA, THF, −78 °C-RT, overnight (75%–86%); c) LiAlH4, THF, 0 °C, 1 h (95%–98%); d) NaH, 3-Fluoropyridine, DMF, 75 °C, overnight (78%–86%); e) TFA, DCM, RT, 1 h (94%–98%).
Synthesis of compound 15. Reagents and conditions: a) Boc2O, DCM, RT, overnight (98%); b) 3,4-Dichlorobenzyl bromide, LDA, THF, −78 °C-RT, overnight (75%–86%); c) LiAlH4, THF, 0 °C, 1 h (95%–98%); d) NaH, 3-Fluoropyridine, DMF, 75 °C, overnight (78%–86%); e) TFA, DCM, RT, 1 h (94%–98%).
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This work was supported by the Science and Technology Commission of Shanghai Municipality (No 15431900100) and the National Natural Science Foundation of China (No 81473076).
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Shen, J., Yang, Xc., Yu, Mc. et al. Discovery, synthesis, biological evaluation and structure-based optimization of novel piperidine derivatives as acetylcholine-binding protein ligands. Acta Pharmacol Sin 38, 146–155 (2017). https://doi.org/10.1038/aps.2016.124
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DOI: https://doi.org/10.1038/aps.2016.124
