Discovery, synthesis, biological evaluation and structure-based optimization of novel piperidine derivatives as acetylcholine-binding protein ligands

Article abstract of DOI:10.1038/aps.2016.124

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

Full text of DOI:10.1038/aps.2016.124

Acta Pharmacologica Sinica 2016: 1–10
© 2016 CPS and SIMM All rights reserved 1671-4083/16
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Original Article
Discovery, synthesis, biological evaluation and
structure-based optimization of novel piperidine
derivatives as acetylcholine-binding protein ligands
Jian SHEN#, Xi-cheng YANG#, Ming-cheng YU, Li XIAO, Xun-jie ZHANG, Hui-jiao SUN, Hao CHEN, Guan-xin PAN, Yu-rong YAN,
*
*
*
Si-chen WANG, Wei LI , Lu ZHOU , Qiong XIE, Lin-qian YU, Yong-hui WANG, Li-ming SHAO
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
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.
Keywords: α7 nAChR; acetylcholine-binding protein ligands; piperidine derivatives; molecular modeling; psychotic disorders
Acta Pharmacologica Sinica advance online publication, 5 Dec 2016; doi: 10.1038/aps.2016.124
human brain, could be activated to suppress neuroinflamma-
Introduction
Acetylcholine is an important neurotransmitter in the central tion[9, 10], oxidative stress[9, 11] and neuropathic pain[12] in ani-
nervous system (CNS). Dysfunctional acetylcholine signal- mal models. Moreover, improvement in the triad of positive
ing is associatedwith significant pathological conditions (eg, symptoms, negative symptoms and cognitive dysfunction in
Alzheimer’s disease[1], Parkinson’s disease[2–5] and schizophre- schizophrenia is shown to be mediated by the α7 receptor in
nia[6–8]). Its cholinergic effects are mediated through either both preclinical and clinical studies[13–15]. Several α7 nAChR
muscarinic (mAChR) or nicotinic (nAChR) receptors. Cumu- agonists (eg, tropisetron[16, 17], GTS-21[18, 19], TC-5619 (bradani-
lative evidence suggests that the homomeric α7 receptor, one cline)[20–22], EVP-6124[23], ABT-126[24], AQW051[25], Figure 1) have
subtype of the most widely expressed nicotinic receptor in the entered clinical trials as anti-Alzheimer’s or anti-psychotic
therapies. FORUM Pharmaceuticals Inc recently completed a
# These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail limingshao@fudan.edu.cn (Li-ming SHAO);
wei-li@fudan.edu.cn (Wei LI);
zhoulu@fudan.edu.cn (Lu ZHOU)
Received 2016-07-21 Accepted 2016-09-20
phase-III clinical trial of EVP-6124 as an adjunctive pro-cogni-
*
tive 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

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Shen J et al
Acetylcholine-binding protein (AChBP) is a soluble protein
separated from Lymnaea stagnalis[32], 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 antago-
nists, such as acetylcholine, nicotine, d-tubocurarine and
α-bungarotoxin[33]. 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
interactions[34–37] and to identify ligands with potential nAChR-
like activities[38–40]
.
Based on the previously described AChBP-based binding
assay[41], 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 sub-
structure for typical nAChR agonists (such as nicotine and
epibatidine) and methoxypyridineis also common in nAChR
ligands[42–44], 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.
Figure 1. Structural formulae of α7 nAChR agonists under clinical
development.
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
hypotheses[26–28] and theoretical models[29–31] have been devel-
oped.
Materials and methods
Chemistry
Chemicals were purchased from Sigma Aldrich (Sigma-
Aldrich Shanghai Trading Co Ltd, Shanghai, China),
Scheme 1. The discovery path of compound 15 as a potent AChBP ligand.
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AcrosOrganics (Fisher Scientific Worldwide (Shanghai) Co, reaction mixture was quenched by the addition of saturated
Ltd, Shanghai, China), Energy Chemical (Sun Chemical Tech- ammonium chloride solution (10 mL) at 0 °C. After dilution
nology (Shanghai) Co, Ltd, Shanghai, China) and Shanghai with water, the crude product was extracted with ethyl acetate
Chemical Reagent Company (Shanghai, China) and were used (100 mL×2). The combined organic extracts were washed
directly without further purification. All compounds were with water (100 mL) and brine (100 mL), dried over Na2SO4,
purified by silica gel thin-layer chromatography (>95%), and filtered, and concentrated to yield compounds 2c–15c. The
their structures were determined with nuclear magnetic reso- crude product was used without further purification.
nance spectra and low-resolution mass spectra. 1H spectra
Detailed analytical information can be found in the support-
were recorded in DMSO-d6 or CDCl3 on Varian Mercury-300 ing information.
or Varian Mercury-400 instruments. Mass spectra were gener-
ated with electric ionization (ESI) produced by an HP5973N General procedure for the preparation of 2d–15d
analytical mass spectrometer.
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
General procedure for the preparation of 2a and 15a
A solution of commercially available substituted piperidine room temperature. Then, 3-fluoropyridine (3.45 mmol) was
derivative (12.7 mmol) in anhydrous DCM (100 mL) was added and the reaction mixture was heated to 75°C for 10 h.
cooled to 0 °C. Tert-butyldicarbonate (2.6 g, 12.1 mmol) in The reaction mixture was quenched by the addition of satu-
DCM (20 mL) was added drop-wise into solution. After being rated ammonium chloride solution (30 mL) at 0 °C. After
stirred for 10 min, the reaction mixture was allowed to adjust dilution with water, the crude product was extracted with
to room temperature and stirred overnight. After dilution ethyl acetate (30 mL×2). The combined organic extracts were
with water, the crude product was extracted with DCM (100 washed with water (10 mL) and brine (10 mL), dried over
mL×2). The combined organic extracts were washed with Na2SO4, filtered, and concentrated. The crude product was
hydrochloric acid (0.5 mol/L, 50 mL), and brine (100 mL), purified by column chromatography over silica using petro-
dried over Na2SO4, filtered, and concentrated to yield com- leum ether/ethyl acetate (5/1) to yield compounds 2d–15d.
pounds 2a and 15a. The crude product was used without fur-
ther purification.
Detailed analytical information can be found in the support-
ing information.
Detailed analytical information can be found in the support-
ing 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
General procedure for the preparation of 2b–15b
A solution of commercially available lithium diisopropyl- stirred at room temperature for 1 h and then concentrated.
amide (2 mol/L, 21.7 mmol) in anhydrous THF (100 mL) was The crude product was dissolved in DCM (50 mL) and basi-
cooled to -78 °C. Compound 2a (12.1 mmol) in anhydrous fied with sodium hydroxide solution (20%, 2 mL). Organic
THF (20 mL) was added drop-wise into the solution. After extracts were dried over Na2SO4, filtered, and concentrated.
being stirred for 10 min, the reaction mixture was allowed to The crude product was purified by column chromatography
adjust to -30 °C and stirred for 30 min. Various substituted over silica using DCM/methanol (10/1) to yield compounds
benzyl bromides (24.2 mmol) in anhydrous THF (20 mL) were 2–15.
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 3-[(4-benzylpiperidin-4-yl)methoxy]pyridine (2)
adjusted to room temperature and stirred overnight. The Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 9.80–9.00
reaction mixture was quenched by the addition of saturated (brs, 1H), 8.33–8.30 (brs, 1H), 8.22–8.19 (brs, 1H), 7.24–7.14 (m,
ammonium chloride solution (30 mL) at 0 °C. After dilu- 5H), 7.02–6.98 (m, 2H), 3.67 (s, 2H), 3.40–3.20 (m, 4H), 2.86 (s,
tion with water, the crude product was extracted with ethyl 2H), 1.94–1.88 (m, 4H). ESI-MS (m/z): 283 [M+H]+. HRMS
acetate (200 mL×2). Combined organic extracts were washed (m/z): calculated for C18H22N2O [M+H]+, 283.1810; observed,
with water (100 mL) and brine (100 mL), dried over Na2SO4, 283.1812.
filtered, and concentrated. The crude product was purified by
column chromatography over silica using petroleum ether/ 3-{[4-(2-chlorobenzyl)piperidin-4-yl]methoxy}pyridine (3)
ethyl acetate (15/1) to yield compounds 2b–15b.
Detailed analytical information can be found in the support- (m, 1H), 8.20–8.16 (m, 1H), 7.28–7.17 (m, 1H), 7.16–7.06 (m,
Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 8.23–8.21
ing information.
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;
General procedure for the preparation of 2c–15c
A solution of compounds 2b–15b (1.39 mmol) in anhydrous observed, 317.1415.
THF (50 mL) was cooled to 0°C. Lithium aluminum hydride
(1 mol/L, 1.39 mmol) in anhydrous THF was added drop- 3-{{4-[2-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine (4)
wise into the solution. After being stirred at 0°C for 1 h, the Pale yellow oil 99%. 1H NMR (400 MHz, CDCl3) δ 8.30–8.19
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Shen J et al
(s, 2H), 7.68–7.62 (m, 1H), 7.48–7.41 (m, 1H), 7.40–7.35 (m, 1H), 3-{[4-(4-methylbenzyl)piperidin-4-yl]methoxy)}pyridine (11)
7.35–7.25 (m, 1H), 7.18–7.10 (m, 1H), 3.98–3.92 (m, 2H), 3.38– Pale yellow oil 96%. 1H NMR (400 MHz, CD3OD) δ 8.32–8.31
3.34 (m, 2H), 3.12–3.09 (m, 2H), 2.01–2.00 (m, 4H). ESI-MS (m, 1H), 8.20–8.17 (m, 1H), 7.51–7.47 (m, 1H), 7.43–7.39 (m,
(m/z): 351 [M+H]+. HRMS (m/z): calculated for C19H21F3N2O 1H), 7.06–7.00 (m, 4H), 3.83 (s, 2H), 3.42–3.36 (m, 2H), 3.34–
[M+H]+, 351.1684; observed, 351.1669.
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-(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, 3-{{4-[(1,1’-biphenyl)-4-ylmethyl]piperidin-4-yl}methoxy}pyridine
1H), 7.18–7.08 (m, 4H), 3.98 (s, 2H), 3.28–3.14 (m, 4H), 2.97 (s, (12)
2H), 2.32 (s, 3H), 2.01–1.94 (m, 2H), 1.92–1.82 (m, 2H). ESI- Pale yellow oil 99%. 1H NMR (400 MHz, CD3OD) δ 8.33–8.31
MS (m/z): 297 [M+H]+. HRMS (m/z): calculated for C19H24N2O (m, 1H), 8.19–8.17 (m, 1H), 7.56–7.53 (m, 2H), 7.49–7.45 (m,
[M+H]+, 297.1967; observed, 297.1962.
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
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/z): calculated for C24H26N2O [M+H]+, 359.2123; observed,
(m, 1H), 7.95–7.75 (m, 2H), 7.07–6.97 (m, 2H), 6.92-6.79 (m, 359.2102.
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): 3-{[4-(2,3-dichlorobenzyl)piperidin-4-yl]methoxy}pyridine (13)
calculated for C18H21ClN2O [M+H]+, 317.1421; observed, Pale yellow oil 96%. 1H NMR (400 MHz, CD3OD) δ 8.20–8.15
317.1417.
(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,
3-{{4-[3-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine (7) 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for
Pale yellow oil 95%. 1H NMR (600 MHz, CDCl3) δ 8.35–8.33 C18H20Cl2N2O [M+H]+, 351.1031; observed, 351.1008.
(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-{[4-(3,4-dichlorobenzyl)piperidin-4-yl]methoxy}pyridine (14)
3.68 (s, 2H), 3.18–3.12 (m, 2H), 3.05–3.00 (m, 2H), 2.96 (s, 2H), Pale yellow oil 98%. 1H NMR (400 MHz, CD3OD) δ 8.31–8.29
1.78–1.73 (m, 4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): (m, 1H), 8.19–8.17 (m, 1H), 7.42–7.37 (m, 3H), 7.26–7.25 (m,
calculated for C19H21F3N2O [M+H]+, 351.1684; observed, 1H), 7.07–7.03 (m, 1H), 3.81 (s, 2H), 3.22–3.15 (m, 2H), 3.12–
351.1674.
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-{[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, 3-{[3-(3,4-dichlorobenzyl)piperidin-3-yl]methoxy}pyridine (15)
1H), 7.01–6.99 (m, 1H), 6.87–6.85 (m, 2H), 4.46–4.45 (brs, 1H), Pale yellow oil 96%. 1H NMR (400 MHz, CDCl3) δ 8.36–8.34
3.70 (s, 2H), 3.16–3.11 (m, 2H), 3.06–3.01 (m, 2H), 2.85 (s, 2H), (m, 1H), 8.25–8.22 (m, 1H), 7.28–7.19 (m, 4H), 6.94–6.90 (m,
2.21 (s, 3H), 1.76–1.72 (m, 4H). ESI-MS (m/z): 297 [M+H]+. 1H), 3.82–3.78 (m, 1H), 3.72–3.68 (m, 1H), 2.90–2.82 (m, 3H),
HRMS (m/z): calculated for C19H24N2O [M+H]+, 297.1967; 2.78–2.69 (m, 3H), 2.36–2.22 (brs, 1H), 1.70–1.54 (m, 3H), 1.50–
observed, 297.1957.
1.42 (m, 1H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calcu-
lated for C18H20Cl2N2O [M+H] +, 351.1031; observed, 351.1023.
3-{[4-(4-chlorobenzyl)piperidin-4-yl]methoxy}pyridine (9)
Pale yellow oil 95%. 1H NMR (600 MHz, CD3OD) δ 8.72–8.21 AChBP-based [3H]-epibatidine binding assay
(m, 1H), 8.11–8.05 (m, 1H), 7.10–7.08 (m, 1H), 7.06–6.81 (m, The inhibition of [3H]-epibatidine binding with Ls-AChBP was
5H), 3.52 (s, 2H), 3.31–3.25 (m, 2H), 3.21–3.12 (m, 2H), 2.70 (s, conducted according to previously reported methods[41]. In
2H), 1.91–1.78 (m, 4H). ESI-MS (m/z): 317 [M+H]+. HRMS brief, Ls-AChBP was diluted in PBS-Tris binding buffer (final
(m/z): calculated for C18H21ClN2O [M+H]+, 317.1421; observed, concentration of 1.4 mmol/L KH2PO4, 4.3 mmol/L Na2HPO4,
317.1407.
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
3-{{4-[4-(trifluoromethyl)benzyl]piperidin-4-yl}methoxy}pyridine 1.3 ng per well. AChBP was incubated with 10-4–10-11 mol/L
(10)
ligands in the presence of approximately 1.5 nmol/L [3H]-
Pale yellow oil 95%. 1H NMR (400 MHz, CD3OD) δ 8.32–8.31 epibatidine. Displacement of [3H]-epibatidine was recorded to
(m, 1H), 8.20–8.18 (m, 1H), 7.56–7.53 (m, 2H), 7.51–7.47 (m, represent binding to AChBP. Five or six log-dilution concen-
1H), 7.43–7.39 (m, 1H), 7.37–7.34 (m, 2H), 3.86 (s, 2H), 3.33– trations of representative compounds were tested in duplicate.
3.28 (m, 2H), 3.24–3.16 (m, 2H), 3.05 (s, 2H), 1.94–1.80 (m, The IC50 (concentration of the compound that produces 50%
4H). ESI-MS (m/z): 351 [M+H]+. HRMS (m/z): calculated for inhibition of binding) was determined by least squares non-
C19H21F3N2O [M+H]+, 351.1684; observed, 351.1669.
linear regression using GraphPad Prism software. Nicotine
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served as the positive control. The results of the binding assay ylate yielded compound 2a. The Boc-protect compound 2a
revealed that the affinity of nicotine was similar to a previous reacted with various substituted benzyl bromide in the pres-
report (Ki=777.7 nmol/L in our article vs Ki=478.0 nmol/L in ence of LDA to afford the corresponding nucleophilic substi-
reported article).
tution 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
Molecular modeling
All molecular models were implemented based on the molec- 2c–14c with 3-fluoropyridine and subsequent deprotection of
ular modeling packages in Schrodinger Maestro 10.1. The the Boc group by TFA furnished the target compounds 2–14.
structures of epibatidine, compound 14, (S)-15, and (R)-15
The synthesis of compound 15 is described in Scheme 3 uti-
were built and optimized. The crystal complex of AChBP in lizing a procedure similar to that used for compounds 2–14.
complex with epibatidine was acquired from the RCSB pro-
tein data bank (PDB ID: 2BYQ) and appropriately treated by Biological evaluation
the protein preparation wizard module. The Glide function, The binding data of the synthesized compounds are shown
available in the packages, was employed for molecular dock- in Table 1. Compared with nicotine, most of the compounds
ing. The procedures were as follows: (1) ligand preparation displayed slightly higher binding affinities for AChBP at a
(the Ligprep module was used to create several conformations test concentration of 100 nmol/L. Compound 14 displayed a
of the ligand), (2) receptor grid generation, and (3) ligand 7-fold higher binding affinity when compared to nicotine.
docking. For the receptor grid generation procedure, the resi-
dues Tyr 55, Tyr 188, Ser 189 and Tyr 195 were chosen as the Molecular docking
‘Rotatable Groups’ because the exact conformations of these To elucidate the binding mode of compound 14, the molecular
residues were flexible within several reported structures.
docking strategy was utilized using the epibatidine-bound
To validate the docking methodology, epibatidine was Ls-AChBP (PDB: 2BYQ). As shown in Figure 2, compound
chosen as the test ligand and the RMSD value was calculated 14 was completely buried within the protein at the interface
using the core pattern comparison. The RMSD value (1.33–2.5) between the principal side of the interface and the comple-
confirmed the reliability of the docking method. Finally, com- mentary side.
pound 14 was docked to AChBP to provide putative docked
Each subunit consisted of an N-terminal α-helix, two short
models.
α3 helices, and a 10-stranded β-sandwich core. On the princi-
The hydrogen bond, cation-π interactions, π–π interactions, pal side, the nitrogen atom, which served as the protonation
as well as the measurements of the distances of certain atoms, center in the piperidine ring of compound 14 at physiologi-
were elucidated by the software Pymol 1.5.0.3. The 2D dia- cal pH, resided within hydrogen-bonding distance of the
gram of the putative binding mode was produced by Schro- backbone carbonyl atom of Tyr 93. Moreover, three cation-π
dinger Maestro 10.1.
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 complemen-
Results
Chemistry
The piperidine derivatives were prepared in a five-step pro- tary side, the 3,4-dichloro aryl entered into a hydrophobic sub-
cedure as demonstrated in Scheme 2. The addition of a Boc pocket formed by Ile 106, Phe 117, Ala 107, Met 116, and Val
group to a commercially available ethyl piperidine-4-carbox- 108. Alternatively, the pyridine ring occupied another pocket
Scheme 2. Synthesis of the piperidine derivatives. Reagents and conditions: a) Boc2O, DCM, RT, overnight (98%); b) substituted benzyl bromide, LDA,
THF, -78ºC-RT, overnight (75%–86%); c) LiAlH4, THF, 0ºC, 1 h (95%–98%); d) NaH, 3-Fluoropyridine, DMF, 75ºC, overnight (78%–86%); e) TFA, DCM, RT,
1 h (94%–98%).
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Scheme 3. Synthesis of compound 15. Reagents and conditions: a) Boc2O, DCM, RT, overnight (98%); b) 3,4-Dichlorobenzyl bromide, LDA, THF, -78ºC-
RT, overnight (75%–86%); c) LiAlH4, THF, 0ºC, 1 h (95%–98%); d) NaH, 3-Fluoropyridine, DMF, 75ºC, overnight (78%–86%); e) TFA, DCM, RT, 1 h (94%–
98%).
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.
Table 1. Binding affinities of compounds 1–15 for AChBP at 100 nmol/L.
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 com-
pound 1, we used compound 2 as the prototype compound
for further optimization. A series of compounds with differ-
ent 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. Epibati-
dine was reported to have the highest binding affinity with
AChBP in natural products comprising an aromatic ring and
an endocyclic secondary amino group[45]. These two groups
were positioned at an optimal distance and an optimal relative
spatial orientation to provide ideal interactions with the resi-
dues at the binding site[36, 46]. Minimal changes in the chemi-
cal structure of these compounds have been found to exert
profound effects on their pharmacological properties[42]. 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 back-
Binding affinity
(%, inhibition
at 100 nmol/L)
Compound
R
IC50
(nmol/L)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
17.2
21.5
25.0
17.5
19.2
29.6
27.4
27.4
23.6
28.3
30.7
26.5
30.8
47.4
96.9
21.5
95.8
H
o-Cl
o-CF3
o-CF3
m-Cl
m-CF3
m-CF3
p-Cl
p-CF3
p-CF3
p-Phenyl
2,3-diCl
3,4-diCl
105.6±0.8
2.8±0.2
777.7±8.1
3.6±0.1
Nicotine
Epibatidine
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Shen J et al
7
Figure 2. 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.
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 com-
pound 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 hydro-
gen-bond with Trp 147, it is possible that Trp 147 was a key
residue for ligand-receptor interactions.
Figure 3. 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.
bone 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 nitro-
gen atom in aliphatic ring of compound 14 with the nitrogen
atom in aliphatic ring of epibatidine was calculated. The
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
Acta Pharmacologica Sinica

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8
Shen J et al
Figure 4. 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.
orientation between the protonation center and the aromatic ing affinity (Ki=105.6 nmol/L). Using the molecular docking
rings. Thus, changing the position of the protonated nitrogen results of compound 14 with Ls-AChBP, a 2nd of optimization
atom may also improve the affinity of this compound.
was implemented with a structure-based strategy, which led
In conclusion, pyridine-based compound 1 was identified to the discovery of the highly affinitive compound 15 (Ki=2.8
from our in-house CNS-biased compound library by manual in nmol/L) as a potent, novel, piperidine-based acetylcholine-
vitro screening. With introduction of the privileged pyridine, binding protein ligand. The binding mode of compound 15
compound 2 (21.5% inhibition at 100 nmol/L) was selected as was then predicted to explain the drastic increase in binding
the prototype compound for further optimization. Within the affinity.
1st of optimization, compound 14 displayed the highest bind-
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Shen J et al
9
tive stress. PLoS One 2014; 9: e105711.
Acknowledgements
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Systemic administration of an alpha-7 nicotinic acetylcholine agonist
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amine-induced schizophrenia-like deficits in rats. Neuropharmacology
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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).
Author contribution
Li-ming SHAO, Lu ZHOU, and Wei LI conceived the research;
Jian SHEN screened the in-house library based the biologi-
cal assay which was set up by Lu ZHOU; Jian SHEN and
Xi-cheng YANG designed and synthesized the target com-
pounds, 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 partici-
pated 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.
18 Freedman R, Olincy A, Buchanan RW, Harris JG, Gold JM, Johnson L,
et al. Initial phase 2 trial of a nicotinic agonist in schizophrenia. Am J
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Supplementary information
The supplementary information is available on the Acta Phar-
macologica Sinica′s website.
20 Hosford D, Dvergsten C, Beaver J, Segreti AC, Toler S, Farr MG, et al.
Phase 2 clinical trial of TC-5619, an alpha 7 nicotinic receptor agonist
in the treatment of negative and cognitive symptoms in schizophrenia.
Eur Neuropsychopharm 2014; 24: S531–2.
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