Varying Chirality Across Nicotinic Acetylcholine Receptor Subtypes: Selective Binding of Quinuclidine Triazole Compounds

Article abstract of DOI:10.1021/acsmedchemlett.6b00146

The novel quinuclidine anti-1,2,3-triazole derivatives T1-T6 were designed based on the structure of QND8. The binding studies revealed that the stereochemistry at the C3 position of the quinuclidine scaffold plays an important role in the nAChR subtype selectivity. Whereas the (R)-enantiomers are selective to α7 over α4β2 (by factors of 44-225) and to a smaller degree over α3β4 (3-33), their (S)-counterparts prefer α3β4 over α4β2 (62-237) as well as over α7 (5-294). The (R)-derivatives were highly selective to α7 over α3β4 subtypes compared to (RS)- and (R)-QND8. The (S)-enantiomers are 5-10 times more selective to α4β2 than their (R) forms. The overall strongest affinity is observed for the (S)-enantiomer binding to α3β4 (K_i, 2.25-19.5 nM) followed by their (R)-counterpart binding to α7 (K_i, 22.5-117 nM), with a significantly weaker (S)-enantiomer binding to α4β2 (K_i, 414-1980 nM) still above the very weak respective (R)-analogue affinity (K_i, 5059-10436 nM).

Full text of DOI:10.1021/acsmedchemlett.6b00146

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Letter
Varying Chirality Across Nicotinic Acetylcholine Receptor
Subtypes – Selective Binding of Quinuclidine Triazole Compounds
Jiradanai Sarasamkan, Matthias Scheunemann, Nattayaporn Apaijai, Siripong Palee,
Warisara Parichatikanond, Kuntarat Arunrungvichian, Steffen Fischer, Siriporn Chattipakorn,
Winnie Deuther-Conrad, Gerrit Schüürmann, Peter Brust, and Opa Vajragupta
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00146 • Publication Date (Web): 09 Aug 2016
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ACS Medicinal Chemistry Letters
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Varying Chirality Across Nicotinic Acetylcholine Receptor Sub-
types–Selective Binding of Quinuclidine Triazole Compounds
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†,‡,
§
‡
y
y
Jiradanai Sarasamkan, Matthias Scheunemann, Nattayaporn Apaijai, Siripong Palee, Warisara Pariꢀ
œ
†
‡
y
chatikanond, Kuntarat Arunrungvichian, Steffen Fischer, Siriporn Chattipakorn, Winnie Deutherꢀ
Conrad, Gerrit Schüürmann, Peter Brust, Opa Vajragupta *
∇ #
,
†
‡
,
‡
†
Center of Excellence for Innovation in Drug Design and Discovery, Faculty of Pharmacy, Mahidol University, 447 Sriꢀ
Ayutthaya Road, Bangkok 10400, Thailand
‡
Department of Neuroradiopharmaceuticals, Institute of Radiopharmaceutical Cancer Research, HelmholtzꢀZentrum Dresꢀ
denꢀRossendorf, Permoserstraße15, 04318 Leipzig, Germany
§National Cyclotron and PET Centre, Chulabhorn Hospital, 54 Kamphaengphet 6 Road, Bangkok 10210, Thailand
y
Neurophysiology Unit, Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai Univerꢀ
sity, Chiang Mai, 50200, Thailand
œ
Department of Pharmacology, Faculty of Pharmacy, Mahidol University, 447 SriꢀAyutthaya Road, Bangkok 10400, Thaiꢀ
land
∇Department of Ecological Chemistry, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße15, 04318
Leipzig, Germany
#Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Leipziger Straße29, 09596 Freiberg, Germaꢀ
ny
ABSTRACT: The novel quinuclidine antiꢀ1,2,3ꢀtriazole derivatives T1-T6
were designed based on the structure of QND8. The binding studies revealed
that the stereochemistry at the C3 position of the quinuclidine scaffold plays
an important role in the nAChR subtype selectivity. Whereas the (R)ꢀ
enantiomers are selective to α7 over α4β2 (by factors of 44ꢀ225) and to a
smaller degree over α3β4 (3ꢀ33), their (S)ꢀcounterparts prefer α3β4 over
α4β2 (62ꢀ237) as well as over α7 (5ꢀ294). The (R)ꢀderivatives were highly
selective to α7 over α3β4 subtypes compared to (RS)ꢀ and (R)ꢀQND8. The
(S)ꢀenantiomers are 5–10 times more selective to α4β2 than their (R) forms.
The overall strongest affinity is observed for the (S)ꢀenantiomer binding to
α3β4 (Ki, 2.25ꢀ19.5 nM) followed by their (R)ꢀcounterpart binding to α7 (Ki, 22.5ꢀ117 nM), with a significantly weaker (S)ꢀenantiomer
binding to α4β2 (Ki, 414ꢀ1980 nM) still above the very weak respective (R)ꢀanalog affinity (Ki, 5059ꢀ10436 nM).
KEYWORDS: Nicotinic acetylcholine receptor, positron emission tomography, quinuclidine anti-1,2,3-triazole, click chemistry
Nicotinic acetylcholine receptors (nAChRs) are members of the
Cysꢀloop receptor superfamily of ligandꢀgated ion channels that
mediate fast synaptic transmission in the central (CNS) and peꢀ
ripheral (PNS) nervous systems.1ꢀ4 They are pentameric structures
of homomeric or heteromeric subunits symmetrically arranged
around a central ion channel. There are 16 homologous mammaliꢀ
an nAChR subunits encoded by this multigene family. These
subunits combine to form many different nAChR subtypes with
various expression patterns, diverse functional properties, and
different pharmacological characteristics.1 Currently, there are at
least 12 known nAChR subunits (α2ꢀα10 and β2ꢀβ4) expressed in
the brain.4 The most predominant nAChRs found in the brain,
associated with cognition, memory formation and behavior, are
the α4β2 and α7 subtypes.3 The α4β2 nAChR in mammalian
brain accounts for the majority of the high affinity sites for nicoꢀ
tine4 and is mainly found in the thalamus, cortical regions, striaꢀ
tum and colliculi whereas the distribution pattern of the α7 nAꢀ
ChR with high affinity for αꢀbungarotoxin is rather diffuse with
focally high density in thalamus and rather low expression in the
cerebellum.3 A further important receptor subtype in the central
nervous system (CNS) is the α3β4 nAChR because of its inꢀ
volvement in drug addiction and depression pathways.5 In contrast
to α4β2 and α7, this subtype is mainly expressed in the autonomꢀ
ic ganglia and only in selected brain regions (subsets of neurons in
the medial habenula, nucleus interpeduncularis, dorsal medulla,
pineal gland and retina).4 The nAChRs are well recognized as key
receptors involved not only in a variety of neuropsychiatric and
neurodegenerative disorders, including Parkinson’s disease (PD),
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attention deficit hyperactivity disorder (ADHD), schizophrenia,
3 of the quinuclidine scaffold was investigated by individual
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Alzheimer’s disease (AD), depression, epilepsy, nicotine and drug
addiction,6ꢀ12 but also in cancer.13,14 The multiplicity of nAChR
subtypes, their localization in the CNS and periphery along with
their pharmacological, physiological and kinetic properties afford
the opportunities to develop novel subtypeꢀspecific nAChR ligꢀ
ands for treatment of these diseases.14,15
analysis of the two enantiomers of the racemic compounds. Quiꢀ
nuclidineꢀcontaining compounds are well recognized for nAChR
interaction and the selectivity of their (R)ꢀ enantiomers for α7
nAChR. However, detailed information with regard to their (S)ꢀ
enantiomers was missing at the beginning of this study.18,29 Thus,
both enantiomers were prepared to investigate the influence of
chirality on the selectivity as well as the affinity for nAChR.
Second, the orientation of the hydrophobic aromatic ring was
explored by altering the substitution from para to meta. With
regard to α7 nAChR, the binding affinity of quinuclidine derivaꢀ
tives was found to prefer the metaꢀphenyl substitution, compared
to the paraꢀposition.18 The mꢀphenyl substitution was included to
verify this notion. Third, the hydrophobic side chains were exꢀ
tended to enhance the binding affinity and selectivity, based on
the increase in size as well as the lipophilicity for receptor bindꢀ
ing. The expansion of the structure was conducted via an ether
linkage (R in Figure 1B), where the phenol of QND8 resides.
Finally, the fluorine atoms were introduced into the structure for
further development as potential positron emission tomography
(PET) radioligands for imaging the nAChR in the brain.
Several compounds have been designed and synthesized based
on known nAChR ligand recognition components covering the
protonated amine that triggers the cationꢀπ interaction, the hydroꢀ
gen bond acceptor and the hydrophobic stabilization. The availaꢀ
bility of aromatic moieties in this hydrophobic part may particiꢀ
pate in favorable electronꢀπ interactions which can be either
electrostatic or dispersive. The tertiary quinuclidine amine is one
of the most promising core structures used for the design and
development of α7 nAChR ligands (Figure 1A). Besides the core
structure, the effective pharmacophore linkers at position 3 of the
quinuclidine scaffold are ether, carbamate, urea, amide, sulfonaꢀ
mide, oxazole, oxadiazole, and triazole.16,17 Another approach in
the design of α7 ligands was the synthesis of conformational
restricted analogues such as oxazolidinone ARꢀR17779.16 Among
them, the compounds containing the quinuclidine antiꢀ1,2,3ꢀ
triazole skeleton bind with high affinity and selectivity to the α7
nAChR.18 This hydrophobic motif in particular plays an important
role in receptor subtype recognition through the interaction with
amino acid residues of the complementary face which has been
demonstrated as an essential part for ligand selectivity among
various nAChR subtypes.19ꢀ21 We recently reported three series of
novel potent and selective α7 nAChR agonists featuring the quiꢀ
nuclidine antiꢀ1,2,3ꢀtriazole containing molecules (IND8 and
QND8) that demonstrated cognitive enhancement in mice.22
There is not only the pharmacological benefit of using the antiꢀ
1,2,3ꢀtriazole as the linker, but the triazole moiety itself is stable
and largely resistant to metabolic degradation.23,24
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Herein we report the synthesis and the binding affinity of quiꢀ
nuclidine derivatives T1-T6 (RS, R, S) at neuronal α7, α3β4 and
α4β2 nAChR subtypes. Promising compounds of high α3β4
binding affinity and good selectivity towards other nAChRs were
further screened in functional assays using HEK cells expressing
human nAChR subtypes.
Scheme 1. Synthesis of Azide (A), Terminal Alkynes (B)
and the Quinuclidine anti-1,2,3-Triazole Containing Mole-
cules (C)a
aReagents and conditions: (a) TfN3, K2CO3, CuSO4.5H2O,
CH3OH, H2O, rt, overnight, (b) 1ꢀfluoroꢀ2ꢀiodoethane for 12 and
1ꢀfluoroꢀ3ꢀiodopropane for 13, K2CO3, CH3CN, 70 °C, 10 h (c)
benzyl chloride for 14 and 4ꢀfluorobenzyl chloride for 15, K2CO3,
CH3CN, 80 °C, 6 h (d) 5 mol% CuSO4.5H2O, 10 mol% sodium
ascorbate, tꢀBuOH, H2O, rt, overnight.
The general procedure for the preparation of the designed comꢀ
pounds started with the synthesis of the azide building block by a
diazo transfer reaction and synthesis of terminal alkynes through
the Sonogashira crossꢀcoupling reaction, followed by the copperꢀ
catalyzed azideꢀalkyne cycloaddition (CuAAC) reaction resulting
Figure 1. Structure of some nAChR ligands containing quinuꢀ
clidine scaffold (A)18,25ꢀ29 and design strategy (B).
In the present design, four different approaches were applied to
the lead compound, (RS)ꢀQND8 (Figure 1B) to improve the poꢀ
tency and selectivity profiles at the most important neuronal
nAChR subtypes (α7, α3β4, α4β2). First, the chirality at position
in
the
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ACS Medicinal Chemistry Letters
Table 1. In Vitro Binding Affinities of 15 Quinuclidine anti-1,2,3-Triazole Derivatives Towards nAChRsa
1
2
3
4
5
6
7
8
Inhibition constant Ki in nM; mean ± SD Selectivityb (inverse of respective Ki ratio)
R
Compd
α7c
α3β4d
α4β2d
α7 vs α3β4 α7 vs α4β2 α3β4 vs α4β2
9
(RS)ꢀT1
(R)ꢀT1
72.8±13
8.50±0.50
449±127
1/8.6
14
6.2
143
3.0
5.6
44
53
10
10
11
12
13
14
15
16
17
18
19
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21
22
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58
59
60
73.0±15
174.5±66
133±40
117±4
1,010±162
3.09±0.10
5.24±0.35
362±27
10,436±1943
515±64
pꢀF
(S)ꢀT1
1/56
1/25
3.1
167
143
14
(RS)ꢀT2
(R)ꢀT2
748±114
5,201±412
519±20
mꢀF
(S)ꢀT2
660.5±139
98.7±39
38.8±8
2.25±0.42
20.9±0.7
558±34
1/294
1/4.7
14
1/1.3
20
231
94
(RS)ꢀT3
(R)ꢀT3
1,962±228
7,050±200
1,262±187
3,894±252
9,010±5,034
1,980±117
668±7
182
17
13
mꢀO(CH2)2F
mꢀO(CH2)3F
mꢀOBn
(S)ꢀT3
74.9±20
74.6±14
62.3±10
96.9±17
91.3±9
11.8±0.3
44.4±8.0
1,628±11
19.5±0.4
7.57±2.9
631±206
6.67±0.7
13.9±2.8
1,090±163
7.17±1.2
3.44±0.04
138±0
1/6.3
1/1.7
26
107
88
(RS)ꢀT4
(R)ꢀT4
52
145
20
5.5
102
88
(S)ꢀT4
1/5.0
1/12
28
(RS)ꢀT5
(R)ꢀT5
7.3
225
1.5
8.0
193
3.6
65
22.5±9
5,059±374
414±59
8.0
62
(S)ꢀT5
279±31
127±5
1/42
1/9.1
33
(RS)ꢀT6
(R)ꢀT6
1,013±107
6,392±230
537±11
73
33.2±7
5.9
75
mꢀOCH2C6H4F
(S)ꢀT6
149±42
9.61±1.47
10.9±1.42
29.3±0.18
1/21
1/2.8
12.7
1/11.8
(RS)ꢀQND8
(R)ꢀ QND8
(S)ꢀ QND8
627±52
182
54
pꢀOH
7389±42
461±89
678
16
2.48±0.04
186
aBinding affinities are represented by the inhibition constant Ki determined for individual compounds in two separate experiments each
b
c
performed as triplicate. Selectivity is reported in terms of the reciprocal of the ratio of the respective Ki values. Human α7 nAChR in
stably transfected SHꢀSY5Y cells, with radiotracer [3H]methyllycaconitine (0.5ꢀ1 nM), Kd = 2.0 nM. Human α4β2 and α3β4 nAChR in
d
stably transfected HEKꢀ293 cells, with radiotracer [3H]epibatidine (0.5ꢀ1 nM), Kd = 0.025 nM for hα4β2 nAChR, Kd = 0.117 nM for hα3β4
nAChR.
formation of the final antiꢀ1,2,3ꢀtriazoleꢀcontaining ligands
(Scheme 1). The diazo transfer reaction for the preparation of
azidoquinuclidine compounds (RS)ꢀ8a, (R)ꢀ8b and (S)ꢀ8c were
achieved, respectively, by using 3ꢀaminoquinuclidine dihydroꢀ
chloride (RS)ꢀ7a, (R)ꢀ7b and (S)ꢀ7c interacted with freshly preꢀ
pared trifluoromethanesulfonyl azide (TfN3). The 3ꢀethynylphenol
11 was used as a starting material for the Williamson etherificaꢀ
tion with 1ꢀfluoroꢀ2ꢀiodoethane, 1ꢀfluoroꢀ3ꢀiodopropane, benzyl
chloride and 4ꢀfluorobenzyl chloride to afford the 3ꢀethynylꢀ
phenylethers 12, 13, 14, and 15, respectively. The final comꢀ
pounds (RS)ꢀT1, (R)ꢀT1 and (S)ꢀT1 were synthesized by the
reaction of the respective azidoquinuclidines (RS)-8a, (R)-8b and
(S)-8c with 4ꢀfluoroꢀphenylacetylene 9 under CuAAC conditions.
Cycloaddition of these azidoquinuclidines with 3ꢀfluorophenylꢀ
acetylene 10 yielded the final compounds (RS)ꢀT2, (R)ꢀT2 and
(S)ꢀT2. CuAAC or click reactions between 8a-c and terminal
alkyne 12 resulted in the formation of its corresponding T3 comꢀ
pounds. The remaining quinuclidine antiꢀ1,2,3ꢀtriazoles T4-T6
were prepared by the same synthetic route as described above.
The bloodꢀbrain barrier (BBB) penetration of CNS drugs deꢀ
pends on several physicochemical properties including molecular
weight (MW), pKa, hydrophobicity (logP & logD) and topological
polar surface area (TPSA).These parameters were calculated for
all synthesized compounds using the ACD/ADME Suite software,
and the predicted values fall within the ranges of CNS drugꢀlike
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properties. Calculated logBB values of ≥ ꢀ1.0 indicate sufficient
By more detailed investigation of the interaction of the herein
BBB permeation of all compounds30 (Supplemental Table S1).
Additional support for a good BBB penetration of the compounds
is provided by calculated pKa values in the range of 7.5ꢀ10.5
which furthermore suggest sufficient protonation of the comꢀ
pounds in the CNS for cationꢀπ or other electrostatic interactions
with conserved amino acid residues or residues comprising elecꢀ
tronꢀrich aromatic moieties in the binding sites of nAChRs.
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presented ligands with α7 nAChR, the fluorineꢀsubstituted enanꢀ
tiomers (R)ꢀT1 and(R)ꢀT2, bearing F, at pꢀ and mꢀposition of the
phenyl ring, respectively, showed binding profiles comparable to
their racemic mixture with (R)ꢀT1 being 3ꢀfold more selective for
α7 over α4β2 than (R)ꢀT2 (factors 143 vs. 44). The binding affinꢀ
ity increases about twoꢀfold when replacing the (R)ꢀT4 phenyl
substituent mꢀO(CH2)3F (Ki= 62.3 nM) by the smaller and less
hydrophobic mꢀO(CH2)2F group ((R)ꢀT3, Ki = 38.8 nM) as well
as by the larger and more hydrophobic groups mꢀOBn ((R)ꢀT5, Ki
= 22.5 nM) and mꢀOCH2C6H4F ((R)ꢀT6, Ki = 33.2 nM). A possiꢀ
ble explanation would be that the respective variation in Ki might
be triggered by the presence or absence of a spatially suitable
electron donor functionality of the ligand (e.g. as Hꢀbond accepꢀ
tor), either through its F lone pair electrons (T4) or through the
benzylic πꢀelectrons (T5, T6).
The in vitro binding affinities of all modified compounds for
the nicotinic receptor subtypes were examined in competitive
radioligand displacement studies using [3H]methyllycaconitine
and [3H]epibatidine as selective radioligands for human α7 nAꢀ
ChR as well as human α4β2 and α3β4 nAChRs, respectively
(supporting information). The binding affinities of the compounds
tested are presented in Table 1. The racemic compounds showed
Ki values at α7 nAChR in the range of 73ꢀ133 nM. In addition, all
of them bound with higher affinity towards α3β4 nAChR. Reꢀ
placement of the hydroxyl group of the lead compound QND8 by
a fluorine atom decreases the binding affinity on α7 as the Ki
value of (RS)-T1 at α7 nAChR is 7 times higher than that of (RS)-
QND8 (73 vs. 10 nM). However, this modified compounds
showed better selectivity at α7 over α3β4.
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For the corresponding (S)ꢀenantiomers, (S)ꢀT2 shows a 5ꢀfold
higher selectivity for α3β4 over α7 in comparison to (S)ꢀT1.
Regarding further substituents, the following pattern is observed
for the affinity of the (S)ꢀenantiomers to α3β4 nAChR. Here, mꢀ
O(CH2)2F ((S)ꢀT3 with an Ki of 11.8 nM) is again ~2ꢀfold more
potent than mꢀO(CH2)3F ((S)ꢀT4, Ki = 19.5 nM), but now the two
phenylꢀcontaining (S)ꢀT5 (Ki = 6.67 nM) and (S)ꢀT6 (Ki = 7.17
nM) yield a further ~2ꢀfold increase in binding affinity. These
results support the suggestion that a ligand πꢀelectron donor capaꢀ
bility at a site distant from the quinuclidine triazole moiety may
contribute to stabilizing the ligandꢀreceptor complex. Interestingꢀ
ly, (S)ꢀT2 and (S)ꢀT1 as the two most promising α3β4 ligands
(Ki= 2.25 vs. 3.09 nM) are the only derivatives with an Fꢀ
substituted phenyl ring, where mꢀF (S)ꢀT2 yields the overall highꢀ
est α3β4 selectivity over both α7 and α4β2 as discussed above.
The significant difference in the selectivity profiles of the Fꢀ
substituted isomers (S)ꢀT1 and (S)ꢀT2 suggests the following
tentative explanation. In (S)ꢀT1, the electronegative F is in pꢀ
position to the electronꢀrich triazole, the latter of which may
partly compensate the electron shift from the benzene ring to F.
By contrast, the respective conjugative substitution pattern is
missing in (S)ꢀT2, thus probably resulting in a stronger loss of πꢀ
electron density through inductive charge transfer to mꢀF. From
this viewpoint it is likely that an electronꢀpoor character of the
benzylic moiety in compound (S)ꢀT2 triggers for a strong α3β4
selectivity, with Kiꢀbased selectivity of 294 (over α7) and 231
(over α4β2), respectively. Interestingly, it was apparent that (S)ꢀ
enantiomers were more selective to α3β4 (47ꢀ326 times) and
α4β2 nAChR (4.5ꢀ20 times) than their corresponding (R)ꢀ
counterparts. Up until now, the selective binding of (S)ꢀ
quinuclidine based compounds on α3β4 and α4β2 subtypes have
never been reported before.
The most important finding was the significant dependence of
the nAChR subtype affinity on the handedness of the ligands. The
chirality of the binding sites of the different nAChR subtypes
offers a rationale for the enantioselective affinity profiles of chiral
ligands for α7, α4β2 and α3β4 nAChRs. More specifically, the
experimentally determined affinities (Table 1) reveal the followꢀ
ing pattern: Firstly, all (R)ꢀenantiomers showed a significant
selectivity for α7 over α4β2 and to a lesser degree also over
α3β4. They bind with low nanomolar affinity at α7, with high
nanomolar affinity at α3β4 and with micromolar affinities at
α4β2. The resulting selectivity values in the range of 3.1ꢀ33 and
44ꢀ225 for α7 vs. α3β4 and α4β2, respectively, indicate preferꢀ
ence for the α7 nAChR binding site. Atypically, (R)ꢀT2 provides
only weak preference for α7 over α3β4 (selectivity of 3.1). Secꢀ
ondly, all (S)ꢀenantiomers are highly selective for α3β4 over
α4β2 and mostly to a lesser degree also over α7. They bind with
low nanomolar affinity at α3β4 and with high nanomolar to miꢀ
cromolar affinities at α7 and α4β2. The selectivity values of α3β4
vs. α7 and α4β2 were estimated in the range of 5ꢀ294 and 62ꢀ231
and indicate preference of the (S)ꢀenantiomers of quinuclidineꢀ
antiꢀ1,2,3ꢀtriazole containing nAChR ligands for the binding site
of α3β4 nAChR. Compound T2 plays again a special role, with
its (S)ꢀenantiomer showing the overall highest selectivity (294)
for α3β4 over α7. Notably AT-1001 previously reported as a
high affinity and selective α3β4 nAChR antagonist,31 also falls
into this category, although with slightly lower affinity and selecꢀ
tivity than T2. However, two other recently reported ligands for
the α3β4 nAChR, CP-60193232 and an anabaseine analogue33
showed modest selectivity of binding to α3β4 over α4β2 and
α3β4 over α7, respectively (Figure 2). Thirdly, the preference of
the (S)ꢀenantiomers for α3β4 over α4β2 (selectivity 62ꢀ231) is
more pronounced than the preference of the (R)ꢀenantiomers for
α7 over α3β4 (selectivity 3ꢀ33). Fourthly, the overall highest
binding affinities for the herein assessed nAChR subtypes were
observed for binding of the (S)ꢀenantiomers to α3β4 (Ki = 2.25ꢀ
19.5 nM) followed by the binding of the (R)ꢀenantiomers to α7
(Ki= 22.5ꢀ117 nM). Concerning binding to α4β2, the affinity of
the (S)ꢀenantiomers is higher than of the (R)ꢀenantiomers (414ꢀ
1980 nM vs.5059ꢀ10436 nM).
Four compounds with good affinity (Ki < 10 nM) which are (S)ꢀ
enantiomers of T1, T2, T5 and T6 were evaluated for the binding
at related receptors, these 4 compounds had no significant effect
on Nꢀmuscle type nAChR (2ꢀ12% inhibition at 1 ꢀM). For the 5ꢀ
HT3 receptor, (S)ꢀenantiomers of T1, T2, T5 and T6 were tested
at 100 nM, all except (S)ꢀT6 showed no significant binding at the
5ꢀHT3 receptor.
To see whether the designed compounds are agonist as QND8
or antagonist as AT-1001, the functional assays of the potential
compounds T1 and T2 in both (R) and (S) forms were performed
using SHꢀSY5Y cells stably expressing human α7 nAChR and
HEK 293 cells stably expressing human α3β4 nAChR. The
screening results indicated that (R)-T1, (R)-T2, (S)ꢀT1 and (S)ꢀT2
are α7 nAChR agonists (Supplemental Figure S1). Compounds
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ACS Medicinal Chemistry Letters
(R)-T2 and (S)ꢀT1 are α3β4 nAChR agonists whereas (R)-T1 and
ABBREVIATIONS
1
2
3
4
5
6
7
8
(S)ꢀT2 are α3β4 nAChR antagonists (Supplemental Figure S2).
However, the selectivity cannot be concluded due to the high
testing concentration (10 ꢀM) in the screening assay for functionꢀ
al effects.
nAChR, nicotinic acetylcholine receptor; PET, positron
emission tomography; CuAAC, copperꢀcatalyzed azideꢀ
alkyne cycloaddition; PD, Parkinson’s disease; ADHD,
attention deficit hyperactivity disorder; AD, Alzheimer’s
disease
Overall, according to the binding data of all novel analogues
presented herein, the stereo center at C3 position of the quinuꢀ
clidine motif has significant impact on the selectivity of modified
compounds to nAChR subtypes. (R)ꢀenantiomers bind preferably
towards α7 while (S)ꢀenantiomers were more selective for the
α3β4 nAChR. Among all, (R)ꢀT5 was the most favorable ligand
for the α7 nAChR, (S)ꢀT2 and (S)ꢀT1 for the α3β4 nAChR with
almost equal affinities, and (although significantly less strong)
(S)ꢀT5 was the most promising α4β2 nAChR ligand.
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In conclusion, we have successfully designed two potent comꢀ
pounds (S)ꢀT1 and (S)ꢀT2 that bind selectively to α3β4 nAChR
over α7 nAChR. Among the (R) and (S)ꢀenantiomers of the quiꢀ
nuclidine base compounds, the binding affinities of (S)ꢀ
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finding may have implications for the treatment of several brain
diseases such as drug addiction and dependence. Compounds (R)ꢀ
QND8 and (R)ꢀT6 which displayed higher affinity for the α7
nAChR and higher selectivity towards the α3β4 nAChR than its
parent (RS)ꢀQND8 might be useful candidate molecules in further
drug development for the treatment of cognitive disorders. In
addition, the introduction of a fluorine substituent in the structure
makes it possible to develop the designed compounds as PET
radioligands for brain imaging to localize the α3β4 and α7 nAꢀ
ChR subtypes in the brain and monitor drug efficacy.
Figure 2. Structures of selected α3β4 nAChR ligands.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and analytical data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +66ꢀ2ꢀ644ꢀ8695. Eꢀmail: opa.vaj@mahidol.ac.th.
Funding Sources
This work was supported by Thailand Research Fund (TRF)
through the Royal Golden Jubilee Ph.D. Program (Grant
No.PHD/0272/2552) to J.S. and O.V., BRG 5780016 to S.C., the
Office of the High Education Commission, Thailand; Thailand
Research Fund (TRF) and Faculty of Pharmacy, Mahidol Univerꢀ
sity (IRG578007) to O.V.
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Notes
The authors declare no competing financial interest.
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