Dual Nicotinic Acetylcholine Receptor α4β2 Antagonists/α7 Agonists: Synthesis, Docking Studies, and Pharmacological Evaluation of Tetrahydroisoquinolines and Tetrahydroisoquinolinium Salts

Article abstract of DOI:10.1021/acs.jmedchem.7b01895

We describe the synthesis of tetrahydroisoquinolines and tetrahydroisoquinolinium salts together with their pharmacological properties at various nicotinic acetylcholine receptors. In general, the compounds were α4β2 nAChR antagonists, with the tetrahydroisoquinolinium salts being more potent than the parent tetrahydroisoquinoline derivatives. The most potent α4β2 antagonist, 6c, exhibited submicromolar binding K_i and functional IC₅₀ values and high selectivity for this receptor over the α4β4 and α3β4 nAChRs. Whereas the (S)-6c enantiomer was essentially inactive at α4β2, (R)-6c was a slightly more potent α4β2 antagonist than the reference β2-nAChR antagonist DHβη. The observation that the α4β2 activity resided exclusively in the (R)-enantiomer was in full agreement with docking studies. Several of tetrahydroisoquinolinium salts also displayed agonist activity at the α7 nAChR. Preliminary in vivo evaluation revealed antidepressant-like effects of both (R)-5c and (R)-6c in the mouse forced swim test, supporting the therapeutic potential of α4β2 nAChR antagonists for this indication.

Full text of DOI:10.1021/acs.jmedchem.7b01895

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Article
Dual Nicotinic Acetylcholine Receptors #4#2 Antagonists/#7 Agonists:
Synthesis, Docking Studies and Pharmacological Evaluation of
Tetrahydroisoquinolines and Tetrahydroisoquinolinium Salts
François Crestey, Anders A. Jensen, Christian Soerensen, Charlotte Busk
Magnus, Jesper T. Andreasen, Günther H. J. Peters, and Jesper L. Kristensen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01895 • Publication Date (Web): 31 Jan 2018
Downloaded from http://pubs.acs.org on February 1, 2018
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Dual Nicotinic Acetylcholine Receptors α4β2 Antagonists/α7 Agonists:
Synthesis, Docking Studies and Pharmacological Evaluation of
Tetrahydroisoquinolines and Tetrahydroisoquinolinium Salts
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François Crestey,†,§ Anders A. Jensen,†,§ Christian Soerensen,‡ Charlotte Busk Magnus,†,‡ Jesper T.
Andreasen,† Günther H. J. Peters‡ and Jesper L. Kristensen*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of
Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
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‡Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800 Kongens Lyngby,
Denmark
ABSTRACT: We describe the synthesis of tetrahydroisoquinolines and tetrahydroisoquinolinium salts
together with their pharmacological properties at various nicotinic acetylcholine receptors. In general
the compounds were α4β2 nAChR antagonists, with the tetrahydroisoquinolinium salts being more
portent than the parent tetrahydroisoquinoline derivatives. The most potent α4β2 antagonist 6c,
exhibited submicromolar binding Ki and functional IC50 values and high selectivity for this receptor
over the α4β4 and α3β4 nAChRs. Whereas the (S)ꢀ6c enantiomer was essentially inactive at α4β2, (R)ꢀ
6c was a slightly more potent antagonist than the reference β2ꢀnAChR antagonist DHβΕ. The
observation that the α4β2 activity resided exclusively in the (R)ꢀenantiomer was in full agreement with
docking studies. Several of tetrahydroisoquinolinium salts also displayed agonist activity at the α7
nAChR. Preliminary in vivo evaluation revealed antidepressantꢀlike effects of both (R)ꢀ5c and (R)ꢀ6c in
the mouse forced swim test supporting the therapeutic potential of α4β2 nAChR antagonists for this
indication.
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Keywords: nAChRs, dual α4β2 antagonist/α7 agonist, tetrahydroisoquinolines, quaternary ammonium
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salts, chiral resolution.
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INTRODUCTION
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The nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of ligandꢀgated ion channels
widely distributed throughout the peripheral and central nervous systems, are involved in a broad range
of psychiatric and neurodegenerative disorders such as schizophrenia, attention deficit hyperactivity
disorder, depression, Alzheimer’s and Parkinson’s diseases, pain and substance abuse.1–4 Considerable
efforts have been put into the design of agonists (based on scaffolds such as nicotine, varenicline,
cytisine and epibatidine) as well as positive allosteric modulators targeting the neuronal nAChRs.5–9 In
comparison, antagonists are far less studied, despite their substantial therapeutic potential.10–20 Most of
the available nAChR antagonists are natural products such as methyllycaconitine (MLA), αꢀ
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bungarotoxin, ibogaine, dꢀtubocurarine, αꢀconotoxins and dihydroꢀβꢀerythroidine (DHβE), the latter
being a widely used selective antagonist of β2ꢀcontaining heteromeric nAChRs and a semisynthetic
member of the Erythrina alkaloid family.21,22 We recently reported the design, synthesis and
pharmacological evaluation of 21 analogs of aromatic Erythrina alkaloids as nAChR antagonists and
found that the structurally simple tetrahydroisoquinoline 1 (also known as Oꢀmethylcorypalline)23,24
displayed submicromolar binding affinity at the α4β2 nAChR and more than 300ꢀfold binding
selectivity over the α4β4, α3β4 and α7 subtypes (see Figure 1A).25
Ligands containing quaternary nitrogens have previously been shown to possess high activity at the
nAChR.27–30 For example, several known nAChR antagonists and neuromuscular blocking agents are
monoꢀ and bisꢀquaternary ammonium derivatives,31 and Crooks and coꢀworkers have investigated Nꢀ
substituted nicotine analogs and bisꢀazaaromatic quaternary ammonium ligands at the α4β2 and α7
receptors.32–36 Furthermore, introduction of a methyl group in cytisine (which provides caulophylline)
has been shown to dramatically reduce its affinity at the α4β2 receptor, while a second Nꢀmethylation
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restores the affinity.37 Finally, the nAChR antagonism exhibited by a broad range of synthetic and
natural quaternary derivatives of curareꢀlike alkaloids has been described.38
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In view of the promising properties of Oꢀmethylcorypalline (1) in our previous study,25 we decided to
pursue a series of quaternary ammonium salts (series 2) based on this scaffold where the length and the
size of the Nꢀsubstituent was varied. Based on their pharmacological properties, two subsequent series
of tetrahydroisoquinolines 3 and 5 were targeted to provide Nꢀmethyl tetrahydroisoquinolinium iodides
4 and 6, respectively (see Figure 1B).
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RESULTS AND DISCUSSION
Synthetic chemistry. As depicted in Scheme 1, treatment of tetrahydroisoquinoline 1 with the
appropriate alkyl or benzyl halides led to the corresponding quaternary ammonium salts 2a–e in 67–
96% yield. NꢀMethyl tetrahydroisoquinolinium iodides 4a–f and 6a–g were obtained in 63–96% yield
after reaction of the corresponding tetrahydroisoquinolines 3a–f and 5a–g with methyl iodide in dry
acetone. Besides, racemic ligands 5c and 5d were obtained via a threeꢀstep protocol as depicted in
Scheme 2.39 First, phenethylamines 7a–b were heated in neat
γꢀbutyrolactone at 150 °C for 15 min
under MW conditions providing amides 8a–b which cyclized upon treatment with POCl3 at 150 °C for
15 min once again under MW conditions. Subsequent reduction of the intermediate iminium salt with
NaBH4 gave tetrahydroisoquinolines 5c and 5d in 39% and 21% overall yield, respectively.40
The two enantiomerically pure compounds (R)ꢀ5d and (S)ꢀ5d which are also known as (R)ꢀ(+)ꢀcrispine
A and (S)ꢀ(–)ꢀcrispine A, respectively, as well as (S)ꢀ5c and (R)ꢀ5c were resolved via separation on
chiral HPLC.41,42 The absolute configurations of (S)ꢀ5c and (R)ꢀ5c (and consequently (S)ꢀ6c and (R)ꢀ6c)
were established as detailed in the Supporting Information. As shown in Scheme 2, (S)ꢀ5c, (R)ꢀ5c, (S)ꢀ
5d and (R)ꢀ5d were quaternized using methyl iodide in acetone to provide (S)ꢀ6c, (R)ꢀ6c, (S)ꢀ6d and
(R)ꢀ6d in 60%, 54%, 42% and 44% yield, respectively.
Computational chemistry: A docking study of the aforementioned ligands was performed using
Glide43 in extra precision mode based on the recently published Xꢀray structure of the human α4β2
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nAChR44 with a critical water molecule modelled into the binding site.45 Figure 2 shows the spacial
limitations of the binding pocket with bicyclic derivatives 2a–c. Thus, the quaternization of the amine,
responsible for important πꢀcation interactions, with substituents larger than a methyl backbone leads to
serious steric clashes which are substantiated by the affinities of the abovementioned ligands.
Interestingly, the chiral carbon atom next to the amine showed consistent difference as there was a clear
tendency of ligands with a Rꢀconfiguration to yield higher binding affinity originating from πꢀcation
interactions between the charged ligand nitrogen and receptor residues as well as a hydrogenꢀbond
between the moiety derived from a catechol function and the water molecule. The specific binding of
the ligands was mediated by the two mentioned pharmacophores where the hydrogenꢀbond acceptor
moiety was often the differentiator between the two enantiomers, as the ligands tended to twist which
resulted in an increased hydrogen bond acceptorꢀdonor distance. As shown in Figure 3A, the position of
the amine moiety of (R)ꢀ6c (colored in dark green) and (S)ꢀ6c (colored in purple) seems to be regulated
by the absolute configuration in order to obtain optimal fit into the binding site. This is confirmed by the
poses of (R)ꢀ6c and (S)ꢀ6c depicted in Figure 3B. Although both ligands appear very uniform, small
changes regarding the amine position are critical for the affinity and the number of interactions.
With a coefficient of determination of 0.58 between the docking scores and the in vitro data of the
enantiopure ligands, the model correlates well with the experimentally determined affinities. This was
further supported by reꢀdocking nicotine into the binding site yielding a rootꢀmeanꢀsquare deviation
value of 0.66 Å. The generated poses indicated that the position of the amine moiety was essential as it
entailed 2ꢀ4 πꢀcation interactions to receptor residues depending on the compound as expected based on
previous studies (for more details see the Supporting Information).46
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In vitro evaluation: The binding properties of the compounds were determined using membranes from
the stable rα3β4ꢀ, rα4β4ꢀ and rα4β2ꢀHEK293 cell lines in a [3H]epibatidine binding assay. The
functional properties of the compounds were determined using the mα4β2ꢀHEK293Tꢀ and rα3β4ꢀ
HEK293ꢀcell lines in the FLIPR Membrane Potential Blue (FMP) assay essentially as previously
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described,16,18,25,26 whereas the functional characterization of selected ligands at the human α7 nAChR
was performed at the stable hα7Ricꢀ3/NACHOꢀHEK293 cell line in the Ca2+/Fluoꢀ4 assay in the presence of
the α7 nAChR PAM PNUꢀ120596 (3 ꢀM). (S)ꢀNicotine (EC70ꢀEC90) was used as agonist in the
antagonist experiments at mα4β2 and rα3β4 in the FMP assay, and acetylcholine (EC70ꢀEC90) was used
as agonist in the antagonist experiments at hα7 in the Ca2+/Fluoꢀ4 assay. All ligands were tested both as
agonists and antagonists.
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Binding and functional properties of the analogs at the α4β2, α4β4 and α3β4 nAChRs. The methyl
tetrahydroisoquinolinium derivative 2a was found to be equipotent with the parent compound as an
α4β2 nAChR antagonist. The Nꢀethylation of Oꢀmethylcorypalline (1) which provided derivative 2b
was also well tolerated although its affinity at the α4β2 nAChR was about 10ꢀfold lower when
compared to the methyl derivative 2a. A further increase in the bulk on the nitrogen when growing
through propyl, allyl and benzyl led to decreases in both the affinities and antagonistic potencies of the
analogs at the α4β2 nAChR (Table 1) as suggested by the docking studies. All of these compounds
displayed negligible binding affinities at the α4β4 and α3β4 nAChRs. In view of this, we proceeded
with the quaternization of structurally related scaffolds with Nꢀmethyl groups. Derivatives 3d and 4d
displayed no significant binding affinity at any of the tested subtypes, suggesting that substitution on Cꢀ
1 is detrimental to nAChR activity, at least when a rather bulky substituent is introduced in this
position.25 In contrast, the binding affinities at α4β2 were increased for all bicyclic derivatives with the
presence of the quaternary nitrogen. Overall, the tetrahydroisoquinolinium salts 4 exhibited ~5ꢀfold
higher binding affinities at α4β2 than the parent tetrahydroisoquinolines 3, with all of these analogs
displaying negligible activity at the α4β4 and α3β4 nAChRs (Table 1, Figure 4A). For example, 4c
displayed a similar Ki value at the α4β2 nAChR (0.38 ꢀM) as that displayed by DHβE and ~80ꢀ and
~130ꢀfold binding selectivity for α4β2 over α4β4 and α3β4 nAChRs, respectively. In contrast, the IC50
values displayed by the tetrahydroisoquinolinium compounds compared to their respective
tetrahydroisoquinolines at the α4β2 nAChR in the FMP assay were largely comparable, and thus the
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introduction of the methyl group on the nitrogen only seemed to slightly increase the functional
inhibitory potencies of some of these analogs. We propose that this difference in the relative binding
affinities and antagonist potencies of the bicyclic derivatives 3 and 4 could arise from the fact that the
measurement of binding affinities and functional inhibitory potencies most likely are performed at
different α4β2 nAChR conformations. In terms of understanding of the SAR of these compounds, no
clear conclusions with respect to the substitution pattern on the phenyl ring of the tested bicyclic ligands
could be extracted from these series.
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Interestingly, the increase in α4β2 binding affinity brought on by quaternization of the nitrogen with a
methyl side chain was reproduced when moving from the bicyclic scaffold to the tricyclic ring system,
albeit to a smaller extent than for the bicyclic analogs (Table 2). Thus, quaternization of 5a–g generally
led to ligands (6a–g) exhibiting higher binding affinities at the α4β2 nAChR, and this was also
generally accompanied by weak binding affinities to the α4β4 and α3β4 nAChRs (Table 2, Figure 4A).
For example, the tetrahydroisoquinolinium derivative 6c displayed a 5ꢀfold lower Ki value (0.14 ꢀM)
than DHβE at α4β2 and displayed the highest degree of binding selectivity for α4β2 over the α4β4 and
α3β4 nAChRs (360ꢀ and 210ꢀfold, respectively) of the analogs in the series. Compounds 5e and 6e
constituted interesting outliers from this overall α4β2 selectivity, as both ligands displayed comparable
binding affinities to the α4β2 and α4β4 nAChRs and considerably weaker binding affinities to the α3β4
subtype. When tested at the α4β2 nAChR in the FMP assay, several of the tetrahydroisoquinolinium
analogs displayed significantly higher antagonist potencies than the corresponding
tetrahydroisoquinoline analogs, the IC50 values of 6a, 6b, 6c and 6e at the α4β2 nAChR being 5ꢀ10 fold
lower than those of 5a, 5b, 5c and 5e, respectively (Table 2, Figure 4A). With a functional IC50 value of
0.52 ꢀM, 6c was the most potent α4β2 nAChR antagonist emerging from this series, and just as the
other derivatives in this study 6c displayed negligible activity at the α3β4 nAChR in the FMP assay.
Inspired by the findings in the computational chemistry investigation and in a previous study on a α4β2ꢀ
selective bridgedꢀnicotine antagonist,18 we next investigated whether the two sets of enantiomers of 5c
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and 5d, i.e. ligands (S)ꢀ5c, (R)ꢀ5c, (S)ꢀ5d and (R)ꢀ5d and their corresponding quaternized analogs (S)ꢀ
6c, (R)ꢀ6c, (S)ꢀ6d and (R)ꢀ6d, respectively, would exhibit different pharmacological properties at the
nAChRs. Characterization of (S)ꢀ5d and (R)ꢀ5d revealed that the α4β2 activity resides in the (R)ꢀ
enantiomer, (R)ꢀ5d displaying Ki values of 2.5, ~100 and ~100 µM at the α4β2, α4β4 and α3β4,
respectively (Table 3). (S)ꢀ6d and (R)ꢀ6d displayed similar tendencies with (R)ꢀ6d exhibiting Ki values
of 2.4, ~25 and ~25 µM at the α4β2, α4β4 and α3β4 receptors, respectively. As observed in Table 3,
compounds (S)ꢀ5c and (R)ꢀ5c displayed higher affinity for the α4β2 receptor than (S)ꢀ5d and (R)ꢀ5d
[(S)ꢀ and (R)ꢀcrispineꢀA, respectively]. Moreover, (R)ꢀ5c exhibited ~25ꢀfold higher binding affinity (Ki
= 0.17 µM) than (S)ꢀ5c at this subtype. The corresponding quaternary ammonium salts were also tested,
and here the α4β2 activity was also found to reside in one enantiomer as (R)ꢀ6c displayed Ki values of
0.045, 2.7 and 11 µM at the α4β2, α4β4 and α3β4, respectively. Notably, (R)ꢀ6c exhibited ~10ꢀfold
higher binding affinities than DHβE itself at all of the three tested nAChRs (α4β2, α4β4 and α3β4)
(Table 3, Figure 4B). These differences in the α4β2 activity between the (S)ꢀ and (R)ꢀenantiomers were
mirrored in the functional properties as (R)ꢀ5c, (R)ꢀ5d, (R)ꢀ6c and (R)ꢀ6d displayed ~23ꢀ, >6ꢀ, >450ꢀ
and >14ꢀfold lower IC50 values, respectively, than their respective (S)ꢀenantiomers at the receptor in the
FMP assay (Table 3). (R)ꢀ6c was the most potent α4β2 antagonist in the series, displaying an IC50 value
of 0.22 µM and ~230 fold selectivity for this receptor over the α3β4 subtype (Table 3, Figure 4B).
The tricyclic derivative (R)ꢀ6c displayed significantly higher binding affinity and somewhat higher
antagonist potency at the α4β2 nAChR than DHβE. Since (R)ꢀ6c also exhibited higher binding affinities
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at α4β4 and α3β4 nAChRs and also is a fairly potent α7 nAChR agonist, it cannot be claimed to be a
more selective β2ꢀnAChR antagonist than DHβE (Table 3, Figures 4B and 4C). However, considering
its high antagonist potency at α4β2 and being a much more accessible scaffold for derivatization efforts
than DHβE, we propose that this ligand could be an interesting lead structure for the future development
of β2ꢀnAChR selective antagonists. Alternatively, some of the several potent and truly selective α4β2
antagonists identified in this study (for example, analogs 3e–f and 5c) could be applied in such efforts.
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Functional properties of the analogs at the α7 nAChR. The functional properties of the ligands at the α7
nAChR were investigated at a HEK293 cell line stably coꢀexpressing the receptor with the Ricꢀ3 and
NACHO proteins in the Ca2+/Fluoꢀ4 assay.47,48 Agonistꢀinduced responses through the α7 nAChR in
these cells could not be detected in the assay unless the assay buffer was supplemented with PNUꢀ
120596 (3 ꢀM), an α7 nAChR PAM that exerts its modulatory effects by dramatically slowing down the
extremely fast desensitization of the receptor.49,50 Thus, the presence of PNUꢀ120596 in the assay means
that the functional properties of the ligands were determined at essentially nonꢀdesensitizing α7
receptors. Nevertheless, the functionalities as well as the rank orders of agonist and antagonist potencies
exhibited by a selection of 8 reference agonists and 4 reference antagonists at the receptor in the assay
were found to be in good agreement with the pharmacological properties reported for the ligands in the
literature (see the Supporting Information for more details). Hence, while the presence of PNUꢀ120596
in the assay certainly should be kept in mind and caution should be taken when it comes to the absolute
values for potencies and efficacies displayed by the ligands in the assay, we propose that the basic
functionalities exhibited by the ligands as well as the rank order of their potencies are likely to reflect
their true pharmacological characteristics at the receptor.
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In concordance with the SAR displayed by compounds 1–6 at the α4β2 nAChR, the
tetrahydroisoquinolinium salts (2a–e, 4a–f, 6a–g) were consistently more potent ligands at the α7
nAChR than their corresponding tetrahydroisoquinolines (1, 3a–f, 5c–g) (Tables 1–3). In fact, the
differences in the activities exhibited by the respective analogs at the α7 receptor were even more
pronounced than at the α4β2 nAChR. With the exception of 5a–b that displayed weak but significant α7
antagonism, all tetrahydroisoquinolines (1, 3a–f, 5c–g) displayed negligible activity at the α7 nAChR.
In contrast, the tetrahydroisoquinolinium salts (2a–e, 4a–f, 6a–g) were not only more potent α7 ligands
but displayed a wide range of receptor functionalities, ranging from being moderately potent antagonists
(2e, IC50 = 2.0 ꢀM) over fairly potent agonists (for example 4e and 6e with EC50 values of 0.99 ꢀM and
1.2 ꢀM, respectively) to other apparently potent agonists that displayed notable biphasic concentrationꢀ
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response curves (4a–b, 6a–b). The most potent agonists in the series displayed EC50 values comparable
to those exhibited by ACh, (S)ꢀnicotine and (–)ꢀcytisine at the receptor, while not being nearly as potent
as other reference nAChR agonists such as (±)ꢀepibatidine and varenicline or as the α7ꢀselective
agonists TCꢀ1698 and PNUꢀ282987 (see the Supporting Information for more details).
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As described above, quaternization of the tetrahydroisoquinoline scaffold yielded compounds with
increased α7 nAChR activity. As an example of this, the inactivity of 1 at the receptor (both as agonist
and antagonist) was contrasted by the pronounced agonist activity displayed by the corresponding
tetrahydroisoquinolinium salt 2a (EC50 value of 9.0 ꢀM). The introduction of an ethyl, propyl or allyl
group on the nitrogen completely eliminated the α7 activity (2b–d), which could be a reflection of steric
clashes between these bigger aliphatic substituents and some of the residues forming the orthosteric
binding site. However, the benzylꢀsubstituted analog 2e displayed potent antagonist activity at the
receptor (IC50 value of 2.0 ꢀM). Thus, the aromatic substituent is either able to fit into the binding
pocket or alternatively protrudes into a vestibule adjacent to the orthosteric site. Whichever way 2e
accommodates binding to α7, it is clearly not able to trigger channel gating in the receptor, in contrast to
the methyl analog 2a. Furthermore, judging from the negligible activity displayed by 2e at α4β2 and the
other heteromeric nAChRs, this analog could be an interesting lead compound for future development
of selective α7 nAChR antagonists. In this respect, it is interesting to note that ligands 4c and 4e (the
6,7ꢀmethylenedioxy and 7ꢀhydroxyꢀ6ꢀmethoxy analogs of 2a, respectively) are considerably more
potent α7 agonists than 2a with EC50 values of 2.6 and 0.99 ꢀM, respectively. Thus, introduction of a
benzyl group on the nitrogen in this series could potentially yield more potent antagonists.
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In 4a–f, the 6ꢀ and 7ꢀsubstituents on the tetrahydroisoquinolinium scaffold were varied compared to the
6,7ꢀdimethoxy analog 2a. As mentioned above, the 7ꢀhydroxyꢀ6ꢀmethoxy 4e and 6,7ꢀmethylenedioxy
4c analogs were both considerably more potent α7 agonists than 2a, whereas 6ꢀmethoxy 4f essentially
was equipotent with 2a at the receptor (Table 1). Analogously to the inactivity of 4d at the heteromeric
nAChRs, introduction of a dMBn group in the C1–position also almost completely eliminated its α7
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activity. Interestingly, introduction of benzyloxy substituents in either the 6ꢀposition or the 7ꢀposition of
the tetrahydroisoquinolinium scaffold resulted in derivatives 4a–b that while displaying agonist
activities in the same concentration ranges as ligands 4c and 4e also displayed distinctly biphasic
concentrationꢀresponse curves. It is tempting to ascribe the decreased agonist responses observed at
higher concentrations of these analogs to increased degrees of receptor desensitization, despite the
presence of PNUꢀ120596 in the assay and even though the other agonists in the series did not exhibit
this characteristic. However, in view of the rather coarse measurement of α7 nAChR signaling provided
by this assay, solid conclusions on the underlying basis for these signaling characteristics will have to
await electrophysiology studies. Nevertheless, it is interesting to note that 6a–b, the tricyclic derivatives
corresponding to 4a–b, display very similar biphasic concentrationꢀresponse curves and thus this
signaling phenotype was exclusively observed for derivatives comprising a benzyloxy substituent.
The agonist properties displayed by the tricyclic analogs 6a–g were comparable to those exhibited by
the bicyclic derivatives 4a–f with similar substituents on the catechol moiety (Tables 1–2). Thus, 6c–g
displayed lowꢀmicromolar EC50 values as α7 nAChR agonists and 6a–b displayed similar biphasic
concentrationꢀresponse curves at the receptors as the bicyclic analogs 4a–b.
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In vivo evaluation. Since the compounds (R)ꢀ5c and (R)ꢀ6c displayed the highest antagonist potencies
at the α4β2 nAChR in vitro, and given the fact that a similar ligand has been found to possess
antidepressantꢀlike activity,25 these two analogs were selected for preliminary in vivo evaluations
(Figure 5). (R)ꢀ5c and (R)ꢀ6c were tested in the mouse forced swim test,51 showing that both compounds
significantly increased swimming activity. Ligand (R)ꢀ5c showed the most pronounced effect
(ANCOVA: significant main effect of treatment (F3,32=5.98; p<0.01)), showing a doseꢀdependent
increase in swimming behavior. Pairwise comparisons revealed a nearꢀsignificant effect of 1 (p=0.08)
and 3 mg/kg (p=0.06) and a significant effect of 10 mg/kg (p<0.001). For compound (R)ꢀ6c, there was
no significant main effect of treatment (F3,32=1.87; p=0.154), but pairwise comparisons revealed that
swimming was significantly increased by 10 mg/kg (p<0.05). Compound 1, previously shown to exhibit
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antidepressantꢀlike properties in the mFST, albeit at higher doses, is included in Fig. 5 for comparison
(Crestey et al., 2013).
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These findings are in line with several previous studies showing antidepressantꢀlike effects in mice
following antagonism of nAChRs,52,53 and the antidepressantꢀlike effect of mecamylamine was revealed
to depend on both β2ꢀ and α7ꢀ subunit containing nAChRs.53 Female NMRI mice similar to those used
in the present study have previously shown antidepressantꢀlike responses to the nonꢀselective nAChR
antagonist mecamylamine as well as α4β2ꢀ and α7ꢀselective nAChR antagonists.54 Although the α7
nAChR agonist PNUꢀ282987 by itself did not affect swimming in the mFST, it enhanced the effects
mediated by the selective serotonin reuptake inhibitor citalopram and of the selective norepinephrine
reuptake inhibitor reboxetine.51,54,55 Therefore, it is possible that α7 nAChR agonism counteracts the
antidepressantꢀlike effect of α4β2 nAChR antagonism, causing the combined α4β2 antagonism/α7
agonism profile of (R)ꢀ6c to be less efficacious than the more selective α4β2 nAChR antagonist (R)ꢀ5c.
Another explanation could be that the fixed positive charge on (R)ꢀ6c inhibits transport across the
bloodꢀbrain barrier.
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CONCLUSIONS
We have investigated the effects of quaternizing several series of tetrahydroisoquinoline derivatives in
the search of new nAChR ligands. We found that the Nꢀmethylation of Oꢀmethylcorypalline (1) was
well tolerated whereas quaternization with larger substituents led to reduced activity at the α4β2
nAChR. Subsequent quaternization of similar ligands with methyl iodide provided compounds
displaying increased binding affinities and antagonist potencies at the α4β2 nAChR. The most potent
compound (6c) was resolved and we found that the pharmacological activity at the α4β2 nAChR resides
solely in the (R)ꢀenantiomer. The in vitro data at the α4β2 nAChR were in good agreement with the
results arising from the docking studies, providing an excellent starting point for the design and
synthesis of new ligands. Preliminary in vivo evaluations indicated antidepressantꢀlike effect of (R)ꢀ5c
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and (R)ꢀ6c in the mouse forced swim test which were consistent with previous reports of antidepressant
action of nAChR antagonists.
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The 40 ligands investigated in this study revealed new compounds with interesting profiles at the
nAChRs. We identified potent and selective α4β2 nAChR antagonists displaying negligible activities at
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the other major neuronal nAChRs and several dual α4β2/α7 nAChR ligands displaying potent α4β2
antagonism and potent α7 agonism. With α4β2 being the only β2ꢀcontaining nAChRs included in this
study, it remains to be clarified whether the compounds, analogously to DHβE, also possess activity at
other β2ꢀcontaining subtypes.
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EXPERIMENTAL SECTION
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Chemistry – Material and Methods. Reagents were obtained from commercial suppliers and used
without further purifications. Syringes which were used to transfer anhydrous solvents or reagents were
purged with nitrogen prior to use. Other solvents were analytical or HPLC grade and were used as
received. Yields refer to isolated compounds estimated to be > 95 % pure as determined by HPLC and
LCꢀMS. Thinꢀlayer chromatography (TLC) was carried out on silica gel 60 F254 plates from Merck
(Germany). Visualization was accomplished under UV lamp (254 nm). Flash column chromatography
was performed on chromatography grade, silica gel 60 Å particle size 35–70 micron from Fisher
Scientific using the solvent system as stated. Microwaveꢀassisted synthesis was carried out in a Biotage
Initiator apparatus operating in single mode; the microwave cavity producing controlled irradiation at
2.45 GHz (Biotage AB, Uppsala, Sweden). The reactions were run in sealed vessels. These experiments
were performed by employing magnetic stirring and a fixed hold time using variable power to reach
(during 1–2 min) and then maintain the desired temperature in the vessel for the programmed time
period. The temperature was monitored by an IR sensor focused on a point on the reactor vial glass. The
IR sensor was calibrated to internal solution reaction temperature by the manufacturer. 1H and 13C NMR
spectra were recorded on Varian 300 (Mercury and Gemini instruments) or on Bruker (400 and 600
MHz) instruments, using CDCl3 or DMSOꢀd6 as deuterated solvents and with the residual solvent as the
internal reference. For all NMR experiments the deuterated solvent signal was used as the internal lock.
Coupling constants (J values) are given in Hertz (Hz). Multiplicities of 1H NMR signals are reported as
follows: s, singlet; d, doublet; dd, doublet of doublets; dt, doublet of triplets; t, triplet; q, quartet; m,
multiplet; br, broad signal. Melting points (mp) were determined using an MPA100 Optimelt melting
point apparatus and are uncorrected. Highꢀresolution mass spectra (HRMS) were obtained using a
Bruker Daltonics MicroTOF instrument.
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Synthesis and Analytical Data of Representative Compounds.
6,7-Methylenedioxy-2,2-dimethyl-1,2,3,4-tetrahydroisoquinolin-2-ium iodide (4c). To a solution of
6,7ꢀmethylenedioxyꢀ2ꢀmethylꢀ1,2,3,4ꢀtetrahydroisoquinoline 3c (191 mg, 1 mmol, 1 equiv) in dry
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acetone (3 mL) was added methyl iodide (623 ꢀL, 10 mmol, 10 equiv) at room temperature. The
mixture was stirred for 12 h in the dark and then filtered. The resulting solid was washed with dry
acetone to lead to pure 4c as a white solid (297 mg, 96 %); dec 216 °C; 1H NMR (300 MHz, DMSOꢀd6):
δ 6.85 (s, 1H), 6.75 (s, 1H), 6.00 (s, 2H), 4.45 (s, 2H), 3.57−3.68 (m, 2H), 3.12 (s, 6H), 2.98−3.08 (m,
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2H); 13C NMR (75 MHz, DMSOꢀd6): δ 147.9, 147.1, 123.5, 120.5, 109.1, 107.2, 102.0, 63.0, 59.0, 51.3
(2C), 24.2; HRMS (APPI): M+ found 206.1176. C12H16NO2 requires 206.1182.
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8,9-Methylenedioxy-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline (5c).25 To a MW vial were
successively added compound 8a (1.51 g, 6.01 mmol, 1 equiv), acetonitrile (13.2 mL) and POCl3 (4.61
g, 30.05 mmol, 5 equiv) at room temperature. The MW vial was sealed and heated under MW
conditions for 15 min at 150 °C. Volatiles were removed under reduced pressure and the resulting
material was dissolved in an AcOH–MeOH (1:12, 13 mL) mixture prior to addition of NaBH4 (0.91 g,
24.04 mmol, 4 equiv) portionwise at 0 °C with resulting gas evolution. Once the effervescence
vanished, the resulting mixture was transferred into a new MW vial which was sealed and heated under
MW conditions for 15 min at 90 °C. The reaction mixture was quenched with water (25 mL) and
volatiles were removed under reduced pressure. The aqueous layer was extracted with DCM (2 x 40
mL) then the combined organic layers were successively washed with a saturated aqueous solution of
sodium bicarbonate and brine, dried over MgSO4, filtered and concentrated under reduced pressure. The
resulting crude material was purified by column chromatography on silica gel using EtOAc–MeOH–
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TEA (40:10:1) as eluent to provide 5c as a pale yellow oil which slowly solidified (0.68 g, 52%); H
NMR (600 MHz, CDCl3): δ 6.58 (s, 1H), 6.55 (s, 1H), 5.88 (s, 2H), 3.34 (br t, J = 8.4, 1H), 3.14−3.18
(m, 1H), 3.06−3.10 (m, 1H), 2.97−3.04 (m, 1H), 2.72 (br dt, J = 16.3 and J = 3.8, 1H), 2.52 (q, J = 8.7,
1H), 2.25−2.32 (m, 1H), 1.88−1.97 (m, 1H), 1.81−1.86 (m, 1H), 1.65−1.73 (m, 1H).
In Vivo Pharmacology – Methods and Data Analysis.
Methods: Mice (n = 9−10) were individually placed in a beaker (16 cm in diameter) filled to a height of
20 cm of water maintained at 23.5−24.5 °C. Total swim distance during the 6 min test period was
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automatically recorded by a camera mounted above the cylinders and stored on a computer equipped
with Ethovision (Noldus, The Netherlands). Twentyꢀfour hours prior to drug testing a preꢀtest was
performed to establish baseline swim distance for each mouse. Compounds (R)ꢀ5c and (R)ꢀ6c were
dissolved in saline (0.9% NaCl) and given subcutaneously 15 min prior to testing in an injection volume
of 10 mL/kg. Data analysis: The first minute was omitted from the data before statistical analysis. This
is because animals generally swim extensively for the first minute, irrespective of treatment; hence, any
true treatment effect only becomes apparent after one minute. Swim distance was analyzed using a oneꢀ
way analysis of covariance (ANCOVA) and followed by pairwise comparisons of the predicted means
using the Planned Comparisons procedure. To ensure variance homogeneity and normality, data were
logꢀtransformed before statistical analysis. Differences were considered significant for p < 0.05.
AUTHOR INFORMATION
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Corresponding author
*J.L.K.: Phone: +45 3533 6487. Eꢀmail: jesper.kristensen@sund.ku.dk
ORCID
François Crestey: 0000ꢀ0001ꢀ7206ꢀ6939
Jesper Langgaard Kristensen: 0000ꢀ0002ꢀ5613ꢀ1267
Author contributions
F.C. and J.L.K. conceived and designed the project. C.B.M. and F.C. performed the organic/analytical
chemistry and analyzed all the synthesized compounds. A.A.J. performed and analyzed the data from
the in vitro pharmacology experiments. J.T.A. performed and analyzed the data from the in vivo
pharmacology experiments. C.B.M., C.S. and G.H.J.P. performed and analyzed the data from the
docking study. The manuscript was written through contributions of all authors who gave approval to
the final version of the manuscript. § A.A.J. and F.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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Generous financial support from the Danish Council for Independent Research − Medical Sciences, the
Lundbeck Foundation, the Augustinus Foundation and the Novo Nordisk Foundation are gratefully
acknowledged. The authors also thank Kasper Skytte Harpsøe, Karla Frydenvang, Camilla Koch and
Birgitte Nielsen for experimental assistance and helpful discussions.
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ABBREVIATIONS USED
HPLC: highꢀperformance liquid chromatography; MW: microwave; DCM: dichloromethane; AcOH:
acetic acid; MeOH: methanol; TEA: triethylamine; EtOAc: ethyl acetate; Bn: benzyl; dMBn: 3,4ꢀ
dimethoxybenzyl; ee: enantiomeric excess; DHβE: dihydroꢀβꢀerythroidine; nAChR: nicotinic
acetylcholine receptor; MLA: methyllycaconitine; HEK: human embryonic kidney; PAM: positive
allosteric modulator; SAR: structureꢀactivity relationship; FMP: FLIPR Membrane Potential Blue;
S.E.M.: standard error of the mean.
ASSOCIATED CONTENT
Supporting Information. Complete biological evaluation data and full experimental details on the
synthesis of the reported compounds, Molecular Formula Strings, chiral HPLC separation, optical
rotation measurements and additional docking data are provided − including copies of 1H and 13C NMR
spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
REFERENCES
(1) Jensen, A. A.; Frølund, B.; Liljefors, T.; KrogsgaardꢀLarsen, P. Neuronal Nicotinic Acetylcholine
Receptors: Structural Revelations, Target Identifications, and Therapeutic Inspirations. J. Med.
Chem. 2005, 48, 4705–4745.
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(2) Arneric, S. P.; Holladay, M.; Williams, M. Neuronal Nicotinic Receptors: A Perspective on Two
Decades of Drug Discovery Research. Bio. Pharmacol. 2007, 74, 1092–1101.
1
2
3
4
5
6
(3) Philip, N. S.; Carpenter, L. L.; Tyrka, A. R.; Price, L. H. Nicotinic Acetylcholine Receptors and
7
8
9
Depression: A Review of the Preclinical and Clinical Literature. Psychopharmacology 2010, 212,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1–12.
(4) Lemoine, D.; Jiang, R.; Taly, A.; Chataigneau, T.; Specht, A.; Grutter, T. LigandꢀGated Ion
Channels: New Insights into Neurological Disorders and Ligand Recognition. Chem. Rev. 2012,
112, 6285–6318.
(5) Jensen, A. A.; Mikkelsen, I.; Frølund, B.; BräunerꢀOsborne, H.; Falch, E.; KrogsgaardꢀLarsen, P.
Carbamoylcholine Homologs: Novel and Potent Agonists at Neuronal Nicotinic Acetylcholine
Receptors. Mol. Pharmacol. 2003, 64, 865–875.
(6) Gündisch, D. Nicotinic Acetylcholine Receptor Ligands as Potential Therapeutics. Expert Opin.
Ther. Patents 2005, 15, 1221–1239.
(7) Gündisch, D.; Eibl, C. Nicotinic Acetylcholine Receptor Ligands, a Patent Review (2006ꢀ2011).
Expert Opin. Ther. Patents 2011, 21, 1867–1896.
(8) Freedman, R. α7ꢀNicotinic Acetylcholine Receptor Agonists for Cognitive Enhancement in
Schizophrenia. Annu. Rev. Med. 2014, 65, 245–261.
(9) Grupe, M.; Grunnet, M.; Bastlund, J. F.; Jensen, A. A. Targeting α4β2 Nicotinic Acetylcholine
Receptors in Central Nervous System Disorder: Perspectives on Positive Allosteric Modulation as
a Therapeutic Approach. Basic Clin. Pharmacol. Toxicol. 2015, 116, 187–200, and references
cited therein.
(10) Dwoskin, L. P.; Crooks, P. A. Competitive Neuronal Nicotinic Receptor Antagonists: A New
Direction for Drug Discovery. J. Pharmacol. Exp. Ther. 2001, 298, 395–402.
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(11) Dwoskin, L. P.; Bardo, M. T. Targeting Nicotinic Receptor Antagonists as Novel
Pharmacotherapies for Tobacco Dependence and Relapse. Neuropsychopharmacology 2009, 34,
244–246.
1
2
3
4
5
6
7
8
9
(12) IturriagaꢀVásquez, P.; Carbone, A.; GarcíaꢀBeltrán, O.; Livingstone, P. D.; Biggin, P. C.;
Cassels, B. K.; Wonnacott, S.; ZapataꢀTorres, G.; Bermudez, I. Molecular Determinants for
Competitive Inhibition for α4β2 Nicotinic Acetylcholine Receptors. Mol. Pharm. 2010, 78, 366–
375.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
(13) Carroll, F. I.; Blough, B. E.; Mascarella, S. W.; Navarro, H. A.; Eaton, J. B.; Lukas, R. J.;
Damaj, M. I. Synthesis and Biological Evaluation of Bupropion Analogues as Potential
Pharmacotherapies for Smoking Cessation. J. Med. Chem. 2010, 53, 2204–2214.
(14) Ambrus, J. I.; Halliday, J. I.; Kanizaj, N.; Absalom, N.; Harpsøe, K.; Balle, T.; Chebib, M.;
McLeod, M. D. Covalent Attachment of Antagonists to the α7 Nicotinic Acetylcholine Receptor:
Synthesis and Reactivity of Substituted Maleimides. Chem. Commun. 2012, 48, 6699–6701.
(15) FaundezꢀParraquez, M.; FariasꢀRabelo, N.; GonzalezꢀGutierrez, J. P.; EtcheverryꢀBerrios, A.;
AlzateꢀMorales, J.; AdasmeꢀCarreño, F.; Varas, R.; Bermudez, I.; IturriagaꢀVásquez, P.
Neonicotinic Analogues: Selective Antagonists for α4β2 Nicotinic Acetylcholine Receptors.
Bioorg. Med. Chem. 2013, 21, 2687–2694.
(16) Jepsen, T. H.; Jensen, A. A.; Lund, M. H.; Glibstrup, E.; Kristensen, J. L. Synthesis and
Pharmacological Evaluation of DHβE Analogues as Neuronal Nicotinic Acetylcholine Receptor
Antagonists. ACS Med. Chem. Lett. 2014, 5, 766–770.
(17) Crooks, P. A.; Bardo, M. T.; Dwoskin, L. P. Nicotinic Receptor Antagonists as Treatment for
Nicotine Abuse. Adv. Pharmacol. 2014, 69, 513–551.
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(18) Petersen, I. N.; Crestey, F.; Jensen, A. A.; Indurthi, D. C.; Pedersen, H.; Andreasen, J. T.; Balle,
T.; Kristensen, J. L. Tying up Nicotine: New Competitive Antagonist of the Neuronal Nicotinic
Acetylcholine Receptors. ACS Med. Chem. Lett. 2015, 6, 472–475.
1
2
3
4
5
6
7
8
9
(19) Jaikhan, P.; Boonyarat, C.; Arunrungvichian, K.; Taylor, P.; Vajragupta, O. Design and
Synthesis of Nicotinic Acetylcholine Receptor Antagonists and Their Effect on Cognitive
Impairment. Chem. Biol. Drug Des. 2016, 87, 39–56.
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52
53
54
55
56
57
58
59
60
(20) Ondachi, P. W.; Castro, A. H.; Sherman, B.; Luetje, C. W.; Damaj, M. I.; Mascarella, S. W.;
Navarro, H. A.; Carroll, F. I. Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro
and in Vivo Pharmacological Properties of 2´ꢀFluoroꢀ(substituted thiophenyl)ꢀdeschloroepibatidine
Analogues. ACS Chem. Neurosci. 2017, 8, 115–127, and references cited therein.
(21) Reimann, E. Synthesis Pathways to Erythrina Alkaloids and Erythrina Type Compounds.
Progress in the Chemistry of Organic Natural Products 2007, 88, 1–62.
(22) Shahsavar, A.; Kastrup, J. S.; Nielsen, E. Ø.; Kristensen, J. L.; Gajhede, M.; Balle, T. Crystal
Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHβE
Suggests a Unique Mode of Antagonism. PLoS One 2012, 7, e40757.
(23) Theuns, H. G.; Vlietstra, E. J.; Salemink, C. A. Corypalline and OꢀMethylcorypalline, Two
Alkaloids from Papaver bracteatum. Phytochemistry 1983, 22, 247–250.
(24) Menachery, M. D.; Lavanier, G. L.; Wetherly, M. L.; Guinaudeau, H.; Shamma, M. Simple
Isoquinoline Alkaloids. J. Nat. Prod. 1986, 49, 745–778.
(25) Crestey, F.; Jensen, A. A.; Borch, M.; Andreasen, J. T.; Andersen, J.; Balle, T.; Kristensen, J. L.
Design, Synthesis and Biological Evaluation of Erythrina Alkaloid Analogues as Neuronal
Nicotinic Acetylcholine Receptor Antagonists. J. Med. Chem. 2013, 56, 9673–9682.
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(26) Jepsen, T. H.; Glibstrup, E.; Crestey, F.; Jensen, A. A.; Kristensen, J. L. Strategic Approach to
[6,6]ꢀBicyclic Lactones: Application Towards the CD Fragment of DHβE. Beilstein J. Org. Chem.
2017, 13, 988–994.
1
2
3
4
5
6
7
8
9
(27) Søkilde, B.; Mikkelsen, I.; Stensbøl, T. B.; Andersen, B.; Ebdrup, S.; KrosgaardꢀLarsen, P.;
Falch, E. Analogues of Carbacholine: Synthesis and Relationship Between Structure and Affinity
for Muscarinic and Nicotinic Acetylcholine Receptors. Arch. Pharm. 1996, 329, 95–104.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
(28) Papke, R. L.; Bencherif, M.; Lippiello, P. An Evaluation of Neuronal Nicotinic Acetylcholine
Receptor Activation by Quaternary Nitrogen Compounds Indicates that Choline is Selective for the
α7 Subtype. Neurosci. Lett. 1996, 213, 201–204.
(29) Coates, K. M.; Flood, P. Ketamine and its Preservative, Benzethonium Chloride, Both Inhibit
Human Recombinant α7 and α4β2 Neuronal Nicotinic Acetylcholine Receptors in Xenopus
Oocytes. Br. J. Pharmacol. 2001, 134, 871–879.
(30) IturriagaꢀVásquez, P.; Pérez, E. G.; Slater, E. Y.; Bermúdez, I.; Cassels, B. K. Aporphine Metho
Salts as Neuronal Nicotinic Acetylcholine Receptor Blockers. Bioorg. Med. Chem. 2007, 15,
3368–3372.
(31) For example, pancuronium bromide (trademarked as Pavulon) and atracurium besilate
(trademarked as Tracrium) are still used for euthanasia and anaesthesia, respectively. Moreover,
vecuronium bromide (trademarked as Norcuron), doxacurium chloride and mivacurium chloride
are used as skeletal muscle relaxation during surgery. Hexamethonium chloride, used to treat
chronic hypertension, inhibits the ganglionic nicotinic receptors.
(32) Dwoskin, L. P.; Leibee, L. L.; Jewell, A. L.; Fang, Z.ꢀX.; Crooks, P. A. Inhibition of
[3H]Dopamine Uptake into Rat Striatal Slices by Quaternary NꢀMethylated Nicotine Metabolites.
Life Sci. 1992, 50, 233–237.
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Journal of Medicinal Chemistry
(33) Crooks, P. A.; Ravard, A.; Wilkins, L. H.; Teng, L.ꢀH.; Buxton, S. T.; Dwoskin, L. P. Inhibition
of NicotineꢀEvoked [3H]Dopamine Release by Pyridino NꢀSubstituted Nicotine Analogues: A New
Class of Nicotinic Antagonists. Drug Dev. Res. 1995, 36, 91–102.
1
2
3
4
5
6
7
8
9
(34) Ayers, J. T.; Dwoskin, L. P.; Deaciuc, A. G.; Grinevich, V. P.; Zhu, J.; Crooks, P. A. Bisꢀ
Azaaromatic Quaternary Ammonium Analogues: Ligands for α4β2* and α7* Subtypes of
Neuronal Nicotinic Receptors. Bioorg. Med. Chem. Lett. 2002, 12, 3067–3071.
10
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57
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59
60
(35) Wilkins, Jr., L. H.; Grinevich, V. P.; Ayers, J. T.; Crooks, P. A.; Dwoskin, L. P. Nꢀnꢀ
Alkylnicotinium Analogs, a Novel Class of Nicotinic Receptor Antagonists: Interaction with
α4β2* and α7* Neuronal Nicotinic Receptors. J. Pharmacol. Exp. Ther. 2003, 304, 400–410.
(36) Grinevich, V. P.; Crooks, P. A.; Sumithran, S. P.; Haubner, A. J.; Ayers, J. T.; Dwoskin, L. P. Nꢀ
nꢀAlkylpyridinium Analogs, a Novel Class of Nicotinic Receptor Antagonists: Selective Inhibition
of Nicotine Evoked [3H]Dopamine Overflow from Superfused Rat Striatal Slices. J. Pharmacol.
Exp. Ther. 2003, 306, 1011–1020.
(37) Imming, P.; Klaperski, P.; Stubbs, M. T.; Seitz, G.; Gündisch, D. Syntheses and Evaluation of
Halogenated Cytisine Derivatives and of Bioisosteric Thiocytisine as Potent and Selective nAChR
Ligands. Eur. J. Med. Chem. 2001, 36, 375–388.
(38) Lukas, R. J. Pharmacological Distinctions between Functional Nicotinic Acetylcholine
Receptors on the PC12 Rat Pheochromocytoma and the TE671 Human Medulloblastoma. J.
Pharmacol. Exp. Ther. 1989, 251, 175–182.
(39) Rowles, I.; Malone, K. J.; Etchells, L. L.; Willies, S. C.; Turner, N. J. Directed Evolution of the
Enzyme Monoamine Oxidase (MAOꢀN): Highly Efficient Chemoꢀenzymatic Deracemisation of
the Alkaloid (±)ꢀCrispine A. ChemCatChem 2012, 4, 1259–1261.
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(40) Performing the reduction with NaBH4 (4 equiv) at room temperature in a mixture MeOH–AcOH
(12:1) overnight provided tetrahydroisoquinoline derivatives 5c and 5d within the same range of
yields (or even higher) than when the reduction was performed under MW conditions
1
2
3
4
5
6
7
8
9
(41) The ee of (S)ꢀ5d after chiral HPLC separation was 95.9% while the ee of (R)ꢀ5d was 99.3%. See
the Supporting Information for more details regarding the chiral HPLC separation as well as the
optical rotation measurements in order to confirm the absolute configuration of the two
enantiomers.
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(42) The absolute configuration of the two enantiomers (S)ꢀ5c and (R)ꢀ5c was correctly assigned
throughout the whole article although at the time of the study the absolute configuration was not
formally yet established. The ee of (S)ꢀ5c after chiral HPLC separation was 97.0% while the ee of
(R)ꢀ5c was 99.1%. See the Supporting Information for more details.
(43) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.;
Sanschagrin, P. C.; Mainz, D. T. Extra Precision Glide: Docking and Scoring Incorporating a
Model of Hydrophobic Enclosure for Protein–Ligand Complexes. J. Med. Chem. 2006, 49, 6177–
6196.
(44) MoralesꢀPerez, C. L.; Noviello, C. M.; Hibbs, R. E. Xꢀray Structure of the Human α4β2
Nicotinic Receptors. Nature 2016, 538, 411–415.
(45) Amiri, S.; Sansom, M. S. P.; Biggin, P. C. Molecular Dynamics Studies of AChBP with
Nicotine and Carbamylcholine: the Role of Water in the Binding Pocket. Protein Eng., Des. Sel.
2007, 20, 353–359.
(46) Beers, W. H.; Reich, E. Structure and Activity of Acetylcholine. Nature 1970, 228, 917–922.
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(47) Williams, M. E.; Burton, B.; Urrutia, A.; Shcherbatko, A.; ChavezꢀNoriega, L. E.; Cohen, C. J.;
Aiyar, J. Ricꢀ3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7
Subunit in Mammalian Cells. J Biol Chem 2005, 280, 1257–1263.
1
2
3
4
5
6
7
8
9
(48) Gu, S.; Matta, J. A.; Lord, B.; Harrington, A. W.; Sutton, S. W.; Davini, W. B.; Bredt, D. S.
Brain alpha7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO. Neuron 2016, 89,
948–955.
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(49) Hurst, R. S.; Hajós, M.; Raggenbass, M.; Wall, T. M.; Hidgon, N. R.; Lawson, J. A.; Rutherfordꢀ
Root, K. L.; Berkenpas, M. B.; Hoffmann, W. E.; Piotrowski, D. W.; Groppi, V. E.; Allaman, G.;
Ogier, R.; Bertrand, S.; Bertrand, D.; Arneric, S. P. A Novel Positive Allosteric Modulator of the
α7 Neuronal Nicotinic Acetylcholine Receptor: In Vitro and In Vivo Characterization. J Neurosci
2005, 25, 4396–4405.
(50) Grønlien, J. H.; Håkerud, M.; Ween, H.; ThorinꢀHagene, K.; Briggs, C. A.; Gopalakrishnan, M.;
Malysz, J. Distinct Profiles of α7 nAChR Positive Allosteric Modulation Revealed by Structurally
Diverse Chemotypes. Mol Pharmacol 2007, 72, 715–724.
(51) In this behavorial test, a mouse which has received the tested compound is place in an
inescapable tank which is filled with water; the distance swum by the animal is measured. An
increase in the swimming distance is interpreted as an antidepressantꢀlike effect of the tested
compound. For more details, see: Slattery, D. A.; Cryan, J. F. Using the Rat Forced Swim Test to
Assess Antidepressantꢀlike Activity in Rodents. Nature Protoc. 2012, 7, 1009–1014.
(52) Caldarone, B. J.; Harrist, A.; Cleary, M. A.; Beech, R. D.; King, S. L.; Picciotto, M. R. Highꢀ
Affinity Nicotinic Acetylcholine Receptors Are Required for Antidepressant Effects of
Amitriptyline on Behavior and Hippocampal Cell Proliferation. Biol. Psychiatry 2004, 56, 657–
664.
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(53) Rabenstein, R. L.; Caldarone, B. J.; Picciotto, M. R. The Nicotinic Antagonist Mecamylamine
has Antidepressantꢀlike Effects in WildꢀType but not β2ꢀ or α7ꢀNicotinic Acetylcholine Receptor
Subunit Knockout Mice. Psychopharmacology (Berl). 2006, 189, 395–401.
1
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4
5
6
7
8
9
(54) Andreasen, J. T.; Olsen, G. M.; Wiborg, O.; Redrobe, J. P. Antidepressantꢀlike Effects of
Nicotinic Acetylcholine Receptor Antagonists, but not Agonists, in the Mouse Forced Swim and
Mouse Tail Suspension Tests. J Psychopharmacol. 2009, 23, 797–804.
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(55) Andreasen, J. T.; Nielsen, E. Ø.; Christensen, J. K.; Olsen, G. M.; Peters, D.; Mirza, N. R.;
Redrobe, J. P. SubtypeꢀSelective Nicotinic Acetylcholine Receptor Agonists Enhance the
Responsiveness to Citalopram and Reboxetine in the Mouse Forced Swim Test. J
Psychopharmacol. 2011, 25, 1347–1356.
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Journal of Medicinal Chemistry
FIGURES, SCHEMES AND TABLES TITLES
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9
Scheme 1. Synthesis of tetrahydroisoquinolinium derivatives 2a–e, 4a–f and 6a–g. Reagents and
conditions: (i) RX (10 equiv), acetone, rt or 35 °C, 12 h, 67–96% for 2a–e, 65–96% for 4a–f, 63–81%
for 6a–g. dMBn = 3,4ꢀdimethoxybenzyl; Bn = benzyl.
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Scheme 2. Synthesis of racemic derivatives 5c and 5d and tetrahydroisoquinolinium derivatives (S)ꢀ6c,
(R)ꢀ6c, (S)ꢀ6d and (R)ꢀ6d. Reagents and conditions: (i) γꢀbutyrolactone (1.1 equiv), MW, 150 °C, 15
min, 76% for 8a, 70% for 8b; (ii) a) POCl3 (5 equiv), CH3CN, MW, 150 °C, 15 min; b) NaBH4 (4
equiv), MeOH–AcOH (12:1), MW, 90 °C, 15 min, 52% for 5c, 30% for 5d (within 2 steps); (iii) CH3I
(15 equiv), acetone, rt, 2 h, 60% for (S)ꢀ6c, 54% for (R)ꢀ6c, 42% for (S)ꢀ6d, 44% for (R)ꢀ6d.
Table 1. Pharmacological properties of bicyclic compounds 1, 2, 3 and 4 at nAChRs.
Table 2. Pharmacological properties of tricyclic compounds 5 and 6 at nAChRs.
Table 3. Pharmacological properties of enantiopure tricyclic compounds 5 and 6 at nAChRs.
Figure 1. (A) Structures of DHβE,26 erysodine,12 erysotrine12 and Oꢀmethylcorypalline (1).25 In
parentheses are the Ki values of the compounds at α4β2 nAChR subtype determined in a
[3H]epibatidine (for DHβE and 1) or [3H]cytisine (for erysotrine and erysodine) binding assay. (B)
Retrosynthetic strategy towards tetrahydrosioquinolinium salts 2, 4 and 6 from derivatives 1, 3 and 5,
respectively.
Figure 2. GlideXP docking of ligands 2a (green), 2b (orange) and 2c (red) explaining the order of
affinity of the three ligands as a consequence of the limited space in the binding pocket. Docking
experiments are based on the Xꢀray structure of the human α4β2 nicotinic receptors (PDB ID: 5KXI).44
Figure 3. GlideXP docking of (R)ꢀ6c (green) and (S)ꢀ6c (purple) seen from two different perspectives:
(A) Here the interactions with three residues in the binding pocket are higligted. Hydrogenꢀbonds are
shown in yellow dashed line while πꢀcation interactions are shown in green dashed lines. (B) Here the
interactions with the water molecule and the size of the binding pocket are highligted. Hydrogenꢀbonds
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are shown in yellow dashed line while πꢀcation interactions are shown in green dashed lines. Docking
experiments are based on the Xꢀray structure of the human α4β2 nicotinic receptors (PDB ID: 5KXI).44
Figure 4. In vitro pharmacological properties of tetrahydroisoquinolines and tetrahydroisoquinolinium
salts at the nAChRs. (A) Pharmacological properties of the tetrahydroisoquinolines and
tetrahydroisoquinolinium derivatives at the α4β2 nAChR in the [3H]epibatidine binding and FMP
assays. Left and middle: Comparison between the binding affinities (pKi ± S.E.M., left) and antagonist
potencies (pIC50 ± S.E.M., middle) displayed by the tetrahydroisoquinolines 1, 3a–f, 5a–g, (R)ꢀ5c, (S)ꢀ
5c, (R)ꢀ5d and (S)ꢀ5d and the corresponding tetrahydroisoquinolinium salts 2a, 4a–f, 6a–g, (R)ꢀ6c, (S)ꢀ
6c, (R)ꢀ6d and (S)ꢀ6d at the α4β2 nAChR. Right: Correlation between the binding affinities (pKi ±
S.E.M.) and antagonist potencies (pIC50 ± S.E.M.) displayed by all active tetrahydroisoquinolines and
tetrahydroisoquinolinium salts the α4β2 nAChR. (B) Binding properties of DHβE, (R)ꢀ5c and (R)ꢀ6c at
1
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nAChRs. Concentrationꢀinhibition curves for DHβE, (R)ꢀ5c and (R)ꢀ6c at α4β2, α3β4 and α4β4 in the
[3H]epibatidine binding assay. (C) Functional properties of DHβE, (R)ꢀ5c and (R)ꢀ6c at nAChRs. Left:
Concentrationꢀinhibition curves for DHβE, (R)ꢀ5c and (R)ꢀ6c at α4β2 and α3β4 in the FMP assay and
for DHβE at α7 in the Ca2+/Fluoꢀ4 assay. Right: Concentrationꢀresponse curves for ACh, (R)ꢀ5c and
(R)ꢀ6c at α7 in the Ca2+/Fluoꢀ4 assay (obtained in the presence of 3 ꢀM PNUꢀ120596). Figures B and C
depict data from single representative experiment performed as described in the Supporting Information
and error bars are omitted for clarity.
Figure 5. Effects of (R)ꢀ5c (A) and (R)ꢀ6c (B) in the mouse forced swim test. The effect of the
previously published25 compound 1 is included for comparison (C). *p<0.05; ***p<0.001 (n=9ꢀ10).
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Journal of Medicinal Chemistry
Table 1a
1
2
3
4
5
Binding Ki (ꢀM)
Functional (ꢀM)
6
7
8
Compound α4β2 α4β4 α3β4 α4β2 (IC50) α3β4 (IC50)
α7 (IC50)
α7 (EC50)
9
~100
DHβE
1
0.65
0.87
0.63
9.7
~25
~100
0.60
12
~100
~100
>300
>300
>300
>300
>300
~100
>300
~300
>100
~100
>100
>300
>300
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
~300 ~300
>100
ꢀ
>100
~50
~100
11
9.0
2a
2b
2c
>300 >300
~300 ~300
~300 >300
>300 ~300
~300 ~300
~300 ~300
11
ꢀ
weak agonistb
14
~30
~30
~30
24
>100
>100
2.0
>100
~30
~30
2.9
>100
2d
2e
ꢀ
~30
~30
>100
>100
>100
>100
ꢀ
ꢀ
3a
3b
3c
15
23
ꢀ
>100
17
~50
~300
2.6
>100
4.4
6.2
7.2
~30
~100
1.7
>100 >100
~100 ~100
>100
3d
3e
>100
2.6
~25
~50
17
~100
~30
>100
3f
0.47
4.5
agonist (biphasic)d
agonist (biphasic)d
4a
4b
~30
ꢀ
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0.38
>100
0.40
0.68
~30
~50
12
>100
1.2
>300
>100
>100
>100
ꢀ
ꢀ
ꢀ
ꢀ
2.6
weak agonistc
0.99
4c
4d
4e
4f
1
2
3
4
5
6
7
8
>100 >100
8.9
1.5
~25
9.3
9
1.4
4.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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a
The binding properties of the compounds were determined with membranes from the stable rα3β4ꢀ, rα4β4ꢀ and rα4β2ꢀ
HEK293 cell lines in a [3H]epibatidine binding assay, while the functional properties of the compounds were determined
using the mα4β2ꢀHEK293Tꢀ and rα3β4ꢀHEK293ꢀcell lines in the FMP assay. The functional characterization of selected
ligands at the human α7 nAChR was performed at the stable hα7Ricꢀ3/NACHOꢀHEK293 cell line in the Ca2+/Fluoꢀ4 assay in the
presence of 3 ꢀM PNUꢀ120596. The data were the means of 3–5 individual experiments performed in duplicate. The
complete data set for this table (i.e., Ki [pKi ± S.E.M.] values from the binding experiments, IC50 [pIC50 ± S.E.M.], EC50
b,c
[pEC50 ± S.E.M.] and Rmax ± S.E.M. values from the functional experiments are given in the Supporting information.
Agonistꢀconcentration response curves not complete within the tested concentration range. Significant agonist responses
observed at concentrations of 30 ꢀMb or 10 ꢀMc. Agonistꢀconcentration response curves were biphasic. The compounds
d
elicited significant and concentrationꢀdependent agonist responses at lower concentrations, whereas higher concentrations
elicited smaller responses. Significant agonist responses that increased with increasing concentrations were observed for 1
and 3 ꢀM (compound 4a) and for 0.3, 1 and 3 ꢀM (compound 4b). At 10 ꢀM and higher concentrations, the agonistꢀinduced
responses decreased substantially.
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Table 2a
1
2
3
4
5
Binding Ki (ꢀM)
Functional (ꢀM)
6
7
8
Compound α4β2 α4β4 α3β4 α4β2 (IC50) α3β4 (IC50)
α7 (IC50)
α7 (EC50)
9
~100
DHβE
5a
5b
5c
0.65
6.3
~25
~100
0.60
~30
~50
2.3
~100
~100
>100
~100
~100
>100
>100
>100
~300
~300
~30
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
~300 ~100
~300 ~300
5.7
ꢀ
~50
0.50
4.6
11
ꢀ
9.3
>100
ꢀ
weak agonistb
~100 ~100
~30
9.1
>100
>100
5d
5e
1.4
3.3
~100
ꢀ
weak agonistb
~25
8.5
~100 >100
~100 ~100
~300 ~300
~300 ~300
~100
15
>100
>100
5f
~100
>100
5g
6a
6b
6c
2.1
3.1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
agonist (biphasic)c
11
5.7
agonist (biphasic)c
0.14
2.4
~50
~30
0.92
12
~30
~50
9.3
0.52
~30
1.8
2.6
7.5
1.2
5.2
2.2
~100
~50
6d
6e
0.23
3.6
~25
~50
~30
~50
~100
>100
6f
17
~25
6g
ACS Paragon Plus Environment
29