Synthesis and biological investigation of new equatorial (β) stereoisomers of 3-aminotropane arylamides with atypical antipsychotic profile

Article abstract of DOI:10.1016/j.bmc.2016.06.038

A series of novel 3β-aminotropane derivatives containing a 2-naphthalene or a 2-quinoline moiety was synthesised and evaluated for their affinity for 5-HT_1A, 5-HT_2Aand D₂receptors. Their affinity for the receptors was in the nanomolar to micromolar range. p-Substitution (6c, 6f, 6i, 6l, 6o), as well as substitution with chlorine atoms (6g, 6h, 6i), led to a significant increase in binding affinity for D₂receptors with compounds 6f (K_i?=?0.6?nM), 6c and 6i (K_i?=?0.4?nM), having the highest binding affinities. m-Substituted derivatives were the most promising ligands in terms of 5-HT_2Areceptor binding affinity whereas 2-quinoline derivatives (10a, 10b) displayed the highest affinity for 5-HT_1AR and were the most selective ligands with K_i?=?62.7?nM and K_i?=?30.5?nM, respectively. Finally, the selected ligands 6b, 6d, 6e, 6g, 6h, 6k, 6n and 6o, with triple binding activity for the D₂, 5-HT_1Aand 5-HT_2Areceptors, were subjected to in vivo tests, such as those for induced hypothermia, climbing behaviour and the head twitch response, in order to determine their pharmacological profile. The tested ligands presented neither agonist nor antagonist properties for the 5-HT_1Areceptors in the induced hypothermia and lower lip retraction (LLR) tests. All tested compounds displayed antagonistic activity against 5-HT_2A, with 6n and 6o being the most active. Four (6b, 6k, 6n and 6o) out of eight tested compounds could be classified as D₂antagonists. Additionally, evaluation of metabolic stability was performed for selected ligands, and introduction of halogen atoms into the benzene ring of 6h, 6k, 6n and 6o improved their metabolic stability. The project resulted in the selection of the lead compounds 6n and 6o, which had antipsychotic profiles, combining dopamine D₂-receptor and 5-HT_2Aantagonism and metabolic stability.

Full text of DOI:10.1016/j.bmc.2016.06.038

Accepted Manuscript
Synthesis and biological investigation of new equatorial (β) stereoisomers of 3-
aminotropane arylamides with atypical antipsychotic profile
Jacek Stefanowicz, Tomasz Słowiński, Martyna Zofia Wróbel, Franciszek
Herold, Anna Edyta Gomó łka, Anna Wesołowska, Magdalena Jastrzębska-
Więsek, Anna Partyka, Marta Andres-Mach, Stanisław Jerzy Czuczwar,
Jarogniew Jacek Łuszczki, Mirosław Zagaja, Agata Siwek, Gabriel Nowak,
Maria Żołnierek, Tomasz Bączek, Szymon Ulenberg, Mariusz Belka, Jadwiga
Turło
PII:
S0968-0896(16)30461-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.038
BMC 13094
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2016
17 June 2016
18 June 2016
Please cite this article as: Stefanowicz, J., Słowiński, T., Wróbel, M.Z., Herold, F., Gomó łka, A.E., Wesołowska,
A., Jastrzębska-Więsek, M., Partyka, A., Andres-Mach, M., Czuczwar, S.J., Łuszczki, J.J., Zagaja, M., Siwek, A.,
Nowak, G., Żołnierek, M., Bączek, T., Ulenberg, S., Belka, M., Turło, J., Synthesis and biological investigation of
new equatorial (β) stereoisomers of 3-aminotropane arylamides with atypical antipsychotic profile, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.038
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Synthesis and biological investigation of new equatorial (β) stereoisomers of 3-
aminotropane arylamides with atypical antipsychotic profile.
a
a
a
a
Jacek Stefanowicz , Tomasz Słowiński , Martyna Zofia Wróbel *, Franciszek Herold ,
b
b
Anna Edyta Gomółka a, Anna Wesołowska , Magdalena Jastrzębska-Więsek , Anna Partyka
c
c,d
b, Marta Andres-Mach , Stanisław Jerzy Czuczwar d,e, Jarogniew Jacek Łuszczki
,
c
f
f
f
g
Mirosław Zagaja , Agata Siwek , Gabriel Nowak , Maria Żołnierek , Tomasz Bączek ,
Szymon Ulenberg g Mariusz Belka g, Jadwiga Turło a
,
aDepartment of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy,
Medical University of Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland
bJagiellonian University Medical College, Department of Clinical Pharmacy, 9 Medyczna
Street, 30-688 Kraków, Poland
cIsobolographic Analysis Laboratory, Institute of Rural Health, 2 Jaczewskiego Street, 20-950
Lublin, Poland
dDepartment of Pathophysiology, Medical University, 8 Jaczewskiego Street , 20-090 Lublin,
Poland
eDepartment of Physiopathology, Institute of Rural Health, 2 Jaczewskiego Street, 20-950
Lublin, Poland
fDepartment of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna
Street, 30-688 Kraków, Poland
gDepartment of Pharmaceutical Chemistry, Medical University of Gdańsk, 107 Al. Gen. J.
Hallera Street, 80-416 Gdańsk, Poland
Abstract
A series of novel 3β-aminotropane derivatives containing a 2-naphthalene or a 2-
quinoline moiety was synthesised and evaluated for their affinity for 5-HT1A, 5-HT2A and D2
receptors. Their affinity for the receptors was in the nanomolar to micromolar range. p-
substitution (6c, 6f, 6i, 6l, 6o), as well as substitution with chlorine atoms (6g, 6h, 6i), led to a
significant increase in binding affinity for D2 receptors with compounds 6f (Ki = 0.6 nM), 6c
and 6i (Ki = 0.4 nM), having the highest binding affinities. m-substituted derivatives were the
most promising ligands in terms of 5-HT2A receptor binding affinity whereas 2-quinoline
derivatives (10a, 10b) displayed the highest affinity for 5-HT1AR and were the most selective
1

ligands with Ki = 62.7 nM and Ki =30.5 nM, respectively. Finally, the selected ligands 6b, 6d,
6e, 6g, 6h, 6k, 6n and 6o, with triple binding activity for the D2, 5-HT1A and 5-HT2A
receptors, were subjected to in vivo tests, such as those for induced hypothermia, climbing
behaviour and the head twitch response, in order to determine their pharmacological profile.
The tested ligands presented neither agonist nor antagonist properties for the 5-HT1A receptors
in the induced hypothermia and lower lip retraction (LLR) tests. All tested compounds
displayed antagonistic activity against 5-HT2A, with 6n and 6o being the most active. Four
(6b, 6k, 6n and 6o) out of eight tested compounds could be classified as D2 antagonists.
Additionally, evaluation of metabolic stability was performed for selected ligands, and
introduction of halogen atoms into the benzene ring of 6h, 6k, 6n and 6o improved their
metabolic stability. The project resulted in the selection of the lead compounds 6n and 6o,
which had antipsychotic profiles, combining dopamine D2-receptor and 5-HT2A antagonism
and metabolic stability.
Keywords: atypical antipsychotics; 3β-aminotropane derivatives; 5-HT1A, 5-HT2A, D2,
dopamine receptor ligands
List of abbreviations: FT-IR, Fourier transform infrared spectroscopy; NMR, nuclear
magnetic resonance; HRMS, high resolution mass spectroscopy; EPS, extrapyramidal
symptoms; 5-HT1AR, serotonin 5-HT1A receptors; (±)8-OH-DPAT, (±)8-hydroxy-2-(di-n-
propylamino)tetralin; DMF, dimethylformamide; TEA, triethylamine; DMSO, dimethyl
sulphoxide; DCM, dichloromethane; DOI, 2,5-dimethoxy-4-iodoamphetamine; HTR, Head
twitch response; LLR, lower lip retraction; APO, apomorphine
* corresponding author, e-mail: martynawrobel.wum@gmail.com
1. Introduction
Schizophrenia was described as a neurobiological disorder almost one hundred years
ago. Nevertheless, the precise explanation of its pathogenesis remains unknown.1
Observations of the antipsychotic efficacy of dopaminergic system inhibitors in the 1950s led
to the oldest and the most widely accepted neurochemical theory of schizophrenia
pathogenesis, which suggests a considerable role for dopaminergic system disturbances.2
First generation antipsychotics (also referred to as typical or classical neuroleptics),
such as chlorpromazine and haloperidol, exert their therapeutic effect mainly on the positive
2

symptoms of schizophrenia and are less effective in alleviation of the negative and cognitive
symptoms of this disorder. Moreover, these drugs have numerous adverse effects, among
which extrapyramidal symptoms (EPS), such as dystonia, akathisia, parkinsonism and tardive
dyskinesia, are of particular importance. Moreover, it is estimated that first generation
neuroleptics are ineffective in about 30% of schizophrenic patients.3 The reversal of positive
symptoms of schizophrenia by these antipsychotics is apparently mediated by blockage of D2
receptors in the mesolimbic region while EPS result from blockage of the corresponding
receptors in the basal ganglia.4
In 1988 Kane et. al. described a new antipsychotic drug — clozapine, which was
effective in patients who did not respond to first generation neuroleptic treatment.5 Moreover,
clozapine was effective not only in treatment of the positive symptoms of schizophrenia, but
also in reducing negative symptoms and cognitive deficits. At the same time it did not induce
the EPS and prolactin; Ki, inhibition constant (PRL) increase commonly seen with first
generation neuroleptics. Many of its atypical properties have been attributed to its affinity for
serotonin receptors, namely 5-HT2A, 5-HT1A, 5-HT2C, 5-HT7, and 5-HT6.6,7
The affinity of clozapine for the D2 dopamine receptor is much lower than that of most first
generation neuroleptics. Other second-generation antipsychotic drugs, also known as atypical
neuroleptics are: olanzapine, risperidone, zotepine, sertindole, quetiapine and ziprasidone.3
Meltzer and co-workers found that compounds belonging to this group should possess a pKi
5-HT2A/D2 ratio (the so-called Meltzer’s index) of ≥ 1.12. Clozapine demonstrates a Meltzer
index of 1.19, whereas chlorpromazine has a Meltzer index of 1.02.8,9
According to some authors other pKi ratios, for example D2/D3 and D2/D4 ,are not relevant in
differentiating between classical and atypical antipsychotics, however, according to other
studies these ratios play a role.10 For drugs with remarkably high affinities for D2 receptors,
even those with significant binding to 5-HT2A receptors do not fit the “atypical”
pharmacological profile.11 It is worth mentioning that compounds that do not affect the
serotonergic system may belong to the group of atypical neuroleptics as well. An example of
such a drug is amisulpride.12
The current state of knowledge suggests that drugs simultaneously targeting dopamine
D2 and serotonin 5-HT1A receptors may be beneficial in pharmacotherapy for schizophrenia.13
However the results of research evaluating the role of the 5-HT1A receptor in the
pathophysiology of this disorder are not entirely consistent.14 An increase in 5-HT1A receptor
binding in the prefrontal cortex (PFC) of patients with schizophrenia was reported in post-
mortem studies.15 On the other hand, this observation was not confirmed by molecular
3

imaging.16–18 In spite of this, clinical studies have demonstrated that co-administration of 5-
HT1A agonists and typical or atypical neuroleptics may enhance the antipsychotic effect of
these drugs especially in terms of reduced cognitive deficits (for review see 19).
A number of studies suggest an important role for 5-HT1A receptors in the control of motor
activity and the alteration of dopamine neurotransmission. Chronic administration of 5-HT1A
receptor agonists led to a substantial dose-related decrease in striatal 5-HT, and these results
suggested that 5-HT has an inhibitory influence on the activity of dopaminergic neurons.20
The dorsal raphe nuclei mediated serotonergic input is reduced in the substantia nigra in
response to the activation of somatodendritic 5-HT1A receptors by selective agonists, which
increases dopamine neurotransmission. Moreover, in the striatum, nigrostriatal dopamine
neurotransmission is released from the inhibitory influence of 5-HT. Activation of
postsynaptic 5-HT1A receptors may also have an important role in improving the negative
symptoms and cognitive deficits associated with schizophrenia, because it causes
enhancement of mesolimbic and mesocortical dopamine neurotransmission.
For these reasons, in the last few years considerable attention has been given to the new
generation of atypical antipsychotics, defined by some researchers as “third-generation
neuroleptics”.21 These include aripiprazole, lurasidone, brexpiprazole, perospirone,
quetiapine, ziprasidone, and cariprazine, all of which exhibit affinity for the 5-HT1A
receptor.22
Progress in the development of antipsychotic drugs is achieved mainly by eliminating
or diminishing side effects, which are typical for this class of drugs. This process was
explicitly presented by Altinbas et al.23 Analysing the binding affinity profiles of the newest
antipsychotics, it seems well-founded that the discovery of ligands with high 5-HT2A/D2
selectivity combined with partial agonistic activity at D2 receptors is still an important aim in
the design of new antipsychotic drugs.24 The impact of 5-HT2A receptor antagonism on the
improved tolerability of atypical antipsychotic drugs is no longer in doubt.25 5-HT1A receptor
agonism or partial agonism has a considerable effect on the cognitive deficits in
schizophrenia.26 This property is observed in the binding affinity profiles of brexpiprazole and
cariprazine, the latest antipsychotic drugs.
The possession of a binding affinity profile such as those discussed above is common for the
latest antipsychotics and is essential for their pharmacological activity and reduced side
effects.27
In a previous publication, we described the synthesis and biological activity of a series
of new 3β-acylamine derivatives of tropane, among which compound A ( Ref. 28) drew our
4

particular attention as it displayed a very high (in the nM range) affinity for each of the
investigated receptors (i.e. 5-HT1A, 5-HT2A and D2) and an ideal Meltzer index (pKi 5-
HT2A/D2 ratio) of 1.21, confirming its atypical antipsychotic profile.28 Analysis of the results
of in vitro radioligand binding assays allowed us to determine the influence of the substituents
at the 3β and N-8 position of the tropane system on the affinity for 5-HT1A, 5-HT2A and D2
receptors in the previously obtained series of compounds.28 A substantial favourable influence
of a 2-naphthyl moiety and a benzyl ring respectively (both present in compound A) on the
binding affinity for 5-HT1A, 5-HT2A and D2 receptors was observed. This suggested that we
should keep the 2-naphtyl moiety unaltered and turn our attention to the modification of the
benzyl substituent at N-8 position. The aim of our work was to examine if the presence of o-,
m-, or p-substituents (CH3, OCH3, Cl, Br, F, CF3) in the benzyl ring would enhance the
biological activity of the investigated analogues of compound A. We decided also to
introduce an quinoline in the place of naphthalene moiety since it is a heterocyclic analogue to
the latter one. This enabled the assessment whether the modification can provide an enhanced
affinity for evaluated receptors. Herein, we report the synthesis and in vitro biological
evaluation of compounds 6a–u and 10a–b, as well as the in vivo characterisation and studies
on the metabolic stability of selected compounds.
N
NH
N-8 position
R3
R1
O
H
R2
X
N
6a-r
NH
O
H
N
X
X
NH
N-3 position
O
H
compound A
6s-u
R1, R2, R3 = H, CH3, OCH3, Cl, Br, F, CF3
Ki [nM] = D2=82.4; 5-HT1A=303.8; 5-HT2A=2.5
X = CH, N
Figure 1. Structure of compound A (in Ref. 28), which was subjected to structural
modifications, leading to the 3β-acylamine derivatives of tropane described in the present
paper (6a–u).
2. Results and discussion
2.1. Chemistry
5

Continuing the search for new dopamine and serotonin receptors ligands in the 3-
aminotropane derivatives group, we performed the synthesis of a series of equatorial (β) 8-
aryl-8-azabicyclo[3.2.1]oct-3-ylamides according to Scheme 1 and Scheme 2.
In the first stage, 8-methyl-8-azabicyclo[3.2.1]octan-3-one was converted into an appropriate
oxime (2) by reaction with hydroxylamine hydrochloride in accordance with the method
29
published in Ref.
(see Supporting information for details). The obtained oxime was
subsequently subjected to stereoselective reduction to give the corresponding β-amine (3)
30
using the Bouveault-Blanc method, described in Ref. (see Supporting information for
details). However, to isolate this compound from the reaction mixture with a very good yield,
it was necessary to use the method of continuous extraction.
In the next stage, the equatorial amine (3) was subjected to acylation with
naphthalene-2-carboxylic acid, using the mixed anhydride method. The obtained product was
N-(8-methyl-8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide (4), which has not been described
in the literature before. Compound (4) was then subjected to demethylation with Olofson's
reagent to give N-(8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide hydrochloride (5).
Intermediate (5) was subsequently alkylated using benzyl chlorides or bromides substituted in
the o-, m- or p-position with a methyl, methoxy or trifluoromethyl group, or a chlorine,
bromine or fluorine atom to give the final amides (6a–f) using Finkelstein protection of KI.
In an analogous manner compounds 6s–u were synthesised using the corresponding
pyridinemethyl chlorides for the alkylation. Reactions c–e (Scheme 1) were performed
retaining the configuration at C3 of the tropane moiety. Therefore they were stereospecific.
In the next step the synthesis of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one (7) was
performed (Scheme 2). For that purpose the Robinson method was applied. The obtained
ketone (7) was subsequently converted to an oxime (8), which was then subjected to a
stereoselective reduction with sodium in butanol to give the equatorial (β) 8-benzyl-8-
azabicyclo[3.2.1]oct-3β-yl-amine (9) (see Supporting information for details). The above
31
reactions were carried out using methods described in the literature . In order to obtain the
planned β-quinolineamides (10a, b) the mixed anhydride method was used. This method
proved to be stereospecific.
The structure and isomerism of all new intermediates and final compounds was
confirmed by analysis of IR 1H NMR and ESI-HRMS spectra. To confirm the composition of
the hydrochloride 10a and oxalate 10b additional elemental analysis (C, H, N) was
performed. The details are presented in the Experimental Section.
6

H3C
H3C
H3C
a
N
N
b
N
NH2
O
NOH
H
3
1
2
H3C
d
N
c
H
N
H
O
COOH
4
Ar
H
N
N
e
H
H
X HCl
ArX
N
N
O
H
O
6a-u
H
5
Ar =
Cl
Br
F
MeO
H3C
o-, m-, p-
6g 6h 6i
o-, m-, p-
6a 6b 6c
o-, m-, p-
6d 6e 6f
o-, m-, p-
6j 6k 6l
o-, m-, p-
6m 6n 6o
N
CF3
N
N
o-, m-, p-
6p 6q 6r
6s
6t
6u
Scheme 1. . Synthesis of the new 3β-aminotropane derivatives 6a–u. Reagents: (a) NH2OH x
HCl, NaHCO3, EtOH; (b) Na/PrOH; (c) ClCOOEt, TEA, DMF; (d) /I/ ClCOOCHClCH3,
DMC, ClCH2CH2Cl, /II/ MeOH; (e) methyl-, methoxy-, chloro-, bromo-, fluoro-,
trifluoromethylbenzyl chlorides or bromides and 2-, 3- or 4-pyridinylmethyl chlorides,
anhydrous K2CO3, KI, acetone.
a
b
N
N
N
NH2
H
NOH
R
O
7
8
9
c
N
H
N
N
H
O
10a (R = -H), 10b (R= -OCH3)
7

Scheme 2. Synthesis of the new 3β-aminotropane derivatives 10a–b. Reagents: (a) NH2OH x
HCl, NaHCO3, EtOH; (b) Na/BuOH; (c) quinoline-2-carboxylic acid or 4-methoxyquinoline-
2-carboxylic acid, ClCOOEt, TEA, DMF.
2.2. Biological evaluation
2.2.1 Radioligand binding assay for D2, 5-HT1A and 5-HT2A receptors
Compounds 6a–u and 10a–b were tested for their in vitro affinity for the D2, 5-HT1A,
and 5-HT2A receptors using a radioligand binding assay. Competition binding studies were
performed according to a previously described procedure in rat brain tissues.28,32 The results
are presented in Table 1.
Table 1
Binding affinities for dopamine D2 and serotonin 5-HT1A / 5-HT2A receptors.
R
N
H
N
Ar
O
H
Ki [nM] ± S.E.M
pKi ratio
5-HT2A/D2
Compound
R
Ar
D2
5-HT1A
5-HT2A
Aa
303.8 ±
11.4
82.4 ± 6.5
2.5 ± 0.8
6a
6b
118.0 ±
14.1
192.7 ±
22.7
854.0 ±
22.4
CH3
182.0 ±
1.0
231.0 ±
39.8
3.3 ± 0.2
0.78
H3C
6c
728.5 ±
81.0
908.0 ±
37.0
H3C
0.4 ± 0.03
6d
919.5 ±
34.5
76.9 ± 3.1 82.5 ± 8.0
0.85
0.81
OMe
6e
212.6 ±
25.7 ± 2.3
663.0 ±
23.3
23.0
MeO
6f
2600 ±
0.6 ± 0.05
509.0 ±
5.3
MeO
300.0
6g
158.0 ±
396.7 ±
4.8
34.8 ± 3.7
2.0
0.86
0.81
Cl
6h
6i
298.0 ±
3.6 ± 0.3
135.7 ±
0.9
23.5
Cl
2500 ±
0.4 ± 0.05
571.0 ±
20.4
Cl
200.0
8

6j
196.3 ±
28.0
326.5 ±
18.5
22.8 ± 1.1
Br
6k
199.5 ±
19.7
105.7 ±
5.4
11.6 ± 0.9
4.5 ± 0.4
0.88
Br
6l
229.7 ±
31.0
298.5 ±
24.0
Br
6m
714.5 ±
40.5
365.5 ±
22.5
141.0 ±
12.0
F
6n
214.0 ±
6.0
210.7 ±
29.0
101.5 ±
0.5
1.05
0.97
F
6o
6p
137.0 ±
15.0
218.0 ±
22.0
209.0 ±
7.8
F
536.7 ±
21.5
476.0 ±
50.0
>10000
CF3
6q
217.3 ±
11.0
130.0 ±
12.9
589.0 ±
93.0
F3C
6r
6s
629.0 ±
71.5
2000 ±
200.0
484.0 ±
18.7
F3C
N
2200.0 ±
600.0
314.8 ±
32.0
1116.0 ±
19.5
N
6t
2400.0 ±
200.0
484.5 ±
87.5
502.0 ±
51.0
6u
2700.0 ±
300.0
2300 ±
200.0
495.0 ±
5.0
N
10a
10b
588.6 ±
8.3
62.7 ±
11.8
1291.0 ±
40.5
N
OMe
2315.0 ±
27.5
>10000
30.5 ± 3.5
N
serotonin
DPAT
9.4
2.3
Mianserine
Clozapine
Quetiapine
18.4
8.9b
220b
130b
180b
140b
230b
a Data from Ref. 28
b Data from Ref. 33
The influence of the type and location of the substituents in the benzene ring of 6a–6r
on the binding affinity for D2, 5-HT1A and 5-HT2A receptors was analysed.
9

In terms of their affinity for D2 receptors, ligands 6a–6r displayed very high (Ki = 0.4 nM for
6c and 6i, Ki = 0.6 nM for 6f) to low affinities for D2 receptors (Ki = 714.5 nM for 6m). It is
worth noting that p-substituted ligands (6c, 6f, 6i, 6l, 6o) proved to be the considerably more
promising compounds than their m- or o-substituted analogues, with compounds 6c (p-CH3),
6f (p-OCH3) and 6i (p-Cl) displaying subnanomolar Ki values. The only exception to this rule
was ligand 6r (p-CF3-substituted). We also observed that m-substituted compounds have
higher affinity for D2 than their o-substituted analogues in all synthesised series of ligands.
Analysing the influence of the type of substituent in the benzene ring, we noted that chlorine
substitution (6g, 6h, 6i) had the most favourable effect on D2 binding affinity, while
introduction of fluorine (6m, 6n, 6o) or a trifluoromethyl substituent (6p, 6q, 6r) negatively
affected D2 receptor binding affinity. Comparing radioligand binding assay results for
compound A and its substituted analogues (6a–6r), we can conclude that the introduction of a
substituent such as a methyl (apart from o-methyl substitution in compound 6a) or methoxy
group, or a chlorine or bromine atom led to an increase in the affinity for the D2 receptor.
As far as affinity for the 5-HT1AR receptor is concerned, p-substituted ligands displayed significantly lower
affinity than the corresponding o- or m- substituted compounds. The only exception was compound 6o (p-
fluoro-substituted), which turned out to has higher affinity for the 5-HT1AR than its o-substituted (6m) analogue
and of similar affinity to its m-substituted (6n) analogue. Comparing Ki values for compound A and ligands 6a–
6r, it can be seen that the introduction of substituents in the o- (except for –F and –CF3 substituents) or m-
position proved to be beneficial in terms of 5-HT1AR binding affinity, with the highest affinities observed for
compounds 6d (Ki = 82.5 nM) R1 = -OCH3; R2, R3 = H and 6q (Ki = 130 nM) R2 = -CF3; R1,R3 = H.
Analysing Ki values for 5-HT2A receptors we found that m-substituted ligands had the highest binding affinities,
with the exception of 6i and 6q. The substituted compounds 6a–6r however displayed significantly lower
binding affinities for 5-HT2A receptors (Ki ranging from 101.5 nM to >10 μM) than the unsubstituted compound
A (Ki = 2.5 nM).
We then studied the effect of replacement of phenyl or naphthyl ring with their heterocyclic analogues,
namely 2-, 3- or 4-pyridine (compound 6s–6u) or 2-quinoline (compounds 10a–10b), respectively.
2-, 3- or 4-pyridine derivatives had significantly lower affinities for D2 receptors than their benzene substituted
analogues, with Ki ranging from 2.2 to 2.7 μM. The highest affinity was observed for a 2-pyridine analogue (6s)
for both of the receptors D2 and 5-HT1A. At the same time the 2-pyridine analogue had the lowest affinity for 5-
HT2AR. Incorporation of a 2-quinoline moiety (10a, 10b) led to a significant decrease or even a complete loss of
affinity for the D2 and 5-HT2A receptors. Interestingly, at the same time affinity for 5-HT1AR was the highest of
all tested compounds with Ki = 62.7 nM for 10a and Ki =30.5 nM for 10b.
The in vitro binding assay showed that in the investigated series the compounds 6b, 6d, 6e, 6g, 6h, 6k, 6n and
6o, displayed triple binding activity for the D2, 5-HT1A and 5-HT2A receptors. That was the criterion for selecting
10

6b, 6d, 6e, 6g, 6h, 6k, 6n and 6o compounds for the behavioural assay. The Meltzer index was calculated for the
tested compounds, see Table 1. The compounds with only significant high D2 receptor affinity was excluded
because they do not fit the “atypical” pharmacological profile.
It is worth mentioning that their affinity for these receptors was comparable or higher than those for
quetiapine and clozapine, very successful antipsychotic drugs. Clozapine is currently the most powerful
antipsychotic drug and has a complex, constantly discussed mechanism of action, being a D2/5-HT2A receptor
antagonist with agonist activity for 5-HT1A receptors.34 Evidence suggests that the side effects of clozapine are
substantial and efforts are still being made to find drugs that would be equally effective and better tolerated.
The mechanism of quetiapine action is also the subject of intense discussion. Some authors suggest it has a
distinct mechanism of action.35 Depending on the dose, quetiapine exerts different effects (hypnotic, sedative or
antidepressant and / or antipsychotic). Undoubtedly, the therapeutic effect is the sum of the activity of quetiapine
and its active metabolite norquetiapine, a potent norepinephrine reuptake inhibitor and a partial 5-HT1A agonist.
The overall pharmacological effect is clearly and precisely explained by Stahl.36 The antipsychotic activity of
quetiapine is closely correlated with D2 receptor saturation. Because of its moderate binding affinity for D2
receptors, the antipsychotic effect occurs only at sufficiently high doses. These data suggest the classic
mechanism of action of this drug — similar to other antipsychotics. Such a mechanism is also postulated by
other authors.37
Assuming the mechanism of action proposed by the above-cited Stahl and Meltzer, examination of the in vivo
properties of the selected derivatives in our study is well justified. As mentioned above, after the rejection of a
further search for ligands with outstandingly high D2 receptor affinity, we focused on compounds having a very
similar binding profile to quetiapine.
2.2.2. In vivo studies
2.2.2.1 General
Experiments were carried out on Swiss mice weighing 20-25 g kept in colony cages in standard
laboratory conditions. Experimental groups were chosen randomly and each animal was used only once. The
compounds studied were suspended in a 1% of Tween 80 (Sigma, St. Louis, MO, USA) and injected
intraperitoneally in a volume of 10 ml/kg.
2.2.2.2 Body temperature in mice
(±)8-OH-DPAT induces hypothermia in mice, which is mediated through presynaptic
5-HT1A receptors.38,39 Its effect is abolished by WAY-100635, as a result of presynaptic 5-
HT1A receptor antagonism.40 The effects of the tested compounds (6b, 6d, 6e, 6g, 6h, 6k, 6n,
6o), given alone at doses of 10 and 20 mg/kg, on the rectal body temperature were recorded
30, 60, 90 and 120 min after administration. None of the studied compounds affected the body
temperature of mice (Table 2).
11

Table 2
Effect of the tested compounds on body temperature in Albino Swiss mice.
Δt ± SEM (°C)
30 min
Dose
(mg/kg)
Treatment
60 min
90 min
120 min
Vehicle
6b
0
10
20
0.33 ± 0.2
-0.02 ± 0.3
0.04 ± 0.3
0.33 ± 0.2
0.73 ± 0.2
0.47 ± 0.1
0.18 ± 0.2
0.35 ± 0.2
0.20 ± 0.3
0.15 ± 0.3
0.27 ± 0.2
0.24 ± 0.1
F(2,16)=0.3041; F(2,16)=1.0350; F(2,16)=0.1131; F(2,16)=0.0787;
NS
NS
NS
NS
vehicle
6d
0
10
20
0.33 ± 0.2
0.23 ± 0.2
-0.34 ± 0.4
0.33 ± 0.2
0.44 ± 0.1
0.01 ± 0.3
0.18 ± 0.2
0.63 ± 0.1
0.16 ± 0.2
0.15 ± 0.3
0.26 ± 0.1
-0.26 ± 0.1
F(2,17)=1.6290; F(2,17)=1.1221; F(2,17)=2.2471; F(2,17)=1.6841;
NS
NS
NS
NS
vehicle
6e
0
10
20
0.31 ± 0.3
-0.03 ± 0.2
-0.10 ± 0.1
0.61 ± 0.3
0.03 ± 0.3
0.57 ± 0.2
0.63 ± 0.2
0.17 ± 0.3
0.81 ± 0.3
1.11 ± 0.2
0.70 ± 0.3
1.26 ± 0.2
F(2,18)=1.0227; F(2,18)=1.5303; F(2,18)=1.7871; F(2,18)=1.5018;
NS
NS
NS
NS
vehicle
6g
0
10
20
0.31 ± 0.3
0.00 ± 0.4
-0.36 ± 0.2
0.61 ± 0.3
0.96 ± 0.2
0.44 ± 0.2
0.63 ± 0.2
0.84 ± 0.2
0.44 ± 0.2
1.11 ± 0.2
1.34 ± 0.1
1.39 ± 0.3
F(2,18)=1.0967; F(2,18)=1.1305; F(2,18)=1.1939; F(2,18)=0.3937;
NS
NS
NS
NS
vehicle
6h
0
10
20
0.20 ± 0.2
0.17 ± 0.4
-0.38 ± 0.2
0.50 ± 0.2
1.00 ± 0.2
0.41 ± 0.2
0.51 ± 0.2
0.84 ± 0.2
0.41 ± 0.2
0.71 ± 0.1
1.16 ± 0.2
1.00 ± 0.2
F(2,18)=1.3183; F(2,18)=1.8677; F(2,18)=1.2289; F(2,18)=1.7140;
NS
NS
NS
NS
vehicle
6k
0
10
20
0.17 ± 0.2
-0.20 ± 0.3
-0.30 ± 0.2
0.47 ± 0.2
0.76 ± 0.1
0.49 ± 0.2
0.49 ± 0.2
0.64 ± 0.2
0.36 ± 0.2
0.97 ± 0.2
1.14 ± 0.3
1.23 ± 0.3
F(2,18)=0.9534; F(2,18)=0.8826; F(2,18)=0.4829; F(2,18)=0.2142;
NS
NS
NS
NS
vehicle
6n
0
10
20
0.17 ± 0.2
-0.17 ± 0.2
-0.36 ± 0.1
0.47 ± 0.2
0.53 ± 0.1
0.43 ± 0.1
0.49 ± 0.2
0.47 ± 0.1
0.20 ± 0.1
0.97 ± 0.2
0.74 ± 0.2
0.81 ± 0.2
F(2,18)=1.5935; F(2,18)=0.0921; F(2,18)=1.0732; F(2,18)=0.3473;
NS
NS
NS
NS
vehicle
6o
0
10
20
0.16 ± 0.2
-0.17 ± 0.2
-0.33 ± 0.1
0.46 ± 0.2
0.50 ± 0.1
0.46 ± 0.1
0.43 ± 0.2
0.44 ± 0.1
0.23 ± 0.1
0.90 ± 0.2
0.59 ± 0.2
0.54 ± 0.1
F(2,18)=1.8670; F(2,18)=0.0265; F(2,18)=0.6587; F(2,18)=1.2082;
NS NS NS NS
The tested compounds were administered i.p. 30 min before the test. The absolute mean body temperatures were
within a range of 36.0±0.6°C; n=6-7 mice per group. One-way ANOVA followed by Bonferroni’s post hoc test,
NS= non-significant.
12

In separate experiments, the effects of 6b, 6d, 6e, 6g, 6h, 6k, 6n, and 6o on (±)8-OH-
DPAT-induced hypothermia in mice were studied, since the decrease in body temperature
evoked by (±)8-OH-DPAT is attributed to stimulation of presynaptic 5-HT1A receptors and is
abolished by WAY-100635, a silent 5-HT1A receptor antagonist (Table 3). In contrast to
WAY-100635, none of the tested compounds inhibited the hypothermia induced by 5-HT1A
agonist (±)8-OH-DPAT (Table 3).
Table 3
Effect of the tested compounds on (±)8-OH-DPAT (5 mg/kg s.c.)-induced hypothermia in
Albino Swiss mice.
Δt ± SEM (°C)
15 min
Dose
(mg/kg)
Treatment
30 min
45 min
60 min
Vehicle +
vehicle
0 + 0
0.28 ± 0.2
0.52 ± 0.3
0.56 ± 0.2
0.70 ± 0.2
Vehicle +
(±)8-OH- 0 + 5
DPAT
-0.78 ± 0.2
-1.66 ± 0.6b
-1.12 ± 0.2a
-0.50 ± 0.2
10 + 5
20 + 5
-0.70 ± 0.2
-1.90 ± 0.4c
-0.97 ± 0.3
-2.08 ± 0.4c
-0.48 ± 0.4
-2.08 ± 0.5c
-0.05 ± 0.3
-1.50 ± 0.6b
6b +
(±)8-OH-
DPAT
F(3,18)=11.6730; F(3,18)=11.0820; F(3,18)=9.1979; F(3,18)=6.1883;
p<0.001
p<0.001
p<0.001
p<0.01
Vehicle +
0 + 0
0.28 ± 0.2
0.52 ± 0.3
0.56 ± 0.2
0.70 ± 0.2
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.66 ± 0.6b
-1.12 ± 0.2a
-0.78 ± 0.2a
-0.50 ± 0.2a
10 + 5
20 + 5
-1.37 ± 0.5a
-1.47 ± 0.3a
-1.77 ± 0.5c
-1.37 ± 0.2b
-1.45 ± 0.5c
-0.92 ± 0.2a
-1.08 ± 0.4c
-0.65 ± 0.1a
6d +
(±)8-OH-
DPAT
F(3,18)=5.6742; F(3,18)=8.9952; F(3,18)=8.0335; F(3,18)=8.0719;
p<0.01
p<0.001
p<0.01
p<0.01
Vehicle +
0 + 0
0.28 ± 0.2
0.52 ± 0.3
0.56 ± 0.2
0.70 ± 0.2
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.66 ± 0.6b
-1.12 ± 0.2a
-0.78 ± 0.2a
-0.50 ± 0.2a
10 + 5
20 + 5
-1.52 ± 0.2b
-1.25 ± 0.5a
-1.32 ± 0.2b
-1.65 ± 0.5c
-0.87 ± 0.2a
-1.33 ± 0.5b
-0.6 ± 0.1
6e +
(±)8-OH-
DPAT
-0.97 ± 0.4b
F(3,18)=6.3865; F(3,18)=7.7894; F(3,18)=6.6475; F(3,18)=5.1691;
p<0.01
p<0.01
p<0.01
p<0.01
Vehicle +
0 + 0
0.27 ± 0.1
0.43 ± 0.2
0.48 ± 0.1
0.58 ± 0.2
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.82 ± 0.2d
-1.36 ± 0.2b
-1.04 ± 0.2a
-0.66 ± 0.2
13

10 + 5
20 + 5
-0.97 ± 0.2b
-2.00 ± 0.4d
-1.17 ± 0.2b
-2.12 ± 0.4c
-0.62 ± 0.4
-2.18 ± 0.4d
-0.12 ± 0.3
-1.53 ± 0.5b
6g +
(±)8-OH-
DPAT
F(3,19)=19.6290; F(3,19)=14.7720; F(3,19)=12.0470; F(3,19)=6.6860;
p<0.00001
p<0.0001
p<0.001
p<0.01
Vehicle +
vehicle
0 + 0
0.27 ± 0.1
0.43 ± 0.2
0.48 ± 0.1
0.58 ± 0.2
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.82 ± 0.2d
-1.36 ± 0.2b
-1.04 ± 0.2a
-0.66 ± 0.2
10 + 5
20 + 5
-1.03 ± 0.2b
-2.12 ± 0.3d
-1.22 ± 0.2b
-2.22 ± 0.4d
-0.85 ± 0.4a
-2.22 ± 0.4d
-0.67 ± 0.1
-1.57 ± 0.5
6h +
(±)8-OH-
DPAT
F(3,19)=24.693; F(3,19)=18.088; F(3,19)=14.239; F(3,19)=8.2417;
p<0.00001
p<0.0001
p<0.0001
p<0.01
Vehicle +
0 + 0
0.27 ± 0.1
0.43 ± 0.2
0.48 ± 0.1
0.58 ± 0.2
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.82 ± 0.2d
-1.36 ± 0.2b
-1.04 ± 0.2a
-0.66 ± 0.2
10 + 5
20 + 5
-1.32 ± 0.1d
-1.42 ± 0.1d
-1.62 ± 0.1d
-1.37 ± 0.1d
-1.00 ± 0.2c
-1.25 ± 0.1c
-0.72 ± 0.1a
-0.58 ± 0.5a
6k +
(±)8-OH-
DPAT
F(3,19)=49.226; F(3,19)=49.226; F(3,19)=30.511; F(3,19)=17.901;
p<0.00001
p<0.00001
p<0.00001
p<0.0001
Vehicle +
0 + 0
0.28 ± 0.2
0.52 ± 0.3
0.56 ± 0.1
0.70 ± 0.2
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
-1.66 ± 0.3a
-1.12 ± 0.2a
-0.78 ± 0.2a
-0.5 ± 0.2a
10 + 5
20 + 5
-1.60 ± 0.3b
-1.67 ± 0.4c
-1.37 ± 0.2b
-1.83 ± 0.4c
-0.97 ± 0.1b
-1.53 ± 0.5c
-0.78 ± 0.1b
-1.08 ± 0.4c
6n +
(±) 8-OH-
DPAT
F(3,18)=13.232; F(3,18)=10.545; F(3,18)=10.618; F(3,18)=8.8983;
p<0.0001
p<0.001
p<0.001
p<0.001
Vehicle +
0 + 0
0.28 ± 0.2
-1.55 ± 0.2c
-1.62 ± 0.3c
0.52 ± 0.3
0.56 ± 0.1
0.70 ± 0.2
vehicle
10 + 5
20 + 5
-1.32 ± 0.1c
-0.92 ± 0.1c
-0.73 ± 0.1b
6o +
(±)8-OH-
-1.60 ± 0.3c
-1.43 ± 0.4c
-1.00 ± 0.4c
F(3,18)=13.631; F(3,18)=12.558; F(3,18)=12.381; F(3,18)=9.2923;
p<0.0001
DPAT
p<0.001
p<0.001
p<0.001
Vehicle +
0 + 0
0.00 ± 0.1
-0.31 ± 0.2
-0.30 ± 0.3
0.00 ± 0.3
vehicle
Vehicle +
(±)8-OH- 0 + 5
DPAT
WAY-
100635 + 0.3 + 0
vehicle
-1.8 ± 0.3c
-0.2 ± 0.2
-1.7 ± 0.2c
-0.1 ± 0.2
-1.3 ± 0.3
-0.1 ± 0.2
-1.3 ± 0.1b
-0.3 ± 0.3
WAY-
100635 +
(±)8-OH-
0.3 + 5
-0.6 ± 0.2C
-0.8 ± 0.2
-0.5 ± 0.2A
-0.2 ± 0.2B
DPAT
F(3,25)=19.577; F(3,25)=12.232; F(3,25)=4.416;
F(3,25)=8.869;
14

p<0.0001
p<0.0001
p<0.05
p<0.001
The tested compounds were administered i.p. 45 min prior 8-OH-DPAT; n=6-7 mice per group. The absolute
mean initial body temperatures were within a range of 36.0±0.6°C. One-way ANOVA followed by Bonferroni’s
post hoc test; ap<0.05, bp<0.01, cp<0.001, dp<0.0001 vs respective vehicle+vehicle group; Ap<0.05, Bp<0.01,
Cp<0.001 vs respective vehicle + (±)8-OH-DPAT group.
2.2.2.3 Lower lip retraction (LLR)
It is commonly accepted that the (±)8-OH-DPAT-induced LLR in normal rats is
connected with the stimulation of postsynaptic 5-HT1A receptors.41 That effect is attenuated
by 5-HT1A receptor antagonists, e.g. WAY-100635 (Table 4). The results of our behavioural
study presented in Table 4 indicate that the tested compounds neither behave like 5-HT1A
receptor agonists nor inhibit LLR produced by (±)8-OH-DPAT, respectively.
Table 4
Induction of lower lip retraction (LLR) by the tested compounds (A) and their effect on the
(±)8-OH-DPAT (1 mg/kg s.c.)-induced LLR (B) in rats.
Treatment
Dose (mg/kg) Mean ± LLR score
A
B
Vehicle
6b
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.7 ± 0.1
2.3 ± 0.3
2.3 ± 0.4
F(2,11)=0.8540; NS
Vehicle
6d
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.8 ± 0.1
2.5 ± 0.4
2.0 ± 0.2
F(2,11)=3.9286; NS
Vehicle
6e
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.7 ± 0.1
2.4 ± 0.4
2.5 ± 0.4
F(2,11)=0.3301; NS
Vehicle
6g
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.8 ± 0.1
2.5 ± 0.3
2.0 ± 0.4
F(2,11)=2.7500; NS
Vehicle
6h
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.8 ± 0.1
2.6 ± 0.2
2.1 ± 0.4
F(2,11)=2.0918; NS
Vehicle
6k
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.8 ± 0.1
2.4 ± 0.4
2.5 ± 0.3
F(2,11)=0.8880; NS
Vehicle
6n
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.8 ± 0.1
2.4 ± 0.4
2.3 ± 0.2
15

F(2,11)=1.9082; NS
2.7 ± 0.1
2.5 ± 0.3
Vehicle
6o
0
10
20
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.4 ± 0.2
F(2,11)=0.4610; NS
Vehicle
0
0.1
0.2
0.0 ± 0
0.0 ± 0
0.0 ± 0
2.7 ± 0.2
1.4 ± 0.3 p<0.05
1.3 ± 0.2 p<0.01
F(2,15)=9.120;
p<0.01
WAY 100635
The tested compounds were administered (i.p.) 15 min before the test (A) or 45 min before (±)8-OH-DPAT
(s.c.), WAY 100635 was injected s.c., n=4-6 rats per group. One-way ANOVA followed by Bonferroni’s post
hoc test, NS= non-significant.
2.2.2.4 Induction of head twitches in mice
The HTR occurs after administration of (±)-2,5-dimethoxy-4-iodoamphetamine ((±)-
DOI) as a result of the activation of 5-HT2AR.42
The obtained results clearly point to 6n and 6o as the most potent compounds
inhibiting the head-twitch response, produced by (±)-DOI at a dose of 2.5 mg/kg, in mice.
Both compounds were still significantly active at 1.25 mg/kg. The weakest potency in this
regard was noted for bromo- and chloro-derivatives, which expressed a significant inhibitory
action on the head-twitch response at the highest dose of 10 mg/kg. 6b retained an
intermediate inhibitory action, being significantly active in the dose range of 2.5–10 mg/kg
(Table 5).
Table 5
Inhibition of (±)-DOI (2.5 mg/kg)-induced head twitches by the investigated compounds 6b;
6d; 6e; 6g; 6h; 6k; 6n; 6o given i.p. at various increasing doses in Albino Swiss mice.
Treatment
Dose (mg/kg) Head twitches
(number of episodes)
Vehicle
0
19.88 ± 2.06
1.25
2.5
5
15.63 ± 2.46
13.00 ± 1.56a
10.00 ± 1.93b
2.38 ± 0.86c
F(4,35)=12.550;
p<0.0001
6b
10
Vehicle
6d
0
2.5
5
19.63 ± 2.04
16.25 ± 1.26
12.25 ± 1.76a
8.25 ± 1.56c
10
16

F(3,28)=8.595;
p=0.0003
Vehicle
6e
0
2.5
5
19.63 ± 2.04
16.63 ± 1.31
14.25 ± 2.70
9.88 ± 1.30b
F(3,28)=4.560;
p=0.0101
10
Vehicle
6g
0
2.5
5
19.63 ± 2.04
20.00 ± 2.50
14.75 ± 2.74
10.75 ± 1.80a
F(3,28)=3.646;
p=0.0245
10
Vehicle
6h
0
2.5
5
19.63 ± 2.04
17.25 ± 1.62
13.13 ± 1.94
8.75 ± 1.88c
F(3,28)=6.490;
p=0.0018
10
Vehicle
6k
0
2.5
5
19.88 ± 2.06
14.50 ± 2.54
13.88 ± 2.04
11.88 ± 2.14a
F(3,28)=2.404;
p=0.0886
10
Vehicle
6n
0
19.63 ± 2.04
17.38 ± 2.22
2.3 ± 0.2
0.625
1.25
2.5
5
11.38 ± 2.35a
3.88 ± 1.51c
2.38 ± 1.45c
F(5,42)=12.210;
p<0.0001
10
Vehicle
6o
0
19.63 ± 2.04
17.63 ± 1.72
12.63 ± 1.38a
5.75 ± 2.22c
5.00 ± 1.93c
5.38 ± 1.95c
F(5,42)=12.070;
p<0.0001
0.625
1.25
2.5
5
10
Values represent the number of head twitches (mean ± SEM) during the 20 min test. n=8 mice per group. One-
way ANOVA followed by Dunnett’s post hoc test; ap<0.05, bp<0.01, cp<0.001 vs.(±)-DOI-treated group.
2.2.2.5 Climbing behaviour
17

Climbing behaviour induced by apomorphine (APO) results from the stimulation of
dopamine D2 receptors in the striatum.43
Out of eight compounds studied, only two (6k and 6b) significantly inhibited the climbing
behaviour within the first 30 min of observation. One was active over the period of 40 min
(6n) and interestingly, one (6o) was able to inhibit the climbing behaviour only at the 60 min
time point. Probably, pharmacokinetic parameters of this compound are responsible for its
peak effect at 60 min and fast decline in its inhibition of climbing behaviour. It is noteworthy
that the binding affinities of the tested compounds do not have to be fully correlated with their
behavioural effects because their penetration through the blood-brain barrier may significantly
differ. A formation of an active metabolite seems unlikely due to the metabolic stability of
this compound (see below). The remaining four compounds (6d, 6e, 6g, and 6h) were
ineffective in this regard (Table 6).
Table 6
Climbing behavior in Albino Swiss mice.
Time APO + APO
APO
APO
APO
APO
APO
APO
APO
(min) vehicle + 6d
+ 6e
+ 6g
+ 6h
+ 6k
+ 6b
+ 6n
+ 6o
(10 mg/kg)
10
8/8
4/8
6/8
4/8
5/8
3/8 a
3/8 a
3/8 a
6/8
20
30
40
50
60
8/8
8/8
8/8
8/8
8/8
5/8
5/8
4/8
5/8
4/8
5/8
5/8
6/8
5/8
7/8
5/8
5/8
5/8
5/8
4/8
5/8
6/8
6/8
5/8
5/8
3/8 a
3/8 a
5/8
2/8 b
3/8 a
4/8
3/8 a
3/8 a
3/8 a
4/8
6/8
5/8
5/8
4/8
2/8 b
6/8
7/8
7/8
7/8
6/8
120
7/8
6/8
8/8
5/8
7/8
7/8
7/8
7/8
4/8
All investigated compounds were administered at a constant dose of 10 mg/kg, 30 min before testing. APO was
administered 10 min before testing. n=8 mice per group. Fisher’s exact probability test with Yates’ correction for
a
b
the control group treated with apomorphine (APO) at the appropriate time. p < 0.05, p < 0.01. vs.
APO+vehicle.
2.2.3. Evaluation of metabolic stability
The results incubating the compounds in the presence of pooled human liver
microsomes and NADPH are shown in Table 7. Metabolic stability is presented as a half-time
value, which allows for easy comparison of the compound structures and their liability to
microsomal enzymes, responsible mainly for phase I biotransformation reactions.
Table 7
Experimental t1/2 values along with corresponding SD and RSD%.
18

Average
t1/2 (n=5)
[min]
Compound
substituent
SD
RSD%
3-CH3
2-OCH3
3-OCH3
2-Cl
3-Cl
3-Br
6b
6d
6e
6g
6h
6k
6n
6o
29.955
41.26
42.90
33.05
>45
>45
>45
>45
4.98
5.93
6.77
8.12
N/A
N/A
N/A
N/A
16.65
14.38
15.80
24.59
N/A
N/A
N/A
N/A
3-F
4-F
SD – standard deviation, RSD% – relative standard deviation, expressed as SD/average*100%.
The half-time assessment in Table 7 shows that compounds 6h, 6k, 6n, and 6o are
stable under the incubation conditions beyond the end point of the experiment. After 45
minutes of incubation, the compound concentration was still more than 50% of the initial
value, preventing a half-time calculation. For compounds 6b, 6d, 6e, and 6g however, half-
time value was easily calculated. Comparison of the structures of these compounds reveals
that a halogen moiety might be the factor responsible for increased metabolic stability of 6h,
6k, 6n, and 6o. 6b was shown to be the compound most liable to biotransformation of all
those studied. The methyl moiety may be responsible for this liability, as 6b is the only
compound among the present derivatives with a methyl group. A methoxyl moiety, present in
compounds 6d and 6e, seems to increase metabolic stability for the presented group of
compounds, yet not in such a significant way as a halogen moiety. Unfortunately, due to the
stability of compounds 6h, 6k, 6n, 6o the direct impact of various halogen groups on
metabolic stability cannot be assessed. Compound 6g is the only halogen derivative, for which
the authors were able to evaluate a half-time. It is also the only halogen derivative possessing
a chlorine moiety in the orto-position. This may lead to the conclusion that not only the type
of moiety but also the position of the moiety plays an important role in increasing the
metabolic stability, with halogen moieties being the most effective substituents and the meta
and para positions affording the most effective substitutions.
Metabolic stability assay was performed on human liver microsomes, as the aim was to
compare susceptibility to biotransformation as the potential drug candidates for humans.
Metabolic stability of compounds 6h, 6k, 6n and 6o proved to be of value high enough to be
beyond the point of incubation, rendering those compounds metabolically stable when
compared to other derivatives in presented set.
3. Conclusions
19

In summary, our efforts in the synthesis and pharmacological evaluation of new 3β-
acylaminotropane derivatives have led to the discovery of novel compounds 6n and 6o with
favourable antipsychotic profiles, combining dopamine D2-receptor and 5-HT2A antagonism
as well as metabolic stability.
SAR studies demonstrate a number of key features of this series: a) The D2, 5-HT1A and 5-
HT2A potencies are sensitive to electronic and stearic modifications on the benzyl ring, and
affinities toward these receptors can be tuned by careful choice of substituents. p-substituted
compounds displayed the highest binding affinities for D2 of all of the tested series of
compounds. However, o- or m- substituted compounds had considerably higher binding
affinities for 5-HT1AR than the corresponding p-substituted ligands, whereas m-substitution
proved to be the most favourable in terms of 5-HT2AR binding affinity. b) Replacement of the
benzene ring with 2-, 3- or 4-pyridine led to a significant decrease in binding affinity for the
D2, 5-HT1A and 5-HT2A receptors in the series. c) Owing to the presence of the 2-quinoline
moiety, compounds in this series are devoid of activity towards the D2 and 5-HT2A receptors,
but at the same time they display the highest affinity for 5-HT1AR of all of the tested
compounds.
The aim of our study was to modify the lead structure (compound A) in order to
enhance its affinity for the D2, 5-HT1A and 5-HT2A receptors. Selected ligands (6b, 6d, 6e, 6g,
6h, 6k, 6n, and 6o) were subjected to in vivo biological evaluation, as their affinity for the
considered receptors was comparable or better than those for quetiapine and clozapine. The
selection of these drugs as a reference was determined by the objective of our work.
The tested compounds neither induced hypothermia in mice nor attenuated the
hypothermia induced by the 5-HT1A agonist (±)8-OH-DPAT. None of the ligands induced
LLR or inhibited LLR produced by (±)8-OH-DPAT. Based on these results we cannot
classify the obtained compounds either as 5-HT1A agonists or as 5-HT1A antagonists. The
explanation for these negative results is not straightforward because many factors could
influence their behaviour in vivo, such as e.g. the moderate affinity of the studied compounds
for 5-HT1A receptors, insufficiency of the doses used, affinity of the studied compounds
toward other receptors involved in thermoregulation such as α1-adrenoceptors, different
classes of dopamine receptors and/or other biological mechanisms. The affinity of the tested
compounds for the 5-HT1A , 5-HT2A and D2 receptors has been determined, which at this stage
of the research restricts a detailed explanation of the lack of positive results. The tested
compounds inhibited the HTR, which allows us to classify them as 5-HT2A antagonists, with
6n and 6o being the most potent in this regard. Climbing behaviour was inhibited by
20

compounds 6b and 6k (within the first 30 min), 6n (within 40 min) and 6o (at 60 min). These
results allow us to classify them as D2 antagonists.
It can be concluded that compounds 6n and 6o are the leading compounds in the series
with an antipsychotic profile. Moreover it should be noted that compounds 10a and 10b
displayed the highest affinities for the 5-HT1A receptor. Therefore in the future, structural
investigations of adequately designed close analogues could help in the discovery of potent
new ligands for the 5-HT1A and D2 receptors among this group of derivatives.
4. Experimental protocols
4.1. General remarks
All solvents and raw materials were purchased from commercial sources. Column
chromatography was carried out using a Merck Silica gel 60A (63–200 µm) column as the
stationary phase and chloroform:methanol (9:1 v/v) as eluent. Melting points were determined
on an Electrothermal 9100 apparatus with open capillary tubes and were uncorrected. Yields
of all carried out reactions were calculated after all purification steps. IR spectra were
obtained using a Shimadzu FTIR-8300 spectrometer. NMR spectra were recorded on a Varian
Inova 500 (500 MHz) spectrometer. Chemical shifts (δ) were expressed in parts per million
(ppm) relative to tetramethylsilane used as the internal reference. The following abbreviations
are used to describe peak patterns when appropriate: s (singlet), bs (broad singlet), d
(doublet), dd (double doublet), t (triplet), td (triple doublet), ps (pseudo triplet), 4d (quartet of
doublets), m (multiplet), q (quartet), qu (quintet), * (conjugation with fluorine nucleus).
Coupling constants (J) are in hertz (Hz). ESI-HRMS spectra were obtained on an LCT TOF
(Micromass) instrument.
Purity (>95%) and homogeneity of the synthesized compounds was confirmed by TLC
1
and H NMR spectra. TLC was performed on Merck silica gel 60 F254 plates with a mobile
phase of toluene, ethanol, and 25% NH4OH (7.0:3.0:0.1, v/v). Spots were detected by their
absorption under UV light (k = 254 nm). Intermediate 2 (Scheme 1) was obtained following
29
the protocol in Ref. . Intermediate 3 (Scheme 1) was obtained following the protocol in Ref.
31
30. Intermediates 7, 8 and 9 (Scheme 1) were obtained according to the protocols in Ref.
.
1
The H NMR spectra of all considered final compounds are presented in Supporting
information.
21

R
9
6'
5"
8"
8
4"
4"a
8"a
1'
6"
7"
5'
3"
11
N
1
2
X 2'
7
NH
X
4
2"
Z
3
R3
5
X
4'
1"
3'
6
R1
O
H
R2
R1, R2, R3 = H, CH3, OCH3, Cl, Br, F, CF3; X = CH; R = H; Z = CH
R1, R2, R3 = H; X= N, CH; R = H, Z = CH
6a-r
6s-u
R1, R2, R3 = H; X = CH; R=H, OCH3; Z = N
10a-b
Figure 2. Numbering system for NMR spectra interpretation.
4.2. (8-Methyl-8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide (4)
A solution of naphthalene-2-carboxylic acid (18.9g, 0.11 mol), ethyl chloroformate
(10.5 mL, 0.11 mol) and triethylamine (16.5 mL, 0.12 mol) in anhydrous DMF (200 mL) was
stirred for 30 min at 0ºC. After this time a solution of amine 3 (16.5g, 0.12 mol) in anhydrous
DMF (40 mL) was added dropwise. The cooling bath was removed and stirring was continued
for 24 h. The solvent was evaporated in vacuo and the residue was dissolved in CH2Cl2 (150
mL). The solution was washed with a 5% aqueous solution of Na2CO3 (3 x 50 mL), then with
5% NaOH (2 x 50 mL) and once with 100 mL of water. The organic layer was dried with
magnesium sulphate, filtered, and concentrated in vacuo. The solid residue was purified by
crystallisation from ethyl acetate. Yield:14.1g (40.7%), m. p. 200.7–201.8ºC;
IR (KBr) cm-1: ν3247 (NH), 1632 (CO); ESI-HRMS m/z calcd for C19H22N2OH (M + H)+
295.1810, found: 295.1812.
4
1H NMR (500 MHz, CDCl3): d 8.22(C1’’H, J =1.0; m 7.87 (C4’’H, C5’’H, C8’’H); dd 7.77
(C3’’H) 3J= 8.5, 4J= 2.0; m 7.53 (C6’’H, C7’’H); d 6.06 (NH), 3J= 8.0; m 4.39 (C3H); pt 3.22
(C1H, C5H); s 2.31(C9H); m 2.08 (C2H(E),C4H(E)); m 1.98 (C6H(E),C7H(E)); m 1.77
(C6H(A),C7H(A)); td 1.66 (C2H(A),C4H(A)), 2J=3J1= 12.5, 3J2= 2.5.
4.3. N-(8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide hydrochloride(5)
To a solution of 10.3g (0.035 mol) (8-methyl-8-azabicyclo[3.2.1]oct-3β-yl)-2-
naphthamide (4) dissolved in a mixture of 150 mL DCM and 100 mL of 1,2-dichloroethane
chilled to 0ºC and maintained at this temperature, 5.25g (4.0 mL, 0.037 mol) 1-chloroethyl
chloroformate in 50 mL of 1,2-dichloroethane was added dropwise. The mixture was then
22

refluxed for 2.5 h. Solvents were removed in vacuo, 100 mL of methanol was added to the
residue and the mixture was refluxed for 90 min. After removing half of the methanol, the
product precipitated. Yield: 8.94g (80.7%), m.p. 160.2–163.4ºC; IR (KBr) cm-1: ν3389 (NH),
1653 (CO); ESI-HRMS m/z calcd for C18H20N2OH (M + H)+ 281.1654, found: 281.1658.
1H NMR (500 MHz, CDCl3): d 8.02 (C1”H); m 7.85 (C5”H,C8”H); d 7.80 (C3”H,3J=8.5); m
7.57 (C4”H,C6”H,C7”H); m 4.28 (C3H); pt 4.13 (C1H,C5H); m 2.16 (C2H(E),C4H(E)); m
2.06 (C6H(E),C7H(E)); m 2.01 (C6H(A),C7H(A)); td 1.85 (C2H(A),C4H(A)),2J=3J1=14.0,
3J2=2.5.
4.4. General procedure for synthesis of amides (6a-u)
N-(8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide hydrochloride (5) 0.48g (1.5 mmol),
1.7 mmol of an appropriate arylmethyl chloride or bromide, K2CO3 0.69g (5.0 mmol) or
(0.97g, 7.0 mmol in case of use 2-, 3- and 4-chloromethylpyridine hydrochloride) and 100mg
of KI were suspended in 30 mL of acetone. The reaction mixture was refluxed with stirring.
The reaction time was determined using TLC. Solvent was removed and the residue was
dissolved in a mixture of 30 mL of DCM and 15 mL of water. The organic layer was washed
with 10 mL of water, dried with MgSO4 and concentrated. Compounds 6a–o and 6s–u were
purified by crystallisation. Compounds 6p–r were purified by column chromatography and
subsequently by crystallisation.
4.4.1. N-[8-(2-methylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6a)
Crystallisation from acetone. Yield: 0.30g (53.6%), m.p. 180.0-181.0ºC; IR (KBr) cm-1:
ν3260 (NH), 1625 (CO); ESI-HRMS m/z calcd for C26H28N2OH (M + H)+ 385.2280, found:
385.2266.
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H); m 7.87 (C4’’H, C5’’H, C8’’H); dd 7.80
3
(C3’’H), J=8.5, 4J=1.5; m 7.53 (C6’’H, C7’’H); dd 7.36 (C3’H), 3J=7.0, 4J=2.5; m 7.16 (C4’H,
C5’H, C6’H); d 6.07 (NH), 3J=8.0; m 4.42 (C3H); s 3.50 (C9H); pt 3.27 (C1H, C5H); s 2,39
(C10H); m 2.10 (C2H(E), C4H(E)); m 1.97 (C6H(E), C7H(E)); m 1.80 (C6H(A), C7H(A)); td
1.65 (C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.2. N-[8-(3-methylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6b)
23

Crystallisation from ethyl acetate. Yield: 0.44g (78.6%), m.p. 189.5-191.0ºC; IR (KBr) cm-1:
ν3247 (NH), 1625 (CO); ESI-HRMS m/z calcd for C26H28N2OH (M + H)+ 385.2280, found:
385.2275.
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H); m 7.87 (C4’’H, C5’’H, C8’’H); dd 7.80
3
4
(C3’’H), J=8.5, J=2.0; m 7.53 (C6’’H, C7’’H); m 7.20 (C2’H, C4’H, C5’H); d 7.06 (C6’H,
3J=6.5; d 6.11 (NH), 3J=8.0; m 4.43 (C3H); s 3.53 (C9H); pt 3.28 (C1H, C5H); s 2.35 (C10H); m
2,09 (C2H(E), C4H(E)); m 1.97 (C6H(E), C7H(E)); m 1.80 (C6H(A), C7H(A)); td 1.69 (C2H(A),
C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.3. N-[8-(4-methylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6c)
Crystallisation from acetone. Yield: 0.22g (39.3%), m.p. 199.0-199.5ºC; IR (KBr) cm-1:
ν3257 (NH), 1623 (CO); ESI-HRMS m/z calcd for C26H28N2OH (M + H)+ 385.2280, found:
385.2294.
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H); m 7.87 (C4’’H, C5’’H, C8’’H); dd 7.79
3
4
3
(C3’’H), J=8.5, J=2.0; m 7.53 (C6’’H, C7’’H); d 7.27 (C3’H, C5’H), J=8.5; d 7.13 (C2’H,
3
3
C6’H), J=8.5; d 6.09 (NH), J=8.0; m 4.42 (C3H); s 3.52 (C9H); pt 3.27 (C1H, C5H); s 2.34
(C10H); m 2,07 (C2H(E), C4H(E)); m 1.95 (C6H(E), C7H(E)); m 1.79 (C6H(A), C7H(A)); td
1.66 (C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.4. N-[8-(2-methoxybenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6d)
Crystallisation from ethanol. Yield: 0.42g (61.8%), m.p. 224.1-225.2ºC; IR (KBr) cm-1:
ν3280 (NH), 1623 (CO); ESI-HRMS m/z calcd for C26H28N2O2Na (M + Na)+ 423.2052,
found: 423.2048.
4
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H), J=1.0; m 7.88 (C4’’H, C5’’H, C8’’H); dd 7.80
3
4
3
4
3
4
(C3’’H), J=8.5, J=1.5; td 7.22 (C4’H), J=8.0, J=1.5; td 6.97 (C5’H), J=7.5, J=1.0; dd 6.85
(C3’H), 3J=8.5, 4J=1.0; d 6.10 (NH), 3J=8.0; m 4.44 (C3’H); s 3,82 (C10H); s 3.58 (C9H); pt 3,31
(C1H, C5H); m 2.12 (C2H(E), C4H(E)); m 1.99 (C6H(E), C7H(E)); m 1.80 (C6H(A), C7H(A));
td 1.72 (C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.5. N-[8-(3-methoxybenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6e)
24

Crystallisation from acetone. Yield: 0.29g (42.6%), m.p. 181.2-182.1ºC; IR (KBr) cm-1:
ν3314 (NH), 1622 (CO); ESI-HRMS m/z calcd for C26H28N2O2H (M + H)+ 401.2229, found:
401.2248.
4
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H), J=1.0; m 7.88 (C4’’H, C5’’H, C8’’H); dd 7.80
3
4
3
4
(C3’’H), J=8.5, J=2.0; m 7.53 (C6’’H, C7’’H); t 7.23 (C5’H), J=7.5; t 7.00 (C2’H), J=2.0; d
6.96 (C6’H), 3J=7.5; d 6.79 (C4’H) , 3J=8.5, 4J1=2.5, 4J2=0.5; d 6.10 (NH), 3J=8.0; m 4.42 (C3H);
s 3.81 (C10H); s 3,55 (C9H); pt 3,27 (C1H, C5H); m 2.07 (C2H(E); C4H(E)); m 1.97 (C6H(E),
C7H(E)); m 1.79 (C2H(A), C4H(A)); td 1.68 (C2H(A), C4H(A)) 2J=3J1=12.0, 3J2=2.5.
4.4.6. N-[8-(4-methoxybenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6f)
Crystallisation from ethanol. Yield: 0.32g (47.1%), m.p. 181.3-182.4ºC; IR (KBr) cm-1:
ν3274 (NH), 1623 (CO); ESI-HRMS m/z calcd for C26H28N2O2H (M + H)+ 401.2229, found:
401.2232.
1H NMR (500 MHz, CDCl3): d 8.23 (C1’’H); m 7.87 (C4’’H, C5’’H, C8’’H); dd 7.79
3
4
3
(C3’’H), J=8.5, J=1.5; m 7.53 (C6’’H, C7’’H); d 7.29 (C2’H, C6’H), J=8.5; dt 6.86 (C3’H,
4
C5’H), 3J=9.0, J=2.5; d 6.09 (NH), 3J=8.0; m 4.42 (C3H); s 3.80 (C10H); s 3,49 (C9H); pt 3,26
(C1H, C5H); m 2.07 (C2H(E), C4H(E)); m 1.96 (C6H(E), C7H(E)); m 1.79 (C6H(A), C7H(A));
td 1.66 (C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.0.
4.4.7. N-[8-(2-chlorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6g)
Crystallisation from ethanol. Yield: 0.47g (70.2%), m.p. 207.5-210.0ºC; IR (KBr) cm-1:
ν3249 (NH), 1633 (CO); ESI-HRMS m/z calcd for C25H25ClN2OH (M + H)+ 405.1734,
found: 405.1732.
1H NMR (500 MHz, CDCl3): d 8.24 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.80 (C3”H),
4
3
4
3
3J=9.0, J=2.0; dd 7.65 (C3’H), J=8.0, J=1.5; m 7.53 (C6”H, C7”H); dd 7.33 (C6’H), J
3
4
3
4
=8.0, 4J =1.5; td 7.25 (C5’H), J=8.0, J=1.5; td 7.17 (C4’H), J=8.0, J=1.5; d 6.10 (NH),
3J=8.5; m 4.44 (C3H); s 3.65 (C9H); pt 3.29 (C1H, C5H); m 2.12 (C2H(E), C4H(E)); m 2.00
2
(C6H(E), C7H(E)); m 1.83 (C6H(A), C7H(A)); td 1.71 (C2H(A), C4H(A)), J=3J1=12.0,
3J2=2.5.
4.4.8. N-[8-(3-chlorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6h)
25

Crystallisation from ethanol. Yield: 0.35g (52.2%), m.p. 195.0-201.2ºC; IR (KBr) cm-1:
ν3241 (NH), 1635 (CO); ESI-HRMS m/z calcd for C25H25ClN2OH (M + H)+ 405.1734,
found: 405.1740.
1H NMR (500 MHz, CDCl3): d 8.24 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.81 (C3”H),
4
3J=8.5, J=2.5; m 7.54 (C6’’H, C7’’H); ps 7.44 (C2’H); m 7.23 (C4’H, C5’H, C6’H); d 6.10
3
(NH), J=8.5; m 4.42 (C3H); s 3.53 (C9H); pt 3.25 (C1H, C5H); m 2.07 (C2H(E), C4H(E));
m 1.98 (C6H(E), C7H(E)); td 1.68 (C2H(A), C4H(A)), 2J=3J1=12.5, 3J2=2.5.
4.4.9. N-[8-(4-chlorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6i)
Crystallisation from ethanol. Yield: 0.47g (70.2%), m.p. 189.4-191.9ºC; IR (KBr) cm-1:
ν3245 (NH), 1639 (CO); ESI-HRMS m/z calcd for C25H25ClN2OH (M + H)+ 405.1734,
found: 405.1737.
1H NMR (500 MHz, CDCl3): d 8.23 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.80 (C3”H),
4
3
4
3J=8.5, J=1.5); m 7.54 (C6”H, C7”H); dt 7.32 (C3’H, C5’H), J=9.0, J=2.0; dt 7.28 (C2’H,
C6’H), 3J=8.5, 4J=2.0; d 6,10 (NH), 3J=8.0; m 4.42 (C3H); s 3.51 (C9H); pt 3,23 (C1H, C5H);
m 2.06 (C2H(E), C4H(E)); m 1.97 (C6H(E), C7H(E)); m 1.80 (C6H(A), C7H(A)); td 1.66
(C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.10. N-[8-(2-bromobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6j)
Crystallisation from ethanol. Yield: 0.40g (54.1%), m.p. 216.5-219.5ºC; IR (KBr) cm-1:
ν3249 (NH), 1637 (CO); ESI-HRMS m/z calcd for C25H25BrN2OH (M + H)+ 449.1229,
found: 449.1216.
1H NMR (500 MHz, CDCl3): d 8.24 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.81 (C3”H),
3
3
3J=8.5, 4J=2.0; dd 7.64 (C3’H), J=8.0, 4J=2.0; dd 7.56 (C6’H), J=8.0,4J=1.5; m 7.53 (C6”H,
C7”H); td 7.30 (C4’H) ,3J=7.5, 4J=1.0; td 7.10 (C5’H), 3J=7.5, 4J=1.5; d 6.10 (NH), 3J=8.0; m
4.44 (C3H); s 3.62 (C9H); pt 3.29 (C1H, C5H); m 2.13 (C2H(E), C4H(E)); m 2.00 (C6H(E),
2
C7H(E)); m 1.83 (C6H(A), C7H(A)); td 1.71 (C2H(A), C4H(A)), J=3J1=12.0, 3J2=2.0.
4.4.11. N-[8-(3-bromobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6k)
26

Crystallisation from ethanol. Yield: 0.35g (47.3%), m.p. 203.7-204.5ºC; IR (KBr) cm-1:
ν3231 (NH), 1635 (CO); ESI-HRMS m/z calcd for C25H25BrN2OH (M + H)+ 449.1229,
found: 449.1246.
1H NMR (500 MHz, CDCl3): d 8.24 (C1”H); m 7.89 (C4”H, C5”H, C8”H); dd 7.81 (C3”H),
3
3J=8.5, 4J=1.5; t 7.60 (C2’H); m7.54 (C6”H, C7”H); 4d 7.37 (C4’H), J=8.0, 4J1=2.0, 4J2=1.0;
3
3
dt 7.29 (C6’H), J=7.5; t 7.18 (C5’H), J3=7.5; d 6.08 (NH), J=8.5; m 4.43 (C3H); s 3.53
(C9H); pt 3.25 (C1H, C5H); m 2.07 (C2H(E), C4H(E)); m 1.98 (C6H(E), C7H(E)); m 1.81
(C6H(A), C7H(A)); td 1.67 (C2H(A), C4H(A)), 2J=3J1=12.5, 3J2=2.5.
4.4.12. N-[8-(4-bromobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6l)
Crystallisation from ethanol. Yield: 0.35g (47.3%), m.p. 185.2-187.6ºC; IR (KBr) cm-1:
ν3297 (NH), 1634 (CO); ESI-HRMS m/z calcd for C25H25BrN2OH (M + H)+ 449.1229,
found: 449.1232.
1H NMR (500 MHz, CDCl3): d 8.23 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.80 (C3”H),
4
3
4
3J=8.5, J=1.5; m 7.54 (C6”H, C7”H); dt 7.43 (C3’H, C5’H); J=8.5, J=2.0; dt 7.26 (C2’H,
C6’H), 3J=9.5, 4J=2.0; d 6.10 (NH), 3J=8.0; m 4.42 (C3H); s 3.50 (C9H); pt 3.23 (C1H, C5H);
m 2.06 (C2H(E), C4H(E)); m 1.97 (C6H(E), C7H(E)); m 1.80 (C6H(A), C7H(A)); td 1.65
(C2H(A), C4H(A)), 2J=3J1=12.5, 3J2=2.5.
4.4.13. N-[8-(2-fluorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6m)
Crystallisation from ethanol. Yield: 0.33g (56.6%), m.p. 200.8-202.1ºC; IR (KBr) cm-1:
ν3253 (NH), 1636 (CO); ESI-HRMS m/z calcd for C25H25FN2ONa (M + Na)+ 411.1851,
found: 411.1849.
1H NMR (500 MHz, CDCl3): bs 8.23 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.79 (C3”H),
4
3
4
4
3J=8.5, J=2.0; m 7.54 (C6’H, C6’’H, C7’’H); m 7.22 (C4’H), J=7.5, JH-F=5.5, J=2.0; td
7.12 (C5’H), 3J=7.5, 4J=1.0; m 7.02 (C3’H), 3JH-F=10.0, 3J=8.0, 4J=1.0; d 6.08 (NH), 3J=8.5; m
4.44 (C3H); s 3.61 (C9H); pt 3.30 (C1H, C5H); m 2.12 (C2H(E), C4H(E)); m 1.98 (C6H(E),
2
C7H(E)); m 1.82 (C6H(A), C7H(A)); td 1.69 (C2H(A), C4H(A)), J=3J1=12.5, 3J2=2.0.
4.4.14. N-[8-(3-fluorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6n)
27

Crystallisation from ethanol. Yield: 0.33g (56.6%), m.p. 180.1-180.7ºC; IR (KBr) cm-1:
ν3247 (NH), 1637 (CO); ESI-HRMS m/z calcd for C25H25FN2OH (M + H)+ 389.2027, found:
389.2029.
1H NMR (500 MHz, CDCl3): bs 8.24 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.80 (C3”H),
4
3
3J=8.5, J=1.5; m 7.54 (C6’’H, C7’’H); m 7.26 (C5’H); d 7.18 (C2’H), JH-F=10.0; d 7.12
3
3
4
3
(C6’H), J=7.5; td 6.93 (C4’H), J=8.0, J=2.0; d 6.10 (NH), J=8.0; m 4.43 (C3H); s 3.55
(C9H); pt 3.25 (C1H, C5H); m 2.07 (C2H(E), C4H(E)); m 1.98 (C6H(E), C7H(E)); m 1.81
(C6H(A), C7H(A)); td 1.68 (C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.15. N-[8-(4-fluorobenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6o)
Crystallisation from ethanol. Yield: 0.35g (60.1%), m.p. 201.1-203.9ºC; IR (KBr) cm-1:
ν3245 (NH), 1636 (CO); ESI-HRMS m/z calcd for C25H25FN2ONa (M + Na)+ 411.1866,
found: 411.1849.
1H NMR (500 MHz, CDCl3): bs 8.23 (C1”H); m 7.88 (C4”H, C5”H, C8”H); dd 7.80 (C3”H),
4
3
4
4
3J=8.5, J=2.0; m 7.54 (C6’’H, C7’’H); m 7.34 (C2’H, C6’H), J=8.0, JH-F=5.5, J=2.0; m
3
4
3
7.00 (C3’H, C5’H), J=7.5, J=5J=2.0; d 6.09 (NH), J=8.0; m 4.42 (C3H); s 3.52 (C9H); pt
3.25 (C1H, C5H); m 2.07 (C2H(E), C4H(E)); m 1.97 (C6H(E), C7H(E)); m 1.80 (C6H(A),
C7H(A)); td 1.66 (C2H(A), C4H(A)), 2J=3J1=12.5, 3J2=2.5.
4.4.16. N-[8-(2-trifluoromethylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6p)
Purified by column chromatography and crystallisation from ethanol. Yield: 0.40g (53.3%),
m.p. 202.0-205.1ºC; IR (KBr) cm-1: ν3275 (NH), 1636 (CO); ESI-HRMS m/z calcd for
C26H25F3N2ONa (M + Na)+ 461.1809, found: 461.1817.
3
1H NMR (500 MHz, CDCl3): d 8.25 (C1”H); d 7.99 (C3’H), J=7.5; m 7.89 (C4”H, C5’’H,
3
4
3
C8’’H); dd 7.81 (C3’’H), J=8.5, J=1.5; d 7.62 (C6’H), J=8.0; m 7.53 (C4’H, C6’’H,
3
3
C7’’H); t 7.32 (C5’H), J=7.5; d 6.08 (NH), J=8.0; m 4.44 (C3H); s 3.73 (C9H); pt 3.26
(C1H, C5H); m 2.10 (C2H(E), C4H(E)); m 2.00 (C6H(E), C7H(E)); m 1.83 (C6H(A),
C7H(A)); td 1.70 (C2H(A), C4H(A)), 2J=3J1=11.5, 3J2=2.5.
4.4.17. N-[8-(3-trifluoromethylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6q)
28

Purified by column chromatography and crystallisation from ethanol. Yield: 0.49g (65.3%),
m.p. 167.0-167.6ºC; IR (KBr) cm-1: ν3257 (NH), 1636 (CO); ESI-HRMS m/z calcd for
C26H25F3N2OH (M + H)+ 439.1997, found: 439.1999.
1H NMR (500 MHz, CDCl3): bs 8.25 (C1”H); m 7.89 (C4’’H, C5’’H, C8’’H); dd 7.81
3
(C3’’H), J=8.5, 4J=1.5; bs 7.71 (C2’H); m 7.48 – 7.58 (C4’H, C6’H, C6’’H, C7’’H); t 7.42
3
3
(C5’H), J=8.0; d 6.10 (NH), J=8.5; m 4.43 (C3H); s 3.61 (C9H); pt 3.25 (C1H, C5H); m
2.08 (C2H(E), C4H(E)); m 1.99 (C6H(E), C7H(E)); m 1.83 (C6H(A), C7H(A)); td 1.68
(C2H(A), C4H(A)), 2J=3J1=12.0, 3J2=2.5.
4.4.18. N-[8-(4-trifluoromethylbenzyl)-8-azabicyclo[3.2.1]oct-3β-yl)]-2-naphthamide (6r)
Purified by column chromatography and crystallisation from ethanol. Yield: 0.46g (61.3%),
m.p. 166.4-166.8ºC; IR (KBr) cm-1: ν3251 (NH), 1635 (CO); ESI-HRMS m/z calcd for
C26H25F3N2OH (M + H)+ 439.1997, found: 439.1995.
1H NMR (500 MHz, CDCl3): d 8.24 (C1”H); m 7.88 (C4’’H, C5’’H, C8’’H); dd 7.80
3
4
(C3’’H), J=8.5, J=1.5; m 7.54 (C2’H, C3’H, C5’H, C6’H, C6’’H, C7’’H); d 6.08 (NH),
3J=8.0; m 4.43 (C3H); s 3.61 (C9H); pt 3.25 (C1H, C5H); m 2.08 (C2H(E), C4H(E)); m 1.99
2
(C6H(E), C7H(E)); m 1.82 (C6H(A), C7H(A)); td 1.68 (C2H(A), C4H(A)), J=3J1=12.0,
3J2=2.0.
4.4.19. N-(8-pyridin-2-ylmethyl-8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide (6s)
Purified by crystallisation from acetone. Yield: 0.24g (42.6%), m.p. 207.8-209.7ºC; IR (KBr)
cm-1: ν3253 (NH), 1638 (CO); ESI-HRMS m/z calcd for C24H25N3OH (M + H)+ 372.2076,
found: 372.2088.
3
4
1H NMR (500 MHz, CDCl3): dt 8.53 (C6H), J=5.0, J=5J=1.0; d 8.23 (C1’’H); m 7.88
(C4’’H, C5’’H, C8’’H); dd 7.80 (C3’’H), 3J=8.5, 4J=2.0; td 7.66 (C4’H), 3J=7.5, 4J=2.0;
3
4
3
m 7.54 (C3’H, C6’’H, C7’’H); td 7.15 (C5’H), J=6.5, J=1.0; d 6.14 (NH), J=8.5; m 4.45
(C3H); s 3.75 (C9H); pt 3.30 (C1H, C5H); m 2.12 (C2H(E), C4H(E)); m 1.97 (C6H(E),
2
3
C7H(E)); m 1.82 (C6H(A), C7H(A)); td 1.74 (C2H(A), C4H(A)), J=3J1=12.5, J2=2.5.
4.4.20. N-(8-pyridin-3-ylmethyl-8-azabicyclo[3.2.1]oct-3β-yl)-2-naphthamide (6t)
29