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Article
3-Aminomethyl derivatives of 2-phenylimidazo[1,2-a]-pyridine as positive
allosteric modulators of GABA receptor with potential antipsychotic activity
A

Monika Marcinkowska, Marcin Kolaczkowski, Krzysztof Kami#ski, Adam Bucki, Maciej Henryk
Pawlowski, Agata Siwek, Tadeusz Karcz, Gabriela Starowicz, Karolina Sloczynska, El#bieta P#kala,
Anna Weso#owska, Jerzy Samochowiec, Pawe# Mierzejewski, and Przemyslaw Bienkowski
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00432 • Publication Date (Web): 17 Feb 2017
Downloaded from http://pubs.acs.org on February 18, 2017

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3 3-Aminomethyl derivatives of 2-phenylimidazo[1,2a]-
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6 pyridine as positive allosteric modulators of GABAA
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8 receptor with potential antipsychotic activity
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14 Monika Marcinkowska‡*, Marcin Kołaczkowski‡, Krzysztof Kamiński‡, Adam
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16 Bucki‡, Maciej Pawłowski‡, Agata Siwek‡, Tadeusz Karcz‡, Gabriela Starowicz‡,
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18 Karolina Słoczyńska‡, Elżbieta Pękala‡, Anna Wesołowska‡, Jerzy Samochowiec#,
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20 Paweł Mierzejewski║, Przemyslaw Bienkowski&
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31 Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna St.,
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30-688 Krakow, Poland
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34 Department of Psychiatry, Pomeranian Medical University, 1 Rybacka St., 70-204
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36 Szczecin, Poland
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Department of Pharmacology, Institute of Psychiatry and Neurology, 9 Sobieskiego
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39 St., 02-957 Warsaw, Poland
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41 Department of Psychiatry, Medical University of Warsaw ul. Nowowiejska 27,
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00-665 Warsaw, Poland
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5 Abstract
6 Schizophrenia is a mental illness characterized by behavioral changes as well as
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8 anatomical and neurochemical abnormalities. There has been a remarkable progress in
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10 the drug discovery for schizophrenia; however, antipsychotics that act through
11 molecular targets, other than monoaminergic receptors, have not been developed. One
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13 of the hypotheses of schizophrenia states that GABAergic dysfunction might be
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15 implemented in the pathophysiology of this disease. Our recent findings and previous
16 clinical observations have suggested that modulation of GABAergic system through
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18 α1-GABAA receptors would represent an original approach for the treatment of
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20 schizophrenia. This study presents the synthesis and biological evaluation of a series
21 of fluorinated 3-aminomethyl derivatives of 2-phenylimidazo[1,2-a]-pyridine as
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23 potential antipsychotic agents. Compound 7 has a high affinity for GABAA receptor
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25 (Ki = 27.2 nM), high in vitro metabolic stability, and antipsychotic-like activity in
26 amphetamine-induced hyperlocomotion test in rats (MED = 10 mg/kg). Compound 7
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28 represents a promising point of entry in the course of development of antipsychotic
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30 agents with a non-dopaminergic mechanism of action.
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35 Graphical abstract
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3 KEYWORDS: schizophrenia, psychosis, positive allosteric modulators of GABAA
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5 receptor, γ-aminobutyric acid (GABA), fluorinated imidazo[1,2-a]pyridines,
6 GABAergic transmission, antipsychotic-like activity, amphetamine-induced
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8 hyperlocomotion, zolpidem
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13 1. Introduction
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15 Schizophrenia is a mental illness that affects ~1% of the worldwide population
16 and is characterized by behavioral changes as well as anatomical and neurochemical
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18 abnormalities.1 The neuropathology of schizophrenia is associated with the
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20 imbalances of various neurotransmitter systems such as dopamine, serotonin,
21 glutamate, and γ-aminobutyric acid (GABA). The first- and the second-generation
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23 antipsychotics that are commonly used in the treatment of schizophrenia primarily
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25 target the subcortical dopamine D2 receptors.2,3 Such mechanism of action reduces the
26 psychotic symptoms in the majority of patients; however ~30%–60% of the patients
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28 remain drug-resistant.4,5
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30 Furthermore, considerable challenge arises when patients refuse to take their
31 medications because of troublesome and unpleasant adverse reactions such as
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33 extrapyramidal side effects, weight gain (metabolic syndrome), nausea,
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35 hyperprolactinemia, agranulocytosis, sexual dysfunction, or increased cardiac risk.6
36 Thus, ~50% of the patients withdraw from the pharmacological treatment. Hence,
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38 there is a growing interest in the development of original antipsychotic agents
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40 involving different mechanisms of action, which do not interact with the
41 dopaminergic receptors.7
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43 In 1972, Eugene Roberts first suggested that the GABA-ergic dysfunction
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45 might be implemented in the pathophysiology of schizophrenia.8 Later, the
46 postmortem studies showed lower brain levels of GABA neurotransmitter in
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48 schizophrenia patients compared to healthy subjects.9 Alternative studies showed an
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50 increased number of GABAA receptors and a correlation between changes in densities
51 of various GABAA receptor subunits and psychical disorders, such as schizophrenia
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53 or bipolar affective disorder.10,11 Moreover, positron emission tomography (PET)
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55 studies using [18F]-fluoroflumazenil revealed reduced binding to GABAA receptor in
56 individuals at ultra-high risk for psychosis in comparison with low-risk subjects.12 In
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58 accordance with these findings, a recent study has shown a lower level of messenger
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3 RNA of the α1 subunit of GABAA receptor in the prefrontal cortices of schizophrenia
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5 patients.13 These findings have suggested that modulation of GABA-ergic system
6 through α1-GABAA receptors would represent a promising approach for the
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8 management of schizophrenia.
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10 Recently, we have found that zolpidem, an α1 selective GABAA positive
11 allosteric modulator displays an antipsychotic-like effect in rats, comparable to the
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13 effect induced by risperidone, a second-generation antipsychotic.14,15 These results are
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15 in line with other clinical observations that have suggested that zolpidem might exert
16 an antipsychotic-like effect.16,17 The aforementioned findings prompted us to further
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18 explore α1-GABAA positive allosteric modulators as candidates for innovative
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20 antipsychotic agents. Previously we have identified a series of fluorinated 2-
21 phenylimidazo[1, 2-a]-pyridines, zolpidem analogs which are positive allosteric
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23 modulators of GABAA receptor.18 The most active compound effectively reduced
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25 hyperlocomotion induced by amphetamine in low dosage (1 mg/kg), confirming the
26 hypothesis that selective positive allosteric modulators of GABAA receptor possess
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28 specific antipsychotic activity, and this seems to be an effective strategy in the design
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30 and development of novel antipsychotics with non-dopaminergic mechanism of
31 action.
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33 In order to provide an enhanced understanding of the structure–activity
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35 relationships (SAR), in this study we designed a series of fluorinated 2-
36 phenylimidazo[1, 2-a]-pyridines, replacing 3-(N,N-dimethylacetamide) moiety with
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38 variously acylated 3-aminomethyl derivatives (Figure 1). We aimed to investigate the
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40 role of “inverse amide moieties” on antipsychotic activity and consequently on
41 pharmacological profile of such molecules. The arrangement of fluorinated
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43 substituents around 2-phenylimidazo[1, 2-a]-pyridine ring was selected on the basis of
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45 the results from our previous studies, which revealed that incorporation of fluorine
46 groups into positions 6, 3′, and 4′ of the 2-phenylimidazo[1, 2-a]-pyridine core
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48 provides analogs with high affinity for the GABAA receptor and increases metabolic
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50 stability of such derivatives.
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Figure 1. Structure of the previously reported fluorinated 2-phenylimidazo[1, 2-a]-pyridines
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and general structure of the novel series.
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23 Herein, we present the synthesis and biological evaluation of a series of
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25 fluorinated 3-aminomethyl derivatives of 2-phenylimidazo[1, 2-a]-pyridine as
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potential antipsychotic agents. All the compounds were tested for affinity for GABAA
28 receptor; the most active compounds were further investigated in the
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30 electrophysiological studies to determine their functional effects, and their metabolic
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stability was examined in in vitro models. Finally, the most promising compound was
33 evaluated in the animal model of psychosis to determine its antipsychotic activity.
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37 2. Results and discussion
38 2.1 Chemistry
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40 The target compounds 6–25 were prepared in a two-step synthesis as
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presented in Scheme 1. In the first step, cyclization between corresponding
43 bromoacetophenones and aminopyridines resulted in 2-phenylimidazo[1,2-a]-
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45 pyridines 1–5.18 Next, the amides 6–25 were obtained in a one-pot Mannich-type
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47 reaction between suitable nitrile and paraformaldehyde in glacial acetic acid along
48 with concentrated sulfuric acid, followed by the subsequent addition of appropriate
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50 imidazo[1,2-a]-pyridines 1–5. The reaction mixture was heated at 75°C for 12 h and
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52 the final compounds were delivered with good yields (70%–98%).
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Scheme 1. Synthesis of the final compounds 6–25. Reagents and conditions: (a) NaHCO3,
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toluene, reflux, 12 h; (b) formaldehyde, 98% H2SO4, glacial CH3COOH, 65°C, 30 min and
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then 75°C, 12 h.
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2.2 Affinity for the GABAA receptor benzodiazepine site
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28 All the final molecules 6–25 were submitted to radioligand binding assay to
29 determine their affinity for the GABAA receptor benzodiazepine site, by measuring
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31 the competitive displacement of [3H]-flunitrazepam, using rat brain tissue. The
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33 majority of the molecules displayed a relatively marked affinity (Ki < 100 nM) for the
34 GABAA receptor. Four compounds, namely 7, 17, 19, and 21, showed a higher
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36 affinity (Ki < 30 nM) in comparison with the reference drug zolpidem. The optimal
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38 imidazo[1,2-a]-pyridine cores were found to be 2-(4-fluorophenyl)-6-
39 fluoroimidazo[1,2-a]pyridine (17) and 2-(3,4-difluorophenyl)-6-fluoroimidazo[1,2-
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41 a]pyridine (21), which gave the most potent analogs of all the series (Ki = 21 ± 2.7
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43 nM and 23 ± 4.0 nM, respectively). Compounds bearing isopropylamide function
44 tended to have a 2–10 times decreased affinity for GABAA receptor, regardless of the
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46 substituents at the imidazo[1,2-a]-pyridine core. The incorporation of a more rigid
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48 substituent cyclopropylamide yielded compounds with acceptable affinities (Ki = 20–
49 136 nM). Further SAR analysis revealed that the introduction of a propanamide
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51 moiety in all combinations of imidazo[1,2-a]-pyridine scaffolds was well tolerated,
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53 and relatively high affinities were observed. For instance, the Ki value of 2-(4-
54 fluorophenyl)-6-methylimidazo[1,2-a]pyridine analog (7) was 27.2 ± 5.9 nM and that
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56 of 2-(3,4-difluorophenyl)-6-fluoroimidazo[1,2-a]pyridine derivative (19) was 24.0 ±
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3 2.0 nM (Table 1). The exchange of propanamide group with a smaller methylamide
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5 function resulted in a small decrease of affinity for GABAA receptor.
6 For further evaluations, compounds 7, 17, 19, and 21 with the Ki values lower
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8 than that for the reference drug zolpidem were selected.
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11 Table 1. GABAA receptor benzodiazepine site affinity data for 3-aminomethyl derivatives of
12 2-phenylimidazo[1,2-a]-pyridine (compounds 6–25).
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23 Compound R1 R2 R3 R4 Ki (nM) ± SEMa
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26 6 CH3 H F CH3 78.4 ± 8.1
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29 7 CH3 H F CH2CH3 27.2 ± 5.9
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32 8 CH3 H F -CH(CH3)2 364.0 ± 14.0
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35 9 CH3 H F -CH(CH2)2 116.0 ± 15.5
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38 10 F H CH3 CH3 44.0 ± 1.2
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11 F H CH3 CH2CH3 36.5 ± 7.0
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12 F H CH3 -CH(CH3)2 87.5 ± 10.0
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13 F H CH3 -CH(CH2)2 55.1 ± 1.0
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51 14 F H F CH3 43.0 ± 8.6
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54 15 F H F CH2CH3 37.0 ± 2.5
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4 17 F H F -CH(CH2)2 20.9 ± 2.7
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7 18 F F F CH3 39.0 ± 3.5
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10 19 F F F CH2CH3 24.0 ± 2.0
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13 20 F F F -CH(CH3)2 72.0 ± 4.4
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16 21 F F F -CH(CH2)2 22.8 ± 4.0
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19 22 F H CF3 CH3 62.7 ± 7.7
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22 23 F H CF3 CH2CH3 48.0 ± 6.1
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24 F H CF3 -CH(CH3)2 86.3 ± 1.5
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25 F H CF3 -CH(CH2)2 67.0 ± 4.7
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Zolpidem 37.8 ± 2.7
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34 Data are expressed as the mean ± SEM of three independent experiments performed in
35 duplicate.
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2.3 Electrophysiological studies
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41 The electrophysiological studies aimed to determine whether the compounds
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44 and thus exert positive allosteric modulation properties. For that purpose, activities of
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46 compounds 7, 17, 19, and 21 at human α1β2γ2-GABAA receptors stably expressed in a
47 HEK293 cell line were evaluated using the automated patch clamp platform
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49 QPatch16X (Sophion Bioscience) as described in the “Methods” section.
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51 The results of the conducted studies showed that the tested compounds (7, 17,
52 19, 21) display weak positive allosteric modulator activity, given the ability to
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54 potentiate the effect of natural agonist GABA. Based on the obtained results, two
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56 compounds 7 and 17, stimulating the highest increase of GABA-evoked current
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5 Table 2. Relative positive allosteric modulator activity of compounds at GABAA receptor,
6 presented as a fold increase of GABA-gated current amplitude compared to the effect of 10
7 µM GABA alone. Data represent mean ± SEM of three experiments performed on distinct
8 cells.
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11 Fold increase of
Compound
12 GABA-gated currents ± SEM
13 Zolpidem 2.56 ± 0.22
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7 1.32 ± 0.10
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16 17 1.32 ± 0.09
17 19 1.02 ± 0.03
18 21 1.17 ± 0.07
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21 2.4 Metabolic stability
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Determination of metabolic stability of compounds at the early stages of drug
25 discovery is important because it helps to increase the rate of success of drug
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27 development.19,20 Recently, in vitro methods using human and animal microsomes
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gained enormous attention since they are time and cost-effective. Therefore,
30 metabolic stability of compounds 7 and 17 was determined in vitro using human and
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32 rat liver microsomes (HLMs and RLMs, respectively).
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Both compounds 7 and 17 exhibited moderate to high in vitro metabolic
35 stability (from 71% to 97%) in different microsomal models of biotransformation
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37 (Table 3). As the test compounds demonstrated high stability in HLMs, their half
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times and intrinsic clearance values were not assessed. In RLMs, the Clint value of
40 compound 7 was in the lower limit of the medium category range (17 µl/mg/min),
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42 whereas the Clint value for compound 17 was within the middle of the medium
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category range (38.5 µl/mg/min) (Table 3). In order to confirm the structures of
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47 chemical route of biotransformation of compounds 17 and 7 was hydroxylation; in
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compound 17, it occurred in the pyridine ring, whereas in compound 7, in the methyl
50 substituent in the pyridine ring (see the supplementary information). Figure 2 depicts
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52 the exemplary HPLC chromatograms of compounds 17 and 7 and their main
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metabolites.
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5 Table 3. Metabolic stability screen of compounds 7 and 17 in human and rat liver microsomes
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% remaining t1/2 (min) Clint
Test compound

Retention time
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Molecular ion
9 at 15 min (µl/mg/min)
(m/z)

(min)
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HLMs

HLMs

HLMs
RLMs

RLMs

RLMs
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15 7 312 3.27 97 93 - 102 - 17.0
16 17 328 3.67 93 71 - 45 - 38.5
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A B
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39 Figure 2. HPLC chromatograms (retention times) of test compounds 17 (A) and 7 (B), their
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Based on these results, compound 7 was selected for further evaluation.
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53 2.5 Antipsychotic-like activity in rats
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the effect of the compound on amphetamine-induced hyperlocomotion was assessed
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3 in rats. Hyperlocomotion induced by dopaminergic substances, including
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5 amphetamine, is mediated through postsynaptic mesolimbic dopamine receptors. In
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8 elicited by amphetamine, reflects the antipsychotic-like activity of new drug
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10 candidates and that the specific effects of potential antipsychotics on the mesolimbic
11 dopamine pathway are essential for successful drug development.21,22 Moreover,
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13 administration of amphetamine can induce GABA deficits in the rat limbic system.23
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15 The latter observation further supports the notion that the model in which psychotic-
16 like effects are induced by amphetamine may be a good tool to examine the
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18 antipsychotic-like activity of GABA-ergic compounds.15
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20 Compound 7 dose-dependently reversed hyperlocomotion induced by
21 amphetamine with MED = 10 mg/kg, p.o. (Figure 3). Notably, we did not observe any
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23 myorelaxant or cataleptogenic effects or any other adverse changes in gross animal
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25 behavior at the same dose. The mean distance traveled by the groups treated with
26 compound 7–amphetamine combination did not fall below the mean locomotor
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28 activity observed in drug-free control rats (see Figure 4 for comparison). In another
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30 control experiment, no decrease in spontaneous locomotor activity was found up to
31 the dose of 10 mg/kg (Figure 4), which supports the specificity of the observed
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33 antipsychotic-like effects of compound 7. Importantly, many currently used
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35 antipsychotic drugs (e.g., haloperidol, olanzapine, and lurasidone) suppress locomotor
36 activity at the antipsychotic-like doses.24 In the present study, compound 7 was tested
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38 under the experimental conditions closely similar to those used in our previous
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40 behavioral experiments with zolpidem (Mierzejewski et al., 2016). Hence, one may
41 wish to compare the antipsychotic-like activity of 7 and its parent compound -
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43 zolpidem. Both molecules (see Fig. 4, the present study, and Mierzejewskiet al., 2016,
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45 Figure 1B)15 significantly reduced amphetamine-induced hyperlocomotion at the
46 dose, which did not alter spontaneous locomotor activity (10.0 mg/kg, p.o., and 0.3
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48 mg/kg, i.p., for 7 and zolpidem, respectively). The effect size calculated for
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50 compound 7 (1.14) was comparable to that calculated for zolpidem (1.33). Thus,
51 although compound 7 was administered p.o. and zolpidem was injected i.p., the effect
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23 hyperlocomotion in rats. The dose of 0 mg/kg refers to rats treated with vehicle and
24 amphetamine (1.0 mg/kg, i.p.). The dotted line reflects the mean spontaneous locomotor
25 activity observed in drug-free control subjects (i.e., rats administered only with vehicle; see
26 Figure 6 for comparison); *p < 0.05.
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3 3. Conclusion
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5 In summary, we have designed and synthesized a series of 3-aminomethyl derivatives
6 of 2-phenylimidazo[1,2-a]-pyridine. The compounds display a high affinity for
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8 GABAA receptor benzodiazepine site and positive allosteric modulator activity. They
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10 also possess moderate to high in vitro metabolic stability in two different microsomal
11 models of biotransformation. The most promising compound 7 has the antipsychotic-
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13 like activity, reversing hyperlocomotion induced by amphetamine, without inducing
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15 significant sedation. The study has also demonstrated that compounds acting as
16 positive allosteric modulators of GABAA receptor might represent a promising
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18 strategy in the development of original antipsychotic agents with a non-dopaminergic
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20 mechanism of action.
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28 4. Methods
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30 4.1 General chemistry methods
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Unless otherwise indicated, all the starting materials were obtained from commercial
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34 suppliers and were used without further purification. All the reactions with air- and
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36 moisture-sensitive components were performed under a nitrogen atmosphere.
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Analytical thin-layer chromatography (TLC) was performed on Merck Kieselgel 60
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39 F254 (0.25 mm) pre-coated aluminum sheets (Merck, Darmstadt, Germany).
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41 Visualization was performed with a 254 nm UV lamp. Column chromatography was
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performed using silica gel (particle size 0.063–0.200mm; 70–230 Mesh ATM)
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44 purchased from Merck. The UPLC-MS or UPLC-MS/MS analyzes were run on
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46 UPLC-MS/MS system comprising Waters ACQUITY® UPLC® (Waters Corporation,
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Milford, MA, USA) coupled with Waters TQD mass spectrometer (electrospray
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49 ionization mode ESI with tandem quadrupole). Chromatographic separations were
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51 carried out using the ACQUITY UPLC BEH (bridged ethyl hybrid) C18 column: 2.1 ×
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100 mm and 1.7 µm particle size. The column was maintained at 40°C and eluted
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54 under gradient conditions using 95% to 0% of eluent A over 10 min, at a flow rate of
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56 0.3 ml/min. Eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic
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acid (0.1%, v/v). A total of 10 µl of each sample were injected, and chromatograms
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3 were recorded using Waters eλ PDA detector. The spectra were analyzed in the range
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5 of 200–700 nm with 1.2 nm resolution and at a sampling rate of 20 points/s. MS
6 detection settings of Waters TQD mass spectrometer were as follows: source
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8 temperature 150°C, desolvation temperature 350°C, desolvation gas flow rate 600 l/h,
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10 cone gas flow 100 l/h, capillary potential 3.00 kV, and cone potential 20 V. Nitrogen
11 was used for both nebulizing and drying. The data were obtained in a scan mode
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13 ranging from 50 to 1000 m/z at 0.5 s intervals; 8 scans were summed up to obtain the
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15 final spectrum. Collision activated dissociation (CAD) analyzes were carried out with
16 the energy of 20 eV, and all the fragmentations were observed in the source.
17
18 Consequently, the ion spectra were obtained in the range from 50 to 500 m/z.
19
20 MassLynx V 4.1 software (Waters) was used for data acquisition. Standard solutions
21 (1 mg/ml) of each compound were prepared in a mixture comprising analytical grade
22
23 acetonitrile/water (1/1, v/v). The UPLC/MS purity of all the test compounds and key
24
25 intermediates was determined to be >95%. 1H NMR, 13C NMR, and 19F NMR spectra
26 were obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA),
27
28 in CDCl3 or DMSO operating at 300 MHz (1H NMR), 75 MHz (13C NMR), and 282
29
30 MHz (19F NMR). Chemical shifts are reported in terms of δ values (ppm) relative to
31 TMS δ = 0 (1H) as internal standard. The J values are expressed in Hertz. Signal
32
33 multiplicities are represented by the following abbreviations: s (singlet), br.s (broad
34
35 singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet).
36 Melting points were determined with a Büchi apparatus and are uncorrected.
37
38
39
40
41 4.2 General procedure for the synthesis of 2-(4-fluorophenyl)-6-
42
43
methylimidazo[1,2-a]pyridine (compound 1)
44 A mixture of 6-methylpyridin-2-amine (10 mmol, 1.08 g), 2-bromoacetophenone (10
45
46 mmol, 2.17 g), and sodium bicarbonate (15 mmol, 1.26 g) in anhydrous toluene (25
47
ml) was stirred under reflux for 12 h. After the removal of toluene under vacuum,
48
49 dichloromethane was added to the residue, and any insoluble material was removed
50
51 by filtration. The crude products were crystallized from methanol or purified by silica
52
gel chromatography using dichloromethane/methanol, 9:0.5 (v/v) as eluent.
53
54 Compound 1: 2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridine
55
56 Yellow solid; yield: 78%; UPLC RT = 3.23; 1H NMR (300 MHz, CDCl3): δ 2.10 (s,
57
3H), 7.02–7.16 (m, 3H), 7.56 (d, 1H), 7.71 (s, 1H), 7.85–7.93 (m, 3H); 19F NMR (282
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3 MHz, CDCl3): δ −142.00 (s, 1F); LC-MS (ESI) calcd for C14H11FN2 227.09 (M+H),
4
5 found 229.2 (M+H)..
6
7 4.3 General procedure for the synthesis of N-((2-(4-fluorophenyl)-6-
8
9
methylimidazo[1,2-a]pyridin-3-yl)methyl)propanamide (compound 7)
10 The solution of propionitrile (1.87 mmol, 0.133 ml), paraformaldehyde (1.87 mmol,
11
12 0.05 g) in 98% H2SO4 (0.05 ml), and glacial CH3COOH (2 ml) was heated at 65°C
13
14
until dissolution of formaldehyde. Next, the solution of 2-(4-fluorophenyl)-6-
15 methylimidazo[1,2-a]pyridine (compound 1) (0.62 mmol, 0.14 g) in glacial
16
17 CH3COOH (4 ml) was added, and the reaction mixture was stirred at 75°C for 12 h.
18
19
Later, the reaction mixture was cooled to room temperature, and 10% solution of
20 KOH was added until pH ~7 was obtained; then, the water layer was extracted 3 times
21
22 with chloroform (3 × 15 ml). The combined organic layer was dried over sodium
23
24
sulfate and concentrated in vacuum, and the crude mixture was purified over column
25 chromatography using chloroform: methanol (98:2) as eluent.
26
27
28
29
30 Compound 7: N-((2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridin-3-
31
32 yl)methyl)propanamide
33
34
Yield 62%, white solid, Mp 220−221 °C, 1H NMR (300 MHz, CD3OD): δ 1.10–1.16
35 (t, 3H, J = 7.7 Hz), 2.18–2.27 (q, 2H, J = 7.69 Hz), 2.35 (d, 3H, J = 1.00 Hz), 4.78 (s,
36
37 2H), 7.15–7.25 (m, 3H), 7.42–7.47 (dd, 1H, J = 0.51 Hz, J = 0.77 Hz), 7.69–7.77 (m,
38
39
2H), 8.14 (d, 1H, J = 1.28 Hz); 19F NMR (282 MHz, CD3OD): δ −116.00–115.88 (m,
40 1F); 13C NMR (75 MHz, CD3OD) δ 9.1, 16.8, 28.6, 32.0, 114.9, 115.2, 116.8, 122.3,
41
42 122.9, 128.8, 129.8, 130.1, 130.2, 142.5, 143.7, 161.1, 164.3, 175.9; Formula
43
44
C18H18FN3O; ESI-MS: 312 [M+H]+
45
46
47
48
49
4.4 Radioligand binding assay for GABAA/BZP using [3H]-flunitrazepam
50 Rat brains were homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH
51
52 7.4) using a ULTRA TURAX homogenizer. The homogenate is then centrifuged at
53
54
20,000 × g for 20 min (0–4°C). The resulting supernatant was discarded, and the
55 pellet was rehomogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4).
56
57 The resulting homogenate was centrifuged as done previously. The pellet was
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3 resuspended and centrifuged further three times. The final pellet was stored at −80°C
4
5 for minimum 18 h. On the day of the assay, the pellet was thawed at room
6 temperature, resuspended in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4)
7
8 and centrifuged at 20,000 × g for 25 min (0–4°C). [3H]Flunitrazepam (spec. act. 80
9
10 Ci/mmol, ARC) was used for labeling GABAA/BZP. Then, 150 µl of the tissue
11 suspension, 50 µl of 10 µM diazepam (displacer), 50 µl of 1 nM [3H]Flunitrazepam,
12
13 and 50 µl of the analyzed compound were incubated at 4°C for 20 min. The
14
15 concentrations of the analyzed compounds ranged from 1.0E−10 M to 1.0E−5 M.
16 The incubation was terminated by rapid filtration over glass fiber filters
17
18 FilterMate B (PerkinElmer, USA) using 96-well FilterMate harvester (PerkinElmer).
19
20 Five rapid washes were performed with ice-cold 50 mM Tris-HCl buffer, pH 7.4.
21 The filter mate was dried in microwave, placed in plastic bag (PerkinElmer),
22
23 and soaked with 10 ml of liquid scintillation cocktail Ultima Gold MV (PerkinElmer),
24
25 and the filter bag was sealed. The radioactivity of the filter was measured in
26 MicroBeta TriLux 1450 scintillation counter (PerkinElmer).
27
28 Radioligand binding data were analyzed using iterative curve fitting routines
29
30 of GraphPad Prism 5.0 software (GraphPad, Inc., La Jolla, CA, USA) using the three
31 built-in parameter logistic model describing ligand competition binding to
32
33 radioligand-labeled sites. The log IC50 (i.e., the log of the ligand concentration that
34
35 reduces a specific radioligand binding by 50%) estimated from the data is used to
36 obtain the Ki values by applying the Cheng–Prusoff approximation.
37
38
39
40 4.5 Electrophysiological studies
41 The electrophysiological experiments were carried out on a QPatch16X automatic
42
43 patch clamp platform (Sophion Bioscience). HEK293 cells, stably expressing the
44
45 α1β2γ2 subunits of the human GABAA receptor, were cultured using standard
46 procedures. On the day of experiment, the cells were collected from the culture flask
47
48 using Detachin solution (VWR) and resuspended in serum-free media. The cell
49
50 suspension was placed in a magnetic stirring tube, located onboard the automated
51 electrophysiology instrument, and allowed to recover for 30 min at room temperature.
52
53 Next, the cells were automatically transferred to a built-in centrifuge, spun down, and
54
55 washed in extracellular Ringer’s solution. Then, the cells were applied to the pipetting
56 wells of a disposable 16-channel planar patch chip plates (QPlate 16×, with 10 patch
57
58 clamp holes per measurement site), and gigaseals were formed upon execution of a
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3 combined suction/voltage protocol. Further suction lead to whole-cell configuration.
4
5 GABAA receptor chloride currents were recorded for 7 s after the addition of each
6 compound. During whole-cell recording, the holding potential was set to −90 mV; the
7
8 recordings were performed at room temperature. The extracellular solution consisted
9
10 of (in mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 4 KCl, 145 NaCl, 10 glucose (pH 7.4, 300
11 mOsm); and the intracellular solution consisted of (in mM): 140 CsF, 1 EGTA, 5
12
13 CsOH, 10 HEPES, 20 NaCl (pH 7.2, 320 mOsm).
14
15 In the performed assays, the sequential application of 10 µM GABA
16
17 (reference agonist) (AG1); 1 µM tested compound (T1); 1 µM tested compound, co-
18
19
administered with 10 µM GABA (T2); second addition of 10 µM GABA (AG2); 10
20 µM bicuculine (reference antagonist) in combination with 10 µM GABA (ATG) was
21
22 set in the instrument software. The interval between the additions of particular
23
24
compounds was minimum 60 s. Typically, 5 µl of the ligand was added to the cells,
25 which was followed after 3 s by washout with extracellular solution (two times 5 µl).
26
27 In the allosteric modulator/antagonist mode (parallel addition of GABA and tested
28
29
compound or reference antagonist), the cells were preincubated with tested allosteric
30 modulator/antagonist alone for minimum 50 s, before the addition of combination
31
32 with agonist.
33
34 The data were analyzed using QPatch Assay Software (v5.0, Sophion
35
36 Bioscience); they represent the mean of three experiments carried out on distinct cells.
37 Validation criteria for each experiment were: current amplitude evoked by the
38
39 addition of GABA should be higher than 500 pA and the difference between the cell
40
41 response to both the GABA applications (AG1 and AG2) should not be greater than
42 25%. Relative compound efficacy was calculated as baseline-corrected ratio of
43
44 maximal current amplitudes evoked in the presence of tested compounds and
45
46 reference agonist (T1-ATG / AG1-ATG or T2-ATG / AG1-ATG).
47
48
49
50
4.6 Metabolism
51 Human liver microsomes (HLMs), rat liver microsomes (RLMs), glucose-6-
52
53 phosphate, NADP, glucose-6-phosphate dehydrogenase, levallorphan, and
54
propranolol were supplied by Sigma-Aldrich (Poznan, Poland).
55
56 Microsomal incubations were conducted in duplicate; the incubations were composed
57
58 of test compound (20 µM), appropriate microsomes (HLMs, RLMs, 0.4 mg/ml),
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3 NADPH-regenerating system, and potassium phosphate buffer (100 mM, pH 7.4).
4
5 NADPH-regenerating system was composed of NADP, glucose-6-phosphate,
6 glucose-6-phosphate dehydrogenase, and potassium phosphate buffer (100 mM, pH
7
8 7.4). First, the reaction mixtures containing microsomes, test compound, and buffer
9
10 were preincubated at 37°C for 15 min before the addition of NADPH-regenerating
11 system. The resulting mixture was incubated for different time points (5–240 min) at
12
13 37°C. Next, an internal standard (20 µM, levallorphan for compound 17 and
14
15 propranolol for compound 7) was added, and the reaction was terminated by the
16 addition of perchloric acid. Similarly, all the procedures were performed in control
17
18 samples, except that NADPH-regenerating system was replaced with phosphate
19
20 buffer.25–27 The samples were then centrifuged to pellet the precipitated protein, and
21 the supernatants were analyzed by using UPLC/MS (Waters Corporation). The in
22
23 vitro half-times (t1/2) for 17 and 7 were determined from the slope of the linear
24
25 regression of ln % of parent compound remaining versus incubation time. The
26 calculated t1/2 was incorporated into the following equation to obtain intrinsic
27
28 clearance (Clint) = (volume of incubation [µl]/ protein in the incubation [mg]) × 0.693
29
30 / t1/2.28
31
32
33
34
35 4.7 In vivo pharmacology
36 4.7.1 Subject
37
38 Drug-naive male Wistar rats were used (n = 8 rats per group). They were housed in
39
40 four per standard plastic cage and kept in a room with constant environmental
41
conditions (temperature: 22 ± 1°C, humidity: 60%, a 12:12 light–dark cycle with
42
43 lights on at 07:00 AM). The animals were supplied by the breeder 2 weeks before the
44
45 onset of the behavioral procedures. During this time, they were weighted and handled
46
several times. Tap water and standard lab chow (Labofeed H, WPIK, Kcynia, Poland)
47
48 were available ad libitum.
49
50 Treatment of rats in the present study was in accordance with the ethical
51
standards laid down in respective Polish and European (Directive no. 2010/63/EU)
52
53 regulations. All procedures were reviewed and approved by a local ethics committee.
54
55 4.7.2 Open-field test, amphetamine-induced hyperlocomotion
56
57
58
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3 All the tests were performed in a sound-attenuated experimental room between 09:00
4
5 AM and 3:00 PM. Within 24 h prior to testing, the rats were transferred from their
6 home cages to the experimental room and allowed to habituate for 60 min. On the
7
8 next day, the open-field test was performed as described below: 15,24
9
10 Locomotor activity was assessed in black octagonal cages (80 cm in diameter,
11 30 cm high) under dim light and continuous white noise (65 dB). Each animal was
12
13 placed in the central part of the open field and allowed to freely explore the whole
14
15 area for 30 min. Forward locomotion (cm/30 min) was registered and analyzed with
16 the help of the computerized motor tracking system (Videomot; TSE, Bad Homburg,
17
18 Germany). The rats were administered p.o. with compound 7 30 min before the start
19
20 of the open-field test. Amphetamine (1.0 mg/kg, i.p.) was injected 15 min before the
21 start of the open-field test.
22
23 4.7.3 Drugs
24
25 D-Amphetamine (Sigma-Aldrich) was dissolved in sterile physiological saline (0.9%
26 NaCl; Baxter, Warsaw, Poland) and administered i.p. in a volume of 1.0 ml/kg.
27
28 Compound 7 was suspended in 1.5% Tween and administered p.o. in a volume of 2.0
29
30 ml/kg. All the solutions were prepared immediately prior to use and protected from
31 the light.
32
33 4.7.4 Data presentation and analysis
34
35 Forward locomotion (cm/30 min) was analyzed with the help of one-way analysis of
36 variance (ANOVA). The Newman–Keuls test was used for individual post hoc
37
38 comparisons. The results of the post hoc analyzes (*p < 0.05, **p < 0.01) are shown
39
40 in Figures 5 and 6. p-Values <0.05 were considered significant. The Statistica 12.0
41 software package for Windows (StatSoft, Tulsa, OK, USA) was used to analyze the
42
43 behavioral data.
44
45
46
47
48
49
50
51
52
53
54
55
56 Abbreviations
57
58 GABA- γ-aminobutyric acid
59
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3 MED- minimal effective dose
4
5 PET- positron emission tomography
6 HLM- human liver microsomes
7
8 RLM- rat liver microsomes
9
10
11
12
13 Associated content
14
15 Supporting information
16
17
18 Detailed synthetic procedures, compounds characterization, electrophysiological
19
20 studies, metabolic stability including fragmentation analysis.
21
22
23 Author Information
24
25 *Corresponding Author:
26 Phone: (+48)126205460; Fax: (+48)126205458
27
28 E-mail: monika.marcinkowska@uj.edu.pl
29
30
31
32
33 Acknowledgment
34
35 We would like to acknowledge the financial support from National Science Center,
36
37 Poland (Grant No 5887/B/P01/2011/40).
38
39
40 Author Contributions:
41
42 Monika Marcinkowska: design and synthesis of the final compounds, data analysis,
43 preparation of the manuscript, final compound characterization
44
45 Marcin Kołaczkowski: contribution to the design of final compounds, structure-activity
46 relationship discussion, preparation of the supporting information
47
48 Krzysztof Kamiński: synthesis of the key building blocks, resynthesis of compound 7 for in
49 vivo studies, compound characterization
50
51 Adam Bucki: purification of the final molecules
52
53 Maciej Pawłowski: discussion of the chemical synthesis
54
55 Agata Siwek: radioligand binding assay
56
57
Tadeusz Karcz: electrophysiological studies, description of the results in the manuscript
58
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3
4 Gabriela Starowicz: electrophysiological studies
5
6 Karolina Słoczyńska: metabolic stability experiments, description of the results in the
7 manuscript
8
9 Elżbieta Pękala: design and discussion of the metabolic stability experiments
10
11 Jerzy Samochowiec: in vivo results discussion
12
13 Anna Wesolowska: in vivo results discussion
14
15 Paweł Mierzejewski: in vivo experiments, data analysis,
16
17 Przemysław Bienkowski: in vivo data analysis, description of the in vivo results in the
18 manuscript
19
20
21
22
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