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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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ACS Chemical Neuroscience

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

Monika Marcinkowska‡*, Marcin Kołaczkowski‡, Krzysztof Kamiński‡, Adam Bucki‡, Maciej Pawłowski‡, Agata Siwek‡, Tadeusz Karcz‡, Gabriela Starowicz‡, Karolina Słoczyńska‡, Elżbieta Pękala‡, Anna Wesołowska‡, Jerzy Samochowiec#, Paweł Mierzejewski║, Przemyslaw Bienkowski&

‡

Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna St.,

30-688 Krakow, Poland #

Department of Psychiatry, Pomeranian Medical University, 1 Rybacka St., 70-204

Szczecin, Poland ║

Department of Pharmacology, Institute of Psychiatry and Neurology, 9 Sobieskiego

St., 02-957 Warsaw, Poland &

Department of Psychiatry, Medical University of Warsaw ul. Nowowiejska 27,

00-665 Warsaw, Poland

1 ACS Paragon Plus Environment


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Abstract Schizophrenia is a mental illness characterized by behavioral changes as well as anatomical and neurochemical abnormalities. There has been a remarkable progress in the drug discovery for schizophrenia; however, antipsychotics that act through molecular targets, other than monoaminergic receptors, have not been developed. One of the hypotheses of schizophrenia states that GABAergic dysfunction might be implemented in the pathophysiology of this disease. Our recent findings and previous clinical observations have suggested that modulation of GABAergic system through α1-GABAA receptors would represent an original approach for the treatment of schizophrenia. This study presents the synthesis and biological evaluation of a series of fluorinated 3-aminomethyl derivatives of 2-phenylimidazo[1,2-a]-pyridine as potential antipsychotic agents. Compound 7 has a high affinity for GABAA receptor (Ki = 27.2 nM), high in vitro metabolic stability, and antipsychotic-like activity in amphetamine-induced hyperlocomotion test in rats (MED = 10 mg/kg). Compound 7 represents a promising point of entry in the course of development of antipsychotic agents with a non-dopaminergic mechanism of action.

Graphical abstract


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KEYWORDS: schizophrenia, psychosis, positive allosteric modulators of GABAA receptor,

γ-aminobutyric

GABAergic

transmission,

acid

(GABA),

fluorinated

antipsychotic-like

imidazo[1,2-a]pyridines,

activity,

amphetamine-induced

hyperlocomotion, zolpidem

1. Introduction Schizophrenia is a mental illness that affects ~1% of the worldwide population and is characterized by behavioral changes as well as anatomical and neurochemical abnormalities.1 The neuropathology of schizophrenia is associated with the imbalances of various neurotransmitter systems such as dopamine, serotonin, glutamate, and γ-aminobutyric acid (GABA). The first- and the second-generation antipsychotics that are commonly used in the treatment of schizophrenia primarily target the subcortical dopamine D2 receptors.2,3 Such mechanism of action reduces the psychotic symptoms in the majority of patients; however ~30%–60% of the patients remain drug-resistant.4,5 Furthermore, considerable challenge arises when patients refuse to take their medications because of troublesome and unpleasant adverse reactions such as extrapyramidal

side

effects,

weight

gain

(metabolic

syndrome),

nausea,

hyperprolactinemia, agranulocytosis, sexual dysfunction, or increased cardiac risk.6 Thus, ~50% of the patients withdraw from the pharmacological treatment. Hence, there is a growing interest in the development of original antipsychotic agents involving different mechanisms of action, which do not interact with the dopaminergic receptors.7 In 1972, Eugene Roberts first suggested that the GABA-ergic dysfunction might be implemented in the pathophysiology of schizophrenia.8 Later, the postmortem studies showed lower brain levels of GABA neurotransmitter in schizophrenia patients compared to healthy subjects.9 Alternative studies showed an increased number of GABAA receptors and a correlation between changes in densities of various GABAA receptor subunits and psychical disorders, such as schizophrenia or bipolar affective disorder.10,11 Moreover, positron emission tomography (PET) studies using [18F]-fluoroflumazenil revealed reduced binding to GABAA receptor in individuals at ultra-high risk for psychosis in comparison with low-risk subjects.12 In accordance with these findings, a recent study has shown a lower level of messenger 3 ACS Paragon Plus Environment


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RNA of the α1 subunit of GABAA receptor in the prefrontal cortices of schizophrenia patients.13 These findings have suggested that modulation of GABA-ergic system through α1-GABAA receptors would represent a promising approach for the management of schizophrenia. Recently, we have found that zolpidem, an α1 selective GABAA positive allosteric modulator displays an antipsychotic-like effect in rats, comparable to the effect induced by risperidone, a second-generation antipsychotic.14,15 These results are in line with other clinical observations that have suggested that zolpidem might exert an antipsychotic-like effect.16,17 The aforementioned findings prompted us to further explore α1-GABAA positive allosteric modulators as candidates for innovative antipsychotic agents. Previously we have identified a series of fluorinated 2phenylimidazo[1, 2-a]-pyridines, zolpidem analogs which are positive allosteric modulators of GABAA receptor.18 The most active compound effectively reduced hyperlocomotion induced by amphetamine in low dosage (1 mg/kg), confirming the hypothesis that selective positive allosteric modulators of GABAA receptor possess specific antipsychotic activity, and this seems to be an effective strategy in the design and development of novel antipsychotics with non-dopaminergic mechanism of action. In order to provide an enhanced understanding of the structure–activity relationships (SAR), in this study we designed a series of fluorinated 2phenylimidazo[1, 2-a]-pyridines, replacing 3-(N,N-dimethylacetamide) moiety with variously acylated 3-aminomethyl derivatives (Figure 1). We aimed to investigate the role of “inverse amide moieties” on antipsychotic activity and consequently on pharmacological profile of such molecules. The arrangement of fluorinated substituents around 2-phenylimidazo[1, 2-a]-pyridine ring was selected on the basis of the results from our previous studies, which revealed that incorporation of fluorine groups into positions 6, 3′, and 4′ of the 2-phenylimidazo[1, 2-a]-pyridine core provides analogs with high affinity for the GABAA receptor and increases metabolic stability of such derivatives.


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Figure 1. Structure of the previously reported fluorinated 2-phenylimidazo[1, 2-a]-pyridines and general structure of the novel series.

Herein, we present the synthesis and biological evaluation of a series of fluorinated 3-aminomethyl derivatives of 2-phenylimidazo[1, 2-a]-pyridine as potential antipsychotic agents. All the compounds were tested for affinity for GABAA receptor;

the

most

active

compounds

were

further

investigated

in

the

electrophysiological studies to determine their functional effects, and their metabolic stability was examined in in vitro models. Finally, the most promising compound was evaluated in the animal model of psychosis to determine its antipsychotic activity.

2. Results and discussion 2.1 Chemistry The target compounds 6–25 were prepared in a two-step synthesis as presented in Scheme 1. In the first step, cyclization between corresponding bromoacetophenones and aminopyridines resulted in 2-phenylimidazo[1,2-a]pyridines 1–5.18 Next, the amides 6–25 were obtained in a one-pot Mannich-type reaction between suitable nitrile and paraformaldehyde in glacial acetic acid along with concentrated sulfuric acid, followed by the subsequent addition of appropriate imidazo[1,2-a]-pyridines 1–5. The reaction mixture was heated at 75°C for 12 h and 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, toluene, reflux, 12 h; (b) formaldehyde, 98% H2SO4, glacial CH3COOH, 65°C, 30 min and then 75°C, 12 h.

2.2 Affinity for the GABAA receptor benzodiazepine site All the final molecules 6–25 were submitted to radioligand binding assay to determine their affinity for the GABAA receptor benzodiazepine site, by measuring the competitive displacement of [3H]-flunitrazepam, using rat brain tissue. The majority of the molecules displayed a relatively marked affinity (Ki < 100 nM) for the GABAA receptor. Four compounds, namely 7, 17, 19, and 21, showed a higher affinity (Ki < 30 nM) in comparison with the reference drug zolpidem. The optimal imidazo[1,2-a]-pyridine

cores

were

found

to

be

2-(4-fluorophenyl)-6-

fluoroimidazo[1,2-a]pyridine (17) and 2-(3,4-difluorophenyl)-6-fluoroimidazo[1,2a]pyridine (21), which gave the most potent analogs of all the series (Ki = 21 ± 2.7 nM and 23 ± 4.0 nM, respectively). Compounds bearing isopropylamide function tended to have a 2–10 times decreased affinity for GABAA receptor, regardless of the substituents at the imidazo[1,2-a]-pyridine core. The incorporation of a more rigid substituent cyclopropylamide yielded compounds with acceptable affinities (Ki = 20– 136 nM). Further SAR analysis revealed that the introduction of a propanamide moiety in all combinations of imidazo[1,2-a]-pyridine scaffolds was well tolerated, and relatively high affinities were observed. For instance, the Ki value of 2-(4fluorophenyl)-6-methylimidazo[1,2-a]pyridine analog (7) was 27.2 ± 5.9 nM and that of 2-(3,4-difluorophenyl)-6-fluoroimidazo[1,2-a]pyridine derivative (19) was 24.0 ±


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2.0 nM (Table 1). The exchange of propanamide group with a smaller methylamide function resulted in a small decrease of affinity for GABAA receptor. For further evaluations, compounds 7, 17, 19, and 21 with the Ki values lower than that for the reference drug zolpidem were selected. Table 1. GABAA receptor benzodiazepine site affinity data for 3-aminomethyl derivatives of 2-phenylimidazo[1,2-a]-pyridine (compounds 6–25).

Compound

R1

R2

R3

R4

Ki (nM) ± SEMa

6

CH3

H

F

CH3

78.4 ± 8.1

7

CH3

H

F

CH2CH3

27.2 ± 5.9

8

CH3

H

F

-CH(CH3)2

364.0 ± 14.0

9

CH3

H

F

-CH(CH2)2

116.0 ± 15.5

10

F

H

CH3

CH3

44.0 ± 1.2

11

F

H

CH3

CH2CH3

36.5 ± 7.0

12

F

H

CH3

-CH(CH3)2

87.5 ± 10.0

13

F

H

CH3

-CH(CH2)2

55.1 ± 1.0

14

F

H

F

CH3

43.0 ± 8.6

15

F

H

F

CH2CH3

37.0 ± 2.5

16

F

H

F

-CH(CH3)2

308.7 ± 17.1



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17

F

H

F

-CH(CH2)2

20.9 ± 2.7

18

F

F

F

CH3

39.0 ± 3.5

19

F

F

F

CH2CH3

24.0 ± 2.0

20

F

F

F

-CH(CH3)2

72.0 ± 4.4

21

F

F

F

-CH(CH2)2

22.8 ± 4.0

22

F

H

CF3

CH3

62.7 ± 7.7

23

F

H

CF3

CH2CH3

48.0 ± 6.1

24

F

H

CF3

-CH(CH3)2

86.3 ± 1.5

25

F

H

CF3

-CH(CH2)2

67.0 ± 4.7

Zolpidem

37.8 ± 2.7

a

Data are expressed as the mean ± SEM of three independent experiments performed in duplicate.

2.3 Electrophysiological studies The electrophysiological studies aimed to determine whether the compounds are capable of enhancing the effect of reference GABA agonist on GABAA receptor and thus exert positive allosteric modulation properties. For that purpose, activities of compounds 7, 17, 19, and 21 at human α1β2γ2-GABAA receptors stably expressed in a HEK293 cell line were evaluated using the automated patch clamp platform QPatch16X (Sophion Bioscience) as described in the “Methods” section. The results of the conducted studies showed that the tested compounds (7, 17,

19, 21) display weak positive allosteric modulator activity, given the ability to potentiate the effect of natural agonist GABA. Based on the obtained results, two compounds 7 and 17, stimulating the highest increase of GABA-evoked current amplitude, were selected for further studies. 8 ACS Paragon Plus Environment

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Table 2. Relative positive allosteric modulator activity of compounds at GABAA receptor, presented as a fold increase of GABA-gated current amplitude compared to the effect of 10 µM GABA alone. Data represent mean ± SEM of three experiments performed on distinct cells.

Compound

Fold increase of GABA-gated currents ± SEM

Zolpidem 7 17 19 21

2.56 ± 0.22 1.32 ± 0.10 1.32 ± 0.09 1.02 ± 0.03 1.17 ± 0.07

2.4 Metabolic stability Determination of metabolic stability of compounds at the early stages of drug discovery is important because it helps to increase the rate of success of drug development.19,20 Recently, in vitro methods using human and animal microsomes gained enormous attention since they are time and cost-effective. Therefore, metabolic stability of compounds 7 and 17 was determined in vitro using human and rat liver microsomes (HLMs and RLMs, respectively). Both compounds 7 and 17 exhibited moderate to high in vitro metabolic stability (from 71% to 97%) in different microsomal models of biotransformation (Table 3). As the test compounds demonstrated high stability in HLMs, their half times and intrinsic clearance values were not assessed. In RLMs, the Clint value of compound 7 was in the lower limit of the medium category range (17 µl/mg/min), whereas the Clint value for compound 17 was within the middle of the medium category range (38.5 µl/mg/min) (Table 3). In order to confirm the structures of generated metabolites, the ion fragment analysis was performed. The dominant chemical route of biotransformation of compounds 17 and 7 was hydroxylation; in compound 17, it occurred in the pyridine ring, whereas in compound 7, in the methyl substituent in the pyridine ring (see the supplementary information). Figure 2 depicts the exemplary HPLC chromatograms of compounds 17 and 7 and their main metabolites.



HLMs

97 93

93 71

-

102 45

-

A

RLMs

RLMs

Clint (µl/mg/min)

HLMs

3.27 3.67

t1/2 (min)

RLMs

312 328

% remaining at 15 min HLMs

7 17

Retention time (min)

Molecular ion (m/z)

Table 3. Metabolic stability screen of compounds 7 and 17 in human and rat liver microsomes (HLMs and RLMs, respectively). Test compound

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17.0 38.5

B

Figure 2. HPLC chromatograms (retention times) of test compounds 17 (A) and 7 (B), their main metabolites (M1–M5), and internal standards.

Based on these results, compound 7 was selected for further evaluation.

2.5 Antipsychotic-like activity in rats In order to evaluate the potential antipsychotic-like activity of compound 7, the effect of the compound on amphetamine-induced hyperlocomotion was assessed


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in

rats.

Hyperlocomotion

induced

by

dopaminergic

substances,

including

amphetamine, is mediated through postsynaptic mesolimbic dopamine receptors. In accordance with this finding, it has been proposed that inhibition of hyperactivity, elicited by amphetamine, reflects the antipsychotic-like activity of new drug candidates and that the specific effects of potential antipsychotics on the mesolimbic dopamine pathway are essential for successful drug development.21,22 Moreover, administration of amphetamine can induce GABA deficits in the rat limbic system.23 The latter observation further supports the notion that the model in which psychoticlike effects are induced by amphetamine may be a good tool to examine the antipsychotic-like activity of GABA-ergic compounds.15 Compound 7 dose-dependently reversed hyperlocomotion induced by amphetamine with MED = 10 mg/kg, p.o. (Figure 3). Notably, we did not observe any myorelaxant or cataleptogenic effects or any other adverse changes in gross animal behavior at the same dose. The mean distance traveled by the groups treated with compound 7–amphetamine combination did not fall below the mean locomotor activity observed in drug-free control rats (see Figure 4 for comparison). In another control experiment, no decrease in spontaneous locomotor activity was found up to the dose of 10 mg/kg (Figure 4), which supports the specificity of the observed antipsychotic-like effects of compound 7. Importantly, many currently used antipsychotic drugs (e.g., haloperidol, olanzapine, and lurasidone) suppress locomotor activity at the antipsychotic-like doses.24 In the present study, compound 7 was tested under the experimental conditions closely similar to those used in our previous behavioral experiments with zolpidem (Mierzejewski et al., 2016). Hence, one may wish to compare the antipsychotic-like activity of 7 and its parent compound zolpidem. Both molecules (see Fig. 4, the present study, and Mierzejewskiet al., 2016, Figure 1B)15 significantly reduced amphetamine-induced hyperlocomotion at the dose, which did not alter spontaneous locomotor activity (10.0 mg/kg, p.o., and 0.3 mg/kg, i.p., for 7 and zolpidem, respectively). The effect size calculated for compound 7 (1.14) was comparable to that calculated for zolpidem (1.33). Thus, although compound 7 was administered p.o. and zolpidem was injected i.p., the effect sizes calculated for the behaviorally-selective doses tend to be similar for 7 and for zolpidem.



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Figure 3. Effects of compound 7 (1–30 mg/kg, p.o.) on amphetamine-induced hyperlocomotion in rats. The dose of 0 mg/kg refers to rats treated with vehicle and amphetamine (1.0 mg/kg, i.p.). The dotted line reflects the mean spontaneous locomotor activity observed in drug-free control subjects (i.e., rats administered only with vehicle; see Figure 6 for comparison); *p < 0.05.

Figure 4. Effects of compound 7 (1–10 mg/kg, p.o.) on spontaneous locomotor activity in rats.


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3. Conclusion In summary, we have designed and synthesized a series of 3-aminomethyl derivatives of 2-phenylimidazo[1,2-a]-pyridine. The compounds display a high affinity for GABAA receptor benzodiazepine site and positive allosteric modulator activity. They also possess moderate to high in vitro metabolic stability in two different microsomal models of biotransformation. The most promising compound 7 has the antipsychoticlike activity, reversing hyperlocomotion induced by amphetamine, without inducing significant sedation. The study has also demonstrated that compounds acting as positive allosteric modulators of GABAA receptor might represent a promising strategy in the development of original antipsychotic agents with a non-dopaminergic mechanism of action.

4. Methods 4.1 General chemistry methods Unless otherwise indicated, all the starting materials were obtained from commercial suppliers and were used without further purification. All the reactions with air- and moisture-sensitive components were performed under a nitrogen atmosphere. Analytical thin-layer chromatography (TLC) was performed on Merck Kieselgel 60 F254 (0.25 mm) pre-coated aluminum sheets (Merck, Darmstadt, Germany). Visualization was performed with a 254 nm UV lamp. Column chromatography was performed using silica gel (particle size 0.063–0.200mm; 70–230 Mesh ATM) purchased from Merck. The UPLC-MS or UPLC-MS/MS analyzes were run on UPLC-MS/MS system comprising Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) coupled with Waters TQD mass spectrometer (electrospray ionization mode ESI with tandem quadrupole). Chromatographic separations were carried out using the ACQUITY UPLC BEH (bridged ethyl hybrid) C18 column: 2.1 × 100 mm and 1.7 µm particle size. The column was maintained at 40°C and eluted under gradient conditions using 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 ml/min. Eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). A total of 10 µl of each sample were injected, and chromatograms 13 ACS Paragon Plus Environment


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Page 14 of 23

were recorded using Waters eλ PDA detector. The spectra were analyzed in the range of 200–700 nm with 1.2 nm resolution and at a sampling rate of 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 150°C, desolvation temperature 350°C, desolvation gas flow rate 600 l/h, cone gas flow 100 l/h, capillary potential 3.00 kV, and cone potential 20 V. Nitrogen was used for both nebulizing and drying. The data were obtained in a scan mode ranging from 50 to 1000 m/z at 0.5 s intervals; 8 scans were summed up to obtain the final spectrum. Collision activated dissociation (CAD) analyzes were carried out with the energy of 20 eV, and all the fragmentations were observed in the source. Consequently, the ion spectra were obtained in the range from 50 to 500 m/z. MassLynx V 4.1 software (Waters) was used for data acquisition. Standard solutions (1 mg/ml) of each compound were prepared in a mixture comprising analytical grade acetonitrile/water (1/1, v/v). The UPLC/MS purity of all the test compounds and key intermediates was determined to be >95%. 1H NMR, 13C NMR, and 19F NMR spectra were obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3 or DMSO operating at 300 MHz (1H NMR), 75 MHz (13C NMR), and 282 MHz (19F NMR). Chemical shifts are reported in terms of δ values (ppm) relative to TMS δ = 0 (1H) as internal standard. The J values are expressed in Hertz. Signal multiplicities are represented by the following abbreviations: s (singlet), br.s (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet). Melting points were determined with a Büchi apparatus and are uncorrected.

4.2

General

procedure

for

the

synthesis

of

2-(4-fluorophenyl)-6-

methylimidazo[1,2-a]pyridine (compound 1) A mixture of 6-methylpyridin-2-amine (10 mmol, 1.08 g), 2-bromoacetophenone (10 mmol, 2.17 g), and sodium bicarbonate (15 mmol, 1.26 g) in anhydrous toluene (25 ml) was stirred under reflux for 12 h. After the removal of toluene under vacuum, dichloromethane was added to the residue, and any insoluble material was removed by filtration. The crude products were crystallized from methanol or purified by silica gel chromatography using dichloromethane/methanol, 9:0.5 (v/v) as eluent. Compound 1: 2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridine Yellow solid; yield: 78%; UPLC RT = 3.23; 1H NMR (300 MHz, CDCl3): δ 2.10 (s, 3H), 7.02–7.16 (m, 3H), 7.56 (d, 1H), 7.71 (s, 1H), 7.85–7.93 (m, 3H); 19F NMR (282 14 ACS Paragon Plus Environment

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MHz, CDCl3): δ −142.00 (s, 1F); LC-MS (ESI) calcd for C14H11FN2 227.09 (M+H), found 229.2 (M+H)..

4.3

General

procedure

for

the

synthesis

of

N-((2-(4-fluorophenyl)-6-

methylimidazo[1,2-a]pyridin-3-yl)methyl)propanamide (compound 7) The solution of propionitrile (1.87 mmol, 0.133 ml), paraformaldehyde (1.87 mmol, 0.05 g) in 98% H2SO4 (0.05 ml), and glacial CH3COOH (2 ml) was heated at 65°C until dissolution of formaldehyde. Next, the solution of 2-(4-fluorophenyl)-6methylimidazo[1,2-a]pyridine (compound 1) (0.62 mmol, 0.14 g) in glacial CH3COOH (4 ml) was added, and the reaction mixture was stirred at 75°C for 12 h. Later, the reaction mixture was cooled to room temperature, and 10% solution of KOH was added until pH ~7 was obtained; then, the water layer was extracted 3 times with chloroform (3 × 15 ml). The combined organic layer was dried over sodium sulfate and concentrated in vacuum, and the crude mixture was purified over column chromatography using chloroform: methanol (98:2) as eluent.

Compound

7:

N-((2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridin-3-

yl)methyl)propanamide Yield 62%, white solid, Mp 220−221 °C, 1H NMR (300 MHz, CD3OD): δ 1.10–1.16 (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, 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, 2H), 8.14 (d, 1H, J = 1.28 Hz); 19F NMR (282 MHz, CD3OD): δ −116.00–115.88 (m, 1F); 13C NMR (75 MHz, CD3OD) δ 9.1, 16.8, 28.6, 32.0, 114.9, 115.2, 116.8, 122.3, 122.9, 128.8, 129.8, 130.1, 130.2, 142.5, 143.7, 161.1, 164.3, 175.9; Formula C18H18FN3O; ESI-MS: 312 [M+H]+

4.4 Radioligand binding assay for GABAA/BZP using [3H]-flunitrazepam Rat brains were homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using a ULTRA TURAX homogenizer. The homogenate is then centrifuged at 20,000 × g for 20 min (0–4°C). The resulting supernatant was discarded, and the pellet was rehomogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4). The resulting homogenate was centrifuged as done previously. The pellet was 15 ACS Paragon Plus Environment


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resuspended and centrifuged further three times. The final pellet was stored at −80°C for minimum 18 h. On the day of the assay, the pellet was thawed at room temperature, resuspended in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) and centrifuged at 20,000 × g for 25 min (0–4°C). [3H]Flunitrazepam (spec. act. 80 Ci/mmol, ARC) was used for labeling GABAA/BZP. Then, 150 µl of the tissue suspension, 50 µl of 10 µM diazepam (displacer), 50 µl of 1 nM [3H]Flunitrazepam, and 50 µl of the analyzed compound were incubated at 4°C for 20 min. The concentrations of the analyzed compounds ranged from 1.0E−10 M to 1.0E−5 M. The incubation was terminated by rapid filtration over glass fiber filters FilterMate B (PerkinElmer, USA) using 96-well FilterMate harvester (PerkinElmer). Five rapid washes were performed with ice-cold 50 mM Tris-HCl buffer, pH 7.4. The filter mate was dried in microwave, placed in plastic bag (PerkinElmer), and soaked with 10 ml of liquid scintillation cocktail Ultima Gold MV (PerkinElmer), and the filter bag was sealed. The radioactivity of the filter was measured in MicroBeta TriLux 1450 scintillation counter (PerkinElmer). Radioligand binding data were analyzed using iterative curve fitting routines of GraphPad Prism 5.0 software (GraphPad, Inc., La Jolla, CA, USA) using the three built-in parameter logistic model describing ligand competition binding to radioligand-labeled sites. The log IC50 (i.e., the log of the ligand concentration that reduces a specific radioligand binding by 50%) estimated from the data is used to obtain the Ki values by applying the Cheng–Prusoff approximation.

4.5 Electrophysiological studies The electrophysiological experiments were carried out on a QPatch16X automatic patch clamp platform (Sophion Bioscience). HEK293 cells, stably expressing the α1β2γ2 subunits of the human GABAA receptor, were cultured using standard procedures. On the day of experiment, the cells were collected from the culture flask using Detachin solution (VWR) and resuspended in serum-free media. The cell suspension was placed in a magnetic stirring tube, located onboard the automated electrophysiology instrument, and allowed to recover for 30 min at room temperature. Next, the cells were automatically transferred to a built-in centrifuge, spun down, and washed in extracellular Ringer’s solution. Then, the cells were applied to the pipetting wells of a disposable 16-channel planar patch chip plates (QPlate 16×, with 10 patch clamp holes per measurement site), and gigaseals were formed upon execution of a 16 ACS Paragon Plus Environment

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combined suction/voltage protocol. Further suction lead to whole-cell configuration. GABAA receptor chloride currents were recorded for 7 s after the addition of each compound. During whole-cell recording, the holding potential was set to −90 mV; the recordings were performed at room temperature. The extracellular solution consisted of (in mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 4 KCl, 145 NaCl, 10 glucose (pH 7.4, 300 mOsm); and the intracellular solution consisted of (in mM): 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, 20 NaCl (pH 7.2, 320 mOsm). In the performed assays, the sequential application of 10 µM GABA (reference agonist) (AG1); 1 µM tested compound (T1); 1 µM tested compound, coadministered with 10 µM GABA (T2); second addition of 10 µM GABA (AG2); 10 µM bicuculine (reference antagonist) in combination with 10 µM GABA (ATG) was set in the instrument software. The interval between the additions of particular compounds was minimum 60 s. Typically, 5 µl of the ligand was added to the cells, which was followed after 3 s by washout with extracellular solution (two times 5 µl). In the allosteric modulator/antagonist mode (parallel addition of GABA and tested compound or reference antagonist), the cells were preincubated with tested allosteric modulator/antagonist alone for minimum 50 s, before the addition of combination with agonist. The data were analyzed using QPatch Assay Software (v5.0, Sophion Bioscience); they represent the mean of three experiments carried out on distinct cells. Validation criteria for each experiment were: current amplitude evoked by the addition of GABA should be higher than 500 pA and the difference between the cell response to both the GABA applications (AG1 and AG2) should not be greater than 25%. Relative compound efficacy was calculated as baseline-corrected ratio of maximal current amplitudes evoked in the presence of tested compounds and reference agonist (T1-ATG / AG1-ATG or T2-ATG / AG1-ATG).

4.6 Metabolism Human liver microsomes (HLMs), rat liver microsomes (RLMs), glucose-6phosphate,

NADP,

glucose-6-phosphate

dehydrogenase,

levallorphan,

and

propranolol were supplied by Sigma-Aldrich (Poznan, Poland). Microsomal incubations were conducted in duplicate; the incubations were composed of test compound (20 µM), appropriate microsomes (HLMs, RLMs, 0.4 mg/ml), 17 ACS Paragon Plus Environment


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NADPH-regenerating system, and potassium phosphate buffer (100 mM, pH 7.4). NADPH-regenerating system was composed of NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and potassium phosphate buffer (100 mM, pH 7.4). First, the reaction mixtures containing microsomes, test compound, and buffer were preincubated at 37°C for 15 min before the addition of NADPH-regenerating system. The resulting mixture was incubated for different time points (5–240 min) at 37°C. Next, an internal standard (20 µM, levallorphan for compound 17 and propranolol for compound 7) was added, and the reaction was terminated by the addition of perchloric acid. Similarly, all the procedures were performed in control samples, except that NADPH-regenerating system was replaced with phosphate buffer.25–27 The samples were then centrifuged to pellet the precipitated protein, and the supernatants were analyzed by using UPLC/MS (Waters Corporation). The in vitro half-times (t1/2) for 17 and 7 were determined from the slope of the linear regression of ln % of parent compound remaining versus incubation time. The calculated t1/2 was incorporated into the following equation to obtain intrinsic clearance (Clint) = (volume of incubation [µl]/ protein in the incubation [mg]) × 0.693 / t1/2.28

4.7 In vivo pharmacology 4.7.1 Subject Drug-naive male Wistar rats were used (n = 8 rats per group). They were housed in four per standard plastic cage and kept in a room with constant environmental conditions (temperature: 22 ± 1°C, humidity: 60%, a 12:12 light–dark cycle with lights on at 07:00 AM). The animals were supplied by the breeder 2 weeks before the onset of the behavioral procedures. During this time, they were weighted and handled several times. Tap water and standard lab chow (Labofeed H, WPIK, Kcynia, Poland) were available ad libitum. Treatment of rats in the present study was in accordance with the ethical standards laid down in respective Polish and European (Directive no. 2010/63/EU) regulations. All procedures were reviewed and approved by a local ethics committee. 4.7.2 Open-field test, amphetamine-induced hyperlocomotion


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All the tests were performed in a sound-attenuated experimental room between 09:00 AM and 3:00 PM. Within 24 h prior to testing, the rats were transferred from their home cages to the experimental room and allowed to habituate for 60 min. On the next day, the open-field test was performed as described below: 15,24 Locomotor activity was assessed in black octagonal cages (80 cm in diameter, 30 cm high) under dim light and continuous white noise (65 dB). Each animal was placed in the central part of the open field and allowed to freely explore the whole area for 30 min. Forward locomotion (cm/30 min) was registered and analyzed with the help of the computerized motor tracking system (Videomot; TSE, Bad Homburg, Germany). The rats were administered p.o. with compound 7 30 min before the start of the open-field test. Amphetamine (1.0 mg/kg, i.p.) was injected 15 min before the start of the open-field test. 4.7.3 Drugs D-Amphetamine

(Sigma-Aldrich) was dissolved in sterile physiological saline (0.9%

NaCl; Baxter, Warsaw, Poland) and administered i.p. in a volume of 1.0 ml/kg. Compound 7 was suspended in 1.5% Tween and administered p.o. in a volume of 2.0 ml/kg. All the solutions were prepared immediately prior to use and protected from the light. 4.7.4 Data presentation and analysis Forward locomotion (cm/30 min) was analyzed with the help of one-way analysis of variance (ANOVA). The Newman–Keuls test was used for individual post hoc comparisons. The results of the post hoc analyzes (*p < 0.05, **p < 0.01) are shown in Figures 5 and 6. p-Values

pyridine as Positive Allosteric Modulators of G - ACS Publications

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