European Journal of Medicinal Chemistry 92 (2015) 221e235
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 5-HT6 receptor antagonists/D2 receptor partial agonists targeting behavioral and psychological symptoms of dementia Marcin Kołaczkowski a, b, *, Monika Marcinkowska b, Adam Bucki b, b , Maciej Pawłowski b, Grzegorz Kazek b, Agata Siwek b, Joanna Sniecikowska Magdalena Jastrze˛ bska-Wie˛ sek b, Anna Partyka b, Anna Wasik b, Anna Wesołowska b, Paweł Mierzejewski c, Przemyslaw Bienkowski c ko w 149, 05-152 Czosno w, Poland Adamed Ltd., Pien Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, 30-688 Cracow, Poland c Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 19 December 2014 Accepted 24 December 2014 Available online 24 December 2014
We describe a novel class of designed multiple ligands (DMLs) combining serotonin 5-HT6 receptor (5HT6R) antagonism with dopamine D2 receptor (D2R) partial agonism. Prototype hybrid molecules were designed using docking to receptor homology models. Diverse pharmacophore moieties yielded 3 series of hybrids with varying in vitro properties at 5-HT6R and D2R, and at M1 receptor and hERG channel antitargets. 4-(piperazin-1-yl)-1H-indole derivatives showed highest antagonist potency at 5-HT6R, with 7-butoxy-3,4-dihydroquinolin-2(1H)-one and 2-propoxybenzamide derivatives having promising D2R partial agonism. 2-(3-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)piperazin-1-yl)propoxy)benzamide (47) exhibited nanomolar affinity at both 5-HT6R and D2R and was evaluated in rat models. It displayed potent antidepressant-like and anxiolytic-like activity in the Porsolt and Vogel tests, respectively, more pronounced than that of a reference selective 5-HT6R antagonist or D2R partial agonist. In addition, 47 also showed antidepressant-like activity (Porsolt's test) and anxiolytic-like activity (open field test) in aged (>18-month old) rats. In operant conditioning tests, 47 enhanced responding for sweet reward in the saccharin self-administration test, consistent with anti-anhedonic properties. Further, 47 facilitated extinction of non-reinforced responding for sweet reward, suggesting potential procognitive activity. Taken together, these studies suggest that DMLs combining 5-HT6R antagonism and D2R partial agonism may successfully target affective disorders in patients from different age groups without a risk of cognitive deficits. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Designed multiple ligand (DML) D2R partial agonist 5-HT6R antagonist Dementia BPSD
1. Introduction Dementia affects 5% of people aged over 65, and half of those aged over 85 years old [1]. Since the world's population is aging
Abbreviations: DML, designed multiple ligand; BPSD, behavioral and psychological symptoms of dementia; 5-HT6R, serotonin 5-HT6 receptor; D2R, dopamine D2 receptor; SAR, structureeactivity relationship; M1R, muscarinic M1 receptor; b2R, adrenergic b2 receptor; D3R, dopamine D3 receptor; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; TMH, transmembrane helix; IFD, induced fit docking; MED, minimum effective dose. * Corresponding author. Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, 30-688 Cracow, Poland. E-mail addresses: [email protected], marcin.kolaczkowski@ uj.edu.pl (M. Kołaczkowski). http://dx.doi.org/10.1016/j.ejmech.2014.12.045 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
rapidly, it is predicted that by the year 2050, 115 million people will suffer from this condition [2]. Dementia patients experience serious cognitive deficits but up to 90% of them also show behavioral and psychological symptoms (BPSD). The spectrum of BPSD is diverse and includes: psychosis, depression, anxiety, verbal and physical aggression, agitation, irritability, wandering etc. Approximately 50% of patients with Alzheimer's disease and other dementias develop depression and anxiety [3]. Behavioral and psychological symptoms of dementia significantly decrease quality of patients’ life, place a heavy burden on caregivers and were found to be even more disturbing than cognitive decline [4]. Since dementia patients necessitate constant care and frequent hospitalization, they constitute a very significant social cost [5]. Antipsychotic drugs are a mainstay of
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psychopharmacological treatment of BPSD [6,7]. However, these medications display only partial therapeutic efficacy and can also cause further cognitive impairment [8]. In addition, long-term therapy with currently available antipsychotics can cause side effects including: weight gain and metabolic dysfunction, extrapyramidal syndrome (EPS), QTc interval prolongation, sedation and anticholinergic effects. Consequently, antipsychotic drugs are not currently approved for the treatment of BPSD [9,10]. In view of the lack of a disease-modifying treatment for dementia, one purpose of symptomatic treatment is to improve behavioral and psychological disturbances without worsening cognitive function or inducing other life-threatening adverse effects. There is currently no pharmacotherapeutic that has been designed and approved for treatment of BPSD, and development of such a therapy therefore remains an unmet medical need [8]. In the past decade, numerous pharmacological studies revealed engagement of 5-HT6 serotonin receptors (5-HT6Rs) in regulation of cognitive functions, and indicated that their antagonism may result in procognitive effects of potential therapeutic significance [11,12]. Recent results of phase-II clinical trials with selective 5-HT6R antagonists used alone or in combination with an acetylcholinesterase inhibitor confirmed their utility in symptomatic treatment of cognitive impairment in Alzheimer's disease [13,14]. Moreover, 5HT6R antagonists exert considerable anxiolytic and antidepressant effects in animal models [15]. Nevertheless, despite the encouraging findings with 5-HT6R selective antagonists described above, it seems unlikely that a disorder as complex as dementia, including both cognitive decline and behavioral and psychological symptoms, might be effectively treated with a drug acting at just a single target. On the other hand, although some nonselective drugs are effective in addressing CNS disorders like psychosis or depression, undesirable crossreactivity with targets eliciting deleterious side effects have made them unsuitable for treatment of elderly patients with dementia-related disturbances. A promising strategy to identify novel pharmacotherapeutics is the discovery of designed multiple ligands (DMLs) that interact with 2 or more biological targets that are physiologically relevant for the disease. Such a combination could show enhanced effectiveness compared with single target agents, while avoiding side effects characteristic of “dirty”, nonselective molecules [16e19]. A growing body of evidence suggests a high therapeutic potential of D2 receptor (D2R) partial agonists (pAg) as both antipsychotic and antidepressant agents [20e22]. The most notable representatives of this class of compounds is the atypical antipsychotic drug, aripiprazole, and its analogue brexpiprazole, a clinical candidate for treatment of major depressive disorder. The beneficial therapeutic profile of D2R partial agonists is characterized also by a benign safety profile, with an especially low occurrence of extrapyramidal side effects (EPS) [23,24]. The pharmacological profile of aripiprazole and brexpiprazole involves low level partial agonism at D2 receptors combined with marked agonism at 5-HT1A receptors and modest antagonism of 5-HT2A and 5-HT7 receptors that may contribute to their therapeutic properties, but involves no interactions with 5-HT6 receptors. Recently, our research focused on discovering new potential drug candidates that would be suitable for treatment of behavioral and psychological symptoms in the fragile population of elderly people with dementia. Therefore our efforts focused on designing novel chemical entities that would have potential efficacy in BPSD without exacerbating e or possibly even improving e preexisting cognitive deficits. In recently published studies we presented the discovery of multimodal ligands displaying combined 5-HT6/5-HT7 and D2 receptor antagonism. Such compounds proved to be efficacious in rat models of psychosis and depression without inducing
cognitive or motor deficits, in contrast to the available antipsychotic drugs tested in the same conditions [25e27]. Considering the fact that 5-HT6R antagonists display procognitive effects as well as promising mood-modulating properties, while D2R partial agonists exert antipsychotic and antidepressant activities with a benign safety profile, it could be beneficial to combine these activities in order to obtain novel compounds with suitable efficacy and safety for treatment of BPSD. The therapeutic potential of such a combination has also recently been suggested by other authors [28]. In the present study we describe a novel series of DMLs, acting primarily as 5-HT6R antagonists and D2R partial agonists, a functional profile that has not been reported previously. Computer-aided design, chemical synthesis and in vitro activity characterization are described, accompanied by extended pharmacological evaluation of antidepressant, anxiolytic and procognitive properties of the most promising compound in animal models. 2. Results and discussion 2.1. Design of dual ligands In the design of ligands acting on both 5-HT6R and D2R we utilized a knowledge-based approach relying on structures of both the known ligands and the binding sites of targeted receptors. Since our primary goal was to achieve ligands of dual activity, we chose the “designing in” approach that incorporates activity at both targets into a single molecule [16]. The design process was started by detailed analysis of both targets’ binding sites and binding modes of 5-HT6R antagonists and D2R partial agonists. To this end we utilized homology models that were developed using the approach described recently by our group [27,29]. Our primary objective therefore, was to assess the orientation of ligands of both receptors in their binding sites and thus to combine the pharmacophores of both types of ligands in a hybrid molecule. Series of ligands exhibiting high affinity at both the receptors (D2R partial agonists and 5-HT6R antagonists e Fig. 2) were docked to the respective binding sites: Fig. 1A e D2R and Fig. 1B e 5-HT6R. Based on the obtained ligand-receptor complexes, structurebased pharmacophore models were developed (Fig. 1C). The procedure enables creating receptor-based excluded volumes, which define regions banned from ligand mapping. This functionality is advantageous compared to ligand-based pharmacophores, remarkably facilitating ligand design. It allows recognition of fragments of a ligand that are likely to be extended, in the context of receptor structure. Receptor-based pharmacophore models developed for D2 and 5-HT6 receptor ligands were then superimposed to determine the common fragments and tolerant regions allowing for merging the chemical moieties required for dual activity (Fig. 1D). Superimposition was achieved by displaying the models developed on pre-aligned binding sites of both receptors. This allowed us to determine pharmacophore features common for both groups of ligands, which were: (i) protonable nitrogen atom and (ii) aryl ring, that occupied the same positions in both binding sites, next to the conserved residues Asp3.32 and Phe6.52, which are known as important interaction points for monoaminergic ligands [30,31]. Tolerant regions were also identified next to the merging point. They were attributed to particular pharmacophore features that were not common, but characteristic for 5-HT6R antagonists or D2R partial agonists, respectively. The additional aromatic moiety of 5-HT6R antagonists, usually attached to the main aryl ring by a sulfonamide fragment, was found to penetrate the hydrophobic cavity between TMHs 4 and 5 (interacting with Phe5.38), whereas the aromatic ring accompanied by h-bond acceptor (HBA) and donor (HBD) features of D2R partial agonists expanded towards TMHs 2 and 7, interacting with Glu2.65 and
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Fig. 1. Design of hybrid molecules. Proposed binding modes presented for D2R partial agonists (A) and 5-HT6R antagonists (B). Structure-based pharmacophore models generated using previously docked ligands (C). Superimposition of pharmacophore models, mapped on representative D2R and 5-HT6R ligands (D). Prototype hybrid molecule satisfying the common pharmacophore model e compound 34 (E). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks. Dotted yellow lines represent H-bonds with polar residues. For the sake of clarity, extracellular loops were hidden (A and B). TMH, transmembrane helix.
Ser7.36 (Fig. 1A and B). As a result, a prototype hybrid molecule was constructed, that possessed all the features present in the merged pharmacophore hypotheses (Fig. 1E). Based on that, a series of analogs was designed, that explored a chemical space within the obtained hybrid pharmacophore hypothesis. More detailed descriptions of both the active sites of D2R and 5-HT6R, as well as ligand binding modes were presented previously [27,29].
2.2. Synthesis of the first series of hybrid molecules In a first series of hybrid molecules of presumed dual activity, we explored various moieties responsible for introducing 5-HT6R antagonist properties. To this end we chose a group of fragments
that were previously described as having 5-HT6R affinity and were characterized by relatively low molecular weight. While exploring the “5-HT6 fragment” of a hybrid molecule, we maintained the “D2 fragment” unchanged, initially using butoxy-3,4-dihydroquinolin2(1H)-one, since it was present in the structure of the best characterized D2 partial agonists, such as aripiprazole. The synthesis of series I of hybrid molecules started with the preparation of N1-substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole derivatives (5, 6) and N1-substituted 3-(piperidin-4-yl)1H-indole derivatives (7, 8) (Scheme 1). To this end, commercially available Boc-protected 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles or Boc-protected 3-(piperidin-4-yl)-1H-indoles were reacted with (bromomethyl)benzene or benzenesulfonyl chloride in the
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Fig. 2. Ligands engaged in structure-based pharmacophore development.
Scheme 1. Synthesis of N1-substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles and 3-(piperidin-4-yl)-1H-indoles. Reagents and conditions: (a) t-BuOK, 0 C, 18-crown-6, 88e95%; (b) 6 N HCl/dioxane, 88e89%. D e double bond, S e single bond.
presence of t-BuOK at 0 C affording corresponding derivatives 1e4 in good yields (88e95%). Next, deprotection of Boc group under acidic conditions (4 N HCl/dioxane) afforded compounds 5e8. N1-substituted 4-(piperazin-1-yl)-1H-indoles were prepared in a similar manner, affording key intermediates 9e20 (Scheme 2). 2-(1-Benzyl-1H-indol-3-yl)ethanamine (21) and 2-[1-(phenylsulfonyl)-1H-indol-3-yl]ethanamines (22) were synthesized as described by Cole et al. [32]. The synthesis of final compounds 33e44 was based on the Nalkylation of the corresponding indole derivatives (5e8 and 15e22) with commercially available 7-(4-bromobutoxy)-3,4dihydroquinolin-2(1H)-one (23) refluxing in acetonitrile in the presence of base and potassium iodide (Scheme 3). Compounds 33e44 were prepared in good overall yields of >75% and high purity >95% HPLC-grade.
2.3. In vitro studies with series I The first series of hybrid molecules was tested for affinity at 5HT6 and D2 receptors using radioligand binding (see experimental section). All the tested compounds showed significant affinity for both targeted receptors, in most cases resulting in Ki values 95%. Column chromatography was performed on Merck silica gel 60 (63e200 mm). Analytical thin layer chromatography was done using aluminum sheets precoated with silica gel 60 F254. 4.3. Synthetic procedures The synthesis of the most promising compound 47 is presented below, while the data for all the other compounds are available in Supplementary data. 4.3.1. 1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-indole (16) To a solution of t-BuOK (1 M sol. in THF, 0.86 mL, 1.3 eq) in 5 mL THF dry and 18-crown-6 (0.13 mmol, 35 mg, 0.2 eq), a solution of commercially available Boc-protected 3-(piperidin-4-yl)-1H-indole (0.66 mmol, 198 mg, 1.0 eq) in 5 mL of THF dry was added dropwise at 0 C. After 20 min, benezenesulfonyl chloride (0.99 mmol, 0.13 ml, 1.50 eq) was added and resulted slurry was stirred overnight at room temperature. After that time, THF was evaporated under reduced pressure, the mixture was diluted with EtOAc (10 mL) and extracted with water (10 mL). Organic layer was dried over sodium sulfate and evaporated, the crude mixture was purified over column chromatography. Next, a mixture of the obtained product 10 (0.59 mmol, 259 mg) and 5 mL of 4 M HCl solution in dioxane were stirred at room temperature for 2 h, then dioxane was removed under reduced pressure and dried under the vacuum for 1 h. The intermediate 16 was used directly to the next step without further purification. 4.3.2. 2-(3-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)piperazin-1-yl) propoxy)benzamide (47) A mixture of commercially available 2-(3-chloropropoxy) benzamide 26 (0.46 mmol, 98 mg, 1.0 eq) and 16 (0.52 mmol,
4.4. Radioligand binding assays for 5-HT6 and D2 receptors
4.5. Functional assays for 5 HT1A, 5 HT2A, 5-HT6 and D2 receptors 1 mM stock solutions of the tested compounds were prepared in DMSO. Serial dilutions were prepared in 96-well microplate in assay buffers using automated pipetting system epMotion 5070 (Eppendorf). Two independent experiments in duplicates were performed and 6 to 10 concentrations were tested. Cellular aequorin-based functional assay was performed with g-irradiated recombinant CHO-K1 cells expressing mitochondrially-targeted Aequorin, human GPCR and the promiscuous G protein a16 for 5-HT6 and 5-HT2A receptor, Gaqi/5 for D2 receptor (PerkinElemer). Assay was performed according to the standard protocol provided by the manufacturer. After thawing, cells were transferred to assay buffer (DMEM/HAM's F12 with 0.1% protease-free BSA) and centrifuged. Cell pellet was resuspended in assay buffer and coelenterazine h was added at final concentrations of 5 mM. Cell suspension was incubated at 21 C, protected from light with constant agitation, for 4 h and
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Table 7 Radioligand binding assay conditions for 5 HT6 and D2 receptors. Receptor source)
Radioligand (final concentration/Kd)
Nonspecific binding
Assay buffer
Incubation condition
5-HT6 (human recombinant, HEK-293 cells)
[3H]LSD (2.5/2.0 nM)
10 mM methiothepin
60 min, 37 C
D2 (human recombinant, CHOeK1 cells)
[3H]N-methylspiperone (0.4/0.2 nM)
10 mM (þ)-butaclamol
50 mM Tris, 10 mM MgCl2, 0.5 mM EDTA, pH 7.4 50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, pH 7.4
then diluted with assay buffer to a concentration of 250,000 cells/ml. After 1 h incubation 50 ml cell suspension was dispensed using automatic injectors built in radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque 96-well microplate preloaded with tested compounds. Immediate light emission generated following calcium mobilization (agonist response) was recorded for 30e60 s. In antagonist mode, after 15e30 min incubation reference agonist was added to the above assay mix and light emission was recorded again. Final concentration of reference agonist was equal EC80: serotonin 40 nM for 5-HT6 receptor, amethylserotonin 30 nM for 5-HT2A receptor, 100 nM serotonin for 5-HT1A receptor and apomorphine 30 nM for D2 receptor. IC50 and EC50 were determined by non-linear regression analysis using GraphPad Prism 6.0 software. The logIC50 was used to obtain the Kb by applying the Cheng-Prusoff approximation. The functional assays for 5-HT1A, 5-HT2A, 5-HT6 and D2 receptors presented in Tables 2 and 5 were performed at Faculty of Pharmacy, Jagiellonian University Collegium Medicum. 4.6. Extended in vitro studies of compound 47 Extended pharmacological profile in vitro for compound 47 (with the exception of functional studies for 5-HT1A, 5-HT2A, 5-HT6 ^que, and D2 receptors), was performed by Cerep (Le Bois l'Eve Poitiers, France). An outline of methodologies is shown in Tables 4 and 5. Further methodological details are available on the company website (www.cerep.fr). Data from all experiments were analyzed using non-linear curve fitting programs and results are given as Ki values for binding affinity or Kb values for antagonist potency. Blockade of hERG-mediated potassium currents was carried out by ChanTest (Cleveland, Ohio) and expressed as mean % of inhibition at 1.0E-06 M (Table 2) or IC50 value (Table 4). Ability to block hERG potassium channels was determined using the electrophysiological method and cloned hERG potassium channels (KCNH2 gene, expressed in CHO cells) as biological material. The effects were evaluated using IonWorks™ Quattro system (Molecular Devices Corporation, Union City CA). hERG current was elicited using a pulse pattern with fixed amplitudes (conditioning pre-pulse: 80 mV for 25 ms; test pulse: þ40 mV for 80 ms) from a holding potential of 0 mV. hERG current was measured as a difference between the peak current at 1 ms after the test step to þ40 mV and the steady-state current at the end of the step to þ40 mV. Data acquisition and analyses was performed using the IonWorks QuattroTM system operation software (version 2.0.2; Molecular Devices Corporation, Union City, CA). Data were corrected for leak current. The hERG block was calculated as: % Block ¼ (1 I TA/IControl) 100%, where IControl and ITA were the currents elicited by the test pulse in control and in the presence of a test compound, respectively. All assays for the lead compound 47 (shown in Tables 4 and 5) were performed in 3e4 independent experiments. The Ki and Kb values were determined from 6 concentrations from 1.0E-06 M to 1.0E-10 M. All the assays were carried out in duplicate (n ¼ 2).
60 min, 37 C
4.7. In vivo studies 4.7.1. Animals Male Wistar rats (Charles River, Sulzfeld, Germany) were kept four per standard plastic cage and housed in a room with constant environmental conditions (temperature: 22 ± 1 C, humidity: 60%, a 12-h lightedark cycle with lights on at 6:00 a.m.). Depending on the test, three- (300e500 g) or twenty-four month old male Wistar rats (600e900 g) were used. Animals were weighted and handled twice per week, for 3 weeks before the onset of experimental procedures. Standard lab chow (Labofeed H, WPiK, Kcynia, Poland) and tap water was available ad libitum unless otherwise stated. Separate groups of drug- and alcohol-naïve subjects (n ¼ 7e10) were used in all experiments. All tests were performed between 9:00 a.m. and 4:00 p.m. in soundproof experimental rooms under dim light and continuous white noise (65 dB). Behavioral parameters were recorded by microcomputers or scored by blinded observers. The tested compounds were administered intraperitoneally (i.p.) in all studies. Treatment of rats in the present study was in full accordance with the ethical standards laid down in respective European (Directive no. 86/609/EEC) and Polish regulations. The study protocol was reviewed and approved by a local ethics committee. 4.7.2. Porsolt's forced swim test The forced swim test is widely used to screen psychotropic medications for their anti-depressant-like properties [52]. The test was conducted as described by Porsolt et al. with some minor modifications [53,54]. Rats were individually placed in glass cylinders (diameter: 18 cm, height: 40 cm) filled with water (temperature: 23 C) at a height that made it impossible to reach the bottom with hind paws. There were two swimming sessions separated by 24 h: a 15-min. pre-test and a 5-min. test. The duration of immobility (s) in the test session was recorded by an observer from the adjacent room with the aid of a video camera. A rat was considered immobile when it floated not moving except to keep the head above water surface [53,54]. A decrease in the immobility time is thought to reflect anti-depressant-like effects of a tested drug. 4.7.3. Conflict drinking test (Vogel test) Anxiety Monitoring System “Vogel test” produced by TSE Systems was used. It was consisted of a polycarbonate cage (dimensions 26.5 15 42 cm), equipped with a grid floor made from stainless steel bars and a drinking bottle containing tap water. Experimental chambers (two) were connected to PC software by control chassis and a device that generates electric shocks. On the first day of the experiment, the rats were adapted to the test chamber for 10 min. After the adaptation period, the animals were deprived of water for 24 h and were then placed in the test chamber for another 10-min adaptation period during which they had free access to the drinking bottle. Afterwards, they were allowed a 30min free-drinking session in their home cages. After another 24-h water deprivation period, the rats were placed again in the test chamber. Recording data started immediately after the first lick and
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every 20 licks rats were punished with an electric shock (0.5 mA, lasting 1 s). The impulses were released via the spout of the drinking bottle. If a rat was drinking when an impulse was released, it received a shock. The number of licks and the number of shocks received throughout a 5-min experimental session were recorded automatically. A studied compound was administered to 10 rats per treatment group. 4.7.4. Hot plate and free-drinking tests To control possible drug-induced changes in shock sensitivity or the thirst drive (which may contribute to animals’ behavior in the Vogel conflict drinking test), stimulus threshold and water consumption during a free-drinking session were determined in separate groups of rats. In either of those two studies, the rats were manipulated similarly to the Vogel conflict drinking test, including two 24-h water deprivation periods separated by 10 min adaptation session in experimental cages and 30 min of water availability in their home cages. In the free-drinking test, each animal was allowed to freely drink from the water spout and the amount of water (g) consumed during 5 min was recorded for each rat. The pain threshold was investigated using hot plate test (Commat Ltd, Turkey) in rats [55]. The plate was enclosed with a transparent Plexiglass cylinder (35 cm high) to keep the animal on the heated surface of the plate. The latency to pain reaction (lick a hind paw or jumping) when the rat was placed on a hot plate (52.5 ± 0.5 C, 19 cm diameter) was measured. The rat was removed from the plate immediately upon visible pain reaction or if no response occurred within 30 s. 4.7.5. Open field test The open field test is widely used to screen psychotropic medications for their anxiolytic-like properties [53,56]. The open field apparatus consisted of four black, octagonal, stainless steel arenas (diameter: 80 cm) with 30-cm high walls. Each arena was divided into a peripheral and central sector. The peripheral sector was defined as the region within 20 cm from the walls. The approximate light intensity was 15 lx in the in the central point of the arena and 4 lx close to its walls. A rat was placed in the central point and the experiment started immediately afterwards. Locomotor activity was recorded for 10 min with the PC-based VideoMot System (TSE, Bad Homburg, Germany). The arenas were cleaned between tests with 20% (v/v) alcohol and allowed to dry. The following parameters were analysed: forward locomotion (cm), central visits, defined as the number of entries into the central sector with all four paws, and central time (s) defined as the total time spent in the central sector [56,57]. An increase in the number of central visits (and/or time spent in the central sector) is thought to reflect anxiolytic-like effects of a tested drug. 4.7.6. Responding for sweet reward: saccharin self-administration As decreased appetite and gustatory anhedonia are thought to be among the cardinal features of depression [37], drugs with antidepressant properties are expected to increase preference for sweet solutions in animal models of depressive symptomatology [58,59]. Saccharin is thought to be highly palatable to various animal species. Vigorous self-administration of low-concentrated saccharin solutions can be easily obtained in rodents [38]. In the present study, rats were trained to lever press for saccharin (0.1%; w/v) in 8 operant chambers (Skinner boxes; Coulbourn, Inc., Allentown, PA, USA) as described by Mierzejewski et al. [38]. The operant chambers consisted of stainless-steel test cages (E10-10 TC, Coulbourn) enclosed within sound-attenuating cubicles with fans for ventilation and background white noise. A white house light
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was centered near the top of the front wall of the cage. The start of each session was signaled by turning the house light on. The cage was also equipped with two response levers separated by the liquid delivery system (the liquid dipper; E14-05, Coulbourn). The liquid dipper and the response levers were mounted on the front wall of the cage, 4 cm above a stainless-steel grid floor. The liquid dipper consisted of an arm ending in a 0.1-ml stainless-steel cup, an electric engine to raise the arm, and a 100ml fluid reservoir located outside the test cage. Contacts with the liquid dipper were registered by small photocells located inside the dipper entrance. Only one lever (the active lever) activated the liquid dipper. Presses on the other lever (the inactive lever) were recorded but had no consequences. The location of the active lever (left vs. right) was randomized across all the subjects. The liquid dipper presented saccharin solution in a volume of 0.1-ml for 5 s. A white stimulus light (4 W) located inside the liquid dipper hole was on during the entire 5-s dipper activation. After that time, the liquid dipper and its stimulus light were switched off. Programming of every experimental session as well as data recording (active and inactive lever presses, contacts with the dipper entrance) was made using the L2T2 Software package (Coulbourn) running on an IBMcompatible PC. All self-administration sessions lasted 30 min and one session was given each day between 1:00 and 3:00 p.m. During the first 3 days of training, the rats were deprived of water for 22 h/day and shaped to lever press for 0.1% saccharin on the fixed ratio (FR) 1 schedule of reinforcement where one response on the active lever activated the liquid dipper. As soon as lever pressing was established, water started to be freely available in the home cages. Thus, except for the initial phase of training, the rats were never deprived of food or water. The subjects were allowed to stabilize their saccharin selfadministration in a maintenance phase which lasted 10 days. Increase in the number of active lever presses was treated as a possible indication of antidepressant-like effects of a tested drug.
4.7.7. Extinction of non-reinforced responding for sweet reward Extinction of saccharin seeking was tested as described by Mierzejewski et al. and Radwanska et al. [38,39]. Rats successfully trained to lever press for 0.1% saccharin as described above were randomly assigned to one of experimental groups administered with vehicle or 47. In extinction sessions, animals were exposed for 30 min to the Skinner boxes where they had previously been trained to selfadminister saccharin. Responding on the active or inactive lever was never reinforced with any reward. Three 30-min. extinction sessions were run on three consecutive days with 24-h intervals. A progressive decrease in the number of non-reinforced responses during subsequent extinction sessions is thought to reflect learning and memory processes associated with the absence of expected reward [38,39].
Acknowledgment This study was financially supported by Adamed Ltd. and Polish Agency for Enterprise Development (PARP), Warsaw, Poland. Grant nr UDA-POIG.01.04.00-14-055/10-00.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.12.045.
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Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 5-HT6 receptor antagonists/D2 receptor partial agonists targeting behavioral and psychological symptoms of dementia Marcin Kołaczkowski a, b, *, Monika Marcinkowska b, Adam Bucki b, b , Maciej Pawłowski b, Grzegorz Kazek b, Agata Siwek b, Joanna Sniecikowska Magdalena Jastrze˛ bska-Wie˛ sek b, Anna Partyka b, Anna Wasik b, Anna Wesołowska b, Paweł Mierzejewski c, Przemyslaw Bienkowski c ko w 149, 05-152 Czosno w, Poland Adamed Ltd., Pien Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, 30-688 Cracow, Poland c Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 19 December 2014 Accepted 24 December 2014 Available online 24 December 2014
We describe a novel class of designed multiple ligands (DMLs) combining serotonin 5-HT6 receptor (5HT6R) antagonism with dopamine D2 receptor (D2R) partial agonism. Prototype hybrid molecules were designed using docking to receptor homology models. Diverse pharmacophore moieties yielded 3 series of hybrids with varying in vitro properties at 5-HT6R and D2R, and at M1 receptor and hERG channel antitargets. 4-(piperazin-1-yl)-1H-indole derivatives showed highest antagonist potency at 5-HT6R, with 7-butoxy-3,4-dihydroquinolin-2(1H)-one and 2-propoxybenzamide derivatives having promising D2R partial agonism. 2-(3-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)piperazin-1-yl)propoxy)benzamide (47) exhibited nanomolar affinity at both 5-HT6R and D2R and was evaluated in rat models. It displayed potent antidepressant-like and anxiolytic-like activity in the Porsolt and Vogel tests, respectively, more pronounced than that of a reference selective 5-HT6R antagonist or D2R partial agonist. In addition, 47 also showed antidepressant-like activity (Porsolt's test) and anxiolytic-like activity (open field test) in aged (>18-month old) rats. In operant conditioning tests, 47 enhanced responding for sweet reward in the saccharin self-administration test, consistent with anti-anhedonic properties. Further, 47 facilitated extinction of non-reinforced responding for sweet reward, suggesting potential procognitive activity. Taken together, these studies suggest that DMLs combining 5-HT6R antagonism and D2R partial agonism may successfully target affective disorders in patients from different age groups without a risk of cognitive deficits. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Designed multiple ligand (DML) D2R partial agonist 5-HT6R antagonist Dementia BPSD
1. Introduction Dementia affects 5% of people aged over 65, and half of those aged over 85 years old [1]. Since the world's population is aging
Abbreviations: DML, designed multiple ligand; BPSD, behavioral and psychological symptoms of dementia; 5-HT6R, serotonin 5-HT6 receptor; D2R, dopamine D2 receptor; SAR, structureeactivity relationship; M1R, muscarinic M1 receptor; b2R, adrenergic b2 receptor; D3R, dopamine D3 receptor; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; TMH, transmembrane helix; IFD, induced fit docking; MED, minimum effective dose. * Corresponding author. Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, 30-688 Cracow, Poland. E-mail addresses: [email protected], marcin.kolaczkowski@ uj.edu.pl (M. Kołaczkowski). http://dx.doi.org/10.1016/j.ejmech.2014.12.045 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
rapidly, it is predicted that by the year 2050, 115 million people will suffer from this condition [2]. Dementia patients experience serious cognitive deficits but up to 90% of them also show behavioral and psychological symptoms (BPSD). The spectrum of BPSD is diverse and includes: psychosis, depression, anxiety, verbal and physical aggression, agitation, irritability, wandering etc. Approximately 50% of patients with Alzheimer's disease and other dementias develop depression and anxiety [3]. Behavioral and psychological symptoms of dementia significantly decrease quality of patients’ life, place a heavy burden on caregivers and were found to be even more disturbing than cognitive decline [4]. Since dementia patients necessitate constant care and frequent hospitalization, they constitute a very significant social cost [5]. Antipsychotic drugs are a mainstay of
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psychopharmacological treatment of BPSD [6,7]. However, these medications display only partial therapeutic efficacy and can also cause further cognitive impairment [8]. In addition, long-term therapy with currently available antipsychotics can cause side effects including: weight gain and metabolic dysfunction, extrapyramidal syndrome (EPS), QTc interval prolongation, sedation and anticholinergic effects. Consequently, antipsychotic drugs are not currently approved for the treatment of BPSD [9,10]. In view of the lack of a disease-modifying treatment for dementia, one purpose of symptomatic treatment is to improve behavioral and psychological disturbances without worsening cognitive function or inducing other life-threatening adverse effects. There is currently no pharmacotherapeutic that has been designed and approved for treatment of BPSD, and development of such a therapy therefore remains an unmet medical need [8]. In the past decade, numerous pharmacological studies revealed engagement of 5-HT6 serotonin receptors (5-HT6Rs) in regulation of cognitive functions, and indicated that their antagonism may result in procognitive effects of potential therapeutic significance [11,12]. Recent results of phase-II clinical trials with selective 5-HT6R antagonists used alone or in combination with an acetylcholinesterase inhibitor confirmed their utility in symptomatic treatment of cognitive impairment in Alzheimer's disease [13,14]. Moreover, 5HT6R antagonists exert considerable anxiolytic and antidepressant effects in animal models [15]. Nevertheless, despite the encouraging findings with 5-HT6R selective antagonists described above, it seems unlikely that a disorder as complex as dementia, including both cognitive decline and behavioral and psychological symptoms, might be effectively treated with a drug acting at just a single target. On the other hand, although some nonselective drugs are effective in addressing CNS disorders like psychosis or depression, undesirable crossreactivity with targets eliciting deleterious side effects have made them unsuitable for treatment of elderly patients with dementia-related disturbances. A promising strategy to identify novel pharmacotherapeutics is the discovery of designed multiple ligands (DMLs) that interact with 2 or more biological targets that are physiologically relevant for the disease. Such a combination could show enhanced effectiveness compared with single target agents, while avoiding side effects characteristic of “dirty”, nonselective molecules [16e19]. A growing body of evidence suggests a high therapeutic potential of D2 receptor (D2R) partial agonists (pAg) as both antipsychotic and antidepressant agents [20e22]. The most notable representatives of this class of compounds is the atypical antipsychotic drug, aripiprazole, and its analogue brexpiprazole, a clinical candidate for treatment of major depressive disorder. The beneficial therapeutic profile of D2R partial agonists is characterized also by a benign safety profile, with an especially low occurrence of extrapyramidal side effects (EPS) [23,24]. The pharmacological profile of aripiprazole and brexpiprazole involves low level partial agonism at D2 receptors combined with marked agonism at 5-HT1A receptors and modest antagonism of 5-HT2A and 5-HT7 receptors that may contribute to their therapeutic properties, but involves no interactions with 5-HT6 receptors. Recently, our research focused on discovering new potential drug candidates that would be suitable for treatment of behavioral and psychological symptoms in the fragile population of elderly people with dementia. Therefore our efforts focused on designing novel chemical entities that would have potential efficacy in BPSD without exacerbating e or possibly even improving e preexisting cognitive deficits. In recently published studies we presented the discovery of multimodal ligands displaying combined 5-HT6/5-HT7 and D2 receptor antagonism. Such compounds proved to be efficacious in rat models of psychosis and depression without inducing
cognitive or motor deficits, in contrast to the available antipsychotic drugs tested in the same conditions [25e27]. Considering the fact that 5-HT6R antagonists display procognitive effects as well as promising mood-modulating properties, while D2R partial agonists exert antipsychotic and antidepressant activities with a benign safety profile, it could be beneficial to combine these activities in order to obtain novel compounds with suitable efficacy and safety for treatment of BPSD. The therapeutic potential of such a combination has also recently been suggested by other authors [28]. In the present study we describe a novel series of DMLs, acting primarily as 5-HT6R antagonists and D2R partial agonists, a functional profile that has not been reported previously. Computer-aided design, chemical synthesis and in vitro activity characterization are described, accompanied by extended pharmacological evaluation of antidepressant, anxiolytic and procognitive properties of the most promising compound in animal models. 2. Results and discussion 2.1. Design of dual ligands In the design of ligands acting on both 5-HT6R and D2R we utilized a knowledge-based approach relying on structures of both the known ligands and the binding sites of targeted receptors. Since our primary goal was to achieve ligands of dual activity, we chose the “designing in” approach that incorporates activity at both targets into a single molecule [16]. The design process was started by detailed analysis of both targets’ binding sites and binding modes of 5-HT6R antagonists and D2R partial agonists. To this end we utilized homology models that were developed using the approach described recently by our group [27,29]. Our primary objective therefore, was to assess the orientation of ligands of both receptors in their binding sites and thus to combine the pharmacophores of both types of ligands in a hybrid molecule. Series of ligands exhibiting high affinity at both the receptors (D2R partial agonists and 5-HT6R antagonists e Fig. 2) were docked to the respective binding sites: Fig. 1A e D2R and Fig. 1B e 5-HT6R. Based on the obtained ligand-receptor complexes, structurebased pharmacophore models were developed (Fig. 1C). The procedure enables creating receptor-based excluded volumes, which define regions banned from ligand mapping. This functionality is advantageous compared to ligand-based pharmacophores, remarkably facilitating ligand design. It allows recognition of fragments of a ligand that are likely to be extended, in the context of receptor structure. Receptor-based pharmacophore models developed for D2 and 5-HT6 receptor ligands were then superimposed to determine the common fragments and tolerant regions allowing for merging the chemical moieties required for dual activity (Fig. 1D). Superimposition was achieved by displaying the models developed on pre-aligned binding sites of both receptors. This allowed us to determine pharmacophore features common for both groups of ligands, which were: (i) protonable nitrogen atom and (ii) aryl ring, that occupied the same positions in both binding sites, next to the conserved residues Asp3.32 and Phe6.52, which are known as important interaction points for monoaminergic ligands [30,31]. Tolerant regions were also identified next to the merging point. They were attributed to particular pharmacophore features that were not common, but characteristic for 5-HT6R antagonists or D2R partial agonists, respectively. The additional aromatic moiety of 5-HT6R antagonists, usually attached to the main aryl ring by a sulfonamide fragment, was found to penetrate the hydrophobic cavity between TMHs 4 and 5 (interacting with Phe5.38), whereas the aromatic ring accompanied by h-bond acceptor (HBA) and donor (HBD) features of D2R partial agonists expanded towards TMHs 2 and 7, interacting with Glu2.65 and
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Fig. 1. Design of hybrid molecules. Proposed binding modes presented for D2R partial agonists (A) and 5-HT6R antagonists (B). Structure-based pharmacophore models generated using previously docked ligands (C). Superimposition of pharmacophore models, mapped on representative D2R and 5-HT6R ligands (D). Prototype hybrid molecule satisfying the common pharmacophore model e compound 34 (E). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks. Dotted yellow lines represent H-bonds with polar residues. For the sake of clarity, extracellular loops were hidden (A and B). TMH, transmembrane helix.
Ser7.36 (Fig. 1A and B). As a result, a prototype hybrid molecule was constructed, that possessed all the features present in the merged pharmacophore hypotheses (Fig. 1E). Based on that, a series of analogs was designed, that explored a chemical space within the obtained hybrid pharmacophore hypothesis. More detailed descriptions of both the active sites of D2R and 5-HT6R, as well as ligand binding modes were presented previously [27,29].
2.2. Synthesis of the first series of hybrid molecules In a first series of hybrid molecules of presumed dual activity, we explored various moieties responsible for introducing 5-HT6R antagonist properties. To this end we chose a group of fragments
that were previously described as having 5-HT6R affinity and were characterized by relatively low molecular weight. While exploring the “5-HT6 fragment” of a hybrid molecule, we maintained the “D2 fragment” unchanged, initially using butoxy-3,4-dihydroquinolin2(1H)-one, since it was present in the structure of the best characterized D2 partial agonists, such as aripiprazole. The synthesis of series I of hybrid molecules started with the preparation of N1-substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole derivatives (5, 6) and N1-substituted 3-(piperidin-4-yl)1H-indole derivatives (7, 8) (Scheme 1). To this end, commercially available Boc-protected 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles or Boc-protected 3-(piperidin-4-yl)-1H-indoles were reacted with (bromomethyl)benzene or benzenesulfonyl chloride in the
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Fig. 2. Ligands engaged in structure-based pharmacophore development.
Scheme 1. Synthesis of N1-substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles and 3-(piperidin-4-yl)-1H-indoles. Reagents and conditions: (a) t-BuOK, 0 C, 18-crown-6, 88e95%; (b) 6 N HCl/dioxane, 88e89%. D e double bond, S e single bond.
presence of t-BuOK at 0 C affording corresponding derivatives 1e4 in good yields (88e95%). Next, deprotection of Boc group under acidic conditions (4 N HCl/dioxane) afforded compounds 5e8. N1-substituted 4-(piperazin-1-yl)-1H-indoles were prepared in a similar manner, affording key intermediates 9e20 (Scheme 2). 2-(1-Benzyl-1H-indol-3-yl)ethanamine (21) and 2-[1-(phenylsulfonyl)-1H-indol-3-yl]ethanamines (22) were synthesized as described by Cole et al. [32]. The synthesis of final compounds 33e44 was based on the Nalkylation of the corresponding indole derivatives (5e8 and 15e22) with commercially available 7-(4-bromobutoxy)-3,4dihydroquinolin-2(1H)-one (23) refluxing in acetonitrile in the presence of base and potassium iodide (Scheme 3). Compounds 33e44 were prepared in good overall yields of >75% and high purity >95% HPLC-grade.
2.3. In vitro studies with series I The first series of hybrid molecules was tested for affinity at 5HT6 and D2 receptors using radioligand binding (see experimental section). All the tested compounds showed significant affinity for both targeted receptors, in most cases resulting in Ki values 95%. Column chromatography was performed on Merck silica gel 60 (63e200 mm). Analytical thin layer chromatography was done using aluminum sheets precoated with silica gel 60 F254. 4.3. Synthetic procedures The synthesis of the most promising compound 47 is presented below, while the data for all the other compounds are available in Supplementary data. 4.3.1. 1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-indole (16) To a solution of t-BuOK (1 M sol. in THF, 0.86 mL, 1.3 eq) in 5 mL THF dry and 18-crown-6 (0.13 mmol, 35 mg, 0.2 eq), a solution of commercially available Boc-protected 3-(piperidin-4-yl)-1H-indole (0.66 mmol, 198 mg, 1.0 eq) in 5 mL of THF dry was added dropwise at 0 C. After 20 min, benezenesulfonyl chloride (0.99 mmol, 0.13 ml, 1.50 eq) was added and resulted slurry was stirred overnight at room temperature. After that time, THF was evaporated under reduced pressure, the mixture was diluted with EtOAc (10 mL) and extracted with water (10 mL). Organic layer was dried over sodium sulfate and evaporated, the crude mixture was purified over column chromatography. Next, a mixture of the obtained product 10 (0.59 mmol, 259 mg) and 5 mL of 4 M HCl solution in dioxane were stirred at room temperature for 2 h, then dioxane was removed under reduced pressure and dried under the vacuum for 1 h. The intermediate 16 was used directly to the next step without further purification. 4.3.2. 2-(3-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)piperazin-1-yl) propoxy)benzamide (47) A mixture of commercially available 2-(3-chloropropoxy) benzamide 26 (0.46 mmol, 98 mg, 1.0 eq) and 16 (0.52 mmol,
4.4. Radioligand binding assays for 5-HT6 and D2 receptors
4.5. Functional assays for 5 HT1A, 5 HT2A, 5-HT6 and D2 receptors 1 mM stock solutions of the tested compounds were prepared in DMSO. Serial dilutions were prepared in 96-well microplate in assay buffers using automated pipetting system epMotion 5070 (Eppendorf). Two independent experiments in duplicates were performed and 6 to 10 concentrations were tested. Cellular aequorin-based functional assay was performed with g-irradiated recombinant CHO-K1 cells expressing mitochondrially-targeted Aequorin, human GPCR and the promiscuous G protein a16 for 5-HT6 and 5-HT2A receptor, Gaqi/5 for D2 receptor (PerkinElemer). Assay was performed according to the standard protocol provided by the manufacturer. After thawing, cells were transferred to assay buffer (DMEM/HAM's F12 with 0.1% protease-free BSA) and centrifuged. Cell pellet was resuspended in assay buffer and coelenterazine h was added at final concentrations of 5 mM. Cell suspension was incubated at 21 C, protected from light with constant agitation, for 4 h and
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Table 7 Radioligand binding assay conditions for 5 HT6 and D2 receptors. Receptor source)
Radioligand (final concentration/Kd)
Nonspecific binding
Assay buffer
Incubation condition
5-HT6 (human recombinant, HEK-293 cells)
[3H]LSD (2.5/2.0 nM)
10 mM methiothepin
60 min, 37 C
D2 (human recombinant, CHOeK1 cells)
[3H]N-methylspiperone (0.4/0.2 nM)
10 mM (þ)-butaclamol
50 mM Tris, 10 mM MgCl2, 0.5 mM EDTA, pH 7.4 50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, pH 7.4
then diluted with assay buffer to a concentration of 250,000 cells/ml. After 1 h incubation 50 ml cell suspension was dispensed using automatic injectors built in radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque 96-well microplate preloaded with tested compounds. Immediate light emission generated following calcium mobilization (agonist response) was recorded for 30e60 s. In antagonist mode, after 15e30 min incubation reference agonist was added to the above assay mix and light emission was recorded again. Final concentration of reference agonist was equal EC80: serotonin 40 nM for 5-HT6 receptor, amethylserotonin 30 nM for 5-HT2A receptor, 100 nM serotonin for 5-HT1A receptor and apomorphine 30 nM for D2 receptor. IC50 and EC50 were determined by non-linear regression analysis using GraphPad Prism 6.0 software. The logIC50 was used to obtain the Kb by applying the Cheng-Prusoff approximation. The functional assays for 5-HT1A, 5-HT2A, 5-HT6 and D2 receptors presented in Tables 2 and 5 were performed at Faculty of Pharmacy, Jagiellonian University Collegium Medicum. 4.6. Extended in vitro studies of compound 47 Extended pharmacological profile in vitro for compound 47 (with the exception of functional studies for 5-HT1A, 5-HT2A, 5-HT6 ^que, and D2 receptors), was performed by Cerep (Le Bois l'Eve Poitiers, France). An outline of methodologies is shown in Tables 4 and 5. Further methodological details are available on the company website (www.cerep.fr). Data from all experiments were analyzed using non-linear curve fitting programs and results are given as Ki values for binding affinity or Kb values for antagonist potency. Blockade of hERG-mediated potassium currents was carried out by ChanTest (Cleveland, Ohio) and expressed as mean % of inhibition at 1.0E-06 M (Table 2) or IC50 value (Table 4). Ability to block hERG potassium channels was determined using the electrophysiological method and cloned hERG potassium channels (KCNH2 gene, expressed in CHO cells) as biological material. The effects were evaluated using IonWorks™ Quattro system (Molecular Devices Corporation, Union City CA). hERG current was elicited using a pulse pattern with fixed amplitudes (conditioning pre-pulse: 80 mV for 25 ms; test pulse: þ40 mV for 80 ms) from a holding potential of 0 mV. hERG current was measured as a difference between the peak current at 1 ms after the test step to þ40 mV and the steady-state current at the end of the step to þ40 mV. Data acquisition and analyses was performed using the IonWorks QuattroTM system operation software (version 2.0.2; Molecular Devices Corporation, Union City, CA). Data were corrected for leak current. The hERG block was calculated as: % Block ¼ (1 I TA/IControl) 100%, where IControl and ITA were the currents elicited by the test pulse in control and in the presence of a test compound, respectively. All assays for the lead compound 47 (shown in Tables 4 and 5) were performed in 3e4 independent experiments. The Ki and Kb values were determined from 6 concentrations from 1.0E-06 M to 1.0E-10 M. All the assays were carried out in duplicate (n ¼ 2).
60 min, 37 C
4.7. In vivo studies 4.7.1. Animals Male Wistar rats (Charles River, Sulzfeld, Germany) were kept four per standard plastic cage and housed in a room with constant environmental conditions (temperature: 22 ± 1 C, humidity: 60%, a 12-h lightedark cycle with lights on at 6:00 a.m.). Depending on the test, three- (300e500 g) or twenty-four month old male Wistar rats (600e900 g) were used. Animals were weighted and handled twice per week, for 3 weeks before the onset of experimental procedures. Standard lab chow (Labofeed H, WPiK, Kcynia, Poland) and tap water was available ad libitum unless otherwise stated. Separate groups of drug- and alcohol-naïve subjects (n ¼ 7e10) were used in all experiments. All tests were performed between 9:00 a.m. and 4:00 p.m. in soundproof experimental rooms under dim light and continuous white noise (65 dB). Behavioral parameters were recorded by microcomputers or scored by blinded observers. The tested compounds were administered intraperitoneally (i.p.) in all studies. Treatment of rats in the present study was in full accordance with the ethical standards laid down in respective European (Directive no. 86/609/EEC) and Polish regulations. The study protocol was reviewed and approved by a local ethics committee. 4.7.2. Porsolt's forced swim test The forced swim test is widely used to screen psychotropic medications for their anti-depressant-like properties [52]. The test was conducted as described by Porsolt et al. with some minor modifications [53,54]. Rats were individually placed in glass cylinders (diameter: 18 cm, height: 40 cm) filled with water (temperature: 23 C) at a height that made it impossible to reach the bottom with hind paws. There were two swimming sessions separated by 24 h: a 15-min. pre-test and a 5-min. test. The duration of immobility (s) in the test session was recorded by an observer from the adjacent room with the aid of a video camera. A rat was considered immobile when it floated not moving except to keep the head above water surface [53,54]. A decrease in the immobility time is thought to reflect anti-depressant-like effects of a tested drug. 4.7.3. Conflict drinking test (Vogel test) Anxiety Monitoring System “Vogel test” produced by TSE Systems was used. It was consisted of a polycarbonate cage (dimensions 26.5 15 42 cm), equipped with a grid floor made from stainless steel bars and a drinking bottle containing tap water. Experimental chambers (two) were connected to PC software by control chassis and a device that generates electric shocks. On the first day of the experiment, the rats were adapted to the test chamber for 10 min. After the adaptation period, the animals were deprived of water for 24 h and were then placed in the test chamber for another 10-min adaptation period during which they had free access to the drinking bottle. Afterwards, they were allowed a 30min free-drinking session in their home cages. After another 24-h water deprivation period, the rats were placed again in the test chamber. Recording data started immediately after the first lick and
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every 20 licks rats were punished with an electric shock (0.5 mA, lasting 1 s). The impulses were released via the spout of the drinking bottle. If a rat was drinking when an impulse was released, it received a shock. The number of licks and the number of shocks received throughout a 5-min experimental session were recorded automatically. A studied compound was administered to 10 rats per treatment group. 4.7.4. Hot plate and free-drinking tests To control possible drug-induced changes in shock sensitivity or the thirst drive (which may contribute to animals’ behavior in the Vogel conflict drinking test), stimulus threshold and water consumption during a free-drinking session were determined in separate groups of rats. In either of those two studies, the rats were manipulated similarly to the Vogel conflict drinking test, including two 24-h water deprivation periods separated by 10 min adaptation session in experimental cages and 30 min of water availability in their home cages. In the free-drinking test, each animal was allowed to freely drink from the water spout and the amount of water (g) consumed during 5 min was recorded for each rat. The pain threshold was investigated using hot plate test (Commat Ltd, Turkey) in rats [55]. The plate was enclosed with a transparent Plexiglass cylinder (35 cm high) to keep the animal on the heated surface of the plate. The latency to pain reaction (lick a hind paw or jumping) when the rat was placed on a hot plate (52.5 ± 0.5 C, 19 cm diameter) was measured. The rat was removed from the plate immediately upon visible pain reaction or if no response occurred within 30 s. 4.7.5. Open field test The open field test is widely used to screen psychotropic medications for their anxiolytic-like properties [53,56]. The open field apparatus consisted of four black, octagonal, stainless steel arenas (diameter: 80 cm) with 30-cm high walls. Each arena was divided into a peripheral and central sector. The peripheral sector was defined as the region within 20 cm from the walls. The approximate light intensity was 15 lx in the in the central point of the arena and 4 lx close to its walls. A rat was placed in the central point and the experiment started immediately afterwards. Locomotor activity was recorded for 10 min with the PC-based VideoMot System (TSE, Bad Homburg, Germany). The arenas were cleaned between tests with 20% (v/v) alcohol and allowed to dry. The following parameters were analysed: forward locomotion (cm), central visits, defined as the number of entries into the central sector with all four paws, and central time (s) defined as the total time spent in the central sector [56,57]. An increase in the number of central visits (and/or time spent in the central sector) is thought to reflect anxiolytic-like effects of a tested drug. 4.7.6. Responding for sweet reward: saccharin self-administration As decreased appetite and gustatory anhedonia are thought to be among the cardinal features of depression [37], drugs with antidepressant properties are expected to increase preference for sweet solutions in animal models of depressive symptomatology [58,59]. Saccharin is thought to be highly palatable to various animal species. Vigorous self-administration of low-concentrated saccharin solutions can be easily obtained in rodents [38]. In the present study, rats were trained to lever press for saccharin (0.1%; w/v) in 8 operant chambers (Skinner boxes; Coulbourn, Inc., Allentown, PA, USA) as described by Mierzejewski et al. [38]. The operant chambers consisted of stainless-steel test cages (E10-10 TC, Coulbourn) enclosed within sound-attenuating cubicles with fans for ventilation and background white noise. A white house light
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was centered near the top of the front wall of the cage. The start of each session was signaled by turning the house light on. The cage was also equipped with two response levers separated by the liquid delivery system (the liquid dipper; E14-05, Coulbourn). The liquid dipper and the response levers were mounted on the front wall of the cage, 4 cm above a stainless-steel grid floor. The liquid dipper consisted of an arm ending in a 0.1-ml stainless-steel cup, an electric engine to raise the arm, and a 100ml fluid reservoir located outside the test cage. Contacts with the liquid dipper were registered by small photocells located inside the dipper entrance. Only one lever (the active lever) activated the liquid dipper. Presses on the other lever (the inactive lever) were recorded but had no consequences. The location of the active lever (left vs. right) was randomized across all the subjects. The liquid dipper presented saccharin solution in a volume of 0.1-ml for 5 s. A white stimulus light (4 W) located inside the liquid dipper hole was on during the entire 5-s dipper activation. After that time, the liquid dipper and its stimulus light were switched off. Programming of every experimental session as well as data recording (active and inactive lever presses, contacts with the dipper entrance) was made using the L2T2 Software package (Coulbourn) running on an IBMcompatible PC. All self-administration sessions lasted 30 min and one session was given each day between 1:00 and 3:00 p.m. During the first 3 days of training, the rats were deprived of water for 22 h/day and shaped to lever press for 0.1% saccharin on the fixed ratio (FR) 1 schedule of reinforcement where one response on the active lever activated the liquid dipper. As soon as lever pressing was established, water started to be freely available in the home cages. Thus, except for the initial phase of training, the rats were never deprived of food or water. The subjects were allowed to stabilize their saccharin selfadministration in a maintenance phase which lasted 10 days. Increase in the number of active lever presses was treated as a possible indication of antidepressant-like effects of a tested drug.
4.7.7. Extinction of non-reinforced responding for sweet reward Extinction of saccharin seeking was tested as described by Mierzejewski et al. and Radwanska et al. [38,39]. Rats successfully trained to lever press for 0.1% saccharin as described above were randomly assigned to one of experimental groups administered with vehicle or 47. In extinction sessions, animals were exposed for 30 min to the Skinner boxes where they had previously been trained to selfadminister saccharin. Responding on the active or inactive lever was never reinforced with any reward. Three 30-min. extinction sessions were run on three consecutive days with 24-h intervals. A progressive decrease in the number of non-reinforced responses during subsequent extinction sessions is thought to reflect learning and memory processes associated with the absence of expected reward [38,39].
Acknowledgment This study was financially supported by Adamed Ltd. and Polish Agency for Enterprise Development (PARP), Warsaw, Poland. Grant nr UDA-POIG.01.04.00-14-055/10-00.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.12.045.
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