Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice

Accepted Manuscript Title: Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice Author: Anna Dziubina Karina Szmyd Małgorzata Zygmunt Jacek Sapa Magdalena Dudek Barbara Filipek Anna Drabczy´nska Michał Załuski Karolina Pytka Katarzyna Kie´c-Kononowicz PII: DOI: Reference:

S1734-1140(16)30095-0 http://dx.doi.org/doi:10.1016/j.pharep.2016.07.008 PHAREP 535

To appear in: Received date: Revised date: Accepted date:

11-3-2016 27-6-2016 22-7-2016

Please cite this article as: Anna Dziubina, Karina Szmyd, Małgorzata Zygmunt, Jacek Sapa, Magdalena Dudek, Barbara Filipek, Anna Drabczy´nska, Michał Załuski, Karolina Pytka, Katarzyna Kie´c-Kononowicz, Evaluation of antidepressant-like and anxiolyticlike activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice, http://dx.doi.org/10.1016/j.pharep.2016.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice

Anna Dziubinaa*, Karina Szmyda, Małgorzata Zygmuntb, Jacek Sapab, Magdalena Dudeka, Barbara Filipeka, Anna Drabczyńskac, Michał Załuskic, Karolina Pytkaa , Katarzyna KiećKononowiczc

a

Department of Pharmacodynamics, Jagiellonian University Medical College, Kraków, Poland

b

Department of Pharmacological Screening, Chair of Pharmacodynamics, Jagiellonian University

Medical College, Kraków, Poland c

Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian

University Medical College, Kraków, Poland

*Correspondence: Anna Dziubina, Department of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland E-mail address: [email protected] Tel/fax: +48-12- 6205535/ +48-12- 6205552

Abstract Background: It has recently been suggested that the adenosine A2A receptor plays a role in several animal models of depression. Additionally, A2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. Methods: In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with

affinity to A2A receptors but poor A1 affinity were evaluated for their

antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. Results: The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. Conclusion: Available data support the proposition that the examined compounds with adenosine A2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents.

Keywords: adenosine A2A receptor, antidepressant activity, imidazo[2,1-f]purinediones, pyrimido[2,1-f]purinediones

Introduction In recent years, much attention has been paid to the role of adenosine A2A receptors in Parkinson‟s disease (PD) and comorbid depression and anxiety. A2A receptors mediate excitatory actions on the nervous system by coupling with Gs proteins, which stimulate adenylyl cyclase [1].These frequently occur in the central nervous system, where they are localized on neurons [2] and glial cells [3]. In the brain, they are concentrated in the basal ganglia especially in areas with dopaminergic nigrostriatal and mesolimbic pathways [4]. Within the striatum, A2A receptors are localized on GABA-ergic striatopallidal neurons, where they are colocalized with dopamine D2 receptors. They are located presynaptically and control the release of a variety of neurotransmitters [2]. Sarges et al. [5] suggested that A2 selective non-xanthine adenosine antagonists induce activity in the swim test by a prolongation of escape-directed behavior, rather than by a locomotor stimulant effect. The A2A receptor knockout mice showed increased mobility in forced swim and tail suspension tests [6,7]. There is considerable evidence that adenosine A2A receptors are involved not only in behavioral despair [6,7,8], but also in learned helplessness [14], and cytokine- and reserpineinduced depression models [10,11]. In these models, adenosine A2A antagonists reverse behavioral deficits and exhibit a profile similar to that of tricyclic antidepressants (TCAs). Moreover, adenosine A2A receptor antagonists reverse reserpine- and haloperidol-induced motor deficits in animals [12]. It has been suggested that istradefylline (KW-6002, adenosine A2A antagonist) may have beneficial effects on depression as well as on motor symptoms in patients with PD [13,14]. Furthermore, similarly to desipramine, long-term caffeine (nonselective A1/A2A adenosine antagonist) exposure exerts an antidepressant effect in chronic unpredictable stress (CUS) [15]. Moreover, long treatment with A2A antagonists reversed the mood and synaptic dysfunction caused by CUS [15]. Some antidepressants may have different impacts on anxiety. For instance, imipramine (IMI) is devoid of effects on anxiety, and, venlafaxine, and citalopram additionally have anxiolytic action. However, drugs such as desipramine, or maprotiline show anxiogenic effects [16,17]. Clinical pharmacological studies and models of genetically modified rodents have implicated caffeine in the modulation of

different types of anxiety [18,19,20]. Given the antidepressant effect of purinedionederivatives, we have attempted to determine their impact on anxiety. The synthesis and pharmacological properties of some derivatives of purinediones have been described [21-23,27-29]. In the present paper, we report the results from pharmacological research on the activity of N-substituted 1,3- dimethyl- or 1,3- dipropyltetrahydropyrimido[1,2-a] purinediones and 1,3- dimethyl- dihydroimidazo-[1,2-a]-purinedione (Fig. 1). We investigated the effect of these new compounds on depression- and anxiolytic-like behavior, using selected animal test. Also, the effect of compounds on spontaneous locomotor activity was studied in order to exclude false-positive results in the assessment of antidepressant and anxiolytic-like activity. Materials and methods Animals The experiments were carried out on male Swiss Albino mice (CD-1) weighing 18-25 g. The animals were housed in groups of 15 in plastic cages (60 cm x 38 cm x 20 cm) with free access to water and food ad libitum, and kept at room temperature 22 ± 2 °C, under a light/dark (12:12) cycle. The mice were used after at least one day of acclimatization to the housing conditions and used only once in each test. Each group consisted of 8-10 mice. All the procedures were approved by the First Local Ethics Committee (68/2015).

Chemicals used in pharmacological assays

The following examined annelated xanthenes were obtained: KD 66, (C16H23O23N5; MW= 317.38) 9-cyclohexyl-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[1,2-a]purine2,4(1H,3H)-dione [30,32]; KD167 (C24H27O2N5;MW = 417.49) 9-(naphthalen-2-yl)-1,3dipropyl-6,7,8,9-tetrahydropyrimido[1,2-a]purine-2,4(1H,3H)-dione [34]; and KD 206, (C17H25O2N5; MW = 331.41) 8-cyclooctyl-1,3-dimethyl-7,8-dihydro-1H-imidazo[1,2a]purine-2,4(3H,6H)-dione [30]. These compounds were tested for A1/A2A adenosine receptor binding affinity: KD 66 rKiA2A= 0.81 ± 0.01μM, hKiA2A= 1.61 ± 0.41μM, rKiA1 >25 μM; KD 167 rKiA2A= 0.398 ± 0.111 μM, hKiA2A=1.39 ± 0.34 μM, rKiA1=1.29 ± 0.32 μM; KD 206 rKiA2A=0.33 ± 0.09 μM, hKiA2A=0.306 ± 0.024 μM, rKiA1 >25 μM [33,34], respectively. For the behavioral

experiments, the compounds were suspended in 0.5% methylcellulose (Loba Chemie, Germany) solution and administered by the intraperitoneal (ip) route 60 min before the test on mice. Control animals were given an appropriate amount of vehicle: 0.5% methylcellulose suspension, (ip) 60 min before the test. Diazepam (Polfa-Kutno, Poland) ampoules (concentration 5 mg/1 ml) were diluted with 0.9% saline to obtain a final concentration of 2 mg/10 ml and administered (ip) 60 min before the test. IMI and caffeine (Sigma-Aldrich, Germany) were dissolved in distilled water and were injected (ip) 60 min before the test. CSC 8- (3-chlorostyryl-caffeine) (Sigma Aldrich, Germany), used a reference drug, was suspended in 0.5% methylcellulose suspension and protected from the light. All solution were injected at a volume of 10 ml/kg. The tail suspension test in mice

The experiment was carried out according to the method described by Steru et al. [33]. Mice were suspended on the edge of a shelf above a table top by adhesive tape placed approximately 1 cm from the tip of the tail, with a minimum distance of 15 cm between the mice and other objects and hanging 75 cm above from the floor. The duration of immobility was performed by direct observation for a period for 6 min. A mouse was recognized as immobile when it hung completely limp. The forced swim test in mice

The experiment was carried out according to the method described by Porsolt et al. [34] with some minor modifications. Mice were individually placed into a transparent glass cylindrical tank (height 25 cm, diameter 10 cm) filled with water to a height of 10 cm, at a temperature of 23–25 °C. After 2 min of vigorous activity, the actual measurement began and this lasted 4 minutes, and included the time of measuring mouse immobilization. A mouse was regarded as immobile when floating motionless in the water, making only the necessary movements to keep its head above the water surface. The first group of mice was injected with test compounds once on a given day while the other group was injected with the compound for 13 days, and on day 14 the test was carried out 60 min after administration.

The four-plate test

The anxiolytic-like activity was evaluated in a four-plate apparatus (Bioseb, France) according to the method described by Aron [35]. The apparatus consists of a cage (25×18×16 cm) floored with four metal plates (11×8 cm) separated from one another by a 4 mm gap. The plates are connected to a power source, which generates electric foot shocks (0.6 mA; 0.5 s) whenever the investigator presses a button. After a 15 s habituation period each mouse was subjected to an electric shock when crossing from one plate to another (two limbs on one plate and two on another). The number of crossings was recorded during a 60 s test period. An increase of spontaneous punished behavior was used as a measure to determine the anxiolytic effects of compounds.

Spontaneous locomotor activity test

The locomotor activity was measured individually for each mouse in activity cages (Ugo Basile, Italy), supplied with I.R. horizontal beam emitters connected to a counter for the recording of light-beam interruptions. After the 60 min habituation period the number of crossings of photobeams (ambulation) was counted between 2nd and 6th min (i.e. time equal to the observation period in the behavioral test).

Statistical analysis

The results were statistically evaluated using one-way analysis of variance (ANOVA), followed by Dunnett's post -hoc comparison or Student's t-test to compare the results obtained in drug-treated and vehicle treated mice. Significance levels were set at p < 0.05.

Results Chemistry Tricyclic xanthine derivatives with the third heterocyclic ring fused to the f-bond and an additional aromatic or saturated ring substituent in the N-9 position were synthesized according to previously described procedures [30-32], (Fig. 1). Theophylline was used as the starting material for the synthesis of 1,3-dimethyl-imidazo- (KD 206) and -pyrimido[1,2-a]purinedione (KD 66) derivatives. Dipropylurea and cyanoacetic acid were employed as starting materials to obtain 1,3-dipropylxanthine under procedures

based on a modified version of Traube's method; 1,3-dipropylxanthine was then used for the synthesis of a 1,3-dipropylpyrimido[1,2-a]purinedione derivative (KD 167) [30-32], (Fig.1a). Subsequently, theophylline and dipropylxanthine were oxidatively brominated, and alkylated in position N-7 by 1,2-dibromoethane or 1-bromo-3-chloropropane in a phase transfer catalysis reaction. In the last step, 8-bromo-7-bromoalkyl-1,3-dialkylxanthine derivatives were condensed with amine, thus obtaining the final compounds [33-35] (Fig. 1b). Antidepressant-like activity in the tail suspension test in mice KD 66 and KD 206 (5, 10, 20 mg/kg) significantly reduced the duration of the immobility time by 61.5%, 45%, 65.8% [F(3,38) = 17.61, p < 0.001] and by 52.1%, 40.7%, 42.1%, [F(3,34) = 11.98, p < 0.001], respectively. KD 167 only at 5 mg/kg reduced the immobility time by 51.1%, [F(3,36) = 4.28, p < 0.05]. In contrast, KD 167 (10 and 20 mg/kg) did not decrease the immobility time. IMI (5, 10 and 20 mg/kg), significantly reduced immobility (by 63%, 69% and 95%, respectively) [F(3,38) = 43.67, p < 0.001]. Likewise, CSC (selective A2A antagonist) (1 and 5 mg/kg) reduced immobility (by 41.5% and 61%, respectively) [F(2,20) = 10.53, p < 0.001] (Fig. 2).

Antidepressant-like activity in the forced swim test after acute administration

Single treatment with KD 66 and KD 206 at all doses (5, 10, 20 mg/kg) (by 32%, 37%, 39% and by 29%, 31%, 33%, respectively), [F(3,33) = 6.29, p < 0.01] and [F(3,33) = 5.11, p < 0.01], respectively and KD 167 (5 and 20 mg/kg) (by 31% and 28%, respectively) [F(3,37) = 4.27, p < 0.05] decreased the immobility time. Only KD 167 at a dose of 10 mg/kg did not significantly reduce the immobility time. One-way ANOVA revealed significant effect of IMI at doses of 10 and 20 mg/kg [F(3,34) = 5.03, p < 0.01, respectively]. CSC (1 and 5 mg/kg) significantly reduced immobility (by 29% and 34%, respectively) [F(2,27) = 5,29, p < 0.05] (Fig. 3). The antidepressant efficacy of KD 66 (5-20 mg/kg), KD 206 (5-20 mg/kg), and KD 167 (5, 20 mg/kg) was similar to that of CSC (1, 5 mg/kg) and to the of IMI (10, 20 mg/kg).

Antidepressant-like activity in the forced swim test after chronic administration

All tested compounds (KD 66, KD 206, KD 167) (5 mg/kg) showed a statistically significant reduction of the time of immobility (by 43.5%, 52.4%, 25%, respectively), (p < 0.001, p < 0.001, p < 0.01, respectively). Simultaneously, CSC (1 mg/kg) (p < 0.05) and IMI (10, 20 mg/kg) significantly decreased the immobility [F(2,27) = 23.15, p < 0.001]. At 5 mg/kg KD 167 and 10 mg/kg IMI these compounds had the same antidepressant efficacies (about 25%). In turn, the antidepressant efficacy of KD 206 was comparable to that of IMI 20 mg/kg (53% and 60%, respectively) (Fig. 4).

Anxiolytic-like activity in the four- plate test KD 66 (5 mg/kg but not 10 and 20 mg/kg) increased the number of crossings (by 51%, [F(3,35) = 5.56, p < 0.05]). KD 206 (5-20 mg/kg) not significantly potentiated the number of crossings. KD 167 (5 and 10 mg/kg but not 20 mg/kg) reduced the number of crossings (by 39% and 66%, respectively) [F(3,38) = 10.50, p < 0.05]. Caffeine (10 mg/kg), significantly increased the number of punished crossings by 112% while caffeine (100 mg/kg) decreased by 57%, [F(2,28) = 8.26, p < 0.01]. Diazepam (2 mg/kg), used as a positive control increased the number of punished crossings by 87%, with p < 0.001. CSC (1 mg/kg) had no effect on the number of crossings (Fig. 5).

Influence on locomotor activity in mice Neither KD 66 nor KD 206 increased the mobility. Only KD 167 decreased the locomotor activity after acute [F(3,24) = 12.23, p < 0.001] and chronic administration, p < 0.05. CSC (1 mg/kg) and IMI (5-20 mg/kg) did not affect locomotor activity after acute and chronic administration. However, only CSC (5 mg/kg) and caffeine (10 mg/kg) increased locomotor activity after acute administration (Table 1). Discussion The present study investigated the antidepressant- and anxiolytic-like activity of two N-substituted tetrahydropyrimido[1,2-a]purinediones, KD 66 and KD 167 (differing in substituents at positions 1, 3 and 9), and N-substituted dihydroimidazo[1,2-a] purinedione KD 206, with affinity to the A2A receptor but poor A1 affinity (Fig. 1). Our previous studies showed that some of the derivatives of imidazo- and pyrimido-purinediones demonstrate antiparkinsonian activity in “oxotremorine” and “reserpine” tests [23] and attenuate the

cataleptic effects of haloperidol [24]. There is evidence that the adenosine A2A receptor plays a role in several animal models of depression [10,11]. Additionally, A2A antagonists reverse behavioral deficits and their effect resembles that of classical antidepressants [9,13]. Further, the adenosine A2A receptor antagonist block the effects of antidepressants compounds such creatine, ketamine, adenosine and inosine, but not fluoxetine [25,26] and at the same time exhibit antidepressant effects [6]. The forced swim test and tail suspension test are highly predictive for antidepressant drug activity. Immobility produced in both tests is taken as paradigm of depression and antidepressant drugs reduce the immobility period. But the tail suspension test differs from the closely related forced swim test. Firstly, there is considerably less stressful to experimental animals than forced swim test because no hypothermia is induced as compared to forced swim test in which animal is immersed in water. Secondly, the tail suspension test detect not only typical ADs but also atypical and new potential ones, like adenosine A2A antagonist [6,7,34,36]. Furthermore, antidepressant response depends on the enhancement of central catecholamine and serotonin neurotransmission in the both tests but the involvement of different neuronal mechanisms has been suggested [36]. Using the both tests we showed that acute administration of KD 66 (5-20 mg/kg), KD 206 (5-20 mg/kg) and KD 167 (5 mg/kg), and repeated administration of lowest doses of the compounds exerted antidepressant effect. Acute administration of compounds as well as chronic treatment significantly reduced immobility, with an efficacy comparable to that obtained by chronic treatment with TCAs such as IMI. However, the strongest antidepressant efficacy of IMI was obtained only after one dose of 20 mg/kg in the tail suspension test. Only KD 167 produced distinct effect in both tests while the antidepressant-like effect of KD 66 and KD 206 was comparable. Although KD 167 and KD 66 belong to the same chemical group, they reduce the duration of immobility to varying degrees. KD 167 decreased locomotor activity, which suggests that might influence its effect on the duration of immobility time in the tests. In both tests, CSC (similarly to istradefylline (KW-6002) [9,13] showed dose-dependent antidepressant-like activity comparable to that of IMI. This date may suggest that the examined compounds have rapid onset of antidepressant activity and the mice do not develop tolerance to the antidepressant-like effects after chronic drug-treatment. However, it was to suggested that neither serotonergic nor adrenergic neurotransmission was involved in the antidepressant-like effect of A2A antagonist [9,13].

Moreover, compounds such KD 66 and KD 206 demonstrated in the tail suspension test the tendency to bell- shape dose-response. This effect is common for antidepressants with different mechanisms of action, and in this case it may be a result of some non-specific activity of the compounds. It is only hypothesized that the lack of typical dose-dependence, and the reduction of the effectiveness of a higher dose of KD 167 indicate that some nonspecific activity of KD 167 prevents a further decrease in immobility time. This may be due to its sedative properties (related to the chemical structure and the speed of the penetration to the central nervous system), which prevent further decrease of immobility at higher doses (1020 mg/kg), or that all A2A receptors seem to be saturated with lower dose of KD 167, and that further augmentation of its dose has no effect on the immobility time. The reliability of the results is supported by the fact that none of the investigated compounds increased spontaneous locomotor activity. This means that the demonstrated antidepressant-like activity of the compounds is not a false positive. However, a concomitant reduction in locomotor activity of mice was observed, which could have contributed to the prolongation of immobility time in the forced swim test. Only CSC (5 mg/kg) increased the mobility, that suggests, that its anti-depressive effect was non-specific. Similarly, caffeine-induced mobility greatly complicates the interpretation of these results [8,37,39]. It is also relevant that treatment with caffeine may yield false positive results in the forced swim test [7]. Summing up -investigated compounds with antidepressant activity possess strikingly different structure in comparison to typical antidepressants. Typical antidepressants are tricyclic structures, thanks middle unsaturated ring not planar, containing basic center. Although investigated structures are also tricyclic their organization of coupled rings is quite different, the main structure core imidazo- and pyrimido[1,2-a]purinediones is planar. Further the structures are characterized by the lack of basic center. Such different structures may promise quite different profile of pharmacological activity. The second part of this study was aimed at an evaluation of anxiolytic behavior. For this purpose, we used a four-plate test to investigate the acute effect of diazepam-the most potent benzodiazepine (BZD), caffeine, CSC and KD 66, KD 206 and KD 167. The four-plate test allows for the detection not only of the anxiolytic effect of BDZs, but also of other nonBZD anxiolytic compounds [35]. It should also be mentioned that not all antidepressants are active in the four -plate test [16]. KD 66 at lowest dose (5 mg/kg) showed anxiolytic-like activity while KD 167 (5 and 10 mg/kg) caused anxiogenic effect. KD 206 (structurally different from other compounds)

similarly as CSC presented a lack of anxiolytic-like properties. The anxiolytic-like efficacy of KD 66 was weaker than that of diazepam. This discrepancy is not clearly explained at the moment. Currently, only some selective A2A antagonists have been reported to ameliorate anxiety-like behaviors in rodents, while others do not affect these behaviors [38]. The role of the A2A receptor in the mechanisms of the formation of the anxiety response has not been precisely explained [19,38]. Such various influence of the investigated compounds on an anxiety can be the result of other chemical structure but also can be related to differences in potency, efficacy, and/or selectivity for modulating the A2A receptor function in the brain or differences in the effects on motor activity. The strong inhibition of locomotor activity in mice may give „false‟ negative results in four-plate test. Thus, the sedative properties of KD 167 (at the doses tested) can influence on its lack of anxiolytic-like activity. Affinities for other receptors, channels, transporters related to serotoninergic, gabaergic and purinergic neurotransmission don't seem to have essential, because the anxiolytic-like action of istradefylline (KW-6002) depend only on the blockade A2A receptor [38]. Further, certain from these effects could depend on the doses. Caffeine at the lowest dose induces an anxiolytic-like effect, whereas at a higher dose it stimulates anxiogenic-like behavior. Similar effects were observed after administration of KD 206 (Fig. 5). Solving this problem requires further study. Conclusion To conclude, in the present paper we have shown the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. In addition, compound KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Available data support the proposition that the examined compounds with adenosine A2A receptor affinity may be an interesting target for the development of antidepressant and/or anti-anxiety agents.

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target drugs for the potential treatment of neurodegenerative diseases. Bioorg Med Chem 2013;21:7435-7452. [30]

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Legends Fig. 1. Chemical structures and syntheses of the test compounds: KD 66, KD 167, KD 206 Fig.1a. Synthesis of dipropylxanthine. a - (CH3CO)2O, CH3COOH; b - NaNO2, 50% CH3COOH; c Na2S2O4, NH3 aq; d - 40 % HCOOH, NaOH aq. Fig.1b. Synthesis of imidazo- or pyrimido-[2,1-f]purinedione derivatives. i - 40% HBr, NaClO3,AcOH; ii - X(CH2)nX, TEBA, K2CO3; iii - R2-NH2; X - Cl, Br; n - 2, 3

Fig. 2. The effect of KD 66, KD 206 and KD 167 on immobility time in tail suspension tests in mice.

These compounds and imipramine (IMI), CSC were given (ip), 60 min before the test. Statistical analysis: one-way ANOVA, Dunnett's post- hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective vehicle-treated group (0.5% methylcellulose), n= 8-10 mice.

Fig. 3. The effect of KD 66, KD 206 and KD 167 on the immobility time in the forced swim test. These compounds and imipramine (IMI), CSC were given (ip), acutely (1x), 60 min before the test. Statistical analysis: one-way ANOVA, Dunnett's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective vehicle-treated group (0.5% methylcellulose), n = 8–10 mice.

Fig. 4. The effect of KD 66, KD 206 and KD 167 on the immobility time in forced swim test. These compounds and imipramine (IMI), CSC were given (ip), daily for 14 days. The last doses of treatments were injected 60 min before the test (on day 14 of the experiment). Statistical analysis: one-way ANOVA, Dunnett's post-hoc test; t-Student test: ***p < 0.001 (KD 66, KD 206), **p < 0.01 (KD 167). *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective vehicle-treated group (0.5% methylcellulose), n = 8–10 mice.

Fig. 5. The effect of KD 66, KD 206 and KD 167 on punished passages in four-plate test. These compounds and diazepam, caffeine, CSC were administered (ip), 60 min before the test. Statistical analysis: one-way ANOVA, Dunnett's post-hoc, t-Student test for diazepam, *p < 0.05, **p < 0.01,***p < 0.001 vs. respective vehicle-treated group (0.5% methylcellulose), n = 8–10 mice.

Table 1. The influence of KD 66, KD 206, KD 167, IMI, caffeine and CSC on locomotor activity in mice; A. acute administration, B. chronic administration; *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective vehicle-treated group (0.5% methylcellulose); n= 8 mice per group. A. Compounds

(%) of control

5 10 20 5 10 20 5 10 20 5 10 20 1 5 10 50 100

Number of crossings during 2-6 (min) 297.6 ± 31.14 207 ± 41.30 266.83 ± 37.79 228.67 ± 45.92 257.67 ± 21.91 347.33 ± 34.67 257.33 ± 27.02 60.00 ± 16.41*** 63.33 ± 28.43*** 114.8 ± 51.85** 354.0 ± 28.20 189.0 ± 27.89 279.3 ± 46.37 229.2 ± 73.9 441 ± 35* 360 ± 21.18 285.75 ± 50.71 136.25 ± 68.76*

Vehicle

-

358.3 ± 80.1

100

KD 66 KD206 KD167

5 5 5 10 20 1

319.5 ± 53.8 307 ± 89.1 223 ± 24.1* 286.3 ± 47.5 209.7 ± 49.5* 218.2 ± 43.2

89.2 85.7 62.2 79.9 58.5 60.9

Vehicle KD 66

KD 206

KD 167

IMI

CSC Caffeine

Dose (mg/kg)

100 69.6 89.7 76.8 85.6 116.7 86.5 20.2 21.3 38.6 119 63.5 93.9 77 148.6 121 96 45.8

B.

IMI CSC

Funding This study was partly supported by a Polish National Science Center grant, given on the basis of decision no. DEC-2012/04/M/NZ4/00219 and Jagiellonian University grant number K/ZDS/004125.

Conflict of interest The authors have declared no conflicts of interest

Figure 1a

b

a

d

c

Figure 1b

ii

i

iii

KD 66, KD 167; KD 206 KD 66: R1- methyl; R2- cyclohexyl; n- 2; KD 167: R1- propyl; R2- 2-naphthyl; n- 2; KD 206: R1- methyl; R2- cyclooctyl; n- 1;

Figure 2

Figure 3

Figure 4

Figure 5