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European Journal of Medicinal Chemistry 60 (2013) 42e50

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European Journal of Medicinal Chemistry

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Original article

Antidepressant and antipsychotic activity of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole targeting serotonin 5-HT1A/5-HT2A/5-HT7and dopamine D2/D3 receptors

Pawe1 Zajdel a,*, Krzysztof Marciniec b, Andrzej Ma�slankiewicz b, Katarzyna Grychowska a,Grzegorz Sata1a c, Beata Duszy�nska c, Tomasz Lenda c, Agata Siwek d, Gabriel Nowak d, Anna Partyka e,Dagmara Wróbel e, Magdalena Jastrzebska-Wiesek e, Andrzej J. Bojarski c, Anna Weso1owska e,Maciej Paw1owski a

aDepartment of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, PolandbDepartment of Organic Chemistry, Medical University of Silesia, 4 Jagiello�nska Street, 41-200 Sosnowiec, Polandc Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Kraków, PolanddDepartment of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, PolandeDepartment of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland

a r t i c l e i n f o

Article history:Received 15 October 2012Received in revised form23 November 2012Accepted 29 November 2012Available online 5 December 2012

Keywords:Quinoline-sulfonamidesIsoquinoline-sulfonamidesLong-chain arylpiperazines2,3-DichlorophenylpiperazineSerotoninDopamine5-HT2A antagonistD2 partial agonist5-HT7 antagonistAripiprazoleDepressionSchizophrenia

* Corresponding author. Tel.: þ48 12 620 54 50; faE-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.11.042

a b s t r a c t

A series of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole was synthesized toexplore the influence of two structural features e replacement of ether/amide moiety with sulfonamideone, and localization of a sulfonamide group in the azine moiety. In contrast to aripiprazole, compound33 (N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)quinoline-7-sulfonamide) and 39 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-3-sulfonamide) displaying multireceptor 5-HT1A/5-HT2A/5-HT7/D2/D3 profile, and behaving as 5-HT1A agonists, D2 partial agonists, and 5-HT2A/5-HT7antagonists, produced significant antidepressant activity in FST in mice. On the other hand, their 4-isoquinolinyl analog 40 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-4-sulfonamide),with similar receptor binding and functional profile, additionally displayed remarkable antipsychoticproperties in the MK-801-induced hyperlocomotor activity in mice.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Affective disorders, e.g. major depressive disorder (MDD),bipolar disorder and schizophrenia, are highly debilitating diseaseswhose treatment, despite development of novel therapy strategies,is still unsatisfactory because of poor efficiency and unwantedeffects of antidepressant/antipsychotic drugs. Furthermore, themajority of patients with MDD fail to achieve full remission with

x: þ48 12 620 54 05.

son SAS. All rights reserved.

currently available antidepressants. Additional unmet clinicalneeds include long time of onset of action, sexual dysfunction,nausea/emesis, weight gain, and/or cardiovascular effects.

In a development of CNS active agents, the multitarget strategyseems to be one of the most valuable approaches. Accordingly,several recently launched antidepressant/antipsychotic drugs e.g.amisulpride and aripiprazole, or compounds investigated in clinicaltrials (vortioxetine) [1] display mixed serotonin (5-HT) and dopa-mine (D) receptor profile. It was recently found, that these drugsbehave as 5-HT7 receptor antagonists, and this mechanism isresponsible for their antidepressant properties [2]. Moreover, themultimodal receptor profile (5-HT1A, 5-HT2A, 5-HT7, D2, D3) of

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50 43

aripiprazole, and its functional profile e partial agonist of D2 and 5-HT1A receptors and antagonist of 5-HT2A and 5-HT7 sites, mightunderline its board efficacy e antipsychotic, antidepressant, andanxiolytic effects.

Extending a concept of mixed 5-HT/D receptor agents as novelantipsychotics, Butini et al. [3] discovered a series of aripiprazoleanalogs combining high affinity for 5-HT1A, 5-HT2A receptors, andlow affinity for D2 sites, at the same time displaying high affinityfor D3 receptors and antagonistic properties at these sites.Structures of compounds were based, among others, around 2,3-dichlorophenylpiperazine core structure, which was functional-ized with isoquinoline-amide and quinoline- and isoquinoline-ether moieties (I, II; Fig. 1).

We have previously explored multireceptor strategy and re-ported on quinoline- and isoquinoline-sulfonamide derivatives oflong-chain arylpiperazines (LCAP) with 3- or 4-chloro-phenyl-piperazine moieties, as potential antidepressant, anxiolytic, andantipsychotic agents. Among them, compound III (Fig. 2)withmixed5-HTandD2, D3 receptor profile, behaved as antagonist of 5-HT1A, 5-HT2A, 5-HT7, D2 postsynaptic sites, and produced antidepressant-like effect in the forced-swim test in mice. On the other hand,a semi-rigid compound IV containing4-isoquinolinylmoiety (Fig. 2),that behaved as 5-HT2A, 5-HT7, and D2 antagonist, and showedaffinity for D3, andD4 receptors, produced antidepressant-like effectand additionally displayed anxiolytic activity in plus-maze test andantipsychotic properties in the MK-801 induced hyperlocomotoractivity inmice, amodel related tonegative symptoms and cognitiveimpairments observed in schizophrenia [4].

Continuing our study in a group of azinesulfonamide derivativesof LCAP [4,5], and to extend the studies aimed at verification of theimpact of localization of sulfonamide group in the azine moiety andthe length of the linker, we designed a series of quinoline- andisoquinoline-sulfonamide analogs of aripiprazole. Herein, wereport on their synthesis, and evaluation for selected serotoninergic(5-HT1A, 5-HT2A, 5-HT6, 5-HT7), dopaminergic (D2, D3), adrenergica1 receptors, and serotonin transporter (SERT), as well as deter-mination of their intrinsic activity. We also present results ofpreliminary evaluation of selected compounds in animal models ofdepression and schizophrenia aimed at validation of the multi-receptor approach and identification of structural elementsresponsible for in vivo activity.

2. Results and discussion

2.1. Chemistry

The synthesis of target compounds (31e40) was carried out asshown in Scheme 1, in reaction of the primary amines (29, 30) withappropriate azinesulfonyl chloride (22e28) in the presence ofHunig’s base. The quinoline- and isoquinoline-sulfonyl chlorides

Aripiprazol

HNO O

I

N ON

NCl

Cl

Fig. 1. Aripiprazole and its quinoline- and

(22e28) were prepared from respective bromoquinolines andbromoisoquinolines, according to the previously reported method[6]. For the pharmacological evaluation compounds 31e40 wereconverted into their water-soluble hydrochloride salts.

2.2. Pharmacology

2.2.1. In vitro evaluationRadioligand binding assays were employed for determining the

affinity and the selectivity profile of the synthesized compounds forcloned 5-HT1A, 5-HT2A, 5-HT6, 5-HT7 and dopamine D2 receptors.This was accomplished by displacement of respective radioligandsfrom cloned human receptors, all stably expressed in HEK293 cells:[3H]-8-OH-DPAT for 5-HT1AR, [3H]-ketanserin for 5-HT2AR, and[3H]-LSD for 5-HT6R, [3H]-5-CT for 5-HT7R and [3H]-raclopride forD2R. Displacement of [3H]-methylspiperone from the clonedhuman receptor stably expressed in HEK293 cells was used in D3Rbinding experiments (Cerep, Le Bois l’Eveque, 86600 Celle L’Ev-escault, France) [7].

Compounds affinity for a1-adrenergic receptors and serotonintransporter (SERT) was accomplished by determining their abilityto displace [3H]-prazosin and [3H]-citalopram, respectively, fromrat cerebral cortex.

2.2.2. Functional in vitro and in vivo evaluationThe functional activity of the selected compounds (33, 34, 37, 39,

40) on 5-HT1A and 5-HT7 receptors, on intracellular cAMP levels,was determined at Cerep according to the previously publishedmethods [8,9]. Assays were carried out in CHO and HEK-293 cells,which stably expressed the human 5-HT1A and 5-HT7 receptors,respectively, and using 8-OH-DPAT and methiothepin as referencecompounds.

The functional activity of the selected compounds (33, 34, 37, 39,40) at D2 and 5-HT2A receptors was tested in the commonly usedin vivo models. To determinate D2 receptor antagonistic effect ofthe tested compounds, their ability to abolish the apomorphine-induced climbing test in mice was tested. The antagonistic 5-HT2Aproperties were assessed in the (�)DOI-induced head twitchresponses in mice [10].

2.2.3. Behavioral evaluationIn the successive phase of our investigations the most in vitro

active derivatives, i.e. 33, 34, 37, 39 and 40 were tested in vivo inbehavioral tests commonly used to predict antidepressant- andantipsychotic-like activity. To evaluate the potential antidepressantproperties of the investigated compounds, the forced swim test(FST) in mice was employed [11]. The influence of the effectivedoses recorded in the FST was studied in spontaneous locomotoractivity in mice in order to exclude the possibility of competingbehaviors such as general locomotor activity. Furthermore,

e

NN

ClCl

II

HN

NN

ClClO

N

isoquinoline amide and ether analogs.

N

SN

OO

NN

Cl

IVN

SNH

OO

III

NN Cl

Fig. 2. The structure of III, a mixed 5-HT1A/5-HT2A/5-HT7/D2/D3 ligand and IV, a mixed 5-HT2A/5-HT7/D2/D3/D4 agent.

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e5044

spontaneous locomotor activity and the hyperactivity induced byMK-801 were used to evaluate specific antipsychotic-like activity inmice. Citalopram and aripiprazole were used as reference drugs.

2.3. Structureeactivity relationship studies

Development of CNS active compoundswas often abandoned bylowaqueous solubility, which greatly hindered in vivo evaluation ofarene derivatives [12,13]. Starting from a series of arene- andquinoline-sulfonamides of LCAP derivatives [5] we have recentlyreported on the design, synthesis and pharmacological evaluationof a series of quinoline- and isoquinoline-sulfonamides of flexibleor semi-rigid LCAP derivatives [4]. In contrast to limited watersolubility of parent arene derivatives, a series of azinesulfonamidesdisplayed improved solubility, still exerting high therapeuticpotential.

In view of the literature data presenting four receptor systems e5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 e as targets for effective treat-ment of affective disorders, all the synthesized compounds 31e40were evaluated for these sites (Table 1). Quinoline- andisoquinoline-sulfonamides displayed high affinity for 5-HT1A, 5-HT2A, and 5-HT7Rs (14e112 nM, 22e115 nM and 12e124 nM,respectively), and moderate-to-low for 5-HT6R (220e1331 nM).Generally, in the evaluated quinoline derivatives of 2,3-dichlorophenylpiperazines, elongation of propylene spacer tobutylene one, decreased affinity for 5-HT1A receptor, simulta-neously increased affinity for 5-HT2A sites, and either retained ordecreased affinity for 5-HT7R.

It was previously found, in a series of 2-methoxy-phenyl-piperazine (PhP) and 3-ClePhP derivatives of quinoline- andisoquinoline-sulfonamides, that localization of the sulfonamidebond in the pyridine ring of quinoline was preferential for inter-action with 5-HT1AR. Although the previously established rankorder (3-quinolinyl > 6-quinolinyl > 8-quinolinyl) remained

X = N, CHn = 1, 2z = 3-Q, 6-Cl-3-Q, 6-Q, 7-Q8-Q, 3-isoQ, 4-isoQ

zSN

OO

nHXX

31 - 40

zBr

XX

1 - 7

zSNa

XX

8 - 14

i ii

Scheme 1. Synthesis of azinesulfonamide derivatives of LCAP 31e40. Reagents and conditio30 min (iv) DIEA, CH2Cl2, 0 �C / rt.

unchanged within both the propylene and butylene derivatives of2,3-diClePhPs, the 7-quinolinyl fragment, revealed to be the mostfavorable for interaction with 5-HT1ARs. It thus confirmed thestructural analogy for dihydroquinolin-2-one moiety of parentaripiprazole. As observed previously, introduction of halogen atomto the 3-quinolinyl moiety led to decreased affinity for 5-HT1AR,and had little effect on the 5-HT2A, and 5-HT7R affinity; this wasexemplified by 35, whose binding constant for 5-HT1AR decreasedup to 4 folds.

Furthermore, compounds 33 and 36 containing 7-quinolinylfragment displayed the highest affinity for 5-HT2A and 5-HT6Rs inthe quinoline set. This quinolinyl fragment was also the mostpreferable, among propylene analogs 31e33, for binding to 5-HT7R.On the other hand, such preference was not observed withinbutylene derivatives; compounds 34, 37, and 38, with 3-quinolinyl,7-quinolinyl, and 8-quinolinyl fragments, respectively, displayedcomparable affinity for 5-HT7Rs.

Replacement of the 8-quinolinyl moiety 38 with its a-positioncounterpart e 4-isoquinolinyl, resulted in compound 40, and up to2-folds increased affinity for 5-HT1A and 5-HT6Rs, and up to 3-foldsdecreased affinity for 5-HT2ARs.

Interestingly, 3-isoquinolinyl derivative 39 and 4-isoquinolinylone 40 displayed opposite preference for 5-HT1A and 5-HT7receptors. This may suggest, that additional hydrogen bondsformed between sulfonamide moiety in position 3 of isoquinolineand an endocyclic nitrogen atom, which stabilizes compoundsstructure in the terminal fragment, may account for higher affinityof 39 for 5-HT1AR.

In line with the previous studies revealing new insights intotherapeutic role of 5-HT1AR in treating of various CNS disorders[15], and extending our aim to discover compounds combiningmultiple pharmacological action, in the next move of in vitroevaluation, compounds with high affinity for 5-HT1A sites(Ki < 60 nM), were investigated for D2, D3 receptors. For these

N

iv

N

zSCl

OO

XX

29, 30

ClCl

zSH

XX

15 - 21 22 - 28

iii

H N Nn N

ClCl

ns: (i) MeSNa (5 mol equiv.), DMF boiling temp., 4 h (ii) conc. HCl, 0 �C (iii) NaOCl, 5 �C,

Table 1The binding data of the quinoline- and isoquinoline-sulfonamides (31e40) for 5-HTreceptors.

Compd Azinyl n Ki � SEM [nM]

5-HT1A 5-HT2A 5-HT6 5-HT7

31 6-Cle3-Quinolinyl 1 23 � 2 115 � 18 901 � 8 46 � 332 6-Quinolinyl 1 25 � 3 65 � 7 326 � 27 31 � 233 7-Quinolinyl 1 14 � 2 47 � 1 257 � 19 12 � 134 3-Quinolinyl 2 23 � 4 34 � 3 1331 � 94 30 � 335 6-Cle3-Quinolinyl 2 88 � 6 42 � 2 555 � 42 41 � 236 6-Quinolinyl 2 79 � 9 23 � 2 484 � 30 124 � 737 7-Quinolinyl 2 17 � 3 22 � 1 301 � 16 31 � 238 8-Quinolinyl 2 112 � 16 29 � 4 598 � 76 27 � 439 3-Isoquinolinyl 2 34 � 1 35 � 2 454 � 37 56 � 840 4-Isoquinolinyl 2 59 � 2 90 � 14 220 � 12 21 � 1Aripiprazole e 5.6 � 0.8a 21 � 1 90 � 1 26 � 2

a Data for 5-HT1A taken from Ref. [14].

Table 2The binding data of the selected quinoline- and isoquinoline-sulfonamides fordopamine receptors.

Compd Azinyl n Ki � SEM [nM]

D2 D3a SERT a1

33 7-Quinolinyl 1 16 � 2 90/99 224 � 6.2 186 � 0.534 3-Quinolinyl 2 9 � 1 94/99 1200 � 12 170 � 1.637 7-Quinolinyl 2 11 � 1 99/101 788 � 25 136 � 2.639 3-Isoquinolinyl 2 17 � 1 95/98 335 � 6.2 255 � 21.540 4-Isoquinolinyl 2 8 � 1 93/95 783 � 24 160 � 3.9Aripiprazole 0.8 � 0.1 9.7 � 5.4b 1080 � 180b 25.7 � 5.0b

a Screening procedure e displacement % at 10�7/10�6.b Data taken from Ref. [14].

Table 3Effects of 33, 34, 37, 39, 40 on (A) cAMP accumulation (agonistic effect) and (B) 5-HT-induced cAMP accumulation in vitro (antagonistic effect).

Compound 5-HT1A EC50 (M) 5-HT7 IC50 (M)

33 7.4 � 10�6 2.8 � 10�6

34 1.65 � 10�6 7.8 � 10�6

37 8.65 � 10�7 3.8 � 10�6

39 1.55 � 10�5 5.6 � 10�6

40 1.5 � 10�5 8.2 � 10�7

8-OHeDPAT 1.5 � 10�8 e

Methiothepine e 2.2 � 10�9

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50 45

assays one representative containing propylene spacer (33) andfour compounds with butylene spacer (34, 37, 39, 40) were selected(Table 2). The binding data revealed that all compounds displayedhigh affinity for D2 and D3 receptors. Furthermore, compoundsshowed moderate-to-low affinity for SERT. Among them, thehighest affinity still in moderate range (224e335 nM), displayedcompound 33 and 39. Since a1 receptors may be considered aspotential source of undesirable cardiovascular effects, and addi-tionally blockade of these receptors may decrease effectiveness ofantidepressant drugs, selected compounds were also tested forthese sites (Table 2).

Generally, the compounds selected for extended in vitro eval-uation may be classified as multimodal serotonin and dopaminereceptor ligands with high affinity for 5-HT1A/5-HT2A/5-HT7 andD2/D3 receptors, and moderate affinity for SERT and a1. Incomparison to the previously reported by Butini et al. [3], ether oramide analogs of aripiprazole, an introduction of azinesulfonamidegroup remarkably increased affinity for D2 receptors.

2.4. Functional evaluation

Among compounds selected for functional evaluation 34, 37, 39,40 behaved as weak 5-HT1A receptor agonist, while 33 displaying58% of maximal effect of 5-HT, was classified as partial agonist(Table 3A) [8]. Evaluated compounds inhibited cAMP accumulationinduced by 5-HT, and thus were classified as weak antagonists atthe h5-HT7Rs (Table 3B) [9].

The same compounds and aripiprazole revealed some agonisticproperties for D2 receptor as well, evoking climbing behavior inmice. Assuming, that the effect produced by apomorphine (3 mg/kg) is 100%, aripiprazole (1 mg/kg), 33 (5 mg/kg), 34 (5 mg/kg), 37(5 mg/kg), 39 (1.25 mg/kg) and 40 (5 mg/kg) displayeda maximum agonist effect of about 50, 50.5, 49.3, 43.6, 49.1 and44.6%, respectively. Moreover, dose-dependent and considerablereduction of the apomorphine-induced climbing behavior of micewere observed for agents 34 (10e40 mg/kg), 37 (10e30 mg/kg),39 (10e30 mg/kg), 40 (5e20 mg/kg) and aripiprazole (0.03e3 mg/kg). Compound 33 (1.25e30 mg/kg) decreased that effectin statistically significant manner only in a dose of 2.5 mg/kg(Table 4).

All tested derivatives 33 (5e30 mg/kg), 34 (10e30 mg/kg), 37(5e20 mg/kg), 39 (5e20 mg/kg) and 40 (1.25e20 mg/kg) as well asaripiprazole (2.5e10 mg/kg) dose-dependently inhibited the (�)DOI-induced head twitches in mice (Table 4) [10]. The highestactivity in apomorphine-induced climbing as well as in DOI-induced head twitches tests displayed compound 40(ID50 ¼ 11.29 mg/kg and 5.3 mg/kg, respectively).

Summarizing the results of our in vitro and in vivo functionalstudy it was found that the investigated compounds behaved as 5-HT1A receptor agonists, 5-HT2A, 5-HT7 receptor antagonists and D2

receptor partial agonists.

2.5. Behavioral evaluation

Regarding many classes of 5-HT receptors described in CNS,much attention has been devoted to the role of 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptor subtypes in many psychiatric disorders, e.g.depression. Behavioral effects similar to those of SSRIs have beenproduced by agonists of 5-HT1A and 5-HT6 receptors. Also, antag-onists of 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors have beenreported to display antidepressant-like responses [16].

The obtained results indicate that all of the tested compounds33 (2.5e10 mg/kg), 34 (5e20 mg/kg), 37 (5e20 mg/kg), 39 (2.5e20 mg/kg), 40 (10e30 mg/kg) and citalopram (1.25e5 mg/kg),used as a reference drug, revealed antidepressant-like properties,shortening to a various extent the immobility time of mice. On theother hand, aripiprazole (0.01e0.1 mg/kg), used as a referenceantipsychotic drug, with structural similarity to the investigatedcompounds was inactive in that test (Fig. 3). The doses of aripi-prazole used in our investigations are based on the active doses inFST and TST (tail suspension test) reported by Sarkisyan et al., who

Table 4Effects of the tested compounds and aripiprazole on (A) the apomorphine-inducedclimbing behavior and (B) on the (�)DOI-induced head-twitches in Swiss albinomice.

Compound A (ID50a mg/kg i.p.) B (ID50

a mg/kg i.p.)

33 e 10.22 (9.30e11.13)34 20.74 (13.9e27.57) 20.21 (16.99e23.43)37 19.94 (18.18e21.71) 4.58 (4.07e5.09)39 19.90 (15.11e24.70) 9.03 (3.94e14.12)40 11.29 (5.53e17.05) 5.30 (3.75e6.74)Aripiprazole 1.58 (0.38e3.54) 9.89 (Wide range)

a ID50 e the dose inhibiting climbing behavior or head twitches in mice by 50%;confidence limits (95%) given in parentheses (Graph Pad Prism 5 Software).

Fig. 4. Effects of the tested compounds and aripiprazole on the MK-801-inducedhyperlocomotor activity in CD-1 mice.

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e5046

emphasized 5-HT7 receptor antagonistic properties as a potentialmechanism of antidepressant efficacy [2]. Similarly to our study,the lack of aripiprazole’s antidepressant-like activity in FST wasreported by Bourin et al. [17], who indicated that at the dose of0.125 mg/kg aripiprazole decreased the locomotor activity of mice.These discrepancies may be caused by different strains of mice usedin the laboratories.

Compounds 34 and 37 administered at higher doses of 10 and20mg/kg reduced by 31% and 30% (34) and by 48% and 41% (37), theimmobility time of mice in FST. Compound 40 was active only ina medium dose of 20 mg/kg, having significantly decreased thisparameter by 44%. The most active compounds were 33 and 39.Derivative 33 administered at doses of 5 and 10mg/kg considerablyreduced the immobility time of animals by 57% and 79%, respec-tively, and compound 39 at doses of 5, 10 and 20 mg/kg decreasedthis parameter in statistically significant manner by 43%, 53% and83%, respectively. It is worth noting, that antidepressant-likeactivity of 33 and 39 given in the highest doses (10 mg/kg and20 mg/kg, respectively) was stronger in terms of its potency thanthat of citalopram. In fact, citalopram in doses of 1.25, 2.5 and 5mg/kg substantially shortened (by 32%, 51% and 61%, respectively) theimmobility time of mice in FST (Fig. 3).

Antidepressant doses of derivatives 33, 34, 39 and 40 did notinfluence the spontaneous locomotor activity of mice during initial2e6-min session (i.e., at the time identical to the observationperiod in the FST). Merely the antidepressant-like effect ofcompound 37 given at the highest dose of 20 mg/kg was notspecific, since it increased the locomotor activity of animals(Table 1-SM).

Both clinical and preclinical studies demonstrate the role of thedopaminergic system in thepathophysiologyof depression and in itstreatment. Moreover, some newer atypical antipsychotics (olanza-pine, quetiapine, aripiprazole, risperidone) exert antidepressanteffects and have been recently launched into the monotherapy and/

Fig. 3. Effects of the tested compounds, aripiprazole and citalopram on the immobilitytime in the forced swim test in Swiss albino mice.

or adjunctive treatment of bipolar disorder, major depressivedisorder and/or treatment-resistant depression [18e20]. In view ofthese facts, and considered that compounds 33, 34, 37, 39 and 40displayhighaffinity fordopamineD2 and/orD3 receptors (Table 3), atthe successive stage their antipsychotic potential was studied in theMK-801-induced locomotor hyperactivity test, a model of thenegative and cognitive symptoms of schizophrenia [21,22].

Among the evaluated compounds, only 40, given at doses of 10and 20 mg/kg, considerably attenuated the MK-801-evokedhyperactivity in mice (Fig. 4). Its effect seems to be specific, since40 administered at effective doses had no influence on the spon-taneous locomotor activity in mice. Derivatives 33 in a dose of20 mg/kg and 37 in a dose of 10 mg/kg significantly increasedhyperactivity induced by MK-801, while compounds 34 and 39produced no effects on the hyperactivity evoked by MK-801. It isnoteworthy that 33 and 37 in above-mentioned doses visiblyaugmented the spontaneous locomotor activity of mice, whereasderivatives 34 and 39 remained without significant effect (Table 1-SI). By comparison, aripiprazole in a dose of 0.5 mg/kg remarkablyattenuated the hyperactivity evoked by MK-801, and its effectseems to be specific, since a dose of 1 mg/kg per se was theminimum sedative one in the spontaneous locomotor activitymodel (Fig. 4).

3. Conclusions

Extending our studies in a group of azinesulfonamide analogs ofaripiprazole allowed identifying some potent, multireceptor 5-HT1A/5-HT2A/5-HT7/D2/D3 ligands, which behaved as 5-HT1Areceptor agonists/5-HT2A and 5-HT7 receptor antagonists/D2receptor partial agonists. The results of pharmacological in vivostudies showed that all of the selected compounds (33, 34, 37, 39,40), but not aripiprazole, displayed some antidepressant-likeactivity in a mouse model of depression (FST). On the other hand,only compound 40, similarly to aripiprazole, was active in MK-801-evoked hyperactivity test in mice.

Regardless receptor binding profile, we found the behavioralactivity dependent upon localization of sulfonamide group in theazine moiety; the highest antidepressant response displayedcompounds containing 7-quinolinyl (33, 37) and 3-isoquinolinyl(39) moieties in sulfonamide fragment. This peculiar relationshipmay be attributed to localization of sulfonamide group in b-posi-tion of azinemoiety. The same substitution patternwas observed inthe structure of aripiprazole. On the other hand, still in line withour previous findings [4], antipsychotic effect was detected in

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50 47

compound containing sulfonamide group in a-position of azinemoiety (4-isoquinolinyl analog, 40).

Receptor binding and functional activity data, followed bybehavioral assays results with these compounds indicate a complexrelationship between structure and these properties. Furtherstudies in the area of multifunctional compounds might be bene-ficial to both depression and schizophrenia, and will addressreceptor selectivity and pharmacokinetic.

4. Experimental protocols

4.1. Chemistry

Organic transformations were carried out at ambient tempera-ture, unless indicated otherwise. Organic solvents (from SigmaeAldrich and Chempur) were of reagent grade and were usedwithout purification. All other reagents were from SigmaeAldrichand Alfa Aesar.

Purity of the synthesized compounds was confirmed by TLCperformed on Merck silica gel 60 F254 aluminum sheets (Merck,Darmstadt, Germany). Spots were detected by their absorptionunder UV light (l ¼ 254 nm).

Analytical HPLCwere run on aWaters Alliance HPLC instrument,equipped with a Chromolith SpeedROD column (4.6 � 50 mm).Standard conditions were eluent system A (water/0.1% TFA), systemB (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a gradient of(0e100)% B over 3 min were used. Detection was performed ona PDA detector. Retention times (tR) are given in minutes.

1H NMR spectra were recorded at 300 MHz (Varian BB 200spectrometer) using TMS (0.00 ppm) and chloroform-d1, J valuesare in hertz (Hz), and splitting patterns are designated as follows: s(singlet), d (doublet), t (triplet), m (multiplet).

Mass spectrometry analyses: Samples were prepared in aceto-nitrile/water (10/90 v/v) mixture. The LC/MS system consisted ofa Waters Acquity UPLC, coupled to Waters TQD mass spectrometer(electrospray ionization mode ESI-tandem quadrupole). All theanalyses were carried out using an Acquity UPLC BEH C18,50� 2.1 mm reversed-phase column. A flow rate of 0.3 mL/min anda gradient of (5e95)% B over 5 min was used. Eluent A: water/0.1%HCO2H; eluent B: acetonitrile/0.1% HCO2H. Nitrogen was used forboth nebulizing and drying gas. LC/MS data were obtained byscanning the first quadrupole in 0.5 s in a mass range from 100 to700 m/z; 8 scans were summed up to produce the final spectrum.

Elemental analyses were found within �0.4% of the theoreticalvalues. Melting points (mp) were determined with a Büchi appa-ratus and are uncorrected. Column chromatography separationswere carried out on column with Merck Kieselgel 60.

4.2. Synthesis of azinesulfonyl chloride 22e28

A mixture of bromoazine (1e7) (4 mmol), sodium meth-anethiolate (20 mmol) and dry DMF (12 mL) was boiled with stir-ring under argon atmosphere for 4 h. It was then cooled to 70 �Cand the volatile components were evaporated under vacuum fromwater bath. The remaining crude sodium azinethiolates (8e14),were cooled down on an ice-water bath (under argon atmo-sphere), and carefully acidified with conc. hydrochloric acid(12mL). Then, CHCl3 (12mL) was added and 6% aqueous solution ofsodium hypochlorite (19 mL, 13.3 mmol) was dropped within30 min to the well-stirred (cold 5 �C) mixture of hydrochloric acidsolution of mercaptoazines (15e21) and CHCl3 at such a rate thattemperature was maintained below 5 �C. The mixture was pouredinto 30 g of ice. The chloroform layer was separated, and aqueouslayer was extracted with CHCl3 (3 � 10 mL). The chloroformextracts were combined, washed with water and dried over

anhydrous sodium sulfate. CHCl3 was evaporated to leave solidresidue. The residue was recrystallized from benzene to givechlorosulfonylazines (22e28) [6].

4.2.1. Isoquinoline-3-sulfonyl chloride (20)Creamy solid, 755 mg (83% yield) following crystallization.

C9H6ClNO2S, MW 227.67, monoisotopic mass 226.98,[MþH]þ ¼ 228.0. 1H NMR (CDCl3) d (ppm) 7.91e7.93 (m,1H), 7.94e7.96 (m,1H), 8.10e8.11 (dd,1H), 8.16e8.19 (dd,1H), 8.55 (s,1H), 9.38(s, 1H). Mp ¼ 133e134 �C. Anal. Calc for C9H6ClNO2S (277.67): C,47.48; H, 2.66; N, 6.15; found C, 47.52; H, 2.65; N, 6.14.

4.3. General procedure for the preparation of compounds 31e40

The starting N-(u-aminoalkyl)-2,3-dichlorophenylpiperazineswere synthesized according to the Gabriel method. A mixture ofthe appropriate N-(u-aminoalkyl)-2,3-dichlorophenylpiperazine(1.0 mmol) in CH2Cl2 (7 mL) and DIEA (2.4 mmol) was cooleddown (ice bath), and azinesulfonyl (22e28) (1.2 mmol) was addedat 0 �C in one portion. The reaction mixture was stirred for 2e6 hunder cooling. Then, the solvent was evaporated and thesulfonamides were separated by column chromatography usingSiO2 and a mixture of CH2Cl2/MeOH ¼ 9/0.7 or 9/0.5, as aneluting system. Free bases were then converted into thehydrochloride salts by treatment of their solution in anhydrousethanol with 1 M HCl in dioxane. The LC/MS of the identifiedcompounds 31e40 exceeded purity of 98%.

4.3.1. 6-Chloro-N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)quinoline-3-sulfonamide (31)

White solid, 307 mg (60% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.32.C22H23Cl3N4O2S, MW 513.87, monoisotopic mass 512.06,[MþH]þ ¼ 512.8. 1H NMR (CDCl3) d (ppm) 1.89e1.98 (m, 2H), 2.95e3.15 (m, 6H), 3.18e3.31 (m, 4H), 3.45e3.50 (m, 2H), 6.84e6.88 (d,1H,J¼7.4Hz, J¼2.1Hz), 6.99e7.08 (m, 3H), 7.78e7.82 (dd,1H, J¼9.1Hz,J ¼ 2.2 Hz), 8.05e8.08 (m, 2H), 8.90e8.91 (d, 1H, J ¼ 2.3 Hz), 9.19e9.20 (dd, 1H, J ¼ 4.6 Hz, J ¼ 1.5 Hz). C22H23Cl3N4O2S$HCl,Mp ¼ 212.5e214.1. Anal. Calc for C22H23Cl3N4O2S HCl (550.33): C,48.01; H, 4.40; N, 10.18; found C, 47.89; H, 4.42; N, 10.20.

4.3.2. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-6-sulfonamide (32)

White solid, 320 mg (67% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.14.C22H24Cl2N4O2S, MW 479.42, monoisotopic mass 478.10,[M þ H]þ ¼ 479.0. 1H NMR (DMSO) d (ppm) 1.86e1.91 (m, 2H),2.86e2.92 (m, 2H), 3.08e3.23 (m, 6H), 3.35e3.39 (m, 2H), 3.46e3.54 (m, 2H), 7.15e7.18 (dd, 1H, J ¼ 6.8 Hz, J ¼ 2.5 Hz), 7.33e7.34(m, 2H), 7.84e7.88 (q, 1H, J ¼ 4.4 Hz), 8.14e8.16 (m, 1H), 8.18e8.21 (dd, 1H, J ¼ 9.00 Hz, J ¼ 2.1 Hz), 8.34e8.37 (dd, 1H), 8.66e8.67 (d, 1H, J ¼ 2.1 Hz), 8.86e8.89 (d, 1H, J ¼ 7.7 Hz), 9.17e9.19(dd, 1H, J ¼ 4.6 Hz, J ¼ 1.5 Hz). C22H24Cl2N4O2S$HCl, Mp ¼ 144.2e145.9 �C. Anal. Calc for C22H24Cl2N4O2S HCl (515.88): C, 51.22; H,4.88; N, 10.86; found C, 51.18; H, 4.86; N, 10.89.

4.3.3. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-7-sulfonamide (33)

Yellow solid, 301 mg (63% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.21.C22H24Cl2N4O2S, MW 479.42, monoisotopic mass 478.10,[M þ H]þ ¼ 479.3. 1H NMR (DMSO) d (ppm) 1.87e1.96 (m, 2H),2.86e2.92 (m, 2H), 3.05e3.23 (m, 6H), 3.35e3.39 (m, 2H), 3.47e3.51 (m, 2H), 5.92 (br s, 1H), 7.15e7.18 (dd, 1H J ¼ 6.8 Hz,J ¼ 2.6 Hz), 7.30e7.38 (m, 2H), 7.79e7.83 (q, 1H, J ¼ 4.4 Hz), 8.00e

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e5048

8.03 (dd,1H, J¼ 8.7 Hz, J¼ 1.8 Hz), 8.17e8.21 (t, 1H, J¼ 5.9 Hz), 8.50(br s,1H), 8.65e8.68 (d,1H, J¼ 8.0 Hz), 9.12e9.15 (dd,1H, J¼ 4.5 Hz,J¼ 1.5 Hz). C22H24Cl2N4O2S$HCl, Mp¼ 211.1e212.9 �C. Anal. Calc forC22H24Cl2N4O2S HCl (515.88): C, 51.22; H, 4.88; N, 10.86; found C,51.36; H, 4.89; N, 10.83.

4.3.4. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-3-sulfonamide (34)

Yellow solid, 344 mg (70% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.38.C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,[MþH]þ ¼ 493.1. 1H NMR (DMSO) d (ppm) 1.35e1.37 (m, 4H), 2.17e2.25 (m, 2H), 2.34 (br s, 4H), 2.84e2.87 (m, 6H), 7.04e7.09 (m, 1H),7.26e7.32 (m, 2H), 7.72e7.77 (m, 1H), 7.90e7.95 (m, 2H), 8.11e8.13(m, 1H), 8.22e8.25 (m, 1H), 8.86 (s, 1H), 9.17 (s, 1H).C23H26Cl2N4O2S$HCl, Mp ¼ 148.0e149.7 �C. Anal. Calc forC23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,52.23; H, 5.15; N, 10.53.

4.3.5. 6-Chloro-N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)quinoline-4-sulfonamide (35)

Yellow solid, 305 mg (58% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.54.C23H25Cl3N4O2S, MW 527.89, monoisotopic mass 526.08,[M þ H]þ ¼ 527.2. 1H NMR (DMSO) d (ppm) 1.43e1.50 (m, 2H), 1.72(m, 2H), 2.84e2.90 (m, 2H), 3.03e3.24 (m, 6H), 3.35e3.50 (dd, 4H,J¼ 31.0 Hz, J¼ 12.0 Hz), 7.16e7.19 (m,1H), 7.33e7.35 (m, 2H), 7.93e7.97 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.3 Hz), 8.13e8.16 (d, 1H, J ¼ 9.23 Hz),8.18e8.20 (m, 1H), 8.41e8.42 (d, 1H, J ¼ 2.3 Hz), 8.88e8.89 (d, 1H,J ¼ 2.0 Hz), 9.21e9.22 (d, 1H, J ¼ 2.3 Hz). C23H25Cl3N4O2S$HCl,Mp ¼ 191.2e192.7 �C. Anal. Calc for C23H25Cl3N4O2S$HCl (564.35):C, 48.95; H, 4.64; N, 9.93; found C, 48.81; H, 4.62; N, 9.95.

4.3.6. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-6-sulfonamide (36)

Yellow solid, 305 mg (62% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.20.C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,[M þ H]þ ¼ 493.2. 1H NMR (DMSO) d (ppm) 1.38e1.46 (m, 2H),1.64e1.75 (br s, 2H), 2.79e2.84 (q, 2H, J ¼ 6.4 Hz), 3.04e3.16 (m,6H), 3.37e3.49 (m, 4H), 7.17e7.20 (m, 1H), 7.33e7.36 (m, 2H), 7.81e7.86 (m, 1H), 8.02e8.08 (m, 1H), 8.16e8.20 (dd, 1H, J ¼ 8.9 Hz,J ¼ 2.1 Hz), 8.32e8.38 (d, 1H, J ¼ 9.0 Hz), 8.63e8.64 (d, 1H,J ¼ 1.8 Hz), 8.82e8.88 (d, 1H, J ¼ 7.7 Hz), 9.15e9.18 (dd, 1H,J ¼ 4.6 Hz, J ¼ 1.5 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 147.1e148.8 �C.Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,10.57; found C, 52.31; H, 5.14; N, 10.60.

4.3.7. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-7-sulfonamide (37)

Yellow solid, 319 mg (65% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.27.C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,[MþH]þ ¼ 493.0. 1H NMR (CDCl3) d (ppm) 1.62 (br s, 4H), 2.42 (br s,2H), 2.69 (br s, 4H), 3.06 (br s, 2H), 3.16e3.19 (t, 4H, J ¼ 4.6 Hz),7.03e7.06 (m, 1H), 7.16e7.18 (m, 2H), 7.51e7.56 (q, 1H, J ¼ 4.1 Hz),7.95 (m, 3H), 8.20e8.23 (m, 1H), 8.63 (s, 1H), 9.02e9.04 (dd, 1H,J ¼ 4.4 Hz J ¼ 1.8 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 142.8e144.1 �C.Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,10.57; found C, 51.94; H, 5.13; N, 10.60.

4.3.8. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-8-sulfonamide (38)

White solid, 255 mg (52% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.5);tR ¼ 1.37.

C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,[M þ H]þ ¼ 493.2. 1H NMR (DMSO) d (ppm) 1.29 (br s, 4H), 2.06e2.10 (m, 2H), 2.33 (br s, 4H), 2.82e2.88 (m, 6H), 7.09e7.13 (m,1H), 7.21e7.33 (m, 3H), 7.69e7.73 (m, 2H), 8.27e8.32 (m, 2H), 8.53e8.56 (dd, 1H, J ¼ 8.5 Hz, J ¼ 1.8 Hz), 9.06e9.08 (m, 1H).C23H26Cl2N4O2S$HCl, Mp ¼ 131.1e131.9 �C. Anal. Calc forC23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,52.29; H, 5.16; N, 10.53.

4.3.9. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-3-sulfonamide (39)

White solid, 334 mg (68% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.33.C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,[M þ H]þ ¼ 492.7. 1H NMR (CDCl3) d (ppm) 1.58e1.60 (m, 4H),2.42e2.44 (m, 2H), 2.72 (br s, 4H), 3.03 (br s, 2H), 3.18e3.21 (m, 4H),6.99e7.04 (m, 1H), 7.08e7.16 (m, 2H), 7.74e7.85 (m, 2H), 7.94e7.97(d,1H, J¼ 8.0 Hz), 8.04e8.07 (dd,1H, J¼ 7.82 Hz, J¼ 0.8 Hz), 8.43 (s,1H), 8.65 (br s, 1H), 9.26 (s, 1H). C23H26Cl2N4O2S$HCl, Mp ¼ 185.3e186.7 �C. Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H,5.14; N, 10.57; found C, 52.03; H, 5.12; N, 10.59.

4.3.10. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-4-sulfonamide (40)

Yellow oil, 344 mg (70% yield) following chromatographicpurification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.28.C23H26Cl2N4O2S, MW 493.45, Monoisotopic Mass 492.12,[M þ H]þ ¼ 493.0. 1H NMR (CDCl3) d (ppm) 1.61 (br s, 4H), 2.42e2.46 (t, 2H, J ¼ 5.1 Hz), 2.72 (br s, 4H), 3.03e3.06 (m, 2H), 3.19e3.22 (t, 4H, J ¼ 4.5 Hz), 7.02e7.05 (q, 1H, J ¼ 3.2 Hz), 7.15e7.19 (m,2H), 7.70e7.77 (m, 1H), 7.80e7.86 (m, 2H), 8.08e8.10 (d, 1H,J ¼ 7.7 Hz), 8.70e8.73 (d, 1H, J ¼ 9.2 Hz), 9.11 (s, 1H), 9.39 (s, 1H).C23H26Cl2N4O2S$HCl, Mp ¼ 145.1e147.4 �C. Anal. Calc forC23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,52.16; H, 5.13; N, 10.53.

4.4. In vitro pharmacology

4.4.1. Cell cultureAll receptor cDNAs were obtained from the Missouri S&T cDNA

Resource Center (www.cdna.org). Human serotonin 5-HT2AR cDNAwas cloned into the pcDNA5/FRT/TO vector (Invitrogen) andtransfected into the HEK293 cells. Cell line with tetracycline-dependent expression of 5-HT2AR was developed using the FLP-IN T-REx system according to manufacturers protocols (Invi-trogen). HEK293 cells with stable expression of human serotonin 5-HT1AR, 5-HT6, 5-HT7bR or dopamine D2LR were obtained with theuse of Lipofectamine 2000 (Invitrogen). Cells were maintained at37 �C in a humidified atmosphere with 5% CO2 and were grown inDulbecco’s Modified Eagle Medium containing 10% dialyzed fetalbovine serum under selective conditions (75 mg/ml hygromycin and10 mg/ml blasticidin for the 5-HT2AR cell line and 500 mg/ml G418sulfate for the other cells). 48 h prior to cell culture harvest theexpression of the 5-HT2A receptor was induced by addition of the2 mg/ml tetracycline. For membranes preparations, cells weresubcultured into 150 cm2 cell culture flasks, grown to 90% conflu-ence, washed twice with prewarmed to 37 �C phosphate bufferedsaline (PBS) and were pelleted by centrifugation (200 g) in PBScontaining 0.1 mM EDTA and 1 mM dithiothreitol, and storedat �80 �C.

4.4.2. 5-HT1A/5-HT2A/5-HT6/5-HT7/D2L radioligand binding assaysMembrane preparation and general assay procedures for cloned

receptors were adjusted to 96-microwell format based on protocolswas described by us previously [4,23,24].

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50 49

Cell pellets were thawed and homogenized in 20 volumes ofassay buffer using an Ultra Turrax tissue homogenizer and centri-fuged twice at 35,000 g for 20 min at 4 �C, with incubation for15 min at 37 �C in between. The composition of the assay bufferswas as follows: for 5-HT1AR: 50 mM TriseHCl, 0.1 mM EDTA, 4 mMMgCl2, 10 mM pargyline and 0.1% ascorbate; for 5-HT2AR: 50 mMTriseHCl, 0.1 mM EDTA, 4 mM MgCl2 and 0.1% ascorbate; for 5-HT6R: 50 mM TriseHCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM TriseHCl, 4 mM MgCl2, 10 mM pargyline and 0.1%ascorbate; for dopamine D2LR: 50 mM TriseHCl, 1 mM EDTA, 4 mMMgCl2, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate.

All assays were incubated in a total volume of 200 mL in 96-wellmicrotitre plates for 1 h at 37 �C, except for 5-HT1AR and 5-HT2ARwhich were incubated at room temperature for 1 h and 1.5 h,respectively. The process of equilibration was terminated by rapidfiltration through Unifilter plates with a 96-well cell harvester andradioactivity retained on the filters was quantified on a Microbetaplate reader (PerkinElmer, USA).

For displacement studies the assay samples contained as radi-oligands (PerkinElmer, USA): 1.5 nM [3H]-8-OH-DPAT (135.2 Ci/mmol) for 5-HT1AR; 2 nM [3H]-ketanserin (53.4 Ci/mmol) for 5-HT2AR; 2 nM [3H]-LSD (83.6 Ci/mmol) for 5-HT6R; 0.6 nM [3H]-5-CT (39.2 Ci/mmol) for 5-HT7R or [3H]-raclopride (76.0 Ci/mmol)for D2LR.

Non-specific binding was defined with 10 mM of 5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas 10 mM of chlor-promazine, 10 mMof methiothepine or 1 mM of (þ)butaclamol wereused in 5-HT2AR, 5-HT6R and D2L assays, respectively.

Each compound was tested in triplicate at 7e8 concentrations(10�11e10�4 M). The inhibition constants (Ki) were calculated fromthe ChengePrushoff equation [25]. Results were expressed asmeans of at least two separate experiments.

4.4.3. Adrenergic a1 competition binding assayTissue (rat cortex) was homogenized in 20 volumes of ice-cold

50 mM TriseHCl buffer, pH 7.6 using an Ultra Turrax T25B (IKA)homogenizer. The homogenate was centrifuged at 20,000� g for20 min. The resulting supernatant was decanted and pellet wasresuspended in the same buffer and centrifuged again in the sameconditions. The final pellet was resuspended in appropriate volumeof buffer (10 mg/1 mL). [3H]-Prazosin (spec. act. 85 Ci/mmol, Per-kinElmer, USA) was used for labeling a1-receptor. 240 ml of thetissue suspension, 30 ml of 10 mM phentolamine (displacer), 30 ml of0.2 nM [3H]-Prazosin and 30 ml of the analyzed compound wereincubated at 30 �C for 30 min. The concentrations of analyzedcompounds ranged from 10�10 to 10�5 M.

The incubation was terminated by rapid filtration over glassfiber filters FilterMate B (PerkinElmer, USA) using 96-well Filter-Mate harvester (PerkinElmer, USA). Five rapid washes were per-formed with ice-cold 50 mM TriseHCl buffer, pH 7.6. Filter matewas dried in microwave and placed in plastic bag (PerkinElmer,USA) and soaked in 10mL of liquid scintillation cocktail Ultima GoldMV (PerkinElmer, USA). After even distribution of scintillationcocktail filter bag was sealed. The radioactivity on the filter wasmeasured in MicroBeta TriLux 1450 scintillation counter (Perki-nElmer, USA). The inhibition constants (Ki) were calculated fromthe ChengePrushoff equation [25]. Results were expressed asmeans of at least two separate experiments.

4.4.4. SERT competition binding assayThe assay was performed according to the method of Owens

et al. [26] with slight modifications. Rat cerebral cortex washomogenized in 30 volumes of ice-cold 50mM TriseHCl containing150 mM NaCl and 5 mM KCl, pH ¼ 7.7 at 25 �C and centrifuged at20,000� g for 20min. The supernatant was decanted and pellet was

resuspended in 30 volumes of buffer and centrifuged again. Theresulting pellet was resuspended in the same quantity of the bufferand centrifuged third time in the same conditions. [3H]-Citalopram(spec. act. 50 Ci/mmol, NEN Chemicals) was used for labeling 5-HT-transporter. 240 ml of the tissue suspension, 30 ml of 1 nM [3H]-Citalopram and 30 ml of the analyzed compound or 30 ml of 1 mMimipramine (displacer) were incubated at 22 �C for 1 h. Theconcentrations of analyzed compounds ranged from 10�10 to10�5 M. Incubations were terminated by vacuum filtration over GF/B filters and washed 5 times with 200 ml of ice-cold buffer. Radio-activity was measured in a MicroBeta TriLux e liquid scintillationcounter (PerkinElmer, USA). The inhibition constants (Ki) werecalculated from the ChengePrushoff equation [25]. Results wereexpressed as means of at least two separate experiments.

4.5. Pharmacology

4.5.1. SubjectsThe experimentswere performed onmale Swiss albinomice (22e

26 g) purchased from a licensed breeder Staniszewska (Ilkowice,Poland) or male CD-1 mice (accredited animal facility JagiellonianUniversity Medical College Kraków, Poland) and mice were kept ingroups of ten to Makrolon type 3 cages (dimensions26.5 � 15 � 42 cm). Male Wistar rats weighing 205e225 g obtainedfromaccreditedanimal facility JagiellonianUniversityMedicalCollege(Kraków, Poland) were housed in polycarbonate Makrolon type 3cages (dimensions 26.5 � 15 � 42 cm) in groups of four. All animalswere kept in an environmentally controlled rooms (ambienttemperature 22 � 2 �C; relative humidity 50e60%; 12:12 light:darkcycle, lights on at 8:00). They were allowed to acclimatize with theenvironment for one week before commencement of the experi-ments. Standard laboratory food (Ssniff M-Z) and filtered water werefreely available. All the experimental procedures were approved bythe I Local EthicsCommissionat the JagiellonianUniversity inKraków.

4.5.2. Experimental proceduresAll the experiments were conducted in the light phase between

09.00 and 14.00 h. Each experimental group consisted of 5e10animals/dose, and the animals were used only once in each test.The experiments were performed by an observer unaware of thetreatment administered.

4.5.3. Head twitch response in Swiss albino miceIn order to habituate mice to the experimental environment,

each animal was randomly transferred to a 12 (diameter � 20 cm)(height) glass cage, lined with sawdust 20 min before the treat-ment. Head twitches in mice were induced by (�)DOI (2.5 mg/kg).Immediately after treatment, the number of head twitches wascounted during 20 min [10]. ID50 (the dose inhibiting the headtwitches in mice by 50%) was calculated using Graph Pad Prism 5Software.

4.5.4. Apomorphine-induced climbing behavior in Swiss albinomice

For observation, mice were placed in separate cages with wallsmade of metal bars. Apomorphine (3 mg/kg) was injected 10 minafter the drugs. Twenty minutes after injection of apomorphine,time of climbing was determined for 2 min. Climbing time wasdefined as the period during which the animal held the 2, 3 or 4paws on the wall. ID50 (the dose inhibiting climbing behavior inmice by 50%) was calculated using Graph Pad Prism 5 Software.

4.5.5. Forced swim test in Swiss albino miceThe experiment was carried out according to the method of

Porsolt et al. [11]. Briefly, mice were individually placed in a glass

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e5050

cylinder (25 cm high; 10 cm in diameter) containing 6 cm of watermaintained at 23e25 �C, andwere left there for 6min. Amousewasregarded as immobile when it remained floating on the water,making only small movements to keep its head above it. The totalduration of immobility was recorded during the last 4 min ofa 6-min test session.

4.5.6. Locomotor activity in miceThe locomotor activity was recorded with an Opto M3 multi-

channel activity monitor (MultiDevice Software v. 1.3, ColumbusInstruments). The Swiss albino mice were individually placed inplastic cages (22 � 12 � 13 cm), and then the crossings of eachchannel (ambulation) were counted from 2 to 6 min, i.e. the timeequal to the observation period in the forced swim test and during30-min experimental sessions. The CD-1 mice were individuallyplaced in plastic cages (22 � 12 � 13 cm) for 30 min habituationperiod, and then ambulation was counted during 1 h with datarecording every 5 min. The cages were cleaned up with 70% ethanolafter each mouse.

4.5.7. MK-801-induced hyperlocomotor in CD-1 miceThe locomotor activity was recorded according to the method

described above.

4.5.8. DrugsThe following drugs were used: imipramine (hydrochloride,

SigmaeAldrich), MK-801 (hydrogen maleate, SigmaeAldrich), ari-piprazole (Adamed Pharmaceuticals), apomorphine (hydrochlo-ride, Tocris), 2,5-dimethoxy-4-iodoamphetamine (DOI,hydrochloride, Adamed Pharmaceuticals) and tested compounds:33, 34, 37, 39, 40. Imipramine, apomorphine, DOI and MK-801 weredissolved in distilled water; remaining compounds were sus-pended in a 1% aqueous solution of Tween 80 immediately beforeadministration. All the compounds were administered intraperi-toneally at a volume of 10 ml/kg 30 min before the test, excludingMK-801 and apomorphine which were injected 15 min and 20 min,respectively, before testing. Control animals received a vehicleinjection according to the same schedule.

4.5.9. StatisticsAll the data are presented as the mean � SEM. The statistical

significance of the results was evaluated by a one-way ANOVA,followed by Bonferroni Comparison Test.

Acknowledgments

This study was partly supported by the Polish Ministry ofScience and Higher Education (MNiSW), Grant No. N N405 378437.Radioligand binding experiments were financially supported by theNorwegian Financial Mechanism as part of the Polish-NorwegianResearch Fund, Grant No. PNRFe103eAI-1/07.

Appendix A. Supplementary material

Supplementary material related to this article can be found athttp://dx.doi.org/10.1016/j.ejmech.2012.11.042.

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