Design, Sustainable Synthesis and Biological Evaluation of a Novel Dual α2A/5-HT7 Receptor Antagonist with Antidepressant-Like Properties

The complex pathophysiology of depression, together with the limits of currently available antidepressants, has resulted in the continuous quest for alternative therapeutic strategies. Numerous findings suggest that pharmacological blockade of α2-adrenoceptor might be beneficial for the treatment of depressive symptoms by increasing both norepinephrine and serotonin levels in certain brain areas. Moreover, the antidepressant properties of 5-HT7 receptor antagonists have been widely demonstrated in a large set of animal models. Considering the potential therapeutic advantages in targeting both α2-adrenoceptors and 5-HT7 receptors, we designed a small series of arylsulfonamide derivatives of (dihydrobenzofuranoxy)ethyl piperidines as dually active ligands. Following green chemistry principles, the designed compounds were synthesized entirely using a sustainable mechanochemical approach. The identified compound 8 behaved as a potent α2A/5-HT7 receptor antagonist and displayed moderate-to-high selectivity over α1-adrenoceptor subtypes and selected serotonin and dopaminergic receptors. Finally, compound 8 improved performance of mice in the forced swim test, displaying similar potency to the reference drug mirtazapine.


Introduction
Depressive disorder is a common and disabling illness characterized by the presence of behavioral and emotional symptoms (i.e., sleep disturbances, low self-esteem, sadness as well as suicidal ideation) [1]. Although different pharmacotherapeutic options are available (e.g., selective serotonin reuptake inhibitors SSRIs-; serotonin/noradrenaline reuptake inhibitors SNRIs; monoamine receptors modulators), the treatment of depressive disorders is still limited. Currently available antidepressants display a delay of therapeutic action, which lasts up to a few weeks in some patients after numerous antidepressant drugs, and numerous unacceptable side effects [2]. Here, we present a medicinal mechanochemistry approach to the generation of a focused library of arylsulfonamides of (aryloxyethyl)piperidines ( Figure 1). The impact of the applied modifications on the affinity for α2-AR and 5-HT7R was first assessed in vitro in radioligand binding studies. The selectivity of the most promising derivatives over structurally related off-target GPCRs (α1-AR, 5-HT1AR, 5-HT2AR, 5-HT6R and D2R) was Here, we present a medicinal mechanochemistry approach to the generation of a focused library of arylsulfonamides of (aryloxyethyl)piperidines ( Figure 1). The impact of the applied modifications on the affinity for α 2 -AR and 5-HT 7 R was first assessed in vitro in radioligand binding studies. The selectivity of the most promising derivatives over structurally related off-target GPCRs (α 1 -AR, 5-HT 1A R, 5-HT 2A R, 5-HT 6 R and D 2 R) was then investigated, followed by a determination of their antagonistic properties at α 2A -AR and 5-HT 7 R in cellular assays. Finally, the selected derivatives improved performance of mice in the forced swim test.

Chemistry
In the last decade, mechanochemistry, and in particular medicinal mechanochemistry [22,23], has been recognized as an innovative technique for the generation of various organic compounds as well as producing pharmaceutically relevant fragments and functionalities [24][25][26]. Taking into account the benefits of employing a solid-state approach over classic in-batch procedures (i.e., increased reaction yield, reduction of time, decreased use of organic solvents) [27,28], we developed a mechanochemical approach for the synthesis of the designed derivatives 6-19 (Scheme 1). Initially, alkylation of Boc-protected 4-aminopiperidine with the commercially available 7-(2-bromoethoxy)-2,2-dimethyl-2,3dihydrobenzofuran 1 was carried out in a 10 mL PTFE jar with a 1.5 cm diameter stainless steel ball using a Retsch vibratory ball mill (vbm) operating at 30 Hz. The use of a slight excess of amine, K 2 CO 3 as base, in the presence of KI, allowed us to achieve high conversion rates for intermediate 2 after a milling time of 140 min (for more details see Table S1). Further scale-up optimization was required to translate reaction conditions to a 35 mL PTFE jar (see Table S1). Increasing the loading of base (from 2 to 3 eq) together with elongation of reaction time (from 140 to 210 min) enabled us to reach a 97% conversion of substrates. After an acidic extraction at pH 3.5 to remove unreacted Boc-protected alicyclic amine, intermediate 2 was isolated with a high 95% purity and 84% yield. Having identified the optimal reaction conditions, the same mechanochemical protocol was applied for the alkylation of Boc protected 4-aminomethylpiperidine to obtain intermediate 3 (96% purity) in a satisfactory yield (81%) (see Table S2). In the next step, primary amine derivatives 4 and 5, obtained upon treatment of Boc-derivative 2 and 3 with gaseous HCl [29,30], reacted in a ball mill with different substituted arylsulfonyl chlorides to generate the designed sulfonamide derivatives.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 20 then investigated, followed by a determination of their antagonistic properties at α2A-AR and 5-HT7R in cellular assays. Finally, the selected derivatives improved performance of mice in the forced swim test.

Chemistry
In the last decade, mechanochemistry, and in particular medicinal mechanochemistry [22,23], has been recognized as an innovative technique for the generation of various organic compounds as well as producing pharmaceutically relevant fragments and functionalities [24][25][26]. Taking into account the benefits of employing a solid-state approach over classic in-batch procedures (i.e., increased reaction yield, reduction of time, decreased use of organic solvents) [27,28], we developed a mechanochemical approach for the synthesis of the designed derivatives 6-19 (Scheme 1). Initially, alkylation of Boc-protected 4-aminopiperidine with the commercially available 7-(2-bromoethoxy)-2,2-dimethyl-2,3-dihydrobenzofuran 1 was carried out in a 10 mL PTFE jar with a 1.5 cm diameter stainless steel ball using a Retsch vibratory ball mill (vbm) operating at 30 Hz. The use of a slight excess of amine, K2CO3 as base, in the presence of KI, allowed us to achieve high conversion rates for intermediate 2 after a milling time of 140 min (for more details see Table S1). Further scale-up optimization was required to translate reaction conditions to a 35 mL PTFE jar (see Table S1). Increasing the loading of base (from 2 to 3 eq) together with elongation of reaction time (from 140 to 210 min) enabled us to reach a 97% conversion of substrates. After an acidic extraction at pH 3.5 to remove unreacted Boc-protected alicyclic amine, intermediate 2 was isolated with a high 95% purity and 84% yield. Having identified the optimal reaction conditions, the same mechanochemical protocol was applied for the alkylation of Boc protected 4-aminomethylpiperidine to obtain intermediate 3 (96% purity) in a satisfactory yield (81%) (see Table S2). In the next step, primary amine derivatives 4 and 5, obtained upon treatment of Boc-derivative 2 and 3 with gaseous HCl [29,30], reacted in a ball mill with different substituted arylsulfonyl chlorides to generate the designed sulfonamide derivatives. Scheme 1. Mechanochemical synthesis of final compounds 6-19. Reagents and conditions: (i) vbm 30 Hz, ϕball = 1.5 cm, total mass of reagents = 500 mg, 35 mL PTFE jar, alkylating agent (1 eq) Boc-protected alicyclic diamine (1.2 eq), K2CO3 (3 eq), KI (0.5 eq), 210 min; (ii) HClgas, 2 h; (iii) vbm 30 Hz, ϕball = 1.5 cm, total mass of reagents = 125 mg, 10 mL PTFE jar, primary amine (1 eq), arylsulfonyl chloride (1 eq), K2CO3 (2 eq), 1-10 min.
Hence, the final compounds 6-19 were obtained by milling equimolar amounts of starting materials in the presence of K 2 CO 3 in moderate-to-high yields (65-94%). According to our previously reported findings on sulfonamide bond formation in the solid-state [29], sulfonylation of the primary amine function was significantly influenced by the nature of the substituent on the phenyl ring of the sulfonyl chloride. Regardless of the type of central amine core, the presence of 4-F and 3-Cl substituents enabled the formation of compounds 6, 7, 14 and 15 with high conversion rates in a relatively shorter time (1 min) than all other tested analogs (see Table S3). The introduction of a second substituent at the 3-chlorophenyl moiety in both subsets (5-Cl, 2-F and 5-Cl, 2-MeO) required longer milling times to guarantee similar conversion rates for the generation of derivatives 8-10 and 16-17. Notably, compounds 11, 12 and 18 bearing 1-naphtyl and 2-naphtyl moieties displayed the lowest conversion rates amongst the series (<70%) after 10 min of reaction. Prolongation of the milling time for these derivatives did not increase the formation of desired products while causing degradation of substrates, which was not detected in the solution. In contrast, milling isoquinolyl-4-sulfonyl chloride and primary amines 4 and 5 for 5 min furnished final compounds 13 and 19 with the highest conversion rate amongst the series (90 and 99%, respectively).

In Vitro Pharmacology
All synthesized compounds were evaluated in 3 [H]clonidine and 3 [H]5-CT binding experiments for their affinity toward α 2 -AR and 5-HT 7 R, respectively (Table 1). The tested compounds showed high-to-low affinity for α 2 -AR (K i = 80-1194 nM) and for 5-HT 7 R (K i = 30-727 nM). Structure-activity relationship (SAR) studies were firstly focused on the impact of the central amine core on the affinity for both biological targets. Compounds with a 4-aminopiperidine scaffold displayed a higher affinity for both α 2 -AR and 5-HT 7 R, than their 4-aminomethylpiperidine analogs (6 vs. 14, 9 vs. 17 and 11 vs. 18).
Next, the impact of the kind of substituents at the arylsulfonamide moiety was analyzed. Based on our data, reporting on the impact of monosubstituted benzenesulfonyl moiety on the affinity for α-AR and 5-HT 7 R, the analysis was limited to the 3-Cl substitution pattern [31,32]. Although replacement of the 4-F substituent present in the pilot compound 6 with the 3-Cl one (compound 7) decreased the affinity for α 2 -AR 5-fold (K i = 138 and 649, respectively), this modification was tolerated for interaction with the 5-HT 7 R. Regardless of the kind of central amine core, introducing a fluorine atom in the meta position to the 3-Cl-phenylsulfonyl moiety (i.e., 5-Cl, 2-F substitution pattern) improved the affinity of compounds 7 and 15 for both biological targets. In contrast to our previous findings [33], the replacement of one or both halogens with the electron-donating methoxy substituent decreased the affinity for α 2 -AR and 5-HT 7 R ligands (8 vs. 9 and 10 and 16 vs. 17). Table 1. The binding data of the synthesized compounds 6-19 for α 2 -AR and 5-HT 7 R. compounds 6, 7, 14 and 15 with high conversion rates in a relatively shorter time (1 min) than all other tested analogs (see Table S3). The introduction of a second substituent at the 3-chlorophenyl moiety in both subsets (5-Cl, 2-F and 5-Cl, 2-MeO) required longer milling times to guarantee similar conversion rates for the generation of derivatives 8-10 and 16-17. Notably, compounds 11, 12 and 18 bearing 1-naphtyl and 2-naphtyl moieties displayed the lowest conversion rates amongst the series (<70%) after 10 min of reaction. Prolongation of the milling time for these derivatives did not increase the formation of desired products while causing degradation of substrates, which was not detected in the solution. In contrast, milling isoquinolyl-4-sulfonyl chloride and primary amines 4 and 5 for 5 min furnished final compounds 13 and 19 with the highest conversion rate amongst the series (90 and 99%, respectively).
Next, the impact of the kind of substituents at the arylsulfonamide moiety was analyzed. Based on our data, reporting on the impact of monosubstituted benzenesulfonyl moiety on the affinity for α-AR and 5-HT7R, the analysis was limited to the 3-Cl substitution pattern [31,32]. Although replacement of the 4-F substituent present in the pilot compound 6 with the 3-Cl one (compound 7) decreased the affinity for α2-AR 5-fold (Ki = 138 and 649, respectively), this modification was tolerated for interaction with the 5-HT7R. Regardless of the kind of central amine core, introducing a fluorine atom in the meta position to the 3-Cl-phenylsulfonyl moiety (i.e., 5-Cl, 2-F substitution pattern) improved the affinity of compounds 7 and 15 for both biological targets. In contrast to our previous findings [33], the replacement of one or both halogens with the electron-donating methoxy substituent decreased the affinity for α2-AR and 5-HT7R ligands (8 vs. 9 and 10 and 16 vs. 17).  [34] with binding experiments performed in rat cerebral cortex; e Data taken from [35] with binding experiments performer in cloned human receptors.
The bicyclic 1-naphtyhyl and 2-naphthyl moiety displayed no significant improvement over the substituted phenyl ring. An exception was observed when the naphthyl substituent was replaced with the 4-isoquinolyl fragment (11 and 12). In line with our previously reported studies demonstrating the preferential α-position of the azinylsulfonyl moiety for interaction with biological targets [36][37][38], this modification highly increased the affinity of compound 13 for α 2 -AR up to four-fold, without drastically reducing activity at the 5-HT 7 R sites (K i = 80 and 91 nM for α 2 -AR and 5-HT 7 R, respectively).
Compounds 6, 8 and 13, which displayed the most balanced activity towards α 2 -AR and 5-HT 7 R among the series, were further evaluated in vitro for their selectivity over structurallyclosed monoaminergic receptors, to assess the risk of evoking CNS or cardiovascular side effects (i.e., hallucinations, body temperature, Parkinsonian-like effects, hypotension).
The tested compounds 6, 8 and 13 displayed high selectivity over adrenergic α 1 -AR, serotonin 5-HT 2A R and 5-HT 6 R while showing moderate affinity and selectivity for 5-HT 1A R and D 2 R subtypes (K i = 221-388 nM) ( Table 2). Considering the high and specific distribution of α 2A -AR in CNS [5], and its engagement in controlling noradrenaline/serotonin release in the hippocampus and the corticolimbic structures involved in affective, cognitive and memory processes [39], targeting α 2A -AR subtype might provide more beneficial therapeutic effects than other isolated α 2 -AR isoforms. Thus, the functional activity of 6, 8 and 13 at α 2A -AR and selectivity over α 2B -AR subtypes were assessed in fluorescence-based cellular assays (Table 3) [40]. The evaluated compounds were classified as potent α 2A -AR antagonists (K b = 12-40 nM). Although the observed potencies were lower than those of reference α 2 -AR antagonist yohimbine, compounds 6 and 8 displayed higher functional selectivity over α 2B -AR subtype (up to seven-and four-fold, respectively). In contrast, 4-isoquinolyl derivatives 13 did not show any preference for the tested α 2 -AR subtype. Table 3. The antagonist activity of selected compounds 6, 8, 13 and reference yohimbine at α 2A -AR, α 2B -AR and 5-HT 7 R. Next, the antagonistic properties of 6, 8 and 13 at 5-HT 7 R were confirmed in HEK-293 cells, which stably over-express the 5-HT 7 R (Table 3). All tested derivatives inhibited the cAMP production promoted by the administration of the agonist 5-CT, thus behaving as potent antagonists in this cellular setting. To exclude pharmacological effects associated with interaction with 5-HT 1A R, further functional profiling of 6, 8 and 13 was performed at Eurofins (Eurofins Scientific, France), revealing low agonist activity (EC 50 > 4 µM) and no antagonist property at 1 µM in cAMP-based assays.

In Vivo Pharmacology
In view of the findings that the modulation of noradrenergic/serotonin transmissions by targeting α 2 -AR and the blockade of 5-HT 7 R are involved in behavioral changes responsible for antidepressant-like effects observed in preclinical models [7,10,41], selected compounds 6, 8 and 13 were assessed for their potential antidepressant properties in the forced swim test using Albino Swiss mice. The clinically used antidepressant mirtazapine was tested as reference. Although mirtazapine displays high-to-moderate affinity for 5-HT 2A/2C , 5-HT 3 and5-HT 7 Rs, its antagonism at presynaptic α 2A -AR, which enhances noradrenaline and serotonin release, is mainly related to the observed in vivo antidepressant effect [42].
All tested compounds (administered at dose range of 1-8 mg/kg, ip) shortened the  Although sedation may be perceived as being therapeutically beneficial in certain stress-related mood disorders [43], it represents an unacceptable side effect, mainly related to some antidepressants. Indeed, about 20% of patients treated with mirtazapine for their depression or anxiety report sedation as a side effect [20].
To evaluate potential sedative activity at the doses used in the behavioral experiments, the influence of tested compounds on the spontaneous locomotor activity of mice was assessed. The data sets for the activity showed normal distribution (α = 0.05). Among the studied compounds, only 8 produced an antidepressant-like effect with no influence Although sedation may be perceived as being therapeutically beneficial in certain stress-related mood disorders [43], it represents an unacceptable side effect, mainly related to some antidepressants. Indeed, about 20% of patients treated with mirtazapine for their depression or anxiety report sedation as a side effect [20].
To evaluate potential sedative activity at the doses used in the behavioral experiments, the influence of tested compounds on the spontaneous locomotor activity of mice was assessed. The data sets for the activity showed normal distribution (α = 0.05). Among the studied compounds, only 8 produced an antidepressant-like effect with no influence on the spontaneous locomotor activity of mice (ANOVA, F(5,33) = 21.98, p < 0.001) ( Figure 2B). Compound 6 (4, 8 mg/kg) and mirtazapine (16 mg/kg) tested in doses effective in the FST induced sedation (ANOVA, F(5,32) = 25.99, p < 0.0001) ( Figure 2D). Despite the fact that compound 13 showed antidepressant-like properties at the lowest dose (1 mg/kg) without affecting locomotor activity, it caused sedation at higher doses (ANOVA, F(7,44) = 20.4, p < 0.001) ( Figure 2E). This may partially explain the lack of pharmacological effect of compound 13 at the doses of 4 and 8 mg/kg in the FST.

General Chemical Methods
All commercially available reagents were of the highest purity (from Sigma-Aldrich, Fluorochem, AlfaAesar). The milling treatments were carried out in a vibratory ball-mill Retsch MM400 operated at 30 Hz. The milling load was defined as the sum of the mass of the reactants per free volume in the jar and was equal to 15 mg/mL. All reactions using the vibratory ball mill were performed under air. 1 H and 13 C NMR spectra were recorded on a JEOL JNM-ECZR500 RS1 (ECZR version) at 500 and 126 MHz, respectively, and were reported in ppm using deuterated solvent for calibration (CDCl 3 ). The J values were reported in hertz (Hz), and the splitting patterns were designated as follows: br s. Mass spectra were recorded on a UPLCMS/MS system consisting of a Waters AC-QUITY UPLC (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 mm × 100 mm, and 1.7 µm particle size, equipped with Acquity UPLC BEH C18 Van Guard precolumn; 2.1 mm × 5 mm, and 1.7 µm particle size. The column was maintained at 40 • C and eluted under gradient conditions from 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 mL min −1 . Eluent A: water/formic acid (0.1%, v/v), Eluent B: acetonitrile/formic acid (0.1%, v/v).
Melting points (mp) were determined with a Büchi apparatus and are uncorrected.
Elemental analyses for C, H, N and S were carried out using the elemental Vario EL III Elemental Analyser (Hanau, Germany). All values are given as percentages and were within ±0.4% of the calculated values.

Alkylation of Boc-Protected 4-Aminopiperidine and 4-Aminomethylpiperidine in Ball Mill (Procedure B)
Commercially available bromine derivative 1 (1 eq) and Boc-protected alicyclic diamine (1.2 eq) were introduced in two 35 mL PTFE jars (milling load 15 mg/mL) with one stainless steel ball (φ ball = 1.5 cm), followed by the addition of previously ground K 2 CO 3 (3 eq) and KI (0.5 eq). The reaction was carried out for 210 min at rt. Then, the product was solubilized in CH 2 Cl 2 (25 mL), and the organic phase was washed with KHSO 4 aqueous solution at pH = 3.5 (3 × 10 mL) and saturated NaCl solution (1 × 10 mL), dried over Na 2 SO 4 , and finally filtered and concentrated under reduced pressure. To obtain the desired amount of product, the reaction was carried out twice (4 × 35 mL).

Sulfonylation of Primary Amine (Procedure E) for the High-Scale Preparation of Selected Final Compounds (6, 8 and 13)
Intermediate 4 or 5 (1 eq), selected arylsulfonyl chloride (1 eq), and previously ground K 2 CO 3 (2 eq) were introduced in a 35 mL PTFE jar (milling load 15 mg/mL) with one stainless steel ball (φ ball = 1.5 cm). The reaction was carried out for 1−5 min at rt. Then, the crude mixture was solubilized in AcOEt (15 mL), and the organic phase was washed with KHSO 4 aqueous solution at pH = 3.5 (3 × 10 mL), saturated NaCl solution (1 × 10 mL), dried over Na 2 SO 4 , and finally filtered and concentrated under a vacuum. To obtain the desired amount of product, the reaction was carried out twice (2 × 15 mL).

Determination of the Intrinsic Activity of the Test Compounds at the α 2 -AR Subtypes
Intrinsic activity assays for α 2A -adrenergic receptor were performed according to the instructions of the manufacturer of the assay kit containing the ready to use cells with stable expression of the α 2A -adrenoceptor (Invitrogen, Life Technologies, Waltham, MA, USA). Tango™ ADRA2A-bla U2OS DA cells (10,000 cells/well) were plated in a 384-well format and incubated for 20 h. Cells were exposed to Yohimbine (Sigma-Aldrich, Merck, Darmstadt, Germany) for 30 min, then stimulated with an EC 80 concentration of UK14,304 (Sigma-Aldrich) in the presence of 0.1% DMSO for 5 h. Cells were then loaded with LiveBLAzer™-FRET B/G substrate for 2 h. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the % inhibition plotted against the indicated concentrations of Yohimbine.
The intrinsic activity assays for the α 2B -adrenergic receptor were assessed by luminescence detection of calcium mobilization using the recombinant expressed jellyfish photoprotein, aequorin. Measurements were performed with adrenergic α 2B AequoScreen cell line (PekinElmer). The cell density in 96-well format measurements was 5000 cells per well. Cell harvesting, coelenterazine h (Invitrogen, cat. no. C 6780) loading and preparation were done according to instructions presented in the AequoScreen Starter Kit Manual (PerkinElmer). Compound concentration series (50 µL/well) were diluted in 0.1% BSA (Intergen) containing assay buffer (D-MEM/F-12, Invitrogen cat. no. 11039) and prepared in white 1 2 Area Plate-96 well microplates (PerkinElmer,). The cell suspension was dispensed on the ligands using the POLARstar optima reader injectors. For the antagonist assay, cells were injected (50 µL) into the assay plate with antagonists (50 µL) using the POLARstar optima reader. The antagonist dilution series with four replicates were prepared as instructed in the AequoScreen Starter Kit Manual at the concentrations from 10 -11 to 10 -6 M/L. The agonist used for α 2A -AR cells was Oxymetazoline (Sigma, cat. no. O2378), which at a single concentration was injected (50 µL, final concentration EC 80 ) on the preincubated (15-20 min) mixture of cells and antagonist, and the emitted light was recorded for 20 s.

Determination of the Intrinsic Activity of the Test Compounds at the 5-HT 7 R
Cells (prepared with the use of Lipofectamine 2000) were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and grown in Dulbeco's Modifier Eagle Medium containing 10% dialyzed fetal bovine serum and 500 mg/mL G418 sulphate. For functional experiments, cells were subcultured in 25 cm diameter dishes, grown to 90% confluence, washed twice with pre-warmed to 37 • C phosphate buffered saline (PBS) and were centrifuged for 5 min (160× g). The supernatant was aspirated, then the cell pellet was resuspended in stimulation buffer (1 × HBSS, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA). The cAMP level was measured using the LANCE cAMP detection kit (PerkinElmer), according to the manufacturer's directions. For the investigation of the antagonist effect on 5-HT 7 R, the agonist 5-carboxyamidotryptamine (5-CT; EC 50 = 1 nM) was used in submaximal concentration (10nM) to stimulate cAMP production. Cells (5 µL) were incubated with compounds (5 µL) for 30 min at room temperature in 384-well white opaque microtiter plate. After incubation, the reaction was stopped, and cells were lysed by the addition of 10 µL working solution (5 µL Eu-cAMP and 5 µL ULight-anti-cAMP). The assay plate was incubated for 1 h at room temperature. Time-resolved fluorescence resonance energy transfer (TR-FRET) was detected by an Infinite M1000 Pro (Tecan) using instrument settings from LANCE cAMP detection kit manual. K b values were calculated from the Cheng-Prusoff equation specific for the analysis of functional inhibition curves: K b = IC 50 /(1+A/EC 50 ) where A is the agonist concentration, IC 50 is the concentration of the antagonist producing a 50% reduction in the response to the agonist, and EC 50 is the agonist concentration which causes half of the maximal response [47].

Animals
Adult male Albino Swiss mice (CD-1, 8 weeks old, 25-30 g: Jagiellonian University Medical College, Krakow, Poland) were used in the study. Animals were housed in groups of 8 in a transparent plastic cage (382 × 220 × 150 mm) at room temperature (22 ± 2 • C), on a 12 h light/dark cycle with ad libitum access to food and water. Mice were handled for one week before starting the experimental procedures. The separate groups of animals were used in the forced swim test and in the locomotor studies. All studies were approved by the Institutional Animal Care and Ethics Committee of the Jagiellonian University (approval no.: 80/2015).

Drug Administration
Mirtazapine (16 mg/kg or 4 mg/kg, Sigma-Aldrich) was dissolved in DMSO and diluted to the appropriate dose with 1% Tween 80, immediately before use (the maximal final DMSO concentration was 2%). Tested compounds were dissolved with 1% Tween 80.
Solutions of mirtazapine and the tested compounds were administered intraperitoneally (ip) 30 min prior to the experiment. The control animals were given ip injections of the 2% DMSO in 1% Tween 80 (vehicle). The volume of vehicle or drug solutions was 10 mL/kg.

Forced Swim Test
The experiment was performed on mice according to the method previously described [48,49]. Mice were forced to swim individually in the glass cylinders (height 25 cm, diameter 10 cm) filled with water at 24 ± 1 • C to a depth of 10 cm and left there for 6 min. Following a 2 min habituation period, total time spent immobile was recorded over the next 4 min. The animal was regarded as immobile when it remained floating passively in the water, making only small movements to keep its head above the water.

Spontaneous Locomotor Activity
Locomotor activity was recorded with an Opto M3 multichannel activity monitor, photoresistor actometers connected to a counter for the recording of light-beam interruptions (MultiDevice Software v1.3, Columbus Instruments, Columbus, OH, USA). The mice, after being placed into the cages individually, had their activity evaluated between the 2nd and the 6th minute. The chosen time period corresponded with the time interval considered in the FST. Spontaneous locomotor activity was evaluated as the distance travelled plus the movements of climbing by animals.

Statistical Analysis
Statistical calculations were performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). The normality of data sets was determined using Shapiro-Wilk test. Comparisons between experimental and control groups were performed by one-way ANOVA, followed by Dunnett post hoc.

Conclusions
Based on the finding that concurrent blockade of α 2 -AR and 5-HT 7 R might be beneficial in the treatment of depressive disorders, we elaborated a medicinal mechanochemical approach to provide a limited series of arylsulfonamides of (dihydrobenzofuranoxy)ethyl piperidines as dual acting α 2 /5-HT 7 R antagonists. Sustainable solid-state protocol furnished designed compounds 6-19 in high yields and purities, limiting the amount of organic solvents as well as the formation of by-products. Further focused SAR studies revealed that the presence of a 4-aminopiperidine central core, together with dihalogenated substituents or a 4-isoquinolyl moiety at the sulfonamide fragment, were responsible for the high affinity of tested compounds for both biological targets. Finally, the study identified 5-chloro-2-fluoro-N-{1-[2-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yloxy)ethyl]piperidin-4-yl}benzenesulfonamide (compound 8) as a potent α 2A /5-HT 7 R antagonist, which produced an antidepressant-like effect in FST in mice. The effect was similar to that produced by mirtazapine used in a two-fold higher dose, without inducing sedation. Preliminary data for compound 8 are promising enough to warrant further efficacy and safety studies on the potential of dual-acting α 2A /5-HT 7 R antagonists in the treatment of affective disorders.

Supplementary Materials:
The following are available online: optimization of mechanochemical reactions (Tables S1-S3); MS, 1 H-NMR and 13 C-NMR spectra of all intermediates and final compounds ( Figures S1-S54).