Synthesis and Pharmacological Evaluation of Novel Silodosin-Based Arylsulfonamide Derivatives as α1A/α1D-Adrenergic Receptor Antagonist with Potential Uroselective Profile

Benign prostatic hyperplasia (BPH) is the most common male clinical problem impacting the quality of life of older men. Clinical studies have indicated that the inhibition of α1A-/α1D adrenoceptors might offer effective therapy in lower urinary tract symptoms. Herein, a limited series of arylsulfonamide derivatives of (aryloxy)ethyl alicyclic amines was designed, synthesized, and biologically evaluated as potent α1-adrenoceptor antagonists with uroselective profile. Among them, compound 9 (3-chloro-2-fluoro-N-([1-(2-(2-(2,2,2-trifluoroethoxy)phenoxy]ethyl)piperidin-4-yl)methyl)benzenesulfonamide) behaved as an α1A-/α1D-adrenoceptor antagonist (Ki(α1) = 50 nM, EC50(α1A) = 0.8 nM, EC50(α1D) = 1.1 nM), displayed selectivity over α2-adrenoceptors (Ki(α2) = 858 nM), and a 5-fold functional preference over the α1B subtype. Compound 9 showed adequate metabolic stability in rat-liver microsome assay similar to the reference drug tamsulosin (Clint = 67 and 41 µL/min/mg, respectively). Compound 9 did not decrease systolic and diastolic blood pressure in normotensive anesthetized rats in the dose of 2 mg/kg, i.v. These data support development of uroselective agents in the group of arylsulfonamides of alicyclic amines with potential efficacy in the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.


Introduction
α 1 -Adrenergic receptors (α 1 -ARs) belong to the G-protein-coupled receptor superfamily. They generally mediate their actions through G q/11 proteins, which stimulate the activation of phospholipase C, via generation of the inositol triphosphate and diacylglycerol, liberation of calcium from the endoplasmic reticulum, and/or activation of genes. To date, three subtypes of α 1 -AR, i.e., α 1A , α1A, α1B, and α1D have been identified in human tissues [1]. Although these subtypes display high structural homology, they differ in biological structure, tissue distributions, and pharmacological actions [2]. Several studies revealed that α1-AR subtypes are highly expressed in blood vesselsmainly α1B-ARs, in the urogenital area (prostate, urethra, bladder, ureter)-mainly α1A and α1D-ARs, and central nervous system [3]. α1-ARs play an important role in the pathogenesis of hypertension and benign prostatic hyperplasia (BPH) [4,5].
An increased α1-adrenergic prostate smooth muscle tone together with enhanced prostate volume are recognized causes of the disease [6]. BPH clinically manifests with lower urinary tract symptoms (LUTS) as storage (irritative) symptoms (nocturia, urgency, incontinence, altered bladder sensations, increased frequency) or obstructive (voiding) symptoms (hesitancy, slow stream, intermittency, splitting, straining, terminal dribble) [7]. Some of them commonly occur secondary to obstructive symptoms, and result from exaggerated, spontaneous detrusor contractions (known as bladder overactivity) [7,8]. BPH affects the majority of men with increasing frequency as they get older [9]. LUTS, if left untreated, result in significant impairment of quality of life and lead to longterm complications, such as recurrent urinary tract infections or renal insufficiency [10].
Despite several classes of BPH medications available, studies have shown that α1-adrenolitics are considered as the first-line drug treatment [11]. It has been suggested that enhanced, three-tonine-fold greater expression of α1A-and α1D-ARs in an enlarged prostate and bladder neck, comparing to healthy tissue, remains in strong contribution with LUTS occurrence [12]. Consequently, an α1Aand α1D-AR blockade relieves obstructive and voiding symptoms by relaxation of the smooth muscle in the prostate and bladder detrusor, respectively [13].
In contrast, a blockade of α1B-ARs, which are predominantly expressed in vascular smooth muscle [14], results in vasodilation of blood vessels leading to cardiovascular side effects, especially orthostatic hypotension [15]. The old α1-adrenolitics, bearing quinazoline scaffold, i.e., doxazosin or terazosin, display nonspecific interaction with all α1-AR subtypes [5]. On the other hand, naftopidil, tamsulosin, and silodosin (Figure 1), displaying relatively high α1A-and α1D-AR subtype selectivity, effectively relieve symptoms related to BHP/LUTS disease without undesirable side effects on blood pressure [16][17][18]. Integrating a concept of arylpiperazine biomimetics recently adapted for development of selective and potent 5-HT7R antagonists [19], we explored the common chemical space with tamsulosin to propose modifications leading to increased α1A-AR properties. Associating arylsulfonamide and aryloxylalkyl fragments identified compound I, which behaved as an α1A-AR antagonist and displayed a moderate selectivity receptor profile over α1B-AR subtype [20]. In an attempt to further increase the uroselective profile, a limited series of compounds integrating silodosin-derived chemical scaffold was designed ( Figure 2). Selection of the central amine core (4aminomethyl-piperidine and 3-amino-pyrrolidine), as well as a kind of substituent at the arylsulfonamide moiety, was based on our previously reported data presenting their preference for α1A-AR over 5-HT1A, and 5-HT7R [20][21][22].
All the synthesized derivatives were in vitro evaluated to assess their affinity for α1-AR and selectivity over α2-AR subtypes. Then, antagonist properties of selected derivatives against α1A-, α1B-, and α1D-AR subtypes were determined in cellular functional assays. The most representative compounds with uroselective functional profile were submitted under extended in vitro screening towards off-targets responsible for potential side effects, and were evaluated in metabolic stability in in vitro assay to assess their susceptibility to biotransformation. Finally, selected compounds were administered to normotensive Integrating a concept of arylpiperazine biomimetics recently adapted for development of selective and potent 5-HT 7 R antagonists [19], we explored the common chemical space with tamsulosin to propose modifications leading to increased α 1A -AR properties. Associating arylsulfonamide and aryloxylalkyl fragments identified compound I, which behaved as an α 1A -AR antagonist and displayed a moderate selectivity receptor profile over α 1B -AR subtype [20]. In an attempt to further increase the uroselective profile, a limited series of compounds integrating silodosin-derived chemical scaffold was designed ( Figure 2). Selection of the central amine core (4-aminomethyl-piperidine and 3-amino-pyrrolidine), as well as a kind of substituent at the arylsulfonamide moiety, was based on our previously reported data presenting their preference for α 1A -AR over 5-HT 1A , and 5-HT 7 R [20][21][22].
All the synthesized derivatives were in vitro evaluated to assess their affinity for α 1 -AR and selectivity over α 2 -AR subtypes. Then, antagonist properties of selected derivatives against α 1A -, α 1B -, and α 1D -AR subtypes were determined in cellular functional assays. The most representative compounds with uroselective functional profile were submitted under extended in vitro screening towards off-targets responsible for potential side effects, and were evaluated in metabolic stability in in vitro assay to assess their susceptibility to biotransformation. Finally, selected compounds were administered to normotensive anesthetized rats to evaluate their effects on blood pressure as a measure of their potential in vivo uroselectivity and to exclude hypotensive effects unfavorable to the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia. anesthetized rats to evaluate their effects on blood pressure as a measure of their potential in vivo uroselectivity and to exclude hypotensive effects unfavorable to the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.

Chemistry
The multistep protocol for synthesis of compounds 8-18 in outlined in Schemes 1 and 2. Initially, (2,2,2-trifluoroethoxy)phenol (3) was synthesized by alkylation of commercially available guaiacol 1, followed by demethylation of intermediate 2 in the presence of boron tribromide (Scheme 1). anesthetized rats to evaluate their effects on blood pressure as a measure of their potential in vivo uroselectivity and to exclude hypotensive effects unfavorable to the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.
Next, the alkylation of phenol 3 under biphasic conditions yielded the corresponding (aryloxy)ethyl bromide 4. Subsequently, this alkylating agent reacted with selected Boc-protected alicyclic amines (4-aminomethyl-piperidine, R-3-amino-pyrrolidine, and S-3-amino-pyrrolidine), giving intermediates 5-7. Removal of the protecting group, followed by the treatment with selected arylsulfonyl chloride, yielded final arylsulfonamide derivatives 8-18. anesthetized rats to evaluate their effects on blood pressure as a measure of their potential in vivo uroselectivity and to exclude hypotensive effects unfavorable to the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.

Radioligand Binding and Functional Evaluation
The pharmacological profile of the new compounds was assessed in radioligand-binding assays as the ability to displace [ 3 H]-Prazosin or [ 3 H]-Clonidine from α 1 -and α 2 -ARs, respectively, on rat cerebral cortex [23]. The inhibition constants (K i ) were calculated from the Cheng-Prusoff equation [24].
The intrinsic activity at α 1A -ARs of the selected compounds was assessed by fluorescence detection (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) of β-lactamase reporter genes using a FRET-enabled substrate. The intrinsic activity at α 1B -ARs and α 1D -ARs was determined by luminescence detection (PerkinElmer, Zaventem, Belgium) of calcium mobilization using the recombinant-expressed jellyfish photoprotein (Aequorin).
The most representative compounds, 9 and 10, with the highest functional selectivity were further tested to determine the affinity for 5-HT 1A and 5-HT 7 Rs in screening radioligand-binding studies using , respectively. Experiments were performed using membranes from CHO-K1 cells stably transfected with the human 5-HT 1A and 5-HT 7 Rs according to the methods previously described [25].
Finally, the percentage of inhibition for selected compounds 9 and 10 for off-target histaminic H 1 R, muscarinic M 1 R, adrenergic β 1 -AR and potassium ion channel hERG were assessed at Eurofins (Celle-Lévescault, France) according to the procedure online at www.eurofins.com [26].

Metabolic Stability
In vitro biotransformation assays of selected compounds 9 and 10 were performed using rat-liver microsomes (RLM), potassium-phosphate buffer, NADPH-regenerating system (NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase), and levallorphan as internal standard, according to the previously published procedure [27]. Compound I and the drug tamsulosin were used as reference standard. UPLC/MS analysis (Waters Corporation, Milford, MA, USA) was performed to determine the quantity of the starting material left in solution. The in vitro half-time (t 1/2 ) for test compounds was determined from the slope of the linear regression of ln % parent compound remaining versus incubation time. The calculated t 1/2 was incorporated into the following equation to obtain intrinsic clearance: (Cl int ) = (volume of incubation [µL]/protein in the incubation [mg]) × 0.693/t 1/2 .

In Vivo Pharmacology
Compounds 9 and 10, which displayed the highest α 1B /α 1A selectivity profile, were selected for in vivo evaluation to determine their influence on blood pressure of normotensive anaesthetized rats after acute administration at single dose of 2 mg/kg (i.v.). The experiments were performed to our previously reported method.

Results and Discussion
All synthesized compounds were in vitro evaluated in binding assays for their affinity for α 1 -AR and selectivity over α 2 -AR subtype. Compounds showed high-to-moderate affinity for α 1 -ARs (K i = 19-171 nM), and low-to-moderate selectivity over α 2 -AR subtype ( Table 1). Analysis of the influence of substituent in position-2 at the aryloxy fragment showed that an increase of its volume by replacing the isopropoxy group present in compound I and II with the 2,2,2-trifluoroethoxy one (present in a new series) only slightly increased the affinity for α 1 -ARs (I vs. 9, II vs. 16).
It is well known that a blockade of α1A-and α1D-ARs relaxes the enhanced prostate and bladder detrusor smooth muscle tone, whereas α1B-AR antagonism is involved in blood-pressure regulation. Normal detrusor, obtained from surgical patients, expresses predominantly α1D-ARs. Some pharmacological experiments revealed that highly selective α1A-AR antagonists are effective in relaxing prostate smooth muscle and therefore improving urine flow in men in this area. However, relaxation of smooth muscle of the prostate alone does not alter reported LUTS scores in men with BPH. Reduction of these symptoms is reported only when pharmacotherapy includes drugs with both α1A-and α1D-AR antagonistic activity. Such activity improves bladder-based symptoms in Further modifications involved the introduction of different electron-withdrawing or electron-donating substituents at the phenyl ring of sulfonamide moiety. A fluorine atom in 4-position was sufficient for obtaining a potent α 1 -AR ligand 8 (K i = 20 nM) among the 4-aminomethyl-piperidine subset, but did not significantly improve the affinity of pyrrolidine derivatives 13 and 14 for α 1 -AR (K i = 188 and 134 nM, respectively). Interestingly, the presence of the 4-F substituent in both series led to derivatives with the highest selectivity over the α 2 -AR subtype (S α2/α1 ≥ 46). An introduction of two halogen substituents did not affect the affinity for α 1 -AR while decreasing the selectivity over the α 2 -AR subtype (8 vs. 9 and 10, 13 vs. 15, and 14 vs. 16). Replacing one of the halogen substituents (e.g., 2-F) in compound 10, with an electron-donating substituent as the 2-methoxy, up to 4-fold reduced both affinity for α 1 -AR and selectivity over α 2 -AR (10 vs. 11). Finally, compounds 12, 17, and 18, with two methoxy groups in 3,4-position at the phenyl ring of sulfonamide moiety, showed higher affinity for α 1 -AR than the 4-F direct analogs (8 vs. 12, 13 vs. 17, and 14 vs. 18); however, this modification decreased the selectivity over α 2 -AR subtype. Selected compounds with the highest affinity for α 1 -ARs (K i ≤ 50 nM) and selectivity ratio, which equals >15-fold over α 2 -AR subtype, behaved as potent antagonists at α 1A -, α 1B -, and α 1D -ARs in in-vitro functional tests (Table 2). Compounds 8, 9, and 10 were classified as more potent antagonists than previously reported compound I at all tested α 1A -, α 1B -, and α 1D -ARs. Compounds 9 and 10, bearing two halogen atoms in ortho and meta position (i.e., 3-Cl,2-F and 5-Cl,2-F) at the phenyl ring of sulfonamide moiety displayed the highest α 1B /α 1A selectivity ratio. An introduction of the strong electron-donating substituent in meta and para position (e.g., 3,4-diOMe), switched the functional-selectivity profile of compound 12, which behaved as a selective α 1B -AR antagonist (IC 50 = 0.022 nM).
It is well known that a blockade of α 1A -and α 1D -ARs relaxes the enhanced prostate and bladder detrusor smooth muscle tone, whereas α 1B -AR antagonism is involved in blood-pressure regulation. Normal detrusor, obtained from surgical patients, expresses predominantly α 1D -ARs. Some pharmacological experiments revealed that highly selective α 1A -AR antagonists are effective in relaxing prostate smooth muscle and therefore improving urine flow in men in this area. However, relaxation of smooth muscle of the prostate alone does not alter reported LUTS scores in men with BPH. Reduction of these symptoms is reported only when pharmacotherapy includes drugs with both α 1A -and α 1D -AR antagonistic activity. Such activity improves bladder-based symptoms in humans and is used in LUTS pharmacotherapy [13]. Compounds 8, 9, 10, and 12 in the intrinsic activity studies showed strong antagonistic properties against the α 1D -AR subtype with EC 50 in the range of 1.1 to 2.7 nM. However, among the tested compounds, only 9 and 10 showed a similar inhibitory effect on intrinsic signal transduction in cells with stable expression of human α 1A -and α 1D -ARs. Some pieces of evidence suggest an involvement of serotonin 5-HT 1A and 5-HT 7 Rs in regulation of rodent bladder and urethral-sphincter contractions in both in in vitro and in vivo models [28,29]. Thus, 5-HT 1A and 5-HT 7 R ligands may be regarded as adjunctive agents in alleviating LUTS associated to BPH. Compounds 9 and 10 displayed high-to-moderate affinity for 5-HT 1A and 5-HT 7 Rs (Table 3). The same compounds were further evaluated for their affinity for "off-target" receptor panels at Eurofins Cerep, and displayed weak affinity for histamine H 1 , muscarinic M 1 , adrenergic β 1 , and hERG channels (<50% @ 1 µM) [26]. These may suggest a low risk of compounds to evoke undesirable cardiovascular or CNS side effects. An initial assessment of the metabolic fate in liver was subsequently performed in an in-vitro RLM model. Compounds 9 and 10 showed relatively low clearances (Cl int = 67 and 91.7 µL/min/mg, respectively, Table 4), with values similar to those of reference compound I and the drug tamsulosin (Cl int = 87 and 41 µL/min/mg, respectively). The values of internal clearance calculated for the tested compounds are in line with the value of clearance of reference drugs (i.e., propranolol, verapamil) reported in the literature [30,31]. Identified compounds 9 and 10 with favorable α 1B /α 1D profile and acceptable metabolic stability were selected for in vivo tests to evaluate their influence on blood pressure. Hypotensive activity was determined after one time i.v. administration to normotensive anaesthetized rats at single doses of 2 mg/kg according to our previously reported method [32].
It thus seems that compound 9 revealed a potential uroselective profile, comparable to tamsulosin, without evoking hypotension as a side effect. These data warrant further investigation of compound 9 in ex vivo preclinical models of BPH disease.
It thus seems that compound 9 revealed a potential uroselective profile, comparable to tamsulosin, without evoking hypotension as a side effect. These data warrant further investigation of compound 9 in ex vivo preclinical models of BPH disease.
It thus seems that compound 9 revealed a potential uroselective profile, comparable to tamsulosin, without evoking hypotension as a side effect. These data warrant further investigation of compound 9 in ex vivo preclinical models of BPH disease.

Conclusions
By combining the 2-(2,2,2-trifluoroethoxy)phenoxy fragment of silodosin with an alicyclic amine core functionalized with arylsulfonamide moiety, derived from previously reported compound I, we designed and synthesized a new series of arylsulfonamides of (aryloxy)ethyl pyrrolidines and piperidines as α 1 -AR antagonists. Structure-activity relationship studies revealed that the 4-aminomethylpiperidine core was preferential for binding with the α 1 -AR over the 3-aminopyrrolidine analog. Additionally, a kind of substituent at the phenyl ring of sulfonamide significantly impacted the selectivity of evaluated compounds over α 1B -and α 2 -AR subtypes. The study allowed the identification of compound 9 as a potent and metabolically stable α 1A -AR antagonist with improved α 1B /α 1A selectivity ratio, comparing with previously reported series. Moreover, compound 9 showed α 1D -AR antagonistic activity that may be beneficial in terms of LUTS therapy. In contrast to the reference drug tamsulosin, the tested compound did not decrease blood-pressure parameters after acute administration at the dose of 2 mg/kg (i.v.) in rats. Preliminary data for compound 9 are promising enough to warrant its further detailed mechanistic studies as a potential uroselective α 1A -and α 1D -AR antagonist in the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia.

General Chemical Methods
Organic transformations were carried out at ambient temperature unless indicated otherwise. Organic solvents (Sigma-Aldrich, Merck Group, Darmstadt, Germany) used in this study were of reagent grade and were used without purification. All other commercially available reagents were of the highest purity (Sigma-Aldrich). All workup and purification procedures were carried out with reagent-grade solvents under ambient atmosphere.
Mass spectra were recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY ® 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 × 100 mm, and 1.7 µm particle size, equipped with Acquity UPLC BEH C18 VanGuard precolumn (Waters Corporation, Milford, MA, USA); 2.1 × 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). Chromatograms were made using Waters eλ PDA detector. Spectra were analyzed in the 200-700 nm range with 1.2 nm resolution and sampling rate 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 150 • C, desolvation temperature 350 • C, desolvation gas flow rate 600 L·h −1 , cone gas flow 100 L·h −1 , capillary potential 3.00 kV, cone potential 40 V. Nitrogen was used for both nebulizing and drying gas. The data were obtained in a scan mode ranging from 50 to 1000 m/z in time 0.5 s intervals. Data acquisition software was MassLynx Elemental analyses for C, H, N and S were carried out using the elemental Vario EL III Elemental Analyser (Elementar Analysensysteme GmbH, Hanau, Germany). All values are given as percentages, and were within ±0.4% of the calculated values.
Melting points (mp) were determined with a Büchi apparatus (Flawil, Switzerland) and are uncorrected.
The general procedures used for the synthesis of intermediate and final compounds were in accordance with previously reported methodology [20].
Spectroscopic data (MS, 1 H-NMR and 13 C-NMR spectra) for representative final compounds are presented in Supplementary Materials. 7.1.2. Preparation of 1-Methoxy-2-(2,2,2-trifluoroethoxy)benzene (2) 2-Methoxy-phenol 1 (5.19 g, 0.04 mol) was dissolved in DMF (25 mL), after addition of K 2 CO 3 , (16.6 g, 0.12 mol) a mixture that was heated to 90 • C. Then 2-iodo-1,1,1-trifluoroethane (4.2 mL, 0.05 mol) was added dropwise in 30 min. The reaction mixture was then heated under reflux for 24 h. Inorganic residues were filtered off and organic mixture was concentrated under reduced pressure. The obtained crude product was purified using silica gel with AcOEt/Hexane (1/9, v/v) as an eluting system (isolated yield 65%). Yellow oil (5.6 g); UPLC/MS purity 99%, t R = 6.52. C 9 H 9 F 3 O 2 , MW 206. 16 Phenol 3 (4.8 g, 0.025 mol) was dissolved in acetone (30 mL). Then K 2 CO 3 (10.4 g, 0.075 mol) and catalytic amount of KI (0.08 g, 0.0005 mol) were added, followed by dropwise addition of 1,2-dibromoethane (12.9 mL, 0.15 mol). The reaction was refluxed for 48 h. Inorganic residues were filtered off and organic mixture was concentrated under reduced pressure. The obtained crude product was purified using silica gel with AcOEt/Hexane (0.5/9, v/v) as an eluting system (isolated yield 75%). Yellow oil (5.61 g); UPLC/MS purity 97%, t R = 7.41. C 10  Commercial Boc-protected amines (1 eq) were dissolved in acetone (15 mL). Then, K 2 CO 3 (3 eq) and a catalytic amount of KI (0.02 eq) were added, followed by dropwise addition of (aryloxy)ethyl bromide 4 (1.2 eq) in 30 min. The reaction was heated under reflux for 48 h. Inorganic residues were filtered off and organic mixture was concentrated under reduced pressure. The obtained crude products were purified according to the methods described below (isolated yields 68-75%). Intermediates 5-7 were converted into their TFA salts by treatment with a mixture of TFA/CH 2 Cl 2 (4 mL/1 mL). The excess reagent and solvent were removed under reduced pressure and maintained overnight under vacuum. A mixture of the appropriate (aryloxy)ethyl alicyclic amine (1 eq) in CH 2 Cl 2 (3 mL) and TEA (3 eq) was then cooled in an ice bath, and the proper arylsulfonyl chloride (1.2 eq) was added at 0 • C (the entire amount was added at the same time). The reaction mixture was stirred for 2-6 h under cooling. The solvent was evaporated, and the sulfonamides were a purified silica-gel column with CH 2 Cl 2 /MeOH (9/0.7, v/v) as an eluting system (isolated yields 55-87%). were applied. The incubation was terminated by rapid filtration over glass fiber filters (Whatman GF/C, Sigma-Aldrich) using a vacuum manifold (Millipore). The filters were then washed twice with the assay buffer and placed in scintillation vials with a liquid-scintillation cocktail. Radioactivity was measured in a WALLAC 1409 DSA liquid-scintillation counter (BioSurplus, San Diego, CA, USA). All the assays were made in duplicate. Binding experiments were conducted in 96-well microplates in a total volume of 250 µL of appropriate buffers. The composition of the assay buffers was as follows: 50 mM Tris-HCl, 0.1 mM EDTA, 10 mM MgCl 2 . The reaction mix included 50 µL solution of test compound, 50 µL of radioligand, and 150 µL of diluted membranes. All assays were incubated for 1 h (5-HT 1A Rs) or 2 h (5-HT 7 Rs) at 37 • C. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer, Waltham, MA, USA). Nonspecific binding is defined with 10 µM of 5-HT and 10 µM of methiothepine in 5-HT 1A R and 5-HT 7 R binding experiments, respectively. Each compound was tested in screening assay at two final concentrations of 10 µM and 1 µM.

Determination of the Intrinsic Activity of the α 1A -ARs
Intrinsic activity assay was performed according to the manufacturer of the assay kit (Invitrogen, Thermo Fisher Scientific corporation, Carlsbad, CA, USA). The cells were harvested and suspended in Assay Medium to a density of 312,500 cells/mL. Of the cell suspension, 32 µL per well was added to the Test Compound wells, the Unstimulated Control wells, and Stimulated Control wells and incubated per 16-24 h. To perform an agonist assay, 8 concentrations of 8 µL of the tested compound (10 −4 -10 −11 M), e.g., in 5-fold higher concentration in comparison to the final tested concentration in the well, were added to the cells. To perform an antagonist assay, 8 concentrations of 4 µL of the tested compound (10 −4 -10 −11 M), e.g., in 10-fold higher concentration in comparison to the final tested concentration in the well, were added to the cells. Then, after 30 min, 4 µL of standard agonist in EC 80 (10-fold higher concentration in comparison to the EC 80 in the well), in Assay Medium, was added to the cells. Then, both the agonist and the antagonist plate were incubated in a humidified 37 • C/5% CO 2 incubator for 5 h. After the incubation 8 µL of LiveBLAzer™-FRET B/G Substrate Mixture (CCF4-AM, Thermo Fisher Scientific corporation) was loaded cells in the absence of direct strong lighting, covered, and incubated at room temperature for 2 h. 7.2.4. Determination of the Intrinsic Activity of the α 1B -ARs and α 1D -ARs Intrinsic activity assay to α 1B -and α 1D -ARs was performed according to the manufacturer of the ready-to-use cells with stable expression of the α 1B -adrenoreceptors and α 1D -adrenoreceptors, respectively (PerkinElmer, Zaventem, Belgium). For measurement, cells (frozen, ready to use) were thawed and resuspended in 10 mL of assay buffer containing 5 µM coelenterazine h. This cells suspension was put in a 10 mL Falcon tube, fixed onto a rotating heel, and incubated for overnight at rt in the dark (8 rpm; 45 • angle). Cells were diluted with Assay Buffer to 5000 cells/20 µL. Agonistic ligands 2 × (50 µL/well), diluted in Assay Buffer, were prepared in 1 2 white polystyrene area plates, and the cell suspension was dispensed in 50 µL volume on the ligands using the injector. The light emitted was recorded for 20 s. Cells with antagonist were incubated for 15 min at room temperature. Thereafter, 50 µL of agonist (3 × EC 80 final concentration) was injected into the mix of cells and antagonist and the light emitted was recorded for 20 s.