The Structural Determinants for α1-Adrenergic/Serotonin Receptors Activity among Phenylpiperazine-Hydantoin Derivatives

Several studies confirmed the reciprocal interactions between adrenergic and serotoninergic systems and the influence of these phenomena on the pathogenesis of anxiety. Hence, searching for chemical agents with a multifunctional pharmacodynamic profile may bring highly effective therapy for CNS disorders. This study presents a deep structural insight into the hydantoin-arylpiperazine group and their serotonin/α-adrenergic activity. The newly synthesized compounds were tested in the radioligand binding assay and the intrinsic activity was evaluated for the selected derivatives. The computer-aided SAR analysis enabled us to answer questions about the influence of particular structural fragments on selective vs. multifunctional activity. As a result of the performed investigations, there were two leading structures: (a) compound 12 with multifunctional adrenergic-serotonin activity, which is a promising candidate to be an effective anxiolytic agent; (b) compound 14 with high α1A/α1D affinity and selectivity towards α1B, which is recommended due to the elimination of probable cardiotoxic effect. The structural conclusions of this work provide significant support for future lead optimization in order to achieve the desired pharmacodynamic profile in searching for new CNS-modulating agents.


Introduction
The α 1 -adrenergic receptors (ARs) belong to the great G-protein coupled receptor's (GPCR's) family and their role is the mediation of the sympathetic nervous system via binding endogenous catecholamines (adrenaline and noradrenaline) [1]. The deep characterization of tissue in the 1980s led to a division of ARs into three subtypes: α 1A -AR, α 1B -AR and α 1D -AR [2]. Thanks to the ability to relax the smooth muscle in the prostate, the α 1 -AR blockers found mainly clinical use in the treatment of benign prostatic hyperplasia (BPH)-the enlargement of the prostate gland, which may further lead to lower urinary tract symptoms (LUTS), significantly decreasing quality of life [3]. The common side effect concerns hypotension, which is most probably due to the result of interactions with the subtype α 1B -AR. For the tamsulosin-the selective α 1A /α 1D -AR antagonist (affinity to α 1A/ α 1D is 10-fold stronger than to α 1B )-a significantly less undesired cardiovascular Potential application of α 1 -AR blockers was indicated as an efficient treatment in cocaine use disorder (CUD) [5]. Additionally, the newest clinical studies confirmed that successful treatment with doxazosin is highly correlated with ADRA1D T-allele [6]. The very recent in vitro studies performed on novel derivatives of naftopidil indicate that α 1 -AR antagonists may be useful in prostate cancer therapy. The authors emphasized the significance of the subtype α 1 -AR selectivity for anticancer properties [7]. Finally, it is suggested that the regulation of α 1 -AR (α 1A and/or α 1B ) may play a neuroprotective role, being potentially useful for the treatment of neurological disorders (anxiety, depression, psychosis) [8]. For example, treatment with prazosin, an α 1 -AR inverse agonist, was successful in improving nightmare symptoms in patients with PTSD (posttraumatic stress disorder) [9]. Moreover, adrenergic receptors variants (including ADRA1A) were recently identified as the susceptibility factor for GAD (generalized anxiety disorders) [10].
Interestingly, several studies showed reciprocal interactions between adrenergic and serotoninergic systems and the influence of this phenomenon on the pathogenesis of anxiety [11,12]. The modulation of serotonin receptors, another protein belonging to the GPCR family, has already been indicated as a potential strategy for dealing with many CNS disorders including anxiety, especially receptors 5-HT 1A [13,14], 5-HT 2A [15] and 5-HT 7 [16,17]. In this light, searching for multitargeted serotonin/adrenergic agents is a promising solution for the above-mentioned CNS dysfunction treatment. The proof of this hypothesis is confirmation of the anxiolytic activity of compound ACH-000029-a multifunctional quinazoline derivative-in animal models (Figure 2), thus highlighting the importance of further research in this field [12]. Arylpiperazine is a very widely investigated chemical scaffold as it meets the main structural requirements of a pharmacophore model for many GPCR ligands (serotonin [18,19], histamine [20], adrenergic [21], dopamine receptors [22,23]). However, the nonpharmacophore moiety of the structure also maintains very important factors influencing Arylpiperazine is a very widely investigated chemical scaffold as it meets the main structural requirements of a pharmacophore model for many GPCR ligands (serotonin [18,19], histamine [20], adrenergic [21], dopamine receptors [22,23]). However, the non-pharmacophore moiety of the structure also maintains very important factors influencing the compound's geometry and, therefore, its pharmacodynamic profile. The structure-activity relationship (SAR) analysis for previously reported arylpiperazine derivatives of 5-arylidene hydantoin with a 2-hydroxypropyl linker ( Figure 3) led to the selection of the most beneficial substituent combinations, which provided high α 1 -AR affinity (for the best derivative: K i = 44.5 nM) [24]. Interestingly, another group of arylpiperazine hydantoins with 2-hydroxypropyl linker ( Figure 3) shows lower α 1 -AR activity (K i = 230 nM, for the best compound) but high and selective serotoninergic 5-HT 7 R activity (3 nM < K i < 94 nM), suggesting that the modulation of hydantoin substitution in position 5 (balancing between sp2 and sp3 hybridization of carbon atom C5) may be one of the main determinants for the serotoninergic/adrenergic affinity profile. Arylpiperazine is a very widely investigated chemical scaffold as it meets the main structural requirements of a pharmacophore model for many GPCR ligands (serotonin [18,19], histamine [20], adrenergic [21], dopamine receptors [22,23]). However, the nonpharmacophore moiety of the structure also maintains very important factors influencing the compound's geometry and, therefore, its pharmacodynamic profile. The structureactivity relationship (SAR) analysis for previously reported arylpiperazine derivatives of 5-arylidene hydantoin with a 2-hydroxypropyl linker ( Figure 3) led to the selection of the most beneficial substituent combinations, which provided high α1-AR affinity (for the best derivative: Ki = 44.5 nM) [24]. Interestingly, another group of arylpiperazine hydantoins with 2-hydroxypropyl linker ( Figure 3) shows lower α1-AR activity (Ki = 230 nM, for the best compound) but high and selective serotoninergic 5-HT7R activity (3 nM < Ki < 94 nM), suggesting that the modulation of hydantoin substitution in position 5 (balancing between sp2 and sp3 hybridization of carbon atom C5) may be one of the main determinants for the serotoninergic/adrenergic affinity profile. Hence, this study concerns the synthesis and in vitro pharmacological evaluation of novel 5-arylidenehydantoins ( Figure 4) with very deep computer-aided insight into the structure-activity relationship in order to deal with the to-date unanswered questions, that is, (i) how the structural stiffening via double bond introduction influences the α1-AR and serotoninergic receptors' activity; (ii) what the role of the hydroxy group in the linker is; (iii) what the best type of substitution of the benzylidene group is; (iv) what the key structural factors for α1-AR sub-type selectivity are. Hence, this study concerns the synthesis and in vitro pharmacological evaluation of novel 5-arylidenehydantoins ( Figure 4) with very deep computer-aided insight into the structure-activity relationship in order to deal with the to-date unanswered questions, that is, (i) how the structural stiffening via double bond introduction influences the α 1 -AR and serotoninergic receptors' activity; (ii) what the role of the hydroxy group in the linker is; (iii) what the best type of substitution of the benzylidene group is; (iv) what the key structural factors for α 1 -AR sub-type selectivity are.  All of the newly synthesized and previously reported derivatives by Czopek et al. (i.e., compound 14) [25] were tested in radioligand binding assays to measure the affinity at α-adrenergic receptors, and the serotoninergic 5-HT1AR, 5-HT6R and 5-HT7R. Additionally, for four compounds with the highest activity at α-ARs (10, 12, 14 and 16), functional affinities at α-AR subtypes (α1A-AR in rat tail artery, at α1B-AR in mouse spleen and at α1D-   [25] were tested in radioligand binding assays to measure the affinity at α-adrenergic receptors, and the serotoninergic 5-HT 1A R, 5-HT 6 R and 5-HT 7 R. Additionally, for four compounds with the highest activity at α-ARs (10, 12, 14 and 16), functional affinities at α-AR subtypes (α 1A -AR in rat tail artery, at α 1B -AR in mouse spleen and at α 1D -AR in rat aorta) were determined. In order to perform a deep structural analysis, the crystallographic studies for representative compound 4 were reported. The molecular docking studies and dynamic simulations were elaborated to analyze interactions within protein-ligand complexes and their stability.

Chemical Synthesis
Compounds 4-16 were synthesized via a three-step chemical pathway according to Scheme 1. 5-Arylidene hydantoins (17)(18)(19)(20) were obtained from hydantoin and a substituted benzaldehyde in Knoevenagel reaction. As we designed derivatives with two types of linkers (branched and unbranched), 2,3-epoxypropan-1-ol was used for N-substitution of position 3 of hydantoin in the Mitsunobu reaction, to obtain compounds 21-23 and 3-chloropropan-1-ol to give compounds 24-27. The final step was a microwave-assisted condensation reaction. In the case of compounds with an epoxide group, no additional reagent was necessary, whereas for compounds with a chloroalkyl group, K 2 CO 3 was used. All of the newly synthesized and previously reported derivatives by Czopek et al. (i.e., compound 14) [25] were tested in radioligand binding assays to measure the affinity at α-adrenergic receptors, and the serotoninergic 5-HT1AR, 5-HT6R and 5-HT7R. Additionally, for four compounds with the highest activity at α-ARs (10, 12, 14 and 16), functional affinities at α-AR subtypes (α1A-AR in rat tail artery, at α1B-AR in mouse spleen and at α1D-AR in rat aorta) were determined. In order to perform a deep structural analysis, the crystallographic studies for representative compound 4 were reported. The molecular docking studies and dynamic simulations were elaborated to analyze interactions within proteinligand complexes and their stability.

Evaluation of Intrinsic Activity towards α1-AR Subtypes
For the representative compounds with the highest α1-AR affinity (10, 12, 14 and 16), intrinsic activity was measured ( Table 2). All the tested compounds act as antagonists towards α1A-, α1B-and α1D-adrenergic receptors. However, compound 14 has significantly lower activity towards α1B in comparison with other investigated derivatives.

Evaluation of Intrinsic Activity towards α 1 -AR Subtypes
For the representative compounds with the highest α 1 -AR affinity (10, 12, 14 and 16), intrinsic activity was measured ( Table 2). All the tested compounds act as antagonists towards α 1A -, α 1B -and α 1D -adrenergic receptors. However, compound 14 has significantly lower activity towards α 1B in comparison with other investigated derivatives. Table 2. Functional activity results of reference and tested compounds 10, 12, 14 and 16 at α 1A -AR in rat tail artery, at α 1B -AR in mouse spleen and at α 1D -AR in rat aorta. Cmpd 19.1 ± 3.4 0.82 ± 0.1 4.0 ± 0.5 * Antagonist potency was presented as IC 50 ± SEM, IC 50 values which were obtained from the linear regression of Schild plot, each value ± SEM of 4-7 experimental results.

ChEMBL-Database-Oriented Searches for Structurally Similar Compounds
The analysis of the similarity coefficients of compounds present in the ChEMBL database [27] allowed us to evaluate their structural novelty. The threshold for the similarity coefficient Tanimoto (Tc) [28] applied was equal to 0.7. Excluding 2, 3 and 14 (compounds previously published [25,26]), no structures within datasets corresponding to considered targets were found to be similar by more than 0.7 (in terms of Tc); which confirmed the high structural novelty of the presented compounds.

Computer-Aided Structure-Activity Relationship towards Adrenergic Receptors
There were two main aims of molecular modeling studies carried out: explanation of the variation of activity of compounds 10, 12, 14 and 16 towards α 1 subtypes (especially the significantly lower activity of compound 14 towards α 1B ), as well as an explanation of compound activity profiles towards serotonin receptors (in particular, the examination of the role of the -OH substituent and hybridization of the hydantoin C5).
At first, the docking studies of 10, 12, 14 and 16 to α 1A , α 1B , and α 1D receptor models were carried out. They were performed with the use of the GPCRdb models [29] (inactive receptor states were used). The obtained docking poses for all the α 1 receptor subtypes are presented in Figure 5. The analysis of the similarity coefficients of compounds present in the ChEMBL database [27] allowed us to evaluate their structural novelty. The threshold for the similarity coefficient Tanimoto (Tc) [28] applied was equal to 0.7. Excluding 2, 3 and 14 (compounds previously published [25,26]), no structures within datasets corresponding to considered targets were found to be similar by more than 0.7 (in terms of Tc); which confirmed the high structural novelty of the presented compounds.

Computer-Aided Structure-Activity Relationship towards Adrenergic Receptors
There were two main aims of molecular modeling studies carried out: explanation of the variation of activity of compounds 10, 12, 14 and 16 towards α1 subtypes (especially the significantly lower activity of compound 14 towards α1B), as well as an explanation of compound activity profiles towards serotonin receptors (in particular, the examination of the role of the -OH substituent and hybridization of the hydantoin C5).
At first, the docking studies of 10, 12, 14 and 16 to α1A, α1B, and α1D receptor models were carried out. They were performed with the use of the GPCRdb models [29] (inactive receptor states were used). The obtained docking poses for all the α1 receptor subtypes are presented in Figure 5. The obtained docking results did not provide much insight into the observed variations in compound activity towards various α1 receptor subtypes. The IC50 values of 14 (in comparison with 10, 12, and 16) are much higher for α1A and α1B (57.6 nM and 182 nM, respectively). However, for this receptor subtype, the conformation of 14 was similar to the orientations of other, more active compounds: for α1A, 14 adopted a similar binding pose to 16, for which the IC50 value was equal to 19.1 nM, whereas for α1B, the orientation of 14 was analogous to the binding pose of 12, whose IC50 was equal to 8.5 nM.
In order to examine the interaction patterns obtained in docking in more detail and verify the stability of the obtained docking poses and indicate contacts which might have The obtained docking results did not provide much insight into the observed variations in compound activity towards various α 1 receptor subtypes. The IC 50 values of 14 (in comparison with 10, 12, and 16) are much higher for α 1A and α 1B (57.6 nM and 182 nM, respectively). However, for this receptor subtype, the conformation of 14 was similar to the orientations of other, more active compounds: for α 1A , 14 adopted a similar binding pose to 16 , for which the IC 50 value was equal to 19.1 nM, whereas for α 1B , the orientation of 14 was analogous to the binding pose of 12, whose IC 50 was equal to 8.5 nM.
In order to examine the interaction patterns obtained in docking in more detail and verify the stability of the obtained docking poses and indicate contacts which might have influence on the observed compound activities, molecular dynamics (MD) simulations were carried out. In addition to the qualitative analysis of the results, the interaction schemes obtained in each MD simulation were quantitatively confronted with experimental data to indicate positions, which can be responsible for the observed activity profiles. The procedure involved the generation of the interaction fingerprints (IFPs) [30] and the calculation of the Pearson correlation [31] coefficient (the correlation was determined between the total number of contacts with a given residue and antagonist potencies gathered in Table 2). The results of this analysis are presented in Figure 6.

Examination of Activity Profiles towards Serotonin Receptor Subtypes
The examination of correlations between contact frequency and the outcome of experimental studies indicate that there are positions for which a very high correlation exists. This is reflected not only by high values of the Pearson's correlation coefficients, but also the examination of the correlation charts and interaction diagrams from MD simulations confirms respective tendencies.
The highest number of highly correlated residues was indicated for α 1B R. Interestingly, one representative of these amino acids was the D3 × 32 (contact formed via α 1B R the piperazine moiety of ligands) position, which is known for the importance of its compound activity. This finding confirms the validity of the approach applied.
Within the six amino acid positions presented in Figure 6, there are also two that belong to the region of extracellular loops (E194 from ECL2 α 1B R, and K378 from ECL3 α 1D R). This indicates the importance of extracellular loops in the specific interaction with ligands and the necessity of the careful consideration of the interaction of compounds with these protein regions when designing new ligands of the desired activity pattern towards α-adrenergic receptors.
The molecular modeling study of serotonin receptors focused on two main aspects: examination of the influence of the hydroxy group in linker and the influence of the hybridization of C5 of hydantoin moiety on the ligand activity profile. In order to answer these questions, a group of compounds was selected for careful examination: 3, 6, 12 and also 7 and 13. The outcome of the docking studies is presented in Figure 7.

. Examination of Activity Profiles towards Serotonin Receptor Subtypes
The molecular modeling study of serotonin receptors focused on two main aspects: examination of the influence of the hydroxy group in linker and the influence of the hybridization of C5 of hydantoin moiety on the ligand activity profile. In order to answer these questions, a group of compounds was selected for careful examination: 3, 6, 12 and also 7 and 13. The outcome of the docking studies is presented in Figure 7.
For all analyzed compounds, the ligands are oriented in such a way that the arylpiperazine moiety is deeply buried in the binding pocket. In general, the compounds share similar fitting in the deeper part of the binding cavity, whereas their orientation varies significantly, when considering the upper part of the pocket. The binding data indicate that the introduction of the hydroxyl group to the linker does not seem to improve compound activity, despite forming strong interactions with respective amino acids. In general, affinity changes when considering OH-substituted ligands and their unsubstituted analogs are higher for 5-HT1AR than for 5-HT7R. Despite sharing the similar position of the arylpiperazine moiety in 5-HT1AR, the hydroxy group makes contacts with different residues. For compound 3, it forms a hydrogen bond with D3 × 32, for 6, with Y7 × 42, and for 7, with N7 × 38. However, although for 12, the respective contacts formed by the OH group are not present, there are other interactions formed, For all analyzed compounds, the ligands are oriented in such a way that the arylpiperazine moiety is deeply buried in the binding pocket. In general, the compounds share similar fitting in the deeper part of the binding cavity, whereas their orientation varies significantly, when considering the upper part of the pocket.
The binding data indicate that the introduction of the hydroxyl group to the linker does not seem to improve compound activity, despite forming strong interactions with respective amino acids. In general, affinity changes when considering OH-substituted ligands and their unsubstituted analogs are higher for 5-HT 1A R than for 5-HT 7 R. Despite sharing the similar position of the arylpiperazine moiety in 5-HT 1A R, the hydroxy group makes contacts with different residues. For compound 3, it forms a hydrogen bond with D3 × 32, for 6, with Y7 × 42, and for 7, with N7 × 38. However, although for 12, the respective contacts formed by the OH group are not present, there are other interactions formed, which strongly keep the compound in the 5-HT 1A R binding site: a hydrogen bond with N7 × 38 formed by the hydantoin moiety, a hydrogen bond with W7 × 39 formed by the methoxy group and a hydrogen bond with D3 × 32 (contact with the piperazine). In addition, pi-pi stacking is observed with W6 × 48, F5 × 45, and Y2 × 63. A similar situation is observed for 13, where several hydrogen bonds are also formed despite the lack of a hydroxy group in the linker. It is also worth indicating that, although compound 7 and 13 differ only in terms of the presence/absence of the hydroxy group in the linker, their docking poses are significantly different (higher pose variation again for 5-HT 1A R, which is also correlated with higher activity change towards this receptor). Interestingly, 7 is located a little bit deeper in the binding pocket of 5-HT 7 R than 13, whereas the relative position of these two ligands towards D3 × 32 of 5-HT 1A R is similar. Summarizing, the presence of the hydroxy group in the linker has no significant influence on 5-HT 7 R affinity, whereas it causes a decrease of 5-HT 1A R affinity, for example, from K i = 45.8 nM (compound 13) to K i = 313 nM (compound 7). Interestingly, the sp 3 hybridization of the C5 atom of hydantoin turned out to be a key factor for high 5-HT 7 R affinity, which provides enhanced structural flexibility (K i = 8 nM for compound 3 and K i = 197 nM for its analogue stiffened with double bond, i.e., compound 6). On the other hand, the structural stiffness achieved by the presence of the sp 2 -hybrydized C5 carbon atom provides a significant increase of general α-AR affinity (with K i = 530 nM for compound 3 and K i = 144.6 nM for compound 6).

Chemical Synthesis
1 H NMR spectra (supplementary materials) for all the final compounds and 13 C NMR spectra for representative compounds (7 and 8) were recorded on a Varian Mercury VX 300 MHz (Varian INC., Palo Alto, CA USA). Chemical shifts are expressed in parts per million (ppm), using the solvent (DMSO) signal as an internal standard. Data are reported using the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; p, pentet; Ar, aromatic, Pp, piperazine, Ph, phenyl (As shown in the Supporting Information). Thin-layer chromatography (TLC) was performed on pre-coated Merck silica gel 60 F254 aluminum sheets (Munich, Germany). The mass of compounds was recorded on a Waters ACQUITYTM UPLC (Waters, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode, EDI-tandem quadrupole). Retention times (t R ) are given in minutes. The UPLC/MS purity of all final compounds was determined (%).

Radioligand Binding Assays: Affinity for α 1 -Receptor
Tissue (rat cortex) was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer, pH 7.6, using an Ultra Turrax T25B homogenizer (IKA, Staufen, Germany). The homogenate was centrifuged at 20,000× g for 20 min. The resulting supernatant was decanted and pellet was resuspended in the same buffer and centrifuged again in the same conditions. The final pellet was resuspended in an appropriate volume of buffer (10 mg/1 mL). The radioactivity on the filter was measured by a MicroBeta TriLux 1450 scintillation counter (PerkinElmer, Waltham, MA, USA).
Radioligand binding data were analyzed using iterative curve fitting routines Graph-Pad Prism 3.0 (GraphPad Software, San Diego, CA, USA) using the built-in three parameter logistic model describing ligand competition binding to radioligand-labeled sites. The log IC 50 (i.e., the log of the ligand concentration that reduces specific radioligand binding by 50%) estimated from the data is used to obtain the K i by applying the Cheng-Prusoff approximation [35].

Radioligand
Binding Assays: Binding Affinities for 5-HT 1A , 5-HT 6 and 5-HT 7 Receptors HEK-293 cells with a stable expression of human 5-HT 1A , 5-HT 6 and 5-HT 7b receptors (prepared with the use of Lipofectamine 2000) were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and grown in Dulbecco's Modifier Eagle Medium containing 10% dialyzed fetal bovine serum and 500 µg/mL G418 sulfate. For membrane preparation, cells were subcultured in 150 cm 3 flasks, grown to 90% confluence, washed twice with phosphate buffered saline (PBS) prewarmed to 37 • C and pelleted by centrifugation (200× g) in PBS containing 0.1 mM EDTA and 1 mM dithiothreitol. Prior to membrane preparation, pellets were stored at −80 • C. Cell pellets were thawed and homogenized in 10 volumes of assay buffer using an Ultra Turrax tissue homogenizer and were centrifuged twice at 35,000× g for 15 min at 4 • C, with incubation for 15 min at 37 • C in-between. The composition of the assay buffers was as follows: for 5-HT 1A R: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl 2 , 10 µM pargyline and 0.1% ascorbate, for 5-HT 6 R-50mM TrisHCl, 0.5 mM EDTA and 4 mM MgCl 2 ; and for 5-HT 7b R: 50 mM Tris HCl, 4 mM MgCl 2 , 10 µM pargyline and 0.1% ascorbate. All the assays were incubated in a total volume of 200 µL in 96-well microtiter plates for 1 h at 37 • C, except those for 5-HT 1A R, which were incubated at room temperature. The process of equilibration was terminated by rapid filtration through Unifilter plates with a FilterMate Unifilter 96 Harvester (PerkinElmer, USA). The radioactivity bound to the filters was quantified on a Microbeta TopCount instrument (PerkinElmer, USA). For competitive inhibition studies, the assay samples contained the following as radioligands were calculated from the Cheng-Prusoff equation [35]. For all the binding assays, results were expressed as the means of at least two separate experiments.

Functional Tests
3.3.1. Determination of the Intrinsic Activity for the α 1A -Ars An intrinsic activity assay was performed according to the manufacturer of the assay kit (Invitrogen, Thermo Fisher Scientific). 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 were incubated per 16-24 h. To perform an agonist assay, eight concentrations of 8 µL of the tested compound (10 −4 -10 −11 M), for example, in five-fold higher concentration in comparison to the final tested concentration in the well, were added to the cells. To perform an antagonist assay, eight concentrations of 4 µL of the tested compound (10 −4 -10 −11 M), for example, in a ten-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 a standard agonist, phenylephrine, in EC 80 (ten-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. Then, cells were loaded with 8 µL of LiveBLAzer™-FRET B/G Substrate Mixture (CCF4-AM, Thermo Fisher Scientific) and incubated at room temperature for 2 h.

Determination of the Intrinsic Activity for the α 1B -ARs and α 1D -Ars
The Aequoscreen technology uses the recombinant cell lines with stable expression of the α 1B or α 1D adrenoreceptor and co-expression of apoaequorin and a GPCR as a system to detect the activation of the receptor, following the addition of an agonist, via the measurement of light emission. For measurement, cells (frozen, ready to use) were thawed and resuspended in 10-mL of assay buffer containing 5 µM coelenterazine h. This cell suspension was put in a 10-mL Falcon tube, fixed onto a rotating wheel and incubated overnight at RT • in the dark. Cells were diluted with Assay Buffer to 5000 cells/20 µL. In the first part, agonistic activity was tested. Potential agonists (standard and tested) 2× (50 µL/well), diluted in Assay Buffer, were prepared in 1/2 white polystryrene 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. In the second part, the agonistic activity was tested. Cells with tested compounds were incubated for 15 min at room temperature. Therefore, 50 µL of standard agonist, phenylephrine (3 × EC80 final concentration) was injected into the mix of cells and the antagonist, and the light emitted was recorded for 20 s.

Docking
The docking was performed with the use of the GPCRdb homology models of the respective receptors (inactive receptor states were considered). Receptors were prepared for docking using the Protein Preparation Wizard from the Schrodinger Suite. The compounds were prepared for docking using LigPrep [38] (protonation states for pH 7.4 were generated) and were docked to the considered receptor models in Glide [39] (grid centering on the aspartic acid from the third transmembrane helix; D3 × 32 according to the GPCRdb numbering) at extra precision mode. The obtained ligand-receptor complexes with the best docking score constituted an input for MD simulations.

Molecular Dynamics
MD simulations were carried out in Desmond [40] using the TIP3P solvent model [41] and POPC (palmitoyl-oleil-phosphatidylcoline) as a membrane model (force-field: OPLS3e, pressure: 1.01325 bars, temperature: 300 K). The box shape was orthorhombic of the size of 10 Å × 10 Å × 10 Å. In each case, the system was neutralized by the addition of the respective number of Cl-ions and relaxed before the simulation; the duration of each simulation was equal to 1000 ns.

Conclusions
The work presented here concerns the deep structural insight into the hydantoinarylpiperazine group and their serotonin/α-adrenergic activity. The computer-aided SAR analysis enabled us to answer the questions about the influence of particular structural fragments on selective vs. multifunctional activity. The obtained results led to conclusions that the hybridization type of the C5 carbon atom of hydantoin and the presence of a hydroxy group in the linker are key structural determinants for balancing between serotonin and α-adrenergic affinity. The selectivity among adrenergic subtypes turned out to be the most challenging issue; however, the results clearly indicate the importance of extracellular loops, which should be considered during the design of novel molecules.
As a result of the performed investigations, the two lead structures that were selected for the further studies were compounds 12 and 14. Compound 12 is a very promising 'lead' for the search of novel multifunctional anxiolytic agents, as it possesses high multifunctional α 1A /α 1D -AR/5-HT 1A R activity and moderates 5-HT 7 R affinity. Compound 14, despite its lower serotoninergic affinity, has high α 1A /α 1D affinity and moderates selectivity towards α 1B , which is recommended due to the elimination of probable cardiotoxic effects [4]. The structural conclusions of this work provide meaningful support for future lead optimization in order to achieve the desired pharmacodynamic profile in the search for new CNS-modulating agents.