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

Abstract

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 α_1A/α_1D affinity and selectivity towards α_1B, 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.

1. Introduction

The α₁-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 α₁-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 extension effect was observed than for the non-subtype selective α₁-AR blocker named terazosin (Figure 1) [4].

Potential application of α₁-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 α₁-AR antagonists may be useful in prostate cancer therapy. The authors emphasized the significance of the subtype α₁-AR selectivity for anticancer properties [7]. Finally, it is suggested that the regulation of α₁-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 α₁-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-HT₇ [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 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 α₁-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 α₁-AR activity (K_i = 230 nM, for the best compound) but high and selective serotoninergic 5-HT₇R 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.

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.

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-HT_1AR, 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 (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.

2. Results and Discussion

2.1. Chemical Synthesis

Compounds 4–16 were synthesized via a three-step chemical pathway according to Scheme 1. 5-Arylidene hydantoins (17–20) 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, K₂CO₃ was used.

2.2. Pharmacology

2.2.1. Radioligand Binding Assays

The radioligand binding results showed that 11 out of the 14 tested compounds (5, 7, 9–16) bind to α-ARs with high affinity (K_i < 100 nM). Compound 14 turned out to be the most active with K_i = 11.9 nM. Interestingly, compounds 12 and 13 are strong dual 5-HT_1AR/α-ARs ligands, whereas compound 5 indicates dual activity 5-HT₇/α-AR (

Table 1. Radioligand binding assays results for compounds 1–16.

Cmpd	Group	R 1	R 2	R 3	Ki [nM]
					α1	5-HT1A	5-HT6	5-HT7
1 *	A	3,4-diOCH3	OH	2-OCH3	44.5	nt	nt	nt
2 *	B	4-F	OH	2-OCH3	230	121	10,790	3
3 *	B	4-OCH3	OH	2-OCH3	530	99	nt	8
4	A	3-OCH3	OH	2-OCH3	187.0 ± 18.6	530	17,610	283
5	A	3-OCH3	OH	2-OC2H5	75.9 ± 2.3	556.7	19,220	79
6	A	4-OCH3	OH	2-OCH3	144.6 ± 13.3	282	20,420	197
7	A	4-OCH3	OH	2-OC2H5	99.1 ± 11.3	313	10,250	120
8	A	4-OCH3	OH	2-F	143.7 ± 7.9	3532	11,730	1036
9	A	3,4,5-triOCH3	OH	2-OC2H5	89.5 ± 9.0	1870	2835	1253
10	A	3-OCH3	H	2-OC2H5	20.2 ± 4.1	103.0	nt	nt
11	A	3-OCH3	H	2-Cl	44.8 ± 3.3	319	1720	152
12	A	4-OCH3	H	2-OCH3	34.6 ± 4.6	20.6	21,990	101
13	A	4-OCH3	H	2-OC2H5	19.5 ± 2.6	45.8	>50,000	114
14	A	3,4-diOCH3	H	2-OCH3	11.9 ± 2.2	128	15,780	189
15	A	3,4,5-triOCH3	H	2-OCH3	25.8 ± 2.9	172	12,320	668
16	A	3,4,5-triOCH3	H	2-OC2H5	14.7 ± 1.1	164.5	15,240	138
Ref	-	-	-	-	15.7 ± 1.5 1	32 2	7 3	62 4

^* Previously reported arylpiperazine hydantoin derivatives [25,26]; ¹ fentolamine, ² buspirone, ³ olanzapine, ⁴ clozapine.

).

2.2.2. 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. 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	IC50 ± SEM [nM] *
	α1A-AR	α1B-AR	α1D-AR
Phenylephrine	30.0 ± 3.4	27.0 ± 4.2	26.2 ± 4.1
Prazosin	4.1 ± 0.5	3.82 ± 0.5	n.d
Terazosin	51.9 ± 0.8	1.73 ± 0.3	0.1 ± 0.02
Tamsulosin	0.07 ± 0.01	0.13 ± 0.02	n.d.
10	7.2 ± 1.1	9.2 ± 1.3	1.2 ± 0.2
12	12.8 ± 2.5	8.5 ± 1.1	1.5 ± 0.3
14	57.6 ± 7.2	182 ± 21.4	3.0 ± 0.5
16	19.1 ± 3.4	0.82 ± 0.1	4.0 ± 0.5

* Antagonist potency was presented as IC₅₀ ± SEM, IC₅₀ values which were obtained from the linear regression of Schild plot, each value ± SEM of 4–7 experimental results.

). 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.

2.3. Cheminformatic Analysis and Molecular Modeling

2.3.1. 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.

2.3.2. 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 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 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 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 IC₅₀ value was equal to 19.1 nM, whereas for α_1B, 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 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.

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_1BR. Interestingly, one representative of these amino acids was the D3 × 32 (contact formed via α_1BR 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_1BR, and K378 from ECL3 α_1DR). 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.

2.3.3. 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-HT_1AR than for 5-HT₇R. Despite sharing the similar position of the arylpiperazine moiety in 5-HT_1AR, 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_1AR 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_1AR, 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-HT₇R than 13, whereas the relative position of these two ligands towards D3 × 32 of 5-HT_1AR is similar. Summarizing, the presence of the hydroxy group in the linker has no significant influence on 5-HT₇R affinity, whereas it causes a decrease of 5-HT_1AR affinity, for example, from K_i = 45.8 nM (compound 13) to K_i = 313 nM (compound 7). Interestingly, the sp³ hybridization of the C5 atom of hydantoin turned out to be a key factor for high 5-HT₇R 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 sp²-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).

3. Materials and Methods

3.1. Chemical Synthesis

¹H NMR spectra (supplementary materials) for all the final compounds and ¹³C 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 (%).

All four 5-arylidene hydantoin derivatives (17–20) and alkylated intermediates (21–27) were already described [25,32,33,34].

3.1.1. (Z)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-(3-methoxybenzylidene)imidazolidine-2,4-dione (4)

White solid. Yield 63%; mp 163–166 °C; LC/MS⁺ purity: 100%, t_R = 4.64, MS calcd for [M + H]⁺: C₂₅H₃₀N₄O₅ m/z: 466.54, found 467.28. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.74 (s, 1H, N¹H), 7.26 (t, J = 7.9 Hz, 1H, Ar), 7.16 (d, J = 7.6 Hz, 1H, Ar), 7.10 (s, 1H, Ar), 6.85 (s, 2H, Ar), 6.82 (d, J = 7.0 Hz, 1H, Ar), 6.79 (d, J = 7.1 Hz, 1H, Ar), 6.72–6.68 (m, 1H, Ar), 6.46 (s, 1H, Ph–CH=), 4.96 (br. s, 1H, OH), 3.98–3.96 (m, 1H, CH-OH), 3.73 (s, 3H, OCH₃), 3.70 (s, 3H, OCH₃), 3.50–3.48 (m, 2H, N³-CH₂), 2.85 (br. s, 4H, Pp-3,5-H), 2.52 (br. s, 2H, Pp-CH₂), 2.40–2.34 (m, 4H, Pp-2,6-H).

3.1.2. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-5-(3-methoxybenzylidene)imidazolidine-2,4-dione (5)

White solid. Yield 41%; mp 120–123 °C; LC/MS⁺ purity: 97.33%, t_R = 4.71, MS calcd for [M + H]⁺: C₂₆H₃₂N₄O₅ m/z: 480.24, found 481.37. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.84 (s, 1H, N¹H), 9.96 (br. s, 1H, OH), 7.35–7.26 (m, 1H, Ar), 7.20 (d, J = 7.7 Hz, 1H, Ar), 7.13–7.12 (m, 1H, Ar), 6.83–6.94 (m, 5H, Ar), 6.49 (s, 1H, Ph–CH=), 4.29–4.25 (m, 1H, CH-OH), 3.96 (q, J = 6.9 Hz, 2H, O-CH₂-CH₃), 3.76 (s, 3H, O-CH₃), 3.64–3.38 (m, 6H, Pp 3,5 H, Pp CH₂), 3.36–2.91 (m, 6H, Pp 2,6 H, N³-CH₂), 1.30 (t, J = 7.0 Hz, 3H, O-CH₂-CH₃).

3.1.3. (Z)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-(4-methoxybenzylidene)imidazolidine-2,4-dione (6)

White solid. Yield 57%; mp 176–178 °C; LC/MS⁺ purity: 100%, t_R = 4.27, MS calcd for [M + H]⁺: C₂₅H₃₀N₄O₅ m/z: 466.22, found 467.21. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.61 (s, 1H, N¹H), 7.55 (d, J = 8.8 Hz, 2H, Ar), 6.91 (d, J = 8.9 Hz, 2H, Ar), 6.88–6.82 (m, 2H, Ar), 6.77– 6.64 (m, 2H, Ar), 6.46 (s, 1H, Ph–CH=), 4.95 (br. s, 1H, OH), 4.02–3.90 (m, 1H, CH-OH), 3.74 (s, 3H, OCH₃), 3.70 (s, 3H, OCH₃), 3.55–3.35 (m, 2H, N³-CH₂), 2.97–2.71 (m, 4H, Pp-3,5-H), 2.63–2.47 (m, 2H, Pp-CH₂), 2.46–2.32 (m, 4H, Pp-2,6-H).

3.1.4. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-5-(4-methoxybenzylidene)imidazolidine-2,4-dione (7)

White solid. Yield 53%; mp 180–183 °C; LC/MS⁺ purity: 100%, t_R = 4.64, MS calcd for [M + H]⁺: C₂₆H₃₂N₄O₅ m/z: 480.24, found 481.30. ¹H NMR (500 MHz, DMSO-d₆) δ 10.65 (br s, 1H, N¹H), 7.57 (d, J = 8.75 Hz, 2H, Ar), 6.90 (d, J = 8.8 Hz, 2H, Ar), 6.83–6.80 (m, 2H, Ar), 6.75–6.70 (m, 1H, Ar), 6.69–6.67 (m, 1H, Ar), 6.46 (s, 1H, Ph–CH=), 4.94 (s, 1H, OH), 3.99–3.97 (m, 1H, CH-OH), 3.91 (q, J = 6.9 Hz, 2H, OCH₂-CH₃), 3.75 (s, 3H, OCH₃), 3.58–3.39 (m, 2H, N³-CH₂), 2.93–2.82 (m, 4H, Pp-3,5-H), 2.52–2.58 (m, 2H, Pp-CH₂), 2.40–2.26 (m, 4H, Pp-2,6-H), 1.27 (t, J = 6.9 Hz, 3H, O-CH₂-CH₃). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]: 165.04, 160.02, 155.93, 151.66, 141.96, 131.66, 125.96, 125.52, 122.69, 121.35, 118.37, 114.84, 113.67, 109.91, 64.09, 63.76, 63.53, 55.80, 54.19, 50.39, 44.10, 15.35.

3.1.5. (Z)-3-(3-(4-(2-fluorophenyl)piperazin-1-yl)-2-hydroxypropyl)-5-(4-methoxybenzylidene)imidazolidine-2,4-dione (8)

White solid. Yield 62%; mp 104–106 °C; LC/MS⁺ purity: 100%, t_R = 4.36, MS calcd for [M + H]⁺: C₂₄H₂₇FN₄O₄ m/z: 454.20, found 455.18. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.59 (s, 1H, N¹H), 7.55 (d, J = 8.6 Hz, 2H, Ar), 7.06–7.02 (m, 1H, Ar), 6.98–6.95 ( t, J = 7.6 Hz, 1H Ar), 6.91 (d, J = 8.6 Hz, 2H, Ar), 6.89–6.86(m, 1H, Ar), 6.83 (t, J = 8.4 Hz, 1H, Ar) 6.46 (s, 1H, Ph–CH=), 4.95–4.94 (m, 1H, OH), 3.97–3.96 (m, 1H, CH-OH), 3.75 (s, 3H, OCH₃), 3.61–3.40 (m, 2H, N³-CH₂), 2.90–2.88 (m, 4H, Pp-3,5-H), 2.60–2.50 (m, 2H, Pp-CH₂), 2.44–2.27 (m, 4H, Pp-2,6-H).¹³C NMR (125 MHz, DMSO-d₆ ) δ [ppm]: 165.04, 160.02, 156.46, 155.94, 154.52, 140.44, 140.37, 131.66, 125.95, 125.51, 125.23, 125.20, 119.66, 119.64, 116.44, 116.28, 114.83, 109.94, 64.11, 63.40, 53.95, 55.58, 50.56, 44.08.

3.1.6. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-5-(3,4,5-trimethoxybenzylidene)imidazolidine-2,4-dione (9)

White solid. Yield 62%; mp 104–106 °C; LC/MS⁺ purity: 100%, t_R = 4.59, MS calcd for [M + H]⁺: C₂₈H₃₆N₄O₇ m/z: 540.62, found 541.25. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.78 (s, 1H, N¹H), 6.83–6.82 (m, 2H, Ar), 6.81 (s, 2H, Ar), 6.78–6.67 (m, 2H, Ar), 6.45 (s, 1H, Ph–CH=), 4.96–4.95 (d, J = 5.4 Hz, 1H, OH), 3.99–3.97 (m, 1H, CH-OH), 3.92 (q, J = 7.0 Hz, 2H, O-CH₂-CH₃), 3.76 (s, 6H, 2xOCH₃), 3.63 (s, 3H, OCH₃), 3.51–3.47 (m, 2H, N³-CH₂), 3.28–3.24 (m, 2H, Pp-CH₂), 2.97–2.76 (m, 4H, Pp-3,5-H), 2.43–2.27 (m, 4H, Pp-2,6-H), 1.26 (t, J = 7.0 Hz, 3H, O-CH₂-CH₃).

3.1.7. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)-5-(3-methoxybenzylidene)imidazolidine-2,4-dione (10)

White solid. Yield 24%; mp 120–122 °C; LC/MS⁺ purity: 100%, t_R = 4.70, MS calcd for [M + H]⁺: C₂₆H₃₂N₄O₄ m/z: 464.24, found 465.37. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.74 (s, 1H, N¹H), 7.28–7.24 (m, 1H, Ar), 7.16 (d, J = 6.9 Hz, 1H, Ar), 7.10–7.09 (m, 1H, Ar), 6.87–6.85 (m, 1H, Ar), 6.83–6.82 (m, 2H, Ar), 6.71–6.81 (m, 2H, Ar), 6.45 (s, 1H, Ph–CH=), 3.94 (q, J = 7.0 Hz, 2H, O-CH₂-CH₃), 3.74 (s, 3H, O-CH₃), 3.52–3.45 (m, 2H, N³-CH₂), 2.97–2.72 (m, 4H, Pp-3,5-H), 2.62–2.49 (m, 2H, CH₂), 2.42–2.27 (m, 6H, Pp-2,6-H, Pp-CH₂), 1.28 (t, J = 6.9 Hz, 3H, O-CH₂-CH₃).

3.1.8. (Z)-3-(3-(4-(2-chlorophenyl)piperazin-1-yl)propyl)-5-(3-methoxybenzylidene)imidazolidine-2,4-dione (11)

White solid. Yield 31%; mp 144–146 °C; LC/MS⁺ purity: 96.78%, t_R = 4.93, MS calcd for [M + H]⁺: C₂₄H₂₇ClN₄O₃ m/z: 454.18, found 455.25. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.77 (s, 1H, N¹H), 7.32 (dd, J = 7.9, 1.5 Hz, 1H, Ar), 7.26 (t, J = 7.9 Hz, 1H, Ar), 7.18 (d, J = 7.7 Hz, 1H, Ar), 7.14 (d, J = 3.3 Hz, 1H, Ar), 7.11 (dd, J = 7.8, 1.7 Hz, 1H, Ar), 6.96–6.92 (m, 2H, Ar), 6.86 (dd, J = 8.6, 2.9 Hz, 1H, Ar), 6.44 (s, 1H, Ph–CH=), 3.74 (s, 3H, O-CH₃), 3.53 (t, J = 6.7 Hz, 2H, N³-CH₂), 2.88–2.86 (m, 4H, Pp-3,5-H), 2.45–2.39 (m, 4H, Pp-2,6-H), 2.35 (t, J = 6.5 Hz, 2H, Pp-CH₃), 1.72 (p, J = 6.5 Hz, 2H, CH₂).

3.1.9. (Z)-5-(4-methoxybenzylidene)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (12)

White solid. Yield 57%; mp 176–179 °C; LC/MS⁺ purity: 96.78%, t_R = 4.52, MS calcd for [M + H]⁺: C₂₅H₃₀N₄O₄ m/z: 450.23, found 451.26. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 7.67 (d, J = 8.4 Hz, 2H, Ar), 6.93–6.82 (m, 5H, Ar), 6.81–6.71 (m, 2H, Ar, Ph-CH=), 3.73 (s, 3H, O-CH₃), 3.71 (s, 3H, O-CH₃), 3.42–3.40 (m, 2H, N³-CH₂), 2.86–2.83 (m, 4H, Pp-3,5-H), 2.41–2.39 (m, 4H, Pp-2,6-H), 2.33–2.28 (m, 2H, Pp-CH₂), 1.73–1.60 (m, 2H, CH₂).

3.1.10. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)-5-(4-methoxybenzylidene)imidazolidine-2,4-dione (13)

White solid. Yield 53%; mp 180–183 °C; LC/MS⁺ purity: 100%, t_R = 4.90, MS calcd for [M + H]⁺: C₂₆H₃₂N₄O₄ m/z: 464.24, found 465.35. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.60 (s, 1H, N¹H), 7.56 (d, J = 9.7 Hz, 2H, Ar), 6.91 (d, J = 8.9 Hz, 2H, Ar), 6.85–6.83 (m, 2H, Ar), 6.75–6.68 (m, 2H, Ar), 6.47 (s, 1H, Ph–CH=), 3.94 (q, J = 7.0 Hz, 2H, O-CH₂-CH₃), 3.75 (s, 3H, O-CH₃), 3.51 (t, J = 6.9 Hz, 2H, N³-CH₂), 3.26–3.24 (m, 4H, Pp-3,5-H), 2.94–2.80 (m, 4H, Pp-2,6-H), 2.32 (t, J = 7.0 Hz, 2H, Pp-CH₂), 1.71 (p, J = 7.0 Hz, 2H, CH₂), 1.27 (t, J = 7.0 Hz, 3H, O-CH₂-CH₃).

3.1.11. (Z)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-(3,4,5-trimethoxybenzylidene)imidazolidine-2,4-dione (15)

White solid. Yield 20%; mp 130–132 °C; LC/MS⁺ purity: 100%, t_R = 4.48, MS calcd for [M + H]⁺: C₂₇H₃₄N₄O₆ m/z: 510.25, found 511.21. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.77 (s, 1H, N¹H), 6.87–6.84 (m, 2H, Ar), 6.82 (s, 2H, Ar), 6.73–6.71 (m, 2H, Ar), 6.46 (s, 1H, Ph–CH=), 3.76 (s, 6H, 2xO-CH₃), 3.70 (s, 3H, O-CH₃), 3.63 (s, 3H, O-CH₃), 3.53 (t, J = 6.7 Hz, 2H, N³–CH₂), 2.86–2-84 (m, 4H, Pp-3,5-H), 2.43–2.32 (m, 6H, Pp-2,6-H, Pp-CH₂), 1.72 (p, J = 6.6 Hz, 2H, CH₂).

3.1.12. (Z)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)-5-(3,4,5-trimethoxybenzylidene)imidazolidine-2,4-dione (16)

White solid. Yield 20%; mp 130–132 °C; LC/MS⁺ purity: 100%, t_R = 4.87, MS calcd for [M + H]⁺: C₂₈H₃₆N₄O₆ m/z: 524.26, found 525.30. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 10.80 (s, 1H, N¹H), 6.97–6.83 (m, 6H, Ar), 6.49 (s, 1H, Ph–CH=), 3.99 (q, J = 7.0 Hz, 2H, O-CH₂-CH₃), 3.81 (s, 6H, 2xO-CH₃), 3.65 (s, 3H, O-CH₃), 3.55–3.49 (m, 6H, N³-CH₂, Pp-3,5-H), 3.20–3.13 (m, 4H, Pp-2,6-H), 2.92–2.87 (m, 2H, Pp-CH₂), 2.01–1.98 (m, 2H, CH₂), 1.31 (t, J = 6.9 Hz, 3H, O-CH₂-CH₃).

3.2. Pharmacology In Vitro

3.2.1. Radioligand Binding Assays: Affinity for α₁-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).

[³H]Prazosin (spec. act. 85 Ci/mmol, Perkin Elmer) was used for labeling the α₁-receptor and 240 μL of the tissue suspension, 30 μL of 10 μM phentolamine (displacer), 30 μL of 0.2 nM [³H]Prazosin and 30 μL of the analyzed compound were incubated at 30 °C for 30 min. The concentrations of analyzed compounds ranged from 10⁻¹⁰ to 10⁻⁵ M.

The incubation was terminated by rapid filtration over glass fiber filters FilterMate B (PerkinElmer, Waltham, MA, USA) using a 96-well FilterMate harvester (PerkinElmer, Waltham, MA, USA). Five rapid washes were performed with ice-cold 50 mM Tris-HCl buffer, pH 7.6.

The FilterMate was dried in a microwave and placed in a plastic bag (PerkinElmer, Waltham, MA, USA) and soaked in 10 mL of a liquid scintillation cocktail Ultima Gold MV (PerkinElmer, Waltham, MA, USA). After even distribution of the scintillation cocktail, the filter bag was sealed.

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 GraphPad 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 IC₅₀ (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].

3.2.2. Radioligand Binding Assays: Binding Affinities for 5-HT_1A, 5-HT₆ and 5-HT₇ Receptors

HEK-293 cells with a stable expression of human 5-HT_1A, 5-HT₆ and 5-HT_7b receptors (prepared with the use of Lipofectamine 2000) were maintained at 37 °C in a humidified atmosphere with 5% CO₂ 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 cm³ 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_1AR: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl₂, 10 μM pargyline and 0.1% ascorbate, for 5-HT₆R—50mM TrisHCl, 0.5 mM EDTA and 4 mM MgCl₂; and for 5- HT_7bR: 50 mM Tris HCl, 4 mM MgCl₂, 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_1AR, 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 (PerkinElmer, USA): 2.5 nM [³H]-8-OH-DPAT (135.2 Ci/mmol) for 5-HT_1AR; 2 nM [³H]-LSD (83.6 Ci/mmol) for 5-HT₆R, and 0.8 nM [³H]-5-CT (39.2 Ci/mmol) for 5-HT₇R. Non–specific binding was defined with 10 μM of 5-HT in 5-HT_1AR and 5-HT₇R binding experiments, whereas 10 mM of methiothepin was used in the 5-HT₆R assays. Each compound was tested in triplicate at seven concentrations (10^–10–10⁻⁴M). The inhibition constants (K_i) 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.

3.3. 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 EC₈₀ (ten-fold higher concentration in comparison to the EC₈₀ 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% CO₂ 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.

3.3.2. 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.

3.4. Molecular Modeling and Cheminformatic Analysis

3.4.1. Searches for Structurally Similar Compounds in the ChEMBL Database

The ChEMBL database v.26 was used as a source of all compounds with experimentally confirmed activity towards the considered targets. All data records referring to 5-HT_1AR, 5-HT₆R, 5-HT₇R, α_1AR, α_1BR, and α_1DR (human-related data) were downloaded. Targets with the following ChEMBLIDs were considered: CHEMBL214 (5-HT_1AR), CHEMBL3371 (5-HT₆R), CHEMBL3155 (5-HT₇R), CHEMBL229 (α_1AR), CHEMBL232 (α_1BR) and CHEMBL223 (α_1DR). The similarity coefficients for all examined compounds towards all the above-described ChEMBL data were determined (Tanimoto similarity was used [28], and ECFP [36] was used for compounds representation) using the InstantJChem v. 16.12.5.0 software [37].

3.4.2. 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.

3.4.3. 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.

4. Conclusions

The work presented here concerns the deep structural insight into the hydantoin-arylpiperazine 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_1AR activity and moderates 5-HT₇R 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.