Synthesis, Docking Studies and Pharmacological Evaluation of Serotoninergic Ligands Containing a 5-Norbornene-2-Carboxamide Nucleus

A new series of 5-norbornene-2-carboxamide derivatives was prepared and their affinities to the 5-HT1A, 5-HT2A, and 5-HT2C receptors were evaluated and compared to a previously synthesized series of derivatives characterized by exo-N-hydroxy-5-norbornene-2,3-dicarboximidenucleus, in order to identify selective ligands for the above-mentioned subtype receptors. Arylpiperazines represents one of the most important classes of 5-HT1AR ligands, and recent research concerning new derivatives has been focused on the modification of one or more portions of such pharmacophore. The combination of structural elements (heterocyclic nucleus, propyl chain and 4-substituted piperazine), known to be critical to the affinity to 5-HT1A receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that Norbo-4 and Norbo-18 were the most active and promising derivatives for the serotonin receptor considered in this study.


Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is one of the major central neurotransmitters involved in many neuropsychiatric and neurological disorders, including depression, schizophrenia, migraine, pain, and Parkinson's disease, to cite a few and, not surprisingly, its receptors have attracted interest as targets for therapeutic intervention. Early studies identified two subtypes of serotonin receptors (denoted S1 and S2) using radiolabeled ligand binding, but molecular biology and gene cloning techniques revealed that there are 14 different subtypes of receptors, divided into different families, based on sequence

Chemistry
The general strategy for the synthesis of the target compounds (Table 1) is summarized in Scheme 1. Briefly, 5-norbornene-2-carboxilic acid reacted with 3-chloropropan-1-amine hydrochloride in acetonitrile in the presence of N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) and triethylamine (TEA) to give the corresponding N-(3chloropropyl)bicyclo [2.2.1]hept-5-ene-2-carboxamide (2). Subsequent condensation of intermediate (2) with the appropriate 4-X-substituted-piperazine performed in acetonitrile (CH 3 CN) with potassium carbonate (K 2 CO 3 ) and sodium iodide (NaI), under reflux, provided the corresponding compounds as a mixture of endo and exo isomers. The purification of the reaction mixture and the separation of the isomers were carried out by silica gel open chromatography and further crystallization from the appropriate solvent. All new compounds were characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. The endo and exo stereoisomerism of the two compounds was determined by comparing their spectroscopic data with those reported in the literature for related compounds. 1 H-NMR, 13 C-NMR, and MS for all final compounds were consistent with the proposed structures. Table 1. Agonist properties of compounds Norbo 1-28 at the 5-HT 1A receptor. Scheme 1. Reagents and conditions: (i) Cl(CH2)3NH2·HCl, DCC, HOBt, TEA, CH3CN, r.t., 24h; (ii) 4-X-substituted-piperazine, K2CO3, NaI, CH3CN, 70 °C, 24 h.

Chemistry
The general strategy for the synthesis of the target compounds (Table 1) is summarized in Scheme 1. Briefly, 5-norbornene-2-carboxilic acid reacted with 3-chloropropan-1amine hydrochloride in acetonitrile in the presence of N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) and triethylamine (TEA) to give the corresponding N-(3-chloropropyl)bicyclo [2.2.1]hept-5-ene-2-carboxamide (2). Subsequent condensation of intermediate (2) with the appropriate 4-X-substituted-piperazine performed in acetonitrile (CH3CN) with potassium carbonate (K2CO3) and sodium iodide (NaI), under reflux, provided the corresponding compounds as a mixture of endo and exo isomers. The purification of the reaction mixture and the separation of the isomers were carried out by silica gel open chromatography and further crystallization from the appropriate solvent. All new compounds were characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. The endo and exo stereoisomerism of the two compounds was determined by comparing their spectroscopic data with those reported in the literature for related compounds. 1 H-NMR, 13 C-NMR, and MS for all final compounds were consistent with the proposed structures. Table 1. Agonist properties of compounds Norbo 1-28 at the 5-HT1A receptor.

Chemistry
The general strategy for the synthesis of the target compounds (Table 1) is summarized in Scheme 1. Briefly, 5-norbornene-2-carboxilic acid reacted with 3-chloropropan-1amine hydrochloride in acetonitrile in the presence of N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) and triethylamine (TEA) to give the corresponding N-(3-chloropropyl)bicyclo [2.2.1]hept-5-ene-2-carboxamide (2). Subsequent condensation of intermediate (2) with the appropriate 4-X-substituted-piperazine performed in acetonitrile (CH3CN) with potassium carbonate (K2CO3) and sodium iodide (NaI), under reflux, provided the corresponding compounds as a mixture of endo and exo isomers. The purification of the reaction mixture and the separation of the isomers were carried out by silica gel open chromatography and further crystallization from the appropriate solvent. All new compounds were characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. The endo and exo stereoisomerism of the two compounds was determined by comparing their spectroscopic data with those reported in the literature for related compounds. 1 H-NMR, 13 C-NMR, and MS for all final compounds were consistent with the proposed structures. Table 1. Agonist properties of compounds Norbo 1-28 at the 5-HT1A receptor.

5-HT1A Receptor G-Protein Stimulation
Compd X pEC50 EC50 (95% CI, nM) Emax (%) ± SEM fication of the reaction mixture and the separation of the isomers were carried out by silica gel open chromatography and further crystallization from the appropriate solvent. All new compounds were characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. The endo and exo stereoisomerism of the two compounds was determined by comparing their spectroscopic data with those reported in the literature for related compounds. 1 H-NMR, 13 C-NMR, and MS for all final compounds were consistent with the proposed structures. fication of the reaction mixture and the separation of the isomers were carried out by silica gel open chromatography and further crystallization from the appropriate solvent. All new compounds were characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. The endo and exo stereoisomerism of the two compounds was determined by comparing their spectroscopic data with those reported in the literature for related compounds. 1 H-NMR, 13 C-NMR, and MS for all final compounds were consistent with the proposed structures. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination. The olefinic protons of the endo compound of the norbornene nucleus resonate at chemical shift δ 5.97 and 6.21 while those of the exo compound resonate at δ 6.05 and 6.11. The comparison of these data with those presents in the literature allowed us to assign the endo and exo configuration to the two compounds. A further confirmation was possible thanks to the analysis of the ROESY spectrum. In the ROESY spectrum of the endo compound, there was a key correlation between the methine proton at δ 2.84 with the methylene bridge proton at δ 1.27, while in the exo compound there was a correlation between the methine proton at δ 1.96 with the olefin proton at δ 6.05 (Figure 1). These correlations together with the comparison of the data present in the literature allowed us to univocally assign the endo and exo stereoisomerism of the two compounds under examination.
The study also identified some inactive derivatives expressing no agonist activity for the 5-HT1A receptor. Namely, within the chlorine substituted analogs, all substitutions of the phenyl ring other than one ortho-positioned chlorine atom (Norbo-9) produced analogs devoid of 5-HT1A agonist activity. Additionally, derivatives with the furoyl (Norbo-27, Norbo-28) terminal fragment also exerted no agonist activity at the 5-HT1A receptor.
The study also identified some inactive derivatives expressing no agonist activity for the 5-HT 1A receptor. Namely, within the chlorine substituted analogs, all substitutions of the phenyl ring other than one orthopositioned chlorine atom (Norbo-9) produced analogs devoid of 5-HT 1A agonist activity. Additionally, derivatives with the furoyl (Norbo-27, Norbo-28) terminal fragment also exerted no agonist activity at the 5-HT 1A receptor.
Finally, none of the compounds tested expressed no agonistic activity for the D 2 receptor (Table 2). When screened for their antagonistic properties, the compounds only showed submilimolar inhibitory potencies against dopamine-induced stimulation of the D 2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 receptor (Table 2). When screened for their antagonistic properties, the compounds only showed submilimolar inhibitory potencies against dopamine-induced stimulation of the D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. 3.98 ± 0.14 105.6 (52-213)

Norbo-3 (exo)
Molecules 2022, 27, 6492 8 of Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. Finally, none of the compounds tested expressed no agonistic activity for the D2 r ceptor (Table 2). When screened for their antagonistic properties, the compounds on showed submilimolar inhibitory potencies against dopamine-induced stimulation of th D2 receptor. 3.99 ± 0.14 100 (51-199) Finally, none of the compounds tested expressed no agonistic activity for the D2 receptor ( Table 2). When screened for their antagonistic properties, the compounds only showed submilimolar inhibitory potencies against dopamine-induced stimulation of the D2 receptor.  Finally, none of the compounds tested expressed no agonistic activity for the D2 receptor ( Table 2). When screened for their antagonistic properties, the compounds only showed submilimolar inhibitory potencies against dopamine-induced stimulation of the D2 receptor.

5-HT 2A and 5-HT 2C Receptor Binding
All of the new compounds were tested for their affinity at 5-HT 2A and 5-HT 2C receptors. Some of the new synthesized derivatives showed interesting affinity values in the nanomolar range towards 5-HT 2A receptors and lower affinities for 5-HT 2C receptors (Table 3) The difference in affinity observed between this new series of derivatives (Norbo 1-28) and the previously described series [14,16] characterized by analog exo-N-oxy-5norbornene-2,3-dicarboximide nucleus linked via two or three methylene spacing unit to 4-substituted piperazines, demonstrated that a simple amide bond (Norbo 1-28) instead of a dicarboximide moiety represents a critical feature in determining differences in binding with 5-HTRs.

5-HT2 Receptor Binding Affinity
The difference in affinity observed between this new series of derivatives (Norbo 1-28) and the previously described series [14,16] characterized by analog exo-N-oxy-5-norbornene-2,3-dicarboximide nucleus linked via two or three methylene spacing unit to 4substituted piperazines, demonstrated that a simple amide bond (Norbo 1-28) instead of a dicarboximide moiety represents a critical feature in determining differences in binding with 5-HTRs.
In fact, regarding these novel derivatives, although they have a lower affinity profile than those previously synthesized, the influence of this original scaffold associated to the appropriate substituents on the phenylpiperazine ring and heterocyclic nucleus were particularly profitable to enhance 5-HT2A and 5-HT2C receptor affinity. Anyway, in order to rationalize the differential binding affinities/activities, molecular docking studies were carried out on the complete series of derivatives.

In Vitro Evaluation of 5-HT-Evoked Contractions
Successively, the compounds Norbo-5, Norbo14, Norbo-16, Norbo-17, Norbo-18, Norbo-19, and Norbo-20 with better affinity/selectivity binding profiles towards 5-HT2A receptors were tested by in vitro assay to determine their activity on 5-HT-evoked contractions. In the rat ileum, 5-HT2A receptors are located on smooth muscles and their activation by 5-HT is known to induce contraction. Consequently, 5-HT2A antagonists depress 5-HT-induced contractions in the rat ileum [24]. According to Briejer and colleagues, we have shown that 5-HT contracted the rat ileum longitudinal muscle. In preliminary experiments we found that the neuronal blocker tetrodotoxin (0.3 µM), the muscarinic receptor antagonist atropine (1 µM), the adrenergic receptor antagonists phentolamine (10 −6 M) plus propranolol (10 −6 M) did not affect the contractions by 5-HT. In contrast, ketanserin (0.1 µM), at concentration that blocks 5-HT2A receptors, depressed the contractions induced by 5-HT. Collectively, these results suggest that 5-HT contracts the ileum by acting on 5-HT2A receptors located on smooth muscle while muscarinic or adrenergic receptors 5 One-way ANOVA, Bonferroni post-hoc test. Statistical significance was marked as follows: & vs. corresponding 4-phenylpiperazin-1-yl substituted isomer. One, two, or three symbols correspond to significance levels of 0.05, 0.01, and 0.001, respectively. Both isomer pairs were equally potent when tested at 5-HT2A and 5-HT2C receptors. None of the compounds tested showed potency superior to ketanserin at the 5-HT2A receptor. Compounds were also less potent than the selective 5-HT2C ligand-RS102221 and non-selective serotonin. pKi-inhibition constant.
The difference in affinity observed between this new series of derivatives (Norbo 1-28) and the previously described series [14,16] characterized by analog exo-N-oxy-5-norbornene-2,3-dicarboximide nucleus linked via two or three methylene spacing unit to 4substituted piperazines, demonstrated that a simple amide bond (Norbo 1-28) instead of a dicarboximide moiety represents a critical feature in determining differences in binding with 5-HTRs.
In fact, regarding these novel derivatives, although they have a lower affinity profile than those previously synthesized, the influence of this original scaffold associated to the appropriate substituents on the phenylpiperazine ring and heterocyclic nucleus were particularly profitable to enhance 5-HT2A and 5-HT2C receptor affinity. Anyway, in order to rationalize the differential binding affinities/activities, molecular docking studies were carried out on the complete series of derivatives.

In Vitro Evaluation of 5-HT-Evoked Contractions
Successively, the compounds Norbo-5, Norbo14, Norbo-16, Norbo-17, Norbo-18, Norbo-19, and Norbo-20 with better affinity/selectivity binding profiles towards 5-HT2A receptors were tested by in vitro assay to determine their activity on 5-HT-evoked contractions. In the rat ileum, 5-HT2A receptors are located on smooth muscles and their activation by 5-HT is known to induce contraction. Consequently, 5-HT2A antagonists depress 5-HT-induced contractions in the rat ileum [24]. According to Briejer and colleagues, we have shown that 5-HT contracted the rat ileum longitudinal muscle. In preliminary experiments we found that the neuronal blocker tetrodotoxin (0.3 µM), the muscarinic receptor antagonist atropine (1 µM), the adrenergic receptor antagonists phentolamine (10 −6 M) plus propranolol (10 −6 M) did not affect the contractions by 5-HT. In contrast, ketanserin (0.1 µM), at concentration that blocks 5-HT2A receptors, depressed the contractions induced by 5-HT. Collectively, these results suggest that 5-HT contracts the ileum by acting on 5-HT2A receptors located on smooth muscle while muscarinic or adrenergic receptors 5 One-way ANOVA, Bonferroni post-hoc test. Statistical significance was marked as follows: & vs. corresponding 4phenylpiperazin-1-yl substituted isomer. One, two, or three symbols correspond to significance levels of 0.05, 0.01, and 0.001, respectively. Both isomer pairs were equally potent when tested at 5-HT 2A and 5-HT 2C receptors. None of the compounds tested showed potency superior to ketanserin at the 5-HT 2A receptor. Compounds were also less potent than the selective 5-HT 2C ligand-RS102221 and non-selective serotonin. pKi-inhibition constant.

In Vitro Evaluation of 5-HT-Evoked Contractions
Successively, the compounds Norbo-5, Norbo14, Norbo-16, Norbo-17, Norbo-18, Norbo-19, and Norbo-20 with better affinity/selectivity binding profiles towards 5-HT 2A receptors were tested by in vitro assay to determine their activity on 5-HT-evoked contractions. In the rat ileum, 5-HT 2A receptors are located on smooth muscles and their activation by 5-HT is known to induce contraction. Consequently, 5-HT 2A antagonists depress 5-HT-induced contractions in the rat ileum [24]. According to Briejer and colleagues, we have shown that 5-HT contracted the rat ileum longitudinal muscle. In preliminary experiments we found that the neuronal blocker tetrodotoxin (0.3 µM), the muscarinic receptor antagonist atropine (1 µM), the adrenergic receptor antagonists phentolamine (10 −6 M) plus propranolol (10 −6 M) did not affect the contractions by 5-HT. In contrast, ketanserin (0.1 µM), at concentration that blocks 5-HT 2A receptors, depressed the contractions induced by 5-HT. Collectively, these results suggest that 5-HT contracts the ileum by acting on 5-HT 2A receptors located on smooth muscle while muscarinic or adrenergic receptors are not involved. Results show the potency (expressed by the IC 50 value) and the efficacy (expressed by the E max value) of the compounds under investigation in inhibiting 5-HT-induced contractions in the rat ileum (a pharmacological assay useful to detect activity towards 5-HT 2A receptors). The compounds under investigation did not significantly inhibit the contractions induced by 5-HT. The rank order of efficacy was: Norbo-16 (3.51

Molecular Docking Studies
In order to study the ligand-receptor interactions at the molecular level and to rationalize the observed structure-activity relationships, compounds Norbo 1-28 were docked to the orthosteric sites of 5-HT 1A , 5-HT 2A , and 5-HT 2C receptors. The visualization of molecular interactions of selected most potent compounds with the receptors under investigation is shown in Figures 1-3. As the studied compounds follow the classical pharmacophore model for the aminergic G protein-coupled receptor (GPCRs) ligands [25], the electrostatic interaction between the protonatable nitrogen atom of the ligand and the conserved Asp 3.32 of the receptor is a key ligand-receptor contact for all studied complexes [26]. In the case of most ligand-receptor complexes, Trp 6.48 and Phe 6.52 are crucial residues engaged in π-π stacking contact with N-aryl moieties of the ligands, as found for many similar complexes [22,23,[27][28][29]. These residues, accompanied by Phe 6.51 and His 6.55, constitute an aromatic microdomain of, i.a., serotonin and dopamine receptors [30]. The importance of these residues for binding ritanserin, an inverse agonist of serotonin 5-HT 2C receptor, was confirmed by X-ray crystallography (PDB ID: 6BQH [6]) and verified by the mutation of Phe 5.47, Phe 6.44, and Trp 6.48. In addition, the serotonin receptor subtype selectivity may be governed by residues from the second extracellular loop (ecl2) [31], which serves for recognition of the "message" part of the ligand [32].
The mode of binding of the studied ligands is more differentiated for 5-HT 1A ( Figure 2) and similar for 5-HT 2A (Figure 3) and 5-HT 2C (Figure 4) receptors and receptors. In the case of most ligands interacting with 5-HT 2A and 5-HT 2C receptors, the ligands adopt an extended conformation parallel to the transmembrane helix bundle. The norbornene moiety of the ligands is directed towards the extracellular vestibule while the N-aryl group penetrates deeper into receptor aromatic microdomain which is in accordance with our previous results [22,23]. This is also in line with the data recently published by Kucwaj-Brysz et al. [33], who identified molecular docking poses of serotonin receptor ligands with the arylpiperazine moiety deeply buried in the binding pocket. Moreover, such a ligand binding mode is in agreement with the one found for ritanserin in 5-HT 2C receptor (PDB ID: 6BQH [6]). In the case of 5-HT 1A receptor, some ligands are able to adopt an extended conformation parallel to the transmembrane bundle similar to that described above (e.g., Norbo-3 and Norbo-4, see Figures 2A and 2B, respectively), some adopt an extended conformation, but they are situated at the angle of 30-60 degrees regarding the transmembrane bundle (e.g., Norbo-8, Figure 2C, Norbo-10, Figure 2E, and Norbo-20, Figure 2F), while others adopt a bent conformation with norbornene moiety, penetrating deeper in the receptor cavity than the N-aryl group, e.g., Norbo-8 ( Figure 2C).
The affinity of the studied ligands to the serotonin receptors is mainly governed by the type of N-aryl group, as previously reported by us [22,23] and by Zagórska et al. [34] for similar series. Many potent compounds bear a halogen substituent in the aryl ring which can be rationalized by the possibility of halogen bond formation as suggested by Partyka et al. [35]. The exo/endo isomerism seems to have a less important effect as it displays no clear trend and is case-specific. This can be explained by the fact that the norbornene moiety of the ligand directs in most complexes towards the extracellular part of the receptor, constituted by flexible loops which can accommodate both isomers. However, regarding the general pharmacological profile of the compounds, the endo isomers are more beneficial, and they have been selected for animal studies (Norbo-4, Norbo-8, Norbo-10,  Norbo-14, Norbo-18, and Norbo-20). The selectivity of the studied ligands to the serotonin receptor subtypes is first governed by the residues from the ecl2. Moreover, in case of 5-HT 1A receptor Phe 3.28, Ser 5.43, and Tyr 7.42 were found to be important in many ligand-receptor complexes. In summary, the performed molecular modeling study can facilitate the design of subsequent compound series with the expected serotonin receptor subtype selectivity or polypharmacology.

In Vivo Behavioral Test
Mental disorders represent a specific group of diseases since their course is miscellaneous in different patients. In fact, each patient should be treated individually, and drugs should possess multidirectional action, which increases the chances of amending the health. There is a great need to improve the treatment and dose selection for patients with mental disorders. Hence, the search for new drugs and effective therapeutic solutions is a constant problem. The affinity of the studied ligands to the serotonin receptors is mainly governed by the type of N-aryl group, as previously reported by us [22,23] and by Zagórska et al. [34] for similar series. Many potent compounds bear a halogen substituent in the aryl ring which can be rationalized by the possibility of halogen bond formation as suggested by Partyka et al. [35]. The exo/endo isomerism seems to have a less important effect as it displays no clear trend and is case-specific. This can be explained by the fact that the norbornene moiety of the ligand directs in most complexes towards the extracellular part of the receptor, constituted by flexible loops which can accommodate both isomers. However, regarding the general pharmacological profile of the compounds, the endo isomers are more beneficial, and they have been selected for animal studies (Norbo-4, Norbo-8, Norbo-10, Norbo-14, Norbo-18, and Norbo-20). The selectivity of the studied ligands to the serotonin receptor subtypes is first governed by the residues from the ecl2. Moreover, in case of 5-HT1A receptor Phe 3.28, Ser 5.43, and Tyr 7.42 were found to be important in many ligand-receptor complexes. In summary, the performed molecular modeling study Behavioral pharmacology research is the cornerstone of understanding the processes underlying the behavior of living organisms, as well as the biological basis of the behavioral, emotional and cognitive disorders that affect humans. Discoveries in this area have helped to explore the potential therapeutic effects of many substances in treating these disorders. Since biochemical abnormalities causing psychiatric disorders are not limited to single signaling pathways, a variety of screening tests are used in experimental pharmacology to reveal new potential drugs. Basic research in laboratory animals is a promising approach to study behavioral abnormalities associated with mental disorders and to identify new pharmacological therapies to address mental health challenges. Knowledge of pharmacology allows us to understand that chemicals with very specific structures and properties that, at controlled doses, can interact with the normal physiology processes to produce health-enhancing effects known as therapeutic effects. However, if dosages are insufficient or excessive, the effects will be useless or harmful (toxic), respectively [36].
Compounds Norbo-4, Norbo-8, Norbo-10, Norbo-14, Norbo-18, and Norbo-20 were selected for further functional in vivo studies. The first part of experiments included: motor coordination and locomotor activity tests, generally accepted as basic procedures in central activity investigations of new agents [37]. Firstly, all compounds were administered at the dose of 30 mg/kg. It was observed that three of them (Norbo-4, Norbo-14 and Norbo-18) disturbed the behavior of mice in the chimney test ( Figure 5A) and impaired motor coordination assessed in the rota-rod test ( Figure 5B). In the locomotor activity test, the same compounds at dose of 30 mg/kg decreased spontaneous motility after 6 and 20 min (Figure 6) of observation. Similarly, Norbo-4 induced the same effect at the dose of 15 mg/kg. In the second stage of this study, we evaluated the antipsychotic ability of the new compounds. Presently, animal models of schizophrenia, commonly employed for preclinical studies of antipsychotic properties of drugs, regard mainly amphetamine and MK-801 models [38]. The first model is based on the manipulation of the dopaminergic system activity, and it may primarily respond to drugs that affect this neurotransmitter system. Many neuroleptics acting as dopaminergic antagonists reverse this effect [38]. Noteworthy, the hyperlocomotion following amphetamine is also sensitive to other classes of drugs [39]. On the other hand, several preclinical tests have pointed to the role of 5-HT 2C ligands in the modulation of monoaminergic systems, including dopaminergic. Indeed, dysfunction in serotoninergic activity could contribute to the alteration of dopaminergic function seen in schizophrenia [40]. Nevertheless, in the amphetamine model, none of the tested compounds at the dose of 15 mg/kg reduced amphetamine-induced hyperactivity of mice ( Figure 7). Furthermore, due to the modulation of the central serotonin neurotransmission, the new compounds may also show anxiolytic and/or antidepressant activity [41,42]. Considering this premise, as well as in vitro data obtained for the tested compounds (mixed 5-HT 1A /5-HT 2C affinity profile for all the compounds), we examined their antidepressant and anxiolytic potential in behavioral models commonly used in mice, i.e., FST and EPM.

In Vivo Behavioral Test
Mental disorders represent a specific group of diseases since their course is miscellaneous in different patients. In fact, each patient should be treated individually, and drugs should possess multidirectional action, which increases the chances of amending the health. There is a great need to improve the treatment and dose selection for patients with mental disorders. Hence, the search for new drugs and effective therapeutic solutions is a constant problem.
Behavioral pharmacology research is the cornerstone of understanding the processes underlying the behavior of living organisms, as well as the biological basis of the behavioral, emotional and cognitive disorders that affect humans. Discoveries in this area have      FST is a simple and fast test established in experimental pharmacology for detecting the antidepressant effect of tested substances [43]. The mouse placed in a beaker with water, without the possibility of escaping, initially makes intensive attempts to get out, but after a short time it gives up and adopts an attitude known as immobility. Selective serotonin reuptake inhibitors (SSRIs) are an example of antidepressants active in this test. They block the serotonin transporter (SERT) protein, which transports serotonin, and reduce its reuptake from the synaptic cleft. Due to their effectiveness, they are one of the most commonly used drugs in the treatment of depression. This group includes, among others, sertraline, which is a 5-HT1A agonist. Sertraline at the dose of 15 mg/kg used in our experiments significantly shortened the immobility time of mice. The tested compound Norbo-4 at three doses of 15, 7.5, and 3.75 mg/kg and Norbo-18 (at the doses of 15 and 7.5 mg/kg) showed an antidepressant effect similar to fluoxetine, which was manifested by a significant reduction of immobility time of mice ( Figure 8). This effect should be considered specific as the tested compound did not show a negative effect on the locomotor activity of mice (in the two lowest used doses) nor induce sedation (at a dose of 15 mg/kg). This suggests that the mice were able to cope with the sedation in this stressful confined space swimming situation. The twice-as-high dose was found to inhibit the CNS so strongly that the immobility time in this group was comparable to the control group. Meanwhile, the other tested compounds, at used doses, remained inactive in this test. Anxiety and stress-related disorders represent severe mental health conditions that affect the performance of daily tasks and represent a high cost to public health. Charles Darwin's preliminary observation that animals and humans have similar traits in expressing emotions opens the possibility to study the mechanisms of mental disorders in other mammals (mainly rodents). The animal test for assessing anxiolytic effects, EPM, is based on the test and Norbo-20 (15 mg/kg) on the amphetamine-induced hyperactivity of mice. Compounds tested were injected 60 min and amphetamine 5 mg/kg 30 min before the test. Locomotor activity was measured for a period of 20 min. Data are expressed as mean ± SEM values of the 1 independent experiment. **** p < 0.001 vs. control. One-way ANOVA did not showed significant changes in the locomotor activity of mice [F(7, 53) = 1601; p = 0.1555]. Dunnett's post hoc test confirmed a significant increase in locomotor activity of mice after the administration of amphetamine (5 mg/kg) (p < 0.001). However, the administered compounds had no influence on amphetamine-induced hyperactivity of mice (p > 0.05).
FST is a simple and fast test established in experimental pharmacology for detecting the antidepressant effect of tested substances [43]. The mouse placed in a beaker with water, without the possibility of escaping, initially makes intensive attempts to get out, but after a short time it gives up and adopts an attitude known as immobility. Selective serotonin reuptake inhibitors (SSRIs) are an example of antidepressants active in this test. They block the serotonin transporter (SERT) protein, which transports serotonin, and reduce its reuptake from the synaptic cleft. Due to their effectiveness, they are one of the most commonly used drugs in the treatment of depression. This group includes, among others, sertraline, which is a 5-HT 1A agonist. Sertraline at the dose of 15 mg/kg used in our experiments significantly shortened the immobility time of mice. The tested compound Norbo-4 at three doses of 15, 7.5, and 3.75 mg/kg and Norbo-18 (at the doses of 15 and 7.5 mg/kg) showed an antidepressant effect similar to fluoxetine, which was manifested by a significant reduction of immobility time of mice ( Figure 8). This effect should be considered specific as the tested compound did not show a negative effect on the locomotor activity of mice (in the two lowest used doses) nor induce sedation (at a dose of 15 mg/kg). This suggests that the mice were able to cope with the sedation in this stressful confined space swimming situation. The twice-as-high dose was found to inhibit the CNS so strongly that the immobility time in this group was comparable to the control group. Meanwhile, the other tested compounds, at used doses, remained inactive in this test. Anxiety and stressrelated disorders represent severe mental health conditions that affect the performance of daily tasks and represent a high cost to public health. Charles Darwin's preliminary observation that animals and humans have similar traits in expressing emotions opens the possibility to study the mechanisms of mental disorders in other mammals (mainly rodents). The animal test for assessing anxiolytic effects, EPM, is based on the test animal's aversion to open spaces [44]. Fear induced inhibition of exploratory activity affects entries into open arms in this task as a significant increase in the percentage of time spent on the open arms and the number of entries into open arms is observed with drugs that are clinically effective anxiolytics [45]. Therefore, this model has been used to assess the anxiolytic-like activity of new putative anxiolytic compounds [46]. Buspirone (5 mg/kg), used as a reference anxiolytic drug, also prolonged the time and increased the percentage of entries into the open arms ( Figure 9). As in the FST test, also in this test, the Norbo-4 compound, used in three lower doses, i.e., 15, 7.5, and 3.75 mg/kg, increased the percentage of open arm exploration time, analogous to buspirone. Additionally, the increase in the percentage of entries into the open arms was statistically significant. The remaining compounds at the used doses were inactive in this test.
animal's aversion to open spaces [44]. Fear induced inhibition of exploratory activ fects entries into open arms in this task as a significant increase in the percentage o spent on the open arms and the number of entries into open arms is observed with that are clinically effective anxiolytics [45]. Therefore, this model has been used to a the anxiolytic-like activity of new putative anxiolytic compounds [46]. Buspiro mg/kg), used as a reference anxiolytic drug, also prolonged the time and increase percentage of entries into the open arms ( Figure 9). As in the FST test, also in this te Norbo-4 compound, used in three lower doses, i.e., 15, 7.5, and 3.75 mg/kg, increase percentage of open arm exploration time, analogous to buspirone. Additionally, t crease in the percentage of entries into the open arms was statistically significan remaining compounds at the used doses were inactive in this test.  . The investigated compounds were administered i.p. 60 min before the test. The values represent means ± SEM of the 1 independent experiment. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control (Dunnett's test). One-way ANOVA showed significant changes in immobility time after administration of the compound Norbo-4 (F(4,35) = 0.386; p < 0.0001) and Norbo-18 (F(3,26) = 16.07; p < 0.0001). Dunnett's post hoc test confirmed a significant reduction in immobility time after the administration of compounds Norbo-4 applied at the doses of 3.75 (p < 0.01), 7.5 (p < 0.0001), 15 (p < 0.001) and 30 mg/kg (p < 0.05), and Norbo-18 applied at the doses of 7.5 (p < 0.0001) and 15 mg/kg (p < 0.01). Also, sertraline (15 mg/kg) induced a significant reduction in the immobility time (p < 0.001).

Conclusions
We have described the synthesis of a new series of arylpiperazines as serotoninergic ligands (Norbo 1-28). The 4-methoxyphenyl and 2,3-dimethylphenyl piperazine derivatives supporting an 5-norbornene-2-carboxamide scaffold as terminal fragment (Norbo-4 and Norbo-8) afforded a favorable agonistic profile for 5-HT 1A receptors (pEC 50 = 6.74 ± 0.08 and pEC 50  The obtained results showed that compounds Norbo-4 and Norbo-18 exerted antidepressantlike effects. Interestingly, the compound Norbo-4 revealed significant anxiolytic properties, and in the EPM test it was almost as efficacious as buspirone. However, further pharmacological studies are necessary to determine detailed mechanism of action and clinical prospective usefulness of the new compounds.
In conclusion, data presented in this study confirm that, as obtained with the series previously synthesized [14,16], the novel synthesized compounds display a general trend of affinity towards serotoninergic receptors investigated. Molecular docking studies supported these results, highlighting some selective and additional interactions of the identified ligands with the investigated receptor subtype.