Design, Synthesis and 5-HT1A Binding Affinity of N-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propyl)tricyclo[3.3.1.13,7]decan-1-amine and N-(3-(4-(2-Methoxyphenyl) piperazin-1-yl)propyl)-3,5-dimethyl-tricylo[3.3.1.13,7]decan-1-amine

Based on previously highlighted structural features, the development of highly selective 5-HT1A receptor inhibitors is closely linked to the incorporation of a 4-alkyl-1-arylpiperazine scaffold on them. In this paper, we present the synthesis of two new compounds bearing the 2-MeO-Phpiperazine moiety linked via a three carbon atom linker to the amine group of 1-adamantanamine and memantine, respectively. Both were tested for their binding affinity against 5-HT1A receptor. N-(3-(4(2-methoxyphenyl)piperazin-1-yl)propyl)tricyclo[3.3.1.13,7]decan-1-amine fumarate (8) and N-(3-(4(2-methoxyphenyl)piperazin-1-yl)propyl)-3,5-dimethyl-tricylo[3.3.1.13,7]decan-1-amine fumarate (10) proved to be highly selective ligands towards 5-HT1A receptor with a binding constant of 1.2 nM and 21.3 nM, respectively, while 5-carboxamidotriptamine (5-CT) (2) was used as an internal standard for this assay with a measured Ki = 0.5 nM.


Introduction
More than one-third of all drugs approved in 2017 by the US Food and Drug Administration (FDA) acted as G protein-coupled receptors (GPCRs) [1]. They are the most studied class of receptors due to their wide involvement in human diseases such as diabetes, obesity, Alzheimer's and several other central nervous system disorders [1,2]. Among the GPCRs belong also the serotonin class of receptors, specifically the serotonin 1A subtype (5-HT 1A ) [3]. This receptor has been tied to a variety of conditions (i.e., anxiety, mood, cognition) and its primary substrate is serotonin (Figure 1), a monoamine neurotransmitter that modulates brain function [3]. Recent therapeutic applications of receptor modulation have expanded towards prostate cancer, gastrointestinal and cardiopulmonary disorders and L-DOPA ( Figure 1) induced dyskinesia [4]. 5-HT 1A was one of the first discovered and still remains one of the most studied serotonin receptors. In the human brain they are abundant with localization both presynaptically on serotonergic neurons and postsynaptically in nonserotonergic neurons [5]. Dysfunctions associated with 5-HT 1A neurotransmission have been directly implicated with stress response, aggressive behavior, psychiatric disorders, anxiety, depression and movement disorders [4,6]. Despite all the progress with 5-HT1A receptors, very little was known about the inhibitor's binding mode in the absence of an available crystal structure, which was only recently published [7]. Therefore, drug design efforts were mostly based in structure-activity relationship data. Some of the most important identified class of drugs inhibiting the 5-HT1A receptor with increased selectivity contains an 4-alkyl-l-arylpiperazine moiety [8]. Representative structures (Figure 1) of marketed drugs are the anxiolytic buspirone [9] and the antipsychotic aripiprazole [10].
Herein we report the synthesis and biological evaluation of two new 5-HT1A inhibitors bearing the 2-MeO-Ph-piperazine moiety linked via a three carbon atom linker to cage-like structures of 1-adamantanamine and memantine. The introduction of this kind of bulky carbocyclic ring increases lipophilicity and biological membrane crossings [11] Hence, the chemistry of adamantane continues to attract research interest due to the significant pharmacological activity of its derivatives [12][13][14][15].
Herein we report the synthesis and biological evaluation of two new 5-HT 1A inhibitors bearing the 2-MeO-Ph-piperazine moiety linked via a three carbon atom linker to cage-like structures of 1-adamantanamine and memantine. The introduction of this kind of bulky carbocyclic ring increases lipophilicity and biological membrane crossings [11]. Hence, the chemistry of adamantane continues to attract research interest due to the significant pharmacological activity of its derivatives [12][13][14][15].

Biological Activity
Target compounds were screened for their affinity against the serotonin 5-HT1A receptor. The exhibited activities were both in low nM magnitude, with (8) and (10) having a Ki of 1.2 nM and 21.3 nM respectively. Taken together, these data indicate that a (2methoxyphenyl)piperazine motif delivers highly potent 5-HT1A ligands.
As an internal control for the 5-HT1A receptor, compound 5-CT (2) was used, based on Hamon et al. [16,17], which returned a Ki value of 0.5 nM.

Biological Activity
Target compounds were screened for their affinity against the serotonin 5-HT 1A receptor. The exhibited activities were both in low nM magnitude, with (8) and (10) having a K i of 1.2 nM and 21.3 nM respectively. Taken together, these data indicate that a (2methoxyphenyl)piperazine motif delivers highly potent 5-HT 1A ligands.
As an internal control for the 5-HT 1A receptor, compound 5-CT (2) was used, based on Hamon et al. [16,17], which returned a K i value of 0.5 nM.
The new compound (8) presented in this study exhibits a 4-fold drop for its binding constant (K i = 1.2 nM) as measured over 5-HT 1A receptor when compared to the value reported for the known drug aripiprazole (K i = 5.0 nM) [21]. The docking pose of (8) gave a −12.81 Chemgauss4 scoring function. As shown in Figure 3, there are two major hydrogen bonds forming for the compound. One is between the protonated tertiary piperazine amine and Asp116, while the second is between the adamantane amine with Tyr390. On the other hand, (10) gave a −12.64 Chemgauss4 scoring function with exactly the same interaction network (Figure 4), since the only difference in their structure is on the branched carbocycles. The measured constant in that case is approximately four-fold higher (K i = 21.3 nM). The difference in their affinities is due to the methyl groups of the memantine core, since one of the methyl groups is situated in the hydrophobic cavity formed by Ala93, Trp387 and Tyr390, while the other is directed towards Tyr96, introducing hindering effects. These results are in accordance with both in vitro and in silico results and they justify the high affinity of the 4-alkyl-l-arylpiperazine moiety towards 5-HT 1A receptor [8].
Co-crystallized aripiprazole reveals only one important bond forming between the tertiary amine of the piperazine and Asp116 residue. Interestingly the etheric oxygen and the quinolinone do not show any apparent interactions with neighboring receptor residues, without excluding the possibility of a water-mediated bond network. However, as a fact there are no solvent molecules included in the deposited structure. Therefore it is rational that during the model validation, conducted by also performing the docking experiment for aripiprazole (scoring function −12.77 Chemgauss4, see also supporting information Table S1), we witnessed a flipped quinolinone orientation (see Figure 5) in order for the molecule to form hydrogen bonds with Gln97 residue. This deviance from the co-crystallized pose resulted in an acceptable (RMSD ≤ 2.0) [22] but high RMSD value (i.e., 1.986) [23]. The new compound (8) presented in this study exhibits a 4-fold drop for its binding constant (Ki = 1.2 nM) as measured over 5-HT1A receptor when compared to the value reported for the known drug aripiprazole (Ki = 5.0 nM) [21]. The docking pose of (8) gave a −12.81 Chemgauss4 scoring function. As shown in Figure 3, there are two major hydrogen bonds forming for the compound. One is between the protonated tertiary piperazine amine and Asp116, while the second is between the adamantane amine with Tyr390. On the other hand, (10) gave a −12.64 Chemgauss4 scoring function with exactly the same interaction network (Figure 4), since the only difference in their structure is on the branched carbocycles. The measured constant in that case is approximately four-fold higher (Ki = 21.3 nM). The difference in their affinities is due to the methyl groups of the memantine core, since one of the methyl groups is situated in the hydrophobic cavity formed by Ala93, Trp387 and Tyr390, while the other is directed towards Tyr96, introducing hindering effects. These results are in accordance with both in vitro and in silico results and they justify the high affinity of the 4-alkyl-l-arylpiperazine moiety towards 5-HT1A receptor [8].    Co-crystallized aripiprazole reveals only one important bond forming between the tertiary amine of the piperazine and Asp116 residue. Interestingly the etheric oxygen and the quinolinone do not show any apparent interactions with neighboring receptor residues, without excluding the possibility of a water-mediated bond network. However, as a fact there are no solvent molecules included in the deposited structure. Therefore it is rational that during the model validation, conducted by also performing the docking experiment for aripiprazole (scoring function −12.77 Chemgauss4, see also supporting information Table S1), we witnessed a flipped quinolinone orientation (see Figure 5) in order for the molecule to form hydrogen bonds with Gln97 residue. This deviance from the cocrystallized pose resulted in an acceptable (RMSD ≤ 2.0) [22] but high RMSD value (i.e., 1.986) [23]. To conclude, based on the scoring function order (see supporting information Tabl S1) and biological activity, these go hand in hand. Thus, the docking model showcases desired mutual compatibility, providing important information for the mode of activity.

Chemistry
Melting points were determined using a Büchi capillary apparatus and are uncor rected. NMR experiments (see Supplementary Materials) were performed to elucidate th structure and determine the purity of the newly synthesized compounds. 1 H-NMR and 2D NMR spectra (COSY, HSQC-DEPT, HMBC) were recorded on a Bruker Ultrashiel™ Plus Avance III 600 spectrometer (150.9 MHz, 13 C-NMR) and a Bruker DRX400 spectrom To conclude, based on the scoring function order (see supporting information Table S1) and biological activity, these go hand in hand. Thus, the docking model showcases a desired mutual compatibility, providing important information for the mode of activity.

Chemistry
Melting points were determined using a Büchi capillary apparatus and are uncorrected. NMR experiments (see Supplementary Materials) were performed to elucidate the structure and determine the purity of the newly synthesized compounds. 1 H-NMR and 2D NMR spectra (COSY, HSQC-DEPT, HMBC) were recorded on a Bruker Ultrashiel™ Plus Avance III 600 spectrometer (150.9 MHz, 13 C-NMR) and a Bruker DRX400 spectrometer (100.62 MHz, 13 C-NMR). Chemical shifts δ (delta) are reported in parts per million (ppm) downfield from the NMR solvent, with the tetramethylsilane or solvent (DMSO-d 6 ) as internal standard. Data processing, including Fourier transformation, baseline correction, phasing, peak peaking and integrations, were performed using MestReNova software v.12.0.0. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; td, triplet of doublets; m, multiplet; complex m, complex multiplet. Coupling constants (J) are expressed in units of Hertz (Hz). The spectra were recorded at 293 K (20 • C) unless otherwise specified. The solvent used to obtain the spectra was deuterated DMSO, DMSO-d 6 (quin, 2.50 ppm, 1 H-NMR; septet, 39.52 ppm, 13 C-NMR). Analytical thin-layer chromatography (TLC) was used to monitor the progress of the reactions, as well as to authenticate the compounds. TLCs were conducted on aluminum sheets precoated with normal-phase silica gel (Silica gel 60 F 254 , Merck, Darmstadt, Germany) (layer thickness 0.2 mm), aluminum sheets precoated with reverse phase silica gel (Silica gel 60 RP-18 F 254 s, Merck) and precoated aluminum oxide plates (TLC Aluminum oxide 60 F 254 , neutral). Developed plates were examined under a UV light source at wavelengths of 254 nm or after being stained by iodine vapors. The Retention factor (R f ) of the newly synthesized compounds, which is equal to the distance migrated over the total distance covered by the solvent, was also measured on the chromatoplates. Elemental analyses (C, H, N) were performed by the Service Central de Microanalyse at CNRS (France) and were within ±0.4% of the theoretical values. Elemental analysis results for the tested compounds correspond to >95% purity. The HRMS spectra were acquired in the negative ionization mode, employing a QTOF-MS (Maxis Impact, Bruker Daltonics, Bremen, Germany) using a resolving power of 40,000. The commercial reagents were purchased from Alfa Aesar, Sigma-Aldrich and Merck, and were used without further purification. Solvent abbreviations: ACN, acetonitrile; AcOEt, ethyl acetate; Et 2 O, diethyl ether; EtOH, ethanol; MeOH, methanol. (6): A solution of 1, 3-dichloropropane (1.13 g, 10 mmol) in acetonitrile was added to a stirring solution of 1-(2-methoxyphenyl)piperazine (1.28 g, 6.67 mmol) and K 2 CO 3 (2.69 g, 20.00 mmol) in 25 mL acetonitrile at 70 • C. The mixture was refluxed for 14 h. After it had been cooled to room temperature, the solvent was removed under vacuum. The crude product was purified by column chromatography on silica gel eluting with 1% MeOH in DCM (R f = 0.75). Light yellow oil, yield: 70%.

Ligand and Protein Preparation
The 5-HT 1A crystal structure (PDB entry 7e2z [7]) was downloaded over the protein databank [28]. The PDB file was prepared with the OEDocking suite program MAKE RECEPTOR v4.0.0.0 (OpenEye Scientific Software, Inc., Santa Fe, NM, USA; www.eyesopen. com (accessed on 10 March 2021) [29,30] to provide the respective oedu extension file, which is used later in simulation experiments. The search space was centered around the crystalized small molecule inhibitor. This generated an initial box of 12,411 Å 3 , which after a balanced site-shape creation resulted in an inner docking space of 1026 Å 3

Model Confidence Experiment
The aripiprazole structure was included for confidence reasons regarding the model generated. Hence, the docked solution of the inhibitor was subjected to RMSD measurement online using the DockRMSD utility [23]. The input format of the corresponding structures are Sybyl MOL2.

Biology
Affinity of compounds was measured in cell membranes prepared in-house from a HEK-293 cell line transfected with human 5-HT 1A receptors. 10 µg of membrane suspension were incubated with 2 nM [ 3 H]-8-hydroxy-DPAT (PerkinElmer) in assay buffer (50 mM Tris-HCl, 5 mM MgSO 4 , 5 mM MgCl 2 ; pH = 7.4) for 120 min at 37 • C in multiscreen FC 96well plate (Millipore, Burlington, MA, USA). Non-specific binding was determined in the presence of 10 µM 5-HT. After the incubation time, samples were filtered and radioactivity was detected in a Microbeta Trilux reader.
Data were fitted to a 4-parameter logistic equation employing GraphPad Prism 5.1, and K i values were calculated by employing the Cheng-Prusoff equation [31].
Supplementary Materials: The following are available online. Copies of NMR spectra; Figure S1: (A) Distant green representation of the 5-HT1A receptor with the presence of crystallized Aripiprazole as magenta stick, (B) 90 • rotation of the 5-HT1A receptor from the Y towards the Z axis revealing the whole interior of it, (C) 2D representation of Aripiprazole crystal structure with the formed binding network, (D) overlay of co-crystalized Aripiprazole as magenta colored sticks and docked solution of it as pink colored sticks. Brick red dashed lines represent HB between protein and the substrate, Table S1: Molecular modeling results. Comparative presentation of in vitro & in silico results of compounds versus 5-HT1A receptor.
Author Contributions: Conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-original draft preparation, writing-review and editing, supervision, project administration, funding acquisition, G.Z.; conceptualization and draft editing, M.C.; investigation, data curation and draft editing M.I.L. All authors have read and agreed to the published version of the manuscript.