Design, Synthesis and Pharmacological Evaluation of Novel Conformationally Restricted N-arylpiperazine Derivatives Characterized as D2/D3 Receptor Ligands, Candidates for the Treatment of Neurodegenerative Diseases

Most neurodegenerative diseases are multifactorial, and the discovery of several molecular mechanisms related to their pathogenesis is constantly advancing. Dopamine and dopaminergic receptor subtypes are involved in the pathophysiology of several neurological disorders, such as schizophrenia, depression and drug addiction. For this reason, the dopaminergic system and dopamine receptor ligands play a key role in the treatment of such disorders. In this context, a novel series of conformationally restricted N-arylpiperazine derivatives (5a–f) with a good affinity for D2/D3 dopamine receptors is reported herein. Compounds were designed as interphenylene analogs of the drugs aripiprazole (2) and cariprazine (3), presenting a 1,3-benzodioxolyl subunit as a ligand of the secondary binding site of these receptors. The six new N-arylpiperazine compounds were synthesized in good yields by using classical methodologies, and binding and guanosine triphosphate (GTP)-shift studies were performed. Affinity values below 1 μM for both target receptors and distinct profiles of intrinsic efficacy were found. Docking studies revealed that Compounds 5a–f present a different binding mode with dopamine D2 and D3 receptors, mainly as a consequence of the conformational restriction imposed on the flexible spacer groups of 2 and 3.


Introduction
Dopamine is a key neurotransmitter involved in several physiological processes for the full functioning of the body, such as voluntary movements, affection, sleep, attention, memory, learning, hormonal regulation and cardiovascular and immune functions [1][2][3]. The degeneration of dopaminergic neurons in the substantia nigra causes an inhibition of dopaminergic signaling, which can generate rigor, tremor, bradykinesia and postural instability, the main symptoms of Parkinson's disease (PD) [4][5][6]. However, the mesolimbic pathway is directly involved with the mechanisms of emotion control and reward. Thus, Figure 1. Compounds acting on D2 and D3 receptors: haloperidol (1), aripiprazole (2), cariprazine (3) and indole derivative (4). Design concept of a new series of conformationally restricted Nphenylpiperazines (5a-f).
Other analog N-phenylpiperazine compounds, now showing intrinsic efficacy as antagonists, have also been developed to act in the treatment of dependence and drug addiction [27][28][29]. Among these compounds, compound (4) (Ki D3 = 0.118 nM; Ki D2 = 12.9 nM), a D2/D3 receptor antagonist developed by Boateng et al. (2015), guards, in its chemical structure, important similarities with cariprazine (3), in the presence of an arylpiperazine subunit (also present in aripiprazole) and an amide group. However, compound (4) also presents differences such as the introduction of an alkyl spacer that gives greater conformational freedom to the compound in relation to cariprazine (3). Furthermore, the presence of an indole subunit appears to be responsible for the change Other analog N-phenylpiperazine compounds, now showing intrinsic efficacy as antagonists, have also been developed to act in the treatment of dependence and drug addiction [27][28][29]. Among these compounds, compound (4) (Ki D 3 = 0.118 nM; Ki D 2 = 12.9 nM), a D 2 /D 3 receptor antagonist developed by Boateng et al. (2015), guards, in its chemical structure, important similarities with cariprazine (3), in the presence of an arylpiperazine subunit (also present in aripiprazole) and an amide group. However, compound (4) also presents differences such as the introduction of an alkyl spacer that gives greater conformational freedom to the compound in relation to cariprazine (3). Furthermore, the presence of an indole subunit appears to be responsible for the change in intrinsic efficacy from a partial agonist in cariprazine (3) to an antagonist in derivative (4) (Figure 1) [30].
In this context, considering the multifactorial behavior of these neurodegenerative diseases and the importance of finding novel compounds that combine the structural requirements of just one molecule to act on dopamine D 2 /D 3 receptors with a fine-tuning adjustment of intrinsic efficacies, we reported herein a new series of conformationally restricted N-arylpiperazine derivatives (5a-f) presenting moderate affinity for D 2 /D 3 dopamine receptors. The compounds were designed as interphenylene analogs of the drugs aripiprazole (2) and cariprazine (3), having a 1,3-benzodioxole subunit (A) as a ligand for the secondary binding site of these receptors. The pharmacophoric arylpiperazine subunit was preserved in the design concept of the new series of derivatives (5a-f) (Figure 1). Replacement of the alkyl spacer with an interphenylene spacer was proposed, bringing a conformational restriction [31]. In addition, the amide group present in the amide derivative (4) was replaced by a sulfonamide, which has additional points capable of interacting with the bioreceptor [32]. Aromatic substituents such as phenyl, 2-methoxyphenyl and 2,3-dichlorophenyl at position 4 of the piperazine ring were used to evaluate the ortho effect on the coplanarity between the piperazine ring and the aromatic ring [33]. The 1,3-benzodioxole subunit A (Figure 1) attached to a sulfonamide group was chosen due to isosteric relationships with the indoleamide subunit present in compound (4), as previously described by our laboratory [34].
Classic synthetic methodologies, molecular modeling studies, and binding and GTPshift experiments were performed. Six new N-phenylpiperazine derivatives (5a-e) were obtained, with affinity values below 1 µM and distinct profiles of intrinsic efficacy ( Figure 1).

Chemistry
All commercially available reagents and solvents were used without further purification. Reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (F245 Merck plates), and the products were visualized with an ultraviolet (UV) lamp (254 and 365 nm). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were determined in dimethyl sulfoxide (DMSO)-d6 solutions using a VARIAN 500-MR spectrometer (Varian, Palo Alto, CA, USA) operating at 500 and 125 MHz, respectively. The chemical shifts are given in parts per million (δ) from solvent residual peaks, and the coupling constant values (J) are given in Hz. Signal multiplicities are represented by s (singlet), d (doublet), dd (double doublet), t (triplet), m (multiplet) and br (broad signal).
Infrared spectra were obtained using a Thermo Nicolet Avatar 330 FTIR (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer equipped with a smart endurance diamond ATR unit for direct measurements. The melting points (MPs) were determined on a Quimis Model Q340.23 apparatus in triplicate.
Microanalyses were carried out using a Thermo Scientific Flash EA 1112 series CHN-Analyzer, using a Mettler MX5 electronic balance.
The purity of the synthesized compounds was determined by high-performance liquid chromatography (HPLC), which was performed in a Shimadzu LC20AD apparatus (Shimadzu, Tokyo, Japan) using a Kromasil 100-5C18 column (4.6 mm × 250 mm) (Kromasil, Bohus, Sweden) and an SPD-M20A detector (Diode Array) at wavelengths ranging from 238 to 287 nm for analyte quantification and a constant flow rate of 1 mL/min. The automatic injector was programmed so that the volume of sample injected per analysis corresponded to 20 µL. The mobile phases used were 60% acetonitrile and 40% water, 60% methanol and 40% water, and 80% ethanol and 20% water; the pH of the mobile phase was adjusted to 3 and 6.5 according to the type of compound to be analyzed. The solvents used for HPLC-PDA analysis were HPLC purity grade (Tedia ® ).

General Procedure for the Synthesis of 4-Nitrobenzyl-phenylpiperazine Intermediates 8a-c
In a 125-mL flask, 0.151 g (1 mmol) of 4-nitrobenzaldehyde (10) was dissolved in 30 mL of absolute ethanol. Then, 1 equivalent of the respective phenylpiperazines (9a-c) and 0.5 equivalents of zinc chloride (ZnCl 2 ; 0.07 g; 0.5 mmol) were added to the solution. The reaction mixture was kept under constant stirring at 60 • C. After 2 h, 3.2 equivalents of sodium cyanoborohydride (NaBH 3 CN; 0.2 g; 3.2 mmol) was added in 2 portions every 1 h. The complete consumption of starting materials was evidenced 24 h after the addition of the reducing agent by TLC using a mixture of hexane:ethyl acetate (60:40) as the eluent.
The isolation of the product was carried out by extraction in a separatory funnel using ethyl acetate and saturated sodium bicarbonate solution. The organic phase was separated, dried with anhydrous sodium sulfate and concentrated at reduced pressure, furnishing the desired 4-nitrobenzyl-phenylpiperazines (8a-c), as described next.
The progress of the reaction was monitored by TLC using hexane:ethyl acetate (60:40) as the eluent. The end of the reaction was observed after 1 h. Then, the reaction mixture was filtered out hot through Celite, and isolation was carried out by extraction in a separatory funnel using ethyl acetate and saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate and filtered and concentrated under reduced pressure, furnishing the desired 4-((4-phenylpiperazin-1-yl)methyl)anilines (6a-c), as described next.

4-((4-Phenylpiperazin-1-yl)methyl)aniline (6c)
Intermediate 6c was obtained with 60% yield as a yellowish oil. 1  In a G30-type microwave tube containing a solution with 0.08 g of the respective 4-((4-phenylpiperazin-1-yl)methyl)anilines (6a-c) and 10 mL of ethanol, 1 equivalent of the desired sulfonyl chloride (7a or 7b) was added. The reaction mixture was irradiated in a microwave oven for 30 min at 100 • C. For the isolation of the products, the reaction medium was concentrated and extracted in a separatory funnel using ethyl acetate and water at pH = 10 (adjusted with 10% NaOH solution). The organic phase was dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was submitted to a purification step by silica gel column chromatography using hexane/ethyl acetate as the mobile phase in a gradient (90:10 to 70:30). Target compounds 5a-f were obtained in moderate to good yields, as described below.
N-Phenylpiperazine derivative 5a was obtained in 36% yield as a white solid, with a melting point of 154-156 • C. 1

Molecular Modeling
Molecular docking studies were performed using Genetic Optimization for Ligand Docking (GOLD) v. 5.6 [35][36][37][38][39][40]. Crystallographic structures of the D 3 and D 2 receptors were selected from the Protein Data Bank (PDB; http://www.rcsb.org, accessed on 22 June 2022) protein database. The crystallographic structure with the 3PBL code (resolution 2.89 Å) in PDB was selected for the D 3 receptor [41], whereas the chosen crystallographic structure of the D 2 receptor was that with the 6CM4 code (2.86 Å) [42]. Such structures were the only structures available in the PDB for these subtypes of dopamine receptors when this work was done.
Based on the structure of the cocrystallized ligand with the D 2 receptor (risperidone), the validation of the methodology that would be used for molecular docking studies was carried out. Risperidone was chosen because it is structurally similar to the compounds designed for this work.
Hydrogen atoms were added to the protein, and the location of the binding site was defined using the cocrystalized ligand (risperidone) and all amino acids 6 Å away from it as a reference.
To carry out both the redocking and further studies, risperidone and other proposed molecules were built in the ChemDraw program, and then the protonation state of the molecules was analyzed using the Percepta program. Subsequently, the equilibrium geometry was calculated by the semiempirical method PM6 (Parametric Method 6) for the lowest energy conformers, which were then used for docking [43].
Since the available crystallographic structure 6CM4 does not present water molecules, redocking of risperidone in the D 2 receptor was performed in the absence of them. Redocking results were evaluated by root-mean-square deviation (RMSD) calculation (more details in the Supplementary Material). For a punctual evaluation of the interaction profile presented by the compounds, we performed a protein-ligand interaction profile analysis (PLIP-https://plip-tool.biotec.tudresden.de/plip-web/plip/index, accessed on 4 August 2022) [44].

Binding and GTP-Shift
Membrane preparations of recombinant Chem-1 cells (ChemiscreenTM, Millipore, Burlington, MA, USA), transfected by a process using human cDNAs encoding the D 3 isoform of the dopaminergic receptor, were used.
Test substances were solubilized in 100% DMSO (stock solution) and then serially diluted in water. Nonspecific binding was measured in the presence of 30 µM sulpiride (a selective antagonist of the central dopamine receptors: D 2 , D 3 , and D 4 ). To evaluate the intrinsic efficacy, 50 mM Tris-HCl and 5 mM KCl buffer were used [45]. In this protocol, we used a medium containing a high concentration of divalent cations (MgCl 2 5 mM and CaCl 2 1.5 mM), which favors the binding of agonists to the receptor, or a medium with high concentrations of sodium and guanosine triphosphate (GTP) (154 mM NaCl and GTP 1 mM), which hinders the binding of agonists.
Finally, to evaluate the selectivity of the substances, classic binding to D 2 -like receptors was performed using rat striatal membranes. Adult male Wistar rats (2.5-3 months) were killed by decapitation, their brains were immediately removed on ice and the striatum was dissected and stored in liquid nitrogen until use. This procedure was approved by the Institutional Ethical Committee for Animal Care from the Federal University of Rio de Janeiro (CEUA no. 052/19; 30 April 2019). The striatum was homogenized in a motorized Potter-type apparatus with a Teflon piston at 4 • C at 20 volumes per gram of tissue in ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 8 mM MgCl 2 and 5 mM ethylenediaminetetraacetic acid (EDTA). The resulting suspension was ultracentrifuged at 48,000× g at 4 • C for 20 min. The pellet was resuspended in 20 volumes of the same buffer and incubated at 37 • C for 10 min to remove endogenous neurotransmitters. This suspension was cooled and ultracentrifuged at 48,000× g for 20 min at 4 • C [46]. The final pellet was resuspended and stored in liquid nitrogen until use.
The affinity of substances for D 3 R and D 2 -like receptors was evaluated through classical competition assays to determine the IC 50 value. Data were analyzed by nonlinear regression using the GraphPad Prism ® program (version 5.00) and the "binding-one site competition" model to adjust the curve and calculate the mean inhibitory concentration (IC 50 ). For D 2 R, the K i value was calculated from the Cheng-Prusoff equation: Analysis of intrinsic efficacy for D 3 R was performed through the displacement caused by sodium with GTP and the ratio of the IC 50 obtained (in this condition) by the IC 50 obtained in the medium with MgCl 2 and CaCl 2 . As a control, an experiment was carried out with dopamine, the endogenous agonist of this receptor.

Binding Affinity, Intrinsic Energy and Molecular Modeling Studies
The new N-arylpiperazine derivatives bind to both D 2 and D 3 receptors with similar micromolar affinities ( Table 1). The presence of aromatic ring systems and basic nitrogen appears to make the N-phenylpiperazine scaffold the main molecular recognition element for the binding site of aminergic G-protein-coupled receptors (GPCRs) [51]. This hypothesis is qualitatively supported by the interaction profile of the compounds. The N-phenylpiperazine subunit occupies the region of the orthosteric site of the D 2 and D 3 receptors, both for aripiprazole and cariprazine (Figure 2A-D) and for the new derivatives, represented here by Compound 5a (Figure 3A,B). According to docking studies, interactions at the orthosteric site are hydrophobic and involve amino acid residues such as serine, tryptophan and phenylalanine. However, by analysis of the specific molecular interactions, we can observe some important differences between them for each of the compounds. For example, in D 3 receptors, both aripiprazole and cariprazine show interactions with three identical amino acid residues of the OBS, Asp110, Phe245 and Phe246, but 5a interacts only with Asp110, in addition to presenting a hydrophobic interaction with Ile183, similarly to cariprazine (Figure 4A-C). In D 2 receptors, both the prototypes and 5a present a salt bridge interaction with the Asp114 residue. However, in relation to the other interactions, in comparative terms, especially when we compare 5a and aripiprazole, we perceive a profile that involves different residues. Aripiprazole interacts with residues such as Thr119, Trp386 and Phe390, and 5a does not interact with any of them ( Figure 5A-C). Table 1. Affinities and intrinsic efficacies of the new derivatives. The IC 50 values were calculated from the mean curves of two to six experiments [n] performed in triplicate and are expressed with their 95% confidence intervals (in parentheses). For D 2 R, the K i value was calculated from the Cheng-Prusoff equation. For D 3 R, the Na+-shifts were calculated by dividing the IC 50 value obtained in the medium containing NaCl and GTP(II) by the IC 50 value obtained in the medium containing MgCl 2 and CaCl 2 (I). Na+-shifts lower than one indicate that the compounds are weak inverse agonists. Na+-shifts similar to one indicate that the compounds act as antagonists, whereas agonists have higher values.

Compounds
Ki D

Binding Affinity, Intrinsic Energy and Molecular Modeling Studies
The new N-arylpiperazine derivatives bind to both D2 and D3 receptors with similar micromolar affinities ( Table 1). The presence of aromatic ring systems and basic nitrogen appears to make the N-phenylpiperazine scaffold the main molecular recognition element for the binding site of aminergic G-protein-coupled receptors (GPCRs) [51]. This hypothesis is qualitatively supported by the interaction profile of the compounds. The N-phenylpiperazine subunit occupies the region of the orthosteric site of the D2 and D3 receptors, both for aripiprazole and cariprazine (Figure 2A-D) and for the new derivatives, represented here by Compound 5a (Figure 3A,B). According to docking studies, interactions at the orthosteric site are hydrophobic and involve amino acid residues such as serine, tryptophan and phenylalanine. However, by analysis of the specific molecular interactions, we can observe some important differences between them for each of the compounds. For example, in D3 receptors, both aripiprazole and cariprazine show interactions with three identical amino acid residues of the OBS, Asp110, Phe245 and Phe246, but 5a interacts only with Asp110, in addition to presenting a hydrophobic interaction with Ile183, similarly to cariprazine ( Figure 4A-C). In D2 receptors, both the prototypes and 5a present a salt bridge interaction with the Asp114 residue. However, in relation to the other interactions, in comparative terms, especially when we compare 5a and aripiprazole, we perceive a profile that involves different residues. Aripiprazole interacts with residues such as Thr119, Trp386 and Phe390, and 5a does not interact with any of them ( Figure 5A-C).     The presence of substituents on the phenyl, linked to the piperazine ring, did not significantly modify the affinity for these receptors.
In addition to the orthosteric site, a secondary binding site (SBP) was identified in dopaminergic receptors, which seems to be related not only to the affinity that ligands may have for these receptors but also to the selectivity between the different subtypes [29,30].
According to the docking studies carried out here, both the prototype compounds (2 and 3) and the new derivatives showed hydrophobic interactions in the SBP of the D 2 and D 3 receptors. However, such interactions took place in different regions of the SBP, which can be explained by two structural characteristics. The first one is related to the different chemical subunits present in the analyzed compounds. In aripiprazole (2), we have a dihydroquinoline, whereas in cariprazine (3), a butyramide, and in the new derivatives (5a-f), a 1,3-benzodioxole subunit. Furthermore, the conformational differences caused by the different spacer groups also contribute to the interactions in different regions of the SBP. While in aripiprazole, the alkyl spacer has great conformational freedom, in the new derivatives, the interphenylene spacer is conformationally restricted (Figure 2A-D and Figure 3A,B). We can note that, in the SBP of D3, aripiprazole and cariprazine have two interactions in common (Leu89, Phe106), as does 5a (Leu89, Glu90), but only the prototypes interact with Phe106 ( Figure 4A-C). In D 2 receptors, 5a has no interaction in common with 2 and 3 in the SBP ( Figure 5A-C).
For both receptors, the interaction pattern of 2 and 3 appears to be more "hydrophobic", while 5a performs a greater number of hydrogen bonds. Since hydrophobic interactions are strongly related to the displacement of water molecules located around the hydrophobic groups of the ligand and the binding site, when they interact with each other [52], these differences could indicate a more entropic interaction profile for the prototypes, while compound 5a would have a more enthalpic profile, as well as the other compounds analyzed. This difference in interaction profile could explain the difference in affinity at both receptors for the prototype compounds (2 and 3) versus the new derivatives.
As efficacy is as important as affinity for the therapeutic effect of a drug, we initially decided to estimate the intrinsic efficacy of the new compounds for the D 3 receptor using a functional binding assay. The classic GTP-shift assay is based on the ternary complex model for GPCRs and has been validated for the D 3 receptor [42]. This assay is based on the difference in affinity measured for agonists in the absence and presence of a high concentration of GTP (or a lower concentration of a nonhydrolyzable GTP analog) that is capable of destabilizing the ternary complex ARG (high affinity state of the receptor), which is formed by the agonist (A), the receptor (R) and the G protein (G).    Figure 6A shows the profiles of competition curves for the binding of [ 3 H]-spiperone to D 3 receptors using a full agonist (dopamine, Figure 6A) for validation purposes. In the presence of 154 mM NaCl and 1 mM GTP, the dopamine competition curve was shifted to the right, indicating a loss of affinity for D 3 receptors. When an antagonist is used as a competitor, the addition of GTP has no effect, as was observed for derivative 5a, since the competition curves in the absence and presence of GTP were superimposed, indicating that 5a is a D 3 receptor antagonist ( Figure 6B). Very similar behavior was found for the other N-phenylpiperazine derivatives presenting R 1 groups as methyl groups, such as 5c and 5b, which were classified as weak inverse agonists. However, in compounds 5d and 5f, where the steric hindrance promoted by the presence of a methyl group in the 1,3-benzodioxole ring was abolished, we found an intrinsic efficacy as a partial agonist. This kind of influence of a methyl group in the bioactive conformation of drugs and drug candidates is well discussed in a previous paper by our group [53]. Figure 6A shows the profiles of competition curves for the binding of [ 3 H]-spip to D3 receptors using a full agonist (dopamine, Figure 6A) for validation purposes. presence of 154 mM NaCl and 1 mM GTP, the dopamine competition curve was s to the right, indicating a loss of affinity for D3 receptors. When an antagonist is use competitor, the addition of GTP has no effect, as was observed for derivative 5a, sin competition curves in the absence and presence of GTP were superimposed, indi that 5a is a D3 receptor antagonist ( Figure 6B). Very similar behavior was found f other N-phenylpiperazine derivatives presenting R1 groups as methyl groups, such and 5b, which were classified as weak inverse agonists. However, in compounds 5 5f, where the steric hindrance promoted by the presence of a methyl group in th benzodioxole ring was abolished, we found an intrinsic efficacy as a partial agonis kind of influence of a methyl group in the bioactive conformation of drugs and dru didates is well discussed in a previous paper by our group [53].
These results are consistent with the structural requirements for D3 receptor an nists, namely, an arylpiperazine subunit, a hydrogen-bonded donor/acceptor grou aryl subunit and a suitable spacer [29,30].
The competition curves for the other compounds are presented in the Suppleme Material ( Figure S28). Although the new N-phenylpiperazine derivatives (5a-f) present similar bindi finities for D2 and D3, the best Compounds 5e and 5f combined the presence of an substituted phenyl group attached to the N-phenylpiperazine subunit with the abse a methyl group in the 1,3-benzodioxole ring, which could favor the interactions vicinal sulfonamide group with both target receptors. Interestingly, Compounds 5 These results are consistent with the structural requirements for D 3 receptor antagonists, namely, an arylpiperazine subunit, a hydrogen-bonded donor/acceptor group, an aryl subunit and a suitable spacer [29,30].
The competition curves for the other compounds are presented in the Supplementary Material ( Figure S28).
Although the new N-phenylpiperazine derivatives (5a-f) present similar binding affinities for D 2 and D 3 , the best Compounds 5e and 5f combined the presence of an orthosubstituted phenyl group attached to the N-phenylpiperazine subunit with the absence of a methyl group in the 1,3-benzodioxole ring, which could favor the interactions of the vicinal sulfonamide group with both target receptors. Interestingly, Compounds 5e and 5f were able to modulate D 3 receptors with different intrinsic efficacies as antagonists and partial agonists, respectively.

Conclusions
As concluding remarks, this work described a new series of substituted N-phenylpiperazines (5a-f) designed as interphenylene analogs of the antipsychotic drugs aripiprazole (2) and cariprazine (3), presenting a 1,3-benzodioxole group as a ligand of the secondary binding pocket of dopamine D 2 and D 3 receptors. The target compounds were synthesized in good yields by using classical methodologies, and their binding to both D 2 and D 3 receptor subtypes, as well as GTP shift studies, were performed. The best, Compounds 5e and 5f, presented affinity values of 0.1 and 0.2 µM (Ki for D 2 ) and 0.2 and 0.2 µM (IC 50 for D 3 ), respectively, and distinct profiles of intrinsic efficacy. Docking studies revealed that Compounds 5a-f present a different binding mode with dopamine D 2 and D 3 receptors, mainly as a consequence of the conformational restriction imposed on the flexible spacer groups of 2 and 3. Although the prototypes (2 and 3) and the new compounds are predicted to interact at the same binding sites, detailed analysis of the interaction profile indicate that the difference in affinity at both receptors for the prototype compounds versus the new derivatives could be related to differences in interactions with binding site residues. Taken together, these results indicated that the N-phenylpiperazine derivatives 5e and 5f are promising dual ligands of dopamine D 2 and D 3 receptor candidates for further studies in animal models of schizophrenia and drug addiction.  Figure S23: Infrared (ATR) of 5f; Figure S24: Reversed-phase chromatogram in MetOH:H2O (60:40) at 254 nm of 5d; Figure S25: Overlap of risperidone structure of the crystallographic structure (PDB 6CM4) in orange, and the result obtained after redocking by the ChemPLP function in purple; Figure S26: Interaction profile of the proposed compounds on D 3 (gray) and D 2 (blue) receptors. A and B: 5a (light blue); C and D: 5b (pink); E and F: 5c (yellow); Figure S27: Interaction profile of the proposed compounds on D 3 (gray) and D 2 (blue) receptors. A and B: 5d (orange); C and D: 5e (pink); E and F: 5f (purple); Figure S28: PLIP analysis for the compound 5b, at D 2 receptors; Figure S29: PLIP analysis for the compound 5b, at D 3 receptors; Figure S30: PLIP analysis for the compound 5c, at D 2 receptors; Figure S31: PLIP analysis for the compound 5c, at D 3 receptors; Figure S32: PLIP analysis for the compound 5d, at D 2 receptors; Figure S33: PLIP analysis for the compound 5d, at D 3 receptors; Figure S34: PLIP analysis for the compound 5e, at D 2 receptors; Figure S35: PLIP analysis for the compound 5e, at D 3 receptors; Figure S36: PLIP analysis for the compound 5f, at D 2 receptors; Figure S37: PLIP analysis for the compound 5f, at D 3 receptors; Figure S38: Estimation of the affinity of of 5a (A), 5b (B), 5c (C), 5d (D), 5e (E) and 5f (F) on [ 3 H]-YM-09151-2 binding to rat striatal D2 receptor (D2R). Data are means (±S.E.) from two or three independent experiments, each performed in triplicate. The data were fitted assuming a single population of binding sites and curves were drawn using the parameters fitted by nonlinear regression (see details in the Materials and Methods); Figure S39: Estimation of the intrinsic efficacy of 5b (A), 5c (B), 5d (C), 5e (D), and 5f (E) at the human D 3 in membrane preparations of recombinant Chem-1 cells. Competition curves were performed using the antagonist radioligand (0.5 nM [ 3 H]-spiperone) in the presence of 5 mM MgCl 2 and 1.5 mM CaCl 2 (black) or 154 mM NaCl and 1 mM GTP (blue). Each curve represents the averaged curve (±S.E.) from two or three independent paired experiments (in triplicate); Table S1: RMSD values for each of the GOLD program function; Table S2: Values of the scores of the results obtained through the docking calculation from the GOLD program, using the ChemPLP function.  Institutional Review Board Statement: The animal study protocol was approved by the Institutional Ethical Committee for Animal Care from the Federal University of Rio de Janeiro, CEUA no. 052/19, 30 April 2019.