Structure–Affinity Relationship Optimization of SYA16263 Yields New Ligands with Improved Receptor Affinity and Antipsychotic-Like Properties

Uma M Gonela; Barbara A Bricker; Chandrashekhar Voshavar; Edem K Onyameh; Matilda E Adzimah; Edith B Kuudaar; Alexus A Morgan; Ismail L Jones; Eljaye W Tellis; Seth Y Ablordeppey

doi:10.1021/acsomega.6c02255

. 2026 Jul 20;11(31):46009–46029. doi: 10.1021/acsomega.6c02255

Structure–Affinity Relationship Optimization of SYA16263 Yields New Ligands with Improved Receptor Affinity and Antipsychotic-Like Properties

Uma M Gonela ¹, Barbara A Bricker ¹, Chandrashekhar Voshavar ¹, Edem K Onyameh ¹, Matilda E Adzimah ¹, Edith B Kuudaar ¹, Alexus A Morgan ¹, Ismail L Jones ¹, Eljaye W Tellis ¹, Seth Y Ablordeppey ^1,^*

PMCID: PMC13470933 PMID: 42598466

Abstract

Central nervous system (CNS) disorders are multifactorial in nature. Dopamine (DA) and serotonin (5-HT) receptors are the prime targets for antipsychotic drug development. In particular, D₂-like (D₂, D₃ and D₄), 5-HT_1A and 5-HT_2A receptors are strongly implicated in the pathophysiology of many neuropsychiatric conditions such as anxiety, schizophrenia, mood and hyperactivity disorders. However, selectivity of the ligands at different receptor subtypes and crosstalk between them complicates the scenario. Development of new ligands that exhibit selectivity toward specific receptor subtypes may aid in understanding their interplay in various CNS disease pathologies. Previously, we reported SYA16263 (6) with high binding affinities toward the D₄ and 5-HT_1A receptors and moderate binding affinities at the D₂, D₃, 5-HT_2A and 5-HT_7A receptors. Further, we have shown that compound 6 exhibits functional selectivity, producing antipsychotic-like effects in rodents without inducing the side effect of catalepsy. In this work, an extended structure-affinity relationship (SAfiR) investigation was carried out with structural modifications at the p-fluorophenyl moiety of 6 by varying the aryl/heteroaryl groups, tweaking the linkage connectivity, and the connecting chain length at the nonpiperazinyl aryl region to improve receptor affinity at D₂R, and 5-HT_1AR. Our SAfiR studies resulted in compounds 35 and 36, with enhanced affinities at D₂R, D₃R, and 5-HT_1AR compared to 6. In addition, compound 35 exhibited improved binding affinity at 5-HT_2AR while compound 36 showed enhancement at 5-HT_7AR. Docking studies were performed with compounds 6, 36 and reference drug, lurasidone to gain structural insight into the ligand–receptor interactions at both the D₂ and 5-HT_2A receptors. Subsequently, pharmacological evaluation of 35 and 36 in the mouse model of schizophrenia using the apomorphine-induced climbing behavior study produced a dose-dependent reduction in climbing behavior and its effects were compared against lurasidone, a standard antipsychotic drug. Further, 35 and 36 did not induce catalepsy in rats even at their respective 3 x 3ED₅₀ doses. Thus, 35 and 36 have the potential to produce antipsychotic-like results in humans in a manner similar to lurasidone. Our future studies are planned to include preclinically evaluating pharmacological responses in specific genetic models to strengthen findings on antipsychotic effects.

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1. Introduction

G-protein coupled receptors (GPCRs) play an important role in regulating several CNS functions including mood, appetite, pain, vision, immune responses, cognition, and several others, and they constitute the most targeted receptors in developing treatment options for neurodegenerative and psychiatric illnesses. Of these receptors, the brain serotonin (5-HT) and dopamine (DA) receptors have been associated with mood regulation, cognition, memory, learning, sleep, movement, motivation and pleasure. − The role of these receptors in various psychiatric conditions such as schizophrenia and depression is strongly implicated. Therefore, dopamine and serotonin receptors have been prime targets for the development of antipsychotics. , It is also reported that these receptors undergo heteromerization with functional crosstalk between several of them. , The complex nature of CNS illnesses often requires the need to target multiple receptors to obtain effective therapies. , Thus, identifying new ligands that bind to multiple receptors and are selective to specific receptor-subtypes may help in understanding the signal transduction mechanisms across dopamine and serotonin receptor systems and their roles in CNS function. Such ligands hold great promise in identifying the potential interaction of heteroreceptors and their subsequent utility in obtaining more effective therapies as antipsychotic and antidepressant agents.

DA receptor subtypes have been widely explored as targets for drug development. The D₂R in particular has been the mainstay for drug development for several neuropsychiatric illnesses including schizophrenia. In fact, there is a school of thought suggesting that drug development for schizophrenia must involve the D₂R. The discovery that SEP-363856 exerts antipsychotic activity by acting as an agonist at trace amine-associated receptor 1 (TAAR1) and 5-HT_1A receptors without binding to D2 receptors , gave the impression that antipsychotic agents could be found which have no association with the D₂R. However, SEP-363856, when evaluated in clinical trials for efficacy and safety, did not produce the desired outcome in acutely psychotic adults with schizophrenia. On the other hand, targeting D₂R has been associated with extrapyramidal and endocrine related side effects , and suggests the need for identifying other mechanisms that might alleviate the problem.

Serotonin receptors are classified into seven classes, 5-HT₁ through 5-HT₇. All the 5-HT receptors belong to the GPCR family except for 5-HT₃R which belongs to the nicotinic acetylcholine receptor family. These receptors are widely distributed on neurons of the dorsal and median raphe nuclei of the brainstem that project into other brain regions such as the hypothalamus, thalamus, hippocampus, and cortex. Among 5-HT receptors, the 5-HT₁ receptor family is the largest class with high sequence homology (∼40–60%) among their subtypes. , The 5-HT_1A receptor is extensively distributed in the brain and is predominantly found in the hippocampus, cerebral cortex, amygdala, raphe nucleus, and septum. It is also present in the regions of basal ganglia and thalamus. Several studies have indicated that 5-HT_1AR is implicated in the pathophysiology of anxiety, schizophrenia, mood and hyperactivity disorders. ,, In addition, it has been reported that 5-HT_1AR activation reduces extrapyramidal side effects (EPS) in rodent models. , Similarly, recurring evidence suggests that targeting the 5-HT_2AR may ameliorate neuropsychiatric symptoms, particularly in conditions like schizophrenia. Emerging evidence also supports the notion that 5-HT_7A receptors are potential therapeutic targets for treating cognitive deficits and negative symptoms in schizophrenia and other psychotic disorders. In particular, antagonism of 5-HT_7A receptors, found in key brain regions like the hippocampus and cortex, modulates dopamine and serotonin pathways, with drugs like lurasidone utilizing 5-HT_7A antagonism to improve cognitive function. − Therefore, selective blocking of 5-HT_2A and 5-HT_7A receptors is a key component of many antipsychotic medications. Thus, we have hypothesized that the most important receptors to be targeted for developing antipsychotic agents may include the D₂R, 5-HT_1AR, 5-HT_2AR and 5-HT_7AR. Therefore, our previous disclosure of SYA16263 (6) with antipsychotic properties but no catalepsy at 19 times its ED₅₀ value should be revisited.

SYA16263 (6), a pyridinyl-piperazine linked with a butyl chain to p-fluoro phenyl group was reported from our group with high binding affinities toward D₄ and 5-HT_1A receptors (K _i, nM = 3.5 and 1.1 respectively) and moderate binding affinities at the D₂, D₃, 5-HT_2A and 5-HT_7A receptors (K _i, nM = 124, 86, 50, and 90 respectively). , Compound 6 is functionally selective and produces antipsychotic-like behavior but without inducing catalepsy in rats. Based on its pharmacological profile, we selected SYA16263 (6) to study its structure-affinity relationship (SAfiR) with a view to obtaining new analogs that display enhanced receptor subtype selectivity.

In the initial SAfiR study, the primary structural modifications involved replacing the pyridinyl-piperazine moiety with a 3,8-diazabicyclo[3.2.1]octane unit and introducing an alkyl linker bearing a restricted carbonyl function. Evidence from the literature indicates that the 1-aryl-piperazine scaffold is widely utilized for its favorable engagement with D₂-like receptors, particularly when linked through an appropriate spacer to a lipophilic aromatic or heteroaromatic moiety (Figure ). Compounds in which an N-heterocyclic lipophilic group is tethered to an aryl piperazine via a suitable hydrocarbon chain have demonstrated notable affinity for D₂-like, 5-HT_1A, and 5-HT_2A receptors, as exemplified by compound 6.

Structures of some antipsychotic drugs in use endowed with the 1-aryl piperazine pharmacophore, and SYA16263 (6).

Building on the previous findings, we expanded the SAfiR studies by introducing structural modifications (Figure ) at the p-fluorophenyl moiety of compound 6, incorporating diverse aryl and heteroaryl frameworks. Variations in the linker connectivity and chain length at the nonpiperazinyl aryl region were explored to enhance affinity for D₂R, 5-HT_1A, and 5-HT_2A receptors. These studies resulted in the identification of new compounds 35 and 36, which displayed improved binding affinity at the D₂, and 5-HT_1A receptors (Figure ). Compound 35 also exhibited enhanced binding affinities at the 5-HT_2AR and compound 36 at the D₃ and 5-HT_7AR. Molecular docking studies were performed with 6, and 36 at the orthosteric pockets of D₂ and 5-HT_2A receptors and compared their interactions with lurasidone (4), a well-known atypical antipsychotic used to treat schizophrenia and several other mental disorders (Figures and and S1–S4). Compounds 35 and 36 were evaluated for their pharmacological properties alongside the standard antipsychotic drug lurasidone (4) in a mouse model of schizophrenia and a rat model of EPS side effects. The assessments involved the reversal of apomorphine-induced climbing behavior in mice (Figure ), and the catalepsy bar and CLP behavioral tests in rats (Figure ).

Structural modifications in the core structure of SYA16263 (6) for the present study.

Docked poses (left) and binding interactions (right) of lurasidone (4), 6, and 36 within the D₂ receptor orthosteric pocket. Similar to lurasidone, both ligands form a salt bridge with ASP114 and engage in π–π stacking with TRP386 interactions commonly associated with antagonist activity.

Docked poses (left) and binding interactions (right) of lurasidone (4), compounds 6 and 36 within the 5-HT_2A receptor orthosteric site. Lurasidone (4) forms the salt bridge with ASP155. The key interaction with TRP336, the toggle switch and π–π with PHE340, to block the conformational rearrangement within the receptor for antagonist activity. Compound 6 forms a salt bridge interaction with ASP155 and hydrophobic interaction with TRP336 accompanied by PHE339 and PHE340 while compound 36 shows a salt bridge and a hydrogen bond with ASP155 and π–π interaction with TRP336 and PHE340 residues which are critical for antagonist activity.

Compounds 35, 36, and lurasidone (4) inhibit Apo-induced climbing behavior in mice. N = 3–5 mice per dose. Error bars are SEM. One-way ANOVA with Dunnett’s post tests. *, **, ***, and **** indicate p < 0.05, 0.01, 0.001, and 0.0001 respectively compared with the vehicle group.

Compounds 35 and 36 did not produce catalepsy at the ED₅₀, 2ED₅₀ or 3ED₅₀ doses (A) and at the ED₅₀, or 3ED₅₀ doses (B) in the Bar and CLP Tests. N = 3–5 rats per dose. Error bars are SEM. One-way ANOVA indicated no statistically significant difference between vehicle and compound 35 or vehicle and compound 36 at the p < 0.05 level.

2. Chemistry

Compounds 6–9, 11, 12, 31 and 32 were previously reported. ,− Azaperone (10) is a commercially available compound. The syntheses of compounds 13–30, 33–36 discussed in the present study are depicted in the Schemes –. Our previously reported routine alkylation methods were employed in the preparation of some of the compounds. Briefly, compounds 13–21 were synthesized from commercially available hydroxy pyridines or substituted hydroxylpyridines, with alkylation using either 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in the presence of K₂CO₃, and acetonitrile as solvent to obtain the corresponding alkylating agents (i–v). These linker compounds (i–v) were then used to N-alkylate the appropriate heteroaryl piperazines in the presence of K₂CO₃/KI and acetonitrile to yield compounds, 13–21 (Schemes and ). Compounds, 22–27 were prepared by reacting quinolin-2-ol or 6-chloroquinolin-2-ol or 2-methylbenzo[d]thiazol-5-ol with 1-bromo-4-chlorobutane followed by N-alkylation with appropriate heteroaryl piperazines using the conditions mentioned above (Schemes and ). For compounds 28 and 29, 2-methylpyridine was initially treated with 1-bromo-3-chloropropane in the presence of LDA to afford 2-(4-chlorobutyl) pyridine. However, this intermediate upon treatment with base and pyridinyl-piperazine, did not produce the desired product, possibly due to the formation of an intramolecular pyridinyl-salt. Therefore, 4-(pyridin-2-yl)butan-1-ol was converted to the corresponding aldehyde using Dess–Martin periodinane and the obtained aldehyde (ix) was subjected to reductive amination with heteroaryl piperazine using sodium triacetoxy borohydride to yield compounds 28 and 29 (Scheme ). Compound 30 was synthesized by reacting picoline with 1-bromo-4-chlorobutane in the presence of LDA followed by N-alkylation with pyridinyl-piperazine (Scheme ). For 33 and 34, 4-chloro-4′-fluorobutyrophenone was subjected to the Grignard reaction using methyl magnesium bromide in anhydrous diethyl ether followed by reacting the obtained secondary alcohol intermediate (xi) with the appropriate aryl-piperazine (Scheme ). Compounds 35 and 36 were obtained by a Grignard reaction of 4-chloro-4′-fluorobutyrophenone and 4-fluorophenylmagnesium bromide in anhydrous THF followed by reacting the obtained tertiary alcohol of the alkyl halide intermediate (xii) with 1-(pyridin-2-yl)piperazine and 1-(2-methoxyphenyl)piperazine, respectively (Scheme ).

^a Reagents and conditions: (a) K₂CO₃, CH₃CN, reflux; (b) K₂CO₃, KI, Aryl piperazine, CH₃CN, reflux.

^a Reagents and conditions: (a) 4-Fluorophenylmagnesium bromide in THF, Anhyd. THF, 0 °C, 4 h; (b) Pyridyl piperazine or 1-(2-methoxyphenyl)piperazine, K₂CO₃, KI, CH₃CN, reflux, 24 h.

^a Reagents and conditions: (a) K₂CO₃, CH₃CN, 1-bromo-4-chlorobutane, reflux; (b) K₂CO₃, KI, Aryl piperazine, CH₃CN, reflux.

^a Reagents and conditions: (a) DMP, DCM, 0 °C, 3 h; (b) Aryl piperazine, NaBH(OAc)₃, MeOH, rt, 12 h.

^a Reagents and conditions: (a) LDA, THF, 1-bromo-4-chlorobutane, 0 °C to rt, 6 h; (b) Pyridyl piperazine, K₂CO₃, Toluene, reflux, 16 h.

^a Reagents and conditions: (a) CH₃MgBr in Et₂O, 0 °C to RT, 4 h; (b) Aryl piperazine, KI, CH₃CN, reflux, 12–24 h.

3. Results and Discussion

3.1. Structure–Affinity Relationship (SAfiR) Studies

SYA16263 (6) is 1-(4-(4-fluorophenyl)butyl)-4-(pyridin-2-yl)piperazine which has shown remarkable antipsychotic properties without catalepsy even at 19 times its ED₅₀ value. It has moderate binding affinity at D₂R (K _i = 124 nM), strong affinity at 5-HT_1AR (K _i = 1.1 nM) and moderate affinity at the 5-HT_2A and 5-HT_7A receptors (K _i = 50 nM and 90 nM respectively). However, it is unclear what properties in the drug are responsible for its desirable in vivo profile. Our previous study of the structure affinity relationship of the scaffold did not yield new insight and a clear understanding of the properties contributing to the profile. In the current study we have sought to identify improved binding profiles at the D₂-like (D₂, D₃ and D₄), and serotonin (5-HT_1A and 5-HT_2A) receptors which might result in improved efficacy without catalepsy.

In this SAfiR study, structural modifications of SYA16263 (6) commenced with the replacement of the pyridine ring by a pyrimidine ring (7) or a chloropyridine moiety (8), resulting in increased or decreased polarity of the respective analogs (Table ). However, neither modification led to improved binding affinity at the target receptors (Table ). Similarly, the introduction of a carbonyl group as in the butyrophenone antipsychotics and its replacement with sulfur and oxygen as displayed in 9–12 did not produce improvement in the binding affinity to the D₂-like receptors, except for 12, which exhibited an improvement at the D₄R (K _i = 1.8 nM) (Table ). Compounds 9–12 also failed to produce notable improvements at the 5-HT_1AR. In compound 13, substitution of the p-fluorophenyl group in 12 with a 3-pyridinyloxy moiety, and in its analog 14, replacement of the pyridinyl-piperazine with a pyrimidinyl-piperazine unit, led to diminished binding affinities at the D₂-like and 5-HT₁A receptors for 13 while 14 exhibited complete loss of binding at the D₂ and D₃ receptors and reduced affinity at the D₄ and 5-HT_1A receptors (K _i = 147 nM and 190 nM, respectively; Table ).

1. Binding Affinity Constants [Mean K _i ± SEM, nM (pKi ± SEM)] at Important Dopamine (DA) and Serotonin (5-HT) Receptors .

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Results from NIMH PDSP.

Numbers in parentheses refer to the reference.

MTprimary assay <50% (missed 50% threshold inhibition at 10 μM concentration). K _i values without the ± value are less than 20% SEM.

Several compounds (15–27) incorporating a fragment analogous to the aryloxyalkyl moiety of aripiprazole were synthesized and evaluated for binding at D₂-like and 5-HT₁A receptors. None of these analogs demonstrated improved affinity at either receptor class. This series included derivatives of compounds 13 and 14 bearing an additional methylene unit in the alkyl chain (15–21) and various substitutions on the pyridinyloxy moiety (19–21). However, these structural modifications did not yield any enhancement in binding affinity at the targeted receptors.

Next, replacing the 2-pyridinyloxy of 17 and 18 with a 2-quinolinoxy group (22 and 23) or a 6-chloro substituted 2-quinolinoxy group (24 and 25) also did not produce any significant impact on the binding profiles at the D₂-like and 5-HT_1A receptors. Compounds 26 and 27 with the introduction of 2-methyl-1,3-benzothiazol-5-oxy group in place of 2-quinolinoxy group of 22 and 23 showed no improvement in binding affinities as well. Analogs of compounds 13 and 14 with a butyl linkage without an ether functionality in the alkyl region (28 and 29) also failed to enhance the binding affinity to the D₂-like and 5-HT_1A receptors. Extending the butyl linkage in 28 to a pentyl linkage as in 30 and substitution on the phenyl ring to form 31 and 32 all failed to significantly improve binding affinity to the D₂-like and 5-HT_1A receptors.

At this point, we returned to modification of the butyrophenone moiety in compound 10 by reducing the carbonyl group to the alcohol (33), a modification which resulted in the introduction of a chiral center, which did improve the binding somewhat at the D₂ and D₃ receptors (K _i = 166 nM and 50.2 nM respectively, Table ), while similar binding was observed at the D₄ and 5-HT_1A receptors (K _i = 7.27 nM and 8.83 nM respectively, Table ). The pyrimidine analog 34, however, showed reduced binding at the D₂ and 5-HT_1A receptors compared to 33 (K _i = 279 nM and 43.1 nM respectively, Table ).

While compound 33 exhibited an improved binding profile, its chiral center necessitated enantiomeric separation and independent evaluation of each isomer’s receptor affinities. To streamline the design and eliminate this stereochemical complication, a bis-fluorophenyl butanol moiety was introduced, yielding compound 35. Remarkably, compound 35 represented a pivotal breakthrough in the SAfiR series, demonstrating a striking enhancement in binding affinity not only across the D₂-like receptors (D₂ K _i = 23.2 nM, D₃ K _i = 41.9 nM, D₄ K _i = 32.1 nM) but also exhibiting subnanomolar and low-nanomolar affinities at the 5-HT_1A (K _i = 0.67 nM) and 5-HT₂A (K _i = 11.4 nM) receptors respectively (Table ). Next, substitution of the pyridyl-piperazine moiety in compound 35 with a 1-(2-methoxyphenyl)piperazine group produced compound 36, resulting in a marked increase in D₂R binding affinity (K _i = 1.24 nM), representing 100-fold and ∼19-fold improvements relative to SYA16263 (6) and compound 35 respectively. Further, compound 36 displayed subnanomolar and low nanomolar affinities at the 5-HT_1AR and D₃R (5-HT_1A K _i = 0.67 nM, D₃ K _i = 5.78 nM). Although compound 36 exhibited an approximately 3-fold reduction in binding affinity at the 5-HT_2AR (K _i = 144.2 nM) relative to SYA16263 (6), it showed a notable improvement in affinity at the 5-HT_7AR (K _i = 19.9 nM) (Table ).

3.2. Comparison of Structure–Affinity Relationship (SAfiR) among D₂-Like and 5-HT Receptor Subtypes

DA and 5-HT receptor subtypes often share highly conserved binding pockets, and slight differences in amino acid sequences, particularly in extracellular loops allow for divergent SAfiR trends. Thus, minor structural modifications such as chain length, connecting atom, substitutions on the aromatic rings can dramatically increase affinity for one subtype while having negligible or inverse effects on another. Therefore, structure-affinity relationship (SAfiR) comparisons within receptor subtypes are essential for establishing the selectivity at the important DA (D₂, D₃ and D₄) and 5-HT (5-HT_1A, 5-HT_2A, 5-HT_2c, 5-HT_7A) receptors. Compound 6 displayed strong selectivity within D₂-like receptor subtypes, exhibiting the highest affinity for D₄R (K _i = 3.5 nM) compared to D₂R (K _i = 124 nM), and D₃R (K _i = 86 nM). Among 5-HT receptors, it showed preferential binding to 5-HT_1AR (K _i = 1.1 nM) over 5-HT_2A (K _i = 50 nM) and 5-HT_7A receptors (K _i = 90 nM). Compound 7, the pyrimidine analog of 6, retained a similar selectivity profile but with reduced overall affinity at D₂-like receptor subtypes (D₂ K _i = 269 nM, D₃ K _i = 262 nM, D₄ K _i = 6.2 nM) compared to 6 (Table ). For 5-HT receptors, 7 showed higher binding affinity at 5-HT_1A and optimal affinity at 5-HT_2A with significant reduction in binding affinity at 5-HT_7A. Both compounds exhibited negligible interaction with 5-HT_2C (Primary Assay <50% threshold inhibition at 10 μM concentration). Introduction of 5-chloro substituent on pyridine ring of 6 (Compound 8) preserved D₄ receptor selectivity (K _i = 4.8 nM) over D₂ and D₃ receptors (D₂ K _i = 193 nM, D₃ K _i = 83 nM). Interestingly, compound 8 showed enhanced affinity at 5-HT_2A (K _i = 9.2 nM) as compared to 5-HT_1A, 5-HT_2C, and 5-HT_7A (K _i = 75 nM, 1024 nM and 159 nM respectively). The 4′-fluorobutyrophenone analog of 8 (Compound 9) maintained a similar D₄ preference with significant affinity (K _i = 5.1 nM) compared to D₂, and D₃ receptors (D₂ K _i = 287 nM, D₃ K _i = 86 nM) while exhibiting moderate binding at 5-HT_1A, and 5-HT_2A (K _i = 34 nM and 46 nM respectively) with lower affinity at 5-HT_2C and 5-HT_7A receptors (K _i = 207 nM and 1540 nM respectively). Compounds 10–12 consistently demonstrated high binding profiles at D₂-like receptor subtypes with significant affinity at D₄ (10, K _i = 6.7 nM; 11, K _i = 5.7 nM; and 12, K _i = 1.8 nM respectively) over D₂ and D₃ receptors (K _i in the range of 89–229 nM). Similarly, compounds 10–12 showed improved binding at 5-HT_1A (10, K _i = 8.1 nM; 11, K _i = 6.2 nM; and 12, K _i = 3.6 nM respectively) and moderate affinity for 5-HT_2A (10, K _i = 62.6 nM; 11, K _i = 26 nM; and 12, K _i = 18 nM respectively). Affinity for 5-HT_7A receptors was modest with K _i in the range of 120–291 nM, and 5-HT_2C binding remained minimal (K _i = 1580–8097 nM). The pyridinyl-piperazine (13) and pyrimidinyl-piperazine (14) analogs linked with a three methylene spacer to the 3-pyridinyloxy moiety exhibited preferential binding affinity at the D₄ receptor (13, K _i = 124 nM; 14, K _i = 147 nM respectively) compared to D₂ and D₃ receptors (13, D₂ K _i = 4500 nM; D₃ K _i = 4500; 14, D₂ and D₃ K _i = MT respectively). Compounds 15 and 16, which are four methylene spacer analogs of 13 and 14, displayed similar binding affinity toward D₄ receptor (15, K _i = 369 nM; 16, K _i = 125 nM respectively) over D₂ and D₃ receptors (15, D₂ and D₃ K _i = MT; 16, D₂ K _i = MT; and D₃ K _i = 766 nM respectively). For 5-HT receptors, compounds 13–16 exhibited preference to 5-HT_1A receptors over 5-HT_7A and 5-HT_2A receptors and minimal binding at the 5-HT_2C receptor (Table ). The 2-pyridinyloxy analogs of compounds 15 and 16, also demonstrated better binding affinity for the D₄ receptor (17, K _i = 32 nM; 18, K _i = 74 nM respectively) compared to D₂ and D₃ receptors (17, D₂ K _i = 1080 nM; D₃ K _i = 519; 18, D₂ K _i = 2410; and D₃ K _i = 1030 nM respectively). These compounds also maintained preferential binding to 5-HT_1A receptors (17, K _i = 7.6 nM; and 18, K _i = 30 nM) over 5-HT_7A, 5-HT_2A and 5-HT_2C receptors (Table ). Compound 19, a 5-chloro-2-pyridinyloxy analog of 17, showed minimal and low binding affinities at the D₂ and D₃ receptors (D₂ K _i = MT; D₃ K _i = 3010 nM, respectively) compared to the D₄ receptor (K _i = 55.3 nM), while it showed comparable affinity at 5-HT_1A and 5-HT_7A receptors (K _i = 16.3 nM; and K _i = 13.4 nM, respectively) and minimal affinity at the 5-HT_2A, and 5-HT_2C receptors. Replacing the 5-chloro group in 19 with a methyl ester (20) lost binding affinity at the D₄ receptor in addition to the D₂ and D₃ receptors (D₄ K _i = 2092 nM; D₂ K _i = MT, and D₃ K _i = > 10, 000 nM, respectively). However, it showed significant affinity at the 5-HT_1A receptor (K _i = 23.6 nM) over other 5-HT receptors (5-HT_7A K _i = 479 nM; 5-HT_2A and 5-HT_2C K _i = MT, respectively). The pyrimidinyl-piperazine analog (21) of compound 20 exhibited a shift in the binding preference toward the D₂ receptor (K _i = 3560 nM) with minimal affinity at the D₂ and D₄ receptors (D₂ and D₄ K _i = MT). Compound 21 showed better affinity at the 5-HT_1A receptor (K _i = 139 nM) over the 5-HT_7A (K _i = 1270 nM) and other 5-HT receptors (5-HT_2A and 5-HT_2C K _i = MT). Compounds 22 and 23, which are 2-quinolinoxy analogs of 17 and 18 regained their preferential binding toward the D₄ receptor (22, K _i = 111 nM; 23, K _i = 198 nM) over the D₂ and D₃ receptors (22, D₂ K _i = MT; D₃ K _i = 2550 nM; 23, D₂ K _i = MT; D₃ K _i = 2860 nM). These compounds also demonstrated significant affinity at 5-HT_1A (22, K _i = 11 nM; 23, K _i = 59 nM) and 5-HT_7A receptors (22, K _i = 8.5 nM; 23, K _i = 34 nM) compared to 5-HT_2A (22, K _i = 197 nM; 23, K _i = 951 nM) and 5-HT_2C (22 and 23 K _i = MT). 6-Chloro substituted 2-quinolinoxy analogs (24 and 25) showed improved affinity at D₄ (24, K _i = 76 nM; 25, K _i = 97.7 nM, respectively) over D₂ and D₃ receptors. While compound 24 showed preferential binding toward 5-HT_1A (K _i = 10 nM) and 5-HT_7A receptors (K _i = 68 nM) over 5-HT_2A (K _i = 249 nM) and 5-HT_2C (K _i = 3050 nM), compound 25 exhibited binding affinity only at 5-HT_1A (K _i = 58.4 nM) with minimal binding at the remaining 5-HT receptors. The 2-methyl-1,3-benzothiazol-5-oxy analogs (26 and 27) and 2-pyridinyl group with butyl chain linked to pyridinyl-piperazine (28) and pyrimidinyl-piperazine (29) also demonstrated significant binding affinity at the D₄ receptor (26, K _i = 43.5 nM; 27, K _i = 30.4 nM; 28, K _i = 31.3 nM; 29, K _i = 33.8 nM) compared to the D₂ and D₃ receptors (Table ). These compounds showed higher binding affinity at 5-HT_2A (26, K _i = 81.8 nM; 27, K _i = 85.4 nM; 28, K _i = 5.39 nM; 29, K _i = 28.4 nM) compared to other 5-HT receptors. Compound 30, an analog of 28 with a pentyl chain in the alkyl region showed comparable affinity at the D₂, D₃ and D₄ receptors (D₂ K _i = 2820 nM; D₃, K _i = 3620 nM; and D₄, K _i = 1570 nM) with a slight preference toward the D₄ receptor. It also showed preference at the 5-HT_1A receptor (K _i = 20.4 nM) followed by the 5-HT_7A receptor (K _i = 67.3 nM). An analog of 29 with a p-amino substituted phenyl ring (31) showed affinity at the D₃ (K _i = 391 nM), D₂ (K _i = 674 nM), and D₄ (K _i = 1872 nM) receptors. At the 5-HT receptors, it showed preference at 5-HT_1A (K _i = 13 nM) over other 5-HT receptors. Similar to 31, an amide analog 32 also showed a similar trend with preferential affinity at the D₃ and 5-HT_1A receptors over D₂, D₄ receptors and other 5-HT receptors. Compound 33, with the introduction of an alcohol and methyl groups at the carbonyl group of compound 10, demonstrated significant improvement in binding affinity at the D₄ receptor (K _i = 7.27 nM) and at the D₃ receptor (K _i = 50.2 nM) compared to the D₂ receptor (K _i = 166 nM). It also showed a better affinity at 5-HT_1A receptor (K _i = 8.83 nM) over other 5-HT receptors. The pyrimidine analog 34 also exhibited preferential binding at the D₄ (K _i = 16 nM) and 5-HT_1A receptors (K _i = 43.1 nM) compared to other D₂-like and 5-HT receptors. The bis-fluorophenyl butanol analog 35 showed comparable binding affinities at the D₂-like receptors (D₂ K _i = 23.2 nM; D₃ K _i = 41.9 nM; D₄ K _i = 32.1 nM, respectively) while there was a significant enhancement observed at the 5-HT_1A receptor (K _i = 0.67 nM) compared to the 5-HT_2A, 5-HT_2C, and 5-HT_7A receptors (K _i = 11.4 nM, 696 nM, and 108 nM, respectively). Compound 36, the 1-(2-methoxyphenyl)piperazine analog of 35 showed a drastic shift in preference among D₂-like receptors with significant binding affinity at the D₂ receptor (K _i = 1.24 nM) compared to the D₃ and D₄ receptors (D₃ K _i = 5.78 nM; D₄ K _i = 39.4 nM) while maintaining its binding affinity at 5-HT_1A receptor (K _i = 0.67 nM) and improved affinity at the 5-HT_7A receptor over the remaining 5-HT receptors (Table ).

3.3. Molecular Docking Studies

Induced fit docking (IFD) studies were conducted to evaluate the predicted binding affinities of lurasidone (4) and compounds 6 and 36 at both the dopamine D₂ receptor (PDB: 6CM4) and the 5-HT₂A receptor (PDB: 8UWL and 6A94). Unlike rigid docking approaches, IFD accounts for the flexibility of the protein binding site, allowing key residues to adjust in response to ligand binding. This provides a more realistic representation of how these interactions may occur under physiological conditions. Results from the IFD studies showed that lurasidone (4) forms a salt bridge with ASP 114 within the orthosteric pocket of the D₂ receptor and a π–π stacking interaction with TRP386 was also noted with IFD Score: −17735.52 (Figure ). These interactions are generally observed in compounds with antagonist activity at the D₂ receptor. Compounds 6 and 36 likewise exhibited similar binding interactions, including formation of a salt bridge with ASP114 and π–π stacking with TRP386 (IFD Score: −15886.62 and −17739.05 for 6 and 36 respectively) suggesting potential D₂ receptor antagonist activity (Figure ).

When validating antagonist activity, it is critical to compare ligand interaction within the appropriate protein with the desired conformational binding site. In many aminergic GPCRs, hydrogen bonding with serine residues is often associated with agonist activity, as these interactions help stabilize the active conformation. Initially, lurasidone (4) was docked into the 5-HT_2A receptor inactive (PDB: 6A94) and active (PDB: 8UWL) binding sites to observe the binding modes at these sites (Figures S1–S4). Results showed a clear distinction in both binding orientation and residue interactions of lurasidone at the active and inactive sites. Within the active state structure, lurasidone (4) was positioned further away from the transmembrane 6 (TM6) and tightly knit into the transmembrane helix 2 and transmembrane helix 3 (TM2 and TM3). Such interactions allow the TM6 to undergo conformational changes needed to activate the Gα_q/11, activating phospholipase C (PLC) to increase intracellular calcium and produce inositol trisphosphate and diacylglycerol. However, lurasidone (4) in the inactive state structure is closer to the TM6 accompanied by a π–π interaction with TRP336the “toggle switch”. This slight shift in placement and residue interaction at the inactive site stabilizes the conformational rearrangement required for antagonistic activity. These structural insights are essential when evaluating compounds 6 and 36 and helps to appropriately classify them. Using lurasidone (4) as a reference antagonist, compounds, 6 and 36 were docked at the 5-HT_2A receptor orthosteric binding site. Results showed that lurasidone forms a key salt bridge with ASP155 along with other key interactions involving TRP336 the toggle switch and π–π with PHE340 (IFD Score: −15886.62, Figure ) demonstrating that the stabilization of the inactive receptor state is associated with interactions near TM6 and π–π stacking with toggle switch residues. Compound 6 showed a salt bridge interaction with ASP155 and a hydrophobic interaction with TRP336 accompanied by PHE339 and PHE340 (IFD Score: −15929.54) while compound 36 displayed a salt bridge and a hydrogen bond with ASP155. It also demonstrated an important π–π interaction with TRP336 and PHE340 residues which are critical for antagonist activity (IFD Score: −15924.48, Figure ). Overall, these docking studies provide preliminary structural insights into ligand–receptor interactions of compounds 6 and 36 at the D₂ and 5-HT_2A receptors. Compound 36, in particular, exhibited antagonist-like binding interactions at both receptor systems. At the D₂ receptor, it maintained key interactions such as the salt bridge with ASP114 and π–π stacking with TRP386, comparable to lurasidone. Similarly, within the 5-HT_2A receptor, compound 36 preserved critical interactions with ASP155 and TRP336, supporting its role in stabilizing the inactive receptor conformation.

It should be noted that compound 35 was excluded from the molecular docking studies because compound 36 emerged as the more advanced lead compound, exhibiting substantially higher D₂ receptor affinity (K _i = 1.24 nM vs 23.2 nM for 35), improved D₃ and 5-HT₇ receptor affinities, and superior in vivo antipsychotic-like potency (ED₅₀ = 0.632 mg/kg vs 5.91 mg/kg for 35). Consequently, docking analyses were focused on compound 36 and the parent lead SYA16263 (6) to provide mechanistic insight into the structural basis for the marked improvement in receptor binding and pharmacological activity observed during lead optimization.

3.4. Animal Behavioral Studies

Having identified compounds that met the desired binding profile and having demonstrated in silico their similarity to the standard drug lurasidone, we next sought to characterize their pharmacological properties in vivo using animal models of schizophrenia. Accordingly, compounds 35 and 36 were evaluated alongside the standard antipsychotic lurasidone (4) in the reversal of apomorphine-induced climbing behavior in male albino Swiss-Webster mice. This assessment was subsequently followed by studies in male Sprague–Dawley rats to determine the compound’s potential to induce catalepsy.

3.4.1. Evaluation of 35, 36, and Lurasidone (4) in an In Vivo Mouse Model of Schizophrenia: Reversal of Apomorphine-Induced Climbing

The apomorphine-induced climbing paradigm remains a cornerstone behavioral assay for evaluating antipsychotic activity, grounded in the dopaminergic hypothesis of schizophrenia. Apomorphine, a potent nonselective dopamine receptor agonist, triggers characteristic climbing behavior in mice through activation of D₂-like receptors (D₂, D₃, and D₄), with minor contribution from D₁-like subtypes (D₁ and D₅). At doses ≥0.5 mg/kg, stimulation of postsynaptic D₂-like receptors elicits hyperlocomotion and stereotyped climbing, reflecting dopaminergic hyperactivity analogous to the positive symptoms of schizophrenia. Compounds possessing D₂R antagonist properties effectively suppress this behavior, serving as a robust preclinical indicator of potential antipsychotic efficacy. ,

Accordingly, compounds 35, 36 and the reference antipsychotic lurasidone (4) were assessed in the apomorphine-induced climbing assay to probe their ability to attenuate dopaminergic hyperactivity. , Compounds 35 and 36 produced clear, dose-dependent inhibition of climbing behavior, with increasing doses yielding greater suppression (one-way ANOVA for compound 35: [F(7,31) = 25.37; p < 0.0001] and one-way ANOVA for compound 36 [F(7,31) = 28.89; p < 0.0001]). Lurasidone exhibited a similar antagonistic profile [F(7,29) = 7.221; p < 0.0001]. The calculated ED₅₀ values were 5.91 mg/kg (14.0 μmol/kg) for 35, 0.632 mg/kg (1.39 μmol/kg) for compound 36, and 1.59 mg/kg (3.01 μmol/kg) for lurasidone (Figure ), confirming that 35 and 36 possess in vivo antipsychotic-like activity, with compound 36 having the lowest ED₅₀ value among all three compounds. Compound 36 exhibited ED₅₀ values more than 8-fold and 2-fold lower than those of SYA16263 (6; ED₅₀ = 3.88 mg/kg or 12.4 μmol/kg) and lurasidone (ED₅₀ = 1.59 mg/kg or 3.01 μmol/kg), respectively.

Lurasidone (4) is known to display high affinity for D₂-like receptors (D₂ K _i = 1.2–1.7 nM; D₃ K _i = 15.7 nM; D₄ K _i = 30 nM) and for serotonergic sites (5-HT_1A K _i = 6.7 nM; 5-HT_2A K _i = 0.5–2 nM; 5-HT_7A K _i = 0.5 nM). Remarkably, compound 35 exhibited a closely aligned binding profile across the D₂-like, 5-HT_1A, and 5-HT_2A receptors, with slightly lower affinities overall but notably stronger interaction at 5-HT_1AR (Table ). Interestingly, compound 36 displayed an even better binding profile than compound 35 at the D₂-like, 5-HT_1A, and 5-HT_7A receptors (Table ). This serotonergic enhancement, coupled with dopaminergic antagonism, suggests that 35 and 36 may reproduce the therapeutic spectrum of lurasidone (4) while potentially offering an improved side-effect profile. Together, these findings establish 35 and 36 as promising multifunctional ligands, capable of addressing the dopaminergic dysregulation and serotonergic modulation implicated in schizophrenia pathophysiology.

3.4.2. Evaluation of 35 and 36 for Induction of Catalepsy in Rats

Catalepsy, characterized by rigid immobility, is a well-established behavioral correlate of extrapyramidal side effects observed with antipsychotic drugs in humans. First-generation antipsychotics (FGAs) such as haloperidol reliably induce catalepsy in rodents, paralleling clinical manifestations of dystonia, akathisia, and Parkinsonism. These motor disturbances arise primarily from excessive D₂R blockade accompanying the therapeutic antipsychotic effect. In contrast, atypical or aripiprazole-like agents produce little or no catalepsy, likely due to partial agonism at D₂ receptors that preserves dopaminergic tone. ,,− Accordingly, the catalepsy model remains a key preclinical tool for assessing motor liability and identifying novel antipsychotic candidates with reduced EPS potential.

In this regard, compounds 35 and 36 were evaluated for cataleptogenic potential in rats using the bar and crossed-leg position (CLP) tests (Figure ) at doses corresponding to the ED₅₀, 2 × ED₅₀, and 3 × ED₅₀ for 35 and at ED₅₀ and 3 × ED₅₀ for 36 respectively, determined from the apomorphine-induced climbing assay (Figure ). No significant cataleptic response was observed in either test compared with vehicle controls as confirmed by one-way ANOVA (p > 0.05). All animals successfully righted themselves during the righting reflex test, indicating the absence of marked impairment. The maximum cutoff time for the catalepsy tests was 30 s, as indicated on the Y-axis (Figure ). Even at the highest tested dose (3 × ED₅₀), compounds 35 and 36 failed to induce catalepsy, suggesting a minimal propensity to elicit extrapyramidal side effects. This absence of EPS liability represents a notable pharmacological advantage for compound 35 and 36, as motor side effects remain a major limitation in the clinical management of schizophrenia.

4. Materials and Methods

4.1. Chemical Syntheses

4.1.1. Materials and Methods for Synthesis and Characterization

All starting materials were purchased from Sigma-Aldrich and utilized without further purification, unless otherwise indicated. Air and/or moisture sensitive reactions were carried out in anhydrous solvents under an atmosphere of argon/nitrogen in an oven or flame-dried glassware. Anhydrous solvents used for the reactions were distilled prior to use: THF and diethyl ether (from Na and benzophenone), toluene, dichloromethane, DMF. Commercial reagents obtained from Sigma-Aldrich and other vendors were used without purification. Flash chromatography was performed using a Biotage Isolera flash chromatographic system with Davisil grade 634 silica gel. All NMR spectra were obtained on a Varian 300 MHz Mercury Spectrometer or 600 MHz Bruker Avance III HD Spectrometer and the free induction decay (FID) data were processed using Mestrelab’s Mnova NMR software (version 8.1) to obtain the reported NMR data. For NMR data, ¹H and ¹³C NMR chemical shifts are reported in ppm downfield from tetramethylsilane (TMS) and coupling constants (J) are reported in hertz (Hz). The following abbreviations were used to designate signal multiplicity: s = singlet, d = doublet, dd = doublet of doublets, dtd = doublet of triplet of doublets, m = multiplet. ¹H and ¹³C NMR data obtained for previously reported intermediates are consistent with our reported NMR spectral data of the intermediates. Melting points of the synthesized compounds were determined on a Gallenkamp (UK) apparatus and are included as uncorrected. Elemental analyses of the final compounds (free base forms, hydrochloride and oxalate salts) for C, H and N were carried out by Atlantic Microlab, Inc., Norcross, GA, and the results were found to be within 0.4% of theoretical values as calculated using ChemDraw Professional software unless otherwise noted.

4.1.2. Synthesis of Compounds 13–30, and 33–36

4.1.2.1. General O-Alkylation Procedure A

To a stirred solution of appropriate hydroxy-compound (10 mmol, 1 equiv) and K₂CO₃ (20 mmol, 2 equiv) in CH₃CN (10 mL), appropriate alkyl halide (15 mmol, 1.5 equiv) was added at room temperature and refluxed for 12–24 h. The reaction progress was monitored by TLC, and at completion, the mixture was cooled to rt, the solvent removed, and the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane to give the pure desired products. The yields for O-alkylation intermediates were in the range of 65–75% and were not optimized.

4.1.2.2. General N-Alkylation Procedure B

A mixture of the appropriate alkylating agent (1.2 mmol), the appropriate amine (1.0 mmol), KI (100 mg), and K₂CO₃ (2.4 mmol) in CH₃CN or toluene (5 mL) was heated to reflux for 12–24 h. The reaction progress was monitored by TLC, and at completion, the mixture was cooled to rt, solvent removed, the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane/MeOH to give the pure desired products. The yields for the N-alkylation compounds were in the range of 45–80% and were not optimized.

The hydrochloride salts were prepared by dissolving each compound in an appropriate solvent, adding excess anhydrous ethereal HCl solution, removing solvent under reduced pressure, and recrystallizing the residue from an appropriate solvent. For the oxalate salt (29), the free base was dissolved in ethyl acetate and 4 equiv of oxalic acid was added and stirred for 30 min. Excess oxalic acid was removed by washing with ether, followed by recrystallization in MeOH–Et₂O.

4.1.2.3. 1-(Pyridin-2-yl)-4-(3-(pyridin-3-yloxy)propyl)piperazine Hydrochloride (13)

The alkylating group (i) was prepared following general O-alkylation procedure A. Pyridin-3-ol was treated with 1-bromo-3-chloropropane in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 8.09 (s, 1H), 7.98 (s, 1H), 6.95 (d, J = 1.0 Hz, 2H), 4.04–3.71 (m, 2H), 3.65–3.32 (m, 2H), 2.17–1.76 (m, 2H).

¹³C NMR (75 MHz, CDCl₃): δ 154.6, 142, 123.8, 137.9, 120.6, 64.3, 41.1, 31.8.

The N-alkylation reaction was carried out according to the general procedure B. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 13. Yield: 94%.

Mp: 210–215 °C.

¹H NMR (600 MHz, DMSO): δ 8.67 (d, J = 2.6 Hz, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.25–8.14 (m, 1H), 8.12–8.05 (m, 1H), 8.05–7.90 (m, 2H), 7.42 (d, J = 9.1 Hz, 1H), 7.01 (t, J = 6.5 Hz, 1H), 4.60 (s, 2H), 4.38 (t, J = 6.0 Hz, 2H), 3.70 (d, 4H), 3.41–3.31 (m, 2H), 3.28 (s, 2H), 2.42–2.24 (m, 2H).

¹³C NMR (151 MHz, DMSO): δ 1570, 153.4, 143.6, 139.7, 135.1, 131.6, 130.2, 128.2, 114.4, 112.5, 67.4, 52.9 (2C), 50.4 (2C), 43.7, 23.3.

Calcd for C₁₇H₂₂N₄O·3HCl + 0.45 H₂O: C, 49.10; H, 6.28; N, 13.47. Found: C, 49.10; H, 6.29; N, 13.41.

4.1.2.4. 2-(4-(3-(Pyridin-3-yloxy)propyl)piperazin-1-yl)pyrimidine Hydrochloride (14)

Mp: 203–207 °C.

¹H NMR (600 MHz, DMSO): δ 8.70 (d, J = 2.4 Hz, 1H), 8.54 (t, J = 8.5 Hz, 1H), 8.49 (d, J = 4.9 Hz, 2H), 8.31–8.17 (m, 1H), 8.02 (dd, J = 8.8, 5.5 Hz, 1H), 6.95–6.72 (m, 1H), 4.70 (d, J = 14.2 Hz, 2H), 4.39 (t, J = 6.0 Hz, 2H), 3.58 (dd, J = 27.2, 13.2 Hz, 4H), 3.29 (s, 2H), 3.12 (s, 2H), 2.41–2.21 (m, 2H).

¹³C NMR (151 MHz, DMSO): δ 159.6, 158.4 (2C), 157.2, 134.4, 132.6, 129.4, 128.5, 111.6, 67.6, 53.0, 50.8 (2C), 41.0 (2C), 23.2.

Calcd for C₁₆H₂₁N₅O·3HCl + H₂O: C, 45.03; H, 6.14; N, 16.41. Found: C, 45.02; H, 6.09; N, 16.23.

4.1.2.5. 1-(Pyridin-2-yl)-4-(4-(pyridin-3-yloxy)butyl)piperazine Hydrochloride (15)

The alkylating group (ii) was prepared following general O-alkylation procedure A. Pyridin-3-ol was treated with 1-bromo-4-chlorobutane in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 8.09 (s, 1H), 7.98 (s, 1H), 6.95 (d, J = 1.0 Hz, 2H), 4.04–3.71 (m, 2H), 3.65–3.32 (m, 2H), 2.17–1.76 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 154.6, 142.0, 123.8, 137.9, 120.6, 64.3, 41.1, 31.8, 27.4.

Mp: 232–235 °C.

¹H NMR (600 MHz, DMSO): δ 8.69 (d, J = 2.8 Hz, 1H), 8.58–8.45 (m, 1H), 8.27–8.09 (m, 2H), 8.03–7.87 (m, 2H), 7.33 (d, J = 8.9 Hz, 1H), 6.98 (t, J = 6.4 Hz, 1H), 4.53 (d, J = 13.6 Hz, 2H), 4.26 (t, J = 6.1 Hz, 2H), 3.83–3.47 (m, 4H), 3.23–3.11 (m, 4H), 2.02–1.70 (m, 4H).

¹³C NMR (151 MHz, DMSO): δ 157.1, 154.3, 142.7, 141.1, 135.5, 131.1, 130.7, 128.0, 114.4, 111.8, 69.3, 55.5, 50.4 (2C), 43.3 (2C), 26.0, 20.2.

Calcd for C₁₈H₂₄N₄O·3HCl + 0.65H₂O: C, 49.87; H, 6.58; N, 12.92; O, 6.09. Found: C, 49.85; H, 6.57; N, 12.69.

4.1.2.6. 2-(4-(4-(Pyridin-3-yloxy)butyl)piperazin-1-yl)pyrimidine Hydrochloride (16)

The reaction was carried out according to the general procedure B. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 16. Yield: 60%.

Mp: 222–226 °C.

¹H NMR (600 MHz, DMSO): δ 8.71 (s, 1H), 8.53 (d, J = 4.3 Hz, 1H), 8.45 (d, J = 4.5 Hz, 2H), 8.22 (d, J = 7.4 Hz, 1H), 8.01 (s, 1H), 6.77 (t, J = 4.4 Hz, 1H), 4.68 (d, J = 13.4 Hz, 2H), 4.27 (s, 2H), 3.52 (dd, J = 29.4, 13.1 Hz, 4H), 3.16 (s, 2H), 3.02 (d, J = 10.0 Hz, 2H), 1.94 (s, 2H), 1.84 (s, 2H).

¹³C NMR (151 MHz, DMSO): δ 160.9, 158.69 (2C), 157.39, 134.7, 132.1, 129.8, 128.4, 111.7, 69.5, 55.5, 50.8 (2C), 40.7 (2C), 26.0, 20.2.

Calcd for C₁₇H₂₃N₅O·3HCl: C, 48.30; H, 6.20; N, 16.57. Found: C, 48.27; H, 6.36; N, 16.38.

4.1.2.7. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yloxy)butyl)piperazine Hydrochloride (17)

The alkylating group (iii) was prepared following general O-alkylation procedure A. Pyridin-2-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 8.10–7.96 (m, 1H), 7.52–7.34 (m, 1H), 6.86–6.53 (m, 2H), 4.24 (tt, J = 12.9, 4.5 Hz, 2H), 3.63–3.39 (m, 2H), 1.98–1.51 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 163.7, 146.8, 138.4, 116.6, 111.0, 64.7, 44.7, 29.3, 26.4.

Mp: 147–151 °C.

¹H NMR (600 MHz, MeOD): δ 8.49 (t, J = 8.1 Hz, 1H), 8.40 (d, J = 5.9 Hz, 1H), 8.20 (dd, J = 8.8, 7.5 Hz, 1H), 8.11 (d, J = 6.2 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.58 – 7.49 (m, 2H), 7.19 (t, J = 6.6 Hz, 1H), 4.59 (t, J = 5.7 Hz, 2H), 4.48 (s, 2H), 3.85 (s, 4H), 3.50 – 3.37 (m, 4H), 2.18 – 2.02 (m, 4H).

¹³C NMR (151 MHz, MeOD): δ 160.58, 152.24, 148.25, 145.13, 139.24, 136.63, 118.36, 114.42, 113.25, 111.30, 70.38, 56.12, 50.18 (2C), 43.27 (2C), 25.27, 20.17.

Calcd for C₁₈H₂₄N₄O·3HCl + 1.8H₂O: C, 47.60; H, 6.79; N, 12.34. Found: C, 47.68; H, 6.81; N, 12.27.

4.1.2.8. 2-(4-(4-(Pyridin-2-yloxy)butyl)piperazin-1-yl)pyrimidine (18)

The N-alkylation reaction was carried out according to the general procedure B. The free base product was then recrystallized from ethyl acetate to give the pure compound 18. Yield: 16%

Mp: 56–59 °C.

¹H NMR (300 MHz, CDCl₃): δ 8.35–8.14 (m, 2H), 8.11–7.93 (m, 1H), 7.68–7.34 (m, 1H), 6.89–6.51 (m, 2H), 6.46–6.22 (m, 1H), 4.23 (dt, J = 24.9, 6.4 Hz, 2H), 3.86–3.58 (m, 4H), 2.48–2.32 (m, 6H), 1.88–1.47 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 163.9, 161.6, 157.6 (2C), 146.8, 138.4, 116.5, 111.0, 109.7, 65.5, 58.3, 53.1 (2C), 43.6 (2C), 27.1, 23.4.

Calcd for C₁₇H₂₃N₅O + 0.5H₂O: C, 63.33; H, 7.50; N, 21.72. Found: C, 63.31; H, 7.48; N, 21.48.

4.1.2.9. 1-(4-((5-Chloropyridin-2-yl)oxy)butyl)-4-(pyridin-2-yl)piperazine Hydrochloride (19)

The alkylating group (iv) was prepared following general O-alkylation procedure A. 5-Chloro pyridin-2-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 7.48–7.08 (m, 2H), 6.68–6.34 (m, 1H), 4.00–3.83 (m, 2H), 3.64–3.45 (m, 2H), 1.96–1.67 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 161.0, 140.3, 134.8, 121.9, 112.3, 76.6, 44.2, 29.3, 26.6.

Mp: 136–141 °C.

¹H NMR (600 MHz, DMSO): δ 8.18–8.06 (m, 1H), 8.07–7.96 (m, 2H), 7.47 (dt, 1H), 7.37 (d, J = 9.0 Hz, 1H), 7.00 (t, J = 6.4 Hz, 1H), 6.43 (d, J = 9.7 Hz, 1H), 4.51 (d, J = 13.2 Hz, 2H), 4.05–3.97 (m, 4H), 3.95–3.88 (m, 2H), 3.25 (s, 4H), 3.15 (dd, J = 13.3, 5.9 Hz, 2H), 1.84–1.57 (m, 2H).

¹³C NMR (151 MHz, DMSO): δ 160.5, 153.6, 143.5, 140.9, 137.3, 121.2, 114.4, 114.2, 112.4, 110.9, 50.3, 43.5 (2C), 43.4, 42.3 (2C), 26.3, 20.6.

Calcd for C₁₈H₂₃ClN₄O·3HCl + H₂O: C, 45.59; H, 5.95; N, 11.81. Found: C, 45.12; H, 5.89; N, 11.90.

4.1.2.10. Methyl 6-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)nicotinate Hydrochloride (20)

The alkylating group (v) was prepared following general O-alkylation procedure A. Substituted pyridin-2-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 8.35–8.03 (m, 1H), 7.81 (dd, J = 9.5, 2.5 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H), 4.08–3.91 (m, 2H), 3.90–3.79 (m, 3H), 3.61–3.44 (m, 2H), 2.04–1.66 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 164.6, 162.4, 142.6, 138.5, 119.8, 109.8, 76.6, 52.1, 44.2, 29.4, 26.7.

The N-Alkylation reaction was carried out according to the general procedure B. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 20. Yield: 34%.

Mp: 197–201 °C.

¹H NMR (300 MHz, DMSO): δ 8.54 (d, J = 2.3 Hz, 1H), 8.15–7.90 (m, 2H), 7.76 (dd, J = 9.5, 2.5 Hz, 1H), 7.40 (d, J = 9.1 Hz, 1H), 7.00 (t, J = 6.5 Hz, 1H), 6.41 (d, J = 9.5 Hz, 1H), 4.55 (d, J = 13.7 Hz, 2H), 3.97 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 3.68 (d, J = 12.3 Hz, 2H), 3.57 (d, J = 11.6 Hz, 2H), 3.13 (s, 4H), 1.80–1.61 (m, 4H).

¹³C NMR (151 MHz, DMSO): δ 164.8, 161.9, 153.1, 144.7, 143.8, 139.4, 138.8, 119.3, 114.3, 112.5, 108.9, 55.3, 52.3 (2C), 50.2 (2C), 49.0, 43.7, 26.4, 20.6.

Calcd for C₂₀H₂₆N₄O₃·2HCl + 0.8H₂O: C, 52.47; H, 6.52; N, 12.24. Found: C, 52.47, H, 6.54, N, 12.00.

4.1.2.11. Methyl 6-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butoxy)nicotinate Hydrochloride (21)

The reaction was carried out according to the general procedure B. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 21. Yield: 35%.

MP: 222–226 °C.

¹H NMR (600 MHz, DMSO): δ 8.56 (d, J = 2.5 Hz, 1H), 8.46 (d, J = 4.8 Hz, 2H), 7.79 (dd, J = 9.5, 2.6 Hz, 1H), 6.78 (t, J = 4.8 Hz, 1H), 6.44 (d, J = 9.5 Hz, 1H), 4.68 (d, J = 14.8 Hz, 2H), 4.00 (t, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.49 (dd, J = 26.1, 12.5 Hz, 4H), 3.13–2.89 (m, 4H), 1.83–1.54 (m, 4H).

¹³C NMR (151 MHz, DMSO): δ 164.8, 161.9, 160.6, 158.5 (2C), 144.7, 138.8, 119.3, 111.7, 108.9, 55.4, 52.3, 50.8 (2C), 49.0, 49.00, 40.8, 26.4, 20.6.

Calcd for C₁₉H₂₅N₅O₃·2HCl + 0.6H₂O: C, 50.14; H, 6.24; N, 15.58. Found: C, 50.21; H, 6.19; N, 15.18.

4.1.2.12. 2-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (22)

The alkylating group (vi) was prepared following general O-alkylation procedure A. Quinolin-2-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 7.65 (d, J = 9.5 Hz, 1H), 7.61–7.49 (m, 2H), 7.35 (d, J = 8.5 Hz, 1H), 7.29–7.10 (m, 1H), 6.67 (d, J = 9.5 Hz, 1H), 4.31 (s, 2H), 3.55 (ddd, J = 40.2, 9.4, 5.3 Hz, 2H), 1.99–1.81 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 162.1, 139.2, 139.0, 130.7, 129.1, 122.1, 121.6, 121.0, 114.0, 44.6, 41.3, 29.7, 24.9.

Mp: 125–129 °C.

¹H NMR (600 MHz, DMSO): δ 8.12–8.01 (m, 2H), 7.90 (t, J = 12.6 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.61 (q, J = 8.5 Hz, 2H), 7.43 (d, J = 9.1 Hz, 1H), 7.26 (t, J = 7.1 Hz, 1H), 7.03 (t, J = 6.5 Hz, 1H), 6.61 (d, J = 9.4 Hz, 1H), 4.56 (d, J = 13.8 Hz, 2H), 4.32–4.18 (m, 2H), 3.74 (t, J = 12.6 Hz, 2H), 3.59 (d, J = 11.9 Hz, 2H), 3.17 (d, J = 18.9 Hz, 4H), 1.89 (dt, J = 15.6, 7.9 Hz, 2H), 1.66 (dt, J = 14.9, 7.6 Hz, 2H).

¹³C NMR (151 MHz, DMSO): δ 161.5, 153.0, 144.1, 140.0, 139.2, 138.9, 131.4, 129.6, 122.4, 121.5, 120.9, 115.0, 114.3, 112.9, 55.5, 50.2 (2C), 43.7 (2C), 41.3, 25.0, 20.9.

Calcd for C₂₂H₂₆N₄O·3HCl + H₂O: C, 53.94; H, 6.38; N, 11.44. Found: C, 53.97; H, 6.58; N, 11.37.

4.1.2.13. 2-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (23)

Mp: 143–147 °C.

¹H NMR (600 MHz, DMSO): δ 8.47 (t, J = 5.4 Hz, 2H), 7.92 (d, J = 9.4 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.69–7.52 (m, 2H), 7.26 (t, J = 7.2 Hz, 1H), 6.79 (t, J = 4.7 Hz, 1H), 6.61 (d, J = 9.4 Hz, 1H), 4.68 (d, J = 14.0 Hz, 2H), 4.26 (t, J = 7.5 Hz, 2H), 3.59–3.36 (m, 4H), 3.22–3.09 (m, 2H), 3.02 (dd, J = 21.2, 9.4 Hz, 2H), 1.97–1.76 (m, 2H), 1.66 (dt, J = 15.0, 7.7 Hz, 2H).

¹³C NMR (151 MHz, DMSO): δ 161.5, 160.4, 158.5 (2C), 140.0, 139.2, 131.4, 129.5, 122.4, 121.5, 120.9, 115.0, 111.7, 55.6, 50.8 (2C), 49.0, 41.3, 40.9, 25.0, 21.0.

Calcd for C₂₁H₂₅N₅O·3HCl + 0.4H₂O: C, 52.54; H, 6.05; N, 14.59. Found: C, 52.56; H, 6.02; N, 14.81.

4.1.2.14. 6-Chloro-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (24)

The alkylating group (vii) was prepared following general O-alkylation procedure A. Substituted Quinolin-2-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 7.48 (ddd, J = 14.7, 11.3, 6.0 Hz, 3H), 7.35–7.19 (m, 1H), 6.67 (d, J = 9.5 Hz, 1H), 4.25 (t, J = 6.9 Hz, 2H), 3.65–3.49 (m, 2H), 1.95–1.77 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 161.7, 138.0, 137.6, 130.6, 128.1, 127.5, 122.9, 121.9, 115.5, 76.6, 44.5, 29.6, 24.8.

Mp: 242–246 °C.

¹H NMR (300 MHz, DMSO): δ 8.19–8.06 (m, 1H), 7.98 (t, J = 8.0 Hz, 1H), 7.87 (dd, J = 9.7, 5.6 Hz, 2H), 7.68–7.56 (m, 2H), 7.35 (d, J = 9.1 Hz, 1H), 6.98 (t, J = 6.5 Hz, 1H), 6.66 (d, J = 9.5 Hz, 1H), 4.52 (d, J = 13.8 Hz, 2H), 4.32–4.15 (m, 2H), 3.80–3.44 (m, 4H), 3.14 (s, 4H), 1.86 (s, 2H), 1.64 (d, J = 7.2 Hz, 2H).

¹³C NMR (151 MHz, DMSO): δ 161.2, 153.8, 143.3, 140.4, 138.9, 138.0, 130.9, 128.4, 126.5, 122.8, 122.2, 117.2, 114.3, 112.1, 55.5, 50.3 (2C), 43.5 (2C), 41.5, 24.9, 20.9.

Calcd for C₂₂H₂₅ClN₄O.2.6HCl: C, 53.74; H, 5.66; N, 11.39. Found: C, 53.75; H, 5.95; N, 11.28.

4.1.2.15. 6-Chloro-2-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (25)

The reaction was carried out according to the general procedure B. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 25. Yield: 34%.

Mp: 204–207 °C.

¹H NMR (300 MHz, DMSO): δ 8.47 (d, J = 4.8 Hz, 2H), 7.83 (dd, J = 16.2, 5.5 Hz, 2H), 7.67–7.48 (m, 2H), 7.05–6.69 (m, 1H), 6.62 (d, J = 9.5 Hz, 1H), 4.68 (d, J = 13.8 Hz, 2H), 4.18 (t, J = 7.1 Hz, 2H), 3.54 (t, J = 12.9 Hz, 4H), 3.05 (dd, J = 20.8, 10.3 Hz, 4H), 1.84 (s, 2H), 1.59 (s, 2H).

¹³C NMR (151 MHz, DMSO): δ 161.2, 159.0, 158.2, 138.9, 137.9, 130.8 (2C), 128.3, 126.4, 122.8, 122.1, 117.1, 111.5, 55.5, 50.6 (2C), 41.5, 41.2 (2C), 24.9, 20.9.

Calcd for C₂₁H₂₄ClN₅O·3HCl: C, 49.72; H, 5.37; N, 13.81. Found: C, 49.88; H, 5.52; N, 13.81.

4.1.2.16. 2-Methyl-5-(4-(4-(pyridin-2-yl)piperazin-1-yl)butoxy)benzo[d]thiazole Hydrochloride (26)

The alkylating group (viii) was prepared following general O-alkylation procedure A. 2-Methylbenzo[d]thiazol-5-ol was treated with 1-bromo-4-chlorobutane, in the presence of K₂CO₃ in CH₃CN at reflux temperature.

¹H NMR (300 MHz, CDCl₃): δ 7.60 (dd, J = 5.8, 2.9 Hz, 1H), 7.33 (d, J = 35.7 Hz, 1H), 7.04–6.81 (m, 1H), 4.03 (dd, J = 27.4, 4.3 Hz, 2H), 3.73–3.41 (m, 2H), 2.75 (dd, J = 3.0, 1.7 Hz, 3H), 2.04–1.77 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 168.2, 158.0, 154.5, 127.4, 121.6, 114.9, 105.8, 67.3, 44.7, 29.3, 26.6, 20.1.

Mp: 243–247 °C.

¹H NMR (600 MHz, DMSO): δ 8.10 (d, J = 5.8 Hz, 1H), 8.04 (t, J = 8.0 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.46 (s, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.08–6.99 (m, J = 8.8 Hz, 2H), 4.52 (d, J = 13.5 Hz, 2H), 4.08 (t, J = 5.8 Hz, 3H), 3.63 (d, J = 11.4 Hz, 3H), 3.20 (s, 4H), 2.76 (s, 3H), 1.95 (t, J = 15.8 Hz, 2H), 1.87–1.77 (m, 2H).

¹³C NMR (151 MHz, DMSO): δ 169.2, 160.2, 158.4 (2C), 158.2, 153.8, 127.1, 122.9, 115.2, 111.6, 106.1, 106.0, 67.8, 55.7, 50.7 (2), 40.9 (2), 26.4, 20.5, 20.0.

Calcd for C₂₁H₂₆N₄OS·3HCl + H₂O: C, 49.47; H, 6.13; N, 10.99. Found: C, 49.57; H, 6.02; N, 10.99.

4.1.2.17. 2-Methyl-5-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)butoxy)benzo[d]thiazole Hydrochloride (27)

Mp: 268–271 °C.

¹H NMR (600 MHz, DMSO): δ 8.44 (d, J = 3.1 Hz, 2H), 7.87 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.87–6.66 (m, 1H), 4.66 (d, J = 13.8 Hz, 2H), 4.18–3.94 (m, 2H), 3.50 (dd, J = 23.5, 12.2 Hz, 4H), 3.12 (s, 2H), 3.06–2.93 (m, 2H), 2.75 (s, 3H), 1.91 (d, J = 6.5 Hz, 2H), 1.84–1.68 (m, 2H).

¹³C NMR (151 MHz, DMSO): δ 168.8, 158.1, 154.5, 153.2, 143.9, 139.3, 127.3, 122.8, 115.0, 114.3, 112.7, 106.4, 67.8, 55.7, 50.3 (2), 43.6(2), 26.4, 20.5, 20.2.

Calcd for C₂₀H₂₅N₅OS 0.3HCl + H₂O: C, 47.02; H, 5.92; N, 13.71. Found: C, 47.00; H, 5.98; N, 13.68.

4.1.2.18. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yl)butyl)piperazine Hydrochloride (28)

Dess–Martin periodinane (1,1,1-Tris(acetyloxy)-1,1-dihydro-1,2-benziodoxo3(1H)-one) (1.2 g, 2.9 mmol) was added to a solution of pyridinylbutanol (0.3 g, 1.98 mmol) in dichloromethane (5 mL) and the mixture was stirred at room temperature for 2 h. The mixture was filtered through a plug of silica gel, washing with dichloromethane, and the solvent was evaporated under reduced pressure to obtain pyridinylbutyraldehyde (ix) and proceeded immediately to further reaction. Yield: 43%

To a mixture of pyridinylbutyraldehyde (ix), obtained from previous reaction and 1-(pyridin-2-yl)piperazine (0.48 g, 3 mmol) in CH₂Cl₂ (5 mL) was added sodium triacetoxyborohydride (1.2 g, 6 mmol). After the mixture was stirred for 16 h at room temperature, saturated NaHCO₃ solution and CH₂Cl₂ were added. The organic layer was dried (Na₂SO₄) and evaporated and the residue was purified by flash chromatography to give desired product. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 28. Yield: 33%

Mp: 164–168 °C.

¹H NMR (600 MHz, MeOD): δ 7.96 (d, J = 5.6 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.29 (t, J = 6.5 Hz, 2H), 7.18 (dt, J = 13.1, 6.4 Hz, 1H), 6.71 (d, J = 9.1 Hz, 1H), 6.36 (t, J = 6.3 Hz, 1H), 3.26 (m, 3H), 2.81 (s, 3H), 2.56 (d, J = 7.1 Hz, 2H), 2.50 (s, 2H), 2.41 (t, J = 6.5 Hz, 2H), 1.18 (s, 4H).

¹³C NMR (151 MHz, MeOD): δ 156.5, 152.4, 146.9, 145.0, 141.0, 136.9, 127.4, 125.0, 114.5, 113.2, 56.0, 50.5 (2), 43.3 (2), 32.2, 25.6, 22.9.

Calcd for C₁₈H₂₄N₄·3HCl + 1.7H₂O: C, 49.54; H, 7.02; N, 12.84. Found: C, 49.45; H, 6.91; N, 12.87.

4.1.2.19. 2-(4-(4-(Pyridin-2-yl)butyl)piperazin-1-yl)pyrimidine oxalate (29)

To a mixture of pyridinylbutyraldehyde (ix), obtained from reaction mentioned in 2.2.18 and pyrimidyl-piperazine (0.48 g, 3 mmol) in CH₂Cl₂ (5 mL) was added sodium triacetoxyborohydride (1.2 g, 6 mmol). After the mixture was stirred for 16 h at room temperature, saturated NaHCO₃ solution and CH₂Cl₂ were added. The organic layer was dried (Na₂SO₄) and evaporated and the residue was purified by flash chromatography to give desired product. The product was converted into the oxalate salt, followed by crystallization from MeOH–Et₂O to give the pure compound 29. Yield: 24%

Mp: 137–140 °C.

¹H NMR (600 MHz, MeOD): δ 8.67 (t, J = 16.5 Hz, 1H), 8.39 (d, J = 4.7 Hz, 2H), 8.31 (t, J = 7.7 Hz, 1H), 7.82 (t, J = 12.1 Hz, 1H), 7.73 (t, J = 6.5 Hz, 1H), 6.71 (t, J = 4.6 Hz, 1H), 3.50–3.33 (m, 4H), 3.31 (s, 2H), 3.23 (s, 3H), 3.08 (d, J = 6.3 Hz, 3H), 1.87 (s, 4H).

¹³C NMR (151 MHz, MeOD): δ 163.9, 161.0, 157.9, 157.9, 143.8, 143.4, 126.0, 123.8, 111.3, 56.2, 51.4 (2), 40.7 (2), 33.4, 26.0, 22.9.

Calcd for C₁₇H₂₃N₅.2.5C₂H₂O₄: C, 50.57; H, 5.40; N, 13.40. Found: C, 50.86; H, 5.580; N, 13.64.

4.1.2.20. 1-(Pyridin-2-yl)-4-(5-(pyridin-2-yl)pentyl)piperazine Hydrochloride (30)

The intermediate x was synthesized using reported literature with adjusted reaction conditions. To an anhydrous THF solution (10 mL) of the 2-methylpyridine (1 g, 10.7 mmol), LDA solution (6.42 mL, 12.8 mmol in 2 M THF solution) was added dropwise under argon at 0 °C. After 15 min, 1-bromo-4-chlorobutane (1.6 mL, 16 mmol) was added to the dark-red solution and stirred for 1 h, allowing it to warm to room temperature. Then, the reaction mixture was refluxed for 6 h. After completion of the reaction, the reaction mixture was quenched with H₂O, then extracted with EtOAc (3 × 10 mL). The combined organic phases were dried over Na₂SO₄ and the solvent was concentrated in vacuo. The crude product was purified by flash-chromatography to give desired product (x) in 6% yield.

Mp: 142–145 °C.

¹H NMR (600 MHz, MeOD): δ 8.75 (d, J = 3.0 Hz, 1H), 8.59 (t, J = 7.8 Hz, 1H), 8.17 (t, J = 7.9 Hz, 1H), 8.11 (d, J = 5.2 Hz, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.97 (s, 1H), 7.51 (d, J = 9.1 Hz, 1H), 7.17 (d, J = 6.0 Hz, 1H), 4.45 (s, 2H), 3.81 (s, 4H), 3.31 (d, J = 14.8 Hz, 4H), 3.17 (t, J = 7.3 Hz, 2H), 1.95 (d, J = 7.3 Hz, 4H), 1.70–1.49 (m, 2H).

¹³C NMR (151 MHz, MeOD): δ 157.2, 152.5, 146.8, 144.9, 140.9, 137.1, 127.3, 124.8, 114.5, 113.0, 56.4, 50.4 (2), 43.3 (2), 32.6, 28.0, 25.4, 23.0.

Calcd for C₁₉H₂₆N₄·3HCl + 1.5H₂O: C, 51.07; H, 7.22; N, 12.54. Found: C, 51.03; H, 7.31; N, 12.26.

4.1.2.21. 2-(4-Fluorophenyl)-5-(4-(pyridin-2-yl)piperazin-1-yl)pentan-2-ol Hydrochloride (33)

To an anhydrous THF solution (20 mL) of 4-chloro-4′-fluorobutyrophenone (2 g, 10 mmol), commercially available 1 M MeMgBr in Et₂O solution (10 mL, 10 mmol) was added dropwise under argon at 0 °C. After 4 h stirring at RT, the reaction mixture was quenched with H₂O, then extracted with Et₂O (3 × 10 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and the solvent was concentrated in vacuo. The crude product was purified by flash-chromatography (silica gel, 80:20, hexane/Et2O), to give desired intermediate (xi) in 72% yield.

¹H NMR (300 MHz, CDCl₃): δ 7.49–7.44 (m, 2H), 7.06 (m, 2H), 3.47–3.42 (m, 2H), 2.17–2.16 (m, 2H), 1.95–1.88 (m, 2H), 1.57 (s, 3H).

¹³C NMR (75 MHz, CDCl₃): δ 162.10 (d, J = 244.6 Hz), 143.1, 126.5 (2C), 115.0, 114.7, 74.0, 45.4, 41.5, 30.5, 27.4.

A mixture of the appropriate alkylating agent (xi, 1 mmol), 1-(pyridin-2-yl)piperazine (1.2 mmol), and KI (100 mg) in CH₃CN (5 mL) was heated to reflux for 12–24 h. The reaction progress was monitored by TLC and at completion, the mixture was cooled to rt, solvent removed, the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane/MeOH to give the pure desired product in 81% yield. The free base product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 33.

Mp: 164–168 °C.

¹H NMR (600 MHz, MeOD): δ 8.21 (t, J = 8.0 Hz, 1H), 8.13 (t, J = 7.6 Hz, 1H), 7.60–7.55 (m, 1H), 7.49–7.44 (m, 2H), 7.20 (t, J = 6.7 Hz, 1H), 7.06 (q, J = 8.4 Hz, 2H), 5.77 (t, J = 6.6 Hz, 1H), 4.72–4.35 (m, 2H), 4.20–3.69 (m, 6H), 3.63–3.35 (m, 4H), 2.84 (t, J = 12.8 Hz, 2H), 2.10 (s, 3H).

¹³C NMR (151 MHz, MeOD): δ 162.10 (d, J = 244.6 Hz), 152.1, 145.3, 139.2, 138.0, 136.7, 127.4, 120.8, 114.7, 114.5, 113.4, 55.9, 50.5 (2), 50.4, 43.5 (2), 43.5, 23.4, 15.3.

Calcd for C₂₀H₂₆FN₃O·2HCl: C, 57.69; H, 6.78; N, 10.09. Found: C, 57.97; H, 6.61; N, 10.15.

4.1.2.22. 2-(4-Fluorophenyl)-5-(4-(pyrimidin-2-yl)piperazin-1-yl)pentan-2-ol Hydrochloride (34)

A mixture of the alkylating agent (xi, 1 mmol) obtained in the previous reaction (Section ), pyrimidyl-piperazine (1.2 mmol), and KI (100 mg) in CH₃CN (5 mL) was heated to reflux for 12–24 h. The reaction progress was monitored by TLC, and at completion, the mixture was cooled to rt, solvent removed, the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane/MeOH to give the pure desired product in 80% yield. The product was converted into the HCl salt, followed by crystallization from MeOH–Et₂O to give the pure compound 34.

Mp: 201–204 °C.

¹H NMR (600 MHz, DMSO): δ 8.46 (d, J = 3.9 Hz, 2H), 7.66–7.37 (m, 2H), 7.28–7.05 (m, 2H), 6.78 (s, 1H), 5.75 (t, J = 6.6 Hz, 1H), 4.73–4.60 (m, 4H), 3.59 (d, J = 11.9 Hz, 2H), 3.52 (t, J = 13.1 Hz, 2H), 3.17 (s, 2H), 3.12–3.01 (m, 2H), 2.71 (d, J = 7.4 Hz, 2H), 2.02 (s, 3H).

¹³C NMR (151 MHz, DMSO): δ 162.6, 160.8 (F–C, d, J = 243.71 Hz), 158.5 (2C), 139.3, 139.3, 136.6, 127.9–127.8 (2C), 122.7, 115.4–115.3 (2C), 111.7, 55.0, 50.7, 50.7 (2), 40.9 (2), 40.5, 23.4, 16.2.

Calcd for C₁₉H₂₅FN₄O·2.5HCl + 0.5H₂O: C, 51.33; H, 6.46; N, 12.60. Found: C, 51.38; H, 6.10; N, 12.62.

4.1.2.23. 1,1-Bis(4-fluorophenyl)-4-(4-(pyridin-2-yl)piperazin-1-yl)butan-1-ol (35)

To an anhydrous THF (10 mL) solution of 4-chloro-4′-fluorobutyrophenone (1 g, 5 mmol), 5 mL of the commercially available 4-fluorophenylmagnesium bromide in THF solution (5 mmol) was added dropwise under argon at 0 °C. After 4 h stirring at RT, the reaction mixture was quenched with H₂O, then extracted with Et₂O (3 × 10 mL). The combined organic phases were dried over Na₂SO₄ and the solvent was concentrated in vacuo. The crude product was purified by flash-chromatography (silica gel, 80:20, hexane/Et₂O) to give the desired intermediate (xii).

¹H NMR (300 MHz, CDCl₃): δ 7.55–7.18 (m, 4H), 7.14–6.78 (m, 4H), 3.75–3.35 (m, 2H), 2.37 (ddd, J = 18.7, 11.3, 7.7 Hz, 2H), 2.23 (d, J = 3.6 Hz, 1H), 1.87–1.58 (m, 2H).

¹³C NMR (75 MHz, CDCl₃): δ 163.4, 160.1, 142.3, 142.3, 128.2, 128.1, 127.8, 127.7, 115.5, 115.2, 114.9 (2C), 78.0, 45.5, 39.3, 27.1.

A mixture of the alkylating agent (xii, 1 mmol), 1-(pyridin-2-yl)piperazine (1.2 mmol), and KI (100 mg), in CH₃CN (5 mL) was heated to reflux for 12–24 h. The reaction progress was monitored by TLC and at completion, the mixture was cooled to rt, the solvent removed, and the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane (MeOH as additive on flash chromatography) to give the pure desired product, 35 in 20% yield. The free base product was then recrystallized from ethyl acetate to give the pure compound.

Mp: 123–125 °C.

¹H NMR (300 MHz, CDCl₃): δ 8.32–8.12 (m, 1H), 7.46 (ddd, J = 14.0, 7.1, 1.6 Hz, 5H), 6.97 (t, J = 8.6 Hz, 4H), 6.64 (dd, J = 7.6, 5.2 Hz, 2H), 3.56 (s, 4H), 2.44 (dt, J = 8.2, 5.0 Hz, 8H), 1.66 (m, 2H).

¹³C NMR (151 MHz, CDCl₃): δ 162.3, 160.6 (F–C, d, J = 244.9 Hz), 159.3, 148.0 (2C), 144.0, 144.0, 137.6, 127.8 (2C), 127.7 (2C), 114.8 (2C), 114.7 (2C), 113.6, 107.1, 76.0, 59.2, 52.6 (2C), 44.6 (2C), 42.8, 21.6.

Calcd for C₂₅H₂₇F₂N₃O: C, 70.90; H, 6.43; F, 8.97; N, 9.92; O, 3.78. Found: C, 70.63; H, 6.39; N, 9.89.

4.1.2.24. 1,1-Bis(4-fluorophenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-ol (36)

A mixture of the alkylating agent (xii, 1 mmol), 1-(2-methoxyphenyl)piperazine (1.2 mmol), and KI (100 mg) in CH₃CN (5 mL) was heated to reflux for 12–24 h. The reaction progress was monitored by TLC and at completion, the mixture was cooled to rt, the solvent removed, and the resulting residue loaded onto a cartridge and purified by flash chromatography using EtOAc/hexane/MeOH to give the pure desired product in 22% yield. The free base was recyrstallized from EtOAc to give the pure compound 36.

Mp: 132–135 °C.

¹H NMR (600 MHz, DMSO): δ 7.45 (s, 4H), 7.22–7.00 (m, 4H), 6.98–6.77 (m, 4H), 3.73 (s, 3H), 3.04–2.80 (m, 4H), 2.35 (d, J = 29.4 Hz, 4H), 2.25 (t, J = 30.7 Hz, 4H), 1.40 (s, 2H).

¹³C NMR (151 MHz, DMSO): δ 161.9, 160.3 (F–C, d, J = 242.3 Hz), 152.4, 145.1(2), 141.7, 128.2 (3), 128.2 (2), 122.8, 121.30, 118.4, 114.9 (2), 114.8 (2), 112.4, 76.2, 58.5, 55.8, 53.3 (2), 50.3 (2), 40.3, 21.4.

Calcd for C₂₇H₃₀F₂N₂O₂: C, 71.66; H, 6.68; N, 6.19. Found: C, 71.40; H, 6.75; N, 6.12.

4.2. Pharmacology

4.2.1. Receptor Binding Affinity Studies

K _i determinations, and receptor binding profiles data were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # 75N95023C00021 (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. The experimental procedures, including the cell lines, radioligands, and binding assay protocols, were conducted as previously detailed and per NIMH PDSP assay protocol book. , Detailed experimental methods can be obtained at https://pdsp.unc.edu/ims/investigator/web/. A list of receptors and radioligands used in the binding affinity evaluations is presented in Table .

2. List of Cell Lines and Radioligands Used for Binding Studies at the Dopamine (DA) and Serotonin Receptors (5-HT) Reported in the Present Study .

receptor	parental cells	radioligand
5-HT_1A	CHO	[³H]-WAY100635
5-HT_2A	HEK T*	[³H]-Ketanserin
5-HT_2C	HET T	[³H]-Mesulergine
5-HT_7A	HEK	[³H]-LSD
D₂	fibroblast	[³H]-N-methylspiperone
D₃	HEK T*	[³H]-N-methylspiperone
D₄	HEK	[³H]-N-methylspiperone

Open in a new tab

Note: All clones are stable lines and are of human origin unless noted. Transient cells are marked with “*”.

For radioligand binding assays, membranes were prepared from stable or transiently transfected cells for the respective receptors. To make membrane fractions from stably transfected cell lines, cells were subcultured into 15 cm dishes and grown to 90% confluency. For 5-HT receptors, cells were incubated overnight in either serum-free medium or medium containing 1% dialyzed fetal bovine serum. The next day, the cells were rinsed with PBS, scraped off into 50 mL conical tubes, and pelleted by centrifugation (1000g, 10 min at 4 °C). The cell pellet was resuspended in chilled (4 °C) lysis buffer (50 mM Tris HCl buffer, pH 7.4) and triturated gently for hypotonic lysis. The suspension was then centrifuged at 21,000g for 20 min (4 °C) to obtain a crude membrane fraction pellet. The fresh membrane pellet was then resuspended in cold lysis buffer with 3× volumes of the pellet size and was immediately subjected to the Bradford protein assay to determine protein concentration, followed by a saturation binding assay to determine receptor expression level and the affinity of the selected radioligand. Based on the receptor expression level and the K_d value, fresh membrane suspensions were stored at −80 °C in small aliquots such that one aliquot is enough for one 96-well plate to have at least 500 cpm/well when assayed at 0.5–1.0× K _d value of the hot ligand.

All the final compounds were initially subjected to primary radioligand binding assays at the selected DA and 5-HT receptors. In primary binding assays, compounds were tested at a single concentration (10 μM) in quadruplicate in 96-well plates. Compounds showing a minimum of 50% inhibition at 10 μM were tagged for secondary radioligand binding assays to determine equilibrium binding affinity at specific receptors. In secondary binding assays, compounds that showed a minimum of 50% inhibition at 10 μM were tested at different concentrations (0.1, 0.3, 1, 3, 10, 30, 100, 300 nM, 1, 3, 10 μM) in triplicate (3 sets of 96-well plates). Both primary and secondary radioligand binding assays were carried out in a final volume of 125 μL per well in appropriate binding buffers. The hot ligand concentration is usually at a concentration close to the K _d (unless otherwise indicated). Total binding and nonspecific binding were determined in the absence and presence of 10 μM of the appropriate reference compound, respectively. In brief, plates were usually incubated at room temperature and in the dark for 90 min (unless otherwise indicated). Reactions were stopped by vacuum filtration onto 0.3% polyethylenimine (PEI) soaked 96-well filter mats using a 96-well Filtermate harvester, followed by three washes with cold wash buffers. Scintillation cocktail was then melted onto the microwave-dried filters on a hot plate and radioactivity was counted in a Microbeta counter. Results were processed using nonlinear regression analysis to determine IC₅₀ values, which are then converted to K _i values. Binding affinities are reported in nanomolar (nM) units, with the mean ± standard error of the mean (SEM).

4.2.2. Animal Behavioral Studies

4.2.2.1. Drugs and Chemicals

Chemicals and reagents used for the behavioral experiments were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Lactic acid (Baker), ascorbic acid, and sodium chloride (Fisher) were ACS reagent grade. The water used to make solutions was HPLC grade. Compounds 35 (CLog P = 4.18) and 36 (CLog P = 4.18) (Figure ) used in the animal studies were synthesized and characterized by us at Florida A&M University. The purity of the compounds 35 and 36 was confirmed using Elemental (C, H, N) analysis with C, H, and N values within 0.4% of theoretical values using ChemDraw Professional software (version 19.1, PerkinElmer Informatics, Inc.). Compounds 35, 36 and the reference drug, lurasidone HCl (4), were prepared in 1% lactic acid vehicle filtered through a 0.22 μm membrane filter and used for all the in vivo studies. Apomorphine hydrochloride hemihydrate (98.5%) was freshly prepared on the day of testing by dissolving it in HPLC-grade water, followed by addition of ascorbic acid (0.1% w/w) and sodium chloride (0.9% w/w). The final apomorphine solution (Apo) was transferred to an amber vial to protect it from light until use. Doses used for the animal studies for lurasidone HCl (4) and compound 35 and 36 are expressed as the lurasidone HCl (4) salt and free bases, respectively. The test compounds, 35, 36 and the reference drug were administered by intraperitoneal (ip) route in a volume of 10 mL/kg while the apomorphine solution was administered by subcutaneous (sc) route. The theoretical CLog P values reported here were calculated using ChemDraw Professional software.

4.2.2.2. Animals

Animal handling protocols for the ethical use of rodents and the experimental procedures for these studies were approved by the Florida A & M University Institutional Animal Care and Use Committee (IACUC), Animal Welfare Assurance number A-3581-01. All animals were purchased from Envigo and housed in the AAALAC-accredited Florida A & M University Animal Care facility under environmentally controlled conditions (12 h light/dark cycle, temperature kept in the range of 68–79 °F, and 30–70% relative humidity) with free access to food and water. Animals were acclimated in their home cages for at least 5 days before testing, then fasted the night before the behavioral experiments. Male albino Swiss-Webster mice weighing 21–34 g and between 5 and 6 weeks old were used for the reversal of apomorphine-induced climbing assays. Male Sprague–Dawley rats weighing 133–153 g at approximately 6 weeks age were used for the catalepsy test.

4.2.2.3. Reversal of Apomorphine-Induced Climbing

A modified apomorphine-induced climbing experiment , was conducted in Swiss-Webster mice to evaluate potential antipsychotic activity. Inhibition or reduction of the apomorphine-induced climbing behavior following apomorphine administration is considered indicative of antipsychotic effects. Groups of five mice first received intraperitoneal (ip) injections of 35, 36, lurasidone (4), or vehicle and were returned to their home cages. After 30 min, the same mice were administered apomorphine (1.5 mg/kg, s.c., free-base equivalent), placed individually in cylindrical wire cages (12 cm diameter × 14 cm height), and observed at 10 and 20 min after the apomorphine injection for their climbing behavior. Scoring was performed as follows: mice with all four paws up on the cage wall, not on the floor, received a score of 2 (0% inhibition); those with two or three paws on the cage wall were assigned a score of 1; and mice with one or no paws on the cage wall received a score of 0 (100% inhibition). Scores were converted to mean percent climbing inhibition and are presented in Figure . ED₅₀ values were derived using the nonlinear regression software in Prism version 10.4 from GraphPad.

4.2.2.4. Catalepsy Bar Test

The catalepsy bar test was used with rats to assess the potential for extrapyramidal side effects (EPS) in humans. Rats were held by the tail and gently lowered until their forepaws could grasp a metal bar and the hind paws and tail touched the floor. The duration in seconds that the rat continued to grasp the bar with 1–2 paws while keeping the hind paws on the floor was measured with stopwatches as previously described. As a second measure, we also used a catalepsy test that is reported to be sensitive to the anticataleptic actions of 5HT_1A receptor agonists, the crossed-legs position (CLP) test. , Following both catalepsy assessments, a righting reflex test , was conducted to evaluate sedation or other nonspecific behavioral effects.

Five rats per dose group (unless otherwise noted) initially received an intraperitoneal (ip) injection of vehicle, test compound, or control, and were tested 60 min later, beginning with the bar test (30 s), followed by the CLP test (30 s), and then the righting test. The rats were then returned to their home cages. The testing sequence was repeated at 3 and 6 min after the initial evaluation. For each rat, the mean of the three time-point measurements for each test was calculated in seconds. The average mean time in seconds for the five rats was calculated for each dose group and test. , Results are graphed in Figure .

4.2.2.5. Catalepsy Crossed-Legs Position (CLP) Test

For the CLP catalepsy assessment, rats were positioned on the stainless-steel floor of the catalepsy testing chamber with their abdomen facing downward or toward the floor. The hind paws were gently moved forward and the forepaws backward so that the ipsilateral hind paws rested on top of the corresponding forepaws. The duration that the animal maintained this posture, whether with one or both paw sets, was recorded using a stopwatch for up to 30 s. ,

4.2.2.6. Righting Test

Following completion of the bar and CLP tests, each rat was gently positioned on its back and observed immediately. If the animal was able to independently return to a normal upright posture without assistance, the response was recorded as “righted”. ,

4.2.2.7. Statistical Analyses

Statistical analyses were conducted using GraphPad Prism version 10.4 (GraphPad Software, LLC). Data are presented as mean ± standard error of the mean (SEM). For the apomorphine-induced climbing experiments, one-way ANOVA followed by Dunnett’s post hoc test was applied. Significance levels are denoted as *, **, ***, and **** corresponding to p < 0.05, 0.01, 0.001, and 0.0001, respectively, versus the vehicle group. For the catalepsy test, one-way ANOVA analysis was used to compare each treatment group with the vehicle, with P < 0.05 considered statistically significant.

4.3. Molecular Docking Studies

4.3.1. Protein Preparation

Protein structures for the Dopamine D₂ Receptor (PDB: 6CM4) and the 5-hydroxytryptamine 2A (5-HT_2A) receptor (PDB: 8UWL and PDB: 6A94 ) were retrieved from the protein databank and imported into Schrödinger Modeling Small-Molecule Drug Discovery Suite 2024–4, utilizing the Maestro interface version 14.2 (Schrödinger, LLC, New York, NY, 2024). Lurasidone (CID: 213046) was retrieved from the National Center for Biotechnology Information (NCBI). The Protein Preparation wizard was utilized to prepare each protein by filling in missing side chains, correcting formal charges and bond orders, adding missing hydrogens, capping protein termini with ACE and NMA residues. Water molecules located within 5 Å of cocrystallized ligands were removed. The proteins were optimized and subsequently energy minimized using OPLS4 force field. ,

4.3.2. Ligand Preparation

Compounds 6, 36 and lurasidone (4) were sketched in ChemDraw, imported into the Schrodinger’s Modeling Suite and prepared using the LigPrep tool. This preparation ensures the ligands are converted from 2D structures into 3D molecules with proper tautomeric and ionization states, adding hydrogens, perform energy minimization using OPLS4all necessary to produce high-quality 3D molecules for docking studies.

4.3.3. Binding Site Identification and Docking Validation

Orthosteric binding sites were validated using SiteMap. To validate the docking protocol, cocrystallized ligands were used as a reference to generate the grid receptor and Glide docking was used to validate their respective receptors. Next, each receptor was further validated using lurasidone (4), a known potent high affinity D₂R and 5-HT_2AR antagonist for the induced fit docking (IFD) studies.

4.3.4. Docking

The individual compounds in their protonated forms were docked into the orthosteric sites of the D₂, and 5-HT_2A receptors using the Induced Fit Docking (IFD) workflow. ,

5. Conclusion

In summary, we performed an extended SAfiR investigation with significant structural modifications on the nonpiperazinyl alkyl-aryl region of SYA16263 which exhibited strong binding affinities for the D₄ and 5-HT_1A receptors, while demonstrating moderate binding affinities at the D₂, D₃, and 5-HT_2A receptors. Our efforts aimed at enhancing selectivity for D₂-like and serotonin receptors (5-HT_1A, 5-HT_2A and 5-HT_7A) through systematic optimization led to the identification of compounds 35 and 36 which exhibited subnanomolar binding affinities at the 5-HT_1A receptor and low nanomolar binding affinities at the D_2‑like, 5-HT_2A and 5-HT_7A receptors. Further, IFD studies indicated that compound 36 exhibits key interactions of dual antagonist activity comparable to lurasidone (4) at the D₂ and the 5-HT_2A receptors. However, receptor functional studies are necessary to corroborate these computational based observations. Pharmacological evaluation of compounds 35 and 36 in an animal model relevant to schizophrenia displayed significant reversal of apomorphine-induced climbing behavior in mice along with lurasidone, but at a higher ED₅₀ dose for compound 35 and at a lower ED₅₀ dose for compound 36 relative to lurasidone (4). Further, compounds 35 and 36 did not produce catalepsy as determined by the Bar and CLP tests in acute studies in rats. Although our initial results are promising, further preclinical studies are warranted that mimic the clinical complexity involving cognitive and social behavioral aspects in animal models. Further, evaluating the lead compounds in chronic psychosis-like models with longitudinal behavioral tests will address their long-term efficacy potential. Our future studies are focused on investigating the lead compounds, 35 and 36 in diversified genetic and neurodevelopmental preclinical models that reflect the human disease condition and progression. Thus, we report new compounds, 35 and 36 with enhanced binding affinities at the D₂-like, 5-HT_1A, 5-HT_2A and 5-HT_7A receptors that exhibit antipsychotic-like properties without inducing catalepsy in rats. We anticipate that these compounds may demonstrate significant antipsychotic-like properties with sustained efficacy in long-term preclinical evaluations.

Supplementary Material

ao6c02255_si_001.pdf^{(5.7MB, pdf)}

Acknowledgments

This work was financially supported by an NIH/NIGMS R16 grant number 1R16GM145581-01 and an NIGMS Endowment grant in support of an Eminent Scholar Chair to SYA. The research was also partly supported with funding from the U.S. Department of Education, Title III Part B, Strengthening Historically Black Graduate Institutions Programs (HBGI), awarded to “Florida A&M University” and an RCMI grant # NIMHD U54 MD 007582. K _i determinations and receptor binding assays were performed by the Psychoactive Drug Screening Program (PDSP) under Prof Bryan Roth at UNC. Funding sources acknowledged had no involvement in the study design, data collection and interpretation, or article preparation and submission of this manuscript.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c02255.

¹H and ¹³C NMR data, elemental (C, H, N) analysis data for final compounds, and molecular docking study figures (PDF)

U.M.G. synthesized most of the compounds tested in this manuscript. C.V. and E.K.O. contributed to the synthesis and characterization of the compounds and the writing of the manuscript. B.A.B. contributed to the experimental design, the writing of the manuscript, and the collection, analysis, and interpretation of the in vivo studies data. M.E.A., I.L.J., E.B.K., A.A.M., and E.W.T. participated in the in vivo studies and assisted in the collection of the in vivo data. A.M.M also conducted the molecular modeling studies. S.Y.A. originated the ideas expressed in the manuscript, contributed to the experimental design, analyzed and interpreted the data, wrote and edited the manuscript.

The authors declare no competing financial interest.

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PERMALINK

Structure–Affinity Relationship Optimization of SYA16263 Yields New Ligands with Improved Receptor Affinity and Antipsychotic-Like Properties

Uma M Gonela

Barbara A Bricker

Chandrashekhar Voshavar

Edem K Onyameh

Matilda E Adzimah

Edith B Kuudaar

Alexus A Morgan

Ismail L Jones

Eljaye W Tellis

Seth Y Ablordeppey

Abstract

1. Introduction

1.

2.

3.

4.

5.

6.

7.

2. Chemistry

1. Synthesis of Compounds 13–16 .

8. Synthesis of Compounds 35 and 36 .

2. Synthesis of Compounds 17–21 .

3. Synthesis of Compounds 22–25 .

4. Synthesis of Compounds 26, 27 .

5. Synthesis of Compounds 28, 29 .

6. Synthesis of Compound 30 .

7. Synthesis of Compounds 33 and 34 .

3. Results and Discussion

3.1. Structure–Affinity Relationship (SAfiR) Studies

1. Binding Affinity Constants [Mean K i ± SEM, nM (pKi ± SEM)] at Important Dopamine (DA) and Serotonin (5-HT) Receptors .

3.2. Comparison of Structure–Affinity Relationship (SAfiR) among D2-Like and 5-HT Receptor Subtypes

3.3. Molecular Docking Studies

3.4. Animal Behavioral Studies

3.4.1. Evaluation of 35, 36, and Lurasidone (4) in an In Vivo Mouse Model of Schizophrenia: Reversal of Apomorphine-Induced Climbing

3.4.2. Evaluation of 35 and 36 for Induction of Catalepsy in Rats

4. Materials and Methods

4.1. Chemical Syntheses

4.1.1. Materials and Methods for Synthesis and Characterization

4.1.2. Synthesis of Compounds 13–30, and 33–36

4.1.2.1. General O-Alkylation Procedure A

4.1.2.2. General N-Alkylation Procedure B

4.1.2.3. 1-(Pyridin-2-yl)-4-(3-(pyridin-3-yloxy)­propyl)­piperazine Hydrochloride (13)

4.1.2.4. 2-(4-(3-(Pyridin-3-yloxy)­propyl)­piperazin-1-yl)­pyrimidine Hydrochloride (14)

4.1.2.5. 1-(Pyridin-2-yl)-4-(4-(pyridin-3-yloxy)­butyl)­piperazine Hydrochloride (15)

4.1.2.6. 2-(4-(4-(Pyridin-3-yloxy)­butyl)­piperazin-1-yl)­pyrimidine Hydrochloride (16)

4.1.2.7. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yloxy)­butyl)­piperazine Hydrochloride (17)

4.1.2.8. 2-(4-(4-(Pyridin-2-yloxy)­butyl)­piperazin-1-yl)­pyrimidine (18)

4.1.2.9. 1-(4-((5-Chloropyridin-2-yl)­oxy)­butyl)-4-(pyridin-2-yl)­piperazine Hydrochloride (19)

4.1.2.10. Methyl 6-(4-(4-(Pyridin-2-yl)­piperazin-1-yl)­butoxy)­nicotinate Hydrochloride (20)

4.1.2.11. Methyl 6-(4-(4-(Pyrimidin-2-yl)­piperazin-1-yl)­butoxy)­nicotinate Hydrochloride (21)

4.1.2.12. 2-(4-(4-(Pyridin-2-yl)­piperazin-1-yl)­butoxy)­quinoline Hydrochloride (22)

4.1.2.13. 2-(4-(4-(Pyrimidin-2-yl)­piperazin-1-yl)­butoxy)­quinoline Hydrochloride (23)

4.1.2.14. 6-Chloro-2-(4-(4-(pyridin-2-yl)­piperazin-1-yl)­butoxy)­quinoline Hydrochloride (24)

4.1.2.15. 6-Chloro-2-(4-(4-(pyrimidin-2-yl)­piperazin-1-yl)­butoxy)­quinoline Hydrochloride (25)

4.1.2.16. 2-Methyl-5-(4-(4-(pyridin-2-yl)­piperazin-1-yl)­butoxy)­benzo­[d]­thiazole Hydrochloride (26)

4.1.2.17. 2-Methyl-5-(4-(4-(pyrimidin-2-yl)­piperazin-1-yl)­butoxy)­benzo­[d]­thiazole Hydrochloride (27)

4.1.2.18. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yl)­butyl)­piperazine Hydrochloride (28)

4.1.2.19. 2-(4-(4-(Pyridin-2-yl)­butyl)­piperazin-1-yl)­pyrimidine oxalate (29)

4.1.2.20. 1-(Pyridin-2-yl)-4-(5-(pyridin-2-yl)­pentyl)­piperazine Hydrochloride (30)

4.1.2.21. 2-(4-Fluorophenyl)-5-(4-(pyridin-2-yl)­piperazin-1-yl)­pentan-2-ol Hydrochloride (33)

4.1.2.22. 2-(4-Fluorophenyl)-5-(4-(pyrimidin-2-yl)­piperazin-1-yl)­pentan-2-ol Hydrochloride (34)

4.1.2.23. 1,1-Bis­(4-fluorophenyl)-4-(4-(pyridin-2-yl)­piperazin-1-yl)­butan-1-ol (35)

4.1.2.24. 1,1-Bis­(4-fluorophenyl)-4-(4-(2-methoxyphenyl)­piperazin-1-yl)­butan-1-ol (36)

4.2. Pharmacology

4.2.1. Receptor Binding Affinity Studies

2. List of Cell Lines and Radioligands Used for Binding Studies at the Dopamine (DA) and Serotonin Receptors (5-HT) Reported in the Present Study .

4.2.2. Animal Behavioral Studies

4.2.2.1. Drugs and Chemicals

4.2.2.2. Animals

4.2.2.3. Reversal of Apomorphine-Induced Climbing

4.2.2.4. Catalepsy Bar Test

4.2.2.5. Catalepsy Crossed-Legs Position (CLP) Test

4.2.2.6. Righting Test

4.2.2.7. Statistical Analyses

4.3. Molecular Docking Studies

4.3.1. Protein Preparation

4.3.2. Ligand Preparation

1. Binding Affinity Constants [Mean K _i ± SEM, nM (pKi ± SEM)] at Important Dopamine (DA) and Serotonin (5-HT) Receptors .

3.2. Comparison of Structure–Affinity Relationship (SAfiR) among D₂-Like and 5-HT Receptor Subtypes

4.1.2.3. 1-(Pyridin-2-yl)-4-(3-(pyridin-3-yloxy)propyl)piperazine Hydrochloride (13)

4.1.2.4. 2-(4-(3-(Pyridin-3-yloxy)propyl)piperazin-1-yl)pyrimidine Hydrochloride (14)

4.1.2.5. 1-(Pyridin-2-yl)-4-(4-(pyridin-3-yloxy)butyl)piperazine Hydrochloride (15)

4.1.2.6. 2-(4-(4-(Pyridin-3-yloxy)butyl)piperazin-1-yl)pyrimidine Hydrochloride (16)

4.1.2.7. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yloxy)butyl)piperazine Hydrochloride (17)

4.1.2.8. 2-(4-(4-(Pyridin-2-yloxy)butyl)piperazin-1-yl)pyrimidine (18)

4.1.2.9. 1-(4-((5-Chloropyridin-2-yl)oxy)butyl)-4-(pyridin-2-yl)piperazine Hydrochloride (19)

4.1.2.10. Methyl 6-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)nicotinate Hydrochloride (20)

4.1.2.11. Methyl 6-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butoxy)nicotinate Hydrochloride (21)

4.1.2.12. 2-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (22)

4.1.2.13. 2-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (23)

4.1.2.14. 6-Chloro-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (24)

4.1.2.15. 6-Chloro-2-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)butoxy)quinoline Hydrochloride (25)

4.1.2.16. 2-Methyl-5-(4-(4-(pyridin-2-yl)piperazin-1-yl)butoxy)benzo[d]thiazole Hydrochloride (26)

4.1.2.17. 2-Methyl-5-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)butoxy)benzo[d]thiazole Hydrochloride (27)

4.1.2.18. 1-(Pyridin-2-yl)-4-(4-(pyridin-2-yl)butyl)piperazine Hydrochloride (28)

4.1.2.19. 2-(4-(4-(Pyridin-2-yl)butyl)piperazin-1-yl)pyrimidine oxalate (29)

4.1.2.20. 1-(Pyridin-2-yl)-4-(5-(pyridin-2-yl)pentyl)piperazine Hydrochloride (30)

4.1.2.21. 2-(4-Fluorophenyl)-5-(4-(pyridin-2-yl)piperazin-1-yl)pentan-2-ol Hydrochloride (33)

4.1.2.22. 2-(4-Fluorophenyl)-5-(4-(pyrimidin-2-yl)piperazin-1-yl)pentan-2-ol Hydrochloride (34)

4.1.2.23. 1,1-Bis(4-fluorophenyl)-4-(4-(pyridin-2-yl)piperazin-1-yl)butan-1-ol (35)

4.1.2.24. 1,1-Bis(4-fluorophenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-ol (36)