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. Author manuscript; available in PMC: 2021 Dec 14.
Published in final edited form as: Bioorg Med Chem. 2020 Dec 14;30:115943. doi: 10.1016/j.bmc.2020.115943

A study of the structure-affinity relationship in SYA16263; is a D2 receptor interaction essential for inhibition of apormorphine-induced climbing behavior in mice?

Edem K Onyameh 1, Barbara A Bricker 1, Suresh VK Eyunni 1, Chandrashekhar Voshavar 1, Uma M Gonela 1, Edward Ofori 1,1, Andrea Jenkins 1, Seth Y Ablordeppey 1,*
PMCID: PMC8670397  NIHMSID: NIHMS1762376  PMID: 33338898

Abstract

Dopamine (DA) and serotonin (5-HT) receptors are prime targets for the development of antipsychotics. The specific role of each receptor subtype to the pharmacological effects of antipsychotic drugs remains unclear. Understanding the relationship between antipsychotic drugs and their binding affinities at DA and 5-HT receptor subtypes is very important for antipsychotic drug discovery and could lead to new drugs with enhanced efficacies. We have previously disclosed SYA16263 (5) as an interesting compound with moderate radioligand binding affinity at the D2 & D3 receptors (Ki = 124 nM & 86 nM respectively) and high binding affinities towards D4 and 5-HT1A receptors (Ki = 3.5 nM & 1.1 nM respectively). Furthermore, we have demonstrated SYA16263 (5) is functionally selective and produces antipsychotic-like behavior but without inducing catalepsy in rats. Based on its pharmacological profile, we selected SYA16263 (5) to study its structure-affinity relationship with a view to obtaining new analogs that display receptor subtype selectivity. In this study, we present the synthesis of structurally modified SYA16263 (5) analogs and their receptor binding affinities at the DA and 5-HT receptor subtypes associated with antipsychotic action. Furthermore, we have identified compound 21 with no significant binding affinity at the D2 receptor subtype but with moderate binding affinity at the D3 and D4 receptors subtypes. However, because 21 is able to demonstrate antipsychotic-like activity in a preliminary test, using the reversal of apomorphine-induced climbing behavior experiment in mice with SYA16263 and haloperidol as positive controls, we question the essential need of the D2 receptor subtype in reversing apomorphine-induced climbing behavior.

Keywords: Dopamine receptors, Serotonin receptors, Pyridinyl piperazine, D2 subtype receptors, 5-HT subtype receptors, SYA16263, Apomorphine-induced climbing behavior

1. Introduction

There is overwhelming evidence in the literature that dopamine (DA) and serotonin (5-HT) receptors play significant roles in the action of antipsychotic drugs.1,2 However, it remains unclear what the specific contribution of each receptor subtype of the dopamine and serotonin receptors is and the primary roles they play in the activity of current antipsychotic drugs. Aripiprazole (Abilify, 1),3–5 the highest selling antipsychotic drug, along with some of the newest antipsychotic drugs on the market, brexpiprazole (Rexulti, 2),6 cariprazine (Vraylar, 3)7 and lurasidone (Latuda, 4),8 (Fig. 1) do interact with very high affinities to one or more of the D2, D3 and D4 receptor subtypes and have high affinities for several 5-HT receptor subtypes. Indeed, it has been reported that atypical antipsychotic activities also correlate with D2 receptor binding affinities.9 In view of this, our lab has been working on the development of DA and 5-HT receptor ligands with specificities for the subtype receptors to allow for further exploration of their contributions.10–13 In particular, there has been a persistent indication for the last several decades or so that, the dopamine D2 receptor (D2R) is essential for antipsychotic activity and apparently no clinically useful antipsychotic agent is devoid of D2 binding affinity.14,15 In a recent article, the authors have shown that SEP-363856, a non-D2 receptor binding compound displayed antipsychotic activity through its agonist activity at trace amine–associated receptor 1 (TAAR1) and 5-HT1A receptors.16,17 SEP-363856 is currently in clinical trials for efficacy and safety evaluation in acutely psychotic adults with schizophrenia.18 Taking cues from such reports including our own work, we wanted to further explore the role of individual receptor subtypes of DA and 5-HT receptors (D2, D3, D4, 5-HT1A, 5-HT2A and 5-HT7 in particular). In a recent publication, we have reported the identification of SYA16263 (5) (Fig. 1) which showed moderate radioligand binding affinity at the D2 & D3 receptors (Ki = 124 nM & 86 nM respectively) and high binding affinities at D4 and 5-HT1A receptors (Ki = 3.5 nM & 1.1 nM respectively) and displayed antipsychotic-like potential without catalepsy in rats even after nineteen times its ED50 value.19 Given its interesting overall profile, we have begun focusing on its structural elements with a view to understanding their contribution to subtype selectivity and the pharmacological activity displayed. To this end, using SYA16263 (5) as a lead compound, we have carried out a structure-affinity relationship (SAR) study by synthesizing a series of related compounds and evaluating their binding affinities at the dopamine and serotonin receptor subtypes to understand the role of structural modifications on receptor binding affinities associated with antipsychotic properties.

Fig. 1.

Fig. 1.

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Structures of some antipsychotic drugs in use, along with SYA16263 and azaperone.

2. Chemistry

The syntheses of compounds 9–11 and 18–22 (Series I to V) are depicted in Schemes 1–4. Compound 5 was previously reported by us20 while compound 6 (Fig. 1), azaperone, is a commercially available agent. All the target compounds were obtained by reacting various alkylating agents with unsubstituted/substituted pyridinyl piperazines or homopiperazines (1,4-diazepanes) or 3,8-diazabicyclo[3.2.1]octanes under reflux conditions or carrying out a microwave-assisted reaction in dimethoxyethane (DME) or isopropanol (iPrOH) in the presence of K2CO3 or NaHCO3 as a base and a catalytic amount of KI. Syntheses of compounds 7, 8 & 12–17 were previously reported by us.20–23 Compound 9 was obtained in three steps (Scheme 1) by initially reacting piperazine (23) with 2-bromo-5-methylpyridine (24) in ethylene glycol (EG) to give 1-(5-methylpyridin-2-yl)piperazine (25) using the previously reported method24 and then subjecting 4-chloro-4′-fluorobutyrophenone (26) to a Clemmensen reduction (Zn/Hg, conc. HCl) procedure to give alkylating agent 1-(4-chlorobutyl)-4-fluorobenzene (27) as reported previously.23 Subsequent N-alkylation of 25 with alkylating agent 27 gave the desired final compound 9. A three-step reaction procedure was used to synthesize compounds 10 & 11 (Schemes 2). Commercially available tert-butyl 4-(5-bromopyridin-2-yl)piperazine-1-carboxylate (28) and tert-butyl 4-(6-bromopyridin-2-yl)piperazine-1-carboxylate (29) were reacted with phenyl boronic acid (30) in the presence of Pd catalyst via Suzuki reaction to give intermediates 31 and 32 respectively. BOC (tert-butyloxycarbonyl) deprotection of 31 and 32 with trifluoroacetic acid (TFA) in dichloromethane (DCM) yielded 33 and 34 (Scheme 2). N-alkylation of the amine intermediates, 33 and 34 with alkylating agent 27 afforded compounds 10 & 11 as indicated in Scheme 2. A three-step procedure was followed for the synthesis of compounds 18 & 19 (Scheme 3). First, tert-butyl 3,8-diazabicyclo[3.2.1] octane-8-carboxylate (35) was reacted with 2,5-dichloropyridine in the presence of triethylamine under refluxing conditions to give intermediate 36 which upon BOC removal with conc. HCl in MeOH yielded intermediate 37 as the hydrochloride salt. N-alkylation of intermediate 37 with alkylating agents 27 and 26 yielded compounds 18 & 19 respectively as depicted in Scheme 3. These free base compounds (18 & 19) were converted to their corresponding oxalate and HCl salts respectively. The synthesis of compound 20 was achieved in a single step by reacting alkylating agent 26 with 1-(pyridin-3-yl)piperazine (38) in the presence of K2CO3 and a catalytic amount of KI under reflux/microwave conditions in (DME) solvent as shown in (Scheme 4). Similarly, compounds 21 & 22 were obtained by N-alkylation of 1-(pyridin-3-yl) piperazine (38) and 1-(pyridin-4-yl)piperazine (39) with alkylating agent 27 under the above mentioned general alkylating conditions (Scheme 4). The free base forms of compounds 20–22 were subsequently converted to their oxalate salts.

Scheme 1.

Scheme 1.

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Reagents and conditions: (a) Ethylene glycol (EG), 140 °C, 2 h; (b) Zn(Hg), conc. HCl, toluene, reflux, 5 h; c) CH3CN, K2CO3 and KI, reflux, 12 h.

Scheme 4.

Scheme 4.

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Reagents and conditions: (a) K2CO3, KI, DME, Microwave assisted reaction (MW)/ reflux; 38 = 1-(pyridin-3-yl)piperazine; 39 = 1-(pyridin-4-yl)piperazine.

Scheme 2.

Scheme 2.

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Reagents and conditions: (a) Pd(OAc)2, K+(CH3)3CO−, EG, 80 °C, 4 h; (b) TFA, CH2Cl2, 0 °C, 12 h.; (c) CH3CN, K2CO3 and KI, reflux, 12 h, 33 = 1-(5-phenylpyridin-2-yl)piperazine; 34 = 1-(6-phenylpyridin-2-yl)piperazine.

Scheme 3.

Scheme 3.

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Reagents and conditions: (a) 2,5-dichloropyridine, Et3N, CH3CN, reflux, 12 h; (b) MeOH, conc. HCl, rt, 12 h; (c) 27, KI, NaHCO3, iPrOH, reflux, 12 h; (d) 26, KI, NaHCO3, iPrOH, reflux, 12 h.

3. Results and discussion

3.1. Receptor binding SAR

SYA16263 is 1-(4-(4-fluorophenyl)butyl)-4-(pyridin-2-yl)piperazine (5) with the pyridinyl piperazine moiety as a key pharmacophore impacting its interesting pharmacology. SYA16263 showed potential antipsychotic action through inhibition of apomorphine-induced climbing behavior in mice with an ED50 value of 3.88 mg/kg IP without producing any extra-pyramidal side effects (EPS) up to 19 times the ED50 value. SYA16263 has high blood brain barrier (BBB) permeability, activates both G-protein and β-arrestin signaling at the D2R which is similar to aripiprazole at the D2R and most importantly SYA16263 binds with a higher affinity at the 5HT1AR than at the D2R with selective activation and β-arrestin recruitment to the 5HT1AR without any significant interaction at the G-Protein dependent signaling pathway.19 In this study, we sought to study the structure-affinity relationships that impart the binding affinities at the dopamine and serotonin receptors often associated with antipsychotic properties. The structure is divided into 3 segments (A-C) (Fig 2) for purposes of evaluating the contributions of the selected moieties to affinity at the selected receptors.

Fig. 2.

Fig. 2.

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SYA16263 segmented for purposes of evaluation of its structure–activity relationships.

We began the study by focusing on the pyridine moiety (Series I), obtaining several analogs (7–11), and then screening them at selected receptors. The results are reported in Table 1. Introduction of a 5-chloro atom on the pyridine ring (7) did not produce any significant changes to binding affinities at the selected DA receptor subtypes (D2, D3 & D4). Interestingly, there was a shift in the binding affinity from 5-HT1A to 5-HT2A receptor subtypes for 7 (Ki = 75.0 nM & 9.2 nM for 5-HT1A and 5-HT2A respectively) when compared to the binding profile of SYA16263 (Ki = 1.1 nM & 50 nM for 5-HT1A and 5-HT2A respectively).

Table 1.

aBinding affinities of series I analogs at DA and 5-HT receptors, and SERT.

graphic file with name nihms-1762376-t0001.jpg
Comp Ki nM/(mean pKi ± SEM)
D2 D3 D4 5-HT1A 5-HT2A 5-HT2C 5-HT7 SERT
5 b 124 ± 10 86 ± 4 3.5 ± 0.2 1.1 ± 0.05 50 ± 3 MT 90 ± 4 MT
6 229.0 (6.64 ± 0.06) 89.7 (7.12 ± 0.09) 6.7 (8.2 ± 0.07) 8.1 (8.12 ± 0.05) 62.7 (7.34 ± 0.06) 1,540.3 (5.87 ± 0.07) 120.5 (6.94 ± 0.1) MT
7 193.0 (6.71 ± 0.03) 83.0 (7.08 ± 0.03) 4.8 (8.32 ± 0.04) 75.0 (7.13 ± 0.07) 9.2 (8.04 ± 0.03) 1,024.0 (5.99 ± 0.04) 159.0 (6.8 ± 0.1) MT
8 287.0 (6.54 ± 0.03) 86.0 (7.06 ± 0.03) 5.1 (8.29 ± 0.04) 34.0 (7.47 ± 0.07) 46.0 (7.34 ± 0.03) 1,540.0 (5.81 ± 0.04) 207.0 (6.68 ± 0.1) MT
9 1861.0 (5.7 ± 0.1) 156.0 (6.8 ± 0.1) 23.0 (7.64 ± 0.06) 92.0 (7.03 ± 0.05) 138.0 (6.86 ± 0.08) 1607.0 (5.79 ± 0.08) 60.0 (7.2 ± 0.1) 2987.0 (5.52 ± 0.1)
10 >10,000 (<5) 2089.0 (5.7 ± 0.1) 2256.0 (5.65 ± 0.08) 1461.0 (5.84 ± 0.06) 147.0 (6.83 ± 0.07) >10,000 (<5) >10,000 (<5) 2667.0 (5.57 ± 0.1)
11 641.0 (6.19 ± 0.09) 58.0 (7.2 ± 0.1) 84.0 (7.08 ± 0.07) 155.0 (6.81 ± 0.05) 261.0 (6.58 ± 0.08) 2959.0 (5.53 ± 0.05) 160.0 (6.8 ± 0.1) >10,000 (<5)
Abilify c 3.3 ± 0.1 9.7 ± 5.4 510 ± 93 5.6 ± 0.8 8.7 ± 2.0 180 ± 37 10.3 ± 3.7 1080 ± 180
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a

Each result represents the mean of at least two determinations, each in triplicates.

b

Compound 5 Ki values, previously reported in reference17.

c

Aripiprazole (Abilify) data was obtained from reference4. MT = missed primary assay threshold of 50% inhibition at 10 μM.

Similar observation was made where the alkyl group on the 5-chloropyridinyl piperazine moiety is the butyrophenone group (8) or when the butyrophenone is on a deschloropyridinyl piperazine moiety, compound 6. Replacing the electron withdrawing chloro with an electron donating methyl group (9) led to 4–10-fold decrease in binding at the dopamine receptor subtypes evaluated. Replacing the methyl group with a bulky but flat phenyl group (10) essentially abolished binding at the D2, 5HT2C and 5-HT7A receptors while also decreasing binding at all other receptors reported in Table 1. However, moving the phenyl group to the 6-position (11) restored much of the lost affinity to all the receptors. These observations would suggest that there is a limit to the hydrophobic pocket in which the pyridine ring of 5 resides as the para-substituted phenyl ring appears to bump into parts of the receptor. We intend to test this hypothesis in future studies.

Next, section B and C (Series II, III and IV) of the scaffold were modified whereby the piperazine ring was replaced either by a homopiperazine or a diazabicyclooctane ring in order to compare their contributions to binding affinities at the selected receptors reported in Table 2. Comparing homopiperazine 12 to the corresponding piperazine analog (7) shows a very significant difference in binding affinities across most of the receptors evaluated (Table 1 and Table 2). In the case of the 5-HT1AR, the difference is about 500-fold in favor of 7. Introducing a 5-chloro on the pyridine ring of 13 resulted in an improved binding at all the D2-like receptors. In addition, this compound demonstrated high affinity for the 5-HT2AR (Ki = 8.9 nM) and SERT (Ki = 37.0 nM) and appears to make compound 13 a potential agent for further exploration.

Table 2.

aBinding affinities of series II, III, and IV analogs at DA and 5-HT receptors, and SERT.

graphic file with name nihms-1762376-t0002.jpg
Comp Ki nM/(mean pKi ± SEM)
D2 D3 D4 5-HT1A 5-HT2A 5-HT2C 5-HT7 SERT
12 181 (6.7 ± 0.1) 3447 (5.46 ± 0.08) 257 (6.6 ± 0.1) 1964 (5.72 ± 0.07) 1.2 (8.91 ± 0.07) 337 (6.47 ± 0.08) 569 (5.81 ± 0.08) MT
13 41.0 (7.39 ± 0.03) 26.0 (7.58 ± 0.03) 37.0 (7.43 ± 0.06) 259.0 (6.59 ± 0.06) 8.9 (8.05 ± 0.05) 392.0 (6.41 ± 0.04) 214.0 (6.67 ± 0.05) 37.0 (7.43 ± 0.06)
14 649 (6.19 ± 0.06) 2287 (5.6 ± 0.1) 191 (6.72 ± 0.04) 886 (6.1 ± 0.1) 59 (7.23 ± 0.05) MT 58 (7.23 ± 0.07) 949 (6.03 ± 0.05)
15 599 (6.22 ± 0.06) 774 (6.1 ± 0.1) 104 (6.98 ± 0.04) 614.5 (6.23 ± 0.1) 43 (7.37 ± 0.05) 963 (6.02 ± 0.08) 267 (6.57 ± 0.07) 64 (7.19 0.05)
16 364 (6.44 ± 0.03) 125.0 (6.90 ± 0.03) 36 (7.44 ± 0.06) 209 (6.68 ± 0.06) 13 (7.88 ± 0.03) 355 (6.45 ± 0.04) 58 (7.24 ± 0.04) 24 (7.63 ± 0.06)
17 1021 (5.99 ± 0.06) 590 (6.23 ± 0.04) 37 (7.43 ± 0.05) 415 (6.38 ± 0.05) 192 (6.72 ± 0.04) 3775 (5.42 ± 0.08) 72 (7.15 ± 0.06) 24 (7.62 ± 0.07)
18 MPA 840.3 ± 90.4 44.0 ± 5.0 94.2 ± 48.2 94.0 ± 7.6 MT 339.4 ± 16.8 158.4 ± 3.21
19 1249 (5.9 ± 0.1) 1009 (6.0 ± 0.1) 119.0 (6.92 ± 0.09) 78.0 (7.11 ± 0.05) 51.0 (7.29 ± 0.05) 1362 (5.87 ± 0.07) 335.0 (6.48 ± 0.06) 241.0 (6.6 ± 0.1)
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a

Each result represents the mean of at least two determinations, each in triplicates. MT = missed primary assay threshold of 50% inhibition at 10 μM.

When the carbonyl in 13 was restricted by the introduction of a fluoro-indanone moiety (14), affinity at the D2R was reduced by over 3-fold while that at 5-HT2AR was reduced by almost 30-fold. Compound 15 without the carbonyl did not result in any significant improvements in binding affinities at the same receptors. Compound 16, with a chloro atom on the pyridine ring, resulted in improvement in affinity across all receptors. Replacing the chloropyridine moiety with a chlorophenyl moiety (17) did not affect binding to the D4, 5-HT7 or SERT receptors, but significantly resulted in reduction at the other receptors evaluated. Disappointingly, replacement of the piperazine ring in compound 5 with a diazabicyclooctane ring to form the 5-chloropyridine analog (18) or the butyrophenone analog of 18 (compound 19) did not result in any significant improvements in binding affinities at the indicated receptors (Table 2), except for a moderate D4 and 5-HTR subtype binding. Given the diazabicyclooctane ring can be considered a restricted piperazine ring in the chair conformation, such a conformational restriction appears relatively unproductive as a strategy to improve binding at dopamine and serotonin receptors.

Table 3 reports the binding affinities of other pyrido-piperazine analogs of azaperone and SYA16263 (Series V) with the position of the nitrogen atom in the pyridine ring moved from the 2 to the 3-position of azaperone (20) or SYA16263 (21) and to the 4-position of SYA16263 (22). Screening of compound 20 at the receptors of interest shows that affinity was decreased by about 3–24 fold across all receptors and similarly for compound 21. However, the binding profile of compound 21 is very interesting as this compound is able to differentiate among binding at the D2-like receptors. For example, while there is essentially no binding affinity at the D2R, 21 binds moderately to the D3, D4 and 5-HT1A receptors. This observation led us to explore the question; is the D2R essential for the display of APO-induced climbing behavior inhibition of antipsychotics as demonstrated by SYA16263? It is also intriguing to observe that the pyridine ring with the nitrogen atom at the 4-position (22) resulted in essentially no binding affinity at any of the dopamine and serotonin receptors. Given the high homology in the amino acid sequence among D2-like receptor subtypes (D2, D3 and D4),25,26 these observations could form the basis of identifying new agents that distinguish binding at the D2-like receptors.

Table 3.

aBinding affinities of series V analogs at DA and 5-HT receptors, and SERT.

graphic file with name nihms-1762376-t0003.jpg
Comp Ki nM /(mean pKi ± SEM)
D2 D3 D4 5-HT1A 5-HT2A 5-HT2C 5-HT7 SERT
6 228 (6.64 ± 0.1) 89.7 (7.12 ± 0.1) 6.7 (8.2 ± 0.1) 8.1 (8.12 ± 0.1) 62.7 (7.34 ± 0.1) 1,540.3 (5.87 ± 0.1 120.5 (6.94 ± 0.1) MT
20 1168 (5.93 ± 0.06) 140 (6.85 ± 0.05) 30 (7.53 ± 0.05) 97 (7.01 ± 0.07) 305 (6.52 ± 0.05) MT 147 (6.87 ± 0.1) MT
21 MT 54 (7.27 ± 0.05) 16 (7.78 ± 0.07) 18 (7.76 ± 0.07) 338 (6.50 ± 0.06) MT 267 (6.63 ± 0.09) MT
22 MT MT 2054 (5.69 ± 0.05) MT MT MT MT 2204 (5.66 ± 0.08)
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a

Each result represents the mean of at least two determinations, each in triplicates. MT = missed primary assay threshold of 50% inhibition at 10 μM.

3.2. In-vivo reversal of apomorphine-induced climbing

Apomorphine is a mixed dopamine agonist which activates D2-like receptors and to a lesser extent of D1-like receptors. Apomorphine at doses higher than 0.5 mg/kg is known to stimulate motor activity due to the stimulation of postsynaptic DA receptors (D1-like and D2-like). However, the same apomorphine at doses lower than 0.5 mg/kg prevents motor activity owing to its presynaptic D2-like receptors activation. Such activation results in climbing and stereotyped behaviors in mice. These apomorphine-induced climbing behaviors are usually antagonized by typical antipsychotic drugs either by DA receptor blockade or activation of other receptors.27 To explore the question of, whether D2 receptor binding is required for a compound to reverse apomorphine-induced climbing behavior, we evaluated the effect of compound 21 on this behavior in mice and compared it to haloperidol and SYA16263 as displayed in Fig 3. The results indicate that compound 21 inhibits APO-induced climbing behavior in a dose dependent manner with an ED50 of 7.31 mg/kg (95% CI 6.54 to 8.17 mg/kg, n = 5 mice per dose) compared to that of SYA16263 of 3.88 mg/kg and haloperidol of 0.06 mg/kg.12 Given the report by Davis et al.28 which suggests both D1 and D2 receptors may play a role in the inhibition of apomorphine-induce climbing behavior in mice, we screened compound 21 and showed that it binds poorly to the D1 receptor with a mean Ki > 2000 nM and therefore is unlikely to be a factor in this inhibition. Thus, these results suggest that compound 21, even without any significant binding to the D2 receptor but with moderate binding affinities at the D3 and D4 receptors, produces similar pharmacological effect albeit with lower potency, in an animal model often used to investigate antipsychotic activity. This result has intensified our quest to obtain compounds which very selectively interact with the dopamine receptor subtypes with a view to further identifying their true contributions to antipsychotic drug development.

Fig. 3.

Fig. 3.

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Inhibition of APO-induced climbing behavior in Swiss-Webster mice, n = 5/dose, by compound 21, SYA16263 and haloperidol.

4. Experimental protocols

4.1. Chemical syntheses

4.1.1. Materials and methods

Melting points were determined on a Gallenkamp (UK) apparatus and are uncorrected. All NMR spectra were obtained on a Varian 300 MHz Mercury 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. Elemental analyses were carried out by Atlantic Microlab, Inc., Norcross, GA, and are within 0.4% of theory unless otherwise noted. Flash chromatography was performed using CombiFlash with Davisil grade 634 silica gel. Starting materials were obtained from Sigma–Aldrich and were used without further purification. All microwave assisted syntheses (MW) were carried out using a Biotage Initiator. Yields reported are not optimized values.

4.1.2. Synthesis of compounds 9–11, general

The alkylating agent 27 was previously reported by us20 and was used to alkylate 1-(5-methylpyridin-2-yl)piperazine 25, 1-(5-phenylpyridin-2-yl)piperazine 33, and 1-(6-phenylpyridin-2-yl)piperazine 34, to afford compounds 9–11. Syntheses of compounds 7, 8 & 12–17 were reported previously.20, 22, 23 and azaperone (6) was obtained from Sigma-Aldrich (USA).

4.1.3. 1-(4-(4-fluorophenyl)butyl)-4-(5-methylpyridin-2-yl)piperazine (9)

To obtain compound 25 (1-(5-methyl-pyridin-2-yl)piperazine), the method reported by Asagarasu et al.24 was followed. Briefly, piperazine (23) (10.0 mmol) was dissolved in ethylene glycol (30 mL) and 2-bromo-5-methylpyridine (24) (1.0 mmol) was added. The mixture was stirred at 140 °C for 2 h and allowed to cool to room temperature. Thereafter, saturated aqueous sodium hydrogen carbonate solution was added until the solution turned basic and the mixture was extracted with DCM (3 × 50 mL). The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a Teledyne CombiFlash instrument to elute the desired compound 25 with DCM:MeOH = 2:1 in a quantitative yield. Compound 25 was dissolved in CH3CN (10 mL), K2CO3 (5 eq) and KI (0.05 g) were added, and the mixture was stirred for 30 min. 1-(4-chrorobutyl)-4-fluorobenzene (27) in CH3CN (5 mL) was slowly added at room temperature to the reaction mixture and the mixture was refluxed overnight. The reaction was brought to room temperature, filtered, and the filtrate concentrated to obtain a residue. The residue was dissolved in EtOAc (50 mL), washed with brine twice (2 × 25 mL). The organic layer was collected, dried over anhydrous Na2SO4, filtered, the filtrate evaporated and the residue subjected to column chromatography (CombiFlash) starting with hexane as the eluent and gradually increasing the polarity with EtOAc. At 70% EtOAc/30% hexane, the product, 1-(4-(4-fluorophenyl)butyl)-4-(5-methylpyridin-2-yl)piperazine (9) was isolated in the pure form as white crystals in a yield of 26%.

Mp. 74 °C. 1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 2.7 Hz, 1H), 7.29, dd, J = 8.7 & 2.7 Hz, 1H), 7.14–7.09(m, 2H), 6.98–6.92 (m, 2H), 6.59 (d, J = 8.7 Hz, 1H), 3.50–3.46 (m, 4H), 2.63–2.52 (m, 6H), 2.39 (t, J = 7.8 Hz, 2H), 2.18 (s, 3H), 1.69–1.55 (m, 4H). 13C NMR (300 MHz, CDCl3) δ 162.78, 159.56, 158.11, 147.67, 138.38, 137.93, 129.68, 122.37, 115.12, 114.84, 106.99, 58.59, 53.09 (2C), 45.64 (2C), 34.98, 29.51, 26.34, 17.35. Anal. Calc. for C20H26N3F: C, 73.36; H, 8.00; N, 12.83. Found: C, 73.79; H, 8.28; N, 12.47.

4.1.4. 1-(4-(4-fluorophenyl)butyl)-4-(5-phenylpyridin-2-yl)piperazine (10) and 1-(4-(4-fluorophenyl)butyl)-4-(6-phenylpyridin-2-yl)piperazine hydrochloride (11)

4.1.4.1. General procedure for the synthesis of intermediates, 1-(5-phenylpyridin-2-yl)piperazine; (33) and 1-(6-phenylpyridin-2-yl)piperazine (34).

Boc-protected piperazines 28 and 29 (2.73 mmol) were each dissolved in ethylene glycol (15 mL), and then phenylboronic acid (30) (4.1 mmol), potassium tert-butoxide (5.46 mmol), and a catalytic amount of Pd(OAc)2 (0.27 g) were added. The mixture was heated at 80 °C for 4 h. The reaction was stopped and allowed to return to room temperature when brine (10 mL) and Et2O (25 mL) were added, and the resulting suspension was filtered over celite. The organic layer was separated, dried over anhydrous Na2SO4, filtered, and the filtrate concentrated. The resulting residue was subjected to Combi-flash column chromatography, starting with hexane and increasing the polarity with EtOAc until at 30% EtOAc the Boc protected compounds 31 and 32 eluted and were then isolated in pure forms.

4.1.4.1.1. tert-butyl 4-(5-phenylpyridin-2-yl)piperazine-1-carboxylate (31).

Viscous liquid., 1H NMR (300 MHz, CDCl3) δ 8.45 (s, 1H), 7.81–7.70 (m, 1H), 7.55–7.49 (m, 2H), 7.45–7.40 (m, 2H), 7.34–7.30 (m, 1H), 6.75 (d, J = 8.4 Hz, 1H), 3.58 (s, 8H), 1.49 (s, 9H).

4.1.4.1.2. tert-butyl 4-(6-phenylpyridin-2-yl)piperazine-1-carboxylate (32).

Viscous liquid., 1H NMR (300 MHz, CDCl3) δ 8.01–7.92 (m, 2H), 7.6–7.56 (m, 1H), 7.47–7.38 (m, 3H), 7.15 (d, J = 7.5 Hz, 1H), 6.64–6.61 (m, 1H), 3.65–3.58 (m, 8H), 1.49 (s, 9H).

4.1.4.1.3. Deprotection of 31 and 32.

Compounds 31 and 32 were each deprotected by dissolving in CH2Cl2 (5 mL), addition of TFA (1 mL) at 0 °C and the reaction mixture was stirred overnight. Reaction was stopped, solvent evaporated, and the residue dissolved in DCM (10 mL). The residue was washed with saturated NaHCO3 solution, the organic layer collected, dried over anhydrous Na2SO4, filtered, and the filtrate concentrated to give the desired compounds in 62% overall yield. Without further purification, the crude products, 33 and 34 were used as such for the next reaction.

4.1.4.1.4. 1-(5-Phenylpyridin-2-yl)piperazine (33).

Viscous Liq., 1H NMR (300 MHz, CDCl3) δ 8.38 (d, J = 2.7 Hz, 1H), 7.65 (dd, J = 8.7 Hz & 2.4 Hz, 1H), 7.47–7.43 (m, 2H), 7.38–7.33 (m, 2H), 7.27–7.23 (m, 1H), 6.66 (d, J = 8.7 Hz, 1H), 3.50 (t, J = 5.1 Hz, 4H), 2.96 (t, J = 5.1 Hz (4H).

4.1.4.1.5. 1-(6-Phenylpyridin-2-yl)piperazine (34).

Viscous Liq., 1H NMR (300 MHz, CDCl3) δ 7.94–7.91 (m, 2H), 7.47–7.42 (m, 1H), 7.38–7.22 (m, 3H), 7.02 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 8.7 Hz), 3.51 (t, J = 5.1 Hz, 4H), 2.91 (t, J = 5.1 Hz, 4H).

4.1.4.2. 1-(4-(4-fluorophenyl)butyl)-4-(5-phenylpyridin-2-yl)piperazine (10) and 1-(4-(4-fluorophenyl)-butyl)-4-(6-phenylpyridin-2-yl)piperazine HCl (11).

1-(5-Phenylpyridin-2-yl)piperazine (33) or 1-(6-phenylpyridin-2-yl)piperazine (34) were each reacted with alkylating agent, 1-(4-chlorobutyl)-4-fluorobenzene (27) under general alkylating conditions as described above to give the target compound 10 as a white crystal with a yield of 56%, and target compound 11 as a white solid with a yield of 42% after conversion to the hydrochloride salt.

4.1.4.2.1. 1-(4-(4-fluorophenyl)butyl)-4-(5-phenylpyridin-2-yl)piperazine (10).

Yield: 56%, mp. 130.5 °C. 1H NMR (300 MHz, CDCl3) δ 8.45 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.7 & 2.7 Hz, 1H), 7.53–7.49 (m, 2H), 7.44–7.40 (m, 2H), 7.33–7.28(m, 1H), 7.15–7.11(m, 2H), 7.0–6.93(m, 2H), 6.73 (d, J = 8.7 Hz, 1H), 3.61 (m, 4H), 2.64–2.55 (m, 6H), 2.42 (t, J = 7.5 Hz, 2H), 1.69–1.50 (m, 4H). 13C NMR (300 MHz, CDCl3) δ 162.80, 159.58, 158.66, 146.16, 138.33 (2C), 137.95, 137.91, 128.73, 128.63, 128.90, 126.76, 126.26, 126.20, 115.14, 114.87, 106.81, 58.56, 53.05 (2C), 45.18 (2C), 34.97, 29.48, 26.30. Anal. Calc. for C25H28N3F: C, 77.09; H, 7.25; N, 10.79. Found: C, 76.84; H, 7.30; N, 10.68.

4.1.4.2.2. 1-(4-(4-fluorophenyl)butyl)-4-(6-phenylpyridin-2-yl)piperazine, HCl (11).

Yield: 42% mp. 88–89 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.10 (s, 1H), 8.04 (d, J = 7.2 Hz, 2H), 7.69 (t, J = 7.5 Hz, 1H), 7.45–7.20 (m, 6H), 7.09 (t, J = 8.9 Hz, 2H), 6.93 (d, J = 8.1 Hz, 1H), 4.49 (d, J = 13.2 Hz, 2H), 3.54–3.33 (m, 5H), 3.08–3.01 (m, 5H), 1.8–1.52 (m, 4H). 13C NMR (300 MHz, DMSO-d6) δ 162.68, 159.49, 158.03, 154.32, 139.50, 139.21, 138.19, 138.15, 130.57, 130.47, 129.34, 129.06, 126.84, 115.55, 115.28, 110.76, 107.10, 55.65, 50.79 (2C), 42.21 (2C), 34.05, 28.60, 23.00. Anal. Calc. for C25H28N3F.2HCl·0.7H2O: C, 63.21; H, 6.37; N, 8.85. Found: C, 63.23; H, 6.80; N, 8.78.

4.1.5. 3-(5-chloropyridin-2-yl)-8-(4-(4-fluorophenyl)butyl)-3,8-diazabicyclo[3.2.1]octane (18)

4.1.5.1. tert-butyl 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (36).

A mixture of tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (35) (0.75 g, 3.5 mmole), 2,5-dichloropyridine (0.63 g, 4.3 mmole), Et3N (2 mL) in CH3CN (10 mL) was refluxed with stirring for 12 hrs. After cooling to rt, the mixture was diluted with EtOAc (200 mL), followed by washing with water (2 × 50 mL). The organic layer was dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to dryness, followed by column chromatography on silica gel to afford tert-butyl 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]-octane-8-carboxylate (36) (0.76 g) in a yield of 67%.

1H NMR (CDCl3, 300 MHz): 8.09 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 2.7, 9.3 Hz, 1H), 6.49 (d, J = 9.3 Hz, 1H), 4.34 (brs, 2H), 3.81 (brs, 2H), 1.96–1.92 (m, 2H), 1.78–1.71 (m, 2H), 1.46 (s, 9H).

4.1.5.2. 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octane hydrochloride (37).

To a solution of tert-butyl 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (36) (0.76 g, 2.3 mmole) in MeOH (10 mL) was added with stirring conc. HCl (4 mL) dropwise at room temperature. The solution was stirred at room temperature for 12 hrs and the solvent was removed in vacuo till dryness. The residue was dried under vacuum for 24 hrs and the product, 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octane hydrochloride (37) was used for the following coupling reaction without further purification.

1H NMR (DMSO-d6, 300 MHz): 9.59 (brs, 1H), 9.50 (brs, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 3.0, 9.3 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 4.10 (brs, 2H), 4.02 (d, J = 13.5 Hz, 2H), 3.22 (d, J = 12.9 Hz, 2H), 1.98–1.92 (m, 2H), 1.81–1.74 (m, 2H).

4.1.5.3. 3-(5-chloropyridin-2-yl)-8-(4-(4-fluorophenyl)butyl)-3,8-diazabicyclo[3.2.1]octane (18).

A mixture of 1-(4-chlorobutyl)-4-fluorobenzene (27) (1.0 g, 5.4 mmol), 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]-octane hydrochloride (37) (1.2 g, 4.6 mmol), KI (100 mg), and NaHCO3 (1.0 g, 11.9 mmol) in iPrOH (10 mL) was heated to reflux under N2 for 12 h. After cooling to rt, the mixture was diluted with EtOAc (500 mL) and then washed with water (2 × 300 mL). The organic layer was removed, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to dryness, and column chromatography on silica gel afforded 3-(5-chloropyridin-2-yl)-8-(4-(4-fluorophenyl)butyl)-3,8-diazabicyclo[3.2.1]octane (18). The free base was converted to the oxalate salt and the crystals were obtained by recrystallization from MeOH/Et2O in a yield of 18%.

Mp: 175–176 °C, 1H NMR (DMSO-d6, 300 MHz): 8.12 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 3.0, 12.0 Hz, 1H), 7.23 (dd, J = 6.0, 8.1 Hz, 2H), 7.08 (t, J = 8.7 Hz, 2H), 6.84 (d, J = 9.3 Hz, 1H), 4.12–3.95 (m, 4H), 3.37–3.26 (m, 2H), 3.07–2.88 (m, 2H), 2.65–2.57 (m, 2H), 3.27–2.08 (m, 2H), 1.86–1.79 (m, 2H), 1.72–1.68 (m, 2H), 1.64–1.54 (m, 2H). 13C NMR (DMSO-d6, 150 MHz): 164.2 (2C), 161.6 (d, JC-F = 239.8 Hz), 157.9, 145.9, 138.2 (d, JC-F = 3.3 Hz, 2C), 137.7, 130.4 (d, JC-F = 7.7 Hz, 2C), 120.3, 115.4 (d, JC-F = 20.8 Hz), 108.8, 60.1 (2C), 50.3 (2C), 48.2, 34.1, 28.5, 24.1 (2C), 23.9. Anal. Calc. for C24H28ClFN3O6: C 56.64, H 5.55, N 8.26; Found: C 56.67, H 5.57, N 8.30.

4.1.6. 4-(3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-(4-fluorophenyl)butan-1-one (19)

A mixture of 4-chloro-1-(4-fluorophenyl)butan-1-one (26) (1.1 g, 5.5 mmol), 3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octane hydrochloride (37) (1.2 g, 4.6 mmol), KI (100 mg), NaHCO3 (1.0 g, 11.9 mmol) in iPrOH (10 mL) was heated to reflux under N2 for 12 h. After cooling to rt, the mixture was diluted with EtOAc (500 mL) and then washed with water (2 × 300 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo to dryness. Purification by column chromatography on silica gel afforded 4-(3-(5-chloropyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-(4-fluorophenyl)butan-1-one (19). The free base was converted to HCl salt. The crystals were obtained by recrystallization from MeOH/Et2O in a yield of 21%.

Mp: 263–264 °C, 1H NMR (DMSO-d6, 300 MHz): 11.08 (brs, 1H), 8.13 (d, J = 3.0 Hz, 1H), 8.05 (dd, J = 6.3, 8.7 Hz, 2H), 7.66 (dd, J = 3.0, 9.0 Hz, 1H), 7.36 (t, J = 9.0 Hz, 2H), 6.87 (d, J = 9.6 Hz, 1H), 4.21 (brs, 2H), 4.08 (d, J = 13.5 Hz, 2H), 3.55 (d, J = 12.6 Hz, 2H), 3.23–3.19 (m, 2H), 3.08–3.00 (m, 2H), 2.16–2.08 (m, 4H), 1.87–1.80 (m, 2H). 13C NMR (DMSO-d6, 150 MHz): 197.9, 165.5 (d, JC-F = 250.1 Hz), 158.0, 145.9, 137.8, 133.7, 131.3 (d, JC-F = 9.5 Hz, 2C), 120.4, 116.2 (d, JC-F = 21.8 Hz, 2C), 108.9, 60.7 (2C), 50.9 (2C), 48.8, 35.7, 23.9 (2C), 18.9. Anal. Calc. for C21H24Cl2FN3O·0.5H2O: C 58.21, H 5.82, N 9.70; Found: C 58.43, H 5.58, N 9.72.

4.1.7. Synthesis of compounds (20–22)

4.1.7.1. General procedure.

Alkylating agent 26 was reacted with 1-(pyridin-3-yl) piperazine (38), and alkylating agent 27 was reacted with either 1-(pyridin-3-yl) piperazine (38) or 1-(pyridin-4-yl)piperazine (39) under the general alkylation conditions, i.e. in the presence of K2CO3 and a catalytic amount of KI under reflux or under microwave conditions in dimethoxyethane to afford compounds 20–22 respectively. The free base forms of compounds 20–22 were subsequently converted to their oxalate salts.

4.1.7.2. 1-(4-fluorophenyl)-4-(4-(pyridin-3-yl)piperazin-1-yl)butan-1-one oxalate (20).

Yield: 48%, mp: 137–138 °C; 1H NMR (300 MHz, CD3OD) δ 8.48 (s, 1H), 8.22 (d, J = 5.1 Hz, 1H), 8.11–8.00 (m, 4H), 7.77 (dd, J = 8.6, 5.1 Hz, 1H), 7.22 (t, J = 8.5 Hz, 2H), 3.75 (s, 5H), 3.54 (s, 4H), 3.23 (d, J = 7.2 Hz, 3H), 2.20 (s, 2H). 13C NMR (151 MHz, CH3OD) δ 197.28, 166.77, 165.09, 162.74, 162.30, 161.37 (2C), 147.60, 133.23, 133.13, 133.11, 130.67, 130.61, 130.20, 129.07, 126.51, 115.35, 115.21, 56.11, 44.40 (4C), 34.55, 17.93. Anal. Calcd. for C25H30FN3O14: C, 48.78; H, 4.91; N, 6.83. Found: C, 48.55; H, 4.58; N, 6.55.

4.1.7.3. 1-(4-(4-fluorophenyl)butyl)-4-(pyridin-3-yl)piperazine oxalate (21).

Yield: 43%, mp: 134–135 °C; 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 8.13 (s, 1H), 7.20–7.09 (m, 4H), 7.00–6.93 (m, 2H), 3.40 (s, 5H), 2.85 (s, 3H), 2.63 (t, J = 7.1 Hz, 4H), 1.68 (s, 4H). 13C NMR (151 MHz, MeOD) δ 163.36 (4C), 147.27, 137.27, 137.25, 134.83, 131.80, 129.68, 129.63, 127.97, 125.97, 114.68, 114.54, 56.53, 50.98 (2C), 44.63 (2C), 33.80, 28.05, 22.97. Anal. Calcd. for C24.4H29.4FN3O10.8: C, 52.66; H, 5.32; N, 7.55. Found: C, 52.68; H, 5.26; N, 7.58.

4.1.7.4. 1-(4-(4-fluorophenyl)butyl)-4-(pyridin-4-yl)piperazine oxalate (22).

White crystals. Yield: 48%, MP: 167–169 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.06 (s, 5H), 8.29 (s, 2H), 7.33–6.95 (m, 6H), 3.99–3.56 (m, 4H), 3.01–2.95 (m, 2H), 2.85–2.75 (m, 2H), 2.68–2.51 (m, 2H), 2.48 (t, J = 3.8 Hz, 2H), 1.73–1.39 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 164.13 (4C), 162.62, 159.43, 156.86, 141.32, 138.36, 138.32, 130.50, 130.40, 115.49, 115.21, 108.34, 56.38, 51.21 (2C), 44.40 (2C), 34.17, 28.78, 24.18. Anal. Calc. for C19H24FN3·2.6(COOH)2: C, 53.09; H, 5.38; N, 7.67. Found: C, 53.11; H, 5.54; N, 7.75.

4.2. Pharmacology

4.2.1. Receptor binding

Receptor binding affinities reported in Tables 1, 2 and 3 were conducted by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) and details of the methods and radioligands used for the binding assays were previously reported.4

4.2.2. Materials and methods

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Compound 21 (CLog P = 4.36) was synthesized and characterized by us at Florida A & M University with CHN values within 0.4% of theoretical values as determined by CHN analysis. The compounds were dissolved in filtered (0.22μ) 1% lactic acid vehicle for all of the animal studies. Haloperidol and apomorphine hydrochloride hemihydrate (APO), were obtained from Sigma-Aldrich. Haloperidol and aripiprazole were dissolved in filtered (0.22μ) 1% lactic acid. APO was dissolved in HPLC grade water, followed by ascorbic acid (0.1% w/ w) dissolution followed by sodium chloride (0.9% w/w) on the morning of the experiments in an amber vial. Lactic acid (ACROS), ascorbic acid, phosphate buffered saline (PBS) (Fisher) and sodium chloride (Fisher) were ACS reagent grade. The d-amphetamine hemisulfate salt was from Sigma-Aldrich, and the water used to make solutions was HPLC grade. Doses are expressed as the free base for all compounds, and were given in a volume of 10 mL/kg by intraperitoneal (ip) injection, except for apomorphine, which was given by subcutaneous (sc) injection. The CLog P values in this report were calculated using ChemDraw Ultra, version 12.0 obtained from CambridgeSoft.

4.2.3. Animals

Reversal of apomorphine-induced climbing behavior experiments were performed using male, albino, Swiss-Webster mice (24–31 g), (5–7 weeks old). All animals were from Harlan Laboratories, Inc. Animals were housed in the Florida A & M University Animal Care facility which is fully AAALAC accredited, and operates with a 12 h light/dark cycle and controlled temperature (24 ± 2 °C). Animals were given free access to food and water and at least 5 days to adjust before experiments were begun. Animals were fasted the night before each experiment. All experimental procedures were performed in accordance with protocols approved by the Florida A & M University Institutional Animal Care and Use Committee.

4.2.4. Reversal of apomorphine-induced climbing

A modified climbing test29,30 was used with Swiss-Webster mice to predict potential antipsychotic-like activity. Inhibition of the apomorphine-induced climbing indicates antipsychotic-like properties. Five mice per dose were injected (IP) first with haloperidol, SYA16263, compound 21, or vehicle and returned to their home cage, then thirty minutes later injected (SC) with 1.5 mg/kg (as free base) apomorphine, and placed in cylindrical wire cages (12 cm diameter, 14 cm height) and observed for climbing behavior at 10 and 20 min after the apomorphine injection. Climbing behavior was assessed as follows: For all 4 paws up on the cage wall, not on the floor, a score of 2 was assigned (0% inhibition). For 3 or 2 paws on the cage wall, a score of 1 was assigned. For 1 or 0 paws on the cage wall, a score of 0 was assigned (100% inhibition). Scores were expressed as mean percent climbing inhibition and plotted in Fig. 3. Prism 5.03, GraphPad software, Inc., non-linear regression software was used to calculate ED50.

5. Conclusion

This manuscript describes the structure-affinity relationship study and a search for compounds that have selective binding affinities for D2-like and serotonin receptors. We now report one such compound, 21 which does not display significant binding affinity at the D2 receptor subtype and yet displays an inhibition of apomorphine-induced climbing behavior. This behavior has been used as a predictive test for potential antipsychotic agents. Thus, this observation calls for further evaluation of the use of this testing paradigm for the discovery of new antipsychotic agents based on their affinities for the D2 receptor alone.

Acknowledgements

This work was financially supported by an NIH/NIGMS SCORE grant number 2SC1GM116724 and a Title III Grant to Florida A&M University. We also acknowledge the financial support from Anxiolytech toward the ongoing studies. The work was also supported in part by the Pharmaceutical Research Center NIH/NCRR 1C06-RR12512-01 Grant. Ki determinations and receptor binding assays were generously carried out by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. Funding sources acknowledged had no involvement in the study design, data collection and interpretation, or article preparation and submission of this manuscript.

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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