Synthesis and dual D₂ and 5-HT_1A receptor binding affinities of 5-piperidinyl and 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones

Abstract

A series of new 5-piperidinyl and 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones have been synthesized and evaluated for dual D₂ and 5-HT_1A receptor binding affinities. The synthesized ligands are structurally related to bifeprunox, a potential atypical antipsychotic, having potent D₂ receptor antagonist and 5-HT_1A receptor agonist properties. The Suzuki–Miyaura reaction of cyclic vinyl boronate with appropriate aryl halide yielded arylpiperidine, which was eventually transformed to piperidinyl-1H-benzo[d]imidazol-2(3H)-one. The reductive amination of the latter with appropriate biarylaldehdyes rendered the synthesis of 5-piperidinyl-1H-benzo[d]imidazol-2(3H)-ones. Likewise, the Buchwald–Hartwig coupling reactions of 1-boc-piperazine with appropriate aryl halide and subsequent removal of the boc group rendered arylpiperazine. The reductive amination of the latter with appropriate biarylaldehdyes accomplished the synthesis of 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones. The structure–activity relationship studies showed that cyclopentenylpyridine and cyclopentenylbenzyl groups contribute significantly to the dual D₂ and 5-HT_1A receptor binding affinities of these compounds.

• 5-HT_1A receptor
• 5-piperidinyl-1H-benzo[d]imidazol-2(3H)-ones
• 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones
• D₂ receptor
• schizophrenia

Introduction

Schizophrenia is a severe psychiatric illness afflicting 1% of the population worldwide. Diagnosis is based on diverse and variably expressed symptoms which can be grouped as positive and negative. The positive symptoms include disorganized thought, delusions and auditory hallucinations, whereas the most characteristic negative symptoms are emotional flattening, poverty of speech and motivational deficits1. Although various molecular mechanisms have been proposed to provide antipsychotic activity2, antagonism of the dopamine D₂ receptor subtype remains the cornerstone of antipsychotic activity3,4. Chlorpromazine (1) and haloperidol (2a), the first-generation antipsychotics or typical antipsychotics, are dopamine antagonists and exhibit robust control of positive symptoms of schizophrenia, for instance hallucinations, agitation and delusions but fail to control the negative symptoms such as blunted affect, emotional withdrawal and cognitive deficits. In addition, motor retardation remains uncontrolled with these therapeutics for most schizophrenic patients5. Moreover, selective D₂ receptor antagonists block nigrostriatal dopaminergic activity, which leads to extrapyramidal symptoms (EPS), such as dystonia and dyskinesia, and also result in the blockade of pituitary-located D₂ receptors that control prolactin release, leading to hyperprolactinemia6^a. The “second-generation” or atypical antipsychotics, such as clozapine (2b), combine D₂ receptor antagonism with activity at other receptors, on the premise that a suitable balance of pharmacological activity should broaden the spectrum of therapeutic efficacy and reduce EPS. Clozapine exhibits partial agonist efficacy for 5-HT_1A receptor-mediated stimulation of G-protein activation6^b, which accounts for part of the activity of clozapine in a model of anxiolytic-like activity: clozapine inhibited stress-induced ultrasonic vocalization in rats, an effect attenuated by selective 5-HT_1A antagonist WAY-1006356^c. With respect to classical neuroleptics, clozapine shows significantly greater efficacy, including an improved effect on negative symptoms, and causes a marked increase in dopamine output in the prefrontal cortex7. Clozapine, however, is associated with its own set of serious side effects, including weight gain, diabetes and an increased risk of seizures and agranulocytosis8.

Although the utility of 5-HT_1A receptor agonism in the treatment of schizophrenia is clearly evident, the optimal level of activation of this target is debatable. To achieve improved overall therapeutic benefit, combining D₂ receptor blockade with 5-HT_1A receptor activation rather than antagonism has been a subject of recent research attention9,10. However, robust protection from EPS elicited by potent D₂ receptor blockade likely requires high-efficacy agonism at 5-HT_1A receptors11^a; whereas lower efficacy at 5-HT_1A receptors should be sufficient when combined with D₂ receptor partial agonism. Several preclinical observations suggest that combining 5-HT_1A and D₂ receptor properties may provide a mutually complementary balance of pharmacological activity with reduced undesirable responses11^b. Indeed, numerous mechanistic considerations12–14 and preclinical evidences15–17 support the potential of such a combination. Consequently, adoprazine (3) (SLV-313) and bifeprunox (4), bearing potent D₂ receptor antagonist and 5-HT_1A receptor agonist properties, were developed18 (Figure 1). Although compound 4 completed phase III clinical trials, its antipsychotic efficacy was determined to be inferior to that of risperidone and olanzapine9 and, despite a satisfactory tolerance profile, the US Food and Drug Administration (FDA) did not grant marketing approval. The lack of sufficient antipsychotic efficacy of 4 likely reflects its marked agonism at D₂ receptors. Thus, 4 suppresses basal firing rates of dopaminergic neurons in the ventral tegmental area and elicits circling behavior in rats unilaterally lesioned with 6-OH-DA9. In addition, the failure of 3 and 4 to oppose phencyclidine-induced social interaction deficits suggested that an appropriate “balance” of activity at these sites is necessary for activity in this model19. Thus, the need to discover compounds having varying ratios of D₂ and 5-HT_1A activities continued20.

Figure 1. Chlorpromazine, haloperidol, clozapine, adoprazine and bifeprunox.

In an ongoing effort to develop new antipsychotics21,22, we have synthesized a series of 5-piperidinyl-1H-benzo[d]imidazol-2(3H)-ones (5a–f) and 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones (6a–f), which are structural analogs of bifeprunox (4) (Figure 2). Herein, we wish to disclose the synthesis, dual D₂ and 5-HT_1A receptor binding affinities and structure–activity relationship (SAR) of these compounds.

Figure 2. 5-piperidinyl-1H-benzo[d]imidazol-2(3H)-ones (5a–f) and 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones (6a–f).

Materials and methods

Chemistry

Instrumentation: Melting points were determined on a Büchi apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. Elemental analysis was carried out on a Perkin Elmer Elemental Analyzer Series 11 Model 2400 (PerkinElmer Inc., Waltham, MA). IR spectra were recorded on a Perkin Elmer 16F PC FTIR spectrophotometer (PerkinElmer Inc., Waltham, MA). ¹H and ¹³C NMR spectra were measured in CDCl₃ and d₆-DMSO using TMS as internal standard on a JEOL JNM-LA 500 MHz spectrometer (JEOL USA Inc., Peabody, MA). Mass spectra were recorded on Agilent Technologies 6890N GC-MS system (Agilent Technologies Inc., Santa Clara, CA). Analytical TLC was carried out on silica gel 60 F₂₅₄ plates (E. Merck, Darmstadt, Germany); column chromatography was carried out on Merck silica gel (200–400 mesh, E. Merck).

di-tert-Butyl 5-bromo-2-oxo-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate (11): To a solution of compound 10 (0.4 g, 1.88 mmol) in anhydrous THF (15 mL) di-tert-butyl dicarbonate was added (1.64 g, 7.52 mmol) followed by the addition of 4-dimethylaminopyridine (0.23 g, 1.88 mmol) and the mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate (25 mL) and washed successively with water (15 mL), brine (15 mL), dried over Na₂SO₄ and evaporated. Column chromatography on silica gel, eluting with ethyl acetate:hexanes (10:90) and then the ratio was changed to 20:80 to obtain the title compound 11. Yield: 94%, colorless amorphous solid, m.p. 109 °C–110 °C. IR (KBr, cm⁻¹) 3041 (Ar-H), 2978 (Alph-H), 1691 (C=O), 1612, 1442 (C=C), 1222 (C-N), 1175 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.63 (s, 18H, OC(CH₃)₃), 7.33 (dd, J = 2.3, 8.4 Hz, 1H, H-6), 7.74 (d, J = 8.4 Hz, 1H, H-7), 8.08 (d, J = 2.4 Hz, 1H, H-4). ¹³C NMR (125.7 MHz, CDCl₃) δ 27.76 (OC(CH₃)₃), 85.66 (OC(CH₃)₃), 115.26 (C-4), 117.26 (C-7), 125.15 (C-5), 127.08 (C-6), 146.73 (C-8), 148.17 (C-3), 154.68 (CO), 155. 46 (CO). Anal. Calcd for C₁₇H₂₁BrN₂O₅ (%): C, 49.41; H, 5.12; N, 6.78. Found (%): C, 49.35; H, 5.17; N, 6.68.

tert-Butyl 4-(4-amino-3-nitrophenyl)-5,6-dihydropyridine-1(2H)-carboxylate (14): To a nitrogen flushed flask containing the boronate (12) (1.39 g, 4.5 mmol), K₂CO₃ (1.86 g, 13.5 mmol) and PdCl₂dppf (0.23 g, 0.28 mmol) was added a solution of the bromide (8) (1.03 g, 4.74 mmol) in DMF (30 mL). The mixture was heated to 80 °C and stirred under N₂ overnight when TLC indicated completion of the reaction. The reaction was cooled to room temperature and filtered through a pad of celite. The filtrate was added to ethyl acetate (50 mL) and washed successively with water (20 mL), brine (3 × 15 mL), dried over Na₂SO₄ and evaporated. Column chromatography of the brown oily material on silica gel, eluting with ethyl acetate:hexanes (30:70) and then the ratio was changed to 50:50 to obtain compound 14. Yield: 52%, light yellow amorphous solid, m.p. 111 °C–112 °C. IR (KBr, cm⁻¹) 3420, 3310 (NH₂), 3030 (Ar-H), 2979 (Alph-H), 1690 (CO), 1612, 1515, 1414 (C=C), 1254 (C-N), 1159 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.45 (s, 9H, OC(CH₃)₃), 2.44 (br s, 2H, CH₂), 3.59 (m, 2H, CH₂), 4.08 (br s, 2H, CH₂), 5.95 (br s, 1H, CH), 6.20 (br s, 2H, NH₂), 6.76 (d, J = 8.8 Hz, 1H, H-5), 7.41 (dd, J = 2.8, 8.8 Hz, 1H, H-6), 8.02 (br s, 1H, H-2). ¹³C NMR (125.7 MHz, CDCl₃) δ 26.88 (CH₂), 28.39 (OC(CH₃)₃), 29.36 (CH₂), 42.86 (CH₂), 79.99 (OC(CH₃)₃), 118.87 (CH), 119.14 (C-5), 121.47 (C-2), 129.49 (C-1), 131.65 (C-3), 132.36 (C-6), 143.89 (C-4), 154.73 (CO). Anal. Calcd for C₁₆H₂₁N₃O₄ (%): C, 60.17; H, 6.63; N, 13.16. Found (%): C, 60.10; H, 6.68; N, 13.08.

tert-Butyl 4-(3,4-diacetamidophenyl)piperidine-1-carboxylate (15): Ra-Ni (20% w/w) was added to a solution of compound 14 (1.4 g, 4.39 mmol), acetic anyhydride (2.48 mL, 26.28 mmol) and sodium acetate (1.44 g, 17.52 mmol) in a mixture of THF (10 mL) and EtOH (20 mL) and the mixture was subjected to hydrogenation in a Parr apparatus at 60 psi for 8 h. After filtering over the pad of celite, the solution was concentrated to get a brown oily material, which was resolved over silica column eluting with ethyl acetate:hexanes (30:70) and then the ratio was changed to 60:40 to get 15. Yield: 82%, light brown gum. IR (KBr, cm⁻¹) 3420, 3338 (NH₂), 3033 (Ar-H), 2978 (Alph-H), 1691 (CO), 1620, 1524, 1408 (C=C), 1221 (C-N), 1154 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.47 (s, 9H, OC(CH₃)₃), 1.71 (m, 2H, CH₂), 2.03 (s, 3H, COCH₃), 2.09 (s, 3H, COCH₃), 2.57 (m, 1H, CH), 2.75 (br s, 2H, CH₂), 3.41 (br s, 2H, CH₂), 4.19 (br s, 2H, CH₂), 6.96 (dd, J = 2.4, 8.2 Hz, 1H, H-6), 7.18 (d, J = 2.3 Hz, 1H, H-2), 7.29 (d, J = 8.2 Hz, 1H, H-5), 8.80 (s, 1H, NH), 8.86 (s, 1H, NH). ¹³C NMR (125.7 MHz, CDCl₃) δ 23.81 (COCH₃), 24.83 (COCH₃), 24.93 (CH₂), 28.51 (OC(CH₃)₃), 33.04 (CH), 42.06 (CH₂), 79.77 (OC(CH₃)₃), 123.51 (C-2/C-5), 124.49 (C-2/C-5), 125.60 (C-6), 128.70 (C-4), 130.66 (C-3), 143.82 (C-1), 154.91 (CO), 170.08 (CO). Anal. Calcd for C₂₀H₂₉N₃O₄ (%): C, 63.98; H, 7.79; N, 11.19. Found (%): C, 63.90; H, 7.85; N, 11.10.

tert-Butyl 4-(3,4-diaminophenyl)piperidine-1-carboxylate (16): To a solution of compound 15 (0.75 g, 2 mmol) in ethanol (20 mL) 4 M solution of KOH in H₂O was added (4 mL, 16 mmol) and the mixture was stirred at 90 °C for 12 h. The mixture was concentrated under reduced pressure to get a brown oily material, which was resolved over silica column eluting with ethyl acetate:hexanes (50:50) and then the ratio was changed to 90:10 to get compound 16. Yield: 83%, dark brown gum. IR (KBr, cm⁻¹) 3420, 3338 (NH₂), 3033 (Ar-H), 2978 (Alph-H), 1691 (CO), 1620, 1524, 1408 (C=C), 1221 (C-N), 1154 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.49 (s, 9H, OC(CH₃)₃), 1.81 (m, 2H, CH₂), 2.07 (m, 2H, CH₂), 2.72–2.82 (m, 5H, CH, CH₂), 4.24 (br s, 4H, 2NH₂), 7.04 (dd, J = 2.5, 8.5 Hz, 1H, H-6), 7.32 (d, J = 2.4 Hz, 1H, H-2), 7.45 (d, J = 8.5 Hz, 1H, H-5). ¹³C NMR (125.7 MHz, CDCl₃) δ 28.40 (OC(CH₃)₃), (CH), 29.95 (CH₂), 33.72 (CH), 42.78 (CH₂), 79.52 (OC(CH₃)₃), 123.50 (C-2/C-5), 123.66 (C-2/C-5), 126.60 (C-6), 127.43 (C-4), 140.29 (C-3), 154.96 (CO). Anal. Calcd for C₁₆H₂₅N₃O₂ (%): C, 65.95; H, 8.65; N, 14.42. Found (%): C, 65.87; H, 8.70; N, 14.33.

tert-Butyl 4-(3,4-diaminophenyl)-5,6-dihydropyridine-1(2H)-carboxylate (18): To a solution of compound 14 (2.1 g, 6.58 mmol) in a mixture of THF (15 mL) and EtOH (30 mL) Pd-C was added (0.21 g, 10% wet basis) and the mixture was subjected to hydrogenation in the Parr apparatus at 10 psi for 3 h. After filtering over the pad of celite, the solution was concentrated and loaded over silica column, eluting with ethyl acetate:hexanes (50:50) and then the ratio was changed to 90:10 to get compound 18. Yield 96%, light brown gum. IR KBr, cm⁻¹) 3429, 3330 (NH₂), 3036 (Ar-H), 2983 (Alph-H), 1691 (CO), 1628, 1520, 1415 (C=C), 1229 (C-N), 1160 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.46 (s, 9H, OC(CH₃)₃), 2.40 (br s, 2H, CH₂), 3.43 (m, 2H, CH₂), 4.02 (br s, 2H, CH₂), 4.38 (br s, 4H, 2NH₂), 5.93 (br s, 1H, CH), 7.06 (dd, J = 2.6, 8.2 Hz, 1H, H-6), 7.30 (d, J = 2.5 Hz, 1H, H-2), 7.46 (d, J = 8.2 Hz, 1H, H-5). ¹³C NMR (125.7 MHz, CDCl₃) δ 26.89 (CH₂), 28.33 (OC(CH₃)₃), 29.39 (CH₂), 42.84 (CH₂), 79.99 (OC(CH₃)₃), 118.77 (CH), 119.19 (C-5), 120.37 (C-2), 129.19 (C-1), 130.65 (C-3), 131.16 (C-6), 141.82 (C-4), 154.63 (CO). Anal. Calcd for C₁₆H₂₃N₃O₂ (%): C, 66.41; H, 8.01; N, 14.52. Found (%): C, 66.33; H, 8.07; N, 14.43.

tert-Butyl 4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate (13): A mixture of compound 18 (0.58 g, 2 mmol) and carbonyldiimidazole (CDI) (0.34 g, 2.1 mmol) in THF (10 mL) was stirred at 60 °C for 6 h. The solvent was removed in vacuo and the residue was triturated with diethyl ether and the resultant off-white solid was filtered, washed with diethyl ether to obtain compound 13. Yield: 68%, off-white solid, m.p. 244 °C–245 °C. IR (KBr, cm⁻¹) 3433, 3191 (CONH), 3037 (Ar-H), 2978 (Alph-H), 1713, 1689 (C=O), 1604, 1433 (C=C), 1244 (C-N), 1178 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.39 (s, 9H, OC(CH₃)₃), 2.48 (br s, 2H, CH₂), 3.54 (m, 2H, CH₂), 3.93 (br s, 2H, CH₂), 5.97 (br s, 1H, CH), 6.86 (d, J = 8.2 Hz, 1H, H-7), 6.95 (d, J = 2.4 Hz, 1H, H-4), 6.99 (dd, J = 2.4, 8.2 Hz, 1H, H-6), 10.60 (br s, 2H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 26.84 (CH₂), 28.28 (OC(CH₃)₃), 32.33 (CH), 42.80 (CH₂), 79.08 (OC(CH₃)₃), 105.16 (CH), 108.52 (C-4), 117.67 (C-7), 129.20 (C-6), 130.04 (C-8), 133.28 (C-5/C-9), 134.81 (C-5/C-9), 154.73 (CO), 155.67 (CO). Anal. Calcd for C₁₇H₂₁N₃O₃ (%): C, 64.74; H, 6.71; N, 13.32. Found (%): C, 64.68; H, 6.76; N, 13.24.

tert-Butyl 4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)piperidine-1-carboxylate (17): (1) Synthesis from 16. Following the same procedure adopted for the synthesis of 13, the title compound 17 was obtained from the reaction of compound 16 with CDI. Yield: 62%, off-white solid. (2) Synthesis from 13. To a solution of compound 13 (0.94 g, 3 mmol) in CH₃OH (30 mL) Ra-Ni was added (0.20 g, 10% wet basis) and the mixture was subjected to hydrogenation in the Parr apparatus at 60 psi for 12 h. After filtering over the pad of celite, the solution was concentrated and recrystallized from diethyl ether to get compound 17. Yield: 90%, off-white solid, m.p. > 250 °C. IR (KBr, cm⁻¹) 3290, 3175 (CONH), 3030 (Ar-H), 2989 (Alph-H), 1691 (CO), 1613, 1515, 1416 (C=C), 1220 (C-N), 1167 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.38 (s, 9H, OC(CH₃)₃), 1.86 (m, 2H, CH₂), 2.06 (m, 2H, CH₂), 2.40–2.48 (m, 3H, CH, CH₂), 3.44 (obscured by H₂O, 2H, CH₂), 6.70–6.80 (m, 3H, H-4, H-6, H-7), 10.49 (br s, 1H, NH), 10.53 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 28.15 (OC(CH₃)₃), 29.25 (CH₂), 33.24 (CH₂), 41.72 (CH₂), 78.72 (OC(CH₃)₃), 106.93 (C-4), 108.48 (C-7), 119.0 (C-6), 128.09 (C-8), 129.89 (C-9), 138.49 (C-5), 154.05 (CO), 155.61 (CO). Anal. Calcd for C₁₇H₂₃N₃O₃ (%): C, 64.33; H, 7.30; N, 13.24. Found (%): C, 64.25; H, 7.35; N, 13.15.

5-(Piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5): To a solution of compound 17 (0.95 g, 3 mmol) in a mixture of CH₂Cl₂ (15 mL) and CH₃OH (20 mL) trifluoroacetic acid was added (3 mL) at 0 °C and the mixture was stirred for 6 h at room temperature. Solvents were evaporated under reduced pressure and triturating with diethyl ether gave the title compound 5. Yield: 90%, light yellow solid, m.p. 214 °C–215 °C. IR (KBr, cm⁻¹) 3238 (CONH), 3043 (Ar-H), 2956 (Alph-H), 1692 (CO), 1612, 1446 (C=C), 1180 (C-N), 1126 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.73–188 (m, 3H, CH, CH₂), 2.77 (m, 2H, CH₂), 2.97 (m, 2H, CH₂), 3.44 (obscured by H₂O, 2H, CH₂), 4.46 (m, 1H, CH), 6.74–6.84 (m, 3H, H-4, H-6, H-7), 10.50 (br s, 1H, NH), 10.54 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 30.04 (CH₂), 33.37 (CH), 43.86 (CH₂), 106.66 (C-4), 118.43 (C-7), 118.94 (C-6), 128.37 (C-8), 129.87 (C-9), 137.24 (C-5), 155.56 (CO). Anal. Calcd for C₁₄H₁₆F₃N₃O₃ (%): C, 50.76; H, 4.87; N, 12.68. Found (%): C, 50.70; H, 4.92; N, 12.60.

tert-Butyl 4-(4-acetamido-3-nitrophenyl)piperazine-1-carboxylate (21): To an oven-dried flask, 1-boc-piperazine (3.19 g, 17.1 mmol), Cs₂CO₃ (5.82 g, 17.86 mmol), Pd₂(dba)₃ (1.44 g, 1.57 mmol), rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.89 g, 1.43 mmol), toluene (8 mL) and compound 7 (3.68 g, 14.26 mmol) were added. While stirring the reaction mixture at room temperature, the air in the flask was removed and replaced by N₂. This process was repeated three times. The reaction temperature was brought to 110 °C and stirred for 8 h. Ethyl acetate (40 mL) was added to the mixture at room temperature, washed with H₂O (15 mL), brine (10 mL), dried over Na₂SO₄ and evaporated. The brown oily material was chromatographed on a silica column, eluting with hexanes:ethyl acetate (3:7) and then the ratio was changed to 1:1 to obtain compound 18. Yield: 37%, light red solid. IR (KBr, cm⁻¹) 3310, 3168 (NH), 3176 (Ar-H), 1709, 1678 (CO), 1519, 1416 (C=C), 1210 (C-N), 1151 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.49 (s, 9H, OC(CH₃)₃), 2.17 (s, 3H, COCH₃), 3.15 (m, 4H, 2CH₂), 3.60 (m, 4H, 2CH₂), 7.22 (dd, J = 2.5, 8.5 Hz, 1H, H-6), 7.62 (d, J = 2.5 Hz, 1H, H-2), 7.57 (d, J = 8.5 Hz, 1H, H-5), 9.98 (br s, 1H, NH). ¹³C NMR (125.7 MHz, CDCl₃) δ 25.59 (COCH₃), 28.40 (OC(CH₃)₃), 48.92 (CH₂), 52.31 (CH₂), 79.94 (OC(CH₃)₃), 111.29 (C-2), 123.52 (C-5/C-6), 124.31 (C-5/C-6), 127.45 (C-4), 137.45 (C-3), 147.11 (C-1), 154.63 (CO), 168.78 (CO). Anal. Calcd for C₁₇H₂₄N₄O₅ (%): C, 56.03; H, 6.64; N, 15.38. Found (%): C, 55.96; H, 6.69; N, 15.30.

tert-Butyl 4-(4-amino-3-nitrophenyl)piperazine-1-carboxylate (22): Following the same procedure adopted for the synthesis of 16, the title compound 22 was obtained from the basic hydrolysis of compound 21. Yield: 90%, blood red solid, m.p. 128 °C–129 °C. IR (KBr, cm⁻¹) 3477, 3325 (Ar-NH₂), 3073 (Ar-H), 1673 (CO), 1516, 1412 (C=C), 1211 (C-N), 1161 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.47 (s, 9H, OC(CH₃)₃), 3.00 (m, 4H, 2CH₂), 3.57 (m, 4H, 2CH₂), 5.92 (br s, 2H, NH₂), 6.79 (d, J = 8.1 Hz, 1H, H-5), 7.15 (m, 1H, H-6), 7.56 (d, J = 2.1 Hz, 1H, H-2). ¹³C NMR (125.7 MHz, CDCl₃) δ 28.42 (OC(CH₃)₃), 43.21 (CH₂), 50.49 (CH₂), 79.97 (OC(CH₃)₃), 111.63 (C-2), 119.85 (C-5), 128.88 (C-6), 131.89 (C-4), 140.00 (C-3), 142.69 (C-1), 154.61 (CO). Anal. Calcd for C₁₅H₂₂N₄O₄ (%): C, 55.89; H, 6.88; N, 17.38. Found (%): C, 55.81; H, 6.94; N, 17.30.

tert-Butyl 4-(3,4-diaminophenyl)piperazine-1-carboxylate (23): Following the same procedure adopted for the synthesis of 18, the title compound 23 was obtained from compound 22. Yield: 71%, light brown solid, m.p. 118 °C–119 °C. IR (KBr, cm⁻¹) 3427, 3336 (NH₂), 3030 (Ar-H), 2973 (Alph-H), 1693 (CO), 1623, 1522, 1405 (C=C), 1249 (C-N), 1162 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.37 (s, 9H, OC(CH₃)₃), 2.77 (br s, 4H, 2CH₂), 3.37 (br s, 4H, 2CH₂), 3.99 (br s, 2H, NH₂), 4.34 (br s, 2H, NH₂), 6.02 (dd, J = 2.5, 8.6 Hz, 1H, H-6), 6.21 (d, J = 2.6 Hz, 1H, H-2), 6.38 (d, J = 8.6 Hz, 1H, H-5). ¹³C NMR (125.7 MHz, DMSO d₆) δ 28.26 (OC(CH₃)₃), 39.99 (CH₂), 50.82 (CH₂), 79.18 (OC(CH₃)₃), 105.38 (C-2), 106.78 (C-6), 115.65 (C-5), 129.12 (C-4), 136.05 (C-3), 144.06 (C-1), 154.19 (CO). Anal. Calcd for C₁₅H₂₄N₄O₂ (%): C, 61.62; H, 8.27; N, 19.16. Found (%): C, 61.55; H, 8.31; N, 19.08.

4-Bromo-1,2-dinitrobenzene (24): To a cold solution of trifluoroacetic acid (25 mL) at 0 °C compound 8 was added (4.96 g, 22.96 mmol). After being stirred for 10 min, a solution of 33% hydrogen peroxide (13.75 mL, 133 mmol) was added and the mixture was stirred at room temperature for 0.5 h followed by stirring at 50 °C for 2 h. The mixture was poured into ice water and the solid obtained was filtered and washed with cold water to get the titled compound 24. Yield: 76%, yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 7.85–7.92 (m, 2H, H-5, H-6), 8.04 (br s, 1H, H-3). ¹³C NMR (125.7 MHz, CDCl₃) δ 126.39 (C-2), 127.91 (C-3), 128.09 (C-4), 136.29 (C-5), 141.26 (C-1), 143.42 (C-2). Anal. Calcd for C₆H₃BrN₂O₄ (%): C, 29.18; H, 1.22; N, 11.34. Found (%): C, 29.12; H, 1.26; N, 11.26.

tert-Butyl 4-(3,4-dinitrophenyl)piperazine-1-carboxylate (25): To an oven-dried flask, 1-boc-piperazine (1.59 g, 8.5 mmol), Cs₂CO₃ (2.91 g, 8.93 mmol), Pd₂(dba)₃ (0.72 g, 0.78 mmol), rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.44 g, 0.71 mmol), toluene (6 mL) and compound 24 (1.76 g, 7.14 mmol) were added. While stirring the reaction mixture at room temperature, the air in the flask was removed and replaced by N₂. This process was repeated three times. The reaction mixture was further stirred at room temperature for 0.5 h. Ethyl acetate (50 mL) was added to the mixture at room temperature, washed with H₂O (15 mL), brine (10 mL), dried over Na₂SO₄ and evaporated. The brown oily material was chromatographed on a silica column, eluting with hexanes:ethyl acetate (30:70) and then the ratio was changed to 40:60 yield compound 25. Yield: 78%, yellow solid. IR (KBr, cm⁻¹) 3073 (Ar-H), 1683 (CO), 1528, 1432 (C=C), 1226 (C-N), 1151 (C-O); ¹H NMR (500 MHz, CDCl₃) δ 1.48 (9H, s, OC(CH₃)₃), 3.02 (m, 4H, 2CH₂), 3.58 (m, 4H, 2CH₂), 7.17 (dd, J = 2.6, 8.8 Hz, 1H, H-6), 7.24 (d, J = 2.5 Hz, 1H, H-2), 7.71 (d, J = 8.5 Hz, 1H, H-4). ¹³C NMR (125.7 MHz, CDCl₃) δ 28.30 (OC(CH₃)₃), 48.24 (CH₂), 51.39 (CH₂), 80.12 (OC(CH₃)₃), 124.23 (C-2), 124.92 (C-6), 127.49 (C-5), 128.17 (C-4), 141.62 (C-3), 146.94 (C-1), 154.60 (CO). Anal. Calcd for C₁₅H₂₀N₄O₆ (%): C, 51.13; H, 5.72; N, 15.90. Found (%): C, 51.07; H, 5.78; N, 15.82.

tert-Butyl 4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)piperazine-1-carboxylate (20): Following the same procedure adopted for the synthesis of 13, the title compound was obtained by the reaction of compound 23 with CDI. Yield: 66%, colorless solid, m.p. 250 °C–251 °C. IR (KBr, cm⁻¹) 3292, 3165 (CONH), 3031 (Ar-H), 2979 (Alph-H), 1692 (CO), 1610, 1509, 1411 (C=C), 1222 (C-N), 1177 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.38 (9H, s, OC(CH₃)₃), 2.91 (m, 4H, 2CH₂), 3.42 (m, 4H, 2CH₂),), 6.52 (m, 2H, H-4, H-6), 6.78 (d, J = 8.6 Hz, 1H, H-7), 10.42 (br s, 1H, NH), 10.50 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 28.37 (OC(CH₃)₃), 40.01 (CH₂), 50.66 (CH₂), 79.30 (OC(CH₃)₃), 99.01 (C-4), 109.03 (C-6), 110.55 (C-7), 123.93 (C-8), 130.52 (C-3), 146.58 (C-5), 154.76 (CO), 155.89 (CO). Anal. Calcd for C₁₆H₂₂N₄O₃ (%): C, 60.36; H, 6.97; N, 17.60. Found (%): C, 60.28; H, 7.03; N, 17.51.

5-(piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6): Following the same procedure adopted for the synthesis of 5, the title compound was obtained from the treatment of compound 20 with trifluoroacetic acid. Yield: 93%, light purple solid, m.p. 110 °C–111 °C. IR (KBr, cm⁻¹) 3220 (CONH), 3053 (Ar-H), 2936 (Alph-H), 1695 (CO), 1609, 1444 (C=C), 1186 (C-N), 1120 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 3.19 (m, 4H, 2CH₂), 3.22 (m, 4H, 2CH₂),), 6.59 (m, 2H, H-4, H-6), 6.80 (d, J = 8.8 Hz, 1H, H-7), 8.86 (br s, 2H, NH), 10.40 (br s, 1H, NH), 10.53 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 43.10 (CH₂), 47.73 (CH₂), 98.97 (C-4), 108.84 (C-6), 110.25 (C-7), 124.54 (C-8), 130.57 (C-3), 145.19 (C-5), 155.76 (CO). Anal. Calcd for C₁₃H₁₅F₃N₄O₃ (%): C, 46.99; H, 4.55; N, 16.86. Found (%): C, 46.93; H, 4.60; N, 16.76.

5-(1-(biphenyl-4-ylmethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5a).

Representative procedure: To a solution of compound 5 (0.15 g, 0.45 mmol) and biphenyl-4-carbaldehyde (a) (0.1 g, 0.55 mmol) in DMSO (2 mL) at 0 °C Et₃N was added (0.13 mL, 0.97 mmol). After being stirred for 0.5 h at room temperature, NaBH(OAc)₃ (0.11 g, 0.53 mmol) was added and the mixture was stirred for 6 h. The reaction mixture was added to a saturated NaHCO₃ solution (10 mL) and stirred for 15 min, followed by the addition of ethyl acetate (30 mL). The organic layer was separated and washed with saturated NaHCO₃, brine, and dried over Na₂SO₄ and evaporated. The light yellow solid was crystallized from diethyl ether to obtain the title compound 5a. Yield: 51%, off-white solid, m.p. > 260 °C. IR (KBr, cm⁻¹) 3429 (CONH), 3055 (Ar-H), 2966 (Alph-H), 1703 (CO), 1620, 1518, 1422 (C=C), 1226 (C-N), 1145 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.62–1.71 (m, 4H, 2CH₂), 2.04 (m, 2H, CH₂), 2.48 (m, 1H, CH), 2.92 (m, 2H, CH₂), 3.51 (s, 2H, NCH₂), 6.77–6.81 (m, 3H, 3Ar-H), 7.32 (m, 1H, Ar-H), 7.40–7.45 (m, 4H, 4Ar-H), 7.59–7.64 (m, 4H, 4Ar-H), 10.43 (br s, 1H, NH), 10.48 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 33.64 (CH₂), 41.86 (CH), 53.66 (CH₂), 59.45 (NCH₂), 106.91 (Ar-C), 108.42 (Ar-C), 118.99 (Ar-C), 126.56 (Ar-C), 126.66 (Ar-C), 127.42 (Ar-C), 127.98 (Ar-C), 129.03 (Ar-C), 129.06 (Ar-C), 129.63 (Ar-C), 138.92 (Ar-C), 140.11 (Ar-C), 155.59 (C=O). Anal. Calcd for C₂₅H₂₅N₃O (%): C, 78.30; H, 6.57; N, 10.96. Found (%): C, 78.22; H, 6.63; N, 10.86.

5-(1-((4′-Fluorobiphenyl-4-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5b): Following the same procedure adopted for the synthesis of 5a, the title compound 5b was obtained from the reductive amination of compound 5 and aldehyde (b). Yield: 54%, off-white solid, m.p. > 260 °C. IR (KBr, cm⁻¹) 3419 (CONH), 3045 (Ar-H), 2976 (Alph-H), 1702 (CO), 1622, 1511, 1428 (C=C), 1236 (C-N), 1170 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.66–1.76 (m, 4H, 2CH₂), 2.16 (br s, 2H, CH₂), 2.95 (m, 2H, CH₂), 3.49 (s, 2H, NCH₂), 6.77–6.82 (3H, m, 3Ar-H), 7.25 (m, 2H, 2Ar-H), 7.41 (d, J = 7.6 Hz, 2H, 2Ar-H), 7.58 (m, 2H, 2Ar-H), 7.66 (m, 2H, 2Ar-H), 10.44 (br s, 1H, NH), 10.49 (br. s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 33.22 (CH₂), 41.51 (CH), 53.55 (CH₂), 61.67 (NCH₂), 106.88 (Ar-C), 108.42 (Ar-C), 115.64 (Ar-C), 115.87 (Ar-C), 118.98 (Ar-C), 126.48 (Ar-C), 126.57 (Ar-C), 128.01 (Ar-C), 128.62 (Ar-C), 128.69 (Ar-C), 129.86 (Ar-C), 136.49 (Ar-C), 138.11 (Ar-C), 138.65 (Ar-C), 155.56 (C=O), 160.94 (Ar-C), 162.87 (Ar-C). Anal. Calcd for C₂₅H₂₄FN₃O (%): C, 74.79; H, 6.03; N, 10.47. Found (%): C, C, 74.70; H, 6.07; N, 10.38.

5-(1-((5-phenylpyridin-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5c): Following the same procedure adopted for the synthesis of 5a, the title compound 5c was obtained from the reductive amination of compound 5 and aldehyde (c). Yield: 46%, off-white solid, m.p. 236 °C–237 °C. IR (KBr, cm⁻¹) 3430, 3192 (CONH), 3048 (Ar-H), 2946 (Alph-H), 1693 (CO), 1629, 1516, 1423 (C=C), 1197 (C-N), 1115 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.62–1.70 (m, 4H, 2CH₂), 2.08 (m, 2H, CH₂), 2.92 (m, 2H, CH₂), 3.60 (s, 2H, NCH₂), 6.77–6.81 (m, 3H, 3Ar-H), 7.42 (m, 1H, Ar-H), 7.50 (m, 2H, 2Ar-H), 7.71 (m, 2H, 2Ar-H), 7.96 (s, 1H, H-4′), 8.50 (s, 1H, H-2′), 8.76 (s, 1H, H-6′), 10.44 (br s, 1H, NH), 10.48 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 33.59 (CH₂), 41.78 (CH), 53.66 (CH₂), 59.45 (NCH₂), 106.91 (Ar-C), 108.37 (Ar-C), 118.98 (Ar-C), 126.95 (Ar-C), 127.02 (Ar-C), 127.96 (Ar-C), 128.26 (Ar-C), 129.22 (Ar-C), 129.26 (Ar-C), 134.02 (Ar-C), 134.66 (Ar-C), 138.86 (Ar-C), 146.35 (Ar-C), 149.07 (Ar-C), 155.56 (C=O). Anal. Calcd for C₂₄H₂₄N₄O (%): C, 74.97; H, 6.19; N, 14.57. Found (%): C, 74.89; H, 6.25; N, 14.48.

5-(1-((5-(4-Fluorophenyl)pyridin-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5d): Following the same procedure adopted for the synthesis of 5a, the title compound 5d was obtained from the reductive amination of compound 5 and aldehyde (d). Yield: 62%, light yellow solid, m.p. 241 °C–242 °C. IR (KBr, cm⁻¹) 3410, 3191 (CONH), 3042 (Ar-H), 2956 (Alph-H), 1688 (CO), 1628, 1512, 1430 (C=C), 1170 (C-N), 1115 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.59–1.70 (m, 4H, 2CH₂), 2.05 (m, 3H, CH, CH₂), 2.91 (m, 2H, CH₂), 3.58 (s, 2H, NCH₂), 6.76–6.81 (m, 3H, 3Ar-H), 7.31 (m, 2H, 2Ar-H), 7.76 (m, 2H, 2Ar-H), 7.77 (s, 1H, H-4′), 8.48 (s, 1H, H-2′), 8.73 (s, 1H, H-6′), 10.43 (br s, 1H, NH), 10.47 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 33.59 (CH₂), 41.79 (CH), 53.68 (CH₂), 59.45 (NCH₂), 106.91 (Ar-C), 108.38 (Ar-C), 116.02 (Ar-C), 116.21 (Ar-C), 119.0 (Ar-C), 127.98 (Ar-C), 129.07 (Ar-C), 129.13 (Ar-C), 129.20 (Ar-C), 129.84 (Ar-C), 133.57 (Ar-C), 134.02 (Ar-C), 134.33 (Ar-C), 134.64 (Ar-C), 138.87 (Ar-C), 146.28 (Ar-C), 149.07 (Ar-C), 155.59 (C=O). Anal. Calcd for C₂₄H₂₃FN₄O (%): C, 71.62; H, 5.76; N, 13.92. Found (%): C, 71.51; H, 5.83; N, 13.82.

5-(1-(3-cyclopentenylbenzyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5e): Following the same procedure adopted for the synthesis of 5a, the title compound 5e was obtained from the reductive amination of compound 5 and aldehyde (e). Yield: 66%, light yellow solid, m.p. 238 °C–239 °C. IR (KBr, cm⁻¹) 3322 (CONH), 3051 (Ar-H), 2966 (Alph-H), 1690 (CO), 1636, 1512, 1428 (C=C), 1223 (C-N), 1172 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.59–1.69 (m, 5H, CH, CH₂), 1.93 (m, 2H, CH, CH₂), 2.04 (m, 2H, CH, CH₂), 2.62 (m, 2H, CH, CH₂), 2.87 (m, 2H, CH₂), 6.24 (s, 1H, CH), 6.76–6.82 (m, 3H, 3Ar-H), 7.16 (m, 1H, Ar-H), 7.24 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.38 (m, 1H, Ar-H) 10.44 (br s, 1H, NH), 10.48 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 22.89 (CH₂), 32.83 (CH₂), 32.96 (CH₂), 33.49 (CH₂), 39.00 (CH₂), 41.79 (CH), 53.68 (CH₂), 62.49 (NCH₂), 106.90 (Ar-C), 108.38 (Ar-C), 118.96 (Ar-C), 124.26 (Ar-C), 126.10 (Ar-C), 127.81 (Ar-C), 128.29 (Ar-C), 129.84 (Ar-C), 136.08 (Ar-C), 139.05 (Ar-C), 142.00 (Ar-C), 155.66 (C=O). Anal. Calcd for C₂₄H₂₇N₃O (%): C, 77.18; H, 7.29; N, 11.25. Found (%): C, 77.10; H, 7.36; N, 11.18.

5-(1-((5-cyclopentenylpyridin-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (5f): Following the same procedure adopted for the synthesis of 5a, the title compound 5f was obtained from the reductive amination of compound 5 and aldehyde (f). Yield: 42%, light yellow solid, m.p. 249 °C–250 °C. IR (KBr, cm⁻¹) 3342 (CONH), 3040 (Ar-H), 2956 (Alph-H), 1691 (CO), 1634, 1508, 1440 (C=C), 1221 (C-N), 1177 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.60–1.69 (m, 4H, CH, CH₂), 1.95–2.05 (m, 6H, CH, CH₂), 2.66 (m, 2H, CH₂), 2.85 (m, 2H, CH₂), 3.50 (s, 2H, NCH₂), 6.41 (s, 1H, CH), 6.76–6.80 (m, 3H, 3Ar-H), 7.73 (s, 1H, H-4′), 8.34 (s, 1H, H-2′), 8.54 (s, 1H, H-6′), 10.43 (br s, 1H, NH), 10.47 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO-d₆) δ 22.77 (CH₂), 32.48 (CH₂), 33.08 (CH₂), 33.55 (CH₂), 40.40 (CH₂), 41.77 (CH), 53.65 (CH₂), 59.51 (NCH₂), 106.90 (Ar-C), 108.38 (Ar-C), 118.98 (Ar-C), 127.96 (Ar-C), 128.39 (Ar-C), 129.82 (Ar-C), 131.30 (Ar-C), 133.11 (Ar-C), 133.51 (Ar-C), 138.86 (Ar-C), 139.14 (Ar-C), 145.46 (Ar-C), 148.52 (Ar-C), 155.58 (C=O). Anal. Calcd for C₂₃H₂₆N₄O (%): C, 73.77; H, 7.00; N, 14.96. Found (%): C, 73.69; H, 7.06; N, 14.88.

Table 1. Structures and affinities (K_i, nM) of compounds 5a–5f and 6a–6f on D₂ and 5-HT_1A receptors. Binding affinity values are expressed as means ± SEM of separate experiments, each performed in duplicate.

		Ki ± SEM
Compound	Structure	D2	5-HT1A
Haloperidol		2.92 ± 0.7	–
5-HT		–	10.0 ± 1.2
Adoprazine		8.40 ± 0.03	8.64 ± 0.01
Bifeprunox		8.81 ± 0.04	7.19 ± 0.14
5a		96.2 ± 3.0	34.2 ± 0.6
5b		34.4 ± 1.8	16.22 ± 0.7
5c		18.5 ± 2.5	6.72 ± 0.6
5d		24.6 ± 1.8	9.42 ± 0.6
5e		8.12 ± 1.4	1.2 ± 0.5
5f		4.26 ± 2.2	6.3 ± 1.1
6a		42.22 ± 2.0	46.26 ± 0.5
6b		62.6 ± 4.2	27.24 ± 1.2
6c		12.6 ± 2.5	5.3 ± 1.4
6d		82.0 ± 2.0	11.4 ± 1.3
6e		10.4 ± 1.8	3.1 ± 0.6
6f		3.1 ± 1.2	1.7 ± 0.4

5-(4-(biphenyl-4-ylmethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6a): Following the same procedure adopted for the synthesis of 5a, the title compound 6a was obtained from the reductive amination of compound 6 and aldehyde (a). Yield: 46%, off-white solid, m.p. > 260 °C. IR (KBr, cm⁻¹) 3430 (CONH), 3060 (Ar-H), 2966 (Alph-H), 1705 (CO), 1622, 1511, 1438 (C=C), 1223 (C-N), 1169 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 2.51 (br s, 4H, 2CH₂), 2.99 (br s, 4H, 2CH₂), 3.53 (s, 2H, NCH₂), 6.49 (m, 2H, 2Ar-H), 6.74 (m, 1H, Ar-H), 7.37–7.44 (m, 5H, 5Ar-H), 7.57–7.63 (m, 4H, 4Ar-H), 10.26 (br s, 1H, NH), 10.38 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 50.13 (CH₂), 52.80 (CH₂), 61.68 (NCH₂), 97.83 (Ar-C), 108.56 (Ar-C), 109.39 (Ar-C), 123.93 (Ar-C), 124.49 (Ar-C), 127.28 (Ar-C), 128.90 (Ar-C), 129.49 (Ar-C), 130.38 (Ar-C), 136.72 (Ar-C), 137.86 (Ar-C), 139.94 (Ar-C), 146.42 (Ar-C), 155.56 (C=O). Anal. Calcd for C₂₄H₂₄N₄O (%): C, 74.97; H, 6.29; N, 14.57. Found (%): C, 74.90; H, 6.35; N, 14.48.

5-(4-((4′-fluorobiphenyl-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6b): Following the same procedure adopted for the synthesis of 5a, the title compound 6b was obtained from the reductive amination of compound 6 and aldehyde (b). Yield: 55%, off-white solid, m.p. > 260 °C. IR (KBr, cm⁻¹) 3439 (CONH), 3065 (Ar-H), 2936 (Alph-H), 1707 (CO), 1632, 1501, 1448 (C=C), 1226 (C-N), 1179 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 2.52 (br s, 4H, 2CH₂), 2.99 (br s, 4H, 2CH₂), 3.53 (s, 2H, NCH₂), 6.49 (m, 2H, 2Ar-H), 6.74 (d, J = 3.2 Hz, 1H, Ar-H), 7.26 (m, 2H, 2Ar-H), 7.39 (m, 2H, 2Ar-H), 7.58 (m, 2H, 2Ar-H), 7.67 (m, 2H, 2Ar-H), 10.26 (br s, 1H, NH), 10.37 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 50.13 (CH₂), 52.79 (CH₂), 61.65 (NCH₂), 97.86 (Ar-C), 108.60 (Ar-C), 109.41 (Ar-C), 115.59 (Ar-C), 123.25 (Ar-C), 126.39 (Ar-C), 128.49 (Ar-C), 129.53 (Ar-C), 130.40 (Ar-C), 136.46 (Ar-C), 137.38 (Ar-C), 137.83 (Ar-C), 146.41 (Ar-C), 155.59 (C=O), 160.90 (Ar-C), 162.17 (Ar-C). Anal. Calcd for C₂₄H₂₃FN₄O (%): C, 71.62; H, 5.76; N, 13.92. Found (%): C, 71.55; H, 5.83; N, 13.86.

5-(4-((5-phenylpyridin-3-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6c): Following the same procedure adopted for the synthesis of 5a, the title compound 6c was obtained from the reductive amination of compound 6 and aldehyde (c). Yield: 44%, off-white solid, m.p. 241 °C–242 °C. IR (KBr, cm⁻¹) 3433, 3195 (CONH), 3038 (Ar-H), 2936 (Alph-H), 1683 (CO), 1639, 1506, 1443 (C=C), 1177 (C-N), 1119 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 2.51 (br s, 4H, 2CH₂), 2.99 (br s, 4H, 2CH₂), 3.62 (s, 2H, NCH₂), 6.50 (m, 2H, 2Ar-H), 6.73 (d, J = 3.2 Hz, 1H, Ar-H), 7.41 (m, 1H, Ar-H), 7.47 (m, 2H, 2Ar-H), 7.70 (m, 1H, Ar-H), 7.96 (s, 1H, H-4′), 8.51 (s, 1H, H-2′), 8.77 (s, 1H, H-6′), 10.26 (br. s, 1H, NH), 10.38 (br. s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO-d₆) δ 50.11 (CH₂), 52.67 (CH₂), 59.01 (NCH₂), 97.88 (Ar-C), 108.58 (Ar-C), 109.44 (Ar-C), 123.29 (Ar-C), 126.93 (Ar-C), 127.09 (Ar-C), 128.16 (Ar-C), 129.15 (Ar-C), 130.40 (Ar-C), 133.57 (Ar-C), 134.56 (Ar-C), 135.19 (Ar-C), 136.99 (Ar-C), 146.37 (Ar-C), 149.04 (Ar-C), 155.58 (C=O). Anal. Calcd for C₂₃H₂₃N₅O (%): C, 71.67; H, 6.01; N, 18.17. Found (%): C, 71.60; H, 6.06; N, 18.08.

5-(4-((5-(4-fluorophenyl)pyridin-3-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6d): Following the same procedure adopted for the synthesis of 5a, the title compound 6d was obtained from the reductive amination of compound 6 and aldehyde (d). Yield: 42%, off-white solid, m.p. 246 °C–247 °C. IR (KBr, cm⁻¹) 3413, 3190 (CONH), 3048 (Ar-H), 2946 (Alph-H), 1686 (CO), 1629, 1506, 1433 (C=C), 1176 (C-N), 1113 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 2.52 (br s, 4H, 2CH₂), 2.99 (br s, 4H, 2CH₂), 3.61 (s, 2H, NCH₂), 6.50 (m, 2H, 2Ar-H), 6.73 (d, J = 3.2 Hz, 1H, Ar-H), 7.31 (m, 2H, 2Ar-H), 7.76 (m, 2H, 2Ar-H), 7.96 (s, 1H, H-4′), 8.50 (s, 1H, H-2′), 8.76 (s, 1H, H-6′), 10.26 (br s, 1H, NH), 10.38 (br s, 1H, NH). ¹³C NMR (125.7 MHz, DMSO d₆) δ 50.08 (CH₂), 52.64 (CH₂), 58.98 (NCH₂), 97.90 (Ar-C), 108.58 (Ar-C), 109.44 (Ar-C), 115.91 (Ar-C), 116.07 (Ar-C), 123.65 (Ar-C), 129.01 (Ar-C), 129.08 (Ar-C), 130.21 (Ar-C), 133.38 (Ar-C), 134.31 (Ar-C), 134.89 (Ar-C), 146.35 (Ar-C), 149.04 (Ar-C), 155.42 (C=O), 161.15 (Ar-C). Anal. Calcd for C₂₃H₂₂FN₅O (%): C, 68.47; H, 5.50; N, 17.36. Found (%): C, 68.46; H, 5.54; N, 17.28.

5-(4-(3-cyclopentenylbenzyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6e): Following the same procedure adopted for the synthesis of 5a, the title compound 6e was obtained from the reductive amination of compound 6 and aldehyde (e). Yield: 62%, off-white solid, m.p. 244 °C–245 °C. IR (KBr, cm⁻¹) 3410, 3183 (CONH), 3050 (Ar-H), 2948 (Alph-H), 1689 (CO), 1625, 1516, 1430 (C=C), 1178 (C-N), 1115 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.93 (m, 2H, CH₂), 2.49 (br s, 2H, CH₂), 2.63 (br s, 2H, CH₂), 2.98 (br s, 4H, 2CH₂), 3.49 (s, 2H, NCH₂), 6.24 (br. s, 1H, CH), 6.49 (m, 2H, 2CH), 6.74 (d, J = 3.1 Hz, 1H, Ar-H), 7.18 (m, 1H, Ar-H), 7.28 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.38 (m, 1H, Ar-H), 10.27 (br s, 1H, NH), 10.39 (br s, 1H, NH). 13C NMR (125.7 MHz, DMSO d₆) δ 22.86 (CH₂), 32.78 (CH₂), 32.94 (CH₂), 50.11 (CH₂), 52.77 (CH₂), 62.08 (NCH₂), 97.95 (Ar-C), 108.70 (Ar-C), 109.49 (Ar-C), 124.28 (Ar-C), 126.08 (Ar-C), 127.75 (Ar-C), 128.29 (Ar-C), 130.44 (Ar-C), 136.07 (Ar-C), 141.94 (Ar-C), 146.42 (Ar-C), 155.66 (C=O). Anal. Calcd for C₂₃H₂₆N₄O (%): C, 73.77; H, 7.00; N, 14.96. Found (%): C, 73.68; H, 7.05; N, 14.86.

5-(4-((5-cyclopentenylpyridin-3-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one (6f): Following the same procedure adopted for the synthesis of 5a, the title compound 6f was obtained from the reductive amination of compound 6 and aldehyde (f). Yield: 39%, off-white solid, m.p. 255 °C–256 °C. IR (KBr, cm⁻¹) 3332 (CONH), 3030 (Ar-H), 2951 (Alph-H), 1689 (CO), 1644, 1506, 1450 (C=C), 1222 (C-N), 1176 (C-O); ¹H NMR (500 MHz, DMSO d₆) δ 1.95 (m, 2H, CH₂), 2.51 (br s, 2H, CH₂), 2.66 (m, 2H, CH₂), 2.98 (br s, 4H, 2CH₂), 3.54 (m, 2H, NCH₂), 6.42 (br s, 1H, CH), 6.50 (m, 2H, 2Ar-H), 6.74 (m, 1H, Ar-H), 7.74 (s, 1H, H-4′), 8.36 (s, 1H, H-2′), 8.57 (s, 1H, H-6′), 10.26 (br s, 1H, NH), 10.40 (br s, 1H, NH). 13C NMR (125.7 MHz, DMSO d₆) δ 22.69 (CH₂), 32.40 (CH₂), 33.01 (CH₂), 50.06 (CH₂), 52.66 (CH₂), 59.06 (NCH₂), 97.92 (Ar-C), 108.61 (Ar-C), 109.46 (Ar-C), 123.31 (Ar-C), 128.31 (Ar-C), 130.40 (Ar-C), 131.21 (Ar-C), 133.04 (Ar-C), 139.03 (Ar-C), 145.56 (Ar-C), 146.37 (Ar-C), 148.52 (Ar-C), 155.61 (C=O). Anal. Calcd for C₂₂H₂₅N₅O (%): C, 70.38; H, 6.71; N, 18.65. Found (%): C, 70.30; H, 6.75; N, 18.55.

Pharmacology

Rat-cloned D_2L dopaminergic receptors

Human-cloned dopamine D_2L receptors stably expressed in C6 rat glioma cells (kindly donated by Professor Roberto Maggio, Università di L'Aquila, Italy) were radiolabeled with [³H]spiroperidol according to Scarselli et al. with minor modifications23. The incubation buffer (120 mM NaCl, 5.0 mM KCl, 5.0 mM MgCl₂, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4) contained 100 µg of dopamine D_2L receptor membranes, 0.30–0.50 nM [³H]spiroperidol (K_d = 0.093 nM) and six to nine concentrations of drug solution in a final volume of 500 µL. The samples were incubated for 120 min at 25 °C, then the incubation was stopped by rapid filtration through Whatman GF/C glass fiber filters (presoaked in 0.5% polyethylenimine for 60 min). The filters were washed three times in 1 mL of ice-cold 50 mM Tris, 0.9% NaCl, pH 7.4. Nonspecific binding was determined in the presence of 10 µM haloperidol. The radioactivity bound to the filters was measured by liquid scintillation using a LS6500 Multi-Purpose scintillation Counter, Beckman.

Human-cloned 5-HT_1A receptors

Human-cloned 5-HT_1A serotonin receptors stably expressed in HEK293-EBNA cells (Perkin-Elmer, Waltham, MA) were radiolabeled with 1.0 nM [³H]-8-OH-DPAT24. Samples containing 32 μg of membrane protein, different concentrations of each compound ranging from 0.1 nM to 10 μM were incubated in a final volume of 500 μL of 50 mM Tris-HCl pH 7.4, 5 mM MgSO₄ for 120 min at 37 °C. After this incubation time, samples were filtered through Whatman GF/C glass microfiber filters pre-soaked in polyethylenimine 0.5% for at least 30 min prior to use. The filters were washed twice with 1 mL of ice-cold buffer (50 mM Tris-HCl, pH 7.4). Nonspecific binding was determined in the presence of 10 μM 5-HT. The radioactivity bound to the filters was measured by liquid scintillation using the LS6500 Multi-Purpose scintillation Counter, Beckman.

Determination of K_i values

K_i values of the compounds were calculated by the Cheng–Prusoff equation25. Binding affinity values are expressed as means ±SEM of separate experiments each performed in duplicate.

Results and discussion

The synthesis of compounds 5a–f required the synthesis of intermediate 5, which was to be synthesized from 13. The synthesis of the latter was envisioned by the Suzuki–Miyaura reaction of bromo-1H-benzo[d]imidazol-2(3H)-one (10) with boronate (12) [22]. Preparation of 10 was commenced by the basic hydrolysis of acetamide (7) to produce nitroaniline (8), which in turn was transformed to benzenediamine (9) by the action of stannous chloride26. Reaction of 9 with CDI finally produced 10 in 62% yield. The Suzuki–Miyaura reaction of cyclic vinyl boronate (12)22, derived from the vinyl triflates of N-protected tetrahydropyridines, with 10 under a variety of conditions (such as PdCl₂dppf, KOAc, DMF, 80 °C; Pd(PPh₃)₄, toluene, ethanol, Na₂CO₃, reflux) were failed; the desired 13 could not be obtained rather the starting 10 was recovered (Scheme 1). The poor solubility of 10 in a variety of solvents is considered to be one of the factors for its lack of reactivity. Thus, intermediate 10 was protected with di-tert-butyl dicarbonate to furnish 11, which in turn was employed in the Suzuki–Miyaura reaction with boronate (12), under different conditions as described in the case of 10; however the desired product 13 was not detected.

Scheme 1. Synthesis of intermediate 13.

In an alternate approach, boronate (12) was reacted with nitroamine (8) under the Suzuki–Miyaura conditions to produce intermediate 14, which in turn was subjected to hydrogenation in the Parr apparatus at 60 psi. The hydrogenation required extended period of time (20 h) and was unclean, yielding the desired diamine (15) in a very low yield (20%) after column purifications. Thus, hydrogenation of compound 14 was performed in ethanol in the presence of acetic anhydride (six equivalents) and sodium acetate (four equivalents), which rendered reduction of both nitro group and double bond with concomitant acetylation of the amino groups, yielding intermediate 15 in 82% yield. Basic hydrolysis of the latter with aqueous KOH in ethanol ultimately generated diamine (16) in a good yield (83%). Reaction of 16 with CDI in THF, heating the mixture at 80 °C for 6 h, finally gave access to the key intermediate 17 in an overall yield of 42% from 14. Alternatively, intermediate 17 was also synthesized from 14 by hydrogenation of 14 over Pd-C at 10 psi to produce diamine (18), which was treated with CDI in THF to generate intermediate 13. Hydrogenation of the latter over Ra-Ni in methanol furnished intermediate 17 in an overall higher yield of 59% from 14. This approach did not require column purifications of intermediates (13 and 17) as was the case with intermediates 15 and 16 (Scheme 2). Exposure of intermediate 17 to trifluoroacetic acid in a mixture of methanol and dichloromethane finally furnished the desired key intermediate 5.

Scheme 2. Synthesis of intermediate 13, an alternative route.

The synthesis of compounds 6a–f required the synthesis of intermediate 6, which was to be accomplished from intermediate 20. The Buchwald–Hartwig coupling of bromides 10 or 11 with tert-butyl piperazine-1-carboxylate 19, under different reaction conditions (PdCl₂dppf, KOAc, DMF, 80 °C; Pd(PPh₃)₄, toluene, ethanol, Na₂CO₃, reflux) were not successful; the desired 20 was not observed in any case (Scheme 3).

Scheme 3. Synthesis of intermediate 20.

In an alternate approach, acetamide (7) was coupled with 19 under the Buchwald–Hartwig reaction condition to construct the adduct 21 in relatively low yield (37%). Hydrolysis of the latter with aqueous KOH gave nitroaniline (22), which in turn was subjected to hydrogenation under the Parr apparatus over Ra-Ni at 15 psi to produce diamine (23) in an overall yield of 31% from 727. Alternatively, intermediate 23 was also synthesized from the oxidation of 8 with trifluoroperacetic acid, generated in situ from hydrogen peroxide and trifluoroacetic acid, to produce dinitrobenzene (24), which was coupled with 19 under the Buchwald–Hartwig reaction condition at room temperature to produce adduct 252⁸. Hydrogenation of intermediate 25, as described for the preparation of 23 from 22, finally produced compound 23 in an overall yield of 51% from 8. It is worth mentioning that reaction of 24 with 19 without the addition of catalyst at room temperature did not progress, the starting materials were intact or degraded over the course of longer reaction time, whereas gentle heating the reaction mixture to 40 °C resulted in a complex mixture of side products; the desired 19 was not observed. This was attributed to a thermal instability of 24 since gentle heating the reaction mixture under the Buchwald–Hartwig reaction condition did not yield the desired 19 either, only side products were observed. Reaction of 23 with CDI in THF at 80 °C yielded the desired intermediate 20, which was exposed to acid to produce the key intermediate 6 in 93% yield (Scheme 4).

Scheme 4. Synthesis of intermediate 6.

Having the desired intermediates 5 and 6 in hands, we next performed reductive amination of 5 and 6 with aldehydes (a–f)21 in DMSO, using NaBH(OAc)₃ as reducing agent to accomplish the synthesis of final compounds (5a–f) and (6a–f), respectively (Scheme 5).

Scheme 5. Synthesis of 5a and 6a, a representative example.

Biological results

SAR of D₂ receptor binding

The SAR studies on D₂ receptor binding of compounds revealed that compounds 6f, 5f and 5e were the most potent compared to others, which have moderate affinity (Table 1). Compound 5a (piperidine series) exhibited week affinity whereas an introduction of fluoro group at C-4′ in the biphenyl moiety, to produce 5b, improved its affinity to three-fold. However, introducing fluorine at C-4′ in biphenyl of 6a (piperazine series), to yield 6b, resulted in decrease in affinity. In case of compounds having phenylpyridine as a biaryl functionality, the introduction of fluoro group at C-4′ reduced the affinities both in piperidine and piperazine series. For instance, in case of piperidine series the affinity of 5d (K_i = 24.6 nM) was slightly reduced compared to its non-fluoro analogue 5c (K_i = 18.5 nM), whereas in case of piperazine series the affinity of 6c was reduced to six-fold when it was compared with 6d. The role of piperidine and piperazine moiety in the D₂ receptor affinity was evident with the observations that affinity generally increased in compounds having piperazine moiety with biphenyl and phenylpyridine substituents as compared with piperidine counterpart (5a versus 6a and 5c versus 6c). However, the role of piperazine and piperidine moieties is reversed, i.e. affinity decreased, when these moieties bear 4-fluorobiphenyl and 4-fluorophenylpyridine substituents. For instance, the affinity was decreased to two-fold in case 5b versus 6b and four-fold in case of 5d versus 6d. As stated earlier that the most active compounds were 6f, 5f and 5e, which features cyclopentenylpyridine and cyclopentenylbenzene moieties in their structures. Such observation suggests that pyridine nitrogen of cyclopentenylpyridine may play a role in the high affinity of 6f and 5f. Also, relatively high affinity of 6f compared to 5f is indicative of important contribution of piperazine moiety.

SAR of 5-HT_1A receptor binding

The compounds exhibited, in general, higher affinity to 5-HT_1A receptor than that of D₂. Compound 5e, being the most potent (K_i = 1.2 nM), reveals that piperidine with cyclopentenylbenzene is the right combination for optimal 5-HT_1A affinity. This was also evident when 5e was compared with its counterpart piperazine analog 6e, which posses high 5-HT_1A receptor affinity (K_i = 3.1 nM). Likewise compounds 5a, 5b, 6a and 6b exhibited moderate affinity. Both in piperidine and piperazine series with cyclopentenylbenzene moiety, the fluoro analogs of these compounds (5b and 6b) showed higher affinity than their non-fluoro counterparts (5a and 6a). However, in case of compounds bearing cyclopentenylpyridine moiety an opposite trend was observed. For instance, the affinity of 5c (K_i = 6.72 nM) was decreased almost half-fold when it was compared with its fluoro counterpart 5d (K_i = 9.42 nM); likewise the affinity was decreased to two-fold when 6c was compared with 6d. In addition, compounds with piperidine moiety (5a and 5b) were more active than compounds with piperazine moiety (6a and 6b). Furthermore, in the piperidine series, the presence of pyridine nitrogen of phenylpyridine moiety did not play a significant role in improving the binding affinity of 5-HT_1A in contrast to that of D₂; the affinity of 5e (K_i = 8.12 nM) was decreased as compared to 5f (K_i = 4.26 nM). However, in case of piperazine series, pyridine nitrogen improved the affinity of 6f (K_i = 3.1 nM) when it was compared with 6e (K_i = 10.4 nM).

Conclusion

A series of new 5-piperidinyl and 5-piperazinyl-1H-benzo[d]imidazol-2(3H)-ones have been synthesized. The evaluation of binding affinities for D₂ and 5-HT_1A receptors of these compounds revealed that, in general, compounds with cyclopentenylbenzene and cyclopentenylpyridine moieties have the higher affinities for both D₂ and 5-HT_1A receptors. In the piperidine series, 5f and 5e showed the highest affinities for D₂ and 5-HT_1A receptors, respectively. However, in case of piperazine series, 6f exhibited the highest affinity for both D₂ and 5-HT_1A receptors.

Declaration of interest

The financial support from King Abdulaziz City for Science and Technology (KACST) Project No: AR-28-38 is gratefully acknowledged.

Acknowledgements

The facilities provided by KFUPM are sincerely acknowledged.