Synthesis and spectral data2-(4-(4-Fluorophenyl)piperazin-1-yl)-1H-benz[d]imidazole (5a)

Yield: 56%; m.p: 216–218 °C; IR Ranges (ATR, cm−1); N–H stretch: 3337.25, C–H aromatic: 3041.76, C–H aliphatic: 2813.17, C–H aliphatic bend (CH2):1433.00, C=C stretch: 1600.32, C–N aromatic: 1210.90, C–F stretch: 999.71. H1 NMR Shifts; Aromatic C–H: (m 4H): 7.04–7.20, Piperazine C–H: (m 8H): 3.16–3.50, Aromatic C–H: (m 4H): 7.63–7.72, Benzimidazole N–H: (s 1H): 13.04. MASS: MOLECULAR ION PEAK (297.1523).

2-((4-(4-Fluorophenyl)piperazin-1-yl)methyl)-1H-benz[d]imidazole (5b)

Yield: 60%; m.p: 159–163 °C; IR Ranges (ATR, cm−1); N–H stretch: 3491.91, C–H aromatic: 3025.29, C–H aliphatic: 2908.47, C–H aliphatic bend (CH2): 1475, C=C stretch: 1650.50, C–N aromatic: 1204.77, C–F stretch: 1011.75. H1 NMR Shifts; Aromatic C–H: (m 4H): 7.16–7.21, Piperazine C-H: (m 8H): 3.29–3.88, Aromatic C–H: (m 4H): 7.44–7.63, methylene C–H: (s 2H): 3.89, Benzimidazole N–H: (s 1H): 13.61. MASS: MOLECULAR ION PEAK (311.1670).

2-((4-(3-Fluorophenyl)piperazin-1-yl)methyl)-1H-benz[d]imidazole (5c)

Yield: 70%; m.p: 122–126 °C; IR Ranges (ATR, cm−1); N–H stretch: 3337.25, C–H aromatic: 3041.55, C–H aliphatic: 2813.17, C–H aliphatic bend (CH2): 1433.00, C=C stretch: 1600.32, C–N aromatic: 1210.90, C–F stretch: 999.71. H1 NMR Shifts; Aromatic C–H: (m 4H): 7.05–7.17, Piperazine C–H: (m 8H): 3.39–3.88, Aromatic C–H: (m 4H): 7.17–7.44, methylene C–H: (s 2H): 3.89, Benzimidazole N–H: (s 1H): 13.61. MASS: MOLECULAR ION PEAK (311.1660).

2-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-1H-benz[d]imidazole (5d)

Yield: 71%; m.p: 116–119 °C; IR Ranges (ATR, cm−1); N–H stretch: 3481.78, C–H aromatic: 3038.96, C–H aliphatic: 2809.86, C–H aliphatic bend (CH2): 1433.42, C=C stretch: 1601.73, C–N aromatic: 1233.37, C–F stretch: 1016.61. H1 NMR Shifts; Aromatic C–H: (m 4H): 6.99–7.17, Piperazine C–H: (m 8H): 3.30–3.50, Aromatic C–H: (m 4H): 7.17–7.61, methylene C–H: (s 2H): 3.89, Benzimidazole N–H: (s 1H): 13.61. MASS: MOLECULAR ION PEAK (311.1675).

2-((4-Phenylpiperazin-1-yl)methyl)-1H-benz[d]imidazole (5e)

Yield: 57%; m.p: 162–165 °C; IR Ranges (ATR, cm−1); N–H stretch: 3481.78, C–H aromatic: 3038.96, C–H aliphatic: 2809.86, C–H aliphatic bend (CH2): 1433.42, C=C stretch: 1601.73, C–N aromatic: 1233.37. H1 NMR Shifts; Aromatic C–H: (s 1H, m 3H): 6.99–7.20, Piperazine C–H: (m 8H): 3.19–3.46, Aromatic C–H: (m 4H): 7.50–7.57, methylene C–H: (s 2H): 4.07, Benzimidazole N–H: (s 1H): 12.50. MASS: MOLECULAR ION PEAK (280.1690).

2-(4-(3-Fluorophenyl)piperazin-1-yl)-1H-benz[d]imidazole (5f)

Yield: 64%; m.p: 195–199 °C; IR Ranges (ATR, cm−1); N–H stretch: 3337.25, C–H aromatic: 3041.55, C–H aliphatic: 2813.17, C=C stretch: 1600.32, C–N aromatic: 1210.90, C–F stretch: 999.71. H1 NMR Shifts; Aromatic C–H: (m 4H): 7.07–7.20, Piperazine C–H: (m 8H): 3.16–3.50, Aromatic C–H: (m 4H): 7.20–7.63, Benzimidazole N–H: (s 1H): 13.04. MASS: MOLECULAR ION PEAK (297.1505).

The thin layer chromatography (TLC) characterization data for the synthesized substituents and derivatives, as well as spectra for infra-red (IR), proton nuclear magnetic resonance (1H NMR), and Mass analyses of the synthesized derivatives, are provided in the Additional files 1 and 2.

Biological activity (anti-anxiety activity)

The anxiolytic activity of the compounds was assessed through two tests: the Hole Board Test and the EPM Test. The experimental protocol for both tests was as follows: Group I received vehicle treatment (0.5% carboxymethyl cellulose in water). At the same time, Group VIII served as the standard reference with diazepam administration at 4 mg/kg intraperitoneally (i.p). Groups II to VII received test compounds 5a–f (synthesized compounds) at 50 mg/kg orally (p.o). Comparisons were made between Groups II to VIII and Group I, and the results were subjected to statistical analysis using GraphPad Prism 5 software, employing one-way ANOVA followed by Dunnett’s test.

Hole-board test

Each of the eight groups, consisting of five mice per group, was individually monitored for the number and duration of pocking in the hole board apparatus over 5 min. A higher number and longer pocking duration indicate the compound's anti-anxiety properties being evaluated. The mean ± standard error of the mean (SEM) for each group is presented in Table 1.

Table 1 Data for Hole board test

Compounds within Groups III, IV, and VII (Derivatives 5b, 5c, and 5f) exhibited very highly significant anxiolytic activity (***p < 0.001) in comparison to the control group, as assessed by the number of pocking. Compounds across Group II-VII (Derivatives 5a–f) demonstrated highly significant anxiolytic activity relative to the control group, as determined by the duration of pocking.

Figure 2 presents a graphical depiction of the data obtained from the hole board test. It is evident from the graph that all derivatives exhibit significant anxiolytic effects compared to both the control and standard groups.

Fig. 2

Graphical representation of anxiolytic activity by Hole board apparatus. (A Number of pocking; B duration of pocking, *significant, **highly significant, ***very highly significant by ANOVA and Dunnett’s test)

EPM test

T Each of the eight groups, comprising five mice per group, was individually monitored for the number of entries and the duration of entries into the open arms of the EPM apparatus over a 5-min duration. An increased number and duration of entries into the open arms of the EPM are indicative of the anti-anxiety properties of the compound being evaluated. The mean ± standard error of the mean (SEM) for each group is presented in Table 2.

Table 2 Data for EPM test