Synthesis, characterization and screening for antidepressant and anticonvulsant activity of 4,5-dihydropyrazole bearing indole derivatives

2.1 Chemistry

All solvents and chemicals used in the synthesis were procured from commercial suppliers and purified when necessary. All microwave assisted reactions were performed using a scientific microwave oven (Catalyst electromagnetic System) with a power of 800 W specially designed for organic synthesis. The completion of the reaction was monitored by thin layer chromatography performed on Merck precoated silica gel F254 plates. Silica gel (60–120 mesh size, Merck) was used for column chromatography. Melting points were recorded on an Elico Melting Point Apparatus using open capillary tube and were uncorrected. All NMR spectra were recorded on a Bruker Advance III, 400 MHz spectrometer with CDCl₃/DMSO as solvent using tetramethyl silane (TMS) as internal reference standard, chemical shifts are reported in ppm (δ). The IR spectrum of synthesized compounds was recorded on a Schimadzu IR 48 Spectrophotometer with DRS technology. Elemental analyses were performed on a Vario-EL III CHNOS elemental analyzer. Mass spectra were recorded on a 410 Prostar Binary LC with 500 MS IT PDA detectors with direct infusion mass with ESI and APCI Negative and Positive mode ionization.

2.2 General procedure for the synthesis of indolyl pyrazoline derivatives (2a–m) using microwave

To a slurry of 1-(substituted 1H-indol-3-yl)-3-(substituted aryl)-prop-1-en-3-ones (1 mmol) and phenyl hydrazine hydrochloride (2 mmol) in 20 ml EtOH was added basic alumina (20 g) in a 100 ml beaker and mixed thoroughly. The reaction mixture was air dried and subjected to microwave irradiation for specified time at 455 W with 30 s pulse and cooled in between. The reaction progress was monitored by TLC. After completion of reaction as indicated by TLC, the reaction mixture was cooled to room temperature and extracted into an organic solvent. The in-organics were filtered off. On standing the filtrate afforded crystals of desired pyrazolines (2a–m), which were purified by column chromatography (Chloroform/Acetone 16:1 as eluant). The structures of the isolated compounds were characterized by spectral methods and microanalyses. Melting point of compounds 2a, 2b, 2c, 2e, 2g, 2k and 2m was confirmed as reported in the literature (Tsukerman et al., 1969).

2.3 Antidepressant activity

The synthesized compounds were screened for their antidepressant activity using the forced swimming test (FST) (Porsolt et al., 1977). Adult male albino Swiss-Webster (20 ± 2 g) mice were used with free access to food and water. They were housed in a group of six. The approval of the Institutional Animal Ethics Committee (IAEC) of R. C. Patel Institute of Pharmaceutical education and Research Shirpur (Maharashtra, India) was taken prior to the start of the experiments. On the test day, mice were dropped one at a time into glass cylindrical container (diameter 10 cm, height 25 cm), containing approximately 20 cm of water at 25±1 °C temperature. Water was replaced between every trial. On this day, mice were assigned into different groups (n = 6–9 for each group). The synthesized compounds (100 mg/kg), Imipramine (20 mg/kg), and Fluoxetine (20 mg/kg) suspended in aqueous Tween 80 (0.5%) were injected as intraperitoneally (ip) [n = 6]. Control animals received 0.5% aqueous solution of Tween 80. Then, after 1/2 h the mice were dropped individually into the glass cylinder and left in the water for 6 min. After the first 2 min of the initial vigorous struggling, the animals were immobile. A mouse was judged immobile if it floated in the water in an upright position and made only slight movements in order to prevent sinking. The duration of immobility was recorded during the last 4 min of the 6-min test. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test to evaluate the results. The results of FST are summarized in Fig. 1.

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Figure 1

The effect of treatment with 2a–m (100 mg/kg, i.p.), imipramine (20 mg/kg, i.p.) and fluoxetine (20 mg/kg, i.p.) on the immobility time in FST (Data were analysed by one way ANOVA followed by Dunnet’s test. n = 6. Values are represented as mean ± S.E.M. compared with control group. P < 0.05 was considered statistically significant, ^∗∗∗P < 0.001, ns = not significant. # indicates compounds 2b, 2e and 2k were employed at the dose of 20 mg/kg.

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2.4 Anticonvulsant activity

The title compounds were tested for anticonvulsant activity against the PTZ induced convulsions in mice according to the reported method (Turner, 1965; Krall et al., 1978). All the solutions of standard drugs and PTZ were prepared in 0.9% sodium chloride solution. The test compounds were administered in the form of suspension made up of 0.5% Tween 80 in 0.9% sodium chloride solutions. All of the test compounds (100 mg/kg) were administered intra-peritoneally in a volume of 0.01 ml/g for mice at doses. Pentylenetetrazole (80 mg/kg) dissolved in 0.9% sodium chloride solution was administered in the posterior midline of the mice and the onset and severity of convulsions were noted for the control group. The test group was administered with the selected compounds 0.5 h prior to the administration of PTZ. The absence or presence of an episode of clonic convulsions was taken as the end point. The data were analysed by one-way ANOVA followed by Dunnett’s test using Graph Pad Prism software. All the values were expressed as mean ± SEM. Comparison of anticonvulsant activity by the PTZ induced seizure method (compared with standard) for compounds 2a–m are shown in Table 2.

Table 1 Physical data of 5-(substituted-1H-Indol-3-yl)-3-(substitutedaryl)-4, 5-dihydropyrazoline derivatives 2a–m synthesized via Scheme 1.

Compound	Ar	R	Yield (%)a	m.p. (°C)
2a		H	85.46	178–180
2b		H	84.52	175–177
2c		H	88.26	160–161
2d		H	74.65	138–141
2e		H	67.36	154–156
2f		H	71.26	160–161
2g		H	79.43	203–204
2h		Br	72.85	197–198
2i		OCH3	77.94	187–188
2j		H	70.36	201–202
2k		H	68.82	192–194
2l		H	77.13	173–174
2m		H	75.39	179–181

a All yields refer to products purified by column chromatography (Chloroform:Acetone 16:1).

2.5 Neurotoxicity screening

Rotarod test has been performed to detect the minimal motor deficit in mice (Vogel, 2002). The animal was placed on a 3.2 cm diameter knurled rod rotating at 6 rpm. Normal mice can remain on a rod rotating at this speed indefinitely. Neurological toxicity is defined as the failure of the animal to remain on the rod for 1 min (Table 2).

3.1 Chemistry

The synthesis of the titled compounds (2a–m) has been carried out as being depicted in (Scheme 1). The starting compound 3 formyl indole (Organic Syntheses, 1963) and substituted indolyl chalcones 1a–m (Panda and Chaudhari, 2008) were prepared according to the known procedures. Finally, the mixture of substituted chalcones 1a–m and phenyl hydrazine hydrochloride was irradiated under microwave without solvent to afford corresponding indolyl pyrazoline derivatives 2a–m. All the synthesized compounds were characterized by their physical (Table 1), and spectral data.

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Scheme 1

Synthesis of 3-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)-1H-indole derivatives (2a-m) under microwave.

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The IR spectra of the pyrazoline derivatives 2a–m exhibited C⚌N stretching vibration in the region of 1626–1516 cm⁻¹. Disappearance of the carbonyl (C⚌O) peak at around 1710–1650 cm⁻¹ revealed the formation of indolyl pyrazolines. In the ¹H NMR spectrum, C4 (H_4a, H_4b) and C5 (H_5c) protons of the pyrazoline ring exhibited doublets of doublets at 3.29–3.39 (δ ppm, H_4a), 3.80–3.87 (δ ppm, H_4b) and 5.60–5.69 (δ ppm, H_5c), respectively, confirms the formation of the pyrazoline ring.

3.2 Antidepressant activity

The forced swimming test is a behavioral test used to predict the efficacy of antidepressant treatments (Porsolt et al., 1977). It has good predictive value for antidepressant potency in humans because of its sensitivity and selectivity (Willner and Mitchell, 2002). As depicted in Fig. 1, all of the compounds except 2d significantly reduced the immobility times at a dose of 100 mg/kg compared with a control (P < 0.001). The immobility time of the mice treated with 2d did not statistically differ from the control value (control, 93.17 ± 3.94 s; 2d, 80.67 ± 7.43 s (P < 0.05). An analysis of the antidepressant activities of compounds 2a–m (Fig. 1) showed the subsequent structure–activity relationships. Amongst the indolyl pyrazolines derivatives, the substituent group on the benzene ring appeared to greatly influence the FST activity. The presence of electron releasing groups (OCH₃, CH₃ and OH) on the phenyl ring of indolyl pyrazolines showed promising antidepressant activity. Whereas substitution of the methoxy group at the 5th position of the indole ring retains the antidepressant activity. Amongst the compounds, 5-(1H-Indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydropyrazoline (2e), 5-(1H-Indol-3-yl)-3-(4-methylphenyl)-4,5-dihydropyrazoline (2b) and 5-(1H-Indol-3-yl)-3-(2-thienyl)-4,5-dihydropyrazoline (2k) were found to be the most active antidepressant compounds and significantly reduced the duration of immobility times at 100 mg/kg dose level when compared to the control (P < 0.001). Moreover active compounds, 2b, 2e and 2k at the dose of 20 mg/kg i.p also exhibited significant antidepressant activity compared with imipramine and fluoxetine (P < 0.001). Comparing derivatives with different halogen substituents on the benzene ring, their activity order was Cl > Br > NO₂ and it resulted in a decrease in antidepressant effect of existing pyrazoline derivatives. Atom bromine on the phenyl ring gave the same contribution to the antidepressant activity compared to Br atom at the 5th position of indole of the target compounds. Replacement of phenyl ring by heterocyclic nucleus on the proposed scaffold increases antidepressant activity and their activity order was thienyl > pyrrole > pyridine. Derivatives with an electron donating group on the phenyl ring of indolyl pyrazoline appear to be a more promising structural feature than with an electron withdrawing group for antidepressant like effects.

3.3 Anticonvulsant activity

The synthesized compounds 2a–m were also evaluated for anticonvulsant activity against PTZ induced seizures in mice and the results from these experiments are shown in Table 2.

Compounds 2c and 2d exhibited the most protective class of the tested compounds and other compounds 2f, 2j, 2h, 2k and 2m exhibited a moderate protection against clonic seizures induced by injection of scPTZ. While evaluating the anticonvulsant activity, it was observed that compounds carrying an electron withdrawing group (2c, 2d and 2m) on the phenyl ring C3 of pyrazoline had shown profound activity in comparison to compounds having electron releasing group (2b, 2e and 2l). Some selected compounds have been further tested for their neurotoxicity using the rotarod toxicity test. None of the synthesized compounds showed neurotoxicity as shown in Table 2.