INTRODUCTION:

Depression, also known as “clinical depression” or sometimes as “depressive disorder” is a mood disorder that may cause stressful symptoms that usually affect how you feel, think, or handle daily actions, like sleeping, eating, or working¹. Depression can come about along with other serious illnesses like diabetes, cancer, heart disease and Parkinson’s disease. Depression can lead to the worsening of these conditions and vice versa. Sometimes, even the medications taken for treatment of these illnesses may lead to side effects that pave way to symptoms of depression². Depression is the initial expression and might vary from mild condition, adjoining on customariness to serve depression occasionally termed psychotic depression that may be accompanied by hallucination and delusions^3-6.

Heterocyclic molecules are widely synthesized and investigated for their CNS effects especially antidepressant action^7-13. Indoles are electron rich heterocyclic systems in which the pyrrole and benzene rings are fused at position 2 and 3 of pyrrole.

Vilazodone, is an indole based molecule that has already been developed for managing depression. Over the past few decades, several researchers have synthesized indole derivatives for the CNS effects including anticonvulsant, antianxiety and antidepressant^14,15. Recently, a few indole alkaloids viz. harman, harmol, harmine, harmalol, harmaline and mitragynine have been isolated and were found to possess good antidepressant action.

Owing to the growing interest in antidepressant potential of indole derivatives, it was envisioned to synthesize a few 2-phenylindole derivatives and evaluate them for antidepressant potential.

MATERIAL AND METHODS:

Substituted acetophenones, phenyl hydrazine, and polyphosphoric acid were obtained from Avra Chemicals. Ethyl chloroacetate were purchased from Sigma. All solvents and reagents were procured from qualigens. Melting points are uncorrected and determined using open capillary in a melting apparatus (BioTechnics). Thin layer chromatography was used to reach the completion of the reaction and purity of the compounds synthesized.

The route for synthesis of the target indole molecules has been adapted from previously reported procedures (Scheme 1)^16,17.

Scheme 1. Synthetic route for 2-phenyl indole congeners

General method for synthesis of substituted phenyl hydrazine:

A mixture of 0.334mol of appropriate acetophenone and 0.334mol of phenyl hydrazine was prepared in 120mL ethanol and a few drops of glacial acetic acid were added to it. The mixture was cooled to 0°C using an ice bath to obtain a solid. The solid was filtered and washed with dilute HCl and then by rectified spirit. The product was recrystallized using ethanol and white product obtained was filtered and stored in air tight container.

General method for synthesis of substituted phenyl indole:

0.15mol of the substituted phenyl hydrazone was placed in a beaker containing excess of polyphosphoric acid (180g). The mix was heated on a water bath, stirring the mixture and maintaining the temperature at 100-120°C for duration of 10min. In the mixture was added 450mL cold water and it was well stirred in order to dissolve the polyphosphoric acid completely. The solid obtained was filtered at pump and washed using ice-cold water several times to remove any trace of acid. The solid was refluxed with 300mL of rectified spirit and a little amount of decolorizing charcoal was added to it and filtered. The filtrate was cooled to room temperature to obtain the white crystals of phenyl indole which were dried in desiccator over anhydrous calcium chloride and stored in air tight container.

General method for synthesis of N- ethyl acetate-2-phenyl indole derivatives:

Anhydrous potassium carbonate (K₂CO₃) (0.006mol) was added to a solution of the appropriate phenyl indole (0.003mol) and ethylchloroacetate (0.003mol) dissolved in anhydrous DMF (10mL) in a round bottom flask and refluxed for 1-2h. The mixture was poured onto crushed ice to precipitate the solid product. The product was filtered at pump using Buchner funnel. The physicochemical characters were determined using reported methods¹⁸.

Pharmacological Study:

Animal:

The in vivo antidepressant activity of the synthesized compounds was performed in male albino mice of weight between 25–30g by Forced Swim Test (FST) and Tail Suspension Test (TST) methods.

The animal were kept segregated in polyacrylic cages and housed in the animal house of the institute under standard conditions (14h light/10 h dark; 27±2°C; %RH 44-56%) with openaccess to use standard diet and potable water ad libitum for a week.

Animal were grouped in 7 groups of 6 animals in each for conducting the procedures. Group I (control) was administered normal saline, group II, III, IV,V and VI were administered 50mg/kg (i.p) of the test compounds, whereas group VII served as positive control and was administered with fluoxetine, 10mg/kg (i.p).

Acute Toxicity Study:

A total of three animals were used which received a single oral dose (2000mg/kg) of each compound. Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24h and daily thereafter for a period of 14 days. Once daily observations were made for changes in skin and fur, eyes and mucous membrane (nasal) and also respiratory rate, circulatory (heart rate and blood pressure), autonomic (salivation, perspiration, urinary incontinence, and defecation) and central nervous system (drowsiness, tremors and convulsion) changes. Mortality, if any, was also observed over the period of 2 weeks.

Forced Swim Test¹⁹:

The synthesized compounds and ﬂuoxetine were suspended in DMSO and injected intraperitoneally (0.05 mL per 20 g body weight), 30 minutes prior to the test. To determine the effect of the test compound mice were individually kept in a glass cylinder (25cm high and 10 cm wide) filled with water (22-25°C) up to 10cm of the cylinder. Each mouse was allowed to swim 6 min during the test, and the duration of inaction or immobility was noted down during the final 4 min of the test. The time spent by the mice floating in the water without struggling and making only those movements necessary to keep its head above water was regarded as the immobility period.

The animals were dried using towel and returned back to their housing conditions.

Tail Suspension Test¹⁹:

The synthesized compounds and ﬂuoxetine were dispersed in DMSO and injected intraperitoneally (0.05 mL per 20g body weight), 30 minutes prior to the test. To determine the effect of the test compound mice were suspended by tail using clamp (2cm from the tip of the tail) in a box (25 × 25 × 30cm) with the head 5cm from the bottom. Minimal background noise was maintained and the test was done in dark room. The total time of keeping animals suspended by tail was 6 minutes, and the duration of inaction or immobility was noted during the final 4 minutes of the test. Animal was considered immobile when they hung completely motionless.

RESULTS AND DISCUSSION:

Synthesis:

In the initial steps, 2-phenyl indole derivatives were prepared by condensation of phenyl hydrazine and aromatic ketone followed by Fisher indole cyclization procedure in acidic catalyst. In the final step, the nitrogen of the 2-phenyl indole derivatives was substituted with ethyl acetate by reaction with ethyl chloroacetate.

Five derivatives of phenylindole were synthesized using five different acetophenones and each was substituted at 1-postion with ethylacetate. The compounds were confirmed by TLC as well as FTIR. The ¹H-NMR and mass spectra studies were carried out to affirm the preparation of molecules. All the compounds were found to be insoluble in water and dimethyl formamide whereas only partially soluble in methanol. The compounds were easily soluble in chloroform.

Ethyl 2-(2-phenyl-1H-indol-1-yl) acetate, 3a

Color: Brown; Yield (%): 62; R_f value: 0.66; M.P (°C): 235-238;¹H NMR Spectra (d, 300 MHz, CDCl₃): 7.2-7.5 (H aromatic), 6.5 (H-pyrrole ring), 3.60 (H-methyl); IR (KBr): 3420.33 (C-H), 3161.06 cm^-1 (CH Ar), 2603.54 (-CH₂-), 1600-1700 cm^-1 (C-C Ar), 1485.76 cm^-1 (C=C), 1197.04 cm^-1 (C-N); m/z: 279.3

Ethyl 2-(2-(3-hydroxyphenyl)-1H-indol-1-yl) acetate, 3b

Color: Yellow; Yield (%): 68; R_f value: 0.45; M.P (°C): 204-206; ¹H NMR Spectra (d, 300 MHz, CDCl₃): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH-Benzene), 2.5 (CH₂), 7.0 (CH-Benzene), 7.1 (CH-Benzene); IR (KBr): 3410.90 (O-H), 3121.21 (C-H Ar), 2602.10 (C-H), 1500-1650 cm^-1 (C-C Ar), 1481.52 (C=C), 1282.15 (C-N), 1069.29 (C-O); m/z: 295.3

Ethyl 2-(2-(4-hydroxyphenyl)-1H-indol-1-yl) acetate, 3c

Color: Yellow; Yield (%): 69; R_f value: 0.62; M.P (°C): 248-251; ¹H NMR Spectra (d, 300 MHz, CDCl₃): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH-Benzene), 2.5 (CH₂), 7.0 (CH-Benzene), 7.1 (CH-Benzene); IR (KBr): 3240.81 (O-H), 3119.55 (C-H Ar), 2981.44 (C-H), 1500-1650 cm^-1 (C-C Ar), 1484.01 (C=C), 1396.53 (O-H bending), 1294.08 (C-N), 1092.37 (C-O); m/z: 295.3

Ethyl 2-(2-p-tolyl-1H-indol-1-yl) acetate, 3d

Color: Brown; Yield (%): 71; R_f value: 0.53; M.P (°C): 269-271; ¹H NMR Spectra (d, 300 MHz, CDCl₃): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH-Benzene), 2.5 (CH₃), 7.0 (CH-Benzene), 7.1 (CH-Benzene); IR (KBr): 3113.84 (C-H Ar), 2943.23 (C-H), 1500-1650 cm^-1 (C-C Ar), 1461.21 (C=C), 1282.19 (C-N); m/z: 293.3

Ethyl 2-(2-(4-nitrophenyl)-1H-indol-1-yl) acetate, 3e

Color: Brown; Yield (%): 74; R_f value: 0.42; M.P (°C): 257-260; ¹H NMR Spectra (d, 300 MHz, CDCl₃): 7.1-7.4 (H Aromatic), 8.25 (H adjacent to NO₂), 3.6 (H – methyl); IR (KBr): 3116.02 (C-H Ar), 2978.17, 2809.22 (C-H), 1500-1650 cm^-1 (C-C Ar), 1525.49 (N-O), 1454.47 (C=C), 1285.93 (C-N); m/z: 324.3

The IR spectra of all the compounds exhibited the stretching vibration peaks due to C-N, C=C, C-H Ar, CH aliphatic at 1300-1100 cm^-1, 1500-1700 cm^-1, 3000-3200 cm^-1 and 2600-3000 cm^-1 respectively. The other vibrations that appeared in the spectra included those from C-O (1000-1100 cm^-1), O-H (3200-3500 cm^-1) and N-O (1400-1500 cm^-1).

The ¹HNMR spectra obtained displayed the peaks of aliphatic CH and aromatic CH as well as peak of O-H in the corresponding compounds (3b, 3c). The mass spectra exhibited the molecular ion peak or the isotopic mass peaks.

Acute toxicity study:

None of the animals presented any symptom of toxicity or mortality and hence the dose of 2000mg/kg was found to be safe for the animals. A 1/20^th dose (50 mg/kg) was considered for determination of CNS depressant action in rodent.

CNS depressant action:

The antidepressant action of the synthesized compounds was tested using two animal models and the results obtained are depicted in Figure 1 and 2. The immobility time was observed in TST while the swimming frequency in FST was statistically analyzed using one way ANOVA followed by Dunnett’s multiple comparison test.

Figure 1: Effect of indoles (50mg/kg) and fluoxetine (10mg/kg) on swimming frequency of mice in FST. *p<0.05, ***p<0.001, ns –not significant; Values are represented as mean ± SD, (n = 6)

As it can be seen from Figure 1 the swimming frequency for the compounds 3d and 3e was much higher than the control group and was comparable to that of fluoxetine (10mg/kg). On the other hand the results obtained by compounds 3a, 3b and 3c were not as significant suggesting the importance of presence of hydrophobic substitution on the phenyl ring.

Figure 2: Effect of indoles (50mg/kg) and fluoxetine (10 mg/kg) on immobility time of mice in TST. ***p<0.05, ns-not significant, Values are represented as mean±SD, (n = 6)

The results of TST (Figure 2) reinforce the inference that compounds 3d and 3e with more hydrophobic substituents were able to reduce the immobility time exhibiting an increase in alertness in the animals in comparison to others. It was also evident that the presence of hydroxyl substitution on the 2-phenyl ring of indole was detrimental for its antidepressant action. Also it was concluded that the hydroxyl substituent on para position was more detrimental to the activity of the compounds.

CONCLUSION:

As indole containing molecule vilazodone is a widely prescribed antidepressant agent, the present work focused on synthesizing 2-phenyl indole derivatives possessing antidepressant potential. The synthesized compounds with diverse substitution pattern were able to exhibit antidepressant action. Further studies on new compounds of similar structure would be carried out in order to derive a relation between the structure and activity of the nucleus.

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Received on 27.09.2022 Modified on 21.12.2022

Research J. Pharm. and Tech 2023; 16(6):2715-2718.

DOI: 10.52711/0974-360X.2023.00446