Synthesis Insilico Screening and Evaluation of Novel Indole Derivatives for Possible Biological Activity

The widespread use of antibiotics has significantly improved public health by reducing the incidence and severity of bacterial infections. However, the inappropriate and excessive use of these agents has accelerated the emergence of antibiotic-resistant bacterial strains, thereby diminishing the effectiveness of conventional antimicrobial therapies. The rapid development of antimicrobial resistance has become a major global health concern and highlights the urgent need for the discovery of novel antibacterial agents. Among the promising heterocyclic scaffolds, benzimidazole and indole derivatives have attracted considerable attention because of their broad spectrum of biological activities. These aromatic heterocyclic compounds serve as valuable structural frameworks in medicinal chemistry and are extensively utilized for the design, synthesis, and structural modification of new pharmaceutical agents with enhanced therapeutic potential.

 The indole nucleus is a pharmacologically important bicyclic heterocyclic ring with nitrogen atom which exhibits a broad spectrum of biological activities. Owing to its versatile chemical structure, indole has been extensively employed in medicinal chemistry to enhance the antibacterial potential of therapeutic compounds. Furthermore, indole derivatives have demonstrated significant pharmacological activities, including antitumor, antimicrobial, antibacterial, antiviral, antifungal, and anti-HIV effects.

MATEERIALS AND METHODS

Phenylhydrazine, 4-bromo acetophenone, Ethanol, Glacial acetic acid, Dilute HCL, Rectified spirit, Polyphosphoric acid, Activated charcoal, Potassium carbonate, Dimethyl formamide, Alkyl halides (Chloro fluro benzene, 2-chloro ethanol, Chloroacetonitrile, 2-chloro acetamide, Trifluoromethyl).

All the reactions werw performed in dried borosilicate glass beakers, round bottemed flask, Heating mantle with reflux condenser.   Precoated silica gel plates (MERCK) were useed for TLC to monitor progress of the reaction. Compounds melting points were determined by capillary method and are uncorrected. JASCO UV chamber was used for detection of spots in TLC. IR spectra were recorded on BRUKER FTIR spectrometer. 1H NMR spectra were recorded on 400MHZ spectrometer using DMSO as solvent. The chemical shift data were expressed as values relative to TMS in ppm. Mass spectra were recoded on a 3-5kvESI-MS instrument. 13C NMR spectra were recoded on 100MHZ spectrometer using DMSO as solvent. Elemental analyses (C,H, and N) of the compounds were obbtained from perki-Elmer 240B analyzer and were within ± 0.4% of the theoretical values.

Experimental

In Silico Screening

Lipinski’s rule of 5 filtration

The molecular structures were prepared and uploaded in one of the supported formats, including *.pdb, *.mol, *.mol2, *.xyz, *.sdf, or *.smiles. The input file names were carefully checked to ensure they contained no blank spaces or unnecessary characters. After uploading the files, the pH was adjusted within the range of 0–14 according to the study requirements. The files were then submitted, and the molecular property results were generated following the method described by Lipinski (2004).

OSIRIS Property Explorer

In the present study, OSIRIS Property Explorer Version 2, a Java-based software application, was employed to predict the physicochemical and toxicity properties of the designed compounds. The chemical structures were either drawn directly within the software or imported in SMILES format for analysis. The software generated property predictions using a color-coded display, where green indicated a favorable or non-toxic property, while red signified a potential toxicity risk. This approach facilitated the preliminary assessment of the drug-likeness and safety profile of the synthesized compounds prior to further computational and experimental investigations.

Prediction of Activity Spectra for Substances (PASS)

Compounds that satisfied the Lipinski rule of five were further evaluated using the online PASS (Prediction of Activity Spectra for Substances) software to predict their potential biological activities. The prediction was based on the probability of activity (Pa) and probability of inactivity (Pi) values, which range from 0.000 to 1.000. These values were used as the selection criteria for assessing the likelihood of various biological activities of the designed molecules.

Molsoft Property Explorer

In the present study, Molsoft Property Explorer (Version 3.7-2) was employed to evaluate the physicochemical properties of the designed compounds. The molecular structures were either drawn directly within the software interface or imported in MOL, InChI, or SMILES formats. The software was used to calculate key molecular descriptors, including MlogP (octanol/water partition coefficient) and MlogS (aqueous solubility), which are important parameters for assessing the drug-likeness of the compounds.

Molecular Docking

Molecular docking studies were performed using AutoDock 4.2 to investigate the binding interactions of the synthesized indole derivatives (5a–5e) with the bacterial target enzyme Dihydrofolate Reductase (DHFR). The crystal structure of DHFR was retrieved from the Protein Data Bank (PDB) and prepared by removing water molecules and heteroatoms, followed by the addition of polar hydrogens and Kollman charges. The synthesized compounds and the standard drug, Norfloxacin, were drawn using ChemDraw, converted into 3D structures, energy minimized, and saved in PDBQT format. Docking simulations were carried out using the Lamarckian Genetic Algorithm (LGA) in AutoDock 4.2. The docked complexes were analyzed based on binding energy, hydrogen bonding, hydrophobic interactions, and interacting amino acid residues. The compound with the lowest binding energy was considered the most stable, and the results were compared with Norfloxacin to evaluate the antibacterial potential of the synthesized indole derivatives.

Chemistry

Synthesi of (E)-1-(4-bromophenyl)ethanone phenylhydrazone

A solution of 4-bromoacetophenone (1 mmol) in ethanol (10 mL) was treated with phenylhydrazine (1 mmol), followed by the addition of 2–3 drops of glacial acetic acid as a catalyst. The reaction mixture was refluxed at 70–80°C for 2–4 h, and the progress was monitored by TLC. After completion, the mixture was cooled to room temperature and then in an ice bath to induce precipitation of the hydrazone. The solid product was collected by filtration, washed successively with dilute hydrochloric acid and rectified spirit, and recrystallized from ethanol to afford pure (E)-1-(4-bromophenyl)ethanone phenylhydrazone.

Synthesis of 2-(4-bromophenyl)-1H-indole

Polyphosphoric acid (PPA) was gently warmed until liquefied, and (E)-1-(4-bromophenyl)ethanone phenylhydrazone (1 mmol) was added portion-wise with continuous stirring. The reaction mixture was heated at 100–120°C for 20–30 min, and the progress was monitored by TLC. After completion, the mixture was cooled and poured onto crushed ice to precipitate the crude indole derivative. The solid was filtered, washed thoroughly with cold water, and dried. The crude product was purified by recrystallization from hot ethanol using activated charcoal, filtered, and the filtrate was allowed to evaporate to obtain the purified indole derivative, which was stored in an airtight container.

Synthesis of N- substituted-2-(4-bromophenyl)-1H indole derivatives (1a-e)

2-(4-Bromophenyl)-1H-indole (1 mmol) was dissolved in dry DMF (10 mL), followed by the addition of K₂CO₃ (1.5 mmol). After stirring for 30 min at room temperature, the appropriate alkyl halide (1.2 mmol) was added dropwise. The reaction mixture was heated at 80–90°C for 6–8 h with continuous stirring, and the progress was monitored by TLC. Upon completion, the reaction mixture was cooled and poured into ice-cold water to precipitate the product. The crude solid was collected by filtration, washed with water, dried, and recrystallized from ethanol to afford the pure N-substituted indole derivatives.

SCHEME:

TABLE 3

Compound	Toxicity	Clogp	Solubility	Molecular weight (grams)	TPSA	Drug Likeness	Drug Score
5a	NO	5.74	-8.36	365	4.93	-0.93	0.2
5b	NO	3.64	-3.91	315	25.16	1.46	0.68
5c	Irritant	3.81	-4.57	310	28.72	-2.93	0.21
5d	NO	2.89	-3.97	328	48.02	2.62	0.75
5e	Irritant	6.52	-6.06	339	4.93	-5.52	0.14
Standard Norfloxacin	NO	-1.65	-2.86	319	72.88	2.24	0.86

 

 

 

 

Molsoft Property Explorer

 

 

                          5a                                                         5b                                                      5c

 

                        5d                                                        5e                                   Standard(Norfloxacin)

Physical Characterisation for Synthesized Derivatives

TABLE 6

Compound	Structure	Molecular formula	Molecular weight	Melting point	Rf value	Percentage Yeild
5a		C20H13BrFN	365	188	0.62	78%
5b		C16H14BrNO	315	153	0.46	80%
5c		C16H11BrN2	310	172	0.58	82%
5d		C16H13BrN2O	328	198	0.40	78%
5e		C15H10BrF3N	339	118	0.68	84%

TABLE 7: Zone oF Inhibition (Concentration µ/ml )

Sr. No	Compound	Staphylococcus aureas				Bacillus subtilis
		25 µg/mL	50 µg/mL	75 µg/mL	100 µg/mL	25 µg/mL	50 µg/mL	75 µg/mL	100 µg/mL
1	5a	-	-	14 ±0.2	18 ± 0.3	-	12 ± 0.3	16 ± 0.3	20 ± 0.3
2	5b	-	-	12 ±0.3	16 ± 0.3	-	-	14 ± 0.3	18 ± 0.3
3	5c	-	12 ±0.3	16 ±0.2	20 ± 0.3	-	14 ± 0.3	18 ± 0.3	22 ± 0.3
4	5d	14±0.3	18 ±0.3	22 ±0.3	26 ± 0.3	16 ± 0.3	20 ± 0.3	24 ± 0.3	28 ± 0.3
5	5e	-	12 ±0.3	15 ±0.3	20 ± 0.3	-	14 ± 0.3	18 ± 0.3	22 ± 0.3
6	Standard norfloxacin	16±0.3	20 ±0.3	24 ±0.2	38 ± 0.3	18 ± 0.3	22 ± 0.3	28 ± 0.3	40 ± 0.3
7	DMSO	-	-	-	No mimimal inhibition	-	-	-	No mimimal inhibition

TABLE 8: Minimum inhibitory concentration

Sr. No	Compound	MIC (µ/ml)
		Staphylococcus aureas	Bacillus subtilis
1	5a	75	50
2	5b	75	75
3	5c	50	50
4	5d	25	25
5	5e	50	50
6	Standard norfloxacin	25	25
7	DMSO	-	-

 

CONCLUSION

The present study successfully designed, synthesized, and characterized a series of novel indole derivatives (5a–5e). The synthesized compounds were confirmed by spectroscopic techniques such as FTIR, ^1H NMR, ^13C NMR, and mass spectrometry. Biological evaluation against Staphylococcus aureus and Bacillus subtilis demonstrated that several derivatives exhibited promising antibacterial activity, with compound 5d showing the Potent inhibitory effect. Molecular docking studies with Novel indole derivative, Standard Norfloxacin and  the DHFR (3SRQ) target protein further supported the experimental findings, as the most active compounds exhibited favorable binding affinities and stable interactions within the active site. Overall, the combined experimental and computational results indicate that the synthesized indole derivatives possess significant potential as antibacterial agents and may serve as promising lead molecules for the development of new antimicrobial drugs. Further optimization and in vivo studies are recommended to establish their therapeutic efficacy and safety.

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