In Silico Drug Design, Molecular Docking, Synthesis and Characterization of 2-Phenyl Indole Derivatives

1.1 Indole

Benzo pyrrole, which is produced by combining indigo with oleum, is known by the trivial term indole. Baeyer and Knop reduced indigo in 1866 to get two compounds, dihydroxy indole and oxindole, which they later proposed as the term "Indole." They believed that indigo was a derivative of C8H7N.Different pharmacological actions are present in heterocyclic compounds. Five or six members of an atom of nitrogen, sulphur, or oxygen are substituted and fused.[1] Heterocyclic compounds have a significant role in drug development and medical chemistry. 5-7 A fused heterocyclic compound, the indole nucleus consists of a five-membered pyrrole ring and a six-membered benzene ring. The majority of commercially accessible medications are heterocyclic molecules that contain nitrogen. In medicinal chemistry, the indole nucleus found in the derivatives plays a significant role in pharmacological actions such as anticancer, antiviral, antihypertensive, anti-inflammatory, analgesic, antibacterial, antifungal, and antidepressant properties.

Figure 1 : Biological activity of 2-phenyl indole

Indole is a crucial neurotransmitter that contains a heterocyclic nucleus, the backbone of the natural hormone melatoin, the neuro transmitter serotonin, and the vital amino acid tryptophan. The derivatives of indole have either been synthesized or extracted from natural sources. Indole alkaloids such as vincristine, vinblastine, and mitomycin, as well as their counterparts, are utilized in antibacterial and cancer chemotherapy, respectively. They are a good starting point for the creation of novel antibacterial and antifungal drugs since they contain indole. The main aim of this research is to design and synthesize a series of 2-phenyl indole derivatives with promising anticancer activity. Through various techniques, such as chemical synthesis, characterization, and in-silico modeling, the study aims to discover and evaluate novel compounds that could potentially be developed into effective anticancer drugs.[2,3,4]

MATERIALS USED

2.1 In-silico drug design studies

2.1.1 ChemDraw

Designing a molecule using ChemDraw is a powerful tool for creating chemical structures and visualizing molecular designs. ChemDraw allows you to represent organic and inorganic molecules in various formats. It offers a user-friendly interface and a range of tools and features that facilitate the creation of high-quality chemical diagrams.[5]

2.1.2 IBM RXN chemistry for Reaction prediction

A cloud-based platform called IBM RXN for Chemistry was created by IBM and uses machine learning (ML) and artificial intelligence (AI) methods to forecast and produce chemical reactions. It is intended to help researchers and chemists find and improve synthetic pathways and reactions[6].

2.1.3 PubChem for Molecular library

The National Centre for Biotechnology Information (NCBI), a division of the National Library of Medicine in the United States, is responsible for maintaining the free online database PubChem. It offers a thorough compilation of chemical data, such as chemical structures, characteristics, biological activity, and citations to academic publications. Chemists, researchers, and the general public can explore and access chemical and biological data with the help of PubChem[7]

2.1.4 RCSB Protein Data Bank (PDB)

The RCSB Protein Data Bank (PDB) is a comprehensive and widely-used online resource that provides access to three-dimensional structures of biological macromolecules, including proteins, nucleic acids, and complex assemblies. It is a collaborative effort between the Research Collaboratory for Structural Bioinformatics (RCSB), which includes Rutgers University, the University of California, San Diego, and the University of California, San Francisco.[8]

Schrodinger Suite for Docking studies

Schrodinger Suite, developed by Schrödinger, Inc., is a comprehensive software package widely used in computational chemistry and drug discovery. It provides a range of tools and modules for molecular modeling, simulations, structure-based drug design, and data analysis.[9]

Swiss ADME

SWISS ADME (Absorption, Distribution, Metabolism, and Excretion) is a web-based tool developed by the Swiss Institute of Bioinformatics (SIB) that provides predictions and assessments of various pharmacokinetic and drug-like properties of small molecules. It aids in the early stages of drug discovery and optimization by providing insights into the potential ADME characteristics of compounds.[10,11]

2.2 Molecular Docking

2.2.1 Software’s used

Table 1 Showing list of Software used for study

2.3 Synthesis

2.3.1 Equipment’s & apparatus used

The synthesis of 2-phenyl indole can be accomplished using various methods and reagents. The specific equipment and apparatus required may vary depending on the chosen synthetic route.[12,13,14,15]

Table 2 Showing list of Laboratory Equipment used in the study

Table 3 Showing list of Laboratory Apparatus used for the study

METHODS

Scheme Preparation   

Figure 2 : Scheme I

Figure 3: Scheme II

Scheme I: Reaction with 2-Phenyl indole: 2-Phenyl indole reacts with Para amino benzoic acid to yield 4 -(2-phenyl-1H indol- 1-yl) benzoic acid(4PI)

Scheme II: 2-Phenyl indole reacts with Thioglycolic acid to yield [(2 -phenyl-4H-indol-4- yl)sulfanyl]acetic acid.

Scheme I

Step-1: Preparation of Acetophenone phenyl hydrazone[16,17,18] 5.15 g of acetophenone (0.042mol) was taken in a conical flask containing 5 mL of Ethanol and 1 mL of glacial acetic acid. To the above mixture 4.53 g of phenyl hydrazine was added dropwise with constant swirling. Heat the reaction mixture in sand bath for 10 minutes and cool the resulting mixture in ice bath, allow the product to precipitate. Collect the precipitate by filtration on filter paper and wash with dilute hydrochloric acid (3 mL) followed by cold ethanol (5 mL). Allow the precipitate to air dry on the filter paper and recrystallized to get pure product from ethanol.

Step-2: Preparation of 2-Phenyl indole

Place crude acetophenone phenyl hydrazone beaker containing 15 mL of ortho phosphoric acid and 5 ml. of concentrated sulphuric acid. Heat the resulting mixture on the water bath for 20 minutes at 100-120°C with constant stirring. Pour the hot reaction mixture into 50 mL of cold water and wash the beaker with few mL of water. Collect the precipitated crude product on a filter paper, wash it with ethanol and allow it to air dry on the filter paper. Recrystallize the crude product from ethanol-water, using about g of decolorizing carbon and filter hot calculate the yield and obtain melting point.

Step-3: preparation of 4-(2-phenyl-1H-indol-1-yl) benzoic acid

A mixture of 2-phenyl indole (1.89g 0.01mol) and Para amino benzoic acid (1.37g 0.01mol) in ethanol (40ml) were refluxed for 4hours and excess of solvent was removed by distillation process. The resulting solid was dried and recrystalised from dilute ethanol to obtain the product.

SPECTRAL ANALYSIS: The synthesized derivatives were characterized using advanced analytical techniques like, Nuclear Magnetic Resonance (NMR) spectroscopy & Mass Spectrometry (MS)

RESULTS & DISCUSSION

5.1 In Silico Drug Design: The initial phase of the study involved the utilization of in silico techniques to design and predict the properties of novel 2-phenyl indole derivatives with potential therapeutic effects. Computational tools such as molecular docking, virtual screening, and pharmacokinetic analysis were employed to identify potential drug candidates. The results of the Insilco drug design phase revealed several compounds with favourable binding affinities and drug-like properties, suggesting their potential as effective drug molecules.

Sr. No.	Code	Compound	Docking score	No of H- bonding
1.	1PI	[(2-phenyl-2,3-dihydro-1H-indol-4-yl)sulfanyl]acetic acid	-6	4
2.	3PI	4-(2-phenyl-1H-indol-1-yl)benzoic acid	-7	3
3.	n5PI	[2-(4-chlorophenyl)-3-ethyl-5-(methanesulfonyl)-1H indol-1-yl](phenyl)methanone	-6	7
4.	n4PI	benzyl 2-(3-chloro-4-methylphenyl)-2,3-dihydro-1H indole-3-carboxylate	-7	1
5.	n7PI	4-({5-chloro-2-[4-(methanesulfonyl)phenyl]-1H-indol-1- yl}methyl)phenol	-5	5
6.	n8PI	1-[(4-hydroxyphenyl)methyl]-2-[4- (methanesulfonyl)phenyl]-1H-indole-5-carboxylic acid	-5	8
7.	25PI	4-({2-[4-(methanesulfonyl)phenyl]-1H-indol-1- yl}methyl)phenol	-7	5

Standard drug

8.

STD

N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1H- indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3- carboxamide

-5

Result showing Physichochemical Properties, Lipophilicity, Druglikeness, Water Solubility, Pharmacokinetics, Medicinal Chemistry Parameter for each newly developed derivatives.

Table 4.3 ; 4-(2-phenyl-1H-indol-1-yl)benzoic acid

SMILE : OC(=O)c1ccc(cc1)n1c(cc2c1cccc2)c1ccccc1
Physicochemical Properties		Water Solubility
Formula	C21H15NO2	Log S (ESOL)	-5.29
Molecular weight	313.35 g/mol	Solubility	1.62e-03 mg/ml ; 5.16e-06 mol/
Num. heavy atoms	24	Class	Moderately soluble
Arom, heavy atoms	21	Log S (Ali)	-5.47
Fraction Csp3	0.00	Solubility	1.06e-03 mg/ml ; 3.38e-06 mol/l
Rotatable bonds	3	Class	Moderately soluble
H-bond acceptors	2	LogS(SILICOS- IT)	-6.85
H-bond donors	1	Solubility	4.45e-05 mg/ml ; 1.42e-07 mol/l
Molar Refractivity	95.67	Class	Poorly soluble
Lipophilicity		Druglikeness
Log Po/w (iLOGP)	2.61	Lipinski	Yes; 0 violation
Log Po/w (XLOGP3)	4.85	Ghose	Yes
Log Po/w (WLOGP)	5.00	Veber	Yes
Log Po/w (MLOGP)	3.98	Egan	Yes
Log Po/w (SILICOS)	4.00	Muegge	Yes
Consensus Log Po/w	4.09	Bioavailability	0.85
Pharmacokinetics		Medicinal Chemistry
GI absorption	High	PAINS	0 alert
BBB permeant	Yes	Brenk	0 alert
P-gp substrate	No	Leadlikeness	No; 1 violation: XLOGP3>3.5
CYP1A2 inhibitor	Yes	Synthetic accessibility	2.30

Table 4.4 ;benzyl 2-(3-chloro-4-methylphenyl)-2,3-dihydro-1H-indole-3- carboxylate

SMILE : O=C(C1C(Nc2c1cccc2)c1ccc(c(c1)Cl)C)OCc1ccccc1
Physicochemical Properties			Water Solubility
Formula	C23H20ClNO2	Log S (ESOL)	-5.82
Molecular weight	377.86 g/mol	Solubility	5.67e-04 mg/ml ; 1.50e- 06 mol/l
Num. heavy atoms	27	Class	Moderately soluble
Arom, heavy atoms	18	Log S (Ali)	-6.08
Fraction Csp3	0.17	Solubility	3.11e-04 mg/ml ; 8.24e- 07 mol/l
Rotatable bonds	5	Class	Poorly soluble
H-bond acceptors	2	LogS(SILICOS-	-8.61
		IT)
H-bond donors	1	Solubility	9.28e-07 mg/ml ; 2.45e- 09 mol/l
Molar Refractivity	111.38	Class	Poorly soluble
Lipophilicity		Druglikeness
Log Po/w (iLOGP)	3.68	Lipinski	Yes; 1 violation: MLOGP>4.15
Log Po/w (XLOGP3)	5.52	Ghose	Yes
Log Po/w (WLOGP)	4.59	Veber	Yes
Log Po/w (MLOGP)	4.50	Egan	Yes
Log Po/w (SILICOS)	5.40	Muegge	No; 1 violation: XLOGP3>5
Consensus Log Po/w	4.74	Bioavailability	0.55
Pharmacokinetics		Medicinal Chemistry
GI absorption	High	PAINS	0 alert
BBB permeant	Yes	Brenk	0 alert

Table4.5; [2-(4-chlorophenyl)-3-ethyl-5-(methanesulfonyl)-1H-indol-1- yl](phenyl)methanone

4.2 Reference compound sunitinib in the binding site of EGFR Kinase

Fig 4.3 showing binding of n4PI with Amino acids of EGFR Kinase

By analyzing the docking studies 3PI and n4PI molecule was found to be the better molecule among all other molecules. The n4PI molecule exhibited strong binding affinity with EGFR Kinase protein (PDB ID: 3POZ) with binding energy of -7 kcal/mol and thus turned out to be the most active 2-Phenyl indole derivative against EGFR Kinase protein. The results of the Insilco drug design phase demonstrated the successful utilization of computational techniques in identifying potential drug candidates. The selected compounds exhibited favourable binding affinities towards the target receptors, suggesting their potential therapeutic effects. The Insilco predictions served as a guide for the subsequent synthesis and evaluation of the compounds. The synthesis of the novel 2-phenyl indole derivatives was achieved through a well-established synthetic pathway.[19,20]

SYNTHESIS

Synthesis of Novel 2-Phenyl Indole Derivatives: Following the in-silico drug design, the selected compounds were synthesized using standard organic synthesis techniques. The synthetic pathway involved the modification of the indole core structure by introducing various substituents at different positions on the phenyl ring. The synthetic reactions were carried out under controlled conditions, and the desired compounds were obtained in good yields. The compound with high docking score (-7kcal/mol) 4-(2- phenyl-1H-indol-1-yl) benzoic acid were synthesised in Laboratory.[21,22] The melting point of the compounds were determined with the aid of Digital melting point apparatus.

Table 4.6. Melting point of Synthesized compound

Spectral analysis

Mass Spectra

The molecular ion peak appeared at m/z value is 313g/mol, corresponding to the molecular weight of the compound. The molecule 4-(2-phenyl-1 H–indole–1-yl ) benzoic acid has indole ring, phenyl group and benzoic acid group.[23]

Fragmentation pattern

• Loss of phenyl group or benzyl group (77 Da): The phenyl group leading to a fragmentation ion at m/z at 237 Da, which forms the base peak.

313 - 77 (phenyl group) = 237 Da

Because of the presence of nitrogen atom in the indole ring the compound is highly stable and forms base peak at 237 Da.

• Loss of Benzoic acid (121Da): The Benzoic acid leading to a fragmentation ion at m/z at 194 Da, which also forms a peak.

313 - 121 (Benzoic acid) = 194 Da

FTIR Spectra: Based on the FTIR data and the known structure of the compound, we can make some observations: The presence of peaks around 1600 cm^-1 suggests the presence of C-O stretching vibrations, which align with the carbonyl group in the aromatic ring of the indole moiety. The presence of peaks around 1309.03 cm^-1 and 1128.37 cm^-1 could indicate the C-N stretching vibrations in the indole ring. Peaks around 3000 cm^-1 and 3100 cm^-1 might correspond to various C-H bending and stretching vibrations in the aromatic rings and aliphatic groups. The peaks around 3484.72 cm^-1 suggest the presence of O-H stretching vibrations, which could be due to the hydroxyl group in the carboxylic acid or possibly hydrogen bonding interactions. Peaks in the range of 2544.14 cm^-1 and 1907.60 cm^- 1 might correspond to various functional groups like C≡C stretching vibrations and C≡N stretching vibrations. Overall, the observed peaks in the FTIR data do align with the expected functional groups present in 4-(2-phenyl-1H-indol-1-yl) benzoic acid.

FTIR Spectrum of 4-(2-phenyl-1H-indol-1-yl) benzoic acid