Molecular Docking, Synthesis and Pharmacological Screening of Indole-3-Mannich bases as EGFR Kinase Inhibitors to Combat non-small-cell Lung Cancer

Sonal Pathak, Archana S. Gurjar*

Dept. of Pharmaceutical Chemistry, Principal K. M. Kundnani College of Pharmacy, Mumbai - 400005, India.

*Corresponding Author E-mail: as.gurjar@kmkcp.edu.in

ABSTRACT:

The epidermal growth factor receptor (EGFR) has been identified as a clinically relevant target for the treatment of advanced non-small-cell lung cancer patients (NSCLC). EGFR-directed tyrosine kinase inhibitors (TKIs) have demonstrated efficacy in patients with advanced NSCLC. The drugs Erlotinib and Osimertinib are commonly prescribed for the NSCLC treatment so docking studies were carried out on the two X-ray crystallographic Protein Data Bank (PDB) IDs, 6LUD and 1M17, with co-crystallized molecules Osimertinib and Erlotinib, respectively. Similar to indole scaffold present in Osimertinib, the series was designed with substitutions at third position on indole with different aldehydes and secondary amines to form mannich bases. On docking of the designed series in both active sites, IM_23, IM_24, IM_25, IM_44, IM_45, IM_50, IM_52, IM_57, and IM_63 demonstrated interactions with active site residues in both the pdb structures.  In silico ADME studies were performed to judge the drug likeliness. The analogs with favourable computational results were synthesized utilizing green, benign method and characterized. The synthesized analogs were assessed for anti-proliferative properties on the human lung cancer A549 cell line in vitro. IM_23 and IM_45 exhibited prominent activity. Correlation of docking and screening results indicated that electron-donating substituents on phenyl ring of the analogues showcased more interactions with crucial residues in active site such as Met 793 with better activity profile than electron-donating groups. In conclusion, some analogues exhibit potential as EGFR inhibitors to target non-small cell lung cancer. Further, the findings may offer perceptions into the possible EGFR binding properties, which may aid in the optimization and further development of more promising analogs.

KEYWORDS: Epidermal growth factor receptor, Non-small-cell lung cancer, Tyrosine kinase inhibitors, Sulforhodamine B assay.

1. INTRODUCTION: 

For both men and women, lung cancer is amongst the most lethal malignancies and small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) are its two primary subtypes with incidence of around 10% to 15% and 80% to 85% respectively¹. The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that is frequently activated in non-small cell lung cancer².

Cancer is frequently linked to the upregulation of EGFR activity brought on by overexpression or mutation³. To inhibit EGFR signaling, tyrosine kinase inhibitors (TKIs) and anti-EGFR antibodies can be utilized⁴. The EGFR has been identified as a clinically relevant target for the treatment of advanced NSCLC patients^5,6. Afatinib, erlotinib, and gefitinib have all been approved as first-line treatment for patients with advanced EGFR mutation-positive NSCLC⁷. Osimertinib (Fig. 1) is a third-generation EGFR TKI that is irreversible and active against exon 19 deletions as well as the L858R mutation, regardless of the presence of the T790M mutation18⁸. This TKI has less activity than the aforementioned TKIs against the wild-type EGFR protein and forms a covalent bond to the cysteine 797 residue⁹. A docking analysis was carried out to determine which developed hybrid had the optimum affinity for the EGFR tyrosine kinase domain as potential ligands¹⁰. Molecular docking is an effective in silico tool to judge the interactions of potential ligands within the active site of the receptor¹¹. Docking on pdb 6LUD by researchers, revealed the four quinoxalinone-containing, CPD4, CPD15, CPD16, and CPD21 candidates with promising results as new TK inhibitors of the EGFR (L858R/T790M/C797S). The IC₅₀ values 3.04±31.24nM, 6.50±3.02nM, 10.50±1.10nM, and 3.81± 1.80nM, respectively, by the enzyme-based assay, which are at a similar level to, osimertinib (8.93±.01nM)¹². We intend to utilize PDB ids 6LUD and 1M17 with marketed EGFR TKIs osimertinib and erlotinib as ligands to explore designed series of indole analogs for plausible interactions within active site of third generation and first-generation EGFR TKIs respectively. Osimertinib possesses an indole scaffold on similar lines we chose indole scaffold to design, dock, synthesize and screen analogs as potential EGFR TKIs for NSCLC (Fig.1)¹³.

Fig 1: Osimertinib, Erlotinib and Indole-3-mannich bases.

2. MATERIAL AND METHODS:

2.1 Computational Studies:

2.1.1 Design of ligands:

The substituted indole series comprising of 72 ligands were sketched in the 2D sketcher of Schrödinger’s Maestro release 2018-1, minimized using LigPrep with OPLS-2005 force field using Epik and Macro Model features at user-defined pH 6-8¹⁴.

2.1.2 Protein preparation:

Epik, Schrödinger, LLC, New York Protein Preparation Wizard; Epik, Schrödinger, LLC, New York was used to prepare the protein 6LUD and 1M17 with osimertinib and erlotinib as ligands respectively. Ligands were removed and then protein was energy minimized. Superimposition of respective ligands of 1M17 and 6LUD was used to validate the results.

2.1.3 Molecular docking:

Molecular Docking studies, GOLD v5.2.2 (Genetic Optimizations for Ligand Docking) software (CCDC, UK) employed to assess the interaction between the designed series of substituted indole molecules with the targets involved in the study. Docking was performed for 20 genetic algorithms (GA) runs with default parameters and gold score as fitness function. The best-ranked ligand pose was examined to determine the type of binding and interactions with the target protein sites ¹⁵.

2.1.4 In silico ADME:

LigPrep processed minimized 3D structures employed as input for QikProp run and results interpreted¹⁶.

2.2 Synthesis and Characterization: 2.2.1 Commercial suppliers provided all synthetic grade chemicals and reagents. The progress of chemical reaction, monitored using Merck silica gel F₂₅₄ plates. The physical constant was determined using traditional method of Thiele’s tube. For structural elucidation, infrared spectroscopy and nuclear magnetic resonance studies ¹HNMR were carried out on Bruker alpha FT-IR spectrometer and a Bruker BioSpin GmbH at 400 MHz in DMSO and CDCl₃, respectively.

2.2.2 Procedures for the synthesis of indole-3-mannich bases:

Method 1:

A mixture of aldehyde (1mmol), secondary amine (1 mmol), and EG (Ethylene glycol) (1mmol) was stirred under reflux at 70°C for 15 minutes before adding indole (1 mmol) to the reaction mixture. The reaction's progress was monitored using two TLC systems (chloroform and n-hexane: ethyl acetate 3:2). The reaction mixture was acidified with conc. HCl on completion. The crude product was purified by recrystallization with a hydroalcoholic mixture (50:50). All compounds were characterized using M.P, IR and ¹HNMR.

Method 2:

A 50mL beaker was charged with secondary amine (1 mmol), ethylene glycol (1mmol, and aldehyde (1mmol) in the water bath ultrasonicator (40 Hz, 35° C). After 15 minutes, (1mmol) of indole was added and the reaction mixture was irradiated. Two TLC systems were used to monitor the reaction's progress (chloroform and n-hexane: ethyl acetate 3:2). After completion of reaction, the mixture was acidified with conc. HCl and precipitate was filtered. Recrystallization of crude product with a ethanol: water (50:50) was used for purification. M.P, IR and ¹HNMR were used to characterize all compounds.

2.3 Sulforhodamine B (SRB) Assay:

Cell lines were cultured in a medium with 10% foetal bovine serum and 2mM L-glutamine. For the screening, 5000 cells per well were inoculated into 96-well microtiter plates and incubated for 24 hours at 37°C, 5% CO₂, 95% air, and 100% humidity¹⁷. 100mg/ml stock solution of experimental drugs were diluted to 1 mg/ml, further diluted to 100-800μg/ml for testing. Aliquots were added to the wells to achieve final drug concentrations of 10-80μg/ml. After 48hours of incubation, assay was terminated with cold trichloroacetic acid (TCA), and cells were stained with Sulforhodamine B (SRB). The absorbance was measured at 540nm, and percent growth was calculated by comparing test wells to control wells. Percentage growth inhibition was determined using absorbance values at different drug concentrations.

3. RESULTS AND DISCUSSION:

3.1 Molecular Docking Studies on EGFR Kinase:

A docking investigation was carried out to determine the interaction between the active site residues and the series. Docking studies on two X-ray crystallographic Protein Data Bank (PDB) IDs, 6LUD¹⁸ and 1M17¹⁹ with co-crystallized ligands Osimertinib and Erlotinib, respectively were undertaken to rationalize our targeting technique. The PDB was refined for docking based on the pH value, resolution, most recent PDB, and co- crystallized ligand. The 6LUD was selected for docking from amongst recent 13 PDB ids¹⁸. 6LUD was preferred as it contains a co-crystallized ligand^{, Osimertinib with an IC}₅₀ ^{value of 28nM20,} a marketed third-generation drug that is effectively used in first-line treatment for NSCLC. The PDB 6LUD (2.05 Å resolution) and osimertinib ligand with an indole ring is analogous to the designed indole scaffold series selected for our study. To substantiate docking protocol, PDB 1M17 (resolution ^{is 2.60 Å)} with first-generation marketed drug, Erlotinib ^(IC₅₀ ^23nM) as co-crystallized ligand was also used²⁰. Interaction and validation results shown in (Table 1).

^{Series of designed indole analogues was docked within the active site of PDB  6LUD and 1M17 utilizing the same validated protocol.}  To design different 72 indole-mannich bases (Fig. 1, Table 2), first the aliphatic secondary amines were considered with increase in alkyl chain such as methyl, ethyl and they exhibited no hydrogen bonds. While on studying interaction with easily available, non-aromatic heterocyclic secondary amine such as pyrrolidine, piperidine, morpholine, interactions were observed. On reducing ring size or decreasing number of hetero atoms such as piperidine, pyrrolidine did not show interaction with Met793 (active site residue) except IM_63. Pyrrolidine and piperidine containing compounds depicted interaction with other residues.

For the R_{2 -} aldehyde group of mannich bases, when formaldehyde was considered, resulted in a methylene bridge with no hydrogen bond interaction. Substitution on methylene hydrogen with phenyl e.g., benzaldehyde displayed no hydrogen bond interaction while substitution on phenyl ring at ortho, meta, para position with different substituents such as nitro, methoxy, hydroxy, halogen, substituted amines depicted hydrogen bond interaction. The NH₂ group of lysine (Lys745) which is a basic amino acid forms the hydrogen bond with the electronegative oxygen atom of nitro, methoxy, hydroxy and furfural substituents of the compounds with morpholine and piperidine both being as the six membered secondary amines. Whereas, threonine forms a hydrogen bond with the oxygen of hydroxy and methoxy present on para position, with all the compounds containing five membered secondary amines with the exception IM_17. IM_23 having morpholine as secondary amine showcased hydrogen bond with Met793, Lys745 (Fig 2). Whereas IM_45, IM_49, IM_50 possess pyrrolidine as secondary amine showed hydrogen bond with Thr854, Lys745, Asp855. IM_63 has piperidine moiety which depicted hydrogen bond with Lys745. Thus, in ^{the series it was observed that seventeen analogues exhibited} interactions analogous to 6LUD, nine analogues i.e., IM_23, IM_24, IM_25, IM_44, IM_45, IM_50, IM_52, IM_57, ^{IM_63 revealed interactions in common with both the PDB id 6LUD and 1M17 having ligands Osimertinib and Erlotinib respectively}. The analogues showcasing interactions similar to both the ligands comprise of different secondary amines as R₂ substituent such as morpholine, pyrrolidine and piperidine and R₁ with different aldehyde moiety consisting of benzaldehyde phenyl ring substituted at ortho, meta and para position primarily with methoxy groups viz. IM_23, IM_24, IM_50, IM_52 and IM_63. The benzaldehyde with -NO₂ group as substituent at ortho, meta and para position also exhibited promising interactions with active residues in both PDB ids viz. IM_44, IM_45, IM_57. IM_25 with morpholine and furfural moiety.

Table 1: Interaction with Crucial active site residues, conformation and validation results

PDB Id	Ligand	Interacting residues	Conformation	Validation RMSD (Å)
6LUD	Osimertinib	N-lobe: Leu718, Val726, Leu792, Ala743 C-lobe: Leu844, Lys745, Pro794, Ser797 ATP binding site/ hinge region: Met793, Met790	DFG-in/ αC- helix-in	0.8403
1M17	Erlotinib	N-lobe: Gly695-Gly700, Activation loop/ catalytic site: Arg812-Asn818, Val852, Asp813, C-lobe: Asp831-Gly833,	DFG-in/ αC- helix-in	0.7987

In Osimertinib, four hydrogen bonds were observed (Fig 2). The pyrimidine ring 4th nitrogen, the NH bridge, the terminal tertiary amine and covalent head exhibited hydrogen bond with Met793, Met793, Leu718 and Ser797, respectively. Pyrimidine ring with two hetero atoms exhibited hydrogen bond with Met793. Analogously morpholine non-aromatic with two hetero atoms in our designed indole series exhibited interaction with Met793. Thus, some designed and docked mannich bases possess potential for the target as projected by their docking results.

3.2 In silico ADME studies:

In silico ADME (absorption, distribution, metabolism, excretion) studies play a crucial role in the success, safety and efficacy of a drug candidate. ADME processes together describe a drug’s overall disposition via pharmacokinetics²¹. The molecular weight of all the analogues was within the range of 130 to 725 Da. For analogues, number of rotatable bonds and hydrogen bond donors (HBD) was ≤3 whereas, the hydrogen bond acceptor (HBA) was ≤4 and logP value <6.5, all obeyed the Lipinski’s rule of five so seem to have drug likeliness (Table 2). LogP value is an important factor for passive membrane partitioning. An increase in logP enhances permeability²². Caco-2 cell line is a popular surrogate for the human intestinal epithelium to estimate in vivo drug permeability. Membrane permeability is a requirement for good oral bioavailability. The analogues were evaluated for their CaCo-2 and MDCK permeability. All the analogues complied within the recommended range of great permeability >500 except the nitro and hydroxy-substituted analogues which projected moderate permeability <500 for both CaCo-2 and MDCK. Percentage of human oral absorption for all the analogues ranged from 80 to 100% indicating that they had good bioavailability. PSA (Polar Surface Area) also affects the bioavailability; the analogues possessed the PSA value in the range 17 to 111. SASA (solvent accessible surface area) influences partition co-efficient, in turn, affecting the aqueous solubility of all the analogues complied within the QikProp range from 450-655. Solubility of drug affects the in silico ADME properties. All the designed mannich bases were within the recommended range of QikProp -3.6 to -5.6. QPLogHERG predicts the IC₅₀ value for blockage of K⁺ channels, the analogues were in the recommended range below -5. All these values were within the accepted criteria for the selected parameters (Table 2). Thus, designed indole-3-mannich bases display favourable pharmacokinetic profile and can be considered further for synthesis and screening.

Osimertinib IM_23

Fig. 2: Docking pose of Osimertinib and IM_23 in active site of PDB 6LUD

Table: 2 In silico ADME results of Indole-3-mannich bases

Title	R1	R2	MW^a	SASA^b	HBD	HBA	QP log Po/w	QPlog HERG	QPP Caco	QPP MDCK	% HOA^c	Rule  Of Five
Qik prop range			130-725	300-1000	0-6	2. - 20.0	6.5 to -2	below -5	<25 poor, >500 great	<25 poor, >500 great	>80% high, <25% poor	4
Osimertinib			499.61	868.95	2	8.75	4.92	-7.64	371.10	187.46	100	0
Erlotinib			393.45	772.73	1.5	7.4	4.474	-7.155	4495.22	2511.29	100	0
IND	-	-	117.6	312.34	1	0	2.14	-3.93	5321.28	3013.61	100	0
IM_17	-morpholino	-4-NO2	337.38	581.00	1	4.7	2.799	-5.994	158.71	74.89	82.72	0
IM_20	-morpholino	-4-OH	308.38	554.97	2	4.45	2.747	-5.9	402.38	204.59	89.65	0
IM_21	-morpholino	-4-N(CH3)2	335.45	609.53	1	4.7	3.882	-5.91	1284.00	717.09	100	0
IM_22	-morpholino	-4-OCH3	322.41	567.63	1	4.45	3.543	-5.692	1328.69	744.11	100	0
IM_23	-morpholino	-3,4,5-tri-OCH3	382.46	642.21	1	5.95	3.752	-5.648	1328.88	744.22	100	0
IM_24	-morpholino	-3,4-di-OCH3	352.43	606.32	1	5.2	3.605	-5.707	1328.71	744.17	100	0
IM_25	-morpholino		282.34	492.85	1	4.2	2.759	-5.181	1328.68	744.10	100	0
IM_44	-pyrrolidinyl	-2-NO2	321.38	569.33	1	3	3.612	-5.962	294.81	146.22	92.21	0
IM_45	-pyrrolidinyl	-4-NO2	321.38	585.45	1	3	3.513	-6.089	154.88	72.81	86.71	0
IM_48	-pyrrolidinyl	-4-OH	292.38	562.31	2	2.75	3.41	-6.079	401.40	204.05	93.51	0
IM_49	-pyrrolidinyl	-4-N(CH3)2	319.45	624.20	1	3	4.647	-6.213	1258.25	701.56	100	0
IM_50	-pyrrolidinyl	-4-OCH3	306.41	581.08	1	2.75	4.299	-5.977	1300.78	727.23	100	0
IM_52	-pyrrolidinyl	-3,4-di-OCH3	336.43	618.81	1	3.5	4.41	-5.993	1302.18	728.08	100	0
IM_57	-piperidinyl	-2-NO2	335.41	611.14	1	3	3.8	-6.306	159.57	75.29	88.62	0
IM_63	-piperidinyl	-3,4,5-tri-OCH3	380.49	673.25	1	4.25	4.837	-5.957	1334.8	747.81	100	0
IM_69	-morpholino	-3,4-di-F	361.27	573.67	1	3.7	4.358	-5.612	1328.95	3795.68	100	0
IM_70	-morpholino	-4-F	310.37	552.54	1	3.7	3.729	-5.916	1328.52	1346.39	100	0

^a g/mol; ^b SASA: solvent accessible surface areas; ^c %HOA: Human oral absorption

Synthetic scheme 1: Synthesis of indole-3-mannich bases

3.3 Synthesis:

For efficient synthesis of heterocycles with significant medicinal value, multicomponent reactions (MCR) are used²³. One such MCR is the mannich reaction, which offers a very flexible technique for the concurrent production of C-C and C-N bonds to synthesize a wide range of medicines and natural compounds²⁴.

An indole, benzaldehyde, and pyrrolidine model reaction were carried out in a variety of solvents at 80°C in the absence of a catalyst to discover an effective and long-lasting technique to synthesize 3-amino alkylated indoles. Through a Mannich-type reaction, ethylene glycol enhanced the multi-component synthesis of indole-3- mannich bases using ultrasonicator and conventional method scheme 1. All synthesized analogues anticipated structures were confirmed by spectral characterization.

3.4 Antiproliferative activity:

The Sulforhodamine B (SRB) assay technique was employed to assess the synthesized indole-3-mannich bases, in vitro anti-proliferative properties on the human lung cancer A549 cell line²⁵. Doxorubicin and gefitinib were utilized as positive controls having GI₅₀ less than 10µM. the given analogues for antiproliferative activity demonstrated GI₅₀ values less than 55µM. IM_23 and IM_45 exhibited prominent activity with GI₅₀ 32.2µM and 39.5µM respectively. While IM_63 gave % control growth of 51.5µM (Table 3). Non substituted indole ring had the lowest inhibition % greater than 80µM. As the substitution on the indole increased the rate of inhibition increased. The study of structure activity relationship of the tested analogues indicated that IM_23 bearing morpholino group as substituted amine showed greater inhibition than IM_45 and IM_63 which possess pyrrolidinyl and piperidinyl moiety as substituted amines respectively²⁶ (Table 2).

Table 3: Anti-proliferative activities against A549 cell line.

	Drug concentrations (µg/ml) calculated from graph
A-549 cell line	LC50	TGI	GI50*
IM-23	NE	>80	32.2
IM-45	NE	>80	39.5
IM-63	NE	>80	51.5
IND	NE	>80	>80
Gef	NE	>80	<10
ADR	NE	<10	<10

LC₅₀ = 50% lethal concentration, GI₅₀ = 50% inhibition concentration for cell growth, TGI = total inhibition of cell growth, ADR = Adriamycin,

4. CONCLUSION:

Osimertinib interacts with ATP binding site Met793 residue and C- lobe Lys745 within the active site, analogously the synthesised analogues exhibited hydrogen bonds with these amino acids. On comparative analysis of the results of docking studies and in silico ADME, it was found that nine analogues viz. IM_23, IM_24, IM_25, IM_44, IM_45, IM_50, IM_52, IM_57, IM_63 depicted in common interactions with active site residues of both the PDB 6LUD and 1M17. From this it can be envisaged that these mannich bases may have potential over the different generations of EGFR inhibitors. On the human lung cancer A549 cell line, the synthesised analogs were evaluated in vitro for their ability to inhibit cell proliferation. Both IM_23 and IM_45 had substantial activity. The results of docking studies and invitro studies project that electron donating groups present on substituted aromatic aldehydic part of the analogues depicted more interactions in active site with better activity as compared to the electron withdrawing groups. As a result, analogues that interact with these residues could be potential inhibitors of non-small-cell lung cancer. Additionally, the results could provide insights into potential EGFR binding characteristics, which could help with the optimisation and subsequent creation of more promising analogues.

5. CONFLICT OF INTEREST:

The authors confirm that there is no conflict of interest related to the manuscript.

6. ACKNOWLEDGEMENTS:

We acknowledge the facility under DST-FIST Funding (Letter SR/FST/College-264 dated 18th November, 2015).

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Received on 22.01.2024      Revised on 12.07.2024

Accepted on 11.12.2024   Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1147-1153.

DOI: 10.52711/0974-360X.2025.00165

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