Synthesis, Characterization, In-Silico Anticancer Evaluation & In-Vivo Antioxidant Evaluation of Novel Benzothiazole Derivatives

Materials

List of Chemical Used

Methodology

Molecular Docking Study

Molecular docking studies were performed to investigate the binding affinity, molecular interactions, and possible binding orientations of the synthesized benzothiazole derivatives (3a–f) against selected breast cancer–related target proteins. Docking analysis helps in understanding the interaction behavior of synthesized compounds within the active binding cavity of receptor proteins and predicts their potential biological activity at the molecular level.

Fig 4.1 Molecular Docking

The docking study performed using AutoDock Vina, which is widely used for predicting ligand–protein interactions based on binding free energy calculations and conformational flexibility. The synthesized derivatives were evaluated against two important breast cancer–associated molecular targets, namely the EGFR kinase domain and Human Estrogen Receptor Alpha.

Selection of Target Proteins

The current molecular docking study's target proteins were linked to the advancement of breast cancer and cellular proliferation. The Protein Data Bank (PDB) provided the proteins' three-dimensional crystal structures.

The selected target proteins were:

Target 1: EGFR kinase domain (PDB ID: 2J6M)

The Epidermal Growth Factor Receptor (EGFR) is a membrane-bound receptor possessing intrinsic tyrosine kinase activity and is critically involved in regulating several cellular processes such as proliferation, survival, differentiation, migration, and angiogenesis. Dysregulation or excessive expression of EGFR has been frequently observed in multiple malignancies, particularly breast cancer, where it contributes to uncontrolled tumor growth and metastatic progression. Due to its significant role in cancer cell signaling pathways, EGFR has emerged as an important molecular target for the development of anticancer agents aimed at suppressing tumor progression through inhibition of kinase activity.

Target 2: Human Estrogen Receptor Alpha Ligand (PDB ID: 3ERT)

Human Estrogen Receptor Alpha (ERα) is a ligand-activated nuclear transcription factor that plays a crucial role in the initiation and progression of hormone-responsive breast cancer. Upon interaction with estrogenic compounds, ERα regulates the expression of genes associated with cellular growth, proliferation, and survival. Abnormal activation of this receptor has been closely linked with estrogen-dependent breast tumor development. Consequently, targeting ERα through receptor antagonism or modulation has become a well-established therapeutic strategy in the management of estrogen receptor–positive breast cancer.

Ligand Preparation

The chemical structures of all synthesized benzothiazole derivatives (3a–f) were drawn using ChemDraw software. The two-dimensional structures were converted into three-dimensional conformations and subjected to geometry optimization in order to obtain energetically stable molecular structures suitable for docking analysis.

The optimized structures were saved in MOL format, then Open Babel was used to transform them into PDB format. Hydrogen atoms were added during ligand preparation, and energy minimization was used to eliminate undesirable conformations and steric hindrance. Ultimately, all ligand structures were transformed into PDBQT format, which is necessary for docking simulations using Auto Dock Vina.

Protein Preparation

The crystal structures of the selected proteins were downloaded in PDB format from the Protein Data Bank. Prior to docking, protein structures were prepared carefully to ensure proper ligand binding analysis.

Protein preparation involved the following steps:

• Removal of co-crystallized ligands and heteroatoms
• Deletion of water molecules present in the crystal structure
• Addition of polar hydrogen atoms
• Assignment of Kollman charges
• Correction of missing atoms and bond orders where necessary
• Conversion of protein structures into PDBQT format

Protein preparation done by AutoDock tools to obtain receptor files compatible with AutoDock Vina.

Active Site Identification

The active binding site of each target protein was identified based on the binding position of the co-crystallized ligand present in the crystal structure. The amino acid residues surrounding the ligand-binding cavity were selected for grid box generation.

For EGFR kinase domain (2J6M), important active site residues included Leu718, Val726, Ala743, Lys745, Thr790, Leu788, and Leu844, which are known to participate in ATP-binding and kinase inhibition.

For Human Estrogen Receptor Alpha (3ERT), important amino acid residues involved in ligand binding included Glu353, Arg394, His524, Leu387, and Phe404.

Docking Procedure

Docking simulations were performed using AutoDock Vina to evaluate the interaction of synthesized compounds with the selected receptor proteins. A grid box was generated around the active site region of each protein to allow ligand flexibility and proper exploration of the binding cavity.

During docking, the ligands were allowed to adopt different conformations and orientations inside the receptor binding pocket. AutoDock Vina calculated the binding affinity based on intermolecular interactions such as hydrogen bonding, hydrophobic interactions, van der Waals forces, and electrostatic interactions.

The docking score obtained from AutoDock Vina was expressed in kcal/mol. Lower binding energy values indicated stronger ligand–protein interaction and better binding stability. The best docking pose for each ligand was selected based on minimum binding energy and favorable interaction profile.

Post-Docking Analysis and Visualization

The docked ligand–protein complexes were analyzed for intermolecular interactions and binding orientation using PyMOL and BIOVIA Discovery Studio Visualizer.

Two-dimensional and three-dimensional interaction analyses were performed to identify:

• Hydrogen bond interactions
• Hydrophobic interactions
• π–π stacking interactions
• π–alkyl interactions
• van der Waals interactions
• Electrostatic interactions

The amino acid residues involved in ligand stabilization inside the active site were carefully examined and compared with the interactions shown by the standard drug.

Standard Drug for Comparative Docking

The docking results of synthesized compounds were compared with the standard anticancer drug Tamoxifen. Tamoxifen was selected as the reference ligand because of its established role in breast cancer therapy and its reported interaction with estrogen receptor and related oncogenic targets.

The comparative docking study helped in evaluating the potential anticancer activity of the synthesized benzothiazole derivatives and identifying compounds with promising receptor binding characteristics for further biological evaluation.

Synthetic Procedures

General Procedure for the Synthesis of Substituted Benzothiazol-2amine Derivatives (2a–f)

The substituted benzo[d]thiazol-2-amine derivatives (2a–f) were prepared by an oxidative cyclization method using suitably substituted aniline derivatives as starting materials in the presence of potassium thiocyanate under acidic conditions. In the initial step, potassium thiocyanate (8 g, 0.08 mol) along with the corresponding substituted aniline derivative (1.45 g, 0.01 mol) was dissolved in 20 mL of pre-chilled glacial acetic acid under constant stirring to obtain a homogeneous reaction mixture. The reaction temperature was carefully controlled at a low level with the help of an ice–salt bath in order to minimize excessive heat evolution and ensure smooth progression of the cyclization process.

A bromine solution prepared by dissolving bromine (1.6 mL) in glacial acetic acid (6 mL) was added slowly to the reaction mixture in a dropwise manner over approximately 105 minutes with constant stirring. Careful addition of bromine was necessary to control the exothermic nature of the cyclization process. After complete addition, the reaction mixture was stirred continuously for an additional 2 hours under cold conditions and then allowed to stir at room temperature for nearly 10 hours. The reaction mixture was subsequently kept undisturbed overnight to ensure completion of cyclization.

After completion of the reaction, the mixture was heated at approximately 85 °C with the addition of 6 mL of distilled water and filtered while hot to obtain the first filtrate. The remaining residue was further extracted using 10 mL of glacial acetic acid at the same temperature and filtered again to obtain a second filtrate. Both filtrates were combined and allowed to cool to room temperature. The combined solution was then neutralized carefully using concentrated ammonia solution until the pH reached nearly 6, leading to the formation of a precipitated solid product.

The separated crude product was collected by filtration, washed appropriately, and subjected to purification using activated charcoal treatment. Final purification was carried out by recrystallization from a benzene–ethanol solvent mixture (1:1), affording pure substituted benzo[d]thiazol-2-amine derivatives (2a–f) in crystalline form.

Synthesis of N-(Substitutedbenzo[d]thiazol-2-yl)-3-phenylprop-2-en-1-imine Derivatives (3a–f)

The Schiff base derivatives of benzothiazole, namely N-(substitutedbenzo[d]thiazol-2-yl)-3-phenylprop-2-en1-imine (3a–f), were synthesized by condensation of substituted benzo[d]thiazol-2-amines with cinnamaldehyde under reflux conditions. Equimolar quantities of substituted benzo[d]thiazol-2-amine and cinnamaldehyde were dissolved in absolute ethanol in a clean round-bottom flask equipped with a reflux condenser. To promote the condensation reaction and facilitate imine bond formation, a few drops of glacial acetic acid were introduced as a catalyst.

The reaction mixture was heated under reflux for approximately 3–4 hours with continuous stirring. The reaction progress is observed using TLC using a suitable mobile phase system. Formation of a new spot corresponding to the desired product confirmed completion of the reaction.

After completion, the reaction mixture was allowed to attain room temperature and was then transferred slowly into ice-cold distilled water with constant stirring. A colored solid precipitate separated out gradually, indicating formation of the Schiff base derivative. The obtained precipitate was collected by vacuum filtration and washed thoroughly with distilled water to remove traces of catalyst and any unreacted reactants. The crude products were dried under reduced pressure and subsequently purified by recrystallization using ethanol as the solvent. Pure crystalline derivatives of N-(substitutedbenzo[d]thiazol-2yl)-3phenylprop-2-en-1imine (3a–f) were obtained after recrystallization and preserved in airtight containers for further characterization and biological evaluation.

Purity Determination of Synthesized Compounds

Purity of synthesizd compounds was determine by perfoerming Thin Layer Chromatography and recrystallization technique.

Characterization of Synthesized Compounds

The synthesized benzothiazole derivatives were characterized using various physicochemical and spectral analytical techniques to confirm their identity, purity, and structural features. The characterization studies included melting point determination, FT-IR, ¹H-NMRspectroscopy, mass spectral analysis, and elemental analysis. These analytical techniques provided detailed information regarding the structural framework, functional groups, molecular weight, and composition of the synthesized compounds.

Evaluation of Antioxidant Activity by DPPH Free Radical Scavenging Assay

The antioxidant potential of the selected synthesized benzothiazole derivatives was assessed by employing the DPPH free radical scavenging method. This assay is widely recognized for its simplicity, sensitivity, and reliability in determining the ability of compounds to donate hydrogen atoms or electrons for neutralization of free radicals. The method is based on reduction of the stable DPPH radical, which undergoes a visible color change upon interaction with antioxidant molecules.

DPPH is a stable nitrogen-centered free radical that exhibits a deep violet coloration in methanolic solution due to its strong absorption around 517 nm. When an antioxidant compound reacts with DPPH, the radical accepts a hydrogen atom or electron from the test compound and becomes reduced to a non-radical form. This reduction is accompanied by fading of the violet color to pale yellow, which can be quantitatively measured spectrophotometrically. The degree of discoloration reflects the radical scavenging efficiency of the tested compounds.

RESULTS & DISCUSSION

Molecular Docking Analysis of Synthesized Benzothiazole Derivatives Against EGFR Kinase Domain (PDB ID: 2J6M)

The synthesized derivatives (3a–3f) were subjected to molecular docking analysis against the EGFR kinase domain using the crystal structure of EGFR tyrosine kinase (PDB ID: 2J6M). The docking study was performed to evaluate the binding affinity and molecular interactions of the synthesized compounds within the active binding pocket of the receptor. The obtained docking scores were compared with the standard drug Tamoxifen.

The docking results revealed that all synthesized derivatives exhibited appreciable binding affinity toward the EGFR kinase domain, with binding energies ranging from −7.5 to −7.8 kcal/mol, which were comparable to or better than the standard drug tamoxifen (−7.4 kcal/mol). Among all tested compounds, derivative 3d demonstrated the highest binding affinity with a docking score of −7.8 kcal/mol, indicating strong interaction and favorable accommodation within the receptor active site.

Table: 2D Interaction and Binding Affinity of Benzothiazole Derivatives Against EGFR Kinase Domain (PDB ID: 2J6M)

Comp	Binding Affinity (kcal/mol)	H-Bond Interactions	π–π / Hydrophobic Interactions	2D Interaction
3a	-7.6	Leu788	Val726, Ala743, Leu844, Leu718
3b	-7.5	-	Leu718, Thr790, Ala743, Leu788, Leu844, Val726, Lys745
3c	-7.7	-	Leu718, Thr790, Ala743, Leu788, Leu844, Val726, Lys745
3d	-7.8	-	Leu718, Thr790, Leu788, Ala743, Leu844, Val726, Lys745
3e	-7.6	Pro794	leu718, Thr790, Leu788, Ala743, Leu844, Val726, Lys745
3f	-7.6	-	Leu718, Thr790, Val726, Leu788, Lys745, Ala743, Leu844
Std (Temoxifen) -7.4		-	Leu844, Leu718, Ala743, Val726

The molecular interaction analysis showed that hydrophobic interactions played a significant role in stabilizing the ligand–receptor complexes. The amino acid residues Leu718, Thr790, Ala743 Leu788, Leu844Val726, and Lys745 were commonly involved in ligand binding for most of the synthesized derivatives. These residues are considered important components of the ATP-binding pocket of EGFR kinase and contribute substantially to ligand stabilization through hydrophobic and van der Waals interactions.

Table: Binding Affinity of Synthesized compounds (3a-e) with target EGFR Kinase Domain (PDB ID: 2J6M)

Compound 3a showed a binding affinity of −7.6 kcal/mol and formed a hydrogen bond with Leu788 along with hydrophobic interactions involving Val726, Ala743, Leu844, and Leu718. Compounds 3b and 3c exhibited docking scores of −7.5 and −7.7 kcal/mol, respectively, with major hydrophobic interactions involving Leu718, Thr790, Ala743, Leu788, Leu844, Val726, and Lys745. Among all derivatives, compound 3d demonstrated the highest binding affinity (−7.8 kcal/mol) due to extensive hydrophobic interactions with key active-site residues. Compound 3e showed a docking score of −7.6 kcal/mol and formed a hydrogen bond with Pro794 along with several hydrophobic contacts. Similarly, compound 3f exhibited a binding affinity of −7.6 kcal/mol with interaction patterns comparable to the standard drug. The standard Tamoxifen showed a docking score of −7.4 kcal/mol with mainly hydrophobic interactions. Overall, several synthesized derivatives displayed better binding affinity than tamoxifen, indicating promising EGFR inhibitory potential.

The docking study suggested that the synthesized derivatives possess favorable interaction profiles within the EGFR kinase active site. The higher binding affinities and extensive hydrophobic interactions observed for compounds particularly 3c and 3d indicate their potential for further biological evaluation as prospective EGFR-targeted anticancer agents.

Molecular Docking Analysis of Synthesized Benzothiazole Derivatives Against Human Estrogen Receptor α (PDB ID: 3ERT)

The synthesized benzothiazole derivatives (3a–3f) were evaluated for their binding affinity and interaction pattern against the Human Estrogen Receptor Alpha ligand-binding domain using the crystal structure of Human Estrogen Receptor Alpha. The docking results were compared with the standard drug Tamoxifen to assess their potential as estrogen receptor-targeted anticancer agents.

Table: 2D Interaction and Binding Affinity of Benzothiazole Derivatives Against Human Estrogen Receptor Alpha Ligand (PDB ID: 3ERT)

Comp	Binding Affinity (kcal/mol)	H-Bond Interactions	π–π / Hydrophobic Interactions	2D Interaction
3a	-8.5	-	Leu354, Trp383, Leu536, Ala350, Leu525, Leu346
3b	-7.3	Thr347	Leu525, Lys529, Ala350, Leu391, Leu346, Leu387
3c	-7.4	Thr347	Leu525, Leu387, Leu346, Leu391, Ala350,      Met 343
3d	-7.6	-	Leu346, Ala350, Leu378, Leu391, Leu525, Met528
3e	-7.7	Phe404	Leu391, Leu346, Leu387, Leu525, Leu384, Trp383, Ala350
3f	-7.6	-	Trp383, Ala350, Leu525, Met388, Phe404,  Met421, Leu346
Std (Temoxifen) -8.6		-	Ala350, Leu387, Leu346, Leu525, Met388, Met421

The molecular docking study against Human Estrogen Receptor Alpha revealed that all synthesized benzothiazole scaffolds shows favorable binding interactions within the active site of the receptor, with docking scores −7.3 to −8.5 kcal/mol. Among the tested compounds, derivative 3a showed the highest binding affinity (−8.5 kcal/mol), which was comparable to the standard drug Tamoxifen (−8.6 kcal/mol), indicating strong ligand–receptor interaction. Hydrophobic interactions played a major role in stabilization of the ligand–protein complexes, involving key amino acid residues such as Leu346, Ala350, Leu387, Leu391, Leu525, Trp383, Met388, and Phe404. Compound 3a interacted hydrophobically with Leu354, Trp383, Leu536, Ala350, Leu525, and Leu346. Compounds 3b and 3c formed hydrogen bonds with Thr347 along with multiple hydrophobic interactions, while compound 3e formed a hydrogen bond with Phe404 and showed extensive hydrophobic contacts. Compounds 3d and 3f mainly exhibited hydrophobic interactions with important active-site residues. The standard drug tamoxifen interacted predominantly through hydrophobic contacts involving Ala350, Leu387, Leu346, Leu525, Met388, and Met421.

Table: Binding Affinity of Synthesized compounds (3a-e) with target Human Estrogen Receptor Alpha Ligand (PDB ID: 3ERT)

The docking study indicated that the synthesized benzothiazole derivatives possess favorable binding characteristics against Human Estrogen Receptor Alpha. In particular, compound 3a exhibited excellent binding affinity and interaction profile, indicating its potential for further investigation as a prospective anticancer agent targeting estrogen receptor-positive breast cancer.

Synthesis Result

Benzothiazol-2amine Derivatives (2a–f)

Table: Physiochemical Properties of Synthesized derivatives (2a-f)

Synthesis of N-(Substitutedbenzo[d]thiazol-2yl)-3-phenylprop-2-en-1imine Derivatives (3a–f)

5nitro-2((1E,3E)-4phenylbuta-1,3-dien-1yl)benzothiazole (3a)

TLC Solvent: Chloroform: Methanol (9:1 v/v)

IR (KBr, cm⁻¹):

¹H NMR (δ, ppm):

Mass Spectra:

6methoxy-2-((1E,3E)-4phenylbuta-1,3dien-1-yl)benzothiazole (3b)

IR (KBr, cm⁻¹):

¹H NMR (δ, ppm):

6-chloro-2-((1E3E)-4phenylbuta-1,3dien-1yl)benzothiazole (3c)

IR (KBr, cm⁻¹):

¹H NMR (δ, ppm):

6-methyl-2-((1E3E)-4phenylbuta-1,3-dien-1yl)benzothiazole (4d)

IR (KBr, cm⁻¹):

¹H NMR (δ, ppm):

2-((1E,3E)-4phenylbuta-1,3-dien-1-yl)benzothiazol-6ol (4e)

¹H NMR (δ, ppm):

5,6-dichloro-2((1E3E)-4phenylbuta-1,3-dien-1yl)benzothiazole (3f)

IR (KBr, cm⁻¹):

Mass Spectra:

Antioxidant Activity by DPPH Assay

The DPPH free radical scavenging assay was used to assess the antioxidant activity of the synthesized benzothiazole scaffolds (3a–3f), and the results were compared with those of the conventional antioxidant Ascorbic Acid. The assay was carried out at various doses between 20 and 100 µg/mL, and the % inhibition of DPPH radicals was measured using UV at 517 nm. All synthesized compounds demonstrated concentration-dependent free radical scavenging activity. An increase in percentage inhibition was observed with increasing concentration of the test compounds, indicating effective antioxidant potential of the synthesized benzothiazole derivatives. The reduction in absorbance of the DPPH solution from deep violet to pale yellow confirmed the hydrogen-donating and radical-neutralizing ability of the compounds.

Table:  Absorption of Synthesized compounds (3a-f)

Among the synthesized benzothiazole derivatives, compound 3e exhibited the highest antioxidant activity, showing 38.7–88.2% inhibition over the concentration range of 20–100 µg/mL, which may be attributed to the presence of the hydroxyl substituent that enhances hydrogen donation and free radical stabilization. Compound 3a also demonstrated considerable antioxidant potential with 75.2% inhibition at 100 µg/mL, possibly due to the nitro substituent and conjugated benzothiazole Schiff base system. Compound 3d showed moderate activity, while compounds 3b and 3f exhibited intermediate free radical scavenging effects. In contrast, compound 3c displayed comparatively lower antioxidant activity (61.4% inhibition at 100 µg/mL), which may be associated with the electron-withdrawing nature of the chloro substituent. The standard antioxidant Ascorbic Acid showed the highest activity with 96.8% inhibition at 100 µg/mL. Overall, the synthesized derivatives demonstrated appreciable antioxidant potential, with activity significantly influenced by the nature of substituents attached to the benzothiazole nucleus.

The results suggested that antioxidant activity of the synthesized compounds was significantly influenced by the nature and position of substituents attached to the benzothiazole nucleus. Electron-donating groups such as hydroxyl substituents enhanced the antioxidant potential, whereas electron-withdrawing groups exhibited comparatively lower activity. The presence of conjugated aromatic systems and Schiff base linkage may also contribute toward stabilization of free radicals through electron delocalization mechanisms.

Table: Percentage Inhibition of Synthesized compounds (3a-f)