Design, Synthesis and Anticancer Activity of Substituted

1, 3-Thiazolidin-4-One Derivatives

Ganesh D. Barkade¹*, Ramesh L. Sawant²

¹Assistant Professor, Department of Pharmaceutical Chemistry,

Dr. Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Ahmednagar, Maharashtra, India - 414111.

²Vice-principal; Senior Professor and Head of PG Studies, Department of Pharmaceutical Chemistry,

Dr. Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Ahmednagar, Maharashtra, India - 414111.

*Corresponding Author E-mail: ganeshbarkade7@gmail.com, sawantrl@yahoo.com

ABSTRACT:

Background: Cancer is the global cause of death worldwide. Anticancer drug development is the need in today’s scenario. Thiazolidine is the nucleus that shows several pharmacological activities like anticancer, anti-inflammatory, antioxidant, antibacterial, antifungal, antidiabetic, antihyperlipidemic, and antiarthritic activity. In the present work molecular docking Glide module (Schrodinger Inc., USA) has been used for ligand docking against the Polo-like kinase-1. The series of substituted 3-Benzothiazol-2-yl-2-phenyl)-thiazolidin-4-ones were synthesized by the microwave-assisted synthesis system (CEM, USA) and characterized by melting point, FT-IR, ¹H NMR, ¹³C NMR, and HR-MS analysis. Results: Molecular docking studies shows good docking score as well as interactions. Among the synthesized compounds, BG2 had the highest docking score of -8.381, followed by BG8 (-8.19) and BG1 (-8.156). All the newly synthesized compounds were examined for their in vitro anticancer activity against breast cancer cell line MCF-7 by Sulforhodamine B (SRB) assay. Conclusion: BG1, BG2, BG3, BG4, BG5, BG8, BG9, BG10, BG11, BG12, and BG13 (GI50: <80 µg/ml) exhibited significant cell growth inhibitory activity. These results indicate that compounds showing in-vitro activity by molecular docking studies and SRB assay could be lead compounds for further development of anticancer agents and suitable candidates for in-vivo anticancer activity.

KEYWORDS: Anticancer Agents, Breast Cancer, Cancer, Docking, MCF7, Polo-like kinase-1 (PLK1), SRB Assay.

INTRODUCTION: 

Cancer is when healthy body cells uncontrollably change into malignant cells. According to the World Health Organization (WHO), cancer is the second leading cause of death globally. Breast and lung cancer are the most hazardous compared to other cancer types. In 2018, there were around 2.09million new cases and 6, 27,000 deaths from breast cancer.^1-2.

Mitotic checkpoint regulation is greatly influenced by PLK1. Because of this, overexpressing this kinase can override checkpoints, resulting in aneuploidy, an immature cell division characteristic of cancer³. PLK1 overexpression promotes excessive cell proliferation through the dysregulation of checkpoint functions, which aids in cancer development⁴. For the treatment of cancer, more than 51 kinase inhibitors are now approved⁵. Some PLK1 inhibitors are now undergoing preclinical and clinical testing⁶. The biological response of 1, 3-thiazolidine-4-ones is diverse, including anticancer⁷, anti-inflammatory⁸, antioxidant⁹, antibacterial¹⁰, antifungal¹¹, antidiabetic¹², and antihyperlipidemic¹³ activity. The literature study reveals that PLK1 plays important role in breast cancer cell growth and hence in present study PLK1 is targeted for the anticancer activity. We can stop the development of breast cancer by blocking PLK1 overexpression. Some Thiazolidineanalogues are used to treat cancer by inhibiting the PLK1 enzyme.

METHODOLOGY:

MOLECULAR DOCKING:

The molecular docking Glide module (Schrodinger Inc., USA) has been used for ligand docking against the Polo-like kinase-1. Polo-like kinase-1 receptor's X-ray crystallographic structure was derived using accession ID 3DBC from the protein databank. The "protein preparation wizard" panel was used to create the returned protein structure. Bond ordering, missing hydrogen addition, disulphide bond formation, and modification of missing side chains and loops were all accomplished by the use of prime during the pr-processing steps. The OPLS3 force field has been employed in the last refinement step to achieve complete energy optimization, with the RMSD of heavy atoms set to 0.3. The LigPrep panel was used to create the 3D structures for every compound that was synthesized. Each ligand structure's ionization state was determined at a physiological pH of 7.2±0.2. The active side grid was allocated by centralizing the cognate ligand in the crystal structure and using the default box dimension. Last but not least, a molecular docking investigation utilising Schrodinger's glide was conducted. The receptor grid file and the ready minimal energy 3D structure of the ligands were put into Maestro's work area, and the ligands were docked using extra precision (SP) docking methods.^14-17.

SYNTHESIS:

In a round bottom flask, a combination of sodium hydroxide (0.01mol) in ethanol (10ml), 2-aminobenzthiazole (0.01mol), substituted aldehydes (0.01mol), and thioglycolic acid (0.01mol) was added. The combinations were well mixed before being exposed to microwave radiation at 30W for a predetermined amount of time in the microwave synthesis system (CEM, USA). The purity of the compounds that were identified under UV light and iodine vapours was evaluated, together with the amount of untreated starting material, using thin-layer chromatography (TLC, Silica gel 60 F254, Merck). After cooling, the mixture was dissolved into ethyl acetate and water. The layer of ethyl acetate was dried. Flash chromatography was used to purify the final reaction product (BUCHI, Switzerland). The chemicals were bought in bulk from Spectrochem Chemicals, Loba Chemicals, S. D. Fine Chemicals, and Sigma Aldrich. Flash chromatography was used to clean the produced substances. Gallenkamp electric melting point equipment was used to calculate the physical constant (Melting point).¹⁸ The route of synthesis is mentioned in scheme-I.

Scheme-1: Synthesis of substituted 3-Benzothiazol-2-yl-2-phenyl)-thiazolidin-4-ones

STRUCTURAL ELUCIDATION:

Flash chromatography was used to clean the produced substances. Gallenkamp's electric melting point equipment was used to calculate melting point. To check the development, determine the quantity of untreated starting material, and evaluate the purity of the chemicals found under UV light and iodine vapours, thin layer chromatography (TLC, Silica gel 60 F254, Merck) was utilised. By using ¹H NMR, ¹³C NMR FT-IR, and Mass spectroscopy, all synthetic substances were characterized. KBr disc FT-IR spectra were recorded using the Jasco Infrared Affinity-1 spectrophotometer.¹H NMR, spectra recorded in DMSO-d6 by using Bruker 500 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) 500 MHz in chloroform (CDCl₃) and dimethyl sulfoxide (DMSO-d6) used as a solvent, tetramethylsilane (TMS) was used as an internal standard. Chemical shifts are shown as δ values (ppm). Signals are represented by the letters s (singlet), d (doublet), t (triplet), q (quintet), or m. (multiplet). A Waters Q-TOF premier mass spectrometer was used to record HR-MS spectrum data. The majority of the compounds' mass spectra reveal that the molecular ion is (M+1)¹⁹.

ANTICANCER ACTIVITY (SRB ASSAY):

In the proper medium, which contains 10% foetal bovine serum and 2mM L-glutamine, the cell lines were cultured. For the current screening test, 100µL of 5000 cells/well were added to 96-well microtiter plates. The microtiter plates were incubated with the cells for 24 hours at 37°C, 5% CO₂, 95% air, and 100% relative humidity before the addition of the experimental synthesized compounds. Prior to usage, experimental drug were stored frozen after being solubilized in the appropriate solvent at a concentration of 100mg/ml and diluted to 1mg/ml with water. An aliquot of frozen concentrate (1mg/ml) was thawed and diluted with complete medium including test material to 100g/ml, 200g/ml, 400g/ml, and 800g/ml at the time of drug administration. The required final drug concentrations, i.e. 10g/ml, 20g/ml, 40g/ml, and 80g/ml, were achieved by adding aliquots of 10 l of each of these various drug dilutions to the corresponding microtiter wells that already contained 90µL of medium.

Plates were incubated at standard temperatures for 48 hours following drug addition, at which point cold TCA was added to end the test. Cells were fixed in situ by gently adding 50µl of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50µl) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. Unbound dye was collected after staining, and the remaining dye was eliminated by washing with 1% acetic acid five times. The plates were dried using air. After the bound dye was eluted with 10m Mtrizma base, the absorbance was measured on a plate reader at 540 nm with a reference wavelength of 690nm. For test wells compared to control wells, plate-by-plate percentage increase was determined. The percentage growth was calculated as the test well's average absorbance divided by the control wells' average absorbance multiplied by 100. The percentage growth at each of the four drug concentration levels was computed using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the four concentration levels (Ti)] [20-24]. Percentage growth inhibition was calculated as:

[Ti/C] x 100 %

RESULTS:

STRUCTURAL ELUCIDATION:

3-Benzothiazol-2-yl-2-phenyl-thiazolidin-4-one (BG1): Pale yellow solid; Yield 84.10 %; R_fvalue 0.78; M.P.:85-88⁰C; FT-IR: (KBr,cm-1):3452, 3053, 2857, 1693, 1611, 1206 ;¹H NMR (500 MHz, DMSO):8.98 (d, 1H,Ar-H C4 of benzthiazole),7.73 (d, 1H, Ar-H C7 of benzthiazole), 7.51 (t, 2H, Ar-H C5 of benzthiazole), 7.20-7.36 (m, 5H,Ar-H), 6.90 (s, 1H, CH, aliphatic, thiazolidione), 3.35-3.87 (dd, 2H, CH2, aliphatic thiazolidinone);¹³CNMR (500 MHz, DMSO): 166.8 (C=O of thiazolidinone ring), 165.0 (C=N, C2, aromatic ring benzothiazole), 155.3 (C= , C9, aromatic ring benzothiazole) 139.7 (CH=, C1 linked to thiazolidinone), 130.60 (C= , C8, aromatic ring benzothiazole), 126.7 (CH=, C2 aromatic ring linked to thiazolidinone), 126.1 (CH=, C3 aromatic ring linked to thiazolidinone), 125.8 (CH=, C5 aromatic ring benzothiazole), 121.9 (CH=, C6 aromatic ring benzothiazole), 121.2 (CH=, C7 aromatic ring benzothiazole), 118.1 (CH=, C4 aromatic ring benzothiazole), 69.2 (S-CH-N aliphatic thiazolidinone), 39.4 (S-CH₂ aliphatic thiazolidinone); MS (ESI): m/z 313.0469 (M+1).

3-Benzothiazol-2-yl-2- (2-chloro-phenyl)-thiazolidin-4-one (BG2): Pale yellow solid; Yield 72.20 %; R_fvalue 0.65; M.P.:85-87⁰C; FT-IR: (KBr, cm^-1):3060, 2900, 1698, 1610, 1210, 750; ¹H NMR (500 MHz, DMSO): 8.2 (d, 1H, Ar-H C4 of benzothiazole), 8.1 (d, 1H, Ar-H C7 of benzothiazole), 7.7 (d, 1H, C3 chlorobenzene ring), 7.5 (t, 2H, Ar-H C5,6 of benzothiazole), 7.2-7.3 (m, 3H, chlorobenzene ring), 6.7 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-4.0 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO): 171.1 (C=O of thiazolidinone ring), 165.1 (C=N, C2, aromatic ring benzothiazole), 148.0 (C= , C9, aromatic ring benzothiazole), 132.0 (C-Cl, aromatic ring linked to thaizolidinone), 130.1 (C= , C8, aromatic ring benzothiazole), 128.4 (CH=, C3-C6 aromatic ring linked to thiazolidinone), 126.1 (CH=, C5,C6 benzothiazole), 121.5 (CH=, C7 aromatic ring benzothiazole), 119.1 (CH=, C4 aromatic ring benzothiazole), 60.78 (S-CH-N aliphatic thiazolidinone), 32.0 (S-CH₂ aliphaticthiazolidinone); MS (ESI): m/z 347.0080 (M+1).

3-Benzothiazol-2-yl-2- (3-chloro-phenyl)-thiazolidin-4-one (BG3): Pale yellow solid; Yield 74.18 %; R_fvalue 0.60; M.P.:75-78⁰C; FT-IR: (KBr, cm^-1):3055 (C-H, Aromatic stretch, sharp, medium), 2860 (C-H, Aliphatic stretch, sharp, medium), 1690 (C=O, amide carbonyl stretch, sharp, strong), 1600 (C-C, Aromatic stretch), 1205 (C=N, stretch), 755 (C-Cl); ¹H NMR (500 MHz, DMSO):8.3 (d, 1H, Ar-H C4 of benzothiazole, 8.2 (d, 1H, Ar-H C7 of benzothiazole), 7.5-7.6 (m, 3H, C5,C6 of benzothiazole and C2 chlorobenzene ring), 7.3 (m, 2H, chlorobenzene ring), 7.2 (d, 1H, C6 chlorobenzene ring), 6.4 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-3.6 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO):171.2 (C=O of thiazolidinone ring), 165.20 (C=N, C2, aromatic ring benzothiazole), 148.0 (C= , C9, aromatic ring benzothiazole), 142.3 (C- aromatic ring linked to thiazolidinone), 134.6 (C-Cl , aromatic ring linked to thiazolidinone), 130.9 (C=, C8 of benzothiazole and C5 of chlorobenzene ring), 128.41 (CH=, C5, C6 of benzothiazole and C2, C3 and C6 of chlorobenzene ring), 121.2 (CH=, C7 benzothiazole), 118.1 (CH=, C4 of benzothiazole), 62.4 (S-CH-N aliphatic thiazolidinone) , 33.3 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z)347.0080 (M+1)

3-Benzothiazol-2-yl-2- (4-chloro-phenyl)-thiazolidin-4-one (BG4): Pale yellow solid; Yield 78.82 %; R_fvalue 0.62; M.P.:98-101⁰C; FT-IR (cm^-1): 3045 (C-H, Aromatic stretch, sharp, medium), 2855 (C-H, Aliphatic stretch, sharp, medium), 1692 (C=O, amide carbonyl stretch, sharp, strong), 1605 (C-C, Aromatic stretch), 1210 (C=N, stretch), 762 (C-Cl) ); ¹H NMR (500 MHz, DMSO): 8.9 (d, 1H, Ar-H C4 of benzothiazole), 7.9 (d, 1H, Ar-H C7 of benzothiazole), 7.5 (m, 2H, C5,C6 of benzothiazole ), 7.4 (d, 2H, C3,C5 chlorobenzene ring), 7.2 (d, 2H, C2, C6 chlorobenzene ring) , 6.9 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-4.0 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO): 172.1 (C=O of thiazolidinone ring), 164.9 (C=N, C2, aromatic ring benzothiazole), 148.0 (C= , C9, aromatic ring benzothiazole), 139.8 (C- aromatic ring linked to thiazolidinone), 132.1 (C-Cl aromatic ring linked to thiazolidinone), 130.9 ( C2, C6 chlorobenzene ring),126.0 (CH=, C5, C6 of benzothiazole ), 121.2 (CH=, C7 benzothiazole), 119.0 (CH=, C4 of benzothiazole), 66.3 (S-CH-N aliphatic thiazolidinone) , 32.3 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 347.0075 (M+1)

3-Benzothiazol-2-yl-2- (2-nitro-phenyl)-thiazolidin-4-one (BG5): Yellow solid; Yield 82.30 %; R_fvalue 0.82; M.P.:94-97⁰C; FT-IR (cm^-1): 3052 (C-H, Aromatic stretch, sharp, medium), 2858 (C-H, Aliphatic stretch, sharp, medium), 1690 (C=O, amide carbonyl stretch, sharp, strong), 1600 (C-C, Aromatic stretch), 1540 (N-O stretch), 1210 (C=N, stretch); ¹H NMR (500 MHz, DMSO): 7.8 (d, 1H, Ar-H C4 of benzothiazole), 7.6 (d, 1H, Ar-H C7 of benzothiazole), 7.5 (d, 1H, C3 Ar-H of nitrobenzene ring), 7.5 (m, 1H, C5 Ar-H, nitrobenzene ring), 7.4-7.5 (m, 4H, C5,C6 of benzothiazole and C5 and C6 of nitrobenzene ring), 6.5 (s, 1H, CH, aliphatic, thiazolidinone), 3.5-3.7 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO): 170.9 (C=O of thiazolidinone ring), 166.1 (C=N, C2, aromatic ring benzothiazole), 147.8 (C= , C9, aromatic ring benzothiazole, C2-NO2 of nitrobenzene ring), 135.4 (C-1, C5 of nitrobenzene ring), 130.0 (C3 and C5 of nitrobenzene ring), 124.9 ( C5,C6 of benzothiazole and C3 of nitrobenzene ring ), 121.2 (CH=, C7 benzothiazole), 118.1 (CH=, C4 of benzothiazole), 64.6 (S-CH-N aliphatic thiazolidinone), 32.3 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 357.0857 (M+1)

3-Benzothiazol-2-yl-2- (3-nitro-phenyl)-thiazolidin-4-one (BG6): Yellow solid; Yield 80.34 %; R_fvalue 0.70; M.P.:107-109⁰C; FT-IR (cm^-1): 3050 (C-H, Aromatic stretch, sharp, medium), 2870 (C-H, Aliphatic stretch, sharp, medium), 1686 (C=O, amide carbonyl stretch, sharp, strong), 1605 (C-C, Aromatic stretch), 1545 (N-O stretch), 1215 (C=N, stretch); ¹H NMR (500 MHz, DMSO): 8.4 (m, 2H, C2,C4 of nitrobenzene ring), 8.0 (d, 1H, Ar-H C4 of benzothiazole), 7.9 (d, 1H, Ar-H C7 of benzothiazole), 7.7 (d, 1H, C6 Ar-H of nitrobenzene ring), 7.6 (m, 1H, C5 Ar-H, nitrobenzene ring), 7.4 (m, 2H, C5,C6 of benzothiazole), 6.5 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-4.0 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO): 172.5 (C=O of thiazolidinone ring), 165.1 (C=N, C2, aromatic ring benzothiazole), 148.3 (C= , C9, aromatic ring benzothiazole), 148.0 (C3-NO2 of nitrobenzene ring), 142.5 (C-1 of nitrobenzene ring), 133.9 (C6 of nitrobenzene ring), 130.0 (C8 of benzothiazole, C5 of nitrobenzene ring), 125.8 ( C2 of nitrobenzene ring ), 122.4 (CH=, C7 benzothiazole and C4 of nitrobenzene ring), 118.1 (CH=, C4 of benzothiazole), 62.3 (S-CH-N aliphatic thiazolidinone) , 34.6 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 358.0316 (M+1)

3-Benzothiazol-2-yl-2- (4-nitro-phenyl)-thiazolidin-4-one (BG7): Yellow solid; Yield 82.10 %; R_fvalue 0.72; M.P.:130-132⁰C; FT-IR (cm^-1):3044 (C-H, Aromatic stretch, sharp, medium), 2896 (C-H, Aliphatic stretch, sharp, medium), 1692 (C=O, amide carbonyl stretch, sharp, strong), 1608 (C-C, Aromatic stretch), 1544 (N-O stretch), 1210 (C=N, stretch); ¹H NMR (500 MHz, DMSO): 8.2 (m, 3H, C2,C4 of nitrobenzene ring and C4 of benzothiazole), 8.0 (d, 1H, ArH, C7 of benzothiazole ), 7.5-7.6 (m, 4H, Ar-H C5, C6 of benzothiazole and C2,C6 of nitrobenzene ring), 6.5 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-3.6 (dd, 2H, CH₂ aliphatic, thiazolidinone); ¹³CNMR (500 MHz, DMSO): 171.0 (C=O of thiazolidinone ring), 166.8 (C=N, C2, aromatic ring benzothiazole), 149.0 (C= , C9, aromatic ring benzothiazole), 147.9 (C4-NO2 of nitrobenzene ring), 147.5 (C-1 of nitrobenzene ring), 131.1 (C8 of benzothiazole and C2,C6 of nitrobenzene ring), 124.9 (C5,C6 of benzothiazole, and C3, C5 of nitrobenzene ring), 121.2 ( C7 benzothiazole ), 118.1 (CH=, C4 of benzothiazole), 62.2 (S-CH-N aliphatic thiazolidinone), 32.3 (S-CH₂ aliphatic thiazolidinone; HR-MS (m/z) 358.0320 (M+1)

3-Benzothiazol-2-yl-2- (2-bromo-phenyl)-thiazolidin-4-one (BG8): Pale yellow solid; Yield 70.20 %; R_fvalue 0.52; M.P.:76-78⁰C; FT-IR (cm^-1): 3044 (C-H, Aromatic stretch, sharp, medium), 2896 (C-H, Aliphatic stretch, sharp, medium), 1692 (C=O, amide carbonyl stretch, sharp, strong), 1608 (C-C, Aromatic stretch), 1210 (C=N, stretch), 450 (C-Br stretch); ¹H NMR (500 MHz, DMSO): 9.1 (d, 1H, C4 of benzothiazole), 7.7 (d, 1H, ArH, C7 of benzothiazole ), 7.6 (d, 1H, C3 of bromobenzene ring), 7.6 (m, 2H, C5, C6 of benzothiazole), 7.3 (m, 1H, C5 of bromobenzene ), 7.1 (m, 1H, C6 of bromobenzene), 7.0 (C4 of bromobenzene ), 6.9 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-3.4 (dd, 2H, CH₂ aliphatic, thiazolidinone) ;¹³CNMR (500 MHz, DMSO): 172.3 (C=O of thiazolidinone ring), 166.8 (C=N, C2, aromatic ring benzothiazole), 148.0 (C= , C9, aromatic ring benzothiazole), 139.2 (C-1 of bromobenzene ring), 129.0 (C3, C4, C6 of bromobenzene ring), 127.3 (C5 of bromobenzene ring), 125.8 ( C5, C6 of benzothiazole), 121.4 ( C7 benzothiazole ), 118.2 (CH=, C4 of benzothiazole), 67.0 (S-CH-N aliphatic thiazolidinone), 32.0 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 392.9550 (M+1)

3-Benzothiazol-2-yl-2- (3-bromo-phenyl)-thiazolidin-4-one (BG9): Pale yellow solid; Yield 72.35%; R_fvalue 0.54; M.P.:84-86⁰C; FT-IR (cm^-1): 3055 (C-H, Aromatic stretch, sharp, medium), 2890 (C-H, Aliphatic stretch, sharp, medium), 1688 (C=O, amide carbonyl stretch, sharp, strong), 1610 (C-C, Aromatic stretch), 1206 (C=N, stretch), 460 (C-Br stretch); ¹H NMR (500 MHz, DMSO): 7.9 (d, 1H, C4 of benzothiazole), 7.8 (d, 1H, ArH, C7 of benzothiazole ), 7.5 (m, 4H, C5,C6 of benzothiazole, and C2,C4 of bromobenzene ring), 7.3 (m, 2H, C5,C6 of bromobenzene ), 6.4 (s, 1H, CH, aliphatic, thiazolidinone), 3.3-3.7 (dd, 2H, CH₂ aliphatic, thiazolidinone);¹³CNMR (500 MHz, DMSO): 171.2 (C=O of thiazolidinone ring), 165.2 (C=N, C2, aromatic ring benzothiazole), 152.1 (C= , C9, aromatic ring benzothiazole), 142.3 (C-1 of bromobenzene ring), 133.6 (C2 of bromobenzene ring), 129-130 (C8 of benzothiazole and, C4, C6 of bromobenzene ring), 122-125 (C5,C6 of benzothiazole and C5 of bromobenzene ring), 122.0 ( C3-Br of benzothiazole), 121.2 ( C7 benzothiazole ), 118.1 (CH=, C4 of benzothiazole), 60.8 (S-CH-N aliphatic thiazolidinone), 33.9 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 390.9496 (M+1)

3-Benzothiazol-2-yl-2- (4-bromo-phenyl)-thiazolidin-4-one (BG10): Pale yellow solid; Yield 71.20 %; R_fvalue 0.55; M.P.:78-80⁰C;FT-IR (cm^-1): 3045 (C-H, Aromatic stretch, sharp, medium), 2878 (C-H, Aliphatic stretch, sharp, medium), 1692 (C=O, amide carbonyl stretch, sharp, strong), 1612 (C-C, Aromatic stretch), 1210 (C=N, stretch), 455 (C-Br stretch);¹H NMR (500 MHz, DMSO): 8.0 (d, 1H, C4 of benzothiazole), 7.9 (d, 1H, ArH, C7 of benzothiazole ), 7.8 (d, 2H, C3,C5 of bromobenzene ring), 7.5 (m, 2H, C5, C6 of benzothiazole), 7.2 (d, 2H, C2,C6 of bromobenzene ), 6.9 (s, 1H, CH, aliphatic, thiazolidinone), 3.6-3.7 (dd, 2H, CH₂ aliphatic, thiazolidinone);¹³CNMR (500 MHz, DMSO):172.1 (C=O of thiazolidinone ring), 166.8 (C=N, C2, aromatic ring benzothiazole), 148.0 (C= , C9, aromatic ring benzothiazole), 141.1 (C-1 of bromobenzene ring), 130.8 (C8 of benzothiazole and C2,C6 of bromobenzene ring), 125.88 (C5,C6 of benzothiazole ), 121.3 (C7 of benzothiazole and C4-Br of bromobenzene ring), 118.1 (CH=, C4 of benzothiazole), 62.6 (S-CH-N aliphatic thiazolidinone), 32.3 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 392.9546 (M+1)

3-Benzothiazol-2-yl-2- (3-methoxy-phenyl)-thiazolidin-4-one (BG11): Greenish yellow solid; Yield 72.25 %; R_fvalue 0.74; M.P.:127-129⁰C; FT-IR (cm^-1): 3052 (C-H, Aromatic stretch, sharp, medium), 2885 (C-H, Aliphatic stretch, sharp, medium), 1689 (C=O, amide carbonyl stretch, sharp, strong), 1610 (C-C, Aromatic stretch), 1305 (C-O, stretch), 1215 (C=N, stretch);¹H NMR (500 MHz, DMSO): 8.0 (d, 1H, C4 of benzothiazole), 7.7 (d, 1H, ArH, C7 of benzothiazole ), 7.5 (m, 2H, C5,C6 of benzothiazole ring), 7.2 (m, 1H, C5 of methoxybenzene), 7.1 (d, 1H, C6 of methoxybenzene), 7.0 (s, 1H, C2 of methoxybenzene), 6.9 (d, C4 of methoxybenzene), 6.3 (s, 1H, CH, aliphatic, thiazolidinone), 3.8-3.9 (dd, 2H, CH₂ aliphatic, thiazolidinone), 3.7 (s,3H, OMe);¹³CNMR (500 MHz, DMSO): 171.1 (C=O of thiazolidinone ring), 165.0 (C=N, C2, aromatic ring benzothiazole),   159.6 (C3-OMe, methoxybenzene), 141.8 (C= , C9, aromatic ring benzothiazole), 141.9 (C-1 of methoxybenzene ring), 130.8 (C8 of benzothiazole and C5 of methoxybenzene ring), 125.8 (C5,C6 of benzothiazole ), 121.4(C7 of benzothiazole ), 119.4 (CH=, C4 of benzothiazole and C6 of methoxybenzene), 61.4 (S-CH-N aliphatic thiazolidinone), 55.8 (C-3, OMe), 35.3 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z)343.0573 (M+1)

3-Benzothiazol-2-yl-2- (4-methoxy-phenyl)-thiazolidin-4-one (BG12): Dark yellow solid; Yield 68.20 %; R_fvalue 0.69; M.P.:100-102⁰C; FT-IR (cm^-1):3048 (C-H, Aromatic stretch, sharp, medium), 2891 (C-H, Aliphatic stretch, sharp, medium), 1690 (C=O, amide carbonyl stretch, sharp, strong), 1612 (C-C, Aromatic stretch), 1215 (C=N, stretch), 1280 (C-O, stretch);¹H NMR (500 MHz, DMSO): 8.1 (d, 1H, C4 of benzothiazole), 8.0 (d, 1H, ArH, C7 of benzothiazole ), 7.9 (d, 2H, C2,C6 of methoxybenzene ring), 7.5 (m, 2H,C4, C5 of benzothiazole), 6.9 (d, 2H, C3,C5 of methoxybenzene ), 6.8 (s, 1H, CH, aliphatic, thiazolidinone), 3.8-3.9 (dd, 2H, CH₂ aliphatic, thiazolidinone), 3.8 (s, 3H, OMe);¹³CNMR (500 MHz, DMSO): 170.9 (C=O of thiazolidinone ring), 166.2 (C=N, C2, aromatic ring benzothiazole), 161.1 (C4-OMe, methoxybenzene), 151.7 (C= , C9, aromatic ring benzothiazole), 136.1 (C-1 of methoxybenzene ring), 131.8 (C1 of methoxybenzene ring), 129.4 (C2, C6 of methoxybenzene), 125.7 (C5,C6 of benzothiazole ), 121.7 (C7 of benzothiazole ), 118.1 (CH=, C4 of benzothiazole), 116.1 (C3, C5 of methoxybenzene), 60.0 (S-CH-N aliphatic thiazolidinone), 55.9 (C-4, OMe), 32.7 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 343.0575 (M+1).

3-Benzothiazol-2-yl-2- (3, 4-dimethoxy-phenyl)-thiazolidin-4-one (BG13): Yellow solid; Yield 70.15 %; R_fvalue 0.80; M.P.:86-88⁰C; FT-IR (cm^-1): 3055 (C-H, Aromatic stretch, sharp, medium), 2888 (C-H, Aliphatic stretch, sharp, medium), 1692 (C=O, amide carbonyl stretch, sharp, strong), 1610 (C-C, Aromatic stretch), 1290 (C-O, stretch), 1215 (C=N, stretch), 1200 (C-O stretch);¹H NMR (500 MHz, DMSO): 8.2 (d, 1H, C4 of benzothiazole), 8.0 (d, 1H, ArH, C7 of benzothiazole ), 7.6 (s, 1H, C2 of dimethoxybenzene), 7.4 (m, 3H,C4, C5 of benzothiazole, and C6 of dimethoxybenzene), 6.8 (d, 1H,C5 of dimethoxybenzene ), 6.4 (s, 1H, CH, aliphatic, thiazolidinone), 3.8-3.9 (dd, 2H, CH₂ aliphatic, thiazolidinone), 3.7 (s, 3H, 3-OMe), 3.6 (s, 3H, 4-OMe);¹³CNMR (500 MHz, DMSO): 171.0 (C=O of thiazolidinone ring), 166.8 (C=N, C2, aromatic ring benzothiazole),   149.6 (C= , C9, aromatic ring benzothiazole), 149.0 (C3-, dimethoxybenzene), 148.9 (C4, dimethoxybenzene), 132.0 (C-1 of dimethoxybenzene ring), 130.1 (C8 of benzothiazole ring), 125.8 (C5,C6 of benzothiazole ), 122.4 (C6 of dimethoxybenzene), 121.2 (C7 of benzothiazole ), 118.1 (CH=, C4 of benzothiazole), 112.0 (C2 of dimethoxybenzene), 111.4 (C5 of dimethoxybenzene), 63.2 (S-CH-N aliphatic thiazolidinone), 56.3 (C3, OMe of dimethoxybenzene), 55.8 (C4, OMe of dimethoxybenzene), 35.1 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 373.0673 (M+1)

3-Benzothiazol-2-yl-2- (3, 4, 5-trimethoxy-phenyl)-thiazolidin-4-one (BG14): Yellow solid; Yield 73.20 %; R_fvalue 0.72; M.P.:80-82⁰C; FT-IR (cm^-1): 3050 (C-H, Aromatic stretch, sharp, medium), 2896 (C-H, Aliphatic stretch, sharp, medium), 1690 (C=O, amide carbonyl stretch, sharp, strong), 1600 (C-C, Aromatic stretch), 1300 (C-O, stretch), 1220 (C-O stretch), 1215 (C=N, stretch);¹H NMR (500 MHz, DMSO): 8.0 (d, 1H, C4 of benzothiazole), 8.0 (d, 1H, ArH, C7 of benzothiazole ), 7.5 (m, 2H, C5,C6 of benzothiazole), 7.3 (s, 2H, C2,C6 of trimethoxybenzene), 6.6 (s, 1H, CH, aliphatic, thiazolidinone), 3.8-3.9 (dd, 2H, CH₂ aliphatic, thiazolidinone), 3.7 (s, 6H, 3,5-diOMe), 3.6 (s, 3H, 4-OMe);¹³CNMR (500 MHz, DMSO): 170.7 (C=O of thiazolidinone ring), 164.5 (C=N, C2, aromatic ring benzothiazole), 152.2 (C3-OMe, C5-OMe of trimethoxybenzene), 137.2 (C4-OMe, trimethoxybenzene), 133.2 (C1 of trimethoxybenzene), 130.3 (C8 of benzothiazole ring), 125.8 (C5,C6 of benzothiazole ), 121.3 (C6 of trimethoxybenzene), 121.2 (C7 of benzothiazole ), 118.1 (CH=, C4 of benzothiazole), 109.0 (C2,C6 of trimethoxybenzene), 71.9 (S-CH-N aliphatic thiazolidinone), 60.8 (C4, OMe of trimethoxybenzene), 56.6 (C3,C5, diOMe of trimethoxybenzene), 39.4 (S-CH₂ aliphatic thiazolidinone); HR-MS (m/z) 403.0782 (M+1)

Physico-chemical properties of substituted 3-Benzothiazol-2-yl-2-phenyl)-thiazolidin-4-ones and reaction time of the reaction was mentioned in Table 1.

DISCUSSION:

MOLECULAR DOCKING:

Serine/threonine kinase Polo-like Kinase-1 (PLK1) phosphorylates various target substrates and modifies their activity. PLK1 controls DNA replication following stress and acts as a pleiotropic master regulator of mitosis. It is a desirable therapeutic target because of its high levels of expression, which are associated with poor prognoses in many malignancies, including rhabdomyosarcomas.^25-27. A docking study has been performed by SP-docking mode using the 3DBC PDB ID from Protein Data Bank. All the synthesized compounds have a better docking score than the standard compound Doxorubicin (-6.711) (Table 1). Among the synthesized compounds, BG2 had the highest docking score of -8.381, followed by BG8 (-8.19) and BG1 (-8.156) (Table 2). The Phe 169 amino acid showed π-π interaction with the benzothiazole ring and phenyl ring attached to the thiazolidine ring in all synthesized compounds (Figure 1-3). Similarly, the ketone functional group of thiazolidine interacted with Cys 119 among all the synthesized compounds (Figure 1-3). These results were compared with the standard compound Doxorubicin, and it showed bidentate hydrogen bond had been observed between the sugar moiety attached with glycosidic linkage and Asp 180 amino acid. Another bidentate hydrogen bonding has been observed between the hydroxy group present at the anchor region of Doxorubicin with Glu 126 and Lys 129. An aromatic hydrogen bond has been observed between the anthraquinone ring of the Doxorubicin and Phe 169 residue and Gly 46 (Figure 4).

Table 1: Reaction time and physico-chemical properties of substituted 3-Benzothiazol-2-yl-2-phenyl)-thiazolidin-4-ones

Sr. No.	Compound	-R	Reaction Time (Min)	% yield	M. P. (^0C)	Rf Value
1	BG1	H	15	84.10	108-110	0.78
2	BG2	2-Cl	09	72.20	118-120	0.65
3	BG3	3-Cl	08	74.18	110-112	0.60
4	BG4	4-Cl	08	78.82	114-116	0.62
5	BG5	2-NO2	10	82.30	80-82	0.82
6	BG6	3-NO2	12	80.34	83-85	0.70
7	BG7	4-NO2	11	82.10	74-76	0.72
8	BG8	2-Br	09	70.20	126-128	0.52
9	BG9	3-Br	09	72.35	130-135	0.54
10	BG10	4-BR	08	71.20	136-138	0.55
11	BG11	3-OCH3	14	72.25	119-121	0.74
12	BG12	4-OCH3	15	68.20	106-108	0.69
13	BG13	3,4-dimethoxy	13	70.15	104-106	0.80
14	BG14	3,4,5-trimethoxy	13	73.20	155-157	0.72

Table 2: Docking score of the synthesized compounds towards the Polo-like kinase-1 by SP Docking method and of anticancer activity (GI₅₀ value (µg/ml) on MCF-7 human breast cancer cell line

Compound Code	Docking Score	Glide energy	MCF-7	Drug concentrations (µg/ml) calculated from graph
BG2	-8.381	-41.785	BG1	GI50*
BG8	-8.190	-42.71	BG2	27.4
BG1	-8.156	-39.518	BG3	42.9
BG12	-8.139	-41.876	BG4	57.9
BG3	-8.122	-40.493	BG5	55.9
BG4	-8.012	-40.406	BG6	>80
BG5	-8.012	-40.406	BG7	>80
BG9	-7.903	-40.294	BG8	43.7
BG10	-7.856	-42.288	BG9	54.0
BG13	-7.296	-45.539	BG10	66.3
BG11	-7.137	-41.099	BG11	72.4
BG6	-7.116	-43.316	BG12	62.7
BG7	-7.057	-44.187	BG13	73.1
BG14	-6.85	-46.741	BG14	>80
Doxorubicin	-6.711	-46.595	ADR	<10

Figure 1: Interaction of BG1 with Polo-like kinase-1

Figure 2: Interaction of BG2 with Polo-like kinase-1

Figure 3: Interaction of BG8 with Polo-like kinase-1

Figure 4: Interaction of Doxorubicin with Polo-like kinase-1

ANTICANCER ACTIVITY (SRB ASSAY)

BG1, BG2, BG3, BG4, BG5, BG8, BG9, BG10, BG11, BG12 and BG13 (GI₅₀: <80 µg/ml) exhibited significant cell growth inhibitory activity. Cell cytotoxicity determined by sulforhodamine B assay (Table 2). The title compounds (BG1 to BG14) tested with various concentrations on MCF-7 (Human breast cancer cell line) and compared with standard drug of Adriamycin. Graph showed growth curve of human breast cancer cell line MCF-7 (Figure 5).

Figure 5: Cell cytotoxicity determined by sulforhodamine B assay. The title compounds (BG1 to BG14) tested with various concentrations on MCF-7 (Human breast cancer cell line) and compared with standard drug of Adriamycin. Graph showed growth curve of human breast cancer cell line MCF-7

Structure Activity Relationship (SAR):

Based on literature survey, molecular docking and biological evaluation it has been observed that for significant activity substitutions at R₁ and R₂ positions of 1, 3-Thiazolidin-4-one should be as shown in Figure 6. As per shown in SAR substitutions at R₁ and R₂ positions of 1, 3-Thiazolidin-4-one dependent on each other for significant activity. The R₁ group should be bulky groups or hetero-aromatic rings substitution with or without linker is significant for activity. Secondly, on R2 position bulky groups or aromatic rings substituted but aliphatic substitutions are not effective.

Figure 6: Structure activity relationship (SAR) of title compounds (BG1 to BG14).

CONCLUSION:

According to molecular docking studies compound BG2 was having the highest docking score of -8.381, followed by BG8 (-8.19) and BG1 (-8.156). The sequence of highest docking score was found to be BG2, BG8, BG1, BG12, BG3, BG4 and BG5 respectively were illustrated in Table 2. The scheme was designed on the basis of molecular docking score and interactions. The anticancer activity of compounds (BG1 to BG14) was determined by Sulforhodamine (SRB) assay with MCF-7 cell lines for breast cancer. The anticancer drug Adriamycin was used as the reference standard. Results were summarized in Table 2 and are expressed as GI₅₀ (50% growth inhibitory concentration) values. The compoundsBG1, BG2, BG3, BG4, BG5, BG8, BG9, BG10, BG11, BG12 and BG13 (GI₅₀: < 80 µg/ml) exhibited significant cell growth inhibitory activity. The mechanism of PLK1 inhibition was postulated on the basis of molecular docking studies.

CONFLICTS OF INTEREST:

The authors have no conflict of interest regarding this investigation.

ACKNOWLEDGEMENTS:

Authors are really thankful to Dr. Jyoti Kode and Dr. Nirmal Kumar, Tata Memorial Centre’s Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Mumbai India for providing Anticancer Screening Facility.

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Received on 31.08.2023            Modified on 14.12.2023

Accepted on 17.02.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(3):1150-1158.

DOI: 10.52711/0974-360X.2024.00179