Description: Background of the invention:
In 1883, Knorr synthesized antipyrine which has analgesic and antipyretic activity that results in explosion in pyrazolone chemistry (Eur. J. Med. Chem. 2015, 97, 786–815). 1H-pyrazol-3-ol and pyrazolin-5-one, mainly known as pyrazolones have gained importance because of their diverse medicinal applications (Eur. J. Med. Chem. 2009, 44, 3852-3857). Nowadays, various FDA approved drugs containing pyrazolone nucleus were explored like eltrombopag (used for adults having low blood platelet counts mainly known as idiopathic chronic immune thrombocytopenia), metamizole (strongest antipyretic used for injury, cancer pain, perioperative pain), aminophenazone (mainly used as anti-inflammatory, antipyretic and in breath test to measure cytochrome P-450 for liver function evaluation), edaravone (first free radical scavenger used as neuroprotective agents), dichloralphenazone (used for vascular headaches and to relieve stress) (Eur. J. Med. Chem. 2020, 186, 111893). Literature survey reported that pyrazolone motif fused with thiazole nucleus have various pharmacological activity like anticancer, anti-inflammatory, antiviral, antioxidant, antidiabetic, and antiviral. Interestingly, this class of compounds induce apoptosis in tumour cell line so considered as potential anticancer agents (Chin. J. Chem. 2013, 31, 1133-1138; Bioorg. Med. Chem. 2015, 25, 5797–5803). The compounds which have potential to inhibit kinase activity of EGFR were considered as potential antitumor agents (Curr. Med. Chem. 1999, 6, 825–843). It was evident from literature that thizolyl-pyrazolines derivatives depicted EGFR TK inhibitory activity which was further supported by molecular docking results (BMC Chem. 2019, 13, 116). Therefore, developing an anti-cancer drug that is both effective and inexpensive is a significant project (US Patent No. 10975102B1, 2021; US Patent 3914266, 1975). Considering the information mentioned above, it is always beneficial to design and develop new compounds that have anticancer potential. Therefore, thiazolyl-pyrazolines were considered as suitable anticancer agents and we synthesized some new thiazolyl-pyrazolone derivatives to investigate their anticancer potential against A549 lung cancer cells.
Summary of the invention:
The present invention discloses the anticancer screening of some new thiazolyl-pyrazole derivatives against lung cancer cell line (A549). The compounds 1c, 1d, 1h, 1k, 1j and 1l were found to display excellent potential than the standard drug, Carboplatin against the lung cancer cell line (A549)
Detailed description of the invention:
Anticancer Activity Evaluation
To demonstrate the versatility of the synthesized compounds (Fig.1), we evaluated their anti-cancer potential against lung cancer cell line.

Fig. 1. Various substitution patterns on the thiazolyl-pyrazole moiety of the
compounds (1a-m)
All the synthesized compounds were evaluated for their cytotoxicity on A549 (human lung adenocarcinoma cell) and Vero (African green kidney monkey cells) cell lines and compared their cytotoxicity to clinically approved chemotherapy drug, Carboplatin. To investigate further, A549 cells were treated with compounds of varying concentrations starting from 1000 µM for 48 h; DMSO (0.25%) was served as negative control. After screening, it was evident that all the novel agents depicted the inhibition of A549 lung adenocarcinoma cells in dose dependent manner (Fig. 2). The inhibitory role on cell viability by these compounds suggested the presence of potent anticancer activity against lung cancer cells. The 50% inhibitory concentration of the compounds were calculated and compared with the standard anticancer drug, Carboplatin. The results revealed that 1h showed the highest anti-proliferative activity on A549 cells among all the compounds with IC50 value 37.83 ± 0.6 µM. Compounds 1d and 1g showed similar inhibitory concentrations with 63.15 ± 1.4 and 63.78 ± 0.5 µM, respectively followed by 1c (IC50 = 86.79 ± 0.6 µM), 1k (IC50 = 98.96 ± 1.2 µM) and 1l (IC50 = 106.63 ± 0.5 µM). Furthermore, compound 1j found to be equally effective as compared to Carboplatin. All these compounds showed higher efficacy than the standard drug, Carboplatin (IC50 = 123.87 ± 0.7 µM) (Table 1). The compounds that showed anti-proliferative activities with IC50 values lower than 200 µM were 1b and 1e. In contrast, highest toxicity against noncancerous kidney Vero cells shown by 1g followed by 1k. Rest of the compounds have shown moderate to very less or no cytotoxicity on Vero cells. Hence, 1h, 1c, 1d, 1k, 1j and 1l were found to be suitable agents with higher anti-proliferative potential and with lesser toxicity on Vero cells. The SAR study revealed that anticancer potential was found remarkable in case of 4-trifluoromethoxyphenyl (1h) followed by 4-trifluoromethylphenyl (1g) substituted thiazole ring, however, 1g found to be highly cytotoxic to normal cells as compared to other substituents.
Table 1. Influence of the compounds 1a-m on A549 lung adenocarcinoma and Vero cells IC50 (µM)
Compound IC50 (µM) ± SEM
A549 Cells IC50 (µM) ± SEM
Vero Cells
1a 495.73 ± 1.9 479.17 ± 3.7
1b 176.87 ± 0.7 794.63 ± 4.0
1c 86.79 ± 0.6 673.33 ± 1.6
1d 63.15 ± 1.4 225.6 ± 0.9
1e 162.97 ± 1.8 141.67 ± 1.4
1f 212.1 ± 1.8 541.97 ± 2.1
1g 63.78 ± 0.5 64.73 ± 0.5
1h 37.83 ± 0.6 521.47 ± 1.5
1i 424.23 ± 0.5 731.50 ± 0.6
1j 127.6 ± 2.5 238.17 ± 0.7
1k 98.96 ± 1.2 192.43 ± 0.3
1l 106.63 ± 0.5 643.63 ± 1.9
1m 228.77 ± 0.5 356.40 ± 1.4
Carboplatin 123.87 ± 0.7 229.07 ± 1.2

(A) (B)
Fig. 2. Percentage of A549 (A) and Vero (B) viable cells on treatment with varying concentration of compounds 1a-m.
Outcome of the Invention:
Compounds 1c, 1d, 1h, 1j, 1k & 1l showed growth inhibitory effects on lung cancer cell line, with IC50 values 86.79 ± 0.6, 63.15 ± 1.4, 37.83 ± 0.6, 127.6 ± 2.5, 98.96 ± 1.2 & 170.03 ± 7.6, respectively. Therefore, compounds 1c, 1d, 1h, 1j, 1k & 1l have emerged as potential therapeutics for lung cancer in future.
Highlights of the Invention:
? Explored new thiazolyl-pyrazole derivatives 1c, 1d, 1h, 1j, 1k & 1l as new anticancer agents for lung cancer.
Experimental:
Anticancer activity
Cell Culture
A549 (human lung adenocarcinoma cell) and Vero (African green kidney monkey cells) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), GibcoTM media supplemented with 10% Fetal bovine serum (FBS), GibcoTM and 100 U/mL Pen-Strap antibiotics solution. Cells were maintained in CO2 incubator with continuous supply of 5% CO2 at 37?.
Cell viability assay
In-vitro cytotoxicity assay the confluent adherent cells were harvested by 0.25% trypsin-EDTA solution. Cells with seeding density of 1 x 104 cells/well were seed on 96 well plates followed by incubation for 24 h for complete adherence. After incubation, the stock concentration of test compounds prepared in DMSO was added to the wells with concentration 1mM as initial concentration in initial wells with 0.2% DMSO as final concertation followed by 2-fold serial dilutions. The cells were treated with the compound for 48 h in CO2 incubator followed by MTT assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) salt was used to validate the cytotoxicity effect of the test compounds on A549 human lung cancer cells and Vero, normal kidney cells. On the day of MTT assay, the media was replaced with fresh media along with 0.5% MTT solution (5mg/mL stock in 1X PBS) followed by incubation for 3 h at 37?. The insoluble formazan crystals formed were dissolved in DMSO and incubated in shaker for 30 minutes at room temperature. The absorbance of 96 well plates were then read at 492 nm on ELISA microplate reader and percentage of cell viability was calculated.
, Claims:I/We claim
1. The compounds of the formula I, where Ar is C6H5, 4-F-C6H4, 4-Cl-C6H4, 4-Br-C6H4, 4-CH3-C6H4, 4-CH3O-C6H4, 4-NO2-C6H4, 4-CF3-C6H4, 4-CF3O-C6H4, 4-C6H5-C6H4, 4-CN-C6H4, Chromenyl, Naphthyl as anticancer agents for lung cancer cell line (A549) at different concentrations from 1000 µM to 125 µM.

2. Compounds as claimed in claim 1, wherein the said compound reduces cell viability of lung cancer cells (A549) up to 22.21% at 1000 µM at 48 h of treatment, whereas the Vero cells (normal cells) possessed viability up to 40.37%.
3. The compounds as claimed in claim 1, wherein the said compounds reduce cell viability of lung cancer cells (A549) up to 25.11% at 500 µM after 48 h treatment whereas the Vero cells (normal cells) possessed viability up to 55.25%.
4. The compounds as claimed in claim 1, wherein the said compounds reduce cell viability of lung cancer cells (A549) up to 29.09% at 125 µM after 48 h treatment whereas the Vero cells (normal cells) possessed viability up to 72.15%.