Description:Background:
Drug designers and discoverers have reported investigated metal complexes as potential therapeutic possibilities since the discovery of cisplatin (Ann. Intern. Med., 1984, 100, 704-713) and auranofin (U.S. Patent US4133952A1979; Curr. Med. Chem., 2016, 23, 3374-3403). According to the findings, herein, analyses have shown the application of Cu(II) complexes as anticancer agents that can cause DNA damage, reactive oxygen species (ROS) production, and proteosome inhibition as ways to kill cancer cells. Prior to recently being suggested by Wehbe et al., Cu compounds employed as medicinal agents had several disadvantages, including solubility problems (Dalton Trans., 2020, 49, 14626-14639; Drugs, 1993, 46, 360-377). Nonetheless, copper-based compounds have recently gained significant attention once again in studies on inorganic synthesis, where scientists have developed Cu(II) complexes for a range of therapeutic purposes (Dalton Trans., 2017, 46, 10758-10773; Curr. Med. Chem., 2006, 13, 525-537; Appl. Organomet. Chem., 2020, 34, e5610; Arabian J. Chem., 2020, 13, 6379-6394; US Patent 7,128,923 B2, 2003). Cu(II)-indomethacin complexes, for example, have been used in veterinary medicine as an anti-inflammatory drug (Coord. Chem. Rev., 2002, 232, 95-126; Inorg. Chem., 1999, 38, 1736-1744). The revival is partially due to copper's inherent presence in numerous natural biological processes that can regulate copper levels and metabolize it significantly (Dalton Trans., 2017, 46, 10758-10773; ACS Omega, 2021, 6, 13704-13718).
In the past few decades, lifestyle-related disorders such as cancer, diabetes, hypertension etc. have increased significantly due to changes in the socioeconomic status of human beings. According to a WHO report, an estimated 20 million new cases and 10 million cancer-related deaths occurred worldwide. Over the next 20 years, the cancer burden will rise by almost 60%, placing an additional load on healthcare systems, individuals, and communities. By 2040, it is anticipated that there will be over 30 million additional instances of cancer worldwide, with the largest increases occurring in low- and middle-income nations. Designing an affordable and efficient cancer medication is, therefore, a major task (US Patent No. 10975102B1, 2021; US Patent 3914266, 1975). In view of above mentioned facts, it is always worthwhile to plan and develop new alternatives. Therefore, we synthesized new 1,3,4-trisubstituted pyrazoles containing Schiff's bases and their Cu(II) complexes in response to an unmet requirement in the field, and further explored their anti-lung cancer potential Against A549 lung cancer cells.
Summary of the invention:
The present invention discloses anticancer screening of new 1,3,4-trisubstituted pyrazole derivatives and their Cu(II) complexes against lung cancer cell lines (A549). Interestingly, all synthesized compounds were found to display excellent potential than standard drugs, Carboplatin & Cisplatin against the lung cancer cells (A549).
Detailed Description of the Invention:
Anticancer Activity Evaluation
To demonstrate the versatility and application of the synthesized compounds in the field of medicinal chemistry (Scheme-1), we evaluated their anti-cancer potential against lung cancer cell line (A549). The cytotoxicity assay on A549 lung adenocarcinoma cells, revealed the presence of high antiproliferative activity on lung cancer cells shown by all the tested compounds.

Scheme-1: Synthesis of 1,3,4-Trisubstituted pyrazole derivatives and their Cu(II) complexes
All the compounds inhibited the proliferation of A549 cells in concentration dependent manner (Fig. 1 (A)(B)). The compound 4d showed the highest anti-proliferative activity against the lung cancer cells with IC50 value 3.81 ± 0.1 µg/mL followed by 4b, 4a, 4e, and 4c with less than 5 µg/mL IC50 values (Table 1). The three positive control anticancer drugs, Cisplatin, Carboplatin and Dexamethasone (DEX) were used and revealed antiproliferative activity with IC50 values 13.93 ± 0.3 µg/mL, 73.05 ± 2.2 µg/mL, and 136.55 ± 19.0 µM, respectively. It is important to mention here that the compound 3b showed the inhibition of A549 cells similar as Cisplatin. All the tested compounds revealed the significant antiproliferative against lung adenocarcinoma inhibiting the growth of A549 cells.

(A) (B)
Figure 1. The graph shows: (A) Percentage of A549 viable cells in column group of 3a-e, (B) Percentage of A549 viable cells in column group of 4a-e.
The structure activity relationship (SAR) study revealed that substitution on the 4th position of the aromatic ring linked to 3rd position of pyrazole moiety increases the anticancer potential of the compound.
Table 1. IC50 values of the lung adenocarcinoma cells & Vero-African green monkey kidney cells (Cytotoxicity) in µg/mL.
Test Compounds IC50 values in µg/mL (Mean ± S.E.M)
A549 Cancer cells Vero cells
3a 30.57 ± 1.1 85.23 ± 0.3
3b 14.43 ± 3.0 47.02 ± 0.4
3c 71.23 ± 0.3 97.58 ±1.7
3d 42.93 ± 2.2 95.60 ± 1.5
3e 62.35 ± 5.3 284.45 ±1.8
4a 4.00 ± 0.2 22.82 ± 0.3
4b 3.84 ± 0.0 51.8 ± 1.0
4c 4.24 ± 0.5 29.97 ± 0.4
4d 3.81 ± 0.1 28.90 ± 1.9
4e 4.08 ± 0.0 31.02 ± 0.9
Carboplatin 73.05 ± 2.2 187.75 ± 1.5
Cisplatin 13.93 ± 0.3 18.92 ± 0.5
Dexamethasone 136.55 ± 19.0 31.45 ± 0.3

It was also observed that in case of aryl substitution at position-3 of pyrazole nucleus, anticancer potential increases in order of Br ? Me ? F ? Cl. It is also important to highlight here that incorporation of copper into 3a-e antiproliferative activity of compounds and thus Cu(II) complexes (4a-e) were explored as excellent lung cancer agents.
Outcome of the Invention:
All tested compounds showed growth inhibitory effects on lung cancer cell line, with IC50 values in the range of 3.81-71.23 µg/ml. Cu(II) complexes exhibited higher anticancer potential than ligands with IC50 values 3.81-4.08 µg/ml & 14.43-71.23 µg/ml, respectively. Therefore, all compounds have been emerged as potential therapeutics for lung cancer in future.
Highlights of the Invention:
? Explored 1,3,4-Trisubstituted pyrazole based Schiff's bases and their Cu(II) complexes as new Lung cancer agents. Results indicated that Cu(II) complexes (4a-e) displayed excellent antiproliferative activity than ligands (3a-e).
Experimental:
Anticancer activity Evaluation
Cell Culture
Human lung adenocarcinoma cells, A549 cell was maintained in Dulbecco’s Modified Eagle Medium (DMEM). Culture media was supplemented with 10 % fetal bovine serum (FBS: Gibco), and Pen-Strep. Culture was continuously maintained in 5 % CO2 at 37? with constant humidity.
Cell Viability assay or Anti-tumor Activity
Cell viability was investigated with MTT assay using yellow 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) salt. Cells were harvested with 0.25% trypsin-EDTA when it reached to the confluency of 75% to 80% with the process trypsinization. Hemocytometer was used for counting the cells. 1 x 104 cells/well were seeded in 96 well plates followed by the incubation for 24 h in CO2 incubator. After 24 h, test compounds with varying concentrations of serial dilutions (500 µg/mL was taken as highest concentrations for compounds 3a-e and Dexamethasone, Cisplatin, Carboplatin; 50 µg/mL was taken as the highest concentration for compounds 4a-e were added in the 96 well plate culture and incubated for 48 h. Dexamethasone (10 mM stock), Carboplatin & Cisplatin were used as standard anti-proliferative drugs. DMSO was used as a solvent for compounds with the final concentrations of 0.05%.
MTT assay
The viability of treated cell culture with test compounds were assessed after 48 hr of incubation. 96 well culture plates were gentle washed with 1X phosphate buffer saline (PBS) to remove the compounds. MTT salt solution (5mg/mL in 1X PBS) was added to the 96 wells with the final concentration of 0.5 mg/mL in each well. After 3 h of incubation, DMSO was used to solubilize the formazan crystals and incubated for 30 min. The 96 well plates were then read for absorbance in Synergy H1 microplate reader at 570 nm. All the experiments were performed in triplicate and IC50 values were calculated for each test compound in GraphPad Prism 8.0.1 by fitting the experimental results to sigmoidal equation.
In-vitro Investigation of Cytotoxicity on Vero cells
Culture of Animal Cell Line
Vero cell (African green monkey kidney cell) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS: Gibco), and Pen-Strep with continuous supply of 5 % CO2 at 37? on CO2 incubator.
Cell Viability assay or Anti-tumor Activity
Cell viability was investigated with MTT assay using yellow 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) salt. Cells were harvested with 0.25% trypsin-EDTA when it reached to the confluency of 75% to 80% with the process trypsinization. Hemocytometer was used for counting the cells. 1 x 104 cells/well were seeded in 96 well plates followed by the incubation for 24 h in CO2 incubator. After 24 h, test compounds with varying concentrations (starting from 500 ug/mL as highest concentrations) were added in the 96 well plate culture and incubated for 48 h. DMSO was used as solvent for compounds with the final concentrations of 0.05%.
MTT assay
The viability of treated cell culture with test compounds were assessed after 48 h of incubation. 96 well culture plates were gentle washed with 1X phosphate buffer saline (PBS) to remove the compounds. MTT salt solution (5mg/mL in 1X PBS) was added to the 96 wells with the final concentration of 0.5 mg/mL in each well. After 3 h of incubation, DMSO was used to solubilize the formazan crystals and incubated for 30 min. The 96 well plates were then read for absorbance in Synergy H1 microplate reader at 570 nm. IC50 values were calculated for each test compound in GraphPad Prism 8.0.1 by fitting the experimental results to sigmoidal equation.
Results
The in-vitro cytotoxicity of the compounds was studied on Vero-African green monkey kidney cells. The cytotoxicity study revealed the presence toxicity against Vero cells with concentration dependent manner. Among all compounds, 3b showed the highest toxicity with IC50 values 47.02 ± 0.04 µg/mL and compounds 3a-e are slightly toxic as compared to Carboplatin, while compounds 4a-e possess less toxicity as compared to Cisplatin (Table 1). Compounds, 4b and 4e are less toxic and found to be more effective as potential drug condidates as compared to both standard drugs.

C D
Figure 2. The graph shows: (C) Percentage of viable Vero cells in sigmoidal curve of 3a-e; (D) Percentage of viable Vero cells in sigmoidal curve of 4a-e. , Claims:I/We claim

1. The compounds of Formula I and Formula II, where Ar is C6H5, p-FC6H4, p-ClC6H4, p-BrC6H4, p-CH3C6H4 as anticancer agents for lung cancer cell line (A549) at different concentrations.

2. The compounds as claimed in claim 1, wherein the said compounds (Formula I & Formula II) can be used with concentrations up to 500 µM reduces cell viability of lung cancer cells (A549) up to 99 % in 48 h cell viability assay.
3. The compounds (3a-e) as claimed in claim 1, wherein the compounds with Formula I reduce cell viability of lung cancer cells (A549) up to 16-58 % at 15.62 µM while compounds with Formula II, reduce cell viability 91-97 % after 48 h treatment whereas the standard drugs, Carboplatin & Cisplatin reduce up to 26- 58 % cell viability.
4. The compounds as claimed in claim 1, wherein the compounds with Formula I reduce cell viability of lung cancer cells (A549) up to 63-89 % at 125 µM while compound with Formula II reduce cell viability 97-98 % after 48 h treatment whereas the standard drug, Carboplatin & Cisplatin reduce up to 67-74% cell viability.
5. These compounds having same basic skeleton as claimed in claim 1.