Description:Field of Invention:
The present invention reported the development of new 3-Methyl-4-(3'-methylisoxazol-5'-yl)-1-aryl-1H-pyrazol-5-ol derivatives as anti-lung cancer agents. The study has been carried on the basis of anticancer screening of the newly synthesized isoxazolyl pyrazoles against A549 lung cancer cell line.
Background:
Cancer is becoming the most common cause of mortality for serious illnesses at global level. Developing novel medications with minimum side effects for its treatment is now the most challenging issue in front of all researchers (Curr Med Chem Anticancer Agents, 2020, 20, 2150-2168). Compounds containing heteroatoms, such as oxygen, nitrogen, and sulfur, strengthen the drug by forming H-bonds with DNA. Furthermore, an optimum interaction takes place when the chemical structure contains one or more nitrogen atoms and gives the intercalating chromophore a polarized property (Eur. J. Med. Chem. 2017, 125, 143-189). Pyrazole is a five- membered heterocyclic compound containing two nitrogen atoms at neighbouring positions and has been formed in many anticancer drugs like Crizotinib used for treatment of lungs cancer, Niraparib used for ovarian cancer, Darolutamide used to treat Prostate cancer (Res Chem Intermed 2016, 42, 6881- 6892; Clin Lymphoma Myeloma Leuk, 2013, 13, 638-45; Lung Cancer, 2014, 83, 37–43; ACS Omega, 2020, 5, 10089-10098).
Furthermore, Isoxazole is also a five-membered heterocyclic compound containing two heteroatoms, one nitrogen and one oxygen atom at the neighbouring positions. It was reported that after the addition of isoxazole moiety in a compound it enhances efficiency, decrease toxicity, and increase pharmacokinetics profiles of the compounds (Mini-Rev. Med. Chem., 2014, 14, 623–627). Isoxazole derivative show insecticidal, antibacterial, antibiotic, antitumor, antifungal, antituberculosis, anticancer, and ulcerogenic activities (Eur. J. Med. Chem., 2021, 221, 113511; Bioorg Med Chem, 2018, 26, 3065-3075; Eur. J. Med. Chem., 2014, 77, 121-133) Risperidone is a drug used for the treatment of schizophrenia in adults. NVP-AUY922 is an experimental cancer treatment drug developed by Vernalis and it has shown promising activity in preclinical testing against multiple types of tumor (J. Med. Chem., 2014, 57, 2136; Curr. Org. Chem., 2011, 15, 1423).
There are some compounds which contain isoxazole as well as pyrazole rings and show good biological activity (Anti-Cancer Agents Med. Chem., 2019, 19, 948-959; Molecules, 2022, 27, 3752; J Heterocyclic Chem., 2022, 59, 341–350). In considering the various applications and as part of our ongoing research in order to find and develop pharmacologically potent compounds, we intended to synthesize new isoxazole-pyrazole hybrids for exploring their anti-lung cancer potential Against A549 lung cancer cells.

Summary of the invention:
The present invention reported the development of new 3-Methyl-4-(3'-methylisoxazol-5'-yl)-1-aryl-1H-pyrazol-5-ol derivatives as anticancer agents against lung cancer cell lines (A549). Interestingly, eleven compounds from the series 5a, 5c, 5e, 5f, 5h, 5i, 5j, 5l, 5m, 5n, 5o were found to be more potent than the standard drug, Carboplatin.
Detailed Description of the Invention:
Anticancer Activity Evaluation
To exhibit the synthetic compounds versatility and potential uses in medicinal chemistry, 3-Methyl-4-(3'-methylisoxazol-5'-yl)-1-aryl-1H-pyrazol-5-ol (5a-p) derivatives were synthesized in our lab by using pyrazolyl-1,3-diketone derivatives (4a-p) and hydroxylamine hydrochloride as reactants and further evaluated their anti-cancer potential against lung cancer cell line (A549) (Scheme-1).

All the synthesized compounds were also evaluated for their cytotoxicity on A549 (human lung adenocarcinoma cell) (Fig. 1 (A)(B)). To investigate further, A549 cells were treated with compounds of varying concentrations starting from 1000 µM for 48 h; DMSO was served as negative control. The 50% inhibitory concentration of the compounds were calculated and compared with the standard anticancer drug, Carboplatin. All the compounds inhibited the proliferation of A549 cells in concentration dependent manner eleven compounds from the series 5a, 5c, 5e, 5f, 5h, 5i, 5j, 5l, 5m, 5n, 5o were found to be more potent than the standard drug, Carboplatin with IC50 value 259.9± 2.5 µM (Table 1). Compound 5a showed the highest cytotoxicity against A549 lung cancer cell with IC50 value 137.55± 0.35 µM followed by compound 5e with IC50 value 147.5 ± 0.2 µM. Also, the seven compounds 5a, 5e, 5f, 5i, 5j, 5l and 5m inhibited the cell proliferation with IC50 values lower than 200 µM.

Compounds a b c d e f g h
R H 4-F 3-F 2-F 4-Br 3-Br 2-Br 4-Cl
Compounds i j k l m n o p
R 3-Cl 2-Cl 4-CH3 3-CH3 2-CH3 4-OCH3 3-OCH3 2-OCH3

Scheme 1. Synthesis of 3-Methyl-4-(3'-methylisoxazol-5'-yl)-1-aryl-1H-pyrazol-5-ol derivatives
Table 1. Influence of the compounds 5a-p on A549 lung adenocarcinoma cells IC50 (µM)
Compound Molecular Weight
(g/mol) IC50 values
(µM) ± S.E.M
5a 255.28 137.55 ± 0.35
5b 273.27 261.7 ± 0.5
5c 273.27 223.55 ± 0.35
5d 273.27 397.7 ± 0.4
5e 334.17 147.5 ± 0.2
5f 334.17 157.5 ± 0.2
5g 334.17 294.65 ± 0.25
5h 289.72 246.05 ± 0.15
5i 289.72 182.15 ± 0.15
5j 289.72 160 ± 0.3
5k 269.12 268.4 ± 0.3
5l 269.12 190.65 ± 0.25
5m 269.12 148.2 ± 0.8
5n 285.30 232.7 ± 0.6
5o 285.30 255.15 ± 1.45
5p 285.30 366.9 ± 0.9
Carboplatin 259.9 ± 2.5

(A) (B)
Figure 1. Growth of A549 cells at 48 hours on treatment with varying µM concentrations of test compounds- (A) 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h and Carboplatin; (B) 5i, 5j, 5k, 5l, 5m, 5n, 5o, 5p and Carboplatin.

Outcome of the Invention:
Compounds 5a, 5c, 5e, 5f, 5h, 5i, 5j, 5l, 5m, 5n, 5o showed good inhibitory effects on lung cancer cell line, with IC50 values 137.55 ± 0.35, 223.55 ± 0.35, 147.5 ± 0.2, 157.5 ± 0.2, 182.15 ± 0.15, 160 ± 0.3, 190.65 ± 0.25, 148.2 ± 0.8 respectively. Therefore, compounds have emerged as potential anticancer agents in future.
Highlights of the Invention:
The compounds having pyrazole and isoxazole rings show good antiproliferative activity. So, these compounds (5a-p) may be used as new anti-lung cancer agents A549 lung cancer cell line.
Experimental:
Anticancer activity
Cell Culture
A549 (lung adenocarcinoma cell) was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), GibcoTM and 100 U/ml Pen- Strap antibiotic solution. Cells are maintained in CO2 incubator with continuous supply of 5% CO2 at 37°C.
Cell viability assay
In-vitro cytotoxicity assay of the confluent adherent cells was harvested by 0.25% trysin-EDTA solution. Cells with seeding density of 1x104 cells/well were seeded on 96 well plates followed by incubated for 24 h for complete adherence. After incubation, the stock concentration of test compounds prepared in DMSO was added to the wells with concentration of 1mM as initial concentration in initial wells with 0.2% DMSO as final concentration followed by 2-fold serial dilutions. The cells were treatment with the compounds for 48 h in CO2 incubator followed by MTT assay. 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) salt was used to validate the cytotoxicity effect of the test compounds on A549 human lung cancer 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 3h at 37?. The insoluble formazan crystal formed was 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.

Statistical analysis
All the experimental data are in triplicate and IC50 value analysed in Graph Pad Prism 8.0.1.
, Claims:I/We claim

1. The compounds of Formula I where R may be H, 2-F, 3-F, 4-F, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-Cl, 3-Cl, 4-Cl, 2-CH3, 3-CH3, 4-CH3, 2-OCH3, 3-OCH3, 4-OCH3 as anticancer agents for lung cancer cell line (A549) at different concentrations.

Formula-I

2. The compounds as claimed in claim 1, wherein the said compounds reduce cell viability of lungs cancer cells (A549) up to 87.5% to 1000 µM after 48 hours treatment.
3. The compounds as claimed in claim 1, wherein the said compounds reduce cell viability of lungs cancer cells (A549) up to 70% to 500 µM after 48 hours treatment.
4. The compounds as claimed in claim 1, wherein the said compounds reduce cell viability of lungs cancer cells (A549) up to 50% to 250 µM after 48 hours treatment.