Design, synthesis and biological evaluation of 3-(2-aminooxazol-5-yl)-2H-chromen-2-one derivatives

Background In view of wide range of biological activities of oxazole, a new series of oxazole analogues was synthesized and its chemical structures were confirmed by spectral data (Proton/Carbon-NMR, IR, MS etc.). The synthesized oxazole derivatives were screened for their antimicrobial and antiproliferative activities. Results and discussion The antimicrobial activity was performed against selected fungal and bacterial strains using tube dilution method. The antiproliferative potential was evaluated against human colorectal carcinoma (HCT116) and oestrogen- positive human breast carcinoma (MCF7) cancer cell lines using Sulforhodamine B assay and, results were compared to standard drugs, 5-fluorouracil and tamoxifen, respectively. Conclusion The performed antimicrobial activity indicated that compounds 3, 5, 6, 8 and 14 showed promising activity against selected microbial species. Antiproliferative screening found compound 14 to be the most potent compound against HCT116 (IC50 = 71.8 µM), whereas Compound 6 was the most potent against MCF7 (IC50 = 74.1 µM). Further, the molecular docking study has been carried to find out the interaction between active oxazole compounds with CDK8 (HCT116) and ER-α (MCF7) proteins indicated that compound 14 and 6 showed good dock score with better potency within the ATP binding pocket and may be used as a lead for rational drug designing of the anticancer molecule.

Molecular docking studies provide the most detailed possible view of drug-receptor interaction and have created a new rational approach to drug design. The CDKs (cyclin dependent kinase) is an enzyme family that plays an important role in the regulation of the cell cycle and thus is an especially advantageous target for the development of small inhibitory molecules. Selective inhibitors of the CDKs can be used for treating cancer or other diseases that cause disruptions of cell proliferation [24]. Estrogen receptor alpha (ERα) is the major driver of ~ 75% of all breast cancers. Current therapies for patients with ER+ breast cancer are largely aimed at blocking the ERα signaling pathway. For example, tamoxifen blocks ERα function by competitively inhibiting E2/ERα interactions and fulvestrant promotes ubiquitin-mediated degradation of ERα. Endocrine therapies are estimated to have reduced breast cancer mortality by 25 ± 30% [25].
On the basis of the information obtained from literature survey (Fig. 2), in the present work we hereby report the synthesis, antimicrobial and antiproliferative potentials of oxazole derivatives.

Antimicrobial activity
The in vitro antimicrobial potential of the prepared oxazole derivatives was determined by tube dilution technique ( and these compounds may be used as a lead compound to discover novel antimicrobial agents.

Anticancer activity
The synthesized derivatives were also screened for their cytotoxic effect using Sulforhodamine B (SRB) assay [26] against two cancer cell lines-human colorectal carcinoma (HCT116) and oestrogen-positive human breast carcinoma (MCF7). In the case of HCT116, compound 14 exhibited good activity with IC 50 = 71.8 µM. In the case of MCF7, compound 6 exhibited good activity with IC 50 = 74.1 µM. Reference drugs used in the study were 5-flourouracil (for HCT116) and tamoxifen (MCF7). They had yielded IC 50 values of 12.7 µM and 4.3 µM, respectively and these compounds may be used as a lead compound to discover novel anticancer agents. Results are displayed in Table 3.

Molecular docking results
The mammalian cyclin-dependent kinase 8 (cdk8) protein which is a component of the RNA polymerase has been one of the proteins responsible for acute lymphoblastic leukaemias. CDK-8 is a heterodimeric kinase protein responsible for regulation of cell cycle progression, transcription and other functions. CDK-8 phosphorylates the carboxyterminal domain of the largest subunit of RNA polymerase II like protein kinases. Therefore, the inhibition of CDK-8 protein may be crucial for controlling cancer [27]. Since compounds were screened through ATP binding pocket so, ATP was used as docking control to compare the binding affinity of compounds within the binding pocket. The synthesized oxazole compounds showed good docking score and were found to interact with important amino acids for the biological function of CDK-8 protein.
Molecular docking were carried out to analyse the binding mode of the most active compound 14 and compound 6 against human colorectal carcinoma HCT116 and oestrogen-positive human breast carcinoma MCF7 cancer cell lines respectively. The molecular docking study was carried out on GLIDE docking program. The compound 14 was docked in the active site of the cyclin dependent kinase cdk8 (PDB: 5FGK) co-crystallized wit 5XG ligand. The results were analysed based on the docking score obtained from GLIDE. Ligand interaction diagram and displayed the binding mode of compound 14 in the active site of cdk8 having co cystallised ligand 5XG and 5-fluorouracil (the standard inhibitor of cancer) is having a different binding mode to that of active compound (Figs. 6 and 7).
The compound 6 was docked in the active site of the ER-alpha of MCF-7 (PDB: 3ERT) co-crystallized wit OHT (Tamoxifen) ligand. The results were analysed based on the docking score obtained from GLIDE. Ligand interaction diagram and show the binding mode of compound 6 in the active site of ER apha having co cystallised ligand OHT and Tamoxifen (the standard inhibitor of cancer) is having a different binding mode to that of active compound (Figs. 8 and 9). The docking scores were demonstrated in terms of negative energy; the lower the binding energy, best would be the binding affinity. The results depend on the statistical evaluation function according to which the interaction energy in numerical values as docking scores. The 3D pose of the ligand interaction with receptor can be visualized using different visualization tools [28]. Based on the molecular docking study the selected compounds with           (Table 5). Thus the docking results suggest that the oxazole derivatives can act as of great interest in successful chemotherapy. CDK-8 may be the target protein of oxazole derivatives for their anticancer activity at lower micromolar concentrations. Based on the docking analysis it is suggested that more structural modifications are required in compounds 6 and 14 to make them more active against cancer cells and to have activity comparable to the standards 5-fluorouracil and tamoxifen.

Structure activity relationship
From the antimicrobial and anticancer activities results following structure activity can be derived (Fig. 10): • The different substitution of aldehydes were used to synthesized the final derivatives of 3-(2-aminooxazol-5-yl)-2H-chromen-2-one derivatives played an important role in improving the antimicrobial and anticancer activities. Presence of electron releasing group (-CH(CH 3 ) 2 ) at para position of the substitution part of the synthesized compound 8, increased the antibacterial activity against B. subtilis. Presence of para-(phenoxy-methyl)benzene group (compound 3), enhanced the antibacterial activity against E. coli and S. aureus as well antifungal activity against C. albicans whereas (Compound 5) also improved the antifungal activity against C. albicans. • Presence of electron releasing group (OH, OCH 3 ) at meta and para position of the substitution portion of the synthesized compound 14, increased the antibacterial activity against P. aeruginosa and also increased anticancer activity against HCT116 cancer cell line whereas electron withdrawing groups (-Cl) at paraposition of the synthesized compound 6, improved the antimicrobial activity against S. enterica and A. niger as well as anticancer activity against MCF7 cancer cell line. These compounds may be used as a lead compound to discover novel antimicrobial and anticancer agents.

Experimental part
The chemicals procured were of analytical grade and were further used without any purification. Melting point (m.p.) was determined in open glass capillaries on a Stuart scientific SMP3 apparatus. Reaction progress of every synthetic step was confirmed by TLC plates on silica gel sheets. 1 H and 13 C-NMR spectra were determined by Bruker Avance III 600 NMR spectrometer in appropriate deuterated solvents and are expressed in parts per million (δ, ppm) downfield from tetramethylsilane (internal standard). Proton NMR spectra are given as multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) and number of protons. Infrared (IR, KBr, cm −1 ) spectra were recorded as KBr pellets on Shimadzu FTIR 8400 spectrometer. Waters Micromass Q-ToF Micro instrument was used for obtaining the Mass spectra.

In vitro antimicrobial assay
Tube dilution method [31] was used for evaluating the antimicrobial potential of the compounds and the standard drugs used were cefadroxil (antibacterial) and fluconazole (antifungal

In vitro anticancer assay
The cytotoxic effect of oxazole derivatives was determined against two different cancer cell lines-human colorectal carcinoma [HCT116] and oestrogen-positive human breast carcinoma (MCF7) using Sulforhodamine-B assay. HCT116 was seeded at 2500 cells/well (96 well plate) whereas MCF7 was seeded at 3000 cells/well (96 well plate). The cells were allowed to attach overnight before being exposed to the respective compounds for 72 h. The highest concentration of each compound tested (100 µg/mL) contained only 0.1% DMSO (non-cytotoxic). Sulforhodamine B (SRB) assay [26] was then performed. Trichloroacetic acid was used for fixing the cells and then staining was performed for 30 min with 0.4% (w/v) sulforhodamine B mixed with 1% acetic acid. After five washes of 1% acetic acid solution, protein-bound dye was extracted with 10 mM tris base solution. Optical density was read at 570 nm and IC 50 (i.e. concentration required to inhibit 50% of the cells) of each compound

Molecular docking study
The protein target for oxazole derivatives was identified through the literature. Since the oxazole nucleus has vast medicinal properties, so the targets enzymes/ receptors were found targeted with anticancer effect of oxazole compounds were collected for selection [33]. Cyclin-dependent kinase-8 (PDB Id: 5FGK) co-crystallized wit 5XG ligand and ER-alpha of MCF-7 (PDB: 3ERT) co-crystallized wit OHT (Tamoxifen) ligand excellent target against cancer [34], was retrieved from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) for docking of potent synthesized oxazole compounds. Docking score obtained from GLIDE and ATP binding site was targeted and the grid was created. The active site grid covered the important amino acids interacting with ATP [35].

Conclusion
A series of oxazole derivatives was designed, synthesized and evaluated for its antimicrobial and antiproliferative activities. The biological screening results indicated that the compounds 3, 5, 6, 8 and 14 had the best antimicrobial activity and had MIC values comparable to the standard drugs whereas in the case of anticancer activity, compound 14 was found to be moderate activity against HCT116 while compounds 6 was moderate activity against MCF7. Further molecular docking study indicated that compound 14 showed the best dock score (− 7.491) with better potency (71.8 µM) within the ATP binding pocket. Compound 6 showed the best dock score (− 6.462) with better potency (74.1 µM) within the ATP binding pocket. Hence these compounds may be taken as lead compound for further development of novel antimicrobial and anticancer agents.