Synthesis, biological evaluation and docking studies of 1,2,4-oxadiazole linked 5-fluorouracil derivatives as anticancer agents

Background 1,2,4-oxadiazole derivatives exhibited significant anti-cancer activity when they were evaluated, against human cancer cell lines. They also showed anti-inflammatory, analgesic, diabetic, immunosuppressive, α,β3-receptor antagonist, antimicrobial, anti-helminthic, histamine-H3 and antiparasitic properties. A pyrimidine analog, 5 fluoro-uracil is a chemotherapeutic drug used for treating multiple solid malignant tumors. But its application is limited, as it has side effects like low bioavailability and high toxicity. Molecular docking is an exemplary tool, helps in identifying target and designing a drug containing high bio-availability and minimum toxicity. Results A set of 1,2,4-oxadiazole linked 5-fluoruracil derivatives (7a–j) were synthesized and their structures were confirmed by 1HNMR, 13CNMR and Mass spectral analysis. Further, these compounds were investigated for their anticancer activity towards a panel of four human cancer cell lines such as (MCF-7, MDA MB-231), lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Among them, compounds 7a, 7b, 7c, 7d and 7i demonstrated more promising anticancer activity than standard. Conclusion Synthesized derivatives (7a–j) of 1,2,4-oxadiazole linked 5-fluorouracil and investigated for their anticancer activity towards a panel of four human cancer cell lines. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-021-00757-y.

The above biological findings and the continuous of demand for the new anticancer agents prompted us to design and synthesize a set of 1,2,4-oxadiazole linked 5-fluorouracil derivatives (7a-j). Their structures were confirmed by 1 HNMR, 13 CNMR and mass spectral data. Their anticancer activity towards four human cancer cells such as breast cancer (MCF-7, MDA MB-231), lung cancer (A549) and prostate cancer (DU-145) were evaluated.

In vitro cytotoxicity
The target compounds (7a-j) were examined for their anticancer activity against a panel of four human cancer cell lines including breast cancer (MCF-7, MDA MB-231), lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Etoposide was used as reference standard and the obtained results were summarized in Table 1.

Molecular docking
The docking studies of the potent 1,2,4-Oxadiazole linked 5-Fluorouracil derivatives (7a-7j) were performed using Molegro Virtual Docker (MVD). The crystal structure of Human VGEFR-2 enzyme (PDB ID: 1YWN) along with the crystal ligand imatinib was downloaded from protein databank [40]. Human VGEFR-2 enzyme is the key enzyme in angiogenesis, hematopoiesis and vasculogenic. All the chemical structures were prepared by using Marvin sketch and minimized and saved in a single file as SDF format. MVD was used to perform computational studies, cavity prediction, assigning bond orders, defining the active binding sites of the Human VGEFR-2 enzyme, structure refinement and preparation. The protein preparation was carried out with MVD and the chain was treated to add missing hydrogen, assign proper bond orders and deleted water molecules. The structure output format was set to pose viewer file so as to view the output of resulting docking studies and hydrogen bond interactions of different poses with the protein. The 2D and 3D interactions were generated by using Discovery Studio Visualizer.

Molecular docking studies
The docking studies of the potent compounds 7a-7j were performed using Molegro Virtual Docker (MVD). The crystal structure of human Vascular Endothelial Growth Factor Receptors (VEGFR-2) enzyme (PDB ID: 1YWN) along with the co-crystal ligand was downloaded from protein databank. Design of VEGFR-2 could be potential target for inhibition of blood vessel formation and leads to the development of anti-angiogenesis agents. All the chemical structures were prepared by using Marvin sketch and minimized. Mol file format structures were converted into Mol2 file format by using Discovery studio. MVD was used to perform computational studies, cavity prediction, assigning bond orders, structure refinement, defining the active binding sites of the VEGFR-2 and structure preparation. The protein preparation was carried out with MVD and the chain was treated to add missing hydrogen, assign proper bond orders and deleted water molecules. The structure output format was set to pose viewer file so  Table 2 In silico ADMET prediction of 1,2,4-oxadiazole linked 5-fluorouracil derivatives (7a-7j) In the present study, the synthesized compounds were docked into X-ray crystal structures of Human VEGFR-2 enzyme (PDB ID: 1YWN) to understand the possible target mechanism of action. The cavities were detected with MVD and the following are the active residues involved with co-crystal ligand, as it forms hydrogen bond interactions with Glu883, Glu915, Cys917, Asp1044. Arg1049 residues and hydrophobic interactions with Leu838, Val846, Val897, Val914, Cys1043, and Asp1044, residues. The docking has validated and found same interactions with Moldock score − 199.37 and H-bond energy − 6.24 kcal/mol (Fig. 2).

Compd
All the synthesized compounds form hydrogen bond interactions with Asp1044 and have shown same interaction with crystal ligand. The binding affinity of the docked compounds were expressed in negative binding energy kcal/mol (Moldock score). The ligands with more negative value of Moldock score will have more affinity with protein binding. 3,5-dinitro substituted compounds (7j and 7h)  also form same hydrophobic interactions with Arg840, Val846, Asp1044, Gly1046, and Arg1049 amino acid residues (Figs. 3, 4). Compound 7c oxygen of oxadiazole group forms a hydrogen bond (NH-O) with amino hydrogen of Gly1046 with a H-bond energy of − 2.59 kcal/mol (Fig. 5) and hydrophobic interactions with Val846, Arg840, Gly1046, Arg1049 amino acid residues. All the docked ligands have exhibited same interactions with active site of VEGFR. From the data it is revealed that compounds of the series 7j (Moldock score − 156.20 kcal/mol) and 7h (Moldock score − 157.88 kcal/mol) showed good inhibitory constant and excellent free energy of binding, which might be the reason for anticancer activity.

Pharmacophore prediction
All the synthesized compounds generated pharmacophore features by using PharmaGist web server [42]. It will predict the spatial arrangement of features like hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic center (HY) and positive ionisable (PI) which are essential for a ligand to interact with specific target protein. All compounds were submitted to PharmaGist and the pharmacophoric score is found to be 75.17 with 10 Spatial Features, 4 Aromatic, 1 Donor and 5 Acceptor features for all set of ligands.

MTT assay
Individual wells of a 96-well tissue culture micro titer plate were inoculated with 100 µl of complete medium containing 1 × 10 4 cells. The plates were incubated at 37 °C in a humidified 5% CO 2 incubator for 18 h prior to the experiment. The results are shown in Table 3.

Conclusion
In conclusion, we have synthesized a library of 1,2,4-oxadiazole linked 5-fluorouracil derivatives (7a-j) and all these compounds were characterized by 1 HNMR, 13 CNMR and Mass spectral analysis. Further, these compounds were investigated for their anticancer activity towards a panel of four human cancer cell lines such as (MCF-7, MDA MB-231), lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Among them, compounds 7a, 7b, 7c, 7d and 7i were demonstrated more promising anticancer activity than