Oxadiazoles-Insimulation to repercussion in malignancy

Dr. Sundhararajan, Seraphine Joyce  J*,  Gopinath C, Gowtham B, Guruprasath. R

Mohamed Sathak A.J.College of Pharmacy,Sholinganallur,Chennai-119

*Correspondence: theresa_sera@yahoo.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4306  

INTRODUCTION

Owing to their intriguing anticancer properties, heterocycles are ever focused scaffolds. Of the heterocycles, oxadiazoles have compelling anti-tumor potential among heterocyclic chemicals, and their derivatives are undergoing clinical trials to treat cancer [1] Thimidine phosphorylase on the other  hand   attracts number of researchers to explore  this enzyme due to its anti –apoptic properties and the repercussion in malignancy [2] The purpose of this research is to determine potent oxadiazoles via insimulation, to synthesise and analyze the selected compounds and carry out  invitro activity and thereby analyzing the impact of oxadiazoles in malignancy

MATERIALS AND METHODS

We conducted the in-silico work in this study using a variety of bio-informatics technologies. We used the following offline tools in our current work: PyRx for molecular docking experiments, PDB, the PubChem database, SPDBV, and Protein-ligand interaction profile (PLIP). We also used Marvin Sketch for molecular sketching.[3]

Protein preparation

We retrieved the targeted protein human MAO-A (PDB ID: 2Z5X) from the online program PDB website and the protein preparation were started from the removal of water molecules, and followed this we added the missing H-atoms, ionization and energy minimization of proteins. The energy minimization was done by applying force filed through SPDBV software and it was validated by Ramachandran plot [4]

Identification of active sites

After preparation of protein, it was subjected to identify the active amino acid present in its structure by Protein- ligand interaction profile. By using PLIP we found the active amino acid residue present in the protein. Preparation of Ligands The 3D and 2D structure of designed derivatives were sketched by using Marvin sketch software. The sketched molecules are optimized and save as .pdb format for further processing. Molecular Docking The PyRx software was used for the docking process. The docking process was performed using molecular docking engine of PyRx [5] using grid resolution. During the docking process the default setting was used for the calculation

Table 1. PDB file of Thymidine Phosphorylase (TP)

Fig:(2)2D and 3D of docking fit of OX1

                                    Fig:(3)2D and 3D of docking fit of OX2

Table :2 Docking scores of Oxadiazole compounds (OX)

Table: 3 Inhibitory potential of Oxazole compounds

Synthetic Methods:

Step 1: Synthesis of N-(5-amino-1,3,4-oxadiazol-2-yl)Benzamide (A) Equimolar quantities of benzoyl isothiocyanate (1 mmol) and different semicabazide (2 mmol) was refluxed in alcohol for 4h in the presence of few drops of acetic acid. The reaction mixture on cooling was poured into cold water,filtered and dried. The crude solid was recrystallized in DMF-water mixture to give the products. Step 2: Title compound A mixture of N-(5-amino-1,3,4-oxadiazol-2-yl) Benzamide (1 mmol) and substituted aldehyde (2 mmol) was refluxed in NaoH (25 ml) for 8–10 h. After completion of reaction excess benzene was evaporated in vacuum. The resulting residue was neutralized with saturated NaHCO3 solution until CO2 evolution ceased. The solid product was washed with water, dried and recrystallized from DMF–water mixture.

Fig: 4 Synthetic scheme

Table: 4 Synthesised Compounds

OX1-Chemical Formula: C15H11N5O2

Exact Mass: 293.09 Molecular Weight: 293.28 m/z: 293.09 (100.0%), 294.09 (18.1%), 295.10 (1.7%)

Elemental Analysis: C, 61.43; H, 3.78; N, 23.88; O, 10.91

¹H NMR (500 MHz, DMSO) δ 9.69 (s, 1H), 8.67 (s, 1H), 8.51 (s, 1H), 7.87 (s, 1H), 7.85 – 7.78 (m, 3H), 7.51 (s, 1H), 7.45 (s, 1H), 7.43 – 7.37 (m, 2H).

¹³C NMR (125 MHz, DMSO) δ 172.19, 164.61, 162.02, 157.30, 155.02, 148.96, 139.02, 134.97, 131.75, 128.57, 128.23, 126.19, 122.28.

OX2-

Chemical Formula: C17H14N4O2

Exact Mass: 306.11 Molecular Weight: 306.32 m/z: 306.11 (100.0%), 307.12 (18.6%), 308.12 (2.0%), 307.11 (1.5%)

Elemental Analysis: C, 66.66; H, 4.61; N, 18.29; O, 10.45

¹H NMR (500 MHz, DMSO) δ 8.37 (s, 1H), 7.87 – 7.75 (m, 2H), 7.45 (s, 1H), 7.43 – 7.37 (m, 2H), 7.24 – 7.09 (m, 4H), 2.36 – 2.32 (m, 3H).

¹³C NMR (125 MHz, DMSO) δ 172.19, 166.06, 164.61, 157.30, 143.65, 134.97, 131.87, 131.75, 130.18, 129.53, 128.57, 128.23, 21.12.

In –Vitro studies

In vitro anti-cancer evaluation The in-vitro cytotoxicity activity of synthesized compounds was evaluated by MTT assay method using MCF-7 cell line and the standard protocol was followed to carry out the experiments . MTT Assay The concept states that MTT (3-(4,5 dimethyl thiazole- 2yl)-2,5-diphenyl tetrazolium bromide) tetrazolium salt needs to be cleaved. It was discovered that there was a relationship between the number of cells employed and the amount of formazan that they generated. To achieve 1.0105 cells/mL, the cell culture was adjusted using a DMEM medium containing 10% FBS. A 96- well flat-bottom microtitre plate was filled with 100μL of distilled cell suspension, or around 10,000 cells per well. Following a 24-hour period during which the cell population was deemed sufficient, the cells underwent centrifugation, and the pellets were subsequently suspended in a maintenance medium that included 100μL of diverse test sample concentrations. The plates were kept in an incubator with 5% CO2 at 37 degrees Celsius for 48 hours, and observations were taken every 24 hours. After 48 hours, MTT (2 mg/mL) in MEM-PR (MEM without phenol red) was added. The plates were incubated (5 percent CO2 atmosphere) for two hours at 37°C. After adding 100μL of DMSO, the plates were stirred to solubilize the formazan that had been formed. At 540 nm, the absorbance was measured

RESULT AND DISCUSSION

Ø  The Pdb file of thymidine phosphorylase was downloaded from protein databank library, the best 2- dimensional and 3-dimensional view of fit was recorded (figure 1 and figure 2)

Ø  The docking score was documented

Ø  The following is docking scan of oxadiazole

Ø  Doxorubicin was taken as standard

The bulleted points should be presented like this:

Ø  The selected oxazole derivatives were synthesized and characterized for structural properties.

Ø  All of the freshly synthesized derivatives' structures were verified using spectroscopic techniques (IR, 1H-NMR, ¹³C NMR, and mass).

Ø  The suggested structures and the spectrum data of the synthesized derivatives were completely compatible.

Ø  Every other aliphatic and aromatic proton was found to be within the anticipated ranges.

Ø  The structures of the synthesized derivatives were also supported by ¹³C data. The base peak and medium-intensity molecular ions in the mass spectra typically corresponded to the corresponding acylium

CONCLUSION

Oxadiazole moiety hybridization with other heterocyclic pharmacophores is a promising strategy to address a number of issues with existing anticancer medications, including toxicity, drug resistance, and other adverse effects. Oxadiazole derivatives were screened and inhibitory potential for thymidine phosphorylase was recorded and sorted based on their docking score, the in-simulated scores were documented with an intention to compare with the invitro inhibitory potential of oxazoles in inhibiting thymidine phosphorylase enzyme in tumour cells. Compounds OX 1 and OX 2 demonstrated more inhibitory potential in insilico simulation, which was in line with their in vitro inhibitory effect and indicated an anticipated impact on malignancy. A greater number of chemicals for comparison will still be needed for more values to arrive at statistical facts, even though it provides a reasonable sense of comparing insilico and invitro values. The synthesis, characterization, and in vitro thymidine phosphorylase inhibition of compounds OX3, OX4, and Ox5 are scheduled for the near future and will be published subsequently.

ACKNOWLEDGEMENT

The authors of this article would like to thank the faculty team of Mohamed Sathak A.J. College of Pharmacy for their support in this work.

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