Alireza Heidari^1,2,3,4*, Zahra Torfeh⁵

¹Faculty of Chemistry, California South University, 14731 Comet St. Irvine, CA 92604, USA.

²BioSpectroscopy Core Research Laboratory (BCRL), California South University, 14731 Comet St. Irvine, CA 92604, USA.

³Cancer Research Institute (CRI), California South University, 14731 Comet St. Irvine, CA 92604, USA.

⁴American International Standards Institute (AISI), Irvine, CA 3800, USA.

⁵An Independent, Volunteer and Unaffiliated Researcher.

*Corresponding Author: Alireza Heidari, Faculty of Chemistry, California South University, 14731 Comet St. Irvine, CA 92604, USA, BioSpectroscopy Core Research Laboratory (BCRL), California South University, 14731 Comet St. Irvine, CA 92604, USA, Cancer Research Institute (CRI), California South University, 14731 Comet St. Irvine, CA 92604, USA, American International Standards Institute (AISI), Irvine, CA 3800, USA,Tel: +1 408-816-2779; Fax: 00-40-253-210 +1 408-816-2779 432; E-mail: scholar.researcher.scientist@gmail.com;  alireza.heidari@calsu.us; central@aisi-usa.org

Citation: Alireza Heidari, Zahra Torfeh (2023) Design of Novel Cancer Inhibitors Using Molecular Docking, Dynamics Simulation and 3D-QSAR and 3D-QSPR Studies. Arch Mol Med & Gen 5: 113.

Received: January12, 2023; Accepted: January 20, 2023; Published: January 23, 2023.

Copyright: © 2023 Alireza Heidari, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Considering the importance of oxadiazole derivatives as effective anti–cancer Nano drugs on cancer cells and various other therapeutic effects, in this research, the effect of new oxadiazole derivatives called 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on single–stranded DNA/RNA in a solution. We studied the use of different spectroscopic methods. The present study investigated the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on single–stranded DNA/RNA in laboratory conditions. The results show that the absorption rate of single–stranded DNA/RNA increases due to the interaction with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at 210 and 260 (nm) wavelengths. The emission spectrum of single–stranded DNA/RNA increases in a concentration–dependent trend of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, which indicates the binding of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with chromophores present in single–stranded DNA/RNA. The present study investigated the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on single–stranded DNA/RNA in laboratory conditions. The results show that the absorption rate of single–stranded DNA/RNA increases due to the interaction with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at 210 and 260 (nm) wavelengths. The emission spectrum of single–stranded DNA/RNA increases in a process dependent on the concentration of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, which indicates the binding of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with chromophores present in single–stranded DNA/RNA. The results obtained from the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on single–stranded DNA/RNA can provide useful information in the field of designing anti–cancer Nano drugs with oxadiazole derivatives with more anti–tumor effect and less side effects.

Figure: 1

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Figure: 3

Schematic of the 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole as anti–cancer Nano drugs’ effect and delivery mechanism on DNA/RNA in human breast cancer cells.

Molecular structures of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Molecular Dynamics, Simulation, Perception, Binding Affinity, Performance, Nano Synthesized, DNA/RNA, Human Cancer Cells, Biospectroscopic Methods and Techniques.

Despite the efforts of scientists and the advancement of science and technology, cancer is still one of the deadliest diseases of mankind, and therefore, research on this disease and its treatment methods are of great interest. Therefore, it can be important to identify the mechanism of action of anti–cancer Nano drugs in order to use them more effectively in the treatment of various types of cancer. The nucleus is the most important organelle that exists in eukaryotic cells, and the target of many new compounds as well as anti–cancer Nano drugs is the cell nucleus. DNA/RNA inside the nucleus of eukaryotic cells during replication or transcription is bare [1–38]. Normally, it is accompanied by histone and non–histone proteins, which together form structures called nucleosomes, which are called chromatin. Therefore, anti–tumor Nano drugs can target either single–stranded or double–stranded DNA/RNA to perform their action. Oxadiazoles are widely found in biology, medicine and polymer science. The most well–known and important oxadiazole are 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, which is prescribed in low doses as a blood thinner. Several oxadiazole are used as anti–cancer Nano drugs in modern and recent medicine. Oxadiazole and its derivatives also show a wide range of different physiological activities, including anti–inflammatory, anti–bacterial, anti–cancer, anti–clotting and anti–HIV activities. Oxadiazole and its derivatives also have a wide range of activities. They show various physiological properties such as anti–inflammatory, anti–bacterial, anti–cancer, anti–clotting, anti–HIV, anti–viral, anti–viral activities [39–57]. They are also used as ingredients in perfumes, cosmetics, food additives, pharmaceuticals, in the preparation of insecticides, optical brighteners, and diffused and laser fluorescent dyes. become oxadiazoles have been noticed due to their toxicity and carcinogenicity. In addition, they show photodynamic effects and are useful intermediates for the synthesis of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole. Tetrazoles participate in the pharmacokinetics and metabolism of anti–cancer Nano drug delivery [58–76]. Therefore, they are always a good candidate for binding and acting on protein and DNA/RNA. 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole are oxadiazole derivatives successfully synthesized via domino Knoevenagel. These oxadiazole derivatives participate in the formation of free radicals in skin cells and causes damage to the structure of DNA/RNA and protein in the cell, which then causes cancer in humans. Considering the above, the purpose of this experiment is to investigate the effect of these oxadiazole derivatives on the single–stranded DNA/RNA of calf thymus gland. The use of calf thymus gland, due to the high amount of DNA/RNA compared to protein, allowed us to investigate the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on pure DNA/RNA [77–114].

3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole as oxadiazole are oxadiazole derivatives that are synthesized through the reaction of active carbonylated compounds such as Isatin derivatives, aromatic aldehydes with Malononitrile through a multicomponent domino reaction. (Knoevenagel condensation/dipolar 1 and 3 ring addition) is performed without using any catalysis in water solvent at a temperature of 50 degrees Celsius. 2 mg of oxadiazole derivatives were combined with 1 ml Tris buffer solution purchased from Merck, Germany, and the solution was kept in the refrigerator. Thymus single–stranded DNA/RNA was purchased from SIGMA. To prepare the DNA/RNA solution, 2 mg of DNA/RNA powder was mixed with 1 ml of 0.01 M Tris buffer with pH 7.4 and the solution was kept in the refrigerator. The interaction of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA was carried out using 10 mM Tris acid buffer (pH 7.2, at room temperature and away from light). For this purpose, a constant concentration of single–stranded DNA/RNA was prepared and incubated with different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro 1,2,4–oxadiazole and  3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole for one hour, and after the desired time, it was used for UV–Vis spectroscopy studies. The absorbance of the solution resulting from the interaction of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA and also the absorbance of different concentrations of the anti–cancer Nano drug in acidic Tris buffer at 210 and 260 (nm) using the spectrophotometer Carry 100 model Bio–Varian and the production of the country of Australia was read and after performing the necessary calculations, the relevant curves were drawn. In order to conduct fluorescence spectroscopy studies, first, a fixed concentration of single–stranded DNA/RNA was incubated with different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at room temperature and away from light for one hour. After a certain period of time has passed, the solution resulting from the interaction of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA is excited at a wavelength of 258 (nm) and their emission spectrum is in the range of 200 to 700 (nm) using a spectrofluorometer device. Carry Eclipse, Bio–Varian model, made in Australia. The solution resulting from the interaction of single–stranded DNA/RNA with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole was also drawn at the excitation wavelength of 258 (nm) and its fluorescence emission spectrum was drawn in the range of 500–550 (nm). Also, the emission spectrum of the standard samples of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole was drawn in the mentioned range and then it was subtracted from the emission of the treated samples. During the experiments, excitation and emission slits were considered to be 10 and 5 (nm), respectively, and a quartz cuvette with a width of one centimeter was used. After drawing the curves resulting from the interaction, according to the obtained information, the Io–I/Io×100 curve was drawn against different concentrations of the anti–cancer Nano drug. In this formula, Io is the intensity of fluorescence emission in the absence of anti–cancer Nano anti–cancer Nano drug and I is the intensity of fluorescence emission in the presence of different concentrations of the anti–cancer Nano drug. Also, the constant of the Stern–Volmer equation (Ksv) was calculated to estimate the amount of fluorescence quenching. In the Stern–Volmer equation Io/I = 1+ Ksv [Q], Io and I are respectively the amount of intrinsic emission in the absence and presence of the quencher 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, [Q] the quenching concentration Ksv is the Stern–Volmer quenching constant of quenching–exposed Fluorines. Accordingly, Ksv for 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole as the slope of the Io/I plot is different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole were obtained. In order to study the second structure change, after the interaction of single–stranded DNA/RNA with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, CD spectroscopy was used. CD study in the Near–UV region and preferably in the wavelength range of 200–320 (nm) and using AVIV model 215 spectropolarimeter with a quartz cuvette with a width of 1 cm for the near region and a cuvette with a width of 1 mm for It was carried out in the remote area under a continuous flow of nitrogen gas and at a temperature of 23 degrees Celsius. The spectrum of interaction samples from the spectrum of standard samples of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole anti–cancer Nano drug that were drawn under the same conditions, the fraction and data under the title (Molar Ellipticity) and in the form of [?]: deg×cm2×dmol–1 were reported (Figures 1–9).

Figure1: Modelling of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure2:Molecular dynamics simulation of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure3:Binding affinity performance for Nano synthesized (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure 4: Pharmacokinetics (PK) of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure5: Pharmacodynamics of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure 6:Quantitative Structure Activity Relationship (QSAR) and Quantitative Structure Properties Relationship (QSPR) of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure 7: Toxico and enzyme kinetics of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

Figure8: Effect of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on DNA/RNA in human cancer cells.

Figure9: Interaction of (a) 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and (b) 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with DNA/RNA in human cancer cells.

Absorption spectroscopy is one of the suitable methods for studying the structure of macromolecules. In order to investigate the spectroscopic properties of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and 3, 5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, first the absorption spectrum of this compound was drawn between 200 and 700 (nm) wavelength. The absorption spectrum of this anti–cancer Nano drug has a long absorption peak at 210 (nm) and several short absorption peaks at 208, 280, 320 and 439 (nm). Therefore, according to the absorption spectrum, the wavelength of 210 (nm) was chosen as the absorption index of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and 3, 5–bis–(4–chlorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole.In order to investigate the absorption changes of the samples incubated with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and 3, 5–bis–(4–chlorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole, UV/vis absorption spectrometry was used. Absorption spectroscopy is a useful method for studying the structure of different macromolecules, and on the other hand, this method is one of the simplest and most common methods for studying ligand–macromolecule interaction. First, the amount of absorption of standard samples of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole in each concentration is subtracted from the amount of absorption of samples treated with the same concentration of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and the graph of changes in absorption of single–stranded DNA/RNA according to concentration. Obtained results show the changes in the absorbance of solutions resulting from the interaction of the anti–cancer Nano drug 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA at wavelengths of 260 (nm) (A)) and 210 (nm) (B) and as can be seen, the changes in single–stranded DNA/RNA absorption follow a similar process. In a comparative way, the changes in the absorbance of single–stranded DNA/RNA after incubation with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at a wavelength of 260 (nm). In different concentrations, the amount of absorption of single–stranded DNA/RNA increases, and in higher concentrations, absorption also increases. While the decrease in absorption due to anti–cancer Nano drug interaction with DNA/RNA was not observed in any wavelength. As a result of the interaction of the anti–cancer Nano drug 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA at a wavelength of 210 (nm) in the presence of all concentrations of the anti–cancer Nano drug, absorption shows an increasing trend gives A closer observation shows that in low and high concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, the absorption rate increases in different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.Next, in order to investigate the interaction of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA, the fluorescence emission spectrum method was used. For this purpose, a certain concentration of single–stranded DNA/RNA was incubated separately with different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole in the dark and at a temperature of 23 degrees Celsius for one hour and after the end of the time, the sample control and treated samples were excited at 258 (nm) wavelength and their absorption spectra were drawn. Then, the amount of fluorescence absorption of the standard samples of the anti–cancer Nano drug at each concentration was subtracted from the amount of absorption of the samples treated with the anti–cancer Nano drug at the same concentration, and the graph related to single–stranded DNA/RNA was drawn in the range of 500–550 (nm).At an excitation wavelength of 258 (nm) and in the absence of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, single–stranded DNA/RNA has an absorption peak at 520 (nm), which corresponds to DNA/RNA chromophores (DNA/RNA bases) and by increasing the concentration of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, the absorption rate of single–stranded DNA/RNA chromophores increases without any shift in the spectrum of single–stranded DNA/RNA. According to the fluorescence results, the I/Io×100 Io curve related to single–stranded DNA/RNA was also drawn against different anti–cancer Nano drug concentrations. Io is the fluorescence emission intensity of single–stranded DNA/RNA sample in the absence of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and I is the fluorescence emission intensity of single–stranded DNA/RNA sample in the presence of different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole. It can be seen that at a low concentration of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole (12.50 μg/ml), the affinity of the anti–cancer Nano drug for single–stranded DNA/RNA is almost low, and at higher concentrations (50–100 μg/ml) ml) of single–stranded DNA/RNA shows a greater affinity for 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole. Stern–Volmer diagram shows the quenching effect of the anti–cancer Nano drug 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole. The slope of this graph is the Stern–Volmer constant, which is expressed in molar terms, and its value for single–stranded DNA/RNA is equal to 1.42×103 M^–1, which indicates that the anti–cancer Nano drug tends too much like single–stranded DNA/RNA. Obtained results show the binding constant of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole to single–stranded DNA/RNA M^–1 = 68.35×103Ka and the number of binding site n = 72.In order to study the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on the second structure of single–stranded DNA/RNA molecule, the circular dipole (CD) method was used in the near ultraviolet range. For this purpose, single–stranded DNA/RNA was incubated with different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at room temperature and away from light for one hour. After a certain time, the CD spectrum of the samples was drawn in the absence of single–stranded DNA/RNA. Then the CD spectrum of different anti–cancer Nano drug concentrations in each concentration was subtracted from the spectrum of samples treated with 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole at the same concentration. As seen in the Figures (1–9), the spectrum of single–stranded DNA/RNA in the absence of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole has two positive regions, one at 220 (nm) and the other at 275 (nm), and a negative region at 245 (nm). Increasing the concentration of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole affects the ellipticity of the single–stranded DNA/RNA molecule in all regions including at 275, 245 and 220 (nm). At 275 (nm) and in the presence of the anti–cancer Nano drug, the ellipticity increases and shifts to shorter wavelengths. At 245 (nm), with the addition of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, the ellipticity becomes more positive and the graph remains unchanged in the wavelength, and finally, at 220 (nm), in addition to increasing the ellipticity, there is some shift towards shorter wavelengths.

The anti–tumor activity of natural and synthetic oxadiazole derivatives has been widely investigated and it has been proven that oxadiazoles, depending on their structure, can act on different tumor cells by different mechanisms of action such as telomerase enzyme inhibition, protein activity inhibition Kinase and downregulation of oncogene expression also induce caspase mediated apoptosis, and suppress cancer cell proliferation by arresting the cell cycle in the G0/G1 phase, G2/M phase. DNA/RNA in the nucleus of eukaryotic cells is naked during replication and may be targeted by many different compounds or enzymes, but it is not naked in the usual state and is connected to histone and non–histone proteins, which together make the nucleoprotein composition Creates the name chromatin. On the other hand, in the structure of chromatin, DNA/RNA forms complex histone octamer and nucleosome. The DNA/RNA compounds in the cell nucleus are among the most important targets of many anti–cancer Nano drugs after they enter the cell nucleus, and so far, many studies have been conducted on the effect of anti–tumor Nano drugs on the structure of chromatin and DNA/RNA. There is no information about the effect of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole on single–stranded DNA/RNA, and there is no available data about the mentioned compound on DNA/RNA structure. For this reason, it was decided that the effect of this anti–cancer Nano drug on soluble single–stranded DNA/RNA was investigated and the results of its interaction with single–stranded DNA/RNA were investigated. For this purpose, a constant concentration of single–stranded DNA/RNA with different concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole was incubated at room temperature and away from light, and after the desired period of time, various spectroscopic studies were performed on them. The results obtained from the increase in absorption of single–stranded DNA/RNA at 210 and 260 (nm) depend on the concentration and indicate the participation of phosphate groups and DNA/RNA bases in the interaction with the anti–cancer Nano drug. At high concentrations of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, the structure of single–stranded DNA/RNA is opened and as a result, the absorption rate increases. The increased intensity of absorption in the case of single–stranded DNA/RNA indicates the greater tendency of the anti–cancer Nano drug to bind to single–stranded DNA/RNA. Fluorescence spectroscopy is more efficient, sensitive and more complex than absorption spectroscopy. Using the fluorescence spectroscopy method, important information about the macromolecule structure can be obtained. The increase in the fluorescence intensity of single–stranded DNA/RNA indicates the opening of DNA/RNA chromophores in the presence of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole, and the linearity of the Stern–Volmer curve also confirms this. The I?–I/I?×100 curve of single–stranded DNA/RNA also shows the high affinity of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole to single–stranded DNA/RNA. On the other hand, the opening of single–stranded DNA/RNA chromophores can be attributed to the intercalation of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4, 5–dihydro–1, 2, 4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole inside these structures. In a study where oxadiazole derivatives such as 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole 35 (Ksv=25.61×103 M^–1) were performed on bovine serum albumin (BSA) and also in another study that investigated another oxadiazole derivative called 4–methyl–7–hydroxy oxadiazole was studied in the presence of BSA and HSA, Ksv is equal to 1.80×103 M^–1 and 4.97×103 M^–1, respectively, which means that 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole (M^–1 = 103×42/1Ksv) has a lower Stern–Volmer equation constant than these oxadiazole derivatives. In another study where the effect of oxadiazole was performed in the presence of ethidium bromide and acridine orange, the Ksv was 0.21×103 M^–1 and 0.56×103 M^–1, respectively, which is the Ksv corresponding to 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole are less. CD spectroscopy is a powerful method in studying the conformational properties of single–stranded DNA/RNA molecules and is used in studying the interaction of nucleic acids with proteins and ligands. The change in the ellipticity of the DNA/RNA molecule in the region of 245 (nm) indicates the change in the orientation of B–DNA/RNA. In addition, anti–cancer Nano drug interaction at 220 and 275 (nm) causes an increase in ellipticity. In general, the changes at 220, 245 and 275 (nm) indicate the effect of the anti–cancer Nano drug on the stacking of bases, which causes a change in the B conformation in the DNA/RNA structure and a decrease in the second structures of the DNA/RNA molecule, and possibly changes B–DNA/RNA to A or C–DNA/RNA(Figure 10).

Figure 10: Raman spectra of DNA/RNA linked to 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole in (a and b) human normal cells and (c and d) human cancer cells.

According to the results of the interaction of 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole with single–stranded DNA/RNA, which indicates the high tendency of the anti–cancer Nano drug to bind to single–stranded DNA/RNA, the involvement of single–stranded DNA/RNA is probably one of the most important The most important targets in chromatin structure are 3–(4–chlorophenyl)–5–(4–fluorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole and 3,5–bis–(4–chlorophenyl)–4–phenyl–4,5–dihydro–1,2,4–oxadiazole.

This study was supported by the Cancer Research Institute (CRI) Project of Scientific Instrument and Equipment Development, the National Natural Science Foundation of the United Sates, the International Joint BioSpectroscopy Core Research Laboratory (BCRL) Program supported by the California South University (CSU), and the Key project supported by the American International Standards Institute (AISI), Irvine, California, USA.

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