Description:Field of Invention:
The present invention discloses the DNA protection ability of the newly synthesized bis-pyrazoles under UV-irradiations. Among them, 1a-j depicted concentration dependent protection of supercoiled form of DNA as compared to control (C) in which DNA is converted into open circular form supercoiled form.
Background of the invention:
Genetics is usually controlled by nucleic acids (complex macromolecule) in living organisms. Secrets of cellular metabolism like cellular functioning and abnormalities were solved by discovery of deoxyribonucleic acid. Genes that indirectly involved in coding of proteins which are building blocks for cellular systems are essential part of DNA (Biochem. Mol. Bio. J. 2018, 4, 5; Bioresour. Bioprocess. 2019, 6, 2). Genetic information, disease, and defects depend on types of DNA, their structure and functions in human body. Functionalities in DNA were modulated by several factors like environmental, synthetic chemicals, UV-rays, and genetic defects which are posing threat at cellular level which results in noticeable changes in living organisms which may lead to certain kinds of diseases (Chem. Biol. Interact. 2018, 279, 73–83). During cellular exposure to radiations communication mechanism plays pivotal role (Human. Exp. Toxicol. 2004, 23, 81–86). Various genetic defects occurred due to continuous or long time exposure to damaging factors. Nowadays, radiations are used for treatment of various diseases for betterment of human race (Br. Med. Bull. 2003, 68, 259–275). In contrast, exposure to radiations have deteriorating effects that result in early aging problem, DNA damage and cellular death at some levels (Radiat. Res. Appl. Sci. 2015, 8, 247–254). Radiations are powerful factors which may show adverse effects even at DNA level. Cellular defense mechanism is initiated by body when someone exposed to radiations; (1) apoptosis phenomenon (elimination of dead and old cells), (2) dismutase and catalase activation (detoxifying enzymes), (3) antioxidants production (Radiat. Res. 1992, 131, 117–123; Dose. Response. 2008, 6, 333–351). Moreover, serious disorders such as retinal degradation, hyperpigmentation, skin cancer, cataract formation, edema and erythema due to generation of reactive nitrogen species and reactive oxygen species are deteriorating effects of UV-irradiation (Micron. 2002, 33, 179–187; J. Photochem. Photobiol. 2014, 136, 12–18). For betterment of living systems it would be important to develop DNA-protective agents. Therefore, in continuation of our work to synthesize bioactive heterocyclic motifs, we developed new derivatives of bis-pyrazoles as DNA protecting agents.
Summary of the invention:
The present invention discloses the DNA protective screening new Bis-pyrazole derivatives with DNA. It was evident that at a very low concentration (25 µg) and (50 µg) all of the synthesized compounds protected the supercoiled form of DNA under UV-irradiation.
Detailed description of the invention:
To explore the versatility of the synthesized compounds (Fig. 1), DNA damage protecting potential against pBR322 DNA has been evaluated.
DNA protecting activity

Fig. 1. Various substituted bis-pyrazole derivatives (1a-j)

All the synthesized compounds 1a-j were examined against plasmid DNA using agarose gel electrophoresis method under UV-irradiation.
It was evident from literature that upon single strand breaking, DNA transforms from supercoiled form (SC) to open circular form (OC) which is more relaxed form, in contrast, while on double strand breaking it will change into liner form (LC). It was revealed from this investigation that in absence of UV-irradiation, it will exist in supercoiled form (lane 1) whereas in case of UV light, plasmid DNA is completely converted into OC form as shown in lane 2. All test compounds potential was compared with bands appeared in control and test samples in presence of UV-irradiation. Fig. 1 represents the outcome of agarose gel electrophoresis.
It was observed that all the compounds have tendency to protect supercoiled form of DNA at 50 µg concentration under UV-irradiation as compared to control which completely degraded the supercoiled form (Fig. 3). However, 1c, 1d and 1h shown photocleavage effect also but intensity of OC form is negligible as compared to band intensity in control. At lower concentration 25 µg (Fig. 2), all compounds exhibited equipotent DNA protective efficacy as compared to band intensity at 50 µg concentration. However all of the compounds protected the supercoiled form even at lower concentration as intensity of SC form was comparable to untreated control (A). Therefore, all of the compounds protected the supercoiled form of DNA from 50 µg to 25 µg concentration as compared to control (C).

Fig. 2. Effects of compounds 1a–j (25 µg) on plasmid DNA under UV-irradiation: lane 1 DNA + DMSO without UV, lane 2 DNA + DMSO + UV, lane 3 DNA + 1a + UV, lane 4 DNA + 1b + UV, lane 5 DNA + 1c + UV, lane 6 DNA + 1d + UV, lane 7 DNA + 1e + UV, lane 8 DNA + 1f + UV, lane 9 DNA + 1g + UV, lane 10 DNA + 1i + UV, lane 11 DNA + 1h + UV, lane 12 DNA + 1j + UV

Fig. 3. Effects of compounds 1a–j (50 µg) on plasmid DNA under UV irradiation: lane 1 DNA + DMSO without UV, lane 2 DNA + DMSO + UV, lane 3 DNA + 1a + UV, lane 4 DNA + 1b + UV, lane 5 DNA + 1c + UV, lane 6 DNA + 1d + UV, lane 7 DNA + 1e + UV, lane 8 DNA + 1f + UV, lane 9 DNA + 1g + UV, lane 10 DNA + 1i + UV, lane 11 DNA + 1h + UV, lane 12 DNA + 1j + UV
Outcome of the Invention:
All of the synthesized compounds act as DNA protective agents. Effectiveness of compounds is concentration dependent and considered suitable drug candidates or material in near future as UV protective agents in sunscreens to protect skin cancers. Toxicology study is under progress.
Highlights of the Invention:
? Explored new bis-pyrazole derivatives as new DNA protective agents.
Experimental:
Treatment of DNA with the samples
The synthesized chemical compounds (1a-j) were dissolved in DMSO to prepare a stock solution of 10 mg/ml. Different concentrations of each compound (1 µg-50 µg) from the above stock solutions were mixed with plasmid DNA and treated with UV using UV-illuminator at 360 nm for 30 minutes at room temperature. Two controls were maintained; Plasmid (A): plasmid in Tris EDTA buffer, and DMSO + UV control (C): plasmid mixed with DMSO followed by UV treatment, to clearly observe the effects of different compounds on plasmid DNA under UV treatment. After UV treatment, the treated plasmid DNA and the controls were incubated at 37? for 1 hour.
Agarose gel electrophoresis
Agarose gel (1% wt./vol.) was prepared by mixing 1 g agarose in 100 mL of 1X Tris-acetate EDTA (TAE) buffer and heated to dissolve the agarose. After cooling down to 55?, ethidium bromide with a final concentration of .5µg/ml was mixed and gel was cast in a tray fitted with a comb. After gel solidification, gel was placed in electrophoresis chamber and flooded with 1X TAE buffer followed by careful removal of the comb. Treated plasmid DNA and controls were mixed with loading dye (0.25% bromothymol blue and 30% glycerol). Each well was loaded with 9 µL of the above mixture and gel electrophoresis was carried out at 90 mV for 1 hour. The gel was visualized using InGenius3 Gel Documentation System (Syngene, UK).
, Claims:I/We claim

1. The compounds of the formula I, where Ar is C6H5, p-F-C6H4, p-Cl-C6H4, p-Br-C6H4, p-CH3-C6H4, p-CH3O-C6H4, p-NO2-C6H4, p-COOH-C6H4, o-CH3O-C6H4 and o-NO2-C6H4 as DNA protective agents at different concentrations.

2. The compounds as claimed in claim 1, wherein the said compounds as claimed in claim 1, acted as DNA protecting agents at 25 µg to 50 µg concentration for effective protecting supercoiled form of DNA.