Synthesis of some novel 3- (4-ethoxyphenyl) -5- (4-substituted) -4,5-dihydro-1H-pyrazole derivatives as potent antioxidant agents

Pyrazolines are the most useful heterocyclic moiety in Pharmaceutical and Chemical ields and as the most potential molecules for the design of new chemical entities. Nitrogen-containing heterocyclic compounds, pyrazolines and their derivatives showed a variety of pharmacological activities, including antioxidant properties. In the present study, eleven novel ethoxylated pyrazoline derivatives were synthesized by condensing chalcones with electron releasing ethoxy group at one end and different electron-donating, electronwithdrawing groups in another end with hydrazine hydrate and alcohol. The compounds synthesized were structural elucidated by their spectroscopic studies. All the compounds synthesized were evaluated for their in vitro antioxidant potential by 2,2’-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide free radical scavenging assaymethods. Some of thesemolecules possessmoderate to good antioxidant activity when compared to standard ascorbic acid. The compound with methoxy group (EH2) exhibits potent antioxidant activity with IC50 value of 9.02 and 9.44μg/ml in DPPH and hydrogen peroxide assay methods respectively and the compound with hydroxy group (EH9) also showed potent antioxidant activity with IC50 value of 12.41 and 14.56μg/ml in DPPH and hydrogen peroxide free radical scavenging assay methods respectively when compared to standard. The compounds containing electron-donating substituents were found to be good antioxidants when compared to standard ascorbic acid.

Generation of free radicals from the reactions of the imbalance between oxidative stress and antioxidant homeostatic phenomenon, which can leads to many pathological conditions in the body (Ashok and Tiwari, 2001). An elevated level of oxidative stress is one of the reasons for many diseases. Many biological damage due to the reactions of free radicals like H 2 O 2 , hydroxyl radicals (OH), oxygen anions (O 2 ) free radicals, which leads to the process of chain reactions. Much reactive free radical plays a major role in the pathological condition of many diseases like heart disease, cancer, diabetes, strokes, Parkinson's diseases and allergies (Süzen, 2007).
The electron-donating CH 3 cluster substituted in the second position of 2-pyrazoline showed greater antioxidant property, whereas the electronwithdrawing Group (EWG) and high electrondonating groups were not increase free radicals inhibition . Indole based pyrazole with an electron-donating group (EDG) has been proved increased antioxidant activity then EWG (Sharath et al., 2013). The presence of 2-quinolone with EWG in a pyrazoline ring showed increased antioxidant activity . Nitrogen-containing heterocyclic compounds with the electron-withdrawing nature of halogen groups showed greater antioxidant property (Hossain et al., 2009).

Figure 1: Graphical abstract of the study
On the basis of these considerations, present research work was done to synthesize 1H-pyrazole derivatives with various EDG and EWG and evaluate them for antioxidant potential by DPPH & H 2 O 2 assay methods. Graphical abstract of the present study was given in Figure 1.

Chemicals and reagents
Analytical grade of reagent & chemicals were used in this study. All para-substituted aldehydes, hydrogen hydrate and other chemicals were procured from Sigma-Aldrich private limited.

Synthesis and characterization of ethoxylated pyrazolines
Eleven compounds of ethoxylated chalcones were prepared and evaluated their biological activities and reported (Lakshminarayanan et al., 2019). Continuation of the research, all the titled compounds (EH1 -EH11) were obtained by condensation of appropriate chalcones (E1 -E11) with 1.35 ml of hydrazine hydrate in the presence of 60 ml methanol and 15 ml acetic acid. The above mixture re luxed gently in a water bath for 6 -8 hours. Then cool and acidi ied with conc. HCl. The product obtained was iltered and washed with water and recrystallized by ethanol. The open capillary method was used to determine the melting point and they were uncorrected. The purity was checked with TLC (hexane: ethyl acetate, 3:7) and melting point test and the route for the synthesis of titled molecules were given in Scheme 1. All prepared molecules were characterized by their spectral studies.

In vitro antioxidant activity DPPH free radical scavenge activity
All titled molecules were selected for their in vitro antioxidant potential by scavenging of DPPH free radical (Kaushik et al., 2016). Standard ascorbic acid and synthesized compounds were prepared in different concentrations from 10 to 50 µg/ml from the stock solution (100µg/ml) with distilled carbinol. In an individual test tube, one ml of each compound solution was taken; 4 ml of 0.004% methanol solution of DPPH was added and shaken vigorously. Keep the mixtures in a dark room for 30 minutes at room temperature and measure the absorbance of all the solution at 517 nm using a UV-VISIBLE spectrophotometer (Shimadzu UV-1800). Percentage radical scavenging activity was calculated with the formula [(Ab 0 -Ab 1 )/Ab 0 ] x 100, where Ab 0 is the absorbance of blank, Ab 1 is the absorbance of synthesized compounds/standard. The calibration curve graph was plotted between percent inhibition and concentrations of the test/standard to get the amount of antioxidants need to decrease 50% from the starting concentration of DPPH free radicals. Half maximal inhibition values were calculated from the calibration curve. (Malladi et al., 2014;Kaushik et al., 2015).

Hydrogen peroxide free radical scavenging activity
Hydrogen peroxide free radical scavenging activities of titled compounds were done by the described method with a small modi ication (Babu et al., 2001;Jayaprakasha et al., 2004). Solutions of 20 mM hydrogen peroxide were prepared in phosphate buffer saline (PBS) and adjust the pH to 7.4. Standard ascorbic acid and synthesized compounds were prepared in different concentrations from 10 -50 µg/ml with distilled carbinol. Each compound (1 ml) was taken in test tubes and adds 2 ml of H 2 O 2 solution to all test tubes. Absorbance was measured at 230 nm after 10 minutes against a blank. The % scavenging activity and IC 50 values measured and intended by using the formula mentioned in the DPPH assay method.

RESULTS AND DISCUSSION
All the newly synthesized compounds were conirmed by spectroscopic studies.

In vitro antioxidant potency
Newly synthesized compounds (EH1 -EH11) are subjected to evaluate for antioxidant activities by  All the synthesized compounds contains electrondonating -O-CH 2 -CH 3 group at one end and different electron-donating and electron-withdrawing groups at another end. Antioxidant potentials of all compounds were in luenced by the substituent on an aromatic ring in each compound. The better antioxidant activities of EH2 and EH9 may be owing to the presence of electron giving nature of -OCH 3 and -OH group. On the other hand, the introduction of electron retreating substituents like -Cl, -F, -Br, -CF 3, -NO 2 in compounds EH6, EH7, EH8, EH11 and EH5 has led to the lower the activity when compared with standard. Antioxidant activity of all the compounds may be owed to the presence of electron giving -O-CH 2 -CH 3 on one of an aromatic circle in all compounds.

CONCLUSIONS
In conclusion, a new class of nitrogen-containing heterocyclic compounds with the ethoxy group were prepared and screened for their antioxidant potency. Among the compounds, EH2 and EH9 showed better antioxidant activity by both assay methods. Compounds with methoxy and hydroxy groups possess good antioxidant activities when compared to standard.