Design, Synthesis, Characterization, Computational Study and In-vitro Antioxidant and Anti- inflammatory Activities of Few Novel Pyrazol-3-one Derivatives

Aim: To design, synthesize and perform computational study on a few Novel pyrazol-3-one derivatives. Study design: Experimental study. Methodology: A series of 6-aryl substituted pyrimidine azodyes were synthesized by coupling phenyl pyrimidine 2-amine with different aromatic amines. The synthetic compounds were screened for their in-vitro antioxidant and anti-inflammatory activities. The Computational study of Original Research Article Unnisa et al.; JPRI, 33(28B): 220-231, 2021; Article no.JPRI.68692


INTRODUCTION
Heterocyclic compounds are cyclic compounds that have atoms of at least two different elements as members of their ring [1]. A pyrazolone is a group of heterocyclic compounds having great importance because of their broad spectrum of biological activities and their wide-ranging use as synthetic tools. Pyrazolone is a five-membered lactam ring containing two adjacent nitrogens and a keto group in its structure [2].
Pyrazolone is an interesting template for medicinal, and combinatorial chemistry because of its simple synthesis and wide range of biological activities which include The literature review revealed the studies conducted for various pharmacological activities of pyrazoles such as cytotoxic activity [3,4,5], antimicrobial activity [6,7,8], antioxidant activity [3,9], anti-Inflammatory activity [10,11], analgesic activity [12,13], Sars-Corona virus 3c-Like protease inhibitors [14], hypoglycemic activity [15,16], antiviral activity [17,18]. Pyrazolone was first reported by Knorr in 1883 who also reported its derivative-Antipyrine which was approved for clinical use. Many other NSAIDs"s like oxyphenbutazone, phenylbutazone, propyphenazone, aminophenazone, etc. contain pyrazolone as their basic ring and are widely used as anti-inflammatory, analgesic, and antipyretic drugs. However, the clinical use of pyrazolones is not free of controversies because of their association with serious side effects like gastrointestinal irritation, ulceration, etc. but their benefits and a range of clinical applications have kept the interest of the research community alive.

The method employed was one-pot synthesis (Knovenegal condensation)
An equimolar mixture of hydrazine dichloride, ethyl acetoacetate, acetic anhydride, acetic acid, and aromatic aldehyde was taken in a conical flask and stirred with a magnetic stirrer. The temperature of the reaction mixture was maintained at 60º c. The reaction mixture formed after continuous stirring for 30 mins was transferred into a beaker containing crushed ice and neutralized with sodium bicarbonate. The product was precipitated and the precipitate was filtered, washed, dried, and weighed. The obtained products were recrystallized with methanol. The progress of the reaction was assessed by thin-layer chromatography and the purity of the compounds was ensured by melting points and thin-layer chromatography. The compounds synthesized were characterized by spectroscopic techniques [18]. yes, Rf value: 0.7, IR data: FTIR (γ max, cm-1), 1702(-C=O stretch), 1552(-NO 2 ), 1519(-C=N), 1200(-C-N stretch).

Molinspiration [18]
Molinspiration offers a broad range of Cheminformatics software tools supporting molecule manipulation and processing, including SMILES and SD file conversion, normalization of molecules, generation of tautomer's, molecule fragmentation, calculation of various molecular properties needed in QSAR, molecular modeling and drug design, high-quality molecule depiction, molecular database tools supporting substructure and similarity searches. Our products support also fragment-based virtual screening, bioactivity prediction, and data visualization.
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Data warrior [21]
Data Warrior combines dynamic graphical views and interactive row filtering with chemical intelligence. Scatter plots, box plots, bar charts, and pie charts not only visualize numerical or category data but also show trends of multiple scaffolds or compound substitution patterns. Chemical descriptors encode various aspects of chemical structures, e.g. the chemical graph, chemical functionality from a synthetic chemist's point of view, or 3-dimensional pharmacophore features. These allow for fundamentally different types of molecular similarity measures, which can be applied for many purposes including row filtering and the customization of graphical views.
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OCHEM [22]
The Online Chemical Modeling Environment is a unique and web-based platform on the Web that aims to automate and simplify the typical steps required for QSAR modeling. The platform consists of two major subsystems: the database of experimental measurements and the modeling framework. The database is user-contributed and contains a set of tools for easy input, search, and modification of thousands of records.
The OCHEM database is based on the wiki principle and focuses on data quality and verification. The database is tightly integrated with the modeling framework, which supports all the steps required to create a predictive model: data search, calculation, and selection of a vast variety of molecular descriptors, application of machine learning methods, validation, analysis of the model, and assessment of the applicability domain.

Swiss ADME [23]
A free web tool to evaluate pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small molecules. The software used in the processes of drug discovery and development where a large number of molecular structures are evaluated according to very diverse parameters to steer the selection of which chemicals to synthesize, test, and promote, with the final goal to identify those with the best chance to become an effective medicine for the patients.
The ADME parameters (for Absorption, Distribution, Metabolism, and Excretion) can be evaluated separately by dedicated methods. It has been demonstrated that early estimation of ADME in the discovery phase reduces drastically the fraction of pharmacokinetics-related failure in the clinical phases. Computer models have been fostered as a valid alternative to experimental procedures for the prediction of ADME, especially at initial steps, when investigated chemical structures are numerous but the availability of compounds is scarce.
The Swiss ADME web tool is freely accessible and meant for user-friendly submission and easy analysis of the results, also for a non-expert in CADD. Swiss ADME includes different input methods, computation for multiple molecules, and the possibility to display, save and share results per individual molecule or through global intuitive and interactive graphs. Finally, Swiss ADME is integrated into the Swiss Drug Design workspace Swiss target Prediction has been primarily developed for identifying targets of molecules known to be bioactive. Swiss target Prediction will suggest some targets, based on the assumption that if the molecule is active, it will likely bind to some protein. For molecules with unknown bioactivity, this assumption is not valid per the molecule may not bind to any protein, in which case all predicted targets are false positives. In particular, inactive compounds can sometimes exhibit good similarity with active molecules if they have been obtained by modifying an active compound at some key position that was crucial for its interactions. This is a known limitation of ligand-based approaches when applied to any kind of compounds and therefore target predictions should be interpreted with care in the absence of indication of bioactivity.

In-Vitro
Study of the Pharmacological Activities

Anti-inflammatory activity: (By Heat hemolysis method) [24,25]
In-Vitro Anti-Inflammatory Activity was performed using the human red blood cell (HRBC) membrane stabilization method. The blood was collected from a healthy human volunteer who had not taken any non-steroidal antiinflammatory drugs for 2 weeks before the experiment and mixed with saline solution and centrifuged at 3,000 rpm. Various concentrations of compounds were prepared (100,250 and 500 μg/ml) using methanol to obtain 3ml and 3 ml of HRBC suspension were added. It was incubated at 60 for 30 min and centrifuged at 3,000 rpm for 20 min. and the hemoglobin content of the supernatant solution was estimated on a UV spectrophotometer at 560 nm. Aspirin (100 and 250,500 µg/ml) was used as the reference standard and HRBC was used as control.
The % inhibition of hemolysis was determined using the below equation.

% inhibition = (O.D of Control-O. D of test)/ (O.D of control) X 100
IC-50 values were determined from the % Inhibition and were tabulated.

RESULTS AND DISCUSSION
The titled compounds (4E)-2-acetyl-4benzylidene-5-methyl-2, 4-dihydro-3H-pyrazol-3one derivatives (C1-C13) were synthesized by one-pot condensation of ethyl acetoacetate, hydrazine dichloride, and aromatic aldehydes under acidic condition. Thirteen compounds were synthesized by condensation reaction (figure 1) and were characterized by IR, NMR spectroscopic methods. The IR spectra revealed the presence of CH stretching, carbonyl peak in the range 1650-1700 -1 , C=N peak between 1500-1550 cm -1 . NMR spectra revealed the presence of aromatic protons and the presence of C=C protons in the region between 6.5-8 ppm.

Fig. 1. Scheme and structures of the synthesized (4E)-2-acetyl-4-benzylidene-5-methyl-2, 4dihydro-3H-pyrazol-3-one derivatives
From the in-silico evaluation of the synthesized compounds, it was observed that compounds obey Lipinski rule of five with high saturation and lower values of flexibility, size, polarity, and solubility and found to be active inhibitors' of kinase, GPCR ligand, and enzyme targets.
The concept of a BOILED egg representing the Brain or Intestina L Estimate D permeation method (Fig. 2) represented that the synthesized compounds can penetrate the blood-brain barrier and are not substrates of PgP carrier.      CYP3A4  CYP2D6  CYP2C19  CYP2C9  CYP1A2  1  Active  -----2  Active  -----3  Active  -----4  Active  -----5  Active  -----6  Active  -----7  Active  -----8  Active  --+  -+  9  Active  -----10  Active  -----11  Active  -----12  Active  -----13 Active The scavenging effect of a chemical by the DPPH radical assay method is a quick and reliable parameter to assess the in vitro antioxidant activity. All compounds under this study were effective scavengers of free radicals except the compounds 1, 2, 6, 10. The result has shown that the free radical scavenging activity of these compounds was concentration-dependent. It could be seen that all compounds of the present study are in agreement with Lipinski's Rule of Five, which is important for the further development of these synthesized drugs and their analogs. Furthermore, the replacement of the ring with aromatic phenol sharply enhanced the antioxidant potency. The antioxidant efficacy depends strongly on its reducing property, the compounds 11, 12 might have the higher reducing potential. Due to this extra stabilization, radicals obtained from compounds 11 and 12 would have a higher aptitude to trap free radicals at a faster rate than the other similar type of molecules [26]. The results obtained from anti-inflammatory Invitro studies have shown that the compounds (6,7,8,9,13) active toward heat hemolysis (Table  4).

CONCLUSION
The findings of the study inferred the design, functioning, and target identification of synthesized compounds rendering them as lead molecules for further development of newer agents with greater efficacy and safety.