Microwave Assisted Synthesis, Biological Evaluation and In-silico Molecular docking and Pharmacokinetic Studies of Novel Heterocyclic Hybrid Dihydropyrazol-1-yl-2-(Quinolin-8-yloxy) Derivatives

A novel heterocyclic hybrid dihydropyrazol-1-yl-2-(quinolin-8-yloxy) derivatives were synthesized. By reacting 8-hydroxyquinoline 1 in absolute ethanol with potassium carbonate followed ethyl chloroacetate by conventional and microwave irradiation methods, precursor 2 were synthesized, further treated with hydrazine hydrates to achieve precursors 3 and by Claisen-Schmidt condensation methods, different chalcone derivatives 6(a-g) were prepared and to these precursors 3 was then cyclized to give the target compounds 7(a-g). The structures of the target compounds were evaluated by the spectral and elemental method of analysis. The screened compounds 7a and 7g produced better action than other compounds but somewhat lesser than the standard drugs. Furthermore molecular docking studies have been carried out against PDB codes: 5MTX (p38 MAPK) using Biovia Discovery Studio 2021. The compounds 7a (-8.947 Kcal/mol) and 7g (-8.994 Kcal/mol) had shown remarkable binding affinities which were greater than the binding affinity of recommended drugs; diclofenac sodium (-6.8 kcal/mol), and nimesulide (-7.8 kcal/mol). Original Research Article Singh and Manna; JPRI, 34(26B): 16-31, 2022; Article no.JPRI.84894 17


INTRODUCTION
Quinoline or 1-aza-naphthalene or benzo [b]pyridine is a nitrogen-containing heterocyclic aromatic compound [1]. The quinoline nucleus possesses an extensive range of biological activities such as antimalarial, [2] anti-inflammatory, [3] antibacterial, [4] and anticancer agents, [5] and this brings it to intense interest and essential aspect in the field of synthetic and medicinal chemistry [6]. Molecular docking studies have been widely explored to understand and predict the protein/enzyme interaction with the synthesized moieties to visualize the probable interaction with amino acids of the target site. This will give insight advantage in the clinical stage for finding potential moieties, and it has been the core of heterocyclic chemistry by reducing the time and costs [7,8]. It exhibited free energy of binding value in Kcal/mole. Investigation of drug binding to mutated receptors and interpretation of conformation of drug-receptor complexes is nowadays regulating the development of safer drugs. Novel drug/drug candidates are needed urgently, and in silico simulation/virtual screening, can be vigorous and fast predictions to scrutinize to yield individualized drugs conferring to pharmacogenomics desirable [9,10]. Mitogen-activated protein kinase (MAPK) is the crucial intermediaries of signal transduction in mammalian cells and is a group of profoundly conserved intracellular signaling molecules involving serine-threonine kinases that are consecutively phosphorylated by upstream kinases, mitogen-activated protein kinase kinase kinases (MAPKKK), and mitogen-activated protein kinase kinase (MAPKK). The MAPK signally peers extracellular signals to the intracellular progression that regulates growth, reproduction, relocation, and program cell death [11][12][13]. Inflammation is a serious constituent of cancer evolution. Various cancers originate from local infection, prolonged aggravation, and inflammation. The tumor ambient, fundamentally organized by inflammatory cells, is a requisite associate in the neoplastic proceeding, promoting proliferation, endurance, and voyage [ 14] . The activation p38 MAPK signalling pathway plays a vital character in the progress of tumours incursion, voyaging, and the outbreak of cancer cells. It has been associated with intricate living processes like cell reproduction, differentiation, mortality, voyage, and incursion.
The p38 MAPK works a binary nature as it modulates cell death and intercedes continuances of a cell to live or die not only on the activation type although also in the cell nature distinct mode. In summation to accentuating of the cell to live, an imperative task of p38 MAPK in cell movement and incursion management gives another possibility to target this pathway regarding cancer metastasis [15][16][17][18].
Synthesis using microwave-assisted development is a safe, non-toxic, environmental remediation, eco-friendly, cost-effective, and green chemistry method. The result of green synthesis by this route/ method is increasing in recent decades because of their various applications in the fields of bio-analytical or medicals, impurity detections, novel drug delivery systems, cancer therapy, water remediation, biomembranes, nanocapsules, gene modification, targeted therapy, biochipsdiagnosis systems, regenerative medicine, and molecular devices [19][20][21][22]. Exploring new drug discovery and development approaches in medicinal chemistry, heterocyclic hybridization gains more interest due to improved efficiency and affinity for the new targets. This new approach is based on the synthesis of two or more active compounds with known bioactive moieties. The present paper deals with the study protocol for the design and synthesis of some novel heterocyclic hybrid dihydropyrazol-1-yl-2-(quinolin-8-yloxy) scaffold.

MATERIALS AND METHODS
All the chemicals, solvents, and reagents used in the synthesis were acquired from Sisco Research Laboratories Pvt. Ltd. (SRL)-India, Fisher Scientific (India), ACROS ORGANICS manufacturers, SD-FINE Chem Limited (India) and Merck (India) and were used without further purification due to ISO certified, FDA approved and GMP certified. The purity of the compounds was determined by using Silica Gel coated aluminium-backed TLC sheets. Method B: Microwave assisted synthesis of 8-Quinolinoxyacetic acid hydrazide, 2: Ethyl 2-(quinolin-8-yloxy) acetate (0.01mol.) 2 was dissolved in required amount of absolute alcohol and then added 0.04mol. of hydrazine hydrate into the 10 mL of glass reaction vial equipped with micro magnetic stirrer and caped tightly. The reaction vial was then placed into closed-vessel Anton Paar Monowave microwave synthesizer and set temperature at 95 o C and reaction was carried out for 4.5 min. After completion of the reaction, the reaction mixture was poured into ice-cold water. The product was collected by filtration and recrystallized from ethanol. The yields of compound 3 were 88%.

Procedure for the Synthesis of
Chalcone Derivatives, 6a-g (Scheme 1) [29] A solution of 2.19 g (0.055 mol.) of sodium hydroxide in 20ml of distilled water and 10ml of alcohol are taken into a 250ml bottle, lightly shielded with a cardboard disk, furnished by an adequate mechanical stirrer, and keep in a large container to allow cooling with broken ice. In the basic solution, 8.5 g (0.043 mol.) of pbromoacetophenone 4 was decanted; the bottle was rapidly surrounded with cracked ice and constant stirring. To these 0.043 mol. of substituted aromatic aldehydes 5 was then added to prepared different chalcone derivatives. The temperature of the mixture was maintained between 15°C to 30°C during the reaction. After two to three hours, the mixture becomes so thick that no more in effect against stirring. The stirrer was later stopped, and the mixture was kept in an icebox for nearly ten hours. The mixture composed a thick paste of small shot-like grains suspended in an almost transparent liquid. It was cooled in a chilled mixture, separated on a Buchner funnel, rinsed with water continuously until neutral to litmus, and ultimately washed with alcohol, which had earlier been reduced to 0°C. Subsequent air drying, the crude product was purified by recrystallization from alcohol. By this process, different chalcone derivatives, 6a-g, were prepared with yield of 57-87%.

Synthesis of Pyrazoline Quinoline
Derivatives, 7a-g (Scheme 1) [30,31] Method A: A solution of compound 3 (0.01 mol.) in 7ml acetic acid and with substituted chalcone derivatives, 6a-g (0.01mol.), was added with continuous stirring. The reaction mixture was then heated for 7 h. along with a mechanical stirrer. The reaction rate was examined by TLC (Mobile phase: methanol: acetone; 6:4). After completing the reaction, the subsequent reaction mixture was transferred toward smashed ice. The crude product obtained was filtrated, rinsed with water, dried, and recrystallized from alcohol to get the compounds 7a-g.
Method B: Microwave assisted synthesis of pyrazoline quinoline derivatives, 7a-g: Equimolar quantities (0.01 mol) of substituted chalcone derivatives, 6a-g was taken into a 10 mL of glass reaction vial equipped with micro magnetic stirrer and then added equimolar quantities (0.01 mol) of acetic acid then caped properly. The reaction vial was then placed into closed-vessel Anton Paar Monowave microwave synthesizer and set temperature at 132 o C and reaction was carried out for 17-28 min. After the end of the reaction, the reaction mixture was quenched in cracked ice, and solid was collected by filtration and dried at room temperature, recrystallized from ethanol to obtained pyrazoline quinoline derivatives 7a-g. The yields of pyrazoline quinoline derivatives 7a-g were obtained more than 53%.

In vitro Study
Anti-oxidant activity [32,33]: The synthesized compounds 7a-g was evaluated for in vitro antioxidant activities against 2,2-diphenyl-1picrylhydrazyl (DPPH). 1ml of 0.1mM of DPPH (2, 2 diphenyl-2-picrylhydrazyl) in methanol was added in 1ml of references standard (10µg/ml, 50µg/ml, 100µg/ml, 250µg/ml, and 500µg/ml) and test solution of different concentration (10µg/ml, 50µg/ml, 100µg/ml, 250µg/ml, and 500µg/ml). It was kept in dark for 30 minutes to protect from light and the absorbance was measured at λ-max 517nm in UV Visible spectrophotometer (SHIMADZU). The assay was performed in triplicate. Ascorbic acid was used as a reference standard. All solution was freshly prepared. The test solution was prepared by dilution with methanol from stock solution of 10mg/ml. The DPPH scavenging was measured by the following equation: Where, A 0 = absorbance of control (containing all measurement except test).
A t = absorbance of test solution. The percentage inhibition after 30 min was plotted against concentration; calculated data for antioxidant are presented as means ± SD of triplicate.
Anti-inflammatory activity [34,35]: All the target compounds were studied for in vitro antiinflammatory activity by albumin denaturation method. The standard and targeted compounds were dissolved in the least quantity of DMF and diluted to phosphate buffer saline (pH 7.4). The target compounds were studied for antiinflammatory activity by using the inhibition of the albumin denaturation method. The standard drug and targeted compounds were dissolved in the least quantity of DMF and diluted with phosphate buffer saline (pH 7.4) so that the concentration of DMF in all solutions was less than 2.5%. Targeted compounds solution (1 ml, 100 mg/ml) was blended with 1 ml of 1% albumin solution in phosphate buffer saline and incubated at 27 ± 1 o C for 15 min. Denaturation was influenced by retaining the reaction mixture at 60 ± 1 o C in a water bath for 10 min. After cooling, the turbidity was measured at 660 nm with a UV visible spectrophotometer. The percentage of inhibition of denaturation was calculated from control, where no drug was added. Each experiment was done in triplicate, and the average was taken. The diclofenac sodium was used as a standard drug. The percentage of inhibition was calculated using the following equation: Where, A t = absorption of test. A c = absorption of control.
p38 MAPK assay [36,37]: Nonradioactive immunosorbent p38 kinase activity assay method has been followed which was applicable for routine screening of small molecule p38 kinase inhibitors. In the method activating transcription factor-2 (ATF-2) was used as a substrate for phosphorylation, which showed linearity in 6-24 ng/well range, based on this 12 ng/well and 60 minute incubation period was optimized. Microtiter plates were coated with 50 µL/well of the p38 kinase substrate ATF-2 (10 µg/mL in TBS) for 1.5 h at 37 ˚C. After washing three times with distilled water, remaining open binding sites were blocked with blocking buffer (BB; 0.05% Tween 20, 0.25% BSA, 0.02% NaN 3 in TBS) for 30 min at room temperature. Plates were washed again, 50 µL of the respective test solution was filled into the wells and the plates were incubated for 1 h at 37 ˚C. Test solutions containing 12 ng/well p38 MAPK were diluted in kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl 2 , 10 mM β-glycerophosphate, 100 µg/mL BSA, 1mM dithiothretol, 0.1 mM Na 3 VO 4 , 100 µM rATP) with or without test substance (10 -4 to 10 -8 M). Test substances were dissolved in dimethyl sulfoxide to form stock solution of 10 -2 M, all further dilutions were carried out in kinase buffer. After subsequent washing, plates were blocked again with BB for 15 min followed by a fourth washing step. Wells were filled with 50 µL of the specific anti-bis-(Thr 69/71) -phospho-ATF-2 (1. AB, 1:500 in BB) and incubated for 1 h at 37 ˚C followed by washing and consecutive incubation with 50 µL of the secondary antibody (2. AB (alkaline phosphatase conjugated), 1:1400 in BB). Then 100 µL of 4-NPP (N-Phenethyl-4piperidinone is a derivative of 4-piperidinone) was pipetted in each well after a final washing step and colour development was measured 1.5-2 h later with an enzyme linked immunosorbent assay reader at 405 nm.

In Silico Study
Molecular Docking studies [38][39][40][41][42]: Molecular docking studies were performed using Biovia Discovery Studio 2021 (v21.1.0.20298) software to determine the possible interaction modes of the target compounds in the p38 mitogen activated protein kinase (p38 MAPK). The X-ray crystallographic structure of the enzymes p38 MAPK, was downloaded from the protein data bank, [43], PDB codes: 5MTX respectively. While the bound substances (ligand and cofactors) and solvent molecule associated with the receptor were removed. The chemical structure of the molecules were drawn with Chemdraw ultra Version 8.0 and further pre-optimized for docking. Furthermore the prepared ligands were docked with the prepared structure of p38 MAPK inhibitors using Biovia Discovery Studio 2021 (v 21.1.0.20298). [44,45]: The targeted compounds were study for pharmacokinetics and drug-likeness in silico using swissADME software web-based (http://www.swissadme.ch/index.php). Chemical structures of the compounds (2D) were drawn in ChemDraw ultra 8.0 versions (Cambridge Software), and relevant SMILES of each compound were converted. Virtual SMILES and structure generator initiate in online tool SwissADME transformed each compound SMILE into molfiles. The prediction was made to investigate that the synthesized compounds were inhibitors of CYP450 isoforms (CYP1A2, CYP2C19, and CYP2C9). To predict pharmacokinetics like gastrointestinal absorption, P-glycoprotein (P-gp), and blood-brain barrier (BBB), and drug-likeness like Lipinski, Ghose, and Veber rules and bioavailability score. The molecular weight, LogP, number of hydrogen bond acceptors (HBA), and number of hydrogen bond donors (HBD) predicted according to Lipinski, Ghose, and Veber rules to measure the drug-likeness properties whether the compounds has to be bioactive.

Statistical Analysis
All statistical analyses were performed with the help of SPSS 17.0 software. The level of statistical significance was expressed by p-value. The in-vitro antioxidant, anti-inflammatory, and p38 MAPK assay were expressed by mean ± S.E.M.

RESULTS AND DISCUSSION
Chemistry 7(a-g): The reaction sequences leading to the different chalcone substituted quinoline are outlined in the Scheme 1. The synthesis of Ethyl 2-(quinoline-8-yloxy) acetate 2 was carried out by reaction of 8-hydroxyquinoline (0.1mole) 1 in 60 ml of absolute ethanol with potassium carbonate (0.1 moles) in 20 ml of absolute ethanol followed ethyl chloroacetate (0.1 moles) by conventional and microwave irradiation methods and filtered. The filtrate was poured into ice-cold water, and separated ester was extracted with ether and dried. Ethyl 2-(quinoline-8-yloxy) acetate 2 was formed, and the product was converted into their hydrazine derivatives 3 by treatment with hydrazines hydrates and then cyclized with different chalcone derivatives 6(a-g) to give the chalcone substituted quinolines analogs 7(a-g). The physical data, reaction time, yields and melting point of the target compounds 7(a-g) via both methods are presented in Table 1.       1H, J=8,4Hz). 13 13

Biological assay:
All the synthesized compounds 7(a-g) were evaluated for in-vitro antioxidant, anti-inflammatory, and p38 MAKP inhibitory activities. The screened compounds 7a and 7g produced better action than other compounds but somewhat lesser than the standard drugs.

In-vitro anti-inflammatory activity:
The protein denaturation method was employed to determine the in-vitro anti-inflammatory activities of the target compounds 7(a-g). In this methodology, protein's tertiary and secondary structure would vanish when an external molecule or pressure was implemented. When denatured, the biological role of most biological proteins would be disappeared. The strength of the title targeted compounds was measured to inhibit protein denaturation (in triplicate). Diclofenac sodium was used as a standard drug. In this examination, 7a provided 67.26% inhibition and 7g provided 75.35% inhibition and diclofenac sodium exhibited 91.89% inhibition of protein denaturation. The in-vitro anti-inflammatory activity targeted compounds are provided in Table 3.
In vitro p38 MAPK assay: To gain more discernment into the interactions of the target compounds molecular docking studies were carried out using Biovia Discovery Studio 2021, using p38 MAPK proteins to correlate the binding mode. The targeted compound's inhibitory strengths on p38α MAPK were assessed in a non-radioactive immunosorbent p38R MAPK enzyme assay. ATP competed with the prohibited for the equivalent binding pocket catalytic site of p38 α MAPK and associated with that exerted by the well-known p38 MAPK inhibitor SB203580. According to the efficacy of the inhibitor and the resulting p38 α kinase activity, the phosphorylation reaction of the activating transcription factor 2 (ATF-2), the endogenous protein substrate of p38 MAPK, is prevented to a more immense or minor amount so that the amount of phosphorylated ATF-2 contrarily correlates with the inhibitory action of the p38 α MAPK inhibitor. Compounds with 7a (IC 50 value 22.93 mM) and 7g (IC 50 value 30.36 mM) shown to be endowed with moderate inhibitory activity as compared to other synthesized compounds, with an IC 50 in the range of 22.93 to 67.46 mM. The reference compound SB203580 showed IC 50 value 0.66 mM against p38 kinase. The in-vitro p38 MAPK activity of the targeted compounds is provided in Table 4.

Structure-activity relationship:
From the invitro antioxidant studies, it was found that target compounds exhibit good antioxidant activities which might be due to the presence of electron withdrawing substituents like 3,4,5trimethoxybenzene (7a), phenylacetaminde (7b), 4-nitrobenzene (7c), 4-flourobenzene (7d) on the phenyl ring attached at the C-5 of the pyrazole nucleus. Compounds consisting electrondonating substituents like N,Ndimethylbenzenamine (R=N(CH 3 ) 2 C 6 H 5 ) (7g), possesses good antioxidant and antiinflammatory activity at phenyl ring attached at the C-5 of the pyrazole nucleus. Compounds 7e and 7f also possess good antioxidant activity but have lessen anti-inflammatory activity as compared to other target compounds due to the presence of 4-phenoxymethylbenzen (7e) and 10-anthracene (7f) at the phenyl ring attached at the C-5 of the pyrazole nucleus. The structural activity relationship of the target compound is shown in Fig. 1.

In Silico Study
Molecular Docking studies: Molecular docking study of the target compounds was carried out against p38 MAP kinase enzyme and is displayed in Table. 5. Due to the flexibility of (linker) connecting pyrazole and quinoline moieties, the target compounds can be housed more favourably and effectively in p38 MAP kinase binding site. Compounds 7a-g, with best binding were visualized and analysed using Discovery Studio. The binding affinity values for all the target compounds range from -8.081 Kcal/mol to -8.994 Kcal/mol with the target site against p38 MAP kinase enzyme. The target compounds 7a (-8.947 Kcal/mol), 7b (-8.200 Kcal/mol), and 7g (-8.994 Kcal/mol) had shown remarkable binding affinities which were greater than the binding affinity of recommended drugs; diclofenac sodium (-6.8 kcal/mol), and nimesulide (-7.8 kcal/mol).  The recommended drugs, diclofenac sodium (std-1) (Fig. 3), account for two hydrogen bonds (LYS152, ASN155) while hydrophobic bonds were observed with ILE141, ILE166, LEU138, LEU151, VAL209, and VAL83 and account hydrophilic interaction with ASP168 of the target site of p38 MAPK. Nimesulide (std-2) (Fig. 3) accounts for two hydrogen bonds (GLY170, LEU171), while hydrophobic bonds were observed with ILE166, LEU51, and LEU138, while hydrophilic interaction accounts with ASP205 of the target site.  The synthesized compounds were performed in silico SWISS ADME for pharmacokinetics studies and the results are shown in Table. 6. Predicted logP values, reveals that out of 7 synthesized compounds, 6 compounds namely 7a,7b, 7c, 7d, 7e, & 7g logP values were above 3.6, whereas 7f logP values lies within the range of 1.6 to 3.6. This parameter could be the probability to depict the compounds as a drug like candidates those showing the logP values ranges within 1.6 to 3.6. Among the synthesized compounds some of them possess favourable ADME properties. 7g predicted to have a good GIT absorption based on WlogP to tPSA. Among the target compounds none of them penetrate blood-brain-barrier (BBB). Among the target compounds, none of them predicted to act on P-gp substrate. Most of the compounds inhibits to CYP450 isoforms. The target compounds interact with the isoform CYP2C19 and CYP2C9 are found to be the major isoform [46][47][48][49][50][51].
At early research phase of drug discovery and development, drug-likeness is the main principles in screening for the drug candidates. This unveil the physicochemical parameter of the compound i.e., bioavailability of per oral route along with their pharmaceutical characteristics in human body. Also the prediction for drug-likeness conducted on the bioavailability score, Veber rules, Lipinski, and ghose. The Lipinski's Rule of five states that the molecular weight under 500 g/mol, will be more likely to be absorb and permeate, logP value must be lower than 5, number of Hydrogen bond donor has utmost 5 and 10 hydrogen bond acceptor atoms. Druglikeness constraints define by Ghose filter (Amgen) are calculated logP values should be lie between -0.4to 5.6, MW of the molecule is in between 160-480, molar refractivity between 40-130, and the total number of atoms 20 to 70. Veber, rules for drug-likeness defines that the rotatable bond count should be less than or equal to 10 and polar surface area (PSA) should be less than or equal to 140 [46][47][48][49][50][51]. According to Lipinski Rule of Five, screening process of the compounds reveals that out of total 7 compounds, only 3 compounds 7a, 7b and 7d were meet the criteria of druglikeness with one violation (Table. 7) i.e. MW>500. Whereas 4 compounds (7c, 7e, & 7g) were rejected with two violations. According to Ghose rules all 7 compounds were rejected with two. According to Veber rules, all the 7compounds meet the criteria of drug likeness (Table. 7). For good bioavailability TPSA value should be <140. Another significance parameter for good oral bioavailability is total polar surface area (TPSA) and those compound passively absorbed with TPSA<140 have low oral bioavailability [46][47][48][49][50][51].  "RB": rotatable bonds; "HBD": hydrogen bond acceptors; "HBD": hydrogen bond donors; "MR": molar refractivity; "Std-1": standard drug -1: sodium diclofenac; "Std-2": standard; drug-2: nimesulide; "MW": molecular weight; "TPSA": topological polar surface area; "BA score": Bioavailability score

CONCLUSION
The present paper reported that the novel heterocyclic hybrid scaffolds 7(a-g) was synthesized using microwave irradiation techniques, showing better yield and lesser time than the conventional synthesis method. The insilico and in-vitro biological screening revealed that the compounds 7a and 7g showed better activity than other target compounds and had shown remarkable binding affinities greater than the recommended drugs. However, further extensive research may lead to the generation of ideal pharmaceuticals to treat inflammation shortly.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

ETHICAL APPROVAL
The institutional ethical committee approved the protocol adopted for the study (CPCSEA Registration No.: 1167/GO/a/12/ CPCSEA).