Microwave-assisted synthesis, computational studies and antibacterial/ anti-inflammatory activities of compounds based on coumarin-pyrazole hybrid

An efficient, high-yield and rapid synthesis of (E)-1,5-dimethyl-4-((2-((substituted-2-oxo-2H-chromen-4-yl)methoxy)naphthalen-1-yl)methyleneamino)-2-phenyl-1,2-dihydropyrazol-3-one derivatives (3a–3i) containing Schiff base structures under microwave-irradiation has been described. Schiff base is a potential target to discover anti-inflammatory chemotherapeutics, material science, catalysis and molecular magnetism. All the newly synthesized compounds (3a–3i) have been characterized by elemental analysis and spectroscopic techniques. The synthesized compounds (3a–3i) were evaluated for their antibacterial activity by agar-well diffusion method and anti-inflammatory activity by egg albumin denaturation method. The compounds (3e) and (3i) exhibit antibacterial effect with minimum inhibitory concentration (MIC) 0.78 µg ml−1 and MIC 1.562 µg ml−1 against Gram-positive Staphylococcus aureus bacterial strain compared with standard ciprofloxacin drug (MIC 6.25 µg ml−1). The compounds (3c) and (3f) exhibited an inhibition of heat-induced protein denaturation at the concentration (31.25 µg ml−1) as 53.65% and 67.27%, respectively, and these compounds are more active than standard aceclofenac drug (5.50%). Molecular docking study has been performed for all the synthesized compounds with S. aureus dihydropteroate synthetase and results obtained are quite promising.


Introduction
Microbial infections are becoming the most pressing issue for global health and the economy [1]. In recent years, method on a Buchi apparatus and are uncorrected. IR spectra were recorded on a Nicolet 6700 FT-IR instrument (Nicolet, Madison, WI, USA) using KBr pellets. 1 H and 13 C NMR spectra were recorded on Bruker 400 MHz FT NMR spectrometer using CDCl 3 and DMSO-d 6 as solvent and TMS as internal standard. All chemical shifts were reported as δ values (ppm). Mass spectra were recorded using Shimadzu GCMSQP2010S. The elemental analysis was carried out using Hereaus CHN rapid analyser. Microwave irradiation synthesis was carried out under CEM-Discover Focused Microwave system. Purity of the compound was checked by TLC.
The synthesized compounds were dissolved in dimethyl sulfoxide (the stock solution 1 mg ml −1 ). Furthermore, the dilutions were prepared at the required quantities of 100, 50 and 25 µg ml −1 concentrations. To ensure that the solvent had no effect on bacterial growth, control test was also performed containing disc loaded with only DMSO at the same dilution used in our experiment. Test compound solutions prepared in DMSO were serially diluted and loaded (10 µl) to sterile filter paper discs (6 mm diameter), which finally contained (25, 50 and 100 µg ml −1 ) of the compound per disc, respectively. Impregnated discs were then dried for 1 h and placed on inoculated plates. plates were incubated at 37°C for 16 h. The radii of inhibition zones (in mm) were measured and the percentage inhibition of test compounds was related to the standard drug whose zone of inhibition was taken as 100%. The results of minimum inhibitory concentrations (MICs) of the synthesized compounds against bacterial species are determined.

Anti-inflammatory activity
Egg albumin denaturation method. All the synthesized compounds were subjected to anti-inflammatory effect against denaturation of hen's egg albumin method [35] at the concentration (31.25 µg ml −1 ) with standard aceclofenac drug (31.25 µg ml −1 ). The mixture (5 ml) consisted of 0.2 ml of egg albumin (from fresh hen's egg), 2.8 ml of phosphate buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of coumarin-based pyrimidine compounds so that final concentrations become 31.25, 62.5, 125, 250, 500, 1000 µg ml −1 and similar volume of double-distilled water served as control. Then the mixtures were incubated at (37 ± 2)°C in an incubator (Bio-technics, India) for 15 min and then heated at 70°C for 5 min. After cooling, their absorbance was measured at 660 nm (SHIMADZU, UV-1800 Spectrophotometer) by using vehicle as blank. Aceclofenac sodium at the final concentration (31.25, 62.5, 125, 250, 500, 1000 µg ml −1 ) was used as reference drug and treated similarly for the determination of absorbance. The percentage inhibition of protein denaturation was calculated by using the following formula: % Inhibition = 100 × Abs of control − Abs of sample Abs of control .

Computational studies
For the docking of ligands to protein active sites and for estimating the binding affinities of docked compounds, Surflex-Dock module, a fully automatic docking tool available on Sybyl X-2.0 v., was used in this study.

Docking simulations
The X-ray crystal structure of S. aureus dihydropteroate synthetase (PDB ID: 1AD4) enzyme [36] was obtained from the Protein Data Bank in PDB format as starting point. The synthesized compounds and the standard compounds tested in this study were docked to S. aureus dihydropteroate synthetase (PDB ID: 1AD4) enzyme using Surflex-Dock program in Sybyl-X software by incremental construction approach of building the structure in the active site so as to favour the binding affinity [37]. Finally, the docked ligands were ranked based on a variety of scoring functions that have been compiled into the single consensus score (C-score) [38].
All the newly synthesized compounds were characterized using FTIR, 1 H NMR, 13         section. The spectral data of the newly synthesized coumarin derivatives (3a-3i) are in accordance with the assigned structures of the compounds and are provided in the Experimental section. The 1 H and 13 C NMR spectra of all the compounds are given in the electronic supplementary material and are in good agreement with the proposed structure of the compounds.
In the case of compound (3b), the IR spectrum exhibited two characteristic bands at 1724 cm −1 for lactone of the coumarin and 3444 cm −1 for NH stretching. The formation of the product was established using the 1 H NMR spectrum (400 MHz, CDCl 3 ) wherein a sharp singlet at δ 1.32 ppm corresponds to the tert-butyl of coumarin. Two singlets at δ 2.51 and 3.18 ppm correspond to the = C-CH 3   pyrazole. Furthermore, the presence of two singlet at δ 5.48 and 6.83 ppm corresponds to the CH 2 and C 3 -H of the coumarin. One doublet was observed at δ 9.26 ppm with J = 8.4 Hz which corresponds to the Ar-H and a characteristic singlet for the imino proton (-CH = N-) at 10.57 ppm confirming the formation of the product.
The 13 C NMR spectrum provides additional support for the structure of the compound (3b), wherein the lactone carbonyl resonated at δ 156.34 ppm, pyrazole carbonyl resonated at δ 160.92 ppm and imino carbon resonated at δ 160.78 ppm. The two methyl carbons of N-CH 3 and CH 3 of pyrazole resonated at δ 36.06 and 10.51 ppm respectively. Tert-butyl and CH 2 of coumarin resonated at δ 31.45 and 68.08 ppm, respectively. The molecular ion peak at 571 [M] + in the mass spectrum confirmed the proposed structure of the compound (3b). The rest of the compounds gave satisfactory analytical and spectroscopic data which were in accordance with their assigned structures.
The presence of the methylene protons approximately δ 5.2-5.4 ppm from derivatives led us to conclude that the initially formed ethers are stable and did not undergo a further intramolecular carbanion addition across the azomethine group located at close spatial proximity (ortho position) leading to the formation of 2,3-dihydronapthofurans by an intramolecular aldol addition followed by dehydration.

Computational studies
The newly synthesized compounds have exhibited excellent antibacterial activity, in particular against Gram-positive bacteria S. aureus. The S. aureus can cause a range of illnesses from minor skin infection to life-threatening diseases and has become resistant to many commonly used antibiotics. Ciprofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class and is a second-generation antibacterial agent, which kills bacteria by inhibiting the enzyme DNA-gyrase.
To understand the mechanism of antibacterial activity of newly synthesized compounds, molecular modelling and docking studies were performed on X-ray crystal structure of the dihydropteroate synthetase (DHPS) complexed with OH-CH 2 -pterin-pyrophosphate from S. aureus (PDB ID: 1AD4, X-ray diffraction, 2.4 Å). Molecular docking was used to clarify the binding mode of the compounds to provide straightforward information for further structural optimization. The docking study was obtained from the Protein Data Bank by using Surflex-Dock program of Sybyl-X software. All the 10 compounds were docked into the active site of the DHPS (figure 4), the predicted binding energies and the observed C-score values of all the compounds are ranging from 4.27 to 10.33, the score values are listed in table 2.
The proteins were prepared for docking by adding polar hydrogen atom with Gasteiger-Huckel charges and water molecules were removed. The 3D structure of the ligands was generated by the SKETCH module implemented in the SYBYL program (Tripos Inc., St. Louis, USA) and its energy-minimized conformation was obtained with the help of the Tripos force field using Gasteiger-Huckel [41] charges, molecular docking was performed with Surflex-Dock program that is interfaced with Sybyl-X2.0, and other miscellaneous parameters were assigned with the default values given by the software.   The binding interaction of standard ciprofloxacin with DHPS active sites shows three bonding interactions and the docked view of the same has been depicted in figure 7. The comparative molecular docking study of synthesized compounds and standard ciprofloxacin drug highlighted that the synthesized compounds exhibited high C-score value. C-score value (5.01) of ciprofloxacin was lower than those of all the nine compounds. Figure 8a,b represents the hydrophobic and hydrophilic amino acids surrounded by the studied compounds (3i) and (3e).

Pharmacological screening
It is evident from the above fact that compounds possessing coumarin and antipyrine moiety are capable of exhibiting biological activities. When this ring system is fused or coupled with other heterocycles, the resulting compounds would exhibit enhanced biological properties. Hence, novel coumarin-antipyrine derivatives (3a-3i) have been synthesized in the course of present investigation. These compounds have been screened for their potential in vitro antibacterial activity by agar-well diffusion method and antiinflammatory activity by egg albumin denaturation method.

In vitro antibacterial screening
The newly synthesized compounds were screened for in vitro antibacterial activity by agar-well diffusion method [34] against two Gram-positive (Bacillus subtilis (ATCC no. 23857) and Staphylococcus aureus (ATCC-29213)) and two Gram-negative (Escherichia coli (ATCC-25922) and P. aeruginosa (ATCC No. 25619)) bacterial strains. The minimum inhibitory concentration (MIC) of the synthesized compounds (3a-3i) and ciprofloxacin was compared, it revealed that almost all the newly synthesized compounds showed excellent antibacterial activity against Gram-positive S. aureus bacterial strain. Screening results are summarized in table 3. The best antibacterial effect has compounds (3e) and (3i) with MIC 0.78 µg ml −1 and MIC 1.562 µg ml −1 , respectively, against Gram-positive S. aureus bacterial strain. Similarly compounds (3a) and (3c) showed better activity against Gram-positive and Gram-negative bacteria. While remaining compounds (3b, 3d, 3f, 3g, 3i) lead to weak antibacterial activity. The results are also represented in bar diagram of figure 9.

In vitro anti-inflammatory activity
The outcome of anti-inflammatory screening of compounds (3a-3j), by using egg albumin denaturation method, is summarized in table 4. The percentage inhibition of all the synthesized compounds was very highly active against the denaturation of protein. Among these compounds, (3c) and (3f) exhibited an excellent inhibition of heat-induced protein denaturation 53.65% and 67.27%, respectively, and these compounds are almost 10 times more active than standard aceclofenac drug (5.50%). Whereas compounds (3a) and (3h) show less activity and the remaining compounds showed good inhibitory anti-inflammatory activity against the denaturation of protein method. The synthesized compounds have shown significant anti-inflammatory activity in protein denaturation method. The results are also represented in the bar diagram of figure 10. These results suggest that an electron releasing group (-OCH 3 ) and hydrogen bonding group (-OH) increases the anti-inflammatory potency.

Structural activity relationship study
Even though the number of compounds tested here is limited, a few key features regarding structural requirements for these coumarin-pyrazole hybrids (3a-3i) to exert their antibacterial activity may be determined. Our initial strategy was to identify the key subunity required for activity such as coumarin      (3a-3i). and 7-position (3a) exhibit increased antibacterial activity with MIC of 3.125 µg ml −1 . The results from the preliminary structure-activity analysis have led to the determination of some key structural requirements for the coumarin-pyrazole hybrids to exert their antibacterial activity, which provide insights into further structural modification.

Conclusion
To explore different scaffold structures, we have described environmentally benign, simple and efficient protocol for the synthesis of coumarin-based pyrazole derivatives (3a-3i) with high yields under microwave-irradiation in shorter reaction time (8-12    method. Antibacterial screening revealed that compounds (3e) and (3i) exhibited potent activity against S. aureus bacterial strain with MIC 0.78 µg ml −1 and 1.562 µg ml −1 , respectively. The compound (3f) exhibited an inhibition of heat-induced protein denaturation at the concentration (31.25 µg ml −1 ) as 67.27%. Among all these synthesized scaffolds, compounds (3e) and (3i) are highly active and more potent in both biological and molecular docking simulation studies. The biological activities of coumarins after combining with pyrazole have been enhanced due to synergistic effect. The results also suggested a new and potential route in the discovery of drug against antibacterial and anti-inflammatory activities.
Ethics. The bioassay was prepared in the laboratory of Department of Biotechnology, Karnatak University Dharwad-580003. We have obtained the necessary approvals from the institutional ethics committee before conducting the research.