Synthesis, antimicrobial activity, pharmacophore modeling and molecular docking studies of new pyrazole-dimedone hybrid architectures

Background Design and synthesis of pyrazole-dimedone derivatives were described by one-pot multicomponent reaction as new antimicrobial agents. These new molecular framework were synthesized in high yields with a broad substrate scope under benign conditions mediated by diethylamine (NHEt2). The molecular structures of the synthesized compounds were assigned based on different spectroscopic techniques (1H-NMR, 13C-NMR, IR, MS, and CHN). Results The synthesized compounds were evaluated for their antibacterial and antifungal activities against S. aureus ATCC 29213, E. faecalis ATCC29212, B. subtilis ATCC 10400, and C. albicans ATCC 2091 using agar Cup plate method. Compound 4b exhibited the best activity against B. subtilis and E. faecalis with MIC = 16 µg/L. Compounds 4e and 4l exhibited the best activity against S. aureus with MIC = 16 µg/L. Compound 4k exhibited the best activity against B. subtilis with MIC = 8 µg/L. Compounds 4o was the most active compounds against C. albicans with MIC = 4 µg/L. Conclusion In-silico predictions were utilized to investigate the structure activity relationship of all the newly synthesized antimicrobial compounds. In this regard, a ligand-based pharmacophore model was developed highlighting the key features required for general antimicrobial activity. While the molecular docking was carried out to predict the most probable inhibition and binding mechanisms of these antibacterial and antifungal agents using the MOE docking suite against few reported target proteins. Electronic supplementary material The online version of this article (10.1186/s13065-018-0399-0) contains supplementary material, which is available to authorized users.


Background
Nosocomial infections caused by antibiotic-resistant gram-positive bacteria have become a serious medical problem with an alarming increasing rate worldwide. Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) are of particular concern among various hospital-acquired infections [1]. Accordingly, emerging investigations have provided new insights into developing novel, safe and effective antibacterial agents. Within this scope, pyrazole based antibacterial agents attracted great interest [2]. Generally, pyrazoles display innumerable pharmacological activities ranging from analgesic, antipyretic, antimicrobial, anti-inflammatory, anticancer effects to antidepressant, anticonvulsant, and selective enzyme inhibitory activities [2][3][4][5][6][7][8][9][10][11]. Recently, Barakat et al, have been reported novel pyrazole hybrid architectures as efficient antibacterial agents. Various pharmacophores were linked to the pyrazole core to build bioactive scaffolds [12,13]. Within this approach, cyclic dicarbonyl compounds of the type dimedone have attracted our interest. Dimedone has been utilized successfully as pharmacophoric building block in various antimicrobial agents such as xanthenes [14,15], substituted chromenes [16], macrocyclic metal complexes [17], quinazoline derivatives [18], tetrahydro quinolone diones [19] and acridine based compounds [20]. Recognizing these facts and in continuation of our previous work [12,13] new hybrid molecules incorporating pyrazoles and dimedone in a single molecular framework were designed and synthesized. We subjected our target compounds to pharmacophore modeling and molecular docking on different target proteins to explore their mode of action.

Chemistry
The designed bioactive scaffolds were synthesized utilizing green approach. The pyrazole-dimedone derivatives were prepared as shown in Scheme 1 via one pot Knoevenagel condensation Michael addition of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one, 1,3-dicarbonyl compound (dimedone) and various aldehydes mediated by aqueous NHEt 2 . This one pot multicomponent reaction afforded the final targets as hybrid frameworks 4a-o in good yields (40-78%) with substrate tolerance of pyrazoledimedone derivatives. The chemical structures of all the synthesized compounds were assigned by the aid of physical and spectroscopic methods ( 1 H-NMR, 13 C-NMR, IR, and elemental analyses).
The suggested mechanisms for obtaining the target compounds are shown in Scheme 2. Olefin is formed by Knoevenagel condensation of aryl aldehyde 1 and 1,3-diketone 2 to give benzylidenecyclohexandione intermediate which acts as a Michael acceptor. This Michael acceptor is attached by 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 3 (Michael donor) to give the requisite final targets 4a (Path A). Another bath way is Knoevenagel condensation between aryl aldehyde 1 and 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 3 to generate benzylidenepyrazolone intermediate which acts as a Michael acceptor. This Michael acceptor is attacked by 1,3-diketone 2 (Michael donor) to afford the final product 4a (Path B).

Antimicrobial activity
The synthesized pyrazole-dimedone derivatives showed various antibacterial activities. Results of the bactericidal activity are shown in Table 1; the minimum inhibitory concentration (MIC) results are expressed as µg/L inhibition.

Antibacterial activity against gram positive bacteria
The antibacterial activity of the novel pyrazole-dimedone compounds were evaluated against gram positive bacteria including E. faecalis ATCC29212, S. aureus ATCC 29213, and B. subtilis ATCC 10400. Ciprofloxacin was used as standard drug.
The results listed in Table 1 revealed that all pyrazoledimedone compounds were active against the testedstrains including S. aureus, E. faecalis, and B. subtilis. Pyrazole-dimedone 4k was the most active compound against B. subtilis with MIC value of 8 µg/L. Compounds 4e and 4l having 3-methyl and 4-trifluromethyl substituents on the phenyl ring respectively exhibited good activity against S. aureus with MIC value of 16 µg/L.
Scheme 1 Substrate scope of the cascade reaction: variation of pyrazole-dimedone adducts

Structure activity relationship profiling via pharmacophore modeling
First of all, to predict the structure activity relationship (SAR) of all the newly synthesized antimicrobial compounds, a ligand-based pharmacophore model was developed. This is the most reliable way to design new potent active molecules having similar scaffolds by utilizing their biological data in computational predictions. In this study, the selected pharmacophore including one hydrogen bond acceptor (F1: Acc& ML), one hydrogen bond donor (F2: Don, Acc& ML) and one hydrophobic feature with an aromatic center (F3: ML/Hyd/Aro) ( Fig. 1a) was mapped over active compounds (Fig. 1b). The mapping was evaluated on the basis of their lowest RMSD between query and matching annotations (Fig. 1c, d).
The lowest RMSD indicates better compound fitness to the selected model. Results in Table 2 showed that all the active compounds were able to satisfy the pharmacophoric features of the generated model with RMSD values ranging from 0.3907 to 0.6571 Å along with their most suitable alignment of each compound over query. These results indicated the critical role of aromatic ring substitution which greatly effects the spatial orientation of cyclohexane ring with respect to the pyrazole moiety. This might be the best explanation to understand the differences in their respective antimicrobial activity profile.

Docking simulation to predict the mode of inhibition
After SAR profiling, docking studies were carried out to predict the most suitable binding pose and inhibition mechanism of newly synthesized derivatives. But before docking, based on the principle that similar Compounds tend to bind to the same proteins, we predicted few protein targets reported against reference compounds (ciprofloxacin and fluconazole) and docked our active compounds against them. Binding DB brought in seven different target proteins i.e. Dihydrofolate Reductase (DHFR) (PDB ID 4HOF), Secreted Aspartic Protease and Sortase A (PDB ID 2MLM) from S. aureus as bacterial target. Among all these seven proteins, only two proteins i.e. one proteins (Thymidylate Kinase) from S. aureus [21] and one protein (N-myristoyl transferase) from C. albican [22] presented good binding affinity, while all other targets showed very few or no interactions with these derivatives.
The potencies of these newly synthesised derivatives were measured computationally in terms of their dock Scores. Dock score which is actually the strength of the non-covalent interactions among multiple molecules within the binding pocket of a target protein. The more negative the score is, the more favorable interactions between compound and the target protein are. Here in our study, the compound 4l being the most potent antibacterial agent against TMK (ID: 4QGG) from S. aurues, displayed the highest negative score of − 6.86 kcal/mol which is comparable of the standard drug ciprofloxacin with the score of − 6.9 kcal/mol. Similarly, 4o being the most potent antifungal agent displayed good docking score of − 8.7 kcal/mol and molecular interactions with N-myristoyl transferase (NMT) enzyme from C. Albicans.
Among all derivatives, compound 4l displayed the same electrostatic and hydrophobic interactions with crucial residues of TMK protein from S. aureusas presented by co-crystallized ligand. As illustrated in Fig. 2, the substituted part of compound 4l moved inside the cavity where both chlorine atoms at 2 and 4 positions were engaged in the formation of two halogen bonds with the amino groups of Arg70 and Gln101 at 2.14 Å and 2.53 Å, respectively. Moreover, dichloro substituted benzene ring along with the pyrazole ring displayed various π-π and π-cation interactions with the crucial residues Phe66 and Arg92 of the target protein. Apart from it, the carbon atom located at R position and methyl of pyrazole ring were observed to establish hydrophobic interactions with Arg48 and Phe66 of TMK protein that might be responsible for their potent antibacterial activity.
Comparatively, compound 4k being the most active against B. subtilis species showed less or very few interactions with the TMK protein (4QGG) from S. aureus origin (Fig. 3).
Similarly, the molecular visualization of 4o revealed a number of significant electrostatic and hydrophobic interactions with the crucial residues of NMT. Figure 4 showed that the hydroxyl moiety attached at dimedone ring presented visible hydrogen bond with Tyr107 at a distance of 2.48 Å. Apart from it, three π-π interactions were observed among phenyl and thiol and hotspot residues Phe117, Tyr225 and Tyr 354. Simultaneously, several hydrophobic interactions were also noticed among compound 4o and the crucial residues i.e. Tyr107, Phe 117, Tyr119, Tyr225, Tyr335. These results predicted TMK (S. aureus) and NMT (C. albicans) as the most probable targets for the antibacterial and antifungal activity of these newly synthesized agents.

Conclusions
By using one-pot green protocol a series of pyrazoledimedone derivatives (4a-o) were synthesized in high yields with a broad substrate scope under mild reaction conditions in water mediated by NHEt 2 . The requisite compounds were evaluated for their antibacterial and antifungal activities. After experimental investigations, Fig. 1 a Best query displaying pharmacophoric features shared by active lead compounds as colored spheres (cyan for hydrogen bond acceptor function with metal ligator (F1: Acc& ML), pink for hydrogen bond acceptor/donor function with metal ligator (F2: Don, Acc& ML) as well as cyan for hydrophobic region with aromatic centre, hydrogen bond acceptor or metal ligator function (F3: ML/Hyd/Aro/Acc). b Validation of the selected query; mapping of previously reported active compounds 4a and 4n [12] as well as 4a and 4f [13], showing RMSD values in acceptable range (0.2823-0.4993). c Mapping of compound 4k on pharmacophore model. d Mapping of compound 4o on pharmacophore model structure-activity relationship profiling was predicted by ligand-based pharmacophore modeling highlighting three features as a requirement for their antimicrobial activity. While Molecular docking predicted the molecular mechanisms of these derivatives with seven different target proteins. Among them, TMK from S. aureus and NMT protein from C. albicans were predicted as the most suitable targets for the antibacterial and antifungal activities of these newly synthesized derivatives.

Materials and methods General
"All the chemicals were purchased from Aldrich, Sigma-Aldrich, Fluka etc., and were used without further purification, unless otherwise stated.