Synthesis of Pyrazole-Thiobarbituric Acid Derivatives: Antimicrobial Activity and Docking Studies

A one-pot reaction was described that results in various pyrazole-thiobarbituric acid derivatives as new pharmacophore agents. These new heterocycles were synthesized in high yields with a broad substrate scope under mild reaction conditions in water mediated by NHEt2. The molecular structures of the synthesized compounds were assigned based on different spectroscopic techniques. The new compounds were evaluated for their antibacterial and antifungal activity. Compounds 4h and 4l were the most active compounds against C. albicans with MIC = 4 µg/L. Compound 4c exhibited the best activity against S. aureus and E. faecalis with MIC = 16 µg/L. However, compounds 4l and 4o were the most active against B. subtilis with MIC = 16 µg/L. Molecular docking studies for the final compounds and standard drugs were performed using the OpenEye program.

anticancer agents, enzyme inhibitors, neuromodulators, antibiotics, herbicides, and plant growth regulators, antibacterial, and anti-oxidant agents ( Figure 1). Structure activity relationship SAR development led to the discovery of new, selective, and potent inhibitors that have attracted much interest from chemists. Molecular docking plays an important role in the rational design of drugs. In the field of molecular modeling, docking is a tool that predicts the best orientation of one molecule to a second when bound to each other to form a stable complex. Molecular docking can be defined as an optimization problem that would describe the "best-fit" orientation of a ligand that binds to a particular protein of interest to allow it to perform reliable virtual screening processes, and help us to understand the mechanism of action for tested compounds [33,34].
In this paper, we synthesized a new series of pyrazole-thiopyrimidine-rione derivatives via a one-pot multi-component reaction in aqueous media to identify new drugs as antimicrobial agents. We subjected our target compounds to molecular docking study with different target proteins to explore their mode of action.

Results
As part of our continuing efforts on the synthesis of bioactive scaffolds using green protocols, we envisioned that pyrazole-thiobarbituric acid derivatives having various substituents were achieved in high yields (63%-88%), as shown in Scheme 1, Table 1. Cascade Aldol-Michael addition of N,N-diethyl thiobarbituric acid, 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one and aldehyde was mediated by aqueous NHEt2 to afford 4a-o. Notably, the present one-pot transformation provides an efficient method for the flexible and rapid synthesis with substrate tolerance of pyrazole-thiobarbituric acid derivatives. The chemical structures of all the synthesized compounds were assigned with the aid of physical and spectroscopic methods.

Results
As part of our continuing efforts on the synthesis of bioactive scaffolds using green protocols, we envisioned that pyrazole-thiobarbituric acid derivatives having various substituents were achieved in high yields (63%-88%), as shown in Scheme 1, Table 1. Cascade Aldol-Michael addition of N,N-diethyl thiobarbituric acid, 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one and aldehyde was mediated by aqueous NHEt 2 to afford 4a-o. Notably, the present one-pot transformation provides an efficient method for the flexible and rapid synthesis with substrate tolerance of pyrazole-thiobarbituric acid derivatives. The chemical structures of all the synthesized compounds were assigned with the aid of physical and spectroscopic methods.
anticancer agents, enzyme inhibitors, neuromodulators, antibiotics, herbicides, and plant growth regulators, antibacterial, and anti-oxidant agents ( Figure 1). Structure activity relationship SAR development led to the discovery of new, selective, and potent inhibitors that have attracted much interest from chemists. Molecular docking plays an important role in the rational design of drugs. In the field of molecular modeling, docking is a tool that predicts the best orientation of one molecule to a second when bound to each other to form a stable complex. Molecular docking can be defined as an optimization problem that would describe the "best-fit" orientation of a ligand that binds to a particular protein of interest to allow it to perform reliable virtual screening processes, and help us to understand the mechanism of action for tested compounds [33,34].
In this paper, we synthesized a new series of pyrazole-thiopyrimidine-rione derivatives via a one-pot multi-component reaction in aqueous media to identify new drugs as antimicrobial agents. We subjected our target compounds to molecular docking study with different target proteins to explore their mode of action.

Results
As part of our continuing efforts on the synthesis of bioactive scaffolds using green protocols, we envisioned that pyrazole-thiobarbituric acid derivatives having various substituents were achieved in high yields (63%-88%), as shown in Scheme 1, Table 1. Cascade Aldol-Michael addition of N,N-diethyl thiobarbituric acid, 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one and aldehyde was mediated by aqueous NHEt2 to afford 4a-o. Notably, the present one-pot transformation provides an efficient method for the flexible and rapid synthesis with substrate tolerance of pyrazole-thiobarbituric acid derivatives. The chemical structures of all the synthesized compounds were assigned with the aid of physical and spectroscopic methods. A tandem Aldol-Michael reaction is shown in Scheme 2. In the first step of the reaction, olefin is produced by Aldol condensation between aryl aldehyde 1 and 2 to give an intermediate that acts as a Michael acceptor and is attached by 3 (the Michael donor) to afford the final product 4a (Bath A). Alternatively, Aldol condensation between aryl aldehyde 1 and 3 gives an intermediate that acts as a Michael acceptor and is attached by 2 (the Michael donor) to afford the final product 4a (Bath B). [19]. A tandem Aldol-Michael reaction is shown in Scheme 2. In the first step of the reaction, olefin is produced by Aldol condensation between aryl aldehyde 1 and 2 to give an intermediate that acts as a Michael acceptor and is attached by 3 (the Michael donor) to afford the final product 4a (Bath A). Alternatively, Aldol condensation between aryl aldehyde 1 and 3 gives an intermediate that acts as a Michael acceptor and is attached by 2 (the Michael donor) to afford the final product 4a (Bath B). [19].

Scheme 2.
Possible mechanism of the tandem Aldol-Michael reaction. Scheme 2. Possible mechanism of the tandem Aldol-Michael reaction.

Antimicrobial Activity
Results of the biological activity were displayed in Table 2; results are expressed as µg/L inhibition.

Antibacterial Activity against Gram-Positive Bacteria
The antibacterial activity of synthesized compounds was elucidated against six strains including E. faecalis ATCC29212, S. aureus ATCC 29213, E. coli ATCC 25922, B. subtilis ATCC 10400, P. aeruginosa ATCC 27857, and P. s vulagris ATCC 6380. Their activities were compared with the known Ciprofloxacin.
The results summarized in Table 2 show that all compounds are sensitive to the tested strains including E. faecalis, S. aureus, and B. subtilis except for compounds 4d-f and 4o, which were not active against S. aureus and E. faecalis, respectively. Compound 4c was the most active compound against

Antifungal Activity
The new pyrazole-thiobarbituric acid derivatives were evaluated for their antifungal activity against fungi) C. albicans ATCC 2091) by the diffusion method and serial dilution method (BSAC, 2015).
Their activities were compared with the known antifungal agent Fluconazole. Compounds 4h and 4l were the most active compounds against C. albicans, with MIC values of 4 µg/L, followed by compounds 4a and 4o with MIC values of 8 µg/L. The order of activity was compounds 4b, 4c, 4k, 4m, 4n, and 4j with MIC values of 16 µg/L, then compounds 4f, 4g, and 4i with MIC values of 32 µg/L. Compounds 4d and 4e were the least active compounds with MIC values of 36 µg/L. Among those compounds most active as antimicrobial, we can conclude that the aryl part is either incorporated by the electron withdrawing group (EWG) at the para position or is replaced by heterocyles (compound 4o).

Molecular Docking as Antifungal
Comparative consensus score of synthesized compounds were listed in Table 3. The synthesized compounds and fluconazole were docked with Lanosterol 14 α-demethylase (CYP51A1) (PDB ID 4WMZ) [35].
The standard drug, fluconazole, with a consensus score of 57, forms two hydrogen bonds with the receptor; with THR318:A through the nitrogen of triazole moiety (HB acceptor) and with Val 311:A through the hydroxyl functionality (HB donor) ( Figure 2). Compound 4l, with a consensus score of 48, interacts with the same receptor through hydrophobic-hydrophobic interaction without HB formation. Compound 4h, with a consensus score of 55, forms a hydrogen bond with receptor THR 318:A (as fluconazole did) through the sulfur of the pyrimidin moiety (HB acceptor), ( Figure 3).
Our hypothesis was directed at the study of another kind of protein as these compounds may have a mode of action that differs from the fluconazole mechanism. After intensive study, synthesized compounds presented docking interaction with dihydrofolate reductase (DHFR), correlated with their biological activity.

Molecular Docking as Gram-Positive Bacteria Antagonist
Molecular modeling gave us the comparative consensus scores of synthesized compounds with two targets: DNA topisomerase II (PDB ID 5BTC) [37] and gyrase B (PDB ID 4URM) proteins [38].
The consensus scores for the tested compounds with these two proteins are listed in Table 4. The standard drug ciprofloxacin has a consensus score of 1 through hydrophobic-hydrophobic interaction and forms HB with ARG:128:A through the oxygen of its carbonyl ( Figure 6). The docking mode for the most active compounds showed hydrophobic-hydrophobic interaction with the receptor. Compound 4c with a consensus score of 29, compound 4o with a consensus score of 10, and compound 4l with a consensus score of 54 showed exhibited hydrophobic-hydrophobic interactions and overlay each other (Figure 7). Our attention was directed to explore another kind of protein. Visual representation of 4a overlay with 4o, 4d, 4g, and 4i, showing the hydrophobic-hydrophobic interaction at the binding site of 3Q70, as shown by Vida.

Molecular Docking as Gram-Positive Bacteria Antagonist
Molecular modeling gave us the comparative consensus scores of synthesized compounds with two targets: DNA topisomerase II (PDB ID 5BTC) [37] and gyrase B (PDB ID 4URM) proteins [38].
The consensus scores for the tested compounds with these two proteins are listed in Table 4. The standard drug ciprofloxacin has a consensus score of 1 through hydrophobic-hydrophobic interaction and forms HB with ARG:128:A through the oxygen of its carbonyl ( Figure 6). The docking mode for the most active compounds showed hydrophobic-hydrophobic interaction with the receptor. Compound 4c with a consensus score of 29, compound 4o with a consensus score of 10, and compound 4l with a consensus score of 54 showed exhibited hydrophobic-hydrophobic interactions and overlay each other (Figure 7). Our attention was directed to explore another kind of protein.  Ciprofloxacin forms a hydrogen bond with ASP 225:A through the NH of the piperidine moiety with a consensus score of 4. Compound 4c (consensus score: 24) showed HB interaction with ASN 145:A through the sulfur atom of the thiobarbiturate ring and overlay with 4a, 4d, and 4f with the same HB. However, 4a showed extra HB with GLN 196:A through the N of pyrazole ( Figure 8). Compounds 4l (consensus score 17) and 4m (consensus score 41) showed hydrophobic-hydrophobic interaction and overlay with 4b; however, compound 4b (consensus score: 24) exhibits three kinds of HB ( Figure 9).
The antimicrobial activity of these compounds might be attributed to the presence of different pharmacophores in the molecules. The sulfur and OH groups in the substrates actively participate in the biological activity, as is corroborated by the formation of a hydrogen bond with the amino acid in the receptor. The site and kind of substituent located in the aldehyde part affects the dissociation rate, required for the liberation of free compounds from its salt form, and participates in the ligandprotein interaction. In this regard, the presence of an electron donating group (EDG) retards activity, while an electron withdrawing group (EWG) represents the best activity. Among the compounds having EWG, the para position is the best site for activity and the electronic and steric effect has a role in activity (compound 4f is inactive). However, the difference in activity between compounds 4l and 4m could be explained by their 3D structures during the docking study by using Omega application. In compound 4m, the chloride atom in position 6 was incorporated closely with the hydroxyl groups, while in the case of compound 4l its presence at position para meant the chloride atom was not incorporated near the hydroxyl group. This evidence represents the importance of EWG at the para position (see the Supplementary Materials).  Ciprofloxacin forms a hydrogen bond with ASP 225:A through the NH of the piperidine moiety with a consensus score of 4. Compound 4c (consensus score: 24) showed HB interaction with ASN 145:A through the sulfur atom of the thiobarbiturate ring and overlay with 4a, 4d, and 4f with the same HB. However, 4a showed extra HB with GLN 196:A through the N of pyrazole (Figure 8). Compounds 4l (consensus score 17) and 4m (consensus score 41) showed hydrophobic-hydrophobic interaction and overlay with 4b; however, compound 4b (consensus score: 24) exhibits three kinds of HB (Figure 9).
The antimicrobial activity of these compounds might be attributed to the presence of different pharmacophores in the molecules. The sulfur and OH groups in the substrates actively participate in the biological activity, as is corroborated by the formation of a hydrogen bond with the amino acid in the receptor. The site and kind of substituent located in the aldehyde part affects the dissociation rate, required for the liberation of free compounds from its salt form, and participates in the ligandprotein interaction. In this regard, the presence of an electron donating group (EDG) retards activity, while an electron withdrawing group (EWG) represents the best activity. Among the compounds having EWG, the para position is the best site for activity and the electronic and steric effect has a role in activity (compound 4f is inactive). However, the difference in activity between compounds 4l and 4m could be explained by their 3D structures during the docking study by using Omega application. In compound 4m, the chloride atom in position 6 was incorporated closely with the hydroxyl groups, while in the case of compound 4l its presence at position para meant the chloride atom was not incorporated near the hydroxyl group. This evidence represents the importance of EWG at the para position (see the Supplementary Materials). Ciprofloxacin forms a hydrogen bond with ASP 225:A through the NH of the piperidine moiety with a consensus score of 4. Compound 4c (consensus score: 24) showed HB interaction with ASN 145:A through the sulfur atom of the thiobarbiturate ring and overlay with 4a, 4d, and 4f with the same HB. However, 4a showed extra HB with GLN 196:A through the N of pyrazole (Figure 8). Compounds 4l (consensus score 17) and 4m (consensus score 41) showed hydrophobic-hydrophobic interaction and overlay with 4b; however, compound 4b (consensus score: 24) exhibits three kinds of HB (Figure 9).
The antimicrobial activity of these compounds might be attributed to the presence of different pharmacophores in the molecules. The sulfur and OH groups in the substrates actively participate in the biological activity, as is corroborated by the formation of a hydrogen bond with the amino acid in the receptor. The site and kind of substituent located in the aldehyde part affects the dissociation rate, required for the liberation of free compounds from its salt form, and participates in the ligand-protein interaction. In this regard, the presence of an electron donating group (EDG) retards activity, while an electron withdrawing group (EWG) represents the best activity. Among the compounds having EWG, the para position is the best site for activity and the electronic and steric effect has a role in activity (compound 4f is inactive). However, the difference in activity between compounds 4l and 4m could be explained by their 3D structures during the docking study by using Omega application. In compound 4m, the chloride atom in position 6 was incorporated closely with the hydroxyl groups, while in the case of compound 4l its presence at position para meant the chloride atom was not incorporated near the hydroxyl group. This evidence represents the importance of EWG at the para position (see the Supplementary Materials).

Materials and Methods
"All the chemicals were purchased from Sigma-Aldrich (Riedstraße, Germany) Fluka (Buchs, Switzerland), etc., and were used without further purification, unless otherwise stated. All melting points were measured on a Gallenkamp melting point apparatus (Bibby Scientific Limited, Beacon Road, Stone, Staffordshire, UK) in open glass capillaries and are uncorrected. IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). The NMR spectra were recorded on a Jeol-400 NMR spectrometer (Tokyo, Japan). 1 H-NMR (400 MHz), and 13 C-NMR (100 MHz) were run in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (δ) are referred to in terms of ppm and J-coupling constants are given in Hz. Mass spectra were recorded on a Jeol of JMS-600 H (Santa Clara, CA, USA). Elemental analysis was carried out on an Elmer 2400 Elemental Analyzer (Waltham, MA, USA) in the CHN mode".

Materials and Methods
"All the chemicals were purchased from Sigma-Aldrich (Riedstraße, Germany) Fluka (Buchs, Switzerland), etc., and were used without further purification, unless otherwise stated. All melting points were measured on a Gallenkamp melting point apparatus (Bibby Scientific Limited, Beacon Road, Stone, Staffordshire, UK) in open glass capillaries and are uncorrected. IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). The NMR spectra were recorded on a Jeol-400 NMR spectrometer (Tokyo, Japan). 1 H-NMR (400 MHz), and 13 C-NMR (100 MHz) were run in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (δ) are referred to in terms of ppm and J-coupling constants are given in Hz. Mass spectra were recorded on a Jeol of JMS-600 H (Santa Clara, CA, USA). Elemental analysis was carried out on an Elmer 2400 Elemental Analyzer (Waltham, MA, USA) in the CHN mode".

Materials and Methods
"All the chemicals were purchased from Sigma-Aldrich (Riedstraße, Germany) Fluka (Buchs, Switzerland), etc., and were used without further purification, unless otherwise stated. All melting points were measured on a Gallenkamp melting point apparatus (Bibby Scientific Limited, Beacon Road, Stone, Staffordshire, UK) in open glass capillaries and are uncorrected. IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). The NMR spectra were recorded on a Jeol-400 NMR spectrometer (Tokyo, Japan). 1 H-NMR (400 MHz), and 13 C-NMR (100 MHz) were run in deuterated dimethylsulphoxide (DMSO-d 6 ).
Chemical shifts (δ) are referred to in terms of ppm and J-coupling constants are given in Hz. Mass spectra were recorded on a Jeol of JMS-600 H (Santa Clara, CA, USA). Elemental analysis was carried out on an Elmer 2400 Elemental Analyzer (Waltham, MA, USA) in the CHN mode".

Agar Cup Plate Method
"The tested bacterial strains were grown in Cation Adjustment Mueller-Hinton (CAMH) broth (Merck ® , Darmstadt, Germany), while C. albicans strain was grown in Sabauraud Dextrose Broth (SDB) to mid-log phase. The bacterial and fungal suspension was measured by spectrophotometery using Spectrophotometer (LKB ® Ultrospec, Madison, WI, USA) t 625 nm to give absorbance 0.12 (1 × 10 8 CFU/mL). The suspension was diluted 1:100 in CAMH broth to obtain 1 × 10 6 CFU/mL. This suspension was swabbed on a CAMH agar plate (Merck ® , Darmstadt, Germany) and allowed to dry completely. Mueller-Hinton Agar and Sabauraud Dextrose Agar were used for bacteria and fungi, respectively. Four wells (7 mm in diameter) were made in agar plate using a cork borer. One milliliter of stock solution (5120 µg/mL) was diluted two-fold in 1 mL DMSO to obtain 2560 µg/mL. One hundred microliters (256 µg) of the tested compound was poured into the well using a calibrated pipette. The plates were kept in a refrigerator at 4 • C for half an hour to allow diffusion of the compound in the agar. Then, the plates were incubated at 37 • C for 24 h. After the incubation period, the diameter of the inhibition zone was measured and recorded in mm with the aid of a ruler. Ciprofloxacin (10 µg/cup) and fluconazole (10 µg/mL) were used as positive controls for antibacterial and antifungal activity, respectively. The experiment was carried out in duplicate and the mean diameter was taken" [39].

Determination of MIC
"MIC was determined for the compounds that showed antimicrobial activity by cup plate method. Briefly, 2 mL of CAMH broth (for bacterial strains) and 2 mL of SAB (for fungal strain) were dispensed into 7-mL Peju sterile tubes. For each compound, 14 tubes were used. Tubes 13 and 14 were used as a positive growth control (no tested compound) and a negative control for medium sterility (no microorganisms), respectively. One milliliter of stock solution (5120 µg/mL) was 10-fold diluted in 9 mL CAMH to obtain 512 µg/mL. Two milliliters of the tested compounds (512 µg/mL) were pipetted into the first tube and mixed well. Then 2 mL were withdrawn from the 1st tube and added to the 2nd tube to make a two-fold dilution. This procedure was repeated down to the 12th tube to reach a concentration of 0.125 µg/mL. Two milliliters were discarded from the 12th tube. A volume of 2 mL of inoculums (1 × 10 6 CFU/mL) was added to all tubes except number 14 to give a final concentration of 1 × 10 6 CFU/mL. Ciprofloxacin and fluconazole were used as positive controls for antibacterial and antifungal activity, respectively. The inoculated tubes were incubated at 37 • C for 20 h. After the incubation period, the results of MIC were recorded manually and interpreted according to the guidelines of the British Society of Antimicrobial Chemotherapy (BSAC)" [39].

Docking Study
The docking study was performed using the OpenEye program [40] and its applications Omega, Oedcocking, Fred, and Vida. This software package is able to perform consensus scoring, which is an essential filtering technique used to obtain more accurate predictions, i.e., the lower the consensus score, the better the binding affinity of the ligands towards the receptor. Both the ligand input file (compile all .pdb files) and the receptor input file were passed into Fred to perform the molecular docking simulations. Multiple scoring functions were employed in order to obtain a consensus structure and score in the final output. A virtual library of target compounds and fluconazole as the standard drug for antifungal activity and ciprofloxacin as the standard for Gram-positive antibacterial activity was prepared using Chem Office 2012 (OpenEye Scientific software Inc., Santa Fe, NM, USA) and energy minimized with PM3 minimization. All calculations were performed on a PC running Windows 7 ultimate. Two different target proteins were downloaded from the Protein Data Bank, namely Lanosterol 14 α-demethylase (CYP51A1) (PDB ID 4WMZ) and secreted aspartic protease (PDB ID 3Q70), and were chosen as antifungal targets against Candia albicans. In order to understand the antibacterial activities of these newly synthesized compounds, DNA topisomerase II (PDB ID 5BTC) and gyrase B (PDB ID 4URM) were selected as antibacterial targets. The catalytic domain for each protein was prepared for docking using Open Eye (OpenEye Scientific software Inc., Santa Fe, NM, USA).

Generation of Conformers Using OMEGA
OMEGA (OpenEye Scientific software Inc., Santa Fe, NM, USA) was used to generate multi-conformer structure databases with high speed and reliability, in order to achieve flexible ligand docking, to be used in the docking simulations. The energy-minimized structures were then converted into .pdb files, maintaining all heavy atoms. Each .pdb file was concatenated into one continuous .pdb file to be used as an input for Omega.

Receptor (PDB file) Preparation Using OEDCKING Application
The receptor PDB files were downloaded from the Protein Data Bank (PDB): Lanosterol 14 α-demethylase (CYP51A1) (PDB ID 4WMZ), and secreted aspartic protease (PDB ID 3Q70), and were chosen as antifungal targets against Candia albicans. Two different target proteins were downloaded from the Protein Data Bank in order to understand the antibacterial activities of these newly synthesized compounds. DNA topisomerase II (PDB ID 5BTC), and gyrase B (PDB ID 4URM) were selected as antibacterial targets and were prepared using the make_receptor command. This step will be followed by finding the potential binding sites on the receptor of interest. The application uses a molecular probe to comb the receptor molecule, identifying all the possible binding interactions. The application allows you to create a grid box in the mode selection pane and adjust its size using the mode controls. The box size should never exceed 50,000-60,000 A • .

FRED Docking Application
Both the ligand input file and the receptor input file were passed into FRED to perform the molecular docking simulations. Multiple scoring functions were employed in order to obtain a consensus structure and score in the final output.

Vida Application
Vida is a graphical user interface that visualizes, analyzes, and manages corporate collections of molecular structures and information. Snapshots were taken to visualize and obtain the main interaction forces between the analogs and the receptor of interest.

Conclusions
A series of pyrazole-thiobarbituric acid derivatives (4a-o) were synthesized using a one-pot method with a broad substrate scope under mild reaction conditions in water, mediated by NHEt 2 . The compounds were evaluated for their antifungal and antibacterial activity. The chemical structures of the newly synthesized molecules were characterized by spectroscopic methods (IR, 1 H-NMR, 13 C-NMR, MS) and elemental analysis. The results of the current study revealed that compounds 4h and 4l were the most active against C. albicans, with an MIC value of 4 µg/L. Next were compounds 4a and 4o, which showed activity of MIC 8 µg/L, as compared with the reference drug Fluconazole with a MIC value of 0.5 µg/L. Furthermore, all synthesized compounds were docked inside the active site for two kinds of proteins, namely Lanosterol 14 α-demethylase (CYP51A1) (PDB ID 4WMZ) and secreted aspartic protease (PDB ID 3Q70). Compound 4h docked with 4WMZ and showed hydrogen bonding interactions with THR 318:A, a binding interaction similar to that of standard Fluconazole. Most of the compounds showed hydrophobic-hydrophobic interaction towards the binding site of 3Q70 and overlay each other.
However, among the synthesized compounds, compound 4c exhibited marked activity against S. aureus and E. faecalis, with MIC values of 16 µg/L. Compounds 4l and 4o were the most active compounds against B. subtilis with MIC = 16 µg/L in comparison to standard ciprofloxacin, with MIC values of <0.25 µg/L against all tested bacteria. The target compounds were subjected to dock with DNA topisomerase II (PDB ID 5BTC) and gyrase B (PDB ID 4URM) proteins to explore their mode of interaction in Grampositive bacteria.
Based on a docking study of the antimicrobial activity of the tested compounds, the sulfur atom and OH functionality in the thiobarbiturate moiety, the OH group in the pyrazole fragment, and the presence of an EWG group in the p-position of the aryl part participate in the compound's interaction with the tested receptors. These pharmacophores may contribute, at least in part, to the activity of the tested compounds as antimicrobial agents.