As Antimicrobial Agents: Synthesis, Structural Characterization and Molecular Docking study of Barbituric Acid Derivatives from Phenobarbital

In spite of phenobarbital has been used in various medical fields as hypnotics, anxiolytics, and anticonvulsants, it also contains active functional groups that can be reacted to form other products as dyes, polymers, antimicrobial and anti-antioxidants agents. A series of barbituric acid derivatives containing 1,2,3,4-Tetrazoline moiety were synthesized from phenobarbital. Phenobarbital 1 as raw starting material was reacted with acrylonitrile compound to give diacetonitrile derivative 2, this compound was treated in two ways, urea and thiourea to form barbituric acid derivatives containing oxadiazole and thiadiazole ring 3, 4 respectively. The Schiff bases derivatives 5, 6 (a-c) were synthesized from reacting the latter compounds with three aromatic aldehydes. In the final step, the barbituric acid derivatives containing 1,2,3,4-Tetrazoline moiety 7, 8 (a-c) were prepared by cycloaddition reaction between different Schiff bases derivatives and sodium azide. The compounds were characterized by Melting point, 13 C-NMR, 1 H-NMR and FTIR techniques. Also, the result compounds were tested against two kinds of bacteria and two kinds of fungi. Most of the prepared derivatives were showed a high and clear effect against different types of bacteria and fungi. Molecular docking of final barbituric acid derivatives 7, 8 (a, b) were investigated with Molegro Virtual Docker (MVD).


Introduction
Due to their diverse biological effects, a significant number of barbituric acid derivatives have piqued the interest of the pharmaceutical community for more than a century [1]. Barbituric acids and its derivatives are organic substances which are used in pharmacology as hypnotics, sedatives, antihypertensive drugs, anesthetics, anticancer, anticonvulsant, antioxidants, antifungal agent, antibacterial, and Alpha-glycosidase enzyme inhibitors [2][3][4][5][6][7]. Chemically, via the donor-acceptor process of protons in the building-block structures of barbituric acid, the presence of five atoms (Three Oxygen and Two Nitrogen) with roles in coordination chemistry allows for the possible development and stability of supramolecular arrangements [8]. Barbituric acid can thus be defined as an organic building block that can behave intramolecularly and intermolecularly due to the presence of these O and N atoms [9]. Phenobarbital (PHB) or 5-ethyl, 5-phenyl barbituric acid as a starting material, is one of the most common barbituric acid derivatives. In their 1904 paper, Fischer and Dilthey listed phenobarbital compound as one of the compounds they discovered, Structure 1 [10].

Structure 1. Phenobarbital Compound
In organic chemistry, Cycloaddition reactions are one of the most significant processes with synthetic and mechanistic importance. The simplicity of these cycloaddition reactions, as well as their exceptional stereochemistry, makes them useful [11]. 1,3-dipolar cycloaddition reactions have been commonly used in the synthesis of natural products and bioactive organic compounds, as well as in the construction of various five-membered heterocyclic. The majority of 1,3-dipolar compounds are heteroatom based diazoalkanes, azomethine ylides, azides, nitrile ylides, nitrile imines, nitrile oxides, nitrones, carbonyl ylides, and carbonyl imines [12]. Tetrazole and its derivatives have a wide spectrum of biological activities, including antimicrobial, antifungal, HCV (Hepatitis C virus) inhibitor, potent hypoglycemic agent, and cholinesterase inhibitors [13][14][15][16][17]. Tetrazoline or substituted tetrazoline compounds have just one isomer and one carbon atom, but the tetrazoline has two tautomers, Structure 2 [18].

Experimental
The chemical materials and solvents were supplied and purchased from BDH (England), Fluke RDH (Switzerland), and Merck (Germany) Companies, Baghdad city. The Electrothermal technique (SMP30) type was used to identify the melting points of the synthesized samples. The TLC test was measured to determine the progress of the reactions by using (glass TLC 1020 GS -Silica gel-60). The synthesized compounds were characterized using (Shimadzu-8400S for IR; Kufa University, Bruker-75 MHZ and 400 MHZ for NMR; Teheran, Iran). Biological activities were measured by using petri dishes, Muller Hinton agar for bacteria test, potato agar for fungi test, and DMSO as solvent.

Chemical Part
In the presence of (0.012 mol) triethylamine, a mixture of (0.006 mol) phenobarbital 1 and (0.012 mol) of acrylonitrile was mixed with (25 ML) of absolute ethanol. The contents were refluxed for 5 hrs. After that, the mixture was acidified by (0.1N) of HCL to neutralize the mixture. The result product (85%) was filtered, washed and re-crystallized from acetone. Melting point: 170-172 0 C, R f : 0.55, TLC (4:1, Benzene: Methanol).

Biological part
The solutions were diluted by dissolving the chemically prepared compounds with dimethyl sulfoxide solvent to (0.05 gm.\ML). After the sterilization process of petri dishes at 140 0 C for one hour, every Petri dish was punctured into three equal holes (6 mm diameter) by a Cork borer. The dilute prepared compounds were placed in the holes for 24 hours at 37 0 C. Finally, the inhibition zones were measured by the ruler and compared the results with stander compounds [26][27][28]. The medium Muller Hinton Agar and potato dextrose agar were added to the petri dish as an active medium for the growth of the bacterial and fungal types respectively. The prepared compounds were tested against two kinds of bacteria ((Staphylococcus aureus (+) & Escherichia coli (-)) and two kinds of fungi (Aspergillus flavus & Candida Albicans).

Molecular docking Methodology
Virtual molecular docking studies were used to investigate the inhibitory potential of produced compounds [29]. Virtual screening was performed using Molegro Virtual Docker (MVD), which was installed on an Intel Core i7 9700k processor with 120 GB SSD, 1 TB hard drive, and an NVIDA GeForce GTX 1050 graphics card [30,31].

Preparation of Ligand
Following characterization, all synthesized structures were drawn using ChemDraw Professional 19.1 (PerkinElmer Inc, USA), and bond length and energy minimization were run on Chem3D Ultra 19.1.0.8 with the MMFF94 force field and imported to the MVD interface in mol2 format [32,33].

Homology Modelling
The protein sequence of Aspergillus flavus DNA-dependent RNA polymerase (RNAP) was retrieved from the NCBI database [34]. It was sent to the automated modeling website MODWEB, which was a comparative protein modeling web service for generating protein sequence modeling of RNAP by comparing the sequence to a template protein from Schizosaccharomyces pombe (PDB ID 3h0g) [35]. After computations, the theoretically modelled structure was evaluated and uploaded to MVD for docking [36], Table 3.
Criteria for the selection of protein was based on the resolution greater than 1.5 A and containing the gene code of same bacterial and fungal specie, whose inhibition was required [39]. Protein co-crystal structure of same bacterial and fungal species were retrieved from PDB site which was also lined up for in vitro activities [36]. Table 3, depict the summary of proteins utilized in molecular docking [40]. Discovery studio visualizer was used for removing solvent/water molecules, along with heteroatom removal. As MVD executed the docking into active sites of protein structure, therefore extra chains were removed from protein [33,36,39]. Binding site sphere was generated, and protein was imported to MVD graphical interface. Surface was created and by using detects cavity preparation tools cavities were generated [41].

Molecular Docking and Scoring Function Assessment
Molegro Virtual Docker has user friendly graphical interface and relies on differential algorithm, which measures the protein-ligand interaction in the form of functional factor Rerank score for best docked pose [42,43]. While total energy of interaction between proteinligand is measured in the form of MolDock score [44], based on PLP (pairwise linear potential) [33]. Grid resolution for docking was fixed to 0.30A, and binding site center coordinates was selected from user defined cavity 1 parameters [37]. Maximum iteration to 2000 and 50 maximum population size [29,30]. Discovery Studio Visualizer was used for generation of 3D and 2D protein-ligand interactions [31,45].

Chemistry
The main goal of the study is to prepare and characterize new barbituric acid derivatives that contain tetrazoline moiety, which is an active moiety in pharmaceutical compounds. The first step involved preparing a 3,3'-(5-ethyl-2,4,6-trioxo-5-phenyldihydropyrimidine-1,3(2H,4H)diyl)dipropanenitrile from the sreaction of phenobarbital with acrylonitrile in the presence of triethylamine as base catalyst, Scheme 1.

Biological Activity in vitro
The main goal of the work is the results of the applications of the prepared compounds; the tetrazoline derivatives have many applications in the field of medicine. From this point, these new derivatives were prepared to give high efficacy against bacteria and fungi types. The chemical prepared derivatives 7, 8 (a, b) were examined against two kinds of bacteria ((Staphylococcus aureus (+) & Escherichia coli (-)) and two kinds of fungi (Aspergillus flavus & Candida Albicans) by using Muller Hinton Agar and Potato Dextrose Agar. The biological results of prepared compounds with standard compounds are recorded in the following table 4 and figure 1.

Figure1. Antimicrobial test of some prepared compounds 7, 8 (a, b)
It is noted in the Table 3 and Figure 1 that the raw material represented by phenobarbital compound gave a low active against bacteria types, while in the fungi types it was inactive. All compounds 7,8 (a, b) gave high activity against types of fungi and equal to the standard compound (Fluconazole). The compounds (7a, 8a-b) also gave moderately active against all bacteria types, while the compound (7b) was inactive at (0.05 gm.\ML). In molecular docking antimicrobial targets was selected based on known mechanism of action of marketed antibiotic drugs.

Molecular Modelling
All synthesized compounds (7a, 7b, 8a, and 8b) were docked to the binding cavity 1 of DNA gyrase, topoisomerase and dihydropteroate synthetase of E. coli and S. aureus respectively. While antifungal in silico studies were carried out into the active sites of RNA polymerase, dihydrofolate reductase of A. flavus. Active sites of antifolates and dihydrofolates from C. albicans were inhibited by docking the synthesized compounds. Table 5 and 6 display the interaction energies in the form of MolDock score and number of protein-ligand interactions in the form of Re-rank score. Best established docked pose was selected based on MolDock score, number of hydrogen bond interactions, hydrophobic and Van der Waals (VdW) interactions [37].
Compound 7a behave as potential lead against S. aureus, as it showed maximum MolDock score for topoisomerase ATPase and DNA gyrase having three hydrogen bond interactions with DNA gyrase and ten hydrogen bond interactions with topoisomerase ATPase. Also showed best MolDock score against dihydrofolate reductase of C. albican with twelve hydrogen bond interactions. All these interactions involved oxygen, nitrogen, methylene hydrogens and hydrogen of diethyamino moieties [46].
Compound 7b showed high MolDock score against RNA polymerase and dihydrofolate reductase of A. flavus. Thus, possessing best inhibiting potential for A. flavus. As these results were in accordance with in vitro antimicrobial activity. It had four hydrogen bond interactions to the active sites of RNA polymerase from oxygen and nitrogen atoms. With dihydrofolate reductase 7b has six hydrogen bond interactions. 7b also showed alkyl and pi-alkyl interactions with the bromo atom [38].
Compound 8a act as lead compound with highest MolDock score for inhibiting the growth of antifolates of A. flavus and dihydropteroate synthetase of S. aureus respectively. 8a showed six hydrogen bond interactions along with six different types of interactions comprising VdW, hydrogen bond, pi-donor, pi-lone pair, and alkyl interactions with dihydropteroate synthetize. Antifolate showed total four hydrogen bond interactions [31].
Compound 8b inhibited the growth of DNA gyrase of E. coli, having six hydrogen bond interactions involving oxygen and nitrogen atoms. While other interactions involved halogen, pi-donor and pi-alkyl [41].      . Three-dimensional view of compounds 7a-8b from 29A-32A into the binding cavity of 6drs, 2D view by Discovery studio of compounds 7a-8b from 29B-32B for 4hof (dihyrofolate reductase of C. albicans).

Conclusions
Barbituric acid and tetrazole or tetrazoline compounds are important in various medical applications. Phenobarbital as raw material was used to make a series of barbituric acid derivatives containing 1,2,3,4-Tetrazoline moiety. The resulting compounds were characterized by various techniques as (Melting point, TLC, FT-IR, 1 H-NMR and 13 C-NMR).
In the antifungal test, all results were given high activity against types of fungi and equal to the standard compound (Fluconazole), however in the antibacterial test; they showed a wide range of activities and low values when compared to the standard compound (Ampicillin). All these results corroborate the in vitro antimicrobial activity also, as the compounds which were inactive in antimicrobial activity also showed less MolDock score in in silco study also. As these compounds showed best hydrophobic interactions along with hydrogen bond thus hypothesizing that these interactions were important for stabilization of protein ligand complex [47]. After antimicrobial docking simulation, we concluded that compound 7a can be antimicrobial lead compound as inhibitor of DNA and nucleic acid synthesis [48].