Design, synthesis, docking studies and antibiotic evaluation (in vitro) of some novel (E)-4-(3-(diphenylamino)phenyl)-1-(4-methoxyphenyl)-2-methylbut-3-en-1-one and their analogues

Antibiotic resistance has risen as a result of a variety of conditions, prompting researchers to look for new compounds that can combat multidrug-resistant organisms. Over the last two decades, chalcones have been proved to be attractive moieties in drug discovery. Various substituted acetophenones, propiophenones and 4-(Diphenylamino) benzaldehyde were combined, using the Aldol condensation reaction to obtain eight novel triphenylamine chalcones. The compound’s antimicrobial properties were investigated (in vitro). With the non-mutant X-ray Human cytochrome P450 21A2 Hydroxyprogesterone retrieved from Protein Data Bank (PDB: 5VBU), molecular docking experiments were also carried out to analyse the most favourable conformation and find the orientation that maximizes interaction and minimize energy. Eight novel triphenylamine chalcones were successfully synthesized and recrystallized using ethanol, the percentage yield of the compounds were between 30 and 92%. The activity against different pathogens revealed that, all synthesized compounds showed marked antimicrobial activity against the tested microorganisms. (E)-3-(4-(diphenylamino)phenyl)-1-(3′-nitrophenyl)prop-2-en-1-one (1b) showed the highest zone of inhibition against Aspergillus niger, measuring 30 mm. The minimum inhibitory concentration (MIC) results revealed that (E)-1-(4′-bromophenyl)-3-(4-(diphenylamino)phenyl)prop-2-en-1-one (1a), (E)-3-(4-(diphenylamino)phenyl)-1-(3′-nitrophenyl)prop-2-en-1-one (1b), (E)-1-(4′-chlorophenyl)-3-(4-diphenylamino)phenyl)prop-2-en-1-one (1c), (E)-3-(4-diphenylamino)phenyl)-1-(4′-fluorophenyl)prop-2-en-1-one (1d) and (E)-4-(3-(diphenylamino)phenyl)-1-(4-fluorophenyl)-2-methylbut-3-en-1-one (2d) had the lowest MIC and inhibit Aspergillus niger growth at 12.5 µg/ml. All the synthesized compounds showed minimum bactericidal concentration and minimum fungicidal concentration (MBC/MFC) effect against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger at 50 µg/ml. The docking studies of the synthesized chalcones with the binding site of the Human cytochrome P450 21A2 Hydroxyprogesterone (PDB: 5VBU) reveal that the binding affinity of the synthesized chalcones was in the range of − 11.2 to − 9.4 kcal/mol and showed highest binding score compared to that of the standard drugs (Fluconazole and Ciproflaxacin), with docking scores of − 7.9 and − 7.3 kcal/mol, respectively. The investigation reveals that compound 1b showed the highest ZOI of 30 mm, least MIC and MBC/MFC of 12.5 and 50 µg/ml against Aspergillus niger, respectively. Therefore, displayed better antifungal potential as compared to the rest of the compounds. The outcome of the docking analysis revealed that (E)-4-(3-(diphenylamino)phenyl)-1-(4′-hydroxyphenyl)-2-methylbut-3-en-1-one (2a) showed a better binding affinity of -11.2 kcal/mol, which is higher than the remaining compounds and the control drugs (fluconazole and ciproflaxacin).


Background
The chemistry of chalcones has generated a bustle of research around the globe. The synthesis, chemical reactions, and biological applications of these compounds have captivated the attention of researchers (Singh et al. 2014;Kumara et al. 2017). Chalcones are characterized by a conjugated double bond on both benzene rings and a totally delocalized π-electron system. (Maidur and Patil 2018), which is dependent on the presence of other auxochromes. β-phenyl-α-benzoyl-ethylene is another name for chalcones (Ugwu et al. 2015). Chalcones are abundantly found in spices, tea, fruits, and vegetables as one of the principal classes of natural products (Kumar et al. 2021). Claisen-Schmidt condensation of acetophenones with benzaldehydes is the most prevalent and efficient methods for producing chalcones. The yields are typically excellent, and reaction takes place at lower temperature. The duration for reaction varies from 3 to 20 h depending on the substitution pattern and the solvent/base system utilized, (Bukhari et al. 2012;Adnan et al. 2020).
Due to the continued resistance and resurgence of pathogens against available antibiotics, the constant search for potentially novel drug candidates has emerged. Simultaneously, researchers developed the concepts of protein purification and crystallography, allowing researchers to learn more about the protein and ligand interactions. Today, computational methodologies are pervading many facets of drug development (Walters et al. 1998;Bajorath, 2002;Jorgensen, 2004). Molecular docking investigations have become important tools in the search for new drugs candidates (Langer and Hoffmann, 2001). This technique represents atomic level interface with target protein, allowing us to define small molecule behaviour in target protein binding sites (McConkey et al. 2002). The process also determines the ligand structure, location, and orientation within the binding site, as well as the binding affinity (Kitchen et al. 2004;Prabhudeva et al. 2019).
Human cytochrome P450 21A2 Hydroxyprogesterone (PDB: 5VBU) is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone to 11-deoxycortisol. Deficiency of this enzyme is involved in approximately 95% cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis, Pradeep et al. (2015). To our knowledge, there is no report on synthesis, antibiotic evaluation and molecular docking studies of triphenylamine chalcones. Hence, present research intended to synthesize, screen antimicrobial potential and study the molecular docking of novel triphenylamine chalcones.

Synthesis of the novel triphenylamine chalcones analogues (1a-d, 2a-d)
To 25 ml ethanol taken in 100 ml round bottomed flask equipped with a magnetic stirrer, were transferred equimolar quantities of 4-(diphenylamino)benzaldehyde (0.4 g, 1.5 mmol) and substituted acetophenones/propiophenone (0.4 g, 2 mmol) and stirred for 30 min. A solution of sodium hydroxide (10 mL, 10%) was added dropwise while stirring. The temperature for the reaction was maintained between 20 and 24 ℃ using cold water bath for 4-5 h. The progress and completion of the reaction were monitored using thin-layer chromatographic technique (TLC). On completion, the reaction mixture was placed in a refrigerator for 10 h. Formation of precipitate was observed, which was filtered, washed severally with water (100 ml), dried in air and purify by recrystallization from ethanol (30 ml) to obtained a triphenylamine chalcone, (1a-d, 2a-d). Hongtian et al. (2019). Table 1 shows the structures of the synthesized triphenylamine chalcones and their percentage yields (Schemes 1, 2). The synthesis of the target novel triphenylamine chalcones was achieved via the conventional Claisen-Schmidt condensation reaction, where different equimolar substituted acetophenones/propiophenones and 4-(Diphenylamino) benzaldehyde in the presence of NaOH (10%) were stirred. This reaction was originated by the abstraction of proton from the alpha-carbon of the acetophenones/propiophenones to generate the resonance stabilized enolate ion by the base. The second stage was the nucleophilic attack on the electron deficient carbonyl carbon of 4-(Diphenylamino) benzaldehyde, which results in the formation of a new C-C bond. This connects the alpha-carbon of the acetophenones/ propiophenones to the aldehydic or carbonyl carbon of 4-(Diphenylamino) benzaldehyde to which form an intermediate. The reaction was completed by protonation and deprotonation by the hydroxyl ion of the base to form the targeted α,β-unsaturated triphenylamine chalcone.

Zone of inhibition (ZOI)
The (ZOI) of the synthesized triphenylamine chalcones was carried out according to the procedure as described by Karou et al. (2006).

Minimum inhibitory concentration (MIC)
The (MIC) of the synthesized triphenylamine chalcones was determined by using the broth dilution method as reported by Bruton et al. (2007).

Minimum bactericidal/fungicidal concentration (MBC/MFC)
The MBC/MFC of the synthesized triphenylamine chalcones was determined according to the procedure, as described by CLSI, 2015 (CLSI = Clinical and Laboratory Standard Institute).

Molecular docking studies
Molecular Docking Analysis for eight (8) ligands (novel triphenylamine chalcones analogues) and controls (Fluconazole and Ciproflaxacin) was carried out to examine the most favourable interaction and identify the orientation which maximizes interactions and minimizes energy with the target receptor (PDB: 5VBU) (www. rcsb. org) (Fig. 1).

Preparation of the receptor
The three-dimensional structure of target receptor ( Fig. 1) retrieved from the PDB was prepared by removing the water molecules and other heteroatoms, before minimization for the docking study. Discovery Studio Visualizer software v.21.1.0.20298 was used for the preparation. The treated target receptor (Fig. 2) was saved in PDB file format and transferred to Pyrx software for docking.

Preparation of ligand
All the eight (8) ligands (triphenylamine chalcones analogues) and controls (Fluconazole and Ciproflaxacin) studied were designed and synthesized as mentioned earlier. ChemDraw ultra 8.0 software was used to generate the two-dimensional structures of the synthesized chalcones (Li et al. 2004). Spartan software (Spartan'20 v.1.1 /2020) was used to convert the 2D structures to 3D. Geometrical optimization using the AM1 semiempirical method was performed on all the compounds using the Spartan software and saved as pdb files. Polar hydrogens were added before computing Gasteiger charges, using the BIOVIA DiscoveryStudio2021.

Molecular docking
PyRx virtual screening software was used to study the ligand-receptor interactions between the target receptor (PDB: 5VBU) and the eight (8) synthesized ligands (novel triphenylamine chalcones analogues) and

Results of antimicrobial studies
See Tables 2, 3 and 4.

Antimicrobial studies
All the synthesized compounds were shown to possess remarkable activities against the tested microbes, by showing a significant zone of inhibitions relative to that of the standard drugs used as shown in  Table 3 presents the results of the MIC and showed that compounds 1a, 1b, 1c, 1d and 2d possess the least MIC and inhibit the growth of Aspergillus niger at 12.5 µg/ml. While compounds 1b, 1c, 1d, 2a, 2b, 2c and 2d inhibit the growth of Candida albicans, Bacillus        Table 4.

Molecular docking studies
Each ligand/chalcones was successively docked to the binding site of the receptor (PDB: 5VBU), in order understand the mode of interaction of the triphenylamine chalcones with the target receptor. The analysis of the docking investigations provided us with an insight into the interactional relation of the novel triphenylamine chalcone analogues and human cytochrome P450 21A2 Hydroxyprogestrerone complex. Table 5 shows the results of the binding affinity of the synthesized triphenylamine chalcones/ligands, which ranges between − 11.2 and − 9.4 kcal/mol. Compound 2a showed the highest docking score of − 11.2 kcal/ mol, followed by compounds/ligands 1b and 2b which showed the binding energy of − 10.7 kcal/mol. While compounds 1c, 2c, 2d and 1a showed the docking scores of − 10.4, − 10.3, − 10.3 and − 10.1 respectively. Compound/ligand 1d showed the least binding score of − 9.4. The eight (8) ligands (novel triphenylamine chalcones analogues) showed highest binding score compared to that of the standard ligand/compounds (fluconazole and ciproflaxacin), which showed the docking scores of − 7.9 and − 7.3 kcal/mol, respectively.
Ligand/triphenylamine chalcone (2a) showed the highest binding affinity (− 11.2 kcal/mol) compared to other compounds. The interaction of the ligand/ triphenylamine chalcone (2a) with the target receptor (PDB: 5BVU) is shown in Figs. 3, 4 and 5. The interaction was observed with one hydrogen, one electrostatic attraction and six hydrophobic interactions (2.5449, 4.0569, 3.73562, 4.34337. 5.2863, 5.48907, 5.44454 and 5.35682 Å) with (ARG427, LYS121, VAL470, TRP202, VAL360, LEU364, LEU108, LEU430) as shown in Figs. 6  and 7. The two-dimensional structural form of the ligandreceptor complex is shown in Fig. 8. One hydrogen bond interaction with ARG427 was formed, due to the presence of OH group in the triphenylamine chalcone/chalcone (2a). The electrostatic interaction of the ligand/ triphenylamine chalcone was detected with LYS121. Also, the hydrophobic interactions were noticed with VAL470, TRP202, VAL360, LEU364, LEU108 and LEU430 of the target receptor. The areas which represent the both hydrogen bond and hydrophobic interactions between the target receptor and the ligand/triphenylamine chalcone are shown in Figs. 6 and 7. Therefore, ligand/triphenylamine chalcone (2a) fits perfectly into the binding site of the receptor (PDB: 5VBU). All type of interactions, amino acids and bond length are shown in Table 6.

Correlation of molecular docking studies and antimicrobial studies
The docking studies of the synthesized chalcones with the binding site of the Human cytochrome P450 21A2 Hydroxyprogesterone (PDB: 5VBU) reveal that the binding affinity of the synthesized chalcones was in the range of − 11.2 to − 9.4 kcal/mol and showed highest binding score compared to that of the standard drugs (fluconazole and ciproflaxacin), with docking scores of − 7.9 and − 7.3 kcal/mol, respectively. Compound 2a displayed best docking score of -11.2 kcal/mol. The binding affinity, hydrogen bond and hydrophobic interactions of the synthesized chalcones and the standard controls (fluconazole and ciproflaxacin) are summarized in Tables 5  and 6

Conclusions
Synthesis of eight novel triphenylamine chalcones was successively carried out via the conventional Claisen-Schmidt condensation reactions, and the compounds were successfully characterized using FT-IR and NMR spectroscopic analyses. The results of the antimicrobial screening of the synthesized chalcones as indicated by zone of inhibition (ZOI) showed that all the synthesized compounds possess remarkable activities against the tested microbes, by showing a significant zone of inhibitions relative to that of the standard drugs used. Compound 1b showed the highest ZOI of 30 mm, least MIC and MBC/MFC of 12.5 and 50 µg/ml against Aspergillus niger. The outcome of the docking studies revealed that compound 2a showed marked docking score with binding affinity of − 11.2 kcal/mol, which is higher relative to other compounds and the standard controls (fluconazole and ciproflaxacin). Therefore, compounds 1b and 2a which showed better antifungal and highest binding affinity could be potential candidates in drug design.