ISSN: 2410-4477 Novel Butan-2-ylidene Benzohydrazides; Synthesis, Antimicrobial Evaluation and Molecular

Enugu State, Nigeria Abstract: A variety of butan-2-ylidene benzohydrazides were synthesized via the reaction of benzohydrazides with 4,4,4-triflouro-1-phenyl-1,-3-butanedione/4,4,4 – triflouro -1, 2, - (2 – napthyl) -1, 3 – butanedione. Acetylation and benzoylation of butan-2-ylidene benzohydrazides gave the derivatives previously unknown. The compounds were characterized by spectral analyses (Mass spec., IR, and NMR), and were evaluated for their in vitro antimicrobial activity against some clinical isolates of interest. The in vitro screening revealed that some of the compounds possessed strong inhibitory potentials against the isolates with IZD in the range of 7-14 mm. Predicted ADMET ensured the druggability properties of the synthesized compounds. The molecular docking study indicated compounds 7a and 9a to possess the highest docking store and established significant interactions in the active site of


Introduction
Human exposure to pathogens has led to bacterial and fungal infections which have been one of the pivotal global health issues in recent memory. The treatment of these broad ranges of microbial infections is complicated because lots of microbes including bacteria, viruses, and fungi are becoming resistant due to inordinate and extended use of standard marketed antimicrobial and antibiotics drugs (Cassir et al. 2014). Antimicrobial resistance (AMR) and the tampering off of potent antimicrobial drugs are considered a major terror to human health. Multidrug resistance of bacteria and fungi such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus aureus (VRSA), Vancomycin-resistant enterococcus (VRE) and Fluconazole-resistant Candida have attained horrifying level becoming a global life-threatening medical issue (Mishra et al. 2013, Cetinkaya et al. 2000, Gulshan et al. 2007). The fight against drugresistant microorganisms involves the strategic development of new, cheap, and more powerful innovative antimicrobial agents with minimal adverse effects (Khan et al. 2005, Klenke et al. 2003). Therefore, the need to develop novel broad-spectrum antibacterial motifs against both Gram-positive (G+) and Gram-negative (G-) bacteria for the treatment of bacterial infections, especially drug-resistant strains remains a practicable objective of most chemists.
Moreover, in vitro metabolism analyses of hydrazide biological functionalities indicate that they can readily experience hydrolytic reactions, which is an advantage to treat several infectious diseases (Kömürcü et al. 1995, Ulgen et al. 1997. Inspired by these findings, we decided to design some new libraries of benzohydrazides for their antimicrobial investigation. We had earlier reported other biological motifs as likely antimicrobial agents (Ezeokonkwo et al. 2018(Ezeokonkwo et al. , 2019. In continuation of our effort to construct potent antimicrobial agents, we herein report the synthesis of (Z)-(substituted)-N'- The structural characterization of the synthesized compounds was done using UV, FTIR, NMR, and mass spectroscopy. The antimicrobial potentials of the synthesized compounds were evaluated in vitro via agar-well dilution method. The compounds were also assessed for their drug-likeness, and molecular docking was carried out to find out the binding affinities of the compounds and their bonding interactions with the target protein.
Compounds (3a-c) were converted to their corresponding acetyl derivatives (7a-b) by acetylation using hydrated sodium acetate (6), and benzoyl derivatives (9a-c) via benzoylation using benzoylchloride (8) at room temperature. In the representative compound 7a, the absorption bands at 1649 and 1495 cm -1 in the FTIR spectrum are due to C=O and C=N stretching respectively. The 1 H NMR spectrum of compound 7a showed peaks at 3.70 ppm assigned to the methyl protons (H-24), 2.33 ppm assigned to methylidyne proton (H-12), while aromatic protons were observed in the range of 7.99-7.08 ppm. The 13 C NMR spectrum of compound 7a showed peaks at 195.54, 179.33 and 170.67 ppm for C=O (C-13, C-7, and C-22 respectively), 154.08 ppm for C=N (C-11), 113.02 (C-15), while aromatic protons were observed at peaks in the range of 150.01-124.74 ppm.
In the representative compound 9b, the stretching for C=O, C=N, and C-O in the FTIR spectrum was observed at 1651, 1498, and 1094 cm -1 respectively. In the 1 H NMR spectrum of compound 9b, the peaks in the range of 8.14-7.07 ppm are due to aromatic protons, while the peaks at 3.92 and 3.31 are due to methyl protons (H-22) and methylidyne proton (H-12). The 13 C NMR spectrum of compound 9b showed signals at 172.67 pmm due to C=O (C-13), 167.84 ppm due to C=O (C-7 and C-23). Other signals shown are in agreement with the proposed structure. Furthermore, the reaction of benzohydrazides (1a-b) and 4,4,4-trifluoro-1-(naphthalen-2-yl)butane-1,3dione (4)

in acetonitrile at room temperature produced the (Z)-(substituted)-N'-(1,1,1-trifluoro-4-
The compounds were obtained in good to excellent yields. The FTIR spectrum of compound 5a for example showed absorption bands at 3445 cm -1 assigned to N-H, 1651 cm -1 assigned to C=O, and 1497 assigned to C=N stretching. The signals observed in the 1 H NMR and 1 C NMR spectra of compound 5b also support the proposed structure.

Antimicrobial activity 2.2.1 Antifungal
The synthesized compounds were evaluated for their antifungal activity against C. albican and A. niger. The result of the sensitivity test is presented in Table  1. Compounds 3c, 7a-b, 9a showed marked antifungal activity against C. albican, with compound 7a being the most active with the IZD of 14 mm. All the compounds were active against A. niger except compounds 3b, 7a and 9c, with compound 5a showing the strongest activity with the IZD of 11mm.

Antibacterial
The synthesized compounds were screened for their antibacterial potential against gram +ve and gram -ve bacterial strains.
All the compounds except compound 7b were active against S. typhi with the IZD in the range of 7-11 mm. Compound 5b showed marked activity against E. coli with the IZD of 11.5 mm. Compounds 5a and 7a revealed the highest inhibition against S. aureus and B. subtilis with the IZD of 10.5 and 10 mm respectively, while compound 5b (IZD: 9 mm) had a comparable antibacterial inhibitory potential to Gentamicin standard (IZD: 10 mm) against Shingella. sp.

In silico ADMET properties
The drug-likeness of the synthesized compounds was evaluated using SwissADME free online tool (http://www.swissadme.ch/). Lipinski's rule of five is a vital assessment of a drug-like molecule. According to the rule (MW ≤500, HBD ≤5, HBA ≤10, Log p ≤5, RBC ≤10) (Lipinski 2000), the synthesized compounds possessed good drug-like properties with zero violation of the rule, Table 2. Also, the compounds possessed tPSA in the range of 58.53-112.63 Å 2 (Veber rule: tPSA ≤140 Å 2 ) (Veber et al. 2002), which further validates their good oral bioavailability and cell permeability.   All the synthesized compounds except 9c revealed stronger binding affinity with the protein, exhibiting better binding energy than ciprofloxacin standard and the native ligand, Table 3. Compounds 7a and 9a had the highest binding energy with -10.10 and -9.80 Kcal/mol respectively. The binding interactions of the most active compounds 7a and 9a against S. typhi have been shown in Figures 4 and 5. Compounds 7a and 9a are well stabilized in the active pocket of the target protein forming significant hydrogen bonding and other hydrophobic and electrostatic interactions. Compound 7a established hydrogen bonding with VAL 1198, ARG 1208 and LYS 1023 residues, while compound 9a formed hydrogen bonding with ARG 1232, ARG 1208, TRY 1159 and LYS 1023 residues. These strong chemical interactions may explain the relatively high inhibitory potentials of these compounds against S. typhi.

Conclusions
We have synthesized novel benzohydrazides and evaluated their antimicrobial potentials against clinical isolates of interest. Some of the target compounds demonstrated weak to excellent inhibition against the tested microorganisms. All the compounds synthesized were active against S. typhi, with compounds 7a and 9a showing the highest sensitivity. In silico drug-like prediction favoured the oral bioavailability of the synthesized compounds. Molecular docking results revealed that the compounds had better binding affinity than ciprofloxacin thereby depicting good inhibition potential. Though the target compounds demonstrated lower in vitro antimicrobial inhibition compared to Gentamicin and Ciprofloxacin standard, the results suggest that these compounds are a good starting point for the rational development of novel antimicrobial agents.

Chemistry
All commercial chemicals were used as received from Sigma-Aldrich and all solvents were purified using standard procedures prior to use. For thin-layer chromatography (TLC) analysis, Merck pre-coated plates (silica gel 60 F254) were used and spots were visualized under UV light. Melting points (m.p) determination was performed using Guoming Melting point tester and are uncorrected. Ultraviolet and visible spectra were recorded on UV-VIS G6860A spectrophotometer using matched 1 cm quart cell; absorption maxima were given in nanometers (nm). The IR spectra were recorded on 630 FT-IR spectrometer. 1 H NMR spectra were recorded on a Bruker Avance II 400 MHz and 13 C NMR spectra on Varian Mercury-400 100 MHz in DMSO as a solvent and tetramethylsilane (TMS) as an internal standard. Mass spectra were scanned on a Bruker Daltonics micrOTOF spectrometer at School of Science and Engineering Laboratory, University of Wakaito, Hamilton, New Zealand. Chemical shifts were reported in ppm units with the use of δ scale. The antimicrobial screening was done at the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka. All the products were purified through repeated recrystallization using suitable solvent(s).

Antimicrobial assay
Antimicrobial activity potentials of the synthesized benzohydrazides were determined by agar-well dilution method (Sridhar et al. 2016 Commercially available antibiotics Ciprofloxacin and Gentamicin were used as reference drugs.

In Silico Molecular Prediction
SwissADME, a free online tool (http://www.swissadme.ch/) developed and maintained by the molecular modeling group of the Swiss Institute of Bioinformatics (SIB) was used to evaluate the drug-likeness of the synthesized compounds (Daina et al. 2017). The molecular descriptors calculated include molecular weight (MW), partition coefficient (log P), hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), topological polar surface area (tPSA), number of rotatable bond (RBC).

Molecular Docking Study
Auto Dock permits the understanding of the molecular interactions between a ligand and a receptor in terms of free binding energy and bonding interactions. Molecular docking was carried out to determine the binding energy and bonding interactions of the synthesized benzohydrazide against a S. typhi receptor. 3D crystal structure of the target receptor (PDB code: 1WVG) was retrieved from the protein data bank with resolution 3.46 Å and prepared using BIOVIA discovery studio 2017 R2 version 17.2.0.16349. The preparations included deleting of multiple chains, water of crystallization, energy minimization and defining of binding site. The grid box size of the binding site was determined by checking the binding site attributes (X= 30.055917, Y= 26.457861, Z= 39.084694, radius = 9.163073). The structures of synthesized compounds were drawn using ChemDraw and converted to their 3D form using Discovery studio. The synthesized compound was docked into the active site of the target receptor using Autodock/Autodock vina (Trott and Olson 2010). The docking results were analyzed using BIOVIA discovery studio. The binding modes with significant binding scores were selected (Lengaur and Rarey 1996, Reetu 2012).
interests financially or otherwise

Funding:
Authors declare that no specific fund was received for this research