Novel Alanine-based Antimicrobial and Antioxidant Agents: Synthesis and Molecular Docking

Objective: To synthesize new alanine-based phenyl sulphonamide derivatives with significant antimicrobial and antioxidant activities. Methods: The reaction of alanine with benzenesulphonyl chloride afforded compound 3a. The ammonolysis of its N-acylated derivative gave the carboxamide which yielded the aryl/heteroaryl derivatives compounds 3d, 3e, and 3f via Buchwald–Hartwig nickel catalyzed amidation reaction. Structures agreed with the spectra data. Their antimicrobial activities, antioxidant activities, and molecular docking interactions were evaluated. Findings: Compounds 3f and 3a were the best antimicrobial agents with minimum inhibitory concentration (MIC) range of 0.5–1.0 μg/ml while compound 3a displayed the highest in vitro antioxidant percentage inhibition of 95.70% and the best 50% inhibitory concentration (IC50) value of 1.072 ± 0.002 μg/ml comparable to ascorbic acid of 96.38% antioxidant percentage inhibition and 0.999 ± 0.001 μg/ml IC50 value. Compounds 3c, 3a, and 3f had the highest in silico antibacterial, antifungal, and antioxidant binding energies of −10.03, −11.79, and −13.13 kcal/mol, respectively. Novelty/improvement: Alanine was found to potentiate the antimicrobial and antioxidant actions of benzenesulphonamide and carboxamide derivatives.


Introduction
The world is faced with the enormous challenge of combating microbial and oxidative stress-related diseases; these two disease conditions have brought untold misery to millions of individuals around the world [1][2][3]. Their relationship is based on the fact that oxidative stress causes a decline in immunity thereby making the body vulnerable to microbial infections [4]. Amino acids possess antimicrobial and antioxidant abilities and therefore could be explored as antimicrobial and antioxidant agents [5][6]. Obviously, alanine is an excellent target for the design of antimicrobial drugs because of its ability to be metabolized by bacteria and alanine being an α-amino acid is utilized in protein biosynthesis and glucosealanine cycle [7][8]. Alanine plays a central role in the conversion of simple sugar to energy [9], processing of vitamin B [7,10], and as a precursor of numerous biomolecules [11].
The ability of microorganisms to resist the preventive and curative effect of drugs that were previously efficacious is worrisome and the increasing dominance of these pathogenic microorganisms is a matter of concern. The global report on surveillance by the World Health Organization (WHO) in 2014 showed that more than 700,000 deaths caused by microbial infections occur annually across the globe of which 23,000 is found in the United States [27][28]. Obviously, the intake of natural antioxidants reduces the risk of oxidative stress-related cases [29][30], yet these diseases are still prevalent. The best solution to these disease conditions is to synthesize drug compounds working on new and strategic target drug sites [31]. The increasing public demand for global effort to combat antimicrobial recalcitrance and effectively tackle oxidative stress-related ailments [32] necessitates the need for the development of new alanine-based antimicrobial and antioxidant agents with enhanced drug properties. This study exploited the synergistic drug actions offered by the coupling of alanine, benzenesulphonamide, carboxamide and aryl/heteroaryl moieties into single drug compounds in order to enhance drug potency to eliminate or minimize the antimicrobial resistance.

Instrumentation
The basic chemicals were imported from Sigma Aldrich. Melting point was ascertained by melting point apparatus. The infrared spectra were obtained with 8400s Fourier Transform Infrared in Ahmedu Bello University, Nigeria. Both 1 H-NMR and 13 C-NMR spectroscopy were conducted with 400 MHz NMR spectrophotometer at Chemistry Department, Indian Institute of Technology, Kanpur. Chemical shifts were determined with reference to tetramethylsilane. Inert conditions were provided with nitrogen gas. Compounds were obtained in analytical grade. The antimicrobial and antioxidant studies were done at the Microbiology and Biochemistry Departments, respectively, University of Nigeria, Nsukka.

Acylation of 2-[(Phenylsulphonyl)amino]propanoic acid (3a)
2 g of compound 3a was transferred to a 100 ml beaker containing 9 ml HCl, 25 ml distilled water and 13 ml acetic anhydride. A solution of 16.0 g of Na 2 CO 3 and 50 ml distilled water was added to the solution of compound 3a stirred, cooled to 0 °C and filtered to obtain compound (3b) in 94.3% yield.

Bis(triphenylphosphine)nickel(II)chloride
Venanzi method [33] of reaction protocol was employed it involves combining a solution of Nickel(II)chloride hexahydrate (10 mmol) in 2 ml distilled water and 50 ml glacial acetic and triphenylphosphine (20 mmol) dissolved in 25 ml glacial acetic acid. The crystals were filtration and dried in desiccators.

Antimicrobial Evaluation
Wiegand et al agar dilution method [34] was used. Test microbes were various bacteria and fungi obtained from the Department of Pharmaceutical Microbiology and Biotechnology laboratory, University of Nigeria, Nsukka.

Standardization of the Test Microbes' Suspension
The test microbes were standardized using 0.

Control Test (Standard)
Ofloxacin and Fluconazole were the standard antimicrobial agents.

Experimental
Various concentrations of the compound used were 0.9 mg/ml, 0.8 mg/ml, 0.7 mg/ml, 0.6 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml, and 0.1 mg/ml. The sample containing molten agar plates were allowed to gel and the plates were divided into seven. Microorganisms were streaked on the divisions and the culture plates incubated at 37 °C for 24 h, and at 25 °C for 2 days. The results were recorded.

Antioxidant Activity by DPPH Method
Using Blois method [35], the compounds' inhibition of stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical [37] informed their antioxidant activities. The DPPH solution was obtained by dissolving 1.9 mg of DPPH in 100 ml of methanol. Then 50, 100, and 200 µg/ ml concentrations of the DPPH solution were also prepared. Similarly, 50, 100, and 200 µg/ml of the title compounds were prepared. Using the same method, standard solution of ascorbic acid was prepared. Then, 1 ml of DPPH solution was added to 2 ml solution of the title compounds and ascorbic acid. The reaction mixture was stirred and left in the dark at room temperature for 30 min after which the absorbance was recorded at the wavelength of 517 nm against the corresponding blank solution spectrophotometrically in triplicate. The percentage inhibitions of scavenging DPPH free radical were calculated using the following formula: ( ) t t DPPH radical scavenging activity % 100 − = × con rol sample con rol

Abs
Abs control = the absorbance of DPPH radical and n-hexane/methanol, Abs sample = the absorbance of DPPH radical and sample/standard.

Molecular Docking
The molecular docking studied some bacterial infections, fungal infections, and oxidative stress using their appropriate drug target whose 3-dimensional structures have gotten from the Protein Data Bank (PDB), (http://www.pdb.org) database. The drug targets for antibacterial was (PDB code: 5MMN); antifungal was (PDB code: 1WS3), and antioxidant was (PDB code: 1HD2). The prepared compounds interacted with each of the receptors through molecular docking using PyRx. The best conformation for each compound was selected and visualized using the Discovery studio.

Chemistry
The synthetic route of alanine-based sulphonamide derivatives is shown in Scheme 1.

Chemistry
As shown in Scheme 1, the acylation was a protection strategy to prevent the amino group from side reactions with oxidizing agents and electrophiles and ensure regioselectivity. Chlorination was to generate a reactive intermediate and ammonolysis was carried out in order to form the carboxamide from the carboxylic acid end of the alanine amino acid [36].

Antimicrobial Activities
The antimicrobial activities ( Table 1) show that all the title compounds exhibited considerable antimicrobial activities. Compounds 3f and 3a possessed the best antimicrobial activities. The best antimicrobial activities displayed by compound 3f could possibly be due to the coupled aminopyrimidine moiety because aminopyrimidines are known to possess good antimicrobial activities [23]. Moreover, amino acids such as alanine were found to potentiate the antimicrobial activities of sulphonamide derivatives [37].

Drug-likeness and Oral Bioavailability of Compound
From compounds was evaluated. Compounds with TPSA ≤140 Å 2 can penetrate the cell [38], and compounds with TPSA ≤90 Å 2 can penetrate the blood-brain-barrier (BBB) and affect the central nervous system (CNS). The results showed that while all the title compounds 3a-3e can penetrate the cell, only compound 3a of TPSA 83.47 Å 2 can penetrate the blood-brain-barriers and therefore could be utilized in treatment of celebral malaria and Alzheimer's diseases which are CNS-related diseases. Lipinski's ro5, stipulated that the drug-likeness of a drug candidate ascertained if the number of hydrogen bond donor (HBD) ≤ 5 lipophilicity (logP) ≤ 5, number of hydrogen bond acceptor (HBA) ≤ 10, molecular weight (MW) ≤ 500. The results in Table 3 revealed that the compounds satisfy Lipinski's rule of 5 (Ro5). Similarly, Van de waterbeemd et al. [38] also stated that the NRB greatly determine the oral bioavailability in rats and NRB ≤10 is a requirement for good oral bioavailability. Summarily, in compliance with these principles earlier mentioned, all the compounds were qualified as likely drugs compounds with good oral bioavailability.

Conclusion
A facile synthesis of alanine-based antimicrobial and antioxidant agents was successfully accomplished. The assigned structures complied with the spectral data. Compound 3f and 3a were found to be the best antibacterial and antifungal agents, respectively. Compound 3a was found to exhibit the best antioxidant activities, could penetrate the blood brain barrier and outperformed Ketoconazole in the in silico antifungal assessment. Compounds 3c and 3f exhibited excellent in silico antibacterial and antioxidant activities comparable to penicillin and α-tocopherol, respectively. The title compounds were found to be likely drugs with good oral bioavailability properties and confirmed to be potential antimicrobial, antioxidant agents.