Synthesis, Molecular Docking and Pharmacological Investigation of Some 4-Methylphenylsulphamoyl Carboxylic Acid Analogs

Melford C Egbujor*1, Uchechukwu C Okoro2, Sunday N Okafor3, Ifeanyi S Amasiatu4, Ugochukwu B Amadi5, Pius I Egwuatu6 1Department of Industrial Chemistry, Renaissance University, Ugbawka, Enugu, Nigeria 2Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka, Nigeria 3Department of Pharmaceutical and Medicinal, University of Nigeria, Nsukka, Nigeria 4Department of Biochemistry, Renaissance University, Ugbawka, Enugu, Nigeria 5Chemistry Unit, Akanu Ibiam Federal Polytechnic Unwana, Ebonyi, Nigeria 6Department of Microbiology, Renaissance University, Ugbawka, Enugu, Nigeria


INTRODUCTION
The urgent need for the eradication of microbial and oxidative stress-related diseases cannot be ignored; the prevalence and negative effect of these diseases are easily noticed around the world (Jos et al., 2013), (Halliwell, 2007). Oxidative stress and microbial infections are related in their mode of in luence on the immune system (Kock et al., 1987). Generally, α-amino acids, when combined with sulphonamides, yield good anti-oxidants and antimicrobials (Egbujor et al., 2019a) , (Egbujor and Okoro, 2019) they are also known as the most excellent category of pharmacologically active amino acids (Young, 1994). It is expected that the utilization of α-amino acids in this study would achieve improved drug potency. When a compound possessing sulphonyl group is directly bonded to an amino group, sulfonamide formed becomes a central framework for drug structures because of their outstanding stability and considerable tolerance in humans (Rosenthal, 1942) , (Shet et al., 2003) . The primary sulphonamide groups are often present in various biologically active compounds that are commonly employed as antimicrobial drugs, antithyroid agents, carbonic anhydrase inhibitors and antibiotics (Suparan, 2008) , (Remko and von der Lieth, 2004). It is important to note that sulphonamides are generally utilized for the treatment of severe infections of the urinary and intestinal tracts in clinical medicine (Gaded et al., 2003). Sulphonamides are antitumor agents because of their ability to bring about an inhibition of carbonic anhydrase, especially those having aromatic and heteroaromatic structures (El-Sayed et al., 2011), (Garcia-Galan et al., 2008). Additionally, sulphonamides are bene icial pharmaceutical compounds because they possess numerous biological properties. (Egbujor et al., 2019b) Scheme 1: 4-methylbenzenesulphamoyl carboxylic acids synthesis However, the pharmaceutical relevance of sulphonamides has been adversely affected because most of the researches have been con ined to the few traditional methods of synthesis and analysis of sulphonamides which have always yielded drugs with drug actions similar to existing ones that have lost ef icacy against microbes (Karch, 2011). Moreover, the use of biologically active α-amino acids in sulphonamide synthesis has also been neglected in recent times (Green ield and Grisanu, 2008), (Fahim and Shalaby, 2019). Biologically, α-amino acids are better than ordinary amines that are conventionally utilized (Nelson and Cox, 2005) . The use of amino acids as coupling partners in drug design and synthesis have been found more biologically useful than the use of ordinary amines because of the consideration of their important drug properties and bioavailability (Bandelin and Malesh, 2006), (Remko and von der Lieth, 2004).
Consequently, the objective of this study was to synthesize new α-amino acid-based 4methylbenzenesulphamoyl carboxylic acids and evaluate their antimicrobial and anti-oxidant activities to improve their potency as drug candidates.

Reagents and Instrumentation
Reagents were supplied by Sigma Aldrich Corporation, United States of America. The melting point ranges of 4-methylphenylsulphamoyl compounds were ascertained using melting point equipment, IA9200 model of Cole-Parmer Ltd, Staffordshire, UK and were uncorrected. FT-IR spectroscopy of the synthesized compounds was determined with Shimadzu 8400s FT-IR of Shimadzu Corporation, Kyoto, Japan. Proton and Carbon Nuclear Magnetic Resonance (NMR), were ascertained with Bruker Avance III 400MHz NMR spectrophotometer of Bruker Corporation, Massachusetts, USA, and results were recorded. Nitrogen gas was utilized for all inert reaction conditions. Compounds were obtained in analytical grade, so chromatographic puri ication was not needed.

Procedure for 4-methylbenzenesulphamoyl carboxylic acids synthesis
Inside a 100ml beaker, α-amino acid (2.2g, 25mmol) was dissolved in water (30ml) and sodium carbonate (5.58g, 52.50mmol) was added. It was stirred thoroughly and cooled to -5 o C and 4-methyl sulphonyl chloride (5.12g, 30mmol) was intermittently added in four parts within 1 hour. This was followed by 4 hours of stirring at normal room temperature. On addition of 2M HCl, crystallization occurred. Reaction steps were monitored strictly using TLC (MeOH/DCM, 1:8). It was kept untouched for at least 12 hours and iltered by suction, and the solid product was washed with tartaric acid (pH2.2) and dried to afford 4-methylbenzenesulphonamoyl carboxylic acids (1-7) as represented in Scheme 1. The title compounds were represented in Scheme 2 .

Mechanism of reaction
Amino acids form zwitterions in a basic aqueous medium as a result of hydrogen ion transfer to another end (Levesque et al., 2010). This enables the nucleophilic attack of the amino group that results in the breaking of the S=O bond. It is followed by the formation and elimination of HCl thereby generating Scheme 2: The target compounds Biological studies

Antimicrobial evaluation
Some pathogenic bacteria and fungi were obtained as clinical isolates and standardized with 0.5 McFarland turbid equivalents, and these pathogenic microorganisms were standardized. O loxacin and luconazole were used as antibacterial and antifungal drugs, respectively. Using agar dilution method (Wiegand et al., 2008), the antimicrobial properties of the 4-methylphenylsulphamoyl analogues were investigated using the procedures outlined by CLSI (Sader et al., 2013), (Lamie et al., 2015).

Anti-oxidant Evaluation Procedure
The anti-oxidative properties of 4-Scheme 3: Mechanism of reaction for 4-methylbenzenesulphonamides synthesis. × 100 Where, Abs control was the absorbance of DPPH radical and n-hexane/methanol, Abs sample was the absorbance of DPPH radical and sample/standard. The graph of percentage inhibition determined the half-maximal inhibitory concentration (IC 50 ) was plotted against the concentration of the 4methylphenylsulphamoyl analogues.
In silico Procedure

Physicochemical properties
The physicochemical parameters were obtained in silico. These are the molecular weight (MW), number of hydrogen bond acceptor (HBA), number of hydrogen bond donor (HBD), number of rotatable bond (NRB), octanol/water partition coeficient logP(o/w), aqueous solubility (SlogP) and topological polar surface area (TPSA). The descriptors calculator in Swiss dock online servers was used in the computation of these parameters. Lipinski's rule of ive was the basis for the drug-likeness evaluation.

Molecular docking Assessments
Microbial infections and oxidative stress were the two disease conditions studied. Drug targets were selected accordingly for molecular assessment. The drug targets for antibacterial:

The In silico Studies of Antibacterial, Antifungal and Anti-oxidative Properties
All the 4methylphenylsulphamoyl analogs had an outstanding binding interaction with the receptors employed in these docking studies. Compounds with the lowest binding energies have the highest binding af inities and vice versa. For clarity, the molecular docking interaction of compound 4 with the receptors was examined as shown in Figures 1,  2 and 3.
The general diagnostic peaks of C=O, N-H and C=C peaks were observed in the FT-IR, 1 H-NMR, 13 C-NMR and elemental analytical data.

Antimicrobial activities
The antimicrobial studies (Table 1) revealed that compounds 1-7 have outstanding antimicrobial properties when compared to standard reference drugs. It was discovered that compounds 1, 3, 5 and 7, possess signi icant antibacterial and antifungal properties similar to o loxacin and luconazole. The antifungal resistance of Candida, Albicans could be because the amino acid intracellular pools in candida albican can reduce the antimicrobial potential of some of these amino acid-based carboxylic acids (Choudary and Rao, 1983). However, many sulfonamide derivatives exhibit good antimicrobial activities (Egbujor et al., 2020a) .

Anti-oxidant activities
The in vitro Anti-oxidative results given in (Table 2) showed that all the 4-methylphenylsulphamoyl analogues had anti-oxidative properties. Compounds 1, 3, and 4 showed impressive antioxidant activities. Compound 1 displayed the best IC 50 value of 1.104±0.001µg/ml comparable to 0.999±0.001µg/ml of ascorbic acid. Amino acids potentiated the anti-oxidant activities of sulphonamide derivatives (Egbujor et al., 2020b). This suggests that further structural modi ication of these potent compounds is required to improve their anti-oxidant activities.

Assessment of the drug-likeness and oral bioavailability
The physicochemical parameters are given in Table 3 above. Lipinski's rule of ive is often required as the assessment reference point in the determination of the drug-likeness of a molecule (Lipinski et al., 2001). According to this rule, a molecule must have a molecular weight value of ≤ 500, hydrogen bond donor ≤ 5, hydrogen bond acceptor ≤ 10, and partition coef icient (Log P) value ≤ 5. When more than one parameter is violated, there could be bio availability problems of the molecule in case of the oral formulation. From Table 3, the 4-methylphenylsulphamoyl analogues synthesized are in perfect agreement with Lipinski's rule of ive and are therefore druggable. Physicochemical parameters have long been used as a reference point for the prediction and estimation of drug pharmacokinetic properties and druglikeness (Karlgren and Bergstrom, 2015), (Petit et al., 2012). It implies that essential factors such as the absorption, distribution, metabolism, and excretion of drugs in humans could be maximized using the physicochemical properties (Almi et al., 2014). A molecule with TPSA 140 Å2 can permeate the cell. All the compounds synthesized had TPSA < 140 and therefore can permeate the cell membranes while compounds 1, 2, 4 and 7 can permeate the blood-brain barrier having TPSA less than 90 Å2.

In silico Antibacterial, Antifungal and Antioxidant studies
In silico antibacterial Activities Table 4 showed that compound 3 and 6 had a better binding af inity of -10.95 and -11.51Kcal/mol, respectively than penicillin(-10.89 kcal/mol). This implies that compounds 3 and 6 are more potent than penicillin as antibacterial agents.

In silico A ntifungal Activities
From Table 4, 1WS3 had a higher binding af inity with compounds 1, 5 and 7 of -11.11, -10.97 and -11.09 Kcal/mol respectively which weremore excellent than ketoconazole (-10.38 kcal/mol). Compound 1is the most potent antifungal agent, and this is supported by the in vitro antimicrobial analysis in which it was the only compound that could inhibit the growth of candida albican.

In silico Anti-oxidant Activities
From Table 4, compound 4 was better than αtocopherol in its interaction with 1HD2 . Compound 4 had a binding af inity of -14.90kcal/mol, which is similar to that of the standard drug α-tocopherol (-14.82 kcal/mol). Compound 4 is the most promising and could serve as anti-oxidant agents if further developed. For clarity, the binding poses of compound 4 in the binding cavities of the drug receptors were represented in Figures 1, 2 and 3. Figures 1 and 2 showed how compound 4 occupied the binding sites of 1SME and its interactions with the amino residues of the receptor. There were distinct H-bond interactions. Firstly, the O-atom of compound 4 formed H-bonds with VAL 78 and SER 79 through intermolecular distances of 3.61 and 3.57 Å respectively. Also, SER 218 interacted with another O-atom of 4 to form H-bond (4.37Å). Other forms of interactions include π-sulphur interaction between the π electrons of PHE 111 and S-atom of 4 (6.84 Å); 7 π-alkyl interactions involving ILE 123, ILE 32, VAL 78, PHE 294, ILE 300, ILE 212 and TYR 193. They interacted with the alkyl groups of 4, and their intermolecular distance of interactions are given in Figure 2. Van der Waals interaction was observed between THR 217 and compound4 (4.04 Å). The π electrons of the 6-membered aromatic ring of 4, through π-anion interaction, interacted with the ASP 214 (5.77Å). Figure 3 gives the picture of the binding interaction compound 4with 1HD2. Compound 4 was found to interact with 1HD2amino acid residues, resulting in the observed anti-oxidative properties in the biological studies. There was H-acceptor interaction between O-17 of 4 and the N GLY 46. The distance of 3.28 Å and energy (-0.5 kcal/mol) of this interaction was recorded. Likewise, O-17 of 4 through H-acceptor interaction, interacted with the NH2 of ARG 127 (3.41Å and -0.6 kcal/mol). The S-18 of 4 formed three H-acceptor bonds with CD1 LEU 116, CD2 LEU 116 and CD1 ILE 119 respectively.

CONCLUSIONS
In this research, a convenient, eco-friendly, and ef icient approach to the synthesis of 4methylphenylsulphonamoyl carboxylic acid analogues (1-7) of medicinal importance has been described. The structures were in agreement with the spectral analysis. In the in vitro antimicrobial activity analysis, compounds 1, 3, 5and 7 had antimicrobial inhibitory concentration range of 0.5-1.0mg/ml comparable with 0.1-2.0mg/ml of o loxacin and luconazole. In the in vitro antioxidant activity study compounds 1, 2 and 6 displayed half-maximal inhibitory concentrations (IC 50 ) of 1.104±0.001 µg/ml, 1.159±0.002µg/ml and1.240±0.001µg/ml respectively comparable with 0.999±0.002µg/ml of ascorbic acid. It was observed that the 4-methylphenylsulphamoyl analogues possessed good antibacterial, antifungal and anti-oxidant properties similar to their reference drugs like penicillin, ketoconazole and α-tocopherol respectively. The 4-methylphenylsulphamoyl analogues were observed to possess antibacterial, antifungal and anti-oxidative properties and therefore could be considered as potential drug candidates.