Novel Leu-Val Based Dipeptide as Antimicrobial and Antimalarial Agents: Synthesis and Molecular Docking

The increase of antimicrobial resistance (AMR) and antimalarial resistance are complex and severe health issues today, as many microbial strains have become resistant to market drugs. The choice for the synthesis of new dipeptide-carboxamide derivatives is as a result of their wide biological properties such as antimicrobial, anti-inflammatory, and antioxidant activities. The condensation reaction of substituted benzenesulphonamoyl pentanamides with the carboxamide derivatives using peptide coupling reagents gave targeted products (8a-j). The in silico antimalarial and antibacterial studies showed good interactions of the compounds with target protein residues and a higher dock score in comparison with standard drugs. In the in vivo study, compound 8j was the most potent antimalarial agent with 61.90% inhibition comparable with 67% inhibition for Artemisinin. In the in vitro antimicrobial activity, compounds 8a and 8b (MIC 1.2 × 10−3 M and 1.1 × 10−3 M) were most potent against S. aureus; compound 8a, 8b, and 8j with MIC 6.0 × 10−3 M, 5.7 × 10−4 M, and 6.5 × 10−4 M, respectively, were the most active against B. subtilis; compound 8b (MIC 9.5 × 10−4 M) was most active against E.coli while 8a, 8b and 8d were the most active against S. typhi. Compounds 8c and 8h (MIC 1.3 × 10−3 M) each were the most active against C. albicans, while compound 8b (MIC 1.3 × 10−4 M) was most active against A. niger.


INTRODUCTION
Infectious diseases and their resistance to many available commercial drugs have remained the most challenging task for human existence. Sulfonamides consist of major drug components called sulfa drugs. The functional group -SO 2 NH 2 in sulfonamides enables it to possess many pharmacological properties such as antibacterial (Qadir et al., 2015), antifungal (Jyothi and Madhavi, 2018), antimalarial (Maloy Kumar et al., 2018), anticancer (Abdelaziz et al., 2015), inhibitors of human carbonic anhydrase I (hCA I) and human carbonic anhydrase II (hCA II, Kilicaslan et al., 2016), anti-HIV (Jiao et al., 2010), and many others. Carboxamides are also ubiquitous functionality GRAPHICAL ABSTRACT | in medicate particles as pharmacophores (Montalbetti and Falque, 2005). Carboxamides have been accounted for as antimicrobial and antioxidant (Eze et al., 2019), carbonic anhydrase enzyme inhibitor and antioxidant (Deniz et al., 2019), anticancer (Kumar et al., 2009), anthelmintic (Ugwu et al., 2018a), antitubercular (Ugwu et al., 2014), antitrypanosomal (Ugwu et al., 2018b), and anti-inflammatory and analgesic (Ugwu et al., 2018c) agents. Day and Greenfield (2004) reported that peptides are resourceful pharmacophores as they play important roles within the physical body and other organisms. As a result of good properties of peptides such as solubility, permeability, and bioavailability, many short peptide derivatives possess the ability to bind to membrane receptors (Qi et al., 2010;Thompson et al., 2012). Khavinson and Anisimov (2000) reported Lys-Glu based dipeptide as an antitumor agent. In 2004, Nitta et al. (2004) reported the neuroprotective effect of Leu-Ile. In the work of Kayser and Meisel (1996), Tyr-Gly was reported to reinforce the proliferation of peripheral blood lymphocytes. Peptides have also been reported as an antimicrobial (Jatinder et al., 2015), carbonic anhydrase I, II, IV, and XII inhibitor (Zehra Küçükbay et al., 2016;Küçükbay et al., 2019), antiplasmodial (Amit et al., 2015;Jatinder et al., 2015), and antihypertensive agent (Kitts and Weiler, 2003). In a continuation of our work, we synthesized novel Leu-Val dipeptide carboxamide scaffolds bearing sulfonamide moieties with potent antimalarial and antimicrobial properties. The quest for leu-val combination skeleton was as a result of antimalarial properties of ala-gly dipeptides (Ugwuja et al., 2019), antimalarial and antioxidant property of val-val dipeptides by Ezugwu et al. (2020), and also an antimalarial property of quinine derivatives containing some amino acid, dipeptide, or tripeptide (Panda et al., 2013).

Experimental General
The chemicals and solvents used were purchased from Aldrich (Sigma-Aldrich) and AVRA Chemicals Pvt. Ltd. (Hyderabad, India) and used without purification. 1 H-NMR and 13 C-NMR spectra were recorded on Advance 300, 400, and 500 MHz spectrometers in DMSO-d 6 using TMS as internal standard.

Synthesis of Carboxamide Derivatives (7a-e)
Dichloromethane/trifluoroacetic acid (1:1%) was added to compounds 6a-c and stirred at room temperature for 1 h (TLC monitored). The products were obtained after evaporating the solvent under reduced pressure. The solid TFA salts were precipitated on the addition of diethylether and dried (Ezugwu et al., 2020).

Physicochemical Properties
The drug-likeness of the synthesized compounds are shown in Table 1. The molecular parameters calculated include molecular weight (MW), partition coefficient (log P), hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), topological polar surface area (TPSA), number of a rotatable bond (nRB), and molar refractivity (MR). The drug-likeness was determined using Lipinski's rule of five.

Molecular Docking
In this study, proteins essential for malaria and bacterial infections were evaluated. The protein targets were plasmepsin II (PDB ID: 1SME) from P. falciparum, and (PDB ID: 5MMN) for antimalarial and antimicrobial studies, respectively, were obtained from the protein data bank. Plasmepsin II from P. falciparum has been used as a novel target for antimalarial drug development because of its role as hemoglobin-degrading enzyme (Silva et al., 1996). Targeting the GyrB/ParE ATP-binding sites is an emerging approach in discovery of resistant bacteria. These ATP-binding sites located on bacterial DNA gyrase (5MMN) and topoisomerase IV, making them potent drug targets (Panchaud et al., 2017).
The 3D crystal structures of protein targets and cocrystallized ligands were from (https://www.rcsb.org/). The co-crystallized ligands were used to validate the docking protocols by redocking them into the active binding sites of the receptors. The structures of the molecules were drawn using ChemSketch. Further purification of the protein and ligands were furnished utilizing the discovery studio to erase various chains, the water of crystallization from the protein, and reduced the energy of the structures. Discovery Studio Visualizer, v16.1.0.15350 was used to envisage the interactions of the prepared ligands into the binding cavity of the protein receptors after docking. SwissADME was used to predict the physicochemical properties of the compounds.

Antimicrobial Evaluation
The microorganisms below were isolated clinically from University of Nigeria, Nsukka at Department of Pharmaceutical Microbiology and Biotechnology laboratory. Staphylococcus

Standardization of the test organism suspension
The standardization of the microorganisms was completed using 0.5 MacFaland turbid equivalent.

Control test (standard)
The standard antibiotics used were ciprofloxacin and fluconazole.

Experimental
Into a sterile petri dish containing a suspension of the stock (4 mL, 50 mg/mL) was added double strength sterile molten agar (16.0 mL) and mixed thoroughly to obtain 1 mg/mL solution. Lower concentrations (0.1-0.9 mg/mL) were calculated from the equation C 1 V 1 = C 2 V 2 . The plates were allowed to gel and divided into seven parts. The test microbes were patterned on the plates, labeled, and kept in an incubator at 37 • C for 24 h and 35 • C for 48 h, respectively, for antibacterial and antifungal activities. Further incubation of the plates for 24 h at 37 • C and 48 h at 25 • C, respectively, used to test for bactericidal and fungicidal activities.

Experimental Design and Treatment of Mice
The antiplasmodial activity were assessed by adopting the methods of Okokon and Nwafor (2009) and Ezugwu et al. (2020).

Chemistry
Sulfonamides and peptides are two important pharmacophores as found in the literature and as such are sorted after functionalities in the drive to combat drug resistance by organisms. The reported compounds showed good binding interaction in the active site of the target proteins as shown in Figures 1, 2. To synthesize the compounds (8a-j) we adopted the use of classical peptide coupling reagent, 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3 ′ -dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) in the amidation of compounds (7ae) with substituted benzenesulfonamides derived from L-leucine. Also, the activation of the carboxylic acid group of leucine was enhanced with HOBt as the EDC.HCI alone could not activate the carboxylic acid functionality. The use of HOBt and EDC.HCl was also recommended to reduce the risk of  racemization. In our work, we synthesized and characterized molecules containing sulfonamide, carboxamide, and Dipeptides moieties. The reaction of substituted benzenesulfonyl chloride (1a-b) with L-leucine afforded substituted benzenesulphonamoyl alkanamides (3a-b) in Scheme 1. The reaction of commercially available Boc-protected valine with substituted amines using EDC.HCl, HOBt, and triethylamine (TEA) in DCM afforded the carbamate derivatives of valine (6a-e) in Scheme 2. Compounds (7a-e) were synthesized through the reaction of compound (6ae) with DCM/ TFA (1:1%) for 1 h, respectively. The amidation of compound (3a-b) with the TFA salt of unprotected amides (7a-e) using peptide coupling reagents EDC.HCl, HOBt, TEA, to afford the desired products (8a-j) in Scheme 3. In the Infrared (IR) spectrum of 8a, bands between ∼3,312 and ∼3,194 cm −1 are for N-H while ∼1,642 and ∼1,640 cm −1 are for the two carbonyls of amide, respectively. In the 1 H-Nuclear magnetic resonance (NMR) spectrum of 8a, the methylene group of leucine displayed a multiplet at δ1.30-1.41 and -methane group of leucine and valine showed multiplet at δ3.95-4.04 due to the interactions with the near amide group protons. The typical NH resonance of the sulfonamide part of the dipeptide conjugates was detected at the δ8.43 ppm region as a doublet peak. The other two NH resonances of the diamide were observed at δ9.91 ppm and 8.12 ppm as singlet and doublet, respectively. The aromatic protons were observed at δ8.33, 8.03 pp, and 7.44 ppm, 7.09 ppm region as doublet and doublet peaks, respectively, for the eight aromatic protons.
In the 13 C-Nuclear magnetic resonance (NMR) spectrum, two peaks at 169.85 and 171.21 ppm for the carbonyl carbons of the amide groups, eight peaks ranging from 119.62 to 149.73 ppm for aromatic carbons, and 10 peaks ranging from 18.69 to 58.78 ppm for aliphatic carbons confirmed the formation of 8a, which was also supported by its high-resolution mass spectrometer (HRMS) peak spectrum with a peak at m/z 505.2125 for [M+H] + . All other compounds were in agreement with their structures.

Physicochemical Properties Results
The pharmacokinetic properties were evaluated in the form of hydrogen bond donor ≤5, hydrogen bond acceptor ≤10, molecular weight value of ≤500, and partition coefficient (Log P) value ≤5. The results ( Table 1) showed that the compounds would not pose oral bioavailability, transport, and permeability problems.
In vitro Antimicrobial Activities Table 4 comprises of minimum inhibitory concentration (MIC) of the synthesized compounds. The in vitro antibacterial properties of the compounds and Ciprofloxacin were evaluated against (staphylococcus aureus and Bacillus subtilis) and (Salmonella typhi and Escherichia coli) by the agar dilution method as Gram-positive and Gram-negative organisms, respectively. The in vitro antifungal properties of leu-val dipeptides synthesized were deduced by the agar dilution method against two fungal strains (Candida albicans and Aspergillus niger) and fluconazole as reference drug. The figures in Table 4 revealed that compound 8a with MIC value of 1.2 × 10 −3 M displayed comparable anti-bacterial activity against S. aureus to standard (9.1 × 10 −4 M). Compounds 8a, 8b, 8g, and 8j with MIC values ranging from (5.7 × 10 −4 M−8.4 × 10 −4 M) have the same or comparable activity with the standard drug (9.1 × 10 −4 M) against B. subtilis. All the compounds showed inhibition against these two Gram-positive organisms except compounds 8f and 8h, which are resistant to S. aureus. For the Gram-negative bacteria, it was revealed compounds 8b with MIC value of 9.5 × 10 −4 M showed activity against E. coli. All other compounds inhibits the growth of E. coli though less than the standard. The data in Table 4 revealed that the synthesized compounds showed good activity against S. typhi though less than the standard except for compounds 8f and 8h that are resistant to S. typhi. Further evaluation of the data revealed that compounds 8a and 8b are more potent as antibacterial agents when compared with other synthesized leu-val dipeptides derivatives. More so, it was revealed that the synthesized compounds showed better activity against C. albicans and A. niger though less active when compared with fluconazole except for compounds 8e, 8f, and 8j that do not have any inhibition against C. albicans and compounds 8e, 8f, 8g, and 8j that are resistant against A. niger.

In vivo Antimalarial
The leu-val dipeptides synthesized were evaluated for in vivo antimalarial activity against P. berghei NK (65 Strain) poison mice. The animal ethics committee, Veterinary Medicine Department, University of Nigeria, Nsukka gave permission and approval for the use of animals in this experiment (PG/PhD/16/80697). The percentage inhibition of parasite was calculated from the equation [(A-B)/A] × 100 (Ugwuja et al., 2019;Ezugwu et al., 2020); where A = parasitemia of the untreated group and B = parasitemia of the tested group. In this study, the compounds with the percentage inhibition below 30 are inactive, 30-40 are partially active, and 40 and above are regarded to be active. From Table 5 it was revealed that compounds 8a, 8b, 8d, 8e, 8g, 8h, 8i, and 8j which have 43.30-61.90% inhibition were active in comparable with the standard drug (with 67% inhibition). The analysis evaluation of the compounds synthesized revealed that compound 8j (61.90%) was the most potent antimalarial when compared with others, looking at the structure-activity relationship on the 4-nitrophenylsulfonamide hybrids (8a-d). The effect of 4methyl, 4-chloro, 4-Isopropyl, and 3-fluoro substituent on the Nphenylacetamide was studied, and it was revealed that 4-methyl-N-phenylacetamide derivative (8a, 60.2%) was the most potent inhibitor, for P. berghei followed by 8b (57.8%) and 8d (43.4%), and 8c were considered moderately active. The effects of 4methyl, 4-bromo, 4-chloro, 4-Isopropyl, and 3-fluoro substituent on the N-phenylacetamide among p-methylbenzenesulfonamide hybrids (8d-j) revealed that compounds 8j and 8h with 61.90 and 49.80% inhibition, respectively, were more active.

CONCLUSION
In this paper, we have described an approach to obtain leu-val based dipeptide derivatives that were tested for their antimalarial and antimicrobial properties. The results obtained showed that among the leu-val dipeptides synthesized, compound 8j was more active against P. berghei. Compound 8b was the most to inhibit growth of E. coli, compound 8a and 8b were most active against S. aureus, compounds 8a, 8b, and 8d were most active against S. typhi, B. subtilis is inhibited most with compounds 8a, 8b, and 8j. Compounds 8c, 8h, and 8b were the most active synthesized compounds against C. albicans and A. niger, respectively.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee, Veterinary Medicine Department, University of Nigeria, Nsukka.