https://orcid.org/0000-0002-0908-9721
Figures
Abstract
The goal of the current work was to create structural analogues of a beta lactam antibiotic that might be possibly effective against bacterial resistant strains. FTIR, 1H NMR, 13C NMR, and CHNS analyses were used to perform the spectroscopic study on the compounds M1–8. The effects of the aforementioned substances on gram-positive and gram-negative bacterial strains were investigated. Most of the eight compounds had antibacterial activity that was lower than or equivalent to that of the original medication, but two molecules, M2 and M3, surprisingly, had stronger antibacterial activity. The findings of synthesized analogues against alpha-glucosidase and DPPH inhibition were found to be modest, whereas M2, M3, and M7 strongly inhibited the urease. To comprehend the potential mode of action, a molecular docking research was conducted against urease and -amylase. The research may help in the quest for novel chemical compounds that would be effective against bacteria that are resistant to antibiotics.
Citation: Khaliq S, Khan MA, Ahmad I, Ahmad I, Ahmed J, Ullah F (2022) Synthesis, antimicrobial and molecular docking study of structural analogues of 3-((5-(dimethylcarbamoyl)pyrrolidin-3-yl)thio)-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. PLoS ONE 17(12): e0278684. https://doi.org/10.1371/journal.pone.0278684
Editor: Mohammad Shahid, Aligarh Muslim University, INDIA
Received: June 8, 2022; Accepted: November 21, 2022; Published: December 27, 2022
Copyright: © 2022 Khaliq et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Worldwide, there is an alarming increase in bacterial resistance that threatens the effectiveness of currently available antibiotics. Therefore, it is still crucial to create new, powerful antibacterial agents. Antibiotic-resistant bacteria are an international issue that raises healthcare expenditures and death. The biggest issue that humanity is now facing is bacterial resistance. Therefore, the time has come to develop new compounds with enhanced antibacterial activity and a novel mode of action [1]. Antimicrobial resistance has harmed human health and had an impact on the economy as a whole [2]. Methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), multidrug-resistant Mycobacterium tuberculosis (MDR-TB), vancomycin-resistant Enterococcus (VRE), and multidrug-resistant Streptococcus pneumonia infections are particularly difficult to treat. To combat bacterial strains that are resistant to antibiotics, this has compelled attention in the creation of novel and powerful antibacterial agents [1]. The largest danger to world health is the rise in multidrug-resistant microbial strains, which is expected to result in >10 million fatalities by 2055. Antibacterial medications from the carbapenem family are a crucial subset of last-resort care for illnesses brought on by bacteria with antibiotic resistance. A significant global danger that is predicted to result in over 10 million fatalities by 2055 is multidrug resistance [3]. Pathogenic bacteria continually develop their resistance mechanisms, which poses a severe challenge to the management of infectious diseases [4]. Due to its low absorption, carbapenem is a subclass of medications that is only used as a last option to treat drug-resistant bacteria. It is commercially accessible as injectables. The compound 3-((5-(dimethylcarbamoyl)pyrrolidin-3-yl)thio) -6- (1-hydroxyethyl) (1-hydroxyethyl) -4-methyl -7-oxo -1-azabicyclo [3.2.0] Since heptane-2 carboxylic acid is the preferred medication and is accessible as an injectable, it is crucial to explore multiple strategies for meropenem oral drug administration. A prodrug strategy might be used to compensate for carbapenem’s limited availability (meropenem). One strategy for creating a prodrug is to hide undesirable drug characteristics such drug instability, low bioavailability, and lack of site-specificity [5]. A common strategy for improving oral absorption in this class of drugs is the production of ester as the prodrug. The oral absorption of such medications can be improved by adding lipophilic moieties at the carbapenem C-3 and pyrrolidine N-1 sites [3]. Strong antibacterial capabilities are handled by all carbapenems since they all include pyrrolidine-3-yl thio groups at the C-2 position in their basic structure. It referred to them as "final line agents" or "antibiotics of last resort" due to their increased effectiveness [6]. Thienamycin, the first carbapenem to be found, was derived from Streptomyces cattleya [7]. One of the most often used medications for illnesses that pose a serious risk of death is meropenem, which was developed in the late 1980s. The quantity of beta-lactamases and dehydropeptidase does not cause meropenem to become inactive [8]. It is effective against extended-spectrum beta-lactamase, gram-positive and gram-negative bacteria (ESBL). Its mode of action centers on the suppression of cell wall synthesis, which leads to ultimate cellular death. The compound The preferred drug of choice for bacterial meningitis, severe skin infections, febrile neutropenia, respiratory infections, and urinary tract infections is 3-((5-(dimethylcarbamoyl)pyrrolidin-3-yl)thio) -6-(1-hydroxyethyl) -4-methyl -7-oxo1-azabicycloheptane-2-carboxylic acid. It is recommended for the treatment of community-acquired pneumonia, pulmonary exacerbations, and gynecological infections [9]. New derivatives of these substances have been found as a result of the rise in drug resistance. Ester derivatives showed improved biological and pharmacological capabilities, according to the literature study. The parent medicine, nalidixic acid, and its ester derivatives have broad antibacterial action against bacteria like Aeromonas hydrophila and Streptococcus pyrogens that are resistant to nalidixic acid in its purest form. Some new beta-lactam compounds shown outstanding antioxidant activity [10]. In addition to being tested for antibacterial activity, newly synthesized 4-alkylidene-b-lactam derivatives shown outstanding radical scavenging activity [11]. Seven out of 17 new quinolones demonstrated a substantial amylase inhibition in studies, indicating that the proline ring is what inhibits amylase [12]. The proline ring is a heterocyclic molecule that has a variety of properties, including antimicrobial, antioxidant, anti-carcinogen, anti-HIV, anti-inflammatory, and -amylase inhibitory active moiety [12]. Urinary tract infections and Helicobacter pylori are the two most dangerous illnesses associated with urease activity [13]. Meropenem is the recommended medication for UTIs, thus derivatives generated in the presence of urease inhibition can demonstrate if innovative compounds have greater capacity to withstand urease inhibition than the original substance [9].
The glycerol esters and diesters of sucrose have stronger antibacterial properties [14], and a number of esters derivatives also have antifungal, anticancer, anti-inflammatory, analgesic, and anesthetic properties When indomethacin is reacted with alcohol and phenol in the presence of 4-dimethylaminopyridine and dicyclohexylcarbodiimide (DCC), ester derivatives of indomethacin are created, which inhibit cox-2 enzymes more effectively and have no gastrointestinal adverse effects [15]. Our research focused on producing 3- ((5- (dimethylcarbamoyl) pyrrolidin-3-yl) thio)-6- ester derivatives of (1-hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] heptane-2-carboxylic acid has better pharmacokinetic characteristics and biological activity.
2. Experimental
2.1 Synthesis
Equimolar mixtures of the appropriate carboxylic acid and meropenem were added to a round-bottomed flask, followed by the addition of 30 ml of 99.8% pure ethanol (34852-M Sigma Aldrich®) and concentrated HCl, and refluxed for 4 hours on the water bath to create M1-8 (Figs 1 and 2). After the reaction was finished, the mixture was held at room temperature before being filtered and evaporated using a rotary process at a lower temperature and pressure. The crude product was recrystallized with the help of the ethanol.
2.2 Spectroscopic and elemental analysis of M1-8 compounds
It was examined if synthetic compounds were soluble in ethanol, methanol, water, chloroform, and DMSO. The melting point apparatus developed by Gallen Kamp was used to determine the melting point. The CHNS analyzer is used to determine the amount of carbon, hydrogen, and nitrogen. Bruker FTIR (Tensor model 27) and NMR 500 MHz are utilized for spectroscopic analysis.
2.3 Biological evaluation
Biological evaluation of M1 to M8 included antibacterial evaluation, enzyme inhibition assay, and antioxidant study.
2.3.1 Antibacterial activity.
Gram-positive and gram-negative bacteria were tested using the agar well diffusion technique, as reported in the literature [16] (e.g. Escherichia coli, Stenotrophomonas maltophilia, Bacillus megatarium, Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, and Serratia marcescens were obtained from the Department of Microbiology at the Islamia University of Bahawalpur in Pakistan. As a positive control, meropenem was utilized, and as a negative control, 5% DMSO.
2.3.2 Enzyme inhibition assays.
The literature-recommended procedures for urease enzyme inhibition and alpha-amylase enzyme inhibition experiments were followed with a few minor modifications, and the findings were represented as mg. Eq/gram of the corresponding positive control [12, 17].
2.3.3 Antioxidant activity.
Using the procedure outlined in the literature, DPPH (1,1-diphenyl-2-picryl hydrazyl) was used to test the scavenging capability of derivatives [18]. The findings were computed using the following formula and expressed as a percentage inhibition of free radicals:
2.4 Molecular docking
2.4.1 Ligand preparation.
Using the semi-empirical quantum mechanical technique PM3, the structures of all meropenem derivatives were constructed and their geometries were optimized. ChemDraw 12.0’s SDF format was used to generate the 3D conformers of the tested compounds for docking. The PyRx 0.08 application imported all tested substances into OpenBabel [19], where they were subjected to energy reduction, in order to change the docking file format from sdf to pdbqt format. With the use of a universal force field (UFF) and an energy difference of less than 0.01 kcal/mol, the conjugate gradient technique was employed to reduce energy. The minimized compounds were then transferred to PDBQT format for further examination.
2.4.2 Protein structure preparation.
The crystal structures of the enzymes urease (PDB ID: 4H9M) [22] and human pancreatic -amylase (PDB code: 5E0F) [20] were retrieved from Protein Data Bank. The protein’s built-in ligands were taken out of the crystal structure. Water molecules were eliminated from the protein by adding polar hydrogens and Kollman charges. In order to make analysis easier at a later stage of the simulation, final files were saved in PDBQT format.
2.4.3 Protocol of docking study.
Auto-Dock version 4.2 was used to perform the docking study. According to the macromolecular target site, the Auto Grid component, for instance, pre-calculates a three-dimensional grid of interaction energies using the AMBER force field. Automated docking tests were carried out to measure the binding free energy of the inhibitor. The most effective conformers were selected using the genetic algorithm. A variety of parameters, including population size and run counts, were specified. The crossover rate was set at 0.8, while the mutation rate was set at 0.02. The positional RMSD (Root Mean Square Deviation) of these findings varied by less than 0.5, and the resulting complex structures had the lowest binding energies.
2.5 ADME prediction
The choice of an agent as a drug is heavily influenced by the physical and molecular properties of substances. The Swiss ADME web server (http://www.swissadme.ch) was used to examine the molecular characteristics of new derivatives in order to confirm their potential as therapeutic target ligands [21]. In order to examine pharmacokinetics factors like as absorption, lipophilicity, and water solubility, each drug was added to the online server as a smile format. The topological polar surface area was used to compute the percentage of absorption (%ABS) of new derivatives:%ABS = 109-(0.345TPSA).
3. Results
3.1 Spectroscopic analysis
3.1.1 M1: 6- (1-((4-aminobenzoyl)oxy)ethyl)-3-((5 (dimethylcarbamoyl) pyrrolidin-3-yl)thio)- 4 methyl-7oxo 1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
Yield (71%); m.p 140–142°C; insoluble in chloroform but soluble in ethanol, methanol, DMSO and water Molecular Formula: C24H30N4O6S and Molecular weight: 502.59 gm/Mol Elemental analysis (calculated) for C24H30N4O6S: C, 57.36; H, 6.02; N, 11.15; (found): C, 57.30; H, 6.06; N, 11.12; FT-IR ν (cm-1), 3649 (COOH), 2886 (CH), 1716 (C = O), 1473 (CH = CH), 3308 (NH), 1280 (C−O), 1280 (C-N), 3586 (NH2). 1H NMR (DMSO, ppm) δ: 3.35–3.36 m, 3.85–3.86 m, 3.87–3.88 t, 6.55–6.56 t, 6.57–6.58 t, 7.43–7.44 m, 7.45–7.46 m, 7.46–7.47 m, 7.89–7.90 m, 7.92–7.93 m, (−CH−), 1.30–1.31 s, 1.09–1.10 s, (−CH2−), 0.58–0.59 s, 1.00–1.01 s, (−CH3−), 10.17 s, (−OH), 8.31 s, (−NH), 5.61 s, (−NH2). 13C NMR (DMSO, ppm) δ: 41.3 (C 1), 44.3 (C 2) (CH3), 46.2 (C 3), 49.8 (C 4), 50.4 (C 5), 51.5 (C 6), 56.9 (C 7), 57.3 (C 8), 59.9 (C 9), 60.5 (C 10), 66.3 (C 11) (CH2), 115.2 (C 12), 116.5 (C 13), 118.0 (C 14), 122.0 (C 15), 123.8 (C 16), 129.3 (C 17), 130.8 (C 18), 130.9 (C 19) (CH), 155.2 (C 20) (C-N), 166.5 (C 21), 166.7 (C 22), 166.9 (C 23), 172.0 (C 24) (C = O).
3.1.2 M2: 6,6’-((oxalylbis(oxy)) bis (ethane-1,1-diyl))-bis-(3-((5-(dimethyl- carbamoyl) pyrrolidin-3-yl) thio)-4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid).
Yield (73%); m. p. 160–161°C, insoluble in chloroform soluble in ethanol, methanol, DMSO, and distilled water. Molecular formula: C36H48N6O12S2 and molecular weight: 820.93 gm/mol. Elemental analysis (calculated) for C36H48N6O12S2: C, 52.67; H, 5.89; N, 10.24 (found) C, 52.62; H, 5.93; N, 10.28. FT-IR ν (cm-1), 3854 (COOH), 2947 (C−H), 1716 (C = O), 1450 (CH = CH), 1106 (C−N), 1206 (C−O), 3253 (N−H). 1H NMR (DMSO, ppm) δ: 3.62–3.63 m, 3.64–3.65 t, 4.16 d, 3.25 t, 3.29 t, 3.64 t, 3.34–3.44 t, 3.45–3.46 m, 3.48–3.49 m, 3.52–3.53 m, 3.55–3.56 m, 3.62–3.63 m, (−CH−), 1.34–1.35 m, 1.32–1.33 m, 1.31–1.32 m, 1.28–1.29 t, (−CH2−), 2.64–2.65 s, 2.62 s, 0.85–0.86 m, 0.82–0.83 m, 1.64–1.65m, 1.62–1.63 m, 1.3–1.4 m, 1.2–1.3 t, (−CH3−), 5.29 s, (−OH), 5.1 s, (−NH). 13C NMR (DMSO, ppm) δ: 26.1(C1), 30.9(C2), 34.50(C3), 36.4(C4), 37.0(C5), 39.1 (C6), 39.4 (C7), 39.6 (C8), 39.9 (C9), 40.2 (C10/11), 40.5 (C12), 40.7 (C13),44.2 (C14)(CH3), 52.4 (C15), 55.7 (C16), 60.8 (C17), 61.0 (C18), 68.5(C19), 70.2 (C20), 71.0 (C21) (CH2), 71.7 (C22), 72.5 (C23), 73.6(C24/25), 75.9(C26),80.9(C27), 81.8(C28), 87.2 (C29), 92.4 (C30/31), 102.5(C32) (CH) 162.7(C33), 165.3(C36)(C = O).
3.1.3 M3: 3-((5-(dimethylcarbamoyl)pyrrolidin-3-yl)thio)-6-(1-((2-hydro- xybenzoyl) oxy) ethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
Yield (75%); m. p. 101–103°C, insoluble in chloroform and distilled water-soluble in ethanol, methanol, and DMSO. Molecular formula: C24H29N3O7S and molecular weight: 503.57gm/mol.
Elemental analysis (calculated) for C24H29N3O7S: C, 57.24; H, 5.80; N, 8.34; (found) C, 57.29; H, 5.75; N, 8.39; FT-IR ν (cm-1), 3885 (COOH), 2864 (C−H), 1721 (C = O), 1484 (CH = CH), 1208 (C−N), 3628 (C−O), 3185 (N−H), 3649 (O−H). 1H NMR (DMSO, ppm) δ: 6.88–6.87 m, 6.93–6.94 m, 6.92–6.94 m, 6.90–6.91 t, 6.91–6.92 t, 1.02–1.03, m 6.88–6.89 t, 4.76–4.77 d, 3.20–3.21 t, (−CH−), 1.00–1.02 d, (−CH2−), 1.5–1.56 d, 0.56–0.57 d, (−CH3−),7.57 s, (−NH), 9.92 s, (−OH). 13C NMR (DMSO, ppm) δ: 41.0(C1), 44.3(C2), 46.2(C3), 49.4(C4) (CH3), 50.44 (C5), 55.9(C6), 57.1(C7),59.8 (C8), 60.16(C9), 65.3 (C10), 66.3(C11) (CH2), 112.9(C12), 115.7(C13), 117.0(C14), 119.1(C15), 123.8(C16), 129.3(C17), 130.2(C18), 135.5(C19)(CH), 159.2 (C20) (C-N), 161.0(C21), 165.4(C22), 171.7(C23), 172.3(C24) (C = O).
3.1.4 M4: 6,6’-((succinylbis(oxy))bis(ethane-1,1-diyl))bis(3-((5-(dimethylcar bamoyl) pyrrolidin-3-yl)thio)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid).
Yield (7079.88%), m.p 120–122°C insoluble in chloroform and distilled water-soluble in ethanol, methanol, and DMSO. Molecular formula: C38H52N6O12S2 and molecular weight: 848.9 gm/mol.
Elemental analysis (calculated) for C38H52N6O12S2: C, 53.76; H, 6.17; N, 9.90; (found): C, 53.70; H, 6.21; N, 9.85; FT-IR ν(cm-1), 3860 (COOH), 2883 (C−H), 1718 (C = O), 1653 (CH = CH), 1208 (C−N), 1023(C−O), 3149 (N−H). 1H NMR (DMSO, ppm) δ: 3.16–3.7 d, 3.38–3.39 t, 3.41–3.42 t, 3.42–3.43 t, 3.44–3.45 t, (−CH−), 1.00–1.01 d, (−CH2−), 2.50–2.51 d, (−CH3−), 5.96 s, (−OH), 7.32 s, (−NH). 13C NMR (DMSO, ppm) δ: 41.29(C 1), 44.3(C 2), 46.2(C 3), 49.8(C 4) (CH3), 50.4 (C 5), 51.5 (C 6), 56.9 (C 7), 57.3 (C 8), 59.9 (C 9), 60.5(C 10), 66.3 (C 11) (CH2), 115.2 (C 12), 116.5(C 13), 118.0 (C 14), 122.0 (C 15), 123.8 (C 16), 129.3 (C 17), 130.8 (C 18), 130.9 (C 19)(CH), 155.2 (C 20) (C-N)), 166.5(C 21), 166.7 (C 22), 166.9 (C 23), 172.0 (C 24) (C = O).
3.1.5 M5:6,6’-(((2-aminopentanedioyl)-bis-(oxy))-bis-(ethane-1,1-diyl))-bis-(3-((5-(dimethyl carbamoyl) pyrrolidin-3-yl)thio)-4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid).
Yield (73%), m.p 133–134°C insoluble in chloroform but soluble in ethanol, methanol, DMSO, and distilled water. Molecular formula: C39H55N7O12S2 and molecular weight: 878.03 gm/mol.
Elemental analysis (calculated) for C39H55N7O12S2: C, 53.35; H, 6.31; N, 11.17; (found): C, 53.40; H, 6.35; N, 11.12; FT-IR ν (cm-1), 3869 (COOH), 2920 (CH), 1711 (C = O), 1638 (CH = CH), 1306 (C−N), 1086 (C−O), 3125 (NH), 3329 (NH2). 1H NMR (DMSO, ppm) δ: 3.12–3.13 t, 3.57–3.58 t, 3.62–3.63 m, 3.91–3.92 t, 3.40–3.41 m, (−CH−), 1.05–1.06 d, (−CH2−), 2.51–2.52 d, 2.32–2.33 d, (−CH3−), 11.02 s, (−OH), 7.82 s, (NH). 13C NMR (DMSO, ppm) δ: 39.1(C1), 39.4 (C2), 39.6 (C3), 39.9 (C4), 40.2 (C5/6), 40.5(C7), 40.7(C8) (CH3), 61.7(C9), 65.2 (C10), 70.4 (C11/12), 74.9 (C13), 79.0 (C14), 79.4 (C15), 79.8 (C16), 81.2 (C17), 85.9 (C18), 92.2 (C19/20) (CH2), 145.4 (C21), 147.0 (C22)(C), 151.2 (C23), 157.7 (C24/25)(C-N), 160.2 (C26), 160.9 (C27/28), 165.2 (C29), 167.3 (C30), 169.9 (C31), 171.1 (C32/33), 173.5(C34), 175.1(C35/36), 176.8(C37), 177.2(C38/39) (C = O).
3.1.6 M6:3-((5-(dimethylcarbamoyl)pyrrolidin-3-yl)thio)-4-methyl-7-oxo-6-(1-((3,4,5trihydroxy-benzoyl) oxy)ethyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
Yield (72%); m.p. 147–149°C insoluble in chloroform but soluble in ethanol, methanol, DMSO, and distilled water. Molecular formula: C24H29N3O9S and molecular weight: 535.57 gm/mol.
Elemental analysis (calculated) for C24H29N3O9S: C, 53.82; H, 5.46; N, 7.85; (found); C, 53.78; H, 5.51; N, 7.81; FT-IR ν (cm-1), 3743 (COOH), 2919 (C−H), 1711 (C = O), 1637 (CH = CH), 1307 (C−N), 1087 (C−O), 3616 (O−H), 3190 (N−H). 1H NMR (DMSO, ppm) δ: 3.33–3.34 d, 4.13–4.14 m,4.17–4.18 m, (−CH−), 1.28–1.29 d,1.33–1.34 m,1.37–1.39 m, (−CH2−) 0.88–0.89 m, 1.23–1.24 m, (−CH3−),4.18 s, (−OH), 7.73 s, (−NH). 13C NMR (DMSO, ppm) δ: 14.2(C1), 14.3(C2), 21.8(C3), 34.1(C4), 40.5 (C5)(CH3), 59.9 (C6)(CH2), 108.4 (C7), 108.5 (C8), 108.7(C9), 119.6 (C10), 120.4 (C11), 138.3 (C12) (CH), 145.3 (C13), 145.5 (C14), 149.9 (C15)(C), 151.3 (C16), 153.4 (C17), 155.9 (C18) (C-N), 158.7 (C19), 161.2 (C20), 163.5 (C21), 165.8 (C22/23), 167.4 (C24) (C = O).
3.1.7 M7:6,6’-(((2,3-dihydroxysuccinyl)bis(oxy))bis(ethane-1,1-diyl))bis(3-((5-(dimethy-lcarbam-oyl)pyrrolidin-3-yl)thio)-4-methyl-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylic acid).
Yield: (75%), m.p. 108–110°C insoluble in chloroform but soluble in ethanol, methanol, DMSO, and distilled water. Molecular formula: CH52N6O14S2 and Molecular weight: 880.98 gm/mol.
Elemental analysis (calculated) for C38H52N6O14S2: C, 51.81; H, 5.95; N, 9.54; (found): C, 51.85; H, 5.92; N, 9.51; FT-IR ν (cm-1), 3853 (COOH), 2991 (C−H), 1734 (C = O), 1653 (CH = CH), 1266 (C−N), 1653 (C−O), 3628 (O−H), 3170 (N−H). 1H NMR (DMSO, ppm) δ: 3.16–3.17 t, 3.52–3.53 t, 3.62–3.63 m, 3.94–3.95 t, 3.97–3.98 t,3.41–3.42 m, (−CH−), 1.2–1.3 d, (−CH2−), 2.49–2.50 d, 2.53–2.54 d, (−CH3−), 8.87 s, (−NH), 14.41 s, (−OH). 13C NMR (DMSO, ppm) δ: 19.0 (C1), 39.3 (C2), 39.5 (C3), 39.7 (C4), 39.9 (C5), 40.2 (C6), 40.6 (C7)(CH3), 56.5 (C8), 75.2 (C9), 77.4 (C10), 79.9 (C11), 85.2(C12), 88.9 (C13), 92.5 (C14), 95.7 (C15), 99.2 (C16) (CH2), 104.2 (C17), 107.1(C18), 111.1 (C19), 112.5 (C20), 120.3 (C21), 122.1 (C22), 124.5 (C23/24), 129.5 (C25) (CH), 130.2 (C26), 130.9 (C27/28)(CH), 138.9 (C29), 157.7 (C30/31/32), 153.4 (C33), 155.2 (C34/35), 158.7 (C36) (C-N), 159.1(C37), 160.3(C38) (C = O).
3.1.8 M8: 6,6’-((phthaloylbis(oxy))bis(ethane-1,1-diyl))bis(3-((5-(dimethyl carbamoyl) pyrrolidin-3-yl)thio)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid).
Yield: (79%); m. p 145–147°C insoluble in chloroform but soluble in ethanol, methanol, DMSO, and distilled water. Molecular formula: C42H52N6O12S2 and molecular weight: 897.03 gm/mol. Elemental Analysis for C42H52N6O12S2 (calculated), C, 56.24; H, 5.84; N, 9.37; found: C, 56.22; H, 5.87; N, 9.31.
FT-IR ν (cm-1), 3853 (COOH), 2990 (C−H), 1716 (C = O), 1473 (CH = CH), 1166 (C−N), 1372 (C−O), 3525 (N−H). 1H NMR (DMSO, ppm) δ: 3.16–3.17 t, 3.52–3.53 t, 3.62–3.63 m, 3.94–3.95 t, 3.41–3.42 m, (−CH−), 1.3–1.4 d, (−CH2−), 2.47–2.48 d, 2.52–2.53 d, (−CH3−), 14.41 s, (−OH), 8.87 s, (−NH). 13C NMR (DMSO, ppm) δ: 40.3(C1), 41.0 (C2), 44.3 (C3), 46.2 (C4), 49.4(C5)(CH3), 50.2 (C6), 52.3 (C7), 57.1 (C8), 59.8 (C9), 60.5 (C10), 61.1 (C11), 66.3 (C12)(CH2), 128.1 (C13), 129.3(C14), 130.7 (C15), 131.2 (C16), 131.3 (C17), 131.4 (C18), 131.5 (C19), 131.9 (C20), 132.0 (C21), 132.3 (C22), 132.4 (C23), 132.7 (C24)(CH), 159.2 (C25), 166.7 (C26/27), 166.8 (C28), 166.8 (C29), 166.9 (30), 167.4 (C31), 167.5, (C32/33), 167.8 (C34), 167.9 (C35/36/37), 168.0 (C38), 168.6 (C39,40), 172.0 (C41),172.3 (C42) ((C = O).
3.2 Biological evaluation
3.2.1. Antibacterial activity.
The results of antibacterial studies were determined and tabulated in Table 1.
3.2.2 Enzyme inhibition activity.
3.2.2.1 Urease Inhibition and alpha-amylase inhibition assays (Table 2).
3.2.3 Antioxidant activity.
The free radical scavenging potential of 3-((5- (dimethylcarbamoyl)pyrrolidin-3-yl) thio)-6- (1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid and its derivatives was determined and results presented in Table 3.
3.3 Molecular docking and ADME pharmacokinetics properties
Tables 4 and 5 show the outcomes of in-silico molecular docking and ADME pharmacokinetics parameters, respectively. The In-silico molecular docking study included 3-((5- (dimethylcarbamoyl) pyrrolidin-3-yl) thio)-6- (1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo [3.2.0] heptane-2-carboxylic acid and its synthesized derivatives against two clinically important enzymes i.e., urease and alpha-amylase.
3.4 ADME pharmacokinetics studies
The predicted ADME parameters of all the tested compounds were determined and tabulated in Table 5.
4. Discussion
In this investigation, Fischer esterification was used to create meropenem ester derivatives. The enhanced PBP and potent action of meropenem were caused by the presence of a pyrrolidine ring in the drug’s structure. Penicillin-binding proteins (PBPs), which are important in the manufacture of bacterial cell walls, attach to it covalently, and when they are inhibited, cell death results [22]. Compared to gram positive bacterial strains, it is more effective against gram negative bacterial strains. Except for PBP3, every PBP in Staphylococcus aureus had a greater affinity for meropenem [23]. Meropenem antimicrobial resistance was primarily caused by changes in PBPs, plasmid-mediated beta-lactamases, and altered bacterial membrane permeability [18]. An exhaustive study of the literature found that aromatic esters have improved anti-microbial and antioxidant effects [24]. Against the gram-negative bacterial strain Bacillus megatarium, M2, M3, M7, and M8 had zones of inhibition that were larger than those of meropenem parent drug. When tested against Bacillus subtilis, the compounds M2, M4, M6, and M8 also showed strong antibacterial properties. Because of PBP3, Staphylococcus aureus displayed the lowest binding affinity to M (std) drug, although compounds M2, M3, M4, M5, M7, and M8 exhibited a greater zone of inhibition (Table 1). As indicated in Table 1, the M7 had little action against Micrococcus luteus, but the M2, M4, and M8 compounds displayed high activity. An increase in lipophilicity, a significant factor in the antibacterial activity that improves the permeability of ester derivatives into the lipid membrane and inhibits bacterial growth, may be the source of an increase in antibacterial activity. According to Table 1, M2 and M3 demonstrated enhanced activity against Escherichia coli, whereas M3, M4, and M8 showed improved activity against Serratia marcescens. According to a review of the literature, clarithromycin’s ability to treat peptic ulcers brought on by Helicobacter pylori is waning. Several studies also showed that blocking the urease enzyme is directly related to bacterial death. Few investigations have found that recently synthesized antibacterial also had anti-diabetic properties. To investigate anti-urease and alpha-amylase inhibition, both enzymes were chosen. When compared to thiourea and the parent drug meropenem, in vitro enzyme inhibition of M1-M8 against jack bean urease exhibited much higher activity. In Table 2, where acarbose was utilized as a positive control, the findings of the -amylase inhibition experiment was not encouraging. Similarly, neither the original drug molecule nor any of its synthesized derivatives, with the exception of M2, M4, M6, and M8, showed any promising antioxidant potential (Table 3). Docking analysis was conducted, as shown in Table 4, to estimate the potential mode of interaction between the substantially active and least active chemical and the target protein. The derivative M4 showed high action against urease and alpha-amylase, according to computational investigations as indicated in Table 4. Compound ADMET characteristics are crucial for drug development and design. All meropenem derivatives had strong anti-urease action, however compounds M4 and M7 were found to be more powerful than thiourea, which was utilized as a positive control. When linked with ALA636, ARG439, and HIS539 at catalytic pocket sites, the compounds M4 and M7 demonstrated robust hydrogen bonding. A salt bridge involving the amino acids ARG439 and ARG639 was seen in M7. ILE411, ALA440, and ALA636 demonstrated hydrophobic interaction with M1, M3, and M6. Fig 3 illustrates the compounds M2 and M5 forming a salt bridge with HIS593 and ARG609. Additionally, docking tests on meropenem derivatives were carried out to look at their affinities for binding to amylase. In the catalytic pocket, the hydrophobic amino acid on the active site appears to be essential for ligand binding. Meropenem and its synthetic analogues did not have attractive docking results, although some of them had hydrophobic interaction, including TRP59, TYR62, ILE162, ALA198, LYS200, and HIS201, as well as hydrogen bonding in HIS195, ILE235, HIS305, and GLY309. Pi-stacking was seen with the compounds M4 and M8 with TYR62, HIS201, and HIS301. Fig 4 depicts a salt bridge between M5 and M8 and ARG195, HIS299, and HIS305. To evaluate the pharmacokinetic and safety profile of the synthesized molecules M1-M8, ADMET properties were estimated. Each compound has noteworthy physicochemical characteristics. Meropenem derivatives in general exhibited no Lipinski violations. The synthesized analogs of meropenem are all shown in Table 5 above, and according to the antibacterial results and ADMET parameters findings, none of them crossed the blood-brain barrier and all compounds have low gastrointestinal absorption, demonstrating that derivatives are good for topical use and can be further utilized for investigation by in vivo studies for better contributions to science and medicine (Fig 5).
5. Conclusion
We synthesized many meropenem ester compounds and investigated their antibacterial, antioxidant, and enzyme-inhibitory properties. M2 and M3 were discovered to have stronger antibacterial effects than the parent medication. The anti-urease potential of M1, M2, M3, and M6 was also notable and found to be in good accord with their molecular docking results, offering important insight into a potential mechanism of action. Thus, it is concluded that there is a large area for more study and compound optimization, which may result in the creation of some potential antibacterial drugs to combat bacterial resistance in a variety of bacterial illnesses.
Supporting information
S1 File.
Part A. Antibacterial activity (Figs 1–7 of S1 File) of synthesized derivatives M1-M8 and meropenem, Part B. FTIR spectrum (Figs 8–16 of S1 File) of synthesized derivatives M1-M8 and meropenem, Part C. 1Proton and 13carbon NMR (Figs 17–33 of S1 File) of synthesized derivatives M1-M8 and meropenem.
https://doi.org/10.1371/journal.pone.0278684.s001
(DOCX)
Acknowledgments
The authors are thankful to the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, and The Islamia University of Bahawalpur for providing all facilities and technical support.
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