Comparative study on Antibacterial efficacy of a series of chromone sulfonamide derivatives against drug-resistant and MDR-isolates

Abstract  

Sulfonamide derivatives have numerous pharmaceutical applications having antiviral, antibacterial, antifungal, antimalarial, anticancer, and antidepressant activities. The structural flexibility of sulfonamide derivatives makes them an excellent candidate for the development of new multi-target agents, although long-time exposure to sulfonamide drugs results in many toxic impacts on human health. However, sulfonamides may be functionalized for developing less toxic and more competent drugs. In this work, sulfonamides including Sulfapyridine (a), Sulfathiazole (b), Sulfamethoxazole (c), and Sulfamerazine (d) are used to synthesize Schiff bases of 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbalde-hyde (1a–1d). The synthesized compounds were spectroscopically characterized and tested against hospital isolates of three Gram-positive (Methicillin-resistant Staphylococcus aureus PH217, Ampicillin-resistant Coagulase-negative Staphylococcus aureus, multidrug-resistant (MDR) Enterococcus faecalis PH007^R) and two Gram-negative bacteria (multidrug-resistant Escherichia coli, and Salmonella enterica serovar Typhi), compared to the quality control strains from ATCC (S. aureus 29213, E. faecalis 25922, E. coli 29212) and MTCC (S. Typhi 734). Two of the four Schiff bases 1a and 1b are found to be more active than their counterpart 1c and 1d; while 1a have showed significant activity by inhibiting MRSA PH217 and MDR isolates of E. coli at the minimum inhibitory concentration (MIC) of 150 μg/mL and 128 μg/mL with MBC of 1024 µg/mL, respectively. On the other hand, the MIC of 1b was 150 μg/mL against both S. aureus ATCC 29213 and Salmonella Typhi MTCC 734, compared to the control antibiotics Ampicillin and Gentamycin. Scanning electron microscopy demonstrated the altered surface structure of bacterial cells as a possible mechanism of action, supported by the in-silico molecular docking analysis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01194-w.

Introduction

Antibiotics are known to fight infections either by killing disease-causing microbes or preventing them to multiply. The potential mechanisms of antibiotics action include: (i) inhibition or prevention of cell wall synthesis; (ii) inhibition of synthesis and functionalities of nucleic acids; (iii) blocking of major enzymes to stop the spread; and (iv) the inhibition of the synthesis of proteins and folic acid [1–5]. Since the beginning of the antibiotic’s era with the introduction of salvarsan (1910), penicillin (1928), and sulfonamides (1937) bacteria are reported to develop resistance to antibacterial [3]. The number of antibiotic-resistant bacteria has increased gradually over time, leading to a global public health threat. One of the ways to overcome the antibiotic resistance crisis is the development of more efficient newer and less toxic drugs [6]. During the 1920-30 s staphylococcal and streptococcal infections were the major killers, when even a minor scratch or scrap became deadly, and young adults were killed by pneumonia and tuberculosis as reported from Europe and the USA [7]. Prontosil (Kl730), a sulfonamide dye, was found to kill bacteria as anti-bacterial agent in 1900’s by the German Pathologist Gerhard Domagk, a Nobel laureate in 1939. Domagk observed the selective ability of prontosil to prevent the infection caused by the bacterium Streptococcus in mice and rabbit [8], and he used the drug to save his daughter, suffering from the streptococci infection. In 1936 the French pharmacist Ernest Fourneau discovered that the pro-drug prontosil converted into active antibacterial sulfonamide in the human body [9] by enzymes of liver cells and microsomes, using NADPH as hydrogen donor, in the presence of excess riboflavin (vitamin B2) either as flavin mononucleotide by adenyltransferases or flavin adenine dinucleotide by riboflavin kinase and a flavoprotein. Flavins serve as a prosthetic group, while unbound hydrogen transport system as a coenzyme [10].

This discovery triggered the identification of several anti-bacterial agents from the Sulfa group, including sulfapyridine for the treatment of pneumonia (1938), sulfa acetamide for urinary tract infections (1941), succinyl-sulfathiazole for gastrointestinal tract infections (1942); and sulfathiazole for wound infection, during Second World War. Moreover, large-scale use of sulfonamides as preventive as well as chemo-therapeutic agents were reported against various ailments. Even today more than thirty different types of sulfa drugs are used as antibacterial, antifungal, antiprotozoal, anti-inflammatory, nonpeptidic vasopressin receptor antagonists, and translation initiation inhibitors [11, 12].

Sulfonamides are known to block the synthesis of folic acid by replacing p-amino benzoic acid (PABA) and thereby hamper the synthesis of bacterial mRNA/DNA [12]; but the selectivity of sulfonamides depended on the enzymatic action of dihydropteroate synthase [13]. While hypersensitivity of sulfonamide drugs is due to the fractional oxidation of the aromatic-NH₂ group to hydroxylamine (SF-NHOH) and/or nitroso-derivative (SF-NO) by human Cytochrome-P450, which results in cellular toxicity [14]. Diverse types of sulfa-drugs or their derivatives are synthesized and therapeutically tested for antibacterial, antifungal, and other activities with safety profiles [15–17]. Some sulfonamides are also used as carbonic anhydrase inhibitors [18]. Coumarins, secondary metabolites of a wide variety of plants, are specific classes of compounds having significant bioactivities [19], but based on different substituents their activities may differ. In coumarins, the benzene ring is fused with the pyrone ring enabling diverse bioactivities including antibacterial, antifungal, anticoagulant, antioxidant, anti-inflammatory, and anticancer [20]. Though coumarins are highly active against both Gram-negative and Gram-positive bacteria, but are more potent against Gram-negatives, by damaging the bacterial outer membrane. Some plants can secrete coumarin-like active molecules as defensive phytochemicals called phytoalexins, to counter the attack from phytopathogens [21]. Phytoalexins are environmentally benign and non-vulnerable to bacterial resistance [21, 22]. Clinically licensed sulfonamides like sulfamate- and sulfamide-drugs, along with substituted benzene-sulfonamides can inhibit the activity of carbonic anhydrase [23]. Thus, sulfonamide derivatives can be useful as a pharmaceutical agent when conventional antibiotics are ineffective. Among the functionalized sulfonamides, the Schiff bases have exhibited significant antibacterial, fungicidal, antioxidant, anti-inflammatory, antitumor, anti-cancer, and herbicidal activities [24–28]. Moreover, microbial resistance has evolved sharply over the last seven decades, and most antimicrobial drugs lost their ‘magic-bullet’ '-like effectiveness [29, 30]. Based on the above background and the higher biological significance of pharmacologically active functionalized sulfonamides, the present work aimed to synthesize four new Schiff bases of Sulfapyridin (a), Sulfathiazole (b), Sulfamethoxazole (c) and Sulfamerazine (d) (Scheme 1) upon condensation with 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde as potential less-toxic antibacterial agent. The plausible mechanism was investigated using Scanning Electron microscopy and in-silico molecular docking studies. The therapeutic potential of four compounds 1a-1d has also been compared with ampicillin and gentamycin.

Scheme 1.

Synthesis of Compound-1a, 1b, 1c, and 1d

Experimental section

Reagents and instruments

The AR grade Resorcinol was procured from M/S Spectrochem; while sulfonamides (Sulfapyridine, Sulfathiazole, Sulfamethoxazole, Sulfamerazine), Mueller–Hinton Broth and Agar–agar were obtained from HiMedia (Mumbai, India). Ethyl acetoacetate was purchased from SRL (India); and Methanol from TCI Chemicals (India). The MTT reagents were purchased from E Merck (India); and Eagle's Minimal Essential Medium (EMEM) for cell culture was from Sigma. The high-purity water used in this study was collected from Milli-Q (Millipore). The 8-Formyl-7-hydroxy-4-methyl coumarin in methanol was prepared following a published protocol [31]. Thin-layer chromatography was performed by using Al sheets (E Merck, India) coated with silica gel 60F254. FTIR spectra on KBr discs (4000–400 cm⁻¹) were taken from Bruker ALPHA-II spectrometer in an attenuated total reflectance (ATR) mode. The ¹H NMR spectra were recorded with Bruker (AC) 300 MHz FT-NMR spectrometer in DMSO-d₆ using TMS as an internal standard; while ESI mass spectra were verified by a Water HRMS XEVO G2QTOF #YCA351 Spectrometer.

Culture Media

Nutrient broth (NB), Peptone water (PW), and Luria broth (LB) are of Oxoid Brand, UK; Triple sugar iron Agar (TSI), MacConkey Agar (MCA), and Mueller Hinton broth (MHB) were from Difco, Detroit, USA; and other basic media were obtained from the HiMedia, Mumbai, India. Peptone agar (PA), nutrient agar (NA), and Mueller Hinton agar (MHA) were prepared in the laboratory by adding the Agar–agar in appropriate amount of the individual liquid media followed by sterilization.

Synthesis of Schiff bases (1b – 1d)

8-Formyl-7-hydroxy-4-methyl coumarin (0.204 g, 1.0 mmol) in methanol (30 mL), was reacted with sulfapyridine (0.249 g, 1 mmol) and refluxed for 4 h. A reddish-colored precipitate, collected by filtration, was washed with cold methanol and dried (yield 391.58 mg, 90%). Other Schiff bases (1b–1d) were prepared by using 8-formyl-7-hydroxy-4-methyl coumarin and sulfathiazole (b), sulfathiazole (c), and sulfamerazine, (d) respectively, as presented in the synthetic Scheme 1.

Micro-analytical data of Compound-1a: C₂₂H₁₇N₃O₅S; Calcd. (Found): C, 60.68 (60.62); H, 3.93 (3.96); N, 9.65 (9.71) %. ¹H-NMR (300 MHz, DMSO-d₆). δ 14.47 (s, 1H), 9.27 (s, 1H),7.77 (d, 4H, J = 8.4), 7.66 (d, 1H, J = 8.7), 7.75 (s, 1H), 7.39 (d, 2H, J = 2.8), 7.21 (s, 1H), 6.97 (m, 2H, J = 9), 6.29 (s, 1H), 2.50 (s, 3H), (Fig. S1). Mass: (M + Na)⁺ 458.00 (Calcd. m/z 435.09) (Fig. S2). IR: 3443.9 cm⁻¹ (aromatic -OH), 1731.4 cm⁻¹ (lactone C = O), 1632.2 cm⁻¹ (Schiff’s bass, C = N), 1388.3 cm⁻¹ (S = O) (Fig. S3).

Micro-analytical data of Compound-1b: C₂₀H₁₅N₃O₅S₂; Calcd. (Found): C, 54.41 (54.38); H, 3.42 (3.46); N, 9.52 (9.51) %. ¹H-NMR (300 MHz, CDCl₃). δ 14.33 (s, 1H), 9.33 (s, 1H),7.99 (d, 2H, J = 7.3), 7.61 (d, 1H, J = 6), 7.40 (d, 2H, J = 8.8), 7.13 (d, 1H, J = 4.8), 6.93 (d, 2H, J = 9), 6.55 (d, 1H, J = 4.8), 6.14 (s, 1H), 2.42 (s, 3H), (Fig. S4). Mass: (M + Na)⁺ 437.04 (Calcd. m/z 413.09) (Fig. S5). IR: 3458.9 cm⁻¹ (aromatic -OH), 1719.4 cm⁻¹ (lactone C = O), 1634 cm⁻¹ (Schiff’s bass, C = N), 1391.1 cm⁻¹ (S = O). Melting points of 1a and 1b are 185 °C, 189 °C respectively (Fig. S6).

Micro-analytical data of Compound-1c: C₂₁H₁₇N₃O₆S; Calcd. (Found): C, 57.40 (57.41); H, 3.90 (3.92); N, 9.56 (9.52) %. ¹H-NMR (300 MHz, DMSO-d₆).δ 14.33 (s, 1H), 11.51 (s, 1H), 9.26 (s, 1H),7.92 (d, 2H, J = 9.3), 7.85 (d, H, J = 9),7.69 (d, 2H, J = 8.3), 6.96 (d, 1H, J = 8.97), 6.27 (s, 1H), 6.15 (s, 1H),2.26 (s, 3H),2.06 (s, 3H) (Fig. S7) Mass: (M + H)⁺ 440.00 (Calcd. m/z 416.08) (Fig. S8). IR: 3445 cm⁻¹ (aromatic -OH), 1729.8 cm⁻¹ (lactone C = O), 1644.4 cm⁻¹ (Schiff’s bass, C = N), 1345.8 cm⁻¹ (S = O), Melting point of 1c is 179 °C (Fig. S9).

Micro-analytical data of Compound-1d: C₂₂H₁₈N₄O₅S; Calcd. (Found): C, 58.66 (58.67); H, 4.03 (4.01); N, 12.44 (12.41) %. ¹H-NMR (300 MHz, DMSO-d₆). δ 14.45 (s, 1H), 10.45 (s, 1H),8.089 (d, 1H, J = 7.8),8.339 (d, 1H, J = 3.6),7.873 (d, 1H, J = 9),7.68 (d, 1H, J = 9.1),7.626 (d, 1H, J = 8.7), 6.994 (d, 1H, J = 9), 6.926 (d, 1H, J = 4. 5), 6.557 (d, 1H, J = 8.4), 6.313 (s, 1H), 5.98 (s, 1H),2.439 (s, 3H), 2.344 (s, 3H) (Fig. S10). IR: 3445 cm⁻¹ (aromatic -OH), 1729 cm⁻¹ (lactone C = O), 1618 cm⁻¹ (Schiff’s bass, C = N), 1341 cm-1 (S = O). The melting point of 1d is 170 °C (Fig. S11).

Determination of Cytotoxic Concentration by MTT assay

The change in Vero cell morphology was examined to determine the cellular toxicity of 1a–1d. Vero cell monolayers were grown on 96-well plates with 1.0 × 10⁵ cells per well. Various concentrations (0–5000 µg/mL) of 1a–1d were administered to each culture well in triplicate at a final volume of 100 µl and incubated at 37 °C in 5% CO₂ for two days. The each well was then added with 10 µl of MTT reagent and incubated for 4 h at 37 °C. The formazan crystal formed by the reaction was then solubilized by adding diluted HCl (0.04 N), and the absorbance was recorded at 570 nm by an ELISA reader with a reference wavelength of 690 nm. The viable cell percentage was equated as:

[(Absorbance from treated well – cell-free sample blank)/ absorbance from cell control)] /100%.

The 50% cytotoxic concentration (CC₅₀), which causes morphological alterations in 50% of Vero cells, was calculated in comparison to cell control [31].

Determination of the antibacterial activity

The compounds 1a–1d were screened in vitro for their antibacterial activity against nine bacterial strains, four Gram negative and five Gran-positive, including two drug-resistant and three multi-drug resistant (MDR) isolates, compared to four quality control strains. The strains include Gram-negative Escherichia coli ATCC 29212, E. coli MDR-isolate, Salmonella enterica serovar Typhi MTCC 734 and Salmonella enterica serovar Typhi MDR-isolate; along with Gram-positive Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 25922, Methicillin Resistance S. aureus (MRSA), Ampicillin-resistant Coagulase Negative Staphylococcus aureus (CONS), and MDR-isolate of Enterococcus faecalis PH007^R. Thus, out of the nine bacterial isolates E. coli, S. typhi and E. faecalis PH007^R were MDR; while ATCC and MTCC strains were included as quality control strains. The antimicrobial susceptibility or resistance of the test bacteria against the commercially used common antibiotic discs was determined by the standard disc diffusion method, following the latest guidelines of the Clinical and Laboratory Standards Institute [32, 33]. A stock solution (1 mg/mL) was prepared by dissolving 1 mg of test compound(s) in 1.0 mL sterile distilled water; or in 100 µl of 0.1% DMSO, and diluted with sterile distilled water. The test compound-impregnated discs of various potency (0–1000 µg/mL), was prepared by absorbing the sterile filter paper (Whatman No 3) discs (5.25 mm diameter) at different amount of stock solution of compounds 1a to 1d or the test antibiotics aseptically, and dried at room temperature [32]. The disks of the desired potency were then aseptically placed on the MHA plate seeded with the test and control bacterium (10⁶ CFU/mL), and incubated at 37 °C for 24 h. The activity was recorded by measuring the clear zones of growth inhibition on the agar surface around the discs [34–36].

Determination of MIC and growth inhibition assay

The minimum inhibitory concentration (MIC) of the test compound(s) was determined against all the test strains including two drug-resistant strains MRSA and ampicillin-resistant CONS, as well as MDR-isolates Enterococcus faecalis PH007^R, Escherichia coli, and Salmonella enterica Serovar Typhi by broth and agar dilution methods, following the National Committee for Clinical Laboratory Standards guidelines [32, 33]. The test compounds were aseptically added at two-fold dilutions (0–1000 µg/mL) in each sterile tube containing MHB, at room temperature. Bacterial isolates were grown overnight in MHB at 37 °C and diluted with the fresh media at a density of 10⁶ CFU/mL to add in each tube containing different concentrations of the test compounds. Aliquots (100 µL) of culture removed from the incubated tubes were inoculated onto MHA plate to record the viable counts at CFU per mL. The agar dilution test was conducted on MHA plates containing a two-fold concentration of the test compounds and antibiotic control along with 10⁶ CFU/mL of inoculum of each bacterium and incubated overnight at 37 °C. The colony count of the plates was made in each dilution. Each test was repeated three times to calculate the mean count of each dilution [33].

Determination of Minimal Bactericidal Concentration (MBC)

The MBC was determined by both dilution methods [32, 35], using freshly prepared MHB tubes containing overnight culture of bacterial inoculum (2.2 × 10⁶ CFU/mL), added with the test compound 1a (2–threefold of MIC). The tubes were incubated at 37 °C for 24 h with shaking (200 rpm) on a platform shaker. At hourly intervals between 0–24 h, 100 µl aliquots were withdrawn and subjected to determine the growth inhibition by measuring the optical density of the culture in a colorimeter at 600 nm, as well as the colony count as CFU/mL in freshly prepared MHA plates. The lowest concentration of the test compounds which did not show any visible growth in tubes or visible growth in MHA plates was considered the MBC [32, 36, 37].

Growth inhibition assay of test compounds on drug-resistant and MDR isolates

The rate and extent of bacterial growth, treated with 1a, were studied by growth inhibition assay and subsequent growth curve analysis. Growing cultures (10⁶ CFU/mL) of drug-resistant MRSA and CONS, as well as MDR-isolates of E. faecalis PH007^R, E. coli, and S. enterica Serovar Typhi in MHB were exposed to the test compound(s) at their MIC, along with a drug-free inoculated medium plate as control. Samples were removed for colony counts at hourly intervals (0–12 h) and at 24 h. Viable counts were determined by the serial dilution of test culture after 24 h of incubation, and plates containing 30 to 300 CFU/mL for each dilution were counted [35]. The procedure was repeated three times for each test organism to record the means, and to plot Log₁₀ CFU/mL vs. time (t). Antibiotic carryover was prevented by serial dilution of the test antibiotics. An antimicrobial agent was considered bactericidal when its lowest concentration can reduce the original inoculum by > 3 log₁₀ CFU/mL (99.9%); while it will be bacteriostatic when the original inoculum was reduced by 0–3 log₁₀ CFU/mL [36].

Effect of compound 1a on bacterial cell Morphology

The bacterial isolates were grown in 5 mL culture media, and incubated with shaking at 198 rpm for 12 h at 37 °C and added with 1a at its MBC. The untreated cells were taken as a negative control. Following centrifugation at 5000 rpm for 5 min, the cells were repeatedly washed with PBS and centrifuges to collect the pellets. The cells were then fixed with 2.5% glutaraldehyde and washed three times with sterile PBS. About 10 µl of the solution was placed on the glass plate and dried under vacuum. After coating, the cell morphology was recorded by a scanning electron microscope (SEM, EVO 18 Special Edition, Carl Zeiss, Germany) [38].

Molecular docking study

The interaction of test compounds (small molecule) with proteins, and assessment of molecules' potency as well as their toxicity was tested by in-silico studies. The bioinformatics study will help to develop a pharmacophore against the target protein, choosing the optimal target, and to comprehend protein inhibition mechanisms through the analysis of interactions [39]. To realize the effect of the investigating compounds on their antibacterial activities, we conducted molecular docking. As sulfonamide drugs commonly inhibit the enzyme dihydropteroate synthetase, we choose the dihydropteroate synthetase (PDB id: 1DHS) for the molecular docking study [40].

Results and discussion

Synthesis and formulation

Four compounds 4-[(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-ylmethylene)-amino]-N-pyridin-2-yl-benzenesulfonamide (1a), 4-[(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-ylmethylene)-amino]-N-(4-methyl-thiazole-2-yl)-benzenesulfonamide (1b), 4-[(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-yl methylene)-amino]-N-(4-methoxazole-2-yl)-benzenesulfonamide (1c) and 4-[(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-ylmethylene)-amino]-N-(4-methyl-pyrimidin-2-yl)-benzenesulfonamide (1d) were successfully synthesized by the condensation reaction between 8-formyl-7-hydroxy-4-methylcoumarin and the corresponding sulfur-amines (sulfapyridine, sulfathiazole, sulfathiazole, sulfamerazine) separately; and were characterize by spectroscopy (NMR, Mass, FTIR). The ¹H NMR spectral data of compound-1a (DMSO-D₆) showed characteristic signals at 14.47 ppm which corresponding to a coumarinyl -OH proton, a sharp singlet at 9.27 ppm indicating the imine (–CH = N–) proton, at 2.50 ppm coumarinyl–CH₃ and another Ar-Hs appear at 6.29–7.77 ppm. The molecular ion peak at 458.00 (M + Na)⁺ (add calculated Mass) from the mass spectrum also supports the molecular identity. The structural characterization was established by IR spectral, which showed characteristic signals corresponding to 3443.9 cm⁻¹ (aromatic -OH), 1731.4 cm⁻¹ (lactone C = O), 1632.2 cm⁻¹ (Schiff’s bass, C = N), and 1388.3 cm⁻¹ (S = O). All the observations strongly support and confirmed the synthesis of compound-1a. Similarly, the structural identification was also made for compound-1b, 1c, and 1d.

Biological evaluation

Determination of cytotoxicity by MTT assay

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; MTT 2003, Sigma-Aldrich, MO, USA) is used to assess the toxicity of the test compounds 1a-1d on Vero cell morphology. Vero cells (1.0 × 10⁶ per well) were cultured onto 96-well plates, and the test compounds (1a-1d) of different concentrations were added to each well at a final volume of 100 µl, in triplicate using DMSO (0.1%) as a negative control [41]. Data were calculated as the percentage (%) of cell viability by the formula: [(sample absorbance cell-free sample blank)/mean media control absorbance)]/100%. Fifty percent cytotoxic concentration (CC₅₀) was calculated from the concentration that causes observable morphological changes in 50% of Vero cells, compared to cell control (Fig. 1). CC₅₀ values for the synthetic compounds 1a-1d are 420 µg/mL, 330 µg/mL, 290 µg/mL, 310 µg/mL, respectively. Changes in Vero cell morphology is presented in Figs. 2 and 3.

Fig. 1.

Cytotoxicity of coumarin functionalized 1a, 1b, 1c, and 1d by MTT Assay. Vero cell morphology does not seem to be affected at CC₅₀ of Schiff base (1a) by exposure, compared to the control

Fig. 2.

Change in Vero cell morphology before and after treatment with 1a; a Cell control, b 50% cytotoxic concentration (CC₅₀), c Vero cell at cytotoxic concentration

Fig. 3.

Antibacterial activity spectrum of 1a against MRSA and Ampicillin-resistant CONS (A); along with Escherichia coli MDR-isolate, and Staphylococcus aureus ATCC 29213 (B)

Determination of Antimicrobial susceptibility by disc diffusion method

The antimicrobial susceptibility of all the test isolates with four quality control strains, were determined against several common antibiotics including ampicillin and gentamycin as the reference drug control for the bacterial strains tested. The antibiogram profile of the test bacteria, presented in Table 2, revealed one Methicillin-resistant S. aureus (MRSA), and another Coagulase-negative ampicillin-resistant S. aureus (CONS), with three multidrug-resistant (MDR) isolates of Enterococcus faecalis, Escherichia coli, and Salmonella enterica Serovar Typhi, as these three are resistant to more than three antibiotics tested. The results revealed that the zone diameter of 1a with MRSA and CONS was 15.9 and 18.2 mm; while it was 10.5, 14.5 and 15.8 against the MDR-isolates of Enterococcus faecalis PH007, Salmonella Typhi and Escherichia coli respectively. Interestingly, the zone diameter with control strains S. Typhi MTCC 734, E. coli ATCC 25922, E. faecalis ATCC 29212 and S. aureus ATCC19213 was 11.3, 15.0, 12.5 and 17.7 mm, respectively. Thus, the disc containing the compound 1a showed significant zone of growth inhibition with drug-resistant isolates than the control strains (Figs. 4 and 5). However, zone diameter produced by 1b containing discs are smaller (12.5–7.4 mm and 8.5–7.2 mm) against the same organisms (Table 1), compared to reference standard ampicillin and gentamicin. From the results it is clear that zone of inhibition with 1c and 1d are comparatively much less than 1a, indicating that their antimicrobial activity in tested concentration was much less and may require higher concentrations to inhibit these test bacteria (Table 1). Further study was carried out with 1a and 1b, as these two compounds showed better antibacterial activity against the selected test bacteria.

Table 2.

MIC of 1a-1d against the test bacteria with Antibiogram of drug-resistant and MDR isolates

Bacteria	No	1a	1b	1c	1d	Resistance profile [a]
		MIC (μg/mL)	MIC (μg/mL)	MIC (μg/mL)	MIC (μg/mL)
Enterococcus faecalis MDR	1	256	350	700	512	A, CIP, Van, C, T, Er, Gm
Escherichia coli MDR	1	128	450	650	650	A, Amc, Gm, Er, CIP
Salmonella Typhi MDR	1	250	220	550	512	A, C, CIP, NA, NOR, T
S. aureus PH217 MRSA	1	150	330	450	700	Mth, Amc, Amp
S. aureus CONS Resistant	1	175	250	350	600	Cft, Amp
Quality Control strains
S. aureus ATCC 29213	1	200	150	450	560	Sensitive
E. coli ATCC 25922	1	250	256	525	720	Sensitive
E. faecalis ATCC 29212	1	220	220	650	600	Sensitive
Salmonella typhi MTCC 734	1	256	150	590	650	Sensitive

[a] Antibiotic discs of: A, ampicillin (10 μg); Amc, amoxicillin with clavulanic acid (20/10 μg); C, chloramphenicol (30 μg); CIP, ciprofloxacin (5 μg); Cft, cefotaxime (30 µg); Er, erythromycin (15 µg); Gm, gentamicin (10 µg); Mth, methicillin (10 µg); NA, nalidixic acid (30 μg); NOR, norfloxacin (10 μg); T, tetracycline (30 μg); Van, vancomycin (16 µg). Lowest MIC values indicate the highest inhibitory effect

Fig. 4.

Zone of inhibition of four bacterial isolates (S. aureus, MRSA, CONS, E. coli) against 1a

Fig. 5.

Zone of inhibition of MRSA and ampicillin-resistant E. faecalis isolates against 1b

Table 1.

Antibacterial activity of coumarin functionalized derivatives 1a-1d and four sulfa-drugs

Bacteria	1a	Sulfapyridine	1b	Sulfathiazole	1c	Sulfathiazole	1d	Sulfamerazine
	IZD# (mm)	IZD# (mm)	IZD# (mm)	IZD# (mm)	IZD# (mm)	IZD# (mm)	IZD# (mm)	IZD# (mm)
Methicillin- resistant S. aureus PH217 (MRSA)R	15.9	13.5	10.5	9.5	8.0	7.5	9.2	8.0
S. aureus CONSR	18.2	16.8	9.0	9.6	5.5	7.6	7.4	7.6
Enterococcus faecalis PH007 MDR	10.5	10.5	9.7	8.7	7.3	7.6	8.2	6.3
Escherichia coli MDR	15.8	5.5	9.2	5.4	9.6	3.5	6.7	3.7
Salmonella Typhi MDR	14.5	4.9	10.5	4.1	8.3	3.9	5.8	3.8
S. aureus ATCC 29213	17.7	15.7	12.5	10.3	9.0	8.4	9.2	8.3
E. faecalis ATCC 29212	12.5	10.8	8.0	8.6	7.3	6.3	8.9	7.1
Escherichia coli ATCC 25922	15.0	13.8	7.4	10.4	8.5	8.3	7.7	7.0
Salmonella typhi MTCC 734	11.3	10.5	8.5	5.5	9.5	7.3	7.5	8.3

IZD*, Inhibition Zone Diameter (mm)

Growth inhibition with determination of MIC of the test compounds

The minimum inhibitory concentration (MIC) of 1a was found to be 150 µg/mL against the MRSA, 175 µg/mL against CONS, 200 µg/mL against S. aureus ATCC 29213, 220 µg/mL against E. faecalis ATCC 29212, and 250 µg/mL against E. coli ATCC 25922. On the other hand, the MIC against MDR strains E. coli, S. enterica serovar Typhi, and E. faecalis PH007 was 128, 250 and 256 µg/mL, respectively; but it was 220, 250 and 256 µg/mL against E. faecalis ATCC 29212, E. coli ATCC 25922, and Salmonella typhi MTCC 734 respectively, at inoculum size of 10⁶ CFU/mL (Table 2). However, the MBC was found to be 2–fourfold higher, than their MIC. The MBC was 1024 µg/mL against E. coli MDR, S. aureus MRSA, S. aureus CONS, E. faecalis MDR, and 512 against S. Typhi MDR; while for control strains MBC was 512 for S. Typhi 734 and E. faecalis 29212; but 1024 µg/mL against S. aureus 29213 and E. coli 25922, respectively (Fig. 3). The growth curve presented in Fig. 3 and Fig. 6 showed that compound 1a is bactericidal at MBC but bacteriostatic at its MIC concentration.

Fig. 6.

Growth inhibition curve of Methicillin-resistant S. aureus (MRSA) and ampicillin-resistant CONS in absence or presence of 1a (A); and Growth inhibition curve of MDR-isolates E. coli and S. enterica serovar Typhi in absence or presence of 1a (B) at its MIC

3.2.4 Mechanistic insights of antibacterial activity of the test compound 1a

The morphology of bacterial cell walls, untreated or treated with 1a, examined by Scanning Electron Microscope (SEM) revealed the altered surface structure of treated bacterial cell, compared to the structure of the untreated cell. Thus, our results indicated that the alteration of bacterial surface structure is the plausible mechanism of action of the test compound 1a. We have used the MDR strains of E. coli as it was highly sensitive to 1a with zone of inhibition of 15.8 mm and MIC of 128 µg/mL. The SEM, used to study the morphological damage [42], demonstrated rod-shaped bacteria with normal topology in untreated control; while the bacteria treated with 1a showed altered ultra-structure presented in Fig. 7 under 25000X magnification of SEM.

Fig. 7.

Cellular morphology of untreated E. coli (Control); and 1a treated E. coli (MIC 128 μg/mL)

by FE-SEM.

The antimicrobial spectrum of Schiff bases (1a-1d) against the test bacteria, evaluated by disc diffusion assay, is presented in Table 1; while their MIC, determined by broth-dilution assay, is in Table 2. Overall, the sulfapyridine-based Schiff base (1a) has exhibited the highest antibacterial activity against S. aureus, similar to the control, indicating that these compounds could be used in developing more effective antibacterial agents against drug-resistant or MDR isolates of E. coli, S. Typhi, S. aureus, MRSA and CONS. It has been reported that after tetracyclines, Sulfa drugs are the second broadly used class of veterinary antibiotics [43–45]. It is known that some Streptococcus species including Streptococcus pyogenes (Group-A ß-hemolytic Streptococcus), that causes hemolysis (termed as hemolytic Streptococcus), toxic shock syndrome and tissue necrosis has been treated with sulfapyridine and sulfathiazole [46]. The sulfapyridine and sulfathiazole are used here as the precursor 1a and 1b, respectively. Moreover, coumarin analogues had also been reported to inhibit other bacterial species [47], which is consistent with our result that coumarin analogue 1a has increased antibacterial activity against both Gram-positive and Gram-negative bacteria tested (Fig. 8).

Fig. 8.

a Docking pose of 1a with DHPS; b non-covalent interactions between 1a and DHPS

Molecular docking study

The molecular docking study was conducted with DHPS available from PDB sources, and our study shows a very high binding affinity with sulfonamide drugs. The binding energy data, account for thermodynamically favored binding (Table 3), revealed –ve ΔG (kcal/mol), via substitution of PABA and the highest stability with 1a (-7.7 kcal/mol).

Table 3.

Binding energy of sulfonamides and their coumarin derivatives using molecular docking study with dihydropteroate synthetase

Compound	ΔG (kcal/mol)	Compound	ΔG (kcal/mol)
8-formyl-7-hydroxy-4-methyl coumarin	-5.8	–	–
Sulfathiazole	-5.6	Sulfamethoxazole	-5.9
1b	-7.3	1c	-7.5
Sulfapyridine	-5.8	Sulfamerazine	-6.1
1a	-7.7	1d	-7.4

Here, coumarin C = O, sulfonamide S = O and pyridine nitrogen are involved in hydrogen bonding with Glycine 283, Valine 285, and Threonine 308, respectively. The first two fragments are also found in non-classical hydrogen bonding with Glycine 284 and Glycine 282. Amino acid residue Alanine 341 interacts with pyridine hydrogen through non-classical hydrogen bonding. Other aromatic parts of the molecule interact with Alanine 238, Glycine 282, Leucine 103, Leucine 281, Valine 285, and Alanine 343 with the help of π-stacking, π-alkyl, and hydrophobic interactions, respectively.

Our results indicate that, the compound 1a may penetrate the outer membrane of E. coli via thinner peptidoglycan layer, and then binds at the nicotinamide-adenine-dinucleotide (NAD) binding site of the dihydropteroate synthase (DHPS), an essential enzyme in the biosynthesis of dihydrofolate in microbes, located on the surface and contains a dihydro-6-hydroxymethylpterin pyro phosphokinase domain at the N terminal. Docking study by Yusuf et al., revealed the active site pocket of nitro benzyl sulfonamide had inhibitory activity against Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa at MIC of 15.625 and 7.81 µg/mL, compared to chloramphenicol [34]. A study by Aragón-Muriel et al. on four benzimidazole-based Schiff bases and their metal complexes revealed antibacterial activity against S. aureus 25923, Listeria monocytogenes 19115, E. coli 25922, and P. aeruginosa 27583 at an MIC of 250 ng/mL, compared to ciprofloxacin at 0.5 ng/mL [48]. Another study reported that the pyrene-based Schiff bases 4-[(5-pyren-1-yl-thiophen-2-ylmethylene)-amino]-phenol and 4-[(4-pyren-1-yl-benzyli-dene)-amino]-phenol had antibacterial activity against P. aeruginosa 9027 and 27853 between 100–500 μg/mL [49].

Conclusion

Antibiotics being one of the great discoveries of twentieth century, pose one of the greatest threat due to steady rise of antibiotic-resistance in hospitals, communities, and the environment concomitant with their use. The MTT assay indicates that the synthetic Schiff bases of sulfonamides 1a-1d, are relatively less-toxic and moderately safe. Compared to parent sulfonamide, the in vitro cytotoxicity of 1a-1d was significantly less. Moreover, their effectiveness against MRSA, CONS, and three MDR-isolates along with their mechanism of action revealed that they had much better activity profile than parent Sulfa drugs. Furthermore, these agents have dual advantages, as the complex structure may help to prevent the bacteria to develop resistance in one hand, and their ability to target multiple sites in bacterial cell on the other hand. Thus, our results indicated that the antimicrobial activities of our synthetic sulfonamide derivatives might be related to their structure–activity relationship, having more binding affinity for the target protein DHPS. Our study also revealed that 1a not only binds to the bacterial cell membrane but also internalized to damage the cellular integrity of bacterial cell, as demonstrated by SEM study with the MDR isolate of E. coli (Fig. 7). We, therefore, conclude that the synthesized biocompatible compound 1a may serve as a promising agent against drug-resistant or MDR bacterial infections that plague our society today.

Supplementary Information

Below is the link to the electronic supplementary material.

Data Availability

We confirm that all data are available in our laboratory.

Declarations

Conflict of interest

The authors confirm that there is no conflict of interest.