Synthesis, molecular modelling, and antibacterial evaluation of new sulfonamide-dyes based pyrrole compounds

In this study, we synthesized new series of 5-oxo-2-phenyl-4-(arylsulfamoyl)sulphenyl) hydrazono)-4,5-dihydro-1H-pyrrole-3-carboxylate hybrids 4a-f with the goal of overcoming sulfonamide resistance and identifying novel therapeutic candidates by chemical changes. The chemical structures of the synthesized hybrids were established over the spectroscopic tools. The frontier molecular orbitals configuration and energetic possessions of the synthesized compounds were discovered utilizing DFT/B3LYP/6-311++ G** procedure. The 3D plots of both HOMO and LUMO showed comparable configuration of both HOMO and LUMO led to close values of their energies. Amongst the prepared analogues, the sulfonamide hybrids 4a-f, hybrid 4a presented potent inhibitory towards S. typhimurium with (IZD = 15 mm, MIC = 19.24 µg/mL) and significant inhibition with (IZD = 19 mm, MIC = 11.31 µg/mL) against E.coli in contrast to sulfonamide (Sulfamethoxazole) reference Whereas, hybrid 4d demonstrated potent inhibition with (IZD = 16 mm, MIC = 19.24 µg/mL) against S. typhimurium with enhanced inhibition against E. Coli, Additionally, the generated sulfonamide analogues’' molecular docking was estimated over (PDB: 3TZF and 6CLV) proteins. Analogue 4e had the highest documented binding score as soon as linked to the other analogues. The docking consequences were fitting and addressed with the antibacterial valuation.

Sulfonamides are an antibacterial sulfa-drug.It is an organic molecule made composed of aniline that has been derivatized with a sulfonamide group as in Fig. 1a [1][2][3] .The Allies in World War II utilized powdered sulfonamide to lower infection rates, which led to a considerable decline in mortality rates compared to previous battles 4,5 .Due to its toxicity and the existence of more efficient sulfonamides, is rarely, if ever, given systemically.Sulfanilamide has been replaced by modern antibiotics on the front lines, but it is still used today in topical treatments for treating vaginal yeast infections, particularly vulvovaginitis, which is brought on by Candida albicans 6,7 .The primary sulfonamide structure, SO 2 NH, is found in a variety of biologically active chemicals that are frequently employed as antibiotics, Anti-hypertension, carbonic anhydrase inhibitors, antithyroid medications, and antimicrobial capsules 8 .Sulfonamides are also very beneficial pharmacological substances because they reveal a wide range of biological properties, such as anti-tumor, anti-inflammatory, and antiviral activity [9][10][11] .Sulfonamide antibiotics are occasionally used in conjunction with other antibiotics to improve their efficacy 12 .They could be coupled, for example, with trimethoprim, another antibiotic that targets a different phase in the folic acid production pathway 13 .Trimethoprim-sulfamethoxazole, often known as co-trimoxazole, is a popular antibiotic used to treat respiratory, urinary, and gastrointestinal infections.Sulfonamido-chrysoidine (prontosil red, Fig. 1b), one of the azo dyes to cure Streptococcus infection in mice, was published by German bacteriologist and pathologist Gerhard Domagk and proved to be very successful 14 .Sulfonamides are used to treat several gastrointestinal and urinary tract infections in clinical settings 15 .Sulfonamides could compete with p-amino benzoic acid (PABA) for inclusion since they are structurally similar to it and may be needed by bacteria to synthesis vitamin Bc 8 .Infections in cattle herds are treated with sulfonamide antibiotics in veterinary medicine 16,17 .Bacterial resistance and sulfonamide adverse effects are two factors that limit the use of sulfonamides in therapy in order to overcome A careful inspection of the synthesized derivatives IR spectra indicated that all derivatives are existing in keto-hydrazo (Fig. 3 and Figure S1) form where: (i) The strong band at 3471-3425 cm −1 with a shoulder at 3265-3200 cm −1 were attributed to the stretching vibrations of the free and H-bonded pyrrole υ(NH) 27 , respectively.The band observed at 1668-1650 cm −1 region was assigned to the ν(C=O) vibration of oxo-pyrrole 27,28 .The spectra displayed another strong band at 1725-1690 cm −1 corresponding to the ν(C=O) of carboxylate group 27 .(ii) Furthermore, the two bands at 3200-3140 and 3130-3100 cm −1 were assigned to the ν(NH) vibrations of sulfonamide 29 and hydrazo 28 groups, respectively.While, the sulfonamide υ(SO 2 ) s and υ(SO 2 ) as were displayed at 1374-1357 and 1144-1135 cm −128 , respectively.(iii) Moreover, the spectral data showed two bands only in the 1640-1630 and 1600-1590 cm −1 regions where the former was attributed to the overlapped hydrazo and heterocyclic ring υ(C=N) vibration 28 while the latter was corresponding to the υ(C=C) of the aromatic rings 28 , respectively.(iv) Also, the spectra displayed several bands at 1560-1555, 1250-1240, 1165-1145 and 735-680 cm −1 owing to the Amide II, Amide III, υ(N=NH) and ρ(NH) vibration [27][28][29] , respectively.
The 1 H NMR spectrum of compound 4a (as an example) disclosed triplet (t) and quartet (q) signals (s) at δ 1.15 and 4.12 ppm belongs the protons of ethoxy group (-OCH 2 CH 3 ).The protons of thiazole ring were detected as two doublet signals at δ 6.78 and 7.19 ppm.The aromatic protons were observed as multiplet and doublet signals in the region from δ 7.45 to 7.74 ppm.Meanwhile, protons of imino-groups were resonated as singlet signals at δ 11.40, 12.66, and 12.99 ppm.

DFT calculation
The DFT geometrical optimization procedures presented resemble angular configuration of the investigated compounds in which the phenylhydrazineylidene pyrrole-3-carboxylate moiety has planar structure (Fig. 4).Whereas, the phenyl pyrrole substituent was tilted one the pyrrole ring plane, i.e.S1).Moreover, both of bond length and angle data displayed noteworthy resemblance with those obtained from the X-ray of hybrid-single crystal 30,31 , where the lengths were longer than the corresponding X-ray by maximum 0.12 Å, RMSD 0.04-0.05,however the angles differences were in range 0.0-18.5°,RMSD = 5.6-6.7.These discrepancies may be attributed to that no intermolecular columbic interactions were considered in the DFT calculations as it carried out for a single molecule in gaseous state, while in practical, there is quite a few interrelating molecules in solid crystal lattice 32 (Tables S2-S3).
Furthermore, the HOMO and LUMO, frontier orbitals, using GaussView, version 5; 2009 program, have substantial role in molecule's affinity to donate and accept electrons 33 , correspondingly, lengthways with molecule's bioactivity that essentially affected by the HOMO-LUMO energy gap [34][35][36] .The graphs of frontier orbitals of deliberate hybrids displayed similar HOMO configuration which was primarily built up of the π-orbitals of conjugated system of the whole molecule as well as lone pairs of heteroatoms.Whereas, their LUMO were constructed predominantly from the π*-orbitals of the 5-oxo-2-phenyl-4-(arylsulfamoyl)phenyl)hydrazono)pyrrole-3-carboxylate moiety (Fig. 5).Accordingly, alike configuration of the HOMO and LUMO led to close values of their energies where the E H ranged from − 5.74 to − 6.00 eV, while the E L were − 4.01 to − 4.28 eV and exhibited the same order, 4a < 4d < 4e < 4f < 4b < 4c.Also, the investigated hybrids revealed low and close energy gap (ΔE H-L ), 1.68-1.75eV, and may be sorted as 4b < 4e < 4c < 4a < 4f < 4d (Table 1).
(1) Vol  1, the synthesized sulfonamide-based pyrrole compounds unveiled the lowest global hardness (η) and highest softness (δ) values and thus it is the furthermost reactive, slightest stable kinetically and softest one.Furthermore, the studied hybrids are powerful electrophile, ω = 13.72-15.50eV, as strong electrophile has electrophilicity index ω > 1.5 eV 37,38 , and follow the order 4a < 4d < 4f < 4e < 4c < 4b.As well, the electron Vol:.( 1234567890 37,38 (Table 1).The Mulliken's atomic charges offered interpretation of charge transfer and electronegativity of the molecule 39 .The atomic charges of the investigated hybrids showed that the pyrrole nitrogen atom, N 1 (Pr) , has a negative charge, − 0.488-0.495,while the C 5 (Pr) atom has positive charge, 0.210-0.216,which may be attributed to the electron withdrawing effect of the nitrogen and attached oxygen atoms.Although the carbon atoms C 3 (Pr) and C 4 (Pr) have negative charge but the former was more negatively charge than the latter, − 0.125-0.152and − 0.078-0.087,respectively, which may be originated from the difference between the carboxylate and phenyl hydrazinylidene groups electron affinity (Table 2).Moreover, the nitrogen and oxygen atoms acquired negative charge whereas the sulfur atoms of the sulfonamide group in addition to those of the thiazole and thiophene rings have positive charge which may be attributed to the presence of adjacent strong electron withdrawing atoms or their involvement in resonance structure of the heterocycle (Table 2).Furthermore, the molecular parameters such as polarizability (α total ), hyperpolarizabilities (β total ), and dipole moment (μ), were calculated [40][41][42] as shown below, as a measure for the molecule's softness and electron density distribution that mainly influence the intermolecular interactions 43 , as well as, optical nonlinearity and response [44][45][46][47] .

In vitro antibacterial activity
In accordance to, both of IZD and MIC methodology, the synthesized sulfonamide hybrids were inspected over antibacterial efficacies crossways Gram+ and − strains.Both of table S4 and Fig. 6 were recognized the antimicrobial actions of the synthesized hybrids over the antibacterial effectiveness since sulfonamides have the competence to stop the generation of folic acid in the bacterial growing 49 .Through the explored hybrids, they unveiled more liable outcomes towards both of "S.aureus and B. subtilis" Gram + and "S.typhimurium and E. coli" Gram -ve bacterial straining cells.Where, sulfonamide hybrids a-f designated respectable activity in general, particularly through Gram + bacteria higher than Gram-proportional to Sulfamethoxazole reference.Temporarily, sulfonamide hybrid 4a have both of amino moiety and thiazole ring exhibited good inhibition zone of (IZD = 17 mm, MIC = 11.31µg/mL) towards B. subtilis, near to the Sulfamethoxazole (IZD = 16 mm, MIC = 11.31µg/mL), and weak inhibition zone of (IZD = 21 mm, MIC = 11.31µg/mL) towards S. aureus.However, sulfonamide derivative 4b have a pyridine ring revealed amazing inhibition (IZD = 16 mm, MIC = 11.33 µg/ mL) against B. subtilis and better inhibition to S. aureus with (IZD = 18 mm, MIC = 11.33 µg/mL) with a match to reference (IZD = 17 mm, MIC = 11.31µg/mL).Though, sulfonamide derivative 4c exhibited a poor inhibition to the Sulfamethoxazole reference towards S. aureus with (IZD = 23 mm, MIC = 11.31µg/mL) and weak inhibition over B. subtilis with (IZD = 25 mm, MIC = 11.31µg/mL).Also, sulfonamide derivative 4d showed the weakest inhibition against S. aureus with (IZD = 26 mm, MIC = 11.31µg/mL) and better inhibition over B. subtilis with (IZD = 18 mm, MIC = 11.31µg/mL).Whereas, sulfonamide derivative 4e have displayed potent inhibitions over     S4).

Structural activity relationship
Because all of the synthesized hybrids contain a sulfonamide moiety, however several compound contain sulphonamide bond play an important role in enhancing the bioactivity 50 , the research was motivated by the intriguing reactivity results of sulfonamide hybrids a-f towards various bacterial strains 5 .The thiazole ring in sulfonamide derivatives plays a crucial role in bacterial death by preventing the manufacture of folic acid, and the hybrids 4a, 4d, and 4e that include it were shown to have acceptable activations against the two bacterial strains as a result 51 , a substance that is necessary for bacterial development and reproduction.Sulfonamides successfully prevent bacterial reproduction by interfering with this metabolic pathway, enabling the immune system to eradicate the infection 52 .Additionally, sulfonamides with pyridine moiety, such as those found in hybrids 4b, 4e, and 4f, work to inhibit the enzyme use PABA, a crucial metabolite for bacterial growth, in order to exhibit their antibacterial actions 53 The pyridine ring also improves these antibiotics' solubility and cellular penetration, which adds to their total antibacterial efficacy.Sulfonamides with a pyridine ring efficiently limit bacterial proliferation and help treat bacterial infections by targeting important metabolic processes and bacterial enzymes 54,55 .

Molecular docking
Tables S5 and S6 provide the fallouts of molecular docking performed on compounds 4a-f by M.O.E "v10.2019.01"computer software.The purpose of this was to develop hypotheses about the binding of the most active compounds to the PABA constituent of S. aureus strain.To create the model for this site, the researchers used the X-ray crystal structures of both a wild type enzyme DHPS (Dihydroptorate Synthase of Versinia pestis, PDB: ID 3TZF) 56 and S. aureus F17L/E208K double mutant DHPS in the ligand-bound conformation with the PDB ID of 6CLV.The study focused on examining sulfonamide-based analogues and their bindings with the amino acids in the 6CLV structure 51 .In the interactions with (BDB:3TZF), hybrid 4a exhibited a binding affinity of − 7.78 kcal/ mol, with interactions spanning hydrogen bonds and π-H interactions across four different residues (Asp96, Ser222, Thr62, and Arg63), indicating a diverse and strong interaction profile, contributing to its high binding affinity.The RMSD value of 1.56 indicates a stable docking posture with distances ranging from 2.68 to 4.37 Å, emphasizing the significance of spatial location for efficient binding.Similarly, hybrid 4b exhibited a binding affinity of − 7.70 kcal/mol, mostly participating in hydrogen acceptor interactions, demonstrating the compound's potential to create stable hydrogen bonds with the targeted protein.Its interactions are defined by distances that permit efficient binding, demonstrating a strong interaction despite having a somewhat lower binding affinity than 4a.Hybrid 4c, with the greatest binding affinity of − 8.09 kcal/mol in the series, had a high tendency for hydrogen bonding, suggesting a very favorable interaction with the protein target.The low RMSD value of 1.28 adds to the dependability of its binding position, making it the most viable choice for future exploration.(Fig. 7).However, Hybrid 4d exhibited a binding affinity of − 7.54 kcal/mol with a combination of hydrogen donor and acceptor interactions, indicating flexible binding capabilities.The contact distances and diversity of bonds indicate a balanced interaction profile, which, despite its lower affinity, highlights the compound's potential for specificity.Through the binding energy, with hybrid 4e exhibiting the most favourable binding energy of − 8.01 kcal/mol.Hybrid 4f, in particular, has the lowest RMSD value of 0.96, indicating a solid and stable docking conformation.While all hybrids exhibit a combination of these interactions, hybrids 4a and 4c stand out for having a more diversified collection of interactions, including π-cation interactions.
The study on the impacts of docking revealed several findings: (1) the primary objective of docking simulation is to identify the most promising interaction between the chosen proteins and its potential partners, while considering other alternatives.(2) The comprehensive molecular docking analysis of the hybrid compounds 4a through 4f has elucidated their interaction mechanisms and binding affinities with a target (BDB:3TZF) protein, revealing significant insights into their potential as inhibitors.3) Among the hybrids tested with the 6CLV protein, those containing pyrimidine substituents (hybrids 4e, 4b, and 4f) had higher binding scores (− 8. were connected to amino acids of 6CLV (Val49, Arg52, Arg204, Arg239, Asp84, and Ser50) through hydrogen bonds and π-H interactions.The analogues were represented in both two and three-images, as well as electron density maps.

In silico ADME analysis
A complete investigation of physicochemical properties for many hybrid compounds was reported in Table S7 utilizing the Swiss ADME program.These characteristics are useful markers of a molecule's pharmacokinetics and drug-likeness.The effect of molecular weight on the ADME characteristics of compounds has been thoroughly studied.Orally active medicines typically have molecular weights ranging from 160 to 480 Da 57 .All of the hybrids described here meet these criteria.Meanwhile, molecular flexibility, as measured by the number of rotatable bonds, can have a considerable influence on molecule bioavailability.Compounds with fewer than ten rotatable bonds have higher oral bioavailability 58 .This requirement is met by all of the hybrids mentioned.Molar refractivity temporarily reveals molecule size and electrical properties.This characteristic has been linked to drug permeability 59 .TPSA also represents the molecule's capacity to interact with biological membranes.Increased TPSA usually indicates a problem with permeability 59 .The hybrid with the greatest TPSA among the hybrids may indicate a relative reduction in permeability.Also MLOGP, a representation of compound lipophilicity, is required because lipophilicity has a significant impact on the pharmacokinetic properties of compounds 60 .
ESOL also provides predictions on the water solubility of compounds.All of the hybrids are labelled MS, which indicates moderate solubility, indicating that solubility may not offer substantial issues for these compounds 61 .The Swiss ADME program was used to assess the physicochemical characteristics of the hybrids, which provided significant insights into their potential drug-like qualities.While certain hybrids have values that are outside of the commonly recognized range for oral medications, it is important to note that there are exceptions, and experimental evidence is required for conclusive findings.In general, these hybrids have an appealing profile, particularly in terms of hydrogen bonding and rotatable bonds.However, more in-vivo and in-vitro investigations would be required to determine their true pharmacokinetic and pharmacodynamic capabilities.

Antibacterial evaluation
The antibacterial effectiveness of the newly prepared sulfonamide hybrids toward ATCC bacterial strains, clinically isolated Gram+ and Gram− bacteria are used.The assigning of antibacterial effectiveness was performed via agar disk diffusion.The results established several antibacterial effectiveness of the newly synthesized sulfonamide derivatives against bacteria determined in this study.The newly synthesized sulfonamide derivatives displayed antibacterial activities against four ATCC bacterial strains B. subtilis ATCC 6633, S. aureus ATCC 25923, S. typhimurium ATCC 14028, and E. coli ATCC 25922 67 .Meanwhile, minimum inhibitory concentration (MIC) was established as the lowest quantity that totally blocks observable bacterial growth.As a result, one loop of the MIC solution that seemed optically clear was grown on agar plates and cultured at 37 °C for 20 h 68 .

Molecular docking
Starting with the X-ray structure, theoretical docking simulation was run to explore the bindings of the newly prepared sulfonamide analogues' ligand structures against two diverse proteins a wild type enzyme DHPS (Dihydroptorate Synthase of Versinia pestis, PDB: ID 3TZF) 69 and S. aureus dihydropteroate synthase (saDHPS) which was represented by (PDB ID: 6CLV)strain rummage-sale in this work 51 .

In silico ADME analysis
Using the publicly available Swiss ADME programme, we investigated the in silico physicochemical and pharmacokinetic features of newly synthesised sulfonamide hybrids with decreased MIC values (16, 18, 20, 21, and 23 µg/mL).This software provides access to a pool of fast yet modest predictive models that use simple molecular and physicochemical descriptors, such as molecular weight 18 , the count of specific types of bonds (the numbers of heavy atoms, aromatic heavy atoms, rotatable bonds, hydrogen-bond acceptors, hydrogen-bond donors), topological polar surface area (TPSA), and several others, all of which are important determinants in predicting good drug/lead-like 70 .

Conclusion
Finally, by inventing and synthesizing a new series of 5-oxo-2-phenyl-4-(arylsulfamoyl)phenyl)hydrazono)-4,5-dihydro-1H-pyrrole-3-carboxylate hybrids 4a-f, this study addressed the difficulty of sulfonamide resistance in antibacterial therapy.Through structural alterations, the goal was to overcome resistance mechanisms and find possible treatment options.The chemical configurations of the prepared hybrids were confirmed using spectroscopic methods.The DFT calculations were rummage-sale to clarify the FMO's construction and energies of the explored hybrids and discovered that they have low HOMO-LUMO energy gap (ΔE H-L ), 1.68-1.75eV, and may be sorted as 4b < 4e < 4c < 4a < 4f < 4d.
The antibacterial activity of sulfonamide hybrid 4a against S. typhimurium was exceptional, with IZD of 15 mm and MIC of 19.24 µg/mL.Furthermore, it inhibited E. coli significantly, with an IZD of 19 mm and MIC of 11.31 µg/mL, surpassing the reference sulfamethazole.Hybrid 4d also demonstrated significant antibacterial activity over S. typhimurium, with by IZD of 16 mm and MIC of 19.24 µg/mL, as well as increased inhibition against E. coli.Furthermore, using the PDB codes 3TZF and 6CLV, docking stimulation technique were achieved to analyze the binding interactions of the produced sulfonamide hybrids.When compared to other hybrids, hybrids 4e and 4c had the highest binding score, indicating robust interactions with the target proteins, respectively.The findings of this study show that these newly synthesized hybrids have the potential to be efficient antibacterial agents against resistant pathogens.Their improved antibacterial activity, as revealed by both experimental testing and molecular docking, provides important insights for the further development and optimization of potential treatment options.This discovery contributes to current efforts to battle bacterial infections by overcoming sulfonamide resistance and establishing a structure-activity link, paving the door for the development of more potent antibacterial drug.

Figure 6 .
Figure 6.MIC of the explored hybrids over both of Gram + and Gram-bacteria.

Table 1 .
The HOMO-LUMO energies and chemical reactivity descriptors (eV) of investigated compounds.