Novel Thiadiazole-Based Molecules as Promising Inhibitors of Black Fungi and Pathogenic Bacteria: In Vitro Antimicrobial Evaluation and Molecular Docking Studies

Novel 1,3,4-thiadiazole derivatives were synthesized through the reaction of methyl 2-(4-hydroxy-3-methoxybenzylidene) hydrazine-1-carbodithioate and the appropriate hydrazonoyl halides in the presence of a few drops of diisopropylethylamine. The chemical structure of the newly fabricated compounds was inferred from their microanalytical and spectral data. With the increase in microbial diseases, fungi remain a devastating threat to human health because of the resistance of microorganisms to antifungal drugs. COVID-19-associated pulmonary aspergillosis (CAPA) and COVID-19-associated mucormycosis (CAM) have higher mortality rates in many populations. The present study aimed to find new antifungal agents using the disc diffusion method, and minimal inhibitory concentration (MIC) values were estimated by the microdilution assay. An in vitro experiment of six synthesized chemical compounds exhibited antifungal activity against Rhizopus oryzae; compounds with an imidazole moiety, such as the compound 7, were documented to have energetic antibacterial, antifungal properties. As a result of these findings, this research suggests that the synthesized compounds could be an excellent choice for controlling black fungus diseases. Furthermore, a molecular docking study was achieved on the synthesized compounds, of which compounds 2, 6, and 7 showed the best interactions with the selected protein targets.


Introduction
Coronavirus disease 2019 (COVID-19) is a severe public health issue plaguing the world. Due to this, it was announced as a pandemic by the World Health Organization (WHO) within a limited timeframe [1]. Other microbial illnesses were observed in COVID-19 individuals [2]. Severe fungal infections such as aspergillosis caused by Aspergillus, Candida, mucoromycetes, and others were also identified in COVID-19 sufferers [3,4]. Mucormycosis, or black fungus, is a fungal ailment primarily caused by Mucorales species including Rhizopus and Mucor [5,6]. As a result, COVID-19-connected mucormycosis (CAM) has been reported in people who have been medicated or recovered [4]. CAM occurrences are on the rise worldwide, especially in Asiatic regions, where COVID-19 sufferers have a high mortality rate [4,7].
The chemical composition of the newly produced target molecules was affirmed by spectral and microanalytical data as disused briefly in the experimental section.

Antimicrobial Activities
In the current practical research, the in vitro zone of inhibition (ZOI) method was applied to appraise the antibacterial action of six newly synthesized compounds ( The chemical composition of the newly produced target molecules was affirmed by spectral and microanalytical data as disused briefly in the experimental section.

Antimicrobial Activities
In the current practical research, the in vitro zone of inhibition (ZOI) method was applied to appraise the antibacterial action of six newly synthesized compounds (2, 3, 4, 5, 6, and 7) against four pathogenic bacteria, namely: K. pneumoniae, P. aeruginosa, S. aureus, and B. subtilis using a disc diffusion assay. The potential antibacterial action towards tested pathogenic microbes was expressed as a diameter of the ZOI. Generally, the obtained results exhibited suitable antimicrobial activities for all compounds (Table 1). The results presented in Table 1 indicate that the highest antibacterial effect was found for compound 7 against all targeted bacterial species, where the diameters of the formed ZOI of the compound 7 against K. pneumoniae, P. aeruginosa, S. aureus, and B. subtilis were 22 ± 0.16, 25 ± 0.14, 18 ± 0.28, and 20 ± 0.43 mm, respectively.
Ciprofloxacin, which is considered one of the most popular antibiotics, was reported to be beneficial against various types of Gram-positive and Gram-negative species [40]. Likewise, it has potent antibacterial properties against E. coli, P. aeruginosa, S. aureus, and Enterococcus faecium [41].
According to this study, the results obtained display superior antifungal action, and azole drugs had greater efficacy than amphotericin B towards Aspergillus species. On the other hand, Azole medicines are believed to be inefficient towards R. oryzae [42].

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Values
To further assess the antibacterial potential of the newly synthesized compounds, each compound's MIC and MBC values were estimated. A series of concentrations (50-300 µg/mL) of six compounds were used to estimate the MIC values and confirm their antibacterial potentials.
The results of the MIC test exhibited that the potent antibacterial effect and MIC values of compound 7 were 75 µg/mL for K. pneumoniae and B. subtilis, 100 µg/mL for P. aeruginosa, and 125 µg/mL for S. aureus (Table 2). Conversely, the lowest antibacterial effect was observed for compound 3, and MIC values were 275 µg/mL for K. pneumoniae and B. subtilis, 250 µg/mL for P. aeruginosa, and 300 µg/mL for S. aureus. Different concentrations of the newly tested compound were then subjected to the MIC test to confirm their antifungal activities further. Interestingly, as shown in Table 2, the entire tested compound revealed antifungal effects against R. oryzae, with different values of MICs. The MIC values were 225, >300, 275, 275, 225, and 150 µg/mL, respectively, for compounds 2, 3. 4, 5, 6, and 7 against R. oryzae. Results revealed that compound 7 showed higher antibacterial and antifungal activities than all studied compounds.
The biological properties of the thiadiazole ring are multifarious due to it having a wide range of pharmacological actions in which 1,3,4-thiadiazole derivatives are one of the most studied thiadiazole isomers, although 1,3,4-triazole derivatives have few therapeutic measures currently in use during medical practice (e.g., antibacterial activity and carbonic anhydrase inhibitory activity). In contrast, its antibacterial activity has been investigated, and other antimicrobial properties, such as antifungal and antituberculosis abilities, may also be explored [43]. Former investigations have discovered that thiadiazole compounds have a variety of medicinal properties, including analgesic, antimicrobial, antitumor, anticonvulsant, and anti-hepatitis B activity [25,44].
Serbanl et al. [9] examined in vitro the antibacterial activity of thiadiazole against S. aureus, Bacillus subtilis, E. coli, and P. aeruginosa and its antifungal activity against R. oryzae and Candida albicans by the disc diffusion technique. The results displayed that decisive antimicrobial action was observed. This phenomenon is attributed to the presence of a sulfur ring in the chemical structure of thiadiazole, which can easily penetrate the cell wall of microbes. The obtained results are in agreement with Er et al. [45] who established that imidazo [2,1-b] [1,3,4]thiadiazole derivatives displayed significantly forceful antibacterial activities with MIC values of 0.03, 0.03, and 0.5 µg/mL against S aureus, B. subtilis, and E. coli, respectively.

Determination of Extracellular Adenosine Triphosphate (ATP) Level
The total levels of ATP-tested microbes before and after exposure to the effective dose of studied compounds were determined and are linearly presented in Figure 1. In PBS, the initial ATP levels produced for K. pneumoniae, P. aeruginosa, S. aureus, B. subtilis, and R. oryzae approximately logged 6 RLU/mL. The total ATP levels decreased rapidly in Gram-negative bacteria and then slowly fell in fungal species throughout the experiment, while for the control vials, the total ATP levels remained relatively constant. The results exhibit that the effective dose of the compound 7 showed higher biocidal effects for all tested microbes, where the ATP level disappeared after 10 h for K. pneumoniae, after 12 h for P. aeruginosa, after 16 h for B. subtilis, and after 18 h for S. aureus and R. oryzae (Figure 1a-e). However, effective concentrations of compound 3 did not affect the fungal strains, indicating that the surfactant did not affect the cellular ATP levels. Similar trends for ATP levels for S. aureus were also observed ( Figure 1c).  This research exhibits that exposure to the studied compounds resulted in a considerable reduction in cellular ATP levels, symbolizing the banning of bacterial growth due to cell destruction. Typically, the consequences in Figure 1 show that the level of ATP that was significantly decreased in the bacterium K. pneumoniae corresponded to the other species tested. In contrast, the decrease level was lower in R. oryzae. Similarly, ATP amounts are an exceptional pointer to microbial activity and vibrancy and their capability to multiply and lead the severe infection. Subjecting Gram-negative bacteria to some antimicrobial compounds may diminish the cellular ATP levels and increase cellular protein levels in the culture, proposing a failure of cell membrane integrity [46].
ATP is an essential biomolecule in microbial species because it functions as a versatile energy source. ATP is considered to reveal the existence of live cells of microbes in the environment. On the other hand, as the intracellular level of ATP was indicated to differ with altering environmental and physiological circumstances, it might be utilized as a proxy to point to the microbial metabolic activity in microorganisms [47]. Interestingly This research exhibits that exposure to the studied compounds resulted in a considerable reduction in cellular ATP levels, symbolizing the banning of bacterial growth due to cell destruction. Typically, the consequences in Figure 1 show that the level of ATP that was significantly decreased in the bacterium K. pneumoniae corresponded to the other species tested. In contrast, the decrease level was lower in R. oryzae. Similarly, ATP amounts are an exceptional pointer to microbial activity and vibrancy and their capability to multiply and lead the severe infection. Subjecting Gram-negative bacteria to some antimicrobial compounds may diminish the cellular ATP levels and increase cellular protein levels in the culture, proposing a failure of cell membrane integrity [46].
ATP is an essential biomolecule in microbial species because it functions as a versatile energy source. ATP is considered to reveal the existence of live cells of microbes in the environment. On the other hand, as the intracellular level of ATP was indicated to differ with altering environmental and physiological circumstances, it might be utilized as a proxy to point to the microbial metabolic activity in microorganisms [47]. Interestingly extracellular ATP was exploited as the primary source of phosphorus for bacterial growth. In contrast, carbon from ATP molecules was not utilized, implying a prospective function of ATP in biological constancy. Indeed, in cells losing viability, the ability to synthesize ATP is misplaced, and many biochemical reactions, including the action of ATPases, speedily reduce any residual ATP from the cytoplasm [48].
Likewise, the liberation of ATP has to be entertained when considering metabolism or viability, as a considerable portion of the ATP in a culture can be extracellular, particularly in the exponential growth stage or when exposed to antimicrobial agents [49]. Accordingly, ATP values have to be handled with respect, notably as their usage is still argued for some applications. Definitely, the understanding of ATP measurements, especially regarding viability and activity, can be sweetened if the other adenylates (ADP and AMP) are taken into account [50].

Toxicological Performance Assay
The conceivable toxicological effects of all studied compounds were successfully measured against the transgenic bacterial cells of the marine bacterium Aliivibrio fischeri. As illustrated in Figure 2, the calculated values of EC 50% are documented and distinguished for all tested compounds. The toxicological result and effective concentration (EC 50% ) level were registered at three time intervals (5, 10, and 15 min). The results displayed that all tested compounds were secure, lacking toxic effects, and safe for living organisms. All the recorded EC 50% readings after 5, 10, and 15 min were less than 100, indicating that these studied compounds were biocompatible and non-toxic ( Figure 2). Toxicity testing of wastes, water streams, contaminated sites, and compounds is critical to avoid harmful effects on living organisms and pollution of the environment [51]. ATP is misplaced, and many biochemical reactions, including the action of ATPases, speedily reduce any residual ATP from the cytoplasm [48]. Likewise, the liberation of ATP has to be entertained when considering metabolism or viability, as a considerable portion of the ATP in a culture can be extracellular, particularly in the exponential growth stage or when exposed to antimicrobial agents [49]. Accordingly, ATP values have to be handled with respect, notably as their usage is still argued for some applications. Definitely, the understanding of ATP measurements, especially regarding viability and activity, can be sweetened if the other adenylates (ADP and AMP) are taken into account [50].

Toxicological Performance Assay
The conceivable toxicological effects of all studied compounds were successfully measured against the transgenic bacterial cells of the marine bacterium Aliivibrio fischeri. As illustrated in Figure 2, the calculated values of EC50% are documented and distinguished for all tested compounds. The toxicological result and effective concentration (EC50%) level were registered at three time intervals (5, 10, and 15 min). The results displayed that all tested compounds were secure, lacking toxic effects, and safe for living organisms. All the recorded EC50% readings after 5, 10, and 15 min were less than 100, indicating that these studied compounds were biocompatible and non-toxic ( Figure 2). Toxicity testing of wastes, water streams, contaminated sites, and compounds is critical to avoid harmful effects on living organisms and pollution of the environment [51]. Furthermore, the MMT experiment was used to determine the cytotoxic effect of the test compounds by calculating the CC50 and safe dose against HEp-2 cell lines; the obtained findings revealed that the CC50 was 825, 811, 835, 843, 801, and 836 µ g/mL, respectively, for C2, C3, C4, C5, C6, and C7, indicating that the synthesized compound had no harmful impact on HEp-2 cells.

Molecular Docking
Phenylalanyl (Phe)-tRNA synthetase (PheRS) is an essential enzyme that catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA(Phe)), an important step in protein biosynthesis [52]. This enzyme is considered a druggable target for thiazole derivatives to develop antimicrobial drugs [53]. In the current study, compounds that Furthermore, the MMT experiment was used to determine the cytotoxic effect of the test compounds by calculating the CC50 and safe dose against HEp-2 cell lines; the obtained findings revealed that the CC50 was 825, 811, 835, 843, 801, and 836 µg/mL, respectively, for C2, C3, C4, C5, C6, and C7, indicating that the synthesized compound had no harmful impact on HEp-2 cells.

Molecular Docking
Phenylalanyl (Phe)-tRNA synthetase (PheRS) is an essential enzyme that catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA(Phe)), an important step in protein biosynthesis [52]. This enzyme is considered a druggable target for thiazole derivatives to develop antimicrobial drugs [53]. In the current study, compounds that could inhibit Gram-negative bacteria in vitro, could also bind to the pocket of PheRS in silico in a manner similar to its co-crystalized inhibitor (formed interactions with phe227, phe171, and ala226) as in Table 3 and Figures 3-6. They could also excel over it by developing H-bonds in the pocket, which varied in position (Arg233, gly228, gly230, gln129), number, and length (from 2.08-3.04 Å). In vitro, compound 7 showed the highest inhibition compared to the others. It did the same in silico by forming the highest number of interactions (four H-bonds and other pi interactions) that qualified it to serve as a potential good inhibitor.     Bacterial DNA gyrase B (GyrB), an enzyme that negatively double-stranded DNA, became an attractive target for the examination of new antibacterial agents. Thiazoles were identified as having an inhibitory effect on the GyrB, with its ATP-binding site [54]. As represented in Table 4 and Figures 7-10, the in silico results confirmed the in vitro results where compounds 2, 4, 5, 6, 7 showed good inhibitory effects on Gram-positive bacteria. Similar to the co-crystalized inhibitor, they could interact with residues such as ile175, ile86, and pro87, including the H-bonds, forming resides, ser55 and asp81. Compounds 2 and 7 formed a higher number of H-bonds (3). The compounds showed better binding compared to the co-crystalized inhibitor of GyrB in terms of binding energy, nature, number, and types of the formed bonds/ interactions. Sterol 14α-demethylase (CYP51) is an essential cytochrome P450 enzyme for sterols biosynthesis in fungi and is a key target of clinical drugs for controlling fungal diseases [55]. In the current study, among the experimentally tested compounds, only compounds 2, 4, 5, 6, and 7 exhibited a promising antifungal activity on the tested fungal strain at roughly close strengths with better advantage for compound 7 over the others. This could be interpreted in the light of the docking results where compounds could form interactions within the pocket of CYP51 as with those developed by the standard inhibitor posaconazole (residues leu376 and tyr118) as shown in Table 5 and Figures 11-14. The new compounds could also form new hydrogen bonds, e.g., ile304, Thours311, his468, tyr132, arg469, which stabilize the compounds in the pocket and make them good inhibitors of CYP51. This represents a good step toward developing new antimicrobial agents for any future antifungal resistance that might emerge for posaconazole, where the compounds formed new interactions different from posaconazole.
(kcal/mol) 2 −7.2 (3): Arg84, gly85, asn54 Glu58, asp57, ile102, ile86, ala61 3 −6.8 (zero) Glu58, ile102, ile86, pro87 4 −6.6 (2): ser55, asn54 asp81, Glu58, ile175, ile86, ala61 5 −6.9 (2): Arg84, ser55 ile86 6 −7.5 (3): ser55, asp81, Glu58 ile175, ile51, pro87, ile102, ile86 7 −7.3 (3): Arg84, ser55, asn54 Glu58, asp57, ile102, pro87 Co-crystalized inhibitor −6.4 (2): ser55, asp81 Val79, ile175, ile86, pro87    Sterol 14α-demethylase (CYP51) is an essential cytochrome P450 enzyme for sterols biosynthesis in fungi and is a key target of clinical drugs for controlling fungal diseases [55]. In the current study, among the experimentally tested compounds, only compounds 2, 4, 5, 6, and 7 exhibited a promising antifungal activity on the tested fungal strain at roughly close strengths with better advantage for compound 7 over the others. This could be interpreted in the light of the docking results where compounds could form interactions within the pocket of CYP51 as with those developed by the standard inhibitor posaconazole (residues leu376 and tyr118) as shown in Table 5 and Figures 11-14. The new compounds could also form new hydrogen bonds, e.g., ile304, Thours311, his468, tyr132, arg469, which stabilize the compounds in the pocket and make them good inhibitors of CYP51. This represents a good step toward developing new antimicrobial agents  Sterol 14α-demethylase (CYP51) is an essential cytochrome P450 enzyme for sterols biosynthesis in fungi and is a key target of clinical drugs for controlling fungal diseases [55]. In the current study, among the experimentally tested compounds, only compounds 2, 4, 5, 6, and 7 exhibited a promising antifungal activity on the tested fungal strain at roughly close strengths with better advantage for compound 7 over the others. This could be interpreted in the light of the docking results where compounds could form interactions within the pocket of CYP51 as with those developed by the standard inhibitor posaconazole (residues leu376 and tyr118) as shown in Table 5 and Figures 11-14. The new compounds could also form new hydrogen bonds, e.g., ile304, Thours311, his468, tyr132, arg469, which stabilize the compounds in the pocket and make them good inhibitors of CYP51. This represents a good step toward developing new antimicrobial agents      In the light of in vitro and silico results, one could recommend compounds 2, 6, and 7 for further steps of drug development as antimicrobial agents.  In the light of in vitro and silico results, one could recommend compounds 2, 6, and 7 for further steps of drug development as antimicrobial agents. In the light of in vitro and silico results, one could recommend compounds 2, 6, and 7 for further steps of drug development as antimicrobial agents.

Experimental Instrumentation
All melting points were determined on an electrothermal apparatus and are uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. 1 H-and 13 C-NMR spectra were recorded in (CD3) 2 SO solutions on a BRUKER 500 FT-NMR system spectrometer, and chemical shifts are expressed in ppm units using TMS as an internal reference. Mass spectra were recorded on a GC-MS QP1000 EX Shimadzu. Elemental analyses were carried out at the Microanalytical Center of Cairo University, Cairo, Egypt.

Synthesis of Compounds General Procedures for Synthesis of Molecules 2-7
Methyl-2-(4-hydroxy-3-methoxybenzylidene)hydrazine-1-carbodithioate (1) acted as key molecule to produce new 1,3,4-thiadiazole derivatives by its reaction with the appropriate hydrazonoyl halides utilizing the grindstone chemistry; in detail, a mixture of 1.28 mg (5 mmol) of compound 1 and the appropriate hydrazonoyl halides (5 mmol) were ground with a pestle in an open mortar at RT with the addition of few drops (2-3) of DIPEA (diisopropyl ethyl amine) as catalytic amount, for 3-5 min till the mixture turned into the melt. The initial syrup grinding continued for 5-15 min, and the reaction was monitored by TLC (Thin Layer Chromatography) using ethyl acetate/petroleum ether (1:2, v/v) as eluent. The solid was collected, washed with water, and then ethanol and recrystallized from the proper solvent to afford the desired molecules 2-7.

Biological Activities
Two strategies were employed in the antibacterial and antifungal assessment. The first was the Kirby-Bauer disc diffusion method, which was utilized to determine the width of the zone of inhibition (ZOI) for each bacterial and fungal strain. The second strategy, microbial growth inhibition, was utilized to define the minimum inhibitory concentration (MICs) values of synthesized compounds on bacterial and fungal strain proliferation.

Preparation of Stock Solution
A standard stock solution of each compound was assembled by dissolving 10 mg of each compound in 1 mL of 10% dimethyl sulfoxide (DMSO) as a solvent [56].

Microorganisms Used
The susceptibility of four distinct microbial species to synthetic compounds was examined. In this research, Rhizopus oryzae was applied as a model for fungal strain. Likewise, four bacterial species were used, namely Pseudomonas aeruginosa and Klebsiella pneumoniae as a model for Gram-negative species, and Staphylococcus aureus and Bacillus subtilis as a model for Gram-positive strains. The conserved bacterial and fungal cultures were re-cultivated by transferring a single colony of each organism into tubes containing 10 mL of nutrient broth for bacteria and Sabouraud Dextrose broth for fungi, and all inculcated tubes were incubated for 18-24 h at 37 • C for bacteria and 3-4 days at 28 • C for fungi [57].

Kirby-Bauer Disc Diffusion Method
Disc diffusion assay was utilized to scrutinize the antibacterial and antifungal potential of the synthesized compounds versus fungal and bacterial species. In a Petri dish (60 mm), 15-20 mL agar of Sabouraud Dextrose agar (SDA) for fungal species and Mueller-Hinton agar (MHA) for bacterial species was deposited. Afterward, broth cultures adjusted to 0.5 McFarland turbidity (10 8 CFU/mL) were implanted onto the agar surface. On the top of the plate inoculated with a standardized suspension of microbes to be investigated, discs containing known amounts (50 µL) of the studied compound were inserted. Negative controls included paper discs containing solely dimethyl sulfoxide (DMSO). Positive controls were ciprofloxacin (30 µg) (Sigma-Aldrich, Steinheim, Germany) for bacteria and amphotericin B (10 µg) (Sigma-Aldrich, Steinheim, Germany) for fungi. A solvent-only control test was also carried out. The MHA and SDA plates were incubated at the optimal conditions for each microorganism; the thickness of the zone of inhibition (ZOI) formed in surrounding discs was measured (mm). All experiments were accomplished in triplicate, and if the results were different, they were repeated [58].

Determination of Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentrations (MICs) were employed to explore antimicrobial activity. The broth dilution technique was operated to estimate the MIC of each compound. A volume of stock solution was prepared formerly of each test compound, and was serially diluted to reach concentrations ranging from 50 to 300 µg/mL for antibacterial potential, while 4-32 mg/mL for antifungal activities. Each concentration of the studied compound was then added to culture media in a test tube at varying concentrations, and different strains were injected at a concentration of 10 8 CFU/mL. Culture media included nutrition agar (for antibacterial) and Sabouraud dextrose agar medium (for antifungal). All tested tubes were incubated at 37 • C (antibacterial) or 30 • C (antifungal) for 3-4 days before being checked for the presence or absence of the tested microbial growth. The MIC values were calculated using the lowest concentration of test compounds at which the tubes stayed clean, signifying that bacterial or fungal proliferation was suppressed entirely [59].

Minimum Biocidal Concentrations (MBCs)
In total, 10 µL aliquots from each test tube that exhibited no microbial proliferation were streaked on MHA and SDA plates and incubated at 37 • C for 24 h (bacteria) and 72 h (fungi) to determine the MBC values.

ATP Bioluminescence Assay
Extracellular ATP levels, which indicate activities and vitals in all microbial cells, were estimated using the luciferin-luciferase process. In total, 30 µL ATP aliquot suspension was mixed with 270 µL luciferin-luciferase aliquot mixture during this direct assay. Finally, the mixed suspension was allocated for spectroscopic analysis as an optical detection system. Using the ATP luminometer, luminescence patterns and luminescence intensity were assessed and expressed as relative illumination units (RLU) [60,61] 4.2.7. Toxicological Performance Assay Microtox ® Model 500 Analyzer (Modern Water, New Castle, DE, USA) was applied to measure each compound's toxicity level and EC 50% . The toxic influences of all studied compounds were ascertained to guarantee their safe and fruitful benefit for pharmaceutical and biomedical purposes without any unfavorable impacts on individuals. The toxicity grade was reckoned at the highest concentration (300 µg/mL) for each studied compound [61].
The cytotoxicity assay on the HEp-2 cell lines by colorimetric MTT assay was applied to determine the percentage of surviving cells. MTT (3,4,5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide) is reduced to its violet formazan product by metabolically active cells; the MTT assay was utilized to determine the compound's cytotoxicity. As indicated above, the cells were subjected to multiple compound concentrations and kept in the incubator for 24 h at 37 • C (Soysa et al. 2014) [62].

Conclusions
The current investigation has potential biomedical implications as the antibacterial and antifungal properties of specific synthetic compounds were successfully evaluated. This serves as a foundation for ongoing studies into alternative antibiotics which are hoped to develop more promising therapies against potential pathogens, especially black fungus and pathogenic bacteria. The results show that compound 7 has remarkable antimicrobial features and potential for pharmaceutical use as a new medication candidate against bacterial and fungal infections. Some of the studied compounds, including compounds 6 and 7, have been ascertained to be promising nominees for additional effectiveness evaluation based on the attained results. Moreover, the computational study revealed that compounds 2, 6, and 7 showed the best interactions with the selected protein targets.