Benzothiazole and 2,3-dihydro-1,5-benzoxazepine Derivatives Demonstrate Antimicrobial Activity: An Antimicrobial and ADMET Study

Abstract

Antimicrobial resistance continues to be a serious public health threat globally, hence the continuous design of new clinical candidates with novel mechanisms of action. Heterocyclic drugs have been a hotspot in antibiotic research. Benzothiazole and 2,3-dihydro-1,5-benzoxazepine are anticancer derivatives. To follow up on their antimicrobial activity, the current work resynthesized 13 benzothiazole, benzimidazole, benzothiazepine, and 2,3-dihydro-1,5-benzoxazepine derivatives, followed by the evaluation of their antimicrobial and antitubercular activity against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumonia, Bacillus subtilis, Streptococcus mutans, Salmonella typhi, and Mycobacterium tuberculosis. In addition, in silico ADMET studies were performed on the compounds using the ADMET Laboratory 2.0 platform. The compounds were found to be active against all the bacterial strains except against S. mutans and S. typhi. 4-[(E)-2-(2-chlorophenyl)ethenyl]-2,2-dimethyl-2,3-dihydro-1,5-benzoxazepine (3) was found to be the most active against E. coli, 2,2,4-trimethyl-2,3-dihydrobenzoxazepine (12) the most active against MRSA, and 4-[(E)-2-(4-methoxyphenyl)ethenyl]-2,2-dimethyl-2,3-dihydro-1,5-benzoxazepine (6) the most active against Klebsiella pneumoniae. The compounds also showed moderate activity against M. tuberculosis. The ADMET analysis predicted largely drug-like properties of the compounds and their suitability as potential drugs. The synthesized compounds showed good activity against some of the selected organisms and, therefore, could be modified to improve their action as antimicrobial agents.

Keywords

benzoxazepines - benzothiazole - anti-microbial - antitubercular - ADMET analysis

Introduction

The evolution of antimicrobial resistance has led to an astronomical increase in mortality rate from infectious diseases.[1] Antibiotics, which are mainly used in infectious disease chemotherapy, have been overexploited through their continuous prescription and indiscriminate use by patients.[2] [3] This practice has led to the continuous emergence of resistant organisms to these medications and consequently accounts for the increase in mortality of ca 1.27 million deaths globally.[4] [5] The common resistance strains according to the WHO report 2024 include the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which represent a highly contagious group.[6] [7] Despite efforts by stakeholders and implementation plans such as Antimicrobial Stewardship policies, the menace is far from over; hence, there is an urgent need to continually seek novel antibiotics with novel mechanisms of action as alternatives for the current chemotherapy.[8] [9]

The search for antibiotics has, in recent times, revolved around heterocyclics because of the ease in optimizing physicochemical properties such as hydrophobicity, plasma solubility, and ionization, the key contributors to the absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile.[10] [11] In drug design, achieving good potency and an ADME profile is important in reducing the attrition rate of compounds that progress through the drug discovery pipeline. For instance, aside from the earlier discovered synthetic penicillins and cephalosphorins, and recent heterocyclic drugs enmetazobactam; contezolid acefosamil, and afabicin, are in various stages of Phase 2 clinical trials. It is in light of this that benzodiazepine derivatives, benzoxazepine-4,7-diones, and several other aryl-containing heterocyclics have been explored due to their pharmacological characteristics, with some of their derivatives showing high activity when tested for their antifungal and antibacterial qualities.[12] [13]

Benzoxazepine derivatives have demonstrated inhibitory effects on a variety of species, including certain malignant cells.[14] Quinolino[3,2-b] and benzoxazepine metal complexes have been shown to have strong antibacterial, antifungal, and antimicrobial properties; the Zn, Co, and Ni complexes exhibit the strongest antibacterial activity.[15] [16] Good antibacterial and antifungal properties were observed in various benzoxazepine, benzothiazepine, and benzodiazepine derivatives that were produced by the multi-component cyclocondensation procedure.[17] In recent times, benzothiazepines conjugated with chloropyrazine have demonstrated strong, versatile antimicrobial properties.[18] Some 1,3,4-oxadiazole-containing benzodiazepines and benzothiazepines have been shown to have antibacterial and antitubercular properties. It was discovered that the benzothiazepine and benzodiazepine derivatives' activity was increased by the incorporation of thiophene, pyridine, and furan heterocyclics into the pharmacophore. The biological, pharmacological, and therapeutic uses of some 1,3,4-thiadiazoles substituted with 1,3-oxazepine and 1,3-benzoxazepine moieties have been investigated.[19] [20]

The diverse pharmacological activities of benzothiazole derivatives are attributed to the ring's high reactivity and ability to interact with various biological targets leading to a wide range of biological activities such as anticancer,[13] antimicrobial,[14] [15] [16] anti-inflammatory, analgesic,[17] antioxidant,[21] anticonvulsant and,[22] neuroprotective,[23] antidiabetic,[24] antiviral,[25] and antitubercular,[26] [27] antimalarial[28] activities.

The design and development of novel medications heavily rely on a compound's ADMET characteristics. A lead compound with good ADMET properties is a good candidate for development into a new drug, especially when it meets the criteria of drug-likeness.[29] [30] Taking inspiration from earlier works on benzodiazepines, the basic scaffold architectures of the compounds in this study are shown in [Fig. 1]. Hence, following up on the research on benzothiazole and 2,3-dihydro-1,5-benzoxazepine that has been found to be highly active against selected breast cancer cells. This study aimed to ascertain their antimicrobial activity and further explore their ADMET properties in silico. The compounds demonstrated good antimicrobial activity against methicillin-resistant S. aureus (MRSA), E. coli, K. pneumoniae, B. subtilis, S. mutans, P. aeruginosa, S. typhi, and Mycobacterium tuberculosis. The ADMET properties of the compounds have been computed and discussed.

Fig. 1 (A) The scaffolds tested against the various disease-causing organisms in this work. (B) Chemical structures of compounds 1–13.

Materials and Methods

Strains

The microbial strains used, which were originally purchased from ATCC Continental (Maryland, United States), were obtained from the Department of Biomedical Sciences of the University of Health and Allied Sciences. Organisms, including Methicillin-resistant S. aureus (ATCC 25923), E. coli (ATCC 25922), K. pneumonia, B. subtilis (ATCC 10004), S. mutans (ATCC 700610), P. aeruginosa (ATCC 4853), and S. typhi 1 (clinical strain), were sub-cultured for 24 hours in nutrient agar at 37°C before the experiment. A prepared inoculum of these strain cultures was then adjusted to obtain a final concentration of 10⁵ CFU/mL using a 0.5 McFarland standard. The M. tuberculosis tests were performed at the Tuberculosis Research Group at the University of Cape Town. Rifampicin was obtained from Sisco Research Laboratories (Mumbai, India), whereas the M. tuberculosis was obtained from ATCC Continental (Maryland, United States).

Chemistry

Compounds 1–13 have been previously reported in our work on their anticancer activity.[31] All reagents and solvents used for the synthesis were analytical-grade. 4-Methylpent-3-en-2-one, 4-methoxybenzaldehyde, 5-bromosalicylaldehyde, 2-chlorobenzaldehyde, 3-methoxylbenzaldehyde, 3-chlorobenzaldehyde, 2-aminophenol, 2-aminothiophenol, and 4-methylbenzaldehyde were obtained from Sigma Aldrich (Johannesburg, South Africa), whereas acetone, ethanol, and methanol were sourced from Merck Chemicals (Johannesburg, South Africa).

The compounds were synthesized and characterized as previously reported.[31] ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance AV 400 MHz spectrometer (Bruker Corporation, Massachusetts, United States) operating at 400 MHz for ¹H and 100 MHz for ¹³C using dimethyl sulfoxide as solvent and tetramethylsilane as internal standard. Chemical shifts (δ) are expressed in ppm. Fourier Transform Infrared Spectroscopy (FT–IR) spectra were recorded on a Bruker Platinum ATR Spectrophotometer Tensor 27 (Bruker Corporation, Massachusetts, United States). Microanalysis was performed using a Vario Elementar Microcube ELIII (Elementar Analysensysteme, Germany). Melting points were obtained using a Stuart Lasec SMP30 (TE equipment, United States), whereas the masses were determined using an Agilent 7890A GC System (Agilent Technologies, California, United States) connected to a 5975C VL-MSC with electron impact as the ionization mode and detection by a triple-Axis detector. The gas chromatography (GC) was fitted with a 30 m × 0.25 mm × 0.25 μm DB-5 capillary column. Helium was used as carrier gas at a flow rate of 1.63 mL·min⁻¹ with an average velocity of 30.16 cm·s⁻¹ and a pressure of 63.73 kPa.

Solubility Tests

The solubility of the compounds was determined by weighing approximately 0.1 g of each compound in a test tube, and approximately 2 mL of the solvent was added to the test tube containing the compound to dissolve. The test tube was shaken until the compound dissolved completely.[32] Where the volume of the solvent was insufficient, approximately 8 mL of the solvent was added and shaken to achieve total dissolution of the compounds. Where the compound still did not dissolve, the test tube was observed for 2 hours, after which the compound's solubility or otherwise in the solvent was recorded.

Micro-Broth Dilution Minimum Inhibitory Concentration Assay: 10-Point Concentration Response Protocol

The synthesized compounds were tested against M. tuberculosis, using the broth microdilution method to obtain the compounds' minimum inhibitory concentration (MIC) against M. tuberculosis.[33] A total of 10 mmol/L of the compounds was used for the tests. A 96-well microtiter plate was used to create duplicate 2-fold serial dilutions of the compounds using 50 µL per well. A total of 50 μL of the diluted M. tuberculosis culture was put into each well in the plate, including the control wells, to bring the final volume to 100 µL per well.[34] The plate was incubated for 24 hours after the addition of the compound. Rifampicin was used as a control. To determine the visual MIC90, the concentration that gave the first color change for the plates was recorded. The visual MIC90 was recorded daily until the seventh day. The relative fluorescence (excitation 540 nm; emission 590 nm) was measured on the seventh day. The data were analyzed using Softmax Pro 6 software. Raw relative fluorescence units (CFU) data were normalised to the minimum and maximum inhibition controls to generate a dose–response curve (% inhibition) from which the MIC90 was calculated.

The Media Used

The MIC analysis for M. tuberculosis was conducted in the Institute of Infectious Disease and Molecular Medicine, University of Cape Town (UCT). Mycobacterium tuberculosis H37RvMA pMSP12:GFP was used to accomplish the usual broth microdilution procedure.[35] The culture (10 mL) was cultivated in glycerol–alanine–salts with 0.05% Tween 80 and iron (0.05%; GAST-Fe) at pH 6.6, achieving an OD600 of 0.6 to 0.7; then, the culture was diluted in GAST-Fe at a 1:100 ratio. Each drug was serially diluted twice in GAST-Fe in a 96-well microtiter plate. Each serial dilution well received 50 μL of the diluted culture, which produced an OD600 of 0.004. The plate arrangement was altered from a previously published technique.[36] [37] [38] [39] All procedures requiring the handling of pathogenic mycobacterial strains were performed in a Biosafety Level III certified and compliant facility.

Plate Preparation and Media Recipes

Serial dilutions were prepared using a programmed automated liquid handler, Hamilton Microlab STARlet. For columns 1 to 11, each well contained 50 µL of the test compounds, while for column 12, each well contained 50 µL of the media and 2 × Rifampicin with a final concentration of 0.150 µmol/L. Row H – was used for a concentration-response assay for the reference drug, rifampicin, with a concentration-response range of 0.150 to 0.0002 µmol/L, whereas a concentration range of 5,000 to 9.76 µmol/L was used for the compounds.

Antimicrobial Susceptibility Testing (Agar Diffusion)

The antimicrobial susceptibility assay was conducted according to previously reported protocols, with a slight modification.[40] [41] About 25 mL of nutrient agar was dispensed into Petri dishes and allowed to set. Using sterile swabs, each of the strains at a final concentration of 1.8 × 10⁸ CFU/ mL acquired was streaked on the agar plates using the three-way method. Wells were bored in each plate using a cork borer (No. 3, 4 mm). Into these wells, 50 µL of 2 mg/mL stock solution of each compound prepared with 20% methanol was pipetted to give a final concentration of 0.1 mg per well, and the last two wells were filled with either 20% methanol as a negative control or 30 µg chloramphenicol/voriconazole as a positive control, respectively. The plates were then subjected to incubation at 37°C for 24 hours, after which the zones of inhibition were measured and recorded. The antimicrobial activity against each test organism was quantified by determining the mean zone of growth inhibition. Each experiment was done in triplicate, and the mean zones of inhibition were recorded.

Minimum Inhibitory Concentration

The MICs of the compounds were determined using the micro broth dilution method using 96-well microtiter plates, following the previously reported protocol, with slight modifications.[42] [43] The stock concentration of each compound was 1,000 µg/mL, prepared in 20% methanol, and used to prepare wells at concentrations ranging from 0.50 to 500 µg/mL. Sterile 96-well plates were prepared, with each well receiving 100 µL of double-strength Mueller–Hinton broth, 80 µL of the test samples, and 20 µL of the inocula of standardized suspensions of the various cultures of test organisms, making the final volume 200 µL. Compounds against the different organisms in column 13 of each plate served as the positive control. The negative control was prepared in a test tube using stock broth plus 20% methanol. The plates were then incubated at 37°C for 24 hours. After incubation, the activity in each well was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. A total of 20 µL of the MTT dye was added to each well, and the results were read after 30 minutes. Each experiment was done thrice, with the MICs recorded.

Determination of Minimum Bactericidal Concentration

After determining the MIC, the MBC was determined by reculturing (subculturing) aliquots of 50 µL from the tubes (broth dilution), which showed no visible bacterial growth, and were seeded on MH broth agar with an antimicrobial agent. This was done by observing pre- and postincubated agar plates for the presence or absence of bacteria. Subsequently, the MBC values were deduced from the wells with the lowest concentrations, indicated by a lack of color change when growth occurred after subculturing for 24 hours of incubation.[44]

Statistical Analysis

The data obtained were recorded in an Excel sheet and analyzed using Microsoft Excel and GraphPad Prism (8). All experiments were performed in triplicate. Results were expressed as mean ± standard error of the mean (SEM). Statistical significance was calculated with Tukey's post hoc test using GraphPad Prism (8). Statistical significance was set at a confidence interval of 95% for data to be statistically significant. For the MIC and MBC values, the goodness of fit (R-squared) value was evaluated to determine the reliability of the model used to fit the dose–response data, which was subsequently used to calculate the MIC and MBC values. Only models with acceptable R-squared values and confidence intervals were used for MIC and MBC determination.

ADMET Method

The physicochemical properties were determined using the ADMET Laboratory 2.0 platform. The structures were drawn using ChemDraw, which was also used to generate the SMILES. The SMILES were submitted to the ADMET laboratory 2.0 platform online, which computed the results,[45] [46] [47] [48] ADMET 2.0 uses machine learning models, including Random Forest (RF) and Support Vector Machine (SVM), to predict these properties. It provides detailed explanations and optimal ranges for each property to facilitate understanding and decision-making. The scoring system is based on a comparison with over 6,000 compounds on the DrugBank database. ADMET 2.0 scoring criteria evaluate various aspects of a compound's adsorption, distribution, metabolism, excretion, and toxicity based on well-defined physicochemical and medicinal chemistry properties. The rule of five developed by Lipinski helps distinguish between the properties of different compounds and their potential as drugs that can be orally absorbed. The five parameters were molecular weight (MW) ≤ 500, octanol/water partition coefficient (A log p) ≤ 5, number of hydrogen bond donors (HBDs) ≤ 5, and number of hydrogen bond acceptors (HBAs) ≤ 10.[49] The rule states that a molecule that violates two or more of the rules would not be orally active.[50] [51] [52] [53] [54]

Results and Discussion

Solubility of Compounds

As shown in [Table 1], all the compounds were found to be insoluble in petroleum ether, but were soluble in ethanol and methanol. Compounds 1, 2, 3, 6, and 9 were partially soluble in 20% methanol, the solvent system for the dissolution of the compounds in the agar diffusion protocol. These compounds gave higher MIC results but lower zones of inhibition, which is consistent with their solubility profile.

Table 1 Solubility of compound 1–13

Compd.	Petroleum ether	Ethanol	Methanol	20% Methanol
1	Insoluble	Soluble	Soluble	Partially soluble
2	Insoluble	Soluble	Soluble	Partially soluble
3	Insoluble	Soluble	Soluble	Partially Soluble
4	Insoluble	Soluble	Soluble	Soluble
5	Insoluble	Soluble	Soluble	Soluble
6	Insoluble	Soluble	Soluble	Partially soluble
7	Insoluble	Soluble	Soluble	Soluble
8	Insoluble	Soluble	Soluble	Soluble
9	Insoluble	Soluble	Soluble	Partially soluble
10	Insoluble	Soluble	Soluble	Soluble
11	Insoluble	Soluble	Soluble	Soluble
12	Insoluble	Soluble	Soluble	Soluble
13	Insoluble	Soluble	Soluble	Soluble

Antitubercular Activity Test

When the visual MIC90 data were plotted, some of the compounds showed antitubercular activity with 2-aminophenol-2,2,4-trimethyl-2,3-dihydrobenzo[b][1,4]oxazepine (1:1) (12) giving the highest antitubercular activity ([Fig. 2]). However using the calculated MIC90 values, eight compounds showed good antitubercular activity with 2,2-dimethyl-4-[(E)-2-(4-methylphenyl)ethenyl]-2,3-dihydro-1,5-benzoxazepine (1) having the highest antitubercular activity ([Fig. 3]).

Fig. 2 Visual antitubercular activity of compounds against Mycobacterium tuberculosis H37Rv incubated at 37°C with 5% CO₂ and humidification for 24 hours using rifampicin as a positive control. Data are expressed as mean ± SEM, one-way ANOVA followed by Tukey's post hoc test were used for multiple comparisons. p < 0.001 relative to DMSO vehicle control. RFP, rifampicin; SEM, standard error of the mean.

Fig. 3 Calculated antitubercular activity of compounds against Mycobacterium tuberculosis H37Rv incubated at 37°C with 5% CO₂ and humidification for 24 hours using rifampicin as a positive control. Data are expressed as mean ± SEM, one-way ANOVA followed by Tukey's post hoc test were used for multiple comparisons. p < 0.01, p < 0.001 relative to DMSO vehicle control. ANOVA, analysis of variance; RFP, rifampicin; SEM, standard error of the mean.

Antimicrobial Activity of the Test Compounds

Antimicrobial Susceptibility Test

By using the well diffusion assay, the activities of the compounds were evaluated; all the test compounds proved to be potential antimicrobial agents, inhibiting the growth of all the organisms except S. typhi and S. mutans when a final concentration of 0.1 mg per well was used. The antibacterial potential was categorised as highly active (>14 mm), moderately active (10–14 mm), slightly active (6–10 mm), and inactive (<6 mm). The zones of inhibition of the tested compounds are reported in [Fig. 4] below. Among the compounds tested, compound 12 recorded the highest activity against MRSA with a zone of inhibition of 31.33 ± 0.67 mm, even greater than the positive control, chloramphenicol, and the lowest activity for all the compounds was against S. typhii and S. mutans, showing no zones of inhibition comparable to the test antibiotic and the negative control used ([Fig. 4]).

Fig. 4 Mean zone of inhibition of E. coli, MRSA, S. mutans, S. typhi, K. pneumonia, P. aeruginosa, and B. subtilis treated with various compounds and incubated at 37°C for 24 hours. Data are expressed as mean ± SEM, two-way ANOVA followed by Tukey's post hoc test were used for multiple comparisons. p < 0.001 relative to NC. ANOVA, analysis of variance; NC, negative control group treated with DMSO instead of the compounds. SEM, standard error of the mean.

Broth Dilution Assay of the Test Compounds

The results from the broth dilution assays indicated that the compounds were active against the test bacteria used in a dose-dependent manner with MICs ranging from 0.5 to 2,000 µg/mL ([Fig. 5]). Based on the previous studies performed on some compounds isolated,[55] the antimicrobial activity of the test compounds in this study can be interpreted as follows: very strong bioactivity <3.515 µg/mL; strong bioactivity 3.515 to 25 µg/mL; moderate bioactivity; 26 to 100 µg/mL; weak bioactivity 101 to 500 mg/mL; very weak bioactivity 500 to 2,000 mg/mL; and no activity above 2,000 µg/mL.[56] [57] From the study, it was observed that the activity of all the compounds was in the range of strong antibacterial activity to weak activity, with compound 3 showing strong activity against B. subtilis ([Fig. 5]). In addition, all the test compounds showed good activity against the resistant strain, MRSA. Furthermore, the definition of a bactericidal agent is one with the ratio of MBC/MIC ≤ 4, whereas a bacteriostatic agent has an MBC/MIC ratio of >4, as reported.[58] [59] The data on the antibacterial activity of the compounds further confirm stronger bactericidal activity for all the compounds against MRSA, K. pneumonia, P. aeruginosa, and B. subtilis ([Table S1], available in the online version). The compounds exhibited variable activity against the different organisms, either bacteriostatic or bactericidal, against the other test bacterial strains ([Fig. 5], [Fig. 6], [Table S1], available in the online version).

Fig. 5 MICs of E. coli, MRSA, S. mutans, S. typhi, K. pneumonia, P. aeroginosa, and B. substilis treated with various compounds using the MTT assay for 24 hours. Data represent the mean of three independent experiments, and error bars represent SEM. p < 0.01 versus DMSO vehicle control. MICs, minimum inhibitory concentrations. SEM, standard error of the mean.

Fig. 6 MBCs of test compounds against E. coli, MRSA, S. mutans, S. typhi, K. pneumonia, P. aeroginosa, and B. substilis treated with various compounds using the MTT assay for 24 hours. Data represent the mean of three independent experiments, and error bars represent SEM. p < 0.01 versus DMSO vehicle control. MICs, minimum inhibitory concentrations. MBC, minimum bactericidal concentration. SEM, standard error of the mean.

Mode of Action

Benzothiazole compounds have been reported to inhibit E. coli and S. aureus by different modes of action depending on the substitution of the aryl group. When tested on bacterial cells and plasmid DNA, the compounds were found to act by binding DNA or perturbing the membrane of the test organism. A cytoplasmic membrane depolarization assay was used to determine membrane-disrupting activities.[60] The compounds in this study have been reported to act by targeting the microtubules of cells. They can inhibit cell growth by affecting cell division. The polar groups in the compounds are reported to interact with the microtubules, leading to their inhibition.[31]

Structure–Activity Relationship

The test compounds proved to be effective against all the bacterial strains used except against S. mutans and S. typhi. Against E. coli, while compounds 3, 5, and 1 were the most effective with zones of inhibitions and MICs of 20.33 mm (250 µg/mL), 19.67 mm (15.63 µg/mL), 15.0 mm (31.25 µg/mL), respectively, compounds 2, 6, and 13 recorded the least activities giving zones of inhibitions of 0 mm and MICs of (250, 125, and 500) µg/mL, respectively. Generally, the benzothiazole derivatives gave smaller zones of inhibition compared with the benzoxazepine derivatives. For compound 3, the presence of the chloro substituent at the 2' position on the phenyl group led to a higher zone of inhibition. The presence of the methoxy substituent at the 3′ position of the phenyl group in compound 5 gave it a high zone of inhibition. The zone of inhibition completely disappears in compound 6, where the methoxy substituent is at the 4' position on the phenyl group; thus, the methoxy substituent at the 3′position of the phenyl group corresponds to a high zone of inhibition of the benzoxazepine scaffold against E. coli.

Against MRSA, while compounds 12, 5, and 4 gave the largest zones of inhibition and low MICs of 31.33 mm (31.25 µg/mL), 25.33 mm (62.50 µg/mL), 25.00 mm (250 µg/mL) respectively, compounds 1, 7, and 11 showed the least activity indicated by their small zones of inhibition and high MICs of 7.0 mm (500 µg/mL), 7.33 mm (250 µg/mL), and 8.33 mm (500 µg/mL). Compound 12, which has the benzoxazepine moiety without the phenyl group, and a 2-aminophenol gave the largest zone of inhibition, followed by compound 5, which is a benzoxazepine with a methoxy substituent at the 3′ position on the phenyl group while compound 4, which is the benzothiazole derivative with the chloro substituent at position 2' also gave a high zone of inhibition. Compound 1, which has a methyl group, gave the least zone of inhibition, followed by compound 7, which is the 5-bromo-2-hydroxyphenyl derivative of the benzoxazepine. Compound 11 also gave a low zone of inhibition, confirming that a methoxy substituent at position 3′ on the phenyl group was required for higher activity of the benzoxazepines against MRSA.

Against K. pneumonia, compounds 6, 3, and 12 demonstrated good zones of inhibition, with MICs being 24.33 mm (15.63 µg/mL), 22.0 mm (125 µg/mL), and 21.0 (62.5 µg/mL). The least activities were observed for compounds 1, 2, and 11 with MICs of 125.0, 250.0, and 62.5 µg/mL, respectively. Compounds 6 and 3, which were the 4-methoxyphenyl and 2-methoxyphenyl derivatives of the benzoxazepine derivative, gave the largest zones of inhibition against K. pneumonia, while compound 1, the 4-methylphenyl benzoxazepine derivative, gave the lowest zone of inhibition against K. pneumonia, confirming that the presence of the methoxy group on the phenyl moiety of the benzoxazepine led to a higher zone of inhibition. The 4-methyl phenyl benzothiazole derivative also gave a low zone of inhibition, confirming that the presence of the 4-methylphenyl group on any of the compounds leads to a low zone of inhibition.

Against P. aeruginosa, compounds 13, 12, and 8 show appreciable zones of inhibition, with MICs being 26.67 mm (>500 µg/mL), 26.33 mm (62.5 µg/mL), and 20.0 mm (250 µg/mL), respectively. Compounds 2, 9, and 10 showed poor activities, with zones of inhibition and MICs being 0 mm (250 µg/mL), 0 mm (250 µg/mL), and 6.67 mm (>500 µg/mL), respectively. Compounds 13 and 8, which are the 4-chlorophenylbenzothiazole and 3-chlorophenylbenzothiazole derivatives, respectively, gave appreciable zones of inhibition; the benzoxazepine derivative also showed good activity. Compounds 2, 9, and 10, which are the 4-methylphenylbenzothiazole, 3-methoxyphenyl benzothiazole, and the 5-bromo-2-hydroxybenzothiazole, gave the lowest zones of inhibition, confirming that the presence of the chloro group in these compounds leads to higher activity against P. aeruginosa.

Against B. subtilis, compounds 11, 3, and 12 showed good activity with zones of inhibition and MICs of 27.0 mm (>500 µg/mL), 26.0 mm (0.5 µg/mL), and 25.0 mm (7.81 µg/mL), respectively. Compounds 9, 13, and 10 showed poor inhibitory effects with zones of inhibition and MICs of 0 mm (>500 µg/mL), 0 mm (>500 µg/mL), and 7.67 mm (500 µg/mL), respectively. Compound 11, which is the benzothiazepine derivative, gave the best activity against B. subtilis followed by compounds 3 and 12, which are the 2-chlorophenylbenzoxazepine and the benzoxazepine derivatives, respectively, while the 3-methoxyphenylbenzothiazole (9), 4-chlorophenylbenzothiazole (13) and 5-bromo-2-hydroxyphenylbenzothiazole (10) gave the least zones of inhibition, indicating that the benzoxazepine scaffold was required for the activity of these compounds against B. subtilis.

ADMET Analysis

The ADMET laboratory 2.0 platform was used to determine the in silico physicochemical properties of the compounds, which showed a range of physicochemical and pharmacokinetic properties ([Table 2]). The compounds were less flexible than Ampicillin, as evidenced by a lower value of topological polar surface area (TPSA) for these compounds compared with Ampicillin. The majority of the compounds were not HBDs, with the exception of compounds 7 (1), 10 (1), 12 (3), and 13 (1), and the HBAs varied from 1 to 4. The majority of the compounds had log S values below −4 logmol/L, except compounds 11 and 12, which had log S values of −2.443 and −2.656, whereas Ampicillin had a log S value of −1.564 logmol/L/L. Based on their TPSA values of ≤68 Å, all of the compounds showed good oral bioavailability and high gastrointestinal absorption; this indicates that they are not p-Glycoprotein (P-gp) substrates, so they likely have good bioavailability because p-glycoprotein will not pump them out of the cell.

Table 2 Molecular properties of compound 1–13. (A) Physicochemical properties. (B) Medicinal chemistry. (C) Absorption. (D) Distribution. (E) Metabolism. (F) Excretion. (G) Toxicity. (H) Environmental toxicity. (I) Tox21 pathway

A. Physicochemical properties
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
Molecular weight	291.16	225.06	311.11	245.01	307.16	307.16	371.05	245.01	241.06	304.95	205.08	298.17	228.05	349.110
Volume	327.50	236.08	325.42	234.00	336.292	336.29	338.28	234.00	244.87	246.86	217.96	323.89	226.48	330.46
Density	0.889	0.953	0.956	1.047	0.913	0.913	1.097	1.047	0.984	1.235	0.941	0.921	1.007	1.056
NHA	2	1	2	1	3	3	3	1	2	2	1	4	2	7
NHD	0	0	0	0	0	0	1	0	0	1	0	3	1	4
nRot	2	1	2	1	3	3	2	1	2	1	0	0	1	5
NRing	3	3	3	3	3	3	3	3	3	3	2	3	3	3
Maxring	11	9	11	9	11	11	11	9	9	9	11	11	9	7
nHet	2	2	3	3	3	3	4	3	3	4	2	4	3	8
fChar	0	0	0	0	0	0	0	0	0	0	0	0	0	0
NRing	19	16	19	16	19	19	19	16	16	16	12	18	16	17
Flexibility	0.106	0.062	0.105	0.062	0.158	0.158	0.105	0.062	0.125	0.062	0	0	0.062	0.294
Stereo centers	0	0	0	0	0	0	0	0	0	0	0	0	0	4
TPSA	21.59	12.89	21.59	12.89	30.82	30.82	41.82	12.89	22.12	33.12	12.36	67.84	28.68	112.73
log S	−6.028	−5.630	−6.149	−5.675	−5.743	−5.783	−5.912	−5.907	−5.384	−5.426	−2.443	−2.656	−5.257	−1.564
log P	4.649	4.506	4.954	4.527	4.184	4.198	4.873	4.673	4.097	4.663	2.534	2.338	4.020	0.899
log D	4.035	4.132	4.049	4.024	3.852	3.840	3.855	4.202	3.881	3.828	2.928	2.983	3.965	0.589
B. Medicinal Chemistry
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
QED	0.731	0.602	0.698	0.610	0.795	0.795	0.756	0.610	0.677	0.721	0.622	0.562	0.673	0.675
Synthetic accessibility score	2.556	1.574	2.639	1.665	2.583	2.529	2.826	1.672	1.650	2.007	2.959	2.571	1.626	3.527
Fsp3	0.250	0.071	0.211	0	0.250	0.250	0.211	0	0.071	0	0.417	0.278	0	0.438
MCE-18	36.720	14.000	36.957	14.000	36.720	36.720	39.130	14.000	14.000	15.000	26.824	30.435	14.000	70.304
NPscore	0.102	−2.034	−0.240	−2.094	0.122	0.207	0.250	−2.396	−1.920	−1.438	−0.331	0.378	−1.365	0.742
Lipinski's Rule	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Pfitzer	No	No	No	No	No	No	No	No	No	No	Yes	No	No	Yes
GSK Rule	No	No	No	No	No	No	No	No	No	No	Yes	No	No	Yes
Golden Triangle	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
C. Absorption
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
Caco-2 permeability	−4.797	−4.689	−4.813	−4.683	−4.818	−4.769	−4.840	−4.690	−4.711	−4.698	−4.643	−4.507	−4.783	−5.910
MDCK permeability	1.6 e − 0.5	1.3e − 05	1.4e − 0.5	1.3e − 0.5	1.3e − 0.5	1.4e − 0.5	1.5e − 0.5	9.1e − 06	1.4e − 0.5	1.2e − 0.5	2.7e − 0.5	3.2e − 05	1.3 e − 0.5	3.1e − 0.5
Pgp inhibitor	Poor	Excellent	Poor	Excellent	Poor	Poor	Poor	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent
Pgp substrate	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent
Human Intestinal Absorption	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent
Human Oral Bioavailability 20%	Poor	Poor	Excellent	Poor	Excellent	Excellent	Excellent	Poor	Poor	Excellent	Excellent	Excellent	Poor	Excellent
Human Oral Bioavailability 30%	Excellent	Poor	Excellent	Poor	Excellent	Excellent	Excellent	Poor	Poor	Excellent	Excellent	Excellent	Excellent	Excellent
D. Distribution
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
Plasma protein binding	98.33%	98.30%	98.86%	98.70%	98.77%	98.43%	98.75%	99.15%	98.42%	98.69%	85.19%	91.09%	98.24%	48.54%
Volume Distribution	0.876	0.841	1.327	0.879	0.981	0.913	1.212	0.978	1.026	0.907	4.673	2.049	1.101	0.222
Blood–brain barrier Penetration	Yes	Yes	No	No	No	No	No	No	No	Yes	Yes	No	Yes	No
Fraction unbound	1.73%	1.42%	1.31%	1.11%	1.65%	1.82%	1.47%	1.22%	1.60%	1.33%	15.63%	8.41%	1.63%	67.51%
E. Metabolism
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
CYP1A2 inhibitor	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	Yes	No
CYP1A2 substrate	No	No	Yes	No	Yes	Yes	Yes	No	Yes	No	Yes	Yes	No	No
CYP2C19 inhibitor	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	No	Yes	No
CYP2C19 substrate	No	No	No	No	Yes	No	No	No	No	No	Yes	Yes	No	No
CYP2C9 inhibitor	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	No	Yes	No
CYP2C9 substrate	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No
CYP2D6 inhibitor	Yes	No	Yes	No	Yes	Yes	Yes	No	Yes	Yes	No	Yes	Yes	No
CYP2D6 substrate	Yes	Yes	Yes	No	Yes	Yes	Yes	No	Yes	Yes	Yes	Yes	No	No
CYP3A4 Inhibitor	Yes	No	Yes	No	Yes	Yes	Yes	No	Yes	No	No	Yes	No	No
CYP3A4 substrate	Yes	No	Yes	No	Yes	Yes	No	No	No	No	No	Yes	No	No
F. Excretion
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
CL	2.918	5.823	2.700	4.260	4.506	4.211	1.372	5.283	6.818	1.943	5.526	9.201	5.404	3.376
T1/2	0.139	0.100	0.096	0.051	0.182	0.149	0.169	0.090	0.124	0.087	0.390	0.726	0.396	0.975
G. Toxicity
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
hERG Blockers	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent
Human hepatotoxicity	Poor	Excellent	Poor	Excellent	Poor	Poor	Excellent	Excellent	Excellent	Excellent	Poor	Poor	Excellent	Excellent
Drug-induced liver injury	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor
AMES Toxicity	Poor	Poor	Poor	Poor	Poor	Poor	Excellent	Poor	Poor	Excellent	Excellent	Excellent	Poor	Excellent
Rat Oral Acute Toxicity	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Poor	Excellent	Excellent
FDAMDD	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Poor
Skin Sensitization	Excellent	Excellent	Excellent	Excellent	Excellent	Poor	Poor	Excellent	Excellent	Excellent	Poor	Poor	Excellent	Excellent
Carcinogenicity	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Excellent
Eye Corrosion	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent
Eye Irritation	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Poor	Excellent
Respiratory Toxicity	Excellent	Excellent	Excellent	Excellent	Excellent	Excellent	Poor	Excellent	Excellent	Excellent	Excellent	Poor	Poor	Excellent
H. Environmental Toxicity
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
Biocentration Factors	2.8874	2.527	2.986	2.678	2.880	2.894	2.237	2.654	2.599	1.930	2.544	0.878	1.998	0.356
IGC50	4.701	4.303	4.839	4.323	4.713	4.71	5.053	4.467	4.299	4.656	3.737	4.007	4.219	2.602
LC50FM	5.320	4.623	5.395	4.776	5.399	5.583	5.714	4.844	4.618	5.224	4.230	4.184	4.834	3.589
LC50DM	5.309	5.179	5.521	5.196	5.786	5.964		5.239	5.378	5.786	4.477	4.88	5.620	3.860
I. Tox21 Pathway
Property	Compound
	1	2	3	4	5	6	7	8	9	10	11	12	13	Ampicillin
Androgen Receptor	Inactive	Inactive	Active	Inactive	Active	Active	Active	Active	Inactive	Inactive	Inactive	Inactive	Active	Inactive
Androgen Receptor Ligand Binding Domain (AR-LBD)	Active	Inactive	Inactive	Inactive	Active	Active	Inactive	Active	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive
Aryl hydrocarbon Receptor (AhR)	Active	Inactive	Active	Inactive	Inactive	Active	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Active	Inactive
Aromatase	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Active	Active	Inactive
Estrogen Receptor Alpha (ER)	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Active	Inactive	Active	Active	Inactive
Estrogen receptor Ligand Binding domain ER-LBD	Active	Active	Inactive	Active	Active	Inactive	Inactive	Active	Active	Inactive	Inactive	Active	Inactive	Inactive
Peroxisome Proliferator Activated Receptor Gamma (PPAR-Gamma)	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Active	Inactive	Active	Inactive	Inactive
SR-ARE	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Active	Active	Inactive
SR-ATAD5	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Inactive	Active	Inactive
Heat shock factor response element (HSE)	Active	Active	Inactive	Inactive	Active	Active	Inactive	Inactive	Active	Active	Inactive	Active	Inactive	Inactive
Mitochondrial membrane protein	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Active	Inactive	Inactive
Phosphoprotein (Tumor Supressor) p53	Active	Active	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive	Active	Inactive	Inactive	Active	Inactive

Abbreviations: fChar, formal charge; log D: the logarithm of the n-octanol/water distribution coefficients at pH = 7.4; log p, the logarithm of the n-octanol/water distribution coefficient; log S, the logarithm of aqueous solubility value; NHA, number of hydrogen bond acceptors; NHD, number of hydrogen bond donors; NHet, number of heteroatoms; NRing, number of rings; NRot, number of rotatable bonds; TPSA, topological polar surface area.

Blood–brain barrier (BBB) permeation property was observed for compounds 1, 2, 10, 11, and 13, but was absent in Ampicillin, which suggests that compounds 1, 2, 10, 11, and 13 would have a greater propensity to cross the BBB. It was discovered that the compounds' molecular weights were within the suggested range of 205 to 380. The 13 compounds' lipophilicity (logP) values were determined to fall between 2.3 and 5.0, which is an acceptable range. According to Lipinski's “Rule of Five,” the majority of molecules with good druglikeness have logP less than or equal to 5.0. MW less than or equal to 500, less than or equal to five HBDs, and fewer than or equal to 10 HBAs. Compounds that fulfil at least three of the four criteria are said to follow Lipinski's “Rule of Five.” All the compounds were found to obey Lipinski's Rule of Five (RO5) with no violations.

Although earlier work showed that the 2,3-dihydro-1,5-benzoxazepines, benzothiazoles, benzimidazoles, and benzothiazepines have some antitumor activity,[31] their antimicrobial potential was investigated in this work. The result showed that they possess weak to strong antibacterial activities against MRSA and other significant bacterial strains with concentrations ranging from 0.5 to 2,000 µg/mL in the broth dilution assay after a preliminary study of their zones of inhibition ([Table S4], available in the online version). This corroborates a similar study performed,[61] which confirmed the activity of some benzoxazepine derivatives against some selected bacterial strains and fungi. The agar well diffusion assay was used to assess the antibacterial activities of the compound. The results suggested that all the test compounds showed appreciable zones of inhibition at a final concentration of 100 µg per hole against MRSA, and some of the compounds gave varied activities against the test bacteria except against S. mutans and S. typhi ([Table S4], available in the online version). This is consistent with the work previously reported.[61]

The MIC of an antibiotic is the lowest concentration that stops the observable growth of a bacterium following incubation. When subcultured onto antibiotic-free media, MBC is the lowest dose that not only inhibits growth but also stops the organism from developing again. Diagnostic laboratories primarily use these measurements to confirm the presence of resistance in bacteria. They are therefore useful research tools for assessing the in vitro activity of antibacterial agents. To guarantee proper antimicrobial dose of the compounds, MICs and MBCs are used to evaluate antimicrobial agents' efficiency against bacteria. The result showed that they exhibit quite promising activities because their MICs and MBCs obtained using broth dilution confirmed their bacteriostatic or bactericidal activities ([Table S5] (available in the online version; [Figs. 5] and [6]), which compared favorably with previous work.[62]

Most compounds inhibited the organism's growth in the antitubercular assay. This is the first time reporting the activity of these derivatives against M. tuberculosis. The compounds could be inhibiting the growth of M. tuberculosis in a couple ways.[63] From the study, compounds 1, 3, 5, 6, 7, 11, and 12 showed antitubercular activity, suggesting that the 2,3-dihydro-1,5-benzoxazepines can be considered potent antimicrobial agents. Further modification of the substituents on the scaffold can greatly improve the activity of the compounds to warrant an in-depth research into the mechanism of action to obtain a better understanding of their mode of action against different microorganisms. The compounds with the highest activity from the antitubercular study were compounds 1 (0.97 ± 0.008), 12 (2.95 ± 1.36), 5 (5.95 ± 2.63), and 3 (8.40 ± 0.83). Of these, compound 12 gave the best physicochemical properties when compared with ampicillin. All the compounds were found to obey the Pfitzer rule and the GSK rule. Only compound 11 obeyed the Lipinski, Pfitzer, GSK, and Golden Triangle rules as ampicillin, making it a better drug candidate in terms of its medicinal chemistry, but it was only moderately active as an antitubercular agent. Compounds 11 and 12 gave good predicted absorption properties compared with ampicillin, whereas compound 12 showed good antituberculativity, and compound 11 was moderately active. In contrast, compounds 1, 3, 5, 6, and 7 were higher in activity than compound 11 but showed poor predicted absorption properties. Compounds 1 and 11 could penetrate the BBB and had overall better distribution properties compared with other compounds. This is consistent with results from the antitubercular work, which shows that the level of activity does not correspond to higher druglikeness. Similarly, for the antimicrobial work, the most active compounds differed based on the organism tested. While compound 2 was the most active against E. coli (20.33 ± 0.30 mm), compound 12 had the highest activity against MRSA. Compounds 6 and 13 had the highest activity against K.pneumonia and P. aeruginosa. These compounds had varying results for physicochemical properties, absorption, distribution, and excretion; the ADMET results were not dependent on the antimicrobial efficacy or the lack of it.

Conclusion

The synthesized 2,3-dihydro-1,5-benzoxazepine, benzothiazepiene, benzimidazole, and benzothiazole derivatives showed moderate activity against S. aureus (MRSA), E. coli, K. pneumonia, B. subtilis, and M. tuberculosis, which presents the possibility of improving their activity through the introduction of different substituents that are known to be active against certain organisms. The compounds can also be used to make co-crystals or co-formulations with known antimicrobial agents or nanoparticles to obtain improved antimicrobial activity through the synergistic activity of these co-formulations. The ADMET analysis gave variable results, indicating that some of the compounds could be good candidates as lead compounds for the development of new drugs.

Supporting Information

Experimental procedure for the synthesis of compounds 1–13, and their structural characterization, including IR, ¹H NMR, ¹³C NMR, DEPT, and mass spectra ([Figs. S1–S65], available in the online version); assement result of bactericidal and bacteriostatic activity the test compounds against E. coli, MRSA, S. mutans, S. typhi, K. pneumonia, P. aeruginosa, and B. substilis ([Table S1], available in the online version), original data of visual MIC90 and calculated MIC90 of the test compounds for Mycobacterium tuberculosis ([Tables S2, S3], available in the online version); mean zone of inhibition of compounds against microorganisms/mm ([Table S4], available in the online version); antibacterial activity grading of the test compounds (MBC/MIC) against the bacterial strains used ([Table S5], available in the online version); MICs and MBCs data of test compounds against microoganisms (ug/mL) ([Table S6], available in the online version) can be found in the “Supporting Information” section of this article's webpage.

Conflict of Interest

None declared.

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Publikationsverlauf

Eingereicht: 10. Januar 2025

Angenommen: 28. September 2025

Artikel online veröffentlicht:
07. November 2025