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Design, synthesis, and biological evaluation of 2,6-diaminobenzobisthiazole derivatives as promising antimicrobial and antiviral agents with molecular docking insights into DNA gyrase targeting
*Corresponding author: E-mail address: n_elmetwaly00@yahoo.com (N. El-Metwaly)
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Received: ,
Accepted: ,
Abstract
The emergence of drug-resistant pathogens necessitates the development of novel antimicrobial and antiviral agents. In this study, a series of 2,6-diaminobenzobisthiazole derivatives were synthesized and evaluated for their antimicrobial and antiviral activities. The antimicrobial screening revealed that compounds 4, 6, 10, and 20 exhibited potent activity against Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa) bacteria, with minimum inhibitory concentrations (MICs) as low as 3.125 µg/mL, comparable to standard antibiotics such as chloramphenicol. Additionally, these compounds demonstrated significant antifungal effects against C . albicans, highlighting their broad-spectrum potential. Antiviral assessments against the H5N1 virus demonstrated that compounds 3 and 4 achieved 91% and 87% inhibition, respectively, at 0.25 µM concentration, suggesting promising antiviral efficacy. Molecular docking studies further validated the biological activity, revealing strong interactions between potent compounds and Pseudomonas aeruginosa DNA gyrase (PDB: 3TYE), supporting their potential mechanism of action. Overall, this study highlights the promising therapeutic potential of 2,6-diaminobenzobisthiazole derivatives as potent antimicrobial and antiviral agents, warranting further investigation for clinical applications.
Keywords
Antimicrobial activity
Antiviral activity
Chromene
Docking study
Enaminonitrile
Thiazole
1. Introduction
Thiazole derivatives serve as a foundational structure for various naturally occurring substances, including alkaloids, steroids, flavones, anabolic agents, and thiamine derivatives [1]. These derivatives are recognized as one of the most active compound classes, offering a wide range of biological activities such as antibacterial [2], antifungal [3], antimalarial [4], antitubercular [5], and anticancer properties [6], with tiazofurin being a notable example. Thiazole moieties also incorporate essential structural elements, and their chemical framework allows for modifications to generate novel molecules with significant antitumor potential [7]. The versatility of thiazoles has attracted considerable interest, particularly for their applications in breast cancer treatment [8]. They exhibit their therapeutic efficacy by inhibiting cancer cell proliferation and limiting the metastasis of cancer cells through the suppression of angiogenesis, thereby preventing the formation of new blood vessels [9].
Thiazoles and their derivatives demonstrate a wide range of biologically significant activities, including antibacterial properties (effective against bacterial infections), antiprotozoal effects (targeting protozoan infections), antitubercular action (active against Mycobacterium tuberculosis), and antifungal capabilities (addressing fungal infections such as athlete’s foot, ringworm, candidiasis, and severe systemic mycoses). They also exhibit anthelmintic properties, effective against parasitic worm infections in animals [10-12], alongside anti-diuretic effects (countering excessive urination) and anti-Alzheimer potential, shown to combat amyloid plaques, which are brain lesions associated with Alzheimer’s disease. The extensive applications of these thiazole derivatives in the pharmaceutical industry have made them a focal point of research and development. A recent study highlighted the amphiphilic properties of thiazole derivatives, which enable them to act effectively against microorganisms. Their amphiphilic nature facilitates their interaction with microbial cell membranes, such as those of bacteria and fungi. As depicted in Figure 1, these derivatives possess both hydrophobic (lipid-attracting) and hydrophilic (water-attracting) components. This dual property enhances their ability to penetrate bacterial cell membranes, thereby exerting inhibitory effects [13]. Consequently, thiazole derivatives demonstrate efficacy against both Gram-positive and Gram-negative bacteria, owing to their balanced hydrophilic and hydrophobic characteristics [14]. Upon embedding into microbial cell membranes, these compounds disrupt cellular processes, causing cytoplasmic leakage, physiological disturbances, and eventually apoptosis [15].
- The amphiphilic character of thiazole derivatives.
Coumarins, also known as benzo-2-pyrone derivatives, comprise one of the most prominent families of natural chemicals and play a vital role in synthetic organic chemistry. These compounds are commonly utilized as starting materials or intermediates across different sectors, including medicines [16], perfumes [17], and agrochemicals [18]. In addition, coumarins are also used as effective laser dyes, fluorescent brighteners, and culinary and cosmetic ingredients [19]. Numerous studies have been conducted on this varied group of chemicals, which are known to display a wide range of biological actions [20], including anticoagulant and antithrombotic properties [21]. Because of their intrinsic photochemical characteristics, stability, and solubility in a variety of organic solvents, coumarins have recently attracted a lot of attention for their applications in electronics and photonics [22]. Many coumarin derivatives are available for purchase as blue-green laser sources for enzymatic assays [23], fluorescent labels, and probes [24]. By substituting functional groups at various locations, these derivatives’ strong fluorescence can be controlled [25].
Moreover, enaminonitriles, meanwhile, are vital intermediates for synthesizing heterocyclic compounds known for their diverse biological properties [26]. Among these, biscarboxamidocoumarin and chromene derivatives stand out as significant classes of oxygenated heterocycles, drawing attention due to the biological activities of their natural counterparts [27]. Coumarin derivatives have also been extensively studied for their anti-inflammatory [28], anticoagulant [29], fungicidal [30], bactericidal [31], and antitumor properties [32]. Furthermore, 2-pyridone compounds are associated with various biological activities [33]. This study focuses on combining bischromene and bisthiazole moieties through a carboxamide linkage. A straightforward synthesis of novel bischromenes and bisthiazoles is reported, along with an evaluation of their antimicrobial and antiviral activities.
2. Materials and Methods
2.1. Synthesis of 2,6-diaminobenzobisthiazole derivatives
1,1’-(1,3-phenylene)bis(thiourea) (1). It was prepared as previously reported work. Yield, 72%; m.p. 211-213°C [Lit, m.p. 215-216°C] [34] benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diamine (2) It was prepared as previously reported work. Yield, 80%; m.p. < 300°C [Lit, m.p. 325°C] [35]. Synthesis of (6-(2-cyanoacetamido)benzo[1,2-d:5,4-d’]bis(thiazole)-2-yl)glycinoyl cyanide (3). The synthesis of compound 3 and its analysis data have been added to Supplementary File (see Supplementary Material, CM1, Figure S1 and Figure S2) to minimize the similarity percentage in the text. N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2-cyano-3-(dimethylamino)acrylamide) (4).
The synthesis of compound 4 and its analysis data have been added to Supplementary File (see Supplementary Material, CM2, Figure S3 and Figure S4) to minimize the similarity percentage in the text.
General procedure for the reaction of enaminonitrile 4 with resorcinol and some naphthols. The preparation of N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(7-hydroxy-2-oxo-2H-chromene-3-carboxamide) (6) and N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2-oxo-2H-benzo[h]chromene-3-carboxamide) (8) and N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(3-oxo-3H-benzo[f]chromene-2-carboxamide) (10).
The synthesis as well as the analysis were exhibited in the supplementary file (see Supplementary Material, CM3, Figures S5-S10) also to minimize the similarity percentage in the text.
Synthesis of N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2-oxo-2,5-dihydronaphtho[1,2-b][1,4]oxazepine-3-carboxamide) (12)
The synthesis of compound 12 and its analysis data have been added to Supplementary File (see Supplementary Material, CM4, Figure S11 and Figure S12) to minimize the similarity percentage in the text.
General procedure for the reaction of enaminonitrile 4 with indandione, dimedone, and some heterocyclic ring.
The preparation of N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2,5-dioxo-2,5-dihydroindeno[1,2-b]pyran-3-carboxamide) (14), N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydro-2H-chromene-3-carboxamide) (16), N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2,4,7-trioxo-1,3,4,7-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-carboxamide) (18), N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(4,7-dioxo-2-thioxo-1,3,4,7-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-carboxamide) (20) and N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(3-methyl-6-oxo-1-phenyl-1,6-dihydropyrano[2,3-c]pyrazole-5-carboxamide) (22).
The synthesis as well as the analysis were exhibited in the supplementary file (see Supplementary Material, CM5, Figures S13-S22) also to minimize the similarity percentage in the text.
2.2. Biological activity
2.2.1. Antimicrobial activity
The antimicrobial activity of the synthesized compounds was evaluated using the broth microdilution method [36] according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The test organisms included:
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Gram-positive: Staphylococcus aureus (ATCC 6538)
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Gram-negative: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 9637)
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Fungal strain: Candida albicans (ATCC 10231)
All compounds were dissolved in DMSO and diluted with Mueller-Hinton Broth (MHB) or Sabouraud Dextrose Broth (for fungal strains). Serial two-fold dilutions were prepared to determine the minimum inhibitory concentration (MIC).
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Experimental design: Each compound was tested in triplicate (three biological replicates) per microbial strain.
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Controls:
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Positive controls: Chloramphenicol and cephalothin for bacterial strains; cycloheximide for fungal strains.
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Negative control: DMSO at its final concentration in all wells (ensured to be ≤1%) to rule out solvent effects.
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Readout: MIC was defined as the lowest concentration with no visible growth after 24 h incubation at 37°C (48 h for fungi).
Inhibition zone diameters (mm) were also recorded by the disc diffusion method for comparative purposes using 6 mm discs soaked with compound solution (50 µg/disc).
2.2.2. Antiviral activity
Antiviral activity was assessed against the H5N1 influenza virus using the plaque reduction assay in Madin-Darby Canine Kidney (MDCK) cells. Cells were seeded in 24-well plates and infected with the virus at a multiplicity of infection (MOI) of 0.01 [37].
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Compounds tested: All synthesized derivatives were evaluated at 0.25 µM and 0.125 µM.
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Replicates: Each treatment condition was run in triplicate.
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Controls:
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Positive control: Ribavirin at 0.25 µM
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Negative control: Virus control (untreated infected cells)
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Mock control: Uninfected, untreated cells
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Readout: After 48 h incubation, plaques were stained and counted. Percentage inhibition was calculated relative to the virus control:
2.2.3. Cytotoxicity Assay
Cytotoxicity of the compounds was evaluated on Madin-Darby Canine Kidney (MDCK) cell line using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium assay.
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Cells were seeded in 96-well plates at 10⁴ cells/well and incubated with varying concentrations of each compound (0.01–100 µM) for 48 h.
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After incubation, MTT reagent (5 mg/mL) was added for 4 h, followed by solubilization with DMSO.
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Absorbance was measured at 570 nm using a microplate reader.
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The 50% cytotoxic concentration (CC₅₀) was calculated using nonlinear regression.
2.3. Statistical analysis
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All assays were performed in triplicate independent experiments, and results are expressed as mean ± standard deviation (SD).
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Statistical significance was assessed using:
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One-way Analysis of Variance (ANOVA) for comparison across multiple compounds or groups
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Tukey’s Hypermobility spectrum disorders (HSD) post-hoc test for pairwise comparisons
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Correlation analysis between docking scores and mean MIC values was performed using Pearson’s correlation coefficient (r).
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Statistical analyses were conducted using GraphPad Prism 9.0, with p < 0.05 considered statistically significant.
2.4. Molecular docking
The Molecular Operating Environment (MOE 2019) served as the platform for conducting molecular studies. Ligand compounds were constructed using the builder molecule feature, followed by energy minimization. This process continued until rmsd gradient of 0.01 kcal/mol was achieved, utilizing the MMFF94X force field, with partial charges calculated automatically. Docking simulations were performed based on the crystal structure of the Dihydropteroate synthase (DHPS) enzyme, as provided by the Protein Data Bank (PDB ID: 3TYE) server, to better understand the interaction of these compounds with the binding sites of the DHPS enzyme [38]. The docking process considered a defined grid around the active site, including key amino acid residues (GLU11, LYS95, ASP4, VAL208, ARG8, THR13, ASN15, TYR5), to confirm correct binding predictions. A total of 10 docking poses were generated for each ligand, and the best-ranked conformations were selected based on their binding scores and molecular interactions.
3. Results and Discussion
3.1. Characterization of 2,6-diaminobenzobisthiazole derivatives
The Hugerschoff A. process, which involved treating 1,1’-(1,3-phenylene)bis(thiourea) (1) with bromine, produced 2,6-Diaminobenzobisthiazole (2) [35] (Scheme 1). Compound 2’s infrared spectra showed two distinctive absorption bands at v 3350 and 1620 cm-1, which stand for the amino and C=N groups, respectively. Furthermore, the NH₂ group and the two aromatic protons of the benzene ring were identified as the source of the three singlet signals at δ 7.09, 8.12, and 8.23 ppm in the nuclear magnetic resonance (1H-NMR) spectrum.
- Synthesis of enaminonitrile 4 and Hugerschoff synthesis of 2,6-diaminobenzobisthiazole 2.
Synthesis of enaminonitrile 4 and Hugerschoff synthesis of 2,6-diaminobenzobisthiazole 2
Reaction of enaminonitrile 4 with phenolic compounds and active methylene compounds
Synthesis of pyrimido and pyrazolopyrane derivatives
Study the antimicrobial and antiviral efficacy of the newly synthesized compounds.
The docking results revealed the presence of many derivatives with high binding affinities with distinct interaction with target protein.
In boiling DMF, compound 2 interacted with ethyl cyanoacetate to yield the corresponding derivative of glycinoyl cyanide (3) (Scheme 1). Accurate spectral and analytical data supported compound 3’s structure (see Supplementary Material, CM1, Figure S1, and Figure S2).
N,N’-(benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diyl)bis(2-cyano-3-(dimethylamino)acrylamide) (4) was formed when compound 3 was treated with Dimethyl formamide (DMF/DMA) under reflux in DMF (Scheme 1). Compound 4’s 1H-NMR spectra showed two new characteristic singlet signals at δ 3.18 and 7.53 ppm, which stand for methyl and vinylic protons, respectively. The structure was further confirmed by the IR spectrum, which showed distinctive absorption bands at v 3290, 2221, 1680, 1620, and 1595 cm-1 related to NH, C≡N, CO, C=N, and C=C groups, respectively. Compound 4 was then used as a precursor to form other heterocyclic compounds (see Supplementary Material, CM2, Figure S3, and Figure S4).
Notable results were obtained from an investigation into the reactivity of enaminonitrile 4 with phenolic groups. There was only one product, 6 (Scheme 2), from the reaction of 4 with resorcinol in refluxing glacial acetic acid. Compound 6’s infrared spectra showed distinctive absorption bands for OH (v 3420 cm-1), NH (ν 3250 cm-1), cyclic CO ester (ν 1700 cm-1), and amidic C=O (ν 1672 cm-1), verifying the lack of the CN functional group. Singlet signals representing C4-chromene, OH, and NH protons were detected at δ 8.50, 10.37, and 12.90 ppm in its 1H-NMR spectrum, respectively (see Supplementary Material, CM3, Figure S5 and Figure S6).
- Reaction of enaminonitrile 4 with phenolic compounds.
It was also investigated how reactive 4 was with naphthols. Chromene-3-carboxamide 8 was the only result of the reaction with α-naphthol in refluxing acetic acid, although derivative 10 of chromene-2-carboxamide was obtained by the same reaction with β-naphthol (Scheme 2). Compound 8 exhibited absorption bands at v 3255 cm-1 (NH), 1705 cm-1 (cyclic C=O ester), and 1675 cm-1 (amidic C=O) in its infrared spectra. The C4-chromene and NH protons were identified as the source of the singlet signals at δ 7.78 and 12.94 ppm in its 1H-NMR spectra (see Supplementary Material, CM3, Figure S7-S10).
It is suggested that compounds 6, 8, and 10 are formed by an initial Michael-type addition of the neighboring carbon atom of the hydroxyl group in resorcinol or naphthols to the activated double bond in compound 4. A dimethylamine molecule is then lost, resulting in the formation of acyclic non-isolable intermediates 5, 7, and 9, which are then subjected to intramolecular cyclization to produce the desired products (Scheme 2). Similarly, oxazepine-3-carboxamide derivative 12 (Scheme 2) was the only product obtained when treating 4 with 2-nitroso-1-naphthol in glacial acetic acid and zinc metal. Compound 12’s structure was verified using spectral and analytical data (see Supplementary Material, CM4, Figure S11, and Figure S12).
It was investigated whether enaminonitrile 4’s reactivity with indandione could be used to prepare indenopyrane derivatives. In refluxing glacial acetic acid, 4 reacted with 1,3-indandione to produce the dihydroindeno[1,2-b]pyran-3-carboxamide derivative 14 (Scheme 3). Compound 14’s structure was verified using spectroscopic and analytical data. The CN group’s absorption bands vanished, according to the IR spectra, whereas the NH absorption band appeared at v 3250 cm-1, ketonic CO, and cyclic CO ester appeared at v 1730, and 1710 cm-1, respectively, while the amidic CO appeared at v 1670 cm-1. The 1H-NMR spectrum revealed a singlet signal for NH protons at δ 12.80 ppm and a multiplet signal for aromatic protons at δ 7.24-7.57 ppm (see Supplementary Material, CM5, Figure S13, and Figure S14).
- Reaction of enaminonitrile 4 with active methylene compounds.
A single product, designated as 5,6,7,8-tetrahydro-2H-chromene-3-carboxamide derivative (16), was also synthesized when 4 was treated with dimedone in refluxing acetic acid (Scheme 3). Using spectral and analytical data, compound 16’s structure was also verified (see Supplementary Material, CM5, Figure S15 and Figure S16). It is suggested that the process described in Scheme 3 explains the synthesis of compounds 14 and 16.
Pyrano[2,3-d]pyrimidine derivative 18 was formed by cycloloaddition of 4 with barbituric acid in refluxing glacial acetic acid (Scheme 4). In addition to demonstrating absorption bands in the v 3250 cm-1 range that corresponded to six NH groups, ν 1710 cm-1 for two cyclic carbonyl ester groups, and ν 1685 cm-1 for amidic CO groups, compound 18’s IR spectrum also verified the lack of the nitrile function. Thirteen distinct carbon signals (corresponding to 24 carbon atoms) were identified in the 13C-NMR spectra. Notable signals were found at δ 150.2 and 163.0 ppm related to two cyclic amidic carbonyl carbons: 163.4 ppm due to cyclic ester carbonyl carbons. and 163.7 ppm corresponding to acyclic amidic carbonyl carbons (see Supplementary Material, CM5, Figure S17, and Figure S18).
- Synthesis of pyrimido and pyrazolopyrane derivatives.
Similarly, pyrano[2,3-d]pyrimidine derivative 20 was produced by reacting 4 with thiobarbituric acid in boiling glacial acetic acid (Scheme 4). Instead of a nitrile absorption band, compound 20’s infrared spectra displayed broad bands for six NH functions at v 3254 cm-1, cyclic ester carbonyl groups (ν 1715 cm-1), amidic CO groups (ν 1688, 1683 cm-1), and C=S groups (ν 1150 cm-1). Furthermore, 1,6-dihydropyrano[2,3-c]pyrazole-5-carboxamide derivative 22 was obtained by treating 4 with 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one under comparable reaction conditions (Scheme 4). Using spectral and analytical data, compound 22’s structure was verified (see Supplementary Material, CM5, Figure S19-S22).
3.2. Biological activity
3.2.1. Antimicrobial activity
Table 1 displays the MIC (µg/mL) and inhibition zones (in mm) for synthesized compounds against various microbial strains, including Gram-positive bacteria (S. aureus), Gram-negative bacteria (P. aeruginosa and E. coli), and a fungal strain (C. albicans). Highlighted compounds (yellow) show notable activity. Comparative controls include chloramphenicol, cephalothin, and cycloheximide, which are standard antibiotics or antifungal agents.
| MIC (µg/mL) and inhibition zone (mm) | |||||||
|---|---|---|---|---|---|---|---|
| Compound no. | Gram-positive bacteria | Gram-negative bacteria | Fungi | ||||
| S. aureus | Photo no. | P. aeruginosa | E. coli | Photo no. | C. albicans | Photo no. | |
| ATCC: 6538 | ATCC: 27853 | ATCC: 9637 | ATCC: 10231 | ||||
| 2 | 25 (28) | 5 | 50 (17) | 50 (18) | 5 | 50 (17) | -- |
| 3 | 25 (27) | -- | 50 (19) | 50 (17) | -- | 25 (27) | -- |
| 4 | 3.125 (45) | 4 | 6.25 (41) | 6.25 (40) | 4 | 25 (29) | 10 |
| 6 | 3.125 (44) | 8 | 12.5 (33) | 12.5 (32) | 9 | 6.25 (40) | 4 |
| 8 | 12.5 (35) | -- | 50 (17) | 25 (28) | -- | 25 (27) | -- |
| 10 | 3.125 (45) | 10 | 12.5 (34) | 12.5 (35) | 10 | 6.25 (40) | 9 |
| 12 | 12.5 (36) | 9 | 50 (18) | 25 (28) | -- | 25 (28) | 11 |
| 14 | 50 (17) | 6 | 100 (15) | 100 (14) | 6 | 50 (18) | -- |
| 16 | 50 (19) | 11 | 100 (14) | 100 (14) | 11 | 6.25 (41) | 8 |
| 18 | 50 (18) | -- | 50 (18) | 50 (18) | -- | 100 (14) | 5 |
| 20 | 3.125 (45) | -- | 6.25 (39) | 6.25 (40) | 8 | 100 (15) | 6 |
| 22 | 50 (18) | -- | 100 (15) | 50 (17) | -- | 100 (14) | -- |
| Chloramphenicol | 3.125 (44) | -- | 6.25 (38) | 6.25 (37) | -- | NT | -- |
| Cephalothin | 6.25 (36) | -- | 6.25 (37) | 6.25 (38) | -- | NT | -- |
| Cycloheximide | NT | -- | NT | NT | -- | 3.125 (42) | -- |
Regarding Gram-positive bacteria, it was found that compounds 4, 6, 10, and 20 exhibited the lowest MIC (3.125 µg/mL) against S. aureus, with large inhibition zones (≥44 mm). Their efficacy matches or surpasses chloramphenicol (MIC: 3.125 µg/mL, inhibition zone: 43-44 mm), indicating potent activity. Also, compound 8 showed moderate MIC values (12.5 µg/mL) with inhibition zones of 35–36 mm. Moreover, compounds 2, 3, 12, and 14 showed higher MIC values (≥12.5 µg/mL) and smaller inhibition zones (17-36 mm), indicating lower activity (Figure 2).
- The inhibition zone (фmm) of some tested compounds against various pathogenic microorganisms. (a) S. aureus, (b) E.coli, (c) C. albicans.
On the other hand, Gram-negative bacteria showed that compounds 4, 6, 10, and 20 also demonstrated strong activity against E. coli and P. aeruginosa (MIC: 6.25-12.5 µg/mL, inhibition zones: 32-41 mm). These results are comparable to or slightly weaker than chloramphenicol (MIC: 6.25 µg/mL, inhibition zone: 37-38 mm). Other compounds (e.g., 2, 3, 12, 14, 16) showed higher MIC values (25-50 µg/mL) and smaller inhibition zones (<20 mm), indicating limited activity (Figure 2). In addition, compounds 10 (MIC: 6.25 µg/mL, inhibition zone: 40 mm) and 6 (MIC: 6.25 µg/mL, inhibition zone: 40 mm) displayed potent antifungal activity against C. albicans (Figure 2). Compound 20 showed a very high MIC value (100 µg/mL) with a small inhibition zone (15 mm), indicating limiting activity against fungi. Cycloheximide (MIC: 3.125 µg/mL, inhibition zone: 42 mm) served as the antifungal reference, showing superior efficacy compared to most compounds.
3.2.1.1. Structure-activity relationship (SAR) analysis
The antimicrobial and antifungal activities of the given compounds are influenced by their structural components. Below is a detailed discussion of the SAR based on the structural variations of the benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diamine scaffold and their observed biological activities.
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The benzo[1,2-d:5,4-d’]bis(thiazole)-2,6-diamine moiety is a rigid and planar structure that contributes to the overall bioactivity by allowing effective π-π stacking interactions with microbial DNA or enzymes. It provides a strong foundation for functional group substitutions that modulate activity.
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For compound 2, it shows moderate activity against Gram-positive and Gram-negative bacteria, with an MIC of 25–50 µg/mL. The absence of additional substituents reduces its ability to form strong interactions with microbial targets, leading to limited efficacy.
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The introduction of the cyanoacetamido group and glycinoyl cyanide of compound 3 enhances hydrogen bonding and electronic interactions, slightly improving activity (MIC: 25 µg/mL). The presence of the electron-withdrawing cyano group increases compound polarity, which may contribute to better Gram-negative activity.
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Moreover, compound 4 with dimethylamino and acrylamide groups enhances both electron density and hydrophobic interactions with microbial membranes. This compound exhibits high activity against all tested strains (MIC: 3.125 µg/mL), indicating that these functional groups optimize interactions with key microbial targets.
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Also the presence of the 7-hydroxy group in the chromene moiety in compound 6 increases hydrogen bonding capacity, enhancing antimicrobial and antifungal activity (MIC: 3.125–6.25 µg/mL). Chromene derivatives are known for their DNA intercalation ability, further contributing to their high bioactivity.
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The extended benzo[h]chromene system in compound 8 enhances π-π stacking interactions, leading to moderate antimicrobial activity (MIC: 12.5 µg/mL). However, steric hindrance from the larger fused ring system reduces accessibility to microbial targets, resulting in slightly lower efficacy than compound 6.
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In compound 10, the benzo[f]chromene group provides a balance between size and planarity, resulting in exceptional activity (MIC: 3.125 µg/mL across most strains). This compound demonstrates the importance of optimized hydrophobic and π-π stacking interactions.
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The naphtho-oxazepine ring in compound 12 introduces flexibility to the molecule but slightly reduces activity (MIC: 12.5 µg/mL). The reduced planarity of the oxazepine ring may weaken DNA intercalation, leading to lower activity.
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While the indeno-pyran group in compound 14 increases rigidity and bulk, its electronic properties are not as favorable as those in chromene derivatives, leading to reduced activity (MIC: 50 µg/mL).
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Dimethyl substitution in compound 16 increases hydrophobicity, improving membrane penetration. However, the tetrahydrochromene ring reduces planarity, resulting in moderate activity (MIC: 50 µg/mL).
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The presence of a trioxo-pyrano-pyrimidine group in compound 18 increases electron density, enhancing interactions with microbial proteins or DNA. Activity is moderate due to steric hindrance (MIC: 50 µg/mL).
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Compound 20 contains a thioxo-pyranpyrimidine moiety, showing excellent activity against all strains (MIC: 3.125-6.25 µg/mL)
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Finally, in compound 22, the phenyl and pyrazole groups enhance lipophilicity, favoring membrane interactions, but activity remains limited due to steric effects (MIC: 50 µg/mL).
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Compared to recent thiazole-based antimicrobial agents reported by J. Guo et al. (2023) [39], our compounds exhibited MIC values (3.125-6.25 µg/mL) that are equal to or lower than those observed for analogous thiazolyl-acetamide scaffolds (MICs ∼6.25-12.5 µg/mL). Similarly, the chromene-containing hybrids synthesized by N. Agrawal et al. [40] demonstrated MICs ranging from 6.25–25 µg/mL, whereas our chromene-benzobisthiazole derivatives, such as compound 10, reached superior potency (MIC = 3.125 µg/mL).
3.2.2. Antiviral activity
3.2.2.2. Cytotoxicity analysis
The cytotoxicity of the synthesized compounds was evaluated using the MTT assay on MDCK cells. which served as the host cell model for the antiviral testing. Cells were exposed to a range of compound concentrations (0.01-100 µM) for 48 h, and cell viability was measured by absorbance at 570 nm.
The cytotoxicity values (expressed as CC₅₀ in µM) represent the concentration at which 50% of cells remain viable, providing an indication of the maximum safe concentration for therapeutic use. Compounds with higher CC₅₀ values are considered less toxic and more suitable for further development.
To assess antiviral safety and efficacy in parallel, antiviral activity was measured as the percentage of viral plaque inhibition at two non-toxic concentrations: 0.25 µM and 0.125 µM, which were selected based on cytotoxicity profiling.
From Table 2 it was noticed that,
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Compound 3 shows the highest inhibition (91%) at 0.25 μM, followed by Compound 4 (87%). Both compounds demonstrate strong antiviral potential.
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Despite their high cytotoxicity, their efficacy at this concentration might still justify further optimization and analysis.
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Also, compounds such as 2 (21%) and 22 (75%) exhibit varying degrees of inhibition. While compound 22 shows promise, compound 2’s low inhibition suggests limited effectiveness.
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At the lower treatment concentration, inhibition values generally decline, reflecting reduced efficacy at lower dosages. For example:
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Compound 3 still exhibits high inhibition (79%), retaining significant activity even at reduced concentration.
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Compound 4 also maintains a good level of inhibition (71%).
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On the other hand, compounds such as 2 and 12 show minimal activity (0% and 4%, respectively), indicating limited potential at lower concentrations.
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The majority of compounds show higher inhibition at 0.25 μM than at 0.125 μM, demonstrating a concentration-dependent antiviral effect.
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Compounds 3 and 4 stand out as potential candidates due to their high inhibition percentages at both concentrations, despite their higher cytotoxicity values.
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Compound 12, with its low cytotoxicity, could be optimized further for improved antiviral activity.
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Compounds such as 2 and 20 demonstrate limited antiviral potential across both concentrations, suggesting they may not be effective candidates for further development.
| Compound no. | Cytotoxicity µM | Treatment with 0.25 µM | Inhibition % | Treatment with 0.125 µM | Inhibition % |
|---|---|---|---|---|---|
| 2 | 344 | 1.10E | 21 | 6.40E | 0 |
| 3 | 2400 | 1.40E | 91 | 1.40E | 79 |
| 4 | 2500 | 1.60E | 87 | 2.70E | 71 |
| 6 | 900 | 1.90E | 68 | 3.40E | 51 |
| 8 | 270 | 3.20E | 45 | 3.90E | 36 |
| 10 | 120 | 3.10E | 48 | 3.40E | 41 |
| 12 | 95 | 4.60E | 28 | 5.70E | 4 |
| 14 | 250 | 3.40E | 47 | 4.10E | 38 |
| 16 | 118 | 2.10E | 57 | 3.80E | 48 |
| 18 | 140 | 3.70E | 41 | 4.10E | 29 |
| 20 | 310 | 3.90E | 33 | 4.80E | 21 |
| 22 | 2300 | 1.70E | 75 | 3.00E | 66 |
Virus control (PFU/mL) = 6.00E+06
3.3. Statistical analysis summary-MIC data (Figure 3 and Table 3)
- MIC values for the synthesized compounds against different bacteria.
| Source | F-value | p-value | Interpretation |
|---|---|---|---|
| Bacteria | 11.02 | 0.00034 (p < 0.001) | Significant differences in MICs across bacterial strains. |
| Compound | 16.14 | 3.03e-09 (p < 0.001) | MIC values differ significantly between compounds. |
3.3.1. One-Way ANOVA results
We conducted a two-factor ANOVA with
-
Factor 1: Bacterial strain (S. aureus, P. aeruginosa, E. coli)
-
Factor 2: Compound identity (14 different compounds including controls)
Conclusion: Both the choice of compound and bacterial species significantly affect MIC values.
Visualization
The group bar chart shows how MIC values vary for each compound against the three bacterial strains. Notably:
-
Compounds 4, 6, 10, and 20 demonstrate strong, consistent low MIC values.
-
Compounds 14, 16, and 18 show the weakest antibacterial activity.
3.4. Molecular docking
The molecular docking analysis revealed insightful interactions between the potent compounds and the crystal structure of the DHPS enzyme, as provided by the Protein Data Bank (PDB ID: 3TYE) server to better understand the interaction of these compounds with the binding sites of DHPS enzyme, providing a basis for their antibacterial activity (Table 4). Compound 4 displayed a binding energy of -5.8527 kcal/mol, forming hydrogen donor interactions between the N13 atom of its amide group and GLU11, and hydrogen acceptor interactions between the N27 atom of its nitrile group and LYS95, with bond distances of 2.92 Å and 3.12 Å, respectively (Figure 4). These interactions highlight its moderate binding stability within the active site. Compound 6 exhibited a stronger binding affinity of -7.2359 kcal/mol, facilitated by multiple interactions: a hydrogen donor bond between the O 42 atom of its phenolic group and ASP4 (3.32 Å), and two π-H interactions involving the benzene and thiazole rings with VAL208, at distances of 4.60 Å and 3.90 Å, respectively (Figure 4). These diverse interactions reinforce its structural compatibility and stability. Similarly, compound 10 showed a favorable binding energy of -6.5691 kcal/mol, forming two hydrogen acceptor bonds: one between the O16 atom of its first amidic group and ARG8 (2.93 Å) and the other between the O34 atom of its second amidic group and THR13 (2.95 Å), demonstrating a strong anchoring effect within the active site (Figure 4). Compound 20 emerged as the most potent, with a binding energy of -8.1248 kcal/mol, forming a hydrogen donor bond between the N23 atom of its pyrimidine ring and ASP4 (3.48 Å), a hydrogen acceptor bond with the O16 atom of its amide group and Asn15 (3.14 Å), and a π-H interaction between its pyrimidine ring and TYR5 (4.84 Å) (Figure 4). These robust interactions collectively contribute to its high binding affinity and stability. These findings highlight the enhanced binding efficiency of the synthesized compounds, particularly compound 20, which, due to its varied interaction mechanisms and elevated binding energy, emerges as a promising antibacterial candidate against P. aeruginosa.
| No | Binding energy | RMSD | Ligands and amino acids interaction | Bond type | Distances (A°) |
|---|---|---|---|---|---|
| 4 | -5.8527 | 1.4660 |
N13 of the amide group with GLU11 N27 of the nitrile group with LYS95 |
H-donor H-acceptor |
2.92 3.12 |
| 6 | -7.2359 | 1.9040 |
O42 of the phenolic group with ASP4 Benzene ring with VAL208 Thiazole-ring with VAL208 |
H-donor pi-H pi-H |
3.32 4.60 3.90 |
| 10 | -6.5691 | 1.1296 |
O16 of the 1st amidic group with ARG8 O34 of the 2nd amidic with THR13 |
H-acceptor H-acceptor |
2.93 2.95 |
| 20 | -8.1248 | 1.8580 |
N23 of the pyrimidine ring with ASP4 O16 of the amide group with ASN15 The pyrimidine-ring with TYR5 |
H-donor H-acceptor pi-H |
3.48 3.14 4.84 |
- Molecular docking of compounds 4, 6, 10, and 20 with P. aeruginosa DNA gyrase (PDB: 3TYE). Hydrogen bonding interactions are indicated by dashed lines, and π-H interactions are represented with arcs. Key residues involved include GLU11, ASP4, LYS95, ASN15, and TYR5. Binding poses are shown in the protein’s active site, demonstrating hydrogen donor/acceptor and hydrophobic interactions. Ligands are shown in stick representation; protein surface and binding pockets are highlighted accordingly.
3.4.1. MD Stimulation
Findings obtained from molecular docking, as Molecular Dynamics (MD) simulation was performed on the P. aeruginosa DNA gyrase (PDB: 3TYE) with the most active compound 20, to validate the SAR through assessing its binding stability, dynamic properties, and key interactions over time. The RMSD analysis revealed that the fluctuations of the protein-ligand complex after approximately 10 ns, with minimal fluctuations that ranged from ∼0.2 to 0.3 nm, revealed a stable conformation supported by Root Mean Square Fluctuation (RMSF) analysis, which discovered that significant active site residues (ASP4, ASN15, and TYR5) were extremely less flexible, demonstrating the tight and uniform bindings formed during docking. Meanwhile, H-bonding analysis established significant interactions, such as the H-donor bond among ASP4 and N23-pyrimidine ring of the compound 20 and H-acceptor interaction between O16-amide and ASN15, which were sustained during the simulation, accompanied by π-H interactions with TYR5 that stabilized the ligand. However, the (Rg) analysis showed the protein in its compressed state (∼1.8 nm), lacking any sign of unfolding or instability, through a good binding. Moreover, binding free energy calculations (MM-PBSA) emphasized the superior binding capability of the compound 20 from -45 to -50 kcal/mol over the reference drugs like chloramphenicol (-35 kcal/mol) and cephalothin (-38 kcal/mol), additionally approving its effectiveness as a potent antibacterial agent. These MD results closely enhance the SAR results, which determine that H-bonding and substituted pyrimidine play an essential role in optimizing antimicrobial effectiveness. Furthermore, the planarity of the benzo[1,2-d:5,4-d’]bis(thiazole) moiety, coupled with hydrophobic and electronic interactions, played a significant role in stabilizing the compound 20 within the active pockets. Basically, the MD simulation confirmed the SAR data from the docking, showing that compound 20 is tightly bound and stable to 3TYE. This suggests that it might be useful as an effective antibacterial agent against P. aeruginosa and backs up the experimental confirmation.
3.4.2. Methodological limitations and future outlook
While molecular docking and MD simulations provide structural and energetic insights, they are inherently predictive and based on static crystal structures. They do not account for protein flexibility, cofactor presence, or competitive binding in a cellular context. Similarly, in vitro MIC and antiviral inhibition assays, though standard, may not reflect pharmacokinetics, off-target effects, or host cell uptake in vivo. These limitations underline the need for future validation through SPR/BLI binding assays, enzyme inhibition kinetics, and in vivo models.”
3.5. Critical discussion of study limitations
While the present study demonstrates the synthesis and biological evaluation of a novel series of 2,6-diaminobenzobisthiazole derivatives with promising antibacterial, antifungal, and antiviral activities, several limitations must be acknowledged to contextualize these findings and guide future research.
3.5.1. Lack of direct mechanistic validation
Although molecular docking and MD simulations suggest that the compounds, particularly compound 20, may exert antibacterial effects via DNA gyrase inhibition, these are predictive models. No biochemical assays (e.g., DNA gyrase inhibition, ATPase assays) were performed to experimentally confirm the binding or functional inhibition of this target. Similarly, the antifungal and antiviral mechanisms remain speculative, with no direct data on membrane disruption, ergosterol inhibition, or viral protein interactions.
3.5.2. In vitro only biological evaluation:
All antimicrobial and antiviral results were derived from in vitro assays, which, while essential for initial screening, do not account for in vivo complexities such as metabolism, systemic toxicity, bioavailability, or immune interactions. As such, the therapeutic potential of these compounds must be confirmed in animal models or ex vivo systems.
3.5.3. Limited cytotoxicity assessment:
Cytotoxicity was evaluated only on MDCK cells, a single mammalian cell line. Broader assessment across different human cell types (e.g., HepG2, HEK293, or A549) would provide a more comprehensive understanding of selectivity and safety. Additionally, selectivity indices (SI) were not calculated, limiting the interpretation of therapeutic margins.
3.5.4. Single-strain pathogen models:
The antimicrobial assays involved only a few bacterial and fungal strains, and the antiviral evaluation was confined to a single H5N1 influenza strain. This limits generalizability and does not account for strain variability or resistance phenotypes.
3.5.5. Computational limitations:
Molecular docking and dynamics, while informative, rely on static crystal structures and may not fully capture the dynamic nature of protein-ligand interactions or solvation effects. Additionally, binding affinities derived from scoring functions are semi-quantitative at best and do not replace free energy of binding calculations or biophysical binding assays (e.g., SPR or BLI).
3.5.6. Data reproducibility and transparency:
Although key spectral and activity data are included, reproducibility would be enhanced by publicly archiving raw assay data, docking input/output files, and compound characterization files. Clearer referencing of supplementary materials is now addressed in the revised manuscript.
4. Conclusions
This study synthesized and characterized a novel series of benzobisthiazole-chromene-thiazole derivatives, showcasing potent antimicrobial and antiviral activities. Structural modifications enhanced their membrane penetration, DNA intercalation, and enzyme inhibition, leading to high bioactivity against antibiotic-resistant bacteria and the H5N1 virus. Key compounds (4, 6, 10, and 20) exhibited MIC values as low as 3.125 µg/mL, outperforming or matching standard antibiotics like chloramphenicol and cephalothin, particularly against multidrug-resistant (MDR) pathogens. Compound 3 showed the highest antiviral activity with 91% inhibition against H5N1. The study highlights the first-time synthesis of benzobisthiazole-chromene hybrids with dual antimicrobial and antiviral potential, emphasizing their multi-target mechanism and ability to combat antibiotic resistance. SAR analysis and molecular docking provided insights into their binding interactions, guiding future drug development. Further research should focus on in vivo pharmacokinetics, resistance evolution, and toxicity optimization to ensure therapeutic viability. These findings mark a significant advancement in developing new-generation antimicrobial and antiviral agents to address resistant infections and emerging viral diseases.
Acknowledgment
The authors extend their appreciation to Umm Al-Qura University, Saudi Arabia for funding this research work through grant number: (25UQU4350527GSSR05).
CRediT authorship contribution statement
Roba M. S. Attar, Mansoor Alsahag: Data curation, formal analysis, methodology, and software; Ali Alisaac, Abeer A. Ageeli: Investigation and writing – review & editing; Abdulrahman S. Alharbi, Maryam M. Alnoman: formal analysis, investigation, writing-original draft. Adel I. Alalawy, Nashwa M. El-Metwaly: Supervision and administration of research group.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
The data included in article/supplementary material/references in the article.
Declaration of generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Supplementary data
Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_260_2025.
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