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. 2026 Jul 2;16(34):33076–33092. doi: 10.1039/d6ra02410f

Design and synthesis of benzothiazole–sulfonate conjugates targeting influenza A (H1N1) and human coronavirus HCoV-229E: in silico and in vitro assessments

Mohamed A Omar a, Aladdin M Srour b,✉, Aisha A K Al-Ashmawy b, Abdou K Allayeh c,d, Ahmed O H El-Nezhawy a,✉
PMCID: PMC13326587  PMID: 42395790

Abstract

A novel series of eighteen benzothiazole–sulfonate conjugates, comprising benzothiazolylphenyl-alkanesulfonates (3a–i) and benzothiazolyl acetohydrazide phenyl sulfonates (7a–i), was synthesized and evaluated for dual antiviral efficacy against Influenza A (H1N1) and human coronavirus (HCoV-229E). Biological assessment via dose–response assays identified several leads with potent inhibitory activity and high safety profiles. Against H1N1, compound 3g emerged as the most selective, yielding a Selectivity Index (SI) value of 20.2, whereas the highest viral replication inhibition% was seen in compound 3i. In HCoV-229E assays, derivative 7a demonstrated superior performance with SI value > 20, and compound 7b showed the highest percentage of inhibition of viral replication. In silico absorption, distribution, metabolism, and excretion (ADME) profiling confirmed that the most active conjugates adhere to drug-likeness parameters, exhibiting favorable absorption and distribution properties. Molecular docking simulations within the H1N1 RNA-dependent RNA polymerase (RdRp) subunits and the HCoV-229E main protease (Mpro) elucidated the molecular basis for these activities, showing strong binding affinities and key hydrogen bonding interactions with essential catalytic residues. These findings position the benzothiazole-sulfonate scaffold as a promising template for the development of broad-spectrum antiviral agents.

New benzothiazole-sulfonate hybrids as dual H1N1/HCoV-229E inhibitors via RdRp/Mpro docking, with favorable ADME profiles.graphic file with name d6ra02410f-ga.jpg

1. Introduction

Viral infections remain a major global health challenge, causing millions of illnesses and deaths annually. According to the World Health Organization, viruses account for nearly 60% of infectious diseases, including influenza, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), hepatitis, and emerging pathogens such as severe acute respiratory syndrome coronavirus-2 SARS-CoV-2.1–6 The coronavirus disease 2019 (COVID-19) pandemic and the growing emergence of drug-resistant viral strains have underscored the urgent need for effective and innovative antiviral therapies.7–10

Antiviral agents play a critical role by targeting key stages of the viral life cycle, including entry, replication, and assembly, thereby reducing viral load and disease transmission.11–13 Advances in molecular virology and structure-based drug design have facilitated the development of more selective and potent antiviral compounds.14–16 Effective antiviral drug design relies on a thorough understanding of virus-host interactions, as viruses depend on host cellular machinery for replication. High selectivity toward viral targets is essential to maximize therapeutic efficacy while minimizing host toxicity. Ongoing research into viral pathogenesis and resistance mechanisms remains crucial for the development of next-generation antiviral therapies.17–19 Both influenza A (H1N1) virus and human coronavirus 229E (HCoV-229E) rely on RNA-dependent RNA polymerase (RdRp) mediated replication, a critical process for viral genome transcription and propagation. In the H1N1 influenza virus, the RdRp is a heterotrimeric complex composed of the PB1, PB2, and PA subunits that cooperatively drive viral RNA synthesis through a cap-snatching mechanism.20,21 On the other hand, HCoV-229E main protease (Mpro) is considered a promising therapeutic target due to many factors. It facilitates the assembly of the replication-transcription complex, which is essential in the viral maturation process, and it offers a high therapeutic index with minimal off-target side effects because it is completely different than the host human protease.22 Moreover, it counteracts the (human) host's innate immune response23 and its catalytic domain is highly conserved among the Coronaviridae family, which implies that its inhibitors may serve as broad-spectrum anti-seasonal coronaviruses.24,25

Heterocyclic compounds such as benzothiazole are significant in organic chemistry and drug discovery due to their broad spectrum of biological activities. Benzothiazole derivatives have been reported to exhibit antibacterial, anti-inflammatory, analgesic, anticonvulsant, antiviral, antioxidant, and anticancer properties. Notably, ethoxzolamide and frentizole are clinically used antimicrobial agents containing a benzothiazole moiety (Fig. 1). Hydrazide-hydrazone derivatives, characterized by the azomethine functionality (–CONHN Created by potrace 1.16, written by Peter Selinger 2001-2019 CH–), also display diverse pharmacological activities, including antimicrobial, anti-inflammatory, analgesic, anticancer, anticonvulsant, antiviral, antiprotozoal, antimalarial, antimycobacterial, vasodilatory, antiplatelet, and anti-schistosomal effects.26,27 These compounds can readily undergo hydrolysis in biological systems, a feature that may contribute to their therapeutic efficacy in serious diseases. Among their reported activities, antimicrobial effects are the most prominent. Well-known chemotherapeutic agents such as nitrofurazone, furazolidone, and nitrofurantoin 28 exemplify clinically important drugs containing the hydrazide-hydrazone motif (Fig. 1).

Fig. 1. Marketed drugs containing benzothiazole, hydrazide-hydrazone, or sulfonate motifs.

Fig. 1

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Sulfonates are widely utilized in medical research due to their significant pharmacological relevance. Their distinctive physicochemical properties confer strong lipid affinity, facilitating membrane penetration and interaction with biological targets. Several aryl sulfonates, such as fenson, chlorfenson, and genite, have long been used as insecticides,29,30 while busulfan, an alkyl sulfonate, is an approved chemotherapeutic agent for the treatment of chronic myeloid leukemia (CML)31 (Fig. 1). Aryl alkanesulfonates, which combine alkyl and aryl substituents, have demonstrated diverse biological activities, including antifungal,32 anticancer,33 and acaricidal effects,34 as well as inhibition of carbonic anhydrase.35

The development of heterocyclic antiviral agents involves an integrated approach combining medicinal chemistry, pharmacology, and structural biology. Advances in synthetic and combinatorial methodologies have accelerated scaffold discovery, while structure–activity relationship (SAR) studies guide the optimization of efficacy and selectivity through insight into key ligand target interactions.36–38

Building on these principles, the fundamental goal of this project was to design and synthesize two innovative series of rationally designed scaffolds, 3a–j and 7a–i, aiming at increasing antibacterial efficacy. We designed and synthesized benzothiazole-based hybrids using a structure-based molecular hybridization strategy. Instead of focusing on a single active moiety, we have directly integrated two distinct antimicrobial pharmacophores, benzothiazole–sulfonate conjugates (3a–i), and via a hydrazide linker to synthesize benzothiazole-hydrazide derivatives (7a–i), which were prepared to enhance antiviral activity and reduce off-target effects. This study highlights their rational design, synthesis, biological evaluation, and potential antiviral mechanisms (Fig. 2).

Fig. 2. The rational design of the targeted benzothiazole–sulfonate conjugates.

Fig. 2

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2. Results and discussion

2.1. Chemistry

A novel series of eighteen benzothiazol–sulfonate conjugates (3a–i) and (7a–i) was designed and synthesized to investigate their potential antiviral properties through systematic structural modification of the benzothiazole-based scaffold. Benzothiazolylphenyl-alkanesulfonate derivatives (3a–i) were prepared via a straightforward condensation reaction between 2-aminobenzenethiol (1) and alkanesulfonyl aryl aldehydes (2a–i) in N,N-dimethylformamide (DMF), catalyzed by sodium metabisulfite (Na2S2O5) under reflux conditions for an appropriate duration, with reaction progress monitored by thin-layer chromatography (TLC). The products precipitated during the hot reaction and were subsequently collected, dried, and recrystallized with suitable solvents to produce compounds 3a–i as colorless microcrystals in good yields (Scheme 1). The structural integrity of all synthesized compounds (3a–i) was confirmed through comprehensive spectroscopic analysis, including infrared spectroscopy (IR), 1H and 13C NMR spectroscopy, and elemental analysis. IR spectroscopy provided key evidence for successful condensation reactions, as indicated by the absence of amino group stretching bands and thiol group signals, confirming complete conversion of the starting materials. Notably, significant SO2 stretching vibrations were observed at 1355–1365 cm−1 and the C Created by potrace 1.16, written by Peter Selinger 2001-2019 N group within the range 1630–1645 cm−1. 1H NMR analysis revealed distinct structural features of the benzothiazolyl alkanesulfonate components, with clear differentiation in the upfield aliphatic region as singlet signals for methyl groups, triplet-quartet patterns for ethyl groups, and triplet-sextet-triplet patterns for propyl groups. Additionally, methoxy groups on vanillin and isovanillin derivatives produced signals at δH = 3.95–4.00 ppm. 13C NMR analysis provided further structural confirmation of the alkanesulfonate carbons (CH3, C2H5, C3H7), appearing in the upfield region at δC = 8.03–52.49 ppm. In comparison, OCH3 groups attached to vanillin and isovanillin rings were observed between δC = 56.26 ppm and δC = 56.47 ppm.

Scheme 1. The synthetic approach towards the preparation of (Benzo[d]thiazol-2-yl)phenyl sulfonate derivatives (3a–i).

Scheme 1

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Furthermore, benzothiazolyl acetohydrazide phenyl sulfonate conjugates (7a–i) were synthesized through a multistep reaction started from the condensation reaction of benzothiazole-2-thiol (4) with methyl 2-bromoacetate in absolute ethanol in the presence of a catalytic amount of K2CO3 to afford methyl benzothiazolylthioacetate (5) that was in situ converted into the corresponding acetohydrazide (6) after being treated with hydrazine hydrate in refluxing ethanol for 3 hours.39 An equimolar quantity of 2-benzothiazolylthio acetohydrazide (6) and appropriate alkane phenyl sulfonates 2a–i in ethanol in the presence of glacial acetic acid were refluxed for a proper time, to eventually afford the desired target conjugates 7a–i (Scheme 2).

Scheme 2. The synthetic pathway to benzothiazolyl acetohydrazide–sulfonate conjugates (7a–i).

Scheme 2

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The chemical structures of derivatives 7a–i were confirmed through various spectroscopic analyses, including IR, 1H and 13C NMR, as well as elemental analysis. The IR spectrum shows that the NH peak appears at 3250–3270 cm−1 and prominent SO2 stretching occurs at 1345–1358 cm−1, along with characteristic C Created by potrace 1.16, written by Peter Selinger 2001-2019 O groups of carbohydrazide, which appear within their respective ranges. The 1H NMR spectra of 7a–i confirmed the presence of alkanesulfonate groups, which appear as singlet signals, triplet-quartet signals, or triplet-sextet-triplet signals in the upfield region for methyl, ethyl, or propyl chains, respectively. Additionally, the 1H NMR spectra of 7a–i show two singlet signals around δH = 3.30 ppm, corresponding to SCH2 protons, while the azomethine protons (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N) are observed in the δH = 8.10–8.17 range. The 13C NMR spectra reveal characteristic peaks corresponding to carbons of the alkanesulfonate groups in the δC = 8.10–52.36 ppm range. The peaks between δC = 39.59 ppm and δC = 40.11 ppm are attributed to methylene carbons (SCH2), while the azomethine carbons (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N) appear between δC = 138.01 ppm and δC = 151.68 ppm. Furthermore, the carbonyl carbon of the acetohydrazide moiety is detected within δC = 165.58–167.12 ppm. Spectral charts for all compounds are provided in the supplementary information (Figures S1–S33).

2.2. Antiviral activity of the tested series 3a–i and 7a–i against H1N1 and HCoV-229E

The antiviral activity of compounds 3a–i and 7a–i was evaluated against influenza A virus (H1N1) and human coronavirus HCoV-229E using a dose–response assay to determine IC50, CC50, and selectivity index (SI) values (Table 1). This approach enabled differentiation between genuine antiviral effects and nonspecific cytotoxicity.

Table 1. Evaluation of Anti-H1N1 and anti-HCoV-229E activities and cytotoxicity of the target compounds 3a–i and 7a–ia.

Sample ID (µg ml−1) Influenza H1N1 virus/MDCK cells HCoV-229E virus/Vero-E6 cells
CC50 IC50 SI CC50 IC50 SI
3a 112.3 8.29 13.5 109.4 18.84 5.8
3b 55.51 22.51 2.4 65.78 25.59 2.57
3c 176.2 9.196 19.2 92.03 23.80 3.86
3d 105.72 15.87 6.66 93.75 26.93 3.48
3e 79.68 9.027 8.82 164.2 15.46 10.62
3f 154 13.9 11.07 209.2 7.629 27.4
3g 154.2 7.63 20.2 137.7 8.279 16.6
3h 72.81 15.07 4.8 211.1 8.810 23.96
3i 105.6 6.7 15.76 40.13 12.47 3.21
7a 51.96 10.49 4.95 199 7.071 28.14
7b 74.39 8.55 8.7 120.3 5.502 21.8
7c 62.24 21.6 2.88 75.81 23.60 3.2
7d 56.08 14.3 3.92 78.72 14.4 5.4
7e 123.1 6.308 19.51 87.25 18.42 4.7
7f 75.86 12.15 6.24 42.26 7.389 5.7
7g 32.44 7.66 4.23 96.72 5.570 17.3
7h 134 16.55 8.09 84.93 7.197 11.8
7i 54.4 11.10 4.9 200.4 13.96 14.3
Favipiravir 76 604 27.31 02.81 38.32 17.60 02.17
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a

TI: therapeutic index, which is a pharmacological term that quantifies the safety of a drug. It is defined as the ratio of the toxic dose (CC50) to the therapeutic dose of a drug (IC50).

Against H1N1, several derivatives exhibited notable antiviral potency with favorable selectivity profiles. In particular, compounds 3a, 3c, 3f, 3g, 3i and 7e demonstrated strong inhibitory activity, reflected by SI values exceeding 10, identifying them as the most promising members of the series. Among these, 3g (SI = 20.20) and 7e (SI = 19.51) showed the highest selectivity compared with SI = 02.81 for Favipiravir, the reference drug used, indicating an optimal balance between antiviral efficacy and cellular safety. Compounds 3d, 3e, 7b, 7f and 7h displayed moderate activity, while the remaining derivatives showed limited inhibition of viral replication.

In the HCoV-229E assay, a higher overall selectivity was observed for several compounds. Derivatives 3f, 3h, 7a, and 7b emerged as the most potent inhibitors, exhibiting high SI values (>20), suggesting strong antiviral effects with minimal cytotoxicity in comparison to SI = 02.17 for the reference drug used, Favipiravir. Additionally, compounds 3e, 3g, 7g, 7h, and 7i showed promising activity, indicating that both structural series retain the capacity to effectively inhibit coronavirus replication. Comparison of the two viral models highlights 3f, 3g, 7a, and 7b as potential broad-spectrum antiviral candidates, as they consistently displayed favorable selectivity across both H1N1 and HCoV-229E. Notably, several compounds demonstrated enhanced selectivity against HCoV-229E relative to H1N1, suggesting possible preferential interactions with coronavirus-related targets or replication pathways. Consequently, the antiviral evaluation identified several compounds with potent and selective activity, particularly derivatives 3f, 3g, 7a and 7e, which demonstrated excellent efficacy and warrant further structure–activity relationship optimization and mechanistic investigations. These findings support the potential of the developed scaffolds as promising candidates for the development of novel antiviral agents. Finally, we acknowledge as a limitation that our study did not include direct benchmarking against oseltamivir or remdesivir, the respective standard-of-care therapies for influenza and HCoV-229E, as our comparator was limited to favipiravir; future studies incorporating side-by-side testing with these approved drugs would provide a more complete assessment of relative antiviral potency.

2.3. Mechanism of anti-H1N1 and anti-HCoV-229E activities

To gain deeper insight into the antiviral behavior of the most active compounds, the mechanism of actions was studied against H1N1 and HCoV-229E at a fixed concentration of 100 µg mL−1. The assay was designed to differentiate whether viral inhibition occurred predominantly at the viral adsorption stage, during intracellular viral replication, or through a direct virucidal effect. The results are summarized in Fig. 3. Against H1N1, all tested compounds (3a, 3c, 3f, 3g, 3i, and 7e) exhibited their highest inhibitory effect during the replication stage, indicating that interference with intracellular viral processes represents the primary mode of action for this series. Notably, compounds 3c and 3i showed the strongest replication inhibition, achieving 90.0% and 92.5% reduction in viral titer, respectively. Derivatives 3g and 7e also demonstrated substantial inhibition (75.0% and 72.0%), whereas 3a and 3f exhibited moderate but significant effects (66.0% and 48.5%). In contrast, inhibition during the adsorption stage was consistently lower, ranging from 24.0% to 55.5%, suggesting a limited ability of these compounds to block viral attachment or entry. The virucidal activity was minimal across all tested derivatives (<30%), indicating that the compounds do not directly inactivate viral particles, but instead exert their antiviral effects after viral entry into host cells (Fig. 3A).

Fig. 3. (A): Mechanisms of action of compounds 3a, 3c, 3f, 3g, 3i, and 7e against H1N1 at 100 µg ml−1. (B): Mechanisms of action of compounds 3e, 3f, 3g, 3h, 7a, 7b, 7g, 7h and 7i against HCoV-229E at 100 µg mL−1.

Fig. 3

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A similar mechanistic trend was observed against HCoV-229E, where the majority of the tested compounds (3e, 3f, 3g, 3h, 7a, 7b, 7g, 7h and 7i) predominantly inhibited the replication stage of the viral life cycle. Compounds 7b and 7a were particularly effective, achieving 86.2% and 78.3% replication inhibition, respectively, followed by 7h (70.5%), 7i (62.8%), and 3f (60.0%). These results are in strong agreement with the high selectivity indices observed for these derivatives in the antiviral screening assay. Moderate inhibition was also detected at the adsorption stage, especially for compounds 7h, 7i, and 3g, which showed adsorption inhibition values exceeding 35%, suggesting a partial contribution of entry inhibition for selected derivatives (Fig. 3B). However, similar to the H1N1 results, virucidal effects remained weak, confirming that the antiviral activity does not arise from direct viral particle destruction.

These outcomes clearly indicate that the synthesized hits act primarily as replication inhibitors against both H1N1 and HCoV-229E. The consistent dominance of replication-stage inhibition across both viral models, combined with minimal virucidal effects, supports the hypothesis that these compounds interfere with conserved intracellular viral targets or host-dependent replication machinery. This mechanistic profile aligns well with the observed selectivity and broad-spectrum antiviral activity of leading candidates such as 3f, 3g, 7a, 7b and 7e, reinforcing their potential as promising antiviral lead compounds.

2.4. SAR structure–activity relationship study

Examination of the antiviral data revealed several structure–activity relationship (SAR) trends that differentiate the two-compound series, namely the benzothiazolyl phenyl sulfonate derivatives (3a–i) and the benzothiazolyl acetohydrazide phenyl sulfonate conjugates (7a–i). Overall, compounds from the 3a–i series, particularly 3a, 3c, 3g and 3i, exhibited stronger antiviral activity and higher selectivity indices against H1N1 compared with many members of the 7a–i series. This trend suggests that the direct linkage between the benzothiazole core and the phenyl sulfonate moiety, without an intervening acetohydrazide spacer, is favorable for anti-influenza activity.

Within the 3a–i series, the nature of the alkyl substituent on the sulfonate group appeared to influence antiviral performance. Derivatives bearing propyl substituents, such as 3i and 3f, generally showed improved selectivity compared with analogues containing shorter ethyl or methyl chains, indicating that moderate hydrophobic extension may enhance interactions with influenza virus–related targets while maintaining acceptable cytotoxicity profiles.

In contrast, several compounds from the 7a–i series demonstrated enhanced activity against HCoV-229E. Notably, derivatives 7a and 7g, which contain methyl sulfonate substituents, exhibited higher selectivity indices against HCoV-229E compared with most members of the 3a–i series. This observation indicates that incorporation of the acetohydrazide linker may favor antiviral activity against coronaviruses, potentially by enabling alternative binding modes or improved adaptability within coronavirus replication-associated environments. Overall, the SAR analysis indicates that removal of the acetohydrazide linker enhances anti-H1N1 activity, whereas its presence favors inhibition of HCoV-229E. These trends suggest virus-dependent differences in compound-target interactions, which warrant further mechanistic investigation.

2.5. In silico study of all the synthesized compounds

2.5.1. The drug likeness and ADMET prediction studies

As a result of the time and money-consuming process of drug discovery, several computational techniques were developed to give us a prediction for some essential physicochemical and pharmacokinetic parameters from the chemical structure of the newly synthesized compound.40 Consequently, all eighteen synthesized derivatives were tested for in silico Drug likeness and ADME (absorption, distribution, metabolism and excretion) prediction study utilizing the Swiss ADME website https://www.swissadme.ch as illustrated in Tables 2 and 341 Moreover, the toxicity (including predicted lethal dose (LD50), organ, and endpoint toxicity) was predicted using ProTox.3 as demonstrated in Table 3.42

Table 2. Swiss ADME website prediction of the drug likeness (Lipinski rule) and some important physicochemical properties of all the final synthesized derivativesa.
Sample ID Lipinski rules Water solubility Pains
MW Heavy atoms Ratable bonds HBA HBD MLOGP TPSA Violation Ali log S Ali class #Alerts
3a 305.37 20 3 4 0 2.68 92.88 0 −5.44 Moderately soluble 0
3b 319.40 21 4 4 0 2.94 92.88 0 −5.83 Moderately soluble 0
3c 333.43 22 5 4 0 3.18 92.88 0 −6.37 Poorly soluble 0
3d 335.39 22 5 4 0 3.18 92.88 0 −6.37 Poorly soluble 0
3e 349.42 23 5 5 0 2.62 102.11 0 −5.76 Moderately soluble 0
3f 363.45 24 6 5 0 2.87 102.11 0 −6.31 Poorly soluble 0
3g 335.40 22 4 5 0 2.37 102.11 0 −5.61 Moderately soluble 0
3h 349.42 23 5 5 0 2.62 102.11 0 −5.76 Moderately soluble 0
3i 363.45 24 6 5 0 2.87 102.11 0 −6.31 Poorly soluble 0
7a 421.51 27 8 6 1 2.06 159.64 0 −6.78 Poorly soluble 0
7b 435.54 28 9 6 1 2.30 159.64 0 −7.17 Poorly soluble 0
7c 449.57 29 10 6 1 2.53 159.64 0 −7.72 Poorly soluble 0
7d 451.54 29 9 7 1 1.78 168.87 0 −6.95 Poorly soluble 0
7e 465.57 30 10 7 1 2.01 168.87 0 −7.33 Poorly soluble 0
7f 479.59 31 11 7 1 2.24 168.87 0 −7.88 Poorly soluble 0
7g 451.54 29 9 7 1 1.78 168.87 0 −6.95 Poorly soluble 0
7h 465.57 30 10 7 1 2.01 168.87 0 −7.33 Poorly soluble 0
7i 479.59 31 11 7 1 2.24 168.87 0 −7.88 Poorly soluble 0
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a

Lipiniski rule: molecular weight ≤ 500; heavy atoms: 20 ≤ atoms ≤ 70; rotatable bonds ≤ 9; lipophilicity: MlogP < 4.15; HBA: hydrogen bond acceptor ≤ 10; HBD: hydrogen bond donor ≤ 5; TPSA (topological polar surface area) 20–130 Å2.

Table 3. The prediction of some pharmacokinetics parameters (ADME), including intestinal absorption, blood–brain barrier permeability, P-glycoprotein substrate, the cytochrome P450 inhibitory profile, the skin permeability and oral bioavailability scorea.
Sample ID GIT BBB Pgp CYP450 inhibitors Skin permeability Bioavailability
Absorption Permeant Substrate CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 Log Kp (cm s−1) Score
3a High No No Yes Yes Yes No No −5.46 0.55
3b High No No Yes Yes Yes No No −5.29 0.55
3c High No No Yes Yes Yes No No −5 0.55
3d High No No Yes Yes Yes No No −5 0.55
3e High No No Yes Yes Yes No No −5.65 0.55
3f High No No Yes Yes Yes No Yes −5.36 0.55
3g High No No Yes Yes Yes No No −5.67 0.55
3h High No No Yes Yes Yes No Yes −5.65 0.55
3i High No No Yes Yes Yes No Yes −5.36 0.55
7a Low No No Yes Yes Yes No Yes −6.22 0.55
7b Low No No Yes Yes Yes No Yes −6.04 0.55
7c Low No No Yes Yes Yes No Yes −5.75 0.55
7d Low No No Yes Yes Yes No Yes −6.42 0.55
7e Low No No Yes Yes Yes No Yes −6.24 0.55
7f Low No No Yes Yes Yes No Yes −5.95 0.55
7g Low No No Yes Yes Yes No Yes −6.42 0.55
7h Low No No Yes Yes Yes No Yes −6.24 0.55
7i Low No No Yes Yes Yes No Yes −5.95 0.55
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a

The higher the negative of log Kp the more the skin impermeability; the score of bioavailability ≥ 0.55 involves a good oral bioavailable compound.

Interestingly, all of the tested derivatives possessed an acceptable drug-like property by obeying Lipinski's rule without any violation. According to Ali's water solubility, all of the compounds were predicted to be poorly soluble except 3a, 3b, 3e, 3g, and 3h, which were assumed to be moderately water soluble. All the derivatives passed the PAIN filter, which measures the presence of any molecular functionality that might interfere with any biological activity.

As shown in Table 3, all the derivatives 3a–i were predicted to be highly absorbed from the gastrointestinal tract. On the other hand, the derivatives 7a–i with the thio-acetyl-hydrazono-methyl linker were expected to be poorly absorbed from the GIT. Favorably, all the derivatives were expected neither to pass through blood–brain barrier nor substrate to the active efflux P-glycoprotein transporter. Concerning the inhibitory profile for some representative cytochrome P450 enzymes, those enzymes that control the compound metabolism and excretion. All of the tested compounds were expected to be good skin penetration, except 7a, 7b, 7d, 7e, 7g and 7h were predicted to be of moderate skin permeability. Also, all of the compounds were anticipated to be oral bioavailable compounds.

As represented in Table 4, all of the derivatives 3a–i were predicted to have a lethal dose (LD50) of 1500 mg Kg−1 but the derivatives 7a–i with the linker moiety had a lower LD50 of 1270 mg kg−1. All of the compounds possessed a good safety profile for neurons and without any carcinogenicity, cytotoxicity or mutagenicity potential except 3b that had a mutagenic potential of 0.51 probability. Unfortunately, all of the compounds were expected to be hepatotoxic with a probability range 0.50–0.56 except 7c, 7f and 7i, which were not hepatotoxic. Compounds 3a–i were predicted to be safe for the kidney; however, the derivatives 7a–i were nephrotoxic with a probability range 0.51–0.56. Concerning the heart, all of the compounds were predicted to be safe except 3e–i, which were cardiotoxic with a probability range of 0.51–0.57. These predictions will be taken into consideration in future in vivo studies to measure the acute and chronic toxicities.

Table 4. Protox.3 predictions of LD50, organ toxicity including liver, neuron, kidney and heart in addition to endpoint toxicity including carcinogenicity, mutagenicity and cytotoxicity.
graphic file with name d6ra02410f-u1.jpg
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2.6. Molecular docking simulation study

2.6.1. H1N1 RNA-dependent RNA polymerase (RdRp)

One of the antiviral targets is the H1N1 RNA-dependent RNA polymerase (RdRp) complex, that is responsible for the influenza virus replication and transcription.43 The RdRp complex consists of three major subunits, including the Endonuclease Polymerase acidic protein (PA), the catalytic subunit Polymerase basic protein (PB1), and Cap-binding subunit Polymerase basic protein (PB2).44 Consequently, the five promising anti-influenza derivatives. 3a, 3c, 3g, 3i, and 7e were selected for the docking study in the three subunits of the H1N1 RdRp complexes (Table 5). The Autodock Vina wizard PyRx software https://pyrx.sourceforge.io was used to carry out the docking study, following the previously reported method.45–47 The PDB code for each protein subunit co-crystalized with native ligand was downloaded from Protein Data Bank https://www.rcsb.org/(PDB ID: 6YA5) for PA subunit, (PDB ID: 6QPG) for PB1 subunit, and (PDB ID: 7AS0) for PB2 subunit.48–50 The proteins were subjected to energy minimization using the YASARA energy minimization server. For validation of the docking, the native ligands Luteolin in PA, Pimodivir in PB2 were re-docked, and the corresponding Root Mean Square Deviation (RMSD) was 0.9 and 0.8 Å respectively.51 On the other hand, the previously reported compounds lead 1 and lead 2, as potent PB1 inhibitors, were used as native ligands, as there was no co-crystallized inhibitor.52 For the generation of 2D and 3D figures, Biovia Discovery Studio 2024 https://discover.3ds.com/was used. The grid box dimensions' table for the docking study was attached in the supplementary file (Table S1).

Table 5. The binding score (kcal mol−1) of 3a, 3c, 3g, 3i, and 7e, in addition to the native ligand in each viral subunit of the RNA-dependent RNA polymerase of H1N1.
Sample ID RNA-dependent RNA polymerase (RdRp)
Endonuclease polymerase acidic protein (PA) (PDB ID: 6YA5) Polymerase basic protein (PB1) catalytic subunit (PDB ID: 6QPG) Cap- binding subunit polymerase basic protein (PB2) (PDB ID: 7ASO)
3a −7.0 −7.5 −7.0
3c −7.5 −7.8 −7.3
3g −7.4 −7.7 −7.0
3i −8.1 −7.9 −7.4
7e −7.4 −7.6 −7.1
Native ligand Luteolin-8.2 Lead 1 = −8.3 Pimodivir-9.0
Lead 2 = −9.8
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2.6.2. RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease subunit (PDB ID: 6YA5)

Interestingly, the 3i derivative possessed excellent binding affinity (−8.1 kcal mol−1), which was very close to the native ligand Luteolin (−8.2 kcal mol−1) in Table 5. As shown in Fig. 4A, this might be due to the formation of a metal-acceptor bond, coordinate covalent bond, between the sulfonyl oxygen and Mn 201, in addition to three hydrogen bonds with Tyr 24 (2.91 Å), Glu 80 (2.69 Å), and the thiazole moiety, as well as Lys 134 (5.10 Å) and the SO functional group. Hydrophobic interactions, including van der Waals, π-alkyl, and Π-Sigma, were also present. The 3i derivative was superimposed over the native ligand Luteolin as depicted in Fig. 4B. The rest of the tested derivatives, along with their 2D interactions, are attached in the supplementary file (Fig. S34). It was noted that a common hydrogen bond interaction exists between the NH of the benzothiazole moiety and Glu 80 in 3a, 3c, and 3g, similar to 3i. Only 3g formed a coordinate covalent bond with Mn 201 as 3i. On the other side, in compound 7e, the presence of thio-acetyl-hydrazono-methyl linker resulted in a slight change in the binding interactions, where the NH of hydrazino moiety formed two hydrogen bonds with Leu 106 and Pro 107, and the propylsulfonate moiety formed two hydrogen bonds with Tyr 24 and Arg 84 residues.

Fig. 4. (A): The predicted 2D-binding interactions of 3i in the Endonuclease Polymerase acidic protein (PA) (PDB code: 6YA5). (B): The expected 3D-binding pose of 3i (cyan) superimposed on the native ligand luteolin (green) in a rose stick endonuclease PA.

Fig. 4

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2.6.3. Polymerase basic protein (PB1) catalytic subunit (PDB ID: 6QPG)

It was reported that the ligand-binding PA-PB1 interface includes Pro 620, Ile 621, Glu 623, Lys 643, Trp 706, Ser 709, and Phe 710 as key amino acid residues.43,44 Compound 3i was predicted to have a binding affinity of 1-fold less than the native lead 1 and 1.2-fold less than the native lead 2, as shown in Table 5. Generally, it was observed that the sulfonyl oxygen atom formed a hydrogen bond with Ser 709 in all tested derivatives, except in 7e, where it formed a hydrogen bond with His 713. Also, the NH of the benzothiazole moiety formed a hydrogen bond with Trp 706 in 3i and 3g, while with Asn 703 in 3c. The phenyl alkyl sulfonate moiety in compound 3i was responsible for several hydrophobic interactions, Pi- Sigma with Trp 706, Pi–Pi with Phe 710, in addition to other van der Waals interactions, as shown in Fig. 5A. Moreover, compound 3i was assumed to be superimposed typically on the native lead 1 and lead 2 as presented in the 3D shape, Fig. 5B. The 2D prediction interactions of the rest tested compounds were attached in the supplementary file (Fig. S35).

Fig. 5. (A) The predicted 2D-binding interactions of 3i in the polymerase basic protein (PB1) (PDB code: 6QPG). (B) The expected 3D-binding pose of 3i (tint) superimposed on the native lead 1 (blue) and lead 2 (green) in a white sticks PB1.

Fig. 5

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2.6.4. Cap-binding subunit polymerase basic protein (PB2) (PDB ID: 7ASO)

As shown in Table 5, compound 3i was predicted to possess a binding affinity of (−7.4 kcal mol−1), which is 1.2-fold less than the native ligand Pimodivir (−9.0 kcal mol−1). As shown in Fig. 6A, the methoxy and o-propylsulfonate moieties formed five hydrogen bonds with Arg 355 and Asn 429, which are common residues in the interactions. Also, the NH of the benzothiazole moiety formed a hydrogen bond with His 357. Moreover, the hydrophobic interactions represented in van der Waals, Pi–Pi with Phe 323 (6.31 Å), Alkyl and Pi–Alkyl with Arg 332, Phe 404, Met 431, and Pi-Sigma with Phe 325. It was observed that His 357 formed Pi–Sulfur interactions in 3i, 3g, and 7e derivatives; the 2D interactions for the rest of the compounds were added in the supplementary file (Fig. S36). Also, 3i, 3g, 7e, and Pimodivir formed hydrogen bonds with Arg 355 and Asn 429, but 3a and 3c formed hydrogen bonds with Asn 429 only. Finally, compound 3i was superimposed on Pimodivir in a 3D plot in Fig. 6B.

Fig. 6. (A) The predicted 2D-binding interactions of 3i in the Cap-binding subunit polymerase basic protein (PB2) (PDB code: 7ASO). (B) The expected 3D-binding pose of 3i (light pink) superimposed on the native ligand Pimodivir (cyan) in a white sticks PB2.

Fig. 6

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In brief, there is a high degree of agreement between the docking results and the viral replication inhibition% biochemical assays, with a higher probability that the promising compound 3i might act by inhibiting the PA endonuclease subunit. Taking into consideration that all the tested derivatives still possessed moderate inhibitory affinities towards the PB1 and PB2 subunits.

2.6.5. HCoV 229E main protease (Mpro, a 3C-like protease, 3CLpro)

One of the promising antiviral coronavirus targets is the main protease Mpro, which is present in its genome, and it processes the viral polyproteins proteolytically in order to produce mature and functioning viral proteins.53 Following the previously reported docking methodology, the protein was downloaded from the Protein Data Bank https://www.rcsb.org/(PDB ID: 8Y4F) complex with Bofutrelvir.54 For validation of the docking, the native ligand Bofutrelvir was re-docked, and the corresponding RMSD was 1.0 Å. The binding affinity scores were presented in Table 6, and the 2D and 3D poses for 7b were illustrated in Fig. 7, and the rest of the compounds were attached in a sup file (Fig. S37).

Table 6. The binding score (kcal mol−1) of 3f, 7a, 7b, 7g, 7h, 7i, in addition to the native ligand Bofutrelvir in HCoV 229E main protease (Mpro).
Sample ID HCoV 229E main protease (Mpro) (PDB ID: 8Y4F)
3f −6.2
7a −6.4
7b −6.5
7g −6.0
7h −6.3
7i −6.3
Native ligand bofutrelvir −8.3
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Fig. 7. A: The predicted 2D-binding interactions of 7b in the HCoV 229E main protease (Mpro) (PDB code: 8Y4F). B: The expected 3D-binding pose of 7b (yellow) superimposed on the native ligand Bofutrelvir (cyan) in a white stick protease Mpro.

Fig. 7

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As shown in Table 6, among the tested derivatives, compound 7b was expected to have the highest binding affinity in protease Mpro with a binding score (−6.5 kcal mol−1), which was 1.2-fold lower than the native ligand Bofutrelvir (−8.3 kcal mol−1). This might be attributed to the failure of our compounds to form a covalent bond with the Cys 144 residue, as the native ligand did. As depicted in Fig. 7a and b, four hydrogen bonds were formed with His 41 (5.04 Å), the hydrazino formed with Glu 165 (3.27 Å) and Leu 166 (6.27 Å), and the NH of the benzothiazole core formed with Glu 165 (3.27 Å). Also, the CO of the carbohydrazide linker formed a carbon–hydrogen bond with Gly 167 (3.49 Å), the phenylalkylsulfonate moiety formed Pi–Sulfur interaction with Cys 144, the phenyl of the benzothiazole moiety formed two Pi–Alkyl interactions with Ile 164 (6.52 Å) and Pro 188 (5.23 Å), in addition to van der Waals hydrophobic interactions. Collectively for compounds 7a, 7g and 7h, their binding interactions were similar, including several hydrogen bonds with His 41, Gln 191, Glu 165, and Glu 167 residues. Compound 3f formed one hydrogen bond with His 41 and the sulfonyl's oxygen atom, and a Pi–Sulfur interaction with Cys 144. Concerning 7i, it formed only three hydrogen bonds with His 41, Glu 165 and Pro 188 with the NH of the benzothiazole core structure. To sum up, the binding scores order of the tested derivatives were in agreement with the in vitro replication inhibition assay results, the lower affinity than the native ligand might due to inability of the synthesized compounds to be covalent inhibitors, although they kept other essential binding interactions that was previously reported by the native ligand including the amino acid residues (His 41, Cys 144, Glu 165). Therefore, one of the mechanisms by which these compounds might inhibit HCoV 229E replication is via the main protease (Mpro) inhibition.

3. Experimental

3.1. General remarks

All chemicals were bought from commercial suppliers. Analytical thin-layer chromatography (TLC) was carried out using UV light visualization on silica gel 60 F245 aluminium plates precoated with silica gel (Merck). Open capillary tubes and a Stuart SMP30 melting point apparatus were used to measure melting points. The National Research Centre's Micro Analytical Laboratory (Cairo, Egypt) and Cairo University's Faculty of Pharmacy in Cairo, Egypt, recorded the elemental analysis and spectral data of the synthesized candidates. A Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a PerkinElmer FT-IR 1650 (spectrophotometer) was used to record infrared spectra (4000–400 cm−1) using KBr pellets. Using DMSO-d6 as a solvent, 1HNMR and 13CNMR spectra were captured on Bruker instruments at 500 (125) MHz. Chemical shifts are expressed in parts per million (ppm) in relation to TMS. Compound purity was assessed by LC-MS, utilizing the area percentage method on the UV trace, which was captured at 254 nm, and it was found to be greater than 95%. A PDA detector, an auto sampler, and a pump make up the Surveyor-LC system. Xcalibur, standard software, was used to operate the system.

3.1.1. General procedure for the synthesis of (Benzo[d]thiazol-2-yl) phenyl sulfonates (3a–i)

To a stirred solution of 2-aminothiophenol (1) (10 mmol) and appropriate alkane phenyl sulfonates 2a–i (10 mmol) in DMF, sodium metabisulfite (Na2S2O5) (12 mmol) was added. The resulting mixture was refluxed for 2 h. After completion of the reaction, as indicated by thin layer chromatography TLC, it was allowed to cool to room temperature before being poured into a beaker containing water (100 mL). The benzothiazole derivatives precipitated out, which were separated and collected by filtration. The products precipitated during the hot reaction and were subsequently collected, dried, and recrystallized with suitable solvents.

3.1.1.1. 4-(Benzo[d]thiazol-2-yl) phenyl methanesulfonate (3a)

Yield: 90%; mp 106–108 °C; IR (KBr) cm−1, ν: 1630 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1356 (SO2); 1H NMR δ (ppm): 3.47 (s, 3H, SO2CH3), 7.57 (ddd, 1H, J = 8.0, 7.5, 1.0 Hz, H-6′), 7.55–7.58 (m, 3H, H-2, H-6 and H-5′), 8.08 (d, 1H, J = 8.0, Hz, H-7′), 8.17 (d, 1H, J = 7.5 Hz, H-4′), 8.21 (dd, 2H, J = 7.0, 2.0 Hz, H-3 and H-5); 13C NMR δ (ppm): 37.64 (SO2CH3), 122.42 (C-4′), 122.99 (C-7′), 123.22 (C-1 and C-6), 125.72 (C-6′), 126.76 (C-5′), 129.05 (C-3 and C-5), 131.79 (C-7′a), 134.67 (C-4), 150.97 (C-1), 153.50 (C-3′a), 165.87 (C-2′); Anal. calc. for C14H11NO3S2 (305.37): C, 55.07; H, 3.63; N, 4.59. Found: C, 55.20; H, 3.74; N, 4.47.

3.1.1.2. 4-(Benzo[d]thiazol-2-yl) phenyl ethanesulfonate (3b)

Yield: 85%; mp 123–125 °C; IR (KBr) cm−1, ν: 1639 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1355 (SO2); 1H NMR δ (ppm): 1.41 (t, 3H, J = 6.0 Hz, SO2CH2CH3), 3.61 (q, 2H, J = 6.0 Hz, SO2CH2CH3), 7.48 (ddd, 1H, J = 6.5, 6.0, 1.0 Hz, H-6′), 7.53 (dd, 2H, J = 7.5, 1.5 Hz, H-2 and H-6), 7.56 (ddd, 1H, J = 6.5, 6.0, 1.0 Hz, H-5′), 8.08 (d, 1H, J = 6.5 Hz, H-7′), 8.16 (d, 1H, J = 6.5 Hz, H-4′), 8.19 (dd, 2H, J = 7.0, 1.5 Hz, H-3 and H-5); 13C NMR δ (ppm): 8.04 (SO2CH2CH3), 44.91 (SO2CH2CH3), 122.41 (C-4′), 122.98 (C-7′), 123.10 (C-1 and C-6), 125.71 (C-6′), 126.76 (C-5′), 129.04 (C-3 and C-5), 131.68 (C-7′a), 134.66 (C-4), 150.82 (C-1), 153.50 (C-3′a), 165.86 (C-2′); Anal. calc. for C15H13NO3S2 (319.39): C, 56.41; H, 4.10; N, 4.39. Found: C, 56.55; H, 4.23; N, 4.53.

3.1.1.3. 4-(Benzo[d]thiazol-2-yl) phenyl propane-1-sulfonate (3c)

Yield: 80%; mp 118–120 °C; IR (KBr) cm−1, ν: 1635 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1360 (SO2); 1H NMR δ (ppm): 1.05 (t, 3H, J = 6.5 Hz, SO2CH2CH2CH3), 1.88 (sextet, 2H, J = 6.5 Hz, SO2CH2CH2CH3), 3.59 (t, 2H, J = 6.5 Hz, SO2CH2CH2CH3), 7.49 (ddd, 1H, J = 6.5, 6.0, 0.5 Hz, H-6′), 7.53 (d, 2H, J = 7.5 Hz, H-2 and H-6), 7.57 (ddd, 1H, J = 6.5, 6.0, 0.5 Hz, H-5′), 8.08 (d, 1H, J = 6.5 Hz, H-7′), 8.17 (d, 1H, J = 6.5 Hz, H-4′), 8.20 (d, 2H, J = 7.5 Hz, H-3 and H-5); 13C NMR δ (ppm): 12.33 (SO2CH2CH2CH3), 16.98 (SO2CH2CH2CH3), 51.57 (SO2CH2CH2CH3), 122.43 (C-4′), 122.98 (C-7′), 123.13 (C-1 and C-6), 125.72 (C-6′), 126.77 (C-5′), 129.05 (C-3 and C-5), 131.67 (C-7′a), 134.66 (C-4), 150.81 (C-1), 153.50 (C-3′a), 165.87 (C-2′); Anal. calc. for C16H15NO3S2 (333.42): C, 57.64; H, 4.53; N, 4.20. Found: C, 57.80; H, 4.41; N, 4.12.

3.1.1.4. 4-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl methanesulfonate (3d)

Yield: 90%; mp 106–108 °C; IR (KBr) cm−1, ν: 1640 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1359 (SO2); 1H NMR δ (ppm): 3.43 (s, 3H, SO2CH3), 4.00 (s, 3H, OCH3), 7.49 (dd, 1H, J = 6.5, 6.0 Hz, H-6′), 7.50 (d, 1H, J = 7.0 Hz, H-6), 7.56 (ddd, 1H, J = 6.5, 6.0, 0.5 Hz, H-5′), 7.70 (dd, 1H, J = 6.5, 1.5 Hz, H-5), 7.85 (d, 1H, J = 1.5 Hz, H-3), 8.10 (d, 1H, J = 6.5 Hz, H-7′), 8.17 (d, 1H, J = 6.5 Hz, H-4′); 13C NMR δ (ppm): 38.52 (SO2CH3), 56.27 (OCH3), 111.32 (C-3), 120.16 (C-6), 122.41 (C-5), 123.00 (C-4′), 124.81 (C-7′), 125.75 (C-6′), 126.78 (C-5′), 132.73 (C-4), 134.73 (C-7′a), 139.92 (C-1), 152.02 (C-2), 153.42 (C-3′a), 166.06 (C-2′); MS (ESI): 336.0 (M++1) (100%); Anal. calc. for C15H13NO4S2 (335.39): C, 53.72; H, 3.91; N, 4.18. Found: C, 53.63; H, 3.79; N, 4.32.

3.1.1.5. 4-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl ethanesulfonate (3e)

Yield: 89%; mp 179–181 °C; IR (KBr) cm−1, ν: 1638 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1362 (SO2); 1H NMR δ (ppm): 1.42 (t, 3H, J = 7.5 Hz, SO2CH2CH3), 3.56 (q, 2H, J = 7.5 Hz, SO2CH2CH3), 4.00 (s, 3H, OCH3), 7.47–7.51 (m, 2H, H-6 and H-6′), 7.57 (ddd, 1H, J = 7.5, 7.0, 1.5 Hz, H-5′), 7.79 (dd, 1H, J = 8.0, 2.0 Hz, H-5), 7.84 (d, 1H, J = 2.0 Hz, H-3), 8.10 (d, 1H, J = 8.0 Hz, H-7′), 8.17 (d, 1H, J = 8.0 Hz, H-4′); 13C NMR δ (ppm): 8.05 (SO2CH2CH3), 45.81 (SO2CH2CH3), 56.26 (OCH3), 111.25 (C-3), 120.15 (C-6), 122.39 (C-5), 122.98 (C-4′), 124.73 (C-7′), 125.73 (C-6′), 126.77 (C-5′), 132.60 (C-4), 134.71 (C-7′a), 139.86 (C-1), 151.95 (C-2), 153.41 (C-3′a), 166.04 (C-2′); Anal. calc. for C16H15NO4S2 (349.42): C, 55.00; H, 4.33; N, 4.01. Found: C, 55.18; H, 4.19; N, 4.15.

3.1.1.6. 4-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl propane-1-sulfonate (3f)

Yield: 88%; mp 191–193 °C; IR (KBr) cm−1, ν: 1645 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1364 (SO2); 1H NMR δ (ppm): 1.05 (t, 3H, J = 6.0 Hz, SO2CH2CH2CH3), 1.90 (sextet, 2H, J = 6.5 Hz, SO2CH2CH2CH3), 3.53 (t, 2H, J = 6.5 Hz, SO2CH2CH2CH3), 3.99 (s, 3H, OCH3), 7.47–7.50 (m, 2H, H-6 and H-6′), 7.57 (dd, 1H, J = 6.5, 6.0 Hz, H-5′), 7.69 (dd, 2H, J = 7.0, 1.5 Hz, H-5), 7.84 (d, 1H, J = 1.5 Hz, H-3), 8.10 (d, 1H, J = 7.0 Hz, H-7′), 8.17 (d, 1H, J = 6.5 Hz, H-4′); 13C NMR δ (ppm): 12.38 (SO2CH2CH2CH3), 17.05 (SO2CH2CH2CH3), 52.49 (SO2CH2CH2CH3), 56.27 (OCH3), 111.24 (C-3), 120.15 (C-6), 122.40 (C-5), 122.99 (C-4′), 124.79 (C-7′), 125.73 (C-6′), 126.77 (C-5′), 132.60 (C-4), 134.72 (C-7′a), 139.86 (C-1), 151.95 (C-2), 153.42 (C-3a'), 166.06 (C-2′); Anal. calc. for C17H17NO4S2 (363.45): C, 56.18; H, 4.71; N, 3.85. Found: C, 56.01; H, 4.58; N, 3.98.

3.1.1.7. 5-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl methanesulfonate (3g)

Yield: 91%; mp 183–185 °C; IR (KBr) cm−1, ν: 1642 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1358 (SO2); 1H NMR δ (ppm): 3.43 (s, 3H, SO2CH3), 3.95 (s, 3H, OCH3), 7.40 (d, 1H, J = 7.0 Hz, H-3), 7.45 (dd, 1H, J = 6.5, 6.0 Hz, H-6′), 7.54 (dd, 1H, J = 6.5, 6.0 Hz, H-5′), 7.00 (d, 1H, J = 1.5 Hz, H-6), 8.03 (dd, 1H, J = 7.5, 1.5 Hz, H-4), 8.05 (d, 1H, J = 6.5 Hz, H-7′), 8.13 (d, 1H, J = 6.5 Hz, H-4′); 13C NMR δ (ppm): 38.39 (SO2CH3), 56.46 (OCH3), 114.29 (C-3), 122.10 (C-6), 122.30 (C-4), 122.68 (C-4′), 125.42 (C-7′), 125.73 (C-6′), 126.67 (C-5′), 127.61 (C-5), 134.39 (C-7′a), 138.09 (C-1), 153.46 (C-2), 153.89 (C-3′a), 165.68 (C-2′); MS (ESI): 336.0 (M++1) (100%); Anal. calc. for C15H13NO4S2 (335.39): C, 53.72; H, 3.91; N, 4.18. Found: C, 53.83; H, 3.77; N, 4.01.

3.1.1.8. 5-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl ethanesulfonate (3h)

Yield: 90%; mp 103–105 °C; IR (KBr) cm−1, ν: 1637 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1361 (SO2); 1H NMR δ (ppm): 1.42 (t, 3H, J = 7.0 Hz, SO2CH2CH3), 3.57 (q, 2H, J = 7.5 Hz, SO2CH2CH3), 3.95 (s, 3H, OCH3), 7.39 (d, 1H, J = 8.5 Hz, H-3), 7.45 (dd, 1H, J = 8.0, 7.0 Hz, H-6′), 7.54 (dd, 1H, J = 8.0, 7.0 Hz, H-5′), 7.96 (s, 1H, H-6), 8.02 (dd, 1H, J = 9.5, 1.5 Hz, H-4), 8.05 (d, 1H, J = 8.0 Hz, H-7′), 8.13 (d, 1H, J = 8.0 Hz, H-4′); 13C NMR δ (ppm): 8.03 (SO2CH2CH3), 45.71 (SO2CH2CH3), 56.43 (OCH3), 114.19 (C-3), 121.98 (C-6), 122.28 (C-4), 122.68 (C-4′), 125.41 (C-7′), 125.70 (C-6), 126.66 (C-5′), 127.47 (C-5), 134.37 (C-7′a), 137.99 (C-1), 153.45 (C-2), 153.81 (C-3′a), 165.66 (C-2′); Anal. calc. for C16H15NO4S2 (349.42): C, 55.00; H, 4.33; N, 4.01. Found: C, 55.17; H, 4.48; N, 4.17.

3.1.1.9. 5-(Benzo[d]thiazol-2-yl)-2-methoxyphenyl propane-1-sulfonate (3i)

Yield: 85%; mp 186–188 °C; IR (KBr) cm−1, ν: 1641 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1357 (SO2); 1H NMR δ (ppm): 1.05 (t, 3H, J = 6.0 Hz, SO2CH2CH2CH3), 1.90 (sextet, 2H, J = 6.5 Hz, SO2CH2CH2CH3), 3.54 (t, 2H, J = 6.0 Hz, SO2CH2CH2CH3), 3.95 (s, 3H, OCH3), 7.38 (d, 1H, J = 7.0 Hz, H-3), 7.45 (dd, 1H, J = 6.5, 6.0 Hz, H-6′), 7.54 (dd, 1H, J = 6.5, 6.0 Hz, H-5′), 7.98 (s, 1H, H-6), 8.12 (d, 1H, J = 7.0 Hz, H-4), 8.04 (d, 1H, J = 7.0 Hz, H-7′), 8.12 (d, 1H, J = 7.0 Hz, H-4′); 13C NMR δ (ppm): 12.38 (SO2CH2CH2CH3), 17.04 (SO2CH2CH2CH3), 52.36 (SO2CH2CH2CH3), 56.45 (OCH3), 114.18 (C-3), 122.07 (C-6), 122.28 (C-4), 122.69 (C-4′), 125.40 (C-7′), 125.70 (C-6′), 126.66 (C-5′), 127.46 (C-5), 134.38 (C-7′a), 137.97 (C-1), 153.46 (C-2), 153.81 (C-3′a), 165.66 (C-2′); Anal. calc. for C17H17NO4S2 (363.45): C, 56.18; H, 4.71; N, 3.85. Found: C, 56.35; H, 4.86; N, 3.69.

3.1.2. General procedure for the synthesis of ((2-(2-(Benzo[d]thiazol-2-ylthio) acetyl)hydrazono)methyl)phenylsulfonates (7a–i)

Equimolar amounts of 2-(benzo[d]thiazol-2-ylthio) acetohydrazide (6) (10 mmol) and appropriate alkane phenyl sulfonates 2a–i (10 mmol) in ethanol (20 mL) and glacial acetic acid (2 drops) were refluxed for 2 h. The solvent was removed, and the reaction mixture was poured into ice-cold water. The crude product obtained was filtered, dried, and crystallized from ethanol.

3.1.2.1. 4-((2-(2-(Benzo[d]thiazol-2-ylthio) acetyl)hydrazono)methyl)phenyl methanesulfonate (7a)

Yield: 89%; mp 201–203 °C; IR (KBr) cm−1, ν: 3250 (NH), 1675 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O),1619 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1350 (SO2); 1H NMR δ (ppm): 3.30 (s, 2H, SCH2), 3.41 (s, 3H, SO2CH3), 7.11 (dd, 1H, J = 8.0, 7.5 Hz, H-5′), 7.30 (dd, 1H, J = 8.0, 7.5 Hz, H-6′), 7.43 (d, 2H, J = 9.0 Hz, H-2 and H-6), 7.49 (d, 1H, J = 8.0 Hz, H-7′), 7.76 (d, 1H, J = 8.0 Hz, H-4′), 7.80 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.17 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.30 (s, 1H, NH); 13C NMR δ (ppm): 36.99 (SO2CH3), 39.59 (SCH2), 121.01 (C-4′), 121.15 (C-6′ and C-7′), 122.20 (C-2 and C-6), 125.45 (C-5′), 127.54 (C-3 and C-5), 128.04 (C-4 and C-7′a), 129.61 (C-3′a), 132.97 (C-1), 149.08 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 166.58 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C17H15N3O4S3 (421.50): C, 48.44; H, 3.59; N, 9.97. Found: C, 48.60; H, 3.75; N, 9.80.

3.1.2.2. 4-((2-(2-(Benzo[d]thiazol-2-ylthio) acetyl)hydrazono)methyl)phenyl ethanesulfonate (7b)

Yield: 81%; mp 182–184 °C; IR (KBr) cm−1, ν: 3255 (NH), 1675 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1632 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1347 (SO2); 1H NMR δ (ppm): 1.39 (t, 3H, J = 7.5 Hz, SO2CH2CH3), 3.30 (s, 2H, SCH2), 3.56 (q, 2H, J = 7.5 Hz, SO2CH2CH3), 7.11 (ddd, 1H, J = 8.0, 7.5, 0.5 Hz, H-5′), 7.30 (ddd, 1H, J = 8.0, 7.5, 0.5 Hz, H-6′), 7.40 (d, 2H, J = 9.0 Hz, H-2 and H-6), 7.43 (d, 1H, J = 8.0 Hz, H-7′), 7.77 (d, 1H, J = 8.0 Hz, H-4′), 7.79 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.16 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.30 (s, 1H, NH); 13C NMR δ (ppm): 8.03 (SO2CH2CH3), 40.11 (SCH2), 44.73 (SO2CH2CH3), 121.53 (C-4′), 121.67 (C-6′ and C-7′), 122.61 (C-2, C-6 and C-5′), 125.96 (C-4 and C-7′a), 128.06 (C-3, C-5 and C-3′a), 133.38 (C-1), 149.44 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 167.09 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); MS (ESI): 436.0 (M++1) (100%); Anal. calc. for C18H17N3O4S3 (435.53): C, 49.64; H, 3.93; N, 9.65. Found: C, 49.80; H, 3.70; N, 9.79.

3.1.2.3. 4-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)phenylpropane-1-sulfonate (7c)

Yield: 79%; mp 157–159 °C; IR (KBr) cm−1, ν: 3260 (NH), 1672 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1632 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1348 (SO2); 1H NMR δ (ppm): 1.04 (t, 3H, J = 7.5 Hz, SO2CH2CH2CH3), 1.86 (sextet, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 3.31 (s, 2H, SCH2), 3.53 (t, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 7.11 (dd, 1H, J = 8.0, 7.5 Hz, H-5′), 7.30 (dd, 1H, J = 8.0, 7.5 Hz, H-6′), 7.40 (d, 2H, J = 9.0 Hz, H-2 and H-6), 7.44 (d, 1H, J = 7.5 Hz, H-7′), 7.76 (d, 1H, J = 7.5 Hz, H-4′), 7.79 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.16 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.28 (S, 1H, NH); 13C NMR δ (ppm): 12.34 (SO2CH2CH2CH3), 16.96 (SO2CH2CH2CH3), 40.11 (SCH2), 51.43 (SO2CH2CH2CH3), 121.53 (C-4′), 121.67 (C-6′and C-7′), 122.63 (C-2, C-6 and C-5′), 125.96 (C-4 and C-7′a), 128.06 (C-3, C-5 and C-3′a), 133.37 (C-1), 149.43 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 167.10 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C19H19N3O4S3 (449.56): C, 50.76; H, 4.26; N, 9.35. Found: C, 50.60; H, 4.19; N, 9.18.

3.1.2.4. 4-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl methanesulfonate (7d)

Yield: 80%; mp 219–220 °C; IR (KBr) cm−1, ν: 3259 (NH), 1668 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1628 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1345 (SO2); 1H NMR δ (ppm): 3.31 (s, 2H, SCH2), 3.37 (s, 3H, SO2CH3), 3.92 (s, 3H, OCH3), 7.11 (dd, 1H, J = 8.0, 7.5 Hz, H-5′), 7.30 (dd, 1H, J = 8.0, 7.5 Hz, H-6′), 7.33 (dd, 1H, J = 8.0, 1.5 Hz, H-5), 7.37 (d, 1H, J = 8.5 Hz, H-6), 7.44 (d, 1H, J = 7.0 Hz, H-7′), 7.50 (d, 1H, J = 1.5 Hz, H-3), 7.77 (d, 1H, J = 7.0 Hz, H-4′), 8.14 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.32 (s, 1H, NH); 13C NMR δ (ppm): 38.38 (SO2CH3), 40.11 (SCH2), 55.94 (OCH3), 110.75 (C-3), 119.15 (C-6), 121.56 (C-4′), 121.69 (C-5 and C-7′), 124.27 (C-5′ and C-6′), 125.97 (C-4, C-7′a), 134.51 (C-1), 138.52 (C-2 and C-3′a), 151.68 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 167.12 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C18H17N3O5S3 (451.53): C, 47.88; H, 3.80; N, 9.31. Found: C, 47.70; H, 3.68; N, 9.17.

3.1.2.5. 4-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl ethanesulfonate (7e)

Yield: 83%; mp 162–164 °C; IR (KBr) cm−1, ν: 3265 (NH), 1668 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1629 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1345 (SO2); 1H NMR δ (ppm): 1.40 (t, 3H, J = 6.5 Hz, SO2CH2CH3), 3.31 (s, 2H, SCH2), 3.51 (q, 2H, J = 6.5 Hz, SO2CH2CH3), 3.91 (S, 3H, OCH3), 7.11 (dd, 1H, J = 7.0, 6.5 Hz, H-5′), 7.30 (dd, 1H, J = 7.0, 6.5 Hz, H-6′), 7.32 (d, 1H, J = 6.5 Hz, H-5), 7.36 (d, 1H, J = 6.5 Hz, H-6), 7.43 (d, 1H, J = 6.0 Hz, H-7′), 7.49 (s, 1H, H-3), 7.77 (d, 1H, J = 6.5 Hz, H-4′), 8.13 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.30 (s, 1H, NH); 13C NMR δ (ppm): 8.06 (SO2CH2CH3), 40.05 (SCH2), 45.65 (SO2CH2CH3), 55.94 (OCH3), 110.68 (C-3), 119.14 (C-6), 121.57 (C-4′), 121.69 (C-5 and C-7′), 124.24 (C-5′ and C-6′), 125.97 (C-4 and C-7′a), 134.39 (C-1), 138.46 (C-2 and C-3′a), 151.63 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 167.11 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C19H19N3O5S3 (465.56): C, 49.02; H, 4.11; N, 9.03. Found: C, 49.18; H, 4.23; N, 9.15.

3.1.2.6. 5-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl propane-1-sulfonate (7f)

Yield: 76%; mp 133–135 °C; IR (KBr) cm−1, ν: 3270 (NH), 1676 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1621 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1355 (SO2); 1H NMR δ (ppm): 1.05 (t, 3H, J = 7.5 Hz, SO2CH2CH2CH3), 1.88 (sextet, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 3.31 (s, 2H, SCH2), 3.48 (t, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 3.92 (s, 2H, OCH3), 7.11 (ddd, 1H, J = 8.0, 7.5, 1.0 Hz, H-5′), 7.30 (ddd, 1H, J = 8.0, 7.5, 1.0 Hz, H-6′), 7.32 (dd, 1H, J = 8.5, 1.5 Hz, H-5), 7.36 (d, 1H, J = 8.0 Hz, H-6), 7.44 (d, 1H, J = 8.0 Hz, H-7′), 7.49 (d, 1H, J = 1.5 Hz, H-3), 7.77 (d, 1H, J = 7.5 Hz, H-4′), 8.13 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.30 (S, 1H, NH); 13C NMR δ (ppm): 12.39 (SO2CH2CH2CH3), 17.04 (SO2CH2CH2CH3), 40.11 (SCH2), 52.36 (SO2CH2CH2CH3), 55.95 (OCH3), 110.67 (C-3), 119.12 (C-6), 121.55 (C-4′), 121.68 (C-5 and C-7′), 124.28 (C-5′ and C-6′), 125.96 (C-4 and C-7′a), 134.38 (C-1), 138.44 (C-2 and C-3′a), 151.63 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-2′), 167.11 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C20H21N3O5S3 (479.58): C, 50.09; H, 4.41; N, 8.76. Found: C, 50.18; H, 4.31; N, 8.60.

3.1.2.7. 5-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl methanesulfonate (7g)

Yield: 86%; mp 178–180 °C; IR (KBr) cm−1, ν: 3263 (NH), 1677 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1621 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1351 (SO2); 1H NMR δ (ppm): 3.31 (s, 2H, SCH2), 3.39 (s, 3H, SO2CH3), 3.89 (s, 3H, OCH3), 7.08 (ddd, 1H, J = 7.5, 7.0, 2.0 Hz, H-5′), 7.26–7.29 (m, 2H, H-3 and H-6′), 7.39 (d, 1H, J = 8.0 Hz, H-7′), 7.60 (d, 1H, J = 1.5 Hz, H-6), 7.60–7.62 (m, 1H, H-4), 7.73 (d, 1H, J = 7.5 Hz, H-4′), 8.10 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.04 (s, 1H, NH); 13C NMR δ (ppm): 38.38 (SO2CH3), 40.07 (SCH2), 56.45 (OCH3), 113.68 (C-3), 117.25 (C-5), 121.07 (C-6), 121.49 (C-4′), 121.51 (C-7′), 125.89 (C-4), 126.89 (C-6′), 127.51 (C-5′), 128.87 (C-7′a), 138.01 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-1), 142.92 (C-2), 149.31 (C-3′a), 152.31 (C-2′), 166.93 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C18H17N3O5S3 (451.53): C, 47.88; H, 3.80; N, 9.31. Found: C, 47.70; H, 3.69; N, 9.18.

3.1.2.8. 5-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl ethanesulfonate (7h)

Yield: 78%; mp 173–175 °C; IR (KBr) cm−1, ν: 3269 (NH), 1677 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1635 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1356 (SO2); 1H NMR δ (ppm): 1.42 (t, 3H, J = 6.5 Hz, SO2CH2CH3), 3.30 (s, 2H, SCH2), 3.52 (q, 2H, J = 6.5 Hz, SO2CH2CH3), 3.90 (S, 3H, OCH3), 7.10 (ddd, 1H, J = 7.5, 7.0, 1.0 Hz, H-5′), 7.27–7.30 (m, 2H, H-3 and H-6′), 7.40 (d, 1H, J = 7.5 Hz, H-7′), 7.62 (d, 1H, J = 1.5 Hz, H-6), 7.62–7.63 (m, 1H, H-4), 7.74 (d, 1H, J = 8.0 Hz, H-4′), 8.11 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.04 (s, 1H, NH); 13C NMR δ (ppm): 8.00 (SO2CH2CH3), 40.09 (SCH2), 45.70 (SO2CH2CH3), 56.40 (OCH3), 113.70 (C-3), 117.28 (C-5), 121.09 (C-6), 121.51 (C-4′), 121.53 (C-7′), 125.91 (C-4), 126.91 (C-6′), 127.53 (C-5′), 128.90 (C-7′a), 138.02 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-1), 142.95 (C-2), 149.35 (C-3′a), 152.33 (C-2′), 166.96 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C19H19N3O5S3 (465.56): C, 49.02; H, 4.11; N, 9.03. Found: C, 49.19; H, 4.22; N, 9.16.

3.1.2.9. 4-((2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)hydrazono)methyl)-2-methoxyphenyl propane-1-sulfonate (7i)

Yield: 82%; mp 198–200 °C; IR (KBr) cm−1, ν: 3266 (NH), 1676 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1635 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1358 (SO2); 1H NMR δ (ppm): 1.06 (t, 3H, J = 7.5 Hz, SO2CH2CH2CH3), 1.90 (sextet, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 3.31 (s, 2H, SCH2), 3.50 (t, 2H, J = 7.5 Hz, SO2CH2CH2CH3), 3.89 (s, 2H, OCH3), 7.09 (ddd, 1H, J = 7.5, 7.0, 1.0 Hz, H-5′), 7.26–7.30 (m, 2H, H-3 and H-6′), 7.41 (d, 1H, J = 8.0 Hz, H-7′), 7.61 (d, 1H, J = 2.0 Hz, H-6), 7.63–7.64 (m, 1H, H-4), 7.75 (d, 1H, J = 8.0 Hz, H-4′), 8.10 (s, 1H, HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 12.05 (s, 1H, NH); 13C NMR δ (ppm): 12.41 (SO2CH2CH2CH3), 17.05 (SO2CH2CH2CH3), 40.11 (SCH2), 52.35 (SO2CH2CH2CH3), 56.24 (OCH3), 113.71 (C-3), 117.29 (C-5), 121.10 (C-6), 121.52 (C-4′), 121.54 (C-7′), 125.92 (C-4), 126.92 (C-6′), 127.54 (C-5′), 128.90 (C-7′a), 138.02 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N and C-1), 142.95 (C-2), 149.34 (C-3′a), 152.34 (C-2′), 166.96 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); Anal. calc. for C20H21N3O5S3 (479.58): C, 50.09; H, 4.41; N, 8.76. Found: C, 50.19; H, 4.30; N, 8.61.

3.1.3. Biological evaluations

The evaluation of antiviral activity for target hybrids (3a–i and 7a–i) against H1N1 and HCoV-229E, along with cytotoxicity assays involving MDCK and Vero E6 cells, was conducted as detailed in prior studies.55–57 This investigation, carried out by the virology laboratory at the National Research Centre in Egypt, utilized antiviral assays employing both human influenza H1N1 and human CoV-229E. The assessment involved the crystal violet and plaque methods to effectively screen for antiviral activity and elucidate the mechanisms of action. Detailed procedural information is available in the SI, ensuring transparency and reproducibility of the methodologies employed.

4. Conclusion

Two related series of benzothiazole–sulfonate conjugates were successfully designed and synthesized and subsequently evaluated for their antiviral potential against influenza A virus (H1N1) and human coronavirus HCoV-229E. The biological results revealed that several derivatives exhibited potent antiviral activity combined with low cytotoxicity, as reflected by favorable selectivity index values. Notably, compound 3g demonstrates the highest selectivity against H1N1, while 3i possessed the topmost viral replication inhibition%. Alternatively, derivative 7a had the highest selectivity against HCoV-229E, and compound 7b had the utmost viral replication inhibition%. Structure–activity relationship analysis indicated that removal of the acetohydrazide linker enhanced anti-H1N1 activity, whereas its presence appeared beneficial for HCoV-229E inhibition, suggesting differences in viral target engagement or intracellular interactions. In silico ADME predictions supported the drug-like properties of the synthesized compounds, and molecular docking studies showed good agreement with the experimental antiviral data. Docking analysis predicted that compounds 3a, 3c, 3g, 3i, and 7e inhibited H1N1 RNA-dependent RNA polymerase complex, in which 3i might exert its antiviral effect through inhibition of the PA endonuclease subunit; weighing that all of the tested compounds still possessed moderate inhibitory affinities towards the PB1 and PB2 subunits. On the other side, compounds 3f, 7a, 7b, 7g, 7h, 7i might inhibit HCoV 229E replication via the main protease (Mpro) inhibition. These findings highlight benzothiazole–sulfonate conjugates as promising antiviral scaffolds and provide valuable insights for further structural optimization and mechanistic investigations aimed at developing novel broad-spectrum antiviral agents.

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

RA-016-D6RA02410F-s001

Acknowledgments

The authors thank National Research Centre, Cairo, Egypt, for supporting this research (project number 13010139).

Data availability

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon a reasonable request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra02410f.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-016-D6RA02410F-s001
RA-016-D6RA02410F-s001.pdf (1.8MB, pdf)

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon a reasonable request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra02410f.

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