Experimental and In Silico Evaluation of New Heteroaryl Benzothiazole Derivatives as Antimicrobial Agents

In this manuscript, we describe the design, preparation, and studies of antimicrobial activity of a series of novel heteroarylated benzothiazoles. A molecular hybridization approach was used for the designing compounds. The in vitro evaluation exposed that these compounds showed moderate antibacterial activity. Compound 2j was found to be the most potent (MIC/MBC at 0.23–0.94 mg/mL and 0.47–1.88 mg/mL) On the other hand, compounds showed good antifungal activity (MIC/MFC at 0.06–0.47 and 0.11–0.94 mg/mL respectively) with 2d being the most active one. The docking studies revealed that inhibition of E. coli MurB and 14-lanosterol demethylase probably represent the mechanism of antibacterial and antifungal activities.


Introduction
The growing problem in the community and in hospitals is resistance to pathogenic bacteria. Thus, the search for novel agents to fight against bacterial resistant is very attractive for scientists.
The antimicrobial potential of benzothiazoles is of great importance against the backdrop of the global aggravating problem of antimicrobial resistance and multidrug resistance that cause significant mortality in the world (about 700,000 annual deaths with the prospect of an increase in more than an order of magnitude) [10].
The antimicrobial activity of benzothiazole derivatives is widely presented in the literature. Thus, Singh et al. [22] synthesized and evaluated the antimicrobial activity of several novel benzothiazole based 4-thiazolidinones. Some compounds appeared to be the very potent against E. coli and C. albicans with MIC values in the range of 15.6-125 microg/mL. Haroun et.al. [5] synthesized new benzothiazole based thiazolidinone and found that all synthesized derivatives expressed better activity than ampicillin against most of the studied strains as well as more than streptomycin against several strains. On the other hand, compounds showed very good antifungal activity higher than reference drugs ketoconazole and bifonazole with very low toxicity (LD50 350-1000 mg/kg). Morsy most of the studied strains as well as more than streptomycin against several strains. On the other hand, compounds showed very good antifungal activity higher than reference drugs ketoconazole and bifonazole with very low toxicity (LD50 350-1000 mg/kg). Morsy et al. [4] evaluated antimicrobial activity of benzothiazole derivatives with MIC for antibacterial and antifungal one at 25-250 mg/mL. Nishad et al. [6] synthesized substituted N-(benzo[d]thiazol-2-yl)-2-chloroacetamides among which compound B4 was the most potent against all the tested strains with low MIC values.
It is noteworthy that there are many drugs with benzothiazole scaffold, such as Ethoxzolamide, a sulfonamide medication acting as carbonic anhydrize inhibitor against glaucoma and duodenal ulcers being a diuretic agent; Frentizole, an FDA-approved immunosuppressive drug, a novel inhibitor of the Aβ-ABAD interaction; Riluzole, a medication for the treatment of amyotrophic lateral sclerosis; and Zopolrestat, an aldose reductase inhibitor for the treatment of antidiabetic drug. [23]. Additionally, there are many benzothiazole derivatives known to be studied in clinical trials [24] (Figure 1). On the other hand, phthalazine core is also mentioned in the literature to possess antimicrobial activity. Mourad et al. [25] prepared a series of phthalazine derivatives and studied their antimicrobial activity towards three bacterial strains. It was found that many of the compounds exhibited excellent inhibition against the tested pathogens. Rayes et al. [26] reported antimicrobial activity of phthalazinedion-based derivatives. Moreover, the antitumor approved drug Dasatinib contains phthalazine moiety. Consequently, the design and development of new benzothiazole-based phthalazine derivatives is a promising option in the creation of novel antimicrobial agents.
Taking all these into account, and as a continuation of our outgoing project on search for new compounds with antimicrobial activity, we synthesized novel derivatives incorporating benzothiazole and substituted phthalazine heterocycle through different linkers in the frame of one molecule. It is known that the combination of two or more molecules in one [27] is a promising strategy for enhancing of the activity as well as diminishing the side effects [28].
Herein we report the synthesis, evaluation of antimicrobial activity, as well as molecular docking studies of new heteroarylated benzothiazole derivatives ( Figure 2). On the other hand, phthalazine core is also mentioned in the literature to possess antimicrobial activity. Mourad et al. [25] prepared a series of phthalazine derivatives and studied their antimicrobial activity towards three bacterial strains. It was found that many of the compounds exhibited excellent inhibition against the tested pathogens. Rayes et al. [26] reported antimicrobial activity of phthalazinedion-based derivatives. Moreover, the antitumor approved drug Dasatinib contains phthalazine moiety. Consequently, the design and development of new benzothiazole-based phthalazine derivatives is a promising option in the creation of novel antimicrobial agents.
Taking all these into account, and as a continuation of our outgoing project on search for new compounds with antimicrobial activity, we synthesized novel derivatives incorporating benzothiazole and substituted phthalazine heterocycle through different linkers in the frame of one molecule. It is known that the combination of two or more molecules in one [27] is a promising strategy for enhancing of the activity as well as diminishing the side effects [28].
Herein we report the synthesis, evaluation of antimicrobial activity, as well as molecular docking studies of new heteroarylated benzothiazole derivatives ( Figure 2).

Chemistry
New heteroarylated benzothiazoles were synthesized according to five routs (Schemes 1-5) and their antimicrobial activity studied against a panel of pathogens.

Chemistry
New heteroarylated benzothiazoles were synthesized according to five routs (Schemes 1-5) and their antimicrobial activity studied against a panel of pathogens.

Chemistry
New heteroarylated benzothiazoles were synthesized according to five routs (Schemes 1-5) and their antimicrobial activity studied against a panel of pathogens.

Antibacterial Activity
Title derivatives were studied for their antibacterial activity against several selected bacterial pathogens by microdilution method. Compounds showed moderate to good potency (MIC/MBC at 0.23 to >3.75 mg/mL and 0.35->3.75 mg/mL, respectively; Table 1) following the order: Table 1. Antibacterial activity of heteroaryl derivatives of benzothiazole (mg/mL).

Antibacterial Activity
Title derivatives were studied for their antibacterial activity against several selected bacterial pathogens by microdilution method. Compounds showed moderate to good potency (MIC/MBC at 0.23 to >3.75 mg/mL and 0.35->3.75 mg/mL, respectively; Table 1) following the order: The most potent among compounds tested appeared 2j with MIC and MBC at 0.23-0.94 mg/mL and 0.47-1.88 mg/mL, respectively, while compound 2f was the less potent. Some compounds demonstrate quite high potency against some bacterial strains. Thus, compounds 2b and 2e exhibit good activity against S. typhimurium with MIC 0.23 mg/mL, while compounds 2g and 2j exhibit good activity against E. coli with the same MIC. Compounds 2d, 2k and 8 were potent against B. cereus (MIC 0.23 mg/mL), whereas 2k also exhibit good activity against L. monocytogenes. On the other hand, activity of compounds Antibiotics 2022, 11, 1654 7 of 21 2a, 2i and 5 against the same strain was a little bit lower (MIC 0.35 mg/mL). En. cloacae appeared to be very sensitive to these derivatives in the contrast to the resistant S. aureus.
According to structure-activity relationships studies the presence of 4-(3,4-dimethylphenyl)-2-methylphthalazin-1(2H)-one connected to benzothiazole via 2-mercapto-N-methylacetamide linker (2j) is beneficial for antibacterial activity. Replacement of 4-(3,4-dimethylphenyl)-2methylphthalazin-1(2H)-one as substituent by 1-phenylphthalazine connected to benzothiazole by S as linker (2c) decreased a little the activity. Introduction of 1-phenylpthalazine N,N-dimethylsulfonic amide as substituent gave compound 2g with less potency in comparison with 2c. N,N-diethyl-4-(phthalazin-1-yl)benzamide as substituent was detrimental not only for the group of compounds with S-linker but for all tested compounds. From all mentioned above, it seems that the important role for antibacterial activity play the substituent of benzothiazole ring as well as the linker.

Antifungal Activity
The evaluation of antifungal activity was performed via a microdilution method, with bifonazole and ketoconazole being used as the reference drugs. According to obtained results (Table 2) all compounds demonstrated good antifungal activity except compound 10. The order of activity can be presented as follows: Compound 2d with MIC/MFC at 0.08-0.17 mg/mL and 0.11-0.23 mg/m, respectively, exhibited the highest potency, whereas compound 10 was the less active.
Several derivatives appeared to be more active than the reference drugs towards some fungal strains. Thus, compounds 2d, 2i, 3b, and 6 showed better potency against T viride compared with ketoconazole and bifonazole with MIC/MFC at 0.06/0.11 mg/mL. Good potency was also expressed in compounds 2a, 2b, 2h, 2i, 2l, 3a, and 5, with minimal inhibitory and fungicidal concentrations at 0.11/0.23 mg/mL, comparable with both reference drugs against T. viride, while 2i, 2m, and 3a were found to be active also against A. niger. On the other side, compounds 2a, 2j, 2m, and 2o were potent against A. versicolor, while 2a was also potent with MIC at 0.11mg/mL against P. cyclopium var. verucosum. It should be mentioned that T. viride demonstrated high sensibility toward our compounds, while A. fumigatus, followed by P. funiculosum. were the most resistant ones.
The structure-activity relationship study showed that the presence of 1-(p-tolyl)phthalazine substituent linked to benzothiazole ring through S-linker (2d) is favorable for antifungal activity. Replacement of 1-(p-tolyl)phthalazine by 2,5-dimethoxy-1,4-phenyl linked to two benzothiazole rings via sulfamethylene linker give less active compound 3b, while introduction of methyl group (3a) instead of methoxy ones (3b) resulted in lesser active compound compared with previous one (3b). It should be mentioned that ten compounds (2b, 2d, 2h, 2i, 2j, 2l, 2m, 2o, 3a, and 6) showed activity better than that of ketoconazole against A. niger mostly, while all compounds exhibited higher activity than ketoconazole against T. viride. Furthermore, derivatives 2b, 2d, 2h, 2i, 2j, 2l, 2m, and 6 were more potent also than bifonazole against T. viride. The general observation is that fungi are more sensitive to tested than bacterial strains. It should be noticed that the response of fungi and bacteria to the compounds tested is different. This behavior is probably due to some differences between bacteria and fungi organization of prokaryotic organisms, organization of DNA genetic material and finally in composition of the cell wall which are made from peptidoglycans (bacteria) and chitin (fungi). Both are prokaryotic organisms, but bacteria are unicellular, while fungi multicellular. On the other hand, despite both containing DNA as genetic material, the genetic material of bacteria is organized in cytoplasm, while in fungi it is organized inside the nucleus. Bacteria do not contain membrane-bound organelles in comparison with fungi which contain membrane-bound. Finally, the cell wall of bacteria is made up of peptidoglycans, whereas the cell wall of fungi is made up of chitin. The only common response of bacteria and fungi to compounds tested was observed for compounds 10 and 2f which were among the less active.

In Silico Studies to Antibacterial Targets
Compounds were docked to different antibacterial targets, aiming for a prediction of possible mechanisms of action.
To this direction, we used the following enzymes for docking studies: responsible for the most common mechanisms of activity of antibacterial agents such as E. coli DNA gyrase, Thymidylate kinase, E. coli Primase, E. coli MurA and E. coli MurB enzymes.
According to the results of the docking studies, the lowest Free Energy of Binding was observed to E. coli MurB (Table 3), suggesting inhibition of this enzyme as putative mechanism of antibacterial activity. One of the most active compounds, 2d, binds E. coli MurB enzyme forming three favorable hydrogen bond interactions. These are between the oxygen atom of COOH group, of compound and residue Ser50 (2.27 Å), and the oxygen atom of the C=O group and Ile173 residue (2.74 Å), and the last one between S atom of the compound and residue Ser116 (3.56 Å). Moreover, hydrophobic interactions between Ile122, Ile110, Ile119, Val52, Ala85 and Ile45 and the compound were detected, contributing to the stability of the complex ligand-enzyme ( Figure 3).
It was observed that the most active compounds bind to MurB in a similar way to FAD, interacting with the residues such as Ser50, Arg213, Arg158 and Ser229 (Figure 3). The similarity in binding mode with FAD is probably the reason of comparable to ampicillin potency of these derivatives. Finally, the docking pose of a known inhibitor of MurB enzyme also co-crystalized with it in the X-ray structure and showed that it binds MurB in a completely different way from our compounds. This inhibitor fit into the binding center of the enzyme away from the binding cavity of substate FAD, while our compounds seem to bind MurB in the FAD cavity of the enzyme, interacting with crucial for the enzyme activity residues (Figures 3  and 4). This observation confirms the better binding energy of our compounds and by extension their higher inhibition over this inhibitor.

In Silico Studies to Antifungal Targets
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans and DNA topoisomerase IV (Table 4) in order to explore the possible mechanism of antifungal activity of compounds.  Finally, the docking pose of a known inhibitor of MurB enzyme also co-crystalized with it in the X-ray structure and showed that it binds MurB in a completely different way from our compounds. This inhibitor fit into the binding center of the enzyme away from the binding cavity of substate FAD, while our compounds seem to bind MurB in the FAD cavity of the enzyme, interacting with crucial for the enzyme activity residues (Figures 3  and 4). This observation confirms the better binding energy of our compounds and by extension their higher inhibition over this inhibitor. It was found that the most active compound, 2d, binds the enzyme alongside the heme group, interacting with it throughout its benzene ring forming aromatic and hydrophobic interactions. The most active compound 2d binds the 14a-lanosterole demethylase enzyme at the side of the heme group, forming aromatic and hydrophobic interactions with its benzene ring. Moreover, hydrophobic interactions between Tyr118, Leu121, Tyr122, Thr311, Leu376, Phe380, Met508 and the compound were detected. Aromatic interaction with the heme group was also observed with the benzene ring of ketoconazole (Figures 5 and 6). This property may account for the good antifungal activity of compound 2d.

In Silico Studies to Antifungal Targets
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans and DNA topoisomerase IV (Table 4) in order to explore the possible mechanism of antifungal activity of compounds. It was found that the most active compound, 2d, binds the enzyme alongside the heme group, interacting with it throughout its benzene ring forming aromatic and hydrophobic interactions.
The most active compound 2d binds the 14a-lanosterole demethylase enzyme at the side of the heme group, forming aromatic and hydrophobic interactions with its benzene ring. Moreover, hydrophobic interactions between Tyr118, Leu121, Tyr122, Thr311, Leu376, Phe380, Met508 and the compound were detected. Aromatic interaction with the heme group was also observed with the benzene ring of ketoconazole (Figures 5 and 6). This property may account for the good antifungal activity of compound 2d.

Drug Likeness
The bioavailability and drug-likeness scores of all compounds are shown in Table 5. According to prediction results, the bioavailability score of all compounds was about 0.55. Moreover, all compounds displayed good-to-excellent Drug-likeness scores (−0.60-0.79). Figure 7 presents the bioavailability radar of most active compound 2j. The best in the insilico predictions results was achieved for compound 5 with a Drug-likeness score of 0.79 and with no violation of any rule. According to predicted results all compounds except 2g, 2h, 2j, 2m, 2n, 3a, and 3b can be orally absorbed since their TPSA are < 120 Å.

Drug Likeness
The bioavailability and drug-likeness scores of all compounds are shown in Table 5. According to prediction results, the bioavailability score of all compounds was about 0.55. Moreover, all compounds displayed good-to-excellent Drug-likeness scores (−0.60-0.79). Figure 7 presents the bioavailability radar of most active compound 2j. The best in the insilico predictions results was achieved for compound 5 with a Drug-likeness score of 0.79 and with no violation of any rule. According to predicted results all compounds except 2g, 2h, 2j, 2m, 2n, 3a, and 3b can be orally absorbed since their TPSA are < 120 Å.

Drug Likeness
The bioavailability and drug-likeness scores of all compounds are shown in Table 5. According to prediction results, the bioavailability score of all compounds was about 0.55. Moreover, all compounds displayed good-to-excellent Drug-likeness scores (−0.60-0.79). Figure 7 presents the bioavailability radar of most active compound 2j. The best in the in-silico predictions results was achieved for compound 5 with a Drug-likeness score of 0.79 and with no violation of any rule. According to predicted results all compounds except 2g, 2h, 2j, 2m, 2n, 3a, and 3b can be orally absorbed since their TPSA are < 120 Å.

Chemistry-General Information
NMR 1 H spectra of all compounds were recorded on a spectrometer Bruker 400 (400 MHz); for compounds 2a, 2b, 5-on Bruker AC-300 in DMSO-d6 and spectra are presented in Supplementary Material File S1. Chemical shifts of nuclei 1 H were measured relatively the residual signals of deuteron solvent (δ = 2.50 ppm). Coupling constants (J) are reported in Hz. Τhe assignment was based on 2D NMR techniques. Melting points were determined using the Fisher-Johns Melting Point Apparatus (Fisher Scientific) and are uncorrected. Elemental analysis was performed by the classical method of microanalysis. The

Chemistry-General Information
NMR 1 H spectra of all compounds were recorded on a spectrometer Bruker 400 (400 MHz); for compounds 2a, 2b, 5-on Bruker AC-300 in DMSO-d 6 and spectra are presented in Supplementary Material File S1. Chemical shifts of nuclei 1 H were measured relatively the residual signals of deuteron solvent (δ = 2.50 ppm). Coupling constants (J) are reported in Hz. The assignment was based on 2D NMR techniques. Melting points were determined using the Fisher-Johns Melting Point Apparatus (Fisher Scientific) and are uncorrected. Elemental analysis was performed by the classical method of microanalysis. The reaction and purity of the obtained compounds were monitored by TLC (plates with Al 2 O 3 III activity grade, eluent CHCl 3 , development of TLC plates by exposition to iodine vapors in "iodine chamber"). The solvents were purified according to standard procedures. The starting compounds-4-substituted 1-chlorophthalazine (for 2b-2o)-were provided by InterBioscreen Ltd. (Russia); benzo[d]thiazole-2-thiol 1 (for 2a, 3a, 3b), benzo[d]thiazole-6-carboxylic acid 7 (for 8), and 4-methyl-1-oxophthalazine-2(1H)-carboxylic acid 9 (for 10) are commercially available. L, 2-(Piperidin-4-yl)benzo[d]thiazole was obtained similarly to the procedure described in [29,30]