Synthesis of N-(6-Arylbenzo[d]thiazole-2-acetamide Derivatives and Their Biological Activities: An Experimental and Computational Approach

A new series of N-(6-arylbenzo[d]thiazol-2-yl)acetamides were synthesized by C-C coupling methodology in the presence of Pd(0) using various aryl boronic pinacol ester/acids. The newly synthesized compounds were evaluated for various biological activities like antioxidant, haemolytic, antibacterial and urease inhibition. In bioassays these compounds were found to have moderate to good activities. Among the tested biological activities screened these compounds displayed the most significant activity for urease inhibition. In urease inhibition, all compounds were found more active than the standard used. The compound N-(6-(p-tolyl)benzo[d]thiazol-2-yl)acetamide was found to be the most active. To understand this urease inhibition, molecular docking studies were performed. The in silico studies showed that these acetamide derivatives bind to the non-metallic active site of the urease enzyme. Structure-activity studies revealed that H-bonding of compounds with the enzyme is important for its inhibition.


Introduction
Benzothiazoles consist of a benzene ring fused with a thiazole ring. Various benzothiazole derivatives serve as drugs, dyes and industrial chemicals [1][2][3]. Benzothiazoleand its derivatives such as esters have also been reported as active against Gram-positive and Gram-negative bacteria Benzothiazole derivatives have also found to possess anticancer, antifungal and antibacterial activities [5,6]. 2-Aminobenzothiazole and a number of other aminobenzothiazole derivatives have been reported as muscle relaxants [7,8]. A literature survey performed for the current study revealed that 6-substituted-2-aminobenzothiazole derivatives such as 6-methyl, 6-methoxy, 6-ethoxy and 6-isopropoxy show antibacterial, anti-inflammatory and analgesic properties [9]. Various other derivatives are found to be cytotoxic against various tumors [10,11].
Suzuki cross coupling reactions are remarkable methods for C-C bond formation, utilized for the synthesis of agrochemicals, advanced materials and pharmaceuticals at both the industrial and laboratory scale [12][13][14][15][16][17].
The purpose of this study was to synthesize novel N- (6-arylbenzo[d]thiazol-2-yl)acetamides employing the Pd(0)-catalyzed Suzuki cross coupling methodology. This article describes our optimized experiments for the synthesis of N-protected 6-bromobenzothiazoles. The biological activities of these newly synthesized molecules were studied with the intention to explore their potential as future drugs. We investigated urease inhibition, nitric oxide scavenging, haemolytic and antibacterial activities. Molecular docking studies were performed to determine how they bind to the urease enzyme. To the best of our knowledge, all the studies reported in the current manuscript have not been reported in the literature to date.

Chemistry
Majo et al. reported low to moderate yielding one-step Suzuki cross coupling reactions using various boronic acids/ester with 2-bromobenzothiazole under thermal conditions [18]. We have previously reported Pd(0)-catalyzed reactions of 2-amino-6-bromobenzothiazole with different arylboronic pinacol esters/arylboronic acids using Suzuki cross coupling methodology with moderate yields [19]. We have not been able to achieve better yields as the amino moiety present in the benzothiazole molecule is basic and nucleophilic. In the current study, we have tried to enhance the yield of the synthesized molecules. In order to achieve high yields, the amino group was protected via acylation, which led to substantially enhanced yields of the products 3a-3h ( Figure 1) compared to unprotected benzothiazole derivatives as reported in literature [19]. Furthermore, we also optimized other reaction parameters like catalyst loading, solvent, temperature and base used in the reactions producing 3a-3h. Thus, we tried various solvents like toluene, dimethylformamide (DMF) and 1,4-dioxane at different temperatures (80-100 °C). It was noted (in Table 1) that the solvent has an effect on overall reaction yield. 1,4-Dioxane was found to be the best solvent because of its better solvation of the reactants. In our studies, these cross coupling reactions progressed efficiently, even in the presence of known sensitive groups such as CN, to give the desired products in very good yields. Finally after optimization, we investigated the coupling of 2 with different arylboronic pinacol esters/acids in the presence of Pd(PPh3)4 as catalyst.
Product 3a was prepared with 80% yield, when 2 was coupled with phenyl boronic acids under the set reaction conditions. The highest yield obtained in this series of reactions (85%) corresponded to product 3b. Product 3h was also obtained in excellent yield (83%) with this cross coupling method. It was seen that the product 3c with an electron withdrawing moiety showed a high yield (81%). Our studies showed that overall the acetylated 2-amino-6-bromobenzothiazole (2) gave good yields in these coupling reactions. Furthermore, we also optimized other reaction parameters like catalyst loading, solvent, temperature and base used in the reactions producing 3a-3h. Thus, we tried various solvents like toluene, dimethylformamide (DMF) and 1,4-dioxane at different temperatures (80-100˝C). It was noted (in Table 1) that the solvent has an effect on overall reaction yield. 1,4-Dioxane was found to be the best solvent because of its better solvation of the reactants. In our studies, these cross coupling reactions progressed efficiently, even in the presence of known sensitive groups such as CN, to give the desired products in very good yields. Finally after optimization, we investigated the coupling of 2 with different arylboronic pinacol esters/acids in the presence of Pd(PPh 3 ) 4 as catalyst.
Product 3a was prepared with 80% yield, when 2 was coupled with phenyl boronic acids under the set reaction conditions. The highest yield obtained in this series of reactions (85%) corresponded to product 3b. Product 3h was also obtained in excellent yield (83%) with this cross coupling method.
It was seen that the product 3c with an electron withdrawing moiety showed a high yield (81%). Our studies showed that overall the acetylated 2-amino-6-bromobenzothiazole (2) gave good yields in these coupling reactions.

Urease Inhibitory Activity
Urease is a nickel-containing metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbamate, which further decomposes to yield a second equivalent of ammonia and carbon dioxide [20]. Bacterial ureases have been reported as an important virulence factor in the development of many harmful clinical conditions for human and animal health. Urease is directly involved in the formation of infectious stones and contributes to pathogenesis [21]. It is the major cause of pathogenesis induced by Helicobacter pylori, which plays an important role in peptic ulcers and may lead to stomach cancer. In recent years, a number of compounds have been proposed as urease inhibitors [22,23], which are considered interesting new targets for anti-ulcer drugs and for the treatment of infections caused by urease producing bacteria [24].
In the current study, urease inhibitory activity assays were performed following a previously reported protocol [25]. Thiourea was used as standard in the assay with an IC 50 value of 23.1 µg/mL. All of the synthesized benzothiazole derivatives were examined for their urease inhibitory activities at concentration of 15 and 40 µg/mL. All of our synthesized compounds exhibit good to excellent urease inhibitory activities (

Urease Inhibitory Activity
Urease is a nickel-containing metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbamate, which further decomposes to yield a second equivalent of ammonia and carbon dioxide [20]. Bacterial ureases have been reported as an important virulence factor in the development of many harmful clinical conditions for human and animal health. Urease is directly involved in the formation of infectious stones and contributes to pathogenesis [21]. It is the major cause of pathogenesis induced by Helicobacter pylori, which plays an important role in peptic ulcers and may lead to stomach cancer. In recent years, a number of compounds have been proposed as urease inhibitors [22,23], which are considered interesting new targets for anti-ulcer drugs and for the treatment of infections caused by urease producing bacteria [24].
In the current study, urease inhibitory activity assays were performed following a previously reported protocol [25]. Thiourea was used as standard in the assay with an IC50 value of 23.1 μg/mL. All of the synthesized benzothiazole derivatives were examined for their urease inhibitory activities at concentration of 15 and 40 μg/mL. All of our synthesized compounds exhibit good to excellent urease inhibitory activities (  Each values is mean ± Standard deviation of three parallel measurements.

Urease Inhibitory Activity
Urease is a nickel-containing metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbamate, which further decomposes to yield a second equivalent of ammonia and carbon dioxide [20]. Bacterial ureases have been reported as an important virulence factor in the development of many harmful clinical conditions for human and animal health. Urease is directly involved in the formation of infectious stones and contributes to pathogenesis [21]. It is the major cause of pathogenesis induced by Helicobacter pylori, which plays an important role in peptic ulcers and may lead to stomach cancer. In recent years, a number of compounds have been proposed as urease inhibitors [22,23], which are considered interesting new targets for anti-ulcer drugs and for the treatment of infections caused by urease producing bacteria [24].
In the current study, urease inhibitory activity assays were performed following a previously reported protocol [25]. Thiourea was used as standard in the assay with an IC50 value of 23.1 μg/mL. All of the synthesized benzothiazole derivatives were examined for their urease inhibitory activities at concentration of 15 and 40 μg/mL. All of our synthesized compounds exhibit good to excellent urease inhibitory activities (  Each values is mean ± Standard deviation of three parallel measurements.   Urease enzyme has an active binding site and it was believed that the newly synthesized benzothiazole derivatives have the capability to bind to these active sites of urease enzyme. In this way the hydrolysis of enzyme is stopped and activity of enzyme is inhibited. Compound 3b showed the highest urease inhibition activity (90.51˘0. 19) with an IC 50 value of 16.5 µg/mL at 40 µg/mL.
Compounds 3h and 3c exhibited excellent urease inhibitory activities (90.07˘0.20 and 90.33˘0.20 at 40 µg/mL) with IC 50 values of 17.2 µg/mL and 18.4 µg/mL, respectively. Molecules 3a, 3d, 3e, 3f and 3g exhibited good urease inhibition with IC 50 values of 18.6, 17, 17.2, 19.2 and 18.9 µg/mL, respectively. Notably we observed that the presence of electron donating methyl functional groups produced high urease inhibition. We also noted that different functional groups are responsible for variable antiurease activities of the compounds.

In Silico Studies with Urease
To understand the binding of the synthesized compounds with urease in-silico studies were performed. Only compounds with IC 50 values were analyzed for docking studies using the freely available software AutoDock 4.2 and others as described in the Experimental Section. All the compounds were screened at different sites (A & B) of the enzyme. The nickel-containing catalytic site A is the most commonly tested site in the literature [26], while site B is less commonly targeted [20,27]. Our compounds bind more strongly to Site B than to site A. The docking studies results were compared with the experimental results and listed in Tables 3 and 4. Binding energy, inhibition constant and moldock scores are reported in Table 3, whereas the moldock H-binding energy and number of H-bonding interactions between the test compounds and enzyme are reported in Table 4.  The number of H-bonding interactions was estimated from the most stable complex formed between the test compound and the enzyme. We found a linear correlation between the experimental IC 50 values and the calculated binding energy, as shown in Figure 3. This linear correlation of results is in agreement with the data reported in the literature [26,28]. More interestingly a better correlation was found between IC 50 values and the moldock H-binding energy ( Figure 4).
is in agreement with the data reported in the literature [26,28]. More interestingly a better correlation was found between IC50 values and the moldock H-binding energy ( Figure 4). These results show that the H-bonding is more important, in urease inhibition mechanism, than other factors involved in this biological reaction. Compound 3b, with the highest in vitro activity, is represented here for modeling analysis and its most active conformations are explained in the following paragraphs. As urease is a nickel-dependent enzyme, the active site A of the enzyme shows weak hydrophobic interaction with compound 3b therefore, a drug with hydrophobic substituents would be able to bind strongly as it would project into the hydrophobic grooves of the enzyme and thus effectively inhibit its activity. The LIGPLOT interaction images show that compound 3b has a total of seven interactions with enzyme site A (Figure 5a). The amino acids His593, Met637, Ala636, Gln635, Met588 and Asp494 form hydrophobic interactions with compound 3b. The hydrophobic interactions favor ligand binding with proteins having metal ions. Furthermore, the study showed hydrogen bonding (2.66 Å, N-H-O type) between compound 3b and PO4 group of the enzyme nickel catalytic site A. Figure 6a represents the most interacting conformation of 3b in the active pocket (site A) of the enzyme at the electrostatic surface. Figure 6 is generated by Molegro Molecular viewer and analyzed by moldock score. The moldock analysis shows that there are two H-bonding interactions in both cases these are also reported in Table 4. These results show that the H-bonding is more important, in urease inhibition mechanism, than other factors involved in this biological reaction. Compound 3b, with the highest in vitro activity, is represented here for modeling analysis and its most active conformations are explained in the following paragraphs. As urease is a nickel-dependent enzyme, the active site A of the enzyme shows weak hydrophobic interaction with compound 3b therefore, a drug with hydrophobic substituents would be able to bind strongly as it would project into the hydrophobic grooves of the enzyme and thus effectively inhibit its activity. The LIGPLOT interaction images show that compound 3b has a total of seven interactions with enzyme site A (Figure 5a). is in agreement with the data reported in the literature [26,28]. More interestingly a better correlation was found between IC50 values and the moldock H-binding energy (Figure 4). These results show that the H-bonding is more important, in urease inhibition mechanism, than other factors involved in this biological reaction. Compound 3b, with the highest in vitro activity, is represented here for modeling analysis and its most active conformations are explained in the following paragraphs. As urease is a nickel-dependent enzyme, the active site A of the enzyme shows weak hydrophobic interaction with compound 3b therefore, a drug with hydrophobic substituents would be able to bind strongly as it would project into the hydrophobic grooves of the enzyme and thus effectively inhibit its activity. The LIGPLOT interaction images show that compound 3b has a total of seven interactions with enzyme site A (Figure 5a). The amino acids His593, Met637, Ala636, Gln635, Met588 and Asp494 form hydrophobic interactions with compound 3b. The hydrophobic interactions favor ligand binding with proteins having metal ions. Furthermore, the study showed hydrogen bonding (2.66 Å, N-H-O type) between compound 3b and PO4 group of the enzyme nickel catalytic site A. Figure 6a represents the most interacting conformation of 3b in the active pocket (site A) of the enzyme at the electrostatic surface. Figure 6 is generated by Molegro Molecular viewer and analyzed by moldock score. The moldock analysis shows that there are two H-bonding interactions in both cases these are also reported in Table 4. The amino acids His593, Met637, Ala636, Gln635, Met588 and Asp494 form hydrophobic interactions with compound 3b. The hydrophobic interactions favor ligand binding with proteins having metal ions. Furthermore, the study showed hydrogen bonding (2.66 Å, N-H-O type) between compound 3b and PO 4 group of the enzyme nickel catalytic site A. Figure 6a represents the most interacting conformation of 3b in the active pocket (site A) of the enzyme at the electrostatic surface. Figure 6 is generated by Molegro Molecular viewer and analyzed by moldock score. The moldock analysis shows that there are two H-bonding interactions in both cases these are also reported in Table 4. All of the synthesized compounds 3a-3h show better interaction at site B. The LIGPLOT interaction diagram of compound 3b illustrates that this inhibitor has better interactions with the protein. The LIGPLOT interaction images show that the compound 3b has a total of eight interactions with enzyme site B (Figure 5b). The amino acids Lys208, Asp206, Thr158, Glu254, Phe182, Lys156 and Asp183 form cationic-π interactions with compound 3b, while Glu252 interacts via hydrogen bonding (2.84Å, N-H-O type) with compound 3b. Figure 5b presents the most interacting conformation of compound 3b in the active pocket of the enzyme (site B). The diagram shows that the enzyme provides enough space for the accommodation of 3b inside the pocket. The backbone dose sterically favors the 3b molecule to interact with catalytic site.
As shown in Figure 4, a strong correlation with experimental results is found between the IC50 values and H-bonding data calculated by moldock in the Molegero docking software at site B of enzyme. In the moldock analysis for 3b at site A two H-bonding interactions (N-H-O and N-H-N type) were observed between 3b and PO4844, His593, respectively, while two strong H-bonding interactions between 3b and enzyme residues Glu252 and Lys156 were observed at site B. The H-bonding distances and moldock scores for all compounds, at site B, are listed in Table 4. Figure 7 shows All of the synthesized compounds 3a-3h show better interaction at site B. The LIGPLOT interaction diagram of compound 3b illustrates that this inhibitor has better interactions with the protein. The LIGPLOT interaction images show that the compound 3b has a total of eight interactions with enzyme site B (Figure 5b). The amino acids Lys208, Asp206, Thr158, Glu254, Phe182, Lys156 and Asp183 form cationic-π interactions with compound 3b, while Glu252 interacts via hydrogen bonding (2.84Å, N-H-O type) with compound 3b. Figure 5b presents the most interacting conformation of compound 3b in the active pocket of the enzyme (site B). The diagram shows that the enzyme provides enough space for the accommodation of 3b inside the pocket. The backbone dose sterically favors the 3b molecule to interact with catalytic site.
As shown in Figure 4, a strong correlation with experimental results is found between the IC 50 values and H-bonding data calculated by moldock in the Molegero docking software at site B of enzyme. In the moldock analysis for 3b at site A two H-bonding interactions (N-H-O and N-H-N type) were observed between 3b and PO 4 844, His593, respectively, while two strong H-bonding interactions between 3b and enzyme residues Glu252 and Lys156 were observed at site B. The H-bonding distances and moldock scores for all compounds, at site B, are listed in Table 4. Figure 7 shows All of the synthesized compounds 3a-3h show better interaction at site B. The LIGPLOT interaction diagram of compound 3b illustrates that this inhibitor has better interactions with the protein. The LIGPLOT interaction images show that the compound 3b has a total of eight interactions with enzyme site B (Figure 5b). The amino acids Lys208, Asp206, Thr158, Glu254, Phe182, Lys156 and Asp183 form cationic-π interactions with compound 3b, while Glu252 interacts via hydrogen bonding (2.84Å, N-H-O type) with compound 3b. Figure 5b presents the most interacting conformation of compound 3b in the active pocket of the enzyme (site B). The diagram shows that the enzyme provides enough space for the accommodation of 3b inside the pocket. The backbone dose sterically favors the 3b molecule to interact with catalytic site.
As shown in Figure 4, a strong correlation with experimental results is found between the IC50 values and H-bonding data calculated by moldock in the Molegero docking software at site B of enzyme. In the moldock analysis for 3b at site A two H-bonding interactions (N-H-O and N-H-N type) were observed between 3b and PO4844, His593, respectively, while two strong H-bonding interactions between 3b and enzyme residues Glu252 and Lys156 were observed at site B. The H-bonding distances and moldock scores for all compounds, at site B, are listed in Table 4. Figure 7 shows the H-bonding interactions of all of the compounds (except 3h) with the active residue of urease enzyme at site B. Compound 3h does not show any H-bonding interaction with the active site residue. In the linear correlation with in vitro IC50, stronger H-bonding is found for compounds with lower IC50 value. All the compounds have H-bonding distances in the 2.696 Å-3.544 Å range and H-bonding energies in the −1.674-−3.45 a.u. range.

Nitric Oxide Scavenging Percentage Assay
The literature contains reports on the antioxidant activities of 6-flourobenzothiazole-substituted triazoles using DPPH assays [29]. A survey of the literature showed that benzothiazole molecules along with pyrozoline rings showed the highest antioxidant activities. Having a phenyl ring on the pyrozoline increased the antioxidant activity in the ferric ion reduction and DPPH solution methods [30]. Our newly synthesized N-protected benzothiazole derivatives exhibit nitric oxide scavenging activities. Ascorbic acid was used as a standard in the assay with 38.5 ± 0.16 and 84.1 ± 0.12 percent nitric oxide scavenging at 20 μg/mL and at 50 μg/mL with an IC50 value of 50.43 μg/mL. Synthesized compounds with their calculated IC50 values are listed in Table 5 (Figures 8 and 9).
It was found that acetyl-protected amino group products were more active in the nitric oxide scavenging assay, than the previously reported non-acetylated compounds [19]. Molecules 3a, 2b, 3e, 3g and 3h were found to be the most active for nitric oxide scavenging activity, with percentage inhibitions of 57.75 ± 0.12, 69 ± 0.12, 55 ± 0.31, 51.25 ± 0.15 and 60.5 ± 0.1 at 40μg/mL with IC50 values of 32.7,26.4, 37.1, 39.1 and 32.3, respectively. Compound 3f, however, was found to be inactive in the nitric oxide scavenging assay. We are unable to account for this inactivity.

Nitric Oxide Scavenging Percentage Assay
The literature contains reports on the antioxidant activities of 6-flourobenzothiazole-substituted triazoles using DPPH assays [29]. A survey of the literature showed that benzothiazole molecules along with pyrozoline rings showed the highest antioxidant activities. Having a phenyl ring on the pyrozoline increased the antioxidant activity in the ferric ion reduction and DPPH solution methods [30]. Our newly synthesized N-protected benzothiazole derivatives exhibit nitric oxide scavenging activities. Ascorbic acid was used as a standard in the assay with 38.5˘0.16 and 84.1˘0.12 percent nitric oxide scavenging at 20 µg/mL and at 50 µg/mL with an IC 50 value of 50.43 µg/mL. Synthesized compounds with their calculated IC 50 values are listed in Table 5 (Figures 8 and 9).
It was found that acetyl-protected amino group products were more active in the nitric oxide scavenging assay, than the previously reported non-acetylated compounds [19]. Molecules 3a, 2b, 3e, 3g and 3h were found to be the most active for nitric oxide scavenging activity, with percentage inhibitions of 57.75˘0.12, 69˘0.12, 55˘0.31, 51.25˘0.15 and 60.5˘0.1 at 40µg/mL with IC 50 values of 32.7,26.4, 37.1, 39.1 and 32.3, respectively. Compound 3f, however, was found to be inactive in the nitric oxide scavenging assay. We are unable to account for this inactivity. Each value is mean˘Standard deviation of three parallel measurements. NC stands for not calculated due to less activity.

Haemolytic Activity
The haemolytic activity of benzothiazole derivatives has already been reported. A literature survey reveals that amino-substituted derivatives of benzothiazole have high cytotoxicites. Benzothiazole compounds with halogen substitutions show cytotoxicity towards cancer cell lines [31]. The haemolytic activity of the newly synthesized benzothiazole derivatives were studied against Triton X-100 by a reported method [32].
The newly synthesized benzothiazole derivatives exhibit moderate to high haemolytic activities (see in Table 6, Figure 10). Compound 3cexhibits the highest haemolytic activity (47.089 ± 0.130). Fluorinated analog 2d also displayed the highest toxicity among all the tested compounds. The antitumor activity of a compound might be considered as corresponding to the highest haemolytic activity. It was observed that substitution does not markedly affected the haemolytic activity of these newly synthesized N-protected benzothiazole derivatives. Compounds 2, 3b and 3d-3g showed good haemolytic activities. The lowest haemolytic activity was found for compound 3h. It was concluded that halogen substitution on N-protected benzothiazole molecules promotes haemolytic activity. These compounds have potential to be used as future anticancer agents.

Haemolytic Activity
The haemolytic activity of benzothiazole derivatives has already been reported. A literature survey reveals that amino-substituted derivatives of benzothiazole have high cytotoxicites. Benzothiazole compounds with halogen substitutions show cytotoxicity towards cancer cell lines [31]. The haemolytic activity of the newly synthesized benzothiazole derivatives were studied against Triton X-100 by a reported method [32].
The newly synthesized benzothiazole derivatives exhibit moderate to high haemolytic activities (see in Table 6, Figure 10). Compound 3cexhibits the highest haemolytic activity (47.089 ± 0.130). Fluorinated analog 2d also displayed the highest toxicity among all the tested compounds. The antitumor activity of a compound might be considered as corresponding to the highest haemolytic activity. It was observed that substitution does not markedly affected the haemolytic activity of these newly synthesized N-protected benzothiazole derivatives. Compounds 2, 3b and 3d-3g showed good haemolytic activities. The lowest haemolytic activity was found for compound 3h. It was concluded that halogen substitution on N-protected benzothiazole molecules promotes haemolytic activity. These compounds have potential to be used as future anticancer agents.

Haemolytic Activity
The haemolytic activity of benzothiazole derivatives has already been reported. A literature survey reveals that amino-substituted derivatives of benzothiazole have high cytotoxicites. Benzothiazole compounds with halogen substitutions show cytotoxicity towards cancer cell lines [31]. The haemolytic activity of the newly synthesized benzothiazole derivatives were studied against Triton X-100 by a reported method [32].
The newly synthesized benzothiazole derivatives exhibit moderate to high haemolytic activities (see in Table 6, Figure 10). Compound 3c exhibits the highest haemolytic activity (47.089˘0.130). Fluorinated analog 2d also displayed the highest toxicity among all the tested compounds. The antitumor activity of a compound might be considered as corresponding to the highest haemolytic activity. It was observed that substitution does not markedly affected the haemolytic activity of these newly synthesized N-protected benzothiazole derivatives. Compounds 2, 3b and 3d-3g showed good haemolytic activities. The lowest haemolytic activity was found for compound 3h. It was concluded that halogen substitution on N-protected benzothiazole molecules promotes haemolytic activity. These compounds have potential to be used as future anticancer agents.

Antibacterial Activity
The synthesized benzothiazole derivatives were examined for their anti-bacterial activity against two Gram positive-bacterial strains (Baccilus subtiles, Staphylococcus aureus) and four Gram-negative strains (Escherichia coli, Psuedomonas aeruginosa, Shigella dysenteriae, Salmonella typhae) at concentrations of 40 and 80 μg/mL (Tables 7 and 8). It was concluded that the potent antibacterial activities of these compounds might be due to electron withdrawing groups present on the aryl moiety in N-protected benzothiazole molecule. Similar observations are also reported by other groups which suggest that the presence of electron releasing and electron withdrawing groups substantially affects the antibacterial activity [33]. Each value is mean ± standard deviation of three parallel measurements. The results show that the benzothiazole compounds 3a-3h exhibit higher activities than the standard against some species. Functional group changes in the benzothiazole molecule led to differences in activity. The newly synthesized compounds were found to be inactive against Baccilus

Antibacterial Activity
The synthesized benzothiazole derivatives were examined for their anti-bacterial activity against two Gram positive-bacterial strains (Baccilus subtiles, Staphylococcus aureus) and four Gram-negative strains (Escherichia coli, Psuedomonas aeruginosa, Shigella dysenteriae, Salmonella typhae) at concentrations of 40 and 80 µg/mL (Tables 7 and 8). It was concluded that the potent antibacterial activities of these compounds might be due to electron withdrawing groups present on the aryl moiety in N-protected benzothiazole molecule. Similar observations are also reported by other groups which suggest that the presence of electron releasing and electron withdrawing groups substantially affects the antibacterial activity [33].  The results show that the benzothiazole compounds 3a-3h exhibit higher activities than the standard against some species. Functional group changes in the benzothiazole molecule led to differences in activity. The newly synthesized compounds were found to be inactive against Baccilus subtilis and Staphylococcus aureus. Only compound 3f showed activity against Psuedomonas aeruginosa with a very small value (7.54˘0.6). These newly synthesized compounds do not exhibit considerable antibacterial activity and the highest value (3.51˘0.43) was observed for compound 3h against Shigella dysenteriae at concentration of 40 µg/mL. These new benzothiazole molecules showed weak activities against Salmonella typhae at 40 µg/mL. It was found that all newly synthesized benzothiazole compounds gave good to very good activity against E. coli at 40 µg/mL. Compounds 3c and 3ge exhibited very good activities against the E. coli strain with values of 49.05˘0.32 and 45.93˘0.3, respectively. These differences in activities may be attributed to the presence of electron loving atoms/groups on the aryl moiety of these N-protected benzothiazole derivatives.
The synthesized compounds were also checked for antibacterial activities at 80 µg/mL and compared against ampicillin. It was shown that these compounds showed moderate activities against Bacillus subtiles with the highest value (12.66˘0.531) corresponding to compound 3e. The authors concluded that these benzothiazole derivatives showed non-significant activity against Staphylococcus aureus. In addition, these compounds were to be found active against Shigella dysenteriae and Salmonella typhae at 80 µg/mL. These compounds displayed very good activity against E. coli at 80 µg/mL. The benzothiazole derivatives were discovered to be the most potent against the E. coli strain. Compounds 3a, 3b and 3g proved to be the most potent at the concentration of 80 µg/mL with the highest antibacterial activities with values of 57.97˘0.25, 57.84˘0.25 and 56.13˘0.32, respectively. The results of this study revealed that electron withdrawing group substitution on the aryl moiety on the benzothiazole molecule enhanced the antimicrobial activity of the compounds.

Procedure for the Preparation of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 3a-3h
The preparation of products 3a-3h was carried out under a nitrogen atmosphere. Compound 2 (synthesized by literature reported method [19]) (2.183 mmol) and 5 mol % Pd(PPh 3 ) 4 in 1,4-dioxane (20 mL) was mixed and stirred for 30 min. After 30 min K 3 PO 4 (4.366 mmol), aryl boronic pinacol esters/aryl boronic acids (2.401 mmol), and H 2 O (1.5 mL) was added under inert atmospheric conditions. The mixture was stirred for 30 h at 95˝C and cooled down to room temperature. Ethyl acetate was used for work up and the organic layer was separated and dried under vacuum. For purification purposes, column chromatography was done. The desired product was obtained by using ethylacetate and n-hexane (20% and 80%respectively) as eluents. The desired products were characterized by various spectroscopic techniques [34].

Procedure for Urease Inhibition Activity
Urease inhibitory assay of newly synthesized compounds 3a-3h were determined as follows: Enzyme (1 unit) in phosphate buffer (200 µL, pH 7) was combined with a particular stock solution (20 µL, a test compound or thiourea) and phosphate buffer (230 µL). The solution was incubated for 5 min at 25˝C. After incubation period 400 µL of urea stock solution (20 mM) was added to the solution. Calibration solution was synthesized without urea solution. For the action of urease, test tubes were incubated for 10 min at 40˝C. The solution of phenol hypochlorite reagent (1150 µL) was added. These tubes were incubated for 25 min at 56˝C. Absorbance of the blue colored compound was noted at 625 nm after 5 min of cooling. Then percentage urease inhibition was determined. While EZ-fit kinetic data base was used to obtain IC 50 values [25,35].

Molecular Docking Study
The PDB structure of 3LA4 was retrieved for docking purposes as a complex co-crystallized with inhibitor 2-amino-3-(2-(2-hydroxyethyl)disulfanyl)propan-1-ol and (5-amino-6-hydroxyhexyl)carbamic acid at the nickel-containing catalytic site [36]. Then, the amino acid chain was retained and the water molecules and co-crystallized ligands were removed and subsequently the missing atom types were repaired using Modeller 9.11 (University of California San Francisco, San Francisco, CA, USA). Afterwards, the polar hydrogen was added to the receptor and the resulting protein was subjected to minimization using OPLS 2005 force field. The prepared protein was saved in pdbqt format using Autodock Tools 1.5.4 [21,[37][38][39]. The ligand coordinates were generated using MarvineSketch 5.8.3, 2012 (ChemAxon LLC, Cambridge, MA, USA) [40], which was converted to 3D structure using Openbabel version (2.3.1). Finally the pdbqt formats (The input format of docking software) of the ligands were prepared with Autodock Tools 1.5.4 using default parameters. AutodockVina ver. 1.1.1 (The Scripps Research Institute, La Jolla CA, USA) was used for docking calculations with default parameters except for exhaustiveness that was set to 80. For all the docking calculations, a grid box size of 40ˆ40ˆ40, centered at the geometrical center of co-crystallized ligands was used. Co-crystalized ligands were attached at two different sites, one near the nickel catalytic site (A) and the other site where the inhibitor 2-amino-3-(2-(2-hydroxyethyl)disulfanyl)propan-1-ol was attached (Site B) [20].
The coordinates x, y, z for the center of grid box were (Site A)´39.86,´45.06, 72.52 and (Site B)´75.03, 20.84, 81.83 respectively. To validate our docking procedure, the co-crystallized ligands were re-docked into their respective site of the enzyme and the reasonable RMSD value of 1.947 A was obtained. Finally, the conformations with the most favorable free energy of binding were selected for analyzing the interactions between urease and its inhibitor. All of the 3D models are generated using the Molegro Molecular Viewer 2.5 (CLC bio company, Aarhus N, Denmark) [41] and LigPlot + (The European Bioinformatics Institute, Hinxton, Cambridge, UK) [42] software.

Nitric Oxide Scavenging Activity
The activity of newly synthesized benzothiazole derivatives was determined using the Garrat method. Griess reported the Garrat method which is followed by a diazotization reaction [43]. Under acidic conditions, this method utilizes sodium nitroprusside as the source of nitric oxide, sulfanilamide and N-1-naphthylethylenediamine dihydrochloride to detect NO 2´, produced at the expense of nitric oxide. A known amount of tested compounds 3a-3h was dissolved in sodium nitroprusside solution (20 mM, 100 µL) and then the volume was made up to 1000 µL with phosphate buffer (200 mM, pH 7.4). This solution was incubated for 2 h at 37˝C and Griess reagent (100 µL) was added. This solution was stored for 20 min at room temperature. At 528 nm, optical density of this colored solution was observed. For positive control, ascorbic acid was used. Negative control was used to form the standard curve [43].

Haemolytic Activity
Haemolytic activity of newly synthesized benzothiazole derivatives 3a-3h was determined using a reported method [44]. Solutions of compounds were prepared at concentrations of 1 mg/mL in 10% DMSO with 90% water Heparinized human fresh blood (3 mL) was used that was homogeneously mixed and added into a 15 mL sterile Falcon tube. It was centrifuged for 5 min and the supernatant was removed. Chilled sterile isotonic phosphate buffer saline solution (5 mL, 7.4 pH) at 4˝C was used three times. Washed red blood cells were suspended in chilled RBS (20 mL). A haemacytometer was used for counting erythrocytes. For each assay 7.068ˆ10 8 red blood cells per mL count were maintained and then diluted blood cells (180 µL) were added to the test compound (20 µL) and suspended in Eppendorf tubes. It was incubated for 35 min at 37˝C then the tubes were kept in an ice bath for 5 min and centrifuged again for 5 min. After centrifugation, the obtained supernatant was collected carefully and diluted with 900 µL of chilled PBS. All these tubes were kept in ice bath and solution (200 µL) was added into 96 well plates from each Eppendrof tube. For each essay, Triton X-100 (0.1%) was taken as positive control. For negative control, phosphate buffer was used. A microplate reader was used for determining the absorbance at 576 nm [32].

Antibacterial Activity
These newly benzothiazole derivatives were tested for their antibacterial activities against two Gram positive strains (Baccilus subtilis, Staphylococcus aureus) and four Gram negative strains (Escherichia coli, Psuedomonas aeruginosa, Shigella dysenteriae and Salmonella typhae) using a reported protocol [45]. Streptomycin was used as positive control. The 96 well plate method was optimized for measuring the antibacterial activities of these compounds. In each well, sterilized broth (175 µL) was added and inoculated with glycerol stock (5 µL) of a specific bacterial strain. The initial absorbance was observed between 0.12-0.19. The bacteria were allowed to grow in an incubator overnight. After a certain waiting time (12 h), test sample (20 µL) was added to the pre-determined wells. Concentration of test sample was 20 µg/well. Total volume was 200 µL in each well. These plates were incubated for 16-24 h at 37˝C. Absorbance was observed at 630 nm by using an ELISA plate reader. The difference in absorbance values were observed and were used as an index of bacterial growth. The following formula was used to calculate percentage inhibition: Percentage Inhibition " pO.D of`ve control´O.D of sampleˆ100q{O.D of`ve control (1)

Conclusions
This study reports C-C coupling reactions of 2 with various arylboronic pinacol ester/aryl boronic acids using palladium catalyst. These new products 3a-3h were prepared in moderate to good yields. These Suzuki coupling benzothiazole derivatives were checked for their biological (urease inhibitory, nitric oxide scavenging, haemolytic and antibacterial) activities. The urease inhibition results showed that product 3b was an excellent urease inhibitor. Products with electron releasing groups on the aryl moiety of the benzothiazole molecule showed the highest inhibition of urease activity. Molecular docking studies of the urease inhibitory activity showed that the H-bonding ability present in these N-protected benzothiazoles prevents the catalytic activity of the enzyme. Nitric oxide scavenging assays were done for these compounds. Compound 3b also exhibited highest nitric oxide scavenging activity. All newly synthesized compounds showed haemolytic activity. It was found that electron withdrawing substitution on the aryl produced the highest haemolytic activity. The newly synthesized benzothiazole derivatives 3a-3h showed excellent antibacterial activities against E. coli.