Synthesis and Antimicrobial Evaluation of a New Series of N-1 , 3-Benzothiazol-2-ylbenzamides

Enterococcus faecalis is a Gram-positive commensal inhabitant of the intestinal tract of humans, animals, and insects. However, it is also an opportunistic pathogen and has emerged as a leading cause of hospital-acquired extraintestinal infections. Fluoroquinolones have been frequently used to treat E. faecalis infections, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. Thus, the identifications of new antibiotics specifically directed to E. faecalis may be envisaged. In this paper, a new series of N-1,3-benzothiazol-2-ylbenzamides have been designed, synthesized, and evaluated for their in vitro antimicrobial activities. Among the tested compounds, 3i was active against E. faecalis.


Introduction
Drug resistance to therapeutic antibiotics poses a challenge to the identification of novel targets and drugs for the treatment of infectious diseases.Infections caused by Enterococcus faecalis are a major health problem.Thus, studies for the identification of novel targets and drugs for the treatment of infectious diseases are at the forefront.E. faecalis is a Grampositive opportunistic pathogen which has emerged as a leading cause of hospital-acquired extraintestinal infections, including urinary tract infections (UTIs), bacteremia, wound infections, and endocarditis [1][2][3].Normally, a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics.We recently determined the X-ray crystallographic structure of E. faecalis thymidylate synthase, which should be a potential target for antibacterial therapy [4].Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis (QREF) strains has recently been reported in several countries [5].Thus, the development of new and different antimicrobial drugs, in particular acting against E. faecalis, is a very important goal, and most of the research program efforts in this field are directed towards the design of new agents.During the past decade, combinatorial chemistry has provided access to chemical libraries based on privileged structures [6], with heterocyclic structures receiving special attention as they belong to a class of compounds with proven utility in medicinal chemistry [7][8][9][10].Many heterocyclic nuclei, such as 1,3,4-thiadiazole [11], benzimidazole [12], 1,3,5-triazine [13], and benzothiazole [14], have been recently reviewed as antimicrobial agents.Our attention was focused on the benzothiazole nucleus.Benzothiazole derivatives possess a wide spectrum of biological applications such as antitumor [15][16][17][18], antimicrobial [19][20][21], schistosomicidal [22], antiinflammatory [23,24], anticonvulsants [25][26][27], antidiabetic [28,29], antipsychotic [30,31], neuroprotective [32], and diuretic [33] activities.In the past, we were interested in a series of 2-mercapto-1,3-benzothiazole derivatives showing antimicrobial activity [19].It is disclosed that the SH moiety at the C2 position of the heterocyclic nucleus led to a remarkable antibacterial activity against Gram positive and negative.Actually, in order to improve SAR studies on 1,3benzothiazoles, we decided to investigate the isosteric relationship between 2-mercapto and 2-amino substitutions.Moreover, in this study we also aimed at widening SAR Figure 1 indications about the effects of halogen substitutions on several amides of 1,3-benzothiazole previously reported (N-1,3-benzothiazol-2-ylbenzamides, 1, Figure 1).We found that these compounds exerted antifungal activity against C. albicans comparable to that of the reference compound sorbic acid, that is, with MIC values in the order of 250-500 g/mL [34].Nowadays, it is known that sorbic acid is no longer the best reference compound to be used to study antifungal activity; it is preferable to use antibiotics, such as amphotericin or fluconazole, which are much more active showing MIC values in the order of 0.5-1 g/mL; thus, the compounds previously reported can be considered inactive against fungi.

Results and Discussion
Compounds 5a, b and 3a-l were prepared as reported in Scheme 1.
5-F most active derivative giving the best antibacterial activity against E. faecalis with an MIC value of 8 g/mL, while compounds 3a, b, f-h, and k displayed moderate activity towards the same bacteria strain (MIC: 32 g/mL).In order to better define structure activity relationships, it is possible to consider two subseries of compounds: the former, 6chloro-4-fluorobenzothiazole benzamides (3a-f); the latter, 6-chloro-5-fluorobenzothiazole benzamides (3g-l).Among all the tested compounds, with the exception of the highly active compound 3i, the best substitutions of the acyl moiety seem to be 2,3-difluoro and 2,4-difluoro in both series (3a, b and 3g, h) and the worst substitution seems to be 2,6-difluoro in both series (3d and 3j).Moreover, the 3,4-difluoro and 3,5difluoro substitutions had different effects in the two series: in particular, the former decreases activity in the first series (3e), while it increased activity in the second (3k), and the opposite was true for the latter substitution (3f was active, and 3l was inactive).Then, 2,5-difluorosubstitution determined a very high increase in activity in the second series giving the best compound tested (3i) while decreasing the activity in the first series (3c; MIC: 128 g/mL).Finally, 2,6-difluoro substitution was detrimental in both series: compounds 3d and 3j showed no activity or low activity against E. faecalis (MIC > 512 g/mL and MIC: 128 g/mL, resp.).All the compounds did show very low or no activity against S. aureus and E. coli and all the strains of Candida.These results are in agreement with what had been found for a series of isomers of our compounds previously reported [34].Moreover, intermediates 5a, b were tested in order to compare results obtained with their isomers recently reported, in which the halogen atoms were inverted in their position [35].These   two compounds did show no antimicrobial activity, while results found as antifungals are similar to their corresponding isomers.In particular, compound 5a was identical to its isomer against Gram-positive and -negative bacteria and against C. parapsilosis, while it was slightly more active than their isomers previously reported [35]

Conclusion
In conclusion, we report the synthesis and antimicrobial activity of a series of 1,3-benzothiazole derivatives (3a-l and 5a, b).All the compounds did not show activity against S. aureus, E. coli and C. albicans, C. parapsilosis, and C. tropicalis in these tracing results previously obtained for the corresponding position isomers previously reported (1, 2, Figure 1) [34,35].Interestingly, compounds 3a, b, f, g, h, i, and k exerted moderate to high activity against E. faecalis (MIC between 8 and 32 g/mL).In particular, compound 3i was the most potent of the series (MIC: 8 g/mL) and the most promising compound, while the other showed an MIC value of 32 g/mL.[36].Stock solutions of the tested compounds were obtained in DMSO.Stock solutions of lower concentrations were prepared for those substances which did not dissolve well.Then twofold serial dilutions in the suitable test medium between 512 and 0.5 g/mL were plated.To be sure that the solvent had no adverse effect on bacterial growth, a control test was carried out by using DMSO at its maximum concentration along with the medium.Bacteria strains available as freeze-dried discs, belonging to the ATCC collection, were used: Gram-positive strains such as Staphylococcus aureus 29213 and Enterococcus faecalis 29212 and Gram-negative one such as Escherichia coli 25922.To preserve the purity of cultures and to allow the reproducibility, a series of cryovials of all microbial strains in 10% glycerol medium was set up and stored at −80 ∘ C. Precultures of each bacterial strain were prepared in cation-adjusted mueller-hinton broth (CAMHB) and incubated at 37 ∘ C until the growth ceased.

Experimental
The turbidity of bacterial cell suspension was calibrated to 0.5 McFarland Standard by spectrophotometric method (625 nm, range 0.08-0.10),and further the standardized suspension was diluted 1 : 100 with CAMHB to have 1-2 × 10 6 CFU/mL.All wells were seeded with 100 L of inoculum.
A number of wells containing only inoculated broth as control growth were prepared.The plates were incubated at 37 ∘ C for 24 h, and the MIC values were recorded as the last well containing no bacterial growth.The MICs were determined by using an antibacterial assayed repeated twice in triplicate.Oxacillin and norfloxacin were used as reference drugs.

Antifungal Studies.
Antifungal studies [37] were carried out against Candida albicans 10231, Candida parapsilosis 22019, Candida tropicalis 750, and Candida krusei 6258, belonging to the ATCC collection.Preparation of stock solutions and purity of cultures preservation were obtained as previously described for antibacterial studies.Pre-cultures of each yeast strain were prepared in Sabouraud broth (SAB) 2% glucose, and incubated at 37 ∘ C until the growth ceased.The turbidity of yeast stock suspension was calibrated to 0.5 McFarland Standard by spectrophotometric method (530 nm, range 0.12-0.15),and further the standardized suspension was diluted first 1 : 50 with SAB and then 1 : 20 in the same medium to have 1-5 × 10 6 CFU/mL.All wells were seeded with 100 L of inoculum.A number of wells containing only inoculated broth as control growth were prepared.The plates were incubated at 37 ∘ C for 24-48 h, and the MIC values were recorded as the last well containing no fungal growth.The MICs were determined by using an antifungal assay repeated twice in triplicates.Fluconazole was used as a reference drug.