Synthesis and biological evaluation of sulfonamide thiazole and benzothiazole derivatives as antimicrobial agents

Several thiazoles and benzothiazoles carrying a benzenesulfonamide moiety at position 2 of the heterocyclic nucleus were synthesised and tested as antimicrobial agents. All sulfanilamides and some of the nitro substituted sulfonamides have effective antibacterial properties against Gram positive bacteria (MIC 0.3-100 μg/mL) such as several bacilli, staphylococci and streptococci, including methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains. On the contrary, no inhibition of Gram negative Escherichia coli and fungi is detected up to the concentration of 100 μg/mL. Synergistic inhibitory activity occurs when the active antibacterial sulfonamides are tested in combination with trimethoprim against both Bacillus subtilis and Staphylococcus aureus.


Chemistry
The general method which is employed to prepare the final compounds is outlined in Scheme 1.
The designed sulfonamides were prepared by heating the appropriate heteroarylamine with the selected benzensulfonylchlorides in pyridine for several hours.Nucleophilic addition of the NH 2 group to the sulfonyl function of the benzenlsulfonylchlorides takes place.Difficulties connected with the poor reactivity of some starting materials were overcome by increasing the duration of heating.4-Aminobenzenesulfonamides 1 and 6-8 were prepared from the corresponding 4-acetamidobenzenesulfonamides by refluxing the last one in ethanol containing small amount of concentrated HCl.
During the course of this study we obtained some sulfonamides in good yield and some others in moderate to low.The structures of the synthesized compounds were confirmed by elemental analysis and by MS, IR and 1 H NMR spectral data.

Antimicrobial activity
The thiazole/benzothiazole sulfonamides were assayed in vitro for their antimicrobial activity against a panel of selected Gram positive and Gram negative bacteria, yeasts and mould and the minimal inhibitory concentrations that inhibited the growth of the tested microorganisms (MIC) were detected.In order to elucidate the kind of the exhibited antimicrobial activity, when MIC values were lower than 100 µg/mL, the minimal bactericidal concentrations (MBCs) and the minimal fungicidal concentrations (MFCs) were determined.The results of antimicrobial testing are reported in Table 1 and are compared with those of standards ampicillin and sulfamethoxazole as antibacterial agents and miconazole as an antifungal drug.Some of the tested compounds display good inhibition of the growth of Gram positive bacteria, being Bacillus subtilis the most sensitive one.It should be noted that compound 6 shows the best activity among all tested compound against Bacillus subtilis (MIC 1.5 µg/mL) as well as Staphylococcus aureus (MIC 12 µg/mL).Interesting results are also obtained for compound 8 towards the same microorganisms at 3 and 12 µg/mL respectively.These compounds are even more potent than sulfamethoxazole but less than ampicillin, while inhibitory properties comparable or slightly inferior to those of sulfamethoxazole are exhibited by compounds 1, 7 and 10 against both Gram positive bacteria and by compounds 2, 4 and 9 against Bacillus subtilis only.The antimicrobial properties of the active compounds were further investigated towards a wide spectrum of Gram positive bacteria, including methicillin-resistant strains.Results, presented in Table 2, confirm the high inhibitory potency of compound 6 extended to all the tested bacilli, Sarcina, staphylococci and several streptococci at concentrations of 0.3-12 µg/mL.Furthermore, sulfanilamides 1, 7 and 8 exhibit promising activity having a range of MIC values almost similar to that of sulfamethoxazole.A good inhibition can be observed also for nitro derivative 10 towards most of the tested bacteria and 2 towards bacilli.It is worth noting that some sulfonamides show significant activity also against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains.The lack of activity of either compounds 4 and 9 in the extended spectrum studies makes these substances selective against Bacillus subtilis (Table 1).
In all the cases, MBC values are two-fold or more higher than MICs, suggesting that the effectiveness of these substances has a bacteriostatic character.
None of the tested compounds exhibits any activity against Gram negative Escherichia coli, yeasts Candida tropicalis and Saccharomyces cerevisiae, and mould Aspergillus niger up the concentration of 100 µg/mL.
In addition, with the aim to investigate the action mechanism of the detected antibacterial activity, we evaluated by synergism studies the effectiveness of the new sulfonamides against the sensitive microorganisms in the presence of trimethoprim.It is, in fact, well-known that sulfonamides exert their antibacterial effect by inhibiting the biosynthesis of dihydrofolic acid and their activity is enhanced by the combination with trimethoprim, a competitive inhibitor of dihydrofolate reductase, an enzyme reducing dihydrofolic to tetrahydrofolic acid. 24So, both Bacillus subtilis and Staphylococcus aureus were used for sulfanilamides 1 and 6-8 and for the nitroderivative 10, while only Bacillus subtilis was used for compounds 2, 4 and 9.The results were, then, compared with the antibacterial activity of the substances used individually.Sulfamethoxazole-trimethoprim mixture was taken as positive control.
The paper strip diffusion determinations show that the antibacterial activity of all the tested sulfonamides is potentiated by trimethoprim.As evidenced in Figure 2, the size of inhibition zones of Bacillus subtilis is greatest where the compounds 4 and 6 overlap with trimethoprim, as observed for trimethoprim-sulfamethoxazole combination.A similar behaviour could be detected for compounds 1, 2 and 7-10 against the same microorganism and for compounds 1, 6-8 and 10 against Staphylococcus aureus (data not shown).
Since the paper strip test has a qualitative character, the antimicrobial synergism was then quantitatively assessed by the two fold dilution method in the checkerboard titration.Antibacterial sulfonamides plus trimethoprim were tested together in a large number of dilutions to determine the concentrations, of each compound in combination with trimethoprim, that inhibit the growth of Bacillus subtilis and Staphylococcus aureus.The interactions were detected in comparison with sulfamethoxazole-trimethoprim mixture and are described by the isobolograms of Figures 3 and 4. The obtained results confirm that subinhibitory concentrations of trimethoprim potentiate the antibacterial activity of all the tested compounds by strongly reducing their MIC values.Figures 3 and 4 exhibit concave isobols and fractional inhibitory concentration indices significantly lower than 0.5.In particular, FICIs range from 0.124 to 0.281 for Bacillus subtilis and from 0.124 to 0.312 for Staphylococcus aureus.This synergistic effect is comparable to that produced by the reference sulfamethoxazole-trimethoprim mixture and suggests that the studies sulfonamides act by inhibiting the synthesis of folic acid.

Structure-activity relationship
As regards the relationships between the structure of the heterocyclic scaffold and the detected antibacterial properties, it seems that there is no difference.Probably in this case the nature of the heterocyclic ring is not so important for antimicrobial activity.Moreover, the presence of substituents in different positions of both thiazole and benzothiazole moieties causes a certain change of activity.Among benzothiazoles, 4-amino-and 4-nitrosulfonamides having a halogen as substituent (compounds 7, 12 and 13) or carrying an ethoxy group (compounds 8 and 11) exhibit inhibitory properties lower than those of the corresponding methyl substituted ones (compounds 6 and 10, respectively).On the other hand, for the latter compound a decreased activity towards Bacillus subtilis and an enhancing inhibition of Staphylococcus aureus can be detected, when compared with unsubstituted compound 9.As concerns thiazole derivatives, the introduction in compound 2 of a lipophilic methyl group (compound 3) abolishes the activity against Bacillus subtilis, while the presence of a bulky group (-CH 2 COOC 2 H 5 ) in compound 4 does enhance its inhibition properties.
From the obtained results it is clear that the major role for antibacterial activity is played by the substituent in the 4-position of the aromatic ring of sulfonamides.It is evident that sulfonamides with an amino group in the C-4 position of the phenyl ring are the most active compounds.Thus, the replacement of the amino group with nitro leads to a dramatic decrease in antibacterial activity (compounds 6 and 10, 7 and 13, 8 and 11).No activity is exhibited by 4chloro substituted sulfonamides 14 and 15 and by the 4-methyl derivative 5.
In addition, synergism studies suggest that an enzymatic reduction to amino derivatives involves the 4-nitrosulfonamides. 25 Experimental Section Chemistry General Procedures.Melting points (mp°C) were determined with a Buchi 512 apparatus or with a Boetius apparatus and are uncorrected.Elemental analysis was performed on a ThermoQuest (Italia) FlashEA 1112 Elemental Analyser, for C, H, N, and S. The values found for C, H, N, S were within ±0.4% of the theoretical ones.IR spectra, such as KBr pellets, were recorded on a Jasco FT-IR 300E spectrophotometer (Jasco Ltd., Tokyo, Japan).Mass spectra were recorded on a VG-250instrument (VG Labs Tritech, England) with ionization energy maintained. 1 H NMR spectra of the newly synthesized compounds, in DMSO-d 6 solutions, were recorded on a Bruker AC 300 instrument at 298 °K.Chemical shifts are reported as δ (ppm) relative to TMS as internal standard; coupling constants J are expressed in Hertz.The progress of the reactions was monitored by thin layer chromatography with F 254 silica-gel precoated sheets (Merck, Darmstadt, Germany).UV light was used for detection.Solvents, unless otherwise specified, were of analytical reagent grade or of the highest quality commercially available.Synthetic starting material, reagents and solvents were purchased from Aldrich Chemical Co. and from Fluka.

General procedure for synthesis of thiazole/benzothiazole sulfonamides
To the starting amine (6 mmol) suspended in anhydrous pyridine (10 mL), appropriate sulfonyl chloride (6.3 mmol) was added gradually.The reaction mixture was heated at 60 o C for 1-4 hours, then poured into ice water and acidified with 1N HCl.The solid product was filtered, washed well with water, and recrystallized from ethanol.

Microbiology Antimicrobial activity evaluation
The new sulfonamides were tested in vitro for their antimicrobial properties against the microorganisms listed in Tables 1 and 2 by using the serial double dilution method. 26tock solutions of the compounds were prepared by dissolving them in dimethyl sulfoxide.Then, the solutions were diluted in the media (Tryptose Phosphate Broth for Streptococcus pyogenes, Mueller Hinton broth for other bacteria and Sabouraud liquid medium for fungi) so as to achieve concentrations of compound ranging from 0.001 to 100 µg/mL.Ampicillin, sulfamethoxazole and miconazole, at the same concentrations of the text compounds, were used as standard antibacterial and antifungal drugs, respectively.Dimethyl sulfoxide was employed as solvent control.Bacteria were inoculated at the concentration of 5x10 4 CFU/mL and fungi at 1x10 3 CFU/mL.After an incubation period of 24 h at 37°C (bacteria) and of 48 h at 30°C (fungi), the minimum inhibitory concentrations (MIC, µg/mL) were detected as the lowest concentrations of compound that didn't show microbial growth.The minimum bactericidal concentrations (MBC, µg/mL) and the minimum fungicidal concentrations (MFC, µg/mL) were determined by subculturing on fresh medium 100 µL of liquid from each suspension remaining clear and incubating the samples obtained at 37°C for 24 h (bacteria) or at 30°C for 48 h (fungi).MBC and MFC values represent the lowest concentration of drug needed for the reduction of the initial inoculum of 99.9 %.All the experiments were performed in triplicate and the reported results were obtained from three independent measurements.

Synergism studies
The effect of combinations of the active sulfonamides with trimethoprim (Sigma, Milano, Italy) against Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 25923 was detected in vitro by using the paper strip diffusion method and the checkerboard titration technique. 11,27The antimicrobial combination of sulfamethoxazole with trimethoprim was used as positive standard control.The solutions of the standard drugs were prepared in water by adding the minimal amount of 2.5 M NaOH (for sulfamethoxazole) or 0.05 M HCl (for trimethoprim) to dissolve them.In the paper strip diffusion technique, the effectiveness of the drug interaction was determined by comparing the zones of inhibition of the bacterial growth produced by sulfonamides alone with those produced in the presence of trimethoprim.Iso-Sensitest Agar plates were inoculated by fresh cultures of the microorganism to be tested suspended in molten top agar at the concentration of 5x10 5 CFU/mL to produce confluent growth.Filter paper strips (40 mm long, 3 mm wide) were impregnated with 25 mL of solutions of the sulfonamides or trimethoprim at concentrations, respectively, slightly lower than or equal to MICs and placed on the agar surface at right angles to one another.The plates were incubated at 4°C for 4 h to allow the drugs to diffuse into the agar medium and, then, transferred at 37°C for 18 h.The combined effect of the sulfonamides with trimethoprim was examined by observing the inhibition halos produced by the substances alone and at the junction of the filter papers and by comparing them with those produced by sulfamethoxazole with trimethoprim.Synergistic interactions are evidenced by the presence of an increased inhibition zone at the junction point, as shown for the combination sulfamethoxazole-trimethoprim.On the contrary, no enhanced halos result for additive or indifferent interactions, while truncated zones of inhibition reveal antagonism.As regards the checkerboard titration technique, sulfonamides were tested at concentrations ranging from 0.15 to 100 µg/mL and trimethoprim from 0.0003 to 3 µg/mL.The checkerboard consisted of columns containing decreasing concentration of sulfonamides and of rows containing increasing concentrations of trimethoprim, so that drug-containing solutions can be mixed to produce all possible combinations of compound concentrations in the range of the dilutions used.The inoculum of the bacterial suspension added to each well was 5x10 4 bacteria/mL.The wells were incubated at 37°C for 24 h and the MICs of the sulfonamides and trimethoprim alone and combined were read.The effect of the studied combinations was expressed by the fractional inhibitory concentration (FIC) of each tested compound (S, sulfonamides; T, trimethoprim).The FIC indices (FICI) for the most effective combinations were then calculated by summing the separate FIC values of each sulfonamide and trimethoprim: FICI = FIC S + FIC T FIC S = MIC of each sulfonamide in combination / MIC of each sulfonamide alone FIC T = MIC of trimethoprim in combination / MIC of trimethoprim alone.Drug interactions are defined as synergistic when the FIC index is ≤ 0.5, additive when the FIC index is >0.5 and ≤ 1, while values >1 and <4 are classified indifference and values ≥ 4 antagonism. 28he results were summarized graphically on the isobolograms constructed taking along the xaxis the FIC of trimethoprim and along the y-axis the corresponding FIC values of each sulfonamide.The isobolograms were constructed by connecting with a line (isobol) the series of points generated for each drug combination.Synergistic interaction is diagrammed as concave isobol, additive effect as straight line and antagonism as a convex isobol.All tests were performed in triplicate and the results were confirmed by three separate experiments.

Figure 1 .
Figure 1.Structure of the synthesized compounds.

Table 1 .
Antimicrobial activity expressed as MIC (µg/mL) and, in brackets, as MBC and MFC (µg/mL) e Not tested.