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Journal of Chemistry
Volume 2013 (2013), Article ID 181758, 7 pages
http://dx.doi.org/10.1155/2013/181758
Research Article

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

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro,” Via E. Orabona 4, 70125 Bari, Italy

Received 30 May 2013; Accepted 2 September 2013

Academic Editor: Gabriel Navarrete-Vazquez

Copyright © 2013 Domenico Armenise et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.

1. 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 Gram-positive opportunistic pathogen which has emerged as a leading cause of hospital-acquired extraintestinal infections, including urinary tract infections (UTIs), bacteremia, wound infections, and endocarditis [13]. 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 [710]. 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 [1518], antimicrobial [1921], schistosomicidal [22], anti-inflammatory [23, 24], anticonvulsants [2527], 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 1,3-benzothiazoles, we decided to investigate the isosteric relationship between 2-mercapto and 2-amino substitutions. Moreover, in this study we also aimed at widening SAR 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 μg/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 μg/mL; thus, the compounds previously reported can be considered inactive against fungi. In this paper, we report a series of trifluoro-substituted-N-(6-chloro-1,3-benzothiazol-2-yl) benzamides (3, Table 1), that are position isomers of those previously reported [34]. Moreover, 2-amino-1,3-benzothiazoles (5, Table 1) were also synthesized and tested, and the results were compared to those obtained with their isomers previously reported (2, Figure 1) [35].

tab1
Table 1: Structures of compounds 3a-l and 5a, b.
181758.fig.001
Figure 1

2. Results and Discussion

Compounds 5a, b and 3a-l were prepared as reported in Scheme 1. The appropriate fluoro-4-chloro aniline (4a, b) was reacted with bromine and potassium thiocyanate to give the corresponding 2-amino benzothiazoles 5a, b, which were reacted with the suitable difluorobenzoyl chloride to give all possible isomeric compounds 3a-l. The antimicrobial activity of compounds 3a-l was evaluated in vitro against Gram-positive and Gram-negative bacteria (Staphylococcus aureus 29213, and Enterococcus faecalis 29212, Escherichia coli 25922) and fungi strains (Candida albicans 10231, Candida parapsilosis 22019, and Candida tropicalis 750) belonging to the ATCC collection [36, 37]. All MIC determinations were carried out according to the clinical laboratory standards institute (CLSI) guidelines. MIC values are given in μg/mL and were compared to MIC values for the standard antibacterial drugs oxacillin and norfloxacin and antifungal fluconazole. Screening results are summarized in Table 2. The combined data showed that seven compounds, out of twelve, exerted interesting inhibitory activity against E. faecalis with MIC values between 8 and 32 μg/mL. In particular, compound 3i was the most active derivative giving the best antibacterial activity against E. faecalis with an MIC value of 8 μg/mL, while compounds 3a, b, f–h, and k displayed moderate activity towards the same bacteria strain (MIC: 32 μg/mL). In order to better define structure activity relationships, it is possible to consider two subseries of compounds: the former, 6-chloro-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 3,5-difluoro 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 μg/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 μg/mL and MIC: 128 μg/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] on C. albicans and C. tropicalis (MIC: 128 μg/mL versus 256 μg/mL). Compound 5b was identical to its isomer against Gram-negative and C. tropicalis, slightly more active against S. aureus (MIC: 128 μg/mL versus 512 μg/mL) and E. faecalis (MIC: 128 μg/mL versus 256 μg/mL) and C. albicans (MIC: 64 μg/mL versus 128 μg/mL), and slightly less active against C. parapsilosis (MIC: 128 μg/mL versus 64 μg/mL).

tab2
Table 2: Antimicrobial activity results of benzothiazole derivatives 3a-l and 5a, b (MIC, μg/mL).
181758.sch.001
Scheme 1: Reagents and conditions: (i) Br2, KSCN, AcOH, 30–35°C; (ii) suitable difluorobenzoyl chloride, Et3N, dioxane, 50–60°C.

3. 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 μg/mL). In particular, compound 3i was the most potent of the series (MIC: 8 μg/mL) and the most promising compound, while the other showed an MIC value of 32 μg/mL.

4. Experimental

4.1. General Experimental Details

Chemicals were purchased from Sigma-Aldrich or Lancaster. Yields refer to purified products and were not optimized. The structures of the compounds were confirmed by routine spectrometric and spectroscopic analyses. Only spectra for compounds not previously described are given. Purity of compounds was assessed by GC analysis. Melting points were determined on a Gallenkamp apparatus in open glass capillary tubes and are uncorrected. Infrared spectra were recorded on a Perkin-Elmer (Norwalk, CT) Spectrum One FT spectrophotometer, and band positions are given in reciprocal centimeters cm−1. 1H NMR spectra were recorded on a Varian VX Mercury spectrometer operating at 300 MHz using CDCl3 and DMSO- as solvents. Chemical shifts are reported in parts per million (ppm) relative to the residual nondeuterated solvent resonance: CDCl3, δ 7.26 and DMSO- , δ 2.48. J values are given in Hz. GC-MS was performed on a Hewlett-Packard 6890-5973 MSD at low resolution. Chromatographic separations were performed on silica gel columns by flash chromatography (Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt, Germany) as previously reported [3841]. TLC analyses were performed on precoated silica gel on aluminum sheets (Kieselgel 60 F254, Merck). GC analyses were performed on a Varian 3800 gas chromatograph equipped with a flame ionization detector and a Jew Scientific DB-5 capillary column (30 m length × 0.25 mm ID, 0.25 μm film thickness) [42].

4.1.1. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-2,3 difluorobenzamide (3a)

A mixture of 5a (0.61 g, 3.0 mmol) and triethylamine (0.30 g, 3.0 mmol) in dry dioxane (30 mL) was stirred for 30 min at 50–60°C. A solution of 2,3-difluorobenzoyl chloride (0.53 g, 3.0 mmol) in dry dioxane (30 mL) was added dropwise. The mixture was stirred for 2 h and then poured into crushed ice. The resulting solid, so separated, was collected by filtration and washed with 1% potassium bicarbonate aqueous solution. The crude residue was purified by column chromatography on silica gel (EtOAc/petroleum ether 3 : 7) to give 0.32 g (31%) of 3a as a white solid: mp 240–242°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 38), 141 (100); 1H NMR: δ 7.18–7.25 (m, 1H, Ar HC-5), 7.26–7.35 (m, 1H, Ar HC-3′), 7.42–7.55 (m, 1H, Ar HC-4′), 7.63 (s, 1H, Ar HC-5′), 7.96 (t, J = 6.3 Hz, 1H, Ar HC-7), 10.08 (br s, 1H, NH, exch D2O); IR (KBr): 3405 (NH), 1686 (C=O) cm−1.

4.1.2. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-2,4-difluorobenzamide (3b)

Prepared as reported previously for 3a starting from 5a and 2,4-difluorobenzoyl chloride. Yield: 33%; white solid: mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 18), 141 (100); 1H NMR (DMSO- ): δ 7.12–7.55 (m, 3H, Ar HC-5 + Ar HC-3′,5′), 7.85–8.05 ppm (m, 2H, Ar HC-7 +Ar HC-6′); IR (KBr): 3418 (NH), 1673 (C=O) cm−1.

4.1.3. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-2,5-difluorobenzamide (3c)

Prepared as reported previously for 3a starting from 5a and 2,5-difluorobenzoyl chloride. Yield: 24%; white solid: mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 17), 141 (100); 1H NMR: δ 7.00–7.25 (m, 2H, Ar HC-5 + HC-4′), 7.50–7.65 (m, 1H, Ar HC-6′), 7.70–7.85 (m, 1H, Ar HC-3′), 8.02 (d, J = 5.2 Hz, 1H, Ar HC-7), 10.22 ppm (br s, 1H, NH); IR (KBr): 3410 (NH), 1677 (C=O) cm−1.

4.1.4. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-2,6-difluorobenzamide (3d)

Prepared as reported previously for 3a starting from 5a and 2,6-difluorobenzoyl chloride. Yield: 12%; white solid: mp 245–247°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 18), 141 (100); 1H NMR: δ 7.01 (t, J = 8.5 Hz, 2H, Ar HC-3′,5′), 7.14 (dd, J = 10.1, 1.8 Hz, 1H, Ar HC-5), 7.44–7.58 (m, 1H, Ar, HC-4′), 7.63 (s, 1H Ar HC-7), 10.37 ppm (br s, 1H, NH); IR (KBr): 3409 (NH), 1694 (C=O) cm−1.

4.1.5. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-3,4-difluorobenzamide (3e)

Prepared as reported previously for 3a starting from 5a and 3,4-difluorobenzoyl chloride. Yield: 14%; slightly yellowish solid: mp 226–228°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 26), 141 (100); 1H NMR: δ 7.19 (dd, J = 9.9, 1.6 Hz, 1H, Ar HC-5), 7.25–7.40 (m, 1H, Ar HC-5′), 7.62 (s, 1H Ar HC-7), 7.80–7.88 (m, 1H, Ar HC-2′), 7.90–8.02 ppm (m, 1H, Ar HC-6′); IR (KBr): 3410 (NH), 1679 (C=O) cm−1.

4.1.6. -(6-Chloro-4-fluoro-1,3-benzothiazol-2-yl)-3,5-difluorobenzamide (3f)

Prepared as reported previously for 3a starting from 5a and 3,5-difluorobenzoyl chloride. Yield: 34%; white solid: mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 36), 141 (100); 1H NMR: δ 7.10 (tt, J = 8.2, 2.2 Hz, 1H, Ar HC-4′), 7.21 (dd, J = 9.9, 1.9 Hz, 1H, Ar HC-5), 7.47–7.57 (m, 2H, Ar HC-2′,6′), 7.62–7.66 ppm (m, 1H, Ar HC-7); IR (KBr): 3414 (NH), 1681 (C=O) cm−1.

4.1.7. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-2,3-difluorobenzamide (3g)

Prepared as reported previously for 3a starting from 5b and 2,3-difluorobenzoyl chloride. Yield: 64%; white solid: mp 241–243°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 25), 141 (100); 1H NMR: δ 7.25–7.38 (m, 1H, Ar HC-4′), 7.42–7.52 (m, 1H, Ar HC-5′), 7.59 (d, J = 9.3 Hz, 1H, Ar HC-4), 7.86 (d, J = 7.1 Hz, 1H, Ar HC-7), 7.93–8.02 ppm (m, 1H, Ar HC-6′); IR (KBr): 3414 (NH), 1673 (C=O) cm−1.

4.1.8. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-2,4-difluorobenzamide (3h)

Prepared as reported previously for 3a starting from 5b and 2,4-difluorobenzoyl chloride. Yield: 64%; beige solid, mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 17), 141 (100); 1H NMR (DMSO- ): δ 7.16–7.32 (m, 1H, Ar HC-3′), 7.40–7.56 (m, 1H, Ar HC-5′), 7.80–7.96 (m overlapping d at 7.84 ppm, 1H, Ar HC-6′), 7.84 (d overlapping m at 7.80–7.96 ppm, J = 10.2 Hz, 1H, Ar HC-4), 8.32 (d, J = 7.7 Hz, 1H, Ar HC-7), 13.04 ppm (br s, 1H, NH); IR (KBr): 3420 (NH), 1671 (C=O) cm−1.

4.1.9. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-2,5-difluorobenzamide (3i)

Prepared as reported previously for 3a starting from 5b and 2,5-difluorobenzoyl chloride. Yield: 64%; white solid, mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 15), 141 (100); 1H NMR: δ 7.15–7.40 (m, 2H, Ar HC-3′,5′), 7.59 (d, J = 9.3 Hz, 1H, Ar HC-4), 7.87 (d, J = 6.9 Hz, 1H, Ar HC-7), 7.90–7.98 (m, 1H, Ar HC-6′), 10.05 ppm (br s, 1H, NH); IR (KBr): 3496 (NH), 1682 (C=O) cm−1.

4.1.10. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-2,6-difluorobenzamide (3j)

Prepared as reported previously for 3a starting from 5b and 2,6-difluorobenzoyl chloride. Yield: 70%; white solid, mp 247–249°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 18), 141 (100); 1H NMR: δ 7.03 (t, J = 9.0 Hz, 1H, Ar HC-3′,5′), 7.34 (d, J = 9.3 Hz, 1H, Ar HC-4), 7.45–7.58 (m, 1H, Ar HC-4′), 7.85 ppm (d, J = 6.9 Hz, 1H, Ar HC-7); IR (KBr): 3430 (NH), 1687 (C=O) cm−1.

4.1.11. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-3,4-difluorobenzamide (3k)

Prepared as reported previously for 3a starting from 5b and 3,4-difluorobenzoyl chloride. Yield: 62%; white solid, mp > 250°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 33), 141 (100); 1H NMR: δ 7.25–7.40 (m, 1H, Ar HC-5), 7.48 (d, J = 9.3 Hz, 1H, Ar HC-4), 7.76–7.85 (m, 1H, Ar HC-2′), 7.86 (d, J = 6.9 Hz, 1H, Ar HC-7), 7.87–7.98 ppm (m, 1H, Ar HC-6′); IR (KBr): 3408 (NH), 1682 (C=O) cm−1.

4.1.12. -(6-Chloro-5-fluoro-1,3-benzothiazol-2-yl)-3,5-difluorobenzamide (3l)

Prepared as reported previously for 3a starting from 5b and 3,5-difluorobenzoyl chloride. Yield: 55%; white solid, mp 235–237°C; GC-MS (70 eV, electron impact) m/z (%) 342 (M+, 39), 141 (100); 1H NMR (DMSO- ): δ 7.55–7.65 (m, 1H, Ar HC-4′), 7.70–7.90 (m, 3H, HC-4,-2′,6′), 8.33 (d, J = 7.4 Hz, 1H, Ar HC-7), 13.2 ppm (br s, 1H, NH); IR (KBr): 3401 (NH), 1679 (C=O) cm−1.

4.1.13. 6-Chloro-4-fluoro-1,3-benzothiazol-2-amine (5a)

A mixture of 4a (15 g, 103 mmol) and potassium thiocyanate (20 g, 206 mmol) in glacial acetic acid (250 mL) was stirred for 5 min. Bromine (24 g, 150 mmol) in glacial acetic acid (250 mL) was added dropwise to this mixture, with the temperature being kept below 30–35°C throughout the addition. Stirring was continued for an additional 1 h after addition of bromine. After cooling, the residue was removed by filtration. The filtered solution was made alkaline with 28% ammonium hydroxide, and the solid precipitate was collected and washed with water. The combined water layers were made alkaline with 28% ammonium hydroxide, and the resulting precipitate was combined with that previously collected. The combined precipitates were extracted with EtOAc. The organic layers were separated, dried (Na2SO4), and evaporated under vacuum. The crude residue was purified by column chromatography on silica gel (hexane/EtOAc 3 : 7) to give 14.6 g (70%) of 5a as a slight green solid: mp 243–245°C; GC-MS (70 eV, electron impact) m/z (%) 202 (M+, 100); 1H NMR: δ 7.08 (dd, J = 10.2, 1.9 Hz, 1H, Ar HC-7), 7.30–7.40 (m, 1H, HC-5), 5.63 ppm (br s, 1H, NH2); IR (KBr): 3461, 3074 (NH2) cm−1.

4.1.14. 6-Chloro-5-fluoro-1,3-benzothiazol-2-amine (5b)

Prepared as reported previously for 5a starting from 4b. Yield: 65%; white solid: mp 234–236°C; GC-MS (70 eV, electron impact) m/z (%) 202 (M+, 100); 1H NMR: δ 7.30 (d, J = 9.9 Hz, 1H, Ar HC-4), 7.56 (d, J = 6.9 Hz, 1H, Ar HC-7), 5.45 ppm (br s, 1H, NH2); IR (KBr): 3473, 3083 (NH2) cm−1.

4.2. Biology
4.2.1. Antibacterial Studies

The in vitro minimum inhibitory concentrations (MICs, μg/mL) were assessed by the broth microdilution method, using 96-well plates, according to CLSI guidelines [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 μg/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. 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 × 106 CFU/mL. All wells were seeded with 100 μL 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.

4.2.2. 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 × 106 CFU/mL. All wells were seeded with 100 μL 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.

Acknowledgment

This work was accomplished thanks to the financial support of the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR).

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