Novel Benzoxazoles: Synthesis and Antibacterial, Antifungal, and Antitubercular Activity against Antibiotic-Resistant and -Sensitive Microbes

(1)

Introduction

Tuberculosis, gonorrhea, malaria, and child- hood ear infections are just a few diseases that have become hard to treat with antibiotic drugs.

Especially tuberculosis still remains a major pub- lic health problem, and it is estimated that new infections of humans by Mycobacterium tubercu- losis are greater than 8 million annually and more than 3 million people die from this disease each year (Kochi et al., 2002). The struggle against bacterial infections has resulted in the development of a wide variety of antibiotics. After years of mis- use of antibiotics bacteria have become antibiotic-resistant, resulting in a potential global health crisis. Also many hospital-acquired infections are resistant to the most powerful antibiotics available, like methicillin and vancomycin. These drugs are reserved to treat only the most intractable infections to slow the development of resistance to them (Fridgin and Gaynes, 1999). So, there is an urgent need for new classes of antitubercular and antimicrobial agents.

Benzoxazoles are structural isosters of natural nucleotides which interact easily with biopoly- mers, and they constitute an important class of heterocyclic compounds with antitubercular, antimicrobial, and antibiotic activities (Prudhomme et al., 1986; Haansuu et al., 2001; Kochi et al., 2002;

Temiz-Arpaci et al., 2002a, b; Sarma et al., 2003;

Vinsova et al., 2005, 2006). The benzoxazole de- rivative calcimycin is a carboxylic polyether antibiotic from a strain of Streptomyces chartreusis (NRRL 3882). It is highly active against Gram- positive bacteria including some Bacillus and Mi- crococcus strains (Prudhomme et al., 1986).

In recent years, we have described the synthesis of various derivatives of some 2,5-disubstituted benzoxazoles and their in vitro antimicrobial activity against some Gram-positive and Gram-negative bacteria and the fungus Candida albicans (Temiz-Arpaci et al., 2002a, b, 2008;

Alper-Hayta et al., 2008; Arisoy et al., 2012). In the present study, we aimed to develop new ef- fective antitubercular and antimicrobial agents possessing benzoxazole nuclei in their structure.

Antitubercular Activity against Antibiotic-Resistant and -Sensitive Microbes

Mustafa Arisoy^a, Ozlem Temiz-Arpaci^a,*, Fatma Kaynak-Onurdag^b, and Selda Ozgen^b

a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100, Tandogan, Ankara, Turkey. Fax: + 90 (312) 213 10 81. E-mail: temiz@pharmacy.ankara.edu.tr

b Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06330, Etiler, Ankara, Turkey

* Author for correspondence and reprint requests

Z. Naturforsch. 68 c, 453 – 460 (2013); received November 2, 2012/November 11, 2013 A new series of 5-(p-substituted benzamido/phenylacetamido)-2-(p-tert-butylphenyl)benzoxazole derivatives were synthesized and evaluated for their antibacterial, antifungal, and antimycobacterial activities against antibiotic-resistant and -sensitive Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuber- culosis as well as against Candida albicans and Candida krusei. The compounds possessed broad-spectrum activity against all of the tested Gram-positive and Gram-negative bacteria and yeasts, their minimum inhibitory concentrations (MICs) ranging between 16 – 128 μg/ml.

One compound exhibited signiﬁ cant antibacterial activity (16 μg/ml) against an antibiotic- resistant Enterococcus faecalis isolate, having twice the potency of the compared standard drugs vancomycin and gentamycin sulfate. The compounds also showed moderate antitubercular activity with MIC values between 8 – 128 μg/ml against Mycobacterium tuberculosis and its clinical isolate.

Key words: Benzoxazoles, Antibacterial Activity, Antifungal Activity, Antitubercular Activity

(2)

Here, we describe the synthesis of a series of 5-(p-substituted benzamido/phenylacetamido)- 2-(p-tert-butylphenyl)benzoxazole derivatives as a new class of synthetic antitubercular and antimicrobial agents along with results of testing their in vitro antitubercular and antimicrobial activities against some Gram-negative and Gram-positive bacteria as well as Mycobacterium tuberculosis, Candida albicans, and Candida krusei. We put a p-tert-butylphenyl moiety in position 2 of the benz oxazole ring. We considered that this lipophilic group might improve the antitubercular activity, because it permits an easier penetration of the molecule through the lipophilic mycobacterial cell walls.

Material and Methods

Chemicals and analytical methods

The chemicals and solvents were purchased from Sigma-Aldrich (Munich, Germany) and Fisher Scientifi c (Pittsburgh, PA, USA); they were used without purifi cation. Silica gel HF254 chroma- toplates (0.3 mm) were used for thin-layer chro- matography, and the mobile phase was chloro- form/methanol (10:0.5, v/v). Melting points were recorded on a Stuart Scientifi c SMP 1 instrument (Bibby Scientifi c Limited, Stone, Staffordshire, UK) and are uncorrected. NMR spectra were recorded on a Varian Mercury 400 MHz NMR spectrometer (Palo Alto, CA, USA) in CDCl3 or dimethylsulfoxide (DMSO-d6); tetramethylsilane (TMS) was used as an internal standard. The mass spectra were recorded on a Waters ZQ Micro- mass LC-MS spectrometer (Milford, MA, USA) using the ESI(+) method.

Materials for microbiology

Materials used in the microbiology study were;

Mueller Hinton agar (MHA) (Merck, Darm- stadt, Germany), Mueller Hinton broth (MHB) (Merck), Sabouraud dextrose agar (SDA) (Merck), RPMI-1640 medium with L-glutamine (Sigma-Aldrich), 3-(N-morpholino)-propane- sulfonic acid (MOPS) (Sigma-Aldrich), 96-well microplates (BD, Franklin Lakes, NJ, USA), ampicillin (Mustafa Nevzat Pharmaceuticals, Is- tanbul, Turkey), gentamycin sulfate (Paninkret Chem.-Pharm. Werk GmbH, Westerhorn, Ger- many), oﬂ oxacin (Zhejiang Huangyan East Asia Chemical Co. Ltd., Huangyan, Zhejiang, China),

vancomycin (Mayne Pharma, Salisbury South, SA, Australia), meropenem (Astra Zeneca, Istan- bul, Turkey), ﬂ uconazole (Sigma-Aldrich), amphotericin B (Riedel de Haen, Seelze, Germany), isoniazid (Sigma-Aldrich), ethambutol (Sigma- Aldrich), DMSO (Riedel de Haen).

Microorganisms

Microorganisms used in the assay were; Kleb- siella pneumoniae clinical isolate [extended beta lactamase spectrum (ESBL)], Escherichia coli isolate (ESBL), Enterococcus faecalis isolate [resistant to vancomycin (VRE)], and Staphylococcus aureus isolate [resistant to methicilline (MRSA)], Klebsiella pneumoniae RSKK 574, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Can- dida albicans ATCC 10231, Candida krusei ATCC 6258, Mycobacterium tuberculosis H37RV ATCC 27294, and a clinical isolate of Mycobacterium tu- berculosis. Reference strains and clinical isolates were provided by Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Micro- biology, Culture Collection, Ankara, Turkey, and Gazi University Hospital Microbiology Labora- tory, Ankara, Turkey, respectively.

General procedure for the preparation of the 5-(p-substituted benzamido/phenylacetamido)-2- (p-tert-butylphenyl)benzoxazoles 6 – 20

Firstly, 5-amino-2-(p-tert-butylphenyl)benzoxazole (3) was synthesized by heating 0.02 mol 2,4-diaminophenol · 2 HCl (1) with 0.02 mol p- tert-butylbenzoic acid (2) in 25 g polyphosphoric acid (PPA) and stirring the resulting mixture for 3 – 4 h. At the end of the reaction period, the resi- due was poured into an ice/water mixture, and the solution was neutralized with 10% NaOH.

The resulting precipitate was fi ltered, washed with distilled water, dissolved in boiling ethanol with 0.2 g charcoal, and fi ltered. Distilled water was slowly added to the fi ltrate in order to stimu- late crystallization. The crude compound 3 was obtained by fi ltering and drying the crystalline material (Temiz-Arpaci et al., 2008). Then, appropriate carboxylic acids 4 (0.5 mmol) and thionyl chloride (1.5 ml) were refl uxed in benzene (5 ml) at 80 °C for 3 h. Excess thionyl chloride was re- moved in vacuo. The appropriate carboxylic acyl chlorides 5 were dissolved in diethyl ether (10 ml), and this solution was added, during 1 h,

(3)

to a stirred, ice-cold mixture of 5-amino-2-(p-tert- butylphenyl)benzoxazole (3) (0.5 mmol), sodium bicarbonate (0.5 mmol), diethyl ether (10 ml), and water (10 ml). The mixture was kept stirred overnight at room temperature and then ﬁ ltered. The precipitate was washed with water, 2 M HCl, and water, respectively, and ﬁ nally with diethyl ether to give compounds 6 – 20. The products were re- crystallized from ethanol/water as needles which were dried in vacuo. Physical and spectral data of compounds 6 – 20 are reported in Table I.

Microbiological assays

For microbiological assays, the standard antibio tics were dissolved in the appropriate solvents recommended by Clinical and Laboratory Standards Institute guidelines (CLSI, 2006, 2008).

Stock solutions of the test compounds were prepared in DMSO.

Bacterial susceptibility testing was performed according to the guidelines of CLSI (M100-S18;

CLSI, 2008). MHB was added to each well of the microplates. The bacterial suspensions used for inoculation were prepared at 10⁶ CFU/ml by diluting fresh cultures at McFarland 0.5 density. Suspensions of the bacteria at 10⁶ CFU/ml were inoculated to the two-fold diluted solution of the compounds. A 10-μl bacterium inoculum was added to each well of the microplates. There were 10⁵ CFU/ml bacteria in the wells after in-

oculations. Microplates were incubated at 37 °C overnight.

Fungal susceptibility testing was performed according to the guidelines of CLSI (M27-A3; CLSI, 2006). RPMI-1640 medium with L-glutamine buf- fered to pH 7 with MOPS was added to each well of the microplates. The colonies were suspended in sterile saline, and the resulting suspension was adjusted to McFarland 0.5 density. A working suspension was prepared by appropriate dilution of the stock suspension. A 10-μl inoculum was added to each well of the microplates resulting in 10³ CFU/ml yeasts in the wells. Microplates were incubated at 35 °C for 24 – 48 h.

After incubation, the lowest concentration of the compounds that completely inhibited mac- roscopic growth was determined and reported as minimum inhibitory concentration (MIC). All solvents and diluents, pure microorganisms, and pure media were used in control wells. All experi- ments were done in three parallel series. The data on the antimicrobial activity of the compounds and the control drugs as MIC values (μg/ml) are given in Table II.

Results and Discussion

The synthetic pathway for preparation of the target compounds is shown in Scheme 1. First, 5-amino-2-(p-tert-butylphenyl)benzoxazole (3)

Fig. 1. Synthesis of the target compounds 6 – 20.

(4)

was obtained by heating p-tert-butylbenzoic acid (2) with 2,4-diaminophenol (1) in polyphosphoric acid (PPA) (Temiz-Arpaci et al., 2008). Com- pounds 6 – 20 were obtained by reaction of 3 with p-substituted benzoic or phenylacetic acyl chlo- rides 5 that were synthesized by treating appropriate benzoic or phenylacetic acids with thionyl chloride (Temiz-Arpaci et al., 2002a). The struc- tures of the 2,5-disubstituted benzoxazoles 6 – 20 synthesized were elucidated by IR, mass, and ¹H NMR spectroscopy. Physical and spectral data of the synthesized benzoxazole derivatives are given in Table I.

The IR spectra of the ﬁ nal compounds showed the characteristic C=O stretching band in the 1636 – 1670 cm^-1region. Besides, C=N stretching bands were observed in the 1488 – 1544 cm^-1 region. In the ¹H NMR spectra of compounds 7 – 10, 12 – 14 the signal of the NH proton was invisible, and in the other compounds the signal of the NH proton was observed at δH 10.77 – 8.95 ppm as a singlet band. Benzylic CH2 protons were observed at δH 3.89 – 3.60 ppm as a singlet band for compounds 7, 12, 15, and 18 – 20. Aromatic me- thyl protons appeared at δH 3.00 and 2.26 ppm for compounds 13 and 15, respectively, as a singlet band. Aromatic protons were observed in the ex- pected regions. Mass spectra of the compounds showed [M⁺ + H] peaks, since the electrospray ionization method was employed.

In vitro antimicrobial and antitubercular activi- ty results of the compounds tested are given in Table II. The synthesized compounds were found to possess MIC values between 32 – 128 μg/ml for Gram-negative bacteria. Compounds 17, 19, and 20 were the most potent derivatives (MIC 32 μg/

ml) against the drug-resistant Escherichia coli isolate, having twice the potency of the standard drug oﬂ oxacin. Thus, the compounds having a p- cyanophenylacetamido, p-nitrophenylacetamido or p-bromophenylacetamido group in position 5 of the benzoxazole ring had higher activity against the Escherichia coli isolate than the other tested compounds. However, all compounds showed lower antibacterial activity against the antibiotic- sensitive Escherichia coli strain (MIC 32 – 64 μg/

ml) than did the control drugs. All synthesized compounds were found to have a more potent antibacterial activity (MIC 64 – 128 μg/ml) against

the drug-resistant Klebsiella pneumoniae isolate than did gentamycin sulfate (MIC 256 μg/ml).

As is evident from Table II, the synthesized compounds showed activity with MIC values of 16 – 128 μg/ml against the Gram-positive bacteria, such as Staphylococcus aureus and Enterococcus faecalis and their drug-resistant isolates. Com- pound 20 was the most active one (MIC 16 μg/

ml) against the Enterococcus faecalis isolate, having twice the potency of the standard drugs vancomycin and gentamycin sulfate, and compound 19 showed the same activity as these standard drugs. These results indicate that the presence of the phenylacetamido moiety with a bromine atom or nitro group as the substituent at position 5 increases the potency against the drug-resistant Enterococcus faecalis isolate.

Moreover, all of the synthesized compounds exhibited weak antifungal activity against Can- dida albicans and Candida krusei with MIC val- ues of 64 μg/ml and were thus less active than the tested reference drugs amphotericin B and ﬂ uconazole. Compounds 6 – 20 showed the same activity against Candida krusei as did the standard drug ﬂ uconazole.

The synthesized compounds possessed in vit- ro activity against Mycobacterium tuberculosis and its clinical isolate. The MIC values were in the range of 8 – 128 μg/ml. The most active com- pounds in the series – 6, 7, and 15 – had MIC values of 8 μg/ml. It can be concluded that the compounds having a hydrogen atom or ﬂ uorine atom or a methyl group as the substituent R of the benzamido/phenylacetamido moiety have increased potency against Mycobacterium tuber- culosis. However, none of the synthesized compounds showed better antitubercular activity than did the standard drugs isoniazid and ethambutol.

These observations may serve as a starting point for the design of further antimicrobial and antitubercular drugs.

Acknowledgements

This work was supported by Ankara Universi- ty Research Fund (Grant No. 12B 3336001). The Central Laboratory of the Faculty of Pharmacy of Ankara University supported the acquisition of the NMR and mass spectra and elemental analy- ses in this work.

(5)

Table I. Physical properties and spectral data of the compounds 6 – 20. CompoundRXFormulaM.p. [°C]Yield (%)IR [cm-1]

1 H NMR (δ in ppm,J in Hz)MS [m/z (%)] 6a H-C24H22N2O223847

3278, 3066, 2958, 1636, 1541, 1478, 1287, 925 – 552 8.95 (1H, s), 8.24 – 8.22 (2H, d,J = 8.0), 8.18 – 8.16 (1H, d, J = 8.0), 8.05 – 7.95 (3H, m), 7.57 – 7.50 (6H, m), 1.38 (9H, s)371 (57) [M+ + H] 7FCH2C25H23FN2O219832

3257, 3068, 2962, 1645, 1509, 1479, 1229, 864 – 553 8.17 – 8.15 (2H, d, J = 8.0), 7.88 (1H, s), 7.80 (1H, s), 7.54 – 7.44 (3H, m), 7.34 – 7.31 (2H, d,J = 8.8), 7.07 – 7.03 (2H, d,J = 8.8), 3.74 (2H, s), 1.36 (9H, s), -NH invisible

403 (100) [M+ + H] 8a F-C24H21FN2O221745

3311, 3063, 2962, 1644, 1571, 1509, 1473, 1231, 885 – 550 8.18 – 8.17 (1H, d, J = 2.0), 8.16 – 8.15 (1H, d, J = 2.0), 7.98 – 7.97 (1H, d, J = 2.0), 7.93 – 7.90 (2H, m), 7.63 – 7.62 (1H, d,J = 8.0,J' = 2.0), 7.56 – 7.53 (3H, m), 7.19 – 7.14 (2H, m), 1.35 (9H, s), -NH invisible

389 (100) [M+ + H] 9Br-C24H21BrN2O222063

3310, 3065, 2961, 1644, 1566, 1488, 1267, 936 – 550 8.18 – 8.15 (2H, d,J = 8.4), 7.99 (1H, s), 7.93 (1H, s), 7.78 – 7.62 (2H, d,J = 8.0), 7.64 – 7.60 (2H, m), 7.56 – 7.53 (3H, m), 1.37 (9H, s), -NH invisible

449 (97) [M+ + H], 451 (100) [M+ + H + 2] 10C2H5-C26H26N2O222332

3290, 2958, 1637, 1552, 1479, 1346 – 1319, 1252, 1186 – 1163, 870 – 508 8.17 – 8.15 (2H, d,J = 8.4), 8.01 – 7.99 (2H, d, J = 2.0), 7.83 – 7.81 (2H, d,J = 8.4), 7.66 – 7.63 (1H, dd,J = 8.0,J' = 2.0), 7.55 – 7.52 (3H, m), 7.31 – 7.29 (1H, d,J = 7.6), 2.74 – 2.69 (2H, q), 1.37 (9H, s), 1.28 – 1.24 (3H, t), -NH invisible

399 (100) [M+ + H] 11NO2-C24H21N3O426936

3299, 3112, 2968, 1644, 1571, 1472, 1268, 879 – 553 10.77 (1H, s), 8.42 – 8.39 (2H, d,J = 8.8), 8.28 – 8.22 (3H, m), 8.16 – 8.14 (2H, d,J = 8.4), 7.82 – 7.77 (2H, dd,J = 8.4,J' = 1.6), 7.66 – 7.64 (2H, d,J = 8.4), 1.35 (9H, s)

416 (100) [M+ + H] 12aHCH2C25H24N2O215631

3248, 3063, 2962, 1663, 1538, 1473, 1260, 877 – 556 8.16 – 8.14 (2H, dd,J = 8.4,J' = 1.6), 7.85 – 7.84 (1H, d,J = 2.0), 7.54 – 7.52 (2H, dd,J = 8.8, J' = 2.0), 7.48 – 7.34 (7H, m), 3.79 (2H, s), 1.37 (9H, s), -H invisible

385 (100) [M+ + H] 13aCH3-C25H24N2O223435

3309, 3027, 2959, 1665, 1537, 1481, 1246, 890 – 501 8.18 – 8.15 (2H, dd,J = 8.0, J' = 2.0), 8.01 (1H, s), 7.99 – 7.98 (1H, d,J = 2.0), 7.81 – 7.79 (2H, d, J = 8.0), 7.66 – 7.63 (1H, dd,J = 8.8,J' = 2.4), 7.56 – 7.52 (3H, m), 7.29 – 7.27 (1H, d,J = 8.4), 3.00 (3H, s), 1.37 (9H, s), -NH invisible

385 (100) [M+ + H]

(6)

CompoundRXFormulaM.p. [°C]Yield (%)IR [cm-1]

1 H NMR (δ in ppm,J in Hz)MS [m/z (%)] 14a Cl-C24H21ClN2O222156

3308, 3105, 2963, 1643, 1568, 1472, 1267, 885 – 527 8.19 – 8.17 (2H, d,J = 8.0), 8.00 (1H, s), 7.91 – 7.84 (3H, m), 7.64 – 7.55 (3H, m), 7.50 – 7.48 (2H, d, J = 8.0) 1.37 (9H, s), -NH invisible

405 (100) [M+ + H], 407 (37) [M+ + H + 2] 15CH3CH2C26H26N2O219255

3223, 2966, 1645, 1612, 1516, 1477, 1267, 839 – 535 10.30 (1H, s), 8.12 – 8.09 (3H, dd,J = 8.8,J' = 2.4), 7.70 – 7.50 (4H, m), 7.24 – 7.22 (2H, d,J = 8.0), 7.13 – 7.11 (2H, d,J = 8.0) 3.61 (2H, s), 2.26 (3H, s), 1.31 (9H, s)

399 (100) [M+ + H] 16C4H9-C28H30N2O222451

3234, 2958, 2935, 2860, 1640, 1610, 1510, 1475, 1269, 952 – 567 10.32 (1H, s), 8.26 – 8.256 (1H, d,J = 2.0), 8.13 – 8.11 (2H, d,J = 8.8), 7.91 – 7.89 (2H, d, J = 8.0), 7.73 – 7.77 (2H, d,J = 1.2), 7.63 – 7.61 (2H, d,J = 8.8), 7.36 – 7.34 (2H, d,J = 8.0), 3.31 (2H, t), 2.63 – 2.67 (2H, m), 1.61 – 1.54 (2H, m), 1.30 (9H, s), 0.91 – 0.88 (3H, t)

427 (100) [M+ + H] 17CN-C25H21N3O226643

3363, 3074, 2962, 2910, 1670, 1618, 1544, 1479, 1280, 1247, 964 – 547 10.67 (1H, s), 8.28 – 8.27 (1H, d,J = 1.6), 8.16 – 8.13 (4H, m), 8.07 – 8.05 (2H, d,J = 8.0), 7.78 – 7.73 (2H, m), 7.66 – 7.64 (2H, d,J = 8.0), 1.34 (9H, s)396 (100) [M+ + H] 18OCH3CH2C26H26N2O320747

3307, 3167, 3082, 2953, 1668, 1608, 1514, 1475, 1250, 950 – 613 10.32 (1H, s), 8.13 – 8.11 (3H, d,J = 8.8), 7.72 – 7.70 (1H, d,J = 8.8), 7.65 – 7.62 (2H, d,J = 8.4), 7.54 – 7.52 (1H, d,J = 8.4), 7.29 – 7.27 (2H, d, J = 8.0), 6.91 – 6.89 (2H, d,J = 8.4), 3.74 (3H, s), 3.60 (2H, s), 1.35 (9H, s)

415 (100) [M+ + H] 19NO2CH2C25H23N3O426335

3257, 3080, 2962, 1658, 1604, 1530, 1469, 1340, 1230, 925, 844 – 533

10.49 (1H, s), 8.24 – 8.22 (2H, d,J = 8.4), 8.14 – 8.11 (3H, m), 7.74 – 7.62 (5H, m), 7.54 – 7.52 (1H, dd, J = 8.4,J' = 2.0), 3.89 (2H, s), 1.33 (9H, s)430 (100) [M+ + H] 20bBrCH2C25H23BrN2O220065

3244, 3076, 2958, 2864, 1647, 1610, 1523, 1475, 1267, 1012, 958 – 578 10.37 (1H, s), 8.13 – 8.11 (3H, m), 7.73 – 7.71 (1H, d, J = 8.0), 7.64 – 7.62 (2H, d,J = 8.0), 7.56 – 7.51 (3H, m), 7.33 – 7.31 (2H, d,J = 8.0), 3.70 (2H, s), 1.31 (9H, s)

463 (97) [M+ + H], 465 (100) [M+ + H + 2] a These derivatives are commercially available. b Bhagyasree et al. (2013).

Table I continued.

(7)

Table II. Antimicrobial and antitubercular activity (MIC in μg/ml) of the synthesized compounds 6 – 20 and the standard drugs. CompoundMicroorganisma E.cE.c.*K.p.K.p.*S.a.S.a.*E.f.E.f.*C.a.C.k.M.t.M.t.* 6326464646412864128646488 732643212812812864128646488 83264321281281286412864641632 9326432128128128641286464128128 103264321281281286412864643232 113264321281281286412864643216 12326432128128128641286464832 13326432128128128641286464168 143264321281281286412864646416 1532643212812812864128646488 163264321281281286412864643216 173232321281281286412864646432 1864646464128128646464646416 1964326464128128643264643232 206432646412812864166464168 Vancomycinn.d. bn.d.n.d.n.d.11132n.d.n.d.n.d.n.d. Gentamycin sulfate110240.52560.12532432n.d.n.d.n.d.n.d. Meropenem0.0625< 0.06250.03< 0.06250.125-20.5n.d.n.d.n.d.n.d. Oﬂ oxacin< 0.0625640.1250.50.1250.2514n.d.n.d.n.d.n.d. Ampicillin2> 20482> 20480.5> 204810.5n.d.n.d.n.d.n.d. Amphotericin Bn.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.0.252n.d.n.d. Fluconazolen.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.164n.d.n.d. Isoniazidn.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.< 0.25< 0.25 Ethambutoln.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.22 a E.c., Escherichia coli ATCC 25922; E.c.*, Escherichia coli isolate (ESBL); K.p., Klebsiella pneumoniae RSKK 574; K.p.*,Klebsiella pneumoniae isolate (ESBL); S.a., Staphylococcus aureus ATCC 29213; S.a.*, Staphylococcus aureus isolate (MRSA); E.f., Enterococcus faecalis ATCC 29212; E.f.*, Enterococcus faecalis isolate (VRE); C.a., Candida albicans ATCC 10231;C.k., Candida krusei ATCC 6258; M.t.,Mycobacterium tuberculosis H37RV ATCC 27294; M.t.*,Mycobacterium tuberculosis isolate. b n.d., not determined (microbiological assays were not performed due to following reasons: antibacterial drugs were not assayed against fungi and M. tuberculosis; antifungal drugs were not assayed against bacteria and M. tuberculosis; antitubercular drugs were not assayed against bacteria and fungi; Gram-negative bacteria employed in the study are naturally resistant to vancomycin).

(8)

Alper-Hayta S., Arisoy M., Temiz-Arpaci O., Yildiz I., Akı E., Özkan S., and Kaynak F. (2008), Synthesis, antimicrobial activity, pharmacophore analysis of some new 2-(substituted phenyl/benzyl)-5-[(2-benzo- furyl)carboxamido]benzoxazoles. Eur. J. Med. Chem.

45, 2568 – 2578.

Arisoy M., Temiz-Arpaci O., Kaynak-Onurdag F., and Ozgen S. (2012), Synthesis and antimicrobial activity of novel benzoxazoles. Z. Naturforsch. 67c, 466 – 472.

Bhagyasree J. B., Samuel J., Varghese H. T., Panicker C.

Y., Arisoy M., and Temiz-Arpaci O. (2013), Synthe- sis, FT-IR investigation and computational study of 5-(4-bromo-phenylacetamido)-2-(4-tert-butylphenyl) benzoxazole. Spectrochim. Acta A 115, 79 – 91.

CLSI (2006), Reference method for broth dilution antifungal susceptibility testing yeast; approved standard, M27-A3. Clinical and Laboratory Standards Insti- tute, Wayne, PA, USA.

CLSI (2008), Performance standards for antimicrobial susceptibility testing; 16th informational supplement, CLSI M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA, USA.

Fridgin S. K. and Gaynes R. P. (1999), Antimicrobial resistance in intensive care units. Clin. Chest. Med.

20, 303 – 316.

Haansuu J. P., Klika K. D., Söderholm P. P., Ochaveranko V. V., Pihlaja K., Haahtela K. K., and Vuorela P. M.

(2001), Isolation and biological activity of franki- amide. J. Ind. Microbiol. Biotechnol. 27, 62 – 66.

Kochi J., Klimesova V., Waisser K., Kaustova J., Dahse H. M., and Möllmann U. (2002), Heterocyclic benz- azole derivatives with antimycobacterial in vitro activi ty. Bioorg. Med. Chem. Lett. 12, 3275 – 3278.

Prudhomme M., Guyot J., and Jeminet G. (1986), Semi- synthesis of A23187 (calcimycin) analogs. IV. Cation carrier properties in mitochondria of analogs with modiﬁ ed benzoxazole rings. Antimicrobial activity. J.

Antibiot. 39, 934 – 937.

Sarma H. K., Sharma B. K., and Tiwari S. C. (2003), A novel calcimycin antibiotic from Gram-positive actinomycete Frankia microsymbiont. Curr. Sci. 85, 1401 – 1403.

Temiz-Arpaci O., Aki-Sener E., Yalçin I., and Altanlar N.

(2002a), Synthesis and antimicrobial activity of some 2-[p-substituted-phenyl]benzoxazol-5-yl-arylcarboxy- amides. Arch. Pharm. (Weinheim) 335, 283 – 288.

Temiz-Arpacı O., Akı-Şener E., Yalçın İ., and Altan- lar N. (2002b), Synthesis and microbiological activity of some novel N-[2-(p-substituted phenyl)- 5-benzoxazolyl]-cyclohexyl carboxamide, -cyclohexyl acetamide and -cyclohexyl propionamide derivatives.

Farmaco 57, 771 – 775.

Temiz-Arpaci O., Yildiz I., Ozkan S., Kaynak F., Aki- Sener E., and Yalçin I. (2008), Synthesis and biological activity of some new benzoxazoles. Eur. J. Med.

Chem. 43, 1423 – 1431.

Vinsova J., Horak V., Buchta V., and Kaustowa J. (2005), Highly lipophilic benzoxazoles with potential antibacterial activity. Molecules 10, 783 – 793.

Vinsova J., Cermakova K., Tomeckova A., Ceckova M., Jampilek J., Cermak P., Kunes J., Dolezal M., and Staud F. (2006), Synthesis and antimicrobial evalu- ation of new 2-substituted-5,7-di-tert-butylbenzoxa- zoles. Bioorg. Med. Chem. 14, 5850 – 5865.