Synthesis and antimicrobial activities of novel 6-(1,3-thiazol-4-yl)-1,3- benzoxazol-2(3H)-one derivatives

Article abstract of DOI:10.1515/hc-2013-0147

Synthesis, characterization and investigation of antimicrobial activities of seven new 6-(1,3-thiazol-4-yl)-1,3-benzoxazol-2(3H)-ones are presented. Their structures were determined using ¹H NMR, ¹³C NMR and elemental analyses. The compounds possess some biological activity against Gram-positive bacteria, especially against Micrococcus luteus belonging to opportunistic pathogens, with an MIC of 31.25 μg/mL.

Full text of DOI:10.1515/hc-2013-0147

ꢁꢁꢁꢂ
ꢀHeterocycl. Commun. 2014; 20(1): 41–46
DOI 10.1515/hc-2013-0147ꢀ
Krzysztof Z. Łączkowski*, Konrad Misiura, Anna Biernasiuk, Anna Malm and Izabela Grela
Synthesis and antimicrobial activities of novel
6-(1,3-thiazol-4-yl)-1,3-benzoxazol-2(3H)-one
derivatives
Abstract: Synthesis, characterization and investigation of (S-14080) have been studied in Phase II clinical trials as
antimicrobial activities of seven new 6-(1,3-thiazol-4-yl)- analgesic drugs (Figure 1) [12].
1,3-benzoxazol-2(3H)-ones are presented. Their structures
Another group of very important pharmacophores are
1
13
were determined using H NMR, C NMR and elemental thiazoles and their derivatives, which have been widely
analyses. The compounds possess some biological activ- studied in medicinal chemistry because of their varied
ity against Gram-positive bacteria, especially against Mic- biological activities such as antibacterial [13–15], antifun-
rococcus luteus belonging to opportunistic pathogens, gal [16–18], anticancer [19] and antiproliferative activities
with an MIC of 31.25 μg/mL.
[20]. This class of drugs prevents conversion of lanosterol
into ergosterol, and accumulation of 14α-methyl sterols
Keywords: antimicrobial drugs; benzoxazolones; through inhibition of the fungal cytochrome P450 enzyme
DFT calculation; Gram-positive bacteria; thiazoles; 14α-demethylase [21, 22].
thiosemicarbazones.
Considering the above suggestions and continuing
our previous investigation on the synthesis and properties
of biologically active heterocycles [23–26], it was decided
to prepare a new series of biologically active agents con-
taining both benzoxazolone and thiazole pharmaco-
phores. These compounds were evaluated for their in vitro
antibacterial and antifungal activities against a panel of
reference strains of 25 microorganisms.
*Corresponding author: Krzysztof Z. Łączkowski, Faculty of
Pharmacy, Department of Chemical Technology and Pharmaceuticals,
Collegium Medicum, Nicolaus Copernicus University, Jurasza 2, 85-089
Bydgoszcz, Poland, e-mail: krzysztof.laczkowski@cm.umk.pl
Konrad Misiura: Faculty of Pharmacy, Department of Chemical
Technology and Pharmaceuticals, Collegium Medicum, Nicolaus
Copernicus University, Jurasza 2, 85-089 Bydgoszcz, Poland
Anna Biernasiuk and Anna Malm: Faculty of Pharmacy, Department
of Pharmaceutical Microbiology, Medical University, Chodźki 1,
20-093 Lublin, Poland
Results and discussion
Izabela Grela: Faculty of Chemical Technology and Engineering,
University of Technology and Life Sciences, Seminaryjna 3,
85-326 Bydgoszcz, Poland
Chemistry
Initially, thiosemicarbazones were obtained by the reac-
tion of appropriate ketones with thiosemicarbazide
in absolute ethyl alcohol in the presence of catalytic
Introduction
The observed increasing number of multidrug-resistant amounts of acetic acid and under reflux. The desired ben-
pathogens leads to the unprecedented interest in synthe- zoxazolone-thiazole conjugates 6–12 were synthesized via
sis of new antimicrobial drugs [1]. Benzoxazolone deriva- the Hantzsch condensation reaction of appropriate thio-
tives are an important class of heterocyclic compounds semicarbazones with 6-(2-chloroacetyl)-benzo[d]oxazol-
widely studied in medicinal chemistry because they show 2(3H)-one (13) in absolute ethyl alcohol and under reflux
a broad spectrum of activity against Gram-positive and with good yields (48–84%) and high chemical purity. The
Gram-negative bacteria and fungi [2–8], human immuno- reaction pathway is summarized in Scheme 1.
deficiency virus (HIV)-1 reverse transcriptase [9] and mul-
The product 6 was obtained as a mixture of isomers
tidrug-resistant cancer cells [10, 11]. Various derivatives of E/Z in a 68:32 ratio; however, a careful separation using
benzoxazolone are commercially available, for example, column chromatography gave pure, more stable (E)-iso-
benzolon 1 (myorelaxant), paraflex 2 (sedative anelgesic) mer of compound 6. Additionally, geometry optimization
and vinizene 3 (topical antiseptic) [12]. The benzoyl-ben- of 6 was carried out at the DFT B3LYP/6-311++G** level
zoxazolone 4 (10194 CERM) and its sulfur bioisoster 5 of theory using the Gaussian 09 code [27]. The resulting

ꢁꢁꢁꢂ
42ꢀ
ꢀK.Z. Łączkowski et al.: 6-(1,3-Thiazol-4-yl)-1,3-benzoxazol-2(3H)-one derivatives
H
N
O
O
H
N
O
13
O
R
HN
R
S
Cl
N
O
HN
EtOH, ∆, 2 h
NH2
S
6-12
Me
6: R =
7: R =
8: R =
9: R =
10: R =
11: R =
N
N
N
N
N
12: R =
Me
N
Me
N
Scheme 1ꢀSynthesis of benzoxazolone-thiazole conjugates 6–12.
Me
N
the Boltzmann constant, and T being the temperature
(Tꢀ= ꢀ298.15 K) [29]. The E- and the Z-isomers were shown
to constitute 81% and 19% of the population, respec-
tively. This result is in a good qualitative agreement with
the experimental one (68% and 32%, respectively). The
structures and purity of all compounds (6–12) were con-
H
N
H
N
Cl
Cl
O
O
O
O
O
O
Br
1
2
3
H
N
H
N
O
O
1
13
Ph
Ph
firmed by H NMR, C NMR, elemental analyses and by
TLC on silica gel. The NMR spectral data were fully con-
O
S
O
O
4
5
1
sistent with the assigned structures. H NMR spectra of
compounds show a singlet at δ 7.17–7.22 due to the thia-
zole-5H proton and a singlet at δ 10.80–11.95 indicating
the presence of hydrazide NH proton, which confirms
Figure 1ꢀCommercial benzoxazolone derivatives 1–5.
electronic energy of the (Z)-isomer was 0.87 kcal/mol the conversion of substrates to the expected products.
higher than that of the (E)-isomer. The fractional popula- All compounds gave satisfactory elemental analysis. All
tion X of isomers j (E, Z) (Figure 2) was calculated using reactions were repeated at least two times and are fully
the Boltzmann’s distribution [28] as:
reproducible.
exp(-∆jE/kT)
Xj =
exp(-∆EE/kT) + exp(-∆ZE/kT)
H
H
N
N
Me
O
O
O
N
HN
N
HN
N
N
O
Me
S
S
6 (Z)
6 (E)
Relative ∆E [kcal/mol]
Theoretical E/Z ratio
Experimental E/Z ratio
0.87
81
0
19
32
68
Figure 2ꢀThe fractional population X of isomers j (E, Z).
with ΔjE denoting the relative electronic energy of
isomer j (calculated with regard to the energy of the
Microbiology
lower-energy isomer E, i.e., the relative energy is 0 and Potency was defined as follows: no bioactivity with
0.87 kcal/mol for isomers E and Z, respectively), k being MIC ꢀ> ꢀ1000 μg/mL, mild bioactivity with MIC in the

ꢂꢁꢁꢁ
K.Z. Łączkowski et al.: 6-(1,3-Thiazol-4-yl)-1,3-benzoxazol-2(3H)-one derivativesꢀ
ꢀ43
a
Table 1ꢁAntibacterial activity data in MIC (MBC) (μg/mL) for benzoxazolone-thiazoles 6–12.
Species
ꢂ
ꢂ
Benzoxazolone-thiazoles
6ꢂ
7ꢂ
8ꢂ
9ꢂ
10ꢂ
11ꢂ
12ꢂ
CIP
M. luteus
ATCC 10240
ꢃ
250 (ꢀ> ꢀ1000)ꢃ
1000 (ꢀ> ꢀ1000)ꢃ
500 (ꢀ> ꢀ1000)ꢃ
–ꢃ
1000 (ꢀ> ꢀ1000)ꢃ
31.25 (250)ꢃ
31.25 (250)ꢃ
0.976
aThe standard antibiotic, ciprofloxacin (CIP) was used as a positive control.
range of 501–1000 μg/mL, moderate bioactivity with number of carbon atoms in the ring is completely inactive.
MIC in the range of 126–500 μg/mL, good bioactivity The bioactivity results provide good starting templates for
with MIC in the range of 26–125 μg/mL, strong bioac- further structural optimization of this type of derivative.
tivity with MIC in the range of 10–25 μg/mL and very
strong bioactivity with MIC ꢀ< ꢀ10 μg/mL. The antibacterial
studies of compounds 6–8, 10–12 against Gram-positive
Experimental
bacteria (staphylococci, streptococci, micrococci and
Bacillus spp.) revealed some bioactivity against Staph-
Materials and methods
ylococcus aureus strains (i.e., S. aureus ATCC 25923,
S. aureus ATCC 43300, S. aureus ATCC 6538, S. aureus NIL
1, S. aureus NIL 2) and Staphylococcus epidermidis ATCC
12228 with MIC values ranging from 250 to 1000 μg/mL.
The MBC for the tested staphylococci was ꢀꢀ≥ ꢀꢀ1000 μg/mL.
The compounds 6–8, 10–12 show similar activity against
Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 10876
with MIC between 500 and 1000 μg/mL and MBC ꢀꢀ≥ ꢀꢀ1000
μg/mL. Among obtained compounds, 6–8 and 10 are
also active against Micrococcus luteus ATCC 10240 with
MICꢀ= ꢀ250–1000 μg/mL and MBC ꢀꢀ≥ ꢀꢀ1000 μg/mL. In turn,
11 and 12 show good bioactivity with bacteriostatic effect
against M. luteus ATCC 10240 with MICꢀ= ꢀ31.25 μg/mL,
MBCꢀ= ꢀ250 μg/mL and MBC/MICꢀ= ꢀ8 (Table 1).
All experiments were carried out under air atmosphere. Reagents
were generally the best quality commercial grade products and were
used without further purification. H NMR (400 MHz) and 13C NMR
1
(100 MHz) spectra were recorded in DMSO-d6 on a Bruker Ascend
400 multinuclear instrument. Melting points were determined in
open glass capillaries and are uncorrected. Silica gel 60, E. Merck
230–400 mesh, was used for preparative column chromatography.
Analytical TLC was performed using Macherey-Nagel Polygram Sil
G/UV254 0.2 mm plates eluting with dichloromethane/methanol (9:1).
6-(2-Chloroacetyl)benzo[d]oxazol-2(3H)-one, thiosemicarbazide and
appropriate ketones were commercial materials.
General procedure for the synthesis of
compounds 6–12
Moreover, minimum concentrations of 7, 8, 11, 12,
which inhibit the growth of streptococci range from 500 to
1000 μg/mL and MBC ꢀꢀ≥ ꢀꢀ1000 μg/mL.
Our results indicate that the tested compounds
possess some activity against Gram-positive bacteria,
especially against M. luteus belonging to the opportunistic
pathogens. The tested compounds 6–12 have no influence
on the growth of the reference strains of Gram-negative
bacteria and of fungi belonging to Candida spp.
A mixture of the appropriate carbonyl compound (20 mmol), thio-
semicarbazide (20 mL) and a catalytic amount of acetic acid (1 mL) in
absolute ethyl alcohol (20 mL) was magnetically stirred for 24 h and
heated under reflux. A precipitate of the desired thiosemicarbazone
was filtered, crystallized from a suitable solvent and dried. This thio-
semicarbazone (1.0 mmol) was added to a stirred solution of 6-(2-chlo-
roacetyl)benzo[d]oxazol-2(3H)-one (13, 0.212 g, 1.0 mmol) in absolute
ethyl alcohol (15 mL), and the mixture was stirred under reflux for 2 h.
Afer cooling to room temperature, a colorless solid of 6–12 began to
separate. The product was filtered off, washed with ethyl alcohol, then
suspended in water and the mixture was neutralized with a NaHCO3
solution. The crude product was subjected to silica gel column chro-
matography (230–400 mesh) using dichloromethane/methanol (9:1)
as an eluent. Chromatography afforded a pure (E)-6 isomer.
Conclusions
An efficient and economic method for the synthesis of
benzoxazolone-thiazol conjugates was developed. The in
vitro antimicrobial effects of the synthesized compounds
on various pathogenic bacteria and fungi were evaluated.
The tested compounds possess some biological activ-
ity against Gram-positive bacteria, especially against
M. luteus, MIC 31.25 μg/mL belonging to opportunistic
pathogens. Interestingly, compound 9 containing an odd
(E)-6-(2-(2-Ethylidenehydrazino)thiazol-4-yl)benzo[d]oxazol-
2(3H)-one (6)ꢁYield 0.16 g, (58%); Rf ꢀ= ꢀ 0.57; mp 250–253°C; 1H NMR:
δ 1.91 (d, 3H, CH3, J ꢀ= ꢀ 5 Hz), 7.10 (d, 1H, CH, J ꢀ= ꢀ 8 Hz), 7.19 (s, 1H, CH),
7.40 (q, 1H, CH, J ꢀ= ꢀ 5 Hz), 7.65 (dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz), 7.70 (m,
1H, CH), 11.75 (bs, 1H, NH, D2O exchangeable), 12.19 (bs, 1H, NH, D2O
exchangeable); 13C NMR: δ 18.4, 102.4, 106.9, 109.8, 121.7, 126.9, 130.5,
143.7, 145.2, 154.5, 164.8, 168.6. Anal. Calcd for C12H10N4O2S: C, 52.54; H,
3.67; N, 20.43. Found: C, 52.48; H, 3.55; N, 20.62.

ꢁꢁꢁꢂ
44ꢀ ꢀK.Z. Łączkowski et al.: 6-(1,3-Thiazol-4-yl)-1,3-benzoxazol-2(3H)-one derivatives
6-(2-[2-(Propan-2-ylidene)hydrazino]thiazol-4-yl)benzo[d]oxa-
In vitro antimicrobial assay
1
zol-2(3H)-one (7)ꢁYield 0.24 g (84%); Rf ꢀ= ꢀ 0.26; mp 255–256°C; H
NMR: δ 1.95 (d, 3H, CH3, J ꢀ= ꢀ 7 Hz), 7.11 (d, 1H, CH, J ꢀ= ꢀ 11 Hz), 7.21 (s, 1H,
CH), 7.65 (dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz), 7.72 (m, 1H, CH), 10.90 (bs,
1H, NH, D2O exchangeable), 11.77 (bs, 1H, NH, D2O exchangeable); 13C
NMR: δ 18.1, 24.8, 102.6, 106.8, 109.9, 121.1, 127.3, 130.4, 143.8, 146.5,
153.1, 154.5, 169.7. Anal. Calcd for C13H12N4O2S: C, 54.15; H, 4.20; N,
19.43. Found: C, 54.23; H, 4.17; N, 19.48.
Compounds 6–12 were screened in vitro for antibacterial and anti-
fungal activities using the broth microdilution method accord-
ing to European Committee on Antimicrobial Susceptibility
Testing (EUCAST) [30] and Clinical and Laboratory Standards
Institute guidelines [31] against a panel of reference strains of 25
microorganisms, including Gram-positive bacteria (Staphylococcus
aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococ-
cus aureus ATCC 43300, Staphylococcus aureus NIL1, Staphylococcus
aureus NIL 2, Staphylococcus epidermidis ATCC 12228, Streptococcus
pyogenes ATCC 19615, Streptococcus pneumoniae ATCC 49619, Strep-
tococcus mutans ATCC 25175, Bacillus subtilis ATCC 6633, Bacillus
cereus ATCC 10876, Micrococcus luteus ATCC 10240), Gram-negative
bacteria (Escherichia coli ATCC 3521, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453,
Bordetella bronchiseptica ATCC 4617, Salmonella typhimurium ATCC
14028, Enterobacter cloacae NIL, Pseudomonas aeruginosa ATCC
9027, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aerugi-
nosa NIL) and fungi belonging to yeasts (Candida albicans ATCC
2091, Candida albicans ATCC 10231, Candida parapsilosis ATCC
22019). These microorganisms were obtained from the American
Type Culture Collection (ATCC), routinely used for the evaluation of
antimicrobials, and from the National Medicines Institute in Warsaw
(NIL). All microbial cultures were first subcultured on nutrient agar
or Sabouraud agar at 35°C for 18–24 h or 30°C for 24–48 h for bacteria
and fungi, respectively.
The surface of Mueller-Hinton agar or Mueller-Hinton agar with
sheep blood (for bacteria) and RPMI 1640 with MOPS (for fungi)
were inoculated with the suspensions of bacterial or fungal species.
Microbial suspensions were prepared in sterile saline (0.85% NaCl)
with an optical density of McFarland standard scale 0.5 – approxi-
mately 1.5ꢀ× ꢀ108 CFU/mL for bacteria and 0.5 McFarland standard scale
– approximately 5ꢀ× ꢀ105 CFU/mL for fungi. Samples containing exam-
ined compounds 6–12 were dissolved in DMSO and were dropped
into the wells on the above-mentioned agar media. The agar plates
were preincubated at room temperature for 1 h, and then they were
incubated at 37°C for 24 h and 30°C for 48 h for bacteria and fungi,
respectively. The well containing DMSO without any compound was
used as the negative control and ciprofloxacin or fluconazole (Sigma)
as positive controls.
6-(2-[2-Cyclobutylidenehydrazino)thiazol-4-yl]benzo[d]oxazol-
1
2(3H)-one (8)ꢁYield 0.20 g; (67%); Rf ꢀ= ꢀ 0.86; mp 258–259°C; H
NMR: δ 1.87–1.99 (m, 2H, CH2), 2.93 (q, 4H, 2CH2, J1 ꢀ= ꢀ 7 Hz, J2 ꢀ= ꢀ 14 Hz),
7.10 (d, 1H, CH, J ꢀ= ꢀ 11 Hz), 7.18 (s, 1H, CH), 7.65 (dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz,
J2 ꢀ= ꢀ 8 Hz), 7.72 (m, 1H, CH), 10.95 (bs, 1H, NH, D2O exchangeable),
11.72 (bs, 1H, NH, D2O exchangeable); 13C NMR: δ 13.2, 33.4, 33.3, 102.2,
106.8, 109.8, 121.5, 128.6, 130.0, 143.7, 148.5, 154.5, 156.0, 169.2. Anal.
Calcd for C14H12N4O2S: C, 55.99; H, 4.03; N, 18.65. Found: C, 56.06; H,
4.10; N, 18.71.
6-(2-(2-Cyclopentylidenehydrazino)thiazol-4-yl)benzo[d]oxa-
zol-2(3H)-one (9)ꢁYield 0.19 g (60%); Rf ꢀ= ꢀ 0.72, mp 250°C (with
1
decomp.); H NMR: δ 1.70–1.75 (m, 2H, CH2), 1.77–1.83 (m, 2H, CH2),
2.37–2.43 (m, 4H, 2CH2), 7.12 (d, 1H, CH, J ꢀ= ꢀ 11 Hz), 7.22 (s, 1H, CH), 7.65
(dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz), 7.72 (m, 1H, CH), 10.95 (bs, 1H, NH,
D2O exchangeable), 11.82 (bs, 1H, NH, D2O exchangeable); 13C NMR: δ
24.5, 24.6, 29.2, 32.9, 102.5, 106.8, 109.9, 121.5, 127.2, 130.4, 143.8, 146.4,
154.5, 156.1, 169.4. Anal. Calcd for C15H14N4O2S: C, 57.31; H, 4.49; N,
17.82. Found: C, 57.25; H, 4.40; N, 17.93.
6-(2-(2-Cyclohexylidenehydrazino)thiazol-4-yl)benzo[d]oxa-
zol-2(3H)-one (10)ꢁYield 0.16 g (49%); Rf ꢀ= ꢀ 0.74; mp 248°C (with
1
decomp.); H NMR: δ 1.55–1.70 (m, 6H, 3CH2), 2.26 (t, 2H, CH2, J ꢀ= ꢀ 7
Hz), 2.45 (t, 2H, CH2, J ꢀ= ꢀ 6 Hz), 7.11 (d, 1H, CH, J ꢀ= ꢀ 11 Hz), 7.20 (s, 1H,
CH), 7.65 (dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz), 7.68 (m, 1H, CH), 10.80 (bs,
1H, NH, D2O exchangeable), 11.73 (bs, 1H, NH, D2O exchangeable);13C
NMR: δ 25.0, 25.6, 26.8, 27.8, 34.7, 102.7, 106.9, 109.9, 121.6, 126.3, 130.6,
143.8, 144.9, 154.4, 159.7, 169.8. Anal. Calcd for C16H16N4O2S: C, 58.52; H,
4.91; N, 17.06. Found: C, 58.60; H, 4.86; N, 17.15.
6-(2-(2-Cyclododecylidenehydrazino)thiazol-4-yl)benzo[d]oxa-
1
zol-2(3H)-one (11)ꢁYield 0.30 g (73%); Rf ꢀ= ꢀ 0.71; mp 220–223°C; H
NMR: δ 1.15–1.45 (m, 14H, 7CH2), 1.50–1.60 (m, 12H, CH2), 1.65–1.75 (m,
2H, CH2), 2.32 (t, 2H, CH2, J ꢀ= ꢀ 6 Hz), 2.40 (t, 2H, CH2, J ꢀ= ꢀ 6 Hz), 7.10 (d,
1H, CH, J ꢀ= ꢀ 11 Hz), 7.17 (s, 1H, CH), 7.65 (dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz),
7.71 (m, 1H, CH), 11.95 (bs, 1H, NH, D2O exchangeable), 11.72 (bs, 1H,
NH, D2O exchangeable); 13C NMR: δ 21.79, 22.33, 22.46, 22.62, 23.04,
23.14, 23.58, 25.58 (two signals), 28.63, 30.58, 102.36, 106.65, 109.8,
121.4, 128.9, 129.9, 143.8, 148.8, 154.5, 170.3. Anal. Calcd for C22H28N4O2S:
C, 64.05; H, 6.84; N, 13.58. Found: C, 64.11; H, 6.82; N, 13.65.
Subsequently, minimal inhibitory concentration (MIC) of the
compounds 6–12 was examined by the microdilution broth method,
using their 2-fold dilutions in Mueller-Hinton broth or RPMI 1640
broth with MOPS prepared in 96-well polystyrene plates. Final con-
centrations of the compounds ranged from 1000 to 0.488 μg/mL.
Microbial suspensions were prepared in sterile saline (0.85% NaCl)
with an optical density of 0.5 McFarland standard. The bacterial or
fungal suspension was added per each well containing broth and
various concentrations of the examined compounds. Afer incuba-
6-(2-(2-Adamantylidenehydrazino)thiazol-4-yl)benzo[d]oxazol- tion, the MIC was assessed spectrophotometrically as the lowest
1
2(3H)-one (12)ꢁYield 0.16 g; (42%); Rf ꢀ= ꢀ 0.49; mp 241–244°C; H concentration of the samples showing complete bacterial or fungal
NMR: δ 1.70–1.80 (m, 6H, 3CH2), 1.85–2.00 (m, 6H, 3CH2), 2.54 (m, 2H, growth inhibition. Appropriate DMSO, growth and sterile controls
CH2), 3.37 (m, 2H, CH2), 7.10 (d, 1H, CH, J ꢀ= ꢀ 11 Hz), 7.19 (s, 1H, CH), 7.65 were carried out. The medium with no tested substances was used
(dd, 1H, CH, J1 ꢀ= ꢀ 2 Hz, J2 ꢀ= ꢀ 8 Hz), 7.71 (m, 1H, CH), 11.95 (bs, 1H, NH, as the control.
D2O, exchangeable), 11.74 (bs, 1H, NH, D2O exchangeable). 13C NMR:
The MBC (minimal bactericidal concentration) or MFC
δ 18.6, 27.2 (three signals), 31.8, 35.8, 37.3 (two signals), 56.1, 102.6, (minimal fungicidal concentration) are defined as the lowest
106.9, 109.9, 121.6, 126.7, 130.5, 143.7, 145.5, 154.5, 165.6, 169.9. Anal. concentration of the compounds that is required to kill a particu-
Calcd for C20H20N4O2S: C, 63.14; H, 5.30; N, 14.73. Found: C, 63.19; H, lar bacterial or fungal species. MBC or MFC was determined by
5.33; N, 14.81.
removing the culture using MIC determinations from each well

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K.Z. Łączkowski et al.: 6-(1,3-Thiazol-4-yl)-1,3-benzoxazol-2(3H)-one derivativesꢀ
ꢀ45
and spotting onto appropriate agar medium. After incubation, the
lowest compound concentration with no visible growth observed
was assessed as a bactericidal/fungicidal concentration. All
experiments were repeated three times and representative data
are presented [32].
The MBC/MIC or MFC/MIC ratios were calculated to determine
the bactericidal/fungicidal (MBC/MIC ꢀꢀ≤ ꢀꢀ4, MFC/MIC ꢀꢀ≤ ꢀꢀ4) or bacte-
riostatic/fungistatic (MBC/MIC ꢀ> ꢀ4, MFC/MIC ꢀ> ꢀ4) effect of the tested
compounds.
Acknowledgments: This study was supported by the
Nicolaus Copernicus University (project no. 844/2012).
The authors thank Dr. Angelika Baranowska-Łączkowska
from the Institute of Physics, Kazimierz Wielki University,
Bydgoszcz, Poland for comments on the manuscript.
Received September 5, 2013; accepted November 10, 2013; previ-
ously published online January 8, 2014
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