K. Liaras et al. / Bioorg. Med. Chem. 19 (2011) 3135–3140
3139
300 MHz): 2.50 (s, 3H, thiazole 40-CH3), 2.82 (s, 3H, N–CH3), 7.27
4.3. Biological evaluation
4.3.1. Antifungal activity
(d, J = 15.6 Hz, 1H, CO–CH), 7.42–7.50 (m, 3H, Ar 30 and 50, Ar-
CH), 7.74 (d, J = 8.4 Hz, 2H, Ar 20 and 60). Anal. Calcd for
C
14H13ClN2OS (MW 292.5): C, 57.43; H, 4.48; Cl, 12.11; N, 9.57.
For the antifungal bioassays, eight fungi were used: Aspergillus
ochraceus (ATCC 12066), Aspergillus fumigatus (plant isolate),
Aspergillus niger (ATCC 6275), A. versicolor (ATCC 11730), Penicil-
lium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC
9112), Trichoderma viride (IAM 5061) and F. sporotrichoides
(IMT 496). The organisms were obtained from the Mycological Lab-
oratory, Department of Plant Physiology, Institute for Biological Re-
Found: C, 57.40; H, 4.30; N, 9.68.
4.2.5. (E)-3-(3-Chlorophenyl)-1-(4-methyl-2-(methylamino)
thiazol-5-yl)prop-2-en-1-one (5)
Yield: 61%, mp: 209–212 °C. IR (Nujol) 3200 (NH), 1685 (C@O),
1651 (CH@CH), 1602, 1075 (Cl) cmꢀ1 1H NMR (d ppm, DMSO-d6,
;
300 MHz): 2.48 (s, 3H, thiazole 40-CH3), 2.80 (s, 3H, N–CH3), 7.32–
7.46 (m, 4H, CO–CH, Ar 20, Ar 50, Ar 40), 7.72 (dd, 2H, Ar 60, Ar-CH),
8.52 (s, 1H, NH). Anal. Calcd for C14H13ClN2OS (MW 292.5): C,
57.43; H, 4.48; Cl, 12.11; N, 9.57. Found: C, 57.44; H, 4.40; N, 9.60.
search ‘Siniša Stankovic’, Belgrade, Serbia.
´
The micromycetes were maintained on malt agar and the cul-
tures stored at 4 °C and sub-cultured once a month.33 In order to
investigate the antifungal activity of the compounds, a modified
microdilution technique was used.34–36 The fungal spores were
washed from the surface of agar plates with sterile 0.85% saline
containing 0.1% Tween 80 (v/v). The spore suspension was adjusted
with sterile saline to a concentration of approximately 1.0 ꢁ 105 in
4.2.6. (E)-3-(2-Chlorophenyl)-1-(4-methyl-2-(methylamino)
thiazol-5-yl)prop-2-en-1-one (6)
Yield: 32%, mp: 185–187 °C. IR (Nujol) 3200 (NH), 1680 (C@O),
1650 (CH@CH), 1602, 1025 (Cl) cmꢀ1
;
1H NMR (d ppm, DMSO-d6,
a final volume of 100 ll per well. The inocula were stored at 4 °C
300 MHz): 2.50 (s, 3H, thiazole 40-CH3), 2.81 (s, 3H, N–CH3), 7.3
(d, J = 15.3 Hz, 1H, CO–CH), 7.36 (dd, 2H, Ar 40, Ar 50), 7.49 (d,
J = 9.3 Hz, 1H, Ar 60), 7.76 (d, J = 15.6 Hz, 1H, Ar-CH), 7.93 (d,
J = 9.3 Hz, 1H, Ar 30). MS: (m/z): 292 (M+, 100%), 291 (28%), 257
(43%), 236 (9%), 201 (9%), 182 (8%), 181 (73%), 167 (13%), 165
(9%), 155 (33%), 137 (13%), 128 (9%), 102 (14%), 101 (22%), 86
(15%), 81 (14%), 75 (9%), 69 (25%), 57 (9%). Anal. Calcd for
for further use. Dilutions of the inocula were cultured on solid malt
agar to verify the absence of contamination and to check the valid-
ity of the inoculum.
Minimum inhibitory concentration (MIC) determinations were
performed by a serial dilution technique using 96-well microtiter
plates. The compounds investigated were dissolved in 5% DMSO
solution containing 0.1% Tween 80 (v/v) (1 mg/ml) and added in
broth Malt medium with inoculum. The microplates were incu-
bated at Rotary shaker (160 rpm) for 72 h at 28 °C. The lowest con-
centrations without visible growth (at the binocular microscope)
were defined as MICs.
C14H13ClN2OS (MW 292.5): C, 57.43; H, 4.48; Cl, 12.11; N, 9.57.
Found: C, 57.41; H, 4.45; N, 9.55.
4.2.7. (E)-3-(4-Methoxyphenyl)-1-(4-methyl-2-(methylamino)
thiazol-5-yl)prop-2-en-1-one (7)
The fungicidal concentrations (MFCs) were determined by serial
Yield: 29%, mp: 219–223 °C. IR (Nujol) 3200 (NH), 1684 (C@O),
subcultivation of a 2
and inoculated for 72 h, into microtiter plates containing 100 l
broth per well and further incubation 72 h at 28 °C. The lowest
concentration with no visible growth was defined as MFC indicat-
ing 99.5% killing of the original inoculum. DMSO was used as a neg-
ative control, commercial fungicides, bifonazole (Srbolek, Belgrade,
Serbia) and ketoconazole (Zorkapharma, Šabac, Serbia), were used
l
l of tested compounds dissolved in medium
l of
1654 (CH@CH), 1604, 1584, 1250 C-O–C) cmꢀ1
;
1H NMR (d ppm,
DMSO-d6, 300 MHz): 2.61 (s, 3H, thiazole 40-CH3), 2.93 (s, 3H, N–
CH3), 3.87 (s, 3H, OCH3), 7.05 (d, J = 8.7 Hz, 2H, Ar 30 and 50), 7.22
(d, J = 15.3 Hz, 1H, CO–CH), 7.58 (d, J = 15.6 Hz, 1H, Ar-CH), 7.77
(d, J = 8.7 Hz, 2H, Ar 20 and 60). Anal. Calcd for C15H16N2O2S (MW
288): C, 62.48; H,5.50; N, 9.71. Found: C,62.45; H, 5.52; N, 9.71.
as positive controls (1–3000
All experiments were performed in duplicate and repeated
three times.
lg/ml).
4.2.8. (E)-3-(2-Methoxyphenyl)-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one (8)
Yield: 36%, mp: 213–215 °C. IR (Nujol) 3200 (NH), 1670 (C@O),
1643 (CH@CH), 1607, 1247 (C-O–C) cmꢀ1; 1H NMR (d ppm, DMSO-
d6, 300 MHz): 2.54 (s, 3H, thiazole 40-CH3), 2.86 (s, 3H, N–CH3),
3.88 (s, 3H, OCH3), 7.05 (m, 2H, CO–CH, Ar 30), 7.41 (dd, 2H, Ar 50,
Ar 40), 7.75 (dd, 2H, Ar 60, Ar-CH). Anal. Calcd for C15H16N2O2S
(MW 288): C, 62.48; H,5.50; N, 9.71. Found: C,62.47; H, 5.53; N, 9.70.
4.3.2. Antibacterial activity
The following Gram-negative bacteria were used: Escherichia
coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmo-
nella typhimurium (ATCC 13311), E. faecalis (human isolate) and
the following Gram-positive bacteria: Bacillus cereus (clinical
isolate), M. flavus (ATCC 10240), L. monocytogenes (NCTC 7973),
and Staphylococcus aureus (ATCC 6538). The organisms were
obtained from the Mycological Laboratory, Department of Plant
4.2.9. (E)-3-(2,6-Dichlorophenyl)-1-(4-methyl-2-(methylamino)
thiazol-5-yl)prop-2-en-1-one (9)
´
Yield: 84%, mp: 195–197 °C. IR (Nujol) 3200 (NH), 1690 (C@O),
Physiology, Institute for Biological Research ‘Siniša Stankovic’,
Belgrade, Serbia.
1650 (CH@CH), 1607 cmꢀ1
;
1H NMR (d ppm, DMSO-d6, 80 MHz):
2.50 (s, 3H, thiazole 40-CH3), 2.91 (s, 3H, N–CH3), 7.15–7.8 (m,
5H, Ar, alkene). Anal. Calcd for C14H12Cl2N2OS (MW 327): C,
51.39; H, 3.70; Cl, 21.67; N, 8.56; O, 4.89; S, 9.80. Found: C,
51.40; H, 3.73; N, 8.58.
The antibacterial assay was carried out by a microdilution
method34,35 in order to determine the antibacterial activity of com-
pounds tested against the human pathogenic bacteria.
The bacterial suspensions were adjusted with sterile saline to a
concentration of 1.0 ꢁ 105 CFU/ml. The inocula were prepared dai-
ly and stored at +4 °C until use. Dilutions of the inocula were cul-
tured on solid medium to verify the absence of contamination and
to check the validity of the inoculum.
4.2.10. (E)-3-(2,4-Dichlorophenyl)-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one (10)
Yield: 76%, mp: 239–240 °C. IR (Nujol) 3200 (NH), 1680 (C@O),
1650 (CH@CH), 1602, 1100 (Cl) cmꢀ1
;
1H NMR (d ppm, DMSO-d6,
All experiments were performed in duplicate and repeated
three times.
300 MHz): 2.56 (s, 3H, thiazole 40-CH3), 2.88 (s, 3H, N–CH3), 7.39
(d, J = 15.3 Hz, 1H, CO–CH), 7.51 (d, J = 8.4 Hz, 1H, Ar 50), 7.74–
7.78 (m, 2H, Ar 30, Ar-CH), 8.05 (d, J = 8.3 Hz, 1H, Ar 60), 8.64 (s,
1H, NH). Anal. Calcd for C14H12Cl2N2OS (MW 327): C, 51.39; H,
3.70; Cl, 21.67; N, 8.56. Found: C, 51.43; H, 3.69; N, 8.55.
4.3.3. Microdilution test
The minimum inhibitory and bactericidal concentrations (MICs
and MBCs) were determined using 96-well microtiter plates. The