Synthesis and Antibacterial Activity of Analogs of 5-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiazoli-din-4-one

Article abstract of DOI:10.3390/molecules190913577

In an effort to develop new antimicrobial agents, 3-(4-methylcoumarin-7- yloxyacetylamino)-2-thioxo-1,3-thiazolidin-4-one (4) was synthesized by reaction of thiocarbonylbisthioglycolic acid with ethyl (4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (3), which was prepared in turn from 7-hydroxy-4-methylcoumarin (1). The condensation of compound 4 with different aromatic aldehydes afforded a series of 5-(arylidene)-3-(4-methylcoumarin-7-yloxyacetyl-amino)-2-thioxo-1,3-thiozolidin-4-one analogs 5a-h. The structures of these synthetic compounds were elucidated on the basis of IR, 1H-NMR and 13C-NMR spectral data and ESI-MS spectrometric analysis. Compounds 5a-h were examined for their antibacterial activity against several strains of Gram-positive and Gram-negative bacteria.

Full text of DOI:10.3390/molecules190913577

Molecules 2014, 19, 13577-13586; doi:10.3390/molecules190913577
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antibacterial Activity of Analogs of
5
1
-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-
,3-thiazoli-din-4-one
1
1
1
2,
Nguyen Tien Cong , Huynh Thi Nhan , Luong Van Hung , Tran Dinh Thang * and
3
,
Ping-Chung Kuo *
1
Department of Chemistry, Ho Chi Minh City University of Education, Ho Chi Minh City 70000,
Vietnam; E-Mails: congchemist@gmail.com (N.T.C.); nhanhuynhsp@gmail.com (H.T.N.);
luonghung365@gmail.com (L.V.H.)
2
Department of Chemistry, Vinh University, Vinh 42000, Vietnam
3
Department of Biotechnology, National Formosa University, Yunlin 63201, Taiwan
*
Authors to whom correspondence should be addressed; E-Mails: thangtd@vinhuni.edu.vn (T.D.T.);
pcckuoo@sunws.nfu.edu.tw (P.-C.K.); Tel.: +886-5-6315491 (P.-C.K.);
Fax: +886-5-6315502 (P.-C.K.).
Received: 30 June 2014; in revised form: 26 August 2014 / Accepted: 27 August 2014 /
Published: 1 September 2014
Abstract: In an effort to develop new antimicrobial agents, 3-(4-methylcoumarin-7-
yloxyacetylamino)-2-thioxo-1,3-thiazolidin-4-one (4) was synthesized by reaction of
thiocarbonylbisthioglycolic acid with ethyl (4-methyl-2-oxo-2H-chromen-7-yloxy)aceto-
hydrazide (3), which was prepared in turn from 7-hydroxy-4-methylcoumarin (1).
The condensation of compound 4 with different aromatic aldehydes afforded a series of
5
-(arylidene)-3-(4-methylcoumarin-7-yloxyacetyl-amino)-2-thioxo-1,3-thiozolidin-4-one analogs
1
5a–h. The structures of these synthetic compounds were elucidated on the basis of IR, H-NMR
13
and C-NMR spectral data and ESI-MS spectrometric analysis. Compounds 5a–h were
examined for their antibacterial activity against several strains of Gram-positive and
Gram-negative bacteria.
Keywords: coumarin; 2-thioxo-1,3-thiazolidin-4-one; spectral elucidation; antibacterial
activity

Molecules 2014, 19
. Introduction
Bacterial disease control, including the food safety issue, has continuously attracted researchers’
13578
1
attention from various fields. The use of preservatives and pathogen antagonists had been reported as a
means of protecting the microbiological safety of fresh and processed food products [1–4]. Although
some antagonists exhibited significant inhibition of bacterial growth, they were too toxic to be utilized
long term. In the present study, we hoped to explore new lead compounds with natural skeletons which
could be modified for further investigation as antimicrobial agents applied to food preservation.
7
-Hydroxy-4-methylcoumarin derivatives with heterocyclic moieties possess diverse biological
properties such as antibacterial [5–7], antifungal [8,9], anticancer [10], enzyme-inhibitory [11], and
antioxidant activities [7,9]. On the other hand, thiazolidin-4-ones are important compounds due to their
broad range of biological activities including anticancer [12–14], virus-inhibitory [15], HIV-inhibitory [16],
and enzyme-inhibitory activities [14]. These observations prompted our interest in synthesizing some
new 7-hydroxy-4-methylcoumarin derivatives bearing 2-thioxo-1,3-thiozolidin-4-one substituents and
evaluate their antibacterial potential.
2
. Results and Discussion
2
.1. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h
The synthetic route for the preparation of the target compounds was presented in Scheme 1.
Compounds 1–3 were synthesized according to the corresponding published procedures [6,7,9–11] and
characterization of these synthetic compounds was achieved by comparison of their physical and
spectral data with those reported in the previous literature [17]. Then compound 3 was reacted
with thiocarbonylbisthioglycolic acid in ethanol to obtain new compound 4, which was converted into
the series of N-(5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-
acetamides 5a–h by Knoevenagel condensation.
In the IR spectrum of compound 4, in addition to the lactone and amide carbonyl group absorption
−
1
−1
at 1709 cm , the stretching band in a high frequency region (1771 cm ) indicated the presence of
1
C=O bonds in a thiazolidine ring. A new signal with intensity of 2H appearing at 4.47 ppm in the H-NMR
spectrum was attributed to the methylene group of the thiazolidine ring. Another signal also with intensity
of 2H appearing in the downfield region at 4.96 ppm was attributed to the oxymethylene protons
(
OCH ). In terms of unexpected results, the signals of the methylene protons in 4 were split instead of
2
being a singlet. The splitting of these signals could be explained by a non-first order splitting effect.
1
3
Comparing the C-NMR spectra of 3 [7] and 4, three more signals appeared in 4, two of which
were at around 160 ppm corresponding to the signals of the carbon atoms in the thioxo group and
carbonyl group, whereas the last one was at 33.4 ppm corresponding to the signal of the saturated
carbon atom of the thiazolidine ring. The spectral data of 4 as well as the agreement of the predicted
mass with molecular mass determined by HR-MS confirmed that a 4-oxo-2-thioxothiazolidine ring
was formed.

Molecules 2014, 19
Scheme 1. Synthetic route for the preparation of compounds 5a–h.
13579
CH3
H
3
C
C
O
CH
2
C
O
OC
2
H
5
O
O
OH
HO
OH
(
1)
ClCH
2
2 5
COOC H
CH
O
3
CH
3
H
2
N
NH
2
O
2 2 5
OCH COOC H
O
O
OCH
SCH
2
2
CONHNH
COOH
2
(
2)
(
3)
S
C
SCH COOH
2
CH3
X
O
CHO
NH
N
O
O
O
O
S
CH
O
3
(
4)
S
O
NH
O
O
N
O
S
S
X
(
5a-h)
(
(
a) X = 4-N(CH3)2
e) X = 4-Br
(c)
(g)
X = 4-H
(b) X = 4-OH
X =4-Cl
X = 4-OCH
X = 2-NO2
3
(d)
(h)
(
f)
X = 4-NO2
In the IR spectrum of compounds 5a–h, there were shifts of the absorption of the carbonyl group at
−
1
−1
1
771 cm to lower frequencies (1721–1755 cm ), in agreement with the formation of a conjugated
1
system between the carbonyl group and the benzylidene moiety. Comparison of the H-NMR spectra
1
of 5a–h with the H-NMR of 4 showed not only the disappearance of the methylene proton’s signal at
4
.47 ppm, but also appearance of additional aromatic proton signals at 6.84–8.36 ppm and methylidene
proton signals at 7.82–8.86 ppm. The signal of the oxymethylene protons (OCH ) in compounds 5a–h
2
appeared at 5.00–5.04 ppm. The signals of these protons, like the signals of the methylene protons
2
OCH in compound 4, were also split by a non–first order splitting effect. Therefore, they did not
appear as singlets.
2.2. Determination of the in Vitro Antimicrobial Activity
Compounds 5a–h were examined for antimicrobial activity against Escherichia coli, Pseudomonas
aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive
bacteria) at concentrations of 0.1% and 0.2% according to the reported method with minor
modifications [18]. As shown in Table 1, most of the compounds 5a–g at 0.1% exhibited low
antimicrobial activity, with antimicrobial inhibition zone diameters of less than 15 mm. However, at
the concentration of 0.2%, most of these compounds showed average activity (the antimicrobial

Molecules 2014, 19
13580
diameters were 15 mm to 20 mm) against certain bacteria including Escherichia coli, Pseudomonas
aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive
bacteria). In addition, the synthetic compounds 3 and 4 did not show any significant inhibition of
bacterial growth in our preliminary screening and therefore the data were not included.
Table 1. Antimicrobial inhibition zone diameters (d) of compounds 5a–h.
X
4
-N(CH
3
)
2
4-OH 4-OCH
3
4-H
(5d)
d (mm)
14
4-Br
(5e)
4-Cl 2-NO
2
4-NO
(5h)
2
Bacteria
Conc.
(
5a)
(5b)
(5c)
(5f)
(5g)
a
0
0
0
0
0
0
0
0
.1%
.2%
.1%
.2%
.1%
.2%
.1%
.2%
14
16
13
15
15
17
13
15
15
17
19
23
15
19
14
16
13
16
12
15
14
16
14
18
15
16
15
18
18
21
17
20
12
15
15
17
14
18
14
16
12
15
14
16
13
16
15
17
15
18
18
20
14
17
16
18
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
16
17
19
15
18
16
Staphylococcus aureus
17
a
D − d ≥ 25 mm: Very high activity; D − d ≥ 20 mm: High activity; D − d ≥ 15 mm: Average activity; D − d ≤ 15 mm: Low
activity. Each experiment was performed in triplicate.
The minimum inhibitory concentration (MIC) value is a measure to define the antibacterial activity
of a compound and is defined as the lowest concentration of drug that inhibits visible growth.
Compounds 5a–h were subjected to examination of their MIC values according to the reported
method [19] and the data are shown in Table 2. The two-fold microdilution broth method was used
and all of the tested samples demonstrated inhibitory effects in a concentration-dependent manner.
However, only 5c, 5g and 5h exhibited any significant inhibition against S. aureus with MIC values of
5
0 μg/mL.
Table 2. The minimum inhibitory concentrations (MICs) of 5a–h against bacteria.
MIC (μg/mL)
Bacteria
4-N(CH3)2 4-OH
4-OCH3
4-H
4-Br
4-Cl
2-NO2
4-NO2
(
5a)
(5b)
(5c)
(5d)
(5e)
(5f)
(5g)
(5h)
a
Escherichia coli
P. aeruginosa
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Bacillus subtilis
-
-
-
Staphylococcus aureus
50
50
50
a
MIC > 50 μg/mL and not determined.
3. Experimental Section
3.1. General Procedures
All starting materials were purchased from Merck (Darmstadt, Germany) and used without
purification. Melting points were measured in open capillary tubes on a Gallenkamp melting point

Molecules 2014, 19
13581
apparatus. The structures of all compounds were confirmed by IR, NMR and HR-MS spectra. IR
1
spectra were recorded on a Shimadzu FTIR-8400S spectrometer using KBr pellets. The H-NMR
6
spectra were recorded on a Bruker Avance spectrometer at 500 MHz using DMSO-d as solvent, while
13
the C-NMR, HSQC, HMBC spectra were recorded at 125 MHz. The data are given in parts per
million (ppm) and are referenced to an internal standard of tetramethylsilane (TMS, δ 0.00 ppm).
The spin-spin coupling constants (J) are given in Hz. Peak multiplicities are reported as s (singlet), d
(
doublet), dd (double-doublet), t (triplet), q (quartet), and m (multiplet). The MS spectra were recorded
on a Bruker microTOF-Q 10187 spectrometer or on a Varian FT-ICR-MS 910 spectrometer.
3
.2. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h
3
.2.1. Synthesis of 4-Oxo-2-thioxothiazolidine Derivatives
Compounds 1–3 were prepared using the corresponding reported methods [6,7,9–11] as shown in
Scheme 1.
3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta-
mide (4)
A mixture of (4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (3, 0.01 mol) and
thio-carbonylbisthioglycolic acid (0.01 mol) in ethanol (5 mL) was refluxed for 8 h. After cooling the
resulting solid was filtered off, dried and recrystallized from HOAc/DMF to give compound 4 as a
−1
yellowish powder in 74.0% yield; mp: 248–249 °C; IR (ν, cm ): 3258, 3100, 2901, 1771, 1709, 1622,
1
3
1
491, 1425, 1391, 1358, 1298, 1245; H-NMR (ppm) δ 11.41 (1H, s, NH), 7.71 (1H, d, J = 9.0 Hz,
3
4
4
H-5), 7.04 (1H, dd, J = 9.0 Hz, J = 2.5 Hz, H-6), 7.01 (1H, d, J = 2.5 Hz, H-8), 6.23 (1H, s, H-3),
13
4.96 (2H, OCH
2
), 4.47 (2H, CH2 thiazolidine ring), 2.39 (3H, s, CH
3
); C-NMR (ppm) δ 199.7 (C=S),
2
170.1 (NH–CO–CH ), 165.8 (>N–CO), 160.3 (C-7), 160.0 (O–C=O), 154.4 (C-9), 153.3 (C-4), 126.5
(
C-5), 112.5 (C-6), 113.8 (C-10), 111.6 (C-3), 101.8 (C-8), 66.0 (OCH
2
), 33.4 (SCH
2
), 18.1 (CH
3
);
+
HR-ESI-MS m/z 365.0266 [M+H] (calcd. for C H N O S , 365.0266).
15
13
2
5 2
3
2
.2.3. General Procedure for Synthesis of N-(5-Arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-
-oxo-2H-chromen-7-yloxy)acetamides 5a–h
Equimolar amounts of 4 (5.0 mmol), anhydrous sodium acetate (5.0 mmol) and an appropriate
aromatic aldehyde (5.0 mmol) in glacial acetic acid (5 mL) were refluxed for 5 h. The reaction mixture
was cooled and the solid separated was filtered and recrystallized to give compounds 5a–h.
N-{5-[4-(Dimethylamino)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}-2-(4-methyl-2-oxo-2H-chromen-
−
1
7
-yloxy)acetamide (5a): Red powder; Yield: 69.0; mp. 265–266 °C; IR (ν, cm ): 3314, 3088, 2915,
1
1
721, 1616, 1574, 1530, 1441, 1385, 1302, 1263; H-NMR (δ, ppm): 3.06 (6H, s, –N(CH
3
3
)
2
), 2.41
4
(
3H, s, CH
3
), 5.00 (2H, OCH
2
); 6.25 (1H, s, 3-H), 7,05 (1H, d, J = 2.5, 8-H), 7.07 (1H, dd, J = 9.0,
4
3
3
J = 2.5, 6-H), 7.72 (1H, d, J = 8.5, 5-H), δ 7.79 (1H, s, =CH-), 11.6 (1H, s, NH), 7.50 (2H, d, J = 9.0)
3
13
and 6.84 (2H, d, J =9.0) (Hbenzene ring); C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C),
8.1 (CH ), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1
1
3

Molecules 2014, 19
13582
(
OCH ), 166.0 (–NH–CO–CH ), 163.2 (>N–CO), 113.9 (>C= thiazolidine ring), 189.6 (C=S), 136.3 (=CH–),
2 2
1
4
19.6, 133.6, 112.6 and 112.3 (Cbenzene ring), 152.2 (Carom–N(CH
3
)
2
), 39.6 (–N(CH
3
)
2
); HR-ESI-MS:
96.1001 (M+H), calcd. for (C24
21 3 5 2
H N O S ): 495.0923.
N-(5-(4-Hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
−1
acetamide (5b): Brown yellow powder; yield: 64%; mp: 284–285 °C; IR (ν, cm ): 3340, 2920, 2851,
1
1
732, 1709, 1678, 1572, 1510, 1483, 1397, 1364, 1242, 1229; H-NMR (δ, ppm): 10.70 (1H, s, OH),
4
3
2
4
.41 (3H, s, CH ), 5.01 (2H, OCH ), 6.25 (1H, s, 3-H), 7.05 (1H, d, J = 2.5, 8-H), 7.06 (1H, dd, J = 9.0,
3 2
3
3
J = 2.5, 6-H), 7.73 (1H, d, J = 8.5, 5-H), 7.86 (1H, s, =CH-), 11.7 (1H, s, NH), 7.58 (2H, d, J = 9.0)
3
13
and 6.95 (2H, d, J = 9.0) (Hbenzene ring); C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.4 (4-C),
8.1 (CH ), 126.5 (5-C), 112.7 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1
1
3
2 2
(OCH ), 166.1 (–NH–CO–CH ), 163.2 (>N–CO), 114.3 (>C= thiazolidine ring), 190.1 (C=S), 135.7 (=CH–),
1
23.8, 133.7 and 116.7 (Cbenzene ring), 161.2 (Carom–OH); HR-ESI-MS: 469.0497 (M+H), calcd. for
(
C H N O S ): 468.0450.
22 16 2 6 2
N-(5-(4-Methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
−1
acetamide (5c): Yellow powder; yield: 71.0%; mp: 239–240 °C; IR (ν, cm ): 3205, 3080, 2936, 1736,
1
4
1
699, 1618, 1510, 1479, 1389, 1314, 1258, 1229, 1180, 1134; H-NMR (δ, ppm): 6.26 (1H, d, J = 1.0,
4
3
3
4
3-H), 2.41 (3H, d, J = 1.0, CH
3
), 7.72 (1H, J = 8.5, 5-H), 7.08 (1H, dd, J = 8.5; J = 2.5, 6-H), 7.05
4
3
(1H, d, J = 2.5, 8-H), 5.02 (2H, OCH
2
), 11.70 (1H, s, NH), 7.92 (1H, s, =CH-), 7.68 (2H, d, J = 8.5) and
3
13
7
1
6
.15 (2H, d, J = 8.5) (Hbenzene ring), 3.85 (3H, s, OCH
3
); C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C),
3
), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C),
53.3 (4-C), 18.1 (CH
6.1 (OCH ), 166.0 (–NH–CO–CH ), 163.2 (>N–CO), 115.6 (>C= thiazolidine ring), 190.0 (C=S), 135.2
2
2
(
=CH–), 125.2, 133.3 and 115.2 (Cbenzene ring), 161.9 (Carom–OCH
3 3
), 55.6 (OCH ); HR-ESI-MS: 505.0494
(
18 2 6 2
M+Na), calcd. for (C23H N O S ): 482.0606.
N-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide (5d):
−
1
Yellow powder; yield: 54.0%; mp: 249–250 °C; IR (ν, cm ): 3376, 3080, 1740, 1719, 1622, 1564,
1
1
479, 1427, 1370, 1296, 1256, 1220; H-NMR (δ, ppm): 2.41 (3H, s, CH
3
), 5.02 (2H, OCH
2
); 6.25
4
3
4
3
(
1H, s, 3-H), 7.05 (1H, d, J = 2.5, 8-H), 7.08 (1H, dd, J = 8.5, J = 2.0, 6-H), 7.74 (1H, d, J = 8.5,
3
13
5
-H), 7.96 (1H, s, =CH-), 7.69 (2H, d, J =7.0) and 7.57 (3H, m) (Hbenzene ring); C-NMR (δ, ppm):
60.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH ), 126.5 (5-C), 112.6 (6-C), δ 160.3 (7-C), 101.9
), 166.1 (–NH–CO–CH ), 163.1 (>N–CO), 119.8
1
3
(
(
8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH
2
2
>C= thiazolidine ring), 190.1 (C=S), 135.1 (–CH=), 132.7, 130.9, 131.4 and 129.6 (Cbenzene ring); HR-ESI-MS:
53.0478 (M+H), calcd. for (C22
4
16 2 5 2
H N O S ): 452.0501.
N-(5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
−1
acetamide (5e): Yellow powder; yield: 73.0%; mp: 279–280 °C; IR (ν, cm ): 3214, 3100, 1736, 1697,
1
1
618, 1605, 1578, 1487, 1440, 1387, 1269, 1258, 1240; H-NMR (δ, ppm): 2.41 (3H, s, CH
3
), 5.02
4
3
4
(
2
2H, OCH ); 6.30 (1H, s, 3-H), 7.04 (1H, d, J = 2.5, 8-H), 7.06 (1H, dd, J = 8.5, J = 2.5, 6-H), 7.74
3
3
3
(
1
1H, d, J = 8.5, 5-H), 7.97 (1H, s, =CH–), 7.76 (2H, d, J = 9.0) and 7.62 (2H, d, J = 9.0) (Hbenzene ring);
3
C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH
60.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH ), 166.1 (–NH–CO–CH
3
), 126.5 (5-C), 112.6 (6-C),
), 163.0 (>N–CO),
1
2
2

Molecules 2014, 19
13583
1
19.9 (>C= thiazolidine ring), 189.8 (C=S), 133.8 (=CH–),131.8 and 132.6 (Cbenzene ring), 125.1 (Carom–Br);
HR-ESI-MS: 530.9671 (M+H) and 532.9651 [M+H+2], calcd. for (C22
and 531.9606.
2 5 2
H15BrN O S ): 529.9606
N-(5-(4-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
−1
acetamide (5f): Yellow powder; yield: 70.0%; mp: 275–276 °C; IR (ν, cm ): 3215, 3084, 2,934, 1736,
1
1
697, 1605, 1584, 1489, 1476, 1441, 1387, 1370, 1269, 1258, 1242, 1132; H-NMR (δ, ppm): 6.30
3
3
4
(
1H, s, 3-H), 2.41 (3H, s, CH ), 7.74 (1H, d, J = 8.5; 5-H), 7.07 (1H, dd, J = 8.5, J = 2.5, 6-H), 7.05
3
4
3
(1H, d, J = 2.5, 8-H), 5.02 (2H, OCH
2
), 11.7 (1H, s, NH), 7.97 (1H, s, =CH–), 7.72 (2H, d, J = 8.5)
3
13
and 7.64 (2H, d, J = 8.5) (Hbenzene ring); C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C),
1
8.1 (CH ), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0
3
2 2
(OCH ), 166.1 (–NH–CO–CH ), 163.0 (>N–CO), 119.8 (>C= thiazolidine ring), 189.8 (C=S), 136.1 (=CH–),
1
31.5, 132.5 and 129.6 (Cbenzene ring), 133.7 (Carom–Cl); HR-ESI-MS: 509.0004 (M+Na), calcd. for
(
C H ClN O S ): 486.0111.
22 15 2 5 2
2
-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(5-(2-nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
−1
acetamide (5g): Pale pink powder; yield: 57.0%; mp: 260–261 °C; IR (ν, cm ): 3316, 3080, 2940,
1
1
755, 1717, 1616, 1522, 1483, 1387, 1343, 1298, 1271; H-NMR (δ, ppm): 6.30 (1H, s, 3-H), 2.41
3
3
4
4
(3H, s, CH
3
), 7.74 (1H, d, J = 8.5, 5-H), 7.07 (1H, dd, J = 8.5, J = 2.5, 6-H), 7.05 (1H, d, J = 2.5,
3
8
-H), 5.03 (2H, OCH
2
), 11.6 (1H, s, NH), 8.22 (1H, s, =CH–), 7.81 (1H, d, J = 7.5), 7.79 (1H, dd,
3
3
3
3
3
13
J
1
= J
2
= 7.5), 7.91 (1H, dd, J
1
= J
2
= 7.5) and 8.26 (1H, d, J = 7.5) (Hbenzene ring); C-NMR (δ,
), 126.6 (5-C), δ 112.6 (6-C), 160.3 (7-C),
), 166.1 (–NH–CO–CH ), 162.3 (>N–CO), 123.4
), 131.7, 125.7, 129.6, 132.3 and 128.3
): 497.0351.
ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH
3
1
01.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0 (OCH
2
2
(>C= thiazolidine ring), 190.3 (C=S), 134.8 (=CH–),147.8 (Carom–NO
2
(
C
benzene ring); HR-ESI-MS: 498.04295 (M+H), calcd. for (C22H N O S
15 3 7 2
2
-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(5-(4-nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
−1
acetamide (5h): Yellow powder; yield: 60%; mp: 273–274 °C; IR (ν, cm ): 3235, 3084, 2940, 1736,
1
1
697, 1609, 1526, 1474, 1442, 1387, 1346, 1269, 1238, 1200; H-NMR (δ, ppm): 6.30 (1H, s, 3-H),
3
3
4
2
.41 (3H, s, CH ), 7.73 (1H, d, J = 9.0, 5-H), 7.07 (1H, dd, J = 9.0, J= 2.5, 6-H), 7.04 (1H, d,
3
4
3
J = 2.5, 8-H), 5.04 (2H, OCH
2
), 11.7 (1H, s, NH), 8.08 (1H, s, =CH–), 8.36 (2H, d, J = 8.5) and 7.95
13
3
(2H, d, J = 8.5) (Hbenzene ring); C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1
(
CH
66.1 (–NH–CO–CH
), 138.7, 124.4 and 131.7 (Cbenzene ring); HR-ESI-MS: 520.0239 (M+Na), calcd. for
): 497.0351.
3
), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0 (OCH
2
),
1
2
), 162.9 (>N–CO), 123.4 (>C= thiazolidine ring), 189.6 (C=S), 132.2 (=CH–), 147.9
(
(
Carom–NO
2
22 15 3 7 2
C H N O S
3.3. Determination of the in Vitro Antimicrobial Activity
The compounds 5a–h at concentrations of 0.1% and 0.2% were examined for antimicrobial activity
against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25923) (Gram-negative
bacteria), Bacillus subtilis (ATCC 11774) and Staphylococcus aureus (ATCC 11632) (Gram-positive
bacteria) according to the reported method with minor modifications [18]. A mixture of meat extract

Molecules 2014, 19
13584
(5.0 g), peptone (5.0 g), NaCl (5.0 g), agar (20.0 g) and distilled water (1000 mL) was stirred to
dissolve the ingredients and then sterilized in an autoclave to give the MPA environment for growing
the bacteria. The mixture was poured to Petri dishes. The Petri dishes then were put in a sterile cabinet
for 24 h. After infusion of the particular bacteria into the MPA environment in the Petri dishes, a hole
was drilled in the center of the dish. DMSO solution (0.1 mL) of the particular chemical at a
concentration of 0.1% or 0.2% was dripped into the hole. The samples were placed in a refrigerator for
4–8 h, and then incubated at room temperature for 24 h. The inhibiting zone was measured by the (D − d)
value expressed in millimeters (mm), where D was the diameter of inhibited zone and d was the diameter
of the hole. The evaluation was based on the following criteria: D − d ≥ 25 mm: very strong antibacterial
activity; D − d ≥ 20 mm: strong antibacterial activity; D − d ≥ 15 mm: medium antibacterial activity;
D − d ≤ 15 mm: weak antibacterial activity. Each experiment was performed in triplicate.
3.4. Minimum Inhibitory Concentration (MIC) Determination
The amount of growth in the wells containing test samples was compared with the amount of growth
in the control wells when determining the growth end points. When a single skipped well occurred, the
highest MIC was read. Each experiment was performed in triplicate. Streptomycin and tetracyclin were
used as positive controls for Gram-positive bacteria and Gram-negative bacteria, respectively.
4
. Conclusions
Eight new 5-arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiozolidin-4-one analogs
1
5
a–h were successfully synthesized. The structures of these compounds were determined by IR, H-NMR,
1
3
C-NMR and HR-ESI-MS spectral data. Most of the compounds 5a–h exhibited significant activity
against Bacillus subtilis and Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa at
a concentration of 0.2%. Further structural modification could be performed to improve the bioactivity
and may prove useful in developing new therapeutic anti-microbial agents.
Acknowledgments
The authors are thankful to the MOST, Vietnam (15/2012/HĐ-NĐT) for the financial support of the
present research. This work was also supported in part by the MOST, Taiwan, ROC.
Author Contributions
Nguyen Tien Cong, Huynh Thi Nhan, and Luong Van Hung performed the research and recorded
the spectra. Tran Dinh Thang and Ping-Chung Kuo designed research and wrote the paper. All authors
read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2014, 19
13585
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Sample Availability: Samples of the compounds 1–4 and 5a–h are available from the authors.
©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(