Antibacterial properties of 5-substituted derivatives of rhodanine-3-carboxyalkyl acids

Article abstract of DOI:10.1007/s00044-017-1852-7

A series of rhodanine 3-carboxyalkanoic acid derivatives possessing 4′-(N,N-dialkyl-amino or diphenylamino)-benzylidene moiety as a substituent at the C-5 position were synthesised and their antibacterial activity was screened. All the rhodanine derivatives showed bacteriostatic or bactericidal activity to the reference gram-positive bacterial strains, but lack of activity to the reference Gram-negative bacterial strains and yeast strains was observed.

Full text of DOI:10.1007/s00044-017-1852-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1852-7
ORIGINAL RESEARCH
Antibacterial properties of 5-substituted derivatives of rhodanine-
3-carboxyalkyl acids
Waldemar Tejchman1 Izabela Korona-Glowniak2 Anna Malm2 Marek Zylewski3
Piotr Suder4,5
●
●
●
●
Received: 31 October 2015 / Accepted: 2 March 2017
© The Author(s) 2017; This article is published with open access at Springerlink.com
Abstract A series of rhodanine 3-carboxyalkanoic acid
derivatives possessing 4′-(N,N-dialkyl-amino or diphenyla-
mino)-benzylidene moiety as a substituent at the C-5 posi-
tion were synthesised and their antibacterial activity was
screened. All the rhodanine derivatives showed bacterio-
static or bactericidal activity to the reference gram-positive
bacterial strains, but lack of activity to the reference Gram-
negative bacterial strains and yeast strains was observed.
Introduction
The 2-thiazolidine-4-one derivatives traditionally named
rhodanine have been known for over 100 years, and due to
their fascinating properties they are still examined (Lesyk
and Zimenkovsky 2004). These compounds have a broad
spectrum of biological effects (Jain et al. 2012). Rhodanine
derivatives show antimalarial (Kumar et al. 2007), anti-
tubercular (Alegaon et al. 2012), cytotoxic (Chandrappa
et al. 2009), antitumor (Rao et al. 2011; Lesyk et al. 2011),
antiviral (Kaminskyy 2015), and antibacterial activity
(Bhatti et al. 2013; Kavitha et al. 2006; Song et al. 2014).
The research to obtain new antibacterial compounds is
vitally important. Recently, due to excessive and improper
use of antibiotics, there has been an increasing rate of
antibiotic resistance in the bacterial strains (Woodford
2003), thus new groups of compounds which may be useful
as antibacterial agents have been examined. A few reports
has been published regarding the rhodanine derivatives with
a carboxyalkyl acid moiety at the N-3 position (Xu et al.
2012). Biological activity of hybrid compounds possessing
chalcone and rhodanine-3-acetic acid has been also studied
(Chen et al. 2010). Such hybrids demonstrated synergistic
effect. Antibacterial activity of rhodanine derivatives and
their oxygen analogues derived from 2,4-thiazolidinedione
was also compared (Zvarec et al. 2012). However, the
results of present study suggested that rhodanine derivatives
showed greater antibacterial activity than their analogues
from the 2,4-thiazolidinedione group having at the C-2
position exocyclic oxygen atom. It was shown that the
activity of the rhodanine derivative correlates with the size
of the substituent at the C-5 position (Pardasani et al. 2001).
The research conducted by Miao et al. (2013) and Patel
et al. (2013) indicated that antibacterial activity of the acid
derivatives occurred when a major hydrophobic group was
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Keywords Rhodanine Thiazolidine-4-one Rhodanine-3-
●
acetic acid Antibacterial activity
* Izabela Korona-Glowniak
iza.glowniak@umlub.pl
1
Departament of Chemistry, Institute of Biology, Pedagogical
University of Cracow, Podchorazych 2, Kraków 30-084,
Poland
2
Department of Pharmaceutical Microbiology, Medical University
of Lublin, Chodzki 1, Lublin 20-093, Poland
3
Jagiellonian Center of Innovation, NMR Laboratory,
Bobrzyńskiego 14, Kraków 30-348, Poland
4
Department of Biochemistry and Neurobiology, AGH University
of Science and Technology, Mickiewicza 30, Kraków 30–059,
Poland
5
Academic Centre for Materials and Nanotechnology, AGH
University of Science and Technology, Mickiewicza 30, Kraków
30–059, Poland

Med Chem Res
S
C
introduced to the arylidene substituent at the C-5 position.
The best results were achieved when an aryl group addi-
tionally with an electron-withdrawing group was intro-
duced. The rhodanine derivatives possessing a 4-(N,N-
dimethylamino)-benzylidene substituent at the C-5 position
were also examined. These compounds acted as β-lactamase
inhibitors (Grant et al. 2000). Taking into account the data
presented by other authors, we decided to synthesise a series
of derivatives having carboxyalkyl (acetic, propionic,
butyric) acid fragment at N-3 position and benzylidene
para-substituent with dimethyloamino, diethylamino, dibu-
thyloamino or diphenylamino group at C-5 position.
(CH2) -COOK
n
H2N-(CH2) -COOH + CS2 + 2KOH
n
KS
N
H
S
(CH2) -COOK
n
+ ClCH2COOH
HCl
KS
C
N
H
O
HOOC-(CH2)
N
S
n
(CH2) -COOK
HOOCCH -
S
C
N
H
S
2
n
S
/1a - d/
1a - n = 1
1b - n = 2
1c - n = 3
1d - n = 5
Chemistry
Scheme 1 . 3-Carboxyalkylrhodanine acids synthesis
Our initial research proved that the antibacterial activity of
the rhodanine derivatives which have carboxyalkyl frag-
ment at N-3 position was more effective than the com-
pounds with a substituent containing an amino group at C-5
position. We synthesised a series of rhodanine derivatives
with a carboxyalkyl acid radical at N-3 position (acetic,
propionic, butyric, caproic). The synthesis of the 3-
carboxyalkylrhodanine acids (Scheme 1) was conducted
according to the modified procedure proposed by Körner
(1908) at the beginning of the 20th century.
The synthesised compounds underwent Knoevenagel con-
densation with 4-diethylaminobenzoic, 4-dibutylaminobenzoic
aldehydes and 4-diphenylaminobenzoic aldehyde with trie-
thylamine as a catalyst. Quaternary ammonium salts, the
intermediates obtained during reactions, were not isolated but
transformed to appropriate acids with hydrochloric acid
(Scheme 2).
O
R
R
O
H
HOOC-(CH2)
N
n
(C2H5)3N
+
N
C
S
S
/1/
/2/
O
(C2H5)3NHOOC-(CH2)
N
n
R
HCl
N
CH
S
S
R
O
HOOC-(CH2)
N
n
R
R
N
CH
S
S
R = -C2H5, -C4H9, -C6H5
n = 1, 2, 3 or 5
/3a-d, 4a-c, 5a-c/
3a - n = 1, R = -C2H5
3b - n = 2, R = -C2H5
3c - n = 3, R = -C2H5
3d - n = 3, R = -C2H5
4a - n = 1, R = -C4H9
4b - n = 2, R = -C4H9
4c - n = 3, R = -C4H9
5a - n = 1, R = -C6H5
5b - n = 2, R = -C6H5
5c - n = 3, R = -C6H5
Scheme 2 Rhodanine-3-carboxyalkyl acid condensation with
aldehydes
Material and methods
All reagents for the synthesis of rhodanine derivatives were
purchased from Sigma-Aldrich and used without further
purification.
analyser with the aid of helium gas. The collision energy
was set to ca. 1 eV. The samples were introduced into the
mass spectrometer in a CH3OH:CHCl3 1:1 solution with
0.1% HCOOH acidification.
Melting point (uncorrected) has been determined on the
Boetius apparatus. The IR spectrum has been recorded with
Jasco FT IR-670 Plus spectrophotometer in the KBr disk.
The NMR spectra were obtained in CDCl3 on the Bruker
Avance III HD spectrometer operating at 400.17 MHz (1H)
and 100.62 MHz (13C) and the Varian Mercury-VX
300 spectrometer operating at 300.08 MHz (1H) and
75.46 MHz (13C), the chemical shifts (ppm) have been
referenced to lock out the signal of the solvent, J has been
expressed in Hz.
General procedure of rhodanine-3-alkanoic acids
synthesis
The solution of 11.22 g (0.2 mol) potassium hydroxide in
50 cm3 of water was added to the suspension of 0.1 mol of
the appropriate amino acid (aminoethanoic acid, 3-
aminopropanoic acid, 4-aminobutanoic acid and 6-
aminohexanoic acid). The resulting solution was cooled to
5 °C and 7.6 g (0.1 mol) carbon disulphide was added. The
content of the flask was mixed at 5 °C for 7 h. The cooling
bath was removed and mixing was continued in room
temperature for 20 h.
The MS analyses were obtained on the AmaZon ETD
mass spectrometer (Bruker Daltonics, Bremen, Germany).
Scan parameters: scan range 100–1000 m/z, positive ioni-
sation mode. CID fragmentation were in the ion trap

Med Chem Res
The solution of 9.45 g (0.1 mol) chloroacetic acid in 50
cm3 water was added to the resulting solution. The solution
was mixed for 7 h at the temperature below 15 °C. Next, the
solution of 60 cm3 hydrochloric acid in 100 cm3 of water
was added to the flask content. The resulting mixture was
heated to 90 °C and kept at the temperature for 20 min.
After cooling, a sediment was received, which was drained
and crystallised from water.
(Ar–C), 133.72 (Ar–C), 135.23 (=CH–Ar), 149.82 (S–
C=CH), 167.76 (N–C=O), 175.59 (HOOC–), 192.98
(S=C–S)
3c/ 5-(4′-N,N-diethylaminobenzylidene)-rhodanine-3-
butyric acid
m.p. 155–157 °C, yield 17.06%, MS [M+1]+—379.1, IR
cm-3: 1716.3C=O, 1693.2C=O conj., 1610.2C=C exo.,
1
1340.3 C–N, 1194.7C=S, H NMR(400 MHz, CDCl3), δ
General procedure of rhodanine-3-alkanoic acids
condensation with aldehydes
ppm, 7.67 (s, 1H,=CH–Ar), 7.39 (d, J = 8.84 Hz, 2H,
Ar–H), 6,72 (d, J = 9.04 Hz, 2H, Ar–H), 4.21 (t, 2H,
HOOC–CH2–CH2–CH2–N), 3.45 (q, 4H, N(CH2CH3)2),
2.45 (t, 2H, HOOC–CH2–CH2–CH2–N), 2.09 (q, 2H,
0.005 mol
of
appropriate
rhodanine-3-alkanoic
acid, 5 g molecular sieves 4 A, 25 cm3 isopropyl alcohol,
0.0055 mol appropriate aldehyde and 2.53 g (0.025 mol)
triethylamine were placed in a flask. The mixture was
heated under a reflux condenser for 5 h in nitrogen. After
heating, the solution was filtered hot. The permeate was
cooled and 50 cm3 of 2M hydrochloric acid solution was
added. The resulting sediment was filtered using Büchner
funnel and crystallised from isopropyl alcohol or glacial
acetic acid.
HOOC–CH2–CH2–CH2–N), 1.26 (t, 6H, N(CH2CH3)2) 13
C
NMR (101 MHz, CDCl3), δ ppm, 12.58 (N(CH2CH3)2),
22.28 (CH2–CH2–CH2–N), 31.17 (CH2–CH2–CH2–N),
43.43 (CH2–CH2–CH2–N), 44.67 (N(CH2CH3)2), 111.66
(Ar–C), 114.74 (Ar–C), 120.22 (Ar–C), 133.66 (Ar–C),
134.94 (=CH–Ar), 149.75 (S–C=CH), 168.23 (N–C=O),
177.79 (HOOC–), 193.38 (S=C–S)
3d/ 5-(4′-N,N-diethylaminobenzylidene)-rhodanine-3-
caproic acid
3a/ 5-(4′-N,N-diethylaminobenzylidene)-rhodanine-3-acetic
acid
m.p. 135–137 °C, yield 32.96%, MS [M+1]+—407.1, IR
cm-3: 1717.3C=O, 1702.8C=O conj., 1615.1C=C exo.,
1327.7 C–N, 1195.7C=S, 1H NMR(400 MHz, CDCl3),
δ ppm,11.25 (br. s HOOC–) 7.66 (s, 1H,=CH–Ar), 7.39
(d, J = 8.97 Hz, 2H, Ar–H), 6,72 (d, J = 8.48 Hz, 2H,
Ar–H), 4.13 (t, 2H, HOOC–CH2–CH2–CH2–CH2–
CH2–N), 3.45 (q, 4H, N(CH2CH3)2), 2.39 (t, 2H, HOOC–
CH2–CH2–CH2–CH2–CH2–N), 1.74 (m, 4H, HOOC–CH2–
CH2–CH2–CH2–CH2–N), 1.43 (m, 2H, HOOC–CH2–CH2–
CH2–CH2–CH2–N), 1.25 (t, 6H, N(CH2CH3)2) 13C NMR
(101 MHz, CDCl3), δ ppm, 12.58 (N(CH2CH3)2), 24.19
m.p. 239–241 °C, yield 43.48%, MS [M+1]+—351.1, IR
cm-3: 1719.3C=O, 1699.9C=O conj., 1612.2, C=C exo.,
1322.9
C–N,
1185.1C=S,
1H
NMR(400 MHz,
CDCl3+MeOD), δ ppm, 7.66 (s, 1H,=CH–Ar), 7.37 (d, J
= 8.97 Hz, 2H, Ar–H), 6,70 (d, J = 9.00 Hz, 2H, Ar–H),
4.81 (s, 2H, HOOC–CH2–N), 3.43 (q, 4H, N(CH2CH3)2),
1.20 (t, 6H, N(CH2CH3)2) 13C NMR (101 MHz,
CDCl3+MeOD), δ ppm, 12.35 (N(CH2CH3)2), 44.61
(CH2–N), 47.70 (N(CH2CH3)2), 111.67 (Ar–C), 114.09
(Ar–C), 119.93 (Ar–C), 133.78 (Ar–C), 135.65 (=CH–Ar),
150.01 (S–C=CH), 167.68 (N–C=O), 168.23 (HOOC–),
193.16 (S=C–S)
(CH2–CH2–CH2–CH2–CH2–N),
26.20
(CH2–CH2–
CH2–CH2–CH2–N), 26.62 (CH2–CH2–CH2–CH2–CH2
–N), 44.21((CH2–CH2–CH2–CH2–CH2–N), 44.73 (N(CH2
CH3)2), 111.72 (Ar–C), 115.10 (Ar–C), 120.30 (Ar–C),
133.59 (Ar–C), 134.64 (=CH–Ar), 149.60 (S–C=CH),
168.14 (N–C=O), 179.47 (HOOC–), 193.33 (S=C–S)
3b/ 5-(4′-N,N-diethylaminobenzylidene)-rhodanine-3-
propionic acid
m.p. 202–204 °C, yield 54.70%, MS [M+1]+—365.1, IR
cm-3: 1716.2C=O, 1700.9C=O conj., 1610.3, C=C exo.,
1331.6 C–N, 1199.5C=S, H NMR(400 MHz, CDCl3), δ
ppm, 7.69 (s, 1H,=CH–Ar), 7.40 (d, J = 9.00 Hz, 2H,
Ar–H), 6,72 (d, J = 9.09 Hz, 2H, Ar–H), 4.45 (t, 2H,
HOOC–CH2–CH2–N), 3.46 (q, 4H, N(CH2CH3)2), 2.85 (t,
2H, HOOC–CH2–CH2–N), 1.24 (t, 6H, N(CH2CH3)2) 13C
NMR (101 MHz, CDCl3), δ ppm, 12.57 (N(CH2CH3)2),
30.97 (CH2–CH2–N), 39.45 (CH2–CH2–N), 44.70(N
(CH2CH3)2), 111.70 (Ar–C), 114.51 (Ar–C), 120.17
4a/ 5-(4′-N,N-dibutylaminobenzylidene)-rhodanine-3-
acetic acid
1
m.p. 191–194 °C, yield 23.0%, MS [M+1]+—407.1, IR
cm-3: 1716.3C=O, 1698.0C=O conj., 1636.3C=C exo.,
1
1324.9 C–N, 1184.1C=S, H NMR(400 MHz, CDCl3), δ
ppm, 7.72 (s, 1H,=CH–Ar), 7.40 (d, J = 8.96 Hz, 2H,
Ar–H), 6,72 (d, J = 7.64 Hz, 2H, Ar–H), 4.95 (s, 2H,
HOOC–CH2–N), 3.37 (t, 4H, N(CH2CH2CH2CH3)2), 1.63
(q, 4H, N(CH2CH2CH2CH3)2), 1.39 (m, 4H,
N

Med Chem Res
(CH2CH2CH2CH3)2), 0.99 (t, 6H, N(CH2CH2CH2CH3)2)
13C NMR (101 MHz, CDCl3),
ppm, 13.92 (N
5a/ 55-(4′-N,N-diphenylaminobenzylidene)-rhodanine-3-
acetic acid
δ
(CH2CH2CH2CH3)2), 20.26 (N(CH2CH2CH2CH3)2), 29.32
(N(CH2CH2CH2CH3)2), 44.25 (–CH2–N), 51.01 (N
(CH2CH2CH2CH3)2), 112.02 (Ar–C), 114.11 (Ar–C),
120.22 (Ar–C), 133.68 (Ar–C), 135.70 (=CH–Ar), 150.17
(S–C=CH), 167.31 (N–C=O), 170.42 (HOOC–), 192.85
(S=C–S)
m.p. 240–242 °C, yield 64.27%, MS [M+1]+—447.1, IR
cm-3: 1724.1C=O, 1706.7C=O conj., 1634.4C=C exo.,
1329.7 C–N, 1192.7C=S, H NMR(400 MHz, CDCl3), δ
ppm, 7.76 (s, 1H,=CH–Ar), 7.53 (d, J = 8.96 Hz, 2H,
Ar–H), 7.44–7.40 (m. 6H Ar), 7.25–7.18 (m, 6H Ar), 4.73
(s, 2H, HOOC–CH2–N) 13C NMR (101 MHz, CDCl3), δ
ppm, 43.46 (–CH2–N), 39.87 (CH2–CH2–N), 117.67,
118.64, 119.67, 124.97, 125.85. 125.94, 125.66, 126.87,
129.05, 130.47, 130.50, 131.76, 133.32, 134.47, 166.92,
(Ar–C), 145.98 (=CH–Ar), 150.65 (S–C=CH), 167.63 (N–
C=O), 191.02 (HOOC–), 193.33 (S=C–S)
1
4b/ 5-(4′-N,N-dibutylaminobenzylidene)-rhodanine-3-
propionic acid
m.p. 163–165 °C, yield 21.99%, MS [M+1]+—421.2, IR
cm-3: 1727.9C=O, 1698.0C=O conj., 1610.3C=C exo.,
1
1336.4 C–N, 1189.9C=S, H NMR(400 MHz, CDCl3), δ
5b/ 5-(4′-N,N-diphenylaminobenzylidene)-rhodanine-3-
ppm, 7.68 (s, 1H,=CH–Ar), 7.38 (d, J = 9.18 Hz, 2H,
Ar–H), 6,69 (d, J = 8.80 Hz, 2H, Ar–H), 4.46 (t, 2H,
HOOC–CH2–CH2–N), 3.37 (t, 4H, N(CH2CH2CH2CH3)2),
2.86 (t, 2H, HOOC–CH2–CH2–N), 1.62 (q, 4H, N
(CH2CH2CH2CH3)2), 1.39 (q, 4H, N(CH2CH2CH2CH3)2),
1.02 (t, 6H, N(CH2CH2CH2CH3)2) 13C NMR (101 MHz,
CDCl3), δ ppm, 13.94 (N(CH2CH2CH2CH3)2), 20.27 (N
(CH2CH2CH2CH3)2), 29.37 (N(CH2CH2CH2CH3)2), 30.95
propionic acid
m.p. 212–215 °C, yield 76.09%, MS [M+1]+—461.1 IR
cm-3: 1733.7C=O, 1706.7C=O conj., 1637.3C=C exo.,
1338.4
C–N,
1193.7C=S,
1H
NMR(400 MHz,
CDCl3+MeOD), δ ppm, 7.61 (s, 1H,=CH–Ar), 7.32–7.23
(m. 6H Ar), 7.14–7.12 (m, 6H Ar), 6,98 (d, J = 8.76 Hz,
2H, Ar–H), 4.37 (t, 2H, HOOC–CH2–CH2–N), 2.71
(t, 2H, HOOC–CH2–CH2–N) 13C NMR (101 MHz, CDCl3
+MeOD), δ ppm, 30.97 (–CH2–CH2–N), 39.87 (CH2–
CH2–N), 118.24, 120.30, 124.97, 125.22, 126.05, 129.66,
132.39, 133.77 (Ar–C), 146.04 (=CH–Ar), 150.46 (S–
C=CH), 167.76 (N–C=O), 172.81 (HOOC–), 192.95
(S=C–S)
(CH2–CH2–N),
39.45
(CH2–CH2–N),
50.87
(N
(CH2CH2CH2CH3)2), 111.82 (Ar–C), 114.41 (Ar–C),
120.06 (Ar–C), 133.64 (Ar–C), 135.23 (=CH–Ar), 150.22
(S–C=CH), 167.77 (N–C=O), 175.49 (HOOC–), 192.96
(S=C–S)
4c/ 5-(4′-N,N-dibutylaminobenzylidene)-rhodanine-3-
5c/ 5-(4′-N,N-diphenylaminobenzylidene)-rhodanine-3-
butyric acid
butyric acid
m.p. 134–136 °C, yield 25.23%, MS [M+1]+—435.1,
IR cm-3: 1710.5C=O, 1691.3C=O conj., 1637.3C=C
exo., 1333.5 C–N, 1193.7C=S, 1H NMR(400 MHz,
CDCl3), δ ppm, 11.00 (br. s, 1H, HOOC–) 7.66 (s, 1H,
=CH–Ar), 7.38 (d, J = 8.97 Hz, 2H, Ar–H), 6.67
(d, J = 9.05 Hz, 2H, Ar–H), 4.21 (t, 2H, HOOC–
CH2–CH2–CH2–N), 3.36 (t, 4H, N(CH2CH2CH2
CH3)2), 2.45 (t, 2H, HOOC–CH2–CH2–CH2–N),
2.09 (t, 2H, HOOC–CH2–CH2–CH2–N), 1.62 (q, 4H,
N(CH2CH2CH2CH3)2), 1.39 (m, 4H, N(CH2CH2CH2
CH3)2), 0.99 (t, 6H, N(CH2CH2CH2CH3)2) 13C NMR
m.p. 137–140 °C, yield 75.33%, MS [M+1]+—475.1, IR
cm-3: 1721.2C=O, 1700.9C=O conj., 1638.23C=C
exo., 1330.6 C–N, 1193.7C=S, 1H NMR(400 MHz,
CDCl3), δ ppm, 10.90 (br. s, HOOC–), 7.67 (s, 1H,=
CH–Ar), 7.37–7.33 (m, 6H Ar), 7.19–7.16 (m, 6H Ar),
7.05 (d, J=8.80 Hz, 2H, Ar–H), 4.23 (t, 2H, HOOC–
CH2–CH2–CH2–N), 2.47 (t, 2H, HOOC–CH2–CH2–
CH2–N), 2.10 (q, 2H, HOOC–CH2–CH2–CH2–N) 13C
NMR (101 MHz, CDCl3), δ ppm, 22.24 (CH2–CH2–CH2
–N), 31.10 (CH2–CH2–CH2–N), 43.50 (CH2–CH2–CH2
–N), 118.50, 120.43, 124.99, 125.41, 126.09, 129.72,
132.40, 133.58 (Ar–C), 146.14 (=CH–Ar), 150.40 (S–
C=CH), 168.12 (N–C=O), 177.75 (HOOC–), 193.27
(S=C–S)
(101 MHz, CDCl3),
δ ppm, 13.94 (N(CH2CH2CH2
CH3)2), 20.27 (N(CH2CH2CH2CH3)2), 22.27 (CH2–
CH2–CH2–N), 29.38 (N(CH2CH2CH2CH3)2), 31.21 (CH2–
CH2–CH2–N), 43.43 (CH2–CH2–CH2–N), 50.85 (N
(CH2CH2CH2CH3)2), 111.78 (Ar–C), 114.66 (Ar–C),
120.13 (Ar–C), 133.58 (Ar–C), 134.93 (=CH–Ar), 150.15
(S–C=CH), 168.23 (N–C=O), 178.12 (HOOC–), 193.35
(S=C–S)
Antibacterial activity assay in vitro
The 5-substituted derivatives of rhodanine-3-carboxyalkyl
acids were screened for antibacterial and antifungal

Med Chem Res
activities by micro-dilution broth method using Mueller-
Hinton broth and Mueller-Hinton broth with 5% lysed
sheep blood for growth of non-fastidious and fastidious
bacteria, respectively or Mueller-Hinton broth with 2%
glucose for growth of fungi. Minimal inhibitory con-
centration (MIC) of the tested derivatives were evaluated for
the panel of the reference microorganisms from American
Type Culture Collection (ATCC), including Gram-negative
bacteria (Escherichia coli ATCC 25922, Salmonella typhi-
murium ATCC14028, Klebsiella pneumoniae ATCC
13883, Pseudomonas aeruginosa ATCC 9027, Proteus
mirabilis ATCC 12453), gram-positive bacteria (Staphylo-
coccus aureus ATCC 25923, Staphylococcus aureus ATCC
6538, Staphylococcus epidermidis ATCC 12228, Micro-
coccus luteus ATCC 10240, Bacillus subtilis ATCC 6633,
Bacillus cereus ATCC 10876, Streptococcus pyogenes
ATCC 19615, Streptococcus pneumoniae ATCC 49619,
Streptococcus mutans ATCC 25175), and fungi (Candida
albicans ATCC 10231, Candida parapsilosis ATCC
22019).
yeast strains—30 °C for 48 h), the MICs were assessed
visually as the lowest concentration of the 5-substituted
derivatives of rhodanine-3-carboxyalkyl acids showing
complete growth inhibition of the reference microbial
strains. Appropriate DMSO control (at a final concentration
of 10%), a positive control (containing inoculum without
the tested derivatives) and negative control (containing the
tested derivatives without inoculum) were included on each
microplate.
Minimal bactericidal concentration (MBC) or minimal
fungicidal concentration (MFC) was determined by sub-
culturing 100 μl of the microbial culture from each well that
showed through growth inhibition, from the last positive
one and from the growth control onto the recommended
agar plates. The plates were incubated at 35 °C for 24 h and
the MBC/MFC was defined as the lowest concentration of
the 5-substituted rhodanine-3-carboxyalkyl acids without
growth of microorganisms. Ciprofloxacin and vancomycin
were used as the standard drugs (Table 1). Each experiment
was repeated in triplicate. Representative data is presented.
The 5-substituted derivatives of rhodanine-3-
carboxyalkyl acids dissolved in dimethylosulfoxide
(DMSO), were first diluted to the concentration (1000 µg/
mL) in an appropriate broth medium recommended for
bacteria or yeasts. Then, using the same media, serial two-
fold dilutions were made in order to obtain final con-
centrations of the tested derivatives ranged from 0.98 to
1000 µg/mL. The sterile 96-well polystyrene microtitrate
plates (Nunc, Denmark) were prepared by dispensing 200 µl
of appropriate dilution of the tested derivatives in broth
medium per well. The inocula were prepared with fresh
microbial cultures in sterile 0.85% NaCl to match the tur-
bidity of 0.5 McFarland standard and 2 μl were added to
wells to obtain final density of 1.5 × 106 CFU/ml for bac-
teria and 5 × 104 CFU/ml for yeasts; CFU—colony forming
units. After incubation (bacterial strains—35 °C for 24 h,
Results and discussion
Chemistry
All the resulting 3-carboxyalkanoic acid derivatives occur-
red as crystalline solids red in colour. They were char-
acterised by high solubility in polar solvents (alcohols,
glacial acetic acid).
The comparison of the condensation reaction yield of all
three groups of the compounds /3a-d/, /4a-c/ and /5a-c/
indicated that 4-N,N-diphenylaminobenzoic aldehyde had
the highest activity in condensation reactions among the
aldehydes used. 4-dibutylaminobenzoic aldehyde was
characterised by the lowest activity.
Table 1 MIC (µg/mL), MBC
Microorganisms
Vancomicin
Ciprofloxacin
(µg/mL) of vancomicin and
ciprofloxacin towards reference
Gram-positive bacterial strains
MIC (µg/ MBC (µg/ MBC/MIC MIC (µg/ MBC (µg/ MBC/MIC
mL)
mL)
ratio
mL)
mL)
ratio
S. aureus ATCC6538
S. aureus ATCC25923
0.49
0.98
0.98
1.95
4
8
1
0.24
0.49
0.49
0.24
0.49
0.49
1
1
1
7.81
0.98
S. epidermidis
ATCC12228
M. luteus ATCC10240
B. subtilis ATCC6633
B. cereus ATCC10876
0.12
0.24
0.98
0.12
0.49
1
2
0.98
0.03
0.12
–
1.95
0.12
0.12
–
2
4
1
–
–
15.6
16
2
S. pyogenes ATCC19615 0.24
0.49
0.49
S. pneumoniae
0.24
2
–
–
ATCC49619
S. mutans ATCC25175
0.98
0.98
1
–
–
–

Med Chem Res
The characteristic bands deriving from the stretching
C=O and C=S groups vibrations were present in the IR
spectra of all the researched compounds. The C=O group
vibrations ranged from 1727.9 to 1710.5 cm-1, whereas
C=S group vibrations ranged from 1195.7 to 1184.1 cm-1.
The MS spectra were very simple. The highest intensity
had always the [M+1]+ ion peak. In most cases it reached
100%.
The 1H NMR spectra contained a very characteristic
signal deriving from the proton in =CH–Ar unit. It was a
singlet, which was present in the 7.61–7.76 ppm range of
chemical shifts. Position of the signal from a methine proton
in this range showed that the condensation reaction carried
out to Z isomers (Hardej et al. 2010). The 13C NMR spectra
were characterised by the signal from the carbon atom
bound with exocyclic sulphur atom. It was present in the
192.85–193.38 ppm range of chemical shifts.
Antibacterial activity
The antimicrobial activity of rhodanines has been known
for over 50 years. The design and synthesis of antibacterial
agents based on this heterocycle have been reported in
numerous studies (Pardasani et al. 2001; Grant et al. 2000;
Gandhe and Gautam 2004; Tomasic and Peterlin
Masic 2012). The 5-ylidene-4-thiazolidinones and 4-
thiazolidinone-3-carboxylic acids are the most studied and
promising 4-thiazolidinones in the context of creating new
drug-like molecules (Lesyk and Zimenkovsky 2004; Lesyk
et al. 2011). It was shown that introduction of substituents
(mainly those containing a carboxyl group) in position N3 is
the chemical path to the design of new compounds with a
significant biological activity and decreased toxicity (Bhat
et al. 2004). In present study, the antimicrobial assay of the
novel 5-substituted derivatives of rhodanine-3-carboxyalkyl
acids was carried out towards reference strains using a serial
dilution method to obtain the MIC. None of the tested
derivatives had activity against gram-negative bacteria
(Escherichia coli ATCC25922, Salmonella typhimurium
ATCC14028, Klebsiella pneumoniae ATCC13883, Pseu-
domonas aeruginosa ATCC9027, Proteus mirabilis
ATCC12453), and yeasts (Candida albicans ATCC10231,
Candida parapsilosis ATCC22019) (MIC > 1000 µg/mL,
data not shown). Tables 2–4 summarised the results
obtained for the MICs of the 10 target compounds (3a–d,
4a–c, 5a–c) to the gram-positive bacteria: staphylococi
(Staphylococcus aureus ATCC25923, Staphylococcus aur-
eus ATCC6538, Staphylococcus epidermidis ATCC12228);
micrococci (Micrococcus luteus ATCC10240), bacilli
(Bacillus subtilis ATCC6633, Bacillus cereus ATCC10876)
and streptococci (Streptococcus pyogenes ATCC19615,
Streptococcus pneumoniae ATCC49619, Streptococcus
mutans ATCC25175). Ciprofloxacin and vancomycin were

Med Chem Res
Table 3 MIC (µg/mL), MBC (µg/mL) of 4a, 4b, and 4c derivatives towards reference gram-positive bacterial strains
Microorganisms
4a
4b
4c
MIC (µg/
MBC (µg/ MBC/MIC MIC (µg/ MBC (µg/ MBC/MIC MIC (µg/ MBC (µg/ MBC/MIC
mL)
mL)
ratio
mL)
mL)
ratio
mL)
mL)
ratio
S. aureus ATCC6538
S. aureus ATCC25923
7.8
3.9
3.9
15.6
62.5
62.5
2
3.9
3.9
3.9
>1000
>1000
7.8
>256
>256
2
3.9
3.9
3.9
>1000
>1000
>1000
>256
>256
>256
16
16
S. epidermidis
ATCC12228
M. luteus ATCC10240
B. subtilis ATCC6633
B. cereus ATCC10876
3.9
3.9
3.9
125
32
1
3.9
7.8
3.9
3.9
500
500
2
2
1
4
4
3.9
>1000
3.9
>256
3.9
1.95
3.9
1.95
3.9
2
125
32
>8
4
3.9
1
S. pyogenes ATCC19615 125
>1000
500
125
125
500
500
>1000
1000
Nd
2
S. pneumoniae
125
ATCC49619
S. mutans ATCC25175
>1000
>1000
Nd
1000
>1000
Nd
1000
>1000
Nd
Table 4 MIC (µg/mL), MBC (µg/mL) of 5a, 5b, and 5c derivatives towards reference gram-positive bacterial strains
Microorganisms
5a
5b
5c
MIC (µg/ MBC (µg/ MBC/MIC MIC (µg/ MBC (µg/ MBC/
MIC (µg/ MBC (µg/ MBC/MIC
mL)
mL)
ratio
mL)
mL)
MICratio
mL)
mL)
ratio
S. aureus ATCC6538
S. aureus ATCC25923
1.95
1.95
1.95
7.8
7.8
7.8
4
4
4
1.95
1.95
1.95
31.5
31.5
31.5
16
16
16
15.6
1.95
1.95
>1000
125
>64
64
S. epidermidis
125
64
ATCC12228
M. luteus ATCC10240
B. subtilis ATCC6633
B. cereus ATCC10876
1.95
1.95
1.95
7.8
4
2
4
4
2
1.95
1.95
1.95
125
125
15.6
1.95
15.6
>1000
500
16
1
3.9
125
32
1
3.9
1.95
3.9
1.95
62.5
>1000
500
7.8
8
16
>8
4
S. pyogenes ATCC19615 125
1000
500
>8
4
125
125
S. pneumoniae
250
ATCC49619
S. mutans ATCC25175 500
>1000
Nd
>1000
>1000
Nd
>1000
>1000
Nd
used as positive controls (Table 1). Mild to moderate
activity (MIC 125–1000 µg/mL) of the all synthesised
derivatives was observed towards streptococci. The new
rhodanine compounds showed different activity from
moderate to very strong against other tested gram-positive
bacteria, i.e., staphylococci, micrococci, and bacilli,
depending on the strain and the synthesised compound. The
first group of derivatives /3a–d/ was less active towards the
tested gram-positive strains (MIC 15.6–250 µg/mL) as
compared to the second group /4a–c/ of derivatives (MIC
1.95–7.8 µg/mL) and the third group /5a–c/ of derivatives
(MIC 1.95–15.6 µg/mL). The most active compounds were
/5a/ and /5b/ showing very strong bioactivity with MIC
1.95 µg/mL. The low values of MBC/MIC ratio (2–4) for
/5a/ suggested its bactericidal power in contrast to higher
values (8–16) for /5b/ indicating bacteriostatic activities
except for bactericidal activity of /5b/ against B. subtilis
ATCC 6633 (MBC/MIC 1). The remaining derivatives
showed bactericidal (MBC/MIC ≤ 4) or bacteriostatic
activity against the tested bacteria (MBC/MIC > 4),
depending on the strain and the rhodanine compound.
In the present study, most of synthesised compounds
(4a–c and 5a–c) exhibited strong antibacterial activity
amongst the tested gram-positive bacteria, although the
mechanism of action is not yet clearly understood. How-
ever, rhodanines seem to be inhibitors of the bacterial
enzyme MurB (Andres et al. 2000). The enzyme MurB, an
NADPH dependant enolpyruvyl reductase, is responsible
for the second committed step of bacterial peptidoglycan
biosynthesis; it means that rhodanines could be expected to
be bactericidal. Peptydoglycan is an essential component of
the cell wall of both Gram-positive and Gram-negative
bacteria and enzyme MurB is found in both of them. It
would be expected that rhodanines might possess a broad

Med Chem Res
spectrum of antibacterial activity. The differences in bio-
logical activity of rhodanines to gram-positive and
gram–negative bacteria could be explained by the differ-
ences in their cell wall structure and thus in the perme-
ability. Peptydoglycan is major component (90%) of the
gram-positive cell wall, whereas in Gram-negative bacteria,
peptydoglycan, constituting 10% of cell wall, lies between
cytoplasmic membrane and the outer lipid byliayer con-
taining lipopolysaccharide, porins, adhesins which create
additional barrier to cross by.
In this study, the preliminary remarks of the structure
activity dependence can be noted. Comparison the MIC
values determined for the newly synthesised rhodanine
derivatives allowed to state that the basic factor increasing
the activity to prevent bacteria growth is the size of the
substituent at the C-5 position. The number of the carbon
atoms present in the connector between carboxylic group
and the 2,4-thiazolidinedione core is of much less impor-
tance. The influence of the connector length on the activity
to suppress bacterial growth is noticeable when 5 atoms of
carbon are present in the connector. The derivatives which
have an acetic, propionic and butyric acid fragment at N-3
position and have the same substituent at C-5 position,
demonstrated similar ability to suppress the growth of
Gram-positive bacteria. In many cases the activity was
identical.
It was observed that there was a dependency between the
growth of the substituent size at C-5 position of the rho-
danine ring and the antifungal activity growth. The deri-
vatives having a p-N,N-bezylidenediphenylamine fragment
at the C-5 position were characterised by the highest anti-
bacterial activity. The increase of activity was probably
caused by higher hydrophobicity of the aryl groups in
comparison to the alkyl groups, which has been suggested
by previous research (Miao et al. 2013; Patel et al. 2013). It
was also established that the size of the connector between
the carboxylic group and rhodanine ring had a limited
influence on the antibacterial activity. The results have
indicated the future direction of the research aiming at
synthesis of the compounds characterised by higher anti-
bacterial activity.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no competing
interests.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits use, duplica-
tion, adaptation, distribution, and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s)
and the source, provide a link to the Creative Commons license, and
indicate if changes were made.
Determining the MBC value allowed to establish the
activity to kill bacteria or inhibit its growth. Antimicrobial
agent are usually regarded as bactericidal if MBC value is
higher no more than four times the MIC value (French
2006).
It was established that /5a/ and /4b/ 5-(4′-dibutylamino-
benzylidene)-4-oxo-2-thioxo-3-thiazolidine acetic acid, as
well as 5-(4′-diphenylaminobenzylidene)-4-oxo-2-thioxo-3-
thiazolidine acetic acid and 5-(4′-diphenylaminobenzyli-
dene)-4-oxo-2-thioxo-3-thiazolidine propionic acid had
antibacterial effect on the majority of the gram-positive
bacteria strains.
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