Antimicrobial activity of some new thioureides derived from 2-(4-chlorophenoxymethyl)benzoic acid

Article abstract of DOI:10.3390/molecules13030567

We report here the characterisation of eight newly synthesized thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid and the evaluation of the in vitro antimicrobial activity of the new compounds against Gram-positive [Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis], Gram-negative [Psedomonas aeruginosa, Escherichia coli, Salmonella enteritidis], as well as Candida spp., using both reference and clinical multidrug resistant strains to establish the minimal inhibitory concentration (MIC) values. Our results showed that the tested compounds exhibited specific antimicrobial activities, both concerning the spectrum of antimicrobial activity and the corresponding MIC values, which ranged widely between 1024 and 32 μg/mL, depending on the nature and position of the substituents on the benzene ring. The most active compounds were N-[2-(4-chlorophenoxymethyl)-benzoyl]-N′-(2,6- dichlorophenyl)-thiourea (5g) and N-[2-(4-chlorophenoxymethyl)-benzoyl]- N′-(4-bromophenyl)-thiourea (5h), which showed a broad spectrum of antimicrobial activity against enterobacterial strains (E. coli and S. enteritidis), P. aeruginosa, S. aureus and Candida spp. All the tested compounds except 5f were highly active against S. aureus (MIC=32 μg/mL), suggesting their possible use in the treatment of MRSA infections. Four of compounds also exhibited antifungal activity (MIC =256-32 μg/mL) against C. albicans, but L. monocytogenes as well as B. subtilis were resistant to all tested compounds. Our studies thus demonstrated that among other biological activities, the thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid also exhibit selective and effective antimicrobial properties that could lead to the selection and use of these compounds as efficient antimicrobial agents, especially for the treatment of multidrug resistant infections.

Full text of DOI:10.3390/molecules13030567

Molecules 2008, 13, 567-580
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Antimicrobial Activity of Some New Thioureides Derived from
2-(4-Chlorophenoxymethyl)benzoic Acid
Carmen Limban 1, Mariana-Carmen Balotescu Chifiriuc 2,*, Alexandru-Vasile Missir 1, Ileana
Cornelia Chiriţă 1 and Coralia Bleotu 3
1
Pharmaceutical Chemistry Department, Faculty of Farmacy, “Carol Davila” University of Medicine
and Pharmacy, Traian Vuia 6, sect. 2, 020956, Bucharest, Romania; E-mails:
carmen_limban@yahoo.com (Carmen Limban, Cornelia Chirita, Alexandru Vasile Missir)
2
Microbiology-Immunology Department, Faculty of Biology, University of Bucharest, Ale.
Portocalelor 1-3, sect. 5, Bucharest, Romania; E-mail: carmen_chifiriuc@yahoo.com (Mariana-
Carmen Balotescu Chifiriuc)
3
Institute of Virology “Prof. St. Nicolau”, Sos. M. Bravu, 36, sect.2, Bucharest, Romania; E-mails:
cbleotu@yahoo.com (Coralia Bleotu)
* Author to whom correspondence should be addressed; E-mail: carmen_balotescu@yahoo.com
Received: 29 January 2008; in revised form: 29 February 2008 / Accepted: 29 February 2008 /
Published: 4 March 2008
Abstract: We report here the characterisation of eight newly synthesized thioureides of 2-
(4-chlorophenoxymethyl)-benzoic acid and the evaluation of the in vitro antimicrobial
activity of the new compounds against Gram-positive [Listeria
monocytogenes,
Staphylococcus aureus, Bacillus subtilis], Gram-negative [Psedomonas aeruginosa,
Escherichia coli, Salmonella enteritidis], as well as Candida spp., using both reference and
clinical multidrug resistant strains to establish the minimal inhibitory concentration (MIC)
values. Our results showed that the tested compounds exhibited specific antimicrobial
activities, both concerning the spectrum of antimicrobial activity and the corresponding
MIC values, which ranged widely between 1024 and 32 μg/mL, depending on the nature
and position of the substituents on the benzene ring. The most active compounds were N-
[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(2,6-dichlorophenyl)-thiourea (5g) and N-[2-(4-
chlorophenoxymethyl)-benzoyl]-N'-(4-bromophenyl)-thiourea (5h), which showed a broad
spectrum of antimicrobial activity against enterobacterial strains (E. coli and S. enteritidis),
P. aeruginosa, S. aureus and Candida spp. All the tested compounds except 5f were highly

Molecules 2008, 13
active against S. aureus (MIC=32 μg/mL), suggesting their possible use in the treatment of
568
MRSA infections. Four of compounds also exhibited antifungal activity (MIC =256-32
μg/mL) against C. albicans, but L. monocytogenes as well as B. subtilis were resistant to
all tested compounds. Our studies thus demonstrated that among other biological activities,
the thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid also exhibit selective and
effective antimicrobial properties that could lead to the selection and use of these
compounds as efficient antimicrobial agents, especially for the treatment of multidrug
resistant infections.
Keywords: Thioureides, 2-(4-chlorophenoxymethyl)-benzoic acid, antimicrobial activity
Introduction
Thioureides are associated with a broad spectrum of biological activities including antibacterial,
antifungal, tuberculostatic, anthelmintic, antitumor, anticonvulsant, diuretic, insecticidal and pesticidal
properties. In light of the therapeutical importance of some N-acylated thiourea derivates, especially as
antibacterials and antifungals, we have evaluated the in vitro activity of some new thioureides derived
from 2-(4-chlorophenoxymethyl)-benzoic acid. We have focused on a qualitative screening assay of
the antimicrobial properties (adapted disk diffusion method), as well as a quantitative assay of the
antimicrobial activity (binary microdilution method), to establish the minimal inhibitory concentration
(MIC) values of these compounds against microbial Gram-positive, Gram-negative and fungal strains.
We also studied the cytototoxicity of the tested compounds by flow cytometry and annexin V assay. In
previous papers [1-4] we have presented the synthesis and characterization of some other thioureides
of 2-phenoxymethylbenzoic acid, either unsubstituted or substituted with a methoxy or methyl group.
We have now extended our research to the preparation and evaluation of the antimicrobial activity of
some new thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid. The reaction conditions, yields and
some of the physical properties were established. NMR and IR spectral parameters and the elemental
analysis have confirmed the structure of the synthesized compounds.
Results and Discussion
Synthesis
The synthesis of the new compounds was completed in three stages:
I. The synthesis of 2-(4-chlorophenoxymethyl)-benzoic acid (1)
The best yields in the synthesis of compound 1 were obtained when the intermediate compound
used was the phthalide 2, which was treated with potassium p-chlorophenoxide in xylene under reflux.
This gives the potassium salt 3 of 2-(4-chlorophenoxymethyl)-benzoic acid, which has good solubility
in 10% aqueous sodium hydroxide solution, allowing its facile separation from xylene. The acid 1 is

Molecules 2008, 13
569
then precipitated using a mineral acid solution. The potassium p-chlorophenoxide was obtained
through the reaction of p-chlorophenol with potassium hydroxide in xylene. The resulting water was
removed by azeotropic distillation. These reactions are presented in Scheme 1.
Scheme 1. The synthesis of 2-(4-chlorophenoxymethyl)-benzoic acid (1).
O
CH2
-
+
O K
Cl
+
O
-
+
Cl
COO K
O
2
3
O
CH2
HCl
- KCl
Cl
COOH
1
II. The synthesis of 2-(4-chloro-phenoxymethyl)-benzoyl chloride (4)
2-(4-Chlorophenoxymethyl)-benzoyl chloride (4) was prepared by refluxing acid 1 with thionyl
chloride, using anhydrous 1,2-dichlorethane as reaction medium (Scheme 2).
Scheme 2. The synthesis of 2-(4-chlorophenoxymethyl)-benzoyl chloride (4).
O
O
CH2
CH2
SOCl2
Cl
Cl
COOH
COCl
1
4
III. The synthesis of the new thioureides 5a-h
The new compounds 5a-h were prepared by refluxing 2-(4-chlorophenoxymethyl)-benzoyl
isothiocyanate (6) with primary aromatic amines in dry acetone. The isothiocyanate 6 was obtained
through the reaction between 2-(4-chlorophenoxymethyl)-benzoyl chloride (4) and ammonium
thiocyanate in dry acetone, as shown in Scheme 3. The isothiocyanate was not isolated and the
appropriate amines were directly added to the reaction mixture to give the thioureides.

Molecules 2008, 13
570
Figure 3. The synthesis of the new thioureides 5a-h.
O
O
CH2
CH2
NH4SCN
Cl
Cl
C-Cl
O
C-N=C=S
O
4
6
Cl
O
CH2
H2N-R
C-NH-C-NH-R
O
S
5
R= -C H , -C H CH ( ), -C H OCH ( ), -C H Cl ( ),
4
4
4
6
5
6
4
3
6
4
3
6
4
-C H Cl (
), -C H Cl (
), -C H Cl (
), -C H Br ( ),
2,6 4
6 4
2,5
2,4
6
3
2
6
3
2
6
3
2
The new thioureides are white or light yellow crystalline solids, soluble at room temperature in
acetone and chloroform, by heating in lower alcohols, benzene, toluene and xylene and insoluble in
water. The structure, molecular formulae, molecular weights, melting points and yields of the new
thioureides are presented in Table 1.
Table 1. Characteristics of the new compounds.
O
Cl
CH2
C-NH-C-NH-R
5a- h
O
S
Molecular
weight
396.893
410.913
426.913
431.336
465.786
465.786
465.786
475.799
Melting point
(0C)
No.
R
Molecular formula
Yield (%)
5a
5b
5c
5d
5e
5f
-C6H5
C21H17ClN2O2S
C22H19Cl N2O2S
C22H19ClN2O3S
C21H16Cl2N2O2S
C21H15Cl3N2O2S
C21H15Cl3N2O2S
C21H15Cl3N2O2S
C21H16ClBrN2O2S
145.2- 147.6
135.7- 138.1
132.3- 135.4
154.8- 157.4
152.5- 155.0
152.6- 155.2
201.9- 204.6
163.5- 165.2
64.3
62.9
69.4
73.2
71.6
78.5
65.7
75.3
-C6H4CH3 (4)
-C6H4OCH3 (4)
-C6H4Cl (4)
-C6H3Cl2 (2,4)
-C6H3Cl2 (2,5)
-C6H3Cl2 (2,6)
-C6H4Br (4)
5g
5h

Molecules 2008, 13
571
The calculated formulae for the proposed structures were in good agreement with the elemental
analysis results (Table 2).
Table 2. Elemental analysis data.
O
Cl
CH2
C-NH-C-NH-R
5a- h
O
S
C%
H%
N%
S%
No.
R
c.
e.
c.
e.
c.
e.
c.
e.
5a.
5b.
5c.
5d.
5e.
5f.
-C6H5
63.49
64.24
61.83
58,42
54.10
54.10
54.10
52.96
63.36
63.99
61.64
58.70
54.20
54.03
54.09
52.64
4.28
4.62
4.45
3.70
3.22
3.22
3.22
3.36
4.25
4.60
4.49
3.70
3.24
3.29
3.18
3.29
7.05
6.81
6.56
6.49
6.01
6.01
6.01
5.88
6.99
6.78
6.45
6.37
5.97
5.92
5.99
5.76
8.06
7.78
7.49
7.42
6.87
6.87
6.87
6.72
7.94
7.82
7.54
7.41
6.70
6.71
6.82
6.65
-C6H4CH3 (4)
-C6H4OCH3 (4)
-C6H4Cl (4)
-C6H3Cl2 (2,4)
-C6H3Cl2 (2,5)
-C6H3Cl2 (2,6)
-C6H4Br (4)
5g.
5h.
c = calculated; e = experimental
Spectral data
1
13
1
All new compounds were characterized by H- and C-NMR and IR spectroscopy. The H-NMR
spectral values (δ, ppm, J, Hz) for the new compounds are presented in Table 3. In Table 4, we present
the 13C-NMR assigments for the new thioureides. The caracteristic IR bands (cm-1) for the 2-(4-chloro-
phenoxymethyl)-benzoic acid thioureides are presented in Table 5.
Table 3. 1H-NMR data (δ, ppm, J, Hz).
14
13
8
12
9
4
O
Cl
CH2
3
5
6
10
11
2
1
R
C
C
NH
NH
7
15
S
O
-CH3
-OCH3
H4-
H7
H8
H10
H11
H13
H14
H17
H18
H19
H20
H21 -NH-
R
18
20
17
21
11.84
7.28-
7.68
m
7.01 7.30 7.30 7.01 7.28- 7.28- 7.28- 7.28- 7.28-
7.68 7.68 7.68 7.68 7.68
5.32
s
s
12.40
s
11.79
sl
12.33
sl
11.79
s
19
d
d
d
d
-
16
17
(7.9) (7.9) (7.9) (7.9)
m
m
m
m
m
18
2.30 s
7.56-
7.62
m
7.31 7.00 7.00 7.31 7.45 7.20
7.20 7.45
19 CH3
5.30
s
d
d
d
d
d
d
-
d
d
16
(8.9) (9.0) (9.0) (8.9) (8.6) (8.2)
(8.2) (8.6)
21
17
20
18
7.45-
7.66
m
7.02 7.46 7.46 7.02 7.32 6.96
6.96 7.32
19 O-CH3
5.31
s
d
d
d
d
d
d
-
d
d
16
21
12.26
s
(8.9) (9.0) (9.0) (8.9) (8.8) (8.8)
(8.8) (8.8)
20
3.77 s

Molecules 2008, 13
572
Table 3. Cont.
18
17
11.90
7.42-
7.63
m
7.00 7.30 7.30 7.00 7.46 7.62
7.62 7.46
19
5.32
s
s
12.38
s
Cl
Cl
d
d
d
d
d
d
-
-
d
d
-
-
16
(9.0) (9.0) (9.0) (9.0) (8.8) (8.8)
(8.8) (8.8)
21
20
18
Cl
7.81
7.96
dd
12.12
17
7.42-
7.68
m
6.99 7.30 7.30 6.99
7.73
d
(2.3)
5.30
s
s
12.43
s
19
d
d
d
d
-
-
d
(8.8;
(8.8)
2.3)
16
(9.1) (9.1) (9.1) (9.1)
21
20
Cl
17
18
7.39
dd
(8.9;
2.6)
12.16
7.40-
7.70
m
7.01 7.28 7.28 7.01
7.40-
7.70
m
8.11
5.30
s
s
12.53
s
19
d
d
d
d
-
d
-
16
(8.9) (8.9) (8.9) (8.9)
(2.6)
21
20
Cl
Cl
17
21
18
7.37
dd
(8.0;
7.4)
11.05
7.42-
7.62
m
7.00 7.32 7.32 7.00
7.54
d
(8.0)
7.54
d
(8.0)
5.28
s
s
11.39
s
19
d
d
d
d
-
-
-
-
16
(8.9) (8.9) (8.9) (8.9)
20
19
Cl
18
17
11.89
7.45-
7.70
m
7.00 7.31 7.31 7.00
5.34
s
7.57 7.57
7.57 7.57
s
s
12.37
s
Br
d
d
d
d
-
16
s
s
s
(8.9) (8.9) (8.9) (8.9)
20
21
Table 4. 13C-NMR data (δ, ppm).
14
13
8
12
9
4
O
NH
18
Cl
CH2
3
5
6
10
11
2
1
C
C
R
NH
7
15
S
O
18
20
17
21
17
18
20
18
20
17
17
21
19 CH3
19 O-CH3
Cl
19
19
R
16
21
16
16
21
16
20
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
169.57
134.99
137.64
127.56
133.10
128.26
128.17
67.73
156.83
116.37
130.73
125,9
130.73
116.37
178.65
134.9
123.77
128.88
124.58
128.88
123.77
170.05
131.04
135.64
124.19
129.22
128.41
127.88
67.79
170.02
133.37
135.24
127.9
131.05
128.56
128.44
67.8
157.05
116.50
129.25
125.9
129.25
116.50
179.05
130.7
169.98
131.1
136.8
126.1
133.3
127.9
127.9
67,82
157.03
116,49
129.3
128.5
129.3
116,49
179.18
135.3
124.7
128.4
130.2
128.4
124.7
157.0
116.44
129.05
124.68
129.05
116.44
178.83
135.22
124.09
128.55
133.32
128.55
124.09
124.74
113.78
157.44
113.78
124.74

Molecules 2008, 13
573
Table 4. Cont.
20.60
-CH3
-
-
-OCH3
55.30
Cl
Cl
Cl
17
18
17 18
18
20
17
17
18
20
19
19
19
19
Br
R
Cl
16
21
16
16
16
20
21
20
21
21
Cl
Cl
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
170.11
133.29
135.33
128.56
131.37
128.76
128.69
68.13
157.13
116.66
129.26
127.95
129.26
116.66
180.12
134.60
128.91
129.17
131.14
127.27
124.93
170.32
131.19
135.25
128.05
131.10
128.69
128.65
67.94
157.01
116.41
129.20
127.71
129.20
116.41
179.95
136.5
169.50
133.14
134.93
128.18
130.86
128.29
128.69
67.79
156.83
116.45
128.9
127.78
128.9
116.45
180.51
133.53
127.88
129.46
128.18
129.46
124.58
169.98
133.23
137.25
127.90
131.46
128.58
128.44
67.80
157.02
116.48
129.24
126.41
129.24
116.48
179.10
135.3
126.88
130.78
126.99
133.22
124.74
124.73
131.13
118.49
131.13
124.73
-CH3
-
-
-
-
-OCH3
Table 5. IR data for the new compounds.
O
Cl
CH2
C-NH-C-NH-R
O
S
-CH2-O-
-CO-NH-
R
ν CH2 ν CH2 νC-O-C νC-O-C
δ CH2
ν C-N
sym
asym
sym
asym
1243.0
1243.9
1241.1
1243.1
1242.7
1243.6
1240.3
1242.9
-C6H5
2852.2 2921.7
2852.2 2921.7
2829.0 2910.1
2848.3 2929.4
2856.0 2921.7
2852.2 2921.7
2856.0 2917.8
2852.2 2921.7
1008.2
1012.7
1036.6
1035.1
1036.0
1036.3
1036.5
1033.4
1491.4 1553.6
1491.1 1550.6
1488.5 1553.1
1491.3 1554.3
1491.5 1540.0
1491.0 1552.7
1486.9 1553.3
1490.5 1553.9
1243.0
1243.9
1241.1
1243.1
1242.7
1243.6
1240.3
1242.9
-C6H4CH3 (4)
-C6H4OCH3 (4)
-C6H4Cl (4)
-C6H3Cl2 (2,4)
-C6H3Cl2 (2,5)
-C6H3Cl2 (2,6)
-C6H4Br (4)

Molecules 2008, 13
574
-NH-CS-
NH-
-CO-NH-
aromatic rings
C-halogen
R
ν C=O ν N-H ν =CH
ν C=C ν C=S ν C-Cl ν C-Br
-C6H5
1682.9 3442.7
1683.5 3458.1
1683.4 3436.9
1679.8 3356.4
1684.0 3381.1
1679.1 3442.7
1693.1 3439.4
1678.6 3360.7
3018.1
3029.7
3002.7
3029.7
3099.2
3025.9
3030.2
3030.6
1598.7
1596.8
1598.6
1603.2
1584.2
1585.0
1597.8
1600.7
1166.7
1164.0
1167.4
1151.4
1152.9
1168.2
1168.3
1151.1
749.5
749.1
746.7
744.2
744.8
751.6
753.2
755.9
-
-C6H4CH3 (4)
-C6H4OCH3 (4)
-C6H4Cl (4)
-C6H3Cl2 (2,4)
-C6H3Cl2 (2,5)
-C6H3Cl2 (2,6)
-C6H4Br (4)
-
-
-
-
-
-
607.7
Qualitative antimicrobial activity screening
The qualitative method used for the screening of the antimicrobial activity for all tested compounds
was an adaptation of the disk diffusion method (see Figure 1). The results correlated well with those of
the quantitative antimicrobial activity assays.
Figure 1. The aspect of inhibition zones obtained for the tested compounds on different
pathogens in the qualitative screening assay by the adapted Kirby- Bauer method.

Molecules 2008, 13
575
Figure 1. Cont.
Quantitative antimicrobial activity assay
The quantitative assay results for the antimicrobial activity of the new thioureides are presented in
Table 6. These results show that the tested compounds exhibited a specific antimicrobial activity,
depending on the nature of the substituents and their position on the benzene ring, both concerning the
microbial spectrum and the MIC values, which ranged widely between 1024 and 32 μg/mL. The most
active compounds proved to be N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(2,6-dichlorophenyl)-
thiourea (5g) and N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(4-bromophenyl)-thiourea (5h), which
showed a broad spectrum of antimicrobial activity against enterobacterial strains (E. coli and S.
enteritidis), P. aeruginosa, S. aureus and Candida spp. All the tested compounds were highly active
against S. aureus (MIC= 32 μg/mL), suggesting their possible use in the treatment of MRSA
infections. Four of the tested compounds exhibited antifungal activity (MIC= 256-32 μg/mL), and L.
monocitogenes as well as B. subtilis were resistant to all tested compounds.

Molecules 2008, 13
Table 6. The quantitative antibacterial and antifungal activity test results of the new
576
thioureides and the respective MIC values.
Compounds
Organisms
5a
> 1024
256
5b
32
5c
32
5d
32
32
5e
5f
5g
256
128
32
5h
128
128
32
E. coli
> 1024 > 1024
> 1024 > 1024
S. enteritidis
P. aeruginosa
L. monocytogenes
S. aureus
> 1024 > 1024
> 1024 > 1024 > 1024 > 1024 > 1024 > 1024
> 1024 > 1024 > 1024 > 1024
512
512
> 1024 > 1024
32 32
32
> 1024 > 1024
256 64
32
32
32
32
256
B. subtilis
1024
512
> 1024 > 1024 > 1024 > 1024 > 1024
512 > 1024 > 1024
32 32
C. albicans
Flow cytometry and annexin V assay
The cytotoxicity of the most active antimicrobial compounds, 5g and 5h, was tested with their
possible use in much more in depth studies for the design of new antimicrobial chemical compounds in
mind.
Table 7. Cytotoxic effects of the 5g and 5h on HeLa cells quantified by flow cytometry assay.
HeLa cells
1
2
3
4
(% necrosis)
(% secondary necrosis)
(% viable cells)
(% apoptosis)
Cell control
3.42
13.89
6.77
0.70
9.10
0.70
95.00
84.00
95.00
0.19
0.15
0.15
5g
5h
The cytotoxicity assay has demonstrated that the two selected compounds exhibited low
cytotoxicity levels, comparable to those obtained for the cell control (Table 7, Figure 2), demonstrating
the possibility to be configured for the in vivo antimicrobial use.
Figure 2. Flow cytometry dot-plots of effects on HeLa cells: a) Control; b) 5g; c) 5h.
b
c
a

Molecules 2008, 13
577
Conclusions
In summary, our studies have demonstrated that among other biological activities, the thioureides
of 2-(4-chlorophenoxymethyl)-benzoic acid also exhibit selective and effective antimicrobial
properties that could lead to the selection and use of these compounds as efficient antimicrobial agents,
especially for the treatment of multidrug resistant infections.
Experimental
General
The new compounds were prepared in three steps, as described above, and their physical properties
1
13
and chemical structures further characterized and confirmed by elemental analysis, H-NMR, C-
NMR and IR. All melting points were recorded with an Electrothermal 9100 apparatus and are
uncorrected. Elemental analysis was realized using a Perkin Elmer CHNS/O Analyser Series II 2400
apparatus. The NMR spectra were recorded on a Gemini 300BB instrument, operating at 300 MHz for
13
1H and 75 MHz for C, using solutions in DMSO-d6 as solvent and tetramethylsilane as internal
reference. The IR spectra were recorded using potassium bromide tablets on a Biorad FTS 135
apparatus.
Synthesis of 2-(4-chloro-phenoxymethyl)-benzoic acid (1)
A solution of freshly distilled p-chlorophenol (mol. wt. 128.61, 6.43 g, 0.05 mol) in xylene (30
mL) was placed in a round-bottomed flask equipped with a Dean-Stark trap device, then Subsequently,
potassium hydroxide (mol. wt. 56.11, 3.10 g, 0.055 mol) was added. The reaction mixture was
refluxed while the resulting water was removed by azeotropic distillation and potassium p-chloro-
phenoxide precipitated. Phthalide (mol. wt. 134.14, 6.7 g, 0.05 mol) was added and the mixture was
refluxed until it solidified. The precipitate was heated with 10% potassium hydroxide solution until id
dissolved and it then was diluted with water. The aqueous phase was separated and acidulated (pH= 3)
with 1M hydrochloric acid solution, whereupon the 2-(4-chlorophenoxymethyl)-benzoic acid (mol. wt.
262.687, 6.5 g, 49.6% yield) precipited. The precipitate was crystallized from water-ethanol (1: 3). It
showed a m.p. of 167- 169°C.
Synthesis of 2-(4-chloro-phenoxymethyl)-benzoyl chloride (4)
2-(4-chlorophenoxymethyl)-benzoic acid (1, 5.25 g, 0.02 mol), dry 1,2-dichlorethane (30 mL) and
20
thionyl chloride (mol. wt. 119, d4 1.6385 g/mL, 3 mL, 0.042 mol) were placed in a round-bottomed
flask equipped with a condenser and drying tube. The mixture was refluxed for 3 hours. The excess
thionyl chloride and the solvent were removed under reduced pressure and the crude acid was used
directly in the next step.

Molecules 2008, 13
578
General procedure for the synthesis of the new thioureides 5a-5h
To a solution of ammonium thiocyanate (mol. wt. 76.13, 0.76 g, 0.01 mol, dried by heating at
100°C) in dry acetone (5 mL, dried over potassium carbonate) was added a solution of 2-(4-
chlorophenoxymethyl)-benzoyl chloride (4, mol. wt. 281.39, 0.01 mol) in dry acetone (15 mL). The
reaction mixture was refluxed one hour in a round-bottomed flask equipped with a condenser and a
drying tube. After cooling, dry and freshly distilled primary aromatic amine (0.01 mol) in dry acetone
(2 mL) were added by stirring, to the reaction mixture. The mixture was then refluxed for one hour.
The product was precipitated after the cooled reaction mixture was poured into water (500 mL). The
crude thioureides obtained were crystallised from isopropanol containing active carbon.
Microbial strains
The antimicrobial activity of the investigated compounds was tested against bacterial and fungal
strains recently isolated from clinical specimens as well as reference strains belonging to the following
genera and species: Gram positive (methicillin resistant Staphylococcus aureus, Listeria
monocytogenes, Bacillus subtilis), Gram-negative (Escherichia coli, Salmonella enteritidis,
Pseudomonas aeruginosa) and Candida albicans. The microbial strains were identified by aid of
VITEK I automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were
inoculated and incubated according to the manufacturer's recommendations. The results were
interpreted by using software version AMS R09.1. Bacterial suspensions of 1.5x108 CFU/mL or 0.5
McFarland density obtained from 15-18 h bacterial cultures developed on solid media were used in the
experiments. The antimicrobial activity was tested on Mueller-Hinton medium recommended for the
bacterial strains and Yeast Peptone Glucose (YPG) medium for Candida albicans. We used the
solutions of the new compounds in DMSO with 2048 μg/ mL concentration. The cell cultures used in
cytotoxicity assay were represented by HeLa (ECACC # 93021013) cells. The cells were grown in
Dulbecco’s Modified Essential medium DMEM (Sigma) supplemented with 10 % fetal calf serum
(Sigma) at 37°C, 5% CO2, in a humid atmosphere [5, 6].
Qualitative screening of the antimicrobial properties of the tested compounds
The qualitative screening was performed by an adapted disk diffusion method. Petri dishes with
Mueller Hinton (for bacterial strains)/YPG (for yeasts) media were seeded with bacterial inoculums as
for the classical antibiotic susceptibility testing disk diffusion method (Kirby-Bauer); 5 mm diameter
paper filter disks were placed on the seeded medium, at 30 mm distance. Subsequently, the disks were
impregnated with 5 μL tested compound solution (1024 µg/mL concentration). The plates were left at
room temperature for 20-30 minutes and then incubated at 37oC for 24 hrs. The positive results were
read as the occurrence of an inhibition zone of microbial growth around the disk [7-9].

Molecules 2008, 13
579
Quantitative assay of the antimicrobial activity
These tests were performed by the binary micro dilution method, in 96 multi-well plates, in order
to establish the minimal inhibitory concentration (MIC) [10-12]. For this purpose, serial binary
dilutions of the tested compounds (ranging between 1024 and 16 µg/ mL) were performed in a 200 μL
volume of nutrient broth and each well was seeded with 50 μL microbial inoculum. The plates were
incubated for 24 hrs at 37oC, and MICs were read as the lowest concentration of compound which
inhibited the microbial growth.
Flow cytometry and annexin V assay
HeLa cells were grown in DMEM supplemented with 10% foetal calf serum (FCS) and 24 h later 5
μg/ml of the tested compound were added. Cells from the supernatant and monolayer were harvested
and 1×105 cells were stained with annexin V and propidium iodide using the Immunotech Annexin V-
FITC Kit (Beckman Coulter Company, France) following the manufacturer’s instructions. Cells were
analyzed by flow cytometry using a Coulter EPICS XL flow cytometer (Beckman Coulter). Green
fluorescence (525 nm; FITC annexin V) and red fluorescence (613 nm; propidium iodide) were
measured. The experiment was repeated three times [5, 6].
References
1. Limban, C.; Missir, Al.; Chiriţă, I. Noi tioureide ale acidului 2-fenoximetil-benzoic. Nota I.
Farmacia 2000, 6, 73- 78.
2. Limban, C.; Missir, Al.; Chiriţă, I. Noi tioureide ale acidului 2-fenoximetil-benzoic. Nota II.
Farmacia 2004, 5, 7- 12.
3. Limban, C.; Missir, Al.; Chiriţă, I. Missir, C. The synthesis and characterization of some new
thioureides of 2-(4-methylphenoxymethyl)-benzoic acid with potential pharmacological activity.
Romanian International Conference on Chemistry and Chemical Engineering-RICCCE XIV,
Bucarest, Rumania, 22-24 September 2005; Vol. 4, S-02-99.
4. Limban, C.; Chiriţă, I.; Missir, Al. Synthesis and characterization of some new thioureides of 2-
(4-methoxyphenoxymethyl)-benzoic acid with potential pharmacological activity. Timisoara Med.
J. 2005, 55, 42-44.
5. Mutiu, A.; Alexiu, I.; Chivu, M.; Petica, M.; Anton, G.; Bleotu, C.; Diaconu, C.; Popescu, C.;
Jucu, V.; Cernescu, C.; Detection of human papillomavirus gene sequence in cell lines derived
from laryngeal tumors. J. Cell. Mol. Med. 2001, 5, 49-59.
6. Diaconu, C.; Bleotu, C.; Chivu, M.; Alexiu, A.; Petrusca, D.; Anton, G.; Achim, R.; Ruta, S.M.;
Cernescu, C. The development of larger cells that spontaneously escape senescence – a step
during the immortalization of a human cancer cell line. J. Cell. Mol. Med. 2004, 8, 93-101.
7. Olar, R.; Badea, M.; Cristurean, E.; Lazar, V.; Cernat, R.; Balotescu, M.C. Thermal behavior,
spectroscopic and biological characterization of Co (II), Zn (II) and Pt (II) complexes with N,N-
dimethylbiguanide. J. Therm. Anal. Calorim. 2005, 80, 451-455.

Molecules 2008, 13
580
8. Lazar, V.; Balotescu, M.C.; Moldovan, L.; Vasilescu, G.; Petrache, L.M.; Bulai, D.; Cernat, R.
Comparative evaluation of qualitative and quantitative methods used in the study of antifungal and
antibacterial activity of hydroalcoholic vegetal extracts. Roum. Biotechnol. Lett. 2005, 10, 2225-
2232.
9. Balotescu, M.C.; Oprea, E.; Petrache, L.M.; Bleotu, C.; Lazar, V. Antibacterial, antifungal and
cytotoxic activity of salvia officinalis essential oil and tinctures. Roum. Biotechnol. Lett. 2005, 10,
2481-2481.
10. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial
Susceptibility Testing; Seventeenth Informational Supplement, M100-S17, Vol. 27 No. 1,
Replaces M100-S16, Vol. 26 No. 3, Informational Supplement. CLSI: Wayne, PA, USA, 2007.
11. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically. Approved standard M7-A2. CLSI: Wayne, PA, USA, 1999.
12. Balotescu, M.C.; Limban, C.; Missir, A.V.; Chirita, I.C.; Nitulescu, M.G. The Synthesis and
Biological Activities Of Some New 2-(4-Methoxyphenoxymethyl)benzoic Acid Thioureides. Rev.
Chim. (Bucuresti), 2007, 58, 1064-1068.
Sample availability: Contact the authors.
© 2008 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.