Synthesis and antimicrobial properties of new 2-((4-ethylphenoxy)methyl) benzoylthioureas

Article abstract of DOI:10.2478/s11696-010-0092-9

New acylthiourea derivatives, 2-((4-ethylphenoxy)methyl)-N- (phenylcarbamothioyl)benzamides, were tested by qualitative and quantitative methods on various bacterial and fungal strains and proved to be active at low concentrations against Gram-positive and Gram-negative bacteria as well as fungi. These compounds were prepared by the reaction of 2-((4-ethylphenoxy) methyl)benzoyl isothiocyanate with various primary aromatic amines, and were characterised by melting point and solubility. The structures were identified by elemental analysis, ¹H and ¹³C NMR, and IR spectral data. The level of antimicrobial activity of the new 2-((4-ethylphenoxy)methyl) benzoylthiourea derivatives was dependent on the type, number and position of the substituent on the phenyl group attached to thiourea nitrogen. The iodine and nitro substituents favoured the antimicrobial activity against the Gram-negative bacterial strains, while the highest inhibitory effect against Gram-positive and fungal strains was exhibited by compounds with electron-donating substituents such as the methyl and ethyl groups.

Full text of DOI:10.2478/s11696-010-0092-9

Chemical Papers 65 (1) 60–69 (2011)
DOI: 10.2478/s11696-010-0092-9
ORIGINAL PAPER
Synthesis and antimicrobial properties of new
2-((4-ethylphenoxy)methyl)benzoylthioureas
a
a
aCarmen Limban*, Alexandru V. Missir, Ileana C. Chirita,
aGeorge M. Nitulescu, Miron T. Caproiu, Mariana C. Chifiriuc, Anca M. Israil
b
c
c
aPharmaceutical Chemistry Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy,
Traian Vuia 6, Bucharest, 020956, Romania
bOrganic Chemistry Centre of Romanian Academy “Costin D. Nenitescu”,
Splaiul Independentei, 202B, Bucharest, 060023, Romania
cNational Institute for Research in Microbiology and Immunology Cantacuzino,
Splaiul Independentei, 103, Bucharest, 050096, Romania
Received 25 March 2010; Revised 17 September 2010; Accepted 27 September 2010
New acylthiourea derivatives, 2-((4-ethylphenoxy)methyl)-N-(phenylcarbamothioyl)benzamides,
were tested by qualitative and quantitative methods on various bacterial and fungal strains and
proved to be active at low concentrations against Gram-positive and Gram-negative bacteria as well
as fungi. These compounds were prepared by the reaction of 2-((4-ethylphenoxy)methyl)benzoyl
isothiocyanate with various primary aromatic amines, and were characterised by melting point and
solubility. The structures were identified by elemental analysis, 1H and 13C NMR, and IR spectral
data. The level of antimicrobial activity of the new 2-((4-ethylphenoxy)methyl)benzoylthiourea
derivatives was dependent on the type, number and position of the substituent on the phenyl
group attached to thiourea nitrogen. The iodine and nitro substituents favoured the antimicrobial
activity against the Gram-negative bacterial strains, while the highest inhibitory effect against
Gram-positive and fungal strains was exhibited by compounds with electron-donating substituents
such as the methyl and ethyl groups.
c
ꢀ 2011 Institute of Chemistry, Slovak Academy of Sciences
Keywords: acylthiourea, 2-((4-ethylphenoxy)methyl)benzoic acid, 1H NMR, 13C NMR, antimi-
crobial activity
Introduction
Kurt et al., 2009; Saeed et al., 2008, 2009; Jia et al.,
1993; Zhong et al., 2008). Some acylthiourea deriva-
tives exhibit antiviral (Sun et al., 2006), antihelmintic
(Chow, 1984; Zikán et al., 1988), and herbicidal (Ke
& Xue, 2006) activities.
It is known that the thiourea derivatives con-
taining both the carbonyl and thiocarbonyl groups
are efficient ambidentate donor ligands to transi-
tion metal ions. Their antimicrobial activity can
be explained by the chelating effect of some metal
ions at the level of the thioureidic fraction; the
molecular complex obtained can block the metal
Increasing incidence of bacterial drug resistance re-
quires an improvement to existing antimicrobial drugs
and the development of new ones. Antimicrobial resis-
tance of pathogenic microorganisms is spreading both
in the community and in hospitals leading to an in-
creased number of deaths, and increased illness and
costs. Thiourea derivatives, and especially their N-
acylated derivatives, are of great pharmacological im-
portance, with proven antimicrobial activity (Cunha
et al., 2007; Desai & Desai, 1989; Kapoor et al., 1991;
*Corresponding author, e-mail: carmen limban@yahoo.com

C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
61
ions in certain enzymes and disturb the formation
of some metalloenzymes. This is the premise be-
hind our study of this series. In our previous pa-
pers, we presented the synthesis of some thiourei-
des of 2-(phenoxymethyl)benzoic acid (Limban et
al., 2000, 2004), 2-((4-chloro-phenoxy)methyl)benzoic
acid (Limban et al., 2008a), 2-((4-methoxyphenoxy)
methyl)benzoic acid (Limban et al., 2008b), 2-((4-
methylphenoxy)methyl)benzoic acid (Limban et al.,
2008c, 2008d), 2-((4-ethylphenoxy)methyl)benzoic
acid (Limban et al., 2009a), 2-((4-fluorophenoxy)
methyl)benzoic acid (Limban et al., 2009b) and their
antimicrobial (Balotescu et al., 2007) and antiparasitic
activity (Mu¨ller et al., 2009). Several transition metal
complexes of these acylthioureas were also prepared
(Nacea et al., 2005; Bo¸scencu et al., 2007).
As part of our interest in identifying a larger num-
ber of bioactive acylthioureas, we have synthesised
new 2-((4-ethylphenoxy)methyl)benzoylthioureas by
the addition of various primary aromatic amines to 2-
((4-ethylphenoxy)methyl)benzoyl isothiocyanate. The
structures were identified by spectral data and elemen-
tal analyses The antimicrobial activity of the synthe-
sised compounds was performed by in vitro assay and
compared with the reference and clinical multidrug
resistant strains.
lows: a solution of 2-((4-ethylphenoxy)methyl)benzoyl
chloride (0.01 mol) in dry acetone (15 mL) was added
to a solution of ammonium thiocyanate (0.76 g, 0.01
mol) in dry acetone (5 mL) and the reaction mixture
was kept under reflux for 1 h. After cooling, primary
aromatic amine (0.01 mol) in dry acetone (2 mL) was
added to the reaction mixture upon stirring and kept
under reflux for 1 h. After completion of the reaction
(detected by TLC), the mixture was poured into cold
water (500 mL). The precipitated thioureas were sep-
arated by filtration and recrystallised (after decolouri-
sation by active carbon) from an appropriate solvent.
Antimicrobial activity assay
Qualitative screening of the susceptibility spectra
of various microbial strains to the compounds was
performed by modified diffusion techniques, while the
quantitative assay of minimal inhibitory concentration
(MIC, µg mL−1) was based on serial micro-dilutions
with liquid medium. The compounds were solubilised
in DMSO to a final concentration of 1000 µg mL−1
.
The effects from in vitro biological screening were
tested on a microbial inoculum of ∼ 1.5 × 108 CFU
per cm3, corresponding to 0.5 McFarland density, rep-
resented by bacteria, Enterobacteriaceae (Escherichia
coli and Klebsiella pneumoniae), Pseudomonadaceae
(Pseudomonas aeruginosa), Micrococcaceae (Staphy-
lococcus aureus), Bacillaceae (Bacillus subtilis), and
fungi, Candida albicans and Aspergillus niger. The ref-
erence strains, K. pneumoniae IC 13420, E. coli IC
13529, S. aureus IC 13204, P. aeruginosa IC 13202,
B. subtilis IC 12488, C. albicans IC 249, and A. niger
IC 13534 from the Cantacuzino Institute Culture Col-
lection Laboratory, were susceptible to all the antibi-
otics recommended by Clinical and Laboratory Stan-
dards Institute (CLSI) (formerly National Commit-
tee for Clinical Laboratory Standards, NCCLS) while
the strains recently isolated from clinical samples ex-
hibited multiresistance patterns (S. aureus 1263 –
methicilin-resistant MRSA, K. pneumoniae 1204 and
E. coli 13147 – producing an extended spectrum of
β-lactamases, conferring the microorganisms with re-
sistance to all β-lactams, including 3rd generation
cephalosporins as well as other classes of antibiotics,
such as quinolones and aminoglycosides), P. aerugi-
nosa 1246 (multiresistant to all antibiotics except col-
istin) and C. albicans 101404, resistant to fluconazole.
The microbial strains were identified using VITEK I
automatic system. VITEK cards for the identification
and susceptibility testing (GNS-522) were inoculated
and incubated according to the manufacturer’s recom-
mendations. The results were interpreted using soft-
ware version AMS R09.1.
Experimental
All chemicals were purchased (Merck, Fluka, Ger-
many) and used as received, except for 4-ethylphenol
which was distilled prior to its use. Acetone and am-
monium thiocyanate were dried before use. Melting
points were recorded with an Electrothermal 9100 ap-
paratus and are uncorrected. Elemental analyses were
carried out using a Perkin–Elmer CHNS/O Analyzer
Series II 2400 apparatus and the results were within
0.4 % of theoretical values.
The reaction progress was observed by thin layer
chromatography performed on silica gel 60 F254 (0.2
mm thick) plates (Merck, Germany) using chloro-
form/ethyl acetate (ϕr = 4 : 6) as a mobile phase
with visualisation by ultraviolet light.
NMR spectra (in DMSO-d6) were recorded using a
Gemini 300BB instrument operating at 300 MHz for
1H and 75 MHz for 13C, and a Unity Inova 400 instru-
1
ment, operating at 400 MHz for H and 100 MHz for
13C, respectively. TMS was used as the internal stan-
dard. IR spectra were recorded using a FT-IR Bruker
Vertex 70 apparatus by ATR technique. The IR band
intensities are denoted as w – weak; m – medium; s –
strong; vs – very strong.
2-((4-Ethylphenoxy)methyl)benzoic acid (I ) and
2-((4-ethylphenoxy)methyl)benzoyl chloride (IV ) were
prepared according to procedures described by Lim-
ban et al. (2009a).
Acylthioureas Va–Vn were synthesised according
to the general method (Limban et al., 2009a) as fol-
The qualitative screening was performed using the
disc diffusion method. Petri dishes with Mueller Hin-
ton (for bacterial strains) and YPG (Yeast Peptone
Glucose) (for yeasts) media were inoculated with mi-

62
C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
Et
Et
Et
O
O
O
SOCl2
HCl
O + Et
OK
COOH
I
COOK
III
COCl
IV
O
II
Fig. 1. Synthesis of 2-((4-ethylphenoxy)methyl)benzoic acid and its chloride.
13
15
15
CH3
'
12
Et
Et
14
8
4
7
3
2
11
5
9
O
RNH2
22 21
O
R1
NH4SCN
10
O
1
16
C
NH
C
NH 17
6
20
C
O
N
C
S
COCl
R2
19
18
O
S
IV
VI
Va–Vn
Fig. 2. Synthesis of new thioureides.
croorganisms as in the classical antibiotic susceptibil-
ity testing disc diffusion method (Kirby–Bauer); 6 mm
diameter paper filter discs were placed on the inocu-
lated medium at a distance of 30 mm. Subsequently,
the discs were impregnated with 5 µL of the solution
of the compound tested (1000 µg mL−1 concentra-
tion). The plates were left at room temperature for
20–30 min and then incubated at 37◦C for 24 h. The
positive results were read as the occurrence of an inhi-
bition zone of microbial growth around the disc (Olar
et al., 2005; Lazar et al., 2005; Balotescu et al., 2005).
The quantitative screening was performed using
the binary micro-dilution method, in 96-well culture
plates, in order to establish the MIC (CLSI, 2007; NC-
CLS, 2000). For this purpose, serial binary dilutions
of the compounds tested (ranging from 1000 µg mL−1
to 7.8 µg mL−1) were performed in 200 µL of nutrient
broth and each well was filled with 50 µL of micro-
bial inoculum. The plates were incubated at 37◦C for
24 h, and MICs were read as the lowest concentra-
tion of the compound which inhibited the microbial
growth. The results were interpreted using standard
antibiotics (aminoglycosides – amikacin for the Gram-
positive and ceftriaxone for the Gram-negative strains
(CLSI, 2007), fluconazole for Candida, and voricona-
zole for Aspergillus (NCCLS, 2002) as controls.
iocyanate (VI ) (obtained from the corresponding chlo-
ride IV ) with primary aromatic amines in dry acetone
under reflux (Fig. 2, Limban et al., 2009a). All these
acylthioureas (Table 1) are white or light-yellow crys-
talline solids, insoluble in water and soluble in acetone
and chloroform at normal temperature and in short
chain aliphatic alcohols, benzene, toluene, and xylene
at higher temperatures. Their structures were identi-
fied by elemental analyses, IR, and NMR spectral data
(Table 2).
In the 1H NMR spectra, the ethyl group exhib-
ited a characteristic quartet at δ 2.48–2.50 and triplet
at δ 1.07–1.19. The methylene group attached to the
oxygen atom showed a singlet at δ 5.24–5.27. The sig-
nals of aromatic protons were observed in a range of δ
6.82–7.96, with the exception of Vj, where the signal
for H-22 was shifted to δ 8.76 and Vn where a shift to
δ 8.25 was recorded for H-19 (H-21). The NH protons
exhibited two characteristic broad singlets at δ 11.74–
12.04 and δ 11.86–12.70, respectively. In the 13C NMR
spectra, the thiocarbonyl carbon showed a character-
istic signal at δ 176.88–180.33, the carbonyl carbon at
δ 169.67–170.49, the methylene carbon of CH2O group
at δ 67.29–67.72, and the carbons of ethyl group at δ
26.98–27.34 (CH2) and δ 13.84–15.94 (CH3).
In the IR spectra, the bands (sharp peaks of weak
intensity) due to stretching vibrations ν(N—H) of the
Results and discussion
amide group were in the region of 3352–3238 cm−1
.
The thioamide group showed a band of stretching vi-
brations at 3181–3144 cm−1 and there is a high level
of probability that the band situated at 1394–1344
cm−1 can be attributed to the thioamide group. As to
antisymmetric stretching vibrations, the methyl and
methylene groups exhibited a saturated (sp3) ν(C—H)
Synthesis and spectral characterisation
2-((4-Ethylphenoxy)methyl)benzoic acid (I ) and
2-((4-ethylphenoxy)methyl)benzoyl chloride (IV )
were prepared according to the reaction scheme shown
in Fig. 1 (Limban et al., 2009a).
stretching at 2974–2954 cm−1 and 2933–2918 cm−1
,
The new thioureides, Va–Vn, were prepared by
heating the 2-((4-ethylphenoxy)methyl)benzoyl isoth-
respectively. These bands are typical of aromatic com-
pounds containing some saturated carbon. These com-

C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
Table 1. Characteristics of the compounds prepared
63
wi(calc.)/%
wi(found)/%
Yield
M.p.
◦C
Compound
R1
R2
Formula
Mr
C
H
N
S
%
68
70
73
65
79
82
71
81
85
73
79
82
71
68
Va
Vb
Vc
Vd
Ve
Vf
2-CH3
2-CH3
3-CH3 C25H26N2O2S 418.55
4-CH3 C25H26N2O2S 418.55
5-CH3 C25H26N2O2S 418.55
6-CH3 C25H26N2O2S 418.55
4-CH3 C25H26N2O2S 418.55
5-CH3 C25H26N2O2S 418.55
71.74
71.89
71.74
71.67
71.74
71.86
71.74
71.66
71.74
71.59
71.74
71.43
71.74
71.48
71.74
71.94
71.74
71.71
69.10
69.02
69.10
68.97
69.10
68.92
53.49
53.39
63.43
63.59
6.26
6.21
6.26
6.30
6.26
6.30
6.26
6.25
6.26
6.17
6.26
6.19
6.26
6.29
6.26
6.21
6.26
6.15
6.03
5.99
6.03
6.14
6.03
5.95
4.10
4.02
4.86
4.78
6.69
6.57
6.69
6.89
6.69
6.79
6.69
6.51
6.69
6.73
6.69
6.70
6.69
6.81
6.69
6.56
6.69
6.78
6.45
6.59
6.45
6.39
6.45
6.52
5.43
5.49
9.65
9.67
7.66
7.81
7.66
7.69
7.66
7.51
7.66
7.80
7.66
7.74
7.66
7.75
7.66
7.83
7.66
7.60
7.66
7.62
7.38
7.46
7.38
7.47
7.38
7.40
6.21
6.23
7.36
7.31
136–139
134–136
131–133
149–150
106–108
139–140
90–91
2-CH3
2-CH3
3-CH3
3-CH3
Vg
Vh
Vi
2-C2H5
3-C2H5
4-C2H5
2-OC2H5
3-OC2H5
4-OC2H5
4-I
H
H
H
H
H
H
H
H
C25H26N2O2S 418.55
C25H26N2O2S 418.55
C25H26N2O2S 418.55
C25H26N2O3S 434.55
C25H26N2O3S 434.55
C25H26N2O3S 434.55
C23H21N2O2SI 516.40
C23H21N3O4S 435.49
91–92
136–138
93–95
Vj
Vk
Vl
123–125
101–102
123–126
173–176
Vm
Vn
4-NO2
—
pounds were characterised by the ν(C O) vibrations
ited by the compound tested. At lower concentrations
of the compounds tested, the microbial culture be-
came visible. The lowest concentration which inhib-
ited the visible microbial growth was considered to be
the MIC (µg mL−1) value of the compound tested.
In the adjacent wells, containing the standard culture
growth control wells, the medium became cloudy as
a result of microbial growth. In the sterility control
wells, the culture medium had to remain clear for an
obvious reason. From the last well without any vis-
ible microbial growth and from the first with a mi-
crobial growth, Gram-stained smears were performed
to confirm the results. The solvent used for evalua-
tion of the compounds exhibited no antimicrobial ac-
tivity. This property represented a practical advan-
tage for the antimicrobial evaluation of these water-
insoluble compounds. The in vitro screening of the
antimicrobial properties of the new compounds was
performed by an modified diffusion method, followed
by the broth micro-dilution method, in order to es-
tablish the MIC for Gram-positive (S. aureus, B. sub-
tilis), Gram-negative bacteria (P. aeruginosa, E. coli,
K. pneumoniae), as well as fungi (C. albicans and A.
niger), using both reference and clinical, multidrug
resistant strains. Our results showed that the com-
—
in the region of 1690–1669 cm−1. The medium inten-
—
—
sity band of ν(C O) vibration of all the compounds
appeared at 1690–1669 cm−1 which was lower than
the intensity of an ordinary carbonyl absorption (1730
cm−1). The formation of intramolecular H-bonds led
to an increase in their polarity decreasing the strength
of their double bond and moving the absorption to
a lower wavenumber. A very intense band of ν(N—
H) stretching was observed at 1518–1506 cm−1. These
compounds also showed a typical band corresponding
to alkylaryl ether: at 1254–1224 cm−1 for the anti-
symmetric vibration and at 1042–1019 cm−1 for the
—
symmetric vibration. The bands of ν(C S) stretch-
—
ing vibrations were in the range of 1178–1148 cm−1
,
which is in agreement with the literature data. The
presence of iodine at the aryl ring was indicated by
the band of stretching vibration ν(C—I) at 498 cm−1
.
Antimicrobial activity
The MIC values were read by observation: in the
first wells containing high concentrations of the com-
pounds tested, the growth of microorganisms was
completely suppressed, as they were killed or inhib-

64
C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
Table 2. Spectral characterisation of newly prepared compounds
Compound
Spectral data
IR, ν˜/cm−1: 436 (w), 546 (w), 568 (w), 614 (w), 654 (w), 692 (w), 726 (m), 763 (m), 791 (w), 828 (m), 858 (w), 890
Va
(w), 951 (w), 1021 (m), 1071 (w), 1091 (w), 1169 (s), 1235 (s), 1300 (w), 1333 (w), 1389 (m), 1461 (s), 1508 (vs),
1584 (w), 1608 (w), 1673 (m), 2871 (w), 2929 (m), 2965 (m), 3028 (w), 3155 (m), 3325 (w)
1H NMR (DMSO-d6), δ: 1.14 (t, 3H, H-15ꢀ, J = 7.5 Hz), 2.01 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.50 (q, 2H, H-15,
J = 7.5 Hz), 5.27 (s, 2H, H-8), 6.90 (d, 2H, H-10, H-14, J = 8.1 Hz), 7.10–7.12 (m, 4H, H-11, H-13, H-20, H-22),
7.21 (t, 1H, H-21, J = 6.2 Hz), 7.46 (td, 1H, H-6, J = 1.7 Hz, J = 7.9 Hz,), 7.54 (td, 1H, H-5, J = 1.7 Hz, J = 7.9
Hz), 7.57 (dd, 1H, H-4, J = 1.7 Hz, J = 7.9 Hz), 7.63 (dd, 1H, H-7, J = 1.7 Hz, J = 7.9 Hz), 11.87 (br, s, 1H, NH),
12.05 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 13.84 (C-15ꢀ), 15.52 (CH3), 20.01 (CH3), 27.34 (C-15), 67.60 (C-8), 114.55 (C-10, C-14),
124.74, 125.37, 127.83, 128.53, 128.53, 128.53, 128.69 (C-11, C-13), 130.91, 132.40, 133.67, 135.72, 136.22, 136.87,
137.20, 156.33 (C-9), 170.37 (C-1), 180.14 (C-16)
Vb
IR, ν˜/cm−1: 442 (w), 541 (w), 627 (w), 663 (w), 735 (m), 824 (m), 852 (w), 919 (w), 954 (w), 1042 (w), 1089 (w),
1121 (w), 1150 (s), 1231 (s), 1253 (m), 1267 (w), 1301 (w), 1340 (m), 1391 (w), 1449 (w), 1507 (vs), 1533 (s), 1584
(w), 1605 (w), 1671 (m), 2870 (w), 2924 (w), 2961 (w), 3011 (w), 3149 (m), 3329 (w)
1H NMR (DMSO-d6), δ: 1.13 (t, 3H, H-15ꢀ, J = 7.5 Hz), 2.08 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.50 (q, 2H, H-15,
J = 7.5 Hz), 5.26 (s, 2H, H-8), 6.89 (d, 2H, H-10, H-14, J = 8.1 Hz), 7.03 (m, 2H, H-19, H-21), 7.09 (d, 2H, H-11,
H-13, J = 8.1 Hz), 7.35–7.63 (m, 5H, H-4, H-5, H-6, H-7, H-22), 11.85 (br, s, 1H, NH), 12.06 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.52 (C-15ꢀ), 17.09 (CH3), 20.25 (CH3), 26.98 (C-15), 67.31 (C-8), 114.26 (C-10, C-14),
125.93, 126.24, 127.46, 128.16, 128.23, 128.30 (C-11, C-13), 130.54, 130.56, 132.67, 133.28, 133.94, 135.39, 135.87,
135.92, 156.01 (C-9), 169.93 (C-1), 179.62 (C-16)
Vc
IR, ν˜/cm−1: 435 (w), 470 (w), 544 (w), 576 (w), 617 (w), 649 (w), 664 (w), 695 (w), 742 (m), 827 (w), 862 (w), 901
(w), 956 (w), 1019 (m), 1072 (w), 1089 (w), 1147 (m), 1172 (m), 1239 (s), 1268 (s), 1300 (m), 1336 (w), 1381 (w),
1460 (m), 1509 (vs), 1583 (w), 1606 (w), 1679 (m), 2872 (w), 2928 (w), 2966 (w), 3027 (w), 3157 (m), 3326 (w)
1H NMR (DMSO-d6), δ: 1.13 (t, 3H, H-15ꢀ, J = 7.5 Hz), 2.08 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.50 (q, 2H, H-15,
J = 7.5 Hz), 5.26 (s, 2H, H-8), 6.89 (d, 2H, H-10, H-14, J = 8.1 Hz), 7.01 (dd, 1H, H-20, J = 1.6 Hz, J = 8.0 Hz),
7.10 (d, 2H, H-11, H-13, J = 8.1 Hz), 7.14 (d, 1H, H-19, J = 8.0 Hz), 7.31 (dd, 1H, H-22, J = 1.6 Hz, J = 8.4 Hz),
7.46 (td, 1H, H-6, J = 1.5 Hz, J = 8.4 Hz), 7.57 (td, 1H, H-5, J = 1.5 Hz, J = 8.4 Hz), 7.58 (dd, 1H, H-4, J = 1.6
Hz, J = 8.4 Hz), 7.62 (dd, 1H, H-7, J = 1.5 Hz, J = 8.4 Hz), 11.85 (br, s, 1H, NH), 12.06 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.49 (C-15ꢀ), 16.76 (CH3), 20.18 (CH3), 27.00 (C-15), 67.35 (C-8), 114.25 (C-10, C-14),
126.48, 127.34, 127.48, 128.14, 128.22, 128.30 (C-11, C-13), 129.88, 130.57, 133.07, 133.29, 134.86, 135.37, 135.89,
136.26, 156.03 (C-9), 170.00 (C-1), 179.53 (C-16)
Vd
Ve
Vf
IR, ν˜/cm−1: 439 (w), 546 (w), 615 (w), 653 (w), 754 (m), 830 (w), 869 (w), 951 (w), 1014 (m), 1031 (w), 1053 (w),
1075 (w), 1089 (w), 1126 (m), 1151 (m), 1167 (m), 1215 (m), 1234 (s), 1297 (w), 1334 (w), 1377 (w), 1507 (vs),
1602 (m), 1685 (m), 2928 (w), 2966 (w), 3028 (w), 3160 (m), 3325 (w)
1H NMR (DMSO-d6), δ: 1.14 (t, 3H, H-15ꢀ, J = 7.6 Hz), 2.11 (s, 6H, CH3), 2.50 (q, 2H, H-15, J = 7.6 Hz), 5.27
(s, 2H, H-8), 6.90 (d, 2H, H-10, H-14, J = 8.6 Hz), 7.10 (d, 2H, H-11, H-13, J = 8.6 Hz), 7.12 (d, 2H, H-19, H-21),
7.15 (t, 1H, H-20), 7.47 (td, 1H, H-6, J = 1.4 Hz, J = 7.5 Hz), 7.55 (td, 1H, H-5, J = 1.6 Hz, J = 7.5 Hz), 7.59 (dd,
1H, H-4, J = 1.4 Hz, J = 7.5 Hz), 7.63 (dd, 1H, H-7, J = 1.6 Hz, J = 7.5 Hz), 11.74 (br, s, 1H, NH), 11.86 (br, s,
1H, NH)
13C NMR (DMSO-d6), δ: 15.94 (C-15ꢀ), 17.80 (CH3), 27.32 (C-15), 67.57 (C-8), 114.58 (C-10, C-14), 127.38, 127.84,
128.59, 128.62, 128.64 (C-11, C-13), 128.72, 130.91, 133.75, 135.11 (C-18, C-22), 135.63, 136.02, 136.19, 156.27 (C-9),
170.06 (C-1), 180.18 (C-16)
IR, ν˜/cm−1: 424 (w), 442 (w), 520 (w), 545 (w), 617 (w), 668 (w), 728 (m), 826 (w), 854 (w), 874 (w), 902 (w),
1033 (m), 1049 (w), 1125 (m), 1151 (s), 1668 (m), 1233 (s), 1303 (w), 1330 (m), 1382 (m), 1448 (s), 1508 (vs), 1531
(vs), 1583 (m), 1606 (w), 1671 (m), 2868 (w), 2918 (m), 2963 (s), 3032 (w), 3063 (w), 3151 (m)
1H NMR (DMSO-d6), δ: 1.12 (t, 3H, H-15ꢀ, J = 6.1 Hz), 2.22 (s, 6H, CH3), 2.50 (q, 2H, H-15, J = 6.1 Hz), 5.26
(s, 2H, H-8), 6.89 (d, 2H, H-10, H-14, J = 8.8 Hz), 7.09 (d, 2H, H-11, H-13, J = 8.8 Hz), 7.15 (d, 1H, H-21, J = 8.0
Hz), 7.28 (d, 1H, H-18, J = 2.1 Hz), 7.35 (dd, 1H, H-22, J = 2.1 Hz, J = 8.0 Hz), 7.46 (td, 1H, H-6, J = 7.2 Hz, J
= 1.9 Hz), 7.55 (td, 1H, H-5, J = 7.2 Hz, J = 1.3 Hz), 7.58 (dd, 1H, H-4, J = 1.9 Hz, J = 7.2 Hz), 7.61 (dd, 1H,
H-7, J = 7.2 Hz, J = 1.3 Hz), 11.79 (br, s, 1H, NH), 12.33 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.84 (C-15ꢀ), 18.95 (CH3), 19.39 (CH3), 27.30 (C-15), 67.55 (C-8), 114.60 (C-10, C-14),
125.06, 125.14, 127.71, 128.27, 128.50, 128.64 (C-11, C-13), 129.46, 130.94, 133.48, 134.28, 135.81, 136.19, 136.53,
139.47, 156.32 (C-9), 170.30 (C-1), 178.81 (C-16)
IR, ν˜/cm−1: 435 (w), 489 (w), 540 (w), 580 (w), 607 (w), 659 (w), 673 (m), 708 (w), 742 (w), 759 (w), 803 (w), 822
(w), 851 (w), 895 (w), 961 (w), 1040 (m), 1122 (m), 1150 (m), 1180 (s), 1247 (w), 1305 (m), 1326 (w), 1388 (m),
1443 (vs), 1511 (vs), 1528 (w), 1582 (w), 1612 (w), 1669 (m), 2867 (w), 2925 (w), 2959 (m), 3030 (w), 3144 (m),
3327 (w)
1H NMR (DMSO-d6), δ: 1.12 (t, 3H, H-15ꢀ, J = 7.6 Hz), 2.27 (s, 6H, CH3), 2.50 (q, 2H, H-15, J = 7.6 Hz), 5.26 (s,
2H, H-8), 6.89 (d, 2H, H-10, H-14, J = 8.8 Hz), 7.10 (d, 2H, H-11, H-13, J = 8.8 Hz), 7.21 (d, 2H, H-18, H-22, J =
1.4 Hz), 7.46 (td, 1H, H-6, J = 1.7 Hz, J = 7.5 Hz), 7.55 (td, 1H, H-5, J = 1.2 Hz, J = 7.5 Hz), 7.58 (dd, 1H, H-4,
J = 1.7 Hz, J = 7.5 Hz), 7.61 (dd, 1H, H-7, J = 1.2 Hz, J = 7.2 Hz), 11.81 (br, s, 1H, NH), 12.35 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.83 (C-15ꢀ), 20.87 (CH3), 27.31 (C-15), 67.58 (C-8), 114.59 (C-10, C-14), 121.71, 127.62,
127.75, 128.36, 128.50, 128.65 (C-11, C-13), 130.96, 133.48, 135.81, 136.19, 137.63, 137.82 (C-19, C-21), 156.35 (C-9),
170.36 (C-1), 178.79 (C-16)

C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
65
Table 2. (continued)
Compound
Spectral data
Vg
IR, ν˜/cm−1: 436 (w), 546 (w), 610 (w), 657 (w), 727 (m), 762 (w), 790 (w), 827 (m), 846 (w), 869 (w), 887 (w), 948
(w), 1019 (m), 1050 (w), 1089 (w), 1161 (s), 1240 (s), 1300 (w), 1333 (w), 1382 (m), 1458 (s), 1509 (vs), 1604 (m),
1676 (m), 2875 (w), 2933 (w), 2964 (m), 3027 (w), 3158 (m), 3325 (w)
1H NMR (DMSO-d6), δ: 1.04 (t, 3H, CH2CH3, J = 7.5 Hz), 1.13 (t, 3H, H-15ꢀ, J = 7.6 Hz), 2.50 (m, 4H, H-15,
CH2CH3), 5.26 (s, 2H, H-8), 6.90 (d, 2H, H-10, H-14, J = 8.8 Hz), 7.10 (d, 2H, H-11, H-13, J = 8.8 Hz), 7.20–7.30
(m, 3H, H-19, H-20, H-21), 7.40–7.60 (m, 4H, H-4, H-5, H-6, H-22), 7.63 (dd, 1H, H-7, J = 1.2 Hz, J = 7.2 Hz),
11.92 (br, s, 1H, NH), 12.17 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 14.26 (CH2CH3), 15.90 (C-15ꢀ), 23.97 (CH2CH3), 27.32 (C-15), 67.60 (C-8), 114.47 (C-10,
C-14), 126.01, 127.20, 127.54, 127.87, 128.52, 128.64 (C-11, C-13), 130.94, 133.54, 135.65, 136.19, 137.18, 138.98,
156.33 (C-9), 170.49 (C-1), 180.33 (C-16)
Vh
IR, ν˜/cm−1: 446 (w), 543 (w), 566 (w), 613 (w), 684 (m), 711 (m), 744 (w), 778 (w), 797 (m), 823 (w), 856 (w),
886 (w), 1040 (m), 1120 (m), 1151 (s), 1168 (m), 1237 (s), 1309 (m), 1348 (s), 1386 (w), 1448 (s), 1511 (vs), 1529
(vs), 1566 (s), 1591 (m), 1612 (m), 1672 (m), 2869 (m), 2929 (m), 2961 (s), 3032 (m), 3238 (m)
1H NMR (DMSO-d6), δ: 1.12 (t, 3H, CH2CH3, J = 7.6 Hz), 1.18 (t, 3H, H-15ꢀ, J = 7.6 Hz), 2.50 (q, 2H, H-15, J =
7.6 Hz), 2.61 (q, 2H, CH2CH3, J = 7.6 Hz), 5.26 (s, 2H, H-8), 6.90 (d, 2H, H-10, H-14, J = 8.8 Hz), 6.98 (dd, 1H,
H-20, J = 1.7 Hz, J = 7.8 Hz), 7.09 (d, 2H, H-11, H-13, J = 8.8 Hz), 7.10 (dd, 1H, H-22, J = 1.7 Hz, J = 7.8 Hz),
7.30 (t, 1H, H-21, J = 7.8 Hz), 7.39 (t, 1H, H-18, J = 1.7 Hz), 7.46 (td, 1H, H-6, J = 1.8 Hz, J = 7.2 Hz), 7.56 (td,
1H, H-5, J = 1.2 Hz, J = 7.2 Hz), 7.58 (dd, 1H, H-4, J = 1.8 Hz, J = 7.2 Hz), 7.61 (dd, 1H, H-7, J = 1.2 Hz, J =
7.2 Hz), 11.83 (br, s, 1H, NH), 12.39 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.46 (CH2CH3), 15.82 (C-15ꢀ), 27.30 (C-15), 28.01 (CH2CH3), 67.57 (C-8), 114.60 (C-10,
C-14), 121.58, 123.46, 125.73, 127.74, 128.35, 128.42, 128.50, 128.64 (C-11, C-13), 130.97, 133.45, 135.81, 136.19,
137.88, 144.39 (C-19), 156.33 (C-9), 170.34 (C-1), 178.89 (C-16)
Vi
IR, ν˜/cm−1: 541 (w), 604 (w), 638 (w), 666 (w), 737 (m), 808 (w), 833 (m), 852 (w), 885 (w), 959 (w), 1031 (m),
1048 (w), 1066 (w), 1121 (m), 1145 (s), 1178 (w), 1224 (s), 1263 (w), 1302 (m), 1344 (m), 1453 (m), 1508 (vs), 1555
(m), 1600 (m), 1671 (m), 2870 (w), 2928 (m), 2963 (m), 3039 (w), 3352 (w)
1H NMR (DMSO-d6), δ: 1.12 (t, 3H, CH2CH3, J = 7.6 Hz), 1.19 (t, 3H, H-15ꢀ, J = 7.6 Hz), 2.50 (q, 2H, H-15, J
= 7.6 Hz), 2.61 (q, 2H, CH2CH3, J = 7.6 Hz), 5.27 (s, 2H, H-8), 6.90 (d, 2H, H-10, H-14, J = 8.6 Hz), 7.09 (d, 2H,
H-11, H-13, J = 8.6 Hz), 7.23 (d, 2H, H-19, H-21, J = 8.2 Hz), 7.46 (td, 1H, H-6, J = 2.0 Hz, J = 7.4 Hz), 7.49 (d,
2H, H-18, H-22, J = 8.2 Hz), 7.55 (td, 1H, H-5, J = 1.4 Hz, J = 7.4 Hz), 7.58 (dd, 1H, H-4, J = 2.0 Hz, J = 7.4
Hz), 7.62 (dd, 1H, H-7, J = 1.4 Hz, J = 7.4 Hz), 11.81 (br, s, 1H, NH), 12.37 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.54 (CH2CH3), 15.85 (C-15ꢀ), 27.31 (C-15), 27.74 (CH2CH3), 67.53 (C-8), 114.59 (C-
10, C-14), 124.23, 127.73, 127.87, 128.34, 128.52, 128.64 (C-11, C-13), 130.99, 133.41, 135.84, 136.20, 144.39 (C-20),
156.32 (C-9), 170.22 (C-1), 178.90 (C-16)
Vj
IR, ν˜/cm−1: 431 (w), 458 (w), 481 (w), 507 (w), 536 (w), 602 (m), 640 (m), 674 (w), 746 (s), 794 (w), 807 (w), 827
(m), 852 (m), 920 (m), 946 (w), 1031 (m), 1117 (m), 1148 (m), 1254 (s), 1289 (m), 1306 (m), 1335 (m), 1378 (w),
1439 (s), 1454 (s), 1507 (vs), 1541 (m), 1681 (m), 2874 (w), 2929 (w), 2963 (m), 3030 (w), 3163 (w), 3415 (s), 3485
(w)
1H NMR (DMSO-d6), δ: 1.09 (t, 3H, H-15ꢀ, J = 7.5 Hz), 1.29 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.49 (q, 2H, H-15, J
= 7.4 Hz), 4.05 (q, 2H, OCH2CH3, J = 7.0 Hz), 5.25 (s, 2H, H-8), 6.86 (d, 2H, H-10, H-14, J = 8.6 Hz), 6.97 (m,
1H, H-21), 7.03 (d, 2H, H-11, H-13, J = 8.6 Hz), 7.08 (dd, 1H, H-19, J = 1.4 Hz, J = 8.2 Hz), 7.18 (m, 1H, H-20),
7.46 (td, 1H, H-6, J = 2.0 Hz, J = 7.4 Hz), 7.55 (td, 1H, H-5, J = 1.4 Hz, J = 7.4 Hz), 7.58 (dd, 1H, H-4, J = 2.0
Hz, J = 7.4 Hz), 7.62 (dd, 1H, H-7, J = 1.4 Hz, J = 7.4 Hz), 8.76 (dd, 1H, H-22, J = 1.7 Hz, J = 8.2 Hz), 11.78
(br, s, 2H, NH)
13C NMR (DMSO-d6), δ: 14.09 (OCH2CH3), 15.43 (C-15ꢀ), 26.93 (C-15), 64.11 (OCH2CH3), 67.29 (C-8), 111.98
(C-19), 114.31 (C-10, C-14), 119.40 (C-21), 121.67, 125.86, 127.13, 127.44, 128.18 (C-11, C-13), 133.04, 135.46,
135.85, 149.10 (C-18), 155.96 (C-9), 169.67 (C-1), 176.88 (C-16)
Vk
IR, ν˜/cm−1: 440 (w), 481 (w), 545 (w), 607 (w), 676 (w), 738 (m), 778 (m), 836 (m), 866 (w), 929 (w), 960 (w),
1027 (m), 1049 (m), 1142 (s), 1165 (m), 1189 (w), 1226 (s), 1251 (m), 1293 (m), 1313 (m), 1347 (w), 1387 (w), 1455
(m), 1506 (vs), 1559 (s), 1593 (m), 1675 (m), 2867 (w), 2926 (w), 2956 (w), 2978 (w), 3031 (w), 3339 (w)
1H NMR (DMSO-d6), δ: 1.11 (t, 3H, H-15ꢀ, J = 7.5 Hz), 1.33 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.50 (q, 2H, H-15,
J = 7.4 Hz), 4.02 (q, 2H, OCH2CH3, J = 7.0 Hz), 5.26 (s, 2H, H-8), 6.82 (dd, 1H, H-20, J = 2.0 Hz, J = 8.2 Hz),
6.89 (d, 2H, H-10, H-14, J = 8.6 Hz), 7.08 (d, 2H, H-11, H-13, J = 8.6 Hz), 7.09 (m, 1H, H-22), 7.28 (t, 1H, H-21,
J = 8.2 Hz), 7.35 (t, 1H, H-18, J = 2.0 Hz), 7.46 (td, 1H, H-6, J = 2.0 Hz, J = 7.4 Hz), 7.55 (td, 1H, H-5, J = 1.4
Hz, J = 7.4 Hz), 7.58 (dd, 1H, H-4, J = 2.0 Hz, J = 7.4 Hz), 7.62 (dd, 1H, H-7, J = 1.4 Hz, J = 7.4 Hz), 11.83 (br,
s, 1H, NH), 12.27 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 14.60 (OCH2CH3), 15.80 (C-15ꢀ), 27.28 (C-15), 63.19 (OCH2CH3), 67.53 (C-8), 114.20
(C-20), 114.58 (C-10, C-14), 116.12 (C-18), 125.91, 127.75, 128.37, 128.51, 128.64 (C-11, C-13), 129.45, 131.02,
133.32, 135.83, 136.21, 138.90, 156.31 (C-9), 158.54 (C-19), 170.18 (C-1), 178.70 (C-16)

66
C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
Table 2. (continued)
Compound
Spectral data
Vl
IR, ν˜/cm−1: 444 (w), 492 (w), 522 (w), 539 (w), 563 (w), 620 (w), 645 (w), 673 (w), 735 (w), 766 (s), 823 (m), 859
(w), 921 (w), 952 (w), 1042 (s), 1072 (m), 1116 (s), 1164 (s), 1236 (vs), 1270 (m), 1299 (m), 1334 (m), 1394 (m),
1461 (m), 1508 (vs), 1536 (s), 1592 (m), 1670 (m), 2868 (w), 2974 (m), 3039 (m), 3154 (m)
1H NMR (DMSO-d6), δ: 1.12 (t, 3H, H-15ꢀ, J = 7.4 Hz), 1.33 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.50 (q, 2H, H-15,
J = 7.4 Hz), 4.03 (q, 2H, OCH2CH3, J = 7.0 Hz), 5.26 (s, 2H, H-8), 6.89 (d, 2H, H-10, H-14, J = 8.6 Hz), 6.93 (d,
2H, H-19, H-21, J = 9.0 Hz), 7.10 (d, 2H, H-11, H-13, J = 8.6 Hz), 7.45 (d, 2H, H-18, H-22, J = 9.0 Hz), 7.46 (td,
1H, H-6, J = 2.0 Hz, J = 7.4 Hz), 7.55 (td, 1H, H-5, J = 1.4 Hz, J = 7.4 Hz), 7.58 (dd, 1H, H-4, J = 2.0 Hz, J =
7.4 Hz), 7.61 (dd, 1H, H-7, J = 1.4 Hz, J = 7.4 Hz), 11.78 (br, s, 1H, NH), 12.27 (br, s, 1H, NH)
13C NMR (DMSO-d6), δ: 14.40 (OCH2CH3), 15.56 (C-15ꢀ), 27.05 (C-15), 63.04 (OCH2CH3), 67.33 (C-8), 114.02
(C-19, C-21), 114.41 (C-10, C-14), 125.62 (C-18, C-22), 127.49, 128.13, 128.22, 128.39 (C-11, C-13), 130.40, 130.74,
133.17, 135.58, 136.01, 156.09 (C-20), 156.49 (C-9), 169.88 (C-1), 178.78 (C-16)
Vm
IR, ν˜/cm−1: 498 (w), 544 (w), 613 (w), 649 (w), 739 (m), 818 (w), 872 (w), 944 (w), 1002 (m), 1028 (m), 1054 (w),
1078 (w), 1117 (m), 1163 (m), 1241 (s), 1324 (w), 1390 (w), 1451 (m), 1481 (m), 1518 (vs), 1584 (w), 1607 (w),
1688 (m), 1876 (m), 2864 (w), 2922 (w), 2954 (w), 3024 (w), 3175 (m)
1H NMR (DMSO-d6), δ: 1.10 (t, 3H, H-15ꢀ, J = 7.5 Hz), 2.49 (q, 2H, H-15, J = 7.5 Hz), 5.24 (s, 2H, H-8), 6.87 (d,
2H, H-10, H-14, J = 8.5 Hz), 7.07 (d, 2H, H-11, H-13, J = 8.5 Hz), 7.40 (d, 2H, H-18, H-22, J = 8.7 Hz), 7.39–7.60
(m, H-4, H-7), 7.73 (d, 2H, H-19, H-21, J = 8.7 Hz), 11.87 (s, 1H, NH), 12.36 (s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.73 (C-15ꢀ), 27.30 (C-15), 67.72 (C-8), 90.87 (C-20), 114.74 (C-10, C-14), 126.35 (C-18,
C-22), 126.47 (C-4), 127.87 (C-6), 128.46 (C-7), 128.53 (C-11, C-13), 131.14 (C-5), 131.27 (C-2), 135.88 (C-12),
137.10 (C-17), 137.41 (C-19, C-21), 143.32 (C-3), 156.37 (C-9), 170.13 (C-1), 178.74 (C-16)
Vn
IR, ν˜/cm−1: 546 (w), 611 (w), 652 (w), 708 (m), 737 (m), 828 (w), 851 (w), 879 (w), 950 (w), 1031 (w), 1078 (w),
1111 (m), 1161 (m), 1235 (s), 1314 (w), 1337 (w), 1394 (w), 1452 (s), 1513 (vs), 1607 (m), 1690 (m), 1894 (m), 2868
(w), 2923 (w), 2956 (w), 3019 (w), 3181 (w)
1H NMR (DMSO-d6), δ: 1.07 (t, 3H, H-15ꢀ, J = 7.4 Hz), 2.48 (q, 2H, H-15, J = 7.4 Hz), 5.26 (s, 2H, H-8), 6.87
(d, 2H, H-10, H-14, J = 8.7 Hz), 7.05 (d, 2H, H-11, H-13, J = 8.7 Hz), 7.42–7.65 (m, H-4, H-7), 7.96 (d, 2H, H-18,
H-22, J = 9.2 Hz), 8.25 (d, 2H, H-19, H-21, J = 9.2 Hz), 12.04 (s, 1H, NH), 12.70 (s, 1H, NH)
13C NMR (DMSO-d6), δ: 15.73 (C-15ꢀ) 27.29 (C-15), 67.65 (C-8), 114.68 (C-10, C-14), 123.90 (C-18, C-22), 124.27
(C-20), 127.88 (C-6), 128.52 (C-7), 128.66 (C-11, C-13), 128.71 (C-4), 131.25 (C-5), 133.20 (C-2), 135.98 (C-12),
136.38 (C-17), 143.00 (C-19, C-21), 143.91 (C-3), 156.34 (C-9), 170.15 (C-1), 179.15 (C-16)
Table 3. Antimicrobial activity (MIC/(µg mL−1)) of acylthioureas Va–Vn
Compound
Vh Vi
Microorganisms
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vj
Vk
Vl
Vm Vn Standard
S. aureus 1263
62.5 125 125 125 125 62.5 125 500 125 500 500 500 125 125
62.5 125 125 125 125 125 125 500 500 500 500 500 250 250
62.5 250 125 125 125 125 125 125 125 125 125 125 250 250
1000 125 62.5 125 1000 125 125 1000 125 1000 125 1000 62.5 62.5
256a
16a
16a
1024b
8b
S. aureus IC 13204
B. subtilis IC 12488
K. pneumoniae 1204
K. pneumoniae IC 13420 125 125 125 1000 1000 1000 125 125 1000 1000 1000 1000 62.5 62.5
E. coli 13147
E. coli IC 13529
P. aeruginosa 1246
P. aeruginosa IC 13202
C. albicans 101404
C. albicans IC 249
A. niger IC 13534
62.5 250 62.5 62.5 125 125 125 125 62.5 62.5 62.5 62.5 62.5 62.5
62.5 125 62.5 62.5 125 125 125 125 62.5 125 125 125 31.2 31.2
1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 62.5 62.5
125 1000 1000 1000 1000 1000 1000 1000 125 1000 1000 1000 62.5 62.5
62.5 62.5 31.2 31.2 62.5 31.2 31.2 31.2 31.2 125 125 31.2 62.5 62.5
62.5 62.5 7.8 62.5 31.2 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5
15.6 15.6 31.2 31.2 31.2 31.2 62.5 62.5 15.6 15.6 31.2 31.2 62.5 62.5
1024b
8b
512b
8b
128c
32c
5d
a) Amikacin; b) ceftriaxone; c) fluconazole; d) voriconazole.
pounds tested exhibited specific antimicrobial activ-
ities as far as the microbial spectrum and the MIC
value were concerned (Table 3).
MIC values ranging between 1000 µg mL−1 and 31.2
µg mL−1 and showed antifungal activity at the MIC
in the range of 125–7.8 µg mL−1
.
Compared with the active-substance charge ap-
plied onto standard antibiotic discs (i.e. up to 30 µg
per disc), it was considered that a low concentration of
7.8 µg mL−1 represented a strong effect and a concen-
tration of 250 µg mL−1 represented a moderate effect.
All the compounds inhibited bacterial growth at the
The level of antimicrobial activity of the new 2-
((4-ethylphenoxy)methyl)benzoylthiourea derivatives
was dependent on the type, number and position of
the substituent on the phenyl ring attached to the
thiourea nitrogen which probably exhibited a steric
and an electronic effect on the nitrogen’s electron den-

C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
67
sity, modifying its capacity to form intramolecular H-
bonds, thus influencing the compound’s ability to pen-
etrate the microbial cell.
aureus and C. albicans IC 249 over the monomethyl
derivatives. Replacement of the methoxy group with
the ethoxy group increased the efficiency against E.
coli and B. subtilis.
The 4-nitro or 4-iodo substituted compounds (Vm,
Vn) showed improved antimicrobial effect on E. coli,
P. aeruginosa, S. aureus, and C. albicans clinical
strains than their 2-((4-methoxyphenoxy)methyl)ben-
zoic acid congeners (Balotescu et al., 2007).
A comparative study of the MIC values in-
dicated that the 2-((4-ethylphenoxy)methyl)benzoic
acid thioureides exhibited higher antimicrobial activ-
ity against E. coli, B. subtilis, and C. albicans than the
2-((4-chlorophenoxy)methyl)benzoic acid thioureide
derivatives, but lower against S. aureus (Limban
et al., 2008a). Analysis of the effect of the sub-
stituent position on the arylthiourea part showed
that the para position favours the antimicrobial ac-
tivity against E. coli in both series. These 2-((4-
ethylphenoxy)methyl)benzoic acid thioureides present
better antimicrobial activity when compared with
those containing the fluorine atom in position 4 at the
phenoxymethyl moiety. In general, the substitution
in position 4 of the phenoxymethyl moiety with the
ethyl group is better for the antibacterial effect than
the substitution with the methyl, methoxy, chlorine or
fluorine group. These compounds show an even higher
antifungal activity and the same structure-activity re-
lationship.
The lowest MIC values, thus the most evident an-
timicrobial activity against Gram-negative bacterial
strains, were noticed for the compound Vm with io-
dine in position 4 and for Vn with the nitro group
in position 4. These compounds are significantly more
active than the antibiotic used as a standard on clin-
ical strains, but they are less efficacious on stan-
dard strains. The presence of the iodine substituent
in Vm produces inductive electron-withdrawing and
resonance electron-donating effects, determining a de-
crease in the nitrogen’s electron density. Similarly, the
nitro group, by inductive and electron-withdrawing ef-
fects, reduces the nitrogen’s electron density. These ef-
fects are responsible for a better internalisation of the
compounds tested through the Gram-negative nega-
tively charged outer membrane.
By contrast, the compounds with electron-donat-
ing substituents, like methyl and ethyl, are more ac-
tive against Gram-positive strains (S. aureus and B.
subtilis). The most efficient compound, exhibiting the
lowest MIC values (62.5 µg mL−1) against the Gram-
positive bacterial strains, was Va, with two methyl
substituents at positions 2 and 3 of the phenyl ring.
Compared with amikacin, the activity of Va was bet-
ter only on the isolated clinical strains.
As for the antifungal activity of the compounds
tested, they exhibited higher MIC values against A.
niger; in comparison with variconazole the most ac-
tive compounds were Va, Vb, Vi, and Vj (MIC 15.6
µg mL−1). All the compounds tested proved to be
more active against C. albicans 101404 strain than
fluconazole. With C. albicans IC249, only compounds
Vc and Ve were more effective than fluconazole.
The results obtained indicated Vc with two methyl
substituents as the most active antifungal compound.
These data are consistent with previous results (Ham-
mam et al., 2007); they show that electron-donating
groups, such as alkyl and alkoxy groups, increase the
fungicidal activity, On the other hand, the introduc-
tion of electron-withdrawing groups decreases this ac-
tivity. The specific antifungal activity exhibited by the
substances tested could be due to easier internalisation
of the compound tested into the eukaryotic pathogens,
as compared with the prokaryotic ones, leading to the
intracellular accumulation of the antimicrobial sub-
stances resulting in an antifungal effect at low MIC
values.
In the future, we intend to extend our research into
this class of (phenoxy)methylbenzoylthioureas by syn-
thesising new alkyl or alkoxy-substituted compounds
and metal complexes with these ligands in order to ob-
tain better antifungals and to better understand the
SARs in this thioureide class.
Conclusions
In order to obtain new compounds with im-
proved antimicrobial activity, fourteen new 2-((4-
ethylphenoxy)methyl)benzoylthioureas were synthe-
sised. Their structures were identified by spectral (IR,
NMR) data and by elemental analysis. The com-
pounds tested exhibited specific antimicrobial activity
against different bacterial and fungal strains, recently
isolated from clinical samples and exhibiting resis-
tance to conventional antibiotics, with MIC values in a
range of 7.8–1000 µg mL−1. The level of antimicrobial
activity of the new 2-((4- ethylphenoxy)methyl)benzo-
ylthiourea derivatives was dependent on the type,
number and position of substituents on the phenyl ring
attached to the thiourea nitrogen atom. The iodine
and nitro substituents favoured the antimicrobial ac-
tivity against Gram-negative bacterial strains, while
the highest inhibitory effect against Gram-positive
and fungal strains was exhibited by the compounds
with electron-donating substituents, like methyl and
ethyl.
Compared with other 2-((4-ethylphenoxy)methyl)
benzoylthioureas investigated previously (Limban et
al., 2009a), the new compounds proved to be more ac-
tive antifungals. The introduction of a methyl group
into different positions of the benzene ring was not
a major determinant in the activity against K. pneu-
moniae, E. coli, P. aeruginosa, and B. subtilis but
represented an advantage in the activity against S.

68
C. Limban et al./Chemical Papers 65 (1) 60–69 (2011)
Acknowledgements. The authors acknowledge support for
this work from the Ministry of Education, Research and Inno-
vation through the PN-II 41-043/2007 grant.
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