Antimicrobial activity and structural study of disubstituted Thiourea Derivatives

Article abstract of DOI:10.1007/s00706-007-0600-y

The antimicrobial activity of six N-phenyl- and fourteen N-benzoylthiourea derivatives were evaluated from their Minimal Inhibitory Concentration (MIC) values using the microdilution procedure against ten microorganisms. Most of the compounds exhibited selective activity against fungi and Gram-positive bacteria, which were very effectively inhibited by some of the tested thioureas. Additionally, SAR considerations and four novel X-ray diffraction structures of N-benzoylthioureas are included. Springer-Verlag 2007.

Full text of DOI:10.1007/s00706-007-0600-y

Monatshefte fu¨r Chemie 138, 511–516 (2007)
DOI 10.1007/s00706-007-0600-y
Printed in The Netherlands
Antimicrobial Activity and Structural Study of Disubstituted Thiourea
Derivatives
Silvio Cunha1;ꢀ, Fernando C. Macedo Jr.1, Giselle A. N. Costa1, Manoel T. Rodrigues Jr.1,
Rosival B. V. Verde1, Lourdes C. de Souza Neta1, Ivo Vencato2;3, Carlito Lariucci3,
3
´
and Fernando P. Sa
1
´
Instituto de Quımica, Universidade Federal da Bahia, Campus de Ondina, Salvador-BA, Brazil
´
2
3
´ ´
Ciencias Exatas e Tecnologicas, Universidade Estadual de Goias, Anapolis-GO, Brazil
^
Instituto de Fısica, Universidade Federal de Goias, Goiania-GO, Brazil
´
´
^
Received July 19, 2006; accepted (revised) August 14, 2006; published online March 15, 2007
# Springer-Verlag 2007
Summary. The antimicrobial activity of six N-phenyl- and
[8], have been reported. Thiourea-based non-nucleo-
side inhibitors of HIV reverse transcriptase have also
been described [9–12].
fourteen N-benzoylthiourea derivatives were evaluated from
their Minimal Inhibitory Concentration (MIC) values using
the microdilution procedure against ten microorganisms. Most
of the compounds exhibited selective activity against fungi
and Gram-positive bacteria, which were very effectively in-
hibited by some of the tested thioureas. Additionally, SAR
considerations and four novel X-ray diffraction structures of
N-benzoylthioureas are included.
Although the antimicrobial properties of thioureas
have been related [7, 8, 15], they are limited both in
terms of microorganisms’ strains and structural di-
versity. To the best of our knowledge, no systematic
screening has been reported for this class of com-
pounds. Among the thioureas, the N-phenyl and
N-benzoyl derivatives caught our attention because
previously published results concerning the low tox-
icity of these thiourea derivatives [14, 15] suggested
their potential as antibiotic drugs.
Keywords. Benzoylthioureas; Phenylthioureas; X-ray
structure.
Introduction
The search for compounds with antimicrobial activ-
ity is a theme of continuous interest because of the
increased antimicrobial resistance developed by im-
portant pathogens [1]. The emergence of multidrug-
resistant strains of Gram-positive bacterial pathogens
is a problem of ever increasing significance.
Thiourea derivatives have attracted the attention
of several research groups due to their potential in
medicinal chemistry [2–8]. The broad spectra of
biological activity of thiourea derivatives and their
metal complexes have been investigated and diverse
bioactivities, such as antifungal [7] and anti-malarial
As part of our interest in the synthesis and screen-
ing of potentially bioactive compounds [13], we re-
port herein our results concerning the antimicrobial
evaluation of thioureas against a diversity of bacteria
and fungi strains. Additionally, SAR considerations
and the conformational bias from novel X-ray diffrac-
tion structures of N-benzoylthioureas are discussed.
Results and Discussion
Chemistry
To evaluate a representative set of thiourea deriva-
tives with a diverse substitution pattern, two series of
ꢀ
Corresponding author. E-mail: silviodc@ufba.br

512
S. Cunha et al.
N1,N2-disubstituted thioureas were prepared by
reaction of primary and secondary amines with
both phenyl and benzoyl isothiocyanate, affording
N-phenyl- 1a–1e and N-benzoylthioureas 2a–2m,
Scheme 1 [16]. Similarly, the N1-benzoyl-N2-benz-
amidethiourea derivative 2n was obtained by treat-
Scheme 1

Disubstituted Thiourea Derivatives
513
Table 1. Minimal inhibitory concentration (MIC=ꢀg cmꢁ3) of thiourea derivatives 1a–1e and 2a–2n
Entry
Thiourea
Microorganisms#
Bacteria tested
´
S. choleraesuıs
B. subtilis
S. aureus
M. luteus
S. mutans
E. coli
P. aeruginosa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
50
>100
>100
100
>100
>100
>100
>100
>100
>100
100
>100
>100
3.1
>100
>100
100
100
50
100
50
12.5
25
>100
12.5
100
12.5
>100
100
>100
6.3
100
>100
3.1
0.78
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
125
>100
100
50
>100
>100
25
2m
2n
PC|
50
6.3
3.1
6.3
6.3
6.3
3.1
Entry
Thiourea
Fungi tested
C. albicans
A. niger
C. cladosporioides
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
100
>100
100
>100
100
>100
100
100
>100
100
50
50
50
>100
100
100
>100
>100
25
100
50
100
>100
>100
>100
>100
100
>100
100
100
100
100
100
>100
3.1
12.5
100
>100
100
100
100
100
>100
>100
>100
100
>100
>100
25
>100
>100
100
100
>100
>100
>100
100
25
100
>100
3.1
6.3
2m
2n
PC|
6.3
#
Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6638, Micrococcus luteus ATCC 10240, Streptococcus mutans
ATCC 24175, Salmonella choleraesuis ATCC 14028, Escherichia coli ATCC 94863, Pseudomonas aeruginosa, Candida albicans
ATCC 18804, Aspergillus niger ATCC 16404, Cladosporium cladosporioides IMI 178517
|
PC Positive control (chloramphenicol for bacteria, and ciclopirox olamine for fungi)

514
S. Cunha et al.
ment of ethyl benzoate with hydrazine followed by
reaction of the formed hydrazide with benzoyl iso-
thiocyanate, while benzaldehyde 4-phenylthiosemi-
carbazone 1f was prepared by condensation of
4-phenyl thiosemicarbazide with benzaldehyde (see
Experimental).
2g. However, the substitution of the N2-aromatic ring
by a corresponding aliphatic group led to an even fur-
ther decrease of the MIC (6.3 ꢀg=cm3 for 2l) against
the later microorganism. Conversely, the N2 second-
ary benzoylthioureas were, in general, more effective
against B. subtilis (see componds 2i, 2l and 2m).
Biological Results
Antifungal Activities
The minimal inhibitory concentrations of all obtained
N1,N2-disubstituted thioureas were evaluated in bio-
assays involving Gram-positive and Gram-negative
bacteria as well as fungi. As can be verified, all the
compounds showed antimicrobial activity in some
tested concentration (Table 1, shadowed entries).
The inhibitory activities of the thiourea derivatives
were broad and more uniform for fungi than for bac-
terias. Similarly to Gram-positive bacterial strains,
2n was the most bioactive compound against fungi
cultures, followed by benzoylthioureas 2a and 2k,
which showed low inhibition concentrations for
C. cladosporioides.
Antibacterial Activities
X-Ray Analysis
The active thioureas showed a clear selectivity against
the Gram-positive bacterias (B. subtilis ATCC 6633,
S. aureus ATCC 6638, M. luteus ATCC 10240) even
for the most potent tested compound 2n, to which
very low MIC values (3.1–6.3 ꢀg=cm3) were ob-
served for these strains. Analysis of Table 1 also
suggests that, in general, benzoylthioureas were more
active than corresponding phenyl- ones as can be
verified directly from comparison of the MIC values
between 1c=2k and 1a=2b. M. luteus seems to be the
most susceptible bacterial strain and was particularly
inhibited by benzoylthioureas bearing a para-substi-
tuted aromatic ring at N2 as observed for 2b, 2c, 2e,
To gain insight into the conformational bias, crystal
structures of some of the obtained thioureas were
analyzed by X-ray crystallography. The ORTEP-3
[17] representations of 2c, 2j, 2l, and 1f are shown
in Fig. 1. The solid state structural features observed
for the N2 secondary derivative 2l confirms its pre-
viously predicted structure [18] using B3LYP=6-
31G. Despite four planar conformations are possi-
ble for N1,N2-disubstituted thioureas 2c, 2j, and 1f
[19], only the cis-trans conformation was observed.
Regarding the crystal structure of 2c, 2j, and 2l,
there is one intramolecular hydrogen bond involving
Fig. 1. ORTEP drawings for 2e, 2j, 2c, and 1f. Displacement ellipsoids are drawn at the 30% probability level and H atoms are
shown as spheres of arbitrary radii. The intramolecular H-bonds are shown with dashed lines

Disubstituted Thiourea Derivatives
515
N-(Benzamido)-N0-phenylthiourea (2n)
A mixture of 105 mmol 50% aqu hydrazine in 10cm3 isopro-
panol and 7 mmol ethyl benzoate was refluxed for 3 h to afford
the benzoylhydrazine as colorless hygroscopic solid after
solvent evaporation. To a solution of this product in 30cm3
isopropanol, 7 mmol benzoyl isothiocyanate were added drop-
wise at room temperature until TLC showed the completion of
the reaction. The mixture was filtered and the colorless solid
was washed with isopropanol yielding product (91%) with mp
170–172ꢃC (Ref. [16m] 172–174ꢃC).
Fig. 2. SAR summary of the thiourea derivatives
atoms N1H1ꢂ ꢂ ꢂO6 and a short contact interaction of
the type CHꢂ ꢂ ꢂS5 in each structure, providing two
pseudo six-membered rings (see Fig. 1). Curiously,
this later weak intramolecular interaction is not ob-
served for 1f. Intermolecular hydrogen bonds in-
volving N3H3ꢂ ꢂ ꢂS5 are observed for 2c, 2j, and 1f,
while for 2l this interaction involves N3H3ꢂ ꢂ ꢂO6.
In conclusion, thiourea derivatives obtained by
simple and inexpensive manner display selective
antimicrobial activities against fungi and Gram-posi-
tive bacterial strains. Among the tested compounds,
some benzoyl derivatives presented promising activity
against B. subtilis, M. luteus, or C. cladosporioides.
MIC values of the two series of thioureas against
bacterial strains indicated the importance of the sub-
stitution pattern on N2, Fig. 2. However, at this stage,
the influence of the electronic nature of the N2-group
of the N1-benzoyl-N2-substituted thioureas and sub-
stitution at the phenyl ring are not clear. Thus, struc-
ture activities relationship studies on other series of
thioureas inspired on structures 2a, 2b, 2k, and par-
ticularly 2n are currently underway.
Benzaldehyde 4-phenylthiosemicarbazone (1f)
Phenyl isothiocyanate 18 mmol was added dropwise to a so-
lution of 54mmol 50% aqu hydrazine in 30cm3 isopropanol
under stirring at room temperature for 1 h. The colorless solid
was decanted and washed with cold isopropanol giving the 4-
phenylthiosemicarbazide (70%). Then, 5 mmol benzaldehyde
were added dropwise to a suspension of 5 mmol 4-phenyl-
thiosemicarbazide in 25cm3 ethanol under stirring at room
temperature for 4 h. The mixture was filtered and the colorless
solid was washed with cold ethanol yielding 1f (81%) with mp
189–191ꢃC (lit. 189–191ꢃ; B 12 413).
Determination of Minimal Inhibitory Concentrations [20]
Values are means of three experiments. The bacteria cultures
used were grown for 24h at 35ꢃC on nutrient agar. The fungi
and yeasts were cultivated for 72h at 26ꢃC on malt extract
agar and yeast malt agar. The inocula for the assays were
prepared by cells suspensions according to McFarland scale
0.5, except to filamentous fungi to which a modified method
was used [11]. Broth microdilution method was carried out to
determine the MIC of the compounds against the microrgan-
isms in sterile 96-well microplates. The 20% DMSO aqueous
stock solutions of the compounds were transferred into the
first well from which serial dilutions were performed so that
concentrations ranged from 100 to 0.78 ꢀg=cm3. Chloramphen-
icol and olamine ciclopirox were used as the reference drugs
against bacteria and fungi. Aqueous DMSO (20%) was used
as negative control. The inoculum was added to all wells and
the plates were incubated in the appropriate conditions. After
incubation, microorganisms growth were observed by the
presence of turbidity on the well. MIC was defined as the
lowest concentration of the substances that inhibited visible
growth.
Experimental
´
Melting points were determined on a Microquımica MQAPF
301 hot plate apparatus. Infrared spectra were recorded with
KBr discs on a FT-IR BOMEM MB100 instrument. NMR
spectra were obtained for 1H at 300 MHz and for 13C at
75MHz using a Varian Gemini 300 spectrometer. All com-
pounds had physical properties identical with reported values.
Crystallographic Data Collection and Structure
Typical Synthesis Procedures
Determination of 2j, 2l, 2c, and 1f
N-Phenylthioureas 1a–1e and N-Benzoylthioureas 2a–2n
The suitable isothiocyanate (5mmol) was added dropwise to a
solution of 5 mmol amine in 5 cm3 benzene under stirring and
ice bath cooling. The reaction mixture was left for 3 h at room
temperature after which time the solvent was evaporated and
the crude solid was triturated with petroleum ether. Recrys-
tallization with CH2Cl2:petroleum ether (4:1) afforded the
thiourea. Its structure was confirmed by mp and spectroscopic
data compared to literature values (1e, 2f [13]; 1b, 1d, 2d, 2e,
and 2h [16b]; 2b, 2c [16c]; 2l [16e]; 2i, 2m [16i]; 2k [16j]; 2j
[16k]; 2g [16l]; 1a, 1c [16n]).
Single crystals X-ray diffraction data were collected at room
temperature using a Nonius CAD-4 diffratometer [12] with
˚
CuKꢁ radiation (l ¼ 1.54180 A). The structures were solved
by direct methods and refined anisotropically with full-matrix
least-squares on F2 using SHELXL97 [13]. The hydrogen atoms
were placed at calculated positions except those involved in
H-bonds and weak interactions, found on difference maps and
refined with riding constraints. The crystallographic data were
deposited at the Cambridge Crystallographic Data Center
under the numbers CCDC 290675, CCDC 290676, CCDC
290677, and CCDC 606380 for 2j, 2l, 2c, and 1f. Copies of

516
S. Cunha et al.: Disubstituted Thiourea Derivatives
the data can be obtained, free of charge via www.ccdc.cam.
ac.uk=conts=retrieving.html (or from the Cambridge Crystal-
lographic Data Centre, CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK; (Fax: þ44 1223 336033; or E-mail: deposit@
ccdc.cam.ac.uk).
[13] Cunha S, Oliveira SM, Rodrigues MT Jr, Bastos RM,
Ferrari J, Oliveira CMA, Kato L, Napolitano HB, Vencato
I, Lariucci C (2005) J Mol Struct 752: 32
[14] Baltabaev UA, Makhsumov AG, Zakirov UB, Babaev
ID, Shukurullaev K (2002) Pharm Chem J 36: 77
[15] Yadava BP (1989) Curr Sci 58: 1026
[16] For representative papers on synthesis, see: (a)
Kascheres A, Ueno M (1991) J Heterocyclic Chem
28: 2057; (b) Sarkis GY, Faisal ED (1985) J Heterocyclic
Chem 22: 137; (c) Rasmussen CR, Villani FJ Jr, Weaner
LE, Reynolds BE, Hood AR, Hecker LR, Nortey SO,
Hanslin A, Costanzo MJ, Powell ET, Molinari AJ (1988)
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TJ (2004) Tetrahedron Lett 45: 8091; (e) Katritzky AR,
Kirichenko N, Rogovoy BV, Kister J, Tao H (2004)
Acknowledgements
The authors gratefully acknowledge the financial support of
´
´
Conselho Nacional de Desenvolvimento Cientıfico e Tecnolo-
`
gico – CNPq and Fundac°a˜o de Amparo a Pesquisa do Estado
da Bahia – FAPESB. We also thank FAPESB for fellowship
to G.A.N.C. and CNPq for fellowships to F.C.M. Jr., L.C.S.N.,
M.T.R. Jr., R.B.V.V. (PIBIC-UFBA-CNPq), I.V., and for a
research fellowship to S.C.
´
Synthesis 1799; (f) Vasquez J, Bernes S, Reyes Y, Moya
M, Sharma P, Alvarez C, Gutierrez R (2004) Synthesis
`
´
1955; (g) Katritzky AR, Ledoux S, Witek RM, Nair SK
(2004) J Org Chem 69: 2976; (h) Herr RJ, Kuhler JL,
Meckler H, Opalka CJ (2000) Synthesis 1569; (i) Plutin
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