Synthesis and microbiological activity of thiourea derivatives of 4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione

Article abstract of DOI:10.1007/s12272-010-2223-9

A series of thiourea derivatives of 4-azatricyclo[5.2.2.0 ^2,6]undec-8-ene-3,5-dione were synthesized. The compounds were investigated for antibacterial activity, including Gram-positive cocci, Gram-negative rods, and antifungal activity. Compounds 1b, 2b, 4b showed significant inhibition against Gram-positive cocci. Research was carried out over 10 standard strains and 20 hospital strains. Synthesized compounds were evaluated for their cytotoxicity and anti-HIV-1 activity in MT-4 cells.

Full text of DOI:10.1007/s12272-010-2223-9

Arch Pharm Res Vol 33, No 1, 47-54, 2010
DOI 10.1007/s12272-010-2223-9
Synthesis and Microbiological Activity of Thiourea Derivatives of 4-
Azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione
Marta Struga1, Szymon Rosolowski1, Jerzy Kossakowski1, and Joanna Stefanska2
2
1Medical University, Department of Medical Chemistry, 3 Oczki Str., 02-007 Warsaw, Poland and Medical University,
Department of Pharmaceutical Microbiology, 3 Oczki Str., 02-007 Warsaw, Poland
(Received September 1, 2009/Revised October 20, 2009/Accepted October 20, 2009)
A series of thiourea derivatives of 4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione were synthe-
sized. The compounds were investigated for antibacterial activity, including Gram-positive
cocci, Gram-negative rods, and antifungal activity. Compounds 1b, 2b, 4b showed significant
inhibition against Gram-positive cocci. Research was carried out over 10 standard strains and
20 hospital strains. Synthesized compounds were evaluated for their cytotoxicity and anti-
HIV-1 activity in MT-4 cells.
Key words: Synthesis, Thiourea derivatives, Antimicrobial activity, Cytotoxicity
terial, antifungal, tuberculostatic, antitumor, anticon-
vulsant and antiviral properties (Limbau et al., 2008;
INTRODUCTION
Bacterial infections have increased dramatically in Venkatachalan et al., 2004; Aramakada et al., 2007;
recent years. Bacteria have been the cause of the most Bloom et al., 2004; Seth et al., 2004; Lee et al., 2003;
deadly diseases and widespread epidemics in human Kaymakciglu et al., 2005; Esteves-Souza et al., 2006).
civilization. Introduction of antibiotics in the 1940s
Here we report the synthesis and antimicrobial
was thought to have eliminated the scourge of all activity of compounds containing the thiourea system
infectious diseases. However, due to the widespread attached to polycyclic imide.
use and misuse of antibiotics, bacterial resistance to
antibiotics has become a serious public health problem.
Some of these resistant strains, such as vancomycin-
resistant enterococci (VRE) and multidrug resistant
Staphylococcus aureus (MRSA), are capable of surviv-
MATERIALS AND METHODS
Chemistry
Melting points were determined in a Kofler’s ap-
1
ing the effect of most, if not all, antibiotics currently in paratus and are uncorrected. The H NMR spectra
use (Hooper, 2001; Galimand et al., 2003; Tenover, were recorded on a Bruker AVANCE DMX400 spec-
2001; Leclerq et al., 2002; Nagai et al., 2002). With the trometer, operating at 400 MHz. Chemical shift
increase of bacterial resistance to antibiotic treatment, values are expressed in ppm relative to TMS as an
attention has focused on developing novel approaches internal standard. Elemental analyses were recorded
to antimicrobial therapy (Fung-Tomc et al., 2002; with a CHN model 2400 Perkin-Elmer. Mass spectral
Chopra, 2001; Nagano et al., 2000; Capobianco et al., ESI measurements were carried out on Waters ZQ
2000; Choudhry et al., 2003; Colca et al., 2003; Oliva Micromass instruments with quadrupol mass analyzer.
et al., 2003).
Spectra were performed in the positive ion mode at a
Thiourea derivatives are associated with a broad declustering potential of 40–60V. Sample was pre-
spectrum of biological activities including antibac- viously separated on a UPLC column (C18) using
UPLC ACQUITYTM system by Waters connected with
Correspondence to: Marta Struga, Medical University, Depart-
ment of Medical Chemistry, 3 Oczki Str., 02-007 Warsaw, Poland
Tel./Fax: 48 0226280679
E-mail: marta.struga@wum.edu.pl (M. Struga), joanna.stefan-
ska@wum.edu.pl (J. Stefanska)
DPA detector.
Flash chromatography was performed on Merck
silica gel 60 (200400 mesh) using chloroform/methanol
(19 : 1 vol) mixture as eluent. Analytical TLC was
47

48
M. Struga et al.
carried out on silica gel F254 (Merck) plates (0.25 mm Hz); 7.04 (t, 1H, CHarom.
,
J
= 7.5 Hz); 7.26 (t, 1H,
thickness).
CHarom.
J
= 7.6 Hz); 7.53 (d, 1H, CHarom., J = 8.1 Hz);
7.73 (d, 1H, CHarom.
8.07 (br. s, 1H, NH). Anal. Calcd for C21H24N3O2SBr:
,
J
= 8.1 Hz); 7.76 (br. s, 1H, NH);
Thiourea derivatives of 4-azatricyclo[5.2.2.02,6]
undec-8-ene-3,5-dione
C 54.55, H 5.23, N 9.09. Found: C 54.60, H 5.28, N
General procedure: A solution of 4-amino-tricyclic 9.08. ESI MS: m/z = 486.1 [M + Na]+ (100%).
imide (0.0025 mole) in acetonitrile (10 mL) was treat-
ed with appropriate isothiocyanate (0.003 mol) and 1-(3-bromophenyl)-3-(1,7,8,9,10-pentamethyl-3,5-
the mixture was refluxed for 6 h. Then the solvent dioxo-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea
was removed on rotary evaporator. The residue was (2b)
1
purified by column chromatography (chloroform : Yield 82%. Mp. 129oC. HNMR (CDCl3)
δ (ppm): 0.63
= 6.3 Hz); 1.36 (s, 6H, CH3); 1.51 (s, 6H,
methanol; 9.5:0.5 vol.). The compound was crystallized (d, 3H, CH3,
J
from ethanol.
CH3); 1.51-1.65 (m, 1H, CH); 3.06 (d, 2H, CH, = 3.9
J
Hz); 7.7.14-7.38 (m, 4H, CHarom.); 8.22 (br. s, 1H, NH);
1-(2-bromophenyl)-3-(1,7,8,9-tetramethyl-3,5-dioxo-
8.47 (br. s, 1H, NH). Anal. Calcd for C21H24N3O2SBr:
4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea (1a) C 54.55, H 5.23, N 9.09. Found: C 54.65, H 5.28, N
Yield 82%. Mp. 136oC. HNMR (CDCl3)
δ
(ppm): 1.49 9.06. ESI MS: m/z = 486.1 [M + Na]+ (100%).
1
(s, 6H, CH3); 1.45 (s, 6H, CH3); 1.43-1.58 (m, 1H, CH2);
2.04 (s, 2H, CH); 7.14 (t, 1H, CHarom.
(t, 1H, CHarom. J = 7.8 Hz); 7.61 (d, 1H, CHarom.
Hz); 7.76 (br. s, 1H, NH); 7.83 (d, 1H, CHarom.
,
J = 7.9 Hz); 7.35 1-(4-bromophenyl)-3-(1,7,8,9,10-pentamethyl-3,5-
,
,
J
J
= 7.5 dioxo-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea
= 7.8 (3b)
Hz); 7.76 (br. s, 1H, NH). Anal. Calcd for C20H22N3O2SBr: Yield 82%. Mp. 154oC. HNMR (CDCl3)
δ (ppm): 0.62
1
C 53.57, H 4.95, N 9.37. Found: C 53.81, H 5.02, N (d, 3H, CH3,
J
= 6.3 Hz); 1.35 (s, 6H, CH3); 1.53 (s, 6H,
CH3); 1.54-1.62 (m, 1H, CH); 3.04 (d, 2H, CH, = 3.6
Hz); 7.25 (m, 2H, CHarom. = 7.5 Hz); 7.26 (t, 1H,
9.18. ESI MS: m/z = 472.0 [M + Na]+ (100%).
J
,
J
1-(3-bromophenyl)-3-(1,7,8,9-tetramethyl-3,5-dioxo-
CHarom. J = 7.6 Hz); 7.53 (d, 1H, CHarom., J = 8.1 Hz);
4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea (1b) 7.73 (d, 1H, CHarom.); 7.46-7.50 (m, 2H, CHarom.); 7.79
1
Yield 72%. Mp. 169oC. HNMR (CDCl3)
δ (ppm): 1.48 (br. s, 1H, NH); 8.06 (br. s, 1H, NH). Anal. Calcd for
(s, 6H, CH3); 1.53 (s, 6H, CH3); 1.43-1.59 (m, 1H, CH2); C21H24N3O2SBr: C 54.55, H 5.23, N 9.09. Found: C
3.12 (s, 2H, CH); 7.2-7.45 (m, 4H, CHarom.); 7.57 (br. s, 54.68, H 5.28, N 9.06. ESI MS: m/z = 486.1 [M + Na]+
1H, NH); 8.19 (br. s, 1H, NH). Anal. Calcd for (100%).
C20H22N3O2SBr: C 53.57, H 4.95, N 9.37. Found: C
53.61, H 4.98, N 9.28. ESI MS: m/z = 472.0 [M + Na]+ 1-(2-bromo-phenyl)-3-(10-isopropyl-8-methyl-3,5-
(100%).
dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-
thiourea (3a)
1
1-(4-bromophenyl)-3-(1,7,8,9-tetramethyl-3,5-dioxo-
Yield 82%. Mp. 136oC. HNMR (CDCl3)
δ
(ppm): 0.82
4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea (1c)
(d, 3H, CH3, J = 6.3 Hz); 0.91 (d, 3H, CH3, J = 6.3 Hz);
1
Yield 91%. Mp. 154oC. HNMR (CDCl3)
δ (ppm): 1.47 1.02-1.16 (m, 2H, CH2); 1.27-1.39 (m, 1H, CH); 1.65 (s,
(s, 6H, CH3); 1.52 (s, 6H, CH3); 1.41-1.59 (m, 1H, CH2); 3H, CH3); 1.74-1.86 (m, 1H, CH); 2.93-2.98 (m, 3H,
3.013 (s, 2H, CH) 7.25 (m, 2H, CHarom. = 7.5 Hz); CH); 3.24 (d, 1H, CH, = 6 Hz); 5.74 (d, 1H, CH=,
7.26 (t, 1H, CHarom. J = 7.6 Hz); 7.27 (d, 2H, CHarom. 6 Hz); 7.16 (t, 1H, CHarom. = 7.2 Hz); 7.36 (t, 1H,
= 5.7 Hz); 7.45 (d, 2H, CHarom. J = 7.8 Hz); 7.97 (br. s, CHarom. = 7.2 Hz); 7.63 (d, 1H, CHarom.
1H, NH); 8.21 (br. s, 1H, NH). Anal. Calcd for 7.77 (br. s, 1H, NH); 7.81 (d, 1H, CHarom.
,
J
J
J =
,
J
, J
,
J
,
,
J
J
= 8.1 Hz);
= 7.5 Hz);
C20H22N3O2SBr: C 53.57, H 4.95, N 9.37. Found: C 8.32 (br. s, 1H, NH). Anal. Calcd for C21H24N3O2SBr:
53.64, H 4.98, N 9.32. ESI MS: m/z = 472.0 [M + Na]+ C 54.55, H 5.23, N 9.09. Found: C 54.60, H 5.20, N
(100%).
9.09. ESI MS: m/z = 486.1 [M + Na]+ (100%).
1-(2-bromophenyl)-3-(1,7,8,9,10-pentamethyl-3,5-
dioxo-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)thiourea
1-(3-bromo-phenyl)-3-(10-isopropyl-8-methyl-3,5-
dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-
(2a)
thiourea (3b)
1
1
Yield 82%. Mp. 166oC. HNMR (CDCl3)
δ
(ppm): 0.54 Yield 82%. Mp. 160oC. HNMR (CDCl3)
δ
(ppm): 0.82
= 6.6 Hz);
= 4.5 1.01-1.15 (m, 2H, CH2); 1.25-1.38 (m, 1H, CH); 1.70 (s,
(d, 3H, CH3,
CH3); 1.49-1.54 (m, 1H, CH); 2.98 (d, 2H, CH,
J = 6.2 Hz); 1.28 (s, 6H, CH3); 1.46 (s, 6H, (d, 3H, CH3, J = 6.3 Hz); 0.91 (d, 3H, CH3, J
J

Synthesis and Microbiological Activity of New Tiourea Derivatives
49
3H, CH3); 1.75-1.86 (m, 1H, CH); 2.98 (br. s, 3H, CH); 1-(4-bromo-phenyl)-3-(1-isopropyl-7-methyl-3,5-
3.21 (d, 1H, CH, = 6 Hz); 5.71 (d, 1H, CH=,
= 5.1 dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thi-
Hz); 7.21 (d, 2H, CHarom. = 8.1 Hz); 7.36 (d, 2H, ourea (4c)
CHarom.
= 8.1 Hz); 7.60 (br. s, 1H, NH); 8.25 (br. s, Yield 82%. Mp. 131oC. HNMR (CDCl3)
1H, NH). Anal. Calcd for C21H24N3O2SBr: C 54.55, H (d, 3H, CH3, = 6.9 Hz); 1.10 (d, 3H, CH3,
J
J
,
J
1
,
J
δ
(ppm): 0.98
J = 6.6 Hz);
J
5.23, N 9.09. Found: C 54.63, H 5.25, N 9.03. ESI MS: 1.21-1.32 (m, 2H, CH2); 1.36-1.57 (m, 2H, CH2); 1.48
m/z = 486.1 [M + Na]+ (100%).
(s, 3H, CH3); 2.47-2.58 (m, 1H, CH); 2.75 (d, 1H, CH,
= 8.1 Hz); 3.12 (d, 1H, CH, = 8.1 Hz); 5.96 (dd, 2H,
CH=, = 8.2 Hz); 7.30 (d, 2H, CHarom. = 8.7 Hz);
= 8.7 Hz); 7.95 (br. s, 1H, NH);
J
J
1-(4-bromo-phenyl)-3-(10-isopropyl-8-methyl-3,5-
J
, J
dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)- 7.40 (d, 2H, CHarom.
, J
thiourea (3c)
Yield 82%. Mp. 131oC. HNMR (CDCl3)
(d, 3H, CH3, = 6.3 Hz); 0.90 (d, 3H, CH3,
8.49 (br. s, 1H, NH). Anal. Calcd for C21H24N3O2SBr:
1
δ
(ppm): 0.81 C 54.55, H 5.23, N 9.09. Found: C 54.69, H 5.28, N
J
J
= 6.6 Hz); 9.09. ESI MS: m/z = 486.1 [M + Na]+ (100%).
1.05-1.13 (m, 2H, CH2); 1.28-1.36 (m, 1H, CH); 1.69 (s,
3H, CH3); 1.78-1.85 (m, 1H, CH); 2.94 (br. s, 3H, CH);
Biology
3.20 (d, 1H, CH,
Hz); 7.28 (d, 2H, CHarom.
CHarom. = 8.7 Hz); 8.20 (br. s, 1H, NH); 8.44 (br. s, a series of Gram-positive bacteria: Staphylococcus
J
= 6 Hz); 5.68 (d, 1H, CH=,
J
= 5.7
In vitro evaluation of antimicrobial activity:
,
J = 8.7 Hz); 7.45 (d, 2H, Antibacterial activity of compounds was tested against
,
J
1H, NH). Anal. Calcd for C21H24N3O2SBr: C 54.55, H aureus ATCC 4163, Staphylococcus aureus ATCC
5.23, N 9.09. Found: C 54.63, H 5.25, N 9.03. ESI MS: 25923, Staphylococcus aureus ATCC 29213, Staphylo-
m/z = 486.1 [M + Na]+ (100%).
coccus aureus ATCC 6538, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus
cereus ATCC 11778, Enterococcus hirae ATCC 10541,
1-(2-bromo-phenyl)-3-(1-isopropyl-7-methyl-3,5-
dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thi- Micrococcus luteus ATCC 9341, Micrococcus luteus
ourea (4a)
Yield 82%. Mp. 135oC. HNMR (CDCl3)
(d, 3H, CH3, = 6.9 Hz); 1.10 (d, 3H, CH3,
1.25-1.35 (m, 2H, CH2); 1.42-1.54 (m, 2H, CH2); 1.49 4635, Pseudomonas aeruginosa ATCC 15442, Pseudo-
(s, 3H, CH3); 2.51-2.60 (m, 1H, CH); 2.75 (d, 1H, CH, monas aeruginosa NCTC 6749, Pseudomonas aeru-
= 8.4 Hz); 3.12 (d, 1H, CH, = 8.1 Hz); 5.98 (dd, 2H, ginosa ATCC 27853, Bordetella bronchiseptica ATCC
CH=, = 8.1 Hz); 7.15 (t, 1H, CHarom. = 7.8 Hz); 7.36 4617. Antifungal activity was tested against yeasts:
(t, 1H, CHarom. = 7.8 Hz); 7.65 (d, 1H, CHarom.
8.1 Hz); 7.8 (br. s, 1H, NH); 7.80 (d, 1H, CHarom.
ATCC 10240 and Gram-negative rods: Escherichia
(ppm): 0.97 coli ATCC 10538, Escherichia coli ATCC 25922,
J = 6.6 Hz); Escherichia coli NCTC 8196, Proteus vulgaris NCTC
1
δ
J
J
J
J
, J
,
J
,
,
J
J
=
=
Candida albicans ATCC 10231, Candida albicans
ATCC 90028, Candida parapsilosis ATCC 220191.
8.1 Hz); 7.93 (br. s, 1H, NH). Anal. Calcd for Microorganisms used in this study were obtained from
C21H24N3O2SBr: C 54.55, H 5.23, N 9.09. Found: C the collection of the Department of Pharmaceutical
54.69, H 5.28, N 9.09. ESI MS: m/z = 486.1 [M + Na]+ Microbiology, Medical University of Warsaw, Poland.
(100%).
Media, growth conditions and antimicrobial
activity assays: Antimicrobial activity was examined
by the disc diffusion and MIC method, under standard
1-(3-bromo-phenyl)-3-(1-isopropyl-7-methyl-3,5-
dioxo-4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thi- conditions, using Mueller–Hinton II agar medium
ourea (4b)
Yield 82%. Mp. 120oC. HNMR (CDCl3)
(d, 3H, CH3, = 6.9 Hz); 1.08 (d, 3H, CH3,
1.25-1.34 (m, 2H, CH2); 1.42-1.56 (m, 2H, CH2); 1.46 containing the tested agents were prepared in methanol
(s, 3H, CH3); 2.46-2.55 (m, 1H, CH); 2.75 (d, 1H, CH, or DMSO. For the disc diffusion method, sterile paper
= 8.1 Hz); 3.11 (d, 1H, CH, = 8.1 Hz); 5.95 (dd, 2H, discs (9 mm diameter, Whatman No. 3 chromatography
CH=, = 7.8 Hz); 7.17 (t, 1H, CHarom. = 7.9 Hz); 7.28 filter paper) were dripped with the compound solu-
tions tested to obtain 400
(d, 1H, CHarom. = 8.1 Hz); 7.37 (d, 1H, CHarom. g of substance per disc. Dry
7.8 Hz); 7.64 (s, 1H, CHarom.); 8.30 (br. s, 1H, NH); 8.86 discs were placed on the surface of an appropriate
(d, 1H, CHarom. = 8.1 Hz); 7.93 (br. s, 1H, NH). Anal. agar medium. The results (diameter of the growth
(Becton Dickinson) for bacteria and RPMI agar with
(ppm): 0.97 2% glucose (Sigma) for yeasts, according to CLSI (pre-
J = 6.6 Hz); viously NCCLS) guidelines [CLSI, 2006a]. Solutions
1
δ
J
J
J
J
, J
,
J
,
J
=
µ
,
J
Calcd for C21H24N3O2SBr: C 54.55, H 5.23, N 9.09. inhibition zone) were read after 18 h of incubation at
Found: C 54.69, H 5.28, N 9.09. ESI MS: m/z = 486.1 35oC. Minimal Inhibitory Concentration (MIC) was
[M + Na]+ (100%).
examined by the twofold serial agar dilution techni-

50
M. Struga et al.
que [CLSI, 2006b]. Concentrations of tested compounds
RESULTS AND DISCUSSION
Chemistry
in solid medium ranged from 3.125 to 400 µg/mL. The
final inoculum of studied organisms was 104 CFU/mL
(colony forming units per mL), except the final in-
The preparation of new twelve thiourea derivatives
oculum for E. hirae ATCC 10541, which was 105 CFU/ of 1,7,8,9-tetramethyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-
mL. Minimal inhibitory concentrations were read off 3,5-dione, 1,7,8,9,10-pentamethyl-4-azatricyclo[5.2.1.02,6]
after 18 h (for bacteria) and 24 h (for yeasts) of dec-8-ene-3,5-dione, 10-isopropyl-8-methyl-4-azatricyclo
incubation at 35oC.
[5.2.2.02,6]undec-8-ene-3,5-dione and 1-isipropyl-7-
methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione is
described (Scheme 1 and Table I). N-amino imides
Antiviral assay procedures
Antiviral investigations of compounds were per- obtained from imides in reaction with hydrazine
formed in Dipartamento di Scienze e Tecnologie hydrate were used as starting materials (Struga et al.,
Biomediche, Universita di Cagliari.
Compounds were solubilized in DMSO at 200 mM subjected to the reaction with isothiocyanate in order
and then diluted into culture medium. to be transformed into the corresponding thiourea
2007; Struga et al., 2008). N-amino imides were
Cell lines were purchased from American Type derivatives (Scheme 1). Obtained compounds were
Culture Collection (ATCC). The absence of mycoplasma purified by flash chromatography. Elemental analysis
1
contamination was checked periodically by the Hoechst and MS, H NMR spectra confirmed the identity of
staining method. Cell lines supporting the multiplica- the products. The general synthetic pathway and
tion of RNA viruses were the following: CD4+ human structure of the investigated compounds is given in
T-cells containing an integrated HTLV-1 genome (MT- Scheme 1 and Table I.
4).
All obtained compounds were tested in vitro against
a number of bacteria, including Gram-positive cocci,
Gram-negative rods and Candida albicans. Micro-
Cytotoxicity assay
For cytotoxicity evaluations, exponentially growing organisms used in this study have common applica-
cells derived from human haematological tumors tion in the antimicrobial tests for many substances
[CD4+ human T-cells containing an integrated HTLV- like antibiotics, disinfectants and antiseptic drugs or
1 genome (MT-4)] were seeded at an initial density of in research on new antimicrobial agents (Struga et al.,
1 ×
105 cells/mL in 96 well plates in RPMI-1640 medi- 2008; Stefanska et al., 2009). All the compounds were
um supplemented with 10% fetal calf serum (FCS), screened for their antimicrobial activity by the disc
100 units/mL penicillin G and 100
µg/mL strepto- diffusion method (CLSI, 2006a). Compounds showing
mycin. Cell cultures were then incubated at 37oC in a significant activity test were next examined for their
humidified 5% CO2 atmosphere in the absence or minimal inhibitory concentration (MIC) (CLSI, 2006b).
presence of serial dilutions of test compounds. Cell Test results of activity for compounds are summarized
viability was determined after 96 h at 37oC by the 3- in Tables II-V.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT) method (Pauwels et al., 1988).
Preliminary test by disc-diffusion method showed
antimicrobial activity against standard Gram-positive
cocci, therefore the next step was evaluation of com-
pounds’ MIC values for standard and hospital strains.
The research was carried out over 10 standard strains
Antiviral assay
Activity of compounds against Human Immunodefi-
ciency virus type-1 (HIV-1) was based on inhibition of and 20 hospital stains of Staphylococcus epidermidis
virus-induced cytopathogenicity in MT-4 cells acutely used for routine antimicrobial media susceptibility
infected with a multiplicity of infection (m.o.i.) of 0.01. testing. Hospital strains were isolated from different
Briefly, 50
added to each well of flat-bottom microtitre trays con- the Warsaw Medical School Hospitals. Among them,
taining 50 L of RPMI, with or without serial dilu- 10 strains showed methicilin susceptibility (MSSA)
tions of test compounds. Then, 20 L of an HIV-1
µL of RPMI containing 2 ×
104 MT-4 were biological materials of patients hospitalized in one of
µ
µ
suspension containing 100 CCID50 were added. After a
4-day incubation, cell viability was determined by the
MTT method.
Scheme 1. Synthesis of obtained compounds

Synthesis and Microbiological Activity of New Tiourea Derivatives
51
Table I. Structure of investigated compounds
R
R1
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
Table II. Antibacterial and antifungal in vitro activity expressed as diameter of growth inhibitory zone (GIZ, mm) for
tested compounds (applied 400 µg per disc)
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
E. coli ATCC 10538
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
14
17
16
20
18
17
18
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
13
−
−
−
18
−
−
−
−
−
−
−
11
11
12
12
−
12
11
−
11
17
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
16
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
13
−
−
13
−
−
−
−
−
−
−
19
20
20
20
15
18
17
−
18
22
−
−
−
−
−
−
−
−
−
−
−
11
11
11
11
−
12
12
−
−
15
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
19
20
19
20
17
17
18
13
18
20
−
−
−
−
−
−
−
−
−
11
11
−
11
−
−
11
−
−
18
−
−
−
−
−
−
−
19
25
−
−
−
−
−
−
−
−
−
−
−
E. coli ATCC 25922
E. coli NCTC 8196
P. vulgaris NCTC 4635
P. aeruginosa ATCC 5442
P. aeruginosa NCTC 6749
P. aeruginosa ATCC 7853
B. bronchiseptica ATCC 4617
C. albicans ATCC 10231
C. albicans ATCC 90028
C. parapsilosis ATCC 22019
−
−
−
−
−
−
−
−
−
−
−
−
−
−
Lack of the growth inhibition zone.
and 10 strains showed methicilin resistance (MRSA). between structure and biological activity is interest-
MIC value of standard Gram-positive strain as in the ing. Activity depends only on a phenyl subtituent. The
range 400-6.25
phylococcus epidermidis (MSSA) the MIC value chang- starting imide possessed antimicrobial activity like
ed from 400 to 6.25 g/mL and average value was 25 compounds 1,7,8,9-tetramethyl-and 1,7,8,9,10-penta-
g/mL (Table IV). MRSA strains were more resistant methyl-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione
to investigated compounds, the range of MICE value (Struga et al., 2008), its thiourea derivatives with dif-
changed from 400 to 50 g/mL (Table V). Comparison ferent then bromophenyl substituent did not always
µg/mL (Table III). For hospital Sta- type of tricyclic imide’s part is not important. Even if
µ
µ
µ

52
M. Struga et al.
Table III. Activities of obtained compounds against Gram-positive bacteria-minimal inhibitory concentrations (MIC, µg/
mL)
Ciprofloxacin
1b
2a
2b
3a
3b
3c
4b
4c
(5
µg per disc)
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
025
025
025
025
025
025
012.5
400
025
012.5
100
100
100
100
200
100
100
>400>
100
050
050
050
050
025
200
050
025
>400>
050
025
400
>400>
>400>
400
400
400
400
>400>
400
12.50 0>50
06.25 0>50
06.25 0>50
06.25 0>50
06.25 >200
12.50 >100
12.50 0>12.5
500.0 >400
12.50 >200
06.25 0>12.5
12.50
12.50
12.50
12.50
12.50
12.50
12.50
500.0
06.25
06.25
200
200
200
200
200
200
200
>400>
200
200
0.500
0.500
0.500
0.500
0.500
<0.125>
0.500
400.0
200.0
100.0
400
Table IV. Activity of compounds against hospital strains
S. epidermidis susceptive of methicillin (MSSA)-minimal
Table VI. Cytotoxicity and anti-HIV activity of obtained
compounds
inhibitory concentrations (MIC,
µg mL)
aCC50
MT-4
bEC50
Compounds
1b
025
025
025
025
025
025
012.5
400
025
2a
2b
3b
4b
HIV-1
311/08
315/08
316/08
317/08
318/08
340/09
341/09
342/09
343/09
377/09
100
100
100
100
200
100
100
>400>
100
050
050
050
050
025
200
050
025
>400>
050
025
12.50
06.25
06.25
06.25
06.25
12.50
12.50
500.0
12.50
06.25
12.50
12.50
12.50
12.50
12.50
12.50
12.50
500.0
06.25
06.25
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
>100
66
>100.000
>66.000
>56.000
>80.000
>57.000
>58.000
>100.000
>59.000
>55.000
>100.000
>55.000
>61.000
0.002
56
80
57
58
>100
59
55
>100
55
012.5
Table V. Activity of compounds against hospital strains
S. epidermidis resistance of methicillin (MRSA)-minimal
61
37
EFV
inhibitory concentrations (MIC,
µg mL)
aCompound concentration (mM) required to reduce the
viability of mock-infected MT-4 cells by 50%, as deter-
mined by the MTT method.
1b
2a
2b
3b
4b
275/08
277/08
306/08
307/08
309/08
329/08
355/09
356/09
357/09
376/09
50
50
50
50
50
50
50
50
50
50
100
100
400
400
400
100
100
400
400
400
50
50
50
50
50
50
50
50
50
50
>400
>400
>200
>100
>400
>100
>400
>400
>400
>100
50
50
50
50
50
50
50
50
50
50
bCompound concentration (mM) required to achieve 50%
protection of MT-4 cells from the HIV-1 induced cyto-
pathogeneticy, as determined by the MTT method.
Some anti HIV-1 agents as NNRTIs (nucleoside
reverse transcriptase inhibitors) possess thiourea
parts (TIBO, Trovirdine). Consequently all thiourea
derivatives were tested on anti HIV-1 activity.
The synthesized compounds were evaluated for
their cytotoxicity and anti-HIV-1 activity in MT-4
possess antimicrobial activity. Changing bromine atom cells. None of the synthesized compounds showed
of phenyl substituent on F, Cl, I and OCH3 removed activity against HIV-1. The range of cytotoxicity was
antimicrobial activity of tested compounds (Struga et from 100 to 37
al., 2007; Struga et al., 2009a, 2009b). The comparison
µM (Table VI).
of position of bromine atom of a phenyl substituent
showed that the position C-3 favours antimicrobial
activity.
ACKNOWLEDGEMENTS
The authors wish to thank Professor Paolo La Colla,

Synthesis and Microbiological Activity of New Tiourea Derivatives
53
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