Synthesis and Antimicrobial Activity of 4-Chloro-3-Nitrophenylthiourea Derivatives Targeting Bacterial Type II Topoisomerases

Article abstract of DOI:10.1111/cbdd.12723

A series of novel 4-chloro-3-nitrophenylthiourea derivatives were synthesized and evaluated for their antimicrobial, antibiofilm and tuberculostatic activities. Most of compounds exhibited high antibacterial activity against both standard and hospital strains (MIC values 0.5-2 μg/mL), as compared to Ciprofloxacin. Derivatives with 3,4-dichlorophenyl (11) and 3-chloro-4-methylphenyl (13) substituents were the most promising towards Gram-positive pathogens. Both of them exhibited antibiofilm potency and effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Two N-alkylthioureas (20, 21) showed twofold to fourfold increase in in vitro potency against isolates of Mycobacterium tuberculosis, as compared to Isoniazid. An action of 7, 10, 11, 13, 20 and 21 against activity of topoisomerases isolated from Staphylococcus aureus was studied. Synthesized compounds were found as non-genotoxic.

Full text of DOI:10.1111/cbdd.12723

Chem Biol Drug Des 2016; 87: 905–917
Research Article
Synthesis and Antimicrobial Activity of 4-Chloro-3-
Nitrophenylthiourea Derivatives Targeting Bacterial
Type II Topoisomerases
1,
ꢀ2
The type II topoisomerase includes topoisomerase IV and
DNA gyrase that share significant sequence similarity,
however play different functions in DNA replication. Gyrase
introduces the negative supercoiling into DNA, whereas
topoisomerase IV preferentially binds and relaxes positive
supercoils (1,2). Type II topoisomerases possess ATP
binding site that could be the intracellular target for potent
antibacterials (3). Topoisomerase poisons can also trap
cleavage complexes by binding at the enzyme-DNA inter-
face. Usually gyrase and topoisomerase IV inhibitors repre-
sent the class of dual-targeting inhibitors, that is both
block ATP binding and interfere with DNA binding/strand
passage (2). Several urea derivatives with high antistaphy-
lococcal (4–6) activities were described as dual-targeting
ATP-site inhibitors. On the other hand, antimycobacterial
potency of this class of compounds is the result of DNA
gyrase B inhibition (6).
Anna Bielenica *, Karolina Stepien ,
z
3
ꢀ
Agnieszka Napiorkowska , Ewa Augustynowicz-
ꢀ3
4
Kopec , Sylwester Krukowski , Marta
Włodarczyk5 and Marta Struga5
1Chair and Department of Biochemistry, Medical
University, 02-097 Warszawa, Poland
2Department of Pharmaceutical Microbiology, Medical
University, 02-007 Warszawa, Poland
3Department of Microbiology, National Tuberculosis and
Lung Diseases Research Institute, 01-138 Warszawa,
Poland
4Department of Inorganic and Analytical Chemistry, Faculty
of Pharmacy, Medical University, 02-097 Warszawa,
Poland
5Department of Pharmacogenomics, Faculty of Pharmacy,
Medical University, 02-097 Warszawa, Poland
*Corresponding author: Anna Bielenica,
abielenica@wum.edu.pl
Bacteria growing in slime-enclosed aggregates known as
biofilms are dominant in chronic bacterial infections (7). In
contrast to planktonic forms of bacteria responsible mainly
for acute infections, biofilm-based diseases are extremely
resistant to antibiotics and other conventional antimicrobial
agents (8,9). What is more, they are insensitive to host
immune response, allowing them to persist and promote
continued infection despite intensive antibiotic therapy
(10). One of the most common aetiologies of device-
related infections is staphylococci, either coagulase-nega-
tive strains or Staphylococcus aureus (11). Up to now, only
several chemical scaffolds have been described that can
inhibit or disperse biofilm of these type of strains (12,13).
Their action is based either on inhibition of intercellular
communication (quorum sensing) and signalling pathways
(14,15) or enzymatic degradation of the integrity of extra-
cellular matrix required for the biofilm formation (16).
A series of novel 4-chloro-3-nitrophenylthiourea deriva-
tives were synthesized and evaluated for their antimi-
crobial, antibiofilm and tuberculostatic activities. Most
of compounds exhibited high antibacterial activity
against both standard and hospital strains (MIC values
0.5–2 lg/mL), as compared to Ciprofloxacin. Derivatives
with 3,4-dichlorophenyl (11) and 3-chloro-4-methylphe-
nyl (13) substituents were the most promising towards
Gram-positive pathogens. Both of them exhibited anti-
biofilm potency and effectively inhibited the formation of
biofilms of methicillin-resistant and standard strains of
Staphylococcus epidermidis. Two N-alkylthioureas (20,
21) showed twofold to fourfold increase in in vitro
potency against isolates of Mycobacterium tuberculo-
sis, as compared to Isoniazid. An action of 7, 10, 11, 13,
20 and 21 against activity of topoisomerases isolated
from Staphylococcus aureus was studied. Synthesized
compounds were found as non-genotoxic.
Antimicrobial activity of thiourea derivatives has been the
subject of numerous investigations. Structure-activity rela-
tionship (SAR) study of both N-phenylurea and thiourea
derivatives showed that these containing electron-with-
drawing substituents exert higher activity as compared to
other analogues (17,18). Moreover, several thiourea-con-
taining compounds exhibit potent antimycobacterial
(19,20), antiviral (21), antimalarial (22) and analgesic
properties (23).
Key words: antimicrobial activity, antitubercular activity,
biofilm, thiourea derivatives, topoisomerase
Received 27 November 2015, revised 7 January 2016 and
accepted for publication 7 January 2016
Bacterial topoisomerases are an attractive target for the
discovery and development of new antibacterial agents.
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12723
905

Bielenica et al.
Taking into consideration significant antimicrobial activity of
thioureas, to continue our work on the evaluation of the
bioactivity of thiourea-containing compounds (24,25), we
designed and synthesized a series of derivatives combin-
ing 4-chloro-3-nitrophenylthiourea moiety and various aro-
matic substituents.
1338.7, 1255.7, 1126.8, 1047.6, 869.4, 695.2. 1H NMR
(300 MHz, DMSO) d: 10.26 (s, 2H, NH), 8.33 (d, 1H,
J = 2.4 Hz, H-2), 7.80 (dd, 1H, J1 = 2.7 Hz, J2 = 2.4 Hz,
H-6), 7.72 (m, 1H, J = 9.0 Hz, H-5), 7.65 (t, 1H,
J = 1.2 Hz, H-5’), 7.41–7.35 (m, 2H, H-2’, H-4’), 7.26–
7.19 (m, 1H, H6’). 13C NMR (75.4 MHz, DMSO) d: 179.81
(C=S), 146.89, 140.43, 139.51, 132.69, 131.36, 130.21,
128.51, 124.63, 123.34, 122.29, 119.92, 119.70. ESI MS:
m/z = 340.0 [M-2H]- (100%).
Experimental
Chemistry
All reagents and solvents were purchased from Alfa Aesar
(Lancashire, UK), Sigma Aldrich (Saint Louis, MO, USA) or
POCh (Polskie Odczynniki Chemiczne). The IR spectra
were obtained on Perkin Elmer Spectrum 1000 spectrom-
eter (Perkin Elmer, Waltham, MA, USA). The NMR spectra
were recorded on Varian VNMRS 300 Oxford NMR spec-
trometer (Varian, Palo Alto, CA, USA), using TMS as the
internal reference. Mass spectral ESI measurements were
carried out on Waters ZQ Micro-mass instruments
(Waters, Milford, MA, USA) with quadruple mass analyser,
at a declustering potential of 40–60 V. Flash chromatogra-
phy was performed on Merck silica gel 60 (200–400 mesh)
(Merck, Darmstadt, Germany) using chloroform eluent.
1-(4-chloro-3-nitrophenyl)-3-(4-chlorophenyl)
thiourea (3)
Yield 64%, m.p. 176–178 °C. FT-IR (KBr, cm-1): 3318.5,
3177.0, 3083.4, 1589.0, 1533.7, 1481.7, 1406.3, 1357.5,
1240.5, 1191.5, 1160.5, 1088.5, 866.0, 709.5. 1H NMR
(300 MHz, DMSO) d: 10.21 (s, 2H, NH), 8.33 (d, 1H,
J = 2.4 Hz, H-2), 7.80 (dd, 1H, J1 = J2 = 2.4 Hz, H-6),
7.71 (d, 1H, J = 8.7 Hz, H-5), 7.50 (d, 2H, J = 9.0 Hz,
H3’, H-5’), 7.43–7.40 (m, 2H, H2’, H-6’). 13C NMR
(75.4 MHz, DMSO) d: 179.79 (C=S), 146.85, 139.61,
137.83, 131.31, 128.94, 128.51, 128.42, 125.59, 119.80,
119.57. ESI MS: m/z = 340.0 [M-2H]- (100%).
1-(2-bromophenyl)-3-(4-chloro-3-nitrophenyl)
thiourea (4)
General procedure for the synthesis of N-
substituted (4-chloro-3-nitrophenyl)thiourea
derivatives (1‒21)
A solution of 4-chloro-3-nitroaniline (0.0029 mol) in dry ace-
tonitrile (10 mL) was treated with appropriate isothiocyanate
(0.0029 mol), and the mixture was stirred at room tempera-
ture for 12 h. Then, the solvent was evaporated and the
solid residue was either crystallized from acetonitrile (chloro-
form) or purified by column chromatography (chloroform).
Yield 63%, m.p. 147–148 °C. FT-IR (KBr, cm-1): 3205.8,
3166.4, 2991.9, 1582.6, 1533.5, 1475.7, 1403.2, 1323.5,
1
1296.2, 1125.5, 1043.6, 879.5, 725.2. H NMR (300 MHz,
DMSO) d: 10.30 (s, 1H, NH), 9.81 (s, 1H, NH), 8.43 (d, 1H,
J = 2.4 Hz, H-2), 7.88 (dd, 1H, J1 = J2 = 2.7 Hz, H-6),
7.73–7.68 (m, 2H, H-5, H-3’), 7.52 (dd, 1H,
J1 = J2 = 1.5 Hz, H-5’), 7.44–7.39 (m, 1H, H-6’), 7.27–
7.21 (m, 1H, H-4’). 13C NMR (75.4 MHz, DMSO) d: 180.50
(C=S), 146.81, 139.52, 137.32, 132.76, 131.37, 130.29,
128.53, 128.38, 128.06, 121.56, 119.74, 119.65. ESI MS:
m/z = 384.9 [M-2H]- (100%).
The structures of compounds along with atom numbering
are presented in Supporting information section (Table S1).
1-(4-chloro -3-nitrophenyl)-3-(2-chlorophenyl)
thiourea (1)
1-(4-chloro-3-nitrophenyl)-3-(3-bromophenyl)
thiourea (5)
Yield 68%, m.p. 146–148 °C. FT-IR (KBr, cm-1): 3198.3,
3165.3, 2996.2, 1585.6, 1533.2, 1475.5, 1401.4, 1317.2,
1262.2, 1122.8, 1042.5, 879.2, 722.5. H NMR (300 MHz,
Yield 45%, m.p. 136–138 °C. FT-IR (KBr, cm-1): 3248.0,
3086.4, 3012.5, 1585.5, 1548.9, 1475.7, 1403.9, 1335.6,
1254.9, 1128.3, 1049.4, 871.3, 700.0. H NMR (300 MHz,
1
1
DMSO) d: 10.31 (s, 1H, NH), 9.84 (s, 1H, NH), 8.41 (d, 1H,
J = 2.7 Hz, H-2), 7.84 (dd, 1H, J1 = J2 = 2.7 Hz, H-6),
7.78–7.70 (m, 1H, H-5), 7.60–7.53 (m, 2H, H-3’, H-5’),
7.40–7.28 (m, 2H, H-4’, H-6’). 13C NMR (75.4 MHz, DMSO)
d: 180.57 (C=S), 146.82, 139.54, 135.85, 131.37, 130.44,
129.89, 129.63, 128.37, 128.13, 127.45, 119.73, 119.65.
ESI MS: m/z = 340.0 [M-2H]- (100%).
DMSO) d: 10.28–10.24 (m, 2H, NH), 8.32 (d, 1H, J = 2.4 Hz,
H-2), 7.81–7.76 (m, 2H, H-6, H-4’), 7.72 (d, 1H, J = 8.7 Hz,
H-5), 7.44 (dt, 1H, J1 = 1.95 Hz, J2 = 1.8 Hz, H-5’), 7.38–
7.34 (m, 1H, H-2’), 7.30 (d, 1H, J = 7.5 Hz, H-6’). 13C NMR
(75.4 MHz, DMSO) d: 179.82 (C=S), 146.89, 140.57, 139.50,
131.36, 130.48, 128.51, 127.52, 126.20, 122.74, 121.03,
119.91, 119.68. ESI MS: m/z = 384.9 [M-2H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-(3-chlorophenyl)
thiourea (2)
1-(4-bromophenyl)-3-(4-chloro-3-nitrophenyl)
thiourea (6)
Yield 30%, m.p. 140–142 °C. FT-IR (KBr, cm-1): 3247.0,
3189.7, 3089.0, 3031.3, 1587.4, 1549.5, 1476.5, 1404.5,
Yield 52%, m.p. 182–184 °C. FT-IR (KBr, cm-1): 3319.9,
3185.5, 3074.8, 2978.6, 1585.9, 1534.2, 1479.3, 1403.8,
906
Chem Biol Drug Des 2016; 87: 905–917

Antibacterial Profile of Disubstituted Thioureas
1
1355.4, 1296.4, 1191.8, 1157.2, 1068.2, 867.4, 710.0. H
1249.3, 1126.6, 1051.8, 872.6, 703.1. 1H NMR
(300 MHz, DMSO) d: 10.24–10.19 (m, 2H, NH), 8.31 (d,
1H, J = 2.4 Hz, H-2), 7.79 (dd, 1H, J1 = 2.4 Hz,
J2 = 2.1 Hz, H-6), 7.75–7.70 (m, 2H, H-5, H-5’), 7.42–
7.40 (m, 2H, H-2’, H-6’). 13C NMR (75.4 MHz, DMSO) d:
180.17 (C=S), 154.18, 146.92, 139.47, 136.06, 131.37,
128.67, 126.28, 125.04, 120.04, 119.76, 118.93, 116.67.
ESI MS: m/z = 358.0 [M-2H]- (100%).
NMR (300 MHz, DMSO) d: 10.21 (s, 2H, NH), 8.32 (d, 1H,
J = 2.4 Hz, H-2), 7.79 (dd, 1H, J1 = 2.7 Hz, J2 = 2.4 Hz,
H-6), 7.71 (d, 1H, J = 8.7 Hz, H-5), 7.57–7.50 (m, 2H,
H3’, H-5’), 7.46–7.42 (m, 2H, H2’, H-6’). 13C NMR
(75.4 MHz, DMSO) d: 179.71 (C=S), 146.85, 139.59,
138.26, 131.43, 131.31, 128.41, 125.87, 119.79, 119.58,
117.10. ESI MS: m/z = 384.9 [M-2H]- (100%).
1-(4-chloro -3-nitrophenyl)-3-(2-fluorophenyl)
thiourea (7)
1-(4-chloro-3-nitrophenyl)-3-(3,4-dichlorophenyl)
thiourea (11)
Yield 60%, m.p. 151–153 °C. FT-IR (KBr, cm-1): 3323.6,
3152.7, 3089.6, 2985.4, 1593.0, 1543.4, 1483.4, 1405.3,
1340.5, 1250.0, 1134.2, 1105.3, 1046.6, 864.5, 709.7. H
Yield 85%, m.p. 173–175 °C. FT-IR (KBr, cm-1): 3242.4,
3177.5, 3080.9, 3038.4, 1581.0, 1545.2, 1470.5, 1335.4,
1255.6, 1129.4, 1048.0, 868.4, 702.8. 1H NMR
(300 MHz, DMSO) d: 10.31 (s, 2H, NH), 8.31 (d, 1H,
J = 2.4 Hz, H-2), 7.84 (d, 1H, J = 2.4 Hz, H-5’), 7.80 (dd,
1H, J1 = J2 = 2.4 Hz, H-6), 7.72 (d, 1H, J = 8.7 Hz, H-5),
7.61 (m, 1H, J = 8.7 Hz, H-2’), 7.44 (dd, 1H, J1 = 2.1 Hz,
J2 = 2.7 Hz, H-6’). 13C NMR (75.4 MHz, DMSO) d:
179.88 (C=S), 146.93, 139.38, 139.12, 131.41, 130.65,
130.38, 128.62, 126.70, 125.27, 123.97, 120.05, 119.86.
ESI MS: m/z = 373.9 [M-3H]- (100%).
1
NMR (300 MHz, DMSO) d: 10.29 (s, 1H, NH), 9.87 (s, 1H,
NH), 8.39 (d, 1H, J = 2.4 Hz, H-2), 7.84 (dd, 1H,
J1 = 2.4 Hz, J2 = 2.7 Hz, H-6), 7.71 (d, 1H, J = 8.7 Hz,
H-5), 7.58–7.53 (m, 1H, H-3’), 7.33–7.18 (m, 3H, H-4’,
H-5’, H-6’). 13C NMR (75.4 MHz, DMSO) d: 180.78 (C=S),
156.42, 146.82, 139.61, 131.35, 128.72, 128.35, 127.92,
126.39, 124.35, 119.69, 119.63, 115.93. ESI MS: m/
z = 324.0 [M-H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-(3-fluorophenyl)
thiourea (8)
1-(4-chloro-3-nitrophenyl)-3-(2,4-dichlorophenyl)
thiourea (12)
Yield 45%, m.p. 162–164 °C. FT-IR (KBr, cm-1): 3250.5,
3152.2, 3009.4, 1593.4, 1549.7, 1486.1, 1404.5, 1337.7,
1256.5, 1141.4, 1131.5, 1049.5, 870.1, 705.7. 1H NMR
(300 MHz, DMSO) d: 10.28–10.26 (m, 2H, NH), 8.34 (d, 1H,
J = 2.4 Hz, H-2), 7.80 (dd, 1H, J1 = J2 = 2.4 Hz, H-6), 7.72
(d, 1H, J = 8.4 Hz, H-5), 7.48 (dt, 1H, J1 = 2.1 Hz,
J2 = 2.25 Hz, H-5’), 7.43–7.36 (m, 1H, H-4’), 7.27–7.24 (m,
1H, H-2’), 7.03–6.97 (m, 1H, H-6’). 13C NMR (75.4 MHz,
DMSO) d: 179.70 (C=S), 161.72, 146.89, 140.66, 139.53,
131.34, 130.19, 128.53, 119.93, 119.68, 119.37, 111.39,
110.40. ESI MS: m/z = 324.0 [M-H]- (100%).
Yield 53%, m.p. 154–155 °C. FT-IR (KBr, cm-1): 3361.7,
3338.3, 3100.7, 1576.8, 1512.5, 1475.6, 1322.5, 1251.9,
1144.6, 1127.9, 1054.0, 822.5, 689.3. 1H NMR
(300 MHz, DMSO) d: 10.34 (br. s, 1H, NH), 9.87 (br. s,
1H, NH), 8.39 (d, 1H, J = 2.4 Hz, H-2), 7.84 (dd, 1H,
J1 = 2.7 Hz, J2 = 2.4 Hz, H-6), 7.73–7.71 (m, 2H, H-5, H-
3’), 7.58 (d, J = 8.4 Hz, 1H, H-5’), 7.45 (dd, 1H,
J1 = J2 = 2.4 Hz, H-6’). 13C NMR (75.4 MHz, DMSO) d:
180.57 (C=S), 146.83, 139.39, 135.14, 131.62, 131.53,
131.40, 131.09, 129.11, 128.45, 127.61, 119.83. ESI MS:
m/z = 373.9 [M-3H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-(4-fluorophenyl)
thiourea (9)
1-(3-chloro-4-methylphenyl)-3-(4-chloro-3-
nitrophenyl)thiourea (13)
Yield 45%, m.p. 172–173 °C. FT-IR (KBr, cm-1): 3319.7,
3181.4, 2996.7, 1587.3, 1536.4, 1504.5, 1404.4, 1352.3,
1310.0, 1152.6, 1138.2, 1043.1, 866.5, 703.3. 1H NMR
(300 MHz, DMSO) d: 10.12 (s, 2H, NH), 8.34 (d, 1H,
J = 2.1 Hz, H-2), 7.80 (dd, 1H, J1 = J2 = 2.4 Hz, H-6),
7.71 (d, 1H, J = 8.7 Hz, H-5), 7.48–7.43 (m, 2H, H-3’,
H-5’), 7.20 (t, 2H, J = 8.7 Hz, H-2’, H-6’). 13C NMR
(75.4 MHz, DMSO) d: 180.07 (C=S), 159.48, 146.85,
139.73, 135.12, 131.28, 128.42, 126.50, 119.79, 119.45,
115.31. ESI MS: m/z = 324.0 [M-H]- (100%).
Yield 65%, m.p. 239–240 °C. FT-IR (KBr, cm-1): 3363.1,
3180.0, 3108.5, 1601.0, 1536.8, 1483.5, 1394.8, 1346.9,
1298.5, 1130.0, 1045.2, 875.6, 706.6. 1H NMR
(300 MHz, CDCl3) d: 9.29 (br. s, 1H, NH), 8.99 (br. s, 1H,
NH), 8.29–8.29 (m, 1H, H-2), 7.68–7.61 (m, 3H, H-5, H-6,
H-5’), 7.28–7.20 (m, 2H, H-2’, H-6’), 2.27 (s, 3H, H-4’a).
13C NMR (75.4 MHz, DMSO) d: 180.17 (C=S), 147.46,
139.70, 138.25, 133.08, 131.77, 131.18, 128.89, 123.21,
118.59, 117.41, 116.80, 114.40, 18.80. ESI MS: m/
z = 354.0 [M-2H]- (100%).
1-(3-chloro-4-fluorophenyl)-3-(4-chloro-3-
nitrophenyl)thiourea (10)
1-(5-chloro-2-methylphenyl)-3-(4-chloro-3-
nitrophenyl)thiourea (14)
Yield 70%, m.p. 142–144 °C. FT-IR (KBr, cm-1): 3240.3,
3146.2, 3026.8, 1593.6, 1551.5, 1480.6, 1402.8, 1337.5,
Yield 61%, m.p. 148–150 °C. FT-IR (KBr, cm-1): 3220.4,
3115.5, 3076.9, 2971.5, 1582.5, 1537.4, 1479.6, 1445.6,
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Bielenica et al.
1
1
1404.6, 1334.0, 1256.2, 1126.3, 1047.5, 862.4, 709.8. H
NMR (300 MHz, DMSO) d: 10.17 (s, 1H, NH), 9.77 (s, 1H,
NH), 8.36 (d, 1H, J = 2.4 Hz, H-2), 7.83 (dd, 1H,
J1 = J2 = 2.4 Hz, H-6), 7.71 (d, 1H, J = 9.0 Hz, H-5),
7.38–7.36 (m, 1H, H-3’), 7.32–7.29 (m, 1H, H-4’), 7.28–
7.23 (m, 1H, H-6’), 2.21 (s, 3H, H-2’a). 13C NMR
(75.4 MHz, DMSO) d: 180.48 (C=S), 146.83, 139.63,
138.60, 133.95, 131.90, 131.30, 129.89, 128.54, 127.60,
126.64, 119.94, 119.61, 17.25. ESI MS: m/z = 354.0 [M-
2H]- (100%).
1334.6, 1250.1, 1178.9, 1114.2, 1046.3, 849.5, 708.6. H
NMR (300 MHz, DMSO) d: 10.66–10.54 (m, 2H, NH), 8.33
(dd, 1H, J1 = J2 = 2.4 Hz, H-2), 8.26–8.21 (m, 2H, H-3’,
H-5’), 7.87–7.73 (m, 4H, H-5, H-6, H-2’, H-6’). 13C NMR
(75.4 MHz, DMSO) d: 179.62 (C=S), 146.70, 145.30,
142.53, 138.95, 131.23, 128.44, 124.17, 121.90, 119.88,
119.66. ESI MS: m/z = 351.0 [M-H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-(4-cyanophenyl)
thiourea (19)
Yield 57%, m.p. 173–175 °C. FT-IR (KBr, cm-1): 3202.2,
3067.2, 2996.8, 2237.6, 1591.2, 1540.5, 1483.2, 1363.4,
1309.3, 1193.5, 1141.2, 1045.2, 871.5, 689.9. 1H NMR
(300 MHz, DMSO) d: 10.47 (s, 2H, NH), 8.33 (d, 1H,
J = 2.4 Hz, H-2), 7.81–7.71 (m, 6H, H-5, H-6, H-2’, H-3’,
H-5’, H-6’). 13C NMR (75.4 MHz, DMSO) d: 179.35 (C=S),
146.94, 143.71, 132.82, 131.41, 128.43, 122.69, 119.80,
119.69, 118.95, 105.78. ESI MS: m/z = 331.0 [M-H]-
(100%).
1-(4-chloro-3-nitrophenyl)-3-[3-(trifluoromethyl)
phenyl]thiourea (15)
Yield 42%, m.p. 147–149 °C. FT-IR (KBr, cm-1): 3255.6,
3160.6, 3098.8, 3047.9, 1591.2, 1557.5, 1481.1, 1407.9,
1
1331.5, 1259.4, 1173.5, 1122.8, 1071.6, 882.4, 698.2. H
NMR (300 MHz, DMSO) d: 10.35 (s, 2H, NH), 8.32 (d, 1H,
J = 2.4 Hz, H-2), 7.91 (m, 1H, H-5’), 7.79 (dd, 1H,
J1 = 2.7 Hz, J2 = 2.4 Hz, H-6), 7.76–7.71 (m, 2H, H-5, H-
4’), 7.60–7.52 (m, 2H, H-2’, H-6’). 13C NMR (75.4 MHz,
DMSO) d: 180.03 (C=S), 146.97, 139.88, 139.40, 131.43,
129.71, 129.11, 128.53, 127.68, 123.87, 121.20, 120.14,
119.94, 119.77. ESI MS: m/z = 374.0 [M-H]- (100%).
1-benzyl-3-(4-chloro-3-nitrophenyl)thiourea (20)
Yield 55%, m.p. 150–152 °C. FT-IR (KBr, cm-1): 3228.2,
3184.5, 3028.5, 2955.9, 2907.8, 1583.3, 1543.5, 1480.4,
1454.7, 1409.4, 1339.2, 1253.1, 1155.6, 1129.4, 1048.5,
877.3, 695.8. 1H NMR (300 MHz, DMSO) d: 10.02 (br. s,
1H, NH), 8.55 (br. s, 1H, NH), 8.42 (d, 1H, J = 2.1 Hz, H-
2), 7.76 (dd, 1H, J1 = J2 = 2.7 Hz, H-6), 7.68 (d, 1H,
J = 8.7 Hz, H-5), 7.35 (d, 4H, J = 4.5 Hz, H-3’, H-4’, H-
6’, H-7’), 7.30–7.24 (m, 1H, H-5’), 4.76 (d, 2H,
J = 5.4 Hz, H-1’). 13C NMR (75.4 MHz, DMSO) d: 180.76
(C=S), 146.84, 139.79, 138.35, 131.35, 128.32, 127.49,
127.36, 127.03, 118.80, 118.62, 47.13. ESI MS: m/
z = 320.0 [M-H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-[4-(trifluoromethyl)
phenyl]thiourea (16)
Yield 40%, m.p. 172–173 °C. FT-IR (KBr, cm-1): 3246.5,
3203.4, 3094.1, 3039.2, 1593.6, 1552.2, 1478.5, 1404.9,
1
1329.6, 1255.6, 1162.2, 1114.5, 1053.7, 868.6, 684.6. H
NMR (300 MHz, DMSO) d: 10.41 (br. s, 2H, NH), 8.34 (d,
1H, J = 2.7 Hz, H-2), 7.81 (dd, 1H, J1 = 2.7 Hz,
J2 = 2.4 Hz, H-6), 7.75 (m, 1H, H-5), 7.72 (m, 4H, H-2’,
H-3’, H-5’, H-6’). 13C NMR (75.4 MHz, DMSO) d: 179.80
(C=S), 146.92, 142.76, 139.44, 131.41, 129.64, 128.48,
125.74, 124.50, 124.26, 123.27, 119.87, 119.79. ESI MS:
m/z = 374.0 [M-H]- (100%).
1-(4-chloro-3-nitrophenyl)-3-(1-phenylethyl)
thiourea (21)
Yield 30%, m.p. 145–147 °C. FT-IR (KBr, cm-1): 3406.5,
3215.6, 3092.9, 3033.4, 1592.4, 1543.5, 1480.3, 1448.2,
1409.4, 1345.6, 1306.3, 1126.5, 1051.7, 871.2, 695.2. H
1-(4-chloro-3-nitrophenyl)-3-(4-iodophenyl)thiourea
(17)
1
Yield 40%, m.p. 184–186 °C. FT-IR (KBr, cm-1): 3363.1,
3317.5, 3177.5, 2980.6, 1581.8, 1537.2, 1478.9, 1400.7,
1353.5, 1299.4, 1196.8, 1147.8, 1053.6, 869.2, 712.4. H
NMR (300 MHz, DMSO) d: 10.20 (s, 2H, NH), 8.33 (d, 1H,
J = 2.7 Hz, H-2), 7.80 (dd, 1H, J1 = J2 = 2.7 Hz, H-6),
7.72–7.68 (m, 3H, H-5, H-3’, H-5’), 7.32–7.28 (m, 2H, H-
2’, H-6’). 13C NMR (75.4 MHz, DMSO) d: 179.59 (C=S),
146.85, 139.62, 138.75, 137.30, 131.31, 128.42, 125.97,
119.80, 119.56, 89.40. ESI MS: m/z = 431.9 [M-2H]-
(100%).
NMR (300 MHz, DMSO) d: 9.83 (br. s, 1H, NH), 8.56 (d,
1H, J = 7.2 Hz, H-2), 8.42 (br. s, 1H, NH), 7.74 (dd, 1H,
J1 = 2.4 Hz, J2 = 2.7 Hz, H-6), 7.66 (d, 1H, J = 8.7 Hz,
H-5), 7.39–7.32 (m, 3H, H-3’, H-5’, H-7’), 7.30–7.23 (m,
2H, H-4’, H-6’), 5.92 (br.s, 1H, H-1’), 1.48 (d, 3H,
J = 6.9 Hz, H-1’a). 13C NMR (75.4 MHz, DMSO) d:
179.59 (C=S), 146.78, 143.36, 139.86, 131.30, 128.34,
126.93, 126.23, 118.56, 118.36, 108.98, 52.70, 21.74.
ESI MS: m/z = 334.0 [M-H]- (100%).
1
In vitro evaluation of antimicrobial activity
1-(4-chloro-3-nitrophenyl)-3-(4-nitrophenyl)
thiourea (18)
Yield 59%, m.p. 160–162 °C. FT-IR (KBr, cm-1): 3329.9,
3239.2, 3084.8, 3025.0, 1587.6, 1533.7, 1477.4, 1402.6,
The antimicrobial activity of the compounds was tested on
Gram-positive bacteria (Staphylococcus aureus NCTC
4163, Staphylococcus aureus ATCC 25923, Staphylococcus
aureus ATCC 6538, Staphylococcus aureus ATCC 29213,
908
Chem Biol Drug Des 2016; 87: 905–917

Antibacterial Profile of Disubstituted Thioureas
Staphylococcus epidermidis ATCC 12228, Staphylococcus
epidermidis ATCC 35984, Enterococcus faecalis ATCC
29212, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC
11778, Micrococcus luteus ATCC 9341, Micrococcus
luteus ATCC 10240), Gram-negative rods (Escherichia coli
ATCC 10538, Escherichia coli ATCC 25922, Escherichia
coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseu-
domonas aeruginosa ATCC 15442, Pseudomonas aerugi-
nosa NCTC 6749, Pseudomonas aeruginosa ATCC 27863,
Bordetella bronchiseptica ATCC 4617) and yeasts (Candida
albicans ATCC 10231, Candida albicans ATCC 90028,
Candida parapsilosis ATCC 22019). Other micro-organisms
used were obtained from the collection of the Department
of Pharmaceutical Microbiology, Medical University of War-
saw, Poland.
14 days old cultures of slowly growing strains. Solutions of
compounds in DMSO were tested. Stock solutions con-
tained 10 mg of compounds in 1 mL. Dilutions (in geomet-
ric progression) were prepared in Youmans’ medium. The
medium containing no investigated substances and con-
taining Isoniazid (INH) as the reference drug were used for
comparison. Incubation was performed at 37 °C. The MIC
values were determined as minimum concentration inhibit-
ing the growth of tested tuberculous strains in relation to
the probe with no tested compound. The influence of the
compound on the growth of bacteria at a certain concen-
tration 3.1, 6.25, 12.5, 25, 50 and 100 lg/mL was evalu-
ated.
Genotoxicity studies
Antibacterial activity was examined by the disc-diffusion
method under standard conditions using Mueller-Hinton II
agar medium (Becton Dickinson and Co, Sparks, MD,
USA) according to CLSI (previously NCCLS) guidelines
(26). Antifungal activities were assessed using Mueller-Hin-
ton agar +2% glucose and 0.5 lg/mL Methylene Blue Dye
Medium (27). Sterile filter paper discs (9 mm diameter,
Whatman No 3 chromatography paper) were dripped with
tested compound solutions (in DMSO) to load 400 lg of a
given compound per disc. Dry discs were placed on the
surface of appropriate agar medium. The results (diameter
of the growth inhibition zone) were read after 18 h of incu-
bation at 35 °C. Minimal inhibitory concentration (MIC) was
tested by the twofold serial microdilution method (in 96-
well microtitre plates) using Mueller-Hinton Broth medium
(Beckton Dickinson) for bacteria or RPMI-1640 medium for
Candida species according to CLSI guidelines (28,29). The
stock solution of tested agent was prepared in DMSO and
diluted in sterile water. Concentrations of tested agents
ranged from 0.125 to 512 lg/mL. The final inoculum of all
studied micro-organisms was 105 CFU/mLÀ1 (colony-
forming units per mL). Minimal inhibitory concentrations
(the lowest concentration of an tested agent that prevents
visible growth of a micro-organism) were read after 18 h
(bacteria) or 24 h (yeasts) of incubation at 35 °C.
DNA-damaging activity of compounds was tested by rec-
assay using two genetically modified Bacillus subtilis
strains: M45 (recÀ) and H17 (rec+) (32,33). Tested com-
pounds were dissolved in DMSO, and 10 lL of each
solution was dripped onto sterile cotton discs (Rotilabo
Carl Roth GmbH & Co. KG Karlsruhe, Germany) to load
256 lg of a given compound per 9 mm disc. Discs were
placed on the surface Nutrient agar plates (Difco, Detroit,
MI, USA) inoculated with 100 lL of bacterial overnight
culture and incubated 24 h at 35 °C. After incubation, the
growth inhibition zones were measured. 4-Nitroquinoline
N-oxide (NOQ) was used as reference genotoxin (concen-
tration 2 lg per disc). Results of the genotoxicity test
were estimated after 18 h of incubation at 35 °C by com-
paring the diameter of the inhibition zone on the B. sub-
tilis M45 (recÀ) strain with that on the B. subtilis H17
(rec+) strain.
Inhibition of bacterial topoisomerase:
Decatenation assay
The assay was performed using S. aureus topoisomerase
IV (topoIV) decantation kit (Inspiralis Ltd., Norwich, UK).
Kinetoplast DNA (kDNA) was the substrate for topoIV. 1 U
of topoIV decatenated 200 ng of kDNA in the dedicated
decantation assay buffer supplied by the manufacturer.
Enzyme activity was detected by incubation for 30 min at
37 °C in a total reaction volume of 30 lL in the presence
of tested compounds and the reference Ciprofloxacin at
concentrations 0.25, 0.5, 1, 2, 4, 8 and 32 lg/mL. The
reactions were terminated by adding of an equal volume
of STEB buffer (40% sucrose, 100 mM Tris-HCl pH 8,
1 mM EDTA, 0.5 mg/mL bromophenol blue), followed by
extraction with 1 volume of chloroform/isoamyl alcohol
(24:1).
Antitubercular activity
The synthesized compounds were examined in vitro for
their tuberculostatic activity against the M. tuberculosis
H37Rv strain (ATCC 25618) and two ‘wild-type’ strains iso-
lated from patients with tuberculosis: one (Spec. 210)
resistant to p-aminosalicylic acid (PAS), isonicotinic acid
hydrazide (Isoniazid, INH), etambutol (ETB) and rifampicin
(RMP) and the another (Spec. 192) fully sensitive to the
administrated tuberculostatics, as well as atypical strains
(M. bovis, M.scrofulaceum, M. fortuitum, M. avium,
M. kansasii, M. xenopi, M. intracellulare). Investigations
were performed by a classical test-tube method of suc-
cessive dilution in Youmans’ modification of the Proskauer
and Beck liquid medium containing 10% of bovine serum
(30,31). Bacterial suspensions were prepared from
Then, 20 lL of the aqueous phase of each sample was
loaded onto a 1% agarose gel. Electrophoresis was con-
ducted in Tris-acetate-EDTA buffer for 1 h at 120 V. Gels
were stained with ethidium bromide and visualized under
UV light in a transilluminator (ChemiDoc MP, Bio-Rad, Her-
cules, CA, USA).
Chem Biol Drug Des 2016; 87: 905–917
909

Bielenica et al.
The assay was also performed using S. aureus Gyrase
Supercoiling Assay Kit (Inspiralis). Supercoiled pBR322
plasmid DNA (500 ng) was incubated with 1 unit of gyrase
in the 19 assay buffer in the presence of varying concen-
trations of the tested compounds. The reaction was car-
ried out at 37 °C for 1 h and then terminated by the
addition of equal volume of STOP buffer (40% sucrose,
100 mM Tris-HCL pH 8, 1 mM EDTA and 0.5 mg/mL bro-
mophenol blue) and chloroform/izoamyl alcohol. Samples
were vortexed, centrifuged and run through a 1% agarose
gel in TAE buffer for 3 h at 50V. Gel was stained with
ethidium bromide and visualized under UV. The IC50 val-
ues of the studied compounds were determined by
plotting the results obtained from the densitometric analy-
ses of the gel images using IMAGE LAB software (Bio-Rad).
Means and SEM were calculated using STATISTICA 10
software (StatSoft, Cracow, Poland).
(high biofilm-producing) control, respectively. Ciprofloxacin
was used as the reference antimicrobial compound;
its final concentration ranged from
½ to 8 9 MIC
(0.125–512 lg/mL). The initial steps for the preparation of
bacterial strains are the same as for biofilm inhibitory
assay. Tested strains were cultured on microplates and
then incubated at 37 °C, 24 h for biofilm development
(without shaking). After incubation, the supernatant con-
taining non-adherent cells was removed, and 200 lL of a
solution of a tested compound was re-filed into the wells
in the microtitre plate, and the whole was incubated for
24 h at 37 °C. Further procedure corresponded to that
described for biofilm inhibitory assay.
Results and Discussion
Chemistry
In the present study, new asymmetric 1,3-disubstituted
thioureas 1-21 were synthesized from commercially avail-
able 4-chloro-3-nitroaniline and variety of isothiocyanates
(Scheme 1, Table 1). Reactions were carried out in anhy-
drous acetonitrile at room temperature. The structures of
compounds were determined using different spectroscopic
methods (1H NMR, 13C NMR, MS and IR).
Biofilm inhibitory and biofilm eradication assay
Four hospital isolates of methicillin-resistant and two stan-
dard strains of Staphylococcus epidermidis (ATCC 12228,
ATCC 35984) were cultured overnight in Tryptone Soy
Broth supplemented with 0.25% glucose. The solution of
tested compounds in TSB-glucose medium was mixed
(1:1) with the bacterial inoculums (106 CFU/mL) in sterile
96-well polystyrene microtitre plates (Karell – Medlab, Italy)
and incubated at 37 °C for 24 h. The final concentra-
tions of tested compounds ranged from 1/2 to 8 9 MIC
(0.5–512 lg/mL).
Biological studies
In vitro antimicrobial activity
All the newly synthesized compounds 1-21 were evaluated
for their in vitro antimicrobial activity against a number of
bacteria and fungi. The preliminary screening by the disc-
diffusion method (26,27) was aimed to select derivatives
with significant antimicrobial properties. As all of them
exhibited this potency against Gram-positive strains
(GIZ ≥ 13 mm), they were further tested for their minimal
inhibitory concentration (MIC) by the twofold serial microdi-
lution method (28,29).
The negative control was TSB-glucose medium, and the
positive control (biofilm formation) was bacterial culture in
TSB-glucose. After incubation, medium was removed from
wells and washed twice with sterile phosphate-buffered
saline (PBS) to remove the non-adherent bacteria. Alive
bacterial cells in each well of the microtitre plate were
stained with 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-
tetrazolium bromide (MTT; 0.5% in PBS) for 2 h at 37 °C
(protected from light). Adherent bacterial cells, which usu-
ally formed biofilm on wells, were uniformly stained with
MTT. After incubation, the solution was removed and bac-
terial biofilm was solubilized by DMSO with glycine buffer
and mixed 15 min at room temperature. The absorbance
(A554) was measured at 554 nm using spectrophotometer
(PowerWave XS, BioTek, Winooski, VT, USA). The IC50
values were calculated from the concentration–response
curves by nonlinear regression analysis. All the experi-
ments were carried out in quadruplicates. The biofilm sur-
vival (%) was determined using the formula: (%) = {(At–
Ab)/(Ac–Ab)} 9 100%, where At = Absorbance value of
biofilm growth in the presence of a tested compound,
Ab = Absorbance value of blank, Ac = Absorbance value
of biofilm growth without any compound.
The results revealed that 18 of 21 investigated compounds
exhibited high and broad antibacterial activity, especially
against standard staphylococcal and M. luteus strains
(Table 1). Within this group, the observed MIC values were
in the range 0.5–4 lg/mL. The remaining three derivatives
were able to display moderate activity against tested
Gram-positive cocci. Derivative 11 was equipotent to
Ciprofloxacin against S. aureus ATCC 25923 (MIC 0.5 lg/
NO2
NO2
Cl
Cl
S
MeCN
12h,
20 °C
R-NCS
+
NH2
R
NH
NH
R = aryl
Staphylococcus epidermidis strain ATCC 12228 and
S. epidermidis ATCC 35984 were used in eradication
assays as a negative (low biofilm-producing) and positive
1-21
Scheme 1: Synthetic scheme of thiourea derivatives 1-21.
Chem Biol Drug Des 2016; 87: 905–917
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Antibacterial Profile of Disubstituted Thioureas
Chem Biol Drug Des 2016; 87: 905–917
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Bielenica et al.
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Antibacterial Profile of Disubstituted Thioureas
Table 3: Activity of compounds 20 and 21 against Mycobac-
mL), while compounds 4-7, 10, 12, 13, 15, 17, 19 showed
50–25% activity of the standard antibiotic against various
S. aureus isolates (MIC 1 lg/mL). Further, derivatives 4, 7,
10, 11, 13, 19 showed 25% of the potency of Ciprofloxa-
cin (MIC 2 lg/mL) against S. epidermidis representatives.
Noticeable inhibitory effect towards tested Staphylococci
was also observed for analogues 2, 3, 20, 21. However,
5, 7, 10-15, 17, 19 were equally or more potent against
B. subtilis than the standard compound (MIC 0.5–2 lg/
mL). Derivative 13 acted four times stronger for the
selected M. luteus isolate than Ciprofloxacin. Compounds
were not active against Gram-negative rods and fungi. On
the other hand, the starting 4-chloro-3-nitroaniline exerted
no relevant activity against both types of staphylococcal
strains, being moderately active only against B. cereus,
M. luteus, P. vulgaris and C. albicans (GIZ 12–14 mm). It
confirms that high biological activity of the synthesized
group is the result of the appropriate substitution at
thiourea nitrogen.
terium tuberculosis strains
(MIC, lg/mL)
–
minimal inhibitory concentrations
MIC (lg/mL)
Strain
20
21
*Ref.
M. tuberculosis H37Rv ATCC 25618
M. tuberculosis Spec. 192
M. tuberculosis Spec. 210
M. bovis
6.25
6.25
6.25
25
50
100
>100
50
12.5
6.25
6.25
12.5
25
50
100
50
50
12.5
12.5
12.5
50
M.scrofulaceum
M. fortuitum
M. avium
25
100
100
12.5
50
M. kansasii
M. xenopi
M. intracellulare
25
50
50
100
*Ref. – Isonicotinic acid hydrazide (INH).
Table 4: Affinity of selected compounds towards bacterial type II
topoisomerases, expressed as IC50 Æ SEM (lg/mL)
Next, the activity of thioureas against hospital methicillin-
resistant strains of S. epidermidis (MRSE) was assigned.
While chosen bacterial strains were not susceptible to the
presence of control Ciprofloxacin, almost all new deriva-
tives had strong influence on the bacterial growth
(Table 2). For the most active compounds (6, 11-13, 15,
17), MIC values ranged from 1 to 2 lg/mL and were 16–
64 times lower than these achieved for the reference.
Only one hospital strain (S. epidermidis MRSE 406/11)
was more resistant to the presence of tested com-
pounds.
*IC50
S. aureus
Topoisomerase IV
Comp.
S. aureus DNA Gyrase
7
10
46.25 Æ 2.65
283.07 Æ 8.94
3.35 Æ 0.41
19.55 Æ 1.22
18.38 Æ 0.89
50.35 Æ 0.96
59.40 Æ 1.55
24.45 Æ 0.96
39.75 Æ 1.40
3.43 Æ 0.28
11
13
36.40 Æ 1.22
4.48 Æ 0.43
20
21
283.68 Æ 6.93
22.20 Æ 1.30
Ciprofloxacin
The highest potency against standard Gram-positive
strains was achieved with derivatives possessing 3,4-
dichlorophenyl (11) and 3-chloro-4-methylphenyl ring (13)
(MIC 0.5–1 lg/mL), as well as with 3-chloro-4-fluorophenyl
(10) and 2-fluorophenyl (7) analogues (MIC 1 lg/mL for
most of the tested strains). Next, the linear decrease in
activity was observed in the direction of the following
substituents at the benzene ring: 3-(trifluoro)methyl = 4-
iodo > 2-bromo = 4-bromo > 2,4-dichloro = 4-cyano > 3-
bromo >3-chloro = 4-chloro > 5-chloro-2-methyl > 4-
fluoro. Moderate activity (MIC 4–8 lg/mL) was denoted for
the group of N-alkylthiourea derivatives (20, 21). The
results of compounds screening against hospital methi-
cillin-resistant strains of S. epidermidis proved that disub-
stituted thioureas (11-13) with at least one chlorine atom
at the C3 position at the ring, as well as 4-iodophenyl
derivative (17), were the most effective antibacterial agents
(MIC 1 lg/mL). Introducing 4-bromophenyl (6) or 3-(tri-
fluoro)methylphenyl (15) terminal fragment at the thiourea
chain also resulted in high inhibitory potency against hos-
pital isolates (MIC 1–2 lg/mL).
*Concentration (lg/mL) of tested compound required to inhibit
50% of enzyme.
The optimal activity towards M. tuberculosis was achieved
for derivatives 20 and 21, possessing alkylphenyl substituents
at the thiourea branch (Table 3). Compound 20 was found
to be equipotent or even stronger inhibitor than INH against
M. tuberculosis H37Rv, M. tuberculosis 210 and M. tubercu-
losis 192 (MIC 6.25 lg/mL). Derivative 21 possessed the
same as the reference drug or 50% of its potency, depend-
ing on the type of M. tuberculosis isolate. It has been shown
to be the most effective inhibitor against atypical Mycobac-
terium strains, possessing a significant twofold to fourfold
increase in the potency against M. bovis, M. fortuitum,
M. xenopi and M. intracellulare, as compared to INH. The
analogue 20 was able to produce noticeable growth inhibi-
tory effect against M. xenopi and M. intracellulare, which
represents twice of the INH activity.
To exclude potential carcinogens, the most active com-
pounds (2-7, 10-15, 17, 19) were tested for DNA-dama-
ging activity by rec-assay. For this purpose, two
genetically modified B. subtilis strains were used, and no
significant differences were observed between diameters
of the inhibition zones between them (Table S2). It led to
The encouraging results from our antibacterial studies
prompted us to investigate the title compounds for their anti-
tubercular potency. For derivatives, 1-19 moderate to weak
activity was observed (MIC 25–100 lg/mL, data not shown).
Chem Biol Drug Des 2016; 87: 905–917
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Bielenica et al.
Table 5: Biofilm inhibitory (as IC50 Æ SD values (lg/mL)) and biofilm eradication activity (as EC50 Æ SD values (lg/mL)) of compounds 11
and 13 against standard and hospital methicillin-resistant strains of S. epidermidis
11
13
***Ref
11
13
***Ref
Strain
*IC50 [lg/mL]
Strain
**EC50 [lg/mL]
#S. e. ATCC 35984 19.31 Æ 0.31 5.14 Æ 0.55
1.31 Æ 0.33 S. e. ATCC 35984 6.44 Æ 0.50 7.09 Æ 1.51
1.08 Æ 0.50 S. e. ATCC 12228 2.87 Æ 0.77 5.97 Æ 0.58
0.55 Æ 0.48
S. e. ATCC 12228
S. e. 404/11
S. e. 405/11
S. e. 411/11
S. e. 412/11
7.53 Æ 0.18 4.25 Æ 0.15
1.10 Æ 0.68
2.16 Æ 1.16 2.07 Æ 1.44 355.84 Æ 1.03 S. e. 404/11
4.65 Æ 0.62 4.97 Æ 1.04 173.44 Æ 0.17
5.31 Æ 0.30 1.93 Æ 0.37
9.78 Æ 1.30 6.15 Æ 0.89
29.28 Æ 1.29 S. e. 405/11
34.52 Æ 1.61 S. e. 411/11
3.25 Æ 1.39 4.29 Æ 0.14
3.81 Æ 0.46 3.80 Æ 0.28
17.08 Æ 1.46
30.48 Æ 0.40
8.72 Æ 0.86 6.11 Æ 0.60 355.84 Æ 0.51 S. e. 412/11
9.00 Æ 0.96 4.92 Æ 0.96 240.00 Æ 0.73
*Concentration (lg/mL) of tested compound required to inhibit 50% of biofilm formation under the assay conditions. **Concentration (lg/
mL) of tested compound required to eradicate 50% of established biofilm under the assay conditions. ***Ref. – Ciprofloxacin. #S. e. – Sta-
phylococcus epidermidis.
the conclusion that examined thiourea derivatives are non-
genotoxic and their antimicrobial potency is not an effect
of their genotoxic activity.
They were tested at doses ranging from ½ 9 MIC to
8 9 MIC (according to Tables 1 and 2). Both compounds
considerably inhibited biofilm formation of hospital strains
at most of used concentrations (1–8 9 MIC), in contrast to
Ciprofloxacin (Fig. S3-S5, Supporting Information). Deriva-
tives 11 and 13, applied at 1 lg/mL (1 9 MIC), in above
50% prevented from biofilm formation of S. epidermidis
404/11 isolate. It is also noteworthy that for this particular
strain the doses of both compounds needed for 40% bio-
film formation inhibition (1/2 9 MIC) were lower than for
planktonic cells inhibition. The concentration of compound
13 required to inhibit biofilm growth of S. epidermidis 405/
11 strain was also definitely lower for biofilm than for free-
floating cultures. Low-biofilm producers (S. epidermidis
411/11 and 412/11) were susceptible only to the highest
concentrations of thiourea compounds (4–8 9 MIC). The
inhibitory potency of derivative 13 against the formation of
biofilm of standard pathogens was equal to Ciprofloxacin.
The ATCC 35984 isolate appeared to be the least suscep-
tible to the presence of tested compounds, probably due
to its highest biofilm-producing level.
Type II topoisomerase inhibition assay
Enzyme inhibition profiles were assessed on a panel of
bacterial topoisomerases comprising DNA gyrase and
topoIV isolated from S. aureus strains (Figures S2–S6).
The aim of these studies was to verify the hypothesis that
antimicrobial activity of thioureas depended on the level of
their inhibitory effect on bacterial type II topoisomerases.
According to the results of the in vitro antibacterial assay,
chosen N-aryl derivatives 7, 10, 11 and 13 gave the most
favourable effect; on the other hand, N-alkylphenylthiour-
eas 20 and 21 were the strongest inhibitors of growth of
M. tuberculosis pathogens, being also quite potent
towards Gram-positive pathogens.
The obtained results (Table 4) demonstrated that
antistaphylococcal activity of studied compounds is not
strictly dependent on the degree of their activity to DNA
gyrase and topoIV. Even if thioureas 11 and 20 inhibited
DNA gyrase stronger than the reference drug, derivative
20 has lower antibacterial activity in terms of MIC, as com-
pared to 11 and the control compound. However, the
strongest antitubercular potency of the alkylthiourea 20
could be explained just by the high level of gyrase inhibi-
tion. Its profile could be similar to Moxifloxacin, the third
generation of quinolones, inhibitor of S. aureus and
S. pneumonia growth that is also active against M. tuber-
culosis, which lacks topoIV (34). Derivatives 10 and 21
acted by visible topoIV inhibition, whereas thioureas 7 and
13 are dual class inhibitors.
Calculated IC50 values indicate that derivative 13 was the
most potent among evaluated thioureas (Table 5). As
compared to the standard drug, these values were from
5.6 (S. epidermidis 411/11) to 172 (S. epidermidis 404/11)
times lower. Similarly, compound 11 inhibited by 50% the
formation of biofilm of S. epidermidis 404/11 at concentra-
tion 165 times lower than standard, being also multiple
times more potent for other clinical staphylococcal strains.
Next, compounds 11 and 13 were examined for their influ-
ence on mature biofilm (Figures S6–S8). The strongest
activity was observed only for compound 11, which at
higher concentrations (2–8 9 MIC) has eradicated biofilm in
40–80%. Its potency was similar to the reference Ciproflox-
acin; however, the activity described as EC50 for S. epider-
midis clinical strains was even stronger (Table 5). Although
Ciprofloxacin was the strongest inhibitor of standard
staphylococci growth (EC50 0.55–1.10 lg/mL), compounds
11 and 13 showed higher activity against hospital isolates –
up to 37–49 times higher than the reference drug.
Biofilm inhibitory and biofilm eradication studies
Preliminary antibacterial studies revealed that derivatives
11 and 13 are the most potent against free-floating plank-
tonic cells of standard and clinical S. epidermidis strains;
therefore, they were further studied for their ability to inhibit
the formation of bacterial biofilm of the same pathogens.
914
Chem Biol Drug Des 2016; 87: 905–917

Antibacterial Profile of Disubstituted Thioureas
anti-pathogenic surface coating nanofluid, obtained by
combining Fe3O4/C12 nanostructures and 2-((4-ethyl-
phenoxy)methyl)-N-(substituted-phenylcarbamo-thioyl)-
benzamides. Nanoscale Res Lett;7:513.
Acknowledgments
Research subject carried out with the use of CePT infras-
tructure financed by the European Union – the European
Regional Development Fund within the Operational Pro-
gramme ‘Innovative economy for 2007–2013’.
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Conflict of Interest
The authors confirm that this article content has no conflict
of interests.
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on agarose gel. Lane 1: Control with 2% DMSO. Lane 2:
negative control without enzyme, containing diluted buffer.
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Supporting Information
Figure S6. The effect of tested compounds on relaxation
of S. aureus topoisomerase IV. Decreasing amounts of 7,
10 and 13 were incubated with 200 ng kDNA and run on
agarose gel. Lane 1: Control with 2% DMSO. Lane 2:
negative control without enzyme, containing diluted buffer.
Lane 3-8: compound 10 (at concentration 32, 8, 4, 2, 1,
0.5 lg/mL, respectively). Lane 9-14: compound 7 (at
concentration 32, 8, 4, 2, 1, 0.5 lg/mL, respectively).
Lane 15-20: compound 13 (at concentration 32, 8, 4, 2,
1, 0.5 lg/mL, respectively).
Additional Supporting Information may be found in the
online version of this article:
Table S1. Structures and atom numbering of thiourea
derivatives 1-21.
Table S2. Mutagenic activity of tested compounds in
Bacillus subtilis rec-assay, expressed as diameter of
growth inhibitory zone (GIZ, mm).
Figure S1. The influence of tested compounds on S. aur-
eus gyrase DNA activity. Decreasing amounts of 11 or 20
Figure S7. Biofilm inhibitory activity of compound 11 –
Chem Biol Drug Des 2016; 87: 905–917
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Antibacterial Profile of Disubstituted Thioureas
the percentage of S. epidermidis biofilm survival in the
presence of the tested compound. (compared to drug-
free control).
Figure S10. Percentage survival of S. epidermidis biofilm
after 24 h growth and after 24 h treatment with com-
pound
Figure S8. Biofilm inhibitory activity of compound 13 –
the percentage of S. epidermidis biofilm survival in the
presence of the tested compound. (compared to drug-
free control).
Figure S11. Percentage survival of S. epidermidis biofilm
after 24 h growth and after 24 h treatment with com-
pound 13.
Figure S12. Percentage survival of S. epidermidis biofilm
after 24 h growth and after 24 h treatment with Ciprofloxacin.
Figure S9. Biofilm inhibitory activity of Ciprofloxacin – the
percentage of S. epidermidis biofilm survival in its pres-
ence.
Chem Biol Drug Des 2016; 87: 905–917
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