Antimicrobial and anti-biofilm activity of thiourea derivatives incorporating a 2-aminothiazole scaffold

Article abstract of DOI:10.1248/cpb.c14-00837

A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans . Compounds were also tested for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H₃₇Rv strain, as well as two 'wild' strains isolated from tuberculosis patients. Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital strain). The range of MIC values is 2-32 μg/mL. Products 3 and 9 effectively inhibited the biofilm formation of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3^rd position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4 showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against MT-4 cells (CC₅₀<10 μM).

Full text of DOI:10.1248/cpb.c14-00837

Vol. 63, No. 3
Chem. Pharm. Bull. 63, 225–236 (2015)
225
Regular Article
Antimicrobial and Anti-biofilm Activity of Thiourea Derivatives
Incorporating a 2-Aminothiazole Scaffold
,b
Joanna Stefanska,a Grażyna Nowicka,b Marta Struga,* Daniel Szulczyk,c Anna Eugenia Koziol,d
Ewa Augustynowicz-Kopec,e Agnieszka Napiorkowska,e Anna Bielenica,f Wojciech Filipowski,g
Anna Filipowska,g,h Aleksandra Drzewiecka,i Gabriele Giliberti,j Silvia Madeddu,j Stefano Boi,j
Paolo La Colla,j and Giuseppina Sannaj
b
a Department of Pharmaceutical Microbiology, Medical University; Warszawa 02–007, Poland: Department of
c
Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw; Warszawa 02–097, Poland: Department of
d
Medical Chemistry, Medical University; Warszawa 02–007, Poland: Faculty of Chemistry, Maria Curie-Sklodowska
e
University; Lublin 20–031, Poland: Department of Microbiology, National Tuberculosis and Lung Diseases Research
f
Institute; Warszawa 01–138, Poland: Chair and Department of Biochemistry, Medical University of Warsaw;
g
Warszawa 02–097, Poland: Silesian University of Technology, Faculty of Automatic Control, Electronics and
h
Computer Science, Institute of Electronics; Gliwice 44–102, Poland: Silesian University of Technology, Faculty of
Biomedical Engineering, Department of Biosensors and Processing of Biomedical Signals; Gliwice 44–100, Poland:
j
i Institute of Physics, Polish Academy of Sciences; Warszawa 02–668, Poland: and Department of Biomedical
Science, University of Cagliari; Monserrato (CA) 09042, Italy.
Received December 9, 2014; accepted January 6, 2015
A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds
were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative
rods and Candida albicans. Compounds were also tested for their in vitro tuberculostatic activity against the
Mycobacterium tuberculosis H37Rv strain, as well as two ‘wild’ strains isolated from tuberculosis patients.
Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital
strain). The range of MIC values is 2–32µg/mL. Products 3 and 9 effectively inhibited the biofilm formation
of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3rd
position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained
compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including
Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4
showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against
MT-4 cells (CC50<10 µM).
Key words antimicrobial activity; antibiofilm activity; antiviral activity; 2-aminothiazole
1,3-Thiazoles are an important group of compounds due an effective anti-tuberculosis drug clinically used against a
to their wide range of application as pharmaceutical agents. range of multidrug-resistant strains of Mycobacterium tuber-
Especially 2-amino-1,3-thiazole scaffold (fused and non-fused) culosis.16) Isoxyl inhibits M. bovis with 6h of exposure, which
would serve as a privileged structure due to their prevalence is similar to isoniazid and ethionamide, two other prominent
in antibacterial agents and other biologically active mol- anti-tuberculosis drugs.16)
ecules.1–5) Database of biologically active substances6) reveals
Isoniazid, ethionamide and isoxyl inhibit biosynthesis of
the extensive use of the 2-aminothiazole structural motif in mycolic acids (fatty acids). Isoniazid and ethionamide stimu-
numerous applied drugs as well as in preclinical and clinical late biosynthesis of short chain fatty acids, while isoxyl inhib-
candidates (e.g., Sulfatizole, Cetraxone, Aztreonam, Riluzole). its their synthesis.17)
Moreover, the literature surrey shows that 2-aminothiazole-
Analogues of isoxyl, with symmetrical and unsymmetrical
based compounds serve as muscarinic and serotonergic modifications of the molecule, have significantly enhanced ac-
ligands.7–9) Therefore 2-amino-1,3-thiazoles are perspective tivity against both Mycobacterium tuberculosis and M. bovis
scaffolds for designing new families of compounds with thera- BCG.18)
peutic importance.
Although the twentieth century has been characterized
The structure of 2-amino-1,3-thiazole can be considered by a drastic reduction in the mortality caused by infectious
as a compound having thiourea system built into the five diseases and a rise in the control of neoplastic pathologies,
membered ring. In our previous studies,10,11) we reported the the treatment of cancer and of infectious diseases caused
synthesis and biological activity of thiourea derivatives. In the by viruses still remains an important and challenging prob-
recent literature, the thioureas are described as most useful lem. As a matter of fact cancer remains the leading cause of
class of agents with large number of activities including anti- death in economically developed countries and AIDS is the
viral,12,13) antibacterial14) and high density lipoprotein (HDL)- major health problem with 34 million people were living with
elevating properties.15)
Human immunodeficiency virus (HIV)19); therefore, despite
Isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea)—an- the considerable progress made, there is continued interest in
other example of the thiourea pharmacofore is known to be developing therapeutic agents.20)
*To whom correspondence should be addressed. e-mail: marta.struga@wum.edu.pl
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
On the basis of the biological properties previously de- ferent biological materials of patients hospitalized in Warsaw
scribed, we have accomplished the synthesis of 22, 10 of them Medical University Hospitals.
new, thiourea derivatives of 1,3-thiazole and the evaluation of
their antibacterial and antiviral activities.
MIC values of hospital S. epidermidis strain were in the
range 128—2µg/mL (Table 2). For the most active compounds
3, 6 and 9 the MICs were in the range 16—2µg/mL.
The biological activity depends on the structure of com-
Chemistry
The preparation of 10 new thiourea derivatives is described. pounds, viz. on an aryl substituent. Based on Table 2, methoxy
1,3-Thiazol-2-amine was subjected to the reaction with iso- (12) and trifluoromethyl (10) derivatives are less potent than
thiocyanates in order to be transformed into the correspond- Ciprofloxacin, while compounds 3 and 9 are more active
ing thiourea derivatives (Chart 1). Obtained compounds were than the reference drug. It led to the conclusion that activity
1
purified by a flash chromatography. MS, H- and 13C-NMR depends on the type of the substituent on phenyl ring. Fur-
1
spectra confirmed the identity of the products. In H-NMR thermore, the position of the substitution is also important.
spectra labile proton of NH group of thiourea branch was not For example, compound 5 is the least active compound, sug-
observed for compound 1. The molecular structure of 9 (Fig. gesting that halogen on meta position of benzene ring does not
1) was determined by an X-ray crystal structure analysis.
favor activity against S. epidermidis.
Generally, the phenyl group substituted with the fluorine,
bromine or chlorine atoms, and methoxy or trifluoromethyl
Results and Discussion
All obtained compounds were tested in vitro against a num- groups increases the antimicrobial activity in comparison to
ber of microorganisms, including Gram-positive cocci, Gram- the aliphatic substituents. The most active compound was 9
negative rods and Candida albicans. Microorganisms used in which is the 3-chloro-4-fluorophenyl substituted derivative.
this study have common application in the antimicrobial tests
Preliminary test by disc-diffusion method showed antimi-
for many substances like antibiotics, disinfectants and anti- crobial activity against standard strain of fungi C. albicans
septic drugs or in research on new antimicrobial agents.21) All for five compounds, therefore the next step was evaluation of
compounds were screened for their antimicrobial activity by a compounds’ MIC values for standard and hospital strains. The
disc diffusion method.22) Compounds showing significant ac- MIC values for these compounds were higher than 64µg/mL.
tivity in the test were next examined for their minimal inhibi-
tory concentration (MIC).23) The results of activity for these static activity against the M. tuberculosis H37Rv strain and
compounds are summarized in Table 1. two ‘wild’ strains isolated from tuberculosis patients: one
Compounds were also tested for their in vitro tuberculo-
Preliminary test by a disc-diffusion method showed antimi- (Spec. 210) resistant to p-aminosalicylic acid (PAS), isonico-
crobial activity against standard Gram-positive cocci, there- tinic acid hydrazide (INH), ethambutol (ETB) and rifampicin
fore the next step was evaluation of compounds’ MIC values (RMP), and the other (Spec. 192) fully sensitive to the admin-
for standard and hospital strains. The research was carried istrated tuberculostatics. The MIC values were determined as
out over 20 standard strains and 15 hospital strains of Staphy- the minimum concentration inhibiting the growth of tested
lococcus epidermidis used for routine antimicrobial media tuberculous strains. INH was used as the reference drugs.
susceptibility testing. Hospital strains were isolated from dif- The results of tuberculostatic activity for these compounds are
summarized in Table 3.
The most active compounds were 11 and 17 which are
the phenyl and the 4-iodophenyl substituted derivatives. The
activity against three strains of M. tuberculosis is similar
for all compounds, indicating that these compounds could be
promising drug candidates for multidrug-resistant tuberculosis
R=2-bromophenyl (1), 3-bromophenyl (2), 3,4-dichlorophenyl (3), cyclohexyl (4),
2-fluorophenyl (5), 3-fluorophenyl (6), 2-chlorophenyl (7), 3-chloro-4-methylphenyl
(8), 3-chloro-4-fluorophenyl (9), 3-trifluorophenyl (10), phenyl (11), 4-metoxy-
phenyl (12), 4-chlorophenyl (13), 4-methylphenyl (14), 4-fluorophenyl (15), 4-bro-
mophenyl (16), 4-iodophenyl (17), benzyl (18), benzoyl (19), 3-chlorophenyl (20),
5-chloro-2-methylphenyl (21), ethoxycarbonyl (22).
treatment.
Antimicrobial activity can be related to cytotoxicity of
compounds (e.g., 2, 3, 16). However it is not a rule because
compounds 1 and 5 that significantly inhibit the growth of
Gram-positive cocci, turned out cytotoxic in the high micro-
molar range (CC50 above 100µM and 38.5µM).
Chart 1. Synthesis of Studied Compounds
Preliminary antibacterial studies revealed that halogen
derivatives of thiourea 3 and 9 have shown the most prom-
ising activity against free-swimming (planktonic) forms of
staphylococcal species. In general MIC values for standard
and medically relevant methicillin-resistant strains of S.
aureus and S. epidermidis ranged from 16 to 4µg/mL. These
thiourea conjugates were further studied for their ability to
inhibit the formation of biofilms of eight methicillin-resistant
strains of S. epidermidis (MRSE) and two standard strains of
S. epidermidis (ATCC 12228, ATCC 35984).
Based on their absorbance, the tested strains of S. epider-
midis were divided into two groups: the first that form biofilm
on low level (A554 <0.7) and high level biofilm forming (A554
<1.9). Strains that produced slime, formed black colonies
Fig. 1. View of the Molecule 9

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
227
Table 1. Activities of Obtained Compounds against Gram-Positive Bacteria and C. albicans—Minimal Inhibitory Concentations (MIC, µg/mL) and
Diameter of Growth Inhibitory Zone (GIZ, mm, Applied 400µg per Disc)
2
3
5
6
8
9
10
11
32
12
15
16
Ref.a)
Ref.b)
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
S. epidermidis ATCC 35984
E. hirae ATCC 10541
E. faecalis ATCC 29212
M. luteus ATCC 10541
M. luteus ATCC 9341
B. subtilis ATCC 6633
B. cereus ATCC 11778
C. albicans ATCC 10231
C. albicans ATCC 90028
C. albicans ATCC 22019
32
16
(15)
16
512
(11)
512
(13)
256
(12)
256
(11)
516
(12)
516
(12)
256
−
32
(13)
16
32
(12)
16
4
32
(14)
32
32
(13)
32
32
(12)
16
16
(13)
16
0.25
(26)
0.5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.5
(20)
0.5
(29)
0.5
(25)
(13)
32
(26)
4
(13)
32
(12)
32
(20)
16
(15)
64
(15)
32
(27)
8
(15)
32
(13)
32
(14)
32
(14)
32
(13)
16
(26)
0.25
(28)
0.5
(11)
16
(17)
4
(15)
64
(15)
16
(26)
8
(14)
32
(11)
32
(14)
32
(12)
16
(13)
16
(15)
16
(18)
8
(18)
16
(19)
2
(25)
4
(16)
16
(12)
32
(13)
4
(11)
16
(13)
8
(22)
0.25
(30)
0.25
(32)
1
(15)
16
(17)
4
(16)
16
(14)
4
(26)
4
(14)
16
(13)
16
(13)
8
(13)
16
(16)
8
(15)
>512
(11)
>512
−
(21)
32
(19)
512
−
(20)
32
(30)
16
(20)
128
(13)
128
−
(14)
>512
−
>512
−
(18)
64
(16)
>512
−
(15)
>512
−
>512
−
(17)
32
(11)
32
(23)
16
(11)
64
(18)
1
516
512
512
−
16
−
−
16
−
8
(20)
4
−
16
−
16
(15)
2
16
516
(17)
128
(30)
128
(17)
256
(17)
256
(16)
256
(19)
256
(20)
32
16
16
(18)
16
(24)
16
(18)
16
(19)
4
(29)
8
(19)
16
(17)
32
(17)
4
(17)
16
(16)
8
(22)
1
(13)
2
(20)
8
(20)
16
(17)
4
(31)
2
(14)
4
(15)
8
(14)
8
(16)
4
(14)
8
(24)
<0.12
(38)
0.25
(26)
−
−
−
−
−
(21)
32
(24)
16
(24)
16
(24)
16
(36)
4
(25)
16
(20)
32
(23)
32
(20)
32
(18)
16
(11)
−
−
−
−
(14)
−
−
−
−
(24)
−
−
−
−
(13)
−
−
−
−
(22)
128
(11)
64
(13)
−
−
−
−
(12)
512
(20)
512
(10)
512
(11)
(13)
−
−
−
−
(13)
256
(20)
128
(11)
128
(15)
(12)
512
(14)
512
(11)
512
(11)
(12)
64
−
−
−
−
−
−
−
−
−
−
−
−
(18)
−
a) Ref.—ciprofloxacin (GIZ—5µg/9mm disc). b) Ref.—Fluconazole (GIZ—25µg/9mm disc). − Lack of the growth inhibition area.
Table 2. Activity of Compounds against Hospital Strains of S. epidermidis—Minimal Inhibitory Concentrations (MIC, µg/mL)
2
3
5
6
8
9
10
11
12
15
16
Cipa)
S. epidermidis 517/12
S. epidermidis 518/12
S. epidermidis 519/12
S. epidermidis 520/12
S. epidermidis 521/12
S. epidermidis 523/12
S. epidermidis 524/12
S. epidermidis 526/12
S. epidermidis 527/12
S. epidermidis 528/12
S. epidermidis 529/12
S. epidermidis 530/12
S. epidermidis 531/12
S. epidermidis 532/12
S. epidermidis 533/12
32
32
16
128
16
32
32
32
32
32
32
32
32
16
16
4
4
2
4
2
8
4
4
4
8
4
4
4
4
4
128
128
128
256
128
128
128
128
128
128
128
128
128
128
128
8
32
32
8
8
8
4
8
4
4
4
4
8
8
4
4
4
4
4
64
64
32
64
32
64
32
32
64
64
32
64
32
32
32
64
64
16
64
32
64
32
32
64
64
32
64
32
16
16
32
32
16
128
32
32
32
32
64
128
32
32
32
8
32
32
16
32
16
32
16
32
32
32
16
32
32
16
16
32
32
8
32
32
0.25
16
32
64
8
8
4
16
8
32
8
32
16
32
16
32
32
32
16
32
32
8
8
32
8
4
4
16
32
32
8
4
16
8
0.25
32
4
4
8
32
8
32
8
8
8
8
0.5
0.25
8
8
32
32
a) Cip—ciprofloxacin.
with a dry crystalline consistency on Congo Red agar (CRA), 1 to 16µg/mL. Both thiourea connections exhibited good bio-
whereas slime-negative isolates were pinkish-red with darken- film inhibitory activity against the aforesaid standard and hos-
ing at the centre (Fig. 2). In MTT test slime-positive strains pital bacterial strains, regardless of the level on which biofilm
were high biofilm-producers, slime-negative—low biofilm was formed.
producers.
The compounds 3 and 9 were found to be more promising
The compounds were tested at concentrations ranging from with IC50 values of 0.35–7.32µg/mL. For most of tested strains

228
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
the highest concentrations of compounds (for 3—4µg/mL, for IC50 for ciprofloxacin were much higher than for both tested
9—8µg/mL) inhibited biofilm formation in above 70% (Figs. thiourea derivatives (Table 4).
3, 4), as compared to the control.
Due to the previously reported anti-HIV activities of thio-
To compare, the reference compound, ciprofloxacin, in its urea derivatives,24,25) title compounds were tested in the cell-
highest concentration (16µg/mL) inhibited biofilm formation based assay against the human immunodeficiency virus type-1
of four tested strains of S. epidermidis in approximately 40% (HIV-1), using Efavirenz as reference inhibitor. The cytotoxic-
(Fig. 5). For the two and only hospital strain (S. epidermidis ity against the MT4 cells was evaluated in parallel with the
519/12 and 533/12) the highest concentration of the standard antiviral activity (Table 5).
drug blocked biofilm formation in 80%. Observed values of
Only compound 4 showed anti-HIV activity, although not
very potent (EC50=36µM), associated with lack of cytotoxicity
(CC50>100 µM).
Interestingly, seven compounds (2, 3, 8–10, 16, 17) turned
out cytotoxic for exponentially growing MT4 in a low mi-
cromolar range (CC50=7.8–9.0 µM). As indicated in Table 5,
selected compounds showed high level of cytotoxicity, also
against other cell monolayers used to support the multiplica-
tion of different viruses (MDBK, BHK, Vero-76) in stationary
growth.
Thiourea derivatives of 1,3-thiazole were also tested in
cell-based assays against representative members of several
virus families. Among ssRNA+ viruses, were: bovine viral
diarrhoea virus [BVDV] and yellow fever virus [YFV] (Flavi-
viridae), two Picornaviridae, human enterovirus B [coxsackie
virus B5, CVB-5] and human enterovirus C [polio virus
type-1, Sb-1]. Among ssRNA-viruses, we tested vesicular
stomatitis virus [VSV] (Rhabdoviridae). Among dsRNA vi-
ruses, we tested reovirus type-1 [Reo-1] (Reoviridae). Finally,
representatives of two DNA virus families were also included:
vaccinia virus [VV] (Poxviridae) and human herpes virus 1
[herpes simplex type-1, HSV-1] (Herpesviridae).
In order to be able to establish whether test compounds
were endowed with selective antiviral activity, their cytotoxic-
ity was evaluated in parallel assays with uninfected cell lines.
2′-C-Methyl-guanosine, 2′-C-methyl-cytidine, 2′-C-ethynyl-
cytidine, mycophenolic acid and acyclo-guanosine (acyclovir)
were used as reference inhibitors.
Table 3. In Vitro Tuberculostatic Activity of Compounds 1–9 and 11–21
MICa) [µg/mL]
Compdb)
M. tuberculosisc)
H37Rv
Spec. 192
Spec. 210
1
2
50
25
50
25
50
12.5
25
3
25
25
4
100
25
100
25
100
25
5
6
50
25
25
7
50
25
25
8
50
25
50
9
25
25
12.5
12.5
12.5
50
11
12
13
14
15
16
17
18
19
20
21
12.5
25
12.5
25
50
25
25
25
25
50
50
50
nt
nt
nt
12.5
50
25
12.5
50
50
100
50
100
50
100
50
50
50
50
a) Minimum inhibitory concentrations for mycobacterial strains were determined
by two-fold classical test-tube method of successive dilution. b) INH, isoniazid; nt, no
tested. c) Mycobacterium tuberculosis H37Rv (ATCC 25618), Spec. 192, Spec. 210.
Three compounds (4, 19, 22) resulted not cytotoxic against
all cell lines used (MT4, MDBK, BHK, Vero-76), while other
Fig. 2. Detection of Slime Production on Congo Red Agar
(A) Black colonies of slime-positive (high biofilm-producer) S. epidermidis ATCC 35984, (B) Red colonies of slime-negative (low biofilm-producer) S. epidermidis
ATCC 12228.

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
229
Fig. 3. Inhibitory Effect of the Compound 3 for Biofilm Formation by Standard and Selected Hospital Methicillin-Resistant Strains S. epidermidis
All presented results are mean from experiments performed in quadruplicate S.D.
Fig. 4. Inhibitory Effect of the Compound 9 for Biofilm Formation by Standard and Selected Hospital Methicillin-Resistant Strains S. epidermidis
All presented results are mean from experiments performed in quadruplicate S.D.

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Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
The structure–activity relationship was also observed when
the positional weighed electronic effect of substituent is ana-
lyzed (Table 6).
Subsequently, some physicochemical parameters as molar
weight (M), volume (V), surface area (SA), surface area grid
(gSA), logarithm of the partition coefficient (logP), the differ-
ence between higest occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO) energy levels
(HLG), the HOMO, the LUMO, hardness (η) obtained from
the equation η=(HOMO−LUMO)/2, Mullkien electronegativ-
ity (χ) obtained from the equation χ=−(HOMO+LUMO)/2,
total energy (ET), binding energy (EB), isolated atomic energy
(EIA), electronic energy (EE), core–core interaction (IC–C), heat
of formation (HF), refractivity (Rf), polarizability (α) were
studied (Table 6). However, there was no close correlation
between such important parameters as logP, HOMO, LUMO.
On the other hand, the relationship between ET (total energy),
EIA (isolated atomic energy) and antibacterial activity was
observed. As seen in Table 6, the most active compounds (3,
9, 10) were characterized by lower value of ET and EIA param-
eters in comparison with inactive derivatives.
Fig. 5. Inhibitory Effect of the Ciprofloxacin for Biofilm Formation by
Standard and Selected Hospital Methicillin-Resistant Strains S. epider-
midis
All presented results are mean from experiments performed in quadruplicate S.D.
Table 4. Antibiofilm Activity of Compounds 3 and 9 against Standard
and Hospital Methicillin-Resistant Strains of S. epidermidis
Test compounds
Compd. 3
Compd. 9
Ciproflox.
Bacterial strain
IC50 values IC50 values IC50 values
Conclusion
(µg/mL)
(µg/mL)
(µg/mL)
On the basis of predicted biological activities, reported
literature and results of our previous studies, we designed,
synthesized and tested 22 thiourea derivatives of 1,3-thiazole,
with the aim of developing new therapeutic agents with anti-
microbial activity.
The antimicrobial screening for the most of tested thiourea
derivatives exhibited significant antibacterial effect. On the
basis of structural activity relationship (SAR) it was estab-
lished that compounds with electron withdrawing halogens,
such as fluorine and chlorine (3, 6, 9) displayed strong anti-
bacterial potency. The presence of 3-chloro-4-fluorophenyl
(9) moiety resulted in the highest activity against standard
and hospital Gram-positive cocci among all investigated com-
S. epidermidis ATCC 12228
S. epidermidis ATCC 35984
S. epidermidis 517/12
S. epidermidis 519/12
S. epidermidis 523/12
S. epidermidis 526/12
S. epidermidis 528/12
S. epidermidis 531/12
S. epidermidis 532/12
S. epidermidis 533/12
3.71
2.55
7.32
2.52
2.53
1.61
2.50
2.83
3.49
1.96
0.35
—
11.89
18.55
—
0.66
2.50
3.14
4.95
1.10
1.13
2.21
2.88
1.65
—
9.28
5.00
6.59
6.73
1.90
compounds showed a different degree of cytotoxicity. Very in- pounds.
terestingly compound 4, in addition to anti-HIV-1 activity al-
Two compounds (3, 9), the most active against planktonic
ready described, showed selective activity also against CVB-5 forms of staphylococcal species, significantly affected the
virus, with EC50 value of 14µM, and lack of cytotoxicity for tested staphylococcal biofilm, in most of cases at concentra-
BHK cell lines. None of other tested compounds showed an- tions next to the MIC observed against the planktonic form.
tiviral activity with the exception of compound 19, resulted
The effect on tuberculostatic activity was especially appar-
weakly active against CVB-5.
ent for the phenyl (11), 4-iodophenyl (17), as well as 3, 9, 10
As presented in Tables 3 and 4, compounds with electron- thiourea derivatives.
withdrawing substituents, such as fluoro or chloro-fluoro (3, 6,
We explore their biological activities also in antiviral as-
9, 10) were found as the most potent antibacterial agents. For says, against HIV-1 and other RNA and DNA viruses causing
most of Gram-positive bacteria, it is reasonable that disubsti- infectious diseases.
tuted compounds (3, 9) are more potent than monosubstituted
When tested against representatives of ssRNA+, ssRNA−,
compounds, because the first group produce stronger elec- dsRNA and DNA virus families, compound 4 have proved to
tronegativity effect. However, substituent groups on different be active against HIV-1 and interestingly also against CVB-5,
positions definitely resulted in various degrees of effects. The a member of Enterovirus genus (Picornaviridae family) that
halogen atom at meta position of phenyl ring improved po- are important human pathogens that cause both acute and
tency against hospital S. epidermidis as the electronegativity chronic diseases in infants, young children and immuno-
increases (compounds 6 and 2). On the other hand all para- compromised individuals.26)
substituted compounds showed similar activity, even with the
electron donating methoxy group (compounds 15, 13, 16, 17,
12). The latter suggests the activity against S. epidermidis is
Experimental
Chemistry The NMR spectra were recorded on a Bruker
not sensitive to 4-substituted groups. For M. tuberculosis, the AVANCE DMX400 spectrometer, operating at 300MHz
3-halogen atoms did not show different effects (compounds 6, (1H-NMR) and 75MHz (13C-NMR). The chemical shift values
2), but 4-halogen atoms decreased activity as electronegativity are expressed in ppm relative to TMS as an internal standard.
increases (compounds 17>15, 13).
Mass spectral ESI measurements were carried out on Waters

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
231
Table 5. Cytotoxicity and Antiviral Activity of Thiourea Derivatives of 1,3-Thiazole against Representatives of ssRNA+ (HIV-1, BVDV, YFV, CBV-5,
Sb-1), ssRNA− (VSV), dsRNA (Reo-1) and dsDNA (VV, HSV-1) Viruses*
MT-4
CC50
HIV-1 MDBK BVDV
BHK
CC50
YFV
EC50
Reo-1 VERO 76 CVB-5
Sb-1
EC50
VSV
EC50
VV
EC50
HSV-1
EC50
Compounds
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
m)
EC50
CC50
EC50
85.4
>52
>12
EC50
CC50
EC50
1
2
>100 >100
>100
52
14
>14
>14
>9.4
>7.0
>100
>51
>9.2
>35
>9.5
>12
>7.0
44
20
>44
>20
>13
14
>44
>20
>13
>100
>100
>49
>100
>33
>15
>13
>45
>100
>12
>47
>49
>20
>50
>100
>100
>100
>100
>100
>44
>20
>13
>100
>100
>49
>100
>33
>15
>13
>45
>100
>12
>47
>49
>20
>50
>100
>100
>100
>100
>100
>44
>20
>13
>100
>100
>49
>100
>33
>15
>13
>45
>100
>12
>47
>49
>20
>50
>100
>100
>100
>100
>100
>44
>20
>13
>100
>100
>49
>100
>33
>15
>13
>45
>100
>12
>47
>49
>20
>50
>100
>100
>100
>100
>100
9.0
>9.0
>9.0
36
9.4
7.0
>100
51
9.2
35
9.5
12
7.0
>9.4
>7.0
>100
>51
>9.2
>35
>9.5
>12
>7.0
>32
>63
>9.0
>38
9.2
3
9.0
12
13
4
>100
>100 >100
>100 >100
>100
>100
49
5
38.5 >38.5
10.8 >10.8
20.8 >20.8
8.5
7.8
9.0
>100
>49
>100
>33
>15
>13
>45
>100
>12
>47
49
6
46
>100
26
>46
78
7
≥100
33
8
>8.5
>7.8
>9.0
>26
>12
>12
9
12
15
10
11
12
13
14
15
16
17
18
19
20
21
22
12
13
50
>50
>100 >100
>100 >100
32
63
>32
>63
>9.0
45
>100 >100
>100 >100
>100 >100
10.8 >10.8
>100
12
65
≥100
46
>65
38
9.0
>38
>46
8.0
6.0
42
47
>47
>9.2
20
>46
>32
>79
>9.2
>8.0
>6.0
>42
>100
>21
9.0
>9.0
>9.0
32
>8.0
>6.0
>42
>100
>21
>17
>20
>50
>100
40
9.0
79
50
>100 >100
>100 >100
40.0 >40.0
43.8 >43.8
>100 >100
≥100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100
>100
≥100
≥100
>100
>100
21
>100
>100
>100
17
>17
>100
>100
>100
Efavirenz
40
0.002
2′-C-Methyl-
guanosine
>10
1.1
>10
1.9
2′-C-Methyl-
cytidine
>100
16
2′-C-Ethynyl-
cytidine
>100
27
23
Mycophenolic acid
Acycloguanosine
≥12.5
>100
1.5
3
*Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%. a) Compound concentration (µM) required to re-
duce the proliferation of mock-infected MT-4 cells by 50%, as determined by the MTT method. b) Compound concentration (µM) required to achieve 50% protection of MT-4
cells from HIV-1 induced cytopathogenicity, as determined by the MTT method. c) Compound concentration (µM) required to reduce the viability of mock-infected MDBK
cells by 50%, as determined by the MTT method. d) Compound concentration (µM) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity,
as determined by the MTT method. e) Compound concentration (µM) required to reduce the viability of mock-infected BHK cells by 50%, as determined by the MTT method.
f, g) Compound concentration (µM) required to achieve 50% protection of BHK cells from YFV-induced(f) and Reo-1-induced(g) cytopathogenicity, as determined by the MTT
method. h) Compound concentration (µM) required to reduce the viability of mock-infected VERO-76 cells by 50%. as determined by the MTT method. i–m) Compound con-
centration (µM) required to reduce the plaque number of CVB-5(i), Sb-1(j), VSV(k), VV(l), HSV-1(m) by 50% in VERO-76 monolayers.
ZQ Micro-mass instruments with quadrupol mass analyzer. 0.69g) in acetonitrile (25mL) was treated with appropriate
The spectra were performed in the positive ion mode at a isothiocyanate (0.0075mol) and the mixture was refluxed
declustering potential of 40–60V. The sample was previously for 8h. Then solvent was removed on rotary evaporator. The
separated on a UPLC column (C18) using UPLC ACQUITY™ residue was purified by column chromatography (chloroform–
system by Waters connected with DPA detector. Flash chro- methanol; 9.5:0.5, v/v). The compound was crystallized from
matography was performed on Merck silica gel 60 (200–400 acetonitrile.
mesh) using chloroform–methanol (19:1, v/v) mixture as elu-
ent. Analytical TLC was carried out on silica gel F254 (Merck) 81%. mp: 163–165°C. H-NMR (DMSO-d6) δ (ppm): 7.08 (brs,
plates (0.25mm thickness). 1H, CHarom.); 7.16–7.21 (t, 1H, CHarom., J=7.5 Hz); 7.38–7.45
1-(2-Bromophenyl)-3-(1,3-thiazol-2-yl)thiourea (1) Yield
1
The intensity measurements of diffraction reflections were (m, 2H, CHarom.); 7.67–7.70 (d, 1H, CHarom., J=7.8Hz);
carried out at 200K with a Xcalibur CCD diffractometer, 7.76–7.79 (d, 1H, CHarom., J=6.9Hz); 12.31 (brs, 1H, NH).
using graphite monochromated CuKα radiation (λ=1.54178 Å) 13C-NMR (DMSO-d6) δ (ppm): 111.45, 119.64, 127.69, 127.77
and ω scan mode. Crystal structure wase solved by the (2C), 129.01, 132.51 (2C), 137.44, 180.12. Electrospray ioniza-
SHELXS-97 program and refined by full-matrix least squares tion (ESI)-MS: m/z=338.0 [M+Na+H]+ (100%).
on F2 using the SHELXL-97 program.27) All non-hydrogen
1-(3-Bromophenyl)-3-(1,3-thiazol-2-yl)thiourea (2) Yield
atoms were refined with anisotropic displacement parameters. 90%. mp: 160–161°C. 1H-NMR (DMSO-d6) δ (ppm): 6.99
Hydrogen atoms were positioned geometrically and allowed to (brs, 1H, CHarom.); 7.22–7.27 (m, 2H, CHarom.); 7.46–7.48
ride on their parent atoms, with Uiso(H)=1.2Ueq(C, N).
(d, 1H, CHarom., J=4.5Hz); 7.82 (brs, 1H, CHarom.); 7.98
Thiourea Derivatives of 1,3-Thiazol-2-amine Gen- (s, 1H, CHarom.); 10.18 (brs, 1H, NH); 12.97 (brs, 1H, NH).
eral procedure: A solution of 1,3-thiazol-2-amine (0.0069mol, 13C-NMR (DMSO-d6) δ (ppm): 110.11, 120.47, 121.13 (2C),

232
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
Table 6. SAR Study of Compounds 1–22
M
V
SA
logP
HOMO
LUMO
η
χ
ET
EB
EIA
EE
IC–C
Rf
Compd.
gSA
HLG
HF
α
1
2
314.22
711.27
314.22
722.90
304.21
740.53
241.37
715.94
253.31
667.58
253.31
670.34
269.77
691.27
283.79
749.51
287.76
711.68
303.32
736.17
235.32
661.34
265.35
739.07
269.77
702.90
249.35
712.64
253.31
669.83
314.22
722.01
361.22
738.70
249.35
721.31
263.33
706.94
269.77
702.99
283.79
740.67
231.29
639.58
362.87
441.17
384.31
455.11
407.18
464.01
360.18
451.11
339.35
423.74
352.03
424.83
354.08
429.89
412.97
466.09
381.77
446.71
381.99
462.55
340.44
414.73
399.64
463.53
375.68
444.73
384.09
450.71
352.46
425.68
384.83
450.14
396.00
457.56
366.90
455.83
330.12
440.15
375.17
441.57
390.00
453.32
343.64
405.65
4.54
−7.269
4.54
−8.752
−1.483
−8.835
−1.566
−8.779
−1.611
−8.692
−1.297
−8.786
−1.521
−8.855
−1.587
−8.745
−1.506
−8.718
−1.513
−8.870
−1.645
−8.663
−1.506
−8.697
−1.458
−8.503
−1.416
−8.711
−1.548
−8.610
−1.435
−8.808
−1.579
−8.829
−1.579
−8.679
−1.547
−8.719
−1.313
−8.704
−1.624
−8.806
−1.541
−8.726
−1.524
−9.178
−1.642
3.635
5.118
3.635
5.201
3.584
5.195
3.698
4.995
3.633
5.154
3.634
5.221
3.620
5.126
3.603
5.116
3.613
5.258
3.579
5.085
3.620
5.078
3.544
4.960
3.582
5.130
3.588
5.023
3.615
5.194
3.625
5.204
3.566
5.113
3.703
5.016
3.540
5.164
3.633
5.174
3.601
5.125
3.768
5.410
−58072
−2514
−58074
−2516
−64178
−2514
−52451
−2913
−60075
−2558
−60076
−2559
−57228
−2531
−60681
−2816
−67026
−2541
−83144
−2882
−50278
−2549
−60492
−2922
−57229
−2532
−53731
−2833
−60077
−2559
−58073
−2515
−56578
−2501
−53727
−2829
−59783
−2816
−57229
−2532
−60679
−2815
−55641
−2262
−55558
−342363
−55558
−335941
−61664
−374979
−49538
−340912
−57518
−346844
−57518
−340740
−54697
−342990
−57865
−373931
−64485
−379595
−80262
−474922
−47730
−299463
−57570
−374310
−54697
−334594
−50898
−333631
−57518
−339181
−55558
−334383
−54077
−331966
−50898
−338045
−56967
−376258
−54697
−336222
−57865
−380319
−53379
−319919
284291
75.29
30.04
75.29
30.04
77.28
31.27
68.61
28.00
67.88
27.33
67.88
27.33
72.47
29.35
77.51
31.18
72.69
29.26
73.64
28.98
67.67
27.42
74.13
29.89
72.47
29.35
72.71
29.25
67.88
27.33
75.29
30.04
80.08
32.45
72.21
29.25
69.44
29.34
72.47
29.35
77.51
31.18
58.64
23.99
110.17
277867
108.63
310801
88.90
288461
49.24
286769
58.79
280663
57.57
285762
95.25
313250
85.40
312569
52.40
391778
−57.16
249185
100.83
313818
62.52
277365
94.25
279900
91.32
279104
57.51
276309
108.78
275388
122.41
284318
95.53
316475
64.35
278993
94.32
319639
86.92
264277
−6.07
−7.269
4.78
3
−7.168
3.51
4
−7.395
3.89
5
−7.265
3.89
6
−7.268
4.26
7
−7.239
4.73
8
−7.205
4.40
9
−7.225
4.63
10
11
12
13
14
15
16
17
18
19
20
21
22
−8.663
3.75
−7.239
3.49
−7.087
4.26
−7.163
4.21
−7.175
3.89
−7.229
4.54
−7.250
5.00
−7.132
3.70
−7.406
5.45
−7.080
4.26
−7.265
4.73
−7.202
2.31
−7.536
Surfaces and refractivity in Å−2, volumes and polarizability in Å−2, energies in kcal/mol.
123.61, 125.60, 130.23 (2C), 141.61, 179.79. ESI-MS: m/z=338.0 5H, CH2cyclo.); 1.53–1.67 (m, 3H, CH2cyclo.); 1.90–1.93 (m,
[M+Na+H]+ (100%).
2H, CH2cyclo.); 4.08–4.11 (m, 1H, CHcyclo.); 7.09–7.11 (d, 1H,
1-(3,4-Dichlorophenyl)-3-(1,3-thiazol-2-yl)thiourea (3) Yield CHarom., J=3.6Hz); 7.38–7.40 (d, 1H, CHarom., J=6.0Hz);
1
81%. mp: 163–165°C. H-NMR (DMSO-d6) δ (ppm): 6.99–7.01 9.70 (brs, 1H, NH); 11.42 (s, 1H, NH). 13C-NMR (DMSO-d6)
(d, 1H, CHarom., J=4.5Hz); 7.48–7.51 (t, 2H, CHarom., δ (ppm): 24.00 (2C), 25.03, 31.42 (2C), 52.28, 112.02, 137.17,
J=4.5Hz); 7.75–7.78 (d, 1H, CHarom., J=6.6Hz); 8.11–8.12 (d, 161.79, 176.35. ESI-MS: m/z=264.1 [M+Na]+ (100%).
1H, CHarom., J=4.5Hz); 10.29 (brs, 1H, NH); 13.02 (brs, 1H,
1-(2-Fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (5) (PubChem
NH). 13C-NMR (DMSO-d6) δ (ppm): 110.05, 121.16, 122.20, Compound ID: 36777148; CCA 002249—Sigma-Aldrich)
124.09, 129.99 (2C), 130.55 (2C), 140.21, 182.25. ESI-MS: Yield 87%. mp: 167–169°C. 1H-NMR (DMSO-d6) δ (ppm):
m/z=325.9 [M+Na]+ (100%).
7.04 (brs, 1H, CHarom.); 7.19–7.28 (m, 3H, CHarom.); 7.42 (s,
1-Cyclohexyl-3-(1,3-thiazol-2-yl)thiourea (4) Yield 92%. 1H, CHarom.); 7.78 (brs, 1H, CHarom.); 12.31 (brs, 1H, NH);
1
mp: 154–156°C. H-NMR (DMSO-d6) δ (ppm): 1.28–1.41 (m, 12.39 (s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 115.88,

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
233
124.12 (2C), 126.93, 127.43, 128.51 (2C), 154.63, 157.89, 181.51. ylcarbamothioyl)carbamate (22)36) have been synthesized as
ESI-MS: m/z=276.0 [M+Na]+ (100%).
described previously.
Biology
In Vitro Evaluation of Antimicrobial Activity The anti-
1-(3-Fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (6) have been
synthesized as described previously.28)
1-(2-Chlorophenyl)-3-(1,3-thiazol-2-yl)thiourea (7) Yield bacterial activity of compounds was tested against a series of
91%. mp: 160–161°C. 1H-NMR (DMSO-d6) δ (ppm): 7.10 Gram-positive bacteria: Staphylococcus aureus ATCC 4163,
(brs, 1H, CHarom.); 7.23–7.28 (t, 1H, CHarom., J=6.6Hz); Staphylococcus aureus ATCC 25923, Staphylococcus aureus
7.34–7.38 (t, 1H, CHarom., J=7.5Hz); 7.44–7.54 (m, 2H, CHa- ATCC 29213, Staphylococcus aureus ATCC 6538, Staphy-
rom.); 7.82–7.84 (d, 1H, CHarom., J=7.8Hz); 10.39 (brs, 1H, lococcus epidermidis ATCC 12228, Bacillus subtilis ATCC
NH); 12.35 (brs, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 6633, Bacillus cereus ATCC 11778, Enterococcus hirae
111.54, 127.31 (3C), 128.65 (2C), 129.48 (2C), 136.10, 180.21. ATCC 10541, Micrococcus luteus ATCC 9341, Micrococcus
ESI-MS: m/z=292.0 [M+Na+H]+ (100%).
luteus ATCC 10240, and Gram-negative rods: Escherichia
1-(3-Chloro-4-methylphenyl)-3-(1,3-thiazol-2-yl)thiourea (8) coli ATCC 10538, Escherichia coli ATCC 25922, Escherichia
1
Yield 79%. mp: 158–159°C. H-NMR (DMSO-d6) δ (ppm): coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas
2.28 (s, 3H, CH3); 6.98–6.99 (d, 1H, CHarom., J=3.9Hz); aeruginosa ATCC 15442, Pseudomonas aeruginosa NCTC
7.23–7.26 (d, 1H, CHarom., J=8.4Hz); 7.45–7.56 (d, 1H, CHa- 6749, Pseudomonas aeruginosa ATCC 27853, Bordetella
rom., J=4.2Hz); 7.56–7.59 (d, 1H, CHarom., J=8.1Hz); 7.84 bronchiseptica ATCC 4617. Antifungal activity was tested
(s, 1H, CHarom.); 10.31 (brs, 1H, NH); 12.70 (brs, 1H, NH). against yeasts: Candida albicans ATCC 10231, Candida al-
13C-NMR (DMSO-d6) δ (ppm): 18.96, 110.22, 120.59, 121.77, bicans ATCC 90028, Candida parapsilosis ATCC 220191.
122.56 (2C), 129.46, 130.72, 132.62, 139.07, 180.81. ESI-MS: Microorganisms used in this study were obtained from the
m/z=306.0 [M+Na]+ (100%).
1-(3-Chloro-4-fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (9) Medical University of Warsaw, Poland.
collection of the Department of Pharmaceutical Microbiology,
(PubChem Compound ID: 21008657) Yield 83%. mp:
171–172°C. H-NMR (DMSO-d6) δ (ppm): 6.99–7.01 (d, 1H, against the M. tuberculosis H37Rv strain (ATCC 25618) and
The tuberculostatic activity of compounds was tested
1
CHarom., J=4.2Hz); 7.35–7.38 (d, 1H, CHarom., J=7.5Hz); two ‘wild’ strains isolated from tuberculosis patients (Spec.
7.47–7.49 (d, 2H, CHarom., J=4.5Hz); 8.06 (s, 1H, CHarom.); 192, Spec. 210). All strains were obtained from the collection
10.42 (brs, 1H, NH); 12.81 (brs, 1H, NH). 13C-NMR (DMSO- of the Department of Microbiology, National Tuberculosis and
d6) δ (ppm): 110.21, 117.67, 119.34, 125.39, 127.50, 129.48 (2C), Lung Diseases Research Institute, Warsaw, Poland.
130.74, 140.74, 182.26. ESI-MS: m/z=310.1 [M+Na]+ (100%).
Media, Growth Conditions and Antimicrobial Activ-
Crystal data for 9: monoclinic space group P21/c, unit cell ity Assays Antimicrobial activity was examined by the
dimensions a=10.091(2)Å, b=3.949(1) Å, c=28.384(6)Å, disc diffusion and MIC method under standard conditions,
3
β=95.45(3)°, V=1126.0(4) Å , Z=4, dcalc=1.697 g/cm3, μ= using Mueller–Hinton II agar medium (Becton Dickinson) for
6.430mm, F(000)=584. Crystal size 0.15×0.03×0.02mm3; re- bacteria and RPMI agar with 2% glucose (Sigma) for yeasts,
flections collected/independent/observed 3754/2025/1393; Good- according to CLSI (previously NCCLS) guidelines.22) Solu-
ness-of-fit on F2=1.031; final R indices [I>2σ(I)]R1=0.0913, tions containing the tested agents were prepared in methanol
−3
wR2=0.2286; Δρ max/min 0.99/−0.67eÅ .
or dimethyl sulfoxide (DMSO). For the disc diffusion method,
The experimental details and final atomic parameters have sterile paper discs (9mm diameter, Whatman No. 3 chroma-
been deposited with the Cambridge Crystallographic Data tography filter paper) were dripped with the compound solu-
Centre as supplementary material (CCDC No. 994080). Cop- tions tested to obtain 400µg of substance per disc. Dry discs
ies of the data can be obtained free of charge by emailing were placed on the surface of an appropriate agar medium.
data_request@ccdc.cam.ac.uk or on request via www.ccdc. The results (diameter of the growth inhibition zone) were read
cam.ac.uk/data_request/cif.
after 18h of incubation at 35°C. Minimal inhibitory concentra-
1-(1,3-Thiazol-2-yl)-3-[3-(trifluoromethyl)phenyl]thiourea tion (MIC) were examined by the twofold serial agar dilution
(10) Yield 81%. mp: 1741–1775°C. 1H-NMR (DMSO-d6) technique.23) Concentrations of the tested compounds in solid
δ (ppm): 6.98–6.99 (d, 1H, CHarom., J=4.2Hz); 7.29–7.35 medium ranged from 3.125 to 400µg/mL. The final inoculum
(t, 1H, CHarom., J=9.3Hz); 7.47–7.48 (d, 1H, CHarom., of studied organisms was 104 (colony forming units per milli-
J=4.2Hz); 7.66–7.71 (m, 2H, CHarom.); 7.99–8.02 (dd, 1H, liter (CFU/mL)), except the final inoculum for E. hirae ATCC
CHarom., J=4.5Hz); 10.29 (brs, 1H, NH); 12.90 (brs, 1H, 10541, which was 105 CFU/mL. MICs were read off after 18h)
NH). 13C-NMR (DMSO-d6) δ (ppm): 110.25, 116.27 (2C), of incubation at 35°C.
116.56 (2C), 118.70, 118.95, 122.14, 123.23, 137.34, 182.17. ESI-
The synthesized compounds were examined in vitro for
their tuberculostatic activity. Investigations were performed
MS: m/z=326.0 [M+Na]+ (100%).
1-Phenyl-3-(1,3-thiazol-2-yl)thiourea (11),29) 1-(4-methoxy- by a classical test-tube method of successive dilution in You-
phenyl)-3-(1,3-thiazol-2-yl)thiourea (12), 1-(4-chlorophenyl)-3- mans’ modification of the Proskauer and Beck liquid medium
(1,3-thiazol-2-yl)thiourea
(13),
1-(4-methylphenyl)-3-(1,3- containing 10% of bovine serum.37,38) Bacterial suspensions
thiazol-2-yl)thiourea (14), 1-(4-fluorophenyl)-3-(1,3-thiazol-2- were prepared from 14d old cultures of slowly growing
yl)thiourea (15), 1-(4-bromophenyl)-3-(1,3-thiazol-2-yl)thiourea strains. Solutions of compounds in DMSO were tested. Stock
(16),30) 1-(4-iodophenyl)-3-(1,3-thiazol-2-yl)thiourea (17),31) solutions contained 10mg of compounds in 1 mililitre. Dilu-
1-(4-benzylophenyl)-3-(1,3-thiazol-2-yl)thiourea (18),32) N-(1,3- tions (in geometric progression) were prepared in Youmans’
thiazol-2-ylcarbamothioyl)benzamide(19),33) 1-(3-chlorophenyl)-3- medium. The medium containing no investigated substances
(1,3-thiazol-2-yl)thiourea (20),34) 1-(3-chloro-6-methylphenyl)- and containing isoniazid (INH) as reference drugs were used
3-(1,3-thiazol-2-yl)thiourea (21)35) and ethyl(1,3-thiazol-2- for comparison. Incubation was performed at 37°C. The MIC

234
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
values were determined as minimum concentration inhibiting low fever virus (YFV) [strain 17-D vaccine (Stamaril Pasteur
the growth of tested tuberculous strains. The influence of the J07B01)] and bovine viral diarrhoea virus (BVDV) [strain
compound on the growth of bacteria at the certain concentra- NADL (ATCC VR-534)]; (iii) Picornaviridae: human entero-
tion, 3.1, 6.2, 12.5, 25, 50 and 100µg/mL, were evaluated.
virus B [coxsackie type B5 (CVB-5), strain Ohio-1 (ATCC
Biofilm Inhibitory Assay Inhibition of bacterial biofilm VR-29)], and human enterovirus C [poliovirus type-1 (Sb-1),
formation was screened using method, described previously,39) Sabin strain Chat (ATCC VR-1562)]. Viruses representative
with some modification. Eight hospital isolates of methicillin- of negative-sense, single-stranded RNAs (ssRNA−) were:
resistant and two standard strains of Staphylococcus epidermi- (iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab
dis (ATCC 12228, ATCC 35984) were cultured overnight in strain Indiana (ATCC VR 1540)]. The virus representative of
Tryptone Soy Broth with 0,5% glucose. The solution of tested double-stranded RNAs (dsRNA) was reovirus type-1 (Reo-1)
compounds in TSB-glucose medium were mixed (1:1) with [simian virus 12, strain 3651 (ATCC VR-214)], Reoviridae
the bacterial inoculums (107 CFU/mL) in sterile 96-well poly- family. DNA virus representatives were: (v) Poxviridae:
styrene microtiter plates (Karell-Medlab, Italy) and incubated vaccinia virus (VV) [vaccine strain Elstree-Lister (ATCC
at 37°C for 24h. The final concentrations of tested compounds VR-1549)]; vi) Herpesviridae: human herpes 1 (HSV-1) [strain
ranged from 1 to 16µg/mL. The positive control—biofilm for- KOS (ATCC VR-1493)].
mation, was bacterial culture in TSB-glucose, negative control
Cytotoxicity Assays Exponentially growing MT-4 cells
was TSB-glucose medium. After incubation, medium was re- were seeded at an initial density of 1×105 cells/mL in 96-well
moved from wells and washed with sterile phosphate buffered plates in RPMI-1640 medium, supplemented with 10% fetal
saline (PBS) to remove the non-adherent bacteria. Alive bacte- bovine serum (FBS), 100units/mL penicillin G and 100µg/mL
rial cells in each well were stained with 0.5% MTT (3-(4,5-di- streptomycin. Cell viability was determined after 96h at 37°C
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) for by the MTT method.40)
2h at 37°C. Adherent bacterial cells, which usually formed
As far as stationary monolayers (analogous to those which
biofilm on wells, were uniformly stained with MTT. After in- support the replication of the other RNA and DNA viruses)
cubation, the solution was removed and bacterial biofilm was are concerned, MDBK and BHK cells were seeded in 24-
solubilized by DMSO with glycine buffer and mixed 15min well plates at an initial density of 6×105 and 1×106 cells/mL,
at room temperature. The absorbance (A554) was recorded at respectively, in minimum essential medium with Earle’s salts
554nm using spectrophotometer (PowerWave XS, BioTek).
(MEM-E), L-glutamine, 1mM sodium pyruvate and 25mg/L
The biofilm-inhibition results were interpreted from concen- kanamycin, supplemented with 10% horse serum (MDBK) or
trations-response curve. IC50 value is defined as the concentra- 10% foetal bovine serum (FBS) (BHK). Cell viability was de-
tions of tested compounds required to inhibit 50% of biofilm termined after 48–96h at 37°C by the MTT method.
formation under the assay conditions. All the experiments
were performed in four replicates. Statistical analysis was per- density of 4×105 cells/mL, in Dulbecco’s modified Eagle’s
formed using program Statistica 5.0 PL. medium (DMEM) with L-glutamine and 25mg/L kanamycin,
Vero-76 cells were seeded in 24-well plates at an initial
S. epidermidis strain ATCC 12228 and S. epidermidis supplemented with 10% FBS. Cell viability was determined
ATCC 35984 were used in assays as a negative (low biofilm- after 48–96h at 37°C by the crystal violet staining method.
producing) and positive (hgh biofilm-producing) control, re-
spectively. Ciprofloxacin was used as reference antibacterial fied, 5% CO2 atmosphere, in the absence or presence of se-
compound.
rial dilutions of test compounds in culture medium. Before
All cell cultures were then incubated at 37°C in a humidi-
All strains of S. epidermidis (hospital and standard) were dilutions, compounds were dissolved in dimethyl sulfaxide
also tested for slime production on Congo Red Agar (CRA) (DMSO) at 100mM.
supplemented with 0.8g/L of Congo red (Sigma) and 50g/L
Antiviral Assays Compound’s activity against HIV-1
sucrose (Sigma). Plates were incubated for 24h at 37°C and was based on inhibition of virus-induced cytopathogenicity in
for 24h at room temperature, in the dark.
exponentially growing MT-4 cell acutely infected with a mul-
Cell-Based Assays
tiplicity of infection (m.o.i.) of 0.01. Briefly, 50µL of RPMI
Cells and Viruses Cell lines were purchased from Ameri- containing 1×104 MT-4 cells were added to each well of flat-
can Type Culture Collection (ATCC). The absence of myco- bottom microtitre trays, containing 50µL of RPMI without
plasma contamination was checked periodically by the Hoechst or with serial dilutions of test compounds. Then, 20µL of a
staining method. Cell lines supporting the multiplication of HIV-1 suspension containing 100 CCID50 were added. After
RNA and DNA viruses were the following: CD4+ human T- a 4-d incubation at 37°C, cell viability was determined by the
cells containing an integrated HTLV-1 genome (MT-4); Madin MTT method.
Darby Bovine Kidney (MDBK) [ATCC CCL 22 (NBL-1) Bos
Compound’s activity against YFV and Reo-1 was based on
Taurus]; Baby Hamster Kidney (BHK-21) [ATCC CCL 10 inhibition of virus-induced cytopathogenicity in BHK-21 cells
(C-13) Mesocricetus auratus] and Monkey kidney (Vero 76) acutely infected with a m.o.i. of 0.01.
[ATCC CRL 1587 Cercopithecus Aethiops].
Compound’s activity against BVDV was based on inhibition
Viruses were purchased from American Type Culture Col- of virus-induced cytopathogenicity in MDBK cells acutely
lection (ATCC), with the exception of Yellow Fever Virus infected with a m.o.i. of 0.01. Briefly, BHK and MDBK cells
(YFV), and Human Immunodeficiency Virus type-1 (HIV-1). were seeded in 96-well plates at a density of 5×104 and
Viruses representative of positive-sense, single-stranded 3×104 cells/well, respectively, and were allowed to form con-
RNAs (ssRNA+) were: (i) Retroviridae: the IIIB laboratory fluent monolayers by incubating overnight in growth medium
strain of HIV-1, obtained from the supernatant of the persis- at 37°C in a humidified CO2 (5%) atmosphere. Cell monolay-
tently infected H9/IIIB cells (NIH 1983); (ii) Flaviviridae: yel- ers were then infected with 50µL of a proper virus dilution

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
235
in maintenance medium [MEM-Earl with L-glutamine, 1mM between HOMO and LUMO energy levels (HLG), the higest
sodium pyruvate and 0.025g/L kanamycin, supplemented occupied molecular orbital (HOMO), the lowest unoccupied
with 0.5% inactivated FBS] to give an m.o.i of 0.01. After 1h, molecular orbital (LUMO), hardness (η) obtained from the
50µL of maintenance medium, without or with serial dilutions equation η=(HOMO−LUMO)/2, Mullkien electronegativity
of test compounds, were added. After a 3-/4-d incubation at (χ) obtained from the equation χ=−(HOMO+LUMO)/2, total
37°C, cell viability was determined by the MTT method.
energy (ET), binding energy (EB), isolated atomic energy (EIA),
Compound’s activity against YFV and Reo-1 was based on electronic energy (EE), core–core interaction (IC–C), heat of
inhibition of virus-induced cytopathogenicity in BHK-21 cells formation (HF), refractivity (Rf), polarizability (α) were col-
acutely infected with a m.o.i. of 0.01.
lected in Table 6.
Compound’s activity against BVDV was based on inhibition
of virus-induced cytopathogenicity in MDBK cells acutely
Acknowledgments We gratefully acknowledge the Sar-
infected with a m.o.i. of 0.01. Briefly, BHK and MDBK cells dinia Regional Government for the financial support of Silvia
were seeded in 96-well plates at a density of 5×104 and Madeddu through his Ph.D. scholarship (P.O.R. Sardegna
3×104 cells/well, respectively, and were allowed to form con- F.S.E. Operational Programme of the Autonomous Region of
fluent monolayers by incubating overnight in growth medium Sardinia, European Social Found 2007–2013).
at 37°C in a humidified CO2 (5%) atmosphere. Cell monolay-
ers were then infected with 50µL of a proper virus dilution
Conflict of Interest The authors declare no conflict of
in maintenance medium (MEM-E with L-glutamine, supple- interest.
mented with 0.5% inactivated FBS, 1mM sodium pyruvate
and 0.025g/L kanamycin) to give an m.o.i of 0.01. After 1h,
50µL of maintenance medium, without or with serial dilutions
of test compounds, were added. After a 3–4d incubation at
37°C, cell viability was determined by the MTT method.
Compound’s activity against CVB-5, Sb-1, VV and HSV-1
was determined by plaque reduction assays in infected cell
monolayers. To this end, Vero 76-cells were seeded in 24-well
plates at a density of 2×105 cells/well and were allowed to
form confluent monolayers by incubating overnight in growth
medium (DMEM with L-glutamine and 4.5g/L D-glucose and
0.025g/L kanamycin, supplemented with 10% FBS) at 37°C
in a humidified CO2 (5%) atmosphere. Then, monolayers were
infected for 2h with 250µL of proper virus dilutions to give
50–100PFU/well. Following removal of unadsorbed virus,
500µL of maintenance medium (DMEM with L-glutamine
and 4.5g/L D-glucose, supplemented with 1% inactivated
FBS) containing 0.75% methyl-cellulose, without or with se-
rial dilutions of test compounds, were added. Cultures were
incubated at 37°C for 2 (Sb-1 and VSV) or 3d (CVB-5, VV
and HSV-1) and then fixed with PBS containing 50% ethanol
and 0.8% crystal violet, washed and air-dried. Plaques were
then counted.
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