Disubstituted thiourea derivatives and their activity on CNS: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.07.020

A series of new thiourea derivatives of 1,2,4-triazole have been synthesized. The difference in structures of obtained compounds are directly connected with the kind of isothiocyanate (aryl/alkyl). The ¹H NMR, ¹³C NMR, MS methods wer

Full text of DOI:10.1016/j.ejmech.2012.07.020

European Journal of Medicinal Chemistry 55 (2012) 205e213
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Disubstituted thiourea derivatives and their activity on CNS: Synthesis
and biological evaluation
Joanna Stefanska a, Daniel Szulczyk b, Anna E. Koziol c, Barbara Miroslaw c, Ewa Kedzierska d,
Sylwia Fidecka d, Bernardetta Busonera e, Giuseppina Sanna e, Gabriele Giliberti e, Paolo La Colla e,
,
Marta Struga b
*
a Department of Pharmaceutical Microbiology, Medical University, 02-007 Warsaw, Poland
b Department of Medical Chemistry, Medical University, 3 Oczki Str., 02-007 Warsaw, Poland
c Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
d Department of Pharmacology and Farmacodynamics, Medical University, 20-059 Lublin, Poland
e Department of Biomedical Science and Technology, University of Cagliari, 09042 Monserrato (CA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new thiourea derivatives of 1,2,4-triazole have been synthesized. The difference in structures
of obtained compounds are directly connected with the kind of isothiocyanate (aryl/alkyl). The 1H NMR,
13C NMR, MS methods were used to confirm structures of obtained thiourea derivatives. The molecular
structure of (1, 17) was determined by an X-ray analysis. Two of the new compounds (8 and 14) were
tested for their pharmacological activity on animal central nervous system (CNS) in behavioural animal
tests. The results presented in this work indicate the possible involvement of the serotonergic system in
the activity of 8 and 14. In the case of 14 is also a possible link between its activity and the endogenous
opioid system. All obtained compounds were tested for antibacterial activity against Gram-positive cocci,
Gram-negative rods and antifungal activity. Compounds (1, 2, 5, 7, 9) showed significant inhibition
against Gram-positive cocci. Microbiological evaluation was carried out over 20 standard strains and 30
hospital strains. Selected compounds (1e13) were examined for cytotoxicity, antitumor, and anti-HIV
activity.
Received 19 March 2012
Received in revised form
4 July 2012
Accepted 15 July 2012
Available online 27 July 2012
Keywords:
CNS activity 5-HT system connection
Antibacterial activity
Cytotoxicity
Antitumor activity
Thiourea derivatives of 1,2,4-triazole
X-ray crystal structure analysis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
triazoles are perspective scaffolds for designing new families of
compounds with therapeutic importance.
Triazoles are an important group of compounds due to their
wide range of applications as pharmaceutical agents. The thera-
peutic application of 1,2,4-triazole derivatives as widely used drugs
is well documented; they show broad spectrum of activity, i.e.:
antifungal [1e7], antiviral [8,9] antidepressant [10,11] and anti-
migrain [12].
In our previous study [30,31], we reported the synthesis and
biological activity of thiourea derivatives.
In the recent literature, the thioureas are described as most
useful class of agents with large number of activities including
antiviral [32,33] antibacterial [34] and HDL-elevating properties
[35].
Various new obtained derivatives of 1,2,4-triazole have been
reported to possess interesting biological activity such as antimi-
crobial [13e17], antitumor [18e23], anti-inflamatory [24e27]
antioxidant [28] and anticonvulsant [29]. Structural properties of
triazoles, like moderate dipole character, hydrogen bonding capa-
bility, rigidity and stability under in vivo conditions are main
reasons for their superior pharmacological activities. Therefore
Numerous compounds containing a thiourea group are selective
ligands for 5-HT family of receptors, including 5-HT2A, 5-HT2Band
5-HT2c [36e39]. In addition, structural analyses of bioactive thio-
ureas have shown that many of these compounds possessing two
hydrophobic moieties and one central hydrophilic part form
a butterfly-like conformation [40]. Anti-HIV agents also adapt this
type of conformation (e.g. the nucleoside reverse transcriptase
inhibitors (NNRTIs) [40].
Literature survey shows that only one connection between
1,2,4-triazole and thiourea was described. N-(1,2,4-triazol-3-yl)-
N0arylthioureas act as effective uncouplers of oxidative phosphor-
ylation in mitochondria [41].
* Corresponding author. Tel./fax: þ48 0226280679.
E-mail addresses: joanna.stefanska@wum.edu.pl (J. Stefanska), marta.struga@
wum.edu.pl, strugam@wp.pl (M. Struga).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.020

206
J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
Table 1
Prompted by these facts, we decided to design new compounds
Structure and molar weight of the investigated compounds 1e18.
containing above-mentioned moieties viz. 1,2,4-triazole and thio-
urea, to explore their possible biological activities.
Symbol
R
Mol. weight (g/mol)
1
2
3
4
5
6
7
8
Phenyl
219.06
249.07
253.02
233.07
237.05
296.97
296.97
269.97
344.95
286.98
247.05
237.05
237.05
233.07
225.10
247.09
197.07
215.05
4-Methoxyphenyl
4-Chlorophenyl
4-Methylphenyl
4-Fluorophenyl
2-Bromophenyl
4-Bromophenyl
3-Bromophenyl
4-Iodophenyl
3,4-Dichlorophenyl
Benzoyl
2-Fluorophenyl
3-Fluorophenyl
Benzyl
Cyclohexyl
Phenethyl
2. Result and discussion
2.1. Chemistry
The preparation of 18 thiourea derivatives is described, however
12 of them are new. 3-Amino-1,2,4-triazole was subjected to
reaction with isothiocyanates in order to be transformed into the
corresponding thiourea derivatives (Scheme 1 and Table 1). The
chemical character of isothiocyanate substituent determined
product of the reaction. When aryl derivative of isothiocyanate was
used as reagent, the amino group of 3-amino-1,2,4-triazole was
substituted. When substituent is bonded to isothiocyanate through
the Csp3 atom, the imine N2-atom of 1,2,4-triazole take part in the
reaction and the products have unsubstituted amino group. Theo-
retical calculations [42] confirm this mechanism only for a hydro-
philic solvent, like acetonitrile. In a hydrophobic solvent two forms
of the product are expected.
9
10
11
12
13
14
15
16
17
18
Methallyl
Ethyl formate
did not affect hyperactivity induced by administration of amphet-
amine, which seems to rule out the involvement of the catechol-
amine system in their action (Figs. 2 and 5).
The tested compounds, used at a dose equivalent to 0.1 ED50 and
at half that dose, i.e. 0.05 ED50, significantly decreased the number
of writhing episodes (Fig. 6).
Obtained compounds were purified by flash chromatography.
MS, 1H NMR and 13C NMR spectra confirmed the identity of the
products. The molecular structure of 1 and 17 (Fig. 1) was deter-
mined by an X-ray crystal structure analysis.
Their antinociceptive activity has been confirmed at the tested
doses (Fig. 7).
2.2. Pharmacology
The use of naloxone inhibited only the antinociceptive action of
14, causing a repeated increase in the number of writhing episodes
(from 6.7 ꢀ 1.4 to 13.2 ꢀ 0.99, respectively (p < 0.05)), which may
indicate a link between this activity and the endogenous opioid
system (Fig. 7).
The tested compounds, used at a dose of 0.1 ED50, caused no
coordination disorders, as they did not change the behaviour of
mice in either test. The results obtained in all groups were similar to
the values in the control group, indicating a lack of a depressive
effect on the striated muscles and coordination (Figs. 3 and 4).
In the pentetrazol seizure test, none of the tested compounds
clearly reduced the severity of clonic or tonic seizures, or protected
the animals from dying.
In this study we tested properties of two new thiourea deriva-
tives, 8 and 14. The connections between 1,2,4-triazole and thio-
urea have not been tested on central nervous system (CNS) yet. The
first compound (8) possessed free thiourea system connected to
1,2,4-triazole whereas in a second (14) thiourea system was built-in
1,2,4-triazole. In this connection compounds 8 and 14 formed
a good comparative group.
The spontaneous activity and amphetamine hyperactivity were
evaluated. The effect on body temperature and behaviour of
animals caused by administration of L-5-HTP, motor coordination
as well as nociceptive and anticonvulsant activity was estimated.
These allowed preliminary determination of the impact of the new
substances on the CNS of experimental animals.
Tests were also carried out to evaluate the effect of the new
thiourea derivatives on the “head-twitch” response in mice caused
by administration of a serotonin precursor, L-5-HTP, which may
indicate the involvement of the serotonergic system in the
observed effects (Fig. 8). The drug-elicited head twitch response
(HTR) [43,44] is a selective behavioural model for 5-HT2 agonist
activity in rodents, and several previous studies have established
that direct and indirect 5-HT agonists induce this effect [45e52].
Additionally, 5-HT2 receptor antagonists selectively block HTR
[52e54], and their potency is highly correlated with the antago-
nist’s affinity for 5-HT2 receptors [45,55].
The tested compounds, used at a dose equivalent to 0.1 ED50, did
not statistically significantly change motor activity of animals and
Among the compounds tested, only 14 significantly (p < 0.05)
reduced the number of the “head-twitch” episodes. The activity of 8
was also evident, but the result did not reach statistical significance.
Since 5-HT2 receptors mediate the occurrence of a typical “head-
twitch” response caused by L-5-HTP, obtained data seem to suggest
interactions with these receptors in the brain.
Tests of the effect of the new compounds on body temperature
in normothermic mice showed that 8 and 14 only reduced it
episodically at 90 min (Fig. 9).
The results presented in this work, based on literature data,
indicate the possible involvement of the serotonergic system in the
activity of the thiourea series compounds. 5-HT2 and/or 5-HT1A
receptors are also probably involved, which seems to be confirmed
Scheme 1. Synthesis of studied compounds.

J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
207
Fig. 1. Molecular structure of 1 (left) and 17 (right). Displacement ellipsoids drawn at 50% probability level.
16
250
200
150
100
50
Control
Control
0.1 ED50
14
0.1 ED50
12
10
8
6
4
2
0
0
8
14
8
14
Fig. 2. The influence of 8 and 14 on spontaneous motor activity.
Fig. 4. The influence of 8 and 14 on motor coordination evaluated in chimney test.
by the inhibition of the “head twitch” response by 14. In the case of
14, one should also remember about the possible link between its
activity and the endogenous opioid system, as the presence of free
amino group should cause additional influence on the endogenous
opioid system.
Behavioural animal tests directed on central nervous system
(CNS) were a part of larger project of research. Up to now six new
compounds composed of urea and thiourea system were tested.
Previously tested compounds formed a connection of urea or
thiourea system and tricyclic imide (1-(,7,8,9,10-pentamethyl-3,5-
dioxo-4-aza-tricyclo[5.2.1.02,6]dec-8-en-4-yl)-3-phenylurea and 1-
(4-iodophenyl)-3-(1,7,8,9-tetramethyl-3,5-dioxo-4-azatricyclo
[5.2.1.02,6]dec-8-en-4-yl)thiourea) [31,43] or thiourea system as
our previous research on central nervous system (CNS) for urea [56]
or thiourea [31] and 1,3-thiazepine derivatives [30] of tricyclic
imide and described in this paper connection of thiourea and 1,2,4-
triazole, we could conclude that this group of compounds possesses
significant influence on central nervous system (CNS) of laboratory
animals. Comparison of urea and thiourea derivatives shows that
urea has influence on “head twitch response” only. However thio-
urea and 1,3-thiazepine derivatives of tricyclic imides were active
in “head twitch response” test similarly to urea derivatives and
have influence on spontaneous motor activity and decrease body
temperature of normothermic mice. Thiourea derivatives of 1,2,4-
triazole are similar in influence on “head twitch response” and
decrease body temperature of normothermic mice but significantly
decreased the number of writing episodes and possessed anti-
noceptive activity. Based on performed research we could conclude
that urea derivatives were less active than thiourea derivatives.
Probably the urea or thiourea part only has influence on CNS
activity. The replacement of imide moiety bound to thiourea with
1,2,4-triazole has no influence on the activity. As well as the
difference in number of methyl groups connected to an imide has
a
part of thiazepine ring (10-isopropyl-8-methyl-4-{2-[(Z)-4-
methylphenylimino]-1,3-thiazepan-3-yl}-4-azatricyclo [5.2.2.02,6
undec-8-ene-3,5-dione and 1-isopropyl-7-methyl-4-{2-[(Z)-3,4-
dichlorophenylimino]-1,3-thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6
]
]
undec-8-ene-3,5-dione) [30]. In the present work tricyclic imide
connected to thiourea system is replaced by 1,2,4-triazole. Base on
60
Control
0.1 ED50
2500
Control
Amphetamine 5mg/kg
***
50
40
30
20
10
0
2000
0.1 ED50
1500
0.1 ED50
+
amphetamine
5 mg/kg
1000
500
0
*** p < 0.001 vs control
14
8
14
8
Fig. 3. The influence of 8 and 14 on motor coordination evaluated in rota-rod test.
Fig. 5. The influence of 8 and 14 on amphetamine induced hyperactivity.

208
J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
Control
Control
8
°C
35
20
15
10
5
0.1 ED50
0.05 ED50
14
34
0.05 ED50+ naloxone 5 mg/kg
^
* p<0.05 vs control
^ p<0.05 vs compound
*** p<0.001 vs control
33
*
*
32
***
0
31
8
14
***
Fig. 6. The influence of naloxone, 5 mg/kg s.c. on antinociceptive activity of 8 and 14
evaluated in ‘writhing’ test. Note: the results are expressed as mean ꢀ S.E.M. of a group
of 8e10 mice.
30
0
30
60
90
120
150
180
210
min
Fig. 9. The influence of 8 and 14 on the body temperature of mice. Note: each point
represents the mean for a group of 10 mice.
Control
20
0.1 ED50
and Candida albicans. Microorganisms used in this study have
common application in the antimicrobial tests for many
substances like antibiotics, disinfectants and antiseptic drugs or in
research on new antimicrobial agents [57]. All the compounds
were screened for their antimicrobial activity by disc diffusion
method [58]. Compounds showing significant activity test were
next examined for their minimal inhibitory concentration (MIC)
[59]. The results of activity for compounds are summarized in
Tables 2 and 3.
15
0.05 ED50
0.025 ED50
* p < 0,05
** p < 0,01
vs control
10
*
**
*
5
**
0
Preliminary test by disc-diffusion method showed antimicrobial
activity against standard Gram-positive cocci, therefore the next
step was evaluation of compounds’ MIC values for standard
hospital strains. The research was carried out over 20 standard
strains, 20 hospital stains of Staphylococcus aureus and 10 hospital
strains of Staphylococcus epidermidis used for routine antimicrobial
media susceptibility testing. Hospital strains were isolated from
different biological materials of patients hospitalized in Warsaw
Medical University Hospitals.
8
14
Fig. 7. The antinociceptive effects of 8 and 14 assessed in the ‘writhing’ test in mice.
Note: the results are expressed as mean ꢀ S.E.M. of a group of 8e10 mice.
not differentiated compounds on CNS activity. The molar masses of
examinated compounds have fluctuated among 233 g molꢁ1 to
507 g molꢁ1 and this factor really has not influenced on pharma-
cological activity in this case. Even if the thiourea was closure in 1,3-
thiazepine ring, the activity on CNS was comparable with the free
thiourea. Only a free amino group connected to 1,2,4-triazole,
which was part of thiourea, has an impact on the CNS activity.
Comparison the activity on CNS of thiourea connected of tricyclic
imides and 1,2,4-triazole derivatives could lead to the conclusion
that generally thiourea compounds indicate the possible involve-
ment of the serotonergic system but further 1,2,4-triazole with
amino group added to thiourea should cause additional influence
on the endogenous opioid system.
Among S. aureus 10 strains showed methicilin susceptibility
(MSSA) and 10 strains showed methicilin resistance (MRSA). MIC
value of standard Gram-positive strain as in the range 400e12.5
mg/
ml (Table 3).
MIC value of hospital S. aureus strain as in the range 50e12.5
mg/
ml (Table 4).
Table 2
Antibacterial and antifungal in vitro activity expressed as diameter of growth
inhibitory zone (GIZ, mm) for tested compounds (applied 400 g per disc).
m
1
3
4
5
6
7
8
9
12 13
2.3. Microbiology
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
E. coli ATCC 10538
E. coli ATCC 25922
E. coli NCTC 8196
P. vulgaris NCTC 4635
P. aeruginosa ATCC 15442
P. aeruginosa NCTC 6749
P. aeruginosa ATCC 27853
B. bronchiseptica ATCC 4617
C. albicans ATCC 10231
C. albicans ATCC 90028
C. parapsilosis ATCC 22019
e
e
e
23
23
15
e
e
e
e
e
14 11 17
18 13 22
14 12 16
11 12 16
16 12 16
e
12 12 16
13 19 34
12 12 15
13 12 18
e
e
e
e
All obtained compounds were tested in vitro against a number
of bacteria, including Gram-positive cocci, Gram-negative rods
11 17
12 20
e
11 22
16 22 13 22 17 30 16 18 20 32
20
12 16
23
14 26
e
e
e
16 12 18
11 13 25
19 16 28 15 13 24 36
e
13 11 14
13 16
Control
18
e
e
e
0.1 ED50
16
12 30 12 23 15 29 14 16 38 40
14
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
* p < 0.05 vs control
12
10
8
6
*
4
2
0
14
8
Fig. 8. The influence of
8 and 14 on ‘head twitch’ response evoked by
5-hydroxytryptophan (5-HTP). The results are expressed as mean ꢀ SEM (n ¼ 10).
e, Lack of the growth inhibition zone.

J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
209
Table 3
Activities of obtained compounds against Gram-positive bacteria e minimal inhibitory concentations (MIC,
m
g/ml).
1
3
5
6
7
9
12
13
50
50
100
50
50
50
50
400
50
Ciprofloxacin (5 mg per disc)
S. aureus NCTC 4163
100
100
100
100
50
25
25
25
25
50
50
50
25
50
50
50
50
25
12.5
25
400
6.25
6.25
50
50
50
25
25
25
25
25
200
200
200
200
200
100
100
400
100
100
0.5
0.5
0.5
0.5
0.5
<0.125
0.5
4
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
25
25
25
25
50
12.5
12.5
200
25
12.5
6.25
400
6.25
6.25
12.5
12.5
400
25
12.5
12.5
400
12.5
12.5
100
200
50
2
1
50
25
12.5
50
Table 5
MRSA strains were more resistant to investigated compounds,
the range of MIC value changed from 400 to 12.5 g/ml (Table 5).
For hospital S. epidermidis (MRSA) the MIC value changed from
400 to 12.5 g/ml and average value was 50 g/ml (Table 6). The
Activity of compounds against hospital methicillin-resistant strains of S. aureus
(MRSA) e minimal inhibitory concentrations (MIC, g/ml).
m
m
1
3
5
6
7
9
12
13
m
m
275/08
277/08
306/08
307/08
309/08
329/08
325/09
356/09
357/09
376/09
100
50
50
50
50
50
50
50
50
50
25
50
25
25
25
25
25
25
50
25
100
50
50
50
50
50
50
50
50
50
100
50
50
50
50
50
50
50
50
50
100
50
50
50
50
50
50
50
50
50
50
25
50
50
25
25
25
50
50
50
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
25
comparison between structure and biological activity is interesting.
The activity depends only on an aryl substituent. Phenyl substituted
fluorine, bromine, iodine atom and methoxy group is profitable for
antimicrobial activity.
Due to the previously reported anti-HIV activities of thiourea
derivatives [60,61], title compounds were tested in cell-based assay
against the human immunodeficiency virus type-1 (HIV-1), using
efavirenz as reference inhibitor. The cytotoxicity was evaluated in
parallel with the antiviral activity.
12.5
12.5
12.5
12.5
12.5
12.5
12.5
25
25
None of test compounds showed selective antiviral activity
against HIV-1. However, one of them, compound 14, turned out
cytotoxic for exponentially growing MT4 cells in the low micro-
previously separated on
a UPLC column (C18) using UPLC
ACQUITYÔ system by Waters connected with DPA detector. Flash
chromatography was performed on Merck silica gel 60 (200e400
mesh) using chloroform/methanol (19:1 vol) mixture as eluent.
Analytical TLC was carried out on silica gel F254 (Merck) plates
(0.25 mm thickness).
The intensity measurements of diffraction reflections were
carried out for 1 and 17 at 296 K with a KM4 diffractometer, using
graphite monochromated CuK
scan mode. Crystal structures were solved by the SHELXS-97
program and refined by full-matrix least squares on F2 using the
SHELXL-97 program [62]. All non-hydrogen atoms were refined
with anisotropic displacement parameters. Hydrogen atoms were
positioned geometrically and allowed to ride on their parent atoms,
with Uiso(H) ¼ 1.2 Ueq(C) and 1.5 for methyl groups.
molar range (CC50 ¼ 7.4 ꢀ 0.6
mM) (Table 7). The antiproliferative
activity against this CD4þ human T cell line derived from an hae-
matological human tumor, prompted us to evaluate the anti-
proliferative activity of 14 also for a panel of other human
haematological and solid tumours and for cell lines derived from
normal human tissues. Interestingly, a CC50 (11 ꢀ 0.7
mM) compa-
rable to that obtained with MT-4 cells was found also for the cell
line derived from a human prostate carcinoma (Tables 8 and 9).
ꢀ
a
radiation (
l
¼ 1.54178 A) and
u/2q
3. Experimental protocol
3.1. Chemistry
The NMR spectra were recorded on a Bruker AVANCE DMX400
spectrometer, operating at 300 MHz (1H NMR) and 75 MHz (13
C
3.1.1. General procedure
NMR). The chemical shift values are expressed in ppm relative to
TMS as an internal standard. Mass spectral ESI measurements were
carried out on Waters ZQ Micro-mass instruments with quadrupol
mass analyzer. The spectra were performed in the positive ion
mode at a declustering potential of 40e60 V. The sample was
A solution of 1H-1,2,4-triazol-3-amine (0.0069 mol, 0.58 g) in
acetonitrile (25 mL) was treated with appropriate isothiocyanate
(0.0075 mol) and the mixture was refluxed for 8 h. Then solvent
was removed on rotary evaporator. The residue was purified by
Table 6
Table 4
Activity of compounds against hospital methicillin-resistant strains of S. epidermidis
(MRSA) e minimal inhibitory concentrations (MIC, mg/ml).
Activity of compounds against hospital methicillin-susceptible strains of S. aureus
(MSSA) e minimal inhibitory concentrations (MIC,
mg/ml).
1
3
5
6
7
9
14
15
1
3
5
6
7
9
12
13
311/08
315/08
316/08
317/08
318/08
340/09
341/09
342/09
343/09
377/09
100
100
100
100
50
25
25
25
25
25
12.5
12.5
200
25
25
50
50
100
50
50
50
100
50
50
100
100
100
100
50
50
100
50
50
50
50
100
25
25
50
25
25
25
50
50
50
50
12.5
25
50
25
50
25
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
25
12.5
25
256/08
261/08
267/08
268/08
269/08
338/08
339/09
367/09
369/09
372/09
50
50
50
50
50
50
50
50
50
50
25
25
25
25
25
25
25
25
25
25
50
50
50
100
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
25
25
25
50
25
50
50
50
50
25
25
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
25
25
25
25
12.5
24
12.5
12.5
12.5
25
25
25
25
25
50
50
25
50
25
25
12.5
12.5
25
25
12.5
25
50
100
200
50
50
50
50
50

210
J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
Table 7
collected. The 4216 unique reflections [R(int) ¼ 0.0635] were used
for the refinement of 272 parameters, including extinction coeffi-
cient [x ¼ 0.027(2)]. Final R indices on F2 for 2975 observed
Cytotoxicity and anti-HIV-1 activity of 1e11, 14 and 15.
Compds
MT-4
HIV-1
reflections [I > 2
s
(I)] were: R1 ¼ 0.0514, wR2 ¼ 0.1361, goodness-
a
b
CC50
EC50
ꢀꢁ3
of-fit 1.038, and largest difference peak/hole 0.52/ꢁ0.35 e A
.
1
2
3
4
5
6
7
8
>100
>100
47
>100
>100
>47
3.1.4. 1-(4-Methoxyphenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (2)
Yield 91%. Mp. 199e200 ꢂC. 1H NMR (DMSO-d6)
(ppm): 3.70 (s,
59
>50
d
>100
>100
>100
50
46
47
>100
7.4 ꢀ 0.6
>100
37
>100
>100
>100
>50
>46
>47
>100
>7.4
>100
0.002 ꢀ 0.001
3H, OCH3); 6.85 (d, 2H, CHarom, J ¼ 9.0 Hz); 7.32 (d, 2H, CHarom
,
J ¼ 9.0 Hz); 8.34 (s, 1H, CH]). 13C NMR (DMSO-d6)
d (ppm):
55.12, 113.92 (2C), 119.86 (2C), 124.25, 128.46, 132.88, 152.89,
154.28. ESI MS: m/z ¼ 273.1 [M þ Na þ H]þ (100%).
9
10
11
14
15
EFV
3.1.5. 1-(4-Chlorophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (3)
Yield 87%. Mp. 206 ꢁ 200 ꢂC. 1H NMR (DMSO-d6)
d (ppm): 7.44
(d, 2H, CHarom, J ¼ 8.7 Hz); 7.67 (d, 2H, CHarom, J ¼ 9.0 Hz); 8.55 (s,
1H CH]); 11.08 (s, 1H, NH); 11.26 (s, 1H, NH); 14.01 (s, 1H, NH). 13C
Data represent mean values for three independent determinations.
a
NMR (DMSO-d6)
d (ppm): 126.02 (2C), 128.41 (2C), 129.52, 137.76,
Compound concentration (
mM) required to reduce the viability of mock-infected
146.23, 153.11, 157.68. ESI MS: m/z ¼ 276.2 [M þ Na]þ (100%).
MT-4 cells by 50%, as determined by the MTT method.
b
Compound concentration (mM) required to achieve 50% protection of MT-4 cells
from the HIV-1-induced cytopathogenicity, as determined by the MTT method.
3.1.6. 1-(4-Methylphenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (4)
Yield 76%. Mp. 219e220 ꢂC. 1H NMR (DMSO-d6)
d
(ppm): 2.30 (s,
3H, CH3); 7.18 (d, 2H, CHarom, J ¼ 8.1 Hz); 7.47 (d, 2H, CHarom
J ¼ 8.4 Hz); 8.50 (s, 1H, CH]); 11.12 (s, 1H, NH); 11.71 (s, 1H, NH);
13.94 (s, 1H, NH). 13C NMR (DMSO-d6)
(ppm): 20.51, 124,31 (2C),
,
Table 8
Antiproliferative activity of 14 against human leukaemia/lymphoma cell lines.
d
Compds
MT-4
CCRF-CEM
WIL-2NS
CCRF-SB
128.91 (2C), 134.72, 136.16, 157.12, 159.42, 177.08. ESI MS:
a
CC50
m/z ¼ 256.0 [M þ Na]þ (100%).
14
7.4 ꢀ 0.6
39 ꢀ 2
52 ꢀ 1
42 ꢀ 2
Camptotecin 0.004 ꢀ 0.0004 0.003 ꢀ 0.0005 0.005 ꢀ 0.0007 0.004 ꢀ 0.0005
3.1.7. 1-(4-Fluorophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (5)
Yield 81%. Mp. 211e212 ꢂC. 1H NMR (DMSO-d6)
d (ppm):
Data represent mean values for three independent determinations.
a
Compound concentration (mM) required to reduce cell proliferation by 50%
7.18e7.24 (m, 2H, CHarom); 7.57e7.62 (m, 2H, CHarom); 8.54 (s, 1H,
under conditions allowing untreated controls to undergo at least three consecutive
rounds of multiplication, as determined by the MTT method.
CH]); 11.24 (s, 1H, NH); 11.00 (s, 1H, NH); 14.00 (s, 1H, NH). 13C
NMR (DMSO-d6)
d (ppm): 114.99 (2C), 115.29 (2C), 126.89, 153.07,
158.02, 161.24, 177.63. ESI MS: m/z ¼ 260.0 [M þ Na]þ (100%).
column chromatography (chloroform: methanol; 9.5:0.5 vol.). The
compound was crystallized from acetonitrile.
3.1.8. 1-(2-Bromophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (6)
Yield 86%. Mp. 241e242 ꢂC. 1H NMR (DMSO-d6)
d (ppm):
3.1.2. 1-Phenyl-3-(1H-1,2,4-triazol-3-yl)thiourea (1)
1-Phenyl-3-(1H-1,2,4-triazol-3-yl)thiourea (1) has been
synthesized as described previously [63].
7.18e7.24 (m, 1H, CHarom); 7.38e7.46 (m, 1H, CHarom); 7.68e7.71
(m, 1H, CHarom); 7.80e7.83 (m, 1H, CHarom); 8.54 (s, 1H, CH]);
11.41 (s, 1H, NH); 11.68 (s, 1H, NH); 14.02 (s, 1H, NH). 13C NMR
Yield 83%. 1H NMR (DMSO-d6)
d
(ppm): 7.22 ( t, 1H, CHarom.
,
(DMSO-d6) d (ppm): 119.53, 127.57, 128.05, 129.69 (2C), 132.41,
J ¼ 7.5 Hz); 7.39 ( t, 2H, CHarom., J ¼ 7.8 Hz); 7.62 ( d, 2H, CHarom.
,
133.24, 137.39, 178.05. ESI MS: m/z ¼ 321.9 [M þ Na þ H]þ (100%).
J ¼ 7.8 Hz); 8.5 ( s, 1H, CHarom.); 11.78 (s,1H, NH). 13C NMR (DMSO-
d6) d (ppm): 124.26 (2C), 127.96, 128.47 (2C), 138.72, 148.79, 157.43,
3.1.9. 1-(4-Bromophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (7)
177.09. ESI MS: m/z ¼ 218.2 [M]þ (100%).
Yield 71%. Mp. 223e224 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
7.54 ꢁ 7.63 (m, 4H, CHarom); 8.51 (s, 1H, CH]); 11.28 (s, 1H, NH);
11.79 (s, 1H, NH); 13.89 (s, 1H, NH). 13C NMR (DMSO-d6)
(ppm):
3.1.3. Crystal data
d
Crystal system triclinic, space group P-1, unit cell dimensions
118.24, 126.97 (2C), 127.61 (2C), 132.75, 134.52, 137.22, 178.76. ESI
MS: m/z ¼ 321.9 [M þ Na þ H]þ (100%).
ꢀ
a ¼ 8.690(2), b ¼ 9.495(2), c ¼ 14.226(3) A,
a
¼ 72.05(3),
3
ꢀ
b
¼
86.85(3),
g
¼
67.20(3)ꢂ,
V
¼
1026.6(4) A ;
Z
¼
4,
dc ¼ 1.419 g cmꢁ3
,
m
¼ 2.591 mmꢁ1, F(000) ¼ 456. A crystal of
3.1.10. 1-(3-Bromophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (8)
dimensions 0.45 ꢃ 0.18 ꢃ 0.15 mm was used for intensity
Yield 88%. Mp. 203e204 ꢂC. 1H NMR (DMSO-d6)
d (ppm):
measurements.
Within
the
q
range
3.27e80.26ꢂ
7.31 ꢁ 7.42 (m, 2H, CHarom); 7.53e7.57 (m, 1H, CHarom); 8.04 (t,
[ꢁ10 ꢄ h ꢄ 10, ꢁ11 ꢄ k ꢄ 11, ꢁ11 ꢄ l ꢄ 18] 4353 reflections were
1H, CHarom, J ¼ 1.8 Hz); 8.54 (s, 1H, CH]); 11.31 (s, 1H, NH); 11.86
Table 9
Antiproliferative activity of 14 against solid tumour and “normal” cell lines. Data represent mean values for three independent determinations.
Compds
SK-MEL-28
MCF7
SKMES-1
HepG2
DU145
MRC-5
CRL7065
a
CC50
14
72 ꢀ 4
49 ꢀ 1
0.08 ꢀ 0.01
85 ꢀ 6
78 ꢀ 5
11 ꢀ 0.7
0.08 ꢀ 0.0004
43 ꢀ 3
47 ꢀ 1
Camptotecin
0.07 ꢀ 0.01
0.05 ꢀ 0.004
0.04 ꢀ 0.01
0.2 ꢀ 0.1
0.3 ꢀ 0.1
a
Compound concentration (mM) required to reduce cell proliferation by 50% under conditions allowing untreated controls to undergo at least three consecutive rounds of
multiplication, as determined by the MTT method.

J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
211
(s, 1H, NH); 13.98 (s, 1H, NH). 13C NMR (DMSO-d6)
d
(ppm): 120.86,
3.1.18. 5-Amino-N-(2-phenylethyl)-1H-1,2,3-triazole-1-
carbothioamide (16)
126.50, 130.38 (2C), 134.85, 136.37, 139.07, 141.56, 179.44. ESI MS:
m/z ¼ 321.9 [M þ Na þ H]þ (100%).
Yield 87%. Mp. 192e193 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
2.91 ꢁ 2.96 (t, 2H, CH2, J ¼ 7.8 Hz); 3.75e3.82 (q, 2H, CH2,
3.1.11. 1-(4-Iodophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (9)
J ¼ 7.1 Hz); 7.19e7.33 (m, 5H, CHarom); 8.20 (s, 1H, CH]);
Yield 79%. Mp. 207e208 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
10.05e10.09 (t, 2H, NH, J ¼ 5.5 Hz). 13C NMR (DMSO-d6)
d (ppm):
7.45e7.50 (m, 2H, CHarom); 7.69e7.74 (m, 2H, CHarom); 8.51 (s, 1H,
33.15, 45.22, 126.31, 128.44 (2C), 128.56 (2C), 138.60, 148.80,
CH]); 11.27 (s, 1H, NH); 11.79 (s, 1H, NH); 13.95 (s, 1H, NH). 13C
157.00, 173.78. ESI MS: m/z ¼ 270.1 [M þ Na]þ (100%).
NMR (DMSO-d6)
d (ppm): 87.21, 125.68, 126.68 (2C), 137.07, 137.14
(2C), 138.63, 177.03. ESI MS: m/z ¼ 367.9 [M þ Na]þ (100%).
3.1.19. 5-Amino-N-(2-methylallyl)-1H-1,2,4-triazole-1-
carbothioamide (17)
3.1.12. 1-(3,4-Dichlorophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea
Yield 92%. Mp. 173e174 ꢂC. 1H NMR (DMSO-d6)
d (ppm): 1.71(s,
(10)
3H, CH3); 4.12e4.18 (m, 2H, CH2); 4.73 (s, 1H, CH2]); 4.82 (s, 1H,
CH2]); 8.19 (s, 1H, CH]); 9.86 (s, 1H, NH2); 10.17 (s, 1H, NH2).
Yield 93%. Mp. 128e129 ꢂC. 1H NMR (DMSO-d6)
d (ppm): 7.38 (s,
1H, CHarom); 7.55e7.59 (dd, 1H, CHarom); 7.61e7.64 (d, 1H, CHarom);
8.50 (s, 1H, CH]); 11.43 (s, 1H, NH); 11.81 (s, 1H, NH); 13.88 (s, 1H,
13C NMR (DMSO-d6)
d (ppm): 30.34, 58.56, 120.44, 149.92, 158.82,
167.03, 184.41. ESI MS: m/z ¼ 198.1 [M þ H]þ (100%).
NH). 13C NMR (DMSO-d6)
d (ppm): 124.25, 125.47, 130.30 (2C),
130.52, 139.00, 143.29, 156.61, 177.39. ESI MS: m/z ¼ 309.9
3.1.20. Crystal data
[M þ Na]þ (100%).
Crystal system triclinic, space group P-1, unit cell dimensions
ꢀ
a ¼ 6.181(1), bꢂ¼ 8.287(2), c ¼ 10.239(2) A,
a
¼ 88.65(1), ¼ 83.16(1),
b
3
¼ 68.19(2) , V ¼ 483.3(2) A ; Z ¼ 2, dc ¼ 1.355 g cmꢁ3
,
ꢀ
3.1.13. 1-Benzoyl-3-(1H-1,2,4-triazol-3-yl)thiourea (11)
1-Benzoyl-3-(1H-1,2,4-triazol-3-yl)thiourea (11) has been
synthesized as described previously [63].
g
m
¼
2.678 mmꢁ1
,
F(000)
¼
208. A crystal of dimensions
0.40 ꢃ 0.20 ꢃ 0.18 mmwas used for intensity measurements. Within
Yield 83%. 1H NMR (DMSO-d6)
CHarom.); 7.60e7.65
8.03e8.06 ( d, 2H, CHarom, J ¼ 7.5 Hz); 11.94 (s, 2H, NH); 13.60 (s,
1H, NH); 13C NMR (DMSO-d6)
(ppm): 128.02 (2C), 128.49 (2C),
d
(ppm): 7.48e7.56 ( m, 2H,
the
q
range 4.35e67.64ꢂ [ꢁ10 ꢄ h ꢄ 10, ꢁ11 ꢄ k ꢄ 11, ꢁ11 ꢄ l ꢄ 18]
(
m, 1H, CHarom.); 7.81 (s, 1H, CH]);
4353 reflections were collected. The 4216 unique reflections
[R(int) ¼ 0.0218] were used for the refinement of 120 parameters,
including extinction coefficient [x ¼ 0.021(2)]. Final R indices on F2
d
132.40, 133.84, 147.15, 154.24, 165.47, 178.65. ESI MS: m/z ¼ 270.0
for 1471 observed reflections [I > 2
s
(I)] were: R1 ¼ 0.0359,
[M þ Na]þ (100%).
wR2 ¼ 0.1016, goodness-of-fit 1.053, and largest difference peak/
ꢀꢁ3
hole 0.21/ꢁ0.32 e A
.
3.1.14. 1-(2-Fluorophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (12)
Yield 83%. Mp. 183e184 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
3.1.21. 5-Amino-N-(2-ethoxycarbonyl)-1H-1,2,4-triazole-1-
carbothioamide (18)
7.21e7.31 (m, 4H, CHarom); 8.00e8.05 (t, 1H, CH], J ¼ 7.8 Hz);
8.52 (s, 1H, NH); 11.42 (s, 1H, NH); 11.70 (s, 1H, NH). 13C NMR
5-Amino-N-(2-ethoxycarbonyl)-1H-1,2,4-triazole-1-carbothioa-
mide (18) has been synthesized as described previously [65].
(DMSO-d6)
d (ppm): 115.38, 123.99, 126.53, 127.32, 127.42, 143.29,
153.84, 157.10, 177.98. ESI MS: m/z ¼ 260.0 [M þ Na]þ (100%).
Yield 81%. Mp. 157e158 ꢂC. 1H NMR (DMSO-d6)
d (ppm):
1.23e1.27 (t, 3H, CH3, J ¼ 7.05 Hz); 4.17e4.24 (q, 2H, CH2,
3.1.15. 1-(3-Fluorophenyl)-3-(1H-1,2,4-triazol-3-yl)thiourea (13)
J ¼ 7 Hz); 8.53 (s, 1H, CH ¼ ); 11.46 (s, 2H, NH2). 13C NMR (DMSO-
Yield 89%. Mp. 179e180 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
d6) d (ppm): 14.08, 63.88, 143.62, 149.93, 151.11, 171.89. ESI MS:
6.88e7.06 (m, 1H, CHarom); 7.24e7.54 (m, 3H, CHarom); 7.78e7.82
(s, 1H, CH]); 8.29 (s, 1H, NH); 8.49 (s, 1H, NH); 11.87 (s, 1H, NH).
m/z ¼ 238.1 [M þ Na]þ (100%).
13C NMR (DMSO-d6)
d
(ppm): 110.63, 111.76, 119.72, 130.00,
3.2. Biology
140.44, 143.23, 160.07, 163.28, 177.12. ESI MS: m/z ¼ 260.0
[M þ Na]þ (100%).
3.2.1. In vitro evaluation of antimicrobial activity
The antibacterial activity of compounds was tested against
a series of Gram-positive bacteria: S. aureus ATCC 4163, S. aureus
ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, S. epi-
dermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus
ATCC 11778, Enterococcus hirae ATCC 10541, Micrococcus luteus
ATCC 9341, M. luteus ATCC 10240 and Gram-negative rods:
Escherichia coli ATCC 10538, E. coli ATCC 25922, E. coli NCTC 8196,
Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa ATCC 15442, P.
aeruginosa NCTC 6749, P. aeruginosa ATCC 27853, Bordetella bron-
chiseptica ATCC 4617. Antifungal activity was tested against yeasts:
C. albicans ATCC 10231, C. albicans ATCC 90028, Candida parapsilosis
ATCC 220191. Microorganisms used in this study were obtained
from the collection of the Department of Pharmaceutical Microbi-
ology, Medical University of Warsaw, Poland.
3.1.16. 5-Amino-N-benzyl-1H-1,2,4-triazole-1-carbothioamide (14)
5-Amino-N-benzyl-1H-1,2,4-triazole-1-carbothioamide
(14)
has been synthesized as described previously [64].
Yield 88%. Mp. 185e186 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
4.78e4.80 (d, 2H, CH2, J ¼ 6.0 Hz); 7.22e7.30 (m, 1H, CHarom);
7.31e7.33 (m, 2H, CHarom); 7.34e7.43 (m, 2H, CHarom); 8.19 (s, 1H,
CH]); 10.55 (s, 1H, NH2). 13C NMR (DMSO-d6)
d (ppm): 46.80,
127.08, 127.51 (2C), 128.24 (2C), 137.17, 148.88, 157.08, 174.33. ESI
MS: m/z ¼ 256.0 [M þ Na]þ (100%).
3.1.17. 5-Amino-N-cyclohexyl-1H-1,2,4-triazole-1-carbothioamide
(15)
5-Amino-N-cyclohexyl-1H-1,2,4-triazole-1-carbothioamide
(15) has been synthesized as described previously [64]
Yield 86%. Mp. 169e170 ꢂC. 1H NMR (DMSO-d6)
d
(ppm):
3.2.1.1. Media, growth conditions and antimicrobial activity
assays. Antimicrobial activity was examined by the disc diffusion and
MIC method under standard conditions, using MuellereHinton II
agar medium (Becton Dickinson) for bacteria and RPMI agar with 2%
glucose (Sigma) for yeasts, according to CLSI (previously NCCLS)
guidelines [58]. Solutions containing the tested agents were prepared
1.05e1.18 (m, 1H, CH2); 1.22e1.36 (m, 2H, CH2); 1.44e1.62 (m, 3H,
CH2); 1.70e1.85 (m, 4H, CH2); 4.10e4.20 (m, 1H, CH); 8.18 (s, 1H,
CH]); 9.64 (d, 2H, NH2, J ¼ 8.4 Hz). 13C NMR (DMSO-d6)
d (ppm):
34.65 (2C), 34.84, 40.33 (2C), 63.38, 158.66, 167.07, 182.48. ESI
MS: m/z ¼ 248.1 [M þ Na]þ (100%).

212
J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
in methanol or DMSO. For the disc diffusion method, sterile paper
discs (9 mm diameter, Whatman No. 3 chromatography filter paper)
were dripped with the compound solutions tested to obtain 400 mg of
a HIV-1 suspension containing 100 CCID50 were added. After
a 4-day incubation at 37 ꢂC, cell viability was determined by the
MTT method [66].
substance per disc. Dry discs were placed on the surface of an
appropriate agar medium. The results (diameter of the growth inhi-
bition zone) were read after 18 h of incubation at 35 ꢂC. Minimal
Inhibitory Concentration (MIC) were examined by the twofold serial
agar dilution technique [59]. Concentrations of the tested compounds
3.2.2.6. Linear regression analysis. The extent of cell growth/
viability and viral multiplication, at each drug concentration tested,
were expressed as percentage of untreated controls. Concentra-
tions resulting in 50% inhibition (CC50 or EC50) were determined by
linear regression analysis.
in solid medium ranged from 3.125 to 400 mg/mL. The final inoculum
of studied organisms was 104 CFU/mL (colony forming units per mL),
except the final inoculum for E. hirae ATCC 10541, which was 105 CFU/
mL. Minimal inhibitory concentrations were read off after 18 h s) of
incubation at 35 ꢂC.
3.2.3. Pharmacology
The experiments were carried out on male Albino Swiss mice
(18e30 g). The animals were kept 8e10 to a cage under standard
laboratory conditions (at a temperature of 20 ꢀ 1 ꢂC and a 12 h
light/dark cycle) with free access to food (LSM, Motycz, Poland) and
water. All experiments were performed between 9:00 a.m. and
4:00 p.m. The experiments were performed in accordance with the
opinion of Local Ethics Committee for Animal Experimentation.
The substances investigated, marked as 8 and 14, in all tests
were administered intraperitoneally (i.p.), as a suspensions in
aqueous solution of 0.5% methylcellulose (tylose) and were injected
60 min before the tests. All substances were administered in
a volume of 10 ml/kg. The control animals received an equivalent
volume of the solvent at the respective time before the test. All tests
performed, suggested by Vogel and Vogel [67], are generally
accepted as basic in investigation of the central activity by behav-
ioural methods. The acute toxicity of the compound was assessed in
mice acc. to Litchfield and Wilcoxon method [68], as the ED50
calculated as “the lost of righting reflex” within 48 h. The
compounds were injected in doses equivalent to 0.1 LD50
(120.1 mg/kg for 8 and 18.06 mg/kg for 14). In addition, the activity
of compounds was assessed in the following tests:
3.2.2. Cell-based assays
Cell-based assays were performed at Dipartimento di Scienze e
Tecnologie Biomediche, Università di Cagliari, Monserrato, Italy.
3.2.2.1. Test compounds. Compounds were dissolved in DMSO at
100 mM and then diluted in culture medium.
3.2.2.2. Cells and viruses. Cell line and viruses were purchased from
American Type Culture Collection (ATCC). The absence of myco-
plasma contamination was checked periodically by the Hoechst
staining method. Cell line supporting the multiplication of Human
Immunodeficiency Virus type-1 (HIV-1) was the CD4þ human T-
cells containing an integrated HTLV-1 genome (MT-4).
3.2.2.3. Cytotoxicity assays. Cytotoxicity assays were run in parallel
with antiviral assays. Exponentially growing MT-4 cells were
seeded at an initial density of 1 ꢃ 105 cells/ml in 96-well plates in
RPMI-1640 medium, supplemented with 10% fetal bovine serum
(FBS), 100 units/mL penicillin G and 100 mg/mL streptomycin. Cell
cultures were then incubated at 37 ꢂC in a humidified, 5% CO2
atmosphere, in the absence or presence of serial dilutions of test
compounds. Cell viability was determined after 96 h at 37 ꢂC by the
- locomotor activity was measured for single mice in photo-
resistor actometers (circular cages, diameter 25 cm, two light
beams; the number of crossed light beams by the mice was
recorded) for 30 min as:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
(MTT) method [66].
bromide
a) spontaneous activity
b) amphetamine-induced hyperactivity: mice received
subcutaneously (s.c.) 5 mg/kg of amphetamine 30 min
before the test;
- nociceptive reactions were studied in the acetic acid (0.6%)-
induced ‘writhing’ test. The number of writhing episodes was
measured for 10 min starting 5 min after i.p. administration of
acid solution;
3.2.2.4. Antiproliferative assays. Cell lines derived from human
haematological tumours [CD4þ human T-cells containing an inte-
grated HTLV-1 genome (MT-4); CD4þ human acute T-lympho-
blastic leukaemia (CCRF-CEM), human splenic B-lymphoblastoid
cells (WIL-2NS), human acute B-lymphoblastic leukaemia (CCRF-
SB)] were seeded at an initial density of 1 ꢃ 105 cells/ml in 96 well
plates in RPMI-1640 medium supplemented with 10% foetal calf
- motor coordination was evaluated in rota rod test [69] and
chimney test;
serum (FCS), 100 units/ml penicillin G and 100 mg/ml streptomycin.
Cell lines derived from human solid tumours [skin melanoma
(SK-28), breast adenocarcinoma (MCF-7), lung squamous carci-
noma (SK-MES-1), hepatocellular carcinoma (HepG-2), prostate
carcinoma (DU-145)] or normal tissues [foreskin fibroblasts
(CRL-7065), lung fibroblasts (MRC-5)] were also seeded at
1 ꢃ 105 cells/ml in 96 well plates in specific media supple-
mented with 10% FCS and antibiotics, as above. Cell cultures
were then incubated at 37 ꢂC in a humidified, 5% CO2 atmo-
sphere in the absence or presence of serial dilutions of test
compounds. Cell viability was determined after 96 h at 37 ꢂC by
the MTT method [66].
- body temperature in normothermic mice was measured in the
rectum by thermistor thermometer;
- pentylenetetrazole (110 mg/kg, s.c.)-induced convulsions were
evaluated as the number of mice with clonic seizures, tonic
convulsions and dead animals;
- ‘head twitch’ responses after 5-hydroxytryptophan (5-HTP),
acc. to Corne et al. [43]. Mice received 5-HTP (180 mg/kg, i.p.)
and the number of head twitches was recorded in 6 two-
minute intervals (4e6, 14e16, 24e26, 34e36, 44e46,
54e56 min).
3.2.2.5. Antiviral assays. Compound’s activity against HIV-1 was
based on inhibition of virus-induced cytopathogenicity in MT-4 cell
acutely infected with a multiplicity of infection (m.o.i.) of 0.01.
3.2.4. Statistics
Obtained data were calculated by c2 test with Yates correction
(pentylenetetrazole-induced seizures), and one-way ANOVA (other
tests). Subsequent comparisons between treatment and control
groups were carried out using a post hoc Dunnett’s test, when
p < 0.05.
Briefly, 50
m
L of RPMI containing 1 ꢃ 104 MT-4 cells were added to
each well of flat-bottom microtitre trays, containing 50
mL of RPMI
without or with serial dilutions of test compounds. Then, 20
mL of

J. Stefanska et al. / European Journal of Medicinal Chemistry 55 (2012) 205e213
213
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