Synthesis, cytotoxicity and effects of some 1,2,4-triazole and 1,3,4-thiadiazole derivatives on immunocompetent cells

Article abstract of DOI:10.1016/j.ejmech.2008.03.006

Novel derivatives of 4,5-substituted-1,2,4-triazole-thiones and 2,5-substituted-1,3,4-thiadiazoles were synthesized and evaluated for their cytotoxicity. The biological study indicated that compounds 4-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-di

Full text of DOI:10.1016/j.ejmech.2008.03.006

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 63e69
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity and effects of some 1,2,4-triazole and
1,3,4-thiadiazole derivatives on immunocompetent cells
b
c
c
Anelia Ts. Mavrova a, , Diana Wesselinova , Yordan A. Tsenov , Pavletta Denkova
*
a Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Boulevard, 1756 Sofia, Bulgaria
b Institute of Parasitology and Experimental Pathology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
c Institute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Received 3 August 2007; received in revised form 6 March 2008; accepted 13 March 2008
Available online 25 March 2008
Abstract
Novel derivatives of 4,5-substituted-1,2,4-triazole-thiones and 2,5-substituted-1,3,4-thiadiazoles were synthesized and evaluated for their
cytotoxicity. The biological study indicated that compounds 4-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione 13, N-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-1,3,4-thiadiazol-2-amine 16, 4-amino-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,
4-dihydro-3H-1,2,4-triazole-3-thione 20 and 4-amino-5-(5-phenylthien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 21 possessed high cytotox-
icity in vitro against thymocytes. The corresponding IC50 values were 0.46 mM, 5.2 ꢀ 10ꢁ6 mM, 0.012 mM and 1.0 ꢀ 10ꢁ6 mM. Most toxic
against lymphocytes was compound 21, IC50 e 0.012 mM. The tested compounds showed a general stimulation effect on B-cells’ response.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2,4-Triazoles; 1,3,4-Thiadiazoles; Cytotoxicity; PFC; LIF
1. Introduction
Several derivatives of 1,3,4-thiadiazole were prepared start-
ing from the precursors, used for the synthesis of 1,2,4-triazoles
and large diversity of effects such as anti-inflammatory,
antituberculosis, anticonvulsant, and antibacterial were proved
[13e16]. A number of N-substituted 2-amino-1,3,4-thiadia-
zoles were synthesized and evaluated for their antiproliferative
activities [17e20].
A large number of compounds containing 1,2,4-triazole
system have been investigated as therapeutically interesting
drug candidates because of their properties both as selective
COX-2 inhibitors [1] and as anti-acetylcholinesterase [2],
antimicrobial and antimycotic agents [3e5].
The efficacy of anastrozole and letrozole as aromatase in-
hibitors and their use as non-steroidal drugs for the treatment
of estrogen-dependent cancer as well as the anticancer proper-
ties of ribavirine led to the investigation of many 1,2,4-triazole
derivatives in laboratorial conditions for their anti-tumor activ-
ity [6e8].
Among the 1,2,4-triazole derivatives, the mercapto- and the
thione-substituted 1,2,4-triazole ring systems have been stud-
ied and so far a variety of anti-tumor properties have been
reported for a large number of these compounds [9e12].
The similarity in the structure of the 2-amino-1,3,4-thiadia-
zole and the mercapto- and thion-substituted 1,2,4-triazole
ring systems assumes similar biological properties. Series of
heterocyclic mercaptans incorporating 1,3,4-thiadiazole- and
1,2,4-triazole rings have been prepared and investigated for
antiproliferative activity against human cancer cell lines and
tumor-associated carbonic anhydrase isoenzymes I, II, and
IX [21,22].
Many types of chemotherapeutic agents have been shown
to be effective against cancer and tumor cells, but many of
these agents also destroy the normal cells. Despite advances
in the field of cancer treatment the discovery of cytotoxic
agents that have specificity for cancer and tumor cells remains
pendant. Unfortunately, none have been found and instead of
* Corresponding author. Tel.: þ359 28163207; fax: þ359 2685488.
E-mail address: anmav@abv.bg (A.Ts. Mavrova).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.006

64
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
that, substances whose target is especially rapid dividing cells
have been used. Alternatively the substances that were
cytotoxic to tumor cells while exerting mild effects on normal
cells would be desirable.
In ethanol solution of potassium hydroxide carbohydrazides
3aec interacted with carbon disulfide to potassium hydrazine-
carbodithionates 18, 19, which were used as precursors for the
synthesis of 4-amino-1,2,4-triazoles 20, 21. The refluxing of
18, 19 and hydrazine hydrate in molar ratio 1:2 led to the prep-
aration of 4-amino-1,2,4-triazol-yl-3-thiones.
Therefore, the search and synthesis of new 3-mercapto-5-
substituted-1,2,4-triazole and 2,5-disubstituted-1,3,4-tiadia-
zole derivatives are determined by the above mentioned facts.
Previously we have reported the synthesis and the cytotox-
icity of some 1,2,4-triazoles against Graffi tumor cells [23].
Having in view the high cytotoxicity of these compounds,
we undertook synthesis of some derivatives of 3-mercapto-
1,2,4-triazoles and 2-amino-1,3,4-thiadiazoles and in vitro
investigation of their cytotoxicity against thymocytes and lym-
phocytes as well as an estimation of their influence on T- and
B-cells’ response.
The chemical structures of the compounds were established
1
by elemental analyses, IR-, H NMR and 13C NMR spectra
and the results are presented in Section 6. The elemental anal-
yses indicated by the symbols of the elements were within
ꢂ0.4% of theoretical values.
3. Pharmacology
3.1. Cytotoxicity
The choice of these structures was in accordance with the
fact that the thiophene heterocycle takes part in the structure
of substances possessing anti-tumor activity, therefore, it was
of pharmacological interest to incorporate a thiophene or a tet-
rahydrobenzothiophene cycle in the structure of 3-mercapto-
1,2,4-triazoles and 2-amino-1,3,4-thiadiazoles [24e26].
The structures of 5-aryl-1,2,4-triazoles and 1,3,4-triazoles
give the opportunity to realize changes in two directions: in-
troduction of some substituents either in the 1,2,4-triazole
and 1,3,4-thiadiazole cycle or in the aromatic ring. That alter-
ation in the structure of the main model compounds may
enhance the interaction of these molecules with the biological
targets.
Compounds 10, 11, 13, 16, 20, and 21 were evaluated for
their cytotoxicity using thymocytes and lymphocytes, derived
from sexually mature hamsters, bearing the solid form of the
Graffi myeloid tumor.
Migration, rosette forming and plaque forming tests were
accomplished on spleen cells, thymocytes and lymphocytes
using compounds 10, 11, 13, 16, 20, and 21 and the effect
on T- and B-cells’ response was estimated.
4. Results and discussion
The derivatives of 1,2,4-triazole and 1,3,4-thiadiazoles con-
taining ethyl or amino group at 4th position of 1,2,4-triazole,
respectively, ethylamino-group at second position of 1,3,
4-thiadiazole cycle as well as 4,5,6,7-tetrahydro-1-benzothio-
phene-2- ,5-phenylthiophene-2- and 5-(4-methylphenyl)thio-
phene-2-substituents at the 5th position of 1,2,4-triazole,
respectively, 1,3,4-thiadiazole cycles were obtained in order to es-
timate their cytotoxicity and influenceon T- andB-cells’response.
The yields of 4-amino-1,2,4-triazoles were in the range 78e96%
and that of 4-ethylamino-1,2,4-triazoles accordingly 78e98%,
while the yield of N-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-
yl)1,3,4-thiadiazol-2-amine was only 43% and that of N-ethyl-5-
(5-phenylthien-2-yl)-1,3,4-thiadiazol-2-amine e 80%.
2. Chemistry
The synthesis of the 1,2,4-triazole and 1,3,4-thiadiazole
derivatives is illustrated and outlined in Fig. 1.
3-Chloro-acrylaldehydes were synthesized according to the
procedure described in Ref. [27]. Because of their instability
the aldehydes have to be kept only for few hours in refrigerator
and must be used as soon as possible.
The interaction of ethyl thioglycolate with 3-chloro-acrylal-
dehydes 1e3 in pyridine medium in the presence of triethyl-
amine led to ethyl thiophene-2-carboxylates 4e6 [28]. The
hydrazides 7e9, obtained by refluxing of ethyl carboxylates
4e6 with hydrazine hydrate in ethanol medium, were used
as precursors for the synthesis of N-ethyl hydrazinecarbothioa-
mides 10e12 as well as for the preparation of potassium aryl-
carbonylhydrazinecarbodithionate 18, 19.
The reaction of hydrazides 7e9 with ethyl isothiocyanate
resulted in N-ethyl hydrazinecarbothioamides 10e12. Thiose-
micarbazides undergo different cyclization reactions to give
five member heterocycles. The product of cyclization depends
on the reagent used. This cyclization leads to the formation of
1,3,4-oxadiazole, 1,3,4-thiadiazole and 1,2,4-triazole deriva-
tives. When the N-ethyl hydrazinecarbothioamides 10e12
were refluxed with solution of sodium hydroxide for 6e12 h,
4-ethyl-5-aryl-1,2,4-triazole-3-thiones 13e15 were synthe-
sized. In sulfuric acid medium the hydrazinecarbothioamides
4.1. Cytotoxicity
The exclusive trypan blue test for the estimation of cytotox-
icity in vitro was performed with compounds 10, 11, 13, and
16 as well as 20, 21 according to the method given in
Ref. [29] on thymocytes and blood lymphocytes, derived
from sexually mature hamsters. The compounds were dis-
solved in DMSO at the concentration of 0.5 mg/ml. The inves-
tigation was carried out by dilution of the stock solution in
ratio 1:10, 1:100, 1:1000 and 1:100,000. To 0.1 ml cell solution
was added 0.1 ml of the corresponding series of compounds.
The preparations were incubated for 24 h at 37 ꢃC under hu-
midified atmosphere in the presence of 5% carbon dioxide.
Trypan blue, 0.1 ml of a 0.2% solution was added to each prep-
aration. The cells’ survival was evaluated by means of elec-
tronic microscope and the percentage of cytotoxicity was
10e12 formed
amines 16, 17.
N-ethyl-5-substituted-1,3,4-thiadiazol-2-

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
65
R1
O
O
R
R
R1
b
a
R1
C
C
O
S
O
Cl
R
4 - 6
1 - 3
4: R = R1 = -(CH2)4;
5: R = C6H5; R1 = H;
6: R = 4-CH3-C6H4;
1: R = R1 = -(CH2)4;
2: R = C6H5; R1 = H;
3: R = 4-CH3-C6H4;
c
R1
R1
R1
R
N
N
NH
HN
d
e
S
R
NH
NH
R
S
S
S
S
NH2
N
H
O
O
7 - 9
10 - 12
13 - 15
7: R = R1 = -(CH2)4;
10: R = R1 = -(CH2)4;
11: R = C6H5; R1 = H;
12: R = 4-CH3-C6H4;
13: R = R1 = -(CH2)4;
14: R = C6H5; R1 = H;
15: R = 4-CH3-C6H4;
8: R = C6H5; R1 = H;
9: R = 4-CH3-C6H4;
f
g
R1
N
N
16: R = R1 = -(CH2)4;
17: R = C6H5; R1 = H;
S
S
R
NH
16 - 17
R1
R1
N
N
NH
KS
h
R
NH
S
S
R
S
S
N
H
H2N
O
18 - 19
20 - 21
20: R = R1 = -(CH2)4;
21: R = C6H5; R1 = H;
18: R = R1 = -(CH2)4;
19: R = C6H5; R1 = H;
Fig. 1. Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Regents and conditions: (a) DMF, POCl3, trichloroethylene; (b) thioglycolate, triethylamine,
pyridine, 10e15 ꢃC; (c) hydrazine hydrate, ethanol, reflux; (d) ethyl isothiocyanate, ethanol, reflux; (e) 10% NaOH, reflux; (f) concentrated sulfuric acid, 0 ꢃC; (g)
NaOH, CS2, absolute ethanol; (h) hydrazine hydrate, reflux.
calculated. The obtained results were plotted and IC50 was es-
timated. The data are given in Table 1. The proved compounds
exhibited relative high cytotoxicity against thymocytes (IC50
values were in the range 0.1e2.43 mM) but lower cytotoxicity
against blood lymphocytes (IC50 e 1.64e2.67 mM).
Because after treatment in vivo the experimental animals
can develop a response reaction, it was interesting to estimate
the cytotoxicity of the studied compounds on cells, derived
from preliminary treated hamsters. That is why a second group
of hamsters, bearing the solid form of the Graffi myeloid
Table 1
In vitro cytotoxicity
Compound
IC50 ꢂ SE (mM)
Type of cells
Thymocytes
After treatment IC50 ꢂ SE (mM)
Type of cells
Blood lymphocytes
Thymocytes
Blood lymphocytes
10
11
13
16
20
21
0.11 ꢂ 8.23
2.10 ꢂ 9.43
0.12 ꢂ 12.69
0.26 ꢂ 11.51
2.20 ꢂ 7.47
0.24 ꢂ 16.20
NA
0.011 ꢂ 14.89
0.014 ꢂ 15.81
0.32 ꢂ 13.77
0.29 ꢂ 17.93
0.41 ꢂ 13.77
0.14 ꢂ 12.68
0.012 ꢂ 16.53
1.64 ꢂ 5.68
1.81 ꢂ 10.87
0.32 ꢂ 9.69
2.67 ꢂ 9.65
NA
3.5 ꢂ 14.37
0.46 ꢂ 17.19
5.2 ꢀ 10ꢁ6 ꢂ 13.17
0.012 ꢂ 14.13
1.0 ꢀ 10ꢁ6 ꢂ 14.75
NA e cytotoxicity not determined.

66
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
tumor was treated in vivo with the substances 10, 11, 13, 16,
20, and 21 with a dose at concentration of 0.5 mg/ml, i.p.
The in vitro effect of the compounds on thymocytes and lym-
phocytes, extracted from the hamsters five days after treatment
was determined. The results for cytotoxicity of compounds 10,
11, 13, 16, 20, and 21 in vitro after treatment of the experi-
mental hamsters are presented in Table 1. Each value repre-
sents a mean value of three parallel samples in three
independent experiments. As it can be seen that the cytotoxic
effects of the compounds on thymocytes increased and the
IC50 values of compounds 13, 16, 20 and 21 varied between
0.46 mM and 1.0 ꢀ 10ꢁ6 mM. The highest activity against
blood lymphocytes exhibited compound 21, IC50 e 0.012 mM.
It was important for us to establish if the studied com-
pounds can cause T- and B-cells’ response. Migration, rosette
forming and plaque forming tests were accomplished on
spleen cells, thymocytes and lymphocytes extracted from
treated hamsters as described in Refs. [30e32]. As control
cells were used cells from not intact animals. The data present
a mean value of three individual experiments and are given in
Table 2. Rosette formation was not observed for all of the
tested compounds. The number of the plaque forming cells
for compound 21 in the test with thymocytes was counted as
haemolytic zone and compared to those of the healthy con-
trols, was the highest e 19,200. Regarding the control cells
all studied compounds, excluding compound 10, exhibited
higher value number of the plaque forming cells in screening
with blood lymphocytes. In comparison to the control group
compound 21 showed the highest value number of PFCe
19,920, followed by compound 16 e 14,288, but these com-
pounds were not active in the LIF test regarding to spleen cells
and blood lymphocytes, while compound 20 showed the high-
est LIF against blood lymphocytes 1.654.
The above-mentioned facts indicated that the compounds
have a general stimulating effect on the B-cells’ response,
but additional experiments are required to elucidate their effi-
cacy. When there is a stimulation of the immunological
response it may be supposed that the resistance of the organ-
ism to the tumor cells will increase.
Statistical significant differences in the level of cells in both
control and experimental groups were determined ( p ꢄ 0.05).
5. Conclusion
New derivatives of 3,4,5-substituted-1,2,4-triazole and 2,5-
substituted-1,3,4-thiadiazoles were synthesized using as
precursors 4,5,6,7-tetrahydro-1-benzothiophene-2, as well as
5-phenylthiophene-2, and 5-(4-methylphenyl)thiophene-2-
carbohydrazide.
The initial biological screening in vitro showed that the
studied compounds possessed relative high cytotoxicity
against thymocytes and low cytotoxicity against blood lym-
phocytes, derived from sexually mature hamsters.
After treatment of the experimental animals with a dose of
the tested compounds at and following estimation of the cyto-
toxicity in vitro, the results showed increase in the cytotoxicity
of compounds 13, 16, 20 and 21 against thymocytes. IC50
values were in the range 0.46e1.0 ꢀ 10ꢁ6 mM. With respect
to blood lymphocytes the most cytotoxic was compound 21,
IC50 was 0.012 mM.
The PFC, LIF and the migration tests’ study indicated that
the compounds revealed a general stimulating effect on the B-
cells’ response. Compound 20 exhibited a general stimulation
regarding blood lymphocytes, LIF e 1.654, while compound
21 showed the highest value number of PFC, which surpasses
29 times than that of the control cells.
The above results also confirmed the hypothesis that the
introduction of a 5-phenylthiophene-2- and tetrahydrobenzo-
thiophene-2-substituent at 5th position in the structure of 3-
mercapto-1,2,4-triazoles and 2-amino-1,3,4-thiadiazoles are
auspicious to the interaction of these molecules with the bio-
logical targets.
Table 2
Effects of the compounds in vitro on the immunocompetent cells
Compound
Cells
Migration
(mm2)
LIF
PFC
10
Spleen cells
Thymocytes
NA
Full lys.
2040
768
146
0.08
NA
Blood lymphocytes
Spleen cells
Thymocytes
NA
11
13
16
20
21
822
202
NA
0.104
NA
2010
7800
5200
3700
5287
4992
3928
15,300
14,288
3920
7250
5869
4700
19,200
19,920
5400
6033
680
6. Experimental part
Blood lymphocytes
Spleen cells
Thymocytes
NA
Melting points (mp) were determined on an Electrothermal
AZ 9000 3MK4 apparatus and were uncorrected. The thin
layer chromatography (TLC, Rf values) was performed on
Al2O3 60 plates F254 or silica gel plates (Merck, 0.2 mm thick)
using mobile phase benzene/ethanol e 2:0.5, respectively,
benzene/ethanol e 4:2, and visualization was effected with ul-
traviolet light. IR spectra were recorded on a Specord 71 IR
spectrophotometer as potassium bromide discs. All NMR
spectra were recorded on a Bruker Avance DRX 250 spec-
trometer (Bruker, Faelanden, Switzerland) operating at
250.13 MHz for 1H and 62.89 MHz for 13C, using a dual
5 mm 1H/13C probehead. Chemical shifts were expressed
relative to tetramethylsilane (TMS) and were reported as
d (ppm). The measurements were carried out at ambient
1823
997
0.00
0.518
0.001
NA
Blood lymphocytes
Spleen cells
Thymocytes
NA
1788
1118
NA
0.581
NA
NA
Blood lymphocytes
Spleen cells
Thymocytes
1286
1024
1800
2332
1836
NA
0.532
1.654
NA
Blood lymphocytes
Spleen cells
Thymocytes
0.954
NA
NA
Blood lymphocytes
Spleen cells
Thymocytes
Control
cells
NA
1924
1088
NA
NA
Blood lymphocytes
NA e not response determined.

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
67
temperature (300 K). The microanalyses for C, H, N and S
were performed on PerkineElmer elemental analyzer.
68.26; H, 5.73; O, 12.99; S, 13.02; Found: C, 68.16; H,
5.83; O, 12.79; S, 13.18.
6.1. Chemistry
6.1.3. General procedure for compounds 7e9
A solution of 0.01 mol 4e6 and 0.02 mol hydrazine hydrate
in 30 ml ethanol was refluxed for 6 h. The hydrazide was crys-
tallized after cooling. TLC: mobile phase benzeneeethanol e
2:0.4, Al2O3 plates.
6.1.1. General procedure for compounds 1e3
Phosphoric chloride 0.044 mol was dropped to 0.054 mol
DMF dissolved in 10 ml dry trichloroethylene by stirring at
10 ꢃC. In absence of humidity a solution of 0.041 mol cyclo-
hexanone in 10 ml trichloroethylene was added dropwise at
ambient temperature for 30 min. The reaction mixture was
stirred at 60 ꢃC for 3 h. After cooling the solution was poured
out in 50 ml water containing 14 g sodium acetate dihydrate.
The organic layer was separated and the water layer was ex-
tracted with trichloroethylene. The combined layers were
dried with sodium sulfate and the solvent was evaporated in
vacuum. The aldehydes were distillated under reduced pres-
sure (5 mm/Hg). Because of their instability they have to be
kept in refrigerator only for a few hours.
6.1.3.1. 4,5,6,7-Tetrahydro-1-benzothiophene-2-carbohydra-
zide 7. Yield e 85%; mp e 146e148 ꢃC; Rf ¼ 0.54; 1H
NMR (C5D5N): 1.76 (t, 4H, 2CH2); 2.35 (t, 2H, CH2); 2.45
(t, 2H, CH2); 5.10 (bs, 2H, NH2, exchangeable with D2O);
6.90 (s, 1H, Th); 11.4 (bs, 1H, NH, exchangeable with
D2O); Analysis: Calc.: C, 55.08; H, 6.15; N, 14.27; S,
16.34; Found: C, 55.18; H, 6.05; N, 14.40; S, 16.18.
6.1.3.2. 5-Phenylthiophene-2-carbohydrazide 8. Yield e 75%;
1
mp e 164e166 ꢃC; Rf ¼ 0.63; H NMR (C5D5N): 5.20 (bs,
2H, NH2, exchangeable with D2O); 6.93 (d, 1H, Th); 7.25
(m 3H, Bz); 7.5 (m 2H, Bz); 7.78 (d 1H, Th); 10.74 (s, 1H,
NH, exchangeable with D2O); Analysis: Calc.: C, 60.53; H,
4.62; N, 12.83; S, 14.69; Found: 60.48; H, 4.66; N, 12.77;
S, 14.78.
6.1.2. General procedure for compounds 4e6
To a cooled solution of 0.025 mol of 2-chloro-acrylalde-
hyde 1e3 and 0.03 mol ethyl thioglycolate in pyridine
5.1 ml triethylamine was added at 10 ꢃC drop by drop by stir-
ring. The temperature must be maintained between 10 ꢃC and
15 ꢃC. The solution colored darker and triethylamine hydro-
chloride was formed. The mixture was stirred for 1 h more.
After cooling with ice 48% potassium hydroxide (5.12 ml)
was added by stirring for 15 min and the reaction mixture
was diluted with 150 ml water. The organic layer was sepa-
rated and the water layer was extracted with chloroform.
The combined organic layers were dried with Na2SO4. The
solvent was removed in vacuum evaporator and the compound
was distilled under reduced pressure.
6.1.3.3. 5-(4-Methylphenyl)thiophene-2-carbohydrazide9. Yield:
92%; mp e 154e157 ꢃC; Rf ¼ 0.45; 1H NMR (C5D5N): 2.31 (s,
3H, CH3); 5.32 (bs, 2H, NH2, exchangeable with D2O); 6.94 (m,
3H, 2H-Bz, 1H-Th); 7.41 (m, 2H, Bz); 7.71 (m, 1H, Th); 10.90
(s, 1H, NH, exchangeable with D2O); Analysis: Calc.: C, 62.04;
H, 5.21, N, 12.06; S, 13.80; Found: C, 62.10; H, 5.16, N, 12.16;
S, 13.88.
6.1.4. General procedure for compounds 10e12
6.1.2.1. Ethyl 4,5,6,7-tetrahydro-1-benzothiophene-2-carboxyl-
ate 4. Yield e 60%; mp e 30e31 ꢃC, re-crystallized with eth-
anol; NMR (CDCl3): 1.22 (t, 3H, CH3); 1.74 (t, 2H, 2CH2);
2.42 (t, 2H, CH2); 2.61 (t, 2H, CH2); 4.18 (q, 2H, CH2); 7.1
(s, 1H, 1H-Th); Analysis: calc.: C, 62.83; H, 6.71; O, 15.22;
S, 15.25; Found: C, 62.73; H, 6.61; O, 15.15; S, 15.15.
Hydrazides 7e9, 0.023 mol, were suspended in 25 ml eth-
anol and 0.028 mol of ethyl isothiocyanate was added. The
mixture was refluxed for 3 h. The product was isolated after
cooling. TLC: mobile phase benzene/ethanol e 4:2.
6.1.4.1. N-Ethyl-2-(4,5,6,7-tetrahydro-1-benzothien-2-ylcarbo-
nyl)hydrazinecarbothioamide 10. Yield e 64%; mp e 189e
190 ꢃC; Rf ¼ 0.57; 1H NMR (DMSO): 1.02 (t, 3H, CH3);
1.74 (m, 4H, 2CH2); 2.53 (m, CH2); 2.72 (t, 2H, CH2); 3.44
(q, 2H, eCH2eN); 7.48 (s, 1H, Th); 8.04 (bs, 1H, NH,
exchangeable with D2O); 9.20 (s, 1H, NH, exchangeable
with D2O); 10.14 (s, 1H, NH exchangeable with D2O); Anal-
ysis: Calc.: C, 50.85; H, 6.05; N, 14.83; S, 22.63; Found: C,
50.75; H, 6.01; N, 14.76; S, 22.58.
6.1.2.2. Ethyl 5-phenylthiophene-2-carboxylate 5. Yield e
40%; mp e 29 ꢃC, re-crystallized with ethanol; NMR
(CDCl3): 1.24 (t, 3H, CH3); 4.22 (q, 2H, CH2); 6.98 (m,
1H, Th); 7.22 (m, 1H, Bz); 7.44 (m, 2H, Bz); 7.54 (m, 2H,
Bz); 7.63 (m, 1H, Th); Analysis: Calc.: C, 67.21; H, 5.21;
O, 13.77; S, 13.80 Found: C, 67.11; H, 5.31; O, 13.57; S,
13.68.
6.1.2.3. Ethyl 5-(4-methylphenyl)thiophene-2-carboxylate 6.
Yield: 56%; mp e 85e88 ꢃC, re-crystallized with ethanol;
Rf ¼ 0.63 mobile phase benzene/ethanol e 3:1, Al2O3 plates;
NMR (CDCl3): 1.42 (t, 3H, CH3); 2.30 (s, 3h, CH3); 4.24
(q, 2H, CH2); 6.88 (m, 1H, Th); 7.16 (m, 2H, Bz); 7.34 (m,
6.1.4.2. N-Ethyl-2-[(5-phenylthien-2-yl)carbonyl]hydrazine-
carbothioamide 11. Yield e 85%; mp e 205e207 ꢃC;
1
Rf ¼ 0.53; H NMR (DMSO): 1.12 (t, 3H, CH3); 3.49 (q, 2H,
CH2); 6.97 (m, 1H, Th); 7.18 (m, 3H, Bz), 7.38 (m 1H, Bz);
8.04 (bs, 1H, NH, exchangeable with D2O); 9.20 (s, 1H, NH ex-
changeable with D2O); 10.14 (s, 1H, NH, exchangeable with
3
2H, Bz, J ¼ 8.02 Hz); 7.58 (m, 1H, Th); Analysis: Calc.: C,

68
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
D2O); Analysis: Calc.: C, 55.06; H, 4.95; N, 13.76; S, 21.00;
Found: C, 55.00; H, 4.91; N, 13.81; S, 21.10.
exchangeable with D2O); Analysis: Calc.: C, 54.31; H, 5.70;
N, 15.83; S, 24.16; Found: C, 54.20; H, 5.68; N, 15.64; S, 24.31.
6.1.4.3. N-Ethyl-2-{[5-(4-methylphenyl)thien-2-yl]carbonyl}-
hydrazinecarbothioamide 12. Yield e 47%; mp e 210e
213 ꢃC; Rf ¼ 0.61; 1H NMR (C5D5N): 1.18 (t, 3H, CH3); 2.31
(s, 3H, CH3); 3.50 (q, 2H, CH2); 7.11 (m, 3H, 2H-Bz, 1H-Th);
7.37 (m, 2H-Bz); 7.54 (d, 1H, Th); 7.78 (bs, 1H, NH, exchange-
able with D2O); 9.1 (s, 2H, NH, exchangeable with D2O); Anal-
ysis: Calc.: C, 56.40; H, 5.36; N, 13.15; S, 20.08; Found: C,
56.34; H, 5.42; N, 13.25; S, 20.18.
6.1.6.2. N-Ethyl-5-(5-phenylthien-2-yl)-1,3,4-thiadiazol-2-
1
amine 17. Yield e 80%; mp e 145e147 ꢃC; Rf ¼ 0.48; H
NMR (C5D5N): 1.2 (t, 3H, CH2eCH3); 3.9 (q, 2H, CH2e
CH3); 7.2 (m, 3H, 2H-Bz, 1H-Th); 7.4 (m, 2H,Bz); (d, 1H,
Th); Analysis: Calc.: C, 58.51; H, 4.56; N, 14.62; S, 22.31;
Found: C, 58.42; H, 4.46; N, 14.71; S, 22.42.
6.1.7. General procedure for the synthesis of
compounds 17e19
To a solution of 0.023 mol carbohydrazides 7, 8 and
0.027 mol of potassium hydroxide in 20 ml absolute ethanol
was added 0.034 mol of carbon disulfide. The solution became
orange colored and was stirred for 2e10 h. The obtained pre-
cipitate was filtered. TLC: mobile phase: benzene/ethanol e
5:2, silica gel plates (Merck, 0.2 mm thick).
6.1.5. General procedure for compounds 13e15
To 0.01 mol of compounds 10e12 was added 2 ml 10%
NaOH and the mixture was refluxed for 5e12 h. After cooling
the solution was acidified with concentrated hydrochloric acid
and the obtained precipitate was filtered and re-crystallized
with ethanol. TCL was performed on silica gel plates using
mobile phase benzene/ethanol e 2:0.5.
6.1.7.1. Potassium 2-(4,5,6,7-tetrahydro-1-benzothien-2-ylcar-
bonyl)hydrazinecarbodithionate 18. Yield e 66%; mp e
268e270 ꢃC; Rf ¼ 0.59
6.1.5.1. 4-Ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione 13. Reaction time: 5 h.
Yield e 97%; mp e 183e185 ꢃC; Rf ¼ 0.63; 1H NMR
(DMSO): 1.23 (t, 3H, CH3); 1.71 (m, 4H, 2CH2); 2.54 (m,
2H, CH2); 2.71 (m, 2H, CH2); 4.18 (q, 2H, CH2); 7.37 (s,
1H, Th); 13,88 (s, 1H, NH, exchangeable with D2O); Analysis:
Calc.: C, 54.31; H, 5.70; N, 15.83; S, 24.16; Found: C, 54.15;
H, 5.45; N, 15.66; S, 24.00.
6.1.7.2. Potassium2-[(5-phenylthien-2-yl)carbonyl]hydrazinecar-
bodithionate 19. Yield e 66%; mp e 253e255 ꢃC; Rf ¼ 0.57.
6.1.8. General procedure for compounds 20, 21
To a solution of 0.012 mol of potassium hydrazinecarbodi-
thionate 18, 19 in 3 ml of water was added 0.024 mol hydra-
zine hydrate and the mixture was heated on steam bath for
1 h. After cooling the solution was quenched with 10 ml water
and acidified with acetic acid. The obtained solid was filtered
and re-crystallized with water.
6.1.5.2. 4-Ethyl-5-(5-phenylthien-2-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione 14. Yield e 87%; mp e 195e197 ꢃC;
1
Rf ¼ 0.63; H NMR (DMSO): 1.32 (t, 3H, CH2eCH3); 4.28
(q, 2H, CH2eCH3); 7.06 (d, 1H, Th); 7.2 (m, 2H, Ar), 7.48
(m,1H-Th, 3H, Ar); Analysis: Calc.: C, 58,51; H, 4.56; N,
14.62; S, 22.31; Found: C, 58.37; H, 4.67; N, 14.41; S, 22.47.
6.1.8.1. 4-Amino-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione 20. Yield: 78%; mp e
181e183 ꢃC; Rf ¼ 0.4, mobile phase: benzene/ethanol e 3:0.5,
1
silica gel plates (Merck, 0.2 mm thick); H NMR (DMSO-
6.1.5.3. 4-Ethyl-5-[5-(4-methylphenyl)thien-2-yl]-2,4-dihydro-
3H-1,2,4-triazole-3-thione 15. Yield e 98%; mp e 226e
228 ꢃC; Rf ¼ 0.63; 1H NMR (C5D5N): 1.34 (t, 3H, CH3);
2.30 (s, 3H, CH3); 4.38 (q, 2H, CH2); 7.10 (m, 1H, Th);
7.58 (m, 5H, 1H-Th, 4H, Ar); 9.41(bs, 1H, NH, exchangeable
with D2O); Analysis: Calc.: C, 59.77; H, 5.02; N, 13.94; S,
21.28; Found: C, 59.49; H, 5.15; N, 14.08; S, 21.37.
d6): 1.75 (m, 4H, 2CH2); 2.37 (m, 2H, CH2); 2.73 (m, 4H,
2CH2); 7.57 (s, 1H, Th); 13.78 (s, 2H, NH2, exchangeable
with D2O); 14.54 (bs, 1H, NH, exchangeable with D2O); Anal-
ysis: Calc.: C, 47.59; H, 4.79; N, 22.20; S, 25.41; Found: C,
47.71; H, 4.92; N, 22.09; S, 25.23.
6.1.8.2. 4-Amino-5-(5-phenylthien-2-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione 21. Yield: 96%; mp
e
185e187 ꢃC;
6.1.6. General procedure for compounds 16, 17
Rf ¼ 0.66, mobile phase: benzene/ethanol e 3:0.5, silica gel
To 1 mlcooled concentratedsulfuric acidwas added 0.01 mol
carbohydrazides 10, 11 for 90 min in portions by cooling (0 ꢃC)
and stirring. The solution was allowed to stay for 2 h at ambient
temperature by stirring. The yellowish orange colored solution
was poured into ice by stirring. The obtained precipitate was fil-
tered, washed with water and re-crystallized with ethanol.
1
plates (Merck, 0.2 mm thick); H NMR (C5D5N): 6.5 (s, 2H,
NH2, exchangeable with D2O); 7.15 (m, 1H, Th); 7.3 (m,
5H, Bz); 7.5 (d, 1H, Th); 8.1 (s, 1H, NH, exchangeable with
D2O); Analysis: Calc.: C, 52.53; H, 3.67; N, 20.42; S,
23.37; Found: C, 52.61; H 4.55; N, 20.54; S, 23.48.
6.1.6.1. N-Ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-1,3,4-
thiadiazol-2-amine 16. Yield e 43%; mp e 137e139 ꢃC;
Rf ¼ 0.56; 1H NMR (C5D5N): 1.2 (t, 3H, CH2eCH3); 1.52 (m,
4H, 2CH2); 2.50 (m, 2H, CH2); 2.74 (m, 2H, CH2); 3.82 (q,
2H, CH2eCH3); 7.71 (s, 1H, Th); 9.16 (bs, 1H, NH,
6.2. Biological evaluation
6.2.1. Cell preparation
Cells from thymus and spleen were obtained by grinding
the tissue in cold PBS, pH 7.2 in Potter homogenizator and

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 44 (2009) 63e69
69
filtering the cells through a 60 mesh and after 10 min centrifu-
Acknowledgements
gation at 1800 rpm the thin white over layer was aspirated and
used in the experiments. Peripheral blood lymphocytes were
obtained from heparinized blood centrifuged at 1100 rpm for
15 min and the white ring was taken out after that laid on
LYMPHOPREP (‘‘Nyccomed’’, Norway) and centrifuged for
further 30 min at 20 ꢃC and 2800 rpm. All the lymphocytes
were finally washed 2 times in cold PBS. The cell vitality
was measured with 0.2% trypan blue and was found to be
85e96%. The number of the immunocompetent cells (RFC
and PFC) was calculated among 106 lymphocytes.
Thanks are due to National Science Fund/Ministry of Edu-
cation and Science grant X1408 and Science Fund/Chemical
Technology and Metallurgy grant 10366 for financial support
of this work.
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