Synthesis and antiproliferative activity of some new thieno[2,3-d ]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety

Article abstract of DOI:10.1016/j.ejmech.2014.09.032

Some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety were synthesized using thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazides as precursors in order to determine their cytotoxicity. Compounds 5, 7-8 and 10-18 wer

Full text of DOI:10.1016/j.ejmech.2014.09.032

Accepted Manuscript
Synthesis and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones
containing 1,2,4-triazole and 1,3,4-thiadiazole moiety
Anelia Ts. Mavrova, Diana Wesselinova, Jordan A. Tsenov, Lubomir A. Lubenov
PII:
S0223-5234(14)00850-2
10.1016/j.ejmech.2014.09.032
EJMECH 7338
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 February 2014
Revised Date: 7 September 2014
Accepted Date: 8 September 2014
Please cite this article as: A.T. Mavrova, D. Wesselinova, J.A. Tsenov, L.A. Lubenov, Synthesis
and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-
triazole and 1,3,4-thiadiazole moiety, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.09.032.
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ACCEPTED MANUSCRIPT
Synthesis and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones
containing 1,2,4-triazole and 1,3,4-thiadiazole moiety
Anelia Ts. Mavrova,a1Diana Wesselinova, b Jordan A. Tsenov c, Lubomir A. Lubenov c
aUniversity of Chemical Technology and Metallurgy, 8Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bInstitute of Experimental Morphology, Pathology and Anrthropology with Museum, Bulgarian Academy of
Science, 1113 Sofia, Bulgaria,
cInstitute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Graphical abstract
Novel thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety were
synthesized and evaluated for their cytotoxicity.
O
R
NH
O
R1
N
NHNH C NH R2
S
S
HT-29
HeLa
Lep-3
O
N
R
N
N
NH
R1
R3
S
X
HepG2
MDA-MB-231
1
Corresponding author: Anelia Mavrova, Department of Organic Synthesis, University of Chemical
Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
e-mail anmav@abv.bg; tel. 0035928163207; fax 003592685488

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones
containing 1,2,4-triazole and 1,3,4-thiadiazole moiety
Anelia Ts. Mavrova,a1 Diana Wesselinova, b Jordan A. Tsenov c, Lubomir A. Lubenov c
aUniversity of Chemical Technology and Metallurgy, 8Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bInstitute of Experimental Morphology, Pathology and Anrthropology with Museum, Bulgarian Academy of Science,
1113 Sofia, Bulgaria,
cInstitute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Abstract
Some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole
moiety were synthesized using thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazides as precursors in
order to determine their cytotoxicity. Compounds 5, 7 - 8 and 10 – 18 were evaluated for their
cytotoxical effect on four cancer cell lines: human colorectal cancer cell line HT-29, breast cancer
cells MDA-MB-231, cervical cancer cells HeLa, human liver carcinoma HepG2 and human normal
diploid cell line Lep3. Exclusively high cytotoxic activity of compounds 8, 16 and 17 against
MDA-MB-231 cells was ascertained and the calculated IC50 values were 3.91.10-2, 1.2.10-3 and 3.74
10-2 ꢀM respectively. Thienopyrimidinones 10, 15 and 17 exhibited high cytotoxicity against HT-29
cell and the IC50 values were in the range 1.56.10-3 ꢀM to 0.13 ꢀM. To HeLa cell lines cytotoxicity
demonstrated compounds 8, 10, 11, 13 and 15-18 but the substance 13 was the most toxic with IC50
- 9.5 10-4 ꢀM. Distinctly high antiproliferative activity of derivatives 10, 14-15 and 17-18 was
estimated against Hep G2, compound 15 showed IC50 – 0.21 ꢀM. Proliferative effects to Lep 3
demonstrated compounds 5, 7-8, 11-14, 16, 18 whose EC50 values were from 0.12 to 2.21 ꢀM. The
1
Corresponding author: Anelia Mavrova, Department of Organic Synthesis, University of
Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
e-mail anmav@abv.bg; tel. 0035928163207; fax 003592685488
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biological data highlighted that the nature and the position of the substituents influence both the
cytotoxicity to the cancer cells and the proliferation properties to Lep3 of the tested compounds.
Keywords: thieno[2,3-d]pyrimidin-4(3H)-ones; 1,2,4-triazoles; 1,3,4-thiadiazoles; cytotoxicity;
anticancer activity;
1. Introduction
The thieno[2,3-d]pyrimidines are compounds of pharmacological interest because of their
similarity to the biogenic pyrimidines. The presence of a pyrimidine ring in the basic building
scaffolds of DNA and RNA modules - thymine, cytosine and uracil is probably the reason for
diversity of their biological activity. Many thieno[2,3-d]pyrimidines has been investigated for their
anticancer activity. A series of thienopyrimidines was identified as a new class of compounds
possessing significant antitumor activities. Among them the cytoxic agent R-253 [N-cyclopropyl-2-
(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent
antiproliferative agent is structurally unique and destabilizes microtubules both in vivo and in vitro
[1]. Some thienopyrimidine derivatives were synthesized as antioxidant and antitumor agents and
had showed significant in-vitro cytotoxic activity against hepatocellular carcinoma (Hep G-2)
compared to the reference drug Doxorubicin [2,3]. 5,6,7,8-Tetrahydrobenzothieno[2,3-d]pyrimidin-
4(3H)-one was identified as a highly selective and potent agent displaying an IC50 of 91 nM toward
the p21-deficient cell line and 2-amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-
d]pyrimidines were potent inhibitors of both thymidylate synthase and dihydrofolate reductase [4,
5, 6]. It was found a 4-morpholino-2-phenylquinazoline derivative to be selective for p110α over
other PI3K isoforms and protein kinases, making it the first example of a selective PI3K p110α
inhibitor [7].
On the other hand the 1,2,4-thriazole heterocycle is of great value as a building block in the
structure of several drug candidates. A series of 3,5-diarylsubstituted-1,2,4-triazole derivatives were
synthesized and it was estimated that the compounds exhibited remarkable anticancer potential in
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screening tests with 60 human cancer cell lines [8]. Some 1,5-disubstituted 1,2,4-triazoles were
synthesized as cis-restricted combretastatin analogues [9] and their effect was accompanied by
apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species,
activation of caspase-3, and PARP cleavage. Diverse 1,2,4-triazoles analogues have been
synthesized and the evaluation for their cytotoxicity against six human cancer cell lines revealed
that some of them displayed promising activity [10]. 1,2,4-Triazole derivatives containing 1,4-
benzodioxan have been screened and have showed good antitumor activity against HEPG2 cancer
cell line [11]. The application of anastrozole and letrozole as aromatase inhibitors for the treatment
of estrogen-dependent cancer as well as the anticancer properties of ribavirine led to the
investigation of many 1,2,4-triazole derivatives in laboratorial conditions for their anti-tumor
activity [12,13].
The 1,3,4-thiadiazoles are of pharmacologic interest because of their antimicrobial,
antifungal [14-15], anti-acetyl- and anti-butyrylcholinesterase [16], antiviral [17] and antioxidant
[18] activities. Many 1,3,4-thiadiazoles which could be seen as isosters of 5-mercapto-substituted
1,2,4-triazoles were synthesized and investigated for their anticancer properties [19-22]. Some
thiadiazole compounds containing acetyl and ethoxy carbonyl groups were described as a new class
of antitumor agents [23]. Having in view the known chemotherapeutic activities both of 1,2,4-
triazoles and 1,3,4-thiadiazoles as anticancer agents, it was of pharmacological interest to
incorporate that moieties into the parent thieno[2,3-d]pyrimidin-4-ones backbone structure to obtain
more active and/or less toxic anticancer agents. The choice of these structures was in conformity
with the fact that the thienopyrimidine heterocycle takes part in the structure of substances
possessing anti-tumor activity.
Considering the above mentioned data and as a continuation of our previous investigation
over 1,2,4-triazoles and 1,3,4-thiadiazoles and as well as thienopyrimidinones, we decided to
synthesize some new thieno[2,3-d]pyrimidin-4-ones containing in their structure 1,2,4-triazole
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respectively 1,3,4-thiadiazole heterocycles in order to study their effects on four human cancer cell
lines.
2. Chemistry
The synthesis of thieno[2,3-d]pyrimidin-4(3H)-one derivatives, containing 1,2,4-triazole or
1,3, 4-thiadiazole ring is illustrated and outlined in Figure1.
O
O
R
R
OC2H5
COOC2H5
NH2
R
b
a
NH
O
a
R1
R1
HCl .HN
R1
S
S
NH
O
S
N
O
C
.HCl
O
1 - 2
3 - 4
3: R = R1 = (CH2)4;
4: R = CH3, R1 = COOC2H5
1: R = R1 = (CH2)4;
2: R = CH3, R1 = COOC2H5
O
N
O
R
R
c
NH
O
NH
O
R1
R1
NHNH C NH R2
S
S
S
N
NHNH2
5 - 6
7 - 10
5: R = R1 = (CH2)4;
6: R = CH3, R1 = COOC2H5
7: R = R1 = (CH2)4; R2 = C2H5
8: R = CH3, R1 = COOC2H5;R2 = C2H5
9: R = R1 = (CH2)4; R2 =C6H5
10: R = CH3, R1 = COOC2H5;R2 = C6H5
e
d
O
R
O
R
N
N
NH
R1
N
N
NH
R1
R2
SH
NH
S
N
R2
N
S
S
N
11 - 14
15 - 18
11: R = R1 = (CH2)4; R2 = C2H5;
15: R = R1 = (CH2)4; R2 = C2H5;
12: R = CH3, R1 = COOC2H5; R2 = C2H5;
13: R = R1 = (CH2)4; R2 = C6H5;
16: R = CH3, R1 = COOC2H5; R2 = C2H5;
17: R = R1 = (CH2)4; R2 = C6H5;
14: R = CH3, R1 = COOC2H5; R2 = C6H5;
18: R = CH3, R1 = COOC2H5; R2 = C6H5;
Scheme 1: Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives of thieno[2,3-d]pyrimidin-
4(3H)-ones; Regents and conditions: a) ethyl cyan acetate, dry HCl gas, ethanol; b) hydrazine
hydrate, ethanol, reflux; c) isothiocyanate, ethanol, reflux; d) 10% NaOH, reflux, hydrochloric acid;
e) sulfuric acid, 0°C, NH4OH;
The starting 3-ethoxycarbonyl-2-amino-thiophenes 1-2 were synthesized according to the
method, described by Gewaldt [24]. Thieno[2,3-d]pyrimidin-4(3H)-ones 3-4 were obtained by
passing a stream of dry hydrogen chloride gas through a solution of the appropriate 2-amino-3-
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ethoxycarbonyl-thiophene in ethyl cyanoacetate. The interaction of the esters 3-4 with hydrazine
hydrate led to the hydrazides 5-6, which by treatment with the corresponding isothiocyanates gave
the semicarbazides 7-10 with yields of 80%. The corresponding 2-(1,2,4-triazol-3-yl)methyl-
thieno[2,3-d]pyrimidin-4(3H)-ones 11-14 were synthesized by refluxing of the relevant
thiosemicarbazide in water solution of sodium hydroxide and following acidifying with
hydrochloric acid. The thiadiazole containing thieno[2,3-d]pyrimidin-4(3H)-ones 15-18 were
obtained from compounds 7-8 in cooled sulfuric acid and neutralization of the obtained solution.
1
The structures of all new compounds were established by IR, H NMR as well as elemental
analysis. Detailed assignment of the 1H NMR and some of the 13C NMR spectra of the synthesized
compounds is given in the Experimental part. The elemental analyses indicated by the symbols of
the elements were within ± 0.4% of the theoretical values.
3. Pharmacology
Twelve of the synthesized compounds (5, 7 - 8 and 10 – 18) were evaluated for their
cytotoxicity to human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231,
cervical cancer cells HeLa, human liver carcinoma HepG2 and human normal diploid cell line
Lep3 by using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrasolium inner salt) - test [25].
4. Result and discussion
To accomplish our aims to synthesize new thienopyrimidinone derivatives we carried out a
study of the reaction between the corresponding 2-aminothiophene and ethyl cyan acetate. By
passing hydrogen chloride gas through a solution of corresponding 2-amino-thiophene in ethyl cyan
acetate were obtained the corresponding esters as precursor for the synthesis of the target
compounds. It was found that the reaction proceeds through amidine hydrochloride formation at
ambient temperature. The reaction of the esters 3 and 4 with excess hydrazine hydrate in ethanol gave
the acetohydrazides 5-8 which upon treatment with the appropriate isothiocyanates afforded the
corresponding thiosemicarbazide derivatives in good yields – 79 – 83 %. The known synthetic
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methods of 1,2,4-triazole or 1,3,4-thiadiazole fragment formation are based on two main approaches:
the first approach is the transforming of thiosemicarbazides in basic medium into triazoles, and the
second approach is the conversion of thiosemicarnazide in thiadiazole derivatives. The yields of the
obtained triazole and thiadiazole compounds were in the range from 75-84 % and 79-87 % respectively.
It should be noted that upon receiving of the products 12 and 14, the ester group in the starting
compounds undergoes hydrolysis yielding the corresponding carboxilyc derivatives.
The in vitro screening of compounds 5, 7 - 8 and 10 – 18 was performed in order to estimate
their effects towards HT-29, MDA-MB-231, HeLa, HepG2 and Lep3 cell lines using the MTS as
described in [20]. The cytotoxicity respectively proliferative effect was assessed by MTS assay,
which is based on the reduction of yellow tetrazolium salt by metabolically active viable cells to a
formazan product that can be measured spectrophotometrically. Hence, the intensity of the colour in
the solution is directly proportional to cell viability [26-27]. The bigger released amount of
formazan indicates to a higher vitality of the cells (proliferation). The low vitality demonstrates a
cytotoxic influence of the experimental compounds. Relative cell viability, expressed as a
percentage of the untreated control (100% viability), was calculated for each concentration. The
obtained results were plotted and IC50 and EC50 were calculated. The data are given in Table 1 and
Table 2.
The examination of the compounds showed that individual cell lines had different sensitivity
towards synthesized compounds. Most of them possessed relatively high cytotoxic effect to the
different tumor cell lines. Thus, compounds 10, 15 and 17 exhibited cytotoxicity against HT-29 cell
line, whereby the IC50 values varied between 1.56.10-3 ꢀM to 0.13 ꢀM. In respect to Lep-3 cell line
the same compounds revealed also cytotoxic effect but in concentrations, which are many times
lower than those at which the compounds excited cytotoxic effect on HT-29 cells (Table 2).
Compounds 8, 10, 15, 16 and 17 showed relative high cytotoxic activity against MDA-MB-
231 cells. Most toxic was compound 16 possessing IC50 value of 1.2.10-3 ꢀM, followed by
compounds 17 and 8 with IC50 - 3.74 10-2 and IC50 - 3.91.10-2 ꢀM respectively.
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Table 1. In vitro cytotoxicity against HeLa, Hep G2, HT-29, MDA-MB-231 and Lep 3cells
Comp
IC50±SE (µM)
.
HT-29
MDA-MB-231 HeLa
3.91.10-2 ± 0.19 1,7 ± 0.03
Hep G2
Lep3
-
8
1.56.10-3 ± 0.02 1.21 ± 0.17
9,3 ± 0,21
1.47 ± 0.05
-
1.5.10.3 ± 0.19
10
11
13
14
15
16
17
18
-
-
1.69 ± 0.18
-
-
9.5. 10-4 ± 0.08 1.8.10 3 ± 0.024
0.29 ± 0.018
9.7.10 -2 ± 0.21 1.26 ± 0.41
3.8 ± 0. 13
0.21 ± 0.16
0.44 ± 0.08
3.17 ± 0.11
1.2.10 -3 ± 0.15 3.31± 0.41
3.74 10-2 ± 0.15 1.85 ± 0.09
0.13 ± 0.17
-
1.23 ± 0.06
0.88 ± 0.04
-
2.3 ± 0.08
To HeLa cell lines cytotoxicity demonstrated the thiosemicarbazides 8 and 10, the triazole
derivatives 11 and 13 and as well as the four studied thiadiazoles 15, 16, 17, 18. The IC50 values of
the thiadiazoles varied from 1.85 to 3.8 ꢀM, but most toxic to HeLa cells was the triazole 13 with
IC50 - 9.5 10-4 ꢀM. It should be pointed out that at the same time compound 13 showed proliferative
activities against Lep 3 with EC50 - 0.46 ꢀM. The calculated IC50 values of compounds 10, 14-15
and 17-18 in test with Hep G2 were from 1.47 to 0.21 ꢀM and most toxic was the thiadiazole 15.
Among the thiosemicarbazides compound 10 was toxic against all cancer cell lines, used in that
study, while it revealed cytotoxicity to Lep 3 in a very high concentration – EC50 - 1.5.10.6 ± 0.19M.
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Table 2. The proliferative activity (EC50) of the studied compounds
Comp.
EC50 ± SE (µM)
HeLa
HT-29
MDA-MB-231
1.54 ± 0.28
1.8 ± 0.24
-
Hep G2
Lep3
2.12 ± 0.53
4.10-2 ± 0.13
8.9.10 -2 ± 0.26
2.43 ± 0.16
0.91 ± 0.59
1.8 ± 0.11
2.52 ± 0.15
2.18 ± 0.31
2.21 ± 0.43
0.12 ± 0.25
2.5.10-2 ± 0.08
1.14 ± 0.38
1.4 ± 0.32
0.46 ± 0.12
1.21 ± 0.11
0.31 ± 0.19
0.26 ± 0.12
5
7
3.2.10 -2 ± 0.14 1.02 ± 0.03
-
0.19 ± 0.32
8
0.13 ± 0.09
1.16 ± 0.26
1.2± 0.16
-
2.08 ± 0.24
11
12
13
14
16
18
2.34 ± 0.18
1.40 ± 0.45
-
-
0.86 ± 0.14
0.42 ± 0.28
0.30 ± 0.20
0.120 ± 0.24
-
-
-
1.26 ± 0.29
-
1.5 ± 0.09
1.8.10-3 ± 0.11
The similarity in the structure of the 2-amino-1,3,4-thiadiazole and the mercapto-1,2,4-
triazole ring systems presumes similar biological properties, but the data, obtained by the MTS-test
accentuate that both the nature of the thienopyrimidine moiety and that of the substituents at the 4-
th position of 1,2,4-triazoles as well as these at the 5-th place of the 1,3,4-thiadiazoles exert
different effects not only on the cytotoxicity but also on the proliferative activity of the studied
compounds. If the effects of the triazole derivatives are taken in to consideration it could be pointed
out that the introduction of a phenyl substituent instead of the ethyl group at the 4-th position of the
1,2,4- triazole 13 leads to increasing of the cytotoxicity towards HeLa cells (the IC50 of compound
11 is 1.69 ꢀM, while that of triazole 13 is 9.5.10-4 ꢀM). The presence of thienopyrimidine ring
bearing a methyl and an ethoxycarbonyl groups in the thiophene heterocycle provoked proliferative
effects of compounds 12 and 14 against HeLa cell lines independently whether an ethyl or a phenyl
group is at the 4-th place of the triazole heterocycle. The biological data indicated that the nature
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and the position of the substituents on the 1,3,4-thiadiazole ring greatly influence both the
cytotoxicity to the cancer cells and the proliferation properties to Lep3 of the tested compounds.
The contribution of the substituents in the structure of theinopyrimidine moiety as well as in
the structure of 1,3,4-thiadiazole ring can be traced through the effects of compounds 15-16 and 17-
18. Compounds 15 and 16 possessed ethylamino group at 4-th place of the thiadazoles ring but
compound 15 containing tetrahydrobenzothienylpyrimidinyl-methyl group in the 2-nd position
showed cytotoxicity to all studied cells. At the same time the presence of ethyl 5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate in the structure of 16 provoked the appearance of
cytotoxicity against MDA-MB-231 and HeLa with IC50 1.2.10 -3 and IC50 3.31 ꢀM respectively and
proliferative activity towards Lep3 ( Figures 1-3).
Figure 1. Viability of HeLa cells (%) after treatment with compounds 15 and 16
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Figure 2. Viability of Hep G2 (%) cells after treatment with compounds 15 and 16
Figure 3. Viability of Lep 3cells (%) after treatment with compounds 15 and 16
The same relationship was observed by compounds 17 and 18. Compound 17 was cytotoxic to all
cell lines, but thiadiazole 18 showed cytotoxicity to HeLa and HepG2 and proliferative activity in
regards to Lep3 (Figures 4-6). As it can be seen from the figures the vitality of all cells treated with
the tested compounds do not exceed 100%. To display more precise the relationship between
structure and activity of the synthesized compounds it is necessary to be carried out both further
research on other derivatives and to be continued the biological research in vivo.
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Figure 4. Viability of HeLa cells after treatment with compound 17 and 18
Figure 5. Viability of Hep G2 cells after treatment with compound 17 and 18
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Figure 6. Viability of Lep 3 cells after treatment with compound 17 and 18
5. Conclusion
New thieno[2,3-d]pyrimidin-4(3H)-ones containing different substituted 1,2,4-triazoles and
1,3,4-thisdiazoles heterocycles were synthesized in good yields using the corresponding thieno[2,3-
d]pyrimidin-3(4H)-yl)acetohydrazides as precursors under optimized reaction conditions.
The initial biological screening in vitro showed that the studied triazole containing
compound 13 possessed relative high cytotoxicity against HeLa cells, the IC50 value was 9.5.10-4
ꢀM but towards the other cells it exhibited proliferative effects, the EC50 values varied from 0.466
to 1,8 µM. Among the thienopyrimidinones containing 1,3,4-thiadiazole ring compounds 15 and 17
revealed cytotoxicity to all used in the screening cells (IC50 - 3.74 10-2 - 3.8 ꢀM) while
compounds16 and 18 demonstrated selective cytotoxicity to the cancer cell lines but showed
proliferative effects on human diploid cells, the EC50 values were in the range 0.26 – 0.31 ꢀM .
The obtained results indicated that the introduction of triazole respectively thiadiazole ring in the
pyrimidinone skeleton prove the necessity for further study to estimate the features related to the
antitumor potential of the tested compounds.
6. Experimental part
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 F254 or silica
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gel plates F254 (Merck, 0.2 mm thick) and visualization was effected with ultraviolet light. IR
spectra were recorded on a Bruker Equinox 55 spectrophotometer as potassium bromide discs. All
NMR spectra were recorded on a Bruker Avance DRX 250 spectrometer (Bruker, Faelanden,
1
13
1
Switzerland) operating at 250.13 MHz for H and 62.89 MHz for C, using a dual 5 mm H/13C
probehead. Chemical shifts were expressed relative to tetramethylsilane (TMS) and were reported
as δ (ppm). The measurements were carried out at ambient temperature (300 K). The microanalyses
for C, H, N and S were performed on Perkin-Elmer elemental analyzer.
6.1. General procedure for the preparation of 1- 2: The 2-amino-thiophenes (1-2) were synthesized
by condensation of cyclohexanone or ethyl acetoacetate with ethyl cyanoacetate and sulfur in the
presence of diethyl amine according to [19].
6.1.1 Ethyl 2-amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate 1: Yield - 82%; Mp – 119 -
-1
1
121ºC, re-crystallization with ethanol; IR, (cm ): 3342, 3232 (ν NH2); 1644.5 (ν C=O); H NMR
(CDCl3) δ (ppm): 1.22 (t, 1H, CH3); 1,76 (m, J3 = 7.10, 4H, 2CH2); 2.45 (m, 2CH2); 2.66 (m, J3 =
8,84 Hz, m 2H, 2CH2); 4.21 (q, 2H, CH2); 6.84 (bs, 2H, NH2, exchangeable with D2O);
6.1.2. Diethyl 5-amino-3-methylthiophene-2,4-dicarboxylate: Yield - 82%; Mp – 119 -121ºC, re-
crystallization with ethanol; Yield – 65 %; Mp – 107 - 109ºC, re-crystallization with ethanol; IR,
(cm -1): 3251.5, 3384.2 (ν NH2); 1680.5 (ν C=O); 1H NMR (CDCl3) δ (ppm): 1,24 (dt, 6H, 2CH3);
2.62 (s, 3H, CH3); 4.33 (m, J = 6.83 Hz, 4H, 2CH2); 6.44 (s, 2H, NH2, exchangeable with D2O);
6.2. General procedure for the preparation of 3- 4:
A stream of dry hydrogen chloride gas was passed through a solution of 0.044 mol of compound
1
or in ethyl cyanoacetate (0.94 mol) for 2 hours at ambient temperature by stirring. At the
2
beginning of the reaction a formation of amidine hydrocloride has been observed, which is turned
later in the reaction solution, forming the thienopyrimidinone ring. The reaction solution was
allowed to stand 24 hours, the excess of ethyl cyan acetate was removed under reduced pressure and
the thienopyrimidinone crystallized. The obtained precipitate was filtered and recrystallized with
ethanol.
13

ACCEPTED MANUSCRIPT
6.2.1. Ethyl (4-oxo-3,4,5,6,7,8-hexahydro[1]benzothieno[2,3-d]pyrimidin-2-yl)acetate 3: Yield -
82%; Mp – 184-186 ºC, re-crystallization with ethanol; Rf = 0.41, mobile phase:
-1
benzene/chloroform/ethanol – 4:1:3; IR, (cm ): 3140.4 (ν NH); 1750.5.5 (ν C=O); 1680.2 (ν
1
C=O); H NMR (DMSO-d6) δ (ppm): 1.14 (t, 3H, CH3); 1.72 (t, 4H, 2CH2); 2.48 (t, J= 8.38 Hz,
2H, CH2); 2.80 ( m, J = 5.56 Hz, 2H, CH2); 4.05 (s, 2H, CH2); 12.36 (s, 1H, NH, exchangeable with
D2O); Analysis: Calc. for C14H16N2O3S: C, 57.52; H, 5.52; N, 9.58; O, 16.42; S, 10.97; Found: C,
57.54; H, 5.52; N, 9.60; O, 16.44; S, 10.95;
6.2.2
Ethyl
2-(2-ethoxy-2-oxoethyl)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxylate 4: Yield – 78 %; Mp – 178-180 ºC, re-crystallization with ethanol; Rf = 0.52, mobile
-1
phase: benzene/chloroform/ethanol – 4:1:3; IR, (cm ): 3180.2 (ν NH); 1753.5.5 (νC=O); 1680.2
1
(νC=O); H NMR (DMSO - d6) δ (ppm): 1.15 (t, 3H, CH3); 1.25 (t, 3H, CH3); 2.76 (s, 3H, CH3);
3.72 (s, 2H, CH2); 4.10 (q, 2H, CH2); 4.21 (q, 2H, CH2); 12.67 (s, 1H, NH, exchangeable with D2O)
Analysis: Calc. for C14H16N2O5S: C, 51.84; H, 4.97; N, 8.64; O, 24.66; S, 9.89; Found: C, 51.81; H,
4.94; N, 8.67; O, 24.68; S, 9.92;
6.3. General procedure for the preparation of compounds 5-6:
To a solution of 0.04 mol of the corresponding thieno[3,2-d]pyrimidinones 3-4 in 60 ml ethanol ,
8.7 ml (0.16 mol ) 98% hydrazine hydrate was added and the mixture was refluxed for 8 hours.
After cooling the obtained solid was filtered and re-crystallized with ethanol.
5.3.1. 2-(4-oxo-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazide 5:
Yield – 90 %; Mp – 278 - 280 ºC, re-crystallization with ethanol; Rf = 0.43, mobile phase: benzene/
ethanol –8:1; IR, (cm -1): 3380.2 , 3183.3 (ν NH2); 1660.2 (ν C=O); 1H NMR (DMSO-d6) δ (ppm):
1.78 (m , 4H, 2CH2); 2.74( t, 2H, CH2); 2.82 (t, 2H, CH2); 3.83 (s, 2H, NH2); 4.15 (s, 2H,CH2); 9.2
(s, 1H, NH); 11.98 (bs, 1H, NH); Analysis: Calc. for C12H14N4O2S: C, 51.78; H, 5.07; N, 20.13; O,
11.50; S, 11.52; Found: C, 51.81; H, 5.04; N, 20.17; O, 11.54; S, 11.56;
6.3.2. Ethyl 3-(2-hydrazino-2-oxoethyl)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxylate 6: Yield – 80 %; Mp – 350 ºC (decomp.), re-crystallization with ethanol; Rf = 0.73,
14

ACCEPTED MANUSCRIPT
mobile phase: benzene/ ethanol – 3:1; IR, (cm -1): 3280.2 , 3203.3 (ν NH2); 1690.5 (ν C=O); 1641,6
1
(ν C=O); H NMR (DMSO- d6) δ (ppm): 1.26 (t, J = 6.82 Hz, 3H,CH3); 2.54 (s, 3H, CH3); 3.98
(s.2H, NH); 4.20 (s 2H, CH2); 4.32 (q, 2H, CH2); 9.45(s, 1H, NH), 12.56 (s,1H, NH); 13C NMR
(DMSO- d6): 14.04 (CH3); 47.20 (CH3); 62.62 (CH2); 118.27 (CH); 118.26 (9-C); 120.05 (7-C);
137.81 (8-C); 155.37 (4-C); 158.76 (6-C); 160.85 (15-C); 166.86 (2-C); 170.26 (11-C); Analysis:
Calc. for C12H14N4O4S: C, 46.44; H, 4.55; N, 18.05; O, 20.62; S, 10.33; Found: C, 46.41; H, 4.59;
N, 18.01; O, 20.60; S, 10.37;
6.4. General procedure for the preparation of compounds 7-10: To a suspension of 0.013 mol of
compounds 5-6 in 40 ml ethanol was added 0.015 mol of the respective isothiocyanate and the
mixture was refluxed by stirring for 4-5 hours. The thiosemicarbazides crystallized after cooling.
6.4.1.
N-Ethyl-2-[(4-oxo-3,4,5,6,7,8-hexahydro[1]benzothieno[2,3-d]pyrimidin-2-
yl)acetyl]hydrazinecarbothioamide 7: Yield – 81 %; Mp – 255-257 ºC (decomp.), re-crystallization
with ethanol; Rf = 0.48, mobile phase: benzene/ ethanol – 2:1; IR, (cm -1): 3218.1 (ν NH); 1651.5 (ν
1
C=O) H NMR (DMSO- d6) δ (ppm): 1.15 (t, 3H, CH3); 1.76 (m, 4H, 2CH2); 2.82 (t, J = 7.14 Hz,
2H, CH2); 2.90 (t, J = 12.40 Hz, 2H, CH2); 3.55 (q, J = 7.30 Hz, 2H, CH2); 4.09 (s, 2H, 2CH2);
12.21 ( bs, 2H, NH); 13.02 (s, 1H, NH); Analysis: Calc. for C15H19N5O2S2: C, 49.30; H, 5.24; N,
19.16; O, 8.76; S, 17.55; Found: C, 49.34; H, 5.26; N, 19.12; O, 8.80; S, 17.57;
6.4.2.
Ethyl
2-(2-{2-[(ethylamino)carbonothioyl]hydrazino}-2-oxoethyl)-5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate 8: Yield – 79.5 %; Mp –210 – 212 ºC (decomp.), re-
crystallization with ethanol; Rf = 0.53, mobile phase: benzene / ethanol – 3:1; IR, (cm -1): 3380.3 (ν
NH); 1740.4; 1703. 1; 1680.7 (ν C=O); 1H NMR (DMSO- d6) δ (ppm): 1.14 (t, 3H, CH3); 1.28 (t,
3H, CH3); 2.55 (s, 3H, CH3); 3.50 (q, J = 7.30 Hz, 2H, CH2); 4.11 (s, 2H, CH2); 4.32 (q, J = 6.82
Hz, 2H, CH2); 12.15 (bs, 3H, NH); 13.18 (s, 1H, NH); Analysis: Calc. for C15H19N5O4S2: C, 45.33;
H, 4.82; N, 17.62; O, 16.10; S, 16.13; Found: C, 45.35; H, 4.84; N, 17.59; O, 16.14; S, 16.17;
6.4.3.
2-[(4-Oxo-3,4,5,6,7,8-hexahydro[1]benzothieno[2,3-d]pyrimidin-2-yl)acetyl]-N-
phenylhydrazinecarbothioamide 9: Yield – 79.5 %; Mp –215 – 217 ºC (decomp.), re-crystallization
15

ACCEPTED MANUSCRIPT
-1
with ethanol; Rf = 0.63, mobile phase: benzene / ethanol – 4:1; IR, (cm ): 3234.6 (ν NH); 1675.6
(ν C=O); 1H NMR (DMSO- d6) δ (ppm): 1.78 (t, 4H,2CH2); 2.77 (t, 2H,CH2, J = 8.0); 2.88 (t, 2H,
CH2, J=7.15); 4.34 (s, 2H-CH2); 6.98 (t, 1H, Bz, J= 7.37); 7.31 (m, 2H, Bz, J= 8.04); 7.48 (d, 2H,
Bz, J= 7.84); 10.31 (s,2H, NH); 11.42(s,1H, NH); 12.56 (s,1H, NH); Analysis: Calc. for
C19H19N5O2S2: C, 55.19; H, 4.63; N, 16.94; O, 7.74; S, 15.51; Found: C, 55.23; H, 4.60; N, 16.96;
O, 7.71; S, 15.48;
6.4.4
Ethyl
2-{2-[2-(anilinocarbonothioyl)hydrazino]-2-oxoethyl}-5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate 10; : Yield – 83 %; Mp –257 – 259 ºC, re-
crystallization with ethanol; Rf = 0.53, mobile phase: benzene / ethanol – 3:1; IR, (cm -1): 3228.4 (ν
NH); 1740.3, 1704.1, 1681.4 (ν C=O); 1H NMR (DMSO- d6) δ (ppm): 1.29 (t, 3H, CH3); 2.56 (s,
3H,CH3); 4.28 (m, 2H, CH2-O, J = 6.83); 4.39 (s, 2H, CH2); 7.12 (t, J=7.37, 1H, Bz); 7.36 (m, J =
7.85, 2H, Bz); 7.59 (d, 2H, Bz); 10.34 (s, 1H, NH); 11.34 (s, 2H, NH); 12.52 (s, 1NH); Analysis:
Calc. for C19H19N5O4S2: C, 51.22; H, 4.30; N, 15.72; O, 14.36; S, 14.39; Found: C, 51.27; H, 4.32;
N, 15.69; O, 14.32; S, 14.43;
6.5. General procedure for the preparation of compounds 11-14: To 0.005 mol of relevant
thiosemocarbazide were added 20 ml of 10% solution of sodium hydroxide and the solution was
refluxed for 5-6 hours. After completing the reaction the solution was cooled and acidified with
hydrochloric acid, whereupon the target compound crystallized.
6.5.1. 2-[(4-Ethyl-5-mercapto-4H-1,2,4-triazol-3-yl)methyl]-5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4(3H)-one 11: Yield – 84 %; Mp > 350 ºC, re-crystallization with ethanol; Rf = 0.48,
-1
1
mobile phase: benzene / ethanol – 3:1; IR, (cm ): 3240.4 (ν NH); 1681.4 (ν C=O); H NMR
(DMSO- d6) δ (ppm): 1.10 (t, 3H, CH3); 1.71 (s, 4H, 2CH2); 2.66 (s, 2H, CH2); 2.80 (s, 2H, CH2);
3.88 (q, 2H, CH2-N); 4.15 (s, 2H, CH2) 12.54 (s, 1H, NH); 13.64 (s1H, SH); Analysis: Calc. for
C15H17N5OS2: C, 51.85; H, 4.93; N, 20.16; O, 4.60; S, 18.46; Found: C, 51.83; H, 4.96; N, 20.19;
O, 4.58; S, 18.44;
16

ACCEPTED MANUSCRIPT
6.5.2. 2-[(4-Ethyl-5-mercapto-4H-1,2,4-triazol-3-yl)methyl]-5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxylic acid 12: Yield – 76.1 %; Mp - 275ºC (decomp), re-crystallization with
-1
ethanol; Rf = 0.42, mobile phase: benzene / ethanol – 2:1; IR, (cm ): 2375 – 3433.5 (ν COOH),
1
3240.4 (ν NH); 1700.4 (ν C=O), 1673.7 (ν C=O); H NMR (DMSO- d6) δ (ppm): 1.19 (t, 3H,
CH3); 2.79 (s, 3H, CH3); 3.95 (q, 2H, CH2); 4.22 (s, 2H, CH2); 12.50 (s, 1H, OH); 12.81 (s, 1H,
NH); 13.69(s, 1H, SH); Analysis: Calc. for C13H13N5O3S2: C, 44.43; H, 3.73; N, 19.93; O, 13.66; S,
18.25; Found: C, 44.41; H, 3.76; N, 19.91; O, 13.64; S, 18.28;
6.5.3.
2-[(5-Mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one 13: Yield –81 %; Mp -334 -336 ºC; Rf =
-1
0.42, mobile phase: benzene / ethanol – 5:1; IR, (cm ): 3287.4 (ν NH); 1670.3 1H NMR (DMSO-
d6) δ (ppm): 1.76 (t, 4H, 2CH2); 2.72 (t, 2H, CH2); 2.79 (t, J= 8.40 Hz, 2H, CH2); 3.96 (s 2H,
CH2); 7.32 (m, J= 8.04 Hz, 2H, Bz,); 7.48 (m, J= 7.54 Hz, 3H, Bz,); 12.22 (s,1H, NH); 13.89 (s,1H,
SH); Analysis: Calc. for C19H17N5OS2: C, 57.70; H, 4.33; N, 17.71; O, 4.05; S, 16.21; Found: C,
57.66; H, 4.36; N, 17.74; O, 4.09; S, 16.18;
6.5.4.
2-[(5-Mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]-5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylic acid 14: Yield – 81 %; Mp -316 -318 ºC; Rf = 0.52,
-1
mobile phase: benzene / ethanol – 4:1; IR, (cm ): 2366 – 3413.5 (ν COOH), 3249.5 (ν NH);
1715.2 (ν C=O), 1683 (ν C=O); 1H NMR (DMSO- d6) δ (ppm): 2.75 (s, 3H, CH3, Th); 4.02 (s, 2H,
CH2); 7.02 (s, 1H, OH); 7.33 (m, 2H, Bz, J= 7.65); 7.45 (m, 3H, Bz, J= 7.56); 12.51 (s, 1H NH);
13.92 (s, 1H, SH); Analysis: Calc. for C17H13N5O3S2: C, 51.12; H, 3.28; N, 17.53; O, 12.02; S,
16.05; Found: C, 51.16; H, 3.30; N, 17.51; O, 12.06; S, 16.08;
6.6. General procedure for the preparation of compounds 15-18:
To a cooled to 0 ºC 98% sulfuric acid (12 ml) was added portionwise 0.003 mol of the appropriated
thiosemicarbazide. Each successive portion was added after complete dissolution of the preceding
one. The reaction mixture was allowed to equilibrate to ambient temperature. The obtained solution
was slowly poured in 20 ml water/ice and neutralized with ammonium hydroxide.
17

ACCEPTED MANUSCRIPT
6.6.1.
2-{[5-(Ethylamino)-1,3,4-thiadiazol-2-yl]methyl}-5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4(3H)-one 15: Yield – 84 %; Mp -166 - 168 ºC; Rf = 0.59, mobile phase: benzene /
ethanol – 4:1; IR, (cm -1): 3189.5 (ν NH); 1663.4 (ν C=O); 1H NMR (DMSO- d6) δ (ppm):
1.10 (t, 3H, CH3); 1.77 (t,2H, 2CH2, J=8.04); 2.72 (t,2H,CH2, J=5.54); 2.86 (t, J = 8.38 Hz, 2H,
CH2); 3.51 (t, 2H, CH2); 3.59 (s, J = 7.13 Hz, 2H, CH2,); 9.54 (bs, 1H, NH); 10.40 (bs 1H, NH);
Analysis: Calc. for C15H17N5OS2: C, 51.85; H, 4.93; N, 20.16; O, 4.60; S, 18.46; Found: C, 51.89;
H, 4.94; N, 20.12; O, 4.66; S, 18.48;
6.6.2.
Ethyl
2-{[5-(ethylamino)-1,3,4-thiadiazol-2-yl]methyl}-5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate 16: : Yield – 87.5 %; Mp - 180 ºC (decomp); Rf =
0.66, mobile phase: benzene / ethanol – 3:1; IR, (cm -1): 3361.5 (ν NH); 1720.2 (ν C=O), 1682.1, (ν
C=O); 1H NMR (DMSO- d6) δ (ppm): 1.30 (t, 6H, 2CH3); 2.82 (s, 3H, CH3); 3.99 (s, 2H, CH2);
4.31 (q, 4H, 2CH2); 9.33 (s, 1H, NH); 12.6 (bs, 1H, NH); Analysis: Calc. for C15H17N5O3S2: C,
47.48; H, 4.52; N, 18.46; O, 12.65; S, 16.90; Found: C, 47.50; H, 4.54; N, 18.42; O, 12.63; S,
16.94;
6.6.3.
2-[(5-Anilino-1,3,4-thiadiazol-2-yl)methyl]-5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4(3H)-one 17: Yield – 87.5 %; Mp – 238-241 ºC Rf = 0.46, mobile phase: benzene /
ethanol – 3:1; IR, (cm -1): 3291.5 (ν NH); 1680.1, (ν C=O); 1H NMR (DMSO- d6) δ (ppm): 1.77 (t,
4H, 2CH2);
2.72 (t, 2H, J = 5.54 Hz CH2,); 2.82 (t, J= 8.40 Hz, 2H, CH2); 4.34 (s, 2H-CH2); 6.98 (t, J = 8.04 Hz
1H, Bz); 7.31 (m, 2H, Bz); 7.48 (d, J= 7.54 Hz; 2H, Bz); 10.31 (s, 1H, NH); 12.56. (s, 1H, NH);
Analysis: Calc. for C19H17N5OS2: C, 57.70; H, 4.33; N, 17.71; O, 4.05; S, 16.21; Found: C, 57.67;
H, 4.31; N, 17.74; O, 4.08; S, 16.18;
6.6.4. Ethyl 2-[(5-anilino-1,3,4-thiadiazol-2-yl)methyl]-5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxylate 18: Yield – 79.5 %; Mp –258-260 ºC Rf = 0.51, mobile phase: benzene
/ ethanol – 3:1; IR, (cm -1): 3331.2 (ν NH); 1742.5 1686.1 (ν C=O); 1H NMR (DMSO- d6) δ (ppm):
18

ACCEPTED MANUSCRIPT
1.30 (t, 3H, CH3); 2.83 (s, 2H,1H, CH3); 4.28 (m, 2H,CH2-O); 4.39 (s, , J = 6.83 Hz, 2H, CH2); 7.01
(t, J= 8.05 Hz, 1H, Bz); 7.36 (m, J= 7.53 Hz, 2H, Bz); 7.59 (d, 2H, Bz); 10.34 (s,1H, NH); 12.52 (s,
NH); Analysis: Calc for C19H17N5O3S2: C, 53.38; H, 4.01; N, 16.38; O, 11.23; S, 15.00; Found: ,
53.37; H, 4.05; N, 16.41; O, 11.26; S, 15.04;
6. 7. Biological assay
The compounds were dissolved in DMSO at the concentration of 4 mg/ml. The investigation was
carried out by dilution of the stock solution in ratio 1:10, 1:100, 1:1000 and 1:10 000. Samples of
cells, grown in non-modified medium served as a control. After 24 h of incubation of the samples
MTS colorimetric assay of cell survival was performed. The wells were treated with MTS solution
and incubated for 2 hours at 37oC under 5% carbon dioxide and 95% air atmosphere. The
absorbance of each well at 490 nm was read by an automatic microplate reader (“Tecan”, Austria).
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List of captions
Scheme 1 Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives of thieno[2,3-d]pyrimidin-
4(3H)-ones; Regents and conditions: a) ethyl cyan acetate, dry HCl gas, ethanol; b) hydrazine
hydrate, ethanol, reflux; c) isothiocyanate, ethanol, reflux; d) 10% NaOH, reflux, hydrochloric acid;
e) sulfuric acid, 0°C, NH4OH;
Figure 1. Viability of HeLa cells after treatment with compound 15 and 16
Figure 2. Viability of Hep G2 cells after treatment with compound 15 and 16
Figure 3. Viability of Lep 3cells after treatment with compound 15 and 16
Figure 4. Viability of HeLa cells after treatment with compound 17 and 18
Figure 5. Viability of Hep G2 cells after treatment with compound 17 and 18
Figure 6. Viability of Lep 3 cells after treatment with compound 17 and 18
Table 1. In vitro cytotoxicity against HeLa, Hep G2, HT-29, MDA-MB-231 and Lep 3cells
Table 2. The proliferative activity (EC50) of the studied compounds
23

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200
150
100
50
15
16
0
16
15
4mg/ml
0,4
0,04
0,004
0,0004
Figure 1. Viability of HeLa cells after treatment with compound 15 and 16

ACCEPTED MANUSCRIPT
O
O
N
R
R
OC2H5
COOC2H5
NH2
R
b
a
NH
O
a
R1
R1
R1
S
S
NH
C
O
S
O
.HCl
HCl .HN
O
1 - 2
3 - 4
3: R = R1 = (CH2)4;
4: R = CH3, R1 = COOC2H5
1: R = R1 = (CH2)4;
2: R = CH3, R1 = COOC2H5
O
O
R
R
c
NH
O
NH
O
R1
R1
S
S
N
NHNH C NH R2
S
N
NHNH2
5 - 6
7 - 10
5: R = R1 = (CH2)4;
6: R = CH3, R1 = COOC2H5
7: R = R1 = (CH2)4; R2 = C2H5
8: R = CH3, R1 = COOC2H5;R2 = C2H5
9: R = R1 = (CH2)4; R2 =C6H5
10: R = CH3, R1 = COOC2H5;R2 = C6H5
e
d
O
R
O
R
N
N
NH
R1
N
N
NH
R1
R2
SH
NH
S
N
R2
N
S
S
N
11 - 14
15 - 18
11: R = R1 = (CH2)4; R2 = C2H5;
15: R = R1 = (CH2)4; R2 = C2H5;
12: R = CH3, R1 = COOC2H5; R2 = C2H5;
13: R = R1 = (CH2)4; R2 = C6H5;
16: R = CH3, R1 = COOC2H5; R2 = C2H5;
17: R = R1 = (CH2)4; R2 = C6H5;
14: R = CH3, R1 = COOC2H5; R2 = C6H5;
18: R = CH3, R1 = COOC2H5; R2 = C6H5;
Scheme 1: Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives of thieno[2,3-
d]pyrimidin-4(3H)-ones; Regents and conditions: a) ethyl cyan acetate, dry HCl gas, ethanol;
b) hydrazine hydrate, ethanol, reflux; c) isothiocyanate, ethanol, reflux; d) 10% NaOH, reflux,
hydrochloric acid; e) sulfuric acid, 0°C, NH4OH;

ACCEPTED MANUSCRIPT
120
100
80
60
40
20
0
15
16
16
15
4 mg/ml
0,4
0,04
0,004
0,0004
Figure 2. Viability of Hep G2 cells after treatment with compound 15 and 16

ACCEPTED MANUSCRIPT
120
100
80
60
40
20
0
15
16
16
15
4 mg/ml
0,4
0,04
0,004
0,0004
Figure 3. Viability of Lep 3cells after treatment with compound 15 and 16

ACCEPTED MANUSCRIPT
350
300
250
200
150
100
50
17
18
0
4 mg
0,4
0,04
0,004
0,0004
0,00004
Figure 4. Viability of HeLa cells after treatment with compound 17 and 18