Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones

Article abstract of DOI:10.1016/j.ejmech.2016.07.022

Some derivatives of 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized using ethyl 2-aminothiophene-3-carboxylates as precursors in order to estimate their cytotoxicity, respectively proliferative activity. Thienopyrimidinones containing thiosemicarbazide as well as 1,3,4-thiadiazole moieties were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 as well as human diploid cell line Lep-3. Compounds 5b, 6a and 6b revealed cytotoxicity to the four studied cancer cell lines. The highst cytotoxicity against MDA-MB-31 exhibited the thiosemicarbazide 5b with IC₅₀2.31.10^?4?μM, but most active towards HT-29?cell lines was thienopyrimidine 6c with IC₅₀0.001?μM. Compound 6a showed the highest inhibitory activity with IC₅₀- 0.99?μM to human liver carcinoma HepG2 cells and low cytotoxicity towards Lep3 (IC₅₀?=?191?μM). The thienopyrimidine derivative linked to thiadiazole 6b was toxic to the four studied cancer cell lines, especially to HeLa (IC50–0.83?μM), and besides that the compound demonstrated toxicity to Lep 3?cells at very high concentration 89?×?10³?μM. The solid-state photostability of the derivatives 5a-c and 6a-c was tested by irradiation with UV light. All of the studied compounds show solid-state photostability in 240?min of irradiation. Using MOE software molecular docking of the three ligands 5b, 6b and 7 was accomplished into an internal pocket formed by the activation segment and the P-loop of^V599EB-Raf. It was established that the binding of the ligands to^V599EB-Raf promotes an inactive conformation of the enzyme.

Full text of DOI:10.1016/j.ejmech.2016.07.022

Accepted Manuscript
Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-
thieno[2,3-d]pyrimidin-4(3H)-ones
Anelia Ts Mavrova, Stefan Dimov, Denitsa Yancheva, Miroslav Rangelov, Diana
Wesselinova, Jordan A. Tsenov
PII:
S0223-5234(16)30570-0
10.1016/j.ejmech.2016.07.022
EJMECH 8740
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 February 2016
Revised Date: 10 July 2016
Accepted Date: 12 July 2016
Please cite this article as: A.T. Mavrova, S. Dimov, D. Yancheva, M. Rangelov, D. Wesselinova,
J.A. Tsenov, Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-
thieno[2,3-d]pyrimidin-4(3H)-ones, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.07.022.
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ACCEPTED MANUSCRIPT
Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-thieno[2,3-
d]pyrimidin-4(3H)-ones
Anelia Ts. Mavrova,a1 Stefan Dimov a, Denitsa Yancheva b, Miroslav Rangelov b, Diana Wesselinova c
Jordan A. Tsenov b
aUniversity of Chemical Technology and Metallurgy, Department of Organic Synthesis, 8Kliment Ohridski Blvd., 1756
Sofia, Bulgaria
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev
Str., build. 9, 1113 Sofia, Bulgaria
c Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Science, 1113
Sofia, Bulgaria
Graphical abstract
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, anticancer activity and photostability of novel 3-ethyl-2-mercapto-thieno[2,3-
d]pyrimidin-4(3H)-ones
Anelia Ts. Mavrova,a1 Stefan Dimov a, Denitsa Yancheva b, Miroslav Rangelov b, Diana Wesselinova c
Jordan A. Tsenov b
aUniversity of Chemical Technology and Metallurgy, Department of Organic Synthesis, 8Kliment Ohridski Blvd., 1756
Sofia, Bulgaria
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad.
G. Bonchev Str., build. 9, 1113 Sofia, Bulgaria
Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy
of Science, 1113 Sofia, Bulgaria
Abstract
Some derivatives of 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized
using ethyl 2-aminothiophene-3-carboxylates as precursors in order to estimate their cytotoxicity,
respectively proliferative activity.
Thienopyrimidinones containing thiosemicarbazide as well as 1,3,4-thiadiazole moieties were
evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-
231, HeLa, HepG2 as well as human diploid cell line Lep-3. Compounds 5b, 6a and 6b revealed
cytotoxicity to the four studied cancer cell lines. The highst cytotoxicity against MDA-MB-31
exhibited the thiosemicarbazide 5b with IC50 2.31.10-4 ꢀM, but most active towards HT-29 cell lines
was thienopyrimidine 6c with IC50 0.001 ꢀM. Compound 6a showed the highest inhibitory activity
with IC50 - 0.99 ꢀM to human liver carcinoma HepG2 cells and low cytotoxicity towards Lep3 (IC50 =
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
1

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191 ꢀM). The thienopyrimidine derivative linked to thiadiazole 6b was toxic to the four studied cancer
cell lines, especially to HeLa (IC50 - 0.83 ꢀM), and besides that the compound demonstrated toxicity
to Lep 3 cells at very high concentration 89.103 ꢀM.
The solid-state photostability of the derivatives 5a-c and 6a-c was tested by irradiation with UV
light. All of the studied compounds show solid-state photostability in 240 min of irradiation.
Using MOE software molecular docking of the three ligands 5b, 6b and 7 was accomplished
into an internal pocket formed by the activation segment and the P-loop of V599EB-Raf. It was
established that the binding of the ligands to V599EB-Raf promotes an inactive conformation of the
enzyme.
Keywords: thieno[2,3-d]pyrimidin-4(3H)-ones, DFT, cytotoxicity, photo stability, anticancer activity,
B-Raf
1. Introduction
It is well known that the pyrimidine structure is closely related to three of the four nucleobases
uracil, thymine and cytosine, which fact makes pyrimidines essential building blocks of all living cells
[1]. Thieno[d]pyrimidines are found to have broad spectrum of biological activity as anticancer [2],
antibacterial [3], antiviral [4], antioxidant [5], antihistaminic [6], anti-inflammatory [7], analgesic [8,
9].
It was found that some thieno[3,2-d]pyrimidines are bioisosters of lapatimib (IC50 120nМ) [10]
and some of them have shown better inbitiory activity (IC50 11nМ) towards epidermal growth factor
receptor tyrosine kinase (EGFR/ErbB-2), compared to the 4-aminoquinazoline derivative – lapatimib.
Many thieno[2,3-d]pyrimidines have showed significant in vitro cytotoxic activity against
hepatocellular carcinoma (Hep G-2) compared to the reference drug Doxorubicin [11, 12]. Wang et al.
have found that some 5,6,7,8-tetrahydrothieno[2,3-d]pyrimidine-4-ones preferentially affect p21-
deficient cancer cells (IC50 2.3ꢀМ) [13]. The further SAR analysis indicated that one of the important
structural-activity recommendations is the presence of oxygen in C-4 position of the pyrimidine ring.
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The 1,3,4-thiadiazole (TDA) derivatives are known because of their pharmacological activity
as anti-inflammatory [14], antibacterial [15], antitumor [16]. The anticancer effect of 1,3,4-thiadiazoles
is mainly related to the high electron-donating ability of the two nitrogen atoms to form H-bonds. 2-
Amino-1,3,4-thiadiazoles have a variety of anticancer activity against melanoma, glioblastoma,
lymphosarcoma [17]. Some thiadiazole derivatives have shown anticancer activity against HepG-2
[18, 19] and HeLa cell lines compairable with staurosporine, resulting from H-bond between the
nitrogen atom from the 1,3,4-thiadiazole and the amino hydrogen of GLY505 amino acid − part of the
ATP binding site of focal adhesion kinase [20]. There was reported a series of 2-amino-1,3,4-
thiadiazoles that display growth inhibition in A549 lung carcinoma cells by inhibiting ERK1/2
pathway and cell cycle progression through G1 into S phase [21]. In addition, same TDA-based
compounds are shown to possess significant anticancer activity against prostate cancer (LnCaP,
DU145 and PC3) and breast cancer cell lines (MCF-7 and MDA-MB-231) [22, 23].
The mesoionic nature of thiadiazoles makes these compounds to be more able to cross cellular
membranes [24]. Therefore, combining TDA with an effective anticancer adenine and guanine based
pyrimidine bioisoster as thieno[2,3-d]pyrimidine-4-ones could lead to potential inhibiting effect on
purine synthesis and less toxic anticancer agents. In the literature it has been described that the 1,3,4-
thiadiazole containing 5,6,7,8-tetrahydrothieno[2,3-d]pyirimidine-4-ones possess anticancer activity
[25, 26]. This fact has drawn our attention for further investigation of that bipharmacophoric
compounds as potential anticancer drugs on four human cancer cells and one non-tumorogenic cell
line.
2. Chemistry
The synthesis of N-ethyl-thieno[2,3-d]pyrimidine-4-one derivatives, containing 1,3,4-
thiadiazole moiety is illustrated on Scheme 1.
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O
C O
O
R
R
R
N
O
b
a
SH
NH2
R1
R1
N
R1
S
S
2a: R = CH3; R1 = CH3;
2b: R= CH3; R1 =COOC2H5;
2c: R = R1 = (CH2)4
1a: R = CH3; R1 = CH3;
1b: R= CH3; R1 =COOC2H5;
1c: R = R1 = (CH2)4
c
O
O
R
R
N
N
O
d
C
S
R1
S
N
O
S
R1
N
O
S
C
NH
NH2
3a: R = CH3; R1 = CH3;
3b: R= CH3; R1 =COOC2H5;
3c: R = R1 = (CH2)4
4a: R = CH3; R1 = CH3;
4b: R= CH3; R1 =COOC2H5;
4c: R = R1 = (CH2)4
e
O
R
O
R
f
N
N
N
NH
N
NH
C
S
C
S
S
R1
N
S
R1
N
O
S
NH
S
NH
6a: R = CH3; R1 = CH3;
6b: R= CH3; R1 =COOC2H5;
6c: R = R1 = (CH2)4
5a: R = CH3; R1 = CH3;
5b: R= CH3; R1 =COOC2H5;
5c: R = R1 = (CH2)4
Scheme 1 Synthesis of semicarbazide and 1,3,4-thiadiazole derivatives of thieno[2,3-d]pyrimidine-4-
ones: a) ethyl cyanacetate, HNEt2, S8, ethanol; b) ethyl isothiocyanate, NaOH, DMF; c) ethyl 2-
chloroacetate, NEt3, benzene, reflux; d) hydrazine hydrate, ethanol, reflux; e) ethyl isothiocyanate,
ethanol, reflux; f) 98% sulfuric acid, 0°C, NH4OH
The 2-aminothiophene esters 1a-c used as starting materials were synthesized by Gewald
reaction which is a one-pot reaction method for 2-aminothiophene synthesis [27-30]. The synthesis of
N-ethyl-2-mercapto-thieno[2,3-d]pyrimidine-4-ones 2a-c was accomplished by alkaline catalized
cyclocondensation of the 2-aminoesters 1a-c with ethyl isothiocyanate. The S-nucleophilic substitution
between the 2-mercapto-thienopyrimidine-2-ones and ethyl chloroacetate, using triethylamine as a
organic base gave thioacetate derivatives 3a-c. The interaction of the esters 3a-c with hydrazine
hydrate in absolute alcohol led to the obtaining of the hydrazides 4a-c. The latter, by treatment with
the corresponding ethyl isothiocyanate under reflux gave the semicarbazides 5a-c with high yeilds.
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The cyclocondensation to the corresponding semicarbazides 5a-c was carried out in water solution of
sodium hydroxide under reflux, followed by acidification with hydrochloric acid. The thiadiazole
containing thieno[2,3-d]pyrimidin-4(3H)-ones 6a-c were obtained from compounds 5a-c in cooled
concentrated sulfuric acid.
3. Pharmacology
The semicarbazides 5a-c and thiadiazole containing thieno[2,3-d]pyrimidin-4(3H)-ones 6a-c
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 cell line 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 [31].
4. Results and discussion
A series of 2-aminothiophenes has been prepared for the aim of our research via a
multicomponent condensation between appropriate ketone, ethyl cyanoacetate, sulfur and diethyl
amine under conditions, described by Gewald [27]. The pyrimidine ring formation of 3-ethyl-2-
mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones 2a-c was performed by basic catalyzed heterocyclic
cyclocondensation of the 2-aminothiophenes 1a-c and ethyl isothiocyanate. It is known that
equilibrium between the thiol and thione form exists, but in an alkaline medium the presence of the
thiol form is advantageous, therefore the acidic 2-SH group of the thienopyrimidin-2-thiols was
alkylated with ethyl chloroacetate in presence of triethyl amine. The reaction of esters 3a-c with excess
amount of hydrazine hydrate in absolute ethanol led to acetohydrazides 4a-c. Within that group, the
synthesis of 4c occured with the highest yield − reaching 94%. The aforementioned compounds were
treated with 12-fold access of ethyl isothiocyanate giving thiosemicarbazides 5a-c with excellent
yields from 88% to 96%. 1,3,4-Thiadiazoles 6a-c were obtained from thiosemicarbazides 5a-c by
dehydrative cyclization in acidic medium. The yields of the final thiadiazole 6b and 6c were in the
range from 90 to 95%.
The chemical structures of the new compounds were established by elemental analyses, IR-, 1H
NMR and the results are presented in the Experimental part. The elemental analyses indicated by the
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symbols of the elements were within ± 0.4% of theoretical values. In the 1H NMR spectra, particularly
meaningful are characteristic SCH2CO signals (singlets) of the substituded in 2 place compounds as
well as the as the signals of S-CH2 between the both heterocycles, shifted downfield due to deshielding
effect of both S-atom and the CO group respectively the pyrimidine and thiadiazole rings. The
chemical shift values varied in the range from 3.99 to 7.43 ppm for the esters, 4.08- 4.11 ppm for the
thiosemicarbazides and from 4.45 to 4.84 ppm depending of the solvents and substituents. The labile
NH protons are not characteristic and their chemical shifts depend on the water quantity in the solvent.
Some 1H NMR spectra are given in the Supplementary material.
The thiosemicarbazide (5a-c) and 1,3,4-thiadiazole (6a-c) thieno[2,3-d]pyrimidine-4-one
derivatives were subjected to in vitro screening in order to estimate their effects towards human cancer
HT-29, MDA-MB-231, HeLa, HepG2 and normal human Lep3 cell lines.
The cell proliferation was established using the MTS assay, based on the reduction of the
tetrazolium salt into a coloured, aqueous soluble formazan product by mitochondrial activity of viable
cells at 37°C. The released amount of formazan produced by dehydrogenase enzymes is proportional
to the number of living cells in the culture and can be measured spectrophotometrically at λmax = 492
nm [32]. The higher levels of formazan indicate to a higher vitality of the cells, whereas the low
amount of formazan is an indicator for the cytotoxicity of the tested compounds [33, 34]. In addition,
the relative cell viability, expressed as a percentage of the untreated control (100% viability) was
calculated for each concentration. All data points represent an average of three independent assays.
The obtained results were plotted and EC50 (Table 1) and IC50 (Table 2) were calculated. Statistical
significant differences in the level of cells in both control and experimental groups were determined (p
≤ 0.05).
Table 1 The proliferative activity (EC50) of the studied compounds
EC50 ± SE (µM)
Compounds
HT-29
0.085 ± 0.20
1.13 ± 0.16
−
MDA-MB-231
8.86 ± 0.10
9.33 ± 0.12
HeLa
Hep G2
Lep3
0.43 ± 0.03
7.97 ± 0.72
9.28 ± 0.03
−
2.72 ± 0.31
−
−
8.25 ± 0.24
−
5a
5c
6c
0.001 ± 0.14
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Table 2 In vitro cytotoxicity against HeLa, Hep G2, HT-29, MDA-MB-231 and Lep 3 cells
IC50 ± SE (µM)
Compounds
HT-29
−
7.38 ± 0.71
3.58 ± 0.25
9.12 ± 0.37
0.001 ± 0.11
MDA-MB-231
−
HeLa
−
8.71 ± 0.09
7.42 ± 0.22
0.83 ± 0.18
−
Hep G2
−
8.71 ± 0.58
0.99 ± 0.52
9.51 ± 0.03
9.8 ± 0.18
Lep3
0.01 ± 0.22
0.8 ± 0.18
191 ± 0.03
89.103 ± 0.27
9.77 ± 0.09
5a
5b
6a
6b
6c
2.31.10-4 ± 0.04
9.4 ± 0.15
2.13. ± 0.13
−
It can be seen that the most of the examined compounds exhibited wide inhibitory
concentration range for all tested human cell lines. Among the thieno[2,3-d]pyrimidine-4-ones,
containing thiosemicarbazide moiety, compound 5b was toxic against all cancer cell lines, used in that
study, especially against breast cancer cells MDA-MB-231 (IC50 = 2,31.10−4 ꢀM), while in the same
time it revealed cytotoxicity to Lep3 in a much higher concentration with IC50 = 0,8 ꢀM.
In the group of the thienopyrimidine derivatives linked to thiadiazole ring, compound 6b was
toxic to the four studied cancer cell lines, especially to HeLa cells (IC50 - 0.83 ꢀM), besides the
compound demonstrated toxicity to Lep 3 cells at very high concentrations, the determined IC50 value
was 89.103 ꢀM. The low cytotoxic profile to Lep3 can provide high selectivity towards all four cancer
cell lines at small concentrations.
Apparently, the most active against human colorectal cancer cell line HT-29 was compound 6c
with IC50 0,001 ꢀM, followed by 6a with IC50 3.58 ꢀM, respectively, but derivative 6c exibits about
100-fold lower cytotoxicity against normal diploid cell line Lep3.
With respect to human liver carcinoma HepG2 cells compound 6a showed the highest
inhibitory activity with IC50 - 0.99 ꢀM and the second lowest cytotoxicity towards Lep3 (IC50 = 191
ꢀM).
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Fig. 1 Viability of Lep 3 cells (%) after treatment
In summary, it can be noted that thieno[2,3-d]pyirimidine-4-ones containing thiosemicarbazide
(5a-c) and thiadiazole moiety (6a-c) exhibit high effect on cell proliferation towards all tested human
cancer and normal cell lines, whereat compound 6b showed comparatively much lower toxicity against
normal Lep3 cells (Fig. 1). This makes the thiadiazole derivative 6b efficient anticancer agent against
all tested tumor cells at low concentration, and at the same time the compound will not demonstrate
any cytotoxicity upon normal human cells. It was observed that the variation of substituents at 5-C and
6-C position of the thiophene ring leads to a major decrease of Lep3 cell line vitality, whereas the only
significant increase (1000-fold) of the apoptotic effect was indicated for the more lipophilic tetrahydro-
substitued compound 6c against MDA-MB-231 and HT-29 cancer cells.
The term ‘photostability’ includes not only the degradation caused by exposure to light, but
also processes as radical formation, energy transfer and luminescence [35]. A large number of drug
substances absorb radiation in the ultraviolet and/or visible area and are thus sensitive to light. They
undergo photodegradation in liquid media or in solid state on exposure to light [36]. To the
photodegradation factors of drug dosage forms in the solid state belong the particle sizes, the surface
area, the colour and crystal structure, the photodimerization or isomerization. There is correlation
between the transparency of the products and the radiation being used. The radiation to which a sample
is exposed may be reflected, scattered, transmitted or absorbed. Only the absorbed radiation
participates in photodegradation. There are many results for the photodegradation of substances in
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aqueous solution, but relatively few studies are done on photostability in solid state. Correlations
between photochemical behaviour in solution and in solid state are not clearly established.
Fig. 2: (a) Solid-state photostability ratiometric analysis of 5a (C=3,128.10-5±0,125 mol/L), 5b
(C=2,185.10-5±0,087 mol/L), 5c (C=3,916.10-5±0,158 mol/L), 6a (C=5,242.10-5±0,209 mol/L), 6b
(C=2,275.10-5±0,091 mol/L), 6c (C=4,089.10-5±0,164 mol/L) in DMF solution; (b) Absorbtion
spectrum of compound 5a;
The solid-state photostability of the novel thieno[2,3-d]pyrimidine-4-one derivatives 5a-c and
6a-c, was tested by irradiation with UV light in a SUNTEST equipment. The kinetics of the compound
photodegradation was monitored colorimetrically. As no changes were established in the absorption
maxima (λA) of compound 5b-c and 6a-c during the irradiation, the correlation between the drug
concentration and the time of irradiation was constant value (Fig. 2a). The 240 min irradiation of
thiosemicarbazide 5a resulted with nearly 30% photodegradation (Fig. 2b).
For promising pharmacological potential, the studied thieno[2,3-d]pyrimidine-4-ones should
possess favourable pharmacokinetic properties in vivo i.e. sufficient bioavailability and transportation
through the different membranes to the desired receptor binding site, as well as an optimal
metabolization and elimination profile. Calculated molecular properties such as lipophilicity,
molecular size, flexibility and presence of hydrogendonor and acceptors could provide useful
information in this relation.
It can be seen from the SAR analysis (Table 3), made by using Molinspiration tool [37], that
all of the examined compounds 5a-c and 6a-c respond to Lipinski’s ‘‘Rule of Five’’ [38]. It appeared
that compounds 6a and 6c possessing the one and the same low topologic polar surface area (TPSA) of
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72.71 Å2 and a equal number of hydrogen donors (NHD) and acceptors (NHA), were the most active
against HT-29 cell line. The low TPSA provides a sufficient intestinal absorbtion on the drug in
humans [39] and the higher number of NHA provides higher solubility, which explains their citotoxicity
against HT-29 and makes 6a and 6c promising candidates for in vivo analysis. The least toxic
compounds against human normal diploid cell line Lep3 6a and 6b have the TPSA = 72.71 Å2 and
99.01 Å2 respectively.
Table 3 Calculated molecular properties of the tested compounds: partition coefficients (logP),
molecular weight (MW) [g/mol], topologic polar surface area (TPSA) [Å2], molecular volume (Vol.)
[Å3], sum of OH and NH H-bond donors (NHD) and sum of O and N H-bond acceptors (NHA)
NHD
Compounds
logP
M.W.
TPSA
Vol
NHA
2.14
2.36
2.71
2.98
3.21
3.56
1.28
399.57
457.60
425.61
381.55
439.59
407.59
397.48
88.05
114.35
88.05
72.71
99.01
72.71
125.21
337.77
382.54
361.01
319.93
364.70
343.17
330.67
7
9
7
6
8
6
9
3
3
3
1
1
1
4
5a
5b
5c
6a
6b
6c
Aa)
a) The compound is previously reported in [23]; for molecular structure see Fig. 3.
The most active to MDA-MB-231 cells compound (5b) has the highest sum of H-bond donor (NHD)
and acceptor (NHA) groups of all examined compounds as well as the highest TPSA, 114.35 Å
respectively. It is well known that to show an acceptable bioavaibility one compound should have
TPSA lower than 140–150 Å2. These molecular properties coincide well with those characterizing the
structurally related thieno[2,3-d]pyrimidine-4-one A (ethyl 2-(2-{2-[(ethylamino)carbonothioyl]
hydrazino}-2-oxoethyl)-5-methyl-4-oxo-3,4-dihydrothieno[2,3d]pyrimidine-6-carboxylate Fig. 3), that
shows MDA-MB-231 selectivity and was recently reported under number 8 in [23]. It can be assumed
that the high TPSA, molecular weight (M.W.) and volume (Vol.) of 5b contribute to a better fitting
with the active center, inhibiting cancer MDA-MB-231 cell line growth. The high cytotoxicity of 5b
should be attributed to the alkylation of the amide group in the pyrimidine ring and to the replacement
of the aliphatic spacer with thiomethylene group (Fig. 3).
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S
NHC2H5
O
O
C2H5
S
H
S
NHC2H5
H3C
H3C
N
N
CONHNH
CONHNH
O
O
C
N
N
H2
S
S
C2H5O
C2H5O
5b
IC50 0.23 nM
A
IC50 39 nM
Fig. 3 Structures of MDA-MB-231 cell line growth inhibitors 5b and A
Based on the structures of 5b and 7 (Fig. S14), optimized at DFT B3LYP/6-311++G** level of
theory [40,41], it can be presumed, that the replacement of the methylene group at 2-nd position of the
pyrimidine with sulfur spacer leads to a much flatter molecular conformation of 5b. That fact,
combined with the N-alkylation at 3-rd position, results in larger molecular volume of 5b than A.
Protein tyrosine kinases are key regulators of cell function, which represent one of the largest
and most functionally different gene families with particular relevance to many human diseases,
including cancer. The kinases are particularly eminent in signal transduction and coordination of
complex functions at the cell cycle. Beside that fact in the literature it has been reported that the highly
selective carbamide derivative (sorafenib, S. Fig. S15) shows evidence for tumor regressions after 9
days of oral dosing against breast cancer MDA-MB-231 cells [42, 43]. In addition a series of
thieno[2,3-d]pyrimidines was synthesized and identified as inhibitors to the most critical isoform of the
Raf family – B-Raf [44-46]. It is assumed that such cell line selectivity of the carbamide and
respectively thiosemicarbazides (5b, 6b and A) share the same bioisosteric mechanism of action by
affecting tumor growth through blocking two cellular processes essential for tumor development:
inhibition of the MAPK pathway and inhibition of tumor angiogenesis.
In order to clarify the possible binding modes of 5b, 6b and A with B-Raf, the compounds were
docked into the crystal structures of human oncogenic V599EB-Raf in complex with sorafenib (PDB
code: 1UWJ [43]) using MOE software [47].
The kinase domain of V599EB-Raf has a bilobal structure typical also for other members of the
protein kinase family group [43]. Similarly to sorafenib (Fig. S15) the three ligands are bound to the
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interfacial cleft, buried between the N and C lobes of the kinase domain. The docking conformations
of 5b, A and 6b are shown in Fig. 4(a), 5(a) and 6(a).
In all cases, the N-ethyl-thieno[2,3-d]pyrimidine-4-one fragment occupies a pocket formed by
residues Val503, Leu504, Ile571, Phe594, Lys482 and Gly592, with only the ethoxy group exposed to
the solvent (Fig. 4(b), 5(b) and 6(b)). The van der Waals interactions dominate the contacts of 5b and 7
with V599EB-Raf as it could be seen from the 2D representations of the ligands in the pocket (Fig. 4(b)
and 5(b)).
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(a)
(b)
Fig. 4: (a) Docking conformation of 5b with shown amino acid residues Thr528, Asp593, Lys482,
Glu500, Gly592; (b) 2D representation of the interactions of 5b with V599ЕB-Raf
Polar interactions to the catalytic Glu500, Gly592 from the activation segment of the DFG
motif and Lys482 or Thr528 also contribute to the stabilization of the ligand-pocket binding. The
thiosemicarbazides moiety of 5b is connected via two hydrogen bonds to Thr528 and Asp593. The
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other MDA-MB-231 selective thieno[2,3-d]pyrimidine-4-one 7 forms hydrogen bonds to Lys482 and
Asp593.
(a)
(b)
Fig. 5: (a) Docking conformation of A with shown amino acid residues Thr528, Asp593, Lys482,
Glu500, Gly592; (b) 2D representation of the interactions of A with V599EB-Raf
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In the case of 6b, one of the N-atoms of the thiadiazole moiety acts as hydrogen bond acceptor
to Thr528, while the N-ethyl substituent is oriented towards the polar Glu500 and Asp593.
(a)
(b)
Fig. 6: (a) Docking conformation of 6b with shown amino acid residues Thr528, Asp593, Lys482,
Glu500, Gly592; (b) 2D representation of the interactions of 6b with V599ЕB-Raf
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These polar interactions along with the hydrophobic interaction to Phe594 connect the three
ligands to the activation segment of the DFG motif. On the other side, as it was mentioned above a
number of contacts to the catalytic residues Lys482, Glu500 is formed. In that way the binding of the
ligands to V599EB-Raf promotes an inactive conformation of the enzyme by holding the activation
segment of the DFG motif and hindering its phosphorylation.
5. Conclusion
A serie of eight new thieno[2,3-d]pyrimidin-4(3H)-ones containing different substituted
thiosemicarbazide, 1,2,4-triazoles and 1,3,4-thiadiazoles heterocycles were synthesized using the
corresponding 2-(thieno[2,3-d]pyrimidin-2-yl)thioacetohydrazides as precursors under optimized
reaction conditions with good yields.
The initial biological screening in vitro, using MTS tetrazolium assay has showed that the
studied thiosemicarbazide containing thieno[2,3-d]pyirimidine-4-one 5b, connected through sulfur
linker possess high cytotoxicity against MDA-MB-231 cells. The cytotoxic effect of 5b towards Lep3
cells was comparatively much lower. Among the thienopyrimidinones containing 1,3,4-thiadiazole
ring compounds 6b revealed cytotoxicity to all screening cancer cells with IC50 from 9,51.10−5
ꢀM
against Hep G2 to 0.83 M againt HeLa cell line, while at the same time indicated relatively high
ꢀ
vitality levels of Lep3 with IC50 - 89.103 ꢀM. Compound 6c demonstrated selective cytotoxicity to HT-
29 and Hep G2 cancer cell lines, and also the 5,6-dimethyl substituted thienopyrimidone 6a showed
cytotopxicity to all four cancer cells, however both thiadiazole derivatives showed proliferative effects
on human diploid cells. In contrast to 5a, all of the examined compounds show solid-state
photostability in 240 min period of irradiation.
Based on molecular docking study, the binding modes of 5b, 6b and a structurally related
compound A were clarified. By association to catalytic amino acid residues Lys482, Glu500 and
Gly592, Asp593 and Phe594 from the activation segment of V599EB-Raf, the ligands promote an
inactive conformation of the enzyme and hinder its phosphorylation.
The obtained activity results towards MDA-MB-231 cells indicate that the thiosemicarbazide
moiety (5b), connected through a sulfur atom in the pyrimidinone skeleton as well as the results
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exhibited by 6b to HeLa and Lep3 prove the necessity for further investigation in vivo to estimate the
exact inhibition pathway in the cellular processes, essential for tumor development related to the
antitumor potential of the tested compounds.
6. Experimental part
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 gel
plates F254 (Merck, 0.2 mm thick) and visualization was effected with ultraviolet light. IR spectrawere
recorded on a Bruker Equinox 55 spectrophotometer as potassium bromide discs. All 1H-NMR spectra
were recorded on a Bruker Avance DRX 250 spectrometer (Bruker, Faelanden, Switzerland) operating
at 250.13 MHz and a Bruker Avance DRX 600 spectrometer (Bruker, Faelanden, Switzerland)
operating at 600 MHz. 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 PerkineElmer elemental analyzer.
6.1. General procedure of the synthesis of ethyl 2-aminothiophene-3-carboxylates 1a-c
Diethylamine (0.1 mol) was added dropwise for 30 min to a suspension of the appropriate
ketone (0.1 mol), ethyl cyanacetate 0.1 mol (11 ml) and 3.2 g sulfur in 30 ml ethanol and the reaction
mixture was stirred at room temperature for about 75 min. When the synthesis was completed the
mixture was cooled. The obtained 2-aminothiophene crystalized in the form of yellow powder. The
precipitate was filtrated, washed with water and recrystallized from ethanol to give the 2-
aminothiophene derivatives [27].
6.1.1. Ethyl 2-amino-4,5-dimethylthiophene-3-carboxylate (1a): Yield: 61%; Mp. 97–100°C [1]; IR
(KBr): 3300 cm-1 (NH), 2920 cm-1 (CH3), 2860 cm-1 (CH3), 1640 cm-1 (C=O), 1160 cm-1 (C−O);
Analysis: Calc. for C9H13NO2S: C, 54.25; H, 6.58; N, 7.03; O, 16.06; S, 16.09; Found: C, 54.30; H,
6.55; N, 7.08; O, 16.10; S, 16.10.
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6.1.2. Diethyl 5-amino-3-methylthiophene-2,4-dicarboxylate (1b): Yield: 65%; Mp. 104–106°C [1]; IR
(KBr): 3300 cm-1 (NH), 2960 cm-1 (CH3), 2860 cm-1 (CH2), 1640 cm-1 (C=O), 1230 cm-1 (C−O);
Analysis: Calc. for C11H15NO4S: C, 51.35; H, 5.88; N, 5.44; O, 24.87; S, 12.46; Found: C, 51.33; H,
5.85; N, 5.52; O, 24.86; S, 12.46.
6.1.3.
Еthyl 2-amino-4,5,6,7-tetrahydrothiophene-3-carboxylate (1c): Yield: 82%; Mp. 114–115°C
[1]; IR (KBr): 3300 cm-1 (NH), 2920 cm-1 (CH3), 2800 cm-1 (CH2), 1640 cm-1 (C=O), 1260 cm-1
(C−O); Analysis: Calc. for C11H15NO2S: C, 58.64; H, 6.71; N, 6.22; O, 14.20; S, 14.23; Found: C,
58.74; H, 6.73; N, 6.26; O, 14.19; S, 14.29.
6.2. Synthesis of 3-ethyl-2-mercaptothieno[2,3-d]pyrimidin-4(3H)-ones 2a-c
To a solution of 0.025 mol of 2-aminothiophene 1a-c and 0.025 mol of NaOH in DMF (30 ml)
0.025 mol ml ethyl isothiocyanate (2.18 ml) was added dropwise. The mixture was stirred for 3h and
after that poured in 750 ml water. The solution was acidified to pH~5 by use of 50% (v/v) acetic acid.
The formed precipitate was filtered, washed with water and recrystallized from ethanol.
6.2.1. 3-ethyl-2-mercapto-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (2a): Yield: 95%; Mp. 253–
255°C; IR (KBr): 3250 cm-1 (NH), 2900 cm-1 (CH3), 1660 cm-1 (CONH); Analysis: Calc. for
C10H12N2OS2: C, 49.97; H, 5.03; N, 11.66; O, 6.66; S, 26.68; Found: C, 50.02; H, 5.05; N, 11.65; O,
6.71; S, 26.69.
6.2.2. Ethyl 3-ethyl-2-mercapto-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxylate
(
2b): Yield: 96%; Mp. 252–254°C; IR (KBr): 3195 cm-1 (NH), 2950 cm-1 (CH3), 1740 cm-1 (COOEt),
1680 cm-1 (CONH), 1100 cm-1 (C−O); Analysis: Calc. for C12H14N2O3S2: C, 48.30; H, 4.73; N, 9.39;
O, 16.09; S, 21.49; Found: C, 48.33; H, 4.78; N, 9.41; O, 16.09; S, 21.45.
6.2.3. 3-ethyl-2-mercapto-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (2c): Yield:
86%; Mp. 263–266°C [9]; IR (KBr): 3190 cm-1 (NH), 2915 cm-1 (CH3), 2810 cm-1 (CH2), 1640 cm-1
(CONH); 1H NMR (CDCl3): 1.35 (t, 3H, CH3), 1.70 (m, 4H, (CH2)2), 2.48 (s, 2H, CH2), 2.70 (s, 2H,
CH2), 3.84 (q, 2H, CH2), 6.30 (s, 1H, NH, exchangeable with D2O); Analysis: Calc. for C12H14N2OS2:
C, 54.11; H, 5.30; N, 10.52; O, 6.01; S, 24.07; Found: C, 54.14; H, 5.40; N, 10.59; O, 6.01; S, 23.99.
6.3. General procedure for the synthesis of thieno[2,3-d]pyrimidin-2-yl-thioacetates 3a-c
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A solution of 2-mercapto-thieno[2,3-d]pyrimidin-4-one 2a-c (0.01 mol), ethyl 2-chloroacetate
(0.01 mol) and NEt3 (0.01 mol) in 30 ml benzene was refluxed for 4h. After the reaction was
completed, the solid triethylamine hydrochloride was removed through hot filtration. The solvent was
removed under reduced pressure and the obtained solid was recrystallized with methanol.
6.3.1. Ethyl 2-((3-ethyl-5,6-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)thio) acetate (3a):
Yield: 81%; Mp. 91–95°C; IR (KBr): 2920 cm-1 (CH3), 2880 cm-1 (CH3), 2820 cm-1 (CH2), 1740 cm-1
1
(COOEt), 1670 cm-1 (CONH), 1120 cm-1 (C−O); H NMR (CDCl3): 1.24 (t, 9H, 3CH3), 2.24 (s, 3H,
CH3), 2.30 (s, 3H, CH3), 3.84 (s, 2H, CH2), 4.00 (q, 4H, 2CH2); Analysis: Calc. for C14H18N2O3S2: C,
51.51; H, 5.56; N, 8.58; O, 14.70; S, 19.65; Found: C, 51.54; H, 5.61; N, 8.56; O, 14.73; S, 19.66.
6.3.2. Ethyl 2-((2-ethoxy-2-oxoethyl)thio)-3-ethyl-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-
6-carboxylate (3b): Yield: 78%; Mp. 119–122°C; IR (KBr): 2990 cm-1 (CH3), 2820 cm-1 (CH2), 1750
cm-1 (COOEt), 1720 cm-1 (COOEt), 1690 cm-1 (CONH), 1120 cm-1 (C−O); 1H NMR (CDCl3): 1.35 (t,
9H, 3CH3), 2.91 (s, 3H, CH3), 3.99−4.31 (dq, 6H, 3CH2), 4.73 (s, 2H, CH2); Analysis: Calc. for
C16H20N2O5S2: C, 49.98; H, 5.24; N, 7.29; O, 20.81; S, 16.68; Found: C, 49.88; H, 5.33; N, 7.31; O,
20.71; S, 16.70.
6.3.3.
Ethyl
2-((3-ethyl-4-oxo-3,4,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)
acetate (3c): Yield: 82%; Mp. 130–133°C ; IR (KBr): 2950 cm-1 (CH3), 2900 cm-1 (CH3), 2820 cm-1
(CH2), 1740 cm-1 (COOEt), 1670 cm-1 (CONH), 1100 cm-1 (C−O); 1H NMR (DMSO-d6): 1.19 (t, 3H,
CH3), 1.25 (t, 3H, CH3), 1.75 (m, 4H, (CH2)2), 2.69 (t, 2H, CH2), 2.70 (t, 2H, CH2), 4.05 (s, 2H, CH2),
4.12 (t, 2H, CH2); Analysis: Calc. for C16H20N2O3S2: C, 54.52; H, 5.72; N, 7.95; O, 13.62; S, 18.19;
Found: C, 54.56; H, 5.75; N, 7.97; O, 13.70; S, 18.22.
6.4. Synthesis of thieno[2,3-d]pyrimidin-2-yl-thioacetohydrazides 4a-c
A mixture of 0.01 mol thieno[2,3-d]pyrimidin-2-yl-thioacetate 3a-c and 0.04 mol hydrazine
hydrate in 40 ml absolute ethanol was refluxed for 8h. The reaction mixture was cooled and the
formed crystaline product was filtered and recrystallized with ethanol.
6.4.1.
4a): Yield: 69%; Mp. 175–178°C; IR (KBr): 3200 cm-1 (NH2), 2920 cm-1 (CH3), 2880 cm-1 (CH2),
19
2-((3-ethyl-5,6-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)thio)acetohydrazide
(

ACCEPTED MANUSCRIPT
1660 cm-1 (CONH); Analysis: Calc. for C12H16N4O2S2: C, 46.13; H, 5.16; N, 17.93; O, 10.24; S,
20.53; Found: C, 46.16; H, 5.26; N, 17.92; O, 10.28; S, 20.51.
6.4.2.
Ethyl
3-ethyl-2-((2-hydrazinyl-2-oxoethyl)thio)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxylate (4b): Yeild: 63%; Mp. 180–185°C; IR (KBr): 3240 cm-1 (NH2), 2950 cm-1
(CH3), 2920 cm-1 (CH2), 1720 cm-1 (COOEt), 1670 cm-1 (CONH), 1250 cm-1 (C−O); 1H NMR
(CDCl3): 1.36 (dt, 6H, 2CH3), 2.89 (s, 3H, CH3), 4.01 (s, 2H, CH2), 4.15 (q, 2H, N-CH2CH3), 4.38 (q,
2H, O-CH2CH3) 7.73 (s, 1H, NH, exchangeable with D2O); Analysis: Calc. for C14H18N4O4S2: C,
45.39; H, 4.90; N, 15.12; O, 17.28; S, 17.31; Found: C, 45.44; H, 4.91; N, 15.15; O, 17.31; S, 17.39.
6.4.3. 2-((3-ethyl-4-oxo-3,4,5,6,7,8-tetrahydrohydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)aceto
hydrazide (4c): Yield: 94%; Mp. 210–214°C; IR (KBr): 3250 cm-1 (NH2), 2950 cm-1 (CH3), 2880 cm-1
(CH2), 2820 cm-1 (CH2), 1660 cm-1 (CONH); Analysis: Calc. for C14H18N4O2S2: C, 49.68; H, 5.36; N,
16.55; O, 9.45; S, 18.95; Found: C, 49.71; H, 5.46; N, 16.61; O, 9.38; S, 19.00.
6.5. Synthesis of thieno[2,3-d]pyrimidin-2-yl-thiosemicarbazides 5a-c
To a suspension of the appropriate hydrazide 4a-c (0.003 mol) in 20 ml absolute ethanol ethyl
isothiocyanate (0.036 mol) was added. The mixture was refluxed for 5h. After cooling the formed
precipitate was filtered and recrystallized with ethanol.
6.5.1. N-ethyl-2-(2-(((3-ethyl-5,6-dimethyl-4-oxo-3,4-dihydro-thieno[2,3-d]pyrimidin-2-yl)thio)methyl)
hydrazinyl)-2-oxoethanethioamide (5a): Yield: 88%; Mp. 197–200°C; UV (DMF) λmax= 322 nm; IR
(KBr): 3300 cm-1 (NH), 2920 cm-1 (CH3), 2880 cm-1 (CH3), 2800 cm-1 (CH2), 1680 cm-1 (CONH); 1H
NMR (DMSO-d6): 1.02 (t, 3H, CH3), 1.26 (t, 3H, CH3), 2.33 (s, 3H, CH3), 2.36 (s, 3H, CH3), 4.08 (m,
6H, 3CH2), 7.88 (s, 1H, NH, exchangeable with D2O), 9.30 (s, 1H, NH, exchangeable with D2O),
10.14 (s, 1H, NH, exchangeable with D2O); Analysis: Calc. for C15H21N5O2S3: C, 45.09; H, 5.30; N,
17.53; O, 8.01; S, 24.08; Found: C, 45.11; H, 5.40; N, 17.56; O, 8.11; S, 24.13.
6.5.2. Ethyl 3-ethyl-2-(((2-(2-(ethylamino)-2-thioxoacetyl)hydrazinyl)methyl)thio)-5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate (5b): Yield: 91%; Mp. 182–184°C; UV (DMF) λmax
=
394 nm; IR (KBr): 3180 cm-1 (NH), 2940 cm-1 (CH3), 2900 cm-1 (CH3), 2880 cm-1 (CH2), 1690 cm-1
(CONH), 1270 cm-1 (C−O); 1H NMR (DMSO-d6): 1.04 (t, 3H, CH3), 1.26 (m, 6H, 2CH3), 2.79 (d, 3H,
20

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CH3), 4.00 (m, 2H, CH2), 4.09 (s, 2H, CH2), 4.27 (dq, 4H, 2CH2), 8.04 (s, 1H, NH, exchangeable with
D2O), 9.35 (d, 1H, NH, exchangeable with D2O), 9.89 (d, 1H, NH, exchangeable with D2O); Analysis:
Calc. for C17H23N5O4S3: C, 44.62; H, 5.07; N, 15.30; O, 13.99; S, 21.02; Found: C, 44.63; H, 5.11; N,
15.40; O, 13.95; S, 21.03.
6.5.3.
N-ethyl-2-(2-(((3-ethyl-4-oxo-3,4,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)
thio)methyl)hydrazinyl)-2-oxoethanethioamide (5c): Yield: 96%; Mp. 188–192°C; UV (DMF) λmax
=
322 nm; IR (KBr): 3200 cm-1 (NH), 2920 cm-1 (CH3), 1670 cm-1 (CONH); 1H NMR (DMSO-d6): 1.02
(t, 3H, CH3), 1.25 (m, 3H, CH3), 1.77 (m, 4H, (CH2)2), 2.70 (d, 2H, CH2), 2.85 (d, 2H, CH2), 4.08 (m,
6H, 3CH2), 7.90 (s, 1H, NH, exchangeable with D2O), 9.29 (s, 1H, NH, exchangeable with D2O),
10.15 (s, 1H, NH, exchangeable with D2O). Analysis: Calc. for C17H23N5O2S3: C, 47.98; H, 5.45; N,
16.46; O, 7.52; S, 22.60; Found: C, 48.02; H, 5.39; N, 16.49; O, 7.51; S, 22.62.
6.6. Cyclocondensation of thieno[2,3-d]pyrimidin-2-yl-thiosemicarbazides to 1,3,4-thiadiazoles 6a-c
The thiosemicarbazide 5a-c (0.001 mol) was added portionwise to 3 ml 98% sulfuric acid at
5°C by stirring. The reaction mixture was cooled and poured into ice-water and neutralized with
NH4OH. The formed precipitate was filtered and recrystalized wit methanol.
6.6.1.
3-ethyl-2-(((5-(ethylamino)-1,3,4-thiadiazol-2-yl)methyl)thio)-5,6-dimethylthieno[2,3-d]
pyrimidin-4(3H)-one (6a): Yield: 63%; Mp. 208–212°C; UV (DMF)
λ
max= 322 nm; IR (KBr): 3300
1
cm-1 (NH), 2920 cm-1 (CH3), 2880 cm-1 (CH3), 2800 cm-1 (CH2), 1670 cm-1 (CONH); H NMR
(DMSO-d6 + CDCl3): 1.38 (t, 6H, 2CH3), 2.24 (s, 6H, 2CH3), 3.10 (q, 2H, CH2), 3.86 (q, 2H, CH2),
4.41 (s, 2H, CH2), 6.85 (s, 1H, NH, exchangeable with D2O); Analysis: Calc. for C15H19N5OS3: C,
47.22; H, 5.02; N, 18.36; O, 4.19; S, 25.21; Found: C, 47.26; H, 5.03; N, 18.44; O, 4.17; S, 25.23.
6.6.2. Ethyl 3-ethyl-2-(((5-(ethylamino)-1,3,4-thiadiazol-2-yl)methyl)thio)-5-methyl-4-oxo -3,4-dihydro
thieno[2,3-d]pyrimidine-6-carboxylate (6b): Yield: 95%; Mp. 188–193°C; UV (DMF) λmax= 394 nm;
IR (KBr): 3420 cm-1 (NH), 3350 cm-1 (NH), 2920 cm-1 (CH3), 2880 cm-1 (CH3), 2820 cm-1 (CH2),
1
1720 cm-1 (COOEt), 1680 cm-1 (CONH); H NMR (DMSO-d6): 1.27 (dt, 9H, 3CH3), 3.02 (s, 3H,
CH3), 4.01−4.29 (dq, 6H, 3CH2), 4.71 (s, 2H, CH2), 8.13 (s, 1H, NH, exchangeable with D2O);
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Analysis: Calc. for C17H21N5O3S3: C, 46.45; H, 4.82; N, 15.93; O, 10.92; S, 21.88; Found: C, 46.45; H,
4.88; N, 15.95; O, 10.93; S, 21.91.
6.6.3.
3-ethyl-2-(((5-(ethylamino)-1,3,4-thiadiazol-2-yl)methyl)thio)-5,6,7,8-tetrahydrobenzo[4,5]
max= 324 nm; IR
thieno[2,3-d]pyrimidin-4(3H)-one (6c): Yield: 90%; Mp. 202–204°C; UV (DMF)
λ
(KBr): 3180 cm-1 (NH), 2920 cm-1 (CH3), 2880 cm-1 (CH3), 2820 cm-1 (CH2), 1670 cm-1 (CONH); 1H
NMR (Pyridine-d6): 1.27 (t, 6H, 2CH3), 1.66 (t, 4H, (CH2)2), 2.61 (t, 2H, CH2), 3.05 (s, 2H, CH2), 3.56
(q, 2H, HN-CH2CH3), 4.01 (q, 4H, N-CH2CH3), 4.84 (s, 2H, CH2), 7.20 + 7.57 (ds, 1H, NH,
exchangeable with D2O); Analysis: Calc. for C17H21N5OS3: C, 50.10; H, 5.19; N, 17.18; O, 3.93; S,
23.60; Found: C, 50.08; H, 5.21; N, 17.23; O, 3.83; S, 23.60.
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 h at 37 °C 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).
6.8. Photostability
The study on the photodegradation of the compounds was conducted in a solar simulator
SUNTEST CPS equipment (Heraeus, Germany), supplied with an arc aircooled Xenon lamp (Hanau,
1.1 kW, 765Wm−2), at ambient temperature. The irradiation was performed in solid state of the
compounds. The UV–vis absorption spectra were recorded on a spectrophotometer Hewlett Packard
8452A in DMF solution at concentration 10–5 mol/L.
6. 9. Computational details
The most probable conformers of the studied molecules (including thione-thiol and lactam-
lactime tautomerism) were constructed and energy minimized using Avogadro’s software [48]. Based
on the energy analysis the most stable conformers were selected and optimized by density functional
theory (DFT). The theoretical calculations were performed using the Gaussian 09 package [40] of
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programs. Geometry and vibrational frequencies of the studied species were performed by analytical
gradient technique without any symmetry constraint. The results were obtained, employing the B3LYP
(Becke’s three-parameter non-local exchange [40] and Lee et al. correlation [41] potentials) in
conjunction with the 6-311++G** basis set. The optimized structures were further characterized by
analytical computations of harmonic vibrational frequencies at the same level.
6.10. Molecular docking
Initial molecular modeling coordinates of the enzyme were taken from the crystal structure of
oncogenic mutant V599EB-Raf kinase domain in complex with sorafenib (PDB code: 1UWJ [43]). The
inhibitors were doked into an internal pocket formed by the activation segment and the P-loop of
V599EB-Raf (PDB entry code 1UWJ) using MOE software [47]. Conformational search for preparation
of the ligands was carried out by LowModelMD method which performs molecular dynamics
perturbations along with low frequency vibrational modes with energy window 7 kCal/mol, and
conformational limits of 1000. Placement of conformers was prepared according to alpha-triangle
method on selected pharmacophores. Scoring of docking poses was performed by affinity dG,
calculated with MMFF94x force field. VMD [49] was used to produce 3-D figures of binding modes.
Acknowledgements: The financial support of this work by the National Science Fund of Bulgaria
(Contracts RNF01/0110) and Science Fund / Chemical Technology and Metallurgy University grant
11429.
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