Synthesis of Substituted Thieno[2,3-d]pyrimidin-4-ones and Their Testing for Evaluation of Cytotoxic Activity on Mammalian Cell Models
Hindawi Publishing Corporation
Journal of Chemistry
Volume 2013, Article ID 976715, 6 pages
http://dx.doi.org/10.1155/2013/976715
Research Article
Synthesis of Substituted Thieno[2,3-d]pyrimidin-4-ones and
Their Testing for Evaluation of Cytotoxic Activity on Mammalian
Cell Models
Kh. A. Bozorov,1 N. Z. Mamadalieva,1 B. Zh. Elmuradov,1 D. Triggiani,2 D. Egamberdieva,3
A. Tiezzi,2 H. A. Aisa,4 and Kh. M. Shakhidoyatov1
1
S.Yu. Yunusov Institute of the Chemistry of Plant Substances AS RUz, Mirzo Ulugbek Street 77, Tashkent 100170, Uzbekistan
Department for the Innovation in Biological, Agrofoods and Forests Systems (DIBAF), Tuscia University, Laboratory of Plant Cytology
and Biotechnology, Largo dell Università, Blocco D, 01100 Viterbo, Italy
3
Department of Biotechnology and Microbiology, National University of Uzbekistan, Tashkent 100174, Uzbekistan
4
Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Urumqi 830011, China
2
Correspondence should be addressed to B. Zh. Elmuradov;
Received 7 September 2012; Accepted 12 November 2012
Academic Editor: Hakan Arslan
Copyright © 2013 Kh. A. Bozorov et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
From 2-amino-3-ethoxycarbonyl-4,5-dimethyl-, -polymethylenethiophenes (1-4) were synthesized 2,3-disubstituted thieno[2,3d]dihydropyrrolo-, tetrahydropyrido-, and tetrahydroazepino[1,2-a]pyrimidin-4-ones (5-16) for pharmacological investigations.
e 12 compounds (5-16) were individually evaluated for their antiproliferative activities on mammalian cancer cell models. All
tested compounds showed weak affection on human cervix adenocarcinoma cells (HeLa) whereas some of the tested compounds
exhibited more consistent inhibition of cell growth on murine myeloma cells (P3X). In both cases some compounds enhanced cell
proliferation.
1. Introduction
ieno[2,3-d]pyrimidin-4-ones are a large group of heterocyclic compounds [1] and some of them show antiviral
[2], antimicrobial [3–10], and antibacterial properties [11].
Fused tri- and tetracyclic thieno[2,3-d]pyrimidin-4-ones are
synthesized by many methods and among them some compounds have fungicidal, antibacterial, and antiin�ammatory
activities [12–19], and their substituted derivatives were
reported as 17𝛽𝛽-HSD1 inhibitors [20].
ese �ndings clearly show the potential importance
of such molecules as active principles of new pharmaceuticals and therefore the development of effective methods
of synthesis and searching of biological activities among
new synthesized compounds are a very important direction.
Prompted by the various biological activities of thieno[2,3d]pyrimidin-4-ones and its substituted derivatives, we envisioned our approach towards the synthesis of a novel series
of thieno[2,3-d]pyrimidin-4-ones derivatives and to evaluate
their possible cytotoxic activity on mammalian cell models.
2. Materials and Methods
2.1. Chemicals and Reagents. 1 H NMR spectra were recorded
on Unity 400+ (400 MHz) in CDCl3 . Chemical shis of 1 H
were measured relative to HMDS as internal standard. IR
spectra were recorded as KBr pellets on an IR Fury System
2000 (Perkin-Elmer). Melting points were measured on a
Boetius (Germany) and MEL-TEMP (USA) apparatus and
are not uncorrected. Reactions were monitored by TLC on
Sorb�l (Russia) and �hatman UV-254 (Germany) (eluent:
benzene-methanol, 2 : 1 (A) and benzene-methanol, 5 : 1 (B)),
which were visualized under UV radiation.
Cell culture media, supplements, and dimethyl sulfoxide (DMSO) were purchased from Roth and Greiner
Journal of Chemistry
Living cells (%)
2
180
160
140
120
100
80
60
40
20
0
Control 5
formed crystals of formazan were dissolved in DMSO and
the absorbance was determined by a plate lecturer. Cytotoxic
activity of substances was determined by percent of discolor
in control samples at 595 nm. Cytotoxic activity is determined
by the change in absorbance relative to the controls and has
been expressed as cell viability rate (%).
6
7
8
9
10 11 12 13 14 15 16
Compounds
500 g/mL 250 g/mL 125 g/mL
Living cells (%)
F 1: Cytotoxic action of compounds 5–16 on HeLa cells aer
24-hour culturing (MTT test).
160
140
120
100
80
60
40
20
0
Control 5
6
7
8
9
HN- HN- HN-
10 11 12 13 14 15 16
Compounds
F 2: Cytotoxic action of compounds 5–16 on P3X cells aer
24-hour culturing (MTT test).
Labortechnik (Italy). e 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from
Sigma and Gibco (Invitrogen, Italy).
2.1.1. Cytotoxic Activity. e evaluation of cytotoxic activity
of the tested samples was carried out on HeLa (human
epithelial cervical cancer, ATCC CCL-2) and P3X (murine
myeloma, ATCC CRL-1580) cell models. For this purpose
cells were cultured at 37∘ C, 90% of humidity and 5% CO2 in
96-well plates (5 × 104 cells/well for HeLa and 1 × 105 for P3X
cells) in DMEM medium culture (pH 7.4), containing 10%
fetal bovine serum and 50 units/mL penicillin/streptomycin.
Cells were treated for 24 hours with the individual substance
(in concentrations of 500, 250, and 62.5 𝜇𝜇g/mL, resp.). Controls consisted of untreated cancer cells incubated for 24
hours. e obtained results were recalculated in percentage
with respect to the control. All experiments were conducted
in 3-time frequency.
2.1.2. MTT Assays. Cytotoxic activity was evaluated by using
the MTT test based on reducing [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] (MTT) with the
participation of cytoplasmatic dehydrogenases in insoluble
formazan, the quantity of which correlates with metabolic
activity of cells and a level of cell proliferation [21]. e
medium was replaced 4 hrs before the incubation period
(100 𝜇𝜇L DMEM), containing 0.5 mg/mL MTT, and incubated
for 3 hours at 37∘ C. At the end of the experiment the
3. Experimental Part
3.1. Sample Preparation. Compounds 1–4 were synthesized
following to the procedure of Gewald et al., as already
reported [17] with some minor modi�cations. To a suspension containing of 0.4 mole ketone, 0.4 mole ethyl cyanoacetate, and 0.44 g sulphur powder in 120 mL ethanol, 40 mL
diethylamine was added dropwise and the temperature of the
reaction mixture was kept at 45–50∘ C for 3 h; the reaction
solution was allowed to stand overnight at +5∘ C (in Gewalds
procedure [17], a reactionary mixture leave in a refrigerator
at some times). 700 mL Distilled water was added and was
mixed for 3 h at room temperature. e formed crystals were
�ltered off and washed with water and dried. e products
1–4 were puri�ed by recrystallization from corresponding
solvent. Data on the prepared compounds 1–4 are given in
Table 1.
Compounds 5–16 were obtained by modi�ed method,
which were reported previously [19, 22]. 2-Amin (...truncated)
