Synthesis of new pyrimidine derivatives and their antiproliferative activity against selected human cancer cell lines

6-Amino-2-thiouracil (1) was condensed with benzenesulfonyl chloride and p-toluenesulfonyl chloride in presence of pyridine as an acid binder to give sulfonamides 2a, b, which could be methylated in basic medium to give methylmercapto derivatives 3a, b, which in turn reacted with bromine in glacial acetic acid to yield 5-bromo derivatives 4a, b. On the other hand, compounds 2a, b were cyclocondensed with monochloroacetyl chloride, p-tolualdehyde in glacial acetic acid/pyridine, and ethyl bromoacetate to give the corresponding thiazolopyrimidines 5a, b, 6a, b, and 7a, b, respectively; also compounds 2a, b were hydrazinolyzed to compounds 8a, b, which could be cyclized to triazolopyrimidines 9a, b in presence of formic acid. They could also be condensed with p-anisaldehyde to give hydrazones 10a, b. In another pathway, compounds 2a, b were reacted with monochloroacetic acid in basic medium to give acetic acid derivatives 11a, b. It can be deduced from the preliminary screening results that the cell lines most sensitive to the antiproliferative activity of tested compounds are human liver HEPG2 and colon cancer HT-29. All selected compounds exhibited moderate to strong growth inhibition activity against the HEPG2 cell line with 50 % inhibitory concentration (IC50) ranging between 1 and 10 μg/ml. The most active compounds, which revealed antiproliferative activity also against human colon HT-29 and breast MCF-7 cell lines, were 3a, 3b, 4a, and 10a.

Introduction 5-Fluorouracil (5-FU) is one of the oldest chemotherapy drugs [1] and has been in use for decades. It is an active medicine against many cancers including colon, rectal, pancreas, head, neck, and liver cancers [2]. It is also used topically as a cream or solution to treat basal cell cancer of skin and actinic keratoses, thus becoming the first prototype of pyrimidines in this field. Analogously, some 5-halogenated thiouracils have been synthesized and screened for anticancer activity. It has been reported that the tested compounds had comparable activity to that of uracils [3]. From 1957 to 2011, different research laboratories investigated the anticancer activity of some 5-and 6-substituted 2-thiouracils and reported that the tested compounds were found to inhibit DNA synthesis [4][5][6][7][8][9][10][11]. Based on these findings, the present work aims to synthesize a new group of pyrimidine compounds incorporated with different heterocycles to investigate whether the resulting compounds have better biological activity as antiproliferative agents against cancer, since all the selected pyrimidine derivatives were soluble in dimethyl sulfoxide (DMSO) at sufficient concentrations to allow cell experiments. The in vitro biological activity of these compounds was evaluated by their growth inhibitory potency against different cancer cell lines. The cytotoxic potency of compounds 3a, 3b, 4a, 5b, 6a, 7b, 8a, 9b, 10a, and 11a was studied in comparison with the known anticancer drugs 5-FU and cisplatin (CIS).
In continuation of our work on the synthesis of some 2-thiouracils [12], we report here the synthesis of novel thiouracil derivatives based on the diverse medicinal uses and biological activities of thiouracils as anticancer [12], antithyroid [13], and anti-infective agents [13][14][15]. In this work we screened some of the prepared compounds as anticancer agents in analogy to 5-FU, and we also develop a program to incorporate the thiouracil nucleus into thiazole and triazole nuclei to compare their activities with 5-FU as a reference standard, finally concluding that 5-and 6-substituted 2-thiouracils may have activity comparable to 5-FU.

Chemistry
All melting points are uncorrected and were determined in capillary tube on a Boetius melting-point microscope. Infrared (IR) spectra (KBr, cm -1 ) were recorded from KBr discs. 1 H nuclear magnetic resonance (NMR) spectra were obtained on a Joel EX 270-MHz spectrometer with tetramethylsilane (TMS) as internal standard. Mass spectra were recorded on a SSQ 7000 mass spectrometer at 70 eV. All reactions were followed and checked by thin-layer chromatography (TLC) using chloroform/methanol (3:1), and spots were examined by ultraviolet (UV) lamp.
An amount (0.05 mol) of 2a, b was dissolved in 25 ml 10 % alc. NaOH with stirring, then methyl iodide (0.05 mol) was added with stirring for 4 h, then the reaction mixture was neutralized with dil. HCl, filtered, dried, and recrystallized from methanol. An amount (0.05 mol) of 3a, b was stirred with 0.05 mol bromine in 25 ml glacial acetic acid for 24 h, then the produced solid was filtered off, washed several times with water, dried, and recrystallized from methanol.
N-(2,5-Dioxo-2,3,8,8a-tetrahydro-5-H- [1,3]thiazolo[3,2-a]pyrimidin-7yl)benzenesulfonamide and N-(2,5-dioxo-2,3,8,8a-tetrahydro-5-H- To an ice-cold solution of 2a, b (0.01 mol) in 40 ml dry DMF, triethylamine (0.01 mol) and chloroacetyl chloride (0.01 mol) were added successively. The reaction mixture was heated in a water bath at 70°C for 6 h, cooled, poured into ice/cold water, and extracted with 3 9 30 ml methylene chloride; the organic layer was dried on anhydrous sodium sulfate and filtered off, and the filtrate was evaporated to dryness. The residue was washed with 10 % sodium carbonate solution, and the undissolved solid was filtered off, and crystallized from DMF/ water. A mixture of 2a, b (0.001 mol) and p-tolualdehyde (0.001 mol) in 30 ml glacial acetic acid/acetic anhydride mixture was refluxed for 7 h, then cooled, and the produced solid was filtered off, dried, and crystallized from DMF/water. To an ice-cold solution of 2a, b (0.01 mol) in 40 ml dry DMF, triethylamine (0.01 mol) and ethyl bromoacetate (0.01 mol) were added successively. The reaction mixture was heated in a water bath at 70°C for 6 h, cooled, poured into ice/cold water, and extracted with methylene chloride (3 9 30 ml); the organic layer was dried on anhydrous sodium sulfate and filtered off, and the filtrate was evaporated to dryness. The residue was washed with 10 % sodium carbonate solution, and the undissolved solid was filtered off, and crystallized from DMF/ water.           To a solution of monochloroacetic acid (0.03 mol) and sodium hydroxide (0.03 mol) in 5 ml water, a solution of 2a, b (0.03 mol) and sodium hydroxide (0.03 mol) was added. The reaction mixture was stirred at ambient temperature for 3 h, then rendered acidic with hydrochloric acid (6 N). The separated solid was filtered off, washed with water, dried, and crystallized from methanol.

Biological investigation
On the basis of monitoring the inhibition of the growth of human cancer cells, a series of novel pyrimidine derivatives possessing a broader spectrum of antitumor activity and fewer toxic side-effects than traditional anticancer drugs have been studied. Ten selected pyrimidine derivatives (3a, 3b, 4a, 5b, 6a, 7b, 8a, 9b, 10a, and 11a) were subjected to screening for investigation of their antiproliferative potency against human A549 (lung), MCF-7 (breast), HT-29 (colon), and HEPG2 (liver) cancer cell lines.
The results indicate that the cell lines most sensitive to the antiproliferative activity of tested compounds are human liver HEPG2 and colon HT-29. In the case of human liver HEPG2, compounds 4a, 3a, 5a, and 10a showed the highest In this study, we have identified some pyrimidine derivatives as a novel class of compounds with antiproliferative activity. The lead compounds were the most potent in the biological assay employed (showing, e.g., improved growth inhibition potential as compared with the reference anticancer drugs).
These experimental findings may provide support for use of these novel compounds as new weapons in the fight against different types of cancer.
(2) The second group: HEPG2 (human liver cancer cell line), is maintained at the National Institute of Cancer, Cairo University, Cairo, Egypt.

Antiproliferative assay in vitro
At 24 h before addition of the tested compounds, the cells were placed in 96-well plates (Sarstedt, Germany) at density of 1 9 10 4 cells per well. An assay was performed after 72 h of exposure to varying concentrations of the tested agents. The in vitro cytotoxic effect of all agents was examined using the sulforhodamine B (SRB) assay [18].

SRB cytotoxicity test
Cells were attached to the bottom of plastic wells by fixing them with cold 50 % trichloroacetic acid (TCA; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) on the top of the culture medium in each well. The plates were incubated at 4°C for 1 h, then washed five times with tap water. The cellular material fixed with TCA was stained with 0.04 % sulforhodamine B (SRB; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) dissolved in 1 % acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (49) in 1 % acetic acid. The proteinbound dye was extracted with 10 mM unbuffered Tris base (POCH, Gliwice, Poland) for determination of the optical density (k = 540 nm) in a computerinterfaced, 96-well microtiter plate reader (Multiskan RC photometer; Labsystems, Helsinki, Finland).
The results were calculated as an IC 50 (50 % inhibitory concentration) value, i.e., the concentration of tested agent which inhibits proliferation of 50 % of the cancer cell population. IC values were calculated for each experiment separately, and mean values with standard deviation (SD) are presented in the table. Each compound at each concentration was tested in triplicate in a single experiment, which was repeated 3-7 times. Table 1 indicates the cytotoxic activity of the newly synthesized derivatives 3a, 3b, 4a, 5b, 6a, 7b, 8a, 9b, 10a, and 11a against various cancer cell lines. Their activity was compared with the traditional anticancer drugs 5-FU and CIS. It can be deduced from the preliminary screening results that the cell lines most sensitive to the antiproliferative activity of tested compounds are human liver HEPG2 and colon cancer HT-29. All selected compounds exhibited moderate to strong growth inhibition activity against HEPG2 cell line with IC 50 values ranging between 1 and 10 lg/ml. The most active compounds, which revealed antiproliferative activity also against human colon HT-29 and breast MCF-7 cell lines, were 3a, 3b, 4a, and 10a.

Discussion
Cytotoxic drugs remain the mainstay of cancer chemotherapy, being administered in novel therapeutic approaches such as signal inhibition. It is therefore important to discover novel cytotoxic agents with spectra of activity and toxicity that differ from currently used agents. It is well known that chemotherapy aims to destroy cancer cells using various types of chemicals. The substances used are supposed to target mainly cancer cells, and doses are calculated to minimize collateral damage to surrounding tissues, which nevertheless occurs. This type of treatment increases the entropy of the organism, suppresses the immune system, and forms a toxic cell environment which may destroy surrounding healthy cells. So, it is important to minimize effective doses to the least amount, as well as to try to minimize the sideeffects of these drugs. Therefore, novel pyrimidine derivatives with a comparable spectrum of antiproliferative activity to 5-FU, but potentially fewer side-effects, were developed. The antiproliferative activities of such compounds were assessed against the HEPG2 cancer cell line in comparison with the traditional anticancer drugs 5-FU and CIS. In the antiproliferative activity study, some of the selected compounds showed reasonable antiproliferative activity in comparison with 5-FU and CIS. Moreover, study of the biochemical parameters induced in mice by the tested compounds showed insignificant differences relative to the control group, which indicates a moderate margin of safety for the selected compounds. Compared with 5-FU and doxorubicin (DOX), dose augmentation of compounds 10a and 11a, in a search for possibly higher potency, therefore seems realizable without undesirable implications. Furthermore, the selected compounds have important potential advantages over 5-FU and DOX because of their lower toxicity and their ability to induce lower biochemical parameters. These results are in agreement with the work of many authors who have reported novel derivatives of 5-FU with a broader spectrum of antitumor activity and fewer toxic side-effects than 5-FU.

Conclusions
Various compounds were selected for further structural modification in an effort to increase their potency. The goal of the present work is to explore the significance of the spacer for the respective biological anticancer activity. The anticancer activity data indicate that the cell lines most sensitive to the activity of tested compounds are human liver HEPG2 and colon cancer HT-29. In the case of human liver HEPG2, compounds 3a, 3b, 4a, and 10a showed the highest antiproliferative activity compared with other compounds. On the other hand, in the case of colon cancer HT-29, only compound 3a showed moderate activity in comparison with other compounds.