Evaluation of antiprolifErativE activity of pyrazolothiazolopyrimidinE dErivativEs

The research aim was to test cytotoxic effects in vitro of seven novel pyrazolothiazolopyrimidine derivatives in targeting several lines of tumor and pseudo-normal mammalian cells. We demonstrated that cytotoxic effects of these derivatives depended on the tissue origin of targeted cells. Leukemia cells were found to be the most sensitive to the action of compounds 2 and 7. Compound 2 demonstrated approximately two times higher toxicity towards the multidrug-resistant sub-line of HL-60/ADR cells compared to the Doxorubicin effect. Antiproliferative action of compounds 2 and 7 dropped in the order: leukemia > melanoma > hepatocarcinoma > glioblastoma > colon carcinoma > breast and ovarian carcinoma cells. These compounds were less toxic than Doxorubicin towards the non-tumor cells. The novel pyrazolothiazolopyrimidine, compound 2, demonstrated high toxicity towards human leukemia and, of special importance, towards multidrug-resistant leukemia cells, and low toxicity towards pseudo-normal cells.

The research aim was to test cytotoxic effects in vitro of seven novel pyrazolothiazolopyrimidine derivatives in targeting several lines of tumor and pseudo-normal mammalian cells.We demonstrated that cytotoxic effects of these derivatives depended on the tissue origin of targeted cells.Leukemia cells were found to be the most sensitive to the action of compounds 2 and 7. Compound 2 demonstrated approximately two times higher toxicity towards the multidrug-resistant sub-line of HL-60/ADR cells compared to the Doxorubicin effect.Antiproliferative action of compounds 2 and 7 dropped in the order: leukemia > melanoma > hepatocarcinoma > glioblastoma > colon carcinoma > breast and ovarian carcinoma cells.These compounds were less toxic than Doxorubicin towards the non-tumor cells.The novel pyrazolothiazolopyrimidine, compound 2, demonstrated high toxicity towards human leukemia and, of special importance, towards multidrug-resistant leukemia cells, and low toxicity towards pseudo-normal cells.

K e y w o r d s: pyrazolothiazolopyrimidines, Doxorubicin, antiproliferative activity in vitro.
C hemotherapy is commonly used for cancer treatment or palliation.However, the chemotherapeutic agents may influence not only tumor but also normal cells, and thus, possess low specificity and/or selectivity [1].Drug resistance is another major problem of the chemotherapy.Therefore, new drugs should be developed that avoid the above noted problems in their action.
Thus, pyrazolothiazolopyrimidine derivatives are attractive heterocycles for pharmaceutical and medicinal chemists who design new potent anticancer agents [14,20].
Doxorubicin is a chemotherapeutic anthracycline drug commonly used to treat different types of cancer, such as breast, ovarian tumors, bladder cancer, lymphomas, leukemia, and others.Doxorubicin intercalates into the DNA molecule and inhibits its biosynthesis.Doxorubicin induces the apoptosis via AMP-activated protein kinase (AMPK), Bcl-2/ Bax, p53 pathways and caspases activation [21][22][23].Doxorubicin can also affect the production of free radicals [21].It should be noted that Doxorubicin is not a specifically targeting drug [21,24].Dilated cardiomyopathy is the most dangerous side effect of Doxorubicin [21][22][23].
The aim of our study was to test the cytotoxic effects in vitro of seven novel pyrazolothiazolopyrimidine derivatives that target several mammalian tumor cell lines and for comparison, pseudo-normal cell lines.
cell culture.Human myeloid leukemia HL-60 and the multidrug resistant sub-line of human myeloid leukemia HL-60/ADR cells (MRP1 over-expression), human myeloid leukemia K-562 cells, human hepatocarcinoma HepG2 cells, and human colon carcinoma HCT116 cells were from a collection of the Institute of Cancer Research at Vienna Medical University (Vienna, Austria).Human ovarian carcinoma SKOV3 cells were from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were donated by Dr. O. Stasyk (Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine).Human glioblastoma U-251 MG cells, human HaCaT keratinocytes and murine monocyte macrophage J774.2 cells were from a collection at the Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine (Kyiv, Ukraine).Human breast adenocarcinoma MCF-7 cells, human melanoma SK-MEL-28 cells and human embryonic kidney cells of HEK293 line were from Cell Collection of R.E.Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine).Cells were grown in RPMI-1640 (APP, Vienna, Austria) or Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) culture medium supplemented with 10% fetal bovine serum (APP, Vienna, Austria).Cells were cultivated at 37 ºC in an atmosphere of 5% CO 2 .
MTT assay for cell proliferation.The antineoplastic activity of the synthesized compounds and Doxorubicin towards cell lines of different tissue origin was examined using the MTT (Sigma-Aldrich, St. Louis, MO, USA) test.Briefly, cells were seeded overnight into 96-well plates in 100 μl at concentrations of 5,000 cells/well (substrate-dependent cells) or 10,000 cells/well (suspension cells).Aliquots of 100 µl of experimental compounds (0-50 µM) were added to culture medium and cells were incubated for the next 72 h.The 72 h term of compound exposure was used in order to analyze the possible cytotoxic activity of studied compounds without time-related limitation.The MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to the cells, as described previously [26].The results of the reaction were determined by an Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, VT, USA).The IC 50 of tested compounds was calculated as the drug concentration that reduced cell viability to 50%.
Data analysis.The results were analyzed and illustrated using GraphPad Prism 6 software (Graph-Pad Software, La Jolla, CA, USA).All data are presented as the mean ± standard deviation (SD) of three replications in two parallels (n = 6).A t test and the column statistics of GraphPad Prism 6 software were used for statistical analysis.Statistical significance was identified at p ≤ 0.05.

results and discussion
The anti-proliferative activities of seven novel pyrazolothiazolopyrimidine derivatives were evaluated in human tumor cell lines of different tissue origin (leukemia (K-562, HL-60, HL-60/ADR), mela noma (SK-MEL-28), glioblastoma (U-251 MG), colon (HCT116), hepatocyte (HepG2), ovary (SKOV3) and breast (MCF-7)) and towards non-tumor cells (HEK293, HaCaT and J774.2) using the MTT test in order to identify if the cytotoxicity of the pyrazolothiazolopyrimidine derivatives is tissue depen dent.Doxorubicin that is widely used for treatment of solid tumors [27,28] was used as a positive control.The anticancer activity of the tested compounds was expressed as the IC 50 value.
Our data showed that leukemia cells were the most sensitive to the action of pyrazolothiazolopyrimidine derivatives.All tested compounds inhibited human myeloid leukemia K-562 cells proliferation to a different extent.Compounds 2 and 7 demonstrated the highest effect in cell viability inhibition (IC 50 was 3.0 and 3.1 μM, respectively, Fig. 1, Table ).The IC 50 value for compound 3 was 5.7 μM; for 5 was 8.8 μM; and for 4 was 10.4 μM.Compounds 6 and 1 possessed the least pronounced toxicity for K-562 cells (IC 50 of 21.1 and 43.8 μM, respectively, Fig. 1, Table ).Doxorubicin was less toxic for K-562 cells (IC 50 was 10.2 μM).
These data showed that all compounds possess similar anti-proliferative activity towards human myelogenic leukemia HL-60 cells, compared with the action of Doxorubicin.The cytotoxic action of pyrazolothiazolopyrimidines and Doxorubicin towards HL-60 cells (Fig. 1, Table )  Rapid development of multidrug resistance is one of the main problems in leukemia treatment.The mechanisms of such resistance can be associated with the over-expression of the ABC transporters (e.g.multidrug resistance gene 1 [MDR1], such as P-glycoprotein and multidrug resistance protein 1 [MRP1]) [29].Here, we have studied the anti-neoplastic activity of the pyrazolothiazolopyrimidine derivatives towards human myeloid leukemia HL-60/ADR cells (a multidrug resistant sub-line that over-expresses MRP1) [29].We found that compound 2 possessed the highest cytotoxic action towards HL-60/ADR cells (IC 50 = 3.9 μM), and such effect was more prominent than the effect of Doxorubicin (IC 50 = 8.9 μM) (Table ).Compound 7 showed a lesser growth inhibition effect on the HL-60/ADR cells (IC 50 = 11.2 μM).Compounds 6 and 5 were also capable of inhibiting cell proliferation of HL-60/ ADR cells (IC 50 = 45.8 and 46.1 μM, respectively).Compounds 1, 3 and 4 were poorly toxic towards HL-60/ADR cells in a dose up to 50 μM and inhibited cells growth by 28.7, 36.9 and 42.6%, respectively (Fig. 1).
Cytotoxic activity of compounds 2 and 7 towards human hepatocyte carcinoma HepG2 cells was observed at their high concentrations (IC 50 = 25.2 and 26.4 μM, respectively; Fig. 2, Table).Doxorubicin was the most toxic when acting towards HepG2 cells (IC 50 was 0.9 μM).Compounds 1, 3, 4, 5 and 6 did not reach the IC 50 value even at a 50 μM dose.The inhibition of growth of HepG2 cells equaled in the range of 38.5-46.0%at the action of compounds 4, 1, 3, 5 and 6.
Human breast adenocarcinoma MCF-7 cells and human ovarian carcinoma SKOV3 cells demonstrated the weakest sensitivity to the action of the pyrazolothiazolopyrimidines.None of tested compounds had an IC 50 value at 50 μM (Fig. 2, Table).We observed the maximum of 32.1% growth inhibition of MCF-7 cells under the effect of compound 2 and minimum of 10% under the effect of compounds 4 and 6 (Fig. 2).Similar toxicities of the pyrazolothiazolopyrimidines were found for SKOV3 cells: 27.2% of growth inhibition under the effect of compound 7 and 7.2% for the action of compound 6 (Fig. 2).Measurement of the IC 50 for Doxorubicin in MCF-7 and SKOV3 cells showed the highest cytotoxic effect of that agent, 1.38 and 2.6 μM, respectively.
Thus, compounds 2 and 7 were the most cytotoxic agents towards a majority of tumor cell lines.Compound 2 contains a trifluoromethyl group in its structure, while compound 7 does not have polar groups in its structure.Therefore, compound 7 possesses better solubility because of its lipophilicity due to the propyl group.The noted structural peculiarities might increase uptake of these compounds by the cell and provide their high activity [14,16].
In order to evaluate a potential side effect of the seven pyrazolothiazolopyrimidine derivatives, we studied their toxicity towards non-tumor cells (Fig. 3, Table ).).The toxicity of the studied compounds and Doxorubicin towards HaCaT cells in decreasing order are as follows: Doxorubicin (IC 50 = 1.7 μM) > compound 6 (IC 50 = 12.7 μM) > 5  ).Thus, the pyrazolothiazolopyrimidine derivatives were less toxic than Doxorubicin towards non-tumor human embryonic kidney HEK293 cells, human keratinocytes of the HaCaT line, and murine macrophages of the J774.2 line.

Fold
. Finiuk, Yu.V. Ostapiuk, V. P. Hreniukh et al. 10 times lower compared to the effect of Doxorubicin.Compounds 2 and 7 inhibited the viability of human glioblastoma U-251 MG cells, human colon carcinoma HCT116 cells, and human hepatocyte carcinoma HepG2 cells with values of IC 50 under 21 μM, and possessed less toxicity compared with Doxorubicin.Human breast adenocarcinoma MCF-7 cells and human ovarian carcinoma SKOV3 cells demonstrated the weakest sensitivity to the pyrazolothiazolopyrimidines.It should be mentioned that the pyrazolothiazolopyrimidine derivatives were less toxic than Doxorubicin towards non-tumor human embryonic kidney HEK293 cells, human keratinocytes of the HaCaT line, and murine macrophages of the J774.2 line.The next steps in our study will be focused on the potential mechanisms (induction of apoptosis, DNA single-strand breaks, effect on cell cycle distribution, intercalation into DNA molecule, genotoxic potential, binding with proteins) of the action of compounds 2 and 7 towards HL-60/ADR cells.We plan in vivo experiments on the action of pyrazolothiazolopyrimidine derivatives.The results of such experiments will provide information needed for starting clinical trials.Conflicts of interest.The authors declare no conflict of interest.acknowledgement This research was supported by the Ministry of Education and Science of Ukraine grant (registration number 0116U001533).The authors thank Dr. O. Stasyk (Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) for donating human ovarian carcinoma SKOV3 cells.We thank Cedars -Sinai Medical Center's International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields

Fig. 3 .
Fig. 3.The results of measuring the antiproliferative activity of seven pyrazolothiazolopyrimidines and Doxorubicin (Dox) towards different non-tumor cell lines (HEK293, HaCaT, J774.2). Cell viability was examined using the MTT assay after 72 h of the compound exposure.Dox was used as a positive control