CK2 Inhibition and Antitumor Activity of 4,7-Dihydro-6-nitroazolo[1,5-a]pyrimidines

Today, cancer is one of the most widespread and dangerous human diseases with a high mortality rate. Nevertheless, the search and application of new low-toxic and effective drugs, combined with the timely diagnosis of diseases, makes it possible to cure most types of tumors at an early stage. In this work, the range of new polysubstituted 4,7-dihydro-6-nitroazolo[1,5-a]pyrimidines was extended. The structure of all the obtained compounds was confirmed by the data of 1H, 13C NMR spectroscopy, IR spectroscopy, and elemental analysis. These compounds were evaluated against human recombinant CK2 using the ADP-GloTM assay. In addition, the IC50 parameters were calculated based on the results of the MTT test against glioblastoma (A-172), embryonic rhabdomyosarcoma (Rd), osteosarcoma (Hos), and human embryonic kidney (Hek-293) cells. Compounds 5f, 5h, and 5k showed a CK2 inhibitory activity close to the reference molecule (staurosporine). The most potential compound in the MTT test was 5m with an IC50 from 13 to 27 µM. Thus, our results demonstrate that 4,7-dihydro-6-nitroazolo[1,5-a]pyrimidines are promising for further investigation of their antitumor properties.


Introduction
Cancer is one of the world s leading causes of death, with an estimated number of 10 million deaths in 2020 [1]. However, many types of cancer are curable with early diagnosis and treatment. The establishment of alternative ways to treat tumor diseases allows for the use of new effective and low-toxic drugs in the early stages of the disease. One of the current trends is the inhibition of biological targets responsible for the growth, proliferation, and survival of tumor cells. From this point of view, type 2 casein kinase is a promising target for chemotherapy. The overexpression of casein kinase 2 (CK2) is closely associated with several cancers, including cancers of the head and neck, breast, kidney, lung, etc. [2][3][4][5][6][7][8][9], thus making CK2 a promising target for chemotherapy [10][11][12][13][14]. The ATP binding site of CK2 is smaller than that of most other kinases due to the presence of unique bulky residues, such as Val66 and Ile174, which create the prerequisites for the development of selective small molecule ATP-competitive inhibitors [15,16]. In the review article of CK2 and its inhibitors [17] by Iegre and colleagues, compounds of the azolo [1,5-a]pyrimidine series are noted as one of the most significant types of inhibitors over the past decade [18,19], along with azole derivatives (Figure 1) [20,21]. of CK2 and its inhibitors [17] by Iegre and colleagues, compounds of the azolo [1,5-a]pyrimidine series are noted as one of the most significant types of inhibitors over the past decade [18,19], along with azole derivatives (Figure 1) [20,21].  [19], B [20], C [21], D [18].

Synthesis
In the present work, we studied compounds of the 4,7-dihydro-6-nitroazolo[1,5-a]pyrimidine 5a-o and 6a-e series. These compounds were obtained by a multicomponent reaction between aminoazoles 1,2, 1-morpholino-2-nitroalkenes 3, and aldehydes 4 (Scheme 1). It was shown [31] that an initial reaction occurs between 1-morpholino-2-nitroalkenes 3 and aminoazoles 1,2, followed by heterocyclization to products 5, and the interaction of boron trifluoride etherate with the morpholinenitroalkene 3 leads to the formation of a corresponding alkyne and morpholinium tetrafluoroborate. The structure of all the obtained products 5,6 was confirmed by the data of 1 H, 13 C NMR spectroscopy, IR spectroscopy, and elemental analysis. The signals H-7 and C-7 are the characteristic for products 5,6 in the corresponding NMR spectra. It is interesting to note that in compounds 5a-d obtained from 3-aminopyrazole 3a, in the 1 H spectra, the H-7 signal is in the region of 5.43-5.84 ppm, while in the 13 C spectra, the characteristic C-7 signal is in the region of 34-40 ppm. In all other structures 5e-o, 6a-d, these signals are shifted to a weaker region of the spectrum in the region of 6.44-6.94 and 55-60 ppm, respectively (see Supplementary Materials). Apparently, the substituent and heteroatom in the azole ring affect the position of these signals.

CK2 Inhibition
Once in hand, target compounds were evaluated against human recombinant CK2 using the ADP-GloTM assay (Table 1). Initial screening at 50 µM revealed that compounds 5a, 5c, 5g, 5m, 5o, 6a, 6c, and 6d paradoxically enhance CK2 activity. Moderate inhibition was demonstrated by compounds 5f, 5h, 5k, 5l, and 6e. Derivatives 5f, 5h, and 5k were the most active inhibitors. One can notice that compounds bearing alkyl or alkylthio substituents at position C-2 and at position C-6 simultaneously tend to be more active, though the high structural similarity in this series does not allow us to define more comprehensive SAR. A dose-response study confirmed that compounds 5f, 5h, 5k, and 5l are micromolar CK2 inhibitors, while 6e has a low potency (Table 2). A hill coefficient around (−1) indicates that lead compounds as well as staurosporine behave like classical inhibitors that bind to a single kinase site.

CK2 Inhibition
Once in hand, target compounds were evaluated against human recombinant CK2 using the ADP-GloTM assay (Table 1). Initial screening at 50 µM revealed that compounds 5a, 5c, 5g, 5m, 5o, 6a, 6c, and 6d paradoxically enhance CK2 activity. Moderate inhibition was demonstrated by compounds 5f, 5h, 5k, 5l, and 6e. Derivatives 5f, 5h, and 5k were the most active inhibitors. One can notice that compounds bearing alkyl or alkylthio substituents at position C-2 and at position C-6 simultaneously tend to be more active, though the high structural similarity in this series does not allow us to define more comprehensive SAR. A dose-response study confirmed that compounds 5f, 5h, 5k, and 5l are micromolar CK2 inhibitors, while 6e has a low potency ( Table 2). A hill coefficient around (-1) indicates that lead compounds as well as staurosporine behave like classical inhibitors that bind to a single kinase site.

Antitumor Activity
The IC 50 parameters were calculated based on the results of the MTT test (Table 3). The values are defined in the range from 13 µM to >650 µM. It should be noted that the study of the 5a, 5b, and 5c cytotoxic effects was limited to exclude because of their low solubility. Compounds 5j and 6a possessed the least pronounced cytotoxic effect on cells (in all cases IC 50 > 0.4 mM), while the greatest decrease in the viability of tumor cells was noted with the addition of compounds 5m, 5o, 6c, and 6d. It is important to note that these azolopyrimidine compounds are characterized by a more pronounced suppression of the viability of tumor cells A-172, Rd, and Hos in comparison with the effect on human embryonic kidney cells Hek-293 (Figures 3 and 4).
It was found that the IC 50 for compounds 5m, 5o, 6c, and 6d in tumor cell lines studies, mostly, did not exceed 50 µM, whereas the cytotoxicity index on embryonic cells was higher than 169 µM (5m, 5o, and 6c). It should be noted that compounds 6d containing a triazole fragment with a CF 3 -substituent in the structure had similar micromolar IC 50 values for kidney cells and tumor cells. The least cytotoxic effect on non-tumor cells was determined for compound 6c (227.50 µM). At the same time, our results indicate that compound 5m may have the most pronounced antitumor properties.
Meanwhile, the mechanisms for the suppression of cultured cells growth remain unclear and require further research. We can assume, that experimental data of cytotoxic action are not fully explained by the effect on CK2. On the one hand, it was noted that azolo[1,5a]pyrimidines 5k, 5l, and 6e inhibit both the enzymatic activity of CK2 and the viability of tumor cells. On the other hand, compounds 5m, 5o, 6c, and 6d significantly inhibit the growth of neoplastic cells without affecting CK2. We can assume that the cytotoxic effect of the synthesized compounds may be due to the effect on other intracellular targets.
Compounds 5j and 6a possessed the least pronounced cytotoxic effect on cells (in all cases IC50 >0.4 mM), while the greatest decrease in the viability of tumor cells was noted with the addition of compounds 5m, 5o, 6c, and 6d. It is important to note that these azolopyrimidine compounds are characterized by a more pronounced suppression of the viability of tumor cells A-172, Rd, and Hos in comparison with the effect on human embryonic kidney cells Hek-293 (Figures 3 and 4).   Compounds 5j and 6a possessed the least pronounced cytotoxic effect on cells (in all cases IC50 >0.4 mM), while the greatest decrease in the viability of tumor cells was noted with the addition of compounds 5m, 5o, 6c, and 6d. It is important to note that these azolopyrimidine compounds are characterized by a more pronounced suppression of the viability of tumor cells A-172, Rd, and Hos in comparison with the effect on human embryonic kidney cells Hek-293 (Figures 3 and 4).

Chemical Experiment
Unless stated otherwise, all solvents and commercially available reactants/reagents were used as received. Non-commercial starting materials were prepared as described below or according to literature procedures. One-dimensional 1 H and 13 C NMR spectra, as well as two-dimensional 1 H-13 C HMBC experiments were acquired on a Bruker DRX-400 instrument (400 and 101 MHz, respectively) or a Bruker Avance NEO 600 instrument (600 and 151 MHz, respectively), equipped with a Prodigy broadband gradient cryoprobe, utilizing DMSO-d 6 as solvent and TMS as internal standard. IR spectra were recorded on a Bruker Alpha FTIR spectrometer equipped with a ZnSe ATR accessory. Elemental analysis was performed on a PerkinElmer 2400 CHN analyzer. The reaction progress was controlled by TLC on Silufol UV-254 plates, eluent-EtOAc. Melting points were determined on a Stuart SMP3 apparatus at the heating rate of 7 • C/min. 1-Morpholino-2-nitroethylenes 3 were prepared according to a literature procedure [34]. 4,7-Dihydro-6-nitroazolo[1,5-a]pyrimidines 5,6; General procedure 1. A total of 3 Mmol (1.5 equiv., 0.37 mL) of BF 3 ·Et 2 O was added to a suspension 2 mmol (1.0 equiv.) of corresponding aminoazole 1,2, 2 mmol (1.0 equiv.) of nitroalkene 3, and 2 mmol (1.0 equiv.) of aldehyde 4 in 5 mL n-BuOH. The reaction mixture was heated on oil bath at 120 • C for 2 h. The resulting solution was cooled to room temperature and stirred 15 min. The obtained precipitate was filtered off, washed with 15 mL of i-PrOH. The precipitate was suspended in 50 mL of water, stirred for 5 min, filtered off again, and washed with 15 mL of water. 4,7-Dihydro-6-nitroazolo[1,5-a]pyrimidines 5,6; General procedure 2. A total of 3 Mmol (1.5 equiv., 0.37 mL) of BF 3 ·Et 2 O was added to a suspension 2 mmol (1.0 equiv.) of corresponding aminoazole 1,2, 2 mmol (1.0 equiv.) of nitroalkene 3, and 2 mmol (1.0 equiv.) of aldehyde 4 in 5 mL n-BuOH. The reaction mixture was heated on oil bath at 120 • C for 2 h. After heating, the resulting solution was concentrated under reduced pressure. To the residue, 20 mL of 2M Na 2 CO 3 and 50 mL of water was added and stirred for 20 min. Solution was extracted twice with 20 mL of EtOAc. To a water phase, 15 mL of hexane was added and mixture was neutralized by diluted HCl to pH 7. Resulting mixture was stirred for 30 min, filtered off, and washed with water.

Viability Assessment
The compounds were dissolved in DMSO. The solutions were diluted with DMEM/F-12 culture medium with 10% fetal bovine serum to the studied concentrations: 8, 16, 32, 64, 128, 256, 512, and 1024 µM. In all cases, the concentration of DMSO in the final solution did not exceed 1%. Cisplatin (cPt) was used as a positive control.
Cells were seeded in 96-well plates at a concentration of 4 × 10 3 cells per well. After 24 h, test compounds were added to the wells in a given concentration range. Then the cells were incubated for 72 h, after which a solution of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to the cultures at 20 µL (5 mg/mL) to the well. After 2.5 h, the medium was removed from the wells and 200 µL of a mixture of DMSO/i-PrOH 1/1 was added. Optical density was measured on a plate spectrophotometer at a wavelength of 570 nm.

Statistical Analysis
Statistical data processing was carried out in the RStudio program (Version 1.4.1106 © 2022-2021 RStudio, PBC, Boston, MA, USA) using the R package (version 4.1.2). The cytotoxicity index (IC 50 ) was calculated by plotting dose-response curves using the "drc" package [41].

Conclusions
Thus, in this work we extended the library of the 4,7-dihydro-6-nitroazolo[1,5a]pyrimidine series, and also studied their antitumor properties. The inhibitory activity of these compounds against CK2 has been established, as well as their cytotoxic effect. Compounds of this series are comparable in inhibitory activity with the reference drug and exhibit a cytotoxic effect on tumor cells at micromolar concentrations. It is evident that the herein reported 4,7-dihydro-6-nitroazolo[1,5-a]pyrimidines have the potential to be studied as a new class of antitumor compounds.