6-(Tetrazol-5-yl)-7-aminoazolo[1,5-a]pyrimidines as Novel Potent CK2 Inhibitors

In this work, we describe the design, synthesis, and structure-activity relationship of 6-(tetrazol-5-yl)-7-aminoazolo[1,5-a]pyrimidines as inhibitors of Casein kinase 2 (CK2). At first, we optimized the reaction conditions for the azide-nitrile cycloaddition in the series of 6-cyano-7-aminoazolopyridimines and sodium azide. The regioselectivity of this process has been shown, as the cyano group of the pyrimidine cycle was converted to tetrazole while the nitrile of the azole fragment did not react. The desired tetrazolyl-azolopyrimidines were obtained in a moderate to excellent yields (42–95%) and converted further to water soluble sodium salts by the action of sodium bicarbonate. The obtained 6-(tetrazol-5-yl)-7-aminopyrazolo[1,5-a]pyrimidines 2a–k and their sodium salts 3a–c, 3g–k showed nano to low micromolar range of CK2 inhibition while corresponding [1,2,4]triazolopyrimidines 10a–k were less active (IC50 > 10 µM). The leader compound 3-phenyl-6-(tetrazol-5-yl)-7-aminopyrazolo[1,5-a]pyrimidine 2i as CK2 inhibitor showed IC50 45 nM.


Introduction
Casein Kinase 2 (CK2) is a highly conserved polyfunctional serine/threonine protein kinase that plays an important role in the regulation of the processes of several cells, such as proliferation, differentiation and survival [1]. It is considered that CK2 has been implicated in the manifestation of some diseases, including multiple sclerosis [2], inflammation [3], hypertension [4], and viral infections [5]. The role of CK2 has been extensively studied in the development of malignant tumors and it was proved as a key regulator of multiple oncogenic pathways, including the PI3K/Akt, JAK/STAT, IL-6 and NF-jB signaling cascades [6]. In turn, CK2 is a key suppressor of cell apoptosis [7], which determines its role in oncogenesis of several tumors with overexpression of CK2, including breast carcinoma, adenocarcinoma of the lung, prostate carcinoma and lymphomas [8]. It can be noted that Silmitasertib has been approved by the FDA for the treatment of cholangiocarcinoma as CK2 inhibitor [9]. Thus, the development of novel CK2 inhibitors as chemotherapeutic agents against cancer and other nosologies where this type of kinases is involved is a relevant task.
Previously, a wide variety of different molecules have been described as CK2 inhibitors, including polyhalogenated benzimidazole and benzotriazole derivatives [10], nitrogen-containing heterocycles [11][12][13] and their polycondensed analogues [14], as well as condensed coumarin derivatives [15] (Figure 1). Azoloazines heterocycles with bridge nitrogen atom are of considerable interest, since many representatives of this class are known to inhibit CK2 in the low nanomolar range. However, most of the currently available CK2 inhibitors lack the potency, physiochemical, and pharmacological properties required to be successful in clinical trials. known to inhibit CK2 in the low nanomolar range. However, most of the currently available CK2 inhibitors lack the potency, physiochemical, and pharmacological properties required to be successful in clinical trials. Figure 1. Examples of pyrazoloazines and other molecules with high affinity for CK2 [11][12][13][14][15].
At the same time, a nitro group or carboxylic fragment should present within heterocyclic scaffold for this useful activity to be formed ( Figure 2). On the other hand, the tetrazole cycle is a metabolically stable bio-isostere of the carboxyl group and the cis-amide fragment due to the similar electronic structure [21][22][23][24][25]. The corresponding similarity for the carboxylic anion and the nitro group can be noted and one can consider the tetrazolyl fragment as an isostere for both of them ( Figure 3a). However, only one example of azoloazines containing tetrazole cycle has been published to date-2-nitro-6-(1H-tetrazol-5-yl)- [1,2,4]triazolo[1,5-a]pyrimidin-7-amine was considered as nitrogen-rich energetic material [26]. known to inhibit CK2 in the low nanomolar range. However, most of the currently available CK2 inhibitors lack the potency, physiochemical, and pharmacological properties required to be successful in clinical trials.
At the same time, a nitro group or carboxylic fragment should present within heterocyclic scaffold for this useful activity to be formed ( Figure 2). On the other hand, the tetrazole cycle is a metabolically stable bio-isostere of the carboxyl group and the cis-amide fragment due to the similar electronic structure [21][22][23][24][25]. The corresponding similarity for the carboxylic anion and the nitro group can be noted and one can consider the tetrazolyl fragment as an isostere for both of them ( Figure 3a). However, only one example of azoloazines containing tetrazole cycle has been published to date-2-nitro-6-(1H-tetrazol-5-yl)- [1,2,4]triazolo[1,5-a]pyrimidin-7-amine was considered as nitrogen-rich energetic material [26].  [16][17][18]20]. Figure 2. Examples of biologically active azolopyrimidines [16][17][18]20].  In this work we propose the introduction of tetrazolyl fragment into azolopyrimidine scaffold as promising structural modification to search for novel CK2 inhibitors ( Figure  3b).

Chemistry
We have developed a versatile approach to the synthesis of 6-cyano-7-aminoazolo [1,5-a]pyrimidines and obtained a library of corresponding heterocycles [27] which are good precursors for azide-nitrile cycloaddition. Herein, 3-Ethoxycarbonyl-6-cyano-7aminopyrazolo [1,5-a]pyrimidine 1g was used as model substrate to study this process and evaluate different reaction conditions while sodium azide served as the source of the azide fragment (Scheme 1).
. Scheme 1. Model reaction of cyanoderivative 1g with sodium azide for condition optimization.
The mechanism of this process has been studied extensively by DFT calculations and it was shown that energy barrier for the reaction of the azide anion with nitriles is considerably lower than the barrier for the attack of the neutral hydrazoic acid [28]. At the same time, experimental data revealed that the reaction is strongly accelerated by Brønsted acids such as AcOH and ammonium salts [29,30]. Lewis acids [31], specific organocatalysts [32], and ionic liquids [33] could serve as catalyst in azide-nitrile cycloaddition as well.
It was found that formation of tetrazole cycle by reaction of sodium azide with nitrile derivative 1g proceeded smoothly in polar aprotic solvents (DMF, MeCN) in the presence of ammonium salts (entry 1-6 and 8, Table 1), AcOH (entry 7, Table 1), ZnCl2 (entry 10, Table 1), or 1-butyl-3-methylimidazolium chloride (entry 9, Table 1). The highest yield (78%) of the desired product 2g was observed in the case AcOH while control experiment where 1g reacted with sodium azide in DMF without any additive resulted in 82% yield of tetrazole 2g (entry 11, Table 1). Optimal conditions required heating at 120 °C for 8 h of a 0.25 molar solution of 1g in DMF with 1.1 equiv. of NaN3 and further treatment of water suspension of 3g with conc. HCl to provide 90% yield of 2g (entry 13, Table 1). It is worth noting that the formation of the tetrazole cycle was not observed in protic polar solvents (H2O, MeOH) both with catalysis (entry 2 and 6, Table 1) and without (entry 18, Table 1) by TLC analysis of the reaction mixture as well as by NMR analysis of the isolated products. In this work we propose the introduction of tetrazolyl fragment into azolopyrimidine scaffold as promising structural modification to search for novel CK2 inhibitors (Figure 3b).

Chemistry
We have developed a versatile approach to the synthesis of 6-cyano-7-aminoazolo [1,5a]pyrimidines and obtained a library of corresponding heterocycles [27] which are good precursors for azide-nitrile cycloaddition. Herein, 3-Ethoxycarbonyl-6-cyano-7-aminopyrazolo pyrimidine 1g was used as model substrate to study this process and evaluate different reaction conditions while sodium azide served as the source of the azide fragment (Scheme 1).  In this work we propose the introduction of tetrazolyl fragment into azolopyrimidine scaffold as promising structural modification to search for novel CK2 inhibitors ( Figure  3b).

Chemistry
We have developed a versatile approach to the synthesis of 6-cyano-7-aminoazolo [1,5-a]pyrimidines and obtained a library of corresponding heterocycles [27] which are good precursors for azide-nitrile cycloaddition. Herein, 3-Ethoxycarbonyl-6-cyano-7aminopyrazolo[1,5-a]pyrimidine 1g was used as model substrate to study this process and evaluate different reaction conditions while sodium azide served as the source of the azide fragment (Scheme 1).
. Scheme 1. Model reaction of cyanoderivative 1g with sodium azide for condition optimization.
The mechanism of this process has been studied extensively by DFT calculations and it was shown that energy barrier for the reaction of the azide anion with nitriles is considerably lower than the barrier for the attack of the neutral hydrazoic acid [28]. At the same time, experimental data revealed that the reaction is strongly accelerated by Brønsted acids such as AcOH and ammonium salts [29,30]. Lewis acids [31], specific organocatalysts [32], and ionic liquids [33] could serve as catalyst in azide-nitrile cycloaddition as well.
It was found that formation of tetrazole cycle by reaction of sodium azide with nitrile derivative 1g proceeded smoothly in polar aprotic solvents (DMF, MeCN) in the presence of ammonium salts (entry 1-6 and 8, Table 1), AcOH (entry 7, Table 1), ZnCl2 (entry 10, Table 1), or 1-butyl-3-methylimidazolium chloride (entry 9, Table 1). The highest yield (78%) of the desired product 2g was observed in the case AcOH while control experiment where 1g reacted with sodium azide in DMF without any additive resulted in 82% yield of tetrazole 2g (entry 11, Table 1). Optimal conditions required heating at 120 °C for 8 h of a 0.25 molar solution of 1g in DMF with 1.1 equiv. of NaN3 and further treatment of water suspension of 3g with conc. HCl to provide 90% yield of 2g (entry 13, Table 1). It is worth noting that the formation of the tetrazole cycle was not observed in protic polar solvents (H2O, MeOH) both with catalysis (entry 2 and 6, Table 1) and without (entry 18, Table 1) by TLC analysis of the reaction mixture as well as by NMR analysis of the isolated products. The mechanism of this process has been studied extensively by DFT calculations and it was shown that energy barrier for the reaction of the azide anion with nitriles is considerably lower than the barrier for the attack of the neutral hydrazoic acid [28]. At the same time, experimental data revealed that the reaction is strongly accelerated by Brønsted acids such as AcOH and ammonium salts [29,30]. Lewis acids [31], specific organocatalysts [32], and ionic liquids [33] could serve as catalyst in azide-nitrile cycloaddition as well.
It was found that formation of tetrazole cycle by reaction of sodium azide with nitrile derivative 1g proceeded smoothly in polar aprotic solvents (DMF, MeCN) in the presence of ammonium salts (entry 1-6 and 8, Table 1), AcOH (entry 7, Table 1), ZnCl 2 (entry 10, Table 1), or 1-butyl-3-methylimidazolium chloride (entry 9, Table 1). The highest yield (78%) of the desired product 2g was observed in the case AcOH while control experiment where 1g reacted with sodium azide in DMF without any additive resulted in 82% yield of tetrazole 2g (entry 11, Table 1). Optimal conditions required heating at 120 • C for 8 h of a 0.25 molar solution of 1g in DMF with 1.1 equiv. of NaN 3 and further treatment of water suspension of 3g with conc. HCl to provide 90% yield of 2g (entry 13, Table 1). It is worth noting that the formation of the tetrazole cycle was not observed in protic polar solvents (H 2 O, MeOH) both with catalysis (entry 2 and 6, Table 1) and without (entry 18, Table 1) by TLC analysis of the reaction mixture as well as by NMR analysis of the isolated products.  With the optimized reaction conditions in hand, a series of 6-(tetrazol-5-yl)pyrazolopyrimidines 2a-k were synthesized in good to excellent yields (60-95%) (Scheme 2). The cycloaddition of sodium azide to the C6-nitrile fragment in this series 1a-k proceeded without competing processes. Thus, in the case of dinitriles 1f and 1k it was observed that only one cyano group reacted with azide to form tetrazole cycle as there were CN characteristic absorption peaks in the region of 2217-2231 cm −1 in IR spectra of the obtained products 2f and 2k. The same results were obtained in the reaction of 1f and 1k with 3 equiv. of sodium azide by the analysis of reaction products with 1 H and IR spectroscopy. We have tried to obtain 3,6-di(tetrazol-5-yl)pyrazolopyrimidine 6 by independent synthesis via two steps starting from 3-amino-4-cyanopyrazole 4 (Scheme 3). It was showed that the heating of compound 4 with sodium azide in DMF both with ammonium chloride and in the absence of it did not lead to tetrazolyl heterocycle 5. Subsequently, [28] 0. With the optimized reaction conditions in hand, a series of 6-(tetrazol-5-yl) pyrazolopyrimidines 2a-k were synthesized in good to excellent yields (60-95%) (Scheme 2). With the optimized reaction conditions in hand, a series of 6-(tetrazol-5-yl)pyrazolopyrimidines 2a-k were synthesized in good to excellent yields (60-95%) (Scheme 2). The cycloaddition of sodium azide to the C6-nitrile fragment in this series 1a-k proceeded without competing processes. Thus, in the case of dinitriles 1f and 1k it was observed that only one cyano group reacted with azide to form tetrazole cycle as there were CN characteristic absorption peaks in the region of 2217-2231 cm −1 in IR spectra of the obtained products 2f and 2k. The same results were obtained in the reaction of 1f and 1k with 3 equiv. of sodium azide by the analysis of reaction products with 1 H and IR spectroscopy. We have tried to obtain 3,6-di(tetrazol-5-yl)pyrazolopyrimidine 6 by independent synthesis via two steps starting from 3-amino-4-cyanopyrazole 4 (Scheme 3). It was showed that the heating of compound 4 with sodium azide in DMF both with ammonium chloride and in the absence of it did not lead to tetrazolyl heterocycle 5. Subsequently, Scheme 2. Scope of 6-(tetrazol-5-yl)-7-aminopyrazolo[1,5-a]pyrimidines 2a-k and corresponding sodium salts 3a-c, 3g-k.
The cycloaddition of sodium azide to the C6-nitrile fragment in this series 1a-k proceeded without competing processes. Thus, in the case of dinitriles 1f and 1k it was observed that only one cyano group reacted with azide to form tetrazole cycle as there were CN characteristic absorption peaks in the region of 2217-2231 cm −1 in IR spectra of the obtained products 2f and 2k. The same results were obtained in the reaction of 1f and 1k with 3 equiv. of sodium azide by the analysis of reaction products with 1 H and IR spectroscopy. We have tried to obtain 3,6-di(tetrazol-5-yl)pyrazolopyrimidine 6 by independent synthesis via two steps starting from 3-amino-4-cyanopyrazole 4 (Scheme 3). It was showed that the heating of compound 4 with sodium azide in DMF both with ammonium chloride and in the absence of it did not lead to tetrazolyl heterocycle 5. Subsequently, pyrazole 4 has been converted to 3-cyano-7-aminopyrazolopyrimidine 7 [34], but the latter also did not react with sodium azide under different conditions and starting material 7 was isolated after workup of the reaction mixture (Scheme 3). These findings support regioselectivity of the azide-nitrile cycloaddition process in the series of 3,6-dicyanopyrazolopyrimidines as only nitrile group in the pyrimidine ring converts into tetrazole fragment.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 15 pyrazole 4 has been converted to 3-cyano-7-aminopyrazolopyrimidine 7 [34], but the latter also did not react with sodium azide under different conditions and starting material 7 was isolated after workup of the reaction mixture (Scheme 3). These findings support regioselectivity of the azide-nitrile cycloaddition process in the series of 3,6-dicyanopyrazolopyrimidines as only nitrile group in the pyrimidine ring converts into tetrazole fragment. The structure of the obtained heterocycles 2a-k and 10a-k was confirmed by 1 H NMR spectroscopy (the signal of C5H proton was shifted downfield (∆δ ≈ 0.5 ppm) in comparison with starting material), 13 C NMR technic (characteristic signal around δ ≈ 85-91 ppm, probably, it can be attributed to C6 atom, while other aromatic carbons located in the region of δ ≈ 145-160 ppm), IR spectroscopy (absence of CN absorption peak in the region of 2100-2300 cm −1 in comparison with starting material), mass-spectrometry (a molecular ion peaks were detected) and elemental analysis (see Supporting Information).
We converted obtained 6-(1H-tetrazol-5-yl)-7-aminoazolo[1,5-a]pyrimidines 2 and 10 to the corresponding sodium salts by the reaction with sodium bicarbonate (Schemes 2 and 4) based on the NH-acidity of the tetrazole ring [35]. These sodium salts 3a-c, 3g-k and 11a-e, 11g, 11h, 11j possess high water solubility which is an undoubted advantage for testing its biological activity in the CK2 assay and further experiments. The unidentified oily products were isolated when thiopropargyl containing derivative 9l was introduced in the reaction, probably due to the side azide-alkyne cycloaddition to form 1,2,3-triazole.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 15 pyrazole 4 has been converted to 3-cyano-7-aminopyrazolopyrimidine 7 [34], but the latter also did not react with sodium azide under different conditions and starting material 7 was isolated after workup of the reaction mixture (Scheme 3). These findings support regioselectivity of the azide-nitrile cycloaddition process in the series of 3,6-dicyanopyrazolopyrimidines as only nitrile group in the pyrimidine ring converts into tetrazole fragment.
We converted obtained 6-(1H-tetrazol-5-yl)-7-aminoazolo[1,5-a]pyrimidines 2 and 10 to the corresponding sodium salts by the reaction with sodium bicarbonate (Schemes 2 and 4) based on the NH-acidity of the tetrazole ring [35]. These sodium salts 3a-c, 3g-k and 11a-e, 11g, 11h, 11j possess high water solubility which is an undoubted advantage for testing its biological activity in the CK2 assay and further experiments.  11a-e, 11g, 11h, 11j. The structure of the obtained heterocycles 2a-k and 10a-k was confirmed by 1 H NMR spectroscopy (the signal of C5H proton was shifted downfield (∆δ ≈ 0.5 ppm) in comparison with starting material), 13 C NMR technic (characteristic signal around δ ≈ 85-91 ppm, probably, it can be attributed to C6 atom, while other aromatic carbons located in the region of δ ≈ 145-160 ppm), IR spectroscopy (absence of CN absorption peak in the region of 2100-2300 cm −1 in comparison with starting material), mass-spectrometry (a molecular ion peaks were detected) and elemental analysis (see Supporting Information).

CK2 Inhibition
Once in hand target compounds were evaluated against human recombinant CK2 using luminescent ADP-GloTM platform. Initial screening performed at 50 µM compound concentration revealed scaffold as a rich source of CK2 inhibitors. Confirmation experiments were run in a concentration-dependent manner to obtain IC 50 values ( Table 2). Structure-activity relationship analysis (Figure 4) suggests that 6-(tetrazol-5-yl)-[1,2,4] triazolo[1,5-a]pyrimidines 10a-j generally have lower activity than corresponding pyrazolopyrimidines 2a-k reflecting in IC 50 values in higher micromolar range. Notably, in the triazolopyrimidine series, compounds 10a and 10h are the most active (IC 50 23.78 and 11.81 µM, correspondingly), while any other substituents in the triazole ring resulted in the decrease of affinity towards CK2.  In turn, derivatives of 6-(tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine series 2a-k and 3ac, 3f, 3g, 3i demonstrated rather improved potency. Compounds 2a and its sodium salt 3a are micromolar inhibitors. Introduction of SMe (2c) or Ph (2d) group in the C2-position of heterocyclic scaffold is beneficial, while the smaller Me-substituent at this position led to less active compound 2b. Evaluation of substituents in position C3 of the pyrazolopyrimidine system indicates non-additive SAR. Thus, both electron-withdrawing and electrondonating groups resulted in low micromolar inhibitors 2f-i with leader compound 2i demonstrated IC50 = 45 nM. At the same time, the combination of 2-methylsulfanyl group with 3-ethoxycarbonyl or 3-nitrile substituents also revealed compounds 2j and 2k with good affinity to CK2. It is worth noting that in most cases sodium salts 3 were surprisingly less active than NH-form of tetrazolyl containing heterocycles 2 excluding potent sodium 5-(7-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)tetrazol-1-ide 3f with IC50 = 65 nM.

Chemistry
Commercial reagents were obtained from Sigma-Aldrich, Acros Organics, or Alfa Aesar and used without any further purification. All workup and purification procedures were carried out using analytical grade solvents. One-dimensional 1 H, 19 F, and 13 С NMR spectra were acquired on a Bruker DRX-400 instrument (400, 376, and 101 MHz, respectively), utilizing DMSO-d6 as solvent and as an external reference. The following abbreviations are used for multiplicity of NMR signals: s-singlet, d-doublet, t-triplet, qquartet, dd-doublet of doublets, dt-doublet of triplets, m-multiplet, br-broaded. Mass spectroscopy studies were performed on a Shimadzu GCMS-QP2010 Ultra (EI, 70 eV). IR spectra were recorded on a Bruker Alpha spectrometer equipped with a ZnSe ATR accessory. Elemental analysis was performed on a PerkinElmer PE 2400 elemental analyzer. Melting points were determined on a Stuart SMP3 and are uncorrected. The monitoring of the reaction progress was performed by using TLC on Silufol UV254 plates (eluent is AcOEt). All synthesized compounds are >95% pure by elemental analysis.
A suspension of 0.01 mol (1 equiv.) of the corresponding 6-cyano-7-aminoazolo[1,5a]pyrimidine (1a-k, 9a-k) and 0.011 mol (1.1 equiv.) of sodium azide in 40 mL of DMF was stirred at 120 °C for 8 hours under air atmosphere (TLC control, AcOEt as eluent, starting material Rf ≈ 0.6-0.7, tetrazole products Rf ≈ 0.0). The reaction mixture was cooled to 25 °C, evaporated at reduced pressure, residue was dissolved in 30 ml of H2O and acidified with conc. HCl to pH≈1. The obtained precipitate was filtered off and washed with 100 ml of H2O to give the corresponding product.  In turn, derivatives of 6-(tetrazol-5-yl)pyrazolo [1,5-a]pyrimidine series 2a-k and 3a-c, 3f, 3g, 3i demonstrated rather improved potency. Compounds 2a and its sodium salt 3a are micromolar inhibitors. Introduction of SMe (2c) or Ph (2d) group in the C2-position of heterocyclic scaffold is beneficial, while the smaller Me-substituent at this position led to less active compound 2b. Evaluation of substituents in position C3 of the pyrazolopyrimidine system indicates non-additive SAR. Thus, both electron-withdrawing and electron-donating groups resulted in low micromolar inhibitors 2f-i with leader compound 2i demonstrated IC 50 = 45 nM. At the same time, the combination of 2-methylsulfanyl group with 3-ethoxycarbonyl or 3-nitrile substituents also revealed compounds 2j and 2k with good affinity to CK2. It is worth noting that in most cases sodium salts 3 were surprisingly less active than NH-form of tetrazolyl containing heterocycles 2 excluding potent sodium 5-(7-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)tetrazol-1-ide 3f with IC 50 = 65 nM.

Chemistry
Commercial reagents were obtained from Sigma-Aldrich, Acros Organics, or Alfa Aesar and used without any further purification. All workup and purification procedures were carried out using analytical grade solvents. One-dimensional 1 H, 19 F, and 13 C NMR spectra were acquired on a Bruker DRX-400 instrument (400, 376, and 101 MHz, respectively), utilizing DMSO-d 6 as solvent and as an external reference. The following abbreviations are used for multiplicity of NMR signals: s-singlet, d-doublet, t-triplet, q-quartet, dd-doublet of doublets, dt-doublet of triplets, m-multiplet, br-broaded. Mass spectroscopy studies were performed on a Shimadzu GCMS-QP2010 Ultra (EI, 70 eV). IR spectra were recorded on a Bruker Alpha spectrometer equipped with a ZnSe ATR accessory. Elemental analysis was performed on a PerkinElmer PE 2400 elemental analyzer. Melting points were determined on a Stuart SMP3 and are uncorrected. The monitoring of the reaction progress was performed by using TLC on Silufol UV254 plates (eluent is AcOEt). All synthesized compounds are >95% pure by elemental analysis.