Design, Synthesis and Structure–Activity Relationship Studies of Protein Kinase CK2 Inhibitors Containing a Purine Scaffold

Abstract

Protein kinase CK2 (CK2) is involved in the suppression of gene expression, protein synthesis, cell proliferation, and apoptosis, thus making it a target protein for the development of therapeutics toward cancer, nephritis, and coronavirus disease 2019. Using the solvent dipole ordering-based method for virtual screening, we identified and designed new candidate CK2α inhibitors containing purine scaffolds. Virtual docking experiments supported by experimental structure–activity relationship studies identified the importance of the 4-carboxyphenyl group at the 2-position, a carboxamide group at the 6-position, and an electron-rich phenyl group at the 9-position of the purine scaffold. Docking studies based on the crystal structures of CK2α and inhibitor (PDBID: 5B0X) successfully predicted the binding mode of 4-(6-carbamoyl-8-oxo-9-phenyl-8,9-dihydro-7H-purin-2-yl) benzoic acid (11), and the results were used to design stronger small molecule targets for CK2α inhibition. Interaction energy analysis suggested that 11 bound around the hinge region without the water molecule (W1) near Trp176 and Glu81 that is frequently reported in crystal structures of CK2α inhibitor complexes. X-ray crystallographic data for 11 bound to CK2α was in very good agreement with the docking experiments, and consistent with activity. From the structure–activity relationship (SAR) studies presented here, 4-(6-Carbamoyl-9-(4-(dimethylamino)phenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl) benzoic acid (12) was identified as an improved active purine-based CK2α inhibitor with an IC₅₀ of 4.3 µM. These active compounds with an unusual binding mode are expected to inspire new CK2α inhibitors and the development of therapeutics targeting CK2 inhibition.

Introduction

Protein kinase CK2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase selective for serine and threonine that can phosphorylate hundreds of different substrates. CK2 functions as a hetero-tetrameric holoenzyme consisting of two catalytic subunits (CK2α and/or CK2α′) and two identical regulatory subunits (CK2β).¹⁾ CK2 substrates are involved in many important cellular functions, including signal transduction and gene expression,^2–5) thus, CK2 is known to be implicated in many important aspects of cancer, including inhibition of apoptosis, regulation of signaling pathways, DNA damage response, and cell cycle regulation. The overexpression of CK2 is often associated with a poor prognosis⁶⁾; consequently, CK2 is an attractive target for cancer therapy. CK2 is also known to be associated with the progression of glomerulonephritis,^7–9) neurodegenerative diseases (such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis),¹⁰⁾ viral infections (such as human immunodeficiency virus type 1,¹¹⁾ and severe acute respiratory syndrome coronavirus 2),^12,13) and regulation of the circadian clock.¹⁴⁾ Therefore, CK2 inhibitors are expected to show therapeutic activity for the diseases listed above.

Currently, there is a global coronavirus disease 2019 pandemic, and the discovery of therapeutic agents operating under a different mechanism from currently approved therapeutics is urgently needed.¹⁵⁾ Many inhibitors have been reported, and compounds derived from natural products that act as ATP-competitive inhibitors include apigenin,¹⁶⁾ emodin,¹⁷⁾ and ellagic acid.¹⁸⁾ Many synthetic inhibitors have also been reported, including the halogenated heteroaryl compounds (4,5,6,7-tetrabromo-2-azabenzimidazole,¹⁹⁾ 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole,⁴⁾ and 5,6-dichloro-1-D-ribofuranosyl-benzimidazole,²⁰⁾ carboxylic acid derivatives [5-oxo-5,6-dihydro-indolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA),²¹⁾ and CX-4945 (silmitasertib),²²⁾ 2,6-disubstituted pyrazine derivatives including CC-4791,^7–9) and arylazole derivatives, including pyridazine-3-carboxylic acid derivatives.²³⁾ The structures of some synthetic inhibitors are shown in Fig. 1. Recently, SRPIN803-rev, a compound containing a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold,²⁴⁾ and triazole-based compound GO289¹⁴⁾ have been reported as possessing phenol groups without carboxy groups. SGC-CK2-1, which has a benzamide structure, has also been reported to be a selective chemical probe against CK2.^25,26) As just demonstrated, many small molecule compounds with diverse chemical structures have been reported for the treatment of the above diseases, including COVID-19, many of which have planar structures, intramolecular carboxy groups, and ionic interactions with Lys68; however, only silmitasertib has been approved by the U. S. Food and Drug Administration as an orphan drug for advanced cholangiocarcinoma.²⁷⁾

Fig. 1. Some Examples of Synthetic CK2 Inhibitors with a Carboxylic Group and Its IC₅₀ Values in the Parentheses (µM)

As mentioned above, except silmitasertib, small molecule CK2 inhibitors have not yet been clinically tested; therefore, much work remains for discovering CK2 inhibiting small molecules with new chemical scaffolds. Our group identified a novel protein kinase CK2 inhibitor with a distinct scaffold using a virtual screening method.²⁸⁾ Most of the identified compounds were lead-like and dissimilar to known inhibitors. However, many of the identified compounds demonstrated weak activity, indicating improvements were necessary before they could be tested as lead compounds. As shown in Fig. 2, two of the inhibitors identified from virtual screening had a purine scaffold (1 and 2). Notably, these two compounds had the same purine scaffold with polar groups (6-carboxamide, 7-NH, and 8-oxo) that might interact with protein kinase CK2. Additionally, the two aromatic groups at the 2- and 9-positions caused changes in inhibition potency lead us to believe that further improvements in inhibition activity could be achieved by experimenting with the substituents on these two aromatic rings.

Fig. 2. Structures of CK2 Inhibitors with a Purine Scaffold and IC₅₀ Values in Parentheses (µM)

To improve clinical success rate, compliance with Lipinski's Rule of 5^29,30) and high Fsp^3,31) in the structure of compounds are desired. However, before investigating these factors, in this study, we synthesized compounds with new purine scaffold, predicted binding modes by docking simulations³²⁾ with CK2α, and structure–activity relationship (SAR) studies were performed by introducing various aryl substituents at positions 2 and 9 of the purine scaffold. The binding structures of potent compounds and the essential portions of the scaffold were also investigated.

Results and Discussion

Design, Synthesis, and CK2α Inhibitory Activities of Compounds Modified at the Purine Scaffold 2-Position

Since the CK2α inhibitory activities of 1 and 2 were still weak and could be improved by changing the polarity and/or size of the two aryl groups (Fig. 3), aryl groups with various 3- or 4-substituents were introduced at the 9-position (Ar¹) and 2-position (Ar²) of the purine scaffold. Starting with two unsubstituted phenyl groups, Compound 3 was found not to be an active CK2α inhibitor (Table 1). While holding Ar¹ as a phenyl group, the Ar² groups (compounds 4–11) were explored. Although compound 4 is structurally similar to compound 1, their inhibitory performance was quite different. This indicates that an interaction between the nitrogen atom of the pyridyl group and the surrounding amino acid residues was assumed, but it was weak, and substituents on the aryl group of Ar¹ are also required for the expression of activity. Interestingly, only compound 11, with a 4-carboxy group, showed weak potency (IC₅₀: 12 µM). Compound 10, with a 4-nitro group, was inactive, despite the 4-nitro and 4-carboxy groups possessing similar shapes and electrostatic potentials. This suggested that the carboxy group was involved in a salt bridge with the Lys68 sidechain of CK2α, similar to other known CK2α inhibitors displaying a carboxy group. Therefore, Ar² was fixed to the 4-carboxyphenyl group for the study of the Ar¹ fragment. Before the further compound screening, we next performed docking studies of compound 11 with the CK2α active site to select more judiciously Ar¹ screening fragments.

Fig. 3. Purine Scaffold with Numbering

Ar¹ and Ar² are defined as variable fragments explored in this SAR study.

Table 1. CK2α Inhibitory Activities of Purine Derivatives Where Ar¹ Is Fixed to Phenyl

Compound	Ar1	Ar2	IC50 (µM)
3	Phenyl	Phenyl	>30
4	Phenyl	4-Pyridyl	>30
5	Phenyl	4-Methylphenyl	>30
6	Phenyl	4-Fluorophenyl	>30
7	Phenyl	4-Hydroxyphenyl	>30
8	Phenyl	4-Hydroxymethylphenyl	>30
9	Phenyl	4-Methoxycarbonylphenyl	>30
10	Phenyl	4-Nitrophenyl	>30
11	Phenyl	4-Carboxyphenyl	12

Docking Simulation of 11 with CK2α

To predict the binding mode of 11 to CK2α, the docking study for 11 was performed using the reported crystal structure of CK2α (PDB ID: 5B0X).³³⁾ The water molecule (W1) near Trp176 and Glu81 is often present in co-crystallized CK2α–inhibitor complex structures,^21–23,33) Therefore, docking simulations were performed with and without W1. The docking structure of 11 with CK2α in the presence of W1 is presented in Fig. 4. To determine whether the W1 was necessary, a more detailed analysis was performed. Molecular dynamics simulations for both with and without W1 complexes were conducted and the root mean square deviation (RMSD) values from the initial structure of 11 and differences in binding free energy (ΔG_bind) were estimated by the Molecular Mechanics/Poisson–Boltzmann and surface area (MM/PBSA) method³⁴⁾ combined with entropy corrections using the Interaction Entropy (IE) method.³⁵⁾ The averaged values over 5 runs are presented in Table 2.

Table 2. Comparison of Binding Stability for the Complexes with and without Crystalline Water (W1)

	Without W1	With W1	Difference
RMSD (Å)	3.3	2.1	1.2
Eint (kcal/mol)	−170.3	−182.2	+11.9
Esolv (kcal/mol)	133.9	146.6	−12.7
EPBSAa) (kcal/mol)	−36.4	−35.6	−0.8
−TdSIEb) (kcal/mol)	29.5	31.5	−2.0
ΔGbindc) (kcal/mol)	−6.9	−4.1	−2.8

a) Estimated values by MM-PBSA method. (E_PBSA = E_int+E_solv). b) Estimated values by IE method. (T = 300 K). c) Estimated values by MM-PBSA combined with IE method. (ΔG_bind = E_PBSA−TdS_IE).

Fig. 4. Docking Structures of 11 with Crystalline Water W1 (Cyan) and without W1 (Magenta)

The RMSD value of 3.3 Å without W1 implies a rather unstable binding mode from the initial structure, while that of 2.1 Å with W1 implies a relatively stable binding mode. Thus, the interaction energy (E_int) without W1 was less strong than the E_int with W1. Considering the solvent effect, there was little enthalpic energy (E_PBSA) difference between the presence and absence of W1 and the binding free energy (ΔG_bind) was 2.8 kcal/mol more stable in the absence of W1. The RMSD was smaller in the presence of water because W1 occupies a space in the substrate binding pocket and prevents the inhibitor’s movement through strong interactions. In other words, the interaction in the narrower pocket caused by the presence of W1 led to strong enthalpic attraction. On the other hand, it was large disadvantageous in terms of binding entropy (−TdS_IE) due to the restricted movement. These results indicate that the absence of water is more favorable for the binding of 11 and W1 should not be expected in the CK2α–compound 11 inhibitory interaction.

As for the binding mode, the carboxamide at the 6-position of the purine scaffold was expected to be hydrogen bonded to Glu114 and Val116 in the hinge region regardless of W1. Although the position was slightly different, the carboxyphenyl group at the 2-position formed a polar interaction with Lys68, and the phenyl ring at the 9-position formed a CH–π interaction with hydrophobic residues, such as Leu45, Gly46, and Val53. This CH–π interaction was especially noticeable in the binding mode without W1.

Crystal structure revealed that 11 bound to the CK2α with a similar manner to the computed docked structure (Fig. 5). The water molecule (W1), highly conserved and ligated with Trp176 and Glu81 in CK2α crystal structures, was replaced by the carboxy group of 11. Inevitably, the carboxy group of 11 was located near Lys68 and Glu81. The oxygen atom of one of the carboxy groups in 11 is in close proximity to the carboxyl group of Glu81 (2.4 Å). Although this distance of 2.4 Å indicates a possibility of repulsive interaction, we can assume that there is no serious problem with this structure due to the following reasons. First, this crystal structure was solved at high resolution of 1.70 Å and the electron density assured the coordinates of the carboxyl group of 11, Glu81, and Lys68. In addition, as reported by Kleywegt and Brünger,³⁶⁾ it is known that even with a high resolution a positional error of 0.2 Å should be included. Therefore, this distance of 2.4 Å is within the range of hydrogen bonding. Second, the carboxy group of 11 is electrostatically neutralized by forming a salt bridge with Lys68, possibly allowing the carboxy group of 11 and Glu81 to be in close each other via the salt-bridging hydrogen atom of Lys68. We believe that the development of CK2 inhibitors would be facilitated by understanding the interactions among the carboxy group of 11, Glu81, and Lys68 based upon their hydrogenation states by advanced structural calculations or neutron crystallography analysis on the 11/CK2α complex.

Fig. 5. Crystal Structure of CK2α with 11 (Pink) (PDB ID: 7BU4)

Design, Synthesis and CK2α Inhibitory Activities of Compounds Modified at the Purine Scaffold 9-Position Guided by Virtual Docking Studies

As shown in the docking simulation results presented above, Ar¹ has a CH–π interaction with the hydrophobic residues of the CK2α active site. Therefore, Ar² was set to the 4-carboxyphenyl group and compounds 12–15 were synthesized with an electron-donating group at Ar¹ that would facilitate the CH–π interaction. Conversely, for completeness of the SAR study, compounds 16–19 were synthesized with an electron-withdrawing group that would inhibit the interaction.

As expected, compounds with an electron-donating group (12–14, except for the 3-methoxy group of 15) showed higher potency, while those with electron-withdrawing substitutions (16–19) lost their inhibitory activity (Table 3). Therefore, a favorable interaction between the π-electrons of Ar¹ and the amino acid residues of the ATP pocket was successfully verified.

Table 3. CK2α Inhibitory Activities of Purine Derivatives Based on the Docking Simulation

Compound	Ar1	Ar2	IC50 (µM)
12	4-N,N-Dimethyaminophenyl	4-Carboxyphenyl	4.3
13	4-Methoxyphenyl	4-Carboxyphenyl	9.1
14	4-Methylphenyl	4-Carboxyphenyl	8.9
15	3-Methoxyphenyl	4-Carboxyphenyl	14
16	4-Fluorophenyl	4-Carboxyphenyl	>30
17	4-Chlorophenyl	4-Carboxyphenyl	>30
18	4-Cyanophenyl	4-Carboxyphenyl	>30
19	4-Nitrophenyl	4-Carboxyphenyl	>30

Quantitative Structure–Activity Relationship (QSAR) Study

Based on the above results, a QSAR study was conducted on compounds with an electron-donating group on the Ar¹ phenyl group. Despite the range of activity being within the same order of magnitude, a very narrow window compared to the applicable range of a traditional QSAR study, a qualitative understanding of the changes in activity was obtained. Since the electronic effects of the substituent on the Ar¹ phenyl group were considered to affect the activity, Hammett plots were prepared by plotting Hammett substituent constants (σ)³⁷⁾ of the substituent on Ar¹ versus the pIC₅₀ of the inhibitors (Table 4 and Fig. 6). A clear correlation showing good linearity (R² = 0.987) between the electronic effects of the substituents and the pIC₅₀ was observed. These results also suggest that, in the case of 3-substituted compounds and compounds with electron-withdrawing groups substituted at the 4-position of Ar¹, the relationship between the activity value and the substituent is affected by both electronic and steric factors.

Table 4. QSAR Analysis

Ar1 (Compound)	pIC50 (exp.)	pIC50 (calc.)	σ	SMR†
4-Dimethylaminophenyl (12)	5.37	5.36	−0.83	4.08
4-Methoxyphenyl (13)	5.04	5.07	−0.27	3.30
4-Methylphenyl (14)	5.05	5.01	−0.17	3.12
Phenyl (11)	4.92	4.88	0.00	2.64
3-Methoxyphenyl (15)	4.85	4.97	0.12	3.30

^† SMR value of the substitution group on Ar¹.

Fig. 6. Hammett Plots of pIC₅₀ Values versus σ Values

As a measure of steric bulkiness for a QSAR parameter,^38,39) the Hansch–Fujita method was used to estimate volume and London dispersion effects and a molar refractivity (MR) parameter was calculated. For the Ar¹ of each compound, MRs of the substituent (SMR) were calculated (Table 4) and multiple regression analysis using σ and SMR was performed. The pIC₅₀ (calc.) was obtained using the following equation: pIC₅₀ (calc.) = −0.26 × σ + 0.18 × SMR + 4.40, and a correlation of R² = 0.871 was obtained with the measured value⁴⁰⁾ (Fig. 7). These results suggest that both electronic and steric factors affected Ar¹ group activity.

Fig. 7. Multiple Linear Regression Analysis of σ Values and SMR Combination versus pIC₅₀ Values

Conclusion

In conclusion, we discovered novel purine-based CK2α inhibitors using the solvent dipole ordering-based method for virtual screening and conducted SAR studies. Compounds with a 4-carboxyphenyl group at the 2-position of the purine skeleton showed high activity against CK2α. In many compounds, a correlation was observed between the electron-donating nature of the substituent on the phenyl group at the 9-position and the activity, suggesting both electronic effects and steric factors affect the activity. The predicted and experimental binding poses of 11 validated that the molecular design to strengthen the interaction between the Ar¹ substituent and the surrounding amino acid residues was reasonable. The X-ray crystallographic data for 11 bound to CK2α was in good agreement with the computed docked structure. From the SAR studies presented here, compound 12 was identified as an improved active purine-based CK2α inhibitor with an IC₅₀ of 4.3 µM. We are currently investigating the creation of even more potent inhibitors based on these results.

Experimental

Chemistry

General Methods

All reagents and solvents were of commercial quality and used without further purification. NMR spectra were recorded on JEOL JNM-AI400 (¹H 400 MHz, ¹³C 100 MHz) spectrometers in CDCl₃ with tetramethylsilane as an internal standard. Melting points were taken on J-SCIENCE RFS-10 micro melting point equipment and not corrected. Mass spectra (electrospray ionization (ESI)) were measured by Thermo Fisher Scientific EXECUTIVE Plus. IR spectra were recorded on a Jasco FT/IR-460 Plus spectrophotometer. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. TLC was carried out on a Merck precoated Silica gel 60 F₂₅₄. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. All spectroscopic data for known compounds were in complete accord with literature values.

General Procedure for Purine Analogues

Purine derivatives were synthesized according to the method reported by Proença and colleagues⁴¹⁾ To a stirred solution of diaminomaleonitrile in dry acetonitrile under nitrogen atmosphere, aryl isocyanate was added dropwise in an ice bath, and the reaction was carried out for 24 h. Since the desired product was obtained in an almost pure state, purification was not required. The resulting derivative and aryl aldehyde were stirred in methanol and triethylamine was added to give purine analogues. And the sodium salts of carboxylic acid (10) were then obtained by treating each methanol solution of 10 with 20% NaOH aq.

8-Oxo-2,9-diphenyl-8,9-dihydro-7H-purine-6-carboxamide (3)

Pale yellow solid, 75%, mp 288.4 °C; ¹H-NMR (600 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 7.45–7.48 (m, 3H), 7.49 (t, 1H, J = 7.3 Hz), 7.62 (t, 2H, J = 7.9 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.98 (s, 1H), 8.44–8.46 (m, 2H), 8.50 (s, 1H), 11.78 (s, 1H); ¹³C-NMR (150 MHz, DMSO-d₆) δ: 166.1, 155.1, 153.4, 153.3, 137.3, 133.4, 133.2, 130.6, 129.4, 128.9, 128.4, 128.0, 126.8, 120.4; IR (KBr) cm⁻¹ 3459.67 (s, N–H), 3268.75 (m, N–H), 3217.65 (s, N–H), 751.05 (m, C=O), 1684.52 (s, C=O), 1610.27 (s, N–H); High-resolution negative-ion ESIMS: Calcd for C₁₈H₁₂O₂N₅ [M − H]⁻: 330.0986 Found: 330.0992; High-resolution positive-ion ESIMS: Calcd for C₁₈H₁₄O₂N₅ [M − H]⁺: 332.1142 Found: 332.1132

8-Oxo-9-phenyl-2-(pyridin-4-yl)-8,9-dihydro-7H-purine-6-carboxamide (4)

White solid, 58%, mp 348.7 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.49 (1H, t, J = 7.8 Hz), 7.59 (2H, d, J = 7.8 Hz), 7.74 (2H, d, J = 7.8 Hz), 7.76 (1H, s), 8.29 (2H, d, J = 6.4 Hz), 8.35 (1H, s), 8.68 (2H, d, J = 6.4 Hz), 11.6 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.3, 153.0, 152.9, 150.1, 141.1, 133.2, 132.8, 128.9, 127.9, 126.1, 121.6, 121.4, 121.0; IR (KBr) cm⁻¹ 3449 (m, NH₂), 1687 (s, C=O), 1508 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₇H₁₁O₂N₆ [M − H]⁻: 331.0949 Found: 331.0945.

8-Oxo-9-phenyl-2-(p-tolyl)-8,9-dihydro-7H-purine-6-carboxamide (5)

White solid, 75%, mp 334.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.35 (s, 3H), 7.26 (2H, d, J = 7.8 Hz), 7.48 (1H, t, J = 6.8 Hz), 7.60 (2H, d, J = 7.8 Hz), 7.72 (2H, d, J = 7.8 Hz), 7.97 (1H, s), 8.33 (2H, d, J = 7.8 Hz), 8.48 (1H, s), 11.7 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.6, 154.8, 152.8, 152.7, 139.7, 134.2, 132.9, 132.7, 129.0, 128.9, 127.8, 127.5, 126.3, 119.6, 20.9; IR (KBr) cm⁻¹ 3456 (s, NH), 1686 (s, C=O), 1508 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₄O₂N₅ [M − H]⁻: 334.1142 Found: 344.1148.

2-(4-Fluorophenyl)-8-oxo-9-phenyl-8,9-dihydro-7H-purine-6-carboxamide (6)

Pale yellow solid, 89% yield, mp 324.5 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.28 (2H, t, J = 8.7 Hz), 7.49 (2H, t, J = 7.3 Hz), 7.61 (2H, t, J = 7.8 Hz), 7.72 (2H, d, J = 8.3 Hz), 7.99 (1H, s), 8.51 (2H, d, J = 8.3 Hz), 8.57 (1H, s), 11.8 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.6, 163.6 (J_HF = 245 Hz), 153.8, 152.9, 152.8, 133.4 (J_HF = 2.4 Hz), 132.9, 132.6, 129.9 (J_HF = 8.3 Hz), 129.0, 128.0, 126.3, 119.8, 115.3 (J_HF = 21 Hz)

IR (KBr) cm⁻¹ 3457 (s, NH), 1700 (s, C=O), 1508 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₈H₁₁O₂N₅ [M − H]⁻: 348.0891 Found: 348.0901.

2-(4-Hydroxyphenyl)-8-oxo-9-phenyl-8,9-dihydro-7H-purine-6-carboxamide (7)

White solid, 36%, mp 382.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.83 (2H, d, J = 8.7 Hz), 7.49 (1H, t, J = 7.6 Hz), 7.61 (2H, t, J = 7.8 Hz), 7.71 (2H, d, J = 7.8 Hz), 7.96 (1H, s), 8.29 (2H, d, J = 8.5 Hz), 8.45 (1H, s), 9.84 (1H, s), 11.7 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.9, 159.6, 155.3, 153.0, 152.9, 133.1, 132.9, 129.5, 129.0, 128.0, 127.9, 126.5, 119.1, 115.2; IR (KBr) cm⁻¹ 3470 (m, NH₂), 3334 (m, NH₂), 3247 (m, OH), 1732 (s, C=O), 1688 (m, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₈H₁₄O₃N₅ [M + H]⁺: 348.1097 Found: 348.1091.

2-(4-(Hydroxymethyl)phenyl)-8-oxo-9-phenyl-8,9-dihydro-7H-purine-6-carboxamide (8)

White solid, 45%, mp 385.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.56 (2H, d, J = 5.7 Hz), 4.56 (1H, t, J = 5.7 Hz), 7.84 (1H, t, J = 7.3 Hz), 7.61 (2H, t, J = 7.3 Hz), 7.96 (1H, s), 8.28 (2H, d, J = 8.3 Hz), 8.45 (1H, s), 9.84 (1H, s), 11.7 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.7, 159.4, 155.1, 152.8, 152.7, 132.9, 132.7, 129.3, 128.9, 127.9, 127.8, 126.3, 118.9, 115.1; IR (KBr) cm⁻¹ 3451 (br.m, OH, NH₂), 1653.52 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₄O₃N₅ [M − H]⁻: 360.1091 Found: 360.1101.

Methyl 4-(6-carbamoyl-8-oxo-9-phenyl-8,9-dihydro-7H-purin-2-yl)benzoate (9)

White solid, 74% yield, mp 360.2 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.49 (1H, t, J = 7.8 Hz), 7.59 (2H, d, J = 7.8 Hz), 7.62 (1H, s), 7.74 (2H, d, J = 7.8 Hz), 8.02 (2H, d, J = 8.3 Hz), 8.30 (1H, s), 8.52 (2H, d, J = 8.3 Hz), 11.5 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 166.1, 165.4, 153.9, 153.0, 143.6, 141.3, 133.2, 132.9, 131.0, 129.2, 128.9, 127.9, 127.7, 126.2, 122.1, 51.9; IR (KBr) cm⁻¹ 3453 (s, NH), 1733 (s, C=O), 1687 (s, C=O), 1508 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₀H₁₄O₄N₅ [M − H]⁻: 388.1051 Found: 388.1053.

2-(4-Nitrophenyl)-8-oxo-9-phenyl-8,9-dihydro-7H-purine-6-carboxamide (10)

Pale yellow solid, 84%; mp 357.3 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.49 (t, 1H, J = 7.3 Hz), 7.61 (d, 2H, J = 5.9 Hz), 7.78 (s, 1H) 7.75 (d, 2H, J = 5.9 Hz), 8.26 (d, 2H, J = 8.8 Hz), 8.37 (s, 1H), 8.64 (d, 2H, J = 8.8 Hz), 11.61 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 165.3, 153.0, 152.9, 152.7, 148.6, 142.9, 133.2, 132.8, 128.9, 128.7, 127.9, 126.2, 123.4, 120.7;IR (KBr) cm⁻¹ 3434 (m, C=O), 3349 (m, NH₂), 1682 (s, C=O), 1417 (s, NO₂); High-resolution negative-ion ESIMS: Calcd for C₁₈H₁₁O₄N₅ [M − H]⁻: 375.0842 Found: 375.0843.

4-(6-Carbamoyl-8-oxo-9-phenyl-8,9-dihydro-7H-purin-2-yl)benzoic Acid (11)

White solid, 62%, mp 382.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.45 (1H, t, J = 7.3 Hz), 7.56 (2H, dd, J = 7.3 Hz), 7.66 (2H, d, J = 7.3 Hz), 7.95 (2H, d, J = 8.2 Hz), 7.96 (1H, s), 8.50 (2H, d, J = 8.2 Hz), 8.51 (1H, s), 11.9 (1H, s), 13.0 (bs, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 126.5, 127.6, 128.0, 129.0, 129.4, 131.9, 132.6, 132.6, 132.9, 140.8, 146.8, 148.2, 152.9, 153.5, 165.5, 167.1; IR (KBr) cm⁻¹ 3435.56 (s, NH₂), 3157 (s, COOH), 1703 (s, C=O), 1684 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₂O₄N₅ [M − H]⁻: 374.0895 Found: 374.0893.

4-(6-Carbamoyl-9-(4-(dimethylamino)phenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (12)

Dark solid, 61% yeild, mp >400 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.00 (s, 6H), 6.89 (2H, d, J = 8.8 Hz), 7.43 (2H, d, J = 8.8 Hz), 8.00 (1H, s), 8.01 (2H, d, J = 8.8 Hz), 8.54 (1H, s), 8.55 (2H, d, J = 8.8 Hz), 11.8 (1H, s) 13.0 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 167.1, 165.6, 153.5, 153.4, 153.4, 150.0, 140.9, 132.5, 131.8, 129.4, 127.6, 127.4, 120.8, 120.2, 112.1, 40.1; IR (KBr) cm⁻¹ 3408 (s, COOH), 1717 (s, C=O), 1669 (s, C=O), 1522 (m, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₁H₁₇O₄N₆ [M − H]⁻: 417.1306 Found: 417.1320, High-resolution positive-ion ESIMS: Calcd for C₂₁H₁₉O₄N₆ [M + H]⁺: 419.1462 Found: 419.1461.

4-(6-Carbamoyl-9-(4-methoxyphenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (13)

Pale yellow solid, 50% yeild, mp 377.1 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.86 (s, 3H), 7.16 (2H, d, J = 8.2 Hz), 7.60 (2H, d, J = 8.2 Hz), 8.00 (2H, d, J = 7.8 Hz), 8.01 (1H, s), 8.54 (1H, s), 8.55 (2H, d, J = 7.8 Hz), 11.8 (1H, s), 13.0 (bs, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 167.1, 165.5, 158.7, 153.5, 153.2, 153.1, 140.8, 132.6, 131.8, 129.3, 128.0, 127.5, 125.0, 120.3, 114.2, 55.4; IR (KBr) cm⁻¹ 3448 (s, COOH), 1708 (s, C=O), 1519 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₀H₁₄O₅N₅ [M − H]⁻: 404.1000 Found: 404.1001.

4-(6-Carbamoyl-8-oxo-9-(p-tolyl)-8,9-dihydro-7H-purin-2-yl)benzoic Acid (14)

Brown solid, 30% yield, mp 372.3 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.40 (s, 3H), 7.39 (2H, d, J = 7.8 Hz), 7.56 (2H, d, J = 7.8 Hz), 7.99 (2H, d, J = 6.4 Hz), 8.00 (1H, s), 8.53 (2H, d, J = 6.4 Hz), 8.54 (1H, s), 11.8 (1H, s), 13.0 (bs, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 167.1, 165.5, 153.5, 153.0, 152.9, 140.8, 137.5, 132.8, 131.8, 129.9, 129.5, 129.4, 127.6, 126.3, 120.3, 20.8; IR (KBr) cm⁻¹ 3227 (s, COOH), 1757 (s, C=O), 1711 (s, C=O), 1611 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₀H₁₄O₄N₅ [M − H]⁻: 388.1051 Found: 388.1054.

4-(6-Carbamoyl-9-(3-methoxyphenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (15)

Pale yellow solid, 80%, mp 362.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.83 (s, 3H), 7.08 (1H, dd, J = 1.9 Hz, J = 8.2 Hz), 7.34–7.30 (m, 2H), 7.52 (1H, t, J = 8.2 Hz), 8.02 (2H, d, J = 8.7 Hz), 8.03 (1H, s), 8.55 (1H, s), 8.56 (2H, d, J = 8.7 Hz), 11.8 (1H, s), 13.0 (bs, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 167.0, 165.4, 159.5, 153.5, 152.8, 152.7, 140.7, 133.6, 132.8, 131.8, 129.7, 129.4, 127.5, 120.3, 118.5, 113.7, 112.2, 55.4; IR (KBr) cm⁻¹ 3449 (s, COOH), 1742 (s, C=O), 1684 (s, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₀H₁₄O₅N₅ [M − H]⁻: 404.1000 Found: 404.1000.

4-(6-Carbamoyl-9-(4-fluorophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (16)

Pale yellow solid, 67%, mp 379.9 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.47 (dd, 2H, J = 8.7 Hz, J_HF = 6.8 Hz), 7.77 (dd, 2H, J = 9.2 Hz, J_HF = 5.3 Hz), 8.01 (d, 2H, J = 8.2 Hz), 8.02 (s, 1H), 8.56 (d, 2H, J = 8.2 Hz), 8.57 (s, 1H), 11.8 (s, 1H), 13.0 (bs, 1H)

¹³C-NMR (100 MHz, DMSO-d₆) δ: 167.0, 165.4, 162.4, 159.9, 153.5, 152.9, 140.7, 132.8, 131.8, 129.4, 128.7, 127.6, 120.3, 116.0, 115.8; IR (KBr) cm⁻¹ 3448.10 (s, COOH), 1753.94 (s, C=O), 1708.62 (s, C=O), 1515.77 (s, C=O), 1239.04 (s, F); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₁O₄N₅F [M − H]⁻: 392.0801 Found: 392.0801

4-(6-Carbamoyl-9-(4-chlorophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (17)

Pale yellow solid, 30%, mp 374.4 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.64 (2H, d, J = 8.2 Hz), 7.65 (bs, 1H), 7.81 (2H, d, J = 8.2 Hz), 8.01 (2H, d, J = 8.2 Hz), 8.21 (bs, 1H), 8.49 (2H, d, J = 8.2 Hz), 11.5 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 120.5, 127.6, 128.1, 129.1, 129.5, 131.6, 132.0, 132.3, 133.1, 136.5, 140.7, 152.8, 153.5, 165.5, 167.2; IR (KBr) cm⁻¹ 3691 (s, COOH), 1758 (s, C=O), 1707.66 (m, C=O), 1093 (s, Cl); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₁O₄N₅Cl [M − H]⁻: 408.0505 Found: 408.0504.

4-(6-Carbamoyl-9-(4-cyanophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (18)

Pale orange solid, 24%, mp 394.6 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 8.02 (2H, d, J = 8.3 Hz), 8.03 (1H, s), 8.04 (2H, d, J = 8.3 Hz), 8.12 (2H, d, J = 8.3 Hz), 8.59 (2H, d, J = 8.3 Hz), 8.60 (1H, s), 12.02 (1H, s), 13.08 (1H, s); IR (KBr) cm⁻¹ 3217 (s, COOH), 2234 (s, CN), 1751 (m, C=O), 1706 (m, C=O), 1516 (m, C=O); High-resolution negative-ion ESIMS: Calcd for C₂₀H₁₁O₄N₆ [M − H]⁻: 399.0836 Found: 399.0848.

4-(6-Carbamoyl-9-(4-nitrophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)benzoic Acid (19)

Pale orange solid, 35%; mp 376.1 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.81 (1H, s), 8.03 (2H, d, J = 8.3 Hz), 8.20 (2H, d, J = 8.3 Hz), 8.35 (1H, s), 8.47 (2H, d, J = 8.3 Hz), 8.56 (2H, d, J = 8.3 Hz), 11.75 (1H, s); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 166.5, 164.8, 153.6, 152.0, 151.7, 145.7, 140.1, 138.2, 133.4, 131.9, 128.9, 127.2, 125.8, 123.8, 120.0

IR (KBr) cm⁻¹ 3447 (m, COOH), 1761 (m, C=O), 1684 (s, C=O), 1521 (s, C=O), 1351 (s, NO₂); High-resolution negative-ion ESIMS: Calcd for C₁₉H₁₁O₆N₆ [M − H]⁻: 419.0735 Found: 419.0747.

CK2α Kinase Assay by Off-Chip Mobility Shift Assay Method

CK2α inhibitory activities were evaluated by the off-chip mobility shift assay by the QuickScout service from Carna Bioscience (Kobe, Japan). Full-length human CK2α1 [1–391(end) amino acids of accession number NP_001886.1] was co-expressed as N-terminal glutathione-S-transferase (GST)-fusion protein (72 kDa) with human His-tagged CK2β [1–215 amino acids of accession number NP_001311.3] using baculovirus expression system. GST-CK2α1 was purified by using glutathione sepharose chromatography. Each chemical in DMSO at different concentrations was diluted fourfold with reaction buffer [20 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.5), 0.01% Triton X-100, 2 mM dithiothreitol]. For CK2 reactions, a combination of the compound, 1 µM CK2tide, 5 mM MgCl₂, 5 µM ATP in reaction buffer (20 µL) were incubated with each CK2 in PP 384-well plates at room temperature for 1 h (n = 2). The reaction was terminated by the addition of 70 µL of termination buffer (Carna Biosciences). Substrate and product were separated by electrophoretic means using the Lab-Chip3000 system (PerkinElmer, Inc., U.S.A.). 4,5,6,7-Tetrabromo-1H-benzotreazole (TBB) was used as a positive control. The kinase reaction was evaluated by the product ratio, which was calculated from the peak heights of the substrate (S) and product (P): [P/(P + S)]. Inhibition data were calculated by comparing with no-enzyme controls for 100% inhibition and no-inhibitor reactions for 0% inhibition. IC₅₀ values were calculated using GraphPad Prism 9 software.

Computational Chemistry

Descriptor Calculation for pIC₅₀ Prediction

The molar refractivity (MR) parameters were calculated atom-based method⁴⁰⁾ implemented in Chemical Computing Group’s Molecular Operating Environment (MOE). Mulliken charges were calculated using Gaussian09 with HF/6–31G* level.

Modeling and Docking Simulation

The crystal structure of a complex of CK2α and 4-[2-[(4-methoxyphenyl)carbonylamino]-1,3-thiazol-5-yl]benzoic acid was retrieved from the Protein Data Bank (PDB ID: 5B0X).³³⁾ Modeling was performed in Protonate 3D implemented in MOE, and only the carboxy group of Glu81 in the active site was manually modified to COO⁻.

Docking models for 11 to protein structure with/without the conserved crystalline water molecule were constructed using MOE dock. Geometry optimization was performed on the candidate 100 structures with good London dG scores, and each structure with the best interaction energy was adopted as the binding structure model with/without the water molecule.

Crystallization Procedure and Structure Determination of the Complex

The structure of CK2α complexes with 11 was determined by the similar procedures used for the structure determination of CK2α complex reported previously.²³⁾ In this study, the C-terminal truncated form of CK2α was cloned into the pGEX6P-1 expression vector (GE Healthcare, England) and expressed in Escherichia coli strain HMS174 (DE3) as a GST-fused protein at the N-terminus. The GST tag was cleaved by PreScission protease (GE Healthcare) on the column at 277 K for 18 h. The CK2α protein lacking the GST tag was eluted with 25 mM Tris–HCl pH 8.5, 1 mM dithiothreitol. The protein was purified by the Glutathione sepharose 4B resin (GE Healthcare) and the MonoQ columns using an AKTA explorer system (GE Healthcare). Crystals of the CK2α complex were prepared by soaking with 11 according to published methods⁴²⁾ X-ray diffraction data sets were collected at the BL44XU beamline of the SPring-8 at 95 K. The data sets were processed with the program XDS package.⁴³⁾ The structures of the complexes were solved by the molecular replacement method, carried out with the program Phaser⁴⁴⁾ in Phenix⁴⁵⁾ using the 3WAR⁴⁶⁾ structure as a starting model. All refinements and model modifications were performed using the programs WinCoot⁴⁷⁾ and Phenix. Data collection and refinement statistics are shown in Table 5. The coordinates of the CK2α complex with compound 11 have been deposited in the Protein Data Bank with the accession code 7BU4.

Table 5. Crystallographic Data for the CK2α -11 Complex (PDB Code: 7BU4)

Data collection		Refinement statistics
Space group	P212121	Resolution (Å)	41.4–1.70 (1.74–1.70)
Unit cell (Å)	a = 48.27	Reflections	35474 (2346)
	b = 82.82	Total atoms	3037
	c = 79.132	RMSD bond lengths (Å)	0.007
Observations	231927	RMSD bond angles (°)	1.045
Unique reflections	35489	R-factor (%)	19.2 (29.3)
Resolutions (Å)	50.07–1.70 (1.81–1.70)	Rfree (%)	22.2 (30.4)
Completeness (%)	99.9 (99.8)
Rmerge (%)a)	8.8 (87.6)
I/σ	16.9 (2.1)

Values in parentheses are for the highest-resolution shell. a) R_merge = Σ_hΣ_j|I_hj− < I_h > |/Σ_hΣ_j|I_hj|, where h represents a unique reflection and j represents symmetry-equivalent indices. I is the observed intensity and < I > is the mean value of I.

Acknowledgments

This study was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1411037, 2014–2018) and Antiaging center, Kindai University. Diffraction data collection was carried out on the Osaka University beamline BL44XU at SPring-8 (Proposal No. 2019A6913).

Conflict of Interest

The authors declare no conflict of interest.

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