Pyrimidine Carboxylic Acids Linked through 1,4,5-Trisubstituted 1,2,3-Triazoles: Synthesis and RNase A Inhibition Studies

Abstract

A series of triazolylated nucleoside carboxylic acid derivatives have been synthesized using a 1,3-dipolar cycloaddition reaction. The ribonuclease A (RNase A) inhibitory properties of the synthesized molecules have been studied by experimental and theoretical techniques. The compounds were found to inhibit RNase A in a reversible competitive manner. The inhibitory potency was significantly enhanced by the incorporation of a larger number of carboxylic acid groups in the inhibitor. A theoretical justification of the observed results was provided by an investigation of the docked structures of the enzyme–inhibitor complexes.

Key words

pyrimidinecarboxylic acids - triazoles - ribonuclease A - 1,3-dipolar cycloaddition - enzyme inhibitor - docking studies

• References and Notes

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For the biological importance of 1,2,3-triazoles, see:
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• 7 Triazolylation Reaction: General Procedure A well-stirred solution of the appropriate azide (1 mmol) and DMAD (1.5 mmol) in CCl₄ (5 mL) was refluxed for 24 h (see the SI for details).
• 8 Ester Hydrolysis: General Procedure Compound 7, 11, 15, or 18 was hydrolyzed under basic conditions at 0 °C. The resulting residue was dissolved in H₂O, and the solution was neutralized with acidic Amberlite resin (see the SI for details).
• 9 Compound 8 Prepared by the general procedure from compound 7 (0.23 g, 0.56 mmol) as a white solid; yield: 0.17 g (78%); mp 196–198 °C. ¹H NMR (200 MHz, DMSO-d₆ ): δ = 1.82 (s, 3 H), 2.02–2.19 (m, 2 H), 4.22–4.30 (m, 2 H), 4.95–5.18 (m, 2 H), 6.15 (t, J = 7.4 Hz, 1 H), 7.55 (s, 1 H), 11.30 (s, 1 H). ¹³C NMR (50 MHz, DMSO-d₆ ): δ = 12.0, 38.0 (CH₂), 51.3 (CH₂), 71.1, 84.3 (2 C), 109.7, 132.9, 136.1, 140.6, 150.4, 159.2, 161.2, 163.7. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₆N₅O₈: 382.0999; found: 382.0887. Compound 12 Prepared by the general procedure from 11 (0.18 g, 0.44 mmol) as a white solid; yield: 0.11 g (64%); mp 118–120 °C. ¹H NMR (600 MHz, DMSO-d ₆): δ = 3.17 (s, 1 H), 4.01–4.02 (t, J = 4.8 Hz, 1 H), 4.15–4.16 (t, J =5.4 Hz, 1 H), 4.19–4.21 (m, 1 H), 5.62–5.63 (d, J = 8.4 Hz, 1 H), 5.72–5.73 (d, J = 5.4 Hz, 1 H), 7.61–7.63 (m, 2 H), 11.34 (s, 1 H). ¹³C NMR (50 MHz, DMSO-d ₆): δ = 51.3 (CH₂), 70.7, 72.2, 81.8, 88.6, 102.0, 132.5, 140.3, 141.2, 150.7, 159.3, 161.4, 163.1. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₃H₁₄N₅O₉: 384.0792; found: 384.0796. Compound 16 Prepared by the general procedure from 15 (0.09 g, 0.22 mmol) as a white solid; yield: 0.05 g (62%); mp 200 °C. ¹H NMR (600 MHz, DMSO-d ₆): δ = 3.59–3.62 (m, 1 H), 3.69–3.72 (m, 1 H), 4.29–4.32 (m, 1 H), 4.73 (t, J = 4.2 Hz, 1 H), 5.63 (d, J = 7.8 Hz, 1 H), 5.79–5.81 (m, 1 H), 6.34 (d, J = 4.8 Hz, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 11.33 (s, 1 H). ¹³C NMR (150 MHz, DMSO-d ₆): δ = 61.0 (CH₂), 66.2, 75.0, 82.5, 85.7, 100.7, 133.4, 140.8, 142.8, 150.9, 159.7, 161.6, 163.8. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₃H₁₄N₅O₉: 384.0792; found: 384.0786. Compound 19 Prepared by the general procedure from 18 (0.25 g, 0.43 mmol) as a white solid; yield: 61%; mp 196–198 °C. ¹H NMR (400 MHz, DMSO-d ₆): δ = 1.83 (s, 3 H), 2.76–2.80 (m, 2 H), 4.76–4.78 (m, 1 H), 5.15–5.18 (m, 1 H), 5.25–5.31 (m, 1 H), 6.31 (br s, 2 H), 6.45 (t, J = 6.8 Hz, 1 H), 7.61 (s, 1 H), 11.35 (s, 1 H). ¹³C NMR (100 MHz, DMSO-d ₆): δ = 12.1, 36.1 (CH₂), 51.7 (CH₂), 60.5, 82.1, 85.1, 110.0, 132.5, 132.6, 136.3, 140.3, 140.7, 150.4, 159.2, 161.3, 161.4, 163.7 (2 C). HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₈H₁₆N₈NaO₁₁: 543.0836; found: 543.0765.
• 10 Comparative Agarose Gel Assay^{3b – d} A 1 μM aqueous solution of RNase A (20 μL) was mixed with a 0.5 mM aqueous solution of compound 4, 8, 12, 16, or 19 (20 μL), and the mixture was adjusted to a final volume of 50 μL and incubated for 3 h. An aliquots of the incubated mixture (20 μL) was then mixed with a freshly prepared 10.0 mg/mL solution of RNA in RNase-free water (20 μL), and the mixture was incubated for another 30 min. Then, 10 μL of a sample buffer containing 10% glycerol and 0.025% bromophenol blue was added, and a 15 μL aliquot of the resulting mixture was loaded onto a 1.1% agarose gel. The gel was run using 0.04 M Tris–acetic acid–EDTA (TAE) buffer (pH 8.0), and the residual RNA was visualized by staining with ethidium bromide and viewed under a UV lamp.
• 11 Inhibition Kinetics of RNase A The assay¹² was performed in a 0.1 M Mes–NaOH buffer (pH 6.0) containing 0.1 M NaCl with 2′,3′-cCMP as the substrate. The inhibition constants were calculated from the initial velocity data by using a Lineweaver–Burk plot. The slopes from the Lineweaver–Burk double reciprocal plot were plotted against the corresponding inhibitor concentrations to obtain the inhibition constants (K _i).
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• 13 FlexX Docking The crystal structure of RNase A was downloaded from the Protein Data Bank (Accession number: 1FS3).¹⁴ The 3D structures of compounds 4, 8, 12, 16, and 19 were generated in Sybyl 6.92 (Tripos Inc., St. Louis, USA), and their energy-minimized conformations were obtained with the help of the MMFF94 force field using MMFF94 charges with a gradient of 0.005 kcal/mole by 1000 iterations with all other parameters set to the default values. The FlexX software, part of the Sybyl suite, was used for docking of the ligands to the protein. Ranking of the generated solutions was performed by using a scoring function that estimates the free binding energy (ΔG) of the protein–ligand complex considering various types of molecular interactions.¹⁵ Docked conformations were visualized by using PyMol.¹⁶
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• 16 https://github.com/schrodinger/pymol-open-source (accessed Jun 4, 2024)