Synlett
DOI: 10.1055/a-2602-6763
letter
Small Molecules in Medicinal Chemistry

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

Kaustav Chakraborty
,
Swagata Dasgupta
,

The authors thank the Department of Biotechnology, Ministry of Science and Technology, New Delhi for funding (project no. SB/S1/OC-30/2014).


Preview

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.

Supporting Information



Publication History

Received: 21 February 2025

Accepted after revision: 07 May 2025

Accepted Manuscript online:
07 May 2025

Article published online:
07 July 2025

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  • References and Notes

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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 Assay3b 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).
  • 12 Anderson DG, Hammes GG, Walz FG. Biochemistry 1968; 7: 1637
  • 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
  • 14 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. Nucleic Acids Res. 2000; 28: 235
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  • 16 https://github.com/schrodinger/pymol-open-source (accessed Jun 4, 2024)