Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens

Eur J Med Chem. 2024 Jan 15:264:115971. doi: 10.1016/j.ejmech.2023.115971. Epub 2023 Nov 29.


Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Keywords: Breast cancer; Cell viability; Dihydrofolate reductase; Enzyme inhibition; In silico docking; Inhibitor development; Pyrimethamine; Target engagement.

MeSH terms

  • Antineoplastic Agents* / chemistry
  • Antineoplastic Agents* / pharmacology
  • Biology
  • Folic Acid Antagonists* / chemistry
  • Folic Acid Antagonists* / pharmacology
  • Humans
  • Methotrexate / pharmacology
  • Neoplasms*
  • Pyrimethamine / pharmacology
  • Tetrahydrofolate Dehydrogenase / chemistry


  • Pyrimethamine
  • Folic Acid Antagonists
  • Methotrexate
  • Antineoplastic Agents
  • Tetrahydrofolate Dehydrogenase