N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis, Characterization, and Decomposition Profile Under Physiological Conditions

J Pharm Sci. 2018 Nov;107(11):2927-2937. doi: 10.1016/j.xphs.2018.06.019. Epub 2018 Jun 27.


(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2'S,3'S)-N-7-(3',4'-epoxy-2'-hydroxybut-1'-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2'S,3'S)-N-7-(2'3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a β-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.

Keywords: HPLC; NMR spectroscopy; adducts; chemical stability; degradation products; kinetics; mass spectrometry; prodrugs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Alkylating / chemical synthesis
  • Antineoplastic Agents, Alkylating / chemistry*
  • Busulfan / analogs & derivatives*
  • Busulfan / chemical synthesis
  • Busulfan / chemistry
  • Chromatography, High Pressure Liquid
  • Drug Stability
  • Guanine / analogs & derivatives*
  • Guanine / chemical synthesis
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Intercalating Agents / chemical synthesis
  • Intercalating Agents / chemistry*
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Prodrugs / chemical synthesis
  • Prodrugs / chemistry*


  • Antineoplastic Agents, Alkylating
  • Intercalating Agents
  • Prodrugs
  • Guanine
  • treosulfan
  • Busulfan