Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers

Mol Pharm. 2009 Sep-Oct;6(5):1604-11. doi: 10.1021/mp900084v.


We evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers in order to find a suitable linker for prodrugs of carboxylic acids with amino acids. l-Valine and l-phenylalanine prodrugs of model compounds (benzoic acid and phenyl acetic acid) containing methoxy, ethoxy and propylene glycol linkers were synthesized. The hydrolysis rate profile of each compound was studied at physiologically relevant pHs (1.2, 4, 6 and 7.4). Enzymatic hydrolysis of propylene glycol containing compounds was studied using Caco-2 homogenate as well as purified enzyme valacyclovirase. It was observed that the stability of the prodrugs increases with the linker length (propyl > ethyl > methyl). The model prodrugs were stable at acidic pH as compared to basic pH. It was observed that the prodrug with the aliphatic amino acid promoiety was more stable compared to its aromatic counterpart. The comparison between benzyl and the phenyl model compounds revealed that the amino acid side chain is significant in determining the stability of the prodrug whereas the benzyl or phenyl carboxylic acid had little or no effect on the stability. The enzymatic activation studies of propylene glycol linker prodrug in the presence of valacyclovirase and cell homogenate showed faster generation of the parent drug at pH 7.4. The half-life of prodrugs at pH 7.4 was more than 12 h, whereas in the presence of cell homogenate the half-lives were less than 1 h. Hydrolysis by Caco-2 homogenate generated the parent compound in two steps, where the prodrug was first converted to the intermediate, propylene glycol benzoate, which was then converted to the parent compound (benzoic acid). Enzymatic hydrolysis of propylene glycol containing prodrugs by valacyclovirase showed hydrolysis of the amino acid ester part to generate the propylene glycol ester of model compound (propylene glycol benzoate) as the major product. The amino acid prodrugs containing methoxy linker were the least stable while prodrugs containing propylene glycol linker were most stable. This work suggests that the propylene glycol linker is an optimal linker for amino acid prodrugs since it has good chemical stability and is enzymatically hydrolyzed to yield the parent drug. This approach can be further extended to other non-amino acid prodrugs and to provide a chemical handle to modify lead molecules containing carboxylic group(s).

MeSH terms

  • Amino Acids / chemical synthesis
  • Amino Acids / chemistry*
  • Amino Acids / metabolism*
  • Animals
  • Caco-2 Cells
  • Carboxylic Ester Hydrolases / metabolism
  • Drug Carriers / chemical synthesis
  • Drug Carriers / chemistry
  • Drug Carriers / metabolism
  • Drug Evaluation, Preclinical
  • Drug Stability
  • Half-Life
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • In Vitro Techniques
  • Intestinal Mucosa / metabolism
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Prodrugs / chemical synthesis
  • Prodrugs / chemistry*
  • Prodrugs / metabolism*
  • Rats


  • Amino Acids
  • Drug Carriers
  • Prodrugs
  • BPHL protein, human
  • Carboxylic Ester Hydrolases