Synthesis and evaluation of new peptide-linked doxorubicin conjugates as prodrugs activated by prostate-specific antigen

The targeted delivery of cytotoxic agents to prostate cancer cells via selective activation of peptide-linked prodrugs by prostate-specific antigen (PSA) has been previously demonstrated. In our continued efforts to design prodrugs with improved prostate tumor specificity, we developed GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln as promoieties with enhanced PSA specificity starting from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln. Based on their PSA cleavage rates and resistance to non-PSA-mediated hydrolysis in plasma, GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln were selected as optimal promoieties and coupled to doxorubicin (Dox) as PSA-targeted prodrugs, using Ser-Leu linkers. Following 72-h incubations with Dox prodrugs, there was insignificant cytotoxicity in non-PSA-producing DU145 cells. The Dox prodrugs, glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I), glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II) and GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III) demonstrated comparable PSA cleavage rates (t_1/2 values <23 min), and were cytotoxic against PSA-producing LNCaP cells with IC₅₀ values of 0.18, 0.27 and 0.082 μM, respectively. To mitigate neprilysin-mediated hydrolysis of the Ser-Leu linker in prodrugs I–III and further improve PSA specificity, 3-aminooxypropionate was incorporated between the promoiety and Dox (prodrug IV). Despite its slower PSA cleavage rate (t_1/2 value of 67 min), GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH₂-C(Me)₂C(O)-14-O-Dox (IV) was equipotent (IC₅₀ value of 0.19 μM) to prodrug I at killing PSA-producing LNCaP cells due to its ability to release free Dox through a cyclization activation mechanism. Further metabolism and PK/PD studies will be conducted to evaluate the tumor specificity of the novel Dox prodrugs (II–IV) reported herein. [Figure not available: see fulltext.]