Synthesis and evaluation of phosphoramide mustard prodrugs for site-specific activation
Creators: name: Surabhi, Prathima
Publisher: name: No Publisher Supplied
Year: 2007

description: Advanced cancers after metastasis need to be treated systemically using chemotherapy or radiation, which are often associated with debilitating side effects. To improve the selectivity of the chemotherapeutic agents and reduce systemic toxicity, prodrugs with tumor targeted activation mechanisms are being developed. Efforts in this project were focused on designing  phosphoramide mustard analogues incorporating two types of tumor-specific activation mechanisms: one by the reductive activation mechanism by  Escherichia coli nitroreductase and the other by the proteolytic activation mechanism by prostate-specific antigen (PSA) in prostate cancer cells. 5-Nitropyridyl-2-methyl phosphoramide mustard was designed and synthesized to be a substrate of E. coli nitroreductase. In cell culture assays it showed an IC50 value of 9 nM in Chinese hamster V79 cells transfected to express E. coli nitroreductase as compared to 75 ;M in the control V79 cells that do not express E. coli nitroreductase. These results suggest that 5-nitropyridyl-2-methyl phosphoramide mustard is a good candidate for use in combination with nitroreductase for enzyme prodrug therapy.  For proteolytic activation by (PSA) in prostate cancer cells, we designed a peptide conjugate by linking a substrate peptide sequence to phosphoramide mustard through a flexible linker that can be modified to better accommodate the unique structural requirements for binding to and catalysis by the target enzyme and thus improve the tumor selectivity of phosphoramide mustard. Pyridine was introduced to modulate the electron-density, to optimize the stability and rate of 1,6-elimination upon proteolytic cleavage. A new set of conditions were developed for selenocarboxylate/azide amidation reaction in order to avoid the use of unstable basic nuclephilic amine intermediates  during the synthesis of the peptide conjugated analogue. Under the new amidation conditions, the half-life of the selenocarboxylate was increased by 27-fold at room temperature as compared to the previous amidation method using DMSO as a co-solvent. Excellent yields were obtained with electron-deficient azides and much improved yields were obtained with electron-rich azides upon mild heating. The peptide conjugate, glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-pyridyl-2-methyl phosphoramide mustard, was successfully synthesized using our new amidation strategy and was found to be stable with a half-life &gt;5 days at pH 7.4 and 8.0. It was shown to be a good substrate for PSA with a half-life of 46.8 min at an enzyme/substrate molar ratio of 1/100. However, the peptide conjugate did not show any selective toxicity towards PSA-expressing LNCaP cells with IC50 values of 209 ;M and 204 ;M against PSA-expressing LNCaP and non-PSA-expressing DU 145 cell lines, respectively. The low cytotoxicity against LNCaP cells may be due to the inability of the released species to penetrate the tumor membranes. Further studies are underway to identify the activated species released and to design approaches to improve its membrane permeability.