The pyrrolo benzodiazepines PBDs are a
The pyrrolo[2,1-][1,4]benzodiazepines (PBDs) are a family of antitumour antibiotics that includes the natural products anthramycin and DC-81. They exert their cytotoxicity by covalently bonding to the exocyclic C2–NH group of guanine residues in the minor groove of DNA through their N10–C11 imine functionality. This leads to a number of biological effects including the inhibition of transcription, and of enzymes binding to cognate sites., The PBD monomers have significant in vitro cytotoxicity, and it has been demonstrated that joining two PBD moieties through a linker (via their C8-positions) leads to PBD dimers capable of interstrand DNA cross-linking., , One example of a PBD dimer, SJG-136 (, ), is now being evaluated in Phase 1 clinical trials., One interesting property of PBD dimers is that the interstrand cross-linked adducts they form in the minor groove of DNA appear to be highly resistant to repair,, and it is this feature that may lead to ADEPT prodrugs with distinct advantages over ZD2767P (, ). To explore the potential of using these extremely cytotoxic molecules in prodrug systems, we initially demonstrated the possibility of converting PBD monomers into nitroreductase-sensitive prodrugs. We now report the design, synthesis and evaluation of four model self-immolative CPG2 PBD prodrugs (, and ,, ) formed from the PBD monomer and dimer (Pentamethylene linker between PBD units), respectively, potentially suitable for use in CPG2-based ADEPT therapy. Prodrugs of this type should have an advantage over existing mustard-based prodrugs both in terms of potency and/or resistance to DNA repair. The new agents (, and ,) are prodrugs of the known PBD monomer () and dimer (), respectively, where the DNA-interactive N10–C11 functionality necessary for biological activity is masked with an -glutamic GDC-0941 CPG2 substrate attached through either a carbamate or ureidic linkage (i.e., X=O or NH, respectively) to the 4′-position of an N10-benzyloxycarbonyl PBD. In the case of and , cleavage of the substrate by CPG2 releases either a 4′-hydroxy- or 4′-aminobenzyloxycarbonyl intermediate (X=O or NH, respectively) which then undergoes 1,6-elimination to release the cytotoxic PBD (). The PBD dimer prodrugs and release PBD dimer by an identical mechanism, except that two N10-progroups are released rather than one. The known carbamate and urea progroups (X=O) and (X=NH) were prepared by modification of literature procedures, allowing the preparation of gram quantities of each (). These were attached to the monomer and dimer PBD precursors and , prepared using standard methodology developed in our laboratory (, ). This was accomplished by first converting the PBD precursors to isocyanates in situ which were then reacted with the progroups to give the carbamates (,) and ureas (,), respectively. De-protection of the silyl ethers, then cyclisation using either pyridinium dichromate or diacetoxyiodobenzene/TEMPO followed by final de-protection of the diallyl esters using Pd(PPh) gave the target compounds , and , in good yields. Stability studies were performed using high-performance liquid chromatography. Both prodrug/parent pairs (, and ; , and ) could be readily separated and detected using a Phenomenex column (C18 5μM, 25cm×0.46cm) with a mobile phase of HO (with 0.1% trifluoroacetic acid)/acetonitrile 70:30 (1ml/min) and detection at 254nM. Stability was assessed over a 24h period in distilled water at both room temperature and 37°C. Differences in the rate of conversion of prodrugs to parent PBDs were observed between the carbamate and urea series. The urea prodrugs and were unstable at both room temperature and 37°C, and were almost completely converted into the parent PBDs within 24h. Conversely, the carbamate prodrugs and were relatively stable at room temperature and underwent only 7% and 3% conversion, respectively, after 24h at 37°C.