Structure-based design of AP23573, a phosphorus-containing analog of rapamycin for anti-tumor therapy.

2476 The mTOR protein, a PI3K-like kinase that links mitogenic stimuli and nutrient status to cell cycle progression, has emerged as an important target for anti-tumor therapy. Rapamycin is a natural product mTOR inhibitor that functions by inducing an inhibitory complex between the protein and cytoplasmic FKBP. The compound potently inhibits the proliferation of a wide variety of human tumor cell lines. We have used structure-based drug design to guide the synthesis of analogs of rapamycin that have optimal pharmacological properties for anti-tumor use. Computational modeling studies and previous SAR analyses indicated the possibility of replacing the C-43 secondary alcohol moiety of the cyclohexyl group of rapamycin with substituted phosphonate and phosphinate groups with retention of high-affinity binding to FKBP and mTOR. In particular, modeling indicated the existence of multiple surface residues with the potential to form favorable binding interactions with such C-43 substituents. In this study, we synthesized and evaluated a series of phosphorus-containing C43-modified rapamycin analogs. Semi-synthesis of the compounds from rapamycin using either one- or two-step conversions proceeded with high yield (>80%). Compounds were tested for FKBP binding affinity using a fluorescence polarization assay, and for the ability to inhibit the proliferation of human tumor-derived cell lines. Most of the compounds were found to have minimal reductions (less than 3-fold) in FKBP binding affinity, as predicted. In addition, the compounds inhibited the proliferation of the human fibrosarcoma cell line HT1080 with IC50 values of 0.1 –3 nM. Several of the compounds were equipotent to rapamycin in this assay (IC50 = 0.1 nM). Full details of structures and structure-activity relationships will be presented. AP23573, one of the most potent compounds, was selected for further study and development. The C43 modification of AP23573 was found to be stable in organic solvents, aqueous solutions at a variety of pHs, and in plasma and whole blood, both in vitro and in vivo. These stability studies along with in vitro metabolism studies also indicated that AP23573 is not a rapamycin pro-drug for rapamycin. AP23573 has shown potent inhibition of diverse human tumor cells lines in vitro and as xenografts implanted into nude mice. The compound is now being evaluated in phase 1 clinical trials in cancer patients.