We have completed mechanism of action studies on a new class of anticancer nucleosides typified by a novel nucleoside discovered in our lab, MAP-870. In order to study the mechanism of MAP-870, several experiments were completed on a colorectal adenocarcinoma cell line, HT-29, including trypan blue cell count, sulforhodamine B assays, flow cytometry of cell cycle, propidium iodide incorporation, and phosphatidylserine externalization, Caspase-Glo3/7 assays, DNA fragmentation gel, cyclophilin A release gel, PAMPA, and confocal imaging. Sulforhodamine B assays show that MAP-870 does indeed cause growth inhibition and cell death in the model tested. PAMPA assays show that MAP-870 does not appear to enter the cell via passive diffusion. Flow cytometry showed that MAP-870 doe not appear to cause cell cycle arrest or externalization of phosphatidylserine. Caspase-Glo3/7 assays demonstrated that MAP-870 does not appear to cause caspase activation. From confocal microscopy, it appears preliminarily that MAP-870 is taken up by cells, often through pseudopodia. The mechanism of MAP-870 on cancer cells must be further studied to elucidate its mechanism of action. However, preliminarily our data could point to TGFβ as a potential target pathway involving a unique, heretofore never described, cell death mechanism.

Recently the Peterson lab discovered a promising anticancer adenosine derivative-- 2´,3´-bis-O-tert-butyldimethylsilyl-5´-deoxy-5´-[N-(methylcarbamoyl)amino]-N6-(N-phenylcarbamoyl)adenosine. This compound showed selective toxicity against human colon cancer cells in vitro with LC50's = 6--10 µM. It was hypothesized that the lead compound exerted its cytotoxic effects by interacting with a protein kinase. A systematic Structure Activity Relationship (SAR) was undertaken in an attempt to increase the kinase-binding affinity of the lead compound. Many regions of the lead compound were examined: the N6-phenyl urea moiety, the 5´-N-methyl urea group, the 2´,3´-bis-O-TBS groups, the nucleobase, and the ribose sugar. Results of these studies produced some promising new derivatives. In particular, one analogue exhibited potent cancer cell growth inhibition with an average GI50 of 0.58 μM (NCI-60). In addition, another compound showed selective toxicity for the non-small cell adenocarcinoma cell line NCI-H522 with an LC50 of 10 nM. Efficient methods for the preparation of a wide variety of N6-aryl and -alkyl substituted derivatives were developed. One versatile route involved the installation of an N6-ethoxy carbonyl and subsequent displacement with an alkly- or arylamine. Synthetic routes for the preparation of of a variety of 2´,3´-bis-O-acylated analogues were also developed. Nucleoside mono-, di-, and triphosphate bioisosteres in which the phosphoester or phosphoanhydride have been replaced by an unnatural functional group have been extensively investigated. A simple and efficient method was developed for the preparation of carbamoyl analogues of nucleoside mono-, di-, and triphosphate surrogates. This method uses a modified version of the Kočovský reaction to install mono-, di-, and triphosphate mimics in good to excellent yields (ave = 75%).