Deuterium-labeled analogs of the topoisomerase inhibitor batracylin were prepared for metabolism studies to further its evaluation as an antitumor agent. Established syntheses of unlabeled batracylin were adapted for the preparation of deuterated batracylin that was trideuterated in the quinazoline ring (d3-batracylin 5), tetradeuterated in the isoindolo ring (d4-batracylin 11), and heptadeuterated in both rings (d7-batracylin 12). Extensive exchange of deuterium or hydrogen in the quinazoline ring was observed from an intermediate in the final concentrated sulfuric acid promoted deblocking/cyclodehydration step of the synthesis. Introduction of deuterated concentrated sulfuric acid in the final step both retained the label in the quinazoline-labeled product and enabled extended labeling of a more exhaustively deuterated analog. Batracylin itself did not readily exchange aromatic protons under the reaction conditions but did loose and scramble deuterium atoms during mass spectral analysis leading to an under calculation of the deuterium content in the quinazoline ring. These results identify a chemical exchange process that can either undo, maintain, or facilitate the labeling process and also mass spectral analyses issues that must be taken into account to characterize and utilize these analogs and, more broadly, that can be recognized as potentially applicable to other classes of compounds. Copyright © 2010 John Wiley & Sons, Ltd.

3042 Background: Deregulation of c-MET (MET), a receptor tyrosine kinase (RTK), signaling has been implicated in the initiation, progression, and metastasis of human cancers, and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing MET modulation. To support the clinical development of Met inhibitors, we describe development of pharmacodynamic assays to assess in vivo MET kinase inhibition, which will be suitable for use in evaluating biopsy specimens in the clinical trial setting.METHODSTwo novel rabbit monoclonal antibodies specific to phosphorylated Y1235 and Y1356 MET were combined with an N-terminal MET specific capture antibody to develop sandwich immunoassays for intact MET. Wild type recombinant intact MET (P08581) was used to create a calibrator. To demonstrate a fit-for-purpose response, we investigated the growth suppression effect of two potent MET inhibitors in human gastric tumor xenografts, and analyzed the degree of inhibition of pY1235 and pY1356 in the excised tumor biopsies. Drugs were given daily for 10 days and biopsies were collected 4 hours after each dose on days 1, 3, 6, 8 and 10.RESULTSWe developed and validated chemiluminescence immunoassays that provided a robust measurement of MET and pMET with a total analytical error (CV) <20% in tumor biopsy extracts. A single IP dose of PHA665752, 25 mg/kg and 50 mg/kg, reduced intra-tumor pY1235 levels within 4-hours by approximately 60% (p<0.05) and 80% (p<0.001), respectively. A single oral dose of PF02341066 (25 mg/kg) decreased pY1235 MET levels by approximately 70% (p<0.01) within 4 hours. At the highest dose, mean intra-tumor pMET levels of pY1235 and pY1356 were consistently lower (50-80%) in treated mice on days 3, 6, 8, and 10, as compared to pMET levels in vehicle treated group.CONCLUSIONSCurrently, several small molecule inhibitors and therapeutic antibodies are in clinical trials to treat cancer driven by the MET pathway. Our new pharmacodynamic assays provide a means to confirm and quantify the intended mechanism of action of MET inhibitors during early phase clinical trials. Funded by NCI Con#HHSN261200800001E.

First synthesis of gemcitabine triphosphate (dFdCTP) as a tris(triethylammonium) salt is reported.

A series of novel, sterically hindered lipophilic analogs of AG 957 was designed and synthesized as potential protein tyrosine kinase (PTK) inhibitors. The in vitro activity, in vivo anti-leukemia activity, and pharmacology of these PTK inhibitors were studied. Some aspects of the structure-activity relationship associated with the carboxylic acid, phenol ring, and linker modifications are discussed. We have demonstrated that the 1,4-hydroquinone moiety is essential for activity and that sterically hindered esters contribute to enhanced in vivo efficacy. Adaphostin (NSC 680410) has emerged as the improved compound with the maximum in vivo anti-leukemia hollow fiber activity, concordant with the original lead compound AG 957. Currently, adaphostin is undergoing preclinical toxicology studies.

A new tetrazolium salt, XTT, has been synthesized. XTT is reduced by a considerable variety of cell lines to a water‐soluble formazan. XTT appears to merit further investigation as a reagent for broader application to cell culture assay systems.