Terrence W. Doyle

Syntheses and Antitumor Activities of Potent Inhibitors of Ribonucleotide Reductase: 3-Amino-4-Methylpyridine-2-Carboxaldehyde-Thiosemicarbazone (3-Amp), 3-Amino-Pyridine-2-Carboxaldehyde-Thiosemicarbazone (3-Ap) and its Water-Soluble Prodrugs

The reductive conversion of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase (RR) is a crucial and rate-controlling step in the pathway leading to the biosynthesis of DNA, since deoxyribonucleotides are present in extremely low levels in mammalian cells. Mammalian ribonucleotide reductase (RR) is composed of two dissimilar proteins, often referred to as R(1), which contains polythiols and R(2), which contains non-heme iron and a free tyrosyl radical. Both the R(1) and R(2) subunits contribute to the active site of the enzyme. Currently, there are two broad classes of RR inhibitors. The first class includes nucleoside analogs which bind to the R1 subunit of the enzyme, several of which are in development. Among those, Gemcitabine and MDL 101,731 have demonstrated impressive efficacy against various solid tumors. Gemcitabine has now been approved for the treatment of pancreatic cancer and non-small cell lung cancer. The most promising second class of inhibitors of RR includes HCTs [alpha--(N)-heterocyclic carboxaldehyde thiosemicarbazones, e.g., 3-AP and 3-AMP], which exert enzyme inhibitory effect through high affinity binding with non-heme iron. Based on the clinical success achieved by Gemcitabine, it seems reasonable that a strong inhibitor of RR, which is essential for cellular replication, would be a useful addition to the existing therapeutic agents against cancer. In this chapter, we wish to report several highly efficient syntheses for both 3-AP and 3-AMP based upon palladium mediated Stille/Suzuki/Heck coupling reactions. Based upon the in vivo efficacy profile observed with these two agents, 3-AP was chosen over 3-AMP as the candidate for further optimization with the intention to improve its biological and pharmaceutical properties. In this vein, we have completed the synthesis of two water soluble phosphate containing prodrugs and one disulfide-linked prodrug of 3-AP. As expected, bioconversion study using either alkaline phosphatase or glutathione showed that these prodrugs were indeed converted to the parent 3-AP. When evaluated against the murine M-109 lung carcinoma as well as the B16-F10 murine melanoma xenograft models, the newly prepared phosphate prodrugs displayed improved efficacy and safety profiles than that found with the parent. More significantly, the ortho-phosphate prodrug 21 demonstrated impressive antitumor effect using once-a-day dosing regimen. In summary, the results disclosed herein demonstrated that some of 3-AP prodrugs prepared indeed demonstrated improved pharmaceutical, biological and toxicity profiles over the parent 3-AP. Efforts directed towards further optimization of 3-AP prodrugs as novel anticancer agents is clearly warranted.

Synthesis of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)

Abstract Palladium-catalyzed cross-coupling of methylboronic acid with 2-chloro-3-nitropyridine produced 2-methyl-3-nitropyridine 4 in one step in high yield. Oxidation of 4 with selenium dioxide gave aldehyde 5. Alternatively, condensation of 4 with DMFDMA followed by oxidation gave 5 in a two step higher yielding conversion. Subsequent direct coupling of 5 with thiosemicarbazide followed by reduction of the nitro group using stannous chloride or sodium sulfide provided 3-AP (3). Reduction with sodium hydrosulfite gave 3-HAP (8). Finally a route which avoids the reduction of a nitro function was devised. Thus direct coupling of styrene with 2-chloro-3-aminopyridine 9 under Heck reaction conditions gave 16 which was converted to 17, oxidized to the aldehyde 18 and converted to 3-AP (3) with in situ deblocking of the t-Boc functionality.

Synthesis and comparative evaluation of two antiviral agents: β-l-Fd4C and β-d-Fd4C

The synthesis of beta-D-Fd4C was achieved in a stereoselective fashion from D-xylose. The antiviral activity and cytotoxicity of beta-D-Fd4C was compared with that of beta-L-Fd4C and 3TC (Lamivudine). Of the three agents compared, beta-L-Fd4C was found to be the most potent antiviral agent.

Synthesis and biological evaluation of a water soluble phosphate prodrug of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP).

With the aim of improving its biological and pharmaceutical profiles, two water soluble phosphate prodrugs of 3-AP, 3a and 3b were prepared. The detailed synthesis and the preliminary evaluation of these prodrugs are described.

Efficient synthesis of ribonucleotide reductase inhibitors 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) via palladium mediated cross-coupling reactions

Abstract An efficient synthesis of potent ribonucleotide reductases inhibitors 3-amino-pyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methyl-pyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) is described. The synthesis of 3-AP and 3-AMP was achieved in 4 and 5 steps, with overall yields of 61% and 39%, respectively. The synthesis featured a convergent approach utilizing a Stille coupling strategy to prepare vinylpyridine derivatives. A more economic way to synthesize vinylpyridine using Heck reaction was also discussed.

Isolation and Identification of Metabolites of Porfiromycin Formed in the Presence of a Rat Liver Preparation

The isolation and identification of the major metabolites of porfiromycin formed in the presence of a rat liver preparation under aerobic conditions were performed with high-performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by the rat liver preparation in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) containing an NADPH generating system at 37 degrees C. A total of eight metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2-methylamino-7-aminomitosene, 1,2-cis and 1,2-trans-1-hydroxy-2-methylamino-7-aminomitosene, which are consistent with those previously observed in hypoxia using purified rat liver NADPH-cytochrome c reductase. Interestingly, 2-methylamino-7-aminomitosene is a reactive metabolite, which undergoes further activation at the C-10 position by the loss of carbamic acid and then links with the 7-amino group of the primary metabolites to yield two dimeric adducts. In addition, three phosphate adducts, 10-decarbamoyl-2-methylamino-7-aminomitosene-10-phosphate, 1,2-cis and 1,2-trans-2-methylamino-7-aminomitosene-1-phosphate, were also identified in the incubation system. The configurations of the diastereoisomeric metabolites were determined with (1)HNMR and phosphatase digestion. On the basis of the metabolite profile, we propose in vitro metabolic pathways for porfiromycin. The findings provide direct evidence for understanding the reactive nature and hepatic metabolism of the drug currently in phase III clinical trials.