The syntheses of furan and thiophene analogues of tiazofurin (furanfurin and thiophenfurin, respectively) are described. Direct stannic chloride-catalyzed C-glycosylation of ethyl 3-furan-carboxylate (6) or ethyl 3-thiophencarboxylate (18) with 1,2,3,5-tetra-O-acetyl-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivatives. Deprotection of ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)furan-3-carboxylate (9 beta) and ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiophene-3-carboxylate (20 beta) with sodium ethoxide afforded ethyl 5-beta-D-ribofuranosylfuran-3-carboxylate (12 beta) and ethyl 5-beta-D-ribofuranosylthiophene-3-carboxylate (23 beta) which were converted into 5-beta-D-ribofuranosylfuran-3-carboxamide (furanfurin, 4) and 5-beta-D-ribofuranosylthiophene-3-carboxamide (thiophenfurin, 5) by reaction with ammonium hydroxide. The anomeric configuration and the site of glycosylation were established by 1H-NMR and proton-proton nuclear Overhauser effect difference spectroscopy. The structure of compound 23 beta was confirmed by X-ray crystallography. Thiophenfurin was found to be cytotoxic in vitro toward murine lymphocytic leukemia P388 and L1210, human myelogenous leukemia K562, human promyelocytic leukemia HL-60, human colon adenocarcinoma LoVo, and B16 melanoma at concentrations similar to that of tiazofurin. In the same test furanfurin proved to be inactive. Thiophenfurin was found active in vivo in BD2F1 mice inoculated with L1210 cells with a % T/C of 168 at 25 mg/kg. K562 cells incubation with thiophenfurin resulted in inhibition of inosine monophosphate (IMP) dehydrogenase (63%) and an increase in IMP pools (6-fold) with a concurrent decrease in GTP levels (42%). Incubation of adenosine-labeled K562 cells with tiazofurin, thiophenfurin, and furanfurin resulted in a 2-fold higher NAD analogue formulation by thiophenfurin than by tiazofurin. Furanfurin was converted to the NAD analogue with only 10% efficiency. The results obtained support the hypothesis that the presence of S in the heterocycle in position 2 with respect to the glycosidic bond is essential for the cytotoxicity and IMP dehydrogenase activity of tiazofurin, while the N atom is not.

region (UTR); heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1); pteroylglutamate (PteGlu); Dulbecco's PBS (D-PBS); chloramphenicol acetyltransferase (CAT); with a cis-element (+cis); lacking a cis-element (-cis).

Maternal folate deficiency results in selective upregulation of FR and hnRNP-E1 associated with multiple aberrations in fetal tissues that include increased cell loss, architectural anomalies, and premature differentiation. The potential significance of these findings to explain the wide spectrum of folate-responsive birth defects in humans is discussed.

The interaction of an 18-base cis-element in the 5-untranslated region of human folate receptor (FR)-␣ mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539 -25547). This trans-factor was isolated to apparent homogeneity as a 43-and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA ciselement in the 3-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as ␣CP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.

Nicotinamide mononucleotide adenylyltransferease (NMNAT), a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions. NAD is necessary for cell survival in oxidative stress and DNA damage. Based on their localization, three different NMNAT's have been recognized, NMNAT-1 (homohexamer) in the nucleus (chromosome 1 p32-35), NMNAT-2 (homodimer) in the cytoplasm (chromosome 1q25) and NMNAT-3 (homotetramer) in the mitochondria. NMNAT also catalyzes the metabolic conversion of potent antitumor prodrugs like tiazofurin and benzamide riboside to their active forms which are analogs of NAD. NAD synthase-NMNAT acts as a chaperone to protect against neurodegeneration, injury-induced axonal degeneration and also correlates with DNA synthesis during cell cycle. Since its activity is rather low in tumor cells it can be exploited as a source for therapeutic targeting. Steps involved in NAD synthesis are being utilized as targets for chemoprevention, radiosensitization and therapy of wide range of diseases, such as cancer, multiple sclerosis, neurodegeneration and Huntington's disease.

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A series of adenosine derivatives substituted at the 1'-, 2'-, or 3'-position of the ribose ring with a methyl group was synthesized and evaluated for antitumor activity. From this study 3'-C-methyladenosine (3'-Me-Ado) emerged as the most active compound, showing activity against human myelogenous leukemia K562, multidrug resistant human leukemia K562IU, human promyelocytic leukemia HL-60, human colon carcinoma HT-29, and human breast carcinoma MCF-7 cell lines with IC(50) values ranging from 11 to 38 muM. Structure-activity relationship studies showed that the structure of 3'-Me-Ado is crucial for the activity. Substitution of a hydrogen atom of the N(6)-amino group with a small alkyl or cycloalkyl group, the introduction of a chlorine atom in the 2-position of the purine ring, or the moving of the methyl group from the 3'-position to other ribose positions brought about a decrease or loss of antitumor activity. The antiproliferative activity of 3'-Me-Ado appears to be related to its ability to deplete both intracellular purine and pyrimidine deoxynucleotides through ribonucleotide reductase inhibition.

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