Synthesis of 2-(3′-Azido- and 3′-Amino-3′-deoxy-β-D-ribofuranosyl)thiazole-4-carboxamide

Abstract

In view of biological activities of tiazofurin and azido or aminosugar nucleosides, novel azido- and amino-substituted tiazofurin derivatives (1 and 2) were efficiently synthesized starting from 1,2;5,6-di-O-isopropylidene-D-glucose.     

2-(3-Amino-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide: a new tiazofurin analogue with potent antitumour activity

A new tiazofurin analogue, 2-(3-amino-3-deoxy-beta-d-xylofuranosyl)thiazole-4-carboxamide (3), was synthesized starting from d-glucose and evaluated for its in vitro antiproliferative activity against a panel of human tumour cell lines. Compound 3 exhibited the most powerful cytotoxicity against K562 cells, being approximately 100-fold more potent than tiazofurin. This analogue was also active against Jurkat, HT-29 and HeLa malignant cells, with respective IC(50) values being ca. 2-, 27- and 17-fold lower than those observed for tiazofurin. Remarkably, compound 3 did not exhibit any significant cytotoxicity towards normal foetal lung MRC-5 cell line.     

Synthesis of 3'-deoxy-3'-C-hydroxymethyl analogues of tiazofurin and ribavirin

On the basis of potent biological activity of 3'-branched-3'-deoxynucleoside analogues, novel ribavirin and tiazofurin derivatives with 3'-C-hydroxymethyl substituent were synthesized, starting from D-xylose.     

SYNTHESIS OF 3′-DEOXY-3′-C-HYDROXYMETHYL ANALOGUES OF TIAZOFURIN AND RIBAVIRIN

On the basis of potent biological activity of 3′-branched-3′-deoxynucleoside analogues, novel ribavirin and tiazofurin derivatives with 3′-C-hydroxymethyl substituent were synthesized, starting from D-xylose.     

Cytotoxicity of tiazofurin and its arabinose and xylose analogues in K562 cells.

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.     

Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.     

Action of tiazofurin and 8-Cl-cAMP in human colon and pancreatic cancer cells.

Tiazofurin and 8-Cl-cAMP are novel chemotherapeutic agents shown to be effective against various cancer cells in vitro and in vivo. They act through distinct mechanisms that might modulate the signal transduction pathway, which causes growth inhibition, differentiation and down-regulation of c-ras and c-myc oncogene expression. We examined the effects of tiazofurin and 8-Cl-cAMP on colony formation of HT-29 human colon cancer and BxPC-3 and PANC-1 human pancreatic cancer cell lines. The IC50 of 8-Cl-cAMP was 0.1 and 0.2 microM in the pancreatic and colon cancer cell lines, respectively, and tiazofurin yielded IC50s from 4 (PANC-1) to 18 microM (HT-29). Simultaneous incubation with 8-Cl-cAMP and tiazofurin had additive effects on the inhibition of colony formation in the three examined cell lines. These results indicate possible clinical usefulness of a combination of tiazofurin and 8-Cl-cAMP in the treatment of colon and pancreatic carcinomas.     

Synthesis and antiproliferative activity of two new tiazofurin analogues with 2'-amido functionalities

Two novel tiazofurin analogues 2 and 3 were synthesized starting from d-glucose. The key step of the synthesis was the efficient one-step hydrogen sulfide-mediated conversion of 2-azido-3-O-acyl-ribofuranosyl cyanides to the corresponding 2-amido thiocarboxamides. Compounds 2 and 3 were evaluated for their in vitro antiproliferative activity against certain human tumour cell lines. Remarkably, compound 2 was found to be 570-fold more potent than tiazofurin against MCF-7 cells, while compound 3 showed the most powerful cytotoxicity against HT-29 cancer cells, being almost 100-fold more active than tiazofurin.     

Inhibitors of IMP dehydrogenase stimulate the phosphorylation of the antiviral nucleoside 2' ,3'-dideoxyguanosine

The inosinate dehydrogenase (IMPD) inhibitors ribavirin, tiazofurin and mycophenolic acid were found to stimulate by as much as 20-fold the anabolism of the anti-HIV agent 2' ,3'dideoxyguanosine to its 5'-diphosphate (ddGDP) in a human T-cell culture system (Molt-4 cells). Stimulation of the further conversion to ddGTP (the active form of the drug) was lesser in magnitude but still highly significant (up to 4-fold at appropriate concentrations of ribavirin or tiazofurin). In parallel with these increases, the inhibitors also produced increases of up to 35-fold in IMP levels. These results support the proposal that the initial phosphorylation of ddGuo is catalyzed by a phosphotransferase (5'-nucleotidase) which utilizes IMP as its phosphate donor (Johnson and Fridland, [1989] Molec. Pharmacol. 36, 291-295). Concomitant with this increase in 5'-phosphorylation of ddGuo, an increase in its anti-HIV activity of up to 6.5-fold was observed when this agent was combined with ribavirin (5 microM) in the H9 [corrected] cell assay system.     

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.     

Involvement of guanine nucleotides in superoxide release by fluoride-treated neutrophils. Implications for a role of a guanine nucleotide regulatory protein

Previous studies demonstrating hydrolysis of phosphatidylinositol bisphosphate (PIP2) and generation of inositol phosphates in neutrophils exposed to 20.0 mM NaF provide indirect evidence that activation of phospholipase-associated guanine nucleotide regulatory protein, a guanine nucleotide binding protein which regulates the activation of a membrane inositol-specific phospholipase C, is an early event in the neutrophil stimulus-response pathway triggered by fluoride. Consistent with this hypothesis, exposure of a plasma membrane rich preparation isolated from 32P labeled neutrophils to 20.0 mM NaF resulted in hydrolysis of labeled PIP2. Levels of other phospholipids were not affected. Inositol bisphosphate and inositol trisphosphate were detected in extracts of neutrophil plasma membranes exposed to fluoride. To further explore the involvement of guanine nucleotides in functional responses of intact neutrophils triggered by fluoride, we preincubated cells with 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin), a selective inhibitor of inosine monophosphate dehydrogenase, to diminish guanine nucleotide synthesis and then compared superoxide generation induced by FMLP, PMA, digitonin, and 20.0 mM NaF to intracellular levels of guanine nucleotides. Preincubation of neutrophils for 2.5 h at 37 degrees C with tiazofurin resulted in dose-dependent depletion of GTP and GDP. Maximal depletion of guanine nucleotides required relatively high levels of tiazofurin (200 to 400 microM) and resulted in a 55 to 60% reduction of GTP and GDP. The effects of tiazofurin on guanine nucleotides levels were not observed when neutrophils were preincubated at 4 degrees C. AT 37 degrees C, tiazofurin also decreased intracellular ATP and ADP levels but adenine nucleotide depletion was less pronounced than guanine nucleotide depletion for each concentration of tiazofurin used. When tiazofurin was removed by washing cells after incubation, adenine nucleotide quickly returned to preincubation values but guanine nucleotide levels remained depressed. Addition of exogenous guanosine (200 microM) prevented tiazofurin-dependent depletion of guanine nucleotides but had no influence on adenine nucleotide depletion. Superoxide released triggered by FMLP and F- was inhibited to an extent similar to that of guanine nucleotide depletion under different conditions of preincubation. Inhibition of superoxide release was not observed if cells were preincubated at 4 degrees C, was not rapidly reversible, and was not observed when guanosine was added with tiazofurin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic action of taxol with tiazofurin and methotrexate in human breast cancer cells: Schedule-dependence

Taxol (paclitaxel, (NSC 125973)) is an active agent in the treatment of metastatic carcinoma of the breast; however, positive responses were observed in only about 60% of cases. Therefore, drug combinations which might improve the effectiveness are required. Since tiazofurin (2-β-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), methotrexate (MTX) and taxol exert their anticancer action in different phases of the cell cycle and have different biochemical targets, we tested the hypothesis that tiazofurin and methotrexate might be synergistic with taxol. MDA-MB-435 human breast cancer cells were grown in monolayer in flasks. In the growth inhibition assay for tiazofurin, methotrexate and taxol the IC₅₀s were 12.5, 0.5 and 0.016 μM, respectively, and in the clonogenic assay 4.5, 0.065 and 0.004 μM. When taxol was given 6 hr before tiazofurin, antagonism was observed and summation was seen when drugs were given simultaneously. Synergism was obtained in both growth inhibitory and clonogenic assays when tiazofurin was followed 12 hr later by taxol. Methotrexate and taxol had an antagonistic effect when they were added simultaneously or when taxol was given 6 hr before methotrexate; summation was observed when taxol was followed 12 hr later by methotrexate. Synergistic action was obtained in the clonogenic assay when methotrexate was followed 12 hr later by taxol. The protocols yielding synergism should be of value in the design of taxol-based clinical trials for breast cancer.     

Synergistic down-regulation of signal transduction and cytotoxicity by tiazofurin and quercetin in human ovarian carcinoma cells.

Ovarian carcinoma is one of the most common causes of cancer death in women. Tiazofurin, a C-nucleoside, arrests the cell cycle at S phase and reduces the activities of PI (phosphatidylinositol) utilizing enzymes in signal transduction by depleting cellular GTP concentration. Quercetin (QN), a flavonoid, attacks the cell cycle at the G1 and S phase boundary and mainly inhibits PI kinase (1-phosphatidylinositol 4-kinase, EC 2.7.1.67) activity in the signal transduction pathway. Because tiazofurin and QN attack different biochemical targets and arrest different phases of the cell cycle, we tested the hypothesis that the two drugs might be synergistic against human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50s (drug concentration that inhibits 50% of cell proliferation) for tiazofurin and QN were (mean +/- SE) 13 +/- 1.2 and 66 +/- 3.0 microM; in clonogenic assays they were 6 +/- 0.5 and 15 +/- 1.2 microM, respectively. When tiazofurin was added to cells followed 12 h later by QN, synergism was observed in both growth inhibition and clonogenic assays. The combination also yielded synergistic reduction of IP3 (inositol 1,4,5-trisphosphate) concentration in the cells which may explain, at least in part, the synergistic action of tiazofurin and QN in OVCAR-5 cells. The protocols yielding synergism may have implications in the clinical treatment of human ovarian carcinoma.     

Unique bioactivation of tiazofurin--studies with resistant cells

Using resistant cells mechanism of action of new oncolytic nucleoside, tiazofurin (2-beta-D-ribofuranosyl thiazole-4 carboxamide, RTC) was studied in tissue cultured cells of Chinese Hamster Ovary cells (CHO-cells). Tiazofurin got converted in CHO-cells to tiazofurin-monophosphate and to NAD-analogue, a potent inhibitor of inosinate dehydrogenase. Resistant cells produced tiazofurin-5'-monophosphate in vitro but had a much reduced capacity to produce NAD-analogue, indicating absence of any effect of tiazofurin on incorporation of [14C]formate into guanine, inhibition of inosinate dehydrogenase as well as GTP levels in resistant cells. Using competition with various possible substrates it is found that the initial tiazofurin metabolism is catalysed by nicotinamide nucleoside kinase and NAD-analog formation is mediated by NAD-pyrophosphorylase. Decreased activity of the latter enzyme found in tiazofurin resistant cells not only inhibited the NAD analog formation from tiazofurin-5'-monophosphate but also the NAD-formation from nictotinamide-5'-monophosphate.     

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide)

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.     

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.     

Increased Nucleoside Concentrations in Tumors as Markers of Tiazofurin Action

Abstract

Tiazofurin injection (150 mg/kg, i.p.) into rats bearing hepatoma 3924A increased in the tumor the pools of guanine, uridine, thymine, hypoxanthine, inosine, and thymidine 3.5-, 4.4-, 8.0- 18.4-, 18.7- and 42-fold over the controls. There were only minor changes in the host liver. This is the first report showing a selective action of tiazofurin in cancer cells on the concentrations of nucleosides and bases, indicating that these might be used as markers of the impact of tiazofurin in clinical trials.     

A new synthetic methodology for tiazofurin

A new synthesis of tiazofurin is described from 2,3-O-isopropylidene-5-O-benzoyl-beta-D-ribofuranosyl cyanide.