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.     

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.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.     

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.     

Design, synthesis and in vitro antiprotozoal activity of benzimidazole-pentamidine hybrids

A series of ten novel hybrids from benzimidazole and pentamidine were prepared using a short synthetic route. Each compound was tested in vitro against the protozoa Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Leishmania mexicana, and Plasmodium berghei, in comparison with pentamidine and metronidazole. Some analogues showed high bioactivity in the low micromolar range (IC(50)<1 microM) against the first four protozoa, which make them significantly more potent than either standard. 1,5-bis[4-(5-methoxy-1H-benzimidazole-2-yl)phenoxy]pentane (2) was 3- and 9-fold more potent againstG. lamblia than metronidazole and pentamidine, respectively. This compound was 23-, 108-, and 13-fold more active than pentamidine against T. vaginalis, E. histolytica and L. mexicana, respectively. Studying further structure-activity relationships through the use of bioisosteric substitution in these hybrids should provide new leads against protozoal diseases.     

Synthesis and cytotoxic activity of novel 1-((indol-3-yl)methyl)–1H-imidazolium salts

A series of novel 1-((indol-3-yl)methyl)–1H-imidazolium salts were prepared and evaluated in vitro against a panel of human tumor cell lines. The results suggest that the 5,6-dimethyl-benzimidazole ring, and substitution of the imidazolyl-3-position with a naphthylacyl or 4-bromophenacyl group, were vital for modulating inhibitory activity of cell growth. In particular, 1-((N-Boc-indol-3-yl)methyl)-3-(2-naphthylacyl)-1H-5,6-dimethyl-benzimidazolium bromide was found to be the most potent derivative and more selective against myeloid liver carcinoma (SMMC-7721), lung carcinoma (A549) and breast carcinoma (MCF-7), with IC₅₀ values 1.9-fold, 1.7-fold and 4.8-fold lower than DDP. This compound can induce significant cell apoptosis in SMMC-7721 cells.     

Novel thiazonaphthalimides as efficient antitumor and DNA photocleaving agents: effects of intercalation, side chains, and substituent groups

A series of novel antitumor and DNA photocleaving agents was designed and synthesized by fusing a (substituted) thiazole ring to the naphthalimide skeletons. C1, the most active compound against A549, was about 30-fold more cytotoxic than the compound amonafide. A1, the most active compound against P388, was about 6-fold more cytotoxic than amonafide. C2, the most efficient DNA intercalator, showed the strongest DNA photocleaving activity via superoxide anion produced under UV light at 360 nm.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

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.     

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.     

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.     

Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4 h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.     

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)     

Benzodiazepine compounds as inhibitors of the src protein tyrosine kinase: screening of a combinatorial library of 1,4-benzodiazepines.

We screened 1680 spatially separated compounds of a diverse combinatorial library of 1,4-benzodiazepines for their ability to inhibit the kinase activity of protein tyrosine kinases Src, Yes, Abl, Lck, Csk, and fibroblast growth factor receptor. This screening yielded novel ligands for the protein tyrosine kinase Src. In the 1, 4-benzodiazepine-2-one scaffold, the preferred substituent at position R(1) was 4-hydroxyphenylmethyl or a 3-indolemethyl derived from a tyrosine or tyrptophan used in building the benzodiazepine, while the substituent at R(2), introduced by alkylating agents, was preferably aromatic in nature. The preferred ring structure introduced on the bicyclic ring of the scaffold by acid chlorides was a p-hydroxy phenyl group. The lead compound, designated as N-L-Yaa, has a L-4-hydroxyphenylmethyl ring at R(1) and a biphenylmethyl substituent at R(2). The compound has an IC(50) of 73 microM against Src, 2- to 6-fold lower than against other protein tyrosine kinases and >10-fold lower than against other nucleotide-utilizing enzymes. The mechanism of binding of N-L-Yaa to Src is mixed against the peptidic substrate with a K(i) of 35 microM and noncompetitive against ATP-Mg with a K(i) of 17 microM. Multiple inhibition analysis of the lead compound in the presence of other competitive inhibitors demonstrated that the binding of the lead compound is nonexclusive to the other competitive inhibitor. The inhibitor was found to be nontoxic to the AFB-13-human fibroblasts cells and inhibited the colony formation of HT-29 colon adenocarcinoma cells that are dependent on Src activity.     

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 cytotoxic activities of novel hybrid 2-phenyl-3-alkylbenzofuran and imidazole/triazole compounds

A series of novel hybrid compounds of 2-phenyl-3-alkylbenzofuran and imidazole or triazole were prepared and evaluated in vitro against a panel of human tumor cell lines. The results suggest that the 2-ethyl-imidazole ring, and substitution of the imidazolyl-3-position with a 2-bromobenzyl or naphthylacyl group, were vital for modulating inhibitory activity. In particular, hybrid compound 31 was found to be the most potent derivative with IC₅₀ values of 0.08–0.55 μM against five strains human tumor cell lines and was found to be more selective against breast carcinoma (MCF-7) and colon carcinoma (SW480) (IC₅₀ values 40.8-fold and 40.1-fold lower than cisplatin (DDP)).     

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.     

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.     

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.     

De novo synthesis of two new cytotoxic tiazofurin analogues with modified sugar moieties

A divergent synthesis of two novel tiazofurin analogues, 2-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)thiazole-4-carboxamide (2) and 2-(3-acetamido-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide (3), has been achieved starting from D-glucose. Both nucleoside analogues were evaluated for their in vitro cytotoxicity against several human leukaemia and solid tumour cell lines.