Synthesis and antitumor activity of new glycosides of epipodophyllotoxin, analogues of etoposide, and NK 611.

A series of 3-amino- and 3-alkylamino-2-deoxy-beta-D-ribo- and beta-D-arabino-glycosides of 4'-demethylepipodophyllotoxin have been synthesized by means of an improved trimethylsilyliodide procedure for the podophyllotoxin-4'-demethylepipodophyllotoxin conversion, an efficient and high yielding synthesis of silyl glycoside donors of 3-azido-2,3-dideoxy-beta-D-ribo- and beta-D-arabino-hexopyranosides and stereoselective glycosylations. In vitro evaluation of cytotoxic effects against murine L1210 leukemia critically demonstrates the essential role played by a 4,6-acetal for biological activity. Among the most cytotoxic compounds, 3-amino-2,3-dideoxy- and 3-N, N-(dimethylamino)-2,3-dideoxy etoposide analogues, 17 and 27-29 are at least as potent as etoposide on the in vivo P388 (iv/ip) murine leukemia models. However, surprisingly enough, none of these compounds inhibits the human DNA topoisomerases I or II or binds to tubulin to prevent its polymerization and microtubule assembly. Therefore, their mechanism of action remains to be cleared up.

Induction of mitochondrial dysfunction and apoptosis in HeLa cells by bis-pyridinium oximes, a newly synthesized family of lipophilic biscations.

When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3'-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.

Methionine deprivation and methionine analogs inhibit cell proliferation and growth of human xenografted gliomas.

Growth of numerous malignant tumors depends on an exogenous methionine (MET) supply, while endogenously synthesized MET supports normal cell proliferation. Because an antitumor effect should be obtained by aggravating the altered MET metabolism in gliomas, MET dependency of human xenografted gliomas was evaluated and a therapeutic approach using MET deprivation or MET analogs to induce MET starvation was applied. In vitro proliferation inhibition of glioma cell lines by MET deprivation and two MET analogs, ethionine (ETH) and trifluoromethylhomocysteine (TFH), was measured. Proliferation of 7 human glioma cell lines tested was inhibited in MET-free medium, and was poorly or not reversed by homocysteine (HCY). ETH or TFH (concentration range: 0.005-2 mg/ml) inhibited proliferation of all cell lines tested. MET analog-induced inhibition was abolished by MET and enhanced by HCY. Cell-cycle alterations due to MET deprivation were optimally assessed after 30 h of culture and bromodeoxyuridine incorporation. In MET- medium, cells were arrested in the G1-phase. ETH induced a dramatic accumulation of cells in the G2-phase. ATP contents were reduced by MET analogs only in HCY+ medium, suggesting complementary effects of MET analogs and HCY. Human glioma bearing nude mice were fed an amino acid-substituted MET- HCY-supplemented diet (MET-HCY+) and/or treated with MET analogs, injected intraperitoneally daily. Using two human xenografted tumors derived from gliomas, antitumor effects were obtained by subjecting tumor-bearing nude mice to MET starvation. TG-1-MA was more sensitive to MET depletion (40% of growth inhibition, P < 0.10) than TG-8-OZ (no growth inhibition). Antitumor effects of a MET-HCY+ diet and 200 mg/kg of ETH were potentiated when co-administered to glioma-bearing mice (77% GI, P < 0.025 and 67%, P < 0.0057 to TG-1-MA and TG-8-OZ respectively). A dose-response effect with no toxicity was obtained when the ETH dose was increased 10 fold. Potentiation of the effects of ETH and a MET-free diet indicates that they probably act on the same pathway but not the same target. In conclusion, experimentally induced MET deprivation and MET-analog treatment retarded the growth of human gliomas. Combination of MET-analog therapy with MET substitution by HCY enhanced their respective effects.

The linking chain length in bridged aldehyde-substituted pyridinium dimers modulates a slow reversible binding that affects supercoiling of plasmid DNA.

Bridged aldehyde substituted pyridinium dimers bind to DNA. Binding induces retardation of the electrophoretic migration of circular plasmid due to relaxation of supercoiling. Binding to DNA is slow and reversible and depends on the length of linking chain, pH, ionic strength and temperature. It is postulated that release of topological stress results from the disruption of hydrogen bonding between complementary bases, a hypothesis consistent with facilitated attack of DNA by formaldehyde promoted by the bridged compounds.

Photobiological activities of 1,6-dioxapyrene in pro- and eukaryotic cells.

The photobiological effect of a new pyrene derivative, 1,6-dioxapyrene (1,6-DP), was studied in Salmonella typhimurium (strain TA100) and in the diploid strain D7 of the yeast Saccharomyces cerevisiae. In Salmonella, 1,6-DP shows little mutagenicity in the dark in comparison to benzo[a]pyrene (B[a]P). This mutagenic activity decreases in the presence of liver S9 homogenates from Aroclor induced XVIInc/Z mice. However, in combination with 365 nm (UVA) radiation and in the absence of S9 mix, 1,6-DP behaves as an effective photodynamic compound inducing lethal and mutagenic effects in both organisms. In yeast, its activity, like that of B[a]P, is highly dependent on the presence of oxygen. For the same incident dose of UVA, 1,6-DP is, however, at least 6 times more effective than B[a]P in inducing cytotoxic and mutagenic effects. At equitoxic doses, 1,6-DP is as photomutagenic as B[a]P, suggesting that in both cases mutagenicity is due to similar mechanisms. Spectrophotometric measurements indicate physical interaction of 1,6-DP with DNA in the dark. Laser flash photolysis experiments show that 1,6-DP generates singlet oxygen with a quantum yield of 0.17. In vitro 1,6-DP produces oxidative damage to guanine bases specific for singlet oxygen mediated reactions. Alkaline step elution analysis of 1,6-DP plus UVA treated yeast cells indicates a decrease in average molecular weights in DNA and an induction of single strand breaks (ssb) originating from alkali labile sites. This effect is enhanced by D2O and is thus likely to be due to the production of singlet oxygen. The strand breaks appear to differ from those induced by gamma-rays because little, if any, repair of these ssb occurs during 30 min of post-treatment incubation in complete growth medium. These results suggest that the photobiological effects of 1,6-DP are due to oxidative damage in DNA mostly induced by singlet oxygen.

Free radical production during photo-assisted NADPH reduction of four alpha-nitroarenofurans.

Generation of radical anions during NADPH reduction of four mutagenic and genotoxic alpha-nitroarenofurans was examined. ESR showed that free radicals were generated during reduction solely in the presence of light. Computer simulations of ESR spectra were in good agreement with the experimental ones.

The binding of bridged bis-pyridinium oximes to DNA and its relevance to the induction of mitochondrial dysfunction in yeast.

Bis-pyridium oximes and methoximes from a newly synthesized series are weak DNA binders (K = 3.10(4) M-1 under physiological conditions). From the number of binding sites per phosphate, 0.25, the ionic strength dependence of the binding constant and the negative electric dichroism, it is concluded that monointercalation is the mode of association. In contrast to methoxy compounds, the oxime derivatives are able both to induce the mutated "petite" phenotype in yeast S. cerevisiae and to cause "in vitro" extensive condensation of single stranded DNA. This reaction is postulated to be relevant to the mutational process that leads to "peptide" cells. The absence of nuclear mutation is interpreted in terms of sequestration of the drug in mitochondria under the effect of the organelle inner membrane electrochemical potential.

Genotoxic effectivity--comparison of 36 nitrated furan and arenofuran derivatives on a quantitative scale. Statistical comparison of T7 and other short-term tests.

Thirty-six nitrated furan and arenofuran derivatives were measured and quantitatively characterized by the T7 inactivation test. A wide range of substances previously studied allowed us to compare the collected quantitative data with those obtained by other workers using different short-term tests. Based on comparative statistical evaluation of these data a borderline was determined for the genotoxic effect: compounds having in our short-term test mutagenicity index (MI) values smaller than 8.0 are positive while the higher values represent negative genotoxicity. Classification of 36 nitrofuran/nitroarenofuran derivatives is given both on the basis of the quantitative genotoxicity scale and in terms of +/- on the qualitative scale. All but one compound were found to be genotoxic and the genotoxic activities of these compounds were compared with the results of other carcinogenicity or mutagenicity tests.

Mutagenic activity of dichloroethylamino derivatives of nitronaphthofuran and some nitrobenzofurans in the Salmonella/microsome assay.

The mutagenic activity of five dichloroethylamino 2-nitrobenzofuran derivatives and one dichloroethylamino 2-nitronaphthofuran derivative was analysed in the Salmonella/microsome assay. We investigated the influence of the position of the dichloroethylamino and/or the methoxy groups on the mutagenic activity of these nitro arenofurans in S. typhimurium strain TA100 and its variant TA100NR, deficient in nitroreductase. Without metabolic activation 7-[bis(2-chloroethyl)amino]-2-nitronaphtho[2,1-b]furan (1), 4-[bis(2-chloroethyl)amino]-7-methoxy-2-nitrobenzofuran (2), 7-[bis(2-chloroethyl)amino]-4-methoxy-2-nitrobenzofuran (5) and 6-[bis(2-chloroethyl)amino]-2-nitrobenzofuran (6) are mutagenic in TA100, while 4-[bis(2-chloroethyl)amino]-5-methoxy-2-nitrobenzofuran (4) is weakly mutagenic and 5-[bis(2-chloroethyl)-amino]-2-nitrobenzofuran (3) toxic. In the NR deficient strain compounds 1, 3 and 6 are strong mutagens and 4 is weakly positive. The two isomers 2 and 5 are negative in that strain. The naphthofuran derivative 1 is highly mutagenic in the absence of S9 mix in both strains considered, but less than R7000 (7). A decrease in the electronic polarity of compound 1 versus compound 7 according to the hypothesis developed by Royer et al. is a possible explanation. After exogenous metabolic activation by S9 mix all the compounds tested are highly mutagenic in both Salmonella strains. The position of the dichloroethylamino group and/or the presence of a methoxyl on the alpha-nitroarenofuran derivatives seem to modify the activity of bacterial as well as exogenous nitroreductases or other activating enzymes.

Mitochondrial uptake of bridged bis-methylpyridinium aldoximes and induction of the "petite" phenotype in yeast.

The 3,3'-[omega,omega'-alkanediylbis(oxy)]bis[2- (hydroxyimino)methyl]-1-methylpyridinium derivatives bearing a linking chain of 4, 5 and 6 methylene groups are accumulated in mitochondria with increasing efficiency under the effect of the electrical potential. Accumulation does not take place with derivatives carrying a 2 and 3 methylene-long linking chain. The uptake process is saturable. The efficiency of the various derivatives to induce the "petite" phenotype in yeast reflects the uptake rate observed with purified mitochondria.

Disposition and metabolism of acetylcholinesterase reactivators 2PAM-I, TMB4 and R665 in rats submitted to organophosphate poisoning.

1. The dispositions of the acetylcholinesterase reactivators: 2PAM-I, TMB4 and R665, labelled with 14C on the oxime group, have been studied in normal rats and rats poisoned by the organophosphates Soman and A4. 2. For all three compounds, radioactivity was eliminated mostly in the urine (60-90% dose in 24 h). Faecal elimination was low (5.8-17.2% in 72 h). 3. All three compounds concentrated in kidney, but only 2PAM-I and R665 concentrated in liver. TMB4 and R665 concentrated in mucopolysaccharide-containing tissues such as cartilage and intervertebral disc. Other tissues were weakly and uniformly labelled. Soman poisoning does not modify the kinetic parameters of both compounds, but A4 poisoning increases 2PAM-I tissue concentration. 4. Chromatography of urine and plasma showed only unchanged 2PAM-I, TMB4 and R665 in both healthy and poisoned animals. Despite the high concentration of 2PAM-I and R665 in liver, these oximes are not metabolized.

Disposition and metabolism of two acetylcholinesterase reactivators, pyrimidoxime and HI6, in rats submitted to organophosphate poisoning.

1. The dispositions of two acetylcholinesterase reactivators, pyrimidoxime and HI6, labelled with 14C on the oxime group, have been studied in normal rats and rats poisoned by the organophosphates Soman and A4. 2. For both compounds, and for healthy and poisoned rats, radioactivity was eliminated essentially in the urine (85% dose in 24 h). Faecal elimination was low (4% in 72 h). 3. Both compounds were concentrated in kidney and mucopolysaccharide-containing tissues such as cartilage and intervertebral disc. Soman and A4 poisoning do not modify the kinetic parameters of pyrimidoxime, but A4 poisoning increases HI6 tissue concentration. 4. Chromatography of urine and plasma showed only unchanged pyrimidoxime in both healthy and poisoned animals. In contrast, HI6 in plasma and urine was strongly degraded by scission of the quaternary ammonium bond, and formation of 2-pyridine aldoxime.

Genotoxicity testing: phage T7 inactivation test of various furan and arenofuran derivatives.

The genotoxic activities of 28 furan and arenofuran derivatives were tested by the phage T7-inactivation test. The genotoxic activity of the compounds was characterized quantitatively. All the compounds studied have pronounced genotoxic activities in our system. Empirical rules relating structure to genotoxic activity were found. Data obtained with our system were compared with the results of other biological systems (Salmonella assay, SOS Chromotest, CHO/HGPRT, gene amplification) in the case of some compounds included as references.

Macrofilaricidal activity of metabolites of diethylcarbamazine.

The antifilarial compound diethylcarbamazine (N,N-diethyl-4-methyl-2-piperazine, DEC) is known rather for its micro- than macrofilaricidal activity. But in some human filariasis i.e. loaiasis, lymphatic filariasis, the spectrum of DEC activities extends to adult filaria. The potential role of the metabolites of DEC in the action of the parent drug once it had been metabolized in the body of infected animals was investigated. The metabolites were evaluated in a new experimental model on which DEC is active: Molinema dessetae in its natural host, the rodent Proechimys oris. Experimental studies were carried out in vivo and in vitro, on microfilariae, infective larvae and adult filaria. Several other nematodes were also used. The metabolites were DEC itself, N-ethyl-4-methyl-1-piperazine-carboxamide (MEC) and their N-oxides, 4-methyl-piperazine-carboxamide and N,N-diethyl-1-piperazine-carboxamide. In vivo most of the metabolites were found active on microfilariae and both N-oxides active on adults and infective larvae. In vitro, the activity of the metabolites was observed only with high concentrations; the in vitro test could not be used as a screening method for antifilarial chemotherapy with piperazine derivatives. Infective larvae were the most sensitive stage. In the rodent and in man, the antifilarial action of DEC is swift and of short duration. This action is prolonged by the activity of metabolites, especially the N-oxides.

Evaluation of the genotoxic activity of 2-nitroanthrafurans.

We measured the genotoxic activities in two bacterial tests, the Salmonella/histidine assay (a reverse mutation assay) and the SOS chromotest (an assay for SOS induction in E. coli), of three 2-nitroanthrafurans: 2-nitroanthra[1,2-b]furan (R-7688), the isomeric compound 2-nitroanthra[2,1-b]furan (R-7686) and its 8-methoxylated derivative (R-7707). Their genotoxic activities were compared to that of 7-methoxy-2-nitronaphtho[2,1-b]furan (R-7000) which has been studied in previous works (Arnaise et al., 1986). We found that: (1) for all three 2-nitroanthrafurans, as generally observed for other 2-nitrofuran derivatives, the responses were correlated in the 2 tests and were decreased in the presence of an 'activating mixture' and in nitroreductase-deficient strains; (2) in contrast to what is usually observed with other 2-nitrofuran derivatives for which methoxylation increases genotoxic activity, the genotoxic activity of the methoxylated 2-nitroanthrafuran (R-7707) was comparable and may be even lower than that of the unsubstituted 2-nitroanthrafuran (R-7686); (3) the addition of a third ring that leads from 2-nitronaphthofurans to 2-nitroanthrafurans increased slightly the genotoxic activity of these compounds; (4) compounds with the oxygen heteroatom outside the 'bay region', R-7686 and R-7707, gave higher responses than their isomers with the oxygen heteroatom within the 'bay region', R-7688.

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene.

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.

Comparison of the carcinogenic effects of two 2-nitro-naphthofurans injected sub-cutaneously in rats.

7-Methoxy-2-nitro-naphtho[2,1-b]furan (R 7000) and its methylated homolog in position 1 (R 7372) are among the most mutagenic agents presently known, as shown by the results obtained both in the Ames test and in the SOS Chromotest. Their carcinogenic effects were tested in rats. We were able to confirm the carcinogenic effects of these nitro-naphthofurans, the presence of a methyl group--while increasing the mutagenic effect of R 7000 10 times--induces a significant decrease of the carcinogenic effects in R 7372. The discrepancy between the mutagenic effects in bacterial assays and the carcinogenic effects of these two 2-nitro-naphthofurans remains to be explained.

Genotoxic activity of two furan analogues of benzo[a]pyrene and their 2-nitro derivatives.

We measured the genotoxic activities in two bacterial tests, the Salmonella/histidine assay (a reverse mutation assay) and the SOS Chromotest (an assay for SOS induction in E. coli), of two pairs of isomeric furan analogues of benzo[a]pyrene: pyreno[1,2-b]furan (R7490) and pyreno[2,1-b]furan (R7692) and their 2-nitro derivatives, 8-nitro-pyreno[1,2-b]furan (R7489) and 8-nitro-pyreno[2,1-b]furan (R7691). We found that: For all 4 compounds, the responses were correlated in the two tests. For the 2-nitro derivatives, R7489 and R7691, the responses were extremely high, reaching SOS-inducing potencies of 5.2 X 10(3) and 10(5)/nmole in the SOS Chromotest and mutagenic potencies of 6.3 X 10(4) and 3.7 X 10(7) revertants/nmole in the Salmonella/histidine assay (strain TA98), respectively; the responses were only slightly decreased in nitroreductase-deficient strains. The responses to the two pyrenofurans were increased in the presence of an "activating mixture" but were still lower than that to benzo[a]pyrene. In contrast to benzo[a]pyrene and pyreno[2,1-b]furan (R7692), pyreno[1,2-b]furan (R7490) also gave a response in the absence of an "activating mixture". (5) Compounds with the oxygen heteroatom within the "bay region" gave lower responses than their isomers with the oxygen heteroatom outside the "bay region".

Genotoxic activities of 2-nitronaphthofurans and related molecules.

The genotoxic activities of 63, 2-nitronaphthofurans and related molecules were examined using two bacterial short-term tests, the Salmonella mammalian microsome assay test or Mutatest, a mutagenesis assay, and/or the SOS Chromotest, an assay for induction of an SOS function in Escherichia coli. Seven compounds were also investigated in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) test, a mammalian gene mutation assay. Our main conclusions are the following: (a) Simple empirical rules relating structure to mutagenic activity in the Mutatest can be derived for some of the compounds. In particular, they account for the extremely high Mutagenic Potency of 7-methoxy-1-methyl-2-nitronaphtho[2,1-b]furan (R7372), approximately 2 X 10(6) mutants/nmol on strain TA100. (b) There is a good quantitative correlation between the Mutagenic Potency in the Salmonella/mammalian microsomes assay and the SOS-inducing potency in the SOS Chromotest. This, and previous evidence, suggests strongly that the 2-nitronaphthofurans derivatives are essentially recA and thus probably umuDC-dependent mutagens. (c) Four out of seven compounds tested in the CHO/HGPRT assay gave responses correlated with the bacterial responses. One of them, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), is among, or is, the strongest mutagen described for mammalian cells. We briefly discuss the practical and theoretical implications of these results.