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A novel series of 10-benzylidene-9(10H)-anthracenones and 10-(phenylmethyl)-9(10H)-anthracenones were synthesized and evaluated for antiproliferative activity in an assay based on K562 leukemia cells. The 3-hydroxy-4-methoxybenzylidene analogue 9h was found to be the most active compound (IC(50) K562: 20 nM). Structure-activity relationships are also considered. The highly active compound 9h and the 2,4-dimethoxy-3-hydroxybenzylidene analogue 9l were tested against five tumor cell lines using the XTT assay, including multidrug resistant phenotypes. Induction of cell death in a variety of tumor cell lines was determined in a monolayer assay using propidium iodide. Noteworthy, all compounds within the series induced elongations in K562 cells similar to vinblastine-treated cells. The effect of the lead compound 9h on K562 cell growth was associated with cell cycle arrest in G2/M. Concentrations for 50% KB/HeLa cells arrested in G2/M after treatment with 9h and 9l were determined and found to be in the range of 0.2 microM. Additionally, we monitored the dose dependent caspase-3-like protease activity in K562 cells and MCF-7/Casp-3 cells treated with 9h, indicating induction of apoptosis. Western blotting analysis demonstrated that 9h caused a shift in tubulin concentration from the polymerized state found in the cell pellet to the unpolymerized state found in the cell supernatant. Seven compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds such as colchicine, podophyllotoxin, and nocodazole. In general, the antiproliferative activity correlated with inhibition of tubulin polymerization. The most active compounds strongly displaced [(3)H]colchicine from its binding site in the tubulin, yielding IC(50) values 3- to 4-fold lower than that of colchicine. The novel benzylidene-9(10H)-anthracenones described in the present study constitute an interesting group of highly active and easily accessible antimitotic agents that inhibit tubulin polymerization.

A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

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Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of <100 nM. The methylamino analogue 14i was the most active compound in this assay (14i, IC50 K562: 0.05 muM). Antiproliferative activities of selected compounds were additionally evaluated against a panel of 12 tumor cell lines, including multi-drug-resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with selected compounds were arrested in the G2/M phases of the cell cycle. In competition experiments, these compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values lower than that of colchicine. The results demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

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An enzyme has been isolated from Escberichia coli strains harboring the I-like plasmid R64drdll, which is capable of initiating DNA synthesis -on the circular, singlestranded DNA of phages 4X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically witf DNA polymerase III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drdll or ColIdrdl, which are derepressed in functions of conjugational DNA transfer, is severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis. The I-like plasmids (R64, R144, and ColT) of the incompatibility group Ta are large, circular DNA molecules with molecular weights ranging from 62-72 X 106 (1, 2). Transfer properties specified by I-like plasmids are generally repressed, but derepressed (drd) mutants have been isolated (3, 4) and used to study the transfer of conjugative plasmids from donor to recipient cells of Escherichia coli (5-7). Only one of the strands of the plasmid is transferred during conjugational DNA synthesis (5). Conjugational DNA synthesis is independent of the E. coli dnaB and polA functions but requires the dnaE gene product in the recipient cell (6, 7). In addition, drd mutants of plasmids R64, R144, and ColT, but not those of F-like plasmids, partially suppress the phenotype of E. coli dnaG mutants (8).Many conjugative plasmids are able to suppress or enhance the temperature sensitivity of E. coli dnaB mutants (9), and five different R plasmids, including R64drdll and R144drd3, have been used to construct viable R-derivatives of the unsuppressed (sup+) E. coli dnaB266 amber mutant (10). This has been interpreted as evidence for the presence of dnaB analog (ban) genes being associated with these plasmids (10). During our studies in collaboration with V. N. Iyer (Ottawa) designed to identify a ban protein of plasmid R64drdll, we have discovered an enzyme capable of initiating DNA synthesis on circular single-stranded (ss) DNA templates. A similar enzyme activity has subsequently been detected in extracts from strains harboring the plasmids R144 and ColI. The enzyme level in extracts from st...

This study examined the relationship between speech characteristics and psychopathology throughout the course of affective disturbances. Our sample comprised 20 depressive, hospitalized patients who had been selected according to the following criteria: (1) first admission; (2) long-term patient; (3) early entry into study; (4) late entry into study; (5) low scorer; (6) high scorer, and (7) distinct retarded-depressive symptomatology. Since our principal goal was to model the course of affective disturbances in terms of speech parameters, a total of 6 repeated measurements had been carried out over a 2-week period, including 3 different psychopathological instruments and speech recordings from automatic speech as well as from reading out loud. It turned out that neither applicability nor efficiency of single-parameter models depend in any way on the given, clinically defined subgroups. On the other hand, however, no significant differences between the clinically defined subgroups showed up with regard to basic speech parameters, except for the fact that low scorers seemed to take their time when producing utterances (this in contrast to all other patients who, on the average, had a considerably shorter recording time). As to the relationship between psychopathology and speech parameters over time, we found significant correlations: (1) in 60% of cases between the apathic syndrome and energy/dynamics; (2) in 50% of cases between the retarded-depressive syndrome and energy/dynamics; (3) in 45% of cases between the apathic syndrome and mean vocal pitch, and (4) in 71 % of low scores between the somatic-depressive syndrome and time duration of pauses. All in all, single parameter models turned out to cover only specific aspects of the individual courses of affective disturbances, thus speaking against a simple approach which applies in general.