New cytotoxic and antitumor agents. VII. Derivatives of 1-benzylidenisoindolin-3-one and 5,6-dihydro-8H-isoquinolo(2,3-a)phthalasin-5-one.

The in vitro cytotoxic effects on P388 leukemia cells of 19 derivatives and analogs of 1-benzylidenisoindolin-3-one, 5,6-dihydro-8H-isoquinolo(2,3-a)phthalasin-5-one and 4-benzyl-1,2-dihydrophthalasin-5-one were studied. The derivatives of the first group which preferentially inhibited uridine utilization were more effective. The cytotoxic effects of these substances depended on the quality of substituents, on the presence of a nitrogen atom in the 5-membered heterocycle of the molecule as well as on the spatial arrangement of the molecule. (Z)-narceine imide-N-oxide II and (Z)-3-(6-ethyl-2-methoxy-3,4-methylenedioxy) benzyliden-6,7-dimethoxy-isoindolin-3-one III were the most active agents, both of them causing a decrease in the proliferation rate of P388 cells. After its addition to the culture medium, compound III caused irreversible damages leading to death of the cells. The removal of the inhibitor did not lead to the repair of the damages either. Of the other two groups of substances, 5-hydroxy-3,4,12-trimethoxy-8-methyl-10,11-methylenedioxy-8H-isoquino lo (2,3-a)phthalasin (XVIII) had a marked inhibitory effect which, at concentrations of up to 25 micrograms ml-1, inhibited the proliferation rate of P388 cells.

New potential cytotoxic and antitumor substances. II. Effect of hydroxyanthraquinones and their glucosides on Ehrlich ascites carcinoma cells in vitro.

Mono and dihydroxyanthraquinones and their derivatives affected the utilization of 14C-labeled precursors nucleic acid and protein synthesis into Ehrlich ascites carcinoma (EAC) cells. Inhibitory effect of the substances markedly increased when one acetyl group was introduced into the basic molecule. As the tested substances inhibited mainly incorporation of 14C uridine into the cold TCA insoluble fraction of EAC cells, their mechanism of action can be explained by the inhibition of RNA synthesis.

Sacharose density gradient separation of Ehrlich ascites carcinoma and L-5178Y tumor cells in different cell cycle phases.

Optimal conditions were determined for the distribution of Ehrlich ascites carcinoma (EAC) and L-5178Y mouse tumor cells, proliferating in vivo, by their age within the cell cycle by sedimentation in a buffered linear sacharose density gradient. Measurements of cell size, DNA content and incorporation of tritiated thymidine in successive parts of the gradient confirmed the actual separation of cells of different age: in the upper fractions there were cells in G1 phase, in the middle fractions in S phase and in the lower layers of the gradient there were cells in G2 and/or M phase.

New potential cytotoxic and antitumor substances I. In vitro effect of bikaverin and its derivatives on cells of certain tumors.

Bikaverin and its derivatives have been found to affect precursor utilization of nucleic acid and protein synthesis in the cells of Ehrlich ascites carcinoma (EAC). Mainly the uridine incorporation into EAC cells was inhibited. This is in agreement with the known concept that anthraquinones, to which bikaverin may also be assigned, intervene into RNA synthesis. The substances followed exerted a cytotoxic effect on in vitro proliferating cells of the three studied tumors. The ED50 values found for cells of these tumors were: EAC 0.5 mug/ml; leukemia L 5178 1.4 mug/ml; sarcoma 37 4.2 mug/ml.

New potential cytotoxic and antitumor substances. III. In vitro effect of 3-benzazepine derivatives on P388 cells.

3-Benzazepine derivatives manifested cytotoxic effects in in vitro tests on lympholeukemia P388 cells. The most efficient derivative (QF 1), i. e. 7,8-dihydro-3,4,12-trimetoxy-7-dimethylamino-10,11-methylenedioxy-5H-indolo [1,2-b][3]benzazepine-5-on in a concentration as low as 5 microliter/ml, considerably inhibited only the incorporation of labeled uridine into P388 cell fractions. In in vitro experiments, this substance blocked cell proliferation. After its prolonged action, the number of dead cells increased and this also when its interaction with the cells lasted but a short time--the substance being removed from the medium. In all probability, the substance retains the cells in the G1/S phase. A marked synergistic effect of tubercidine was attained in the presence of the substance.

New substances with cytotoxic and antitumor effects. IV. In vitro effect of some veratrum alkaloids and their derivatives on leukemia P388 cells.

Some Veratrum alkaloids and their derivatives exhibited an in vitro cytotoxic effect on leukemia P388 cells, depending on the structure of the skeleton of the molecule, particularly on the type of the heterocycle attached to C-20. Veracintine and 20-(2-methyl-1-pyrrolin-5-yl)-4-pregnen-3-one, which proved to be the most effective, inhibited incorporation of uridine, and to a lesser extent that of L-valine into P388 cells fractions. After a brief reaction (15 min), these substances became irreversibly bound in the P388 cells and stopped their further in vitro proliferation. The cytotoxic effect of veracintine became enhanced by sublethal doses of tubercidine (phase of maximum lethality of G1).

Cytotoxic and mutagenic in vitro effect of 7-O-epoxyalkyl derivatives of daunomycinone. Part IX.

The cytotoxic effect of (7S)- and (7R)-O-epoxyalkyl derivatives of daunomycinone on leukemia P388 cells was followed in in vitro tests and their mutagenicity was determined by means of the bacterial SOS chromotest. The biological effects of the substances were compared with those of daunomycin, carminomycin and nogalamycin. The most efficient derivative proved to be the (7S)-9-acetyl-4-methoxy 7-O-(2,3-epoxypropyl)-7,8,9,10-tetrahydro-6,9,11-trihydroxy-5, 12-naphthacenequinone 10 which inhibited the DNA and RNA synthesis and proliferation of P388 cells on the level of daunomycin or carminomycin. The cytotoxic and mutagenic action of 7-O-epoxyalkyl derivatives of daunomycinone was affected by the length of alkyl and its configuration.

Different sensitivity to antibiotics of the established intermitent in vivo--in vitro tumor cell lines in comparison with the primary cultures.

From original strains of tumors growing only in vivo (I) new lines of strains (II) were obtained, the cells of which proliferated readily both in vitro and in vivo. Sensitivity in all the new strains (II) generally increased but in varying degrees as against individual cancerostatic drugs, as evident from the given ID50 values and sensitivity indices. In addition to an enhanced sensitivity of the strains to inhibitors, their ability to utilize precursors of the synthesis of nucleic acids and proteins in a linear dependence during the course of 24 hours has also been determined.

Novel cytotoxic and antitumor agents. IV. Withaferin A: relation of its structure to the in vitro cytotoxic effects on P388 cells.

In vitro effects of withaferin A and its 9 new derivatives on P388 cells have been studied. The cytotoxicity was calculated from the utilization of precursors in protein and nucleic acid (NA) synthesis and from capacity to suppress cell proliferation. The most potent agents proved to be 4-dehydrowithaferin A and withaferin A diacetate exhibited an equal inhibitory effect on thymidine, uridine, and L-valine incorporation. They stopped cell proliferation and, at the same time, killed the cells. Cytotoxicity was found to be due to a double bond at position C2-3, by dissociating this bond the cytotoxicity markedly decreased in all derivatives. A dissociation of the double bond at C24-25 or a removal of OH group from C27 did not cause any significant changes in the biological effects of the derivatives. An addition of a carbonyl group at C4 increased the effects of the agent. An addition of OH groups to the molecule of withaferin A resulted chiefly in a qualitative change in the action of derivatives manifested by a significant decrease in L-valine inhibition. As withaferin A promptly reacted with L-cysteine, it was presumed that one of the possible target sites in the cell might be the SH groups of enzymes which react with the lactone and epoxide groups of the agent.

Inhibition of Tritrichomonas foetus by vermiculine in vitro.

The macrolide aglycosidic antibiotic vermiculine, added to the cultivation medium to a concentration of 30 micogram/ml, has an in vitro inhibitory effect on Tritrichomonas foetus. The agent interacts rapidly with the cells, causing irreversible changes after several hours of action. The changes are not repaired on removing the agent; the cells suffer from a rapid inhibition of nucleic acid synthesis, the protein synthesis remaining intact.

Antitrichomonadal and other biological effects of sugar hydrazones and phenylosazones.

When investigating the possible antitrichomodal effect of 41 phenylsazones, nitro- and dinitro-phenylhydrazones a significant inhibitory effect on multiplication of Trichomonas foetus could be detected in 14 derivatives; eight derivatives were ineffective, other derivatives exhibited only a slight effect. The inhibitory effect of most compounds increased after acetylation. Toxicity of seven effective compounds was determined in vivo. Mutagenicity of these compounds was followed with microorganisms and their cytotoxicity with tumor EAC cells. Two of the effective compounds exhibited also a significant mutagenic effect, three of eight compounds had a pronounced cytotoxic effect, three of eight compounds had a pronounced cytotoxic effect in vitro on the EAC cells, in which they inhibited mainly the incorporation of adenine.

In vitro effect of duclauxin and derivatives of coumarin on nucleic acid and protein synthesis in Ehrlich's Ascites Carcinoma cells (EAC).

The effect was studied of 32 natural and synthetic derivatives of coumarin, dicoumarin, and 4.4'epoxydicoumarin on the uptake of 14C-labelled precursors in nucleic acid (NA) and protein synthesis by EAC cells in vitro. Of the three tested groups of compounds, the most cytotoxic effect was found in the derivatives of coumarin that inhibited, on the same level, incorporation of the added 14C-adenine, 14C-L-valine and 14C-uridine but not 14C-thymidine. The utilization of the present precursors by EAC cells was not affected by epoxydicoumarins. Derivatives of coumarin and dicoumarin inhibited only NA synthesis in in vitro proliferating EAC cells. Of the evaluated compounds, chiefly duclauxin specifically decreased proliferation of EAC cells in vitro.

7,8-dihydro-5H-isoindolo[1,2-b] [3]benzazepine-5-ones. Part 2: In vitro effect of derivatives substituted at position 7 on leukemia P 388 cells.

The in vitro cytotoxic effect on leukemia P 388 cells of new derivatives, 7,8-dihydro-5H-isoindolo[1,2-b] [3] benzazepine-5-ones substituted at position was evaluated. The most effective were the derivatives which were substituted at position 7 by the dimethylamino, hydroxy and/or ethoxy group. These substances selectively inhibited incorporation only of 14C-uridine into P 388 cells fractions. Substance 7 which was studied more intensively stopped the proliferation of P 388 cells in vitro, but caused only a slight killing effect. The maximum inhibitory effect of this substance on P 388 cells is related to the G1/S phase of the cell cycle. In in vitro test, the substance showed synergism with the cytostatics: cycloheximide, methotrexate and vermiculin, their phase of greatest lethal activity also being G1/S or early S.

[Screening of antitumor antibiotics, formed by molds]. / K voprosu o skrininge protivoopukholevykh antibiotikov, obrazuemykh plesnevymi gribami

A system for screening fungal metabolites with cytotoxic activity against tumor cells is described and the results obtained using this system are discussed. It was found that 35.2 per cent of the strains isolated from uranium mines had a cytotoxic effect on the EAC cells in vitro. As for the strains isolated from other sources only 6.85 and 9.87 per cent of them inhibited the EAC cells in vitro. Five substances, i. e. vermiculline, PSX-I, Frequentine, bikaverin and duclauxin isolated from 227 evaluated cultures showed a strong inhibitory effect on the EAC cells and other tumors in vitro.

In vitro effect of alpha, beta-unsaturated sulphones of the 5-nitrofuran series on the incorporation of C-labelled precursors into Ehrlich's ascites carcinoma cells.

The effect of new 5-nitrofuran (5NF) derivatives on the incorporation of 14C precursors of nucleic acids and protein synthesis into Ehrlich's ascites carcinoma (EAC) cells in vitro has been studied. Of the 32 substances that were tested, 22 markedly inhibited the incorporation of adenine, L-valine, uridine and thymidine. The 5NF derivatives influenced the synthesis of nucleic acids in vitro and the proliferation of EAC cells. The effect of the derivatives depends on the nature and position of the substituents as well as on the spatial arrangement of the whole molecule. Some of the evaluated derivatives showed a weak virostatic effect and induced lysogenicity in E. coli C 600. As most of the tested 5NF inhibited E. coli(rec-) mutants, it can be concluded that the (rec-) gene evidently takes part in the repair of their damage.