Microbial transformations of natural antitumor agents. 23. Conversion of withaferin-A to 12 beta- and 15 beta-hydroxy derivatives of withaferin-A.

Microbial transformation experiments were conducted with the antitumor lactone withaferin-A. Cunninghamella elegans NRRL 1393 transformed withaferin-A (1a) to 15 beta-hydroxywithaferin-A (2a) and 12 beta-hydroxy-withaferin-A (3a). The hydroxylated metabolites were isolated by solvent extraction and were purified by column and thin-layer chromatography. Structures of the hydroxylated metabolites were determined by proton-and carbon-13 NMR, IR and mass spectral analyses, and by the preparation of acylated derivatives. Compounds 2a and 3a inhibited the growth and biochemical functions of in vitro grown P-388 lymphocytic leukemic cells.

Inhibition of mitochondrial functions by the antibiotics, bikaverin and duclauxine.

Four cytotoxic antibiotics, bikaverin, duclauxine, PSX-1 and vermiculine, were examined with respect to their interference with glycolysis and respiration and their possible ionophoric or cytolytic activity. Duclauxine was found to be an inhibitor of respiration and bikaverin an uncoupler of oxidative phosphorylation of tumor cells and of isolated rat liver mitochondria. Bikaverin was also found to be an efficient haemolytic agent. Vermiculine and PSX-1 showed no effect in the systems employed.

Vermixocins A and B, two novel metabolites from Penicillium vermiculatum.

Vermixocins A and B, 3-(1'-hydroxy-3'-methylbutyl)- and 3-(1'-acetoxy-3'-methylbutyl)-11-hydroxy-4-methoxy-9-methyl-5H,7H- dibenzo[c,f][1,5]dioxocin-5-one, respectively, were isolated from the mycelium of Penicillium vermiculatum. Both metabolites showed cytotoxic effects on lympholeukemia cells P388.

Synthesis and biological activity of (7S)-O-epoxyalkyl derivatives of daunomycinone.

Synthesis and antibacterial activity of a number of 7-O-epoxyalkyl derivatives of daunomycinone prepared from 7-O-alkenyl derivatives of daunomycinone are described along with their inhibitory effect on leukemia P 388 cells.

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.

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).

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.

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.

Immunosuppressive properties of the antibiotics cytostipin and vermiculine.

The properties of two antibiotics, cytostipin isolated from Penicillium stipitatum and vermiculine isolated from Penicillium vermiculatum, were examined in two systems for a rapid screening of current immunosuppressive agents [nucleolar test determining the degree of RNA synthesis in nucleoli of individual lymphocyte populations, and the reactivity of mouse lymphocytes to "T" (PHA) and "B" (LPS) mitogens]. Both antibiotics, distinctly suppressed the increase in number of "active" lymphocytes with compact nucleoli in the popliteal lymph node activated by SRBC. Incorporation of 3H-uridine into PHA-stimulated "T" lymphocytes was suppressed by both antibiotics, incorporation into LPS-stimulated "B" lymphocytes was inhibited by cytostipin but stimulated by vermiculine. The antibiotics were also tested in another two "classic" immune systems, the Jerne test and the GVH reaction. Both antibiotics in doses markedly inhibitory for GVH reaction did not suppress but significantly increased the number of the haemolytic plaques in spleens of SRBC-immunized mice.

Regulation of biosynthesis of vermiculin and vermistatin in Penicillium vermiculatum.

Biosynthesis of vermiculin (1) and vermistatin (2) in Penicillium vermiculatum can be controlled by the carbon and nitrogen sources. Glucose and sucrose affect the levels of the two metabolites; cornsteep liquor influences the quality the biosynthesis. The concentrations of Fe3+ and Cu2+ ions also affect the biosynthesis, the effect being dependent on the type of carbon source utilized. The compounds capable of electron transport generally stimulate the production of 1 and 2 but do not influence the biosynthesis qualitatively.

The antibiotic PSX-1 produced by Penicillium stipitatum is identical with botryodiplodin.

Antibiotic PSX-1 exhibiting antitumour activity, formerly isolated from the filtrate of a culture of Penicillium stipitatum, was shown to be identical with botryodiplodin.

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.

Regulation of biosynthesis of thiolutin and aureothricin in Streptomyces kasugaensis.

L-Methionine and DL-ethionine decreased production of thiolutin and aureothricin in Streptomyces kasugaensis. In the presence of L-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration of L-methionine in the medium.

Microbiological and chemical dehydrogenation of withaferin A.

Arthrobacter simplex dehydrogenated withaferin A to 4-dehydrowithaferin A but it was not able to dehydrogenate this substrate in position 27. 27-Dehydrowithaferin A was prepared chemically using pyridinium chlorochromate. Whereas 4-dehydrowithaferin A surpassed in its effect on leukemic (388 cells the original compound and all its derivates synthesized so far, 27-dehydrowithaferin A was biologically inactive.

Production of (-)-mitorubrinic acid byPenicillium vermiculatum.

Biosynthesis of (-)-mitorubrinic acid and (-)-vermistatin byPenicillium vermiculatum strain IV/5 was stimulated by high concentration of glucose and urea as a C and N source, respectively. Effect of phosphate, trace elements and organic acids was also studied.

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.