n-trifluoroacetyladriamycin and valrubicin

Drug-DNA binding is claimed to be the basis by which the antitumor antibiotic adriamycin (doxorubicin) inhibits DNA and RNA synthesis in vitro. However, in preliminary studies the DNA-non-binding adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD 32) showed somewhat greater inhibition of DNA and RNA synthesis than adriamycin under identical conditions. The kinetics of macromolecule synthesis inhibition induced by adriamycin and AD 32, and the two principal DNA-non-binding metabolites of AD 32, N-trifluoroacetyladriamycin (AD 41) and N-trifluoroacetyladriamycinol (AD 92), have now been subjected to comparative study in cultured CEM (human leukemic lymphoblastic) cells. At equimolar concentrations (10 microM), or at concentrations related to their 50% growth-inhibitory values vs CEM cells, AD 32 was consistently found to be more inhibitory than adriamycin of DNA and RNA synthesis, as measured by the incorporation of tritiated thymidine and uridine, respectively, into acid-precipitable fractions relative to untreated controls. Marked inhibitory activity was apparent with 10 microM AD 32 even at the earliest sampling time (15 min); with adriamycin at the same concentration the maximal effect was not achieved until 3 h. AD 32 at 4.8 microM concentration continued to show strong inhibition of nucleic acid synthesis, whereas adriamycin at 1.0 microM was essentially inactive. Like AD 32, AD 41 and AD 92 showed greater inhibition than adriamycin of DNA and RNA synthesis at the early sampling times, although in all instances the effects of AD 32 were more profound. AD 32 at 10 microM concentration produced a moderate but significant inhibition of the incorporation of tritiated methionine into protein compared with adriamycin, which at this concentration was not active. Parallel HPLC analytical studies with similar drug-treated cultures indicated that, while small amounts of adriamycin were found in cells treated with 10 microM AD 32, the amount of adriamycin present at 15 min was only a small fraction (less than 5%) of the amount of adriamycin achieved at 3 h in cultures treated with 1.0 microM adriamycin, a concentration already shown to be only slightly inhibitory of nucleic acid synthesis under the culture conditions. The present study thus confirms the marked DNA and RNA synthesis-inhibitory effects of AD 32, and establishes that this inhibitory activity is not due to conversion of AD 32 into adriamycin. These findings accordingly call into questio

Topics: Cell Survival; Cells, Cultured; DNA; DNA Replication; Doxorubicin; Humans; Protein Biosynthesis; RNA

N-(Trifluoroacetyl)adriamycin 14-valerate (AD 32), a novel DNA nonbinding analogue of adriamycin with superior experimental antitumor activity, has undergone extensive clinical trial, with documentation of antitumor activity and low toxicity in human subjects. However, poor water solubility necessitates that the drug be administered to patients by continuous intravenous infusion at high dilution in a surfactant-containing formulation, with steroid prophylaxis to protect against a chest pain syndrome associated with the vehicle. On the basis of pharmacologic considerations, the title compounds have been prepared as second-generation analogues of N-(trifluoroacetyl)adriamycin 14-valerate with improved aqueous solubility; use is made of the available carboxylic acid function to solubilize the products in dilute aqueous alkaline medium. Target compounds were made by treating N-(trifluoroacetyl)-14-halodaunorubicin (bromo or iodo) with monosodium salts of dibasic acids (malonic, succinic, glutaric, adipic, pimelic, azelaic, sebacic) in aqueous acetone. All of the products showed significant in vivo antitumor activity against the murine P388 leukemia (ip tumor, ip treatment once daily on days 1, 2, 3, and 4); most compounds were superior to the +181% increase in life span afforded by adriamycin (optimal dose 3.0 mg/kg per day), one of two drugs used as positive controls for the assays. Several of the test compounds showed highly curative activity in this system, similar to N-(trifluoroacetyl)adriamycin 14-valerate, the other positive control agent. The hemiadipate product exhibited the most desirable properties of high antitumor efficacy (86% cure rate all P388 tumor-bearing animals through four levels of a 40-70 mg/kg dose-response range), aqueous solubility (60 mg/mL in pH 7.4 phosphate buffer), and solution stability (no decomposition at 4 degrees C, 0.5% hydrolysis at 27 degrees C, over 24 h at pH 7.4).

Topics: Animals; Chemical Phenomena; Chemistry; Daunorubicin; Doxorubicin; Drug Stability; Esters; Hydrogen-Ion Concentration; Leukemia P388; Leukemia, Experimental; Male; Mice; Solubility; Water

The experimental and clinical antitumor activity, as well as the low toxicity, of N-(trifluoroacetyl)adriamycin 14-valerate (AD 32), a non-DNA binding anthracycline analogue, has led us to prepare and evaluate several N-perfluoroacyl analogues of daunorubicin, adriamycin, and N-(trifluoroacetyl)adriamycin 14-valerate. Target compounds were prepared by reaction of the appropriate perfluoroacyl anhydride with daunorubicin in chloroform-ether, with adriamycin in cold pyridine, and with adriamycin 14-valerate in ethyl acetate. In connection with this work, it was found that reaction of perfluoroacyl anhydrides with N-acylated or N-unsubstituted anthracyclines in pyridine at room temperature afforded with ease and in good yield the corresponding 9,10-anhydro-N-acylated derivatives. A number of products showed good to highly significant antitumor activity in vivo against the murine P388 leukemia system. However, the lack of in vivo antitumor activity of the pentafluoropropionyl and heptafluorobutyryl analogues of N-(trifluoroacetyl)adriamycin 14-valerate is noteworthy. The results continue to show that non-DNA binding anthracycline analogues can exhibit in vivo antitumor activity. Loss of the anthracycline 9-carbinol function by dehydration leads to reduction of biological activity as compared to the parent compound.

Topics: Animals; Antineoplastic Agents; Daunorubicin; Doxorubicin; Leukemia L1210; Leukemia P388; Mice; Molecular Conformation

We have used a laser flow cytometer to excite and quantitate the intracellular fluorescence of cells exposed in vitro and in vivo to various anthracyclines. In cells exposed to Adriamycin (ADR), intracellular drug fluorescence appeared slowly and reached a peak after 4 hr of incubation. Cells incubated with 10 micrograms/ml were 5 times more fluorescent than were cells incubated with 1 microgram/ml. Cells exposed to daunomycin were 2 to 4 times more fluorescent than were cells similarly exposed to ADR, and the intracellular appearance of daunomycin fluorescence was much more rapid. Cells exposed to N-trifluoroacetyladriamycin and carminomycin had higher amounts of intracellular fluorescence (2 to 4 times), and peak values were reached much more rapidly than in cells exposed to ADR. In cells exposed to rubidazone, fluorescence increased 2- to 4-fold with increased drug concentration and length of exposure. In contrast, nogalamycin fluorescence reached a peak after 60 min of incubation, and a 10-fold increase in drug concentration increased fluorescence only 2-fold. In animals given injections of ADR (4 mg/kg) and sacrificed after 3 hr, drug fluorescence could be detected in tumor and spleen cells. In contrast, fluorescence in heart nuclei was barely recognizable. However, incubation of isolated nuclei in ADR (1 microgram/ml) showed that bone marrow and heart nuclei had greater amounts of ADR fluorescence (2- to 3-fold) than did spleen or liver nuclei similarly treated. The use of laser flow cytometry for monitoring intracellular anthracycline transport, binding, and efflux is demonstrated.

Topics: Antibiotics, Antineoplastic; Carubicin; Cell Nucleus; Cells, Cultured; Cytoplasm; Daunorubicin; Doxorubicin; Glycosides; Humans; Lasers; Microscopy, Fluorescence; Naphthacenes; Nogalamycin; Spectrometry, Fluorescence