Synthesis and mass-spectrometric analysis of N-cycloalkyl derivatives of carminomycin, daunorubicin and their analogs

Condensation of carminomycin or daunorubicin with glutaric dialdehyde in the presence of NaBH3CN yielded 3'-deamino-3'-piperidinocarminomycin or 3'-deamino-3'-piperidinodaunorubicin and corresponding (13-R, S)-dihydroderivatives. To prepare similar derivatives of 14-hydroxycarminomycin or doxorubicin, 13-dimethylketals of 14-bromocarminomycin or 13-bromodaunorubicin were used in the reaction of reductive alkylation with glutaric or glycolic dialdehyde to give 3'-deamino-3'-piperidino- or 3'-deamino-3'-morpholino derivatives of 13-dimethylketals of 14-bromocarminomycin or daunorubicin, respectively. After deblocking and subsequent hydrolysis of these compounds 3'-deamino-3'-piperidino- and 3'-deamino-3'-morpholino derivatives of 13-hydroxycarminomycin or doxorubicin were prepared. Reduction of the antibiotic derivatives under mass spectrometry conditions was demonstrated.     

Immunodepressive action of carminomycin and rubomycin derivatives]

The immunodepressive effects of carminomycin and its 3 semi-synthetic derivatives, as well as rubomycin and its derivative R-103 were compared. It was found that 14-hydroxycarminomycin was much superior to the other substances in the experiments with synthesis induction suppression of antibodies against sheep red cells in mice. Suppression of the rejection of the skin allogenic grafts in the mice by carminomycin was higher as compared to that by the other substances. Probably different populations of the immune competent cells have selective sensitivity to separate anthracyclines.     

Semisynthetic derivatives of daunorubicin and carminomycin: inhibition of DNA synthesis after intravenous administration to mice

Inhibition of DNA synthesis in the liver, kidneys, spleen and heart of mice after intravenous administration of 0.1 and 0.3 LD50 of semisynthetic derivatives of rubomycin (daunorubicin) and carminomycin was studied. The level of DNA synthesis inhibition was estimated by a decrease in incorporation of (methyl-3H) thymidine. Under the action of 13-trebutoxycarbonyl hydrazone and 14-salicyloiloxy derivatives of rubomycin and carminomycin maximum inhibition of DNA synthesis was reached later while its recovery started earlier as compared to the initial antibiotics.     

Antiviral action of carminomycin and some of its derivatives]

Carminomycin was shown to inhibit the development of both the DNA-containing variolovaccine virus and the RNA-containing grippe virus in chick embryos. Comparison of the effects of rubomycin, carminomycin, 14-oxy-carminomycin and carminomycin complex with bovine serum albumin in experiments with chick embryos showed that the inhibitory effect of carminomycin and its derivatives on the development of the grippe virus was much higher than that of rubomycin. The carminomycin derivatives proved to be much more active in this respect than the initial antibiotic. Carminomycin and its derivatives had a therapeutic effect on mice with experimental grippe pneumonia also on their oral use.     

Synthesis and antitumor properties of carminomycin 13-cyclohexylidenhydrazone

Carminomycin 13-cyclohexylidenhydrazone (CCH) was prepared by interaction of carminomycin 13-hydrazone with cyclohexane. The antiblastomic properties of CCH were studied on mice with transplantable tumors. The preparation was administered intravenously or orally. The studies showed a high antitumor activity of CCH. When CCH was administered intravenously to mice with lymphosarcoma LIO-1, the antitumor effect selectivity of it was practically equal to that of carminomycin. When used in doses equivalent by their toxicity to those of carminomycin, CCH had practically the same inhibitory effect on sarcoma 180 as carminomycin. When used orally in doses equivalent by their toxicity to those of carminomycin, CCH was more effective than carminomycin in treatment of mice with lymphosarcoma LIO-1, sarcoma 180 and lymphadenosis NK/Ly.     

Toxicity and pharmacological properties of carminomycin complex components]

Acute toxicity of the components of the carminomycin complex after intravenous administration to albino mice increased as follows. I less than II less than III. Component II induced a decrease in all the indices of the bone marrow and peripheral blood of the animals. It was most pronounced in dogs. The dogs died after administration of component II in the lethal doses as a result of the bone marrow aplasia. The indices of the functional state of the liver and kidneys in the animals after administration of components I and II changed slightly. Component III administered repeatedly to rabbits even in low doses induced significant impairments in the function of the liver and kidneys. Component II differed from component I by more pronounced cardiotoxicity. On the basis of the experimental data and the results published earlier component I is recommended for clinical trials as the least toxic one.     

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

Study of toxicity and antineoplastic activity of 14-substituted derivatives of rubomycin and carminomycin

Toxicity and antitumor activity of five derivatives of rubomycin and carminomycin were studied in animals. The derivatives were prepared by modification of the methyl C-14 group. These were the following: 14-chlorrubomycin, 14-chlorcarminomycin, 14-salicyloyloxyrubomycin, 14-salicyloyloxycarminomycin and 14-quinaldinoyloxyrubomycin. The chemotherapeutic study revealed that, in their activity, all the compounds were inferior to the starting antibiotics. Unlike the other derivatives, 14-chlorcarminomycin induced a significant inhibition of leukemia P-388 development (the average lifespan of the mice amounted to 165 per cent as compared to the control). However, in the magnitude of its effect, the derivative was inferior to carminomycin.     

New derivatives of carminomycin and rubomycin

For preparing new semisynthetic analogs of anthracycline antibiotics, hydrolysis of 13-dimethylketals of 14-bromrubomycin and 14-brom-arminomycin in solution of diluted hydrochloric acid was studied. It was shown that such hydrolysis yielded 14-chlorrubomycin and 14-chlorcarminomycin. Conditions for separating the mixture of 14-chlor- and 14-bromrubomycins and the mixture of 14-chlor- and 14-bromcarminomycins by HPLC were developed. Interaction of 14-chlorine derivatives of rubomycin and carminomycin with potassium formate in the presence of the crown ether yielded 14-formyloxy derivatives of rubomycin and carminomycin. Interaction of rubomycin and carminomycin with formic acid in the presence of N-oxysuccinimide and dicyclohexylcarbodiimide resulted in formation of N-formyl derivatives of rubomycin and carminomycin.     

Synthesis and characterization of methacrylic derivatives of 5-amino salicylic acid with pH-sensitive swelling properties

The purpose of this study is to develop novel colon-specific drug delivery systems with pH-sensitive swelling and drug release properties. Methacrylic-type polymeric prodrugs with different content levels of 5-amino salicylic acid (5-ASA) were synthesized by free radical copolymerization of metacrylic acid (MAA), polyethylene glycol monomethacrylate (PEGMA), and a methacrylic derivative of 5-ASA (methacryloyloxyethyl 5-amino salicylate [MOES]). The copolymers were characterized, and the drug content of the copolymers was determined. The effect of copolymer composition on the swelling behavior and hydrolytic degradation was studied in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.2). The swelling and hydrolytic behavior of the copolymers was dependent on the content of MAA groups and caused a decrease in gel swelling in SGF or an increase in gel swelling in SIF. Drug release studies showed that increasing content of MAA in the copolymer enhances the hydrolysis in SIF but has no effect in SGF. The results suggest that hydrogen-bonded complexes are formed between MAA and PEG pendant groups and that these pH-sensitive systems could be useful for preparation of a controlled-release formulation of 5-ASA.     

Synthesis of 13-amino costunolide derivatives as anticancer agents

A number of costunolide derivatives (4a-p) have been synthesized and evaluated for their in vitro cytotoxicity against eight tumor and a non-tumor cell lines. Compound 4d showed around 2-fold better cytotoxicity against SW-620 (colon) cell line with improved safety index than costunolide (1). While compounds 4e, 4g, and 4p have shown around 2- to 3-fold better cytotoxicity against MIAPaCa2 (pancreas), K-562 (leukemia) and PA-1 (ovary) cell lines as well as better safety index in comparison to costunolide (1). Compound 4p also exhibited cytotoxicity against HBL100 (breast) cell line with 2-fold better safety index. Structure-activity relationship has been described.     

Suppression of DNA synthesis in mice by the anthracycline antibiotics daunorubicin, carminomycin and doxorubicin

Inhibition of DNA synthesis by rubomycin (daunorubicin), carminomycin and doxorubicin in the spleen, liver, kidneys and heart was studied on mice. The antibiotics were administered intravenously in a dose of 0.3 LD50. The inhibition level was estimated by incorporation of 3H-thymidine. The time courses of DNA synthesis inhibition by daunorubicin, carminomycin and doxorubicin markedly differed, whereas the patterns of their inhibition curves for all the organs were close. The maximum inhibition of DNA synthesis by carminomycin was observed in 6 hours. After that period it gradually restored. Doxorubicin induced the maximum inhibition of DNA synthesis in 24-48 hours after its administration. Daunorubicin induced two maxima in inhibition of DNA synthesis i. e. in 6 and 48 hours. Definite correlation between the levels of DNA synthesis inhibition by the antibiotics and their toxic action was shown.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Interaction with DNA of semisynthetic carminomycin and rubomycin derivatives

The apparent binding constants and the effect of semisynthetic derivatives of carminomycin and rubomycin (anthracycline antibiotics) on DNA fusion were studied. The following semisynthetic derivatives were used. 13-dihydrocarminomycin, 14-hydroxycarminomycin, 13-(4-methylpiperazinyl) imine carminomycin, 13-benzoylhydrazone carminomycin (carminazone), 13-tret-butoxycarbonyl hydrazone rubomycin, 13-(4-methylpiperazinyl) imine rubomycin, 14-(1-hydroxyl-2,2,6,6-tetramethylpiperidyl-4)-acetoxyrubomycin (spin-labeled rubomycin). The above derivatives slightly differed from the initial antibiotics by their affinity to DNA. The binding constants of methylpiperazinyl imines was 2-3 times higher than those of the respective antibiotics.     

Mechanism of the myeloinhibitory effect of carminomycin]

The proliferative activity and level of aberrant mitoses in the cells of the bone marrow were studied experimentally on 223 noninbred mice treated with carminomycin administered intraperitoneally in single (LD50) and repeated doses. When the antibiotic was used in a single dose the values of the mitotic activity of the bone marrow elements did not correspond to the severity of depression and thir quantitative composition, which was explained by an impairement of the mitosis quality and possible interkinetic destruction of a significant part of both erythroid and immature myeloid cells capable of division at early stages after the exposure. At the same time the level of the bone marrow devastation under conditions of the treatment with repeated doses was mainly determined by inhibition of the erythronormoblast proliferative activity.     

Toxicity and cumulative properties of carminomycin, rubomycin and adriamycin at different times of use]

LD50 of karminomycin, rubomycin and adriamycin were determined after their single administration or 3-, 5-, 10- and 15-fold administration once a day to 540 hybrid male mice F1(C57B1 X CBA). Comparison of the cumulative indices for these antibiotics showed that after injections they were close. After 5 injections the cumulative properties were more pronounced for adriamycin. After 10 or 15 injections the cumulative properties were less pronounced for karminomycin.     

Synthesis and selected properties of oligonucleotidyl-(Pm----O)-amino acids

N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was synthesized as a diastereomeric mixture of approx. (1:1) by reacting d(TpT) with 2,4,6-triisopropylbenzenesulfonyl chloride and N-Cbz-Ser-OMe. The phosphoesteric bond of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was found to be stable in acid (1 N HCl, 1 h, 37 degrees C), but labile in alkaline solution (0.1-1 N NaOH, 1 h, 37 degrees C). The products of alkaline hydrolysis were determined to be d(TpT) and the amino acid derivatives. Furthermore, the phosphotriester N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was more labile than the diesteric analogue N-Cbz-Ser (OMe)-pdT. The internucleotide phosphotriester linkage of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was also found to be resistant to enzymatic digestion with spleen and snake venom phosphodiesterases.     

Synthesis and anti-HIV properties of new water-soluble bis-functionalized[60]fullerene derivatives

A new series of bis-functionalized fullerene C₆₀ derivatives bearing two or more solubilizing chains have been evaluated for their activity against HIV-1 and HIV-2 strains. Some of the compounds show activity against HIV-1 type in the low micromolar range. The effect of the positions of the addends on the C₆₀ nucleus has been investigated, indicating that only trans-2 isomers possess promising activity. The presence of a quaternary pyrrolidinium nitrogen is essential to increase solubility.     

Antitumor activity of carminomycin antibiotic used orally]

Antitumor activity of karminomycin used perorally was studied with respect to 3 strains of mouse transplantable tumors, i. e. one ascitic strain of lymphadenosis NK/LI and two solid strains of lymphosarcoma L10-1 and sarcoma 180. Karminomycin was shown to have a high antitumor activity against the above tumors on its oral administration. In the experiments with lymphadenosis NK/LI the efficiency of karminomycin was higher when it was used perorally as compared to its intravenous administration. It was found that karminomycin had practically the same inhibitory effect on growth of lymphosarcoma L10-1 and sarcoma 180 on its peroral and intravenous administration in doses equivalent by their toxicity.     

Antitumor properties and toxicity of a carminomycin and protein complex

The properties of carminomycin complexes with protein, a bovine serum albumin prepared with two different methods using glutaraldehyde or carbodiimine were studied. The complex prepared with the use of carbodiimine was biologically inactive. The complex prepared with the use of glutaraldehyde had a molecular mass of about 15 000 000 dalton, was more toxic than carminomycin and possessed proportionally higher antitumor activity and a wider antitumor spectrum. The studies on the use of the method of carminomycin complex formation with antitumor immunoglobulins are promising.