cc-1065 and adozelesin

We have found that a cyclopropylpyrroloindole antibiotic, compound CC-1065 (benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1, 6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8, 8a-tetrahydro-7-methyl-4-oxocyclopropan[c]pyrrolo[3, 2-e]indol-2(1H)-yl)carbonyl]benzo[1,2-b:4, 3-b']dipyrrol-3(2H)-yl]-carbonyl]-1,6-dihydro-4-hydroxy-5-methoxy, (7bR,8aS)), forms interstrand DNA cross-links of an apparently covalent nature in HeLa S(3) cells. This compound induced interstrand cross-links at concentrations ranging from 0.1 to 1 nM/3 hr in whole cells, but these cross-links were absent or marginally low when the drug was added to cell lysates with inactivated cellular enzymes or isolated nuclei, which suggests that metabolic activation of the drug is a necessary step for DNA cross-linking to occur. In contrast, an analog of CC-1065, Bizelesin, which forms DNA-DNA cross-links by direct alkylation, induced interstrand DNA cross-links in both whole cells and in cell lysates. Interestingly, a demethoxy analog of CC-1065, Adozelesin, did not induce DNA cross-links under the same conditions. CC-1065 was found to be extremely potent in terms of concentrations required to cross-link DNA of tumor cells, and this may be related to its remarkable cytotoxic activity.

Topics: Antibiotics, Antineoplastic; Benzofurans; Cell Division; Cross-Linking Reagents; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA; DNA, Neoplasm; Duocarmycins; HeLa Cells; Humans; Indoles; Leucomycins; Urea

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.), chloroethylmethane sulfonate (CI-EMS, 35 or 50 mg/kg i.p.), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 microg/kg i.v.) and CC-1065 (6 microg/kg i.v.) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg i.v.) were equivocal. Adozelesin, CC-1065, and CI-EMS treatments increased the percentage of T-lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys.

Topics: Animals; Benzofurans; Chromosome Aberrations; Chromosome Mapping; Cyclohexanecarboxylic Acids; Cyclohexenes; Cyclophosphamide; Duocarmycins; Ethyl Methanesulfonate; Hypoxanthine Phosphoribosyltransferase; Indoles; Leucomycins; Macaca fascicularis; Mutagens; T-Lymphocytes

Bizelesin (NSC-615291), a potent, bifunctional analog of the cyclopropylpyrroloindole antitumor antibiotics CC-1065 and adozelesin, has been selected by the National Cancer Institute for evaluation as a potential chemotherapeutic agent. All three compounds bind to and alkylate DNA at the N-3 position of adenine in a sequence-selective manner. Bizelesin is unique among the analogs with bifunctional alkylating capability due to two chloromethyl moieties that are converted to the cyclopropyl alkylating species that interact with DNA. A reverse-phase high-performance liquid chromatography (HPLC) assay and an L1210 cell bioassay were developed for bizelesin and subsequently applied to stability and murine pharmacokinetics studies. Following 48 h of incubation with L1210 cells the 50% growth-inhibitory concentrations (IC50) of bizelesin, adozelesin, and CC-1065 were 2.3, 3.4, and 88.1 pM, respectively. Bizelesin was stable in organic solvents but was less stable in aqueous solutions, with the half-life values obtained in buffers at pH 4, 7, and 10 being 9.6, 2.1, and < 1 h, respectively. By HPLC analysis, bizelesin degradation was associated with the appearance of two peaks, the mono- and dicyclopropyl derivatives formed by base-catalyzed intramolecular alkylation of the chloromethyl groups. Bizelesin and the dicyclopropyl derivative were equipotent in the L1210 cell bioassay. Following i.v. administration of bizelesin (15 micrograms/kg) to male CD2F1 mice, the plasma elimination of cytotoxic activity determined with the bioassay was described by a two compartment open model; the alpha-phase (t1/2 alpha) and beta-phase (t1/2 beta) half-lives, steady-state volume of distribution (VSS), and total body clearance (ClTB) were 3.5 min, 7.3 h, 7,641 ml/kg, and 16.3 ml min-1 kg-1, respectively. The systemic drug exposure following i.p. administration was at least 10 times lower than that resulting from i.v. infusion. Following i.v. or i.p. administration the recovery of material in urine was < 0.1% of the delivered dose.

Topics: Alkylating Agents; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzofurans; Chromatography, High Pressure Liquid; Cyclohexanecarboxylic Acids; Cyclohexenes; Duocarmycins; Indoles; Leucomycins; Leukemia L1210; Male; Mice; Mice, Inbred Strains; Urea

1,2-Dihydro-1-(chloromethyl)-5-hydroxy-8-methyl-3H-furano[3,2-e]in dole (CFI) as a novel replacement of the cyclopropylpyrroloindoline (CPI) alkylation subunit of CC-1065, U-71184, and U-73975 (adozelesin) has been synthesized and incorporated into a series of efficacious antineoplastic agents. A partial solution to an asymmetric synthesis of the CFI alkylation subunit has been achieved by the implementation of an asymmetric hydroboration reaction of an intermediate 3-methyleneindoline (13). Extension to the asymmetric synthesis of the CBI and CI alkylation subunits is presented. The demonstration and comparative study of the sequence-selective DNA alkylation properties of the CFI-based agents are detailed, and the preliminary in vitro and in vivo antineoplastic properties of these agents in the human epidermoid cell lung carcinoma (T222) are described.

Topics: Alkylation; Animals; Antineoplastic Agents; Base Sequence; Benzofurans; Boron Compounds; Carcinoma, Squamous Cell; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA; Drug Screening Assays, Antitumor; Duocarmycins; Female; Furans; Humans; Indoles; Leucomycins; Lung Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Tumor Cells, Cultured

Although considerable work has focused on characterizing the bonding chemistry and sequence selective alkylation of DNA by cyclopropylpyrroloindole compounds, little is known about the molecular consequence of their N-3-adenine adducts in whole animal systems. We have utilized a transgenic mouse system, harboring a lambda phage shuttle vector, to assess the mutagenic potential of the antitumor compounds CC-1065 and adozelesin and, for the first time, to track the in vivo fate of their unique DNA modifications at the nucleotide level. Mice were inoculated with a single therapeutic dose of these agents and sacrificed at either 18 h, 3 days, or 15 days for extraction and analysis of liver DNA. Mutant frequencies obtained from drug treated and control animals were determined by in vitro packaging of the phage vector from genomic DNA followed by a colorimetric plaque assay to screen for phage in which the accompanying lacI repressor gene had mutated. Although undetectable at 18 h posttreatment, by 72 h a 3-fold increase in mutant frequency was observed in drug treated animals such that sequence analysis of drug induced mutations could be performed and a direct comparison made between in vitro and in vivo DNA alkylation. Base substitution involving guanine or cytosine accounted for 64% of the 41 mutations sequenced from drug treated animals. Only 7 of the mutations occurred at a cyclopropylpyrroloindole alkylation site while 23 occurred 1 to 4 nucleotides from a potentially alkylated adenine.

Topics: Alkylation; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Bacteriophage lambda; Base Sequence; Benzofurans; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA; Duocarmycins; Indoles; Leucomycins; Male; Mice; Mice, Transgenic; Molecular Sequence Data; Mutagenesis

Several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with DNA via a cyclopropylpyrroloindole (CPI) group are susceptible to a multidrug resistance (MDR) phenotype in Chinese hamster ovary (CHO) cells. The multidrug resistant CCHR-C5 cell line was 16-, 23- and 13-fold more resistant to the analogs U-73,975, U-77,779 and U-80,244, respectively, although its cytotoxic response to the parent compound CC-1065 was similar to the response of the drug-sensitive wild-type cells (AuxB1). For a sequence of MDR cell lines showing increasing expression of P-glycoprotein (Pgp) there were corresponding increments in the level of resistance to U-73,975, arguing that Pgp is the key determinant in resistance of the MDR cells to CPI agents. MDR cells treated with U-73,975 showed diminished generation of covalent adducts on DNA as well as increased resistance to cytotoxicity.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzofurans; Cell Line; Cell Survival; CHO Cells; Cricetinae; Cricetulus; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA Damage; Drug Resistance; Duocarmycins; Indoles; Leucomycins; Membrane Glycoproteins; Urea

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.

Topics: Alkylation; Animals; Antibiotics, Antineoplastic; Benzofurans; Cattle; Cell Survival; Circular Dichroism; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA; Duocarmycins; Indoles; Leucomycins; Leukemia L1210; Molecular Structure; Solutions; Spectrophotometry, Ultraviolet; Tritium; Tumor Cells, Cultured

U-73,975 (U-73) and U-77,779 (U-77), two analogues of the cyclopropylpyrroloindole antitumor antibiotic CC-1065, are promising novel chemotherapeutic agents which are known to alkylate the N3 position of adenine in a sequence-selective manner. The concentration of U-73 required to produce a 1 log cell kill in 6 human tumor cell lines varied from 20-60 pM. U-77 was more cytotoxic than U-73, with the concentrations required for a 1 log cell kill ranging from 1-20 pM. The cytotoxicity of U-73 and U-77 was found to be independent of the guanine O6-alkyltransferase phenotype. The sensitivity of the BE and HT-29 human colon carcinoma cells was increased when the time of drug exposure was increased from 2 to 6 h. DNA interstrand cross-links, as measured by the technique of alkaline elution, could only be detected when HT-29 or BE cells were exposed to extremely high concentrations of U-77 for 6 h. No other forms of DNA damage were detected in genomic DNA with either compound. U-77 was also found to induce DNA interstrand cross-links in naked DNA, as measured by an agarose gel method. The rate of interstrand cross-linking was extremely rapid with the "second-arm" of the cross-link being completed within 2 h. The mechanism by which these cyclopropylpyrroloindole compounds elicit their cytotoxicity, however, remains to be elucidated.

Topics: Antibiotics, Antineoplastic; Benzofurans; Carcinoma; Cell Death; Colonic Neoplasms; Cross-Linking Reagents; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA Damage; Dose-Response Relationship, Drug; Duocarmycins; Humans; Indoles; Leucomycins; Lung Neoplasms; Plasmids; Tumor Cells, Cultured; Urea