Activity of a novel anthracenyl bishydrazone, 9,10-anthracenedicarboxyaldehyde Bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride, against experimental tumors in mice.

9,10-Anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride (CL 216942; bisantrene hydrochloride; NSC 337766), a member of a new chemical class of compounds with antineoplastic properties, has been evaluated for antitumor activity in experimental murine tumor systems. The compound produced significant increases in life span (LS) and long-term survivors among mice bearing transplantable leukemias and solid tumors. Optimal treatment regimens resulted in an ILS of greater than 173 and 151% in mice with P388 and L1210 leukemia, respectively, an ILS of greater than 85% in mice with Lieberman plasma cell tumor, and an ILS of greater than 200, 150, and 63%, respectively, in mice with B16 melanoma, colon tumor 26, and Ridgway osteogenic sarcoma. An adriamycin-resistant subline of P388 leukemia showed complete cross-resistance to CL of 216942. The compound was active when administered by the i.p., i.v., and s.c. routes, but p.o. activity was not observed. Significant schedule dependency was not observed when the drug was administered once daily for 9 days, once every 4 days, or as a single dose, but single doses typically produced the best effects. CL 216942 was a potent inhibitor of DNA and RNA synthesis in L5178Y lymphoma cells cultured in vitro, and preliminary studies indicated the drug was a DNA-intercalating agent. The drug was cytotoxic for rapidly proliferating and nonproliferating (G0) human colon carcinoma WiDR cells in vitro.U

Molecular and biochemical pharmacology of mitoxantrone.

Evidence has been presented which indicates that Nv: intercalates DNA and additionally causes inter- and intra-strand crosslinking possibly associated with its charged side arms; there is an apparent preference for G-C base pairs; induces single strand and double strand breaks in DNA; strongly inhibits DNA and RNA synthesis; causes nuclear aberrations and chromosomal scattering; induces a block in the G2 phase of the cell cycle with an increase in cellular RNA and polyploidy; is not cell cycle phase-specific with respect to cell kill; does not induce free-radical formation; does not induce lipid peroxidation or superoxide formation; rather it may inhibit ADR-stimulated lipid peroxidation and microsomal superoxide production; does not appear to have a strong potential for cardiotoxicity on the basis of currently postulated mechanisms of action; is capable of inducing cellular resistance in vitro; resistance is associated with an apparent alteration in the cell membrane impairing drug transport into the cell. Although the precise mechanism(s) of tumor cell killing has not been fully defined it is most likely associated with an interaction by Nv with chromosomes resulting in DNA damage, which if not efficiently repaired, will lead to inhibition of nucleic acid synthesis and eventual cell death.

N-phosphoryl derivatives of bisantrene. Antitumor prodrugs with enhanced solubility and reduced potential for toxicity.

The selective phosphorylation of bisantrene (1) affords bis(phosphonoguanidinic acid) 6, a prodrug with enhanced aqueous solubility (as sodium salt 7) at physiological pH. Unlike 1, in a rat tail vein model, no precipitation was observed when bis(phosphonoguanidinic acid) 6 was injected. While in rats 6 hydrolyzed to monophosphonoguanidinic acid 9 with a half-life of ca. 12 min., complete hydrolysis to bisantrene required several hours. The corresponding monophosphonoguanidinic acid 9 was synthesized from bisantrene and also showed good solubility and antitumor activity. While the antitumor activities of 6 in mice were comparable to bisantrene against B-16 melanoma and P-388 and L-1210 leukemias, it was inactive in vitro vs several tumor cell types. Thus, its activity in vivo resulted from its ability to serve as a prodrug for bisantrene.

Antitumor agents. 1. 1,4-Bis[(aminoalkyl)amino]-9,10-anthracenediones.

The condensation of alkylenediamines with quinizarin or with 2,3-dihydro-1,4,5,8-tetrahydroxy-9,10-anthracenedione, followed by oxidation, gave 1,4-bis[aminoalkyl)amino]-9,10-anthracenediones. Some of these compounds and their 2,3-dihydro derivatives were markedly active against both leukemias and solid tumors in mice. Activity was maximal with 5,8-dihydroxylation and 1,4-bis[(2-aminoethyl)amino] substitution, in which the terminal nitrogen atoms were either unsubstituted (compound 50) or carried 2-hydroxyethyl groups (compound 40), indicating the importance of hydrophilicity. Against B-16 melanoma, 50 gave greater than 433% increase in median life span (ILS) with 7/10 80-day survivors. Against P-388 leukemia, 40 gave greater than 500% ILS with 4/5.60-day survivors; its efficacy and therapeutic index equaled or surpassed those of adriamycin, cyclophosphamide, daunorubicin, methotrexate, or 5-fluorouracil. Against L-1210 leukemia, B-16 melanoma, and colon tumor 26, 40 was generally as effective or more effective than adriamycin and is now undergoing preclinical toxicological evaluation.

Synthesis of 3,6-bis(aminoalkoxy)acridines and their effect on the immune System.

A series of 3,6-bis(aminoalkoxy)acridines (2) was prepared and shown to have a protective antiviral effect against an interferon-sensitive virus (Columbia SK) and to partially restore an antibody response to a T-cell-dependent antigen in leukemic immunosuppressed mice. The presence of circulating interferon and the stimulation of natural killer cell activity in mice was observed for 21.

A new family of water-soluble, third generation antitumor platinum complexes.

[1,1-Cyclobutanedicarboxylato(2)-O,O'](1,3-dioxane-5,5-dimethan amine- N,N')platinum(II), 3a, a third generation, very water-soluble platinum complex, has been synthesized along with several of its analogues. All members of the new family contain a 1,3-dioxane or 1,3-dioxolane-1,3-diamine as their basic ligand, a moiety which contributes to their increased water solubility, and a bidentate acid ligand, which is responsible for their good stability. They were all easily crystallized and characterized by 1H NMR and elemental analysis, and the parent complex 3a was further characterized by 13C NMR. Their very desirable physical properties combined with their broad spectrum of antitumor activity and reduced toxicity make them good candidates of further development.

Water-soluble third generation antitumor platinum complexes, [2,2-bis (aminomethyl)-1,3-propanediol-N,N']-[1,1-cyclobutanedicarboxylato (2-)-O,O']platinum(II) and [1,1-cyclobutanedicarboxylato(2-)-O,O'] [tetrahydro-4H-pyran-4,4-dimethanamine-N,N']platinum(II).

The synthesis, stability, and antitumor activity of a series of water-soluble third generation platinum(II) complexes have been described. Among these complexes, [2,2-bis(aminomethyl)-1,3- propanediol-N,N'] [1,1-cyclobutanedicarboxylato(2-)-O,O']platinum(II) and [1,1-cyclobutanedicarboxylate(2-)-O,O'](tetrahydro-4H-pyran-4,4- dimethanamine-N,N'-)platinum(II) have shown the greatest promise for further investigation and are currently under clinical evaluation.

Antitumor agents. 2. Bisguanylhydrazones of anthracene-9,10-dicarboxaldehydes.

9,10-Anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] (bisantrene, VI-1) showed anticancer activity in mice vs. both leukemias and solid tumors. Increases in life span vs. the following neoplasms were: P-388 leukemia, 137%; B-16 melanoma, 122%; Lieberman plasma cell tumor, greater than 85%; colon tumor 26, 150%; Ridgway osteogenic sarcoma, 85%. There were significant numbers of long-term survivors. Both DNA and RNA synthesis were strongly inhibited. The drug was resistant to biodegradation and was bound strongly to tissues; in monkeys the half-life for disappearance from serum was 6 days. Related hydrazones were synthesized, and structure-activity relationships are discussed. Two routes to ring-substituted anthracene-9,10-dicarboxaldehyde intermediates were developed.

Polyphasic taxonomy of the genus Vibrio: polynucleotide sequence relationships among selected Vibrio species.

Polynucleotide relationships among selected Vibrio species were examined by means of deoxyribonucleic acid (DNA) reassociation reactions and chromatography on hydroxyapatite. Relative levels of intraspecific DNA duplex formation (V. cholerae-V. cholerae and V. parahaemolyticus-V. parahaemolyticus) were found to be high at 60 C (>80%), and only minimally reduced at 75 C. Interspecific DNA duplexes between V. cholerae DNA and that of the non-cholera vibrios also exhibited high relative levels of formation at 60 C (>80%) and, with one exception, were only slightly reduced at 75 C. The thermal stability of these duplexes formed at 60 or 75 C was virtually identical to that of homologous V. cholerae DNA duplexes. The degree of reassociation and the thermal stability of V. cholerae-non-cholera vibrio DNA duplexes suggests relatively little evolutionary divergence in these organisms. In all other interspecific DNA reassociation reactions, only low levels of DNA duplex formation were noted at 60 C (<25%), and these were drastically reduced (>50%) at 75 C. The degree of nucleotide sequence divergence indicated by these reactions suggests that these Vibrio species are not significantly related to V. cholerae or V. parahaemolyticus. Reassociation reactions between V. cholerae DNA and the DNA of V. parahaemolyticus indicated these species were not significantly related to each other.

Studies on vaccinia virus-directed deoxyribonucleic acid polymerase.

A vaccinia-directed deoxyribonucleic acid (DNA) polymerase has been partially purified from the cytoplasmic fractions of virus-infected HeLa cells. The utilization of natural and synthetic templates by this enzyme resembles that of the host cell DNA-dependent DNA polymerases. The vaccinia DNA polymerase cannot copy ribopolymers or ribonucleic acid but is very effective with an "activated" DNA as template. An exonuclease preferring single-stranded DNA as substrate is found in the most highly purified preparations of the enzyme. The molecular weight of the vaccinia DNA polymerase seems to be about 110,000. The viral DNA polymerase is also found to be associated with purified, infected cell nuclei, and this association may be due, at least in part, to nonspecific adsorption of the vaccinia DNA polymerase by nuclei.

Properties of two marine bacteriophages.

Chen, Peter K. (Georgetown University, Washington, D.C.), Ronald V. Citarella, Omar Salazar, and Rita R. Colwell. Properties of two marine bacteriophages. J. Bacteriol. 91:1136-1139. 1966.-Various properties have been determined for two bacteriophages, NCMB 384 and 385, and their host, NCMB 397, a Cytophaga sp., isolated from the marine environment. The purified bacteriophages have been subjected to serological analysis, results of which indicate a high degree of relatedness. Purified, highly polymerized deoxyribonucleic acid (DNA) prepared from the host strain showed an overall base composition of 37 moles% guanine + cytosine (buoyant density of 1.696 g/cc). The bacteriophage DNA, in the native configuration, from NCMB 384 and 385 banded at 1.691 g/cc in a CsCl gradient and the denatured bacteriophage DNA demonstrated a bimodal peak. Stability tests of the bacteriophages in various buffers and diluents suggest a requirement for inorganic cations, most likely Na(+) and Mg(++), for retention of viability.

Polynucleotide sequence relationships among members of Enterobacteriaceae.

Polynucleotide relationships were examined among many representatives of the Enterobacteriaceae by means of agar, membrane filter, and hydroxyapatite procedures. The amount of deoxyribonucleic acid (DNA) that reassociated was dependent, especially in interspecific reactions, on the annealing temperature. In only three cases: Escherichia coli-Shigella flexneri, Salmonella typhimurium-S. typhi, and Proteus mirabilis-P. vulgaris, was relative interspecific duplex formation 80% or higher. In most cases interspecies DNA duplex formation was 40% or less of that obtained from intraspecies DNA reassociation reactions. The stability of E. coli-S. flexneri DNA duplexes formed at either 60 or 75 C was virtually identical to that of homologous E. coli DNA duplexes, and the degree of interspecies duplex formation was minimally affected by the temperature increase (86% at 60 C; 77% at 75 C). The thermal stability of DNA duplexes formed at 60 C between DNA from E. coli and DNA from strains of Aerobacter aerogenes, S. typhimurium, S. typhi, and P. mirabilis was about 12 to 14 C below that of reassociated E. coli DNA. At 75 C, the formation of the interspecific DNA duplexes was markedly decreased, but the stability of the DNA able to reassociate at this temperature approximated that of reassociated E. coli DNA. The degree of reassociation and the thermal stability of E. coli-S. flexneri DNA duplexes suggests relatively little evolutionary divergence in these organisms. The other enterobacteria tested, however, have diverged to a point where less than one-half of their DNA can reanneal with E. coli DNA at 60 C and less than 10% reacts at 75 C. The degree of divergence between various enterobacteria does not appear to be uniform along the DNA molecule. Ribosomal ribonucleic acid (RNA)-specific sequences are conserved among most enterobacteria. An examination of messenger RNA relatively specific for the lactose operon suggests that specific chromosomal genes may diverge more or less than the genome as a whole.

Tissue deposition of bisantrene in B16 melanoma tumor-bearing mice.

Single doses of 14C-labeled bisantrene, a new antitumor agent, were administered intravenously at 10 and 100 mg/kg to mice bearing intraperitoneally implanted B16 melanoma. At 24 hr after dosing, the tumors contained relatively high drug concentrations as compared to most of the other tissues. The concentrations averaged 2.4 and 28.3 micrograms/g tumor and the tumor/blood concentration ratios were 40/1 and 29/1 for the low and high doses, respectively.

Modulation of the immune response to tumors by a novel synthetic compound, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763).

CL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These "activated" macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interfering with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.