William A. Remers

1,4-Disubstituted anthracene antitumor agents

Three different types of 1,4-disubstituted anthracenes were synthesized, and their cytotoxicity in a panel of tumor cells was compared with that of the corresponding 9,10-disubstituted anthracenes. The panel contained human myeloma, melanoma, colon, and lung cancer cells and sensitive and multidrug-resistant murine L1210 leukemia cells. These compounds had [[(dimethylamino)ethyl]amino]methyl, N-[(dimethylamino)ethyl]carbamoyl, and carboxaldehyde (4,5-dihydro-1H-imidazol-2-yl)hydrazone side chains. The 1,4-diamide was more potent across the tumor panel than the corresponding 9,10-isomer, but the 1,4-diamine and the 1,4-hydrazone were less potent than their 9,10-isomers. Although the 1,4-hydrazone was active against P388 leukemia in mice, it was inactive against L1210 leukemia. Within each pair of compounds, the one with greater average potency against tumor cells gave a greater increase in the transition melt temperature of DNA.

Preclinical antitumor activity of the azonafide series of anthracene-based DNA intercalators

The azonafides are a series of anthracene-based DNA intercalators which inhibit tumor cell growth in vitro at low nanomolar concentrations and are not affected by the multidrug resistance phenomenon (MDR). Prior studies have described antitumor efficacy in murine tumor models including L-1210 and P-388 leukemias, and B-16 melanoma. The current results extend these cell line observations to human tumors tested in the NCI panel of 56 cell lines, in freshly isolated tumors tested in colony-forming assays in soft agar and in several animal models. In the NCI panel, the overall mean 50% cell kill (LC50) for the unsubstituted azonafide, AMP-1, was 10−5.53 M, with some selectivity noted in melanomas (10−6.22 M). The mean LC50 for the 6-ethoxy substituted analog, AMP-53, was 10−5.53 M, with some selectivity found in non-small cell lung cancer (10−5.91) and renal cell carcinoma (10−5.84). In freshly isolated human tumors tested in soft agar, there was marked activity (mean IC50 in μg/ml) for AMP-53 in four cell types: breast cancer (0.09), lung cancer (0.06), renal cell carcinomas (0.06) and multiple myeloma (0.03). These effects were superior to doxorubicin and to several other azonafides, including AMP-1, AMP-104 and the 6-hydroxyethoxy derivative, AMP-115. Compound AMP-1 was shown to be superior to amonafide in the mammary 16C breast cancer model in B6CF31 mice, but it had little activity in Colon-38 nor in M5076 ovarian sarcomas in vivo. Nine azonafides were evaluated in the Lewis lung cancer model in C57/bl mice, but only AMP-53 demonstrated significant efficacy with a treated/control×100% (T/C) value of 30%. Because AMP-53 demonstrated the greatest breadth of activity, it was then evaluated in several human tumor cell lines growing in mice with severe combined immunodeficiency disease (SCID). Only three tumors were sensitive (T/C<42%), including HL-60 leukemia (T/C=39%), MCF-7 breast cancer (T/C=39%) and A549 non-small cell lung cancer (T/C=37%). Overall, these results demonstrate that the 6-ethoxy substituted azonafide, AMP-53, has consistent (in vitro and in vivo) experimental antitumor activity in human breast and lung cancer, and could be considered for clinical testing in patients with MDR tumors.

Cardiotoxicity of mitomycin A, mitomycin C, and seven N7 analogs in vitro.

SummaryThe alkylating antitumor agents mitomycin A (MMA), mitomycin C (MMC), and sevenN7 analogs were compared in terms of their cardiotoxic and antitumor activity in vitro. Neonatal rat-heart myocytes were sensitive to five of the compounds studied, including MMA, 7-dimethylamidinomitosane (BMY-25282), 7-(N-methyl-piperazinyl)-mitosane (RR-194),N7-(4-iodophenyl)-MMC (RR-208), andN7-(4-hydroxyphenyl)-MMC (M-83) in order of descending molar potency. MMA and RR-208 possessed the greatest cytotoxic potency against 8226 human myeloma tumor cells in vitro. Two of the nine mitomycins studied, BMY-25282 and M-83, showed greater cytotoxic potency for heart cells. For these two agents, the ratio of the 50% inhibitory concentration in heart cells to that in 8226 myeloma cells was 50 and 32, respectively. For the other analogs, the tumor-cell cytotoxic potency was much higher (ranging from 200 to 7,000). For the nine mitomycin compounds, a correlation was found between heart-cell toxicity and low reduction potentials (E1/2 values) ranging from −0.16 to −0.37 V. Thus, as the reduction potential decreased (easier reducibility), the cardiotoxic potency in vitro increased (r = 0.81). In contrast, mitomycins with reduction potentials of higher than −0.37 V were much less potent cardiotoxins. Thus, mitomycin C (E1/2 = −0.45 V) was noncardiotoxic even when tested at concentrations 100-fold above those pharmacologically achievable in humans. Mitomycin C also failed to enhance doxorubicin (Adriamycin) cardiotoxicity in vitro. Importantly, no correlation was found between the reduction potential and the antitumor activity of the nine analogs (n = 0.51), in this small series.

Computer simulation of the binding of quinocarcin to DNA. Prediction of mode of action and absolute configuration

SummaryComputer-based models were derived for the covalent and noncovalent binding of the antitumor antibiotic quinocarcin to a representative DNA segment, d(ATGCAT)2. They showed that a mode of action, involving opening of the oxazolidine ring to give an iminium ion, followed by initial noncovalent binding in the minor groove and subsequent alkylation of the 2-amino group of guanine, was rational and attended by favorable interaction energies in each step. The best model had the aryl ring of quinocarcin lying in the 3′ direction from the covalent binding site and anR configuration at the carbon involved in covalent bond formation. It also showed that the preferred absolute configuration for quinocarcin was the reverse of that arbitrarily assigned in the literature.

Synthesis and biological activity of 6-substituted mitosene analogues of the mitomycins

A series of 1-acetoxymitosene analogues, in which the substituent at C-6 was varied, was prepared by total synthesis and screened for activity against P388 leukemia in mice and induction of lambda phage in Escherichia coli. Among the 6-substituents prepared, none was as effective as the methyl group in conferring biological activity. However, certain N-methylcarbamates were more active than the unsubstituted carbamates.

Computer simulation of the binding of amonafide and azonafide to DNA

SummaryIntercalative binding of the antitumor drugs amonafide and azonafide to the oligonucleotide duplex d(GGCCGGCCGG)·d(CCGGCCGGCC) was compared using molecular dynamics in vacuum with the AMBER force field. A number of reasonable possible binding conformations were obtained, with the azonafide complexes favored over the amonafide complexes in net binding enthalpy. In comparison with amonafide, the larger chromophore of azonafide permits greater DNA distortion and wider side-chain swings, without falling out of the intercalation site. The best model obtained was used for further dynamics on amonafide and azonafide with solvent and counterions present, and again the azonafide complex had a more favorable enthalpy. Furthermore, the enthalpy change on going from solvent into the intercalation site was less unfavorable for azonafide. These results are consistent with the stronger DNA binding of azonafide compared to amonafide, as observed in relative melting transition temperature increases and tumor inhibition in cell cultures.

Intracellular localization of 6- and 7-substituted 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h]isoquino line-1,3-diones (azonafides) is not the limiting factor for their cytotoxicity: an in vitro confocal microscopy study.

The intracellular localization of 14 structurally unique azonafide analogs was studied to determine if intracellular drug distribution is the limiting factor in azonafide cytotoxicity. Using scanning laser confocal microscopy, cytotoxicity of the azonafide analogs studies was observed in Chinese hamster ovary cells immediately after a 1 h exposure. The intracellular drug distribution patterns varied significantly for different analogs. Eight analogs showed primarily nuclear localization, five analogs showed primarily cytoplasmic localization and two analogs displayed perinuclear localization. In general, the type of chemical substitution on the anthracene nucleus determined the distribution pattern. For example, for each analog seven of eight nuclear-localizing analogs were amine-substituted agents, while four of five cytoplasmic-localized agents were ethoxy-substituted analogs. The individual exception within these groups was the 6-[(dimethylamino)ethoxy] agent that was nuclear localized. The two perinuclear-localized agents included the unsubstituted parent, azonafide, and its 6-methyl azonafide analog. Comparison of the cytotoxicity of the azonafides, based on intracellular localization, revealed that none of the localization patterns were associated with increased cytotoxicity. These results show that minor structural changes in the azonafide class of antitumor agents involving substitution along an anthracene chromophore result in substantially different intracellular drug distribution patterns. However, these distribution differences do not determine relative cytotoxic potency in vitro.

In vitro cytotoxicity against fresh human tumors and P388 leukemia predicts the differential in vivo activity of a series of anthracene anticancer drugs.

To date, random anticancer drug screening has proven to be relatively inefficient and non-specific with respect to selecting active compounds for most tumor types (except for leukemia/lymphoma). Although large numbers of compounds from diverse sources were evaluated for many years in the P388 mouse leukemia model, only a few clinically useful drugs have been identified by this in vivo screening method. Thus, there is intense interest in the development of more effective in vitro screening models for new anticancer drugs. In the present paper we have compared the discriminating power for fresh human tumors from patients, human tumor cell lines developed from 11 patients and murine P388 leukemia in tumor colony forming assays as indicators of cytotoxicity for a series of anthracene antitumor agents. Two of a series of 21 novel bisantrene analogs, R6 (N, N1-bis[2-(dimethylamino)ethyl]-9,10-anthracenebis(methylamine)) and R26 (N, N1-bis(1-ethyl-3-piperidinyl)-9,10-anthracene-bis(methylamine)) produced significant cytotoxicity against the 11 human tumor cell lines and were therefore selected for additional in vitro and in vivo studies. R26 was specifically selected for further testing since it had Ion or doxorubicin-resistant 8226 myeiomd ten lines, in contrast to the cell line data, only one of the 22 fresh human tumors showed significant in vitro sensitivity (i.e. <50% survival of tumor colony forming units) to either R6 or R26 tested at high concentrations. Both of these bisantrene analogs also proved inactive at 1.2–1.6 μM concentrations against P388 leukemia in vitro, whereas mitoxantrone and bisantrene were highly active in this model at a concentration of 0.2 μM. In order to compare the in vitro data with antitumor activity in vivo, R26, the most active bisantrene analog, and mitoxantrone, the most active of the two anthracene parent compounds, were tested against P388 leukemia and M5076 ovarian sarcoma in mice. In both models mitoxantrone showed significant activity whereas R26 produced minimal or no antitumor effects. We conclude that fresh human tumors, but not defined human tumor cell lines, predict the in vivo cytotoxicity of a series of anthracene anticancer agents. Although this conclusion may not apply to the screening of other classes of antitumor agents, we propose an in vitro screening process which first utilizes numerous human tumor cell lines of many different biologies (to screen a large number of new compounds each year), followed by confirmatory tests in fresh human tumors using colony forming assays to screen up to a smaller number of 1000 compounds. Finally appropriate in vivo tumor model based on histologic pecificity would be used to screen a few consistently active new compounds for advancement to clinical trials. Thus, the first screening stage would be highly sensitive and non-specific, the second in vitro stage more specific and the third in vivo stage relevant by histologic tumor type.

Synthesis of Mitomycins

This chapter describes mitomycin syntheses published in the period 1984–1988 plus 1989 literature available by August 1. Mitomycin synthesis was intensively investigated in this period. It is characterized by a considerable variety of strategies and novel chemistry. The most significant accomplishment was an efficient new total synthesis of mitomycin С by way of isomitomycin A. Other achievements include syntheses of decarbamoyl 7-methoxymitosane by electrophile-initiated biscyclization and by a copper-catalyzed double cyclization of an azidoquinone. New methods for 9,9a-dihydromitosenes were based on photocyclization of a diene-bearing azidoquinone and on intramolecular Reformatsky or Wittig reactions. A stereoselective synthesis of a 1,2-aziridinomito- sene involved photochemical oxidation-reduction followed by palladium-catalyzed cyclization. Mitosenes were prepared by a variety of strategies including triazole photolysis, “criss-cross” annulation of a 2-[2-(l,3-dioxocyclopentanyl)]aniline, Madelung synthesis, and Dieckmann cyclization of a substituted pyrrolazene. Other approaches were based on thermal isomerization of a 1-(1-pyrrolidinyl)-2-vinylbenzene, palladium-catalyzed cyclization of a 6-allyl-5-allylamino-1,4- benzoquinone, and intramolecular cycloaddition of a nitrile oxide to a vinyl group. A 1,2- cyclopropano derivative was formed by 1,3-dipolar addition of a diazo group to an alkene, followed by elimination of nitrogen.

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the...