SCIENCE
Anticancer Activity of Mitomycins Becomes Clearer Results from several groups of researchers provide more information about metabolic activation and reactions of metabolites with DNA Several groups of chemists have put together different pieces of the puzzle of how the anticancer drugs k n o w n as mitomycins function. Mitomycins themselves are inactive and must be metabolically activated. Thus, detailed knowledge of the activation and of reactions of mitomycin metabolites with deoxyribonucleic acid (DNA) may lead to design of similar new anticancer drugs. One group at the University of Maryland Cancer Center, Baltimore, has demonstrated that a one-electron reduction of the drug suffices to
activate it [/. Am. Chem. Soc, 108, 4158 (1986)]. Previously, other workers had suggested that a two-electron reduction of the quinone to the hydroquinone form occurs. On another front, joint research at Hunter College of the City University of New York and Columbia University has established that activated mitomycin can crosslink DNA by alkylating guanine residues on two different strands through C-l and C-10 of the drug. That work will be published later this year. And chemists at Yale University [/. Am. Chem. Soc, 108, 4648 (1986)] and the University of Houston [/. Am. Chem. Soc, 108,296 (1986)] have deduced some of the reactions following reduction that lead to alkylative crosslinking of DNA. Mitomycins were discovered beg i n n i n g in 1956 by workers at Kyowa Hakko Kogyo Co., a Tokyo d r u g firm, among fermentation
products of Streptomyces caespitosus. The U.S. Food & Drug Administration approved mitomycin C for marketing by Bristol Laboratories, Syracuse, N.Y., in 1974. Some problems about the actions of mitomycins remain to be solved. For example, a mitomycin molecule may also alkylate only one strand of DNA through C-l without crosslinking. This insertion of a mitomycin molecule may be enough to inhibit replication of DNA in malignant cells. Also, reduced mitomycins may reduce oxygen to superoxide ions. Superoxide may also damage malignant DNA. Nicholas R. Bachur, director of the University of Maryland Cancer Center; Su-shu Pan, professor of experimental therapeutics; and graduate student Paul A. Andrews showed the efficacy of a one-electron reduction of mitomycin C by cyclic voltammetry and flow cell reduc-
Reduction activates mitomycins as anticancer drugs Quinone form is inactive toward nucleophiles...
. . . but one-electron reduction triggers formation of active intermediate... Q
CH 2 -0 2 CNH 2
0
r uH. n - 0 CNH rMu P 2 2 2
O ©
CH 2 -0 2 CNH 2
»OCH, H3C O Mitomycin C
Aziridinomitosene C semiquinone
Semiquinone
. . . thereby crosslinking DNA strands through guanine groups
.. which is susceptible to nucleophilic attack by DNA. DNA
oe CCH2-Q>2CNH2
CH 2 -0 2 CNH 2 DNA
26
September 1, 1986 C&EN
and
tion. Their work was supported by the National Cancer Institute. The Baltimore workers saw one electron-transfer process at —0.937 volt and a second at —1.410 volt using a silver chloride electrode in cyclic voltammetry with dimethylformamide solvent. Turning to flow cell reduction, they showed by electron spin resonance that the first process produced a radical anion, whereas the second process yielded a dianion. On adding water to the radical anion, they separated mitomycin C and at least eight other products by high-performance liquid chromatography. The profile of products was similar to that made by action of flavoenzymes on mitomycin C. Flavoenzymes are thought to activate mitomycins in living systems. By contrast, adding water to the dianion preparation gave only two products. Demonstration that mitomycin C could crosslink DNA by alkylation of guanine residues through both C-l and C-10 of the drug was carried out in a collaboration between chemistry professor Maria Thomasz and research assistant Roselyn Lipman at Hunter and organic chemistry professor Koji Nakanishi and graduate student Gregory Verdine at Columbia. In their forthcoming paper, the Hunter and Columbia chemists describe the positions of attachment to guanine, the mode of reductive activation they used, or whether they worked with native DNA or oligonucleotides. Their work was supported by the National Institutes of Health, National Science Foundation, and Public Health Service (PHS). Organic chemistry professor Samuel J. Danishefsky at Yale studied the sequence of changes in molecular structure that mitomycins undergo on activation. This information is crucial to an understanding of the function of activated mitomycins and to the design of similarly acting anticancer drugs of the future. Danishefsky's work has been supported by PHS. Mitomycins have several structural components that are thought to act in a sequence. A quinone ring keeps the drug inactive until re-
Method detects light elements near solid's surface Charles R. Gossett, a physicist at the Naval Research Laboratory, Washington, D.C., adjusts a magnetic spectrometer used in a method he developed to detect the presence of light elements near the surface of a solid. Gossett's technique employs an elastic recoil detection system to identify elements from hydrogen to oxygen near the surface of solids. These lighter elements are nearly impossible to detect in samples containing heavier elements by the standard energetic ion beam method of Rutherford backscattering, Gossett says. His technique measures the number and energy of ions knocked out of the surface region of a sample when the sample is bombarded by an energetic ion beam from an accelerator. It can determine both which elements are being ejected and how deep they come from inside the sample. Such measurements are particularly useful for characterizing materials that have been modified by ion implantation methods, Gossett says. This method is being employed increasingly to tailor the electrical, optical, corrosion, or wear properties of solid materials.
duced. The Maryland work showed that reduction probably happens in a semiquinone rather than a hydroquinone. The quinone, in turn, is part of an indoline nucleus. On reduction, the indoline nitrogen assists in the elimination of a group (methoxy in mitomycins A and C) from the C-9a position to form an indole. The indole form of the drug is called a mitosene. Also part of the molecule is an aziridine ring and a carbamoyloxymethyl group. The new unsaturation introduced on conversion from mitomycin to mitosene transmits electronic effects from the indole nitrogen that render both the aziridine ring susceptible to opening on
nucleophilic attack at C-l and the carbamate group to leaving from C-10. C-l and C-10 form the crosslinks between different DNA strands. Before Danishefsky began his studies, it was widely thought that reduction to a hydroquinone, railed a leucomitomycin, resulted in immediate elimination of the group at C-9a. In fact, it was believed that the leucomitomycin might even be a transition state rather than an intermediate. This is because previous workers who reduced mitomycins and then reoxidized the products got mitosenes and aziridine ring-opened products called apomitosenes. But in work with postdoctoral fellow Marco Ciufolini and graduate September 1, 1986 C&EN
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Science
TECHNOLOGY
student Melissa Egbertson, Danishefsky showed that in N-methylmitomycin A and mitomycins B (hydroxyl at C-9a) and C, hydrogenation followed by reoxidation with oxygen over palladium-charcoal catalyst resulted in recovery of the m i t o m y c i n . Danishefsky credits Egbertson with the leap of thought that explained the discrepancy. She reasoned that in the previous work something must have happened during the reoxidation that expelled the group at C-9a. Egbertson mixed solutions of the quinone and h y d r o q u i n o n e forms of Nmethylmitomycin A, evaporated the mixture to dryness, and got as products the mitomycin and the mitosene. Because mixtures of quinones and hydroquinones yield semiquinones, the Yale chemists reasoned that the semiquinone could be the oxidation state that yields mitosenes. At the University of Houston, meanwhile, organic chemistry professor Harold Kohn and graduate
student Nada Zein have studied how the loss of the carbamoyloxy group at C-10 could be involved in crosslinking DNA. A nucleophilic portion of DNA could displace the carbamoyloxy group directly. Or elimination of the group could produce C-10 as an exocyclic methylene group, and the DNA could add across that double bond. Starting with an aziridine ringopened apomitosene, the Houston chemists hydrogenated the compounds catalytically in deuterium methoxide. They also reduced the apomitosene with d e u t e r i u m in methanol. Only the first experiment resulted in uptake of deuterium by the organic substrate from the solvent. Thus, they conclude that the final crosslinking of DNA by reaction at C-10 is by addition across an exocyclic methylene group rather than by displacement of the carbamoyloxy group. The Houston work was supported by NIH. Stephen Stinson, New York
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September 1, 1986 C&EN
Coal-water feed for combustors to be tested A major problem with burning coal in a pressurized fluidized-bed combustor is getting the coal into it. The Department of Energy is about to join two British organizations in tests of a new way: feeding the coal as a coal-water mixture. Current practice is to feed the coal using valves and bins to receive the coal, raising it to a high pressure, and delivering it in an air stream. But this wears out the valves and erodes the pipes excessively. Disruption of the feed flow by pressure surges from the bed is another problem. The use of a coal-water mixture is an attempt to overcome those difficulties. The tests will be carried out by DOE in cooperation with the U.K/s National Coal Board and Central Electricity Generating Board. They will be conducted in an 18-month program at a 26-MW test facility at Grimethorpe, England—an advanced combustor representative of small commercial units. With a contribution of $5 million, DOE will fund the equipment installation for the coal-water mixture fuel preparation and feeding system, in which coal will be ground to one-eighth inch or smaller and then mixed with water for a 70% coal/30% water mixture. This fuel will be pumped into the combustor at high pressure by commercial pumps, with flow controlled by adjusting the pump speed. Fluidized-bed combustors hold promise for utilization of high-sulfur coal, since the fluid bed of limestone or dolomite captures the sulfur dioxide generated. Also, nitrogen oxide emissions are lower because combustion temperatures are lower than with other systems. Successful results in the upcoming tests would indicate that the cost of coalfeeding equipment for such units could be reduced. Water in the mixture will use up some heat as it turns to steam. But the steam will provide added mass to the gas turbine downstream from the burner, resulting in a boost in power output. Thus, the water will not reduce performance very much. James Krieger, Washington
