- Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts
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Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-lin
- Champeil, Elise,Cheng, Shu-Yuan,Huang, Bik Tzu,Conchero-Guisan, Marta,Martinez, Thibaut,Paz, Manuel M.,Sapse, Anne-Marie
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- Studies on the reactivity of reductively activated mitomycin C
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Mitomycin C (1a), a clinically significant antineoplastic antiobiotic, is considered to be the prototype of bioreductive alkylating agents. It has been reported that, in the absence of DNA, reductive activation of 1a furnished both solvolytic C(1) electro
- Schiltz, Pascal,Kohn, Harold
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- Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts
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Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2′-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.
- Zheng, Maggie,Hwang, Seokjin,Snyder, Timothy,Aquilina, Jake,Proni, Gloria,Paz, Manuel M.,Pradhan, Padmanava,Cheng, Shu-Yuan,Champeil, Elise
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- Reductive activation of mitomycins A and C by vitamin C
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The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.
- Paz, Manuel M.
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- Reaction of reductively activated mitomycin C with aqueous bicarbonate: Isolation and characterization of an oxazolidinone derivative of cis-1-hydroxy-2,7-diaminomitosene
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The reductive activation of mitomycin C in aqueous bicarbonate buffer resulted in the formation of a previously unknown compound, characterized as an oxazolidinone derivative of cis-1-hydroxy-2,7-diaminomitosene. This compound is the result of a cyclization reaction of bicarbonate with the aziridine ring of aziridinomitosene, and was observed at bicarbonate concentrations close to those present in physiological plasma.
- Paz, Manuel M.
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experimental part
p. 31 - 34
(2010/04/02)
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- A mitomycin-N6-deoxyadenosine adduct isolated from DNA.
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A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)n sequence is favored(AT)n for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine-to-alkylation frequency in the model oligonucleotides was 0.3-0.6 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall ratio of adenine to guanine alkylation in calf thymus DNA was 0.03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by further comparison.
- Palom, Jolanda,Lipman, Roselyn,Musser, Steven M.,Tomasz, Maria
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p. 203 - 210
(2007/10/03)
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- Studies on the use of Na2S2O4 for the reductive activation of mitomycin C
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Mitomycin C (1a) is considered to be the prototypical bioreductive alkylating agent. Among the numerous reductive procedures employed for the in vitro activation of mitomycin C, incremental addition of Na2S2O4 has emerged as the method of choice for generating high yields of mitomycin C-DNA adducts. The major products and distinguishing features of the incremental addition Na2S2O4-mitomycin C reductive processes in water (pH 7.4) in the absence of DNA are reported. Key observations included (1) rapid and efficient consumption of mitomycin C, (2) production of high amounts of 7-aminomitosane-9a-sulfonate (1b) in the early stages of the reaction, and (3) generation of significant amounts of C(1) and C(10) sulfonato adducts. The complexity of this transformation has been attributed in part to HSO3-, a byproduct of the Na2S2O4 reduction process. Use of buffered methanol solutions ("pH" 7.4) in place of water simplified the product profile. The poor solubility of Na2S2O4 and NaHSO3 in methanol produced only trace amounts of mitosene sulfonato adducts. There were significant differences between product profiles for the incremental addition Na2S2O4 procedure versus a protocol in which the equivalent amount of Na2S2O4 was added in a single shot. First, higher amounts of C(1) electrophilic versus C(1) nucleophilic products were observed using the single shot technique. Second, C(1) sulfonato adducts composed a larger amount of the C(1) nucleophilic product pool when the Na2S2O4 was added using the single shot protocol than with the incremental addition method. Third, higher amounts of 1a were converted to C(1), C(10) fully functionalized mitosene adducts using the incremental procedure. Select auxiliary experiments provided additional information concerning the Na2S2O4-mediated mitomycin C reductive process. Examination of the reactivity of key C(1), C(9a), and C(10) mitomycin sulfonato products demonstrated that 7-aminomitosane-9a-sulfonate (1b) was efficiently converted to C(1)- and C(10)-functionalized mitosenes under reductive conditions, whereas mitosene C(1) and C(10) sulfonates did not undergo displacement reactions and hence did not function as viable alkylating agents. On the basis of these cumulative studies, we suggest the likely mechanism for the Na2S2O4-mediated mitomycin C reductive process and the beneficial properties accrued by the use of the incremental addition technique. These notions are discussed in light of the pathway that may be operative in in vitro mitomycin C-DNA bonding transformations.
- Schiltz, Pascal,Kohn, Harold
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p. 10497 - 10509
(2007/10/02)
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- Sodium dithionite-mediated mitomycin C reductive activation processes
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Sodium dithionite is the reagent of choice for the reductiue activation of mitomycin C in the presence of DNA. No comprehensive study of this transformation in the absence of DNA has appeared. The major products of this reaction have been determined and the key parameters governing this transformation identified.
- Schiltz, Pascal,Kohn, Harold
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p. 4709 - 4712
(2007/10/02)
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- 7-Aminoaziridinomitosenes: Synthesis, Structure, and Chemistry
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7-Aminoleucoaziridinomitosene (2a) has been proposed as a key intermediate in the reductive activation process for the antineoplastic agent, mitomycin C (1a).Little is known about 2a and its oxidised equivalent, 7-aminoaziridinomitosene (3a).An expedient electrochemical procedure for 3a and the corresponding N-methyl analogue 3b has been developed.NMR spectral studies of 3a in DMF-d7 and DMSO-d6 provided important information concerning the solution-state structure for this adduct.Factors controlling the aziridine ring-opening process under reductive and nonreductive conditions have been determined, as well as evidence for the intermediacy of 2a in the reductive activation cascade of 1a.
- Han, Insook,Kohn, Harold
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p. 4648 - 4653
(2007/10/02)
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- Studies on the Use of Cr(ClO4)2 for the Reductive Activation of Mitomycin C 1
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Cr(ClO4)2 has been shown to be a highly efficient reductant of the anticancer agent, mitomycin C (1). Two different Cr(ClO4)2-mediated reductive techniques were developed and utilized in buffered water and methanolic solutions. In the first procedure, Cr(ClO4)2 (1-2 equiv) was directly added to 1 at various "pH" values. Key observations included the following: (1) Consumption of mitomycin C was rapid and generated as the major products trans- and cis-10-decarbamoyl-1-hydroxy-2,7-diaminomitosenes (11 and 12), and trans- and cis-10-decarbamoyl-1,10-dimethoxymitosenes (16 and 17) in acidic-to-neutral aqueous and methanolic solutions, respectively. (2) Between "pH" 6.0 and 7.0, the difunctionalized mitosene adducts accounted for nearly half of the product profile even though noticeable amounts of unreacted 1 remained. (3) Significant amounts of C-1 electrophilic products were not observed under acidic conditions. The product profiles observed with the second Cr(ClO4)2-mediated reductive procedure were markedly different. Activation of 1 was accomplished by the prior addition of Cr(ClO4)2 to excess cis-10-decarbamoyl-1,10-dimethoxy mitosene (17) to generate the putative mitosene monochromate 20 and mitosene dichromate 21 species in situ, followed by the addition of 1 (1 equiv per Cr(ClO4)2). The products obtained by using this protocol were similar to those observed with conventional reductants in which C-1 electrophilic adducts predominated in acid, C-1 nucleophilic products were the major products under neutral and basic conditions, and little modification of the C-10 site was detected throughout the "pH" range examined. The product profiles coupled with select auxiliary experiments have provided information concerning the mechanism of both reductive procedures. The major products furnished by using the direct Cr(ClO4)2-mediated procedure under acidic and neutral conditions have been attributed to the two one-electron reductions of 1 to give the bis-CrIII-bound species 22. Complexation of the C-5 and C-8 phenolic-type oxygens in reduced 1 is believed to facilitate the loss of methanol at C-9 and C-9a in 1 and the nucleophilic substitution processes at C-1 and C-10 as well as inhibit the electrophilic transformations at both DNA bonding sites. Explanations and supporting data have also been provided to account for the other products detected in these reactions. Correspondingly, the second procedure is conjectured to occur by an outer-sphere electron-transfer process from 20 and/or 21 to 1 to give the uncomplexed hydroquinone (or semiquinone) mitomycin C species 2. Subsequent loss of methanol at C-9 and C-9a yields the activated mitosene capable of furnishing the C-1 functionalized adducts 7 and 9 + 10. The distinctive product profiles observed with the direct addition of Cr(ClO4)2 to 1 and the remarkable high yields of C-1, C-10 dinucleophilic substitution adducts suggest that similar pathways may be operative in the in vivo process to provide the DNA-mitomycin C cross-link adducts. These notions are discussed in light of the DNA sequence selectivity recently observed for the drug monoalkylation bonding process.
- Hong,Kohn, Harold
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p. 4634 - 4644
(2007/10/02)
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- DEVELOPMENT OF A HYDRAZINE-MEDIATED SYSTEM FOR THE REDUCTIVE ACTIVATION OF MITOMYCIN C
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The reactivity of mitomycin C (1) with hydrazines (2) has been examined.Monoarylhydrazines have been shown to efficiently reduce 1 leading to the activation of both proposed DNA binding sites (C-1 and C-10) within the drug.
- Russell, David J.,Kohn, Harold
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p. 223 - 227
(2007/10/02)
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- Nucleotide derivatives of 2,7-diaminomitosene
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Treatment of mitomycin C with pyrimidine nucleotides in acidic media produced derivatives of 2,7-diaminomitosene in which C-1 was covalently bound to the phosphate group of the nucleotides. On reduction, these derivaties liberated the nucleotides and a mitomycin intermediate that alkylated DNA. Their reduction in the presence of 2'-deoxyguanosine produced some bifunctional alkylation as did mitomycin C. They were readily taken up by L1210 leukemia cells, in which they showed potent cytotoxicity. These properties suggest that they are acting as prodrugs capable of conversion into two active species. The uridylate derivative showed activity comparable to that of mitomycin C against P-388 leukemia in mice.
- Iyengar,Dorr,Remers,Kowal
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p. 1579 - 1585
(2007/10/02)
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- Aspects of the chemical stability of mitomycin and porfiromycin in acidic solution
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Aspects of the degradations of mitomycin and porfiromycin were studied. The initial degradation processes of the compounds in an acidic medium were investigated. Influences of pH, buffers, and other additives such as halogenides and dioctyl sodium sulfosuccinate [sodium 1,4-bis(2-ethylhexyl)sulfosuccinate] were studied. The hydrogen ion catalyzes the degradation of both the unchanged and the protonated species. Anions also promote the degradation of the compounds in an acidic medium. Rate constants for all of the catalytic reactions could be determined. From the pH profiles, after correction for buffer influences, accurate pK[a] values for the aziridine nitrogens could be obtained. The protective influence of the dioctyl sulfosuccinate ion could be explained. From the data obtained a plausible mechanism for the initial acidic degradation reactions was developed.
- Underberg,Lingeman
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p. 549 - 553
(2007/10/02)
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