Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

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.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

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-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

Synthesis-enabled probing of mitosene structural space leads to improved IC50 over mitomycin C.

A DNA crosslinking approach, which is distinct but related to the double alkylation by mitomycin C, involving a novel electrophilic spiro-cyclopropane intermediate is hypothesized. Rational design and substantial structural simplification permitted the expedient chemical synthesis and rapid discovery of MTSB-6, a mitomycin C analogue which is twice as potent as mitomycin C against the prostate cancer cells. MTSB-6 shows improvements in its selective action against noncancer prostate cells over mitomycin C. This hypothesis-driven discovery opens novel yet synthetically accessible mitosene structural space for discovering more potent and less toxic therapeutic candidates.

Synthesis of a leucomitosane via a diastereoselective radical cascade.

The preparation of trans-2,3-disubstituted indolines from 1-azido-2-allylbenzene derivatives via a diastereoselective radical cascade using ethyl iodoacetate and triethylborane is described. Further lactamization afforded substituted benzopyrrolizidinones with excellent diastereomeric ratios. The radical cascade/lactamization sequence was efficiently applied to the synthesis of a 3-oxo-leucomitosane related to the mitomycin family of alkaloids.

Gold-catalyzed one-step construction of 2,3-dihydro-1H-Pyrrolizines with an electron-withdrawing group in the 5-position: a formal synthesis of 7-methoxymitosene.

What a ring formation! Bicyclic dihydropyrrolizines with an electron-withdrawing group (EWG) at the 5-position are formed in one step from linear azidoenynes under gold catalysis. This novel route involves the use of azide as a nitrene precursor, electronically-controlled regioselectivity, and the generation of destabilized 1-azapentadienium ions and their pericyclic reactions. This method was used for a formal synthesis of 7-methoxymitosene.

Development of a flexible strategy towards FR900482 and the mitomycins.

FR900482 and the mitomycins are two intriguing classes of alkaloid natural products that have analogous biological mechanisms and obvious structural similarity. Both classes possess potent anticancer activity, a feature that has led to their investigation and implementation for the clinical treatment of human cancer. Given the structural similarity between these natural products, we envisioned a common synthetic strategy by which both classes could be targeted through assembling the mitomycin skeleton prior to further oxidative functionalization. Realization of this strategy with respect to FR900482 was accomplished through the synthesis of 7-epi-FR900482, which displayed equal potency relative to the natural product against two human cancer cell lines. With the challenging goal of a synthesis of either mitomycin or FR900482 in mind, several methodologies were explored. While not all of these methods ultimately proved useful for our synthetic goal, a number of them led to intriguing findings that provide a more complete understanding of several methodologies. In particular, amination via π-allyl palladium complexes for the synthesis of tetrahydroquinolines, eight-membered heterocycle formation via carbonylative lactamization, and amination through late-stage C-H insertion via rhodium catalysis all featured prominently in our synthetic studies.

Synthesis and mechanistic studies of a mitomycin dimer containing an eight-membered cyclic disulfide.

Dimeric DNA alkylating agents have drawn significant interest because these compounds are expected to provide at least two reactive sites and as a result, generate enhanced levels of DNA interstrand cross-link (DNA ISC) adducts compared to their monomeric agents. We report the synthesis and mechanistic studies of a novel mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiocanyl-3″,8″-dimethylenyl)bismitomycin C (8) connected by an eight-membered cyclic disulfide. Mitomycins require prior activation (i.e., transformation to a good electrophile) for DNA adduction and therefore, 8 was aimed to undergo facile nucleophilic activation and produce enhanced levels of DNA ISC. At the core of this function lies a cyclic disulfide in 8. It was expected that disulfide cleavage by an appropriate nucleophile would successively produce two thiols that may trigger activation of two mitomycin rings in a dimer through intramolecular cyclization to quinine rings. Compound 8 was synthesized from mitomycin A (1) and the key intermediate, cyclic disulfide (11), along with the reference diol mitomycin 7-N,7'-N'-(2″,7″-dihydroxy-1″,8″-octanediyl)bismitomycin C (23) which does not contain the disulfide unit. We found that 8 underwent significantly enhanced nucleophilic activation in the presence of Et(3)P compared with 23, and that the disulfide unit in 8 played a key role for the nucleophilic activation. Based on these findings, we proposed a mechanism for nucleophilic activation of 8. We further demonstrated that 8 generated much higher levels of DNA ISC (94%) compared with 23 (4%) and 2 (3%) in the presence of Et(3)P (and L-DTT) leading to the conclusion that 8 is more efficient for DNA ISC processes than 23 and 2 due to the role of disulfide unit.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.

Structure and stereochemistry of some 1,2-disubstituted mitosenes from solvolysis of mitomycin C and mitomycin A.

Starting with mitomycin C (1), a number of solvolytic reactions were investigated and were found to result in opening of the aziridine ring with loss or migration of the 9a-methoxy group. A careful examination of the resulting 1,2-disubstituted 7-aminomitosenes indicated that there was a strong tendency for the azridine ring on opening to furnish mainly one stereoisomer, always with the oxygen stom at C-1 and the nitrogen atom at C-2. Thus the hydrolysis of 1withdition to small amounts of the trans-aminohydrin (10). Mitomycin A (2) BEHAVED ANALOGOUSLY. Both 1 and 2 generated a cis-1-acetoxy-2-acetamide when they were allowed to react with acetic anhydride. Acetolysis of mitomycin C was found to give the cis-1-hydroxy-2-acetamide (5), the trans-1-acetoxy-2-amine (14), and a cis-trans mixture of 1-acetoxy-2-acetamides (4 and 11, respectively). Routes to cis-1-methoxy-2-acetamide (9) were possible through the methanolysis of 1 or through the methylation of 5. For comparison, the trans-1-methoxy-2-acetamide (16) was obtained through a dnown resin-catalyzed methoxy migration from C-9A TO C-1 IN MITOMYCIN C. The use of 1-H nmr spectroscopy to asign configurations to 1,2-disubstituted mitosenes is discussed.

Mitomycin C analogues with aryl substituents on the 7-amino group.

A series of 30 different N7-phenyl-substituted mitomycin C analogues, including 25 new compounds, was prepared from mitomycin A. Seven of these compounds were clearly superior to mitomycin C in activity against P-388 murine leukemia. The para- and the meta-substituted derivatives were subjected to Hansch analysis, which revealed that the lipid-water distribution coefficient pi was the only significant factor in determining antitumor potency (MED). The substituent electronegativity factor sigma was statistically insignificant in determining potency, despite the good correlation of sigma p with the polarographic quinone-reduction potential. These results suggest that diffusion into the tumor cell or access to the receptor is more important than bioreductive activation in determining antitumor potency for this particular group of mitosanes . Fifteen new mitomycin C analogues with heterocycles on the 7-amino group also were prepared. Two of them, containing pyrazolyl and aminopyridyl substituents, were more active than mitomycin C against P-388 murine leukemia. No broad correlations could be made among the antitumor potencies and physicochemical properties for this type of analogue.

Mitomycin antibiotics. Synthesis and activity of 1,2-disubstituted mitosenes.

cis-1-Acetamido-2-acetoxy-7-methoxy-N-methylmitosene was prepared in 11 steps from 7-methoxy-6-methyl-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-one by a route involving bromination of the pyrrolidineenamine or trimethylsilyl enol ether of starting material, displacement of bromide by acetate, oxime formation, and reductive acetylation, followed by elaboration of the quinone and methyl carbamate functions according to previously established methods. An unsubstituted carbamate could not be prepared. The mitosene thus synthesized differs from previously reported 1,2-disubstituted mitosenes, which are derived from the solvolysis of mitomycins, in that it has the opposite arrangement of oxygen and nitrogen substituents at the 1 and 2 positions. It showed antibacterial activities in disk-plate assays superior to those of cis-diacetylapomitomycin A and equivalent to those of certain 1-substituted mitosenes; however, it was less active than mitomycin A in these assays. It was inactive in inducing lambda-bacteriophage in Escherichia coli and inactive against P388 leukemia in mice. In contrast, certain 1-substituted mitosenes were active in prophage induction and 2b and mitomycin A were active in both assays.

Cyclopropamitosenes, novel bioreductive anticancer agents. Synthesis, electrochemistry, and biological activity of 7-substituted cyclopropamitosenes and related indolequinones.

The synthesis of the indolequinones 8 and 9 starting from methyl 4-(benzyloxy)-5-methoxy-indole-2-carboxylate (10) is described. The methoxy group in the indolequinones 1, 2, 4, 5, and 7-9 can be displaced by various nitrogen nucleophiles (ammonia, 2-methoxyethylamine, aziridine, 2-methylaziridine, pyrrolidine) in 22-88% yield. The resulting amino-substituted quinones, together with their methoxy precursors, were studied by cyclic voltammetry to determine their reduction potentials, which, in DMF solution, lie in the range -1.355 to -1.597 V (vs ferrocene). The cytotoxicity of the compounds towards aerobic and hypoxic mammalian cells was also determined; in general, under aerobic conditions, the cyclopropamitosenes are more toxic than the corresponding pyrrolo[1,2-a]indolequinones, which are in turn more toxic than the simple 1,2-dimethylindolequinones, with many of the compounds in each series showing greater toxicity toward hypoxic cells.

Additional nucleotide derivatives of mitosenes. Synthesis and activity against parental and multidrug resistant L1210 leukemia.

Cytidine 5'-monophosphate and 5'-ara-CMP conjugates of 2,7-diaminomitosene, with the phosphate groups linked to C-1, were prepared by treating mitomycin C with the appropriate nucleotides. 5'-UMP conjugates were prepared from mitomycin A, 7 (M-83), and 8 (BMY-25282) by similar procedures. A conjugate could not be prepared from mitomycin C and 6-MPRP, but a sulfur-linked derivative was made with 6-MP ribonucleoside. The corresponding 1-hydroxy-2-aminomitosenes were prepared from the parent mitomycin analogues for structure-activity comparisons. All compounds were tested against L1210 murine leukemia in the MTT tetrazolium dye assay. In general, the conjugates were less potent than the parent mitomycins; however 5'-ara-CMP conjugate 14 derived from mitomycin C was more potent than the parent compound or any mitomycin tested except mitomycin A. It also was more potent than ara-C. This result establishes the value of this approach to prodrugs, at least in cell culture. Against a multi-drug-resistant L1210 cell line, all of the conjugates derived from mitomycin C were more potent than the parent compound. 6-Mercaptopurine ribonucleoside conjugate 15 was more active against the resistant cells than it was against the parental cell line.

Preparation and antitumor activity of new mitomycin A analogues.

A series of 26 mitomycin A analogues including 23 new ones was prepared by a variety of methods. The most useful methods were alkoxide exchange on mitomycin A and treatment of 7-hydroxymitosane with 3-substituted 1-phenyltriazenes. Many of the new analogues were superior to mitomycin C in the P388 leukemia assay and the more stringent subcutaneous B16 melanoma assay both in mice. Four of them gave long-term survivors in the latter assay. Quantitative correlations between log P and antitumor activity were not possible, but some guidelines for future analogue development are proposed.

Conformations of complexes between mitomycins and decanucleotides. 3. Sequence specificity, binding at C-10, and mitomycin analogues.

Molecular mechanics simulation of the interactions of mitomycin C and certain analogues with DNA models are presented. The sequence specificity of mitomycin C binding was investigated by using a d(GCGCGCGCGC)2 decanucleotide duplex, abbreviated herein as GC10, in which the base pair was varied on either side of the covalent binding site. A CGT fragment was favored, although its correlation with the diverse findings in the literature is questionable. A model was derived for the monocovalent binding at C10 of 2,7-diaminomitosene with GC10 and for the noncovalently bound hydroquinone intermediate. Revised models were established for three highly active mitomycin C analogues: M-83, BMY-25282, and RR-150. They involved covalent binding at the 2-amino group of a guanine residue, and they accounted for enhanced noncovalent binding afforded by specific interactions of the C7 substituents with residues in GC10.

Preparation and antitumor activity of 7-substituted 1,2-aziridinomitosenes.

7-Methoxy-1,2-aziridinomitosenes were prepared from mitomycin A and its N-methyl homologue by catalytic reduction followed by air oxidation. Treatment of these products with amines, including ammonia, ethylenimine, 2-methylethylenimine, propargylamine, and furfurylamine gave the corresponding 7-(substituted amino) derivatives. Screening of these compounds against P-388 leukemia in mice revealed some good activities. The more easily reduced compounds gave prolongation of life span comparable to that of mitomycin C, but their optimal doses were higher. Among these compounds, a methyl group on the aziridine nitrogen increased potency. The 7-amino derivatives, which were difficult to reduce to hydroquinones, were essentially inactive. The aziridinomitosenes were subjected to a Hansch-type analysis, but no statistically significant correlation was found.

New potent mitomycin derivatives: synthesis and antitumor activity of 7,7-(ethylenedioxy)mitomycins.

A series of 6,7-dihydro-7,7-(ethylenedioxy)mitomycins was synthesized and evaluated for antitumor and anticellular activities. These compounds were prepared by basic treatment of 7-methoxymitomycins with ethylene glycol, and were structurally novel mitomycin derivatives containing a masked quinone moiety. 5,6-Enol or 6-chloro derivatives of 6,7-dihydro-7,7-(ethylenedioxy)mitomycins were also prepared and the (allyloxy)carbonyl group at the aziridine nitrogen has proved to be an efficient protecting group in chemical modification of mitomycins. Most of these mitomycin derivatives displayed potent antitumor activity against P388 leukemia in mice and anticellular activity against HeLa S3 cells.

Synthesis and antitumor activity of various 6-demethylmitomycins and 6-demethyl-6-halomitomycins.

A series of 6-demethylmitomycins and 6-demethyl-6-halomitomycins having various mitomycin skeletons were synthesized, taking into account the electronic effect toward the quinone moiety and the partition coefficients. Treatment of enones 15 and 16 with selenenamide or N-halosuccinimide-Et2NH afforded the 6-demethyl intermediates 17, 18, and 21-24 via the tandem Michael addition/retro-Mannich reaction sequence. Subsequent conversions into the mitomycin skeletons resulted in the formation of the desired derivatives 7a-c, 8a-c, 11a-c, and 12a,b. These mitomycin derivatives including 3a-c and 4a-c were evaluated for their anticellular activity against HeLa S3 cells and antitumor activity against Sarcoma 180 in mice. The anticellular activity of 1 and 3a-c depends on the substituent at the C-6 position and the order of increasing activity is H < CH3 < Br < Cl. A similar tendency was observed in their antitumor potency (ED50). The activities of 9 and 11a-c also follow a pattern similar to that of 1 and 3a-c. Compounds 4b,c, 8b,c, and 12b having both a halogen at the C-6 position and a methoxy group at the C-7 position did not show the activities because of the instability of the compounds. Interestingly, a correlation between the anticellular activity (IC50) and the partition coefficients (log kappa') determined by HPLC was observed within the compounds studied except the unstable compounds, while their antitumor activity (ED50 or T/C) did not correlate with the quinone reduction potential (E1/2). These results would indicate the importance of the C-6 substituents and the mitomycin skeletons for exhibiting both anticellular and antitumor activities.

Synthesis and antineoplastic activity of 1a-formyl and 1a-thioformyl derivatives of mitomycin C and 2-methylaziridine.

A select number of 1-formyl- and 1-thioformyl-2-methylaziridine derivatives and the corresponding 1a-substituted mitomycin C analogues were synthesized and tested for antineoplastic activity by using an in vivo test with murine P388 leukemia. Select compounds were also tested in vivo with murine melanoma B16. Several of the mitomycin C derivatives displayed activity and some of the mitomycin C analogues were comparable in activity to the parent compound.