A comparison of mechanisms proposed for the conversion of mitomycins into mitosenes

Two mechanisms proposed for the acid-catalyzed conversions of mitomycins into mitosenes were investigated by deuterium incorporation methods. Four different mitomycins, an aziridinomitosene, and an N-acetylmitomycin all underwent the conversion in acetic acid-d with no incorporation of deuterium at C-1. This evidence suggests that the mechanism based on initial elimination of the elements of methanol to give an aziridinomitosene is more likely the correct one. The products of these reactions had considerable variation in the ratios of cis to trans isomers: 7-aminomitosanes gave a predominance of trans and 7-methoxymitosanes gave a predominance of cis. Treatment of mitomycin C with DCl in D2O gave predominantly cis product with about 45% deuterium exchange at C-1. The isomeric 2,7-diamino-1-methoxymitosenes previously obtained by treating mitomycin C in methanol containing acetic acid were found to have stereochemistry opposite to that originally assigned by us.     

Synthesis, DNA cross-linking activity, and cytotoxicity of dimeric mitomycins

Dimeric DNA cross-linking compounds have emerged as important new antitumor agents. We report the synthesis and biochemical evaluation of a select set of dimeric mitomycins in which the two mitomycin units are tethered at either the mitomycin C(7) amino or the aziridine N(1a) positions. Significantly, mitomycin C (1) itself is the prototypical bioreductive DNA cross-linking agent. DNA cross-linking experiments using a denaturing-gel-electrophoresis-based assay showed that the extent of DNA cross-linking for select dimeric mitomycins can exceed that of the parent compound, mitomycin C, and that the reaction proceeds, in part, at the two distal C(1) sites in the mitomycins. The efficiency of DNA cross-linking depended on the nature of the linker and the position of linker unit's attachment. When we compared the efficiency of DNA cross-linking for the dimeric mitomycins with their in vitro cytotoxicities in cultured human tumor cells, we observed a poor correlation. The mitomycins that gave the highest levels of DNA cross-linked adducts displayed the weakest cytotoxicities. These findings determined that the denaturing-gel-electrophoresis-based assay was a poor predictor of cytotoxic activity.     

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.     

New mitomycin analogs produced by directed biosynthesis

When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of mitomycin analogs described as Type I and Type II are produced. Type I analogs are related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogs also contain the same substitutions at C7 but the conformation of the mitosane ring is related to mitomycin B having an OH at positions C9a and a methyl substituted aziridine. The products obtained from the supplementation of the medium with methylamine, ethylamine, propylamine, propargylamine and 2-methylallylamine were isolated and characterized. In all cases the Type I analogs are more active in a prophage induction test and against L1210 lymphatic leukemia in mice. A number of other amines have been tested and shown to yield new products that have not yet been isolated. No secondary amines are incorporated.     

The derivation of 1a-demethylmitomycin G from mitomycin C

Mitomycin G (2) was derived from porfiromycin (10b) in 3 steps via the methanesulfonate (14b) in an overall yield of 39%. On the basis of the established method for the introduction of an exomethylene group in mitomycins with a 9a-methoxy group, the preparation of biologically more important 1a-demethylmitomycin G (5) from mitomycin C (1) was accomplished by the use of a protective acetyl group on the aziridine in an overall yield of 57%. 1a-Demethylmitomycin K (6) was obtained from 5 in a yield of 42%. In a preliminary evaluation of their antitumor activity, compound 5 showed superior activity against sarcoma 180 (sc-ip) to its 1a-methyl congener, i.e., mitomycin G (2).     

Kinetics and mechanism of the degradation of la-acetylmitomycin C in aqueous solution

The acid-catalyzed degradation of mitomycin C is supposed to be governed, to a certain extent, by the protonation status of the aziridine nitrogen in the molecule as well as the protonation degree of the opened aziridine function in a key intermediate species, formed during mitomycin degradation. In order to obtain information about the contribution of the protonation degrees of these functions in controlling the degradation processes, we investigated the degradation of 1a-acetylmitomycin C in acidic aqueous solutions. In the presence of 0.001 mol/l phosphate buffers five 1-hydroxy and mono-acetyl mitosenes are formed, whereas in 1.0 mol/l acetate buffers a total of eight products could be identified, two of them being diacetyl mitosenes. Over the whole pH range studied the formation of 1,2-Z-mitosenes prevails, indicating that, contrary tomitomycin C, a pH-independent factor controls the ultimate 1,2-stereochemistry.     

Effect of cyclodextrins on the chemical stability of mitomycins in alkaline solution.

The effects of cyclodextrins on the chemical stability of several mitomycin antibiotics in an alkaline medium have been investigated. A stability-indicating high-performance liquid chromatographic method was used to determine the overall degradation rate constants. The influence of various parameters such as structural variations of the cyclodextrins and mitomycins, temperature and pH was studied. It appears that complexation is most favourable with gamma-cyclodextrin. All mitomycin-gamma-cyclodextrin complexes degrade at lower rates than those of the free drugs. Moreover, it was shown that gamma-cyclodextrin influences the equilibrium between mitomycin C and its zwitterion mesomer.     

Stability of the 6,14-endo-ethanotetrahydrooripavine analgesics: acid-catalyzed rearrangement of buprenorphine

Buprenorphine (I), a member of the 6,14-endo-ethanotetrahydrooripavine series of analgesics, undergoes an acid-catalyzed rearrangement reaction when exposed to acid and heat. The product was shown by 1H-NMR and GC-MS to have undergone overall elimination of a molecule of methanol with concurrent formation of a tetrahydrofuran ring at C(6)-C(7) of I. Short-term stability studies across a wide range of pH and temperature conditions indicate that I is stable in aqueous solution at pH greater than 3 for 24 h at 36-38 degrees C. Under the more extreme conditions of the autoclave, significant loss of I occurred. Long-term stability studies (10 weeks) of I in aqueous solution (pH 1 and pH 5) at 0-4 degrees C and 26-28 degrees C indicate only minor conversion (4%) to the rearrangement product. Eight other 6,14-endo-ethanotetrahydrooripavine derivatives were subjected to extremes of acid (pH 0) and temperature (autoclave) to determine if similar rearrangement reactions occur. GC-MS indicated that hydrolysis products were produced whose spectra were consistent with the proposed rearrangement structures.     

The derivation of a novel mitomycin skeleton: 3 alpha-alkoxymitomycin

The first example of C-3 alkoxylation in mitomycins has been achieved. 3 alpha-iso-Propoxy-10-O-decarbamoylmitomycin D (4) and 3 alpha-iso-propoxymitomycin D (5) were derived from mitomycin D (3) under decarbamoylation conditions with iso-propoxide. Under similar conditions 3 alpha-iso-propoxy-10-O-decarbamoylporfiromycin (8) and 3 alpha-methoxy-10-O-decarbamoylmitomycin B (11) were also derived from porfiromycin (6) and mitomycin B (9), respectively. The mechanism of generation of these novel analogs was based on the premise that the key intermediate of hydroquinone iminium salt (14) was led through the iminium salt (13), followed by alkoxide addition and oxidation.     

Nuclear localization of NADPH:cytochrome c (P450) reductase enhances the cytotoxicity of mitomycin C to Chinese hamster ovary cells

Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.     

Molecular mechanics simulations on covalent complexes of mitomycin C and its analogues with left-handed DNA duplexes.

We present molecular mechanics simulations on covalent complexes between d(GCGCGCGCGC).d(GCGCGCGCGC) in the left-handed double helical forms (B and Z) and potent antitumor antibiotics mitomycin C and three of its analogues using the all atom force field in the framework of the program AMBER(UCSF). The energy-refined models of the complexes show interesting networks of hydrogen-bonding interactions between the drugs and DNA groups in the minor groove of the left-handed helices. The energy-refined models suggest that mitomycins could bind strongly to left-handed helices. This result might be relevant to the interpretation of earlier experiments which suggested that DNA bound by mitomycin C underwent a transition to a non-Z left-handed structure.     

Transition state effects in the acid-catalyzed hydrolysis of 5-methoxyacenaphthylene 1,2-oxide: implications for the mechanism of acid-catalyzed hydrolysis of cyclopenta[cd]pyrene 3,4-oxide

5-Methoxyacenaphthylene 1,2-oxide (5) was synthesized by the reaction of 5-methoxyacenaphthylene with dimethyldioxirane. The rates and products from the acid-catalyzed and pH-independent reactions of 5 in 50:50 dioxane/water have been determined. The half-life of the pH-independent reaction of this very reactive epoxide in 50:50 dioxane/water is only 22 s. Acid-catalyzed hydrolysis of 5 in 50:50 dioxane/water yields 62% of cis diol 6, 37% of trans diol 7, and approximately 1% of 5-methoxy-1,2-dihydroacenaphthylen-1-one (8). The pH-independent reaction of 5 yields mostly ketone 8 (94%), along with minor amounts of cis and trans diols. The relative stabilities of cis and trans diols 6 and 7 were determined by treating either cis or trans diol with perchloric acid in water solutions and following the approach to an equilibrium cis/trans diol mixture as a function of time. At equilibrium, the ratio of cis and trans diols is 19:81, which establishes that trans diol 7 is more stable than cis diol 6. The acid-catalyzed hydrolysis of epoxide 5 therefore yields the less stable cis diol as the major product. It is concluded that transition state effects therefore selectively stabilize the transition state for attack of water on the intermediate carbocation leading to the less stable cis diol. These results suggest that transition state effects are also responsible for formation of the major cis diol in the acid-catalyzed hydrolysis of cyclopenta[cd]pyrene 3,4-oxide, which has a cyclopenta-fused ring similar to that in 5.     

Synthesis and evaluation of tryprostatin B and demethoxyfumitremorgin C analogues

Tryprostatin B and demethoxyfumitremorgin C are fungal inhibitors of mammalian cell cycle progression at the G(2)/M transition. N-Alkyl derivatives of the L-Trp-L-Pro diketopiperazine were prepared as analogues of tryprostatin B, and two of these were more active than the natural product. A second series of cis- and trans-tetrahydro-beta-carbolines annulated to a diketopiperazine were prepared as analogues of demethoxyfumitremorgin C. The nature of the alkyl substituent, as well as its cis or trans relationship in the tetrahydro-beta-carboline ring, was found to have a significant effect on cytotoxic activity. Small cis-alkyl substituents fall into the demethoxyfumitremorgin C family, whereas bulky benzyl trans compounds appear to act via a different mechanism of action.     

An NMR and theoretical study of the conformation and internal flexibility of butaclamol hydrochloride.

A theoretical (MM2) and experimental (1H and 13C NMR) study of butaclamol hydrochloride in CDCl3 has been done in order to determine preferred conformations and internal molecular flexibility of this molecule. The theoretical calculations suggest the presence of four low-energy conformations, two of which involve a trans junction of the D and E rings, with the other two involving a cis I ring junction. An alternative cis junction (cis II) was excluded on energetic grounds. The 1H NMR data strongly suggest the presence of a trans D-E ring junction and are consistent with a chair conformation of the E ring. 13C spin-lattice relaxation time measurements show that most of the molecule is rigid, although there is some degree of mobility in the seven-membered B ring, associated with rapid flipping of the bridging C8 and C9 carbons between two skewed conformations, which have previously been referred to as conformer A and conformer B (Laus et al. Heterocycles 1984, 22, 311).     

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.     

Mechanism for the reductive activation of diaziquone

In order to understand the redox properties of diaziquone (AZQ) and the reductive alkylation role of its one-electron reduced free radical anion AZQ- in biological systems, we investigated the electrochemical and structural properties of AZQ and its reduced species by cyclic voltammetry, controlled potential electrolysis, optical absorbance spectroscopy and 1H NMR. This study was carried out in aqueous media as well as in Me2SO. In aqueous media AZQ can be reduced by 2 electrons to the dianion AZQ2- which is oxidized back to AZQ- and AZQ. In Me2SO, 1 - e- reduction or oxidation steps are possible. This allowed the characterization of AZQ and its 1 or 2 - e- reduced species by UV-visible absorbance spectroscopy. The redox properties of the aziridine rings were observed by cycle voltammetry. Using 1H NMR, it was possible to follow the structural dependence of AZQ on the nature of the medium. Protonation of the aziridine rings at low pH may facilitate the opening of the ring leading to the carbonium ion, the required species for alkylation. Cyclic voltammetry data indicate that reduction of the quinone facilitates the aziridine ring opening.     

The mechanism of activation of 4-hydroxycyclophosphamide

4-Hydroxycyclophosphamide (2/3) of unknown stereochemistry is the initial metabolite formed after administration of cyclophosphamide (1). Ultimate conversion to the cytotoxic metabolite phosphoramide mustard (6) is initiated by ring opening of 4-hydroxycyclophosphamide to produce aldophosphamide (4). The ring-opening reaction and subsequent equilibration of 2-4 are subject to general-acid catalysis, and the equilibrium composition is independent of buffer structure and pH. In contrast, formation of 6 from 4 proceeds by general-base-catalyzed beta-elimination. trans-4-Hydroxycyclophosphamide undergoes ring opening ca. 4 times faster than the cis isomer, and cyclization of 4 favors the trans isomer by a factor of ca. 3 over the cis isomer. The rapid equilibration of 2-5 and the absence of elimination to give 6 at pH approximately 5 provides a convenient method to prepare a stable equilibrium mixture of activated cyclophosphamide metabolites suitable for in vitro use.     

Benzo[a]pyrene diol epoxide forms covalent adducts with deoxycytidylic acid by alkylation at both exocyclic amino N(4) and ring imino N-3 positions

The carcinogen 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) alkylates DNA at dGuo, dAdo, and dCyd. dCyd adducts, formed in small amounts, elute near the more abundant dGuo adducts. We isolated the dCyd adducts formed with dCMP. Each BPDE enantiomer forms three major adducts with dCMP, two cis and one trans. The trans adduct and one of the cis adducts form by alkylation at exocyclic N(4), while the second cis adduct is a dUrd adduct formed by alkylation at ring N-3 followed by deamination. Epoxide ring-opening geometries were assigned on the basis of halide and temperature effects on adduct yield, the sign of the major CD band, and benzo ring proton NMR coupling constants. One of each set of cis adducts is fluorescent (FL), and the other is nonfluorescent (NF). The trans and FL cis adducts have fluorescence quantum yields 40-50% of that of the BPDE hydrolysis product. The long wavelength UV maxima of the FL and NF cis adducts are red-shifted 1 and 3 nm relative to the trans adduct. (1)H NMR deuterium exchange experiments indicate that in the trans and FL cis adducts N(4)-H is coupled to C10-H. Adduct formation experiments with methyl-protected Cyd derivatives show that NF cis adducts result from alkylation at N-3. MS results, pK(a) measurements, and dUrd alkylation experiments indicate that the N-3 dCyd adducts spontaneously deaminate to dUrd adducts. NMR coupling constants show that in the NF cis adduct the C7 and C8 substituents are quasi equatorial and the C9 substituent is quasi axial, unlike in other cis BPDE adducts. (1)H NOESY spectra of the (-)-BPDE NF cis adduct reveal that it exists in two conformers. Molecular modeling shows that the conformers result from two low-energy conformations of very similar energies with the pyrimidine in opposite orientations, separated by significant barriers to rotation of the uracil moiety.     

新蒽环类抗生素变活霉素小组分的分离和结构测定

从无活性链霉菌1254经诱变所获变株113的发酵液中,除变活霉素A外,还分离得到四种小组分,变活霉素B、C、D及7—脱氧变活霉酮A。基于光谱及质谱证据,它们的结构阐明如式(1)所示。它们均对某些革兰氏阳性菌有抑制作用,其中变活霉素C还对小鼠红细胞白血病的生长有诱导分化和抑制作用。 通过变活霉素B、C、D的CD谱与柔红霉素,柔红霉酮、变活霉索A及变活霉酮A_2的CD谱比较以及NOE差谱的研究确定变活霉素B、C、D的“A”环与变活霉素A的“A”环立体化学相同,亦为7S、9R。“A”环呈半椅式构象,C_9在蒽醌环平面上方。     

Characterization of 7,12-dimethylbenz[a]anthracene-adenine nucleoside adducts

Chromatographic comparisons were made between radioactive adducts derived from the DNA of cells treated with [3H]7,12-dimethylbenz[a]anthracene and adducts derived from calf thymus DNA or nucleotides which had been treated in vitro with the synthetic syn 3,4-dihydrodiol 1,2-epoxide of this same carcinogen. This confirmed that three of the adducts formed in cells were derived from reaction of this particular dihydrodiol epoxide with deoxyadenosine while a fourth adduct was derived from its reaction with deoxyguanosine. After reaction of the dihydrodiol epoxide with polyadenylic acid, two ribonucleoside adducts were characterized by spectroscopic methods and were shown to have arisen from the cis opening of the epoxide ring at C1 by the amino group of adenine residues.