FR66979 requires reductive activation to cross-link DNA efficiently

The activity of FR66979 as a DNA-DNA interstrand cross-linking agent in the absence of exogenous reducing agents was re-examined. Samples of FR66979 prepared by hydrogenation of FR900482 (H₂/Pd-C) were quite active whereas reduction of FR900482 with sodium borohydride afforded inactive or very weakly active FR66979 samples. Rigorous purification of FR66979 from either source abolished the observed cross-linking activity thus indicating that FR66979 does not efficiently cross-link DNA in the absence of exogenous reducing agents. LC-MS analysis of active samples indicated the possible presence of the over-reduced impurity 3, below. The DNA-DNA cross-link arising from the same sample was identical in structure to that formed by reductively activated FR66979.     

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.     

A concise total synthesis of (+)-FR900482 and (+)-FR66979

The concise, enantioselective total synthesis of the potent antitumor antibiotics (+)-FR900482 and (+)-FR66979 are described. Sharpless asymmetric epoxidation technology has been deployed to construct the optically active aziridine-containing fragment that is joined to the aromatic moiety in a highly convergent manner. Dimethyldioxirane effects the remarkable one-step deprotection/oxidative cyclization of an eight-membered ring amino-ketone to the unique hydroxylamine hemiketal ring system that is a distinctive structural motif of FR900482. This reaction has been exploited in a concise 33-step enantioselective total synthesis of FR900482.     

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.     

Synthetic and biosynthetic studies on FR900482 and mitomycin C: an efficient and stereoselective hydroxymethylation of an advanced benzazocane intermediate

We report a simple, efficient, and stereoselective Mukaiyama aldol approach to install the key hydroxymethyl moiety into the benzazocane framework of FR900482. Synthetic investigations revealed that the reaction is highly dependent upon the electronics of the aromatic ring. This approach enabled the economical introduction of a [13C] label to study the biosynthesis of these structurally and biogenetically related natural products. Epimerization of the initially formed beta-hydroxy ketone may enable access to mitomycin C or FR900482 biosynthetic congeners.     

Synthesis and reactivity of 2,3-dihydro-1H-pyrrolo[1,2-a]indole derivatives,analogs of the FR900482 and mitomycin C active intermediates

A series of 2,3-dihydro-1H-pyrrolo[1,2-a]indoles were prepared as analogs of the active intermediates of the natural products, mitomycin C and FR900482, and their reactions with various nucleophiles were studied.     

Differential effects of FR900482 and FK317 on apoptosis, IL-2 gene expression, and induction of vascular leak syndrome

Vascular leak syndrome (VLS) is a harmful side effect that resulted in withdrawal of the antitumor drug FR900482, but not FK317, from clinical trials. Here we present chromatin immunoprecipitation data showing that FK317, like FR900482, crosslinks minor-groove binding proteins to DNA in vivo. However, these drugs differ in how they induce cell death. We demonstrate that, whereas FR900482 induces necrosis, FK317 induces a necrosis-to-apoptosis switch that is drug concentration dependent. Northern blot analyses of drug-treated cells suggest that this "switch" is mediated, at least in part, by modulation of the expression levels of Bcl-2. Additionally, FR900482, in contrast to FK317, induces the expression of known elicitors of both Bcl-2 gene expression and VLS. These findings provide plausible explanations for why these structurally similar drugs have different biological effects, especially with respect to VLS.     

7-N,7'-N'-(1",2"-Dithianyl-3",6"-dimethylenyl)bismitomycin C: synthesis and nucleophilic activation of a dimeric mitomycin

Dimeric alkylating agents that modify complementary DNA strands have engendered significant interest. We have prepared the novel dimeric mitomycin, 7-N,7'-N'-(1",2"-dithianyl-3",6"-dimethylenyl)bismitomycin C (9), in which the mitomycins are bridged by a dithiane unit. Dimer 9, like the clinically tested acyclic disulfides KW-2149 (3) and BMS-181174 (4), was designed to activate under nucleophilic and reductive conditions. Successive nucleophile-mediated disulfide cleavage transformations of 9 are expected to generate thiol species ideally positioned to render the two mitomycin systems vulnerable to nucleophilic attack and permit DNA interstrand cross-link formation. The dithiane linker, strategically positioned between the two mitomycins, distinguished 9 from 3 and 4. Nucleophilic activation of this cyclic disulfide permitted both activated mitomycins to remain tethered to one another. We report the synthesis of 9, and show that the nucleophile Et(3)P markedly enhances the activation and consumption of 9, compared with the reference compound 7-N, 7"-N'-(cyclohexanyl-trans-1",4"-dimethylenyl)bismitomycin C (27). We further demonstrated that provides higher levels of DNA interstrand cross-links than either the dimeric reference compounds, and 7-N,7-N'-(2",5"-dihydroxy-1",6"-hexanediyl)bismitomycin C (28), or the monomeric mitomycins, 1 and 3, when Et(3)P is added to solutions containing EcoRI-linearized pBR322 DNA.     

Design,synthesis and biological evaluation of a novel platinum(II) complex possessing bioreductive groups for cancer therapy

A Pt(Ⅱ) complex possessing bioreductive groups was constructed and evaluated as a more potent antitumor agent than cisplatin.     

Ultrasound-assisted convenient synthesis of hypolipidemic active natural methoxylated (E)-arylalkenes and arylalkanones

An ultrasound-assisted convenient method was developed for the conversion of toxic methoxylated cis-isomer of arylalkenes into its hypolipidemic active trans-isomer. Treatment of cis-isomer or mixture of all three isomers (1a-1j) with ammonium formate and 10% Pd/C gave arylalkanes (2a-2j), which upon oxidation with DDQ in anhydrous dioxane containing a little amount of silica gel, provided (E)-arylalkenes (3a-3g) in 42-72% yield depending upon the substituents attached at the aryl ring. The same method, upon addition of a few drops of water, provided hypolipidemic active arylalkanones (3h-3j) in 59-65% yield.     

Insight Into Factors Governing Formation,Synthesis and Stereochemical Configuration of DNA Adducts Formed by Mitomycins

Mitomycin C, (MC), an antitumor drug used in the clinics, is a DNA alkylating agent. Inert in its native form, MC is reduced to reactive mitosenes in cellulo which undergo nucleophilic attack by DNA bases to form monoadducts as well as interstrand crosslinks (ICLs). These properties constitute the molecular basis for the cytotoxic effects of the drug. The mechanism of DNA alkylation by mitomycins has been studied for the past 30 years and, until recently, the consensus was that drugs of the mitomycins family mainly target CpG sequences in DNA. However, that paradigm was recently challenged. Here, we relate the latest research on both MC and dicarbamoylmitomycin C (DMC), a synthetic derivative of MC which has been used to investigate the regioselectivity of mitomycins DNA alkylation as well as the relationship between mitomycins reductive activation pathways and DNA adducts stereochemical configuration. We also review the different synthetic routes to access mitomycins nucleoside adducts and oligonucleotides containing MC/DMC DNA adducts located at a single position. Finally, we briefly describe the DNA structural modifications induced by MC and DMC adducts and how site specifically modified oligonucleotides have been used to elucidate the role each adduct plays in the drugs cytotoxicity.     

Flash vacuum thermolysis generation and a UV-photoelectron spectroscopy study of the N-substituted iminoacetonitriles

Flash vacuum thermolysis of cis and trans 1-benzyl and 1-allyl-2-cyano-3-phenylzetidines (1a and 1b) at 470 °C resulted in the formation of E and Z isomers of N-benzyl and N-allyl iminoacetonitriles 2a and 2b, respectively, beside small amounts of products 3a and 3b of their [1,3] prototropic shifts. It was found that the thermal fragmentation of the azetidine ring occurred fully regioselectively with a cleavage of the C2-C3 and C4-N bonds, but not the N-C2 and C3-C4 bonds. The UV-photoelectron spectrum of compound 2b was measured and analyzed with the aid of quantum chemical calculations of ionization energies. The strong modification of the electronic structure of the simplest carbon-nitrogen double bond upon the α,β substitution is due to the combined electron-withdrawing effect of the nitrile group and the donor effect of the allyl group.     

Advances in the Synthesis of Ring-Fused Benzimidazoles and Imidazobenzimidazoles

This review article provides a perspective on the synthesis of alicyclic and heterocyclic ring-fused benzimidazoles, imidazo[4,5-f]benzimidazoles, and imidazo[5,4-f]benzimidazoles. These heterocycles have a plethora of biological activities with the iminoquinone and quinone derivatives displaying potent bioreductive antitumor activity. Synthesis is categorized according to the cyclization reaction and mechanisms are detailed. Nitrobenzene reduction, cyclization of aryl amidines, lactams and isothiocyanates are described. Protocols include condensation, cross-dehydrogenative coupling with transition metal catalysis, annulation onto benzimidazole, often using CuI-catalysis, and radical cyclization with homolytic aromatic substitution. Many oxidative transformations are under metal-free conditions, including using thermal, photochemical, and electrochemical methods. Syntheses of diazole analogues of mitomycin C derivatives are described. Traditional oxidations of o-(cycloamino)anilines using peroxides in acid via the t-amino effect remain popular.     

Tandem mass spectrometry for the determination of the sites of DNA interstrand cross-link

Formation of DNA interstrand cross-link is implicated in the mechanism of anticancer activity of some drugs. Here we examined the fragmentation of deprotonated ions of double-stranded oligodeoxynucleotides (ODNs) that are covalently held together with either a mitomycin C or a 4,5',8-trimethylpsoralen. Our results showed that, upon collisional activation, the covalently-bound duplex ODNs cleaved to give a series of wn and [an-base] ions; the sites of interstrand cross-linking could be determined from the mass shifts of some product ions. In addition, compared with the product-ion spectra acquired on an ion trap, those obtained from sustained off-resonance irradiation-collisionally activated dissociation (SORI-CAD) on a Fourier transform mass spectrometer offered high mass-resolving power, which facilitated unambiguous assignment of product ions and made it an effective method for locating the cross-linking sites.     

Photodynamic cross-linking of proteins: IV. Nature of the His–His bond(s) formed in the rose bengal-photosensitized cross-linking of N-benzoyl-L-histidine

The photodynamic (photosensitized) cross-linking of N-benzoyl-L-histidine (Bz-His) as a model system was examined as part of a continuing study of the role of His–His intermolecular cross-links in the photosensitized cross-linking of proteins. The illumination of Bz-His in the presence of rose bengal (RB) bound to water insoluble plastic beads in 0.1 M sodium phosphate buffer of pH 7.4 resulted in the covalent cross-linking of the His derivative. The main dimeric cross-linked product (1) was isolated using a preparative silica gel 60 column and purified by preparative reverse phase HPLC. The chemical structure of the cross-link was determined using MS, 2D NMR spectral methods and other standard techniques. Product 1 was found to be a dimer of two His residues between the δ2-carbon of one residue (photo-oxidized to the carbonyl functionality at the ε1-carbon) and the ε2-nitrogen of the other residue. The formation of His–His cross-links was mediated by singlet oxygen, as would be expected with RB as the sensitizer. A mechanism for the formation of the cross-link was proposed in which the first step was the 1,4-cycloaddition of singlet oxygen to the Bz-His imidazole ring to give an unstable endoperoxide. This then underwent changes followed by nucleophilic addition and the elimination of one molecule of water to give 1.     

Hydroxyquinone O-methylation in mitomycin biosynthesis

Mitomycins are bioreductively activated DNA-alkylating agents. One member of this family, mitomycin C, is in clinical use as part of combination therapy for certain solid tumors. The cytotoxicity displayed by mitomycins is dependent on their electrochemical potential which, in turn, is governed in part by the substituents of the quinone moiety. In this paper we describe studies on the biogenesis of the quinone methoxy group present in mitomycins A and B. An engineered Streptomyces lavendulae strain in which the mmcR methyltransferase gene had been deleted failed to produce the three mitomycins (A, B, and C) that are typically isolated from the wild type organism. Analysis of the culture extracts from the mmcR-deletion mutant strain revealed that two new metabolites, 7-demethylmitomycin A and 7-demethylmitomycin B, had accumulated instead. Production of mitomycins A and C or mitomycin B was selectively restored upon supplementing the culture medium of a S. lavendulae strain unable to produce the key precursor 3-amino-5-hydroxybenzoate with either 7-demethylmitomycin A or 7-demethylmitomycin B, respectively. MmcR methyltransferase obtained by cloning and overexpression of the corresponding mmcR gene was shown to catalyze the 7-O-methylation of both C9beta- and C9alpha-configured 7-hydroxymitomycins in vitro. This study provides direct evidence for the catalytic role of MmcR in formation of the 7-OMe group that is characteristic of mitomycins A and B and demonstrates the prerequisite of 7-O-methylation for the production of the clinical agent mitomycin C.     

Aplyronine A, a potent antitumor macrolide of marine origin, and the congeners aplyronines B and C: isolation, structures, and bioactivities

Aplyronine A (2), a potent antitumor macrolide was isolated from the sea hare Aplysia kurodai together with the congeners aplyronines B (3) and C (4). The absolute stereostructure of aplyronine A (2) was determined by the instrumental analysis (mainly NMR and MS) and the enantioselective synthesis of the fragments obtained from chemical degradation of aplyronine A (2). The structures of aplyronines B (3) and C (4) were also elucidated. Cytotoxicity and antitumor activity of aplyronine A (2) were evaluated.     

Nucleic Acid Binding Behavior and Cytotoxicity Properties of a Ruthenium(II) Polypyridyl Complex

The binding of [Ru(IP)₂(dppz-11-CO₂Me)]²⁺ (1) {IP = imidazo[4,5-f][1,10]phenanthroline, dppz-11-CO₂Me = dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxylic acid methyl ester} to calf thymus DNA and yeast tRNA has been investigated by UV–Vis spectroscopy, fluorescence spectroscopy, viscosity, as well as equilibrium dialysis and circular dichroism. In addition, the antitumor activities of complex 1 have been evaluated by the MTT method. On the basis of the spectroscopic results, the binding mode of complex 1 to CT-DNA and yeast tRNA is intercalation. However, DNA binding with complex 1 is stronger than RNA binding with complex 1, and complex 1 is a better candidate for an enantioselective binder to CT-DNA than to yeast tRNA. These results indicate that the structures of DNA and RNA have significant effects on the binding behaviors of complex 1. Furthermore, complex 1 demonstrates different antitumor activities against selected cancer cell lines in vitro.     

CBS-QB3 computational examination of substituent effects on the interconversion of 1,3,5-cyclooctatriene and bicyclo[4.2.0]-2,4-octadiene

The position of equilibrium between 7-substituted and 7,8-disubstituted 1,3,5-cyclooctatrienes (1) and the corresponding valence isomeric bicyclo[4.2.0]-2,4-octadienes (2) is highly sensitive to the nature of substituents. In particular, the difference between trans and cis-7,8 isomers in this regard has never been explained. These differences have been clarified by computational means. The CBS-QB3 hybrid quantum chemical computational method reproduces the experimental free energy differences between a series of cyclooctatrienes (1) and the corresponding valence isomers (2) with excellent accuracy; the MAD and root mean square (rms) differences are 0.54 kcal/mol and 0.58 kcal/mol, respectively. The energy barriers between several derivatives of 1 and 2 were computed with good accuracy (MAD=1.3 kcal/mol and rms=1.5 kcal/mol) by the same procedure. The dihedral angle between the substituents increases upon electrocyclic conversion of trans-7,8-disubstituted cyclooctatrienes to the bicyclic isomer and decreases for the corresponding cis isomers. This differential effect explains both the higher bicyclic proportion at equilibrium and the faster rate of cyclization in the trans series compared to cis.