Antitumor Antibiotic Streptonigrin Research Articles

A bifunctional methyltransferase in biosynthesis of antitumor antibiotic streptonigrin.

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. STN structurally contains four methyl groups belonging to two types: C-methyl group and O-methyl groups. Here, we report the biochemical characterization of the O-methyltransferase StnQ2 that can catalyze both the methylation of a hydroxyl group and a carboxyl group in the biosynthesis of streptonigrin. This work not only provides a new insight into methyltransferases, but also advances the elucidation of the complete biosynthetic pathway of streptonigrin.

Methyl 1-(7-acetamido-5,8-dimethoxyquinolin-2-yl)-4-methyl-β-carboline-3-carboxylate

The title compound, C27H24N4O5, is an inter­mediate in the synthesis of lavendamycin via a ruthenium-catalysed [2 + 2 + 2] cyclo­addition. An intra­molecular hydrogen-bond bridge from the carboline to the quinoline stabilizes a highly planar geometry [maximum deviation = 0.065 (6) Å] for the two rigid units. This hydrogen-bond-stabilized coplanarity has a very close analogy in the structure of the anti­tumor anti­biotic streptonigrin in the solid state and in solution. Inter­molecular hydrogen-bond bridges of amides groups along the a axis and π–π stacking inter­actions [centroid–centroid distance = 3.665 (9) Å] connect mol­ecules arranged in a parallel manner.

Antitumor antibiotic streptonigrin and its derivatives as inhibitors of nitric oxide-dependent activation of soluble guanylyl cyclase

The influence of streptonigrin on the activity of human platelet guanylyl cyclase was investigated. Streptonigrin (0.1–5 μM) had no effect on the basal activity of the enzyme, but inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet soluble guanylyl cyclase with an IC 50 value of 4.16 μM. Streptonigrin (10 μM) also inhibited (by 28%) the activation of the enzyme by the direct nitric oxide (NO) donor–spermine–NONO (100 μM), but had no influence on the stimulation of soluble guanylyl cyclase by protoporphyrin IX. The absence of a correlation between the inhibition of NO-stimulated guanylyl cyclase activity by streptonigrin ( I) and its derivatives (streptonigrone ( IV), streptonigrone-2′-imine ( V), amide of 1 and 2′-deoxy-2′-amino- d-glucose ( VI), amide of 1 and 2′-deoxy-2′-amino-2′- d-galactose ( VII), amide of 1 and 1- O-methyl-6-deoxy-6-amino- d-glucose ( VIII), diphenylmethyl ester of I ( IX), conjugate of I and daunorubicin ( X)), and the level of cytotoxic effects of these compounds excludes the involvement of guanylyl cyclase in the mechanism of antitumor action of streptonigrin. Inhibition of guanylyl cyclase activation by NO donors but not by protoporphyrin IX represents a new biochemical effect of streptonigrin, which should be taken into account in addition to its antitumor action.

The effect of metal ions on the electrochemistry of the antitumor antibiotic streptonigrin

The effect of transition metal ions on the electrochemistry of 6-methoxy-5,8-quinolinedione (L1), 7-amino-6-methoxy-5,8-quinolinedione (L2) and the antitumor antibiotic streptonigrin (SN) was studied. In 10% methanol/water, the one-electron reduction of quinones L1 and L2 to the corresponding semiquinones is shifted to more positive potentials upon addition of one equivalent of Zn(II), Ni(II), Co(II) or Cd(II) and is consistent with formation of a 1:1 complex involving the quinone(N) and adjacent quinone(O). Similar results are observed for Cu(II) and Mn(II), but the redox chemistry is also complicated by metal-based redox chemistry. The addition of further equivalents of M(II) results in a number of different coordination and electrochemical processes including formation of 1:1 and 2:1 complexes of the quinone, semiquinone and dianion. Under similar conditions, the 1:1 SN 2,2′-bipyridyl metal complex undergoes a reversible one-electron reduction to the semiquinone. The redox potential of the quinone in SN was shifted positive in the presence of the metal ions, but both the magnitude of the shift, and the relative influence of the metals was different to ligands L1 and L2. The changes in redox chemistry of SN compared with L1 and L2 are consistent with the formation of the 2,2′-bipyridyl complexes in which there is weaker coordination to the quinone(O) in ring A of SN. These results suggest that in vivo, metal ions such as Zn(II), Cu(II) and Mn(II) facilitate the initial reduction of streptonigrin to the semiquinone by capturing the semiquinone after SN is reduced by biological reductants.

Ruthenium complexes of analogues of the antitumor antibiotic streptonigrin.

The complexes Ru(L1-CH3)(CO)3Cl, RuL2(CO)2Cl2, and RuL3(CO)2Cl2 (L1= 6-methoxy-5,8-quinolinedione, L2 = 7-amino-6-methoxy-5,8-quinolinedione, L3 = 6,6'-dimethoxycarbonyl-2,2'-bipyridine) were prepared by reaction of L1-L3 with the tricarbonyldichlororuthenium(II) dimer. L1-L3 act as bidentates through the ortho oxygen atoms, the pyridyl nitrogen and the adjacent quinone oxygen, and the bipyridyl nitrogens, respectively. RuL3(CO)2Cl2 is characterized by X-ray crystallography. 15N NMR correlation spectra give upfield shifts of around 60 ppm for the pyridyl nitrogens that are coordinated to the metal, while 13C NMR correlation spectra give a downfield shift of 10 ppm for the quinone carbonyl group that is coordinated to the metal. The electrochemistry of RuL2(CO)2Cl2 is examined, and the implications for the formation of metal complexes of the antitumor antibiotic streptonigrin, which cleaves DNA in the presence of metal ions, are discussed.

Further insight into the Zn 2+-mediated binding of streptonigrin to DNA

The interaction of double- and single-stranded DNA with the antitumor antibiotic streptonigrin (STN) in the presence of zinc ions has been examined by spectrophotometric and circular dichroism techniques. Very little interaction occurs in the absence of the metal ion. When Zn 2+ is present, the binding process exhibits a bell-shaped dependence upon the ion concentration, consistent with the formation of a ternary complex, splitting into two binary complexes at high content of the metal ion. Evidence is presented for the participation of two STN molecules bridged by Zn 2+ in the complex with DNA.

ChemInform Abstract: Interaction of the Antitumor Antibiotic Streptonigrin with Metal Ions and DNA

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NMR Studies of metal complexes and DNA binding of the quinone-containing antibiotic streptonigrin

Optical and 1H NMR techniques have been applied to the study of a few metal complexes (Co2+, Fe2+, and Yb3+) of the antitumor antibiotic streptonigrin (SN) produced by Streptomyces flocculus to elucidate the structure of the complexes. The hyperfine-shifted 1H NMR signals of these paramagnetic complexes were fully assigned by means of relaxation and two-dimensional NMR techniques. These studies revealed that SN binds transition metal and lanthanide ions and forms stable metal–drug complexes, with the metal located at the quinolinequinone–picolinate site. This configuration requires a ≈180° twist of the C2–C2′ bond in the crystal structure of the drug. The hyperfine-shifted 1H NMR signals of the Co2+–SN complex are significantly changed upon addition of calf thymus DNA or poly[dA-dT], indicating direct binding of Co2+–SN complex with DNA.

Interaction of the Antitumor Antibiotic Streptonigrin with Metal Ions and DNA

Streptonigrin is a functionalized 7-aminoquinoline-5,8-dione that is highly active against a range of human cancers. While streptonigrin was in clinical use until 1977, the mechanism of antitumor action of the drug was only investigated in detail after clinical trials were discontinued. In common with other aminoquinone antitumor antibiotics, extensive studies suggest that streptonigrin exerts its antitumor activity by causing DNA strand scission. Metal ions were identified as serving a key role in both the DNA-degradation pathway, either by direct complexation with streptonigrin or reduced streptonigrin and/or catalysing the production of the DNA damaging species, and in enhancing the interaction of the drug with ccc-DNA and calf thymus DNA. While streptonigrin itself interacts only very weakly with DNA, this interaction is strongly enhanced when the drug is reductively activated or in the presence of transition metal ions. This review will cover the literature reported since 1985. Important results that have been recently reported include the solution conformation of the drug, structures of the metal complexes of streptonigrin, characterisation of streptonigrin semiquinone, DNA-footprinting and key structure-activity studies. These reports have clarified some of the mechanistic aspects of streptonigrin-mediated DNA cleavage and provide a basis for rational drug design of streptonigrin analogues with modified therapeutic properties and side effects.

Preparation and ESR spectroscopic characterization of the zinc(II) and cadmium(II) complexes of streptonigrin semiquinone

Semiquinone metal complexes derived from the antitumor antibiotic streptonigrin have been prepared for the first time. They were obtained by reduction of the zinc(II) and cadmium(II) complexes of the parent aminoquinone ligand with sodium borohydride, followed by air oxidation of the intermediate dihydroquinones. Alternatively, N-benzyldihydronicotinamide reduction was used to produce the same Cd(II) complex of the p-semiquinone free radical. Electron spin resonance spectroscopic studies showed that metal binding significantly changes the spin densities of the unpaired electron which is confined to the quinolinesemiquinone moiety of the complexed antibiotic. Complexation with both Cd(II) and Zn(II) perturbs the coupling constants of all atoms involved in delocalization of the unpaired electron, shifting its distribution toward the pyridine ring. The coupling constant of the pyridine ring-proton adjacent to the semiquinone ring increases from 0.31 to 0.43 G in the Cd(II) complex in methanol, while the proton meta to the pyridine nitrogen increases from 1.76 to 1.96 G. Furthermore, the coupling constant of the heterocyclic nitrogen increases from 0.46 to 0.61 G. A similar trend is noted for the Zn(II) complex as well, including the observed decrease in splitting constant of the amino nitrogen from 1.34 to 1.08 G, and perturbation of the previously equivalent amino protons from 0.89 and 0.89 to 1.09 and 0.95 G. The spectral parameters have been confirmed by deuteration. Complexation studies using isotopically enriched 113Cd(II) revealed hyperfine coupling of the unpaired electron of the p-semiquinone and the nuclear spin of 113Cd(II), indicating direct coordination between the metal and the complexing ligand. Although the metal complexes could readily be prepared in a series of different solvent systems, they appear to have substantially shorter half lives than the non complexed p-semiquinone radical (5 to 15 min vs. 2 to 3 wk in sealed ampoules). Formation of tight-binding p-semiquinone metal complexes as here described should provide useful leads for the design of related systems to study p-quinone-metal interactions for mechanistic elucidation of metal ion catalyzed quinone-dependent electron transfer reactions in biological oxidations. © 1997 Elsevier Science B.V. All rights reserved.

The structure of streptonigrin semiquinone in solution

Streptonigrin semiquinone (SQ ·), a free radical intermediate implicated in the biological functioning of the antitumor antibiotic streptonigrin has been prepared and its structural properties in solution have been characterized. Through the use of electron paramagnetic resonance spectroscopy, the spin densities of the unpaired electron have been determined, indicating that the unpaired electron is largely confined to the quinolinesemiquinone moiety of the antibiotic. Unambiguous assignment of the hyperfine coupling constants was achieved employing isotopically labeled semiquinone radical, INDO molecular orbital calculations, and the study of unsubstituted 5,8-quinolinesemiquinone as a reference system. The assignments point to a negative spin density at the carbon para to the pyridine nitrogen in the radicals derived from both streptonigrin and the unsubstituted 5,8-quinolinequinone. Characterization of the properties of the streptonigrin semiquinone in solution indicate that the radical is stable in solution: it can be conveniently studied in 0.1 M methanolic lithium hydroxide or in aqueous organic solvent mixtures buffered with 0.06 M K 3PO 4 at pH 12.0. Under these conditions, the semiquinone shows completely reversible spectral changes between −10 to 60°C. Lowering the pH from 12.0 to 7.0 in aqueous DMSO decreases the lifetime of the radical from two weeks to a few minutes. Changes in structural properties of streptonigrin semiquinone in solution have been found to occur mainly due to variation in solvation and freedom of rotation of the amino group. Decreasing the temperature of SQ · solution in methanol from 60 to −10°C leads to an increase in the hyperfine coupling constant to the amino nitrogen from 1.28 to 1.40 G, and those of the two amino protons from 0.73 and 0.73 to 1.02 and 1.11 G respectively, while the other coupling constants change less than 3%. Greater electron spin delocalization onto the -NH 2 group has been found throughout the solvent systems examined, yet the temperature at which the two amino protons become equivalent changes with the nature of the solvent (e.g., from 25°C in MeOH to 40°C in aq. DMSO). The effective rotational diffusion constant as measured from EPR spectra in a series of solvents with fixed polarity ( E T) follows the Stokes–Einstein equation only in solvent mixtures of low to moderate viscosities (10–12.4 cP) suggesting that in addition to the viscosity of the medium a more specific mechanism (e.g., hydrogen bonding to the solvent) restricts the motion of the amino group. This hydrogen bonding mechanism is further supported by the fact that the degree of inequivalence in the amino proton hyperfine coupling constants varies monotonically with the spin density at the amino nitrogen. Studies of SQ · in aqueous micellar dispersions using neutral, cationic, and anionic sufractants indicate that SQ · is located at the surface of these aggregates. The structure of SQ · changes little at neutral and anionic micellar surfaces; however, more severe structural changes occur in cationic micelles which appear to be consistent with a conformational change induced by the positively charged surface.

Selective killing of tumor cells in vitro by immunotoxin composed of antitumor antibiotic streptonigrin and polyclonal specific antibodies

Streptonigrin N-hydroxysuccinimide ester (STN-COONSu) was obtained by carbodiimide synthesis. Poly- L-lysine (PLL) was loaded with STN-COONSu and conjugated to polyclonal rabbit immunoglobulin G (IgG) activated with sodium periodate. Non-specific IgG and IgG against Ehrlich carcinoma cells were used to construct non-specific and specific immunotaxins. Immunotaxins contained 100 molecules of streptonigrin per 1 molecule of IgG. The streptonigrin concentration that caused 50% of inhibition of [ 3H]thymidine incorporation in Ehrlich carcinoma cells (IC 50) was 0.8 μg/ml for specific immunotoxin, 16 μg/ml for non-specific immunotoxin, and 20 μg/ml for the poly- L-lysine-streptonigrin conjugate (PLL-STN) used as the initial water-soluble form of antibiotic. Our results demonstrate that the toxicity for target cells of streptonigrin conjugated to specific IgG was 25 times higher than that of the initial water soluble form of antibiotic. This specific immunotoxin was non-toxic for non-target cells.

Electron spin resonance of the semiquinone of the antitumor antibiotic streptonigrin and its metal complexes.

Mechanistic elucidation of the redox chemistry of aminoquinone antibiotics exhibiting potent antitumor activity has become one of the most timely problems of cancer chemotherapy today1,2. Specifically, streptronigrin (Figure 1a), is currently regarded as one of the most effective agents for the treatment of a broad spectrum of human cancers3, yet its clinical application is greatly limited due to severe cytotoxicity of the drug. It has been reported that streptonigrin is activated in vivo by one-electron reduction forming the semiquinone4–6 and that the mechanism of antitumor action of streptonigrin depends on participation of metal ions7.

Biological properties of streptonigrin derivatives. II. Inhibition of reverse transcriptase activity.

Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies. Against AMV reverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH, with the ID50 values ranging 2-8 μg/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but not STN-CONH(CH2)3N(CH3)2. Doxorubicin-resistant murine lymphoblastoma L5178Y cells showed collateral sensitivity to both STN-COOH and STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.

Synthetic studies of the antitumor antibiotic streptonigrin. 3. Synthesis of the C-D ring of streptonigrin by an unsymmetrical Ullmann reaction

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthetic studies of the antitumor antibiotic streptonigrin. 3. Synthesis of the C-D ring of streptonigrin by an unsymmetrical Ullmann reactionPhilip J. Wittek, Tsung Kai Liao, and Chia Chung ChengCite this: J. Org. Chem. 1979, 44, 5, 870–872Publication Date (Print):March 1, 1979Publication History Published online1 May 2002Published inissue 1 March 1979https://doi.org/10.1021/jo01319a052RIGHTS & PERMISSIONSArticle Views191Altmetric-Citations24LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (463 KB) Get e-Alerts Get e-Alerts

Streptonigrin-transition metal complexes: Binding to DNA and biological activity

Summary Two effects of transition metal divalent cations on the behavior of the antitumor antibiotic streptonigrin are reported: (1) Metal ions such as Mn++, Zn++, and Cd++ promote an association of the antibiotic with double-stranded DNA. The metal ion causes a depression and slight red shift of the 365 nm absorption maximum of the antibiotic. Addition of DNA to the zinc-streptonigrin complex causes a further depression and red shift of the absorption maximum, indicating the formation of a DNA-streptonigrin-zinc complex. (2) These metal cations affect the biological activity of streptonigrin, in that they greatly enhance the lethal effect of the antibiotic on Escherichia coli . It is likely that the two effects are related.

Synthetic studies of the antitumor antibiotic streptonigrin. II. Synthesis of the C‐D ring portion of streptonigrin

AbstractPartial mclhylalion of gallopropiophenonc (4) followed by benzylation and base‐catalyzed condensation with ethyl acetate yielded 3‐(2‐benzyloxy‐3,4‐din]etlK>xy)benzoyl‐2‐butanonc (6). Animation of the latter and subsequent base‐catalyzed cyclization with ethyl cyanoacetate gave 4‐(2‐benzyloxy‐3,4‐dimethoxy)phenyl‐5,6‐dimethyl‐2‐oxo‐1,2‐dihydropyridine (8). Removal of the 2‐oxo group of 8 through chlorination and dechlorination and stepwise conversion of the 5‐cyano and 2‐methyl groups into the 5‐amino and 2‐carboxylic acid groups, respectively, with introduction and removal of protecting groups at proper stages, yielded the C‐D ring moiety of the antitumor antibiotic streptonigrin.

Synthetic studies of the antitumor antibiotic streptonigrin. I. Synthesis of the A‐B ring portion of streptonigrin

AbstractBromination of 6‐methoxy‐5,8‐quinolinedione gave the 7‐bromo derivative in quantitative yield. Treatment of the bromo compound with sodium azide followed by hydrogenation yielded 7‐amino‐6‐methoxy‐5,8‐quinolinedione, the A‐B ring portion of the antitumor antibiotic strep tonigrin. The corresponding 2‐methyl homolog was prepared in a similar manner from 6‐methoxy‐2‐methyl‐8‐nitroquinoline, which in turn, was obtained by a Skraup synthesis from 2‐nitro‐anisidine and crotonaldehyde.

Studies related to antitumor antibiotics. Part VII. Synthesis of streptonigrin analogues and their single strand scission of DNA

The syntheses of a group of 2-(o-nitrophenyl)- and 2-(o-aminophenyl)-5,8-quinolinediones which are structurally related to the antitumor antibiotic streptonigrin are described. Ambiguities in the position of required nucleophilic displacements are resolved by independent synthesis. The rates of single strand cleavage of PM2 ccc-DNA (covalently-closed circular-DNA) induced by these compounds are compared, which correlates with antitumor activity. The 2-(o-nitrophenyl) derivatives give consistently more rapid DNA cleavage than the 2-(o-aminophenyl) compounds. The autoxidations of the dihydroxyquinolines are subject to selective catalysis by Cu2+ on. 2-(o-Aminophenyl)-7-amino-6-methoxy-5,6-quinolinedione which has a substitution pattern most closely resembling streptonigrin also closely parallels the rate of scission of DNA of the latter in the presence of NADPH.

Studies related to antitumor antibiotics. Part VIII. Cleavage of DNA by streptonigrin analogues and the relationship to antineoplastic activity.

A group of substituted 5,8-quinolinequinones which exhibit antineoplastic activity and which are structurally related to the antitumor antibiotic streptonigrin induce single strand cleavage of PM2 covalently-closed circular-DNA (ccc-DNA) when reductively activated. The cleavage which is detected by an ethidium fluorescence assay is specifically enhanced by cuprous and ferrous ion and is selectively inhibited by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) and by free radical scavengers. Independent generation of the superoxide ion by xanthine-xanthine oxidase (EC 1.2.3.2) also cleaves PM2 DNA and therefore a chemical mechanism for the scission process induced by the streptonigrin analogues is formulated. A correlation between rate of PM2 ccc-DNA cleavage and inhibition of Walker carcinosarcoma 256 is observed.