Chemistry and Properties of Indolocarbazoles.

The indolocarbazoles are an important class of nitrogen heterocycles which has evolved significantly in recent years, with numerous studies focusing on their diverse biological effects, or targeting new materials with potential applications in organic electronics. This review aims at providing a broad survey of the chemistry and properties of indolocarbazoles from an interdisciplinary point of view, with particular emphasis on practical synthetic aspects, as well as certain topics which have not been previously accounted for in detail, such as the occurrence, formation, biological activities, and metabolism of indolo[3,2- b]carbazoles. The literature of the past decade forms the basis of the text, which is further supplemented with older key references.

Role of Thiol Reactivity for Targeting Mutant p53.

Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target thiol groups in mutant p53. Here we have investigated the relationship between thiol reactivity, p53 thermostabilization, mutant p53 refolding, mutant p53-dependent growth suppression, and induction of cell death. Analysis of the National Cancer Institute database revealed that Michael acceptors show the highest selectivity for mutant p53-expressing cells among analyzed thiol-reactive compounds. Further experimental testing demonstrated that Michael acceptors, aldehydes, imines, and primary alcohols can promote thermodynamic stabilization of mutant p53. Moreover, mild thiol reactivity, often coupled with combined chemical functional groups, such as in imines, aldehydes, and primary alcohols, can stimulate mutant p53 refolding. However, strong electrophile activity was associated with cellular toxicity. Our findings may open possibilities for rational design of novel potent and selective mutant p53-reactivating compounds.

Thionation of Tryptanthrin, Rutaecarpine, and Related Molecules with a Reagent Prepared from P4S10 and Pyridine.

Reaction of P4S10 in hot pyridine produces a crystalline solid which can be collected and used for thionations in other solvents such as acetonitrile and sulfolane. The biologically active natural products tryptanthrine, rutaecarpine, 7,8-dehydrorutaecarpine, and some related compounds have now been converted to thionated versions simply by heating the molecules with this thionating reagent in sulfolane (typically at 135 °C for 20 min) followed by a workup in water. No chromatography was necessary.

Evidence for New Light-Independent Pathways for Generation of the Endogenous Aryl Hydrocarbon Receptor Agonist FICZ.

Activation of the aryl hydrocarbon receptor (AhR), a conserved transcription factor best known as a target for highly toxic halogenated substances such as dioxin, under normal xenobiotic-free conditions is of considerable scientific interest. We have demonstrated previously that a photoproduct of tryptophan, 6-formylindolo[3,2-b]carbazole (FICZ), fulfills the criteria for an endogenous ligand for this receptor and proposed that this compound is the enigmatic mediator of the physiological functions of AhR. Here, we describe novel light-independent pathways by which FICZ can be formed. The oxidant H2O2 was shown to convert tryptophan to FICZ on its own in the absence of light. The enzymatic deamination of tryptamine yielded indole-3-acetaldehyde (I3A), which then rearranged to FICZ and its oxidation product, indolo[3,2-b]carbazole-6-carboxylic acid (CICZ). Indole-3-pyruvate (I3P) also produced I3A, FICZ, and CICZ. Malassezia yeast species, which constitute a part of the normal skin microbiota, produce a number of AhR activators from tryptophan. We identified both FICZ and CICZ among those products. Formation of FICZ from tryptophan or I3P produces a complex mixture of indole derivatives, some of which are CYP1A1 inhibitors. These can hinder the cellular clearance of FICZ and thereby increase its power as an AhR agonist. We present a general molecular mechanism involving dehydrogenations and oxidative coupling for the formation of FICZ in which I3A is the important precursor. In conclusion, our results suggest that FICZ is likely to be formed systemically.

Oxidative ring expansion of spirocyclic oxindole derivatives.

Oxidation of the spirocyclic oxindole derivative, isamic acid 1, led to decarboxylation and ring expansion to quinazolino[4,5-b]quinazoline-6,8-dione 7 rather than, as previously believed, its isomer 6. The structure of 7 was confirmed by X-ray crystallography. Condensation of isatin (indole-2,3-dione) and 2-aminobenzamide led to the spirocyclic molecule, spiro[3H-indole-3,2'(1H)quinazoline]-2,4'(1H,3H)dione 8, which was also identified as an intermediate in the oxidation of isamic acid. Mild hydrolysis of 7 gave the 10-membered molecule 22. Isamic acid could easily be converted to N-nitrosoisamic acid, which when heated in ethanol underwent a ring expansion to a hydroximino derivative, 38, of compound 6. The structure of 38 was confirmed by X-ray crystallography.

[Early team assessment relieves the emergency department]. / Tidig team-bedömning avlastar akutmottagningen.

A model for physician-led team triage was evaluated at the Emergency Department at the University hospital of Örebro, Sweden. Data from 1600 patients indicate that this work model reduces length of stay, time to physician assessment, emergency department occupancy, rate of admission and the proportion of patients in need of close monitoring. The project was conducted without any change in the number of physicians, nurses or staff nurses working in the Emergency Department. 

Synthesis of a new intercalating nucleic acid 6H-INDOLO[2,3-b] quinoxaline oligonucleotides to improve thermal stability of Hoogsteen-type triplexes.

A new intercalating nucleic acid monomer X was obtained in high yield starting from alkylation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was coupled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS protected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4'-dimethoxytrityl chloride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,N'-diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phosphoramidite was used to insert the monomer X into an oligonucleotide which was used for thermal denaturation studies of a corresponding parallel triplex.

Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals.

Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, ß-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine.

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. ß-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

Thionations using a P4S10-pyridine complex in solvents such as acetonitrile and dimethyl sulfone.

Tetraphosphorus decasulfide (P(4)S(10)) in pyridine has been used as a thionating agent for a long period of time. The moisture-sensitive reagent has now been isolated in crystalline form, and the detailed structure has been determined by X-ray crystallography. The thionating power of this storable reagent has been studied and transferred to solvents such as acetonitrile in which it has proven to be synthetically useful and exceptionally selective. Its properties have been compared with the so-called Lawesson reagent (LR). Particularly interesting are the results from thionations at relatively high temperatures (∼165 °C) in dimethyl sulfone as solvent. Under these conditions, for instance, acridone and 3-acetylindole could quickly be transformed to the corresponding thionated derivatives. Glycylglycine similarly gave piperazinedithione. At these temperatures, LR is inefficient due to rapid decomposition. The thionated products are generally cleaner and more easy to obtain because in the crystalline reagent, impurities which invariably are present in the conventional reagents, P(4)S(10) in pyridine or LR, have been removed.

One-pot Eschenmoser episulfide contractions in DMSO: applications to the synthesis of fuligocandins A and B and a number of vinylogous amides.

Practical total syntheses of the natural products fuligocandin A (2a) and fuligocandin B (3) have been achieved through a convergent strategy depending on the Eschenmoser episulfide contraction as a key step. Conducting the reaction in DMSO proved to be an efficient and general method for the synthesis of a variety of vinylogous amides, such as azepan-2-ylidenepropan-2-one.

Extract from Asteraceae Brachylaena ramiflora induces apoptosis preferentially in mutant p53-expressing human tumor cells.

The p53 tumor suppressor gene is inactivated by point mutation in a large fraction of human tumors, allowing evasion of apoptosis and tumor progression. p53 mutation is often associated with increased resistance to therapy. Pharmacological reactivation of mutant p53 is an attractive therapeutic strategy. We previously identified p53 reactivation and induction of massive apoptosis, a low-molecular weight compound that suppresses the growth of cancer cells in a mutant p53-dependent manner. Here, we report the identification and characterization of an extract from the terrestrial plant Brachylaena ramiflora (Asteraceae) that preferentially induces apoptosis in human tumor cells expressing mutant p53. Further analysis of this extract and identification of active compounds may provide novel structural scaffolds for the development of mutant p53-targeting anticancer drugs.

PRIMA-1 reactivates mutant p53 by covalent binding to the core domain.

Restoration of wild-type p53 expression triggers cell death and eliminates tumors in vivo. The identification of mutant p53-reactivating small molecules such as PRIMA-1 opens possibilities for the development of more efficient anticancer drugs. Although the biological effects of PRIMA-1 are well demonstrated, little is known about its molecular mechanism of action. We show here that PRIMA-1 is converted to compounds that form adducts with thiols in mutant p53. Covalent modification of mutant p53 per se is sufficient to induce apoptosis in tumor cells. These findings might facilitate the design of more potent and specific mutant p53-targeting anticancer drugs.

Synthesis of 1,4-benzodiazepin-3-ones and 1,5-benzodiazocin-4-ones by addition of Grignard reagents to derivatives of o-aminobenzonitrile.

Addition of organometallics to N-(alpha-haloacyl)-o-aminobenzonitrile resulted in the formation of 2,5-disubstituted 1,4-benzodiazepin-3-ones, whereas N-(beta-haloacyl)-o-aminobenzonitrile gave 2,6-disubstituted 1,5-benzodiazocin-4-ones under similar conditions. Initial cylization of N-(beta-haloacyl)-o-aminobenzonitrile to obtain the corresponding lactam (e.g.alpha,alpha-dimethyl-N-(2-cyanophenyl)-beta-lactam) increased the yield of 1,5-benzodiazocin-4-ones significantly. Somewhat surprisingly, addition of lithium reagents to N-(beta-haloacyl)-o-aminobenzonitrile gave 4,4-disubstituted quinazolines via Grob fragmentation.

Synthesis and biological evaluation of fused thio- and selenopyrans as new indolocarbazole analogues with aryl hydrocarbon receptor affinity.

A series of thio- and selenopyrans having two fused indole units, structurally related to indolocarbazoles, have been prepared and evaluated for aryl hydrocarbon receptor (AhR) affinity, leading to the identification of several new significant AhR ligands. In particular, the parent thiopyrano[2,3-b:6,5-b']diindole and its derivative having a methyl group in the central ring, as well as the two corresponding selenopyrans, displayed the highest potencies of the compounds tested.

The suggested physiologic aryl hydrocarbon receptor activator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in humans.

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

Interactions of antiviral indolo[2,3-b]quinoxaline derivatives with DNA.

Here, we present the synthesis of five novel indoloquinoxaline derivatives and investigate the DNA binding properties of these monomeric as well as dimeric compounds using absorption, fluorescence, and linear dichroism. Several of the mono- and dicationic derivatives presented have previously demonstrated an excellent antiviral effect that is higher than already acknowledged agents against human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). We find that the DNA binding constants of the monomeric and dimeric derivatives are high (approximately 10(6)) and very high (approximately 10(9)), respectively. Results from the spectroscopic measurements show that the planar aromatic indoloquinoxaline moieties upon interaction with DNA intercalate between the nucleobases. Furthermore, we use poly(dA-dT)(2) and calf thymus DNA in a competitive binding experiment to show that all our derivatives have an AT-region preference. The findings are important in the understanding of the antiviral effect of these derivatives and give invaluable information for the future optimization of the DNA binding properties of this kind of drugs.

Synthetic studies of cephalandole alkaloids and the revised structure of cephalandole A.

A synthesis of the originally proposed 2-(1 H-indol-3-yl)-4 H-3,1-benzoxazin-4-one structure of the alkaloid cephalandole A (1) led to a structural revision, and the isolated natural product has now been identified as the previously known compound 3-(1 H-indol-3-yl)-2 H-1,4-benzoxazin-2-one (7). The structural assignment was corroborated by detailed NMR studies. A short synthesis of the related natural compound cephalandole B (2) has also been performed, confirming its structure. In addition some chemical transformations, involving, for example, the related synthetic molecule 2-(1 H-indol-3-yl)-3 H-quinazolin-4-one (9), are presented.