Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.     

Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation.     

Condensation of oxindole with acetoacetic ester and acetylacetone

4-Methylpyrano[2,3-b]indol-2-one is formed on thermal condensation of oxindole with acetoacetic ester. The condensation of oxindole with acetylacetone proceeds in position 3 of oxindole without closure in the pyrane ring. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 8, pp. 1167–1169, August, 2006.     

Zeolite-catalyzed synthesis of nicotinyl-fused indolo-pyrazoles and evaluation of their activities against Anopheles arabiensis and Lipoxygenase

A series of nicotyl-fused indolo-pyrazoles (NFIPs) were synthesized by a one-pot multicomponent reaction of aryl aldehydes, isoniazid, and indole in the presence of zeolite as a catalyst. Structures of all the synthesized compounds were established by IR, ¹H-NMR, ¹³C-NMR, 2D-NMR, TOF-MS, and elemental analysis. The products were obtained in excellent yields and high purity. All 10 compounds were screened for larvicidal and insecticidal properties against Anopheles arabiensis and tested for their lipoxygenase inhibitory activity. Compounds (3-(3-hydroxy-4-methoxyphenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4i ) and (3-(3-bromo-5-hydroxy-4-methoxyphenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4j) displayed highest larvae mortality at a 4 μg/ml dose in 24 h. Compounds (3-(4-methoxyphenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4h ) and (3-(3-hydroxy-4-methoxyphenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4i ) showed a significant knockdown activity after 24 h with 70% mortality. Furthermore, (3-(4-chlorophenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4c ) and (3-(3-bromo-5-hydroxy-4-methoxyphenyl)-2,3-dihydropyrazolo[4,3-b]indol-1(4H)-yl)(pyridin-4-yl)methanone ( 4j ) displayed promising lipoxygenase inhibitory activity with a mortality of 70% and 60%, respectively.     

Synthesis and characterisation of estrogenic carriers for cytotoxic Pt(II) fragments: biological activity of the resulting complexes

This paper describes the synthesis and the spectroscopic characterisation of cis-dichloro[N-(4-(17alpha-ethynylestradiolyl)-benzyl)-ethylenediamine]platinum(II) and cis-diamino[2-(4-(17alpha-ethynylestradiolyl)-benzoylamino)-malonato]platinum(II). These complexes were synthesised in good yield according to multi-step procedures based on the classical and non-classical Sonogashira coupling reaction, respectively. These compounds retain an acceptable degree of relative binding affinity (RBA) for the alpha form of estrogen receptor. Combined treatment of breast cancer cell lines, namely hormone-sensitive MCF-7 and hormone-insensitive MDA-MB-231 cell lines, indicates that these compounds maintain agonistic activity so that the potential advantage in vehiculation of the cytotoxic moiety by means of the receptor system is counteracted by the proliferative effect of the estrogenic component of the entire molecule, especially at low concentrations.     

Co2(CO)8-catalyzed intramolecular hetero-Pauson-Khand reaction of alkynecarbodiimide: synthesis of (+/-)-physostigmine

[reaction: see text] Herein we describe a novel Co(2)(CO)(8)-catalyzed intramolecular aza-Pauson-Khand-type reaction of alkynecarbodiimide derivatives affords pyrrolo[2,3-b]indol-2-one ring systems in reasonable yields. This is the first reported Co(2)(CO)(8) successfully applied in the hetero-Pauson-Khand reaction. Significantly, the transformation of one of our pyrrolo[2,3-b]indol-2-one derivatives into the indole alkaloid, (+/-)-physostigmine, was completed in a highly stereoselective manner.     

Synthesis and properties of 4-(3-amino-2-benzofuranyl)-coumarins

The alkylation of o-cyanophenol by 4-chloromethylcoumarins and subsequent intramolecular condensation by the cyano and methylene groups gives substituted 4-(3-amino-2-benzo-furanyl)coumarins. We studied the reactions of these compounds with acylating agents as well as with aldehydes, which lead to the 6H-[1]benzofuro[3,2-b]chromeno[4,3-d]pyridin-6-one system as the result of consecutive transformations.     

Synthesis of epigallocatechin trimer, (epigallocatechin)2-epicatechin,and (epigallocatechin)2-catechin via a Lewis acid mediated one-pot condensation and their antitumor activities in prostate cancer cells

Syntheses of epigallocatechin trimer, (epigallocatechin)₂-epicatechin and (epigallocatechin)₂-catechin were achieved. The key condensation to form the proanthocyanidin trimer derivatives was accomplished in a one-pot procedure using a dimeric epigallocatechin electrophile, which was prepared in situ by self-condensation of an epigallocatechin derivative, and an epigallocatechin, epicatechin, or catechin derivative as the nucleophile in the presence of a Lewis acid. The epigallocatechin monomer to trimer compounds containing a pyrogallol group significantly suppressed cell proliferation in PC-3 prostate cancer cells.     

Identification of estrogenic/anti-estrogenic compounds in diesel exhaust particulate extract

Diesel exhaust particulate extract (DEPE) was obtained from diesel exhaust particulates with Soxhlet extraction using dichloromethane. After separating DEPE into 11 fractions by liquid-liquid extraction, the neutral fraction (N) showed anti-estrogenic activity and the weak acid (phenol) fraction (WA(P)) showed estrogenic and anti-estrogenic activities by a yeast two-hybrid assay system expressing human estrogen receptor alpha. Both fractions were thoroughly fractionated by silica gel column chromatography and reversed-phase HPLC. In the WA(P) fraction, 3-methyl-4-nitrophenol and 2,6-dimethyl-4-nitrophenol were identified by LC-MS/MS as estrogenic compounds. This is the first study to identify 2,6-dimethyl-4-nitrophenol in DEPE and the first study to show that it is an estrogenic compound. In the N fraction, 1-hydroxypyrene was also identified by LC-MS/MS as an anti-estrogenic compound.     

Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [³H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4′,7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.     

Reaction of indoles with aldehydes. Synthesis of dihydropyrrolo[3,4-b]indoles

Aromatic aldehydes react with amides of 1-methylindole-2-carboxylic acid under acid catalysis conditions to give 1-aryl-4-methyldihydropyrrolo[3,4-b]indol-3-ones. The intermediate 1-methyl-2-CONHR-3(-X-benzyl)indoles, which are subsequently converted to the indicated cyclic compounds, were isolated. o-Acetyl derivatives were obtained by the action of acetic anhydride on derivatives of unsubstituted amides. Dihydropyrrolo[3,4-b] indol-3-ones were reduced by LiAlH₄ to the corresponding dihydropyrrolo[3,4-b] indoles. A mechanism for the formation of dihydropyrrolo[3,4-b] indoles is proposed.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 11, pp. 1516–1523, November, 1976.     

Psilocin analogs II. Synthesis of 3-[2-(dialkylamino)ethyl]-, 3-[2-(N-methyl-N-alkylamino)ethyl]-, and 3-[2-(cycloalkylamino)ethyl]indol-4-ols

Structural alteration of the N^b-substituents of psilocin (3-[2-dimethylamino)ethyl]indol-4-ol) ( 12a ) has led to a number of compounds containing known pharmacophoric groups. Further, it is hoped that the subtle changes in the nature of these substituents may lead to a clearer understanding of the structure-activity relationships of the 4-hydroxytryptamine hallucinogens.     

Synthesis and evaluation as NOP ligands of some spiro[piperidine-4,2'(1'H)-quinazolin]-4'(3'H)-ones and spiro[piperidine-4,5'(6'H)-[1,2,4]triazolo[1,5-c]quinazolines

Some spiro[piperidine-4,2'(1'H)-quinazolin]-4'(3'H)-ones 3 and spiro[piperidine-4,5'(6'H)-[1,2,4]triazolo[1,5-c]quinazolines] 4 were synthesized and evaluated as ligands of the nociceptin receptor. The examined compounds showed partial agonistic activity, except compounds 3, 4n that proved to be pure antagonists.     

Transformations of 3-formylindoles under the action of acids

Under the influence of acids 3-formylindole forms urorosein (the salt of di(indol-3-yl)methylium) which is unstable in solution and decomposes to give a series of indole derivatives, among which 6-(indol-3-yl)-5H,7H-indolo[2,3-b]carbazole and tri(indol-3-yl)methylium salt predominate. N,N′-Dimethylurorosein in solution also forms a mixture of indole derivatives, from which tri(1-methylindol-3-yl)methylium salt, 5N,11N-dimethylindolo[3,2-b]carbazole and its 6-(1-methylindol-3-yl) derivative were isolated. Research Institute of New Antibiotics, RAMN (Russian Academy of Medical Science), Moscow 119867, Russia; Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 631–639, May, 1999.     

Synthesis and properties of 4-(3-aminothieno-[2,3-b]pyridin-2-yl)coumarins

Substituted 4-[(3-cyanopyridin-2-yl)thiomethyl]coumarins have been synthesized by the alkylation of 2-mercapto-4,6-dimethylnicotinonitrile with 4-chloromethylcoumarins. Substituted 4-(3-amino-thieno[2,3-b]pyridin-2-yl)coumarins have been obtained by a subsequent intramolecular condensation of the methylene and cyano groups. 6H-Chromeno[4,3-d]thieno[3,2-b:5,4-b']dipyridin-6-ones were isolated as a result of the interaction of these compounds with aldehydes, and their aminomethyl derivatives were synthesized.     

1,1-Disubstituted-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indolecarboxylic acid esters and ketones. The base catalyzed transformation of 1-(2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-[1H]pyrido[3,4-b]indol]-2-yl)alkanones into 2-(4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)-1 -alkylcyclohexanols

Condensation of 1H-indole-3-ethanamines 1 with cyclic β-keto esters 2 under azeotropic conditions followed by acid-catalyzed ring closure of the resulting enamines 3 gave 2′,3′,4′,9′-tetrahydrospiro[piperidine-3,1′,-[1H]pyrido[3,4-b]indole] -4-carboxylic acid alkyl esters 4 . Condensation of 1 with 2-acylcycloalkanones 8 gave two types of enamines, 10 and 11 , respectively. Enamines 10 on treatment with acid gave 1-(2′,3′,4′,9′-tetrahydro-3H-pyrido[3,4-b]indol-1-yl)-1-alkylcyclohexanols 17 . Compounds 17 were further dehydrated to give cycloalkane derivatives 19.     

Preparation of new nitrogen-bridged heterocycles. 54.increased arene-arene interactions of 3-(bicyclic and tricyclic arylmethylthio)thieno[3,4-b]indolizine derivatives

Some thieno[3,4-b]indolizine derivatives having a 1-naphthylmethylthio, 2-methyl-1-naphthylmethylthio, 2-naphthylmethylthio, or 9-anthrylmethylthio group at the 3-position were prepared and their intramolecular arene-arene interactions were investigated. In comparison with 3-(methylthio)thieno[3,4-b]indolizines which have no such interactions, the (1)H-NMR spectra of title compounds showed large high-field shifts (delta 0.06-0.89 ppm) for the protons of the pyridine ring in the thieno[3,4-b]indolizine, and these values were considerably larger than those (delta <0.3 ppm) in 3-(benzylthio)thieno[3,4-b]indolizines. The UV spectra also exhibited a characteristic absorption band near 425 nm attributable to the arene-arene interaction. In the X-ray analyses of some compounds, however, the presence of both the gauche and the anti conformers at the sulfide spacer were confirmed.     

Cascade cyclization of 3(5)-aminopyrazoles with aromatic aldehydes and cyclohexanediones

Three-component condensation of 5(3)-amino-3(5)-methylpyrazole with aromatic aldehydes and 1,3-cyclohexanedione afforded mixtures of 3-methyl-4-aryl-2,4,6,7,8,9-hexa hydro-5H-pyrazolo[3,4-b]quinolin-5-ones and 2-methyl-9-aryl-5,6,7,9-tetrahydro pyrazolo[5,1-b]quinazolin-8(4H)-ones. The reaction of 3-aminopyrazolo-4-carbonitrile and ethyl 3-aminopyrazolo-4-carboxylate with aldehydes and cyclo hexanedione or dimedome is regioselective and leads to the formation of partially hydrogenated pyrazolo [5,1-b]quinazolin-8-one systems. In all compounds the dihydroazine ring exists in the enamine tautomeric form.     

Characterization of AhR agonist compounds in roadside snow

Aryl hydrocarbon receptor (AhR) agonistic contaminants were identified in roadside snow samples. Snow was collected in Oslo, Norway, and compared to a background sample collected from a mountain area. The water and particulate fractions were analysed for AhR agonists using a dioxin-responsive, chemically activated luciferase expression (CALUX) cell assay and by gas chromatography coupled to high-resolution time-of-flight mass spectrometry with targeted analysis for polycyclic aromatic hydrocarbons (PAHs) and broad-spectrum non-target analysis. The AhR agonist levels in the dissolved fractions in the roadside samples were between 15 and 387 pg/L CALUX toxic equivalents (TEQ(CALUX)). An elevated AhR activity of 221 pg TEQ(CALUX) per litre was detected in the mountain sample. In the particle-bound fractions, the TEQ(CALUX) was between 1,350 and 7,390 pg/L. One possible explanation for the elevated levels in the dissolved fraction of the mountain sample could be the presence of black carbon in the roadside samples, potentially adsorbing dioxin-like compounds and rendering them unavailable for AhR interaction. No polychlorinated dibenzodioxins and dibenzofurans or polychlorinated biphenyls were detected in the samples; the occurrence of PAHs, however, explained up to 9 % of the AhR agonist activity in the samples, whilst comprehensive two-dimensional gas chromatography coupled to mass spectrometry GCxGC-ToF-Ms identified PAH derivatives such as polycyclic aromatic ketones and alkylated, nitrogen sulphur and oxygen PAHs in the particle fractions. The (large) discrepancy between the total and explained activity highlights the fact that there are other as yet unidentified AhR agonists present in the environment.     

Convenient synthetic method for 3-(3-substituted indol-2-yl)quinoxalin-2-ones as VEGF inhibitor

It has already been reported that 3-(indol-2-yl)quinoxalin-2-ones have a potent inhibitory effect on the growth of tumor cells based on anti-angiogenesis activity. We have also carried out a structure-activity relationship (SAR) study of 3-(indol-2-yl)quinoxalin-2-ones, which showed a potent inhibitory activity toward the vascular endothelial growth factor (VEGF)-induced proliferation of human mesangial cells and the VEGF-induced auto-phosphorylation of human umbilical vein endothelial cells. Moreover, one of these compounds has a potent medicinal effect based on anti-angiogenic action, by oral administration (Chart 1, 9). However, since the existing synthetic methods for the preparation of 3-(indol-2-yl)quinoxalin-2-ones consist of multiple steps some of which require strict anhydrous conditions, a convenient and simple synthetic method in place of the existing method is desirable. As a result of the investigations into the synthetic procedures, 3-(3-substituted indol-2-yl)quinoxalin-2-ones can be easily prepared by the condensation of 3-substituted indoles with quinoxalin-2-ones in the presence of trifluoroacetic acid (TFA). Herein, we report the examination of these reaction conditions and the application of this new synthetic method to the synthesis of the derivatives as VEGF inhibitors.