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Biologically active 6-aminophenanthridines were prepared in a single step procedure: Metal amides in liquid ammonia promoted the condensation of anilines with 2-chloro-benzonitriles. 6-Aminophenanthridines were isolated in moderate yield.

International audience; We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs. The method allowed us to identify several compounds effective against budding yeast prions responsible for the [PSI+] and [URE3] phenotypes. These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine. Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay. The compounds isolated here were also active in promoting mammalian prion clearance. These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans.

This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. We measured Cl− transport as the amplitude of short-circuit current in response to the membrane permeable cAMP analogue 8-(4-chlorophenylthio)adenosine-3′-5′-cyclic monophosphate (CPT-cAMP) alone or in combination with a CFTR opener. The correction of G551D-CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. In both types of epithelia, cAMP caused activation of Cl− transport that was inhibited by glibenclamide and not by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. Addition of genistein (10–200 μM), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1–100 μM) or of the benzo[c]quinolizinium MPB-07 (10–200 μM) to FRT-G551D epithelia pre-treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. The genistein dose-response curve was bell-shaped due to inhibitory activity at the highest concentrations. The dose-dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl− transport defect on G551D patients. British Journal of Pharmacology (2002) 137, 504–512. doi:10.1038/sj.bjp.0704882

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca2+-activated Cl– transport, whereas the swelling-activated Cl– transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca2+,Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. The CFTR chloride channel activity was blocked by glibenclamide (100 μM) but not by DIDS (100 μM). Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels. British Journal of Pharmacology (1998) 123, 683–693; doi:10.1038/sj.bjp.0701648

A simple synthesis of biologically active 6-aminophenanthridines was achieved by a Suzuki–Miyaura coupling reaction. Condensation of 2-(4,4,5,5-tetramethyl-1,3-dioxaborolan-2-yl)aniline with 2-chlorobenzonitriles afforded 6-aminophenanthridines useful as prions inhibitors in a mild one-step procedure. The intermediate 2-amino-2′cyanobiphenyls could not be isolated.

The synthesis and in vitro pharmacology of a series of triaryl-1,4-dihydropyridines were investigated. Molecules containing a nitro group on the phenyl ring had a higher affinity for specific [ 3 H](+)-PN 200-110 binding sites. All the compounds possessed a radical scavenging effect and an antiperoxidant activity. These properties were more marked for molecules with 2-pyridinyl and 2-thienyl substituents. The synthesis of new triaryl-1,4-dihydropyridines with both a higher binding affinity and antioxidant activity might be effective in cerebral ischemic disease treatment.

Heteroarylmethylation of halogenobenzonitriles in the presence of air (O 2 ) gives 1-hydroxy-3-oxo-1-aryl-1H,3H-isoindoles in liquid ammonia. A one-pot or two-step synthesis is described and a mechanism is proposed

The synthesis and in vitro pharmacology of a series of triaryl-1,4-dihydropyridines were investigated. Molecules containing a nitro group on the phenyl ring had a higher affinity for specific [ 3 H](+)-PN 200-110 binding sites. All the compounds possessed a radical scavenging effect and an antiperoxidant activity. These properties were more marked for molecules with 2-pyridinyl and 2-thienyl substituents. The synthesis of new triaryl-1,4-dihydropyridines with both a higher binding affinity and antioxidant activity might be effective in cerebral ischemic disease treatment.

The reaction of balogenobenzonitriles with 2-aminobenzenethiol is a new route, in a “one-pot” or two-step approach, to 11-aminodibenzo[b,f][1,4]thiazepine and fluoro derivatives.

A one-pot synthesis of 1, 3 and 4-cyanophenothiazines and a two-step approach to 2-cyanoisomer have been developed. The condensation of 2-aminobenzenethiol and 2,3 or 3,4-dihalogenobenzonitriles followed by Smiles rearrangement or by intramolecular aromatic substitution gave the desired ring systmes.

The synthesis and in vitro antioxidant activity of 17 new tetraarylpyrroles are investigated by 2 tests highly documented in the literature: capability to prevent Fe2+-induced lipid peroxidation on microsomes, which is a membrane preparation rich in polyunsaturated fatty acids, and direct scavenging effect on a stable free radical, 1,l-diphenyl-2-picryl-hydrazyl (DPPH). For the Fe2+-induced microsomal lipid peroxidation system, the results show that molecules which possess 2-pyrazinyl or 2-pyridyl in the 3- and 4-positions on the pyrrole ring are the most efficient. Introduction of methoxy groups on the phenyl ring in the 2- and 5-positions increases the effects but the higher activity is obtained with 2-furyl or 2-thienyl. The only compounds which possess a direct scavenger effect on trapping the stable free radical DPPH are those which have 2-pyridyl in the 3- and 4-positions and 2-furyl or 2-thienyl in the 2- and 5-positions.

Resume La synthese de thieno- et isothiazolo[3,4-b]quinoxalines par annelation de la quinoxaline a ete codifiee. La stereochimie de la jonction des cycles a ete etablie par RMN. Une etude biologique preliminaire a mis en evidence des activites antiinflammatoires notables: le compose 1 (hexahydro-1,3,3a,4,9,9a thieno[3,4-b] quinoxaline dioxyde-2,2) a, sur le test a la carrageenine, une activite superieure a celle de la phenylbutazone.

A one-pot synthesis of 1, 3 and 4-cyanophenothiazines and a two-step approach to 2-cyanoisomer have been developed. The condensation of 2-aminobenzenethiol and 2,3 or 3,4-dihalogenobenzonitriles followed by Smiles rearrangement or by intramolecular aromatic substitution gave the desired ring systmes.

The reaction of balogenobenzonitriles with 2-aminobenzenethiol is a new route, in a “one-pot” or two-step approach, to 11-aminodibenzo[b,f][1,4]thiazepine and fluoro derivatives.

Cyclin-dependent kinases (CDKs) regulate the cell cycle, apoptosis, neuronal functions, transcription, and exocytosis. The observation of CDK deregulations in various pathological situations suggests that CDK inhibitors may have a therapeutic value. In this article, we report on the identification of 6-phenyl[5H]pyrrolo[2,3-b]pyrazines (aloisines) as a novel potent CDK inhibitory scaffold. A selectivity study performed on 26 kinases shows that aloisine A is highly selective for CDK1/cyclin B, CDK2/cyclin A-E, CDK5/p25, and GSK-3 alpha/beta; the two latter enzymes have been implicated in Alzheimer's disease. Kinetic studies, as well as the resolution of a CDK2-aloisine cocrystal structure, demonstrate that aloisines act by competitive inhibition of ATP binding to the catalytic subunit of the kinase. As observed with all inhibitors reported so far, aloisine interacts with the ATP-binding pocket through two hydrogen bonds with backbone nitrogen and oxygen atoms of Leu 83. Aloisine inhibits cell proliferation by arresting cells in both G1 and G2.

The photodegradation of 6‐hydroxy‐10‐chlorobenzo[c]quinolizinium chloride (MPB‐07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration, and temperature were used. MPB‐07 concentration was determined at regular time intervals by reversed‐phase HPLC. MPB‐07 stability was also studied at pH 7.4 in the dark. Results showed that in all the conditions tested MPB‐07 underwent rapid photodegradation, apparently following first‐order kinetics. Rate constants were dependent on the initial MPB‐07 concentration, temperature, and pH. At pH 7.4, and for concentrations from 1 to 125 μM, half‐lives ranged from 0.681 ± 0.047 to 4.54 ± 0.28 h. The Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ · mol −1 . Analysis by chemical ionization‐mass spectrometry showed that the chlorine atom of the MPB‐07 molecule might be replaced by an OH group during the photodegradation process. In the dark, MPB‐07 in solutions at pH 7.4 was found to be stable over a 6‐week period. In conclusion, MPB‐07 is a highly photolabile molecule that should be carefully protected from light when used.

The synthesis and in vitro antioxidant activity of 17 new tetraarylpyrroles are investigated by 2 tests highly documented in the literature: capability to prevent Fe2+-induced lipid peroxidation on microsomes, which is a membrane preparation rich in polyunsaturated fatty acids, and direct scavenging effect on a stable free radical, 1,l-diphenyl-2-picryl-hydrazyl (DPPH). For the Fe2+-induced microsomal lipid peroxidation system, the results show that molecules which possess 2-pyrazinyl or 2-pyridyl in the 3- and 4-positions on the pyrrole ring are the most efficient. Introduction of methoxy groups on the phenyl ring in the 2- and 5-positions increases the effects but the higher activity is obtained with 2-furyl or 2-thienyl. The only compounds which possess a direct scavenger effect on trapping the stable free radical DPPH are those which have 2-pyridyl in the 3- and 4-positions and 2-furyl or 2-thienyl in the 2- and 5-positions.

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl−current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

A one-pot synthesis of 2-substituted phenylbenzothiazoles is achieved when 2-aminobenzenethiol is treated with an excess of sodium hydride and aromatic nitriles in THF. This method allows to obtain substituted products (F, CF 3 , Cl or OCH 3 ) in each ortho, meta and para position and plurisubstituted products

The reaction of halogenobenzonitriles with 2-aminobenzenethiol gave, by a Smiles rearrangement 2-mercaptocyanodiphenylamines which are cyclised to cyanophenothiazines via a disulfide intermediate. A mechanism is proposed

Heteroarylmethylation of halogenobenzonitriles in the presence of air (O 2 ) gives 1-hydroxy-3-oxo-1-aryl-1H,3H-isoindoles in liquid ammonia. A one-pot or two-step synthesis is described and a mechanism is proposed

L'action du phenyllithium sur la pyrazine, la methyl pyrazine et l'acetonyl pyrazine conduit a des produits d'addition sur une ou sur deux liaisons iminiques du noyau pyrazine. L'addition de benzoate de methyle au milieu reactionnel entraine la formation de produits C ou N condenses a partir des intermediaires lithies formes. Nous n'avons pas isole de produits issus d'une metallation des chaines laterales (- CH3 ou -CH2COCH3). The action of phenyllithium on pyrazine, 2-methyl pyrazine and acetonyl pyrazine gave mono- or di-addition products with azomethines bonds of pyrazine. The adducts can be condensed with methyl benzoate to give C or N condensed derivatives. No product with initial metalation of methyl or acetonyl groups was observed.

Analogues of bromo-levamisole and guanidine derivatives including cimetidine are examined in vitro in order to investigate their comparative inhibition, towards alkaline phosphatase (ALP) from human liver and diamine-oxidase (DAO) from human placenta. Bromo-levamisole, considered as a potent selective uncompetitive inhibitor of ALP (Ki, 2.8 · 10−6M at pH 10.5) is shown to be a non-competitive inhibitor of DAO (Ki = 7 · 10−4 M). According to the structure-inhibition relationship, the imidazole ring is important for ALP and DAO inhibition. The phenyl ring of bromo-levamisole is required for ALP inhibition but not for DAO inhibition, which is mediated mainly by aminoguanidine or guanidine groups. These results have allowed the selection of cimetidine, an H2-antagonist but also an immunomodulating compound, as inhibitor of these two enzymes. Cimetidine is an uncompetitive inhibitor of ALP (Ki = 3.2 · 10−3 M at pH 10.5), and a good inhibitor of DAO ( i 50 = 3.8 · 10 −4 M ). The Ki of ALP is commonly calculated at pH 10.5, but to study the role of the enzyme at the physiological pH, the inhibition has also been performed at pH 7.4. The Ki values are only slightly affected by this pH variation. So far several compounds, including levamisole, imidazole, theophylline and aminoguanidine are known to possess immunomodulating activities in vivo and/or in vitro and inhibit ALP and/or DAO. Therefore, it seems reasonable to assume that the inhibition of enzymes is involved in the immunomodulating effects of these drugs, when the ranges of active concentrations are similar for these properties.

A new route to tetrahydrodibenzofurannes is described. It is based upon Claisen transposition of an aryloxycyclohexene to o .hydroxyphenyl cyclohexene which is, in turn, cyclized by palladium acetate. In the studied example, an overall yield of 45 % is obtained in two steps. The isolation of an unexpected minor isomeric transposed compound sets a problem of mechanism.

L'action d'organolithiens comme le butyllithium et le phenyllithium sur la methyl-2 quinoxaline conduit soit a une metallation du methyle soit a l'addition de l'organolithien sur une (ou sur les deux) liaisons imini-ques du noyau pyrazinique suivie ou non de metallation du methyle. L'addition d'eau (reaction A) au melange reactionnel permet l'isolement de composes stables dihydro-ou tetrahydroquinoxalines. L'addition d'electrophiles comme l'o-chlorobenzonitrile (reaction B) ou le benzoate de methyle (reaction C) livre des produits C ou N condenses a partir des intermediates lithtes formes. Seul le benzoate de methyle donne des produits de condensation a l'azote. The action of organolithium reagents such as phenyllithium or n-bulyllithium on 2-methylquinoxaline gave lithiation of the methyl group which upon reaction with electtropholesphiles produce side chain alkenyl derivatives. On the other hand organolithium reagents react with the quinoxaline azomethine bond to give I-lithio-2-alkyl)or ary-1)-3 methylquinoxalines which can be further loithiated on the methyl group to give 2-alkyl(or aryl)-3-alkenylquinoxaline derivatives. The adducts can be condensed with clectrophiles such as benzonitrile or methlyl benzoate but only methyl benzoate leads to N condensed derivatives. Furthermore substituted 1,2,3,4-terahydroqinoxalines are available via the above lithio intermediates.

Pour introduire des centres polaires dans des systemes pluricycliques plans en vue d'etudier leur action intercalante, on a prepare des derives de benzo[c]quinolizinium fonctionnalises par un groupement OH ou NH2: les precurseurs cetoniques ou imines sont obtenus a partir du picolyl-2 ou du quinaldyllithium; ils subissent ensuite une cyclisation par quaternisation mettant vraisemblablement en jeu un processus concerte. La structure des composes obtenus et de certains de leurs derives est etudiee. The introduction of polar groups in planar polycyclic systems, for the purpose of studying their intercalating action, has been realized in the benzo[c]quinolizinium series. A functionalizing synthesis has been developed from 2-picolyl-and quinaldyllithium. Intermediate ketones or imines were cyclized by a quaternization reaction involving a concerted process. Structure and physico-chemical features of the compounds prepared were studied.

The structure of pyrrolo ([4,5-b]-1′,2′,3′,4′-tetrahydroquinoxaline) [1,2,3-Im]- 1 - methyl - 3 - propyl - 1, 3, 3a, 4, 9, 9a - hexahydrothieno[3,4-b]quinoxaline 2,2-dioxide, isolated in the course of a study on a series of thieno[3,4,-b]quinoxalines, was assigned on the basis of a spectral 1H-13C correlated inverse chemical shift experiment.

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50= 85 μM) and activated CFTR in Calu-3 cells (EC50= 47 μM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

O.Chlorobenzonitrile reacts with carbanions from methylpyrazine in liquid ammonia, affording selectively arylmethylpyrazine derivatives.