RESUMEN
Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M1 vs M2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.
Asunto(s)
Diseño de Fármacos , Antagonistas Muscarínicos/farmacología , Pirenzepina/farmacología , Receptores Muscarínicos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Antagonistas Muscarínicos/síntesis química , Antagonistas Muscarínicos/química , Pirenzepina/síntesis química , Pirenzepina/química , Relación Estructura-ActividadRESUMEN
Tagged biologically active molecules represent powerful pharmacological tools to study and characterize ligand-receptor interactions. However, the labeling of such molecules is not trivial, especially when poorly soluble tags have to be incorporated. The classical method of coupling usually necessitates a tedious final purification step to remove the excess of reagents and to isolate tagged molecules. To overcome this limitation, Cu(I)-catalyzed 1,3-dipolar cycloaddition, referred to as "click" chemistry, was evaluated as a tool to facilitate the access to labeled molecules. In order to validate the approach, we focused our attention on the incorporation of a fluorophore (Lissamine Rhodamine B), a nonfluorescent dye (Patent Blue VF), or biotin into a muscarinic antagonist scaffold derived from pirenzepine. The reaction performed in acetonitrile/water, in the presence of CuSO4 and Cu wire, allowed us to obtain three novel pirenzepine derivatives with high purity and in good yield. No coupling reagents were needed, and the quasi-stoichiometric conditions of the reaction enabled the straightforward isolation of the final product by simple precipitation and its use in bioassays. The affinity of the compounds for the human M1 muscarinic receptor fused to EGFP was checked under classical radioligand and FRET binding conditions. The three pirenzepine constructs display a nanomolar affinity for the M1 receptor. In addition, both dye-labeled derivatives behave as potent acceptors of energy from excited EGFP with a very high quenching efficiency.
Asunto(s)
Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/metabolismo , Pirenzepina/química , Pirenzepina/metabolismo , Receptor Muscarínico M1/metabolismo , Catálisis , Línea Celular , Humanos , Ligandos , Estructura Molecular , Antagonistas Muscarínicos/síntesis química , Pirenzepina/síntesis química , Factores de TiempoRESUMEN
The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.
Asunto(s)
Bencenosulfonatos/síntesis química , Benzodiazepinonas/síntesis química , Colorantes/síntesis química , Proteínas Fluorescentes Verdes/metabolismo , Pirenzepina/análogos & derivados , Pirenzepina/síntesis química , Compuestos de Quinolinio/síntesis química , Receptor Muscarínico M1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Bencenosulfonatos/química , Benzodiazepinonas/química , Compuestos de Boro , Línea Celular , Colorantes/química , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Humanos , Ligandos , Pirenzepina/química , Compuestos de Quinolinio/química , Ensayo de Unión Radioligante , Receptor Muscarínico M1/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Following a recent description of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP)-fused human muscarinic M1 receptors and Bodipy-labeled pirenzepine, we synthesized seven fluorescent derivatives of this antagonist in order to further characterize ligand-receptor interactions. These compounds carry Bodipy [558/568], Rhodamine Red-X [560/580], or Fluorolink Cy3 [550/570] fluorophores connected to pirenzepine through various linkers. All molecules reversibly bind with high affinity to M1 receptors (radioligand and energy transfer binding experiments) provided that the linker contains more than six atoms. The energy transfer efficiency exhibits modest variations among ligands, indicating that the distance separating EGFP from the fluorophores remains almost constant. This also supports the notion that the fluorophores may bind to the receptor protein. Kinetic analyses reveal that the dissociation of two Bodipy derivatives (10 or 12 atom long linkers) is sensitive to the presence of the allosteric modulator brucine, while that of all other molecules (15-24 atom long linkers) is not. The data favor the idea that these analogues might interact with both the acetylcholine and the brucine binding domains.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Pirenzepina/análogos & derivados , Pirenzepina/síntesis química , Receptor Muscarínico M1/efectos de los fármacos , Estricnina/análogos & derivados , Regulación Alostérica , Sitios de Unión , Unión Competitiva , Compuestos de Boro/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/farmacología , Proteínas Fluorescentes Verdes , Humanos , Cinética , Ligandos , Proteínas Luminiscentes/genética , Pirenzepina/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Rodaminas/química , Relación Estructura-Actividad , Estricnina/farmacologíaRESUMEN
Various fragments of the hexamethonio-type allosteric agent W84 were linked to the secondary amino group of the muscarinic M(2) acetylcholine receptor-preferring antagonist AF-DX 384 to increase the area of attachment with the allosteric site. Addition of only the phthalimido moiety of W84 gave an allosteric enhancer of NMS binding. Thus, a new lead structure for the development of allosteric enhancers of NMS binding has been discovered.
Asunto(s)
Antagonistas Muscarínicos/síntesis química , Ftalimidas/síntesis química , Pirenzepina/análogos & derivados , Pirenzepina/síntesis química , Regulación Alostérica , Animales , Cobayas , Técnicas In Vitro , Isoindoles , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/farmacología , Miocardio/metabolismo , Ftalimidas/química , Ftalimidas/farmacología , Pirenzepina/química , Pirenzepina/farmacología , Ensayo de Unión Radioligante , Receptores Muscarínicos/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , PorcinosRESUMEN
Enantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1- yl]ethylamino]-carbonyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-(-)-2-[N,N-dipropylaminomethyl]piperidine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the alpha-highly sulphated cyclodextrin (alpha-HSCD) as chiral selector within the running electrolyte. (S)-(+)-(4) was prepared from (S)-(-)-pipecolic acid in a 4-step procedure (overall yield: 30%, ee: 99%) and (R)-(-)-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-(-) isomer exhibited in vitro a 23-fold higher affinity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2.
Asunto(s)
Pirenzepina/análogos & derivados , Animales , Unión Competitiva , Ciclodextrinas , Electroforesis Capilar , Ligandos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Antagonistas Muscarínicos/síntesis química , Antagonistas Muscarínicos/metabolismo , Miocardio/química , N-Metilescopolamina/metabolismo , Parasimpatolíticos/antagonistas & inhibidores , Parasimpatolíticos/síntesis química , Parasimpatolíticos/metabolismo , Pirenzepina/síntesis química , Pirenzepina/metabolismo , Ratas , Solventes , Estereoisomerismo , TritioRESUMEN
A series of tropinyl and piperidinyl esters was synthesized and evaluated for inhibitory activities on the endothelial muscarinic receptors of rat (M3) and rabbit (M2) aorta. Some of the esters (cyclohexylphenylglycolates and cyclohexylphenylpropionates) were found to be better antimuscarinic compounds than standard M2 and M3 inhibitors such as AFDX116 and 4-diphenylacetoxy-N-methylpiperidine (DAMP), with pKEC50 values in the range of 8-9. A few esters were found to be more selective M3 than M2 inhibitors, but these tended to have low activities. The hydrophobic, electronic and steric characteristics of these esters were correlated with antimuscarinic activity by using appropriate parameters representing hydrophobicity (HPLC capacity factor, log kw), size (molecular volume) and electronic character (Taft's polar substituent constant sigma * and 13C chemical shift difference delta delta). Finally, 92% of the M2-inhibitory activities of the esters could be accounted for by the size and electronic character sigma * of the side chain. In contrast, the M3-inhibitory activities of these esters were mainly attributed to the electronic nature (sigma *, delta delta) of the side chain, with good activity being associated with electron-withdrawing groups. Visualization of the comparative molecular field analysis (CoMFA) steric and electrostatic fields provided further confirmation of the structure-activity relationship (SAR) derived from traditional quantitative structure activity relationship (QSAR) approaches.
Asunto(s)
Antagonistas Muscarínicos/síntesis química , Antagonistas Muscarínicos/farmacología , Piperidinas/síntesis química , Piperidinas/farmacología , Receptores Muscarínicos/efectos de los fármacos , Tropanos/síntesis química , Tropanos/farmacología , Animales , Ésteres/síntesis química , Ésteres/química , Ésteres/farmacología , Técnicas In Vitro , Cinética , Masculino , Modelos Moleculares , Antagonistas Muscarínicos/química , Piperidinas/química , Pirenzepina/análogos & derivados , Pirenzepina/síntesis química , Pirenzepina/química , Pirenzepina/farmacología , Conejos , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Tropanos/químicaRESUMEN
Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.
Asunto(s)
Miocardio/metabolismo , Parasimpatolíticos/síntesis química , Pirenzepina/análogos & derivados , Receptores Muscarínicos/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Indicadores y Reactivos , N-Metilescopolamina , Parasimpatolíticos/metabolismo , Parasimpatolíticos/farmacología , Pirenzepina/síntesis química , Pirenzepina/metabolismo , Pirenzepina/farmacología , Prosencéfalo/metabolismo , Ratas , Receptores Muscarínicos/efectos de los fármacos , Derivados de Escopolamina/metabolismo , Relación Estructura-ActividadRESUMEN
The muscarinic antagonists pirenzepine and telenzepine were derivatized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors.
Asunto(s)
Marcadores de Afinidad/síntesis química , Isotiocianatos , Antagonistas Muscarínicos , Parasimpatolíticos/síntesis química , Pirenzepina/análogos & derivados , Acilación , Marcadores de Afinidad/farmacología , Animales , Reactivos de Enlaces Cruzados , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Técnicas In Vitro , Masculino , Parasimpatolíticos/farmacología , Pirenzepina/síntesis química , Pirenzepina/farmacología , Prosencéfalo/efectos de los fármacos , Ratas , TiocianatosRESUMEN
The synthesis of Pyrimido[4,5-b][1,5]benzodiazepine derivatives from 4-(o-aminophenylene)amino-5-ethoxy-carbonyl-1,2-dihydro-6-methyl-2 - oxopyrimidine has been described. Pyrimidobenzodiazepine 5 alkylated with N-methyl-N'-chloroacetyl-piperazine gives a product related to pirenzepine. Compound 5 shows weak antianxiety and antidepressive action.
Asunto(s)
Ansiolíticos/síntesis química , Antidepresivos/síntesis química , Benzodiazepinas , Benzodiazepinonas/síntesis química , Pirenzepina/análogos & derivados , Pirimidinas/síntesis química , Animales , Benzodiazepinonas/farmacología , Temperatura Corporal/efectos de los fármacos , Fenómenos Químicos , Química , Ratones , Pirenzepina/síntesis química , Pirimidinas/farmacologíaRESUMEN
On the basis of the cardioselective muscarinic receptor antagonist AF-DX 116 (2), a series of 11-substituted pyridobenzodiazepinones (9-35) was prepared and screened for their binding affinity to muscarinic receptors located in cardiac (M2) and glandular (M3) tissue. The ratio of IC50 values of the test compounds in the two different tissues was taken as a measure of cardiac (M2) receptor selectivity. Qualitative structure-selectivity relationships point to the fact that it is the spatial orientation of the protonated side-chain nitrogen atom in relation to the tricycle that is the main determinant for receptor subtype recognition and hence is important for the achievement of cardiac (M2) selectivity.