RESUMEN
LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states.
Asunto(s)
Acetatos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Fosfolipasas A/antagonistas & inhibidores , Animales , Ácido Araquidónico/farmacología , Línea Celular , Clonación Molecular , Cricetinae , Fosfolipasas A2 Grupo II , Cobayas , Humanos , Cetoácidos , Masculino , Mesocricetus , Ratones , Ratones Transgénicos , Músculo Liso/efectos de los fármacos , Fosfolipasas A/biosíntesis , Fosfolipasas A/sangre , Fosfolipasas A2 , Pleura/efectos de los fármacos , Pleura/metabolismo , Conejos , Ratas , Proteínas Recombinantes/biosíntesis , Tromboxano A2/biosíntesisRESUMEN
As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.
Asunto(s)
Ácidos Indolacéticos/química , Ácidos Indolacéticos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fosfolipasas A2 , Relación Estructura-ActividadRESUMEN
The preceding papers of this series detail the development of functionalized indole-3-acetamides as inhibitors of hnps-PLA2. We describe here the extension of the structure-activity relationship to include a series of indole-3-glyoxamide derivatives. Functionalized indole-3-glyoxamides with an acidic substituent appended to the 4- or 5-position of the indole ring were prepared and tested as inhibitors of hnps-PLA2. It was found that the indole-3-glyoxamides with a 4-oxyacetic acid substituent had optimal inhibitory activity. These inhibitors exhibited an improvement in potency over the best of the indole-3-acetamides, and LY315920 (6m) was selected for evaluation clinically as an hnps-PLA2 inhibitor.
Asunto(s)
Fosfolipasas A/antagonistas & inhibidores , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/farmacología , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fosfolipasas A2 , Relación Estructura-ActividadRESUMEN
Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácidos Indolacéticos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Cobayas , Humanos , Técnicas In Vitro , Ácidos Indolacéticos/química , Pulmón/efectos de los fármacos , Pulmón/enzimología , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Fosfolipasas A2 , Relación Estructura-ActividadRESUMEN
The primary objective of this study was to develop a functional assay that could provide rapid and reliable information on some pharmacologic characteristics of a novel inhibitor of human secretory phospholipase A2 (sPLA2). Guinea pig bronchoalveolar lavage (BAL) fluid, containing predominantly macrophages, eosinophils and epithelial cells, released thromboxane A2, as measured by thromboxane B2, in a concentration-dependent manner on exposure to recombinant human sPLA2 (rh-sPLA2). Similarly, n-formyl-L-methionyl-L-leucyl-L-phenylalanine (n-F-Met-Leu-Phe) or arachidonic acid also released this lipid mediator. Indomethacin, a cyclooxygenase inhibitor, blocked synthesis of thromboxane in response to these agents. p-Bromophenacylbromide-inactivated rh-sPLA2 was substantially less effective than the untreated enzyme in causing release of thromboxane. LY311727 is a potent indole-derived inhibitor of the isolated enzyme (IC50 = 23 nM). Incubation of this agent with BAL cells, just before addition of rh-sPLA2, reduced release of thromboxane with an IC50 = 1.8 x 10(-6) M. Specificity for sPLA2 was demonstrated in that LY311727, unlike indomethacin, did not reduce synthesis and subsequent release of thromboxane A2 in response to arachidonic acid. Using this technique as a basis, we determined whether LY311727 could sufficiently accumulate in lung after i.v. administration to inhibit rh-sPLA2-induced thromboxane A2 release from BAL cells. The compound, given i.v. to guinea pigs 5 min before collecting BAL fluid, produced a dose-dependent inhibition of rh-sPLA2 with an ED50 = 50 mg/kg. Thus, new in vitro and ex vivo assays were developed that permit functional evaluation of novel sPLA2 inhibitors. These techniques should serve as secondary assays for evaluation of human sPLA2 inhibitory activity from a chemical series and in addition provide initial data related to metabolic stability and distribution to the lung.
Asunto(s)
Fosfolipasas A/metabolismo , Tromboxano A2/metabolismo , Tromboxano B2/metabolismo , Animales , Ácido Araquidónico/farmacología , Lavado Broncoalveolar , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Cobayas , Humanos , Indoles/farmacología , Masculino , Fosfolipasas A2RESUMEN
Transgenic mice were created which overexpress human secretory non-pancreatic phospholipase A2 (sPLA2) pansomatically as a potential disease and drug-testing model. The mice were produced using a DNA construct in which the inducible mouse metallothionein gene promoter drives expression of a human sPLA2 minigene. High levels of sPLA2 were detected in several tissues by immunofluorescence localization. Expression in the testes caused hypospermia and male infertility. Circulating catalytically active sPLA2 could be induced to levels observed in patients undergoing a systemic inflammatory response but had no detectable effect on the mice. Therefore, these results suggest that sPLA2 hyperphospholipasemia alone may have only limited pathophysiological consequences. We further show that 3-[3-acetamide-1-benzyl-2-ethylindolyl-5-oxy]propane phosphonic acid LY311727), a potent new inhibitor of phospholipase A2 catalysis developed by our group, dramatically suppresses the circulating enzyme activity in these animals whereas 3-[3-acetamide-1-benzyl-2-propylindolyl-5-oxy]propane phosphonic acid (LY314024), a substantially less potent LY311727 analog, is without effect. These later results thus motivate the further development of this compound as a potential new therapeutic agent and valuable research tool.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/genética , Animales , Northern Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Indoles/farmacología , Masculino , Ratones , Ratones Transgénicos , Fosfolipasas A/análisis , Fosfolipasas A2 , Testículo/química , Testículo/patologíaRESUMEN
Structural derivatives of LY255283 have been studied as receptor antagonists of leukotriene B4. Substitution of the 2-hydroxyacetophenone subunit of 1 (LY255283) with a 2-arylphenol group provided entry into several new series that feature various mono- and diacidic core functionality. These new analogues, the subject of a broad structure-activity investigation, displayed significantly increased in vitro and in vivo activity as receptor antagonists of LTB4. A series of diaryl ether carboxylic acids demonstrated especially interesting activity and led to the discovery of compound 43b, 2-[2-propyl-3-[3-[2-ethyl-4-(4- fluorophenyl)-5-hydroxyphenoxy]-propoxy]phenoxy]benzoic acid (LY293111), a 2-arylphenol-substituted diaryl ether carboxylic acid which displayed potent binding to human neutrophils (IC50 = 17 +/- 4.6 nM) and guinea pig lung membranes (IC50 = 6.6 +/- 0.71 nM), inhibition of LTB4-induced expression of the CD11b/CD18 receptor on human neutrophils (IC50 = 3.3 +/- 0.81 nM), and inhibition of LTB4-induced contraction of guinea pig lung parenchyma (pKB = 8.7 +/- 0.16). In vivo, 43b demonstrated potent activity in inhibiting LTB4-induced airway obstruction in the guinea pig when dosed by the oral (ED50 = 0.40 mg/kg) or intravenous (ED50 = 0.014 mg/kg) routes. A specific LTB4 receptor antagonist, 43b had little effect on inhibiting contractions of guinea pig lung parenchyma induced by leukotriene D4 (LTD4), histamine, carbachol, or U46619. Compound 43b has been chosen as a clinical candidate and is currently in phase I studies for a variety of inflammatory diseases.
Asunto(s)
Benzoatos/farmacología , Fenoles/farmacología , Receptores de Leucotrieno B4/antagonistas & inhibidores , Obstrucción de las Vías Aéreas/metabolismo , Animales , Benzoatos/síntesis química , Benzoatos/química , Cobayas , Humanos , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fenoles/síntesis química , Fenoles/química , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacologíaRESUMEN
A lead compound obtained from a high volume human non-pancreatic secretory phospholipase A2 (hnps-PLA2) screen has been developed into a potent inhibitor using detailed structural knowledge of inhibitor binding to the enzyme active site. Four crystal structures of hnps-PLA2 complexed with a series of increasingly potent indole inhibitors were determined and used as the structural basis for both understanding this binding and providing valuable insights for further development. The application of structure-based drug design has made possible improvements in the binding of this screening lead to the enzyme by nearly three orders of magnitude. Furthermore, the optimized structure (LY311727) displayed 1,500-fold selectivity when assayed against porcine pancreatic s-PLA2.
Asunto(s)
Diseño de Fármacos , Indoles/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/química , Animales , Sitios de Unión/fisiología , Bioensayo , Calcio/química , Cristalografía por Rayos X , Cobayas , Humanos , Indoles/síntesis química , Indoles/química , Inflamación/tratamiento farmacológico , Cinética , Pulmón/metabolismo , Modelos Moleculares , Estructura Molecular , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Cloruro de Potasio/metabolismo , Conformación Proteica , Relación Estructura-ActividadAsunto(s)
Fenoles/farmacología , Receptores de Leucotrieno B4/antagonistas & inhibidores , Tetrazoles/farmacología , Animales , Antígenos CD/metabolismo , Broncoconstricción/efectos de los fármacos , Antígenos CD11 , Antígenos CD18 , Membrana Celular/metabolismo , Cobayas , Humanos , Leucotrieno B4/farmacología , Pulmón/metabolismo , Estructura Molecular , Contracción Muscular/efectos de los fármacos , Neutrófilos/metabolismo , Fenoles/química , Fenoles/metabolismo , Receptores de Leucotrieno B4/metabolismo , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/metabolismoRESUMEN
We have shown previously that reserpine is an effective "modulator" of P-glycoprotein-associated multidrug resistance (MDR). In addition to enhancing drug cytotoxicity in our multidrug-resistant human leukemia cell line, CEM/VLB100, reserpine strongly competes with a photoactivatible analog of vinblastine, N-(p-azido-3-[125I]iodosalicyl)-N'-(beta-aminoethyl)vindesine, for binding to P-glycoprotein. We also demonstrated previously that there are three substructural domains present in many compounds that modulate P-glycoprotein-associated MDR: a basic nitrogen atom and two planar aromatic rings. In the present study, we wished to test more rigorously the hypothesis that not only are these domains necessary for modulators of MDR but also they must exist in an appropriate conformation. Reserpine is a modulator of MDR in which these domains are present in a well-defined conformation. Accordingly, we prepared eight compounds that vary the spatial orientation of these domains, using either naturally occurring reserpine or yohimbine as chemical templates. When tested for their ability to enhance the cytotoxic activity of natural product antitumor drugs in CEM/VLB100 cells, five compounds that retained the pendant benzoyl function in an appropriate spatial orientation all modulated MDR. By contrast, compounds lacking this moiety failed to do so. These active modulators competed strongly with the 125I-labeled vinblastine analog for binding to P-glycoprotein in plasma membrane vesicles prepared from these cells. Conformational analysis using molecular mechanics revealed the structural similarities of the active modulators. Our results support the hypothesis that the relative disposition of aromatic rings and basic nitrogen atom is important for modulators of P-glycoprotein-associated MDR, and they suggest a ligand-receptor relationship for these agents. These results also provide direction for the definition of an MDR "pharmacophore."
Asunto(s)
Proteínas Sanguíneas/metabolismo , Glicoproteínas de Membrana/metabolismo , Reserpina/análogos & derivados , Reserpina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Línea Celular , Supervivencia Celular/efectos de los fármacos , Resistencia a Medicamentos , Humanos , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Yohimbina/análogos & derivados , Yohimbina/farmacologíaRESUMEN
The ergoline dopamine agonist, [3H]pergolide, binds with pharmacological specificity to particulate fractions of rat and calf brains. Dopamine agonists (apomorphine, 5,6-dihydroxy-2-dimethylaminotetralin, 6.7-dihydroxy-2-methylaminotetralin, lergotrileand bromocriptine) and dopamine antagonists (haloperidol and (+)butaclamol but not its pharmacologically inactive isomer, (-)butaclamol) were potent inhibitors, while monoamines (dopamine, norepinephrine, epinephrine and serotonin) were weaker inhibitors of [3H]pergolide binding. Olfactory tubercle was the only brain region other than striatum which exhibited significant [3H]pergolide binding displaceable by 1 microM (+)butaclamol. Saturable of calf and rat striatum with dissociation constants, kd values, of 1.2 to 3.1 nM. The number of binding sites was enriched in crude synaptosomal fractions. The relationship of [3H]pergolide binding to the binding of other dopaminergic ligands is discussed.
Asunto(s)
Encéfalo/metabolismo , Ergolinas/metabolismo , Receptores Dopaminérgicos/metabolismo , Animales , Bovinos , Cuerpo Estriado/metabolismo , Técnicas In Vitro , Masculino , Pergolida , Ratas , Fracciones Subcelulares/metabolismo , Sinaptosomas/metabolismo , Factores de TiempoRESUMEN
A general method has been developed for removal of the pyrrole ring from the ergolines. Oxidation of various ergolines at the 2,3 double bond gave formamido ketones 10, which afforded the amino ketones 11. These were deaminated to give the ketones 12, from which the carbonyl group was removed by reduction. The resulting depyrroloergolines (9) have, in contrast to the ergolines, little or no dopamine agonist activity in two tests.
Asunto(s)
Ergolinas/farmacología , Quinolinas/síntesis química , Animales , Dopamina/fisiología , Humanos , Masculino , Prolactina/sangre , Pirroles , Quinolinas/farmacología , Ratas , Conducta Estereotipada/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
It is proposed, based upon comparisons with apomorphine, that the rigid pyrroleethylamine moiety of the ergolines is the portion of the molecule responsible for dopamine agonist activity. In support of this hypothesis, bicyclic and tricyclic ergoline partial structures 6, 11, 25, and 35 have been synthesized. In addition, some pyrazole isosters (37, 38, 40, and 45) of these rigid pyrroleethylamines have been made. All of the classes show dopaminergic activity in prolactin inhibition and in lesioned rat turning assays. The most potent drugs, the linear tricyclic pyrazoles 38 (R = Pr) and 40 (R = Pr), are comparable in potency with the highly active ergoline pergolide (41).
Asunto(s)
Dopamina/fisiología , Ergolinas/síntesis química , Pirazoles/síntesis química , Pirroles/síntesis química , Animales , Fenómenos Químicos , Química , Humanos , Modelos Moleculares , Conformación Molecular , Prolactina/antagonistas & inhibidores , Prolactina/sangre , Pirazoles/farmacología , Pirroles/farmacología , Ratas , Conducta Estereotipada/efectos de los fármacosAsunto(s)
Ergolinas/síntesis química , Antagonistas Adrenérgicos alfa , Animales , Fenómenos Químicos , Química , Ergolinas/farmacología , Femenino , Cobayas , Humanos , Técnicas In Vitro , Dosificación Letal Mediana , Ratones , Músculo Liso/efectos de los fármacos , Parasimpatolíticos , Ratas , Serotonina , Antagonistas de la Serotonina , Conducta Estereotipada/efectos de los fármacos , Contracción Uterina/efectos de los fármacosAsunto(s)
Encéfalo/metabolismo , Dopamina/fisiología , Ergolinas/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Encéfalo/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Dopamina/metabolismo , Humanos , Hidroxidopaminas/farmacología , Masculino , Ratas , Receptores Dopaminérgicos/fisiología , Reserpina/farmacología , Conducta Estereotipada/efectos de los fármacos , Factores de TiempoRESUMEN
9,10-Didehydro-6-methyl-8beta-arylergolines 2, in which the carboxyl group of lysergic acid and isolysergic acid is replaced by various aryl groups, were prepared in two steps by alkylation of aromatic substrates with the tetracyclic allylic alcohol 3, followed by aromatization with MnO2. The new ergolines 2 have modest prolactin-inhibiting and rat antimuricidal activities and possess significant alpha-blocking and antiserotonin properties.
Asunto(s)
Ergolinas/síntesis química , Agresión/efectos de los fármacos , Animales , Ergolinas/farmacología , Femenino , Humanos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Prolactina/sangre , Ratas , Antagonistas de la Serotonina , Contracción Uterina/efectos de los fármacosRESUMEN
The ability of a series of 8-beta-carboxamido ergolines, 8-formamido ergolines, and 8-methyl ergolines to cause regressions of established dimethylbenz[a]anthracene-induced mammary carcinomas was compared to some ergot alkaloids. Although most of the ergoline derivatives depressed serum prolactin concentrations in rats, only a few had pronounced effects against the dimethylbenz[a]anthracene-induced mammary carcinoma in rats. Some derivatives from each of the three groups of substituted ergolines gave comparable activities against the dimethylbenz[a]anthracene-induced mammary carcinoma.