RESUMEN
The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/química , Inhibidores Enzimáticos/química , Organofosfonatos/química , Oximas/química , Pseudomonas aeruginosa/efectos de los fármacos , Inhibidores de beta-Lactamasas , Antibacterianos/síntesis química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imipenem/farmacología , Pruebas de Sensibilidad Microbiana , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Oximas/síntesis química , Oximas/farmacología , Tiofenos/química , beta-Lactamasas/metabolismoRESUMEN
4,7-Dichloro-1-benzothien-2-yl sulfonylaminomethyl boronic acid (DSABA, Compound I) was discovered as the first boronic acid-based class D beta-lactamase inhibitor. It exhibited an IC(50) of 5.6 microM against OXA-40. The compound also inhibited class A and C beta-lactamases with sub to low microM IC(50), and synergized with imipenem against Acinetobacter baumannii.
Asunto(s)
Antibacterianos/farmacología , Ácidos Borónicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de beta-Lactamasas , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/química , Ácidos Borónicos/química , Inhibidores Enzimáticos/química , Concentración 50 Inhibidora , Pruebas de Sensibilidad MicrobianaRESUMEN
Estradiol receptors (ER), ERalpha and ERbeta, are ligand-dependent transcription factors that regulate gene expression. Human and murine genetics suggest that ERalpha is the key target for estradiol action on bone, uterus and breast. To date, the molecular mode of action of estradiol and selective estradiol receptor modulators (SERMs) on bone is not fully understood. This is exemplified by a lack of in vitro assays that reliably predict SERM agonist activities in vivo. We hypothesized that ligand-dependent ERalpha transrepression, via protein-protein interactions at AP1, may predict estrogenic effects on bone. We modeled this using the MMP1 promoter, which encodes an AP1 binding site. We show that ICI-182780, raloxifene, 4-hydroxytamoxifen and estradiol all exhibit differential agonistic activities on the MMP1 promoter by suppressing activity by 20-80%. Transrepression efficacy and potency correlated with both uterotrophic (R(2)=0.98) and osteoprotective (R(2)=0.80) potential in the ovariectomized rat. This identifies MMP1 promoter transrepression as an agonist activity commonly shared by AF2 agonists and "antagonists" alike. Mutation analysis showed that the repression by estradiol and SERMs required correct amino acid sequences in the AF-2 domain. For instance, L540Q AF2 mutation did not alter responses to raloxifene, although it greatly increased responses to ICI-182780 (threefold) and reduced estradiol's effect by 20%. Furthermore, all tested ligands repressed the MMP1 promoter through the L540Q mutant with identical efficacy. Together, these data suggest that estradiol and SERMs share common agonist transcriptional activity via protein-protein interactions at AP1.
Asunto(s)
Huesos/efectos de los fármacos , Receptor alfa de Estrógeno/agonistas , Metaloproteinasa 1 de la Matriz/genética , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Útero/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Animales , Huesos/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/fisiología , Femenino , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Proteínas Mutantes/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Células Tumorales Cultivadas , Útero/metabolismoRESUMEN
Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.
Asunto(s)
Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Animales , Benzoquinonas , Células COS , Chlorocebus aethiops , Quinasas Ciclina-Dependientes/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Endometrio/citología , Endometrio/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Receptor alfa de Estrógeno/agonistas , Femenino , Fulvestrant , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas , Metaloproteinasa 1 de la Matriz/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/genética , Tamaño de los Órganos/efectos de los fármacos , Fosforilación , Regiones Promotoras Genéticas , Quinonas/farmacología , Ratas , Ratas Sprague-Dawley , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Serina/metabolismo , Útero/efectos de los fármacos , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
[reaction: see text] During the course of drug metabolism studies, a major metabolite of compound 1 was detected in rhesus monkeys and assigned structure 4. The intriguing biotransformation of 1 leading to 4 was confirmed by a 19-step total synthesis starting from resorcinol (11), the key feature of which was the construction of the oxygen bridge utilizing a phenolic oxidation and trapping sequence. In addition, the synthesis of a related metabolite (5) is described.
Asunto(s)
Receptor alfa de Estrógeno/antagonistas & inhibidores , Osteoporosis/tratamiento farmacológico , Oxatiinas/síntesis química , Oxatiinas/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Biotransformación , Macaca mulatta , Modelos Moleculares , Conformación Molecular , Oxidación-Reducción , Oxígeno/química , Resorcinoles/metabolismoRESUMEN
The discovery and synthesis of dihydrobenzoxathiins as potent, ERalpha subtype selective ligands are described. The most active analogue, 4-D, was found to be 50-fold selective in a competitive binding assay and 100-fold selective in a transactivation assay in HEK-293 cells. The alpha selectivity was postulated to lie in the interaction of the sulfur atom of the benzoxathiin ring with the two discriminating residues in the binding pocket of the receptor isoforms.
Asunto(s)
Oxatiinas/síntesis química , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Animales , Sitios de Unión , Unión Competitiva , Línea Celular , Cristalografía por Rayos X , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Tamaño de los Órganos/efectos de los fármacos , Oxatiinas/química , Oxatiinas/farmacología , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Activación Transcripcional , Útero/efectos de los fármacosRESUMEN
TFA/Et(3)SiH-mediated cyclization of thioketones 5 derived from the reaction of functionalized thiophenols (catechols) with bromo ketones gave syn 2,3-bisaryl(or heteroaryl) dihydrobenzoxathiins and benzodioxane 1 with total diastereoselectivity (>99:1), in excellent yields.
Asunto(s)
Dioxanos/síntesis química , Catecoles/química , Ciclización , Cetonas/química , Oxidación-Reducción , EstereoisomerismoRESUMEN
A novel class of indole ligands for estrogen receptor alpha have been discovered which exhibit potent affinity and high selectivity. Substitution of the bazedoxifene skeleton to the linker present in the HTS lead 1a provided 22b which was found to be 130-fold alpha-selective and acted as an antagonist of estradiol activity in uterine tissue and MCF-7 cancer cells.
Asunto(s)
Receptor alfa de Estrógeno/antagonistas & inhibidores , Indoles/química , Indoles/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Concentración 50 Inhibidora , Ligandos , Útero/efectos de los fármacosRESUMEN
The ring oxygen and sulfur analogs of lasofoxifene, 1a and 1b, were synthesized in an attempt to impart ERalpha selectivity, as found in the closely related dihydrobenzoxathiin compound I, recently discovered in these laboratories. The resulting isochroman and isothiochroman compounds were found to exhibit equipotent binding affinities to the ER isoforms and were less active in the inhibition of estradiol-triggered uterine growth when compared to I and lasofoxifene.
Asunto(s)
Cromanos/síntesis química , Cromanos/farmacología , Receptor alfa de Estrógeno/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Femenino , Humanos , Concentración 50 Inhibidora , Ligandos , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas , Relación Estructura-Actividad , Útero/efectos de los fármacos , Útero/crecimiento & desarrolloRESUMEN
Two novel side chains which had previously been found to enhance antagonist activity in the dihydrobenzoxathiin SERM series were applied to three existing platforms. The novel side chains did not improve the antagonist activity of the existing platforms.
Asunto(s)
Receptor alfa de Estrógeno/antagonistas & inhibidores , Oxatiinas/química , Moduladores Selectivos de los Receptores de Estrógeno/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Ligandos , RatasRESUMEN
Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Microsomas Hepáticos/metabolismo , Oxatiinas/farmacocinética , Moduladores Selectivos de los Receptores de Estrógeno/farmacocinética , Benzoquinonas/metabolismo , Biotransformación , Citocromo P-450 CYP3A , Hepatocitos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Unión ProteicaRESUMEN
An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell assay.
Asunto(s)
Oxatiinas/síntesis química , Receptores de Estrógenos/química , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Antagonistas de Estrógenos/síntesis química , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Oxatiinas/farmacología , Ratas , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Relación Estructura-Actividad , Útero/efectos de los fármacos , Útero/crecimiento & desarrolloRESUMEN
A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.
Asunto(s)
Oxatiinas/farmacología , Pirrolidinas/química , Receptores de Estrógenos/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Ligandos , Modelos Moleculares , Oxatiinas/química , Receptores de Estrógenos/metabolismoRESUMEN
The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).
Asunto(s)
Cromanos/química , Cromanos/farmacología , Receptor alfa de Estrógeno/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Sitios de Unión , Línea Celular , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Modelos Químicos , Estructura Molecular , Tamaño de los Órganos , Unión Proteica , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Útero/efectos de los fármacosRESUMEN
Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/metabolismo , Clorhidrato de Raloxifeno/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/metabolismo , Compuestos de Sulfhidrilo/química , Acetilcisteína/química , Animales , Citocromo P-450 CYP3A , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Cromatografía de Gases y Espectrometría de Masas , Glutatión/química , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Clorhidrato de Raloxifeno/química , Clorhidrato de Raloxifeno/farmacología , Ratas , Ratas Sprague-Dawley , Moduladores Selectivos de los Receptores de Estrógeno/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacologíaRESUMEN
A class of flavanoids exhibiting a high degree of selectivity for ERalpha over ERbeta has been discovered. The most active analogue 6 was found to be 66-fold ERalpha-selective and demonstrated uterine estradiol antagonism.
Asunto(s)
Flavonoides/química , Flavonoides/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Femenino , Humanos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Útero/metabolismoRESUMEN
Dihydrobenzodithiin compounds (1-6) were prepared to explore the expansion of the dihydrobenzoxathiin lead compounds I-III as SERAMs (Selective Estrogen Receptor Alpha Modulators). The dihydrobenzodithiin compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, however, they lacked the in vivo antagonism/agonism activity exhibited by the lead class in an immature rat uterine growth model.
Asunto(s)
Compuestos Heterocíclicos con 2 Anillos/síntesis química , Piperidinas/síntesis química , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Animales , Sitios de Unión , Células Cultivadas , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Concentración 50 Inhibidora , Ligandos , Modelos Biológicos , Piperidinas/farmacología , Ratas , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Relación Estructura-Actividad , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
Dihydrobenzoxathiin analogs (1-11) with modifications on the basic side chain region were prepared and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, similar to the original lead compound I. Many of the compounds also maintained high potency in the inhibition of human carcinoma MCF-7 cell growth. However, all were less potent in the inhibition of estradiol-triggered uterine growth. This work demonstrates the sensitive nature of modification to the antagonist basic side chain region.
Asunto(s)
Antagonistas de Estrógenos/síntesis química , Oxatiinas/síntesis química , Receptores de Estrógenos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/antagonistas & inhibidores , Humanos , Ligandos , Oxatiinas/farmacología , Relación Estructura-ActividadRESUMEN
A series of 3-alkyl, 3-cycloalkyl, and 3-heteroaryl dihydrobenzoxathiin analogs 1 were prepared and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. In general, the compounds were found to exhibit a high degree of selectivity for ER alpha over ER beta, but were less potent than the original lead compound 1a in the inhibition of estradiol-driven uterine proliferation.
Asunto(s)
Receptores de Estrógenos/antagonistas & inhibidores , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Ligandos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Relación Estructura-Actividad , Útero/citologíaRESUMEN
A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.