ABSTRACT
ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.
Subject(s)
Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Cilastatin/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Imipenem/chemistry , beta-Lactamase Inhibitors , Cilastatin/pharmacology , Cilastatin, Imipenem Drug Combination , Crystallography, X-Ray , Drug Combinations , Drug Resistance, Bacterial/drug effects , Imipenem/pharmacology , Inhibitory Concentration 50 , Klebsiella/drug effects , Microbial Sensitivity Tests , Models, Biological , Pseudomonas/drug effects , Structure-Activity RelationshipABSTRACT
The bridged monobactam ß-lactamase inhibitor MK-8712 (1) effectively inhibits class C ß-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C ß-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.
Subject(s)
Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Monobactams/chemical synthesis , beta-Lactamase Inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Binding Sites , Computer Simulation , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Imipenem/pharmacology , Microbial Sensitivity Tests , Monobactams/pharmacology , Protein Structure, Tertiary , Structure-Activity Relationship , beta-Lactamases/metabolismABSTRACT
Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.
Subject(s)
Carbapenems/chemistry , Drug Discovery/methods , Imipenem/chemistry , beta-Lactamase Inhibitors , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbapenems/pharmacology , Drug Combinations , Imipenem/pharmacology , Mice , Pseudomonas Infections/drug therapy , Pseudomonas Infections/enzymology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiology , Structure-Activity Relationship , beta-Lactam Resistance/drug effects , beta-Lactam Resistance/physiology , beta-Lactamases/metabolismABSTRACT
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.
Subject(s)
Bone and Bones/drug effects , Estrogen Receptor alpha/agonists , Matrix Metalloproteinase 1/genetics , Selective Estrogen Receptor Modulators/pharmacology , Uterus/drug effects , Amino Acid Motifs/physiology , Animals , Bone and Bones/metabolism , Down-Regulation/drug effects , Estradiol/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/physiology , Female , Humans , Matrix Metalloproteinase 1/metabolism , Mutant Proteins/physiology , Promoter Regions, Genetic/drug effects , Protein Structure, Tertiary/drug effects , Rats , Rats, Sprague-Dawley , Tumor Cells, Cultured , Uterus/metabolismABSTRACT
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.
Subject(s)
Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/metabolism , Animals , Benzoquinones , COS Cells , Chlorocebus aethiops , Cyclin-Dependent Kinases/drug effects , Cyclin-Dependent Kinases/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Endometrium/cytology , Endometrium/drug effects , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Receptor alpha/agonists , Female , Fulvestrant , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/drug effects , HSP90 Heat-Shock Proteins/metabolism , Humans , Lactams, Macrocyclic , Matrix Metalloproteinase 1/drug effects , Matrix Metalloproteinase 1/genetics , Organ Size/drug effects , Phosphorylation , Promoter Regions, Genetic , Quinones/pharmacology , Rats , Rats, Sprague-Dawley , Selective Estrogen Receptor Modulators/pharmacology , Serine/metabolism , Uterus/drug effects , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
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.
Subject(s)
Oxathiins/chemical synthesis , Receptors, Estrogen/chemistry , Selective Estrogen Receptor Modulators/chemical synthesis , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Dose-Response Relationship, Drug , Estrogen Antagonists/chemical synthesis , Estrogen Antagonists/pharmacology , Female , Humans , Oxathiins/pharmacology , Rats , Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/pharmacology , Structure-Activity Relationship , Uterus/drug effects , Uterus/growth & developmentABSTRACT
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.
Subject(s)
Cytochrome P-450 Enzyme System/physiology , Microsomes, Liver/metabolism , Oxathiins/pharmacokinetics , Selective Estrogen Receptor Modulators/pharmacokinetics , Benzoquinones/metabolism , Biotransformation , Cytochrome P-450 CYP3A , Hepatocytes/metabolism , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Protein BindingABSTRACT
[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.
Subject(s)
Estrogen Receptor alpha/antagonists & inhibitors , Osteoporosis/drug therapy , Oxathiins/chemical synthesis , Oxathiins/pharmacology , Selective Estrogen Receptor Modulators/chemical synthesis , Selective Estrogen Receptor Modulators/pharmacology , Animals , Biotransformation , Macaca mulatta , Models, Molecular , Molecular Conformation , Oxidation-Reduction , Oxygen/chemistry , Resorcinols/metabolismABSTRACT
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.
Subject(s)
Oxathiins/pharmacology , Pyrrolidines/chemistry , Receptors, Estrogen/drug effects , Selective Estrogen Receptor Modulators/pharmacology , Ligands , Models, Molecular , Oxathiins/chemistry , Receptors, Estrogen/metabolismABSTRACT
A series of benzoxathiin SERAMs with bicyclic amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.
Subject(s)
Bridged Bicyclo Compounds/chemistry , Ligands , Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/metabolism , Animals , Bridged Bicyclo Compounds/pharmacology , Female , Kinetics , Models, Molecular , Molecular Conformation , Selective Estrogen Receptor Modulators/chemistry , Selective Estrogen Receptor Modulators/pharmacology , Structure-Activity Relationship , Uterus/drug effectsABSTRACT
A series of benzoxathiin SERAMs with heteroatom-substituted amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.
Subject(s)
Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/metabolism , Animals , Female , Kinetics , Ligands , Models, Molecular , Molecular Conformation , Selective Estrogen Receptor Modulators/chemistry , Selective Estrogen Receptor Modulators/pharmacology , Structure-Activity Relationship , Uterus/drug effectsABSTRACT
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.
Subject(s)
Estrogen Receptor alpha/chemistry , Oxathiins/pharmacology , Selective Estrogen Receptor Modulators/chemical synthesis , Animals , Binding, Competitive , Cell Line, Tumor , Cell Proliferation/drug effects , Estrogen Receptor beta/chemistry , Female , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/pharmacokinetics , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Ligands , Organ Size , Oxathiins/chemical synthesis , Oxathiins/pharmacokinetics , Protein Binding , Rats , Rats, Sprague-Dawley , Selective Estrogen Receptor Modulators/pharmacokinetics , Selective Estrogen Receptor Modulators/pharmacology , Structure-Activity Relationship , Uterus/drug effectsABSTRACT
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.
Subject(s)
Oxathiins/chemical synthesis , Selective Estrogen Receptor Modulators/chemical synthesis , Animals , Binding Sites , Binding, Competitive , Cell Line , Crystallography, X-Ray , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Humans , Ligands , Models, Molecular , Molecular Conformation , Organ Size/drug effects , Oxathiins/chemistry , Oxathiins/pharmacology , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/chemistry , Selective Estrogen Receptor Modulators/pharmacology , Stereoisomerism , Structure-Activity Relationship , Transcriptional Activation , Uterus/drug effectsABSTRACT
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.
Subject(s)
Receptors, Estrogen/antagonists & inhibitors , Selective Estrogen Receptor Modulators/chemical synthesis , Selective Estrogen Receptor Modulators/pharmacology , Animals , Cell Line , Cell Proliferation/drug effects , Female , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Inhibitory Concentration 50 , Ligands , Organ Size/drug effects , Rats , Structure-Activity Relationship , Uterus/cytologyABSTRACT
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.