RESUMO
Amide synthesis is one of the most widely practiced chemical reactions, owing to its use in drug development and peptide synthesis. Despite the importance of these applications, the attendant effort to eliminate waste associated with these protocols has met with limited success, and pernicious α-epimerization is most often minimized but not eliminated when targeting challenging amides (e.g., N-aryl amides). This effort has focused on what is essentially a single paradigm in amide formation wherein an electrophilic acyl donor reacts with a nucleophilic amine. Umpolung amide synthesis (UmAS) emerged from α-halo nitroalkane reactions with amines and has since been developed into a method for the synthesis of enantiopure amides using entirely catalytic, enantioselective synthesis. However, its inability to forge N-aryl amides has been a longstanding problem, one limiting its application more broadly in drug development where α-chiral N-aryl amides are increasingly common. We report here the reaction of α-fluoronitroalkanes and N-aryl hydroxyl amines for the direct synthesis of N-aryl amides using a simple Brønsted base as the promoter. No other activating agents are required, and experiments guided by mechanistic hypotheses outline a mechanism based on the UmAS paradigm and confirm that the N-aryl amide, not the N-aryl hydroxamic acid, is the direct product. Ultimately, select chiral α-amino-N-aryl amides were prepared with complete conservation of enantioenrichment, in contrast to a parallel demonstration of their ability to epimerize using the conventional amide synthesis alternative.
Assuntos
Amidas , Aminas , Catálise , Ácidos Hidroxâmicos , PeptídeosRESUMO
BACKGROUND: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable ß-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.
Assuntos
Displasia Arritmogênica Ventricular Direita , Placofilinas , Retículo Sarcoplasmático , Animais , Arritmias Cardíacas , Displasia Arritmogênica Ventricular Direita/genética , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Condicionamento Físico Animal/efeitos adversos , Placofilinas/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.
Assuntos
Azepinas/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Imidazóis/administração & dosagem , Melanoma/tratamento farmacológico , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirimidinas/administração & dosagem , Pirrolidinas/administração & dosagem , para-Aminobenzoatos/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azepinas/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA/efeitos dos fármacos , Células HCT116 , Humanos , Imidazóis/farmacologia , Melanoma/genética , Camundongos , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , para-Aminobenzoatos/farmacologiaRESUMO
Peptide synthesis is a truly interdisciplinary tool, familiar to a broad group of scientists who do not otherwise overlap scientifically. For this reason, some may perceive even complex peptide synthesis to be a "solved problem", while others might argue that immense opportunity remains untapped or simply inaccessible. At the extreme of complexity, what might a concise assessment of the state-of-the-art in peptide synthesis look like? As one of the most practiced forms of synthetic chemistry by chemists and non-chemists alike, what restrictions remain that constrain access to chemical space? Using popular terminology, what forms of peptide synthesis are appropriately termed "on-demand"? The purpose of this Perspective is to appraise synthetic access to complex peptides, particularly those containing unnatural α-amino amides. Several case studies in complex peptide synthesis are summarized here, each selected to characterize the challenges attendant to unnatural α-amino amide synthesis. As peptidic molecules find increasing value in therapeutic development, especially in clinical applications, their impact will ultimately be determined by efficient preparative methods.
Assuntos
Técnicas de Química Sintética/métodos , Peptídeos/síntese química , Aminoácidos , Desenho de Fármacos , Peptídeos/químicaRESUMO
Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/metabolismo , Azepinas/administração & dosagem , Azepinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacologiaRESUMO
The finding by scientists at Hoffmann-La Roche that cis-imidazolines could disrupt the protein-protein interaction between p53 and MDM2, thereby inducing apoptosis in cancer cells, raised considerable interest in this scaffold over the past decade. Initial routes to these small molecules (i.e., Nutlin-3) provided only the racemic form, with enantiomers being enriched by chromatographic separation using high-pressure liquid chromatography (HPLC) and a chiral stationary phase. Reported here is the first application of an enantioselective aza-Henry approach to nonsymmetric cis-stilbene diamines and cis-imidazolines. Two novel mono(amidine) organocatalysts (MAM) were discovered to provide high levels of enantioselection (>95% ee) across a broad range of substrate combinations. Furthermore, the versatility of the aza-Henry strategy for preparing nonsymmetric cis-imidazolines is illustrated by a comparison of the roles of aryl nitromethane and aryl aldimine in the key step, which revealed unique substrate electronic effects providing direction for aza-Henry substrate-catalyst matching. This method was used to prepare highly substituted cis-4,5-diaryl imidazolines that project unique aromatic rings, and these were evaluated for MDM2-p53 inhibition in a fluorescence polarization assay. The diversification of access to cis-stilbene diamine-derived imidazolines provided by this platform should streamline their further development as chemical tools for disrupting protein-protein interactions.
Assuntos
Amidinas/química , Diaminas/química , Imidazolinas/química , Estilbenos/química , Apoptose , Catálise , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Polarização de Fluorescência , Humanos , Estrutura Molecular , Fenômenos de Química Orgânica , Domínios e Motivos de Interação entre Proteínas , EstereoisomerismoRESUMO
We report that N-(trimethylsilyl)imines serve in the Bis(AMidine)-catalyzed addition of bromonitromethane with a high degree of enantioselection. This allows for the production of a range of protected α-bromo nitroalkane donors (including Fmoc) for use in Umpolung Amide Synthesis (UmAS). Hence, peptide homologation with nonnatural aryl glycine amino acids is achieved in three steps from aromatic aldehydes, which are plentiful and inexpensive. Epimerization during the homologation step is circumvented by avoiding an α-amino acid intermediate.
Assuntos
Aldeídos/química , Aminoácidos/química , Aminoácidos/síntese química , Glicina/química , Peptídeos/química , Peptídeos/síntese química , Compostos de Silício/química , Etano/análogos & derivados , Etano/química , Nitrocompostos/químicaRESUMO
Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more general use. Our disclosure of the first enantioselective synthesis of (-)-Nutlin-3 provided a basis to prepare larger amounts of this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (-20 °C) and low catalyst loadings. Further refinements to the procedure led to the synthesis of (-)-Nutlin-3 in a 17 g batch and elimination of all but three chromatographic purifications.
Assuntos
Amidinas/química , Imidazóis/síntese química , Imidazolinas/química , Piperazinas/síntese química , Animais , Catálise , Humanos , Imidazóis/química , Camundongos , Piperazinas/química , Estereoisomerismo , Temperatura , Células Tumorais CultivadasRESUMO
A concise synthesis of a highly functionalized intermediate lacking only C10 of the mitomycin backbone is described. The key to this development is the Brønsted acid-catalyzed aza-Darzens reaction used to forge the cis-aziridine. Additionally an oxidative ketalization fortuitously occurs during the quinone-enamine coupling step, leading to an orthogonally protected hydroquinone.
Assuntos
Antibióticos Antineoplásicos/síntese química , Aziridinas/síntese química , Mitomicina/síntese química , Antibióticos Antineoplásicos/química , Aziridinas/química , Catálise , Cristalografia por Raios X , Mitomicina/química , Modelos Moleculares , EstereoisomerismoRESUMO
The amide bond is one of nature's most common functional and structural elements, as the backbones of all natural peptides and proteins are composed of amide bonds. Amides are also present in many therapeutic small molecules. The construction of amide bonds using available methods relies principally on dehydrative approaches, although oxidative and radical-based methods are representative alternatives. In nearly every example, carbon and nitrogen bear electrophilic and nucleophilic character, respectively, during the carbon-nitrogen bond-forming step. Here we show that activation of amines and nitroalkanes with an electrophilic iodine source can lead directly to amide products. Preliminary observations support a mechanism in which the polarities of the two reactants are reversed (German, umpolung) during carbon-nitrogen bond formation relative to traditional approaches. The use of nitroalkanes as acyl anion equivalents provides a conceptually innovative approach to amide and peptide synthesis, and one that might ultimately provide for efficient peptide synthesis that is fully reliant on enantioselective methods.