RESUMEN
Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/genética , Ratones Desnudos , Leucina Zippers , Proliferación Celular/fisiología , Recurrencia Local de Neoplasia , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK-protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein-protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.
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Dioxanos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Tiazoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Sitios de Unión/genética , Línea Celular Tumoral , Cisteína/genética , Cisteína/metabolismo , Dioxanos/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/genética , Melanoma/metabolismo , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Simulación de Dinámica Molecular , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Tiazoles/metabolismoRESUMEN
Glycolytic enzyme fructose-bisphosphate aldolase A is an emerging therapeutic target in cancer. Recently, we have solved the crystal structure of murine aldolase in complex with naphthalene-2,6-diyl bisphosphate (ND1) that served as a template of the design of bisphosphate-based inhibitors. In this work, a series of ND1 analogues containing difluoromethylene (-CF2), methylene (-CH2), or aldehyde substitutions were designed. All designed compounds were studied using molecular dynamics (MD) simulations with the AMOEBA force field. Both energetics and structural analyses have been done to understand the calculated binding free energies. The average distances between ligand and protein atoms for ND1 were very similar to those for the ND1 crystal structure, which indicates that our MD simulation is sampling the correct conformation well. CF2 insertion lowers the binding free energy by 10-15 kcal/mol, while CF2 substitution slightly increases the binding free energy, which matches the experimental measurement. In addition, we found that NDB with two CF2 insertions, the strongest binder, is entropically driven, while others including NDA with one CF2 insertion are all enthalpically driven. This work provides insights into the mechanisms underlying protein-phosphate binding and enhances the capability of applying computational and theoretical frameworks to model, predict, and design diagnostic strategies targeting cancer.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Simulación de Dinámica Molecular , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Ratones , Fosfatos/química , Fosfatos/metabolismo , Fosfatos/farmacología , Conformación Proteica , TermodinámicaRESUMEN
The protein kinase MELK is an important kinase in cell signaling and has shown to be a promising anti-cancer target. Recent work has resulted in a novel small molecule scaffold targeting MELK, IN17. However, there has been little structural information or physical understanding of MELK-IN17 interactions. Using Tinker-OpenMM on GPUs, we have performed free energy simulations on MELK binding with IN17 and 11 derivatives. This series of studies provides structural insights into how substitution on IN17 leads to differences in complex structure and binding thermodynamics. In addition, this study serves as an assessment of the current capabilities of the AMOEBA forcefield, accelerated by GPU computing, to serve as a molecular-dynamics-based free energy simulation platform for lead optimization.
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Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Relación Estructura-Actividad Cuantitativa , Fenómenos Biofísicos , Humanos , Indoles/química , Indoles/farmacología , Isomerismo , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , SolventesRESUMEN
Triple negative breast cancers (TNBC) have poor prognosis compared to other breast cancer subtypes and represent 15-20% of breast cancers diagnosed. Unique targets and new molecularly-targeted therapies are urgently needed for this subtype. Despite high expression of Epidermal Growth Factor Receptor, inhibitors such as lapatinib have not shown therapeutic efficacy in TNBC patients. Herein, we report that treatment with the covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death, while these compounds as single agents have little effect. The combination significantly increases survival of mice bearing xenografts of MDA-MB-231 human TNBC cells. Our studies demonstrate that lapatinib treatment increases c-Jun and JNK phosphorylation indicating a mechanism of resistance. Combined, these compounds significantly reduce transcriptional activity of Nuclear Factor kappa B, Activating Protein 1, and Nuclear factor erythroid 2-Related Factor 2. As master regulators of antioxidant response, their decreased activity induces a 10-fold increase in reactive oxygen species that is cytotoxic, and is rescued by addition of exogenous antioxidants. Over expression of p65 or Nrf2 also significantly rescues viability during JNK-IN-8 and lapatinib treatment. Further studies combining JNK-IN-8 and lapatinib may reveal a benefit for patients with TNBC, fulfilling a critical medical need.
RESUMEN
Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.
RESUMEN
Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.
Asunto(s)
Acetanilidas/farmacología , Antineoplásicos/farmacología , Indoles/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Acetanilidas/síntesis química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Indoles/síntesis química , Simulación del Acoplamiento Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Inhibidores de Proteínas Quinasas/síntesis químicaRESUMEN
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.
Asunto(s)
Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Neoplasias/tratamiento farmacológico , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Proteína p300 Asociada a E1A/fisiología , Humanos , Ratones , Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Three new tris(2-pyridylmethyl)amine-based ligands possessing phenylethynyl units have been prepared using Sonogashira couplings and substitution reactions. Copper(ii) complexes of those tetradentate ligands have also been synthesized. Solid-state structures of the six new compounds have been determined by single-crystal X-ray diffraction analyses. Examination of the molecular structures of the ligands revealed the expected triangular geometries with virtually undeformed carbon-carbon triple bonds. While the tertiary nitrogen of the free ligands seem to be prevented from participation in supramolecular non-covalent interactions by the pyridyl hydrogen at the 3-position, the pyridyl nitrogens play a crucial role in the packing mode of the crystal structure. The nitrogens form weak hydrogen bonds, varied in length between 2.32 and 2.66 Å, with the pyridyl hydrogen of its neighbouring molecule. The [NH-C] contacts enforce one-dimensional columnar assemblies on ligands that organize into wall-like structures, which in turn assemble into three-dimensional structures through CH-π interactions. Structural analyses of Cu(ii) complexes of the ligands revealed propeller-like structures caused by steric crowding of three pyridine ligands. The copper complexes of the ligands having three phenylethynyl substituents showed a remarkably deformed carbon-carbon triple bond enforced by a steric effect of the three phenyl groups. Most significantly, a total of seventy non-covalent interactions, classified into twelve types of hydrogen-involving short contacts, were identified in this study. The phenylethynyl substituent participated in forty-two interactions as a hydrogen bond acceptor, and its role was more distinctive in the crystal structures of the Cu(ii) complexes.
RESUMEN
A-484954 is a known eEF2K inhibitor with submicromolar IC50 potency. However, the binding mechanism and the crystal structure of the kinase remains unknown. Here, we employ a homology eEF2K model, docking and alchemical free energy simulations to probe the binding mechanism of eEF2K, and in turn, guide the optimization of potential lead compounds. The inhibitor was docked into the ATP-binding site of a homology model first. Three different binding poses, hypothesis 1, 2, and 3, were obtained and subsequently applied to molecular dynamics (MD) based alchemical free energy simulations. The calculated relative binding free energy of the analogs of A-484954 using the binding pose of hypothesis 1 show a good correlation with the experimental IC50 values, yielding an r (2) coefficient of 0.96 after removing an outlier (compound 5). Calculations using another two poses show little correlation with experimental data, (r (2) of less than 0.5 with or without removing any outliers). Based on hypothesis 1, the calculated relative free energy suggests that bigger cyclic groups, at R1 e.g., cyclobutyl and cyclopentyl promote more favorable binding than smaller groups, such as cyclopropyl and hydrogen. Moreover, this study also demonstrates the ability of the alchemical free energy approach in combination with docking and homology modeling to prioritize compound synthesis. This can be an effective means of facilitating structure-based drug design when crystal structures are not available.
RESUMEN
Protein kinases are mutated or otherwise rendered constitutively active in numerous cancers where they are attractive therapeutic targets with well over a dozen kinase inhibitors now being used in therapy. While fluorescent sensors have capacity to measure changes in kinase activity, surprisingly they have not been utilized for biomarker studies. A first-generation peptide sensor for ERK based on the Sox fluorophore is described. This sensor called ERK-sensor-D1 possesses high activity toward ERK and more than 10-fold discrimination over other MAPKs. The sensor can rapidly quantify ERK activity in cell lysates and monitor ERK pathway engagement by BRAF and MEK inhibitors in cultured melanoma cell lines. The dynamic range of the sensor assay allows ERK activities that have potential for profound clinical consequences to be rapidly distinguished.
RESUMEN
The use of reversible covalent-bonding in a four-component assembly incorporating chiral alcohols was recently reported to give a method for determining the enantiomeric excess of the alcohols via CD spectroscopy. Experiments that probe the mechanism of this assembly, which consists of 2-formylpyridine (2-PA), dipicolylamine (DPA), Zn(II), and alcohols, to yield zinc-complexes of tren-like ligands, are presented. The studies focus upon the mechanism of conversion of a hemi-aminal (1) to a hemi-aminal ether (3), thereby incorporating the fourth component. It was found that molecular sieves along with 3 to 4 equivalents of alcohol are required to drive the conversion of 1 to 3. Attempts to isolate an intermediate in this reaction via addition of strong Lewis-acids led to the discovery of a five-membered ring pyridinium salt (5), but upon exposure to Zn(II) and alcohols gave different products than the assembly. This was interpreted to support the intermediacy of an iminium species. Kinetic studies reveal that the conversion of 1 to 3 is zero-order in alcohol in large excesses of alcohol, supporting rate-determining formation of an intermediate prior to reaction with alcohol. Further, the magnitude of the rate constant for interconversion of 1 and 3 are similar, supporting the notion that there are similar rate-determining steps (rds's) for the forward and reverse reactions. Hammett plots show that the rds involves creation of a negative charge (interpreted as the loss of positive charge), supporting the notion that decomplexation of Zn(II) from the assemblies to generate apo-forms of 1 and 3 is rate-determining. The individual mechanistic conclusions are combined to create a qualitative reaction coordinate diagram for the interconversion of 1 and 3.
RESUMEN
eEF-2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6-diamino-4-(2-fluorophenyl)-4H-thiopyran-3,5-dicarbonitrile (DFTD) as an inhibitor of eEF-2K. Here we describe its mechanism of action against eEF-2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile-containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF-2K with a two-step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF-2K.
Asunto(s)
Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Nitrilos/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nitrilos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.
Asunto(s)
Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinonas/farmacología , Relación Dosis-Respuesta a Droga , Quinasa del Factor 2 de Elongación/metabolismo , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Pirimidinonas/síntesis química , Pirimidinonas/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.
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Quinasa del Factor 2 de Elongación/metabolismo , Ensayos Analíticos de Alto Rendimiento , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Mediciones Luminiscentes/métodos , Péptidos/química , Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
A library of hydrazide derivatives was synthesized to target non-structural protein 1 of influenza A virus (NS1) as a means to develop anti-influenza drug leads. The lead compound 3-hydroxy-N-[(Z)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethylideneamino]naphthalene-2-carboxamide, which we denoted as "HENC", was identified by its ability to increase the melting temperature of the effector domain (ED) of the NS1 protein, as assayed using differential scanning fluorimetry. A library of HENC analogs was tested for inhibitory effect against influenza A virus replication in MDCK cells. A systematic diversification of HENC revealed the identity of the R group attached to the imine carbon atom significantly influenced the antiviral activity. A phenyl or cyclohexyl at this position yielded the most potent antiviral activity. The phenyl containing compound had antiviral activity similar to that of the active form of oseltamivir (Tamiflu), and had no detectable effect on cell viability.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hidrazinas/química , Hidrazinas/farmacología , Virus de la Influenza A/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Línea Celular , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
This short perspective reports on the synthesis and applications of a class of chiral amino carbonyl compounds, masked oxo-sulfinamides where the amine is protected with an N-sulfinyl moiety and the carbonyl group is protected as the ketal or 1,3-dithiane. These polyfunctionalized chiral building blocks are prepared by addition of organometallic reagents to masked oxo-sulfinimines (N-sulfinyl imines) or the addition of oxo-organometallic reagents and lithio-1,3-dithianes to sulfinimines. Because unmasking of the amino and carbonyl groups results in cyclic imines, these chiral building blocks are particularly useful for the asymmetric synthesis of functionalized nitrogen heterocycles, including prolines, pipecolic acids, pyrrolidines, homotropinones, tropinones, and tropane alkaloids such as cocaine and C-1 cocaine analogues.
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Amidas/química , Aminas/química , Técnicas de Química Sintética/métodos , Compuestos Heterocíclicos/química , Iminas/química , Compuestos de Sulfonio/química , Amidas/síntesis química , Aminas/síntesis química , Aminoácidos/síntesis química , Aminoácidos/química , Amino Alcoholes/síntesis química , Amino Alcoholes/química , Cocaína/análogos & derivados , Cocaína/síntesis química , Compuestos Heterocíclicos/síntesis química , Iminas/síntesis química , Organofosfonatos/síntesis química , Organofosfonatos/química , Ácidos Pipecólicos/síntesis química , Ácidos Pipecólicos/química , Prolina/síntesis química , Prolina/química , Pirrolidinas/síntesis química , Pirrolidinas/química , Compuestos de Sulfonio/síntesis química , Tropanos/síntesis química , Tropanos/química , beta-Lactamas/síntesis química , beta-Lactamas/químicaRESUMEN
Proline derivatives with a C(γ)-exo pucker typically display a high amide bond trans/cis (K(T/C)) ratio. This pucker enhances nâπ* overlap of the amide oxygen and ester carbonyl carbon, which favors a trans amide bond. If there were no difference in nâπ* interaction between the ring puckers, then the correlation between ring pucker and K(T/C) might be broken. To explore this possibility, proline conformations were constrained using a methylene bridge. We synthesized discrete gauche and anti 5-fluoro- and 5-hydroxy-N-acetylmethanoproline methyl esters from 3-syn and 3-anti fluoro- and hydroxymethanopyrrolidines using directed α-metalation to introduce the α-ester group. NBO calculations reveal minimal nâπ* orbital interactions, so contributions from other forces might be of greater importance in determining K(T/C) for the methanoprolines. Consistent with this hypothesis, greater trans amide preferences were found in CDCl(3) for anti isomers en-MetFlp and en-MetHyp (72-78% trans) than for the syn stereoisomers ex-MetFlp and ex-MetHyp (54-67% trans). These, and other, K(T/C) results that we report here indicate how substituents on proline analogues can affect amide preferences by pathways other than ring puckering and nâπ* overlap and suggest that caution should be exercised in assigning enhanced pyrrolidine C(γ)-exo ring puckering based solely on enhanced trans amide preference.