RESUMO
The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Masculino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Metionina/metabolismo , Ratos Sprague-Dawley , Purina-Núcleosídeo Fosforilase/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , MultiômicaRESUMO
Treatment of homoallylic N-tosyl amines or allylic N-tosyl hydroxylamines with 1.5 equiv of a malonoyl peroxide provides a stereoselective method to access functionalized pyrrolidines and isoxazolidines. This metal free alkene oxyamination proceeds in 50-85% yield and up to 13:1 trans-selectivity. In addition, the relative stereochemistry of the oxygen and nitrogen substituents can be inverted through an oxidation/reduction sequence or inverting the stereochemistry of the starting alkene. Mechanistic investigations show a higher reactivity for hydroxyl nucleophiles over sulfonamide nucleophiles revealing a preference for dioxygenation over oxyamination.
RESUMO
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Animais , Benzimidazóis/síntese química , Ligação Competitiva , Cálcio/metabolismo , Citrulina/metabolismo , Inibidores Enzimáticos/síntese química , Células HEK293 , Histonas/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Bibliotecas de Moléculas Pequenas , Especificidade por SubstratoRESUMO
The Chan-Evans-Lam reaction is a valuable C-N bond forming process. However, aryl boronic acid pinacol (BPin) ester reagents can be difficult coupling partners that often deliver low yields, in particular in reactions with aryl amines. Herein, we report effective reaction conditions for the Chan-Evans-Lam amination of aryl BPin with alkyl and aryl amines. A mixed MeCN/EtOH solvent system was found to enable effective C-N bond formation using aryl amines while EtOH is not required for the coupling of alkyl amines.
RESUMO
The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.
Assuntos
Neoplasias , Humanos , Entropia , Metionina Adenosiltransferase/metabolismoRESUMO
INTRODUCTION: In methylthioadenosine phosphorylase (MTAP)-deficient tumor cells, reduced S-adenosylmethionine (SAM) levels in the context of elevated methylthioadenosine (MTA) has been hypothesized to lead to inhibition of protein arginine methyltransferase 5 (PRMT5) and tumor growth inhibition. Inhibitors of methionine adenosyltransferase 2A (MAT2a) prevent the synthesis of SAM from methionine and have therefore attracted increasing attention as potential chemotherapeutic agents in cancers characterized by MTAP-loss. AREAS COVERED: This review covers patent applications between January 2018 and December 2021. 18 patent applications from 5 different applicants are evaluated. EXPERT OPINION: Recent advances in the field show a significant interest in the MAT2a therapeutic hypothesis. Agios and Ideaya in particular have capitalized on an allosteric binding mode first published by Pfizer in at least two of the filings during this time period, leading to potent, selective inhibitors. They have advanced MAT2a inhibitors to phase I clinical studies to explore their benefit to patients suffering with MTAP-deficient solid tumors or lymphoma. Whilst the other patent disclosures during this time frame have not led to disclosed candidates, the trials initiated by Agios and Ideaya studies will clearly inform on the potential for such inhibitors as viable therapeutic agents either as single agent or in combination.
Assuntos
Metionina Adenosiltransferase , Neoplasias , Humanos , Metionina/metabolismo , Metionina/uso terapêutico , Metionina Adenosiltransferase/metabolismo , Neoplasias/tratamento farmacológico , Patentes como Assunto , Proteína-Arginina N-Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/uso terapêuticoRESUMO
Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.
Assuntos
Aminoquinolinas/química , Desenho de Fármacos , Proteínas/metabolismo , Administração Oral , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacocinética , Aminoquinolinas/uso terapêutico , Animais , Benzoatos/química , Benzoatos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Meia-Vida , Humanos , Masculino , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Proteínas/antagonistas & inibidores , Ratos , Relação Estrutura-AtividadeRESUMO
Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving >1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound 36 was identified as being 2000-fold selective for BD2 over BD1 (Brd4 data) with >1 mg/mL solubility in FaSSIF media. 36 represents a valuable new in vivo ready molecule for the exploration of the BD2 phenotype.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Piridinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Herein, a series of 2,3-dihydrobenzofurans have been developed as highly potent bromo and extra-terminal domain (BET) inhibitors with 1000-fold selectivity for the second bromodomain (BD2) over the first bromodomain (BD1). Investment in the development of two orthogonal synthetic routes delivered inhibitors that were potent and selective but had raised in vitro clearance and suboptimal solubility. Insertion of a quaternary center into the 2,3-dihydrobenzofuran core blocked a key site of metabolism and improved the solubility. This led to the development of inhibitor 71 (GSK852): a potent, 1000-fold-selective, highly soluble compound with good in vivo rat and dog pharmacokinetics.
Assuntos
Benzofuranos/farmacologia , Proteínas/antagonistas & inibidores , Benzofuranos/síntese química , Benzofuranos/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas/metabolismo , Solubilidade , Relação Estrutura-AtividadeRESUMO
Second-generation bromodomain and extra terminal (BET) inhibitors, which selectively target one of the two bromodomains in the BET proteins, have begun to emerge in the literature. These inhibitors aim to help determine the roles and functions of each domain and assess whether they can demonstrate an improved safety profile in clinical settings compared to pan-BET inhibitors. Herein, we describe the discovery of a novel BET BD2-selective chemotype using a structure-based drug design from a hit identified by DNA-encoded library technologies, showing a structural differentiation from key previously reported greater than 100-fold BD2-selective chemotypes GSK620, GSK046, and ABBV-744. Following a structure-based hypothesis for the selectivity and optimization of the physicochemical properties of the series, we identified 60 (GSK040), an in vitro ready and in vivo capable BET BD2-inhibitor of unprecedented selectivity (5000-fold) against BET BD1, excellent selectivity against other bromodomains, and good physicochemical properties. This novel chemical probe can be added to the toolbox used in the advancement of epigenetics research.
Assuntos
DNA/química , Descoberta de Drogas , Proteínas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Domínios Proteicos/efeitos dos fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Humanos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.
Assuntos
Furanos/farmacologia , Proteínas/antagonistas & inibidores , Pirazóis/farmacologia , Relação Dose-Resposta a Droga , Furanos/química , Humanos , Estrutura Molecular , Proteínas/metabolismo , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Most bromodomain inhibitors mimic the interactions of the natural acetylated lysine (KAc) histone substrate through key interactions with conserved asparagine and tyrosine residues within the binding pocket. Herein we report the optimization of a series of phenyl sulfonamides that exhibit a novel mode of binding to non-bromodomain and extra terminal domain (non-BET) bromodomains through displacement of a normally conserved network of four water molecules. Starting from an initial hit molecule, we report its divergent optimization toward the ATPase family AAA domain containing 2 (ATAD2) and cat eye syndrome chromosome region, candidate 2 (CECR2) domains. This work concludes with the identification of (R)-55 (GSK232), a highly selective, cellularly penetrant CECR2 inhibitor with excellent physicochemical properties.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Sulfonamidas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.
RESUMO
The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.
Assuntos
Anti-Inflamatórios/química , Ligantes , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Amidas/química , Amidas/metabolismo , Amidas/farmacocinética , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Cães , Meia-Vida , Humanos , Ligação de Hidrogênio , Masculino , Simulação de Dinâmica Molecular , Domínios Proteicos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Fatores Imunológicos/farmacologia , Terapia de Alvo Molecular , Fatores de Transcrição/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Fatores Imunológicos/química , Fatores Imunológicos/uso terapêutico , Inflamação/tratamento farmacológico , Neoplasias/tratamento farmacológico , Domínios Proteicos/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains-BRD2, BRD3, BRD4, and BRDT-each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.
Assuntos
Descoberta de Drogas , Proteínas Nucleares/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinoxalinas/química , Quinoxalinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Homologia de SequênciaRESUMO
A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contains an oxazoline capping group and a N-(2-aminophenyl)-benzamide unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent inhibition of HDAC3-NCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and HDAC3-NCoR2 by N-(2-aminophenyl)-benzamide 15k gave respective IC50 values of 80, 110, and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7, and 9: IC50 > 100â¯000 nM; HDAC8: IC50 = 25â¯000 nM; HDAC10: IC50 > 4000 nM; HDAC11: IC50 > 2000 nM) confirmed the Class I selectivity of 15k. 2-Aminoimidazolinyl, 2-thioimidazolinyl, and 2-aminooxazolinyl units were shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)-benzamides previously reported, but the 2-aminooxazolinyl unit was the most potent in inhibiting HDAC3-NCoR2. Many of the new HDAC inhibitors showed higher solubilities and lower binding to human serum albumin than that of Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and PC-3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also lowered cyclin E expression in U937 cells but not in PC-3 cells, indicating underlying differences in the mechanisms of action of the inhibitors on those two cell lines.
Assuntos
Anilidas/química , Benzamidas/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Oxazóis/química , Acetilação , Anilidas/síntese química , Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/síntese química , Benzamidas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imidazolinas/síntese química , Imidazolinas/química , Imidazolinas/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Oxazóis/síntese química , Oxazóis/farmacologia , Permeabilidade , Ligação Proteica , Pirimidinas/farmacologia , Albumina Sérica/metabolismo , Solubilidade , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A highly stereocontrolled total synthesis of the cytotoxic marine macrolide aplyronine C is described. The route exploits aldol methodology to install the requisite stereochemistry and features a crucial boron-mediated aldol coupling of an N-vinylformamide-bearing methyl ketone with a macrocyclic aldehyde to introduce the full side chain. The synthesis of two novel C21-C34 side chain analogs is also reported.
Assuntos
Antineoplásicos/síntese química , Macrolídeos/síntese química , Compostos de Vinila/química , Antineoplásicos/química , Boro/química , Macrolídeos/química , Estrutura Molecular , EstereoisomerismoRESUMO
The synthesis of a novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contain a heterocyclic capping group and a N-(2-aminophenyl)benzamide unit that binds in the active site. In vitro assays for the inhibition of HDAC1, HDAC2, HDAC3-NCoR1, and HDAC8 by the N-(2-aminophenyl)benzamide 24a gave respective IC50 values of 930, 85, 12, and 4100 nM, exhibiting class I selectivity and potent inhibition of HDAC3-NCoR1. Both imidazolinone and thiazoline rings are shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)benzamides previously reported, an example of each ring system at 1 µM causing an increase in histone H3K9 acetylation in the human cell lines Jurkat and HeLa and an increase in cell death consistent with induction of apoptosis. Inhibition of the growth of MCF-7, A549, DU145, and HCT116 cell lines by 24a was observed, with respective IC50 values of 5.4, 5.8, 6.4, and 2.2 mM.