RESUMO
Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Éteres/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Compostos de Anilina/química , Antineoplásicos/química , Linhagem Celular Tumoral , Éteres/química , Humanos , L-Lactato Desidrogenase/químicaRESUMO
The AAA+ ATPase p97 (valosin-containing protein, VCP) is a master regulator of protein homeostasis and therefore represents a novel target for cancer therapy. Starting from a known allosteric inhibitor, NMS-873, we systematically optimized this scaffold, in particular, by applying a benzene-to-acetylene isosteric replacement strategy, specific incorporation of F, and eutomer/distomer identification, which led to compounds that exhibited nanomolar biochemical and cell-based potency. In cellular pharmacodynamic assays, robust effects on biomarkers of p97 inhibition and apoptosis, including increased levels of ubiquitinated proteins, CHOP and cleaved caspase 3, were observed. Compound (R)-29 (UPCDC-30766) represents the most potent allosteric inhibitor of p97 reported to date.
RESUMO
Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-alpha production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-alpha production.
Assuntos
Anti-Inflamatórios/química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Nitrilas/química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinolinas/química , Fator de Necrose Tumoral alfa/sangue , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Feminino , Humanos , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Nitrilas/síntese química , Nitrilas/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas/metabolismo , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA(2)alpha inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA(2)alpha inhibition.
Assuntos
Compostos Benzidrílicos/síntese química , Compostos Benzidrílicos/farmacologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/metabolismo , Quinazolinonas/síntese química , Quinazolinonas/farmacologia , Animais , Ácido Araquidônico/metabolismo , Compostos Benzidrílicos/química , Sangue/efeitos dos fármacos , Sangue/metabolismo , Linhagem Celular , Humanos , Quinazolinonas/química , Ratos , Solubilidade , Relação Estrutura-AtividadeRESUMO
Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.
RESUMO
Tumor progression loci-2 (Tpl2) (Cot/MAP3K8) is a serine/threonine kinase in the MAP3K family directly upstream of MEK. Recent studies using Tpl2 knockout mice have indicated an important role for Tpl2 in the lipopolysaccharide (LPS) induced production of tumor necrosis factor alpha (TNF-alpha) and other proinflammatory cytokines involved in diseases such as rheumatoid arthritis. Initial 4-anilino-6-aminoquinoline-3-carbonitrile leads showed poor selectivity for Tpl2 over epidermal growth factor receptor (EGFR) kinase. Using molecular modeling and crystallographic data of the EGFR kinase domain with and without an EGFR kinase-specific 4-anilinoquinazoline inhibitor (erlotinib, Tarceva), we hypothesized that we could diminish the inhibition of EGFR kinase by substitution at the C-8 position of our 4-anilino-6-aminoquinoline-3-carbonitrile leads. The 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles were prepared from the appropriate 2-substituted 4-nitroanilines. Modifications to the C-6 and C-8 positions led to the identification of compounds with increased inhibition of TNF-alpha release from LPS-stimulated rat and human blood, and these analogues were also highly selective for Tpl2 kinase over EGFR kinase. Further structure-activity based modifications led to the identification of 8-bromo-4-(3-chloro-4-fluorophenylamino)-6-[(1-methyl-1H-imidazol-4-yl)methylamino]quinoline-3-carbonitrile, which demonstrated in vitro as well as in vivo efficacy in inhibition of LPS-induced TNF-alpha production.
Assuntos
Aminoquinolinas/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Imidazóis/síntese química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Modelos Moleculares , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Aminoquinolinas/farmacocinética , Aminoquinolinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Cristalografia por Raios X , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Cloridrato de Erlotinib , Feminino , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Técnicas In Vitro , MAP Quinase Quinase Quinases/biossíntese , MAP Quinase Quinase Quinases/química , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/química , Quinazolinas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/químicaRESUMO
INTRODUCTION: Tumor necrosis factor-alpha (TNFalpha) participates in many inflammatory processes. TNFalpha modulators show beneficial effects for the treatment of many diseases including rheumatoid arthritis. The purpose of this study was to validate a rat pharmacokinetic/pharmacodynamic (PK/PD) model for rapid assessment of drug candidates that intended to interrupt TNFalpha synthesis or release. METHODS: Rats received intravenous (IV) or oral administrations of test article or dose vehicle, followed by LPS challenge. Plasma levels of test article and TNFalpha were determined. The areas under the concentration-time curves (AUC(drug) and AUC(TNFalpha)) were calculated. The overall percentage of inhibition on TNFalpha release in vivo was calculated by comparing AUC(TNFalpha) of the test article treated group against that for the vehicle control group. RESULTS: The dosing vehicles tested in this study did not increase plasma TNFalpha level. At IV dose of up to 100 microg/kg, LPS did not alter the pharmacokinetics of the compound tested. Using a selective TNFalpha converting enzyme (TACE) inhibitor as model compound, this PK/PD model demonstrated its ability to correlate plasma test article concentration with its biological activity of lowering the LPS-induced TNFalpha plasma levels in vivo. DISCUSSION: A rat PK/PD model for evaluation of the effect of drug candidates on LPS-induced TNFalpha synthesis and/or release has been investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test articles.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Sulfonamidas/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Proteína ADAM17 , Animais , Feminino , Humanos , Modelos Biológicos , Ratos , Ratos Endogâmicos Lew , Sulfonamidas/farmacocinéticaRESUMO
A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.
RESUMO
p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Regulação Alostérica , Sítios de Ligação , Microscopia Crioeletrônica , Inibidores Enzimáticos , Humanos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example, EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile (30) was efficacious in a rat model of LPS-induced TNF-alpha production.
Assuntos
Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Ligação Competitiva , Cicloeptanos/química , Cicloeptanos/farmacologia , Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftiridinas/química , Naftiridinas/farmacologia , Nitrilas/química , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.
Assuntos
Sangue/efeitos dos fármacos , Inflamação/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/fisiologia , Monócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Líquido Sinovial/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Catálise , Dinoprostona/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismoRESUMO
The synthesis and structure-activity studies of a series of quinoline-3-carbonitriles as inhibitors of Tpl2 kinase are described. Potent inhibitors of Tpl2 kinase with selectivity against a panel of selected kinases in enzymatic assays and specificity in cell-based phosphorylation assays in LPS-treated human monocytes were identified. Selected inhibitors with moderate activity in human whole blood assay effectively inhibited LPS/D-Gal induced TNFalpha release when administered intraperitoneally in mice.
Assuntos
Artrite Reumatoide/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , Nitrilas/química , Nitrilas/farmacologia , Quinolinas/química , Fator de Necrose Tumoral alfa/biossíntese , Artrite Reumatoide/tratamento farmacológico , Reagentes de Ligações Cruzadas/química , Humanos , Imidazóis/química , MAP Quinase Quinase Quinases/metabolismo , Estrutura Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Nitrilas/síntese química , Relação Estrutura-AtividadeRESUMO
The synthesis and structure-activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.
Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Naftiridinas/química , Nitrilas/química , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Artrite Reumatoide/tratamento farmacológico , Humanos , Naftiridinas/síntese química , Naftiridinas/farmacologia , Nitrilas/síntese química , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-AtividadeRESUMO
Rhizoxin D (2) was synthesized from four subunits, A, B, C, and D representing C3-C9, C10-C13, C14-C19, and C20-C27, respectively. Subunit A was prepared by cyclization of iodo acetal 21, which set the configuration at C5 of 2 through a stereoselective addition of the radical derived from dehalogenation of 21 at the beta carbon of the (Z)-alpha,beta-unsaturated ester. Aldehyde 29 was obtained from phenylthioacetal 24 and condensed with phosphorane 30, representing subunit B, in a Wittig reaction that gave the (E,E)-dienoate 31. This ester was converted to aldehyde 33 in preparation for coupling with subunit C. The latter in the form of methyl ketone 55 was obtained in six steps from propargyl alcohol. An aldol reaction of 33 with the enolate of 55 prepared with (+)-DIPCl gave the desired beta-hydroxy ketone 56 bearing a (13S)-configuration in a 17-20:1 ratio with its (13R)-diastereomer. After reduction to anti diol 57 and selective protection as TIPS ether 58, the C15 hydroxyl was esterified to give phosphonate 59. An intramolecular Wadsworth-Emmons reaction of aldehyde 62, derived from delta-lactone 60, furnished macrolactone 63, which was coupled in a Stille reaction with stannane 68 to give 2 after cleavage of the TIPS ether.
Assuntos
Antineoplásicos/síntese química , Lactonas/síntese química , Rhizopus/química , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Estrutura MolecularRESUMO
The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1-CD28 interaction. In a high-throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure-activity relationships of a series of these compounds.