RESUMO
The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA(4)-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.
Assuntos
Química Farmacêutica/métodos , Diaminas/síntese química , Inibidores Enzimáticos/síntese química , Epóxido Hidrolases/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Diaminas/química , Cães , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Cinética , Modelos Químicos , RatosRESUMO
The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA(4)-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability.
Assuntos
Amidas/química , Inibidores Enzimáticos/farmacocinética , Epóxido Hidrolases/antagonistas & inibidores , Glicina/química , Administração Oral , Aminas/química , Anti-Inflamatórios/farmacologia , Ácido Benzoico/química , Disponibilidade Biológica , Química Farmacêutica , Desenho de Fármacos , Éteres , Concentração Inibidora 50 , Modelos QuímicosRESUMO
Leukotriene B(4) (LTB(4)) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB(4) and possibly identify novel treatments, inhibitors of the LTB(4) biosynthetic enzyme, leukotriene A(4) hydrolase (LTA(4)-h), have been explored. Here we describe the discovery of a potent inhibitor of LTA(4)-h, arylamide of glutamic acid 4f, starting from the corresponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood. This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria.
Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Ácido Glutâmico/síntese química , Ácido Glutâmico/farmacologia , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Ácido Glutâmico/análogos & derivados , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs' therapeutic potential as new agonists for the resolution of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Lipoxinas/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Éteres Fenílicos/farmacologia , Hipersensibilidade Respiratória/prevenção & controle , Acetatos/farmacologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antiasmáticos/farmacologia , Antígenos de Plantas , Ciclopropanos , Modelos Animais de Doenças , Antagonistas de Leucotrienos/farmacologia , Lipoxinas/química , Lipoxinas/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Quinolinas/farmacologia , Receptores de Leucotrienos/fisiologia , Hipersensibilidade Respiratória/imunologia , Relação Estrutura-Atividade , SulfetosRESUMO
Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.
Assuntos
Epóxido Hidrolases/metabolismo , Leucotrieno B4/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Ativação Enzimática , Epóxido Hidrolases/química , Fluorimunoensaio , Humanos , Leucotrieno B4/química , Leucotrieno B4/imunologia , Camundongos , Ligação ProteicaRESUMO
Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 are structurally and functionally distinct eicosanoids, with potent anti-inflammatory and immunomodulatory actions. Therapeutic use of LXA4 is greatly limited by its rapid metabolism in vivo and chemical instability. First-generation synthetic LXA4 analogs such as methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), were designed to minimize metabolism from the omega-end of the molecule. Pharmacokinetic analysis of ATLa revealed beta-oxidation as a novel route for LXA4 metabolism, prompting the development of second-generation 3-oxa-LXA4 analogs with improved pharmacokinetic disposition. Second-generation 3-oxa-LXA4 analogs such as (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-3-oxa-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoic acid (3), have shown potency and efficacy comparable to ATLa in diverse animal models after topical, intravenous or oral delivery. These include several acute (2-24 h) inflammatory reactions: calcium ionophore-induced skin edema and inflammation (topical), LTB4/PGE2-induced skin inflammation and vascular leak (topical), zymosan A-induced peritonitis (i.v. and oral) and ischemia-reperfusion-induced secondary organ injury (i.v.). Remarkably, 3-oxa-LXA4 analogs have potent once daily oral efficacy in preventing and promoting the resolution of established colitis induced by the hapten trinitrobenzene sulphonic acid (TNBS), an acute/chronic 7-14-day model of Crohn's disease. The second-generation 3-oxa-LXA4 analogs thus provide new stable pharmacophores with which to explore the emerging role of lipoxins as a new therapeutic principle for regulating inflammation, allergy and immune dysfunction in preclinical and clinical research.
Assuntos
Inflamação/tratamento farmacológico , Lipoxinas/metabolismo , Administração Oral , Animais , Humanos , Lipoxinas/administração & dosagem , Lipoxinas/química , OxirreduçãoRESUMO
Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter-regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a beta-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 mug/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor alpha, IL-2, and IFN-gamma. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with approximately 75% inhibition of mucosal tumor necrosis factor alpha and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that ATL analogs may be an attractive oral treatment approach for inflammatory bowel diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colite/tratamento farmacológico , Lipoxinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Sequência de Bases , Colite/genética , Colite/imunologia , Colite/patologia , Feminino , Haptenos/toxicidade , Lipoxinas/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ácido Trinitrobenzenossulfônico/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
1. Lipoxins (LX) and aspirin-triggered 15-epi-lipoxins (ATL) exert potent anti-inflammatory actions. In the present study, we determined the anti-inflammatory efficacy of endogenous LXA(4) and LXB(4), the stable ATL analog ATLa2, and a series of novel 3-oxa-ATL analogs (ZK-996, ZK-990, ZK-994, and ZK-142) after intravenous, oral, and topical administration in mice. 2. LXA(4), LXB(4), ATLa2, and ZK-994 were orally active, exhibiting potent systemic inhibition of zymosan A-induced peritonitis at very low doses (50 ng kg(-1)-50 microg kg(-1)). 3. Intravenous ZK-994 and ZK-142 (500 microg kg(-1)) potently attenuated hind limb ischemia/reperfusion-induced lung injury, with 32+/-12 and 53+/-5% inhibition (P<0.05), respectively, of neutrophil accumulation in lungs. The same dose of ATLa2 had no significant protective action. 4. Topical application of ATLa2, ZK-994, and ZK-142 ( approximately 20 microg cm(-2)) prevented vascular leakage and neutrophil infiltration in LTB(4)/PGE(2)-stimulated ear skin inflammation. While ATLa2 and ZK-142 displayed approximately equal anti-inflammatory efficacy in this model, ZK-994 displayed a slower onset of action. 5. In summary, native LXA(4) and LXB(4), and analogs ATLa2, ZK-142, and ZK-994 retain broad anti-inflammatory effects after intravenous, oral, and topical administration. The 3-oxa-ATL analogs, which have enhanced metabolic and chemical stability and a superior pharmacokinetic profile, provide new opportunities to explore the actions and therapeutic potential for LX and ATL.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Lipoxinas/farmacologia , Administração Oral , Administração Tópica , Animais , Aspirina/farmacologia , Orelha Externa/patologia , Inflamação/patologia , Inflamação/prevenção & controle , Injeções Intravenosas , Lipoxinas/administração & dosagem , Pneumopatias/patologia , Pneumopatias/prevenção & controle , Masculino , Camundongos , Peritonite/induzido quimicamente , Peritonite/patologia , Peritonite/prevenção & controle , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Zimosan/toxicidadeRESUMO
Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Lipoxinas/síntese química , Éteres Fenílicos/síntese química , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Calcimicina , Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/etiologia , Estabilidade de Medicamentos , Ionóforos , Lipoxinas/metabolismo , Lipoxinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Éteres Fenílicos/metabolismo , Éteres Fenílicos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.
Assuntos
Amidinas/química , Fator Xa/química , Imidazóis/química , Piridinas/química , Sítios de Ligação , Inibidores do Fator Xa , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Tripsina/químicaRESUMO
Double rotational-echo double resonance (double REDOR) NMR was used to investigate the conformation of a (13)C-, (15)N-, and (19)F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon-fluorine dipolar couplings were measured by (13)C[(19)F] REDOR. Natural abundance carbon signals in the full-echo spectra were removed by (13)C[(15)N] REDOR. Major and minor binding modes were suggested by the NMR data, but only the former had adequate signal to noise for distance determinations. Molecular dynamics simulations restrained by double-REDOR-determined intramolecular (13)C-(19)F distances revealed two models for the dominant binding mode that are consistent with the NMR data. We conclude that ZK-806299 binds similarly to both FXa. Moreover, it appears to bind to FXa in a fashion previously demonstrated for ZK-807834, a more selective FXa inhibitor.
Assuntos
Amidinas/química , Fator Xa/química , Piridinas/química , Sítios de Ligação , Inibidores do Fator Xa , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Tripsina/químicaRESUMO
The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Colite/imunologia , Colite/prevenção & controle , Sulfato de Dextrana/toxicidade , Regulação para Baixo/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/uso terapêutico , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Lipoxinas , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Salmonella typhimurium/imunologia , Índice de Gravidade de DoençaRESUMO
A novel series of diaryloxypyridines have been designed as selective nanomolar factor Xa (fXa) inhibitors for use as anticoagulants. In this paper, we describe our efforts to identify an additional interaction and a replacement for the distal amidine group that binds in the S3/S4 pocket of fXa. Introduction of a hydroxyl group para to the proximal amidine group increases the potency vs fXa by 1-2 orders of magnitude, which is the result of a hydrogen bond to Ser195 of the catalytic triad. A methyl imidazoline and a dimethylamide are good alternatives for the second amidine. These substitutions have increased the selectivity vs the related serine proteases trypsin and thrombin. The synthesis, in vitro activity, and hypothetical modes of binding to fXa based on trypsin crystallographic data are outlined.
Assuntos
Amidinas/síntese química , Inibidores do Fator Xa , Inibidores de Serina Proteinase/síntese química , Amidinas/química , Amidinas/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Fator Xa/química , Humanos , Modelos Moleculares , Ratos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacocinética , Relação Estrutura-Atividade , Trombina/química , Tripsina/químicaRESUMO
A novel potent and selective aminophenol scaffold for fXa inhibitors was developed from a previously reported benzimidazole-based naphthylamidine template. The aminophenol template is more synthetically accessible than the benzimidazole template, which simplified the introduction of carboxylic acid groups. Substitution of a propenyl-para-hydroxy-benzamidine group on the aminophenol template produced selective, sub-nanomolar fXa inhibitors. The potency of the inhibitors is partially explained with the aid of a trypsin complex crystal structure.
Assuntos
Aminofenóis/química , Aminofenóis/síntese química , Inibidores do Fator Xa , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/síntese química , Difração de Raios XRESUMO
Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.
Assuntos
Benzimidazóis/química , Inibidores do Fator Xa , Inibidores de Serina Proteinase/química , Trombina/metabolismo , Tripsina/metabolismo , Benzimidazóis/metabolismo , Inibidores de Serina Proteinase/metabolismoRESUMO
A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.