RESUMO
Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.
Assuntos
Amidoidrolases/antagonistas & inibidores , Benzimidazóis/isolamento & purificação , Benzimidazóis/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Animais , Benzimidazóis/farmacocinética , Cumarínicos , Cristalização , Inibidores Enzimáticos/farmacocinética , Escherichia coli , Humanos , Estrutura Molecular , Ratos , Espectrofotometria Ultravioleta , Espectrometria de Massas em Tandem , Ureia/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/química , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Células Cultivadas , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Estrutura Molecular , Isoformas de Proteínas/química , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The optimization of a series of 8-aza-quinazolinone analogs for antagonist activity against the CXCR3 receptor is reported. Compounds were optimized to avoid the formation of active metabolites and time-dependent-inhibitors of CYP3A4. In addition, antagonists showed potent against CXCR3 activity in whole blood and optimized to avoid activity in the chromosomal aberration assay. Compound 25 was identified as having the optimal balance of CXCR3 activity and pharmacokinetic properties across multiple pre-clinical species, which are reported herein.
Assuntos
Quinazolinas/síntese química , Quinazolinonas/síntese química , Receptores CXCR3/antagonistas & inibidores , Animais , Bleomicina/toxicidade , Aberrações Cromossômicas , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Cães , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Inflamação , Concentração Inibidora 50 , Leucócitos/efeitos dos fármacos , Macaca fascicularis , Camundongos , Modelos Químicos , Quinazolinas/farmacologia , Quinazolinonas/farmacologia , Fatores de TempoRESUMO
Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.
Assuntos
Amidoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/química , Amidoidrolases/metabolismo , Animais , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Lactamas/síntese química , Lactamas/química , Lactamas/farmacocinética , Ratos , Compostos de Espiro/química , Relação Estrutura-Atividade , Ureia/síntese química , Ureia/química , Ureia/farmacocinéticaRESUMO
A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was identified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is evaluated in a mouse lung inflammation model.
Assuntos
Anti-Inflamatórios/química , Benzenoacetamidas/química , Óxidos S-Cíclicos/química , Imidazóis/química , Pirazinas/química , Receptores CXCR3/antagonistas & inibidores , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Benzenoacetamidas/síntese química , Benzenoacetamidas/farmacocinética , Óxidos S-Cíclicos/síntese química , Óxidos S-Cíclicos/farmacocinética , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Camundongos , Conformação Molecular , Pirazinas/síntese química , Pirazinas/farmacocinética , Ratos , Receptores CXCR3/metabolismoRESUMO
A series of six-six and six-five fused heterocyclic CXCR3 antagonists has been synthesized and their activities evaluated in an [(125)I]-IP-10 displacement assay and an ITAC mediated in vitro cell migration assay. The pharmacokinetic properties of several top compounds have also been studied. This effort led to the discovery of compounds with increased potency and improved pharmacokinetic properties that could serve as useful tools to study the role of the CXCR3 receptor in vivo.
Assuntos
Compostos Heterocíclicos/farmacologia , Quinazolinonas/farmacologia , Receptores CXCR3/antagonistas & inibidores , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Quinazolinonas/farmacocinética , Ratos , EstereoisomerismoRESUMO
Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos Alquilantes/síntese química , DNA/química , Dipeptídeos/química , Imunoconjugados/química , Indóis/síntese química , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Catepsina B/química , Linhagem Celular Tumoral , Ciclopropanos/síntese química , Ciclopropanos/química , Ciclopropanos/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Indóis/farmacologia , Camundongos , Relação Estrutura-AtividadeRESUMO
The first nonpeptidic, noncovalent inhibitors of the cysteine protease cathepsin S (CatS) are described. Electronic database searching using the program DOCK generated a screening set of potential CatS inhibitors from which two lead structures were identified as promising starting points for a drug discovery effort. Lead optimization afforded potent (IC(50) < 50 nM) and selective inhibitors of CatS demonstrating cellular activity and reversibility of enzyme inhibition.
Assuntos
Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Catepsinas/química , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Regiões Constantes de Imunoglobulina/efeitos dos fármacos , Regiões Constantes de Imunoglobulina/metabolismo , Concentração Inibidora 50 , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.
Assuntos
Neoplasias/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias/genética , Neoplasias/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.
Assuntos
Acetatos/síntese química , Antineoplásicos/síntese química , Piperidonas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetatos/farmacocinética , Acetatos/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Hepatócitos/metabolismo , Humanos , Macaca fascicularis , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Transplante de Neoplasias , Piperidonas/farmacocinética , Piperidonas/farmacologia , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Transplante Heterólogo , Proteínas rho de Ligação ao GTP/biossínteseRESUMO
A series of competitive, reversible cathepsin S (CatS) inhibitors was investigated. An earlier disclosure detailed the discovery of the 4-(2-keto-1-benzimidazolinyl)-piperidin-1-yl moiety as an effective replacement for the 4-arylpiperazin-1-yl group found in our screening hit. Continued investigation into replacements for the 4-aryl piperazine resulted in the identification of potentially useful CatS inhibitors with enzymatic and cellular activity similar to that of JNJ 10329670 as disclosed in a previous publication.
Assuntos
Compostos Bicíclicos com Pontes/química , Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/química , Piperidinas/química , Animais , Sítios de Ligação , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Camundongos , Piperidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Aldol reactions of methyl ketone lithium enolates proceed via chairlike Zimmerman-Traxler transition states with 7:1 to 50:1 preference over alternative, boatlike transition structures, as determined by studies involving the configurationally stable deuterium-labeled enol silane 18 as the lithium enolate precursor.
Assuntos
Álcoois/síntese química , Aldeídos/química , Cetonas/síntese química , Lítio/química , Pentanonas/química , Deutério , Cetonas/química , Conformação Molecular , Compostos Organometálicos/química , EstereoisomerismoRESUMO
A novel series of competitive, reversible cathepsin S (CatS) inhibitors was discovered and optimized. The 4-(2-keto-1-benzimidazolinyl)-piperidin-1-yl moiety was found to be an effective replacement for the 4-arylpiperazin-1-yl group found in our earlier series of CatS inhibitors. This replacement imparted improved PK properties as well as decreased off-target activity. Optimization of the ketobenzimidazole moiety led to the discovery of the lead compound JNJ 10329670, which represents a novel class of selective, noncovalent, reversible, and orally bioavailable inhibitors of cathepsin S.
Assuntos
Catepsinas/antagonistas & inibidores , Animais , Benzimidazóis/química , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Linhagem Celular , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Modelos Químicos , Estrutura Molecular , Pirazóis/química , Pirazóis/farmacocinética , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
We describe a simple 1H NMR analysis that permits the stereochemistry of beta-hydroxy ketones to be assigned by visual inspection of the ABX patterns for the alpha-methylene unit of the beta-hydroxy ketone in the 1H NMR spectra. This method has been verified by application to a wide range of beta-hydroxy ketones deriving from aldol reactions of chiral aldehydes with a variety of chiral and achiral methyl ketone enolates (see Tables 1 and 2). The stereochemistry of 54 of these compounds have been assigned by rigorous chemical methods.