RESUMEN
In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 1, several series of analogues were made that examined the C-6 aryl substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other acyl functionality at the N-7 position. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonreceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the more thoroughly evaluated members, 32, with IC50 values of 0.21 microM (PDGFr), 0.049 microM (bFGFr), and 0.018 microM (c-Src), was evaluated in in vivo studies against a panel of five human tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon xenograft model.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Urea/análogos & derivados , Urea/síntesis química , Células 3T3 , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Proteína Tirosina Quinasa CSK , División Celular/efectos de los fármacos , Neoplasias del Colon , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Glioma , Humanos , Indicadores y Reactivos , Cinética , Ratones , Conformación Molecular , Estructura Molecular , Fosforilación , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-Actividad , Transfección , Células Tumorales Cultivadas , Urea/química , Urea/farmacología , Familia-src QuinasasRESUMEN
7-substituted 3-(2,6-dichlorophenyl)-1,6-naphthyridin-2(1H)-ones are potent inhibitors of protein tyrosine kinases, with some selectivity for c-Src. The compounds were prepared by condensing 4, 6-diaminonicotinaldehyde with 2,6-dichlorophenylacetonitrile and selectively converting the 2- and 7-amino groups of the product to hydroxy and fluoro groups, respectively, by prolonged diazotization in 50% aqueous fluoboric acid. N-Methylation, followed by treatment with aliphatic diamines, aromatic amines, or their derived lithium anions, gave the desired compounds. Selected isomeric 1, 8-naphthyridin-2(1H)-ones were also prepared in order to evaluate the relative contributions of both ring A aza atoms of the related pyrido[2,3-d]pyrimidin-7(8H)-ones to the inhibitory activity. The compounds were evaluated for their ability to prevent phosphorylation of a model substrate by c-Src, FGF-1 receptor, and PDGF-beta receptor enzymes. Overall, there was a high degree of correlation of the activities against the different kinases, with c-Src being generally the most sensitive to structural changes. 1, 6-Naphthyridin-2(1H)-one analogues bearing basic aliphatic side chains [7-NH(CH(2))(n)()NRR, 7-NHPhO(CH(2))(n)()NRR, or 7-NHPhN(CH(2))(4)NMe] were the most potent against c-Src (IC(50)s of 10-80 nM), showing good selectivity with respect to PDGFR (10-300-fold) but less with respect to FGFR. The 1, 6-naphthyridin-2(1H)-ones showed broadly similar activity to the analogous pyrido[2,3-d]pyrimidin-7(8H)-ones, whereas the 1, 8-naphthyridin-2(1H)-ones were at least 10(3)-fold less potent. These results, indicating that the 3-aza atom in the pyrido[2, 3-d]pyrimidin-7(8H)-ones is mandatory, whereas the 1-aza atom is not, support the published binding model for these compounds to c-Src (J. Med. Chem. 1998, 41, 1752), where the 3-aza and 2-NH atoms form a bidentate H-bond donor-acceptor motif that interacts with Met341 and the 1-aza atom is not involved in specific binding interactions.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Naftiridinas/síntesis química , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Naftiridinas/química , Naftiridinas/farmacología , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Following an earlier discovery of 1-phenylbenzimidazoles as ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR), further structure-activity relationships for analogues (particularly 5-substituted derivatives) are reported. The data are consistent with a binding model (constructed from the homology-modeled structure of the catalytic subunit of the PDGFR using protein kinase A as the template) in which the ligand binds in the relatively narrow ATP site, with the phenyl ring pointing toward the interior of the pocket and the 5-position of the benzimidazole ring toward the mouth of the pocket. The narrow binding pocket allows a maximum torsion angle between the phenyl and benzimidazole rings of about 40 degrees, consistent with that calculated (43.6 degrees) for the minimum-energy conformation of the unsubstituted free ligand. The inactivity of 7- or 2'-substituted analogues is consistent with the greater torsion angle (and thus larger ligand cross-section) of such substituted analogues. There is substantial bulk tolerance for 5-substituents, which protrude out of the mouth of the hydrophobic pocket, with the most effective analogues being those bearing weak bases. On the basis of this model, 5-OR derivatives bearing cationic side chains were prepared as soluble analogues, and these showed sub-micromolar potencies against the isolated PDGFR enzyme. They were also moderately effective inhibitors of autophosphorylation of PDGFR in rat aortic vascular smooth muscle cells, with IC50s in the range 0.1-1 microM.
Asunto(s)
Bencimidazoles/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Bencimidazoles/síntesis química , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Ligandos , Modelos Moleculares , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Relación Estructura-ActividadRESUMEN
Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of three PDGF-dependent tumor lines.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridonas/síntesis química , Pirimidinas/síntesis química , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Células 3T3 , Animales , Disponibilidad Biológica , Proteína Tirosina Quinasa CSK , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Desnudos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Fosforilación , Piridonas/química , Piridonas/farmacocinética , Piridonas/farmacología , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Ratas Wistar , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales Cultivadas , Familia-src QuinasasRESUMEN
Through direct synthetic efforts, we discovered a small molecule that is a nanomolar inhibitor of the human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866, a member of a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines, was identified by screening a compound library with assays that measure protein tyrosine kinase activity. PD 166866 inhibited human full-length FGFR-1 tyrosine kinase with an IC50 value of 52.4 +/- 0.1 nM and was further characterized as an ATP competitive inhibitor of the FGFR-1. In contrast, PD 166866 had no effect on c-Src, platelet-derived growth factor receptor-beta, epidermal growth factor receptor or insulin receptor tyrosine kinases or on mitogen-activated protein kinase, protein kinase C and CDK4 at concentrations as high as 50 microM. PD 166866 was a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 did not inhibit platelet-derived growth factor, epidermal growth factor or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or NIHIR cells, respectively, further supporting its specificity for the FGFR-1. In addition, daily exposure of PD 166866 to L6 cells at concentrations from 1 to 100 nM resulted in a concentration-related inhibition of bFGF-stimulated cell growth for 8 consecutive days with an IC50 value of 24 nM. In contrast, PD 166866 had little effect on platelet-derived growth factor-BB-stimulated growth of L6 cells or serum-stimulated vascular smooth muscle cell proliferation. Finally, PD 166866 was found to be a potent inhibitor of microvessel outgrowth (angiogenesis) from cultured artery fragments of human placenta. These results highlight the discovery of PD 166866, a new nanomolar potent and selective small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Urea/análogos & derivados , Células 3T3 , Animales , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Técnicas In Vitro , Ratones , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Urea/farmacologíaRESUMEN
Through direct synthetic efforts we discovered a small molecule which is a 40 nanomolar inhibitor of the human FGF-1 receptor tyrosine kinase. 1-Tert-butyl-3-[6-(2,6-dichloro-phenyl)-2-(4-diethylamino-butylamino)-py rido[2,3-d]pyrimidin-7-yl]-urea (PD 161570) had about 5- and 100-fold greater selectivity toward the FGF-1 receptor (IC50 = 40 nM) compared with the PDGFbeta receptor (IC50 = 262 nM) or EGF receptor (IC50 = 3.7 microM) tyrosine kinases, respectively. In addition, PD 161570 suppressed constitutive phosphorylation of the FGF-1 receptor in both human ovarian carcinoma cells (A121(p)) and Sf9 insect cells overexpressing the human FGF-1 receptor and blocked the growth of A121(p) cells in culture. The results demonstrate a novel synthetic inhibitor with nanomolar potency and specificity towards the FGF-1 receptor tyrosine kinase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Urea/análogos & derivados , Animales , División Celular/fisiología , Línea Celular , Factores de Crecimiento de Fibroblastos/fisiología , Humanos , Fosforilación , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/metabolismo , Spodoptera , Células Tumorales Cultivadas , Urea/farmacologíaRESUMEN
PD 089828, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido-[2,3-d]pyrimidines, was identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 089828 was found to inhibit human full-length fibroblast growth factor (FGF) receptor-1 (FGFR-1), platelet-derived growth factor (PDGF) receptor beta subunit (PDGFR-beta), Src nonreceptor tyrosine kinase (c-Src) and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 0.15 +/- 0.02 (n = 4), 0.18 +/- 0.04 (n = 3), 1.76 +/- 0.28 (n = 4) and 5.47 +/- 0.78 (n = 6) microM, respectively. PD 089828 was further characterized as an ATP competitive inhibitor of the growth factor receptor tyrosine kinases (FGFR-1, PDGFR-beta and EGFR) but a noncompetitive inhibitor of c-Src tyrosine kinase with respect to ATP. In addition, PD 089828 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular SMC (VSMC) and basic FGF-mediated tyrosine phosphorylation in A121 cells with IC50 values similar to the potencies observed for inhibition of receptor tyrosine kinase activity. The inhibition of PDGF receptor autophosphorylation in VSMC by PD 089828 occurred rapidly, with maximal effects reached within 5 min of drug exposure. Inhibition after single exposure was long lasting but also rapidly reversible, occurring within 5 min after drug removal. The PDGF-induced association of downstream signaling proteins, including phosphoinositide-3-kinase (PI-3K), growth factor receptor binding protein-2 (GRB2), SH-2 domain and collagen like (Shc) and phospholipase Cgamma (PLCgamma), with VSMC PDGF receptors was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 089828. PD 089828 also inhibited the PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms. Moreover, the effects of PD 089828 were demonstrated in functional assays in which PDGF-stimulated DNA synthesis, PDGF-directed migration and serum-stimulated growth of VSMC were all inhibited to the same extent as PDGF receptor autophosphorylation (IC50 = 0.8, 4.5 and 1.8 microM, respectively). These results highlight the biological characteristics of PD 089828 as a novel, broadly active protein tyrosine kinase inhibitor with long-lasting but reversible cellular effects. The potential therapeutic use of these broadly acting, nonselective inhibitors as antiproliferative and antimigratory agents could extend to such diseases as cancer, atherosclerosis and restenosis in which redundancies in growth-signaling pathways are known to exist.
Asunto(s)
Aorta/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Animales , Células Cultivadas , Humanos , Masculino , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
PD 166285, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido[2,3-d]pyrimidines, was synthesized as the most potent and soluble analog of a series of small molecules originally identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 166285 was found to inhibit Src nonreceptor tyrosine kinase, fibroblast growth factor receptor-1, epidermal growth factor receptor and platelet-derived growth factor receptor beta subunit (PDGFR-beta), tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 8.4 +/- 2.3 nM (n = 6), 39.3 +/- 2.8 nM (n = 16), 87.5 +/- 13.7 nM (n = 6) and 98.3 +/- 7.9 nM (n = 16), respectively. PD 166285 also demonstrated inhibitory activity against mitogen-activated protein kinase (IC50 = 5 microM) and protein kinase C (IC50 = 22.7 microM). PD 166285 was further characterized as an ATP competitive inhibitor of Src nonreceptor tyrosine kinase, PDGFR-beta, fibroblast growth factor receptor-1 and epidermal growth factor receptor tyrosine kinases. In addition, PD 166285 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular smooth muscle cells (VSMCs) and A431 cells, respectively, and basic fibroblast growth factor-mediated tyrosine phosphorylation in Sf9 cells, with IC50 values of 6.5 nM, 1.6 microM and 97.3 nM, respectively, further establishing a tyrosine kinase mechanism of inhibition. The inhibition of PDGF receptor autophosphorylation in VSMCs by PD 166285 was long lasting and persisted for 4 days after a single 1-hr exposure followed by extensive washing. The PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 166285 in VSMCs. The effects of PD 166285 were also demonstrated in functional assays of cell attachment, migration and proliferation, in which vascular cell adhesion to vitronectin, PDGF-directed chemotaxis and serum-stimulated cell growth were all potently inhibited with IC50 values of 80 yo 120 nM. Finally, PD 166285 uniquely demonstrated potent inhibition of phorbol ester-induced production of 92-kDa gelatinase A (MMP-9) in VSMC without affecting 72-kDa gelatinase B (MMP-2) as measured by gelatin zymography. These results highlight the biological characteristics of PD 166285 as a broadly active protein tyrosine kinase capable of potently inhibiting a number of kinase mediated cellular functions, including cell attachment, movement and replication. The potential therapeutic utility of this broadly acting inhibitor as an antiproliferative and antimigratory agent could extend to such diseases as cancer, atherosclerosis and restenosis, in which redundancies in protein kinase signaling pathways are known to exist.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridonas/farmacología , Pirimidinas/farmacología , Animales , ADN/biosíntesis , Humanos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Conejos , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Spodoptera , Tirosina/metabolismoRESUMEN
Recent reports suggest that delayed myocardial protection ("second window of preconditioning") occurs 24 hours after brief ischemic or thermal stress. In order to test this hypothesis, we subjected New Zealand White rabbits to a heating regimen (42 degrees C for 15-20 minutes). Twenty four hours later, the effect of heat stress on infarct size was determined by conducting a 30 minute ischemia/3 hour reperfusion protocol. In a separate group of rabbits, Western blot analysis was used to verify that the heating regimen increased expression of HSP72i. The size of the region at risk was delineated by infusion of Unisperse blue and infarcted myocardium was identified by incubation of left ventricular slices in triphenyl tetrazolium chloride. In contrast to expectations, induction of HSP72i with thermal stress was not effective in limiting infarct size in rabbits 24 hours later, calling into question the concept that heat stress induces delayed or "second window" myocardial protection.
Asunto(s)
Trastornos de Estrés por Calor/metabolismo , Proteínas de Choque Térmico/biosíntesis , Infarto del Miocardio/metabolismo , Animales , Western Blotting , Proteínas del Choque Térmico HSP72 , Trastornos de Estrés por Calor/patología , Calor/efectos adversos , Precondicionamiento Isquémico Miocárdico , Infarto del Miocardio/patología , Conejos , Factores de TiempoRESUMEN
Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to other mechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generated to elucidate the specificity in the individual binding pockets with systematic substitutions in the P5-P2, and P2'-P3' based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, a second series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined for both sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, and mouse and human renin. Important specificity-determining interactions were found in the S3 (Glu-13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites.
Asunto(s)
Catepsinas/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/química , Sitios de Unión , Catepsina E , Catepsinas/aislamiento & purificación , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Programas Informáticos , Espectrofotometría Ultravioleta , Estadística como AsuntoRESUMEN
Using molecular modeling and the information derived from the X-ray crystal structure of HIV-1 protease (HIV PR) complexed with the pyran-2-one 1, a series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes was designed and analyzed as novel, nonpeptidic inhibitors of HIV PR. Structure-activity studies led to the discovery of inhibitor 19 having (RS)-1-(cyclopentylthio)-3-methylbutyl functionalization at the C-3 position, which exhibited a Kc of 33 nM. A X-ray crystallographic structure of 19 bound to HIV PR showed that structural water-301 (inhibitor-flap-bridging water) was displaced by the inhibitor. Interestingly, the enol moiety of the pyran-2-one formed a hydrogen bond directly with Asp125 and with Asp25 via a bridging water molecule, thus illustrating a unique mode of active site binding by an HIV PR inhibitor. The pendant cyclopentyl and isobutyl groups of 19 occupied the S1' and S2' binding sites, respectively, whereas the 6-phenyl group occupied a region in between the S1 and S3 pockets of HIV PR. Selected compounds were tested for antiviral activity on H9 cells infected with HIV-1IIIb. A correlation between enzymatic activity and antiviral activity was not found in this series. The best antiviral compound in this series, 18, contained (RS)-3-[cyclopentyl(cyclopentylthio)methyl] functionalization at the C-3 position of the pyran-2-one ring and exhibited a CIC50 of 14 microM and TC50 of 70 microM. These studies demonstrate that potent enzyme inhibition can be achieved by inhibitors that span only three subsites.
Asunto(s)
Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Pironas/síntesis química , Pironas/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Células Cultivadas , Cristalografía por Rayos X , Inhibidores de la Proteasa del VIH/química , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Pironas/química , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
Cl-992, a novel potent inhibitor of primate renin, was tested for blood pressure-lowering efficacy in sodium-restricted, furosemide-treated conscious normotensive cynomolgus monkeys and conscious renal hypertensive monkeys. The hypertensive monkey model provided an opportunity to determine the response to a renin inhibitor in a pathological nonhuman primate model of hypertension without concurrent diuretic treatment or dietary sodium restriction and on repeated oral administration. Cl-992 has IC50 values of 0.58 +/- 0.06 (n = 4) and 0.36 +/- 0.03 nM (n = 8) against human and monkey renin, respectively. In normotensive monkeys, oral Cl-992 at doses of 3, 10 and 30 mg/kg reduced mean arterial blood pressure (MABP) by 8 +/- 2, 15 +/- 7 and 29 +/- 7 mm Hg (n = 5 animals per dose level, P < .05), respectively (base line, 103 +/- 3 mm Hg). Intravenous Cl-992 (0.0001 to 0.1 mg/kg) also caused dose-dependent decreases in MABP and a maximum reduction of 23 +/- 4 mm Hg. The decrease in MABP after Cl-992 was paralleled by an inhibition of plasma renin activity (PRA) and a reduction in immunoreactive angiotensin II. In renal hypertensive monkeys, oral Cl-992 at doses of 3, 10 and 30 mg/kg reduced MABP by 6 +/- 2, 18 +/- 6 and 37 +/- 8 mm Hg (n = 3 or 4, P < .05), respectively (base line, 134 +/- 4 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antihipertensivos/farmacología , Dipéptidos/farmacología , Renina/antagonistas & inhibidores , Administración Oral , Animales , Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Dipéptidos/administración & dosificación , Perros , Humanos , Hipertensión Renal/tratamiento farmacológico , Macaca fascicularis , Masculino , Ratas , Renina/sangreRESUMEN
To understand the differences in the binding specificities within the aspartic proteinase family of enzymes, we have carried out studies to determine the inhibition constants of a set of related compounds with various members of the human enzyme family. The inhibition constants (Ki values) were determined by competitive inhibition of the hydrolysis of chromogenic octapeptide substrates in the pH range of 3-5. For comparison, inhibition of monkey renin was studied by RIA at pH 6.0. All inhibitors were based on the general structure 4-(morpholinylsulfonyl)-L-Phe-P2-(cyclohexyl)Ala psi[isostere]-P1'-P2'. The isosteric replacements of the scissile peptide bond included difluorohydroxyethylene, 1,2-diols, 1,3-diols, and difluoroketones. Side chain substituents in P2 include hydrogen, allyl, ethylthio, (methoxycarbonyl)methyl, N-methylthiouridobutyl, imidazolylmethyl, and 4-amino-2-thiazolylmethyl. Our measurements have identified potent and selective inhibitors which are useful in evaluating the differences in the specificities among selected enzymes of this family.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Dipéptidos/química , Morfolinas/química , Inhibidores de Proteasas/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Catepsina D/antagonistas & inhibidores , Catepsina D/metabolismo , Catepsina E , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Simulación por Computador , Dipéptidos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Morfolinas/metabolismo , Pepsina A/antagonistas & inhibidores , Pepsina A/metabolismo , Inhibidores de Proteasas/química , Renina/antagonistas & inhibidores , Renina/metabolismo , Relación Estructura-ActividadRESUMEN
A dissociation between changes in blood pressure (BP) and plasma renin activity (PRA) has been noted after administration of renin inhibitors. In the present study, the renin inhibitor PD 132002 was given to salt-deplete, anesthetized dogs. PRA was measured at pH 6.0 by a conventional angiotensin I (ANG I) RIA method (PRA-C) and by an ANG I antibody-trapping RIA method (PRA-AT) performed at pH 7.4. PD 132002 at 0.01, 0.1, 1, and 10 mg/kg IV, reduced BP by 3 +/- 2, 9 +/- 2, 24 +/- 4, and 39 +/- 4 mm Hg, respectively, (baseline of 136 +/- 8 mm Hg, N = 5), when infused IV over 30 minutes with a 30 minute recovery between doses. The BP response at 10 mg/kg equaled that of saralasin (20 micrograms/kg/min IV). PRA-AT (baseline of 20 +/- 6 ng ANG l/ml/hr, N = 4) was inhibited by 0%, 28% +/- 12%, 75% +/- 10%, and 97% +/- 1% at 0.01, 0.1, 1, and 10 mg/kg, respectively. Plasma concentrations of immunoreactive ANG II were also reduced dose-dependently and paralleled changes in BP. In contrast, PRA-C (baseline of 13 +/- 4 ng ANG l/ml/hr, N = 4) was inhibited by 82% +/- 8% at 0.01 mg/kg and by > 98% at higher doses. After a single dose of PD 132002 at 10 mg/kg infused over 30 minutes, BP recovery paralleled changes in immunoreactive ANG II and PRA-AT, yet PRA-C inhibition showed no recovery over the same time course. Our data support the conclusion that BP relates better to PRA-AT than PRA-C. Thus the dissociation sometimes observed in studies with renin inhibitors between changes in BP and PRA may be attributed to the assay used to determine PRA.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Dipéptidos/farmacología , Morfolinas/farmacología , Renina/antagonistas & inhibidores , Animales , Presión Sanguínea/fisiología , Dipéptidos/administración & dosificación , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Infusiones Intravenosas , Macaca fascicularis , Masculino , Morfolinas/administración & dosificación , Radioinmunoensayo/métodos , Ratas , Renina/sangreRESUMEN
A series of primate renin inhibitors containing difluorocarbinol and difluoroketone groups at the P1-P1' position have been synthesized and studied both in vitro and in vivo. In vitro, the compounds were evaluated as inhibitors of monkey renin and the closely related aspartic proteinase, cathepsin D (bovine), as a measure of enzyme selectivity. Interestingly, the difluoroketone derivatives showed greatly reduced selectivity compared with the corresponding alcohols. However, selectivity could be enhanced by judicious choice of other substituents. Sites influencing selectivity, included not only P2, which is well-known to strongly affect selectivity, but also the P4, P1-P1', and P2' sites. These results make possible the design of inhibitors with a greater selectivity for either renin versus cathepsin D. In vivo several of the compounds in the difluoroketone series have shown good oral activity in the salt depleted normotensive cynomolgus monkey model.