Matrix metalloproteinase-3 differences in oral and skin fibroblasts.

While skin wounds heal by scarring, wounds of oral mucosa show privileged healing with minimal scar formation. Our hypothesis was that phenotypic differences between oral and skin fibroblasts underlie these differences in healing. The aims of this study were to compare MMP-3 expression by oral and skin fibroblasts and investigate a role for MMP-3 in mediating collagen gel contraction. Oral fibroblasts induced significantly greater gel contraction than did paired skin cells. Inhibition of MMP activity significantly inhibited gel contraction by both cell types. Specific inhibition of MMP-3 activity reduced gel contraction by oral, but not skin, fibroblasts. Oral fibroblasts produced significantly higher levels of MMP-3 than did skin fibroblasts at all levels studied. TGF-beta1 and -beta3 isoforms stimulated MMP-3 expression at mRNA, protein, and activity levels by both fibroblast populations. Results suggest that increased MMP-3 production by oral fibroblasts may underlie the differences in wound-healing outcome seen in skin and oral mucosa.

Structure-activity relationships of 1,4-dihydro-(1H,4H)-quinoxaline-2,3-diones as N-methyl-D-aspartate (glycine site) receptor antagonists. 1. Heterocyclic substituted 5-alkyl derivatives.

A series of 6,7-dichloro-1,4-dihydro-(1H, 4H)-quinoxaline-2,3-diones (1-17) were prepared in which the 5-position substituent was a heterocyclylmethyl or 1-(heterocyclyl)-1-propyl group. Structure-activity relationships were evaluated where binding affinity for the glycine site of the N-methyl-D-aspartate (NMDA) receptor was measured using the specific radioligand [3H]-L-689,560, and functional antagonism was demonstrated by inhibition of NMDA-induced depolarizations of rat cortical wedges. The ability to prevent NMDA-induced hyperlocomotion in mice in vivo was measured for selected compounds. Binding affinity increased significantly if the heterocyclic group, e.g. 1,2,3-triazol-1-yl could participate in accepting a hydrogen bond from the receptor. It was difficult to obtain compounds with adequate aqueous solubility and strategies to improve it were investigated. The most potent compound in this series, 6,7-dichloro-5-[1-(1,2,4-triazol-4-yl)propyl]-1,4-dihydro-(1H, 4H)-quinoxaline-2,3-dione (17) (binding IC50 = 2.6 nM; cortical wedge EC50 = 90 nM), inhibited NMDA-induced hyperlocomotion in mice (6/9 protected at 20 mg/kg iv). Pharmacokinetic parameters, including extent of brain penetration, for 11 and 17 are reported.

Selectivity of inhibition of matrix metalloproteases MMP-3 and MMP-2 by succinyl hydroxamates and their carboxylic acid analogues is dependent on P3' group chirality.

Structure-activity relationships are described for a series of succinyl hydroxamic acids 1a-o and their carboxylic acid analogues 2a-o as inhibitors of matrix metalloproteases MMP-3 and MMP-2. For this series (P1' = (CH2)3Ph, P2' = t-Bu) selectivity for the inhibition of MMP-2 was found to be strongly dependent on P3'.

Discovery of potent and selective succinyl hydroxamate inhibitors of matrix metalloprotease-3 (stromelysin-1).

Structure activity relationships are described for a series of succinyl hydroxamic acids 4a-o as potent and selective inhibitors of matrix metalloprotease-3 (stromelysin-1). Optimisation of P1' and P3' groups gave compound 4j (MMP-3 IC50=5.9nM) which was >140-fold less potent against MMP-1 (IC50=51,000nM), MMP-2 (IC50=1790nM), MMP-9 (IC50=840nM) and MMP-14 (IC50=1900nM).

Novel antagonists of platelet-activating factor. 1. Synthesis and structure-activity relationships of benzodiazepine and benzazepine derivatives of 2-methyl-1-phenylimidazo[4,5-c]pyridine.

Following the discovery of moderately potent antagonist activity platelet-activating factor (PAF) in 2-methyl-1-phenylimidazo[4,5-c]pyridine (2) (IC50 = 840 nM), 19 derivatives (3-21) were prepared which incorporated various lipophilic groups attached to the phenyl 4-position. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit the PAF-induced aggregation of rabbit washed platelets and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. [1,5]Benzodiazepines, e.g., 14 (2,3-dihydro-1-methyl-4-[4-(2-methylimidazo[4,5-c] pyrid-1-yl)phenyl]-1H-[1,5]benzodiazepin-2-one) (IC50 = 4.9 nM, Ed50 = 0.03 mg/kg po), were found to possess equivalent or superior potency to the 1,4-dihydropyridine PAF antagonist UK-74,505 (1,4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl-2- [4-(2-methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2-pyridyl) carbamoyl]pyridine) in vitro and in vivo. Furthermore, a potent benzazepine, 21 (7,8-dichloro-1-methyl-4-[4-(methylimidazo[4,5-c]pyrid-1-yl) phenyl]-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one) (IC50 = 0.5 nM, ED50 = 0.03 mg/kg po), was discovered. These investigations prompted the synthesis and evaluation of additional diazepine derivatives, which are described in the following paper. The relationship between the key PAF antagonist pharmacophores of 2-methyl-1-phenylimidazo[4,5-c]pyridine, a triazolothienodiazepine (WEB2170), and a pyrrolothiazolidine (RP-52,770) is discussed.

Novel antagonists of platelet-activating factor. 2. Synthesis and structure-activity relationships of potent and long-acting heterofused [1,5]benzodiazepine and [1,4]diazepine derivatives of 2-methyl-1-phenylimidazo[4,5-c]pyridine.

The optimization of in vitro activity and oral potency and duration of action in vivo is described for three novel structural types of platelet-activating factor (PAF) antagonist: [1,5]benzodiazepines 5-12 onto which a variety of other heterocyclic rings were fused, pyrido[2,3-b][1,4]-diazepinones 13-26, and pyrazolo[3,4-b][1,4]diazepinones 27-46. Compounds 5-12 were prepared by elaboration of the [1,5]benzodiazepine-2-thiones 47 and 48, and 13-46 were prepared by cyclocondensation reactions of a variety of 2,3-diaminopyridine and 4,5-diaminopyrazole derivatives with ethyl 4'-(2-methylimidazo[4,5-c] pyrid-1-yl)benzoylacetate (53). The presence of imine-enamine tautomerism was observed in certain diazepine derivatives and is discussed. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit PAF-induced aggregation of rabbit washed platelets and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. In addition, the duration of action in conscious dogs as measured by determining the oral dose of selected compounds required to inhibit completely PAF-induced whole blood aggregation ex vivo. The most potent compound was 1,6,7,8-tetrahydro-1,8-dimethyl-5-[4-(2-methylimidazo [4,5-c]pyrid-1-yl)phenyl]-7-oxo-3-(3-pyridyl) pyrazolo[3,4-b][1,4]diazepine (43, UK-91,473) (IC50 = 2.4 nM, ED50 = 0.01 mg/kg po), which was found to be significantly more potent in vivo (murine lethality) than the dihydropyridine PAF antagonist 4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl- 4-[(2-methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5- [N-(2-pyridyl)carbamoyl]pyridine (4, UK-74,505) (ED50 = 0.26 mg/kg po). Compound 43 also possessed a longer duration of action than compound 4 in the conscious dog at one-fourth of the dose. The crystal structure of compound 43, established by X-ray diffraction, is reported.

1,4-Dihydropyridines as antagonists of platelet activating factor. 1. Synthesis and structure-activity relationships of 2-(4-heterocyclyl)phenyl derivatives.

A novel class of 2-(4-heterocyclylphenyl)-1,4-dihydropyridines (2-38) possessing antagonist activity against platelet activating factor (PAF) was prepared by the Hantzsch synthesis from a variety of ethyl 4'-heterocyclic-substituted benzoylacetates, aryl or heteroaryl aldehydes, and substituted 3-aminocrotonamides or 3-aminocrotonate esters. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit the PAF-induced aggregation of rabbit washed platelets, and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. The nature of the substituent at the dihydropyridine 2-position was found to be important for both in vitro and in vivo activity, whereas there was greater flexibility for structural variation at the 4- and 5-positions. The most potent compound was 4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl-2-[4-(2- methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2- pyridyl)carbamoyl]pyridine (17, UK-74,505), IC50 = 4.3 nM, ED50 = 0.26 mg/kg po, which was found to be approximately 33 times more potent in vitro (rabbit platelet aggregation) and about 8 times more potent in vivo (murine lethality) than WEB2086. Compound 17 also exhibited a long duration of action in the dog (inhibition of PAF-induced whole blood aggregation ex vivo was maintained for greater than 24 h following a single oral dose of 75 micrograms/kg) and was highly selective as a PAF antagonist, showing only weak affinity (IC50 = 6600 nM) for the [3H]nitrendipine binding site. As a result of its high oral potency, selectivity, and duration of action, UK-74,505 has been selected for clinical evaluation.