ABSTRACT
OBJECTIVE AND DESIGN: Hydroxamic-and carboxylic-acid based matrix metalloproteinase inhibitors (MMPIs) were compared for their potency against various MMPs, pharmacodynamic properties and in vivo efficacy in a model of cartilage degeneration. MATERIALS AND METHODS: The MMPIs were evaluated for their ability to inhibit human MMPs using the quenched fluorescence assay. The ability of the MMPIs to inhibit the degeneration of the knee joint was evaluated in rats injected intraarticularly with iodoacetate. The amount of MMPI in the plasma and cartilage was determined using liquid chromatography/mass spectrometry/mass spectrometry (LC/ MS/MS). Plasma protein binding was measured by ultrafiltration and unbound MMPI was quantitated using HPLC. RESULTS: The hydroxamic acid based inhibitor PGE-3321996 and the carboxylic acids PGE-2909492 and PGE-6292544 were potent MMP-13 inhibitors, but only the hydroxamic acid PGE 3321996 demonstrated significant inhibition of knee degeneration in the rat iodoacetate model. Both of the carboxylic acids demonstrated superior pharmacokinetic properties and established much higher plasma concentrations than the hydroxamic acid. However, neither of the carboxylic acids was detectable in the cartilage, whereas, the hydroxamic acid was present in both the cartilage and the plasma. The carboxylic acid based MMPIs also demonstrated higher plasma protein binding (>99%) than the hydroxamic acid (79%). CONCLUSIONS: Carboxylic acid-based MMPIs were identified that had superior in vivo plasma exposure compared to a hydroxamic acid inhibitor but lacked in vivo efficacy in the rat iodoacetate model of cartilage degeneration. The lack of in vivo efficacy of the carboxylic acid based MMPIs were probably due to their lack of cartilage penetration which was related to their physicochemical properties.
Subject(s)
Amino Acids/pharmacokinetics , Amino Acids/therapeutic use , Carboxylic Acids , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/therapeutic use , Matrix Metalloproteinase Inhibitors , Osteoarthritis/drug therapy , Phenylalanine/analogs & derivatives , Protease Inhibitors , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic use , Sulfones/pharmacokinetics , Sulfones/therapeutic use , Amino Acids/chemistry , Animals , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacokinetics , Carboxylic Acids/therapeutic use , Cartilage/chemistry , Cartilage/pathology , Disease Models, Animal , Humans , Hydroxamic Acids/chemistry , Iodoacetates/toxicity , Knee Joint/pathology , Male , Molecular Structure , Osteoarthritis/chemically induced , Osteoarthritis/pathology , Phenylalanine/chemistry , Phenylalanine/pharmacokinetics , Phenylalanine/therapeutic use , Plasma/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Protease Inhibitors/therapeutic use , Rats , Rats, Sprague-Dawley , Sulfonamides/chemistry , Sulfones/chemistryABSTRACT
A series of carboxylic acids was prepared based on cyclohexylglycine scaffolds and tested for potency as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors such as compound 18 display low nanomolar potency for MMP-2 and MMP-13, while selectively sparing MMP-1 and MMP-7.
Subject(s)
Enzyme Inhibitors/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Animals , Carboxylic Acids/chemical synthesis , Carboxylic Acids/metabolism , Carboxylic Acids/pharmacology , Collagenases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7/metabolism , Metalloendopeptidases/metabolism , Protein Binding/physiology , Rats , Structure-Activity RelationshipSubject(s)
Biphenyl Compounds/chemical synthesis , Carboxylic Acids/chemical synthesis , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , Biphenyl Compounds/chemistry , Blood Proteins/chemistry , Carboxylic Acids/chemistry , Protease Inhibitors/chemistry , Solubility , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Potent and selective inhibition of matrix metalloproteinases was demonstrated for a series of sulfonamide-based hydroxamic acids. The design of the heterocyclic sulfonamides incorporates a six- or seven-member central ring with a P2' substituent that can be modified. Binding interactions of this substituent at the S2' site are believed to contribute to high inhibitory potency against stromelysin, collagenase-3 and gelatinases A and B, and to provide selectivity against collagenase-1 and matrilysin. An X-ray structure of a stromelysin inhibitor complex was obtained to provide insights into the SAR and selectivity trends observed for the series.
Subject(s)
Heterocyclic Compounds, 1-Ring/pharmacology , Matrix Metalloproteinase Inhibitors , Sulfonamides/pharmacology , Collagenases , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 1-Ring/chemical synthesis , Inhibitory Concentration 50 , Macromolecular Substances , Matrix Metalloproteinase 13 , Sensitivity and Specificity , Structure-Activity Relationship , Sulfonamides/chemical synthesisABSTRACT
A series of carboxylic acids were prepared from a propargylglycine scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for four enzymes within the MMP family. The inhibitors were typically potent against collagenase-3 (MMP-13) and gelatinase A (MMP-2), while they spared collagenase-1 (MMP-1) and only moderately inhibited stromelysin (MMP-3). Compound 40 represents a typical inhibition profile of a compound with reasonable potency. Introduction of polar groups was required in order to generate inhibitors with acceptable water solubility, and this often resulted in a loss of potency as in compound 63. High serum protein binding proved to be a difficult hurdle with many compounds such as 48 showing >99% binding. Some compounds such as 64 displayed approximately 90% binding, but no reliable method was discovered for designing molecules with low protein binding. Finally, selected data regarding the pharmacokinetic behavior of these compounds is presented.
Subject(s)
Alkynes/chemical synthesis , Carboxylic Acids/chemical synthesis , Glycine/analogs & derivatives , Glycine/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Alkynes/chemistry , Carboxylic Acids/chemistry , Glycine/chemistry , Humans , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/chemistry , Models, Molecular , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The synthesis of homochiral functionalized cyclohexylglycines and alpha-methylcyclohexylglycines via chelated Kazmaier-Claisen rearrangement is described. These were shown to be potent scaffolds for the development of MMP inhibitors.
Subject(s)
Glycine/analogs & derivatives , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , Glycine/chemical synthesis , Glycine/chemistry , Glycine/pharmacology , Humans , Molecular Conformation , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacologyABSTRACT
OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) inhibitors in mono-iodoacetate-induced arthritis in rats. DESIGN: The ability of compounds to inhibit MMPs in vitro was assessed kinetically using a quenched fluorescent substrate. Rats were injected with iodoacetate intraarticularly in one knee joint and damage to the tibial plateau was evaluated from digitized images captured using an image analyser and by histology. Collagenase and gelatinase activity in cartilage from iodoacetate injected knees were evaluated using(3)H-rat type I collagen and gelatin zymography, respectively. RESULTS: Collagenase and gelatinase activity significantly increased in the knee cartilage of rats injected with iodoacetate with peak activity by day 7. Three MMP inhibitors were examined for their efficacy in the rat iodoacetate-induced arthritis model. Significant (P< 0.05) inhibition of cartilage damage was observed in animals treated orally with 35 mg/kg b.i.d. of the three different MMP inhibitors. Inhibition of cartilage damage by the MMP inhibitors ranged from 36-42%. CONCLUSION: MMP inhibitors are partially protective against cartilage and subchondral bone damage induced by iodoacetate. These results support an important role for MMPs in mediating the joint damage in this model of arthritis.
Subject(s)
Enzyme Inhibitors/therapeutic use , Matrix Metalloproteinase Inhibitors , Osteoarthritis/drug therapy , Animals , Collagenases/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Gelatinases/metabolism , Image Processing, Computer-Assisted/methods , Injections, Intra-Articular , Iodoacetates/administration & dosage , Iodoacetates/adverse effects , Male , Osteoarthritis/chemically induced , Osteoarthritis/pathology , Rats , Rats, Sprague-Dawley , Up-RegulationABSTRACT
Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.
Subject(s)
Cartilage/chemistry , Protease Inhibitors/analysis , Animals , Autoanalysis , Chromatography, High Pressure Liquid , Mass Spectrometry , Protease Inhibitors/blood , RatsABSTRACT
A new generation of cyclic matrix metalloproteinase (MMP) inhibitors derived from dl-piperazinecarboxylic acid has been described. The design involves: incorporation of hydroxamic acid as the bidentate chelating agent for catalytic Zn(2+), placement of a sulfonamide group at the 1N-position of the piperazine ring to fill the S1' pocket of the enzyme, and finally attachment of diverse functional groups at the 4N-position to optimize potency and peroral absorption. A unique combination of all three elements produced inhibitor 20 with high affinity for MMPs 1, 3, 9, and 13 (24, 18, 1.9, and 1.3 nM, respectively). X-ray crystallography data obtained for MMP-3 cocrystallized with 20 gave detailed information on key binding interactions defining an overall scaffold geometry for piperazine-based MMP inhibitors.
Subject(s)
Metalloendopeptidases/antagonists & inhibitors , Piperazines/chemical synthesis , Protease Inhibitors/chemical synthesis , Sulfonamides/chemical synthesis , Animals , Cartilage/cytology , Cartilage/drug effects , Cattle , Cells, Cultured , Crystallography, X-Ray , Drug Design , Models, Molecular , Piperazines/chemistry , Piperazines/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Recombinant Proteins/antagonists & inhibitors , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
A series of hydroxamates was prepared from an aminoproline scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors, such as compound 47, display broad-spectrum activity with sub-nanomolar potency for some enzymes. Modifications of the P1' portion of the molecule played a key role in affecting both potency and selectivity within the MMP family. Longer-chain aliphatic substituents in this region of the molecule tended to increase potency for MMP-3 and decrease potency for MMP-1, as exemplified by compounds 48-50, while aromatic substituents, as in compound 52, generated broad-spectrum inhibition. The data is rationalized based upon X-ray crystal data which is also presented. While the in vitro peroral absorption seemed to be less predictable, it tended to decrease with longer and more hydrophilic substituents. Finally, a rat model of osteoarthritis was used to evaluate the efficacy of these compounds, and a direct link was established between their pharmacokinetics and their in vivo efficacy.
Subject(s)
Hydroxamic Acids/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Proline/analogs & derivatives , Proline/chemical synthesis , Protease Inhibitors/chemical synthesis , Animals , Cartilage, Articular/pathology , Crystallography, X-Ray , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Iodoacetates , Male , Matrix Metalloproteinase 3/chemistry , Models, Molecular , Osteoarthritis, Knee/chemically induced , Osteoarthritis, Knee/drug therapy , Osteoarthritis, Knee/pathology , Proline/chemistry , Proline/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
The synthesis and enzyme inhibition data for a series of thiazine- and thiazepine-based matrix metalloproteinase (MMP) inhibitors are described. The thiazine- and thiazepine-based inhibitors were discovered by optimization of hetererocyclic sulfonamide-based inhibitors. The most potent series of inhibitors was obtained by modification of the amino acid D-penicillamine. This amino acid provides a gem-dimethyl group on the thiazine or thiazepine ring which has a dramatic effect on the in vitro potency of this series. In particular, the sulfide 4a and the sulfone 5a were potent, broad-spectrum inhibitors of the MMPs with IC(50)'s against MMP-1 of 0.8 and 1.9 nM, respectively. The binding mode of this novel thiazepine-based series of MMP inhibitors was established based on X-ray crystallography of the complex of stromelysin and 4a.
Subject(s)
Cyclic S-Oxides/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Thiazepines/chemical synthesis , Thiazines/chemical synthesis , Crystallography, X-Ray , Cyclic S-Oxides/chemistry , Drug Design , Enzyme Inhibitors/chemistry , Models, Molecular , Structure-Activity Relationship , Thiazepines/chemistry , Thiazines/chemistryABSTRACT
Since their inception during the eighties, MMP inhibitors (MMPIs) have gone through several cycles of metamorphosis. The design of early MMPIs was based on the cleavage site of peptide substrates. The second generation contained a substituted succinate scaffold (e.g., marimastat) coupled to a modified amino acid residue. The lower molecular weight analogs with multiple substitution possibilities produced a series of MMP inhibitors with varying degrees of selectivity for various MMPs. The introduction of sulfonamides in the midnineties added a new dimension to this field. The simplicity of synthesis coupled with high potency (e.g., CGS-27023A, AG-3340) produced a number of clinical candidates. This review highlights some of the key features that contributed to the discovery of this novel series of MMP inhibitors.
Subject(s)
Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Caprolactam/chemical synthesis , Caprolactam/chemistry , Caprolactam/pharmacology , Drug Design , Extracellular Matrix/enzymology , Metalloendopeptidases/chemistry , Phosphinic Acids/chemical synthesis , Phosphinic Acids/chemistry , Phosphinic Acids/pharmacology , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacologyABSTRACT
A new series of hydroxamic acid-based matrix metalloproteinase (MMP) inhibitors containing a unique phosphinamide motif derived from D-amino acid was designed, synthesized, and tested for enzyme inhibition. Compounds with an R configuration at phosphorus were found to be potent MMP inhibitors while molecules with the S configuration were almost inactive. Structure-activity relationship studies of the series led to the discovery of the potent inhibitor 16 with IC50 = 20.5 nM and 24.4 nM against fibroblast collagenase (MMP-1) and stromelysin (MMP-3), respectively. The binding mode of this novel phosphinamide-based series of MMP inhibitors was established based on X-ray crystallography of the complex of stromelysin and 16.
Subject(s)
Hydroxamic Acids/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Organophosphorus Compounds/chemical synthesis , Protease Inhibitors/chemical synthesis , Binding Sites , Crystallography, X-Ray , Drug Design , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/metabolism , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Models, Molecular , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Organophosphorus Compounds/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Recombinant Proteins/antagonists & inhibitors , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The synthesis and structure-activity relationship (SAR) studies of a series of proline-based matrix metalloproteinase inhibitors are described. The data reveal a remarkable potency enhancement in those compounds that contain an sp(2) center at the C-4 carbon of the ring relative to similar, saturated compounds. This effect was noted in compounds that contained a functionalized oxime moiety or an exomethylene at C-4, and the potencies were typically <10 nM for MMP-3 and <100 nM for MMP-1. Comparisons were then made against compounds with similar functionality where the C-4 carbon was reduced to sp(3) hybridization and the effect was typically an order of magnitude loss in potency. A comparison of compounds 14 and 34 exemplifies this observation. An X-ray structure was obtained for a stromelysin-inhibitor complex which provided insights into the SAR and selectivity trends observed within the series. In vitro intestinal permeability data for many compounds was also accumulated.
Subject(s)
Matrix Metalloproteinase Inhibitors , Proline/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Animals , Ileum/drug effects , Ileum/metabolism , In Vitro Techniques , Intestinal Absorption , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Protease Inhibitors/chemical synthesis , Rats , Structure-Activity RelationshipSubject(s)
Metalloendopeptidases/antagonists & inhibitors , Piperazines/chemical synthesis , Crystallography, X-Ray , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/metabolism , Models, Molecular , Molecular Conformation , Piperazines/chemistry , Piperazines/metabolism , Piperazines/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity RelationshipABSTRACT
A novel series of conformationally constrained matrix metalloprotease inhibitors was identified. The potencies observed for these inhibitors were highly dependent upon the substitution pattern on the caprolactam ring as well as the succinate moiety.
