Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer beta-amyloid fibril formation.

One of the major pathological features of Alzheimer's disease is the deposition of beta-amyloid peptide (Abeta). Cellular toxicity has been shown to be associated with fibrillar forms of Abeta; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Abeta fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Abeta fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Abeta and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans.

A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the beta-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to beta-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to beta-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the beta-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on beta-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils.

In vivo model of cartilage degradation--effects of a matrix metalloproteinase inhibitor.

OBJECTIVES: To develop a model of cartilage degradation that (i) enables the testing of synthetic, small molecular weight matrix metalloproteinase (MMP) inhibitors as agents to prevent cartilage erosion, (ii) permits the direct assay of the principal constituents of the extracellular matrix (collagen and proteoglycan) in both the non-calcified articular cartilage and the calcified cartilage compartments, and (iii) is mediated by a chronic, granulomatous tissue that closely apposes intact articular cartilage, and in this respect resembles the pannus-cartilage junction of rheumatoid arthritis. METHODS: Femoral head cartilage was obtained from donor rats, wrapped in cotton and implanted subcutaneously into recipient animals. After a two stage papain digestion procedure, the proteoglycan and collagen contents were measured by assaying for glycosaminoglycans and hydroxyproline, respectively, in both the non-calcified cartilage that comprises the articular surface layer and the calcified cartilage compartment. The incorporation in vitro of [35S]-sulphate into glycosaminoglycans was assayed as a measure of proteoglycan biosynthesis. An osmotic minipump was cannulated to the implanted femoral head cartilage and synthetic MMP inhibitors (MI-1 and MI-2) were infused continuously over a 14 day period. RESULTS: The implanted, cotton wrapped femoral head cartilages provoked a granulomatous response that resulted in the removal of collagen and proteoglycan from the cartilage matrix. The removal of proteoglycan and collagen was exclusively from the non-calcified articular cartilage, whereas the proteoglycan and collagen content of the calcified compartment increased during the experiments. MI-1 reproducibly reduced the degradation of proteoglycan and collagen in implanted femoral head cartilage. CONCLUSIONS: We have described an in vivo model of cartilage degradation that permits the measurement of proteoglycan and collagen in both non-calcified articular cartilage and calcified cartilage compartments. The model can be used to test the effects of agents of unknown systemic bioavailability and pharmacokinetic profile by infusing them directly to the site of cartilage degradation. The removal of cartilage extracellular matrix by granulomatous tissue was inhibited by an MMP inhibitor, thus proving the involvement of this family of proteinases in cartilage catabolism in this model.

A simple in vivo model of collagen degradation using collagen-gelled cotton buds: the effects of collagenase inhibitors and other agents.

A simple in vivo model of collagen degradation has been developed, and the effects of various agents have been tested. Type I collagen was prepared from rat skin and acetylated with either [3H]- or [14C] acetic anhydride. The radiolabelled collagen was added to sterile cotton buds and incubated at 37 degrees C to allow the collagen to form native fibrils that were firmly adsorbed to the cotton matrix. After subcutaneous implantation of the collagen-gelled cotton buds into rats, the radiolabelled collagen was progressively removed over a period of weeks by an infiltrating granuloma. Of the agents that were administered directly into the cotton buds using subcutaneously implanted osmotic mini-pumps, only the synthetic collagenase inhibitors CI-A (containing a hydroxamate moiety as a zinc ligand) and CI-C (containing a thiol moiety as a zinc ligand) were able to prevent the removal of collagen: their efficacy correlated with the level of collagenase inhibitory activity assayed in the exudate fluid sequestered within the cotton bud granuloma. Of the agents that were administered systemically, including anti-inflammatory drugs and other compounds used as therapies for arthritis, only hydrocortisone was able to inhibit the removal of radiolabelled collagen. These results suggest that, in this model, interstitial collagenase, a member of the matrix metalloproteinase family, comprised the major degradative pathway for collagen. The collagen-gelled cotton bud model is a useful test system for delineating those processes that result in collagen catabolism. In addition, the model can be used for testing agents, including those of limited or unknown systemic bioavailability, in order to discover novel therapeutic agents for preventing collagen degradation in connective tissue diseases such as arthritis.

Synthesis of novel N-phosphonoalkyl dipeptide inhibitors of human collagenase.

The synthesis of a series of N-phosphonalkyl dipeptides 6 is described. Syntheses were devised that allowed the preparation of single diastereoisomers and the assignment of stereochemistry. The compounds were evaluated in vitro for their ability to inhibit the degradation of radiolabeled collagen by purified human lung fibroblast collagenase. Several of the compounds were potent collagenase inhibitors and were at least 10-fold more potent than their corresponding N-carboxyalkyl analogues. Activity was lost when the phosphonic acid group P(O)(OH)2 was replaced by the phosphinic acid groups P(O)(H)(OH) and P(O)(Me)(OH). At the P1 position, (R)- or (S)-alkyl groups, especially ethyl and methyl (e.g., 12a,b, 52a,b, and 53a,b), or an (R)-phenethyl moiety (55a) conferred high potency (IC50 values in the range 0.23-0.47 microM). (S)-Stereochemistry was preferred for the P1' isobutyl side chain. Structure-activity relationships were also investigated at the P2' site, and interestingly, compounds with basic side chains, such as the guanidine 57a, were equipotent with more lipophilic compounds, such as 52a. As with other series of collagenase inhibitors, potency was enhanced by introducing bicyclic aromatic P2' substituents. The most potent phosphonic acid of the series was the bicyclic aromatic P2' tryptophan analogue 59a (IC50 0.05 microM).

Synthesis of novel modified dipeptide inhibitors of human collagenase: beta-mercapto carboxylic acid derivatives.

The synthesis of a series of thiol-containing, modified dipeptide inhibitors (8) of human collagenase, which incorporate various carboxylic acid derivatives at the presumed P1 position, beta to the thiol group, is described. The compounds were evaluated, in vitro, for their ability to inhibit the degradation of rat skin type 1 collagen by purified human lung fibroblast collagenase, and structure-activity relationship studies are described. Optimum potency (IC50 values in the nanomolar range) was achieved by incorporating methyl (compounds 43a, 56a, and 57ab) or benzyl esters (44a) at the P1 position. Small amides were also accommodated (e.g. primary amide 47a), but in general, increasing the size of the P1 amide substituent lowered potency. PheNHMe, TrpNHMe, and Tyr(Me)NHMe substituents were found to be approximately equipotent P2'-residues. The results of testing all four diastereoisomers 56a-d of the compound with (S)-TrpNHMe at the P2' position indicated that the S,S,S diastereoisomer 56a possessed highest potency (IC50 2.5 nM) and that the second most potent diastereoisomer was 56d (IC50 12 nM) with the R,R,S configuration. It appeared that the orientation of the P1' and the thiol-bearing centers to each other is a more critical influence on potency than any absolute stereochemical requirements. It is suggested that the high potency of the beta-mercapto carboxylic acid derivatives may be a consequence of bidentate coordination of the thiol and carbonyl groups to the active-site zinc ion in the collagenase enzyme.

Antihypertensive and angiotensin converting enzyme inhibitory activities of a novel dihydrobenzofuran analogue.

1. The biochemical and pharmacological profiles of the novel, orally active angiotensin converting enzyme (ACE) inhibitor, N-[N-[[4-(2, 3-dihydro-2-benzofuranyl)-1-(ethoxycarbonyl)]butyl]-(s)-alanyl]- (s)-proline (BRL 36378), have been compared with those of enalapril and captopril. 2. In the conscious sodium deficient spontaneously hypertensive rat, BRL 36378 and enalapril (0.3-10 mg/kg orally) produced comparable falls in blood pressure; at 3 mg/kg orally, captopril was less active than BRL 36378 and enalapril. 3. In the anaesthetised spontaneously hypertensive rat, enalapril was slightly more potent than BRL 36378 as an inhibitor of angiotensin I (AI) pressor responses whilst BRL 36378 was about twice as potent as captopril in this test (i.v. route used). BRL 36378 and enalapril were equipotent as potentiators of bradykinin depressor responses. 4. In the anaesthetised Wistar rat, the maximum inhibition of AI pressor responses by 0.1 microgram/kg i.v. BRL 36378 and captopril was achieved sooner than after the same dose of enalapril. The inhibitory effect of captopril subsided completely by 40-50 min but the maximum effects of BRL 36378 and enalapril persisted for at least 60 min. 5. In the conscious renal hypertensive cat, captopril was slightly more potent than BRL 36378 or enalapril as a blood pressure lowering agent, over 1-10 mg/kg orally. BRL 36378 was more potent than enalapril as an inhibitor of AI induced pressor responses in this model. Captopril possessed similar inhibitory activity to BRL 36378 although minor differences in time course were apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel 1H-benzimidazol-4-ols with potent 5-lipoxygenase inhibitory activity.

The synthesis and structure--activity profile of 2-substituted benzimidazol-4-ols as inhibitors of cell-free RBL-1 5-lipoxygenase are discussed, and their potency is compared with that of the standard inhibitors phenidone, AA 861, BW 755C, and nordihydroguaiaretic acid. In contrast to the standard compounds, most did not inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vivo when administered at 200 microM ip to rats subjected to peritoneal anaphylaxis, although five compounds containing a methoxylated benzyl group (compounds 36, 39, 42, and 43) or hydroxylated benzyl group (41) showed similar activity to that of phenidone, nordihydroguaiaretic acid, and AA 861. Of the many compounds tested, two, 5-tert-butyl-7-methyl-2-(trifluoromethyl)-1H-benzimidazol-4-ol (57) and 2-(4-methoxybenzyl)-7-methyl-1H-benzimidazol-4-ol (36), like dexamethasone, inhibited monocyte accumulation in a pleural exudate model of inflammation. Standard lipoxygenase inhibitors such as phenidone, BW 755C, and AA 861 were inactive in this system.

7-Aroyl-2,3-dihydrobenzo[b]furan-3-carboxylic acids and 7-benzoyl-2,3-dihydrobenzo[b]thiophene-3-carboxylic acids as analgesic agents.

The synthesis of a series of 7-aroyl-2,3-dihydrobenzo[b]furan-3-carboxylic acids and 7-benzoyl-2,3-dihydrobenzo[b]thiophene-3-carboxylic acids is described. The isomeric 4-benzoyl-1,3-dihydrobenzo[c]furan-1-carboxylic acid was also prepared. Compounds were evaluated for analgesic activity in the mouse phenyl-p-quinone-induced writhing test. Selected compounds were tested for their ability to produce gastric damage in fasted mice and for inhibition of prostaglandin synthetase activity in vitro. Zomepirac was used as a reference. Structure-activity relationships are discussed. One of the compounds, 7-benzoyl-5-chloro-2,3-dihydrobenzo[b]furan-3-carboxylic acid (2c), combined potent analgesic activity with low gastric irritancy.