Non-thiazolidinedione antihyperglycaemic agents. Part 3: The effects of stereochemistry on the potency of alpha-methoxy-beta-phenylpropanoic acids.

Rhizopus delemar lipase catalysed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate 1 affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Absolute stereochemistry was determined by a single crystal X-ray analysis of a related synthetic analogue. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerisation of the (R)-(+) isomer. Binding studies using recombinant human PPARgamma (peroxisomal proliferator activated receptor gamma), now established as a molecular target for this compound class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.

CD23 (FcepsilonRII) release from cell membranes is mediated by a membrane-bound metalloprotease.

CD23 (low-affinity IgE receptor, FcepsilonRII) is expressed as a Type II extracellular protein on a variety of cells such as B-cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in the regulation of IgE synthesis. Here we report that the release of CD23 from the cell surface is mediated by a metalloprotease. An assay utilizing purified CD23 and an neo-epitope antibody specific for one of the known cleavage products is described and used to demonstrate unambiguously the cleavage of CD23 by a distinct protease. Characterization of the mechanism of CD23 processing shows that the protease exists as an integral membrane protein with a functional molecular mass of approx. 63 kDa as determined by gel-filtration chromatography. The CD23-cleaving activity found in enriched plasma membranes from RPMI 8866 cells is inhibited by the metalloprotease inhibitors 1, 10-phenanthroline and imidazole and by the matrix metalloprotease inhibitor batimastat, but not by inhibitors of cysteine proteases, serine proteases or acid proteases. The same or a similar activity that cleaves CD23 to the known 33 kDa fragment and is inhibited by batimastat is present in diverse cell types such as unstimulated fibroblasts and monocytic cell lines not expressing CD23, as well as in the Epstein-Barr virus-transformed B-cell line, RPMI 8866, which constitutively expresses CD23.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Hydroxamate-based inhibitors of low affinity IgE receptor (CD23) processing.

A series of hydroxamic acids related to the non-selective matrix metalloprotease inhibitor Batimastat is described, which inhibits the proteolytic cleavage of the low affinity IgE receptor from cell membrane preparations. Limited SAR studies suggest that the structural requirements for effective inhibition are distinct from those required for the inhibition of collagenase.

Selective inhibition of low affinity IgE receptor (CD23) processing.

A series of hydroxamic acids related to the non-selective matrix metalloproteinase inhibitor Batimastat has been prepared, some members of which are potent inhibitors of the processing of the low affinity IgE receptor (CD 23). Increased activity is obtained by appropriate substitution at the alpha-position, whilst selectivity is gained by use of a P1' benzyl group in conjunction with a C-terminal primary amide.

Protease Inhibition: Design, Technology & Opportunities--First RSC/SCI Symposium on Protease Inhibition.18-19 May 1998, London, UK.

This symposium, organized jointly by the Royal Society of Chemistry and the Society of Chemical Industry, aimed to distil current knowledge on the classification, structure and mechanism of proteolytic enzymes whilst focusing on the approaches and methodology being used to identify potent and selective inhibitors. A wide range of therapeutic targets covering all proteinase classes was presented to a largely medicinal chemical audience of around 130 participants from the UK and mainland Europe. There were no poster sessions.

Medicinal Chemistry--XVth EFMC International Symposium. New technologies in drug discovery. 6-10 September 1998, Edinburgh, Scotland, UK.

This report summarizes the new technologies in drug discovery session of a five-day symposium organized by the Royal Society of Chemistry, Perkin Division, and the Biological and Medicinal Chemistry Sector of the Industrial Affairs Division on behalf of the European Federation of Medicinal Chemistry. The objective of this international symposium, which was attended by over 1000 participants from 48 different countries, was to appeal to all scientists interested in drug discovery from lead identification to lead optimization. The session on new technologies focused on those tools currently being used in these processes, with particular emphasis on new developments. There were seven supporting sessions on specific molecular target classes and one on the prediction of drug metabolism and pharmacokinetics. In addition, there were over 350 supporting posters held in two separate sessions.

Glycochemistry and glycobiology.

This report summarizes the glycochemistry and glycobiology session held on the fourth day of this international symposium organized under the auspices of the European Federation of Medicinal Chemistry. The session objective was to highlight and emphasize the important and diverse functional roles played by glycoproteins and glycolipids and to demonstrate the advances that have been made to exploit their interactions in the development of new therapeutic agents.

Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer.

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc epsilonRII).

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Evaluation of the novel potassium channel activator BRL 55834 as an inhaled bronchodilator in guinea-pigs and rats: comparison with levcromakalim and salbutamol.

The novel potassium channel activator BRL 55834 and the prototype compound levcromakalim have been compared as inhaled bronchodilators in guinea-pigs and rats. Salbutamol was included in the guinea-pig studies. In anaesthetized guinea-pigs, inhaled BRL 55834 [ED50 = 0.9 (0.5-1.5) micrograms per animal] was equipotent with salbutamol and about tenfold more potent than levcromakalim as an inhibitor of the increase in airways resistance in response to iv histamine. In anaesthetized rats, BRL 55834 [ED50 = 0.5 (0.4-0.7) micrograms] was about eightfold more potent than levcromakalim in inhibiting the response to inhaled methacholine. BRL 55834 had no effect on blood pressure in anaesthetized guinea-pigs or rats, whereas levcromakalim lowered blood pressure in rats at a dose level that had less effect on the airways than one tenth of the highest dose of BRL 55834 used. In conscious guinea-pigs, BRL 55834 (ED100 = 5 micrograms) was twice as potent as levcromakalim and one sixth as potent as salbutamol in delaying the onset of dyspnoea in response to inhaled histamine. In each model each compound was effective at the earliest time studied, but the peak effect of BRL 55834 tended to be delayed and it was longer acting than levcromakalim or salbutamol. Thus inhaled BRL 55834 is a potent bronchodilator, with a rapid but prolonged duration of action that lacks significant systemic vascular activity.

Inhibition of cyclic nucleotide phosphodiesterase by derivatives of 1,3-bis(cyclopropylmethyl)xanthine.

Alkylation of the selective type IV phosphodiesterase inhibitor, 8-amino-1,3-bis(cyclopropylmethyl)-xanthine (1, BRL 61063), led exclusively to the N-7 substituted derivatives 2-9, which showed varying selectivities for the PDE type IV isoenzyme relative to PDE Va. The 4-methoxybenzyl derivative 6 in particular was a highly potent PDE Va inhibitor (IC50 0.14 microM) and showed a 24-fold selectivity for this isoenzyme relative to PDE IV. Sulfonation of 1 was more complex, with the product profile being highly dependent on the reaction conditions. As with alkylation, sulfonation at N-7 generally increased potency against PDE Va, especially in the aryl-containing moieties lacking strongly electron-withdrawing substituents (12, 15-17, 19). Bis-arylsulfonation at the exocyclic amino group generally reduced inhibitory potency against both PDE IV and Va. An 8-amidino compound 33, formed by the unusual reaction of 1 with N-methylpyrrolidinone in the presence of benzenesulfonyl chloride, had an IC50 value of 0.05 microM against PDE Va and is believed to be the most potent inhibitor of this isoenzyme reported. No correlation of PDE IV inhibition with displacement of [3H]rolipram from its high-affinity binding site was demonstrated. This suggests that either the catalytic site and the rolipram binding site are not the same or that PDE IV can exist in two conformations, only one of which binds to rolipram with high affinity, and that the compounds described vary in their selectivity for this isoform.

Comparison of the airways relaxant and hypotensive potencies of the potassium channel activators BRL 55834 and levcromakalim (BRL 38227) in vivo in guinea-pigs and rats.

1. BRL 55834, a novel potassium channel activator, has been compared with levcromakalim (BRL 38227) for its relaxant effects in vivo on the airways and vasculature of the guinea-pig and rat. 2. When administered intravenously 2 min prior to challenge, BRL 55834 and levcromakalim each inhibited histamine-induced increases in airways resistance (Raw) in the anaesthetized guinea-pig, with BRL 55834 showing a 4.5 fold greater potency than levcromakalim (ED25 = 2.5 micrograms kg-1 and 11.3 micrograms kg-1 respectively). By contrast, both compounds had similar hypotensive potencies (ED18 = 8.5 micrograms kg-1 and 6.5 micrograms kg-1 respectively). 3. In the same guinea-pig model, intraduodenally administered BRL 55834 (100 and 250 micrograms kg-1) and levcromakalim (500 micrograms kg-1) each protected against histamine-induced changes in Raw and dynamic lung compliance (Cdyn), both compounds showing a rapid onset of action that persisted for more than 50 min. The lower dose of BRL 55834 had a similar bronchodilator effect to that of levcromakalim, yet both doses of BRL 55834 elicited substantially smaller effects than levcromakalim on mean arterial blood pressure. 4. In the anaesthetized rat, BRL 55834 and levcromakalim each evoked a dose-related inhibition of inhaled methacholine-induced changes in Raw and Cdyn when given i.v., with BRL 55834 showing some four fold greater potency than levcromakalim (BRL 55834: Raw ED35 = 3.7 micrograms kg-1, Cdyn ED35 = 5.9 micrograms kg-1; levcromakalim: Raw ED35 = 16 micrograms kg-1, Cdyn ED35 = 23.5 micrograms kg-1). As in the guinea-pig,BRL 55834 had a reduced propensity to lower mean arterial blood pressure (ED11 = 8 microg kg-1 for BRL55834, 11 +/- 3% being its maximum effect; ED11= 16 microg kg-1, maximum effect= 34 +/- 6% for levcromakalim.5. When administered intraduodenally to anaesthetized rats, BRL 55834 (10, 20 and 100 microg kg-1)evoked rapid and dose-related inhibitions of methacholine-induced Raw and Cdyn changes which persisted for over 30 min. At the lower and middle dose there was little effect on mean arterial blood pressure(<10% fall). Levcromakalim (500 microg kg-1) by contrast elicited transient airways responses that diminished rapidly after 5 min, while the effects on blood pressure were well maintained (>20% at 65 min). Levcromakalim (100 microg kg-1) did not affect airways responses but also evoked a marked and sustained fall in blood pressure.6. BRL 55834, administered per os, prolonged the time to histamine-induced dyspnoea in conscious guinea-pigs. The greatest effect of BRL 55834 was observed when it was administered 60 min prior to challenge, a dose of 0.20 mg kg-1 doubling the mean time to collapse. A similar level of protection was afforded by levcromakalim (1.25 mg kg-1), with maximal activity occurring between 30 and 60 min.7. The present studies in guinea-pigs and rats indicate that BRL 55834 is the first potassium channel activator to exhibit greater bronchodilator potency than levcromakalim but reduced tendency to lower arterial blood pressure. It is suggested that BRL 55834 may have greater potential than levcromakalim as a bronchodilator for therapeutic use in man.

Relaxant effects of the potassium channel activators BRL 38227 and pinacidil on guinea-pig and human airway smooth muscle, and blockade of their effects by glibenclamide and BRL 31660.

The airways relaxant effects and mechanism of action of the potassium channel activators BRL 38227 and pinacidil have been compared in guinea-pig and human airways. BRL 38227 was a potent relaxant in guinea-pig isolated trachealis (IC50 = 4.9 x 10(-7) M against spontaneous tone) and human isolated bronchi (IC50 = 4.75 x 10(-7) M against histamine-induced tone) and was eight- and six-fold more potent respectively than pinacidil. The relaxant effects of both compounds were shown to be markedly attenuated by glibenclamide (10(-5) M) and BRL 31660 (10(-5) M), with the nature of the blockade being species/tissue dependent. Glibenclamide (20 mg/kg iv) also inhibited the protective effects of BRL 38227 (50 micrograms/kg iv) and pinacidil (500 micrograms/kg iv) on histamine-induced changes in airways resistance and dynamic compliance in the anaesthetized guinea-pig, although the effects were short-lived. That both BRL 38227 and pinacidil owed their relaxant effects to potassium channel activation was supported by their ability to stimulate 42/43K efflux from guinea-pig trachealis preloaded with the radiotracer at concentrations of 10(-7) - 10(-5) M and 10(-5) M respectively. Pretreatment with either glibenclamide (10(-5) M) or BRL 31660 (10(-5) M) ablated the response to both compounds. These studies show that two mechanistically distinct potassium channel blockers, glibenclamide and BRL 31660, do not substantially differentiate between the actions of BRL 38227 and pinacidil, although differences do occur, particularly at high concentrations in vitro.

The inhibitory effects of cromakalim and its active enantiomer BRL 38227 against various agonists in guinea pig and human airways: comparison with pinacidil and verapamil.

The effects of the potassium channel activators, cromakalim, BRL 38227 and pinacidil, and the calcium antagonist, verapamil, have been compared against various spasmogens on airway responses in vitro and in vivo in the guinea pig and also in human isolated bronchi. In guinea pig tracheal spirals, potassium channel activators generally had a greater inhibitory effect than verapamil against tone induced by a wide range of spasmogens (spontaneous, 5-hydroxytryptamine, leukotriene D4, prostaglandin E2). The potassium channel activators had very little effect against potassium chloride- and carbachol-induced tone in guinea pig tracheal spirals [e.g., cromakalim (20 microM) induced relaxations of 0.21 +/- 0.03 (relative to an isoprenaline maximum = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.77 +/- 0.03 against histamine]. In vivo, the potassium channel activators prevented histamine and 5-hydroxytryptamine-induced bronchoconstrictions, but had little inhibitory effect against acetylcholine. In contrast, in human bronchi, cromakalim was capable of inducing powerful concentration-dependent relaxations against carbachol-induced tone [cromakalim (20 microM) induced relaxations of 0.77 +/- 0.09 (relative to isoprenaline = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.95 +/- 0.04 against histamine]. In human bronchi, all the inhibitory agents were more potent and more effective, except that verapamil did not have an increased maximum response. We conclude that potassium channel activators should be effective at relaxing contractions induced by a wide range of spasmogens in man.

Relaxant activity of 6-cyano-2,2-dimethyl-2H-1-benzopyran-4-carboxamides and -thiocarboxamides and their analogues in guinea pig trachealis.

Structural modifications of the potassium channel activator cromakalim (1) are described in which the amide moiety at C-4 has been replaced by carboxamide and thiocarboxamide functions. Analogues in which the hydroxyl group at C-3 has been oxidized or removed are also disclosed. Such analogues display an interesting profile of smooth muscle relaxant activity in the guinea pig isolated trachea, not all of which appears to result from the opening of potassium channels, but few compounds retain useful in vivo activity. However, one compound in particular, 6-cyano-2,2-dimethyl-N-methyl-2H-1-benzopyran-4-thiocarboxamide (13) was shown to be a potent potassium channel activator in vitro and to provide prolonged protection to guinea pigs from the respiratory effects of inhaled histamine.

Synthesis and smooth muscle relaxant activity of a new series of potassium channel activators: 3-amido-1,1-dimethylindan-2-ols.

The synthesis of a novel series of smooth muscle relaxants which have been shown to act via the opening or activation of potassium channels is described. Compounds have been evaluated for their ability to inhibit spontaneous tone in guinea pig isolated trachealis and structure-activity relationships are discussed. One compound in particular, 1,1-dimethyl-5-nitro-3-(2-pyridon-1-yl)indan-2-ol, (16) was identified as a potent relaxant of airways smooth muscle in vitro with IC50 = 0.15 microM and was found to significantly inhibit histamine-induced dyspnoea in conscious guinea pigs when given orally 30-45 min prior to challenge.

Evaluation of the potassium channel activator BRL 38227 as an inhaled bronchodilator in the guinea-pig: contrast with nifedipine and salbutamol.

The potential of the potassium channel activator cromakalim and its active enantiomer BRL 38227 as inhaled bronchodilators has been evaluated in the guinea-pig, in comparison with nifedipine, salbutamol and aminophylline. Inhaled cromakalim and BRL 38227 prolonged the time before histamine-induced collapse in conscious guinea-pigs, BRL 38227 (ED50 250 to 500 micrograms/mL, roughly 10 to 20 micrograms per animal) being twice as potent as cromakalim. In anaesthetized guinea-pigs, BRL 38227 (inhaled and i.v.) and aminophylline (i.v.) caused similar percentage inhibitions of the increase in airways resistance and decrease in dynamic lung compliance elicited by histamine, whereas salbutamol (inhaled and i.v.) was more effective against resistance. Inhaled BRL 38227 and salbutamol were more potent against inhaled than against i.v. histamine. BRL 38227 inhibited the effects of i.v. and inhaled histamine by 67-78% when nebulized from solutions of 250 and 31 micrograms/mL respectively, but the lowest concentration that lowered blood pressure significantly was 500 micrograms/mL. In contrast, nifedipine had no effect on compliance and caused only a marginal (21%) inhibition of resistance at a dose (200 micrograms/kg i.v.) which lowered blood pressure by 44%. These results show that BRL 38227 is an effective bronchodilator when given by inhalation. It differs from salbutamol in its effects on airways dynamics, and its effect on lung compliance cannot be attributed to a pulmonary vasodilator effect. Furthermore, L-type calcium channels are not significantly involved in histamine-induced bronchoconstriction or therefore in the bronchodilator effect of BRL 38227.

The effects of BRL 38227 on antigen-induced extravasation and cellular infiltration into the peritoneal cavity of actively sensitised rats.

The ability of the potassium channel activator cromakalim to relax smooth muscle of the airways is now well established. The compound has been shown to be an effective bronchodilator in animal models, and more importantly, to attenuate induced bronchoconstriction and nocturnal asthma in man. Evidence now exists to suggest that BRL 38227, the 3S,4R enantiomer of cromakalim, is the predominantly active moiety and shares the same profile of activity. Further evaluation of BRL 38227 in a rat model of active peritoneal anaphylaxis provides evidence that potassium channel activators may exert beneficial effects in terms of inhibition of extravasation and inflammatory cell infiltration.