The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams.

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.

Synthesis of tetrasubstituted bicyclo[3.2.1]octenes as potential inhibitors of influenza virus sialidase.

Several racemic bicyclo[3.2.1]octene derivatives have been synthesised and evaluated as inhibitors of influenza virus sialidases. The 5-acetamido-bicyclo[3.2.1]octenol 4 showed modest activity against influenza A and B virus sialidases.

Efficient syntheses of (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine; a nucleoside analogue with potent biological activity.

(E)-5-(2-Bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) is a potent antiherpesvirus agent and its use in gene therapy as an anticancer agent has recently been described. We here outline 2 efficient methods for the synthesis of S-BVDU. The decision as to which method is to be used depends upon the starting materials available but starting from BVU, an overall yield of beta-nucleoside of 35% can be expected. From 5-ethyl-2'-deoxy-4'-thiouridine, radical bromination using bromine will give a quantitative conversion to S-BVDU if unreacted starting material is recycled (50%) or using N-bromosuccinimide, a one step yield in excess of 80% can be obtained.

Synthesis and anti-herpes virus activity of 2'-deoxy-4'-thiopyrimidine nucleosides.

A series of 5-substituted 2'-deoxy-4'-thiopyrimidine nucleosides was synthesized and evaluated as potential antiviral agents. A number of analogues such as 2'-deoxy-5-propyl-4'-thiouridine (3ii), 2'-deoxy-5-isopropyl-4'-thiouridine (3iii), 5-cyclopropyl-2'-deoxy-4'-thiouridine (3iv), 2'-deoxy-4'-thio-5-vinyluridine (3viii), and 5-(2-chloroethyl)-2'-deoxy-4'-thiouridine (3xx) were found to be highly active against herpes simplex virus type-1 (HSV-1) and varicella zoster virus (VZV) in vitro with no significant cytotoxicity. The compound with the broadest spectrum of activity was 2'-deoxy-5-ethyl-4'-thiouridine (3i) which showed significant activity against HSV-1, HSV-2, and VZV.

Peripherally acting enkephalin analogues. 2. Polar tri- and tetrapeptides.

The design, synthesis, and biological activity of a series of D-Arg2-enkephalin-derived tetrapeptide amides and tripeptide aralkylamides are reported. These polar analogues were designed to be excluded from the central nervous system with their action thus limited to peripheral opioid receptors. The effects of the nature of the aromatic ring, aryl ring substitution, and aralkylamine chain length on activity were investigated; in a number of cases the N-terminal amino group of Tyr1 was converted to a guanidino group to further increase hydrophilicity. The peptides were all synthesized by classical solution methodology. The opioid activity of the peptides was assessed in vitro on the guinea pig ileum and their antinociceptive activity was determined in vivo in chemically induced writhing models (peripheral activity) and in the hot-plate test (central activity), in rodents. That the analgesic effects were predominantly mediated in the periphery was demonstrated by antagonism of antinociception by the peripheral opioid antagonist N-methylnalorphine and by comparison of the activities in the writhing and hot-plate tests. As a class, the tetrapeptides were more potent than the tripeptides; N alpha-amidination generally increased activity. A number of compounds exhibited very potent opioid activity and had the desired pharmacological profile, indicating a high degree of peripheral selectivity.

A hybrid gene to express protein epitopes from both sporozoite and merozoite surface antigens of Plasmodium falciparum.

The DNA coding for parts of the repetitive amino acid sequence of Plasmodium falciparum circumsporozoite protein has been spliced to a sequence encoding part of the precursor to the major merozoite surface antigens, to produce a hybrid gene. Expression in Escherichia coli produces a protein with antigenic determinants from both malaria proteins. Antibodies raised against the expressed material react with both a peptide derived from the circumsporozoite repeat sequence, and the merozoite surface molecule. Hybrid molecules of this type may be the basis of a malaria vaccine.

Peripherally acting enkephalin analogues. 1. Polar pentapeptides.

The design, synthesis, and biological activity of a series of highly polar enkephalin-related pentapeptides are reported. These analogues incorporate structural features that exclude them from the central nervous system and thereby restrict their action to peripherally located receptors. Hydrophilic analogues were obtained by introduction of polar D-amino acid residues at position 2 and, in certain cases, by conversion of the N-terminal amino group of the Tyr residue to a guanidino function. The peptides were synthesized by classical solution methods. All compounds demonstrated in vitro opioid activity in the GPI and all were shown to possess antinociceptive activity in chemically induced writhing models. The analgesic effects were shown to be predominantly peripherally mediated by antagonism of antinociception with the peripheral antagonist N-methylnalorphine. Comparative data obtained in writhing and hot-plate tests were also supportive of a peripheral mode of action. Compound 13a, L-tyrosyl-D-arginylglycyl-L-4-nitrophenylalanyl-L-prolinamide (BW 443C), was identified as having a favorable pharmacological profile, indicating a high level of peripheral selectivity, and worthy of further investigation.

Antinociceptive effects of the novel opioid peptide BW443C compared with classical opiates; peripheral versus central actions.

1. To investigate peripherally mediated antinociceptive effects of opioids, the activity of a novel polar enkephalin analogue H-Tyr-D-Arg-Gly-Phe (4-NO2)-Pro-NH2 (BW443C) has been compared with those of classical tertiary opiates against different nociceptive stimuli in the mouse. 2. In chemically-induced writhing models BW443C, administered subcutaneously, demonstrated dose-related antinociceptive effects less potent than morphine and of a similar order to pethidine and D-propoxyphene. In assays using heat as the noxious stimulus BW443C was markedly less potent than any of the opiates tested. 3. In heat-induced assays, but not in chemically-induced writhing assays, BW443C demonstrated a 'U'-shaped dose-time response relationship. Morphine, pethidine and D-propoxyphene showed simple, approximately linear, dose-time effects in all assays. 4. When given subcutaneously, the inhibitory effects of BW443C and morphine in chemically-induced writhing were antagonized by naloxone given intraperitoneally. The inhibitory effects on writhing of BW443C, but not those of morphine, were also antagonized by prior intraperitoneal administration of the quaternary opioid antagonist N-methyl nalorphine. When this antagonist was administered intracerebroventricularly, the antinociceptive effects in writhing of both BW443C and morphine were antagonized. 5. It is concluded that BW443C, being only poorly able to cross the blood brain barrier, demonstrates peripherally mediated opioid antinociceptive effects in chemically-induced writhing models. In heat-induced models, that detect centrally acting opioids, BW443C is effective only at high doses and at time intervals after dosing sufficient to allow slow penetration of drug into the CNS. It is suggested that the peripheral antinociceptive actions of BW443C are mediated by inhibition of sensory neurones.

BW A575C, a chemically novel agent with angiotensin converting enzyme inhibitor and beta-adrenoceptor-blocking properties.

BW A575C (N-(1-(S)-carboxy-5-[4(3-isopropylamino-2-(R, S)-hydroxypropoxy)indole-2- carboxamido]pentyl)-(R, S)-alanyl-(S)-proline) is a chemically novel agent which exhibits in a single molecule both angiotensin converting enzyme (ACE) inhibition and beta-adrenoceptor-blocking properties. BW A575C produced a competitive blockade of heart rate responses to isoprenaline in a guinea-pig right atrial preparation (pKB 7.18 +/- 0.05, cf. pindolol 8.9 +/- 0.7). BW A575C inhibited a partially purified preparation of ACE obtained from rabbit lung (IC50 10.7 +/- 2.1 nM, cf. enalaprilat, 4.4 +/- 0.8 nM). Intravenous administration of BW A575C (1-100 micrograms kg-1 min-1) to the pithed rat inhibited in a dose-dependent fashion both angiotensin I-induced pressor responses and isoprenaline-induced tachycardia. Dose-ratios obtained from such studies demonstrated that, in this preparation, BW A575C was approximately 100 times more active as an ACE inhibitor than as a beta-adrenoceptor blocking agent. Intravenous administration of BW A575C (1 mg kg-1) to the conscious rat inhibited angiotensin I-induced pressor responses, being approximately equipotent to enalapril and 10 times more potent than captopril. At the same dose, BW A575C had a similar duration of action as an ACE inhibitor to enalapril. Intravenous administration of BW A575C (1 mg kg-1) to either conscious dogs or rats inhibited both angiotensin I-induced pressor responses and isoprenaline-induced heart rate responses. Dose-ratios obtained from such studies demonstrated that in these species, BW A575C was 2-10 times more active as an ACE inhibitor than as a beta-adrenoceptor blocking agent.

BW A575C: pharmacological profile in vivo of a novel angiotensin converting enzyme inhibitor and beta-blocker.

The novel compound BW A575C, N-(1-(S)-carboxy-5-[4-(3-isopropylamino-2-(R,S)-hydroxypropoxy)-indole-2 - carboxamido]pentyl)-(R,S)-alanyl-(S)-proline, is a potent angiotensin converting enzyme (ACE) inhibitor and beta-blocker in vitro. It was therefore of considerable interest to establish whether this novel pharmacological profile was maintained in vivo. In conscious instrumented normotensive rats and dogs, intravenous and oral administration of BW A575C causes a dose-dependent rightward displacement of the pressor dose-response curve to angiotensin I (dose ratio of 29.5 and 16.1 in rats and dogs, respectively, at 1.0 mg/kg i.v.) and the tachycardia dose-response curve to isoprenaline (dose ratio of 3.1 and 8.0 in rats and dogs, respectively, at 1.0 mg/kg i.v.). In these experiments BW A575C is approximately 2-10 times more active as an ACE inhibitor than as a beta-blocker. In conscious instrumented acute renovascular hypertensive dogs, where plasma renin activity is elevated 10-fold, BW A575C (1.0 mg/kg i.v.) causes a reduction in blood pressure of 35% within 10 min of injection, which is sustained for up to 4 h. This reduction in blood pressure is accompanied by a consistent, but nonsignificant, reduction in heart rate. These results confirm the novel pharmacological profile of BW A575C in vivo and demonstrate that this compound is an effective antihypertensive agent in a renin-dependent model of hypertension.

Human renin: a new class of inhibitors.

A new class of human renin inhibitor is described, containing a novel analogue of the peptide bond. High inhibitory potency was observed for octapeptide-length substrate analogues but inhibition progressively weakened as the molecule was shortened from the amino terminal end.

Primary structure of 3-phosphoglycerate kinase from horse muscle. I. Purification of cyanogen bromide peptides and amino acid sequence of peptide CB5 (104 residues).

3-Phosphoglycerate kinase was isolated from horse muscle and subjected to the action of cyanogen bromide. The resulting peptides were separated using gel filtration combined with either ion exchange chromatography on phosphocellulose in 6 M urea or high voltage paper electrophoresis. The sequence of the largest peptide, CB5, has been determined by a combination of automated and manual Edman degradation carried out on the intact peptide and derivatives obtained by proteolytic digestion. The isolation of two peptides derived from CB5 by cleavage of the bond between Asp109 and Pro110 facilitated the sequence analysis of CB5. The sequence analysis of the remaining 13 cyanogen bromide fragments and the complete sequence of the enzyme are described in the second paper of this series (Merrett, M. (1981), J. Biol. Chem. 256, 10293-10305).

Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme.

The fitting of sequenced peptides to a high-resolution X-ray map of phosphoglycerate kinase has yielded the complete sequence and structure of the horse muscle enzyme. Metal ADP and ATP substrates are bound to one of the two widely separated domains in an environment that seems unsuitable for phosphoglycerate binding. The most plausible binding site for the phosphoglycerate substrate is on the other domain about 10 A from the ATP, which implies the possibility of a large scale hinge-bending of the domains to bring the two substrates together in a water-free environment for catalysis.