From disorder to order in marching locusts.

Recent models from theoretical physics have predicted that mass-migrating animal groups may share group-level properties, irrespective of the type of animals in the group. One key prediction is that as the density of animals in the group increases, a rapid transition occurs from disordered movement of individuals within the group to highly aligned collective motion. Understanding such a transition is crucial to the control of mobile swarming insect pests such as the desert locust. We confirmed the prediction of a rapid transition from disordered to ordered movement and identified a critical density for the onset of coordinated marching in locust nymphs. We also demonstrated a dynamic instability in motion at densities typical of locusts in the field, in which groups can switch direction without external perturbation, potentially facilitating the rapid transfer of directional information.

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 2: lead optimization affording selective, orally bioavailable compounds with potent anti-HIV activity.

Investigations of the structure-activity relationships of 1,3,4-trisubstituted pyrrolidine human CCR5 receptor antagonists afforded orally bioavailable compounds with the ability to inhibit HIV replication in vitro.

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 1: discovery of the pyrrolidine scaffold and determination of its stereochemical requirements.

A series of 1,3,4-trisubstituted pyrrolidines was discovered to have the ability to displace [(125)I]-MIP-1alpha from the CCR5 receptor expressed on Chinese hamster ovary (CHO) cell membranes. CCR5 activity was found to be dependent on the regiochemistry and the absolute stereochemistry of the pyrrolidine.

The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets.

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist.

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

Distinct mechanism for antidepressant activity by blockade of central substance P receptors.

The localization of substance P in brain regions that coordinate stress responses and receive convergent monoaminergic innervation suggested that substance P antagonists might have psychotherapeutic properties. Like clinically used antidepressant and anxiolytic drugs, substance P antagonists suppressed isolation-induced vocalizations in guinea pigs. In a placebo-controlled trial in patients with moderate to severe major depression, robust antidepressant effects of the substance P antagonist MK-869 were consistently observed. In preclinical studies, substance P antagonists did not interact with monoamine systems in the manner seen with established antidepressant drugs. These findings suggest that substance P may play an important role in psychiatric disorders.

Characterization of the binding and activity of a high affinity, pseudoirreversible morpholino tachykinin NK1 receptor antagonist.

2(S)-((3,5-Bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl)methyl)morpholine (L-742,694) is a selective morpholino tachykinin NK1 receptor antagonist that inhibits the binding of 125I-substance P to the human tachykinin NK1 receptor with a Kd = 37 pM. Increasing concentrations of L-742,694 added to cells 15 min prior to agonist progressively increase the apparent EC50 of substance P for inducing the synthesis of inositol phosphate in Chinese hamster ovary (CHO) cells expressing human tachykinin NK1 receptor and decrease the maximal level of stimulation observed. In contrast, addition of substance P and L-742,694 to the cells at the same time results in an increase in the EC50 for substance P with no decrease in the maximal level of stimulation. The compound also decreases the apparent number of binding sites for 125I-substance P observed by Scatchard analysis. Analysis of the binding of [3H]L-742,694 to the tachykinin NK1 receptor shows that it associates with the receptor with k(a) = 3.98 x 10(8) M(-1) min(-1), and dissociates with k(d) = 0.026 min(-1) and t1/2 = 27 min at 22 degrees C. The slow rate of dissociation of L-742,694 from the tachykinin NK1 receptor and the observation that altering the order of addition of antagonist and substance P attenuates the effect of the antagonist on the maximal activation suggest that L-742,694 is a competitive antagonist that can behave as a pseudoirreversible antagonist under some experimental conditions. L-742,694 has reduced affinity for tachykinin NK1 receptors in which alanine has been substituted for Gln165, His197 or His265 in transmembrane helices 4, 5 and 6, respectively. These three residues have previously been shown to be present in the binding site of tachykinin NK1 receptor antagonists of several structural classes. In addition, L-742,694 inhibits binding of the quinuclidine antagonist (2S,3S)-cis-2-(diphenyl methyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octane 3-amine ([125I]L-703,606) with the same affinity as it inhibits binding of 125I-substance P. These data indicate that L-742,694 binds to the same site within the transmembrane domain of the receptor as previously described competitive antagonists.

In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists.

The ability of tachykinin NK1 receptor antagonists to inhibit GR73632 (D-Ala-[L-Pro9,Me-Leu8]substance P-(7-11))-induced foot tapping in gerbils was employed as an indirect measure of brain penetration and this was compared with their ability to prevent acute emesis induced by cisplatin in ferrets. (+)-GR203040 ((2S,3S and 2R,3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin- 3-yl)-amine), CP-99,994 ((2S,3S)-cis-3-(2-methoxybenzylamino)-2-phenyl piperidine) dihydrochloride), and L-742,694 (2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-4-(5-(3-oxo-1,2, 4-triazolo)methylmorpholine) potently inhibited GR73632-induced foot tapping (ID50 < or = 0.85 mg/kg), and acute retching induced by cisplatin (ID50 < or = 0.18 mg/kg). RPR100893 ((3aS,4S,7aS)-7,7-diphenyl-4-(2-methoxyphenyl)-2-[(S)-2-(2-m ethoxyphenyl)proprionyl] perhydroisoindol-4-ol) was not a potent antagonist of retching (ID50 4.1 mg/kg) or foot tapping (ID50 > 10 mg/kg). High doses (3-10 mg/kg) of CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-N-[(4-quinolinyl)methyl] -4-piperineamine) dihydrochloride), FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-propyl]-N-methy l-N-phenylmethyl-L-3-(2-naphthyl)-alaninamide), and LY303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(pi peridinyl)piperidin-1-yl)acetyl)amino]propane) were required to inhibit foot tapping; these agents were not anti-emetic in this dose range. SR140333 ((S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane; 3-10 mg/kg) failed to inhibit foot tapping or emesis. Affinities for the human and ferret tachykinin NK1 receptor were highly correlated (r = 0.93, P = 0.0008). Inhibition of foot tapping in gerbils, but not NK1 receptor binding affinity, predicted anti-emetic activity in ferrets (r = 0.75, P < 0.01). These findings confirm that the anti-emetic activity of tachykinin NK1 receptor antagonists is dependent on brain penetration.

Proteoglycan-degrading activity of human stromelysin-1 and leukocyte elastase in rabbit joints. Quantitation of proteoglycan and a stromelysin-induced HABR fragment of aggrecan in synovial fluid and cartilage.

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.

Chemical, biochemical, pharmacokinetic, and biological properties of L-680,833: a potent, orally active monocyclic beta-lactam inhibitor of human polymorphonuclear leukocyte elastase.

A series of potent and highly selective time-dependent monocyclic beta-lactam inhibitors of human polymorphonuclear leukocyte elastase (PMNE, EC 3.4.21.37) is described. The intrinsic potency of these compounds, as exemplified by L-680,833 (k(inactivation)/K(i) of 622,000 M-1.s-1), is reflected at the cellular level where it inhibits generation of the specific N-terminal cleavage product A alpha-(1-21) from the A alpha chain of fibrinogen by enzyme released from isolated polymorphonuclear leukocytes stimulated with fMet-Leu-Phe with an IC50 of 0.06 microM. The inhibitory activity of L-680,833 is also apparent in whole blood stimulated with A23187, where it inhibits formation of A alpha-(1-21) and PMNE-alpha 1-proteinase inhibitor complex formation with IC50 values of 9 microM. Pharmacokinetic studies indicate that after oral dosing L-680,833 is bioavailable in rats and rhesus monkeys. This oral bioavailability is reflected by the inhibition (i) of tissue damage elicited in hamster lungs by intratracheal instillation of human PMNE and (ii) enzyme released from human PMN stimulated after their transfer into the pleural cavity of mice. The properties of L-680,833 allow it to effectively supplement the activity of natural inhibitors of PMNE in vivo, suggesting that this type of low-molecular-weight synthetic inhibitor could have therapeutic value in diseases where PMNE damages tissue.

Mechanism of inhibition of human leucocyte elastase by monocyclic beta-lactams.

The kinetic and catalytic mechanisms of time-dependent inhibition of human polymorphonuclear leukocyte elastase (HLE) by the monocyclic beta-lactams described by Knight et al. [Knight, W.B., et al. (1992) Biochemistry 31, 8160] are investigated in this work. The dependence of the pseudo-first-order rate constant (k(obs)) on inhibitor concentration was saturable. The individual kinetic constants for the inhibition by L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzeneacetic acid, and L-683,845, [S-(R*,S*)]-4-[(1-(((1-(5-benzofuranyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)oxy]benzeneacetic acid, at pH 7.5 were k(inact) = 0.08 and 0.06 s-1 and Ki = 0.14 and 0.06 microM, respectively. The relative potency of this class of compounds as measured by k(inact)/Ki is primarily controlled by the Ki, term which ranged from 6 nM to 8 mM, while K(inact) was relatively insensitive to structural changes and varied by only an order of magnitude. Inactivation by the beta-lactams was efficient, requiring only 1.3 and 1.7 equiv of L-680,833 and L-683,845 to inactivate HLE. These values are indicative of some partitioning between turnover of inhibitor and inactivation. The partition ratio ranged as high as 3.5:1 depending upon the structure of the inhibitors, but this ratio was essentially independent of the availability and identity of a leaving group at C-4 of the lactam ring. Inactivation and partitioning liberate the leaving group when present at C-4. p-Hydroxy-m-nitrophenylacetic acid is liberated from this position at a rate similar to that for enzyme inactivation, suggesting kinetic competence of this process. Other products observed during the interaction of L-680,833 with HLE include a substituted urea, a species previously observed during the base-catalyzed decomposition of this class of compounds, and small amounts of products observed during reactivation of beta-lactam-derived HLE-I complexes. Both the pH dependence of k(inact)/Ki for the inactivation of HLE by [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl - 4-oxo-2-azetidinyl)oxyl]benzoic acid and V/K for HLE-catalyzed substrate hydrolysis indicate that a single ionizable group with a pK of approximately 7 must be deprotonated for both processes. This group is likely the active site histidine. The data are consistent with initial formation of a Michaelis complex, acylation of the catalytic serine, and loss of the leaving group at C-4 of the original beta-lactam ring followed by partitioning between regeneration of active enzyme and production of a stable enzyme-inhibitor complex.(ABSTRACT TRUNCATED AT 400 WORDS)

NK2 receptors mediate plasma extravasation in guinea-pig lower airways.

1. Neurokinin (NK) receptor-mediated extravasation has been examined in guinea-pig airways by use of a recently described marker for microvascular protein leakage, 125I-labelled human fibrinogen. 2. Neurokinin A (NKA) caused a dose-dependent increase in plasma [125I]-fibrinogen extravasation in trachea, main bronchi, secondary bronchi and intraparenchymal airways. In contrast, the NK2 selective agonist [beta-Ala8]NKA(4-10) only caused extravasation in the secondary and intraparenchymal airways. 3. The NK2 selective antagonist, SR 48968, caused a dose-dependent inhibition of NKA and [beta-Ala8]NKA(4-10)-induced extravasation of fibrinogen in guinea-pig secondary bronchi and intraparenchymal airways. SR 48968 was without effect on the NKA-induced extravasation in trachea and main bronchi. 4. NKA- or [beta-Ala8]NKA(4-10)-induced plasma extravasation was not modified by pretreatment with histamine H1- or H2-receptor antagonists. 5. It is concluded that NK2 receptors mediate plasma [125I]-fibrinogen extravasation in guinea-pig secondary bronchi and intraparenchymal airways. This effect is direct and does not depend upon histamine released from mast cells.

Orally active beta-lactam inhibitors of human leukocyte elastase-1. Activity of 3,3-diethyl-2-azetidinones.

A thorough analysis of the mechanism of inhibition of human leukocyte elastase (HLE) by a monocyclic beta-lactam and the mechanism of beta-lactam hydrolysis led to the preparation of potent and highly stable inhibitors of HLE. This work led to the identification of 4-[(4-carboxyphenyl)-oxy]-3,3-diethyl-1- [[(phenylmethyl)amino]carbonyl]-2-azetidinone (2) as the first orally active inhibitor of human leukocyte elastase (HLE). Analogs of 2 with different substituents on the urea N were synthesized and evaluated for their activity in vitro against HLE as well as in vivo in a hamster lung hemorrhage model. Compounds with a methyl or a methoxy group in the para position of the benzene ring were very potent in both assays. The results are discussed on the basis of the proposed model for the binding of this class of inhibitors to HLE and a possible mechanism of inhibition is presented.