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Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the mitigation of cell cytotoxicity. Further systematic structure-activity relationship (SAR) efforts driven by structural biology studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.

A novel class of potent Syk inhibitors has been developed from rational design. Highly potent aminopyridine derivatives bearing a 4-trifluoromethyl-2-pyridyl motif and represented by compound 13b IC50: 0.6 nM were identified. Substitution by a 2-pyrazinyl motif and SAR expansion in position 4 of the central core provided diverse potent non-cytotoxic Syk inhibitors showing nanomolar activity inhibiting human mast cell line LAD2 degranulation.

A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.

The aim of the following study was to characterize a passive systemic anaphylaxis rat model of dinitrophenyl (DNP)-induced plasma extravasation in the trachea to determine if the model is appropriate for the evaluation of new drugs targeting airway mast cells by oral and intratracheal (i.t.) route. To this purpose we have used fluticasone and a range of anti-allergic drugs including compounds either active on mast cell activation, such as cromoglycate and the Syk inhibitor R406, or active on mast cell mediators, such as cetirizine and montelukast. To further characterize the model, the effect of fluticasone, cromoglycate and R406 on rat tracheal mast cell degranulation was also assessed histologically. DNP-induced tracheal plasma extravasation was inhibited by cromoglycate (i.v. and i.t.) and R406 (p.o.), but not by fluticasone (i.t.), cetirizine or montelukast (p.o.). Cromoglycate and R406 also showed inhibition of tracheal mast cell degranulation, whereas fluticasone was inactive. These results suggest that the DNP-induced tracheal plasma extravasation model constitutes a useful animal model for the evaluation, by oral and i.t. route, of new anti-allergic drugs intended to target airway mast cells.

Some heterocyclic systems, called privileged scaffolds, appear frequently in bioactive products and marketed drugs. The combination of a recognized privileged scaffold (hydantoin) and a functional group with high incidence in bioactive molecules (guanidine) guided the design of a library of amphipatic compounds, which allowed the discovery of novel TRPV1 ion channel blockers. The library was synthesized by parallel solid-phase synthesis from an orthogonally protected resin-bound Lys-Lys skeleton. Key steps of the synthetic procedure were the construction of the hydantoin ring, by reaction of the N-terminal amino group with N,N-disuccinimidyl carbonate (DSC) and subsequent base-induced cyclization, and the guanidinylation of the C-terminal Lys side-chain after removal of the Alloc protecting-group. The preliminary biological studies have allowed the identification of some of the key structural features directing the blockage of capsaicin-induced Ca(2+) influx through TRPV1 channels, particularly, the strong preference showed for highly lipophilic acyl groups and substituted guanidine moieties. Active compounds based on this new pharmacophoric scaffold that display in vitro and in vivo inhibitory activity.

Peptides representing the sequence of the immunodominant loop of foot-and-mouth disease virus strain C-S8 (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1) and of several variant viruses have been prepared by solid phase methods. In addition, five peptides with single-residue replacements at Leu147 (Ile, Nle, Val, Ala, Gly) have been synthesized. Tosyl and dinitrophenyl protections for histidine have been compared, the latter being found to give better synthetic products. The peptides have been tested in an immunodot assay against a panel of monoclonal antibodies directed towards the VP1 loop. Immunochemical results are discussed on the basis of the mobility of the region reproduced by the peptides and the nature of the side chain of residue 147.

The vanilloid receptor subunit 1, or transient receptor potential vanilloid 1 (TRPV1), integrates physical and chemical stimuli in the peripheral nervous system, playing a key role in inflammatory pain. Identification of potent TRPV1 antagonists is thus an important goal of current neuropharmacology. Herein, we describe the solid-phase synthesis of a series of indole-based peptoids (N-alkylglycines) and the biological activity of the peptoids as novel TRPV1 antagonists. The potency and selectivity of the compounds were determined by electrophysiological recordings in Xenopus oocytes. The most potent and selective noncompetitive TRPV1 antagonist of the series, compound 7, represents an interesting pharmacophoric structure for analgesic lead optimization.

NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively. The solution structure of NK-lysin comprises five amphipathic α-helices. To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin. CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of α-helical peptides. The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin. One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive. Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them. Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity. These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.

The 2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate system (IBTM) has been proposed as a dipeptide surrogate of type II‘ β-turns. To evaluate which of the 11bR and 11bS diastereomers of IBTM best reproduces the conformational properties of type II‘ β-turns, gramicidin S (GS), a cyclic antibiotic peptide that contains two such units, has been chosen as a test compound and the effect of either diastereomer on both conformation and activity of the resulting peptide analogues has been determined. A conventional approach to the cyclic peptide structure based on solution cyclization of a partially protected precursor was only practicable for the (S)-IBTM diastereomer. As an alternative, a solid phase mediated cyclization approach has been devised and applied successfully to both gramicidin S and its Lys2,2‘ analogue, then extended to the (R)-IBTM-containing analogues. NMR conformational analysis has clearly shown that only the (R) diastereomer of IBTM is a suitable mimic of the type II‘ β-turn confo...

Small peptides patterned after the N-terminus of the synaptosomal protein of 25 kDa (SNAP-25), a member of the protein complex implicated in Ca 2+ -dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107, a 6-mer palmitoylated peptide that blocks the inflammatory recruitment the inflammatory recruitment of ion channels to the plasma membrane of nociceptors, and the release of calcitonin gen-related peptide (CGRP) from primary sensory neurons, displays potent and long-lasting in vivo anti-hyperalgesia and anti-allodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund9s Adjuvant, osteosarcoma, chemotherapy and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent anti-hyperalgesic and anti-allodynic activity that lasted ≥24 h. The compound showed a systemic distribution, characterized by a bicompartmental pharmacokinetic profile. Safety pharmacology studies indicated that the peptide is largely devoid of side effects, and substantiated that the in vivo activity is not due to locomotor impairment. Therefore, DD04107 is a potent and long-lasting anti-nociceptive compound that displays a safe pharmacological profile. These findings support the notion that neuronal exocytosis of receptors and neuronal algogens pivotally contribute to chronic inflammatory and neuropathic pain, and imply a central role of peptidergic nociceptor sensitization to the pathogenesis of pain.

To study the conformational requirements of epitopes from a conserved antigenic area (area II) of respiratory syncytial (RS) virus fusion (F) glycoprotein, peptides of increasing length containing amino acids essential for these epitopes were synthesized. The synthetic peptides were tested for binding to a panel of neutralizing monoclonal antibodies (MAbs) for this area as well as to rabbit hyperimmune and human convalescent antisera. Antibody binding was dependent on peptide length; thus, a 61-residue peptide spanning amino acids 215 to 275 of the F1 subunit (peptide F215-275) reacted with more antibodies than a shorter (41-residue) peptide F235-275, and this one with more than the (21-residue) peptide F255-275. Most human convalescent sera contained antibodies that reacted with peptides F215-275 and F235-275 but failed to react with F255-275. The results of antibody binding could be related to the structure adopted by the peptides in solution, as determined by circular dichroism spectroscopy and susceptibility of peptides to trypsin digestion. Pretreatment of peptide F215-275 with SDS abolished reactivity with certain MAbs, supporting the notion that higher order structures were needed for antibody binding. High titre anti-peptide antisera were induced in rabbits inoculated with the peptides; however, these sera failed to react with the native F molecule. In mice, only the largest F215-275 peptide induced an anti-peptide response, but their sera reacted poorly with the native F protein and the animals were not protected against an RS virus challenge. These results illustrate the potential use of synthetic peptides in studies of the F protein physical and antigenic structures as well as the problems in designing synthetic RS virus vaccines.

Aclidinium bromide is a novel potent, long-acting inhaled muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease. Aclidinium showed subnanomolar affinity for the five human muscarinic receptors (M(1)-M(5)). [(3)H]Aclidinium dissociated slightly faster from M(2) and M(3) receptors than [(3)H]tiotropium but much more slowly than [(3)H]ipratropium. Its association rate for the M(3) receptor was similar to [(3)H]ipratropium and 2.6 times faster than [(3)H]tiotropium. Residence half-life of [(3)H]aclidinium at the M(2) receptor was shorter than at the M(3) receptor, demonstrating kinetic selectivity for the M(3) receptor. In isolated guinea pig trachea, aclidinium showed comparable potency to ipratropium and tiotropium, faster onset of action than tiotropium, and duration of action similar to tiotropium and significantly longer than ipratropium. Nebulized aclidinium inhibited bronchoconstriction induced by acetylcholine in guinea pigs in a concentration-dependent manner with an onset of action faster than tiotropium. Duration of action of aclidinium (t(1/2) = 29 h) was much longer than ipratropium (8 h) but shorter than tiotropium (64 h). In dogs, aclidinium induced a smaller and more transient increase in heart rate than tiotropium at comparable supratherapeutic doses. Therefore, under these conditions, aclidinium showed a greater therapeutic index than tiotropium (4.2 versus 1.6). These results indicate that aclidinium is a potent muscarinic antagonist with a fast onset of action, a long duration of effect, and a favorable cardiovascular safety profile.

The transient receptor potential vanilloid member 1 (TRPV1). an integrator of multiple pain-producing stimuli, is regarded nowadays as an important biological target for the discovery of novel analgesics. Here, we describe the first experimental evidence for the behavior of an old family of analgesic dipeptldes, namely Xaa-Trp(Nps) and Trp(Nps)-Xaa (Xaa=Lys, Arg) derivatives, as potent TRPV1 channel bolckers. We also report the synthesis and biological investigation of a series of new conformatidnally restricted Trp(Nps)-dipeptide derivatives with improved TRPV1/ NMDA selectivity. Compound 15b, Which incorporates an N-terminal 2S-azetidine-derived Arg residue, was the most selective compound in this series. Collectively, a new family of TRPV1 channel blockers emerged from our results, although further modifications are required to fine-tune the potency/selectivity/ toxicity balance.

Vanilloid receptor subunit 1 (TRPV1) is an integrator of physical and chemical stimuli in the peripheral nervous system. This receptor plays a key role in the pathophysiology of inflammatory pain. Thus, the identification of receptor antagonists with analgesic and anti-inflammatory activity in vivo is an important goal of current neuropharmacology. Here, we report that [L-arginyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl) glycinamide (H-Arg-15-15C) is a channel blocker that abrogates capsaicin and pH-evoked TRPV1 channel activity with submicromolar activity. Compound H-Arg-15-15C preferentially inhibits TRPV1, showing marginal block of other neuronal receptors. Compound H-Arg-15-15C acts as a noncompetitive capsaicin antagonist with modest voltage-dependent blockade activity. The compound inhibited capsaicin-evoked nerve activity in afferent fibers without affecting mechanically activated activity. Notably, administration of compound H-Arg-15-15C prevented the irritant activity of a local administration of capsaicin and formalin and reversed the thermal hyperalgesia evoked by injection of complete Freund’s adjuvant. Furthermore, it attenuated carrageenan-induced paw inflammation. Compound H-Arg-15-15C specifically decreased inflammatory conditions without affecting normal nociception. Taken together, these findings demonstrate that compound H-Arg-15-15C is a channel blocker of TRPV1 with analgesic and anti-inflammatory activity in vivo at clinically useful doses and substantiate the tenet that TRPV1 plays an important role in the etiology of chronic inflammatory pain., Supported by grants from the Ministerio de Educacion Ciencia y Deporte (SAF 2003/0509), La Fundación Ramón Areces (01/08-500), and Generalitat Valenciana (GV04B-0398) to A.F.M., PROFIT-090000-2001-70 and CDTI-03-006 (Ministerio de Industria) to DiverDrugs, SL.

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino Ψ[CH 2 NH] and ketomethylene Ψ[COCH 2 ] peptide bond surrogates have been introduced at the position Leu 3 -Tyr 4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu 3 -Phe 4 of [Phe 4 ]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica . The analogue was less active than BLAST-2 when tested in vitro , but more active than it when tested in vivo .