Antinociceptive constituents from Saccharum officinarum L. juice.

This work aimed to investigate the main components of methanol fractions (MFSC and MFSCf) from Saccharum officinarum L. juice and their in vivo antinociceptive potential. After LC-ESI-MS and ESI-MS/MS analysis, phenolic compounds, such as dicaffeoylquinic acid, schaftoside, vicenin-2, stilbene glycoside and the major compound tricin-7-O-(2″- α-L-rhamnopyranosyl)-α-D-galacturonide (1), were identified. MFSC and MFSCf significantly inhibited nociceptive responses in classical mice pain models. The isolated flavone, 1, inhibited strongly the neurogenic phase in formalin test without interfering with the inflammatory one. The co-administration of the opioid antagonist, naloxone, significantly reversed the antinociceptive effects on the neurogenic phase of both methanol fractions and 1, demonstrating the involvement of the opioid system on the antinociceptive effect. This work describes for the first time the antinociceptive effect of flavonoids present in sugarcane juice, highlighting the isolation and the structural elucidation of tricin-7-O-(2″-α-L-rhamnopyranosyl)-α-D-galacturonide through ESI-MS/MS, 1D- and 2D-NMR.

Long-Chain Omega-3 Fatty Acids Supplementation Accelerates Nerve Regeneration and Prevents Neuropathic Pain Behavior in Mice.

Fish oil (FO) is the main source of long chain omega-3 polyunsaturated fatty acids (ω-3 PUFAs), which display relevant analgesic and anti-inflammatory properties. Peripheral nerve injury is driven by degeneration, neuroinflammation, and neuronal plasticity which results in neuropathic pain (NP) symptoms such as allodynia and hyperalgesia. We tested the preventive effect of an EPA/DHA-concentrate fish oil (CFO) on NP development and regenerative features. Swiss mice received daily oral treatment with CFO 4.6 or 2.3 g/kg for 10 days after NP was induced by partial sciatic nerve ligation. Mechanical allodynia and thermal hypernociception were assessed 5 days after injury. CFO 2.3 g/kg significantly prevented mechanical and thermal sensitization, reduced TNF levels in the spinal cord, sciatic MPO activity, and ATF-3 expression on DRG cells. CFO improved Sciatic Functional Index (SFI) as well as electrophysiological recordings, corroborating the increased GAP43 expression and total number of myelinated fibers observed in sciatic nerve. No locomotor activity impairment was observed in CFO treated groups. These results point to the regenerative and possibly protective properties of a combined EPA and DHA oral administration after peripheral nerve injury, as well as its anti-neuroinflammatory activity, evidencing ω-3 PUFAs promising therapeutic outcomes for NP treatment.

LASSBio-1135: a dual TRPV1 antagonist and anti-TNF-alpha compound orally effective in models of inflammatory and neuropathic pain.

LASSBio-1135 is an imidazo[1,2-a]pyridine derivative with high efficacy in screening models of nociception and inflammation, presumed as a weak COX-2 inhibitor. In order to tease out its mechanism of action, we investigated others possible target for LASSBio-1135, such as TNF-α and TRPV1, to better characterize it as a multitarget compound useful in the treatment of chronic pain. TRPV1 modulation was assessed in TRPV1-expressing Xenopus oocytes against capsaicin and low pH-induced current. Modulation of TNF-α production was evaluated in culture of macrophages stimulated with LPS. In vivo efficacy of LASSBio-1135 was investigated in carrageenan and partial sciatic ligation-induced thermal hyperalgesia and mechanical allodynia. Corroborating its previous demonstration of efficacy in a model of capsaicin-induced hyperalgesia, LASSBio-1135 blocks capsaicin-elicited currents in a non-competitive way with an IC50 of 580 nM as well as low pH-induced current at 50 µM. As an additional action, LASSBio-1135 inhibited TNF-α release in these cells stimulated by LPS with an IC50 of 546 nM by reducing p38 MAPK phosphorilation. Oral administration of 100 µmol x Kg(-1) LASSBio-1135 markedly reduced thermal hyperalgesia induced by carrageenan, however at 10 µmol x Kg(-1) only a partial reduction was observed at the 4th h. Neutrophil recruitment and TNF-α production after carrageenan stimulus was also inhibited by the treatment with LASSBio-1135. Modulating TRPV1 and TNF-α production, two key therapeutic targets of neuropathic pain, 100 µmol x Kg(-1) LASSBio-1135 was orally efficacious in reversing thermal hyperalgesia and mechanical allodynia produced by partial sciatic ligation 7-11 days after surgery without provoking hyperthermia, a common side effect of TRPV1 antagonists. In conclusion LASSBio-1135, besides being a weak COX-2 inhibitor, is a non-competitive TRPV1 antagonist and a TNF-α inhibitor. As a multitarget compound, LASSBio-1135 is orally efficacious in a model of neuropathic pain without presenting hyperthermia.

Synthesis and characterization of the atropisomeric relationships of a substituted N-phenyl-bipyrazole derivative with anti-inflammatory properties.

This work describes the atropisomeric relationships of 3-methyl-5-(3-methyl-5-phenyl-1H-pyrazol-1-yl)-1-phenyl-1H-pyrazol-4-amine (2d), which belongs to series 4-aminobipyrazole derivatives designed as anti-inflammatory agents. The (1)H nuclear magnetic resonance spectra obtained in the presence of a chiral lanthanide shift salt associated to chiral high-performance liquid chromatography analysis, X-ray diffraction, and molecular modeling tools confirmed that ortho bis-functionalized bipyrazole 2d exists as a mixture of aR,aS-atropisomers. These results provide useful information to understand the pharmacological profile of this derivative and of other 4-aminobipyrazole analogs.

Novel furfurylidene N-acylhydrazones derived from natural safrole: discovery of LASSBio-1215, a new potent antiplatelet prototype.

We describe herein the discovery of (E)-N-methyl-N'-((5-nitrofuran-2-yl)methylene)benzo[d]( 1 , 3 ) dioxole-5-carbohydrazide (9e), named LASSBio-1215, as a novel antiplatelet agent belonging to the N-methyl-N-acylhydrazone class, which exert their antiaggregating actions on human and rabbit platelets induced by different agonists, through cyclooxygenase-1 (COX-1) or thromboxane synthase inhibition. This compound was elected after screening of a series of functionalized furyl N-acylhydrazone derivatives, synthesized from natural safrole 10. In vitro assays showed that compound 9e presents platelet-aggregating activity in rabbit platelet-rich plasma (PRP) induced by arachidonic acid (IC(50) = 0.7 µM) and collagen (IC(50) = 4.5 µM). Moreover, LASSBio-1215 also inhibited almost completely the second wave of adenosine diphosphate-induced platelet aggregation in human PRP, and this effect was correlated with their ability to block the production of pro-aggregating autacoid thromboxane A(2).

Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative with potent alpha 1A/D-adrenergic receptor blocking properties.

We described herein the discovery of 1-(2-(benzo[d] [1,3]dioxol-6-yl)ethyl)-4-(2-methoxyphenyl) piperazine (LASSBio-772), as a novel potent and selective alpha 1A/1D adrenoceptor (AR) antagonist selected after screening of functionalized N-phenylpiperazine derivatives in phenylephrine-induced vasoconstriction of rabbit aorta rings. The affinity of LASSBio-772 for alpha 1A and alpha 1B AR subtypes was determined through displacement of [(3)H]prazosin binding. We obtained Ki values of 0.14 nM for the alpha 1A-AR, similar to that displayed by tamsulosin (K(i) = 0.13 nM) and 5.55 nM for the alpha 1B-AR, representing a 40-fold higher affinity for alpha 1A-AR. LASSBio-772 also presented high affinity (K(B) = 0.025 nM) for the alpha 1D-AR subtype in the functional rat aorta assay, showing to be equipotent to tamsulosin (K(B) = 0.017 nM).

Novel thienylacylhydrazone derivatives inhibit platelet aggregation through cyclic nucleotides modulation and thromboxane A2 synthesis inhibition.

The aim of this study has been to investigate the antiplatelet activity of a new series of thienylacylhydrazone derivatives analogous to the lead compound LASSBio-294 ((2-thienylidene) 3,4-methylenedioxybenzoylhydrazine). The antiplatelet effect was investigated in rabbit and human platelet rich plasma stimulated by arachidonic acid, collagen, ADP and in washed platelet stimulated by thrombin. The effects on the production of cyclic nucleotides and thromboxane A(2) (TXA(2)) in human platelets were also investigated. Compounds LASSBio-785 (N-Methyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-786 (N-Benzyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-787 ((5-Methyl-2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-788 (N-Allyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine) and LASSBio-789 ((5-Bromo-2-thienylidene) 3,4-methylenedioxybezoylhydrazine) inhibited platelet aggregation induced by arachidonic acid, collagen and ADP. LASSBio-785, LASSBio-788 and LASSBio-789 presented the higher potency in platelet aggregation induced by arachidonic acid (IC(50) values of 0.3, 0.2 and 3.1 microM, respectively) and collagen (IC(50) values of 0.9, 1.5 and 3.4 microM, respectively), with a 20 to 70-fold increase in potency compared to LASSBio-294. They inhibited the ATP release reaction by 95%, the whole blood aggregation by 35-45% and the TXB(2) production was totally abolished. In addition, they presented a significant effect on bleeding time. Qualitative studies in thrombin-induced washed platelet aggregation in the presence of sodium nitroprusside (SNP) suggested a phosphodiesterase-2 (PDE2) like effect for LASSBio-785, LASSBio-788 and LASSBio-789. They were able to increase the cGMP levels in non-stimulated platelets, in SNP-stimulated platelets and in the presence of 1-H- [1, 2, 4] oxadiazolo [4, 3- a] quinoxalin- 1- one (ODQ). The antiplatelet aggregation activity exerted by thienylacylhydrazone derivatives seems to be related to cyclic nucleotides regulation and TXA(2) synthesis inhibition. The structural modification of compound LASSBio-294 led to the optimization of its pharmacological properties and to the discovery of new potent antiplatelet prototypes with an antithrombotic potential.

Design, synthesis and analgesic properties of novel conformationally-restricted N-acylhydrazones (NAH).

A set of six azaheterocycles were designed as conformationally-constrained N-acylhydrazones and tested as analgesics.

Synthesis, pharmacological evaluation and docking studies of new sulindac analogues.

This paper describes the synthesis, pharmacological evaluation and docking studies of a series of new sulindac analogues. Overall, the designed compounds revealed good, in vivo, antinociceptive activity and satisfactory anti-inflammatory profile. Flexible molecular docking with COX-1/COX-2 has shown putative binding modes of the designed compounds while the theoretical evaluation of cell permeability based on Lipinski's rule of five has helped rationalize the biological results.

Evaluating the prophylactic potential of the phtalimide derivative LASSBio 552 on allergen-evoked inflammation in rats.

A previous study showed that the novel tetrazolephtalimide derivative LASSBio 552 (2-4-[3-(1H-1,2,3,4-tetraazol-5-yl)propoxy]phenethyl-1,3-isoindolinedione) prevents LTD(4)-evoked tracheal contraction. This led us to examine the putative anti-inflammatory effect of LASSBio 552 in comparison with the leukotriene CysLT(1) receptor antagonist zafirlukast using a model of allergic pleurisy in rats. Treatment with either LASSBio 552 (24-96 micromol/kg, i.p.) or zafirlukast (9-72 micromol/kg, i.p.), 1 h before challenge, inhibited eosinophil and mononuclear cell influx into the pleural cavity 24 h post-challenge, but failed to alter the increased levels of eotaxin, plasma leakage, mast cell degranulation and neutrophil infiltration noted 6 h post-challenge. CD4(+) T cell recruitment 24 h post-challenge was also sensitive to LASSBio 552. This treatment failed to alter cysteinyl leukotriene production at 6 h, but clearly inhibited the phenomenon 24 h and 48 h post-challenge. In in vitro settings LASSBio 552 inhibited allergen-evoked cysteinyl leukotriene generation from isolated mast cells, while histamine release remained unchanged. It also slightly inhibited cysteinyl leukotriene production by eosinophils and mononuclear cells triggered by Ca(+2) ionophore A23187. A leukotriene CysLT(1) receptor transfected cell-based assay revealed that LASSBio 552 did not prevent LTD(4)-evoked Ca(+2) influx, indicating that it was not a leukotriene CysLT(1) receptor antagonist. These findings indicate that LASSBio 552 is able to inhibit eosinophil influx triggered by allergen chalenge in a mechanism at least partially associated with suppression of CD4(+) T cell influx and cysteinyl leukotriene production.

New class of potent antinociceptive and antiplatelet 10H-phenothiazine-1-acylhydrazone derivatives.

In this work, we reported the synthesis and evaluation of the analgesic, antiinflammatory, and antiplatelet properties of new phenothiazine-attached acylhydrazone derivatives (6), designed exploring the molecular hybridization approach between antipsychotic chlorpromazine (4) and other heterocyclic derivatives (3) and (5) developed at LASSBio. Target compounds were synthesized in very good yields exploiting diphenylamine (7) as starting material, through regioselective functionalization of the C-1 position of 10H-phenothiazine ring. The evaluation of platelet antiaggregating profile lead us to identify a new potent prototype of antiplatelet derivative, that is (6a) (IC(50)=2.3 microM), which acts in arachidonic acid pathway probably by inhibition of platelet COX-1 enzyme. Additionally, the change of para-substituent group of acylhydrazone framework permitted us to identify hydrophilic carboxylate derivative (6g) and hydrophobic bromo derivative (6b) as two new leads of analgesics more active than dipyrone used as standard and with selective peripheral or central mechanism of action.

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives.

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.

Novel phthalimide derivatives, designed as leukotriene D(4) receptor antagonists.

A series of phthalimide acid derivatives was synthesized and evaluated as leukotriene D(4) receptor antagonists. The tetrazolephthalimide LASSBio 552 (7) was shown to be able to inhibit the contractile activity induced by 100 nM of LTD(4) in guinea-pig tracheal strips with an IC(50) = 31.2 microM. In addition, LASSBio 552 (7) has been showed to present a better efficacy than zafirlukast (1) used as standard.

New isoxazole derivatives designed as nicotinic acetylcholine receptor ligand candidates.

In this work we report the synthesis and evaluation of the analgesic properties of new isosteric heterocyclic derivatives, presenting the isoxazole nucleus, designed as nicotinic acetylcholine receptor ligand candidates, analogues to alkaloid epibatidine. Compound 2-(3-methyl-5-isoxazolyl)pyridine (3) presented the best analgesic profile of this series in hot plate test, which was partially prevented by pretreatment with nicotinic receptor antagonist mecamylamine.

Design, synthesis and pharmacological evaluation of novel pyrazolo[3,4-b]thieno[2,3-d]pyridine acid derivatives: a new class of anti-inflammatory and anti-platelet agents.

A series of pyrazolo[3,4-b]thieno[2,3-d]pyridine alkanoic acid derivatives has been synthesized and evaluated as thromboxane synthetase inhibitors and leukotriene D(4) receptor antagonists. The glutaric acid derivative LASSBio341 (6) was shown to be active in arachidonic acid-induced platelet aggregation (IC(50)=0.14 microM) and inhibition of the contraction of guinea pig tracheal strip induced with LTD(4) (IC(50) = 43.7 microM), displaying still in vivo anti-inflammatory profile.

Synthesis and pharmacological evaluation of novel antinociceptive N-substituted-phenylimidazolyl-4-acylhydrazone derivatives.

This paper describes recent results of design, synthesis and pharmacological evaluation of new N-heterocyclic functionalized N-acylhydrazone compounds (NAH), belonging to the N-substituted-phenylimidazolyl-4-acylhydrazone class (3a-o). These compounds were planned by applying the molecular hybridization strategy to propose the structural modifications on the previously described functionalized 2-methyl-imidazolyl-3-acylhydrazone class (2), which presented an important analgesic profile. This new series (3) was synthesized in order to investigate the possible pharmacophoric contribution of the N-heteroaromatic ring and N-acylhydrazone moieties to the analgesic activity. Compounds 3g and 3n are the most potent analgesic agents from this series, at the screening dose of 100 mg/kg p.o. and compounds 3e, 3j and 3o presented the best antiinflammatory properties at the same screening concentration.