Leandro L. da Silva

Discovery of novel analgesic and anti-inflammatory 3-arylamine-imidazo[1,2-a]pyridine symbiotic prototypes

We describe herein the design, synthesis and pharmacological evaluation of novel 3-arylamine-imidazo[1,2-a]pyridine derivatives structurally designed as novel symbiotic prototypes presenting analgesic and anti-inflammatory properties. The derivatives obtained were submitted to in vivo assays of nociception, hyperalgesia and inflammation, and to in vitro assays of human PGHS-2 inhibition. These assays allowed the identification of compound LASSBio-1135 (3a) as an anti-inflammatory and analgesic symbiotic prototype. This compound inhibited moderately the human PGHS-2 enzyme activity (IC(50)=18.5 microM) and reverted the capsaicin-induced thermal hyperalgesia (100 micromol/kg, po) similarly to p38 MAPK inhibitor SB-203580 (2). Additionally, LASSBio-1135 (3a) presented activity similar to celecoxib (1) regarding the reduction of the carrageenan-induced rat paw edema (33% of inhibition at 100 micromol/kg, po). We also discovered derivatives LASSBio-1140 (3c) and LASSBio-1141 (3e) as analgesic and anti-inflammatory prototypes, which were able to attenuate the capsaicin-induced thermal hyperalgesia (100 micromol/kg, po) and reduce the carrageenan-induced paw edema (ED(50)=11.5 micromol/kg (3.3mg/kg) and 14.5 micromol/kg (4.1mg/kg), respectively), being both more active than celecoxib (1), despite the fact that their effects involve a different mechanism of action. Additionally, derivative LASSBio-1145 (3j) showed remarkable analgesic (ED(50)=22.7 micromol/kg (8.9 mg/kg)) and anti-inflammatory (ED(50)=8.7 micromol/kg (3.4 mg/kg)) profile in vivo (100 micromol/kg; po), in AcOH-induced abdominal constrictions in mice and carrageenan-induced rat paw edema models, respectively, being a novel orally-active anti-inflammatory drug candidate that acts as a selective PGHS-2 inhibitor (IC(50)=2.8 microM).

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).

Structure-based design and biological profile of (E)-N-(4-Nitrobenzylidene)-2-naphthohydrazide, a novel small molecule inhibitor of IκB kinase-β.

In this study, we describe the rational design, molecular modeling and pharmacological profile of a novel IKK-β inhibitor (E)-N-(4-nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524). The design based on the IKK-β active site, and a privileged structure template yielded a novel IKK-β inhibitor scaffold with significant selectivity over IKK-α and CHK2, as assessed by an in vitro kinase assay. For a better understanding of the structural requirements of IKK-β inhibition, molecular dynamics simulations of LASSBio-1524 (3) were performed. The NAH derivative LASSBio-1524 (3), was able to suppress arachidonic acid-induced edema formation in a dose-dependent manner, demonstrating an in vivo anti-inflammatory effect. The molecular architecture of this novel, low-molecular weight IKK-β inhibitor is encouraging for further lead optimization toward the development of innovative anti-inflammatory drug candidates.

Lipoxin A4 attenuates endothelial dysfunction during experimental cerebral malaria

A breakdown of the brain-blood barrier (BBB) due to endothelial dysfunction is a primary feature of cerebral malaria (CM). Lipoxins (LX) are specialized pro-resolving mediators that attenuate endothelial dysfunction in different vascular beds. It has already been shown that LXA4 prolonged Plasmodium berghei-infected mice survival by a mechanism that depends on inhibiting IL-12 production and CD8(+)IFN-γ(+) T cells in brain tissue; however, the effects of this treatment on endothelial dysfunction induced during experimental cerebral malaria (ECM) remains to be elucidated. Herein, we investigate the role of LXA4 on endothelial dysfunction during ECM. The treatment of P. berghei-infected mice with LXA4 prevented BBB breakdown and ameliorated behavioral symptoms but did not modulate TNF-α production. In addition, microcirculation analysis showed that treatment with LXA4 significantly increased functional capillary density in brains of P. berghei-infected C57BL/6 mice. Furthermore, histological analyses of brain sections demonstrated that exogenous LXA4 reduced capillary congestion that was accompanied by reduced ICAM-1 expression in the brain tissue. In agreement, LXA4 treatment of endothelial cells stimulated by Plasmodium berghei (Pb)- or Plasmodium falciparum (Pf)-parasitized red blood cells (RBCs) inhibited ICAM-1 expression. Additionally, LXA4 treatment restored the expression of HO-1 that is reduced during ECM. As well, LXA4 treatment inhibits PbRBC and PfRBC adhesion to endothelial cells that was reversed by the use of an HO-1 inhibitor (ZnPPIX). Our results demonstrate for the first time that LXA4 ameliorates endothelial dysfunction during ECM by modulating ICAM-1 and HO-1 expression in brain tissue.

Discovery of Novel Orally Active Anti-Inflammatory N-Phenylpyrazolyl-N-Glycinyl-Hydrazone Derivatives That Inhibit TNF-α Production

Herein, we describe the synthesis and pharmacological evaluation of novel N-phenylpyrazolyl-N-glycinyl-hydrazone derivatives that were designed as novel prototypes of p38 mitogen-activated protein kinase (MAPK) inhibitors. All of the novel synthesized compounds described in this study were evaluated for their in vitro capacity to inhibit tumor necrosis factor α (TNF-α production in cultured macrophages) and in vitro MAPK p38α inhibition. The two most active anti-TNF-α derivatives, (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-((4-(2-morpholinoethoxy)naphthalen-1-yl)methylene)acetohydrazide (4a) and (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-(4-chlorobenzylidene)acetohydrazide (4f), were evaluated to determine their in vivo anti-hyperalgesic profiles in carrageenan-induced thermal hypernociception model in rats. Both compounds showed anti-inflammatory and antinociceptive properties comparable to SB-203580 used as a standard drug, by oral route at a dose of 100 µmol/kg. This bioprofile is correlated with the ability of NAH derivatives (4a) and (4f) suppressing TNF-α levels in vivo by 57.3 and 55.8%, respectively.

Synthesis and analgesic profile of conformationally constrained N-acylhydrazone analogues: Discovery of novel N-arylideneamino quinazolin-4(3H)-one compounds derived from natural safrole

In this work we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and platelet anti-aggregating properties of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a-j), designed as conformationally constrained analogues of analgesic 1,3-benzodioxolyl-N-acylhydrazones (1) previously developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (2) as starting material. The pharmacological assays lead us to identify compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) as new analgesic prototypes, presenting an antinociceptive profile more potent and effective than dipyrone and indomethacin used, respectively, as standards in AcOH-induced abdominal constrictions assay and in the formalin test. These results confirmed the success in the exploitation of conformation restriction strategy for identification of novel cyclic N-acylhydrazone analogues with optimized analgesic profile.

Docking, synthesis and pharmacological activity of novel urea-derivatives designed as p38 MAPK inhibitors.

p38 mitogen-activated protein kinase (p38 MAPK) is an important signal transducing enzyme involved in many cellular regulations, including signaling pathways, pain and inflammation. Several p38 MAPK inhibitors have been developed as drug candidates to treatment of autoimmune disorders, such as rheumatoid arthritis. In this paper we reported the docking, synthesis and pharmacological activity of novel urea-derivatives (4a-e) designed as p38 MAPK inhibitors. These derivatives presented good theoretical affinity to the target p38 MAPK, standing out compound 4e (LASSBio-998), which showed a better score value compared to the prototype GK-00687. This compound was able to reduce in vitro TNF-α production and was orally active in a hypernociceptive murine model sensible to p38 MAPK inhibitors. Otherwise, compound 4e presented a dose-dependent analgesic effect in a model of antigen (mBSA)-induced arthritis and anti-inflammatory profile in carrageenan induced paw edema, indicating its potential as a new antiarthritis prototype.

CYP1A2-mediated biotransformation of cardioactive 2-thienylidene-3,4-methylenedioxybenzoylhydrazine (LASSBio-294) by rat liver microsomes and human recombinant CYP enzymes

We describe herein the metabolic fate of cardioactive 1,3-benzodioxolyl N-acylhydrazone prototype LASSBio-294 (4) and the structural identification of its major phase I metabolite from rat liver microsomal assays. Our results confirmed the hard-metabolic character of N-acylhydrazone (NAH) framework of LASSBio-294 (4). The development of a reproducible analytical methodology for the major metabolite by using HPLC-MS and the comparison with an authentic synthetic sample, allowed us to identify 2-thienylidene 3,4-dihydroxybenzoylhydrazine derivative (7), formed by oxidative scission of methylenedioxy bridge of LASSBio-294, as the main metabolite formed by action of CYP1A2 isoform. The identification of this isoform in the LASSBio-294 in the clearance of LASSBio-294 (4) oxidation was performed by the use of selective CYP inhibitors or human recombinant CYP enzymes.

Development of CoMFA and CoMSIA models of affinity and selectivity for indole ligands of cannabinoid CB1 and CB2 receptors.

This paper describes CoMFA and CoMSIA studies for affinity and selectivity of a series of indole ligands to cannabinoid CB1 and CB2 receptors. The developed models have proven to be predictive, with average q(2) of 0.675 and average r(2) of 0.855, demonstrating a good statistical validation. The obtained results have helped us to understand the structural motifs that are responsible for the affinity and selectivity of some of these derivatives towards each subtype of cannabinoid receptor and have demonstrated that the exploited 3D-QSAR methods could be useful tools for the design of new safer analogues presenting better affinity and selectivity profiles.

Novel Potent Imidazo[1,2-a]pyridine-N-Glycinyl-Hydrazone Inhibitors of TNF-α Production: In Vitro and In Vivo Studies

In this work, we describe the design, synthesis and pharmacological evaluation of novel imidazo[1,2-a]pyridine-N-glycinyl-hydrazone derivatives (1a–k) intended for use as inhibitors of tumor necrosis factor alpha (TNF-α) production. The compounds were designed based on the orally active anti-inflammatory prototype LASSBio-1504 (2), which decreases the levels of the pro-inflammatory cytokine TNF-α in vitro and in vivo. The in vitro pharmacological evaluation of the imidazo[1,2-a]pyridine compounds (1) showed that substitution of the N-phenylpyrazole core present in prototype 2 by a bioisosteric imidazo[1,2-a]pyridine scaffold generated anti-TNF-α compounds that were more potent than the previously described N-phenylpyrazole derivative 2 and as potent as SB-203580, a p38 MAPK inhibitor. The most active derivative (E)-2-(2-tert-butylimidazo[1,2-a]pyridin-3-ylamino)-N’-(4-chlorobenzylidene) acetohydrazide, or LASSBio-1749 (1i) was orally active as an anti-inflammatory agent in a subcutaneous air pouch model, reducing expressively the levels in vivo of TNF-α and other pro-inflammatory cytokines at all of the tested doses.