Pyridazinones and Structurally Related Derivatives with Anti-Inflammatory Activity.

Persistent inflammation contributes to a number of diseases; therefore, control of the inflammatory response is an important therapeutic goal. In an effort to identify novel anti-inflammatory compounds, we screened a library of pyridazinones and structurally related derivatives that were used previously to identify N-formyl peptide receptor (FPR) agonists. Screening of the compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP1-Blue monocytic cells identified 48 compounds with anti-inflammatory activity. Interestingly, 34 compounds were FPR agonists, whereas 14 inhibitors of LPS-induced NF-κB activity were not FPR agonists, indicating that they inhibited different signaling pathways. Further analysis of the most potent inhibitors showed that they also inhibited LPS-induced production of interleukin 6 (IL-6) by human MonoMac-6 monocytic cells, again verifying their anti-inflammatory properties. Structure-activity relationship (SAR) classification models based on atom pair descriptors and physicochemical ADME parameters were developed to achieve better insight into the relationships between chemical structures of the compounds and their biological activities, and we found that there was little correlation between FPR agonist activity and inhibition of LPS-induced NF-κB activity. Indeed, Cmpd43, a well-known pyrazolone-based FPR agonist, as well as FPR1 and FPR2 peptide agonists had no effect on the LPS-induced NF-κB activity in THP1-Blue cells. Thus, some FPR agonists reported to have anti-inflammatory activity may actually mediate their effects through FPR-independent pathways, as it is suggested by our results with this series of compounds. This could explain how treatment with some agonists known to be inflammatory (i.e., FPR1 agonists) could result in anti-inflammatory effects. Further research is clearly needed to define the molecular targets of pyridazinones and structurally related compounds with anti-inflammatory activity and to define their relationships (if any) to FPR signaling events.

1,5,6,7-Tetrahydro-4H-indazol-4-ones as human neutrophil elastase (HNE) inhibitors.

Human neutrophil elastase (HNE) is a serine protease that is expressed in polymorphonuclear neutrophils. It has been recognized as an important therapeutic target for treating inflammatory diseases, especially related to the respiratory system, but also for various types of cancer. Thus, compounds able to inhibit HNE are of great interest in medicinal chemistry. In the present paper, we report the synthesis and biological evaluation of a new series of HNE inhibitors with an innovative 1,5,6,7-tetrahydro-4H-indazol-4-one core that was developed as a molecular modification of our previously reported indazole-based HNE inhibitors. Since the 1,5,6,7-tetrahydro-4H-indazol-4-one scaffold can occur in two possible tautomeric forms, the acylation/alkylation reactions resulted in a mixture of the two isomers, often widely unbalanced in favor of one form. Using analytical techniques and NMR spectroscopy, we characterized and separated the isomer pairs and confirmed the compounds used in biological testing. Analysis of the compounds for HNE inhibitory activity showed that they were potent inhibitors, with Ki values in the low nanomolar range (6-35 nM). They also had reasonable stability in aqueous buffer, with half-lives over 1 h. Overall, our results indicate that the 1,5,6,7-tetrahydro-4H-indazol-4-one core is suitable for the synthesis of potent HNE inhibitors that could be useful in the development of new therapeutics for treating diseases involving excessive HNE activity.

Synthesis, biological evaluation, molecular modeling, and structural analysis of new pyrazole and pyrazolone derivatives as N-formyl peptide receptors agonists.

N-formyl peptide receptors (FPR1, FPR2, and FPR3) play key roles in the regulation of inflammatory processes, and recently, it was demonstrated that FPR1 and FPR2 have a dual role in the progression/suppression of some cancers. Therefore, FPRs represent an important therapeutic target for the treatment of both cancer and inflammatory diseases. Previously, we identified selective or mixed FPR agonists with pyridazinone or pyridinone scaffolds showing a common 4-(bromophenyl)acetamide fragment, which was essential for activity. We report here new pyrazole and pyrazolone derivatives as restricted analogues of the above 6-membered compounds, all exhibiting the same 4-bromophenylacetamide side chain. Most new products had low or absent FPR agonist activity, suggesting that the pyrazole nucleus was not appropriate for FPR agonists. This hypothesis was confirmed by molecular modeling studies, which highlighted that the five-membered scaffold was responsible for a worse arrangement of the molecules in the receptor binding site.

'Proximity frequencies' a new parameter to evaluate the profile of GABAAR modulators.

We reported the synthesis of new 8-methoxypyrazolo[1,5-a]quinazolines bearing an amide fragment at the 3-position. The final compounds, as aromatic (2a-i) and 4,5-dihydro derivatives (3a-i), have been evaluated in vitrofor their ability to modulate the chlorine current on recombinant GABAA receptors of the α1ß2γ2L type (expressed in frog oocytes of the Xenopus laevis species). From electrophysiological test two groups of compounds emerged: positive modulators agonist (2e, h, i and 3e, h) and null modulators antagonist (2a, b, d, f, g and 3a-d, f, g) of GABAA subtype receptor. Using a set of compounds (new derivatives, known products and GABAA subtype receptor ligands from our library) we identify the amino acids at the α+/γ- interface, which could be involved in the agonist or antagonist profile, using the 'Proximity Frequencies', namely the frequencies with which a ligand intercepts two or more binding-site amino acids during the molecular dynamic simulation. The linear discriminant analysis (LDA) evidences that the combination of amino acids αVAL203- γTHR142 and αTYR 160- γTYR 58 allowed to collocate 70.6% of agonists and 72.7% of antagonists in their respective class.

Exploration of nitrogen heterocycle scaffolds for the development of potent human neutrophil elastase inhibitors.

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes but is also involved in a variety of pathologies that affect the pulmonary system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrazolopyridine and pyrrolopyridine derivatives as HNE inhibitors designed as modifications of our previously synthesized indazoles and indoles in order to evaluate effects of the change in position of the nitrogen and/or the insertion of an additional nitrogen in the scaffolds on biological activity and chemical stability. We obtained potent HNE inhibitors with IC50 values in the low nanomolar range (10-50 nM), and some compounds exhibited improved chemical stability in phosphate buffer (t1/2 > 6 h). Molecular modeling studies demonstrated that inhibitory activity was strictly dependent on the formation of a Michaelis complex between the OH group of HNE Ser195 and the carbonyl carbon of the inhibitor. Moreover, in silico ADMET calculations predicted that most of the new compounds would be optimally absorbed, distributed, metabolized, and excreted. Thus, these new and potent HNE inhibitors represent novel leads for future therapeutic development.

Novel Sulfonamide Analogs of Sivelestat as Potent Human Neutrophil Elastase Inhibitors.

Human neutrophil elastase (HNE) is involved in a number of essential physiological processes and has been identified as a potential therapeutic target for treating acute and chronic lung injury. Nevertheless, only one drug, Sivelestat, has been approved for clinical use and just in Japan and the Republic of Korea. Thus, there is an urgent need for the development of low-molecular-weight synthetic HNE inhibitors, and we have developed a wide variety of HNE inhibitors with various chemical scaffolds. We hypothesized that substitution of the active fragment of Sivelestat into these HNE inhibitor scaffolds could modulate their inhibitory activity, potentially resulting in higher efficacy and/or improved chemical stability. Here, we report the synthesis, biological evaluation, and molecular modeling studies of novel compounds substituted with the 4-(sulfamoyl)phenyl pivalate fragment necessary for Sivelestat activity. Many of these compounds were potent HNE inhibitors with activity in the nanomolar range (IC50 = 19-30 nM for compounds 3a, 3b, 3f, 3g, and 9a), confirming that the 4-(sulfamoyl)phenyl pivalate fragment could substitute for the N-CO group at position 1 and offer a different point of attack for Ser195. Results of molecular docking of the these pivaloyl-containing compounds into the HNE binding site supported the mechanism of inhibitory activity involving a nucleophilic attack of Ser195 from the catalytic triad onto the pivaloyl carbonyl group. Furthermore, some compounds (e.g., 3a and 3f) had a relatively good stability in aqueous buffer (t1/2 > 9 h). Thus, this novel approach led to the identification of a number of potent HNE inhibitors that could be used as leads for the further development of new therapeutics.

Novel formyl peptide receptor (FPR) agonists with pyridinone and pyrimidindione scaffolds that are potentially useful for the treatment of rheumatoid arthritis.

The resolution of inflammation is an active response involving the interaction of pro-resolving mediators with specific receptors, such as N-formyl peptide receptor 2 (FPR2). FPRs represent potentially important therapeutic targets for the treatment of some pathologies, including asthma and rheumatoid arthritis. Previously, we identified selective or mixed FPR agonists with a pyridazin-3(2H)-one scaffold, all containing a 4-bromophenylacetamide fragment at N-2. The most effective compounds in this series were EC3, a potent mixed FPR1/FPR2/FPR3 agonist, and EC10, which had a preference for FPR1. We report here a new series of pyridinone and pyrimidindione derivatives containing the 4-(bromophenyl)acetamide substituent that was essential for activity in the pyridazinone series. All new compounds were evaluated for FPR agonist activity in HL60 cells transfected with FPR1 or FPR2 and in human neutrophils. While most of the pyridinone derivatives had reasonable FPR agonist activity in the submicromolar/micromolar range, the pyrimidindione derivatives were less active. Compound 2a (N-(4-bromophenyl)-2-[3-cyano-5-(3-methoxyphenyl)-6-methyl-2-oxopyridin-1(2H)-yl]acetamide) was the most active pyridinone derivative and had a 10-fold preference for FPR2 (EC50 = 120 nM) versus FPR1 (EC50 = 1.6 µM). To assess their therapeutic activity, compounds 2a, EC3, and EC10 were evaluated in vivo using a rat model of rheumatoid arthritis. All three compounds increased the pain threshold and reduced pain hypersensitivity in the treated rats versus control rats, although 2a and EC10 were much more effective than EC3. Thus, these FPR agonists represent potential leads to develop for the treatment of inflammatory diseases such as rheumatoid arthritis.

New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation.

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. Here, we report the further elaboration of our previously reported 3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bearing different substituents at the 4-phenyl ring. The most potent compounds 3a, 3g, and 3h, had IC50 values of 16, 11, and 18 nM, respectively. Molecular modeling studies and molecular dynamic (MD) simulations demonstrated no substantial differences between the 3-methylisoxazole derivatives previously tested and the corresponding 3-unsubstituted derivatives in the snapshot conformations sampled during the MD simulations, which is consistent with their similar levels of HNE inhibitory activity. Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold requirements, and the resulting derivatives are quite potent HNE inhibitors.

Further modifications of 1H-pyrrolo[2,3-b]pyridine derivatives as inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes and is considered to be a multifunctional enzyme. HNE is also involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrrolo[2,3-b]pyridine derivatives of our previously reported potent HNE inhibitors. Our results show that position 2 of the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted, and modifications of this position resulted in loss of HNE inhibitory activity. Conversely, the introduction of certain substituents at position 5 was tolerated, with retention of HNE inhibitory activity (IC50 = 15-51 nM) after most substitutions, indicating that bulky and/or lipophilic substituents at position 5 probably interact with the large pocket of the enzyme site and allow Michaelis complex formation. The possibility of Michaelis complex formation between Ser195 and the ligand carbonyl group was assessed by molecular docking, and it was found that highly active HNE inhibitors are characterized by geometries favorable for Michaelis complex formation and by relatively short lengths of the proton transfer channel via the catalytic triad.

Synthesis of New GABAA Receptor Modulator with Pyrazolo[1,5-a]quinazoline (PQ) Scaffold.

We previously published a series of 8-methoxypirazolo[1,5-a]quinazolines (PQs) and their 4,5-dihydro derivatives (4,5(H)PQ) bearing the (hetero)arylalkylester group at position 3 as ligands at the γ-aminobutyric type A (GABAA) subtype receptor. Continuing the study in this field, we report here the design and synthesis of 3-(hetero)arylpyrazolo[1,5-a]quinazoline and 3-(hetero)aroylpyrazolo[1,5-a]quinazoline 8-methoxy substituted as interesting analogs of the above (hetero)arylalkylester, in which the shortening or the removal of the linker between the 3-(hetero)aryl ring and the PQ was performed. Only compounds that are able to inhibit radioligand binding by more than 80% at 10 µM have been selected for electrophysiological studies on recombinant α1ß2γ2L GABAA receptors. Some compounds show a promising profile. For example, compounds 6a and 6b are able to modulate the GABAAR in an opposite manner, since 6b enhances and 6a reduces the variation of the chlorine current, suggesting that they act as a partial agonist and an inverse partial agonist, respectively. The most potent derivative was 3-(4-methoxyphenylcarbonyl)-8-methoxy-4,5-dihydropyrazolo[1,5-a] quinazoline 11d, which reaches a maximal activity at 1 µM (+54%), and it enhances the chlorine current at ≥0.01 µM. Finally, compound 6g, acting as a null modulator at α1ß2γ2L, shows the ability to antagonize the full agonist diazepam and the potentiation of CGS 9895 on the new α+/ß- 'non-traditional' benzodiazepine site.

1H-pyrrolo[2,3-b]pyridine: A new scaffold for human neutrophil elastase (HNE) inhibitors.

Human neutrophil elastase (HNE) is a potent serine protease belonging to the chymotrypsin family. It is an important target for the development of novel and selective inhibitors for the treatment of inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis and biological evaluation of a new series of HNE inhibitors with a pyrrolo[2,3-b]pyridine scaffold, which is an isomer of our previously reported indazoles, in order to assess how a shift of the nitrogen from position 2 to position 7 influences activity. The majority of new compounds were effective HNE inhibitors and had IC50 values in the micromolar/submicromolar range, with some compounds active in low nanomolar levels. For example, 2a and 2b inhibited HNE with IC50 values of 15 and 14 nM, respectively. Molecular modeling of compounds differing in the position of heteroatom(s) in the bicyclic moiety and in the oxadiazole ring demonstrated that the calculated geometries of enzyme-inhibitor complexes were in agreement with the observed biological activities. Docking experiments showed that orientation of the active pyrrolo[2,3-b]pyridines in the HNE catalytic triad Ser195-His57-Asp102 correlated with effectiveness of the inhibitor interaction with the enzyme. Thus, the pyrrolo[2,3-b]pyridine scaffold represents a novel scaffold for the development of potent HNE inhibitors.

Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors.

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in the nanomolar range (20-70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack.