Amelioration of functional and histopathological consequences after spinal cord injury through phosphodiesterase 4D (PDE4D) inhibition.

Spinal cord injury (SCI) is a life-changing event that severely impacts the patient's quality of life. Modulating neuroinflammation, which exacerbates the primary injury, and stimulating neuro-regenerative repair mechanisms are key strategies to improve functional recovery. Cyclic adenosine monophosphate (cAMP) is a second messenger crucially involved in both processes. Following SCI, intracellular levels of cAMP are known to decrease over time. Therefore, preventing cAMP degradation represents a promising strategy to suppress inflammation while stimulating regeneration. Intracellular cAMP levels are controlled by its hydrolyzing enzymes phosphodiesterases (PDEs). The PDE4 family is most abundantly expressed in the central nervous system (CNS) and its inhibition has been shown to be therapeutically relevant for managing SCI pathology. Unfortunately, the use of full PDE4 inhibitors at therapeutic doses is associated with severe emetic side effects, hampering their translation toward clinical applications. Therefore, in this study, we evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B and PDE4D) on inflammatory and regenerative processes following SCI, as inhibitors selective for these subtypes have been demonstrated to be well-tolerated. We reveal that administration of the PDE4D inhibitor Gebr32a, even when starting 2 dpi, but not the PDE4B inhibitor A33, improved functional as well as histopathological outcomes after SCI, comparable to results obtained with the full PDE4 inhibitor roflumilast. Furthermore, using a luminescent human iPSC-derived neurospheroid model, we show that PDE4D inhibition stabilizes neural viability by preventing apoptosis and stimulating neuronal differentiation. These findings strongly suggest that specific PDE4D inhibition offers a novel therapeutic approach for SCI.

Anticancer Effects of the Novel Pyrazolyl-Urea GeGe-3.

In a screen of over 200 novel pyrazole compounds, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a potential anticancer compound. GeGe-3 displays potent anti-angiogenic properties through the presumptive targeting of the protein kinase DMPK1 and the Ca2+-binding protein calreticulin. We further explored the anticancer potential of GeGe-3 on a range of established cancer cell lines, including PC3 (prostate adenocarcinoma), SKMEL-28 (cutaneous melanoma), SKOV-3 (ovarian adenocarcinoma), Hep-G2 (hepatocellular carcinoma), MDA-MB231, SKBR3, MCF7 (breast adenocarcinoma), A549 (lung carcinoma), and HeLa (cervix epithelioid carcinoma). At concentrations in the range of 10 µM, GeGe-3 significantly restricted cell proliferation and metabolism. GeGe-3 also reduced PC3 cell migration in a standard wound closure and trans-well assay. Together, these results confirm the anticancer potential of GeGe-3 and underline the need for more detailed pre-clinical investigations into its molecular targets and mechanisms of action.

Structure-Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents.

Aminopyrazoles represent interesting structures in medicinal chemistry, and several derivatives showed biological activity in different therapeutic areas. Previously reported 5-aminopyrazolyl acylhydrazones and amides showed relevant antioxidant and anti-inflammatory activities. To further extend the structure-activity relationships in this class of derivatives, a novel series of pyrazolyl acylhydrazones and amides was designed and prepared through a divergent approach. The novel compounds shared the phenylamino pyrazole nucleus that was differently decorated at positions 1, 3, and 4. The antiproliferative, antiaggregating, and antioxidant properties of the obtained derivatives 10-22 were evaluated in in vitro assays. Derivative 11a showed relevant antitumor properties against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28) with micromolar IC50 values. In the platelet assay, selected pyrazoles showed higher antioxidant and ROS formation inhibition activity than the reference drugs acetylsalicylic acid and N-acetylcysteine. Furthermore, in vitro radical scavenging screening confirmed the good antioxidant properties of acylhydrazone molecules. Overall, the collected data allowed us to extend the structure-activity relationships of the previously reported compounds and confirmed the pharmaceutical attractiveness of this class of aminopyrazole derivatives.

Investigations of Antioxidant and Anti-Cancer Activities of 5-Aminopyrazole Derivatives.

To further extend the structure-activity relationships (SARs) of 5-aminopyrazoles (5APs) and identify novel compounds able to interfere with inflammation, oxidative stress, and tumorigenesis, 5APs 1-4 have been designed and prepared. Some chemical modifications have been inserted on cathecol function or in aminopyrazole central core; in detail: (i) smaller, bigger, and more lipophilic substituents were introduced in meta and para positions of catechol portion (5APs 1); (ii) a methyl group was inserted on C3 of the pyrazole scaffold (5APs 2); (iii) a more flexible alkyl chain was inserted on N1 position (5APs 3); (iv) the acylhydrazonic linker was moved from position 4 to position 3 of the pyrazole scaffold (5APs 4). All new derivatives 1-4 have been tested for radical scavenging (DPPH assay), anti-aggregating/antioxidant (in human platelets) and cell growth inhibitory activity (MTT assay) properties. In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated. 5APs 1 emerged to be promising anti-proliferative agents, able to suppress the growth of specific cancer cell lines. Furthermore, derivatives 3 remarkably inhibited ROS production in platelets and 5APs 4 showed interesting in vitro radical scavenging properties. Overall, the collected results further confirm the pharmaceutical potentials of this class of compounds and support future studies for the development of novel anti-proliferative and antioxidant agents.

Recent Advances in the Discovery of SIRT1/2 Inhibitors via Computational Methods: A Perspective.

Sirtuins (SIRTs) are classified as class III histone deacetylases (HDACs), a family of enzymes that catalyze the removal of acetyl groups from the ε-N-acetyl lysine residues of histone proteins, thus counteracting the activity performed by histone acetyltransferares (HATs). Based on their involvement in different biological pathways, ranging from transcription to metabolism and genome stability, SIRT dysregulation was investigated in many diseases, such as cancer, neurodegenerative disorders, diabetes, and cardiovascular and autoimmune diseases. The elucidation of a consistent number of SIRT-ligand complexes helped to steer the identification of novel and more selective modulators. Due to the high diversity and quantity of the structural data thus far available, we reviewed some of the different ligands and structure-based methods that have recently been used to identify new promising SIRT1/2 modulators. The present review is structured into two sections: the first includes a comprehensive perspective of the successful computational approaches related to the discovery of SIRT1/2 inhibitors (SIRTIs); the second section deals with the most interesting SIRTIs that have recently appeared in the literature (from 2017). The data reported here are collected from different databases (SciFinder, Web of Science, Scopus, Google Scholar, and PubMed) using "SIRT", "sirtuin", and "sirtuin inhibitors" as keywords.

New Pyrazolyl Thioureas Active against the Staphylococcus Genus.

To meet the urgent need for new antibacterial molecules, a small library of pyrazolyl thioureas (PTUs) was designed, synthesized and tested against difficult-to-treat human pathogens. The prepared derivatives are characterized by a carboxyethyl functionality on C4 and different hydroxyalkyl chains on N1. Compounds 1a-o were first evaluated against a large panel of Gram-positive and Gram-negative pathogens. In particular, the majority of PTUs proved to be active against different species of the Staphylococcus genus, with MIC values ranging from 32 to 128 µg/mL on methicillin-resistant Staphylococcus strains, often responsible for severe pulmonary disease in cystic fibrosis patients. Time-killing experiments were also performed for the most active compounds, evidencing a bacteriostatic mechanism of action. For most active derivatives, cytotoxicity was evaluated in Vero cells, and at the tested concentrations and at the experimental exposure time of 24 h, none of the compounds analysed showed significant toxicity. In addition, favourable drug-like, pharmacokinetic and toxicity properties were predicted for all new synthesized derivatives. Overall, the collected data confirmed the PTU scaffold as a promising chemotype for the development of novel antibacterial agents active against Gram-positive multi-resistant strains frequently isolated from cystic fibrosis patients.

Imidazo-Pyrazole-Loaded Palmitic Acid and Polystyrene-Based Nanoparticles: Synthesis, Characterization and Antiproliferative Activity on Chemo-Resistant Human Neuroblastoma Cells.

Neuroblastoma (NB) is a childhood cancer, commonly treated with drugs, such as etoposide (ETO), whose efficacy is limited by the onset of resistance. Here, aiming at identifying new treatments for chemo-resistant NB, the effects of two synthesized imidazo-pyrazoles (IMPs) (4G and 4I) were investigated on ETO-sensitive (HTLA-230) and ETO-resistant (HTLA-ER) NB cells, detecting 4I as the more promising compound, that demonstrated IC50 values lower than those of ETO on HTLA ER. Therefore, to further improve the activity of 4I, we developed 4I-loaded palmitic acid (PA) and polystyrene-based (P5) cationic nanoparticles (P5PA-4I NPs) with high drug loading (21%) and encapsulation efficiency (97%), by a single oil-in-water emulsification technique. Biocompatible PA was adopted as an emulsion stabilizer, while synthesized P5 acted as an encapsulating agent, solubilizer and hydrophilic-lipophilic balance (HLB) improver. Optic microscopy and cytofluorimetric analyses were performed to investigate the micromorphology, size and complexity distributions of P5PA-4I NPs, which were also structurally characterized by chemometric-assisted Fourier transform infrared spectroscopy (FTIR). Potentiometric titrations allowed us to estimate the milliequivalents of PA and basic nitrogen atoms present in NPs. P5PA-4I NPs afforded dispersions in water with excellent buffer capacity, essential to escape lysosomal degradation and promote long residence time inside cells. They were chemically stable in an aqueous medium for at least 40 days, while in dynamic light scattering (DLS) analyses, P5PA-4I showed a mean hydrodynamic diameter of 541 nm, small polydispersity (0.194), and low positive zeta potentials (+8.39 mV), assuring low haemolytic toxicity. Biological experiments on NB cells, demonstrated that P5PA-4I NPs induced ROS-dependent cytotoxic effects significantly higher than those of pristine 4I, showing a major efficacy compared to ETO in reducing cell viability in HTLA-ER cells. Collectively, this 4I-based nano-formulation could represent a new promising macromolecular platform to develop a new delivery system able to increase the cytotoxicity of the anticancer drugs.

Antiproliferative Imidazo-Pyrazole-Based Hydrogel: A Promising Approach for the Development of New Treatments for PLX-Resistant Melanoma.

Aiming at developing a dermal formulation against melanoma, the synthesized imidazo-pyrazoles 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylic acid (3-methoxy-4-phenoxy-benzylidene)-hydrazide (4G) and 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylic acid (4-benzyloxy-3-methoxy-benzylidene)-hydrazide (4I) were screened on patient-isolated melanoma cells (MEOV NT) and on Vemurafenib (PLX4032)-resistant (MEOV PLX-R) ones. Since 4I on MEOV PLX-R cells was 1.4-fold more effective than PLX, a hydrogel formulation containing 4I (R4HG-4I) was prepared in parallel with an empty R4-based hydrogel (R4HG) using a synthesized antibacterial resin (R4) as gelling agent. Thanks to its high hydrophilicity, porosity (85%), and excellent swelling capability (552%), R4 allowed to achieve R4HG and R4HG-4I with high equilibrium degree of swelling (EDS) and equilibrium water content (EWC). Chemometric-assisted ATR-FTIR analyses confirmed the chemical structure of swollen and fully dried (R4HG-D and R4HG-4I-D) hydrogels. The morphology of R4HG-D and R4HG-4I-D was examined by optical microscopy and SEM, while UV-vis analyses were carried out to obtain the drug loading (DL%) and the encapsulation efficiency (EE%) of R4HG-4I. Potentiometric titrations were performed to determine the equivalents of NH3+ in both R4HG and R4HG-4I. The swelling and water release profiles of both materials and related kinetics were assessed by equilibrium swelling rate and water loss studies, respectively, while their biodegradability over time was assessed by in vitro degradation experiments determining their mass loss. Rheological experiments established that both R4HG and R4HG-4I are shear-thinning Bingham pseudoplastic fluids with low yield stress, thus assuring easy spreadability in a future topical application. Release studies evidenced a sustained and quantitative release of 4I governed mainly by diffusion. Upon favorable results from further experiments in a more realistic 3D model of melanoma, R4HG-4I could represent a starting point to develop new topical therapeutic options to adjuvate the treatments of melanoma cells also when resistant to currently available drugs.

Novel 5-aminopyrazoles endowed with anti-angiogenetic properties: Design, synthesis and biological evaluation.

The promising anti-angiogenetic properties of previously synthesized pyrazolyl ureas provided the rationale for the synthesis of novel 5-aminopyrazoles 2-5, differently decorated on the pyrazole nucleus. All the derivatives were tested by MTT assays and proved to be non-cytotoxic against eight different tumor cell lines and normal fibroblasts. An EdU proliferation assay was carried out on human foreskin fibroblasts and VEGF stimulated human umbilical vein endothelial cells which confirmed the absence of cytotoxicity of the compounds on human cells up to 20 µM concentration. To evaluate the influence of the newly synthesized pyrazoles on MAPK and PI3K signaling pathways, the phosphorylation of ERK1/2 and Akt was analyzed by Western blots from HFF and HUVEC cell lysates stimulated with growth factors in the presence or absence of the compounds. Pyrazoles 3b and 3c showed a significant inhibition of Akt phosphorylation in both tested cell lines with lower phosphorylation levels than the reference compound GeGe-3 in HUVEC. Furthermore, derivatives 2 and 3 appeared to strongly affect the migration of HFF cells in a wound healing assay, confirming their potential ability to interfere with the angiogenesis process. The new pyrazole library extends the structure-activity relationships of the previously isolated compounds and highlights the attractiveness of this chemical class for pathological cell migration and angiogenesis.

Indole Antitumor Agents in Nanotechnology Formulations: An Overview.

The indole heterocycle represents one of the most important scaffolds in medicinal chemistry and is shared among a number of drugs clinically used in different therapeutic areas. Due to its varied biological activities, high unique chemical properties and significant pharmacological behaviors, indole derivatives have drawn considerable interest in the last decade as antitumor agents active against different types of cancers. The research of novel antiproliferative drugs endowed with enhanced efficacy and reduced toxicity led to the approval by U.S. Food and Drug Administration of the indole-based anticancer agents Sunitinib, Nintedanib, Osimertinib, Panobinostat, Alectinib and Anlotinib. Additionally, new drug delivery systems have been developed to protect the active principle from degradation and to direct the drug to the specific site for clinical use, thus reducing its toxicity. In the present work is an updated review of the recently approved indole-based anti-cancer agents and the nanotechnology systems developed for their delivery.

Ablation of specific long PDE4D isoforms increases neurite elongation and conveys protection against amyloid-ß pathology.

Inhibition of phosphodiesterase 4D (PDE4D) enzymes has been investigated as therapeutic strategy to treat memory problems in Alzheimer's disease (AD). Although PDE4D inhibitors are effective in enhancing memory processes in rodents and humans, severe side effects may hamper their clinical use. PDE4D enzymes comprise different isoforms, which, when targeted specifically, can increase treatment efficacy and safety. The function of PDE4D isoforms in AD and in molecular memory processes per se has remained unresolved. Here, we report the upregulation of specific PDE4D isoforms in transgenic AD mice and hippocampal neurons exposed to amyloid-ß. Furthermore, by means of pharmacological inhibition and CRISPR-Cas9 knockdown, we show that the long-form PDE4D3, -D5, -D7, and -D9 isoforms regulate neuronal plasticity and convey resilience against amyloid-ß in vitro. These results indicate that isoform-specific, next to non-selective, PDE4D inhibition is efficient in promoting neuroplasticity in an AD context. Therapeutic effects of non-selective PDE4D inhibitors are likely achieved through actions on long isoforms. Future research should identify which long PDE4D isoforms should be specifically targeted in vivo to both improve treatment efficacy and reduce side effects.

Targeting USP-7 by a Novel Fluorinated 5-Pyrazolyl-Urea Derivative.

The impact of innovative technologies on the target discovery has been employed here to characterize the interactome of STIRUR 41, a promising 3-fluoro-phenyl-5-pyrazolyl-urea derivative endowed with anti-cancer activity, on neuroblastoma-related cells. A drug affinity responsive target stability-based proteomic platform has been optimized to elucidate the molecular mechanism at the basis of STIRUR 41 action, together with immunoblotting analysis and in silico molecular docking. Ubiquitin Specific Protease 7 (USP-7), one of the deubiquitinating enzymes which protect substrate proteins from proteasomal degradation, has been identified as the most affine STIRUR 41 target. As further demonstrated by in vitro and in-cell assays, STIRUR 41 was able to inhibit both the enzymatic activity of USP-7 and its expression levels in neuroblastoma-related cells, thus laying an encouraging base for the blockade of USP-7 downstream signaling.

Virtual Screening Combined with Enzymatic Assays to Guide the Discovery of Novel SIRT2 Inhibitors.

Sirtuin isoform 2 (SIRT2) is one of the seven sirtuin isoforms present in humans, being classified as class III histone deacetylases (HDACs). Based on the high sequence similarity among SIRTs, the identification of isoform selective modulators represents a challenging task, especially for the high conservation observed in the catalytic site. Efforts in rationalizing selectivity based on key residues belonging to the SIRT2 enzyme were accompanied in 2015 by the publication of the first X-ray crystallographic structure of the potent and selective SIRT2 inhibitor SirReal2. The subsequent studies led to different experimental data regarding this protein in complex with further different chemo-types as SIRT2 inhibitors. Herein, we reported preliminary Structure-Based Virtual Screening (SBVS) studies using a commercially available library of compounds to identify novel scaffolds for the design of new SIRT2 inhibitors. Biochemical assays involving five selected compounds allowed us to highlight the most effective chemical features supporting the observed SIRT2 inhibitory ability. This information guided the following in silico evaluation and in vitro testing of further compounds from in-house libraries of pyrazolo-pyrimidine derivatives towards novel SIRT2 inhibitors (1-5). The final results indicated the effectiveness of this scaffold for the design of promising and selective SIRT2 inhibitors, featuring the highest inhibition among the tested compounds, and validating the applied strategy.

Insights into the Pharmacological Activity of the Imidazo-Pyrazole Scaffold.

In previous studies, we synthesized different imidazo-pyrazoles 1 and 2 with interesting anticancer, anti-angiogenic and anti-inflammatory activities. To further extend the structure-activity relationships of imidazo-pyrazole scaffold and to identify novel antiproliferative/anti-inflammatory agents potentially active with multi-target mechanisms, a library of compounds 3-5 has been designed and synthesized. The chemical modifications characterizing the novel derivatives include: i) decoration of the catechol ring with groups with different electronic, steric and lipophilic properties (compounds 3); ii) insertion of a methyl group on C-6 of imidazo-pyrazole scaffold (compounds 4); iii) shift of the acylhydrazonic substituent from position 7 to 6 of the imidazo-pyrazole substructure (compounds 5). All synthesized compounds were tested against a panel of cancer and normal cell lines. Derivatives 3 a, 3 e, 4 c, 5 g and 5 h showed IC50 values in the low micromolar range against selected tumor cell lines and proved to have antioxidant properties, being able to inhibit ROS production in human platelet. In silico calculation predicted favourable drug-like and pharmacokinetic properties for the most promising compounds. Furthermore, molecular docking and molecular dynamic simulations suggested the ability of most active derivative 3 e to interact with colchicine binding site in the polymeric tubulin α/tubulin ß/stathmin4 complex.

Amino-Pyrazoles in Medicinal Chemistry: A Review.

A pyrazole nucleus is an easy-to-prepare scaffold with large therapeutic potential. Consequently, the search for new pyrazole-based compounds is of great interest to the academic community as well as industry. In the last ten years, a large number of papers and reviews on the design, synthesis, and biological evaluation of different classes of pyrazoles and many pyrazole-containing compounds have been published. However, an overview of pyrazole derivatives bearing a free amino group at the 3, 4, or 5 position (namely, 3-aminopyrazoles, 4-aminopyrazoles, and 5-aminopyrazoles, respectively) and their biological properties is still missing, despite the fact that aminopyrazoles are advantageous frameworks able to provide useful ligands for receptors or enzymes, such as p38MAPK, and different kinases, COX and others, as well as targets important for bacterial and virus infections. With the aim to fill this gap, the present review focuses on aminopyrazole-based compounds studied as active agents in different therapeutic areas, with particular attention on the design and structure-activity relationships defined by each class of compounds. In particular, the most relevant results have been obtained for anticancer/anti-inflammatory compounds, as the recent approval of Pirtobrutinib demonstrates. The data reported here are collected from different databases (Scifinder, Web of Science, Scopus, Google Scholar, and Pubmed) using "aminopyrazole" as the keyword.

Pyrazole-Enriched Cationic Nanoparticles Induced Early- and Late-Stage Apoptosis in Neuroblastoma Cells at Sub-Micromolar Concentrations.

Neuroblastoma (NB) is a severe form of tumor occurring mainly in young children and originating from nerve cells found in the abdomen or next to the spine. NB needs more effective and safer treatments, as the chance of survival against the aggressive form of this disease are very small. Moreover, when current treatments are successful, they are often responsible for unpleasant health problems which compromise the future and life of surviving children. As reported, cationic macromolecules have previously been found to be active against bacteria as membrane disruptors by interacting with the negative constituents of the surface of cancer cells, analogously inducing depolarization and permeabilization, provoking lethal damage to the cytoplasmic membrane, and cause loss of cytoplasmic content and consequently, cell death. Here, aiming to develop new curative options for counteracting NB cells, pyrazole-loaded cationic nanoparticles (NPs) (BBB4-G4K and CB1H-P7 NPs), recently reported as antibacterial agents, were assayed against IMR 32 and SHSY 5Y NB cell lines. Particularly, while BBB4-G4K NPs demonstrated low cytotoxicity against both NB cell lines, CB1H-P7 NPs were remarkably cytotoxic against both IMR 32 and SHSY 5Y cells (IC50 = 0.43-0.54 µM), causing both early-stage (66-85%) and late-stage apoptosis (52-65%). Interestingly, in the nano-formulation of CB1H using P7 NPs, the anticancer effects of CB1H and P7 were increased by 54-57 and 2.5-4-times, respectively against IMR 32 cells, and by 53-61 and 1.3-2 times against SHSY 5Y cells. Additionally, based on the IC50 values, CB1H-P7 was also 1-12-fold more potent than fenretinide, an experimental retinoid derivative in a phase III clinical trial, with remarkable antineoplastic and chemopreventive properties. Collectively, due to these results and their good selectivity for cancer cells (selectivity indices = 2.8-3.3), CB1H-P7 NPs represent an excellent template material for developing new treatment options against NB.

Antimicrobial Evaluation of New Pyrazoles, Indazoles and Pyrazolines Prepared in Continuous Flow Mode.

Multi-drug resistant bacterial strains (MDR) have become an increasing challenge to our health system, resulting in multiple classical antibiotics being clinically inactive today. As the de-novo development of effective antibiotics is a very costly and time-consuming process, alternative strategies such as the screening of natural and synthetic compound libraries is a simple approach towards finding new lead compounds. We thus report on the antimicrobial evaluation of a small collection of fourteen drug-like compounds featuring indazoles, pyrazoles and pyrazolines as key heterocyclic moieties whose synthesis was achieved in continuous flow mode. It was found that several compounds possessed significant antibacterial potency against clinical and MDR strains of the Staphylococcus and Enterococcus genera, with the lead compound (9) reaching MIC values of 4 µg/mL on those species. In addition, time killing experiments performed on compound 9 on Staphylococcus aureus MDR strains highlight its activity as bacteriostatic. Additional evaluations regarding the physiochemical and pharmacokinetic properties of the most active compounds are reported and showcased, promising drug-likeness, which warrants further explorations of the newly identified antimicrobial lead compound.

Inside the Mechanism of Action of Three Pyrazole Derivatives in Human Platelets and Endothelial Cells.

In the effort to obtain multitarget compound interfering with inflammation, oxidative stress, and tumorigenesis, we synthesized a small library of pyrazole compounds, selecting 4a, 4f, and 4g as the most noteworthy being IC50 against platelet ROS production induced by thrombin of about 10 µM. The in vitro antioxidant potential of the three molecules was evaluated, and since they show a remarkable antioxidative activity, their effect on several parameter indicative of oxidative status and on the efficiency of the aerobic metabolism was tested. The three molecules strongly inhibit superoxide anion production, lipid peroxidation, NADPH oxidase activity and almost restore the oxidative phosphorylation efficiency in thrombin-stimulated platelet, demonstrating a protective effect against oxidative stress. This effect was confirmed in endothelial cell in which 4a, 4f, and 4g show an interesting inhibition activity on H2O2-stimulated EA.hy926 cells. At last, antiproliferative activity of 4a, 4f, and 4g was submitted to a large screening at the NCI. The molecules show interesting anticancer activity, among them the most remarkable is 4g able to strongly inhibit the proliferation of both solid tumor and leukemia cells lines. In conclusion, all the three newly synthetized pyrazoles show remarkable antioxidant and antiproliferative effect worthy of further study.

Scouting Different Phosphodiesterase 4 Inhibitor Chemotypes in Silico To Guide the Design of Anti-inflammatory/Antioxidant Agents.

During the last years, we developed a large library of new selective phosphodiesterase 4D inhibitors, maintaining the catechol portion of the well-known PDE4 inhibitor Rolipram, featuring different substitutions in place of the lactam group of this reference compound. Based on the X-ray analysis of PDE4 inhibitors (PDE4Is) previously synthesized by us and of naphthyridine- and naphthyridinone-containing derivatives exhibiting PDE4 inhibitory ability described in the literature, we designed and synthesized new compounds 1-3. All of them were screened in silico as putative PDE4Is, via molecular docking studies to exploit structural variation at the catechol group to gain further contacts especially with the flat aromatic residues (Phe506 and Phe538) of enzyme. Subsequent in silico prediction of ADMET properties and in vitro biological assays on platelets and endothelial cells are in good agreement with our previous data concerning the antioxidant/anti-inflammatory activity exhibited by our previous PDE4Is and similarly to other well-known PDE4Is.