Pills of Multi-Target H2S Donating Molecules for Complex Diseases.

Among the various drug discovery methods, a very promising modern approach consists in designing multi-target-directed ligands (MTDLs) able to modulate multiple targets of interest, including the pathways where hydrogen sulfide (H2S) is involved. By incorporating an H2S donor moiety into a native drug, researchers have been able to simultaneously target multiple therapeutic pathways, resulting in improved treatment outcomes. This review gives the reader some pills of successful multi-target H2S-donating molecules as worthwhile tools to combat the multifactorial nature of complex disorders, such as inflammatory-based diseases and cancer, as well as cardiovascular, metabolic, and neurodegenerative disorders.

3-Nitroatenolol: First Synthesis, Chiral Resolution and Enantiomers' Absolute Configuration.

4-Nitro and 7-nitro propranolol have been recently synthesized and characterized by us. (±)-4-NO2-propranolol has been shown to act as a selective antagonist of 6-nitrodopamine (6-ND) receptors in the right atrium of rats. As part of our follow-up to this study, herein, we describe the first synthesis of (±)-3-nitroatenolol as a probe to evaluate the potential nitration of atenolol by endothelium. Chiral chromatography was used to produce pure enantiomers. By using Riguera's method, which is based on the sign distribution of ΔδH, the absolute configuration of the secondary alcohol was determined.

Design, Synthesis and Evaluation of Novel Molecular Hybrids between Antiglaucoma Drugs and H2S Donors.

Glaucoma is a group of eye diseases consisting of optic nerve damage with corresponding loss of field vision and blindness. Hydrogen sulfide (H2S) is a gaseous neurotransmitter implicated in various pathophysiological processes. It is involved in the pathological mechanism of glaucomatous neuropathy and exerts promising effects in the treatment of this disease. In this work, we designed and synthetized new molecular hybrids between antiglaucoma drugs and H2S donors to combine the pharmacological effect of both moieties, providing a heightened therapy. Brinzolamide, betaxolol and brimonidine were linked to different H2S donors. The H2S-releasing properties of the new compounds were evaluated in a phosphate buffer solution by the amperometric approach, and evaluated in human primary corneal epithelial cells (HCEs) by spectrofluorometric measurements. Experimental data showed that compounds 1c, 1d and 3d were the hybrids with the best properties, characterized by a significant and long-lasting production of the gasotransmitter both in the aqueous solution (in the presence of L-cysteine) and in the intracellular environment. Because, to date, the donation of H2S by antiglaucoma H2S donor hybrids using non-immortalized corneal cells has never been reported, these results pave the way to further investigation of the potential efficacy of the newly synthesized compounds.

Synthesis, Chiral Resolution and Enantiomers Absolute Configuration of 4-Nitropropranolol and 7-Nitropropranolol.

We recently identified 6-nitrodopamine and other nitro-catecholamines (6-nitrodopa, 6-nitroadrenaline), indicating that the endothelium has the ability to nitrate the classical catecholamines (dopamine, noradrenaline, and adrenaline). In order to investigate whether drugs could be subject to the same nitration process, we synthesized 4-nitro- and 7-nitropropranolol as probes to evaluate the possible nitration of the propranolol by the endothelium. The separation of the enantiomers in very high yields and excellent enantiopurity was achieved by chiral HPLC. Finally, we used Riguera's method to determine the absolute configuration of the enantiomers, through double derivatization with MPA and NMR studies.

Synthesis, Docking Studies and Pharmacological Evaluation of Serotoninergic Ligands Containing a 5-Norbornene-2-Carboxamide Nucleus.

A new series of 5-norbornene-2-carboxamide derivatives was prepared and their affinities to the 5-HT1A, 5-HT2A, and 5-HT2C receptors were evaluated and compared to a previously synthesized series of derivatives characterized by exo-N-hydroxy-5-norbornene-2,3-dicarboximidenucleus, in order to identify selective ligands for the above-mentioned subtype receptors. Arylpiperazines represents one of the most important classes of 5-HT1AR ligands, and recent research concerning new derivatives has been focused on the modification of one or more portions of such pharmacophore. The combination of structural elements (heterocyclic nucleus, propyl chain and 4-substituted piperazine), known to be critical to the affinity to 5-HT1A receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that Norbo-4 and Norbo-18 were the most active and promising derivatives for the serotonin receptor considered in this study.

Synthesis, docking studies, and pharmacological evaluation of 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands.

A new series of norbornene and exo-N-hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide derivatives was prepared, and their affinities to the 5-HT1A , 5-HT2A , and 5-HT2C receptors were evaluated and compared with a previously synthesized series of derivatives characterized by the same nuclei, to identify selective ligands for the subtype receptors. Arylpiperazines represent one of the most important classes of 5-HT1A R ligands, and the research of new derivatives has been focused on the modification of one or more portions of this pharmacophore. The combination of structural elements (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine), known to be critical for the affinity to 5-HT1A receptors, and the proper selection of substituents resulted in compounds with high specificity and affinity toward serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that 3e, 4j, and 4n were the most active and promising derivatives for the serotonin receptor considered in this study.

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis.

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists.

Synthesis, docking studies, and pharmacological evaluation of 5HT2C ligands containing the N'-cyanoisonicotinamidine or N'-cyanopicolinamidine nucleus.

N'-Cyanoisonicotinamidine and N'-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5-HT1A , 5-HT2A , and 5-HT2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5-HT1A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1 , D2 , α1 , and α2 ). Compound 8e (Ki = 21.4 nM) was the most affine for the 5-HT2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c, instead, showed an interesting mixed 5-HT1A /5-HT2C activity with Ki values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5-HT2C (4a, 4c, 8b, and 8e) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a, 8b, and 8e exerted antidepressant-like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a, which displayed antipsychotic-, antidepressant- and anxiolytic-like properties. No side effects, like catalepsy, motor-impairment or ethanol-potentiating effects, were observed after the injection of the tested compounds.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

New 5-HT1A, 5HT2A and 5HT2C receptor ligands containing a picolinic nucleus: Synthesis, in vitro and in vivo pharmacological evaluation.

Picolinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors was evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine), known to play critical roles in affinity for serotoninergic receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide (3o) with Ki=0.046nM, was the most affine and selective derivative for the 5-HT1A receptor compared to other serotoninergic dopaminergic and adrenergic receptors. N-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)picolinamide (3b), instead, showed a subnanomolar affinity towards 5-HT2A with Ki=0.0224nM, whereas N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)ethyl)picolinamide (3s) presented an attractive 5-HT2C affinity with Ki=0.8nM. Moreover, the compounds having better affinity and selectivity binding profiles towards 5-HT2A were selected and tested on rat ileum, to determine their effect on 5HT induced contractions. Those more selective towards 5-HT1A receptors were studied in vivo on several behavioral tests.

Development of 1,2,3-Triazole-Based Sphingosine Kinase Inhibitors and Their Evaluation as Antiproliferative Agents.

Two series of N-(aryl)-1-(hydroxyalkyl)pyrrolidine-2-carboxamides (2a-2g and 3a-3g) and 1,4-disubstituted 1,2,3-triazoles (5a-5h and 8a-8h) were synthesized. All the compounds, containing a lipophilic tail and a polar headgroup, were evaluated as sphingosine kinase (SphK) inhibitors by assessing their ability to interfere with the acetylcholine (Ach) induced relaxation of aortic rings pre-contracted with phenylephrine. Moreover, their antiproliferative activity was tested on several cell lines expressing both SphK1 and SphK2. Compounds 5h and 8f, identified as the most efficient antiproliferative agents, showed a different selectivity profile, with 8f being selective for SphK1.

Synthesis, biological evaluation, and docking studies of PAR2-AP-derived pseudopeptides as inhibitors of kallikrein 5 and 6.

A series of protease activated receptor 2 activating peptide (PAR2-AP) derivatives (1-15) were designed and synthesized. The obtained compounds were tested on a panel of human kallikreins (hKLK1, hKLK2, hKLK5, hKLK6, and hKLK7) and were found completely inactive toward hKLK1, hKLK2, and hKLK7. Aiming to investigate the mode of interaction between the most interesting compounds and the selected hKLKs, docking studies were performed. The described compounds distinguish the different human tissue kallikreins with compounds 1 and 5 as the best hKLK5 and hKLK6 inhibitors, respectively.

Synthesis and in vitro pharmacological evaluation of novel 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands.

This paper reports the synthesis of new norbornene and exo-N-hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide derivatives and their binding to the 5-HT1A , 5-HT2A , and 5-HT2C receptors, in order to identify selective ligands for these 5-hydroxytryptamine (5-HT, serotonine) receptor subtypes. The combination of structural elements (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine) known to be critical for affinity to 5-HT1A receptors and the proper selection of substituents led to compounds with high specificity and affinity toward serotoninergic receptors. The most active compounds were selected and further evaluated for their binding affinities to D1 , D2 dopaminergic and α1 , α2 adrenergic receptors. 4-[3-[4-(2-Furoyl)piperazin-1-yl]propoxy-2-ol]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione 3e with Ki = 5.04 ± 0.227 nM was the most active and selective derivative for the 5-HT2C receptor with respect to other serotonin receptors, and the most selective derivative versus dopaminergic and adrenergic receptors.

Isothiocyanate-Corticosteroid Conjugates against asthma: Unity makes strength.

Asthma is a major noncommunicable disease, affecting both children and adults, and represents one of the major causes leading to high health care costs due to the need for chronic pharmacological treatments. The standard gold therapy of inflammation in asthmatic patients involves the use of glucocorticoids even if their chronic use is often related to serious adverse effects. Growing evidence suggests the biological relevance of hydrogen sulfide (H2S) in the pathogenesis of airway diseases. Hence, aiming to associate the beneficial effects of steroidal anti-inflammatory drugs (SAIDs) to H2S biological activity, we designed and synthesized novel multi-target molecules by chemically combining a group of glucocorticoids, usually employed in asthma treatment, with an isothiocyanate moiety, well-known for its H2S releasing properties. Firstly, the synthesized compounds have been screened for their H2S-releasing profile using an amperometric approach and for their in vitro effects on the degranulation process, using RBL-2H3 cell line. The physicochemical profile, in terms of solubility, chemical and enzymatic stability of the newly hybrid molecules, has been assessed at different physiological pH values and in esterase-rich medium (bovine serum albumin, BSA). The selected compound 5c, through both its corticosteroid and H2S releasing component, has been evaluated in vivo in experimental model of asthma. The compound 5c inhibited in vivo all asthma features with a significative effect on the restoration of pulmonary structure and reduction of lung inflammation.

Identification of a pepducin acting as S1P3 receptor antagonist.

Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

Solomonsterol A, a marine pregnane-X-receptor agonist, attenuates inflammation and immune dysfunction in a mouse model of arthritis.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

Prednisone-hydrogen sulfide releasing hybrid shows improved therapeutic profile in asthma.

Introduction: Hydrogen sulfide (H2S) is emerging as an important potential therapeutic option for respiratory inflammatory diseases. In this study, we investigated the effectiveness of a novel corticosteroid derivative, that is chemically linked to an H2S donor, in managing asthma features. Methods: The effects of prednisone (PS), H2S donor (4-hydroxybenzamide; TBZ), and their combination (PS-TBZ) have been evaluated in vitro and in vivo. The in vitro experiments were conducted using lipopolysaccharidestimulated J774 macrophages, while the in vivo experiments utilizing an experimental asthma model. Results: In the in vitro study we found that PS-TBZ exhibited an increased effect compared to the individual parent compounds in modulating the production of inflammatory mediators. TBZ also significantly reduced bronchial contractility and enhanced bronchial relaxation. In the in vivo experiments, where we administered PS, TBZ, or PS-TBZ to ovalbumin-sensitized BALB/c mice, we confirmed that PS-TBZ had a significantly better action in controlling airway hyperreactivity as compared to TBZ or PS alone. Moreover, PS-TBZ was more effective in restoring salbutamol-induced relaxation. The immunohistochemistry analysis demonstrated a significant reduction in the production of α-SMA and procollagen III, indicating the efficacy of PS-TBZ in controlling airway remodeling. Moreover, PS-TBZ also promoted epithelial repair, recovery of the bronchial and parenchyma structure and inhibited mucin production. Discussion: In conclusion, PS-TBZ offers an important opportunity to optimize the beneficial impact of corticosteroids on asthma features.

Design, Synthesis and Biological Evaluation of Novel N-Arylpiperazines Containing a 4,5-Dihydrothiazole Ring.

Arylpiperazines represent one of the most important classes of 5-HT1AR ligands and have attracted considerable interests for their versatile properties in chemistry and pharmacology, leading to the research of new derivatives that has been focused on the modification of one or more portions of such pharmacophore. An efficient protocol for the synthesis of novel thiazolinylphenyl-piperazines (2a-c) and the corresponding acetylated derivatives was used (3a-c). The new compounds were tested for their functional activity and affinity at 5-HT1A receptors, showing an interesting affinity profile with a Ki value of 412 nM for compound 2b. The cytotoxic activity of novel thiazolinylphenyl-piperazines (2a-c) and corresponding N-acetyl derivatives (3a-c) against human prostate and breast cancer cell lines (LNCAP, DU-145 and PC-3, MCF-7, SKBR-3 and MDA-MB231) was investigated according to the procedure described in the literature. The reported data showed a cytotoxic effect for 2a-c and 3a-c compounds (IC50 values ranging from 15 µM to 73 µM) on the investigated cancer cell lines, with no effect on noncancer cells. Future studies will be aimed to investigate the mechanism of action and therapeutic prospects of these new scaffolds.

Synthesis of benzamide derivatives and their evaluation as antiprion agents.

A new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substituted)benzamide and 5-(2-(piperidin-1-yl)acetamido)-N-butyl-2-(substituted) benzamide derivatives were synthesized in which as structural features the 2-(1-pyrrolidinyl)- or 2-(1-piperidyl)acetylamino group or a diphenylether moiety are associated to a benzamide scaffold. Their binding affinity for human PrP(C) and inhibition of its conversion into PrP(Sc) were determined in vitro; moreover, the antiprion activity was assayed by inhibition of PrP(Sc) accumulation in scrapie-infected mouse neuroblastoma cells (ScN2a) and scrapie mouse brain (SMB) cells. The results clearly indicate the benzamide derivatives as attractive lead compounds for the development of potential therapeutic agents against prion disease.

H2S donating corticosteroids: Design, synthesis and biological evaluation in a murine model of asthma.

Introduction: Hydrogen sulfide (H2S) is a fundamental biological endogenous gas-mediator in the respiratory system. It regulates pivotal patho-physiological processes such as oxidative stress, pulmonary circulation, airway tone and inflammation. Objectives: We herein describe the design and synthesis of molecular hybrids obtained by the condensation of several corticosteroids with different hydrogen sulfide releasing moieties. Methods: All the molecules are characterized for their ability to release H2S both via amperometric approach and using a fluorescent probe. The chemical stability of the newly synthesized hybrid molecules has been investigated at differing pH values and in human serum. Results: Prednisone-TBZ hybrid (compound 7) was selected for further evaluations. The obtained results from the in vitro and in vivo studies clearly show evidence in favor of the anti-inflammatory properties of the released H2S. Conclusions: The protective effect on airway remodeling makes the hybrid Prednisone-TBZ (compound 7) as a promising therapeutic option in reducing allergic asthma symptoms and exacerbations.