Synthesis, antiproliferative activity, and molecular modeling of novel 4-methylcoumarin derivatives and/or nitric oxide donor hybrids.

Two new 4-methylcoumarin derivatives (3a-f and 4a-f) were designed, synthesized, and evaluated for their cytotoxic activity. Different spectroscopic methods and elemental analyses confirmed all the synthesized derivatives' characterization. All the prepared compounds were biologically screened against four cancer cell lines (hepatocellular carcinoma HepG-2, colon cancer cell lines HCT-116, breast cancer cell lines MCF-7, and prostate cancer cell lines PC3). The in vitro antiproliferative activity of the target analogues 4b, 4c, 4f, 3b, and 3d against the MCF-7 cancer cell line was significant, with IC50 values of 3.98, 7.80, 10.94, 17.7, and 24.07 µM, respectively. Furthermore, the potent cytotoxic oxime derivative 4b was evaluated for cell cycle analysis showing a significant substantial disruption in cell cycle profile and cell cycle arrest at the S phase boundary with a time-dependent rise in a pre-G cell population, as well as a 22-fold increase in MCF-7 apoptosis compared to control cells. Accordingly, the Bax/Bcl-2 ratio, a critical ratio in controlling cell sensitivity to apoptosis, increased upon treatment with the oxime analog 4b. A docking investigation was conducted within the BcL-2 binding site to explore and anticipate the binding modes of the synthesized compounds. Thus, synthesizing these novel coumarin/nitric oxide hybrids may aid in developing promising antiproliferative agents.

Regioselective Synthesis of NO-Donor (4-Nitro-1,2,3-triazolyl)furoxans via Eliminative Azide-Olefin Cycloaddition.

A facile and efficient method for the regioselective [3 + 2] cycloaddition of 4-azidofuroxans to 1-dimethylamino-2-nitroethylene under p-TSA catalysis affording (4-nitro-1,2,3-triazolyl)furoxans was developed. This transformation is believed to proceed via eliminative azide-olefin cycloaddition resulting in its complete regioselectivity. The developed protocol has a broad substrate scope and enables a straightforward assembly of the 4-nitro-1,2,3-triazole motif. Moreover, synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations, thus providing a novel platform for future drug design and related biomedical applications of heterocyclic NO donors.

NO in Viral Infections: Role and Development of Antiviral Therapies.

Nitric oxide is a ubiquitous signaling radical that influences critical body functions. Its importance in the cardiovascular system and the innate immune response to bacterial and viral infections has been extensively investigated. The overproduction of NO is an early component of viral infections, including those affecting the respiratory tract. The production of high levels of NO is due to the overexpression of NO biosynthesis by inducible NO synthase (iNOS), which is involved in viral clearance. The development of NO-based antiviral therapies, particularly gaseous NO inhalation and NO-donors, has proven to be an excellent antiviral therapeutic strategy. The aim of this review is to systematically examine the multiple research studies that have been carried out to elucidate the role of NO in viral infections and to comprehensively describe the NO-based antiviral strategies that have been developed thus far. Particular attention has been paid to the potential mechanisms of NO and its clinical use in the prevention and therapy of COVID-19.

Exploring Nitric Oxide (NO)-Releasing Celecoxib Derivatives as Modulators of Radioresponse in Pheochromocytoma Cells.

COX-2 can be considered as a clinically relevant molecular target for adjuvant, in particular radiosensitizing treatments. In this regard, using selective COX-2 inhibitors, e.g., in combination with radiotherapy or endoradiotherapy, represents an interesting treatment option. Based on our own findings that nitric oxide (NO)-releasing and celecoxib-derived COX-2 inhibitors (COXIBs) showed promising radiosensitizing effects in vitro, we herein present the development of a series of eight novel NO-COXIBs differing in the peripheral substitution pattern and their chemical and in vitro characterization. COX-1 and COX-2 inhibition potency was found to be comparable to the lead NO-COXIBs, and NO-releasing properties were demonstrated to be mainly influenced by the substituent in 4-position of the pyrazole (Cl vs. H). Introduction of the N-propionamide at the sulfamoyl residue as a potential prodrug strategy lowered lipophilicity markedly and abolished COX inhibition while NO-releasing properties were not markedly influenced. NO-COXIBs were tested in vitro for a combination with single-dose external X-ray irradiation as well as [177Lu]LuCl3 treatment in HIF2α-positive mouse pheochromocytoma (MPC-HIF2a) tumor spheroids. When applied directly before X-ray irradiation or 177Lu treatment, NO-COXIBs showed radioprotective effects, as did celecoxib, which was used as a control. Radiosensitizing effects were observed when applied shortly after X-ray irradiation. Overall, the NO-COXIBs were found to be more radioprotective compared with celecoxib, which does not warrant further preclinical studies with the NO-COXIBs for the treatment of pheochromocytoma. However, evaluation as radioprotective agents for healthy tissues could be considered for the NO-COXIBs developed here, especially when used directly before irradiation.

Effects of albumin-bound nitrosyl iron complex with thiosulfate ligands on lipid peroxidation and activities of mitochondrial enzymes in vitro.

Nitric oxide (NO) mediates diverse physiological processes in living organisms. Small molecular NO donors usually lack stability and have a short half-life in human tissues, limiting the therapeutic application. The anionic tetranitrosyl iron complex with thiosulfate ligands (TNIC) is one of the most promising NO donors. This study shows that bovine serum albumin (BSA) can effectively stabilize the TNIC complex under aerobic (physiological) conditions, which contributes to its prolonged action as NO donor. Our results demonstrated that TNIC-BSA inhibits formation of TBARS - standard biomarker for the lipid peroxidation induced oxidative stress. Also, it was found that TNIC-BSA inhibits the catalytic activity of mitochondrial membrane-bound enzymes: cytochrome c oxidase and monoamine oxidase A. Together, these results demonstrate that, stabilization of TNIC with BSA opens up the possibility of its practical application in chemotherapy of socially significant diseases.

Synthesis and pharmacological evaluation of pomalidomide derivatives useful for sickle cell disease treatment.

Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide-nitric oxide (NO) donor derivatives (3a-f) and evaluated their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging 0.3-30.3%. Compound 3d was the most effective HbF inducer for CD34+ cells, exhibiting an effect similar to that of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-α levels in human mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer prototype with the potential to treat SCD symptoms.

Tuning NO release of organelle-targeted furoxan derivatives and their cytotoxicity against lung cancer cells.

We herein report a study on a set of hybrid compounds in which 3-R-substituted furoxan moieties (R = CH3, CONH2, CN, SO2C6H5), endowed with varying NO-releasing capacities, are joined to a mitochondrial probe, rhodamine B. Each product has been investigated for its ability to release NO both in physiological solution, in the presence of cysteine, and in A549 lung adenocarcinoma cancer cells. The cytotoxicity of all the products against the aforementioned cancer cells has been assessed, including the structurally related compounds with no mitochondrial targeting, which were taken as a reference. In the case of the models bearing the -CH3 and -CONH2 groups at the 3-position on the furoxan, only the targeted models showed a significant cytotoxic activity, and only at the highest concentrations, in accordance with their weak NO-releasing properties. On the contrary, the presence of the strong electron-withdrawing groups, as -CN and -SO2C6H5, at the 3-position gave rise to anticancer agents, likely because of the high NO-releasing and of their capability of inhibiting cellular proteins by covalent binding. In detail, the rhodamine hybrid containing the 3-SO2C6H5 substituted furoxan moiety emerged as the most interesting product as it showed high cytotoxicity over the entire concentration range tested. This substructure was also linked to a phenothiazine scaffold that is able to accumulate in lysosomes. Nevertheless, mitochondrial targeting for these NO-donor furoxan substructures was found to be the most efficient.

Glyceryl Trinitrate: History, Mystery, and Alcohol Intolerance.

Glyceryl trinitrate (GTN) is one of the earliest known treatments for angina with a fascinating history that bridges three centuries. However, despite its central role in the nitric oxide (NO) story as a NO-donating compound, establishing the precise mechanism of how GTN exerts its medicinal benefit has proven to be far more difficult. This review brings together the explosive and vasodilatory nature of this three-carbon molecule while providing an update on the likely in vivo pathways through which GTN, and the rest of the organic nitrate family, release NO, nitrite, or a combination of both, while also trying to explain nitrate tolerance. Over the last 20 years the alcohol detoxification enzyme, aldehyde dehydrogenase (ALDH), has undoubtedly emerged as the front runner to explaining GTN's bioactivation. This is best illustrated by reduced GTN efficacy in subjects carrying the single point mutation (Glu504Lys) in ALDH, which is also responsible for alcohol intolerance, as characterized by flushing. While these findings are significant for anyone following the GTN story, they appear particularly relevant for healthcare professionals, and especially so, if administering GTN to patients as an emergency treatment. In short, although the GTN puzzle has not been fully solved, clinical study data continue to cement the importance of ALDH, as uncovered in 2002, as a key GTN activator.

The Application of Nitric Oxide for Ocular Hypertension Treatment.

Despite of various therapeutic methods for treating ocular hypertension and glaucoma, it still remains the leading cause of irreversible blindness. Intraocular pressure (IOP) lowering is the most effective way to slow disease progression and prevent blindness. Among the ocular hypotensive drugs currently in use, only a couple act on the conventional outflow system, which is the main pathway for aqueous humor outflow and the major lesion site resulting in ocular hypertension. Nitric oxide (NO) is a commendable new class of glaucoma drugs that acts on the conventional outflow pathway. An increasing number of nitric oxide donors have been developed for glaucoma and ocular hypertension treatment. Here, we will review how NO lowers IOP and the types of nitric oxide donors that have been developed. And a brief analysis of the advantages and challenges associated with the application will be made. The literature used in this review is based on Pubmed database search using 'nitric oxide' and 'glaucoma' as key words.

Straightforward Access to the Nitric Oxide Donor Azasydnone Scaffold by Cascade Reactions of Amines.

A novel one-pot cascade method for the assembly of valuable NO-donor azasydnone scaffold has been developed. The construction strategy involves a diazotization/azo coupling/elimination/double rearrangement cascade sequence of readily available amines. The current protocol enables the generation of a diverse array of azasydnones, including previously hardly accessible heteroaryl substituted azasydnones (25 examples, 70-97 % yield) with a good functional group tolerance under very mild conditions. Preliminary NO-releasing studies revealed an ability of azasydnones to produce NO in a wide range of concentrations. This method provides a new approach to nitrogen-oxygen heterocycles with potential applications in medicine and material science.

New generation of nitric oxide-releasing porous materials: Assessment of their potential to regulate biological functions.

Nitric oxide (NO) presents innumerable biological roles, and its exogenous supplementation for therapeutic purposes has become a necessity. Some nanoporous materials proved to be potential vehicles for NO with high storage capacity. However, there is still a lack of information about their efficiency to release controlled NO and if they are biocompatible and biologically stable. In this work, we address this knowledge gap starting by evaluating the NO release and stability under biological conditions and their toxicity with primary keratinocyte cells. Titanosilicates (ETS-4 and ETS-10 types) and clay-based materials were the materials under study, which have shown in previous studies suitable NO gas adsorption/release rates. ETS-4 proved to be the most promising material, combining good biocompatibility at 180 µg/mL, stability and slower NO release. ETS-10 and ETAS-10 showed the best biocompatibility at the same concentration and, in the case of clay-based materials, CoOS is the least toxic of those tested and the one that releases the highest NO amount. The potentiality of these new NO donors to regulate biological functions was assessed next by controlling the mitochondrial respiration and the cell migration. NO-loaded ETS-4 regulates O2 consumption and cell migration in a dose-dependent manner. For cell migration, a biphasic effect was observed in a narrow range of ETS-4 concentration, with a stimulatory effect becoming inhibitory just by doubling ETS-4 concentration. For the other materials, no effective regulation was achieved, which highlights the relevance of the new assessment presented in this work for nanoporous NO carriers that will pave the way for further developments.

Does Pentaerytrithyltetranitrate reduce fetal growth restriction in pregnancies complicated by uterine mal-perfusion? Study protocol of the PETN-study: a randomized controlled multicenter-trial.

BACKGROUND: Affecting approximately 10% of pregnancies, fetal growth restriction (FGR), is the most important cause of perinatal mortality and morbidity. Impaired placental function and consequent mal-perfusion of the placenta is the leading cause of FGR. Although, screening for placental insufficiency based on uterine artery Doppler measurement is well established, there is no treatment option for pregnancies threatened by FGR. The organic nitrate pentaerithrityl tetranitrate (PETN) is widely used for the treatment of cardiovascular disease and has been shown to have protective effects on human endothelial cells. In a randomized placebo controlled pilot-study our group could demonstrate a risk reduction of 39% for the development of FGR, and FGR or death, by administering PETN to patients with impaired uterine artery Doppler at mid gestation. To confirm these results a prospective randomized placebo controlled double-blinded multicentre trial was now initiated. METHOD: The trial has been initiated in 14 centres in Germany. Inclusion criteria are abnormal uterine artery Doppler, defined by mean PI > 1.6, at 190 to 226 weeks of gestation in singleton pregnancies. Included patients will be monitored in 4-week intervals. Primary outcome measures are development of FGR (birth weight < 10th percentile), severe FGR (birth weight < 3rd centile) and perinatal death. Placental abruption, birth weight below the 3rd, 5th and 10th centile, development of FGR requiring delivery before 34 weeks` gestation, neonatal intensive care unit admission, and spontaneous preterm delivery < 34 weeks` and 37 weeks` gestation will be assessed as secondary endpoints. Patient enrolment was started in August 2017. Results are expected in 2020. DISCUSSION: During the past decade therapeutic agents with possible perfusion optimizing potential have been evaluated in clinical trials to treat FGR. Meta-analysis and sub-analysis of trials targeting preeclampsia revealed ASS to have a potential in reducing FGR. Phosphodiesterase-type-5 inhibitors have recently been tested in a worldwide RCT for therapy of established FGR, failing to show an effect on neonatal outcome. The ongoing multicenter trial will, by confirming our previous results, finally provide a therapeutic option in cases at risk for FGR. TRIAL REGISTRATION: DRKS00011374 registered at September 29th, 2017 and NCT03669185 , registered September 13th, 2018.

Design, Synthesis and Study of Nitrogen Monoxide Donors as Potent Hypolipidaemic and Anti-Inflammatory Agents.

Inflammation and oxidative stress are involved in cardiovascular diseases. Nitrogen monoxide participates in the regulation of endothelial processes. Thus, derivatives of classic nonsteroidal anti-inflammatory drugs (NSAIDs), trolox or cinnamic acids esterified with 2-(nitrooxy)ethanol were designed and studied. It was found that the nitrogen monoxide (NO) releasing activity was comparable to that of S-nitroso-N-acetylpenicillamine. The nitrooxy derivatives decreased potently lipid indices in the plasma of hyperlipidaemic rats (30-85%). All compounds presented increased anti-inflammatory activity in vivo, inhibiting carrageenan-induced rat paw oedema as high as 76%, up to six times higher than that of the parent acids. Lipoxygenase inhibitory activity was significant for most of them, although the parent molecules exerted a minor effect (IC50 > 0.2 mM). Those compounds incorporating an antioxidant structure inhibited rat microsomal membrane lipid peroxidation strongly and possessed radical scavenging activity. These results indicated that the described compounds could act at different targets in multifactorial diseases, further limiting the possible adverse effects of drug combinations.

Combination Therapy with DETA/NO and Clopidogrel Inhibits Metastasis in Murine Mammary Gland Cancer Models via Improved Vasoprotection.

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-ß plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-ß were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.

An update on emerging drugs for the treatment of erectile dysfunction.

Introduction: Erectile dysfunction is an extremely frequent and extensively studied condition, currently affecting the lives of tens of millions of men around the globe. The extensive knowledge of its pathophysiology has led to the development of phosphodiesterase 5-inhibitors, which can facilitate sexual intercourse in a large number of patients. However, an ever-increasing number of patients is unresponsive to these drugs due to underlying comorbidities or previous surgery. Different molecular pathways need to be addressed to provide treatment for a larger patient population. Areas covered: In this paper, we will review the underlying molecular pathways, discuss already available treatment options and their limitations and provide an overview of the newest therapeutics in development. Centrally and peripherally acting agents will be discussed separately. Additionally, newest advances in regenerative medicine options will be discussed. Expert opinion: Even though novel drugs have not been tested in a phase III setting, several phase II clinical trial results are eagerly awaited. These newest therapeutics could be applied as monotherapy or combination therapy in the subset of patients unresponsive to traditional treatment options.

Novel primary amine diazeniumdiolates-Chemical and biological characterization.

Hit, Lead & Candidate Discovery Diazeniumdiolates, also known as NONOates, are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release nitric oxide (NO. ) and/or their congeneric nitroxyl (HNO). The purpose of this work was to synthesize a series of primary amine-based diazeniumdiolates as HNO/NO donors and to determine their efficacy as anticancer and antifungal agents in vivo. The seven compounds (3a-3g) were successfully synthesized and characterized, one of which had been previously reported in the literature (3g). Two compounds showed anti-proliferative effects against ovarian (ES2 and SKOV3) and AML monocyte-derived cancer cells (THP-1) when tested with standard MTT assays. Compounds 3a and 3g demonstrated reduced ovarian cancer cell proliferation when treated at doses from 0.033 to 1.0 mg/mL at the 24 hr time point. These compounds also exhibited moderate and selective antifungal activity against Fusarium oxysporum f.sp. lycopersici, one cause of opportunistic infections of immunocompromised patients, inhibiting the growth of the fungi at LD50 at 10 mg/mL. A third compound (3e) did not exhibit similar activities, possibly due to the alkyl chain. Our results suggest that the primary amine diazeniumdiolates may offer a versatile platform for the development of HNO/NO donors for biomedical applications.

Nitric oxide donors increase the pregnancy rate in patients with unexplained infertility undergoing clomiphene citrate stimulation and intrauterine insemination: a randomized controlled pilot study.

This study evaluated the effects of nitric oxide donor's treatment on the pregnancy rate and uterine blood flow in patients with unexplained infertility undergoing clomiphene citrate stimulation and intrauterine insemination. A total of 120 patients were randomly allocated to a control group who received 100 mg clomiphene citrate daily from day 5 to 9 of cycle plus placebo vaginal tablets, and a study group received clomiphene citrate plus isosorbide mononitrate 10 mg vaginal tablets. Vaginal ultrasound was done before treatment and every other day starting from day 12 of cycle to count mature follicles and ovulation was triggered by IM injection of 10 000 IU hCG when one follicle measured 18 ≥ mm followed by intrauterine insemination after 36 h. The endometrial thickness, uterine arteries resistance and pulsation indices, and endometrial vascular flow and vascular flow indices were measured before treatment and at day of hCG injection. Results were analyzed after one cycle treatment using the Mean ± SD, the Student t test and the Fisher Exact test. Significant result was considered at p values <0.05. The study group had significant higher pregnancy rate/cycle, higher endometrial and lower uterine artery blood flow indices (p < 0.05).

Crystal structure of bis-{2-[(E)-(4-meth-oxy-lbenz-yl)imino-meth-yl]phenolato-κ(2) N,O (1)}nickel(II).

The asymmetric unit of the title compound, [Ni(C15H14NO2)2], comprises an Ni(II) cation, lying on an inversion centre, and a Schiff base anion that acts as a bidentate ligand. The Ni(II) cation is in a square-planar coordination environment binding to the imine N and phenolate O atoms of the two Schiff base ligands. The N- and O-donor atoms of the two ligands are mutually trans, with Ni-N and Ni-O bond lengths of 1.9191 (11) and 1.8407 (9) Å, respectively. The plane of the meth-oxy-benzene ring makes a dihedral angle of 84.92 (6)° with that of the phenolate ring. In the crystal, mol-ecules are linked into screw chains by weak C-H⋯O hydrogen bonds. Additional C-H⋯O hydrogen bonds, together with C-H⋯π contacts, arrange the mol-ecules into sheets parallel to the ac plane.

Crystal structure of bis-{2-[(E)-(4-fluoro-benz-yl)imino-meth-yl]phenolato-κ(2) N,O}nickel(II).

The asymmetric unit of the title complex, [Ni(C14H11FNO)2], contains one-half of the mol-ecule with the Ni(II) cation lying on an inversion centre coordinated by a bidentate Schiff base anion. The cationic Ni(II) center is in a distorted square-planar coordination environment chelated by the imine N and phenolate O donor atoms of the two Schiff base ligands. The N and O donor atoms of the two ligands are mutually trans with Ni-N and Ni-O bond lengths of 1.9242 (10) and 1.8336 (9) Å, respectively. The fluoro-phenyl ring is almost orthogonal to the coordination plane and makes a dihedral angle of 82.98 (7)° with the phenolate ring. In the crystal, mol-ecules are linked into screw chains by weak C-H⋯F hydrogen bonds. Additional C-H⋯π contacts arrange the mol-ecules into sheets parallel to the ac plane.

Sunlight-triggered prebiotic nanomotors for inhibition and elimination of pathogen and biofilm in aquatic environment.

Pathogen contamination in drinking water sources causes waterborne infectious diseases, seriously threatening human health. Nowadays, stimuli-responsive self-propelled nanomotors are appealing therapeutic agents for antibacterial therapy in vivo. However, achieving water disinfection using these nanobots is still a great challenge. Herein, we report on prebiotic galactooligosaccharide-based nanomotors for sunlight-regulated water disinfection. The nanomotors can utilize galactooligosaccharide-based N-nitrosamines as sunlight-responsive fuels for the spontaneous production of antibacterial nitric oxide. Such a solar-to-chemical energy conversion would power the nanomotors for self-diffusiophoresis, which could promote the diffusion of the nanomotors in water and their penetration in the biofilm, significantly enhancing the inhibition and elimination of the pathogens and their biofilms in aquatic environments. After water treatments, the prebiotic-based residual disinfectants can be selectively utilized by beneficial bacteria to effectively relieve safety risks to the environment and human health. The low-energy-cost, green and potent antibacterial nanobots show promising potential in water disinfection.