A Novel Prodrug of a nNOS Inhibitor with Improved Pharmacokinetic Potential.

Under different pathological conditions, aberrant induction of neuronal nitric oxide synthase (nNOS) generates overproduction of NO that can cause irreversible cell damage. The aim of this study was to develop an amidoxime prodrug of a potent nNOS inhibitor, the benzhydryl acetamidine. We synthesized the benzhydryl acetamidoxime, which was evaluated in vitro to ascertain the potential NOS inhibitory activity, as well as conducting bioconversion into the parent acetamidine. The prodrug was also profiled for in vitro physicochemical properties, by determining the lipophilicity, passive permeation through the human gastrointestinal tract and across the blood-brain barrier by PAMPA, and chemical, enzymatic, and plasma stability. The obtained data demonstrate that the amidoxime prodrug shows an improved pharmacokinetic profile with respect to the acetamidine nNOS inhibitor, thus suggesting that it could be a promising lead compound to treat all those pathological conditions in which nNOS activity is dysregulated.

Acetamidine-Based iNOS Inhibitors as Molecular Tools to Counteract Inflammation in BV2 Microglial Cells.

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.

Discovery of N-{3-[(ethanimidoylamino)methyl]benzyl}-l-prolinamide dihydrochloride: A new potent and selective inhibitor of the inducible nitric oxide synthase as a promising agent for the therapy of malignant glioma.

In mammalian cells, aberrant iNOS induction may have detrimental consequences, and seems to be involved in the proliferation and progression of different tumors, such as malignant gliomas. Therefore, selective inhibition of iNOS could represent a feasible therapeutic strategy to treat these conditions. In this context, we have previously disclosed new acetamidines able to inhibit iNOS with a very high selectivity profile over eNOS or nNOS. Here we report the synthesis of a new series of compounds structurally related to the leading scaffold of N-[(3-aminomethyl)benzyl] acetamidine (1400 W), together with their in vitro activity and selectivity. Compound 39 emerged as the most promising molecule of this series, and it was ex vivo evaluated on isolated and perfused resistance arteries, confirming a high selectivity toward iNOS inhibition. Moreover, C6 rat glioma cell lines biological response to 39 was investigated, and preliminary MTT assay showed a significant decrease in cell metabolic activity of C6 rat glioma cells. Finally, results of a docking study shed light on the binding mode of 39 into NOS catalytic site.

Non-enzymatic reaction of glycosyl oxazoline with peptides.

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In this work, we made an attempt to identify the side product by using model substrates. Analysis of the product allowed us to propose that the oxazoline ring was attacked by the amino group of lysine, leading to the formation of disubstituted acetamidine.

Comparative theoretical kinetics and thermodynamics study on high-energy insensitive explosive 1,1-diamino-2,2-dinitroethene synthesis.

Two synthesis methods were investigated in this study in order to explore feasible reaction pathways to obtain the target DADNE product: (1) the nitration of tetrahalogen ethene and (2) the reaction of acetamidine hydrochloride with dicarbonyl dichloride. Through theoretical simulation, the findings revealed that synthesis was possible, starting from acetamidine hydrochloride in a hydrated environment, followed by subsequent reaction routes via cyclization of the methoxy-substituted acetamidine anion intermediate with oxalyl chloride to form 2-methoxy-2-methyl-imidazolan-4,5-dione, acid-catalyzed synthesis of 2-methylene-imidazolan-4,5-dione, nitration using nitric acid to obtain 2-dinitromethylene-imidazolan-4,5-dione, and hydrolysis to produce 1,1-diamino-2,2-dinitroethene. A total energy of 1048.4 kJ mol(-1) was needed to carry out the reaction according to calculation of the energy barriers at each stage, as shown by the reaction profiles.

Screening of NOS activity and selectivity of newly synthesized acetamidines using RP-HPLC.

Nitric Oxide Synthase (NOS) inhibitors could play a powerful role in inflammatory and neurodegenerative diseases. In this work, novel acetamidine derivatives of NOS were synthesized and the inhibitor activity was evalued. To screen the activity and selectivity, the l-citrulline residue, after the enzymatic NOS assay, was derivatized with o-phthaldialdehyde/N-acetyl cysteine (OPA/NAC) and then evaluated by RP-HPLC method with fluorescence detection. All compounds did not affect the activity of endothelial and neuronal isoforms, while nine of them possessed a percentage of iNOS activity at 10µM lower than 50%, and were selected for IC50 evaluation. Among them, a compound emerged as a very potent (IC50 of 53nM) and selective iNOS inhibitor.

Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies.

N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region.

Effect of Inducible Nitric Oxide Synthase Inhibitor on Apoptosis in Hypoxic-Ischemic Injury of the Neonatal Rat Brain: 1H Magnetic Resonance Spectroscopy Study / 대한마취과학회지

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed during the late stage of ischemia and may play an important role in the delayed progression of ischemic brain injury. This study was conducted to investigate the effect of N-(3-[aminomethyl] benzyl] acetamidine (1,400 W), a selective inhibitor of iNOS on hypoxic ischemic injury in a neonatal rat model. METHODS: Seven-day old Sprague-Dawley rats were used. The right common carotid artery was ligated under halothane anesthesia. Three 3 hours after recovery, animals were exposed to 8% oxygen in 92% nitrogen. The treatment group (n = 14) received 7 intraperitoneal injection of 20 mg/kg of 1,400 W. The first dose was given 18 hours after the injury and interval between injections was 8 hours. The control group (n = 13) did not receive 1,400 W. The degree of ischemic damage and apoptosis were evaluated 3 days after injury by 1H magnetic resonance spectroscopy (MRS) and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining. The authors obtained Lipid/N-acetyl aspartate (Lip/NAA) and lipid/ creatine (Lip/Cr) ratios as apoptotic markers via 1H MRS, and counted the apoptotic cell number by TUNEL staining in the right hemisphere. Data were analyzed using the unpaired t-test. RESULTS: There were no significant differences in the Lip/NAA or Lip/Cr ratios of the 2 groups, and no significant differences were found in terms of the number of TUNEL positive cells between 2 groups, either. CONCLUSIONS: The iNOS inhibitor investigated did not show protective effects against delayed neuronal damage and apoptosis after the hypoxic-ischemic injury in newborn rats, at least during the 3 days following injury. Our results suggest that NO has different roles in cerebral hypoxia-ischemia according to the stage of neonatal cerebral development.