Thermodynamic destabilization of azurin by four different tetramethylguanidinium amino acid ionic liquids.

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔHunf°) and entropy (ΔSunf°) values, derived from fits of the unfolding data, show that some ILs increase ΔHunf°while others do not significantly change this value. All ILs, however, increase ΔSunf°. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔHunf° values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔSunf° values.

Effects of N-[Imino(1-Piperidinyl)Methyl] Guanidine on the Intensity of Free Radical Processes, Aconitase Activity, and Citrate Level in the Tissues of Rats with Experimental Type 2 Diabetes Mellitus.

Effects of a synthetic biguanide derivative N-[imino(1-piperidinyl)methyl] guanidine (NIPMG) on free radical homeostasis, aconitase activity, and citrate concentration were studied in the liver and blood serum of rats with type 2 diabetes mellitus. Analysis of biochemiluminescence parameters showed that administration of this agent (10 mg/kg body weight) to animals with diabetes reduced the intensity of free radical processes in study tissues relative to the increased values in untreated diabetic animals. Under these conditions, aconitase activity, a principal target of ROS effects, and citrate level in the liver and blood serum of rats approached the control levels. The results show that NIPMG can positively regulate free radical homeostasis and reduce the intensity of oxidative stress in type 2 diabetes mellitus, which was accompanied by normalization of the studied parameters.

N'-3-(Trifluoromethyl)phenyl Derivatives of N-Aryl-N'-methylguanidines as Prospective PET Radioligands for the Open Channel of the N-Methyl-d-aspartate (NMDA) Receptor: Synthesis and Structure-Affinity Relationships.

N-Methyl-d-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric disorders, including Alzheimer's disease, epilepsy, drug addiction, and schizophrenia. A radioligand that could be used with PET to image and quantify human brain NMDA receptors in the activated "open channel" state would be useful for research on such disorders and for the development of novel therapies. To date, no radioligands have shown well-validated efficacy for imaging NMDA receptors in human subjects. In order to discover improved radioligands for PET imaging, we explored structure-affinity relationships in N'-3-(trifluoromethyl)phenyl derivatives of N-aryl-N'-methylguanidines, seeking high affinity and moderate lipophilicity, plus necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18. Among a diverse set of 80 prepared N'-3-(trifluoromethyl)phenyl derivatives, four of these compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of [(3)H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand development.

Emerging nitrogenous disinfection byproducts: Transformation of the antidiabetic drug metformin during chlorine disinfection of water.

As an environmental contaminant of anthropogenic origin metformin is present in the high ng/L- up to the low µg/L-range in most surface waters. Residues of metformin may lead to the formation of disinfection by-products during chlorine disinfection, when these waters are used for drinking water production. Investigations on the underlying chemical processes occurring during treatment of metformin with sodium hypochlorite in aqueous medium led to the discovery of two hitherto unknown transformation products. Both substances were isolated and characterized by HPLC-DAD, GC-MS, HPLC-ESI-TOF, (1)H-NMR and single-crystal X-ray structure determination. The immediate major chlorination product is a cyclic dehydro-1,2,4-triazole-derivate of intense yellow color (Y; C4H6ClN5). It is a solid chlorimine of limited stability. Rapid formation was observed between 10 °C and 30 °C, as well as between pH 3 and pH 11, in both ultrapure and tap water, even at trace quantities of reactants (ng/L-range for metformin, mg/L-range for free chlorine). While Y is degraded within a few hours to days in the presence of light, elevated temperature, organic solvents and matrix constituents within tap water, a secondary degradation product was discovered, which is stable and colorless (C; C4H6ClN3). This chloroorganic nitrile has a low photolysis rate in ambient day light, while being resistant to heat and not readily degraded in the presence of organic solvents or in the tap water matrix. In addition, the formation of ammonia, dimethylamine and N,N-dimethylguanidine was verified by cation exchange chromatography.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46µL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11µL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly µ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%).

Enhanced aqueous Suzuki-Miyaura coupling allows site-specific polypeptide 18F-labeling.

The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki-Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [(18)F]4-fluorophenylboronic acid. These findings now enable site-specific protein (18)F-labeling under biologically compatible conditions using a metal-triggered reaction.

An innovative strategy for sulfopeptides analysis using MALDI-TOF MS reflectron positive ion mode.

Sulfation of tyrosine residues is a key posttranslational modification in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Despite substantial advances, intact sulfated peptides are still difficult to detect by MALDI-MS due to the extreme lability of the sulfo-moiety. The present report demonstrates for the first time how intact sulfated peptides can be directly and specifically detected by MALDI-MS in positive reflectron mode by using pyrenemethylguanidine (pmg) as a noncovalent derivatizing agent and an ionization enhancer. This new method allows the determination of the degree of sulfation of sulfopeptides pure or in mixtures. Moreover, the observation of specific peaks in the mass spectra enables a rapid and unambiguous discrimination between phospho- and sulfopeptides.

[The influence of N-[imino(1-piperidinyl)methyl]guanidine and N-[imino(4-morpholinyl)methyl]guanidine on citrate content, aconitase and citrate synthase activities at ischemia-reperfusion of rats brain].

The influence of some guanidine derivatives on the level of brain citrate, brain activities of aconitase and citrate synthase has been investigated in rats subjected to ischemia-reperfusion. Administration of N-[imino(1-piperidinyl)methyl]guanidine and N-[imino(4-morpholinyl)methyl]guanidine resulted in changes of specific activities of aconitase and citrate synthase towards control values. Under these conditions the citrate level considerably decreased versus rats with untreated ishemia-reperfusion. Treatment with these compounds also decreased the degree of DNA fragmentation markedly increased in rats with ischemia-reperfusion.

A supramolecular fluorescence sensor for pyrovanadate as a functional model of vanadium haloperoxidase.

A novel basket-shaped tris(pyrene guanidinium) receptor was synthesized which binds pyrovanadate and pyrophosphate with Ka > 107 M-1. The binding of both anions is associated with quenching of the excimer fluorescence of the pyrenes. The supramolecular vanadate complex catalyzes the bromination of activated C-H bonds and hence is an enzyme mimic of vanadium haloperoxidases.

A selective human H(4)-receptor agonist: (-)-2-cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-2-y] methylguanidine.

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H(3)- and H(4)-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H(3)-receptor than the H(4)-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H(4)-receptor with a pEC(50) value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H(3)-receptor. As such, 3b and 3c are the first selective H(4) receptor agonists.

New 4-methoxybenzoyl derivatives from the ascidian Polycarpa aurata.

From the hydrophilic extract of the ascidian Polycarpa aurata three new compounds, N-(4-methoxybenzoyl)-N'-methylguanidine (1), butyl 2-(4-methoxyphenyl)-2-oxoacetate (2), and 2-(4-methoxyphenyl)-N-methyl-2-oxoacetamide (3), together with the known compounds methyl 2-(4-methoxyphenyl)-2-oxoacetate (4) and 4-methoxybenzoic acid were isolated. The structures of all isolates were determined from their spectroscopic data (NMR, MS, IR, UV).

Effects of 1,1-dimethylguanidine administration on blood pressure, heart rate and renal sympathetic nerve activity in normotensive and spontaneously hypertensive rats.

1,1-dimethylguanidine (DMG) is an endogenous nitric oxide (NO) synthesis inhibitor. This study investigates the effects of exogenous DMG administration, in anaesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Mean blood pressure (MBP), heart rate (HR) and renal sympathetic nerve activity (SNA) were recorded in 12- to 14-week old, anaesthetized SHR and WKY. Each rat received increasing bolus injections of DMG intravenously (1.03, 2.05, 6.39, 20.45 and 51.15 mg kg-1). In separate experiments, SHR received L-arginine or D-arginine in a dose of 300 mg kg-1 followed by DMG at 6.39 mg kg-1. Thirty minutes later they received the same doses of the respective arginines followed by DMG at 20.45 mg kg-1 DMG induced dose-dependant increases in MBP in SHR and WKY. In SHR, HR increased with increasing doses of DMG (except at the near-toxic doses of 51.15 mg kg-1), whereas in WKY HR decreased with increasing doses of DMG. The net change of renal SNA ranged from -5 +/- 3 to -55 +/- 12% in SHR and from -6 +/- 8 to -66 +/- 8% in WKY. Pre-treatment with L-arginine in SHR partly inhibited the pressor effect and attenuated the inhibition of renal SNA induced by DMG, but had little effect on HR. Thus the administration of NO inhibitor DMG could alter cardiovascular function and sympathetic nerve activity, and subsequently resulted in tachycardia in SHR and bradycardia in WKY.

Synthesis and pharmacological evaluation of N-(2,5-disubstituted phenyl)-N'-(3-substituted phenyl)-N'-methylguanidines as N-methyl-D-aspartate receptor ion-channel blockers.

In the mammalian central nervous system, the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors may play an important role in brain diseases such as stroke, brain or spinal cord trauma, epilepsy, and certain neurodegenerative diseases. Compounds which specifically antagonize the actions of the neurotransmitter glutamate at the NMDA receptor ion-channel site offer a novel approach to treating these disorders. CERESTAT (4, aptiganel CNS 1102) is currently undergoing clinical trial for the treatment of traumatic brain injury and stroke. Previously, we reported that analogues of N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (4) bound to the NMDA receptor ion-channel site with high potency and selectivity. Recently, molecules active at both sigma receptors and NMDA receptor sites were investigated. A series of substituted diphenylguanidines 6 which are structurally related to N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine was prepared. Compounds containing appropriate substitution pattern in one of the phenyl rings of diphenylguanidines displayed high affinity. For example, N-(2,5-dibromophenyl)-N'-(3-ethylphenyl)-N'- methylguanidine (27b, R2 = R5 = Br, R3 = C2H5) exhibited potency at both sigma receptors and NMDA receptor sites; 27b also showed high efficacy in vivo in a neonatal rat excitotoxicity model. Further studies indicated that substituent effects were important in this compound series, and 2,5-disubstituted phenyl was the preferred substitution pattern for high-affinity binding at NMDA receptor sites. Bromo and methylthio were the optimal substituents for the R2 and R5 positions of the 2,5-disubstituted phenyl group, respectively. N-(2-Bromo-5-(methylthio)phenyl)-N'- (3-ethylphenyl)-N'-methylguanidine (34b, R2 = Br, R5 = SMe, R3 = C2H5) was highly active at NMDA receptor sites. We found that the binding affinity of guanidines of type 6 could be further enhanced with the appropriate substitution at R3. Optimal activity in this series are afforded by 43b and 44b (R2 = Cl or Br, R5 = R3 = SCH3). Both 43b and 44b bound to NMDA receptor sites with high potency and selectivity (Ki vs [3H]MK-801: 1.87 and 1.65 nM, respectively); these compounds are active in vivo in various animal models of neuroprotection. The structure--activity relationships for these compounds at the NMDA receptor ion-channel site are discussed.

Estimation of impurity profiles of drugs and related materials Part 15. Identification of minor impurities in cimetidine.

Besides several known impurities in cimetidine, two additional compounds at levels below 0.1% were detected by ion-pair reversed-phase high-performance liquid chromatography (HPLC). The impurities were isolated from crude cimetidine using normal-phase preparative HPLC. 1H and 13C NMR and mass spectrometric investigations revealed the structures of the impurities to be 2,5-bis[(N'-cyano-N"-methyl)guanidinoethylthiomethyl]-4-methylimid azole (VII) and 1,8-bis[(N'-cyano-N"-methyl)guanidino]-3,6-dithiaoctane (VIII). These structures were verified by synthesis of the impurities and comparison of the spectra and chromatographic (HPLC and TLC) retention data of the isolated and synthesized materials.

Inactivation of nitric oxide synthase isoforms by diaminoguanidine and NG-amino-L-arginine.

Diaminoguanidine (DAG) and NG-amino-L-arginine each produced a time- and concentration-dependent inactivation of the citrulline-forming activity of all three NOS isoforms. DAG inactivates both the NADPH-oxidase and the citrulline-forming activities of GH3 pituitary nNOS while NG-amino-L-arginine inactivates only its citrulline-forming activity. The inactivation by DAG of GH3 nNOS NADPH-oxidase and citrulline forming activities is stimulated by (6R)-5,6,7,8-tetrahydrobiopterin (BH4) cofactor, follows pseudo-first-order kinetics and is not substrate saturable. DAG-induced inactivation of the citrulline-forming activity for the iNOS and eNOS isoforms displayed maximal inactivation rates of 0.37 and 0.14 min-1 and Ki values of 385 and 670 microM, respectively. At 1 mM DAG and saturating BH4, half-times of inactivation of 0.7, 8, and 2 min were observed for the nNOS, eNOS, and iNOS isoforms, respectively. NG-Amino-L-arginine-induced inactivation of the citrulline-forming activity of the nNOS, iNOS, and eNOS isoforms displayed maximal inactivation rates of 0.35, 0.26, and 0.53 min-1 and Ki values of 0.3, 3, and 2.5 microM, respectively. The inactivation of the NOS activities by both DAG and NG-amino-L-arginine in preincubations required the presence of oxygen and Ca2+, consistent with an inactivation mechanism that requires active metabolism by NOS. Methylguanidine and 1,1-dimethylguanidine exhibited a reversible inhibition pattern in contrast to all three NOS isoforms. Neither agent exhibited significant isoform selectivity.

Reaction of 3-deoxypentosulose with N-methyl- and N,N-dimethylguanidine as model reagents for protein-bound arginine and for creatine.

Deoxyosones are established key-intermediates in Maillard processes. Due to their dicarbonyl structure, they undergo condensation to form heterocyclic compounds with guanidine derivatives. In biological systems, guanidino functions are present in protein-bound arginine moieties as well as in creatine. The reactivity of such structures towards 3-deoxypentosulose is investigated with N-methyl- and N,N-dimethylguanidine as model substrates. Two diastereoisomers each are isolated from both reactions; they have been characterized unequivocally, respectively, as 4-(2,3-dihydroxypropyl)-2-N-methylamino-2-imidazoline-5-one and 4-hydroxy-5-(2,3-dihydroxypropyl)-2-(N,N-dimethylamino)-5H-imidazole. In aqueous medium as well as in the crystalline state, both diastereoisomer pairs exist in different tautomeric forms.

Inhibition of nitric oxide formation by guanidines.

Aminoguanidine, N,N'-diaminoguanidine, methylguanidine, and 1,1-dimethylguanidine were compared to NG-monomethyl-L-arginine (L-NMMA) for their ability to inhibit nitric oxide (NO) formation by cytokine-inducible and vascular constitutive isoforms of NO synthase. These comparisons were performed by assessing (1) cytokine-induced production of nitrite by RINm5F cells, (2) vasoconstrictor responses of isolated rat mesenteric arteries, and (3) in vivo blood pressure responses following intravenous bolus injection into anesthetized rats. Aminoguanidine and L-NMMA were the most potent inhibitors of cytokine-induced NO formation in RINm5F cells, while the other guanidine compounds were 10 (1,1-dimethylguanidine) to 100 (methylguanidine) times less potent. L-NMMA and 1,1-dimethylguanidine were the most potent inhibitors of the vascular constitutive isoform of NO synthase in both assay systems, while aminoguanidine and N,N'-diaminoguanidine were the least potent. These results (1) confirm the selective inhibition of the inducible isoform of NO synthase by aminoguanidine, (2) indicate that N,N'-diaminoguanidine, while approximately 30 times less potent than aminoguanidine in inhibiting inducible NO synthase, has very little effect on constitutive NO synthase activity, and (3) 1,1-dimethylguanidine, like L-NMMA, is a relatively potent inhibitor of both isoforms of NO synthase.

Interactions of guanidine and a related compound with potassium channels in frog myelinated nerve fibre.

The effects of guanidine and dimethyl guanidine were studied on current- and voltage-clamped nodes of Ranvier, to determine the electrophysiological basis for the guanidine-induced increase in neurotransmitter release. When added to the external solution, guanidine produced positive shifts of K and Na conductance-voltage curves. As a consequence, we observed a decrease of ionic flux through K channels, as well as through Na channels, and the resultant prolongation of the action potential. In addition, guanidine slowed down the time course of K current activation and reduced the percentage of inactivated Na channels. The main effect of guanidine is thus to induce depolarizing shifts of the potential dependence of K and Na channel gating parameters. Guanidine, like divalent cations, may alter the density of fixed negative charges on the outside membrane surface. The results of the action of dimethyl guanidine imply that, in addition to possible unspecific screening of the negative surface charges by cations, some specific interactions are involved. In conclusion, the prolongation of the action potential attributable to the effect of guanidine on external surface charges might contribute to the action of the drug in facilitating transmitter release by allowing the entry of more Ca ions into nerve endings.

Studies on bethanidine and meobentine: direct and indirect effects of antifibrillatory drugs.

The electrophysiological effects of bethanidine and meobentine were studied on isolated canine cardiac tissues and the in situ dog heart using standard techniques. The "direct" electrophysiological effects of bethanidine (in the beta-adrenergic-blocked Purkinje fiber) resemble the effects of meobentine in the normal canine Purkinje fiber; both drugs produce use-dependent decreases of the maximum rate of depolarization of phase 0 and action potential amplitude. In addition, meobentine prolongs action potential duration (100%) of Purkinje fibers. In ventricular muscle cells, the only significant effect of meobentine is a decrease in the maximum rate of depolarization. In studies of ouabain-induced tachycardias and 24-h infarct-induced ventricular arrhythmias, bethanidine tends to increase heart rate and/or exacerbate the ectopic activity (due to its sympathomimetic effects), whereas meobentine tends to reduce heart rate and restore normal sinus rhythm. Both bethanidine and meobentine increase ventricular fibrillation threshold. This increase is evident following bethanidine injection after the subsidence of the sympathomimetic effects. Finally, moderate increases of ventricular fibrillation threshold following treatment with meobentine are accompanied by partial cardiac sympathetic blockade, as indicated by reduced chronotropic responses to stellate ganglion stimulation. The antiarrhythmic and antifibrillatory effects of bethanidine and meobentine may be explained by the use-dependent effects of these drugs on phase 0 of the action potential and by their sympatholytic actions on the autonomic nervous system. Meobentine may, in addition, exert antiarrhythmic effects by decreasing automaticity in partially depolarized cells.

Potassium channel blockade: A mechanism for suppressing ventricular fibrillation.

The suppression of ventricular fibrillation by antidysrhythmic drugs is well correlated with their ability to block potassium channels in nerve and cardiac membranes. Blockade of potassium channels reduces electrical inhomogeneities in action potential and conduction parameters that lead to ventricular fibrillation. These actions tend to effectively decrease the electrical size of the heart, which suggests a mechanism for antifibrillatory drug action. The receptor sites for antifibrillatory drug action (IK blockade) appear to be on the outside of the cardiac membrane whereas receptors for antiarrhythmic drug action (INa blockade) appear to be on the inside of the cardiac membrane.