Structure-activity relationships of pentacycloundecylamines at the N-methyl-d-aspartate receptor

Prompted by our interest in neuroprotective agents with multiple mechanisms of action, we assessed the structure-activity relationship of a series of pentacycloundecylamine derivatives previously shown to have both L-type calcium channel blocking activity and N-methyl-d-aspartate receptor (NMDAR) antagonistic activity. We utilized a functional assay to measure NMDAR channel block using (45)Ca(2+) influx into synaptoneurosomes. The cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6). 0(3,10).0(5,9)]undecane (NPG1-01) proved to be the most potent experimental compound with an IC(50) of 2.98microM, while 8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane had the next most potent IC(50) of 4.06microM. Increasing the polycyclic cage size of NGP1-01 from a pentacycloundecane to a tridecane cage structure, but retaining the N-benzyl moiety decreased potency 10-fold, indicating a limitation on the volume of the cage that can be accommodated in the channel binding site. In the presence of NGP1-01, NMDA/glycine-induced maximal (45)Ca(2+) influx was attenuated by 34% with an insignificant effect on agonist potency. These results are consistent with uncompetitive antagonism for this group of compounds. Radioligand binding studies with [(3)H]MK-801 or [(3)H]TCP showed little or no displacement of these ligands by pentacycloundecylamines, suggesting that the latter compounds bind to a unique site in the NMDAR channel. The pentacycloundecylamines tested represent a novel group of NMDAR antagonists that have potential as therapeutic agents for neurodegenerative diseases including Parkinson's and Alzheimer's disease.     

Physicochemical prediction of a brain-blood distribution profile in polycyclic amines

Recent investigation into the pharmacological character of the pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecyl and related polycyclic amines has revealed interesting facts regarding their possible use as neuroprotective agents. At this stage however, a clear shortcoming in the quest for further development of this novel class of compounds is the lack of concrete data on their ability to cross the blood-brain barrier (BBB). Working towards the aim of predicting BBB permeability, a series of related N-substituted 8-amino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes were synthesised. Compounds were characterised by both experimental and calculative methods, followed by biological assessment and statistical manipulation of the results obtained. In doing so, a simple biological model was established for the comparative evaluation of brain-blood distribution properties within the class. A highly sensitive ESI-MS.MS analytical procedure was developed for the detection of these compounds in biological tissues, indicating significant drug concentrations in the brain after intraperitoneal administration to C57Bl/6 mice. Stepwise multiple linear regression analysis of all data yielded two meaningful models (R(2)=0.9996 and R(2)=0.7749) depicting lipophilicity (log P(oct)), solvent accessible molecular volume (SV), molar refractivity (MR) and system energy as the prime determinants of the brain-blood profile for these amines. The inherently high lipophilicity potential within the series is attributed to strong hydrophobic influences dominating hydrogen bonding effects. A possible conformational and energy dependent preference at the site of permeation is also suggested. The proposed estimations allow for the expedient and reliable prediction of brain partitioning behaviour for related polycyclic amines, facilitating the early rejection of unsuitable candidates and enabling research to focus on neuroprotective activity.     

N-substituted 8-aminopentacyclo[5.4.0.02,6.03,10.05,9]undecanes as σ receptor ligands with potential neuroprotective effects

Several libraries of similarly N-substituted 8-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecanes (9), N-methyl-8-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecanes (14), and N-methyl-11-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecan-8-ones (13) were synthesised and screened against a panel of CNS targets in order to develop structure–affinity relationships for cage-modified trishomocubane σ receptor ligands based on the N-substituted 4-azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecan-3-ol (8) scaffold. In general, compared to the corresponding 4-azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecan-3-ols, compounds of type 9 were potent σ receptor ligands with low levels of subtype selectivity, while the corresponding N-methyl-8-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecanes showed reduced affinity but greater selectivity for σ₂ receptors. The N-methyl-11-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecan-8-ones demonstrated the poorest σ receptor affinities, suggesting that 4-azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecan-3-ols interact with σ receptors in the bridged hemiaminal form rather than as the non-transannular, aminoketone tautomers. Several compounds of type 8, 9, and 14 were assessed for their ability to inhibit nitric oxide release in vitro, and demonstrated comparable or greater efficacy than 4-phenyl-1-(4-phenylbutyl)piperidine (PPBP), an established neuroprotective σ ligand with NOS inhibitory activity.     

Computational study of the conformational preferences of the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide.

alpha-Amino acids are important building blocks for the synthesis of a large number of bioactive compounds and pharmaceutical drugs. However, a literature survey revealed that no theoretical conformational study of alpha-amino acids with cage carbon frameworks has been performed to date. This paper reports the results of a conformational study on the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide (cage monopeptide), using molecular mechanics and ab initio methods. The in vacuo Ramachandran maps computed using the different parameterizations of the AMBER force field show the C7eq structure as the most favourable conformation, in contrast to the C7ax structure, that is the lowest energy conformation at the ab initio level. Analysis of these maps reveals the helical preference for the monopeptide and provides the potential for the cage residue to be incorporated into constrained peptide analogues.     

Pentacycloundecane derived hydroxy acid peptides: a new class of irreversible non-scissile ether bridged type isoster as potential HIV-1 wild type C-SA protease inhibitors

Novel peptides incorporating the PCU derived hydroxy acid (5-hydroxy-4-oxahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane) were synthesized and their activity against the resistance-prone wild type C-South African (C-SA) HIV-protease is reported. The attachment of peptides and peptoids to the PCU derived hydroxy acid resulted in a series of structurally diverse promising HIV-1 protease inhibitors. Amongst the nine novel compounds, 16, 17, 20 and 23 gave IC(50) values ranging from 0.6 to 5.0 μM against the wild type C-SA HIV-1 protease enzyme. Docking studies and molecular dynamic (MD) simulations have been carried out in order to understand the binding mode of the PCU moiety at the active site of the HIV protease enzyme. A conserved hydrogen bonding pattern between the PCU derived hydroxy ether and the active site residues, ASP25/ASP25', was observed in all active compounds.     

Comparison of dopamine uptake and release in vitro in sheep and rat striatum.

Cocaine inhibits tritium-labeled dopamine ([3H]DA) uptake in rat (IC50 approximately 400 nM) and sheep (IC50 approximately 1 microM) striatum. GBR 12909, a selective DA uptake inhibitor, potently inhibits [3H]DA uptake in rat (IC50 less than 10 nM), but is less effective (only 60% of the uptake is inhibited at a concentration of 10 microM) and less potent (IC50 approximately 300 nM) in sheep. [3H]DA release from slices of rat or sheep striatum is stimulated by potassium (15-50 mM). In the presence of nomifensine (10 microM), cocaine (10 microM) had no effect on potassium-stimulated [3H]DA release in either species. [3H]DA release is increased by N-methyl-D-aspartate (NMDA) (10-1000 microM) in rat striatum but NMDA did not stimulate [3H]DA release in sheep striatum. These findings suggest that NMDA receptors either are absent from or do not regulate release of preloaded [3H]DA in sheep striatum.     

Trishomocubane as a scaffold for the development of selective dopamine transporter (DAT) ligands

In our continued exploration of trishomocubane derivatives with central nervous system (CNS) activity, N-arylalkyl-8-aminopentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecanes (10-13) displaying affinity for the sigma (σ) receptor were also found, in several cases, to interact with the dopamine transporter (DAT). Compound 12 was identified as the first trishomocubane-derived high affinity DAT ligand (K_i = 1.2 nM), with greater than 8300-fold selectivity over the monoamine transporters NET and SERT, and only low to moderate affinity for σ₁ and σ₂ receptors.     

Amperozide, a putative anti-psychotic drug: uptake inhibition and release of dopamine in vitro in the rat brain

The effects of amperozide (a diphenylbutylpiperazinecarboxamide derivative) on the uptake and release of 3H-dopamine in vitro were investigated. Amperozide inhibited the amphetamine-stimulated release of dopamine from perfused rat striatal tissue in a dose-dependent manner. With 1 and 10 microM amperozide there was significant inhibition of the amphetamine-stimulated release of dopamine, to 44 and 36% of control. In contrast, 10 microM amperozide significantly strengthened the electrically stimulated release of dopamine from perfused striatal slices. Amperozide 1-10 microM had no significant effect on the potassium-stimulated release of dopamine. 10 microM amperozide also slightly increased the basal release of 3H-dopamine from perfused striatal tissue. These effects on various types of release are similar to those reported for uptake inhibitors (Bowyer et al, 1984). The uptake of dopamine in striatal tissue was inhibited by amperozide with IC50 values of 18 microM for uptake in chopped tissue and 1.0 microM for uptake in synaptosomes. Amperozide also inhibited the uptake of serotonin in synaptosomes from frontal cortex, IC50 = 0.32 microM and the uptake of noradrenaline in cortical synaptosomes, IC50 = 0.78 microM. In conclusion, amperozide shows uptake-inhibiting properties in both release and uptake studies done in vitro on the rat. In the in vivo studies, however, amperozide differs from dopamine uptake inhibitors.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4-Dihydroxyphenylethylamine and [3H]5-Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4-dihydroxyphenylethylamine-([3H]DA), [3H]5-hydroxytryptamine ([3H]5-HT), and [3H]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5-HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4-electron oxidation product of MPTP, i.e., 1-methyl-4-phenyl-pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles

The environmental levels of brominated flame retardants (BFRs) are increasing, but little is known about their toxic effects. In this paper, we show that some of the most important BFRs in commercial use today, have a neurotoxicological potential. Hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) inhibit plasma membrane uptake of the neurotransmitters dopamine, glutamate and gamma-amino-n-butyric acid (GABA) at a concentration level similar to what previously found for polychlorinated biphenyls (PCBs) and even for ecstasy. The IC(50) value for HBCD on dopamine uptake was 4 microM, and the IC(50) values for TBBPA were 9, 6 and 16 microM for dopamine, glutamate and GABA, respectively. HBCD also inhibited glutamate uptake at low concentrations, but never achieved more than 50% inhibition. The inhibition was primarily due to their effect on the membrane potential, measured by the membrane potential marker tetraphenylphosphonium bromide (TPP(+)). Other brominated flame retardants such as octaBDE and decaBDE did not have any effects on uptake. TBBPA, HBCD and even the pentabrominated diphenylether mixture (pentaBDE, DE-71, Great Lakes) also inhibited the vesicular uptake of dopamine with an IC(50) value of 3, 3 and 8 microM, respectively. The neurotoxicological consequences of these findings for environmental contaminants such as BFRs and PCBs are discussed.     

Design and synthesis of 2,6-diprenyl-4-iodophenol TX-1952 with a novel and potent anti-peroxidative activity

The structure-based elucidation of 2,4,6-tri-substituted phenols for their antioxidative and anti-peroxidative effects has been investigated using TX-1952 (2,6-diprenyl-4-iodophenol), TX-1961, TX-1980, BTBP and BHT. In the inhibition of mitochondrial lipid peroxidation, the inhibitory activity of 2,6-di-tert-butyl-4-bromophenol (BTBP) (IC(50)=0.17 microM) was twice as high as that of 2,6-di-tert-butyl-4-methylphenol (BHT) (IC(50)=0.31 microM). This result shows that the 4-halogen group increases inhibitory activity for mitochondrial lipid peroxidation. Besides, TX-1952 (IC(50)=0.60 microM) was the highest inhibitor among 2,6-diprenyl-4-halophenols, followed by TX-1961 (IC(50)=0.93 microM) and TX-1980 (IC(50)=1.2 microM). In 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging experiments, the activity of TX-1952 (IC(0.200)=53.1 microM) was lower than that of BHT (IC(0.200)=33.7 microM) and BTBP (IC(0.200)=16.0 microM), but TX-1952 and BHT showed the same HOMO energy (-8.991 eV). These results suggest that the two prenyl groups at ortho position hinder the phenolic hydrogen abstraction by DPPH radical. These findings demonstrated that TX-1952 was a novel and potent inhibitor for lipid peroxidation.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic analgesics and other phenylpiperidines: effects on uptake and storage of dopamine and other monoamines mouse forebrain tissue

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce degeneration of dopamine (DA) and other central monoamine neurons, leading to Parkinson's disease-like effects in man, monkey, and mouse. MPTP and other substituted phenylpiperidines related to synthetic analgesics including alphaprodine and meperidine were evaluated for potency vs. uptake of 0.1 microM tritiated DA, norepinephrine (NE), or serotonin (5HT) in synaptosomal preparations of mouse striatum or cerebral cortex. The most potent inhibitor of the uptake of 3H-DA was N-methyl-4-phenylpyridinium ion (MPP+; IC50 = 1 microM, Ki = 0.4 microM), a metabolite of MPTP; its effect was competitive and reversible. Other analogs of MPTP: the N-ethylindole AHR-1709, N,N-dimethyl-MPTP, and N-methyl-4-phenylpiperidine were all more potent than MPTP against 3H-DA uptake. N-dealkylation and N-propyl substitution, as well as pyridine ring substitution, decreased affinity for DA uptake while 3',4'-dihydroxyphenyl substitution increased potency and selectivity for catecholamine uptake, and quarternarization of the pyridine ring also increased potency against DA uptake. Active compounds showed higher potency against the uptake of NE than of DA. MPP+ was also more potent than MPTP in releasing endogenous DA from striatal synaptosomes (EC50 = 3 vs. 30 microM), but did not release the cytoplasmic markers tyrosine hydroxylase and lactate dehydrogenase (LDH). In contrast to MPTP, synthetic phenylpiperidine analgesics, their potential metabolites and the experimental neuroleptic agent AHR-1709 all failed to deplete striatal DA in vivo, even if active in vitro against DA uptake.     

Muscarinic Receptors Modulate Dopamine-Activated Adenylate Cyclase of Rat Striatum

We investigated the effect of acetylcholine (ACh) on the activation of adenylate cyclase by dopamine (DA) in a lysed synaptosomal preparation from rat striatum. ACh reduced both basal and the DA-activated adenylate cyclase with an apparent IC50 of approximately 1 microM. From a kinetic analysis it appeared that ACh reduced the Vmax for activation by DA but not the activation constant for DA. For most preparations the Vmax was reduced by 30-40%. The presence of atropine did not affect the activation of the enzyme by DA but it blocked the inhibition by ACh. Following 6-hydroxydopamine lesion of the nigrostriatal pathway, the enzyme became supersensitive to activation by DA and also more sensitive to inhibition by ACh. Inhibition of adenylate cyclase by ACh appeared to be rather specific for activation by DA, as ACh had no effect on activation of adenylate cyclase by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine. These results indicate that some striatal muscarinic and dopaminergic receptors are probably coupled to the same adenylate cyclase domain. Moreover, they suggest a biochemical model for the dynamic balance of cholinergic and dopaminergic neurons that innervate the striatum.     

Inhibition of [3H]dopamine uptake into rat striatal slices by quaternary N-methylated nicotine metabolites.

The effects of quaternary N-methylated nicotine derivatives were examined on in vitro uptake of [3H]dopamine ([3H]DA) in rat striatal slices. Striatal slices were incubated with a 10 microM concentration of the following compounds: N-methylnicotinium, N-methylnornicotinium, N-methylcotininium, N,N'-dimethylnicotinium and N'-methylnicotinium salts. The results clearly indicated that significant (60%) inhibition of [3H]DA uptake occurred with those compounds possessing a N-methylpyridinium group; whereas, compounds that were methylated at the N'-pyrrolidinium position were less effective or exhibited no inhibition of [3H]DA uptake. The results suggest that high concentrations of quaternary N-methylated nicotine metabolites which are structurally related to the neurotoxin MPP+, and which may be formed in the CNS, may protect against Parkinson's Disease and explain the inverse relationship between smoking and Parkinsonism reported in epidemiologic studies.     

Molecular hybridization of 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane-3-ol with sigma (σ) receptor ligands modulates off-target activity and subtype selectivity

A series of N-substituted 4-azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecan-3-ols incorporating the respective arylalkyl subunits from several known sigma (σ) receptor ligands were synthesized and evaluated for their affinity against σ receptors and dopamine receptors. The hybrid trishomocubane-derived ligands (4-6) showed good selectivity for σ₁ and σ₂ receptors over multiple dopamine receptors. The molecular hybrid obtained from haloperidol and 4-azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecan-3-ol (4, σ₁K_i = 27 nM, σ₂K_i = 55 nM) showed reduced affinity for D₁-D₅ dopamine receptors when compared to haloperidol itself. The compound with the greatest σ₁ affinity in the series, benzamide 4 (σ₁K_i = 7.6 nM, σ₂K_i = 225 nM) showed a complete reversal of the subtype selectivity displayed by the highly σ₂ selective parent benzamide, RHM-2 (3, σ₁K_i = 10412 nM, σ₂K_i = 13.3 nM).     

Dynamic Observation of Dopamine Autoreceptor Effects in Rat Striatal Slices

Fast-scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one-, two-, and 50-pulse, 10-Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 microM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 microM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 microM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 microM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one-pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half-time of approximately 17 s was found. The addition of picrotoxin (100 microM) and glutamate (10 microM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 microM) attenuated overflow for all the trains tested.     

The bicuculline-like properties of dopamine sulfate in rat brain

To determine whether the convulsive action of intraventricularly injected dopamine sulfate, a dopamine metabolite present in rat brain and human cerebrospinal fluid, could be due to its interaction with GABAergic pathway, we compared the convulsive effect of dopamine sulfate with that of bicuculline in the conscious rat and determined the interaction of dopamine sulfate with [3H] GABA binding and uptake in rat brain tissues. The results showed that the convulsive effects of dopamine sulfate and of bicuculline could be abolished by GABA agonists diazepam and muscimol, but not by DA antagonists haloperidol and metoclopramide. In addition they were additive. Both dopamine 3-O-sulfate and dopamine-4-O-sulfate, like bicuculline, could displace sodium-independent [3H] GABA binding to rat brain synaptic membranes (IC50 = 400 microM) but had no action on GABA uptake. DA sulfate had no effect on [3H] strychnine binding to rat brain homogenates. This evidence together with the structural resemblance between dopamine sulfate and GABA suggested that the convulsive activity of dopamine sulfate may result from its interaction with central GABA receptors.     

Synthesis and biological evaluation of aroylguanidines related to amiloride as inhibitors of the human platelet Na(+)/H(+) exchanger

Pyridine and benzene bioisosteres of amiloride were synthesized and evaluated for their inhibitory potency against the sodium-hydrogen exchanger (NHE) involved in intracellular pH regulation. The inhibition of NHE was determined by using the platelet swelling assay (PSA) in which the swelling of human platelets was induced by their incubation in an acid buffer (pH 6.7). Additionally, the inhibitory potency of the most active compounds was assessed by measuring the inhibition of the EIPA-sensitive (22)Na(+) uptake (UIA) by human platelets after intracellular acidosis. The results indicated that several benzene derivatives and compounds bearing an carbonylguanidine moiety in the meta position of the pyridine nitrogen were much more potent than amiloride (PSA:IC(50)=43.5 microM; UIA:IC(50)=100.1 microM), but less than EIPA, a pyrazine NHE inhibitor (PSA:IC(50)=0.08 microM; UIA:IC(50)=0.5 microM). In both biological assays (2-amino-5-bromo-pyridine-3-carbonyl)guanidine (32) was the most active molecule (PSA: IC(50)=0.8 microM, UIA : IC(50)=0.8 microM). Our investigations demonstrated that the replacement of the pyrazine ring of amiloride by a pyridine or a phenyl ring improved the NHE inhibitory potency (phenyl >pyridine >pyrazine).     

Fluorescent polycyclic ligands for nitric oxide synthase (NOS) inhibition

In recent years polycyclic compounds have been shown to exhibit pharmacological profiles of importance in the symptomatic and proposed curative treatment of neurodegenerative diseases (e.g., Parkinson's and Alzheimer's disease). These structures also show modification and improvement of the pharmacokinetic and pharmacodynamic properties of drugs in current use. Nitric oxide (NO) is a molecular messenger involved in a number of physiological processes in mammals. It is synthesised by nitric oxide synthase (NOS) from L-arginine and its overproduction could lead to a number of neurological disorders. The aim of this study was to synthesise a series of novel indazole, indole and other fluorescent derivatives conjugated to polycyclic structures for evaluation in NOS assays. NOS is a target system where fluorescent techniques and fluorescently labelled NOS inhibitors can be used for detecting the biophysical properties of enzyme-ligand interactions and thus facilitate development of novel inhibitors of neurodegeneration. This could lead to a greater insight into the neuroprotective mechanism and a possible cure/treatment for neurodegenerative diseases. A series of compounds incorporating polycyclic structures such as 3-hydroxy-4-aza-8-oxoheptacyclo[9.4.1.0.(2,10)0.(3,14)0.(4,9)0.(9,13)0(12,15)]tetradecane and amantadine as well as suitable fluorescent moieties were selected for synthesis. In the biological evaluation the oxyhaemoglobin (oxyHb) assay was employed to determine the activity of the novel compounds at an enzymatic level of NOS. IC(50) values of the novel fluorescent compounds were compared to that of aminoguanidine (AG) and 7-nitroindazole (7-NI), two known NOS inhibitors, and showed moderate to high affinity (IC(50) values ranging from 7.73 microM to 0.291 microM) for the NOS enzyme.