Synthesis and Biological Evaluation of Pentacycloundecylamines and Triquinylamines as Voltage-Gated Calcium Channel Blockers.

Preclinical studies for neurodegenerative diseases have shown a multi-targeted approach to be successful in the treatment of these complex disorders with several pathoetiological pathways. Polycyclic compounds, such as NGP1-01 (7a), have demonstrated the ability to target multiple mechanisms of the complex etiology and are referred to as multifunctional compounds. These compounds have served as scaffolds with the ability to attenuate Ca(2+) overload and excitotoxicity through several pathways. In this study, our focus was on mitigating Ca(2+) overload through the L-type calcium channels (LTCC). Here, we report the synthesis and biological evaluation of several novel polycyclic compounds. We determined the IC50 values for both the pentacycloundecylamines and the triquinylamines by means of a high-throughput fluorescence calcium flux assay utilizing Fura-2/AM. The potential of these compounds to offer protection against hydrogen peroxide-induced cell death was also evaluated. Overall, 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6) .0(3,10) .0(5,9) ]undecane (NGP1-01, 7a) had the most favorable pharmacological profile with an IC50 value of 86 µM for LTCC inhibition and significant reduction of hydrogen peroxide-induced cell death. In general, the triquinylamines were more active as LTCC blockers than the oxa-pentacycloundecylamines. The aza-pentacycloundecylamines were potent LTCC inhibitors, with 8-hydroxy-N-phenylethyl-8,11-azapentacyclo[5.4.0.0(2,6) .0(3,10) .0(5,9) ]undecane (8b) also able to offer significant protection in the cell viability assays.

Psychotropic Drug Development Strategies that Target Neuropsychiatric Etiologies in Alzheimer's and Parkinson's Diseases.

Preclinical Research Neuropsychiatric symptoms are currently recognized as a common burden in patients suffering from Alzheimer's disease (AD), Parkinson's disease (PD), and many other neurodegenerative disorders. Earlier theories positing that these symptoms emerge predominantly in patients with late-stage disease have been largely dismissed. It is now generally accepted that many neuropsychiatric symptoms commonly manifest very early in neurodegenerative disease stages, and in many cases are even considered prodromal indicators. Despite intense research efforts, no reliable drug treatment strategies have been found for the neuropsychiatric symptoms associated with AD and PD. Among the medications commonly used at this stage, many present significant risks for patients in this particular cohort. Transcriptomic tools and proteomic profiling have clearly indicated that neurodegenerative diseases and their associated neuropsychiatric comorbidities are multifactorial in origin. As such, multiple-and in many cases divergent-disease etiologies lead to the neuropsychiatric symptoms associated with AD, PD, and other neurodegenerative disorders. The complexity of these pathways (initiated by a cascade of molecular events that involve several neurotransmitter systems) offer significant challenges to drug discovery efforts aimed at addressing these symptoms. In response to this complexity, a new paradigm has emerged that challenges the widely held assumption that "targeted" drug design is superior to the development of "multi-targeted" drugs as a strategy to address the neuropsychiatric symptoms associated with AD and PD. In this Overview, I offer an overview of drug discovery strategies and investigative drugs currently under development that address multiple CNS etiological targets associated with an array of neuropsychiatric symptoms. Drug Dev Res 77 : 458-468, 2016. © 2016 Wiley Periodicals, Inc.

Methyl Yellow: A Potential Drug Scaffold for Parkinson's Disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease affecting movement. To date, there are no currently available therapeutic agents which can prevent or slow disease progression. Here, we evaluated an azobenzene derivative, methyl yellow (MY), as a potential drug scaffold for PD; its inhibitory activity toward monoamine oxidase B (MAO-B) as well as drug-like properties were investigated. The inhibitory effect of MY on MAO activity was determined by a MAO enzyme inhibition assay. In addition, the in vitro properties of MY as a drug candidate (e.g., blood-brain barrier (BBB) permeability, serum albumin binding, drug efflux through P-glycoprotein (P-gp), drug metabolism by P450, and mitochondrial toxicity) were examined. In vivo effectiveness of MY was also evaluated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian mouse model. MY selectively inhibited MAO-B in a dose-dependent and reversible manner. MY was BBB-permeable, bound relatively weakly to serum albumin, was an unlikely substrate for both systems of P-gp and P450, and did not cause mitochondrial toxicity. Results from the MPTP Parkinsonian mouse model indicated that, upon treatment with MY, neurotoxicity induced by MPTP was mitigated. Investigations of MY demonstrate its inhibitory activity toward MAO-B, compliant properties for drug consideration, and its neuroprotective capability in the MPTP Parkinsonian mouse model. These data provide insights into potential use, optimization, and new design of azobenzene derivatives for PD treatment.

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma.

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (µ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by µ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.

3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor.

Pentacycloundecylamine (PCU) derived compounds have been shown to be promising lead structures for the development of novel drug candidates aimed at a variety of neurodegenerative and psychiatric diseases. Here we show for the first time a 3D quantitative structure-activity relationship (3D-QSAR) for a series of aza-PCU-derived compounds with activity at the sigma-1 (σ1) receptor. A comparative molecular field analysis (CoMFA) model was developed with a partial least squares cross validated (q(2)) regression value of 0.6, and a non-cross validated r(2) of 0.9. The CoMFA model was effective at predicting the sigma-1 activities of a test set with an r(2) >0.7. We also describe here the docking of the PCU-derived compounds into a homology model of the sigma-1 (σ1) receptor, which was developed to gain insight into binding of these cage compounds to the receptor. Based on docking studies we evaluated in a [(3)H]pentazocine binding assay an oxa-PCU, NGP1-01 (IC50=1.78µM) and its phenethyl derivative (IC50=1.54µM). Results from these studies can be used to develop new compounds with specific affinity for the sigma-1(σ1) receptor.

A scaffold hopping approach to identify novel monoamine oxidase B inhibitors.

Monoamine oxidase B (MAO-B) inhibitors are used to treat Parkinson's disease. In this study, we searched for novel MAO-B inhibitors using a scaffold hopping approach based on our experience with the thiazolidinedione (TZD) class of compounds as MAO-B inhibitors. Several novel compounds were identified, with potencies in the low nanomolar and low micromolar range. We also found that derivatives of the natural product sulfuretin are potent MAO-A and MAO-B inhibitors.

Inhibition of monoamine oxidase by derivatives of piperine, an alkaloid from the pepper plant Piper nigrum, for possible use in Parkinson's disease.

A series of compounds related to piperine and antiepilepsirine was screened in a monoamine oxidase A and B assay. Piperine is an alkaloid from the source plant of both black and white pepper grains, Piper nigrum. Piperine has been shown to have a wide range of activity, including MAO inhibitory activity. The z-factor for the screening assay was found to be greater than 0.8 for both assays. Notably, the compounds tested were selective towards MAO-B, with the most potent compound having an IC(50) of 498 nM. To estimate blood-brain barrier (BBB) permeability, we used a PAMPA assay, which suggested that the compounds are likely to penetrate the BBB. A fluorescent bovine serum albumin (BSA) high-throughput screening (HTS) binding assay showed an affinity of 8 µM for piperine, with more modest binding for other test compounds. Taken together, the data described here may be useful in gaining insight towards the design of selective MAO-B inhibitory compounds devoid of MAO-A activity.

Nicotine exacerbates brain edema during in vitro and in vivo focal ischemic conditions.

We have previously shown that nicotine, the addictive component of tobacco products, alters the blood-brain barrier (BBB) Na(+),K(+),2Cl(-) cotransporter (NKCC) during in vitro hypoxia-aglycemia exposure. Attenuation of abluminal NKCC suggests that accumulation of ions in the brain extracellular fluid would result in an increase of fluid or cytotoxic edema in the brain during hypoxia-aglycemia or stroke conditions. To further investigate whether nicotine products have the potential to worsen stroke outcome by increasing edema formation, two separate models to mimic stroke conditions were utilized to decipher the effects of short-term and long-term administrations of nicotine products on brain edema following stroke. Oxygen glucose deprivation (OGD) was studied in rat hippocampal slices with short-term or long-term exposure to nicotine and cigarette smoke constituents. During short-term exposure, the presence of nicotine at a concentration mimicking heavy smokers increased water content of hippocampal slices during OGD. Furthermore, long-term 1-week administration of nicotine increased water content in hippocampal slices that could be attenuated with nicotine acetylcholine receptor (nAChR) antagonists, suggesting nicotine increase edema during OGD via nAChRs. A second model of focal ischemia, middle cerebral artery occlusion, showed an increase of infarct size during short-term exposure to nicotine and an increase of edema during both short-term and long-term administration of nicotine, compared with saline controls. These findings support the paradigm that nicotine products not only increase the incidence of stroke but also have the potential to worsen stroke outcome by increased edema formation.

Identification of novel monoamine oxidase B inhibitors by structure-based virtual screening.

Parkinson's disease is a severe debilitating neurodegenerative disorder. Recently, it was shown that the peroxisome proliferating-activator receptor-gamma agonist pioglitazone protected mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity due to its ability to inhibit monoamine oxidase B (MAO-B). Docking studies were initiated to investigate pioglitazone's interactions within the substrate cavity of MAO-B. Modeling studies indicated that the thiazolidinedione (TZD) moiety was a likely candidate for its specificity to MAO-B. To explore this potential novel MAO-B scaffold, we performed a structure-based virtual screen to identify additional MAO-B inhibitors. Our search identified eight novel compounds containing the TZD-moiety that allowed for a limited study to identify structural requirements for binding to MAO-B. Inhibition assays identified two TZDs (A6355 and L136662) which were found to inhibit recombinant human MAO-B with IC(50) values of 82 and 195 nM, respectively.

Predictive screening model for potential vector-mediated transport of cationic substrates at the blood-brain barrier choline transporter.

A set of semi-rigid cyclic and acyclic bis-quaternary ammonium analogs, which were part of a drug discovery program aimed at identifying antagonists at neuronal nicotinic acetylcholine receptors, were investigated to determine structural requirements for affinity at the blood-brain barrier choline transporter (BBB CHT). This transporter may have utility as a drug delivery vector for cationic molecules to access the central nervous system. In the current study, a virtual screening model was developed to aid in rational drug design/ADME of cationic nicotinic antagonists as BBB CHT ligands. Four 3D-QSAR comparative molecular field analysis (CoMFA) models were built which could predict the BBB CHT affinity for a test set with an r(2) <0.5 and cross-validated q(2) of 0.60, suggesting good predictive capability for these models. These models will allow the rapid in silico screening of binding affinity at the BBB CHT of both known nicotinic receptor antagonists and virtual compound libraries with the goal of informing the design of brain bioavailable quaternary ammonium analogs that are high affinity selective nicotinic receptor antagonists.

Neuroprotection in mice by NGP1-01 after transient focal brain ischemia.

The effect of the polycyclic cage amine NGP1-01, a dual action antagonist at both L-type calcium channels and NMDA receptors, was measured after transient (1 h) focal cerebral ischemia in the mouse middle cerebral artery occlusion (MCAO) model. Mice were left untreated, or received NGP1-01 (20 mg/kg per dose intraperitoneally), memantine (20 mg/kg per dose intraperitoneally), or vehicle (DMSO) at 15 min, 24 h and 48 h after reperfusion. Sensorimotor function was tested daily for two weeks using the "corner test", a proven paradigm for the assessment of functional integrity in rodents. NGP1-01 significantly reduced sensorimotor deficits over the 2-week period (p<0.001, ANOVA). Although memantine was less effective than NGP1-01 (p<0.05), it still significantly attenuated sensorimotor deficits in the animals. In a separate study, brain damage 3 days after stroke was determined histologically in mice receiving no treatment, DMSO, or NGP1-01 (dosages and dosage schedule same as above). Serial brain sections were stained for nonviable neurons with Fluoro-Jade B and the volume of damaged tissue was estimated. NGP1-01 treated mice had a significantly lower volume of brain damage (13+/-7 mm(3), p<0.01) than both control groups (no treatment: 47+/-4 mm(3), DMSO: 50+/-10 mm(3)). In conclusion, at weight-equivalent doses, NGP1-01 was at least as neuroprotective as the established NMDA receptor antagonist memantine. It may be a promising lead structure for the development of novel multiple-action drugs in treating ischemic stroke and other neurodegenerative diseases with an excitotoxic component.

Novel multifunctional anti-Alzheimer drugs with various CNS neurotransmitter targets and neuroprotective moieties.

Traditionally, drug design programs are focused on optimizing the specificity of lead compounds against a carefully selected drug target. Disappointingly, this approach to discover a "magic bullet" drug has not met with the expected success for CNS disorders. Transcriptomics and proteomic profiling of neurodegenerative diseases have indicated that they are poly-etiological in origin and that the processes leading to neuronal death are multifactorial. An emerging concept is the design of drug ligands that modulate multiple drug targets identified for a particular disease. In this review we explore some examples of multifunctional drugs which may be useful in the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

Why should we use multifunctional neuroprotective and neurorestorative drugs for Parkinson's disease?

Parkinson's disease (PD) is a severe neurodegenerative disorder, with no available drugs able to prevent the neuronal cell loss characteristic in brains of patients suffering from PD. Due to the complex cascade of molecular events involved in the etiology of PD, an innovative approach towards neuroprotection or neurorescue may entail the use of multifunctional pharmaceuticals that target an array of pathological pathways, each of which is believed to contribute to events that ultimately lead to neuronal cell death. Here we discuss examples of novel multifunctional ligands that may have potential as neuroprotective and neurorestorative therapeutics in PD. The compounds discussed originate from synthetic chemistry as well as from natural sources where various moieties, identified in research to possess neuroprotective and neurorestorative properties, have been introduced into the structures of several monomodal drugs, some of which are used in the clinic.

Triquinane-based compounds as possible serotonin 5-HT(6) receptor antagonists for the treatment of Alzheimer's disease.

The recently discovered serotonin 5-HT(6) receptor is a novel target for the treatment of cognitive decline associated with neurodegenerative diseases such as Alzheimer's disease (AD). Several studies have shown that antagonism of this receptor leads to improved learning and memory. Two serotonin 5-HT(6) receptor antagonists are currently in Phase II clinical trials for cognitive enhancement. Striking structural similarities between the polycyclic features of some 5-HT(6) antagonists and those of the cis-syn-cis triquinane ring system led us to propose that triquinane-derived compounds may represent a novel class of 5-HT(6) receptor antagonists. Using computational methodologies, we propose a structure with features that may impart potent 5-HT(6) receptor antagonistic activities to the triquinane ring complex. Related compounds, once synthesized, may have possible utility in the treatment of cognitive decline associated with AD.

Optimizing the use of open-source software applications in drug discovery.

Drug discovery is a time consuming and costly process. Recently, a trend towards the use of in silico computational chemistry and molecular modeling for computer-aided drug design has gained significant momentum. This review investigates the application of free and/or open-source software in the drug discovery process. Among the reviewed software programs are applications programmed in JAVA, Perl and Python, as well as resources including software libraries. These programs might be useful for cheminformatics approaches to drug discovery, including QSAR studies, energy minimization and docking studies in drug design endeavors. Furthermore, this review explores options for integrating available computer modeling open-source software applications in drug discovery programs.

Synthesis, structural reassignment, and biological activity of type B MAO inhibitors based on the 5H-indeno[1,2-c]pyridazin-5-one core.

The synthesis and enzyme inhibitor properties of reversible type B monoamine oxidase inhibitors are described. These compounds belong to the 5H-indeno[1,2-c]pyridazine family and possess a hydrophobic benzyloxy or 4,4,4-trifluorobutoxy side chain which, in contrast to a previous assignment, has been unambiguously located at C(8) of the heterocyclic moiety. Investigation of the regioisomeric structures establishes that substitution of the 5H-indeno[1,2-c]pyridazin-5-one core at C(7) vs C(8) dramatically influences the MAO-inhibiting properties of these compounds.

NGP1-01, a lipophilic polycyclic cage amine, is neuroprotective in focal ischemia.

NGP1-01, a member of the pentacycloundecylamine cage compound family, was recently shown to exhibit both NMDA receptor channel blocking and L-type calcium channel antagonism activity. In the present study, focal ischemia was induced in mice by permanent middle cerebral artery occlusion (MCAO) to test for potential neuroprotective properties of the compound. In female CD-1 mice injected 30 min before MCAO, NGP1-01 (20 mg/kg) reduced infarct area by 42.6% (P < 0.05) compared to vehicle-treated controls as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Concomitantly, NGP1-01 reduced brain swelling by 78.3% (P < 0.001), compared to vehicle (DMSO) treated controls. These data identify NGP1-01 and related compounds as potential lead structures to develop neuroprotective compounds based on a dual mechanism of action.

Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2alpha) cells, a simple and rapid in situ assay to investigate mitochondrial toxins.

Most mitochondria-based methods used to investigate toxins require the use of relatively large amounts of material and hence compromised sensitivity in assay. We adopted procedures from methods initially developed to diagnose mitochondrial encephalomyopathies and unified these into a single assay. Eukaryotic cell membranes are selectively permeabilized with digitonin to render a system in which mitochondrial respiration can be measured rapidly and with considerable sensitivity. Mitochondria remain intact, uninjured, and in their natural environment where mitochondrial respiration can be measured in situ under physiologically relevant conditions. This approach furthermore allows measurement of toxin effects on individual mitochondrial complexes. Numerous compounds at varying concentrations can be screened for mitochondrial toxicity, while the site of mitochondrial inhibition can be determined simultaneously. We used this assay to investigate, in murine neuroblastoma (N-2alpha) cells, the mitochondrial inhibitory properties of the parkinsonian-inducing proneurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its neurotoxic monoamine oxidase-B (MAO-B)-generated metabolite, the 1-methyl-4-phenylpyridinium species (MPP(+)). Within the time frame of each measurement (15 min), MPTP (< or = 1 mM) did not interfere with in situ mitochondrial respiration. As expected, MPP(+) was found to be a potent Complex I inhibitor but surprisingly also found to inhibit Complex IV. Optimized conditions for performing this assay are provided.

Rational drug discovery design approaches for treating Parkinson's disease.

INTRODUCTION: Parkinson's disease (PD) is a severe progressive neurodegenerative disorder. As yet, no therapeutic agent can prevent the characteristic neuronal cell loss in PD brain. The introduction of levodopa to the clinic several decades ago has greatly mitigated the symptomatic burden in PD patients. But the discovery of neuroprotective and disease-modifying therapies has lagged behind, becoming one of the most desired prizes in the drug discovery arms race for neurodegenerative disorders, including PD. AREAS COVERED: In this review, the author provides an overview of the rational drug discovery approaches that are designed to prevent the onset or alter the course of the disease, and/or target its non-motor symptoms. EXPERT OPINION: Largely due to the intertwined etiology that is a hallmark of PD's pathology, neuroprotective drug discovery is challenging, while very limited targeting strategies exist for the non-motor symptoms that afflict sufferers of PD. Rational approaches toward PD neurotherapeutics should target previously identified or emerging pathological pathways that are discovered in the course of investigating the underlying mechanisms in PD disease progression. Each of these pathways contributes to events that ultimately lead to the complex disease burden seen in PD and can form the basis for rational and highly targeted drug development.

Screening of novel pentacyclo-undecylamines for neuroprotective activity.

A novel series of pentacyclo-undecylamines with 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (NGP1-01) as the lead compound was synthesised and screened for neuroprotective activity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian mouse model. We hypothesise that these compounds may attenuate excitotoxic neuronal cell death mediated through the NMDA receptor (similar to memantine), and through calcium channel block. The pentacyclo-undecylamines (300 mg/kg) were administered to C57BL/6 mice 30 min before intraperitoneal (i.p.) MPTP administration (35 mg/kg). Striatal dopamine, 3,4-hydroxyphenylacetic acid (DOPAC), and homovanillic acid levels were analysed 10 days later by means of HPLC with electrochemical detection. Increased levels of DOPAC and homovanillic acid were observed when some of the test compounds were administered together with MPTP (compared to animals receiving only MPTP). One compound in the series, 8-phenylethylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane, attenuated MPTP-induced striatal dopamine depletion when compared to animals treated with MPTP only (p<0.05).