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We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B-selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B-selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. We describe here a novel class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory, cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel open probability, slows receptor deactivation, in addition to decreasing both single channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion channel permeation., One Sentence Summary A new generation of NMDA receptor modulators control both channel opening and ionic selectivity of the channel pore.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic ligand-gated glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Several neurological disorders may involve NMDAR hypofunction, which has driven therapeutic interest in positive allosteric modulators (PAMs) of NMDAR function. Here we describe modest changes to the tetrahydroisoquinoline scaffold of GluN2C/GluN2D-selective PAMs that expands activity to include GluN2A- and GluN2B-containing recombinant and synaptic NMDARs. These new analogues are distinct from GluN2C/GluN2D-selective compounds like (+)-(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ) by virtue of their subunit selectivity, molecular determinants of action, and allosteric regulation of agonist potency. The (S)-enantiomers of two analogues (EU1180-55, EU1180-154) showed activity at NMDARs containing all subunits (GluN2A, GluN2B, GluN2C, GluN2D), whereas the (R)-enantiomers were primarily active at GluN2C- and GluN2D-containing NMDARs. Determination of the actions of enantiomers on triheteromeric receptors confirms their unique pharmacology, with greater activity of (S) enantiomers at GluN2A/GluN2D and GluN2B/GluN2D subunit combinations than (R) enantiomers. Evaluation of the (S)-EU1180-55 and EU1180-154 response of chimeric kainate/NMDA receptors revealed structural determinants of action within the pore-forming region and associated linkers. Scanning mutagenesis identified structural determinants within the GluN1 pre-M1 and M1 regions that alter the activity of (S)-EU1180-55 but not (R)-EU1180-55. By contrast, mutations in pre-M1 and M1 regions of GluN2D perturb the actions of only the (R)-EU1180-55 but not the (S) enantiomer. Molecular modeling supports the idea that the (S) and (R) enantiomers interact distinctly with GluN1 and GluN2 pre-M1 regions, suggesting that two distinct sites exist for these NMDAR PAMs, each of which has different functional effects.

We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B–selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B–selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.

The N-methyl-d-aspartate receptor (NMDAR) is an ion channel that mediates the slow, Ca(2+)-permeable component of glutamatergic synaptic transmission in the central nervous system (CNS). NMDARs are known to play a significant role in basic neurological functions, and their dysfunction has been implicated in several CNS disorders. Herein, we report the discovery of second-generation GluN2C/D-selective NMDAR-positive allosteric modulators (PAMs) with a dihydropyrrolo[1,2-a]pyrazin-3(4H)-one core. The prototype, R-(+)-EU-1180–453, exhibits log unit improvements in the concentration needed to double receptor response, lipophilic efficiency, and aqueous solubility, and lowers cLogP by one log unit compared to the first-generation prototype CIQ. Additionally, R-(+)-EU-1180–453 was found to increase glutamate potency 2-fold, increase the response to maximally effective concentration of agonist 4-fold, and the racemate is brain-penetrant. These compounds are useful second-generation in vitro tools and a promising step toward in vivo tools for the study of positive modulation of GluN2C- and GluN2D-containing NMDA receptors.

[Image: see text] The N-methyl-d-aspartate receptor plays a critical role in central nervous system processes. Its diverse properties, as well as hypothesized role in neurological disease, render NMDA receptors a target of interest for the development of therapeutically relevant modulators. A number of subunit-selective modulators have been reported in the literature, one of which is TCN-201, a GluN2A-selective negative allosteric modulator. Recently, it was determined from a cocrystallization study of TCN-201 with the NMDA receptor that a unique active pose exists in which the sulfonamide group of TCN-201 incorporates a π–π stacking interaction between the two adjacent aryl rings that allows it to make important contacts with the protein. This finding led us to investigate whether this unique structural feature of the diaryl sulfonamide could be incorporated into other modulators that act on distinct pockets. To test whether this idea might have more general utility, we added an aryl ring plus the sulfonamide linker modification to a previously published series of GluN2C- and GluN2D-selective negative allosteric modulators that bind to an entirely different pocket. Herein, we report data suggesting that this structural modification of the NAB-14 series of modulators was tolerated and, in some instances, enhanced potency. These results suggest that this motif may be a reliable means for introducing a π–π stacking element to molecular scaffolds that could improve activity if it allowed access to ligand–protein interactions not accessible from one planar aromatic group.

The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has demonstrated anticancer activity in numerous in vivo settings. However, as no Enigmol analog with enhanced potency in vitro has been identified, a new strategy to improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and biological evaluation of two novel fluorinated Enigmol analogs, CF3-Enigmol and CF2-Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue exposures were anticipated to trend with fluorine content, CF2-Enigmol absorbed into tissue at strikingly higher concentrations than CF3-Enigmol. Using mouse xenograft models of prostate cancer, we also show that CF3-Enigmol underperformed Enigmol-mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF2-Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor activity as compared to Enigmol.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

N-methyl-d-aspartate receptors (NMDARs) are an important receptor in the brain and have been implicated in multiple neurological disorders. Many non-selective NMDAR-targeting drugs are poorly tolerated, leading to efforts to target NMDAR subtypes to improve the therapeutic index. We describe here a series of negative allosteric NMDAR modulators with submaximal inhibition at saturating concentrations. Modest changes to the chemical structure interconvert negative and positive modulation. All modulators share the ability to enhance agonist potency and are use-dependent, requiring the binding of both agonists before modulators act with high potency. Data suggest that these modulators, including both enantiomers, bind to the same site on the receptor and share structural determinants of action. Due to the modulator properties, submaximal negative modulators in this series may spare NMDAR at the synapse, while augmenting the response of NMDAR in extrasynaptic spaces. These modulators could serve as useful tools to probe the role of extrasynaptic NMDARs., eLife digest The neurons in the brain form networks that can change in response to experience, causing new connections to form between certain neurons and breaking the connections between others. This remodeling process underlies learning and memory. However, in certain neurological disorders, such as schizophrenia and epilepsy, these networks are disrupted and no longer work correctly. A receptor protein called the NMDA receptor plays an important role in reshaping the networks of neurons. Chemicals called neurotransmitters that are released by one neuron bind to and activate NMDA receptors on a neighbouring neuron to communicate with it. This activation encourages new connections to form between neurons. Drugs that alter the activity of the NMDA receptor could potentially act as treatments for neurological conditions that disrupt how the networks of neurons work. However, few have been approved for use in patients because most of the potential drug compounds investigated so far produce severe side effects. Perszyk et al. have now identified a new group of compounds that can potentially alter the activity of NMDA receptors without fully blocking the response, potentially eliminating the unwanted side effects. The compounds were tested on frog egg cells and human embryonic kidney cells that had been engineered to produce NMDA receptors. Small changes to the chemical structure of these compounds could switch their effect from increasing to decreasing the activity of the receptor. The compounds only interact with active receptors that have a neurotransmitter bound to them, and compete with each other to bind to the receptors. Perszyk et al. also found that some compounds behaved differently depending on how active the NMDA receptor was. These compounds could potentially be used to sense the activity in a network of neurons, which opens up new options for treating neurological conditions that affect the networks. Further experiments are now required to see how these compounds affect the activity of NMDA receptors in neurons and living animals. The range of effects produced by the compounds studied by Perszyk et al. suggests that other related compounds may have different effects on receptor activity. Future work could investigate the properties of these compounds to see if they could treat a different set of neurological disorders.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated, cation-selective channels that mediate a slow component of excitatory synaptic transmission. Subunit-selective positive allosteric modulators of NMDA receptor function have therapeutically relevant effects on multiple processes in the brain. A series of pyrrolidinones, such as PYD-106, that selectively potentiate NMDA receptors that contain the GluN2C subunit have structural determinants of activity that reside between the GluN2C amino terminal domain and the GluN2C agonist binding domain, suggesting a unique site of action. Here we use molecular biology and homology modeling to identify residues that line a candidate binding pocket for GluN2C-selective pyrrolidinones. We also show that occupancy of only one site in diheteromeric receptors is required for potentiation. Both GluN2A and GluN2B can dominate the sensitivity of triheteromeric receptors to eliminate the actions of pyrrolidinones, thus rendering this series uniquely sensitive to subunit stoichiometry. We experimentally identified NMR-derived conformers in solution, which combined with molecular modeling allows the prediction of the bioactive binding pose for this series of GluN2C-selective positive allosteric modulators of NMDA receptors. These data advance our understanding of the site and nature of the ligand-protein interaction for GluN2C-selective positive allosteric modulators for NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC(50) value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some drug-like physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

We have identified a series of positive allosteric NMDA receptor (NMDAR) modulators derived from a known class of GluN2C/D-selective tetrahydroisoquinoline analogues that includes CIQ. The prototypical compound of this series contains a single isopropoxy moiety in place of the two methoxy substituents present in CIQ. Modifications of this isopropoxy-containing scaffold led to the identification of analogues with enhanced activity at the GluN2B subunit. We identified molecules that potentiate the response of GluN2B/GluN2C/GluN2D, GluN2B/GluN2C, and GluN2C/GluN2D-containing NMDARs to maximally effective concentrations of agonist. Multiple compounds potentiate the response of NMDARs with submicromolar EC50 values. Analysis of enantiomeric pairs revealed that the S-(−) enantiomer is active at the GluN2B, GluN2C, and/or GluN2D subunits, whereas the R-(+) enantiomer is only active at GluN2C/D subunits. These results provide a starting point for the development of selective positive allosteric modulators for GluN2B-containing receptors.

Enigmol is a synthetic, orally active 1-deoxysphingoid base analogue that has demonstrated promising activity against prostate cancer. In these studies, the pharmacologic roles of stereochemistry and N-methylation in the structure of enigmols were examined. A novel enantioselective synthesis of all four possible 2S-diastereoisomers of enigmol (2-aminooctadecane-3,5-diols) from l-alanine is reported, which features a Liebeskind-Srogl cross-coupling reaction between l-alanine thiol ester and (E)-pentadec-1-enylboronic acid as the key step. In vitro biological evaluation of the four enigmol diastereoisomers and 2S,3S,5S-N-methylenigmol against two prostate cancer cell lines (PC-3 and LNCaP) indicates that all but one diastereomer demonstrate potent oncolytic activity. In nude mouse xenograft models of human prostate cancer, enigmol was equally effective as standard prostate cancer therapies (androgen deprivation or docetaxel), and two of the enigmol diastereomers, 2S,3S,5R-enigmol and 2S,3R,5S-enigmol, also caused statistically significant inhibition of tumor growth. A pharmacokinetic profile of enigmol and N-methylenigmol is also presented.

The sphingoid base backbones of sphingolipids (sphingosines, sphinganines, 4-hydroxysphinganines and others) are highly bioactive species directly and-in most cases-as their metabolites, the N-acyl-sphingoid bases (ceramides) and sphingoid base 1-phosphates. The complexity of these compounds affords many opportunities to prepare synthetic analogs for studies of sphingolipid metabolism and the functions of the sphingoid bases and metabolites. Described in this review are methods for the preparation of libraries of sphingoid bases, including a series of 1-deoxy-analogs, as well as information about their metabolism and biological activities. Findings with these compounds have uncovered some of the complications of working with compounds that mimic a naturally occurring biomodulator-such as that they are sometimes metabolized by enzymes that handle the endogenous compounds and the products may have potent (and unexpected) biological activities. Through studying such compounds, there is now a greater understanding of the metabolism and mechanism(s) of action of naturally occurring sphingoid bases as well as of these analogs.

Cells in culture often undergo a “burst” of free sphingosine, sphingosine 1-phosphate, ceramide, and other bioactive lipids upon removal of “conditioned” medium, and at least one lipid signaling pathway (protein kinase C) has been shown to be affected by these changes (Smith, E. R. & Merrill A. H., Jr. (1995) J. Biol. Chem. 270, 18749–18758; Smith, E. R., Jones, P. L., Boss, J. M. & Merrill, A. H., Jr. (1997) J. Biol. Chem. 272, 5640–5646). Whereas increases in sphinganine and dihydroceramide are responses to provision of precursors for sphingolipid biosynthesis de novo in the new medium, the sphingosine burst is due to sphingolipid turnover upon removal of suppressive factor(s) in conditioned medium. This study describes the purification and characterization of these suppressive factors. Conditioned medium from J774 cells was fractionated into two components that suppress the burst as follows: ammonium ion, which reaches 2–3 mm within 48 h of cell culture; and a low molecular weight, cationic compound that has been assigned the structure 2,6-bis(ω-aminobutyl)-3,5-diimino-piperazine (for which we suggest the name “batrachamine” based on its appearance) by1H and 13C NMR, Fourier transform infrared spectroscopy, and mass spectrometric analyses. The physiological significance of these compounds as suppressors of sphingolipid metabolism is unclear; however, ammonium ion is a by-product of amino acid catabolism and reaches high concentrations in some tissues. Batrachamine is even more intriguing because this is, as far as we are aware, the first report of a naturally occurring compound of this structural type. Considering the many cell functions that are affected by sphingoid bases and their derivatives, the effects of NH4 and batrachamine on sphingolipid metabolism may have important implications for cell regulation.

Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro- and threo-2-amino, 3-hydroxy- (and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with Vmax/Km of 40-125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4-6 for the threo-isomers. Ceramide synthase also acylates AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a Vmax/Km of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro- and threo-isomers are acylated with the highest apparent Vmax/Km for the erythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins.

As part of an effort to enhance the cellular activity of balanol analogs 2 and 3, we prepared a series of benzophenone ester analogs of varying steric size and hydrolytic stability. These were evaluated for protein kinase C inhibitory activity, with the results showing that both small alkyl esters and the pivaloyloxymethyl ester analogs displayed large increases in cellular PKC inhibition.

Analogs 3–5 of balanol (−)- 1 , a potent protein kinase C (PKC) inhibitor, were synthesized with the perhydroazepine moiety replaced by a smaller ring. Their in vitro inhibitory activities against PKC revealed a significant relationship between ring size and potency.

Plasmodium-infected erythrocytes have been shown to employ sphingolipids from both endogenous metabolism as well as existing host pools. Therapeutic agents that limit these supplies have thus emerged as intriguing, mechanistically distinct putative targets for the treatment of malaria infections. In an initial screen of our library of sphingolipid pathway modulators for efficacy against two strains of the predominant human malaria species Plasmodium falciparum and Plasmodium knowlesi, a series of orally available, 1-deoxysphingoid bases were found to possess promising in vitro antimalarial activity. To better understand the structural requirements that are necessary for this observed activity, a second series of modified analogues were prepared and evaluated. Initial pharmacokinetic assessments of key analogues were investigated to evaluate plasma and red blood cell concentrations in vivo.

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 mumol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 mumol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.

The sphingoid base backbones of sphingolipids are highly bioactive compounds that affect cell growth, differentiation, diverse cell behaviors, and programmed cell death. Therefore, the efficacy of sphingosine (SPH) and the analogs N-acetylsphingosine (NAS), N-methylsphingosine (NMS), octylamine (OCT), and sterylamine (STR) in the prevention of skin cancer was assessed in female Sencar mice by measuring effects on the induction of epidermal ornithine decarboxylase (ODC) activity and hyperplasia by 12-O-tetradecanoylphorbol-13-acetate (TPA) and effects on the induction of skin tumors by 7, 12-dimethylbenz[a]anthracene (DMBA) and TPA. ODC was measured in the shaved dorsal skin of mice treated topically with 0.05-20 mumol of these compounds 30 minutes before application of 8.5 nmol of TPA in 0.2 ml of acetone. ODC activity was inhibited by > or = 5 mumol of SPH and STR, > or = 10 mumol of NAS and NMS, and 20 mumol of OCT. In contrast, the induction of hyperplasia was not inhibited by application of these compounds 30 minutes before TPA. Two carcinogenesis studies were conducted with 10 nmol of DMBA as the initiator and 3.2 nmol of TPA (2x/wk for 15 wk) as the promoter. In the first study, NAS, NMS, OCT, and STR (0.05 and 0.5 mumol) were applied before each TPA application. Papilloma incidence and multiplicity were not inhibited, but NAS (0.05 mumol) and NMS (0.05 and 0.50 mumol) increased cancer-free survival. In the second experiment, SPH, NAS, and NMS (0.05 and 0.5 mumol) were applied 30 minutes before each TPA treatment and twice weekly for 10 weeks after the final TPA treatment. Papilloma incidence and multiplicity were not inhibited; however, the proportion of mice without carcinoma was increased by both doses of SPH and by 0.5 mumol of NAS. Thus low doses of sphingolipids that were not effective in inhibiting ODC activity, reducing hyperplasia, or preventing epidermal papilloma development were, nonetheless, effective in inhibiting carcinoma development.

Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol analogs in which the perhydroazepine moiety is replaced have been synthesized and their biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The results suggested that the activity and the isozyme/kinase selectivity of these compounds are largely related to the conformation about this nonaromatic structural element of the molecules.

Ceramides and sphingoid bases (such as sphingosine and sphinganine) are not only intermediates of complex sphingolipid biosynthesis and turnover, but also highly bioactive compounds that have been proposed to mediate cellular responses to growth factors, cytokines, and other agents (including γ-irradiation and some anti-cancer drugs). In studying the involvement of these compounds in growth, differentiation, diverse cell functions, and cell death, sphingoid bases and/or ceramides are often added to cells exogenously, or the amounts of the endogenous compounds are analyzed. A complicating factor in such analyses is that sphingoid bases and ceramides can be interconverted by acylation and deacylation reactions, respectively, catalyzed by ceramide synthase and ceramidase. This review describes the characteristics of ceramide synthesis and turnover, properties of the enzymes responsible for these reactions, and how inhibitors are useful in studying the function of these compounds. Furthermore, one class of ceramide synthase inhibitor (the fumonisins) are responsible for severe diseases of plants and animals; therefore information is presented about how fumonisins disrupt sphingolipid metabolism, alter cell behavior, and cause disease.

Fumonisins are inhibitors of sphinganine (sphingosine) N-acyltransferase (ceramide synthase) in vitro, and exhibit competitive-type inhibition with respect to both substrates of this enzyme (sphinganine and fatty acyl-CoA). Removal of the tricarballylic acids from fumonisin B1 reduces the potency by at least 10 fold; and fumonisin A1 (which is acetylated on the amino group) is essentially inactive. Studies with diverse types of cells (hepatocytes, neurons, kidney cells, fibroblasts, macrophages, and plant cells) have established that fumonisin B1 not only blocks the biosynthesis of complex sphingolipids; but also, causes sphinganine to accumulate. Some of the sphinganine is metabolized to the 1-phosphate and degraded to hexadecanal and ethanolamine phosphate, which is incorporated into phosphatidylethanolamine. Sphinganine is also released from cells and, because it appears in blood and urine, can be used as a biomarker for exposure. The accumulation of these bioactive compounds, as well as the depletion of complex sphingolipids, may account for the toxicity, and perhaps the carcinogenicity, of fumonisins.

A new bidentate ligand, mbit, [mbit=1,1′-methylenebis (3-methyl-2H-imidazole-2-thione)] was synthesized and characterized along with two new pnictogen complexes - SbCl3mbit and BiCl3mbit. A single crystal X-ray structure of SbCl3mbit gave the following cell parameters: space group P21/c, a = 11.812(9), b = 7.699(4), c = 18.100(9) A, β = 101.29(5)°, Z = 4, V = 1614.2 A3, Do = 1.92 g/cm3, Dc = 1.927 g/cm3 (λ = 0.71069 A). The structure refined to a conventional R=0.042. The local geometry around the antimony atom was distorted octahedral with bridging sulfur atoms creating long zigzag chains of octahedra sharing vertices cis to each other. Two chlorine atoms are trans to each other, and the third chlorine is trans to the bridging sulfur. The Sb—S bridges are 3.41 A in length. There is no strong evidence for a stereoactive lone pair in the coordination sphere of antimony. The ligand forms an eight-membered ring with the antimony atom included. Solid state IR data showed little change in ligand vibrational modes normally sensitive to coordination, and solution state proton NMR gave evidence for a high degree of association.

A stereoselective synthesis of each of the four enantiomers of sphingosine from either L- or D-serine is reported here. The key steps in the sequence involve: (a) the diastereoselective addition of 1-lithiopentadecyne to aldehyde 4 and (b) Mitsunobu inversion of propargyl alcohol 5 .

SummaryStroke remains a significant problem despite decades of work on neuroprotective strategies. NMDA receptor (NMDAR) antagonists are neuroprotective in preclinical models, but have been clinically unsuccessful, in part due to side effects. Here we describe a prototypical GluN2B-selective antagonist with an IC50 value that is 10-fold more potent at acidic pH 6.9 associated with ischemic tissue compared to pH 7.6, a value close to the pH in healthy brain tissue. This should maximize neuroprotection in ischemic tissue while minimizing on-target side effects associated with NMDAR blockade in noninjured brain regions. We have determined the mechanism underlying pH-dependent inhibition and demonstrate the utility of this approach in vivo. We also identify dicarboxylate dimers as a novel proton sensor in proteins. These results provide insight into the molecular basis of pH-dependent neuroprotective NMDAR block, which could be beneficial in a wide range of neurological insults associated with tissue acidification.