The Chemotype of Chromanones as a Privileged Scaffold for Multineurotarget Anti-Alzheimer Agents.

The multifactorial nature of Alzheimer's disease necessitates the development of agents able to interfere with different relevant targets. A series of 22 tailored chromanones was conceptualized, synthesized, and subjected to biological evaluation. We identified one representative bearing a linker-connected azepane moiety (compound 19) with balanced pharmacological properties. Compound 19 exhibited inhibitory activities against human acetyl-, butyrylcholinesterase and monoamine oxidase-B, as well as high affinity to both the σ1 and σ2 receptors. Our study provides a framework for the development of further chromanone-based multineurotarget agents.

Acetylcholinesterase Inhibition of Diversely Functionalized Quinolinones for Alzheimer's Disease Therapy.

In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase (MAO) inhibition of 19 quinolinones (QN1-19) and 13 dihydroquinolinones (DQN1-13) designed as potential multitarget small molecules (MSM) for Alzheimer's disease therapy. Contrary to our expectations, none of them showed significant human recombinant MAO inhibition, but compounds QN8, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent and quite selective non-competitive inhibitor of hrAChE (IC50 = 0.29 µM), with Ki value in nanomolar range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an interesting hit for further investigation.

Synthesis and Pharmacological Characterization of 2-(2,6-Dichlorophenyl)-1-((1S,3R)-5-(3-hydroxy-3-methylbutyl)-3-(hydroxymethyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (LY3154207), a Potent, Subtype Selective, and Orally Available Positive Allosteric Modulator of the Human Dopamine D1 Receptor.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

Chromenones as Multineurotargeting Inhibitors of Human Enzymes.

The complex nature of multifactorial diseases, such as Morbus Alzheimer, has produced a strong need to design multitarget-directed ligands to address the involved complementary pathways. We performed a purposive structural modification of a tetratarget small-molecule, that is contilisant, and generated a combinatorial library of 28 substituted chromen-4-ones. The compounds comprise a basic moiety which is linker-connected to the 6-position of the heterocyclic chromenone core. The syntheses were accomplished by Mitsunobu- or Williamson-type ether formations. The resulting library members were evaluated at a panel of seven human enzymes, all of which being involved in the pathophysiology of neurodegeneration. A concomitant inhibition of human acetylcholinesterase and human monoamine oxidase B, with IC50 values of 5.58 and 7.20 µM, respectively, was achieved with the dual-target 6-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (7).

Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

Synthesis and pharmacological characterization of C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: identification of a potent, selective metabotropic glutamate receptor agonist and determination of agonist-bound human mGlu2 and mGlu3 amino terminal domain structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens.

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.

The ATPase cycle of the mitotic motor CENP-E.

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding. These features endow CENP-E with a high duty cycle, a prerequisite for processivity. However, unlike kinesin 1, neck linker docking in CENP-E is slow, occurring at a rate closer to that for Eg5, a mitotic kinesin that takes only 5-10 steps per processive run. These results suggest that like kinesin 1, features outside of the catalytic domain of CENP-E may also play a role in regulating the processive behavior of this motor.

The role of myosin II in glioma invasion of the brain.

The ability of gliomas to invade the brain limits the efficacy of standard therapies. In this study, we have examined glioma migration in living brain tissue by using two novel in vivo model systems. Within the brain, glioma cells migrate like nontransformed, neural progenitor cells-extending a prominent leading cytoplasmic process followed by a burst of forward movement by the cell body that requires myosin II. In contrast, on a two-dimensional surface, glioma cells migrate more like fibroblasts, and they do not require myosin II to move. To explain this phenomenon, we studied glioma migration through a series of synthetic membranes with defined pore sizes. Our results demonstrate that the A and B isoforms of myosin II are specifically required when a glioma cell has to squeeze through pores smaller than its nuclear diameter. They support a model in which the neural progenitor-like mode of glioma invasion and the requirement for myosin II represent an adaptation needed to move within the brain, which has a submicrometer effective pore size. Furthermore, the absolute requirement for myosin II in brain invasion underscores the importance of this molecular motor as a potential target for new anti-invasive therapies to treat malignant brain tumors.

Investigation of a flexible enantiospecific approach to aziridines.

A flexible approach to functionalized and enantioenriched aziridines has been developed from an intermediate aziridinylmethyl tosylate. This protocol features a Cu-catalyzed Grignard substitution reaction that allows a range of functionalized organic fragments to be incorporated. Moreover, a convenient one-pot procedure is outlined that allows the trityl group to be exchanged for a range of common N-protecting groups.

Leaf angle responds to nitrogen supply in eucalypt seedlings. Is it a photoprotective mechanism?

We examined the adjustment of leaf angle (L theta) and foliar chlorophyll and xanthophyll chemistry in Eucalyptus nitens (Deane and Maiden) Maiden seedlings maintained in various nitrogen (N)-supply treatments over a 6-month period. Adjustment of L theta toward the vertical was greatest under conditions of foliar N deficiency and became incrementally more horizontal with increasing foliar N concentration. Photochemical efficiency (Fv/Fm) and quantum yield were lower in seedlings with low foliar N (low-N seedlings) in winter, but not in autumn. Low-N seedlings generally had low area-based chlorophyll concentrations and high xanthophyll-cycle conversion ratios, particularly during months of low temperature. Under mild temperature conditions, high concentrations of zeaxanthin and antheraxanthin were associated with lower electron transport rates (ETR). Incident light, Fv/Fm, ETR and total chlorophyll concentration were negatively correlated with L theta, with horizontal leaf orientation measured as 0 degrees and vertical leaf orientation as 90 degrees . Xanthophyll conversion ratio was positively correlated with L theta. Adjustments in L theta may play a role in photoprotection of E. nitens seedlings by assisting the leaf to balance its utilization and dissipation of energy.

1-Aryl-3,4-dihydro-1H-quinolin-2-one derivatives, novel and selective norepinephrine reuptake inhibitors.

A novel series of 1-aryl-3,4-dihydro-1H-quinolin-2-ones have been discovered as potent and selective norepinephrine reuptake inhibitors. Efficient synthetic routes have been developed which allow for the multi-gram preparation of both final targets and advanced intermediates for SAR expansion.

Photosynthetic capacity and foliar nitrogen distribution in Eucalyptus nitens is altered by high-intensity thinning.

The changes in photosynthetic rates, light environment and foliar nutrient concentrations following thinning were examined in an 8-year-old Eucalyptus nitens (Deane and Maiden) Maiden plantation. The objectives of the study were to: (1) determine the extent to which maximum photosynthetic rates (Amax) of E. nitens are affected by stand thinning; (2) relate the spatial pattern of Amax within the crown to the changes in light environment caused by thinning; and (3) establish if the responses of Amax to thinning are driven by changes in area-based foliar nitrogen (Na) or phosphorus (Pa) concentrations. Photosynthetic rates measured under saturating light availability increased throughout the crown after thinning. The greatest increases were observed in the lower and middle crown zones. Photosynthetic rate was positively related to foliar N concentration. Thinning increased Na and Pa because of a significant decrease in specific leaf area (SLA) after thinning. Changes in photosynthetic rates, SLA and foliar nutrient distributions with thinning were well correlated with changes in incident solar irradiance throughout the tree crown.

Chilling-dependent photoinhibition, nutrition and growth analysis of Eucalyptus nitens seedlings during establishment.

Effects of chilling-dependent photoinhibition on gas exchange, chlorophyll fluorescence, growth and nutrition of Eucalyptus nitens (Deane and Maiden) Maiden seedlings were assessed for 70 weeks after transplanting 9-month-old seedlings in early winter. One month before transplanting, the seedlings were assigned to fertilized or nutrient-deprived treatments. Immediately after transplanting, half the seedlings in each nutrient treatment were placed in shadecloth tree shelters. The experimental site was at an altitude of 700 m, which is considered marginal for the establishment of E. nitens plantations in Tasmania because of low mean annual minimum temperatures. Overnight frosts followed by sunny morning conditions in the first 20 weeks after transplanting (early June to early October) caused severe photoinhibition. Predawn maximal photochemical efficiency (Fv/Fm) and maximum net photosynthesis (Amax) were depressed in nutrient-deprived seedlings compared with fertilized seedlings, although shading partially alleviated this difference. Neither Fv/Fm nor Amax recovered to values observed before transplanting until > 20 weeks after transplanting. During this period, non-photochemical quenching (NPQ) was high in seedlings in all treatments, although NPQ was lower in shaded, fertilized seedlings than in seedlings in the other treatments. Total foliar nitrogen (N) concentration increased up to 42 weeks after transplanting in the nutrient-deprived seedlings in parallel with increasing relative growth rate (RGR). Fractionation of N- and phosphorus (P)-containing compounds indicated that differences in protein N accounted for the treatment differences in total seedling N. Nucleic acid P increased and inorganic P decreased during growth periods, although total seedling P remained constant. Among treated seedlings, height growth was greatest in shaded seedlings: this was probably a result of apical dominance effects because RGR was higher in unshaded seedlings than in shaded seedlings. Thus, the shade treatment alleviated chilling-dependent photoinhibition and maximized growth during winter, but limited growth during warmer periods and therefore overall growth.

Measured and predicted changes in tree and stand water use following high-intensity thinning of an 8-year-old Eucalyptus nitens plantation.

We investigated changes in the pattern of water use of an 8-year-old Eucalyptus nitens (Deane and Maiden) Maiden plantation soon after thinning. Sap flow sensors using heat pulse technology were deployed across three stands thinned to a final density of 100, 250 or 600 trees ha-1 plus an unthinned control (1250 trees ha-1). Changes in the relationship between tree size and daily water use were measured for 4 to 7 months after thinning. Thinning had no effect on sapwood water content. The increase in tree water use as a result of thinning was driven largely by significant changes in the radial pattern of sap velocity through the sapwood. The use of a canopy fraction factor in the Penman-Monteith equation to account for discontinuous canopies showed promise as a simple and effective method of scaling the model to predict transpiration from thinned plantations.

Whole-tree transpiration and water-use partitioning between Eucalyptus nitens and Acacia dealbata weeds in a short-rotation plantation in northeastern Tasmania.

Whole-tree water use in 4- and 8-year-old plantations of Eucalyptus nitens Deane and Maiden (ex Maiden) in the presence and absence of Acacia dealbata Link. weeds was estimated by the heat pulse velocity technique during a six-week summer period. Maximum sap velocities were recorded between 5 and 15 mm under the cambium for both eucalypt and acacia trees, and marked radial and axial variations in sap velocity were observed. The latter source of variation was most pronounced in mixed stands where crowns were asymmetrical. Mean daily sap flux ranged from 1.4 to 103.6 l day(-1) for eucalypts and from < 0.1 to 8.4 l day(-1) for acacias. Stem diameter explained 98% of the variation in sapwood area for E. nitens and 89% for A. dealbata, and was determined to be a suitable parameter for scaling water use from the tree to stand level. Plot transpiration varied from 1.4 to 2.8 mm day(-1) in mixed 8-year-old plots and was 0.85 mm day(-1) in a mixed 4-year-old plot. The degree of A. dealbata infestation was associated with absolute plot water use and regression models predicted that, in the absence of acacia competition, plot water use for the 8-year-old stand would approach 5-6 mm day(-1) during the growing season.