Christopher Barden

Design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators

In an systematic attempt to develop novel Selective Estrogen Receptor Modulators (SERMs), chiral 1-((4-(2-(dialkylamino)ethoxy)phenyl)(2-hydroxynaphthalen-1-yl)methyl)piperidin-4-ols were designed based on an accepted pharmacophore model. Simpler prototypes, viz. racemic 1-((2-hydroxynaphthalen-1-yl)arylmethyl)piperidin-4-ols, were first synthesized to develop kinetic resolution to pure enantiomers. Simultaneously, a series of racemic 1-((4-(2-(dialkylamino)ethoxy)phenyl)(2-hydroxynaphthalen-1-yl)methyl)piperidin-4-ols were evaluated against estrogen-responsive human MCF-7 breast cancer cells, but the compounds were found to be moderately active. The lack of potency could be due to the molecular bulk resulting in inadequate fit at the receptor. Subsequently, the molecular motif was modified to achiral 1-(4-(2-(dialkylamino)ethoxy)benzyl)naphthalen-2-ols by removing the piperidinol moiety. Bioevaluation of this new series of compounds displayed significantly enhanced cytotoxicity against MCF-7 cells. A representative compound for this series showed estrogen receptor alpha binding activity and the action is that of an antagonist.

ALZHEIMER'S DISEASE AS A DISORDER OF TRYPTOPHAN METABOLISM

cortex (Fig. 1). Conclusions:We have shown Sp1 is an AD-relevant TF with a significant SNP near its gene. From TF activation profiling, we observed several potential TFs that may also affect the amyloid pathway. We continue to investigate the relationship between genetic variants in TFs and AD-related endophenotypes as well as how these changes can be targeted by TF-modulating drugs as a possible strategy in treatment of AD. Supported by grants fromNIH/NIA R01AG051086-01, IADC-Core and ISDH-13609 to DKL.

TARGETING OLIGOMERIC PROTEIN SPECIES WITH SMALL MOLECULES

Marcia Taylor, Scott Banfield, Christopher Barden, Bin Deng, Kunal Keskar, Erhu Lu, Luhze Pan, Mark Reed, Kurt Stover, Braden Sweeting, Yanfei Wang, Derek J. Wilson, Fan Wu, Arun Yadav, Seung-Pil Yang, Shaolong Zhu, Donald F. Weaver, Treventis Corporation, Toronto, ON, Canada; Treventis Corporation, Halifax, NS, Canada; Krembil Research Institute, Toronto, ON, Canada; York University, Toronto, ON, Canada; Treventis, Toronto, ON, Canada; Center for Research inMass Spectrometry, Toronto, ON, Canada. Contact e-mail: dweaver@uhnres.utoronto.ca

DISCOVERY OF SMALL MOLECULE DUAL INHIBITORS OF BOTH ABETA AND TAU OLIGOMERIZATION

viability. Cellular ROS level was significantly decreased after treatment of KST013418. Therapeutic effects of KST013418 were demonstrated from the in vivo assays, as well. Conclusions: KST013418 and its derivatives can be used to activate the neuropretective mechanism by recovering mitochondrial dysfunction. This development of novel TSPO ligands supported that the discovery of potent TSPO ligands must be a meaningful way for curing AD patients.

Inhibition of protein misfolding by optimization of small molecules targeted to both beta-amyloid and tau peptides

with bromochloropropane to obtain an intermediate which was subsequently treated with various cyclic amines (1a-1f). Docking study of the compounds was performed using vlife 4.3 having crystal structure of TcAChE. Results: Docking study of the analogs revealed that they could bind to both the catalytic and peripheral sites as they interacts with various amino acid residues of the gorge like Trp279, Tyr121, Gly118, Gly119, Glu99 and Trp84. Conclusions:Coumarin analogues may alter the progression of disease as the analogs interact with both the sites of enzyme.

DEVELOPMENT OF TRYPTOPHAN-BASED BETA-AMYLOID ANTI-AGGREGANTS

markers. The latter was interpreted as vascular sprouting and/or increase of vascular diameter. Yet, it is not clear, if these mechanisms to increase cerebral blood flow and sustenance can be found in both mice and men, if they are common within mouse models at all, or if there are probably misinterpretations of published data. Methods: Real comparative quantitative data between human AD cases and AD model mice are not available today. Thus we created a setup comparing CAA, vascular diameter and number in APP SLmodel mice at different ages and humanAD cases at different Braak stages of disease. CAA was immunohistochemically evaluated using a colabeling of an amyloid plaque marker (6E10) with Annexin-IV and smooth muscle actin to be able to directly quantify vascular amyloid load on arteries and venules. Results: CAA is related to arteries and does hardly appear at venules in both mice and men. APP SL mice react with an increased vascular diameter on amyloid load rather than with angiogenesis. Also in humans, no increase of the vessel number but rather an increase in vascular diameter of amyloid loaded vessels seems present. Furthermore, and as in mice over age, CAA increases with Braak stage in most cases.Conclusions: Obtained data support the thesis that, CAA is growing with age in APP SL mice and Braak stage in humans, and that the typical reaction to CAA is an increase of blood volume rather than an increase of arterial network. Differences and parallels between mice and men are presented and methodological topics discussed.

CHARACTERIZATION AND OPTIMIZATION OF NEW CHEMICAL ENTITY ANTI-PROTEIN MISFOLDING AGENTS

Background and Hypothesis: Both beta amyloid and tau produce neurotoxic protein misfolding co-conspirators most often implicated in the molecular causation of AD. Moreover, since the clinical presentation of AD is heterogeneous (e.g. extrapyramidal symptoms in some people with AD and dementia symptoms in some people with PD), the clinical overlap amongst neurodegenerative disorders implies that AD is more of a syndrome than a disease and that the suppression of other aberrantly misfolded protein (e.g. alpha synuclein) may afford additional therapeutic benefits. Accordingly, we are seeking to design and develop brain-penetrable small molecule new chemical entities as anti-protein misfolding agents targeting three proteins: beta amyloid 40 and 42; tau and alpha synuclein. Based upon extensive in silico modeling, a family of novel compounds has been identified. A representative compound is TRV101. Methods: Efficacy of TRV101 was measured in a variety of in vitro assays including Thioflavin T beta amyloid aggregation, Thioflavin S tau aggregation, and biotin-beta amyloid 42 oligomerization. TRV101 was incubated with full length tau and the resultant solution was examined by electron microscopy. Additionally, data was collected on blood brain penetrance, protein binding, and longevity in mouse liver microsomes. Results: TRV101 showed anti-aggregation activity against both beta amyloid and tau. TRV has drug like properties, has high brain penetrance, and is stable in various ADME tests. Conclusion: We have developed a new class of compounds capable of inhibiting aggregation of both beta amyloid and tau proteins. Our small molecules are pharmacokinetically stable and are able to reach the target tissue. Our compounds are currently being tested in animal models of both beta amyloid and tau pathologies. In Vivo PK (Mouse, p.o.)  AUC (brain) = 8692 hr*ng/ mL  T1/2 = 5.4h  Cmax = 1217 ng/mL (brain)  Bioavailability = 78.3 %  B/P = 1 MLM T1/2 > 60 min Clint = 0.008 mL/min/mg HLM T1/2 = 57 min Clint = 0.024 mL/min/mg Physical Chemistry Properties  LogD = 3.15  MW: 446 g/mol  tPSA = 60.6 Protein binding  98.9 % (mouse)  97.6 % (human) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 A b s o rb a n c e ( 4 5 0 n m ) In vitro assays Thioflavin S aggregation assay – Thioflavin S fluoresces when in the presence of aggregated tau. The reaction is initiated by addition of heparin and the reaction is followed by fluorescence until the signal change plateaus. Test compounds are measured by change in fluorescence compared to a vehicle control. TRV101 inhibits tau-441 aggregation with an IC50 of 2.5μM Oligomer formation assay – Biotinylated amyloid-β is allowed to self-associate in the presence of compound, vehicle, and 0.1% Tween20. The reaction is stopped by addition of 0.1% Tween-20 and the sample is applied to a neutravidin ELISA plate. Oligomers are detected by addition of streptavidin-HRP, which will only detect unbound biotin label, i.e., oligomers. TRV101 inhibits biotinylated amyloidβ (1-40) oligomerization with an IC50 of 5μM Animal models APP/PS1 (amyloid-β model), n=13 The mice were dosed at 30mg/kg (p.o., qd) commencing at 6 weeks of age with behavioral testing (2d-RAWM and fear conditioning) at ~3.5 months of age followed by ex vivo LTP analysis performed at the CA3/CA1 synapse in the hippocampus.