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The discovery of nerve growth factor (NGF) a half century ago and its role in the maintenance of mature neurons was the starting point in establishing a new area of research dealing with neurotrophins (NTs). Neurotrophins are secreted growth factors responsible for the development and maintenance as well as differentiation of the peripheral and central nervous systems. It was proposed that neurons are competing for a limited amount of NTs. Neurons that fail to obtain sufficient quantities of NTs die by the process of programmed cell death (PCD). Apoptosis is the best known form of PCD and is mainly characterized by nuclear condensation, cytoplasmic shrinkage and phagocytosis. Caspases are a family of cystein proteases that can cleave a variety of cellular substrates and destroy cellular function. Caspase activation triggers apoptosis and this activation can be initiated by intrinsic and extrinsic mechanisms. The extrinsic pathway involves the binding of extracellular ligands to their receptors such as tumor necrosis factor alpha (TNFa) while the intrinsic pathway is initiated through the release of cytochrome c from the intramembrane space of mitochondria which binds to the apoptosis protease activating factor-1 (Apaf-1) in the cytosol and eventually activates caspase-3. In addition to NGF, the family of NTs includes: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-4/5 (NT4/5) and glial-cell derived neurotrophic factor (GDNF). NTs exert their physiological effects through binding to two distinct types of cell surface receptors: the tropomyosin receptor kinase (Trk) and the p75 neurotrophin receptor (p75NTR), the latter of which is a member of the Fas/tumor necrosis factor receptor family. NGF binds TrkA, BDNF and NT-4 bind TrkB, NT-3 binds TrkC, and p75 receptor binds unselectively to all NTs with similar affinity. Binding of NTs with p75 initiates several proapoptotic signaling cascades of which the Mitogen-Activated Protein Kinase (MAPK) pathways are the most important (Figure 1A). The c-Jun NH2-terminl Kinase (JNK) belongs to the MAPK's and upon activation is able to phosphorylate a variety of transcription factors including c-jun and the tumor suppressor transcription factor p53. The activation of JNK following in vitro stimulation of apoptosis was reported. For example, JNK signaling was shown to be activated following trophic factor withdrawal, as well as low potassium treatment.7 JNK was also reported to activate the proapoptotic Bax protein. p53, which is activated via JNK, is an important regulator of the cell cycle. In response to genotoxic stress (i.e. a tumor suppressor), it arrests the cell cycle, activating apoptosis cascades. The role of p53 in neuronal apoptosis is well documented. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75 activation. Significant protection against neuronal death induced by ischemia was reported in mice lacking p53. Moreover, over-expression of p53 is sufficient to cause death of sympathetic neurons in the presence of NGF. The major mechanism for p53 induction of neuronal apoptosis is by inducing the expression of the proapoptotic protein Bax. Bax protein belongs to the Bcl-2 protein family which consists of critical apoptotic regulators that act by controlling the release of cytochrome c.
On the other hand, Trk receptors mediate survival signals. Trk activation results in receptor dimerization and kinase activation which enable several adaptor proteins such as Shc and PLC-g1 to couple these receptors to the survival intracellular signaling cascades. Ras, a small GTPase protein that regulates neuronal differentiation, is activated through the recruitment of the adaptor protein Shc. Activated Ras then activates PI3K which is responsible for the phosphorylation of phosphatidylinositol-4,5-biphosphate, PtdIns(4,5)P2, to phosphatidylinositol-3,4,5-triphosphate, PtdIns(4,5)P3. A key target of PI3K is the promotion of neuronal survival through the serine-threonine kinase Akt (Figure 1B). Akt is a well known survival kinase activator which targets two classes of proteins: proapoptotic and antiapoptotic. One of the proapoptotic proteins that is phosphorylated by Akt, and then sequestered to the cytosol, is the Bcl2 family member BAD (Bcl2/Bclx associated death promoter). BAD in the non-phosphorylated form binds to the Bcl2 proapoptotic protein which eventually activates Bax which assist in the release of cytochrome c from the mitochondrial intermembrane. Akt also phosphorylates glycogen synthase kinase 3b (GSK3b) which has been shown to induce apoptosis in cultured neurons. This means that Akt is silencing the action of GSK3b and BAD. On the other hand, anti-apoptotic proteins are also phosphorylated by Akt. For example, Akt can activate IkB kinas (IKK) that can stimulate the DNA transcription enhancer factor NF-kB. Interestingly, NF-kB is also stimulated through the activation of the p75 receptor suggesting that p75 can also stimulate survival signals. The interesting finding that Ras activation can phosphorylate several proteins in the JNK-p53-Bax apoptotic pathway (such as p53 and Bax) suggests that Trk receptors are silencing the death cascades activated by the p75 receptor (Figure 1B). However, p75 mediated activation of the survival enhancer NF-kB is not silenced by Akt (Trk activation), which supports the idea that p75 collaborate with Trk when Trk is robustly activated. In the case of weakly activated Trk receptor, death cascades predominate and apoptosis is triggered.
Figure 1. Summary of NTs signaling cascades.(modified from ) (A) p75 activation initiate death signaling cascades through the jnk-p53-Bax pathway; while Trk receptors mediate neuronal survival thought activation of Ras-PI3K/Akt pathway. (B) Binding of NTs with Trk initiate survival signaling by silencing the jnk-p53-Bax cascade that is activated by p75.
Although neuronal apoptotic activation is necessary for proper functioning of the nervous system, it can be activated through inappropriate neuronal death which can result in a variety of neurological conditions such as neurodegenerative diseases and ischemic stroke. It is clear that targeting the apoptotic biochemical pathways with potential modulators will protect neurons from degeneration, hence providing neuroprotection. The term "neuroprotection" applies to any mechanism or chemical compound (proteins, synthetic or natural product) that can prevent neuronal dysfunction, degeneration, apoptosis and injury. The effect of the administration of BDNF on several stimuli-induced apoptosis either in vitro or in vivo is well studied. Xia et. al has shown that exogenous application of BDNF prevents phencyclidine-induced apoptosis, in cultured brain slices through stimulation of the ERK and PI3K/Akt signaling cascades. In another report, BDNF was able to protect cortical neurons from the mitochondrial induced apoptosis initiated by 3-nitropropionic acid and this protection proved to be through the activation of the PI3K/Akt signaling pathway and the inhibition of the proapoptotic protein Bim in mitochondria. Moreover, BDNF protects hippocampal neurons from glutamate induced apoptosis in vitro, b-amyloid induced neurotocixity in vitro and in vivo, reduces inflammation as well as apoptosis in animal models. Additionally, BDNF has been shown to rescue basal forebrain cholinergic neurons (BFCNs) after excitotoxic lessoning and reduces infarct size after ischaemic injury. Administration of BDNF in rodent and monkey models of Parkinson's Disease (PD) shows protection of nigrostriatal dopaminergic neurons from neurotoxins. This promising preclinical data suggests that BDNF has the potential to be a neuroprotective agent and is supportive for the therapeutic application of BDNF in a variety of neurological disorders. However, clinical trials were unsuccessful mainly due to the poor pharmacokinetics of BDNF.
C. Preliminary Studies
Small molecules that can activate TrkB receptor and mimic the physiological action of BDNF are in high demand for development as potential neuroprotective agents. NTs selective agonists are the focus of many research groups including Ye's group that has developed a cell-based, caspase-activated assay using the flourecent dye MR(DERD)2 that turns red upon caspase-3 activation. Thousands of natural products and synthetic compounds were screened using this visual method for neuroprotective hits as well as selective TrkA and TrkB agonists. Based on their results deoxygedunin (1) (Figure 2A), a limonoid triterpenoid isolated from the Indian neem tree, showed the most robust protective effect in the primary screening against glutamate induced apoptosis. Deoxygedunin also protected hippocampal neurons from oxygen-glucose-deprivation (OGD), an in vitro model of ischemia, in a dose dependant manner (Figure 2B). This interesting neuroprotetive property of 1 was proven through the activation of TrkB. Immunofluorescent staining assay showed that 1 elicited strong TrkB activation (Figure 2C) which was shown to be in a dose-dependent manner (Figure 2D).
Figure 2. Deoxygedunin protects neurons from glutamate induced apoptosis through the activation of TrkB receptor. (Jang et al, 2010). (A) Chemical structure of deoxygedunin. (B) deoxygedunin inhipits OGD-triggered neuronal apoptosis in a dose dependent manner. (C). Deoxygedunin activates TrkB in primary hippocampal neurons. Hippocampal neurons were treated with 500 nM gedunin derivatives for 30 min and neurons were fixed and immunostained with rabbit polyclonal anti-p- TrkB (816) (1:100) and anti-MAP2. The nuclei were stained with DAPI. (D) Deoxygedunin triggers TrkB activation in primary neurons in dose depenet manner. Rat cortical neurons were treated with various concentrations of deoxygedunin for 30 min. Neuronal lysates were subjected to immunoblotting analysis by mouse monoclonal anti-p-TrkB (817)(1:20,000). Equal amount of TrkB was loaded (anti-TrkB from Biovision, 1:1,000) (lower panel).
In addition, 1 was able to provoke both Erk1/2 and Akt activation, the downstream cascades of TrkB activation, with a time course and in a dose-dependent manner as well. Interestingly, 1 was also able to provoke the activation of TrkB in the brain suggesting that 1 is orally bioavailable and can cross the blood brain barrier and stimulate TrkB activation. Further, 1 was able to prevent degeneration of vestibular ganglion in BDNF (-/-) mice. In addition, administration of 1 into mice showed neuroprotection as well as antidepressant and learning enhancement effects in the forced swim test.26a All of these outstanding in vitro and in vivo neuroprotective activities of 1 support the clinical application of 1. However, the major drawback of 1 is the presence of the metabolically labile furan moiety. Furan containing natural products are potential hepatotoxic agents via bioactivation by various CYP450 enzymes. This would limit the further development of 1 as a neruprotective drug. Thus, lead optimization and structure activity relationship (SAR) studies are needed. However, the furan moiety in 1 is chemically not accessible (cannot be replaced with other heterocycles or modified). So, an efficient synthetic route that would facilitate access to this part of the molecule is needed. Interestingly, no completed total synthesis of 1 has been reported.
Experimental/Research Design & Methods
D.1. Aim 1. The total synthesis of deoxygendunin using free radical-mediated cascade:
In designing a synthetic route toward the triterpenoid 1, we envisioned the common trans chair-chair conformation of the A, B and C rings, that are biosynthesized through a cationic triggered cascade of cyclizations of multiene intermediates. Based on that, we designed a biomimetic retrosynthetic analysis (Scheme 1) that utilizes the penta-ene intermediate II to initiate a free radical cyclization cascade. Intermediate II is basically the result of disconnection of the d-lactone as well as the furan moieties from 1. We envisioned several coupling approaches in analyzing intermediate II that will use simple and commercially available starting materials. Two key sites are valuable for disconnection in II as highlighted in Scheme 1 mainly between the two conjugated systems. This will help break down II into the iodo-enone III, a four carbon fragment IV and the cyanoaldehyde V. Further analysis of III revealed that it could be developed from the biomimetic starting material hydroxyisoprene which is commercially available.
Scheme 1. Retrosynthetic plan for 1
D.1.1. Synthesis of intermediate III:
Building up intermediate III starts with the epoxidation of hydroxyisoprene 2 followed by the silyl protection of the primary hydroxy group which should yield the corresponding protected epoxide 3 in high yield (Scheme 2). Nucleophilic addition of the acetylenide anion to 3 should open the peroxide and give the corresponding alcohol after workup. Protection by methoxymethylbenzene (MOM) group will give the important intermediate 4 which is the reduced form of III. Deprotonation of 4 by lithium diisopropyl amide (LDA) and then addition of the generated acetylenide anion to the commercially available 2-cyanoacetaldehyde will yield the cyanoacetylino compound 5. Two diastereoisomers are expected, however, further oxidation of the generated secondary hydroxy groups followed by enolization by LDA and then methylation will help maximize the yield of the cyano methyl enol 6.
Scheme 2. Synthesis of intermediate III and the installation of the triene functionality.
Trans reduction of the acetylenic functionality in 6 could be achieved via Birch reduction which may also remove the MOM protection group. Thus, reprotection will follow to generate 7. The next stage is to extend the second portion of the molecule. Deprotection of the silyl group followed by a swern oxidation of the unprotected primary hydroxy group should yield the corresponding aldehyde 8 in high yield. Applying the Takai olefination to the aldehyde group in 8 will install the iodovinyl functionality. Both cis and trans isomers are expected and the desired cis conformation 9 will be purified.
D. 1.2. Synthesis of intermediate II: The iodovinyl derivative 9 is perfectly set up for a Stille coupling reaction with the hydroxytinylated compound 10 (Scheme 3). The tinylated partner 10 could be easily obtained from a one-pot reductive tinylation with Red-Al and Bu3SnCl (Scheme 3). Stille cross coupling reaction of 9 with 10 followed by a silyl protection of the secondary hydroxy group will give the important cyano intermediate 11. At this stage we will have now the tetraene fragment that would be used to construct the A and B rings in 1. The cyano group will be used for the installation of the remaining double bond needed for the completion of the tricyclic core of 1. Mild acidic hydrolysis followed by a selective reduction of the generated carboxylic acid will give the corresponding aldehyde (structure not shown). Nucleophilic addition of propenyllithium followed by the oxidation of the two diastereoisomers with pyridinium chlorochromate, then a nucleophilic substitution of the hydroxy group with an iodide will yield the iodo pentaene intermediate 12 (II). Having this iodopentaene compound in hand, the free radical-mediated cyclization cascade could be achieved. Treatment of 12 with a free radical initiator, AIBN, and after workup should deliver the tricyclic core of 1 with the expected trans-trans conformation of the three rings. Although this conformation is very common in triterpenes and several synthetic groups including Corey's group have utilized similar approach for the synthesis of stereoids, other stereoisomers of 13 could be obtained and will have a great value in the structure activity relationship studies (SAR) (discussed in aim 2).
Scheme 3. Synthesis of intermediate II and the cyclized product 13.
D.1.3. Completing the total synthesis:
An unsaturated center will be installed in conjugation with the keto group of 13 through a series of selenation, oxidation and elimination reactions to yield the a,b-unsaturated intermediate 14 (Scheme 4). Alkylative Birch reduction will then be employed to install a two carbon fragment in the a position to the carbonyl 15. Reduction of the newly installed ester group followed by protection of the produced primary hydroxy group would yield compound 16. A typical aldol condensation reaction between 16 and an activated methylene reagent, diethylmalonate, will yield the condensed product which upon mild hydrolysis and heating will undergo a rapid decarboxylation reaction to produce the carboxylic acid 17 (the other isomer could be obtained). Installing the d-lactone moiety is quite tricky since compound 17 has one additional carbon that needs to be truncated first. A series of reactions will be utilized to accomplish this task which start with the reduction of the carboxy group followed by a MOM protection of the produced hydroxy group to give an intermediate that has two primary hydroxy groups with two different protection groups (structure is not shown). Selective deprotection of the silylated hydroxy group with a fluoride source, then further oxidation to the corresponding aldehyde via Swern oxidation would generate the aldehyde 18. Degradation of the aldehyde group to one carbon homolog is achievable target via in-situ enolization of the carbonyl group followed by a rapid dihydroxylation of this enolate and then cleavage by periodate reaction. Utilizing this approach, a one carbon-less homolog 19 will be prepared. Addition of the lithiofuran, generated by the tranmetallation of 3-bromofurane with t-butyl lithium, to the carbonyl group will generate two diastereoisomers in which 20 is the desired product. The other diastereoisomer will be used in aim 2 in the SAR studies. Deprotection of the MOM protective groups in 20 followed by selective oxidation of the primary hydroxy group to the corresponding aldehyde will make the access to the d-lactone moiety possible through the formation of hemiactal intermediate in-situ which will undergo further oxidation to form the d-lactone 21. Compound 21 is actually an analog of 1 that will be used in aim 2. A selective cleavage of the enol methyl ether functionality followed by a stereoselective reduction of the produced carbonyl group and further esterification will finalize the total synthesis of 1.
Scheme 4. Completed synthesis of 1
D.2. Aim 2. Biological Evaluation and Structure activity relationship studies:
D.2.1. Structure activity relationship (SAR) studies:
We will utilize our completed synthetic route to access several parts of the molecule and generate useful SAR studies. Our modification strategy is based on the replacement of the furan ring, an established toxicophore, with other heterocycles and exploring the effect of stereochemistry on the neuroprotective activity of 1. Replacement of the furan ring with other heterocycles could be achieved through the reaction of compound 19 with different arylhalides. This will generate a library of compounds for the SAR studies (Table 1). These analogs will be evaluated for neuroprotection as discussed in section D.2.2 and the positive hits will be further evaluated in vitro for hepatotoxicity. We will take the advantage of all the omitted diastereoisomers in aim 1 at the late stages of the synthesis to build up different diastereoisomers of the potent and less toxic analog in table 1. For example, all the diastereoisomers of the ketonic intermediate 13 will be utilized (Figure 3) as well as diastereoisomers of 15, 20 and 21 (Figure 4).
Table 1. Proposed analogs of 1 with different heterocycles
Figure 3. Possible epimers of 1 that could be generated from the different diastereoisomers of 13
Figure 4. Possible epimers of 1 that could be generated from the different diastereoisomers of 15, 20 and 21
D.2.2. Neuroprotection assays: Cerebellar granule neurons (CGNs) are widely used as a model for neuronal survival and death mechanistic studies. Immature CGNs isolated from newborn rats die by apoptosis when cultured in a media containing a physiological concentration of K+ (~5 mM K+ low K), however, CGNs survive when cultured in fetal bovin serum (FBS) and non-physiologically high extracellular K+ (25 mM). We will utilize CGNs cultured in trophic factor deprived-low K to test our generated analogs of 1 for their neuroprotective properties.
D.2.2.1. Preparation of Cerebellar Granule Cell Cultures: 7 day-old Wistar rats (from Harlan) will be killed by decapitation and the cerebellum will be removed and washed with phosphate-buffered saline (PBS) supplemented with 13.9 mM glucose, 3.2 mM MgSO4, and 3 mg/mL defatted BSA at 4 oC. Cerebellum will be minced into small sections with a No. 11 scalpel blade. Minced tissue will then be incubated in trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) at 37 oC with gentle agitation for 10 min. After trypsinization, the minced tissue will be resuspended and triturated in weak DNase (0.05% final concentration) followed by strong DNase buffer (0.3% final concentration) solutions (the buffer composed of 6 mM MgSO4, 30 units/mL DNase and 50 ug/ml soybean trypsin inhibitor). After centrifugation at room temperature (5 min, 224 xg), the cell pellet will be resuspended in cell culture media (Eagle's minimum essential media with Earle's balanced salt solution, 2 mM glutamine (0.292 g/L), 10% fetal heat-inactivated bovine serum (Lifetech), 6 mg/mL glucose, 25 mM KCl, 10 U/mL penicillin, 10 lg/mL streptomycin). The diluted suspension will be filtered through a 70 mm filter to remove large clumps, washed once and counted using hemocytometer. Cells will be platted on 4-well LabTek chambers previously coated with polyethleneimine (1.5 x 106 per well) and maintained at 37 oC in a humidified atmosphere of 5% CO2/95% air. One day after plating, 10 mM cytosine arabinosefuranoside will be added to prevent replication of non-neuronal cells (glial cell). Neurons will be incubated in culture for 6-10 days and will be used for the evaluation of the analogs.
D.2.2.2. Low potassium initiated apoptosis and incubation conditions: Apoptosis will be initiated by modifying the culture media with (in mM): 137.5 NaCl, 3.5 KCl, and 0.3% w/v fatty acid-free bovine serum albumin (BSA) in place of FBS (trophic factor deprivation (3.5K/BSA, or low K)). Deoxygedunin analogs dissolved in DMSO will be added to a final concentration of 0.5 mM in low K and the treated cells will be incubated for 5 hrs at 37 oC. To normalize the results, and to overcome handling error and variability in cell counts between preparations, the high and low K reference will be run in parallel with the treated cells.
D. 2.2.3. Assessing apoptotic cells and calculating % apoptosis: After the incubation period, treated cells will be perfused 5 min with the low K buffer containing 0.15 mM of the cell-permeable SYTO13 fluorescent dye which exhibits bright green fluorescence upon binding to nucleic acid. The cells will be visualized under the fluorescent microscope (Axiovert 200 M epifluorescent microscope (Zeiss)) using 480 nm excitation. At least 12 random images from different locations on the plate will be taken so that at least 100 cells per treatment per experiment were obtained. Cells with condensed nuclei (punctuate and/or discrete high intensity staining) will be considered as apoptotic cells. Apoptotic cells as well as normal cells will be counted (AxioVision, V 18.104.22.168) and the percentage of apoptosis will be calculated as the mean value of all the percentage in all the random images. Compounds are considered neuroprotective if they show less apoptotic percentage than that calculated in the low K reference. The experiments will be run in triplicate and data will be compared using one-way ANOVA followed by Bonfferoni post hoc analyses (GraphPad Prism 4). Differences were considered statistically significant when p <0.05.
D.2.2.4. Cell-based caspase-3 assay:25a This assay will be done in parallel with the low K-triggered apoptosis in which the treated cells will be exposure to the low K buffer containing 10 mM of MR(DEVD)2, a cell permeable caspase-3 activated fluorescent dye. The cells will be fixed with 4% paraformaldehyde for 15 min and then washed with PBS. The cells will be visualized by fluorescent microscope as discussed early and the percentage of apoptosis will be analyzed in the same way.
D.2.3. Hepatocytotoxicity assay: Positive hits from the neuroprotection assays will be evaluated for hepatocytotoxicity in vitro assay. Primary culture of rat hepatocytes will be used for the evaluation of analogs.
D.2.3.1. Cell Isolation and Culture Preparation: Rat hepatocytes will be isolated from adult Sprague-Dawley rats (from Harlan) using the two step collagenase perfusion method. In brief, the abdomen of the rat will be opened through a midline incision and the liver will be taken out. A portal cannula will be then placed and the liver will be prefused with EDTA solution (0.02%) at 37 oC at flow rate of 30 mL/min for 10 minutes. The collagenase solution will be recirculated through the liver at the same flow rate. After ten minutes, the digested liver parenchyma will be suspended in the ice-cold Hanks's solution (Invitrogen, Carlsbad, CA, USA). The resulting cell suspension will be filtered, washed and cultured in William's E culture media (Invitrogen, Carlsbad, CA, USA) supplemented with 10mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid, 10% U/mL pencillin, 100 ug/mL streptomycin and insulin-transferring-selenuim A supplement. Trypan blue (Invitrogen, Carlsbad, CA, USA) exclusion test via a hemocytometer will be used to ascertain the viability of cultured cells. The cell suspension will be considered valid if the cell viability was greater than 80%. Cells will be platted on 96-well plate at a density of 105 and maintained at 37 oC in a humidified atmosphere of 5% CO2/95% air for 48 hr.
D.2.3.2. Incubation conditions and lactate dehydrogenase (LDH) measurement: LDH activity will be determined by using LDH cytotoxicity assay kit (Abcam Plc). Briefly, hepatocytes cells will be seeded in 96-well plate at density of 105. Deoxygedunin analogs will be dissolved in DMSO and diluted with the media buffer according to the desired concentrations (0.1, 0.2, 0.3, 0.4 and 0.5 mM). The 96-well plat will be incubated at 37 oC in a humidified atmosphere of 5% CO2/95% air for 1 h and the plate will be centrifuged at 250 x g for 10 minutes. 100 mL of each supernatant from each well will be transferred to another 96-well plate that will have 100 mL of the reaction mixture in each well. The plate will be incubated for 30 minutes in the dark at room temperature and the absorbance will be recorded at 490 nm with a plate reader. 1% Triton X-100 will be used as positive control and media without cells will be used as vehicle (negative control). All samples, positive, negative controls and treated cells are run in triplicate and the LDH leakage will be calculated. Dose response curves will be generated by plotting the percentage of LDH leakage versus test concentrations using Graphpad prism 5.0 software. Mean +/- SEM will be calculated from three independent determinations. Statistical and significant differences will be determined by analysis of variance. Data will be considered significant if P values < 0.05. The compound will be considered toxic if the average cell leakage will be 50% or greater.
D.3. Expected results:
We expect to complete the first total synthesis of the neuroprotective natural product, deoxygedunin (1), through an innovative synthetic route utilizing the free radical triggered cyclization cascades. This synthetic route will help the replacement of the toxicophore moiety, the furan ring, with other heterocycles. We will generate a library of analogs and evaluate their neuroprotective activities. Positive hits will be evaluated for their hepatocytotoxicity by using the primary hepatocytes cells. Our long term goal is to synthesize a nontoxic neuroprotective agent that will be ready for the preclinical studies.
D.4. Time Frame:
Year 1: Completing the total synthesis of deoxygedunin
Year 2: Generate a library of analogs and evaluate their neuroprotective properties using CGN low potassium induced apoptosis. Evaluation of the hepatocytotoxicity of the positive hits using primary hepatocyte cells will be completed.