WO2011050481A1

WO2011050481A1 - New eremophilane sesquiterpene lactones from senecio jacobaea

Info

Publication number: WO2011050481A1
Application number: PCT/CA2010/001738
Authority: WO
Inventors: Junzeng Zhang; Xiuhong Jl; Jianhui Liu; Jeff Zidichouski; Yanwen Wang
Original assignee: National Research Council Of Canada
Priority date: 2009-10-30
Filing date: 2010-11-01
Publication date: 2011-05-05

Abstract

New eremophilane sesquiterpene lactones were isolated from the extract of the root of Senecio jacobaea having the general formula I. The structures were elucidated by spectroscopic methods including 2D NMR techniques. Methods of using the compounds as acetylcholinesterase inhibitors and neuroprotective compounds are also described

Description

NEW EREMOPHILANE SESQUITERPENE LACTONES FROM SENECIO /ACOBAEA

FIELD OF THE INVENTION

The present invention relates to plant extracts and uses thereof. More specifically, the invention relates to compounds isolated from plant extracts and their use in the prevention and/or treatment of neurodegenerative diseases.

BACKGROUND OF THE INVENTION

It is well known that acetylcholinesterase (AChE) inhibitors are among the known therapeutic agents for Alzheimer's disease.

Age-related neurodegenerative disorders such as Alzheimer's disease (AD) are becoming major health problems and it was estimated that 25 million people are now affected by AD alone worldwide. This progressive cognitive impairment has caused neuropsychiatric and behavioural disturbances and lowered the quality of life for many senior individuals. With the growing aging population, AD and other age-related brain disorders are becoming one of the major health challenges for the current century. The cause or causes of AD are unknown at present time, and current fields of research point in several directions in terms possible treatment options. Acetylcholine (Ach) is a neurotransmitter implicated in the complex processes underlying cognition, learning and memory formation. It is broken down in the brain to inactive metabolites, and may be in deficit in persons afflicted with AD. In this regard, the ability to consolidate and retrieve new memories the hallmark and first recognizable symptomatic deficit associated with the onset and progression of AD. This forms the basis of the longstanding connection between ACh dimunition and loss of cognitive function. Currently, blocking the breakdown of acetylcholine (ACh) in the brain is an intervention that provides only symptomatic relief for AD sufferers.

A central theme occurring in all neurodegenerative diseases, Including AD is neuronal dysfunction leading to cellular death and the ultimate loss of associated function. The process of neurodegeneration is complex and involves a variety of diverse mechanisms that trigger or lead to a cascade of biochemical events that precede cellular death. For example, excitotoxicity, oxidative stress, inflammation, hyperactivation of the immune system, environmental toxins, viruses, gene activation/inactivation, intracellular calcium homeostasis, have ail been implicated as factors that cause or contribute to processes and mechanisms involved in neuronal death, including those regarded as potential causes of AD. Moreover, the overall process cell death whether it be rapid {eg. subsequent to a stroke) or very slow (due to exposure to a toxin) involves cellular dysfunction that, if not corrected, will ultimately lead to neuronal death and loss or diminution of physiological, structural, and/or biochemically- important function. In the context of treating neurodegenerative-related diseases, it is logical and of high utility be able to detect and intervene at the first evidence of cellular dysfunction. In the case of AD, investigations for biomarkers and/or clinically-detectable endpoints have unfortunately not materialized to date. AD is still clinically diagnosed and confirmed via a post-mortem examination of the brain. Hallmark, postmortum pathologically defined observations of AD are excessive accumulation of A-Beta protein in the form of amyloid plaques, the observation of neurofibrillary tangles comprised of hyperphosphorylated Tau proteins, marked atrophy of the brain, and significant loss of neuronal connections.

Cognitive impairment or deficit due to age-related neurodegeneration is presently managed by a number of different approaches. Firstly, natural antioxidants and phytochemicals such as green tea polyphenols, curcuminolds, blueberry anthocyanlns, soybean isoflavones, resveratrol, gingko flavones, ginsenosides, etc. are found to have beneficial effects in animal studies and clinical trails. Secondly, acetylchoinesterase inhibitors (AChEI, which Inhibit acetylcholinesterase, an enzyme that breaks Ach down to inactive metabolites) are becoming standard therapeutic agents for AD; among AChEI-based AD drugs approved by the FDA are tacrine, donepezil, n^'vastigmine, and galatamine. Huperzine A, an AChEI derived from Chinese herb Huperzia serrta, has been in Chinese market for dementia and is now verified in European clinical trails. The third approach includes the application of N-methyl-D-aspartate (NMDA) antagonists such as memantine for treatment of moderate to severe AD. Combination use of AChEI and NMDA antagonist was also shown to have incremental benefits.

Although some AChEls and NMDA antagonists are currently used as therapeutic agents for AD and other cognitive deficits, most of them are synthetic chemical drugs which have been reported to have safety concerns (for example, tacrine's reversible hepatoxicity) and gastrointestinal symptoms. Plant-derived compounds such as Huperzine A and Galatamine are generally presented at very low level in specific plant species so the cost of production is considerably high.

Unfortunately, the current treatment options available to AD suffers provide only symptomatic relief as they do not modify, slow or stop the progression of AD to its more severe stages ultimately leading to early death. Therefore, the discovery of disease modifying drugs (DMDs), rather than those that treat the disease symptomatically has been an area of intense investigation for many years. The global scope and scale of resources and R&D efforts put towards finding new DMD-based treatments for AD is staggering, but has been met with many late stage clinical failures. However, as the prevalence and incidence of AD is increasing globally and many countries are preparing for a large demographic wave of subjects over the age of 65 in the coming years, the demand and need for the discovery and development of new treatment options for AD as well as other neurodegenerative diseases (such as Parkinson's Disease and Multiple Sclerosis) Is paramount. In particular, multimodal compounds or compositions that interfere with more than one mechanism involved in the etiology of cellular death would be of high utility In treatment of neurodegenerative diseases, including AD.

SUMMARY OF THE INVENTION

The present invention provides a compound comprising the structure of Formula I

Formula t or a pharmaceutically acceptable derivative thereof, wherein R is OH or a derivative thereof, or taken together with R² forms a double bond between C9 and C10; and R³ is OH or a derivative thereof, or taken together with R² forms a double bond between C8 and C9; with the proviso that one of R or R³ is OH or a derivative thereof, but not both. The compound of the present invention may be as described above, wherein the compound comprises the structure of Formula II

Formula II or a pharmaceutically acceptable derivative thereof, wherein when R¹ is OH or a derivative thereof, the stereochemistry at C10 is R; and when R³ is OH or a derivative thereof, the stereochemistry at C8 is R.

In one example, the compound may be as described above, wherein R³ is OH or a derivative thereof, and a double bond is present between C9 and C10. More specifically, the compound may comprise the structure of Formula III

Formula III or a pharmaceutically acceptable derivative thereof.

Alternatively, the compound may be as described in Formula I or II, wherein R¹ is OH or a derivative thereof, and a double bond is present between C8 and C9. More specifically, the compound may comprise the structure of Formula IV

Formula IV or a pharmaceutically acceptable derivative thereof.

The compounds as described herein above may be acetylcholinesterase inhibitors.

The present invention also provides a composition comprising one or more than one compound as described herein and a pharmaceutically acceptable diluent, excipient, or carrier. The composition may further comprise one or more than one neuroprotective compound.

The method further provides a method of Inhibiting aceltylcholinesterase, comprising contacting the acetylcholinesterase with one or more than one eremophifane sesquiterpene.

A method of increasing neuronal cell viability is also provided; the method may comprise contacting the neuronal cell with one or more than one eremophilane sesquiterpene.

The present invention additionally provides a method for treatment or prevention of a neurodegenerative disease comprising administering an effective amount of one or more than one eremophilane sesquiterpene to a patient in need thereof. In the methods described above, the one or more than one eremophilane sesquiterpene may be obtained from Senecio jacobaea. The one or more than one eremophilane sesquiterpene may also be a compound of Formula V

Formula V or a pharmaceutically acceptable derivative thereof, wherein R¹ is OH or a derivative thereof, or taken together with R² forms a double bond between C9 and C10; R³ is OH or a derivative thereof, or taken together with R² forms a double bond between C8 and C9; R⁴ is OH or a derivative thereof; with the proviso that one of R¹ or R³ is OH, but not both. More specifically, the one or more than one eremophilane sesquiterpene may be a compound of Formula VI

Formula VI or a pharmaceuticaliy acceptable derivative thereof, wherein when R¹ is OH or a derivative thereof, the stereochemistry at C10 is R; and when R³ is OH or a derivative thereof, the stereochemistry at C8 is R. In one example, R¹ or R³ is OH or a derivative thereof, and R⁴ is selected from

In one example of the methods described above, the one or more than one eremophilane sesquiterpene may be selected from the group consisting of 6p^-dimethoxy-1 -oxoeremophil- 9(10)-en-8a, 12-olide, 6p-angeloyloxy-8a-hydroxy-1 -oxoeremophil-9(10)-βη-8β,12-olide, 6β- angeloyloxy-1 Oa-hydroxy-1 -oxoeremophil-8(9)-en-8,12-olide, and any combination thereof.

In the methods described above, the neurodegenerative disease may be Alzheimer's disease. Also, the composition or compound in the methods described may be administered orally or parenterally.

The root extract of common plant tansy ragwort (Senecto jacobaea) was found to have AChE inhibitory effect. Bioassay-guided fractionation and purification led to isolation of 3 pure eremophilane sesquiterpene compounds, all possessing potent AChE inhibition activity. The AChE inhibitory activity of these eremophilanes were further confirmed in SH-SY5Y cell assay. In addition, the invention revealed neuroprotective functions of the eremophilane compounds against beta amyloid, glutamate, and hydrogen peroxide induced toxicity in SH-5Y5Y cells. As such, the present invention provides eremophilane sesquiterpenes as a new class of AChE inhibitory and neuroprotective agents for prevention or treatment of neurodegenerative diseases such as Alzheimer's. Extracts or refined fractions from Senecio jacobaea, a very common plant regarded as an invasive weed in certain regions or countries (such as Canada), containing bioactive eremophilane sesquiterpenenes, may also be used as natural health products for the sample applications.

Additional aspects and advantages of the present invention will be apparent in view of the following description. The detailed description and examples, while indicating preferred embodiments of the invention, are given by way of Illustration only, as various changes and modifications within the scope of the invention will become apparent to those skilled in the art in light of the teachings of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein: FIGURE 1 is a flow chart summarizing the extraction and purification process for INH-25-2NA- 1 and INH-25-2NA-5. Raw material was extracted with solvent, and fractioned. The two pure compounds were obtained through steps of further purification such as preparative thin layer chromatography (TLC). FIGURE 2 is a flow chart summarizing the extraction and purification process for LR-49-55-2 and the other two compounds INH-25-2NA-1 and INH-25-2NA-5. This raw material was obtained from a different location, and after extraction, a different fractionation, and purification procedures were applied.

FIGURE 3 is a high resolution mass spectrometry (HRMS) spectrum for INH-25-2NA-1. The molecular formula was determined to be C₁₇H₂₂0₅. MS ion peak 307.1536 for [M+H]⁺ (calculated for C_1rH₂₃0₅ ⁺ 307.1546).

FIGURE 4 is a HRMS spectrum for INH-25-2NA-5. The molecular formula was determined to be C₂₀H₂4O₆. MS ion peak 361.1645 for [M+H]⁺ (calculated for C₂oH₂₅0₆ ⁺ 361.1651).

FIGURE 5 is a HRMS spectrum for INH-25-2NA-5. The molecular formula was determined to be C₂oH₂40₆. MS ion peak 383.1462 for [M+Naf (calculated for C₂oH₂₄0eNa⁺ 383.1671).

FIGURE 6 is a graph illustrating the AChE inhibiting effect of initial fractions of a methanol extract designated as INH-25, and further fractions thereof. The bioactive fraction INH-25-2 was further fractioned, and yield the more potent sub-fraction INH-25-2NA.

FIGURE 7 is a graph illustrating the AChE inhibiting effect of a fraction designated as INH-25-2 and the pure compounds INH-25-2NA-1 and INH-25-2NA-5 thereof.

FIGURE 8 is a bar graph showing the effect of INH-25-2NA-1 on the AChE activity in SH-SY5Y cells. After SH-SY5Y cells were treated with 0, 1 , 10 and 100 μΜ of the compound for 24 h, the cells were lysed and AChE activity was determined with a spectrophotometric method. Data are mean±SD from three independent experiments. **, P<0.01 , and *P<0.05 vs. control. Eserine (18.2 μΜ) was used as a positive control in these experiments.

FIGURE 9 is a bar graph showing the effect of INH-25-2NA-5 on the AChE activity in SH-SY5Y cells. After SH-SY5Y cells were treated with 0, 0.1 , 1 , 10 and 100 μΜ of the compound for 24 h, the cells were lysed and AChE activity was determined with spectrophotometric method. Data are mean±SD from three independent experiments. **, P<0.01 , and ***P<0.001 vs. control. Eserine (18.2 μΜ) was used as a positive control in these experiments. FIGURE 10 is a bar graph showing effect of LR-49-55-2 on the AChE activity in SH-SY5Y cells. After SH-SY5Y cells were treated with 0.1 , 1 , 0 and 100 μ of the compound for 24 h, the cells were lysed and AChE activity was determined with spectrophotometric method. Data are mean±SD from three independent experiments. **, PO.01 vs. control. FIGURE 11 Is a bar graph showing the effect of INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55- 2 on the AChE activity in SH-SY5Y cells. After cells were treated with 10 μ of testing compounds for 24 h, AChE activity was determined. Eserine (18.2 μΜ) was taken as positive control.

FIGURE 12 is a bar graph showing the effect of INH-25-2NA-5 on the cell viability of SH-SY5Y in the presence or absence of glutamate. After the cells were treated with indicated concentration (0.1 , 1 , 10, or 100 μΜ) of INH-25-2NA-5 for 2 h in phenol red- and serum-free media, the cells were incubated for 24 h in the presence or absence of 5 rtiM glutamate. Cell viability was determined with MTT assay.

FIGURE 13 is a bar graph showing the effect of INH-25-2NA-5 on the cell viability of SH-SY5Y in the presence or absence of Αβ1 -42. After the cells were treated with indicated concentration of INH-25-2NA-5 (0.1 , 1 , 10 or 100 μ ) for 2 h in phenol red- and serum-free media, the cells were incubated for 24 h in the presence or absence of 5 μ Αβ1-42. Cell viability was determined with MTT assay.

FIGURE 14 is a bar graph showing the effect of INH-25-2NA-5 on the cell viability of SH-SY5Y in the presence or absence of H2O2. After the cells were treated with indicated concentration of INH-25-2NA-5 (0.1 , 1 , 10 or 100 μΜ) for 2 h in phenol red- and serum-free media, the cells were incubated for 24 h in the presence or absence of 100 μ H₂0₂. Cell viability was determined with MTT assay.

FIGURE 15 is a bar graph showing results of cytotoxicity test of INH-25-2NA-5 in SH-SY5Y cells. After SH-SY5Y cells were treated with 0.1 , 1 , 10 or 100 μΜ of the compound for 24 h, cell viability was determined with MTT assay. Data are shown as mean±SD from three independent experiments.

FIGURE 16 is a bar graph showing a neuroprotective effect of INH-25-2NA-1 using a Glutamate-induced excitoxicity-based MTT assay in SH-SY5Y cells. (** indicates p<0.01 , n=3) FIGURE 17 is a bar graph showing the neuroprotective effects of INH-25-2NA-5 using a Glutamate-induced excitoxicity-based MTT assay and shows dose-dependency up to 10μΜ INH-25-2NA-5. The neuroprotective effect was statistically-significant from controls at 1 (** indicates p<.05, n=3) and 10μΜ INH-25-2NA-5 (^indicates p<.001 , n=3).

FIGURE 18 is a bar graph showing the effects of LR-49-55-2 on Glutamate-induced excitoxicity based MTT assay in SH-SY5Y cells {** indicates p<0.01 , n=3). A highly significant protective effect was observed at the lowest doses tested (10μΜ), but higher doses of the compound appears to not have this effect as dose was increase.

Detailed Description of the Invention

The present invention provides a compound comprising the structure of Formula I

Formula I or a pharmaceutically acceptable derivative thereof, wherein R¹ is OH or a derivative thereof or taken together with R² forms a double bond between C9 and C10; and R³ is OH or a derivative thereof or taken together with R² forms a double bond between C8 and C9; with the proviso that one of R or R³ is OH or a derivative thereof, but not both. Thus, when one of R¹ or R³ is a substituent, the other forms a double bond together with R².

A person of skill in the art would recognize that a number of chiral centres are present in Formula i. The chiral centres may be in either configuration. For example, and without wishing to be limiting in any manner, in one embodiment of the present invention, the compound may comprise the stereochemistry of Formula II

Formula II

or a pharmaceutically acceptable derivative thereof, wherein when R¹ is OH or a derivative thereof, the stereochemistry at C10 is R; and when R³ is OH or a derivative thereof, the stereochemistry at C8 is R.

In a specific, non-limiting example, the compounds of the present invention may be, but are by no means limited to:

• a compound as defined above, where R³ is OH or a derivative thereof, and a double bond is present between C9 and C10. Such a compound includes the structure of

Formula III

Formula III or a pharmaceutically acceptable derivative thereof. The compound of Formula III may also be referred to herein as 6p-angeloyloxy-8a-hydroxy-1-oxoeremophil-9(10)-en-8p,12- olide, or I H-25-2NA-5; or a compound as defined above, where R¹ is OH or a derivative thereof, and a double bond is present between C8 and C9. Such a compound includes the structure of Formula IV

Formula IV or a pharmaceutically acceptable derivative thereof. The compound of Formula IV may also be referred to herein as 6 -angeloyloxy-10a-hydroxy-1-oxoeremophiI-8(9)-en-8,12- olide, or LR-49-55-2.

The compounds of Formula I also encompass chemical derivatives of the compounds as described above. For example, the hydroxyl group at R¹ or R³ may be derivatized by methods commonly used in the art. In a non-limiting example, R¹ or R³ may be derivative to an

-C_1-30ester, -Ci.₃oamido, -Ci_-3ocarboxyamido, CrCso-carboxy, or C C_3crcarbonyl; the carbon-containing substituents of R or R³ may be unbranched or branched. Thus, in the compounds described above, wherein R¹ may be OH, -Ci-3₀alkoxy, -Ci.₃₀acyl, -Ci.3oether, -Chester, -C_1-3oamido,

CvC₃o-carboxy, CrC₃₀-carbonyl, or, taken together with R² forms a double bond between C9 and C10; and R³ may be OH, -Ci-3oalkoxy, -C_1-3oacyl,

-C_1-30amido, -C_1-30carboxyamido, C Cso-carboxy, C-VCso-carbonyl, or taken together with R² forms a double bond between C8 and C9. In one non-limiting example, R¹ or R³may be esters of omega-3 fatty acids such as ct-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) (C18, C20 or C22, respectively. Deriyatization the hydroxyl groups may be accomplished by any suitable organic chemistry method known in the art. Ample texts address derivitization of such groups, for example but not limited to Fruniss et al (1989), Vollhardt and Schore (1998). For example, esters may be prepared by reacting the hydroxyl group with carboxylic acid, acyl chlorides, etc.).

The present invention further provides compositions, also referred to herein as "formulations", comprising a compound of the present invention. The formulations of the present invention may comprise one or more than one of the compounds of the present invention; in particular, and without wishing to be limiting in any manner, the present invention encompasses a composition comprising a mixture (or "cocktail") of the compounds of the present invention. Such a mixture may provide a composition with increased potency. Compositions also include extracts comprising the compounds as described herein; such extracts may include buffers, salts, and other components as listed below.

In addition to the one or more compound of the present invention, the compositions may comprise a pharmaceutically acceptable carrier, diluent or excipient (or "pharmacologically acceptable ingredient"). The carrier, diluent or excipient may be any suitable carrier, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and must not be deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in liquid form, suspension form, powder form (for example, lyophilised), capsule or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH. In a specific, non-limiting example, the pharmaceutically acceptable carrier may be saline. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be so formulated as to deliver the compound to the gastrointestinal tract of the subject, or in a time- release manner. Thus, the composition may comprise encapsulation, time-release, or other suitable technologies for delivery of the antibody or fragment thereof. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.

For example, and without wishing to be limiting in any manner, pharmacologically acceptable ingredients for nutraceutical and/or pharmaceutical compositions, include anti-adherents, binders (e.g. starches, sugars, cellulose, hydroxypropyl cellulose, ethyl cellulose, lactose, xylitol, sorbitol and maltitol), coatings (e.g. cellulose, synthetic polymers, corn protein zein and other polysaccharides), disintegrants (e.g. starch, cellulose, cross-linked polyvinyl pyrrolidone, sodium starch glycolate and sodium carboxymethyl cellulose), fillers/diluents (e.g. water, plant cellulose, dibasic calcium phosphate, vegetable fats and oils, lactose, sucrose, glucose, mannitol, sorbitol and calcium carbonate), flavors and colors, glidants, lubricants (e.g. talc, silica, vegetable stearin, magnesium stearate and stearic acid), preservatives (e.g. vitamin A, vitamin E, vitamin C, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben and propyl paraben), antioxidants, sorbents, sweeteners, and mixtures thereof. The composition as described above may also comprise one or more than one known neuroprotective compound. The one or more than one neuroprotective compound may be any suitable neuroprotective compound known in the art. For example, the neuroprotective compound may be a natural neuroprotective. In a specific, non-limiting example, the one or more than one neuroprotective compound may be chosen from natural antioxidants and phytochemicals such as, but not limited to green tea polyphenols, curcuminoids, blueberry anthocyanins, soybean isoflavones, resveratrol, gingko flavones, ginsenosides, omega-3 fatty acids, CoQ10, phosphatidylserine; or drugs such as, but not limited to AChEls (such as, but not limitied to such as tacrine, donepezil, rivastigmine, and galatamine, huperzine A.), NMDA antagonists (such as, but not limited to memantine), Αβ aggregation blockers, and β- and γ- secretase inhibitors. The present invention additionally provides a method of inhibiting acetylcholinesterase, comprising contacting the acetylcholinesterase with one or more than one eremophilane sesquiterpene. These compounds also provide a neuroprotective effect (i.e., protection against neurodegeneration). Thus, the present invention also provides a method of increasing neuronal cell viability, comprising contacting the neuronal cell with one or more than one eremophilane sesquiterpene.

Eremophilane sesquiterpenes are a class of sesquiterpene compounds with the structural skeleton of eremophilane (Formula VII),

Formula VII

Formula VIII Formula IX In the methods as described above, the one or more than one eremophilane sesquiterpene may be provided in pure form, an extract or a bioactive fragment thereof, or in a composition. The eremophilane sesquiterpene may be obtained from Senecio spp., Ligularia spp., Farfugium spp., Cacalia spp., Petasites spp., Ligulariopsis spp., Stevla spp. or any other suitable plants. Eremophilane sesquiterpenes may also be obtained by fermentation of fungi such as Xylaria spp (Isaka et al, 2010). In a non-limiting example, the eremophilane sesquiterpene may be obtained from Senecio jacobaaa, or more specifically, from the root of

Senecio jacobaea. Methods of extracting eremophilane sesquiterpene are described herein and in the prior art; without wishing to be limiting in any manner, eremophilane sesquiterpene may be obtained by providing tissue from Saneclo spp., Ligularla spp., Farfugium spp., Cacalia spp., Petasites spp., Ligulariopsis spp.,Stevla spp. or any other suitable plants, or from fermentation of fungi such as Xylaria spp. and extracting therefrom by a suitable solvent, an extract, or a bioactive fraction thereof. Thus, it is within the abilities of one skilled in the art to extract eremophilane sesquiterpenes from the plant. Alternatively, the eremophilane sesquiterpenes used in the methods of the present Invention may be synthesized using methods known in the art, for example, but not limited to those described by Srinivas et al, 2008; Harimaya et al, 1998; Jenniskens et al, 1998; and Tatsuta et al, 1997.

In a further example, the one or more than one eremophilane sesquiterpene in the methods of the present invention may be a compound of Formula V

Formula V or a pharmaceutically acceptable derivative thereof, wherein R¹ is OH or a derivative thereof, or taken together with R² forms a double bond between C9 and C10; R³ is OH or a derivative thereof, or taken together with R² forms a double bond between C8 and C9; R⁴ is OH or a derivative thereof; with the proviso that one of R¹ or R³ is OH or a derivative thereof, but not both.

In a more specific example, the one or more than one eremophilane sesquiterpene in the methods of the present invention may be a compound of Formula VI

Formula VI or a pharmaceutically acceptable derivative thereof, wherein when R¹ is OH or a derivative thereof, the stereochemistry at C10 is R; and when R³ is OH or a derivative thereof, the stereochemistry at C8 is R.

The compounds of Formula V and VI, the hydroxyl group at R\ R³, or R⁴ may be derivatized by methods commonly used in the art. In a non-limiting example, R\ R³, or R⁴ may be derivative to an -C_1-3oalkoxy,

-d-Mester, -Cijoamido, -Ci.₃ocarboxyamido, Ci-C3o-carbox , or CrC3o-carbonyl; the carbon-containing substituents of R\ R³, or R⁴ may be unbranched or branched. Thus, in the compounds of Formula V and VI, R¹ may be OH, -C^alkoxy, -C_1-30acyl, -C_1-3oether, -Ci-3oester, -C^amido, -C _-3ocarboxyamido, C C₃o-carboxy, C-i-Cso-carbonyl, or, taken together with R² forms a double bond between C9 and C10; R³ may be OH, -C-i,₃oalkoxy, -C_1-3oacyl, -Ci.₃₀ether,

-Ci_-3oamido, -C^aocarboxyamido, CrC₃o-carboxy, Ci-C₃o-carbonyl, or taken together with R² forms a double bond between C8 and C9; and R⁴ may be OH, -C_1-30alkoxy, -d^oacyl, -C_1-3oether, -Ci.₃₀ester,

-C_1-3ocarboxyamldo, CrCarcarboxy, or Ci-CarCarbonyl. In one non-limiting example, R¹, R³, or R⁴ may be esters of omega-3 fatty acids such as a- linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) (C18, C20 or C22, respectively). Derivatization the hydroxyl groups may be accomplished by any suitable organic chemistry method known in the art. Ample texts address derivitlzation of such groups, for example but not limited to Fruniss et al (1989), Vollhardt and Schore (1998). For example, esters may be prepared by reacting the hydroxyl group with carboxylic acid, acyl chlorides, etc.).

In another non-limiting example, R¹ or R³ is OH, and R⁴ is selected from

, and any combination thereof.

In other words, the one or more than one eremophilane sesquiterpene for use in the methods of the present invention is selected from the group consisting of 6p,8P-dimethoxy-1- oxoeremophil-9(10)-en-8a,12-olide, 6β-angeloyloxy-8cc-hydroxy-1 -oxoeremophil-9(10)-en- 8p,12-olide, and 6 -angeloyloxy-10a-hydroxy-1-oxoeremophil-8(9)-en-8,12-olide, or any combination thereof.

In yet another alternative, the one or more than one eremophilane sesquiterpene for use in the methods described above may be provided in an extract or a bioactive fraction thereof. The extract may be obtained by providing tissue from Senacio spp., Ligularia spp., Farfugium spp., Cacalia spp., Patasites spp., Ligulariopsis spp.,Stevia spp. or any other suitable plants. Eremophilane sesquiterpenes may also be obtained by fermentation of fungi such as Xylaria spp. and extracting therefrom by a suitable solvent, an extract, or a bioactive fraction thereof. There is also provided a method for treatment or prevention of a neurodegenerative disease comprising administering an effective amount of one or more than one eremophilane sesquiterpene to a patient in need thereof. The one or more than one eremophilane sesquiterpene may be as described above. In one non-!lmitlng example, the neurodegenerative disease may be Alzheimer's disease. The compounds and formulations described above of the present invention may be delivered by any suitable route of administration known in the art. For example, and without wishing to be limiting in any manner, the compounds of the present invention or composition thereof may be delivered systemically (orally, nasally, parentally, intravenously, etc.) or may be delivered to the gastrointestinal tract. In a specific, non-limiting example, the compositions of the present invention are administered orally or parentally. Those of skill in the art would be familiar with such methods of delivery.

The compounds and compositions of the present invention may be administered in a suitable amount or dosage form. As would be recognized by one of skill in the art, the particular amount or dosage will vary based on the specific compound, the route of administration, the neurodegenerative disease, ailment and or dysfunction being treated, and the specifics of the patient. However, a suitable amount or dosage may be, but is not limited to the range of about 0.01 to 750 mg/kg of bodyweight per day; for example, the suitable amount may be 0.01 , 0.05, 0.1 , 0.5, 1 , 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg kg of bodyweight per day, or any amount there between. The present invention also encompasses a method of producing a compound of Formula I. The method may comprise the steps of solvent extraction and purification. The extraction solvents could be any suitable method used in natural products extraction, and the process may involve heating, sonicating, microwaving, etc. Purification process may involve various chromatographic approaches (such as normal phase, reverse phase, flash chromatography, ion exchange chromatography, macroporous resin adsorption, preparative HPLC, TLC). In the methods as described above, the eremophilane sesquiterpene may be obtained from Sanacio spp., Ligularia spp., Farfugium spp., Cacalia spp., Patasites spp., Ligulariopsis spp.,Stevia spp. or any other suitable plants. Eremophilane sesquiterpenes may also be obtained by fermentation of fungi such as Xylaria spp.. In a non-limiting example, the eremophilane sesquiterpene may be obtained from Senecio jacobaea, or more specifically, from the root of Senecio jacobaea.

The present invention further provides a kit or commercial packaging comprising the one or more than one compound as described above or a composition comprising the one or more than one compound, along with instructions for use in the methods described herein. The kit or commercial package may optionally include other known neuroprotective compound (such as those described above), buffers, and/or administration aids (such as syringes, needles, antiseptic wipes, etc). For example, when the compound or composition is provided in powder form, the kit may include a buffer for dissolution or suspension of the compound or composition, as well as a suitable container for mixing the two components.

The present invention will be further illustrated In the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Extraction and isolation of INH-25-2NA-1 and INH-25-2NA-5

Extracts were prepared from the root of Senecio Jacobaea. The extraction and purification process is summarized in Figure 1.

Pulverized, air-dried root of Senecio jacobaea (INH-25, 200.22g) was extracted with MeOH twice (overnight each time) at room temperature. After filtration, the filtrates were combined and concentrated using a rotary evaporator under reduced pressure at 40°C. 3.79g of MeOH extract was obtained. The extract (2g) was loaded to a C-18 SPE column (20g C-18), then eluted with 200 mL each of 25% and 50% aqueous MeOH, then 100% MeOH followed by MeOH-CH₂CI₂ (1 :1 ). After evaporation, 4 elution fractions were obtained: INH-25-1 (1.34 g), INH-25-2 (0.16 g), INH-25-3 (0.56 g), and INH-25-4 (0.14 g), respectively.

Based on results of AChE inhibition assay (see Example 4), INH-25-2 was subjected to further purification. INH-25-2 (0.108 g) was extracted twice with 10 mL 2N HCI by sonication. After centrifugation at 4000rpm, the supernatants were combined then extracted via liquid-liquid extraction three times with an equal volume of CH₂CI₂. CH₂CI₂ solutions were combined then evaporated to yield the INH-25-2NA fraction (0.031 g). The aqueous phase was basified to pH 9-10 with ammonium hydroxide. The basic aqueous fraction was then re-extracted with CH₂CI₂. After evaporating CH₂CI₂, the I NH-25-2A fraction (0.028 g) was obtained.

Based on AChE inhibition results (see Example 4) of fractions INH-25-2A and INH-25-2NA, INH-25-2NA was chosen for further purification using preparative TLC on silica gel. INH-25-NA (31 mg) was dissolved in acetone, loaded to 20x20 cm prep-silica gel-TLC plate and then developed with CH₂CI₂-MeOH (100:2). Two compounds were obtained after scratching the silica gel bands and eluting with CH₂CI₂- eOH (3:1). The solvents were then evaporated to yield 1NH-25-2NA-1 (0.84 mg) and INH-25-2NA-5 (2.87 mg). Example 2: Extraction and isolation of LR-49-55-2

Extracts were prepared from the root of Senecio jacobaea. This extraction and purification process is summarized in Figure 2.

Air dried Senecio jacobaea root (752 g, INH-OS-26, collected in July 2009, from Chllllwack, BC, Canada) was milled and extracted with MeOH (1.5 L * 2) at room temperature with the assistance of sonication for 30 min. The combined MeOH extracts were dried under vacuum on a rotary evaporator to obtain MeOH extract (32.6 g). The MeOH extract was suspended in 2N HCI, and then extracted with CH₂CI₂ for four times. The CH₂CI₂ extract (9.0 g dry weight) was coated on Celite, and eluted sequentially with hexane, CH₂CI₂ and EtOAc. After evaporating solvents, the CH₂CI₂ fraction (3.8 g) was subjected to a silica gel column and eluted with hexane, CH₂CI₂ and MeOH. The CH₂CI₂ fraction (1.4 g) was loaded on a 40 g RediSep silica gel column to obtain 8 fractions with a gradient CH₂CI₂-MeOH elution. Fraction 6 (1.1 g) was then further purified on a C-18 column (38.5 g C-18, Sigma-Aldric ) and eluted with water-MeOH in gradient mode to yield 7 fractions. With a repeated C-18 column purification on the newly obtained fraction 4 (184 mg), and further silica gel column purification eluted with CH₂CI₂-MeOH (1000:4), 24.5 mg of LR-49-55-2 was Isolated. Certain quantities of INH-25-2NA-1 (1.6 mg) and INH-25-2NA-5 (55.2 mg) were also obtained from this extraction and purification process.

Example 3: Structure characterization

The molecular formula and structure for each of INH-25-2NA-1 , INH-25-2NA-5, and LR-49-55- 2 were determined based on data from high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Both 1 D (¹H and ¹³C) and 2D NMR (COSY, HSQC, HMBC, NOESY) were used. HRMS data were obtained on a Waters QToF Premier mass spectrometer. The NMR spectra were acquired on a Bruker Avance III 600 MHz or 700 MHz NMR spectrometers . INH-25-2NA-1: The molecular formula of INH-25-2NA-1 was determined to be C^H^Os by high resolution mass spectrometry (HRMS; Figure 3) measured at 307.1536 for [M+Hf (calculated for C ₇H₂₃0₅ ⁺ 307.1546). The structure was determined to be

oxoeremophil-9(10)-en-8a,12-olide based on 1 D and 2D-N R data. The structure is shown below and the NMR data is given in Table 1.

Table 1. NMR data of INH-25-2NA-1

INH-25-2NA-1 was obtained as a colourless gum with [a]²⁷ _D = -67.5 (c = 0.16, CHCI₃). The compound was reported previously from Senecio nemorensis (Meng et al, 2007).

INH-25-2NA-5: The molecular formula of INH-25-2NA-5 was determined to be C2OH_Z4OB by HRMS (Figure 4) measured at 361.1645 for [M+H]⁺ (calculated for C2oH₂₅0₆ ⁺ 361.1651 ). The structure was determined to be 6P-angeloyloxy-8a-hydroxy-1-oxoeremophil-9(10)-en-8p,12- o!ide based on 1 D and 2D-N R data. The structure is shown below and the NMR data is given in Table 2.

Table 2. NMR data of INH-25-2NA-5

INH-25-2NA-5 was obtained as a colourless gum with [a]^Z7 _D = -135.2 (c = 0.17, CHCI₃). LR-49-55-2: The molecular formula was determined to be C20H24O6 by HRMS (Figure 5) measured at 383.1462 for [M+Naf (calculated for C₂oH₂₄0₆ a⁺ 383.1671). The structure was determined to be 6 -angeloyloxy-10a-hydroxy-1-oxoeremophil-8(9)-en-8,12-olide based on 1 D and 2D-NMR data. The structure is shown below and the NMR data is given in Table 3.

Table 3. NMR data of LR-49-55-2

LR-49-55-2 was obtained as a colourless gum with [a]²⁷ _D = -153.2 (c = 0.81 , CHCI₃). Example 4: Acetylcholinesterase inhibition assay

Acetylcholinesterase (AchE) inhibition of various extracts and sub-extracts was determined using a standard inhibition assay.

The Inhibitory activity ef fractions and pure compounds including INH-25-2, INH-25-2NA, INH- 25-2NA-1 , INH-25-2NA-5 and positive control eserine was tested. AChE inhibition activity was measured with a 96-well microplate reader. The wells contained a 20 μΙ_ fraction or compound (in 20% DMSO), positive control (Eserine, in 20% DMSO) or blank (20% DMSO); 200 μΐ of Ellman's solution (0.15 mM DTNB, 5,5'-Dithiobis (2-nitrobenzoic acid) in 0.1 M pH 7.4 phosphate buffer), 20 μΐ AChE solution (0.5 U/mL in 0.01 M pH 7.4 phosphate buffer with 1 mg/mL bovine serum albumin) and 30 μΐ acetylthiochollne solution (6.7 mM in water). Each fraction or compound was tested at eight concentrations in the range of 0.128 g/mL to 10 mg/mL. The increase in absorbance at 412nm was measured during 15min at RT. Measurements were made in duplicate. GraphPad Prism was used for data analysis.

Results are shown in Figures 6 and 7 (concentration 0.148 mg/mL). IC_∞ of these fractions and compounds were 2.94 pg/mL for INH-25-2, 1.11 g mL for INH-25-2NA, 0.16 pg/mL (0.5 μΜ) for INH-25-2NA-1 , 0.98 pg/mL (2.7 μΜ) for INH-25-2NA-5. The !Cso of positive control ©serine was measured to be 0.014 pg/mL (0.05 μΜ). In contrast, commercially available AChE inhibitors inhibitors such as Huperzine A and galantamine have reported IC₅₀ of 0.023 μΜ and 1.07 pM, respectively (Houghton et al, 2006).

The results demonstrate that these eremophllane sesquiterpene compounds INH-25-2NA-1 and INH-25-2NA-5 are potent AChE inhibitors. This class of compounds have not been reported for any bioactivities or pharmacological effects related to AChE inhibition, and thus represent a new class of natural AChE inhibitor. Example 5: AChE inhibition assay in SH-SY5Y cells

The effect of INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55-2 on AChE activity in SH-SY5Y cells was determined.

SH-SY5Y human neuroblastoma cells were purchased from American Type Tissue Culture (Manassas, VA, USA). The cells were cultured in DM EM supplemented with 12% heat- inactivated fetal bovine serum, 100 U/mL penicillin, and 100 pg/mL streptomycin in a humidified incubator with 5% C0₂ at 37°C. Stock cultures of exponentially grown cells were trypsinized, and replated into 6-well plates with 3 *10⁸ cell/well prior to use.

The cells were treated with 0, 0.1 , 1 , 10 and 100 μΜ of compounds for 24 h. Total proteins were extracted from cells by washing with PBS and lysed in 100 μίΛνβΙΙ of ice-cold buffer (1 * RIPA supplied protease inhibitor). After 30 minutes incubation on ice, proteins were collected in supernatants by centrifugation at 1 ,000 g for 10 minutes. AChE activity in the cell lysate was then determined by mixing 40 μΐ cell lysate, 40 μΐ ACh (6.7mM in water), and 200 μΐ_ Ellman's solution (0.15 mM DTNB in 0.1M pH 7.4 phosphate buffer); the absorbance at 412 nm was measured over 15 mins.

After treatment with the compounds for 24 h, INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55-2 showed significantly inhibition of AChE activity In a concentration-dependent manner in SH- SY5Y cells. The IC₅₀ for INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55-2 were determined to be 17.8 μΜ (Figure 8), 20.0 μΜ (Figure 9), and 5.5 μΜ (Figure 10), respectively. The efficiency of inhibition of INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55-2 on AChE activity in H-SY5Y cells was also determined. The results showed that at the same concentration, compound LR-49- 55-2 is the most potent of the three compounds tested (Figure 11).

Example 6: Neuroprotective activity in SH-SY5Y cells

The neuroprotective effect of INH-25-2NA-5 in SH-SY5Y cells was determined by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.

SH-SY5Y human neuroblastoma cells were purchased from American Type Tissue Culture (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 12% heat- inactivated fetal bovine serum, 100 U/mL penicillin, and 100 pg/mL streptomycin in a humidified incubator with 5% C0₂ at 37*C. Stock cultures of exponentially grown cells were trypsinized, and replated into 6-well plates with 3 *10⁵ cell/well prior to use.

SH-SY5Y cells were replaced into 12-well plates one day prior to experiments. After the attached cells were rinsed once with PBS, the media was replaced with phenol red-free and serum-free D E . Prior to β-amyloid (Αβ), glutamate or hydrogen peroxide treatment, the cells were pre-incubated with 0.1 , 1 , 10, or 100 μΜ of 1NH-25-2NA-5 for 2 hours. After the preincubation period cells were then treated in either Αβ1-42 (5μΜ), glutamate (5m ) or hydrogen peroxide (100μ ) for 24 additional hours and then cell viability was determined using the MTT assay. Cells were then were incubated with MTT at 0.5 mg/mL for 2-4 hours at 37°C; formazan salt generated by viable cells as a result of conversion of MTT was dissolved in DMSO and the absorbance was measured at 570 nm with 630 nm as reference to evaluate the neuroprotective activity of INH-25-2NA-5.

The results showed that compound INH-25-2NA-5 increases neuroblastoma cell viability significantly in glutamate-, Αβ1-42-, and In hydrogen peroxide-treated cell based assays after pre-treatment with 0.1 , 1, 10, or 00 μΜ of 1NH-25-2NA-5 for 2 hours for 2 h {see Figures 12, 13, and 14). Thus, INH-25-2NA-5 exhibits a neuroprotective effect.

Possible cytotoxicity of INH-25-2NA-5 was also tested by the MTT assay described above. The compound was shown to have no significant cytotoxicity in SH-SY5Y cells when the concentration was below 100 μΜ (Figure 15).

Example 7: SH-SY5Y High Dose Glutamate Excitotoxicitv Assay

A follow-up study to further validate the findings using glutamate-based neuroprotection assay was performed with serum included in the media.. An excitotoxicity protocol was used where the control cytotoxic effect is based upon the concentration of glutamate for 24 hours that is necessary to cause a 50% or greater reduction in cell viability effect compared to control using SH-SY5Y neuroblastoma cell line. This protocol (higher dose glutamate) was selected to maximize the ability to test and statistically determine the potential neuroprotective effects of the compounds.

Cells were cultured and treated, and cytotoxicity using MTT-based assay as a readout was determined using methods as described in Example 6. SH-SY5Y cells seeded at 0.5 M cells/well exposed to 25mM glutamate for 24 hr was found to cause -50% reduction of the MTT signal. This dose was used for all further high-dose glutamate excitotoxicity studies using doses of INH-25-2NA-1 , INH-25-2NA-5 and LR-49-55-2.

SH-SY5Y cells were cultured in DMEM, supplemented with 10% Fetal Bovine Serum, 100 pg/ml streptomycin and 100 U/ml penicillin, at 37°C with 5% CO_z. Compounds INH-25-2NA-1 ,

INH-25-2NA-5 and LR-49-55-2 were dissolved in dimethylsulfoxide and diluted to working concentrations in DMEM. SH-SY5Y cells were seeded at a density of 500,000 cells per well in

12-well BD polystyrene plates. After 24 h, cells were treated with varying concentrations of compounds (0.1-10 pg/ml) for 2 hours, this was followed by a 24 hr challenge with the addition of 25m glutamate for 24 hours. Mitochondrial activity was measured, as an indication of cell viability, by thiazolyl blue tertrazolium bromide (MTT) colorimetric reduction; specifically, cells were exposed to 0.5 mg/ml MTT for 2 h then lysed with DMSO. MTT levels from triplicate samples derived from each well was determined by measuring absorbance at 570 nm and 630 nm was used as a reference wavelength. The experiment was performed 3 times (3 separate experiments) to test the effects of INH-25-2NA-1 , 25-2NA-5 and LR-49-55-2 on cell viability at various doses. Data was analyzed in Prism with 2-way ANOVA and is expressed as percent increase in neuronal survival vs untreated control (normalized). Figure 16 shows statistically-significant increases in MTT signal at 10 μΜ of 1NH-25-2NA-1 (p< .01 n=3). A small but not-statistically significant effect observed at 0,1 or 1 μΜ..

Figure 17 shows highly significant dose-dependent increases in MTT signal with 1 (p<-05, n=3) and 10 μΜ (p<-001 , n=3) INH-25-2NA-5 and although not reaching statistical significance, an emerging dose-related trend at .01 μΜ appears to be evident.. Figure 18 shows that the addition of LR-49-55-2 resulted in a highly statistically significant increase in MTT signal at the lowest dose tested (0.1 μΜ, p<.01 , n=3) but at higher concentrations, the neuroprotective effect appears to be lost.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.

REFERENCES

All patents, patent applications and publications referred to herein and throughout the application are hereby incorporated by reference.

Fraga, B. M. Natural Sesquiterpenoids, Natural Products Reports, 2008, 25, 1180-1209.

Fruniss, B. S., Hannaford, A. J., Smith, P. W. G., and Tatchell, A. R. Vogel's Textbook of Practical Organic Chemistry (Fifth ed.), Longmen Group UK Ltd., Burnt Mill, England, 1989. Harimaya, K.; Magome, E.; Tabata, Y.; Sasaki, T., Preparation of eremophilane sesquiterpene derivatives having progesterone receptor binding inhibitory activity 1998, US 5817816 A 19981006.

Houghton.P. J. Ren, Y., and Howes, M.-J. Acetylcholinesterase inhibitor from plants and fungi, Natural Products Reports 2006, 23, 181-199.

Isaka M., Chinthanom P., Boonruangprapa T., Rungjindamai N., and Pinruan U., Eremophllane-Type Sesquiterpenes from the Fungus Xylaria sp. BCC 21097, J. Nat. Prod. 2010, 73, 683-687

Jenniskens, L. H. D.; De Groot, A. , Enantioselective synthesis of R-(-)-ligularenolide and the progesterone receptor ligand R-(-)-PF1092C starting from S-(+)-carvone Tetrahedron 1998, 54(21), 5617-5622.

Meng, F.-J., Zhao, H., Xie, W.-D., Zhao, R.-J., Lai, P.-X., Zhou, Y.-X., and iao, Y.-L. New eremophilenolactones from senecio nemorensis, Helvetica Chimica Acta, 90, 2196-2200, 2007. Pinder, A. R. The chemistry of eremophilane and related sesquiterpenes, Fortschritte der Chemie organischer Naturstoffe 1977, 34, 81-186.

Srinivas, P.; Srinivasa Reddy, D.; Shiva Kumar, K.; Dubey, P. K.; Iqbal, Javed; Das, Parthasarathi, A new route to eremophilanes: synthesis of (±)-eremophilenolide, (±)- eremophiledinone, and (±)-deoxyeremopetasidione 2008, Tetrahedron Letters, 49, 6084-6086. Tatsuta, K.; Yasuda, S.; Kurihara, K.; Tanabe, K.; Shinei, R.; Okonogi, T., Total synthesis of progesterone receptor ligands, (-)-PF1092A, B and C Tetrahedron Letters 1997, 38(8), 1439- 1442.

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Claims

CLAIMS:

1. A compound comprising the structure of Formula I

Formula I or a pharmaceutically acceptable derivative thereof, wherein R¹ Is OH or a derivative thereof, or taken together with R² forms a double bond between C9 and C10; and R³ is OH or a derivative thereof, or taken together with R² forms a double bond between C8 and C9; with the proviso that one of R¹ or R³ is OH or a derivative thereof, but not both.

2. The compound of claim 1 , wherein the compound comprises the structure of Formula II

Formula II or a pharmaceutically acceptable derivative thereof, wherein when R is OH or a derivative thereof, the stereochemistry at C10 is R; and when R³ is OH or a derivative thereof, the stereochemistry at C8 is R.

3. The compound of claim 1 , wherein R³ is OH or a derivative thereof, and a double bond is present between C9 and C10.

4. The compound of claim 3, wherein the compound comprises the structure of Formula III

Formula III

or a pharmaceutically acceptable derivative thereof.

5. The compound of claim 1 , wherein R¹ is OH or a derivative thereof, and a double bond present between C8 and C9.

6. The compound of claim 5, wherein the compound comprises the structure of Formula IV

Formula IV or a pharmaceutically acceptable derivative thereof.

7. The compound of any one of claims 1 to 6, wherein the compound is an acetylcholinesterase inhibitor.

8. A composition comprising one or more than one compound of any one of claims 1 to 6 and a pharmaceutically acceptable diluent, excipient, or carrier.

9. The composition of claim 7 or 8, further comprising one or more than one neuroprotective compound. 10. A method of inhibiting acetylcholinesterase, comprising contacting the acetylcholinesterase with one or more than one eremophiiane sesquiterpene.

11. A method of increasing neuronal cell viability, comprising contacting the neuronal cell with one or more than one eremophiiane sesquiterpene.

12. A method for treatment or prevention of a neurodegenerative disease comprising administering an effective amount of one or more than one eremophiiane sesquiterpene to a patient in need thereof. 3. The method of any one of claims 10 to 12, wherein the one or more than one eremophiiane sesquiterpene is obtained from Senecio Jacobaea.

14. The method of any one of claims 10 to 12, wherein the one or more than one eremophiiane sesquiterpene is a compound of Formula V

Formula V or a pharmaceutically acceptable derivative thereof, wherein R¹ is OH or a derivative thereof, or taken together with R² forms a double bond between C9 and C10; R³ is OH or a derivative thereof, or, taken together with R^z forms a double bond between C8 and C9; R⁴ is OH or a derivative thereof; with the proviso that one of R¹ or R³ is OH, but not both.

15. T e method of claim 14, wherein the one or more than one eremophilane sesquiterpene is a compound of Formula VI

16. The method of claim 15, wherein, R¹ or R³ is OH or a derivative thereof, and R⁴ is selected from

17. The method of claim 10 or 11 , wherein the one or more than one eremophiiane sesquiterpene is selected from the group consisting of 6p,8p-dlmethoxy-1 -oxoeremophil-9(10)- en-8a,12-olide, 6p-angeloyloxy-8a-hydroxy-1-oxoeremophil-9(10)-en-8p,12-olide, and 6β- angeloyloxy-10a-hydroxy-1-oxoeremophll-8{9)-en-8,12-olide, or any combination thereof.

18. The method of claim 12, wherein the neurodegenerative disease is Alzheimer's disease.

19. The method of claim 12 wherein the composition or compound is administered orally or parenterally.