WO2013001490A1 - Novel anti-inflammatory dihydrochalcone derivatives and use thereof - Google Patents

Abstract

Disclosed are dihydrochaicone derivatives useful in the treatment of inflammatory and neuro-inflammatory diseases such as Alzheimer and Parkinson or conditions such as stroke. Both a and β carbons of the dihydrochaicone are hydroxylated. Also disclosed are methods of using such compounds in the treatment of inflammatory diseases in mammals and pharmaceutical compositions containing such compounds.

Description

NOVEL ANTI-INFLAMMATORY DIHYDROCHALCONE DERIVATIVES

AND USE THEREOF

[0001] Field of the Invention

[0002] The present invention relates to compounds, and pharmaceutical compositions containing them, that may be used in the treatment of inflammation and neurodegenerative diseases. The present invention also relates to methods for treating inflammation using those compounds.

[0003] Description of the state of the art

[0004] The elevation in life span of the population in the western world has caused an elevated frequency of neurodegenerative diseases, like Alzheimer's and Parkinson's disease. These diseases have multifactorial pathogenesis, and in most of them, a massive neuronal cell death occurs as a consequence of an uncontrolled neuroinflammatory response. This process plays a pivotal role in the initiation and progression of various neurodegenerative diseases and involves the activation of two main cell types in the brain - astrocytes and microglial cells.

[0005] Astrocytes are the most abundant glial cell type in the brain, and play multiple roles in the protection of brain cells. Despite their high antioxidative activities, astrocytes exhibit a high degree of vulnerability, and are not resistant to the effects of reactive oxygen species ( OS). They respond to substantial or sustained oxidative stress with increased intracellular Ca , loss of mitochondrial potential, and decreased oxidative phosphorylation.

[0006] Microglia are a type of glial cell that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of active immune defense in the central nervous system (CNS). Microglia in culture secrete large amounts of ¾0₂ and NO in a process known as 'respiratory burst'.

[0007] Both astrocytes and microglial cells can produce proinflammatory cytokines (such as Tumor necrosis factor alpha, TNF ) and cytotoxic agents in response to ischemia, traumatic and infectious insults, chronic neurodegenerative diseases leading to deterioration of the disease processes.

[0008] It is known that some cytokines are involved in the up and down regulation of immune and inflammatory cells, and in regulation of activity in connective tissue and neural, epithelial, endothelial, and other cell types which are involved in tissue repair and restoration of homeostasis. [0009] In general, normal levels of cytokines are benign in their effects on tissues and cells. However, the overproduction of cytokines can have harmful effects on tissues and can result in cellular damage, capillary leakage and even death. In addition, cytokines have been implicated in neointimal hyperplasia or cell proliferation following surgical procedures, cancer, allergy, infection, angiogenesis, and restenosis. It is anticipated that cytokines present in excessive amounts will result in cellular and tissue damage. Inhibition of the activated cells may provide an effective therapeutic intervention that might alleviate the progression of the neurodegenerative diseases.

[0010] increased oxidative stress has been implicated in the pathology of many diseases as well as of neurodegenerative diseases. Reactive oxygen species (ROS), are produced in excess, and may damage cells through direct oxidation of lipids, proteins, and DNA or can act as a signaling molecule to trigger intracellular pathways leading to cell death. ¾⁽¾ is a major form of reactive oxygen species (ROS).

[0011] Inhibition of the activated cells may provide an effective therapeutic intervention that might alleviate the progression of the neurodegenerative diseases. One of the molecules secreted by these activated cells is nitric oxide (NO), which is a free radical that causes nitrosative stress that plays a role in neurodegenerative diseases. NO serves as a double-edged sword depending on its concentration in the microenvironment, and is involved in both physiological and pathological processes of many organ systems including the brain. Excessive amounts of NO are synthesized by induced NO -synthases (iNOS), secreted by both activated astrocytes and MG and are involved in neuroinflammation and neurodegeneration. Activation of iNOS and NO generation has come to-be accepted as a marker and therapeutic target in neuroinflammatory conditions such as those observed in ischemia, multiple sclerosis, spinal cord injury, Alzheimer's disease and inherited peroxisomal (e.g. X-linked adrenoleukodystrophy; X-ALD) and lysosomal (e.g. Krabbe's disease) disorders.

[0012] There is now evidence that depression, as characterized by melancholic symptoms, anxiety, and fatigue and somatic (F&S) symptoms, is the clinical expression of peripheral cell- mediated activation, inflammation and induction of oxidative and nitrosative stress (IO&NS) pathways and of central microglial activation, decreased neurogenesis and increased apoptosis (Maes et al, Neuro Endocrinol Lett. 201 l;32(l):7-24). According to Maes et al., depression contributes to increased (neuro)inflarnmatory burden and may therefore drive the inflammatory and degenerative progression. Maes et al. concludes that the activation of peripheral and / or central IO&NS pathways may explain the co-occurrence of depression with the above disorders. This shows that depression belongs to the spectrum of inflammatory and degenerative disorders.

[0013] Stroke is an important cause of mortality and morbidity worldwide but effective therapeutic strategy for the prevention of brain injury in patients with cerebral ischemia is lacking. Stroke mediated cell death is a complex interplay of aberrant events involving inflammation, oxidative stress, excitotoxicity, acidosis, peri-infarct depolarization, and apoptosis. Strokes can be subdivided into two categories, namely ischemic and hemorrhagic strokes. Ischemic strokes are more prevalent than hemorrhagic strokes making up approximately 87 % of all cases, and have been the target of most drug trials. A thrombosis results in a restriction of blood flow to the brain and this result in insufficient oxygen and glucose delivery to support cellular homeostasis. These processes share overlapping biochemical abnormalities causing injury to glia, neurons, and endothelial cells. Within the core of ischemic territory, where blood flow is most severely restricted, excitotoxic and necrotic cell death occurred within minutes. This elicits multiple processes that lead to brain injury, such as free radical production, oxidative stress, inflammation, excitotoxicity, ionic imbalance, apoptosis, and peri-infarct depolarization ( Front Biosci (Elite Ed). 2012 Jan 1 ;4:809-17. Traditional Chinese herbal medicine and cerebral ischemia. Chen YF.) [0014] Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in the development of cardiovascular disease, including hypertension, atherosclerosis, diabetes, cardiac hypertrophy, heart failure, ischemia-reperfusion injury, and stroke. See Paravicini TM, Touyz RM: Redox signaling in hypertension (Cardiovasc Res 2006, 71 :247-258; Forbes JM, Coughlan MT, Cooper ME: Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 2008, 57: 1446-1454;3. Huang PL: eNOS, metabolic syndrome and cardiovascular disease. Trends Endocrinol Metab 2009, 20:295-302.

[0015] Pulicaria incisa (Pi) is a desert plant which belongs to the asteraceae family and has been used in traditional medicine for heart diseases (Mansour et al., FITOTERAPIA Volume LXI, No. 1, 1990, Nabiel Saleh, Phytochemistry 63, (2003), 239-241 (Editorial). Pulicaria incisa is commonly referred to as wild tea or desert fleabane and replaces tea for many of the Bedouins in Egypt (Mansour et al., 1990, Nabiel Saleh, Phytochemistry 63, (2003), 239-241 (Editorial). Pi was also stated to decrease total lipid, total cholesterol and triglycerides and was proposed as a hypocholesterolemic agent (Amer et al., Deutsche Lebensmittel-Rundschau, 103. Jahrgang. Heft 7, 2007, 320-327) and as hypoglycaemic (Shabana, Arch Exp Veterinarmed. 1990 44(J):389-94).

[0016] Ramadan et al, J. Verbr. Lebensm. (2009) 4:239-245, examined the antioxidant properties of the methanolic extracts of Pi. Ramadan et al. did not demonstrate any specific active anti-oxidant compound existing in Pi. Moreover, Ramadan et al. showed a mere in vitro inhibition of the oxidation of linoleic acid by methanolic extract of Pi and not in vivo protection of living cells.

[0017] To the best of the authors' knowledge, no prior art teaches that effects of Pi in the context of neurodegenerative diseases.

[0018] Calliste et al., ANTICANCER RESEARCH 21: 3949-3956 (2001) tested the anti- oxidative potential of substituted chalcones with different numbers and different positions of the hydroxy groups. Calliste et al. establishes the importance of the α-β double bond of the chalcones. Compounds of the present invention however, have α-β single bond structure, with the a and the β carbons being substituted, the a and the β carbons of the compounds of the present invention are those that appearing in formula I set forth below.

[0019] Won et al., Eur. J. Med. Chem., 2005, 40, 103 tested synthetic chalcones as potential anti-inflammatory and cancer cheraopreventive agents. In Hsieh et al., J. Pharm. Pharmacol., 2000, 52, 163, series of chalcones, hydroxy- and dihydroxychalcones were synthesized and their inhibitory effects on the activation of mast cells, neutrophils, microglial cells and macrophages were evaluated in-vitro.

[0020] Nevertheless, none of the techniques disclosed in the above art teaches or hints for any kind of hydroxylation, neither of the phenyl rings and nor of the a and β carbons appearing in Formula I.

[0021] Thus, inhibition of the activated immune cells (microglial cells in neurodegenerative diseases, and macrophages and lymphocytes in other diseases), and protection from oxidative stress- induced cell death may provide an effective therapeutic intervention that might prevent or ameliorate various diseases.

SUMMARY OF THE INVENTION

[0022] This invention provides novel dihydrochalcone compounds, and prodrugs thereof, which are useful in the treatment of inflammatory diseases. It has been found that some trihydroxy-dihydrochalcones compounds having specific substituents as described herein are potent anti-inflammatory, anti-neuroinflammatory, and antioxidant drugs. [0023] More specifically, one aspect of the present invention provides compounds including tautomers, metabolites, resolved enantiomers, diastereomers, solvates, prodrugs and pharmaceutically acceptable salts thereof, said compound having the Formula I:

[0024] wherein:

[0025] R¹ is Me, Et, MeO-, EtO, H0CH₂CH₂O, H0CH₂C(Me)₂O, (S)- -, cyclopropyl-CH₂0-, H0CH₂CH₂-_> HOCH₂-,

[0026] Each of R², R³, R⁴ and R⁵ is H, Me, Et, OH, MeO-, EtO-, HOCH₂CH₂0-,

HOC¾C(Me)₂0-, (S)-MeCH(OH)C¾0-, cyclopropyl-CH₂0-, HOCH₂CH₂-₅ HOCH₂~,

[0027] In another embodiment of formula I, R⁵ is H.

[0028] More specifically, the invention relates to a compound selected from 1-propanone-

3-(4-hydroxyphenyl)-2,3-dihydroxy-l-(4,6-dihydroxy-2-methoxyphenyI) and l-(2,4-dihydroxy-6- methoxyphenyl)-3-(3 ,4-dihydroxyphenyI)-2,3-dihydroxypropan~ 1 -one.

[0029] In another aspect, the present invention provides a composition comprising a compound as described herein.

[0030] In another aspect of the Invention, this invention relates to the compounds of formula I, for the treatment or prevention of neuroinflammatory and inflammatory diseases.

[0031 ] In another aspect, this invention relates to the compounds of formula I, for the treatment or prevention of inflammatory CNS disease.

[0032] In a preferred embodiment, this invention relates to the compounds of formula I, for the treatment or prevention of oxidative stress.

[0033] In a preferred embodiment, this invention relates to the compounds of formula I, for reducing the amount of reactive oxygen species (ROS) in cells having oxidative stress condition. In a more preferred embodiment, these cells are glia cells.

[0034J In a further aspect, the present invention provides a method of treating or preventing inflammation in a subject, the method comprising administering to the subject a therapeutically effecti ve amount of a compound as described herein.

[0035] In another aspect, the present invention provides a method of treating or preventing inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein.

[0036] In another aspect, the present invention provides a method of treating a disease or condition characterized by or associated with inflammation, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound as described herein. In a further aspect, the present invention provides a method of treating a disease or condition characterised by or associated with inflammation, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition as described herein.

[0037] In a preferred embodiment, the neuroinflammatory and inflammatory diseases are associated with increased oxidative stress. In a preferred embodiment, the neuroinflammatory and inflammatory diseases are associated with excess amounts of reactive oxygen species (ROS).

[0038] In a still preferred embodiment, the oxidative stress is characterized by ¾(¾- induced cell death.

[0039] In another embodiment, neuroinflammatory and inflammatory diseases are associated with nitrosative stress, characterized by excessive amounts of NO produced by the cells.

[0040] In another embodiment, neuroinflammatory and inflammatory diseases are associated with production of cytokines selected from the group consisting of TNFa, IFNy, IL-6, IL-2, IL-4, IL-10, and IL-12.

[0041] An additional aspect of the invention is the use of compound of the present invention in the preparation of a medicament for the treatment or prevention of neuroinflammatory diseases a warm-blooded animal, preferably a mammal, more preferably a human, suffering from such disorder. More particularly, the invention includes the use of a compound of the invention in the preparation of a medicament for the treatment or prevention of a neuroinflammatory disorder or an inflammatory condition in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

[0042] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate non-limiting embodiments of the present invention, and together with the description, serve to explain the principles of the invention.

[0043] In the Figures:

[0044] Figure 1 shows H₂0₂-induced astrocytic cell death in a dose dependent manner.

[0045] Figure 2 shows the structures of the compounds 7-3028/9 and 7-3028/6.

[0046] Figure 3 shows the effect of compounds 7-3028/6 and 7-3028/9 on ¾02-induced cytotoxicity in astrocytes.

[0047] Figure 4 shows amelioration of H₂0₂ insult by 7-3028/9 in cells that were pre-, co- or post treated with H₂0₂.

[0048] Figure 5A shows induction of ROS production by H₂0₂ in cultured astrocytes in a dose dependent manner.

[0049] Figure 5B shows induction of ROS production by H₂0₂ in cultured astrocytes in a time dependent manner.

[0050] Figure 6 shows the effect of pre-treatment of astrocytes with 7-3028/9 on the ROS levels following treatment of the astrocytes with H2O2.

[0051] Figure 7 shows inhibition of peroxyl radical - induced oxidation of DCFH to DCF in primary astrocytes by 7-3028/9.

[0052] Figure 8 shows inhibition of peroxyl radical - induced oxidation of DCFH to DCF in primary microglial cells by 7-3028/9.

[0053] Figure 9 shows the effect of 7-3028/9 and dexamethasone on the ear edema.

[0054] Figures 10A-H shows the effect of 7-3028/9 and dexamethasone on the levels of different cytokines and MPO activity in the supernatants of ear homogenates from oxazolone- treated mice. A. INF-γ; B. IL-6; C. IL-12; D. TNF-a; E. IL-4; F. IL 0; G. MPO Activity and H. IL-2.

[0055] Figure 11 shows the effect of 7-3028/9 on LPS- induced cytokine secretion for different cytokines from naive splenocytes. A. INF-γ, B. IL-6 and C. IL-2.

[0056] Figure 12 shows the effect of in vivo administration of 7-3028/9 on cytokine secretion from unstimulated splenocytes derived of CFA/KLH-immunized mice. A. IL-6 and B. TNF-α.

[0057] Figure 13 shows the no-effect of 7-3028/9 on cell viability of non-stimulated and

LPS -stimulated splenocytes.

[0058] Figure 14A shows the effect of 7-3028/9 on KLH-stimulated splenocyte proliferation.

[0059] Figure 1.4B shows the effect of 7-3028/9 on LPS -stimulated splenocyte proliferation.

[0060] Figures 15A-D shows the effect of 7-3028/9 on KLH-induced cytokine secretion in different cytokines from splenocytes. A. IL-2; B. INF-γ; C. lL-10 and D. TNF-a.

[0061] Figure 16 shows the effect of 7-3028/9 on LPS-induced cytokine secretion from splenocytes. A. IL-2; B. INF-γ; C. IL-10 and D. TNF-a.

[0062] Figure 17 shows the effect of 7-3028/9 on induced increase in ROS levels following H2O₂ -induced oxidative stress.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention relates to novel trihydroxy-dihydrochalcone compounds according to Formula I. In the compounds of Formula I, the a and β carbons are linked by a single covalent bond and are both substituted with hydroxyl groups.

[0064] Certain compounds of this invention can exist as two or more tautomeric forms. A

"tautomer" is one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another, such as structures formed by the movement of a hydrogen from one site to another within the same molecule. Other tautomeric forms of the compounds may interchange, for example, via enolization/de-enolization and the like. Accordingly, the present invention includes the preparation of all tautomeric forms of compounds of this invention.

[0065] A "pharmaceutically acceptable salt" as used herein, unless otherwise indicated, includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable. A compound of the invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable sale.

[0066] Methods of chemical synthesis are generally known in the art and the compounds of the various embodiments may be prepared employing techniques available in the art using starting materials that are readily available. The skilled person will recognise that known chemical reactions may be readily adapted to prepare compounds of the various embodiments. The synthesis of the compounds of the embodiments may be performed by modifications apparent to the skilled person, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. A list of suitable protecting groups in organic synthesis can be found in T.W. Greene's Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, 1991. Alternatively, other reactions disclosed herein or known in the art will be recognised as having applicability for preparing other compounds of the various embodiments. Reagents useful for synthesising compounds may be obtained or prepared according to techniques known in the art. For example, approaches to synthesising the core decalin structure of the compounds of the present invention are provided in Ley et al. (Chem. Soc, Chem. Commun., 1983, 503 - 505) and references cited therein. Alternatively, the compound may be isolated from a natural source. For example, the compound may be isolated from a plant, in some embodiments, the compound may be isolated from the plant Pulicaria incisa.

[0067] Instructions for the making substitution of groups on the phenyl rings of the dihydrochalchones are described in Hsieh et al., J. Pharm. Pharmacol, 2000, 52, 163 and in Won et al., Eur. J. Med. Chem., 2005, 40, 103, both are incorporated herein by reference in their entirety.

[0068] General procedure for obtaining alkoxychalcones by substituting hydroxyl groups on the phenyl rings of the dihydrochalchones with appropriate alkoxy groups are described in Hsieh et al., 2000. One non-limiting example for this may be obtaining ethoxydihydrochalcone where the starting material is 7-3028/9 or 7-3028/6 hydroxychalcone disclosed in the present invention. A mixture of the hydroxychalcone, equirnolar amount of potassium carbonate and ethyl iodide (in excess) in N,N-dimethylformamide (DMF) is to be stirred at room temperature for 18 h. The mixture is diluted with water and is washed three times with water. The organic phase is dried over sodium sulphate, filtered and concentrated in-vacuo to give the product. Purification is made via silica-gel column chromatography. Alkylation of 7-3028/9 and 7-3028/6 that substitute the hydroxyl groups positioned on the arenes to ethers are well known to a person skilled in the art and is described for example in G. S. Hiers and F. D. Hager (1941), "Anisole", Org. Synth.; Coll. Vol. 1 : 58. Substitution reactions for substituting the hydroxyl groups positioned on the arenes of 7-3028/9 and 7-3028/6 with Me, Et or alcohols are also very well known to a skilled artisan.

[0069] As already mentioned, none of the techniques disclosed in the above art teaches or hints for any kind of hydroxylation, neither of the phenyl rings and nor of the a and β carbons appearing in Formula I.

[0070] This invention also encompasses pharmaceutical compositions containing a compound of the present invention and methods of treating inflammatory, autoimmune and neurodegeneratrive diseases, by administering compounds of the present invention. Compounds of the present invention having free hydroxy groups can be converted into pharmaceutically acceptable prodrugs.

[0071] A "prodrug" is a compound that may be converted under physiological conditions or by solvolysis to the specified compound or to a pharmaceutically acceptable salt of such compound. Prodrugs include free hydroxy groups that may be derivatized as prodrugs by converting the hydroxy group to a phosphate ester, hemisucci nates dimethylaminoacetate, or phosphoryloxymethyloxycarbonyl, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy) ethyl ethers. Prodrugs of this type are described in J Med. Chem., 1996, 39, 10. More specific examples include replacement of the hydrogen atom of the alcohol group with a group such as (Cj-C6)alkanoyIoxymethyl, l-((d-C6)alkanoyloxy)ethyI, 1 -methyl- 1-((C₁ - C₆)alkanoyloxy)ethyl, (Ci-C₆)alkoxycarbonyloxymethyl, N-(Cj -Cejalkoxycarbonylaminomethyl, succinoyl, (Ci-C₆)alkanoyl. a-amino(Ci-C4)alkanoyl, arylacyl and α-aminoacyl, or -aminoacyl- a-aminoacyl, where each cc-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(0)(OH)₂, -P(0)(0(C_rC₆)alkyl)2 or glycosyl (the radical resulting from, the removal, of a hydroxyl group of the hemiacetal form of a carbohydrate).

[0072] Prodrugs of a compound of the present invention may be identified using routine techniques known in the art. Various forms of prodrugs are known in the art. For examples of such prodrug derivatives, see, for example, a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) A Textbook of Drug Design and Development, edited by rogsgaard- Larsen and H. Bundgaard, Chapter 5 "Design and Application of Prodrugs, " by H. Bundgaard p. 113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and e) N. Kakeya, et al., Chem. Pharm. Bull., 32: 692 (1984), each of which is specifically incorporated herein by reference.

[0073] The term "inflammation" as used herein is intended to mean the process by which a subject's immune system coordinates a response to tissue damage, infection, antigenic challenge, etc. Inflammation may be associated with overproduction of reactive oxygen species (ROS). Inflammation may be associated with any one or more of a H₂0₂-oxidative stress induced cell death, overproduction of NO, overproduction of cytokines and MPO activity. Myeloperoxidase (MPO) is a peroxidase enzyme that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂0₂) and chloride anion (CI^") (or the equivalent from a non-chlorine halide).

[0074] "Oxidative stress" is defined as pathologic change seen in living organisms in response to excessive levels of cytotoxic oxidants and free radicals in the environment

[0075] Diagnosis of inflammatory or neuroinflammatory condition in a subject, wherein said inflammatory or neuroinflammatory condition is associated with overproduction of ROS, H₂0₂-oxidative stress induced cell death, overproduction of NO, overproduction of cytokines and MPO activity is a process well known in the art.

[0076] The term "treating" as used herein in relation to inflammation in a subject is intended to mean that the compound or pharmaceutical composition reduces or abrogates the symptoms and/or cause of the inflammation.

[0077] The term "prevention" as used herein in relation to inflammation in a subject is intended to mean that the compound or pharmaceutical composition substantially prevents an inflammatory response and/or reduces the symptoms of the inflammatory response that would otherwise occur had the subject not been treated with the compound or pharmaceutical composition.

[0078] The preparation of such pharmaceutical compositions is known in the art, for example as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S.

[0079] Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa.,

1984). For example, the compound may be prepared into a variety of pharmaceutical compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., and these preparations may be administered as intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions). Compositions containing the compound may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent. Examples of suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilisers are dextran, gelatin, a-tocopherol acetate or alpha- thioglycerin. Examples of suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydro genated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D- sorbitol or D- mannitol. The composition may also contain other constituents or additives such as a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, fiavorant or sweetener, taking into account the physical and chemical properties of the compound being administered.

[0080] The composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, ocularly, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrastemal, and intracranial injection or infusion techniques.

[0081] When administered parenterally, the composition may be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable earner. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenteral ly-acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any fixed oil may be employed including synthetic mono- or di-glycerides, com, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants. The carrier may contain minor amounts of additives, such as substances that enhance solubility, iso tonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

[0082] In the present invention, the anti-neuroinflammatory, anti-inflammatory and antioxidant properties of l-(2,4-dihydroxy-6-methoxyphenyl)-3-(3_J4-dihydroxyphenyl)~2,3- dihydroxypropan-l-one in primary cultures of rat brain glial cells and in vivo in mice have been assessed.

The result indicate that this active compound might be developed as a drug or food additive for the prevention and/or amelioration of various diseases that involve inflammation and oxidative stress, e.g. inflammatory, autoimmune and neurodegeneratrive diseases.

[0083] In another embodiment of the present invention, compounds of the present invention are used to inspire a protective effect on an oxidative stress condition. According to figure 17, 7-3028/9 prevents the induced increase in ROS levels following H2O2 -induced oxidative stress. Astrocytes were preloaded with DCF-DA for 30 min and washed. 7-3028/9 (6 g ml) was added to astrocytes before (-2 h, -1 h), concomitant (0) or after (1 h, 2 h) the addition of H2O2 (175 μΜ). ROS production was measured at the indicated time points.

[0084] Said protective effect appears to be independent of the anti-oxidant property of the compounds. Basis for this statement is clear when comparing Figures 4 and 17. Figure 4 shows that only cells that were at least 1 hour pre-incubated with 7-3028/9 were protected from ¾(¾- induced cell death. However, Figure 17 shows that the timing of the addition of 7-3028/9 relative to the addition of ROS (before, concomitant or after) does not make any difference on neutralizing ROS. This may indicate that the compounds of the invention set the cell into a 'defensive' state that helps it to contend with the oxidative stress, and that it takes about 1 hour to get the cell into that 'defensive' state. Thus, compounds of the invention are not mere oxidant species neutralizers. Possible mechanisms for this may be: binding of the compound to surface-cell receptor and conduction of protective signal, binding a receptor protein inside the cell or binding transcription factor.

[0085] According to the present invention, the inventors have used primary cultures of rat brain astrocytes and microglial cells in the following experimental system:

H₂O₂ inducion of astrocytic cell death.

[0086] Figure 1 shows that H₂0₂ induced astrocytic cell death in a dose dependent manner. Astrocytes (100,000 cells/well of 24 wells plate) were treated with different concentrations of H₂0₂ and cell death was quantified 24 hr later using a method based on the measurements of the enzyme Lactate Dehydrogenase (LDH).

EXAMPLES

[0087] In order to illustrate the invention, the following examples are included. However, it is to be understood that these examples do not limit the invention and are only meant to suggest a method of practicing the invention. Persons skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other anti-neuroinflammatory compounds of the invention, and alternative methods for preparing the compounds of this invention are deemed to be within the scope of this invention. For example, the synthesis of non- exemplified compounds according to the invention may be successfully performed by modifications apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art other than those described, and/or by making routine modifications of reaction conditions.

[0088] Persons skilled in the art will also recognize that bio-assays used to obtain results described herein may be readily applied on non-exemplified compounds of the invention.

[0089] Following are examples to such an application on non-exemplified compounds and methods of the invention. The following procedure examples are general practical routes that enable a person skilled in the art to make and use methods of the invention on any exemplified or non-exemplified compounds of the invention.

General procedure A.

[0090] In order to determine whether a compound of the invention gains a protective activity against H₂0₂-induced cell death in cultured astrocytes, a person skilled in the art should follow the following steps: 1. Produce a primary culture of rat brain glial cells according to the method described in "Protective effects of the essential oil of salvia fruticosa and its constituents on astrocytic susceptibility to hydrogen peroxide-induced cell death". 2009. A. Elmann, S. Mordechay, M. Rindner, O. Larkov, M. Elkabetz, U. Ravid. Journal of agricultural and food chemistry.

57(15):6636-41 ;

2. Separate astrocytes from other cell types in the culture according to the method described in "Protective effects of the essential oil of salvia fruticosa and its constituents on astrocytic susceptibility to hydrogen peroxide-induced cell death". 2009. A. Elmann, S. Mordechay, M. Rindner, O. Larkov, M. Elkabetz, U. Ravid. Journal of agricultural and food chemistry.

57(15):6636-41 ;

3. Incubate the cultured astrocytes (37°C, 5% C02) for 2 hours with freshly diluted compound of the invention (in DMSO);

4. Add 175-200 micromolars of freshly diluted Η₂0₂;

5. Incubate the culture for 20-24 hr (37°C, 5% C02);

6. Measure cell viability and cytotoxicity by the colorimetric lactate dehydrogenase (LDH) assay (Roche Applieed science) according to the manufacturer's instructions, and the crystal violet cell staining as follows:

7. Gently draw out medium from plate;

8. Transfer plate to a chemical hood;

9. Dispense 150μ1 5% formaldehyde (in PBS) to each well and incubate 15 min at RT;

10. Pour out formaldehyde to chemical waste and gently rinse under running tap water;

1 1. Remove excess water by tapping plate on a tissue paper;

12. Dispense 150μ1 crystal violet solution- (10 gr/liter, in water) to each well;

13. Incubate 15 min at RT (in the chemical hood);

14. Pour out crystal violet to chemical waste and gently rinse under running tap water until all residual dye is removed;

15. Tap plate on a tissue paper to remove remaining water;

16. Dispense 150μ1 33% aqueous glacial acetic acid to each well;

17. Measure absorbance using a microplate reader at 540 ran with 690 nm reference filter;

18. A reduction of at least 70% of ROS levels are indication for anti-oxidant activity against HbOrinduced ROS levels; General procedure B.

[0091] In order to determine whether a compound of the invention gains an anti-oxidant activity in H₂0₂ -treated cultured astrocytes, a person skilled in the art may take the following steps:

1. Produce a primary culture of rat brain glial cells according to the method described in "Protective effects of the essential oil of salvia fmticosa and its constituents on astrocytic susceptibility to hydrogen peroxide-induced cell death". 2009. A. Elmann, S. Mordechay, M Rindner, O. Larkov, M. Elkabetz, U. Ravid. Journal of agricultural and food chemistry.

57(15):6636-41 ;

2. Separate astrocytes from other cell types in the culture according to the method described in "Protective effects of the essential oil of salvia fmticosa and its constituents on astrocytic susceptibility to hydrogen peroxide-induced cell death". 2009. A. Elmann, S. Mordechay, M. Rindner, O. Larkov, M. Elkabetz, U. Ravid. Journal of agricultural and food chemistry.

57(15):6636-41;

3. Plate astrocytes (300,000 cells/0.5 ml/well of 24 wells plate) and incubate (37°C, 5% C02) for 24 r as described in:

Elmann, A., Telerman, A., Mordechay, S., Erlank, H., Rindner, M., Ofir, R. and Beit-Yannai, E. (201 1). Extract of Achillea fragrantissima downregulates ROS production and protects astrocytes from oxidative-stress-induced cell death. In: "Neurodegenerative Diseases - Processes,

Prevention, Protection and Monitoring", ISBN 978-953-307-485-6, (Raymond Chuen-Chung Chang, ed). InTech Publisher. http://www.intechopenxorn/arfe^

fragrantissima-dowraegd

4. Replace growth medium with a fresh one containing 20 micromolars 27'- dichlorofluorescein diacetate (DCF-DA);

5. Incubate 30 min (37°C, 5% C02);

6. Wash twice with PBS;

7. Replace with fresh medium;

8. Read fluorescence of "time 0" at 485 nm (Extinction) and 528 nm (Emmision).

9. Incubate the cultured cells for 2 hours with freshly diluted compound of the invention (in DMSO);

10. Add freshly diluted H₂O₂ (175 micromolars); 11. Read fluorescence at 1, 2, 3, 4 hr after the addition of H2O2 (Ex: 485 11m and Em: 528 nm). General procedure C.

[0092] In order to determine whether a compound gains an intracellular anti-oxidant activity in cultured astrocytes containing the peroxyl radical generating molecule ABAP, a person skilled in the art may take the following steps:

1. Plate cells in 24 wells plates (microglial cells: 130,000 cells/well, astrocytes: 300,000 cells/ well);

2. The day after replace medium with fresh medium as described in: Elmann, A.,

Telerman, A., Mordechay, S., Erlank, H., Rindner, M.„ Ofir, R. and Beit-Yannai, E. (2011).

Extract of Achillea fragrantissima downregulates ROS production and protects astrocytes from oxidative-stress-induced cell death. In: "Neurodegenerative Diseases - Processes, Prevention, Protection and Monitoring", ISBN 978-953-307-485-6, (Raymond Chuen-Chung Chang, ed). InTech Publisher: http:// www.intechopen.com/ articles/ show/title/ extract-of-achillea- fragrantissima-downregulates-ros-production_:and-protects-astrocYtes-rrom-oxidatt;

For microglial cells: 36 hr later replace medium with fresh medium as described in Elmann, A., Mordechay, S., Erlank, H., Telerman, A., Rindner, M._f and Ofir, R. 2011 Anti-neuroinflammatory effects of the extract of Achillea fragrantissima. BMC Complement Altern Med 1 1(1):98.

htt :// www, biomedcentral com/ 1472-6882/11/98

3. Incubate cells with freshly diluted (in DMSO) compound of the invention (1 hr, 5% C02, 37 C);

4. Add DCF-DA and incubate cells for additional 30 min (37°C, 5% C02);

5. Wash twice with PBS;

6. Read "time 0" fluorescence (Ex: 485 nm and Em: 528 nm);

7. Add to cells freshly diluted 2^!2'-azobis(2-amidinopropane) dihydrochloride (ABAP, generate peroxyl radicals) to a final concentration of 600 microM, except of "blank " cells (cells w/o antioxidants and w/o ABAP);

8. Read fluorescence at 3, 6, and 24 hr Ex: 485 nm and Em: 528 nm;

9. Measure cell viability 20 hr later using the crystal violet cell staining as described above;

10. A reduction of at least 70% of ROS levels are indication for anti-oxidant activity in the cellular anti-oxidant assay (See Wolfe, .L., & Liu R.H. (2007) Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. Journal of Agriculture and Food Chemistry, Vol.55, No. 22, pp.8896-8907.).

General procedure D.

[0093] In order to determine whether a compound of the invention gains an antiinflammatory activity in LPS-activated splenocytes, a person skilled in the art may take the following steps:

1. Prepare a primary culture of splenocytes from naive female Balb/c mice (-20 gr);

2. Lyse red blood cells with "red blood cells lysis buffer" (BioLegend), and wash cells with PBS;

3. Plate splenocytes (5 ^χ 10⁶ cells/well/ml) on a 24-well tissue culture plate;

4. Treat cells with freshly diluted (in DMSO) compound of the invention;

5. Activate splenocytes with lipopolyscaccharide (LPS, 5 micrograms/ml);

6. Collect conditioned media after 24 hr for the measurement of IL-2 levels, and after 48 hr for the measurement of IFNy and IL-6;

7. Measure the levels of cytokines by ELISA;

8. Inhibition higher than 60% of the induced cytokines are indication for anti-inflammatory activity in this model;

General procedure E.

[0094] In order to determine whether a compound of the invention gains an antiinflammatory activity in delayed type hypersensitivity (DTH) in mice, a person skilled in the art may take the following steps

1. On "day 1 "anesthetize mice (6 weeks old, female, balb/c) with domitor and shave their abdomen. Paint on the abdomen with 150 microliters of 3% oxazolone (4:1) in olive oil;

2. On day 6 freshly prepare the compound of the invention in the vehicle: EthanokTween 20:PBS (5:5:90), and place the vial in a sonication bath for 15 minutes, 250C;

3. On day 6 inject the mice either subcutaneously (s.c, 100 microliters) or intra-peritoneally (i.p., 100 microliters) with the compound of the invention. Thirty minutes later - challenge mice by applying to one ear a total of 20 microliters/ear of 1% oxazolone (in 4:1 aceton/olive oil) onto both sides of one ear topically (10 microliters/side);

4. Two hr after the challenge repeat the injection of the compound of the invention 5. To the "gold standard" group of mice apply Dexamethasone (in acetone, topically, 50 micrograms/20 microliter s/ear, 10 microliters/each side of challenged ear) 1 hr after oxazolone challenge;

6. Six hr after challenge measure ear thickness with a micrometer;

7. Sacrifice mice;

8. Immediately after sacrificing the mice, collect the sensitized ears from each experimental group into a separate pre-weight tube, and freeze immediate in liquid nitrogen;

9. Weigh frozen ears and calculate the total weight of the ears by subtracting the weight of the tube;

10. Add 0.5 ml/ear of PBS containing 0.1% Tween 20 to the tube containing the frozen ears;

11. Freeze and thaw twice;

12. Homogenize ears with polytron;

13. Centrifuged for 5 min at 13,000g, 40C;

14. Collect the supernatants and freeze at -800C;

15. Measure cytokine levels in the supernatant by ELISA kits according to the manufacturer's instructions;

16. Measure myeloperoxidase (MPO) activity levels in the supernatant by a colorimetric assay kit (Bio Vision) according to the manufacturer's instructions;

17. Inhibition of 90% of the induced MPO activity and inhibition of 80% of the induced cytokine levels in the inflamed ear, and inhibition of 40% in ear edema indicates for antiinflammatory activity in the DTH model.

General procedure F.

[0095] In order to determine whether a compound of the invention gains an antiinflammatory activity in Keyhole limpet hemocyanin (KLH) immunized mice, a person skilled in the art should:

1. Freshly prepare the compound of the invention in the vehicle: Ethano Tween 20:PBS (5:5:90), and place the glass via! in a sonication bath for 15 minutes, 25°C;

2. Inject the compound of the invention subcutaneously (s.c.) in 0.1 ml vehicle 1 day and 2 days before and 2 hr before with immunization; 3. Immunize female Balb/c mice (-20 gr) intraperitonealy (i.p.) with KLH (0.25 mg in CFA, 1 : 1 v/v; 200 μΐ total volume);

4. Sacrifice mice on day 8 after immunization;

5. Remove spleens from mice and squeeze to a single ceil suspension;

6. Lyse red blood cells with "red blood cells lysis buffer" (BioLegend), and wash cells with PBS;

7. Plate splenocytes 5 x 10⁶ cells/well/ml on a 24-well tissue culture plate for cytokine detection or lxlO⁶ cells/well/100 μL on a 96 well flat-bottom microtiter plate;

8. Stimulate cells with either LPS (5 μg/mL) or KLH (20 μg mL), in the absence or presence of different concentrations of the compound of the invention (freshly diluted in DMSO). For proliferation assay the final volume should be 200 microliters;

9. After 72 hr, measure cell proliferation by the (3-[4,5~dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT test. Add 22 microliters of MTT solution (5 mg/ml) to each well and incubated for 4 hr;

10. Remove carefully 100 microliters by pipetting;

11. Add 100 μL of Isopropanol.HCl.-Triton X-100 (89:1 :10) for 10 min;

12. Measure absorbance using a plate reader at 570 nm with a 630 nm reference;

13. For cytokine measurement collect conditioned media from the cells plated at the 24 wells plates after 24 hr (for IL-2 and TNFa), 48 hr (for !FNy) or 72 hr (IL-10);

14. Centrifuge twice to discard the pellet, and freeze supernatant in -80°C;

15. Test supernatant for cytokine levels with commercial ELISA kits, according to the manufacturer's instructions;

16. Ninety percent inhibition of proliferation of LPS- and KLH-stimulated splenocytes derived from the KLH (in CFA) immunized mice and 70% inhibition in of the induced cytokine levels of this experimental group are indication for anti-inflammatory activity in this model.

Example 1.

[0096] Purification of the astroprotective compounds from PL

[0097] The astro-protective compounds were purified from Pi by activity guided fractionation using the bioassay of protection of astrocytes from H₂0₂-induced cell death (as described in Fig. IB). The wild plant Pi (56 gr) was homogenized and extracted with ethyl acetate (EA) (3x250 fflL, overnight). The EA crude extract (1.08 gr) was chromatographed on a Sephadex LH-20 column, eluting with Hexane/MeOH/CH₂Cl₂ (2:1 :1) to afford compound 7-3028/9 (described hereinbelow) (16.7 mg) and compound 7-3028/6 (described hereinbelow) (3.5 mg). The cultivated Pi was found also to contain compounds 7-3028/9 and 7-3028/6 in varying yields.

[0098] The structures of these compounds were determined and were found to be

(1) l-propanone-3-(4-hydroxyphenyl)-2,3-dihydroxy-l-(4,6-dihydroxy-2- methoxyphenyl), (d₆Hi₆0₇); molecular weight: 320.29; H-NMR: 7.28d (2), 6.85d (2) , 6.05s (1) , 5.98s (1) , 4.95d (1) , 4.50d (1) , 3.75s (3) ppm. This compound will be designated from now on as 7-3028/6; and

(2) 1 ~(2,4-dihydroxy-6-methoxyphenyl)-3 -(3 ,4-dihydroxyphenyl)-2,3- dihydroxypropan-l-one, C^HieOg); molecular weight: 336.29; H-NMR: 6.91s (1) , 6.80 AB(2) , 5.99s (1) , 5.93s (1) , 4.88d (1) , 4.46d (1) , 3.71 s (3) ppm. This compound will be designated hereinafter as 7-3028/9.

[0099] The pure molecules have similar structure, and differ only in one hydroxyl group

(Figure 2). These molecules belong to the dihydrochalcone family, and due to their low molecular weight they may be able to traverse the Blood Brain Barrier (BBB).

Example 2.

[00100] Protection from ¾0₂-mduced astrocytic ceil death by different concentrations of the purified astroprotective compounds 7-3028/6 and 7-3028/9.

[00101] The dose response of the purified chalcones 7-3028/6 and 7-3028/9 is presented in Figure 3 and shows that these molecules exhibit astroprotective activity at 0.5 or 3 μ^πιΐ (~ 1 or 6 μΜ) respectively. Astrocytes were preincubated for 2 hr with the indicated concentrations of the pure compounds. Η₂0₂ (200 μΜ) was then added and cytotoxicity was measured 20 hr later. The results are the mean±SEM of 1 (the purified compound 7-3028/6) or 2 experiments (the purified compound 7-3028/9), each performed in quadriplicates (n=8).

Example 3.

[00102] Amelioration of I¾0₂ insult by 7-3028/9 in cells with pre-, co- or post treatment with ¾0₂.

[00103] To elucidate the optimal time at which 7-3028/9 ameliorated the ¾<¾ insult, the cells were pre-incubated with 7-3028/9 for 1 h or 2 h, co-treated with ¾0₂, or post treated. ¾<¾ was added and cytotoxicity was measured. The results, presented in Figure 4, demonstrated that 7- 3028/9 acted more efficiently when preincubated with the cells before the insult. The pure compound 7-3028/9 (2 μg/ml, 6.25 uM) was added to astrocytes before (-2, ~1) concomitant (0) or after (+1, +2) the addition of H2O2. Cytotoxicity was determined 20 hr later by the levels of LDH in the conditioned media. The results are the mean±SEM of 3 experiments each performed in quadriplicates (n=12). It is concluded that preincubation of astrocytes with the purified astroprotective compound 7-3028/9 is needed to exert its protective effect against H₂0₂ cytotoxicity.

Example 4.

[00104] Induction of ROS production by H₂0₂ in cultured astrocytes.

[00105] In order to get more insight into the mechanisms by which the purified compounds might exerts their anti-inflammatory and astro-protective effects, the authors tested whether treatment of astrocytes with H2O2 and the purified compounds affects intracellular ROS levels, using 27'-dichlorofluorescein diacetate (DCF). Astrocytes were preloaded with DCF-DA for 30 min and washed. H2O₂ was added and the fluorescence intensity representing ROS productio was measured at the indicated concentrations (A. after 1 hr) or at the indicated time points (B. 175 μΜ H₂0₂). It can be shown that H2O2 induces ROS production in astrocytes in a dose (Figure 5 A) and time (Figure 5B) dependent manner.

Example 5.

[00106] Effect of pre-treatmeni of astrocytes with 7-3028/9 on the ROS levels following treatment of the astrocytes with H₂0₂.

[00107] Astrocytes were preloaded with DCF-DA for 30 min and washed. Astrocytes were then preincubated for 2 hr with different concentrations of 7-3028/9. H2O2 (175 μΜ) was added and the fluorescence intensity was measured. 20 r later. Pretreatment of astrocytes with 7-3028/9 prevented the H₂0₂-induced elevation in the levels of intracellular ROS in a dose dependent manner (Figure 6).

Example 6.

[00108] 7-3028/9 reduces 2,2^,-Azobis(amidinopropane) (ABAP)-mediated peroxyl radicals levels in astrocytes.

[00109] Peroxyl radicals are generated by thermolysis of 2_;2'-Azobis(amidinopropane) (ABAP) at physiological temperature. ABAP decomposes at approximately 1.36xlO^~V^l at 37°C, producing at most IxlO¹² radicals/ml/s. The experiment was carried out with ABAP 0.6 mM, and increasing doses of 7-3028/9. The kinetics of DCFH oxidation in astrocytes and microglial cells by peroxyl radicals generated from ABAP is shown in Figures 7 and 8, respectively. In the experiment shown in Figure 7, astrocytes were incubated with 7-3028/9 (12 microg/ml, A; or with the indicated concentrations, B). Then astrocytes were preloaded with DCF-DA for 30 min and washed. 0.6 mM ABAP was then added and ROS levels were measured at the indicated time points (A) or after 20 hr (B). The results are the mean±SEM of three experiments (n-10). 7- 3028/9 thus inhibits peroxyl radical - induced oxidation of DCFH to DCF in primary astrocytes. Example 7.

[00110] 7-3028/9 reduces 2,2'-Azobis(amidinopropane) (ABAP)-mediated peroxyl radicals levels in microglial cells.

[00111] In the experiment shown in Figure 8, microglial cells were incubated for 1 hr with 7-3028/9. Then microglial cells were preloaded with DCF-DA for 30 min and washed. 0.6 mM ABAP was then added and ROS levels were measured at the indicated time points (A) or after 20 hr (B). The results are the mean±SEM of two experiments (n=8). 7-3028/9 thusinhibits peroxyl radical - induced oxidation of DCFH to DCF in primary microglial cells.

[001 12] Thus, it can be seen that ABAP generates radicals in a time dependent manner and that treatment of cells with 7-3028/9 attenuates this induction in a time and dose dependent manner in astrocytes as well as in microglial cells. Thus, it can be concluded that 7-3028/9 enters the cells and represents an efficient hydroperoxyl radical scavenger.

Example 8.

[00113] Efficiency of 7-3028/9 in the oxazolone-induced Delayed Type Hypersensitivity (DTK) model.

[00114] Compound 7-3028/9 reduced the degree of oxazolone-induced ear edema in mice, as was measured by both ear weight (Figure 9) and thick (Figure 9). The anti-inflammatory effect of this compound was also demonstrated by the reduction of myeloperoxidase (MPO) activity and the levels of various inflammatory factors (TNFa, IFNy, IL-6, IL-2, IL-4, IL-10, IL-12) secreted by different immune cells, in ear homogenates of the experimental mice (Figure 10).

As to the results appearing in Figure 10, cytokine levels were measured in duplicates by ELISA. The data are expressed as mean ± SEM of: (1) IFNy: 4 different experiments (N=23 mice/group); 30 μΐ homogenate/well; (2) IL-2: 3 different experiments (N=16 mice/group); 30 μΐ homogenate; (3) IL-4: 3 different experiments (H=I8 mice/group); 50 μΐ homogenate; (4) IL-12: 2 different experiments (N=l 1 mice/group); 25 μΐ homogenate; (5) TNFot: 4 different experiments (NK23 mice/group); 10 μΐ homogenate/well; (6) IL-6: 3 different experiments ( ~18 mice/group); 25 μΐ homogenate; (7) IL-10: 3 different experiments (N=18 mice/group); 50 μΐ homogenate. (8) MPO activity: 3 different experiments.

Example 9.

[00115] Effect of 7-3028/9 on cytokine secretion by splenocytes from naive mice

[00116] Splenocytes from naive mice were cultured and stimulated with LPS. Treatment with 7-3028/9 inhibited the induced secretion of different cytokines (IFNy, IL-2, IL-6, IL-12 and IL-10) as demonstrated in Figure 9. The results in Figure 9 represent the mean±SEM for 5 different experiments (N=20 mice/group) for ear thickness; and 2 different experiments (N=l 1 mice/group) for ear weight.

Example 10.

[00117] 7-3028/9 down-regulates LPS- induced cytokine secretion from naive splenocytes.

[00118] Spleens were removed from naive mice, pooled and splenocytes were stimulated with LPS in the presence or absence of 7-3028/9. As can be seen in Figure Π, cytokine levels in supernatants were measured in duplicates by ELISA, and the results are presented as Mean±SEM of 2 different in vivo experiments. IL-2, 100 μΕ, collected after 24 h; IFNy (100 uL), IL-10 (100 μΤ), IL-6 (20 μΐ), IL-12 (30 |iL), collected after 48 hr. Results are shown In Figure 1 1. Example 11.

[00119] Effect of 7-3028/9 on inflammatory responses of splenocytes from LH- immunized mice.

[00120] The in vivo effect of 7-3028/9 administration in the KLH-immunization model is demonstrated in Figure 12. Data represent the mean ± SEM from two different experiments (6 mice/group in each experiment; total N=T2). It can be seen that the levels of IL-6 and TNFa secreted from splenocytes originated from CFA -immunized mice are very low, and are enhanced in CFA-KLH-immunized mice. In vivo Administration of 7-3028/9 (10 mg/kg, i.p.) prevented the antigen-induced elevation in the levels of both cytokines. The inventors have also tested cultured splenocytes or lymph node cells that were stimulated in vitro with either LPS or KLH. According to Figure 12, In vivo administration of 7-3028/9 down-regulates cytokine secretion from unstimulated splenocytes derived of CFA/KLH-immunized mice. Example 12.

[00121] 7-3028/9 is not toxic to none-stimulated and LPS-stimulated splenocytes.

[00122] Figure 13 demonstrates that 7-3028/9 is not toxic to none-stimulated and. LPS- stimulated splenocytes. With respect to Figure 13, Splenocytes were prepared from naive (none- immunized mice). Cell viability was measured at the indicated time points by Crystal violet. Data represent the mean ± SEM from two experiments (10 mice), performed in quadriplicates.

Example 13.

[00123] Effect of 7-3028/9 on LPS- and LH-stimulated splenocyte proliferation.

[00124] Figure 14 demonstrate that 7-3028/9 down-regulates the proliferative response of splenocytes to each of the stimulators. With respect to Figure 14, Cell proliferation was measured by the MTT test. Data represent the mean ± SEM from two different experiments (6 mice/group in each experiment; total N=12), each experiment performed in quadriplicates. The inventors have also determined the cytokine profile secreted from splenocytes obtained from the different in vivo experimental groups that were activated by either LPS or KLH and treated (or not) with 7-3028/9. It can be deduced that 7-3028/9 down-regulates the proliferative response of splenocytes.

Example 14.

[00125] Effect of 7-3028/9 on KXH- and LPS- induced cytokine secretion from splenocytes.

As can be seen in Figures 15 and 16, treatment of LPS- or KLH -stimulated splenocytes with 7- 3028/9 also inhibited the induced secretion of different cytokines from these cells.

With respect to Figures 15 and 16, mice (6 mice per group for each experiment) were immunized with KLH in CFA, and treated (s.c.) with 7-3028/9. Eight days later, spleens were removed from mice, pooled and splenocytes were stimulated with either KLH or LPS in the presence or absence of 7-3028/9. Cytokine levels in supernatants were measured in duplicates by ELISA, and the results are presented as MeaniSEM of different in vivo experiments. IL-2, 100 μί, collected after 24 h, 2 experiments; IFNy and TNFa, 100 μL_J collected after 48 h, 2 experiments; IL-10 lOOpL collected after 48 h 3 experiments.

All patents, patent publications, and non-patent publications cited are incorporated by reference herein.

Claims

WHAT IS CLAIMED IS:

1. A compound having the Fo

prodrugs thereof and pharmaceutically acceptable salts thereof, wherein:

R¹ is Me, Et, MeO-, EtO-, HOCH₂CH₂0~, HOC¾C(Me)₂0-, (S)-MeCH(OH)CH₂0-, cyclopropyl-CHaO-, HOCH₂CH₂~, HOC¾-,

each of R², R³ and R⁴ is H, Me, Et, OH, MeO-, EtO-, HOCH₂CH₂0-, HOCH₂C(Me)₂0-, (S)- -, cydopropyl-CH₂0-. H0CH₂CH₂-₅ HOC¾~,

2. The compound of claim 1, wherein R⁵ is H.

3. The compound of claim 1 selected from the group consisting of: l-propanone-3-(4- hydroxyphenyl)-2,3-dihydroxy- 1 -(4,6-dihydroxy-2-methoxyphenyl) and 1 -(2,4~dihydroxy-6- methoxyphenyl)-3-(3,4-dihydroxyphenyl)-2,3-dihydroxypropan-l-one.

4. A compound according to any one of claims 1 to 3 for use as a medicament.

5. A compound according to claim 4 for use in the treatment or prevention of an inflammatory disease.

6. A compound according to claim 5, wherein said inflammatory disease is characterized by a disorder selected from the group consisting of: H₂0₂-oxidative stress induced cell death, overproduction of a cytokine, increased MPO activity, increased levels of ROS, increased levels of peroxyl radicals; and overproduction of NO.

7. A compound according to claim 5, wherein said inflammatory disease is characterized by overproduction of a cytokine, and wherein said cytokine is selected from the group consisting of: TNFa, IFNy, IL-6, IL-2, IL-4, IL-10, and IL-12.

8. A compound according to claim 5, wherein said inflammatory disease is inflammatory CNS disease.

9. A compound according to claim 4, for use in the treatment or prevention of neurodegenerative or autoimmune disease associated with inflammation and oxidative stress.

10. A compound according to claim 4, for use in the treatment or prevention of Alzheimer's or Parkinson's disease.

11. A compound according to claim 4, for use in the treatment or prevention of stroke.

12. A compound, according to any one of claims 1 to 3, for producing an oxidative stress reduction effect in a warm blooded animal.

13. A compound according to claim 5, wherein said inflammatory disease is associated with depression, wherein said depression is characterized by melancholic symptoms, anxiety, and fatigue and somatic (F&S) symptoms.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 in association with a pharmaceutically acceptable carrier.

15. The use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of an inflammatory disease.

16. A use according to claim 12, wherein said inflammatory disease is characterized by a disorder selected from the group consisting of: LtCVoxidative stress induced cell death, overproduction of a cytokine, increased MPO activity and overproduction of NO.

17. A use according to claim 12, wherein said inflammatory disease is characterized by overproduction of a cytokine, and wherein said cytokine is selected from the group consisting of: TNFa, IFNy, IL-6, IL-2, IL-4, IL-10, and IL-12.

18. A use according to claim 12, wherein said disease is inflammatory CNS disease.

19. The use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of neurodegenerative or autoimmune disease associated with inflammation and oxidative stress.

20. The use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of Alzheimer's or Parkinson's disease.

21. The use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of stroke.

22. The use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for producing an oxidative stress reduction effect in a warm blooded animal.

23. A method of producing a reactive oxygen species ( OS) reduction effect in a warmblooded animal, comprising administering to said animal an effective amount of a compound according to any one of claims 1 to 3.

24. A method of producing an effect of down-regulation of proliferation of splenocytes in a warm-blooded animal, comprising administering to said animal an effective amount of a compound according to any one of claims 1 to 3.