WO2006138399A1 - Procedes pour le traitement de maladies oculaires induites par les proteines beta-amyloidiennes - Google Patents

Procedes pour le traitement de maladies oculaires induites par les proteines beta-amyloidiennes Download PDF

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WO2006138399A1
WO2006138399A1 PCT/US2006/023197 US2006023197W WO2006138399A1 WO 2006138399 A1 WO2006138399 A1 WO 2006138399A1 US 2006023197 W US2006023197 W US 2006023197W WO 2006138399 A1 WO2006138399 A1 WO 2006138399A1
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alkenyl
alkyl
alkynyl
compound
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Darrick S. H. L. Kim
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Kim Darrick S H L
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Priority claimed from US11/287,080 external-priority patent/US7728043B2/en
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Priority to EP06773171A priority Critical patent/EP1895849A4/fr
Priority to CA002611490A priority patent/CA2611490A1/fr
Publication of WO2006138399A1 publication Critical patent/WO2006138399A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger

Definitions

  • Plaques are diffusely distributed throughout the cerebral cortex of AD patients, and are the neuropathologic hallmark of the disease. See Seidl et al., Neurosci Lett 232, 49 (1997), Yan et al., Nature 382, 685 (1997), Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994) and Trojanowski et al., Am J Pathol 144, 449 (1994).
  • NTFs are intraneuronal accumulation of paired helical filaments composed mainly of an abnormal form of tau protein, that is a microtubule associated phosphoprotein which can promote microtubule formation. See Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994) and Trojanowski et al., Am J Pathol 144, 449 (1994).
  • NTFs occur primarily in medial temporal lobe structures (hippocampus, entorhinal cortex, and amygdala), and NTFs density appears to correlate with dementia severity.
  • ⁇ A has been suggested as one of the major causes of AD. ⁇ A was shown to exert direct toxic effects on neurons and to inhibit neurite growth in vitro in a dose dependent manner. Thus, therapeutic approaches that can modulate ⁇ A toxicity have been hypothesized to represent important methods for controlling the onset of AD. It is envisioned that if neuronal cells can be protected from ⁇ A/senile plaque-induced toxicity, the onset of AD may be delayed or prevented.
  • nerve growth factor (NGF) (See Hefti, Neurobiol Aging 15 (Suppl 2), S 193 (1994), and Seiger et al., Behav Brain Res 57, 255 (1993)), calcium channel blockers (See Zhou et al., J Neurochem 67, 1419 (1996) and Friedlich et al., Neurobiol Aging 15, 443 (1994)), Zinc (See Cuajungco et al., Neurobiol Dis 4, 137 (1997)), sulfonated compounds (See Pollack et al., Neurosci Lett 197 211 (1995) and Lorenzo, et al., Ann N Y Acad Sci 111, 89 (1996)), triaminopyridine nonopiate analgesic drug (See Muller et al., J Neurochem 68, 2371 (1997)), low molecular lipophilic compounds that can activate neurotrophic factor
  • ROS reactive oxygen intermediates
  • ROS free radical scavenging enzyme superoxide dismutase
  • ⁇ A peptide In addition to ⁇ A peptide-induced ROS mediated neurotoxicity, ⁇ A peptide has been shown to cause neuronal cell death by stimulating microglial expression of tumor necrosis factor ⁇ (TNF ⁇ ). See Tarkowski et al., Neurology 54, 2077 (2000) and Barger et al., Proc Natl Acad Sd USA 92, 9328 (1995). The accumulation of ⁇ A peptide as neuritic plaques is known to be both trophic and toxic to hippocampal neurons, causing apoptosis or necrosis of the neurons in a dose dependent manner.
  • TNF ⁇ tumor necrosis factor ⁇
  • ⁇ A peptide was demonstrated to induce these cellular effects by binding with a receptor for advanced glycation end products (RAGE) that was previously known as a central cellular receptor for advanced glycation endproducts.
  • RAGE receptor for advanced glycation end products
  • EMBO J 23, 4096 (2004) Huttunen et al., J Biol Chem 21 A, 19919 (1999), and Yan etal., Nature 382, 685 (1996).
  • RAGE was suggested to mediate the interaction of ⁇ A peptide with neurons and with microglia, resulting in oxidative stress mediated cytotoxicity. Blocking RAGE with anti-RAGE F( ⁇ ')2 prevented the appearance of TNF ⁇ messenger
  • RNA and diminished TNF ⁇ antigen to levels seen in untreated cells RNA and diminished TNF ⁇ antigen to levels seen in untreated cells.
  • RAGE mediates microglial activation by ⁇ A peptide by producing cytotoxic cytokines that cause neuronal damage in AD patients.
  • RAGE was also demonstrated to specifically bind with ⁇ A peptide and mediate ⁇ A peptide-induced oxidative stress.
  • Cell receptors that bind to ⁇ A peptide have been identified.
  • the low-affinity neurotrophin receptor p75 (p75NTR) which belongs to the family of apoptotic receptors that generate cell-death signals on activation was found throughout the brains of AD patients.
  • ⁇ A peptide was found to be a ligand for p75NTR, and to cause preferential apoptosis of neurons and normal neural crest-derived melanocytes that express p75NTR upon specifically, binding to p75NTR. See Zhang et al., JNeurosci 23, 7385 (2003) and Perini et al., J Exp Med 195, 907 (2002).
  • Basal forebrain cholinergic neurons express the highest levels of p75NTR in the adult human brain and have been shown to be involved in AD.
  • the expression of p75NTR neuronal cells was shown to potentiate ⁇ A peptide-induced cell death.
  • This interaction of ⁇ A peptide with p75NTR to mediate neuronal death in AD suggested a new target for therapeutic intervention. See Zhang et al., JNeurosci 23, 7385 (2003) and Perini et al., J Exp Med 195, 907 (2002).
  • ERAB which is over-expressed in neurons of the AD brain, was shown to bind with ⁇ A peptide to induce neuronal death in AD.
  • Glaucoma and age-related macular degeneration are the most common leading cause of irreversible progressive visual dysfunction that leads to blindness.
  • Glaucoma causes irreversible vision loss worldwide an estimated 66.8 million people.
  • AMD is the leading cause of blindness and vision loss in developing countries due to increased life expectancy and subsequent increase in aged population.
  • VanNewk ⁇ rk et al. Ophthalmol 108, 960 (2001).
  • Glaucoma and AMD have profound effect on the family members and other loved ones who provide most of the care for people having this disease. Unfortunately, the cure for glaucoma and AMD has not yet been discovered.
  • AMD is characterized by abnormal extracellular deposits, known as drusen, the hallmark sign of AMD, that accumulate along the basal surface of the retinal pigmented epithelium.
  • drusen is common in older individuals, large numbers of drusen and/or extensive areas of confluent drusen represent a significant risk factor for AMD.
  • Widespread drusen deposition is associated with retinal pigmented epithelial cell dysfunction and degeneration of the photoreceptor cells. See Johnson et al., Proc Natl Acad Sci USA 99, 11830 (2002).
  • AMD dry and wet.
  • the dry type of AMD is characterized by a geographic atrophy that progresses slowly over many years.
  • choroidal neovascularization occurs that result in a dense fibro vascular scar that may involve the entire macular area.
  • the wet type of AMD is more sight threatening than the dry type and is responsible for 90% of cases of severe visual loss in elderly population. See Chopdar et al., BMJ 326, 485 (2003).
  • Glaucoma is a chronic neurodegeneration of the optic nerve, retinal ganglion cells, that result in irreversible vision loss. See Khaw et al., 5MJ 320, 1619 (2000).
  • ⁇ A beta-amyloid
  • ⁇ A deposition was found an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis and the pathogenesis of AMD. See Johnson et al., Proc Natl Acad Sci USA 99, 11830 (2002).
  • Glaucoma Current glaucoma treatment focuses on lowering intraocular pressure, the major risk factor for the disease. Glaucoma has been treated medically, surgically, or with laser to lower intraocular pressure that can slow the disease progression.
  • Pharmacological treatment approaches are: cholinergic agents (pilocarpine — increases outflow of the aqueous humour; beta blockers — reduce aqueous secretion), oral carbonic anhydrase inhibitors (acetazolamide and dorzolamide — reduces aqueous secretion), al ⁇ ha-2 adrenergic agonists (apraclonidine and brimonidine), and prostaglandin agonists (latanoprost - opens up an alternative pathway for aqueous outflow by altering the resistance of the extracellular matrix).
  • cholinergic agents potocarpine — increases outflow of the aqueous humour
  • beta blockers reduce aqueous secretion
  • One current treatment approach for AMD is a technique called photodynamic therapy that uses verteporfin as the photosensitizer.
  • Long term supplementation with high dose zinc and antioxidant vitamins (A, C, and E) showed a significant reduction in the relative risk of developing neovascular AMD.
  • carotenoids lutein and zeaxanthin which are potent antioxidants found in high concentrations in the macular retina are found to be effective. See Chopdar et al., BMJ 326, 485 (2003).
  • One important pharmacological approach related to ⁇ A-induced neurodegenerative disease preventive and neuroprotective interventions may be antioxidant therapy. See Kumar et al., Int JNeurosci 79, 185 (1994), Lucca, et al., Brain Res 764, 293 (1997), Manelli et al., Brain Res Bull 38, 569 (1995), Parnetti et al., Drugs 53, 752 (1997), Preston et al., NeurosciLett 242, 105 (1998), and Zhou, et al., JNeurochem 67, 1419 (1996).
  • Curcuma longa (Zingiberaceae) has been used as curry spice and a well known constituent of Indonesian traditional medicine. See Nurfina et al., Eur J Med Chem 32, 321 (1997).
  • curcumin that has been known as a natural antioxidant with antitumor activity. See Ruby et al., Cancer Lett 94, 79
  • curcuminoids with antioxidant property have been demonstrated to protect neuronal cells from ⁇ A insult. See Kim DSHL et al., Neurosci Lett 303, 57 and Park SY et al., J Nat Prod 65, 1227 (2002).
  • a representative list of Curcuma sp. include C. longa, C. aromatica, C. domestica, C. xanthorrhiza, and C. zedoaria.
  • Zingiber officinale is one of the world's favorite spices, probably discovered in the tropics of Southeast Asia. Ginger has benefited humankind as a wonder drug since the beginning of recorded history. See Jitoe et al., JAgric Food Chem 40, 1337 (1992), Kikuzaki et al., J Food Sd 58, 1407 (1993) and Schulick, Herbal Free Press, Ltd. (1994). From ginger, shogaols with antioxidant property have also been demonstrated to protect neuronal cells from ⁇ A insult. See Kim et al., Vlanta Medica 68, 375 (2002). A representative list of ' Zingiber sp. include Z.
  • Ginkgo ⁇ Ginkgo biloba (Ginkgoaceae)) is an herbal that has been used to treat neurologic ailment for thousand years as an Asian traditional medicine. Ginkgo leaf extract has shown to exhibit potent antioxidant activity and are widely used in the dietary supplement industry. The antioxidant activity of ginkgo has shown to be primarily contributed by diterpenes such as ginkgolides, bilobilide, flavonoids, and ginkgolic acids. See Hopia et al., J Agric Food Chem 44, 2030 (1996) and Nakatani et al., Agric Biol Chem 47, 353 (1983). [0026] Sage (Salvia officinalis L. (Lamiaceae)) and Rosemary (Rosmarinus officinalis
  • the present invention relates to the identification and isolation of natural compounds present in turmeric, ginger, gingko biloba, sage, and rosemary that exhibit potent anti- ⁇ A peptide activity.
  • the invention further provides novel synthetic compounds exhibiting potent anti- ⁇ A peptide activity which includes but is not limited to, the ability to neutralize amyloid protein mediated cytotoxicity towards retinal cells that relate to the pathogenesis of glaucoma and AMD.
  • the invention provides compounds and pharmaceutical compositions capable of protecting neurons from ⁇ A peptide insult, and methods for treating ⁇ A protein- induced disease with the same.
  • Beta- Amyloid Protein-Induced Disease Including Sage and Rosemary Derived Compounds
  • These compounds have potent anti- Beta-amyloid activity alone and may be combined with the other turmeric, ginger, and ginkgo-biloba derived compounds and their analogs and homologues have anti Beta-amyloid activity to treat beta- Amyloid protein-induced ocular disease including age-related macular degeneration (AMD) and glaucoma.
  • AMD age-related macular degeneration
  • synthetic turmeric, ginger, ginkgo biloba, sage, or rosemary compounds include chemically synthesized versions of naturally occurring turmeric sp., ginger sp., ginko biloba, sage sp., or rosemary sp. compounds respectively as well as analogues and homologues of such naturally occurring compounds which have anti-BA peptide activity.
  • anti-BA peptide activity includes, but is not limited to, the ability to neutralize amyloid protein mediated cytotoxicity including neurotoxicity.
  • the present invention is directed to treating (which when used herein also includes preventing) ⁇ A-involved ocular disease glaucoma and AMD.
  • an extract containing natural compounds found in turmeric may be administered to protect retinal cells from ⁇ A-involved cytotoxicity.
  • Natural compounds that are suitable for use with the invention include, but are not limited to 4' !-(3' "- methoxy-4' ⁇ -hydroxyphenyl)-2' '-oxo-3' '-enebutanyl 3-(3'-methoxy-4'- hydroxyphenyl)propenoate (calebin-A) and l,7-bis(4-hydroxy-3-methoxyphenyl)- 1,4,6- heptatrien-3-one, and seven known compounds, l,7-bis(4-hydroxy-3-methoxyphenyl)-l,6- heptadiene-3,5-dione (curcumin), l-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-l,6- heptadiene-3,5-dione (demethoxycurcumin), l,7-bis(4-hydroxyphenyl)-l,6-heptadiene-3,5- dione (bisdem
  • the invention relates to a method for the treatment of a beta-Amyloid protein-induced disease including but not limited to Alzheimer's Disease (AD), age-related macular degeneration (AMD) and glaucoma comprising administering to a subject suffering from the beta- Amyloid protein-induced disease a therapeutically effective amount of a compound having the formula (I):
  • AD Alzheimer's Disease
  • AMD age-related macular degeneration
  • glaucoma comprising administering to a subject suffering from the beta- Amyloid protein-induced disease a therapeutically effective amount of a compound having the formula (I):
  • Ri is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • Rj is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I.
  • R 1 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 1 is OH.
  • Ri is selected from the group consisting of H and OMe when the dotted configuration of compound (I) is a double bond
  • Ri is selected from the group consisting of H and OH when the dotted configuration is a single bond
  • R 2 is selected from the group consisting of OH, OMe, OR 50 , and X wherein.
  • R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 2 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I.
  • R 2 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 2 is OH. Even more preferably, R 2 is selected from the group consisting of H and OMe when the dotted configuration of compound (I) is a double bond, and R 2 is H when the dotted configuration is a single bond.
  • Other compounds useful for practice of the invention include those of the formula (II):
  • R 3 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 3 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 3 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 3 is H.
  • R 4 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 4 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 4 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 4 is H.
  • R 4 is H when the first dotted configuration of compound (II) is a double bond and the second dotted configuration of compound (II) is a single bond, R 4 is H when both dotted configurations are single bonds, and R 4 is selected from the group consisting of H and OMe when both dotted configurations are double bonds.
  • R 5 is selected from the group consisting of H, OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 5 is selected from the group consisting of H, OH, OMe, OR 50 , and X wherein R 50 is (CHi) n CH 3 and n is 1-7, ans X is F, Cl, Br, or I. More preferably, R 5 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 5 is OH.
  • the invention contemplates the use and production of compounds in either tautomeric form, and as a mixture of the two forms.
  • turmeric-related compounds useful in practice of the invention include those of formula (III):
  • Z is a representation of isosteric variation in which Z is selected from O, S, NH, NR 60 , where R 60 is alkyl, alkenyl, or alkynyl.
  • R 6 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 6 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 6 is selected from the group consisting of OH and OMe. Even more preferably, R 6 is OH. Generally, R 7 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 7 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CHa) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 7 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 7 is H.
  • Rg is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 8 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 8 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 8 is OH. Generally, R 9 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 9 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 9 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 9 is H. .
  • the second set of compounds useful for practice of the invention include natural compounds which can be extracted on otherwise derived from Ginkgo biloba as well as synthetic Ginkgo biloba compounds including biologically active homologues and
  • R is selected from the group consisting of higher alkyl, higher alkenyl, and higher alkynyl.
  • R is and n is 1-7. Even more preferably, R is selected from the group consisting of
  • R is also selected from the group consisting of alkyl, alkenyl, and alkynyl; for example;
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of ; for example;
  • the dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • the third set of compounds useful for practice of the invention include natural compounds which can be extracted on otherwise derived from Zingiber sp. (ginger) as well as synthetic ginger compounds including biologically active homologues and analogues of natural ginger compounds which share anti- ⁇ A activity.
  • Such compounds have the formula (V):
  • R 10 is selected from the group consisting of OH, OMe, OR', and X wherein R' is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I. More preferably, R 10 is selected from the group consisting of OH, OMe, OR", and X wherein R" is (CH 2 ) n CH 3 and n is 1-7, and X is F, Cl, Br, or I. Even more preferably, R 10 is OH.
  • Rn is selected from the group consisting of H, OH, OMe, and OR' wher R' is alkyl, alkenyl, or alkynyl. More preferably, Rn is selected from the group consisting of H, OH, OMe, and OR" wherein R" is (CH 2 ) n CH 3 and n is 1-7. Even more preferably, Rn is selected from the group consisting of H and OMe.
  • R 12 is selected from the group consisting of alkyl, alkenyl, and alkynyl. More preferably, R] 2 is selected from the group consisting of
  • R] 2 is selected from the group consisting of
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of ; for example;
  • dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • Ri 3 is selected from the group consisting of OH, OMe, OR', and X wherein R' is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I. More preferably, Ri 3 is selected from the group consisting of OH, OMe, OR", and X wherein R" is (CH 2 ) n CH 3 and n is 1-7, and X is F, Cl, Br, or I. Even more preferably, Ri 3 is OH.
  • Ri 4 is selected from the group consisting of H, OH, OMe, and OR' wher R' is alkyl, alkenyl, or alkynyl.
  • Ri 4 is selected from the group consisting of H, OH, OMe, and OR" wherein R" is (CH 2 ) n CH 3 and n is 1-7. Even more preferably, Ri 4 is selected from the group consisting of H and OMe.
  • R 15 is selected from the group consisting of alkyl, alkenyl, and alkynyl. More preferably, Ri 5 is selected from the group consisting of
  • R 15 is selected from the group consisting of
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of ; for example;
  • dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • the length of the side chain is important for the expression of biological activity.
  • compounds (11), (12), (13) and (14) the biological activity appears to improve as the compounds' side chain length increases.
  • analogues having different and lengthier side-chains Preferably, shogaol compounds have side chains wherein
  • Ri 2 has five or more carbons. More preferably, Ri 2 has nine or more carbons, and even more preferably, Ri 2 has eleven or more carbons.
  • two of the synthesized shogaol analogue compounds, compounds (45) and (50), also effectively protected cells from ⁇ A peptide insult despite the fact that these compounds have different substituents than the ginger-derived natural product compounds.
  • compound (45) differs from the ginger-derived natural product compounds because it has a saturated hydrocarbon side chain
  • compound (50) differs from the ginger-derived natural product compounds because it does not have a methoxy substituent.
  • alkyl refers to a carbon chain having at least two carbons.
  • alkyl refers to a carbon chain having between two and twenty carbons. More preferably, alkyl refers to a carbon chain having between two and eight carbons.
  • alkenyl refers to a carbon chain having at least two carbons, and at least one carbon-carbon double bond.
  • alkenyl refers to a carbon chain having between two and twenty carbons, and at least one carbon-carbon double bond.
  • alkenyl refers to a carbon chain having between two and eight carbons, and at least one carbon-carbon double bond.
  • alkynyl refers to a carbon chain having at least two carbon atoms, and at least one carbon-carbon triple bond.
  • alkynyl refers to a carbon chain having between two and twenty carbon atoms, and at least one carbon-carbon triple bond.
  • alkynyl refers to a carbon chain having between two and eight carbon atoms, and at least one carbon-carbon triple bond.
  • higher alkyl refers to a carbon chain having at least five carbon atoms.
  • higher alkyl refers to a carbon chain having between five and twenty carbons. More preferably, higher alkyl refers to a carbon chain having between five and twelve carbon atoms.
  • higher alkenyl refers to a carbon chain having at least five carbon atoms, and at least one cabon-carbon double bond.
  • higher alkenyl refers to a carbon chain having between five and twenty carbon atoms, and at least one carbon-carbon double bond. More preferably, higher alkenyl refers to a carbon chain having between five and twelve carbon atoms, and at least one carbon-carbon double bond.
  • higher alkynyl refers to a carbon chain having at least five carbons, and at least one carbon-carbon triple bond.
  • higher alkynyl refers to a . carbon chain having between five and twenty carbon atoms, and at least one carbon-carbon
  • the fourth set of compounds useful for practice of the invention include natural compounds which can be extracted or otherwise derived from Salvia sp. (sage) and Rosmarinus sp. (rosemary) which share anti- ⁇ A activity. Such compounds have the formulas (VII), (VIII), and (IX):
  • combinations of each of the anti-Beta amyloid compounds may be administered as well as combinations of compounds of natural or synthetic compounds selected from the different classes of tumeric, ginkgo biloba, ginger, sage and rosemary compounds may be administered in combination for additive or synergistic effect.
  • the invention also provides methods whereby the plant-derived compounds and homologues and analogues thereof may be combined with other agents including those selected from the group consisting of cholinergic agents (such as pilocarpine, beta blockers), oral carbonic anhydrase inhibitors (such as acetazolaminde and dorzolamide), alpha-2 adrenergic agonists (such as apraclonidine and brimonidine), prostaglandin agonists (latanoprost), carotenoids, lutein and zeaxanthin.
  • cholinergic agents such as pilocarpine, beta blockers
  • oral carbonic anhydrase inhibitors such as acetazolaminde and dorzolamide
  • alpha-2 adrenergic agonists such as apraclonidine and brimonidine
  • prostaglandin agonists such as apraclonidine and brimonidine
  • carotenoids lutein and zeaxanthin.
  • FIG. 1 shows the structures of turmeric-derived natural product compounds that protected PC 12, IMR32, and HUVEC cells from ⁇ A peptide-induced toxicity.
  • FIG. 2 shows a scheme for the synthesis of dihydro- and tetrahydro-curcuminoids.
  • FIG. 3 shows a scheme for the synthesis of symmetric and unsymmetric curcumin analogues and related compounds.
  • FIG. 4 shows a scheme for the synthesis of turmeric-derived natural product compound (6).
  • FIG. 5 shows the structures of curcuminoid compounds that have been synthetically prepared and assayed for biological activity against ⁇ A peptide-induced toxicity.
  • FIG. 6 shows the structures of ginger-derived natural product compounds that protected PC 12, IMR32, and HUVEC cells from ⁇ A peptide-induced toxicity.
  • FIG. 7 shows a scheme for the synthesis of ginger-derived natural product compound (13).
  • FIG. 8 shows a scheme for the synthesis of [9]-dihydroshogaol, compound
  • FIG. 9 shows a scheme for the synthesis of [9]-demothoxyshogaol, compound
  • FIG. 10 shows the structures of ginkgo biloba-derived natural product compounds that protected PC 12 and HUVEC cells from ⁇ A peptide-induced toxicity.
  • FIG. 11 shows a proposed synthesis for ginkolic acids and their analogues.
  • One aspect of the present invention is directed to the use of methanol and other extract of Curcuma sp. (Zingiberaceae), Zingiber sp. (Zingiberaceae), Ginkgo biloba, Salvia sp. (Lamiaceae) and Rosmarinus sp. (Labiatae) to effectively protect cells from ⁇ A insult.
  • the extract is obtained by pharmacologically acceptable solvent that is comprised of but not limited to methanol, ethanol, isopropyl alcohol, butanol etc.
  • the natural product compounds were synthetically prepared. It is necessary and cost efficient to chemically synthesize the compounds in order to perform a thorough bioassay because only a small amount of these compounds are available from the natural sources.
  • the biological activities of the synthesized natural product compounds were identical to those of the natural product compounds isolated from the plants.
  • a series of natural product analogues that protect cells from ⁇ A peptide insult as effectively as the isolated natural product compounds were also synthesized.
  • compositions comprising one or more compounds of the invention and a pharmaceutically acceptable diluent, adjuvant, or carrier are provided.
  • a pharmaceutically acceptable diluent, adjuvant, or carrier are provided.
  • the use of the compounds of the invention for the manufacture of a medicament for treatment of a beta- amyloid protein-induced ocular disease is also disclosed herein.
  • Natural product compounds having the following general formula were isolated from turmeric and were found to protect cells from ⁇ A peptide insult. In addition, several of the natural product compounds described by this general formula were synthetically prepared.
  • R 1 is selected from the group consisting of H, OH, OMe, and OR 50 wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R 1 is selected from the group consisting of H, OH, OMe, and OR 60 wherein R 60 is (CH 2 ) n CH 3 and n is 1-7. More preferably, R 1 is selected from the group consisting of H, OH, and OMe.
  • Ri is selected from the group consisting of H and OMe when the dotted configuration of compound (I) is a double bond
  • Ri is selected from the group consisting of H and OH when the dotted configuration is a single bond
  • R 2 is selected from the group consisting of H, OMe 3 and OR 50 wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R 2 is selected from the group consisting of H, OMe, and OR 60 wherein R 60 is (CHa) n CH 3 and n is 1-7. More
  • R 2 is selected from the group consisting of H and OMe. Even more preferably, R 2 is selected from the group consisting of H and OMe when the dotted configuration of compound (I) is a double bond, and R 2 is H when the dotted configuration is a single bond.
  • Other compounds useful for practice of the invention include those of the formula (II):
  • R 3 is selected from the group consisting of H, OMe, and OR 50 wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R 3 is selected from the group consisting of H, OMe, and OR 6O wherein R 60 is (CH 2 ) I1 CH 3 and n is 1-7. More preferably, R 3 is selected from the group consisting of H and OMe. Even more preferably, R 3 is H.
  • R 4 is selected from the group consisting of H, OH, OMe, and OR 5O wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R 4 is selected from the group consisting of H, OH, OMe, and OR 60 wherein R 60 is (CH 2 )I 1 CH 3 and n is 1-7. More preferably, R 4 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 4 is H when the first dotted configuration of compound (II) is a double bond and the second dotted configuration of compound (II) is a single bond, R 4 is H when both dotted configurations are single bonds, and R 4 is selected from the group consisting of H and OMe when both dotted configurations are double bonds.
  • R 5 is selected from the group consisting of H, OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 5 is selected from the group consisting of H, OH, OMe, OR 60 , and X wherein R 60 is (CHa) n Ch 3 and n is 1- 7, ans X is F, Cl, Br, or I. More preferably, R 5 is selected from the group consisting of H, OH, and OMe. Even more preferably, R 5 is OH.
  • the invention contemplates the use and production of compounds in either tautomeric form, and as a mixture of the two forms.
  • turmeric-related compounds useful in practice of the invention include those of formula (III):
  • Z is a representation of isosteric variation in which Z is selected from O, S, NH, NR 6O , where R 60 is alkyl, alkenyl, or alkynyl.
  • R 6 is selected from the group consisting of OH, OMe, OR 50 , and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 6 is selected from the group consisting of OH, OMe, OR 60 and X wherein R 6 o is (CH 2 ) n CH 3 and n is 1-7 and X is F, Cl, Br, or I. More preferably, R 6 is selected from the group consisting of OH and OMe. Even more preferably, R 6 is OH.
  • R 7 is selected from the group consisting of H, OMe, and OR5 0 wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R 7 is selected from the group consisting of H, OMe and OR 6O wherein R 60 is (CH 2 ) n CH 3 and n is 1-7.
  • R 7 is selected from the group consisting of H and OMe. Even more preferably, R 7 is OMe.
  • R 8 is selected from the group consisting of OH, OMe, OR 50 and X wherein R 50 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I.
  • R 8 is selected from the group consisting of OH, OMe, OR 6 o and X wherein R 60 is (CH 2 ) n CH 3 and n is 1-7, and X is F, Cl, Br, or I. More Preferably, R 8 is selected from the group consisting of OH and OMe. Even more preferably, R 8 is OH.
  • R 9 is selected from the group consisting of H, OMe and OR 50 wherein R 50 is alkyl, alkenyl, or alkynyl.
  • R9 is selected from the group consisting of H, OMe and OR 60 wherein R 6O is (CH 2 ) n CH 3 and n is i -7. More preferably, R 9 is selected from the group consisting of H and OMe. Even more preferably, R 9 is OMe.
  • the second set of compounds useful for practice of the invention include natural compounds which can be extracted on otherwise derived from Ginkgo biloba as well as synthetic Ginkgo biloba compounds including biologically active homologues and analogues of natural Ginkgo biloba compounds which share anti- ⁇ A activity.
  • Such compounds have the formula (IV):
  • R is selected from the group consisting of higher alkyl, higher alkenyl, and higher alkynyl.
  • R is and n is 1-7. Even more preferably, R is selected from the group consisting of and
  • R is also selected from the group consisting of alkyl, alkenyl, and alkynyl; for example;
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of ; for example;
  • the dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • the third set of compounds useful for practice of the invention include natural compounds which can be extracted on otherwise derived from Zingiber sp. (ginger) as well as synthetic ginger compounds including biologically active homologues and analogues of natural ginger compounds which share anti- ⁇ A activity.
  • Such compounds have the formula (V):
  • Rj 0 is selected from the group consisting of OH, OMe, OR', and X wherein R' is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I. More preferably, R 10 is selected from the group consisting of OH, OMe 3 OR", and X wherein R" is (CH 2 ) n CH 3 and n is 1-7, and X is F, Cl, Br, or I. Even more preferably, R 10 is OH.
  • Rj 1 is selected from the group consisting of H, OH, OMe, and OR' wher R' is alkyl, alkenyl, or alkynyl. More preferably, Rn is selected from the group consisting of H, OH, OMe, and OR" wherein R" is (CH 2 ) I1 CH 3 and n is 1-7. Even more preferably, Rn is selected from the group consisting of H and OMe.
  • Rj 2 is selected from the group consisting of alkyl, alkenyl, and alkynyl. More preferably, Ri 2 is selected from the group consisting of and y is 1-9.
  • Ri 2 is selected from the group consisting of
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of; for example;
  • dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • Ri 3 is selected from the group consisting of OH, OMe, OR', and X wherein R 1 is alkyl, alkenyl, or alkynyl, and X is F, Cl, Br, or I. More preferably, Ri 3 is selected from the group consisting of OH, OMe, OR", and X wherein R" is (CH 2 ) n CH 3 and n
  • Ri 4 is selected from the group consisting of H, OH, OMe, and OR 1 wher R' is alkyl, alkenyl, or alkynyl. More preferably, Ri 4 is selected from the group consisting of H, OH, OMe, and OR" wherein R" is (CH 2 ) n CH 3 and n is 1-7. Even more preferably, R] 4 is selected from the group consisting of H and OMe.
  • Ri 5 is selected from the group consisting of alkyl, alkenyl, and alkynyl. More preferably, R 15 is selected from the group consisting of
  • y is 1-9, or having more than one double bond (cis or trans), or triple bond consisting of ; for example;
  • dotted line configuration is optionally a single bond (cis or trans), or a triple bond, wherein the alkyl, alkenyl, and alkynyl group is selected from ethers and/or thioethers or amines; for example;
  • the length of the side chain is important for the expression of biological activity.
  • compounds (11), (12), (13) and (14) the biological activity appears to improve as the compounds' side chain length increases.
  • analogues having different and lengthier side-chains Preferably, shogaol compounds have side chains wherein Ri 2 has five or more carbons. More preferably, Ri 2 has nine or more carbons, and even more
  • Ri 2 has eleven or more carbons.
  • two of the synthesized shogaol analogue compounds, compounds (45) and (50), also effectively protected cells from ⁇ A peptide insult despite the fact that these compounds have different substituents than the ginger-derived natural product compounds.
  • compound (45) differs from the ginger-derived natural product compounds because it has a saturated hydrocarbon side chain
  • compound (50) differs from the ginger-derived natural product compounds because it does not have a methoxy substituent.
  • alkyl refers to a carbon chain having at least two carbons. Preferably, alkyl refers to a carbon chain having between two and twenty carbons. More preferably, alkyl refers to a carbon chain having between two and eight carbons.
  • alkenyl refers to a carbon chain having at least two carbons, and at least one carbon-carbon double bond. Preferably, alkenyl refers to a carbon chain having between two and twenty carbons, and at least one carbon-carbon double bond. More preferably, the term alkenyl refers to a carbon chain having between two and eight carbons, and at least one carbon-carbon double bond.
  • alkynyl refers to a carbon chain having at least two carbon atoms, and at least one carbon-carbon triple bond.
  • alkynyl refers to a carbon chain having between two and twenty carbon atoms, and at least one carbon-carbon triple bond. More preferably, alkynyl refers to a carbon chain having between two and eight carbon atoms, and at least one carbon-carbon triple bond.
  • higher alkyl refers to a carbon chain having at least five carbon atoms.
  • higher alkyl refers to a carbon chain having between five and twenty carbons. More preferably, higher alkyl refers to a carbon chain having between five .
  • higher alkenyl refers to a carbon chain having at least five carbon atoms, and at least one cabon-carbon double bond.
  • higher alkenyl refers to a carbon chain having between five and twenty carbon atoms, and at least one carbon-carbon double bond.
  • higher alkenyl refers to a carbon chain having between five and twelve carbon atoms, and at least one carbon-carbon double bond.
  • higher alkynyl refers to a carbon chain having at least five carbons, and at least one carbon-carbon triple bond.
  • higher alkynyl refers to a carbon chain having between five and twenty carbon atoms, and at least one carbon-carbon triple bond. More preferably, the term higher alkynyl refers to a carbon chain having between five and twelve carbon atoms, and at least one carbon-carbon triple bond.
  • the fourth set of compounds useful for practice of the invention include natural compounds which can be extracted or otherwise derived from Salvia sp. (sage) and Rosmarinus sp. (rosemary) which share anti- ⁇ A activity.
  • Such compounds have the formulas (VII), (VIII) and (IX):
  • the administration of the natural product and natural product analogue compounds of the invention is preferably accomplished with a pharmaceutical composition comprising a therapeutically effective amount of an active compound of the present invention and a pharmaceutically acceptable diluent, adjuvant, or carrier.
  • a compound according to the invention may be administered without or in conjunction with known antibiotics, surfactants, or other therapeutic agents.
  • the pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parentally, intracisternally, intraperitoneally, intraocularly by injection or depot, topically (as by powders, ointments, or drops), intraocularly, bucally, intranasally, or by any other effective route of administration.
  • ⁇ A protein-induced disease is treated in a subject, such as a human or lower mammal, by administering to the subject a therapeutically effective amount of an active compound of the invention in such amounts and for such time as is necessary to achieve the desired results.
  • a subject such as a human or lower mammal
  • administering to the subject a therapeutically effective amount of an active compound of the invention in such amounts and for such time as is necessary to achieve the desired results.
  • beta- Amyloid protein-induced disease refers to disease states that are characterized by the formation and aggregation of beta- Amyloid protein or beta- Amyloid peptide fibril deposits or plaques, such as, for example, Alzheimer's disease, Down's syndrome, age-related macular degeneration (AMD) and glaucoma.
  • AMD age-related macular degeneration
  • the methods for treatment in accordance with the invention encompass the treatment of subjects wherein the ⁇ A protein-induced disease process is ongoing but wherein the subjects do not exhibit manifest outward symptoms
  • the methods for treatment of the present invention contemplate not only treating the common symptoms associated with ⁇ A protein-induced diseases but also treating the pathology of the disease.
  • the methods for treatment provided herein include treating symptoms associated with ⁇ A protein-induced diseases, such as, for example, the memory loss and dementia associated with Alzheimer's disease, but also include preventing senile plaque formations, and/or clearing such formations.
  • the methods of the invention are contemplated to be useful in treating the symptoms associated with ⁇ A- induced ocular diseases such as glaucoma and AMD and further treat the pathology of those diseases.
  • the term "therapeutically effective amount” means that amounts of a compound of the present invention sufficient to alleviate, ameliorate, prevent, and/or clear the symptoms and/or the pathology of ⁇ A protein-induced disease are contemplated for administration. Accordingly, the methods for treatment of AD in accordance with the invention contemplate administration of an active compound of the invention whether ⁇ A protein-induced disease- like symptoms are manifest, or not.
  • the total daily dose of natural product compound (6) of this invention to be administered to a human or other mammal is preferably between 1 to 200 mg/kg body weight. More preferably, the total daily dosage is between 20 to 160 mg/kg body weight. Even more preferably, the total daily dosage is between 40 to 100 mg/kg body weight.
  • One skilled in the art could obtain preferred dosage ranges for the other compounds of the ⁇ invention by extrapolating from the compounds' ED 50 values, such as, for example the ED 50 values presented in Tables 1, 2, 3, and 4. It will be understood that the total daily usage of the compounds and " compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the severity and progression of the disease, the time of administration, the route of administration, the size of the subject, the rate of excretion of the specific compound employed, the duration of the treatment, the additional therapeutic agents used in
  • the mechanism of action of the natural product compounds and the natural product analogue compounds of the invention appears to involve both antioxidant and non-antioxidant pathways. Without intending to be bound by a theory of the mechanism of the invention, it is believed that the compounds and compositions of the invention provide therapeutic and preventive agents that protect neurons from ⁇ A peptide insult by (1) an antioxidant pathway, (2) preventing the aggregation of ⁇ A peptide by directly binding to ⁇ A peptide, thereby altering its structural conformation and rendering it non-toxic, and/or (3) binding to a receptor site on the cell, thereby altering the cell function in such a way that it is protected from ⁇ A peptide toxicity.
  • potent anti-AD natural product compounds that protect cells from ⁇ A peptide-induced toxicity were isolated from turmeric by following bioassay-guided fractionation schemes. Briefly, ground turmeric was extracted with 90% methanol overnight (2x), and the solvent was removed under vacuum at 35°C. The residue was partitioned between petroleum ether/water, dichloromethane/water, and ethyl acetate/water, successively. After removing the solvents under vacuum at 35 0 C, the residues from each partition were screened for inhibitory activity against ⁇ A peptide-induced cytotoxicity using the MTT assay described in this example.
  • the active principles were isolated from the residues of the active fractions by a series of column chromatography using various resins (Amberchrom non-ionic resin and silica gel) and semi-preparative HPLC reverse-phased separation (isopropyl alcohol/water or acetonitrile/water solvent system).
  • Six curcuminoids, natural product compounds (1), (2), (3), (4), (5), and (6) were isolated from turmeric, and their structures were elucidated using NMR (1-D and 2-D 1 H, 13 C, APT, HMBC) and mass spectrum analysis. These compounds are shown in FIG. 1.
  • the inhibitory activity of the residues and of the identified compounds was determined by observing the differences in the cell viability of ⁇ A peptide (both 25-35 and 1-42) treated cells, ⁇ A peptide (both 25-35 and 1-42) treated cells further including a compound according to the invention, and a DMSO control.
  • the degree of ⁇ A insult was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction assay.
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide
  • ⁇ A peptide-induced toxicity inhibitory effects of the compounds were tested on PC12 cells.
  • the cells were incubated with ⁇ A peptide (25-35) (1.0 ⁇ g/ml, made from 1.0 mg/r ⁇ l stock solution in DMSO) or ⁇ A peptide (1-42) (2.0 ⁇ g/ml, made from 1.0 mg/ml stock solution in DMSO) and the test compounds at various concentrations (25, 5.0, 1.0, and 0.2 ⁇ g/ml) in collagen-coated 96-well tissue culture plates for 24 hours.
  • the ⁇ A peptide-induced toxicity inhibitory effect of the compounds was determined by colorimetrically and microscopically evaluating the PC12 cells' potential to reduce MTT against a positive control (1% DMSO only) and a negative control (1.0 ⁇ g/ml ⁇ A peptide in 1% DMSO alone). Cells were incubated in MTT solution (5 mg/ml) at 37°C for 2 hours. During this time, cells were observed under a microscope every 15 min. Cells were incubated in Lysing buffer (100 ⁇ l) overnight at 37°C. Colorimetric determination of MTT reduction was made at 550 nm. The ⁇ A peptide-induced cytotoxicity inhibitory activity of the compounds was also evaluated against IMR32, HUVEC, and primary cortical rat neuronal cells.
  • PC 12 rat pheochromocytoma and IMR32 human neuroblastoma cells were obtained from the American Type Culture Collection (ATCC).
  • HUVEC normal umbilical human vein endothelial cells were obtained from Clonetics (San Diego, CA). Cells were routinely cultured on a tissue culture plate (Corning, New York, NY).
  • PC 12 cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM), 10% horse serum, 5% fetal calf serum, and 1% penicillin/streptomycin.
  • IMR32 cells were grown in 90% DMEM and 10% fetal calf serum with 1% penicillin/ streptomycin.
  • HUVEC cells were grown in EGM-2 Bullet Kit (Clonetics, San Diego, CA). For the bioassay using ⁇ A peptide(25-35)
  • IMR32 and HUVEC cells were chosen to confirm and supplement the anti- ⁇ A peptide activity of the compounds identified by the assay using PC 12 cells.
  • ⁇ A peptide has been reported to be cytotoxic to IMR32 and endothelial cells.
  • Dissociated primary neuronal cell cultures were established from 18-day-old Sprague-Dawley rat fetuses. The pups were delivered by caesarean section while the dam was anesthetized with ether. Hippocampal tissue from embryonic day 18 Sprague-Dawley rat pups was dissected and then rinsed in cold Ca2 + /Mg2 + ⁇ free Hank's balanced salt solution supplemented with 20 mM HEPES, 4.2 mM sodium bicarbonate, ImM pyruvate, and 3 mg/ml bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • fetal bovine serum FBS
  • DMEM fetal bovine serum
  • the cell pellets were resuspended in serum-free DMEM (pH 7.3), supplemented with 2.4 mg/ml BSA and a modification of Brewer's Bl 6 defined components (with 250 nM vitamin B12 and without catalase, glutathione, and superoxide dismutase). Cells were plated at a density of 15,000 cells/cm ⁇ and grown at 37°C.
  • the serum-containing medium was replaced by defined medium with DMEM/F12 containing bovine transferrin (100 ⁇ g/ml), bovine insulin (5 ⁇ g/ml), putrescine (0.1 mM), progesterone (10 nM), sodium selenite (30 nM), sodium pyruvate (1 mM), and potassium bicarbonate (15
  • the inhibitory activity of the turmeric-derived natural product compounds (1), (2), (3), (4), (5), and (6) (shown in FIG. 1) against ⁇ A peptide-induced toxicity was measured by the MTT reduction assay described in example 1.
  • These six turmeric-derived curcuminoids protected PC 12, EVIR32, and HUVEC cells from ⁇ A peptide-induced toxicity (Table 1). These compounds also protected primary cortical neuronal cells at 5 ⁇ g/ml against ⁇ A peptide(l-42) insult (10 ⁇ g/ml).
  • ED 50 values reflect the results from the MTT assay, and represent the sample concentration that is required to achieve 50% cell viability, a mid-point between the positive control values and the negative control values.
  • the samples that gave values as determined by the MTT assay less than or equal to that of ⁇ A peptide treated wells were considered cytotoxic or without desired activity, and are labeled "toxic".
  • lactate dehydrogenase activity released to the extracellular bathing media was also used to assess cell viability in cell culture.
  • LDH activity in the medium was measured. See Kimura et al., Brain Res 1047, 72 (2005) and Loudina et al., Exp Neurol 184, 923 (2003). This assay was used to confirm the ED 50 results obtained in the MTT assay.
  • IC50 values reflect the results of the antioxidant assay described in this example, and represent the sample concentration which is required to scavenge 50% of the
  • DPPH l-Diphenyl-2-picrylhydrazyl
  • the samples were prepared in various concentrations (200, 20, 2.0, and 0.2 ⁇ g/ml) by serial dilution of a stock solution (5 mg/ml) and were tested by the following procedure.
  • Reaction mixtures containing test compounds (dissolved in DMSO) and 300 ⁇ M DPPH ethanolic solution in 96-well microtiter plates were incubated at 37°C for 30 min. and abs ⁇ rbance was measured at 515 nm.
  • Percent inhibition by sample treatment was determined by comparison with a DMSO-treated positive control group.
  • IC 50 values were determined from percent inhibition by sample. IC 50 values denote the concentration of the tested compound that was required to scavenge 50% of the DPPH free radicals.
  • the antioxidant potency of the natural product compounds was evaluated by measuring the compounds' ability to scavenge free radicals in order to elucidate the possible involvement of antioxidant pathways in the compounds ability to protect the cells (Tables 1 and 2). The results showed that only compounds (1) and (2) have strong antioxidant activity, suggesting that the compounds of the invention may be protecting cells from ⁇ A peptide insults through a mechanism that does not involve an antioxidant pathway.
  • curcuminoids and curcuminoid analogues were synthesized.
  • TMS protected acyl chloride, compound (20), dissolved in THF was added.
  • the reaction mixture was stirred at -78 0 C for 15 minutes and slowly warmed to room temperature.
  • the reaction mixture was quenched with water and poured into ethyl acetate.
  • the organic layer was washed three times with water and the water layer was back washed (2x) with ethyl acetate.
  • the organic layers were combined, dried (MgSO 4 ), filtered, and the solvent was removed under vacuum.
  • the residue was stirred in methanol in the presence OfK 2 CO 3 for 30 min to remove TMS protection.
  • the solution was acidified with 2N HCl and poured into ethyl acetate.
  • Natural product compound (6) was synthetically prepared according to the procedure shown in FIG. 4. The alcohol functionalities of acetol, compound (29), and
  • the inhibitory activity of the synthetic curcuminoid analogues against ⁇ A peptide-induced toxicity was measured by the MTT reduction assay described in example 1.
  • Synthesized compounds (1), (3), (4), and (9) shown in FIG. 5 protected the cells from ⁇ A peptide insult (Table 2).
  • Microscopic analyses of ⁇ A peptide treated cells further including synthesized curcuminoid compounds (3) and (4) also demonstrated that these compounds effectively protect cells from ⁇ A peptide insults.
  • the positive control and cells treated with compounds (3) and (4) maintained MTT formazan granules in the cytosole, a sign of viable cells, while the negative control showed extensive MTT formazan spike processes, a sign of nonviable cells.
  • the inhibitory activity of natural product compounds (11), (12), (13), and (14) (shown in FIG. 6) against ⁇ A peptide-induced toxicity was measured by the MTT reduction assay set forth in example 1.
  • These natural product compounds effectively protected PC 12, IMR32, and HUVEC cells from ⁇ A peptide-induced toxicity (Table 2).
  • the results of the MTT assay were confirmed by the LDH methodology set forth in example 2.
  • shogaols and their analogues were successfully synthesized in 100 mg scale.
  • Gingerols were synthesized from zingerone by conversion into the corresponding O-trimethylsilyl ether, deprotonation with lithium bis(trimethylsilyl)amide or lithium diisopropylamide (LDA), and regioselective aldol condensation.
  • Shogaols are gingerol analogues with a 4, 5-double bond, resulting from the elimination of the 5-hydroxy group.
  • the active principles were isolated from the residues of active fractions by a series of column chromatography using various resins (Amberchrom non-ionic resin and silica gel) and semi-preparative HPLC reverse-phased separation (isopropyl alcohol/water or acetonitrile/water solvent system).
  • resins Amberlite non-ionic resin and silica gel
  • HPLC reverse-phased separation isopropyl alcohol/water or acetonitrile/water solvent system.
  • the structures of the compounds were elucidated using
  • the inhibitory activity of natural product compounds derived from ginkgo biloba against ⁇ A peptide-induced toxicity was measured by MTT reduction assay.
  • the two ginkgo biloba- derived natural product compounds that do not possess antioxidant properties, compounds (15) and (16), were found to protect PC12, IMR32, and HUVEC cells from ⁇ A peptide-induced toxicity.
  • the results of the MTT assay were confirmed by following the LDH methodology set forth in example 2. This example
  • FIG. 11 The benzoic acid, compound (60) and an alkyne having a terminal carbon-carbon triple bond, R, are treated with tetrakis(triphenylphosphine)palladium, in the presence of diisopropyl amine and copper(I) iodide to yield an alkyne substituted benzoic acid, compound (61).
  • Compound (61) is treated with LDA in THF, the temperature is lowered to -7 ⁇ °C and the reaction mixture is treated with oxodiperoxymolybdenum (pyridine)-(hexamethylphosphoric triamide) (MoOPh) to yield a hydroxy functionalized product, compound (62).
  • Compound (62) is then reacted with hydrogen gas over a palladium/carbon catalyst, and treated with acetic acid to yield the desired ginkgolic acid product, compound (63).
  • vitamin A As a control study, vitamin A, ⁇ - carotene, vitamin C, and vitamin E were tested for both anti- ⁇ A peptide(25-35) and anti- ⁇ A peptide(l-42) activity. Since these vitamins are suggested for the delaying the onset of AD, the biological activity of the compounds of the invention were compared with these vitamins. Under the experimental conditions, these vitamins did not protect PC 12 cells from ⁇ A peptide insults even at 200
  • the compounds of the invention were evaluated to ascertain if their antioxidant potency was increased when administered in conjunction with reduced glutathione.
  • the synergistic interaction between estrogens and the intracellular antioxidant, reduced glutathione (GSH) was reported to protect neurons from ⁇ A peptide-induced toxicity. See Barkats et al., JNeurochem 75, 1438 (2000) and Muller et al., J Neurochem 68, 2371 (1997).
  • the possible involvement of this mechanism was evaluated using PC 12 cells with the compounds of the invention.
  • the dose of GSH used in this study was comparable to the low micromolar GSH (3.25 ⁇ M) concentrations found in the cerebrospinal fluid and used by Green et al.
  • the ability of the compounds of the invention to pass through the blood brain barrier was measured.
  • the ability of compounds to cross the blood brain barrier is represented by the log of the partition coefficient (P) of a molecule of the invention between water and octane alcohol.
  • Natural product compounds (1) and (3) were found to have log P values of 3.4 and 3.1, respectively. Accordingly, the octane alcohol fraction contained more than 1000 times as much of the compounds as the water fraction.

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Abstract

La présente invention concerne des procédés pour le traitement de maladies oculaires induites par les protéines bêta-amyloïdiennes, et notamment le glaucome et la dégénérescence maculaire liée à l'âge, des composés et compositions pharmaceutiques à cet effet, et l'utilisation de ces composés pour la fabrication d'un médicament à cet effet. L'invention concerne plus particulièrement l'utilisation de composés de produits naturels tirés des espèces Curcuma, Zingiber, Ginkgo biloba, Salvia, et Rosmarinus, et de certains de leurs analogues synthétiques, pour le traitement de maladies oculaires induites par les protéines bêta-amyloïdiennes.
PCT/US2006/023197 2005-06-15 2006-06-14 Procedes pour le traitement de maladies oculaires induites par les proteines beta-amyloidiennes WO2006138399A1 (fr)

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EP06773171A EP1895849A4 (fr) 2005-06-15 2006-06-14 Procedes pour le traitement de maladies oculaires induites par les proteines beta-amyloidiennes
CA002611490A CA2611490A1 (fr) 2005-06-15 2006-06-14 Procedes pour le traitement de maladies oculaires induites par les proteines beta-amyloidiennes

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US11/287,080 2005-11-23
US11/287,080 US7728043B2 (en) 1999-10-22 2005-11-23 Methods for treatment of beta-amyloid protein-induced ocular disease

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2950804A1 (fr) * 2009-10-01 2011-04-08 Oreal Composition, utilisation et procede de conservation
EA027726B1 (ru) * 2014-07-01 2017-08-31 Мухаммед Маджид Синтез калебина-а и его биологически активных аналогов
CN110229056A (zh) * 2019-06-21 2019-09-13 天津科技大学 一种新型姜黄素类似物及其制备方法和应用
WO2023281476A1 (fr) * 2021-07-08 2023-01-12 Neuracle Science Co., Ltd. Inhibiteurs et leurs utilisations
WO2023095001A1 (fr) * 2021-11-23 2023-06-01 Neuracle Science Co., Ltd. Molécules mimétiques de la famille lrrc4 et utilisations diagnostiques associées

Citations (1)

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Publication number Priority date Publication date Assignee Title
US20040101578A1 (en) * 2001-08-03 2004-05-27 Min-Young Kim Compositon containg ginkgo biloba that inhibit angiogenesis and matrix metalloprotinase

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Publication number Priority date Publication date Assignee Title
WO2001030335A2 (fr) * 1999-10-22 2001-05-03 The Board Of Trustees Of The University Of Illinois Compositions pharmaceutiques utiles dans la prevention et le traitement d'une maladie induite par la proteine beta-amyloide

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101578A1 (en) * 2001-08-03 2004-05-27 Min-Young Kim Compositon containg ginkgo biloba that inhibit angiogenesis and matrix metalloprotinase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1895849A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2950804A1 (fr) * 2009-10-01 2011-04-08 Oreal Composition, utilisation et procede de conservation
FR2950806A1 (fr) * 2009-10-01 2011-04-08 Oreal Composition, utilisation et procede de conservation
WO2011039444A3 (fr) * 2009-10-01 2011-05-26 L'oreal Composition, utilisation et procédé de conservation
EA027726B1 (ru) * 2014-07-01 2017-08-31 Мухаммед Маджид Синтез калебина-а и его биологически активных аналогов
CN110229056A (zh) * 2019-06-21 2019-09-13 天津科技大学 一种新型姜黄素类似物及其制备方法和应用
CN110229056B (zh) * 2019-06-21 2022-11-08 天津科技大学 一种新型姜黄素类似物及其制备方法和应用
WO2023281476A1 (fr) * 2021-07-08 2023-01-12 Neuracle Science Co., Ltd. Inhibiteurs et leurs utilisations
WO2023095001A1 (fr) * 2021-11-23 2023-06-01 Neuracle Science Co., Ltd. Molécules mimétiques de la famille lrrc4 et utilisations diagnostiques associées

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EP1895849A4 (fr) 2009-11-11
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