CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims benefit to U.S. provisional application serial no. 61/752,214 filed January 14, 2013, the entire contents of which are incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to antioxidant flavonoids, compositions containing such compounds, and methods of using such compounds and compositions to prevent, treat or ameliorate macular degeneration and other diseases or conditions associated with oxidative damage. The present invention also relates to methods of making such antioxidant flavonoids. More particularly, the present invention relates to methods of making an antioxidant flavonoid containing water-solubilizing amine groups by reacting quercetin or a quercetin glycoside with an amine under Mannich reaction conditions.

BACKGROUND OF THE INVENTION

[0003] The macula is located at the back of the eye in the center of the retina. When the millions of cells in this light-sensitive, multilayer tissue deteriorate central vision is lost along with the ability to perform tasks such as reading, writing, driving, and seeing color. This "macular degeneration" principally affects the elderly and occurs in three out of ten people over the age of 75. In younger populations, macular degeneration is found in individuals with genetic disorders, such as Stargardt, Vitelliform or Best (VMD), Sorsby's Fundus Dystrophy and Malattia Leventinese (Doyne Honeycomb or Dominant Radial Drusen) (1 ). Stargardt Disease is the most common form of inherited juvenile macular degeneration, affecting about 1 in 10,000 children.

[0004] Age related macular degeneration (AMD) is the leading cause of blindness in the United States, leads to photoreceptor cell death and loss of vision. There are no therapies currently available for genetic or dry (non- neovascular) age related macular degeneration. Vitamin supplements and diet changes, such as low fat diets, have been shown to slow disease progression in some clinical studies. However, for the majority of patients, diagnosis is followed by the progressive loss of central vision.

[0005] The above listed macular dystrophies are all marked by the accumulation of lipofuscin, fluorescent deposits, in the retinal pigment epithelium (RPE) cell layer (2- 5). Lipofuscin is a mixture of chromophores, including A2E, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. A2E has been shown to cause RPE cell death, which is thought to lead to photoreceptor cell degeneration and vision loss (6, 7).

[0006] Photooxidation of A2E is linked to RPE cell damage. RPE cells are particularly susceptible to damage by wavelengths within the blue region of the visible spectrum (8, 9). A2E also has a strong absorption in the blue region, and blue-light irradiation of A2E leads to the photooxidation of A2E such that oxygens are added to carbon-carbon double bonds along the side arms of the molecule (10, 1 1). Furthermore, A2E-loaded RPE cells have increased susceptibility to blue-light damage (12). Blue-light irradiation was also found to enhance A2E-mediated damage to mitochondria (13).

[0007] A2E is believed to mediate light damage via various mechanisms. First, A2E can photochemically initiate free-radical reactions (14) and can produce superoxide and peroxyl radicals called reactive oxygen species (ROS) (15). In addition, blue light can chemically modify A2E molecules, and these chemically modified A2E molecules may subsequently be toxic in the dark, because photo- oxidized A2E molecules damage RPE cells in the dark (16).

[0008] Plant-derived anthocyanins may protect against A2E photo-oxidation in retinal pigment epithelial cells (17). Anthocyanins are present in fruits such as blueberries, which contain the common flower petal pigment and which are marketed as herbal remedies for AMD. However, anthocyanin-derived antioxidants are not commercially practical because, inter alia, the anthocyanins are difficult to secure in large quantities and are unstable, and hence not suitable as starting compounds for various chemical modification and derivatization reactions.

[0009] In view of the foregoing, oxidation of A2E in RPE cells may contribute to a decline in cell function followed by vision loss. Accordingly, it would be advantageous to prevent or to provide treatments for AMD and other forms of macular degeneration. In particular, it would be advantageous to provide new chemically stable and readily synthesized compounds and compositions containing water-solubilizing amine groups that may be used to alleviate the effects of diseases or conditions associated with oxidative damage, e.g., by treating, preventing or ameliorating the oxidation damage to A2E molecules in the RPE cells of the retina.

SUMMARY OF THE INVENTION

[0010] One embodiment of the present invention is a method for making an antioxidant flavonoid comprising reacting quercetin or a quercetin glycoside under Mannich reaction conditions sufficient to link a water solubilizing amine group to the quercetin or quercetin glycoside.

[0011] Another embodiment of the invention is a method for making an antioxidant flavonoid comprising:

(a) reacting a compound of formula (I):

wherein R1 is OH or a glycoside under conditions sufficient to link a water- solubilizing amine group to the compound of formula (I).

[0012] A further embodiment of the present invention is a method for treating age-related macular degeneration (AMD) comprising administering to a patient in need thereof an amount of an antioxidant flavonoid made according to the any of the preceding methods.

[0013] Yet another embodiment of the invention is an antioxidant flavonoid of the formula (II):

R1 is OH or a glycoside;

R is -CHR4NR2R3;

R2 and R3 are independently selected from H, C1 -C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C1 - C6 heteroalkyl, C6 - C14 aryl, C1 - C9 heteroaryl, C3- C8 cycloalkyl, C3 - C8 cycloakenyl, and C3 - C8 cycloheteroalkenyl, each of which may or may not be substituted; or R2 and R3 together with the nitrogen atom to which they are attached join to form a 3 - 7 membered heterocycle, which may or may not be substituted and may or may not be fused to another aryl ring and which may be saturated or unsaturated and which may or may not contain additional heteroatoms; R2, R3 or the 3 - 7 membered heterocycle that R2 and R3 may form may be substituted with quercetin or substituents containing quercetin.

R4 is independently selected from H, C1 -C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C1 - C6 heteroalkyl, C6 - C14 aryl, C1 - C9 heteroaryl, C3-C8 cycloalkyl, C3 - C8 cycloakenyl, and C3 - C8 cycloheteroalkenyl, each of which may or may not be substituted.

[0014] Yet another embodiment of the invention is a pharmaceutical composition comprising a carrier and an antioxidant flavonoid according to the present invention.

[0015] Yet another embodiment of the invention is a compound having a structure selected from the group consisting of:

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0016] Yet another embodiment of the invention is a pharmaceutical composition comprising a carrier and compound selected from the group consisting of:

or an N-oxide crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0017] A further embodiment of the invention is a method for treating age- related macular degeneration (AMD) comprising administering to a patient in need thereof an amount of a pharmaceutical composition according to the present invention, which amount is sufficient to reduce oxidation of A2E in the patient's retina.

[0018] Another embodiment of the invention is a method of modulating A2E. This method comprises providing a photooxidation reducing amount of a compound of formula II to, e.g. a cell or a patient, which may be a mammal, preferably a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a reaction scheme showing the synthesis of 8,8'- (piperazine-1 ,4-diylbis(methylene))bis(2-(3,4-dihydroxyphenyl)-3,5,7-trih ydroxy-4H- chromen-4-one (compound 2)

[0020] Figure 2 is a reaction scheme showing the synthesis of 8,8'-(Ethane- 1 ,2-diylbis(methylazanediyl))bis(methylene)-bis(2-(3,4-dihydr oxyphenyl)-3,5,7- trihydroxy-4H-chromen-4-one) (compound 3)

[0021] Figure 3 is a reaction scheme showing the synthesis of 2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxy-8-(morpholinomethyl)-4/-/- chromen-4-one

(compound 4).

[0022] Figure 4 is a reaction scheme showing the synthesis of 8-((6,7- Dihydroxy-3,4-dihydroisoquinolin-2(1 H)-yl)methyl)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxy-4H-chromen-4-one (compound 5).

[0023] Figure 5 contains bar graphs showing the protective effects of antioxidants of this invention on the photooxidation of A2E. The graph of Figure 5A shows the inhibition of in vitro (noncellular) A2E photooxidation by quercetin (1 ) and compounds 2, 3, 4, and 5 at 100 μΜ. The graph of Figure 5B shows the inhibition of in vitro (noncellular) A2E photooxidation upon varying the concentrations of antioxidants 2, 3, 4, 5, and quercetin (1 ). The graph of Figure 5C shows the cellular (RPE cells accumulated antioxidant from a 20 μΜ concentration in medium) inhibition of A2E photooxidation by compounds 2, 3, 4, 5, and quercetin (1 ). DETAILED DESCRIPTION OF THE INVENTION

[0024] One embodiment of the present invention is a method of making an antioxidant flavonoid. This method includes reacting quercetin (compound 1 ) or a quercetin glycoside under conditions sufficient to link an amine containing group to the quercetin or quercetin glycoside. In the present invention, "link" or "linking" preferably means covalently bonded.

[0025] In this embodiment, the reacting step includes contacting 2 equivalents of quercetin (1 ) with 2 equivalents of formaldehyde and one equivalent of piperazine in ethanol to form compound 2:

[0026] In another embodiment, the reacting step includes contacting 2 equivalents of quercetin (1 ) with 2 equivalents of formaldehyde and one equivalent of A/ ¹ ,/V ² -dimethylethane-1 ,2-diamine in ethanol to form compound 3:

[0027] In another embodiment, the reacting step includes contacting quercetin (1 ) with formaldehyde and morpholine in ethanol to form compound 4:

[0028] In another embodiment, the reacting step includes contacting quercetin (1 ) with 2 equivalents of formaldehyde and dopamine in ethanol to form compound 5:

[0029] In another embodiment of the invention, a method is provided for making an antioxidant flavonoid. This method includes reacting a compound of formula (I):

wherein R1 is OH or a glycoside under conditions sufficient to link an amine containing group to the compound of formula (I).

[0030] Preferably, the antioxidant flavonoid formed by this method is purified. The purification step or steps may be any conventionally know purification step, including those set forth in the examples.

[0031] In another embodiment of the invention, a method is provided for making an antioxidant flavonoid. This method includes reacting a compound of formula (I): wherein R1 is OH under conditions sufficient to link an amine to the compound of formula (I), particularly, e.g., at the moiety pendant at R.

[0032] In the present invention, the glycone (sugar) portion of the glycoside may be any physiologically acceptable sugar molecule that can be incorporated with an aglycone to form a glycoside and participate in the reactions described herein to form the antioxidant flavonoids of the present invention. For example, the glycone (sugar) portion of the glycoside may be selected from aldopentose, aldohexose, aldotetrose, ketopentose and ketohexose. Preferably, the glycone (sugar) portion of the glycoside is selected from ribose, arabinose, xylose, lyase; allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, rhamnose, ribulose, xylulose, erythrose, threose, psicose, fructose, sorbose, and tagatose. [0033] Preferably, the antioxidant flavonoid is selected from:

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0034] In another embodiment of the invention, there is provided a method for treating age-related macular degeneration (AMD). This method includes administering to a patient in need thereof an amount of an antioxidant flavonoid made according to any of the methods of the present invention. In this method, the antioxidant flavonoid is as previously defined.

[0035] Preferably, the antioxidant flavonoid is administered as part of a pharmaceutical or a nutraceutical composition. Preferably, the antioxidant flavonoid derivative is administered as a unit dosage form. [0036] Another embodiment of the present invention is a pharmaceutical composition that includes a carrier and one or more antioxidant flavonoids according to the present invention.

[0037] In another embodiment of the invention, there is provided a compound having a structure selected from:

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0038] In a further embodiment of the present invention, there is provided a method for treating age-related macular degeneration (AMD). This method includes administering to a patient in need thereof an amount of a pharmaceutical composition according to the present invention, which is amount sufficient to reduce oxidation of A2E in the patient's retina. [0039] As noted above, the compounds and compositions of the present invention may be used to treat, prevent, or ameliorate diseases and/or conditions associated with oxidation. Non-limiting examples of such diseases/conditions associated with oxidation include injury to the brain, such as a stroke; neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis; cancer; heart disease; prostasis; inflammation; allergies; respiratory diseases such as bronchitis and asthma; and environmental damage caused by, e.g., sun or UV irradiation.

[0040] As noted above, the compositions of the present invention may be administered in a unit dosage form, such as for example, as part of a vitamin supplement or a pharmaceutical formulation. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of animal, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a compound according to the invention will be that amount of the compound, which is the lowest dose effective to produce the desired effect. The effective dose of a compound maybe administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0041] A compound of the present invention may be administered in any desired and effective manner: as a pharmaceutical composition for oral ingestion, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a compound of the present invention may be administered in conjunction with other treatments. A compound or composition of the present invention maybe encapsulated or otherwise protected against gastric or other secretions, if desired.

[0042] While it is possible for a compound of the invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition) or a vitamin supplement. The pharmaceutically acceptable compositions of the invention comprise one or more compounds as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).

[0043] Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0044] The pharmaceutical compositions or vitamin supplements of the invention may, optionally, contain additional ingredients and/or materials commonly used in such pharmaceutical compositions or vitamin supplements. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamidepowder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface- active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

[0045] Pharmaceutical compositions or vitamin supplements suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil- in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

[0046] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type maybe employed as fillers in soft and hard- filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

[0047] Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

[0048] Pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Pharmaceutical compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.

[0049] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active compound may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0050] Pharmaceutical compositions suitable for parenteral administrations comprise one or more compound in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0051] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation or vitamin supplement), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension or amorphous material having poor water solubility.

[0052] The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug may be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

[0053] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0054] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES EXAMPLE 1

Synthesis Of 8,8'-(Piperazine-1 ,4-diylbis(methylene))bis(2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (compound 2)

[0055] To a solution of quercetin dihydrate (200 mg, 0.59 mmol) and piperazine (26 mg, 0.30 mmol) in ethanol (5 mL) was added 37% aqueous formaldehyde (0.044 mL, 0.59 mmol). A precipitate formed slowly over 30 min. After

48 h the reaction mixture was filtered to provide the product as a yellow powder (185 mg, 85% yield): ¹ H NMR (DMSO-d6) δ 2.63 (8H, s, bd), 3.88 (4H, s), 6.19 (2H, s), 6.89 (2H, d, J = 9.0 Hz), 7.58 (2H, dd, J = 2.1 , 9.0 Hz), 7.71 (2H, d, J = 2.1 Hz), 12.52 (2H, s, bd); ^{1 3} C NMR (DMSO-d6) δ 50.9, 52.1 , 98.1 , 99.9, 102.8, 1 15.0, 1 15.7, 1 19.9, 122.2, 135.7, 145.1 , 146.5, 147.7, 153.8, 159.6, 164.0, 176.0; MS m/z MH ⁺ = 715.

EXAMPLE 2

Synthesis of 8,8'-(Ethane-1 ,2-diylbis(methylazanediyl))bis(methylene)- bis(2- (3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4- one) (compound 3).

1 9

[0056] To a solution of quercetin dihydrate (100 mg, 0.30 mmol) and N ,N - dimethylethane-1 ,2-diamine (0.036 mL, 0.33 mmol) in ethanol (2 mL) was added 37% aqueous formaldehyde (0.041 mL, 0.33 mmol). After 48 h the resulting precipitate was collected to provide the product as a yellow powder: H NMR (DMSO-d ₆ ) δ 2.29 (6H, s), 2.86 (4H, s), 3.93 (4H, s), 6.10 (2H, s), 6.86 (2H, d, J =

8.5 Hz), 7.53 (2H, d, J = 8.5 Hz), 7.67 (2H, s), 12.53 (2H, s, bd); ^{1 3} C NMR (DMSO- d ₆ ) 41 .1 , 50.5, 51 .4, 53.9, 79.4, 98.8, 99.6, 101 .8, 1 14.9, 1 15.5, 1 19.9, 122.3, 135.4,

145.4, 146.2, 147.7, 154.3, 160.0, 166.8, 175.6; MS m/z MH ⁺ 717.

EXAMPLE 3

Synthesis of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-8-(morpholinomethyl )-

4H-chromen-4-one (compound 4)

[0057] To a solution of quercetin dihydrate and morpholine in ethanol was added 37% aqueous formaldehyde. The reaction was filtered to provide the product as a solid. EXAMPLE 4

Synthesis of 8-((6,7-Dihydroxy-3,4-dihydroisoquinolin-2(1 H)-yl)methyl)- 2 -(3,4- dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (compound 5).

[0058] To a solution of quercetin dihydrate (100 mg, 0.30 mmol) and dopamine hydrochloride (63 mg, 0.33 mmol) in ethanol (2 ml_) was added triethylamine (0.046 ml_, 0.33 mmol) followed by 37% aqueous formaldehyde (0.025 ml_, 0.33 mmol). After 72 h the reaction was filtered to provide a brown powder (68 mg). Silica gel chromatography (20% MeOH in CH2CI2) provided the product as a yellow solid (38 mg, 43%): ¹ H NMR (DMSO-d6, 300 MHz) δ 2.73 (2H, s, bd) 3.00

(2H, s, bd) 3.79 (2H, s, bd), 4.16 (2H, s, bd), 6.23 (1 H, s), 6.43 (1 H, s), 6.49 (1 H, s), 6.88 (1 H, d, J = 8.4 Hz), 7.55 (1 H, dd, J = 1 .8, 8.4 Hz), 7.74 (1 H, d, J = 1 .8 Hz),

12.56 (1 H, s, bd); MS m/z MH ⁺ 480.

EXAMPLE 5

Quercetin Analogs Reduce Noncellular A2E Photoxidation

[0059] To test the effect of antioxidants on A2E photooxidation, 100 μί of A2E stock (200 μΜ in DMSO) was added to 200 μί of DPBS as described in the literature.^ To these samples was added 1 .5 μί of antioxidant (20 mM in DMSO) to obtain a final concentration of 100 μΜ. These mixtures were irradiated (430 nm; light intensity of 2.8 mW/cm ) for 5 min. Samples were analyzed by reversed-phase HPLC with sample preparation and injection performed under dim red light. To test the effects of varying concentrations of antioxidants on A2E photooxidation, the appropriate volume of antioxidant solution (20 mM in DMSO) was added to the solution prepared by adding 100 μί of A2E stock solution (200 μΜ in DMSO) to 200 μί of DPBS, and the samples were irradiated as described above. Samples were diluted with EtOH (0.5 ml_) and analyzed by reversed-phase HPLC. A2E loss relative to unirradiated samples was interpreted as percent photooxidation.

EXAMPLE 6

Quercetin Analogs Reduce Cellular Photooxidation of A2E

[0060] A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA, USA) that is devoid of endogenous A2E was cultured in 35 mm cell culture dishes (Corning, NY, USA) to confluence for 5 days as previously

fi 1 ft 1 Q

reported. ' The cells were allowed to accumulate synthesized A2E ^σ for 18 days from a 10 μΜ concentration in culture medium that included 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). From day 10 to 18 the cells were also incubated with 20 μΜ antioxidant. Subsequently, the cultures were washed 10 times with DPBS (total 500 μΙ_ volume) and then in DPBS were irradiated at 430 nm (2.8 mW/cm ) for 10 min. The cells were then detached from the culture dish with trypsin, washed, pelleted, and counted three times (Bright Light Counting Chamber, Hausser Scientific Company, Horsham, PA, USA). The cells were dried under vacuum in a desiccator for 2 h following which 100 μί of methanol was added to each vial for homogenization. After centrifugation, 30 μί of supernatant solution was used for HPLC analysis. A2E loss relative to unirradiated samples was interpreted as percent photooxidation.

CITED DOCUMENTS

[0061] The following documents, which have been cited above, are incorporated by reference as if recited in full herein:

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[0062] The scope of the present invention is not limited by the description, examples, and suggested uses herein and modifications can be made without departing from the spirit of the invention. Thus, it is intended that the present invention cover modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.