WO2013130020A1

WO2013130020A1 - Quercetin derivatives, their pharmaceutical composition and use

Info

Publication number: WO2013130020A1
Application number: PCT/SK2013/000002
Authority: WO
Inventors: Milan ŠTEFĚK; Lucia KOVÁČIKOVÁ; Ivana MILÁČKOVÁ; Miroslav Veverka; Emil ŠVAJDLENKA; Eva Veverkovà
Original assignee: Ústav Experimentálnej Farmakológie A Toxikológie Sav
Priority date: 2012-02-29
Filing date: 2013-02-27
Publication date: 2013-09-06
Also published as: SK50062012A3

Abstract

The invention relates to semi-synthetic derivatives of quercetin formula I and pharmaceutically acceptable salts, hydrates and solvates, wherein groups R₁ to R₅, identical or different, are independently H, benzyl, and at least one group is 3-chloro-2,2-dimethylpropanoyl, 4-O-acetylferuloyl, or 2-chloro-1,4-naphthoquinon-3-yl. The invention relates to the use of derivatives of quercetin, the preparation of medicaments for the treatment or prevention of diseases or disorders where oxidative stress and polyol pathway are key etiological factors. The invention also relates to pharmaceutical compositions containing these compounds and their use in the treatment of human and animal health problems in which R₁ to R₅ are independently H, benzyl, and at least one group is selected from 3-chloro-2,2-dimethylpropanoyl, 4-O-acetylferuloyl, or 2-chloro-1,4-naphthoquinon-3-yl.

Description

Quercetin derivatives, their pharmaceutical composition and use

Technical Field

This invention relates to novel semi-synthetic derivates of quercetin of the general formula (I) and pharmaceutically acceptable salts, hydrates, and solvates thereof

in which groups Ri to R₅, identical or different, are independently H, benzyl, and at least one group is selected from 3-chloro-2,2-dimethylpropanoyl, 4-O-acetylferuloyl, or 2-chloro-l,4- naphthoquinon-3-yl. The invention also relates to a pharmaceutical composition that contains quercetin derivatives and their use.

Substances that are the subject of the invention are useful as bifunctional agents combining antioxidant activity with the ability to inhibit aldose reductase, the first enzyme of polyol pathway, for treatment and control of human diseases in which oxidative stress and polyol pathway are key etiological factors, including development of late diabetic complications (macro- and microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy and neuropathy), resistances of hepatic carcinoma to chemotherapy, abnormal cell proliferation of vascular smooth muscles during atherosclerosis and restenosis, inflammatory diseases such as uveitis, sepsis, colorectal cancer, periodontitis, and asthma. The invention also concerns pharmaceutical means containing semi-synthetic derivatives of quercetin of formula I, and therapeutic methods involving the administration to semi-synthetic derivatives of quercetin to mammals. The invention also relates to compounds useful as intermediates in the preparation of semi-synthetic derivatives of quercetin included in this invention.

Background of the Invention

Diabetic patients are susceptible to the development of chronic health complications, tightly connected to hyperglycemia, which are responsible for a significant increase in morbidity and mortality of diabetic patients. Understanding of the mechanisms by which glucose exerts its toxicity is of utmost importance for rational pharmacological interventions to treat diabetic complications (DCs).

The etiology of DCs is multifactorial-multiple hyperglycemia-dependent mechanisms contribute to their development. Oxidative stress and the polyol pathway are considered to have key roles in the etiology of DCs (Brownlee 2005). Under conditions of hyperglycemia, excessive glucose initiates an array of glyco-oxidation changes of the endogenous proteins and glucose becomes a substrate of the aldose reductase, the first enzyme of the polyol pathway (Fig. 1-attachemen). This results in accumulation of advanced glycation endproducts (AGEs), formation of reactive oxygen species and intracellular accumulation of the osmolyte sorbitol [Baynes JW, Thorpe SR. Role of oxidative stress in diabetic Complications: a new perspective on an old paradigm. Diabetes. 48:1-9 (1999); Obrosova IG. Increased sorbitol pathway activity generates oxidative stress in tissue sites for diabetic Complications. Antioxid Redox Signal. 7:1543-1552 (2005); Oates PJ. Aldose reductase, still a compelling target for diabetic neuropathy. Curr Drug Targets. 9:14-36 (2008)]. Depletion of NADPH due to aldose reductase activity reduces intracellular GSH, an endogenous antioxidant, thereby inducing oxidative stress.

Under conditions of diabetes, the need of tight blood glucose control is a key prerequisite to reduce the incidence, progression, and severity of DCs. Yet in the daily regimen of a diabetic patient, periods of hyperglycemia cannot be avoided, with all of the aforementioned deleterious consequences of glucose toxicity. Therefore additional adjunct therapy interfering with the pathological processes at molecular level, e.g. based on antioxidants (AOs), aldose reductase inhibitors (ARIs) and anti-glycation agents, is needed to attenuate the noxious effects of glucose.

Parallel administration of AOs and ARIs can counterbalance the inhibition of the detoxification role of aldose reductase against toxic carbonyl products, significantly enhanced in diabetic tissues [Baynes JW. Role of oxidative stress in development of Complications in Diabetes. Diabetes. 40 (4):405-12 (1991), Thorpe SR, Baynes JW. Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs Aging 9 (2):69-77 (1996), Thornalley PJ. Measurement of protein glycation, glycated peptides, and glycation free adducts. Perit Dial Int. 25 (6):522-33 (2005), Turk Z. Glycotoxines, carbonyl stress and relevance to diabetes and its Complications. Physiol Res. 59 (2): 147-56 (2010)]. In addition, antioxidant activity may suppress processes of advanced glycation (glycoxidation) at the level of free radical intermediate [Brownlee M. The pathobiology of diabetic Complications: a Unifying mechanism. Diabetes 54:1615-1625 (2005), JW Baynes. Role of oxidative stress in development of Complications in Diabetes. Diabetes 40 (4):405-12 (1991), Thorpe SR, Baynes JW.Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs Aging 9 (2):69-77 (1996), GIACC F, Brownlee M. Oxidative stress and diabetic Complications. Circ Res. 107 (9): 1058-70 (2010), Brownlee M. Glycosylation products as toxic Mediators of Diabetic Complications. Annu Rev Med. 42:159-66 (1991), Brownlee M. Negative Consequences of glycation. Metabolism 49 (2 Suppl 1):9-13 (2000)]. In the case of diseases of multifactorial origin, it appears to be clear that modulating a single target, even with a very efficient drug, is unlikely to yield the desired outcome. Innovative„multi-target" strategies are focused on a rational design of chemical entities able to affect simultaneously multiple key mechanisms.^■ This approach increases the chance of successful therapeutic intervention, decreases the risk of side effects and is economical. ARIs possessing AO activity would therefore seem to be desirable.

Examples of the "multi-target" strategy in treatment of diabetic complications are the bifunctional compounds combining the aldose reductase inhibiory activity with the antioxidant effect, including pyrido-pyrimidines [La Motta C, Sartini S, Mugnaini L, Simorini F, Italians S, Salerno S, Marini AM, Da Settimo F, Lavecchia A, Novellino E Cantore M, Failla P Ciuffi M. pyrido (1,2-a) pyrimidin-4-one derivatives as a new class of selective Aldose reductase inhibitors exhibiting antioxidant activity. J Med Chem. 50 (20) :4917-27 (2007)], pyridazines [Coudert P Albuisson E, Boire JY, Duroux E P Bastide, J. Couquelet Synthesis of pyridazine acetic acid derivatives possessing Aldose reductase inhibitor activity and anti-oxidant properties. Eur J Med Chem. 29:471-477 (1994)],, benzopyranes [Constantino L, Rastelli G, Gamberini MC et al. l-benzopyran-4-one antioxidants and Aldose reductase inhibitors. J. Med Chem. 42:1881-1893 (1999)] and carboxymethylated pyridoindoles [Stefek M, Snirc V, Djoubissie PO, Majekova M, Demopoulos V, Rackova L, Bezakova Z, Karasu C, Carbone V, El-Kabbani O. Carboxymethylated pyridoindole antioxidants as Aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling. Bioorg Med Chem. 16:4908-4920 (2008)], compounds combining the aldose reductase inhibiory activity with the ability to attenuate nonenzymatic glycation [Demopoulos VJ, Zaher N, Zika C, Anagnostou C, Mamadou E, Alexiou P, Nicolaou I. Compounds that combine Aldose reductase inhibitor activity and the ability to Prevent glycation (glucation and / or fructation) of proteins as putative Pharmacia- therapeutic agents. "Drug Design Reviews-Online. 2:293-304 (2005)] or compounds combining antioxidant activity with ability to chelate redox reactive metal ions such as iron and copper [Randazzo J, Zhang P, Makita J, Blessing K, Kador PF. Orally Active Multi- Functional Antioxidants Delay Cataract Formation in streptozotocin (Type 1) Diabetic and Gamma-Irradiated Rats. PLoS ONE.6: 4. el8980 (2011)].

Recently, in addition to its involvement in diabetic complications via reducing glucose, aldose reductase was found to efficiently reduce also lipid peroxidation derived aldehydes and their glutathione conjugates [KV Ramana. Aldose reductase: new insights for an old enzyme. Biomat Concepts. 2:103-114 (2011)]. It has been demonstrated that lipid peroxidation derived lipid aldehydes, such as 4-hydroxy-trans-2-nonenal (HNE) and their glutathione conjugates (e.g. GS-HNE), are efficiently reduced by aldose reductase to corresponding alcohols, DHN (1,4-dihydroxy-nonene) and GS-DHN (glutathionyl-1,4- dihydroxy-nonene), which mediate the inflammatory signals. The reduced aldehyde glutathione conjugate GS-DHN is considered a novel signaling intermediate in the transduction of reactive oxygen species-initiated cell signals, leading eventually to inflammation response [Srivastava SK, Yadav UC, Reddy AB, et al. Aldose reductase inhibition suppresses oxidative stress-induced inflammatory disorders. Int. Biol Chem. 191:330-8 (2011)]. Inhibition of aldose reductase was found to prevent significantly the inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens and auto-immune reactions in cellular as well as animal models [Ramana KV, Srivastava SK. Aldose reductase: a novel therapeutic target for inflammatory pathologies. Int J Biochem Cell Biol. 42 (1): 17-20 (2010)].

Inflammatory diseases are the leading causes of morbidity and mortality in populations worldwide. Aldose reductase inhibitors thus present a novel therapeutic approach to treat a wide array of inflammatory diseases such as atherosclerosis, asthma, colon cancer, uveitis, sepsis, arthritis, periodontitis and other injuries that have the potential of stimulating the immune system and generating large amounts of inflammatory cytokines and chemokines [KV Ramana. Aldose reductase: new insights for an old enzyme. BioMol Concepts. 2:103- 114 (2011), Srivastava SK, Yadav UC, Reddy AB, et al. Aldose reductase inhibition suppresses oxidative stress-induced inflammatory disorders. Chem Biol Interaction. 191:330- 8 (2011), Ramana KV, Srivastava SK. Aldose reductase: A novel therapeutic target for inflammatory pathologies. The International Journal of Biochemistry & Cell Biology. 42:17- 20 (2010), Tammali R, Saxena A, Srivastava SK, Ramana KV. Aldose reductase regulates Vascular Smooth Muscle Cell Proliferation by Modulating Gl / S Phase Transition of Cell Cycle. Endocrinology. 151 (5) :2140-2150 (2010), Tammali R, Srivastava SK, Ramana KV. Targeting Aldose reductase for the treatment of cancer. Curr Cancer Drug Targets. 11 (5) :560-571 (2011), PF Kador, JD O'Meara, Blessing K, Marx DB, Reinhardt RA. Efficacy of structurally Diverse Aldose reductase Inhibitors on Experimental Periodontitis in Rats. J Periodontol. 82:926-933 (2011), Yadav UC, Ramana KV, Srivastava SK. Aldose reductase inhibition suppresses airway inflammation. Chem Biol Interact. 191 (1-3) :339-45 (2011), Yadav UC, Srivastava SK, Ramana KV. Understanding the role of Aldose reductase in ocular inflammation. Curr Mol Med. 10 (6) :540-9 (2010)]. Aldose reductase inhibition represents an attractive strategy for future anti-inflammatory therapy. Recently, considerable attention has been devoted to the search for phytochemical therapeutics. A variety of constituents, like vitamins, minerals, fiber, and numerous phytochemicals, including flavonoids, may contribute to the health effects of fruits and vegetables. Indeed, flavonoids are capable of affecting multiple mechanisms or etiological factors responsible for the development of diabetes-related complications including oxidative stress, non-enzymatic glycation and the polyol pathway [Bhimanagouda S, GK Patil, KN Jayaprakash, Chidambaram Murthy, Amit Vikram. Bioactive Compounds: Historical Perspectives, Opportunities, and Challenges. J Agric Food Chem 5:8142-8160 (2009), Majumdar S, Srirangam R. Potential of the bioflavonoids in the prevention / treatment of ocular disorders. J Pharm Pharmacol 62 (8) :951-965 (2010), Kalt W, Hanneken A, Milbury P, Tremblay F. Recent Research on polyphenolics in Vision and Eye Health. J Agric Food Chem 58:4001-4007 (2010), Stefek M. Natural flavonoids as potential multifunctional agents in prevention of diabetic cataract. Interdiscip Toxicol. 4 (2) :69-7 (2011)].

The flavonol quercetin is the most widely consumed flavonoid in the human diet [Boots AW, Haenen GRMM, Bast A. Health effects of quercetin: From antioxidant to nutraceutical. Eur J Pharm. 585:325-337 (2008)]. Quercetin and other flavonoids have been shown to exert protective effects against eye lens opacification [U.S. Patent Publication 2010/0292287 Al, Stefek M, Karasu C. Eye lens in aging and diabetes: effect of quercetin. Rejuvenation Res. 14 (5) :525-534 (2011), Stefek M. Natural flavonoids as potential multifunctional agents in prevention of diabetic cataract. Interdiscip Toxicol. 4 (2) :69-7 (2011)]. In plants, quercetin is present mainly in the form of O-glycosides with a sugar group, such as glucose, galactose, rhamnose, rutinose, or xylose. The glycosidic structure has a large impact on quercetin bioavailability [Arts IC, Sesink AL, Faassen-Peters M, Hollman PC. The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides. Br J Nutr. 91:841-847 (2004), Crozier A, Del Rio D, Clifford MN. Bioavailability of dietary flavonoids and phenolic compounds. Mol Aspects Med. 31:446-467 (2010), Stefek M, Karasu C. Eye lens in aging and diabetes: effect of quercetin. Rejuvenation Res. 14 (5) :525-534 (2011)].

For O-benzyl derivatives of quercetin especially antioxidant effects are declared. U.S. patent U.S. 2,892,846A describes a process for the preparation of 7-O-benzylquercetin, and reports increased antioxidant activity compared to quercetin, data not shown.

Patent GB 1295 606A protects 3,3',4',5,7-O-pentabenzyl quercetin, method of its preparation and its use for the treatment of vascular disorders of patients suffering with diabetic retinopathy, atherosclerosis and other diseases.

Benzyl group is preferably used for the selective protection of the hydroxyl groups of quercetin; e.g. the compound is acylated and benzyl group is subsequently removed e.g. by hydrogenation to give the desired acyl derivatives. These are the synthetic steps with the most frequent variability.

This procedure is used e.g. by U.S. 2011/0034413A1 or U.S. 6,235,294 Bl patents to prepare quercetin esters as cosmetic products with antioxidant properties. U.S. 2002/0106338 Al patent describes quercetin derivatives substituted by mono- to penta- acyl or alkyl groups and their cosmetic and pharmaceutical compositions. The synthesis employs benzyl to protect hydroxyl groups of quercetin.

WO 2009064485 (Al) provides 3 or 7 or 3,7-di-O-propylamino derivatives of quercetin, substituted with ferulic or caffeic acid as effective antioxidant prodrugs; 3 ',4',7 tribenzyl quercetin is used as a source for synthesis of 3 O-propylamino quercetin derivatives.

U.S. 2 892846 A patent describes a quercetin derivative wherein R1-R5 are H, as a starting compound for the preparation of 7-O-benzylquercetin. The document describes a detailed procedure for the preparation of 7-O-benzylquercetin which is characterized with enhanced antioxidant activity in comparison with quercetin.

Document GB 1295606 relates to 3,3',4',5,7-pentabenzyl quercetin, which is a derivative of quercetin, in which R1-R5 are benzyls. The invention describes a process of preparation of 3,3',4',5,7-pentabenzyl quercetin, and its use in the treatment of vascular disorders of patients with diabetic retinopathy, atherosclerosis, chronic glomerulonephritis, and so on. The sequential multistep protection of the different phenolic functions of quercetin using dichlorodiphenylmethane and benzyl bromide gave products in low yield (Bouktaib, M., et al., (2002), Tetrahedron, 58, 10001-10009).

The document U.S. 2011/00344413 Al describes quercetin and its antioxidant activity as well as other biological effects such as vasodilator, antidiabetic, antihistaminic, antiinflammatory efects, immune supporting effect, and other, including the anti-cancer effect. In Example 1 of the patent describes the procedure for synthesis of 3,7-bis-benzyloxy-2-(4- benzyloxy-3 -hydroxyphenyl)-5 -hy droxychromen-4-one .

The document CN 101250176 describes derivatives having flavonoid skeleton, wherein substituents in position 3,3',4',5,7 can independently be represented by hydroxy or benzyloxy groups. The quercetin derivatives described have anticancer effects. 5-O-benzyl quercetin is described in Scheme 6 and its conversion to 3,7-bis-benzyloxy-2-(4-benzyloxy- 3hydroxyfenyl)-5-hydroxy-chrome-4-one.

U.S. 2002/0106338 Al patent describes the 3 ',4',7-tribenzylquecertin (Example 2).

Document U.S. 2003/0055103 Al describes semi-synthetic flavonoids and the method of their preparation. Quercetin and its derivatives are disclosed as effective agents in treating ischemic heart disease and/or brain degenerative disease.

WO 2009064485 Al relates to flavonoids with antioxidant activity, procedure of their preparation and their use. The compounds described are derivatives of quercetin with substituents derived from known antioxidants, located in the position 3, or in position 3 or 7. The compounds are effective in treating neurodegenerative diseases, amyotrophic lateral sclerosis, cancer, heart diseases, inflammatory diseases, asthma and so on.

The document JP 3232851 A describes ability of quercetin to inhibit aldose reductase, and its use for the treatment of complications associated with diabetes such as cataract, retinopathy, kidney disease and neurological disorders.

The document U.S. 2010/0292287 Al reports inhibitory effect of quercetin on aldose reductase and thus its usefulness in the treatment or prevention of diabetic cataracts, neuropathy and other complications associated with diabetes.

The regioselective synthesis of tetra-O-acyl derivatives with the free OH group in position 5 of quercetin was achieved with a high yield by the reaction with ten equivalents of acyl chloride. By careful control of the reaction conditions, it is possible to stop the acylation at the 3,3', 4',7-tetra-O-acyl stage (Mattarei et al., 2010 Molecules, 15, 4722-4736).

Considering pharmaceutical value of derivatives of quercetin, it is apparent that possibilities for optimal modification of quercetin have not been exhausted yet. Derivatives of quercetin may be of lipophilic or hydrophilic nature, depending on the type and position of substituents in the molecule quercetin.

It is important to selectively modify various hydroxyl groups of quercetin since they are not equivalent, neither in the chemical nor bio-functional terms. Further modifications with appropriate substituents, balancing hydrophilic-lipophilic behavior and biological effect, thus provide an alternative to the previously described quercetin derivatives. The inventions US 3,420,815; US 4,202,815; US 5,955,100 and US 6,258,840 describe various chemical modifications of quercetin at the positions 3, 5, 7, 3' and 4' from the perspective of better transport through biological barriers. Surprisingly, it has been shown that quercetin derivatives with 3-chloro-2,2- dimethylpropanoyl, 4-O-acetylferuloyl, or 2-chloro-l,4-naphthoquinon-3-yl group prepared according to the present invention exhibit high inhibitory activity in relation to aldose reductase.

The objective of the invention is to provide new substances with improved inhibitory activity in relation to aldose reductase, increased antioxidant capacity, endowed with high selectivity and optimal bioavailability.

Disclosure of Invention

The present invention relates to derivatives of quercetin of formula (I)

I and pharmaceutically acceptable salts, hydrates and solvates, wherein Ri and R₅ are independently H, benzyl, and at least one group is 3-chloro-2,2-dimethylpropanoyl, 4-O- acetylferuloyl, or 2-chloro-l,4-naphthoquinon-3-yl.

Thus, the present invention relates to derivatives of quercetin of formula I for use in the treatment and control of human diseases in which oxidative stress and polyol pathway factors are key atiological factors to the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy and neuropathy), resistance of hepatic cancer to chemotherapy, abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, inflammatory diseases such as uveitis, sepsis, colon cancer, asthma and periodontitis.

In another aspect the invention relates to the use of derivatives of quercetinof formula I for the preparation of medicaments for the treatment or prevention of diseases or disorders in which oxidative stress and polyol pathway factors are key to the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy and neuropathy), resistance of hepatic cancer to chemotherapy, abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, inflammatory diseases such as uveitis, sepsis, colon cancer, asthma and periodontitis.

The present invention also relates to pharmaceutical compositions comprising at least one compound of formula I and a pharmaceutically acceptable carrier.

According to another aspect of the invention, solid oral formulation or injection form is shown for systemic administration or in the form of drops, pastes, gels or ointments for topical application.

The compounds of the general formula I may be prepared by known alkylation or acylation method per se to the person skilled in the art and are described in standard works such as Houben-Weyl, "Methoden der organischen Chemie", Georg Thieme Verlag, Stuttgart. The conditions under which such acylation reactions with e.g. acyl halogenide are carried out are well known to those skilled in the art; they can be carried in the presence of a base and in the presence of a suitable solvent or alternatively in the presence of an activated carboxylic acid reagent. The acylation reaction can be carried out in conventional manner in the presence of a solvent to allow solubilization of the starting quercetine or selectively benzylated polyphenolic quercetine. This acylation can be carried out on one or more or even all hydroxyl groups of quercetin or benzylated quercetin derivatives (Molecules 2010, 15, 4722).

The quercetin skeleton selectively alkylated in various positions may be obtained by known method (Tetrahedron, 2002, 58, 10001-10009, Beilstein Journal of Organic Chemistry 2009, 5, 60). To allow selective derivatization the acetylation benzylation strategy was adapted (J. Amer. Chem. Soc. 1958, 20, 5531, J. Amer. Chem. Soc.1958, 20, 5527, J. Org. Chem. 1962, 27, 1294). On the other hand, it is well-known the alternative strategies using the direct acylation of quercetin which depends on the relative reactivity of the different hydroxyl position toward an acylating agent gives complex reaction mixtures (US patent 3661890, 6235294B1, WO 200121164) which are easily separated by chromatography based on the different polarity. Thus, occasionally this direct acylation can be used which is exemplified by using 3-chloro-2 ,2-dimethylpropanoyl chloride and by subsequent separation of products.

For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained- release preparations and devices.

The compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile -filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

General experimental procedures

The NMR measurements were performed on a spectrometer VNMRS-600 MHz Varian at temperature 298K and were collected in DMSO-d₆. The working frequency for 1H was 599.773 MHz, for ¹³C was 150.829 MHz. The concentrations of the samples were approximately 50mM. Chemical shifts are given in ppm relative to the residual signal of the solvent. LS/Mass spectra were performed with an Agilent Technologies MSD SL Ion Trap mass spectrometer with ESI APCI source coupled with an 1100 Series HPLC system.

The melting points were measured on an NAGEMA (VEB Muhlenbau Dresden) apparatus and were not corrected.

NADPH, β-mercaptoethanol, D,L-glyceraldehyde, D-glucuronate and sodium valproate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Diethylaminoethyl cellulose DEAE DE 52 was from Whatman International Ltd. (Maidstone, England). Zopolrestat (lot # 43668-12-7F) was supplied as gift samples by Pfizer (Groton, CT, USA). Other chemicals were purchased from local commercial sources and were of analytical grade quality. Total protein in enzyme preparations was determined according to Geiger and Bessman (1972). Male Wistar rats 8 - 9 weeks old, weighing 200 - 230 g, were used as organ donors. The animals came from the Breeding Facility of the Institute of Experimental Pharmacology Dobra Voda (Slovak Republic). The study was approved by the Ethics Committee of the Institute and performed in accordance with the Principles of Laboratory Animal Care (NIH publication 83-25, revised 1985) and the Slovak law regulating animal experiments (Decree 289, Part 139, July 9th 2003). The invention is illustrated by reference to the following examples comprising chemical synthesis procedures, aldose reductase inhibition measurement, selectivity assessment and antioxidant capacity determination of the novel compounds. However, the examples are not intended to limit the scope of the patent claims. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the purpose and interest of this invention.

Best Modes for Carrying out the Invention

Example 1

2-[3-(3-Chloro-2,2-dimethylpropanoyloxy)-4-hydroxyphenyl]-3,5,7-trihydroxychromen-4- one

Step A

3-Chloro-2,2-dimethylpropanoyl chloride (1.05 g) in acetone (30 ml) was added to a mixture of 3,7-bis-benzyloxy-2-(4 '-benzyloxy-3 '-hydroxyphenyl)-5-hydroxy-chromen-4-one (1.0 g, according Tetrahedron, 2002, 58, 10001-10009) and triethylamine (0.69 g) in dry acetone (50 ml). Reaction mixture was stirred for 12 h at 25°C under nitrogen atmosphere. The reaction mixture was filtered and evaporated to dryness to give crude product. 3,7-Bis- benzyloxy-2-(4-benzyloxy-3-(3-Chloro-2,2-dimethylpropanoyloxy)phenyl)-5-hydroxy- chromen-4-one was carefully separated by column chromatography using ethyl acetate/hexane (1:1.5 v/v) as eluent. The required product solidified as off white solid. Yield 1.1 g, m.p. 186-190°C dec.

Step B

To a solution of product from step A (0.8 g) in methanol (80 ml) was added 10% Pd/C on charcoal (0.08 g, 10% w/w). The resulting mixture was shaken under a hydrogen atmosphere (415kPa) for 12 hours at 25 °C. The resultant mixture was filtered over Celite. The filtrate was evaporated to afford crude product. The crude product was purified by flash column chromatography (silica gel) using methylene chloride/methanol (9:0.5, v/v,) as eluent to furnish the title compound. Yield 0.2 g, m.p. 93-100 °C, LC/Mass M⁺ 419, Rf-0.6 (dichloromethane/methanol/formic acid 9:0.5:0.1). The title compound was characterized by IR (v-. cm-i) 3150-3500b, 1732m, 1651s, 1598s, 1134s, elemental analysis, and 1H and ¹³C NMR. The NMR data are included in Table 1.

1 1

Table 1. The H and C chemical shifts, δΗ (J, Hz), of flavone derivative example 1.

Example 2

2-[3,4-bis-(3-chloro-2,2-dimethylpropanoyloxy)-phenyl]-7-(3-chloro-2,2-dimethylpropanoyl- oxy)-3,5-dihydroxychromen-4-one

and 2-(3,4-dihydroxyphenyl)-7-(3-chloro-2,2-dimethylpropanoyloxy)-3,5-dihydroxychromen- 4-one

and 2-[4-(3-chloro-2,2-dimethylpropanoyloxy)-3-hydroxyphenyl]-3,5,7-trihydroxychromen- 4-one

and 2-[3,4-bis-(3-chloro-2,2-dimethylpropanoyloxy)]-3,7-di-(3-chloro-2,2-dimethylpropan- oyloxy)-5 -hydroxychromen-4-one

3-Chloro-2,2-dimethylpropanoyl chloride (4.65 g) in acetone (30 ml) was added to a mixture of quercetine (3.0 g) and DIPEA (4 g) in dry acetone (170 ml) at temperature 5°C. Reaction mixture was stirred for 2 h at 5°C and then 2h at 25°C. The reaction mixture was filtered, evaporated to dryness and washed with water (lOOmL) to give crude product (7.6g). Title products were subsequently separated from an isomer mixture by column chromatography using dichloromethane/methanol (9:0.05 v/v) as eluent. The title 2-[3,4-bis- (3-chloro-2,2-dimethylpropanoyloxy)-phenyl]-7-(3-chloro-2,2-dimethylpropanoyloxy)-3,5- dihydroxy chromen-4-one with Rf-0.71 solidified as off white solid. Yield 0.96 g, m.p. 210-211 °C dec. IR _{(v> cm-1)} 3150-3500b, 1760s, 1647s, 1600s,1158s, 1080s. Rf-0.78 (dichloromethane/methanol 9:0.5). The NMR data are included in Table 2.

Table 2. The 1H and ¹³ C chemical shifts, δΗ (J, Hz). assignment 13_C position δΗ (J, Hz)

2 - 145,13 Q

3 - 138,31 O

4 - 176,88 Q

5 - 160,21 Q

6 6,60 103,87 CH d J₆,8 = 2,l

7 - 155,57 Q

8 7,03 101,24 CH d h,6 = 2,1

The title product 2-(3,4-dihydroxyphenyl)-7-(3-chloro-2,2-dimethylpropanoyloxy)-3,5- dihydroxychromen-4-one with Rf-0.26 (dichloromethane/methanol 9:0.5) solidified off as white solid. Yield 0.6 g, m.p. 173-175 °C dec. The NMR data are included in Table 3.

Table 3. The 1H and ¹³ C chemical shifts, δΗ J, Hz).

The title product 2-[4-(3-chloro-2,2-dimethylpropanoyloxy)-3-hydroxyphenyl]-3,5,7- trihydroxychromen-4-one with Rf-0.24 (dichloromethane/methanol 9:0.5) solidified off as white solid. Yield 0.2 g. The NMR data are included in Table 4.

Table 4. The 1H and ¹³ C chemical shifts, δΗ (J, Hz).

The title product 2-[3,4-bis-(3-chloro-2,2-dimethylpropanoyloxy)]-3,7-di-(3-chloro-2,2- dimethylpropan-oyloxy)-5-hydroxychromen-4-one with Rf-0.85 (dichloromethane/methanol 9:0.5) solidified off as white solid, m.p.140-145 °C. Yield 2.16g. IR ^ _cm-1) 2800-3000b, 1765s, 1649s, 1606s, 1140s, 1084s. The NMR data are included in Table 5.

Table 5. The 1H and ¹³ C chemical shifts, δΗ (J, Hz).

Example 3

3,7-Dihydroxy-2-[4-(2-chloro-l,4-naphthoquinon-3-yloxy)-3-hydroxyphenyl]-5-hydroxy- chromen-4-one

2,3-Dichloro-l,4-naphtoquinone (0.75g) was added to a mixture quercetine (lg) in 2- pentanone-toluene (100ml, l:lv/v). DIPEA (0.45 g) in toluene (5ml) was then added dropwise and the mixture was heated to reflux and stirred for 12hours under inert atmosphere. The resulting precipitate was filtered and washed with hot toluene (10ml). Collected filtrates were evaporated under reduced pressure. Ice -water (50g) and ethanol (20ml) was added to the gummy mass. The resulting precipitate was filtered and dried. The yellowish material was two time purified by silica gel chromatography (first eluent: ethyl acetate/methanol 9:0.1 v/v, and second eluent: dichloromethane/methanol/formic acid 9:0.5:0.05 v/v) to afford 3,7-dihydroxy- 2-[4-(2-chloro-l,4-naphthoquinon-3-yloxy)-3-hydroxyphenyl]-5-hydroxy- chromen-4-one as a yellow solid. The product was stored under nitrogen atmosphere. Yield 0.505 g, m.p. 165- 170 °C dec, LC/Mass M⁺ 492. IR _{(rt cm}._1} 3050-3450b, 1639s, 1622s, 1598s, 1595s, 1497s, 1009s. The title compound was characterized by elemental analysis. Example 4

3 ,7-Di-hydroxy-2-(3 ^'-hydroxy-4 '-(4-0-acetylferuloyloxy)phenyl)-5 -hy droxy-chromen-4-one

Analogous to example 1, but 4-O-Acetylferulic acid chloride (Eur. Food Res. Technol (2006) 222: 492-508) was used. M.p. 176-183 °C, IR _(r, _cm-r) 1763s, 1729s, 1631-1618m, 1506s, 1174d, 1104. The title compound was characterized by elemental analysis, and 1H and ¹³C NMR.

Example 5

Aldose reductase (ALR2) activity measurement in vitro

Preparation of ALR2. ALR2 from rat lens was partially purified using a procedure adapted from Hayman and Kinoshita [Hayman S, Kinoshita J H. Isolation and properties of lens aldose reductase. J Biol Chem. 240: 877-882 (1965)] as follows: lenses were quickly removed from rats following euthanasia and homogenized in a glass homogenizer with a teflon pestle in 5 volumes of cold distilled water. The homogenate was centrifuged at 10 000 g at 0-4 °C for 20 min. The supernatant was precipitated with saturated ammonium sulfate at 40 %, 50 % and then at 75 % salt saturation. The supernatant was retained after the first two precipitations. The pellet from the last step, possessing ALR2 activity, was dispersed in 75 % ammonium sulfate and stored in smaller aliquots in liquid nitrogen container.

Enzyme assay. ALR2 activities were assayed spectrophotometrically [Stefek M, Snirc V, Djoubissie PO, Majekova M, Demopoulos V, L Rackova, Bezakova Z, Karasu C, Carbone V, El-Kabbani O. Carboxymethylated pyridoindoles antioxidants as Aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as decrease of the optical density (O.D.)/s/mg protein. The reaction mixture contained 4.67 mM D,L-glyceraldehyde as a substrate, 0.11 mM NADPH, 0.067 M phosphate buffer, pH 6.2 and 0.05 mL of the enzyme preparation in a total volume of 1.5 mL. The reference blank contained all the above reagents except the substrate D,L-glyceraldehyde to correct for oxidation of NADPH not associated with reduction of the substrate. The enzyme reaction was initiated by addition of D,L-glyceraldehyde and was monitored for 4 min after an initial period of 1 min at 30 °C. Enzyme activities were adjusted by diluting the enzyme preparations with distilled water so that 0.05 ml of the preparation gave an average reaction rate for the control sample of 0.020 ± 0.005 absorbance units/min. The effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank. IC₅o values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarthmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %.

Table 1. Inhibition of aldose reductase (ALR2) isolated from rat lenses

Compound IC™ (uM)

From example 3 2.09 ± : 0.57

From example 1 14.55 ± 4.60

From example 4 14.39 ± 4.94

Quercetin 13.55 ± 3.64 Experimental results are mean ± SD of at least three experiments. Example 6

Aldehyde reductase (ALR1) activity measurement in vitro

Preparation of ALR1. ALR1 from rat kidney was partially purified according to the reported procedure of Costantino et al. [Constantino L Rastelli G Gamberini MC et al. 1- benzopyran-4-one antioxidants and Aldose reductase inhibitors. J. Honey. Chem. 42:1881- 1893 (1999)] as follows : kidneys were quickly removed from rats following euthanasia and homogenized in a knife homogenizer followed by processing in a glass homogenizer with a teflon pestle in 3 volumes of 10 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium salt and 2.5 mM β-mercaptoethanol. The homogenate was centrifuged at 16 000 g at 0-4 °C for 30 min and the supernatant was subjected to ammonium sulfate fractional precipitation at 40 %, 50 % and 75 % salt saturation. The pellet obtained from the last step, possessing ALR1 activity, was redissolved in 10 mM sodium phosphate buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt and 2.0 mM β- mercaptoethanol to achieve total protein concentration of approximately 20 mg/ml. DEAE DE 52 resin was added to the solution (33 mg/ml) and after gentle mixing for 15 min removed by centrifugation. The supernatant containing ALR1 was then stored in smaller aliquots in liquid nitrogen. No appreciable contamination by ALR2 in ALR1 preparations was detected since no activity in terms of NADPH consumption was observed in the presence of glucose substrate up to 150 mM.

Enzyme assay. ALR1 activities were assayed spectrophotometrically [Stefek M, V Snirc, Djoubissie PO Majekova M, Demopoulos V, L Rackova, Bezakova Z, Karasu C, Carbone V, El-Kabbani O. Carboxymethylated pyridoindoles antioxidants as Aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as decrease of the optical density (O.D.)/s/mg protein. The reaction mixture contained 20 mM D-glucuronate as a substrate, 0.12 mM NADPH in 0.1 M phosphate buffer pH 7.2 and 0.05 ml of the enzyme preparation in a total volume of 1.5 ml. The reference blank contained all the above reagents except the substrate D-glucuronate to correct for oxidation of NADPH not associated with reduction of the substrate. The enzyme reaction was initiated by addition of D-glucuronate and was monitored for 4 min after an initial period of 1 min at 37 °C. Enzyme activities were adjusted by diluting the enzyme preparations with distilled water so that 0.05 ml of the preparation gave an average reaction rate for the control sample of 0.020 ± 0.005 absorbance units/min. The effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank. IC ₀ values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarthmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %. Table 2. Inhibition of aldehyde reductase (ALR1) isolated from rat kidney

Compound C (μΜ) I (%)

From example 3 10 54.2 1 0.6

From example 1 10 60.1 ± 4.1

From example 4 10 11.4 ± 2.7

20 17.7

50 33.8

Quercetin 10 61.5 ± 3.2

I ( ): Percentage of aldehyde reductase inhibition. IC50 for example 3 was 11.1 ± 1.1 μιτιοΐ/ΐ. Results are means from two experiments or means ± SD from at least three measurements

Example 7

Measurement of antioxidant activity in vitro

DPPH test. To investigate the antiradical activity of the derivatives, the ethanolic solution of DPPH (50 μΜ) was incubated in the presence of a compound tested (50 μΜ) at laboratory temperature [Stefek M, V Snirc, Djoubissie PO Majekova M, Demopoulos V, L Rackova, Bezakova Z, Karasu C, Carbone V, El-Kabbani O. Carboxymethylated pyridoindoles antioxidants as Aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling. Bioorg Med Chem. 16:4908-4920 (2008)]. The absorbance decrease, recorded at λ _max = 518 nm, during the first 75-s interval was taken as a marker of the antiradical activity.

Table 3. Antiradical activity in a DPPH assay

Compound (-AA_Si8nm/75 S)

From example 3 0.462 ± 0.015 From example 1 0.446 ± 0.024 From example 4 0.040 ± 0.026 Quercetin 0.386 ± 0.025 Trolox 0.520 ± 0,031

The ethanolic solution of DPPH radical (50 μΜ) was incubated in the presence of the compounds tested (50 μΜ). Absorbance decrease at 518 nm during the first 75-s interval was determined. Results are mean values ± SD from at least three measurements. Industrial Utility

Compounds of the present invention can conveniently be administered in a pharmaceutical composition containing the compound in combination with a suitable excipient. Such pharmaceutical compositions can be prepared by methods and contain excipients which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington's Pharmaceutical Sciences by E.W. Martin (Mark Publ. Co., 15th Ed., 1975). The compounds and compositions of the present invention can be administered parenterally (by subcutaneous injection, intravenous, intramuscular, intraperitoneal, intrasternal injection or infusion techniques) topically, orally, or rectally.

Claims

1. Derivatives of quercetin of general formula (I)

I

and pharmaceutically acceptable salts, hydrates and solvates thereof, wherein Ri to R₅ are independently H, benzyl, and at least one group is 3-chloro-2,2-dimethylpropanoyl, 4-0- acetylferuloyl, or 2-chloro-l,4-naphthoquinon-3-yl.

2. Quercetin derivatives according to claim 1 for use in the treatment and control of human diseases in which oxidative stress and polyol pathway are key etiological factors, such as the development of diabetic complications including macro and microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy and neuropathy, resistance of hepatic cancer to chemotherapy , abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, inflammatory diseases such as uveitis, sepsis, colon cancer, asthma and periodontitis.

3. The use of derivatives of quercetin according to claim 1, for the preparation of medicaments for the treatment or prevention of diseases or disorders in which oxidative stress and polyol pathway are key etiological factors, such as the development of diabetic complications including macro and microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy and neuropathy, resistance of hepatic cancer to chemotherapy , abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, inflammatory diseases such as uveitis, sepsis, colon cancer, asthma and periodontitis.

4. A pharmaceutical composition comprising at least one compound of claim 1 and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising at least one compound according to claim 1, formulation of which is a solid oral form or injection form for systemic administration or it is in the form of drops, pastes, gels or ointments for topical administration.