KR20130036493A - A novel compound, 3',4'-difluoroquercetin, preparation method thereof and use thereof - Google Patents

Abstract

PURPOSE: A novel quercetin derivative, 3,4-difluoro quercetin, a manufacturing method thereof, and an usage thereof are provided to increase anticancer activity of cancer cells by increasing safety and bioavailability. CONSTITUTION: A pharmaceutical composition for cancer prevention or treatment comprises a chemical in the first clause or pharmaceutically allowed salt thereof. Preventable and treatable cancers by the pharmaceutical composition for the cancer prevention or the treatment are liver cancer, skin cancer, or breast cancer. A manufacturing method of a quercetin derivative, 3,4-difluoro quercetin, comprises a step of synthesizing 3`,4`-difluoro quercetin by substituting hydroxyl groups located on the third and fourth position of quercetin with fluoro groups, and providing the stability to chemical or enzymatic hydrolysis.

Description

Novel quercetin derivative, 3 ', 4'-difluoro quercetin, preparation method and use thereof {A novel compound, 3', 4'-difluoroquercetin, preparation method about and use}

The present invention relates to a novel quercetin derivative 3 ', 4'-difluoro quercetin having a hydroxyl group at positions 3 and 4 affecting the stability of quercetin and a method for preparing the compound and a pharmaceutical use thereof will be.

Quercetin is a plant-derived flavonoid compound known to have a variety of physiological activities, including antioxidant, anticancer and antiviral effects (Ferry, DR et al. Clin . Cancer . Res . 1996, 2 (4), 659-668; Lamson, DW;.. . Brignall, MS Altern Med Rev 2000, 5 (3), 196-208).

Quercetin has a structure as shown in Figure 1, C2 = C3 double bond, C4-ketone group, C3 hydroxyl group and the catechol structure of the B ring is known to have a critical influence on the antioxidant activity of quercetin. However, quercetin has an unstable disadvantage in aqueous solution because it is easily decomposed through oxidative decomposition as shown in FIG. 2. In addition, quercetin has low water solubility, and has a problem of poor absorption in the small intestine. In addition, the metabolism of ring B (B ring) is the most important target for methylation, sulfonation, and glucuronation by metabolic enzymes, especially in the liver. The sieve does not have the physiological activity of quercetin. Therefore, quercetin has a problem of low bioavailability due to instability in aqueous solution, low water solubility, low cell absorption, and fast metabolism (AJ Day, Y. Bao, MR Morgan, G. Wiliamson, Free). Rad . Biol . Med . 2002, 9 , 1234-1243; Formica, J. V; Regelson, W. Fd . Chem . Toxic . 1995, 33 , 1061-1080; Mariusz KP BioFactors . 2000, 12 , 175-180; O, KA; Day, AJ; Needs, PW; Mellon, FA; O, NM; Williamson, G. Biochem . Pharmacol . 2003, 65 , 479).

In related prior patents, Korean Patent Publication No. 1020100081826 relates to quercetin-amino acid conjugates, wherein quercetin is attached to quercetin with one or more amino acid residues selected from the group consisting of alanine, valine, lysine, phenylalanine, aspartic acid, methionine and glutamic acid Amino acid conjugates are described,

In another related prior art, Korean Patent Publication No. 1020100122287 relates to quercetin prodrugs and a method for preparing the same, which is a quercetin prodrug, 3-dimethylcarbamic acid quercetin ester (3-N, N-dimethyl carbamoyl quercetin; Improved chemical stability and pharmacokinetics properties of DCQ) are described.

The present invention solves the problems described above, and the object of the present invention is to solve the problems of quercetin having anti-cancer activity, such as instability in the intracellular and external environment and extracorporeal discharge by metabolism, etc. It is to provide a novel quercetin derivative that can overcome the problem.

Another object of the present invention is to provide a method for preparing the novel quercetin derivative.

Another object of the present invention is to provide a composition having increased anticancer activity against cancer cell lines.

In order to achieve the above object, the present invention provides a compound represented by the following Chemical Formula 1.

[Formula 1]

The present invention also provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof.

"Pharmaceutically acceptable salts" in the present invention refers to salts of any of the compounds of the present invention that are not toxic or inappropriate for pharmaceutical use while maintaining the biological properties of the compounds of the present invention. Such salts can be derived from and include various organic and inorganic counterions known in the art. Such salts include (1) organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, gluc Taric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, picric acid, cinnamic acid, mandelic acid, Phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzene sulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid , Camphorsulfonic acid, 4methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid , Benzoic acid, glutamic acid, hydroxynaphthoic acid, Acid addition salts formed as florisil acid, stearic acid, sulfamic acid cyclohexylsulfonyl, kwinsan, mu acid; Or (2) acidic protons present in the parent compound (a) metal ions (eg, alkali metal ions, alkaline earth metal ions or aluminum ions), alkali metal or alkaline earth metal hydroxides (eg, sodium, potassium, calcium, magnesium , Aluminum, lithium, zinc and barium hydroxides), ammonia, or (b) an organic base such as aliphatic, cycloaliphatic or aromatic organic amines such as ammonia, methylamine, dimethylamine, diethylamine, picoline , Ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethyl Salts formed when coordinating with amines, N-methylglucamine piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, and the like.

Further examples of salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium salts, and the like, if the compound contains basic functional groups, salts with non-toxic organic or inorganic acids, such as hydrohalides (eg hydro Chloride or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate Bate, Lactate, Malonate, Succinate, Sorbate, Ascorbate, Maleate, Maleate, Fumarate, Tartarate, Citrate, Benzoate, 3- (4-hydroxybenzoyl) benzoate, Peak Late, cinnamate, mandelate, phthalate, laurate, methanesulfonate mesylate), ethanesulfone , 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, Camphorsulfonate, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfide Pate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfate, quinate, muconate salt and the like.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, wherein the cancer is preferably liver cancer, skin cancer or breast cancer, but is not limited thereto.

The term “therapeutic agent (s) or therapeutic composition” as used herein refers to any agent (s) that can be used to treat or prevent a disorder or one or more indications thereof. In certain embodiments, the term "therapeutic agent" refers to a compound of the present invention. In other specific embodiments, the term "therapeutic agent" does not refer to a compound of the present invention. In one embodiment, the therapeutic agent is an agent that is known to be useful for treating or preventing a disorder or one or more indications thereof, has been used for this purpose, or is currently in use.

A "therapeutically effective amount" means an amount of a compound, complex or composition sufficient to have an effect on treating a disease when administered to a subject for treatment of a disease. A "therapeutically effective amount" may vary, in particular, depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.

In one embodiment, “treating” any of the diseases or disorders or “treatment” thereof refers to ameliorating the disease or disorder present in the subject. In another embodiment, “treating” or “treatment” refers to improving one or more body parameters, which a subject may not recognize. In another embodiment, “treating” or “treatment” refers to physically regulating a disease or disorder (eg, stabilizing a recognizable symptom) or physiologically regulating (eg, a body parameter). Stabilization), or both. In another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.

As used herein, the term “prophylactic agent (s)” refers to any agent (s) that can be used to prevent a disorder or one or more symptoms thereof. In certain embodiments, the term "prophylactic agent" refers to a compound of the present invention. In other specific embodiments, the term "prophylactic agent" does not represent a compound of the present invention. For example, a prophylactic agent is an agent that is known to be useful for preventing or delaying the onset, development, progression and / or aggravation of a disorder, or has been used or is currently used for this purpose.

As used herein, the terms “prevent”, “preventing” and “preventing” administer a therapeutic agent (eg, a prophylactic or therapeutic agent) or a combination of therapeutic agents (eg, a combination of prophylactic or therapeutic agents). Thereby preventing the recurrence, onset, or development of one or more signs of the disorder in a subject.

The phrase “prophylactically effective amount” as used herein prevents the development, recurrence or onset of one or more symptoms associated with a disorder, or augments the prophylactic effect (s) of another therapy (eg, another prophylactic agent) or The amount of therapy (eg, prophylactic agent) sufficient to improve is indicated.

The compounds used in the process of the invention preferably use one or more compounds of formula (1), where appropriate in the form of salts, alone, or one or more suitable pharmaceutically acceptable carriers such as diluents or adjuvants Or in the form of a pharmaceutical composition containing in combination with another anticancer agent.

In clinical practice, the compounds of the present invention can be administered by any conventional route, in particular by oral, parenteral, rectal route, or by inhalation (eg in aerosol form).

As solid compositions for oral administration, tablets, pills, hard gelatin capsules, powders or granules can be used. In these compositions, the compounds according to the invention are admixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch. These compositions, in addition to diluents, may contain, for example, lubricants such as magnesium stearate, or controlled release. It may include a material such as a coating for.

As liquid compositions for oral administration, inert diluents such as pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing water or liquid paraffin can be used. These compositions may also include, in addition to diluents, substances such as, for example, wetting agents, sweetening agents or flavoring agents.

The composition for parenteral administration may be an emulsion or a sterile solution. As solvents or vehicles, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil or injectable organic esters such as ethyl oleate can be used. These compositions may also contain adjuvants, especially wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in a number of ways, for example by using a filter for bacteria, by irradiation or by heating. They can also be prepared in the form of sterile solid compositions that, when used, can be dissolved in sterile water or any other injectable sterile medium.

Compositions for rectal administration are suppositories or capsules for rectal administration, which contain, in addition to the active ingredient, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols.

The composition may also be an aerosol. When used in liquid aerosol form, the composition may be a stable sterile solution, or a solid composition which, when used, is dissolved in non-pyrogenic sterile water, saline or any other pharmaceutically acceptable vehicle. When used in anhydrous aerosol form for direct inhalation, the active ingredient is finely divided and mixed with a water soluble solid diluent or vehicle such as dextran, mannitol or lactose.

In one embodiment, the compositions of the invention are pharmaceutical compositions or single unit dosage forms. Pharmaceutical compositions and single unit dosage forms of the invention may be used in a prophylactic or therapeutically effective amount of one or more prophylactic or therapeutic agents (eg, a compound of the invention, or other prophylactic or therapeutic agents), and typically one or more pharmaceutically acceptable Carriers or excipients.

In certain embodiments and contexts, the term "pharmaceutically acceptable" means that it can be used in animals, more specifically in humans, as listed in a pharmacopoeia that has been approved or generally accepted by the government or regulatory bodies of the government. do. The term “carrier” refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete adjuvant)), excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils including oils or synthetic oils of petroleum, animal or vegetable origin, for example peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously.

Saline solutions, and aqueous dextrose and glycerol solutions can also be used as liquid carriers, in particular injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Conventional pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are known to those skilled in the pharmaceutical arts, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, arc flour, silica gel, sodium stearate, glycerol monostearate, Talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like. Whether it is suitable for a particular excipient to be incorporated into a pharmaceutical composition or dosage form depends on various factors known in the art, including the manner in which the dosage form is administered to the subject, the specific active ingredients included in the dosage form, and the like. If desired, the composition or single unit dosage form may also contain small amounts of wetting or emulsifying agents, or pH buffers.

The invention also includes anhydrous pharmaceutical compositions and dosage forms comprising the active ingredient. For example, the addition of water is widely accepted in the pharmaceutical industry as a simulated long term storage means for determining the properties (eg shelf life or stability) of a formulation over time. See, eg, Jens T. Carstensen, Drug Stability: Principles Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379 80. Indeed, water and heat accelerate the decomposition of some compounds. Thus, water and / or moisture typically occur during the manufacture, handling, packaging, storage, transportation and use of the formulation, so the effect of water on the formulation can be very important.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture components and low moisture or low humidity conditions. If it is anticipated to be in substantial contact with moisture and / or moisture during manufacture, packaging and / or storage, pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising primary or secondary amines may be in anhydrous form. Anhydrous pharmaceutical compositions should be prepared and stored so that their anhydrous properties are maintained. Therefore, it is desirable to package using known materials to prevent the anhydrous composition from exposure to water and to include it in a suitable specification kit. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.

The present invention also includes pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of degradation of the active ingredient. Such compounds (referred to herein as "stabilizers") include, but are not limited to, antioxidants (eg, ascorbic acid), pH buffers, or salt buffers.

Pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations and the like. Formulations for oral administration may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Such compositions and dosage forms contain a prophylactic or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, together with a suitable amount of carrier, and will be provided in a form suitable for administration to a subject. The formulation should be appropriate for the mode of administration. In a preferred embodiment, the pharmaceutical composition or single unit dosage form is sterile and is in a form suitable for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

Pharmaceutical compositions of the invention are formulated to be suitable for their intended route of administration. Examples of routes of administration include parenteral routes of administration such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, oral, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, synovial and There is, but is not limited to, a rectal route of administration. In certain embodiments, the composition is formulated into a pharmaceutical composition that has been modified for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to a human according to conventional procedures. In one embodiment, the pharmaceutical composition is formulated into a composition that is administered subcutaneously to a human according to conventional procedures.

Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include solubilizers and local anesthetics (eg lignocaine) to soothe pain at the injection site.

Examples of dosage forms include tablets; Caplets; Capsules such as soft elastic gelatin capsules; Casein; Troches; Rosenji; Dispersion liquids; Suppositories; Ointment; Poultice (foam medicine); paste; Powder; dressing; cream; salve; Solution; patch; Aerosols (eg, nasal sprays or inhalers); Gel; Liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (eg, aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions and elixirs; Liquid dosage forms suitable for parenteral administration to a subject; And sterile solids (eg, crystalline or amorphous solids) that can provide a liquid dosage form suitable for parenteral administration to a subject when restored.

Still other ways of modifying certain dosage forms encompassed by this invention will be apparent to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

The components of the compositions of the present invention are generally anhydrous lyophilized powders or water-free concentrates in sealed containers, such as ampoules or sachets, for example indicative of the amount of active agent, mixed separately or together in a unit dosage form. It is provided in the form. When the composition is administered by infusion, the composition may be dispensed into an infusion container containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, sterile water or saline ampoules for injection can be provided so that the components can be mixed before administration.

Conventional dosage forms of the present invention comprise a compound of the present invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof in an amount ranging from about 0.1 mg to about 1000 mg / day, which is once daily A single dose or, preferably, divided doses with meals throughout the day. Certain dosage forms of the invention comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of a compound of Formula 1 Has

Pharmaceutical compositions of the present invention suitable for oral administration may be in separate dosage forms such as tablets (eg chewing tablets), caplets, capsules and liquids (eg flavor syrups) and the like. Such dosage forms contain a predetermined amount of active ingredient and can be prepared by methods of pharmacy known to those skilled in the art. Generally, literature

See Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). In certain embodiments, oral dosage forms are solid and are prepared under anhydrous conditions using anhydrous ingredients as detailed in the paragraphs above.

However, the scope of the present invention extends to other than anhydrous solid oral dosage forms. Conventional oral dosage forms of the invention are prepared by combining the active ingredient (s) as an intimate mixture with one or more excipients according to conventional pharmaceutical synthesis techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in liquid or aerosol dosage forms for oral administration include, but are not limited to, water, glycols, oils, alcohols, flavors, preservatives and coloring agents. Suitable excipients for use in solid oral dosage forms (eg, powders, tablets, capsules and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants. Does not. When solid excipients are used, they appear in the most advantageous oral dosage unit form because tablets and capsules are easy to administer. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of preparation.

In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then molding the product into the desired shape, if necessary. Tablets can be made by compression or molding. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free flowing form (eg, powder or granules), optionally mixed with excipients. Molded tablets may be made by molding in a suitable machine a mixture of powdered compounds moistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants and lubricants. Suitable binders for use in pharmaceutical compositions and dosage forms include corn starch, potato starch or other starch, gelatin, natural and synthetic gums such as acacia gum, sodium alginate, alginic acid, other alginates, powder tragacanth, guar gum, Cellulose and its derivatives (e.g. ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, (e.g. , 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include talc, calcium carbonate (eg, granules or powders), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, Pregelatinized starch, and mixtures thereof, but is not limited thereto. The binder or filler included in the pharmaceutical composition of the present invention is typically present in an amount of about 50 to about 99 weight of the pharmaceutical composition or dosage form. Suitable forms of microcrystalline cellulose include AVISEL PH 101, AVICEL PH 103, AVICEL RC 581, AVICE PH 105 and are commercially available (FMC Corporation, American Viscose Division, Avicel Sales). Sales; commercially available from Markus Hook, Pennsylvania, USA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold by Avicel RC 581. Suitable anhydrous or low moisture excipients or additives include Avicel PH 103 ™ and starch 1500 LM.

The use of disintegrants in the compositions of the present invention provides tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate upon storage, and tablets containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, a sufficient amount of disintegrant should not be used to form the solid oral dosage form of the present invention, which is neither too much nor too little to deleteriously alter the release of the active ingredient. The amount of disintegrant used depends on the type of formulation and can be readily determined by one skilled in the art. Typical pharmaceutical compositions comprise about 0.5 to about 15 weight percent of disintegrant, specifically about 1 to about 5 weight percent. Disintegrants that can be used in the pharmaceutical compositions and dosage forms of the invention include agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polyacrylic potassium, sodium starch glycolate, potato or tapioca starch, Pregelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in the pharmaceutical compositions and dosage forms of the present invention include calcium stearate, magnesium stearate, mineral oil, mineral diesel, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, Talc, hydrogenated vegetable oils (e.g. peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof There is, but is not limited to this. Additional lubricants include, for example, siloid silica gel (AEROSIL 200, manufactured by WR Grace Co., Baltimore, MD), solidified aerosol of synthetic silica (Degussa Corporation) (Available from Degussa Co., Piano, Tex.), CAB O SIL (pyrogenic silicon dioxide product, from Cabot Co .; Boston, Mass.), And mixtures thereof. . When used, lubricants are typically used in amounts less than about 1% by weight of the pharmaceutical composition or dosage form into which they are incorporated.

The compounds of the present invention can be administered eg by controlled release means or delivery devices known to those skilled in the art. Examples of these include US Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556 and 5,733,566, each of which is incorporated herein by reference in its entirety, but is not limited thereto. Such dosage forms can be, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, particulates, liposomes, microspheres, or combinations thereof to provide a desired release profile at various ratios. Water may be used to delay or release controlled release of one or more active ingredients. Suitable controlled release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the present invention. Accordingly, the present invention includes single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, etc., modified to controlled release.

All controlled release drugs have a common purpose to provide improved drug therapies than are achieved by their corresponding non-controlled release drugs. Ideally, the use of controlled release formulations that are best designed for medical treatment is characterized by the use of a minimum amount of drug substance to cure or control symptoms within a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, a decrease in the number of doses and an increase in compliance of the subject. In addition, controlled release formulations may be used to affect the time of onset of the drug or other properties such as blood levels of the drug, thus affecting the development of side effects (adverse effects).

Most controlled release formulations initially release an amount of drug (active ingredient) that immediately produces the desired therapeutic effect, and release the drug stepwise and continuously in an amount that sustains the level of the therapeutic or prophylactic effect over an extended period of time. It is designed to As such, to maintain a constant drug level in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug that is metabolized and secreted by the human body. Controlled release of the active ingredient can be stimulated by a variety of conditions including pH, temperature, enzymes, water, or other physiological conditions or compounds, and the like.

In certain embodiments, the drug can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump can be used (Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987)); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989). In other embodiments, polymeric materials can be used. In another embodiment, a controlled release system can be located at a suitable site determined by one of skill in the art in a subject, ie only a portion of the systemic dosage is required (eg, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems have been reviewed and discussed by Langer (Science 249: 1527-1533 (1990)). The active ingredient may be a solid internal matrix such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene , Polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate, copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrogels of hydrophilic polymers such as acrylic acid and methacrylic acid, collagen, It can be dispersed in crosslinked polyvinylalcohol and partially hydrolyzed crosslinked polyvinyl acetate, which is an external polymer membrane such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl Acetate copolymer, silicone rubber, polydimethyl siloxane, neo Pren rubber, polyethylene chloride, polyvinylchloride; Vinyl acetate, vinylidene chloride, vinyl chloride copolymer with ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol tripolymer, and ethylene / It is surrounded by vinyloxyethanol copolymer, which is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in the release rate control step. The proportion of active ingredient in such parenteral compositions is highly dependent on the particular nature of the subject as well as the needs of the subject.

The invention also provides parenteral dosage forms. Parenteral dosage forms can be administered to a subject by a variety of routes including subcutaneous, intravenous (including bolus injection), intramuscular and intraarterial routes of administration, and the like. Because these parenteral routes of administration typically bypass the subject's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized prior to administration to the subject. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, anhydrous products that can be dissolved or suspended in pharmaceutically acceptable injectable vehicles, injectable suspensions, and emulsions.

Suitable vehicles are known to those skilled in the art that can be used to provide the parenteral dosage forms of the invention. Examples of these include water for injection (USP); Aqueous vehicles such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, and the like; Water miscible vehicles such as ethyl alcohol, polyethylene glycol, polypropylene glycol, and the like; And non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate, and the like. The solubility of one or more active ingredients disclosed herein is also limiting. Compounds that increase can also be incorporated into the parenteral dosage forms of the invention.

The invention also provides transdermal, topical and mucosal dosage forms. Transdermal, topical and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions or other forms known to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 2000) and Introduction to Pharmaceutical dose form, 4th ed., Lea Febiger, Philadelphia (1985). See.

Suitable dosage forms for treating oral mucosal tissue may be formulated as oral cleaning solutions or oral gel formulations. In addition, transdermal dosage forms include "reservoir" or "matrix" patches, which may be skin or wound The site may be applied for a certain period of time to infiltrate the desired amount of active ingredient.

Suitable excipients (eg, carriers and diluents) included in the present invention, and other materials that can be used to provide transdermal, topical and mucosal dosage forms, are known to those of ordinary skill in the art of pharmacy, and they are given in any given pharmaceutical composition or dosage form. It depends on the specific organization to which it applies. In view of this fact, conventional excipients include water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl, forming lotions, tinctures, creams, emulsions, gels or ointments. Palmitate, mineral oil, and mixtures thereof, and the like, which are non-toxic and pharmaceutically acceptable. If desired, humectants or humectants may also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are known in the art. See, eg, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 2000).

Depending on the particular tissue to be treated, additional ingredients may be used prior to treatment with the active ingredients of the invention, used with the active ingredients of the invention, or after treatment with the active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredient to the tissue. Suitable penetration enhancers include acetone; Various alcohols such as ethanol, oleyl and tetrahydrofuryl; Alkyl sulfoxides such as dimethyl sulfoxide; Dimethylacetamide; Dimethylformamide; Polyethylene glycol; Pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (povidone, polyvidone); Urea; And various water soluble or water insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

In addition, the pH of the pharmaceutical composition or dosage form or the pH of the tissue to which the pharmaceutical composition or dosage form is applied can be adjusted to improve delivery of one or more active ingredients. Similarly, the delivery can be improved by adjusting the polarity of the solvent carrier, its ionic strength or tension. The delivery may also be improved by adding a compound, such as stearate, to the pharmaceutical composition or dosage form to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients. In this regard, stearates may act as liquid vehicles for formulation, emulsifiers or surfactants, and delivery or penetration enhancers. Other salts, hydrates or solvates of the active ingredient can be used to further adjust the properties of the resulting composition.

When treating a human, the physician will determine the dosage that is considered most appropriate, depending on the prophylactic or curative treatment, and the age, weight, stage of infection and other factors specific to the subject to be treated. Generally, for adults, the dosage is about 1 to about 1000 mg / day, about 5 to about 250 mg / day, or about 10 to 50 mg / day. In certain embodiments, the dosage is about 5 to about 400 mg / day, more preferably 25 to 200 mg / day, per adult. Also preferred is a dosage rate of about 50 to about 500 mg / day.

In another aspect, the present invention provides a method for synthesizing 3 ', 4'-difluoro quercetin in which the hydroxyl groups at the 3' and 4 'positions of quercetin are substituted with fluorine groups to impart stability to chemical or enzymatic hydrolysis.

In addition, the present invention synthesizes perbenzylated quercetin by hiding the hydroxyl group of quercetin with a benzyl group, and hydrolyzing the compound to produce 2- (benzyloxy) -1- (2,4-bis (benzyloxy) -6 Obtaining hydroxyphenyl) ethanone; reacting 3,4-difluorobenzoic acid with oxalyl chloride to convert acyl chloride and converting the foreign substance into 2- (benzyloxy) -1. Condensed with-(2,4-bis (benzyloxy) -6-hydroxyphenyl) ethanone to form 3,5-bis (benzyloxy) -2- (2- (benzyloxy) acetyl) phenyl 3,4-diflo Obtaining robenzoate; And cyclizing the 3,5-bis (benzyloxy) -2- (2- (benzyloxy) acetyl) phenyl 3,4-difluorobenzoate compound, followed by hydrolysis of the benzyl group that masked the hydroxyl group of quercetin. It provides a method for preparing a 3 ', 4'-diflolocercetin compound comprising the step of removing using a reaction.

Hereinafter, the present invention will be described.

Much research has been directed towards making prodrugs to improve the pharmacokinetic properties of quercetin. However, in most cases, prodrugs in the form of quercetin esters have the disadvantage of being rapidly converted to quercetin by chemical hydrolysis and enzymatic hydrolysis, and have very limited utility. In order to remedy this drawback, quercetin-glycine carbamate prodrug QC12 (Formula 2), a carbamate-like prodrug that is more stable than ester, has been developed and clinically tested (Mulholland, PJ; Ferry). , DR; Anderson, D .; Hussain, SA; Young, AM; Cook, JE; Hodgkin, E .; Seymour, LW; Kerr, DJ Ann . Oncol . 2001 , 12 , 245).

QC12 has significantly improved water solubility compared to quercetin and has excellent properties as a prodrug, such as slow hydrolysis to quercetin (half-life 16.9 hours) in water but very fast hydrolysis by hydrolase in vivo (half-life 0.39 Time) had limitations that could not be developed for oral administration (Mulholland, PJ; Ferry, DR; Anderson, D .; Hussain, SA; Young, AM; Cook, JE; Hodgkin, E .; Seymour, LW; Kerr , DJ Ann. Oncol. 2001, 12, 245).

[Formula 2]

Quercetin-amino acid conjugate [Formula 3] is (Mi Kyoung Kim, Kwang-su Park, Woon-seok YeO, Hyunah Choo, Youhoon Chong. Bioorg . Med . Chem . 2009, 17 , 1164- 1171) Forming conjugates of quercetin with various amino acids in carbamate form showed an increase in water solubility, ranging from 6-fold (Qu-K) to 52-fold (Qu-E), as well as improved cell permeability. In addition, quercetin-amino acid conjugates were exposed to various hydrolase enzymes contained in cell lysate, and approximately 1 hour 30 minutes (Qu-D, Qu-A and Qu-F) for 3 hours (Qu). -K, Qu-E) showed a marked increase in the stability of quercetin-amino acid conjugates to hydrolase. However, quercetin-amino acid conjugates are chemically hydrolyzed very quickly in 10 to 60 minutes in DMEM (Dulbecco's Modified Eagle's Medium) cell culture (Mi Kyoung Kim, Kwang-su Park, Woon-seok Yeo, Hyunah Choo, Youhoon Chong. Bioorg. Med. Chem. 2009, 17 , 1164-1171), which required a supplement to the chemical stability of quercetin prodrugs.

(3)

Therefore, in the very large effect of ring B on the chemical / metabolic instability of quercetin, in the present invention, examples efforts result of quercetin, such as formula (1) below three times to replace the catechol structure of the ring B of the quercetin in a stable bioisostere And 3 ', 4'-difluoro quercetin substituted with hydroxyl group at position 4 and fluorine group for stability against chemical and enzymatic hydrolysis in the extracellular environment such as phosphate buffer and cell culture solution and various cancer cell lines. By evaluating cytotoxicity, antioxidant effects and the like, the usefulness of quercetin derivative was demonstrated and the present invention was completed.

[Formula 1]

That is, according to the present invention, a quercetin derivative in which hydroxyl groups at positions 3 and 4 affecting the stability of quercetin is substituted with a fluoro group, thereby enhancing stability and bioavailability, thereby increasing anticancer activity against cancer cell lines. Provide new quercetin analogs.

Therefore, the present inventors have designed a quercetin analogue in which a 3 ', 4' hydroxyl group of quercetin is substituted with a fluoro group in order to develop a novel quercetin derivative having increased anticancer activity based on stability in an intracellular or external environment.

As shown in Figure 2, the oxidative decomposition process of quercetin proceeds as the hydroxyl of the ring B loses hydrogen, so the present inventors can improve the stability of the quercetin by replacing the hydroxyl group of the quercetin ring B with a fluoro group. New quercetin derivatives have been devised.

In the present invention by synthesizing a novel quercetin derivative in which the hydroxyl group at the 3 'and 4' position of quercetin ring B is substituted with a fluoro group as shown in [Formula 1] above, in the extracellular environment such as buffer and cell culture medium having different pH By evaluating the stability, solubility, antioxidant activity, cytotoxicity against various cancer cell lines and the like, the usefulness of the anticancer agent more active than quercetin was demonstrated and the present invention was completed.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, 3 ', 4'-difluoro quercetin represented by the above [Formula 1] was synthesized.

Specifically, FIG. 3 shows a process for synthesizing 3 ', 4'-diflolocercetin from commercially available starting materials.

 Hereinafter, the synthesis process of 3 ', 4'-diflolocercetin will be described in detail.

Perbenzylated quercetin ( 2 ) was synthesized by quercetin ( 1 ) as a starting material and the quercetin hydroxyl group was benzyl group (85% yield), and hydrolyzed again under strong basic conditions to obtain compound 3 (40% yield). Meanwhile, 3,4-difluorobenzoic acid ( 7 ) was converted to acyl chloride ( 8 ) by reacting with oxalyl chloride and the foreign material was condensed with compound 3 obtained above to obtain ester ( 4 ) in 84% yield. After the cyclized compound ( 4) was cyclized, the benzyl group occupying the hydroxyl group of quercetin was removed by hydrogenolysis, whereby the desired compound 3 ', 4'-diflolocercetin ( 6 ) was obtained in 41% yield. have.

Hereinafter, the results of analyzing the stability and solubility, antioxidant activity and cytotoxicity of cancer cell lines of the synthesized 3 ', 4'-diflolocercetin ( 6 ) will be described in detail.

First, in order to compare the stability of the synthesized 3 ', 4'-diflolocercetin ( 6 ) and quercetin incubated for 24 hours in buffer and cell culture (37 ℃) of pH 2, pH 7, pH 10, Quercetin is rapidly degraded to less than 1 hour half-life in pH2 buffer conditions, less than 1 hour half-life in pH 10 buffer conditions, and less than 1 hour in cell culture except for pH 7 buffer conditions (half-life 11 hours). Half-life pH 2 buffer, whereas 3 ', 4'-diflolocercetin was found to be very stable with a half-life of more than 24 hours under all conditions (Figure 4).

As a result of measuring the solubility of 3 ', 4'-diflolocercetin ( 6 ), the solubility of quercetin and 3', 4'-diflolocercetin ( 6 ) was observed to be almost the same (Fig. 5).

DPPH (1,1-diphenyl-2-picrydrazyl) free radical scavenging activity was measured to compare the antioxidant activity of quercetin and 3 ', 4'-difluoro quercetin ( 6 ). Vitamin C was used as a control. As can be seen in FIG. 6, quercetin showed antioxidant activity at low concentrations, whereas 3 ', 4'-diflolocercetin showed little antioxidant activity. In the case of quercetin, ring B's 3 and 4 hydroxyl groups react with DPPH free radicals to form a resonance structure with the benzene ring, thereby stabilizing the radicals. When 3 'and 4' are placed on fluoro groups other than hydroxyl groups, fluorobenzene It could not be said that the antioxidant activity was decreased because the radical was not stabilized because it did not react with the radical.

On the other hand, since cancer often occurs due to stress oxidized radicals in one part of the cell, when quercetin is treated on cancer cells, quercetin, which has antioxidant activity, eliminates radicals and prevents the mutation of the cell to prevent cancer. It was difficult to determine with certainty whether it was active or had a clear target, and it was expected that 3 ', 4'-diflolocercetin could be clarified because of its lower antioxidant activity than quercetin. The cytotoxicity of two cancer cell lines (MCF-7, Huh-7) was measured by WST assay. MCF7 is a breast cancer cell line and Huh-7 is a liver cancer cell line. In addition, the experiment was performed using HS27, a human skin cell line, as a control cell line. As a result, 3 ', 4'-diflolocercetin was more toxic than quercetin in all cell lines. In particular, it was confirmed that anticancer activity against breast cancer cell line (MCF-7) was much higher than quercetin (FIG. 7).

As can be seen through the present invention, the novel compounds of the present invention is a quercetin derivative in which hydroxyl groups at positions 3 and 4, which affect the stability of quercetin, are substituted with fluoro groups, thereby enhancing stability and bioavailability, thereby increasing cancer cells. It has the effect of increasing anticancer activity against the state.

1 shows the structure of quercetin.
2 shows the oxidative degradation of quercetin.
Figure 3 shows a summary of the method for synthesizing 3 ', 4'-diflolocercetin.
Figure 4 shows the chemical stability of 3 ', 4'-difluoro quercetin in buffer conditions with different pH and cell free medium.
5 shows the water solubility of 3 ', 4'-difluoro quercetin.
Figure 6 shows the antioxidant activity of 3 ', 4'-diflolocercetin.
Figure 7 shows the cytotoxicity of each cell line of 3 ', 4'-difluoro quercetin.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: 3,5,7-tris (benzyloxy) -2- (3,4-bis (benzyloxy) phenyl) -4H-chromen-4-one ( 2 ):

Quercetin ( 1 ) (10 g, 33.1 mmol), potassium carbonate (45.7 g, 331 mmol), benzylbromide (39.3 ml, 331 mmol) and anhydrous dimethylformamide (150 ml) are stirred at 80 ° C for 3 days. The reaction is filtered using filter paper and the filtrate is concentrated under reduced pressure. Compound distilled through recrystallization by adding dichloromethane and ethyl acetate to the concentrate ( 2 ) (85% yield) in the form of a white powder.

Example 2: 2- (benzyloxy) -1- (2,4-bis (benzyloxy) -6-hydroxyphenyl) ethanone ( 3 ):

The quercetin obtained above was dissolved in a benzyl group ( 2 ) (10 g, 13.3 mmol) in a mixed solution of diethylene glycol (40 ml) and pyridine (40 ml), and 18N potassium hydroxide (20 ml) was added thereto. Stir at 12 ° C. for 12 hours. After the reaction was completed, neutralized by adding 2N hydrochloric acid at 0 ℃. The neutralized solution was extracted three times with dichloromethane (50 ml), magnesium sulfate was added to the organic layer, and the reaction was filtered. The filtrate was concentrated under reduced pressure, and then purified by silica gel column chromatography (16: 1: 1 = nucleic acid: dichloromethane: ethyl acetate) to give compound 3 (40% yield) as a pale yellow powder: NMR (500 MHz). , CDCl ₃ ) δ 13.74 (s, 1H), 7.39-7.21 (m, 15H), 6.18 (s, 1H), 6.06 (s, 1H), 5.05 (s, 2H), 4.98 (s, 2H), 4.56 (s, 2H), 4.52 (s, 2H).

Example 3: 3,5-bis (benzyloxy) -2- (2- (benzyloxy) acetyl) phenyl 3,4-difluorobenzoate ( 4 Synthesis of):

Compound ( 2 ) (500 mg, 1.1 mmol) and Compound ( 8 ) obtained above (300 mg, 1.7 mmol) was dissolved in anhydrous dichloromethane (7 ml), and triethylamine (0.24 ml, 1.7 mmol) was slowly added at 0 ° C., and stirred at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure, and then the concentrate was purified by silica gel column chromatography (30: 30: 1 = nucleic acid: dichloromethane: ethyl acetate) to give a compound ( 4 ) (84% yield) Was obtained in the form of a clear oil: NMR (400 MHz, Acetone_ d 6) δ8.02-7.97 (m, 2H), 7.56-7.49 (m, 5H), 7.43-7.36 (m, 6H), 7.29-7.20 (m, 5H), 6.84 (d, J = 2.1 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 5.24 (s, 2H), 5.20 (s, 2H), 4.51 (s, 2H), 4.40 ( s, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 198.8, 164.0, 162.9, 159.9, 156.3-149.7 (ddd, J = 12.9 Hz, 251.3 Hz, 396.8 Hz, 1C), 151.4, 138.5, 136.3, 129.6, 129.5, 129.4, 129.3, 129.2, 128.8, 128.7, 128.6, 128.5, 127.0, 120.6-118.4 (dd, J = 18.3 Hz, 204.6 Hz, 1C), 115.2, 102.8, 99.4, 76.9, 72.1, 71.4.

Example  4: 3,7- Vis ( Benzyloxy ) -2- (3,4- Difluorophenyl ) -5-hydroxy-4H- Kromen -4--on ( 5 ):

Compound ( 4 ) (450 mg, 0.76 mmol) obtained above was dissolved in anhydrous toluene (5 ml), followed by addition of potassium carbonate (420 mg, 3.04 mmol) and TBAB (367 mg, 1.14 mmol) for 2 hours at 90 ° C. Stir. The reaction was filtered using filter paper and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (50: 50: 1 = nucleic acid: dichloromethane: ethyl acetate) to give a compound ( 5 ) (65% yield) Was obtained as an oil in the form of yellow: NMR (400 MHz, Acetone_ d 6) δ12.94 (s, 1H), 7.97-7.91 (m, 2H), 7.52-7.29 (m, 11H), 6.79 (d, J = 2.00 Hz, 1H), 6.46 (d, J = 2.4 Hz, 1H), 5.27 (s, 2H), 5.20 (s, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.8, 165.8, 163.1, 157.7, 155.1-151.3 (ddd, J = 12.0 Hz, 99.0 Hz, 277.8 Hz, 1C), 139.1, 137.0, 136.7, 130.1, 129.8, 129.6, 129.5, 128.5, 126.6, 119.2-118.3 (dd, 18.5 Hz, 75 Hz, 1C), 107.2, 99.9, 94.1, 75.7, 71.6.

Example  5: 3 ', 4'- Diplolocercetin  ( 6 ):

Compounds obtained above ( 5 ) (250 mg, 0.51 mmol) is dissolved in anhydrous methanol and anhydrous dichloromethane, and then Palladium / Chacoal (10%, 25 mg, 0.051 mmol) is added. The reaction flask is saturated with hydrogen gas and stirred at room temperature for 12 hours. The reaction was washed with acetone and passed through diatomaceous earth, and the filtrate was concentrated under reduced pressure. This concentrate was purified by silica gel column chromatography (8: 1 = dichloromethane: acetone) to give 3 ', 4' difluorolocetine ( 6 ) (41% yield) in the form of a yellow powder: NMR (400 MHz, Acetone_). d ₆ ) δ 12.00 (s, 1H), 8.22 (ddd, J = 1.8, 7.9, 12.4 Hz, 1H), 8.13-8.15 (m, 1H), 7.54 (dd, J = 8.6, 18.9, 1H), 6.60 (s, 1 H), 6.30 (s, 1 H); ^{13 C NMR (100 MHz, DMSO_} d 6) δ177.8, 166.0, 162.3, 159.4, 157.8, 152.8-149.5 (ddd, J = 63.0 Hz, 270.4 Hz, 990.4 Hz), 144.7, 139.1, 130.0, 126.4, 119.5 -118.0 (dd, J = 73.4 Hz, 525.2 Hz), 114.0, 104.8, 100.0, 95.3.

Example  6: 3,4- Dichlorobenzoyl  Chloride ( 8 ):

3, 4-difluorobenzoic acid (270 mg, 1.7 mmol) is dissolved in anhydrous dichloromethane and cooled to zero. After careful addition of oxalyl chloride to this mixture, anhydrous dimethylformamide (0.01 ml, 0.17 mmol) is added. When the temperature of the reaction is room temperature, the mixture is stirred at 45 ° C. for 3 hours. The reaction mass was concentrated under reduced pressure, and then used in the next reaction without further purification.

Example 7 Determination of Chemical Stability of 3 ', 4' Diflolocercetin in Buffer

Synthesized 3 ', 4' diflolocercetin (10 mM) is added to each buffer of different pH (pH 2, pH 7, pH 10) and incubated at 37 ℃. Samples are taken at intervals (1, 2, 3, 4, 5, 6, 12, 24 hours), with the appropriate amount (100 μl) of the sample filtered out and analyzed using HPLC equipped with a C-18 reversed phase column. . Flow rate 1 mL / min; Detection, UV 340 nm; Mobile phase 0-8 minutes (water with 20% acetonitrile and 0.1% formic acid), 8-18 minutes (water with 25% acetonitrile and 0.1% formic acid), 18-40 minutes (35% acetonitrile Water and 0.1% formic acid), 40-45 minutes (water with 80% acetonitrile and 0.1% formic acid), 45-54 minutes (100% acetonitrile and 0.1% formic acid), and 54-60 minutes (20% Water with acetonitrile and 0.1% formic acid).

Example 8 Determination of Chemical Stability of 3 ', 4' Diflolocercetin in Cell-Free Medium

Synthesized 3 ', 4'-diflolocercetin (10 mM) is placed in the cell-free medium (cDMEM) and incubated at 37 ℃. Samples are taken at intervals (1, 2, 3, 4, 5, 6, 12, 24 hours), with the appropriate amount (100 μl) of the sample filtered out and analyzed using HPLC equipped with a C-18 reversed phase column. . HPLC mobile phases were the same as in Example 7.

Example  9: 3 ', 4'- Diplolocercetin  Solubility Verification:

Dissolve 3 ', 4'-diflolocercetin in 1% dimethyl sulfoxide (DMSO), each containing 99% PBS buffer, cell culture medium (DMEM), and serum containing cell culture medium (95% DMEM, 5% Dilution with FBS) to a solution of 100 μM, 250 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM and 2500 μM. Transfer 250 μL of these to a 96 well plate and measure the solubility with a NEPHELOstar instrument.

Example  10: 3 ', 4'- Diplolocercetin  Antioxidant Activity Verification:

Into a 96 well, add DPPH (190 L, 0.1 mM) dissolved in methanol and 3 ', 4'-diflolocercetin dissolved in methanol (1% DMSO) at different concentrations (0.01, 0.05, 0.1, 0.2). , 0.5, 1.0, 2.0 mM) add 10 µl each. After 30 minutes, when the remaining DPPH concentration becomes constant, the absorbance according to the concentration is measured at 517 nm.

Example 11 of 3 ', 4'- diflolocercetin Cytotoxicity measurement for cancer cell lines :

The cytotoxicity of quercetin conjugates against breast cancer (MCF-7) and liver cancer (Huh7) cell lines was verified. Human skin cell line (Hs27) was used as a control. 5 x 10 ³ cells for each cancer cell are prepared in a 96-well plate and incubated for one day under conditions of Dulbecco Modified Eagle Medium (DMEM) medium containing 10% FBS and 1% antibiotic (37 ° C., 5% CO ₂ ). A solution of 3 ', 4'-diflolocercetin in dimethyl sulfoxide (DMSO) was diluted to 7 concentrations (0.1, 0.5, 1, 5, 10, 50 μM) and added to the cultured cell lines, respectively. After culturing for 24 hours, the cell viability is measured by WST assay.

Claims (6)

Claims 1. A compound of formula (1)
[Formula 1]

A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating cancer, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof. The pharmaceutical composition for preventing or treating cancer according to claim 3, wherein the cancer is liver cancer, skin cancer or breast cancer. A method for synthesizing 3 ', 4'-difluoro quercetin, in which the hydroxyl groups at positions 3' and 4 'of quercetin are substituted with fluorine groups, to impart stability to chemical or enzymatic hydrolysis. A perbenzylated quercetin is synthesized by hiding the quercetin hydroxyl group with a benzyl group, and the compound is hydrolyzed to yield 2- (benzyloxy) -1- (2,4-bis (benzyloxy) -6-hydroxyphenyl Obtaining ethanone;
3,4-difluorobenzoic acid is reacted with oxalyl chloride to convert to acyl chloride, and the foreign substance is converted to 2- (benzyloxy) -1- (2,4-bis (benzyloxy) Condensation with -6-hydroxyphenyl) ethanone to obtain 3,5-bis (benzyloxy) -2- (2- (benzyloxy) acetyl) phenyl3,4-difluorobenzoate;
After cyclizing the above-mentioned 3,5-bis (benzyloxy) -2- (2- (benzyloxy) acetyl) phenyl 3,4-difluorobenzoate compound, the hydrolysis reaction of the benzyl group covering the hydroxyl group of quercetin was carried out. Method for producing a 3 ', 4'-diflolocercetin compound comprising the step of removing using.

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