WO2011049400A2 - Pharmaceutical composition containing acid addition salt of n,n-dimethyl imidocarbonimidic diamide for anti-cancer - Google Patents

Abstract

Provided is a use for anticancer of dichloroacetic acid salt or aminooxyacetic acid salt of N,N-dimethyl imidodicarbonimidic diamide (metformin). According to the present invention, the acid addition salt of metformin synergistically causes anticancer effect by AMPK enzyme activation of metformin and anticancer effect of organic acids used as a salt, and thus shows a remarkable anticancer effect. In addition, the acid addition salt has superior physiochemical properties such as solubility, stability, non-hygroscopicity, processibility as a tablet formulation and the like, and has lower toxicity compared with metformin hydrochloride. Therefore, the acid addition salt can be useful as a pharmaceutically acceptable salt.

Description

Anti-cancer pharmaceutical composition comprising acid addition salt of N, N-dimethyl imidodicarbonimidic diamide

The present invention relates to dichloroacetic acid salt and aminooxyacetic acid salt of N, N-dimethyl imidodicarbonimidic diamide, and a pharmaceutical composition for anticancer comprising the same.

Various types of anticancer agents such as alkylating agents, metabolic antagonists, hormones, etc. have been developed and are currently used, but as can be seen in Table 1 below, they inhibit DNA activity of cancer cells, inhibit DNA synthesis, inhibit cell division, and the like. Since the mechanism of action of cancer cells to induce death, the normal cells of the human body to show a variety of side effects such as bone marrow disorders, hair loss, vomiting, diarrhea, neurological disorders.

Table 1

Anticancer drug classification	Mechanism of action	Anticancer drugs
Alkylating agents	Cancer DNA Suppression	Temozolomide, busulfan, cisplatin, etc.
Metabolic antagonists (Antimetabolite)	Inhibition of DNA Synthesis in Cancer Cells	Gemcitabine, methotrexate, fluorouracil, etc.
Hormones	Cause cancer cell DNA disorder	Tamoxifen, anastrozole, flutamide, etc.
Vegetable alkaloids	Inhibition of cancer cell division M stage	Docetaxel, paclitaxel, irinotecan, etc.
Antibiotics	Inhibits cell proliferation by binding to DNA in cancer cells	Doxorubicin, epirubicin, idarubicin, etc.
Cytokines	Immune booster	Interferon alpha-2a, interferon alpha-2b, etc.

On the other hand, the present invention, unlike the existing anticancer drugs, provides a novel mechanism of selectively removing cancer stem cells in cancer tissues, and inhibiting the oncogenesis of cancer cells carrying cancer genes but not changed into cancer cells.

The present invention is a novel mechanism of killing cancer cells by attacking the metabolism of cancer cells by preventing the use of glucose required in cancer unlike conventional anticancer drugs, and in the anticancer pharmaceutical composition in which the anticancer effect of metformin acid addition salts is synergistically enhanced. It is about.

N, N-dimethyl imidodicarbonimide diamide, or metformin, which is used to treat insulin-independent diabetes (type 2 diabetes), is an AMP-activated protein that physiologically regulates carbohydrate and lipid metabolism. It is known that kinase is activated, and when metformin is treated with cancer cells lacking the p53 gene, metformin activates AMPK enzymes in cancer cells to alter energy metabolic pathways, thereby killing cancer cells because they cannot adapt to the altered metabolic pathways. Has been demonstrated [Monica Buzzai, et al. Syntemic Treatment with the Antidiabetic Drug Metformin Selectively Impairs p53-Deficient Tumor Cell Growth, Cancer Res 2007; 67: (14)].

In addition, Josie MM Evans has shown a lower incidence of cancer in patients with type 2 diabetes than those who do not receive metformin [Josie MM, Evans et al. BMJ. 2005 , 330, 1304-1305. Samantha L. Bowker also reported that patients with type 2 diabetes who take metformin have lower cancer-related mortality than patients who take sulfonylureas or take insulin [Samantha L et al. Diabetes Care. 2006, 29, 254-258.

There is increasing clinical evidence that cancer stem cells are involved in the recurrence and metastasis of cancer. Cancer stem cells are less than 0.2% in cancer tissues, but conventional chemotherapy has a problem that can not remove cancer stem cells. Metformin has an anticancer effect on these cancer stem cells and is very well tolerated. In recent studies, metformin eliminates cancer stem cells when cancer stem cells show little change when doxorubicin, an anticancer agent, has been reported [Heather A. Hirsch et al., Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission, Cancer Res 2009; 69: (19). October 1, 2009].

On the other hand, dichloroacetic acid has been used for advanced neurodegenerative disorders (MELAS), a disease caused by defects of mitochondria, and normalizes lactic acidosis caused by progressive neurodegenerative disorders. This is because dichloroacetic acid activates pyruvate dehydrogenase, an enzyme that makes pyruvate the acetyl coenzyme A (Acetyl-CoA) substrate of the TCA pathway. This shifts metabolism towards oxidative phosphorylation in glycolysis and reduces the lactic acid, a byproduct of glycolysis. Due to the metabolic nature of cancer cells that depend on glycolysis and cause lactic acidosis, dichloroacetic acid has attracted attention as a potential cancer treatment. In 2007, Bonnet et al. Confirmed in vitro experiments that dichloroacetic acid reduced cancerous tissues and caused apoptosis (Cancer Cell. 2007 Jan; 11 (1): 37-51).

In addition, Johua et al. Confirmed that aminooxyacetic acid inhibits the growth of breast cancer cells by inhibiting AST (Asparatate aminotransferase), an enzyme that transfers electrons from NADH generated from glycolysis to an electron transport system inside the mitochondria. Cancer Res. 2008; 10 (5): R 84. Epub 2008 Oct 15).

However, there has been no report on the synergistic anti-cancer effect of acid addition salts of metformin with dichloroacetic acid or aminooxyacetic acid added to metformin.

The present invention is to provide an anticancer pharmaceutical composition comprising a dichloroacetic acid salt or aminooxyacetate salt of metformin synergistically enhanced the anticancer effect of metformin killing cancer cells by activating the AMPK enzyme of the cancer cells to change the energy metabolic pathway do.

The present invention also provides an aminooxyacetic acid salt of N, N-dimethyl imidodicarbonimidic diamide and a method for producing the same.

Through the present invention, the anti-cancer efficacy of metformin hydrochloride by using dichloroacetic acid, which activates the oxidative phosphorylation process by activating pyruvate dehydrogenase, which converts pyruvate to acetyl coenzyme A, a substrate of the TCA pathway, as a salt of metformin It has been found that can be significantly enhanced. In addition, the dichloroacetic acid salt of metformin increases anticancer efficacy by preventing the composition of an environment in which lactic acid is accumulated, which is suitable for survival, proliferation and metastasis of cancer cells. In addition, the dichloroacetic acid salt of metformin prevents lactic acidosis, a side effect of metformin.

In addition, by using aminooxyacetic acid, which inhibits ATT (Asparatate aminotransferase), an enzyme required to transfer electrons from NADH generated from glycolysis to the electron transport system inside the mitochondria, as a salt of metformin, supply of energy through glycolysis of cancer cells It was confirmed that it can significantly enhance the anticancer effect compared to metformin hydrochloride.

Therefore, the acid addition salt of metformin according to the present invention can be used as a medicament having a stronger anticancer effect than metformin. The acid addition salt of metformin according to the present invention, unlike the existing anticancer agent, prevents the use of glucose required by cancer, thereby killing cancer cells, and adds an acid addition salt that synergistically enhances the anticancer effect of metformin. It was confirmed to represent. In addition, it was confirmed that only cancer cells, not normal tissues, were selectively removed, and cancer stem cells in cancer tissues could be removed.

In addition, acid addition salts of metformin according to the present invention have increased pharmaceutical and physical properties such as stability, non-hygroscopicity and processability as tablet formulations, while using organic acids having relatively low toxicity compared to conventional metformin hydrochloride prepared using hydrochloric acid. It is suitable for the preparation of pharmaceutical formulations, compared to metformin hydrochloride.

Therefore, the present invention

Pharmaceutical composition for anticancer comprising a pharmaceutically acceptable carrier and metformin acid addition salt having the structure of formula 1 as an active ingredient, the use and treatment of metformin acid addition salt having the structure of formula 1 for the manufacture of the anticancer agent Provided is a method of treating cancer comprising administering to a subject a phase effective amount of metformin acid addition salt having the structure of Formula 1:

 [Formula 1]

Where

A is dichloroacetic acid or aminooxyacetic acid.

The acid addition salt of metformin having the structure of Chemical Formula 1 may be prepared by synthesis through a known method or by applying the method for preparing metformin aminooxyacetic acid salt described below.

In the present invention, the anticancer pharmaceutical composition or the anticancer agent is defined to include an anticancer adjuvant and a cancer preventive agent.

The pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable carrier in addition to the active ingredient. As used herein, 'pharmaceutically acceptable carrier' refers to known pharmaceutical excipients that are useful when formulating pharmaceutically active compounds for administration and which are virtually nontoxic and insensitive under the conditions of use. The exact proportion of such excipients is determined by standard pharmaceutical practices, as well as solubility and chemical properties of the active compound, the route of administration chosen.

The pharmaceutical compositions of the present invention may be formulated in a form suitable for the desired method of administration using suitable and physiologically acceptable excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants, flavoring agents and the like. Can be.

The pharmaceutical composition may be formulated in the form of, but not limited to, tablets, capsules, pills, granules, powders, injections or solutions.

Formulations suitable for oral administration include solid formulations, such as capsules containing tablets, particulates, liquids or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates. Agents, gels, solid solutions, liposomes, films (including muco-adhesives), ovules, sprays and liquid formulations. Liquid formulations include, for example, suspensions, solutions, syrups and elixirs.

For tablet dosage forms, depending on the dosage, the drug may comprise from 1% to about 80%, more typically from about 5% to about 60% by weight of the dosage form. Tablets generally contain disintegrants in addition to drugs. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydrides. Oxypropyl cellulose, starch, pregelatinized starch and sodium alginate. In general, the disintegrant will comprise, but is not limited to, about 1% to about 25% by weight of the dosage form, preferably about 5% to about 20% by weight of the dosage form.

Binders are generally used to impart tack to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycols, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose . The tablets may also be diluted with diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrides, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate. May comprise water.

Tablets may also optionally include surfactants such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. If present, the surfactant may comprise from about 0.2% to about 5% by weight of the tablet, and the lubricant may comprise from about 0.2% to about 1% by weight of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. Lubricants generally comprise from about 0.25% to about 10%, preferably from about 0.5% to about 3% by weight of the tablet.

Other possible ingredients include antioxidants, colorants, flavors, preservatives and taste-blockers.

Tablet formulations may be compressed directly or by roller to form tablets. Alternatively, the tablet blend, or a portion of the blend, may be wet-, dry-, melt-granulated, melt congealed, or extruded before tableting. The final formulation may comprise one or more layers, may or may not be coated and may be encapsulated.

Tablet formulations are discussed in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X).

Solid formulations for oral administration may be formulated in immediate release and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release forms.

Formulations of the pharmaceutical compositions and pharmaceutically acceptable carriers may be appropriately selected according to techniques known in the art, for example, see Urquhart et al., Lancet, 16: 367, 1980; Lieberman et al., PHARMACEUTICAL DOSAGE FORMS-DISPERSE SYSTEMS, 2nd ed., Vol. 3, 1998; Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS, 7th ed., 2000; Martindale, THE EXTRA PHARMACOPEIA, 31 st ed .; Remington's PHARMACEUTICAL SCIENCES, 16th-20th editions; THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, eds., 9th ed., 1996; Wilson and Gisvolds' TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, eds., 10th ed., 1998. In addition, the principles of formulating pharmaceutical compositions are also described, for example, in Platt, Clin Lab Med, 7: 289-99, 1987; Aulton, PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone, NY, 1988; [EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP, 1998], "Drug Dosage," J Kans Med Soc, 70 (1): 30-32, 1969, and the like.

In one embodiment, the pharmaceutical composition of the present invention may be a tablet. Tablets may optionally be film coated. The total amount of drug per dosage unit can be that amount that provides a dosage form of a convenient size to the patient.

In one embodiment, the pharmaceutical compositions of the present invention may be formulated in the form of sustained release tablets.

For the production of sustained-release tablets, a component selected from an enteric polymer, a hydrophobic substance, a hydrophilic polymer, and the like can be used as the matrix base.

The enteric polymer may be a mixture of one or more selected from polyvinylacetate phthalate, methacrylic acid copolymer, hydroxypropylmethylcellulose phthalate, shellac, cellulose acetate phthalate, cellulose propionate phthalate, Eudragit L, Eudragit S, and the like. May be used, and preferably hydroxypropylmethylcellulose phthalate may be used.

The hydrophobic materials are pharmaceutically acceptable polyvinyl acetate, poly (methacrylate, methyl methacrylate) copolymer, poly (ethylacrylate, methyl methacrylate, trimethylaminoethylmethacrylate as polymethacrylate copolymers). Acrylates) copolymers, ethyl cellulose and cellulose acetate, fatty acids and fatty acid esters, fatty alcohols, waxes and inorganic substances, and the like, specifically, glyceryl palmitostearate, Fatty acid alcohols such as glyceryl stearate, glyceryl bihenate, cetyl palmitate, glyceryl monooleate and stearic acid; waxes such as cetostearyl alcohol, cetyl alcohol and stearyl alcohol; carnauba wax, beeswax and microcrystalline wax Talc, precipitated coal as inorganic materials Can be used to select one or more selected from calcium, calcium hydrogen phosphate, zinc oxide, titanium oxide, kaolin, bentonite, montmorillonite and bigeom.

The hydrophilic polymer may be selected from sugars, cellulose derivatives, gums, proteins, polyvinyl derivatives, polymethacrylate copolymers, polyethylene derivatives and carboxyvinyl polymers, and specifically, as the sugars, dextrin, polydextrin, dextran, Pectin and pectin derivatives, alginates, polygalacturonic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose, amylopectin and the like can be selected and used as cellulose derivatives. , Hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl methyl cellulose acetate succinate, hydroxyethyl methyl cellulose can be selected and used as a gum , By Custard bean gum, tragacanta, carrageenan, acacia gum, gum arabic, gellan gum, xanthan gum, and the like can be selected. Gelatin, casein, zein, etc. can be selected and used as proteins. Alcohol, polyvinyl pyrrolidone, polyvinyl acetal diethylamino acetate, and the like can be selected and used as the polymethacrylate copolymer, and poly (butyl methacrylate, (2-dimethylaminoethyl) methacrylate, methyl methacrylate Rate) copolymer, poly (methacrylic acid, methyl methacrylate) copolymer, poly (methacrylic acid, ethyl acrylate) copolymer, etc. can be selected and used, and polyethylene glycol, polyethylene oxide, etc. are selected as a polyethylene derivative. Carbomer may be used as the carboxyvinyl polymer.

The pharmaceutical composition of the present invention may be used alone or in combination with other drugs for anticancer use.

In one embodiment, the pharmaceutical composition may be for use in combination with a second drug.

In the present invention, the "second drug" means another pharmaceutically effective ingredient other than the metformin acid addition salt of the present invention. Metformin acid addition salts of the present invention may be used in combination with a second drug for efficient treatment of cancer. For example, the second drug may be an anticancer agent, an antihyperglycemic agent, or the like.

In one embodiment, the second drug may be an anticancer agent. The anticancer agent for use in combination with metformin acid addition salt according to the present invention may be any known anticancer agent. Examples include known chemotherapeutic agents such as alkylating agents, metabolic antagonists, natural agents, hormones and antagonists, and biological agents such as immunotherapy agents, gene therapy agents, and the like. In one embodiment, the anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin , Cetuximab, biscumalboom, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramastine, gemtuzumab ozogamycin, ibritumab tucetan, heptaplatin, methylaminolevulinic acid, Amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegapur, capecitabine, gimerasin, otheracil, azacytidine , Methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enositabine, decitabine, mercaptopurine, thioguanine, cladribine, carmorpher, raltitrexed, docetaxel, paclitaxel, irinotecan, velotecan Cannes, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactino Mycin, pyrarubicin, aclarubicin, pepromycin, temozolomide, busulfan, iphosphamide, cyclophosphamide, melfaran, altretmin, dacarbazine, cheotepa, nimustine, chlorambucil And one or more drugs selected from the group consisting of mitolactol, romustine and carmustine.

If the metformin acid addition salt according to the invention and the second drug can be administered in the same manner, the metformin acid addition salt may also be provided in the form of a combined formulation formulated with the second drug.

Meanwhile, in the present invention, the 'subject' means a warm-blooded animal such as a mammal suffering from a specific disease, disorder or disease, and for example, human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig. , Chlorine, sheep, and the like, but are not limited to these examples.

The pharmaceutical composition of the present invention may be used in a subject via, for example, oral, intravenous, intraarterial, intraperitoneal, intramuscular, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, intraocular or intradermal routes. It can be administered in a conventional manner.

"Treatment" also includes, but is not limited to, alleviating the symptom, removing the cause of the symptom temporarily or permanently, or preventing or slowing the appearance of the symptom and the progression of the disease, disorder or condition described above.

An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to achieve the treatment of the disease. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. For example, in adults, metformin acid addition salt may be administered in a dose of 50 to 3000 mg in total, once to several times daily.

The present invention also provides an aminooxyacetic acid salt of metformin having the structure of formula (II):

[Formula 2]

In addition, the present invention also

To reacting the metformin free base of Formula 3 with aminooxyacetic acid

It provides a method for preparing the aminooxyacetic acid salt of metformin having the structure of formula (2).

 [Formula 3]

[Formula 2]

The production method can be applied in the same manner to the preparation of acid addition salts of metformin, for example, dichloroacetic acid salt of metformin, to which an organic acid other than aminooxyacetic acid is added.

The free base of metformin used to form the acid addition salt of metformin can be obtained commercially or synthesized by known methods.

In the following examples, a metformin free base is formed by reacting a known metformin acid addition salt such as metformin hydrochloride with an inorganic base. The prior art requires harsh production conditions when synthesizing metformin free base, which is difficult to produce using an ion exchange resin column to remove hydrochloric acid from metformin hydrochloride, and the solvent is heated to reflux and filtered under hot conditions (US Patent 4,080,472). In the present invention, it is possible to synthesize an acid addition salt of metformin at a lower unit cost by simply improving the process so that synthesis in a general production facility without special facilities is possible.

Known metformin acid addition salts used for the formation of the organic base of metformin are, for example, hydrochloric acid, sulfates, nitrates, phosphates, sulfites, dithionates, acetates, benzoates, citrate, glycorate salts, glycosides. Oxylate, mercaptoacetate, gammahydroxybutyrate, pamoate, aspartate, glutamate, pyrrolidone carboxylate, methanesulfonate, naphthalene sulfonate, glucose-1-phosphate, chlorophenoxy acetate, embonate, chloro Phenoxy acetate, maleate, parachlorophenoxyisobutyrate, formate, lactate, succinate, tartrate, cyclohexane carboxylate, hexanoate, octanoate, decanoate, hexadecanoate, octode Decanoate, benzenesulfonate, trimethoxy benzoate, paratoluene Sulfonate, ah it may adamantan carboxylate, glutamate, pyrrolidone carboxylate, words of acid and carbonate, malate, acid addition salt is selected from the oxalate and so on.

In one embodiment, the inorganic base used to form the free base of metformin can be selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydrogen carbonate and potassium hydrogen carbonate.

When reacting a metformin acid addition salt and an inorganic base, water or a normal organic solvent can be used.

The free base of metformin thus prepared is reacted with an organic acid to form an acid addition salt of metformin.

In one embodiment, the metformin free base may react with 0.5 to 4 molar equivalents of organic acid per molar equivalent.

The reaction of the metformin with the free base and the organic acid can be carried out using water or a conventional organic solvent.

Organic solvents that can be used in the process for the preparation of acid addition salts of metformin according to the present invention are not limited to methanol, ethanol, isopropanol, butanol, pentanol, dioxane, dimethylformamide, dimethylsulfoxide, acetone and acetonitrile. It may be selected from the group consisting of.

When the reaction between the free base of metformin and the organic acid is carried out under an organic solvent, the organic acid is added to the free base of metformin, and the resulting solid is filtered, washed, and dried to form a crystalline acid addition salt of metformin.

On the other hand, when the reaction between the free base of metformin and the organic acid is carried out in an aqueous solution, the organic acid is added to the free base of metformin, and then washed and filtered through the drying process such as freeze drying, vacuum drying, hot air drying And drying to form the crystalline acid addition salt of metformin.

The reaction and the solid production temperature may be carried out in a temperature range of -10 to 100 ℃.

The invention also

Diabetes, obesity, hyperlipidemia, fatty liver, coronary artery disease, osteoporosis, polycystic ovary syndrome, metabolic syndrome, myalgia, muscle comprising a pharmaceutically acceptable carrier and aminooxyacetate of metformin having the structure of Formula 2 as an active ingredient Provided are pharmaceutical compositions for the treatment or prevention of diseases selected from the group consisting of cell damage and rhabdomyolysis:

[Formula 2]

It is known in the art that metformin can be used to activate AMPK to treat diseases such as diabetes, obesity, hyperlipidemia, fatty liver, coronary artery disease, osteoporosis, polycystic ovary syndrome, metabolic syndrome, myalgia, muscle cell damage and rhabdomyolysis It is well known. Metformin aminooxyacetate according to the present invention, in addition to this pharmacological effect of metformin, greatly enhances the neutral lipid lowering action, it is more effective than metformin hydrochloride to lower blood sugar, especially fasting as well as postprandial blood sugar lowering and insulin sensitivity Increases the effect.

The pharmacological effects of metformin dichloroacetate and metformin aminooxyacetate according to the present invention can be determined in various ways in vivo and in vitro. As a result, the metformin acid addition salt according to the present invention shows a significantly enhanced anticancer effect compared to metformin. In addition, physicochemical properties such as solubility, stability, non-hygroscopicity, and processability as tablet formulations are superior to metformin hydrochloride, and the toxicity is low, and thus may be usefully used as a pharmaceutically acceptable salt of metformin.

Figure 1 shows the effect of AMPKα activation of metformin dichloroacetate (DCA).

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

EXAMPLE

Example 1 Preparation of Metformin Free Base

16.6 g of metformin hydrochloride and 6.0 g of 93% potassium hydroxide are added to 50 mL of isopropanol and stirred at 50 ° C. for 2 hours. The reaction solution was cooled to 25 ° C., filtered, washed with 20 mL of isopropanol, and washed once more with 20 mL of acetone. The filtrate was concentrated and dried in vacuo to give 12.8 g (yield yield: 98.5%) of a white solid metformin freebase. Melting Point 119.0 ~ 119.5 ℃

¹ H-NMR (600 MHz, D ₂ O) δ (ppm) 3.07 (s, 6H)

¹³ C-NMR (150 MHz, D ₂ O) δ (ppm) 161.05, 158.5, 37.35

Example 2 Preparation of Metformin Dichloroacetate

30 ml of isopropanol was added to 8.0 g (1.0 equiv) of metformin free base prepared in Example 1, and dissolved. Dichloroacetic acid 8.0g (1.0 equiv) was slowly added dropwise while stirring the reaction solution, followed by stirring at 50 ° C for 2 hours. After 200 mL of ethyl acetate was added dropwise to form crystals, the resulting crystals were filtered and washed with 20 mL of isopropanol and 50 mL of acetone. The filtrate was hot-air dried to obtain 14.4 g of a white solid metformin dichloroacetate (yield yield: 90.0%). Melting Point 154.6 ℃

¹ H-NMR (600 MHz, D ₂ O) δ 6.33 (s, 1H), 2.47 (s, 6H)

¹³ C-NMR (150 MHz, D ₂ O) δ (ppm) 170.6, 163.0, 66.2, 34.5

Example 3 Preparation of Metformin Aminooxyacetate

30 ml of isopropanol was added to 8.0 g (1.0 equiv) of metformin free base prepared in Example 1, and dissolved. 5.7 g (1.0 equivalent) of aminooxyacetic acid was slowly added dropwise while stirring the reaction solution, followed by stirring at 50 ° C for 2 hours. After 200 mL of ethyl acetate was added dropwise to form crystals, the resulting crystals were filtered and washed with 20 mL of isopropanol and 50 mL of acetone. The filtrate was hot-air dried to obtain 11.6 g of a white solid metformin oxyacetate (yield yield: 85.0%). Melting Point 164.0 ℃

¹ H-NMR (600MHz, D ₂ O) δ 4.47 (s, 2H), 2.47 (s, 6H)

¹³ C-NMR (150 MHz, D ₂ O) δ (ppm) 172.9, 163.0, 79.8, 34.5

Experimental Example  1: metformin Dichloroacetic acid  Check anticancer effect

Metformin dichloroacetic acid synthesized in the manner described in Example 2 of the present invention was treated with cancer cells to determine the effect of inhibiting cancer cell proliferation. A brief experimental method is as follows.

MCF7 cells derived from human breast cancer were used, and the survival rate of cells by metformin dichloroacetate (DCA) using MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-ditetrazoliumbromide) reagent (%) And the concentration at which cell growth is inhibited to 50% (cell growth inhibitory concentration, GIC50) were measured to determine the cancer cell proliferation inhibitory effect of metformin dichloroacetate (DCA).

MCF7 (Korea Cell Line Bank) cells were placed in a 96 well plate in a DMEM medium containing 10% calf serum so that the number of cells is about 5000 and incubated for about 24 hours. Thereafter, in order to confirm cell viability, metformin dichloroacetate salt (DCA) synthesized in Example 2 was treated with 2 mM and 10 mM of the culture medium, respectively, and cultured for 72 hours. To obtain GIC ₅₀ , metformin dichloroacetate (DCA) was treated with 10 mM, 2 mM, 0.4 mM, 0.08 mM, and 0.016 mM of the culture solution, and cultured for 72 hours.

MTT was added to the cultures and further incubated for 3 hours to identify live cells after metformin dichloroacetate (DCA) treatment. The resulting forazane crystal was dissolved using dimethyl sulfoxide (DMSO) and the absorbance of the solution was measured at 560 nm.

After 72 hours of incubation, the number of cells surviving in the well plate treated with metformin dichloroacetate (DCA) compared to the number of cells cultured in the well plate not treated with metformin dichloroacetate (DCA) was expressed as% cell viability. Indicated. In addition, the concentration of metformin dichloroacetate (DCA) (GIC 50) in which growth was inhibited to 50% was calculated using a cell viability curve to confirm the effect of inhibiting cancer cell proliferation of metformin dichloroacetate (DCA). It is shown in Table 2 and Table 3, respectively.

In addition, metformin hydrochloride or dichloroacetic acid (DCA) was used instead of metformin dichloroacetate (DCA) to obtain cell viability (%) and GIC 50 values, and the results are shown in Tables 2 and 3, respectively.

TABLE 2 Cell survival rate (%)

Cell survival rate (%)	MCF7
	2mM
	10 mM
Metformin dichloroacetate	83.5%	25.6%
Metformin hydrochloride	87.8%	71.9%
Dichloroacetic acid	103.6%	47.3%

TABLE 3 Cell growth inhibitory concentration (GIC 50)

GIC 50	MCF7
Metformin dichloroacetate	6.5mM
Metformin hydrochloride	> 10mM
Dichloroacetic acid	9.9mM

As can be seen from the cell viability data of Table 2, when the MCF7 cells were cultured in the presence of metformin dichloroacetate, the cell viability was lower than when cultured under metformin hydrochloride, from which metformin dichloroacetate metformin It was found that the survival of cancer cells, particularly breast cancer cells, is more effectively suppressed than hydrochloride.

In addition, as can be seen from the GIC 50 value of Table 3, the metformin dichloroacetate treated group can inhibit the growth of MCF7 cells by 50% at a concentration of 6.5 mM, whereas the metformin hydrochloride treated group treated 10 mM Even if the growth of the cells did not inhibit more than 50%. Therefore, the use of metformin dichloroacetate less than metformin hydrochloride can effectively inhibit the growth of cancer cells derived from breast cancer.

Experimental Example  2: metformin Of aminooxyacetates  Check anticancer effect

Using metformin aminooxyacetate (AOA) instead of metformin dichloroacetate, the cell viability (%) and GIC 50 values were determined in the same manner as in Experimental Example 1 and the results are shown in Tables 4 and 5, respectively.

Table 4

Cell survival rate (%)	MCF7
	2mM
	10 mM
Metformin aminooxyacetic acid (AOA) salt	68.0%	40.1%
Metformin hydrochloride	87.8%	71.9%

Table 5

GIC 50	MCF7
Metformin aminooxyacetic acid (AOA) salt	5.1mM
Metformin hydrochloride	> 10mM

As can be seen from the cell viability data of Table 4, when MCF7 cells were cultured in the presence of metformin aminooxyacetic acid (AOA) salt, the cell viability was lower than when cultured under metformin hydrochloride, from which metformin amino It was found that oxyacetic acid (AOA) salts more effectively inhibit the survival of cancer cells, particularly breast cancer cells, than metformin hydrochloride.

In addition, as can be seen from the GIC 50 value of Table 5, the metformin aminooxyacetic acid (AOA) salt treatment group can inhibit the growth of MCF7 cells by 50% at a concentration of 5.1 mM, while the metformin hydrochloride treatment group was 10 mM. Treatment did not inhibit cell growth by 50%. Therefore, the use of metformin aminooxyacetic acid (AOA) salt in a smaller amount than metformin hydrochloride can effectively inhibit the growth of cancer cells derived from breast cancer.

Experimental Example  3: metformin Dichloroacetic acid AMPK Confirmation of α's activation effect

Metformin dichloroacetate (DCA) synthesized in the manner described in Example 2 of the present invention was treated with cells to determine the effect of activating AMPKα. A brief experimental method is as follows.

MCF7 cells derived from human breast cancer cells were used, and AMPKα (5'-AMP-activated protein kinase alpha) activation effect by metformin dichloroacetate (DCA) was activated using the AMPKα immunoassay kit (Invitrogen, catalog No. KHO0651). Confirmed.

MCF7 (Korea Cell Line Bank) cells were cultured in DMEM medium containing 10% calf serum, placed in a 6-well plate so that the number of cells was about 5 × 10 ⁵ , and then in a incubator fed with 5% CO ₂ . Cells were cultured. The culture solution was treated with 2 mM and 10 mM metformin dichloroacetate, respectively, and then incubated for 24 hours. After lysing the cells by the method described in the instructions for use of the AMPKα immunoassay kit (Invitrogen, catalog No. KHO0651), 20 ug of cell lysate was obtained by protein quantification, and then the AMPKα immunoassay kit According to the method, the phosphorylated degree of threonine 172 residue (Thr172) of AMPKα was confirmed from the cell lysate, and the results are shown in Table 6 and FIG. 1. The ratio of the degree of AMPKa activation by metformin dichloroacetate (DCA) is phosphorylated in cells cultured in the presence of metformin dichloroacetate (DCA) compared to phosphorylated AMPKa in cells cultured without metformin dichloroacetate (DCA). The degree of AMPKa is shown.

In addition, the degree of phosphorylation of threonine 172 residue (Thr172) of AMPKa was confirmed in the same manner using metformin hydrochloride instead of metformin dichloroacetate (DCA), and the results are shown in Table 6 below.

Table 6

		0 mM	2mM	10 mM
Metformin dichloroacetate	Measure (unit / ml)	11.95 ± 0.50	19.70 ± 1.27	31.10 ± 3.68
	ratio	1.0	1.6	2.6
Metformin hydrochloride	Measures	11.95 ± 0.50	18.20 ± 0.71	19.95 ± 2.62
	ratio	1.0	1.5	1.7

As can be seen in Table 6 and FIG. 1, it was found that metformin dichloroacetate phosphorylated more threonine 172 residues of AMPKα than metformin hydrochloride at 10 mM. Therefore, metformin dichloroacetate (DCA) activates AMPKα more efficiently than metformin hydrochloride, and is accompanied by metabolic syndrome, which is a combination of diabetes, obesity, hypertension, hyperlipidemia, fatty liver, coronary artery disease, osteoporosis, and polycystic ovary syndrome. It can be seen that it can have an excellent effect on diseases such as diabetes and its complications, cancer, myalgia, muscle cytotoxicity and rhabdomyolysis.

Claims (8)

1. A pharmaceutical composition for anticancer comprising a pharmaceutically acceptable carrier and metformin acid addition salt having a structure of Formula 1 as an active ingredient:

   [Formula 1]

   

   Where

   A is dichloroacetic acid or aminooxyacetic acid.
2. The method of claim 1,

   The pharmaceutical composition is formulated in the form of tablets, capsules, pills, granules, powders, injections or solutions.
3. The method of claim 1,

   The pharmaceutical composition is for use in combination with a second drug.
4. The method of claim 3,

   The second drug is an anticancer agent.
5. The method of claim 4, wherein

   The anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, Biscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramastine, gemtuzumab ozogamycin, ibritumab tucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtu Zumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluidine, pemetrexed, tegapur, capecitabine, gimerasine, oterasyl, azacytidine, methotrexate, uracil, Cytarabine, Fluorouracil, Fludagabine, Enositabine, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Carmofer, Raltitrexed, Docetaxel, Paclitaxel, Irinotecan, Velotecan, Topotecan, Norelvin, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pyrarubicin , Aclarubicin, pepromycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melfaran, altretmin, dacarbazine, chiotepa, nimustine, chlorambucil, mitolactol, romu A pharmaceutical composition that is one or more drugs selected from the group consisting of stin and carmustine.
6. Aminooxyacetic acid salt of metformin having the structure

   [Formula 2]

   
7. To reacting the metformin free base of Formula 3 with aminooxyacetic acid

   A method for producing an aminooxyacetic acid salt of metformin having the structure of Formula 2 below.

   [Formula 3]

   

   [Formula 2]

   
8. The method of claim 7, wherein

   The metformin free base is reacted with 0.5 to 4 molar equivalents of aminooxyacetic acid per molar equivalent.

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