Glutamine metabolism inhibitor

Abstract

The present invention provides: an inhibitor that inhibits α-ketoglutaric acid production, the inhibitor comprising a fluorine-containing glutamine or a pharmacologically acceptable salt thereof as an active ingredient, the fluorine-containing glutamine having a structure in which one or more hydrogen atoms bound to a carbon atom in glutamine are individually substituted with a fluorine atom; an inhibitor that inhibits a glutaminase, the inhibitor comprising a fluorine-containing glutamine or a pharmacologically acceptable salt thereof as an active ingredient, the fluorine-containing glutamine having a structure in which one or more hydrogen atoms bound to a carbon atom in glutamine are individually substituted with a fluorine atom; an inhibitor that inhibits a glutamate dehydrogenase, the inhibitor comprising a fluorine-containing glutamine or a pharmacologically acceptable salt thereof as an active ingredient, the fluorine-containing glutamine having a structure in which one or more hydrogen atoms bound to a carbon atom in glutamine are individually substituted with a fluorine atom; and a pharmaceutical composition comprising any of the above-mentioned inhibitors.

Description

Glutamine metabolism inhibitors

[0001] The present invention relates to an inhibitor that inhibits glutamine metabolism and a pharmaceutical composition containing the inhibitor. This application claims priority to Japanese Patent Application No. 2024-099052, filed on June 19, 2024, the contents of which are incorporated herein by reference.

[0002] Fluorine-containing amino acids have been reported to exhibit unique physiological activities and have attracted attention. For example, it has been reported that 3,3,3-trifluoroalanine and its derivatives act as suicide inhibitors of pyridoxal enzymes (Non-Patent Document 1). It has been reported that alanine racemase in the gram-negative bacterium Salmonella typhimurium and the gram-positive bacterium Bacillus stearothermophilus is inactivated by 3,3,3-trifluoroalanine (Non-Patent Document 2). Fluorine-containing amino acids and peptides containing them are expected to be used as physiologically active substances in the pharmaceutical field.

[0003] In pharmaceuticals, it is important to deliver pharmacologically active substances to target molecules present in target cells. Lipid nanoparticles (Patent Document 1) and cationic polymer nanoparticles (Patent Document 2) are known as drug delivery agents for pharmacologically active substances. Compounds with a polyfluoro structure are known to be stable and low in toxicity in vivo, and to be excellent at being taken up into cells and escaping from endosomes (Non-Patent Document 3).

[0004] On the other hand, glutamine is an important metabolic fuel involved in the supply of ATP, biosynthetic precursors, reducing agents, etc. Glutamine taken up into cells is metabolized to glutamic acid by glutaminase in mitochondria, and then metabolized to α-ketoglutaric acid by glutamate dehydrogenase. α-Ketoglutaric acid is an organic acid that constitutes the TCA cycle and plays an important role in energy metabolism. For example, in rapidly proliferating cells such as cancer cells, glutamine is an important energy source, and they take up large amounts of glutamine to produce energy and necessary substances for active proliferation. It has been reported that in pancreatic ductal adenocarcinoma, enhanced glutaminolysis increases the oxygen consumption rate via mitochondrial oxidative phosphorylation, inducing hypoxia and promoting chemotherapy resistance, and that inhibiting glutaminolysis alleviates hypoxia and improves the efficacy of chemotherapy (Non-Patent Document 4).

[0005] International Publication No. 2011 / 036557 International Publication No. 2017 / 212006

[0006] Sakai et al., Tetrahedron, 1996, vol.52(1), p.233-244.Faraci and Walsh, Biochemistry, 1989, vol.28(2), p.431-437.Zhang et al., MRS Communications, 2018, vol.8, p.303-313.Park et al., CANCER RESEARCH METABOLISM AND CHEMICAL BIOLOGY, 2023, vol.83(5), p.735-752.Kondratov et al., Journal of Fluorine Chemistry, 2018, vol.211, p.100-108.Meffre et 1l., Tetrahedron Letters, 2001, vol.42, p.8625-8627.

[0007] An object of the present invention is to provide an inhibitor that has excellent cell membrane permeability and inhibits glutamine metabolism, and a pharmaceutical composition containing said inhibitor.

[0008] The present inventors have found that fluorine-containing glutamine, in which one or more hydrogen atoms bonded to carbon atoms in the side chain of glutamine are substituted with fluorine atoms, has excellent cell membrane permeability, ability to inhibit the growth of cancer cells, and ability to inhibit glutamine metabolism in cells, and have completed the present invention.

[0009] That is, the present invention is as follows: [1] An inhibitor which inhibits the production of α-ketoglutaric acid, comprising as an active ingredient a fluorine-containing glutamine in which one or more hydrogen atoms bonded to a carbon atom of glutamine have been substituted with a fluorine atom, or a pharmacologically acceptable salt thereof. [2] An inhibitor which inhibits glutaminase, comprising as an active ingredient a fluorine-containing glutamine in which one or more hydrogen atoms bonded to a carbon atom of glutamine have been substituted with a fluorine atom, or a pharmacologically acceptable salt thereof. [3] An inhibitor which inhibits glutamate dehydrogenase, comprising as an active ingredient a fluorine-containing glutamine in which one or more hydrogen atoms bonded to a carbon atom of glutamine have been substituted with a fluorine atom, or a pharmacologically acceptable salt thereof. [4] The inhibitor of any of the above [1] to [3], wherein the fluorine-containing glutamine is one or more selected from the group consisting of 3-monofluoroglutamine, 4-monofluoroglutamine, 3,3-difluoroglutamine, and 4,4-difluoroglutamine. [5] A pharmaceutical composition containing the inhibitor of any of [1] to [3] above. [6] An anticancer agent having as an active ingredient a fluorine-containing glutamine in which one or more hydrogen atoms bonded to a carbon atom of glutamine are substituted with a fluorine atom, or a pharmacologically acceptable salt thereof, wherein the fluorine-containing glutamine is one or more species selected from the group consisting of 4,4-difluoroglutamine and 3,3-difluoroglutamine. [7] A pharmaceutical composition containing the anticancer agent of [6] above. [8] 3,3-difluoroglutamine.

[0010] The inhibitor according to the present invention has excellent cell membrane permeability and glutamine metabolism inhibitory activity, and is useful as an inhibitor of α-ketoglutaric acid production, glutamic acid production by glutaminase, and α-ketoglutaric acid production by glutaminase. The anticancer agent according to the present invention is an excellent anticancer agent that has excellent cell membrane permeability and inhibitory activity against cancer cell proliferation, and can be easily taken up by target cancer cells.

[0011] FIG. 1 shows the results of metabolomic analysis of HeLa cells to which glutamine, (2S,4S)4-fluoroglutamine, or (2S,4R)4-fluoroglutamine was added in Example 3. FIG. 2 shows the results of metabolomic analysis of HeLa cells to which glutamine or 4,4-difluoroglutamine was added in Example 3. FIG. 3 shows the results of metabolomic analysis of AsPC-1 cells to which glutamine or 4,4-difluoroglutamine was added in Example 3.

[0012] In the present invention and this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The term "halogen atom other than a fluorine atom" refers to a chlorine atom, a bromine atom, or an iodine atom. Preferred examples of "halogen atoms other than a fluorine atom" include a chlorine atom or a bromine atom, with a chlorine atom being particularly preferred.

[0013] The inhibitor of the present invention comprises, as an active ingredient, fluorine-containing glutamine, or a pharmacologically acceptable salt thereof, in which one or more hydrogen atoms bonded to a carbon atom of glutamine are substituted with fluorine atoms. Due to the presence of fluorine atoms, the fluorine-containing glutamine has excellent cell membrane permeability. Therefore, the anticancer agent of the present invention can be introduced into target cells simply by contacting the cell surface with the cell. The fluorine-containing glutamine has a high inhibitory activity against enzymes that mediate glutamine metabolic reactions, and is particularly excellent in inhibitory activity against glutaminase (GLS) (EC 3.5.1.2) and glutamate dehydrogenase (GDH) (EC 1.4.1.3). Therefore, in cells into which the inhibitor of the present invention has been incorporated, α-ketoglutaric acid production is inhibited, resulting in a decrease in energy production by the TCA cycle. That is, the inhibitor of the present invention is useful as an α-ketoglutaric acid production inhibitor, glutaminase inhibitor, glutamate dehydrogenase inhibitor, etc.

[0014] When the target enzyme inhibited by the inhibitor according to the present invention is glutaminase, examples of human glutaminase include glutaminase 1 (GLS1), glutaminase 2 (GLS2), etc. When the target enzyme inhibited by the inhibitor according to the present invention is glutamate dehydrogenase, examples of human glutamate dehydrogenase include glutamate dehydrogenase 1 (GLUD1), glutamate dehydrogenase 2 (GLUD2), etc.

[0015] Glutamine metabolism is important for energy metabolism required for cell proliferation. When glutamine metabolism is inhibited and the amount of α-ketoglutaric acid synthesized decreases, energy production by the TCA cycle decreases, which in turn reduces the production of various components required for proliferation and cell proliferation. Therefore, the inhibitor of the present invention is also useful as an anticancer agent. Anticancer agents containing fluorine-containing glutamine as an active ingredient have excellent cell membrane permeability and can be introduced into target cancer cells simply by contacting them with the surface of the cancer cells. The fluorine-containing amino acid has a high ability to inhibit the proliferation of cancer cells and also has an excellent antitumor effect. Note that the fluorine-containing amino acid also exhibits a proliferation inhibitory effect on normal cells, but the effect is weaker than that on cancer cells.

[0016] The fluorine-containing glutamine used as the active ingredient of the inhibitor according to the present invention or the anticancer agent according to the present invention may be a compound in which one or more hydrogen atoms bonded to carbon atoms of glutamine have been substituted with fluorine atoms, but a compound in which one or more hydrogen atoms bonded to carbon atoms constituting the side chain have been substituted with fluorine atoms is preferred, a fluorine-containing glutamine in which 1 to 6 hydrogen atoms bonded to carbon atoms constituting the side chain have been substituted with fluorine atoms is more preferred, and a fluorine-containing glutamine in which 1 or 2 hydrogen atoms bonded to carbon atoms constituting the side chain have been substituted with fluorine atoms is even more preferred. Examples of the fluorine-containing glutamine used in the present invention include (2S,4S)-4-fluoroglutamine ((4S)-4-fluoro-L-glutamine, CAS RN: 1262523-37-4), (2S,4R)-4-fluoroglutamine ((4R)-4-fluoro-L-glutamine, CAS RN: 238418-71-8), 4,4-difluoro-L-glutamine (CAS RN: 401915-19-3), 4,4-difluoro-D-glutamine (CAS RN: 2165985-98-6), 4,4-difluoroglutamine (CAS RN: 175548-95-5), and 3,3-difluoroglutamine.

[0017] The fluorine-containing glutamine used as the active ingredient of the inhibitor according to the present invention or the anticancer agent according to the present invention may be a compound in which one or more hydrogen atoms bonded to a carbon atom of L-glutamine are substituted with fluorine atoms, or a compound in which one or more hydrogen atoms bonded to a carbon atom of D-glutamine are substituted with fluorine atoms, or a mixture of both (racemate). In view of obtaining a higher growth inhibitory effect on cancer cells, the fluorine-containing glutamine used in the present invention is preferably a compound in which one or more hydrogen atoms bonded to a carbon atom of L-glutamine are substituted with fluorine atoms, more preferably a compound in which one or more hydrogen atoms bonded to a carbon atom constituting the side chain of L-glutamine are substituted with fluorine atoms, more preferably a compound in which one to six hydrogen atoms bonded to a carbon atom constituting the side chain of L-glutamine are substituted with fluorine atoms, even more preferably a compound in which one or two hydrogen atoms bonded to a carbon atom constituting the side chain of L-glutamine are substituted with fluorine atoms, and 4,4-difluoro-L-glutamine or 3,3-difluoro-L-glutamine is even more preferred, with 4,4-difluoro-L-glutamine being particularly preferred.

[0018] The method for synthesizing fluorine-containing glutamine is not particularly limited, and it can be synthesized by combining various known synthetic reactions. It is also preferable to use a commercially available fluorine-containing glutamine preparation. For example, monofluoroglutamine can be synthesized by treating glutamine with a fluorinating agent while protecting the carboxyl and amino groups. Protection of the carboxyl and amino groups and treatment with a fluorinating agent can be carried out by conventional methods. N-fluorobenzenesulfonimide (CAS RN: 133745-75-2) or the like can be used as the fluorinating agent. Difluoroglutamine can be synthesized by treating glutamine with a -CF ２ It can be synthesized by a building block synthesis method using a compound containing -.

[0019] In the fluorine-containing glutamine used in the present invention, the hydrogen atoms not substituted with fluorine atoms may be substituted with other substituents, as long as the desired anti-inhibitory effect or anti-tumor effect is not impaired. Examples of such other substituents include halogen atoms other than fluorine atoms, C １－６ Examples thereof include alkyl groups.

[0020] The inhibitor and anticancer agent according to the present invention may contain a pharmacologically acceptable salt of the fluorine-containing amino acid as an active ingredient. Examples of the pharmacologically acceptable salt include salts with bases such as inorganic bases and organic bases, and salts with acids such as inorganic acids and organic acids. Examples of inorganic bases include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum, ammonium, etc. Examples of organic bases include primary amines such as ethanolamine, secondary amines such as diethylamine and diethanolamine, and tertiary amines such as trimethylamine, triethylamine, and triethanolamine. Examples of inorganic acids include hydrochloric acid, phosphoric acid, nitric acid, and sulfuric acid. Examples of organic acids include acetic acid, citric acid, lactic acid, malic acid, maleic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0021] The inhibitor according to the present invention and the anticancer agent according to the present invention may contain one type of fluorine-containing glutamine as an active ingredient, or two or more types of fluorine-containing glutamine.

[0022] The inhibitors and anticancer agents of the present invention have excellent cell membrane permeability, and can be introduced into target cells simply by contacting them with the cells. When the target cells for the inhibitors and anticancer agents of the present invention are cultured cells, the agents can be introduced into the cells simply by adding the agents to a culture medium and culturing them. For animal tissue, the agents can be introduced into the cells that make up the tissue, for example, by spraying or applying a solution in which the inhibitors and anticancer agents of the present invention are dissolved onto the tissue surface.

[0023] The anticancer agent according to the present invention is particularly useful as a therapeutic agent for preventing or treating cancer, including, for example, adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, endometrial cancer, esophageal cancer, Ewing's tumor, gallbladder cancer, Hodgkin's disease, hypopharyngeal cancer, laryngeal cancer, lip and oral cancer, liver cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, testicular cancer, and thyroid cancer. Among these, pancreatic cancer, colon cancer, and breast cancer are particularly preferred, as their cell proliferation has been reported to depend primarily on glutaminolysis.

[0024] The inhibitors and anticancer agents according to the present invention can be used as prophylactic or therapeutic agents for various diseases including cancer in mammals, either as they are or by mixing them with pharmacologically acceptable carriers or the like to form pharmaceutical compositions.

[0025] Here, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used as pharmacologically acceptable carriers, and are compounded as, for example, excipients, lubricants, binders, disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. Pharmaceutical additives such as preservatives, antioxidants, coloring agents, sweeteners, etc. can also be used as needed.

[0026] Suitable examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate.

[0027] Suitable examples of the lubricant include magnesium stearate, calcium stearate, talc, and colloidal silica.

[0028] Suitable examples of binders include pregelatinized starch, sucrose, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and polyvinylpyrrolidone.

[0029] Suitable examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, light anhydrous silicic acid, and low-substituted hydroxypropyl cellulose.

[0030] Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, and cottonseed oil.

[0031] Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0032] Suitable examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, and polyoxyethylene hydrogenated castor oil.

[0033] Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, and glucose.

[0034] Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0035] A suitable example of a soothing agent is benzyl alcohol.

[0036] Suitable examples of the preservative include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0037] Suitable examples of antioxidants include sulfites and ascorbic acids.

[0038] Suitable examples of the coloring agent include water-soluble food tar dyes (e.g., food dyes such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), and natural dyes (e.g., β-carotene, chlorophyll, and red iron oxide).

[0039] Suitable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, and stevia.

[0040] Examples of dosage forms of the pharmaceutical composition include oral preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, and orally disintegrating tablets), capsules (including soft capsules and microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, and films (e.g., orally disintegrating films); and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and drip infusions), topical preparations (e.g., transdermal preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), and eye drops. These can be safely administered orally or parenterally (e.g., topically, rectally, or intravenously). These preparations may be immediate-release preparations or controlled-release preparations such as sustained-release preparations (e.g., sustained-release microcapsules).

[0041] In the case of oral preparations, coating may be carried out as necessary for the purposes of taste masking, enteric coating, or sustained release. Examples of coating bases used for coating include sugar coating bases, water-soluble film coating bases, enteric film coating bases, and sustained-release film coating bases.

[0042] As the sugar coating base, sucrose is used, and one or more substances selected from the group consisting of talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax, etc. may be used in combination.

[0043] Examples of water-soluble film coating bases include cellulose-based polymers such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, and methylhydroxyethyl cellulose; synthetic polymers such as polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E (Eudragit E (trade name)), and polyvinylpyrrolidone; and polysaccharides such as pullulan. Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, carboxymethylethyl cellulose, and cellulose acetate phthalate; acrylic acid-based polymers such as methacrylic acid copolymer L (Eudragit L (trade name)), methacrylic acid copolymer LD (Eudragit L-30D55 ​​(trade name)), and methacrylic acid copolymer S (Eudragit S (trade name)); and natural products such as shellac. Examples of sustained-release film coating bases include cellulose-based polymers such as ethyl cellulose; and acrylic acid-based polymers such as aminoalkyl methacrylate copolymer RS ​​(Eudragit RS (trade name)) and ethyl acrylate-methyl methacrylate copolymer suspension (Eudragit NE (trade name)).

[0044] The above coating bases may be used in combination of two or more kinds in an appropriate ratio. In coating, a light-shielding agent such as titanium oxide or iron sesquioxide may be used.

[0045] Pharmaceutical compositions containing the inhibitors according to the present invention or the anticancer agents according to the present invention can be produced by methods commonly used in the field of pharmaceutical formulation, for example, the methods described in the Japanese Pharmacopoeia, etc. The content of the anticancer agent according to the present invention in the pharmaceutical composition varies depending on the route of administration, dosage form, the intended dose of the anticancer agent according to the present invention, etc., but is, for example, about 0.1 to 100% by mass.

[0046] The dosage of the inhibitor according to the present invention or the anticancer agent according to the present invention varies depending on the subject of administration, the administration route, the target disease, symptoms, etc., but for example, when administered orally or parenterally to an adult patient, the dosage of the anticancer agent according to the present invention per dose is usually about 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.5 to 20 mg / kg body weight.

[0047] The inhibitor of the present invention, the anticancer agent of the present invention, and the pharmaceutical composition containing the same can be used as a single therapeutic agent or in combination with one or more other therapies. Such combined treatments may be performed simultaneously, separately, or over a period of time. For example, the anticancer agent of the present invention is preferably used in combination with one or more other cancer therapies.

[0048] Other cancer treatment methods include surgical therapy for physically removing and excising cancer cells, chemotherapy for administering a chemotherapeutic agent containing an active ingredient other than the anticancer agent of the present invention, immunotherapy, radiotherapy, etc. Examples of such chemotherapeutic agents include alkylating agents, platinum compounds, antimetabolites, topoisomerase inhibitors, microtubule inhibitors, antibiotics, etc. Examples of immunotherapy include a method for administering an immune checkpoint inhibitor.

[0049] The term "concurrently" when referring to a dosage form means that the administration and / or treatment of two or more active ingredients in the dosage form is at approximately the same time; by simultaneous administration, a subject is exposed to the administration and / or treatment of two or more active ingredients at the same time. When administered simultaneously, the two or more active ingredients may be administered as a fixed dose combination, or may be administered as a non-fixed dose combination (e.g., by using two or more different pharmaceutical compositions to be administered at approximately the same time by the same route of administration), or may be administered as a non-fixed dose combination using two or more different routes of administration, which exposes a subject to the administration and / or treatment of two or more active ingredients at substantially the same time.

[0050] "Fixed combination" when referring to a dosage form means that the dosage form is the administration of a single pharmaceutical composition having two or more active ingredients.

[0051] The term "separately" in relation to dosage forms means that the administration and / or treatment of two or more active ingredients is performed at different times in the dosage form. Simultaneous administration leads to a treatment phase in which a subject is simultaneously exposed to the administration and / or treatment of two or more active ingredients (e.g., for at least 1 hour, particularly at least 6 hours, and especially at least 12 hours), while separate administration can also lead to a treatment phase in which a subject is only exposed to one of the administrations and / or treatments of two or more active ingredients for a certain period of time (e.g., for at least 12 hours, particularly at least one day). Separate administration particularly means that at least one of the active ingredients is administered and / or treated at a periodicity that is substantially different from daily administration (e.g., once or twice a day). For example, one active ingredient can be administered and / or treated once or twice a day, and another, for example, every other day, once a week, or at longer intervals.

[0052] Administration "over a period of time" refers to the sequential administration of two or more active ingredients and / or treatments at different times. This term particularly refers to administration in which one active ingredient and / or treatment is fully administered before one or more other active ingredients are administered. In this case, administration of one active ingredient and / or treatment can be for several months before administration of the other active ingredient and / or treatment.

[0053] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0054] [Production Example 1] 3,3-Difluoroglutamine was synthesized by the following reaction.

[0055]

[0056] Compound 1 was synthesized according to the literature (J. Org. Chem. 1995, 60, 6289-6295). To a solution of compound 1 (2.0 g, 6.5 mmol, 1.0 equivalent) in chloroform (22 mL), ammonium bicarbonate (2.6 g, 32.5 mmol, 5.0 equivalents) and 2-ethoxycarbonyl-1,2-dihydroquinoline (2.4 g, 9.8 mmol, 1.5 equivalents) were added in that order, and the mixture was stirred overnight at room temperature. The resulting reaction mixture was diluted with dichloromethane (160 mL) and washed three times with 0.5 mol / L hydrochloric acid (30 mL) and once with saturated brine (30 mL). The organic layer was then dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:2 (volume ratio)). Thereafter, reprecipitation (dichloromethane, hexane) was carried out to obtain the target compound 2 (430 mg, 21%) as a white solid.

[0057] Compound 2: １ H NMR (400MHz, CDCl ３ ) δ 4.88 (dt, J=15.4, 9.8Hz, 1H), 4.42 - 4.20 (m, 2H), 3.10 - 2.88 (m, 1H), 1.48 (s, 9H), 1.32 (t, J=7.1Hz, 3H), 5.48 (s, 1H), 5.53 (s, 1H), 6.61 (s, 1H). １９F NMR (376MHz, CDCl ３ ) δ -102.0 - -102.2 (m, 2F).

[0058]

[0059] Compound 2 (430 mg, 1.4 mmol) was added with 6N hydrochloric acid (5.5 mL) and heated under reflux for 8 hours. After the resulting reaction solution cooled, it was concentrated and reprecipitated (methanol, dichloromethane) to obtain the target compound 3 (60 mg, 24%) as a white solid.

[0060] Compound 3: １ H NMR (400MHz, D ２ O) δ 4.55 (dd, J=24.7, 3.7Hz, 1H), 3.58 - 3.34 (m, 2H). １９ F NMR (376MHz, D ２ O) δ -98.29 (dddd, J=254.3, 23.1, 19.0, 3.8Hz, 1F), -103.71 (dddd, J=254.6, 24.2, 13.3, 10.5Hz, 1F).

[0061] Example 1: The effect of fluorinated glutamine, in which one hydrogen atom bonded to a carbon atom in the side chain of glutamine was replaced with a fluorine atom, on cancer cell proliferation was examined. The fluorinated glutamines used were (2S,4S)4-fluoroglutamine [(2S,4S)Gln(1F)], (2S,4R)4-fluoroglutamine [(2S,4R)Gln(1F)], 4,4-difluoroglutamine [4,4-diF-Gln], and 3,3-difluoroglutamine [3,3-diF-Gln]. (2S,4S)4-fluoroglutamine and (2S,4R)4-fluoroglutamine were purchased from Namiki Shoji Co., Ltd. 4,4-Difluoroglutamine was synthesized according to the method of Kondratov et al. (Non-Patent Document 5). 3,3-Difluoroglutamine was synthesized in Production Example 1.

[0062] The cancer cells used were human cervical cancer-derived HeLa cells, human pancreatic cancer-derived PANC-1 cells, or human metastatic pancreatic adenocarcinoma-derived AsPC-1 cells. All of these were obtained from the American Type Culture Collection (ATCC). NHDF cells obtained from Lonza were used as control non-cancer cells. The culture media used were Dulbecco's Modified Eagle's Medium (D-MEM, Fujifilm Wako Co., Ltd.) supplemented with 10% FBS (hereinafter sometimes referred to as "normal medium"), and a medium prepared by removing glutamine from D-MEM and supplementing with 10% FBS (hereinafter sometimes referred to as "Gln-removed medium").

[0063] Cultured cells were placed in a 96-well culture plate at 3 × 10 ３ After seeding at a density of 1 cell per well and allowing the cells to adhere, the cells were cultured for 24, 48, or 72 hours in normal medium, Gln-depleted medium, or Gln-depleted medium supplemented with monofluoroglutamine or difluoroglutamine to a final concentration of 4 mM. The cell proliferation rate of the cultured cells was measured by SRB assay. As a control, the cell proliferation rate of cells cultured for 0 hours was also measured in the same manner.

[0064] The SRB assay was performed as follows. After cell culture, the medium was removed and 10% (mass / volume) trichloroacetic acid was added to fix the cells. The fixed cells were then washed three times with tap water and stained with 0.4% (wt / vol) sulforhodamine B dissolved in 1% acetic acid. After washing three times with 1% acetic acid, 10 mM Tris solution was added and the absorbance at 540 nm was measured. The absorbance value at 540 nm for each sample was used as the cell mass value, and the relative cell proliferation rate (%) for each sample after X hours of culture was calculated using the following formula:

[0065] [Relative cell proliferation rate (%) after X hours of incubation] = [absorbance at 540 nm of sample after X hours of incubation] / [absorbance at 540 nm of sample after 0 hours of incubation] × 100 (%)

[0066] Table 1 shows the results for HeLa cells supplemented with monofluoroglutamine, Table 2 shows the results for PANC-1 cells supplemented with monofluoroglutamine, Table 3 shows the results for AsPC-1 cells supplemented with monofluoroglutamine, Table 4 shows the results for HeLa cells supplemented with 4,4-difluoroglutamine, Table 5 shows the results for PANC-1 cells supplemented with 4,4-difluoroglutamine, Table 6 shows the results for AsPC-1 cells supplemented with 4,4-difluoroglutamine, Table 7 shows the results for HeLa cells supplemented with 3,3-difluoroglutamine, Table 8 shows the results for PANC-1 cells supplemented with 3,3-difluoroglutamine, and Table 9 shows the results for AsPC-1 cells supplemented with 3,3-difluoroglutamine. In the tables, "DMEM + 10% FBS" indicates the relative cell proliferation rate of cells cultured in normal medium, and "DMEM-Gln + 10% FBS" indicates the relative cell proliferation rate of cells cultured in Gln-depleted medium. In the table, "DMEM-Gln + (2S,4S)Gln (1F) + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-removed medium supplemented with (2S,4S)Gln (1F), "DMEM-Gln + (2S,4R)Gln (1F) + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-removed medium supplemented with (2S,4R)Gln (1F), "DMEM-Gln + 4,4-diF-Gln + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-removed medium supplemented with 4,4-diF-Gln, and "DMEM-Gln + 3,3-diF-Gln + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-removed medium supplemented with 3,3-diF-Gln.

[0067]

[0068]

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[0070]

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[0074]

[0075]

[0076] As shown in Tables 1 to 9, in the case of HeLa cells, PANC-1 cells, and AsPC-1 cells, which are cancer cell-derived culture lines, the relative cell proliferation rate after 72 hours of culture in a Gln-depleted medium supplemented with monofluoroglutamine or difluoroglutamine was less than 100%, indicating that cell proliferation was significantly inhibited by the fluorinated glutamine. These results demonstrate that the fluorinated glutamine has the ability to inhibit cell proliferation of cancer cells.

[0077] Example 2 The anticancer effect of fluorine-containing glutamines, in which one or two hydrogen atoms bonded to the carbon atoms in the side chain of glutamine were substituted with fluorine atoms, on tumor tissue in mice was examined. As in Example 1, (2S,4S)4-fluoroglutamine, (2S,4R)4-fluoroglutamine, 4,4-difluoroglutamine, and 3,3-difluoroglutamine were used as fluorine-containing glutamines.

[0078] (1) Anticancer activity of monofluoroglutamine 1.0 x 10 ７ 0.1 mL of a HeLa cell solution containing 0.1 cells / mL was subcutaneously transplanted into SCID / SCID (immunodeficient) mice. Starting 8 days after cell transplantation, PBS (control group), glutamine [Gln], (2S,4S)4-fluoroglutamine [(2S,4S)Gln(1F)], or (2S,4R)4-fluoroglutamine [(2S,4R)Gln(1F)] (5 mg / kg mouse body weight) was intraperitoneally administered daily for 2 weeks. The growth of tumor tissue was observed over time from cell transplantation, and tumor volume (mm ３ The tumor volume (mm ３ ) was calculated from the major axis (mm) and minor axis (mm) of the tumor tissue using the following formula:

[0079] [Tumor volume (mm ３ ) ]=[tumor long diameter (mm)] ２ × [tumor short axis (mm)] / 2

[0080]

[0081] The results of measuring tumor volume are shown in Table 10. In the (2S,4S)4-fluoroglutamine-administered and (2S,4R)4-fluoroglutamine-administered mice, tumor tissue growth was clearly suppressed compared to the control group and glutamine-administered group. These results confirmed that monofluoroglutamine has the ability to inhibit cancer cell proliferation in vivo.

[0082] (2) Anticancer activity of difluoroglutamine 1.0 x 10 ６ cells / mL of HeLa cell solution or 2.5 x 10 ５ 0.1 mL of AsPC-1 cells (0.1 cells / mL) was subcutaneously transplanted into SCID / SCID (immunodeficient) mice. Starting 8 days after cell transplantation, PBS (control group), 4,4-difluoroglutamine [4,4-diF-Gln], or 3,3-difluoroglutamine [3,3-diF-Gln] (5 mg / kg mouse body weight) was intraperitoneally administered daily for 10 days. The growth of tumor tissue was observed over time from cell transplantation, and tumor volume (mm ) was determined in the same manner as in (1) above. ３ ) was calculated.

[0083]

[0084]

[0085] Table 11 shows the results for HeLa cells, and Table 12 shows the results for AsPC-1 cells. In the difluoroglutamine-administered mice ("3,3-diF-Gln" and "4,4-diF-Gln" in the table), tumor tissue growth was clearly suppressed compared to the control group. These results confirmed that difluoroglutamine has the ability to inhibit cancer cell proliferation in vivo.

[0086] Example 3 Metabolomic analysis of glutamine degradation and the TCA cycle in HeLa cells or AsPC-1 cells into which fluorinated glutamines had been incorporated was performed. As in Example 1, (2S,4S)4-fluoroglutamine [(2S,4S)Gln(1F)], (2S,4R)4-difluoroglutamine [(2S,4R)Gln(1F)], and 4,4-difluoroglutamine [4,4-diF-Gln] were used as fluorinated glutamines.

[0087] (1) Cell Pretreatment Method: Monofluoroglutamine or difluoroglutamine was added to the cell culture medium instead of glutamine, and the cells were cultured for 24 hours. The resulting cells were pretreated as follows before analysis using a CE-TOF-MS system. Methionine sulfone, used as an internal standard for measuring cationic metabolites, and camphorsulfonic acid and 2-(N-morpholino)ethanesulfonic acid, used as internal standards for measuring anionic metabolites, were diluted with methanol to 25 μM to prepare internal standard solutions. The cells were washed twice with 5% mannitol, and 1 mL of the internal standard solution was added. The mixture was then allowed to stand for 10 minutes, after which the sample solution was collected. 400 μL of chloroform and 200 μL of ultrapure water were added and mixed thoroughly. The resulting mixture was centrifuged at 10,000 × g at 4°C for 3 minutes, and the upper layer was ultrafiltered at 9,100 × g at 20°C for 3 hours. The obtained filtrate was dried at 40°C for 2 hours using a centrifugal concentrator, dissolved in 50 μL of ultrapure water containing 3-aminopyrrolidine (3-AP) and 1,3,5-benzenetricarboxylic acid (Trimesate), and 8 μL of the solution was transferred to an analytical vial and subjected to CE-TOF-MS system.

[0088] (2) Analysis by CE-TOF-MS A CE-TOF-MS system was prepared using standard substances, internal standards, reagents, and equipment. Prior to analysis, the capillary column was flushed with running buffer for 30 minutes to equilibrate the inner wall. Then, voltage was gradually applied to confirm that current was flowing before proceeding with the analysis. The CE-TOF-MS system used was a time-of-flight mass spectrometer (6546 Q-TOF, Agilent Technologies) equipped with a capillary electrophoresis column (7100 Capillary Electrophoresis, Agilent Technologies) connected to a pump (1260 Infinity II, Agilent Technologies) in the upstream stage. For cation analysis, a fused silica capillary column was used, and for anion analysis, a COSMO(+) capillary was used. The outer polyvinyl film was burned off approximately 5 mm with a lighter, and the burnt residue was wiped off with methanol, revealing both ends of the inner glass. One end was placed in a dedicated cassette for capillary electrophoresis, and the other end was inserted into the nebulizer of the mass spectrometer. The tip of the capillary column was exposed from the inner needle by approximately one-third the length of the inner needle extending from the nebulizer. A dedicated magnifying glass was used to confirm that the sheath liquid was being released in a mist, and the column was then placed in the sprayer installed in the ionization chamber.

[0089] <Cation analysis> 1 M formic acid was used as the migration buffer for cation mode analysis, and methionine sulfone was used as the internal standard for measuring cationic metabolites. The cationic standards used were those listed in Tables 13 and 14.

[0090]

[0091]

[0092] <Anion Analysis> 50 mM ammonium acetate (pH 8.5) was used as the electrophoresis buffer for anion mode analysis, and camphorsulfonic acid and 2-(N-morpholino)ethanesulfonic acid were used as internal standards for measuring anionic metabolites. The anionic standards used were those listed in Tables 15 and 16.

[0093]

[0094]

[0095] The conditions for measuring metabolites were as shown in Table 17.

[0096]

[0097] Figure 1 shows the results of metabolomic analysis of HeLa cells supplemented with glutamine, (2S,4S)4-fluoroglutamine, or (2S,4R)4-fluoroglutamine. Specifically, the results show the intracellular content (fmol / cell) of molecules involved in glutamine degradation (glutamine, glutamic acid, and α-ketoglutaric acid). In the figure, "-Gln" indicates control cells to which nothing was added, "+Gln" indicates cells to which glutamine was added, "+(2S,4S)Gln(1F)" indicates cells to which (2S,4S)4-fluoroglutamine was added, and "+(2S,4R)Gln(1F)" indicates cells to which (2S,4R)4-fluoroglutamine was added. In the figure, "Gln" is the measurement result of the amount of intracellular glutamine, "Glu" is the measurement result of the amount of intracellular glutamic acid, "αKG" is the measurement result of the amount of intracellular α-ketoglutaric acid, "Gln(1F)" is the measurement result of the amount of intracellular monofluoroglutamine, "Glu(1F)" is the measurement result of the amount of intracellular monofluoroglutamic acid, and "αKG(1F)" is the measurement result of the amount of intracellular monofluoro-α-ketoglutaric acid. In the figure, "N.D." indicates that it was not detected.

[0098] As shown in Figure 1, the contents of glutamine and its metabolic products, glutamic acid and α-ketoglutaric acid, were increased in cells treated with glutamine compared to control cells. On the other hand, the amount of αKG was significantly reduced in cells treated with (2S,4S)Gln(1F) or (2S,4R)Gln(1F) compared to control cells. These results demonstrate that the addition of monofluoroglutamine to cells likely inhibits glutaminolysis, resulting in a reduction in αKG levels.

[0099] The results of metabolomic analysis of HeLa cells supplemented with glutamine or 4,4-difluoroglutamine are shown in Figure 2. Specifically, the results are the intracellular content (fmol / cell) of molecules involved in glutamine degradation (glutamine, glutamic acid, and α-ketoglutaric acid). In the figure, "-Gln" indicates control cells to which nothing was added, "+Gln" indicates cells to which glutamine was added, and "+4,4-diF-Gln" indicates cells to which 4,4-diF-Gln was added. In the figure, "Gln" is the measurement result of the amount of intracellular glutamine, "Glu" is the measurement result of the amount of intracellular glutamic acid, "αKG" is the measurement result of the amount of intracellular α-ketoglutaric acid, "4,4-diF-Gln" is the measurement result of the amount of intracellular difluoroglutamine, "PyroGln(2F)" is the measurement result of the amount of intracellular difluoropyroglutamine, "Glu(2F)" is the measurement result of the amount of intracellular difluoroglutamic acid, and "αKG(2F)" is the measurement result of the amount of intracellular difluoro-α-ketoglutaric acid.

[0100] As shown in Figure 2, the amount of α-ketoglutaric acid was reduced in HeLa cells to which 4,4-difluoroglutamine had been added compared to cells to which glutamine had been added. These results demonstrate that the amount of α-ketoglutaric acid is also reduced by adding difluoroglutamine to cells.

[0101] The results of metabolomic analysis of AsPC-1 cells supplemented with glutamine or 4,4-difluoroglutamine are shown in Figure 3. Specifically, the results show the intracellular content (fmol / cell) of molecules involved in glutamine degradation (glutamine, glutamic acid, and α-ketoglutarate). In the figure, "Gln," "Glu," "αKG," "4,4-diF-Gln," "PyroGln(2F)," "Glu(2F)," and "αKG(2F)" are the same as in Figure 2.

[0102] As shown in Figure 3, in AsPC-1 cells to which 4,4-difluoroglutamine had been added, the amount of α-ketoglutaric acid was much reduced compared to cells to which glutamine had been added. These results demonstrate that the addition of difluoroglutamine also reduces the amount of α-ketoglutaric acid in cancer cells derived from pancreatic adenocarcinoma. Comparing the results of Figures 2 and 3 confirms that difluoroglutamine exerts a stronger inhibitory effect on α-ketoglutaric acid production in pancreatic cancer cells than in cervical cancer cells.

[0103] Example 4: The effect of fluorinated glutamine, in which two hydrogen atoms bonded to the carbon atoms of the glutamine side chain were replaced with fluorine atoms, on cancer cell proliferation was examined. The optically active form of 4,4-difluoroglutamine [4,4-diF-Gln] was used as the fluorinated glutamine. 4,4-Difluoroglutamine was prepared by synthesizing an intermediate (9, benzyl 5-amino-2-[(N,N-di-tert-butoxycarbonyl)amino]-4,4-difluoro-5-oxopentanoate) according to the method of Kondratov et al. (Non-Patent Document 5), separating the R- and S-isomers using a chiral column, and then deriving the target product. The configuration was determined by measuring the specific rotation and comparing it with literature values ​​(Non-Patent Document 6). (2S,4S)4-fluoroglutamine was also used for comparison.

[0104] The cancer cells used were MiaPACA1 cells derived from human pancreatic cancer, PANC-1 cells derived from human pancreatic cancer, or AsPC-1 cells derived from human metastatic pancreatic adenocarcinoma. All of these were obtained from ATCC. NHDF cells obtained from Lonza were used as control non-cancer cells. Normal medium and Gln-depleted medium were used as culture media.

[0105] Cultured cells were placed in a 96-well culture plate at 3 × 10 ３ After seeding at a density of 100 cells / well and allowing them to adhere, the cells were cultured for 72 hours in a Gln-depleted medium supplemented with 4,4-difluoroglutamine, 4,4-difluoro-L-glutamine, 4,4-difluoro-D-glutamine, or (2S,4S)4-fluoroglutamine before optical resolution at a final concentration of 0, 0.01, 0.03, 0.1, 0.3, or 1 mM. The cell proliferation rate of the cultured cells was measured by SRB assay. As a control, the cell proliferation rate of cells cultured in standard medium was also measured in the same manner. The SRB assay was performed in the same manner as in Example 1. The cell proliferation rate of each cell type was calculated relative to the cell proliferation rate of cells cultured in standard medium, which was set at 100%.

[0106] Table 18 shows the results for MiaPaCa cells, Table 19 shows the results for PANC-1 cells, and Table 20 shows the results for AsPC-1 cells. In the tables, "DMEM-Gln + (2S,4S)Gln(1F) + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-free medium supplemented with (2S,4S)4-fluoroglutamine, and "DMEM-Gln + 4,4-diF-Gln(rac) + 10% FBS" indicates the relative cell proliferation rate of cells cultured in a Gln-free medium supplemented with 4,4-difluoroglutamine before optical resolution. "DMEM-Gln+4,4-diF-L-Gln+10% FBS" shows the relative cell proliferation rate of cells cultured in a Gln-free medium supplemented with 4,4-difluoro-L-glutamine, and "DMEM-Gln+4,4-diF-D-Gln+10% FBS" shows the relative cell proliferation rate of cells cultured in a Gln-free medium supplemented with 4,4-difluoro-D-glutamine. The "Concentration" column shows the final concentration of fluorinated glutamine in each medium.

[0107]

[0108]

[0109]

[0110] As shown in Tables 18 to 20, for all cancer cells, the relative cell proliferation rate after 72 hours of culture was lower in the medium supplemented with 4,4-difluoro-L-glutamine than in the medium supplemented with 4,4-difluoro-D-glutamine. These results demonstrate that L-fluorinated glutamine has a stronger inhibitory effect on cancer cell proliferation than D-fluorinated glutamine.

Claims

1. An inhibitor that inhibits the production of α-ketoglutaric acid, the active ingredient of which is fluorine-containing glutamine, in which one or more hydrogen atoms bonded to the carbon atoms of glutamine are replaced with fluorine atoms, or a pharmacologically acceptable salt thereof.

2. An inhibitor that inhibits glutaminase, the active ingredient of which is fluorine-containing glutamine, in which one or more hydrogen atoms bonded to the carbon atoms of glutamine are replaced with fluorine atoms, or a pharmacologically acceptable salt thereof.

3. An inhibitor that inhibits glutamate dehydrogenase, the active ingredient of which is fluorine-containing glutamine, in which one or more hydrogen atoms bonded to the carbon atoms of glutamine are replaced with fluorine atoms, or a pharmacologically acceptable salt thereof.

4. The inhibitor according to any one of claims 1 to 3, wherein the fluorine-containing glutamine is one or more selected from the group consisting of 3-monofluoroglutamine, 4-monofluoroglutamine, 3,3-difluoroglutamine, and 4,4-difluoroglutamine.

5. A pharmaceutical composition containing the inhibitor according to any one of claims 1 to 3.

6. An anticancer agent comprising, as an active ingredient, a fluorine-containing glutamine in which one or more hydrogen atoms bonded to a carbon atom of glutamine are substituted with a fluorine atom, or a pharmacologically acceptable salt thereof, wherein the fluorine-containing glutamine is one or more species selected from the group consisting of 4,4-difluoroglutamine and 3,3-difluoroglutamine.

7. A pharmaceutical composition containing the anticancer agent according to claim 6.

8. 3,3-Difluoroglutamine.

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