Anti-cancer agent

Abstract

Provided are: an anticancer agent having excellent cell membrane permeability; and a pharmaceutical composition containing the anticancer agent. Namely, the present invention provides an anticancer agent having, as an active ingredient, a fluorine-containing amino acid in which one or more hydrogen atoms bonded to a carbon atom of an alpha amino acid are substituted with a fluorine atom, or a pharmacologically acceptable salt thereof, the fluorine-containing amino acid having a monofluoromethyl group, a difluoromethyl group, or a trifluoromethyl group. And a pharmaceutical composition containing any one of these.

Description

Technical Field

[0001] The present invention relates to a novel anticancer agent and a pharmaceutical composition containing the anticancer agent.

[0002] This application claims priority based on Japanese Patent Application No. 2022-203332 filed in Japan on December 20, 2022, and Japanese Patent Application No. 2023-192426 filed in Japan on November 10, 2023, and incorporates their contents herein. Background Art

[0003] It has been reported that fluorinated amino acids exhibit specific physiological activities and have attracted much attention. For example, it has been reported that 3,3,3-trifluoralanine and its derivatives act as suicide inhibitors of pyridoxal enzymes (Non-Patent Document 1). In addition, it has been reported that the alanine racemases of Gram-negative bacterium Salmonella typhimurium and Gram-positive bacterium Bacillus stearothermophilus are inactivated by 3,3,3-trifluoralanine (Non-Patent Document 2). Fluorinated amino acids and peptides containing them are expected to be applied as physiologically active substances in the pharmaceutical field.

[0004] It is known that in pharmaceuticals, it is important to make a pharmacologically active substance reach a target molecule present inside a target cell. As drug delivery agents for pharmacologically active substances, lipid nanoparticles (Patent Document 1) and cationic polymer nanoparticles (Patent Document 2) are known.

[0005] On the other hand, it is known that compounds having a polyfluorinated structure are stable in vivo, have low toxicity, and are excellent in uptake into cells and escape from endosomes (Non-Patent Document 3).

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication No. 2011 / 036557

[0009] Patent Document 2: International Publication No. 2017 / 212006

[0010] Non-Patent Documents

[0011] Non-Patent Document 1: Sakai et al., Tetrahedron, 1996, vol. 52(1), p. 233-244.

[0012] Non-Patent Document 2: Faraci and Walsh, Biochemistry, 1989, vol. 28(2), p. 431-437.

[0013] Non-Patent Document 3: Zhang et al., MRS Communications, 2018, vol. 8, p. 303-313.

[0014] Non-Patent Document 4: Nodwell et al., Journal of the American Chemical Society, 2017, vol. 139, p. 3595-3598. SUMMARY OF THE INVENTION

[0015] An object of the present invention is to provide a novel anticancer agent and a pharmaceutical composition containing the anticancer agent.

[0016] The inventors of the present invention have found that a fluorine-containing amino acid in which one or more hydrogen atoms bonded to a carbon atom in the side chain of an α-amino acid are replaced by fluorine atoms has excellent cell membrane permeability and ability to inhibit the proliferation of cancer cells, and thus completed the present invention.

[0017] That is, the present invention is as described below.

[0018] [1] An anticancer agent comprising, as an active ingredient, a fluorine-containing amino acid in which one or more hydrogen atoms bonded to a carbon atom of an α-amino acid are replaced by fluorine atoms, or a pharmacologically acceptable salt thereof.

[0019] [2] The anticancer agent according to the above [1], wherein the fluorine-containing amino acid has a monofluoromethyl group, a difluoromethyl group, or a trifluoromethyl group.

[0020] [3] The anticancer agent according to the above [1] or [2], wherein the α-amino acid is one or more selected from valine, leucine, isoleucine, and glutamine.

[0021] [4] The anticancer agent according to the above [1], wherein the fluorine-containing amino acid is one or more selected from monofluoro leucine, trifluoro leucine, monofluoro valine, hexafluoro valine, monofluoro isoleucine, monofluoro glutamine, and difluoro glutamine.

[0022] [5] A pharmaceutical composition containing any one of the anticancer agents according to the above [1] to [4].

[0023] The anticancer agent of the present invention has excellent cell membrane permeability and ability to inhibit the proliferation of cancer cells, and is an excellent anticancer agent that can be easily taken up by target cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] In the present invention and the specification of the present application, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. "Halogen atom other than fluorine atom" means a chlorine atom, a bromine atom or an iodine atom. As an example of "halogen atom other than fluorine atom", a chlorine atom or a bromine atom is preferred, and a chlorine atom is particularly preferred.

[0025] In the present invention and the specification of the present application, "C p1－p2 "(where p1 and p2 are positive integers satisfying p1 < p2) means a group having 1 to p2 carbon atoms.

[0026] In the present invention and the specification of the present application, "C 1－6 alkyl" means an alkyl group having 1 to 6 carbon atoms, which may be straight-chain or branched-chain. As an example of C 1－6 alkyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, etc. can be cited.

[0027] The anticancer agent of the present invention contains, as an active ingredient, a fluorine-containing amino acid obtained by substituting one or more hydrogen atoms bonded to the carbon atom of an α-amino acid with a fluorine atom, or a pharmaceutically acceptable salt thereof. Since the fluorine-containing amino acid has a fluorine atom, it has excellent cell membrane permeability. Therefore, the anticancer agent of the present invention can be introduced into target cancer cells only by contacting the surface of the cancer cells. In addition, the fluorine-containing amino acid has a high ability to inhibit the proliferation of cancer cells and excellent antitumor effects. It should be noted that the fluorine-containing amino acid also shows an inhibitory effect on the proliferation of normal cells, but the effect is weaker than that on cancer cells.

[0028] As the fluorine-containing amino acid which is the active ingredient of the anticancer agent of the present invention, any compound obtained by substituting one or more hydrogen atoms bonded to the carbon atom of an α-amino acid with a fluorine atom is acceptable. A compound obtained by substituting one or more hydrogen atoms bonded to the carbon atom constituting the side chain with a fluorine atom is preferred. A fluorine-containing amino acid obtained by substituting 1 to 6 hydrogen atoms bonded to the carbon atom constituting the side chain with a fluorine atom is preferred, and a fluorine-containing amino acid having 1 or 2 monofluoromethyl, difluoromethyl or trifluoromethyl groups is more preferred.

[0029] The fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, is preferably a fluorine-containing amino acid obtained by substituting one or more hydrogen atoms bonded to the carbon atom of an α-amino acid (hereinafter sometimes referred to as "branched-chain α-amino acid") selected from valine, leucine, and isoleucine with fluorine atoms. The fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, is preferably a fluorine-containing amino acid obtained by substituting 1 to 6 hydrogen atoms in the side chain of a branched-chain α-amino acid with fluorine atoms, and more preferably a fluorine-containing amino acid having 1 or 2 monofluoromethyl, difluoromethyl, or trifluoromethyl groups. It should be noted that the side chains of valine, leucine, and isoleucine are isopropyl, isobutyl, and sec-butyl groups bonded to the α-carbon, respectively. The fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, is particularly preferably at least one selected from monofluoro leucine (CAS RN: 857026-04-1, 4-fluoro-L-leucine), trifluoro leucine (CAS RN: 2260931-25-5, 2-amino-5,5,5-trifluoro-4-methylpentanoic acid hydrochloride), monofluoro valine (CAS RN: 43163-94-6, 3-fluoro-L-valine), hexafluoro valine (CAS RN: 16063-80-2, 4,4,4,4’,4’,4’-hexafluoro-DL-valine), monofluoro isoleucine (3-fluoro isoleucine (CAS RN: 79205-62-2, 3-fluoro-isoleucine) and 4-fluoro isoleucine (CAS RN: 2643315-39-1, 4-fluoro-isoleucine)).

[0030] As the fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, α-amino acids with a linear side chain are also preferred. As the fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, a fluorine-containing amino acid obtained by substituting 1 to 6 hydrogen atoms in the side chain of an α-amino acid with a linear side chain with fluorine atoms is preferred, and a fluorine-containing amino acid having 1 or 2 monofluoromethyl, difluoromethyl, or trifluoromethyl groups is more preferred. As the fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, a fluorine-containing amino acid obtained by substituting one or more hydrogen atoms bonded to the carbon atom of glutamine with fluorine atoms is preferred, and monofluoro glutamine obtained by substituting one hydrogen atom bonded to the carbon atom of glutamine with a fluorine atom and difluoro glutamine obtained by substituting two hydrogen atoms bonded to the carbon atom of glutamine with fluorine atoms are more preferred.

[0031] As the fluorine-containing amino acid, which is the active ingredient of the anticancer agent of the present invention, as long as the anti-tumor effect is not impaired, the hydrogen atoms not substituted with fluorine atoms may also be substituted with other substituents. Examples of such other substituents include halogen atoms other than fluorine atoms, C 1－6 alkyl groups, etc.

[0032] The fluorine-containing amino acid contained as the active ingredient of the anticancer agent of the present invention may be one kind or two or more kinds.

[0033] The fluorine-containing amino acid contained as an active ingredient in the anticancer agent of the present invention can be synthesized by treating while protecting the carboxyl group and amino group of the α-amino acid with a fluorinating agent. Protection of the carboxyl group and amino group and treatment based on the fluorinating agent can be carried out by conventional methods. As the fluorinating agent, N-fluorobenzenesulfonimide (CAS RN: 133745-75-2) etc. can be used.

[0034] Since the anticancer agent of the present invention has excellent cell membrane permeability, it can be introduced into target cancer cells only by contacting with the target cancer cells. When the target cells of the anticancer agent of the present invention are cultured cells, the anticancer agent can be introduced into the cells only by adding the anticancer agent to the culture medium and culturing. In addition, for animal tissues, for example, the anticancer agent can be introduced into the cells constituting the tissue by spraying or coating a solution in which the anticancer agent of the present invention is dissolved on the tissue surface.

[0035] The anticancer agent of the present invention can also use a pharmacologically acceptable salt of the above-mentioned fluorine-containing amino acid as an active ingredient. As the pharmacologically acceptable salt, for example, salts with bases such as inorganic bases and organic bases, and salts with acids such as inorganic acids and organic acids can be cited. As the inorganic base, for example, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum, ammonium etc. can be cited. As the organic base, for example, primary amines such as ethanolamine, secondary amines such as diethylamine and diethanolamine, tertiary amines such as trimethylamine, triethylamine and triethanolamine etc. can be cited. As the inorganic acid, for example, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid etc. can be cited. As the organic acid, for example, acetic acid, citric acid, lactic acid, malic acid, maleic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid etc. can be cited.

[0036] The anticancer agent of the present invention is particularly useful as a therapeutic agent for preventing or treating cancer. As the cancer, for example, adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, Hodgkin's disease, hypopharyngeal cancer, laryngeal cancer, lip and oral cancer, liver cancer, non-small cell lung cancer, non-Hodgkin lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, testicular cancer, thyroid cancer etc. can be cited.

[0037] The anticancer agent of the present invention can be directly or mixed with a pharmacologically acceptable carrier etc. to form a pharmaceutical composition and used as a preventive or therapeutic agent for various diseases represented by cancer for mammals.

[0038] Here, as a pharmaceutically acceptable carrier, various organic or inorganic carrier substances that are often used as formulation materials can be used. For example, as excipients, lubricants, binders, and disintegrants in solid preparations; solvents, cosolvents, suspending agents, isotonic agents, buffers, soothing agents, etc. in liquid preparations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed.

[0039] Preferred examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminum silicate.

[0040] Preferred examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica.

[0041] Preferred 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, hydroxypropyl methyl cellulose, polyvinylpyrrolidone.

[0042] Preferred examples of disintegrants include lactose, sucrose, starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, light anhydrous silicic acid, low-substituted hydroxypropyl cellulose.

[0043] Preferred examples of solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil.

[0044] Preferred examples of cosolvents include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate.

[0045] Preferred examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionate, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose; polysorbates, polyoxyethylene hydrogenated castor oil.

[0046] Preferred examples of isotonic agents include sodium chloride, glycerol, D-mannitol, D-sorbitol, glucose.

[0047] As preferred examples of buffers, buffers such as phosphates, acetates, carbonates, and citrates can be mentioned.

[0048] As a preferred example of a soothing agent, benzyl alcohol can be mentioned.

[0049] As preferred examples of preservatives, parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid can be mentioned.

[0050] As preferred examples of antioxidants, sulfites, ascorbates, etc. can be mentioned.

[0051] As preferred examples of coloring agents, water-soluble edible tar pigments (e.g., edible red No. 2 and No. 3, edible yellow No. 4 and No. 5, edible blue No. 1 and No. 2, etc.), water-insoluble lake pigments (e.g., aluminum salts of the above water-soluble edible tar pigments), and natural pigments (e.g., β-carotene, chlorophyll, iron oxide red) can be mentioned.

[0052] As preferred examples of sweeteners, sodium saccharin, dipotassium glycyrrhizinate, aspartame, and stevioside can be mentioned.

[0053] As dosage forms of the above pharmaceutical composition, for example, oral preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films); and non-oral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drip infusions), external preparations (e.g., transdermal preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal preparations, pulmonary preparations (inhalants), eye drops, etc. These can be safely administered orally or non-orally (e.g., topically, rectally, intravenously). These preparations can also be release-controlled preparations such as immediate-release preparations or sustained-release preparations (e.g., sustained-release microcapsules).

[0054] In the case of oral preparations, coating can be carried out as needed for the purpose of odor masking, enteric solubility, or sustained release. As coating bases used for coating, for example, sugar coating bases, water-soluble film coating bases, enteric film coating bases, and sustained-release film coating bases can be mentioned.

[0055] As a sugar coating base, white sugar can be used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax, etc. can be further used in combination.

[0056] As a water-soluble film coating base, for example, there may be mentioned cellulose polymers such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, methylhydroxyethyl cellulose; synthetic polymers such as polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [EUDRAGIT E (trade name)], polyvinylpyrrolidone; polysaccharides such as pullulan polysaccharide.

[0057] As an enteric film coating base, for example, there may be mentioned cellulose polymers such as hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethylethyl cellulose, cellulose acetate phthalate; acrylic polymers such as methacrylic acid copolymer L [EUDRAGIT L (trade name)], methacrylic acid copolymer LD [EUDRAGIT L-30D55 (trade name)], methacrylic acid copolymer S [EUDRAGIT S (trade name)]; natural products such as shellac.

[0058] As a sustained-release film coating base, for example, there may be mentioned cellulose polymers such as ethyl cellulose; acrylic polymers such as aminoalkyl methacrylate copolymer RS [EUDRAGIT RS (trade name)], ethyl acrylate-methyl methacrylate copolymer suspension [EUDRAGIT NE (trade name)].

[0059] Two or more of the above coating bases can be mixed and used in an appropriate ratio. In addition, during coating, for example, opacifying agents such as titanium oxide and iron oxide can be used.

[0060] The pharmaceutical composition containing the anticancer agent of the present invention can be manufactured by methods commonly used in the pharmaceutical formulation field, such as the methods described in the Japanese Pharmacopoeia. It should be noted that the content of the anticancer agent of the present invention in the pharmaceutical composition varies depending on the administration route, dosage form, target dosage of the anticancer agent of the present invention, etc., and is, for example, about 0.1 to 100% by mass.

[0061] The dosage of the anticancer agent of the present invention also varies depending on the administration subject, administration route, target disease, symptoms, etc. For example, in the case of oral or parenteral administration to adult patients, usually in terms of a single dose, the dosage of the anticancer agent of the present invention is 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.

[0062] The anticancer agent of the present invention and the pharmaceutical composition containing the anticancer agent can be used as a single therapeutic agent or in combination with one or two or more other cancer treatment methods. Such combination treatments can be carried out simultaneously, separately, or over a period of time.

[0063] As other cancer treatment methods, there can be mentioned surgical treatment methods for physically removing cancer cells, chemotherapy using chemotherapeutic agents containing agents other than the anticancer agent of the present invention as active ingredients, immunotherapy, radiation therapy, and the like. As such chemotherapeutic agents, for example, there can be mentioned alkylating agents, platinum compounds, antimetabolites, topoisomerase inhibitors, microtubule inhibitors, antibiotics, and the like. In addition, in immunotherapy, for example, there can be mentioned a method of administering an immune checkpoint inhibitor.

[0064] When related to the administration method, the term "simultaneously" means that in this administration method, the administration and / or treatment of two or more active ingredients are almost simultaneous; by simultaneous administration, the subject is simultaneously exposed to the administration and / or treatment of two or more active ingredients. When administering simultaneously, the two or more active ingredients can be administered in the form of a fixed dose combination, or in the form of an equivalent non-fixed dose combination (for example, by using two or more different pharmaceutical compositions that should be administered almost simultaneously through the same administration route), or can also be administered in the form of a non-fixed dose combination using two or more different administration routes. Through these administrations, the subject is substantially simultaneously exposed to the administration and / or treatment of two or more active ingredients.

[0065] "Fixed dose combination", when indicating an administration method, means an administration method of administering one pharmaceutical composition having two or more active ingredients.

[0066] When related to the administration method, the term "separately" means that in this administration method, the administration and / or treatment of two or more active ingredients are carried out at different times. Simultaneous administration leads to a treatment stage in which the subject is simultaneously exposed to the administration and / or treatment of two or more active ingredients (for example, at least 1 hour, especially at least 6 hours, particularly at least 12 hours), while separate administration can lead to a treatment stage in which the subject is exposed to only one of the administration and / or treatment of two or more active ingredients during a certain period of time (for example, at least 12 hours, especially at least 1 day). Separate administration especially means providing at least one of the administration and / or treatment of active ingredients in a cycle that is substantially different from daily administration (such as once or twice a day). For example, the administration and / or treatment of one active ingredient can be administered once or twice a day, and the other active ingredient can be administered, for example, every other day, once a week, or at longer intervals.

[0067] Administration "over a certain period" means the administration and / or treatment of two or more active ingredients continuously at different times. This term particularly refers to the administration method in which the administration and / or treatment of one or two or more other active ingredients is started after the administration and / or treatment of one active ingredient is completely finished. In this case, the administration and / or treatment of another active ingredient can be carried out several months after the administration and / or treatment of one active ingredient.

[0068] Example

[0069] Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

[0070] [Example 1]

[0071] Regarding the fluorine-containing amino acids obtained by substituting one hydrogen atom bonded to the carbon atom of the side chain of a branched-chain α-amino acid with a fluorine atom, the influence on cancer cell proliferation was investigated. As the fluorine-containing amino acids, monofluoroleucine, trifluoroleucine, hexafluoroleucine (CAS RN: 1214114-26-7, 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valeric acid hydrochloride), monofluorovaline, trifluorovaline (CAS RN: 16063-79-9, 2-amino-3-methyl-4,4,4-trifluorobutyric acid) and hexafluorovaline were used. All of these were commercial products obtained from NAMIKI Shoji Co., Ltd.

[0072] As cancer cells, HeLa cells or PANC-1 cells were used. All of these were cells obtained from ATCC. In addition, as non-cancer cells for control, NHDF cells obtained from Lonza were used. As the culture medium, a medium containing 10% FBS in Dulbecco's Modified Eagle Medium (D-MEM, manufactured by Fujifilm Wako Pure Chemical Corporation) (hereinafter, sometimes referred to as "normal medium"), a medium containing 10% FBS in a medium obtained by removing leucine from D-MEM (hereinafter, sometimes referred to as "Leu-deficient medium"), and a medium containing 10% FBS in a medium obtained by removing valine from D-MEM (hereinafter, sometimes referred to as "Val-deficient medium") were used.

[0073] The cultured cells were inoculated into a 96-well culture plate at a density of 3×10 3 cells / well, and after allowing them to adhere, they were cultured for 24, 48 or 72 hours using normal medium, Leu-deficient medium or Val-deficient medium, or a medium containing a fluorine-containing amino acid added to the Leu-deficient medium or Val-deficient medium at a final concentration of 0.8 mM. For the cultured cells, the cell proliferation rate was measured using the SRB assay method. As a control, the cell proliferation rate of cells at 0 hours of culture was also measured in the same manner.

[0074] The SRB detection method is carried out as follows. After cell culture, the culture medium is removed, and 10% (mass / volume) trichloroacetic acid is added to fix the cells. Then, the fixed cells are washed 3 times with tap water, and 0.4% (wt / vol) sulforhodamine B dissolved in 1% acetic acid is added for staining. Next, after washing 3 times with 1% acetic acid, 10 mM Tris solution is added, and the absorbance at 540 nm is measured. The absorbance value at 540 nm of each sample is used as the cell amount value, and the relative cell proliferation rate (%) at culture time X for each sample is calculated according to the following formula.

[0075] [Relative cell proliferation rate (%) at culture time X] = [Absorbance at 540 nm of the sample at culture time X] / [Absorbance at 540 nm of the sample at culture time 0 hours] × 100 (%)

[0076] The results of HeLa cells supplemented with fluoroleucine are shown in Tables 1 - 3, the results of PANC-1 cells supplemented with fluoroleucine are shown in Tables 4 - 6, the results of NHDF cells supplemented with fluoroleucine are shown in Tables 7 - 9, the results of HeLa cells supplemented with fluorovaline are shown in Tables 10 - 12, the results of PANC-1 cells supplemented with fluorovaline are shown in Tables 13 - 15, and the results of NHDF cells supplemented with fluorovaline are shown in Tables 16 - 18. In the tables, "DMEM + 10% FBS" represents the relative cell proliferation rate of cells cultured with normal medium, "DMEM - Leu + 10% FBS" represents the relative cell proliferation rate of cells cultured with Leu-deficient medium, "DMEM - Leu + Leu(1F) + 10% FBS", "DMEM - Leu + Leu(3F) + 10% FBS", and "DMEM - Leu + Leu(6F) + 10% FBS" respectively represent the relative cell proliferation rate of cells cultured with medium supplemented with monofluoro-leucine, trifluoro-leucine, and hexafluoro-leucine in Leu-deficient medium. "DMEM - Val + 10% FBS" represents the relative cell proliferation rate of cells cultured with Val-deficient medium, "DMEM - Val + Val(1F) + 10% FBS", "DMEM - Val + Val(3F) + 10% FBS", and "DMEM - Val + Val(6F) + 10% FBS" respectively represent the relative cell proliferation rate of cells cultured with medium supplemented with monofluoro-valine, trifluoro-valine, and hexafluoro-valine in Val-deficient medium.

[0077] [Table 1]

[0078]

[0079] [Table 2]

[0080]

[0081] [Table 3]

[0082]

[0083] [Table 4]

[0084]

[0085] [Table 5]

[0086]

[0087] [Table 6]

[0088]

[0089] [Table 7]

[0090]

[0091] [Table 8]

[0092]

[0093] [Table 9]

[0094]

[0095] [Table 10]

[0096]

[0097] [Table 11]

[0098]

[0099] [Table 12]

[0100]

[0101] [Table 13]

[0102]

[0103] [Table 14]

[0104]

[0105] [Table 15]

[0106]

[0107] [Table 16]

[0108]

[0109] [Table 17]

[0110]

[0111] [Table 18]

[0112]

[0113] As shown in Tables 1 to 6, for HeLa cells and PANC-1 cells, which are cancer cell-derived culture strains, in the medium supplemented with monofluoro leucine and trifluoro leucine in the Leu-free medium, the relative cell proliferation rate after 72-hour culture was less than 100%, and cell proliferation was significantly inhibited by monofluoro leucine and trifluoro leucine. On the other hand, in the medium supplemented with hexafluoro leucine, the relative cell proliferation rate after 72-hour culture was at the same level as that in the Leu-free medium for HeLa cells, and although it was less than that in the Leu-free medium for PANC-1 cells, it was also close to 200%, and cell proliferation was not inhibited. From these results, it can be known that monofluoro leucine and trifluoro leucine have the ability to inhibit the cell proliferation of cancer cells.

[0114] In addition, as shown in Tables 7 to 9, for NHDF cells, which are non-cancer cell-derived culture strains, the relative cell proliferation rate after 72-hour culture was less than 100% in the medium supplemented with monofluoro leucine, and cell proliferation inhibition was observed, but the effect was less than that of HeLa cells and PANC-1 cells. In addition, in the medium supplemented with trifluoro leucine, although it was less than that in the Leu-free medium, it was also close to 200%, and cell proliferation was not inhibited. The relative cell proliferation rate after 72-hour culture in the medium supplemented with hexafluoro leucine was at the same level as that in the Leu-free medium, and no cell proliferation inhibition was observed. From these results, it is considered that the cell proliferation inhibitory ability of trifluoro leucine in particular is highly selective for cancer cells, and it is particularly preferably used as an active ingredient of an anticancer agent.

[0115] As shown in Tables 10 - 15, for HeLa cells, in the medium lacking Val with hexafluorovaline added, the relative cell proliferation rate after 72 - hour culture was less than 100%, and cell proliferation was significantly inhibited. In the medium lacking Val with monofluorovaline added, the relative cell proliferation rate after 72 - hour culture was 273.6%. Although the cells proliferated, it was significantly less than the relative cell proliferation rate in the medium lacking Val, and cell proliferation was inhibited. On the other hand, for PANC - 1 cells, in the medium lacking Val with monofluorovaline added, the relative cell proliferation rate after 72 - hour culture was about 100%, and cell proliferation was significantly inhibited. In the medium lacking Val with hexafluorovaline added, the relative cell proliferation rate after 72 - hour culture was 141.3%. Although the cells proliferated, it was significantly less than the relative cell proliferation rate in the medium lacking Val, and cell proliferation was inhibited. In contrast, in the medium lacking Val with trifluorovaline added, the relative cell proliferation rates of both HeLa cells and PANC - 1 cells after 72 - hour culture were at the same level as that in the medium lacking Val, and cell proliferation was not inhibited. From these results, it can be known that monofluorovaline and hexafluorovaline have the ability to inhibit the cell proliferation of cancer cells.

[0116] In addition, as shown in Tables 16 - 18, for NHDF cells, the relative cell proliferation rate after 72 - hour culture in the medium with monofluorovaline or hexafluorovaline added was less than that in the medium lacking Val. Although inhibition of cell proliferation was observed, it was significantly weaker compared to the cell proliferation inhibition effects observed in HeLa cells and PANC - 1 cells. From these results, monofluorovaline and hexafluorovaline have the ability to inhibit the cell proliferation of both cancer cells and normal cells, but it can be said that they exhibit a stronger cell proliferation inhibition effect on cancer cells compared to normal cells.

[0117] [Example 2]

[0118] For fluorine-containing amino acids in which one hydrogen atom of the carbon atom bonding to the side chain of a branched-chain α-amino acid is substituted with a fluorine atom, the anticancer effect on tumor tissues in mice was investigated. As the fluorine-containing amino acids, a mixture of trifluoroleucine (CAS RN: 2260931-25-5) (obtained from NAMIKI Shoji Co., Ltd.), 3-fluoroisoleucine (CAS RN: 79205-62-2) and 4-fluoroisoleucine (CAS RN: 2643396-11-4) (hereinafter, sometimes referred to as "Ile(3 / 4F)" or simply "monofluoro isoleucine"), and 5-fluoro-5-methyl-norleucine (CAS RN: 1361228-49-0) was used. Monofluoro isoleucine and 5-fluoro-5-methyl-norleucine were synthesized according to the method of Nodwell et al. (Non-Patent Document 4).

[0119] (1) Anticancer effect of trifluoroleucine

[0120] 0.1 mL of a HeLa cell solution at 1.0×10 7 cells / mL was transplanted subcutaneously into SCID / SCID (immunodeficient) mice. Seven days after the transplantation of HeLa cells, PBS (control group), leucine (5 mg / kg mouse body weight), or trifluoroleucine (5 mg / kg mouse body weight) was intraperitoneally administered daily for 2 weeks. In addition, the proliferation of tumor tissues was observed over time from the transplantation of HeLa cells, and the tumor volume (mm 3 ) was calculated. The tumor volume (mm 3 ) was determined from the major axis (mm) and minor axis (mm) of the tumor tissue by the following formula.

[0121] [Tumor volume (mm 3 )] = [Tumor major axis (mm)] 2 × [Tumor minor axis (mm)] / 2

[0122] [Table 19]

[0123]

[0124] The measurement results of the tumor volume are shown in Table 19. In the table, "Leu" and "Leu(3F)" represent the results of mice administered with leucine or trifluoroleucine, respectively. In the mice in the trifluoroleucine administration group, the increase in tumor tissues was significantly inhibited compared with the control group and the leucine administration group. From this result, it was confirmed that trifluoroleucine also has the ability to inhibit the proliferation of cancer cells in vivo.

[0125] (2) Anticancer effect of monofluoro isoleucine

[0126] Use monofluoro isoleucine or 5-fluoro-5-methyl-norleucine (CAS RN: 1361228-49-0) to replace trifluoro leucine, and start administering PBS, 5-fluoro-5-methyl-norleucine, and monofluoro isoleucine 11 days after transplantation of HeLa cells instead of 7 days after transplantation. Except for this, measure the volume (mm 3 ) of the tumor formed by subcutaneously transplanted HeLa cells over time in the same manner as in (1) above.

[0127] [Table 20]

[0128]

[0129] The measurement results of the tumor volume are shown in Table 20. In the table, "5F-NorLeu" and "Ile(3F / 4F)" represent the results of mice administered 5-fluoro-5-methyl-norleucine or monofluoro isoleucine, respectively. In the mice in the monofluoro isoleucine administration group, the increase in tumor tissue was significantly inhibited compared with the control group and the 5-fluoro-5-methyl-norleucine administration group. Based on this result, it was confirmed that monofluoro isoleucine also has the ability to inhibit the proliferation of cancer cells in vivo.

[0130] [Example 3]

[0131] Regarding monofluoro glutamine (Gln(1F)) in which one hydrogen atom bonded to the carbon atom in the side chain of the linear α-amino acid glutamine (Gln) is replaced by a fluorine atom, investigate the effect on the proliferation of cancer cells. As monofluoro glutamine, (2S,4S) Gln(1F) and (2S,4R) Gln(1F) are used. These are all products obtained from NAMIKI Shoji Co., Ltd.

[0132] (1) Anticancer effect in tumor-bearing mice

[0133] Transplant 0.1 mL of a HeLa cell solution at 1.0×10 7 cells / mL subcutaneously into SCID / SCID (immunodeficient) mice. 7 days after transplantation of HeLa cells, intraperitoneally administer PBS (control group), glutamine (Gln) (5 mg / kg mouse body weight), (2S,4S) Gln(1F) (5 mg / kg mouse body weight), or (2S,4R) Gln(1F) (5 mg / kg mouse body weight) every day for 2 weeks. In addition, observe the proliferation of tumor tissue over time after transplantation of HeLa cells, and calculate the tumor volume (mm 3 ). The tumor volume (mm 3 ) is calculated in the same manner as in Example 2.

[0134] [Table 21]

[0135]

[0136] The measurement results of the tumor volume are shown in Table 21. In the Gln administration group, the tumor volume was larger than that of the control group. In contrast, in both of the monofluoro glutamine administration groups ((2S,4S)Gln(1F) administration group and (2S,4R)Gln(1F) administration group), the increase in tumor tissue was significantly inhibited compared with the control group. Based on this result, it was confirmed that monofluoro glutamine has the ability to inhibit the proliferation of cancer cells in vivo.

[0137] (2) SRB assay method

[0138] In the SRB assay method, a medium containing 10% FBS in a medium obtained by removing glutamine from D-MEM (hereinafter sometimes referred to as "Gln-free medium") was used instead of the Leu-free medium or the like as the medium, and monofluoro glutamine ((2S,4S)Gln(1F) or (2S,4R)Gln(1F)) was used as the fluorine-containing amino acid, and the amount of monofluoro glutamine added to the Gln-free medium was an amount to reach a final concentration of 4 mM, and other than that, the operation was carried out in the same manner as in Example 1.

[0139] The relative cell proliferation rate (%) measured by the SRB assay method (setting the cells cultured for 0 hours as 100%) is shown in Tables 22 to 24.

[0140] [Table 22]

[0141]

[0142] [Table 23]

[0143]

[0144] [Table 24]

[0145]

[0146] As shown in Tables 22 to 23, for HeLa cells and PANC-1 cells as cancer cell-derived cultured strains, in the medium supplemented with monofluoro glutamine in the Gln-free medium, the relative cell proliferation rate after 24-hour culture was less than 100%, and the cell proliferation was significantly inhibited by monofluoro glutamine. In addition, as shown in Table 24, even for NHDF cells as non-cancer cell-derived cultured strains, inhibition of cell proliferation by the addition of monofluoro glutamine was also observed, but the inhibitory effect was smaller than that of the cancer cell-derived cell strains. In particular, (2S,4S)Gln(1F) has a higher selectivity for cancer cells in terms of the ability to inhibit cell proliferation than (2S,4R)Gln(1F), and is considered to be particularly preferably an active ingredient of an anticancer agent.

[0147] [Example 4]

[0148] Regarding di-F-Gln(F), the effect on cancer cell proliferation was investigated.

[0149] (1) Anticancer effect in tumor-bearing mice

[0150] A 0.1 mL solution of HeLa cells at 1.0×10 7 cells / mL was transplanted subcutaneously into SCID / SCID (immunodeficient) mice. Seven days after the transplantation of HeLa cells, PBS (control group), glutamine (Gln) (5 mg / kg body weight of mice), or di-F-Gln(F) (5 mg / kg body weight of mice) was administered intraperitoneally every day for 2 weeks. In addition, the proliferation of tumor tissues was observed over time from the transplantation of HeLa cells, and the tumor volume (mm 3 ) was calculated. The tumor volume (mm 3 ) was determined in the same manner as in Example 2.

[0151] [Table 25]

[0152]

[0153] The measurement results of the tumor volume are shown in Table 25. In the Gln administration group, the tumor volume was larger than that in the control group. In contrast, the administration group of di-F-Gln(F) significantly inhibited the enlargement of tumor tissues compared with the control group. From this result, it was confirmed that di-F-Gln has the ability to inhibit the proliferation of cancer cells in vivo.

[0154] (2) SRB assay method

[0155] In the SRB assay method, a Gln-free medium was used instead of a Leu-free medium, etc. as the medium, and di-F-Gln was used as the fluorine-containing amino acid. The amount of di-F-Gln added to the Gln-free medium was an amount to reach a final concentration of 4 mM. Otherwise, it was carried out in the same manner as in Example 1.

[0156] The relative cell proliferation rates (%) determined by the results of the SRB detection method (with the cells cultured for 0 hours set as 100%) are shown in Tables 26 and 27. The results of HeLa cells supplemented with difluoroglutamine are shown in Table 26, and the results of PANC-1 cells supplemented with difluoroglutamine are shown in Table 27. In the tables, "DMEM + 10% FBS" represents the relative cell proliferation rate of cells cultured with normal medium, "DMEM - Gln + 10% FBS" represents the relative cell proliferation rate of cells cultured with Gln-deficient medium, and "DMEM - Gln + di-F-Gln + 10% FBS" represents the relative cell proliferation rate of cells cultured with the medium supplemented with difluoroglutamine in Gln-deficient medium.

[0157] [Table 26]

[0158]

[0159] [Table 27]

[0160]

[0161] As shown in Tables 26 and 27, for HeLa cells and PANC-1 cells, which are cancer cell-derived culture strains, the relative cell proliferation rate after 72 hours of culture in the medium supplemented with difluoroglutamine in Gln-deficient medium was less than 100%, and cell proliferation was significantly inhibited by difluoroglutamine. From these results, it can be known that difluoroglutamine has the ability to inhibit cell proliferation of cancer cells.

Claims

1. An anticancer agent, which contains a fluorine-containing amino acid or a pharmacologically acceptable salt thereof, in which one or more hydrogen atoms bonded to the carbon atom of an α-amino acid are replaced by fluorine atoms, as an active ingredient.

2. The anticancer agent according to claim 1, wherein, The fluorine-containing amino acid has a monofluoromethyl group, a difluoromethyl group, or a trifluoromethyl group.

3. The anticancer agent according to claim 1, wherein, The α-amino acid is one or more selected from valine, leucine, isoleucine, and glutamine.

4. The anticancer agent according to claim 1, wherein, The fluorine-containing amino acid is one or more selected from monofluoroleucine, trifluoroleucine, monofluorovaline, hexafluorovaline, monofluoroisoleucine, monofluoroglutamine, and difluoroglutamine.

5. A pharmaceutical composition, which contains the anticancer agent according to any one of claims 1 to 4.

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