JP7233669B2 - Poorly soluble substance solubilizer containing zwitterion - Google Patents

Description

The present invention relates to a zwitterion, and a medium additive and a sparingly soluble substance-dissolving agent containing the zwitterion.

Conventionally, when a poorly water-soluble substance such as a drug (hereinafter referred to as "poorly soluble substance") is added to the medium in an assay using cells, dimethyl sulfoxide (DMSO) is often used as a solvent for the poorly soluble substance. . However, it has been known that DMSO exhibits cytotoxicity at high concentrations (Non-Patent Document 1). Therefore, when a sparingly soluble substance is added to a medium, for example, by dissolving it in DMSO so as to be 100 to 1000 times the amount of the added substance and adding the necessary amount to the medium, the DMSO concentration in the medium is increased. It is adjusted to be 1% to 0.1%. However, the adverse effects of DMSO on cells are unavoidable. Therefore, it is desirable to develop a new additive for media used as a solvent that can replace DMSO, has less toxicity to cells, and dissolves sparingly soluble substances well. It is rare.

Yamada et al., "Research on Factors Affecting Proliferation of Animal Cultured Cells", Kyushu University Journal of Agricultural Sciences, Vol.34, No.1, No.2, 1-6 (1979)

Accordingly, the present invention has been made in view of the above problems, and provides a medium additive that has low toxicity to cells and can be used as a solvent in place of the conventional DMSO. It also provides a solubilizer for poorly soluble substances such as medicines.

The present inventors have found that zwitterions, which have both positive and negative charges in one molecule, have low toxicity to cells and can dissolve various poorly soluble substances well. I discovered that and completed the invention. In general, organic ions, especially organic ions recognized as ionic liquids (imidazolium cation, phosphonium cation, ammonium cation, sulfonium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation, morpholinium cation, cyclopropene Lithium cations, piperidinium cations, etc.) are known to exhibit toxicity due to the insertion of cationic alkyl chains (which may contain heteroatoms) into cell membranes (Lim, GS, Zidar, J., Cheong, DW, Jaenicke, S. & Klahn, M. Impact of ionic liquids in aqueous solution on bacterial plasma membranes studied with molecular dynamics simulations. J. Phys. Chem. B 118, 10444-10459 (2014) .). The process consists of the following two steps.
(1) The cation of the organic salt and the phosphoric acid of the lipid bilayer membrane (cell membrane) approach due to electrostatic interaction.
(2) Hydrophobic interaction between the alkyl chain of the cation of the organic salt and the lipid moiety of the lipid bilayer membrane causes the alkyl chain of the cation to be inserted into the lipid bilayer membrane, thereby destroying the cell membrane.
The present inventors thought that toxicity could be suppressed by introducing a highly polar anion into the terminal of the alkyl chain of the cation to form a zwitterion. Specifically, the introduction of an anion causes electrostatic repulsion between the phosphoric acid and the anion, which can inhibit the above (1). Also, by introducing an anion, the polarity becomes very high, and the hydrophobic interaction of the above (2) can also be suppressed. Therefore, the toxicity of organic salts having cations such as imidazolium, phosphonium, ammonium, sulfonium, pyridinium, and pyrrolidinium can be greatly reduced. That is, the gist of the present invention is as follows.

（式中、Ａは、ＳＯ_３ ^－、－ＣＯＯ^－、－ＯＰ＝Ｏ（Ｈ）Ｏ^－、－ＯＰ＝Ｏ（ＣＨ_３）Ｏ^－及び－ＯＰ＝Ｏ（ＯＲ_３）Ｏ^－からなる群から選択されるアニオンであり、Ｒ_１は、分子鎖中に１又は２個の酸素原子を含んでいても良い炭素数１～５個のアルキル基であり、Ｒ_２は、炭素数３～５個のアルキレン基であり、Ｒ_３は、分子鎖中にヘテロ原子を有していても良いアルキル基である。）
で表される上記（１）に記載の培地用添加剤。
（７）前記双性イオンが、下記式（２）

（式中、Ｒ_１及びＲ_２は、上記（６）で定義したとおりである。）
で表される上記（６）に記載の培地用添加剤。
（８）双性イオンを含む難溶性物質溶解剤。
（９）前記双性イオンのカチオン部位が、イミダゾリウムカチオン、ホスホニウムカチオン、アンモニウムカチオン、スルホニウムカチオン、ピラゾリウムカチオン、ピリジニウムカチオン、ピロリジニウムカチオン、モルホリニウムカチオン、シクロプロペニリウムカチオン及びピペリジニウムカチオンからなる群から選択されるカチオンである上記（８）に記載の難溶性物質溶解剤。
（１０）前記双性イオンのカチオン部位が、イミダゾリウムカチオン又はホスホニウムカチオンである上記（８）に記載の難溶性物質溶解剤。
（１１）
前記双性イオンのカチオン部位が、置換基として分子鎖中に１個以上のヘテロ原子を有していても良い炭素数１～５個のアルキル基を１個以上有する上記（８）～（１０）のいずれか１つに記載の難溶性物質溶解剤。
（１２）前記双性イオンのカチオン部位とアニオン部位との間が、分子鎖中に１個以上のヘテロ原子を有していても良い炭素数１～５個の１個以上のアルキレン基を介して結合される上記（８）～（１１）のいずれか１つに記載の難溶性物質溶解剤。
（１３）前記双性イオンが、下記式（１）

（式中、Ａは、ＳＯ_３ ^－、－ＣＯＯ^－、－ＯＰ＝Ｏ（Ｈ）Ｏ^－、－ＯＰ＝Ｏ（ＣＨ_３）Ｏ^－及び－ＯＰ＝Ｏ（ＯＲ_３）Ｏ^－からなる群から選択されるアニオンであり、Ｒ_１は、分子鎖中に１又は２個の酸素原子を含んでいても良い炭素数１～５個のアルキル基であり、Ｒ_２は、炭素数３～５個のアルキレン基であり、Ｒ_３は、分子鎖中にヘテロ原子を有していても良いアルキル基である。）
で表される上記（８）に記載の難溶性物質溶解剤。
（１４）前記双性イオンが、下記式（２）

（式中、Ｒ_１及びＲ_２は、上記（１３）で定義したとおりである。）
で表される上記（１３）に記載の難溶性物質溶解剤。
（１５）下記式（４）

で表される双性イオン。 (1) A medium additive containing a zwitterion.
(2) the cation sites of the zwitterion include imidazolium cations, phosphonium cations, ammonium cations, sulfonium cations, pyrazolium cations, pyridinium cations, pyrrolidinium cations, morpholinium cations, cyclopropenylium cations and pyrazolium cations; The medium additive according to (1) above, which is a cation selected from the group consisting of peridinium cations.
(3) The medium additive according to (1) above, wherein the cationic site of the zwitterion is an imidazolium cation or a phosphonium cation.
(4) The above (1) wherein the cationic site of the zwitterion has at least one alkyl group having 1 to 5 carbon atoms which may have at least one heteroatom in the molecular chain as a substituent. The medium additive according to any one of (3).
(5) between the cation site and the anion site of the zwitterion via one or more alkylene groups having 1 to 5 carbon atoms which may have one or more heteroatoms in the molecular chain; The medium additive according to any one of (1) to (4) above, which is bound by
(6) The zwitterion is represented by the following formula (1)

(wherein A is from the group consisting of SO ₃ ⁻ , —COO ⁻ , —OP=O(H)O ⁻ , —OP=O(CH ₃ )O ^— and —OP=O(OR ₃ )O ^— The selected anion, R ₁ is an alkyl group having 1 to 5 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain, and R ₂ is an alkyl group having 3 to 5 carbon atoms. and _R3 is an alkyl group which may have a heteroatom in the molecular chain.)
The medium additive according to the above (1) represented by.
(7) The zwitterion is represented by the following formula (2)

(Wherein, R ₁ and R ₂ are as defined in (6) above.)
The medium additive according to (6) above, represented by
(8) A sparingly soluble substance-dissolving agent containing a zwitterion.
(9) the cation site of the zwitterion is imidazolium cation, phosphonium cation, ammonium cation, sulfonium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation, morpholinium cation, cyclopropenylium cation and pyrazolium cation; The sparingly soluble substance-dissolving agent according to (8) above, which is a cation selected from the group consisting of peridinium cations.
(10) The poorly soluble substance-dissolving agent according to (8) above, wherein the cationic site of the zwitterion is an imidazolium cation or a phosphonium cation.
(11)
The above (8) to (10) wherein the cationic site of the zwitterion has at least one alkyl group having 1 to 5 carbon atoms which may have at least one heteroatom in the molecular chain as a substituent. ).
(12) between the cation site and the anion site of the zwitterion via one or more alkylene groups having 1 to 5 carbon atoms which may have one or more heteroatoms in the molecular chain; The poorly soluble substance-dissolving agent according to any one of the above (8) to (11), which is bound by
(13) The zwitterion is represented by the following formula (1)

(wherein A is from the group consisting of SO ₃ ⁻ , —COO ⁻ , —OP=O(H)O ⁻ , —OP=O(CH ₃ )O ^— and —OP=O(OR ₃ )O ^— The selected anion, R ₁ is an alkyl group having 1 to 5 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain, and R ₂ is an alkyl group having 3 to 5 carbon atoms. and _R3 is an alkyl group which may have a heteroatom in the molecular chain.)
The sparingly soluble substance-dissolving agent according to (8) above, represented by:
(14) The zwitterion is represented by the following formula (2)

(Wherein, R ₁ and R ₂ are as defined in (13) above.)
The sparingly soluble substance-dissolving agent according to (13) above, represented by
(15) Formula (4) below

A zwitterion represented by

The zwitterion of the present invention has low toxicity to various cells such as human cells, and can be used as a medium additive such as a solvent in place of conventional DMSO. In addition, since the zwitterion of the present invention dissolves various sparingly soluble substances well, it can be used as a dissolving agent for sparingly soluble substances in assays and the like.

Fig. 3 is a graph showing the viability of human fibroblasts after 24 hours of culture (2% by weight). Fig. 3 is a graph showing the viability of human fibroblasts after 24 hours of culture (10% by weight).

The present invention will be described in detail below.
The medium additive of the present invention contains a zwitterion. Zwitterions include substances in which an ionic liquid-like cation site and an ionic liquid-like anion site are linked by a covalent bond. Preferably, the cation site and the anion site of the zwitterion are separated via one or more alkylene groups having 1 to 5 carbon atoms which may have one or more heteroatoms in the branched chain. combined. Here, examples of heteroatoms include oxygen, nitrogen, sulfur, phosphorus and the like. By setting the number of carbon atoms in the alkylene group to 1 to 5, the toxicity of zwitterions to cells can be further weakened.

Examples of zwitterionic ionic liquid-like cations include phosphonium cations having one or more substituents, ammonium cations, imidazolium cations, sulfonium cations, pyrazolium cations, pyridinium cations, pyrrolidinium cations, mol Examples include folinium cations, cyclopropenylium cations and piperidinium cations. Among them, imidazolium cations and phosphonium cations having substituents are preferably used. In addition, each substituent may be the same or different, for example, an alkyl group having 1 to 18 carbon atoms which may have one or more heteroatoms in the molecular chain and an alkyl group having 1 to 18 can be appropriately selected from the alkoxy groups of In particular, the substituent preferably has at least one alkyl group having 1 to 5 carbon atoms which may have at least one heteroatom in the molecular chain. By setting the number of carbon atoms in the alkyl chain to 1 to 5, the toxicity to cells can be further weakened. Here, heteroatoms include oxygen, nitrogen, sulfur, phosphorus and the like.

Examples of zwitterionic liquid-like anions include carboxylate ion -COO ^- , sulfonate ion -SO ₃ ^- , phosphate ion -OP=O(H)O ^- , -OP=O(CH ₃ )O ⁻ , —OP=O(OR ₃ )O ⁻ (here, R ₃ is an alkyl group which may have a heteroatom in the molecular chain) and the like. It is not limited.

で表される双性イオンは好ましく用いられる。式（１）中、Ａは、ＳＯ_３ ^－、－ＣＯＯ^－、－ＯＰ＝Ｏ（Ｈ）Ｏ^－、－ＯＰ＝Ｏ（ＣＨ_３）Ｏ^－及び－ＯＰ＝Ｏ（ＯＲ_３）Ｏ^－からなる群から選択されるアニオンである（ここで、Ｒ_３は、分子鎖中にヘテロ原子を有していても良いアルキル基である）。なお、上記ヘテロ原子としては、酸素、窒素、イオウ、リン等が挙げられる。また、Ｒ_３であるアルキル基の例として、メチル基、エチル基、プロピル基、ブチル基、プロピル基等が挙げられるがこれらに限定されるものではない。すなわち、本発明に係る培地用添加剤に含まれる双性イオンとして、下記の式（２）及び式（３）で表される双性イオンが包含される。好ましくは、Ａは－ＣＯＯ^－である。

In particular, as a zwitterion contained in the medium additive, the following formula (1)

A zwitterion represented by is preferably used. In formula (1), A consists of SO ₃ ^- , -COO ^- , -OP=O(H)O ^- , -OP=O(CH ₃ )O ^- and -OP=O(OR ₃ )O ^- is an anion selected from the group (wherein _R3 is an alkyl group which may have a heteroatom in the molecular chain); In addition, oxygen, nitrogen, sulfur, phosphorus etc. are mentioned as said heteroatom. Examples of the alkyl group for _R3 include methyl group, ethyl group, propyl group, butyl group, and propyl group, but are not limited to these. That is, zwitterions contained in the medium additive according to the present invention include zwitterions represented by the following formulas (2) and (3). Preferably, A is ^-COO- .

In formulas (1) to (3), R ₁ is an alkyl group having 1 to 5 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain, and R ₂ is a carbon 3 to 5 alkylene groups. Specific examples of R ₁ include methyl group, ethyl group, propyl group, butyl group, pentyl group, CH ₃ OCH ₂ CH ₂ —, CH ₃ OCH ₂ CH ₂ OCH ₂ CH ₂ — and the like. Further, specific examples of R ₂ include a propylene group, a butylene group, a pentylene group and the like, but are not limited to these.

In particular, the following zwitterions are preferably used as media additives because of their low cytotoxicity. Among them, OE ₁ imC ₃ C is a novel compound.

In addition, the zwitterion (P _8,8,8 C ₃ S) shown below is a zwitterion whose cation is not an imidazolium ion but a phosphonium ion, and is represented by formulas (1) to (3). Similar to imidazolium zwitterions, it has low cytotoxicity and can be used as a medium additive.

In synthesizing the zwitterion as described above, a general organic synthesis method for those skilled in the art can be appropriately employed. That is, the zwitterion in which the cation represented by the above formula (1) is an imidazolium ion can be obtained, for example, by refluxing 1-alkylimidazole and ethyl bromoalkylate in acetonitrile and mixing this with an anion exchange resin. A zwitterion consisting of imidazolium and carboxylic acid can be obtained by distilling off the solvent under reduced pressure. The imidazole alkyl group may contain one or more heteroatoms, eg 1 or 2 oxygen atoms. Also, by changing 1-alkylimidazole to a trialkylphosphine, trialkylamine, dialkylsulfone, pyridine, N-alkylpyrrolidine, or the like, a zwitterion having a cation other than the imidazolium cation can be synthesized. Alternatively, imidazole having an oligoether chain introduced can be obtained by mixing NaH with tetrahydrofuran and adding imidazole and 1-bromo-2-(2-methoxyethoxy)ethane. Similarly, alkylimidazoles, trialkylphosphines, trialkylamines, dialkylsulfones, pyridines, N-alkylpyrrolidines, etc. having various functional groups can be obtained. ions can be obtained.

As the medium to which the zwitterion is added, conventionally known various media are applicable, and are appropriately selected according to the type of cells to be cultured. Any of synthetic medium, semi-synthetic medium and natural medium may be used, and both liquid medium and solid medium are applicable. Specifically, YM medium, cornmeal medium, glucose-bouillon medium, broth medium, SIM medium and other bacterial media, oatmeal medium, wort, fermentation test medium, starch production medium, yeast extract and other fungal media, Examples include, but are not limited to, LB medium, Davis medium, MS medium, TG medium, DMEM medium, and the like.

Additives for culture media are generally added to culture media in a state in which the below-described poorly soluble substances are dissolved in order to perform various assays and the like.

The concentration of the zwitterion added to the medium can be appropriately set according to the type of cells cultured in the medium. It has low toxicity to cells, so it can be added at higher concentrations if desired. Specifically, it can be added at a concentration of 0.001 to 99.9% by weight, preferably 0.01 to 90% by weight, in the medium, but is not limited to this range.

Moreover, when it is added as a solvent to the medium, the zwitterion may be added alone, or may be added in a state of being mixed with another solvent, if necessary. Other solvents in combination with such zwitterions can include water, DMSO, glycerol, methanol, ethanol, and the like. Among the zwitterions mentioned above, there are compounds that are solid at 100°C, such as OE ₁ imC ₃ C. can be treated in the same way as the zwitterion of Even when mixed with these other solvents, it is preferable to adjust the temperature of the zwitterion in the medium so that it falls within the above range.

The type of cells that can be cultured in the medium is not particularly limited, and any cells can be applied. Examples thereof include animal cells, insect cells, plant cells, yeast cells, bacteria cells and the like. Animal cells include cells of humans, mice, rats, monkeys, pigs, dogs, sheep, goats, and the like. Furthermore, examples of bacteria include lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria.

The type of cells is also not particularly limited, and is appropriately selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, and germ cells, for example. Here, "pluripotent stem cells" is a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells). ), embryonic germ stem cells (EG cells), germ stem cells (GS cells) and the like. ES cells or iPS cells are preferred.

In addition, the term "tissue stem cell" means a stem cell whose differentiation-capable cell lineage is limited to a specific tissue, but which has the ability to differentiate into various cell types (pluripotency). hematopoietic stem cells, neural stem cells, hepatic stem cells, skin stem cells, etc.

The term "somatic cell" refers to cells other than germ cells among the cells that constitute multicellular organisms. Preferably, osteoclasts, fibroblasts, hepatocytes, pancreatic cells, muscle cells, osteocytes, osteoblasts, chondrocytes, adipocytes, skin cells, renal cells, lung cells, lymphocytes, red blood cells, white blood cells, single spheres, macrophages and the like.

"Reproductive cells" include gametes for sexual reproduction, ie eggs, egg cells, sperm, sperm cells, spores for asexual reproduction, and the like.

Cells may be selected from the group consisting of sarcoma cells, cell lines and transformed cells. The term "sarcoma" refers to cancer occurring in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma, malignant bone tumor, and the like. A sarcoma cell is a cell derived from a sarcoma. A “established cell line” means a cultured cell that has been maintained outside the body for a long period of time, has certain stable properties, and is capable of semi-permanent subculturing. Examples include PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human embryonic kidney), HL-60 cells (derived from human white blood cells), HeLa cells (derived from human cervical cancer). ) and the like. A "transformed cell" means a cell into which a nucleic acid (such as DNA) has been introduced from the outside of the cell to change its genetic properties. Transformation of animal cells, plant cells, and bacteria is performed using conventionally known methods.

In addition, the zwitterion of the present invention can be used as a solubilizer for sparingly soluble substances. Thus, various sparingly soluble substances can be dissolved in zwitterions, added to, for example, cell culture media and used to assay the effectiveness of sparingly soluble substances. In this case, since the zwitterion of the present invention has low cytotoxicity, it is preferable because it facilitates administration of a high-concentration poorly soluble substance compared to conventional DMSO.

The sparingly soluble substance is not particularly limited as long as it has the property of being only slightly soluble in water. . Preferably, it is 0.5% by weight or less, and particularly preferably 0.1% by weight or less. Such poorly soluble substances include, for example, pharmaceuticals, veterinary drugs, quasi-drugs, cosmetics, substances that are active ingredients of agricultural chemicals (including candidate substances that can be active ingredients), food additives, biological substances, and They are plant-derived substances and include low-molecular-weight substances and oligomers and macromolecular substances such as oligopeptides, polypeptides, polysaccharides, DNA and RNA.

Poorly soluble drugs include drugs defined in the Japanese Pharmacopoeia as “slightly soluble,” “slightly soluble,” “extremely soluble,” and “hardly soluble.” Specifically, antitumor agents, antibiotics, Antihyperlipidemic agents, antibacterial agents, therapeutic agents for allergic diseases, therapeutic agents for hypertension, therapeutic agents for arteriosclerosis, blood circulation promoting agents, hormone agents, fat-soluble vitamins, therapeutic agents for diabetes, antiandrogens, cardiotonic agents, arrhythmia anti-inflammatory agents, sedatives, tranquilizers, antiepileptic agents, antidepressants, therapeutic agents for digestive system diseases, diuretic agents, local anesthetics, anticoagulants, antihistamines, antimuscarinics, antimycobacteria drugs, immunosuppressants, antithyroid drugs, antiviral agents, anxiolytic sedatives, astringents, beta-adrenergic receptor blockers, myocardial inotropic agents, contrast agents, corticosteroids, cough suppressants, diagnostic agents , diagnostic imaging agents, diuretics, dopamine agonists, lipid regulators, muscle relaxants, parasympathetic agents, thyroid calcitonin, prostaglandin, radiopharmaceuticals, sex hormones, stimulants, appetite suppressants, sympathomimetics , thyroid agents, vasodilators, isoflavones, xanthenes, and the like.

Antitumor agents include methotrexate, taxol, doxorubicin hydrochloride, bleomycin hydrochloride, tamoxifen, cisplatin, carboplatin, cyclosporine, HER2 inhibitors, melphalan, dacarbazine, carmofur, enocitabine, etoposide, 5-fluorouracil, mitoxantrone, mesna, dimesna. , aminoglutethimide, acrolein, cyclophosphamide, lomustine, carmustine, cyclophosphamide, busulfan, para-aminosalicylic acid, mercaptopurine, tegaflu, azathioprine, vinblastine sulfate, mitomycin C, L-asparakinase, Ubenimex, etc. be able to.

Antibiotics include streptomycin, chloramphenicol, gentamicin, tetracycline, penicillin, amikacin, dibekacin, bacitracin, cephalexin, nystatin, erythromycin, fradiomycin sulfate, cefmetazole, tolnaftate and the like.

Antihyperlipidemic agents include cholestyramine, niceritrol, clinofibrate, clofibrate, fenofibrate, bezafibrate, soysterol, tocopherol nicotinate, nicomole, probucol, simvastatin, colestimide, and elastase.

Antibacterial agents include chloramphenicol, rokitamycin, roxithromycin, cefatrizine, ofloxacin, ciprofloxacin hydrochloride, tosufloxacin tosylate, norfloxacin, lomefloxacin hydrochloride, pazufloxacin, cefpodoxime proxetil, midecamycin acetate, and josamycin propionate. , fosfomycin or a salt thereof.

Examples of therapeutic agents for allergic diseases include ebastine, mequitazine, methoxyphenamine, clemastine fumarate, cyproheptadine hydrochloride, fexofenadine hydrochloride, diphenhydramine, methdilamine, clemizole and the like.

Antihypertensive agents include nicardipine hydrochloride, delapril hydrochloride, valnidipine hydrochloride, efonidipine hydrochloride, benidipine hydrochloride, alacepril, captopril, cilnidipine, felodipine, amlodipine besilate, nisoldipine, manidipine hydrochloride, nitrendipine, nilvadipine, trandolapril, valsartan, candesartansi Examples include lexetil, urapidil, carvedilol, prazosin hydrochloride, bunazosin hydrochloride, doxazosin mesilate, reserpine, methyldopa, guanabenz acetate, deserpidine, meptame, meptamate, and the like.

Arteriosclerosis therapeutic agents include elastase, clofibrate, simfibrate, soysterol, nicomole and the like.

Blood circulation promoters include tocopherol acetate, tocopherol nicotinate, benzyl nicotinate, caffeine, tolazoline, verapamil, cyclanderate, acetylcholine and the like.

Hormone agents include dexamethasone, dexamethasone acetate, betamethasone, betamethasone valerate, betamethasone dipropionate, beclomethasone propionate, prednisolone, prednisolone valerate, prednisolone acetate, methylprednisolone, methylprednisolone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone acetate propionate. , amcinonide, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, estriol, fluocinonide, hexestrol, methimazole, estriol propionate, clobetasone acetate, clobetasol propionate, testosterone propionate, testosterone enanthate, fluoxymesterone , dromostanolone propionate, estradiol benzoate, estradiol propionate, estradiol valerate, ethinyl estradiol, mestranol, estriol acetate benzoate, fluorometholone, fludroxycortide, diflucortolone valerate, halcinonide, progesterone, hydroxyprogesterone caproate , pregnanediol, medroxyprogesterone acetate, dimethisterone, norethisterone, allylestrenol, gestonolone caproate, oxendrone, and the like.

Examples of fat-soluble vitamin preparations include vitamin A, vitamin D, vitamin E, vitamin K, and the like.

Antidiabetic agents include gliclazide, tolbutamide, glibenclamide, troglitazone, epalrestat, buformin, metformin and the like.

Anti-androgen agents include gestonolone caproate, osaprone acetate, flutamide, oxendrone, allylestrenol, chlormadinone acetate, bicalutamide and the like.

Cardiotonic agents include digoxin, digotoxin, cobidecarenone, and the like.

Arrhythmic agents include lidocaine, bopindolol malonate, arotinolol hydrochloride, atenolol, pindolol, nadolol, propafenone hydrochloride, amiodarone hydrochloride, disopyramide, carteolol hydrochloride, and the like.

Antiphlogistic agents include glycyrrhetinic acid, dipotassium glycyrrhizinate, aspirin, aluminum aspirin, ibuprofen, ketoprofen, ricyrrhetinic acid, salicylic acid, acetaminophen, methyl salicylate, glycol salicylate, aminopyrine, phenacetin, mefenamic acid, flufenamic acid, and aluminum flufenamate. , tolfenamic acid, acemethacin, indomethacin, alclofenac, diclofenac, ibuprofen piconol, oxyphenbutazone, phenylbutazone, ketophenylbutazone, clofezone, tiaramide hydrochloride, diclofenac sodium, sulindac, naproxen, febufen, flurbiprofen , fenprofen, bufexamac, mepyrizole, perisoxal citrate, graphenin, bucolome, pentazocine, methiazic acid, protidic acid, pranoprofen, fenoprofen calcium, piroxicam, feprazone, fentiazac, bendazac, dimethylisopropylazulene, bufexamac, Bucolome, benzydamine, thiaramide, tinoridine, ethenzamide, tenoxicam, chlortenoxicam, clidanac, naproxen, glycyrrhizin, azulene, camphor, thymol, l-menthol, sazapyrin, alclofenac, diclofenac, suprofen, loxoprofen, diflunisal, tiaprofenic acid, oxaprozin, felbinac, etc. can be mentioned.

Hypnotic sedatives include barbital, amobarbital, amobarbital sodium, phenobarbital, phenobarbital sodium, secobarbital sodium, pentobarbital calcium, hexobarbital, triclophos, bromvaleryl urea, glutethimide, methacarone, perlapine, nitrazepam, estazolam, Flurazepam hydrochloride, flunitrazepam, estazolam and the like can be mentioned.

Examples of tranquilizers include diazepam, lorazepam, oxazolam, and the like.

Antiepileptic agents include phenytoin, carbamazepine, phenobarbital, primidone, phenasemide, ethylphenasemide, ethotoin, phensuccimide, nitrazeban, clonazeban and the like.

Antidepressants include phenelzine, imiplanin, noxiptiline and the like.

Examples of therapeutic agents for gastrointestinal diseases include famotidine, sucralfate, aldioxa, irsogladine maleate, metoclopramide, cimetidine, omeprazole, lansoprazole, enprostil, gefarnate, teprenone, sulpiride, trepibutone, and oxethazain.

Diuretic agents include spironolactone, chlorthalidone, polythiazide, triamterene, hydrochlorothiazide, furosemide and the like.

Local anesthetics include ethyl aminobenzoate, procaine hydrochloride, lidocaine, lidocaine hydrochloride, dipcaine hydrochloride, tetracaine hydrochloride, benzyl alcohol, thecaine, bezocaine, pramoxine hydrochloride, katacaine hydrochloride, butanicaine hydrochloride, piperocaine hydrochloride, chlorobutanol, and the like. be able to.

Examples of anticoagulants include coumarin, heparin, and the like.

Antiviral agents include acyclovir, nevirapine, zidovudine, zanamivir, oseltamivir, favipiravir, and the like.

Isoflavones include ononin, daidzein, biochanin A, glycitein, daidzin, glycitin, genistin and the like. Isoflavones may be aglycones or glycosides.

Poorly soluble substances used as quasi-drugs or cosmetics include dl-α-tocopherol acetate, α-tocopherol (vitamin E), methyl cinnamate, ethyl cinnamate, hexyl laurate, and trichlorocarbanilide. , triazine, benzophenone, triazole, eugenol, isoeugenol, ethyl methylphenylglycidate, geranyl acetate, piperonal, cinnamyl acetate, decyl oleate, terpenyl acetate, anilide, cinnamide, sulfonated benzimidazole, carotene, piroctone olamine, Minoxidil, phytosteside, tocopherol nicotinate, ethinyl estradiol, polypolsterone, ecdysteroids and the like can be mentioned.

As an agricultural chemical, any sparingly soluble agricultural chemical active ingredient having an insecticidal action, a bactericidal action, a herbicidal action, a plant growth regulating action, or the like can be applied. For example, sparingly soluble insecticides include abamectin, acrinathrin, amitraz, azadirachtin, azamethyphos, azinphos-methyl, azocyclotin, methiocarb, thiodicarb, trimetacarb, etofenprox, ethylthiometone, methoxychlor, chlorpyrifosmethyl, chlorfenson, chlorfluazuron, tebufenpyrad, bensultap, bifenthrin, bromopropylate, buprofezin, carbaryl, chlorfenapyr, clofentezene, coumaphos, diazinon, cycloprothrin, cyfluthrin, beta-cyfluthrin, cypermethrin, alpha-cypermethrin, theta-cypermethrin, deltamethrin, diafenthi Ron, dicofol, diflubenzuron, carbosulphan, endosulfan, esfenvalerate, etoxazole, fenazaquin, fenbutatin oxide, fenoxycarb, fenpyroximate, fipronil, fluazuron, flucycloxuron, flufenoxuron, flubendiamide, fenthion, halofenozide, Hexaflumuron, hexythiazox, hydramethylnon, metaflumizone, lufenuron, milbemectin, novaluron, pentachlorophenol, pyridaben, rotenone, sulfluramide, tebufenozide, tebupyrimphos, teflubenzuron, tetrachlorbinphos, tetradifone, benfuracarb, tolfenpyrad, triflumuron, tralomethrin, Furatiocarb and the like can be dissolved.

In addition, sparingly soluble bactericidal substances include bromconazole, carpropamide, diclofen, metconazole, hexaconazole, fentin, mancozeb, maneb, diclomedine, azoxystrobin, isoprothiolane, benalaxil, benomyl, bitertanol, captahol, captan, Carbendazim, quinomethionate, chlorothalonil, clozolinate, cyprodinil, diclofluanid, dichlorane, diclosimet, diethofencarb, dimethomorph, diniconazole, dithianone, thiazinyl, epoxiconazole, famoxadone, fenarimol, fenbuconazole, fenfuram, fenpicronil, fluazinam, fludioxonil, fluoro imido, fluquinconazole, fursulfamide, flutolanil, folpet, hexachlorobenzene, imibenconazole, ipoconazole, iprodione, cresoxime methyl, mepanipyrim, mepronil, metiram, nickel bis(dimethyldithiocarbamate), nuarimol, oxine copper, oxolinic acid, penciclon, phthalide , procymidone, propineb, quintozene, sulfur, tebuconazole, tecloftalam, tecnazene, thifluzamide, thiophenate-methyl, thiram, tolclofos-methyl, triadimefon, tolylfluanid, triadimenol, triazoxide, trifoline, triticonazole, vinclozoline, zineb, ziram etc. can be mentioned.

Poorly soluble herbicides include bromobutide, acronifene, clomethoxyphene, lactofen, promethrin, propazine, azafenidine, thenylchlor, bifenox, sulfentrazone, pyraflufenethyl, flumicrolacpentyl, flumioxazin, atrazine, indanophane, bens Rufuron-methyl, benzofenap, bromophenoxime, chlorbromuron, chlorimuron-ethyl, chlornitrophen, chlorotoluron, chlorthal-dimethyl, clomeprop, daimuron, desmedifam, dichlobenil, diflufenican, dimeflon, dinitramine, diuron, etamethsulfuron-methyl, triaziflam , fenoxapropethyl, flampropmethyl, flazasulfuron, flumetsulam, fluthiacet-methyl, flupoxam, fluridone, flurutamon, oxadiclomefone, isoproturon, isoxaben, isoxapyrifop, lenacyl, linuron, mefenacet, metabenzthiazuron , metobenzuron, naproanilide, nevron, norflurazon, oryzalin, oxadiazone, oxyfluorfen, fenmedifam, prodiamine, propyzamide, pyrazolinate, pyrazosulfuron-ethyl, pyribticarb, quinclorac, quizalofop-ethyl, rimsulfuron, siduron, simazine, terbuthylazine , terbutryn, thiazopyr, trakoxydim, trietadine, and the like.

Poorly soluble plant growth regulators include cyclanilide, flumetralin, 6-benzylaminopurine, forchlorfenurone, inabenfide, 2-(1-naphthyl)acetamide, paclobutrazole, N-phenylphthalamic acid, thidiazuron, Uniconazole and the like can be mentioned.

Furthermore, poorly soluble substances that are food additives include glycyrrhizin, L-ascorbic acid stearate, benzoic acid, isoeugenol, ergocalciferol (vitamin D ₂ ), eugenol, butyl parahydroxybenzoate, isopropyl parahydroxybenzoate, β-carotene, citronellyl formate, cholecalciferol (vitamin D ₃ ), propyl gallate, folic acid, lecithin, cinnamyl acetate, phenethyl acetate, ethyl cinnamate, dibutylhydroxytoluene, allyl hexanoate, methyl β-methylketone, riboflavin butyrate Esters, dl-α-tocopherol and the like can be mentioned.

Poorly soluble plant-derived substances include xylan, lignin, chondroitin sulfate, glucomannan, and the like.

Examples of polypeptides include collagen, casein, albumin, elastin, silk protein, and the like.

Poorly soluble polysaccharides include chitin and chitosan.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

1. Cytotoxicity to Human Cells (1) Synthesis of zwitterionic OE ₂ imC ₃ C 15 g of NaH was mixed with 50 mL of tetrahydrofuran, 14 g of imidazole was added and stirred for one day. 37 g of 1-bromo-2-(2-methoxyethoxy)ethane was added and stirred at 70° C. for 6 hours. The solution was removed and purified by distillation. It was mixed with 250 mL of acetonitrile, mixed with 29 g of ethyl-4-bromobutyrate and refluxed at 80° C. for 16 hours. After washing with diethyl ether, the mixture was mixed with an anion exchange resin, filtered, and the solvent was distilled off under reduced pressure to obtain OE ₂ imC ₃ C.
NMR data: δ=2.13-2.27 (4H, m, CH2CO and CH2CH2CO), 3.37 (3H, s, CH3O), 3.51-3.65 (4H, m, CH3OCH2CH2), 3.86 (2H, t, J = 3.6Hz, OCH2CH2N ), 4.40 (2H, t, J=6.7Hz, NCH2CH2CH2COO), 4.66 (2H, t, J=3.7Hz, OCH2CH2N), 7.29 and 7.49 (2H, t, J=both 1.6 Hz, NCHCHN), 11.00 (1H , s, NCHN).

(2) The following ionic liquid [C ₂ mim]OAc was purchased from Iolitech GmbH.

(3) Synthesis of Ionic Liquid [OE ₂ eim]OAc 50 mL of tetrahydrofuran, 14 g of 1-ethylimidazole, and 26 g of 1-bromo-2-(2-methoxyethoxy)ethane were added and stirred at 80° C. for 4 hours. The ionic liquid phase was removed and washed with diethyl ether. It was mixed with an anion exchange resin, subjected to ion exchange, filtered, and neutralized with acetic acid. A small excess of acetic acid was removed with alumina to obtain [OE ₂ eim]OAc.
NMR data: δ=1.58 (3H, t, J=7.4Hz, CH3CH2N), 1.98 (3H, s, CH3CO), 3.37 (3H, s, CH3O), 3.51-3.67 (4H, m, CH3OCH2CH2), 3.86 ( 2H, t, J=4.0Hz, OCH2CH2N), 4.36 (2H, quin, J=7.4Hz, CH3CH2N), 4.62 (2H, t, J=5.4Hz, OCH2CH2N), 7.34 and 7.55 (2H, t, J= both 1.6 Hz, NCHCHN), 11.23 (1H, s, NCHN).

(4) Culture experiment The above OE ₂ imC ₃ C, [C ₂ mim]OAc, [OE ₂ im]OAc, or dimethyl sulfoxide (DMSO) as an organic solvent was added to Dulbecco's modified MEM medium containing 5% calf serum for 2 hours. The human fibroblasts were cultured for 24 hours after adding to 10% by weight or 10% by weight. The initial seeding number was 2.0×10 ⁴ cells/well. After culturing for 24 hours, the viability of human fibroblasts was examined by a method using Nile Red. The results are shown in FIGS. 1 and 2. FIG.

As shown in FIG. 1, when OE ₂ imC ₃ C was added at 2% by weight, the cells did not die at all, whereas when the non-zwitterion ionic liquid and DMSO were used, the survival rate was halved. It was found to decrease to some extent.

Moreover, as shown in FIG. 2, it was found that when added to 10% by weight, the ionic liquid kills 90% or more of the cells, while the DMSO kills about 70% of the cells. On the other hand, in OE ₂ imC ₃ C, death was only about 20%. From this, it was revealed that OE ₂ imC ₃ C, which is a zwitterion, has much lower cytotoxicity than DMSO and the like.

2. Escherichia coli culture in the presence of sparingly soluble substance After dissolving chloramphenicol as a sparingly soluble substance in OE ₂ imC ₃ C synthesized in 1 above to a final concentration of 100 mg/L and diluting with LB medium, (OE ₂ imC ₃ C final concentration: 10% by weight), E. coli was cultured. _OD600 , an index of bacterial concentration, increased from 0.1 to 3.9 with 24 hours of culture. This indicates that OE ₂ imC ₃ C, which is a zwitterion, has low cytotoxicity to E. coli and can be added to the medium in a state in which a poorly soluble substance is dissolved at a high concentration.

3. Cytotoxicity of phosphonium-based zwitterions to E. coli (1) Synthesis of zwitterion P _8,8,8 C ₃ S 37 g of trioctylphosphine and 12 g of butane sultone were refluxed in 50 mL of hexane at 200° C. for 10 hours, Washing _with hexane and diethyl ether gave _P8,8,8C3S .

(2) Culture experiment Escherichia coli was cultured in _an LB medium supplemented with 1% by weight of _P8,8,8C3S . _OD600 , which is _an index of bacterial concentration, increased from 0.1 to 0.57 after 24 hours of culture, confirming that the bacteria can grow even in the presence of _P8,8,8C3S .

4. Dissolution Experiment of Acyclovir Acyclovir was dissolved as a sparingly soluble substance in the OE ₂ imC ₃ C synthesized in 1 above.
Acyclovir is a therapeutic agent for viral infections that is effective against herpes simplex virus, chickenpox, herpes zoster, and the like. It is used for the treatment of herpes simplex virus infection (herpes simplex), suppression of onset during bone marrow transplantation, and treatment of chickenpox and herpes zoster. Injectable drugs are also indicated for encephalitis and meningitis caused by these viruses. Its chemical properties are white to slightly yellowish white crystalline powder, odorless and bitter in taste. It is freely soluble in dimethyl sulfoxide (DMSO) and sparingly soluble in acetic acid. It is sparingly soluble in water and extremely sparingly soluble in methanol and ethanol. Almost insoluble in acetone, 1-propanol, diethyl ether and hexane. Dissolves in dilute hydrochloric acid, dilute sodium hydroxide TS or ammonia TS.

Experimental results showed that acyclovir was soluble in OE ₂ imC ₃ C at 80° C. at 5% by weight, showing high solubility.

5. Dissolution experiment of various poorly soluble substances Glycyrrhetinic acid, dipotassium glycyrrhizinate, coumarin, ononin, glycyrrhizic acid, liquiritin, collagen, and xylan were added to OE ₂ imC ₃ C synthesized in 1 above, water, and DMSO as poorly soluble substances. , lignin, and chloramphenicol were dissolved respectively. Structural formulas other than chloramphenicol and collagen are shown below.

The results of the experiment are summarized in Table 1. In Table 1, ◯ indicates dissolution, specifically, it was judged to be in a dissolved state when it was a uniform solution when it was stirred for 15 minutes and visually confirmed immediately after. X is insoluble and represents a state other than ◯. The concentration of the sparingly soluble substance was 1% by weight with respect to OE ₂ imC ₃ C, water, and DMSO. As shown in Table 1, the zwitterionic OE ₂ imC ₃ C was able to dissolve more types of sparingly soluble substances better than DMSO.

6. Cytotoxicity against Escherichia coli (1) Synthesis of zwitterionic OE ₂ imC ₅ C Same as for OE ₂ imC ₃ C above, except that ethyl-6-bromohexanoate was used instead of ethyl-4-bromobutyrate. OE ₂ imC ₅ C was synthesized in a similar manner.

(2) Synthesis of zwitterionic OE ₁ imC ₃ C For OE ₂ imC ₃ C above, except 1-bromo-ethylmethyl ether was used in place of 1-bromo-2-(2-methoxyethoxy)ethane. OE ₁ imC ₃ C was synthesized in the same manner as.

(3) Synthesis of zwitterionic C ₁ imC ₃ C 12 g of 1-methylimidazole was mixed with 250 mL of acetonitrile, mixed with 29 g of ethyl-4-bromobutyrate and refluxed at 80° C. for 16 hours. After washing with diethyl ether, the mixture was mixed with an anion exchange resin, filtered, and the solvent was distilled off under reduced pressure to obtain C ₁ imC ₃ C.

(4) Synthesis of zwitterionic C ₄ imC ₃ C C ₄ imC ₃ C was synthesized in the same manner as for C ₁ imC ₃ C above, except that 1-butylimidazole was used instead of 1-methylimidazole. bottom.

(5) Synthesis of zwitterionic C ₁ imC ₃ S 12 g of 1-methylimidazole was mixed with 250 mL of acetonitrile, mixed with 17 g of 1,3-propanesultone, and refluxed at 80° C. for 16 hours. After washing with diethyl ether, the solvent was distilled off under reduced pressure to obtain C ₁ imC ₃ S.

(6) Synthesis of zwitterionic C ₁ imC ₄ S C ₁ imC ₄ S was synthesized in the same manner as for C ₁ imC ₃ S above, except that 1,4-butanesultone was used instead of propanesultone. .

(7) Measurement of OD ₆₀₀ value Each synthesized zwitterion, OE ₂ imC ₃ C used in 1 above, and [C ₂ im] OAc and [OE ₂ eim] OAc, which are ionic liquids, were added to LB medium. Each was added (concentration of zwitterion or ionic liquid in the medium was 0.5 M) and examined for growth of E. coli. The cells were inoculated into LB medium so that _OD600 , which is an indicator of the concentration of E. coli, was 0.1, and the _OD600 was examined after culturing at 37°C for 24 hours. Table 2 shows the results.

As shown in Table 2, the zwitterions of the present invention have higher OD ₆₀₀ values and lower toxicity to E. coli than the two ionic liquids.

7. Cytotoxicity against Escherichia coli at Low Temperature The OE ₂ imC ₃ C used in 1 above was added to LB medium at a concentration of 30% by weight, and the growth of Escherichia coli was examined. E. coli was inoculated into LB medium so that OD ₆₀₀ , which is an index of the concentration of E. coli, was 0.1, and after culturing at -10°C for 10 days, OD ₆₀₀ was 0.2. These results demonstrate that the zwitterions of the present invention do not inhibit the growth of E. coli even at low temperatures.

Claims (2)

Formula (1) below

(Wherein, A is an anion selected from the group consisting of - COO ^- , -OP=O(H)O ^- , -OP=O(CH ₃ )O ^- and -OP=O(OR ₃ )O ^- and R ₁ is a methyl group, ethyl group, propyl group, butyl group, pentyl group, CH ₃ OCH ₂ CH ₂ — or CH ₃ OCH ₂ CH ₂ OCH ₂ CH ₂ — , and R ₂ is the number of carbon atoms 3 to 5 alkylene groups, and R ₃ is an alkyl group.)
A dissolution agent for poorly water-soluble substances (excluding polysaccharides) containing zwitterions represented by The zwitterion is represented by the following formula (2)

(Wherein, R ₁ and R ₂ are as defined in claim 1.)
The agent for dissolving poorly water -soluble substances (excluding polysaccharides) according to claim 1, represented by:

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