Fluorinated compound

Abstract

The present invention provides: a fluorinated compound in which a group represented by general formula (1) (wherein n represents an integer of 1-7; and the black dot represents a bond) is linked, via a linking group, to a functional molecule that acts in cells, and the functional molecule is a low-molecular-weight compound having a molecular weight of 200-1,000 Da; a pharmaceutical composition which contains the fluorinated compound; and a method for transporting a fluorinated compound in cells, the method comprising culturing cells in a culture medium containing the fluorinated compound to transport the fluorinated compound into the cells.

Description

Fluorine-containing compounds

[0001] The present invention relates to a functional molecule that operates intracellularly and has improved cell membrane permeability, a pharmaceutical composition containing the functional molecule, and a method for transporting the functional molecule into cells.This application claims priority based on Japanese Patent Application No. 2024-072312, filed on April 26, 2024, the contents of which are incorporated herein by reference.

[0002] 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.

[0003] For example, compounds having a polyfluoro structure are known to be stable and low in toxicity in vivo, and to have excellent uptake into cells and escape from endosomes (Non-Patent Document 1). Taking advantage of these properties, studies have been conducted to introduce a polyfluoro structure into oligonucleotides or peptide nucleic acids as a moiety capable of penetrating cell membranes (Patent Documents 3 and 4).

[0004] International Publication No. 2011 / 036557 International Publication No. 2017 / 212006 International Publication No. 2012 / 130941 International Publication No. 2021 / 060506

[0005] Zhang et al., MRS Communications, 2018, vol.8, p.303-313.

[0006] An object of the present invention is to provide a functional molecule that has improved cell membrane permeability and operates within cells, and a method for transporting the functional molecule into cells.

[0007] The present inventors have discovered that perfluorophosphoryl crown ethers are highly water-soluble and highly reactive, and can be bound to various functional groups such as maleimide groups, alkyne groups, primary amino groups, and guanidinium groups through nucleophilic addition-elimination reactions, and that modifying a low molecular weight compound that functions within cells with a group derived from a perfluorophosphoryl crown ether improves the cell membrane permeability of the low molecular weight compound and increases the efficiency of introduction into cells, thereby completing the present invention.

[0008] That is, the present invention is as follows: [1] A compound represented by the following general formula (1):

[0009]

[0010] (wherein n represents an integer of 1 to 7, and a black circle represents a bond) is linked to a functional molecule that operates within a cell via a linking group, and the functional molecule is a low molecular weight compound having a molecular weight of 200 to 1000 Da. [2] The fluorine-containing compound according to [1] above, wherein the functional molecule is a fat-soluble molecule. [3] The fluorine-containing compound according to [1] or [2] above, wherein the functional molecule is a low molecular weight compound having a molecular weight of 400 to 800 Da. [4] The fluorine-containing compound according to any of [1] to [3] above, wherein n represents 2, 3, or 4. [5] The fluorine-containing compound according to any one of [1] to [4] above, wherein the linking group is -O-, -S-, -NH-, -C(=O)-, -O-P(=O)(OH)-O-, an alkylene group having 1 to 10 carbon atoms, an alkenylene group having 2 to 10 carbon atoms, or a group combining two or more selected from the group consisting of these organic groups. [6] The fluorine-containing compound according to any one of [1] to [5] above, wherein the functional molecule is a fluorescent substance. [7] The fluorine-containing compound according to any one of [1] to [6] above, wherein the functional molecule is an active ingredient of a pharmaceutical. [8] The fluorine-containing compound according to any one of [1] to [6] above, wherein the functional molecule is an anticancer agent. [9] A pharmaceutical composition comprising the fluorine-containing compound according to any one of [1] to [8] above.

[10] The pharmaceutical composition according to [9] above, wherein the content of the fluorine-containing compound in the pharmaceutical composition is 0.1 to 100% by mass with respect to the total amount of the composition.

[11] A method for intracellular delivery of a fluorine-containing compound, comprising culturing cells in a culture medium containing any one of the fluorine-containing compounds [1] to [8] above, and delivering the fluorine-containing compound into the cells.

[0011] The fluorine-containing compound according to the present invention is a compound in which a functional molecule that functions within a cell is modified with a perfluorophosphoryl crown ether. Therefore, the fluorine-containing compound is an excellent functional molecule that has excellent cell membrane permeability and can be easily taken up by target cells.

[0012] 1 is a diagram showing the results of measuring the relative fluorescence intensity of Hep3B cells cultured with the addition of each NBD derivative in Example 1. FIG. 2 is a diagram showing the results of measuring the relative cell viability for each PPER-OH concentration for Hep3B cells cultured with the addition of PPER-OH in Example 1. FIG. 3 is a diagram showing the results of measuring the relative cell viability for each 5FU concentration for Hep3B cells cultured with the addition of PPER-SS-5FU or 5FU in Example 1. FIG. 4 is a diagram showing the results of measuring the relative cell viability for each DOX concentration for Hep3B cells cultured with the addition of PPER-SS-DOX, PPER-DOX, or DOX in Example 1. FIG. 5 is a diagram showing the results of measuring the relative cell viability for each FU concentration for Hep3B cells cultured with the addition of PPER-SS-FU, PPER-FU, or FU in Example 1. FIG. 6 is a diagram showing the results of measuring the relative cell viability for each FU concentration for Hep3B cells cultured with the addition of PPER-EG-NBD (upper row) or LPFE in Example 2. １ -NBD １ Fluorescence images (left) and transmitted light images (right) of Jurkat cells cultured with fluorescein (FITC), PPER-FITC, rhodamine (Rho-TEG), and PPER-Rho (bottom row) in Example 3. Fluorescence images (top row) and transmitted light images (bottom row) of Hep3B cells cultured with fluorescein (FITC), PPER-FITC, rhodamine (Rho-TEG), and PPER-Rho in Example 3.

[0013] The present invention will be described in detail below.

[0014] <Fluorine-Containing Compound> The fluorine-containing compound of this embodiment is a fluorine-containing compound in which a group represented by the following general formula (1) is linked to a functional molecule that operates within a cell via a linking group. Hereinafter, the group represented by general formula (1) may be referred to as a "PPER group." Furthermore, unless otherwise specified, "functional molecule" refers to "functional molecule that operates within a cell."

[0015]

[0016] In general formula (1), n ​​represents an integer of 1 to 7. In the PPER group contained in the fluorine-containing compound of the present embodiment, n is preferably an integer of 1 to 7, more preferably an integer of 1 to 5, still more preferably an integer of 2 to 4, still more preferably 2 or 3, and particularly preferably 2.

[0017] In general formula (1), a black circle represents a bond. In the fluorine-containing compound of this embodiment, the black circle is bonded to a linking group, and the linking group is also bonded to a functional molecule. The linking group is not particularly limited as long as it is an organic group that can link a PPER group to a functional molecule, and any divalent or higher organic group can be used. Examples of the divalent or higher organic group include -O-, -S-, -NH-, -C(=O)-, -O-P(=O)(OH)-O-, an alkylene group, an alkenylene group, or a group combining two or more selected from the group consisting of these organic groups. Examples of the group combining two or more selected from the group consisting of these organic groups include a polyethylene glycol (PEG) group (-(C ２ H ４ Examples of such linking groups that link a PPER group to a functional molecule include a pyrrole ring, a maleimide (pyrroledione) ring, a pyrazole ring, an imidazole ring, a triazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, an oxazole ring, a thiazole ring, a furan ring, a thiophene ring, a benzene ring, and the like, where n' is a natural number, -C(=O)-O-, -O-C(=O)-, -C(=O)n'-, -C(=O)n'-, -C(=O)NH-, -NH-C(=O)-, -NH-C(=O)-O-, and -O-C(=O)-NH-. Examples of such linking groups that link a PPER group to a functional molecule include a pyrrole ring, a maleimide (pyrroledione) ring, a pyrazole ring, an imidazole ring, a triazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, an oxazole ring, a thiazole ring, a furan ring, a thiophene ring, a benzene ring, and the like, in which two or more hydrogen atoms have been removed.

[0018] When the linking group is an alkylene group, the alkylene group is １－１０ An alkylene group (an alkylene group having 1 to 10 carbon atoms) is preferred, and C １－６ An alkylene group (an alkylene group having 1 to 6 carbon atoms) is more preferred, and it may be a straight chain or a branched chain. When the linking group is an alkenylene group, the alkenylene group is preferably C ２－１０ Alkenylene groups (alkenylene groups having 2 to 10 carbon atoms) are preferred, and C ２－６ Alkenylene groups (alkenylene groups having 2 to 6 carbon atoms) are more preferred, and may be linear or branched. By further removing a hydrogen atom from the alkylene group or alkenylene group, a trivalent or higher valent linking group can be obtained.

[0019] The functional molecule possessed by the fluorine-containing compound of this embodiment is a functional molecule that operates intracellularly and is a low-molecular-weight compound having a molecular weight of 200 to 1000 Da. Because the functional molecule is a low-molecular-weight compound, the fluorine-containing compound of this embodiment achieves high cell membrane permeability due to the PPER group. The molecular weight of the functional molecule possessed by the fluorine-containing compound of this embodiment is preferably 300 to 900 Da, more preferably 400 to 800 Da, and even more preferably 400 to 600 Da.

[0020] The functional molecule contained in the fluorine-containing compound of this embodiment is preferably a fat-soluble molecule, since this allows for higher cell membrane permeability. A fat-soluble molecule refers to a compound having a partition coefficient (n-octanol / water) ([concentration of substance soluble in n-octanol] / [concentration of substance soluble in water]) of greater than 1.0.

[0021] The functional molecule possessed by the fluorine-containing compound of this embodiment is not particularly limited as long as it is a low molecular weight compound that functions within cells. Examples of such low molecular weight compounds include labeling substances such as fluorescent substances and active ingredients of pharmaceuticals. By introducing a PPER group into these functional molecules, cell membrane permeability can be improved, and transport efficiency into cells can be increased.

[0022] Among the functional molecules possessed by the fluorine-containing compound of this embodiment, examples of fluorescent substances include fluoresceins, rhodamines, cyanines, bodipys, dansyls, pyranines, coumarins, acridines, oxazines, benzoxadiazoles, phycocyanins, and indocyanine greens.

[0023] Among the functional molecules possessed by the fluorine-containing compound of this embodiment, examples of active ingredients of pharmaceuticals (including diagnostic agents, in vitro testing agents, etc.) include active ingredients of anticancer agents, anti-inflammatory agents, antipyretic analgesics, antiviral agents, antibacterial agents, etc. Preferred examples of the functional molecules possessed by the fluorine-containing compound of this embodiment include poorly soluble low-molecular-weight compounds such as fluorouracil (5FU), doxorubicin (DOX), paclitaxel, cytogenin, indomethacin, ketoprofen, flurbiprofen, loxoprofen, ketorolac, felbinac, diferonac, ibuprofen, etc.

[0024] The functional molecule possessed by the fluorine-containing compound of this embodiment is also preferably an active ingredient of a research reagent, such as an enzyme inhibitor, a transporter inhibitor, or a transcription inhibitor, which is a fat-soluble low-molecular-weight compound that is incorporated into cultured cells or the like for use in elucidating biological functions.

[0025] The fluorine-containing compound of this embodiment can be synthesized, for example, by using as raw materials a compound in which a PPER group is bonded to a chlorine atom via a bonding bond (hereinafter, sometimes referred to as "PPER-Cl"), a chain molecule having two reactive groups, and a functional molecule, and by forming a covalent bond between one reactive group of the chain molecule and PPER-Cl through an organic synthesis reaction, and then forming a covalent bond between the remaining reactive group of the chain molecule and the functional molecule. The organic synthesis reaction between PPER-Cl and the reactive group, and the organic synthesis reaction between the functional molecule and the reactive group can be carried out by a general organic synthesis reaction.

[0026] PPER-Cl can be produced by reacting a fluorinated polyethylene glycol represented by the following general formula (II) (wherein n is an integer of 1 to 7) with phosphoryl chloride.

[0027] HOCH ２ CF ２ O-(CF ２ CF ２ O) n-CF ２ CH ２ OH (II)

[0028] The reaction of the fluorinated polyethylene glycol with phosphoryl chloride is typically carried out in the presence of a base such as pyridine, triethylamine, N,N-diisopropylethylamine (DIPA), or 1,8-diazacyclo[5.4.0]undec-7-ene (DBU). The amount of phosphoryl chloride used is typically 0.9 to 2 equivalents, preferably 0.9 to 1.2 equivalents, relative to compound (II). The solvent is not particularly limited as long as it does not inhibit the reaction. For example, anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous acetonitrile, anhydrous 1,4-dioxane, or anhydrous dimethylformamide is preferably used. The reaction is initiated at -70°C and is carried out without maintaining that temperature, while allowing the temperature of the reaction solution to return to room temperature over time. In the formula (II), the compound in which n is 1 or 2, i.e., fluorinated triethylene glycol and fluorinated tetraethylene glycol (1H,1H,11H,11H-dodecafluoro-3,6,9-trioxaundecane-1,11-diol), can be commercially available products.

[0029] PPER-Cl is highly reactive and can relatively easily form covalent bonds with various reactive groups such as hydroxyl groups, primary amino groups, thiol groups (mercapto groups), maleimide groups, and alkyne groups through nucleophilic addition-elimination reactions.

[0030] For example, as shown in the chemical reaction below, PPER-Cl can be reacted with a compound having a hydroxyl group and a maleimide group or an alkyne group to introduce a maleimide group or an alkyne group, respectively.

[0031]

[0032] Compounds with introduced maleimide groups can undergo click reactions with various reagents containing thiol groups. Compounds with introduced alkyne groups can undergo click reactions with azide groups (N ３-) can undergo a click reaction. Therefore, a compound in which a maleimide group or an alkyne group has been introduced into PPER-Cl as described above can be linked to various functional molecules via a reaction reagent having a thiol group or a reaction reagent having an azide group. As the reaction reagent having a thiol group or the reaction reagent having an azide group, a reaction reagent that is commercially available as a click-reactive reagent, a reaction reagent produced by a conventionally known method, or the like can be used as appropriate.

[0033] <Method for Intracellular Delivery of Fluorine-Containing Compound> The method for intracellular delivery of a fluorine-containing compound of this embodiment is a method for culturing cells in a culture medium containing the fluorine-containing compound of this embodiment and delivering the fluorine-containing compound into the cells. The fluorine-containing compound of this embodiment has a PPER group introduced therein, and therefore has excellent cell membrane permeability. Therefore, the fluorine-containing compound of this embodiment can be relatively easily taken up into cells simply by contacting the compound with cells, and has high transport efficiency into cells. Therefore, when target cells (cells into which the fluorine-containing compound is to be introduced) are exogenous cells such as cultured cells, the fluorine-containing compound can be delivered into the cells simply by culturing the target cells in a culture medium containing the fluorine-containing compound of this embodiment.

[0034] <Pharmaceutical Composition> The fluorine-containing compound of this embodiment is suitable as an active ingredient of a pharmaceutical composition. In general, the therapeutic effect of a pharmaceutical depends on the amount of the medicinal ingredient absorbed. The fluorine-containing compound of this embodiment has high transport efficiency to target cells, and is therefore expected to have a higher therapeutic effect than functional molecules without a PPER group introduced therein.

[0035] The fluorine-containing compound of this embodiment can be used as a preventive or therapeutic agent for various diseases including cancer, either as is or by mixing it with a pharmacologically acceptable carrier or the like to form a pharmaceutical composition. Furthermore, when the fluorine-containing compound of this embodiment is a fluorescent dye having a PPER group introduced therein, the pharmaceutical composition containing the fluorine-containing compound of this embodiment can also be used as a testing reagent for detecting diseased tissues, etc.

[0036] As pharmacologically acceptable carriers, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used, and are incorporated as excipients, lubricants, binders, disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. Furthermore, pharmaceutical additives such as preservatives, antioxidants, coloring agents, sweeteners, etc. can also be used as necessary.

[0037] 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.

[0038] Suitable examples of the lubricant include magnesium stearate, calcium stearate, talc, and colloidal silica.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, and glucose.

[0045] Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0046] A suitable example of a soothing agent is benzyl alcohol.

[0047] Suitable examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0048] Suitable examples of antioxidants include sulfites and ascorbic acids.

[0049] 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).

[0050] Suitable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, and stevia.

[0051] 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).

[0052] 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.

[0053] 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.

[0054] 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)).

[0055] The coating bases may be used by mixing two or more of them in an appropriate ratio. In addition, a light-shielding agent such as titanium oxide or iron sesquioxide may be used during coating.

[0056] A pharmaceutical composition containing the fluorine-containing compound of this embodiment can be produced by a method commonly used in the technical field of formulation, for example, a method described in the Japanese Pharmacopoeia, etc. The content of the fluorine-containing compound of this embodiment in the pharmaceutical composition varies depending on the route of administration, dosage form, intended dose, etc., but is, for example, about 0.1 to 100% by mass.

[0057] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0058] [Production Example 1] Synthesis of PPER-Cl

[0059]

[0060] Fluorinated tetraethylene glycol (1H,1H,11H,11H-dodecafluoro-3,6,9-trioxaundecane-1,11-diol) (4.52 g, 11.0 mmol) was dissolved in 300 mL of anhydrous dichloromethane in an oven-dried 1 L three-neck round-bottom flask, and the temperature was lowered to -78 °C to obtain a white suspension. A solution of phosphoryl chloride (1.65 g, 1.0 mL, 10.8 mmol) in 200 mL of anhydrous dichloromethane was added dropwise using a dropping funnel. The mixture was stirred for 1 hour while maintaining the temperature at -78 °C, and then the temperature was slowly returned to room temperature. After stirring for an additional 6 hours, anhydrous pyridine (1.87 g, 23.7 mmol) was added dropwise via syringe. The reaction mixture was stirred at room temperature for an additional 24 hours. The solvent was evaporated under a nitrogen stream to obtain the desired PPER-Cl. MALDI-TOF MS in negative ion mode using trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile as the matrix showed a single peak for PPER (PPER-OH) at 471.146 Da, indicating the formation of a reactive PPER-Cl intermediate. The product was purified with CHCl / Hexane (1:1) at −20°C under N ２ It was recrystallized under

[0061] 1 H NMR (CDCl3, 298 K, 600 MHz): δ = 4.39 (m, -CH2 CF2), 4.64 (m, -CH2 CF2) ppm. 13 C NMR (CDCl3, 298 K, 150 MHz): δ = 66.07 (m, -CH2), 114.22 (m, -CF2), 114.50(m, -CF2), 120.21 (td, JC,F = 274.4 Hz, 10.6 Hz -CF2) ppm. 19 F NMR (CDCl3, 298 K, 565 MHz): δ = -77.93 (m), -78.31 (m), -89.24 (m), -89.60 (s), -90.09 (m) ppm. 31P NMR (CDCl3, 298 K, 243 MHz): δ = 5.24. MALDI-TOF MS: for C8H4F 12 O7P [MH] - , calculated: 471.07 Da, found: 471.15 Da

[0062] Example 1 PPER-Cl synthesized in Production Example 1 was linked to a derivative (NBD-OH) of the fluorescent dye nitrobenzoxadiazole (NBD) to synthesize NBD derivatives (PPER-NBD, PPER-NH-NBD, PPER-TEG-NBD, PPER-EG-NBD) incorporating a PPER group, and their cell membrane permeability, etc. were investigated. １ , LPFE １ -NBD １ , LPFE ２ -NBD １ , LPFE-NBD ２ , LPF-NBD ２ , LPFE ２ -NBD ２ , and 2LPFE ２ -NBD ２ was also synthesized.

[0063] (1) Synthesis of NBD derivatives

[0064]

[0065] PPER-EG-NBD was synthesized by combining NBD-OH and PPER-Cl via a nucleophilic addition-elimination reaction.

[0066]

[0067] Crude PPER-Cl was dissolved in anhydrous toluene (200 mL), and pyridine (1.0 mL) and NBD-OH (1.50 g, 5.59 mmol) were added under a nitrogen atmosphere. The reaction solution was heated and stirred at 80°C for 1 hour, and the progress of the reaction was monitored by thin layer chromatography (TLC). The solvent was evaporated under reduced pressure, and the residue was diluted with methylene chloride (100 mL) and washed with water (150 mL). The organic fraction was purified by NaCl. ２ SO ４The mixture was dried at 77°C, filtered, and the solvent was removed under vacuum. The resulting crude product was purified by silica gel column chromatography (φ=3.5 cm, L=20 cm) using methanol / methylene chloride (1 / 20 (volume ratio)) as the mobile phase (Rf=0.20). As a result, PPER-EG-NBD was isolated as a yellow solid (1.55 g, 2.15 mmol, yield: 38%).

[0068] 1 H NMR (CD2Cl2, 298 K, 600 MHz): δ = 3.73 (br, 2H,-CH2), 3.70 (m, 2H, - CH2), 3.87 (m, 2H, -CH2), 4.32 (dd, 3J = 8.4, 5.4 Hz, 2H, ,-CH2), 4.36 (m, 2H, -OCF2CH2O), 4.55 (m, 2H, -OCF2CH2O), 6.52 (d, 3J = 8.8 Hz, 1H, ,NBD-CH), 7.37 (t, 3J = 5.4 Hz 1H, -NH), 8.48 (d, 3J = 8.8 Hz, 1H, ,NBD-CH) ppm. 13 C NMR (CD2Cl2, 298 K, 150 MHz): δ = 44.30 (s, -CH2), 65.97 (d, 3JC,F = 4.5 Hz, -CH2), 68.57 (s, -CH2), 68.60 (s, -CH2), 70.12 (m, -CH2), 99.49 (s, -Ar-C), 114.49 (m, -CF2), 114.81 (m, -CF2), 121.15 (td, 3JC,F = 276, 9.0 Hz, -CF2), 124.18 (s, Ar-C), 136.84 (s, Ar-C), 144.60 (s, -Ar-C), 144.87 (s, - 2C) ppm. 19 F NMR (CD2Cl2, 298 K, 565 MHz): δ = -78.09 (m), -79.07 (m), -88.72 (m), -89.73 (s), -90.67 (m) ppm. 31P NMR (CD2Cl2, 298 K, 243 MHz): δ = -2.56 ppm. MALDI-TOF MS: for C 18 H 16 F 12 N4O 11 P [M+H + ], Calculated: 723.30 Da, found: 723.888 Da (PPER-EG-NBD). HRMS (ESI-MS): C 18 H 14 F 12 N4O 11 P [MH] - , Calculated m / z = 721.0205 Da; m / z found = 721.0204 Da.

[0069] PPER-NH-NBD is PPER-NH ２ and NBD-Cl via a nucleophilic addition-elimination reaction. PC-NBD was synthesized by combining NBD-OH and PC-Cl via a nucleophilic addition-elimination reaction. F4-NBD was synthesized by cyclizing 2,2,3,3-tetrafluoro-1,4-butanediol with phosphoryl chloride and combining it with NBD-OH via a nucleophilic addition-elimination reaction.

[0070] LPFE １ -NBD １ was synthesized by coupling NBD-OH and 1H,1H-tridecafluoro-3,6,9-trioxadecan-1-ol via a nucleophilic addition-elimination reaction.

[0071]

[0072] In an oven-dried 1 L three-necked round-bottom flask, 1H,1H-tridecafluoro-3,6,9-trioxadecan-1-ol (1.00 g, 2.51 mmol) and NBD-OH (0.68 g, 2.54 mmol) were dissolved in anhydrous dichloromethane (250 mL), and the temperature was lowered to 0°C. To the resulting solution, a solution of phosphoryl chloride (0.41 g, 0.25 mL, 2.71 mmol) in anhydrous dichloromethane (200 mL) was added dropwise using a dropping funnel. The mixture was stirred for 1 hour while maintaining the temperature at 0°C, and then the temperature was slowly returned to room temperature. Anhydrous pyridine (0.65 mL, 0.64 g, 8.07 mmol) was then slowly added dropwise under a nitrogen atmosphere. The resulting reaction mixture was heated to reflux for 3 hours. The solvent was distilled off under reduced pressure, 2.0 mL of water was added, and the solvent was distilled off again under reduced pressure. The resulting crude product was purified by elution with a mobile phase of ethyl acetate / methylene chloride (1 / 4 to 1 / 1 (volume ratio)) followed by methanol / methylene chloride (1 / 10 (volume ratio)) (R ｆ = 0.20) and purified by silica gel column chromatography (φ = 3.5 cm, L = 30 cm). This gave LPFE, a yellow solid. １ -NBD １ was isolated (220 mg, 0.30 mmol, yield: 12%).

[0073] 1H NMR (CDCl3, 298 K, 600 MHz): 3.71 (br, 2H, - CH2), 3.80 (m, 2H, -CH2), 3.87 (m, 2H, -OCH2), 4.32 (m, 2H, -OCH2), 4.44 (m, 2H, -OCF2CH2O), 6.20 (d, 3J = 8.8 Hz, 1H, ,NBD-CH), 7.29 (br, 1H, -NH), 8.48 (d, 3J = 8.8 Hz, 1H, NBD-CH) ppm. 13C NMR (CDCl3, 298 K, 150 MHz): δ = 43.92 (m, -CH2), 65.26 (m, -CH2), 68.08 (m, -CH2), 68.60 (s, -CH2), 70.12 (m, -CH2), 99.05 (m, -Ar-C), 114.27 (m, -CF2), 114.58 (m, -CF2), 123.97 (td, 3J C,F = 276, 9.0 Hz, -CF2), 136.37 (s, Ar-C), 136.66 (s, Ar-C), 144.17 (s, -Ar-C), 144.28 (s, -Ar-C), 144.45 (s, -Ar-C) ppm. 19F NMR (CDCl3, 298 K, 565 MHz): δ = -55.28 (m), -78.81 (m), -88.57 (m), -88.60 (s), -88.69, -90.62 (m) ppm. 31P NMR (CDCl3, 298 K, 243 MHz): δ = -0.59 ppm. MALDI-TOF MS: for C 17 H 13 F 13 N4O 11 P [M-H + ], Calculated m / z = 727.0111 Da, m / z found = 726.638 Da (LPFE1-NBD1). HRMS (ESI-MS): C 17 H 13 F 13 N4O 11 P [M-H +], Calculated m / z = 727.0111 Da; m / z found = 727.0111 Da.

[0074] LPFE １ -NBD ２ and LPFE ２ -NBD １ is LPFE １ -NBD １ Obtained as a by-product of the synthesis. ２ is LPF-NBD １ Obtained as a by-product of synthesis. ２ -NBD ２ In the case of 2LPFE, phenylphosphonic dichloride was added to a mixed solution of fluorinated tetraethylene glycol and NBD-OH, and the mixture was reacted in the presence of pyridine. ２ -NBD ２ is LPFE ２ -NBD ２ It was obtained as a by-product during the synthesis of

[0075] (2) Evaluation of cell membrane permeability by flow cytometry. Each NBD derivative was introduced into liver cancer-derived Hep3B cells, and cell membrane permeability was evaluated. MEM medium containing 10% FBS (fetal bovine serum) was used as the culture medium. Hep3B cells were pre-cultured at 37°C for 24 hours, and the medium was replaced with the sample solution (culture medium supplemented with 2.0 μM NBD derivative) and cultured at 37°C for 2 hours. After culturing, the cells were washed three times with PBS (phosphate-buffered saline) and then detached from the culture vessel using 0.05% Trypsin-EDTA (Gibco). The collected cells were then applied to a flow cytometer (a BD model S3 Accuri (registered trademark) C6 flow cytometer, manufactured by BD Biosciences) to measure the fluorescence intensity of green fluorescence (excitation: 488 nm, detection: 533±30 nm) emitted from NBD.

[0076] Cells cultured in a culture medium without the addition of an NBD derivative instead of the sample solution were used as a blank. Cells cultured in a medium containing NBD-OH or PC-NBD were used as a negative control to examine the effect of fluorination.

[0077] The fluorescence intensity of blank cells was set to 1.0, and the relative fluorescence intensity of each cell was calculated. The results are shown in Figure 1. As shown in Figure 1, the relative fluorescence intensity of cells into which PPER groups were introduced, such as PPER-NBD, PPER-NH-NBD, PPER-TEG-NBD, and PPER-EG-NBD, was significantly higher than that of cells into which PC-OH, which does not contain fluorine atoms, and LPFE, which contains a linear perfluorophosphoryl ether group, was introduced. ２ -NBD １ , LPFE ２ -NBD ２ , and 2LPFE ２ -NBD ２ The expression level of the α- and β-actin-dependent ATP ...

[0078] (3) Evaluation of Cytotoxicity by CCK Assay Hep3B cells were cultured in a 96-well culture plate at 1.0 × 10 ４ Cells were seeded at 1 cell / well, allowed to adhere and grow, and cultured for 24 hours. PPER-OH was then added to the culture medium to a final concentration of 5-1000 μM, and the cells were cultured for 24 hours. After culturing, the cell viability of each cell was measured by CCK assay. The CCK assay was performed using a commercially available cytotoxicity measurement kit (manufactured by Dojindo) according to the protocol.

[0079] The relative cell viability of the cells (cell viability of blank cells is set to 100%) was calculated from the CCK assay measurement value (absorbance at 450 nm). The relative cell viability (%) of the cells at each PPER-OH concentration is shown in Figure 2. At all concentrations, the viability was approximately 100%, and no cytotoxicity due to PPER-OH was observed.

[0080] (4) Intracellular delivery of anticancer drugs and evaluation of their efficacy We synthesized a compound (PPER-SS-5FU) in which a PPER group was introduced into 5-fluorouracil (5FU), a known anticancer drug, via a disulfide (SS) bond.

[0081]

[0082] Hep3B cells were cultured in a 96-well culture plate at a density of 1.0 × 10 ４The cells were seeded at 16 cells / well, allowed to adhere and grow, and cultured for 24 hours. Subsequently, PPER-SS-5FU or 5FU was added to the culture medium to a final concentration of 1 to 500 μM in terms of 5FU, and cultured for 24 hours. After the culture, the cells were subjected to a CCK assay in the same manner as in (3) above, to determine the relative cell viability.

[0083] The relative cell viability (%) of cells at each 5FU concentration is shown in Figure 3. The relative cell viability of cells cultured in 5FU-supplemented medium was high, at 60% or higher, whereas the relative cell viability of cells cultured in PPER-SS-5FU-supplemented medium decreased significantly in a dose-dependent manner. These results demonstrate that PPER-SS-5FU has higher cell membrane permeability than 5FU and has a good introduction efficiency into cells, and that introducing PPER groups into anticancer drugs improves cell membrane permeability and results in higher pharmacological efficacy.

[0084] (5) Intracellular delivery of anticancer drugs and evaluation of their efficacy. A compound (PPER-SS-DOX) was synthesized by introducing a PPER group into the anticancer drug doxorubicin (DOX) via a disulfide (SS) bond or a maleimide group.

[0085]

[0086] Hep3B cells were cultured in a 96-well culture plate at a density of 1.0 × 10 ４ The cells were seeded at 1 cell / well, allowed to adhere and grow, and cultured for 24 hours. Subsequently, PPER-SS-DOX or DOX was added to the culture medium to a final DOX concentration of 1 to 10 μM, and the cells were cultured for 24 hours. After the culture, a CCK assay was performed on the cells in the same manner as in (3) above, and the relative cell viability was determined.

[0087] The relative cell viability (%) of cells at each DOX concentration is shown in Figure 4. For cells cultured in a medium supplemented with DOX, the relative cell viability was high, at 40% or higher, even at the maximum addition amount of 10 μM. In contrast, for cells cultured in a medium supplemented with PPER-SS-DOX, the relative cell viability decreased significantly in a dose-dependent manner. These results demonstrate that PPER-SS-DOX has higher cell membrane permeability than DOX and has a better introduction efficiency into cells. Furthermore, the introduction of PPER groups into anticancer drugs improves cell membrane permeability and results in higher efficacy.

[0088] (6) Evaluation of Cell Membrane Permeability of Anticancer Drugs Compounds (PPER-SS-FU, PPER-FU) were synthesized by introducing a PPER group into the anticancer drug 5FU via a disulfide (SS) bond or a maleimide group.

[0089]

[0090] Hep3B cells were cultured in a 96-well culture plate at a density of 1.0 × 10 ４ The cells were seeded at 10 cells / well, allowed to adhere and grow, and cultured for 24 hours. Subsequently, PPER-SS-FU, PPER-FU, or 5FU was added to the culture medium to a final concentration of 10 to 500 μM in terms of 5FU, and cultured for 30 minutes. After culture, the cells were subjected to a CCK assay in the same manner as in (3) above, to determine the relative cell viability.

[0091] The relative cell viability (%) of cells at each 5FU concentration is shown in Figure 5. The relative cell viability of cells cultured in a medium containing 5FU was nearly 100%, whereas the relative cell viability of cells cultured in a medium containing PPER-SS-FU decreased significantly in a dose-dependent manner. Furthermore, the relative cell viability of cells cultured in a medium containing PPER-FU also decreased in a dose-dependent manner, although the decrease was somewhat slower than that of PPER-SS-FU. These results demonstrate that PPER-SS-FU and PPER-FU have higher cell membrane permeability and better cell introduction efficiency than FU, and that the introduction of PPER groups into anticancer drugs improves cell membrane permeability and results in higher efficacy.

[0092] [Example 2] The NBD derivatives synthesized in Example 1 (PPER-EG-NBD and LPFE) １ -NBD １ The cell membrane permeability of the antibody against Jurkat cells derived from human acute leukemia T cells was examined. RPMI medium was used as the culture medium.

[0093] Jurkat cells were cultured in an 8-well culture plate at 1.0 × 10 ４ The cells were seeded at 100 cells / well, allowed to adhere and grow, and cultured for 2 hours. １ -NBD １ was added to the culture medium to a final concentration of 2.0 μM and cultured for 30 minutes. After the culture, the cells were washed with D-PBS, and then the green fluorescence emitted from NBD (excitation: 488 nm, detection: 500-560 nm) was observed and images were taken using a confocal microscope.

[0094] Fluorescence images of each cell are shown in Figure 6. In the figure, the right image is a transmitted light image, and the left image is a fluorescence image. For cells cultured in a medium containing PPER-EG-NBD, green fluorescence was observed from almost all cells within the field of view, confirming that the cell transport efficiency was very high. On the other hand, １ -NBD １ In cells cultured in a medium containing , no green fluorescence was observed, indicating that the antibody had not been introduced into the cells.

[0095] Example 3 PPER-Cl synthesized in Production Example 1 was linked to the fluorescent dye fluorescein (FITC) or rhodamine (Rho) to synthesize an FITC derivative (PPER-FITC) with a PPER group introduced therein and a rhodamine derivative (PPER-Rho) with a PPER group introduced therein, and their cell membrane permeability, etc. were investigated. For comparison, a rhodamine derivative (Rho-TEG) ​​without a PPER group introduced therein was also synthesized.

[0096]

[0097] PPER-FITC was synthesized by the following reaction.

[0098]

[0099] 1-(2-mercaptoethyl)-3-(fluorescein-5-yl)thiourea (70.0 mg, 0.150 mmol) and PPER-MAL (200 mg, 0.336 mmol) were dissolved in anhydrous DMF (20 mL). To the resulting solution, an anhydrous DMF solution (10 mL) of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU, 23.0 mg, 0.151 mmol) was added dropwise under a nitrogen atmosphere. The reaction solution was stirred at 50°C for 1 hour, and the reaction was monitored by TLC. The solvent was then evaporated under reduced pressure, and the residue was purified by HPLC using methanol / methylene chloride (1 / 20 (volume ratio)) as the mobile phase (R ｆ = 0.20) and purified by silica gel column chromatography (φ = 1.5 cm, L = 10 cm), thereby isolating PPER-FITC as an orange solid (120 mg, 0.113 mmol, yield: 75%).

[0100] 1H NMR (CD3OD, 298 K, 600 MHz): δ = 2.83(dd, 3J = 18.2 Hz, 5J = 4.8 Hz, 1H), 3.20 (m, 1H), 3.43 (m, 2H), 4.02 (m, 3H), 4.18 (br, 1H), 4.29 (m, 1H), 4.52 (m, 2H), 4.75 (m, 2H), 4.83 (m, 2H), 6.73 (m, 2H), 6.87 (m, 4H), 7.36 (d, 3J = 8.4 Hz, 1H), 7.95 (d, 3J = 8.4 Hz,1H), 8.33 (s, 1H) ppm. 13C NMR (CD3CN, 298 K, 150 MHz): δ = 30.50 (m), 35.82 (m), 38.78 (m), 39.54 (d, 3J C,F 118 (s, 2C), 121.05 (td, 3J C,F = 276, 9.0 Hz, -CF2), 124.56 (s), 124.40 (s), 127.68 (s), 128.94 (s), 130.66 (s), 140.68 (s), 148.62 (s), 152.82 (s), 160.04 (s, 19F NMR (CD3CN, 298 K, 565 MHz): δ = -79.34 (m), -79.83 (m), -90.22 (m), -90.83 (s), -91.19 (m) ppm. 31P NMR (CD3CN, 298 K, 243 MHz): δ = -2.70 ppm. HRMS (ESI-MS): C 37 H 27 F 12 N3O 14 PS2 [MH] + , Calculated m / z = 1060.0480 Da; m / z found = 1060.0480 Da

[0101] PPER-Rho was synthesized by the following reaction.

[0102]

[0103] Crude PPER-Cl was dissolved in anhydrous toluene (100 mL), and pyridine (1.0 mL) and diethylene glycol (1.50 g, 14.1 mmol) were added under a nitrogen atmosphere. The reaction solution was heated and stirred at 45°C for 1 hour, after which the progress of the reaction was monitored by TLC. The solvent was then evaporated under reduced pressure, and the residue was diluted with methylene chloride (100 mL) and washed with water (150 mL). The organic fraction was purified by NaCl. ２ SO ４ After drying with hexane, the mixture was filtered and the solvent was removed under vacuum. The resulting crude product was purified by elution with ethyl acetate / methylene chloride (1 / 1 (volume ratio)) as the mobile phase (R ｆ = 0.20) and purified by silica gel column chromatography (φ = 3.5 cm, L = 20 cm). ２-OH was isolated (2.60 g, 4.64 mmol, yield: 42%).

[0104] 1H NMR (CD2Cl2, 298 K, 600 MHz): δ =2.99 (m, 1H,-OH), 3.64 (m, 2H, - CH2), 3.74 (m, 4H, - CH2), 4.32 (m, 2H, -OCF2CH2O), 4.51 (m, 4H, - CH2 and -OCF2CH2O) ppm. 13C NMR (CD2Cl2, 298 K, 150 MHz): δ = 61.13 (m, -CH2), 65.37 (m, -CH2), 68.63 (m, -CH2), 69.42 (m, -CH2), 72.68 (m, -CH2), 114.01 (m, -CF2), 114.35 (m, -CF2), 120.68 (td, 3J C,F = 276, 9.0 Hz, -CF2) ppm. 19F NMR (CD2Cl2, 298 K, 565 MHz): δ = -77.71 (m), -78.68 (m), -88.33 (m), -89.45 (s), -90.51 (m) ppm. 31P NMR (CD2Cl2, 298 K, 243 MHz): δ = -2.53 ppm. HRMS (ESI-MS): C 12 H 13 F 12 NaO9P [M+Na] + , Calculated m / z = 583.0003 Da; m / z found = 583.0000 Da.

[0105]

[0106] Into an oven-dried 1 L three-neck round-bottom flask was added rhodamine B (480 mg, 1.00 mmol), 4-(dimethylamino)pyridine (DMAP, 122 mg, 1.00 mmol), and PPER-PEG ２To the resulting solution, a solution of N,N'-dicyclohexylcarbodiimide (DCC, 310 mg, 1.50 mmol) in anhydrous dichloromethane (20 mL) was slowly added dropwise under a nitrogen atmosphere. The resulting reaction solution was stirred at room temperature for 24 hours. The solvent was then evaporated under reduced pressure, and the residue was purified by HPLC using a mobile phase of methanol / methylene chloride (1 / 20 (volume ratio)) containing 1% trimethylamine (R ｆ The crude product was purified by silica gel column chromatography (φ=2.5 cm, L=30 cm) (p = 0.20), resulting in the isolation of solid PPER-Rho (720 mg, 0.71 mmol, yield: 71%).

[0107] 1H NMR (CDCl3, 298 K, 600 MHz): 1.19 (t, 12H, - CH3), 3.50 (br, 10H, - CH2), 3.53 (m, 2H, -CH2), 4.07 (d, 2H, 3J = 11.4 Hz, OCH3), 4.11 (m, 2H, -OCH2), 4.16 (m, 2H, -OCH2), 4.30 (m, 2H, -OCF2CH2O), 6.67 (d, 3J = 2.4 Hz, 1H), 6.96 (dd, 3J = 9.2 Hz, 2.4 Hz, 2H) 6.92 (d, 3J = 9.2 Hz, 2H), 7.16 (d, 3J = 7.8 Hz, 1H), 7.18 (s, 1H), 7.62 (t, 3J = 7.8 Hz, 1H), 7.69 (t, 3J = 7.8 Hz, 1H), 8.19 (d, 3J = 7.8 Hz, 1H) ppm. 13C NMR (CDCl3, 298 K, 150 MHz): δ = 12.56, 45.97, 55.36, 59.47, 64.22, 65.30 (m), 67.40, 96.16, 113.53, 114.00, 114.25 (m, 2C, -CF2), 120.50 (m, -CF2), 129.75, 130.32, 130.39, 131.32, 131.84, 132.60, 133.31, 155.41, 157.78, 159.11, 165.26 ppm. 19F NMR (CDCl3, 298 K, 565 MHz): δ = -77.78 (m), -78.61 (m), -88.59 (m), -89.47 (s), -90.21 (m) ppm. 31P NMR (CDCl3, 298 K, 243 MHz): δ = -2.82 ppm. MALDI-TOF MS: for C 18 H 15 F 12 N4O 11 P [M+H + ], calculated: 723.30 Da, found: 723.888 Da (PPER-Rho). HRMS (ESI-MS): C 40 H 42F 12 N2O 11 P [M-Cl] + , Calculated m / z = 985.2330 Da; m / z found = 985.2332 Da.

[0108] Each NBD derivative was introduced into liver cancer-derived Hep3B cells, and cell membrane permeability was evaluated. MEM medium without FBS was used as the culture medium. Hep3B cells were cultured in an 8-well culture plate at a density of 1.0 × 10 ４ Cells were seeded at 100 cells / well, allowed to adhere and grow, and cultured for 2 hours. Subsequently, PPER-FITC, FITC, PPER-Rho, or Rho-TEG was added to the culture medium to a final concentration of 2.5 μM, and the cells were cultured for 30 minutes. After the culture, the cells were washed with D-PBS and then observed and photographed using a confocal microscope for fluorescence emitted from FITC or Rho (excitation: 488 nm, detection: 500-560 nm).

[0109] Fluorescence images of each cell are shown in Figure 7. In the figure, the lower row is a transmitted light image, and the upper row is a fluorescence image. Green fluorescence was observed in almost all cells within the field of view when cells were cultured in medium supplemented with PPER-FITC and PPER-Rho, confirming that the cell transport efficiency was very high. On the other hand, no green fluorescence was observed in cells cultured in medium supplemented with FITC or Rho-TEG, indicating that the drugs had not been introduced into the cells.

Claims

1. The following general formula (1) (wherein n represents an integer of 1 to 7, and the black circle represents a bond) is linked to a functional molecule that operates within a cell via a linking group, and the functional molecule is a low molecular weight compound having a molecular weight of 200 to 1000 Da.

2. The fluorine-containing compound according to claim 1, wherein the functional molecule is a fat-soluble molecule.

3. The fluorine-containing compound according to claim 1, wherein the functional molecule is a low molecular weight compound having a molecular weight of 400 to 800 Da.

4. The fluorine-containing compound according to claim 1, wherein n represents 2, 3, or 4.

5. The fluorine-containing compound according to claim 1, wherein the linking group is -O-, -S-, -NH-, -C(=O)-, -O-P(=O)(OH)-O-, an alkylene group having 1 to 10 carbon atoms, an alkenylene group having 2 to 10 carbon atoms, or a group formed by combining two or more selected from the group consisting of these organic groups.

6. The fluorine-containing compound according to claim 1, wherein the functional molecule is a fluorescent substance.

7. The fluorine-containing compound according to claim 1, wherein the functional molecule is an active ingredient of a pharmaceutical.

8. The fluorine-containing compound according to claim 1, wherein the functional molecule is an anticancer drug.

9. A pharmaceutical composition comprising the fluorine-containing compound according to any one of claims 1 to 8.

10. The pharmaceutical composition according to claim 9, wherein the content of the fluorine-containing compound in the pharmaceutical composition is 0.1 to 100% by mass based on the total amount of the composition.

11. A method for intracellular delivery of a fluorine-containing compound, comprising culturing cells in a culture medium containing the fluorine-containing compound according to any one of claims 1 to 8, and delivering the fluorine-containing compound into the cells.

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