Peptides and 19F-MRI contrast agents

Abstract

This invention provides high sensitivity 19 A fluorine-containing molecule suitable as an F-MRI contrast agent, and a material containing the fluorine-containing molecule as an active ingredient. 19 We provide F-MRI contrast agents. [Solution] A peptide comprising two or more amino acids linked by peptide bonds, wherein the peptide contains a fluorine-containing alkyl group and an organic radical within the same amino acid residue; the peptide according to the above, wherein the fluorine-containing alkyl group is a perfluoroalkyl group having 1 to 6 carbon atoms; the peptide according to the above, wherein the fluorine-containing alkyl group is a trifluoromethyl group or a nonafluoro-tert-butyl group; the peptide according to the above, wherein the organic radical is an N-oxyl radical: A pharmaceutical composition containing any of the above peptides; and a pharmaceutical composition containing any of the above peptides as an active ingredient. 19 F-MRI contrast agent.

Description

Technical Field

[0001] The present invention relates to a fluorine-containing molecule suitable as a contrast agent for MRI diagnosis using 19 19 F as a detection nucleus, and an MRI contrast agent containing the fluorine-containing molecule as an active ingredient. 19 F-MRI).

Background Art

[0002] Magnetic resonance imaging (hereinafter sometimes referred to as "MRI") diagnosis is one of the imaging diagnostic methods alongside X-ray diagnosis and ultrasonic (US) diagnosis, and is widely used in the medical field both in basic research and clinical applications. Currently, 1H-MRI using 1 1 H as a detection nucleus is used in medical MRI. 1 1H-MRI captures and images the magnetic environment of water molecules present in the living body.

[0003] 19 19F-MRI is 1 expected to be used as a next-generation diagnostic method following 1 19 1H-MRI diagnosis. This is because fluorine is an inexpensive element with a natural abundance ratio of 100%, 1 the detection sensitivity of 19 19 19F is as high as 83% of 1 1 1H, and the magnetic rotation ratio of 19

[0004] 19F is close to that of a proton, so it can be imaged with a conventional 1 19 1H-MRI device. 19 In addition, 19 19 19F that can be detected by MRI hardly exists in the living body. Therefore, by using a compound containing a fluorine atom as a contrast agent, 19 19 19F-MRI diagnosis using 19

[0005] 19 19F as a tracer is possible. For example, by using a fluorine compound that recognizes and accumulates endogenous changes caused by a disease as a contrast agent, . location information of the lesion can be obtained from 19 19 19F-MRI. This method is useful for diagnosing lesions that do not cause morphological changes that could not be detected by conventional imaging diagnostic methods. As a fluorine-containing compound used in contrast agents for MRI diagnostics that use fluorine as a detection nucleus, for example, Patent Document 1 describes a compound having a nitroxide covalently bonded to a fluorine-containing compound. Furthermore, Patent Document 2 discloses a metal chelate composite using branched polyethylene glycol having three or more branches as its basic framework, which can be used as an MRI contrast agent. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 5,362,477 [Patent Document 2] Japanese Patent Publication No. 2015-40242 [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention provides high sensitivity 19 A fluorine-containing molecule suitable as an F-MRI contrast agent, and a material containing the fluorine-containing molecule as an active ingredient. 19 The objective is to provide F-MRI contrast agents. [Means for solving the problem]

[0008] In MRI measurements, signals generated in the vertical (Z) direction are tilted to the horizontal (XY) direction to obtain the signal. The shorter the vertical relaxation time T1 (time constant for signal generation in the Z direction) and the longer the horizontal relaxation time T2 (time constant for signal decay in the XY direction), the stronger the signal intensity obtained per unit time, and the higher the sensitivity.

[0009] The inventors of this invention have found that fluorine atoms have low water solubility. 19We found that when F-MRI contrast agents aggregate in water, the T1 timescale lengthens and the T2 timescale shortens in MRI measurements, resulting in reduced sensitivity. Further research by the inventors revealed that by introducing a fluorine-containing group, which has a structure equivalent to a paramagnetic nitroxide radical and contains many fluorine atoms at an appropriate distance, into the side chain of a peptide, aggregation in water is suppressed. 19 We discovered that sensitivity is enhanced when used as an F-MRI contrast agent, and thus completed the present invention.

[0010] In other words, the present invention includes the following embodiments. [1] A peptide comprising two or more amino acids linked by peptide bonds, wherein a fluorine-containing alkyl group and an organic radical are contained within the same amino acid residue. [2] The peptide according to [1], wherein the fluorine-containing alkyl group is a perfluoroalkyl group having 1 to 6 carbon atoms. [3] The peptide according to [1] or [2], wherein the fluorine-containing alkyl group is a trifluoromethyl group or a nonafluoro-tert-butyl group. [4] Any of the peptides from [1] to [3] above, wherein the organic radical is an N-oxyl radical. [5] The organic radical has a substructure represented by the following formula (1) of any of the peptides [1] to [4] above.

[0011] [ka]

[0012] [6] The side chain is given by the following formula (1-1)

[0013] [ka]

[0014] A peptide having a group represented by [1] to [5] above. [7] The peptide according to any one of [1] to [6], wherein the amino acid residue adjacent to the amino acid residue having a fluorine-containing alkyl group and an organic radical is a hydrophilic amino acid residue. [8] The peptide according to [7], wherein the hydrophilic amino acid residue is a lysine residue. [9] The peptide according to any one of [1] to [8], which consists of 3 to 5 amino acid residues.

[10] A pharmaceutical composition containing the peptide according to any one of [1] to [9].

[11] Using the peptide according to any one of [1] to [9] as an active ingredient, 19 F-MRI contrast agent. [Effect of the Invention]

[0015] The peptide according to the present invention is excellent in dispersibility in an aqueous medium, 19 and by using it as a material for an F-MRI contrast agent, a magnetic resonance image with sufficient sensitivity can be obtained. [[ID=2​​​​​​​​​​​​​​​​​​​​​​Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl groups.

[0020] Furthermore, in the following, "compound n" means "the compound represented by formula (n)".

[0021] [peptide] The peptide of this embodiment is a peptide in which two or more amino acids are linked by peptide bonds, and which contains a fluorine-containing alkyl group and an organic radical within the same amino acid residue. Hereinafter, "amino acid residue having a fluorine-containing alkyl group and an organic radical" may be referred to as "fluorine-containing organic radical amino acid residue".

[0022] High sensitivity 19 To obtain F-MRI, the fluorine-containing compound included in the contrast agent is: 19 It is preferable to use a compound with a short F-spin-lattice relaxation time (T1). The shorter the T1 of the fluorine-containing compound, the shorter the repetition time can be set. This increases the amount of signal obtained per unit time, resulting in a high-sensitivity image. On the other hand, the fluorine-containing compound 19 If the F-spin-spin relaxation time (T2) is too short, the signal intensity decreases.

[0023] Fluorine-containing compounds 19 F-spin-lattice relaxation time (T1) and 19 The F-spin-spin relaxation time (T2) is affected by the paramagnetic relaxation enhancement (PRE) effect. The PRE effect is a phenomenon in which the unpaired electron spins of paramagnetic materials shorten the T1 and T2 times of MRI-observed nuclei near the unpaired electron spins.

[0024] The PRE effect is inversely proportional to the sixth power of the distance between the paramagnetic material and the MRI observation nucleus (fluorine atom in the fluorine-containing compound used as a contrast agent) that is relaxed by the paramagnetic material. Therefore, the closer the distance between the organic radical and the fluorine atom in the fluorine-containing compound used as a contrast agent, the shorter T1 and T2 become. In the peptide of this embodiment, since the fluorine-containing alkyl group and the organic radical are contained within the same amino acid residue, the distance between the organic radical and the fluorine atom in the fluorine-containing alkyl group is appropriate, and T1 can be made sufficiently short.

[0025] The fluorine-containing alkyl group is not particularly limited as long as it is a group in which one or more hydrogen atoms in the alkyl group are substituted with fluorine atoms, but C is preferred because it is easier to adjust the distance from the organic radical to an appropriate range. 1-6 A group in which one or more hydrogen atoms in the alkyl group are substituted with fluorine atoms is preferred, and a perfluoroalkyl group having 1 to 6 carbon atoms is more preferred.

[0026] Also, high sensitivity 19 To obtain 1F-MRI, it is preferable to use a fluorine-containing molecule with a large number of structurally equivalent fluorine atoms as a contrast agent. For this reason, the fluorine-containing alkyl group in the peptide of this embodiment is preferably a group having at least one trifluoromethyl group (-CF3) containing three structurally equivalent fluorine atoms, more preferably a group having one to three trifluoromethyl groups, even more preferably a trifluoromethyl group or a nonafluoro-tert-butyl group, and particularly preferably a nonafluoro-tert-butyl group. Because the fluorine-containing alkyl group has a trifluoromethyl group, the peptide of this embodiment has a sufficiently large number of structurally equivalent fluorine atoms contained in the molecule, and when used as a contrast agent for MRI diagnosis using fluorine as a detection nucleus, a strong signal intensity is obtained and a highly sensitive image is obtained.

[0027] The organic radical is preferably a nitrogen radical or a carbon radical, and more preferably a nitrogen radical, due to its relatively high safety for living organisms. Among these, the N-oxyl radical is preferred from the viewpoint of stability in living organisms, and more preferably an organic radical having a substructure represented by the following formula (1) (hereinafter sometimes referred to as "structure (1)"). In formula (1), the asterisk indicates a binding site with another group in an amino acid residue.

[0028] [ka]

[0029] Organic radicals have a half-occupied orbital (SOMO) containing an unpaired electron between their highest occupied orbital (HOMO) and lowest unoccupied orbital (LUMO). The redox process of organic radicals corresponds to the electron transfer process in the SOMO. The reduction reaction of organic radicals by reducing agents such as ascorbic acid is more likely to occur when the energy difference between the HOMO of the reducing agent and the SOMO of the organic radical is small. Therefore, the lower the energy level of the SOMO of the organic radical, the more easily it is reduced.

[0030] The SOMO of an organic radical having structure (1) has a sufficiently large energy difference with the HOMO of a reducing agent such as ascorbic acid. Therefore, organic radicals having structure (1) are difficult to reduce in vivo and have high stability in vivo. For this reason, when the organic radical in the peptide of this embodiment is an organic radical having structure (1), the peptide of this embodiment is very suitable as a contrast agent with excellent stability in vivo.

[0031] Examples of organic radicals having structure (1) in the peptide of this embodiment include a group in which a linking group is attached to the carbon atom at position 3 of 2,2,5,5-tetramethylpyrrolidine-N-oxide, and a group in which a linking group is attached to the carbon atom at position 4 of 2,2,6,6-tetramethylpiperidine-N-oxide.

[0032] In this embodiment, the fluorine-containing organic radical amino acid residue in the peptide is preferably an amino acid residue consisting of an amino acid in which a fluorine-containing alkyl group, a group having an organic radical, and the α-carbon of an α-amino acid are linked by a trivalent linking group. The trivalent linking group is not particularly limited as long as it is a trivalent organic group. An example of the trivalent linking group is a group in which three divalent organic groups are linked to a trivalent or tetravalent central atom consisting of a carbon atom, a silicon atom, a nitrogen atom, or a phosphorus atom. The divalent organic groups linked to the central atom may be of the same type or different types. Furthermore, if the central atom is a tetravalent atom, a group in which three divalent organic groups and one monovalent organic group are linked can be used as the trivalent linking group. The monovalent organic group is not particularly limited, for example, C 1-6 Examples include alkyl groups, with methyl groups being preferred.

[0033] The three divalent organic groups linked to the central atom are not particularly limited and include, for example, -CH2-, -O-, -S-, -C(=O)-, -C(=S)-, -NH-, -S(=O)2-, -Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group), and may also be groups formed by combining two or more of the same or different types of these divalent groups. An example of a linking group formed by combining these divalent groups is -(CH2) n -,-(CH2) n -O-, -(CH2) n -C(=O)-, -(CH2) n -OC(=O)-, -(CH2) n -C(=O)-O-, -(CH2) n -C(=O)-NH-, -(CH2) n -C(=O)-NH-(CH2) n Examples include -, -C(=O)-O-, -C(=O)-OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-(n is a natural number), etc.

[0034] In this embodiment, the fluorine-containing organic radical amino acid residue in the peptide is particularly preferred if it has a group represented by the following formula (1-1) in its side chain. In the following formula (1-1), the asterisk indicates a binding bond. Hereafter, the "group represented by the following formula (1-1)" may be referred to as "fluorine-containing group A".

[0035] [ka]

[0036] Examples of fluorine-containing organic radical amino acid residues in the peptide of this embodiment include amino acid residues in which a fluorine-containing group A is linked to the α-carbon of an amino acid by a divalent linking group. The divalent linking group is not particularly limited, and the same as described above can be used.

[0037] The fluorine-containing organic radical amino acid residues in the peptides of the embodiments may be amino acids in which any atom constituting the side chain of a natural amino acid is substituted with a fluorine-containing group A. Examples include amino acids in which the carboxyl group of the side chain of aspartic acid or glutamic acid is substituted with a fluorine-containing group A; amino acids in which the hydroxyl group of the side chain of serine, threonine, or tryptophan is substituted with a fluorine-containing group A; amino acids in which the hydrogen atom of the amino group of the side chain of lysine, arginine, or histidine is substituted with a fluorine-containing group A; amino acids in which the hydrogen atom of the thiol group of the side chain of cysteine ​​or methionine is substituted with a fluorine-containing group A; and amino acids in which the hydrogen atom of the aromatic ring of the side chain of tyrosine, phenylalanine, or tryptophan is substituted with a fluorine-containing group A. The substitution of atoms in the side chain of an amino acid with a fluorine-containing group A can be carried out by general synthetic reactions such as esterification reactions.

[0038] By using the peptide of this embodiment as a material for a contrast agent for magnetic resonance imaging using fluorine as the detection nucleus, magnetic resonance images with sufficient sensitivity can be obtained in a short measurement time. The peptide of this embodiment was measured by the method described later. 19The F spin-lattice relaxation time (T1) is preferably 350 ms or less, more preferably 300 ms or less, even more preferably 200 ms or less, even more preferably 150 ms or less, and particularly preferably 100 ms or less. 19 The lower limit of the F spin-lattice relaxation time (T1) is not particularly limited, but is preferably 1 ms or more, more preferably 5 ms or more, and even more preferably 10 ms or more.

[0039] The peptide of this embodiment exhibits excellent dispersibility in aqueous media. Therefore, the peptide of this embodiment can be dispersed at a sufficient concentration in an aqueous media within a living organism without the use of a surfactant. The peptide of this embodiment is preferably one that can be dispersed in water at a concentration of 5 mmol / L, and more preferably one that can be dissolved in water at a concentration of 5 mmol / L.

[0040] When the fluorine portion in the contrast agent aggregates, 19 The F-spin-spin relaxation time (T2) is extremely shortened, making it impossible to obtain an MRI image. For this reason, it is preferable for contrast agents to have suppressed aggregation in aqueous media. Generally, both trifluoromethyl groups and nitroxide radicals are highly hydrophobic, and therefore compounds containing both trifluoromethyl groups and nitroxide radicals in proximity tend to aggregate. In contrast, the peptide of this embodiment has suppressed aggregation in aqueous media and can be dispersed well. The reason why the peptide of this embodiment has good dispersibility in aqueous media is not clear, but it is presumed that because the peptide of this embodiment has fluorine-containing organic radical amino acid residues, aggregation of peptides is suppressed due to steric hindrance by the main chain and other side chains of the peptide.

[0041] In the present invention and this specification, "peptides are not dispersed in the aqueous medium" means that the peptides placed in the aqueous medium are separated within the aqueous medium. "Peptides are dispersed in the aqueous medium" means that the peptides are present in the aqueous medium without being separated. The aqueous medium in which the peptides are dispersed may be transparent or opaque when observed visually in a 2 mL vial. "Peptides are dissolved in the aqueous medium" means that the state is "peptides dispersed in the aqueous medium" and is transparent when observed visually in a 2 mL vial.

[0042] The peptide of this embodiment may be any peptide that contains at least one amino acid residue having a fluorine-containing organic radical amino acid residue in its side chain. If a single peptide molecule has two or more fluorine-containing organic radical amino acid residues in its side chain, it is preferable that the multiple fluorine-containing organic radical amino acid residues are not adjacent to each other. In the peptide of this embodiment, the fluorine-containing organic radical amino acid residue may be at the N-terminus, the C-terminus, or anywhere other than the terminal.

[0043] In this embodiment, the peptide is preferably one in which the amino acid residues adjacent to the fluorine-containing organic radical amino acid residue are hydrophilic amino acid residues whose side chains are hydrophilic groups. The presence of hydrophilic groups near the fluorine-containing organic radical amino acid residue can more effectively suppress the aggregation of peptides. Examples of hydrophilic amino acid residues include lysine residues, arginine residues, histidine residues, aspartic acid residues, and glutamine residues, with lysine residues being particularly preferred. In particular, the peptide in this embodiment is preferably one in which lysine residues are located on both sides of the fluorine-containing organic radical amino acid residue.

[0044] The peptide in this embodiment may be any peptide consisting of two or more amino acids, and peptides consisting of three or more amino acids are also preferred. The number of amino acid residues constituting the peptide in this embodiment is preferably 40 or less, more preferably 30 or less, even more preferably 20 or less, even more preferably 10 or less, and particularly preferably 5 or less. Among these, the peptide in this embodiment is preferably a peptide consisting of 3 to 5 amino acid residues.

[0045] The N-terminus of the peptide in this embodiment may be protected with an amino group protecting group. The N-terminus protecting group is not particularly limited as long as it is an amino group protecting group; for example, amino group protecting groups used in peptide synthesis can be used. Examples of amino group protecting groups include carbamate protecting groups such as tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Alloc) group, and 2,2,2-trichloroethoxycarbonyl (Troc) group. Preferably, the tert-butoxycarbonyl (Boc) group or the 9-fluorenylmethyloxycarbonyl (Fmoc) group can be deprotected under mild conditions.

[0046] The C-terminus of the peptide in this embodiment may be protected with a protecting group. The protecting group for the C-terminus is not particularly limited as long as it is a carboxyl group protecting group, and for example, carboxyl group protecting groups used in peptide synthesis can be used. Specific examples of carboxyl group protecting groups include benzyl group, diphenylmethyl group, triphenylmethyl group, 4-nitrobenzyl group, 4-methoxybenzyl group, 2,4-dimethoxybenzyl group, 3,4-dimethoxybenzyl group, 4-methylbenzyl group, 2,6-dimethylbenzyl group, 3-chlorobenzyl group, 9-anthrylmethyl group, piperonyl group, 2-(9,10-dioxo)anthrylmethyl group, benzyloxymethyl group, phenacyl group, and the like. The protecting group for the C-terminus carboxyl group is preferably a benzyl group or a triphenylmethyl group, and more preferably a benzyl group, because it can be deprotected under mild conditions.

[0047] In the peptide of this embodiment, the amino acid residues other than fluorine-containing organic radical amino acid residues are not particularly limited and may be α-amino acid residues, β-amino acid residues, γ-amino acid residues, or δ-amino acid residues. They may also be L-amino acid residues or D-amino acid residues. In the peptide of this embodiment, the amino acid residues other than fluorine-containing organic radical amino acid residues are preferably amino acid residues of amino acids that constitute proteins, their D-forms, and modified amino acid residues in which the side chains of these are modified.

[0048] Examples of amino acids that make up proteins include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, and histidine. Modified amino acids, which are amino acids that make up proteins, include, for example, amino acids in which the hydrogen atoms of the amino group in the side chain of lysine, arginine, and histidine are replaced with protecting groups or other groups; amino acids in which the hydrogen atoms of the carboxyl group in the side chain of aspartic acid and glutamic acid are replaced with the aforementioned carboxyl group protecting groups or other groups; and amino acids in which the hydrogen atoms of the thiol group of cysteine ​​are replaced with other groups.

[0049] The peptide of this embodiment can be synthesized by peptide synthesis using amino acids from which fluorine-containing organic radical amino acid residues are induced (hereinafter sometimes referred to as "fluorine-containing organic radical amino acids") as raw materials. This peptide synthesis can be carried out by general peptide synthesis methods such as solid-phase peptide synthesis. Solid-phase peptide synthesis is a method of producing peptides by sequentially condensing amino acids with protected amino groups onto an amino acid whose C-terminus is bonded to a solid phase, and then removing the peptide from the solid phase. The peptide of this embodiment can be easily synthesized using an automated peptide synthesizer with fluorine-containing organic radical amino acids as raw materials.

[0050] The target peptide or its intermediate can be isolated and purified by various methods, such as ion chromatography, gel filtration chromatography, reverse-phase chromatography, normal-phase chromatography, recrystallization, extraction, and fractional crystallization. Furthermore, the peptides thus obtained can be converted to their respective salts by conventional methods.

[0051] The protecting groups of the amino or carboxyl groups of the manufactured peptides can be deprotected as needed. Deprotection can be carried out by conventional methods depending on the type of protecting group.

[0052] The peptide of this embodiment exhibits good dispersibility in aqueous media and high safety for humans and other animals. For this reason, the peptide of this embodiment is also useful as an active ingredient in pharmaceutical compositions.

[0053] [Contrast agent] The contrast agent of this embodiment contains the peptide of this embodiment. The contrast agent of this embodiment is a contrast agent for magnetic resonance imaging using fluorine as the detection nucleus. The contrast agent of this embodiment can be manufactured by formulating the peptide of this embodiment into a solid formulation, powder formulation, liquid formulation, or the like using known formulation techniques. The peptide of this embodiment is suitable for use in solvent form because it has good dispersibility in aqueous media. In addition to the peptide of this embodiment, the contrast agent of this embodiment may also contain, as necessary, one or more additives used in known formulations, such as excipients, stabilizers, surfactants, buffers, and electrolytes.

[0054] The contrast agent of this embodiment contains the peptide of this embodiment, and therefore exhibits good dispersibility in aqueous media. Furthermore, by using the contrast agent of this embodiment as a contrast agent for magnetic resonance imaging using fluorine as the detection nucleus, highly sensitive magnetic resonance images can be obtained.

[0055] Although embodiments of the present invention have been described in detail above, the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications to the configurations are possible without departing from the spirit of the present invention. [Examples]

[0056] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0057] In subsequent analyses, nuclear magnetic resonance (NMR) was measured using an NMR spectrometer (JEOL JNM-ECA500 (500MHz)) unless otherwise specified. 1 In 1H-NMR, 0 ppm of tetramethylsilane is used as the reference. 19 For 1F-NMR, -162.9 ppm of hexafluorobenzene was used as the reference.

[0058] The following abbreviations were used in this specification. Fmoc:9-Fluorenylmethyloxycarbonyl Asp: Aspartic acid t Bu:tert-butyl COMU: (1-Cyano-2-Ethoxy-2-Oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate Oxyma: (hydroxyimino) cyanoethyl acetate Gly: Glycine Trt: Triphenylmethyl PEG: Polyethylene glycol Lys: Lysine Pra: Propargylglycine Sar:N-methylglycine

[0059] [Manufacturing Example 1] (Fmoc-Asp(F9-Radical)-OH(compound (1-6)) was synthesized.

[0060] [ka]

[0061] (1) Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (compound (1-1)) Under an argon stream, trimethylolethane (9.988 g, 83.1 mmol) and p-toluenesulfonic acid monohydrate (p-TsOH·H2O) (0.190 g, 1.00 mmol) were dissolved in 100 mL of acetone. To the resulting solution, 2,2-dimethoxypropane (11.2 mL, 91.4 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Potassium carbonate (0.140 g, 1.01 mmol) was added to the resulting reaction solution, and the mixture was filtered through a silica gel column and washed with ethyl acetate. The resulting filtrate was concentrated under reduced pressure to obtain the target compound (1-1) (yield 13.180 g, yield 99%).

[0062] 1 H-NMR(500MHz,CDCl3)δ3.70-3.66(m,4H),3.63-3.59(m,2H),2.23(br,1H),1.44(s,3H),1.40(s,3H),0.83(s,3H).

[0063] (2) Synthesis of 5-(((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)methyl)-2,2,5-trimethyl-1,3-dioxane (compound (1-2)) Under an argon stream, the compound (1-1) (12.112 g, 75.6 mmol) synthesized by the above reaction, triphenylphosphine (PPh3) (27.777 g, 105.9 mmol), molecular sieves 4A (MS4A) (10.000 g), and tetrahydrofuran (THF) (150 mL) were mixed and cooled in an ice bath. Diisopropyl azodicarboxylic acid ester (DIAD) (20.6 mL, 105.9 mmol) was added dropwise to the resulting mixture over 10 minutes, and after stirring for 35 minutes, 25.000 g (105.9 mmol) of nonafluoro-tert-butanol was added all at once, and the mixture was stirred at 45°C for 72 hours. The resulting reaction solution was filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane:ethyl acetate = 85:15~4:1 (volume ratio)) to obtain the target compounds (1-2) (yield 21.826 g, yield 76%).

[0064] 1 H-NMR(500MHz,CDCl3)δ3.71-3.66(m,4H),3.63-3.58(m,2H),2.23(br,1H),1.42(s,3H),1.40(s,3H),0.83(s,3H).

[0065] (3) Synthesis of 2-(((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)methyl)-2-methylpropane-1,3-diol (compound (1-3)) Compound (1-2) (21.826 g, 57.7 mmol) synthesized by the above reaction was dissolved in methanol (MeOH) (100 mL), concentrated hydrochloric acid (conc.HCl) (10 mL) was added, and the mixture was stirred at 65°C for 15 hours. The resulting reaction solution was cooled to room temperature and then concentrated under reduced pressure. Water was added to the resulting concentrate, and after extraction with ethyl acetate, the resulting extract was dried over magnesium sulfate. The dried organic layer (ethyl acetate extract) was concentrated under reduced pressure to obtain the target compound (1-3) (yield 17.540 g, yield 89%).

[0066] 1H-NMR(500MHz,Acetone-d6)δ4.22(s,2H),3.70-3.62(m,4H),0.87(s,3H). 19 F-NMR(470MHz,Acetone-d6)δ-69.19.

[0067] (4) Synthesis of 3-(((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)-2-(hydroxymethyl)-2-methylpropyl-2,2,5,5-tetramethylpiperidine-3-carboxylate-1-oxyl free radical (compound (1-4)) Under an argon stream, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical (4.295 g, 23.1 mmol), 1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (4.869 g, 25.4 mmol), and N,N-dimethylaminopyridine (DMAP) (0.310 g, 2.54 mmol) were dissolved in dichloromethane (CH2Cl2) (90 mL) and cooled in an ice bath. Next, a THF solution (20 mL) of the compound (1-3) (10.923 g, 32.3 mmol) synthesized by the above reaction was added, and the mixture was stirred at room temperature for 19 hours. Water was added to the resulting reaction solution, and after extraction with dichloromethane, it was dried over magnesium sulfate. The dried organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:ethyl acetate = 85:15~2:1 (volume ratio)) to obtain the target compounds (1-4) (yield 6.856 g, yield 59%).

[0068] 19 F-NMR (470 MHz, CDCl3) δ-70.84. LCMS [M] + :m / z calcd for C 18 H 25 F9NO5 + 506.2, found 506.2.

[0069] (5)Fmoc-(L)-Asp(F9-Radical)-O tSynthesis of Bu (compounds (1-5)) Under an argon stream, Fmoc-(L)-Asp-O t Bu (3.189 g, 7.75 mmol), 1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride (1.783 g, 9.30 mmol), and N,N-dimethylaminopyridine (0.114 g, 0.93 mmol) were dissolved in 70 mL of dichloromethane and cooled in an ice bath. Then, a THF solution (15 mL) of the compound (1-4) (3.922 g, 7.75 mmol) synthesized by the above reaction was added, and the mixture was stirred at room temperature for 17 hours. Water was added to the resulting reaction solution, and after extraction with dichloromethane, the mixture was dried over magnesium sulfate. The dried organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:ethyl acetate = 85:15~3:1 (volume ratio)) to obtain the target Fmoc-(L)-Asp(F9-Radical)-O t Bu (compound (1-5)) was obtained (yield 3.688g, yield 53%).

[0070] 19 F-NMR (470 MHz, CDCl3) δ-70, 80. LCMS [M] + :m / z calcd for C 41 H 48 F9N2O 10 + 899.3, found 899.2.

[0071] (6) Synthesis of Fmoc-(L)-Asp(F9-Radical)-OH (compounds (1-6)) Fmoc-(L)-Asp(F9-Radical)-O synthesized by the above reaction tBu(1-5) (3.688 g, 4.10 mmol) was dissolved in dichloromethane (5 mL) and cooled in an ice bath. Trifluoroacetic acid (TFA) (5 mL) was added to the resulting solution and stirred at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:ethyl acetate = 1:1 (volume ratio)) to obtain the target Fmoc-(L)-Asp(F9-Radical)-OH (compound (1-6)) (yield 2.492 g, yield 72%).

[0072] 19 F-NMR (470MHz, CDCl3) δ-68.53. LCMS [M] + :m / z calcd for C 37 H 40 F9N2O 10 + 843.3, found 843.2.

[0073] [Example 1] (H-lys-Asp(F9-Radical)-lys-Gly-OH (tetrapeptide-1) was synthesized.

[0074] [ka]

[0075] Fmoc-Gly-Trt-PEG Resin (Watanabe Chemical Co., Ltd.) (238 mg, 50 μmol) and N,N-dimethylformamide (DMF) (2 mL) were added to a solid-phase synthesis tube and stirred at room temperature for 30 minutes to swell the resin. Next, 2 mL of 20% piperidine / DMF solution was added to the tube and stirred for 3 minutes, followed by washing three times with DMF (2 mL). Then, 2 mL of 20% piperidine / DMF solution was added again and stirred for 12 minutes to deprotect the Fmoc group. Subsequently, Fmoc-(D)-Lys-OH (94 mg, 200 μmol), COMU (86 mg, 200 μmol), Oxyma (28 mg, 200 μmol), diisopropylethylamine (DIPEA) (70 μL, 400 μmol), and DMF (1 mL) were added and stirred at room temperature for 1 hour to carry out the amino acid condensation reaction. Next, the resulting reaction product was washed three times with DMF (2 mL), and then the Fmoc group was deprotected in the same manner as described above. The same procedure was performed on Fmoc-(L)-Asp(F9-Radical)-OH (compound (1-6)) (84 mg, 100 μmol) and Fmoc-(D)-Lys-OH (94 mg, 200 μmol) synthesized by the above reaction. As a subsequent workup, the cleavage cocktail (TFA:triisopropylsilane (TIPS):dichloromethane = 1:5:94 (volume ratio)) (2 mL) was added and stirred at room temperature for 5 minutes, and this process was repeated three times to cleave the product from the resin. After concentrating the cleaved solution under reduced pressure, a deprotection cocktail (hexafluoroisopropanol (HFIP): trifluoroethane (TFE): triethylsilane (TES): dichloromethane = 20:10:5:65 (volume ratio)) (2 mL) was added and the mixture was stirred at room temperature for 1 hour to deprotect it, yielding a crude product containing H-lys-Asp(F9-Radical)-lys-Gly-OH (tetrapeptide-1). The crude product was vacuum-dried and then purified by reverse-phase chromatography (acetonitrile: water: TFA = 15:85:0.1~100:0:0.1 (volume ratio)) to obtain H-lys-Asp(F9-Radical)-lys-Gly-OH (tetrapeptide-1) (yield 9 mg, yield 19%).

[0076] 19F-NMR (470MHz, D2O) δ-69.77. MALDI-TOF MS [M+H] + :m / z calcd for C 36 H 58 F9N7O 11 + 935.41, found 936.15.

[0077] [Comparative Example 1] (Fmoc-(L)-Asp(F9)-OH (compound (1-11)) was synthesized.

[0078] [ka]

[0079] (1) Synthesis of benzyl(2-bromoethyl)carbamate (compounds (1-7)) Under an argon stream, 2-bromoethylamine hydrobromide (10,000 g, 48.8 mmol) and DIPEA (10.3 mL, 73.2 mmol) were dissolved in dichloromethane (100 mL) and cooled in an ice bath. Benzyl chloroformate (25.4 mL, 146 mmol) was added to the resulting solution over 10 minutes, and the mixture was stirred for 18 hours while gradually increasing the temperature to room temperature. The resulting reaction solution was washed with saturated sodium bicarbonate aqueous solution, then with 10% citric acid aqueous solution, and then dried over magnesium sulfate. The dried organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:ethyl acetate = 9:1-4:1 (volume ratio)) to obtain the target compounds (1-7) (yield 11.371 g, 90%).

[0080] 1 H-NMR(500MHz,CDCl3)δ7.40-7.29(m,5H),5.18(br,1H),5.12(s,2H),3.61(t,J=5.7Hz,2H),3.48(t,J=5.7Hz,2H).

[0081] (2) Synthesis of benzyl(2-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)ethyl)carbamate (compound (1-8)) Under an argon atmosphere, the compounds (1-7) (5.162 g, 20.0 mmol) synthesized by the above reaction, sodium nonafluoro-tert-butoxide (5.800 g, 22.5 mmol), and sodium iodide (NaI) (0.300 g, 2.00 mmol) were dissolved in DMF (60 mL) and stirred at 100°C for 16 hours. After the resulting solution was cooled to room temperature, water was added, and the mixture was extracted with chloroform and dried over magnesium sulfate. The dried organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:ethyl acetate = 95:5~4:1 (volume ratio)) to obtain the target compound (1-8) (yield 5.201 g, yield 63%).

[0082] 1 H-NMR (500MHz, CDCl3) δ7.39-7.31(m,5H),5.12(s,2H),5.07(br,1H),4.14-4.08(m,2H),3.53-3.47(m,2H). 19 F-NMR (500 MHz, CDCl3) δ-70.27.

[0083] (3) Synthesis of 2-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)ethane-1-amine (compound (1-9)) Under an argon stream, the compound (1-8) (5.201 g, 12.6 mmol) synthesized by the above reaction was dissolved in THF (20 mL), and 5% palladium-activated carbon (Pd / C) (0.500 g) was added. The reaction vessel was purged with hydrogen (H2) gas, and the reaction solution in the vessel was stirred at room temperature for 18 hours. The reaction solution was filtered through Celite and concentrated under reduced pressure to obtain the target compound (1-9) (yield 1.209 g, yield 34%).

[0084] 1H-NMR(500MHz, CDCl3) δ4.05(t,J=5.2Hz,2H),2.98(t,J=5.2Hz,2H). 19 F-NMR (500 MHz, CDCl3) δ-70.22.

[0085] (4)Fmoc-(L)-Asp(F9)-O t Synthesis of Bu (compounds (1-10)) Under an argon stream, Fmoc-(L)-Asp-O t Bu (1.646 g, 4.00 mmol), (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (2.086 g, 4.00 mmol), and DIPEA (2.23 mL, 12.8 mmol) were dissolved in dichloromethane (20 mL) and DMF (20 mL) and stirred at room temperature for 10 minutes. Then, to the resulting solution, a 10 mL solution of the compound (1-9) (1.209 g, 4.33 mmol) synthesized by the above reaction in dichloromethane:DMF = 1:1 (volume ratio) was added and stirred at room temperature for 18 hours. The resulting reaction solution was concentrated under reduced pressure and reprecipitated with water to obtain the crude product. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 9:1 to 1:1 (volume ratio)) to obtain the target Fmoc-(L)-Asp(F9)-O t Bu (compound (1-10)) was obtained (yield 1.352 g, yield 50%).

[0086] 1 H-NMR(500MHz,CDCl3)δ7.76(d,J=7.5Hz,2H),7.60(d,J=7.5Hz,2H),7.40(t,J=7.4 Hz,2H),7.30(t,J=7.4Hz,2H),6.04(m,1H),5.99(d,J=7.9Hz,1H),4.50-4.44(m,1H) ),4.44-4.37(m,1H),4.34-4.28(m,1H),4.22(t,J=7.1Hz,1H),4.09(s,2H),3.63-3 .47(m,2H),2.90(dd,J=15.8,4.0Hz,2H),2.74(dd,J=15.8,4.0Hz,2H),1.47(s,9H).

[0087] (5) Synthesis of Fmoc-(L)-Asp(F9)-OH (Compound (1-11)) Fmoc-(L)-Asp(F9)-O t Bu (Compound (1-10)) (1.352 g, 2.01 mmol) was dissolved in dichloromethane (10 mL) and cooled in an ice bath. TFA (10 mL) was added to the resulting solution, and the mixture was stirred at room temperature for 17 hours. The resulting reaction solution was concentrated under reduced pressure and purified by reprecipitation with diethyl ether to obtain the target Fmoc-(L)-Asp(F9)-OH (Compound (1-11)) (yield 0.917 g, yield 74%).

[0088] 1 1H-NMR (500 MHz, CD3OD) δ 7.80 - 7.76 (m, 2H), 7.67 - 7.61 (m, 2H), 7.41 - 7.35 (m, 2H), 7.33 - 7.27 (m, 2H), 4.59 - 4.52 (m, 1H), 4.36 - 4.26 (m, 2H), 4.26 - 4.18 (m, 1H), 4.17 - 4.08 (m, 2H), 3.52 - 3.39 (m, 2H), 2.83 - 2.68 (m, 2H). 19 19F-NMR (470 MHz, CD3OD) δ -69.27. LCMS [M] + : m / z calcd for C 25 H 21 F9N2O6 + 617.1, found 617.2.

[0089] (6) Synthesis of Sar-Pra-lys-Asp(F9)-Gly-OH (Pentapeptide-2)

[0090]

Chemical Structure

[0091] To a tube for solid-phase synthesis, Fmoc-Gly-Trt-PEG Resin (manufactured by Watanabe Chemical Industries, Ltd.) (250 mg, 50 μmol) and DMF (2 mL) were added, and the mixture was stirred at room temperature for 30 minutes to swell the resin. Next, 20% piperidine / DMF solution (2 mL) was added to the tube, and after stirring for 3 minutes, it was washed three times with DMF (2 mL). Deprotection of the Fmoc group was carried out by adding 20% piperidine / DMF solution (2 mL) again and stirring for 12 minutes. Subsequently, Fmoc-(L)-Asp(F9)-OH (Compound (1-11)) (61.6 mg, 100 μmol), COMU (43 mg, 100 μmol), Oxyma (14 mg, 100 μmol), DIPEA (35 μL, 200 μmol), and DMF (1 mL) synthesized by the above reaction were added, and the mixture was stirred at room temperature for 1 hour to carry out the condensation reaction of the amino acid. Next, the obtained reaction product was washed three times with DMF (2 mL), and then deprotection of the Fmoc group was carried out in the same manner as above. The same operation was performed for Fmoc-(D)-Lys-OH (94 mg, 200 μmol), Fmoc-(L)-Pra-OH (67 mg, 200 μmol), and Fmoc-Sar-OH (62 mg, 200 μmol). As subsequent post-treatment, a cleavage cocktail (TFA:TIPS:water = 95:2.5:2.5 (volume ratio)) (2 mL) was added, and the mixture was stirred at room temperature for 1 hour to carry out cleavage from the resin. The cleavage solution was concentrated under reduced pressure to obtain a crude product containing Sar-Pra-lys-Asp(F9)-Gly-OH (pentapeptide-2). The crude product was purified by reverse-phase chromatography (acetonitrile:water:TFA = 30:70:0.1 to 100:0:0.1 (volume ratio)) to obtain Sar-Pra-lys-Asp(F9)-Gly-OH (pentapeptide-2) (yield 9 mg, yield 24%).

[0092] 19 F-NMR (376 MHz, D2O) δ -70.55. MALDI-TOF MS [M] + : m / z calcd for C 26 H 36 F9N⑦O⑧ + 7①5.②5, found 7①5.09. Note: In the translation, the tags ,

[0092] , , 19 , , , are preserved exactly as they are because they are 7 - digit tags. For the chemical formulas and notations like , [[ID=]], etc., they are also preserved as per the rules. Regarding the specific chemical notations in the original text such as F9, ⑦, ⑧ which seem to be some custom or specific notations in the original chemical context, they are directly translated and kept in the translated text without further modification as there is no clear indication of how to exactly convert them in a more standard way without losing their original meaning in the given context. If there are specific rules or standard conversions for such notations in the relevant chemical field, the translation might need to be adjusted accordingly. Also, the "F-NMR" is translated as "F-NMR" as it is likely a specific type of NMR measurement related to fluorine and might be a specialized term in the relevant field where the original form is more recognizable.

[0093] [Test Example 1] The peptides obtained in Example 1 and Comparative Example 1 were subjected to a repeating rotation method. 19 The longitudinal relaxation time T1 of the F nucleus was measured. A 2 mM deuterium aqueous solution of each peptide was used as the test sample.

[0094] [ka]

[0095] 19 The measurement conditions for measuring the longitudinal relaxation time T1 of F were as follows: Measurement temperature: 22℃ Pulse series: double_pulse relaxation_delay:10[sec] tau_interval:4,3,2,1,0.8,0.6,0.4,0.2,0.1[seconds],80,60,40,20,10,8,6,4,2[msec] Total number of times: 128

[0096] 19 The measurement results for the longitudinal relaxation time T1 of F are shown in Table 1.

[0097] [Table 1]

[0098] As shown in Table 1, the peptide of Example 1 having a nitroxide radical is superior to the peptide of Comparative Example 1 that does not have a nitroxide radical. 19 The F-spin-lattice relaxation time (T1) was very short. Therefore, the peptide of Example 1 can produce a higher signal per unit time compared to the peptide of Comparative Example 1, and by using this as a contrast agent... 19 High-sensitivity images can be obtained using F-MRI.

Claims

1. A peptide comprising two or more amino acids linked by peptide bonds, wherein a fluorine-containing alkyl group and an organic radical are contained within the same amino acid residue.

2. The peptide according to claim 1, wherein the fluorine-containing alkyl group is a perfluoroalkyl group having 1 to 6 carbon atoms.

3. The peptide according to claim 1, wherein the fluorine-containing alkyl group is a trifluoromethyl group or a nonafluoro-tert-butyl group.

4. The peptide according to claim 1, wherein the organic radical is an N-oxyl radical.

5. The peptide according to claim 1, wherein the organic radical has a substructure represented by the following formula (1). 【Chemistry 1】

6. The side chain is given by the following formula (1-1) 【Chemistry 2】 The peptide according to claim 1, having a group represented by .

7. The peptide according to claim 1, wherein the amino acid residue adjacent to the amino acid residue having the fluorine-containing alkyl group and the organic radical is a hydrophilic amino acid residue.

8. The peptide according to claim 7, wherein the hydrophilic amino acid residue is a lysine residue.

9. The peptide according to claim 1, comprising 3 to 5 amino acid residues.

10. A pharmaceutical composition containing the peptide described in any one of claims 1 to 9.

11. The active ingredient is the peptide described in any one of claims 1 to 9. １９ F-MRI contrast agent.

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