Peptides and methods for producing same

Abstract

To provide a peptide having a fluoroalkyl group on a side chain.SOLUTION: A method for producing a fluoroalkyl group-containing peptide in which after performing reductive reaction to a compound represented by a general formula (4) (in the formula, Rf denotes C1-30 alkyl group substituted by at least two pieces of F; R2 denotes a protecting group of amino group; R1 denotes a protecting group selected from a group expressed by a general formula (p-1) (in the formula, R3 denotes C6-14 aryl group; R4 and R5 each independently denotes a hydrogen atom or a C6-14 aryl group; a dot denotes a bond), 2-(9,10-dioxo)anthrylmethyl group, benzyloxymethyl group, and phenacyl group), R2 is deprotected to synthesize a compound represented by general formula (6-1), and furthermore, the compound is condensed with fluorine-containing amino acid with protected amino group, amino acid with protected amino group, fluorine-containing peptide with protected N terminal, or peptide with protected N terminal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a peptide containing an amino acid residue having a fluoroalkyl group introduced into the side chain, and a method for producing the same. This application claims priority based on Japanese Patent Application No. 2019-124016, filed July 2, 2019, and Japanese Patent Application No. 2019-209908, filed November 20, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] Antibody drugs, peptide drugs, nucleic acid drugs, and the like have the advantage of being highly specific to target molecules and having few side effects. However, all of these drugs have the problem of difficulty in reaching target molecules present within cells. Various methods have been investigated to solve this problem. Among them, cell penetrating peptides (CPPs) are considered promising. Representative CPPs include peptides derived from the TAT protein of the HIV virus (Patent Document 1) and peptides with poly-Arg sequences (Patent Document 2). These can be linked to medicinal peptides to transport the medicinal peptides into cells (for example, Patent Document 3 and Non-Patent Document 1).

[0003] On the other hand, fluorine-containing amino acids have been reported to exhibit unique physiological activities and have attracted attention. For example, it has been reported that 3,3,3-trifluoroalanine and its derivatives act as suicide inhibitors of pyridoxal enzymes (Non-Patent Document 2). It has also been reported that alanine racemase in the gram-negative bacterium Salmonella typhimurium and the gram-positive bacterium Bacillus stearothermophilus is inactivated by 3,3,3-trifluoroalanine (Non-Patent Document 3). Fluorine-containing amino acids and peptides containing them are expected to be used in the pharmaceutical field as physiologically active substances.

[0004] Compounds with a polyfluoro structure are known to be stable and low-toxic in vivo, and to have excellent cellular uptake and endosome escape properties (Non-Patent Document 4). Taking advantage of this property, it has been reported that peptide dendrimers, which use lysine as a constituent amino acid with perfluoroacylated amino groups in the side chain, can be used for gene delivery (Non-Patent Document 5). However, because they are dendrimers, they cannot form hybrids with pharmacologically active peptides, nucleic acids, or proteins that can be used as antibody drugs, as CPPs can. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,316,003 [Patent Document 2] U.S. Patent No. 6,306,993 [Patent Document 3] International Publication No. 2008 / 089491 [Non-patent literature]

[0006] [Non-Patent Document 1] Miyaji et. al., Drug Metabolism and Disposition, 2011, vol.39, p.1946-1953. [Non-patent document 2] Sakai et al., Tetrahedron, 1996, vol.52(1), p.233-244. [Non-patent document 3] Faraci and Walsh, Biochemistry, 1989, vol.28(2), p.431-437. [Non-patent document 4] Zhang et al., MRS Communications, 2018, vol.8, p.303-313. [Non-patent document 5] Cai et al., ACS Applied Materials and Interfaces, 2016, vol.8, p.5821-5832. Summary of the Invention [Problem to be solved by the invention]

[0007] The CPPs described in Patent Document 1 and elsewhere have various problems, such as poor transport efficiency into cells and degradation by peptidases in vivo. An object of the present invention is to provide a peptide containing an amino acid residue having a fluoroalkyl group introduced into the side chain, and a method for producing the same. [Means for solving the problem]

[0008] The present inventors produced a peptide containing an amino acid residue having a fluoroalkyl group introduced into the side chain, and found that the peptide had excellent cell membrane permeability, thereby completing the present invention.

[0009] That is, the present invention is as follows. [1] The following general formula (6-2) or (6-4)

[0010] [ka]

[0011] (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf is a C group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom. 1-30 Alkyl group (C 1-30 Alkyl group is C 2-30 When it is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms), R 2 is a protecting group for the amino group) a compound represented by the formula (I): with a fluorine-containing amino acid having a protected carboxyl group, an amino acid having a protected carboxyl group, a fluorine-containing peptide having a protected C-terminus, or a peptide having a protected C-terminus. [2] The following general formula (6-1) or (6-3)

[0012] [ka]

[0013] (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf is a C group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom. 1-30 Alkyl group (C 1-30 Alkyl group is C 2-30 When it is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms), R 1 is represented by the following general formula (p-1):

[0014] [ka]

[0015] (In the formula, R 3 may be substituted C 6-14 is an aryl group, and R 4 and R 5 are each independently a hydrogen atom or an optionally substituted C 6-14 (The black circle represents a bond.) a protecting group selected from a group represented by the formula: A method for producing a fluoroalkyl group-containing peptide, comprising condensing a compound represented by the formula (I) with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus. [3] The following general formula (7) or (7-1)

[0016] [ka]

[0017] (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf is a C group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom. 1-30 Alkyl group (C 1-30 Alkyl group is C 2-30 When it is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms. A compound represented by After protecting the amino group with a protecting group, the resulting product is condensed with a fluorine-containing amino acid having a protected carboxyl group, an amino acid having a protected carboxyl group, a fluorine-containing peptide having a protected C-terminus, or a peptide having a protected C-terminus, or After protecting the carboxy group with a protecting group, the compound is condensed with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus; A method for producing a fluoroalkyl group-containing peptide. [4] The method for producing a fluoroalkyl group-containing peptide according to any one of [1] to [3] above, further comprising deprotecting the protecting group of the amino group or carboxy group of the produced fluoroalkyl group-containing peptide. [5] A peptide in which two or more amino acids are peptide-bonded, At least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in the side chain. 1-30 Alkyl group (C 1-30Alkyl group is C 2-30 When the alkyl group is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms. [6] The C substituted with at least two fluorine atoms. 1-30 The peptide according to [5] above, wherein the alkyl group may be further substituted with a halogen atom other than a fluorine atom. [7] The C substituted with at least two fluorine atoms. 1-30 The side chain having an alkyl group is represented by the following general formula (f-1) or (f-2):

[0018] [ka]

[0019] (In the formula, Rf P is a fully halogenated C containing at least two fluorine atoms. 1-10 Alkyl group (C 1-10 The peptide according to [5] or [6], wherein the alkyl group is a group represented by (wherein, when the alkyl group has two or more carbon atoms, it may have an ether-bonded oxygen atom between the carbon atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black circle represents a bond). [8] The peptide according to any one of [5] to [7] above, wherein the C-terminus or N-terminus may be protected with a protecting group. [9] The following general formula (101) or (102):

[0020] [ka]

[0021] (In the formula, Rf P is a fully halogenated C containing at least two fluorine atoms. 1-10 Alkyl group (C 1-10 When the alkyl group has two or more carbon atoms, it may have an ether-bonding oxygen atom between the carbon atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and R 11 and R12 are each independently 1-6 an alkyl group or a benzyl group, X is a 9-fluorenylmethyloxycarbonyl group or a tert-butoxycarbonyl group, and Z is C 1-6 an alkoxy group) The peptide according to any one of [5] to [8] above, which is a tripeptide represented by the following formula:

[10] The peptide according to any one of [5] to [9] above, which is cell membrane permeable. [Effects of the Invention]

[0022] According to the production method of the present invention, a peptide having a fluoroalkyl group introduced into the side chain can be produced. Furthermore, the peptide according to the present invention has excellent cell membrane permeability due to the introduction of a fluoroalkyl group into the side chain, and is therefore expected to be used in the pharmaceutical field as a physiologically active substance. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of flow cytometry of HeLa cells treated with peptide fluorescent conjugate 1 (Alexa-Ala-RFAA-Phe-OMe), peptide fluorescent conjugate 3 (Alexa-Ala-Nle-Phe-OMe), or peptide fluorescent conjugate 4 (Alexa-Ala-Ala-Phe-OMe) in Test Example 1. [Figure 2] FIG. 1 shows the results of flow cytometry of HeLa cells treated with peptide fluorescent conjugate 1 (Alexa-Ala-RFAA-Phe-OMe), peptide fluorescent conjugate 2 (Alexa-Ala-RFAA(C8)-Phe-OMe), peptide fluorescent conjugate 3 (Alexa-Ala-Nle-Phe-OMe), or peptide fluorescent conjugate 4 (Alexa-Ala-Ala-Phe-OMe) in Test Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0024] In the present invention and the specification of this application, the "fluorine-containing amino acid" means an amino acid containing at least two fluorine atoms in its side chain. The "fluorine-containing peptide" means a peptide containing an amino acid containing at least two fluorine atoms in its side chain.

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

[0026] In the present invention and the specification of this application, "C 1-10 alkyl group" is an alkyl group having 1 to 10 carbon atoms, which may be linear or branched. "C 2-10 alkyl group" is an alkyl group having 2 to 10 carbon atoms, which may be linear or branched. Examples of the C 1-10 alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group and the like.

[0027] In the present invention and the specification of this application, "C 1-30 alkyl group" is an alkyl group having 1 to 30 carbon atoms, which may be linear or branched. "C 2-30 alkyl group" is an alkyl group having 2 to 30 carbon atoms, which may be linear or branched. C 1-30Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0028] In the present invention and the present specification, "C 1-6 The "alkyl group" is an alkyl group having 1 to 6 carbon atoms, and may be either a straight chain or a branched chain. 1-6 Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, and a hexyl group.

[0029] In the present invention and the present specification, "C 6-14 The "aryl group" is an aromatic hydrocarbon group having 6 to 14 carbon atoms, 6-12 An aryl group is particularly preferred. 6-14 Examples of the aryl group include a phenyl group, a naphthyl group, an anthryl group, and a 9-fluorenyl group, with a phenyl group being particularly preferred.

[0030] In the present invention and the present specification, "optionally substituted C 6-14 The "aryl group" is C 6-14 It is a group in which one or more, preferably 1 to 3, hydrogen atoms bonded to carbon atoms of an aryl group are substituted with other functional groups. When the aryl group has two or more substituents, the substituents may be the same or different from each other. Examples of the substituents include a nitro group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a C 1-6 Alkyl group, C1-6 Examples of the substituted or unsubstituted C include an alkoxy group and a methylenedioxy group (-O-CH2-O-). 6-14 Examples of the "aryl group" include a phenyl group, a naphthyl group, an anthryl group, a 4-nitrophenyl group, a 4-methoxyphenyl group, a 2,4-dimethoxyphenyl group, a 3,4-dimethoxyphenyl group, a 4-methylphenyl group, a 2,6-dimethylphenyl group, a 3-chlorophenyl group, and a 1,3-benzodioxol-5-yl group.

[0031] In the present invention and the present specification, "C 6-14 Aryl-C 1-6 "Alkyl group" is C 1-6 One hydrogen atom attached to a carbon atom of an alkyl group is C 6-14 It is a group substituted with an aryl group. 6-14 Aryl-C 1-6 C in alkyl groups 6-14 Examples of the aryl group include a phenyl group, a naphthyl group, an anthryl group, and a 9-fluorenyl group, with a phenyl group or a 9-fluorenyl group being particularly preferred. 6-14 Aryl-C 1-6 C in alkyl groups 1-6 The alkyl group is C 1-4 Alkyl groups are preferred. 6-14 Aryl-C 1-6 Examples of the alkyl group include a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 2-phenylethyl group, a 9-anthrylmethyl group, and a 9-fluorenylmethyl group.

[0032] In the present invention and this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The term "halogen atom other than a fluorine atom" refers to a chlorine atom, a bromine atom, or an iodine atom. Preferred examples of "halogen atoms other than a fluorine atom" include a chlorine atom or a bromine atom, with a chlorine atom being particularly preferred.

[0033] In the present invention and the present specification, "C 1-6 "Alkoxy group" refers to a C 1-6A group in which an oxygen atom is bonded to the bond terminal of an alkyl group. 1-6 The alkoxy group may be straight-chain or branched. 1-6 Examples of the alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a tert-butoxy group, a pentyloxy group, and a hexyloxy group.

[0034] In the present invention and this specification, an "ether-bonded oxygen atom" refers to an oxygen atom that connects carbon atoms, and does not include oxygen atoms in which oxygen atoms are connected in series. An alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) may have a maximum of Nc-1 ether-bonded oxygen atoms.

[0035] In the following description, "compound n" means a compound represented by formula (n).

[0036] <Synthetic reaction of fluoroalkyl-containing amino acids> A fluoroalkyl group-containing amino acid, which is an amino acid having a fluoroalkyl group introduced into the side chain, can be produced, for example, by the following synthesis reaction.

[0037] [ka]

[0038] Rf is C 1-30 Among the alkyl groups, at least two hydrogen atoms bonded to carbon atoms are substituted with fluorine atoms, and one or more hydrogen atoms bonded to carbon atoms may be further substituted with halogen atoms other than fluorine atoms. 1-30 The alkyl group is C 1-20 Alkyl groups are preferred, and C 1-10 Alkyl groups are more preferred, and C 2-10 Alkyl groups are more preferred, and C 2-8 Alkyl groups are even more preferred. 1-30 Alkyl group is C 2-30When Rf is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between carbon atoms. The number of hydrogen atoms substituted with fluorine atoms in Rf is not particularly limited as long as it is 2 or more, and is, for example, preferably 3 or more, more preferably 6 or more, and even more preferably 7 or more.

[0039] Examples of Rf include a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, a perfluorodecyl group, a difluoromethyl group, a 1,1-difluoroethyl group, a 2,2-difluoroethyl group, a 1,1,2,2-tetrafluoroethyl group, Examples thereof include a 1,1,2,2,3,3-hexafluoropropyl group, a 1,1,2,3,3,3-hexafluoropropyl group, a 1,1,2,2,3,3-hexafluorohexyl group, a 1,1,2,2,3,3-hexafluorooctyl group, a 1,1,2,2,3,3-hexafluorodecyl group, a 1,1,2,2,3,3-hexafluorooctadecyl group, and a 1,1,2,2,3,3-hexafluorohexacosyl group.

[0040] When Rf is a group having 2 carbon atoms, Rf in compound 2 is preferably a group in which at least four of the hydrogen atoms bonded to the carbon atom are substituted with fluorine atoms, such as a pentafluoroethyl group, rather than a 1,1,1-trifluoroethyl group (CF3-CH2-). Also, when Rf is a group having 3 carbon atoms, Rf in compound 2 is preferably a linear group, and in the case of a branched group, a group having 0 or 1 trifluoromethyl group is preferred to a group having two trifluoromethyl groups, such as a 1,1,1,3,3,3-hexafluoropropan-2-yl group ((CF3)2-CH-). When Rf is a group having 4 carbon atoms, Rf in compound 2 is preferably a linear group, and in the case of a branched group, a group in which the hydrogen atoms bonded to the carbon atoms constituting the alkylene group moiety are substituted with fluorine atoms, or a fully fluorinated group, is preferred.

[0041] Specifically, Rf is preferably a group represented by the general formula (f-1) or (f-2) described below.

[0042] R 1 is a protecting group for a carboxy group, specifically a protecting group selected from a group represented by the following general formula (p-1), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group. 3 may be substituted C 6-14 is an aryl group, and R 4 and R 5 are each independently a hydrogen atom or an optionally substituted C 6-14 It is an aryl group. The black circle represents a bond.

[0043] [ka]

[0044] R 1 Examples of the protecting group for the carboxyl group represented by the formula (I) include a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 4-nitrobenzyl group, a 4-methoxybenzyl group, a 2,4-dimethoxybenzyl group, a 3,4-dimethoxybenzyl group, a 4-methylbenzyl group, a 2,6-dimethylbenzyl group, a 3-chlorobenzyl group, a 9-anthrylmethyl group, a piperonyl group, a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group. 1 is preferably a benzyl group or a triphenylmethyl group, more preferably a benzyl group.

[0045] In this production method, the protecting group R of the carboxy group 1 By using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group, R 1 This method is advantageous in that it can deprotect the amino acid residues, and thus allows the synthesis of fluorine-containing amino acids and fluorine-containing peptides to be carried out without decomposing the functional groups of the amino acids.

[0046] R 6 is a silyl protecting group. 6 Examples of R include a trimethylsilyl (TMS) group, a triethylsilyl (TES) group, a triisopropylsilyl (TIPS) group, a tert-butyldimethylsilyl (TBDMS) group, and a tert-butyldiphenylsilyl (TBDPS) group. 6 is the trimethylsilyl (TMS) group.

[0047] R 2 is a protecting group for the amino group. 2 There are no particular limitations on the R protecting group, so long as it is an amino protecting group used in peptide synthesis. Examples of amino 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. R is preferred because it can be deprotected under mild conditions. 2 is preferably a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group.

[0048] [Process 1] Compound 2-2 can be obtained by reacting compound 2 with compound 8 in the presence of a metal fluoride. 6 Compound 8 represented by the following formula can be synthesized in one step from readily available Rf-I (fluoroalkyl iodide), and therefore a wide range of Rf groups can be introduced.

[0049] As the metal fluoride, alkali metal fluorides such as cesium fluoride, lithium fluoride, sodium fluoride, etc. can be used, with cesium fluoride being preferred. The reaction can be carried out in an inert solvent such as tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N,N-dimethylformamide, or N,N-dimethylacetamide, with tetrahydrofuran being preferred.

[0050] The amount of compound 8 is preferably 0.5 to 10 mol per mol of compound 2. The amount of metal fluoride is preferably 0.01 to 2 mol per mol of compound 2. The reaction in step 1 is preferably carried out at a temperature of 10°C or lower. By carrying out the reaction at a temperature of 10°C or lower, compound 2-2 can be produced in high yield. The reaction temperature is preferably -78°C to 10°C, more preferably -50°C to -10°C, and particularly preferably -40°C to -20°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0051] Compound 2 can be produced by diesterifying oxalic acid by a known method, or a commercially available product may be used.

[0052] [Process 1-1] In the reaction of step 1, compound 2-1 (one of the hydroxy groups is R 6 (a compound protected by silyl protecting group R) or a mixture of compound 2-2 and compound 2-1 may be obtained. In this case, the silyl protecting group R 6 Compound 2-2 can be obtained by deprotecting the compound. The reaction in step 1-1 can be carried out in the same manner as in step 1.

[0053] [Step 1-2] Silyl protecting group R of compound 2-1 6 Compound 2-2 can be obtained by deprotecting the compound. The deprotection can be carried out in the presence of a fluoride salt such as tetrabutylammonium fluoride (TBAF), cesium fluoride, or hydrofluoride, or an acid such as hydrochloric acid, acetic acid, or paratoluenesulfonic acid.

[0054] The reaction can be carried out in an inert solvent such as tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N,N-dimethylformamide, or N,N-dimethylacetamide, with tetrahydrofuran being preferred. The reaction is preferably carried out in the presence of acetic acid.

[0055] The amount of the fluoride salt is preferably 0.1 to 10 mol per mol of compound 2-1 (1 mol of the mixture in the case of a mixture of compound 2-2 and compound 2-1). The amount of the acid is preferably 0.1 to 10 mol per mol of compound 2-1 (1 mol of the mixture in the case of a mixture of compound 2-2 and compound 2-1). The reaction in step 1-2 is preferably carried out at a temperature of 50°C or lower. By carrying out the reaction at a temperature of 50°C or lower, compound 2-2 can be produced in a high yield. The reaction temperature is preferably -80°C to 50°C, more preferably -40°C to 30°C, and particularly preferably -20°C to 30°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0056] [Process 2] Compound 3 can be obtained by subjecting compound 2-2 to a dehydration reaction. The dehydration reaction can be carried out in the presence of a dehydrating agent such as diphosphorus pentoxide, concentrated sulfuric acid, calcium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, molecular sieves (synthetic zeolite), or silica gel. Diphosphorus pentoxide is preferred as the dehydrating agent. The amount of dehydrating agent is preferably 10 to 100% by weight relative to 100% by weight of compound 2-2. The dehydration reaction can be carried out by distilling compound 2-2 in the presence of the dehydrating agent. Distillation is preferably carried out at a temperature of 30°C to 150°C. If the distillation temperature is too high, compound 3 may decompose. If the distillation temperature is too low, compound 3 may not be condensed, resulting in a low recovery rate. Distillation can be carried out at any pressure, including reduced pressure, atmospheric pressure, or elevated pressure, and can be appropriately determined so that the boiling point of compound 3 falls within the above-mentioned preferred temperature range. The pressure is preferably 0.1 mmHg to 5 atmospheres (3800 mmHg).

[0057] [Process 3] Compound 4 can be obtained by reacting compound 3 with compound 9 or compound 10.

[0058] In general formula (9), R 2 As described above, R is a protecting group for an amino group. 7 , R 8 and R 9 are each independently 6-14 is an aryl group. 7 , R 8 or R 9 C, represented by 6-14 Examples of the aryl group include a phenyl group and a naphthyl group. Preferably, R 7 , R 8 and R 9 are each a phenyl group.

[0059] The reaction can be carried out in an inert solvent such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, or N,N-dimethylacetamide, with diethyl ether being preferred.

[0060] The amount of compound 9 or compound 10 is preferably 0.5 to 10 moles per mole of compound 3. The reaction temperature is preferably −78° C. to 100° C., more preferably 0° C. to 40° C. The reaction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 4 hours.

[0061] In a preferred embodiment, the protecting group R 2 By using a carbamate protecting group such as a tert-butoxycarbonyl group or a 9-fluorenylmethyloxycarbonyl group, R 2 This allows the synthesis of fluorine-containing amino acids while suppressing decomposition and racemization of the compounds.

[0062] [Step 4] Compound 5 can be obtained by subjecting compound 4 to a reduction reaction. The reduction reaction can be carried out by a method using a reducing agent or a method in the presence of a metal catalyst.

[0063] (1) Method using a reducing agent The reducing agent may be a borohydride reagent such as sodium borohydride, zinc borohydride, sodium cyanoborohydride, lithium triethylborohydride, lithium tri(sec-butyl)borohydride, potassium tri(sec-butyl)borohydride, lithium borohydride, or sodium triacetoxyborohydride. The reducing agent is preferably sodium borohydride or zinc borohydride, more preferably sodium borohydride. The amount of the reducing agent is preferably 0.5 to 10 moles per mole of compound 4.

[0064] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include diethyl ether, tetrahydrofuran, hydrochlorofluorocarbons (HCFCs) (e.g., Asahiklin (registered trademark) AK-225 (a mixture of 3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,3-dichloro-1,1,2,2,3-pentafluoropropane, AGC Inc.)), dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, and diethyl ether is preferred. The reaction temperature is preferably −78° C. to 100° C., more preferably −10° C. to 40° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0065] (2) Reduction in the presence of a metal catalyst Examples of the metal catalyst include palladium catalysts (e.g., palladium on carbon, palladium hydroxide, Pearlman catalyst, Lindlar catalyst, silica gel-supported palladium catalyst, alumina-supported palladium catalyst, palladium oxide), nickel catalysts (e.g., Raney nickel), platinum catalysts (e.g., platinum on carbon, platinum oxide, silica gel-supported platinum catalyst, alumina-supported platinum catalyst), rhodium catalysts (e.g., rhodium on carbon, alumina-supported rhodium catalyst, rhodium oxide), ruthenium catalysts (e.g., ruthenium on carbon, alumina-supported ruthenium catalyst, ruthenium oxide), and cobalt catalysts (e.g., Raney cobalt), with palladium catalysts being preferred. The amount of the metal catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, per 1 mol of compound 4.

[0066] The reaction can be carried out in a solvent inert to the reaction, such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, or N,N-dimethylacetamide.

[0067] The reduction reaction is carried out in the presence of hydrogen gas. The reduction reaction may be carried out at normal pressure or under increased pressure. The hydrogen gas pressure is preferably 0.5 to 10 atmospheres. The reaction temperature is preferably 0 to 100°C, more preferably 10 to 50°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0068] [Step 5-1] Protecting group R of compound 5 2 Compound 6-1 can be obtained by deprotecting the compound. Deprotection is carried out by removing the protecting group R 2 This can be done depending on the type of R 2 When is a Boc group, it can be deprotected under acidic conditions. Examples of the acid to be used include trifluoroacetic acid (TFA), hydrochloric acid, etc. The amount of the acid is preferably 1 to 1000 mol per 1 mol of compound 5.

[0069] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, with dichloromethane and N,N-dimethylformamide being preferred. An acid can also be used as the solvent. Examples of the solvent include inorganic acids and organic acids such as hydrochloric acid, acetic acid, and trifluoroacetic acid, with trifluoroacetic acid being preferred. The reaction temperature is preferably −78° C. to 50° C., more preferably 0° C. to 40° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0070] R 2 When is an Fmoc group, it can be deprotected under basic conditions. Examples of the base to be used include secondary amines such as piperidine, morpholine, and pyrrolidine. The amount of the base is preferably 1 to 100 moles per mole of compound 5. The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide. The reaction temperature is preferably -20°C to 80°C, more preferably 0°C to 40°C. The reaction time is preferably 1 minute to 24 hours, more preferably 5 minutes to 2 hours.

[0071] [Step 6-1] Protecting group R of compound 6-1 1 Compound 7 can be obtained by deprotection of Deprotection is carried out by removing the protecting group R 1 This can be done depending on the type of R 1 When is a benzyl group, triphenylmethyl group, 9-anthrylmethyl group, piperonyl group, 2-(9,10-dioxo)anthrylmethyl group, benzyloxymethyl group, or phenacyl group, deprotection can be performed by reduction in the presence of a metal catalyst. The reduction reaction can be performed in the same manner as the reduction in the presence of a metal catalyst in Step 4.

[0072] [Step 5-2] Protecting group R of compound 5 1 Compound 6-2 can be obtained by deprotecting the compound 6-2. The deprotection can be carried out in the same manner as in step 6-1.

[0073] [Step 6-2] Protecting group R of compound 6-2 2 Compound 7 can be obtained by deprotecting the compound 7. The deprotection can be carried out in the same manner as in step 5-1.

[0074] An optically active fluorine-containing amino acid (a compound containing a fluoroalkyl group) can be synthesized by asymmetric reduction of an imine (compound 4) represented by general formula (4). In the following reaction scheme, an asterisk (*) indicates that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R. In addition, Rf, R 1 , and R 2 is as defined above.

[0075] [ka]

[0076] In this production method, the protecting group R of the carboxy group 1 By using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group, R 1 This is advantageous in that it allows the synthesis of fluorine-containing amino acids and fluorine-containing peptides while maintaining optical activity.

[0077] [Step 7] Compound 4 can be subjected to asymmetric reduction to obtain compound 5-1. The asymmetric reduction reaction can be carried out by reducing compound 4 in the presence of an asymmetric reduction catalyst.

[0078] As the asymmetric reduction catalyst, a transition metal complex in which a chiral ligand is coordinated to a transition metal can be used. Examples of the transition metal include palladium, rhodium, ruthenium, iridium, nickel, cobalt, platinum, and iron. Examples of the transition metal complex include a palladium complex, a rhodium complex, a ruthenium complex, an iridium complex, and a nickel complex.

[0079] Chiral ligands include dpen (1,2-diphenylethylenediamine), daipen (1,1-di(4-anisyl)-2-isopropyl-1,2-ethylenediamine), and optically active phosphine ligands.Optically active phosphine ligands include 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), 2,2'-bis(diphenylphosphino)-5,5',6,6',7,7',8,8'-octahydro-1,1'-binaphthyl (H8-BINAP), 2,2'-bis(di-p-tolylphosphino)-1,1'-binaphthyl (Tol-BINAP), 2,2'-bis[bis(3,5-dimethylphenyl)phosphino]-1,1'-binaphthyl (Xyl-BINAP), 2,2'-bis[bis(3,5-di-t ert-butyl-4-methoxyphenyl)phosphino]-1,1'-binaphthyl (DTBM-BINAP), 1,2-bis(anisylphosphino)ethane (DIPAMP), 2,3-bis(diphenylphosphino)butane (CHIRAPHOS), 1-cyclohexyl-1,2-bis(diphenylphosphino)ethane (CYCPHOS), 1,2-bis(diphenylphosphino)propane (PROPHOS), 2,3-bis(diphenylphosphino)-5-norbornene (NORPHOS), 2,3-O-isopropylidene-2,3 -dihydroxy-1,4-bis(diphenylphosphino)butane (DIOP), 1-[1',2-bis(diphenylphosphino)ferrocenyl]ethylamine (BPPFA), 1-[1',2-bis(diphenylphosphino)ferrocenyl]ethyl alcohol (BPPFOH), 2,4-bis-(diphenylphosphino)pentane (SKEWPHOS), 1,2-bis(substituted phosphorano)benzene (DuPHOS), 5,5'-bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole (SEGPHOS), 5, Examples of suitable phosphino derivatives include 5'-bis[di(3,5-xylyl)phosphino]-4,4'-bi-1,3-benzodioxole (DM-SEGPHOS), 5,5'-bis[bis(3,5-di-tert-butyl-4-methoxyphenyl)phosphino]-4,4'-bi-1,3-benzodioxole (DTBM-SEGPHOS), 1-[2-(disubstituted phosphino)ferrocenyl]ethyl-2-substituted phosphine (Josiphos), and 1-[2-(2'-disubstituted phosphinophenyl)ferrocenyl]ethyl-2-substituted phosphine (Walphos).

[0080] The amount of the asymmetric reduction catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, relative to 1 mol of compound 4. The reaction can be carried out in a solvent inert to the reaction, such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, or N,N-dimethylacetamide. The reduction reaction is carried out in the presence of hydrogen gas. The reduction reaction may be carried out at normal pressure or under increased pressure. The hydrogen gas pressure is preferably 0.5 to 10 atmospheres. The reaction temperature is preferably 0 to 100°C, more preferably 10 to 50°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0081] [Step 8-1] Protecting group R of compound 5-1 2 Compound 6-3 can be obtained by deprotecting the compound 6-3. The deprotection can be carried out in the same manner as in step 5-1.

[0082] [Step 9-1] Protecting group R of compound 6-3 1 Compound 7-1 can be obtained by deprotecting the compound 7-1. The deprotection can be carried out in the same manner as in step 6-1.

[0083] [Step 8-2] Protecting group R of compound 5-1 1 Compound 6-4 can be obtained by deprotecting the compound 6-4. The deprotection can be carried out in the same manner as in step 6-1.

[0084] [Step 9-2] Protecting group R of compound 6-4 2 Compound 7-1 can be obtained by deprotecting the compound 7-1. The deprotection can be carried out in the same manner as in step 5-1.

[0085] The synthesis of optically active fluorine-containing amino acids (fluoroalkyl group-containing compounds) can also be carried out by the following reaction. In the following reaction scheme, an asterisk indicates that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R. In addition, Rf, R 1 , and R 2 is as defined above.

[0086] [ka]

[0087] [Step 10-1] Compound 6-3 can be obtained by optical resolution of compound 6-1. Optical resolution can be carried out by known methods, such as a method using a chiral column, a crystallization method, or a diastereomer method.

[0088] (1) Using a chiral column A racemic compound can be separated into optically active compounds by liquid chromatography or supercritical fluid chromatography (SFC) using a chiral column. Chiral columns such as CHIRALPAK (registered trademark) (Daicel Corporation) and CHIRALCEL (registered trademark) (Daicel Corporation) can be used.

[0089] (2) Crystallization method The racemate is salted with an optically active amine or an optically active acid, and the resulting diastereomeric salt is induced into a crystalline form, which is then fractionally crystallized. Repeated recrystallization can yield a single diastereomeric salt. If necessary, the diastereomeric salt is neutralized to obtain the optically active free form. Examples of optically active amines include brucine, cinchonidine, cinchonine, and 1-phenethylamine. Examples of optically active acids include camphorsulfonic acid, tartaric acid, and mandelic acid.

[0090] (3) Diastereomer method The racemate is reacted with an optically active reagent to obtain a mixture of diastereomers, which are then separated into single diastereomers by fractional crystallization and chromatography.The optically active reagent moiety is then removed from the resulting single diastereomer to obtain the desired optical isomer.

[0091] [Step 11-1] Protecting group R of compound 6-3 1 Compound 7-1 can be obtained by deprotecting the compound 7-1. The deprotection can be carried out in the same manner as in step 6-1.

[0092] [Step 10-2] Compound 6-4 can be obtained by optical resolution of compound 6-2. Optical resolution can be carried out in the same manner as in step 10-1.

[0093] [Step 11-2] Protecting group R of compound 6-4 2 Compound 7-1 can be obtained by deprotecting the compound 7-1. The deprotection can be carried out in the same manner as in step 5-1.

[0094] [Step 12] Compound 7-1 can be obtained by optical resolution of compound 7. Optical resolution can be carried out in the same manner as in step 10-1.

[0095] <Method of producing fluoroalkyl group-containing peptide> The fluoroalkyl group-containing peptide can be produced using an amino acid having a fluoroalkyl group introduced into the side chain as a starting material, for example, Compound 6-1, Compound 6-2, Compound 6-3, or Compound 6-4.

[0096] For example, a fluoroalkyl group-containing peptide can be produced by condensing Compound 6-2 or 6-4 with a fluorine-containing amino acid with a protected carboxy group, an amino acid with a protected carboxy group, a fluorine-containing peptide with a protected C-terminus, or a peptide with a protected C-terminus. Also, a fluoroalkyl group-containing peptide can be produced by condensing Compound 6-1 or 6-3 with a fluorine-containing amino acid with a protected amino group, an amino acid with a protected amino group, a fluorine-containing peptide with a protected N-terminus, or a peptide with a protected N-terminus.

[0097] Alternatively, compound 7 or compound 7-1 can be similarly prepared by protecting the amino or carboxy group thereof. Specifically, the amino group is protected with a protecting group, followed by condensation with a fluorine-containing amino acid with a protected carboxy group, an amino acid with a protected carboxy group, a fluorine-containing peptide with a protected C-terminus, or a peptide with a protected C-terminus. Alternatively, the carboxy group can be protected with a protecting group, followed by condensation with a fluorine-containing amino acid with a protected amino group, an amino acid with a protected amino group, a fluorine-containing peptide with a protected N-terminus, or a peptide with a protected N-terminus.

[0098] Peptides can be produced by a general peptide synthesis method, for example, solid-phase peptide synthesis. Fluoroalkyl group-containing peptides can be easily synthesized using an automated peptide synthesizer from amino acids with fluoroalkyl groups introduced into their side chains.

[0099] Peptides can be produced by sequentially condensing amino acids with protected amino groups with an amino acid whose C-terminus is bound to a solid phase, and then cleaving the peptide from the solid phase. It is preferable to use amino acid raw materials whose amino groups are protected with a Boc group or an Fmoc group. It is preferable to use amino acid raw materials whose side chain functional groups are protected with a protecting group. Examples of protecting groups for side chain functional groups include a Boc group, a triphenylmethyl group, a benzyl group, and a 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group.

[0100] Condensation agents for forming peptide bonds include, for example, N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (WSC), benzotriazol-1-yloxy-trisdimethylaminophosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytrispyrrolizinophosphonium hexafluorophosphate (pyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, etc. Furthermore, N-hydroxybenzotriazole (HOBt) and the above-mentioned condensation agents can also be mixed in a preferred ratio and used.

[0101] Peptide bond formation may be achieved by activating the carboxy terminus, and examples of activating agents include N-hydroxysuccinimide, p-nitrophenyl ester, and pentafluorophenyl ester. Examples of bases used in peptide bond formation include triethylamine and diisopropylethylamine (DIPEA). Examples of solvents used in peptide bond formation reactions include chloroform, dichloromethane, acetonitrile, N,N-dimethylformamide (DMF), and dimethyl sulfoxide.

[0102] The Boc and Fmoc groups, which are protecting groups for the amino-terminal amino group of a peptide or amino acid, can be removed with trifluoroacetic acid or piperidine, respectively. Protecting groups for the side chain functional groups of amino acid residues of a peptide can be removed with, for example, trifluoroacetic acid, hydrogen fluoride (HF), trifluoromethanesulfonic acid, etc.

[0103] In solid-phase peptide synthesis, a peptide having a protecting group attached to the side chain functional group of the peptide or amino acid residue can be removed from the solid-phase peptide synthesis resin using, for example, TFA. The removal of the peptide from the solid-phase peptide resin and the removal of the protecting group from the side chain functional group of the amino acid residue can be carried out simultaneously in the same reaction system. Alternatively, they can be carried out independently. Examples of commercially available resins that can be used for solid-phase peptide synthesis include 4-hydroxymethyl-3-methoxyphenoxybutyric acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, and oxime resin.

[0104] 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, fractional crystallization, etc. Furthermore, the peptide thus obtained can be converted into its respective salt by a conventional method.

[0105] The protecting group of the amino group or carboxy group of the produced fluoroalkyl-containing peptide can be deprotected as needed. The deprotection can be carried out by a conventional method depending on the type of protecting group.

[0106] <Fluoroalkyl group-containing peptides> The fluoroalkyl group-containing peptide according to the present invention is a peptide consisting of two or more amino acids, in which at least one of the amino acid residues constituting the peptide has a C group substituted with at least two fluorine atoms in the side chain. 1-30 It has an alkyl group. C substituted with at least two fluorine atoms 1-30 The alkyl group may be further substituted with a halogen atom other than a fluorine atom. 1-30 Alkyl group is C 2-30 In the case of an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between carbon atoms.

[0107] The fluoroalkyl group-containing peptide according to the present invention includes a peptide having an amino acid residue whose side chain is the Rf. At least one side chain of the amino acid residues constituting the peptide may be Rf, and the side chains of all amino acid residues may be Rf. When a single peptide molecule contains two or more amino acid residues whose side chains are Rf, these multiple Rfs may be the same or different. Furthermore, in the peptide, the amino acid residue whose side chain is Rf may be located at the N-terminus, C-terminus, or other position.

[0108] Rf is preferably a group represented by the following general formula (f-1) or (f-2): P is a fully halogenated C containing at least two fluorine atoms. 1-10 Represents an alkyl group. P is C 1-10 All hydrogen atoms of the alkyl group are substituted with halogen atoms, and at least two of these halogen atoms are fluorine atoms. P When the carbon number is 2 or more, that is, fully halogenated C 2-10 In the case of an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between carbon atoms. P may be the same group or different groups.

[0109] In the following general formula (f-1) or (f-2), n1 is an integer of 0 to 10, and n2 is an integer of 0 to 9. When n1 and n2 are 0, they both represent a single bond. That is, when n1 is 0, the group represented by general formula (f-1) is Rf P When n2 is 0, the group represented by general formula (f-2) is (Rf P )2-CH-.

[0110] [ka]

[0111] When Rf is a group represented by general formula (f-1), Rf is Rf P is preferably a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n1 is an integer of 0 to 4; P is more preferably a group in which n1 is an integer of 0 to 2, and Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n1 is an integer of 0 to 2 (provided that n1 is 1 and Rf P is a trifluoromethyl group) is more preferred.

[0112] When Rf is a group represented by general formula (f-2), Rf is Rf P is preferably a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n2 is an integer of 0 to 4; P is more preferably a group in which n2 is an integer of 0 to 2, and Rf Pis a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n2 is an integer of 0 to 2 (wherein n2 is 0 or 1 and Rf P is a trifluoromethyl group) is more preferred.

[0113] Examples of Rf include a difluoromethyl group, a 1,1-difluoroethyl group, a 2,2-difluoroethyl group, a 1,1,2,2-tetrafluoroethyl group, a 1,1,2,2,3,3-hexafluoropropyl group, and a 1,1,2,3,3,3-hexafluoropropyl group.

[0114] A peptide consisting of two or more amino acids is preferably a peptide consisting of three or more amino acids, more preferably a peptide consisting of 2 to 40 amino acids, and even more preferably a peptide consisting of 3 to 20 amino acids.

[0115] The C-terminus of the fluoroalkyl group-containing peptide of the present invention is R 1 R 1 is preferably a benzyl group. In addition, the N-terminus of the fluoroalkyl group-containing peptide according to the present invention is preferably a 2 The amino group may be protected by a protecting group represented by R 2 is preferably a Boc group or an Fmoc group.

[0116] The fluoroalkyl group-containing peptide according to the present invention includes, for example, a tripeptide represented by the following general formula (101) or (102): 11 and R 12 are each independently 1-6 R is an alkyl group or a benzyl group, each of which is preferably a methyl group or a benzyl group; 11 is a methyl group, and R 12 is particularly preferably a benzyl group. X is Fmoc or Boc. Z is C1-6 Alkoxy groups are preferred, with methoxy groups being particularly preferred.

[0117] [ka]

[0118] In the general formulas (101) and (102), Rf P , n1, and n2 are the same as those in the general formulae (f-1) and (f-2). The group represented by the general formula (101) or (102) includes Rf P Fully fluorinated C 1-10 It is preferable that n1 or n2 is an integer of 0 to 4, and Rf P is more preferably a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, or a perfluorooctyl group, and n1 or n2 is an integer of 0 to 2; P It is more preferable that n1 or n2 is an integer of 0 to 2.

[0119] Fluoroalkyl groups have a high affinity for cell membranes. Therefore, the fluoroalkyl group-containing peptides of the present invention have excellent cell membrane permeability. Furthermore, because their structure is significantly different from that of natural peptides, they are less susceptible to degradation by peptidases. Taking advantage of these properties, the fluoroalkyl group-containing peptides of the present invention are expected to be used in the pharmaceutical field as physiologically active substances. For example, the fluoroalkyl group-containing peptides of the present invention are expected to be used as DDS carriers that deliver medicinal ingredients to target cells. For example, by adding the fluoroalkyl group-containing peptides of the present invention to functional peptides that exhibit some physiological activity upon uptake into target cells in vivo without impairing their function, the uptake efficiency of the functional peptide into target cells can be improved. Furthermore, by substituting some side chains of hydrophobic amino acid residues of a physiologically active functional peptide with Rf, preferably a group represented by the general formula (101) or (102), within a range that does not impair the function of the functional peptide, the cell membrane permeability and intracellular residence time of the functional peptide can be improved. [Example]

[0120] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0121] The NMR apparatus used in the analyses of the Examples and Comparative Examples was a JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd. 1 H NMR showed tetramethylsilane at 0 ppm, 19 For F NMR, C6F6 was used as the reference value at -162 PPM.

[0122] The following abbreviations are used in this specification: Bn: Benzyl Boc: t-butoxycarbonyl All: Allyl Et2O: Diethyl ether Fmoc: 9-fluorenylmethyloxycarbonyl THF: tetrahydrofuran TMS: Trimethylsilyl C4F9: 1,1,2,2,3,3,4,4,4-nonafluorobutyl C8F 17 :1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluorooctyl

[0123] [Manufacturing Example 1] 2-((t-Butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoic acid was synthesized from trimethyl(nonafluorobutyl)silane and dibenzyl oxalate.

[0124] [Process 1]

[0125] [ka]

[0126] A 100 mL two-necked oven-dried flask was equipped with a stir bar, and under a nitrogen atmosphere, dibenzyl oxalate (5.41 g, 20.0 mmol), cesium fluoride (255 mg, 1.68 mmol), and THF (54 mL) were added and stirred. The mixture was then cooled to -30°C, and trimethyl(nonafluorobutyl)silane (4.50 mL, 20.2 mmol) was added, followed by stirring for 24 hours at -30°C. A saturated aqueous solution of ammonium chloride (30 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (3 x 50 mL). The combined organic phase was dried over sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to give a crude mixture of benzyl 2-(benzyloxy)-3,3,4,4,5,5,6,6,6-nonafluoro-2-((trimethylsilyl)oxy)hexanoate and benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2,2-dihydroxyhexanoate, which was used in the next step without further purification.

[0127] [Step 1-2]

[0128] [ka]

[0129] A 100 mL two-necked oven-dried flask was fitted with a stir bar. Under a nitrogen atmosphere, the entire crude product obtained in step 1, tetrabutylammonium fluoride (TBAF) 1 mol / L THF solution (10.5 mL, 10.5 mmol), acetic acid (1 mL), and THF (50 mL) were added and stirred at 0 °C. The mixture was then warmed to room temperature and stirred for 24 h. The mixture was then quenched by the addition of saturated aqueous sodium bicarbonate (30 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with water (50 mL) and saturated brine (50 mL), dried over sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to give crude benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2,2-dihydroxyhexanoate. The crude product was used in the next step without further purification.

[0130] 1 H NMR(400MHz,CDCl3) δ7.39(brs,5H),5.37(s,2H). 19 F NMR(376MHz,CDCl3) δ-80.79(brs,3F),-121.09(brs,2F),-121.24-121.26(m,2F),-126.12-126.21(m,2F).

[0131] [Process 2]

[0132] [ka]

[0133] A stirrer was placed in a 20 mL flask dried in an oven, and the entire crude product obtained in step 2 and phosphorus pentoxide (22% by weight of the crude product) were added under a nitrogen atmosphere, followed by distillation under reduced pressure. The fraction obtained at 2 mmHg and 77°C was collected, yielding benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2-oxohexanoate as a colorless liquid (73% yield throughout steps 1 to 3).

[0134] 1 H NMR(400MHz,CDCl3) δ7.41(brs,5H),5.40(s,2H). 19 F NMR(376MHz,CDCl3) δ-80.79(brs,3F),-117.78-117.850(t,2F,J F-F =13Hz),-122.01(brs,2F),-125.58(brs,2F).

[0135] Benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2-oxohexanoate was obtained as a colorless liquid in the same manner as in steps 1 and 2, except that the temperature in step 1 was changed to 0° C. The yield from step 1 to step 2 was 69%.

[0136] [Process 3]

[0137] [ka]

[0138] A 30 mL oven-dried Schlenk flask equipped with a stir bar was charged with benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2-oxohexanoate (1 g, 2.6 mmol), t-butyl(triphenylphosphaneilidene)carbamate (2.6 mmol), and EtO (10 mL) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 1 hour, then filtered, and the residue was washed with EtO (2 × 2 mL). The combined organic phase was evaporated under reduced pressure to give crude benzyl 2-((t-butoxycarbonyl)imino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate. The resulting crude product was purified by silica gel chromatography (Et2O / hexane = 1 / 4) to give benzyl 2-((t-butoxycarbonyl)imino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate as a colorless liquid (yield 87%).

[0139] 1 H NMR(400MHz,CDCl3) δ7.410-7.352(m,5H),5.350(s,2H),1.504(s,9H). 19F NMR(376MHz,CDCl3) δ-80.76-80.78(t,3F,J F-F =9Hz),-112.37(brs,2F),-121.0(brs,2F),-125.36(brs,2F).

[0140] [Step 4]

[0141] [ka]

[0142] In an oven-dried 30 mL Schlenk flask equipped with a stir bar, benzyl 2-((t-butoxycarbonyl)imino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate (0.2 g, 0.42 mmol) was dissolved in EtO (15 mL) under a nitrogen atmosphere and stirred at 0 °C. Sodium borohydride (0.46 mmol) was added in three portions at 0 °C, and the mixture was then warmed to room temperature and stirred for 24 h. The mixture was quenched with ice water, and 1 mol / L hydrochloric acid was added to adjust the pH to less than 7. The aqueous phase was extracted with EtO (2 × 10 mL), and the combined organic phase was evaporated under reduced pressure to give crude benzyl 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate. The obtained crude product was purified by silica gel chromatography (ethyl acetate / hexane=1 / 4) to obtain benzyl 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate as a colorless liquid (yield 61%).

[0143] 1 H NMR(400MHz,CDCl3) δ7.40-7.33(m,5H),5.41-5.39(d,2H,J H-H =10Hz),5.28-5.20(m,3H),1.45(s,9H). 19 F NMR(376MHz,CDCl3) δ-80.86-80.89(t,3F,J F-F=9Hz),-115.36-118.55(m,2F),-121.50-123.17(m,2F),-125.00-126.77(m,2F).

[0144] A reaction similar to that in Step 4 was carried out using the solvents and reducing agents (equivalent amounts) shown in Table 1 instead of EtO and sodium borohydride. The yields are shown in Table 1. In the table, "AK225" is "ASAHIKLIN (registered trademark) AK-225" (a mixture of 3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,3-dichloro-1,1,2,2,3-pentafluoropropane, AGC Inc.).

[0145] [Table 1]

[0146] [Step 5-2]

[0147] [ka]

[0148] A 25 mL oven-dried two-neck flask was equipped with a stir bar, and benzyl 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoate (73.6 mg, 0.15 mmol), palladium on carbon (Pd 5%, approximately 55% wet with water, 20 mg), ethyl acetate (1 mL), and ethanol (7 mL) were added. The mixture was stirred at room temperature under a hydrogen atmosphere at atmospheric pressure. After stirring for 24 hours at room temperature, the mixture was filtered through Celite. The residue was washed with ethanol (3 × 5 mL), and the combined organic phase was evaporated under reduced pressure to give crude 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoic acid. The obtained crude product was purified by silica gel chromatography (ethyl acetate / hexane=1 / 1) to obtain 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoic acid as a colorless liquid (yield 86%).

[0149] 1 H NMR(400MHz,CDCl3) δ7.72(br,1H),5.49-5.47(d,2H,J H-H =10Hz),5.24-5.15(m,1H),1.45(s,9H). 19 F NMR(376MHz,CDCl3) δ-80.30(brs,3F),-114.64-118.03(m,2F),-120.70-122.40(m,2F),-124.52-126.22(m,2F).

[0150] [Step 5-1]

[0151] [ka]

[0152] A stir bar was placed in an oven-dried 25 mL two-neck flask, and Boc-RFAA-OBn (376 mg, 0.78 mmol) and 4 M HCl in 1,4-dioxane (3 mL) were added at 0°C. After stirring at room temperature for 18 hours, saturated aqueous sodium carbonate solution was added to adjust the pH to greater than 7, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The organic phase was filtered, and the filtrate was evaporated under reduced pressure to obtain the hydrochloride salt of H-RFAA-OBn as a white solid (yield 78%).

[0153] 1 H NMR(400MHz,D2O) δ7.31(brs,2H),6.81-6.77(m,5H),5.27(m,1H),3.71-3.67(m,2H). 19 F NMR(376MHz,D2O) δ-80.38(t,3F),-118.29-121.00(m,2F),-121.00-123.80(m,2F),-126.12-128.12(m,2F).

[0154] Hereinafter, amino acids may be represented by three-letter symbols. For example, "Phe" is phenylalanine and "Gly" is glycine. Peptides are represented as (N-protecting group)-amino acid three-letter symbol-(C-protecting group). "H-AA-OMe" means that the N-terminus is unprotected and the C-terminus is a methyl ester. When the C-terminus is unprotected, "OH" is used instead of "OMe."

[0155] [Example 1] A dipeptide bearing a nonafluorobutyl group was synthesized.

[0156] [ka]

[0157] A 25 mL two-necked oven-dried flask equipped with a stir bar was charged with 2-((t-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,6-nonafluorohexanoic acid (33.4 mg, 0.085 mmol), DIPEA (0.13 mmol), DCM (3 mL), L-phenylalanine methyl ester (0.13 mmol), and benzotriazol-1-ol monohydrate (0.085 mmol) at room temperature. After cooling to 0 °C, BOP (0.085 mmol) was added. After stirring at room temperature for 24 h, the solvent was evaporated under reduced pressure, the mixture was diluted with ethyl acetate, and the organic phase was washed with saturated aqueous citric acid, saturated aqueous sodium carbonate, and saturated brine, and dried over sodium sulfate. The organic phase was filtered, and the filtrate was evaporated under reduced pressure to obtain crude Boc-RFAA-Phe-OMe. The resulting crude product was purified by silica gel chromatography (ethyl acetate / hexane=1 / 4) to give two Boc-RFAA-Phe-OMe diastereomers as colorless liquids (22% yield for the two diastereomers combined).

[0158] Diastereomer A 1 H NMR(400MHz,CDCl3) δ7.31-7.25(m,5H),6.41-6.40(d,NH,J H-H=7Hz), 5.48-5.46(d,1H,J H-H =9Hz),4.99-4.91(m,1H),4.91-4.86(m,1H),3.75(s,3H),3.23-3.10(m,2H),1.47(s,9H). 19 F NMR(376MHz,CDCl3) δ-80.98(t,3F,J F-F =7Hz),-114.56-119.80(m,2F),-121.40-123.22(m,2F),-125.03-127.15(m,2F).

[0159] Diastereomer B 1H NMR (400MHz, CDCl3) δ7.30-7.07(m,5H),6.42-6.40(d,NH,J H-H =9Hz), 5.51-5.48(d,1H,J H-H =8Hz),5.00-4.95(m,1H),4.93-4.88(m,1H),3.74(s,3H),3.15-3.13(m,2H),1.44(s,9H). 19 F NMR(376MHz,CDCl3) δ-80.77(t,3F,J F-F =9Hz),-113.74-119.30(m,2F),-121.22-122.94(m,2F),-124.82-127.05(m,2F).

[0160] Similar reactions were carried out using the amino acid methyl esters listed in Table 2 instead of L-phenylalanine methyl ester. The yields are shown in Table 2. In the table, "DCM" represents dichloromethane, "BOP" represents benzotriazol-1-yloxy-trisdimethylaminophosphonium hexafluorophosphate, and "EDC" represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0161] [Table 2]

[0162] [Example 2] The protecting group on the N-terminus of the peptide synthesized in Example 1 was deprotected.

[0163] [ka]

[0164] A 25 mL two-necked oven-dried flask was equipped with a stir bar and charged with Boc-RFAA-Gly-OMe (21.2 mg, 0.05 mmol), DCM (1.5 mL), and TFA (0.4 mL). After stirring at room temperature for 24 hours, the pH was adjusted to greater than 7 by adding saturated aqueous sodium carbonate, and the mixture was extracted with DCM. The organic phase was washed with saturated brine and dried over sodium sulfate. The organic phase was filtered, and the filtrate was evaporated under reduced pressure to obtain crude H-RFAA-Gly-OMe (yield 66%).

[0165] 1 H NMR(400MHz,CDCl3) δ7.08(brs,1H),4.15-4.07(m,2H),3.79(s,3H). 19 F NMR(376MHz,CDCl3) δ-80.71(t,3F,J F-F =7Hz),-115.13-119.94(m,2F),-119.94-121.93(m,2F),-125.02-126.82(m,2F).

[0166] [Example 3] A tripeptide bearing a nonafluorobutyl group was synthesized.

[0167] [ka]

[0168] H-RFAA-Gly-OMe (10.9 mg, 0.03 mmol), DCM (0.5 mL), DIPEA (0.13 mmol), Fmoc-Gly-OH (0.03 mmol), and benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (0.085 mmol) were added to an oven-dried NMR test tube at room temperature. After standing at room temperature for 24 hours, the solvent was removed by evaporation under reduced pressure, the mixture was diluted with ethyl acetate, and the organic phase was washed with saturated aqueous citric acid, saturated aqueous sodium carbonate, and saturated brine and dried over sodium sulfate. The organic phase was filtered, and the filtrate was evaporated under reduced pressure to obtain crude Fmoc-Gly-RFAA-Gly-OMe.

[0169] 1 H NMR(400MHz,CDCl3) δ7.78-7.27(m,8H),δ7.30(brs,1H),7.13(brs,1H),5.63-5.61(d,1H,J H-H =7Hz),5.64-5.54(m,1H),4.42-4.41(d,2H,J H-H =7Hz),4.24-4.20(m,1H),4.08-3.98(m,4H),3.74(s,3H). 19 F NMR(376MHz,CDCl3) δ-80.78(t,3F,J F-F =10Hz),-114.61-118.74(m,2F),-121.22-123.09(m,2F),-124.89-126.90(m,2F).

[0170] [Example 4] A tripeptide bearing a nonafluorobutyl group (Boc-Ala-RFAA-Phe-OMe) was synthesized.

[0171] [ka]

[0172] A 25 mL two-neck flask equipped with a stir bar was charged with Boc-RFAA-Phe-OMe diastereomer A (DR > 95, 0.11 mmol) and DCM (5 mL). The reaction mixture was cooled to 0 °C, then TFA (1.25 mL) was added and the mixture was warmed to room temperature. After stirring for 4 h, the reaction was quenched by the addition of aqueous sodium bicarbonate. The aqueous phase was extracted with DCM, and the combined organic phase was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate:triethylamine = 2:1:1%) to give H-RFAA-Phe-OMe (33.9 mg, 70.0% yield).

[0173] 1 H NMR(400MHz,CDCl3) δ=1.77(br s,2H),3.10-3.14(m,2H),3.74(s,3H),3.93-3.99(dd,1H),4.90-4.95(dd,1H),6.81-6.83(d,NH),7.27(m,5H) 19 F NMR(376MHz,CDCl3) δ=-126.2-125.7(m,2F),-121.2-119.7(m,2F),-120.5-114.4(m,2F),-80.7(t,3F)

[0174] [ka]

[0175] To one side of a 25 mL two-neck flask equipped with a stir bar, Boc-Ala-OH (1.1 equiv.), 1-hydroxy-7-azabenzotriazole (HOAt, 1.1 equiv.), DIPEA (1.3 equiv.), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 1.1 equiv.), and dipeptide (H-RFAA-Phe-OMe, dr>95:5, 38 μmol) were added to 3 mL of DCM and the temperature was adjusted to 0 °C. The mixture was warmed to room temperature and stirred for 1.5 h. The reaction was then quenched by the addition of 1 N HCl. The reaction mixture was partitioned between 1 N HCl and DCM, and the combined organic phase was evaporated under reduced pressure and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO3, and brine, dried over Na2SO4, and evaporated to give a white solid. The crude mixture was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to give the tripeptide (dr>95:5, 18.2 mg, 75.1% yield).

[0176] 1 H NMR(400MHz,Acetone d6) δ=1.30(d,3H),1.38(s,9H),3.04-3.16(m,2H),3.66(s,3H),4.26-4.29(m,1H),4.68-4. 77(m,1H),5.53-5.61(m,1H),6.23(d,NH),7.19-7.29(m,5H),7.82(d,NH),8.30(d,NH). 19 F NMR(376MHz,Acetone d6) δ=-126.9-126.3(m,2F),-123.1-121.9(m,2F),-120.1-115.0(m,2F),-81.5(t,3F)

[0177] [Example 5] A tripeptide bearing a nonafluorobutyl group (H-Ala-RFAA-Phe-OMe) was synthesized.

[0178] [ka]

[0179] A 25 mL two-neck flask equipped with a stir bar was charged with Boc-RFAA-Phe-OMe diastereomer A (DR > 95, 29 μmol) and DCM (2 mL). The reaction mixture was cooled to 0 °C, then TFA (0.4 mL) was added and warmed to room temperature. After stirring for 4 h, the reaction was quenched by the addition of aqueous sodium bicarbonate. The aqueous phase was extracted with DCM, and the combined organic phase was evaporated under reduced pressure. The resulting mixture was purified by silica gel column chromatography (CHCl3:MeOH = 10:1) to give H-Ala-RFAA-Phe-OMe (DR > 95, 13.8 mg, 90.6% yield).

[0180] 1 H NMR(400MHz,Acetone d6) δ=1.16(d,3H),2.81(br s,2H),3.00-3.14(m,2H),3.67(s,3H),3.94-3.99(m,1H),4.68-4.74(m,1H),5.49-5.56(m,1H),7.19-7.27(m,5H),8.16(d,NH),8.33(d,NH). 19 F NMR(376MHz,Acetone d6) δ=-126.9-125.6(m,2F),-123.1-122.1(m,2F),-120.0-115.3(m,2F),-81.7(t,3F)

[0181] [Example 6] The fluorescent substance Alexa Fluor 647 was fused to the N-terminus of the tripeptide (H-Ala-RFAA-Phe-OMe) having a nonafluorobutyl group synthesized in Example 5.

[0182] To a 1.5 mL black tube, Alexa Fluor 647 (250 μg) dissolved in dry DMSO (15 μL), H-Ala-RFAA-Phe-OMe (1.5 equivalents) dissolved in dry DMSO (15 μL), and DIPEA (1.5 equivalents) were added. The mixture was stirred overnight at room temperature. The mixture was purified by reverse-phase chromatography (acetonitrile / water / TFA = 30:70:0.1 to 95:5:0.1) and lyophilized to give fluorescent conjugate 1 as a blue solid (32.3% yield calculated by fluorimetry). Fluorescence was measured using a Nano Drop® ND-1000 spectrophotometer at an emission wavelength of 650 nm.

[0183] MALDI-TOF MS [M] - :m / z calcd.for C 55 H 63 F9N5O 17 S4 - 1364.2964,found 1364.7252

[0184] [Example 7] A tripeptide bearing a heptadecafluorooctyl group (H-Ala-RFAA(C8)-Phe-OMe) was synthesized.

[0185] [ka]

[0186] The perfluoroalkylation reaction was carried out according to a previous report (Journal of Fluorine Chemistry, 1984, vol. 26, pp. 341-358). The resulting crude product was purified by sublimation (72 °C, 0.5 mmHg). The resulting white solid was directly transferred to a 100 mL three-necked round-bottom flask, dissolved in EtO (10 mL), and reacted with tert-butyl(triphenylphosphanylidene)carbamate (5.5 mmol) at room temperature for 1 hour. The crude product was filtered, and the filtrate was evaporated. The resulting white solid was purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 w / 0.4% NEt) to give the α-imino ester (311 mg, 3-step yield: 8.2%).

[0187] 1 H NMR (400MHz, CDCl3) δ=1.50(s,9H),5.36(s,2H),7.36-7.38(m,5H) 19 F NMR(376MHz,CDCl3) δ=-126.3(m,2F),-122.8(m,2F),-121.8-122.0(m,4F),-121.2(m,2F),-120.2(m,2F),-112.6(m,2F),-81.0(t,3F)

[0188] [ka]

[0189] A 25 mL two-necked round-bottom flask equipped with a stir bar was charged with the α-iminoester (311 mg, 0.47 mmol) and THF (5 mL). Sodium triacetoxyborohydride (0.59 mmol) was added to the reaction mixture at 0°C, followed by stirring at room temperature for 24 hours. The reaction mixture was directly evaporated and partitioned between water and DCM. The combined organic phase was evaporated under reduced pressure to give a crude mixture. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 1:10) to give a white solid (Boc-RFAA(C8)-OBn) (49.6% yield).

[0190] 1 H NMR(400MHz CDCl3) δ=1.28(s,9H),5.07(s,2H),5.137(m,1H),7.18(m,5H) 19 F NMR(376MHz CDCl3) δ=-126.3(m,2F),-122.9(m,2F),-121.5-122.1(m,8F),-115.3-118.8(m,2F),-81.0(t,3F)

[0191] The procedure for the tripeptide was the same as above. Boc-RFAA(C8)-OH was obtained from 0.23 mmol of Boc-RFAA(C8)-OBn purified by HPLC in the same manner as in step 5-2 of Production Example 1. Boc-RFAA(C8)-Pne-OMe was then obtained from Boc-RFAA(C8)-OH in the same manner as in Example 1, and 46 mg of the tripeptide (H-Ala-RFAA(C8)-Phe-OMe) was obtained from Boc-RFAA(C8)-Pne-OMe in the same manner as in Example 4 (total yield 27.8%).

[0192] ESI-MS [M+H] + :m / z calcd.for 726.13,found 726.52

[0193] [Example 8] The fluorescent substance Alexa Fluor 647 (manufactured by Thermo Fisher Scientific) was fused to the N-terminus of the tripeptide having a heptadecafluorooctyl group (H-Ala-RFAA(C8)-Phe-OMe) synthesized in Example 7.

[0194] To a 1.5 mL black tube, Alexa Fluor 647 (125 μg) dissolved in dry DMSO (15 μL), H-Ala-RFAA(C8)-Phe-OMe (1.5 equivalents) dissolved in dry DMSO (15 μL), and DIPEA (1.5 equivalents) were added. The mixture was stirred overnight at room temperature. The mixture was purified by reverse-phase chromatography (acetonitrile / water / TFA = 5:95:0.1 to 10:95:0.1) and lyophilized to give fluorescent conjugate 2 as a blue solid (5.7% yield calculated by fluorimetry, emission wavelength = 650 nm).

[0195] MALDI-TOF MS [M] - :m / z calcd.for C 55 H 72 N5O 17 S4 - 1564.2836,found 1564.5701

[0196] [Comparative Example 1] A dipeptide bearing a butyl group (H-Nle-Phe-OMe) was synthesized.

[0197] [ka]

[0198] Boc-Nle-Phe-Ome was synthesized (yield: 556 mg, 40.2%) according to a previous report (Chemical and Pharmaceutical Bulletin, 1987, vol. 35, p. 468).

[0199] [ka]

[0200] A 25 mL two-neck flask equipped with a stir bar was charged with Boc-Nle-Phe-OMe (1.42 mmol) and DCM (10 mL). The reaction mixture was cooled to 0 °C, then TFA (2 mL) was added and warmed to room temperature. After stirring for 4 h, the reaction was quenched by the addition of aqueous sodium bicarbonate. The aqueous phase was extracted with DCM, and the combined organic phases were evaporated under reduced pressure to give a stoichiometric amount of H-Nle-Phe-OMe. The product was used without further purification.

[0201] 1 H NMR (400MHz, ACETONE-D6) Rotamer A Δ8.09-7.88(m,1H),7.42-7.04(m,5H),4.67(dd,J=11.0,4.6Hz,1H),4.59(t,J=7.5Hz,1H),3.69(s,3H),3.22( dd,J=13.7,4.6Hz,1H),3.00(dd,J=14.0,10.7Hz,1H),2.00-1.80(m,2H),1.48-1.18(m,2H),0.83-0.73(m,3H) Rotamer B 7.60(t,J=7.8Hz,1H),7.42-7.04(m,5H),4.75(t,J=6.9Hz,1H),4.25(t,J=6.4Hz,1H),3.6 4(s,3H),3.10(t,J=7.3Hz,2H),2.00-1.80(m,2H),1.48-1.18(m,2H),0.86(t,J=7.3Hz,3H)

[0202] [Comparative Example 2] H-Ala-Nle-Phe-OMe is a synthetic compound made of H-Ala-Nle-Phe-OMe.

[0203]

change

[0204] A 50 mL two-necked round-bottom flask was charged with Fmoc-Ala-OH (1.1 equiv.), HOAt (1.1 equiv.), and DIPEA (1.3 equiv.). HATU (1.1 equiv.) and the dipeptide (H-N1-Phe-OMe, 1.42 mmol) dissolved in 20 mL of DCM were added to the mixture at 0 °C. The mixture was allowed to warm to room temperature and then stirred for 1.5 h before quenching with HCl (1N). The mixture was partitioned between HCl (1N) and DCM. The combined organic phase was evaporated under reduced pressure and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO3, and brine, dried over Na2SO4, and evaporated to give a white solid. 25 mL of a 20% solution of piperidine in DMF was added to the crude mixture and stirred at room temperature for 1 h. The solvent was removed by vacuum drying to give a white solid, which was purified by silica gel column chromatography (Et2O:DCM=1:3) to give H-Ala-Nle-Phe-OMe (dr>95, 116 mg, 23.0% yield).

[0205] MALDI-TOF MS [M+H] + :m / z calcd.for 364.22,found 364.07 [M+H] + :m / z calcd.for 386.21,found 386.06

[0206] Comparative Example 3 The fluorescent substance Alexa Fluor 647 was fused to the N-terminus of the tripeptide (H-Ala-Nle-Phe-OMe) having a butyl group synthesized in Comparative Example 2.

[0207] To a 1.5 mL black tube, Alexa Fluor 647 (250 μg) dissolved in dry DMSO (15 μL), H-Ala-Nle-Phe-OMe (1.5 equivalents) dissolved in dry DMSO (15 μL), and DIPEA (1.5 equivalents) were added. The mixture was stirred overnight at room temperature. The mixture was purified by reverse-phase chromatography (acetonitrile / water / TFA = 30:70:0.1 to 95:5:0.1) and lyophilized to give the fluorescent conjugate 3 as a blue solid (74.5% yield calculated by fluorimetry, emission wavelength = 650 nm).

[0208] MALDI-TOF MS [M] - :m / z calcd.for C 55 H 72 N5O 17 S4 - 1202.3812,found 1202.1449

[0209] [Test Example 1] Peptide fluorescent conjugate 1 (Alexa-Ala-RFAA-Phe-OMe) synthesized in Example 6 and peptide fluorescent conjugate 3 (Alexa-Ala-Nle-Phe-OMe) synthesized in Comparative Example 3 were contacted with cultured cells to examine the efficiency of intracellular uptake. Furthermore, for comparison, peptide fluorescent conjugate 4 (Alexa-Ala-Ala-Phe-OMe), in which the fluorescent substance Alexa Fluoro 647 was fused to the N-terminus of the tripeptide (H-Ala-Ala-Phe-OMe) lacking a butyl group, was also used.

[0210] HeLa cells were seeded (0.5 × 10 ) onto cover glass chambers 24 h before peptide treatment. 5 cells / well). The cellular uptake assay was performed by replacing the medium (DMEM low-glucose medium containing 10% FBS and 1% penicillin-streptomycin solution) with 0.4% DMSO medium (without additives) containing peptide fluorescent conjugate 1 or 2 at a final concentration of 3.3 μM. After the medium replacement, the cells were incubated at 37°C for 1 hour and washed with cell culture medium and PBS (phosphate-buffered saline).

[0211] The cells were treated with TrypLE™ Express (Gibco) and then harvested. They were then analyzed by flow cytometry (guava easyCyte™ 8). Red 2 fluorescence (661 / 19 nm) was measured. The results are shown in Figure 1. The vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity of each cell. As shown in Figure 1, peptide fluorescent conjugate 1 (Alexa-Ala-RFAA-Phe-OMe) produced approximately twice as many cells emitting Alexa Fluor 647 fluorescence as peptide fluorescent conjugate 3. These results suggest that Alexa-Ala-RFAA-Phe-OMe, a peptide with a fluoroalkyl group, has higher cellular uptake efficiency and superior cell membrane permeability than peptides without a fluoroalkyl group.

[0212] [Test Example 2] The intracellular uptake efficiency of peptide fluorescent conjugate 2 (Alexa-Ala-RFAA(C8)-Phe-OMe) synthesized in Example 8 was investigated. For comparison, peptide fluorescent conjugate 1 (Alexa-Ala-RFAA-Phe-OMe), peptide fluorescent conjugate 3 (Alexa-Ala-Nle-Phe-OMe), and peptide fluorescent conjugate 4 (Alexa-Ala-Ala-Phe-OMe) were also used.

[0213] HeLa cells were seeded (1.0 × 10 ) in 12-well coverglass chambers 24 h before peptide treatment. 5 cells / well). The cellular uptake assay was performed in the same manner as in Test Example 1, except that peptide fluorescent conjugate 1, 2, 3, or 4 was added to the medium to a final concentration of 1.5 μM. Then, the cells were collected and analyzed by flow cytometry in the same manner as in Test Example 1.

[0214] The results are shown in Figure 2. The vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity of each cell. As shown in Figure 2, peptide fluorescent conjugate 2 (Alexa-Ala-RFAA(C8)-Phe-OMe) produced approximately 16 times more cells that emitted Alexa Fluor 647 fluorescence than peptide fluorescent conjugates 3 and 4. Furthermore, peptide fluorescent conjugate 2 produced approximately 6 times more cells that emitted Alexa Fluor 647 fluorescence than peptide fluorescent conjugate 1. These results suggest that Alexa-Ala-RFAA-Phe-OMe, a peptide with a fluoroalkyl group, has higher cellular uptake efficiency and superior cell membrane permeability than peptides without a fluoroalkyl group.

[0215] [Example 9] A tripeptide bearing a tridecafluorohexyl group (H-Ala-RFAA-Phe-OMe) was synthesized.

[0216] [ka]

[0217] Compound 1 (200 mg) dissolved in THF (1 mL) was added to LDA (2.2 equivalents) and kept at -78 °C in dry THF (1 mL) under argon for 30 minutes. Then, RFCH2CH2I (1.1 equivalents) was added to the mixture and stirred for 3 hours. The mixture was then slowly brought to -30 °C and stirred overnight. After that, the reaction was quenched by adding water (5 mL) to the mixture at 0 °C. The mixture was then extracted with CHCl2 (200 mL × 3). The product was columned on alumina to obtain a white solid (yield 54.1%).

[0218] 1 H NMR(400MHz CDCl3) δ=1.45(s,9H),2.78-2.66(m,2H),3.23-3.27(m,2H),4.03(t,1H),7.2-7.7(m,10H) 19 F NMR(376MHz CDCl3) δ=-126.2(m,2F),-123.4(m,2F),-122.9(m,2F),-121.9(m,2F),114.9(m,1F),114.2(m,1F),-80.8(t,3F)

[0219] A solution of compound 2 (50.8 mmol) in HCl (6 M, 50 mL) and 1,4-dioxane (200 mL) was heated at 80° C. for 24 h. The solution was filtered, and the precipitate was washed several times with acetone. The resulting white solid was sufficiently pure to be used in the next step without further purification (yield 50.2%).

[0220] 1 H NMR(400MHz MeOH-d4) δ=2.11(m,2H),2.35(m,1H),2.52(m,1H),3.68(m,1H) 19 F NMR(376MHz MeOH-d4) δ=-127.2(m,2F),-124.4(m,2F),-123.8(m,2F),-122.8(m,2F),-115.7(m,2F),-82.3(t,3F)

[0221] [ka]

[0222] A 50 mL two-necked round-bottom flask was equipped with a stir bar and charged with compound 3 (100 mg, 0.22 mmol) and DCM (10 mL). Fmoc-OSn (0.24 mmol) and DIPEA (1.3 equiv.) were added at room temperature, and the reaction mixture was then stirred at room temperature for 20 h. The reaction mixture was directly evaporated and purified by silica gel column chromatography (MeOH / CHCl3 = 1 / 9) to give a white solid (64.2% yield).

[0223] [ka]

[0224] A 25 mL two-necked round-bottom flask was charged with Fmoc-RFAA-OH (90.4 mg, 0.14 mmol), HOAt (1.2 equiv.), and DIPEA (1.3 equiv.). HATU (1.2 equiv.) and H-Phe-OMe (HCl salt, 1.2 equiv.) dissolved in DCM (5 mL) were added to the mixture at 0 °C, and the mixture was warmed to room temperature and stirred for 4 h. The reaction was then quenched with HCl (1 N), and the mixture was partitioned between HCl (1 N) and DCM. The combined organic phase was evaporated and then diluted with ethyl acetate. The organic phase was washed with HCl (1 N), saturated aqueous NaHCO3, and brine, dried over Na2SO4, and evaporated to give a white solid. 20% piperidine in DMF (25 mL) was added to the crude mixture and stirred at room temperature for 1 h. The solvent was removed by vacuum drying to give a white solid, which was purified by silica gel column chromatography (CHCl3:MeOH=10:1) to give H-RFAA-Phe-OMe (41.4 mg, yield 64.8%).

[0225] 1 H NMR(400MHz Acetone-d6) δ=1.96(m,2H),2.32(m,2H),2.81(br s,NH2),2.99-3.15(m,2H),3.15(s,3H),4.05(t,1H),4.71(m,1H)7.2-7.7(m,5H) 19 F NMR(376MHz Acetone-d6) δ=-126.7(m,2F),-123.8(m,2F),-123.4(m,2F),-122.4(m,2F),-114.6(m,2F),-81.4(t,3F)

[0226] [ka]

[0227] A 25 mL two-necked round-bottom flask was charged with Fmoc-RFAA-OH (44.8 μmol), HOAt (1.2 equiv.), and DIPEA (1.3 equiv.). HATU (1.2 equiv.) and Fmoc-AlaOH (1.2 equiv.) dissolved in DCM (5 mL) were added to the mixture at 0 °C, then the mixture was warmed to room temperature and stirred for 4 h. After quenching the reaction with HCl (1N), the mixture was partitioned between HCl (1N) and DCM. The combined organic phase was evaporated and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO3, and brine, dried over Na2SO4, and evaporated to give a white solid. 5 mL of 20% piperidine in DMF was added to the crude mixture and stirred at room temperature for 1 h. The solvent was removed under reduced pressure to give a white solid, which was purified by HPLC.

[0228] ESI-MS [M+H] + :m / z calcd.for 754.16,found 654.52

[0229] Comparative Example 4 A tripeptide bearing an octyl group (Boc-nOctyl-Phe-OMe) was synthesized.

[0230] [ka]

[0231] Boc-nOctyl-Phe-OMe was prepared as previously described (Liebigs Annalen der Chemie, 1990, 12p, pp. 1175-1183). The Boc deprotection of the dipeptide was carried out using the same standard procedure as above (yield: 100%). The tripeptide was synthesized according to the procedure described above.

[0232] ESI-MS [M+H] + :m / z calcd.for 420.29,found 420.72

[0233] [Example 10] A dipeptide bearing a heptadecafluorooctyl group (Boc-RFAA(C8)-Gly-OMe) was synthesized.

[0234] [ka]

[0235] The crude product of allyl 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-hexadecafluoro-2,2-dihydrodecanoate was obtained in a yield of 80.9% in the same manner as in Example 7, except that diallyl oxalate (3.4 g) was used instead of dibenzyl oxalate. The obtained product was used in the next step without purification.

[0236] 1 H NMR(400MHz Acetone-d6) δ=6.25-5.71(m,1H),5.65-5.03(m,2H),4.97-4.52(m,2H) 19 F NMR(400MHz Acetone-d6) δ=81.72(m,3F),-120.30(s,4F),-122.24(s,6F),-123.26(s,2F),-126.79(d,J=54.5Hz,2F)

[0237] [ka]

[0238] The crude product obtained was purified by sublimation at 64° C. and 2.2 mmHg in the same manner as in Example 7, to obtain allyl 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-hexadecafluoro-2-oxodecanoate in a yield of 60.3%.

[0239] 1 H NMR(400MHz,CDCl3) δ=6.08-5.79(m,1H),5.59-5.13(m,2H),4.89-4.80(m,2H) 19 F NMR(400MHz,CDCl3) δ=-81.03(t,J=10.0Hz,3F),-117.88(t,J=12.9Hz,2F),-121.25(m,4F),-121.96(m4F),-122.85(s,2F),-126.31(d,J=5.7Hz,2F)

[0240] [ka]

[0241] Next, the α-imino ester was obtained in a yield of 95.1% in the same manner as in Example 7, except that silica gel chromatography was performed using a mixture of hexane and ethyl acetate (9:1) to which 1% triethylamine was added as an eluent.

[0242] 1 H NMR(400MHz,CDCl3) δ=6.01-5.82(m,1H),5.49-5.30(m,2H),4.81(d,J=5.9Hz,2H),1.63-1.49(m,9H) 19 F NMR(400MHz,CDCl3) δ=-79.98~-82.00(m,3F),-112.58(m,2F),-120.10(s,2F),-121.11(s,2F),-121.80(m,4F),-122.71(s,2F),-126.38(m,2F)

[0243] [ka]

[0244] To a solution of the resulting imino ester (2.0 g, 3.2 mmol) in dry diethyl ether (30 mL) was added 2-picoline borane (1 equivalent) at 0°C with stirring. The reaction mixture was stirred at room temperature for 1.5 hours and then diluted with HCl (1N) (20 mL). The organic phase was washed twice with HCl (1N) and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (hexane:ethyl acetate=9:1) to give the α-butoxycarbonylamino ester as a yellow solid (54% yield).

[0245] 1 H NMR(400MHz,CDCl3) δ6.01-5.79(m,1H),5.51-4.97(m,4H),4.84-4.62(m,2H),1.57-1.35(m,9H) 19 F NMR(400MHz,CDCl3) δ-80.96(t,J=10.0Hz,3F),-115.81(d,J=280Hz,1F),-118.10(d,J=281Hz,1F),-120.88~-123.39(m,10F),-126.23(m,2F)

[0246] [ka]

[0247] To a solution of the resulting α-butoxycarbonylamino ester (1.2 g, 1.9 mmol) in THF (18 mL), phenylsilane (2 equivalents) and tetrakis(triphenylphosphine)palladium (5 mol%) were added at 0 °C and stirred at room temperature for 2 h. The reaction mixture was diluted with HCl (1 N) (10 mL) and extracted twice with DCM. The organic phase was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (chloroform:methanol = 6:1 / 1% acetic acid) to give 2-((tert-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid as a pale yellow liquid (74% yield).

[0248] 1H NMR(400MHz,CDCl3) δ=5.03(m,J=8.4Hz,1H),1.38(s,9H) 19 F NMR(400MHz,CDCl3) δ=-80.87(t,J=10.0Hz,3F),-115.78(d,J=281.1Hz,1F),-118.12(d,J=281.1Hz,1F),-120.16~-123.43(m,10F),-126.19(s,2F)

[0249] [ka]

[0250] In a dry 25 mL two-necked flask, 2-((tert-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (15.4 mg, 26.0 μmol), DCM (2 mL), DIPEA (57 μmol), glycine methyl ester hydrochloride (29 μmol), and ethyl (hydroxyimino)cyanoacetate(oxyma) (CAS RN: 3849-21-6) (29 μmol) were combined and stirred. The reaction mixture was cooled to 0°C, and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) (CAS RN: 1075198-30-9) (29 μmol) was added. The mixture was then allowed to warm to room temperature and stirred for 1.5 hours. The reaction mixture was then quenched with HCl (1N) and extracted three times with DCM. The combined organic phase was concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated aqueous sodium bicarbonate, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude Boc-RFAA(C8)-Gly-OMe. The crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1 / 3) to obtain Boc-RFAA(C8)-Gly-OMe (92% yield).

[0251] 1H NMR(400MHz,Acetone-d6) δ=8.25(t,J=5.3Hz,1H),6.66(d,J=9.6Hz,1H),5.44-5.19(m,1H),4.07(d,J=5.5Hz,2H),3.68(s,3H),1.42(s,9H) 19 F NMR(400MHz,Acetone-d6) δ=-81.53(s,3F),-115.31(d,J=281.1Hz,1F),-119.15~-120.92(m,1F),-120.92~-124.38(10F),-125.54~-127.82(m,2F)

[0252] [Example 11] A dipeptide bearing a heptadecafluorooctyl group (Boc-RFAA(C8)-Ala-OMe) was synthesized.

[0253] [ka]

[0254] Boc-RFAA(C8)-Ala-OMe (a 47:53 mixture of diastereomers) was obtained from 2-((tert-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (15 μmol) in the same manner as in Example 10, except that alanine methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride (74% yield).

[0255] 1 H NMR(400MHz,Acetone-d6) δ=8.22-8.27(d,J=7.1Hz,1H),6.82-6.43(m,1H),5.44-5.19(m,1H),4.64-4.40(m,1H),3.68(s,3H),1.42(s,9H),1.38-1.40(d,3H) 19F NMR(400MHz,Acetone-d6) δ=-80.91(t,J=10.0Hz,3F),-113.87~-115.77(m,1F),-119.22~-119.98(m,1F),- 120.67~-121.35(m,2F),-121.48(m,6F),-122.03~-122.81(m,2F),-126.00(m,2F)

[0256] [Example 12] A dipeptide bearing a heptadecafluorooctyl group (Boc-RFAA(C8)-Leu-OMe) was synthesized.

[0257] [ka]

[0258] Boc-RFAA(C8)-Leu-OMe (a 49:51 mixture of diastereomers) was obtained from 2-((tert-butoxycarbonyl)amino)-3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (15 μmol) in the same manner as in Example 10, except that leucine methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride and silica gel chromatography was not performed (yield 83%).

[0259] 1 H NMR(400MHz,Acetone-d6) δ=8.19(d,J=7.5Hz,1H),6.64(d,J=10.1Hz,1H),5.45-5.13(m,1H),4.68-4 .47(m,1H),3.68(s,3H),1.81-1.66(m,1H),1.42(s,9H),0.96-0.88(m,6H) 19 F NMR(400MHz,Acetone-d6) δ=-81.56(t,J=10.0Hz,3F),-115.49(m,J=272.51F),-119.40~-120.88(m,1F),-121.33~-123.04(m,10F),-126.63(m,2F)

[0260] [Example 13] A dipeptide bearing a heptadecafluorooctyl group (Boc-RFAA(C8)-Lys(Boc)-OMe) was synthesized.

[0261] [ka]

[0262] Boc-RFAA(C8)-Lys(Boc)-OMe was obtained (yield 69%) in the same manner as in Example 10, except that lysine (Boc) methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride.

[0263] 1 H NMR(400MHz,CDCl3)δ 7.71(d,J=3.7Hz,1H),7.51-7.54(m,1H),6.94(d,J=8.2Hz,1H),5.67(d,J=10.1Hz ,1H),5.13(s,1H),4.57-4.62(m,2H),3.75(s,3H),1.45(s,18H),1.10-1.90(m,6H) 19 F NMR(400MHz,CDCl3)δ -80.63(s,3F),-114.56(d,J=281.1Hz,1F),-119.10(d,J=284.0Hz,1F),-122.61―-120.86(m,10F),-126.02(s,2F)

[0264] LRMS (ESI-TOF) [M+Na] + :calcd.for C 27 H 34 F 17 N3NaO7858.20,found 858.03

[0265] Comparative Example 5 Ac-L-Ala-L-Ala-NHBn was synthesized.

[0266] [ka]

[0267] Compound 220 (61 mg, 0.32 mmol) and compound 222 (68 mg) were dissolved in 3 mL of methanol, and DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride)·3.2H₂O (135 mg) was added to the solution. The reaction mixture was stirred at room temperature for 12 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the solution was washed with 1 M aqueous Na₂CO₃, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo to give compound 240 (92 mg, 83% yield).

[0268] Compound 240 (92 mg, 0.26 mmol) was dissolved in 4 mL of DCM and 1 mL of THF. 1.25 mL of TFA was added to the solution at 0° C., and the mixture was stirred for 15 minutes. The reaction mixture was then warmed to room temperature and stirred for 3 hours. 1 M aqueous NaHCO3 was added to the reaction mixture, and the resulting solution was stirred for 2 hours. The mixture was extracted four times with DCM. The organic phase was dried over Na2SO4 and evaporated in vacuo to give compound 241.

[0269] To compound 241 (73 mg, 0.29 mmol) in 3 mL of DCM was added acetic anhydride (33 μL). The reaction mixture was stirred at room temperature for 24 hours and then evaporated in vacuo. The residue was dissolved in 10 mL of 40% aqueous acetonitrile and purified using a reverse-phase HPLC column to give compound 242 (9 mg, 11% yield).

[0270] 1 H NMR (CD3OD, 400MHz): δ7.33-7.20(m,5H),4.42-4.33(m,3H),4.29(q,J=6.9Hz,1H),1.96(s,3H),1.38(d,J=6.87Hz,3H),1.33(d,J=7.3Hz,3H).

[0271] MS (MALDI-TOF MS.m / z) [M+Na] +:calcd.for C 15 H 21 N3O3Na 314.15,found 313.88.

[0272] [Example 14] Ac-D,L-Ala(F3)-L-Ala-NHBn was synthesized.

[0273] [ka]

[0274] D,L-Trifluoroalanine hydrochloride (compound 243) (52 mg, 0.28 mmol) was dissolved in 3 mL of acetonitrile. DIPEA (57 μL) and di-tert-butyl dicarbonate (77 μL) were added to the solution at 0° C. The reaction mixture was allowed to warm to room temperature over 21 hours. The solution was evaporated in vacuo, and water was added to the residue. The solution was extracted three times with diethyl ether. 1 M aqueous HCl was added to the aqueous phase, and the solution was extracted three times with diethyl ether. The combined organic phases were dried over Na2SO4 and evaporated in vacuo to give compound 244 as a white solid (62 mg, 91% yield).

[0275] Compound 244 (40 mg, 0.16 mmol) and compound 222 (29 mg) were dissolved in 1.5 mL of methanol, and DMTMM·3.2H₂O (62 mg) was added to the solution. The reaction mixture was stirred at room temperature for 11.5 h and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na₂CO₃, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo to give compound 245 (45 mg, 68% yield).

[0276] Compound 245 (45 mg, 0.11 mmol) was dissolved in 4 mL of DCM and 1 mL of THF. 1.25 mL of TFA was added to the solution at 0° C., and the mixture was stirred for 15 minutes. The reaction mixture was then warmed to room temperature and stirred for 3 hours. 1 M aqueous NaHCO3 was added to the reaction mixture, and the resulting solution was stirred for 2 hours. The mixture was extracted four times with DCM. The organic phase was dried over Na2SO4 and evaporated in vacuo to give compound 246 (41 mg, quantitative yield).

[0277] To compound 246 (41 mg, 0.14 mmol) in 3 mL of DCM was added acetic anhydride (67 μL). The reaction mixture was stirred at room temperature for 24 hours and then evaporated in vacuo. The residue was dissolved in 10 mL of 46% aqueous acetonitrile and purified using a reverse-phase HPLC column to give compound 247 as a white solid (0.3 mg, 1% yield). The molecule was obtained as a diastereomeric mixture and used directly in the permeability assay.

[0278] 1 H NMR (CD3OD, 400MHz): δ7.33-7.22(m,5H),5.37-5.29(m,1H),4.45-4.38(m,3H),2.66(s,3H),1.40-1.38(m,3H).

[0279] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 15 H 18 F3N3O3Na 368.12,found 367.92.

[0280] Comparative Example 6 Ac-L-Ala-L-Phe-iBu was synthesized.

[0281] [ka]

[0282] Compound 201 (700 mg, 2.34 mmol) and isobutylamine (181 μL) were dissolved in 23 mL of methanol, and DMTMM·1.3H₂O (838 mg) was added to the solution. The reaction mixture was stirred at room temperature for 5 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na₂CO₃ solution, water, 1 M aqueous HCl solution, water, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 4:6) to give compound 248 (523 mg, 65% yield).

[0283] A collection flask was charged with compound 248 (523 mg, 1.48 mmol), palladium on carbon 10% (55 mg), and 7.4 mL of methanol. H2 was introduced into the flask, and the mixture was stirred at room temperature for 15 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 249 (319 mg, 98% yield).

[0284] Compound 205 (45 mg, 0.2 mmol) and compound 249 (53 mg) were dissolved in 2 mL of methanol, and the solution was stirred at room temperature. To the solution was added DMTMM·1.3H2O (74 mg). The reaction mixture was stirred at room temperature for 23 h and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na2CO3, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na2SO4 and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19:1) to give compound 250 (9.1 mg, 11% yield).

[0285] To a collection flask was added compound 250 (9.1 mg, 21 μmol), 10% palladium on carbon (2.8 mg), and 2 mL of methanol. H2 was introduced into the flask, and the mixture was stirred at room temperature for 22 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 251 (7.4 mg, quantitative yield).

[0286] To compound 251 (4 mg, 14 μmol) in 1 mL DCM and 0.2 mL N-methylpyrrolidone (NMP) was added acetic anhydride (23 μL). The reaction mixture was stirred at room temperature for 1.5 hours and then evaporated in vacuo. The residue was dissolved in 3.5 mL of 14% aqueous acetonitrile and purified using a reverse-phase HPLC column to give compound 252 (2.0 mg, 43% yield).

[0287] 1 H NMR(CDCl3,400MHz):δ7.32-7.19(m,10H),6.62(d,J=7.7Hz,1H),5.90-5.86(m,2H),4.57(q,J=7.7Hz,1H),4.39(dq,J=6.9,7.1Hz, 1H),3.14(dd,J=6.4,13.9Hz,1H),3.06-2.98(m,3H),1.94(s,3H),1.70-1.62(m,1H),1.33(d,J=7.1Hz,3H),0.79(t,J=6.3Hz,6H).

[0288] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 18 H 27 N3O3Na 356.20,found 356.23.

[0289] [Example 15] Ac-D,L-Ala(F3)-L-Phe-iBu was synthesized.

[0290] [ka]

[0291] Compound 244 (41 mg, 0.17 mmol) and compound 249 (45 mg) were dissolved in 1 mL of methanol and 0.7 mL of DCM, and DMTMM·1.3H₂O (59 mg) was added to the solution. The reaction mixture was stirred at room temperature overnight and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous NaHCO₃ solution, saturated aqueous NH₄Cl solution, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19:1) to give compound 253 (47 mg, 63% yield).

[0292] To compound 253 (47 mg, 0.11 mmol) in 3 mL of ethyl acetate was added 4 M HCl in ethyl acetate (3 mL), and the solution was stirred at room temperature for 20 minutes. The solution was evaporated in vacuo to give compound 254 (45 mg, quantitative yield).

[0293] To compound 254 (30 mg, 79 μmol) in 2 mL of DCM was added acetic anhydride (45 μL). The reaction mixture was stirred at room temperature for 3 hours and then evaporated in vacuo. The residue was dissolved in acetonitrile / HO / MeOH (=2.4 mL:3.6 mL:2 mL) and purified using a reverse-phase HPLC column to give compound 255 (9.4 mg, 30% yield). The molecule was obtained as a diastereomeric mixture and used directly in the permeability assay.

[0294] 1 H NMR(CDCl3,400MHz):δ7.34-7.21(m,5H),6.82-6.76(m,1H),6.41(d,J=6.4Hz,1H),5.3(s,1H),5.17(q,J=7.3Hz,1H) ,4.59-4.53(m,1H),3.21-3.12(m,1H),3.01-2.92(m,3H),1.42(dd,J=6.9,12.4Hz,1H),0.76(dd,J=6.4,10.5Hz,6H).

[0295] MS (MALDI-TOF MS.m / z) [M+Na] +:calcd.for C 18 H 24 F3N3O3Na 410.17,found 410.19.

[0296] Comparative Example 7 Ac-L-Val-L-Ala-NMe2 was synthesized.

[0297] [ka]

[0298] N-Carbobenzoxy-L-valine (compound 256) (60 mg, 0.24 mmol) and compound 213 (33 mg) were dissolved in 2.2 mL of methanol, and DMTMM·1.3H₂O (91 mg) was added to the solution. The reaction mixture was stirred overnight at room temperature and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na₂CO₃, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo to give compound 257 (75 mg, 89% yield).

[0299] To a collection flask was added compound 257 (773 mg, 3.10 mmol), palladium on carbon 10% (7.5 mg), and 2.1 mL of methanol. H2 was introduced into the flask, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 258 (35 mg, 76% yield).

[0300] To compound 258 (35 mg, 0.16 mmol) in 0.8 mL of DCM was added acetic anhydride (18 μL). The reaction mixture was stirred at room temperature for 20 hours and then evaporated in vacuo. The residue was dissolved in 4 mL of 10% aqueous acetonitrile and purified using a reverse-phase HPLC column to give compound 259 as a white solid (27 mg, 65% yield).

[0301] 1H NMR(CDCl3,400MHz):δ7.10(d,J=7.3Hz,1H),6.41(d,J=8.7Hz,1H),4.87(quin,J=6.9,1H),4.37(dd,J=6.1,2.6Hz,1 H),3.09(s,3H),2.99(s,3H),2.10-2.02(m,4H),1.34(d,J=6.9Hz,3H),0.94(d,J=6.0Hz,3H),0.92(d,J=5.5Hz,3H).

[0302] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 12 H 23 N3O3Na 280.16,found 280.05.

[0303] [Example 16] Ac-D,L-Val(F6)-L-Ala-NMe2 was synthesized.

[0304] [ka]

[0305] D,L-Hexafluorovaline (compound 260) (50 mg, 0.22 mmol) was dissolved in 2.5 mL of acetonitrile. Di-tert-butyl dicarbonate (49 μL) was added to this solution at 0° C. The reaction mixture was allowed to warm to room temperature over 11.5 hours. DIPEA (38 μL) was added to the solution, and the reaction mixture was stirred at room temperature for 6.5 hours. The solution was evaporated in vacuo, and water was added to the residue. The solution was extracted with diethyl ether. 1 M aqueous HCl was added to the aqueous phase, and the resulting solution was extracted three times with diethyl ether. The combined organic phases were dried over Na2SO4 and evaporated in vacuo to give compound 261 (63 mg, 87% yield).

[0306] Compound 261 (50 mg, 0.15 mmol) and compound 213 (21 mg) were dissolved in 0.7 mL of methanol, and DMTMM·1.3H₂O (56 mg) was added to the solution. The reaction mixture was stirred at room temperature for 17 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na₂CO₃, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo to give compound 262 (50 mg, 77% yield).

[0307] Compound 262 (50 mg, 0.12 mmol) was added with 4 M HCl (2.5 mL) in ethyl acetate, and the solution was stirred at room temperature for 2 hours. Ethyl acetate was added to the reaction mixture, and the resulting solution was extracted twice with 1 M aqueous HCl. 1 M aqueous NaOH was added to the aqueous phase until the pH reached 11. The solution was extracted three times with DCM, and the organic phase was dried over Na2SO4. The solvent was removed under reduced pressure to give compound 263 (38 mg, quantitative yield).

[0308] To compound 263 (38 mg, 0.12 mmol) in DCM (1.2 mL) was added acetic anhydride (13 μL). The reaction mixture was stirred at room temperature for 13 hours and then evaporated in vacuo. The residue was dissolved in 8 mL of 30% aqueous acetonitrile and purified using a reverse-phase HPLC column to give compound 264 as a white solid (27 mg, 63% yield). The molecule was obtained as a diastereomeric mixture and used directly in the permeability assay.

[0309] 1 H NMR (CDCl3,400MHz): δ7.57(d,J=6.4Hz,0.5H),7.39(d,J=6.9Hz,0.5H),6.22(d,J=9.6Hz,0.5H),6.08(d,J=9.6Hz,0.5H),5.41(t,J=9.2 Hz,1H),4.86-4.75(m,1H),4.35-4.22(m,1H),3.07(d,J=5.0Hz,3H),2.98(d,J=3.2Hz,3H),2.14(d,J=2.14Hz,3H),1.32(t,J=7.3Hz,3H).

[0310] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 12 H 17 F6N3O3Na 388.11,found 388.18.

[0311] [Comparative Example 8] Ac-L-Val-L-Phe-iBu was synthesized.

[0312] [ka]

[0313] Compound 256 (52 mg, 0.2 mmol) and compound 249 (53 mg) were dissolved in 2 mL of methanol, and the solution was stirred at room temperature. To the solution was added DMTMM·1.3H2O (72 mg). The reaction mixture was stirred at room temperature for 23 h and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with 1 M aqueous Na2CO3, water, 1 M aqueous HCl, water, and brine. The organic phase was dried over Na2SO4 and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19:1) to give compound 265 (60 mg, 67% yield).

[0314] To a collection flask was added compound 265 (60 mg, 0.13 mmol), palladium on carbon 10% (6 mg), and 1.3 mL of methanol. H2 was introduced into the flask, and the mixture was stirred at room temperature for 22 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 266 (39 mg, 92% yield).

[0315] Compound 266 (15 mg, 47 μmol) was dissolved in 1 mL DCM, 0.4 mL NMP, and 0.2 mL THF. Acetic anhydride (23 μL) was added to the solution. The reaction mixture was stirred at room temperature for 1.5 hours and then evaporated in vacuo. The residue was dissolved in acetonitrile / HO / MeOH (=1.8 mL:2.9 mL:1.5 mL) and purified using a reverse-phase HPLC column to give compound 267 (7.2 mg, 43% yield).

[0316] 1 H NMR (CDCl3,400MHz): δ7.30-7.18(m,5H),6.51(d,J=7.3Hz,1H),5.92(d,J=7. 8Hz,1H),5.75(s,1H),4.58(dt,J=6.0,8.2Hz,1H),4.20(dd,J=6.0,8.2Hz,1H ),3.11(dd,J=6.0,13.7Hz,1H),3.02-2.92(m,3H),2.10-2.03(m,1H),1.97(s ,3H),1.66-1.59(m,1H),0.88(dd,J=6.9,11.5Hz,6H),0.76(t,J=7.3Hz,6H).

[0317] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 20 H 31 N3O3Na 384.23,found 384.13.

[0318] [Example 17] Ac-D,L-Val(F6)-L-Phe-iBu was synthesized.

[0319] [ka]

[0320] Compound 261 (70 mg, 0.22 mmol) and compound 249 (57 mg) were dissolved in 1.2 mL of methanol, and DMTMM·1.3H₂O (83 mg) was added to the solution. The reaction mixture was stirred at room temperature for 5 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was washed with saturated aqueous NaHCO₃, 1 M aqueous HCl, and brine. The organic phase was dried over Na₂SO₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19:1) to give compound 268 (79 mg, 69% yield).

[0321] To compound 268 in 3 mL of ethyl acetate was added 4 M HCl in ethyl acetate (3 mL). The solution was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure to give compound 269 (53 mg, 76% yield).

[0322] To compound 269 (40 mg, 86 μmol) in 2 mL of DCM was added DIPEA (16 μL) and acetic anhydride (45 μL). The reaction mixture was stirred at room temperature for 8.5 hours and then evaporated in vacuo. The residue was dissolved in acetonitrile / HO / MeOH (=3 mL:3 mL:8 mL) and purified using a reverse-phase HPLC column to give compound 270 as a white solid (5.7 mg, 13% yield). The molecule was obtained as a diastereomeric mixture and used directly in the permeability assay.

[0323] 1 H NMR(CD3OD3,400MHz):δ7.29-7.17(m,5H),5.29(dq,J=7.8,23.8Hz,1H),4.63-4.58(m,1H),3.1 5-3.05(m,1H),3.01-2.86(m,3H),2.01(d,J=4.6hz,3H),1.72-1.62(m,1H),0.82-0.78(m,6H).

[0324] MS (MALDI-TOF MS.m / z) [M+Na] + :calcd.for C 20 H 25F6N3O3Na 492.17, found 491.97.

[0325] [Test Example 3] For the peptides synthesized in Examples 15 to 17 and Comparative Examples 6 to 8, a PAMPA assay was performed. Also, for the peptides synthesized in Examples 14, 15, 17 and Comparative Examples 5, 6, 8, an MDCK-II assay was performed. In the MDCK-II assay, Propranolol (CAS No: 318-98-9) was used as a positive control ("PC") and Norfloxacin (CAS No: 70458-96-7) was used as a negative control ("NC").

[0326] <PAMPA (Parallel Artificial Membrane Permeability Assay) Assay> The permeability of the peptide was measured by PAMPA. In the PAMPA assay, 300 μL of PBS containing 5% DMSO was added to each well of an acceptor plate (MultiScreen 96-well transport receiver plate, manufactured by Merck). Next, 150 μL of a peptide solution (20 μM) dissolved in 5% DMSO / PBS was added to each well of a donor plate (MultiScreen-IP filter plate, 0.45 μm, manufactured by Merck). A dodecane solution of 1% lecithin (soybean-derived) was sonicated for 30 minutes before use, and 5 μL of the solution was applied to the membrane support (PVDF) of each well of the donor plate. The donor plate was placed on the acceptor plate, and the plate was left in an incubator at 25 °C for 18 hours. The concentration of the peptide was determined using LC / MS. The experiment was repeated 3 times. The permeability value (P e ) was calculated using the following formula.

[0327] [Equation]

[0328] A: Filter area (0.3 cm 2 ) VD: Volume of donor well (0.15 cm3 ) VA: volume of acceptor well (0.3 cm 3 ) t: incubation time (s) (18 hours = 64800 s) CD(t): Compound concentration in the donor well at time t CA(t): Compound concentration in the acceptor well at time t

[0329] <MDCK-IIアッセイ> 5.04 × 10 MDCK-II cells were cultured on cell culture inserts (Falcon). 4 Cell monolayer assays (MDCK-II assays) were performed 5 days after seeding at 1000 cells / mL. Peptide stock solutions were prepared at 2 mM in DMSO and diluted with HBSS containing 20 mM HEPES, pH 7.5, to prepare 2 μM peptide solutions in 0.1% DMSO / HBSS(+) as the donor solution. The acceptor solution was a 0.1% DMSO solution in HBSS(+) containing 20 mM HEPES, pH 7.5. Apparent permeability (P app ) was determined by incubation with peptide solution from apical to basolateral direction for 2 hours at 37°C, 5% CO2. Peptide concentrations were analyzed by LC / MS. Experiments were performed in triplicate. Permeability values ​​(P app ) was calculated using the following formula:

[0330]

number

[0331] A: Filter area (0.3 cm 2 ) VB: basolateral well volume (0.75 cm 3 ) t: incubation time (s) (2 hours = 7200 s) C0: initial concentration in the apical chamber (2 μM) CB(t): basolateral compound concentration at time t

[0332] The results of the PAMPA assay are shown in Table 3, and the results of the MDCK-II assay are shown in Table 4. As a result, the fluorine-containing peptides of Examples 14 to 17, into which fluorine atoms had been introduced in the side chains of the peptides of Comparative Examples 5 to 8, had improved cell membrane permeability compared to the peptides of Comparative Examples 5 to 8.

[0333] [Table 3]

[0334] [Table 4] [Industrial Applicability]

[0335] The present invention provides a peptide having an amino acid residue bearing a fluoroalkyl group in the side chain, and a method for producing the same. The peptide according to the present invention has excellent cell membrane permeability, and is therefore expected to be used in the pharmaceutical field as a physiologically active substance, for example, as a carrier for introducing a medicinal component into target cells.

Claims

1. The following general formula (4) 【Chemical 1】 (wherein Rf represents a C group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom). １－３０ Alkyl group (the C １－３０ The alkyl group is C ２－３０ When the alkyl group is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms. R ２ is a protecting group for an amino group, R １ is represented by the following general formula (p-1): 【Chemistry 2】 (In the formula, R ３ is an optionally substituted C ６－１４ is an aryl group, and R ４ and R ５ are each independently a hydrogen atom or an optionally substituted C ６－１４ (The black circle represents a bond.) a protecting group selected from a group represented by the formula (I), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group) After subjecting the compound represented by R ２ By deprotecting the compound represented by the following general formula (6-1), 【Chemistry 3】 (Wherein Rf and R １ is the same as above) and further condensing the compound represented by general formula (6-1) with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus.

2. The following general formula (4) 【Chemistry 4】 (wherein Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom (when the C 1-30 alkyl group is a C 2-30 alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms), R 2 is an amino-protecting group; R 1 is a group represented by the following general formula (p-1): 【Chemistry 5】 (In the formula, R 3 is an optionally substituted C 6-14 aryl group, R 4 and R 5 are each independently a hydrogen atom or an optionally substituted C 6-14 aryl group, and the black circle represents a bond.) a protecting group selected from a group represented by the formula (I), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group) is subjected to a reduction reaction, and then R 2 is deprotected to obtain a compound represented by the following general formula (6-1): 【Chemistry 6】 (wherein Rf and R 1 are the same as defined above) and further synthesizing a compound represented by the general formula (6-1) by the reaction of １ is deprotected to obtain a compound represented by the following general formula (7): 【Chemistry 7】 (Rf is the same as above) and further synthesizing a compound represented by the general formula (7): After protecting the amino group with a protecting group, the resulting product is condensed with a fluorine-containing amino acid having a protected carboxyl group, an amino acid having a protected carboxyl group, a fluorine-containing peptide having a protected C-terminus, or a peptide having a protected C-terminus, or After protecting the carboxy group with a protecting group, the compound is condensed with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus; A method for producing a fluoroalkyl group-containing peptide.

3. The following general formula (4) 【Chemistry 8】 (wherein Rf represents a C group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom). １－３０ Alkyl group (the C １－３０ The alkyl group is C ２－３０ When the alkyl group is an alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms. R ２ is a protecting group for an amino group, R １ is represented by the following general formula (p-1): 【Chemistry 9】 (In the formula, R ３ is an optionally substituted C ６－１４ is an aryl group, and R ４ and R ５ are each independently a hydrogen atom or an optionally substituted C ６－１４ (The black circle represents a bond.) a protecting group selected from a group represented by the formula (I), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group) After subjecting the compound represented by R ２ By deprotecting the compound, a compound represented by the following general formula (6-3) can be obtained. 【Chemistry 10】 (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf and R １ is the same as above) and further condensing the compound represented by general formula (6-3) with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus.

4. The following general formula (4) 【Chemistry 11】 (wherein Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms and optionally further substituted with a halogen atom other than a fluorine atom (when the C 1-30 alkyl group is a C 2-30 alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between the carbon atoms), R 2 is an amino-protecting group; R 1 is a group represented by the following general formula (p-1): 【Chemistry 12】 (In the formula, R 3 is an optionally substituted C 6-14 aryl group, R 4 and R 5 are each independently a hydrogen atom or an optionally substituted C 6-14 aryl group, and the black circle represents a bond.) a protecting group selected from a group represented by the formula (I), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group) is subjected to a reduction reaction, and then R 2 is deprotected to obtain a compound represented by the following general formula (6-3): 【Chemistry 13】 (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf and R 1 are the same as above. and synthesizing a compound represented by the general formula (6-3) by the reaction of １ By deprotecting the compound, a compound represented by the following general formula (7-1) can be obtained. 【Chemistry 14】 (wherein the asterisk represents that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R; Rf is the same as above. and further synthesizing a compound represented by the general formula (7-1): After protecting the amino group with a protecting group, the resulting product is condensed with a fluorine-containing amino acid having a protected carboxyl group, an amino acid having a protected carboxyl group, a fluorine-containing peptide having a protected C-terminus, or a peptide having a protected C-terminus, or After protecting the carboxy group with a protecting group, the compound is condensed with a fluorine-containing amino acid having a protected amino group, an amino acid having a protected amino group, a fluorine-containing peptide having a protected N-terminus, or a peptide having a protected N-terminus; A method for producing a fluoroalkyl group-containing peptide.

5. The method for producing a fluoroalkyl group-containing peptide according to any one of claims 1 to 4, further comprising deprotecting the protecting group of the amino group or carboxyl group of the produced fluoroalkyl group-containing peptide.

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