WO2021177336A1 - Peptide and cell membrane permeation agent - Google Patents

Abstract

The present invention provides a peptide including an amino acid residue having a fluorine atom introduced in the side chain thereof. A peptide according to the present invention includes two or more amino acids forming a peptide bond. At least one amino acid residue forming the peptide has, in a side chain thereof, a group represented by any one of general formulae (g-1)-(g-3) [in the formulae, R¹ and R² each independently represent a hydrogen atom, a halogen atom, a carboxy group, a C_6-14 aryl group, a C_5-14 heteroaryl group, or an amino group, R³-R⁶ each independently represent a hydrogen atom, a halogen atom, a C_1-30 alkyl group, a C_6-14 aryl group, or a C_5-14 heteroaryl group, R¹, R², R³, and R⁴ are optionally linked to form a C_6-14 aryl group or a C_5-14 heteroaryl group, and each black circle represent a bond].

Description

Peptides and cell membrane penetrants

The present invention relates to peptides containing amino acid residues in which a fluorine atom has been introduced into the side chain.
The present application claims priority based on Japanese Patent Application No. 2020-037194 filed in Japan on March 4, 2020 and Japanese Patent Application No. 2020-185438 filed in Japan on November 5, 2020. The contents are used here.

Antibody drugs, peptide drugs, nucleic acid drugs, etc. have the advantages of high specificity for target molecules and few side effects. However, all of them have a problem that it is difficult to reach the target molecule existing in the cell. Various methods are being studied to solve this problem. Among them, cell-penetrating peptides (CPP) are promising. Typical examples of the CPP include a peptide derived from the TAT protein of the HIV virus (Patent Document 1) and a peptide having a polyArg sequence (Patent Document 2). This can be combined with a medicinal peptide to transport the medicinal peptide into cells (for example, Patent Document 3 and Non-Patent Document 1).

On the other hand, fluorine-containing amino acids have been reported to exhibit unique bioactivity and are attracting attention. For example, 3,3,3-trifluoroalanine and its derivatives have been reported to act as a suicide inhibitor of pyridoxal enzyme (Non-Patent Document 2). In addition, it has been reported that alanine racemase of Gram-negative bacteria Salmonella typhimurium and Gram-positive bacteria 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.

It is known that a compound having a polyfluoro structure is stable and low in toxicity in vivo, and is excellent in intracellular uptake and escape from endosomes (Non-Patent Document 4). It has been reported that a peptide dendrimer using lysine in which a side chain amino group is perfluoroacylated can be used for gene delivery by utilizing this property (Non-Patent Document 5). However, since it is a dendrimer, it cannot form a hybrid that is bound to a medicinal active peptide, nucleic acid, or protein that is an antibody drug like CPP.

U.S. Pat. No. 6,316,003 U.S. Pat. No. 6,306,993 International Publication No. 2008/089491

The CPP described in Patent Document 1 and the like has various problems such as intracellular transport efficiency and decomposition by peptidase in vivo.
An object of the present invention is to provide a peptide containing an amino acid residue in which a fluorine atom is introduced into a side chain.

The present inventors have prepared a peptide containing an amino acid residue in which a fluorine atom is introduced into a side chain, and a partial structure containing the fluorine atom in the side chain constitutes a hydrogen bond in the molecule together with -NH in the peptide bond. Upon production, the peptide was found to have excellent cell membrane permeability, and the present invention was completed.

That is, the present invention is as follows.
[1] A peptide in which two or more amino acids are peptide-bonded.
At least one of the amino acid residues constituting the peptide is placed in the side chain in the following general formulas (g-1) to (g-3).

[In the formula, R ¹ and R ² may have a hydrogen atom, a halogen atom, a carboxy group, a C _6-14 aryl group, and a substituent, which may have a substituent, independently of each other. It is a C _5-14 heteroaryl group or an amino group which may have a substituent; R ³ to R ⁶ may have a hydrogen atom, a halogen atom and a substituent independently of each other. It has a C _1-30 alkyl group (the C _1-30 alkyl group may have an ether-bonding oxygen atom between carbon atoms when the number of carbon atoms is 2 or more), and a substituent. _May be a C 6-14 aryl group, or a C _5-14 heteroaryl group which ^{may have a substituent; R 1} , R ² , R ³ and R ⁴ are linked to each other to form a substituent. _{It may constitute a C 6-14} aryl group which may have, or a C _5-14 heteroaryl group which may have a substituent; black circles mean binders].
A peptide having a group represented by any of.
[2] At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-1) in the side chain.
In the general formula (g-1), the peptide of the above [1] ^{, wherein the R 1} is a hydrogen atom and the R ² _{is a C 6-14} aryl group which may have a substituent.
[3] At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-1) in the side chain.
In the general formula (g-1), ^{the peptide of the above [1], wherein R 1} and R ² are independently hydrogen atoms or fluorine atoms, respectively.
[4] At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-2) in the side chain.
In the general formula (g-2), the peptide of the above [1] ^{, wherein R 1} and R ² are independently hydrogen atoms or fluorine atoms, and R ³ and R ^{4 are hydrogen atoms, respectively.}
[5] At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-2) in the side chain.
In the general formula (g-2), R ¹ and R ² are independently hydrogen atoms or fluorine atoms, R ³ is a hydrogen atom, and R ⁴ is a trifluoromethyl group. The peptide of the above [1].
[6] At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-3) in the side chain.
In the general formula (g-3), the R ¹ , R ² , R ³ and R ⁴ are linked to each other and have a C _6-14 aryl group or a substituent which may have a substituent. _{It constitutes a C 5-14} heteroaryl group which may be present.
The peptide of [1] above, wherein R ⁵ and R ^{6 are hydrogen atoms.}
[7] The peptide according to any one of [1] to [6] above, wherein the C-terminal or N-terminal may be protected by a protecting group.
[8] The following general formula (p2-1) or general formula (p3-1)

[R ¹¹ and R ¹² are independently C _1-6 alkyl groups or C _6-14 aryl-C _1-6 alkyl groups; Z ¹ is a hydroxy group, C _1-6 alkyl groups, C. _1-6 Alkoxy group, or carboxyl group protective group; Z ² and Z ³ are independent hydrogen atoms, C _1-6 alkyl groups, C _6-14 aryl-C _1-6 alkyl groups, respectively. Alternatively, it is a protective group for an amino group; Rg is a general formula (g-1) to (g-3) below.

(In the formula, R ¹ and R ² may have a hydrogen atom, a halogen atom, a carboxy group, a substituent, and a C _6-14 aryl group, which may have a substituent, independently of each other. It is a C _5-14 heteroaryl group or an amino group which may have a substituent; R ³ to R ⁶ may have a hydrogen atom, a halogen atom and a substituent independently of each other. It has a C _1-30 alkyl group (the C _1-30 alkyl group may have an ether-bonding oxygen atom between carbon atoms when the number of carbon atoms is 2 or more), and a substituent. _May be a C 6-14 aryl group, or a C _5-14 heteroaryl group which ^{may have a substituent; R 1} , R ² , R ³ and R ⁴ are linked to each other to form a substituent. _{It may constitute a C 6-14} aryl group which may have a C 6-14 aryl group or a C _5-14 heteroaryl group which may have a substituent; a black circle means a bonder).
It is a group represented by any of]
The peptide according to any one of the above [1] to [7], which is represented by.
[9] The peptide according to any one of the above [1] to [6], which is a cyclic peptide.
[10] The peptide of [9], wherein the number of amino acid residues constituting the cyclic structure of the cyclic peptide is 4 to 31 residues.
[11] The peptide according to any one of the above [1] to [10], which is cell membrane permeable.
[12] A cell membrane penetrant containing the peptide according to any one of [1] to [10] as an active ingredient.

The peptide according to the present invention has excellent cell membrane permeability because a fluorine atom is introduced into the side chain. Therefore, the peptide is expected to be used in the pharmaceutical field as a physiologically active substance.

In the present invention and the present specification, the "fluorine-containing amino acid" means an amino acid containing at least one fluorine atom in the side chain. The "fluorine-containing peptide" means a peptide containing an amino acid containing at least one fluorine atom in the side chain.

In the present invention and the present specification, the "cyclic peptide" means a peptide having a cyclic structure formed by 4 or more amino acid residues. The cyclic structure is not limited to the bond between the N-terminal amino acid residue and the C-terminal amino acid residue of the linear peptide, and the bond between the terminal amino acid residue and the amino acid residue other than the terminal, or the amino acid other than the terminal. It may be formed by binding the residues together.

In the present invention and the present specification, "C _p1-p2 " (p1 and p2 are positive integers satisfying p1 <p2) means that the group has p1 to p2 carbon atoms.

In the present invention and the present specification, the "C _1-10 alkyl group" is an alkyl group having 1 to 10 carbon atoms, and may be a straight chain or a branched chain. The "C _2-10 alkyl group" is an alkyl group having 2 to 10 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-10 alkyl groups 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- Examples thereof include a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group and the like.

In the present invention and the present specification, the "C _1-30 alkyl group" is an alkyl group having 1 to 30 carbon atoms, and may be a straight chain or a branched chain. The "C _2-30 alkyl group" is an alkyl group having 2 to 30 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-30 alkyl groups 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, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecil group, eicosyl group, heneicosyl group. , Docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group and the like.

In the present invention and the present specification, the "C _1-6 alkyl group" is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-6 alkyl groups 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- Examples include a pentyl group and a hexyl group.

In the present invention and the present specification, the "C _6-14 aryl group" is an aromatic hydrocarbon group having 6 to 14 carbon atoms, and a C _6-12 aryl group is particularly preferable. Examples of the C _6-14 aryl group include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group and the like, and a phenyl group is particularly preferable.

In the present invention and the present specification, the "optionally substituted C _6-14 aryl group" is one or more hydrogen atoms bonded to the carbon atom of the _{C 6-14 aryl group, preferably 1 to 1.} Three are groups substituted with other functional groups. When having two or more substituents, the substituents may be the same kind or different from each other. Examples of the substituent include a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a C _1-6 alkyl group, a C _1-6 alkoxy group, and a methylenedioxy group (-O-CH). _2- O-) and the like can be mentioned. _{Examples of "optionally} substituted C 6-14 aryl groups" are phenyl group, naphthyl group, anthryl group, 4-nitrophenyl group, 4-methoxyphenyl group, 2,4-dimethoxyphenyl group, 3, Examples thereof include 4-dimethoxyphenyl group, 4-methylphenyl group, 2,6-dimethylphenyl group, 3-chlorophenyl group, 1,3-benzodioxol-5-yl group and the like.

In the present invention and the present specification, the "C _6-14 aryl-C _1-6 alkyl group" is a _{C 6-14} aryl group in which one hydrogen atom bonded to the carbon atom of the _{C 1-6 alkyl group is a C 6-14 aryl group.} It is a group substituted with. The _{C 6-14} aryl group in the C _6-14 aryl _{-C 1-6} alkyl group, a phenyl group, a naphthyl group, an anthryl group, can be exemplified a 9-fluorenyl group, a phenyl group or a 9-fluorenyl group is particularly preferred .. As the _{C 1-6} alkyl group in the C _6-14 aryl _{-C 1-6} alkyl _{group, C 1-4} alkyl groups are preferred. Examples of C _6-14 aryl-C _1-6 alkyl groups include benzyl group, diphenylmethyl group, triphenylmethyl group, 2-phenylethyl group, 9-anthrylmethyl group, 9-fluorenylmethyl group and the like. Can be mentioned.

In the present invention and the specification of the present application, the "heteroaryl group" is a cyclic group having aromaticity, and the ring is a group composed of a carbon atom and an atom other than the carbon atom. The heteroaryl group may be a group containing a nitrogen atom (nitrogen-containing heteroaryl group), a group containing an oxygen atom (oxygen-containing heteroaryl group), or a group containing a sulfur atom (sulfur-containing heteroaryl group). It may be a heteroaryl group). Further, the number of atoms other than the carbon atom constituting the aromatic ring may be two or more.

Examples of the C _5-14 nitrogen-containing heteroaryl group include a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridadinyl group, an indolyl group, an isoindryl group, a benzoimidazolyl group and a benzotriazolyl group. Examples thereof include a quinolyl group, an isoquinolyl group, a quinazolyl group and a carbazolyl group. Examples of the C _5-14 oxygen-containing heteroaryl group include a furanyl group, a pyranyl group, a benzopyranyl group, and a xanthenyl group. Examples of the C _5-14 sulfur-containing heteroaryl group include a thienyl group and the like. _{Examples of the C 5-14} heteroaryl group containing two or more heteroatoms include an oxazolyl group, an isooxazolyl group, a thiazolyl group, an isothiazolyl group and the like.

In the present invention and the present specification, the "optionally substituted heteroaryl group" is one or more hydrogen atoms bonded to atoms constituting the aromatic ring of the heteroaryl group, preferably 1 to 3 hydrogen atoms. Is a group substituted with another functional group. When having two or more substituents, the substituents may be the same kind or different from each other. Examples of the substituent include a C _1-6 alkyl group, a C _1-6 alkoxy group, a methylenedioxy group (-O-CH _2- O-), a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom). ), Trihalomethyl group, cyano group, nitro group and the like.

In the present invention and the present specification, the "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The "halogen atom other than the fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. As an example of the "halogen atom other than the fluorine atom", a chlorine atom or a bromine atom is preferable, and a chlorine atom is particularly preferable.

In the present invention and the present specification, the "C _1-6 alkoxy group" refers to a group in which an oxygen atom is bonded to the bond end of _{a C 1-6} alkyl group having 1 to 6 carbon atoms. The C _1-6 alkoxy group may be a straight chain or a branched chain. Examples of the C _1-6 alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a tert-butoxy group, a pentyloxy group, a hexyloxy group and the like.

In the present invention and the present specification, the "amino group which may have a substituent" means that one or two hydrogen atoms bonded to the nitrogen atom of the amino group are replaced with other functional groups. It is a group that has been. When having two substituents, the substituents may be the same kind or different from each other. Examples of the substituent include a C _1-6 alkyl group, a C _1-6 alkoxy group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a trihalomethyl group and the like.

In the present invention and the specification of the present application, the "ether-bonded oxygen atom" is an oxygen atom that connects carbon atoms, and does not include an oxygen atom in which oxygen atoms are connected in series. The maximum number of ether-bonded oxygen atoms that an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have is Nc-1.

In the following, "Compound n" means a compound represented by the formula (n).

<Fluorine-containing peptide>
The peptide according to the present invention is a peptide consisting of two or more amino acids, and at least one of the amino acid residues constituting the peptide has the following general formulas (g-1) to (g-3) in the side chain. ) Is a fluorine-containing peptide having a group represented by any of). Hereinafter, the group represented by any of the general formulas (g-1) to (g-3) is referred to as Rg. Further, in the general formulas (g-1) to (g-3), the black circle means a bond.

In the general formulas (g-1) to (g-3), R ¹ and R ² may have a hydrogen atom, a halogen atom, a carboxy group, and a substituent independently of each other. C _6-14 aryl A C _5-14 heteroaryl group which may have a group, a substituent, or an amino group which may have a substituent. R ¹ and R ² are independent of each other and may have a hydrogen atom, a halogen atom, a carboxy group, a C _6-14 aryl group which may have a substituent, or C _{5 which may have a substituent. -14 heteroaryl groups are preferred; C 6-14} aryl groups, which may have hydrogen atoms, halogen atoms, or substituents independently of each other, are more preferred; independent of each other, hydrogen atoms, fluorine atoms, Alternatively, a phenyl group which may have a substituent is further preferable; a hydrogen atom, a fluorine atom, or a phenyl group is further preferable independently of each other.

In the general formula (g-2), R ³ and R ⁴ _{have a hydrogen atom, a halogen atom, a C 1-30} alkyl group which may have a substituent, and a substituent, which are independent of each other. It may be a C _6-14 aryl group, or a C _5-14 heteroaryl group which may have a substituent. When R ³ and R ⁴ _{are C 1-30} alkyl groups which may have a substituent, the C _1-30 alkyl group has an ether-bonding oxygen between carbon atoms when the number of carbon atoms is 2 or more. It may have an atom. As R ³ and R ⁴ , a hydrogen atom, a halogen atom, a C _1-6 alkyl group which may have a substituent, or a phenyl group which may have a substituent are preferable as independent of each other; _{More preferably, a C 1-4} alkyl group, which may be substituted with a hydrogen atom, a halogen atom, a halogen atom, or a phenyl group, which may have a substituent, independently of each other; , Fluorine atom, methyl group, trihalomethyl group, or phenyl group; independent of each other, hydrogen atom, fluorine atom, methyl group, or trihalomethyl group is even more preferable.

In the general formula (g-3), R ³ to R ⁶ _{have a hydrogen atom, a halogen atom, a C 1-30} alkyl group which may have a substituent, and a substituent independently of each other. It may be a C _6-14 aryl group, or a C _5-14 heteroaryl group which may have a substituent. When R ³ to R ⁶ _{are C 1-30} alkyl groups which may have a substituent, the C _1-30 alkyl group has an ether-bonding oxygen between carbon atoms when the number of carbon atoms is 2 or more. It may have an atom. As R ³ to R ⁶ , a hydrogen atom, a halogen atom, a C _1-6 alkyl group which may have a substituent, or a phenyl group which may have a substituent are preferable as independent of each other; _{More preferably, a C 1-4} alkyl group, which may be substituted with a hydrogen atom, a halogen atom, a halogen atom, or a phenyl group, which may have a substituent, independently of each other; , Fluorine atom, methyl group, trihalomethyl group, or phenyl group; independently of each other, hydrogen atom, fluorine atom, methyl group, or trihalomethyl group is even more preferred.

In the general formula (g-3), R ¹ , R ² , R ³ and R ⁴ are linked to each other and have a C _6-14 aryl group, which may have a substituent, or a substituent. It may _{constitute a C 5-14} heteroaryl group. As the ^{ring structure composed of R 1} , R ² , R ³ and R ⁴ , a phenyl group which may have a substituent or a C _5-14 heteroaryl group which may have a substituent is preferable. , A phenyl group which may have a substituent or a C _5-14 nitrogen-containing heteroaryl group which may have a substituent is more preferable, and a phenyl group, a pyrrolyl group or an indolyl group is further preferable.

In the fluorine-containing peptide according to the present invention, a partial structure containing a fluorine atom of Rg constitutes an intramolecular ring structure via a hydrogen bond together with -NH in the peptide bond. When Rg is a group represented by the general formula (g-1), the fluorine atom (the fluorine atom at the β-position) in the general formula (g-1) and the hydrogen atom bonded to the nitrogen atom in the peptide bond The hydrogen bonds between them form an intramolecular 6-membered ring structure. When Rg is a group represented by the general formula (g-2), the fluorine atom (fluorine atom at the γ position) in the general formula (g-2) and the hydrogen atom bonded to the nitrogen atom in the peptide bond The hydrogen bonds between them form an intramolecular 6-membered ring structure. When Rg is a group represented by the general formula (g-3), it is formed by a hydrogen bond between the terminal fluorine atom in the general formula (g-3) and the hydrogen atom bonded to the nitrogen atom in the peptide bond. , Form an intramolecular 7-membered ring structure. A schematic diagram of the intramolecular ring structure is shown below. It should be noted that these intramolecular ring structures are not ordinary ring structures but contain hydrogen bonds.

The fluorine-containing peptide according to the present invention has excellent cell membrane permeability due to this intramolecular ring structure. Although the relationship between the intramolecular ring structure and cell membrane permeability is not clear, -NH in the peptide bond is suppressed from hydrogen bonding with water molecules in the environment, and the hydrophilicity of the entire peptide molecule is reduced. It is presumed that this is because of it.

When Rg is a group represented by the general formula (g-1), it is preferable that R ¹ and R ² are independently hydrogen atoms or fluorine atoms, respectively; from the viewpoint of easily forming an intramolecular structure; ^{A group in which one of 1} and R ² is a fluorine atom and the other is a hydrogen atom, or a group in which ^{both R 1} and R ² are fluorine atoms is more preferable, ^{and both R 1} and R ² are fluorine atoms. Groups are particularly preferred.

When Rg is a group represented by the general formula (g-1), a group in which R ¹ is a hydrogen atom and R ² _{is a C 6-14} aryl group which may have a substituent is also preferable. Among them, R ¹ is hydrogen atom, group are preferable R ² is a phenyl group which may have a substituent; R ¹ is hydrogen atom, group R ² is a phenyl group is more preferable.

When Rg is a group represented by the general formula (g-2), ^{at least one of R 1} and R ² is a hydrogen atom or a fluorine atom, and R ³ is a hydrogen atom because it is easy to form an intramolecular structure. There, ^{preferably a group in which R 4} is a hydrogen atom or a trifluoromethyl group; ^{at least one of R 1} and R ² is a hydrogen atom, the other is a fluorine atom, R ³ is a hydrogen atom, and R ⁴ More preferably a group in which is a hydrogen atom or a trifluoromethyl group; a group in which ^{both R 1} and R ² are fluorine atoms, R ³ is a hydrogen atom and R ⁴ is a hydrogen atom or a trifluoromethyl group. More preferably; a group ^{in which both R 1} and R ² are fluorine atoms, R ³ is a hydrogen atom and R ⁴ is a trifluoromethyl group is particularly preferred.

When Rg is a group represented by the general formula (g-3), ^{at least one of R 1} and R ² is a hydrogen atom or a fluorine atom, and R ³ is a hydrogen atom because it is easy to form an intramolecular structure. There, preferably a ^{group in which R 4} is a hydrogen atom or a trifluoromethyl group and R ⁵ and R ⁶ are hydrogen atoms; ^{at least one of R 1} and R ² is a hydrogen atom and the remaining one is a fluorine atom. , R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or a trifluoromethyl group, and R ⁵ and R ⁶ are hydrogen atoms, more preferably; ^{both R 1} and R ² are fluorine atoms. More preferably, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or a trifluoromethyl group, and R ⁵ and R ⁶ are hydrogen atoms; ^{both R 1} and R ² are fluorine atoms and R ^A group in which 3 is a hydrogen atom, R ⁴ is a trifluoromethyl group, and R ⁵ and R ⁶ are hydrogen atoms is particularly preferable.

When Rg is a group represented by the general formula (g-3), R ¹ , R ² , R ³ and R ⁴ may be linked to each other and have a substituent C _6-14 aryl group, _{Alternatively, groups constituting a C 5-14} heteroaryl group which may have a substituent ^{and where R 5} and R ⁶ are hydrogen atoms are also preferable; R ¹ , R ² , R ³ and R ⁴ are each other. It is linked to form a phenyl group which may have a substituent, or a pyrrolyl group or an indrill group which may have a substituent, and a group in which R ⁵ and R ⁶ are hydrogen atoms is more. Preferably; R ¹ , R ² , R ³ and R ⁴ are linked to each other to form a phenyl group, pyrrolyl group, or indolyl group, more preferably a group in which ^{R 5} and R ^{6 are hydrogen atoms; R A group in which 1} , R ² , R ³ and R ⁴ are linked to each other to form a phenyl group or an indrill group, and R ⁵ and R ⁶ are hydrogen atoms is even more preferable.

In the fluorine-containing peptide according to the present invention, among the amino acid residues constituting the peptide, at least one side chain may be Rg, and two or more side chains may be Rg, and all amino acids. The side chain of the residue may be Rg. When there are two or more Rg in one molecule, the Rg may be the same kind or different from each other. When one molecule of peptide has two or more amino acid residues having a side chain of Rg, these plurality of Rg may be the same species or different from each other. Further, among the peptides, the amino acid residue whose side chain is Rg may be at the N-terminal, at the C-terminal, or at a position other than the terminal.

The fluorine-containing peptide according to the present invention can be produced using an amino acid [HOOC-CH (Rg) -NH _{2] in which the side chain is replaced with Rg as a raw material.} Examples of amino acids in which the side chain is replaced with Rg include 2-fluoro-L-phenylalanine (CAS No: 19883-78-4), (2S, 4R) -4-fluoroglutamic acid, (2S, 4S) -4. -Fluoroglutamic acid, 4,4-difluoro-L-glutamic acid (CAS No: 130835-20-0), 2-amino-4,4,4-trifluorobutyric acid (CAS No: 15959-93-0), 3- Fluorovaline (CAS No: 43163-94-6), 5', 5', 5'-trifluoroleucine (CAS No: 372-22-5), 4,4-difluoro-DL-ornithine, β-fluoro- DL-alanine (CAS No: 43163-93-5), hexafluoro-DL-valine (CAS No: 16063-80-2), α-difluoromethyl-DL-ornithin (CAS No: 70052-12-9), etc. Can be mentioned.

The fluorine-containing peptide according to the present invention may be a peptide consisting of two or more amino acids, preferably a peptide consisting of 2 to 40 amino acids, more preferably a peptide consisting of 2 to 20 amino acids, or 2 or 3. Peptides consisting of a single amino acid are more preferred.

The fluorine-containing peptide according to the present invention may be a cyclic peptide. The number of amino acid residues forming the cyclic structure is preferably 4 to 31 residues, more preferably 4 to 11 residues, and even more preferably 6 to 7 residues.

When the fluorine-containing peptide according to the present invention is a cyclic peptide, at least one side chain of the amino acid residues forming the cyclic structure may be Rg, and two or more side chains may be Rg. , The side chains of all amino acid residues may be Rg. When the fluorine-containing peptide according to the present invention has two or more Rg in one molecule, these plurality of Rg may be the same kind or different from each other. Further, among the fluorine-containing peptides according to the present invention, the amino acid residue having an Rg side chain may be at the N-terminal, at the C-terminal, or at a position other than the terminal.

Peptide can be produced by a general peptide synthesis method. For example, it can be carried out by a peptide solid phase synthesis method. The fluorine-containing peptide can be easily synthesized using an automatic peptide synthesizer using an amino acid having a fluorine atom introduced into the side chain as a raw material. Cyclic peptides can also be produced by general peptide synthesis methods.

A peptide can be produced by sequentially condensing an amino acid having an amino group protected with an amino acid having a C-terminal bonded to a solid phase and desorbing the peptide from the solid phase. As the amino acid raw material, it is preferable to use one in which the amino group is protected by a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group. As the side chain functional group of the amino acid raw material, it is preferable to use one protected by a protecting group. Examples of the protecting group for the side chain functional group include a Boc group, a triphenylmethyl group, a benzyl group, a 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group and the like.

Examples of the condensing agent that forms a peptide bond include N, N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (WSC), and benzotriazole-1-yloxy-trisdimethyl. Aminophosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (pyBOP), 2- (1H-benzotriazole-1-yl) -1,1,3 Examples thereof include 3-tetramethyluronium hexafluorophosphate (HBTU) and 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium tetrafluorobolate. Further, N-hydroxybenzotriazole (HOBt) and the above condensing agent can be mixed and used in a preferable ratio.

A method of activating the carboxyl terminus may be used for the formation of the peptide bond, and examples of the activator include N-hydroxysuccinimide, p-nitrophenyl ester, pentafluorophenyl ester and the like. Examples of the base used for forming a peptide bond include triethylamine, diisopropylethylamine (DIEA) and the like. Examples of the solvent used in the peptide bond formation reaction include chloroform, dichloromethane, acetonitrile, N, N-dimethylformamide (DMF), dimethyl sulfoxide and the like.

The Boc and Fmoc groups, which are the protecting groups for the amino-terminal amino groups of peptides or amino acids, can be removed with trifluoroacetic acid (TFA) or piperidine, respectively. The protecting group of the side chain functional group of the amino acid residue of the peptide can be removed by, for example, TFA, hydrogen fluoride (HF), trifluoromethanesulfonic acid or the like.

Further, in the peptide solid phase synthesis method, for example, TFA can be used as a method for removing a peptide having a protecting group on the side chain functional group of the peptide or amino acid residue from the peptide solid phase synthetic resin. Desorption of the peptide from the peptide solid phase resin and desorption of the protecting group of the side chain functional group of the amino acid residue can also be performed simultaneously in the same reaction system. Alternatively, each can be performed independently. Examples of the peptide solid phase synthetic resin for peptide solid phase synthesis include 4-hydroxymethyl-3-methoxyphenoxybutyric acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, and oxime resin, which are usually commercially available. Can be used.

The target peptide or its intermediate is isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, and fractional crystallization. be able to. In addition, the peptide thus obtained can be converted into each salt by a conventional method.

The protective group of the amino group or the carboxyl group of the produced fluorine-containing peptide can be deprotected if necessary. Deprotection can be performed by a conventional method depending on the type of protecting group.

In the fluorine-containing peptide according to the present invention, the terminal amino group or carboxyl group may be protected by a protecting group, or may be substituted by a substituent other than the protecting group. Examples of the carboxyl group protecting group include an acetyl group, a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 4-nitrobenzyl group, a 4-methoxybenzyl group, a 2,4-dimethoxybenzyl group, and a 3,4-dimethoxy group. Benzyl group, 4-methylbenzyl group, 2,6-dimethylbenzyl group, 3-chlorobenzyl group, 9-anthrylmethyl group, piperonyl group, 2- (9,10-dioxo) anthrylmethyl group, benzyloxymethyl Benzyl group and the like can be mentioned. Examples of the amino-protecting group include carbamate-based protecting groups such as Boc group, Fmoc group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Alloc) group, and 2,2,2-trichloroethoxycarbonyl (Troc) group. The group is mentioned.

Examples of the fluorine-containing peptide according to the present invention include the following general formula (p2-1) or general formula (p3-1). In the general formula (p2-1) or the general formula (p3-1), Rg is a group represented by the general formula (g-1), the general formula (g-2), or the general formula (g-3). R ¹¹ and R ¹² are independently C _1-6 alkyl groups or C _6-14 aryl-C _1-6 alkyl groups, respectively. Z ¹ is a protecting group for a hydroxy group, a C _1-6 alkyl group, a C _1-6 alkoxy group, or a carboxyl group, and Z ² and Z ³ are independent hydrogen atoms and C _1-6 alkyl, respectively. A group, a C _6-14 aryl-C _1-6 alkyl group, or an amino protecting group.

As the peptides of the general formula (p2-1) or the general formula (p3-1), R ¹¹ and R ¹² are independently C _1-6 alkyl groups or phenyl-C _1-6 alkyl groups, respectively. , Z ¹ is a hydroxy group, a C _1-6 alkyl group, or a C _1-6 alkoxy group, Z ² is a hydrogen atom or a C _1-6 alkyl group, and Z ³ is a hydrogen atom, a Boc group. , Fmoc group, C _1-6 alkyl group, or phenyl-C _1-6 alkyl group are preferred; R ¹¹ and R ¹² are independently C _1-6 alkyl or benzyl groups, respectively. Z ¹ is a hydroxy group, C _1-6 alkyl group, or C _1-6 alkoxy group, Z ² is a hydrogen atom or C _1-6 alkyl group, and Z ³ is a hydrogen atom, Boc group, Peptides that are Fmoc groups, C _1-6 alkyl groups, benzyl groups, or phenylethyl groups are more preferred.

The fluorine-containing peptide according to the present invention has excellent cell membrane permeability. Although the reason is not clear, the fluorine-containing peptide according to the present invention has an inner ring structure in which -NH in the peptide bond is suppressed from hydrogen-bonding to a water molecule in the environment, and the entire peptide molecule is suppressed. It is presumed that this is due to the decrease in hydrophilicity. In addition, since the structure is significantly different from that of natural peptides, it is not easily decomposed by peptidase. Utilizing these properties, the fluorine-containing peptide according to the present invention is expected to be used in the pharmaceutical field as a physiologically active substance. For example, the fluorine-containing peptide according to the present invention can be expected to be used as an active ingredient of a cell membrane penetrating agent or a DDS carrier that carries a medicinal ingredient to a target cell. For example, by adding a fluorine-containing peptide according to the present invention to a functional peptide that exhibits some physiological activity by being taken up into a target cell in a living body so as not to impair its function, the functional peptide can be obtained. The efficiency of uptake into target cells can be improved. In addition, some side chains of the hydrophobic amino acid residues of the functional peptide exhibiting physiological activity are Rg, preferably the general formula (g-1) or the above, as long as the function of the functional peptide is not impaired. By substituting with the group represented by (g-2), the cell membrane permeability of the functional peptide and the residence time in the cell can be improved.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

The NMR apparatus used for the analysis of Examples and Comparative Examples was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd., and tetramethylsilane was set to 0 PPM in ¹ _{H NMR and C 6} F ₆ was set ^{to 162 PPM in 19} F NMR. bottom.

The following abbreviations are used herein.
DCM: dichloromethane DMF: N, N-dimethylformamide NMP: N-methylpyrrolidone EDC · HCl: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride HOBt: 1-hydroxybenzotriazole monohydrate HOAt: 1-Hydroxy-7-azabenzotriazole Oxyma: Ethyl cyano (hydroxyimino) acetate DIC: N, N'-diisopropylcarbodiimide DIPEA: N, N-diisopropylethylamine TFA: Trifluoroacetate DMTMM: 4- (4,6-dimethoxy- 1,3,5-triazin-2-yl) -4-methylmorpholinium chloride THF: tetrahydrofuran PAMPA: Parallel artificial membrane permeability assay MDCK: Madin-Darby canine kidney
WBSS: Hank's balanced salt solution
Boc: tert-butoxycarbonyl group Cbz: benzyloxycarbonyl group Ac: acetyl group Me: methyl group Bn: benzyl group EPh: phenylethyl group (-CH ₂ CH _2- Ph)
HATU: 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate PyAOP: hexafluorophosphoric acid (7-azabenzotriazole- 1-Iloxy) Trispyrrolidinophosphonium PyBOP: Hexafluorophosphoric acid (benzotriazole-1-yloxy) Trispyrrolidinophosphonium HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol TIPS: Triisopropyl Silane

The chemicals and solvents used in this study were purchased from commercial suppliers and used without further purification. Preparative HPLC was performed on a Prominence HPLC system (manufactured by Shimadzu Corporation) equipped with a 5C18-MS-II column (manufactured by Nacalai Tesque, catalog number: 34355-91, inner diameter 10 mm × 150 mm). However, in Example 10, Comparative Example 11 and Example 12, a 5C18-AS-II column (manufactured by Nacalai Tesque, catalog number: 34355-91, inner diameter 10 mm × 150 mm) was used. All HPLC are two solvents (solvent A: 0.1% TFA-containing _H 2 O, solvent B: 0.1% TFA-containing acetonitrile) were performed using. The NMR spectrum was recorded using ECS-400 (manufactured by JEOL Ltd.). LC-MS was carried out using INTSstein AQ-C18 (manufactured by GL Sciences, inner diameter 2.1 mm × 50 mm) and ACQUITYUPLC H-Class / SQD2 (manufactured by Waters). МALDI-TOF-MS was performed by autoflex maX (manufactured by Bruker). MDCK-II cells were purchased from the European Collection of Authenticated Cell Cultures from DS Pharma Biomedical.

In the following, amino acids may be written in three-letter notation, and if the side chain contains a fluorine atom, "(F)" is added after the three-letter notation. For example, "Phe" is phenylalanine and "Gly" is glycine. "L-Phe" is L-phenylalanine, and "Phe (F)" is phenylalanine containing a fluorine atom in the side chain. In addition, the peptide is described as (N-side protecting group) -amino acid three-letter notation- (C-side protecting group). "H-AA-OMe" means that the N-terminal side is unprotected and the C-terminal side is a methyl ester. When the C-terminal side is unprotected, it is expressed as "OH" instead of "OMe".

<PAMPA assay>
Peptide permeability 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). A 150 μL peptide solution (20 μM) dissolved in 5% DMSO / PBS was then added to each well of the donor plate (MultiScreen-IP filter plate, 0.45 μm, manufactured by Merck). A dodecane solution of 1% lecithin (derived from soybean) was sonicated for 30 minutes prior to 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 the incubator at 25 ° C. for 18 hours. The peptide concentration was determined using LC / MS. The experiment was repeated 3 times. The transmittance value ( _Pe ) was calculated using the following equation.

A: Filter area (0.3 cm ² )
VD: Donor well volume (0.15 cm ³ )
VA: Acceptor well volume (0.3 cm ³ )
t: Incubation time (s) (18 hours = 64800s)
CD (t): Donor well compound concentration at time t CA (t): Acceptor well compound concentration at time t

<MDCK-II Assay>
Five days after seeding MDCK-II cells in a cell culture insert (manufactured by Falcon) at 5.04 × 10 ⁴ cells / mL, a cell monolayer assay (MDCK-II assay) was performed. The peptide stock solution is prepared at 2 mM in DMSO solution, diluted with HBSS containing 20 mM HEPES and pH 7.5, and used as a donor solution in a 2 μM peptide solution using 0.1% DMSO / HBSS (+) as a solvent. Was prepared. The acceptor solution was a 0.1% DMSO solution using HBSS (+) containing 20 mM HEPES and pH 7.5 as a solvent. Apparent permeability ( _Papp ) was determined by apical to lateral bottom incubation in peptide solution at 37 ° C., 5% CO _{2 for 2 hours.} Peptide concentration was analyzed by LC / MS. The experiment was repeated 3 times. The transmittance value ( _Papp ) was calculated using the following formula.

A: Filter area (0.3 cm ² )
VB: Side bottom well volume (0.75 cm ³ )
t: Incubation time (s) (2 hours = 7200s)
C0: Initial concentration of apical chamber (2 μM)
CB (t): Compound concentration at the bottom at time t

[Comparative Example 1]
Ac-L-Phe-L-Ala-NHMe was synthesized.

Compound _{1 (62mg, 0.2mmol), H} 2 N-Ala-NHMe · HCl (35mg), and DIPEA (41 [mu] L) was dissolved in methanol 2 mL, the resulting solution was stirred at room temperature. DMTMM 1.6H ₂ O (74 mg) was added to the solution. The resulting reaction mixture was stirred at room temperature overnight and evaporated in vacuo. DCM was then added to the reaction mixture and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water and brine. The resulting organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The obtained residue was purified by silica gel column chromatography (DCM / methanol = 9: 1 (volume ratio)) to obtain Compound 2 (31 mg, yield 40%).

Compound 2 (30 mg, 78 μmol), 10% palladium carbon (6.8 mg), and 3 mL of methanol were added to the recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 37.5 hours. The resulting reaction mixture was filtered through Celite. After removing the solvent under reduced pressure, the residue was dissolved in 6 mL of 10% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 3 (18 mg, 93% yield).

DIPEA (8.6 μL) and acetic anhydride (46 μL) were added to Compound 3 (6.3 mg, 25 μmol) dissolved in 1 mL DCM and 1 mL THF. The reaction mixture was stirred at room temperature for 19 hours and then evaporated in vacuo. The residue was dissolved in 4 mL of 10% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 4 (Ac-L-Phe-L-Ala-NHMe) as a white solid (3.7 mg, 51% yield).

^{1 1} H NMR (DMSO-d 6,400 MHz): δ8.04-7.98 (m, 2H), 7.52 (d, J = 4.1 Hz, 1H), 7.24-7.12 (m, 5H) ), 4.48-4.42 (m, 1H), 4.15 (dq, J = 7.3, 6.9Hz, 1H), 2.96 (dd, J = 4.6, 13.7Hz, 1H), 2.67 (dd, J = 9.6, 13.3Hz, 1H), 2.52 (d, J = 4.6Hz, 3H), 1.70 (s, 1H), 1.14 ( d, J = 7.3Hz, 3H).
MS (LC / ESI-MS.m / z): calcd. for C ₁₅ H ₂₂ N ₃ O ₃ (M + H) ⁺ : 292.17, found: 292.20.

[Example 1]
Ac-L-Phe (F) -L-Ala-NHMe was synthesized.

Under a nitrogen atmosphere, 10 mL anhydrous DCM, 2.4 mL anhydrous DMF, 4.6 mL DIPEA, N-carbobenzoxi-L-alanine (Compound 5) (1.5 g, 6.7 mmol), EDC / HCl. It was added to (2.6 g) and HOBt (1.8 g). The reaction mixture was stirred at room temperature for 10 minutes. 2M Methylamine (5.1 μL) in THF was added and the mixture was stirred overnight. After vacuum evaporation of the reaction mixture, water was added to the residue and the resulting solution was extracted 5 times with ethyl acetate. The organic phase was washed _{twice with saturated NaHCO 3} with 1M aqueous HCl and brine. The resulting organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1: 4 (volume ratio)) to obtain Compound 6 (819 mg, yield 49%).

Compound 6 (819 mg, 3.47 mmol), palladium carbon 10% (84 mg), and 23 mL of methanol were added to the recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 2 days. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 7 (368 mg, quantitative yield).

Compound 8 was synthesized according to the method of Miao et al. (Non-Patent Document 6). Compound 8 (40 mg, 0.13 mmol) and compound 7 (13 mg) were dissolved in 1 mL of methanol, and DMTMM 3.2H ₂ O (53 mg) was added to the solution. The reaction mixture was stirred at room temperature for 18 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 9 (15 mg, 37% yield).

Compound 9 (15 mg, 47 μmol) was dissolved in 2.9 mL of methanol and 2.9 mL of ethanol. The resulting solution was stirred at 0 ° C. Hydrazine monohydrate (28 μL) was added to the solution, and the reaction mixture was stirred at 0 ° C. for 13 minutes. The resulting mixture was warmed to room temperature over 50 minutes. The solvent was removed under reduced pressure and the residue was dissolved in 5 mL of 1% aqueous acetonitrile solution. Purification using an HPLC reversed-phase column gave compound 10 (8.9 mg, 63% yield).

DIPEA (4 μL) and acetic anhydride (22 μL) were added to Compound 10 (8.9 mg, 23 μmol) in 0.9 mL of DCM. The reaction mixture was stirred at room temperature for 14 hours and then evaporated in vacuo. The residue was dissolved in 5 mL of 20% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 11 (Ac-L-Phe (F) -L-Ala-NHMe) as a white solid (0.4 mg, 4% yield).

^{1 1} H NMR (DMSO-d 6,400 MHz): δ7.33-7.20 (m, 5H), 4.42-4-33 (m, 3H), 4.29 (q, J = 6.9 Hz, 1H) ), 1.96 (s, 3H), 1.38 (d, J = 6.87Hz, 3H), 1.33 (d, J = 7.3Hz, 3H).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₅ H ₂₀ FN ₃ O ₃ Na (M + Na) ⁺ : 332.14, found: 332.14.

[Comparative Example 2]
Ac-L-Phe-L-Ala-NMe ₂ was synthesized.

2M dimethylamine in compound 5 (805 mg, 3.60 mmol) and THF (2.0 mL) was dissolved in 36 mL of methanol, then DMTMM 3.2H ₂ O (1.32 g) was added to the solution. .. The reaction mixture was stirred at room temperature for 15 hours and then evaporated in vacuo. DCM was added to the reaction mixture and the solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water and brine. The resulting organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 15: 1 (volume ratio)) to give compound 12 (773 mg, 86% yield).

Compound 12 (773 mg, 3.10 mmol), palladium carbon 10% (77 mg), and 10 mL of methanol were added to a 50 mL recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 37 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure to give compound 13 (277 mg, 63% yield).

Compound 1 (65 mg, 0.2 mmol) and compound 13 (28 mg) were dissolved in 2 mL of methanol and the resulting solution was stirred at room temperature. DMTMM 1.6H ₂ O (75 mg) was added to the solution. The reaction mixture was stirred at room temperature for 30 hours and evaporated in vacuo. DCM was added to the reaction mixture and the solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water and brine. The resulting organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1: 4 (volume ratio)) to obtain compound 14 (37 mg, yield 46%).

Compound 14 (37 mg, 93 μmol), palladium on 10% carbon (8.8 mg), and 2 mL of methanol were added to the recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 37.5 hours. The reaction mixture was filtered through Celite. The solvent was removed under reduced pressure and the residue was dissolved in 6 mL of 10% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 15 (23 mg, 96% yield).

Acetic anhydride (46 μL) was added to compound 15 (12 mg, 44 μmol) in 1 mL DCM and 1 mL THF. The reaction mixture was stirred at room temperature for 19.5 hours and then evaporated in vacuo. The residue was dissolved in 4 mL of 10% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 16 (Ac-L-Phe-L-Ala-NMe ₂ ) as a white solid (11.5 mg, 85% yield).

^{1 1} H NMR (DMSO-d 6,400 MHz): δ8.11 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.23-7.11 (m) , 5H), 4.64 (dq, J = 6.9, 7.3Hz, 1H), 4.49-4.43 (m, 1H), 2.96-2.89 (m, 4H), 2 .77 (s, 3H), 2.68-2.65 (m, 1H), 1.12 (d, J = 6.4Hz, 3H).
MS (LC / ESI-MS.m / z): calcd. for C ₁₆ H ₂₄ N ₃ O ₃ (M + H) ⁺ : 306.18, found: 306.20.

[Example 2]
Ac-L-Phe (F) -L-Ala-NMe ₂ was synthesized.

Compound 8 (40 mg, 0.13 mmol) and compound 13 (15 mg) were dissolved in 1.3 mL of methanol, and DMTMM 3.2H ₂ O (55 mg) was added to the solution. The reaction mixture was stirred at room temperature for 12.5 hours and then evaporated in vacuo. DCM was added to the reaction mixture and the solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1: 1 (volume ratio)) to give compound 17 (20 mg, 38% yield).

Compound 17 (21 mg, 51 μmol) was dissolved in 1 mL of methanol and 1 mL of ethanol. The solution was stirred at 0 ° C., hydrazine monohydrate (30 μL) was added to the solution and the reaction mixture was stirred at 0 ° C. for 13 minutes. The mixture was warmed to room temperature over 50 minutes and then the solvent was removed under reduced pressure. The residue was dissolved in 5 mL of 4% aqueous acetonitrile solution. Purification using an HPLC reversed-phase column gave compound 18 (9.0 mg, 63% yield).

DIPEA (3.9 μL) and acetic anhydride (21 μL) were added to compound 18 (9.0 mg, 23 μmol) in DCM (0.95 mL). The reaction mixture was stirred at room temperature for 14 hours and then evaporated in vacuo. The residue was dissolved in 5 mL of 20% aqueous acetonitrile solution. Purification using an HPLC reverse phase column gave compound 19 (Ac-L-Phe (F) -L-Ala-NMe ₂ ) (2.1 mg, 28% yield).

_{^{1 H NMR (CDCl 3, 400MHz}} ): δ7.41-7.34 (m, 5H), 6.78 (d, J = 6.4Hz, 1H), 6.20 (d, J = 8.2Hz, 1H), 5.83 (dd, J = 4.6, 45.8Hz, 1H), 4.99 (ddd, J = 5.0, 8.2, 17.9Hz, 1H), 4.77 (dq) , J = 6.4, 6.9Hz, 1H), 3.17-3.10 (m, 1H), 3.04 (s, 1H), 2.94 (s, 1H), 2.00 (s) , 1H), 1.26 (d, J = 6.9Hz, 1H).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₆ H ₂₂ FN ₃ O ₃ Na (M + Na) ⁺ : 346.15, found: 346.14.

[Comparative Example 3]
Ac-L-Phe-L-Ala-NHBn was synthesized.

Under a nitrogen atmosphere, 3 mL anhydrous DCM, DIPEA (184 μL), and benzylamine were added to N-Boc-L-alanine (Compound 20) (135 mg, 0.71 mmol) and HOAt (116 mg). The solution was stirred at 0 ° C. HATU (324 mg) was added to the solution and the reaction mixture was warmed to room temperature over 17 hours. A 1M aqueous HCl solution was added to the reaction mixture, and the resulting solution was extracted 3 times with DCM. The solvent was removed under reduced pressure and ethyl acetate was added to the residue. The solution was washed with 1M aqueous HCl, saturated NaHCO ₃ , and brine. The organic phase was _{dried over Na 2} SO ₄ and then evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 21 (156 mg, 79% yield).

Compound 21 (156 mg, 0.56 mmol) was dissolved in 4 mL DCM. 1 mL of TFA was added dropwise to the solution at 0 ° C. and the mixture was stirred for 5 minutes. The reaction mixture was warmed to room temperature and stirred for 1 hour. A saturated _{aqueous solution of NaHCO 3} was added until the pH reached 8. The mixture was extracted 3 times with DCM. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 22 (73 mg, 73% yield).

Under a nitrogen atmosphere, 1 mL of anhydrous DCM was added to Compound 23 (22 mg, 0.70 mmol) and HOAt (12 mg). The solution was stirred at 0 ° C. DIPEA (18 μL), compound 22 (15 mg) and HATU (32 mg) were added to the solution. The solution was warmed to room temperature over 24 hours. A 1M aqueous HCl solution was added to the reaction mixture and the solution was extracted 3 times with DCM. The solvent was removed under reduced pressure and ethyl acetate was added to the residue. The solution was washed with 1M aqueous HCl, saturated NaHCO ₃ , and brine. The organic phase was _{dried over Na 2} SO ₄ and then evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 24 (23 mg, 72% yield).

Compound 24 (9.4 mg, 21 μmol) was dissolved in 0.6 mL of methanol and 0.6 mL of ethanol. Hydrazine monohydrate (12 μL) was added to the solution, and the reaction mixture was stirred at room temperature for 40 minutes. The solvent was removed under reduced pressure, ethyl acetate was added to the residue and the solution was extracted twice with 1M aqueous HCl. A saturated _{aqueous solution of NaHCO 3} was added until the pH reached 8, and the solution was extracted with ethyl acetate. The solvent was removed under reduced pressure and the residue was dissolved in 6.4 mL of 34% aqueous acetonitrile solution and purified using an HPLC reversed phase column to give compound 25 (3.5 mg, 52% yield).

Acetic anhydride (34 μL) was added to compound 25 (3.5 mg, 11 μmol) dissolved in 1 mL DCM and 0.5 mL THF. The reaction mixture was stirred at room temperature for 17 hours and then evaporated in vacuo. The residue was dissolved in 3 mL of 20% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 26 (Ac-L-Phe-L-Ala-NHBn) (2.0 mg, 49% yield). ).

^{1 1} H NMR (CD ₃ OD, 400 MHz): δ8.2 (d, J = 7.6 Hz, 1H), 7.3 (d, J = 7.3 Hz, 1H), 8.02 (s, 1H), 7.33-7.15 (m, 10H), 4.63-4.57 (m, 1H), 4.40-4.30 (m, 1H), 3.11 (dd, J = 6.0) , 13.7Hz, 1H), 2.88 (dd, J = 8.7, 14.2Hz, 1H), 1.89 (s, 3H), 1.35 (d, J = 6.9Hz, 3H) ..
MS (LC / ESI-MS.m / z): calcd. for C ₂₁ H ₂₆ N ₃ O ₃ (M + H) ⁺ : 368.20, found: 368.33.

[Example 3]
Ac-L-Phe (F) -L-Ala-NHBn was synthesized.

Under a nitrogen atmosphere, 0.85 mL anhydrous DCM was added to compound 8 (30 mg, 0.10 mmol) and Oxyma (22 mg). The solution was stirred at 0 ° C. DIC (22 μL) was added to the solution and the reaction mixture was stirred for 8 minutes. The solution was warmed to room temperature over 5 minutes. Compound 22 (26 mg) was added to the solution and the reaction mixture was stirred at room temperature for 14.5 hours. To the solution was added saturated aqueous _NH 4 Cl and the resulting solution was extracted 4 times with DCM. The organic phase was _{washed with 1M NH 4} Cl aqueous solution, 1M NaHCO ₃ and brine. The organic phase was _{dried over Na 2} SO ₄ and then evaporated in vacuo. The residue was dissolved in 6 mL of 50% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 27 (6.7 mg, 15% yield).

Compound 27 (5.0 mg, 11 μmol) was dissolved in 4.75 mL of methanol. To the solution, 34 mM hydrazine monohydrate in ethanol (375 μL) was added dropwise on ice, and the reaction mixture was stirred at 0 ° C. for 10 minutes. The solution was warmed to room temperature over 2 hours. Hydrazine monohydrate (30 μL) was added to the reaction mixture and the resulting solution was stirred for 4.5 hours. The solvent was removed under reduced pressure. The residue was dissolved in 3 mL of 40% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 28 (1.3 mg, 36% yield).

Acetic anhydride (23 μL) was added to compound 28 (0.7 mg, 1.9 μmol) dissolved in 0.5 mL DCM and 0.5 mL THF. The reaction mixture was stirred at room temperature for 18 hours and then evaporated in vacuo. The residue was dissolved in 3 mL of 40% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 29 (Ac-L-Phe (F) -L-Ala-NHBn) (0.2 mg, yield). Rate 25%).

^{1 1} H NMR (CD ₃ OD, 400 MHz): δ7.39-7.252 (m, 10H), 6.35 (d, J = 7.8 Hz, 1H), 6.30 (s, 1H), 6. 06 (d, J = 7.2Hz, 1H), 5.76 (dd, J = 5.0, 46.1Hz, 1H), 4.93 (ddd, J = 5.0, 7.9, 19. 0Hz, 1H), 4.46-4.9 (m, 3H), 1.31 (d, J = 7.1Hz, 1H).
MS (LC / ESI-MS.m / z): calcd. for C ₂₁ H ₂₆ FN ₃ O ₃ (M + H) ⁺ : 386.19, found: 386.20.

[Comparative Example 4]
Ac-L-Phe-L-Leu-NHBn was synthesized.

Compound 30 (802 mg, 3.2 mmol) and benzylamine (420 μL) were dissolved in 16 mL of methanol, and DMTMM 3.2H ₂ O (1.17 g) was added to the solution. The reaction mixture was stirred at room temperature for 4.7 hours and evaporated in vacuo. DCM was added to the residue and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous, 1M HCl aqueous and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 8: 2 (volume ratio)) to give compound 31 (883 mg, yield 86%).

After adding 4M HCl (3 mL) in ethyl acetate to compound 31 (868 mg, 2.71 mmol) in 3 mL of ethyl acetate, the solution was stirred at room temperature for 2.5 hours. The solution was evaporated in vacuo to give compound 32 (38 mg, quantitative yield).

Compound 33 (67 mg, 0.25 mmol), compound 32 (79 mg), and DIPEA (52 μL) were dissolved in 2.5 mL of methanol, and DTMMM 1.3H ₂ O (95 mg) was added to the solution. The reaction mixture was stirred at room temperature for 13 hours and evaporated in vacuo. DCM was added to the residue, and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, 1M HCl aqueous solution, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 34 (99 mg, 84% yield).

4M HCl (3 mL) in ethyl acetate was added to compound 34 (99 mg, 0.21 mmol) in 3 mL of ethyl acetate, and then the solution was stirred at room temperature for 1.5 hours. The solution was evaporated in vacuo to give compound 35 (83 mg, quantitative yield).

DIPEA (25 μL) and acetic anhydride (132 μL) were added to compound 35 (50 mg, 0.14 mmol) in 3.8 mL DCM and 2 mL acetonitrile. The reaction mixture was stirred at room temperature for 13 hours and then evaporated in vacuo. The residue was dissolved in 6 mL of 50% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 36 (Ac-L-Phe-L-Leu-NHBn) (46 mg, 80% yield).

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.35-7.13 (m, 10H), 6.40-6.38 (m, 2H), 6.16 (d, J = 7.3Hz, 1H) , 4.68 (q, J = 7.3Hz, 1H), 4.34-4.32 (m, 3H), 3.03 (d, J = 6.9Hz, 2H), 1.94 (s, 3H), 1.77-1.67 (m, 1H), 1.60-1.44 (m, 2H), 0.89 (t, J = 6.0Hz, 6H).
MS (LC / ESI-MS.m / z): calcd. for C ₂₄ H ₃₂ N ₃ O ₃ (M + H) ⁺ : 410.25, found: 410.20.

[Example 4]
Ac-L-Phe (F) -L-Leu-NHBn was synthesized.

Compound 8 (40 mg, 0.13 mmol) and compound 32 (39 mg) were dissolved in 1.3 mL of methanol, and DMTMM 1.3H ₂ O (49 mg) was added to the solution. The reaction mixture was stirred at room temperature overnight and evaporated in vacuum. DCM was added to the reaction mixture, and the resulting solution was washed with 1M _{aqueous NaHCO 3} solution, 1M aqueous HCl solution, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was dissolved in 4 mL of 50% aqueous acetonitrile solution and purified using an HPLC reverse phase column to give compound 37 (6.3 mg, 10% yield).

Compound 37 (6.3 mg, 12 μmol) was dissolved in 1.5 mL of methanol, and the solution was stirred at 0 ° C. Hydrazine monohydrate (7.3 μL) was added to the solution, and the reaction mixture was stirred at 0 ° C. for 14 minutes. The mixture was warmed to room temperature over 7 minutes and methanol was added to the reaction mixture. The solvent was removed under reduced pressure. The residue was dissolved in acetonitrile / H ₂ O / MeOH (= 2 mL: 6 mL: 2 mL) and purified using an HPLC reverse phase column to give compound 38 (2.4 mg, 51% yield).

Acetic anhydride (46 μL) was added to compound 38 (2.0 mg, 4.0 μmol) in 2 mL DCM. The reaction mixture was stirred at room temperature overnight and then evaporated in vacuo. The residue was dissolved in 4 mL of 30% aqueous acetonitrile solution and purified using an HPLC reverse phase column to give compound 39 (Ac-L-Phe (F) -L-Leu-NHBn) (1.1 mg, yield). Rate 64%).

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.38-7.27 (m, 10H), 6.34-6.32 (m, 2H), 5.87 (d, J = 8.2Hz, 1H) , 5.76 (dd, J = 5.0, 46.3Hz, 1H), 4.93 (ddd, J = 5.0, 7.8, 19.2Hz, 1H), 4.64-4.37 (M, 3H), 1.98 (s, 3H), 1.72-1.65 (m, 1H), 1.53-1.35 (m, 2H), 0.87 (dd, J = 1) 8.8, 6.4Hz, 6H).
MS (LC / ESI-MS.m / z): calcd. for C ₂₄ H ₃₀ FN ₃ O ₃ Na (M + Na) ⁺ : 450.22, found: 450.40.

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

Compound 20 (61 mg, 0.32 mmol) and compound 22 (68 _{mg) were dissolved in 3 mL of methanol, and DMTMM 3.2H 2} O (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 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 40 (92 mg, 83% yield).

Compound 40 (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. _{A 1M aqueous solution of NaHCO 3} was added to the reaction mixture, and the obtained solution was stirred for 2 hours. The mixture was extracted 4 times with DCM. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 41.

Acetic anhydride (33 μL) was added to compound 41 (73 mg, 0.29 mmol) in 3 mL DCM. 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 solution and purified using an HPLC reverse phase column to give compound 42 (Ac-L-Ala-L-Ala-NHBn) (9 mg, 11% yield).

^{1 1} H NMR (CD ₃ OD, 400 MHz): δ7.33-7.20 (m, 5H), 4.42-4-33 (m, 3H), 4.29 (q, J = 6.9 Hz, 1H) ), 1.96 (s, 3H), 1.38 (d, J = 6.87Hz, 3H), 1.33 (d, J = 7.3Hz, 3H).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₅ H ₂₁ N ₃ O ₃ Na (M + Na) ⁺ : 314.15, found: 313.88.

[Example 5]
Ac-D, L-Ala (F3) -L-Ala-NHBn were synthesized.

D, L-trifluoroalanine hydrochloride (Compound 43) (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 warmed to room temperature over 21 hours. The solution was evaporated in vacuo and water was added to the residue. The solution was extracted 3 times with diethyl ether. A 1M aqueous HCl solution was added to the aqueous phase, and the solution was extracted 3 times with diethyl ether. The combined organic phases were _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 44 as a white solid (62 mg, 91% yield).

Compound 44 (40 mg, 0.16 mmol) and compound 22 (29 mg) were dissolved in 1.5 mL of methanol, and DMTMM 3.2H ₂ O (62 mg) was added to the solution. The reaction mixture was stirred at room temperature for 11.5 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 45 (45 mg, 68% yield).

Compound 45 (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. _{A 1M aqueous solution of NaHCO 3} was added to the reaction mixture, and the obtained solution was stirred for 2 hours. The mixture was extracted 4 times with DCM. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 46 (41 mg, quantitative yield).

Acetic anhydride (67 μL) was added to compound 46 (41 mg, 0.14 mmol) in 3 mL DCM. 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 solution and purified using an HPLC reverse phase column to give compound 47 (Ac-D, L-Ala (F3) -L-Ala-NHBn) as a white solid. (0.3 mg, yield 1%). The molecule was obtained as a diastereomix and used as is in the permeability assay.

^{1 1} H NMR (CD ₃ OD, 400 MHz): 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).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₅ H ₁₈ F ₃ N ₃ O ₃ Na (M + Na) ⁺ : 368.12, found: 367.92.

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

Compound 1 (700 mg, 2.34 mmol) and isobutylamine (181 μL) were dissolved in 23 mL of methanol, and DMTMM 1.3H ₂ O (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 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 4: 6 (volume ratio)) to give compound 48 (523 mg, 65% yield).

Compound 48 (523 mg, 1.48 mmol), palladium carbon 10% (55 mg) and 7.4 mL methanol were added to the recovery flask. _{H 2} 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 49 (319 mg, 98% yield).

Compound 5 (45 mg, 0.2 mmol) and compound 49 (53 mg) were dissolved in 2 mL of methanol and the solution was stirred at room temperature. DMTMM 1.3H ₂ O (74 mg) was added to the solution. The reaction mixture was stirred at room temperature for 23 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to obtain Compound 50 (9.1 mg, yield 11%).

Compound 50 (9.1 mg, 21 μmol), 10% palladium carbon (2.8 mg), and 2 mL of methanol were added to the recovery flask. _{H 2} 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 51 (7.4 mg, quantitative yield).

Acetic anhydride (23 μL) was added to compound 51 (4 mg, 14 μmol) in 1 mL DCM and 0.2 mL NMP. 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 a 14% aqueous acetonitrile solution and purified using an HPLC reversed-phase column to give compound 52 (Ac-L-Ala-L-Phe-iBu) (2.0 mg, yield). 43%).

¹ H NMR (CDCl ₃ , 400 MHz): δ7.32-7.19 (m, 10H), 6.62 (d, J = 7.7 Hz, 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).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₈ H ₂₇ N ₃ O ₃ Na (M + Na) ⁺ : 356.20, found: 356.23.

[Example 6]
Ac-D and L-Ala (F3) -L-Phe-iBu were synthesized.

Compound 44 (41 mg, 0.17 mmol) and compound 49 (45 mg) were dissolved in 1 mL of methanol and 0.7 mL of DCM, and DMTMM 1.3H ₂ O (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, the solution 1M NaHCO ₃ aqueous solution obtained, saturated aqueous _NH 4 Cl, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 53 (47 mg, 63% yield).

4M HCl (3 mL) in ethyl acetate was added to compound 53 (47 mg, 0.11 mmol) in 3 mL of ethyl acetate, and then the solution was stirred at room temperature for 20 minutes. The solution was evaporated in vacuo to give compound 54 (45 mg, quantitative yield).

Acetic anhydride (45 μL) was added to compound 54 (30 mg, 79 μmol) in 2 mL DCM. The reaction mixture was stirred at room temperature for 3 hours and then evaporated in vacuo. The residue was dissolved in acetonitrile / H ₂ O / MeOH (= 2.4 mL: 3.6 mL: 2 mL), purified using an HPLC reverse phase column, and compound 55 (Ac-D, L-Ala (F3)- L-Phe-iBu) was obtained (9.4 mg, yield 30%). The molecule was obtained as a diastereomix and used as is in the permeability assay.

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.34-7.21 (m, 5H), 6.82-6.76 (m, 1H), 6.41 (d, J = 6.4 Hz, 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) ).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₈ H ₂₄ F ₃ N ₃ O ₃ Na (M + Na) ⁺ : 410.17, found: 410.19.

[Comparative Example 7]
Ac-L-Val-L-Ala-NMe ₂ was synthesized.

N-Carbobenzoxyl-L-valine (Compound 56) (60 mg, 0.24 mmol) and Compound 13 (33 mg) were dissolved in 2.2 mL of methanol, and DMTMM 1.3H ₂ O (91 mg) was added to the solution. added. 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 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 57 (75 mg, 89% yield).

Compound 57 (773 mg, 3.10 mmol), palladium carbon 10% (7.5 mg), and 2.1 mL of methanol were added to the recovery flask. _{H 2} 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 58 (35 mg, 76% yield).

Acetic anhydride (18 μL) was added to compound 58 (35 mg, 0.16 mmol) in 0.8 mL DCM. 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 solution and purified using an HPLC reversed-phase column to give compound 59 (Ac-L-Val-L-Ala-NMe ₂ ) as a white solid (27 mg, yield). 65%).

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.10 (d, J = 7.3 Hz, 1H), 6.41 (d, J = 8.7 Hz, 1H), 4.87 (quin, J = 6. 9,1H), 4.37 (dd, J = 6.1,2.6Hz, 1H), 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).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₂ H ₂₃ N ₃ O ₃ Na (M + Na) ⁺ : 280.16, found: 280.05.

[Example 7]
Ac-D and L-Val (F6) -L-Ala-NMe ₂ were synthesized.

D, L-Hexafluorovaline (Compound 60) (50 mg, 0.22 mmol) was dissolved in 2.5 mL of acetonitrile. To this solution was added di-tert-butyl dicarbonate (49 μL) at 0 ° C. The reaction mixture was warmed 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. A 1M aqueous HCl solution was added to the aqueous phase, and the resulting solution was extracted 3 times with diethyl ether. The combined organic phases were _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 61 (63 mg, 87% yield).

Compound 61 (50 mg, 0.15 mmol) and compound 13 (21 mg) were dissolved in 0.7 mL of methanol, and DMTMM 1.3H ₂ O (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 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 62 (50 mg, 77% yield).

After adding 4M HCl (2.5 mL) in ethyl acetate to compound 62 (50 mg, 0.12 mmol), 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 solution. A 1 M aqueous NaOH solution was added to the aqueous phase until the pH reached 11. The solution was extracted 3 times with DCM and the organic phase was dried over _{Na 2} SO _4. The solvent was removed under reduced pressure to give compound 63 (38 mg, quantitative yield).

Acetic anhydride (13 μL) was added to compound 63 (38 mg, 0.12 mmol) in DCM (1.2 mL). 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 solution and purified using an HPLC reversed-phase column to give compound 64 as a white solid (27 mg, 63% yield). The molecule was obtained as a diastereomix and used as is in the permeability assay.

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.57 (d, J = 6.4 Hz, 0.5H), 7.39 (d, J = 6.9 Hz, 0.5H), 6.22 (d, J = 9.6Hz, 0.5H), 6.08 (d, J = 9.6Hz, 0.5H), 5.41 (t, J = 9.2Hz, 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).
MS (MALDI-TOF MS.m / z): calcd. for C ₁₂ H ₁₇ F ₆ N ₃ O ₃ Na (M + Na) ⁺ : 388.11, found: 388.18.

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

Compound 56 (52 mg, 0.2 mmol) and compound 49 (53 mg) were dissolved in 2 mL of methanol and the solution was stirred at room temperature. DMTMM 1.3H ₂ O (72 mg) was added to the solution. The reaction mixture was stirred at room temperature for 23 hours and evaporated in vacuo. DCM was added to the reaction mixture, and the resulting solution was _{washed with 1M Na 2} CO ₃ aqueous solution, water, 1M HCl aqueous solution, water, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 65 (60 mg, 67% yield).

Compound 65 (60 mg, 0.13 mmol), palladium carbon 10% (6 mg), and 1.3 mL of methanol were added to the recovery flask. _{H 2} 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 66 (39 mg, 92% yield).

Compound 66 (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 / H ₂ O / MeOH (= 1.8 mL: 2.9 mL: 1.5 mL), purified using an HPLC reverse phase column, and compound 67 (Ac-D, L-Val (F6)). ) -L-Ala-MeOH ₂ ) was obtained (7.2 mg, yield 43%).

^{1 1} H NMR (CDCl ₃ , 400 MHz): δ7.30-7.18 (m, 5H), 6.51 (d, J = 7.3 Hz, 1H), 5.92 (d, J = 7.8 Hz, 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) ..
MS (MALDI-TOF MS.m / z): calcd. for C ₂₀ H ₃₁ N ₃ O ₃ Na (M + Na) ⁺ : 384.23, found: 384.13.

[Example 8]
Ac-D and L-Val (F6) -L-Phe-iBu were synthesized.

Compound 61 (70 mg, 0.22 mmol) and compound 49 (57 mg) were dissolved in 1.2 mL of methanol, and DMTMM 1.3H ₂ O (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 obtained solution was washed with saturated _{aqueous NaHCO 3} solution, 1M aqueous HCl solution, and brine. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel column chromatography (DCM / methanol = 19: 1 (volume ratio)) to give compound 68 (79 mg, 69% yield).

4M HCl (3 mL) in ethyl acetate was added to compound 68 in 3 mL of ethyl acetate. The solution was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure to give compound 69 (53 mg, 76% yield).

DIPEA (16 μL) and acetic anhydride (45 μL) were added to compound 69 (40 mg, 86 μmol) in 2 mL DCM. The reaction mixture was stirred at room temperature for 8.5 hours and then evaporated in vacuo. The residue was dissolved in acetonitrile / H ₂ O / MeOH (= 3 mL: 3 mL: 8 mL), purified using an HPLC reverse phase column, and compound 70 (Ac-D, L-Val (F6) -L-Phe- iBu) was obtained as a white solid (5.7 mg, yield 13%). The molecule was obtained as a diastereomix and used as is in the permeability assay.

^{1 1} H NMR (CD ₃ OD ₃ , 400 MHz): δ7.29-7.17 (m, 5H), 5.29 (dq, J = 7.8, 23.8 Hz, 1H), 4.63-4. 58 (m, 1H), 3.15-3.05 (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).
MS (MALDI-TOF MS.m / z): calcd. for C ₂₀ H ₂₅ F ₆ N ₃ O ₃ Na (M + Na) ⁺ : 492.17, found: 491.97.

[Example 9]
Cbz-Ala (F3) -OH was synthesized.

N-Carbobenzoxioxysuccinimide (72) (245 mg, 0.98 mmol) and TEA (272 μL, 1.96 mmol) were dissolved in 3 mL of THF. In this solution, D, L-trifluoroalanine hydrochloride (71) (160 mg, 0.89 mmol) was dissolved in 6 mL of water and added dropwise at 0 ° C. The reaction mixture was stirred at 0 ° C. for 10 minutes, warmed to room temperature over 7 hours, then N-carbobenzoxoxysuccinimide (72) (222 mg, 0.89 mmol) was added to add 17 to the reaction mixture. Stir for hours.
A 1M _{aqueous NaHCO 3} solution was added to the reaction mixture and the mixture was extracted 3 times with DCM. A 1M aqueous HCl solution was added to the aqueous phase to acidify the solution, and the solution was extracted 3 times with ethyl acetate. The combined organic phases are _{dried on Na 2} SO ₄ and evaporated in vacuum, the residue is purified by silica gel column chromatography (DCM / methanol = 9: 1 (volume ratio)) and an HPLC reversed phase column is used. Purified to give compound 73 (Cbz-Ala (F3) -OH) as a white solid (85 mg, 35% yield).

^{1 1} H NMR (CD ₃ OD, 400 MHz): δ8.17 (d, 1H, J = 9.2 Hz), 7.39-7.29 (m, 5H), 5.15 (s, 2H), 5. 04-4.95 (m, 1H).

A chiral column was used in the HPLC system to separate the racemic Cbz-Ala (F3) -OH. Cbz-L-Ala (F3) -OH was obtained as a white solid (48 mg, 20% yield).

^{1 1} H NMR (CD ₃ OD, 400 MHz): δ7.46-7.32 (m, 5H), 5.21 (s, 2H), 5.10-5.03 (m, 1H).

Fmoc-Tyr-Oallyl was synthesized according to the method of Morimoto et al. (Non-Patent Document 7).

[Example 10]
Cyclic peptide 1_TriF was synthesized.

Compound 74 was manually synthesized on 2-chlorotrityl polystyrene resin (1.92 mmol / g). The resin (62 mg, 0.12 mmol) was swollen with DCM in a 6 mL frit syringe with continuous shaking. By dissolving DIPEA (83 μL, 0.48 mmol) and Fmoc-Leu-OH (42 mg, 0.24 mmol) in 1.2 mL of DCM and shaking the resin at room temperature for 2 hours in the reaction solution. The first residue was extended on the resin. The resin after the reaction was washed 3 times each with DCM / methanol / DIPEA = 17/2/1 (volume ratio) and DCM. The resin was applied to the peptide synthesis of the second and subsequent residues. Fmoc deprotection was performed by incubating the resin with 20% piperidine in DMF for 15 minutes. The resin after the reaction was washed 3 times with DMF.

Coupling reactions other than Cbz-L-Ala (F3) -OH use DMF (3 equivalents), Fmoc-protected amino acids (3 equivalents), HATU (3 equivalents), HOAt (3 equivalents), and DIPEA (3 equivalents). It was reacted with Fmoc-protected amino acids in 0.2 M) for 1 to 3 hours. The resin after the coupling reaction was washed with DMF three times each.
The coupling reaction of Cbz-L-Ala (F3) -OH was carried out using Cbz-L-Ala (F3) -OH (2 eq), Oxyma (2 eq), and DIC (2 eq) using DMF (2 eq). It was reacted with Cbz-L-Ala (F3) -OH in 0.1 M). The resin after the coupling reaction was washed with DMF three times each.

Coupling and deprotection were repeated up to the 6th residue. The synthesized peptide was cleaved from the resin by incubating the resin with 30% HFIP in DCM for 15 minutes three times. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was then washed with DCM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 74.

Compound 74 (34 mg, 36 μmol), 10% palladium carbon (6.8 mg), and 2 mL of methanol were added _{to the recovery flask, H 2} was introduced, and the mixture in the recovery flask was allowed to stand at room temperature for 14.5 hours. Stirred. The resulting reaction mixture was filtered through Celite and then the solvent was removed under reduced pressure to give compound 75.

Compound 75 (19 mg, 15 μmol), HOAt (6.4 mg), PyAOP (24.5 mg), and DMF (6 mL) were added to the recovery flask. The mixture in the recovery flask was stirred at room temperature for 18 hours. The solvent of the resulting reaction mixture was removed under reduced pressure to give compound 76.

Compound 76, TIPS of 50 [mu] L, _H 2 O of 50 [mu] L, and TFA for 1.9 mL, was added dropwise on ice. The resulting reaction mixture was stirred on ice for 10 minutes, warmed to room temperature over 80 minutes, and then the solvent was removed under reduced pressure. The residue was purified using an HPLC reverse phase column to give compound 77 (Cyclic peptide 1_TriF).

MS (LC / ESI-MS.m / z): calcd. for C ₃₅ H ₅₂ F ₃ N ₆ O ₇ (M + H) ⁺ : 725.39, found: 725.81

[Example 11]
Cyclic peptide 1_MonoF was synthesized.

Compound 78 was manually synthesized on 2-chlorotrityl polystyrene resin (1.69 mmol / g). The resin (131 mg, 0.22 mmol) was swollen with DCM in a 6 mL frit syringe with continuous shaking. DIPEA (39 μL) and Fmoc-Leu-OH (162 mg) were dissolved in 2.2 mL of DCM, and the resin was shaken and stirred at room temperature for 4.5 hours in the reaction solution to make the first residue resin. Stretched up. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA = 17/2/1 (volume ratio) and DCM. This resin was applied to the peptide synthesis of the second and subsequent residues. Fmoc deprotection was performed by incubating the resin with 20% piperidine in DMF for 15 minutes. After the reaction, the resin was washed 3 times with DMF.

Coupling reactions other than Cbz-L-Ala (F) -OH, which will be described later, use Fmoc-protected amino acids (4 equivalents), Oxyma (4 equivalents), DIC (4 equivalents), and DIPEA (4 equivalents). The reaction was carried out in DMF (0.2 M for Fmoc-protected amino acid) for 1 to 3 hours.

The coupling reaction of Cbz-L-Ala (F) -OH ((R) -2-(((Benzyloxy) carbonyl) amino) -3-fluoropropanoic acid, manufactured by CLDpharm) is Cbz-L-Ala (F). ) -OH (2 eq), Oxyma (2 eq), and DIC (2 eq) were used to react in DMF (0.2 M to Cbz-L-Ala (F) -OH).

After the reaction, the resin was washed 3 times each with DMF. Coupling and deprotection were repeated up to the 6th residue. The synthesized peptide was cleaved from the resin by incubating the resin with 30% HFIP in DCM for 15 minutes three times. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was washed with DCM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 78.

Compound 78 (120 mg, 0.13 mmol), 10% palladium carbon (12 mg), and 2.7 mL of methanol were added to the recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 15.5 hours. The resulting reaction mixture was filtered through Celite and the solvent was removed under reduced pressure to give compound 79.

Compound 79 (57 mg, 75 μmol), HOAt (15 mg), PyAOP (59 mg), and DMF (7.5 mL) were added to the recovery flask. After stirring the mixture at room temperature for 29 hours, the solvent was removed under reduced pressure to give compound 80.

Compound 80 was added dropwise TIPS of 50 [mu] L, _H 2 O of 50 [mu] L, and TFA in 1.9mL ice. The reaction mixture was stirred on ice for 80 minutes, warmed to room temperature over 80 minutes and then the solvent was removed under reduced pressure. The residue was purified using an HPLC reverse phase column to give compound 81 (Cyclic peptide 1_MonoF).

MS (LC / ESI-MS.m / z): calcd. for C ₃₅ H ₅₃ FN ₆ O ₇ Na (M + Na) ⁺ : 711.39, found: 711.70.

[Comparative Example 9]
Cyclic peptide 1_NonF was synthesized.

Compound 82 was manually synthesized on 2-chlorotrityl polystyrene resin (1.6 mmol / g). The resin (105 mg, 0.17 mmol) was swollen with 2 mL of DCM with continuous shaking in a 6 mL frit syringe. DIPEA (58 μL) and Fmoc-Tyr-OAll (15 mg) were dissolved in 0.4 mL of DCM, and the resin was stirred at room temperature for a long time in the reaction solution to extend the first residue onto the resin. .. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA = 17/2/1 (volume ratio) and DCM. This resin was applied to the peptide synthesis of the second and subsequent residues. Fmoc deprotection was performed by incubating the resin with 20% piperidine in DMF for 15 minutes. After the reaction, the resin was washed 3 times with DMF.

The coupling reaction used Fmoc-protected amino acids (4 eq), HATU (3.8 eq), HOAt (3.8 eq), and DIPEA (6 eq) to 0. The reaction was carried out in 1M) for 1 to 3 hours. After the reaction, the resin was washed 3 times each with DMF. Coupling and deprotection were repeated up to the 6th residue.

The allyl group of the C-terminal Tyr residue was removed by incubating the resin with tetrakis (triphenylphosphine) palladium (1 eq) and triphenylphosphine (3 eq) in anhydrous DCM for 1 hour at room temperature. The resin was washed 3 times each with 0.5% sodium diethyldithiocarbamate in DMF, 0.5% DIPEA in DMF, and DMF.
The resin was incubated with 20% piperidine / DMF for 3 minutes and then with 20% piperidine / DMF for 12 minutes to deprotect the N-terminal Fmoc of the peptide on the resin. After washing the resin 3 times with DMF, the peptides on the resin were combined with PyBOP (3 eq), HOAt (3 eq), DIPEA (6 eq) in anhydrous DMF (0.2 M relative to PyBOP) 19 It was cyclized by incubation for hours. After the reaction, the resin was washed 3 times each with DMF and DCM.
The synthesized peptide was cleaved from the resin by incubating the resin with 5% TFA in DCM for 60 minutes. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was washed twice. All the filtrate and the washing solution were combined, the solvent was evaporated under reduced pressure, and the obtained residue was purified using an HPLC reverse phase column to obtain Compound 82 (Cyclic peptide 1_NonF).

MS (LC / ESI-MS.m / z): calcd. for C ₃₅ H ₅₄ N ₆ O ₇ Na (M + Na) ⁺ : 693.40, found: 693.80.

[Comparative Example 10]
Cyclic peptide 2_NonF was synthesized.

Compound 83 was manually synthesized on 2-chlorotrityl polystyrene resin (1.80 mmol / g). The resin (200 mg, 0.36 mmol) was swollen with DCM in a 6 mL frit syringe with continuous shaking. By dissolving DIPEA (245 μL, 1.44 mmol) and Fmoc-Ala-OH (224 mg, 0.72 mmol) in 3.6 mL of DCM and stirring the resin in the reaction solution at room temperature for 2 hours. The first residue was extended onto the resin. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA = 17/2/1 (volume ratio) and DCM. This resin was applied to the peptide synthesis of the second and subsequent residues. DFmoc deprotection was performed by incubating the resin with 20% piperidine in DMF for 15 minutes. After the reaction, the resin was washed 3 times with DMF.

The coupling reaction was 1 in DMF (0.2 M eq for Fmoc protected amino acids) using Fmoc protected amino acids (4 eq), HATU (4 eq), HOAt (4 eq) and DIPEA (4 eq). It was allowed to react for ~ 3 hours. After the reaction, the resin was washed with DMF three times each. Coupling and deprotection were repeated up to the 7th residue.

The synthesized peptide was cleaved from the resin by incubating the resin with 30% HFIP in DCM for 15 minutes three times. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was then washed with DCM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 83.

Compound 83, HOAt (37 mg), PyAOP (141 mg), DIPEA (138 μL), and DMF (18 mL) were added to the recovery flask. The mixture was stirred at room temperature for 13 hours and then the solvent was removed under reduced pressure to give compound 84.

Compound 84, 100 [mu] L of TIPS, a 100 [mu] L of _{H 2} O and 3.8mL of TFA, it was added dropwise on ice. The reaction mixture was stirred on ice for 10 minutes and then warmed to room temperature over 60 minutes. Then, the solvent of the reaction mixture was removed under reduced pressure, and the obtained residue was purified using an HPLC reverse phase column to obtain Compound 85 (Cyclic peptide 2_NonF).

MS (LC / ESI-MS.m / z): calcd. for C ₃₈ H ₅₃ N ₁₀ O ₁₀ (M + H) ⁺ : 809.40, found: 809.95.

[Example 12]
Cyclic peptide 2_TriF was synthesized.

Compound 86 was manually synthesized on 2-chlorotrityl polystyrene resin (1.80 mmol / g). The resin (200 mg, 0.36 mmol) was swollen with DCM in a 6 mL frit syringe with continuous shaking. DIPEA (245 μL, 1.44 mmol) and Fmoc-Ala-OH (224 mg, 0.72 mmol) are dissolved in 3.6 mL of DCM and the resin is shaken and stirred at room temperature for 2 hours in the reaction solution. The first residue was elongated on the resin. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA = 17/2/1 (volume ratio) and DCM. This resin was applied to the peptide synthesis of the second and subsequent residues. Fmoc deprotection was performed by incubating the resin with 20% piperidine in DMF for 15 minutes. After the reaction, the resin was washed 3 times with DMF.

Coupling reactions other than Cbz-L-Ala (F3) -OH use DMF (Fmoc) using Fmoc-protected amino acids (3 equivalents), HATU (3 equivalents), HOAt (3 equivalents) and DIPEA (3 equivalents). It was reacted with the protected amino acid in 0.2 M) for 1 to 3 hours.
The coupling reaction of Cbz-L-Ala (F3) -OH was carried out using Cbz-L-Ala (F3) -OH (2 eq), Oxyma (2 eq), and DIC (2 eq) using DMF (2 eq). It was reacted with Cbz-L-Ala (F3) -OH in 0.1 M).

After the reaction, the resin was washed 3 times each with DMF. Coupling and deprotection were repeated up to the 6th residue. The synthesized peptide was cleaved from the resin by incubating the resin with 30% HFIP in DCM for 15 minutes three times. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was then washed with DCM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 86.

Compound 86, 10% palladium carbon (12.4 mg), and 2 mL of methanol were added to the recovery flask. _{H 2} was introduced into the flask and the mixture was stirred at room temperature for 14.5 hours. The resulting reaction mixture was filtered through Celite and the solvent was removed under reduced pressure to give compound 87.

Compound 87 (35 mg), HOAt (8.0 mg), PyAOP (28.5 mg), and DMF (5.3 mL) were added to the recovery flask. After stirring the mixture at room temperature for 18 hours, the solvent was removed under reduced pressure to give compound 88.

Compound 88, TIPS of 50 [mu] L, _H 2 O of 50 [mu] L, and TFA for 1.9 mL, was added dropwise on ice. The reaction mixture was stirred on ice for 10 minutes, warmed to room temperature over 80 minutes, and then the solvent was removed under reduced pressure. The obtained residue was purified using an HPLC reverse phase column to obtain Compound 89 (Cyclic peptide 2_TriF).

MS (LC / ESI-MS.m / z): calcd. for C ₃₈ H ₄₈ F ₃ N ₁₀ O ₁₀ (MH) ⁺ : 861.35, found: 861.64.

[Comparative Example 11]
Ac-L-Ala-L-Val-NHEPh was synthesized.

Compound 90 (401 mg, 1.84 mmol) and phenethylamine (278 μL) were dissolved in 18.4 mL of methanol and the resulting solution was stirred at room temperature. DMTMM nH ₂ O (665 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 twice with water. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo to give compound 91 (539 mg, 91% yield).

4M HCl in ethyl acetate was added to the obtained compound 91, and then the solution was stirred at room temperature for 20 minutes. The solution was evaporated in vacuo to give compound 92 (372 mg, quantitative yield).

Boc-Ala-OH (52 mg) and compound 92 (64 mg) were dissolved in 2.5 mL of methanol and the resulting solution was stirred at room temperature. DMTMM · nH ₂ O (92 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 3 times with water. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo. The residue was purified by silica gel chromatography (DCM / methanol = 19/1 (volume ratio)) to give compound 93 (98 mg, 95% yield).

4M HCl (6 mL) in ethyl acetate was added to compound 93 in 6 mL of ethyl acetate, and then the solution was stirred at room temperature for 3 hours. The solution was evaporated in vacuo to give compound 94 (142 mg, quantitative yield).

Compound 94 (142 mg) was dissolved in 8 mL of DCM and DIPEA (150 μL) and acetic anhydride (400 μL) were added. The reaction mixture was stirred at room temperature for 17.5 hours and then evaporated in vacuo. Water was added to the residue and eluted with DCM twice. The organic phase was _{dried over Na 2} SO ₄ and evaporated in vacuo, and the resulting residue was purified by silica gel chromatography (DCM / methanol = 19/1 (volume ratio)). The residue was dissolved in acetonitrile / H ₂ O / methanol (= 2.4 mL / 5.6 mL / 15 mL), purified using an HPLC reverse phase column, and compound 95 (Ac-L-Ala-L-Val-). NHEPh) was obtained (9.2 mg, yield 6%).

^{1 1} H NMR (methanol-d ₄ , 400 MHz): deruta 7.29-7.16 (m, 5H), 4.36 (q, J = 7.3 Hz, 1H), 4.09 (d, 6.9 Hz, 1H), 3.52-3.35 (m, 2H), 2.80 (t, J = 7.3Hz, 2H), 2.02-1.99 (m, 1H), 1.98 (s, 3H), 1.32 (d, J = 7.3Hz, 3H), 0.83 (dd, J = 2.3, 6.9Hz, 6H).
MS (LC-ESI-MS.m / z): calcd. for C ₁₈ H ₂₈ N ₃ O ₃ (M + H) ⁺ : 334.22, found: 334.54.

[Example 13]
Ac-L-Ala (F3) -L-Val-NHEPh was synthesized.

Compound 96 (Ac-L-Ala (F3) -L-Val-NHEPh) was synthesized in the same manner as in Comparative Example 6.

^{1 1} H NMR (methanol-d ₄ , 400 MHz): δ7.29-7.16 (m, 5H), 5.40 (q, J = 7.8 Hz, 1H), 4.12 (d, J = 7. 8Hz, 1H), 3.50-3.38 (m, 2H), 2.80 (t, J = 7.3Hz, 2H), 2.05 (s, 3H), 2.03-1.94 ( m, 1H), 0.88 (dd, J = 5.5, 6.4Hz, 6H).
MS (LC-ESI-MS.m / z): calcd. for C ₁₈ H ₂₅ F ₃ N ₃ O ₃ (M + H) ⁺ : 388.20, found: 388.53.

[Example 14]
Ac-L-Ala (F3) -L-Phe-iBu was synthesized.

Compound 97 (Ac-L-Ala (F3) -L-Phe-iBu) was synthesized in the same manner as in Comparative Example 6.

^{1 1} H NMR (methanol-d ₄ , 400 MHz): deruta 7.27-7.17 (m, 5H), 5.32 (q, J = 7.8 Hz, 1H), 4.61 (t, J = 7. 3Hz, 1H), 3.10-2.86 (m, 4H), 2.01 (s, 3H), 1.66 (m, 1H), 0.80 (dd, J = 6.4,8. 7Hz.6H).
MS (LC-ESI-MS.m / z): calcd. for C ₁₈ H ₂₅ F ₃ N ₃ O ₃ (M + H) ⁺ : 388.19, found: 388.48.

[Comparative Example 12]
Cbz-L-Ala-L-Ala-L-Ala-iBu was synthesized.

Compound 98 was synthesized on 2-chlorotrityl chloride polystyrene resin (1.80 mmol / g). First, dehydrated DCM was added to the resin (237 mg, 0.43 mmol), and the resin was swollen by shaking in a 6 mL frit syringe. Fmoc-Ala-OH (254 mg) and DIPEA (297 μL) were dissolved in 4.3 mL of dehydrated DCM, and the resin was shaken and stirred at room temperature for 2 hours in the obtained reaction solution to make the first residue of the resin. Stretched up. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA (= 17/2/1 (volume ratio)), DCM, and DMF. Peptide elongation after the second residue was performed on the resin.

Fmoc deprotection was performed by incubating the resin in 20% piperidine / DMF for 15 minutes. After the deprotection reaction, the resin was washed 3 times with DMF.

The condensation reaction was carried out using Fmoc-Ala-OH (4 equivalents), Oxyma (4 equivalents), and DIC (4 equivalents) in 4 mL of DMF for 1 to 13 hours. After the condensation reaction, the resin was washed 3 times with DMF.

After stretching and deprotecting to 3 residues, 2 of the resin was dissolved in 1.1 mL of DMF in which N-carbobenzoxoxysuccinimide (5 equivalents) and DIPEA (10 equivalents) were dissolved. The N-terminus was Cbz protected by incubation at room temperature for hours.

The synthesized peptide was cleaved from the resin by incubating the resin in 30% HFIP / DCM three times for 15 minutes each. The filtrate was collected in a flask. The resin was then washed with DCM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 98.

Compound 98 (11 mg) was dissolved in 0.3 mL of DMF, Oxyma (6.4 mg) and DIC (7.0 μL) were added, and the mixture was stirred at room temperature for 10 minutes. Isobutylamine (4.4 μL) was added to the reaction solution, and the mixture was stirred overnight at room temperature. The solution was evaporated under reduced pressure, after dissolved in 40% acetonitrile / _{H 2} O, and purified using reverse phase column HPLC, Compound 99 (Cbz-L-Ala- L-Ala-L-Ala- iBu) was obtained (3.6 mg, yield 29%).

MS (LC-ESI-MS.m / z): calcd. for C ₂₁ H ₃₂ N ₄ O ₅ Na (M + Na) ⁺ : 443.23, found: 443.50

[Example 15]
Cbz-L-Ala (F) -L-Ala (F) -L-Ala (F) -iBu was synthesized.

In a flask, compound 100 (Cbz-L-Ala (F) -OH) (50 mg, 207 μmol) and isobutylamine (22.7 μL) were dissolved in 3 mL of methanol and stirred at room temperature. DMTMM (68 mg) was added to the solution, and the mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure, DCM was added to the obtained residue, and the mixture was washed once with water. After drying the organic phase with Na ₂ SO ₄ , the solvent was removed under reduced pressure to give compound 101 (54 mg).

To compound 101 (54 mg) in the flask, 10% palladium carbon (5.8 mg) and methanol (5 mL) were added, the inside of the flask _{was replaced with H 2} , and then the mixture was stirred at room temperature for 4 hours. The resulting reaction mixture was filtered through Celite and the solvent was removed under reduced pressure to give compound 102 (34 mg).

Cbz-L-Ala (F) -OH (26 mg) was added to compound 102 (34 mg) in the flask, and these were dissolved in 3 mL of methanol and stirred at room temperature for 10 minutes. DMTMM (48 mg) was added to the solution, and the mixture was stirred at room temperature for 13 hours. The solvent was removed under reduced pressure, H _{2 O} was added to the resulting residue, and extracted three times with DCM. After drying the organic phase with Na ₂ SO ₄ , the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (DCM / MeOH = 19/1 (volume ratio)) to obtain compound 103 (44 mg).

To compound 103 (44 mg) in the flask, 10% palladium carbon (7.8 mg) and methanol (4 mL) were added, the inside of the flask _{was replaced with H 2} , and the mixture was stirred at room temperature for 18 hours. The resulting reaction mixture was filtered through Celite and the solvent was removed under reduced pressure to give compound 104 (7.1 mg).

Cbz-L-Ala (F) -OH (6.2 mg) was added to compound 104 (7.1 mg) in the flask, and these were dissolved in 4 mL of methanol and stirred at room temperature for 10 minutes. DMTMM (10 mg) was added to the solution, and the mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, residue was dissolved in 40% acetonitrile / _{H 2} O, and purified using reverse phase column HPLC, Compound 105 (Cbz-L-Ala ( F) -L-Ala (F ) -L-Ala (F) -iBu) was obtained (0.2 mg, yield 1%).

MS (LC-ESI-MS.m / z): calcd. for C ₂₁ H ₂₉ F ₃ N ₄ O ₅ Na (M + Na) ⁺ : 496.47, found: 496.46.

[Comparative Example 13]
Cyclic peptide 3_LK1-L3A was synthesized.

Compound 106 (Cyclic peptide 3_LK1-L3A) was synthesized in the same manner as in Comparative Example 10.

MS (LC-ESI-MS.m / z): calcd. for C ₃₅ H ₅₅ N ₆ O ₇ (M + H) ⁺ : 671.42, found: 671.72

[Example 16]
Cyclic peptide 3_LK1-L3A (F3) was synthesized.

Compound 107 was synthesized on 2-chlorotrityl chloride polystyrene resin (1.80 mmol / g). Dehydrated DCM was added to the resin (300 mg, 0.54 mmol) and swollen by shaking in a 6 mL frit syringe. Fmoc-Leu-OH (391 mg) and DIPEA (378 μL) were dissolved in dehydrated DCM (5.4 mL), and the resin was shaken and stirred at room temperature for 2 hours in the obtained reaction solution to obtain the first residue. It extended on the resin. After the reaction, the resin was washed 3 times each with DCM / methanol / DIPEA (= 17/2/1 (volume ratio)), DCM, and DMF. Peptide elongation after the second residue was performed on the resin.

Fmoc deprotection was performed by incubating the resin in 20% piperidine / DMF for 15 minutes. After the deprotection reaction, the resin was washed 3 times with DMF.

Condensation reactions other than Cbz-l-Ala (F3) -OH, which will be described later, use Fmoc amino acid (4 equivalents), Oxyma (4 equivalents), and DIC (4 equivalents), and DMF (Fmoc amino acid is 0.2 M). The reaction was carried out at room temperature for 1 to 12 hours. After the condensation reaction, the resin was washed 3 times with DMF.

The condensation reaction of Cbz-l-Ala (F3) -OH is Cbz-l-Ala (F3) -OH (2.5 eq), HOBt (2.5 eq), DIC (2.5 eq), and CuCl. ₂ (1 eq) was used and reacted at 4 ° C. for 22 hours in DMF / DCM = 1/1. After the condensation reaction, the resin was washed 3 times with DCM.

The synthesized peptide was cleaved from the resin by incubating the resin in 30% HFIP / DCM three times for 15 minutes each. All filtrates containing the synthesized peptides were collected in recovery flasks. The resin was washed with CM and MeOH. All filtrates and washings were combined and the solvent evaporated under reduced pressure to give compound 107.

To compound 107 (21 mg) in the recovery flask, 10% palladium carbon (5.5 mg) and methanol were added, the inside of the recovery flask _{was replaced with H 2} , and then the mixture was stirred at room temperature for 4 hours. The resulting reaction mixture was filtered through Celite, the solvent was removed under reduced pressure and the resulting residue was purified using an HPLC reversed phase column to give compound 108 (9.8 mg).

Compound 108 (4.9 mg), PyAOP (4.8 mg), HOAt (1.3 mg), and DIPEA (1 μL) were dissolved in DCM / DMF = 1/1 (1.2 mL) and stirred overnight at room temperature. .. The solvent was removed under reduced pressure from the solution, the residue DCM (2.1mL), TIPS (75μL ), and _H 2 O a (75 [mu] L) was added and stirred on ice. Further, TFA (0.9 mL) was added, and the mixture was stirred on ice for 1 hour, and then the solvent was removed under reduced pressure. The obtained residue was purified using an HPLC reverse phase column to obtain compound 109 (Cyclic peptide 3_LK1-L3A (F3)) (1.4 mg, yield 9%).

MS (LC-ESI-MS.m / z): calcd. for C ₃₅ H ₅₂ F ₃ N ₆ O ₇ (M + H) ⁺ : 725.39, found: 725.66.

[Test Example 1]
PAMPA assays were performed on the peptides synthesized in Examples 6-8 and Comparative Examples 6-8. The MDCK-II assay was also performed on the peptides synthesized in Examples 5, 6 and 8 and Comparative Examples 5, 6 and 8. In the MDCK-II assay, Propranolol (CAS No: 318-98-9) was used as the positive control (“PC”) and Norfloxacin (CAS No: 70458-96-7) was used as the negative control (“NC”). ..

The results of the PAMPA assay are shown in Table 1, and the results of the MDCK-II assay are shown in Table 2. As a result, the cell membrane permeability of the fluorine-containing peptides of Examples 5 to 8 into which the fluorine atoms in their side chains were introduced was improved as compared with the peptides of Comparative Examples 5 to 8.

[Test Example 2]
The PAMPA assay was performed on the cyclic peptides synthesized in Examples 10 and 11 and Comparative Example 9.

The results of the PAMPA assay are shown in Table 3. As a result, the cell membrane permeability of the cyclic peptides of Examples 10 and 11 in which a fluorine atom was introduced into the side chain was improved as compared with the cyclic peptide of Comparative Example 9.

The present invention provides a peptide containing an amino acid residue in which a fluorine atom is introduced into a side chain. Since the peptide according to the present invention has excellent cell membrane permeability, it is expected to be used in the pharmaceutical field as a physiologically active substance such as a carrier for introducing a medicinal ingredient into a target cell.

Claims (12)

1. A peptide in which two or more amino acids are peptide-bonded.
   At least one of the amino acid residues constituting the peptide is placed in the side chain in the following general formulas (g-1) to (g-3).
   

   

   [In the formula, R ¹ and R ² may have a hydrogen atom, a halogen atom, a carboxy group, a C _6-14 aryl group, and a substituent, which may have a substituent, independently of each other. It is a C _5-14 heteroaryl group or an amino group which may have a substituent; R ³ to R ⁶ may have a hydrogen atom, a halogen atom and a substituent independently of each other. It has a C _1-30 alkyl group (the C _1-30 alkyl group may have an ether-bonding oxygen atom between carbon atoms when the number of carbon atoms is 2 or more), and a substituent. _May be a C 6-14 aryl group, or a C _5-14 heteroaryl group which ^{may have a substituent; R 1} , R ² , R ³ and R ⁴ are linked to each other to form a substituent. _{It may constitute a C 6-14} aryl group which may have, or a C _5-14 heteroaryl group which may have a substituent; black circles mean binders].
   A peptide having a group represented by any of.
2. At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-1) in the side chain.
   The peptide according to claim 1, wherein in the general formula (g-1), R ¹ is a hydrogen atom and R ² _{is a C 6-14} aryl group which may have a substituent.
3. At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-1) in the side chain.
   The peptide according to claim 1, wherein in the general formula (g-1), R ¹ and R ² are independently hydrogen atoms or fluorine atoms, respectively.
4. At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-2) in the side chain.
   The peptide according to claim 1, wherein in the general formula (g-2), R ¹ and R ² are independently hydrogen atoms or fluorine atoms, and R ³ and R ^{4 are hydrogen atoms, respectively.}
5. At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-2) in the side chain.
   In the general formula (g-2), R ¹ and R ² are independently hydrogen atoms or fluorine atoms, R ³ is a hydrogen atom, and R ⁴ is a trifluoromethyl group. The peptide according to claim 1.
6. At least one of the amino acid residues constituting the peptide has a group represented by the general formula (g-3) in the side chain.
   In the general formula (g-3), the R ¹ , R ² , R ³ and R ⁴ are linked to each other and have a C _6-14 aryl group or a substituent which may have a substituent. _{It constitutes a C 5-14} heteroaryl group which may be present.
   The peptide according to claim 1, wherein R ⁵ and R ^{6 are hydrogen atoms.}
7. The peptide according to any one of claims 1 to 6, wherein the C-terminal or N-terminal may be protected by a protecting group.
8. The following general formula (p2-1) or general formula (p3-1)
   

   

   [In the formula, R ¹¹ and R ¹² are independently C _1-6 alkyl groups or C _6-14 aryl-C _1-6 alkyl groups; Z ¹ is a hydroxy group, C _1-6 alkyl. A group, a C _1-6 alkoxy group, or a protecting group for a carboxyl group; Z ² and Z ³ are independently hydrogen atoms, C _1-6 alkyl groups, and C _6-14 aryl-C _1-6. It is an alkyl group or an amino protecting group; Rg is a general formula (g-1) to (g-3) below.
   

   

   (In the formula, R ¹ and R ² may have a hydrogen atom, a halogen atom, a carboxy group, a substituent, and a C _6-14 aryl group, which may have a substituent, independently of each other. It is a C _5-14 heteroaryl group or an amino group which may have a substituent; R ³ to R ⁶ may have a hydrogen atom, a halogen atom and a substituent independently of each other. It has a C _1-30 alkyl group (the C _1-30 alkyl group may have an ether-bonding oxygen atom between carbon atoms when the number of carbon atoms is 2 or more), and a substituent. _May be a C 6-14 aryl group, or a C _5-14 heteroaryl group which ^{may have a substituent; R 1} , R ² , R ³ and R ⁴ are linked to each other to form a substituent. _{It may constitute a C 6-14} aryl group which may have a C 6-14 aryl group or a C _5-14 heteroaryl group which may have a substituent; a black circle means a bonder).
   It is a group represented by any of]
   The peptide according to any one of claims 1 to 7, which is represented by.
9. The peptide according to any one of claims 1 to 6, which is a cyclic peptide.
10. The peptide according to claim 9, wherein the number of amino acid residues constituting the cyclic structure of the cyclic peptide is 4 to 31 residues.
11. The peptide according to any one of claims 1 to 10, which is cell membrane permeable.
12. A cell membrane penetrant containing the peptide according to any one of claims 1 to 10 as an active ingredient.