WO2022149584A1 - Peptide - Google Patents

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Publication number
WO2022149584A1
WO2022149584A1 PCT/JP2022/000144 JP2022000144W WO2022149584A1 WO 2022149584 A1 WO2022149584 A1 WO 2022149584A1 JP 2022000144 W JP2022000144 W JP 2022000144W WO 2022149584 A1 WO2022149584 A1 WO 2022149584A1
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group
peptide
ome
rfaa
phe
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PCT/JP2022/000144
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English (en)
Japanese (ja)
Inventor
王明 柏木
隆 岡添
光介 相川
淳平 森本
信介 山東
峻輝 三上
Original Assignee
Ａｇｃ株式会社
国立大学法人東京大学
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Publication of WO2022149584A1 publication Critical patent/WO2022149584A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to peptides containing amino acid residues in which a fluoroalkyl group has been introduced into the side chain.
  • 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 membrane penetrating peptides (CPP) are expected to be promising.
  • CPP cell membrane penetrating peptides
  • 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 the medicinal peptide to transport the medicinal peptide into the cell (for example, Patent Document 3 and Non-Patent Document 1).
  • Non-Patent Document 2 Fluorine-containing amino acids and peptides containing them are expected to be used in the pharmaceutical field as physiologically active substances.
  • Non-Patent Document 4 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. 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.
  • the present inventors have produced a peptide containing an amino acid residue having a fluoroalkyl group introduced into the side chain, and found that the peptide has excellent cell membrane permeability, and completed the present invention.
  • the present invention is as follows.
  • the peptide having an oxygen atom of [1] may be further substituted with a halogen atom other than the fluorine atom.
  • [3] 1 to 5 ether bonds between the carbon atoms of the C 1-30 alkyl group substituted with at least two fluorine atoms or the C 2-30 alkyl group substituted with at least two fluorine atoms.
  • the side chain having a group having an oxygen atom of is the following general formula (f-1) or (f-2).
  • Rf P is between carbon atoms of a fully halogenated C 1-10 alkyl group containing at least two or more fluorine atoms or a fully halogenated C 2-10 alkyl group containing at least two or more fluorine atoms.
  • n1 is an integer of 0 to 10
  • n2 is an integer of 0 to 9, and a black circle means a bond.
  • the peptide of the above [1] or [2] which is a group represented by.
  • [4] The peptide according to any one of [1] to [3] above, wherein the C-terminal or N-terminal may be protected by a protecting group.
  • [5] The peptide according to any one of the above [1] to [4], which is permeable to cell membranes.
  • peptide fluorescence conjugate 1 Alexa-Ala-[(R) -RFAA (C8)]-Phe-OMe, diastereomer A
  • peptide fluorescence conjugate 2 Alexa-Ala- [ (S) -RFAA (C8)]-Phe-OMe, Diasteromer B)
  • PFCJ2 Peptide Fluorescence Conjugate 5
  • PFCJ5 Peptide Fluorescence Conjugate HeLa cells treated at 37 ° C.
  • Test Example 1 the figure which showed the result of the flow cytometry of the HeLa cell treated at 37 degreeC for 4 hours in the sample solution containing peptide fluorescence conjugate 1, peptide fluorescence conjugate 2, or peptide fluorescence conjugate 3. be.
  • Test Example 1 the results of flow cytometry of HeLa cells treated at 4 ° C. for 40 minutes in a sample solution containing peptide fluorescent conjugate 1, peptide fluorescent conjugate 2, peptide fluorescent conjugate 3, or fluorescent dye 1 are shown. It is a figure shown.
  • Test Example 3 in a sample solution containing a peptide fluorescence conjugate 7, a peptide fluorescence conjugate 1, a peptide fluorescence conjugate 2, a peptide fluorescence conjugate 5, a peptide fluorescence conjugate 6, or a fluorescent dye 1, 1 at 37 ° C. It is a figure which compared the average fluorescence intensity in the flow cytometry of the time-treated HeLa cells.
  • C p1-p2 (p1 and p2 are positive integers satisfying p1 ⁇ p2) means that the number of carbon atoms is a group of p1 to p2.
  • 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.
  • C 1-10 alkyl groups are 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.
  • Docosyl group tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group and the like.
  • 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 are 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 and a hexyl group.
  • 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.
  • 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.
  • the substituents 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.
  • 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 used. It is a group substituted with.
  • Examples of the C 6-14 aryl group in the C 6-14 aryl -C 1-6 alkyl group include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group and the like, and a phenyl group or a 9-fluorenyl group is particularly preferable. ..
  • C 1-6 alkyl group in the C 6-14 aryl-C 1-6 alkyl group a C 1-4 alkyl group is preferable.
  • Examples of the C 6-14 aryl-C 1-6 alkyl group include a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 2-phenylethyl group, a 9-anthrylmethyl group, a 9-fluorenylmethyl group and the like. Can be mentioned.
  • 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.
  • a chlorine atom or a bromine atom is preferable, and a chlorine atom is particularly preferable.
  • 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-bonding oxygen atoms that an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have is Nc-1.
  • compound n means a compound represented by the formula (n).
  • C 1-30 alkyl group substituted with at least two fluorine atoms (the C 1-30 alkyl group is 1 to 5 between carbon atoms when the number of carbon atoms is 2 or more). It may have an ether-bonding oxygen atom) "means" a C 1-30 alkyl group substituted with at least two fluorine atoms, or a C substituted with at least two fluorine atoms. It means "a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms of a 2-30 alkyl group”.
  • a peptide having a C 1-30 alkyl group substituted with at least two fluorine atoms in the side chain may be referred to as "fluoroalkyl group-containing peptide”.
  • fluoroalkyl group-containing peptide a group in which at least two hydrogen atoms bonded to a carbon atom of the C 1-30 alkyl group are substituted with a fluorine atom.
  • Rf examples include trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, perfluorodecyl group, Difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group , 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,
  • a group having 0 or 1 trifluoromethyl group is preferable to a group having 2 trifluoromethyl groups such as -2-yl group ((CF 3 ) 2 -CH-).
  • Rf is a group having 4 carbon atoms
  • a linear group is preferable as Rf, and in the case of a branched chain group, a hydrogen atom bonded to a carbon atom constituting an alkylene group portion becomes a fluorine atom. It is preferably a substituted group or a fully fluorinated group.
  • a fully halogenated C 1-10 alkyl group containing at least two or more fluorine atoms (the C 1-10 alkyl group is used between carbon atoms when the number of carbon atoms is two or more.” It may have an ether-bonding oxygen atom) "means" a fully halogenated C 1-10 alkyl group containing at least two or more fluorine atoms, or a completely halogen containing at least two or more fluorine atoms.
  • the two Rf Ps may be groups of the same kind or different groups from each other.
  • Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluoro.
  • a hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group preferably a group in which n1 is an integer of 0 to 4, and Rf P is a trifluoromethyl group, a pentafluoroethyl group, or a hepta.
  • Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n1 is an integer of 0 to 2 (where n1 is). 1 and excluding the group in which Rf P is a trifluoromethyl group) is more preferable.
  • Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluoro.
  • a hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group preferably a group in which n2 is an integer of 0 to 4, and Rf P is a trifluoromethyl group, a pentafluoroethyl group, or a hepta.
  • Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n2 is an integer of 0 to 2 (where n2 is). (Excluding the group which is 0 or 1 and Rf P is a trifluoromethyl group) is more preferable.
  • Rf are difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3.
  • -It may be a hexafluoropropyl group, a 1,1,2,3,3,3-hexafluoropropyl group or the like.
  • Examples of the fluoroalkyl group-containing peptide according to the present invention include peptides containing at least one amino acid residue having an Rf side chain.
  • at least one side chain may be Rf, and the side chains of all amino acid residues may be Rf.
  • these plurality of Rfs may be the same species or different from each other.
  • the amino acid residue whose side chain is Rf may be at the N-terminal, at the C-terminal, or at a position other than the C-terminal.
  • the fluoroalkyl group-containing peptide according to the present invention may be a peptide consisting of two or more amino acids, and a peptide consisting of three or more amino acids is also preferable.
  • the fluoroalkyl group-containing peptide according to the present invention is preferably a peptide consisting of 2 to 40 amino acids, and more preferably a peptide consisting of 3 to 20 amino acids.
  • the C-terminus of the fluoroalkyl group-containing peptide according to the present invention may be protected by a protecting group.
  • the C-terminal protecting group the group listed in R 1 below can be used, and a benzyl group is preferable.
  • the N-terminal of the fluoroalkyl group-containing peptide according to the present invention may be protected by a protecting group of an amino group.
  • the N-terminal protecting group the group listed in R2 below can be used, and a Boc group or an Fmoc group is preferable.
  • the amino acid residue having no Rf in the side chain is not particularly limited, and may be an amino acid residue of ⁇ -amino acid, or ⁇ -amino acid. It may be an amino acid residue of ⁇ -amino acid, an amino acid residue of ⁇ -amino acid, or an amino acid residue of ⁇ -amino acid. Further, it may be an amino acid residue of L-amino acid or an amino acid residue of D-amino acid.
  • Amino acid residues that do not have Rf in the side chain contained in the fluoroalkyl group-containing peptide according to the present invention include amino acids constituting proteins, D-forms thereof, and modified amino acids in which these side chains are modified. It is preferably an amino acid residue.
  • amino acids constituting the protein examples include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, and histidine.
  • modified amino acid in which the amino acids constituting the protein are modified include the hydrogen atom of the amino group of the side chain of lysine, arginine, and histidine, the group mentioned in R2 below, and Pbf (N- ⁇ - (2).
  • Amino acid substituted with a group Amino acids substituted with an alkyl group; amino acids in which the hydrogen atom of the thiol group of cysteine is substituted with a benzyl group can be mentioned.
  • Examples of the fluoroalkyl group-containing peptide according to the present invention include tripeptides represented by the following general formula (101) or (102).
  • R 11 and R 12 are preferably C 1-6 alkyl groups or benzyl groups, respectively, and preferably methyl groups or benzyl groups, respectively. It is particularly preferred that R 11 is a methyl group and R 12 is a benzyl group.
  • X is Fmoc or Boc.
  • Z is a C 1-6 alkoxy group, and a methoxy group is particularly preferable.
  • Rf P , n1 and n2 are the same as those in the general formulas (f-1) and (f-2).
  • Rf P is preferably a fully fluorinated C 1-10 alkyl group
  • n1 or n2 is preferably an integer of 0 to 4, preferably Rf P.
  • Rf P is a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, or a perfluorooctyl group, and n1 or n2 is an integer of 0 to 2.
  • the fluoroalkyl group-containing peptide according to the present invention may be carried out by a general peptide synthesis method except that a fluoroalkyl group-containing amino acid, which is an amino acid having a fluoroalkyl group introduced into a side chain, is used as a raw material amino acid.
  • a fluoroalkyl group-containing amino acid which is an amino acid having a fluoroalkyl group introduced into a side chain
  • the fluoroalkyl group-containing peptide can be easily synthesized using an automatic peptide synthesizer using an amino acid having a fluoroalkyl group introduced into the side chain as a raw material.
  • an amino acid having an Rf side chain is preferable.
  • 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.
  • the amino acid raw material it is preferable to use one in which the amino group is protected by a Boc group or an Fmoc group.
  • 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 of 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 forming a peptide bond include N, N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (WSC), and benzotriazole-1-yloxy-trisdimethyl.
  • DCC N-dicyclohexylcarbodiimide
  • WSC 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide
  • benzotriazole-1-yloxy-trisdimethyl benzotriazole-1-yloxy-trisdimethyl.
  • a method of activating the carboxy terminal 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.
  • the base used for forming a peptide bond include triethylamine, diisopropylethylamine (DIPEA) and the like.
  • the solvent used for the peptide bond forming 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 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, trifluoroacetic acid, hydrogen fluoride (HF), trifluoromethanesulfonic acid or the like.
  • 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 done independently.
  • Examples of the peptide solid phase synthetic resin for peptide solid phase synthesis include 4-hydroxymethyl-3-methoxyphenoxybutyic 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.
  • the peptide thus obtained can be converted into each salt by a conventional method.
  • the protecting group of the amino group or the carboxy group of the produced fluoroalkyl group-containing peptide can also be deprotected if necessary. Deprotection can be performed by a conventional method depending on the type of protecting group.
  • the fluoroalkyl group-containing amino acid can be produced, for example, by the following synthetic reaction.
  • Rf a group represented by the general formula (f-1) or (f-2) described later is preferable.
  • R 1 is a protecting group for a carboxy group, and specifically, a group represented by the following general formula (p-1), a 2- (9,10-dioxo) anthrylmethyl group, a benzyloxymethyl group, and the like. And a protecting group selected from phenacyl groups.
  • R 3 is a optionally substituted C 6-14 aryl group
  • R 4 and R 5 are independently hydrogen atoms or optionally substituted C. It is a 6-14 aryl group.
  • the black circle means a bond.
  • R 1 is preferably a benzyl group, a triphenylmethyl group, and more preferably a benzyl group in that it can be deprotected under mild conditions.
  • R1 can be deprotected under mild conditions by using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group as the protecting group R1 of the carboxy group, and the functional group of the amino acid is decomposed. It is advantageous in that it is possible to synthesize a fluorine-containing amino acid and a fluorine-containing peptide.
  • an aralkyl protecting group such as a benzyl group or a triphenylmethyl group
  • R6 is a silyl protecting group.
  • R 6 include a trimethylsilyl (TMS) group, a triethylsilyl (TES) group, a triisopropylsilyl (TIPS) group, a tert-butyldimethylsilyl (TBDMS) group, a tert-butyldiphenylsilyl (TBDPS) group and the like.
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TIPS triisopropylsilyl
  • TDMS tert-butyldimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • R6 is a trimethylsilyl (TMS) group.
  • R 2 is a protecting group for the amino group.
  • R2 is not particularly limited as long as it is a protecting group for an amino group used in peptide synthesis.
  • As the protective group of the amino group tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Allloc) group, 2,2 Examples thereof include a carbamate-based protective group such as a 2-trichloroethoxycarbonyl (Troc) group.
  • R2 is preferably a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group in that it can be deprotected under mild conditions.
  • Step 1 Compound 2-2 can be obtained by reacting compound 2 and compound 8 in the presence of metal fluoride. Since the compound 8 represented by the general formula (8) Rf-R6 can be synthesized from easily available Rf-I (fluoroalkyl iodide) in one step, the range of Rf groups that can be introduced is wide.
  • alkali metal fluorides such as cesium fluoride, lithium fluoride and sodium fluoride can be used, and cesium fluoride is preferable.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide and the like.
  • Tetrahydrofuran is preferred.
  • the amount of compound 8 is preferably 0.5 to 10 mol with respect to 1 mol of compound 2.
  • the amount of metal fluoride is preferably 0.01 to 2 mol with respect to 1 mol of compound 2.
  • the reaction in step 1 is preferably carried out at a temperature of 10 ° C. or lower. By carrying out the reaction at a temperature of 10 ° C. or lower, compound 2-2 can be produced in high yield.
  • the reaction temperature is preferably ⁇ 78 ° C. to 10 ° C., more preferably ⁇ 50 ° C. to ⁇ 10 ° C., and particularly preferably ⁇ 40 ° C. to ⁇ 20 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • Compound 2 can be produced by diesterizing oxalic acid by a known method, or a commercially available product may be used.
  • Step 1-1 In the reaction of step 1 , compound 2-1 (a compound in which one of the hydroxy groups is protected by R6) or a mixture of compound 2-2 and compound 2-1 may be obtained. In that case, compound 2-2 can be obtained by deprotecting the silyl protecting group R6 of compound 2-1.
  • the reaction of step 1-1 can be carried out in the same manner as in step 1.
  • Compound 2-2 can be obtained by deprotecting the silyl protecting group R6 of compound 2-1. Deprotection can be performed in the presence of fluoride salts such as tetrabutylammonium fluoride (TBAF), cesium fluoride, hydrofluoride salts, or acids such as hydrochloric acid, acetic acid, paratoluenesulfonic acid.
  • fluoride salts such as tetrabutylammonium fluoride (TBAF), cesium fluoride, hydrofluoride salts, or acids such as hydrochloric acid, acetic acid, paratoluenesulfonic acid.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and tetrahydrofuran is preferable. It is preferable to add acetic acid.
  • the amount of the fluoride salt is preferably 0.1 to 10 mol with respect to 1 mol of compound 2-1 (in the case of a mixture of compound 2-2 and compound 2-1).
  • the amount of acid is preferably 0.1 to 10 mol with respect to 1 mol of compound 2-1 (1 mol of the mixture in the case of a mixture of compound 2-2 and compound 2-1).
  • the reaction in step 1-2 is preferably carried out at a temperature of 50 ° C. or lower. By carrying out the reaction at a temperature of 50 ° C. or lower, compound 2-2 can be produced in high yield.
  • the reaction temperature is preferably ⁇ 80 ° C. to 50 ° C., more preferably ⁇ 40 ° C. to 30 ° C., and particularly preferably ⁇ 20 ° C. to 30 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • Distillation is preferably carried out at a temperature of 30 ° C to 150 ° C. If the distillation temperature is too high, compound 3 may decompose. If the distillation temperature is too low, compound 3 cannot be condensed and the recovery rate may decrease. Distillation can be carried out at any pressure of reduced pressure, normal pressure and pressure, and can be appropriately determined so that the boiling point of compound 3 falls within the above-mentioned preferable temperature range.
  • the pressure is preferably 0.1 mmHg to 5 atm (3800 mmHg).
  • Compound 4 can be obtained by reacting compound 3 with compound 9 or compound 10.
  • R 2 is a protecting group for an amino group as described above.
  • R 7 , R 8 and R 9 are independently C 6-14 aryl groups. Examples of the C 6-14 aryl group represented by R 7 , R 8 or R 9 include a phenyl group and a naphthyl group. Preferably, R 7 , R 8 and R 9 are phenyl groups, respectively.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and diethyl ether is preferable.
  • the amount of compound 9 or compound 10 is preferably 0.5 to 10 mol with respect to 1 mol of compound 3.
  • the reaction temperature is preferably ⁇ 78 ° C. to 100 ° C., more preferably 0 ° C. to 40 ° C.
  • the reaction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 4 hours.
  • a carbamate-based protecting group such as a tert-butoxycarbonyl group or a 9-fluorenylmethyloxycarbonyl group is used as the amino group protecting group R2 to deprotect R2 under mild conditions. It is possible to synthesize fluorine-containing amino acids while suppressing the decomposition and racemization of compounds.
  • Compound 5 can be obtained by subjecting compound 4 to a reduction reaction.
  • the reduction reaction can be carried out by a method using a reducing agent or a method of reducing in the presence of a metal catalyst.
  • a reducing agent sodium borohydride, zinc borohydride, sodium borohydride cyanoborohydride, lithium triethylborohydride, lithium borohydride (sec-butyl), lithium borohydride, and hydride tri.
  • a boron borohydride reagent such as potassium boron (sec-butyl), lithium boron borohydride, and sodium triacetoxyborohydride can be used.
  • sodium borohydride or zinc borohydride is preferable, and sodium borohydride is more preferable.
  • the amount of the reducing agent is preferably 0.5 to 10 mol with respect to 1 mol of the compound 4.
  • the reaction can be carried out in a solvent inert to the reaction.
  • Solvents include diethyl ether, dichloromethane, hydrochlorofluorocarbon (HCFC) (eg, Asahiclin® AK-225 (3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,1). 3-Dichloro-1,1,2,2,3-pentafluoropropane mixture, AGC Co., Ltd.)), dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, etc.
  • the inert solvent of the above is mentioned, and diethyl ether is preferable.
  • the reaction temperature is preferably ⁇ 78 ° C. to 100 ° C., more preferably ⁇ 10 ° C. to 40 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • metal catalyst examples include palladium catalysts (eg, palladium carbon, palladium hydroxide, Pearlman catalyst, Lindler catalyst, silica gel-supported palladium catalyst, alumina-supported palladium catalyst, palladium oxide) and nickel.
  • Catalysts eg, lane nickel
  • platinum catalysts eg, platinum carbon, platinum oxide, silica gel-supported platinum catalysts, alumina-supported platinum catalysts
  • rhodium catalysts eg, rhodium carbon, alumina-supported rhodium catalysts, rhodium oxide
  • ruthenium catalysts eg, rhodium oxide.
  • the amount of the metal catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, based on 1 mol of the compound 4.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide.
  • the reduction reaction is carried out in the presence of hydrogen gas.
  • the reduction reaction may be carried out under normal pressure or under pressure.
  • the pressure of hydrogen gas is preferably 0.5 atm to 10 atm.
  • the reaction temperature is preferably 0 ° C to 100 ° C, more preferably 10 ° C to 50 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • Step 5-1 Compound 6-1 can be obtained by deprotecting the protecting group R2 of compound 5. Deprotection can be performed according to the type of protecting group R2 . When R 2 is a Boc group, it can be deprotected under acidic conditions. Examples of the acid used include trifluoroacetic acid (TFA) and hydrochloric acid. The amount of acid is preferably 1 to 1000 mol with respect to 1 mol of compound 5.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and dichloromethane, N, N. -Dichloromethane amide is preferred.
  • An acid can also be used as a solvent.
  • the solvent include inorganic acids such as hydrochloric acid, acetic acid and trifluoroacetic acid, and organic acids, and trifluoroacetic acid is preferable.
  • the reaction temperature is preferably ⁇ 78 ° C. to 50 ° C., more preferably 0 ° C. to 40 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • R 2 When R 2 is an Fmoc group, it can be deprotected under basic conditions.
  • the base used include secondary amines such as piperidine, morpholine and pyrrolidine.
  • the amount of the base is preferably 1 to 100 mol with respect to 1 mol of the compound 5.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide.
  • the reaction temperature is preferably ⁇ 20 ° C. to 80 ° C., more preferably 0 ° C. to 40 ° C.
  • the reaction time is preferably 1 minute to 24 hours, more preferably 5 minutes to 2 hours.
  • Step 6-1 Compound 7 can be obtained by deprotecting the protecting group R1 of compound 6-1. Deprotection can be performed according to the type of protecting group R1 .
  • R 1 is a benzyl group, a triphenylmethyl group, a 9-anthrylmethyl group, a piperonyl group, a 2- (9,10-dioxo) anthrylmethyl group, a benzyloxymethyl group or a phenacyl group, in the presence of a metal catalyst. It can be deprotected by the method of reducing with. The reduction reaction can be carried out in the same manner as the method of reduction in the presence of the metal catalyst in step 4.
  • Step 5-2 Compound 6-2 can be obtained by deprotecting the protecting group R1 of compound 5. Deprotection can be performed in the same manner as in step 6-1.
  • Step 6-2 Compound 7 can be obtained by deprotecting the protecting group R2 of compound 6-2. Deprotection can be performed in the same manner as in step 5-1.
  • an optically active fluorine-containing amino acid (fluoroalkyl group-containing compound) can be synthesized.
  • an asterisk indicates that the absolute configuration of the asymmetric carbon atom with an asterisk is S or R.
  • Rf, R1 and R2 are as defined above.
  • R1 In the production method, by using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group as the protecting group R1 of the carboxy group, R1 can be deprotected under mild conditions and the optical activity is maintained. It is advantageous in that it is possible to synthesize a fluorine-containing amino acid and a fluorine-containing peptide.
  • an aralkyl protecting group such as a benzyl group or a triphenylmethyl group
  • Compound 5-1 can be obtained by subjecting compound 4 to an asymmetric reduction reaction.
  • the asymmetric reduction reaction can be carried out by reducing compound 4 in the presence of an asymmetric reduction catalyst.
  • a transition metal complex in which an asymmetric ligand is coordinated to the transition metal can be used.
  • the transition metal include palladium, rhodium, ruthenium, iridium, nickel, cobalt, platinum, iron and the like.
  • the transition metal complex include a palladium complex, a rhodium complex, a ruthenium complex, an iridium complex, a nickel complex and the like.
  • Asymmetric ligands include dpen (1,2-diphenylethylenediamine), daipen (1,1-di (4-anicil) -2-isopropyl-1,2-ethylenediamine), and optically active phosphine ligands. Can be mentioned.
  • Optically active phosphinid ligands include 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl (BINAP), 2,2'-bis (diphenylphosphino) -5,5', 6 , 6', 7,7', 8,8'-octahydro-1,1'-binaphthyl (H8-BINAP), 2,2'-bis (di-p-tolylphosphino) -1,1'-binaphthyl (Tol) -BINAP), 2,2'-bis [bis (3,5-dimethylphenyl) phosphino] -1,1'-binaphthyl (Xyl-BINAP), 2,2'-bis [bis (3,5-di-) tert-butyl-4-methoxyphenyl) phosphino] -1,1'-binaphthyl (DTBM-BINAP
  • the amount of the asymmetric reduction catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, based on 1 mol of the compound 4.
  • the reaction can be carried out in a solvent inert to the reaction.
  • the solvent include inert solvents such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide.
  • the reduction reaction is carried out in the presence of hydrogen gas.
  • the reduction reaction may be carried out under normal pressure or under pressure.
  • the pressure of hydrogen gas is preferably 0.5 atm to 10 atm.
  • the reaction temperature is preferably 0 ° C to 100 ° C, more preferably 10 ° C to 50 ° C.
  • the reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.
  • Step 8-2 Compound 6-4 can be obtained by deprotecting the protecting group R1 of compound 5-1. Deprotection can be performed in the same manner as in step 6-1.
  • Step 9-2 Compound 7-1 can be obtained by deprotecting the protecting group R2 of compound 6-4. Deprotection can be performed in the same manner as in step 5-1.
  • optically active fluorine-containing amino acids fluoroalkyl group-containing compounds
  • the asterisk indicates that the absolute configuration of the asymmetric carbon atom with the asterisk is S or R.
  • Rf, R 1 and R 2 are as defined above.
  • Compound 6-3 can be obtained by optically resolving compound 6-1.
  • Optical resolution can be performed by a known method. For example, it can be performed by a method using a chiral column, a method by crystallization, a diastereomer method, or the like.
  • a racemate can be divided into optically active substances by liquid chromatography or supercritical fluid chromatography (SFC) using a chiral column.
  • SFC supercritical fluid chromatography
  • CHIRALPAK registered trademark
  • CHIRALCEL registered trademark
  • a salt of a racemate and an optically active amine or an optically active acid is formed and induced into a crystalline diasteremer salt for fractional crystallization. By repeating recrystallization, a single diastereomer salt can be obtained. If necessary, the diastereomeric salt is neutralized to obtain a free optically active substance.
  • optically active amines include brucine, cinchonidine, cinchonine, 1-phenethylamine and the like.
  • the optically active acid include camphorsulfonic acid, tartaric acid, mandelic acid and the like.
  • Diastereomer method A racemic mixture is reacted with an optically active reagent to obtain a mixture of diastereomers, which is separated by fractional crystallization and chromatography to separate a single diastereomer. The optically active reagent moiety is removed from the obtained single diastereomer to obtain the desired optical isomer.
  • Step 11-1 Compound 7-1 can be obtained by deprotecting the protecting group R1 of compound 6-3. Deprotection can be performed in the same manner as in step 6-1.
  • Step 11-2 Compound 7-1 can be obtained by deprotecting the protecting group R2 of compound 6-4. Deprotection can be performed in the same manner as in step 5-1.
  • Step 12 Compound 7-1 can be obtained by optically resolving compound 7.
  • the optical resolution can be performed by the same method as in step 10-1.
  • the fluoroalkyl group-containing peptide can be produced from an amino acid having a fluoroalkyl group introduced in the side chain as a raw material.
  • a fluoroalkyl group-containing peptide can be produced by using compound 6-1 as a raw material, compound 6-2, compound 6-3, or compound 6-4 as a raw material.
  • compound 6-2 or 6-4 is condensed with a carboxy group-protected amino acid, a carboxy group-protected amino acid, a C-terminal protected fluorinated peptide, or a C-terminal protected peptide.
  • a fluoroalkyl group-containing peptide By allowing the peptide to be produced, a fluoroalkyl group-containing peptide can be produced.
  • the compound 6-1 or the compound 6-3 may be a fluorine-containing amino acid having an amino group protected, an amino acid having an amino group protected, a fluorine-containing peptide having an N-terminal protection, or a peptide having an N-terminal protection. By condensing, a fluoroalkyl group-containing peptide can be produced.
  • a fluoroalkyl group-containing peptide can be produced in the same manner. Specifically, after protecting the amino group with a protective group, a carboxy group-protected amino acid containing a fluorine, an amino acid having a carboxy group protected, a C-terminal protected fluorine-containing peptide, or a C-terminal protected. Condensate with peptide.
  • Fluoroalkyl groups have a high affinity for cell membranes. Therefore, the fluoroalkyl group-containing peptide according to the present invention has excellent cell membrane permeability. Moreover, since the structure is significantly different from that of the natural peptide, it is not easily decomposed by peptidase. Utilizing these properties, the fluoroalkyl group-containing peptide according to the present invention is expected to be used in the pharmaceutical field as a physiologically active substance. For example, the fluoroalkyl group-containing peptide according to the present invention can be expected to be used as a DDS carrier that carries a medicinal ingredient to a target cell.
  • a fluoroalkyl group-containing peptide according to the present invention for example, by adding a fluoroalkyl group-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 the living body so as not to impair its function, the functionality thereof.
  • the efficiency of peptide uptake into target cells can be improved.
  • a side chain of a part of the hydrophobic amino acid residues of the functional peptide exhibiting physiological activity is Rf, preferably the general formula (101) or (102), as long as the function of the functional peptide is not impaired.
  • Step 1 Benzyl 3,3,4,4,5,5,6,6-nonafluoro-2-oxohexanoate was added in the same manner as in Steps 1 to 2 except that the temperature in Step 1 was changed to 0 ° C. Obtained as a colorless liquid. The yield from step 1 to step 2 was 69%.
  • AK225 refers to "Asahiclean® AK-225" (3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,3-dichloro-1,1,1. Amixture of 2,2,3-pentafluoropropane, AGC Co., Ltd.).
  • amino acids may be written in three-letter notation.
  • “Phe” is phenylalanine and "Gly” is glycine.
  • 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”.
  • Example 1 A dipeptide having a nonafluorobutyl group was synthesized.
  • Example 2 The protecting group on the N-terminal side of the peptide synthesized in Example 1 was deprotected.
  • Example 3 A tripeptide having a nonafluorobutyl group (Fmoc-Gly-RFAA (C4) -Gly-OMe) was synthesized.
  • H-RFAA (C4) -Gly-OMe (10.9 mg, 0.03 mmol), DCM (0.5 mL), DIPEA (0.13 mmol), Fmoc-Gly-OH (0) in an oven-dried NMR test tube. .03 mmol) and benzotriazole-1-yloxy-trisdimethylaminophosphonium salt (0.085 mmol) were added at room temperature. After allowing to stand at room temperature for 24 hours, the solvent was distilled off under reduced pressure, diluted with ethyl acetate, the organic phase was washed with saturated aqueous citrate solution, saturated aqueous sodium carbonate solution, and saturated aqueous sodium carbonate solution, and dried over sodium sulfate. The organic phase was filtered and the filtrate was distilled off under reduced pressure to obtain a crude product of Fmoc-Gly-RFAA-Gly-OMe.
  • Example 5 A tripeptide having a nonafluorobutyl group (H-Ala-[(R) -RFAA (C4)]-Phe-OMe) was synthesized.
  • a stir bar was placed in a 25 mL two-necked flask, and Boc-[(R) -RFAA (C4)]-Phe-OMe (diastereomer A, DR> 95,29 ⁇ mol) and DCM (2 mL) were added to one of them. .. After cooling the reaction mixture to 0 ° C., TFA (0.4 mL) was added and the mixture was warmed to room temperature. After stirring for 4 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction.
  • Example 6 The fluorescent substance Alexa Fluor 647 was fused to the N-terminal of the tripeptide having a nonafluorobutyl group (H-Ala-[(R) -RFAA (C4)]-Phe-OMe) synthesized in Example 5 (Alexa). -Ala-[(R) -RFAA (C4)]-Phe-OMe).
  • AlexaFluor 647 250 ⁇ g
  • dry DMSO 15 ⁇ L
  • H-Ala-[(R) -RFAA (C4)]-Phe dissolved in dry DMSO (15 ⁇ L) in a 1.5 mL black tube.
  • -OMe 1.5 eq
  • DIPEA 1.5 eq
  • the mixture was continuously stirred at room temperature overnight.
  • Boc-Nle-Phe-Ome was synthesized according to the previous report (Chemical and Pharmaceutical Bulletin, 1987, vol.35, p.468) (yield 556 mg, yield 40.2%).
  • Example 7 A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Phe-OMe) was synthesized.
  • the solid of RFAA (C8) contained by X-ray structural analysis was determined to be the (S) form.
  • Rigaku's VariMax Dual Saturn was used for the X-ray structure analysis.
  • Boc-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 98.7 ⁇ mol) was deprotected in the same manner, and H-[(S) -RFAA (C8)]- Phe-OMe (diastereomer B) was obtained (60.1 ⁇ mol, yield 60.8%).
  • Example 8 A tripeptide having a heptadecafluorooctyl group (Boc-Ala-RFAA (C8) -Phe-OMe) was synthesized.
  • a stir bar was placed in a 20 mL two-necked flask, and Boc-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 0.194 mmol) and DCM (5 mL) were added to one of them. .. After cooling the reaction mixture to 0 ° C., TFA (1 mL) was added, the temperature was raised to room temperature, and the mixture was stirred for 2 hours. Then, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction.
  • Boc-Ala-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 45.7 ⁇ mol) was deprotected in the same manner, and H-Ala-[(S) -RFAA ( C8)]-Phe-OMe (diastereomer B) was obtained (21.1 ⁇ mol, yield 46.2%).
  • the fluorescent substance AlexaFluor 647 was bound to the N-terminal of the synthesized deprotected tripeptide (H-Ala-[(R) -RFAA (C8)]-Phe-OMe).
  • Alexa Fluor 647 NHS ester (1.75 mg) dissolved in dry DMSO (175 ⁇ L) and H-Ala-[(R) -RFAA (C8) dissolved in dry DMSO (50 ⁇ L) in a 1.5 mL black tube. )]-Phe-OMe (diastereomer A, 3.0 equivalents), DIPEA dissolved in dry DMSO (71 ⁇ L) (3.0 equivalents), and dried DMSO 124 ⁇ L were added. The mixture was continuously stirred at room temperature overnight.
  • the fluorescence was measured at a emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.
  • Example 9 A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Gly-OMe) was synthesized.
  • Example 10 A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Ala-OMe) was synthesized.
  • Example 11 A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Leu-OMe) was synthesized.
  • Example 18 A tripeptide having a tridecafluorohexyl group (H-Ala-RFAA (C6) -Phe-OMe) was synthesized.
  • H-RFAA (C6) -Phe-OMe (44.8 ⁇ mol), HOAt (1.2 eq), and DIPEA (1.3 eq) were added to a 25 mL two-necked round bottom flask.
  • HATU (1.2 eq) and Fmoc-AlaOH (1.2 eq) dissolved in DCM (5 mL) were added to the mixture at 0 ° C., the mixture was warmed to room temperature and then stirred for 4 hours. After stopping the reaction by adding HCl (1N), the mixture was partitioned between HCl (1N) and DCM. The combined organic phases were evaporated and then diluted with ethyl acetate.
  • Alexa Fluor 647 NHS ester 500 ⁇ g dissolved in dry DMSO (50 ⁇ L) and H-[(R) -RFAA (C6)] -Phe dissolved in dry DMSO (50 ⁇ L) in a 1.5 mL black tube.
  • -OMe (3.0 eq) and DIPEA (3.0 eq) dissolved in dry DMSO (20 ⁇ L) were added.
  • the mixture was continuously stirred at room temperature overnight.
  • the fluorescence was measured at an emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.
  • Example 20 A dipeptide having a tridecafluorohexyl group (Phth-RFAA (C6') -Phe-OMe) was synthesized.
  • Example 21 A tetrapeptide having a heptadecafluorooctyl group (Boc-Cys (Trt) -Ala-RFAA (C8) -Phe-OMe) was synthesized.
  • Tripeptide H-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 27.6 ⁇ mol
  • Boc-Cys Trt
  • flask 1.8 eq
  • COMU 1.8 eq
  • the reaction mixture was cooled to 0 ° C. and DIPEA (1.8 eq) was added. After the addition, the temperature was raised to room temperature and the mixture was stirred for 3.5 hours. Then, the reaction mixture was quenched with HCl (1N) and extracted 3 times with DCM.
  • the aqueous phase was extracted with DCM and the combined organic phase was distilled off under reduced pressure to give a crude product of the deprotected H-Cys-Ala-RFAA (C8) -Phe-OMe.
  • the fluorescent substance AlexaFluor 647 was bound to the terminal SH group of the synthesized peptide after deprotection (H-Cys-Ala-RFAA (C8) -Phe-OMe).
  • AlexaFluor 647 C2 maleimide 500 ⁇ g
  • PBS 50 ⁇ L of phosphate saline
  • H-Cys-Ala-RFAA C8 dissolved in dried DMSO (50 ⁇ L) in a 1.5 mL black tube.
  • -Phe-OMe 3.0 equivalents
  • dried DMSO 20 ⁇ L
  • the mixture was continuously stirred at room temperature overnight.
  • the fluorescence was measured at a emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.
  • the recovered cells were analyzed by measuring and analyzing red 2 fluorescence (661 / 15 nm) for detecting the fluorescent dye (Alexa Fluor 647) introduced into the peptide fluorescence conjugate by flow cytometry (guava easeCyte TM 8). ..
  • the results of flow cytometric analysis of cells cultured at 37 ° C. for 1 hour in the sample solution are shown in FIG.
  • the vertical axis is the number of cells (count), and the horizontal axis is the fluorescence intensity of each cell.
  • the results of comparing the average fluorescence intensities of each sample are shown in FIG.
  • FIG. 3 shows a comparison of average fluorescence intensities in cells cultured at 37 ° C. for 1 hour, 4 hours, and 24 hours.
  • the peptide fluorescent conjugate is referred to as PFCJ
  • the fluorescent dye is referred to as FD.
  • the average value of the fluorescence intensity of Alexa Fluor 647 of the cells is about 9 for the cells treated with the peptide fluorescence conjugate 1 as compared with the cells treated with the peptide fluorescence conjugate 6 or the fluorescent dye 1.
  • the cells treated with the peptide fluorescence conjugate 2 were more than twice as high, and the cells treated with the peptide fluorescence conjugate 5 were more than twice as high. From these results, it was found that peptides having a fluoroalkyl group in the side chain have higher intracellular uptake efficiency and excellent cell membrane permeability as compared with peptides and fluorescent dyes having no fluoroalkyl group. ..
  • peptide fluorescent conjugates 1 and 2 having a large number of carbon atoms and fluorine atoms in the side chain have higher cell membrane permeability than peptide fluorescent conjugate 5 having a relatively small number of carbon atoms and fluorine atoms in the side chain, and are cells.
  • the average value of the fluorescence intensity of Alexa Fluor 647 of the cells was about 4 times that of the cells treated with the peptide fluorescence conjugate 5 as compared with the cells treated with the peptide fluorescence conjugate 5, and the cells treated with the peptide fluorescence conjugate 2 were treated with the peptide fluorescence conjugate 2. It was about 14 times higher in the cells.
  • the peptide fluorescent conjugates 1 and 2 having a heptadecafluorooctyl group were superior in cell membrane permeability to the peptide fluorescent conjugate 5 having a nonafluorobutyl group. Furthermore, by comparing the results of peptide fluorescence conjugates 1 and 2, it was also found that the cell membrane permeability changes depending on the configuration of the asymmetric points present in the fluorine-containing amino acid (RFAA (C8)).
  • the average value of the fluorescence intensity of AlexaFluor 647 of the cells treated with the peptide fluorescence conjugates 1, 2 and 5 containing a fluorine atom in the side chain is the side chain. It was higher than the cells treated with the peptide fluorescent conjugate 6 or the fluorescent dye 1 containing no fluorine atom. From the results shown in FIG. 3, the proportion of cells that emit fluorescence increased over time in the cells treated with any of the peptide fluorescent conjugates, and the amount of peptide fluorescent conjugates taken up by the cells under the test conditions of 37 ° C. was also confirmed to increase over time.
  • the peptide fluorescent conjugate 3 having the same side chain carbon number but a small number of fluorine atoms has a cell membrane permeability equal to or higher than that of the peptide fluorescent conjugate 1 after 1 hour at 37 ° C.
  • the cell membrane permeability was close to that of the peptide fluorescent conjugate 2. From this, it was found that by adjusting the number of carbon atoms in the side chain, the same or higher cell membrane permeability can be obtained even if the number of fluorine atoms is reduced.
  • the average fluorescence intensity of Alexa Fluor 647 of cells after incubation at 37 ° C. for 1 hour, 4 hours, and 24 hours was a fluorescent dye in cells treated with peptide fluorescent conjugates 1 and 4 containing a fluorine atom in the side chain. It was higher than 1 treated cells. Further, in comparison between the peptide fluorescence conjugate 4 derived from the tetrapeptide having the same fluorine-containing alkyl group and the peptide fluorescence conjugate 1 derived from the tripeptide, the peptide fluorescence conjugate was obtained at any of 1 hour, 4 hours and 24 hours. Gate 4 had a higher average fluorescence intensity.
  • Example 22 A tripeptide having a tridecafluorohexyl group (H-Ala-[(R) -RFAA (C6)]-Phe-OMe) was synthesized.
  • the peptide fluorescent conjugate 7 was obtained as a blue solid in the same manner as in Example 8 (calculated by a fluorometer, yield 40% based on the dye).
  • the sample solution was prepared in the same manner as in Test Example 1, and the analysis results by flow cytometry of cells cultured at 37 ° C. for 1 hour in the sample solution are shown in FIG. In addition, the result of comparing the average fluorescence intensity of each sample is shown in FIG.
  • the average fluorescence intensity of Alexa Fluor 647 of cells after incubation at 37 ° C. for 1 hour was 1 for fluorescent dye 1 in all cells treated with peptide fluorescent conjugates 1, 2, 5, and 7 containing a fluorine atom in the side chain. It was higher than the cells treated with the peptide fluorescent conjugate 6 containing no fluorine atom in the side chain.
  • the average fluorescence intensity was higher in the order of peptide fluorescence conjugates 2, 7, and 1.
  • the present invention provides a peptide having an amino acid residue having a fluoroalkyl group in the 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.

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Abstract

La présente invention concerne un peptide ayant un groupe fluoroalkyle en tant que chaîne latérale. La présente invention concerne un peptide dans lequel deux acides aminés ou plus sont liés par peptide, au moins l'un des résidus d'acides aminés constituant le peptide ayant, en tant que chaîne latérale, un groupe alkyle en C1-30 substitué par au moins deux atomes de fluor ou un groupe ayant de 1 à 5 atomes d'oxygène de liaison éther entre des atomes de carbone dans un groupe alkyle en C2-30 substitué par au moins deux atomes de fluor.
PCT/JP2022/000144 2021-01-06 2022-01-05 Peptide WO2022149584A1 (fr)

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