WO2019181901A1 - Compound for improving cell transplantation efficiency - Google Patents
Compound for improving cell transplantation efficiency Download PDFInfo
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- WO2019181901A1 WO2019181901A1 PCT/JP2019/011320 JP2019011320W WO2019181901A1 WO 2019181901 A1 WO2019181901 A1 WO 2019181901A1 JP 2019011320 W JP2019011320 W JP 2019011320W WO 2019181901 A1 WO2019181901 A1 WO 2019181901A1
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- 0 CSc1nc(Cl)c(C=O)c(NCC[N+]2CC[*+](CCNc(nc(nc3Cl)SC)c3/N=N/OCC(NC(CO)C(NC(Cc3ccccc3)C(NC(Cc3ccccc3)C(N)=O)=O)=O)=O)CC2)n1 Chemical compound CSc1nc(Cl)c(C=O)c(NCC[N+]2CC[*+](CCNc(nc(nc3Cl)SC)c3/N=N/OCC(NC(CO)C(NC(Cc3ccccc3)C(NC(Cc3ccccc3)C(N)=O)=O)=O)=O)CC2)n1 0.000 description 3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
Definitions
- the present invention relates to a dispirotripiperazine derivative capable of increasing the efficiency of cell transplant, an anoikis inhibitor containing the derivative, an additive for cell culture or cell transplant, and a cell engraftment promoter.
- cells useful for treatment are transplanted into the body.
- One problem in cell therapy is the low efficiency of cell transplantation. Many of the transplanted cells cannot survive after injection into the patient, and only a small percentage of the cells can survive, requiring enormous numbers of cells to achieve the desired therapeutic effect.
- Syndecan is a cell membrane heparan sulfate proteoglycan in which a core protein penetrates the cell membrane.
- syndecans 1, 2, 3, and 4 are known due to differences in core protein, and are mixed proteoglycans having two sugar side chains, heparan sulfate and chondroitin sulfate.
- Syndecan interacts with growth factors such as extracellular matrix components and fibroblast growth factors via heparan sulfate sugar chains, and contributes to adhesion and migration to the extracellular matrix and recognition of cells. .
- Previous studies have found that adhesamine promotes cell adhesion and proliferation in suspension by binding to cell surface syndecan heparan sulfate.
- Integrin which is a receptor that penetrates the cell membrane and is involved in cell adhesion and cell-cell interaction, recognizes and binds to a specific amino acid sequence RGD possessed by extracellular matrix proteins such as fibronectin.
- Adh-RGDS which is a conjugate of adhesamine and the integrin-binding peptide RGDS
- Fibronectin an extracellular matrix protein, has both an integrin binding site and a heparan sulfate binding site. By cooperating both sites, fibronectin induces a strong signal that promotes cell survival.
- Adh-RGDS also has both an integrin binding site (RGDS) and a heparan sulfate binding site, and has a stronger effect on cell viability than fibronectin.
- An object of the present invention is to solve the problem of anoikis in cell transplantation and cell culture, to provide a compound having an action of inhibiting cell anoikis and increasing cell viability.
- metastatic cancer cells have high cell viability and cell functions such as cell invasion. These are all desirable cell characteristics for cell transplantation. If not only the cell survival rate of the transplanted cells can be increased but also the cell invasion activity can be increased, it is considered that the engraftment of the transplanted cells can be promoted.
- An object of the present invention is to provide a technique for increasing cell viability of transplanted cells such as metastatic cancer cells and inducing cell invasion. Achieving these objectives makes it possible to improve the engraftment efficiency of transplanted cells and reduce the number of cells required for treatment.
- the present inventors have developed a novel dispirotripiperazine derivative in which an aromatic amino acid sequence having a specific length is bound to a dispirotripiperazine derivative such as adhesamine. And it discovered that this novel dispirotripiperazine derivative had a higher effect of increasing cell viability than adhesamine.
- the dispirotripiperazine derivative of the present invention binds to the syndecan heparan sulfate of the cell surface and self-assembles to form submicron particles on the cell surface.
- the formation of this submicron particle results in clustering of syndecan 4 (SDC4), translocation and activation of protein kinase C (PKC) ⁇ , and phosphorylation of mitogen-activated protein kinase (MAPK). It was found that cell survival signal transduction is induced and cell viability is activated.
- the present inventors also synthesized a derivative in which a ligand that specifically binds to a ligand-binding protein is bound to the dispirotripiperazine derivative of the present invention.
- This derivative and matrix metalloproteinase 2 (MMP2) bound to the ligand binding protein can form a complex via the ligand.
- MMP2 matrix metalloproteinase 2
- MMP2 is known to degrade extracellular matrix, particularly type I and type IV collagen, and participate in cancer cell metastasis and invasion.
- the complex of the dispirotripiperazine derivative of the present invention and MMP2 can form submicron particles on the cell surface, and the cell surface can be modified with MMP2.
- Cells modified with MMP2 not only increase cell viability, but also increase cell invasive activity.
- engraftment of transplanted cells can be promoted in cell transplantation.
- a compound represented by the following formula (I) or a salt thereof is the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino; R 1 is formyl or hydroxymethyl; R 2 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2 (Where L is a linker; n is 0, 1 or 2; X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu,
- Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino; R 1 is formyl or hydroxymethyl; R 3 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y (Where L is the first linker; n is 0, 1 or 2; X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may
- a cell transplantation method comprising administering to a subject the compound or salt thereof according to any one of [1] to [7].
- [14] (a) The compound or a salt thereof according to any one of [4] to [7], (B) A method of transplanting a cell, comprising administering to a subject a matrix metalloproteinase 2 (MMP2) bound to the ligand-binding protein, and (c) a cell.
- MMP2 matrix metalloproteinase 2
- the compound represented by the formula (I), (II), (III) or (IV) of the present invention may have an action of inhibiting cell anoikis and increasing cell viability.
- the compounds of the present invention may be useful as anoikis inhibitors, cell engraftment promoters, or additives for cell culture or cell transplantation.
- MMP2 matrix metalloproteinase 2
- the compound represented by the formula (III) or (IV) of the present invention is useful as a compound that enhances the efficiency of cell transplantation.
- FIG. 1 shows the structures of Adh-SFFK and Adh (SFFK) -SFFK.
- (c, d) ⁇ Effect of Adh-SFFK (100 ⁇ M) and Adh (SFFK) -SFFK (100 ⁇ M) on cell viability of NIH3T3 cells incubated in serum-free DMEM containing 1% (v / v) DMSO for 3 days It is a graph which shows. Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF® (100 ⁇ M).
- eGFP-H eGFP-haloalkane dehydrogenase fusion protein
- Adh-SFF-Cl Adh-SFF-Cl
- Cells are eGFP-H (7.5 ⁇ M), Adh-SFF-Cl (50 ⁇ M), a mixture of Adh-SFF-Cl (50 ⁇ M) and eGFP-H (7.5 ⁇ M), or Adh-SFF (50 ⁇ M).
- Adh-SFF-Cl 50 ⁇ M
- eGFP-H 7.5 ⁇ M
- Adh-SFF 50 ⁇ M
- After incubation in serum-free DMEM supplemented with a mixture of eGFP-H (7.5 ⁇ M) the cells were washed with PBS.
- a “Merge” image is an image overlaid with a bright field.
- eGFP-H Construction and evaluation of eGFP-H.
- MMP2-H MMP2-haloalkane dehydrogenase fusion protein
- the rate parameter k cat / K m of the fusion protein (1.33 x 10 4 M -1 s -1 ) was equivalent to the rate parameter of full-length mouse MMP2 (Biolegend 554402, 1.97 x 10 4 M -1 s -1 ) (Both activated with 1 mM 4-aminophenylmercuric acetate).
- FIG. 5 shows cell surface functionalization by MMP2-H using Adh-SFF-Cl / fusion protein system.
- a dispirotripiperazine derivative or a salt thereof A salt of a compound represented by the above formula (I), (II), (III) or (IV) is one monovalent anion or two divalents per compound. Means a salt with one molecule of anion.
- salts include hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate and other inorganic acid salts, oxalate, malonate, succinate, Organics such as maleate, fumarate, lactate, malate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethanesulfonate And acidic amino acid salts such as glutamate and aspartate.
- each group represented by the above formula (I), (II), (III) or (IV) is as follows.
- 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl (particularly 1,3,5-triazinyl).
- Pyrimidinyl is preferred because the anoikis inhibitory activity of the compound of formula (I) or (III) is high.
- Halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
- a chlorine atom is preferred because the anoikis inhibitory activity of the compound of formula (I) or (III) is high.
- Alkyl may be linear or branched, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, heptyl , Octyl, nonyl and decyl. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkyl, more preferably C 1-6 alkyl, and further preferably C 1-4 alkyl. .
- “Hydroxyalkyl” means a group in which alkyl as defined above is substituted with hydroxy. Examples include hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxyisopropyl, hydroxy-n-butyl, hydroxyisobutyl, hydroxy-tert-butyl, hydroxy-n-pentyl, hydroxyisopentyl and hydroxyhexyl. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably hydroxy-C 1-10 alkyl, more preferably hydroxy-C 1-6 alkyl, and still more preferably hydroxy-C 1-4 alkyl.
- Alkoxy may be linear or branched and includes, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy and hexyloxy. . Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkoxy, more preferably C 1-6 alkoxy, and further preferably C 1-4 alkoxy. .
- Alkylthio may be either linear or branched, for example, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio. And hexylthio. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkylthio, more preferably C 1-6 alkylthio, and still more preferably C 1-4 alkylthio. .
- Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, It may be substituted with 1 or 2 substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, the 6-membered aromatic heterocyclic group is preferably 1 or 2 substituents independently selected from a halogen atom and alkylthio May be substituted.
- amino acid residue means a divalent group obtained by removing one hydroxy group from the carboxyl group bonded to the ⁇ -carbon of an amino acid and removing one hydrogen from the amino group bonded to the ⁇ -carbon. Refers to the group.
- the amino acid residue may be either L-form or D-form, and is preferably L-form because high anoikis inhibitory activity is expected and amino acids are easily available.
- amino acid residues and peptides are described with the N-terminus on the left and the C-terminus on the right, according to a conventional method.
- X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n
- each X 1 may be the same or different.
- X 1 is preferably an amino acid residue independently selected from Ser, Ala, and Gly because it has no reactive functional group and facilitates peptide synthesis. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, n is preferably 0 or 1.
- Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, each X 2 is preferably Phe.
- X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When is 2 or 3, each X 3 may be the same or different.
- X 3 is preferably an amino acid residue independently selected from Lys, Gly and Ser because it has no reactive functional group and facilitates peptide synthesis. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, p is preferably 0 or 1.
- amino acid residues represented by X 3 if the amino acid residues located C-terminal side is Lys or Orn, amino side chain of the C-terminal amino acid residue groups may be bonded to a second linker L 2.
- the linker represented by L or the first linker is not particularly limited as long as it can bind ring A 2 and an amino acid residue, and those having various lengths and structures can be used.
- -CH N-O-CH 2 -CO -, - CH 2 NH -, - O -, - S -, - SO -, - SO 2 -, - OSO 2 -, - NH -, - CO -, - CH ⁇ CH—, —C ⁇ C—, —CONH—, —NHCO—, —NHCOO—, —OCH 2 CONH—, —OCH 2 CO— and the like can be mentioned.
- the “ligand binding protein” is not particularly limited as long as it is a protein capable of forming a ligand complex by specifically binding to a specific “ligand”.
- the “ligand” is not particularly limited as long as it is a substance capable of forming a ligand complex by specifically binding to a specific “ligand binding protein”.
- the combination of “ligand binding protein” and “ligand that specifically binds to a ligand binding protein” may be a protein having a predetermined domain structure and a low molecular compound that binds to the predetermined domain structure.
- HaloTag registered trademark
- SNAP-tag protein Covalys
- CLIP-tag protein Covalys
- HaloTag protein is preferably used from the viewpoint of the size of the tag and the reaction rate.
- Examples of the “ligand that specifically binds to a ligand binding protein” represented by Y include, for example, a binding site of a HaloTag ligand (Promega Corporation) that specifically binds to a HaloTag protein, and a SNAP-tag protein (Covalys). And a ligand that specifically binds to CLIP-tag protein (Covalys).
- a specific example of Y is a binding site of a HaloTag ligand that specifically binds to a HaloTag protein.
- the second linker represented by L 2 is not particularly limited as long as it can bind an amino acid residue and a “ligand that specifically binds to a ligand-binding protein”.
- —CO— (CH 2 ) q —CO— (where q is 1, 2, 3 or 4)
- —CO—CH (R 4 ) — can be used.
- NH—CO— (CH 2 ) q —CO— (wherein R 4 is a side chain of an amino acid and q is 1, 2, 3 or 4).
- Specific examples of L 2 include —CO—CH ((CH 2 ) 4 NH 2 ) —NH—CO— (CH 2 ) 2 —CO—.
- Preferable examples of the compound represented by the formula (I) or a salt thereof include the following compounds or salts thereof. [Wherein R 1 and R 2 are as defined above. ]
- the amino acid residue may be either L-form or D-form, but is preferably L-form because of easy availability of amino acids.
- Preferable examples of the compound represented by the formula (III) or a salt thereof include the following compounds or salts thereof. [Wherein R 1 and R 3 are as defined above. ]
- the amino acid residue may be either L-form or D-form, but is preferably L-form because amino acids are easily available.
- the compound represented by the formula (I) or the salt thereof is, for example, a compound represented by the following formula (V) or a salt thereof, and a solid phase synthesis method (for example, Fmoc synthesis).
- the peptide having a desired sequence synthesized by (Method) can be produced by binding via a linker L. (In the formula, ring A 1 , ring A 2 and R 1 are as defined above, and R 5 is formyl or hydroxymethyl.)
- the compound represented by the formula (I) or a salt thereof can be produced by the following method. After condensing [(tert-butoxycarbonyl) aminooxy] acetic acid to the N-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) using a coupling reagent, if necessary Then, the peptide represented by the formula (VI) is obtained by deprotecting the protecting group.
- a solid phase synthesis method for example, Fmoc synthesis method
- Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2 (VI) (Where Aoa is aminooxyacetyl.)
- a compound represented by the formula (I) or a salt thereof can be produced by reacting a peptide represented by the formula (VI) with a compound represented by the formula (V) wherein R 5 is formyl or a salt thereof.
- the compound represented by the formula (III) or a salt thereof can be produced, for example, by the following method.
- a ligand binding site Y is bound to the C-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) via a second linker L2, and the obtained peptide derivative is represented by the formula
- a compound represented by the formula (III) or a salt thereof can be produced by binding the compound of (V) or a salt thereof via the first linker L.
- amino acid residues represented by X 3 if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
- the amino acid residues represented by X 3 if the amino acid residues located C-terminal side is Asp or Glu, the carboxyl group of the side chain of the C-terminal amino acid residues, the binding site Y ligand first You may couple
- the compound represented by the formula (III) or a salt thereof can be produced by the following method.
- the C-terminus of the peptide represented by the formula (VI) by binding the binding site Y ligand via a second linker L 2, to obtain the peptide derivative represented by the formula (VII).
- Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y (VII) (Where Aoa is aminooxyacetyl.)
- a compound represented by the formula (III) or a salt thereof can be produced by reacting a peptide derivative of the formula (VII) with a compound of the formula (V) wherein R 5 is formyl or a salt thereof.
- R 5 is formyl or a salt thereof.
- the amino acid residues represented by X 3 if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
- the binding site Y ligand first You may couple
- the compound of formula (V) or a salt thereof which is a raw material compound in the above production method, can be produced by a method known per se (the method described in WO2009 / 154201) or is easily obtained because it is commercially available. be able to.
- the compound of formula (I) or (III) produced by the above production method or a production method according to these can be isolated and purified according to conventional methods such as chromatography, recrystallization, reprecipitation and the like. It can be converted into a salt by treating with various acids according to a conventional method.
- the compound of formula (I) or (III) can be obtained in the form of a salt depending on the reaction / treatment conditions, etc., but can be converted to the compound of formula (I) or (III) according to a conventional method.
- Anoikis inhibitor The present invention provides an anoikis inhibitor containing the compound represented by the formula (I) or (III) or a salt thereof.
- Anoikis refers to cell death (apoptosis) induced by loss or abnormality of adhesion between cells and extracellular matrix.
- the anoikis inhibitor of the present invention has an activity of inhibiting anoikis of cells.
- the anoikis inhibitor of the present invention can be added to a culture solution or a preservation solution containing cells for the purpose of inhibiting anoikis of the cells.
- the cell is preferably an animal cell, and more preferably a mammalian animal cell.
- mammalian animal cells include cells derived from humans, mice, rats, rabbits and the like.
- the cell may be either an anchorage-dependent cell or a suspension cell.
- the anchorage-dependent cell means a cell that cannot survive or proliferate unless it adheres to the culture vessel.
- fibroblast, epithelial cell, endothelial cell, smooth muscle cell, epidermal cell, hepatocyte, bone examples include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells).
- the floating cell means a cell that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells.
- the cells used in the present invention also include primary cultured cells, and the primary cultured cells may be derived from any tissue.
- the effective concentration of the compound represented by the formula (I) or (III) or a salt thereof when used as an anoikis inhibitor is preferably about 0.1 to 1,000 ⁇ M, more preferably about 50 to 150 ⁇ M.
- the present invention relates to an additive for cell culture or transplanted cells, and a cell engraftment promoter.
- the present invention comprises a compound represented by formula (I) or (III) or a salt thereof, for cell culture or transplanted cells. Additives are provided.
- the present invention also provides a cell engraftment promoter containing a compound represented by formula (I) or (III) or a salt thereof.
- the additive and engraftment promoter of the present invention can be added to a suspension in which cells or transplanted cells are suspended.
- the effective concentration of the compound represented by the formula (I) or (III) or the salt thereof in the suspension is preferably about 0.1 to 1,000 ⁇ M, more preferably about 50 to 150 ⁇ M.
- the cell or transplanted cell in the present invention is preferably an animal cell, more preferably a mammalian animal cell.
- mammalian animal cells include cells derived from humans, mice, rats, rabbits and the like.
- the cell may be either an anchorage-dependent cell or a suspension cell.
- the anchorage-dependent cell means a cell that cannot survive or proliferate unless it adheres to the culture vessel.
- fibroblast, epithelial cell, endothelial cell, smooth muscle cell, epidermal cell, hepatocyte, bone examples include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells).
- the floating cell means a cell that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells.
- the cells used in the present invention also include primary cultured cells, and the primary cultured cells may be derived from any tissue.
- the type of transplanted cell is not particularly limited as long as it is a cell used for cell transplantation (cell therapy), and preferred are corneal endothelial cells, bone marrow-derived cells, fibroblasts and the like.
- a buffer solution or a medium can be used.
- the buffer solution include phosphate buffered saline (PBS) and Hanks solution (HBSS).
- PBS phosphate buffered saline
- HBSS Hanks solution
- a medium suitable for the type of cell may be appropriately selected and used.
- Dulbecco's modified Eagle medium (DMEM) Williams E medium
- Ham F-10 medium F-12 medium
- RPMI -1640 medium MCDB153 medium
- 199 medium etc.
- the medium may be either a medium supplemented with serum or a serum-free medium, but a serum-free medium is preferred.
- the cell therapy in this specification means the therapy which treats a disease by transplanting a cell.
- Cell transplantation method and transplantation kit The present invention provides a cell transplantation method comprising administering a compound represented by the formula (I) or (III) or a salt thereof and a cell.
- the compound represented by the formula (I) or (III) or a salt thereof and cells can be administered to a subject in the form of a therapeutic composition.
- examples of the cells include those described above.
- the therapeutic composition is administered to a subject, that is, a mammal including a human, and is an injection of a suspension containing a compound represented by the formula (I) or (III) or a salt thereof and a transplanted cell. Can be administered parenterally. A buffer solution, a culture medium, etc. can be used for this suspension, What was mentioned above as a suspension and a culture medium is mentioned.
- the therapeutic composition may further comprise matrix metalloprotease 2 (MMP2) bound to the ligand binding protein.
- MMP2 matrix metalloprotease 2
- the effective concentration of the compound represented by the formula (I) or (III) or a salt thereof in the therapeutic composition is preferably about 0.1 to 1,000 ⁇ M, more preferably about 50 to 150 ⁇ M.
- the effective concentration of MMP2 in the therapeutic composition is preferably about 0.1 to 1,000 ⁇ M, more preferably about 7.5 to 22.5 ⁇ M.
- the dosage of the therapeutic composition can be appropriately determined in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
- the present invention (A) a compound represented by the formula (III) or a salt thereof,
- a cell transplantation method comprising (b) MMP2 bound to the ligand binding protein, and (c) administering the cell to a subject.
- the present invention also provides (A) a compound represented by the formula (III) or a salt thereof, and (b) MMP2 bound to the ligand-binding protein.
- a kit comprising:
- the compound represented by the formula (I) of the present invention or a salt thereof self-assembles on a sulfated glycosaminoglycan, forms submicron particles on the cell surface, and cell viability by MAPK signal Can be activated.
- Matrix metalloproteinase 2 is known to degrade extracellular matrix, particularly type I and type IV collagen, and to be involved in cancer cell metastasis and invasion.
- the compound represented by the formula (III) of the present invention or a salt thereof has a ligand site that specifically binds to a ligand-binding protein. Therefore, (a) a compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand binding protein, and (c) a formula conjugated with MMP2 by administering a cell to a subject (
- the compound of III) can form submicron particles on the cell surface and the cell surface can be modified with MMP2.
- the present inventors have found that cells modified with MMP2 have increased cell invasive activity like cancer cells.
- ligand binding protein corresponding to “ligand that specifically binds to ligand binding protein” represented by Y in formula (III) is used.
- a HaloTag ligand Promega Corporation
- a HaloTag protein Promega Corporation
- MMP2 bound to the ligand binding protein can be produced by a method known per se.
- a compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand binding protein, and (c) a cell can be administered to a subject in the form of a therapeutic composition.
- examples of the cells include those described above.
- Adh-SFF 5.97 mg, 4.29 ⁇ mol, 30%
- ESI-HRMS found 525.673463 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-SF 5.76 mg, 6.36 ⁇ mol, 36%) as a white solid.
- ESI-HRMS found 452.139067 calcd 452.139189 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-SFFF (3.64 mg, 3.03 ⁇ mol, 40%) as a white solid.
- ESI-HRMS found 599.207270 calcd 599.207603 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-FF (2.31 mg, 2.39 ⁇ mol, 23%) as a white solid.
- ESI-HRMS found 482.157184 calcd 482.157382 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-SSFF Adh-SSFF (4.68 mg, 4.10 ⁇ mol, 39%) as a white solid.
- ESI-HRMS found 569.189369 calcd 569.189410 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-SSSFF 3.74 mg, 3.05 ⁇ mol, 36%) as a white solid.
- ESI-HRMS found 612.705249 calcd 612.705424 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-FSF 4.83 mg, 4.59 ⁇ mol, 24%) as a white solid.
- ESI-HRMS found 525.673200 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-FFS 525.673387 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Adh-SFFK 4.71 mg, 3.99 ⁇ mol, 27%) as a white solid.
- ESI-HRMS found 589.720952 calcd 589.720877 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Example 7 (1) Synthesis of Aoa-SFFK (alkyl chloride-K-) peptide Peptides were synthesized in the same manner by ordinary Fmoc solid phase peptide synthesis (SPPS) using RINK Amide MBHA resin LL. After condensation of [(tert-butoxycarbonyl) aminooxy] acetic acid, the resin was washed with NH 2 OH.HCl (1.25 g) and imidazole (918 mg) in NMP (5 ml) and CH 2 Cl 2 (1 ml), Treatment at room temperature for 3 hours deprotected 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl (dde).
- SPPS Fmoc solid phase peptide synthesis
- Adh-SFF-Cl 5.79 mg, 3.59 ⁇ mol, 38%) as a white solid.
- ESI-HRMS found 806.338810 calcd 806.338052 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
- Example 8 Adh-AFF
- Adh-GFF Adh-GFF
- Adh-SYY Adh-SWW
- Adh-SFFGK Adh-SFFGK
- Test example 1 Affinity for heparin and cell viability
- the affinity for heparin of the adhesamine derivatives produced in the Examples and Reference Examples was calculated by isothermal titration calorimetry (ITC) (Table 3).
- Adh-SFF showed higher affinity than adhesamine (FIGS. 1c and d, Table 3), but peptide SFF itself showed no affinity for heparin (FIG. 2a).
- Adh-SFF maintained the high selectivity of adhesamine for heparin but did not show affinity for chondroitin sulfate A (FIG. 2b).
- the cells treated with Adh-SFF maintained an unexpectedly high cell viability after culturing for 3 days compared to adhesamine.
- Treatment with peptide SFF alone or a mixture of adhesamine and peptide SFF showed negligible increases in cell viability (FIG. 3a), and the conjugate between adhesamine and SFF peptide increased activity. It is essential to.
- the EC50 that increases the cell viability of Adh-SFF was 38 ⁇ M (FIG. 3b).
- FIG. 3a shows serum-free serum containing 1% (v / v) DMSO supplemented with adhesamine (100 ⁇ M), peptide SFF (100 ⁇ M), a mixture of adhesamine and peptide SFF (100 ⁇ M each) or Adh-SFF (100 ⁇ M) It is a graph which shows the cell viability of the NIH3T3 cell incubated for 3 days in DMEM.
- FIG. 3b is a graph showing the concentration effect of Adh-SFF (0-300 ⁇ M). Cell viability was calculated with the WST-8 assay and normalized to cells treated with Adh-SFF (100 ⁇ M).
- Adh-RGDS a conjugate of adhesamine and the integrin ligand peptide RGDS, also increases cell viability, but has low affinity for heparin (Table 3). This difference may be attributed to the difference in the mechanism of the two adhesamine derivatives, in which Adh-RGDS is dependent on integrin-RGDS binding and Adh-SFF acts by self-assembly on heparan sulfate.
- Test example 2 Self-assembly structure of adhesamine on sulfated GAG
- the inventors have previously shown that adhesamine coordinates to heparan sulfate and forms a self-assembled structure by ⁇ - ⁇ stacking of the pyrimidine moiety.
- the FF peptide is located in the immediate vicinity of the adhesamine pyrimidine and is predicted to form a strong self-assembled structure by ⁇ - ⁇ stacking between aromatic groups.
- NOESY spectra of Adh-SFF in a heparin-containing solution were measured. The results revealed that the pyrimidine part of Adh-SFF is close to the phenyl group as well as the adjacent pyrimidine.
- Adh-SFF The CD spectrum of Adh-SFF also shows that the absorbance at 220 nm decreases and increases around 200 nm when complexed with heparin, suggesting that a ⁇ -turn-like structure is formed. . These results suggest that self-assembly interactions collectively induce ⁇ -turn-like structures when bound to heparan sulfate.
- Test example 3 Intracellular signaling for cell survival induced by Adh-SFF
- SDC4 syndecan 4
- a heparan sulfate known to be involved in cell adhesion It was revealed that biological activity was exhibited by inducing the clustering of syndecan. Immunostaining with anti-SDC4 antibody shows that Adh-SFF treatment induces SDC4 clustering similar to adhesamine (FIG. 6d).
- MAPK activity is controlled by PKC ⁇ activity.
- PKC ⁇ activity In order to identify specific signaling pathways for cell survival induced by Adh-SFF, we monitored MAPK phosphorylation levels after Adh-SFF treatment in a time course experiment by Western blot (Fig. 6g, h).
- Adh-SFF treatment gradually amplified ERK phosphorylation, but inhibited JNK phosphorylation compared with DMSO or adhesamine treatment (FIGS. 6g and h).
- MAPK phosphorylation is controlled in the same manner as cells cultured under normal culture conditions (10% (v / v) FBS, SC on standard plastic culturaing plate) Should.
- Adh-SFF treatment does not cause a significant difference in Akt phosphorylation, which is another cell survival signal transduction pathway.
- FAK phosphorylation was unresponsive to Adh-SFF treatment and resulted in signal transduction similar to DMSO or adhesamine treatment. This is in contrast to cells under SC conditions (which increases FAK phosphorylation from cell adhesion to the dish).
- UO126 which is a selective inhibitor for MEK, an upstream kinase of ERK.
- UCN01 which is a selective PKC ⁇ inhibitor
- triciribine which is an Akt inhibitor
- the inhibitor concentration was optimized to a concentration that does not affect cell viability under normal culture conditions but inhibits the target pathway. Under these conditions, UO126 and UCN01 decreased cell viability caused by Adh-SFF treatment. On the other hand, triciribine did not significantly inhibit cell viability.
- Test example 4 Modification of cell surface of transplanted cells
- GAG such as heparan sulfate
- Further functionalization of this structure is thought to enhance cell transplantation.
- Cancer cells increase their invasive properties in the metastatic process, allowing them to penetrate and engraft at distant sites in the body.
- Non-invasive NIH3T3 has been reported to acquire invasive activity by high expression of active MMP2 after transformation with Tgat (trio-related transforming gene) (Biochemical and Biophysical Research Communications 355, 937 -943 (2007)).
- MMP2 breaks down the extracellular matrix (ECM), particularly type I and type IV collagen, and opens the way for cell migration.
- Adh-SFF alkyl chloride conjugates were synthesized.
- Derivative conjugates with two SFFK peptides, one for each aldehyde of adhesamine (Adh (SFFK) -SFFK) showed a slight increase in cell viability compared to the negative control (FIG. 8b, d).
- Adh-SFFK aldehyde of adhesamine
- a derivative (Adh-SFFK) conjugated with only one SFFK peptide maintained the property of increasing the cell viability of Adh-SFF (FIGS. 8a and 8c).
- Controls Adh-Cl and Adh (Cl) -SFF showed negligible increases in cell viability.
- Adh-SFFK was selected for cell surface modification. Furthermore, by modifying Adh-SFFK with alkyl chloride, Adh-SFF-Cl maintaining the affinity of the parent compound Adh-SFF for heparin and the increase in cell viability was obtained (FIGS. 9a and b, FIG. 10a).
- eGFP-H eGFP-Halo fusion protein
- Adh-SFF-Cl Adh-SFF-Cl to modify the cell surface with eGFP
- the gene encoding eGFP is primer 5'- GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3 '(SEQ ID NO: 1) from pHTN HaloTag (registered trademark) CMV-neo Vector (Promega) / 5′-GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG-3 ′ (SEQ ID NO: 2).
- a pET28b vector in which DNA encoding eGFP was inserted into the restriction enzyme sites Nco I / EcoR I of the pET28b vector encoding Halo-Tag was prepared.
- eGFP was introduced into the N-terminus of the Halo-Tag protein via a GGSGGS linker (FIGS. 11a and b).
- the entire sequence of eGFP-H (SEQ ID NO: 3) is shown in FIG. 11b.
- the recombinant protein eGFP-H was expressed in E. coli BL21 (de3) -RPIL and isolated by Ni-NTA / His tag technology (FIG. 11c). It was confirmed that fluorescence and Halo-Tag activity were maintained with eGFP-H (FIGS. 11d, e, f).
- FIG. 10d is a 2D image
- FIG. 12a is a z-stack image
- FIG. 12b is flow cytometry.
- Treatment with Adh-SFF-Cl or eGFP-H alone and with Adh-SFF and eGFP-H produced no detectable fluorescence signal (FIG. 10d).
- the observed eGFP fluorescence signal overlapped with the SDC4 cluster induced by Adh-SFF-Cl treatment (FIG. 12c).
- MMP2H matrix metalloprotease 2
- Multifunctionalization of cells by this system That is, increase of cell viability and cell invasion activity by Adh-SFF-Cl and MMP2-H were evaluated. Maintained cell viability was confirmed by measuring NADPH dehydrogenase activity (FIG. 14b) and cell invasive activity was measured using Cytoselect® CBA-110® cell® Invasion® assay® kit (FIGS. 14c, 15d, e). This kit measures cells that can migrate from the upper well through the ECM-coated membrane to the lower well, thereby measuring the invasive activity of the cells (FIG. 15c).
- Test Example 5 Skin-muscle infiltration experiment (in vivo experiment) Suspension cells usually have a low engraftment ability to target tissues, which is a major obstacle to regenerative medicine.
- the system of the present invention can solve this problem not only by increasing the viability of the suspension cells but also by giving the suspension cells the ability to migrate to the surrounding tissue.
- skin-muscle infiltration experiments were performed. NIH3T3 cells stably expressing firefly luciferase (NIH3T3 / luc) were injected subcutaneously into ICR mice, and 24 hours later, surviving cells present in the skin and muscle regions were quantified (FIG. 16a).
- mice Male ICR mice aged 5 to 6 weeks were purchased from Sankyo Lab Service Co., Ltd. and maintained under Specific Pathogen Free conditions. The animal experiment protocol was approved by the Animal Experiment Committee of the Faculty of Pharmaceutical Sciences, Tokyo University of Science. All animal experiments were conducted according to the principles and procedures outlined in the National Institutes of Health Guidelines for Laboratory Animal Care and Use.
- NIH3T3 cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS.
- a pEBMulti-Hyg plasmid vector (Wako Pure Chemical Industries, Ltd.) encoding firefly luciferase was prepared as follows.
- a firefly luciferase cDNA fragment was amplified by polymerase chain reaction (PCR) from a previously reported pCMV-Luc plasmid vector (Y. Takahashi et al, Journal of Controlled Release, 105, 332-343 (2005)).
- the forward primer and reverse primer used for amplification were 5'-CGG GGT ACC ATG GAA GAC GCC AAA AAC-3 '(SEQ ID NO: 7) and 5'-TAA AGC GGC CGC TTA CAC GGC GAT CTT-3' ( SEQ ID NO: 8).
- the amplified PCR product and the pEBMulti-Hyg vector were cleaved using Kpn I-HF and Not I-HF restriction enzymes. These cleavage products were separated by electrophoresis and purified by Nucleo Spin Gel and PCR Clean-up (Takara Bio Inc.).
- telomeres were transfected with this plasmid to obtain NIH3T3 / luc cells. These cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS and 500 ⁇ g / mL HygroGold (InvivoGen) under humidified air containing 5% CO 2 .
- mice transplanted with NIH3T3 / luc cells were euthanized by inhalation of isoflurane, and the site of cell transplant was collected.
- Tissue samples were minced, homogenized in cell lysate, and centrifuged at 10000 g for 10 minutes at 4 ° C.
- the luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells.
- the optimal concentration (100 ⁇ M) was applied in the skin-muscle infiltration assay (FIG. 17).
- mice transplanted with NIH3T3 / luc cells were euthanized by isoflurane inhalation, the site of cell transplantation was collected, and the skin and muscle were separated. Muscle samples were minced, homogenized in cell lysate, and centrifuged at 10,000 g for 10 minutes at 4 ° C. The luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells.
- a new strategy is provided to equip cells with selective metastatic properties (cell viability and cell invasion activity).
- the compound represented by the formula (III) of the present invention increases the cell viability by forming a submicron self-assembled structure on the cell, and modifies the cell surface with MMP2. It provides cells with superior functions such as invasion and improved cell viability.
- a cell surface modification system that maintains cell viability after cell transplantation.
- the compound, anoikis inhibitor, additive for cell culture or cell transplant, cell engraftment promoter, cell transplantation method, and kit of the present invention can be useful in the fields of cell transplantation and regenerative medicine.
- SEQ ID NO: 1 (primer): gtcgggatcc gaattcggtg gtagtggtgg tagtatggca gaaatcggta ctgg
- SEQ ID NO: 2 (primer): gacggagctc gaattgttat cgctctgaaa gtacag
- SEQ ID NO: 3 (eGFP-haloalkane dehydrogenase fusion protein): Met Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser
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Abstract
The purpose of the present invention is to provide a compound that acts to inhibit anoikis in cells and enhance cell survival rates, and a compound that induces cell infiltration and enhances cell engraftment. The present invention provides a compound represented by formula (I) or a salt thereof. [In the formula, ring A1 and ring A2 are the same or different and represent a substitutable six-membered aromatic heterocyclic group having 1–3 nitrogen atoms, R1 represents formyl or hydroxymethyl, and R2 is a group represented by -L-(X1)n-(X2)m-(X3)p-NH2 (in the formula: L represents a linker; n represents 0, 1, or 2; m represents 2 or 3; p represents 0, 1, 2, or 3; X1 and X3 each independently represent an amino acid residue selected from among Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn; and the X2 radicals are the same or different and represent an amino acid residue selected from among Phe, Tyr, and Trp).]
Description
本発明は、細胞移植の効率を高めることのできるジスピロトリピペラジン誘導体、並びにそれを含有するアノイキス阻害剤、細胞培養用又は細胞移植用の添加剤、及び細胞生着促進剤に関する。
The present invention relates to a dispirotripiperazine derivative capable of increasing the efficiency of cell transplant, an anoikis inhibitor containing the derivative, an additive for cell culture or cell transplant, and a cell engraftment promoter.
細胞治療・再生医療では、治療に有用な細胞を体内へ移植する。細胞治療における問題の一つは細胞の移植効率が低いことである。移植細胞の多くは患者に注射後、生存することができず、僅かな割合の細胞のみが生存可能であり、所望の治療効果を達成するには膨大な数の細胞が必要となる。
In cell therapy / regenerative medicine, cells useful for treatment are transplanted into the body. One problem in cell therapy is the low efficiency of cell transplantation. Many of the transplanted cells cannot survive after injection into the patient, and only a small percentage of the cells can survive, requiring enormous numbers of cells to achieve the desired therapeutic effect.
細胞の移植効率が低い原因の一つは、細胞移植の際に、細胞が細胞外マトリックスから切り離され、高い確率で細胞死（アポトーシス）が起こることである。細胞と細胞外マトリックス間の接着の喪失又は異常により誘導される細胞死を「アノイキス(anoikis)」という。
One of the causes of low cell transplantation efficiency is that cells are detached from the extracellular matrix during cell transplantation, and cell death (apoptosis) occurs with high probability. Cell death induced by loss or abnormality of adhesion between cells and extracellular matrix is called “anoikis”.
これまでに、アノイキスを阻害し、培養細胞の接着と増殖を促進する小分子化合物として、下記式で表されるアドヘサミン(adhesamine)が見出されている(非特許文献１、特許文献１)。
So far, adhesamine represented by the following formula has been found as a small molecule compound that inhibits anoikis and promotes adhesion and proliferation of cultured cells (Non-patent Document 1, Patent Document 1).
シンデカンは、コアタンパク質が細胞膜を貫通した細胞膜ヘパラン硫酸プロテオグリカンである。コアタンパク質の違いによりシンデカン1, 2, 3, 4の４種類が知られており、ヘパラン硫酸とコンドロイチン硫酸の２種類の糖側鎖をもつ混合型プロテオグリカンである。シンデカンはヘパラン硫酸糖鎖を介して、細胞外マトリックス成分や線維芽細胞増殖因子などの増殖因子と相互作用しており、細胞外マトリックスへの接着や移動、細胞どうしの認識などに寄与している。これまでの研究により、アドヘサミンは、細胞表面のシンデカンのヘパラン硫酸に結合することにより、懸濁液中の浮遊細胞の接着と増殖を促進することが見出されている。
Syndecan is a cell membrane heparan sulfate proteoglycan in which a core protein penetrates the cell membrane. Four types of syndecans, 1, 2, 3, and 4 are known due to differences in core protein, and are mixed proteoglycans having two sugar side chains, heparan sulfate and chondroitin sulfate. Syndecan interacts with growth factors such as extracellular matrix components and fibroblast growth factors via heparan sulfate sugar chains, and contributes to adhesion and migration to the extracellular matrix and recognition of cells. . Previous studies have found that adhesamine promotes cell adhesion and proliferation in suspension by binding to cell surface syndecan heparan sulfate.
また、細胞接着及び細胞間相互作用に関与する、細胞膜を貫通する受容体であるインテグリンは、フィブロネクチンなどの細胞外マトリックスタンパク質が有する特異的なアミノ酸配列RGDを認識して結合する。
アノイキスを阻害する別の化合物として、アドヘサミンとインテグリン結合ペプチドRGDSのコンジュゲートであるAdh-RGDSが報告されている（図１ｂ参照）（非特許文献２、特許文献２）。細胞外マトリックスタンパク質であるフィブロネクチンには、インテグリン結合部位とヘパラン硫酸結合部位の両方がある。この両方の部位が協同することで、フィブロネクチンは細胞の生存を促す強いシグナルを誘導する。Adh-RGDSもインテグリン結合部位(RGDS)とヘパラン硫酸結合部位の両方を有し、フィブロネクチンよりも強く細胞生存率を高める作用を有する。 Integrin, which is a receptor that penetrates the cell membrane and is involved in cell adhesion and cell-cell interaction, recognizes and binds to a specific amino acid sequence RGD possessed by extracellular matrix proteins such as fibronectin.
As another compound that inhibits anoikis, Adh-RGDS, which is a conjugate of adhesamine and the integrin-binding peptide RGDS, has been reported (see FIG. 1b) (Non-patentDocument 2, Patent Document 2). Fibronectin, an extracellular matrix protein, has both an integrin binding site and a heparan sulfate binding site. By cooperating both sites, fibronectin induces a strong signal that promotes cell survival. Adh-RGDS also has both an integrin binding site (RGDS) and a heparan sulfate binding site, and has a stronger effect on cell viability than fibronectin.
アノイキスを阻害する別の化合物として、アドヘサミンとインテグリン結合ペプチドRGDSのコンジュゲートであるAdh-RGDSが報告されている（図１ｂ参照）（非特許文献２、特許文献２）。細胞外マトリックスタンパク質であるフィブロネクチンには、インテグリン結合部位とヘパラン硫酸結合部位の両方がある。この両方の部位が協同することで、フィブロネクチンは細胞の生存を促す強いシグナルを誘導する。Adh-RGDSもインテグリン結合部位(RGDS)とヘパラン硫酸結合部位の両方を有し、フィブロネクチンよりも強く細胞生存率を高める作用を有する。 Integrin, which is a receptor that penetrates the cell membrane and is involved in cell adhesion and cell-cell interaction, recognizes and binds to a specific amino acid sequence RGD possessed by extracellular matrix proteins such as fibronectin.
As another compound that inhibits anoikis, Adh-RGDS, which is a conjugate of adhesamine and the integrin-binding peptide RGDS, has been reported (see FIG. 1b) (Non-patent
本発明は、細胞移植や細胞培養におけるアノイキスの問題を解決し、細胞のアノイキスを阻害し、細胞生存率を高める作用を有する化合物を提供することを目的とする。
An object of the present invention is to solve the problem of anoikis in cell transplantation and cell culture, to provide a compound having an action of inhibiting cell anoikis and increasing cell viability.
また、転移性がん細胞は、高い細胞生存能や細胞浸潤などの細胞機能を有している。これらは全て細胞移植に望ましい細胞の特性である。移植細胞の細胞生存率を高めるだけでなく、細胞浸潤活性を高めることができれば、移植細胞の生着を促進させることが可能になると考えられる。本発明は、転移性がん細胞のように移植細胞の細胞生存率を高め、細胞浸潤を誘導する技術を提供することを目的とする。これらの目的を達成することは、移植細胞の生着効率を改善し、治療に必要な細胞数を減らすことを可能にする。
In addition, metastatic cancer cells have high cell viability and cell functions such as cell invasion. These are all desirable cell characteristics for cell transplantation. If not only the cell survival rate of the transplanted cells can be increased but also the cell invasion activity can be increased, it is considered that the engraftment of the transplanted cells can be promoted. An object of the present invention is to provide a technique for increasing cell viability of transplanted cells such as metastatic cancer cells and inducing cell invasion. Achieving these objectives makes it possible to improve the engraftment efficiency of transplanted cells and reduce the number of cells required for treatment.
本発明者らは、アドヘサミンのようなジスピロトリピペラジン誘導体に特定の長さの芳香族アミノ酸配列を結合させた新規なジスピロトリピペラジン誘導体を開発した。そして、この新規なジスピロトリピペラジン誘導体が、アドヘサミンよりも細胞生存率を高める効果が高いことを見出した。
The present inventors have developed a novel dispirotripiperazine derivative in which an aromatic amino acid sequence having a specific length is bound to a dispirotripiperazine derivative such as adhesamine. And it discovered that this novel dispirotripiperazine derivative had a higher effect of increasing cell viability than adhesamine.
また、本発明のジスピロトリピペラジン誘導体が、細胞表面のシンデカンのヘパラン硫酸と結合して自己集合し、細胞表面上にサブミクロン粒子を形成することを見出した。さらに、このサブミクロン粒子の形成が、シンデカン4(SDC4)のクラスター化、プロテインキナーゼC(PKC)αの細胞膜への移動と活性化、及び分裂促進因子活性化タンパク質キナーゼ(MAPK)のリン酸化を経由する細胞の生存シグナル伝達を誘導し、細胞生存能を活性化することを見出した。
It has also been found that the dispirotripiperazine derivative of the present invention binds to the syndecan heparan sulfate of the cell surface and self-assembles to form submicron particles on the cell surface. In addition, the formation of this submicron particle results in clustering of syndecan 4 (SDC4), translocation and activation of protein kinase C (PKC) α, and phosphorylation of mitogen-activated protein kinase (MAPK). It was found that cell survival signal transduction is induced and cell viability is activated.
本発明者らはまた、本発明のジスピロトリピペラジン誘導体に、リガンド結合タンパク質に特異的に結合するリガンドを結合させた誘導体を合成した。この誘導体と、前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ2(MMP2)は、リガンドを介して複合体を形成し得る。この複合体を用いて、細胞表面をMMP2で修飾することに成功した。
The present inventors also synthesized a derivative in which a ligand that specifically binds to a ligand-binding protein is bound to the dispirotripiperazine derivative of the present invention. This derivative and matrix metalloproteinase 2 (MMP2) bound to the ligand binding protein can form a complex via the ligand. Using this complex, the cell surface was successfully modified with MMP2.
MMP2は、細胞外マトリックス、特にI型及びIV型コラーゲンを分解し、がん細胞の転移・浸潤に関与することが知られている。本発明の方法によれば、本発明のジスピロトリピペラジン誘導体とMMP2との複合体が、細胞表面でサブミクロン粒子を形成し、細胞表面がMMP2で修飾され得る。MMP2で修飾された細胞は、細胞生存率が増大するだけでなく、細胞浸潤活性が増大する。本発明の方法によれば、細胞移植において移植細胞の生着が促進され得る。
MMP2 is known to degrade extracellular matrix, particularly type I and type IV collagen, and participate in cancer cell metastasis and invasion. According to the method of the present invention, the complex of the dispirotripiperazine derivative of the present invention and MMP2 can form submicron particles on the cell surface, and the cell surface can be modified with MMP2. Cells modified with MMP2 not only increase cell viability, but also increase cell invasive activity. According to the method of the present invention, engraftment of transplanted cells can be promoted in cell transplantation.
本発明は、これらの知見に基づき完成されたものであり、以下に関する。
［１］ 下記式(I)で表される化合物又はその塩。
［式中、
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ２は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－ＮＨ２
（式中、
Ｌは、リンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよい。）
で表される基である。］ The present invention has been completed based on these findings and relates to the following.
[1] A compound represented by the following formula (I) or a salt thereof.
[Where:
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 2 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2
(Where
L is a linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When is 2 or 3, each X 3 may be the same or different. )
It is group represented by these. ]
［１］ 下記式(I)で表される化合物又はその塩。
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ２は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－ＮＨ２
（式中、
Ｌは、リンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよい。）
で表される基である。］ The present invention has been completed based on these findings and relates to the following.
[1] A compound represented by the following formula (I) or a salt thereof.
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 2 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2
(Where
L is a linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When is 2 or 3, each X 3 may be the same or different. )
It is group represented by these. ]
［２］ 下記式(II)で表される化合物である、［１］に記載の化合物又はその塩。
［式中、Ｒ１及びＲ２は［１］で定義した通りである。］
[2] The compound or salt thereof according to [1], which is a compound represented by the following formula (II).
[Wherein R 1 and R 2 are as defined in [1]. ]
［３］ 以下の群から選択される、［１］に記載の化合物又はその塩。
及び
[3] The compound according to [1] or a salt thereof selected from the following group.
as well as
［４］ 下記式(III)で表される化合物又はその塩。
［式中、
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ３は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－Ｌ２－Ｙ
（式中、
Ｌは、第一のリンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよく、
Ｌ２は、第二のリンカーであり、
Ｙはリガンド結合タンパク質に特異的に結合するリガンドである。）
で表される基である。］ [4] A compound represented by the following formula (III) or a salt thereof.
[Where:
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 3 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y
(Where
L is the first linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When X is 2 or 3, each X 3 may be the same or different;
L 2 is a second linker;
Y is a ligand that specifically binds to the ligand binding protein. )
It is group represented by these. ]
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ３は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－Ｌ２－Ｙ
（式中、
Ｌは、第一のリンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよく、
Ｌ２は、第二のリンカーであり、
Ｙはリガンド結合タンパク質に特異的に結合するリガンドである。）
で表される基である。］ [4] A compound represented by the following formula (III) or a salt thereof.
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 3 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y
(Where
L is the first linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When X is 2 or 3, each X 3 may be the same or different;
L 2 is a second linker;
Y is a ligand that specifically binds to the ligand binding protein. )
It is group represented by these. ]
［５］ 下記式(IV)で表される、［４］に記載の化合物又はその塩。
［式中、Ｒ１及びＲ３は［４］で定義した通りである。］
[5] The compound or salt thereof according to [4], represented by the following formula (IV):
[Wherein R 1 and R 3 are as defined in [4]. ]
［６］ Ｙが、－ＮＨ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌである、［４］又は［５］に記載の化合物又はその塩。
[6] The compound or salt thereof according to [4] or [5], wherein Y is —NH (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl.
［７］ 下記式（ｋ）で表される、［４］に記載の化合物又はその塩。
[7] The compound or a salt thereof according to [4], represented by the following formula (k).
［８］ ［１］～［７］のいずれか１項に記載の化合物又はその塩を含有する、アノイキス阻害剤。
[8] An anoikis inhibitor containing the compound or salt thereof according to any one of [1] to [7].
［９］ ［１］～［７］のいずれか１項に記載の化合物又はその塩を含有する、細胞培養用又は細胞移植用の添加剤。
[9] An additive for cell culture or cell transplantation containing the compound or salt thereof according to any one of [1] to [7].
［１０］ ［１］～［７］のいずれか１項に記載の化合物又はその塩を含有する、細胞の生着促進剤。
[10] A cell engraftment promoter containing the compound or salt thereof according to any one of [1] to [7].
［１１］ ［１］～［７］のいずれか１項に記載の化合物又はその塩と細胞を対象に投与することを含む、細胞の移植方法。
[11] A cell transplantation method comprising administering to a subject the compound or salt thereof according to any one of [1] to [7].
［１２］ （ａ）［４］～［７］のいずれか１項に記載の化合物又はその塩、及び
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２（MMP2） [12] (a) The compound according to any one of [4] to [7] or a salt thereof, and (b) matrix metalloproteinase 2 (MMP2) bound to the ligand binding protein
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２（MMP2） [12] (a) The compound according to any one of [4] to [7] or a salt thereof, and (b) matrix metalloproteinase 2 (MMP2) bound to the ligand binding protein
［１３］ 細胞移植用キットである、［１２］に記載のキット。
[13] The kit according to [12], which is a cell transplantation kit.
［１４］ （ａ）［４］～［７］ののいずれか１項に記載の化合物又はその塩、
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２（MMP2）、及び
（ｃ）細胞を対象に投与することを含む、細胞の移植方法。 [14] (a) The compound or a salt thereof according to any one of [4] to [7],
(B) A method of transplanting a cell, comprising administering to a subject a matrix metalloproteinase 2 (MMP2) bound to the ligand-binding protein, and (c) a cell.
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２（MMP2）、及び
（ｃ）細胞を対象に投与することを含む、細胞の移植方法。 [14] (a) The compound or a salt thereof according to any one of [4] to [7],
(B) A method of transplanting a cell, comprising administering to a subject a matrix metalloproteinase 2 (MMP2) bound to the ligand-binding protein, and (c) a cell.
本発明の式(I)、(II)、(III)又は(IV)で表される化合物は、細胞のアノイキスを阻害し、細胞生存率を高める作用を有し得る。本発明の化合物は、アノイキス阻害剤、細胞生着促進剤、あるいは細胞培養用又は細胞移植用の添加剤として有用であり得る。また、本発明の式(III)又は(IV)で表される化合物を、リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ2(MMP2)と細胞と共に対象に投与することによって、細胞表面がMMP2で修飾され、MMP2が修飾された細胞の細胞浸潤を誘導し、細胞生着を高めることが可能である。本発明の式(III)又は(IV)で表される化合物は、細胞移植の効率を高める化合物として有用である。
The compound represented by the formula (I), (II), (III) or (IV) of the present invention may have an action of inhibiting cell anoikis and increasing cell viability. The compounds of the present invention may be useful as anoikis inhibitors, cell engraftment promoters, or additives for cell culture or cell transplantation. Further, by administering the compound represented by the formula (III) or (IV) of the present invention to a subject together with a matrix metalloproteinase 2 (MMP2) bound to a ligand-binding protein and a cell, the cell surface is modified with MMP2. It is possible to induce cell invasion of MMP2-modified cells and enhance cell engraftment. The compound represented by the formula (III) or (IV) of the present invention is useful as a compound that enhances the efficiency of cell transplantation.
以下、本発明について詳細に説明する。
ジスピロトリピペラジン誘導体又はその塩
上記式(I)、(II)、(III)又は(IV)で表される化合物の塩とは、化合物1分子に対し、一価のアニオン2分子又は二価のアニオン1分子との塩を意味する。このような塩の具体例としては、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、硫酸塩、過塩素酸塩等の無機酸塩、シュウ酸塩、マロン酸塩、コハク酸塩、マレイン酸塩、フマル酸塩、乳酸塩、リンゴ酸塩、酒石酸塩、安息香酸塩、トリフルオロ酢酸塩、酢酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩、トリフルオロメタンスルホン酸塩等の有機酸塩、及びグルタミン酸塩、アスパラギン酸塩等の酸性アミノ酸塩が挙げられる。 Hereinafter, the present invention will be described in detail.
A dispirotripiperazine derivative or a salt thereof A salt of a compound represented by the above formula (I), (II), (III) or (IV) is one monovalent anion or two divalents per compound. Means a salt with one molecule of anion. Specific examples of such salts include hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate and other inorganic acid salts, oxalate, malonate, succinate, Organics such as maleate, fumarate, lactate, malate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethanesulfonate And acidic amino acid salts such as glutamate and aspartate.
ジスピロトリピペラジン誘導体又はその塩
上記式(I)、(II)、(III)又は(IV)で表される化合物の塩とは、化合物1分子に対し、一価のアニオン2分子又は二価のアニオン1分子との塩を意味する。このような塩の具体例としては、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、硫酸塩、過塩素酸塩等の無機酸塩、シュウ酸塩、マロン酸塩、コハク酸塩、マレイン酸塩、フマル酸塩、乳酸塩、リンゴ酸塩、酒石酸塩、安息香酸塩、トリフルオロ酢酸塩、酢酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩、トリフルオロメタンスルホン酸塩等の有機酸塩、及びグルタミン酸塩、アスパラギン酸塩等の酸性アミノ酸塩が挙げられる。 Hereinafter, the present invention will be described in detail.
A dispirotripiperazine derivative or a salt thereof A salt of a compound represented by the above formula (I), (II), (III) or (IV) is one monovalent anion or two divalents per compound. Means a salt with one molecule of anion. Specific examples of such salts include hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate and other inorganic acid salts, oxalate, malonate, succinate, Organics such as maleate, fumarate, lactate, malate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethanesulfonate And acidic amino acid salts such as glutamate and aspartate.
上記式(I)、(II)、(III)又は(IV)において示される各基は、より具体的にはそれぞれ次の通りである。
More specifically, each group represented by the above formula (I), (II), (III) or (IV) is as follows.
「１～３個の窒素原子を含む６員芳香族ヘテロ環基」の具体例としては、ピリジル、ピラジニル、ピリミジニル、ピリダジニル及びトリアジニル（特に、１，３，５－トリアジニル）が挙げられる。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくは、ピリミジニルである。
Specific examples of the “6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms” include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl (particularly 1,3,5-triazinyl). Pyrimidinyl is preferred because the anoikis inhibitory activity of the compound of formula (I) or (III) is high.
「ハロゲン原子」とは、フッ素原子、塩素原子、臭素原子又はヨウ素原子を意味する。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくは、塩素原子である。
“Halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. A chlorine atom is preferred because the anoikis inhibitory activity of the compound of formula (I) or (III) is high.
「アルキル」とは、直鎖状又は分枝鎖状のいずれでもよく、例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、ヘキシル、ヘプチル、オクチル、ノニル及びデシルが挙げられる。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくはＣ１－１０アルキルであり、より好ましくはＣ１－６アルキルであり、さらに好ましくはＣ１－４アルキルである。
“Alkyl” may be linear or branched, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, heptyl , Octyl, nonyl and decyl. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkyl, more preferably C 1-6 alkyl, and further preferably C 1-4 alkyl. .
「ヒドロキシアルキル」とは、前記定義のアルキルがヒドロキシで置換された基を意味する。例えば、ヒドロキシメチル、ヒドロキシエチル、ヒドロキシ－ｎ－プロピル、ヒドロキシイソプロピル、ヒドロキシ－ｎ－ブチル、ヒドロキシイソブチル、ヒドロキシ－ｔｅｒｔ－ブチル、ヒドロキシ－ｎ－ペンチル、ヒドロキシイソペンチル及びヒドロキシヘキシルが挙げられる。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくはヒドロキシ－Ｃ１－１０アルキルであり、より好ましくはヒドロキシ－Ｃ１－６アルキルであり、さらに好ましくはヒドロキシ－Ｃ１－４アルキルである。
“Hydroxyalkyl” means a group in which alkyl as defined above is substituted with hydroxy. Examples include hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxyisopropyl, hydroxy-n-butyl, hydroxyisobutyl, hydroxy-tert-butyl, hydroxy-n-pentyl, hydroxyisopentyl and hydroxyhexyl. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably hydroxy-C 1-10 alkyl, more preferably hydroxy-C 1-6 alkyl, and still more preferably hydroxy-C 1-4 alkyl.
「アルコキシ」とは、直鎖状又は分枝鎖状のいずれでもよく、例えば、メトキシ、エトキシ、プロポキシ、イソプロポキシ、ブトキシ、イソブトキシ、ｔｅｒｔ－ブトキシ、ペンチルオキシ、イソペンチルオキシ及びヘキシルオキシが挙げられる。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくはＣ１－１０アルコキシであり、より好ましくはＣ１－６アルコキシであり、さらに好ましくはＣ１－４アルコキシである。
“Alkoxy” may be linear or branched and includes, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy and hexyloxy. . Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkoxy, more preferably C 1-6 alkoxy, and further preferably C 1-4 alkoxy. .
「アルキルチオ」とは、直鎖状又は分枝鎖状のいずれでもよく、例えば、メチルチオ、エチルチオ、ｎ－プロピルチオ、イソプロピルチオ、ｎ－ブチルチオ、イソブチルチオ、ｔｅｒｔ－ブチルチオ、ｎ－ペンチルチオ、イソペンチルチオ及びヘキシルチオが挙げられる。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、好ましくはＣ１－１０アルキルチオであり、より好ましくはＣ１－６アルキルチオであり、さらに好ましくはＣ１－４アルキルチオである。
“Alkylthio” may be either linear or branched, for example, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio. And hexylthio. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, it is preferably C 1-10 alkylthio, more preferably C 1-6 alkylthio, and still more preferably C 1-4 alkylthio. .
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよい。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、前記６員芳香族ヘテロ環基は、好ましくは、ハロゲン原子及びアルキルチオから独立して選択される１又は２個の置換基で置換されていてもよい。
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, It may be substituted with 1 or 2 substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, the 6-membered aromatic heterocyclic group is preferably 1 or 2 substituents independently selected from a halogen atom and alkylthio May be substituted.
「アミノ酸残基」とは、アミノ酸のα炭素に結合しているカルボキシル基から一つのヒドロキシ基を除き、且つ該α炭素に結合しているアミノ基から一つの水素を除いて得られる２価の基をいう。アミノ酸残基は、Ｌ体又はＤ体のいずれでもよいが、高いアノイキス阻害活性が期待され、アミノ酸の入手が容易であることから、好ましくはＬ体である。本明細書において、アミノ酸残基及びペプチドは、特に言及のない限り、定法に従って、Ｎ末端を左に、Ｃ末端を右に記載する。
“Amino acid residue” means a divalent group obtained by removing one hydroxy group from the carboxyl group bonded to the α-carbon of an amino acid and removing one hydrogen from the amino group bonded to the α-carbon. Refers to the group. The amino acid residue may be either L-form or D-form, and is preferably L-form because high anoikis inhibitory activity is expected and amino acids are easily available. In this specification, unless otherwise specified, amino acid residues and peptides are described with the N-terminus on the left and the C-terminus on the right, according to a conventional method.
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよい。反応性の官能基を有さず、ペプチドの合成が容易であることから、Ｘ１は、好ましくは、Ser、Ala及びGlyから独立して選択されるアミノ酸残基である。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、ｎは、好ましくは、０又は１である。
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n When X is 2, each X 1 may be the same or different. X 1 is preferably an amino acid residue independently selected from Ser, Ala, and Gly because it has no reactive functional group and facilitates peptide synthesis. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, n is preferably 0 or 1.
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基である。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、各Ｘ２は、好ましくは、Pheである。
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, each X 2 is preferably Phe.
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよい。反応性の官能基を有さず、ペプチドの合成が容易であることから、Ｘ３は、好ましくは、Lys、Gly及びSerから独立して選択されるアミノ酸残基である。式(I)又は(III)の化合物のアノイキス阻害活性が高いことから、ｐは、好ましくは、０又は１である。
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When is 2 or 3, each X 3 may be the same or different. X 3 is preferably an amino acid residue independently selected from Lys, Gly and Ser because it has no reactive functional group and facilitates peptide synthesis. Since the anoikis inhibitory activity of the compound of formula (I) or (III) is high, p is preferably 0 or 1.
式(I)又は(II)のＲ２の定義において、「－ＮＨ２」はＣ末端のカルボキシル基がアミド化されていることを示す。
In the definition of R 2 in formula (I) or (II), “—NH 2 ” indicates that the C-terminal carboxyl group is amidated.
式(III)又は(IV)において、Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がLys又はOrnである場合、当該Ｃ末端アミノ酸残基の側鎖のアミノ基が第二のリンカーＬ２と結合していてもよい。
In formula (III) or (IV), of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Lys or Orn, amino side chain of the C-terminal amino acid residue groups may be bonded to a second linker L 2.
式(III)又は(IV)において、Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がAsp又はGluである場合、当該Ｃ末端アミノ酸残基の側鎖のカルボキシル基が第二のリンカーＬ２に結合していてもよい。
In formula (III) or (IV), of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Asp or Glu, carboxyl of the side chain of the C-terminal amino acid residue group may be bonded to a second linker L 2.
Ｌで表されるリンカー又は第一のリンカーとしては、環Ａ２とアミノ酸残基を結合することが可能なものであれば特に限定されず、種々の長さ及び構造のものを使用することができるが、例えば、
－ＣＨ＝Ｎ－Ｏ－ＣＨ２－ＣＯ－、－ＣＨ２ＮＨ－、－Ｏ－、－Ｓ－、－ＳＯ－、－ＳＯ２－、－ＯＳＯ２－、－ＮＨ－、－ＣＯ－、－ＣＨ＝ＣＨ－、－Ｃ≡Ｃ－、－ＣＯＮＨ－、－ＮＨＣＯ－、－ＮＨＣＯＯ－、－ＯＣＨ２ＣＯＮＨ－、－ＯＣＨ２ＣＯ－などが挙げられる。 The linker represented by L or the first linker is not particularly limited as long as it can bind ring A 2 and an amino acid residue, and those having various lengths and structures can be used. For example,
-CH = N-O-CH 2 -CO -, -CH 2 NH -, - O -, - S -, - SO -, - SO 2 -, - OSO 2 -, - NH -, - CO -, - CH═CH—, —C≡C—, —CONH—, —NHCO—, —NHCOO—, —OCH 2 CONH—, —OCH 2 CO— and the like can be mentioned.
－ＣＨ＝Ｎ－Ｏ－ＣＨ２－ＣＯ－、－ＣＨ２ＮＨ－、－Ｏ－、－Ｓ－、－ＳＯ－、－ＳＯ２－、－ＯＳＯ２－、－ＮＨ－、－ＣＯ－、－ＣＨ＝ＣＨ－、－Ｃ≡Ｃ－、－ＣＯＮＨ－、－ＮＨＣＯ－、－ＮＨＣＯＯ－、－ＯＣＨ２ＣＯＮＨ－、－ＯＣＨ２ＣＯ－などが挙げられる。 The linker represented by L or the first linker is not particularly limited as long as it can bind ring A 2 and an amino acid residue, and those having various lengths and structures can be used. For example,
-CH = N-O-CH 2 -CO -, -
「リガンド結合タンパク質」とは、特定の「リガンド」と特異的に結合することでリガンド複合体を形成し得るタンパク質であれば、特に限定されない。また、「リガンド」としては、特定の「リガンド結合タンパク質」と特異的に結合することでリガンド複合体を形成し得る物質であれば、特に限定されない。例えば、「リガンド結合タンパク質」と「リガンド結合タンパク質に特異的に結合するリガンド」の組み合わせとしては、所定のドメイン構造を持つタンパク質とこの所定のドメイン構造に結合する低分子化合物であってもよい。
The “ligand binding protein” is not particularly limited as long as it is a protein capable of forming a ligand complex by specifically binding to a specific “ligand”. The “ligand” is not particularly limited as long as it is a substance capable of forming a ligand complex by specifically binding to a specific “ligand binding protein”. For example, the combination of “ligand binding protein” and “ligand that specifically binds to a ligand binding protein” may be a protein having a predetermined domain structure and a low molecular compound that binds to the predetermined domain structure.
「リガンド結合タンパク質」としては、例えば、ＨａｌｏＴａｇ（登録商標）タンパク質（プロメガ コーポレイション）、ＳＮＡＰ－ｔａｇタンパク質（Covalys社）、ＣＬＩＰ－ｔａｇタンパク質（Covalys社）などが挙げられる。本発明においては、タグの大きさや反応速度の観点から、好ましくはＨａｌｏＴａｇタンパク質が使用される。
Examples of the “ligand binding protein” include HaloTag (registered trademark) protein (Promega Corporation), SNAP-tag protein (Covalys), CLIP-tag protein (Covalys). In the present invention, HaloTag protein is preferably used from the viewpoint of the size of the tag and the reaction rate.
Ｙで表される「リガンド結合タンパク質に特異的に結合するリガンド」としては、例えば、ＨａｌｏＴａｇタンパク質と特異的に結合するＨａｌｏＴａｇリガンド（プロメガ コーポレイション）の結合部位、ＳＮＡＰ－ｔａｇタンパク質（Covalys社）と特異的に結合するリガンド、ＣＬＩＰ－ｔａｇタンパク質（Covalys社）と特異的に結合するリガンドなどが挙げられる。Ｙの具体例としては、ＨａｌｏＴａｇタンパク質と特異的に結合するＨａｌｏＴａｇリガンドの結合部位である、
－ＮＨ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＮＨ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＣＯ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＣＯ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌなどが挙げられる。 Examples of the “ligand that specifically binds to a ligand binding protein” represented by Y include, for example, a binding site of a HaloTag ligand (Promega Corporation) that specifically binds to a HaloTag protein, and a SNAP-tag protein (Covalys). And a ligand that specifically binds to CLIP-tag protein (Covalys). A specific example of Y is a binding site of a HaloTag ligand that specifically binds to a HaloTag protein.
—NH— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
—NH— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
-CO- (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
—CO— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl and the like can be mentioned.
－ＮＨ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＮＨ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＣＯ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌ、
－ＣＯ－（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌなどが挙げられる。 Examples of the “ligand that specifically binds to a ligand binding protein” represented by Y include, for example, a binding site of a HaloTag ligand (Promega Corporation) that specifically binds to a HaloTag protein, and a SNAP-tag protein (Covalys). And a ligand that specifically binds to CLIP-tag protein (Covalys). A specific example of Y is a binding site of a HaloTag ligand that specifically binds to a HaloTag protein.
—NH— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
—NH— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
-CO- (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl,
—CO— (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl and the like can be mentioned.
Ｌ２で表される第二のリンカーとしては、アミノ酸残基と「リガンド結合タンパク質に特異的に結合するリガンド」を結合することが可能なものであれば特に限定されず、種々の長さ及び構造のものを使用することができるが、例えば、－ＣＯ－（ＣＨ２）ｑ－ＣＯ－（ここで、ｑは１、２、３又は４である）、－ＣＯ－ＣＨ（Ｒ４）－ＮＨ－ＣＯ－（ＣＨ２）ｑ－ＣＯ－（ここで、Ｒ４はアミノ酸の側鎖であり、ｑは１、２、３又は４である）などが挙げられる。Ｌ２の具体例としては、－ＣＯ－ＣＨ（（ＣＨ２）４ＮＨ２）－ＮＨ－ＣＯ－（ＣＨ２）２－ＣＯ－などが挙げられる。
The second linker represented by L 2 is not particularly limited as long as it can bind an amino acid residue and a “ligand that specifically binds to a ligand-binding protein”. For example, —CO— (CH 2 ) q —CO— (where q is 1, 2, 3 or 4), —CO—CH (R 4 ) — can be used. NH—CO— (CH 2 ) q —CO— (wherein R 4 is a side chain of an amino acid and q is 1, 2, 3 or 4). Specific examples of L 2 include —CO—CH ((CH 2 ) 4 NH 2 ) —NH—CO— (CH 2 ) 2 —CO—.
式(I)で表される化合物又はその塩の好適な例としては、以下の化合物又はその塩が挙げられる。
［式中、Ｒ１及びＲ２は前記で定義した通りである。］
Preferable examples of the compound represented by the formula (I) or a salt thereof include the following compounds or salts thereof.
[Wherein R 1 and R 2 are as defined above. ]
式(I)で表される化合物又はその塩の好適な具体例としては、以下の化合物又はその塩が挙げられる。
Preferable specific examples of the compound represented by the formula (I) or a salt thereof include the following compounds or salts thereof.
式(a)から(j)において、アミノ酸残基は、Ｌ体又はＤ体のいずれでもよいが、アミノ酸の入手が容易であることから、好ましくはＬ体である。
In formulas (a) to (j), the amino acid residue may be either L-form or D-form, but is preferably L-form because of easy availability of amino acids.
式(III)で表される化合物又はその塩の好適な例としては、以下の化合物又はその塩が挙げられる。
［式中、Ｒ１及びＲ３は前記で定義した通りである。］
Preferable examples of the compound represented by the formula (III) or a salt thereof include the following compounds or salts thereof.
[Wherein R 1 and R 3 are as defined above. ]
式(III)で表される化合物又はその塩の好適な具体例としては、以下の化合物又はその塩が挙げられる。
Preferable specific examples of the compound represented by the formula (III) or a salt thereof include the following compounds or salts thereof.
式(k)において、アミノ酸残基は、Ｌ体又はＤ体のいずれでもよいが、アミノ酸の入手が容易であることから、好ましくはＬ体である。
In formula (k), the amino acid residue may be either L-form or D-form, but is preferably L-form because amino acids are easily available.
ジスピロトリピペラジン誘導体又はその塩の製造方法
式(I)で表される化合物又はその塩は、例えば、下記式(V)で表される化合物又はその塩と、固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドを、リンカーＬを介して結合することにより製造することができる。
（式中、環Ａ１、環Ａ２及びＲ１は、前記で定義した通りであり、Ｒ５は、ホルミル又はヒドロキシメチルである。）
Production method of dispirotripiperazine derivative or salt thereof The compound represented by the formula (I) or the salt thereof is, for example, a compound represented by the following formula (V) or a salt thereof, and a solid phase synthesis method (for example, Fmoc synthesis). The peptide having a desired sequence synthesized by (Method) can be produced by binding via a linker L.
(In the formula, ring A 1 , ring A 2 and R 1 are as defined above, and R 5 is formyl or hydroxymethyl.)
式(I)で表される化合物又はその塩は、例えば、下記式(V)で表される化合物又はその塩と、固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドを、リンカーＬを介して結合することにより製造することができる。
例えば、Ｌが－ＣＨ＝Ｎ－Ｏ－ＣＨ２－ＣＯ－の場合、式(I)で表される化合物又はその塩は、以下の方法により製造することができる。
固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドのＮ末端に[(tert-ブトキシカルボニル)アミノオキシ]酢酸を、カップリング試薬を用いて縮合させた後、必要に応じて保護基の脱保護処理を行うことにより、式(VI)で表されるペプチドを得る。
Aoa－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－ＮＨ２ (VI)
（式中、Aoaはアミノオキシアセチルである。）
式(VI)のペプチドを、Ｒ５がホルミルである式(V)の化合物又はその塩と反応させることにより、式(I)で表される化合物又はその塩を製造することができる。 For example, when L is —CH═N—O—CH 2 —CO—, the compound represented by the formula (I) or a salt thereof can be produced by the following method.
After condensing [(tert-butoxycarbonyl) aminooxy] acetic acid to the N-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) using a coupling reagent, if necessary Then, the peptide represented by the formula (VI) is obtained by deprotecting the protecting group.
Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2 (VI)
(Where Aoa is aminooxyacetyl.)
A compound represented by the formula (I) or a salt thereof can be produced by reacting a peptide represented by the formula (VI) with a compound represented by the formula (V) wherein R 5 is formyl or a salt thereof.
固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドのＮ末端に[(tert-ブトキシカルボニル)アミノオキシ]酢酸を、カップリング試薬を用いて縮合させた後、必要に応じて保護基の脱保護処理を行うことにより、式(VI)で表されるペプチドを得る。
Aoa－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－ＮＨ２ (VI)
（式中、Aoaはアミノオキシアセチルである。）
式(VI)のペプチドを、Ｒ５がホルミルである式(V)の化合物又はその塩と反応させることにより、式(I)で表される化合物又はその塩を製造することができる。 For example, when L is —CH═N—O—CH 2 —CO—, the compound represented by the formula (I) or a salt thereof can be produced by the following method.
After condensing [(tert-butoxycarbonyl) aminooxy] acetic acid to the N-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) using a coupling reagent, if necessary Then, the peptide represented by the formula (VI) is obtained by deprotecting the protecting group.
Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2 (VI)
(Where Aoa is aminooxyacetyl.)
A compound represented by the formula (I) or a salt thereof can be produced by reacting a peptide represented by the formula (VI) with a compound represented by the formula (V) wherein R 5 is formyl or a salt thereof.
式(III)で表される化合物又はその塩は、例えば、以下の方法により製造することができる。
固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドのＣ末端に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合し、得られたペプチド誘導体を、式(V)の化合物又はその塩と第一のリンカーＬを介して結合することにより、式(III)で表される化合物又はその塩を製造することができる。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がLys又はOrnである場合、当該アミノ酸残基の側鎖のアミノ基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がAsp又はGluである場合、当該Ｃ末端アミノ酸残基の側鎖のカルボキシル基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。 The compound represented by the formula (III) or a salt thereof can be produced, for example, by the following method.
A ligand binding site Y is bound to the C-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) via a second linker L2, and the obtained peptide derivative is represented by the formula A compound represented by the formula (III) or a salt thereof can be produced by binding the compound of (V) or a salt thereof via the first linker L.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Asp or Glu, the carboxyl group of the side chain of the C-terminal amino acid residues, the binding site Y ligand first You may couple | bond together through two linker L2.
固相合成法（例えばＦｍｏｃ合成法）により合成した所望の配列を有するペプチドのＣ末端に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合し、得られたペプチド誘導体を、式(V)の化合物又はその塩と第一のリンカーＬを介して結合することにより、式(III)で表される化合物又はその塩を製造することができる。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がLys又はOrnである場合、当該アミノ酸残基の側鎖のアミノ基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がAsp又はGluである場合、当該Ｃ末端アミノ酸残基の側鎖のカルボキシル基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。 The compound represented by the formula (III) or a salt thereof can be produced, for example, by the following method.
A ligand binding site Y is bound to the C-terminus of a peptide having a desired sequence synthesized by a solid phase synthesis method (for example, Fmoc synthesis method) via a second linker L2, and the obtained peptide derivative is represented by the formula A compound represented by the formula (III) or a salt thereof can be produced by binding the compound of (V) or a salt thereof via the first linker L.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Asp or Glu, the carboxyl group of the side chain of the C-terminal amino acid residues, the binding site Y ligand first You may couple | bond together through two linker L2.
例えば、Ｌが－ＣＨ＝Ｎ－Ｏ－ＣＨ２－ＣＯ－の場合、式(III)で表される化合物又はその塩は、以下の方法により製造することができる。
式(VI)で表されるペプチドのＣ末端に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合することにより、式(VII)で表されるペプチド誘導体を得る。
Aoa－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－Ｌ２－Ｙ (VII)
（式中、Aoaはアミノオキシアセチルである。）
式(VII)のペプチド誘導体を、Ｒ５がホルミルである式(V)の化合物又はその塩と反応させることにより、式(III)で表される化合物又はその塩を製造することができる。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がLys又はOrnである場合、当該アミノ酸残基の側鎖のアミノ基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がAsp又はGluである場合、当該Ｃ末端アミノ酸残基の側鎖のカルボキシル基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。 For example, when L is —CH═N—O—CH 2 —CO—, the compound represented by the formula (III) or a salt thereof can be produced by the following method.
The C-terminus of the peptide represented by the formula (VI), by binding the binding site Y ligand via a second linker L 2, to obtain the peptide derivative represented by the formula (VII).
Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y (VII)
(Where Aoa is aminooxyacetyl.)
A compound represented by the formula (III) or a salt thereof can be produced by reacting a peptide derivative of the formula (VII) with a compound of the formula (V) wherein R 5 is formyl or a salt thereof.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Asp or Glu, the carboxyl group of the side chain of the C-terminal amino acid residues, the binding site Y ligand first You may couple | bond together through two linker L2.
式(VI)で表されるペプチドのＣ末端に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合することにより、式(VII)で表されるペプチド誘導体を得る。
Aoa－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－Ｌ２－Ｙ (VII)
（式中、Aoaはアミノオキシアセチルである。）
式(VII)のペプチド誘導体を、Ｒ５がホルミルである式(V)の化合物又はその塩と反応させることにより、式(III)で表される化合物又はその塩を製造することができる。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がLys又はOrnである場合、当該アミノ酸残基の側鎖のアミノ基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。
Ｘ３で表されるアミノ酸残基のうち、Ｃ末端側に位置するアミノ酸残基がAsp又はGluである場合、当該Ｃ末端アミノ酸残基の側鎖のカルボキシル基に、リガンドの結合部位Ｙを第二のリンカーＬ２を介して結合してもよい。 For example, when L is —CH═N—O—CH 2 —CO—, the compound represented by the formula (III) or a salt thereof can be produced by the following method.
The C-terminus of the peptide represented by the formula (VI), by binding the binding site Y ligand via a second linker L 2, to obtain the peptide derivative represented by the formula (VII).
Aoa- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y (VII)
(Where Aoa is aminooxyacetyl.)
A compound represented by the formula (III) or a salt thereof can be produced by reacting a peptide derivative of the formula (VII) with a compound of the formula (V) wherein R 5 is formyl or a salt thereof.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Lys or Orn, the side chain amino groups of the amino acid residues of the ligand binding site Y a second it may be bonded via a linker L 2.
Of the amino acid residues represented by X 3, if the amino acid residues located C-terminal side is Asp or Glu, the carboxyl group of the side chain of the C-terminal amino acid residues, the binding site Y ligand first You may couple | bond together through two linker L2.
環Ａ１及び環Ａ２の構造中に反応に関与する官能基が存在する場合には、それらを常法に従って保護しておき、反応終了後に保護基を脱離させることが望ましい。
上記製造方法における原料化合物である式(V)の化合物又はその塩は、自体公知の方法（WO2009/154201に記載の方法)により製造することができるか、或いは市販されているので容易に入手することができる。
上記製造方法或いはこれらに準じた製造方法により生成する式(I)又は(III)の化合物は、クロマトグラフィー、再結晶、再沈殿等の常法に従って単離・精製することができる。常法に従って各種の酸と処理することにより塩に導くことができる。また、式(I)又は(III)の化合物は、反応・処理条件等により、塩の形で得られるが、常法に従って式(I)又は(III)の化合物に変換することができる。 When functional groups involved in the reaction are present in the structures of ring A 1 and ring A 2 , it is desirable to protect them according to a conventional method and to remove the protective group after the reaction is completed.
The compound of formula (V) or a salt thereof, which is a raw material compound in the above production method, can be produced by a method known per se (the method described in WO2009 / 154201) or is easily obtained because it is commercially available. be able to.
The compound of formula (I) or (III) produced by the above production method or a production method according to these can be isolated and purified according to conventional methods such as chromatography, recrystallization, reprecipitation and the like. It can be converted into a salt by treating with various acids according to a conventional method. The compound of formula (I) or (III) can be obtained in the form of a salt depending on the reaction / treatment conditions, etc., but can be converted to the compound of formula (I) or (III) according to a conventional method.
上記製造方法における原料化合物である式(V)の化合物又はその塩は、自体公知の方法（WO2009/154201に記載の方法)により製造することができるか、或いは市販されているので容易に入手することができる。
上記製造方法或いはこれらに準じた製造方法により生成する式(I)又は(III)の化合物は、クロマトグラフィー、再結晶、再沈殿等の常法に従って単離・精製することができる。常法に従って各種の酸と処理することにより塩に導くことができる。また、式(I)又は(III)の化合物は、反応・処理条件等により、塩の形で得られるが、常法に従って式(I)又は(III)の化合物に変換することができる。 When functional groups involved in the reaction are present in the structures of ring A 1 and ring A 2 , it is desirable to protect them according to a conventional method and to remove the protective group after the reaction is completed.
The compound of formula (V) or a salt thereof, which is a raw material compound in the above production method, can be produced by a method known per se (the method described in WO2009 / 154201) or is easily obtained because it is commercially available. be able to.
The compound of formula (I) or (III) produced by the above production method or a production method according to these can be isolated and purified according to conventional methods such as chromatography, recrystallization, reprecipitation and the like. It can be converted into a salt by treating with various acids according to a conventional method. The compound of formula (I) or (III) can be obtained in the form of a salt depending on the reaction / treatment conditions, etc., but can be converted to the compound of formula (I) or (III) according to a conventional method.
アノイキス阻害剤
本発明は、式(I)又は(III)で表される化合物又はその塩を含有する、アノイキス阻害剤を提供する。 Anoikis inhibitor The present invention provides an anoikis inhibitor containing the compound represented by the formula (I) or (III) or a salt thereof.
本発明は、式(I)又は(III)で表される化合物又はその塩を含有する、アノイキス阻害剤を提供する。 Anoikis inhibitor The present invention provides an anoikis inhibitor containing the compound represented by the formula (I) or (III) or a salt thereof.
「アノイキス(anoikis)」とは、細胞と細胞外マトリックス間の接着の喪失又は異常により誘導される細胞死（アポトーシス）をいう。本発明のアノイキス阻害剤は、細胞のアノイキスを阻害する活性を有する。本発明のアノイキス阻害剤は、細胞のアノイキスを阻害する目的で、細胞を含有する培養液又は保存液に添加することができる。
“Anoikis” refers to cell death (apoptosis) induced by loss or abnormality of adhesion between cells and extracellular matrix. The anoikis inhibitor of the present invention has an activity of inhibiting anoikis of cells. The anoikis inhibitor of the present invention can be added to a culture solution or a preservation solution containing cells for the purpose of inhibiting anoikis of the cells.
細胞としては、好ましくは動物細胞であり、より好ましくは哺乳類の動物細胞である。哺乳類の動物細胞としては、例えば、ヒト、マウス、ラット、ウサギ等由来の細胞が挙げられる。細胞は、足場依存性細胞及び浮遊性細胞のどちらであってもよい。ここで、足場依存性細胞とは、培養容器に接着できなければ生存できず増殖もできない細胞を意味し、例えば線維芽細胞、上皮細胞、内皮細胞、平滑筋細胞、表皮細胞、肝細胞、骨芽細胞、骨格筋細胞、胚性幹細胞(ES細胞)、人工多能性幹細胞(iPS細胞)等が挙げられる。浮遊性細胞とは、浮遊状態でも増殖できる細胞を意味し、例えば骨髄細胞、リンパ系細胞、腹水ガン細胞等が挙げられる。また、本発明で用いられる細胞としては初代培養細胞も挙げられ、初代培養細胞はいずれの組織に由来するものであってもよい。
The cell is preferably an animal cell, and more preferably a mammalian animal cell. Examples of mammalian animal cells include cells derived from humans, mice, rats, rabbits and the like. The cell may be either an anchorage-dependent cell or a suspension cell. Here, the anchorage-dependent cell means a cell that cannot survive or proliferate unless it adheres to the culture vessel. For example, fibroblast, epithelial cell, endothelial cell, smooth muscle cell, epidermal cell, hepatocyte, bone Examples include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). The floating cell means a cell that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells. The cells used in the present invention also include primary cultured cells, and the primary cultured cells may be derived from any tissue.
アノイキス阻害剤として使用する場合の式(I)又は(III)で表される化合物又はその塩の有効濃度は、好ましくは0.1～1,000μM程度、より好ましくは50～150μM程度である。
The effective concentration of the compound represented by the formula (I) or (III) or a salt thereof when used as an anoikis inhibitor is preferably about 0.1 to 1,000 μM, more preferably about 50 to 150 μM.
細胞培養用又は移植細胞用の添加剤、及び細胞の生着促進剤
本発明は、式(I)又は(III)で表される化合物又はその塩を含有する、細胞培養用又は移植細胞用の添加剤を提供する。 The present invention relates to an additive for cell culture or transplanted cells, and a cell engraftment promoter.The present invention comprises a compound represented by formula (I) or (III) or a salt thereof, for cell culture or transplanted cells. Additives are provided.
本発明は、式(I)又は(III)で表される化合物又はその塩を含有する、細胞培養用又は移植細胞用の添加剤を提供する。 The present invention relates to an additive for cell culture or transplanted cells, and a cell engraftment promoter.The present invention comprises a compound represented by formula (I) or (III) or a salt thereof, for cell culture or transplanted cells. Additives are provided.
本発明はまた、式(I)又は(III)で表される化合物又はその塩を含有する、細胞の生着促進剤を提供する。
The present invention also provides a cell engraftment promoter containing a compound represented by formula (I) or (III) or a salt thereof.
本発明の添加剤及び生着促進剤は、細胞又は移植細胞が懸濁された懸濁液に添加することができる。
The additive and engraftment promoter of the present invention can be added to a suspension in which cells or transplanted cells are suspended.
懸濁液中の式(I)又は(III)で表される化合物又はその塩の有効濃度は、好ましくは0.1～1,000μM程度、より好ましくは50～150μM程度である。
The effective concentration of the compound represented by the formula (I) or (III) or the salt thereof in the suspension is preferably about 0.1 to 1,000 μM, more preferably about 50 to 150 μM.
本発明における細胞又は移植細胞としては、好ましくは動物細胞であり、より好ましくは哺乳類の動物細胞である。哺乳類の動物細胞としては、例えば、ヒト、マウス、ラット、ウサギ等由来の細胞が挙げられる。細胞は、足場依存性細胞及び浮遊性細胞のどちらであってもよい。ここで、足場依存性細胞とは、培養容器に接着できなれば生存できず増殖もできない細胞を意味し、例えば線維芽細胞、上皮細胞、内皮細胞、平滑筋細胞、表皮細胞、肝細胞、骨芽細胞、骨格筋細胞、胚性幹細胞(ES細胞)、人工多能性幹細胞(iPS細胞)等が挙げられる。浮遊性細胞とは、浮遊状態でも増殖できる細胞を意味し、例えば骨髄細胞、リンパ系細胞、腹水ガン細胞等が挙げられる。また、本発明で用いられる細胞としては初代培養細胞も挙げられ、初代培養細胞はいずれの組織に由来するものであってもよい。移植細胞の種類としては、細胞の移植(細胞治療)に使用される細胞であれば特に限定されないが、好ましくは角膜内皮細胞、骨髄由来細胞、線維芽細胞などである。
The cell or transplanted cell in the present invention is preferably an animal cell, more preferably a mammalian animal cell. Examples of mammalian animal cells include cells derived from humans, mice, rats, rabbits and the like. The cell may be either an anchorage-dependent cell or a suspension cell. Here, the anchorage-dependent cell means a cell that cannot survive or proliferate unless it adheres to the culture vessel. For example, fibroblast, epithelial cell, endothelial cell, smooth muscle cell, epidermal cell, hepatocyte, bone Examples include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). The floating cell means a cell that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells. The cells used in the present invention also include primary cultured cells, and the primary cultured cells may be derived from any tissue. The type of transplanted cell is not particularly limited as long as it is a cell used for cell transplantation (cell therapy), and preferred are corneal endothelial cells, bone marrow-derived cells, fibroblasts and the like.
本発明における懸濁液には、緩衝液や培地などを使用することができる。緩衝液としては、リン酸緩衝化生理食塩水(PBS)、ハンクス液(HBSS)などが挙げられる。また、培地としては、細胞の種類により適した培地を適宜選択して使用すればよく、例えばダルベッコの改変イーグル培地(DMEM)、ウィリアムズE培地、HamのF-10培地、F-12培地、RPMI-1640培地、MCDB153培地、199培地などの従来公知の細胞培養用基礎培地に、必要に応じて各細胞の培養に適合した従来公知の成長因子や抗酸化剤などを加えたものを使用することができる。培地は、血清を添加した培地又は無血清培地のどちらでもよいが、無血清培地の方が好ましい。
For the suspension in the present invention, a buffer solution or a medium can be used. Examples of the buffer solution include phosphate buffered saline (PBS) and Hanks solution (HBSS). As the medium, a medium suitable for the type of cell may be appropriately selected and used.For example, Dulbecco's modified Eagle medium (DMEM), Williams E medium, Ham F-10 medium, F-12 medium, RPMI -1640 medium, MCDB153 medium, 199 medium, etc. should be added to conventional well-known medium for cell culture, with addition of conventionally known growth factors, antioxidants, etc. suitable for each cell culture as required Can do. The medium may be either a medium supplemented with serum or a serum-free medium, but a serum-free medium is preferred.
本発明の添加剤を細胞又は移植細胞の懸濁液に添加することで、細胞生存率が増大し、細胞治療(細胞の移植)において優れた治療効果が得られることが期待される。本発明の添加剤は、細胞生着促進作用を有することから、このような効果を得られると考えられる。本明細書における細胞治療とは、細胞を移植することにより疾患を治療する療法のことを意味する。
It is expected that by adding the additive of the present invention to a suspension of cells or transplanted cells, the cell viability is increased and an excellent therapeutic effect can be obtained in cell therapy (cell transplantation). Since the additive of the present invention has a cell engraftment promoting effect, it is considered that such an effect can be obtained. The cell therapy in this specification means the therapy which treats a disease by transplanting a cell.
細胞の移植方法、及び移植用キット
本発明は、式(I)又は(III)で表される化合物又はその塩と細胞を投与することを含む、細胞の移植方法を提供する。 Cell transplantation method and transplantation kit The present invention provides a cell transplantation method comprising administering a compound represented by the formula (I) or (III) or a salt thereof and a cell.
本発明は、式(I)又は(III)で表される化合物又はその塩と細胞を投与することを含む、細胞の移植方法を提供する。 Cell transplantation method and transplantation kit The present invention provides a cell transplantation method comprising administering a compound represented by the formula (I) or (III) or a salt thereof and a cell.
式(I)又は(III)で表される化合物又はその塩と細胞は、治療用組成物の形態で対象に投与することができる。ここで細胞は、前記と同様のものが挙げられる。
The compound represented by the formula (I) or (III) or a salt thereof and cells can be administered to a subject in the form of a therapeutic composition. Here, examples of the cells include those described above.
治療用組成物は、対象、すなわちヒトを含む哺乳動物に投与されるものであって、式(I)又は(III)で表される化合物又はその塩及び移植細胞を含む懸濁液の注射剤の形で非経口的に投与することができる。該懸濁液には、緩衝液や培地などを使用することができ、懸濁液や培地としては前述するものが挙げられる。ある態様において、治療用組成物はさらに、前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２(MMP2)を含んでもよい。治療用組成物中の式(I)又は(III)で表される化合物又はその塩の有効濃度は、好ましくは0.1～1,000μM程度、より好ましくは50～150μM程度である。治療用組成物中のMMP2の有効濃度は、好ましくは0.1～1,000μM程度、より好ましくは7.5～22.5μM程度である。
The therapeutic composition is administered to a subject, that is, a mammal including a human, and is an injection of a suspension containing a compound represented by the formula (I) or (III) or a salt thereof and a transplanted cell. Can be administered parenterally. A buffer solution, a culture medium, etc. can be used for this suspension, What was mentioned above as a suspension and a culture medium is mentioned. In certain embodiments, the therapeutic composition may further comprise matrix metalloprotease 2 (MMP2) bound to the ligand binding protein. The effective concentration of the compound represented by the formula (I) or (III) or a salt thereof in the therapeutic composition is preferably about 0.1 to 1,000 μM, more preferably about 50 to 150 μM. The effective concentration of MMP2 in the therapeutic composition is preferably about 0.1 to 1,000 μM, more preferably about 7.5 to 22.5 μM.
治療用組成物の投与量は、剤型の種類、投与方法、患者の年齢や体重、患者の症状等を考慮して、適宜決定することができる。
The dosage of the therapeutic composition can be appropriately determined in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
本発明は、
（ａ）式(III)で表される化合物又はその塩、
（ｂ）前記リガンド結合タンパク質に結合したMMP2、及び
（ｃ）細胞を対象に投与することを含む、細胞の移植方法を提供する。 The present invention
(A) a compound represented by the formula (III) or a salt thereof,
There is provided a cell transplantation method comprising (b) MMP2 bound to the ligand binding protein, and (c) administering the cell to a subject.
（ａ）式(III)で表される化合物又はその塩、
（ｂ）前記リガンド結合タンパク質に結合したMMP2、及び
（ｃ）細胞を対象に投与することを含む、細胞の移植方法を提供する。 The present invention
(A) a compound represented by the formula (III) or a salt thereof,
There is provided a cell transplantation method comprising (b) MMP2 bound to the ligand binding protein, and (c) administering the cell to a subject.
本発明はまた、
（ａ）式(III)で表される化合物又はその塩、及び
（ｂ）前記リガンド結合タンパク質に結合したMMP2
を含む、キットを提供する。 The present invention also provides
(A) a compound represented by the formula (III) or a salt thereof, and (b) MMP2 bound to the ligand-binding protein.
A kit comprising:
（ａ）式(III)で表される化合物又はその塩、及び
（ｂ）前記リガンド結合タンパク質に結合したMMP2
を含む、キットを提供する。 The present invention also provides
(A) a compound represented by the formula (III) or a salt thereof, and (b) MMP2 bound to the ligand-binding protein.
A kit comprising:
本発明の式(I)で表される化合物又はその塩は、硫酸化グリコサミノグリカン上で自己集合(self-assemble)し、細胞表面でサブミクロン粒子を形成し、MAPKシグナルによって細胞生存能を活性化し得る。
The compound represented by the formula (I) of the present invention or a salt thereof self-assembles on a sulfated glycosaminoglycan, forms submicron particles on the cell surface, and cell viability by MAPK signal Can be activated.
マトリックスメタロプロテアーゼ２(MMP2)は、細胞外マトリックス、特にI型及びIV型コラーゲンを分解し、がん細胞の転移・浸潤に関与することが知られている。本発明の式(III)で表される化合物又はその塩は、リガンド結合タンパク質に特異的に結合するリガンド部位を有する。したがって、（ａ）式(III)で表される化合物又はその塩、（ｂ）前記リガンド結合タンパク質に結合したMMP2、及び（ｃ）細胞を対象に投与することにより、MMP2とコンジュゲートした式(III)の化合物が細胞表面でサブミクロン粒子を形成し、細胞表面がMMP2で修飾され得る。MMP2で修飾された細胞は、がん細胞のように細胞浸潤活性が増大することを本発明者らは見出した。
Matrix metalloproteinase 2 (MMP2) is known to degrade extracellular matrix, particularly type I and type IV collagen, and to be involved in cancer cell metastasis and invasion. The compound represented by the formula (III) of the present invention or a salt thereof has a ligand site that specifically binds to a ligand-binding protein. Therefore, (a) a compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand binding protein, and (c) a formula conjugated with MMP2 by administering a cell to a subject ( The compound of III) can form submicron particles on the cell surface and the cell surface can be modified with MMP2. The present inventors have found that cells modified with MMP2 have increased cell invasive activity like cancer cells.
「リガンド結合タンパク質」としては、式(III)においてＹで表される「リガンド結合タンパク質に特異的に結合するリガンド」に対応する「リガンド結合タンパク質」が使用される。例えば、Ｙとして、ＨａｌｏＴａｇリガンド（プロメガ コーポレイション）を使用する場合、「リガンド結合タンパク質」としては、ＨａｌｏＴａｇタンパク質（プロメガ コーポレイション）が使用される。
As the “ligand binding protein”, “ligand binding protein” corresponding to “ligand that specifically binds to ligand binding protein” represented by Y in formula (III) is used. For example, when a HaloTag ligand (Promega Corporation) is used as Y, a HaloTag protein (Promega Corporation) is used as the “ligand binding protein”.
リガンド結合タンパク質に結合したMMP2は、自体公知の方法により製造することができる。
MMP2 bound to the ligand binding protein can be produced by a method known per se.
（ａ）式(III)で表される化合物又はその塩、（ｂ）前記リガンド結合タンパク質に結合したMMP2、及び（ｃ）細胞は、治療用組成物の形態で対象に投与することができる。ここで細胞は、前記と同様のものが挙げられる。
(A) A compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand binding protein, and (c) a cell can be administered to a subject in the form of a therapeutic composition. Here, examples of the cells include those described above.
以下、本発明を更に詳しく説明するため実施例及び試験例を挙げる。しかし、本発明はこれら実施例等に限定されるものではない。
Hereinafter, examples and test examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples.
製造例１
アミノオキシ酢酸が結合したペプチドの合成
すべてのペプチドは、RINK Amide MBHA樹脂LLを用い、通常のFmoc固相ペプチド合成法(SPPS)により合成した。樹脂(0.1 mmol)を使用前にCH2Cl2中で3時間膨潤させた。Fmoc基の脱保護は、N,N-ジメチルホルムアミド(DMF)中の20%ピペリジンで行った（1分間及び10分間）。連続するアミノ酸のカップリングは、DMF/N-メチル-2-ピロリドン(NMP) = 1/1混合物中で、対応するFmoc-アミノ酸(0.3 mmol, 渡辺化学工業株式会社, 表１)又は[(tert-ブトキシカルボニル)アミノオキシ]酢酸(0.3 mmol, 東京化成工業株式会社)、O-(ベンゾトリアゾール-1-イル)-N,N,N',N'-テトラメチルウロニウム ヘキサフルオロホスファート(HBTU, 0.3 mmol, 渡辺化学工業株式会社)、1-ヒドロキシベンゾトリアゾール(HOBt, 0.3 mmol, 東京化成工業株式会社)、及びジイソプロピルエチルアミン(DIPEA, 0.6 mmol, 和光純薬工業株式会社)を用い、室温で30分間行った。この手順をペプチド配列にしたがって数回繰り返した後、ペプチド結合樹脂をメタノール及びCH2Cl2で洗浄した。減圧下で乾燥後、樹脂をトリフルオロ酢酸(TFA)/トリイソプロピルシラン(TIS)/水 = 95/2.5/2.5の混合物中、室温で3時間処理した。ろ過後、冷却したジエチルエーテルをろ液に添加した。遠心分離(2300 g×5分間)後、上清を廃棄した。この手順を更に2回繰り返した。残渣を減圧下、室温で3時間乾燥して、ペプチド粗生成物を白色固体として得た。
Production Example 1
Synthesis of peptides with aminooxyacetic acid attached All peptides were synthesized by conventional Fmoc solid phase peptide synthesis (SPPS) using RINK Amide MBHA resin LL. Resin (0.1 mmol) was swollen in CH 2 Cl 2 for 3 hours before use. Deprotection of the Fmoc group was performed with 20% piperidine in N, N-dimethylformamide (DMF) (1 min and 10 min). Coupling of consecutive amino acids was performed in a DMF / N-methyl-2-pyrrolidone (NMP) = 1/1 mixture with the corresponding Fmoc-amino acid (0.3 mmol, Watanabe Chemical Co., Table 1) or [(tert -Butoxycarbonyl) aminooxy] acetic acid (0.3 mmol, Tokyo Chemical Industry Co., Ltd.), O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU , 0.3 mmol, Watanabe Chemical Co., Ltd.), 1-hydroxybenzotriazole (HOBt, 0.3 mmol, Tokyo Chemical Industry Co., Ltd.), and diisopropylethylamine (DIPEA, 0.6 mmol, Wako Pure Chemical Industries, Ltd.) at room temperature. I went for 30 minutes. After repeating this procedure several times according to the peptide sequence, the peptide-bound resin was washed with methanol and CH 2 Cl 2 . After drying under reduced pressure, the resin was treated in a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) /water=95/2.5/2.5 at room temperature for 3 hours. After filtration, cooled diethyl ether was added to the filtrate. After centrifugation (2300 g × 5 minutes), the supernatant was discarded. This procedure was repeated two more times. The residue was dried under reduced pressure at room temperature for 3 hours to obtain a crude peptide product as a white solid.
アミノオキシ酢酸が結合したペプチドの合成
すべてのペプチドは、RINK Amide MBHA樹脂LLを用い、通常のFmoc固相ペプチド合成法(SPPS)により合成した。樹脂(0.1 mmol)を使用前にCH2Cl2中で3時間膨潤させた。Fmoc基の脱保護は、N,N-ジメチルホルムアミド(DMF)中の20%ピペリジンで行った（1分間及び10分間）。連続するアミノ酸のカップリングは、DMF/N-メチル-2-ピロリドン(NMP) = 1/1混合物中で、対応するFmoc-アミノ酸(0.3 mmol, 渡辺化学工業株式会社, 表１)又は[(tert-ブトキシカルボニル)アミノオキシ]酢酸(0.3 mmol, 東京化成工業株式会社)、O-(ベンゾトリアゾール-1-イル)-N,N,N',N'-テトラメチルウロニウム ヘキサフルオロホスファート(HBTU, 0.3 mmol, 渡辺化学工業株式会社)、1-ヒドロキシベンゾトリアゾール(HOBt, 0.3 mmol, 東京化成工業株式会社)、及びジイソプロピルエチルアミン(DIPEA, 0.6 mmol, 和光純薬工業株式会社)を用い、室温で30分間行った。この手順をペプチド配列にしたがって数回繰り返した後、ペプチド結合樹脂をメタノール及びCH2Cl2で洗浄した。減圧下で乾燥後、樹脂をトリフルオロ酢酸(TFA)/トリイソプロピルシラン(TIS)/水 = 95/2.5/2.5の混合物中、室温で3時間処理した。ろ過後、冷却したジエチルエーテルをろ液に添加した。遠心分離(2300 g×5分間)後、上清を廃棄した。この手順を更に2回繰り返した。残渣を減圧下、室温で3時間乾燥して、ペプチド粗生成物を白色固体として得た。
Synthesis of peptides with aminooxyacetic acid attached All peptides were synthesized by conventional Fmoc solid phase peptide synthesis (SPPS) using RINK Amide MBHA resin LL. Resin (0.1 mmol) was swollen in CH 2 Cl 2 for 3 hours before use. Deprotection of the Fmoc group was performed with 20% piperidine in N, N-dimethylformamide (DMF) (1 min and 10 min). Coupling of consecutive amino acids was performed in a DMF / N-methyl-2-pyrrolidone (NMP) = 1/1 mixture with the corresponding Fmoc-amino acid (0.3 mmol, Watanabe Chemical Co., Table 1) or [(tert -Butoxycarbonyl) aminooxy] acetic acid (0.3 mmol, Tokyo Chemical Industry Co., Ltd.), O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU , 0.3 mmol, Watanabe Chemical Co., Ltd.), 1-hydroxybenzotriazole (HOBt, 0.3 mmol, Tokyo Chemical Industry Co., Ltd.), and diisopropylethylamine (DIPEA, 0.6 mmol, Wako Pure Chemical Industries, Ltd.) at room temperature. I went for 30 minutes. After repeating this procedure several times according to the peptide sequence, the peptide-bound resin was washed with methanol and CH 2 Cl 2 . After drying under reduced pressure, the resin was treated in a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) /water=95/2.5/2.5 at room temperature for 3 hours. After filtration, cooled diethyl ether was added to the filtrate. After centrifugation (2300 g × 5 minutes), the supernatant was discarded. This procedure was repeated two more times. The residue was dried under reduced pressure at room temperature for 3 hours to obtain a crude peptide product as a white solid.
実施例１
Adh-SFFの合成
アドヘサミン(20.3 mg, 33.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-SFF (9.03 mg, 19.1 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間24分)で精製した。溶出液を凍結乾燥して、Adh-SFF (5.97 mg, 4.29 μmol, 30%)を白色固体として得た。
ESI-HRMS: found 525.673463 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
1H-NMR (300 MHz, DMSO-d6) δ 2.52 (3H, s), 2.55 (3H, s), 2.70-2.84 (2H, m), 2.89-2.96 (1H, dd, J = 4.8 Hz), 3.00-3.06 (1H, dd, J = 4.8Hz), 3.50-3.67 (2H, m), 3.86-4.09 (24H, br, m) , 4.34-4.41 (3H, m, J = 4.8 Hz), 4.64 (2H, s), 5.33 (1H, br), 7.10-7.35 (12H, m), 7.89 (1H, d, J = 7.5 Hz), 8.02 (1H, d, J = 8.1 Hz), 8.33 (1H, d, J = 7.5 Hz), 8.46 (1H, s), 10.1 (1H, s). 13C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 36.8, 37.3, 40.3, 40.9, 41.6, 51.1, 51.2, 54.0, 54.5, 54.6, 61.5, 72.4, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 128.9, 129.0, 137.4, 137.7, 145.9, 159.6, 160.6, 164.3, 168.5, 170.0, 170.2, 170.7, 172.9, 173.3, 186.5.
Example 1
Synthesis of Adh-SFF Adhesamine (20.3 mg, 33.8 μmol) was added to peptide Aoa-SFF (9.03 mg, 19.1 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,retention time 24 minutes). The eluate was freeze-dried to obtain Adh-SFF (5.97 mg, 4.29 μmol, 30%) as a white solid.
ESI-HRMS: found 525.673463 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
1 H-NMR (300 MHz, DMSO-d6) δ 2.52 (3H, s), 2.55 (3H, s), 2.70-2.84 (2H, m), 2.89-2.96 (1H, dd, J = 4.8 Hz), 3.00-3.06 (1H, dd, J = 4.8Hz), 3.50-3.67 (2H, m), 3.86-4.09 (24H, br, m), 4.34-4.41 (3H, m, J = 4.8 Hz), 4.64 ( 2H, s), 5.33 (1H, br), 7.10-7.35 (12H, m), 7.89 (1H, d, J = 7.5 Hz), 8.02 (1H, d, J = 8.1 Hz), 8.33 (1H, d , J = 7.5 Hz), 8.46 (1H, s), 10.1 (1H, s). 13 C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 36.8, 37.3, 40.3, 40.9, 41.6, 51.1, 51.2, 54.0, 54.5, 54.6, 61.5, 72.4, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 128.9, 129.0, 137.4, 137.7, 145.9, 159.6, 160.6, 164.3, 168.5, 170.0, 170.2, 170.7, 172.9, 173.3, 186.5.
Adh-SFFの合成
アドヘサミン(20.3 mg, 33.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-SFF (9.03 mg, 19.1 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間24分)で精製した。溶出液を凍結乾燥して、Adh-SFF (5.97 mg, 4.29 μmol, 30%)を白色固体として得た。
ESI-HRMS: found 525.673463 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
1H-NMR (300 MHz, DMSO-d6) δ 2.52 (3H, s), 2.55 (3H, s), 2.70-2.84 (2H, m), 2.89-2.96 (1H, dd, J = 4.8 Hz), 3.00-3.06 (1H, dd, J = 4.8Hz), 3.50-3.67 (2H, m), 3.86-4.09 (24H, br, m) , 4.34-4.41 (3H, m, J = 4.8 Hz), 4.64 (2H, s), 5.33 (1H, br), 7.10-7.35 (12H, m), 7.89 (1H, d, J = 7.5 Hz), 8.02 (1H, d, J = 8.1 Hz), 8.33 (1H, d, J = 7.5 Hz), 8.46 (1H, s), 10.1 (1H, s). 13C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 36.8, 37.3, 40.3, 40.9, 41.6, 51.1, 51.2, 54.0, 54.5, 54.6, 61.5, 72.4, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 128.9, 129.0, 137.4, 137.7, 145.9, 159.6, 160.6, 164.3, 168.5, 170.0, 170.2, 170.7, 172.9, 173.3, 186.5.
Synthesis of Adh-SFF Adhesamine (20.3 mg, 33.8 μmol) was added to peptide Aoa-SFF (9.03 mg, 19.1 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,
ESI-HRMS: found 525.673463 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
1 H-NMR (300 MHz, DMSO-d6) δ 2.52 (3H, s), 2.55 (3H, s), 2.70-2.84 (2H, m), 2.89-2.96 (1H, dd, J = 4.8 Hz), 3.00-3.06 (1H, dd, J = 4.8Hz), 3.50-3.67 (2H, m), 3.86-4.09 (24H, br, m), 4.34-4.41 (3H, m, J = 4.8 Hz), 4.64 ( 2H, s), 5.33 (1H, br), 7.10-7.35 (12H, m), 7.89 (1H, d, J = 7.5 Hz), 8.02 (1H, d, J = 8.1 Hz), 8.33 (1H, d , J = 7.5 Hz), 8.46 (1H, s), 10.1 (1H, s). 13 C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 36.8, 37.3, 40.3, 40.9, 41.6, 51.1, 51.2, 54.0, 54.5, 54.6, 61.5, 72.4, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 128.9, 129.0, 137.4, 137.7, 145.9, 159.6, 160.6, 164.3, 168.5, 170.0, 170.2, 170.7, 172.9, 173.3, 186.5.
参考例１
Adh-Sの合成
アドヘサミン(12.5 mg, 20.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2.5 ml)中のペプチド Aoa-S (2.09 mg, 10.5 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/35→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh-S (2.93 mg, 3.87 μmol, 37%)を白色固体として得た。
ESI-HRMS: found 378.604964 calcd 378.604982 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference example 1
Synthesis of Adh-S Adhesamine (12.5 mg, 20.8 μmol) was added to peptide Aoa-S (2.09 mg, 10.5 μmol) in a mixture (2.5 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/35 → 25/95,retention time 20 minutes). The eluate was lyophilized to give Adh-S (2.93 mg, 3.87 μmol, 37%) as a white solid.
ESI-HRMS: found 378.604964 calcd 378.604982 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-Sの合成
アドヘサミン(12.5 mg, 20.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2.5 ml)中のペプチド Aoa-S (2.09 mg, 10.5 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/35→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh-S (2.93 mg, 3.87 μmol, 37%)を白色固体として得た。
ESI-HRMS: found 378.604964 calcd 378.604982 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-S Adhesamine (12.5 mg, 20.8 μmol) was added to peptide Aoa-S (2.09 mg, 10.5 μmol) in a mixture (2.5 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/35 → 25/95,
ESI-HRMS: found 378.604964 calcd 378.604982 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例２
Adh-SFの合成
アドヘサミン(17.1 mg, 28.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-SF (5.69 mg, 17.6 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh-SF (5.76 mg, 6.36 μmol, 36%)を白色固体として得た。
ESI-HRMS: found 452.139067 calcd 452.139189 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference example 2
Synthesis of Adh-SF Adhesamine (17.1 mg, 28.5 μmol) was added to peptide Aoa-SF (5.69 mg, 17.6 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,retention time 20 minutes). The eluate was lyophilized to give Adh-SF (5.76 mg, 6.36 μmol, 36%) as a white solid.
ESI-HRMS: found 452.139067 calcd 452.139189 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-SFの合成
アドヘサミン(17.1 mg, 28.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-SF (5.69 mg, 17.6 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh-SF (5.76 mg, 6.36 μmol, 36%)を白色固体として得た。
ESI-HRMS: found 452.139067 calcd 452.139189 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-SF Adhesamine (17.1 mg, 28.5 μmol) was added to peptide Aoa-SF (5.69 mg, 17.6 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,
ESI-HRMS: found 452.139067 calcd 452.139189 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
実施例２
Adh-SFFFの合成
アドヘサミン(4.64 mg, 7.73 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SFFF (4.64 mg, 7.50 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間27分)で精製した。溶出液を凍結乾燥して、Adh-SFFF (3.64 mg, 3.03 μmol, 40%)を白色固体として得た。
ESI-HRMS: found 599.207270 calcd 599.207603 ([M]2+), 純度 >95% (LCMS分析で計算).
Example 2
Synthesis of Adh-SFFF Adhesamine (4.64 mg, 7.73 μmol) was added to peptide Aoa-SFFF (4.64 mg, 7.50 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 27 minutes). The eluate was lyophilized to give Adh-SFFF (3.64 mg, 3.03 μmol, 40%) as a white solid.
ESI-HRMS: found 599.207270 calcd 599.207603 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-SFFFの合成
アドヘサミン(4.64 mg, 7.73 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SFFF (4.64 mg, 7.50 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間27分)で精製した。溶出液を凍結乾燥して、Adh-SFFF (3.64 mg, 3.03 μmol, 40%)を白色固体として得た。
ESI-HRMS: found 599.207270 calcd 599.207603 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-SFFF Adhesamine (4.64 mg, 7.73 μmol) was added to peptide Aoa-SFFF (4.64 mg, 7.50 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 27 minutes). The eluate was lyophilized to give Adh-SFFF (3.64 mg, 3.03 μmol, 40%) as a white solid.
ESI-HRMS: found 599.207270 calcd 599.207603 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
実施例３
Adh-FFの合成
アドヘサミン(8.21 mg, 13.7 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-FF (3.94 mg, 10.2 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間28分)で精製した。溶出液を凍結乾燥して、Adh-FF (2.31 mg, 2.39 μmol, 23%)を白色固体として得た。
ESI-HRMS: found 482.157184 calcd 482.157382 ([M]2+), 純度 >95% (LCMS分析で計算).
Example 3
Synthesis of Adh-FF Adhesamine (8.21 mg, 13.7 μmol) was added to peptide Aoa-FF (3.94 mg, 10.2 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 28 minutes). The eluate was lyophilized to give Adh-FF (2.31 mg, 2.39 μmol, 23%) as a white solid.
ESI-HRMS: found 482.157184 calcd 482.157382 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-FFの合成
アドヘサミン(8.21 mg, 13.7 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-FF (3.94 mg, 10.2 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間28分)で精製した。溶出液を凍結乾燥して、Adh-FF (2.31 mg, 2.39 μmol, 23%)を白色固体として得た。
ESI-HRMS: found 482.157184 calcd 482.157382 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-FF Adhesamine (8.21 mg, 13.7 μmol) was added to peptide Aoa-FF (3.94 mg, 10.2 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 28 minutes). The eluate was lyophilized to give Adh-FF (2.31 mg, 2.39 μmol, 23%) as a white solid.
ESI-HRMS: found 482.157184 calcd 482.157382 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
実施例４
Adh-SSFFの合成
アドヘサミン(9.30 mg, 15.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SSFF (6.10 mg, 10.5 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間22分)で精製した。溶出液を凍結乾燥して、Adh-SSFF (4.68 mg, 4.10 μmol, 39%)を白色固体として得た。
ESI-HRMS: found 569.189369 calcd 569.189410 ([M]2+), 純度 >95% (LCMS分析で計算).
Example 4
Synthesis of Adh-SSFF Adhesamine (9.30 mg, 15.5 μmol) was added to peptide Aoa-SSFF (6.10 mg, 10.5 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 22 minutes). The eluate was freeze-dried to obtain Adh-SSFF (4.68 mg, 4.10 μmol, 39%) as a white solid.
ESI-HRMS: found 569.189369 calcd 569.189410 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-SSFFの合成
アドヘサミン(9.30 mg, 15.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SSFF (6.10 mg, 10.5 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間22分)で精製した。溶出液を凍結乾燥して、Adh-SSFF (4.68 mg, 4.10 μmol, 39%)を白色固体として得た。
ESI-HRMS: found 569.189369 calcd 569.189410 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-SSFF Adhesamine (9.30 mg, 15.5 μmol) was added to peptide Aoa-SSFF (6.10 mg, 10.5 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 22 minutes). The eluate was freeze-dried to obtain Adh-SSFF (4.68 mg, 4.10 μmol, 39%) as a white solid.
ESI-HRMS: found 569.189369 calcd 569.189410 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例３
Adh-SSSFFの合成
アドヘサミン(8.66 mg, 14.4 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SSSFF (5.71 mg, 8.55 μmol)の溶液に添加した。この溶液を55℃で22時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間21分)で精製した。溶出液を凍結乾燥して、Adh-SSSFF (3.74 mg, 3.05 μmol, 36%)を白色固体として得た。
ESI-HRMS: found 612.705249 calcd 612.705424 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference example 3
Synthesis of Adh-SSSFF Adhesamine (8.66 mg, 14.4 μmol) was added to peptide Aoa-SSSFF (5.71 mg, 8.55 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 22 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,retention time 21 minutes). The eluate was freeze-dried to obtain Adh-SSSFF (3.74 mg, 3.05 μmol, 36%) as a white solid.
ESI-HRMS: found 612.705249 calcd 612.705424 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-SSSFFの合成
アドヘサミン(8.66 mg, 14.4 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のペプチド Aoa-SSSFF (5.71 mg, 8.55 μmol)の溶液に添加した。この溶液を55℃で22時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/18→ 21/48→ 25/95, 保持時間21分)で精製した。溶出液を凍結乾燥して、Adh-SSSFF (3.74 mg, 3.05 μmol, 36%)を白色固体として得た。
ESI-HRMS: found 612.705249 calcd 612.705424 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-SSSFF Adhesamine (8.66 mg, 14.4 μmol) was added to peptide Aoa-SSSFF (5.71 mg, 8.55 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 22 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/18 → 21/48 → 25/95,
ESI-HRMS: found 612.705249 calcd 612.705424 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例４
Adh-FSFの合成
アドヘサミン(15.2 mg, 25.3 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-FSF (8.99 mg, 19.0 μmol)の溶液に添加した。この溶液を51℃で22時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間25分)で精製した。溶出液を凍結乾燥して、Adh-FSF (4.83 mg, 4.59 μmol, 24%)を白色固体として得た。
ESI-HRMS: found 525.673200 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference example 4
Synthesis of Adh-FSF Adhesamine (15.2 mg, 25.3 μmol) was added to peptide Aoa-FSF (8.99 mg, 19.0 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 51 ° C. for 22 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95,retention time 25 minutes). The eluate was lyophilized to give Adh-FSF (4.83 mg, 4.59 μmol, 24%) as a white solid.
ESI-HRMS: found 525.673200 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-FSFの合成
アドヘサミン(15.2 mg, 25.3 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-FSF (8.99 mg, 19.0 μmol)の溶液に添加した。この溶液を51℃で22時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間25分)で精製した。溶出液を凍結乾燥して、Adh-FSF (4.83 mg, 4.59 μmol, 24%)を白色固体として得た。
ESI-HRMS: found 525.673200 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-FSF Adhesamine (15.2 mg, 25.3 μmol) was added to peptide Aoa-FSF (8.99 mg, 19.0 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 51 ° C. for 22 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95,
ESI-HRMS: found 525.673200 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
実施例５
Adh-FFSの合成
アドヘサミン(16.5 mg, 27.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-FFS (10.2 mg, 21.6 μmol)の溶液に添加した。この溶液を55℃で20時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間28分)で精製した。溶出液を凍結乾燥して、Adh-FFS (5.80 mg, 5.51 μmol, 26%)を白色固体として得た。
ESI-HRMS: found 525.673387 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
Example 5
Synthesis of Adh-FFS Adhesamine (16.5 mg, 27.5 μmol) was added to peptide Aoa-FFS (10.2 mg, 21.6 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 20 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 28 minutes). The eluate was freeze-dried to obtain Adh-FFS (5.80 mg, 5.51 μmol, 26%) as a white solid.
ESI-HRMS: found 525.673387 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-FFSの合成
アドヘサミン(16.5 mg, 27.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(4 ml)中のペプチド Aoa-FFS (10.2 mg, 21.6 μmol)の溶液に添加した。この溶液を55℃で20時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間28分)で精製した。溶出液を凍結乾燥して、Adh-FFS (5.80 mg, 5.51 μmol, 26%)を白色固体として得た。
ESI-HRMS: found 525.673387 calcd 525.673396 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-FFS Adhesamine (16.5 mg, 27.5 μmol) was added to peptide Aoa-FFS (10.2 mg, 21.6 μmol) in a mixture (4 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 20 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 28 minutes). The eluate was freeze-dried to obtain Adh-FFS (5.80 mg, 5.51 μmol, 26%) as a white solid.
ESI-HRMS: found 525.673387 calcd 525.673396 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例５
ペプチドSFF(peptide SFF)の合成
アセトン(30 μl, 23.6 mg, 40.6 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(1 ml)中のペプチド Aoa-SFF (5.63 mg, 11.9 μmol)の溶液に添加した。この溶液を55℃で6時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間23分)で精製した。溶出液を凍結乾燥して、ペプチド SFF (2.16 mg, 4.20 μmol, 35%)を白色固体として得た。
ESI-HRMS: found 512.250717 calcd 512.250360 ([M+H]+ ), 純度 >95% (LCMS分析で計算). Reference Example 5
Synthesis of peptide SFF
Acetone (30 μl, 23.6 mg, 40.6 μmol) is added to a solution of peptide Aoa-SFF (5.63 mg, 11.9 μmol) in a mixture (1 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added. The solution was stirred at 55 ° C. for 6 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 23 minutes). The eluate was lyophilized to give peptide SFF (2.16 mg, 4.20 μmol, 35%) as a white solid.
ESI-HRMS: found 512.250717 calcd 512.250360 ([M + H] + ), purity> 95% (calculated by LCMS analysis).
ペプチドSFF(peptide SFF)の合成
ESI-HRMS: found 512.250717 calcd 512.250360 ([M+H]+ ), 純度 >95% (LCMS分析で計算). Reference Example 5
Synthesis of peptide SFF
ESI-HRMS: found 512.250717 calcd 512.250360 ([M + H] + ), purity> 95% (calculated by LCMS analysis).
実施例６
Adh-SFFKの合成
アドヘサミン(12.3 mg, 20.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(3.6 ml)中のペプチド Aoa-SFFK (8.81 mg, 14.7 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/55→ 25/95, 保持時間19分)で精製した。溶出液を凍結乾燥して、Adh-SFFK (4.71 mg, 3.99 μmol, 27%)を白色固体として得た。
ESI-HRMS: found 589.720952 calcd 589.720877 ([M]2+), 純度 >95% (LCMS分析で計算).
Example 6
Synthesis of Adh-SFFK Adhesamine (12.3 mg, 20.5 μmol) was added to peptide Aoa-SFFK (8.81 mg, 14.7 μmol) in a mixture (3.6 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time 19 minutes). The eluate was freeze-dried to obtain Adh-SFFK (4.71 mg, 3.99 μmol, 27%) as a white solid.
ESI-HRMS: found 589.720952 calcd 589.720877 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-SFFKの合成
アドヘサミン(12.3 mg, 20.5 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(3.6 ml)中のペプチド Aoa-SFFK (8.81 mg, 14.7 μmol)の溶液に添加した。この溶液を55℃で24時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/55→ 25/95, 保持時間19分)で精製した。溶出液を凍結乾燥して、Adh-SFFK (4.71 mg, 3.99 μmol, 27%)を白色固体として得た。
ESI-HRMS: found 589.720952 calcd 589.720877 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-SFFK Adhesamine (12.3 mg, 20.5 μmol) was added to peptide Aoa-SFFK (8.81 mg, 14.7 μmol) in a mixture (3.6 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time 19 minutes). The eluate was freeze-dried to obtain Adh-SFFK (4.71 mg, 3.99 μmol, 27%) as a white solid.
ESI-HRMS: found 589.720952 calcd 589.720877 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例６
Adh(SFFK)-SFFKの合成
アドヘサミン(4.63 mg, 7.72 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(1 ml)中のペプチド Aoa-SFFK (9.64 mg, 16.1 μmol)の溶液に添加した。この溶液を55℃で20時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/55→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh(SFFK)-SFFK (6.70 mg, 3.80 μmol, 49%)を白色固体として得た。
ESI-HRMS: found 880.369003 calcd 880.368968 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference Example 6
Synthesis of Adh (SFFK) -SFFK Adhesamine (4.63 mg, 7.72 μmol) was added to peptide Aoa-SFFK (9.64 mg, 16.1) in a mixture (1 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. μmol) solution. The solution was stirred at 55 ° C. for 20 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/55 → 25/95,retention time 20 minutes). The eluate was lyophilized to give Adh (SFFK) -SFFK (6.70 mg, 3.80 μmol, 49%) as a white solid.
ESI-HRMS: found 880.369003 calcd 880.368968 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh(SFFK)-SFFKの合成
アドヘサミン(4.63 mg, 7.72 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(1 ml)中のペプチド Aoa-SFFK (9.64 mg, 16.1 μmol)の溶液に添加した。この溶液を55℃で20時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/55→ 25/95, 保持時間20分)で精製した。溶出液を凍結乾燥して、Adh(SFFK)-SFFK (6.70 mg, 3.80 μmol, 49%)を白色固体として得た。
ESI-HRMS: found 880.369003 calcd 880.368968 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh (SFFK) -SFFK Adhesamine (4.63 mg, 7.72 μmol) was added to peptide Aoa-SFFK (9.64 mg, 16.1) in a mixture (1 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. μmol) solution. The solution was stirred at 55 ° C. for 20 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/55 → 25/95,
ESI-HRMS: found 880.369003 calcd 880.368968 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
製造例３
化合物5及び化合物6の合成
(1) tert-ブチル (2-(2-ヒドロキシエトキシ)エチル)カルバマート(1)の合成
ジ-tert-ブチル-ジカルボナート(4.75 g, 21.8 mmol)を、2-(2-アミノエチル)エタノール(2.14 g, 20.4 mmol)の脱水CH2Cl2(30 ml)溶液に添加した。この溶液を室温で2時間撹拌した。溶媒の除去後、残渣に水(100 ml)を添加した。この溶液をCH2Cl2(40 ml x 2)で
抽出し、有機層をNa2SO4で乾燥した。溶媒留去後、残渣を減圧下で乾燥して、化合物1(4.59 g, 定量的)を透明オイルとして得た。
ESI-MS, found 228.00 calcd 228.12 ([M+H]+). Production Example 3
Synthesis ofCompound 5 and Compound 6
(1) Synthesis of tert-butyl (2- (2-hydroxyethoxy) ethyl) carbamate (1) Di-tert-butyl-dicarbonate (4.75 g, 21.8 mmol) was converted to 2- (2-aminoethyl) ethanol (2.14 g, 20.4 mmol) in dehydrated CH 2 Cl 2 (30 ml). The solution was stirred at room temperature for 2 hours. After removal of the solvent, water (100 ml) was added to the residue. This solution was extracted with CH 2 Cl 2 (40 ml × 2) and the organic layer was dried over Na 2 SO 4 . After the solvent was distilled off, the residue was dried under reduced pressure to obtain Compound 1 (4.59 g, quantitative) as a clear oil.
ESI-MS, found 228.00 calcd 228.12 ([M + H] + ).
化合物5及び化合物6の合成
ジ-tert-ブチル-ジカルボナート(4.75 g, 21.8 mmol)を、2-(2-アミノエチル)エタノール(2.14 g, 20.4 mmol)の脱水CH2Cl2(30 ml)溶液に添加した。この溶液を室温で2時間撹拌した。溶媒の除去後、残渣に水(100 ml)を添加した。この溶液をCH2Cl2(40 ml x 2)で
抽出し、有機層をNa2SO4で乾燥した。溶媒留去後、残渣を減圧下で乾燥して、化合物1(4.59 g, 定量的)を透明オイルとして得た。
ESI-MS, found 228.00 calcd 228.12 ([M+H]+). Production Example 3
Synthesis of
ESI-MS, found 228.00 calcd 228.12 ([M + H] + ).
(2) tert-ブチル (2-(2-((6-クロロヘキシル)オキシ)エトキシ)エチル)カルバマート(2)の合成
NaH(1.32 g, 60% in oil)を、化合物1(4.59 g)のTHF(28 ml)及びDMF(14 ml)の混合物中の溶液に添加した。このエマルションを0℃で30分間撹拌し、1-クロロ-6-ヨードヘキサン(4.6 ml, 31.3 mmol)を添加した。室温で13時間撹拌した後、反応混合物を飽和NH4Cl溶液(10 ml)でクエンチした。水(100 ml)の添加後、この溶液をAcOEt(150 ml)で抽出し、飽和食塩水(50 ml)で洗浄した。有機層をNa2SO4で乾燥し、溶媒留去した。残渣をカラムクロマトグラフィー(SiO2, ヘキサン/AcOEt = 4/1)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物2(2.69 g, 8.32 mmol, 41%)を透明オイルとして得た。
ESI-MS, found 346.05 calcd 346.18 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.44 (m, 13H), 1.53-1.64 (m, 2 H), 1.75-1.80 (m, 2H), 3.32 (m, br, 2H), 3.44-3.48 (tr, 2H, J = 6.6 Hz), 3.50-3.61 (m, 8H), 5.02 (br, 1H). (2) Synthesis of tert-butyl (2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) carbamate (2) NaH (1.32 g, 60% in oil) was dissolved in Compound 1 (4.59 g). To a solution in a mixture of THF (28 ml) and DMF (14 ml) was added. The emulsion was stirred at 0 ° C. for 30 minutes and 1-chloro-6-iodohexane (4.6 ml, 31.3 mmol) was added. After stirring at room temperature for 13 hours, the reaction mixture was quenched with saturated NH 4 Cl solution (10 ml). After addition of water (100 ml), the solution was extracted with AcOEt (150 ml) and washed with saturated brine (50 ml). The organic layer was dried over Na 2 SO 4 and evaporated. The residue was purified by column chromatography (SiO 2, hexane / AcOEt = 4/1) was purified. The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 2 (2.69 g, 8.32 mmol, 41%) as a clear oil.
ESI-MS, found 346.05 calcd 346.18 ([M + H] + ). 1 H-NMR (300 MHz, CDCl 3 ) 1.37-1.44 (m, 13H), 1.53-1.64 (m, 2 H), 1.75-1.80 (m, 2H), 3.32 (m, br, 2H), 3.44-3.48 (tr, 2H, J = 6.6 Hz), 3.50-3.61 (m, 8H), 5.02 (br, 1H).
NaH(1.32 g, 60% in oil)を、化合物1(4.59 g)のTHF(28 ml)及びDMF(14 ml)の混合物中の溶液に添加した。このエマルションを0℃で30分間撹拌し、1-クロロ-6-ヨードヘキサン(4.6 ml, 31.3 mmol)を添加した。室温で13時間撹拌した後、反応混合物を飽和NH4Cl溶液(10 ml)でクエンチした。水(100 ml)の添加後、この溶液をAcOEt(150 ml)で抽出し、飽和食塩水(50 ml)で洗浄した。有機層をNa2SO4で乾燥し、溶媒留去した。残渣をカラムクロマトグラフィー(SiO2, ヘキサン/AcOEt = 4/1)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物2(2.69 g, 8.32 mmol, 41%)を透明オイルとして得た。
ESI-MS, found 346.05 calcd 346.18 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.44 (m, 13H), 1.53-1.64 (m, 2 H), 1.75-1.80 (m, 2H), 3.32 (m, br, 2H), 3.44-3.48 (tr, 2H, J = 6.6 Hz), 3.50-3.61 (m, 8H), 5.02 (br, 1H). (2) Synthesis of tert-butyl (2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) carbamate (2) NaH (1.32 g, 60% in oil) was dissolved in Compound 1 (4.59 g). To a solution in a mixture of THF (28 ml) and DMF (14 ml) was added. The emulsion was stirred at 0 ° C. for 30 minutes and 1-chloro-6-iodohexane (4.6 ml, 31.3 mmol) was added. After stirring at room temperature for 13 hours, the reaction mixture was quenched with saturated NH 4 Cl solution (10 ml). After addition of water (100 ml), the solution was extracted with AcOEt (150 ml) and washed with saturated brine (50 ml). The organic layer was dried over Na 2 SO 4 and evaporated. The residue was purified by column chromatography (SiO 2, hexane / AcOEt = 4/1) was purified. The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 2 (2.69 g, 8.32 mmol, 41%) as a clear oil.
ESI-MS, found 346.05 calcd 346.18 ([M + H] + ). 1 H-NMR (300 MHz, CDCl 3 ) 1.37-1.44 (m, 13H), 1.53-1.64 (m, 2 H), 1.75-1.80 (m, 2H), 3.32 (m, br, 2H), 3.44-3.48 (tr, 2H, J = 6.6 Hz), 3.50-3.61 (m, 8H), 5.02 (br, 1H).
(3) 2-(2-((6-クロロヘキシル)オキシ)エトキシ)エタン-1-アミン(3)の合成
TFA(5 ml)を、化合物2(2.69 g)のCH2Cl2(5 ml)溶液に添加した。混合物を室温で4時間撹拌した。溶媒の除去後、残渣をカラムクロマトグラフィー(SiO2, CHCl3/MeOH = 10/1)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物3(2.49 g, 11.1 mmol, 定量的)を淡黄色オイルとして得た。
ESI-MS, found 224.05 calcd 224.14 ([M+H]+). (3) Synthesis of 2- (2-((6-chlorohexyl) oxy) ethoxy) ethan-1-amine (3) TFA (5 ml) was added to compound 2 (2.69 g) in CH 2 Cl 2 (5 ml ) Added to the solution. The mixture was stirred at room temperature for 4 hours. After removal of the solvent, the residue was purified by column chromatography (SiO 2 , CHCl 3 / MeOH = 10/1). The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 3 (2.49 g, 11.1 mmol, quantitative) as a pale yellow oil.
ESI-MS, found 224.05 calcd 224.14 ([M + H] + ).
TFA(5 ml)を、化合物2(2.69 g)のCH2Cl2(5 ml)溶液に添加した。混合物を室温で4時間撹拌した。溶媒の除去後、残渣をカラムクロマトグラフィー(SiO2, CHCl3/MeOH = 10/1)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物3(2.49 g, 11.1 mmol, 定量的)を淡黄色オイルとして得た。
ESI-MS, found 224.05 calcd 224.14 ([M+H]+). (3) Synthesis of 2- (2-((6-chlorohexyl) oxy) ethoxy) ethan-1-amine (3) TFA (5 ml) was added to compound 2 (2.69 g) in CH 2 Cl 2 (5 ml ) Added to the solution. The mixture was stirred at room temperature for 4 hours. After removal of the solvent, the residue was purified by column chromatography (SiO 2 , CHCl 3 / MeOH = 10/1). The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 3 (2.49 g, 11.1 mmol, quantitative) as a pale yellow oil.
ESI-MS, found 224.05 calcd 224.14 ([M + H] + ).
(4) tert-ブチル (2-((2-(2-((6-クロロヘキシル)オキシ)エトキシ)エチル)アミノ)-2-オキソエトキシ)カルバマート(4)の合成
[(tert-ブトキシカルボニル)アミノオキシ]酢酸(240 mg)、O-(7-アザベンゾトリアゾール-1-イル)-N,N,N',N'-テトラメチルウロニウム ヘキサフルオロホスファート(HATU)(506 mg)、及び1-ヒドロキシ-7-アザベンゾトリアゾール(HOAt)(163 mg)のDMF(5 ml)溶液を、化合物3(248 mg)及びDIPEA(700 μl)のDMF(1 ml)溶液に添加した。混合物を室温で2時間撹拌した。AcOEt(70 ml)を添加した後、有機層をNaHCO3溶液、水及び飽和食塩水で洗浄した。有機層をNa2SO4で乾燥し、溶媒留去した。残渣をカラムクロマトグラフィー(SiO2, ヘキサン/AcOEt = 1/2)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物4(144 mg, 364 μmol, 29%)を淡黄色オイルとして得た。
ESI-MS, found 419.15 calcd 419.19 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.43 (m, 4H), 1.49 (s, 9 H), 1.57-1.66 (m, 2H), 1.71-1.80 (m, 2H), 3.54-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.34 (s, 2H), 7.73 (s, 1H), 7.84 (s, 1H). (4) Synthesis of tert-butyl (2-((2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) amino) -2-oxoethoxy) carbamate (4) [(tert-butoxycarbonyl) Aminooxy] acetic acid (240 mg), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HATU) (506 mg), and A solution of 1-hydroxy-7-azabenzotriazole (HOAt) (163 mg) in DMF (5 ml) was added to a solution of compound 3 (248 mg) and DIPEA (700 μl) in DMF (1 ml). The mixture was stirred at room temperature for 2 hours. After adding AcOEt (70 ml), the organic layer was washed with NaHCO 3 solution, water and saturated brine. The organic layer was dried over Na 2 SO 4 and evaporated. The residue was purified by column chromatography (SiO 2, hexane / AcOEt = 1/2) was purified. The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 4 (144 mg, 364 μmol, 29%) as a pale yellow oil.
ESI-MS, found 419.15 calcd 419.19 ([M + H] + ). 1 H-NMR (300 MHz, CDCl 3 ) 1.37-1.43 (m, 4H), 1.49 (s, 9 H), 1.57-1.66 (m , 2H), 1.71-1.80 (m, 2H), 3.54-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.34 (s, 2H), 7.73 (s, 1H), 7.84 (s, 1H ).
[(tert-ブトキシカルボニル)アミノオキシ]酢酸(240 mg)、O-(7-アザベンゾトリアゾール-1-イル)-N,N,N',N'-テトラメチルウロニウム ヘキサフルオロホスファート(HATU)(506 mg)、及び1-ヒドロキシ-7-アザベンゾトリアゾール(HOAt)(163 mg)のDMF(5 ml)溶液を、化合物3(248 mg)及びDIPEA(700 μl)のDMF(1 ml)溶液に添加した。混合物を室温で2時間撹拌した。AcOEt(70 ml)を添加した後、有機層をNaHCO3溶液、水及び飽和食塩水で洗浄した。有機層をNa2SO4で乾燥し、溶媒留去した。残渣をカラムクロマトグラフィー(SiO2, ヘキサン/AcOEt = 1/2)で精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物4(144 mg, 364 μmol, 29%)を淡黄色オイルとして得た。
ESI-MS, found 419.15 calcd 419.19 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.43 (m, 4H), 1.49 (s, 9 H), 1.57-1.66 (m, 2H), 1.71-1.80 (m, 2H), 3.54-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.34 (s, 2H), 7.73 (s, 1H), 7.84 (s, 1H). (4) Synthesis of tert-butyl (2-((2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) amino) -2-oxoethoxy) carbamate (4) [(tert-butoxycarbonyl) Aminooxy] acetic acid (240 mg), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HATU) (506 mg), and A solution of 1-hydroxy-7-azabenzotriazole (HOAt) (163 mg) in DMF (5 ml) was added to a solution of compound 3 (248 mg) and DIPEA (700 μl) in DMF (1 ml). The mixture was stirred at room temperature for 2 hours. After adding AcOEt (70 ml), the organic layer was washed with NaHCO 3 solution, water and saturated brine. The organic layer was dried over Na 2 SO 4 and evaporated. The residue was purified by column chromatography (SiO 2, hexane / AcOEt = 1/2) was purified. The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 4 (144 mg, 364 μmol, 29%) as a pale yellow oil.
ESI-MS, found 419.15 calcd 419.19 ([M + H] + ). 1 H-NMR (300 MHz, CDCl 3 ) 1.37-1.43 (m, 4H), 1.49 (s, 9 H), 1.57-1.66 (m , 2H), 1.71-1.80 (m, 2H), 3.54-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.34 (s, 2H), 7.73 (s, 1H), 7.84 (s, 1H ).
(5) 2-(アミノオキシ)-N-(2-(2-((6-クロロヘキシル)オキシ)エトキシ)エチル)アセトアミド(5)の合成
TFA(1 ml)を、化合物4(144 mg)のCH2Cl2(3 ml)溶液に添加した。混合物を室温で4時間撹拌した。溶媒の除去後、CH2Cl2(1 ml)を添加した。この手順を2回繰り返した。溶媒の除去後、残渣を減圧下で乾燥して、化合物5(160 mg, 定量的)を淡黄色オイルとして得た。
ESI-MS, found 297.05 calcd 297.15 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.46 (m, 4H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 3.49-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.59 (s, 2H), 7.52 (s, 1H), 9.14 (br, 3H). (5) Synthesis of 2- (aminooxy) -N- (2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) acetamide (5) TFA (1 ml) was converted to compound 4 (144 mg) To a solution of CH 2 Cl 2 (3 ml). The mixture was stirred at room temperature for 4 hours. After removal of the solvent, CH 2 Cl 2 (1 ml) was added. This procedure was repeated twice. After removal of the solvent, the residue was dried under reduced pressure to give compound 5 (160 mg, quantitative) as a pale yellow oil.
ESI-MS, found 297.05 calcd 297.15 ([M + H] +). 1 H-NMR (300 MHz, CDCl 3) 1.37-1.46 (m, 4H), 1.61-1.64 (m, 2H), 1.75-1.80 ( m, 2H), 3.49-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.59 (s, 2H), 7.52 (s, 1H), 9.14 (br, 3H).
TFA(1 ml)を、化合物4(144 mg)のCH2Cl2(3 ml)溶液に添加した。混合物を室温で4時間撹拌した。溶媒の除去後、CH2Cl2(1 ml)を添加した。この手順を2回繰り返した。溶媒の除去後、残渣を減圧下で乾燥して、化合物5(160 mg, 定量的)を淡黄色オイルとして得た。
ESI-MS, found 297.05 calcd 297.15 ([M+H]+). 1H-NMR (300 MHz, CDCl3) 1.37-1.46 (m, 4H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 3.49-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.59 (s, 2H), 7.52 (s, 1H), 9.14 (br, 3H). (5) Synthesis of 2- (aminooxy) -N- (2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) acetamide (5) TFA (1 ml) was converted to compound 4 (144 mg) To a solution of CH 2 Cl 2 (3 ml). The mixture was stirred at room temperature for 4 hours. After removal of the solvent, CH 2 Cl 2 (1 ml) was added. This procedure was repeated twice. After removal of the solvent, the residue was dried under reduced pressure to give compound 5 (160 mg, quantitative) as a pale yellow oil.
ESI-MS, found 297.05 calcd 297.15 ([M + H] +). 1 H-NMR (300 MHz, CDCl 3) 1.37-1.46 (m, 4H), 1.61-1.64 (m, 2H), 1.75-1.80 ( m, 2H), 3.49-3.56 (m, 6H), 3.59-3.65 (m, 6H), 4.59 (s, 2H), 7.52 (s, 1H), 9.14 (br, 3H).
(6) 4-((2-(2-((6-クロロヘキシル)オキシ)エトキシ)エチル)アミノ)-4-オキソブタン酸(6)の合成
コハク酸無水物(4.16 g, 41.6 mmol)を、化合物3(2.49 g, 8.32 mmol)の脱水CH2Cl2(60 ml)溶液に添加した。トリエチルアミン(9.7 ml, 69.9 mmol)の添加後、溶液を30℃で21時間撹拌した。溶媒の除去後、残渣をカラムクロマトグラフィー(SiO2, CHCl3/MeOH = 20/1)で2回精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物6(2.27 g, 7.03 mmol, 84%)を淡褐色オイルとして得た。
ESI-MS, found 324.05 calcd 324.16 ([M+H]+). 1H-NMR (600 MHz, CDCl3) 1.21-1.25 (m, 2H), 1.38 (t, J = 7.2 Hz, 2 H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 3.45-3.55 (m, 8H), 2.68 (t, J = 4.8 Hz, 4H), 3.70-3.73 (m, 1H), 6.40 (br, 1H). (6) Synthesis of 4-((2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) amino) -4-oxobutanoic acid (6) Succinic anhydride (4.16 g, 41.6 mmol) Compound 3 (2.49 g, 8.32 mmol) was added to a solution of dehydrated CH 2 Cl 2 (60 ml). After the addition of triethylamine (9.7 ml, 69.9 mmol), the solution was stirred at 30 ° C. for 21 hours. After removal of the solvent, the residue was purified twice by column chromatography (SiO 2 , CHCl 3 / MeOH = 20/1). The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 6 (2.27 g, 7.03 mmol, 84%) as a light brown oil.
ESI-MS, found 324.05 calcd 324.16 ([M + H] + ). 1 H-NMR (600 MHz, CDCl 3 ) 1.21-1.25 (m, 2H), 1.38 (t, J = 7.2 Hz, 2 H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 3.45-3.55 (m, 8H) , 2.68 (t, J = 4.8 Hz, 4H), 3.70-3.73 (m, 1H), 6.40 (br, 1H).
コハク酸無水物(4.16 g, 41.6 mmol)を、化合物3(2.49 g, 8.32 mmol)の脱水CH2Cl2(60 ml)溶液に添加した。トリエチルアミン(9.7 ml, 69.9 mmol)の添加後、溶液を30℃で21時間撹拌した。溶媒の除去後、残渣をカラムクロマトグラフィー(SiO2, CHCl3/MeOH = 20/1)で2回精製した。得られた溶液を溶媒留去し、残渣を減圧下で乾燥して、化合物6(2.27 g, 7.03 mmol, 84%)を淡褐色オイルとして得た。
ESI-MS, found 324.05 calcd 324.16 ([M+H]+). 1H-NMR (600 MHz, CDCl3) 1.21-1.25 (m, 2H), 1.38 (t, J = 7.2 Hz, 2 H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 3.45-3.55 (m, 8H), 2.68 (t, J = 4.8 Hz, 4H), 3.70-3.73 (m, 1H), 6.40 (br, 1H). (6) Synthesis of 4-((2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) amino) -4-oxobutanoic acid (6) Succinic anhydride (4.16 g, 41.6 mmol) Compound 3 (2.49 g, 8.32 mmol) was added to a solution of dehydrated CH 2 Cl 2 (60 ml). After the addition of triethylamine (9.7 ml, 69.9 mmol), the solution was stirred at 30 ° C. for 21 hours. After removal of the solvent, the residue was purified twice by column chromatography (SiO 2 , CHCl 3 / MeOH = 20/1). The obtained solution was evaporated, and the residue was dried under reduced pressure to obtain Compound 6 (2.27 g, 7.03 mmol, 84%) as a light brown oil.
ESI-MS, found 324.05 calcd 324.16 ([M + H] + ). 1 H-NMR (600 MHz, CDCl 3 ) 1.21-1.25 (m, 2H), 1.38 (t, J = 7.2 Hz, 2 H), 1.61-1.64 (m, 2H), 1.75-1.80 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 3.45-3.55 (m, 8H) , 2.68 (t, J = 4.8 Hz, 4H), 3.70-3.73 (m, 1H), 6.40 (br, 1H).
参考例７
Adh-Clの合成
アドヘサミン(8.85 mg, 14.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中の化合物5(4.10 mg, 10.0 μmol)の溶液に添加した。この溶液を56℃で21時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間27分)で精製した。溶出液を凍結乾燥して、Adh-Cl (3.05 mg, 3.47 μmol, 35%)を白色固体として得た。
ESI-HRMS: found 438.142704 calcd 438.142646 ([M]2+), 純度 >95% (LCMS分析で計算).
Reference Example 7
Synthesis of Adh-Cl Adhesamine (8.85 mg, 14.8 μmol) was added to a solution of compound 5 (4.10 mg, 10.0 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added. The solution was stirred at 56 ° C. for 21 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 27 minutes). The eluate was lyophilized to give Adh-Cl (3.05 mg, 3.47 μmol, 35%) as a white solid.
ESI-HRMS: found 438.142704 calcd 438.142646 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
Adh-Clの合成
アドヘサミン(8.85 mg, 14.8 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中の化合物5(4.10 mg, 10.0 μmol)の溶液に添加した。この溶液を56℃で21時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/45→ 25/95, 保持時間27分)で精製した。溶出液を凍結乾燥して、Adh-Cl (3.05 mg, 3.47 μmol, 35%)を白色固体として得た。
ESI-HRMS: found 438.142704 calcd 438.142646 ([M]2+), 純度 >95% (LCMS分析で計算).
Synthesis of Adh-Cl Adhesamine (8.85 mg, 14.8 μmol) was added to a solution of compound 5 (4.10 mg, 10.0 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. Added. The solution was stirred at 56 ° C. for 21 hours. After removing the solvent under a nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 27 minutes). The eluate was lyophilized to give Adh-Cl (3.05 mg, 3.47 μmol, 35%) as a white solid.
ESI-HRMS: found 438.142704 calcd 438.142646 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
参考例８
Adh(Cl)-SFFの合成
Adh-SFF (5.13 mg, 4.87 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(1.8 ml)中の化合物5(2.40 mg, 4.98 μmol)の溶液に添加した。この溶液を55℃で21時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 31/45→ 25/95, 保持時間38分)で精製した。溶出液を凍結乾燥して、Adh(Cl)-SFF (1.39 mg, 1.05 μmol, 22%)を白色固体として得た。
ESI-HRMS: found 664.742973 calcd 664.743256 ([M]2+), 純度 >95% (LCMS分析で計算)
Reference Example 8
Synthesis of Adh (Cl) -SFF Adh-SFF (5.13 mg, 4.87 μmol) was added to compound 5 (2.40 mg, 4.98 in DMF / dioxane / water / TFA = 2/1/2 / 0.0004 mixture (1.8 ml). μmol) solution. The solution was stirred at 55 ° C. for 21 hours. After removing the solvent under nitrogen flow, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 31/45 → 25/95, retention time 38 minutes). The eluate was lyophilized to give Adh (Cl) -SFF (1.39 mg, 1.05 μmol, 22%) as a white solid.
ESI-HRMS: found 664.742973 calcd 664.743256 ([M] 2+ ), purity> 95% (calculated by LCMS analysis)
Adh(Cl)-SFFの合成
Adh-SFF (5.13 mg, 4.87 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(1.8 ml)中の化合物5(2.40 mg, 4.98 μmol)の溶液に添加した。この溶液を55℃で21時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 31/45→ 25/95, 保持時間38分)で精製した。溶出液を凍結乾燥して、Adh(Cl)-SFF (1.39 mg, 1.05 μmol, 22%)を白色固体として得た。
ESI-HRMS: found 664.742973 calcd 664.743256 ([M]2+), 純度 >95% (LCMS分析で計算)
Synthesis of Adh (Cl) -SFF Adh-SFF (5.13 mg, 4.87 μmol) was added to compound 5 (2.40 mg, 4.98 in DMF / dioxane / water / TFA = 2/1/2 / 0.0004 mixture (1.8 ml). μmol) solution. The solution was stirred at 55 ° C. for 21 hours. After removing the solvent under nitrogen flow, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 31/45 → 25/95, retention time 38 minutes). The eluate was lyophilized to give Adh (Cl) -SFF (1.39 mg, 1.05 μmol, 22%) as a white solid.
ESI-HRMS: found 664.742973 calcd 664.743256 ([M] 2+ ), purity> 95% (calculated by LCMS analysis)
実施例７
(1) Aoa-SFFK(アルキルクロリド-K-) ペプチドの合成
ペプチドを、RINK Amide MBHA樹脂LLを用い、通常のFmoc固相ペプチド合成法(SPPS)により同様に合成した。[(tert-ブトキシカルボニル)アミノオキシ]酢酸の縮合後、樹脂をNH2OH・HCl (1.25 g)及びイミダゾール(918 mg)のNMP (5 ml)及びCH2Cl2 (1 ml)溶液で、室温で3時間処理して、1-(4,4-ジメチル-2,6-ジオキソシクロヘキシリデン)エチル (dde)を脱保護した。Fmoc-Lys(Boc)-OH及び化合物6の縮合後、ペプチド結合樹脂をメタノール及びCH2Cl2で2回洗浄した。減圧下で乾燥後、樹脂をトリフルオロ酢酸(TFA)/トリイソプロピルシラン(TIS)/水 = 95/2.5/2.5の混合物中、室温で3時間処理した。ろ過後、冷却したジエチルエーテルをろ液に添加し、生成物を沈殿させた。遠心分離(3500 rpm×5分間)後、上清を廃棄した。この手順を更に2回繰り返した。残渣を減圧下、室温で3時間乾燥して、Aoa-SFFK(アルキルクロリド-K-) ペプチド (91.5 mg)の粗生成物を白色固体として得た。
ESI-MS, found 1033.40 calcd 1033.55 ([M+H]+). Example 7
(1) Synthesis of Aoa-SFFK (alkyl chloride-K-) peptide
Peptides were synthesized in the same manner by ordinary Fmoc solid phase peptide synthesis (SPPS) using RINK Amide MBHA resin LL. After condensation of [(tert-butoxycarbonyl) aminooxy] acetic acid, the resin was washed with NH 2 OH.HCl (1.25 g) and imidazole (918 mg) in NMP (5 ml) and CH 2 Cl 2 (1 ml), Treatment at room temperature for 3 hours deprotected 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl (dde). After condensation of Fmoc-Lys (Boc) -OH and compound 6, the peptide bond resin was washed twice with methanol and CH 2 Cl 2 . After drying under reduced pressure, the resin was treated in a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) /water=95/2.5/2.5 at room temperature for 3 hours. After filtration, cooled diethyl ether was added to the filtrate to precipitate the product. After centrifugation (3500 rpm × 5 minutes), the supernatant was discarded. This procedure was repeated two more times. The residue was dried at room temperature under reduced pressure for 3 hours to obtain a crude product of Aoa-SFFK (alkyl chloride-K-) peptide (91.5 mg) as a white solid.
ESI-MS, found 1033.40 calcd 1033.55 ([M + H] + ).
(1) Aoa-SFFK(アルキルクロリド-K-) ペプチドの合成
ESI-MS, found 1033.40 calcd 1033.55 ([M+H]+). Example 7
(1) Synthesis of Aoa-SFFK (alkyl chloride-K-) peptide
ESI-MS, found 1033.40 calcd 1033.55 ([M + H] + ).
(2) Adh-SFF-Clの合成
アドヘサミン(7.94 mg, 13.2 μmol)を、DMF/ジオキサン/水/TFA = 2/1/2/0.0004の混合物(2 ml)中のAoa-SFFK(アルキルクロリド-K-) ペプチド(9.68 mg, 9.37 μmol)の溶液に添加した。この溶液を51℃で22時間撹拌した。窒素気流下で溶媒を除去した後、残渣をHPLC (ODS-A, 30x150 mm, グラジエント 時間(分)/B(%) = 0/15→ 1/15→ 21/55→ 25/95, 保持時間22分)で精製した。溶出液を凍結乾燥して、Adh-SFF-Cl (5.79 mg, 3.59 μmol, 38%)を白色固体として得た。
ESI-HRMS: found 806.338810 calcd 806.338052 ([M]2+), 純度 >95% (LCMS分析で計算).
1H-NMR (600 MHz, DMSO-d6) δ 1.22-1.32 (6H, m), 1.35-1.41 (4H, m), 1.46-1.52 (6H, m), 1.65-1.72 (4H, m), 2.29-2.37 (4H, m), 2.52 (3H, s), 2.55 (3H, s), 2.70-2.83 (4H, m), 2.94-3.08 (4H, m), 3.18 (2H, q, J = 6.0 Hz), 3.35-3.40 (4H, m), 3.45-3.50 (4H, m), 3.52-3.58 (2H, m), 3.62 (2H, t, J = 6.6 Hz), 3.82-4.14 (26H, m), 4.34 (1H, q, J = 6.0 Hz), 4.42-4.46 (1H, m), 4.50-4.54 (1H, m), 4.64 (2H, s), 7.05 (1H, br), 7.13-7.27 (12H, m), 7.71 (2H, br), 7.89-7.91 (2H, m), 7.96 (1H, t, J = 5.4 Hz), 8.00 (2H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.4 Hz), 8.22 (1H, d, J = 7.8 Hz), 8.44 (1H, s), 10.1 (1H, s).
13C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 22.3, 22.6, 24.8, 26.0, 26.5, 28.7, 28.9, 30.4, 30.5, 31.1, 31.6, 31.9, 36.9, 37.3, 38.4, 38.5, 38.6, 39.9, 40.8, 41.5, 45.3, 51.1, 51.2, 52.3, 52.5, 54.0, 54.7, 61.5, 69.0, 69.3, 69.5, 70.1, 72.3, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 129.0, 129.1, 137.5, 137.6, 145.8, 159.6, 160.6, 164.3, 168.5, 169.7, 170.2, 170.6, 170.7, 171.3, 171.5, 171.6, 173.2, 173.3, 186.5. (2) Synthesis of Adh-SFF-Cl
Adhesamine (7.94 mg, 13.2 μmol) was added to Aoa-SFFK (alkyl chloride-K-) peptide (9.68 mg, 9.37 μmol) in a mixture (2 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. ). The solution was stirred at 51 ° C. for 22 hours. After removing the solvent under nitrogen stream, the residue was HPLC (ODS-A, 30x150 mm, gradient time (min) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time 22 minutes). The eluate was lyophilized to give Adh-SFF-Cl (5.79 mg, 3.59 μmol, 38%) as a white solid.
ESI-HRMS: found 806.338810 calcd 806.338052 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
1 H-NMR (600 MHz, DMSO-d6) δ 1.22-1.32 (6H, m), 1.35-1.41 (4H, m), 1.46-1.52 (6H, m), 1.65-1.72 (4H, m), 2.29 -2.37 (4H, m), 2.52 (3H, s), 2.55 (3H, s), 2.70-2.83 (4H, m), 2.94-3.08 (4H, m), 3.18 (2H, q, J = 6.0 Hz ), 3.35-3.40 (4H, m), 3.45-3.50 (4H, m), 3.52-3.58 (2H, m), 3.62 (2H, t, J = 6.6 Hz), 3.82-4.14 (26H, m), 4.34 (1H, q, J = 6.0 Hz), 4.42-4.46 (1H, m), 4.50-4.54 (1H, m), 4.64 (2H, s), 7.05 (1H, br), 7.13-7.27 (12H, m), 7.71 (2H, br), 7.89-7.91 (2H, m), 7.96 (1H, t, J = 5.4 Hz), 8.00 (2H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.4 Hz), 8.22 (1H, d, J = 7.8 Hz), 8.44 (1H, s), 10.1 (1H, s).
13 C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 22.3, 22.6, 24.8, 26.0, 26.5, 28.7, 28.9, 30.4, 30.5, 31.1, 31.6, 31.9, 36.9, 37.3, 38.4, 38.5, 38.6 , 39.9, 40.8, 41.5, 45.3, 51.1, 51.2, 52.3, 52.5, 54.0, 54.7, 61.5, 69.0, 69.3, 69.5, 70.1, 72.3, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 129.0, 129.1, 137.5 , 137.6, 145.8, 159.6, 160.6, 164.3, 168.5, 169.7, 170.2, 170.6, 170.7, 171.3, 171.5, 171.6, 173.2, 173.3, 186.5.
ESI-HRMS: found 806.338810 calcd 806.338052 ([M]2+), 純度 >95% (LCMS分析で計算).
1H-NMR (600 MHz, DMSO-d6) δ 1.22-1.32 (6H, m), 1.35-1.41 (4H, m), 1.46-1.52 (6H, m), 1.65-1.72 (4H, m), 2.29-2.37 (4H, m), 2.52 (3H, s), 2.55 (3H, s), 2.70-2.83 (4H, m), 2.94-3.08 (4H, m), 3.18 (2H, q, J = 6.0 Hz), 3.35-3.40 (4H, m), 3.45-3.50 (4H, m), 3.52-3.58 (2H, m), 3.62 (2H, t, J = 6.6 Hz), 3.82-4.14 (26H, m), 4.34 (1H, q, J = 6.0 Hz), 4.42-4.46 (1H, m), 4.50-4.54 (1H, m), 4.64 (2H, s), 7.05 (1H, br), 7.13-7.27 (12H, m), 7.71 (2H, br), 7.89-7.91 (2H, m), 7.96 (1H, t, J = 5.4 Hz), 8.00 (2H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.4 Hz), 8.22 (1H, d, J = 7.8 Hz), 8.44 (1H, s), 10.1 (1H, s).
13C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 22.3, 22.6, 24.8, 26.0, 26.5, 28.7, 28.9, 30.4, 30.5, 31.1, 31.6, 31.9, 36.9, 37.3, 38.4, 38.5, 38.6, 39.9, 40.8, 41.5, 45.3, 51.1, 51.2, 52.3, 52.5, 54.0, 54.7, 61.5, 69.0, 69.3, 69.5, 70.1, 72.3, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 129.0, 129.1, 137.5, 137.6, 145.8, 159.6, 160.6, 164.3, 168.5, 169.7, 170.2, 170.6, 170.7, 171.3, 171.5, 171.6, 173.2, 173.3, 186.5. (2) Synthesis of Adh-SFF-Cl
ESI-HRMS: found 806.338810 calcd 806.338052 ([M] 2+ ), purity> 95% (calculated by LCMS analysis).
1 H-NMR (600 MHz, DMSO-d6) δ 1.22-1.32 (6H, m), 1.35-1.41 (4H, m), 1.46-1.52 (6H, m), 1.65-1.72 (4H, m), 2.29 -2.37 (4H, m), 2.52 (3H, s), 2.55 (3H, s), 2.70-2.83 (4H, m), 2.94-3.08 (4H, m), 3.18 (2H, q, J = 6.0 Hz ), 3.35-3.40 (4H, m), 3.45-3.50 (4H, m), 3.52-3.58 (2H, m), 3.62 (2H, t, J = 6.6 Hz), 3.82-4.14 (26H, m), 4.34 (1H, q, J = 6.0 Hz), 4.42-4.46 (1H, m), 4.50-4.54 (1H, m), 4.64 (2H, s), 7.05 (1H, br), 7.13-7.27 (12H, m), 7.71 (2H, br), 7.89-7.91 (2H, m), 7.96 (1H, t, J = 5.4 Hz), 8.00 (2H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.4 Hz), 8.22 (1H, d, J = 7.8 Hz), 8.44 (1H, s), 10.1 (1H, s).
13 C-NMR (150 MHz, DMSO-d6) δ 13.6, 13.8, 22.3, 22.6, 24.8, 26.0, 26.5, 28.7, 28.9, 30.4, 30.5, 31.1, 31.6, 31.9, 36.9, 37.3, 38.4, 38.5, 38.6 , 39.9, 40.8, 41.5, 45.3, 51.1, 51.2, 52.3, 52.5, 54.0, 54.7, 61.5, 69.0, 69.3, 69.5, 70.1, 72.3, 103.6, 107.3, 126.1, 126.2, 127.9, 128.0, 129.0, 129.1, 137.5 , 137.6, 145.8, 159.6, 160.6, 164.3, 168.5, 169.7, 170.2, 170.6, 170.7, 171.3, 171.5, 171.6, 173.2, 173.3, 186.5.
実施例１～６と同様の方法で、実施例８～１３の化合物を製造した。
実施例８：Adh-AFF
実施例９：Adh-GFF
実施例１０：Adh-SYY
実施例１１：Adh-SWW
実施例１２：Adh-SFFGK
実施例１３：Adh-SFFGGK
In the same manner as in Examples 1 to 6, the compounds of Examples 8 to 13 were produced.
Example 8: Adh-AFF
Example 9: Adh-GFF
Example 10: Adh-SYY
Example 11: Adh-SWW
Example 12: Adh-SFFGK
Example 13: Adh-SFFGGK
実施例８：Adh-AFF
実施例９：Adh-GFF
実施例１０：Adh-SYY
実施例１１：Adh-SWW
実施例１２：Adh-SFFGK
実施例１３：Adh-SFFGGK
Example 8: Adh-AFF
Example 9: Adh-GFF
Example 10: Adh-SYY
Example 11: Adh-SWW
Example 12: Adh-SFFGK
Example 13: Adh-SFFGGK
試験例１
ヘパリンに対するアフィニティー及び細胞生存率
等温滴定型カロリメトリー(ITC)により、実施例及び参考例で製造したアドヘサミン誘導体のヘパリンに対するアフィニティーを計算した（表３）。Adh-SFFはアドヘサミンより高いアフィニティーを示したが(図１ｃ及びｄ、表３)、ペプチドSFF自体はヘパリンに対するアフィニティーを示さなかった(図２ａ)。Adh-SFFは、アドヘサミンのヘパリンに対する高選択性を維持したが、コンドロイチン硫酸Ａに対するアフィニティーは示さなかった(図２ｂ)。これらのデータは、コンジュゲートしたFFペプチドが選択性を損なうことなく、アドヘサミンのヘパリンに対する結合を高めることを示す。 Test example 1
Affinity for heparin and cell viability The affinity for heparin of the adhesamine derivatives produced in the Examples and Reference Examples was calculated by isothermal titration calorimetry (ITC) (Table 3). Adh-SFF showed higher affinity than adhesamine (FIGS. 1c and d, Table 3), but peptide SFF itself showed no affinity for heparin (FIG. 2a). Adh-SFF maintained the high selectivity of adhesamine for heparin but did not show affinity for chondroitin sulfate A (FIG. 2b). These data indicate that the conjugated FF peptide enhances the binding of adhesamine to heparin without compromising selectivity.
ヘパリンに対するアフィニティー及び細胞生存率
等温滴定型カロリメトリー(ITC)により、実施例及び参考例で製造したアドヘサミン誘導体のヘパリンに対するアフィニティーを計算した（表３）。Adh-SFFはアドヘサミンより高いアフィニティーを示したが(図１ｃ及びｄ、表３)、ペプチドSFF自体はヘパリンに対するアフィニティーを示さなかった(図２ａ)。Adh-SFFは、アドヘサミンのヘパリンに対する高選択性を維持したが、コンドロイチン硫酸Ａに対するアフィニティーは示さなかった(図２ｂ)。これらのデータは、コンジュゲートしたFFペプチドが選択性を損なうことなく、アドヘサミンのヘパリンに対する結合を高めることを示す。 Test example 1
Affinity for heparin and cell viability The affinity for heparin of the adhesamine derivatives produced in the Examples and Reference Examples was calculated by isothermal titration calorimetry (ITC) (Table 3). Adh-SFF showed higher affinity than adhesamine (FIGS. 1c and d, Table 3), but peptide SFF itself showed no affinity for heparin (FIG. 2a). Adh-SFF maintained the high selectivity of adhesamine for heparin but did not show affinity for chondroitin sulfate A (FIG. 2b). These data indicate that the conjugated FF peptide enhances the binding of adhesamine to heparin without compromising selectivity.
次に、WST-8アッセイを使用してアドヘサミン誘導体による処理が細胞生存率(Cell Survival, %)に影響を及ぼすか調べた（図１ｅ及び表３）。図１ｅは、各アドヘサミン誘導体(100 μM)を添加した1%(v/v)DMSO含有無血清DMEM中で3日間インキュベートしたNIH3T3細胞の細胞生存率を示すグラフである。細胞生存率はWST-8アッセイで計算し、Adh-SFFで処理した細胞に対して正規化した。エラーバーは標準偏差(n=3)である。
Next, it was examined whether treatment with an adhesamine derivative had an effect on cell viability (Cell Survival,%) using the WST-8 assay (FIG. 1e and Table 3). FIG. 1e is a graph showing the viability of NIH3T3 cells incubated for 3 days in serum-free DMEM containing 1% (v / v) DMSO supplemented with each adhesamine derivative (100 μM). Cell viability was calculated with WST-8 assay and normalized to cells treated with Adh-SFF. Error bars are standard deviation (n = 3).
Adh-SFFで処理した細胞は3日間培養後、アドヘサミンと比較して予想外に高い細胞生存率を維持した。ペプチドSFF単独の処理、又はアドヘサミンとペプチドSFFの混合物の処理は、無視できるほどわずかな細胞生存率の増加しか示さず(図３ａ)、アドヘサミンとSFFペプチドの間のコンジュゲートが活性を増加させるのに必須であることを示唆する。Adh-SFFの細胞生存率を高めるEC50は38 μMであった（図３ｂ）。図３ａは、アドヘサミン(100 μM), ペプチドSFF(100 μM), アドヘサミンとペプチドSFFの混合物(各100 μM)又はAdh-SFF(100 μM)を添加した1%(v/v)DMSO含有無血清DMEM中で3日間インキュベートしたNIH3T3細胞の細胞生存率を示すグラフである。図３ｂは、Adh-SFF(0-300 μM)の濃度効果を示すグラフである。細胞生存率はWST-8アッセイで計算し、Adh-SFF(100 μM)で処理した細胞に対して正規化した。
The cells treated with Adh-SFF maintained an unexpectedly high cell viability after culturing for 3 days compared to adhesamine. Treatment with peptide SFF alone or a mixture of adhesamine and peptide SFF showed negligible increases in cell viability (FIG. 3a), and the conjugate between adhesamine and SFF peptide increased activity. It is essential to. The EC50 that increases the cell viability of Adh-SFF was 38 μM (FIG. 3b). FIG. 3a shows serum-free serum containing 1% (v / v) DMSO supplemented with adhesamine (100 μM), peptide SFF (100 μM), a mixture of adhesamine and peptide SFF (100 μM each) or Adh-SFF (100 μM) It is a graph which shows the cell viability of the NIH3T3 cell incubated for 3 days in DMEM. FIG. 3b is a graph showing the concentration effect of Adh-SFF (0-300 μM). Cell viability was calculated with the WST-8 assay and normalized to cells treated with Adh-SFF (100 μM).
次に、ペプチド部分の構造的効果を検討するために、種々のペプチド配列を有するアドヘサミン誘導体の細胞生存率の増加を評価した(図１ｅ及び表３)。結果は、SFF配列が細胞生存率の増加に最も適していたが、Adh-FF、Adh-FSF、及びAdh-FFSも同等であった。ヘパリンに対するアフィニティーも確認した(表３)。図４に示すプロットデータは、ヘパリンに対するアフィニティー（見かけ上の結合定数Ka）と細胞生存率の増加は良好な相関関係(r=0.838)にあることを示し、より高いアフィニティー(Ka > 3 x 107 M-1)が細胞生存率を効果的に高めるといえる。アドヘサミンとインテグリンリガンドペプチドRGDSのコンジュゲートであるAdh-RGDSも細胞生存率を高めるが、ヘパリンに対するアフィニティーは低い（表３）。この相違は、Adh-RGDSはインテグリン-RGDS結合に依存し、Adh-SFFはヘパラン硫酸上の自己集合によって作用するという２つのアドヘサミン誘導体のメカニズムの相違に帰すると考えられる。
Next, in order to examine the structural effects of the peptide moiety, the increase in cell viability of adhesamine derivatives having various peptide sequences was evaluated (FIG. 1e and Table 3). The results showed that SFF sequences were most suitable for increasing cell viability, but Adh-FF, Adh-FSF, and Adh-FFS were equivalent. Affinities for heparin were also confirmed (Table 3). The plot data shown in FIG. 4 shows that the affinity for heparin (apparent binding constant Ka) and the increase in cell viability are in a good correlation (r = 0.838) with higher affinity (Ka> 3 × 10 7 M -1 ) can effectively increase cell viability. Adh-RGDS, a conjugate of adhesamine and the integrin ligand peptide RGDS, also increases cell viability, but has low affinity for heparin (Table 3). This difference may be attributed to the difference in the mechanism of the two adhesamine derivatives, in which Adh-RGDS is dependent on integrin-RGDS binding and Adh-SFF acts by self-assembly on heparan sulfate.
試験例２
硫酸化GAG上のアドヘサミンの自己集合構造(self-assembly structure)
本発明者らは、以前に、アドヘサミンがヘパラン硫酸に配位して、ピリミジン部分のπ－πスタッキングによって自己集合構造を形成することを示した。FFペプチドがアドヘサミンのピリミジンのすぐ近くに位置し、芳香族基間のπ－πスタッキングによって強固な自己集合構造を形成すると予測される。結合構造を検討するために、ヘパリン含有溶液中のAdh-SFFのNOESYスペクトルを測定した。結果は、Adh-SFFのピリミジン部分が近接するピリミジンだけでなく、フェニル基とも近接していることが明らかとなった。また、Adh-SFFのCDスペクトルは、ヘパリンと複合体を形成したとき220 nmの吸光度が減少し、200 nm付近で増加することを示し、これはβターン様構造が形成されることを示唆する。これらの結果は、ヘパラン硫酸に結合したとき、自己集合体の相互作用が集合的にβターン様構造を誘導することを示唆する。 Test example 2
Self-assembly structure of adhesamine on sulfated GAG
The inventors have previously shown that adhesamine coordinates to heparan sulfate and forms a self-assembled structure by π-π stacking of the pyrimidine moiety. The FF peptide is located in the immediate vicinity of the adhesamine pyrimidine and is predicted to form a strong self-assembled structure by π-π stacking between aromatic groups. In order to examine the binding structure, NOESY spectra of Adh-SFF in a heparin-containing solution were measured. The results revealed that the pyrimidine part of Adh-SFF is close to the phenyl group as well as the adjacent pyrimidine. The CD spectrum of Adh-SFF also shows that the absorbance at 220 nm decreases and increases around 200 nm when complexed with heparin, suggesting that a β-turn-like structure is formed. . These results suggest that self-assembly interactions collectively induce β-turn-like structures when bound to heparan sulfate.
硫酸化GAG上のアドヘサミンの自己集合構造(self-assembly structure)
本発明者らは、以前に、アドヘサミンがヘパラン硫酸に配位して、ピリミジン部分のπ－πスタッキングによって自己集合構造を形成することを示した。FFペプチドがアドヘサミンのピリミジンのすぐ近くに位置し、芳香族基間のπ－πスタッキングによって強固な自己集合構造を形成すると予測される。結合構造を検討するために、ヘパリン含有溶液中のAdh-SFFのNOESYスペクトルを測定した。結果は、Adh-SFFのピリミジン部分が近接するピリミジンだけでなく、フェニル基とも近接していることが明らかとなった。また、Adh-SFFのCDスペクトルは、ヘパリンと複合体を形成したとき220 nmの吸光度が減少し、200 nm付近で増加することを示し、これはβターン様構造が形成されることを示唆する。これらの結果は、ヘパラン硫酸に結合したとき、自己集合体の相互作用が集合的にβターン様構造を誘導することを示唆する。 Test example 2
Self-assembly structure of adhesamine on sulfated GAG
The inventors have previously shown that adhesamine coordinates to heparan sulfate and forms a self-assembled structure by π-π stacking of the pyrimidine moiety. The FF peptide is located in the immediate vicinity of the adhesamine pyrimidine and is predicted to form a strong self-assembled structure by π-π stacking between aromatic groups. In order to examine the binding structure, NOESY spectra of Adh-SFF in a heparin-containing solution were measured. The results revealed that the pyrimidine part of Adh-SFF is close to the phenyl group as well as the adjacent pyrimidine. The CD spectrum of Adh-SFF also shows that the absorbance at 220 nm decreases and increases around 200 nm when complexed with heparin, suggesting that a β-turn-like structure is formed. . These results suggest that self-assembly interactions collectively induce β-turn-like structures when bound to heparan sulfate.
さらに、動的光散乱法(DLS)及び電子顕微鏡(EM)により自己集合構造を研究した。DLS測定は、ヘパリンに結合したAdh-SFFがサブミクロン粒子(直径600-1200 nm)を形成することを明らかにした(図６ａ)。Adh-SFFと4 μMコンドロイチン硫酸Aの組合せ、アドヘサミン(100 μM)とヘパリン(4 μM)の混合物のいずれも、粒子を形成しなかった(データは示していない)。次いで、クライオ電子顕微鏡法(Cryo-TEM)を使用して、Adh-SFF(100 μM)とヘパリン(1 μM)が水溶液中で凝集することを観察し(図６ｂ)、Adh-SFF(300 μM)又はヘパリン(3 μM)単独のサンプルではサブミクロン粒子は存在しなかった(図５)。また、電界放出型走査型電子顕微鏡(FE-SEM)を使用して、細胞表面にAdh-SFFの複合体を観察し(図６ｃ)、細胞上の多くの粒子が明らかとなった。これらの結果は、Adh-SFFが細胞表面のへパラン硫酸と結合して細胞表面を凝集粒子でコーティングしていることを示唆する(図６ｉ)。
Furthermore, self-assembled structures were studied by dynamic light scattering (DLS) and electron microscope (EM). DLS measurements revealed that Adh-SFF bound to heparin formed submicron particles (diameter 600-1200 nm) (FIG. 6a). Neither the combination of Adh-SFF and 4 μM chondroitin sulfate A or a mixture of adhesamine (100 μM) and heparin (4 μM) formed particles (data not shown). Next, using Cryo-TEM, it was observed that Adh-SFF (100 μM) and heparin (1 μM) aggregate in an aqueous solution (FIG. 6b), and Adh-SFF (300 μM). ) Or heparin (3 μM) alone had no submicron particles (FIG. 5). In addition, using a field emission scanning electron microscope (FE-SEM), a complex of Adh-SFF was observed on the cell surface (FIG. 6c), and many particles on the cell were revealed. These results suggest that Adh-SFF binds to heparan sulfate on the cell surface and coats the cell surface with aggregated particles (FIG. 6i).
試験例３
Adh-SFFにより誘導される細胞生存のための細胞内シグナル伝達
本発明者らの以前の研究により、アドヘサミンは、シンデカン4(SDC4)（細胞接着に関与することが知られているヘパラン硫酸で修飾されたシンデカン）のクラスター化を誘導することにより生物学的活性を示すことが明らかとなった。抗SDC4抗体による免疫染色は、Adh-SFF処理が、アドヘサミンと同様にSDC4クラスター化を誘導することを示す(図６ｄ)。Adh-SFFで処理したサンプルは、アドヘサミンで処理したサンプルで観察されるSDC4クラスターと比較してより多くのより大きな(> 1.15 μm)SDC4クラスターを示し（図６ｅ）、Adh-SFFがアドヘサミンと比較してより強力なSDC4クラスター化を誘導することを示唆する。 Test example 3
Intracellular signaling for cell survival induced by Adh-SFF According to our previous studies, adhesamine was modified with syndecan 4 (SDC4), a heparan sulfate known to be involved in cell adhesion It was revealed that biological activity was exhibited by inducing the clustering of syndecan. Immunostaining with anti-SDC4 antibody shows that Adh-SFF treatment induces SDC4 clustering similar to adhesamine (FIG. 6d). Samples treated with Adh-SFF show more larger (> 1.15 μm) SDC4 clusters compared to those observed with samples treated with adhesamine (Figure 6e), with Adh-SFF compared to adhesamine Suggesting that it induces stronger SDC4 clustering.
Adh-SFFにより誘導される細胞生存のための細胞内シグナル伝達
本発明者らの以前の研究により、アドヘサミンは、シンデカン4(SDC4)（細胞接着に関与することが知られているヘパラン硫酸で修飾されたシンデカン）のクラスター化を誘導することにより生物学的活性を示すことが明らかとなった。抗SDC4抗体による免疫染色は、Adh-SFF処理が、アドヘサミンと同様にSDC4クラスター化を誘導することを示す(図６ｄ)。Adh-SFFで処理したサンプルは、アドヘサミンで処理したサンプルで観察されるSDC4クラスターと比較してより多くのより大きな(> 1.15 μm)SDC4クラスターを示し（図６ｅ）、Adh-SFFがアドヘサミンと比較してより強力なSDC4クラスター化を誘導することを示唆する。 Test example 3
Intracellular signaling for cell survival induced by Adh-SFF According to our previous studies, adhesamine was modified with syndecan 4 (SDC4), a heparan sulfate known to be involved in cell adhesion It was revealed that biological activity was exhibited by inducing the clustering of syndecan. Immunostaining with anti-SDC4 antibody shows that Adh-SFF treatment induces SDC4 clustering similar to adhesamine (FIG. 6d). Samples treated with Adh-SFF show more larger (> 1.15 μm) SDC4 clusters compared to those observed with samples treated with adhesamine (Figure 6e), with Adh-SFF compared to adhesamine Suggesting that it induces stronger SDC4 clustering.
SDC4のオリゴマー化及びクラスター化は、ホスファチジルセリンやジアシルグリセロールのようなPKCα結合分子の転移反応(re-arrangement)によりPKCαを活性化することが知られている。抗PKCα抗体による免疫染色は、Adh-SFF処理が速やかに細胞質基質(cytosol)から膜へのPKCαの移動を誘導することを示した(図６ｆ)。時間的に、SDC4クラスター化はPKCαの移動より先に起こり、SDC4クラスター化がPKCαの移動を誘導することを示唆する(図７)。無血清DMEMにAdh-SFF(100 μM)を添加後、NIH3T3細胞をAF488コンジュゲート抗SDC4抗体及び抗PKCα抗体/AF568-IgGで染色した。下の列はSDC4(λex = 488 nm, λem = 500-550 nm)、PKCα(λex = 568 nm, λem = BP617/73)、及びDAPI染色(λex = 405 nm, λem = 417-477 nm)を重ねた蛍光染色画像を示す。
It is known that oligomerization and clustering of SDC4 activates PKCα by re-arrangement of PKCα-binding molecules such as phosphatidylserine and diacylglycerol. Immunostaining with anti-PKCα antibody showed that Adh-SFF treatment rapidly induced PKCα transfer from the cytosol to the membrane (FIG. 6f). In time, SDC4 clustering occurs prior to PKCα migration, suggesting that SDC4 clustering induces PKCα migration (FIG. 7). After adding Adh-SFF (100 μM) to serum-free DMEM, NIH3T3 cells were stained with AF488-conjugated anti-SDC4 antibody and anti-PKCα antibody / AF568-IgG. Bottom row is SDC4 (λ ex = 488 nm, λ em = 500-550 nm), PKCα (λ ex = 568 nm, λ em = BP617 / 73), and DAPI staining (λ ex = 405 nm, λ em = 417-477 nm) is a fluorescent staining image superimposed.
MAPK活性はPKCα活性により制御される。Adh-SFFが誘導する細胞生存のための特異的シグナル伝達経路を同定するために、本発明者らは、ウェスタンブロットによる経時変化実験でAdh-SFF処理後のMAPKリン酸化レベルをモニターした(図６ｇ、ｈ)。Adh-SFF処理は徐々にERKリン酸化を増幅したが、DMSO又はアドヘサミン処理と比較してJNKリン酸化を阻害した（図６ｇ、ｈ）。MAPKリン酸化が、通常の培養条件下(standard plastic culturaing plate上で上で10%(v/v) FBSで培養、SCという）で培養された細胞と同様の方法で制御されることは注目すべきである。一方、p38 MAPKリン酸化レベルに処理間で有意な差は見られなかった。一般に、ERKリン酸化は細胞増殖と細胞生存のシグナル伝達を誘導するが、JNKリン酸化は細胞アポトーシスを助ける。これらの結果は、Adh-SFF処理がERK及びJNKリン酸化レベルを制御して、PKCα経路により細胞生存を活性化することを示唆する。
MAPK activity is controlled by PKCα activity. In order to identify specific signaling pathways for cell survival induced by Adh-SFF, we monitored MAPK phosphorylation levels after Adh-SFF treatment in a time course experiment by Western blot (Fig. 6g, h). Adh-SFF treatment gradually amplified ERK phosphorylation, but inhibited JNK phosphorylation compared with DMSO or adhesamine treatment (FIGS. 6g and h). Note that MAPK phosphorylation is controlled in the same manner as cells cultured under normal culture conditions (10% (v / v) FBS, SC on standard plastic culturaing plate) Should. On the other hand, there was no significant difference in p38pMAPK phosphorylation levels between treatments. In general, ERK phosphorylation induces cell proliferation and cell survival signaling, whereas JNK phosphorylation helps cell apoptosis. These results suggest that Adh-SFF treatment regulates ERK and JNK phosphorylation levels and activates cell survival through the PKCα pathway.
さらに、Adh-SFF処理が、別の細胞生存シグナル伝達経路であるAktリン酸化に有意な差をもたらさないことを確認した。FAKリン酸化はAdh-SFF処理に無反応であり、DMSO又はアドヘサミン処理と同様のシグナル伝達の結果であった。これは、SC条件下の細胞（ディッシュへの細胞接着からFAKリン酸化を増加させる）と対照的である。これらのデータは、Adh-SFF処理がAktシグナル伝達により細胞生存を活性化するのではなく、ディッシュへ細胞を接着させるのでもないことを示す。
Furthermore, it was confirmed that Adh-SFF treatment does not cause a significant difference in Akt phosphorylation, which is another cell survival signal transduction pathway. FAK phosphorylation was unresponsive to Adh-SFF treatment and resulted in signal transduction similar to DMSO or adhesamine treatment. This is in contrast to cells under SC conditions (which increases FAK phosphorylation from cell adhesion to the dish). These data indicate that Adh-SFF treatment does not activate cell survival by Akt signaling, nor does it attach cells to the dish.
次いで、ERKの上流キナーゼであるMEKに対する選択的阻害剤であるUO126を使用してERKシグナル伝達の阻害を試験した。また、選択的PKCα阻害剤であるUCN01、及びAkt阻害剤であるトリシリビン(triciribine)を使用した。まず、通常の培養条件下の細胞生存率に影響を及ぼさないが、ターゲットの経路を阻害する濃度に阻害剤濃度を最適化した。これらの条件で、UO126及びUCN01はAdh-SFF処理によって生じる細胞生存率を減少させた。一方、トリシリビンは細胞生存率を有意に阻害しなかった。これらの結果は、図６ｉに示すように、Adh-SFFが主にMAPK制御によって細胞生存率を高めるメカニズムの証拠となる。
Next, inhibition of ERK signaling was tested using UO126, which is a selective inhibitor for MEK, an upstream kinase of ERK. In addition, UCN01, which is a selective PKCα inhibitor, and triciribine, which is an Akt inhibitor, were used. First, the inhibitor concentration was optimized to a concentration that does not affect cell viability under normal culture conditions but inhibits the target pathway. Under these conditions, UO126 and UCN01 decreased cell viability caused by Adh-SFF treatment. On the other hand, triciribine did not significantly inhibit cell viability. These results provide evidence for a mechanism by which Adh-SFF increases cell viability mainly through MAPK control, as shown in FIG. 6i.
試験例４
移植細胞の細胞表面の修飾
本発明者らは、ヘパラン硫酸のようなGAGに結合して、細胞表面にサブミクロン粒子を形成することによるAdh-SFFの自己集合を見出した。この構造に対する更なる機能化が細胞移植を高めると考えられる。がん細胞は転移プロセスにおいてその浸潤特性を増大させ、体内の離れた部位で貫通し生着することを可能にする。非浸潤性のNIH3T3はTgat (trio-related transforming gene)で形質転換した後、活性型MMP2を高発現することによって、浸潤活性を獲得することが報告されている(Biochemical and Biophysical Research Communications 355, 937-943 (2007))。MMP2は細胞外マトリックス(ECM)、特にI型及びIV型コラーゲンを分解し、細胞移動のための道を開ける。さらに、分解されたECMは増殖因子、接着タンパク質、及び血管新生因子を放出し、細胞移動及び細胞生存率を高める。したがって、ターゲット組織での生着のためには、細胞表面をMMP2で修飾することが有効と考えられる。タグの大きさや反応速度の観点から、本発明の目的に最も適したHalo-Tag技術を検討した。 Test example 4
Modification of cell surface of transplanted cells The present inventors have found self-assembly of Adh-SFF by binding to GAG such as heparan sulfate to form submicron particles on the cell surface. Further functionalization of this structure is thought to enhance cell transplantation. Cancer cells increase their invasive properties in the metastatic process, allowing them to penetrate and engraft at distant sites in the body. Non-invasive NIH3T3 has been reported to acquire invasive activity by high expression of active MMP2 after transformation with Tgat (trio-related transforming gene) (Biochemical and Biophysical Research Communications 355, 937 -943 (2007)). MMP2 breaks down the extracellular matrix (ECM), particularly type I and type IV collagen, and opens the way for cell migration. In addition, degraded ECM releases growth factors, adhesion proteins, and angiogenic factors, increasing cell migration and cell viability. Therefore, it is considered effective to modify the cell surface with MMP2 for engraftment in the target tissue. From the viewpoint of tag size and reaction rate, the Halo-Tag technology most suitable for the purpose of the present invention was examined.
移植細胞の細胞表面の修飾
本発明者らは、ヘパラン硫酸のようなGAGに結合して、細胞表面にサブミクロン粒子を形成することによるAdh-SFFの自己集合を見出した。この構造に対する更なる機能化が細胞移植を高めると考えられる。がん細胞は転移プロセスにおいてその浸潤特性を増大させ、体内の離れた部位で貫通し生着することを可能にする。非浸潤性のNIH3T3はTgat (trio-related transforming gene)で形質転換した後、活性型MMP2を高発現することによって、浸潤活性を獲得することが報告されている(Biochemical and Biophysical Research Communications 355, 937-943 (2007))。MMP2は細胞外マトリックス(ECM)、特にI型及びIV型コラーゲンを分解し、細胞移動のための道を開ける。さらに、分解されたECMは増殖因子、接着タンパク質、及び血管新生因子を放出し、細胞移動及び細胞生存率を高める。したがって、ターゲット組織での生着のためには、細胞表面をMMP2で修飾することが有効と考えられる。タグの大きさや反応速度の観点から、本発明の目的に最も適したHalo-Tag技術を検討した。 Test example 4
Modification of cell surface of transplanted cells The present inventors have found self-assembly of Adh-SFF by binding to GAG such as heparan sulfate to form submicron particles on the cell surface. Further functionalization of this structure is thought to enhance cell transplantation. Cancer cells increase their invasive properties in the metastatic process, allowing them to penetrate and engraft at distant sites in the body. Non-invasive NIH3T3 has been reported to acquire invasive activity by high expression of active MMP2 after transformation with Tgat (trio-related transforming gene) (Biochemical and Biophysical Research Communications 355, 937 -943 (2007)). MMP2 breaks down the extracellular matrix (ECM), particularly type I and type IV collagen, and opens the way for cell migration. In addition, degraded ECM releases growth factors, adhesion proteins, and angiogenic factors, increasing cell migration and cell viability. Therefore, it is considered effective to modify the cell surface with MMP2 for engraftment in the target tissue. From the viewpoint of tag size and reaction rate, the Halo-Tag technology most suitable for the purpose of the present invention was examined.
細胞生存率とヘパリン結合を最も維持する誘導体を見つけるために、Adh-SFFアルキルクロリドコンジュゲートの一団を合成した。アドヘサミンの各アルデヒドに1個ずつ、2個のSFFKペプチドを有する誘導体コンジュゲート(Adh(SFFK)-SFFK)は、ネガティブコントロールと比較してわずかな細胞生存率の増大を示した（図８ｂ、ｄ）。これに対して、1個のSFFKペプチドのみをコンジュゲートした誘導体(Adh-SFFK)は、Adh-SFFの細胞生存率を増大させる特性を維持した(図８ａ、ｃ)。コントロールのAdh-Cl及びAdh(Cl)-SFFは、無視できるほどわずかな細胞生存率の増大を示した。これらの結果から、細胞表面の修飾のために、Adh-SFFKを選択した。さらに、Adh-SFFKをアルキルクロリドで修飾することにより、親化合物Adh-SFFのヘパリンに対するアフィニティーと細胞生存率の増大を維持するAdh-SFF-Clを得た(図９ａ及びｂ、図１０ａ)。
In order to find derivatives that most maintain cell viability and heparin binding, a group of Adh-SFF alkyl chloride conjugates was synthesized. Derivative conjugates with two SFFK peptides, one for each aldehyde of adhesamine (Adh (SFFK) -SFFK) showed a slight increase in cell viability compared to the negative control (FIG. 8b, d). ). In contrast, a derivative (Adh-SFFK) conjugated with only one SFFK peptide maintained the property of increasing the cell viability of Adh-SFF (FIGS. 8a and 8c). Controls Adh-Cl and Adh (Cl) -SFF showed negligible increases in cell viability. From these results, Adh-SFFK was selected for cell surface modification. Furthermore, by modifying Adh-SFFK with alkyl chloride, Adh-SFF-Cl maintaining the affinity of the parent compound Adh-SFF for heparin and the increase in cell viability was obtained (FIGS. 9a and b, FIG. 10a).
コントロールシステムとして、eGFP-Halo融合タンパク質(eGFP-H)を構築し、この構築物をAdh-SFF-Clと共に使用して、eGFPで細胞表面を修飾した(図１０ｂ、ｄ及び図１１)。
eGFPをコードした遺伝子はpHTN HaloTag(登録商標) CMV-neo Vector (Promega)からプライマー 5’- GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3’（配列番号１）/5’- GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG -3’（配列番号２）を用いてPCR増幅することで得られた。次にHalo-TagをコードしたpET28bベクターの制限酵素サイトNco I/EcoR IへeGFPをコードしたDNAを挿入したpET28bベクターを調製した。
eGFPをHalo-Tagタンパク質のN末端にGGSGGSリンカーを介して導入した(図１１ａ及びｂ)。eGFP-Hの全配列（配列番号３）を図１１ｂに示す。組み換えタンパク質eGFP-HはE.coli BL21(de3)-RPIL中で発現され、Ni-NTA/His tag技術により単離された(図１１ｃ)。eGFP-Hで蛍光性とHalo-Tag活性が維持されていることを確認した(図１１ｄ、ｅ、ｆ)。 As a control system, an eGFP-Halo fusion protein (eGFP-H) was constructed, and this construct was used with Adh-SFF-Cl to modify the cell surface with eGFP (FIGS. 10b, d and FIG. 11).
The gene encoding eGFP is primer 5'- GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3 '(SEQ ID NO: 1) from pHTN HaloTag (registered trademark) CMV-neo Vector (Promega) / 5′-GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG-3 ′ (SEQ ID NO: 2). Next, a pET28b vector in which DNA encoding eGFP was inserted into the restriction enzyme sites Nco I / EcoR I of the pET28b vector encoding Halo-Tag was prepared.
eGFP was introduced into the N-terminus of the Halo-Tag protein via a GGSGGS linker (FIGS. 11a and b). The entire sequence of eGFP-H (SEQ ID NO: 3) is shown in FIG. 11b. The recombinant protein eGFP-H was expressed in E. coli BL21 (de3) -RPIL and isolated by Ni-NTA / His tag technology (FIG. 11c). It was confirmed that fluorescence and Halo-Tag activity were maintained with eGFP-H (FIGS. 11d, e, f).
eGFPをコードした遺伝子はpHTN HaloTag(登録商標) CMV-neo Vector (Promega)からプライマー 5’- GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3’（配列番号１）/5’- GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG -3’（配列番号２）を用いてPCR増幅することで得られた。次にHalo-TagをコードしたpET28bベクターの制限酵素サイトNco I/EcoR IへeGFPをコードしたDNAを挿入したpET28bベクターを調製した。
eGFPをHalo-Tagタンパク質のN末端にGGSGGSリンカーを介して導入した(図１１ａ及びｂ)。eGFP-Hの全配列（配列番号３）を図１１ｂに示す。組み換えタンパク質eGFP-HはE.coli BL21(de3)-RPIL中で発現され、Ni-NTA/His tag技術により単離された(図１１ｃ)。eGFP-Hで蛍光性とHalo-Tag活性が維持されていることを確認した(図１１ｄ、ｅ、ｆ)。 As a control system, an eGFP-Halo fusion protein (eGFP-H) was constructed, and this construct was used with Adh-SFF-Cl to modify the cell surface with eGFP (FIGS. 10b, d and FIG. 11).
The gene encoding eGFP is primer 5'- GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3 '(SEQ ID NO: 1) from pHTN HaloTag (registered trademark) CMV-neo Vector (Promega) / 5′-GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG-3 ′ (SEQ ID NO: 2). Next, a pET28b vector in which DNA encoding eGFP was inserted into the restriction enzyme sites Nco I / EcoR I of the pET28b vector encoding Halo-Tag was prepared.
eGFP was introduced into the N-terminus of the Halo-Tag protein via a GGSGGS linker (FIGS. 11a and b). The entire sequence of eGFP-H (SEQ ID NO: 3) is shown in FIG. 11b. The recombinant protein eGFP-H was expressed in E. coli BL21 (de3) -RPIL and isolated by Ni-NTA / His tag technology (FIG. 11c). It was confirmed that fluorescence and Halo-Tag activity were maintained with eGFP-H (FIGS. 11d, e, f).
共焦点顕微鏡写真下の観察により、NIH3T3細胞のAdh-SFF-Cl(50 μM)とeGFP-H(7.5 μM)による処理は、細胞表面に分布した明るい蛍光粒子を形成することが示された（図１０ｄは２Ｄ画像、図１２ａはz-stack画像、図１２ｂはフローサイトメトリー）。Adh-SFF-Cl又はeGFP-H単独の処理、及びAdh-SFFとeGFP-Hの処理は、検出可能な蛍光シグナルを生じなかった（図１０ｄ）。免疫染色により、観察されたeGFP蛍光シグナルは、Adh-SFF-Cl処理により誘導されたSDC4クラスターと重なった（図１２ｃ）。また、細胞表面修飾のAdh-SFF-Cl(50 μM)に対するeGFP-Hの比率の効果を調べた。結果は、7.5 μM eGFP-Hが最適であった。このシステムは、Halo-Tag融合タンパク質を設計し、それとAdh-SFF-Clで細胞を処理するだけで、選択したタンパク質で細胞を修飾するために使用できるので、高度に多様化し得る。
Observation under confocal micrographs showed that treatment of NIH3T3 cells with Adh-SFF-Cl (50 μM) and eGFP-H (7.5 μM) formed bright fluorescent particles distributed on the cell surface ( FIG. 10d is a 2D image, FIG. 12a is a z-stack image, and FIG. 12b is flow cytometry. Treatment with Adh-SFF-Cl or eGFP-H alone and with Adh-SFF and eGFP-H produced no detectable fluorescence signal (FIG. 10d). By immunostaining, the observed eGFP fluorescence signal overlapped with the SDC4 cluster induced by Adh-SFF-Cl treatment (FIG. 12c). In addition, the effect of the ratio of eGFP-H to cell surface modified Adh-SFF-Cl (50 μM) was examined. The result was optimal for 7.5 μM eGFP-H. This system can be highly diversified because it can be used to modify cells with a selected protein simply by designing a Halo-Tag fusion protein and treating it with Adh-SFF-Cl.
Adh-SFF-Cl/Halo-Tag細胞表面修飾システムを、細胞表面をマトリックスメタロプロテアーゼ2(MMP2)で修飾するのに適用した。
MMP2のHalo-Tag融合タンパク質(MMP2-H)の設計を図１３ａ、ｂに示す。 The Adh-SFF-Cl / Halo-Tag cell surface modification system was applied to modify the cell surface with matrix metalloprotease 2 (MMP2).
The design of the MMP2 Halo-Tag fusion protein (MMP2-H) is shown in FIGS.
MMP2のHalo-Tag融合タンパク質(MMP2-H)の設計を図１３ａ、ｂに示す。 The Adh-SFF-Cl / Halo-Tag cell surface modification system was applied to modify the cell surface with matrix metalloprotease 2 (MMP2).
The design of the MMP2 Halo-Tag fusion protein (MMP2-H) is shown in FIGS.
このシステムによる細胞の多機能化：すなわち、Adh-SFF-Cl及びMMP2-Hによる細胞生存率の増大、細胞浸潤活性を評価した。維持された細胞生存率を、NADPHデヒドロゲナーゼ活性を測定することにより確認し（図１４ｂ）、Cytoselect CBA-110 cell Invasion assay kitを使用して細胞浸潤活性を測定した（図１４ｃ、１５ｄ、ｅ）。このキットは、上側ウェルからECMでコートされた膜を通過して下側ウェルに移動し得る細胞を測定し、それにより細胞の浸潤活性を測定する(図１５ｃ)。Adh-SFF-ClとMMP2-Hで処理すると細胞浸潤活性が有意に増加するが、Adh-SFF-Cl又はMMP2-H単独の処理では増加しなかった(図１４ｃ、１５ｄ、ｅ)。同様に、Adh-SFFとMMP2-Hの処理は細胞浸潤活性を増強しなかった。これは、浸潤活性の増加が観察されるためにはHalo-Tag技術によるコンジュゲートが必要であることを示す。よく知られたMMP2及びMMP9の阻害剤であるmarimastat (200 nM)で浸潤活性が阻害されたという知見は、浸潤活性をMMP2作用に関連付ける。これらのデータは、Adh-SFF-ClとMMP2-Hのコンジュゲートが、細胞生存率と細胞浸潤活性を高めるための細胞表面の新規な修飾であることを示唆する。
Multifunctionalization of cells by this system: That is, increase of cell viability and cell invasion activity by Adh-SFF-Cl and MMP2-H were evaluated. Maintained cell viability was confirmed by measuring NADPH dehydrogenase activity (FIG. 14b) and cell invasive activity was measured using Cytoselect® CBA-110® cell® Invasion® assay® kit (FIGS. 14c, 15d, e). This kit measures cells that can migrate from the upper well through the ECM-coated membrane to the lower well, thereby measuring the invasive activity of the cells (FIG. 15c). Treatment with Adh-SFF-Cl and MMP2-H significantly increased cell invasive activity, but did not increase with treatment with Adh-SFF-Cl or MMP2-H alone (FIGS. 14c, 15d, e). Similarly, treatment with Adh-SFF and MMP2-H did not enhance cell invasive activity. This indicates that conjugation with the Halo-Tag technology is required in order to observe increased invasive activity. The finding that marimastat® (200 nM), a well-known inhibitor of MMP2 and MMP9, inhibited invasive activity correlates invasive activity with MMP2 action. These data suggest that a conjugate of Adh-SFF-Cl and MMP2-H is a novel modification of the cell surface to increase cell viability and cell invasive activity.
試験例５
皮膚－筋肉浸潤実験(In vivo実験)
懸濁細胞は通常、ターゲット組織に対する生着能が低く、再生医療の大きな障害となっている。本発明のシステムは、懸濁細胞の生存率を高めるのみならず、懸濁細胞に周囲の組織に移動する能力を付与することにより、この問題を解決し得る。これを実証するために、皮膚－筋肉浸潤実験を行った。安定的にホタルルシフェラーゼを発現するNIH3T3細胞(NIH3T3/luc)をICRマウスに皮下注射し、24時間後に皮膚及び筋肉領域に存在する生存細
胞を定量した（図１６ａ）。 Test Example 5
Skin-muscle infiltration experiment (in vivo experiment)
Suspension cells usually have a low engraftment ability to target tissues, which is a major obstacle to regenerative medicine. The system of the present invention can solve this problem not only by increasing the viability of the suspension cells but also by giving the suspension cells the ability to migrate to the surrounding tissue. To demonstrate this, skin-muscle infiltration experiments were performed. NIH3T3 cells stably expressing firefly luciferase (NIH3T3 / luc) were injected subcutaneously into ICR mice, and 24 hours later, surviving cells present in the skin and muscle regions were quantified (FIG. 16a).
皮膚－筋肉浸潤実験(In vivo実験)
懸濁細胞は通常、ターゲット組織に対する生着能が低く、再生医療の大きな障害となっている。本発明のシステムは、懸濁細胞の生存率を高めるのみならず、懸濁細胞に周囲の組織に移動する能力を付与することにより、この問題を解決し得る。これを実証するために、皮膚－筋肉浸潤実験を行った。安定的にホタルルシフェラーゼを発現するNIH3T3細胞(NIH3T3/luc)をICRマウスに皮下注射し、24時間後に皮膚及び筋肉領域に存在する生存細
胞を定量した（図１６ａ）。 Test Example 5
Skin-muscle infiltration experiment (in vivo experiment)
Suspension cells usually have a low engraftment ability to target tissues, which is a major obstacle to regenerative medicine. The system of the present invention can solve this problem not only by increasing the viability of the suspension cells but also by giving the suspension cells the ability to migrate to the surrounding tissue. To demonstrate this, skin-muscle infiltration experiments were performed. NIH3T3 cells stably expressing firefly luciferase (NIH3T3 / luc) were injected subcutaneously into ICR mice, and 24 hours later, surviving cells present in the skin and muscle regions were quantified (FIG. 16a).
(1) 実験動物
５から６週齢の雄性ICRマウスを三協ラボサービス株式会社より購入し、Specific Pathogen Free条件下で維持した。動物実験のプロトコールは、東京理科大学薬学部の動物実
験委員会の承認を得た。全ての動物実験は、アメリカ国立衛生研究所の実験動物の管理と使用に関する指針に概説された原則及び手順に従って行われた。 (1) Experimental animals Male ICR mice aged 5 to 6 weeks were purchased from Sankyo Lab Service Co., Ltd. and maintained under Specific Pathogen Free conditions. The animal experiment protocol was approved by the Animal Experiment Committee of the Faculty of Pharmaceutical Sciences, Tokyo University of Science. All animal experiments were conducted according to the principles and procedures outlined in the National Institutes of Health Guidelines for Laboratory Animal Care and Use.
５から６週齢の雄性ICRマウスを三協ラボサービス株式会社より購入し、Specific Pathogen Free条件下で維持した。動物実験のプロトコールは、東京理科大学薬学部の動物実
験委員会の承認を得た。全ての動物実験は、アメリカ国立衛生研究所の実験動物の管理と使用に関する指針に概説された原則及び手順に従って行われた。 (1) Experimental animals Male ICR mice aged 5 to 6 weeks were purchased from Sankyo Lab Service Co., Ltd. and maintained under Specific Pathogen Free conditions. The animal experiment protocol was approved by the Animal Experiment Committee of the Faculty of Pharmaceutical Sciences, Tokyo University of Science. All animal experiments were conducted according to the principles and procedures outlined in the National Institutes of Health Guidelines for Laboratory Animal Care and Use.
(2) In vivo実験用の細胞培養
NIH3T3細胞を10%(v/v)FBS含有DMEM中、37℃で培養した。ホタルルシフェラーゼ発現NIH3T3(NIH3T3/luc)細胞を構築するために、ホタルルシフェラーゼをコードするpEBMulti-Hygプラスミドベクター（和光純薬工業株式会社）を次のようにして調製した。以前に報告されたpCMV-Lucプラスミドベクター（Y. Takahashi et al, Journal of Controlled Release, 105, 332-343(2005))からホタルルシフェラーゼcDNA断片をポリメラーゼ連鎖反応(PCR)により増幅した。増幅に使用したフォワードプライマー及びリバースプライマーは、それぞれ、5’-CGG GGT ACC ATG GAA GAC GCC AAA AAC-3’（配列番号７）及び5’-TAA AGC GGC CGC TTA CAC GGC GAT CTT-3’（配列番号８）である。増幅されたPCR産物とpEBMulti-HygベクターをKpn I-HF及びNot I-HF制限酵素を使用して切断した。これらの切断産物を電気泳動により分離し、Nucleo Spin Gel and PCR Clean-up（タカラバイオ株式会社）により精製した。最後に、Ligation high Ver.2（東洋紡株式会社）を使用して、PCR産物をpEBMulti-Hygベクターに組み込み、pEBMulti-lucプラスミドを得た。その後、NIH3T3細胞をこのプラスミドでトランスフェクションして、NIH3T3/luc細胞を得た。これらの細胞を、5% CO2含有加湿空気下、10%(v/v)FBS及び500 μg/mL HygroGold (InvivoGen)を含有するDMEM中、37℃で培養した。 (2) Cell culture for in vivo experiments NIH3T3 cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS. In order to construct firefly luciferase-expressing NIH3T3 (NIH3T3 / luc) cells, a pEBMulti-Hyg plasmid vector (Wako Pure Chemical Industries, Ltd.) encoding firefly luciferase was prepared as follows. A firefly luciferase cDNA fragment was amplified by polymerase chain reaction (PCR) from a previously reported pCMV-Luc plasmid vector (Y. Takahashi et al, Journal of Controlled Release, 105, 332-343 (2005)). The forward primer and reverse primer used for amplification were 5'-CGG GGT ACC ATG GAA GAC GCC AAA AAC-3 '(SEQ ID NO: 7) and 5'-TAA AGC GGC CGC TTA CAC GGC GAT CTT-3' ( SEQ ID NO: 8). The amplified PCR product and the pEBMulti-Hyg vector were cleaved using Kpn I-HF and Not I-HF restriction enzymes. These cleavage products were separated by electrophoresis and purified by Nucleo Spin Gel and PCR Clean-up (Takara Bio Inc.). Finally, using Ligation high Ver.2 (Toyobo Co., Ltd.), the PCR product was incorporated into the pEBMulti-Hyg vector to obtain the pEBMulti-luc plasmid. Thereafter, NIH3T3 cells were transfected with this plasmid to obtain NIH3T3 / luc cells. These cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS and 500 μg / mL HygroGold (InvivoGen) under humidified air containing 5% CO 2 .
NIH3T3細胞を10%(v/v)FBS含有DMEM中、37℃で培養した。ホタルルシフェラーゼ発現NIH3T3(NIH3T3/luc)細胞を構築するために、ホタルルシフェラーゼをコードするpEBMulti-Hygプラスミドベクター（和光純薬工業株式会社）を次のようにして調製した。以前に報告されたpCMV-Lucプラスミドベクター（Y. Takahashi et al, Journal of Controlled Release, 105, 332-343(2005))からホタルルシフェラーゼcDNA断片をポリメラーゼ連鎖反応(PCR)により増幅した。増幅に使用したフォワードプライマー及びリバースプライマーは、それぞれ、5’-CGG GGT ACC ATG GAA GAC GCC AAA AAC-3’（配列番号７）及び5’-TAA AGC GGC CGC TTA CAC GGC GAT CTT-3’（配列番号８）である。増幅されたPCR産物とpEBMulti-HygベクターをKpn I-HF及びNot I-HF制限酵素を使用して切断した。これらの切断産物を電気泳動により分離し、Nucleo Spin Gel and PCR Clean-up（タカラバイオ株式会社）により精製した。最後に、Ligation high Ver.2（東洋紡株式会社）を使用して、PCR産物をpEBMulti-Hygベクターに組み込み、pEBMulti-lucプラスミドを得た。その後、NIH3T3細胞をこのプラスミドでトランスフェクションして、NIH3T3/luc細胞を得た。これらの細胞を、5% CO2含有加湿空気下、10%(v/v)FBS及び500 μg/mL HygroGold (InvivoGen)を含有するDMEM中、37℃で培養した。 (2) Cell culture for in vivo experiments NIH3T3 cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS. In order to construct firefly luciferase-expressing NIH3T3 (NIH3T3 / luc) cells, a pEBMulti-Hyg plasmid vector (Wako Pure Chemical Industries, Ltd.) encoding firefly luciferase was prepared as follows. A firefly luciferase cDNA fragment was amplified by polymerase chain reaction (PCR) from a previously reported pCMV-Luc plasmid vector (Y. Takahashi et al, Journal of Controlled Release, 105, 332-343 (2005)). The forward primer and reverse primer used for amplification were 5'-CGG GGT ACC ATG GAA GAC GCC AAA AAC-3 '(SEQ ID NO: 7) and 5'-TAA AGC GGC CGC TTA CAC GGC GAT CTT-3' ( SEQ ID NO: 8). The amplified PCR product and the pEBMulti-Hyg vector were cleaved using Kpn I-HF and Not I-HF restriction enzymes. These cleavage products were separated by electrophoresis and purified by Nucleo Spin Gel and PCR Clean-up (Takara Bio Inc.). Finally, using Ligation high Ver.2 (Toyobo Co., Ltd.), the PCR product was incorporated into the pEBMulti-Hyg vector to obtain the pEBMulti-luc plasmid. Thereafter, NIH3T3 cells were transfected with this plasmid to obtain NIH3T3 / luc cells. These cells were cultured at 37 ° C. in DMEM containing 10% (v / v) FBS and 500 μg / mL HygroGold (InvivoGen) under humidified air containing 5% CO 2 .
(3) 移植後の細胞生存
Adh-SFF-Clが皮膚領域において、効果的に細胞生存能を維持する濃度効果を確認した。
ICRマウスの背部被毛をイソフルラン吸入麻酔下で電気クリッパーと除毛クリームを使用して除去した。NIH3T3/luc細胞(5 x 105 cells)を50又は100 μM Adh-SFF-Clと共に37℃で20分間インキュベートした。1000 gで3分間遠心分離後、上清を除いた。これらの細胞をHBSSに再懸濁し、マウスの背中の2箇所に皮下投与した(5 x 105 cells/site)。24時間後、NIH3T3/luc細胞を移植したマウスをイソフルラン吸入で安楽死させ、細胞移植の部位を集めた。組織サンプルを細切し、細胞溶解液中でホモジナイズし、10000 g、4℃で10分間遠心分離した。サンプルの上清のルシフェラーゼ活性をPicaGene (東洋ビーネット株式会社）を使用して、2104 EnVision Multilabel Plate Readers (PerkinElmer)で測定した。細胞生存率を、投与した細胞に対する残存細胞のパーセンテージとして算出した。最適濃度(100μM)を皮膚－筋肉浸潤アッセイで適用した（図１７）。 (3) Cell survival after transplantation A concentration effect that Adh-SFF-Cl effectively maintains cell viability in the skin region was confirmed.
The back hair of ICR mice was removed using an electric clipper and a hair removal cream under isoflurane inhalation anesthesia. NIH3T3 / luc cells (5 × 10 5 cells) were incubated with 50 or 100 μM Adh-SFF-Cl at 37 ° C. for 20 minutes. After centrifugation at 1000 g for 3 minutes, the supernatant was removed. These cells were resuspended in HBSS and administered subcutaneously at two locations on the back of mice (5 × 10 5 cells / site). After 24 hours, mice transplanted with NIH3T3 / luc cells were euthanized by inhalation of isoflurane, and the site of cell transplant was collected. Tissue samples were minced, homogenized in cell lysate, and centrifuged at 10000 g for 10 minutes at 4 ° C. The luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells. The optimal concentration (100 μM) was applied in the skin-muscle infiltration assay (FIG. 17).
Adh-SFF-Clが皮膚領域において、効果的に細胞生存能を維持する濃度効果を確認した。
ICRマウスの背部被毛をイソフルラン吸入麻酔下で電気クリッパーと除毛クリームを使用して除去した。NIH3T3/luc細胞(5 x 105 cells)を50又は100 μM Adh-SFF-Clと共に37℃で20分間インキュベートした。1000 gで3分間遠心分離後、上清を除いた。これらの細胞をHBSSに再懸濁し、マウスの背中の2箇所に皮下投与した(5 x 105 cells/site)。24時間後、NIH3T3/luc細胞を移植したマウスをイソフルラン吸入で安楽死させ、細胞移植の部位を集めた。組織サンプルを細切し、細胞溶解液中でホモジナイズし、10000 g、4℃で10分間遠心分離した。サンプルの上清のルシフェラーゼ活性をPicaGene (東洋ビーネット株式会社）を使用して、2104 EnVision Multilabel Plate Readers (PerkinElmer)で測定した。細胞生存率を、投与した細胞に対する残存細胞のパーセンテージとして算出した。最適濃度(100μM)を皮膚－筋肉浸潤アッセイで適用した（図１７）。 (3) Cell survival after transplantation A concentration effect that Adh-SFF-Cl effectively maintains cell viability in the skin region was confirmed.
The back hair of ICR mice was removed using an electric clipper and a hair removal cream under isoflurane inhalation anesthesia. NIH3T3 / luc cells (5 × 10 5 cells) were incubated with 50 or 100 μM Adh-SFF-Cl at 37 ° C. for 20 minutes. After centrifugation at 1000 g for 3 minutes, the supernatant was removed. These cells were resuspended in HBSS and administered subcutaneously at two locations on the back of mice (5 × 10 5 cells / site). After 24 hours, mice transplanted with NIH3T3 / luc cells were euthanized by inhalation of isoflurane, and the site of cell transplant was collected. Tissue samples were minced, homogenized in cell lysate, and centrifuged at 10000 g for 10 minutes at 4 ° C. The luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells. The optimal concentration (100 μM) was applied in the skin-muscle infiltration assay (FIG. 17).
(4) 皮膚－筋肉浸潤アッセイ
前記のように、マウスの背部被毛を麻酔下で除去した。その後、100 μM Adh-SFF-Clを15 μM MMP2-Hと37℃で20分間混合し、MMP2-Hの活性化のために3 μM ZnCl2を添加した。NIH3T3/luc細胞(5 x 105 cells)を、活性化したAdh-SFF-Cl-MMP2コンジュゲートと共に37℃で20分間インキュベートした。これらの細胞をマウスの背中の2箇所に皮下投与した(3.3 x 105 cells/site)。24時間後、NIH3T3/luc細胞を移植したマウスをイソフルラン吸入で安楽死させ、細胞移植の部位を集め、皮膚と筋肉を分離した。筋肉サンプルを細切し、細胞溶解液中でホモジナイズし、10000 g、4℃で10分間遠心分離した。サンプルの上清のルシフェラーゼ活性をPicaGene (東洋ビーネット株式会社）を使用して、2104 EnVision Multilabel Plate Readers (PerkinElmer)で測定した。細胞生存率を、投与した細胞に対する残存細胞のパーセンテージとして算出した。 (4) Skin-muscle infiltration assay As described above, the back hair of the mice was removed under anesthesia. Thereafter, 100 μM Adh-SFF-Cl was mixed with 15 μM MMP2-H at 37 ° C. for 20 minutes, and 3 μM ZnCl 2 was added to activate MMP2-H. NIH3T3 / luc cells (5 × 10 5 cells) were incubated with activated Adh-SFF-Cl-MMP2 conjugate at 37 ° C. for 20 minutes. These cells were subcutaneously administered to two places on the back of the mouse (3.3 × 10 5 cells / site). After 24 hours, mice transplanted with NIH3T3 / luc cells were euthanized by isoflurane inhalation, the site of cell transplantation was collected, and the skin and muscle were separated. Muscle samples were minced, homogenized in cell lysate, and centrifuged at 10,000 g for 10 minutes at 4 ° C. The luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells.
前記のように、マウスの背部被毛を麻酔下で除去した。その後、100 μM Adh-SFF-Clを15 μM MMP2-Hと37℃で20分間混合し、MMP2-Hの活性化のために3 μM ZnCl2を添加した。NIH3T3/luc細胞(5 x 105 cells)を、活性化したAdh-SFF-Cl-MMP2コンジュゲートと共に37℃で20分間インキュベートした。これらの細胞をマウスの背中の2箇所に皮下投与した(3.3 x 105 cells/site)。24時間後、NIH3T3/luc細胞を移植したマウスをイソフルラン吸入で安楽死させ、細胞移植の部位を集め、皮膚と筋肉を分離した。筋肉サンプルを細切し、細胞溶解液中でホモジナイズし、10000 g、4℃で10分間遠心分離した。サンプルの上清のルシフェラーゼ活性をPicaGene (東洋ビーネット株式会社）を使用して、2104 EnVision Multilabel Plate Readers (PerkinElmer)で測定した。細胞生存率を、投与した細胞に対する残存細胞のパーセンテージとして算出した。 (4) Skin-muscle infiltration assay As described above, the back hair of the mice was removed under anesthesia. Thereafter, 100 μM Adh-SFF-Cl was mixed with 15 μM MMP2-H at 37 ° C. for 20 minutes, and 3 μM ZnCl 2 was added to activate MMP2-H. NIH3T3 / luc cells (5 × 10 5 cells) were incubated with activated Adh-SFF-Cl-MMP2 conjugate at 37 ° C. for 20 minutes. These cells were subcutaneously administered to two places on the back of the mouse (3.3 × 10 5 cells / site). After 24 hours, mice transplanted with NIH3T3 / luc cells were euthanized by isoflurane inhalation, the site of cell transplantation was collected, and the skin and muscle were separated. Muscle samples were minced, homogenized in cell lysate, and centrifuged at 10,000 g for 10 minutes at 4 ° C. The luciferase activity of the sample supernatant was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Benet Inc.). Cell viability was calculated as the percentage of remaining cells relative to administered cells.
(5) 結果
Adh-SFF-ClとMMP2-Hの混合物で前処理した細胞を皮下注射したとき、注射された細胞の亜集団が皮膚領域で生存しており、その一部が近傍の筋肉領域に移動した。一方、Adh-SFF-Cl単独、又はMMP2-H単独の前処理では、浸潤は全く観察されなかった（図１６ｂ、ｃ）。これらの結果は、Adh-SFF-ClとMMP2-Hを使用する本発明の細胞の移植方法がin vivo条件下で細胞の生着を促進することを示唆する。 (5) Results When cells pretreated with a mixture of Adh-SFF-Cl and MMP2-H were injected subcutaneously, a subpopulation of injected cells survived in the skin area, some of which were in the nearby muscle area Moved to. On the other hand, infiltration with Adh-SFF-Cl alone or MMP2-H alone was not observed at all (FIGS. 16b and 16c). These results suggest that the cell transplantation method of the present invention using Adh-SFF-Cl and MMP2-H promotes cell engraftment under in vivo conditions.
Adh-SFF-ClとMMP2-Hの混合物で前処理した細胞を皮下注射したとき、注射された細胞の亜集団が皮膚領域で生存しており、その一部が近傍の筋肉領域に移動した。一方、Adh-SFF-Cl単独、又はMMP2-H単独の前処理では、浸潤は全く観察されなかった（図１６ｂ、ｃ）。これらの結果は、Adh-SFF-ClとMMP2-Hを使用する本発明の細胞の移植方法がin vivo条件下で細胞の生着を促進することを示唆する。 (5) Results When cells pretreated with a mixture of Adh-SFF-Cl and MMP2-H were injected subcutaneously, a subpopulation of injected cells survived in the skin area, some of which were in the nearby muscle area Moved to. On the other hand, infiltration with Adh-SFF-Cl alone or MMP2-H alone was not observed at all (FIGS. 16b and 16c). These results suggest that the cell transplantation method of the present invention using Adh-SFF-Cl and MMP2-H promotes cell engraftment under in vivo conditions.
本発明によれば、細胞に選択的転移性（細胞生存能及び細胞浸潤活性）を装備するための新しい戦略が提供される。本発明の式(III)で表される化合物（例えば、Adh-SFF-Cl)は、細胞上でサブミクロン自己集合構造を形成して細胞生存率を高め、細胞表面をMMP2で修飾することにより、浸潤及び細胞生存率の向上など優れた機能を細胞に提供する。本発明によれば、細胞移植後の細胞生存率を維持する細胞表面修飾システムが提供される。本発明の化合物、アノイキス阻害剤、細胞培養用又は細胞移植用の添加剤、細胞生着促進剤、細胞の移植方法、及びキットは、細胞移植、再生医療の分野において有用となり得る。
According to the present invention, a new strategy is provided to equip cells with selective metastatic properties (cell viability and cell invasion activity). The compound represented by the formula (III) of the present invention (for example, Adh-SFF-Cl) increases the cell viability by forming a submicron self-assembled structure on the cell, and modifies the cell surface with MMP2. It provides cells with superior functions such as invasion and improved cell viability. According to the present invention, there is provided a cell surface modification system that maintains cell viability after cell transplantation. The compound, anoikis inhibitor, additive for cell culture or cell transplant, cell engraftment promoter, cell transplantation method, and kit of the present invention can be useful in the fields of cell transplantation and regenerative medicine.
配列表
配列番号１（プライマー）：
gtcgggatcc gaattcggtg gtagtggtgg tagtatggca gaaatcggta ctgg
配列番号２（プライマー）：
gacggagctc gaattgttat cgctctgaaa gtacag
配列番号３（eGFP-ハロアルカンデヒドロゲナーゼ融合タンパク質）：
Met Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser Ala Arg Ile Asn Val Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
配列番号４（プライマー）：
aggagatata ccatgcagaa gttctttggg
配列番号５（プライマー）：
cactaccacc gaattcgcag atctccggag tgaca
配列番号６（マトリックスメタロプロテアーゼ 2-ハロアルカンデヒドロゲナーゼ融合タンパク質）：
Thr Met Gln Lys Phe Phe Gly Leu Pro Gln Thr Gly Asp Leu Asp Gln Asn Thr Ile Glu Thr Met Arg Lys Pro Arg Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe Phe Pro Arg Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile Ile Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe Ala Arg Ala Leu Lys Val Trp Ser Asp Val Thr Pro Leu Arg Phe Ser Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn Phe Gly Arg Trp Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe Ala Pro Gly Thr Gly Val Gly Gly Asp Ser His Phe Asp Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn Gly Arg Glu Tyr Ser Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Asp Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser Tyr Asn Ser Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Ala Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala Pro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg Asn Asp Gly Lys Val Trp Cys Ala Thr Thr Thr Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His Glu Phe Gly His Ala Met Gly Leu Glu His Ser Gln Asp Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser His Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr Gly Pro Ser Pro Asp Ala Asp Thr Asp Thr Gly Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu Ile Cys Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Glu Pro Thr Thr Glu Asp Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
配列番号７（プライマー）：
cggggtacca tggaagacgc caaaaac
配列番号８（プライマー）：
taaagcggcc gcttacacgg cgatctt Sequence listing SEQ ID NO: 1 (primer):
gtcgggatcc gaattcggtg gtagtggtgg tagtatggca gaaatcggta ctgg
SEQ ID NO: 2 (primer):
gacggagctc gaattgttat cgctctgaaa gtacag
SEQ ID NO: 3 (eGFP-haloalkane dehydrogenase fusion protein):
Met Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser Ala Arg Ile Asn Val Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
SEQ ID NO: 4 (primer):
aggagatata ccatgcagaa gttctttggg
SEQ ID NO: 5 (primer):
cactaccacc gaattcgcag atctccggag tgaca
SEQ ID NO: 6 (matrix metalloprotease 2-haloalkane dehydrogenase fusion protein):
Thr Met Gln Lys Phe Phe Gly Leu Pro Gln Thr Gly Asp Leu Asp Gln Asn Thr Ile Glu Thr Met Arg Lys Pro Arg Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe Phe Pro Arg Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile Ile Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe Ala Arg Ala Leu Lys Val Trp Ser Asp Val Thr Pro Leu Arg Phe Ser Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn Phe Gly Arg Trp Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe Ala Pro Gly Thr Gly Val Gly Gly Asp Ser His Phe Asp Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn Gly Arg Glu Tyr Ser Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Asp Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser Tyr Asn Ser Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Ala Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala Pro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg Asn Asp Gly Lys Val Trp Cys Ala Thr Thr Thr Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His Glu Phe Gly His Ala Met Gly Leu Glu His Ser Gln Asp Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser His Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr Gly Pro Ser Pro Asp Ala Asp Thr Asp Thr Gly Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu Ile Cys Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Glu Pro Thr Thr Glu Asp Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
SEQ ID NO: 7 (primer):
cggggtacca tggaagacgc caaaaac
SEQ ID NO: 8 (primer):
taaagcggcc gcttacacgg cgatctt
配列番号１（プライマー）：
gtcgggatcc gaattcggtg gtagtggtgg tagtatggca gaaatcggta ctgg
配列番号２（プライマー）：
gacggagctc gaattgttat cgctctgaaa gtacag
配列番号３（eGFP-ハロアルカンデヒドロゲナーゼ融合タンパク質）：
Met Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser Ala Arg Ile Asn Val Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
配列番号４（プライマー）：
aggagatata ccatgcagaa gttctttggg
配列番号５（プライマー）：
cactaccacc gaattcgcag atctccggag tgaca
配列番号６（マトリックスメタロプロテアーゼ 2-ハロアルカンデヒドロゲナーゼ融合タンパク質）：
Thr Met Gln Lys Phe Phe Gly Leu Pro Gln Thr Gly Asp Leu Asp Gln Asn Thr Ile Glu Thr Met Arg Lys Pro Arg Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe Phe Pro Arg Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile Ile Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe Ala Arg Ala Leu Lys Val Trp Ser Asp Val Thr Pro Leu Arg Phe Ser Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn Phe Gly Arg Trp Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe Ala Pro Gly Thr Gly Val Gly Gly Asp Ser His Phe Asp Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn Gly Arg Glu Tyr Ser Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Asp Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser Tyr Asn Ser Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Ala Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala Pro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg Asn Asp Gly Lys Val Trp Cys Ala Thr Thr Thr Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His Glu Phe Gly His Ala Met Gly Leu Glu His Ser Gln Asp Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser His Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr Gly Pro Ser Pro Asp Ala Asp Thr Asp Thr Gly Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu Ile Cys Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Glu Pro Thr Thr Glu Asp Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
配列番号７（プライマー）：
cggggtacca tggaagacgc caaaaac
配列番号８（プライマー）：
taaagcggcc gcttacacgg cgatctt Sequence listing SEQ ID NO: 1 (primer):
gtcgggatcc gaattcggtg gtagtggtgg tagtatggca gaaatcggta ctgg
SEQ ID NO: 2 (primer):
gacggagctc gaattgttat cgctctgaaa gtacag
SEQ ID NO: 3 (eGFP-haloalkane dehydrogenase fusion protein):
Met Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser Ala Arg Ile Asn Val Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
SEQ ID NO: 4 (primer):
aggagatata ccatgcagaa gttctttggg
SEQ ID NO: 5 (primer):
cactaccacc gaattcgcag atctccggag tgaca
SEQ ID NO: 6 (matrix metalloprotease 2-haloalkane dehydrogenase fusion protein):
Thr Met Gln Lys Phe Phe Gly Leu Pro Gln Thr Gly Asp Leu Asp Gln Asn Thr Ile Glu Thr Met Arg Lys Pro Arg Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe Phe Pro Arg Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile Ile Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe Ala Arg Ala Leu Lys Val Trp Ser Asp Val Thr Pro Leu Arg Phe Ser Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn Phe Gly Arg Trp Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe Ala Pro Gly Thr Gly Val Gly Gly Asp Ser His Phe Asp Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn Gly Arg Glu Tyr Ser Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Asp Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser Tyr Asn Ser Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Ala Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala Pro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg Asn Asp Gly Lys Val Trp Cys Ala Thr Thr Thr Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His Glu Phe Gly His Ala Met Gly Leu Glu His Ser Gln Asp Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser His Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr Gly Pro Ser Pro Asp Ala Asp Thr Asp Thr Gly Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu Ile Cys Glu Phe Gly Gly Ser Gly Gly Ser Met Ala Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Glu Pro Thr Thr Glu Asp Leu Tyr Phe Gln Ser Asp Asn Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
SEQ ID NO: 7 (primer):
cggggtacca tggaagacgc caaaaac
SEQ ID NO: 8 (primer):
taaagcggcc gcttacacgg cgatctt
Claims (14)
- 下記式(I)で表される化合物又はその塩。
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ２は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－ＮＨ２
（式中、
Ｌは、リンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよい。）
で表される基である。］ A compound represented by the following formula (I) or a salt thereof.
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 2 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -NH 2
(Where
L is a linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When is 2 or 3, each X 3 may be the same or different. )
It is group represented by these. ] - 下記式(II)で表される化合物である、請求項１に記載の化合物又はその塩。
- 以下の群から選択される、請求項１に記載の化合物又はその塩。
- 下記式(III)で表される化合物又はその塩。
環Ａ１及び環Ａ２は、同一又は異なって、１～３個の窒素原子を含む６員芳香族ヘテロ環基であり、前記６員芳香族ヘテロ環基は、ハロゲン原子、ヒドロキシ、アルキル、ヒドロキシアルキル、アルコキシ、アルキルチオ、シアノ、ニトロ及びアミノから独立して選択される１又は２個の置換基で置換されていてもよく、
Ｒ１は、ホルミル又はヒドロキシメチルであり、
Ｒ３は、－Ｌ－（Ｘ１）ｎ－（Ｘ２）ｍ－（Ｘ３）ｐ－Ｌ２－Ｙ
（式中、
Ｌは、第一のリンカーであり、
ｎは、０、１又は２であり、
Ｘ１は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｎが２のとき、各Ｘ１は、同一又は異なっていてもよく、
ｍは、２又は３であり、
各Ｘ２は、同一又は異なって、Phe、Tyr及びTrpから選択されるアミノ酸残基であり、
ｐは、０、１、２又は３であり、
Ｘ３は、Ser、Ala、Gly、Lys、Val、Leu、Ile、Thr、Cys、Met、Asn、Gln、Pro、Asp、Glu、Arg、His及びOrnから選択されるアミノ酸残基であり、ｐが２又は３のとき、各Ｘ３は、同一又は異なっていてもよく、
Ｌ２は、第二のリンカーであり、
Ｙはリガンド結合タンパク質に特異的に結合するリガンドである。）
で表される基である。］ A compound represented by the following formula (III) or a salt thereof.
Ring A 1 and Ring A 2 are the same or different and are a 6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic group includes a halogen atom, hydroxy, alkyl, Optionally substituted with one or two substituents independently selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino;
R 1 is formyl or hydroxymethyl;
R 3 represents -L- (X 1 ) n- (X 2 ) m- (X 3 ) p -L 2 -Y
(Where
L is the first linker;
n is 0, 1 or 2;
X 1 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His and Orn, n Each X 1 may be the same or different when
m is 2 or 3,
Each X 2 is the same or different and is an amino acid residue selected from Phe, Tyr and Trp;
p is 0, 1, 2 or 3;
X 3 is an amino acid residue selected from Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn, and p When X is 2 or 3, each X 3 may be the same or different;
L 2 is a second linker;
Y is a ligand that specifically binds to the ligand binding protein. )
It is group represented by these. ] - 下記式(IV)で表される、請求項４に記載の化合物又はその塩。
- Ｙが、－ＮＨ（ＣＨ２）２Ｏ（ＣＨ２）２Ｏ（ＣＨ２）６Ｃｌである、請求項４又は５に記載の化合物又はその塩。 The compound or a salt thereof according to claim 4 or 5, wherein Y is -NH (CH 2 ) 2 O (CH 2 ) 2 O (CH 2 ) 6 Cl.
- 下記式（ｋ）で表される、請求項４に記載の化合物又はその塩。
- 請求項１～７のいずれか１項に記載の化合物又はその塩を含有する、アノイキス阻害剤。 An anoikis inhibitor comprising the compound according to any one of claims 1 to 7 or a salt thereof.
- 請求項１～７のいずれか１項に記載の化合物又はその塩を含有する、細胞培養用又は細胞移植用の添加剤。 An additive for cell culture or cell transplant containing the compound or salt thereof according to any one of claims 1 to 7.
- 請求項１～７のいずれか１項に記載の化合物又はその塩を含有する、細胞の生着促進剤。 A cell engraftment promoter comprising the compound or salt thereof according to any one of claims 1 to 7.
- 請求項１～７のいずれか１項に記載の化合物又はその塩と細胞を対象に投与することを含む、細胞の移植方法。 A cell transplantation method comprising administering the compound according to any one of claims 1 to 7 or a salt thereof and a cell to a subject.
- （ａ）請求項４～７のいずれか１項に記載の化合物又はその塩、及び
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２
を含む、キット。 (A) the compound according to any one of claims 4 to 7 or a salt thereof, and (b) a matrix metalloproteinase 2 bound to the ligand-binding protein.
Including a kit. - 細胞移植用キットである、請求項１２に記載のキット。 The kit according to claim 12, which is a cell transplantation kit.
- （ａ）請求項４～７のいずれか１項に記載の化合物又はその塩、
（ｂ）前記リガンド結合タンパク質に結合したマトリックスメタロプロテアーゼ２、及び
（ｃ）細胞を対象に投与することを含む、細胞の移植方法。 (A) the compound or salt thereof according to any one of claims 4 to 7,
(B) A matrix metalloprotease 2 bound to the ligand-binding protein, and (c) a cell transplantation method comprising administering the cell to a subject.
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| JP2018053515A JP2021088504A (en) | 2018-03-20 | 2018-03-20 | Compound for improving cell transplantation efficiency |
| JP2018-053515 | 2018-03-20 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009154201A1 (en) * | 2008-06-18 | 2009-12-23 | 国立大学法人京都大学 | Cell adhesion promoting agent and method of promoting cell adhesion |
| WO2013168807A1 (en) * | 2012-05-11 | 2013-11-14 | 国立大学法人京都大学 | Additive for graft cell suspension, and therapeutic composition |
-
2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009154201A1 (en) * | 2008-06-18 | 2009-12-23 | 国立大学法人京都大学 | Cell adhesion promoting agent and method of promoting cell adhesion |
| WO2013168807A1 (en) * | 2012-05-11 | 2013-11-14 | 国立大学法人京都大学 | Additive for graft cell suspension, and therapeutic composition |
Non-Patent Citations (3)
| Title |
|---|
| FRISCO-CABANOS, H. L. ET AL.: "Synthetic molecules that protect cells from anoikis and their use in cell transplantation", ANGEWANDTE CHEMIE , INTERNATIONAL EDITION, vol. 53, no. 42, 2014, pages 11208 - 11213, XP055637531 * |
| SCHMIDTKE, M. ET AL.: "Synthesis, cytotoxicity and antiviral activity of N,N'-bis-5-nitropyrimidyl derivatives of dispirotripiperazine", ANTIVIRAL RESEARCH, vol. 55, 2002, pages 117 - 127, XP008141707 * |
| TAKASHIMA, I. ET AL.: "Multifunctionalization of Cells with a Self-Assembling Molecule to Enhance Cell Engraftment", ACS CHEMICAL BIOLOGY, vol. 14, no. 4, 26 February 2019 (2019-02-26), pages 775 - 783, XP055637536 * |
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