JP2021088504A - Compound for improving cell transplantation efficiency - Google Patents

Abstract

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.SOLUTION: 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 Ser, Ala, Gly, Lys, Val, Leu, Ile, Thr, Cys, Met, Asn, Gln, Pro, Asp, Glu, Arg, His, and Orn; and each X2 is the same or different and represents an amino acid residue selected from Phe, Tyr, and Trp)].SELECTED DRAWING: None

Description

The present invention relates to a dyspirotripirazine derivative capable of increasing the efficiency of cell transplantation, an anoikis inhibitor containing the same, an additive for cell culture or transplanted cells, and a cell engraftment promoter.

In cell therapy / regenerative medicine, cells useful for treatment are transplanted into the body. One of the problems in cell therapy is the low efficiency of cell transplantation. Many of the transplanted cells cannot survive after injection into the patient, only a small percentage of the cells are viable for engraftment, and a huge number of cells are needed to achieve the desired therapeutic effect. It becomes.

One of the causes of low cell transplantation efficiency is that during cell transplantation, cells are separated from the extracellular matrix 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".

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 and Patent Document 1).

Cindecane is a cell membrane heparan sulfate proteoglycan in which a core protein penetrates the cell membrane. Four types of syndecane 1, 2, 3, and 4 are known depending on the difference in core protein, and it is a mixed proteoglycan having two types of sugar side chains, heparan sulfate and chondroitin sulfate. Cindecane interacts with growth factors such as extracellular matrix components and fibroblast growth factor 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 the adhesion and proliferation of suspended cells in suspension by binding to the heparan sulfate of syndecane on the cell surface.

Integrin, a receptor that penetrates the cell membrane and is involved in cell adhesion and cell-cell interaction, recognizes and binds to the 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 Document 2 and Patent Document 2). Fibronectin, an extracellular matrix protein, has both an integrin binding site and a heparan sulfate binding site. The cooperation of both sites induces fibronectin to induce strong signals that promote cell survival. Adh-RGDS also has both an integrin binding site (RGDS) and a heparan sulfate binding site, and has a stronger effect of increasing cell viability than fibronectin.

International Publication No. 2009/154201 International Publication No. 2013/168807

Chemistry & Biology 16, 773-782 (2009) Angew. Chem. Int. Ed. 53, 11208-11213 (2014)

An object of the present invention is to provide a compound which solves the problem of anoikis in cell transplantation and cell culture, inhibits cell anoikis, and enhances cell viability.

Also, in metastasis, cancer cells increase cell functions such as cell viability and cell infiltration. These are all desirable properties for transplanted cells. If the cell infiltration activity can be enhanced as well as the cell viability of the transplanted cells, it will be possible to promote the engraftment of the transplanted cells. An object of the present invention is to provide a technique for increasing the cell viability of transplanted cells such as metastatic cancer cells and inducing cell infiltration. Achieving these goals 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 dispirotripirazine derivative in which an aromatic amino acid sequence of a specific length is bound to a dispirotripirazine derivative such as adhesamine. They found that this novel dispirotripirazine derivative is more effective in increasing cell viability than adhesamine.

It was also found that the dyspirotripiperazine derivative of the present invention binds to heparan sulfate of syndecane on the cell surface and self-assembles to form submicron particles on the cell surface. In addition, the formation of these submicron particles results in clustering of Cindecane 4 (SDC4), migration and activation of protein kinase C (PKC) α to the cell membrane, and phosphorylation of mitogen-activated protein kinase (MAPK). It was found that it induces cell survival signal transduction via cells and activates cell viability.

The present inventors also synthesized a derivative in which a ligand that specifically binds to a ligand-binding protein is bound to the dispirotripirazine derivative of the present invention. This derivative and matrix metalloproteinase 2 (MMP2) bound to the ligand-binding protein form a complex via the ligand. Using this complex, we succeeded in modifying the cell surface with MMP2.

MMP2 is known to degrade extracellular matrix, especially type I and type IV collagen, and to be involved in metastasis and infiltration of cancer cells. According to the method of the present invention, the complex of the dispiropiperazine derivative of the present invention and MMP2 forms submicron particles on the cell surface, and the cell surface is modified with MMP2. MMP2-modified cells not only increase cell viability, but also increase cell infiltration activity. According to the method of the present invention, engraftment of transplanted cells is promoted in cell transplantation.

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.

[During the ceremony,
Ring A ¹ and ring A ² are the same or different and are 6-membered aromatic heterocyclic groups containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic groups are halogen atoms, hydroxy, alkyl, and. It may be substituted with one or two substituents selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino.
R ¹ is formyl or hydroxymethyl and
R ² is −L − (X ¹ ) _n − (X ² ) _m − (X ³ ) _p −NH ₂
(During the ceremony,
L is a linker and
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, and n When is 2, each X ¹ may be the same or different.
m is 2 or 3
Each X ² is the same or different 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, p. When is 2 or 3, each X ³ may be the same or different. )
It is a group represented by. ]
[2] The compound according to [1] or a salt thereof, which is a compound represented by the following formula (II).

[In the formula, R ¹ and R ² are as defined in [1]. ]
[3] The compound according to [1] or a salt thereof, which is selected from the following group.

as well as

[4] A compound represented by the following formula (III) or a salt thereof.

[During the ceremony,
Ring A ¹ and ring A ² are the same or different and are 6-membered aromatic heterocyclic groups containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic groups are halogen atoms, hydroxy, alkyl, and. It may be substituted with one or two substituents selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino.
R ¹ is formyl or hydroxymethyl and
R ³ is −L − (X ¹ ) _n − (X ² ) _m − (X ³ ) _p −L ² −Y
(During the ceremony,
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, and n When is 2, each X ¹ may be the same or different.
m is 2 or 3
Each X ² is the same or different 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, p. When is 2 or 3, each X ³ may be the same or different L ² is the second linker.
Y is a ligand that specifically binds to a ligand-binding protein. )
It is a group represented by. ]
[5] The compound according to [4] or a salt thereof, which is represented by the following formula (IV).

[In the formula, R ¹ and R ³ are as defined in [4]. ]
[6] The compound according to [4] or [5] or a salt thereof, wherein Y is −NH (CH ₂ ) ₂ O (CH ₂ ) ₂ O (CH ₂ ) _{6 Cl.}
[7] The compound according to [4] or a salt thereof, which is represented by the following formula (k).

[8] An anoikis inhibitor containing the compound according to any one of [1] to [7] or a salt thereof.
[9] An additive for cell culture or transplanted cells, which contains the compound according to any one of [1] to [7] or a salt thereof.
[10] A cell engraftment promoter containing the compound according to any one of [1] to [7] or a salt thereof.
[11] A method for transplanting cells, which comprises administering the compound according to any one of [1] to [7] or a salt thereof and cells.
[12] (a) The compound according to any one of [4] to [7] or a salt thereof, and (b) a matrix metalloprotease 2 bound to the ligand-binding protein.
Including, kit.
[13] The kit according to [12], which is a cell transplantation kit.
[14] (a) The compound according to any one of [4] to [7] or a salt thereof.
(B) A method for transplanting cells, which comprises administering matrix metalloproteinase 2 bound to the ligand-binding protein and (c) cells.

The compound represented by the formula (I), (II), (III) or (IV) of the present invention has an action of inhibiting cell anoikis and increasing cell viability. The compounds of the present invention are useful as anoikis inhibitors, cell engraftment promoters, or additives for cell culture or transplanted cells. In addition, by administering the compound represented by the formula (III) or (IV) of the present invention together with matrix metalloproteinase 2 bound to a ligand-binding protein and cells, the cell surface is modified with MMP2 and cell infiltration is induced. However, it is possible to increase cell engraftment. It is useful as a compound that enhances the efficiency of cell transplantation.

(a) It is a figure which shows the structure of Adhesamine and Adh-SFF (conjugate of Adhesamine and SFF peptide). (b) It is a figure which shows the structure of the adhesamine derivative which bound each peptide. (c, d) Phosphate buffer (50 mM, pH 6.0), 10 mM NaCl, 3% (v / v) DMSO, adhesamine (100 μM) (c) or Adh-SFF (100 μM) (d) It is a figure which shows the result of the isothermal titration calorimetry (ITC) of the heparin solution (100 μM) of. (e) It is a graph which shows the cell viability of NIH3T3 cells incubated for 3 days in serum-free DMEM containing 1% (v / v) DMSO to which each adhesamine derivative (100 μM) was added. Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF. The error bar is the standard deviation (n = 3). (a) Figure showing ITC results of heparin solution (250 μM) in phosphate buffer (50 mM, pH 6.0), 10 mM NaCl, 3% (v / v) DMSO, peptide SFF (150 μM). is there. (b) ITC results of chondroitin sulfate A solution (500 μM) in phosphate buffer (50 mM, pH 6.0), 10 mM NaCl, 3% (v / v) DMSO, Adh-SFF (100 μM) It is a figure which shows. (a) 1% (v / v) DMSO-containing serum-free DMEM supplemented with adhesamine (100 μM), peptide SFF (100 μM), adhesamine and peptide SFF mixture (100 μM each) or Adh-SFF (100 μM) It is a graph which shows the cell viability of the NIH3T3 cell which was incubated in 3 days. (b) It is a graph which shows the concentration effect of Adh-SFF (0-300 μM). Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF (100 μM). It is a graph which shows the correlation between the affinity for heparin of each adhesamine derivative, and the cell viability. A cryo-electron microscope (Cryo-TEM) photograph is shown. Conditions: Heparin alone (a, 3 μM) and Adh-SFF alone (b, 300 μM) in serum-free DMEM containing 1% (v / v). (a) It is a graph which shows the dynamic light scattering method (DLS) analysis result (distribution of particle size in a solution) of Adh-SFF-heparin complex. DLS measurement was performed in serum-free DMEM at 25 ° C. ([Adh-SFF] = 100 μM, [Heparin] = 1 μM). (b) Cryo-EM photography of the Adh-SFF-heparin complex in serum-free DMEM ([Adh-SFF] = 100 μM, [heparin] = 1 μM). (c) Photograph of Adh-SFF-treated or untreated cell surface by field emission scanning electron microscope (FE-SEM). _{Cindecane 4 (SDC4) (d, λ ex} = 488 nm) incubated in 1% (v / v) DMSO-containing serum-free DMEM supplemented with (d, f) adhesamine (100 μM) or Adh-SFF (100 μM) , λ _em = 500-550 nm) and PKCα (f, λ _ex = 568 nm, λ _em = BP617 / 73). (e) Quantification of the number of particles in Fig. 6d classified by size (calculated by ImageJ software). Western blot analysis of MAPK phosphorylation after incubation in serum-free DMEM for 3, 6, 9, 12 hours on ultra low attachment plate with (g, h) adhesamine (100 μM) or Adh-SFF (100 μM) (ERK1 / 2: Thr202 / Tyr204 and JNK: Thr183 / 185). The SC condition was that cells were incubated on a standard plastic plate in 10% (v / v) FBS. (i) It is proposed that a submicron aggregate of Adh-SFF induces intracellular signal transduction and inhibits cell death. It is a micrograph showing the time-dependent localization of Cindecane 4 (SDC4) and PKCα. (a, b) The structures of Adh-SFFK and Adh (SFFK) -SFFK are shown. (c, d) Effect of Adh-SFFK (100 μM) and Adh (SFFK) -SFFK (100 μM) on cell viability of NIH3T3 cells incubated in 1% (v / v) DMSO-containing serum-free DMEM 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 μM). (a) It is a graph which shows the concentration effect of Adh-SFF-Cl on the cell viability of NIH3T3 cells incubated for 3 days in serum-free DMEM containing 1% (v / v) DMSO. Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF (100 μM). (b) ITC results for heparin solution (100 μM) in phosphate buffer (50 mM, pH 6.0), 10 mM NaCl, 3% (v / v) DMSO, Adh-SFF-Cl (100 μM) It is a figure which shows. It is a figure which shows the modification of the cell surface using an eGFP-haloalkane dehydrogenase fusion protein (referred to as "eGFP-H") and Adh-SFF-Cl. (a) The structure of Adh-SFF-Cl is shown. (b) The structural image of eGFP-H is shown. (c) It is a conceptual diagram of the modification of the cell surface using eGFP-H and Adh-SFF-Cl. (d) It is a confocal micrograph of the modification of the cell surface modified with eGFP-H. Cells are with eGFP-H (7.5 μM), Adh-SFF-Cl (50 μM), a mixture of Adh-SFF-Cl (50 μM) and eGFP-H (7.5 μM), or Adh-SFF (50 μM). After incubation in serum-free DMEM supplemented with a mixture of eGFP-H (7.5 μM), it was washed with PBS. The "Merge" image is an image with a bright field of view superimposed. Hoechst 33342 (λ _ex = 405 nm, λ _em = 417-477 nm) and eGFP-H (λ _ex = 488 nm, λ _em = 500-550 nm) Construction and evaluation of eGFP-H. (a) pET28b vector design for expression of eGFP-H. (b) Full sequence of eGFP-H. (c) Electrophoretic analysis of eGFP-H purification (8% (v / v) acrylamide gel). (d) Fluorescence spectrum of eGFP-H (0.5 μM). (e) Reaction trace of eGFP-H and Adh-SFF-Cl. The reaction was almost completed within 4 minutes under the following conditions: [Adh-SFF-Cl] = 50 μM, [eGFP-H] = 7.5 μM, 37 ° C in serum-free DMEM (pH 7.9). (f) MALDI-TOF-MS of eGFP-H (solid line, Mw 68400) and eGFP-H (dashed line, Mw 69600) bound to Adh-SFF-Cl using sinapinic acid as the matrix. It is an image showing the localization of eGFP-H on the cell surface. (a) A z-stack image of the cell surface modified with eGFP-H and Adh-SFF-Cl. Cells were incubated with eGFP-H (7.5 μM) and Adh-SFF-Cl (50 μM) and then washed with PBS. Cells were also stained with Hoechst 33342 (2 μg / ml, λ _ex = 405 nm, λ _em = 417-477 nm). (b) Flow cytometric analysis of fluorescence intensity of NIH3T3 cells pretreated with Adh-SFF / Adh-SFF-Cl (50 μM) and eGFP-H (7.5 μM). I: Adh-SFF-Cl, II: eGFP-H, III: Adh-SFF-Cl + eGFP-H, IV: Adh-SFF + eGFP-H. (c) Co-staining of eGFP-H / Adh-SFF-Cl and anti-cindecane 4 (SDC4) antibody (ab24511) / AF568-IgG with DAPI staining (λ _ex = 405 nm, λ _em = 417-477 nm). Construction and evaluation of MMP2-haloalkane dehydrogenase fusion protein (referred to as "MMP2-H"). (a) pET28b vector design for shortened MMP2-H expression. (b) Complete sequence of fusion proteins. (c) Electrophoretic analysis of MMP2-H purification (8% (v / v) acrylamide gel) and gelatin zymography analysis confirming MMP2 enzyme activity. (d) Enzyme kinetics of MMP2-H (0.5 μM) analyzed with quenched fluorescent peptides (1, 2, 5, 10 μM) in serum-free DMEM (pH 7.9), 50 μM ZnCl _2. The velocity parameter k _cat / K _m of the fusion protein (1.33 x 10 ⁴ M ^-1 s ^-1 ) was comparable to the velocity parameter of full-length mouse MMP2 (Biolegend 554402, 1.97 x 10 ⁴ M ^-1 s ^-1). (Both activated with 1 mM 4-aminophenylmercury acetate). (e) Reaction trace of fusion protein and Adh-SFF-Cl. The reaction was almost complete within 4 minutes. (f) Reaction trace of fusion protein (7.5 μM) and Halo-Tag® TMR ligand (50 μM, Promega Corporation) in serum-free DMEM (pH 7.9) at 37 ° C. (g) Plot of TMR-labeled MMP2-H quantified in Figure 13f over time. (h) MMP2-H (solid line, Mw 81683) and MMP2-H (dashed line, Mw 82413) MALDI-TOF-MS bound to Adh-SFF-Cl using sinapinic acid as the matrix. It is a figure which shows the property of the cell treated with Adh-SFF-Cl and MMP2-H. (a) Immunostaining of MMP2 after modifying the cell surface under the following conditions. In serum-free DMEM containing 1% (v / v) DMSO, [MMP2-H] = 7.5 μM, [Adh-SFF-Cl or Adh-SFF] = 50 μM. (b) Anoikis conditions: Cell viability under 0.5% (v / v) methylcellulose-containing serum-free DMEM for 3 days, Adh-SFF-Cl (50 μM), MMP2-H (7.5 μM). Cell viability was measured by WST-8 assay and normalized to cells treated with Adh-SFF-Cl (50 μM). (c) Cell infiltration activity under each condition, in serum-free DMEM [Adh-SFF-Cl] = 50 μM, [MMP2-H] = 5, 7.5, 10 μM (n = 2, mean value). ^* Indicates a significant difference in student t-test. p <0.05. It is a figure which shows the functionalization of the cell surface by MMP2-H using the Adh-SFF-Cl / fusion protein system. (a) The structural image of MMP2-H is shown. (b) It is a conceptual diagram of the modification of the cell surface by MMP2-H using Adh-SFF-Cl. (c) Cytoselect-kit (Cosmo-Bio) infiltration assay diagram. After adding the cells to the upper well (serum-free DMEM), the cells pass through the extracellular matrix (ECM) coated membrane and migrate to the lower well (10% (v / v) FBS-containing DMEM). (d) Photographs (4x) of infiltrating cells stained with crystal violet after incubation for 24 hours. The inset shows the entire membrane surface. (e) After 24-hour treatment ([Adh-SFF or Adh-SFF-Cl] = 100 μM, [Marimastat (MMP2 inhibitor)] = 0.2 μM, [MMP2-H] = 7.5 μM), wash with PBS. , Infiltrated cells were quantified. After staining with crystal violet, infiltrating cells were lysed and the absorbance at 570 nm was measured. It is a figure which shows the cell graft (skin-muscle infiltration experiment) of NIH3T3 cell expressing luciferase into ICR mouse. (a) A protocol for skin-muscle infiltration experiments. (b) Percentage of transplanted cells as cell viability in skin area 24 hours after subcutaneous injection of NIH3T3 / luc cells pretreated with Adh-SFF-Cl (100 μM) and MMP2-H (15 μM), n = 4-8. (c) Percentage of transplanted cells in muscle area 24 hours after subcutaneous injection of NIH3T3 / luc cells pretreated with Adh-SFF-Cl (100 μM) and MMP2-H (15 μM). Injections contained 3.3x10 ⁵ cells / 200 μl HBSS / site and were calculated as an average of n = 4-8. ^* Statistical significance was evaluated by Dunnett's multiple comparison test after one-way ANOVA. It is a graph which shows the optimization of the Adh-SFF-Cl concentration for the cell viability in an in vivo experiment. Shows the appropriate concentration of Adh-SFF-Cl to maintain cell viability ^{of injected cells (5.0 x 10 5} cells / 200 μl HBSS / site, n = 6) in the skin area 24 hours after subcutaneous injection.

Hereinafter, the present invention will be described in detail.
Dispirotripirazine derivative or salt thereof The salt of the compound represented by the above formulas (I), (II), (III) or (IV) is two monovalent anions or two divalent anions with respect to one molecule of the compound. Means a salt with one molecule of anion. Specific examples of such salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, and perchlorate, oxalate, malonate, and succinate. Organics such as maleate, fumarate, lactate, malate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethanesulfonate, etc. Examples thereof include acid salts and acidic amino acid salts such as glutamate and asparaginate.

More specifically, each group represented by the above formulas (I), (II), (III) or (IV) is as follows.

Specific examples of the "6-membered aromatic heterocyclic group containing 1 to 3 nitrogen atoms" include pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl and triazinyl (particularly 1,3,5-triazinyl). Since the compound of the formula (I) or (III) has a high anoikis inhibitory activity, it is preferably pyrimidinyl.

"Halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Since the compound of the formula (I) or (III) has a high anoikis inhibitory activity, it is preferably a chlorine atom.

The "alkyl" may be linear or branched, and may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, heptyl. , Octyl, nonyl and decyl. Due to the high anoikis inhibitory activity of the compound of formula (I) or (III), it is preferably C _1-10 alkyl, more preferably C _1-6 alkyl, and even more preferably C _1-4 alkyl. ..

"Hydroxyalkyl" means a group in which the alkyl in the above definition is substituted with hydroxy. For example, hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxyisopropyl, hydroxy-n-butyl, hydroxyisobutyl, hydroxy-tert-butyl, hydroxy-n-pentyl, hydroxyisopentyl and hydroxyhexyl can be mentioned. Due to the high anoikis inhibitory activity of the compound of formula (I) or (III), it is preferably hydroxy-C _1-10 alkyl, more preferably hydroxy-C _1-6 alkyl, and even more preferably hydroxy-C. It is _{1-4 alkyl.}

The "alkoxy" may be linear or branched, and examples thereof include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy and hexyloxy. .. Since the compound of the formula (I) or (III) has a high anoikis inhibitory activity, it is preferably C _1-10 alkoxy, more preferably C _1-6 alkoxy, and even more preferably C _1-4 alkoxy. ..

The "alkylthio" may be linear or branched, and may be, for example, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio. And hexylthio. Since the compound of formula (I) or (III) has high anoikis inhibitory activity, it is preferably C _1-10 alkyl thio, more preferably C _1-6 alkyl thio, and even more preferably C _1-4 alkyl thio. ..

Ring A ¹ and ring A ² are the same or different and are 6-membered aromatic heterocyclic groups containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic groups are halogen atoms, hydroxy, alkyl, and. It may be substituted with one or two substituents selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino. Due to the high anoikis inhibitory activity of the compounds of formula (I) or (III), the 6-membered aromatic heterocyclic group is preferably substituted with one or two substituents selected from halogen atoms and alkylthios. May be.

The "amino acid residue" is a divalent amino acid obtained by removing one hydroxy group from the carboxyl group bonded to the α carbon of the 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, but is preferably L-form because high anoikis inhibitory activity is expected and amino acids are easily available. In the present specification, amino acid residues and peptides are described with the N-terminal on the left and the C-terminal on the right according to a conventional method, unless otherwise specified.

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, and n When is 2, each X ¹ may be the same or different. ^{X 1} is preferably an amino acid residue selected from Ser, Ala and Gly because it has no reactive functional groups and is easy to synthesize peptides. Since the compound of the formula (I) or (III) has a high anoikis inhibitory activity, n is preferably 0 or 1.

Each X ² is the same or different amino acid residue selected from Phe, Tyr and Trp. ^{Each X 2} is preferably Phe because of the high anoikis inhibitory activity of the compounds of formula (I) or (III).

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, p. When is 2 or 3, each X ³ may be the same or different. No reactive functional group, since the synthesis of peptides is easy, X ³ is preferably an amino acid residue selected Lys, from Gly and Ser. Since the compound of formula (I) or (III) has high anoikis inhibitory activity, p is preferably 0 or 1.

^{In the definition of R 2 in} formula (I) or (II), "-NH ₂ " indicates that the C-terminal carboxyl group is amidated.

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.

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.

The linker or first linker represented by L, as long as capable of binding a ring A ² and amino acid residues not specifically limited, but the use of a variety of lengths and structures You can, but 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.

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 the "ligand-binding protein" and the "ligand that specifically binds to the ligand-binding protein" may be a protein having a predetermined domain structure and a low-molecular-weight compound that binds to the predetermined domain structure.

Examples of the "ligand-binding protein" include HaloTag (registered trademark) protein (Promega Corporation), SNAP-tag protein (Covalys), CLIP-tag protein (Covalys) and the like. In the present invention, HaloTag protein is preferably used from the viewpoint of tag size and reaction rate.

Examples of the “ligand that specifically binds to the ligand-binding protein” represented by Y include the binding site of the HaloTag ligand (Promega Corporation) that specifically binds to the HaloTag protein, and the SNAP-tag protein (Covalys). Examples thereof include a ligand that specifically binds to CLIP-tag protein (Covalys) and the like. A specific example of Y is the binding site of a HaloTag ligand that specifically binds to the 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.

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", and has various lengths and. Structural ones can be used, for example
-CO- (CH ₂ ) _q- CO- (q is 1, 2, 3 or 4),
-CO-CH (R ⁴ ) -NH-CO- (CH ₂ ) _q -CO- (R ⁴ is the side chain of an amino acid and q is 1, 2, 3 or 4) and the like. Specific examples of L ² include -CO-CH ((CH ₂ ) ₄ NH ₂ ) -NH-CO- (CH ₂ ) ₂ -CO- and the like.

Preferable examples of the compound represented by the formula (I) or a salt thereof include the following compounds or salts thereof.

[In the formula, R ¹ and R ² are as defined above. ]

Suitable specific examples of the compound represented by the formula (I) or a salt thereof include the following compounds or salts thereof.

In formulas (a) to (j), the amino acid residue may be either L-form or D-form, but is preferably L-form because amino acids are easily available.

Preferable examples of the compound represented by the formula (III) or a salt thereof include the following compounds or salts thereof.

[In the formula, R ¹ and R ³ are as defined above. ]

Suitable specific examples of the compound represented by the formula (III) or a salt thereof include the following compounds or salts thereof.

In the 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.

Method for Producing Dispirotripirazine Derivative or Salt thereof The compound represented by the formula (I) or a 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). A peptide having a desired sequence synthesized by the 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.)

For example, when L is −CH = NO—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 at the N-terminal 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. By deprotecting the protecting group, the peptide represented by the formula (VI) is obtained.
Aoa- (X ¹ ) _n- (X ² ) _m- (X ³ ) _p- NH ₂ (VI)
(In the formula, Aoa is aminooxyacetyl.)
Peptides of formula (VI), by R ⁵ is reacted with a compound of the formula (V) is formyl, can be prepared a compound or a salt thereof represented by the formula (I).

The compound represented by the formula (III) or a salt thereof can be produced, for example, by the following method.
To the C-terminus of the peptide having the desired sequence was synthesized by solid-phase synthesis (e.g. Fmoc synthesis), combine the binding site Y ligand via a second linker L ^2, the resulting peptide derivative of the formula By binding to the compound (V) or a salt thereof via the first linker L, the compound represented by the formula (III) or a salt thereof can be produced.
When ^{the amino acid residue located on the C-terminal side of the amino acid residues represented by X 3} is Lys or Orn, the binding site Y of the ligand is second to the amino group of the side chain of the amino acid residue. it may be bonded via a linker L ^2.
When ^{the amino acid residue located on the C-terminal side of the amino acid residues represented by X 3} is Asp or Glu, the binding site Y of the ligand is set at the carboxyl group of the side chain of the C-terminal amino acid residue. It may be bound via the ^second linker L2.

For example, when L is −CH = NO—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)
(In the formula, Aoa is aminooxyacetyl.)
The peptide derivative of formula (VII), by R ⁵ is reacted with a compound of the formula (V) is formyl, can be prepared a compound or a salt thereof represented by the formula (III).
When ^{the amino acid residue located on the C-terminal side of the amino acid residues represented by X 3} is Lys or Orn, the binding site Y of the ligand is second to the amino group of the side chain of the amino acid residue. it may be bonded via a linker L ^2.
When ^{the amino acid residue located on the C-terminal side of the amino acid residues represented by X 3} is Asp or Glu, the binding site Y of the ligand is set at the carboxyl group of the side chain of the C-terminal amino acid residue. It may be bound via the ^second linker L2.

When the functional groups involved in the reaction in the ring A ¹ and the structure of the ring A ² is present, they leave protected according to conventional methods, the protecting group after the reaction to desorb desirable.
The compound of formula (V) or a salt thereof, which is the 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 commercially available and can be easily obtained. be able to.
The compound of formula (I) or (III) produced by the above production method or a production method similar thereto can be isolated and purified according to a conventional method such as chromatography, recrystallization, or reprecipitation. It can be derived into salts by treatment with various acids according to conventional methods. The compound of formula (I) or (III) can be obtained in the form of a salt depending on the reaction / treatment conditions and the like, but can be converted into the compound of formula (I) or (III) according to a conventional method.

Anoikis Inhibitor The present invention provides an anoikis inhibitor containing a 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 cellular anoikis. 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 cell anoikis.

The cell 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 cells may be either scaffold-dependent cells or floating cells. Here, the scaffold-dependent cells mean cells that cannot survive or proliferate unless they can adhere to the culture vessel, for example, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, epidermal cells, hepatocytes, and bones. Examples thereof include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells) and the like. Floating cells mean cells that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells. Further, examples of the cells used in the present invention 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 μM, more preferably about 50 to 150 μM.

Additives for cell culture or transplanted cells, and cell engraftment promoters The present invention contains compounds represented by the formula (I) or (III) or salts thereof, for cell culture or transplanted cells. Provide additives.

The present invention also provides a cell engraftment promoter containing a compound represented by the formula (I) or (III) or a salt thereof.

The additives and engraftment promoters of the present invention can be added to suspensions in which cells or transplanted cells are suspended.

The effective concentration of the compound represented by the formula (I) or (III) or a salt thereof in the suspension is preferably about 0.1 to 1,000 μM, more preferably about 50 to 150 μM.

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 cells may be either scaffold-dependent cells or floating cells. Here, the scaffold-dependent cells mean cells that cannot survive or proliferate unless they can adhere to the culture vessel, for example, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, epidermal cells, hepatocytes, and bones. Examples thereof include blast cells, skeletal muscle cells, embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells) and the like. Floating cells mean cells that can proliferate even in a floating state, and examples thereof include bone marrow cells, lymphoid cells, and ascites cancer cells. Further, examples of the cells used in the present invention include primary cultured cells, and the primary cultured cells may be derived from any tissue. The type of the transplanted cell is not particularly limited as long as it is a cell used for cell transplantation (cell therapy), but is preferably a corneal endothelial cell, a bone marrow-derived cell, a fibroblast, or the like.

A buffer solution, a medium, or the like can be used as the suspension in the present invention. Examples of the buffer solution include phosphate buffered saline (PBS) and Hanks solution (HBSS). As the medium, a medium suitable for the cell type may be appropriately selected and used. For example, Dalveco's modified Eagle medium (DMEM), Williams E medium, Ham's F-10 medium, F-12 medium, and RPMI. Use a conventionally known basal medium for cell culture, such as -1640 medium, MCDB153 medium, or 199 medium, to which a conventionally known growth factor or antioxidant suitable for culturing each cell is added, if necessary. Can be done. The medium may be either a serum-added medium or a serum-free medium, but a serum-free medium is preferable.

By using the additive of the present invention in a suspension of cells or transplanted cells, it is expected that 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 action, it is considered that such an effect can be obtained. Here, the cell therapy in the present specification means a therapy for treating a disease by transplanting cells.

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 cells.

The compound represented by the formula (I) or (III) or a salt thereof and cells can be administered in the form of a therapeutic composition. Here, the cells include the same cells as described above.

Therapeutic compositions are administered to mammals, including humans, and can be administered parenterally in the form of injections of transplanted cell suspensions. A buffer solution, a medium, or the like can be used as the suspension, and examples of the suspension and the medium include those described above. 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 dose of the therapeutic composition can be appropriately determined in consideration of the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, and the like.

The present invention
(A) A compound represented by the formula (III) or a salt thereof,
Provided is a method for transplanting cells, which comprises administering (b) matrix metalloproteinase 2 bound to the ligand-binding protein and (c) cells.

The present invention also
(A) A compound represented by the formula (III) or a salt thereof, and (b) a matrix metalloprotease 2 bound to the ligand-binding protein.
Provide a kit, including.

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 has cell viability by a MAPK signal. To activate.

Matrix metalloproteinase 2 (MMP2) is known to degrade extracellular matrix, especially type I and type IV collagen, and to be involved in metastasis and infiltration of cancer cells. 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, the formula (III) conjugated to MMP2 by administering (a) a compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand-binding protein, and (c) cells. Compounds form submicron particles on the cell surface, and the cell surface is modified with MMP2. The present inventors have found that cells modified with MMP2 have increased cell infiltration activity like cancer cells.

As the "ligand-binding protein", a "ligand-binding protein" corresponding to the "ligand that specifically binds to the ligand-binding protein" represented by Y in the formula (III) is used. For example, when HaloTag ligand (Promega Corporation) is used as Y, HaloTag protein (Promega Corporation) is used as the "ligand-binding protein".

MMP2 bound to the ligand-binding protein can be produced by a method known per se.

The compound represented by the formula (III) or a salt thereof, (b) MMP2 bound to the ligand-binding protein, and (c) cells can be administered in the form of a therapeutic composition. Here, the cells include the same cells as described above.

Therapeutic compositions are administered to mammals, including humans, and can be administered parenterally in the form of injections of transplanted cell suspensions. A buffer solution, a medium, or the like can be used as the suspension, and examples of the suspension and the medium include those described above. The effective concentration of the compound represented by the formula (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.

The dose of the therapeutic composition can be appropriately determined in consideration of the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, and the like.

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 and the like.

Manufacturing example 1
Synthesis of Peptide with Aminooxyacetic Acid All peptides were synthesized using RINK Amide MBHA resin LL by the usual Fmoc solid phase peptide synthesis method (SPPS). The resin (0.1 mmol) was _{swollen in CH 2} Cl ₂ for 3 hours prior to use. Deprotection of the Fmoc group was performed with 20% piperidine in N, N-dimethylformamide (DMF) (1 and 10 minutes). Consecutive amino acid couplings in the DMF / N-methyl-2-pyrrolidone (NMP) = 1/1 mixture include the corresponding Fmoc-amino acids (0.3 mmol, Watanabe Kagaku Kogyo Co., Ltd., Table 1) or [(tert -Butoxycarbonyl) aminooxy] acetic acid (0.3 mmol, Tokyo Kasei Kogyo Co., Ltd.), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) , 0.3 mmol, Watanabe Chemical Industries, Ltd.), 1-Hydroxybenzotriazole (HOBt, 0.3 mmol, Tokyo Kasei Kogyo 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 bond 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 x 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 give the crude peptide product as a white solid.

Example 1
Synthesis of Adh-SFF Adhesamine (20.3 mg, 33.8 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time Purified in 24 minutes). The eluate was lyophilized to give 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 = 4.8 Hz), 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.

Reference example 1
Synthesis of Adh-S Adhesamine (12.5 mg, 20.8 μmol) of peptide Aoa-S (2.09 mg, 10.5 μmol) in a mixture of DMF / dioxane / water / TFA = 2/1/2 / 0.0004 (2.5 ml) Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/35 → 25/95, retention time Purified in 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).

Reference example 2
Synthesis of Adh-SF Adhesamine (17.1 mg, 28.5 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time Purified in 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).

Example 2
Synthesis of Adh-SFFF Adhesamine (4.64 mg, 7.73 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time It was purified in 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).

Example 3
Synthesis of Adh-FF Adhesamine (8.21 mg, 13.7 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / 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).

Example 4
Synthesis of Adh-SSFF Adhesamine (9.30 mg, 15.5 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time Purified in 22 minutes). The eluate was lyophilized to give 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).

Reference example 3
Synthesis of Adh-SSSFF Adhesamine (8.66 mg, 14.4 μmol) was added to the 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 a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/18 → 21/48 → 25/95, retention time 21 minutes). The eluate was lyophilized to give 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).

Reference example 4
Synthesis of Adh-FSF Adhesamine (15.2 mg, 25.3 μmol) was added to the 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, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time Purified in 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).

Example 5
Synthesis of Adh-FFS Adhesamine (16.5 mg, 27.5 μmol) was added to the 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, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time 28 minutes). The eluate was lyophilized to give 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).

Reference example 5
Synthesis of peptide SFF (peptide SFF)

Acetone (30 μl, 23.6 mg, 40.6 μmol) in 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, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time It was purified in 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).

Example 6
Synthesis of Adh-SFFK Adhesamine (12.3 mg, 20.5 μmol) of peptide Aoa-SFFK (8.81 mg, 14.7 μmol) in a mixture of DMF / dioxane / water / TFA = 2/1/2 / 0.0004 (3.6 ml) Added to the solution. The solution was stirred at 55 ° C. for 24 hours. After removing the solvent under a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time Purified in 19 minutes). The eluate was lyophilized to give 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).

Reference example 6
Synthesis of Adh (SFFK) -SFFK Adhesamine (4.63 mg, 7.72 μmol) was added to the peptide Aoa-SFFK (9.64 mg, 16.1) in a mixture of DMF / dioxane / water / TFA = 2/1/2 / 0.0004 (1 ml). It was added to a solution of μmol). The solution was stirred at 55 ° C. for 20 hours. After removing the solvent under a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time Purified in 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).

Manufacturing example 3
Synthesis of compound 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 added to 2- (2-aminoethyl) ethanol (2.14). g, 20.4 mmol) _{was added to a dehydrated CH 2} Cl ₂ (30 ml) solution. The solution was stirred at room temperature for 2 hours. After removal of the solvent, water (100 ml) was added to the residue. The solution was _{extracted with CH 2} Cl ₂ (40 ml x 2) and the organic layer was dried over _{Na 2} SO _4. After distilling off the solvent, the residue was dried under reduced pressure to give compound 1 (4.59 g, quantitative) as a clear oil.
ESI-MS, found 228.00 calcd 228.12 ([M + H] ⁺ ).

(2) Synthesis of tert-butyl (2-(2-((6-chlorohexyl) oxy) ethoxy) ethyl) carbamate (2)
NaH (1.32 g, 60% in oil) was added to the solution of compound 1 (4.59 g) in a mixture of THF (28 ml) and DMF (14 ml). 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 the 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 the solvent was distilled off. The residue was purified by column chromatography (SiO ₂ , hexane / AcOEt = 4/1). The resulting solution was solvent distilled off and the residue was dried under reduced pressure to give compound 2 (2.69 g, 8.32 mmol, 41%) as a clear oil.
ESI-MS, found 346.05 calcd 346.18 ([M + H] ⁺ ). ^{1 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) Synthesis of 2- (2-((6-chlorohexyl) oxy) ethoxy) ethane-1-amine (3)
TFA (5 ml) was added to a solution of compound 2 (2.69 g) in CH ₂ Cl _{2 (5 ml).} 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 resulting solution was solvent evaporated and the residue was dried under reduced pressure to give 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) Synthesis of tert-butyl (2-((2-(2-((6-chlorohexyl) oxy) ethoxy) ethyl) amino) -2-oxoethoxy) carbamate (4)
[(tert-Butoxycarbonyl) Aminooxy] Acetate (240 mg), O- (7-azabenzotriazole-1-yl) -N, N, N', N'-Tetramethyluronium Hexafluorophosphate (HATU) ) (506 mg), and 1-hydroxy-7-azabenzotriazole (HOAt) (163 mg) in DMF (5 ml), compound 3 (248 mg) and DIPEA (700 μl) in DMF (1 ml). Added to the solution. The mixture was stirred at room temperature for 2 hours. After adding AcOEt (70 ml), the organic layer was washed with LVDS ₃ solution, water and saturated saline. The organic layer was _{dried over Na 2} SO ₄ and the solvent was distilled off. The residue was purified by column chromatography (SiO ₂ , hexane / AcOEt = 1/2). The resulting solution was solvent distilled off and the residue was dried under reduced pressure to give compound 4 (144 mg, 364 μmol, 29%) as a pale yellow oil.
ESI-MS, found 419.15 calcd 419.19 ([M + H] ⁺ ). ^{1 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) Synthesis of 2- (aminooxy) -N- (2- (2-((6-chlorohexyl) oxy) ethoxy) ethyl) acetamide (5)
TFA (1 ml) was added to a solution of compound 4 (144 mg) in 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 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) 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 dehydrated CH 2} Cl ₂ (60 ml) solution. 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 resulting solution was solvent distilled off and the residue was dried under reduced pressure to give 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).

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, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/45 → 25/95, retention time It was purified in 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).

Reference example 8
Synthesis of Adh (Cl) -SFF
Adh-SFF (5.13 mg, 4.87 μmol) was added to a solution of compound 5 (2.40 mg, 4.98 μmol) in a mixture (1.8 ml) of DMF / dioxane / water / TFA = 2/1/2 / 0.0004. The solution was stirred at 55 ° C. for 21 hours. After removing the solvent under a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / 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)

Example 7
(1) Synthesis of Aoa-SFFK (alkyl chloride-K-) peptide

Peptides were similarly synthesized using RINK Amide MBHA resin LL by the usual Fmoc solid phase peptide synthesis method (SPPS). After condensation of [(tert-butoxycarbonyl) aminooxy] acetic acid, the resin is mixed with NH ₂ OH · HCl (1.25 g) and imidazole (918 mg) in NMP (5 ml) and CH ₂ Cl ₂ (1 ml) solution. 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 x 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 give a crude product of the Aoa-SFFK (alkyl chloride-K-) peptide (91.5 mg) as a white solid.
ESI-MS, found 1033.40 calcd 1033.55 ([M + H] ⁺ ).

(2) Synthesis of Adh-SFF-Cl

Adhesamine (7.94 mg, 13.2 μmol) in 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. ) Was added to the solution. The solution was stirred at 51 ° C. for 22 hours. After removing the solvent under a nitrogen stream, leave the residue on HPLC (ODS-A, 30x150 mm, gradient time (minutes) / B (%) = 0/15 → 1/15 → 21/55 → 25/95, retention time Purified in 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, br) 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, 16 0.6, 164.3, 168.5, 169.7, 170.2, 170.6, 170.7, 171.3, 171.5, 171.6, 173.2, 173.3, 186.5.

The compounds of Examples 8 to 13 were produced in the same manner as in Examples 1 to 6.
Example 8: Adh-AFF
Example 9: Adh-GFF
Example 10: Adh-SYY
Example 11: Adh-SWW
Example 12: Adh-SFFGK
Example 13: Adh-SFFGGK

Test Example 1
Affinity for heparin and cell viability The affinity of the adhesamine derivatives prepared in Examples and References for heparin 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 did not exhibit affinity for heparin (FIG. 2a). Adh-SFF maintains the high selectivity of adhesamine for heparin and has no 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.

Next, the WST-8 assay was used to investigate whether treatment with adhesamine derivatives affected cell survival (%) (FIGS. 1e and 3). FIG. 1e is a graph showing the cell viability of NIH3T3 cells incubated in 1% (v / v) DMSO-containing serum-free DMEM supplemented with each adhesamine derivative (100 μM) for 3 days. Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF. The error bar is the standard deviation (n = 3).

Cells treated with Adh-SFF maintained an unexpectedly high cell viability compared to adhesamine after culturing for 3 days. Treatment with peptide SFF alone, or a mixture of adhesamine and peptide SFF, shows a negligible increase in cell viability (Fig. 3a), with the conjugate between adhesamine and the SFF peptide increasing activity. Suggests that it is essential for. The EC50 that increased the cell viability of Adh-SFF was 38 μM (Fig. 3b). Figure 3a shows 1% (v / v) DMSO-containing serum-free with adhesamine (100 μM), peptide SFF (100 μM), adhesamine and peptide SFF mixture (100 μM each) or Adh-SFF (100 μM). It is a graph which shows the cell viability of the NIH3T3 cell which was incubated in DMEM for 3 days. FIG. 3b is a graph showing the concentration effect of Adh-SFF (0-300 μM). Cell viability was calculated by WST-8 assay and normalized to cells treated with Adh-SFF (100 μM).

Next, in order to examine the structural effect 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 the SFF sequence was most suitable for increasing cell viability, but so did Adh-FF, Adh-FSF, and Adh-FFS. Affinity for heparin was also confirmed (Table 3). The plot data shown in FIG. 4 show a good correlation (r = 0.838) between affinity for heparin (apparent binding constant Ka) and increased cell viability, with higher affinity (Ka> 3 x 10). ^{It can be said that 7} M ^-1 ) effectively enhances cell viability. Adh-RGDS, a conjugate of adhesamine and the integrin ligand peptide RGDS, also enhances cell viability but has low affinity for heparin (Table 3). This difference is thought to be due to the difference in the mechanism of the two adhesamine derivatives that Adh-RGDS depends on the integrin-RGDS bond and Adh-SFF acts by self-assembly on heparan sulfate.

Test Example 2
Self-assembly structure of adhesamine on sulfated GAG
We have previously shown that adhesamine coordinates with heparan sulfate to form a self-assembling structure by π-π stacking of the pyrimidine moiety. It is predicted that the FF peptide is located in the immediate vicinity of the pyrimidine of adhesamine and forms a strong self-assembling structure by π-π stacking between aromatic groups. The NOESY spectrum of Adh-SFF in heparin-containing solution was measured to study the binding structure. The results revealed that the pyrimidine moiety of Adh-SFF was close not only to the close pyrimidine but also to the phenyl group. The CD spectrum of Adh-SFF also shows that when complexed with heparin, the absorbance at 220 nm decreases and increases near 200 nm, suggesting that a β-turn-like structure is formed. .. These results suggest that the interaction of self-assemblies collectively induces β-turn-like structures when bound to heparan sulfate.

In addition, the self-assembly structure was studied by dynamic light scattering (DLS) and electron microscopy (EM). DLS measurements revealed that heparin-bound Adh-SFF formed submicron particles (600-1200 nm in diameter) (Fig. 6a). None of the combination of Adh-SFF and 4 μM chondroitin sulfate A or the mixture of adhesamine (100 μM) and heparin (4 μM) formed particles (data not shown). Then, using cryo-electron microscopy (Cryo-TEM), we observed that Adh-SFF (100 μM) and heparin (1 μM) aggregated in the aqueous solution (Fig. 6b) and Adh-SFF (300 μM). ) Or heparin (3 μM) alone, no submicron particles were present (Fig. 5). In addition, a field emission scanning electron microscope (FE-SEM) was used to observe the Adh-SFF complex on the cell surface (Fig. 6c), revealing many particles on the cell. These results suggest that Adh-SFF binds to heparan sulfate on the cell surface and coats the cell surface with aggregated particles (Fig. 6i).

Test Example 3
Intracellular signaling for cell survival induced by Adh-SFF According to our previous studies, adhesamine is modified with syndecane 4 (SDC4) (heparan sulfate, which is known to be involved in cell adhesion). It was revealed that the biological activity was exhibited by inducing the clustering of Cindecane). Immunostaining with anti-SDC4 antibody shows that Adh-SFF treatment induces SDC4 clustering as well as adhesamine (Fig. 6d). Samples treated with Adh-SFF show more larger (> 1.15 μm) SDC4 clusters compared to the SDC4 clusters observed in samples treated with adhesamine (Fig. 6e), with Adh-SFF compared to adhesamine. It suggests that it induces stronger SDC4 clustering.

Oligomerization and clustering of SDC4 is known to activate PKCα by rearrangement of PKCα-binding molecules such as phosphatidylserine and diacylglycerol. Immunostaining with anti-PKCα antibody showed that Adh-SFF treatment rapidly induced the transfer of PKCα from the cytosol to the membrane (Fig. 6f). In time, SDC4 clustering precedes 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. The bottom row shows 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) superposed fluorescent stained images are shown.

MAPK activity is regulated by PKCα activity. To identify specific signaling pathways for Adh-SFF-induced cell survival, we monitored MAPK phosphorylation levels after Adh-SFF treatment in Western blot chronological experiments (Figure). 6 g, h). Adh-SFF treatment gradually amplified ERK phosphorylation, but inhibited JNK phosphorylation compared to DMSO or adhesamine treatment (Fig. 6 g, h). Note that MAPK phosphorylation is regulated in a manner similar to cells cultured under normal culture conditions (cultured on a standard plastic culturaing plate on a 10% (v / v) FBS, referred to as SC). Should be. On the other hand, there was no significant difference in p38 MAPK phosphorylation levels between treatments. In general, ERK phosphorylation induces signal transduction of cell proliferation and cell survival, whereas JNK phosphorylation aids in cell apoptosis. These results suggest that Adh-SFF treatment regulates ERK and JNK phosphorylation levels and activates cell survival by the PKCα pathway.

Furthermore, we confirmed that Adh-SFF treatment did not make a significant difference in Akt phosphorylation, another cell survival signaling 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 increase 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 adhere cells to the dish.

Inhibition of ERK signaling was then tested using UO126, a selective inhibitor of MEK, the upstream kinase of ERK. In addition, UCN01, which is a selective PKCα inhibitor, and tricilibine, 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 reduced the cell viability produced by Adh-SFF treatment. On the other hand, tricilibine did not significantly inhibit cell viability. These results provide evidence of the mechanism by which Adh-SFF enhances cell viability primarily by MAPK regulation, as shown in FIG. 6i.

Test Example 4
Modification of Cell Surface of Transplanted Cells We have found self-assembly of Adh-SFF by binding to GAGs 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 infiltrating properties during the metastatic process, allowing them to penetrate and engraft at remote locations in the body. It has been reported that non-invasive NIH3T3 acquires infiltration activity by highly expressing active MMP2 after transformation with Tgat (trio-related transforming gene) (Biochemical and Biophysical Research Communications 355, 937). -943 (2007)). MMP2 degrades extracellular matrix (ECM), especially type I and type IV collagen, paving the way for cell migration. In addition, the degraded ECM releases growth factors, adhesive 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 technique most suitable for the object of the present invention was investigated.

A group of Adh-SFF alkyl chloride conjugates was synthesized to find derivatives that best maintain cell viability and heparin binding. Derivative conjugates (Adh (SFFK) -SFFK) with two SFFK peptides, one for each aldehyde of adhesamine, showed a slight increase in cell viability compared to negative controls (FIGS. 8b, d). ). In contrast, the derivative (Adh-SFFK) conjugated with only one SFFK peptide maintained the property of increasing the cell viability of Adh-SFF (FIGS. 8a, 8c). The controls Adh-Cl and Adh (Cl) -SFF showed a negligible slight increase in cell viability. From these results, Adh-SFFK was selected for modification of the cell surface. Furthermore, by modifying Adh-SFFK with an alkyl chloride, Adh-SFF-Cl was obtained that maintained the affinity of the parent compound Adh-SFF for heparin and the increased cell viability (FIGS. 9a and 9a, FIG. 10a).

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 11).
The gene encoding eGFP is a primer from pHTN HaloTag® CMV-neo Vector (Promega) 5'-GTC GGG ATC CGA ATT CGG TGG TAG TGG TGG TAG TAT GGC AGA AAT CGG TAC TGG -3'(SEQ ID NO: 1) It was obtained by PCR amplification using / 5'-GAC GGA GCT CGA ATT GTT ATC GCT CTG AAA GTA CAG -3'(SEQ ID NO: 2). Next, a pET28b vector was prepared by inserting eGFP-encoding DNA into the restriction enzyme site Nco I / EcoR I of the pET28b vector encoding Halo-Tag.
eGFP was introduced at the N-terminus of the Halo-Tag protein via the GGSGGS linker (FIGS. 11a and 11b). 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 in eGFP-H (Fig. 11d, e, f).

Observations 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 ( 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 treatment with Adh-SFF and eGFP-H did not produce a detectable fluorescent signal (Fig. 10d). The eGFP fluorescent signal observed by immunostaining overlapped with the SDC4 clusters induced by Adh-SFF-Cl treatment (Fig. 12c). We also investigated the effect of the ratio of eGFP-H on cell surface modification Adh-SFF-Cl (50 μM). The best results were 7.5 μM eGFP-H. This system can be highly diversified as it can be used to modify cells with selected proteins simply by designing a Halo-Tag fusion protein and treating the cells with Adh-SFF-Cl.

The Adh-SFF-Cl / Halo-Tag cell surface modification system was applied to modify the cell surface with matrix metalloproteinase 2 (MMP2).
The design of the Halo-Tag fusion protein (MMP2-H) for MMP2 is shown in FIGS. 13a and 13b.

Using this system, cell multifunctionality: that is, the increase in cell viability and cell infiltration activity by Adh-SFF-Cl and MMP2-H were evaluated. Maintained cell viability was confirmed by measuring NADPH dehydrogenase activity (FIG. 14b) and cell infiltration activity was measured using the Cytoselect CBA-110 cell Invasion assay kit (FIGS. 14c, 15d, e). The kit measures cells that can migrate from the upper well through the ECM-coated membrane to the lower well, thereby measuring the infiltration activity of the cells (FIG. 15c). Treatment with Adh-SFF-Cl and MMP2-H significantly increased cell infiltration activity, but treatment with Adh-SFF-Cl or MMP2-H alone did not (FIGS. 14c, 15d, e). Similarly, treatment with Adh-SFF and MMP2-H did not enhance cell infiltration activity. This indicates that a conjugate by Halo-Tag technique is required for the increased infiltration activity to be observed. The finding that infiltration activity was inhibited by the well-known MMP2 and MMP9 inhibitor marimastat (200 nM) correlates infiltration activity with MMP2 action. These data suggest that the conjugate of Adh-SFF-Cl and MMP2-H is a novel modification of the cell surface to enhance cell viability and cell infiltration activity.

Test Example 5
Skin-muscle infiltration experiment (in vivo experiment)
Suspended cells usually have low engraftment ability to target tissues and are a major obstacle to regenerative medicine. The system of the present invention can solve this problem by not only increasing the viability of suspended cells, but also imparting the ability of suspended cells to migrate to surrounding tissues. To demonstrate this, a skin-muscle infiltration experiment was performed. NIH3T3 cells (NIH3T3 / luc) that stably express firefly luciferase were subcutaneously injected into ICR mice, and after 24 hours, surviving cells present in the skin and muscle areas were quantified (Fig. 16a).

(1) Experimental animals Male ICR mice 5 to 6 weeks old 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 studies were performed according to the principles and procedures outlined in the National Institutes of Health Guidelines for the Management and Use of Laboratory Animals.

(2) Cell culture for in vivo experiments
NIH3T3 cells were cultured in DMEM containing 10% (v / v) FBS at 37 ° C. In order to construct firefly luciferase-expressing NIH3T3 (NIH3T3 / luc) cells, a pEB Multi-Hyg plasmid vector (Wako Pure Chemical Industries, Ltd.) encoding firefly luciferase was prepared as follows. Firefly luciferase cDNA fragments were 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 and reverse primers 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: 7), respectively. SEQ ID NO: 8). The amplified PCR product and pEB Multi-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 integrated into the pEBMulti-Hyg vector to obtain a pEBMulti-luc plasmid. NIH3T3 cells were then transfected with this plasmid to give NIH3T3 / luc cells. These cells were cultured _{under humidified air containing 5% CO 2} in DMEM containing 10% (v / v) FBS and 500 μg / mL HygroGold (InvivoGen) at 37 ° C.

(3) Cell survival after transplantation
We confirmed the concentration effect of Adh-SFF-Cl in the skin area to effectively maintain cell viability.
The back coat of ICR mice was removed using an electric clipper and depilatory cream under isoflurane aspiration 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 subcutaneously administered to two sites on the back of mice (5'10 ⁵ cells / site). Twenty-four hours later, mice transplanted with NIH3T3 / luc cells were euthanized by isoflurane aspiration to collect the site of cell transplantation. Tissue samples were shredded, homogenized in cytolysis, and centrifuged at 10000 g at 4 ° C for 10 minutes. The luciferase activity of the supernatant of the sample was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Beanet Co., Ltd.). Cell viability was calculated as the percentage of remaining cells relative to the administered cells. Optimal concentration (100 μM) was applied in the skin-muscle infiltration assay (Fig. 17).

(4) Skin-Muscle Infiltration Assay As described above, the back hair of the mouse was removed under anesthesia. Then 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 for activation of MMP2-H. NIH3T3 / luc cells (5'10 ⁵ cells) were incubated with activated Adh-SFF-Cl-MMP2 conjugates at 37 ° C. for 20 minutes. These cells were subcutaneously administered to two sites on the back of the mouse (3.3'10 ⁵ cells / site). Twenty-four hours later, mice transplanted with NIH3T3 / luc cells were euthanized by isoflurane aspiration, the site of cell transplantation was collected, and skin and muscle were separated. Muscle samples were chopped, homogenized in cytolysis, and centrifuged at 10000 g at 4 ° C for 10 minutes. The luciferase activity of the supernatant of the sample was measured with 2104 EnVision Multilabel Plate Readers (PerkinElmer) using PicaGene (Toyo Beanet Co., Ltd.). Cell viability was calculated as the percentage of remaining cells relative to the administered cells.

(5) Result
When cells pretreated with a mixture of Adh-SFF-Cl and MMP2-H were injected subcutaneously, a subpopulation of the injected cells survived in the skin area, some of which migrated to nearby muscle areas. On the other hand, no infiltration was observed in the pretreatment with Adh-SFF-Cl alone or MMP2-H alone (FIGS. 16b, c). 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.

The present invention provides a new strategy for equipping cells with selective metastasis (cell viability and cell infiltration activity). The compound represented by the formula (III) of the present invention (for example, Adh-SFF-Cl) forms a submicron self-assembling structure on the cell to increase the cell viability, and the cell surface is modified with MMP2. Functionalize into cells with enhanced capacity for infiltration and transplantation. The present invention provides a cell surface modification system that maintains cell viability after cell transplantation. The compounds of the present invention, annoix inhibitors, additives for cell culture or transplanted cells, cell engraftment promoters, cell transplantation methods, and kits can be useful in the fields of cell transplantation and regenerative medicine.

Claims (14)

A compound represented by the following formula (I) or a salt thereof.

[During the ceremony,
Ring A ¹ and ring A ² are the same or different and are 6-membered aromatic heterocyclic groups containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic groups are halogen atoms, hydroxy, alkyl, and. It may be substituted with one or two substituents selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino.
R ¹ is formyl or hydroxymethyl and
R ² is −L − (X ¹ ) _n − (X ² ) _m − (X ³ ) _p −NH ₂
(During the ceremony,
L is a linker and
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, and n When is 2, each X ¹ may be the same or different.
m is 2 or 3
Each X ² is the same or different 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, p. When is 2 or 3, each X ³ may be the same or different. )
It is a group represented by. ] The compound according to claim 1, which is a compound represented by the following formula (II), or a salt thereof.

[In the formula, R ¹ and R ² are as defined in claim 1. ] The compound according to claim 1 or a salt thereof, which is selected from the following group.

as well as

A compound represented by the following formula (III) or a salt thereof.

[During the ceremony,
Ring A ¹ and ring A ² are the same or different and are 6-membered aromatic heterocyclic groups containing 1 to 3 nitrogen atoms, and the 6-membered aromatic heterocyclic groups are halogen atoms, hydroxy, alkyl, and. It may be substituted with one or two substituents selected from hydroxyalkyl, alkoxy, alkylthio, cyano, nitro and amino.
R ¹ is formyl or hydroxymethyl and
R ³ is −L − (X ¹ ) _n − (X ² ) _m − (X ³ ) _p −L ² −Y
(During the ceremony,
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, and n When is 2, each X ¹ may be the same or different.
m is 2 or 3
Each X ² is the same or different 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, p. When is 2 or 3, each X ³ may be the same or different L ² is the second linker.
Y is a ligand that specifically binds to a ligand-binding protein. )
It is a group represented by. ] The compound according to claim 4 or a salt thereof, which is represented by the following formula (IV).

[In the formula, R ¹ and R ³ are as defined in claim 4. ] The compound according to claim 4 or 5, or a salt thereof, wherein Y is −NH (CH ₂ ) ₂ O (CH ₂ ) ₂ O (CH ₂ ) _{6 Cl.} The compound according to claim 4 or a salt thereof, which is represented by the following formula (k).

An anoikis inhibitor containing the compound according to any one of claims 1 to 7 or a salt thereof. An additive for cell culture or transplanted cells, which comprises the compound according to any one of claims 1 to 7 or a salt thereof. A cell engraftment promoter containing the compound according to any one of claims 1 to 7 or a salt thereof. A method for transplanting cells, which comprises administering the compound according to any one of claims 1 to 7 or a salt thereof and cells. (A) The compound according to any one of claims 4 to 7 or a salt thereof, and (b) a matrix metalloprotease 2 bound to the ligand-binding protein.
Including, kit. The kit according to claim 12, which is a kit for cell transplantation. (A) The compound according to any one of claims 4 to 7 or a salt thereof,
(B) A method for transplanting cells, which comprises administering matrix metalloproteinase 2 bound to the ligand-binding protein and (c) cells.

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