JP2013035817A - Mucosal immune regulating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method with which TGDK (Tetra-galloyl-D-Lys(K)) is synthesized via a liquid phase synthesis method, and to examine a new biological action of TGDK.SOLUTION: A new liquid phase synthesis method of N,N-bis[N,N-bis(3,4,5-trihydroxybenzoyl)-L-lysyl]-N-(2-aminoethyl)-L-lysine amide (TGDK), and a mucosal immune regulating agent including the compound are disclosed.

Description

The present invention relates to N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide (TGDK ) Novel liquid phase synthesis method and a mucosal immunity regulator comprising the above compound.

  The mucosal immune system is the largest immune device in the body. The mucosal immunity of the intestinal mucosal immune system (GALT) and nasal mucosal system (NALT) has significant features. The former discriminates and discriminates between safe foods such as food and pathogenic bacteria such as pathogenic bacteria, food is taken in, and pathogenic bacteria are excluded. The latter nasal cavity immune system is essential for removing pathogenic microorganisms such as viruses that invade by inhalation. M cells are present in each epithelial tissue as cells that sense foreign substances such as pathogenic bacteria in these mucosal immune systems. Antigens (pathogenic bacteria, etc.) that invade each mucosal tissue are transcytosed (transferred) to the mucosal lamina propria downstream via M cells present in each mucosal epithelial tissue, and the mucosal innate immune system It plays an extremely important role in the defense of the body. Therefore, it is possible to prevent or treat diseases by efficiently and specifically sending substances involved in biological defense into the M cells.

  The present inventor has reported TGDK (Tetra-galloyl-D-Lys (K)) as a substance that specifically targets this M cell (Non-patent Document 1). Non-Patent Document 1 describes a method of synthesizing TGDK by a solid phase method using oligo D-Lys-resin as a starting material.

Patent Document 1 also discloses Formula 1: TGDK-CH ₂ —CH ₂ —NH—R (where TGDK-CH ₂ —CH ₂ —NH— is 2- [N-α, N-ε-bis ( N-α, N-ε-digalloyllysinyl) lysinyl] aminoethylamino group, R is a hydrogen atom; a group having an active ester group via a peptide bond; a group binding to an SH group via a peptide bond; Peptide, protein, lipid or sugar bound via a peptide: or a peptide, protein, lipid or sugar bound via a Schiff base) is described. Patent Document 1 describes that the above TGDK identifies M cells.

International Publication No. WO2007 / 052641 (International Application No. PCT / JP2006 / 321720)

Misumi et al., Journal of Immunology, 2009, 182, 6061-6070

  The solid phase synthesis of TGDK starts with binding of D-Lys3 molecules to the resin according to the solid phase synthesis method of the peptide. It is a heterogeneous reaction between liquid and solid, yield is extremely low, and the reagent is expensive. There was a problem. The problem to be solved by the present invention is to provide a method for synthesizing TGDK by a liquid phase synthesis method using D-Lys as a starting material. Another problem to be solved by the present invention is to investigate a new biological action of TGDK, find a new action mainly on the innate immune system, and provide a new mucosal immunity regulator.

  As a result of intensive studies to solve the above problems, the present inventor has developed an industrially possible liquid phase synthesis method for TGDK and succeeded in synthesizing TGDK in large quantities. In addition to the function as a conventional mucosal M cell targeting agent, the present inventors have also shown that natural killer cells (NK cells) and natural killer T cells (NKT cells) involved in the bridge between innate immunity and acquired immunity. ) And control of mucosal immunity by acting on regulatory T cells that suppress immune system runaway and excessive reactions. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
[1] N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) from D-lysine methyl ester dihydrochloride comprising the following steps (1) to (6) ) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide.
(1) By reacting D-lysine methyl ester dihydrochloride in the presence of 3,4,5-trimethoxybenzoyl chloride, N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) Producing L-lysine methyl ester;
(2) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine methyl ester obtained in step (1) is treated with alkali and methanol to give N ² , N ⁶ -bis Producing (3,4,5-trimethoxybenzoyl) -L-lysine;
(3) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine obtained in step (2) is replaced with N, N′-dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBT) is reacted in the presence of D-lysine methyl ester and N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester;
(4) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester obtained in step (3) is treated with alkali was treated with methanol, N ^2, N ⁶ - bis [N ^2, N ⁶ - bis (3,4,5-trimethoxybenzoyl) -L- lysyl] process for manufacturing -L- lysine;
(5) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine obtained in step (4) is treated with 2- [ 1H-benzotriazol-1-yl] -1,1,3,3-tetramethyluronium hexafluorophosphate and reaction in the presence of ethylenediamine to give N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide; and (6) N ² , N ⁶ obtained in step (5) -Bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide is removed in the presence of boron tribromide Methylation produces N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide Process.

[2] N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide (TGDK) Or a mucosal immunity regulator comprising, as an active ingredient, a compound having a peptide, protein, lipid or sugar bound thereto.
[3] The mucosal immunity regulator according to [2], which is a mucosal immunity regulator for activating immune cells expressing CD161.
[4] The mucosal immune regulator according to [3], wherein the immune cells expressing CD161 are natural killer cells (NK cells) or natural killer T cells (NKT cells).
[5] Activates floating natural killer cells (NK cells) or natural killer T cells (NKT cells) and suppresses adherent natural killer cells (NK cells) or natural killer T cells (NKT cells) to mucosa The mucosal immunity regulator according to any one of [2] to [4], which controls immunity.

Furthermore, according to the present invention, N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl]-N for the production of mucosal immune regulators There is provided use of-(2-aminoethyl) -L-lysine amide (TGDK) or a compound to which a peptide, protein, lipid or sugar is bound.

Furthermore, according to the present invention, N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L- There is provided a method of controlling mucosal immunity comprising administering lysine amide (TGDK), or a compound having a peptide, protein, lipid or sugar bound thereto, to a mammal including a human.

The liquid phase synthesis method of TGDK according to the present invention is a homogeneous reaction, and the yield is about three times that of the solid phase method (molar yield: solid phase method, 2.6%; liquid phase method, 8.5%). Solid phase method, 1 million yen / 100 mg; liquid phase method, 10,000 yen / 100 mg) is also inexpensive. In addition, the maximum synthesis amount per solid phase method is 10 mg, which takes one month, whereas in liquid phase synthesis, 1000 mg synthesis can be achieved in one week.
Further, according to the present invention, TGDK has been found to act on regulatory T cells that increase the number of natural killer cells and natural killer T cells in the innate immune system and suppress excessive reactions of immune responses. It has become possible to provide a novel mucosal immunity regulator containing.

FIG. 1 shows an MS analysis chart of TGDK. FIG. 2 shows the effect of TGDK on floating bone marrow lymphoid cells. FIG. 3 shows the results of FACS analysis using surface markers of CD16, CD56, CD161 of NK cells and CD3, CD16, CD56, CD161 of NKT cells. FIG. 4 shows the results of FACS analysis of the effect of TGDK on adherent bone marrow lymphoid cells. FIG. 5 shows the results of examining the effect of TGDK on peripheral lymphocyte cells by FACS analysis.

Hereinafter, the present invention will be described in more detail.
According to the present invention, N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl)-L -lysyl]-N- (2- The method for synthesizing (aminoethyl) -L-lysineamide includes the following steps (1) to (6).
(1) By reacting D-lysine methyl ester dihydrochloride in the presence of 3,4,5-trimethoxybenzoyl chloride, N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) Producing L-lysine methyl ester;
(2) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine methyl ester obtained in step (1) is treated with alkali and methanol to give N ² , N ⁶ -bis Producing (3,4,5-trimethoxybenzoyl) -L-lysine;
(3) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine obtained in step (2) is replaced with N, N′-dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBT) is reacted in the presence of D-lysine methyl ester and N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester;
(4) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester obtained in step (3) is treated with alkali was treated with methanol, N ^2, N ⁶ - bis [N ^2, N ⁶ - bis (3,4,5-trimethoxybenzoyl) -L- lysyl] process for manufacturing -L- lysine;
(5) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine obtained in step (4) is treated with 2- [ 1H-benzotriazol-1-yl] -1,1,3,3-tetramethyluronium hexafluorophosphate and reaction in the presence of ethylenediamine to give N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide; and (6) N ² , N ⁶ obtained in step (5) -Bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide is removed in the presence of boron tribromide Methylation produces N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl]-N- (2-aminoethyl)-L-lysine amide Process.

  The above step (1) can be carried out according to the method described in Protocol-1 in the following examples, and similarly, the step (2) can be carried out according to the method described in Protocol-3. Step (3) can be performed according to the method described in Protocol-4, Step (4) can be performed according to the method described in Protocol-6, and Step (5) can be performed according to Protocol-8. The step (6) can be performed according to the method described in Protocol-10.

Furthermore, the present invention relates to N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide ( TGDK), or a mucosal immunity regulator comprising as an active ingredient a compound to which a peptide, protein, lipid or sugar is bound. In the present invention, it can be used to activate immune cells that express CD161. More specifically, natural killer cells (NK cells) or natural killer T cells (NKT cells) that express CD161 are used. Can be used to activate. In particular, the mucosal immune regulator of the present invention can be used to activate floating natural killer cells (NK cells) or natural killer T cells (NKT cells), while adhesive natural killer cells (NK NK cells) or natural killer T cells (NKT cells) can also be used.

As a compound used in the mucosal immunity regulator of the present invention, not only TGDK, but also a compound in which a peptide, protein, lipid or sugar is bound to TGDK may be used. The peptide, protein, lipid, or sugar may be directly bound to TGDK or may be bound via an appropriate linker. As such a compound, for example, TGDK-CH ₂ —CH ₂ —NH—R (in the formula, TGDK-CH ₂ —CH ₂ —NH— is described in International Publication WO2007 / 052641) 2- [N-α, N-ε-bis (N-α, N-ε-digalloyllysinyl) lysinyl] aminoethylamino group, R is a hydrogen atom; a group having an active ester group via a peptide bond; a peptide bond A group that binds to an SH group via a peptide; a peptide, protein, lipid, or sugar linked via a peptide bond: or a peptide, protein, lipid, or sugar linked via a Schiff base). A compound can be mentioned. The compound represented by TGDK-CH ₂ —CH ₂ —NH—R can be produced according to the method described in International Publication WO2007 / 052641.

  The peptide, protein, lipid, polyethylene glycol or sugar described above is not particularly limited as long as it can serve as an antigen, and any one can be used.

  Antigens that can be used for the mucosal immunity regulator of the present invention include, but are not limited to, tumor antigens, pathogen antigens, and allergen antigens. The pathogen antigen as used herein means an antigen specific to a pathogen, and is an antigen obtained from a virus, bacteria, parasite or fungus.

  Specific examples of pathogens include cholera, toxigenic E. coli, rotavirus, Clostridium difficile, Shigella, Salmonella typhi, parainfluenza virus, influenza virus, Streptococcus mutans, Plasmodium falciparum, Staphylococcus aureus, Examples include, but are not limited to, rabies virus and Epstein-Barr virus. Examples of allergen antigens include haptens or antigens derived from pollen, dust, mold, spores, scales, insects and foods.

  In the mucosal immunity regulator of the present invention, the compound (TGDK etc.) used as an active ingredient can be made into a suitable formulation together with a pharmaceutically acceptable carrier. As the carrier, excipients, binders, disintegrants, lubricants and the like can be used. Moreover, you may mix | blend additives, such as antioxidant. The form of the preparation is not particularly limited, and can be any form such as a liquid, tablet, pill, capsule, powder, granule, syrup and the like.

  Examples of the excipient include sugars such as lactose, sucrose, and glucose; starches such as potato starch and wheat starch; celluloses such as crystalline cellulose; inorganic salts such as anhydrous calcium hydrogen phosphate or calcium carbonate, polyethylene, and the like Glycols can be used. As the binder, for example, polyfunctional polyethylene glycols such as crystalline cellulose, pullulan, gum arabic, sodium alginate, or polyvinylpyrrolidone can be used. Examples of disintegrants that can be used include carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl cellulose, hydroxypropyl starch, sodium alginate, and polyethylene glycols. As the lubricant, for example, magnesium stearate, talc, hardened oil, or the like can be used. As the nanoparticulate agent, cholesterol pullulan, multifunctional polyethylene glycols and the like (Nippon Oils & Fats KK) can be used.

  The administration route of the mucosal immunity regulator of the present invention is not particularly limited and may be oral administration or parenteral administration (for example, rectal administration, subcutaneous administration, intramuscular administration, nasal administration, intravenous administration, etc.), preferably oral. Administration.

  The amount of the compound that is an active ingredient contained in the preparation of the mucosal immunity regulator of the present invention can be appropriately set according to the age, weight, symptom, etc. of the administration subject or patient, for example, 1 μg to 1000 mg. / Kg / dose, preferably 10 μg to 100 mg / kg / dose.

  The mucosal immune regulator of the present invention may be administered together with an adjuvant. As the adjuvant, any substance can be used as long as it can enhance the immune response by being administered prior to the administration of the vaccine (antigen). In addition to those having antigenicity such as bactericidal microorganisms, non-antigenic ones such as Alum (aluminum sulfate, potassium, etc.) and mineral oil may be used. In 1947, Freund found that the amount of antibody produced increased when the antigen aqueous solution was mixed with an equal amount of oil (85% mineral oil, 15% surfactant) and injected in the form of an emulsion. This is called incomplete Freund's adjuvant (IFA), and the addition of the Mycobacterium tuberculosis cell components to complete adjuvant complete F's. A. (CFA). In addition, mineral salts such as aluminum hydroxide and aluminum phosphate exhibit an adjuvant effect. In addition, bacterial endotoxins, especially lipopolysaccharides of Gram-negative bacteria, can significantly promote antibody production, and the active ingredient is Lipid A. In the present invention, those described above can be used as an adjuvant. The administration route of the adjuvant is not particularly limited, and may be oral administration or parenteral administration.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(A) Experimental materials (1) Organic synthesis reagents
D-lysine O-methyl ester · 2HCl ((D-LysOMe · 2HCl), 3,4,5-trimethoxybenzoyl chloride (TMBC), N-methylmorpholine (NMM), N, N-dimethylformamide (DMF), Boron tribromide (BBr ₃ ), fluoroisothiocyanate FITC) and fetal calf serum (FCS) are available from Wako Pure Chemical Industries, Ltd. 2- [1H-benzotriazol-1-yl] -1,1,3,3- Tetramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBT), N, N'-dicyclohexylcarbodiimide (DCC) are from Nova biochem, activated polyethylene glycols (PEGs) are oil-based industrial stocks Purchased from the company. As RPMI medium and other general-purpose reagents, products of Nissui Pharmaceutical, Wako Pure Chemical Industries, Sigma, Watanabe Chemical Co., Ltd. were used. The special fluorescent label import antibody was obtained through each import source, Wako Pure Chemicals, Takara Bio Inc., etc.

(2) Cells Human cells and monkey iliac bone marrow cells were used after separating lymphocytes by the Ficoll gradient method according to a conventional method. The effect of TGDK was examined by performing flow cytometer (FACS) analysis on natural killer cells (NK Cell), natural killer T cells (NKT Cell) and regulatory T cells (Treg) using respective cell markers.

(B) Experimental method The outline of the synthesis method and the abbreviations and chemical names of the new compounds in each process are shown below.
(1) Outline of mass production method of TGDK by liquid phase synthesis The outline is shown in Scheme-1, and the chemical name of TGDK is described at the end of scheme-1.

(2) Method of each process
(2-1) Protocol-1 (acid chloride method):
D-LysOMe · 2HCl (12.8 g, Mw 2332.19, 55 mmole) is suspended in 150 ml of DMF (dehydrated), 40 ml of NMM (dehydrated) is added, and the mixture is stirred at room temperature for 10 minutes. To this suspension is added a DCM (dehydrated, 88 ml) solution in which TMBC (28.8 g, MW230.03) is dissolved, and the mixture is stirred at room temperature. After about 48 hours, water is added to the suspension reaction solution (about 280 ml) to make 1 L, and the mixture is stirred at room temperature for 30 minutes. Then, leave it overnight, remove the separated aqueous layer with an aspirator, and dispense 10 ml of the DCM layer into each 50 ml tube. Add water to the tube to make 50 ml, and mix up and down to wash the DCM layer with water. This operation is repeated twice, and the DCM layer is recovered and distilled, a small amount of CH ₃ CN is added, and freeze-dried (2TBOMe). This product is purified according to Protocol-2 and the resulting 3,4,5-Trimethoxybenzoic acid is removed.

(2-2) Protocol-2 (gel purification) method:
Dissolve the lyophilized product (2TBOMe, 3 g) in CH ₃ CN 12 ml, add TFA (0.5 ml), acidify TFA, add Ether to 50 ml, and gel until gelation. Process. The resulting gel is separated, ether is added thereto to make 50 ml, and the gel layer is similarly washed four times with Ether. Remove the Ether layer, add a small amount of CH ₃ CN and freeze-dry. By this operation, 3,4,5-trimetoxybennzoic acid generated by decomposition can be almost completely removed.

(2-3) Protocol-3 (saponification) method:
Dissolve 1 g of purified 2TBOMe in 1 ml of DCM, add 1 ml of KOH saturated MeOH solution and leave at room temperature for 30 min. Then, 5 ml of DCM and 5 ml of water are added, Vortex, Supersonic treatment is performed, and 1 to 2 ml of concentrated HCL is added to make it strongly acidic. Water is added to this solution to make 50 ml, and mixed up and down (Vortex, Supersonic treatment is not performed), the DCM layer is separated, and this DCM layer is washed with water. This operation is performed twice, the DCM of the separated DCM layer is distilled off, a small amount of CH ₃ CN is added, and lyophilized.

(2-4) Protocol-4 (DCC activation method) method:
Place a stir bar in a reaction stir container (made of organic solvent resistant plastic), add 2TBOH 2.25 g and DMF (dehydrated) 4.2 ml to this container, dissolve, dissolve HOBT (567 mg / ml DMF) and DCC (867 mg / ml). Add DMF) and stir at room temperature for 30 minutes. In a few minutes, 460 μL of NMM (dehydrated) was added to D-LysOMe · 2HCl (441 mg / 2 ml DMF) in advance, and the mixture was stirred for 30 minutes at room temperature. Using an Eppendorf pipette with the tip cut off, add the insoluble material together as slowly as possible. After the addition is complete, the reaction stirred suspension is stirred at room temperature for 24 hours.

(2-5) Protocol-5 method:
After 24 hours of reaction stirring in the previous operation, the suspension (about DMF 8.2 ml) was transferred from the stirring vessel to a 50 ml polyethylene tube, and 1 ml of DMF was added to the reaction suspension residue remaining in the stirring vessel. Transfer the entire precipitate to the same tube, then add 1 ml of DCM to the stirring vessel and wash thoroughly, and transfer the DCM solution to the same tube. Add 1 ml of DCM and perform the same operation. Carefully transfer the reaction suspension together with the precipitate from the reaction vessel to the centrifuge tube. The tube is centrifuged (3300 x 3 minutes) to remove the precipitate (DCC Urea), and a supernatant is obtained. After centrifugation, add 1 ml of DMF and 1 ml of DCM to the precipitate remaining at the bottom of the tube, perform the same operation, and add the supernatant obtained by centrifugation to the supernatant obtained first. Pay attention to the amount of DMF in the DCM and DMF mixture in this combined supernatant. If the amount of DMF in this mixture is 5 ml or more and 10 ml or less, mix this mixture with 2 tubes (5 ml / tube). Add water to each to make 50 ml, centrifuge (3300 x 3 min) to separate the DCM layer, and wash this DCM layer three times in the same way. The DCM in the DCM layer obtained by washing is distilled off, a small amount of CH ₃ CN is added, and freeze-dried (4TBOMe). CH ₃ CN insoluble matter is a trace of DCC Urea, so remove it by centrifugation.

(2-6) Protocol-6 method:
Same as Protocol-3.

(2-7) Protocol-7 method:
Dissolve 4TBOH (0.5 g) in 2 ml of CH ₃ CN. If insolubles are present, centrifuge to remove insolubles, add water, and freeze-dry. This operation may be omitted if impurities are not recognized on the MS chart.

(2-8) Method of Protocol-8 (HBTU / EDA solvent method):
Dissolve 4TBOH (Mw1178, 0.26 g, 0.22 mmole) in 2 ml of DMF (dehydrated), add 41 μL of diisopropylethylamine (MW 129.24, d = 0.755), and add HBTU (MW 379.24, 91.8 mg / ml DMF dehydrated). Immediately add dropwise to 1.1 ml of EDA (ethylenediamine) solvent prepared in advance with stirring at a rate of 1 drop / 1 second. During this dripping, the tube is heated with a dryer (so hot to touch). After completion of the dropwise addition, the mixture is similarly heated with a dryer for 10 minutes, and then stirred at room temperature for 60 minutes. After stirring, remove any insoluble material by centrifugation, and use the supernatant as a 4TBA DMF solution.

(2-9) Protocol-9 (4TBA extraction / separation purification):
Add the same amount of DCM as the amount of 4TBA DMF solution. If the amount of DMF exceeds 10 ml, divide the 4TBA DMF solution into two tubes. Add water to these tubes to make 50 ml, and mix up and down. The DCM layer is separated by centrifugation, and the DCM layer is washed with water in the same manner. The DCM is distilled off, CH ₃ CN is added, and lyophilized to obtain 4TBA.

(2-10) Protocol-10 (BBr ₃ decomposition / Ca-Capture method):
4TBA (Mw 1221, 0.28 g, 0.299 mmole) is dissolved in DMF (dehydrated) 0.5 ml, lauric acid DCM solution (Mw 200.32, 2 mmole, 400 mg / ml DCM: dispersant and post-treatment aid) is added dropwise, Crystals of CaCl ₂ · 2 H ₂ 0 (Mw 147.01 1 mmole, 145 mg, demethylation remethylation inhibitor and resulting galloyl group protecting agent) are added directly to form a 4TBA reaction solution. BBr ₃ solution _{(10 mmoleBBr 3/10 ml DCM} ) using 10 ml at a time, carried out twice. This reaction is performed only when the reagent state of BBr ₃ (red-colored reagent and reagent stored for more than 6 months at room temperature after the rainy season is unusable, unopened, reagent stored at 4 ° C or lower after 6 months have passed. It can be used), the humidity of the laboratory (humidity should be 50% or less), and the state of skill of operation (it is important to do it quickly without weighing the reagent etc.).

1) First BBr ₃ decomposition:
_{BBr 3 (10 mmole / 10 ml} DCM solution) of 1 Supoido units 4TBA solution of the above (1 unit amount: In a glass Pasteur pipette fitted with a Teflon cap of 1 ml, taken in one operation the BBr ₃ solution amount , About 1.6 ml), add 4 units quickly over about 3-4 minutes with vigorous stirring. When 4 spoid units are added (about 7 ml out of 10 ml of BBr ₃ solution), stop immediately before the added reaction solution becomes cloudy, close the tube, and stir vigorously for 2-3 minutes. After that, add 1 spoid volume of the 5th time gradually over 2 minutes, the reaction solution gradually becomes cloudy, add 1 spoid volume and the rest of the 6th time, and finish this operation in about 4 minutes. The reaction becomes a suspension and is capped and stirred vigorously for 10 minutes. The first BBr ₃ addition and stirring reaction time takes about 17 to 18 minutes, and the reaction solution changes from a transparent / suspended state to a gel-like / translucent / viscous liquid.

2) Second BBr ₃ decomposition:
To the first BBr ₃ decomposition product, 10 ml of BBr ₃ is divided into several times in one spoid unit amount and added over about 2 minutes to perform the second BBr ₃ decomposition operation. The gel state of the first BBr ₃ reaction solution turns into a light brown, light brown liquid. After the addition, close the cap for 5 minutes and stir vigorously. After closing and stirring for 5 minutes, add 250 μL of EDA (N-methylation inhibitor) little by little. Simultaneously with the addition of EDA, it is neutralized explosively, producing white smoke. After the EDA drop is complete, stir for 3 minutes and move on to the next evaporation / drying process.

3) BBr ₃ decomposition product evaporation / drying operation:
Heat the tube containing the BBr ₃ decomposition product with a drier (heated by hand, feel hot), and evaporate to dryness. This heating / evaporation / drying operation takes about 10 minutes. The standard of the reaction is that the liquid in the tube changes from a smooth semi-liquid to a dry and then dry state, and the dry state is the end of the reaction.

4) BBr ₃ decomposition and water addition decomposition of dried solids:
Water is quickly added to the BBr ₃ decomposition product in a dry state to make 5 ml. This reaction reacts violently with water and generates heat and foams, so be careful. The water addition decomposition product is cooled to room temperature. Centrifuge this solution (3300 x 5 minutes), separate it into a precipitate and a supernatant, and add 1 to 2 ml of water to each precipitate to the supernatant obtained by further centrifuging the supernatant under the same conditions. The supernatant obtained by centrifugation is added and combined.

5) Purification of TGDK using Ca salt coprecipitation by Ca-Capture method:
BBr ₃ demethylates the 3,4,5-trimethoxy group to form a galloyl group, and at the same time, it is a strongly acidic solution containing a large amount of a large excess of BBr ₃ degradation products, boric acid, HBr and the like. The Ca-Capture method was devised as a method for efficiently purifying TGDK present in this strongly acidic solution in a short time.

The same amount of NMM (7.5 ml) as the combined supernatant volume (7.5 ml) of the above-mentioned BBr ₃ degradation product is added to first neutralize strong acidity. Since this neutralization reaction is an exothermic reaction, this solution (15 ml, brown) is allowed to stand at room temperature for a while (about 10 to 20 minutes) and cooled. To this solution is added 2 ml of 1 M CaCl ₂ water to form a precipitate, which is left at -5 ° C. for 5 minutes and then at 4 ° C. for 10 minutes to age the precipitate. The resulting precipitate is centrifuged and collected. By adding CaCl ₂ , the Ca salt of lauric acid is produced, and at the same time, the Ca salt of the TGDK galloyl group is produced. TGDK Ca salt co-precipitated in the precipitate of Ca- (lauric acid) ₂ . Since the target product is easily dissolved in water, this precipitate cannot be washed away with water.

Add DCM (first time) to this precipitate to make 30-35 ml, stir with Vortex, transfer Ca- (lauric acid) ₂ salt to DCM layer and remove. In this case, Sonication is strictly prohibited. Sonication makes it easy to polymerize the target compound and makes it difficult to remove impurities. The DCM-added stirring solution is centrifuged (3300 × 3 minutes), and the resulting upper precipitate (light beige) is collected by decantation (gradient removal). To this pale beige precipitate, add 30-35 ml of DCM (second time), perform Vortex, and centrifuge. Since yellow liquid appears in the upper precipitate, this yellow liquid is carefully removed with a Pasteur pipette, and the lower DCM layer is removed by decantation. This operation is further repeated until no yellow liquid is generated. Add 5 ml of water to the precipitate (brown) that is no longer producing a yellow liquid, centrifuge to separate the precipitate and the supernatant, add 100 μl of TFA to each, and freeze-dry. Pure-TGDK · TFA salt is obtained from the supernatant, and Oligo-TGDK · TFA salt is obtained from the precipitate.

(3) Structural formula, chemical name and abbreviation of the synthesis process

(4) Experimental method of biological action of TGDK:
(4-1) Pretreatment of TGDK:
TGDK synthesized by the liquid phase method is a TFA salt and contains trace amounts of Ca salt of lauric acid. First, dissolve it in a small amount of 100% TFA, add 10 times the amount of ether (dehydrated), and generate The precipitate was redissolved in a small amount of 100% TFA. This operation was repeated several times, and the resulting precipitate was dried (drying under reduced pressure, removing TFA), dissolved in a small amount of 3N HCl, lyophilized and used as a TGDK HCl salt. The sample was used after sterilizing with 70% ethanol, removing ethanol and dissolving in sterile PBS (−), and diluting with a culture solution to a working concentration.

(4-2) Action of TGDK on floating bone marrow lymphoid cells Monkey iliac bone marrow cells were used by separating lymphocyte cells from bone marrow fluid by Ficoll gradient method according to a conventional method. Add 100 μl of TGDK (in culture solution) prepared to a final concentration of 0.1 μM of TGDK to 5 ml of the obtained myeloid lymphoid cells (1 × 10 ⁶ / ml in RPMI1640 containing 10% FCS), and add 12 Cultured for days. During this period, the number of living cells and the number of dead cells (trypan blue method) was determined every 1 day or 2 days, and the action of TGDK on the living cells was observed.

(4-3) Action of TGDK on adherent bone marrow lymphoid cells Adherent cells were not observed from the beginning of culture to the middle stage (7 days), but thereafter, adherent cells attached to the bottom of the incubator were observed. Therefore, after culturing for 12 days, adherent cells were detached by EGTA method, and 20 μl of TGDK (in culture solution, final concentration of action 25 μM) was added to 0.5 ml of the obtained cells (3 × 10 ⁵ cell / ml), 15 FACS analysis was performed by allowing it to act at room temperature for a minute. Natural killer cells (NK cells) and natural killer T cells (NKT cells) and T cell specific cell markers, CD16 (Fc receptor type III) and CD56 (adhesion factors) and CD3, as well as CD161 (C-type lectin family) Receptor) was used.

(4) Action of TGDK on human peripheral lymphocyte cells Human peripheral blood (heparin blood) was isolated from lymphocyte cells according to a conventional method, and was performed according to the experimental conditions of the bone marrow cell system.

(C) Results and Discussion (1) Yield and purity of TGDK by liquid phase synthesis Scheme-3 and Fig. 1 show the yield of each step of the liquid phase synthesis method and the mass spectrometry result of TGDK finally obtained. .

  Mass spectrometry of TGDK obtained by the liquid phase synthesis method confirmed that it was almost homogeneous (Figure 1). The yields of the liquid phase synthesis method and solid phase synthesis are expressed as mole yields relative to the starting materials. When compared, D-Lys (12.8 g) yields TGDK (4.9 g, mole yield 8.5%), which uses a resin. It was found that the yield was higher than that in the solid phase synthesis method (mole yield: 2.6%) (Scheme-3). Manufacturing costs were reduced to about 1/100 compared to the resin method.

(2) Biological action of TGDK
(2-1) Action of TGDK on floating bone marrow lymphoid cells FIG. 2 shows the relative number of floating cells (%) of TGDK (0.1 μM) over time.
When comparing the effect of TGDK with Control and Treated, except for the results of Day 4, 6, and 7, there was no significant change in the number of living cells of both floating bone marrow lymphoid cells, but Day 4 and Day 6, 7 , A significant decrease in the number of viable cells considered to be the effect of TGDK was observed. This reduction effect is unlikely to be due to TGDK's universal cytotoxicity. In general, the decrease in viable cells due to drug cytotoxicity increases with culture time and is completely killed in a few days. However, in TGDK treatment, the number of viable cells decreased in Day 4, 6, and 7 culture, recovered from Day 9 to Control level, and further, on Day 12, cell proliferation was observed, which is not due to TGDK toxicity. Conceivable. This decrease in the number of viable cells is considered to be due to the activation of NK cells by TGDK, and the decrease of viable cells due to injury of the target cells of NK cells.

(2-2) FACS1 analysis of day 12 cultured floating bone marrow lymphoid cells
The results of FACS analysis using surface markers of CD16, CD56, CD161 of NK cells and CD3, CD16, CD56, CD161 of NKT cells are shown in FIGS.

  The percentage of CD16 + and CD56 + double positive cells (%) from 4.24 (Control, Fig.3A) to 4.43 (Fig.3B) with TGDK treatment, and the percentage of CD3 + and CD16 + double positive cells (%) is 2.16 (Control, (Figure 3C) to 2.46 (Figure 3D) with TGDK treatment, and the percentage (%) of CD3 + and CD56 + double positive cells increased from 2.10 (Control, Figure 3E) to 2.50 (Figure 3F) with TGDK treatment. It is thought that. These increased cells are considered to be NK cells or NKT cells based on surface markers. In the case of humans, the total number of NK cells is estimated to be about 5 billion, which is very small, about 1/1000 compared to the total number of human cells of about 60 trillion. NK cells or one NKT cell constantly searches for 1000 cells such as living cells, invading pathogenic cells, different cells (cancer cells) that have changed from the living body, and aged cells, and the foreign body is removed to protect the living body ing. Based on this, 0.1% increase / decrease in NK cells and NKT cells is extremely biologically significant. From these facts, it is considered that TGDK stimulates and proliferates NK cells and NKT cells. .

(2-3) Action of TGDK on Adherent Bone Marrow Lymphoid Cells Bone marrow cells are treated with TGDK (0.1 μM), cultured for 12 days, and attached to the bottom of the culture vessel to proliferate cells (morphologically Monocytes, dendritic cells, macrophages, CD161 +, CD16 + positive cells) were detached with EGTA, treated with TGDK (final concentration, 25 μM) for 15 minutes, and the results of FACS analysis are shown in FIGS. 4A and B.

  The percentage of CD161 + and CD16 + double positive cells from 6.07 (Control, day12 culture) to 1.36% by TGDK (25 μM) treatment, CD161 + positive and CD16-negative cells from 2.11 (Control) to 0.3% Drastically decreased. Bone marrow cells are a large collection of undifferentiated cells, progenitor cells, homing cells, etc., and after 12 days of culture, differentiated macrophages, dendritic cells, monocytes and other cells are adherent, It grows on the bottom. In addition, these cells express C-type lectin on the cell surface, and NK cells and NKT cells having CD161, which is the C-type lectin receptor, bind to these lectin sugar chains and Ca, and the bottom of the incubator It is thought that it adheres to. By adding EGTA to such a state, it is considered that Ca was removed and cells floating from the adherent cells or adherent cell groups having CD161 were used for the experiment. When TGDK (25 μM) is added to these floating cells, TGDK binds to CD161 and masks CD161, so it can no longer bind to anti-CD161 antibody, so in FACS analysis, NK cells expressing CD161 and NKT are expressed. The results show a dramatic decrease in cells. These results are thought to imply the dual nature of TGDK action. In other words, in a floating state such as blood, NK cells and NKT cells are stimulated and proliferated, and pathogenic cells that have invaded the living body or generated foreign cells (such as cancer cells, such as cancer cells, CD161-expressing cancer cells are It shows that it can be removed. It also controls the action of NK cells in a relatively fixed state, such as the mucosal membrane, and activates and immediately removes foreign substances when invading foreign substances. (Runaway) is prevented. TGDK is considered to be very effective for intractable diseases such as ulcerative colitis that are thought to be a runaway innate immune response.

(2-4) Action of TGDK on peripheral lymphocyte cells
Fig. 5 shows the results of FACS analysis using human lymphoid cells (PBMC) cultured in the presence and absence of TGDK for 3 days, using T cells, NK cells, and NKT cell marker molecules CD3, CD16, and CD56 as indicators. It was shown to. The concentration of CD16 and CD56 double positive cells at 0.1 mM (Fig.5B) was about 1.8 times that of TGDK 0 μM (Fig.5A), and TGDK 1 mM (Fig.5C) was about 6 times. Doubled.

  NK cells were found about 35 years ago as lymphocytes that kill tumor cells and virus-infected cells without antigen sensitization, and have a natural killer (natural killer). ). NK cells play an important role in the initial defense of the body. The number of NK cells, which are present in about 5 billion in the whole body, decreases with aging, and decreases to about one-tenth at 60 years of age or older, making it easier to catch a cold and more likely to become cancer. Increasing and revitalizing is important for medical care in old age. The biggest cause of death for elderly people over 75 years old is bacterial infections such as pneumonia, and a society in which resistant bacteria that are ineffective to antibiotics spread is imminent, NK cells and NKT cells are activated, antibiotics We must build a society that does not depend on as soon as possible. However, there is currently no drug that actively activates NK cells, and TGDK can be the first drug. Administration of TGDK activates NK cells and is expected to eradicate cancers and viruses through NK cells, leading to a radical treatment method for intractable diseases such as cancer and AIDS.

  On the other hand, NKT cells have the characteristics of NK cells and T cells, and are deeply involved in innate immunity and acquired immunity. Since TGDK binds to CD161 (C-type lectin) and activates NK cells and NKT cells, it can be an important drug for biological defense. In particular, since TGDK can specifically activate NKT cells, it stimulates innate and acquired immunity and is important for the prevention of pathogenic bacteria and viruses that have invaded the body.

  As described above, NK cells are important for eliminating cancer cells generated by chemicals such as tumors and food additives that occur with aging, and natural environments. In addition, TGDK controls allergic reactions (allergic reactions, ulcerative colitis, cedar pollinosis, etc.) via NKT cells, which can suppress allergic reactions, making it a completely new immune regulator. At the same time, it can be an essential drug in preventive medicine for preventing the living body from falling into a disease.

  TGDK has been developed and created as the only low molecular weight compound that can specifically target M cells. It has already been reported that when TGDK is administered to monkeys, it is specifically taken up by M cells in the intestinal mucosa of monkeys and can send antigens to the immune system of the lamina propria and activate the immune system (Misumi et al., The Journal of Immunology, 2009, 182: 6061-6070). That is, TGDK is an M cell targeting agent and can efficiently deliver a vaccine to the lamina propria.

Moreover, since TGDK is a ligand of C-type lectin molecule as an antagonist and agonist of C-type lectin molecule, it can be applied to the following.
(1) Acts as an effector for cells with C-type lectin molecules.
(2) An inhibitor that binds to C-type lectin, Langerin (Langerhans cells) or DC-SIGN (dendritic cells), inhibits binding to the sugar chain of HIV ENVgp120, and inhibits HIV infection in the mucosa. Function as.
(3) Stimulates and activates Treg cells expressing CD161, suppresses an excessive immune response, and suppresses autoimmune diseases caused by impaired immune tolerance.
(4) Since TGDK can bind to the sugar chain of HIV ENVgp120 glycoprotein via Ca, the surface of the virus can be masked with TGDK, and can be an anti-HIV drug that prevents HIV infection.
(5) TGDK can be an anti-HIV drug that activates NK cells and kills HIV-infected cells in the mucosa.
(6) TGDK activates macrophages expressing CD161 molecule, and can take in and kill TGDK-Masked-HIV opsonic with TGDK.
(7) TGDK can activate NKT cells expressing CD161 and efficiently link to the acquired immune system.
(8) TGDK binds to and suppresses osteoclasts expressing CD161, and is therefore an effective drug for osteoporosis.

Claims (5)

N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L from D-lysine methyl ester dihydrochloride comprising the following steps (1) to (6) -Lysyl] -N- (2-aminoethyl) -L-lysineamide synthesis method.
(1) By reacting D-lysine methyl ester dihydrochloride in the presence of 3,4,5-trimethoxybenzoyl chloride, N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) Producing L-lysine methyl ester;
(2) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine methyl ester obtained in step (1) is treated with alkali and methanol to give N ² , N ⁶ -bis Producing (3,4,5-trimethoxybenzoyl) -L-lysine;
(3) N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysine obtained in step (2) is replaced with N, N′-dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBT) is reacted in the presence of D-lysine methyl ester and N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester;
(4) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine methyl ester obtained in step (3) is treated with alkali was treated with methanol, N ^2, N ⁶ - bis [N ^2, N ⁶ - bis (3,4,5-trimethoxybenzoyl) -L- lysyl] process for manufacturing -L- lysine;
(5) N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -L-lysine obtained in step (4) is treated with 2- [ 1H-benzotriazol-1-yl] -1,1,3,3-tetramethyluronium hexafluorophosphate and reaction in the presence of ethylenediamine to give N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide; and (6) N ² , N ⁶ obtained in step (5) -Bis [N ² , N ⁶ -bis (3,4,5-trimethoxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysine amide is removed in the presence of boron tribromide Methylation produces N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl)-L-lysyl]-N- (2-aminoethyl)-L-lysine amide Process. N ² , N ⁶ -bis [N ² , N ⁶ -bis (3,4,5-trihydroxybenzoyl) -L-lysyl] -N- (2-aminoethyl) -L-lysineamide (TGDK), or this A mucosal immunity regulator comprising as an active ingredient a compound to which a peptide, protein, lipid or sugar is bound. 3. The mucosal immunity regulator according to claim 2, which is a mucosal immunity regulator for activating immune cells expressing CD161. 4. The mucosal immune regulator according to claim 3, wherein the immune cells expressing CD161 are natural killer cells (NK cells) or natural killer T cells (NKT cells). Activates floating-type natural killer cells (NK cells) or natural killer T cells (NKT cells) and suppresses adherent-type natural killer cells (NK cells) or natural killer T cells (NKT cells) to control mucosal immunity 5. The mucosal immunity regulator according to any one of claims 2 to 4.