JP2015040179A - Immunosuppressive agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel immunosuppressive agent useful for autoimmune diseases (connective tissue disease, rheumatism, and the like), allergic diseases, organ transplantation, and the like, having an excellent effect against them.SOLUTION: The immunosuppressive agent contains O-(5-amino-2-phenyl benzoxazole-7-yl)methyl-3,5-dichloro-L-tyrosine or a pharmacologically acceptable salt thereof as an active ingredient. This immunosuppressive agent has also an excellent effect in suppressing cytokine release from T cells, and suppressing T cell proliferation.

Description

  The present invention relates to a novel immunosuppressive agent.

  In clinical practice, a disease that cannot be cured by chemotherapy, and the organ or tissue (eg, kidney, liver, lung, intestinal tract, heart, pancreas, cornea, skin, etc.) or part of the disease or part of it By transplanting, a method for recovery can be selected. What is most concerned about transplantation of organs and tissues from other people is rejection caused by the immunity of the recipient. Therefore, research on immunosuppressive agents has been promoted for the purpose of permanent establishment of transplanted organs.

  Furthermore, even in so-called autoimmune diseases such as collagen disease and rheumatism, an immunosuppressive agent can be an important drug in alleviating the symptoms.

  Here, as immunosuppressive agents, corticosteroids, antimetabolites, alkylating agents, alkaloids, antibiotics, anti-lymphocyte globulin, anti-CD3 monoclonal antibodies, etc. are known, and autoimmune diseases, allergic diseases, It is used as a therapeutic agent for organ transplantation. In recent years, there has been a demand for the development of even better immunosuppressive agents.

JP 2011-016851 A JP 2007-091644 A JP 2005-281235 A JP 2005-126343 A JP 2004-307442 A

  Then, this invention makes it a subject to provide the much more superior immunosuppressive agent.

  The present invention relates to an immunosuppressant comprising O- (5-amino-2-phenylbenzoxazol-7-yl) methyl-3,5-dichloro-L-tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient. It is.

  The immunosuppressive agent according to the present invention has an excellent effect in suppressing cytokine release from T cells and T cell proliferation.

FIG. 1A is a diagram showing a reaction scheme of a compound according to the present invention. FIG. 1B is a diagram showing a reaction scheme of a compound according to the present invention. FIG. 1C is a diagram showing a reaction scheme of a compound according to the present invention. FIG. 2 shows the principle of the cell division measurement method using CFSE. The fluorescence intensity per cell of CFSE taken up into cells is halved by cell division. By measuring fluorescence decay by FACS, the number of cell divisions can be examined. FIG. 3 is a graph showing the actual fluorescence decay of CFSE after T cell stimulation in the presence of the compound according to the present invention. It can be seen that as the concentration of the test compound increases, the population with lower fluorescence intensity decreases (ie, fewer cells divide). FIG. 4 shows the mechanism of T cell activation. T cells are completely activated by stimulation of T cell receptors that recognize foreign substances taken up by antigen-presenting cells and costimulation from CD28. Each stimulus can be replaced with an anti-CD3 antibody or an anti-CD28 antibody. Fully activated T cells activate transcription factors such as NF-kB, NFAT, AP1, release various cytokines while actively proliferating, and activate other immunocompetent cells, Promotes individual immune response. FIG. 5 shows the effect of the compound of the present invention on cytokine production in human T cells. It can be seen that cytokine production is suppressed as the concentration of the compound of the present invention increases.

≪Active ingredient≫
The immunosuppressant according to the present invention comprises O- (5-amino-2-phenylbenzoxazol-7-yl) methyl-3,5-dichloro-L-tyrosine or a pharmacologically acceptable salt thereof as an active ingredient Include as.

  Here, “pharmacologically acceptable salt” means, for example, alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), ammonium salts, organic bases (trimethylamine). , Salts with triethylamine, pyridine, picoline, dicyclohexylamine, dibenzylethylenediamine, etc., organic acids (acetic acid, benzoic acid, succinic acid, fumaric acid, maleic acid, lactic acid, citric acid, tartaric acid, gluconic acid, methanesulfonic acid, Salts with benzenesulfonic acid, formic acid, p-toluenesulfonic acid, trifluoroacetic acid, etc., salts with inorganic acids (hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.), amino acids (arginine, aspartic acid, glutamic acid, etc.) ) And a salt. The compound and the salt thereof may be in the form of a hydrate or a solvate such as ethanolate. In addition, in order to improve water solubility, the compound or a salt thereof is preferably an inclusion body of cyclodextrin (for example, β-cyclodextrin). Here, the compound or a salt thereof: cyclodextrin (mass ratio) is preferably 1:10 to 70, more preferably 1:20 to 50, and particularly preferably 1:25 to 35.

Administration method
The compound and its salt can be administered, for example, orally, transdermally or by injection.

  Tablets, granules and capsules for oral administration are conventional additives such as binders (eg syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone); fillers (eg lactose, sugar, corn starch, calcium phosphate) Sorbitol or glycine); lubricants (eg magnesium stearate, talc, polyethylene glycol or silica); disintegrants (eg potato starch) or wetting agents (eg sodium lauryl sulfate). Tablets, granules and capsules may be formed into a film by a method known in the field of ordinary preparations.

  Liquid formulations for oral administration may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or as lyophilized formulations that dissolve in water or other suitable solvent prior to use. Good. Liquid formulations are conventional additives such as suspending agents (eg sorbitol, syrup, methylcellulose, glucose syrup, gelatin water edible fat); emulsifiers (eg lecithin, sorbitan monooleate or gum arabic); non-aqueous excipients (Eg almond oil, fractionated coconut oil or oily esters such as glycerin, propylene glycol or ethyl alcohol); including preservatives (eg methyl or propyl p-hydroxybenzoate or sorbic acid) and flavoring or coloring agents You may go out.

  When administered transdermally, the active ingredient may take the form of a cream, lotion or ointment. Cream or ointment preparations that can be used as drugs can be prepared by methods well known in the art.

  An injection can be produced by suspending or dissolving the compound or a salt thereof in an appropriate medium. The injection may contain adjuvants such as local anesthetics, preservatives and buffering agents.

≪Dose≫
The dosage of the compound and its salt can be appropriately adjusted according to various factors including the age, weight, general health, sex, administration time, administration route, and severity of the disease of the patient. In general, it is appropriate to administer about 10 to 5000 mg, preferably about 100 to 3000 mg per day for an adult divided into 1 to 5 times.

  Below, the manufacturing method of the said compound is demonstrated in detail by a manufacture example and an Example, and the effect | action etc. as an immunosuppressant are demonstrated in detail by a test example.

Production Example According to the reaction scheme of FIG. 1, β-cyclodextrin of O- (5-amino-2-phenylbenzoxazol-7-yl) methyl-3,5-dichloro-L-tyrosine dihydrochloride according to the present invention Inclusion bodies were prepared. The salt was a pale yellow crystal or crystalline powder, and had a melting point of 180 to 200 ° C. (decomposition).

1) Synthesis of benzoxazole derivatives
Synthesis of (2) 96% sulfuric acid (60.6 ml) was added dropwise at room temperature to a solution of 3-nitrosalicylic acid (1) (201 g, 1.09 mol) in methanol (1600 ml), and then refluxed for 23 hours. The reaction solution was concentrated under reduced pressure to about 1/3 and then cooled in an ice bath. The produced solid was collected by filtration, washed with cold methanol (600 ml) and cold water (600 ml), and dried at 40 ° C. under reduced pressure to give 209.7 g (97%) of a yellow solid as methyl 3-nitrosalicylate (2). Obtained.
Synthesis of (3) 10% Pd-C (15.2 g) was added to a solution of methyl 3-nitrosalicylate (2) (108.2 g, 0.549 mol) in THF (1140 ml) / MeOH (810 ml), and hydrogen was supplied at 1 atm. The catalytic reduction was performed at room temperature for 2 hours. The catalyst was filtered off, washed with THF, and the solvent was distilled off. The residue was washed with diisopropyl ether to give methyl 3-aminosalicylate (3) as a brown solid (166.7 g, 95%).
Synthesis of (4) N, N-dimethylaniline (108.7 g, 0.897 mol) was added to a solution of methyl 3-aminosalicylate (3) (74.8 g, 0.447 mol) in THF (1130 ml) at 5 ° C. for 40 minutes. It was dripped at. After stirring the reaction solution for 15 minutes, a solution of benzoyl chloride (75.7 g, 0.538 mol) in THF (570 ml) was added dropwise over 1 hour. After stirring at the same temperature for 1 hour, water (100 ml) was added and THF was distilled off. Water (900 ml) was added to the residue and the mixture was extracted with ethyl acetate. The organic layer was washed with 10% hydrochloric acid, water and saturated brine, dried over dead glass, and the solvent was distilled off. The obtained crude crystals and the amino compound (3) (90.16 g) were combined with crystals obtained by the same operation and recrystallized from ethyl acetate to obtain 247.1 g (92 of methyl 3-benzoylaminosalicylate (92). %) Obtained as a colorless solid.
Synthesis of (5) Methyl 3-benzoylaminosalicylate (4) (83.2 g, 0.296 mol) in acetic acid (1480 ml) at an internal temperature of 15 ° C. and 69% nitric acid (d = 1.42) (95 ml, 1. 458 mol) was added dropwise. The reaction solution was stirred at room temperature for 3 hours, poured into ice water (300 ml) and stirred for 10 minutes. The produced solid was filtered, washed with water (3 times), ether and then air-dried to obtain 93.4 g (96%) of methyl 3-benzoylamino-5-nitrosalicylate (5) as a yellow solid.
Synthesis of (6) A mixture of methyl 3-benzoylamino-5-nitrosalicylate (5) (72.2 g, 0.260 mol) and 1,2-dichlorobenzene solution of PPSE (780 ml) was stirred at 150 ° C. for 4 hours. . The reaction solution is ice-cooled, and the precipitated crystals are collected by filtration, washed with n-hexane and cold methanol, and dried under reduced pressure to give 5-nitro-2-phenylbenzoxazole-7-carboxylic acid methyl ester (6). Obtained 61.2 g (90%) as a pale yellow solid.
Synthesis of (7) 5-Nitro-2-phenylbenzoxazole-7-carboxylic acid methyl ester (6) (59.2 g, 0.198 mol) in methanol (990 ml) suspension in 1M aqueous sodium hydroxide (297 ml, 0.297 mol) was added dropwise, followed by stirring at room temperature for 19 hours. The reaction solution was acidified with 10% hydrochloric acid under ice cooling and stirred for several minutes. The resulting solid was collected by filtration, washed with water and methanol, and then dried under reduced pressure to obtain 54.2 g (96%) of a colorless solid as 5-nitro-2-phenylbenzoxazole-7-carboxylic acid (7).
Synthesis of (9) 5-Nitro-2-phenylbenzoxazole-7-carboxylic acid (7) (26.4 g, 0.0931 mol) in THF (470 ml) suspension in triethylamine (15.57 ml, 0.112 mol) And stirred at room temperature for 30 minutes. The reaction mixture was cooled to −10 ° C., ethyl chlorocarbonate (10.68 ml, 0.112 mol) was added, and the mixture was stirred at the same temperature for 30 min. After adding sodium borohydride (8.84 g, 233.7 mmol) and stirring for 30 minutes, THF / water (317 ml / 64 ml) was added dropwise at −10 to −6 ° C. over 3 hours. The reaction solution was further stirred for 2 hours, after which ice water was poured, and the precipitated solid was collected by filtration and washed with water. Solids obtained by the same procedure from 5-nitro-2-phenylbenzoxazole-7-carboxylic acid (7) (14.6 g) were combined, suspended in THF (600 ml) / methanol (600 ml), and 1M hydroxylated. A sodium aqueous solution (115 ml) was added, followed by stirring at room temperature for 4.5 hours. The reaction solution was poured into ice water, and the precipitated solid was filtered and air-dried to obtain 24.4 g (63%) of an almost colorless solid 7-hydroxymethyl-5-nitro-2-phenylbenzoxazole (9). .
Synthesis of (10) 7-hydroxymethyl-5-nitro-2-phenylbenzoxazole (9) (46.2 g, 0.171 mol) in THF (1150 ml) suspension in ice-cooled triethylamine (34.9 ml, 0 .250 mol) was added. Methanesulfonyl chloride (23.1 g, 0.200 mol) was added dropwise, and the mixture was stirred for 30 minutes under ice-cooling and then at room temperature for 3 hours. Triethylamine (4.55 ml, 0.033 mol) and methanesulfonyl chloride (1.29 ml, 0.017 mol) were added, and the mixture was further stirred for 30 minutes. The reaction solution was poured into ice water and the precipitated solid was filtered. The obtained solid was washed with water and diisopropyl ether and then dried at 40 ° C. under reduced pressure to give 59.1 g (99%) of pale yellowish 5-nitro-2-phenylbenzoxazol-7-ylmethylmethanesulfonate (10). Obtained as a solid.
2) Synthesis of dichlorotyrosine derivatives
Synthesis of (12) Sulfyl chloride (52.22 ml, 0.65 mol) was added to a suspension of (S) -tyrosine methyl ester hydrochloride (11) (60.15 g, 0.26 mol) in acetic acid (294 ml) at room temperature. The solution was added dropwise and then stirred for 3.5 hours. The reaction solution was concentrated, and the residue was washed thoroughly with ether and dried to obtain (S) -dichlorotyrosine methyl ester hydrochloride (12) as 72.70 g (93%) as a colorless solid.
Synthesis of (13) (S) -dichlorotyrosine methyl ester hydrochloride (12) (72.42 g, 0.24 mol) in water (1.4 l) suspension under water cooling with 2M potassium carbonate aqueous solution (120.5 ml) ) Was added dropwise. Di-t-butyl dicarbonate (58.1 ml, 0.25 mol) in dichloromethane (1.4 l) was added, and the mixture was stirred under ice-cooling for 1.5 hours and at room temperature for 40 hours. The reaction mixture was filtered using celite, and the filtrate was extracted with chloroform. The organic layer was washed with water and saturated brine, dried over dry glass, and the solvent was distilled off. The residue was washed with n-hexane to obtain 76.95 g (88%) of a colorless solid (S) -N-Boc-dichlorotyrosine methyl ester (13).
3) Synthesis of the compound salt according to the present invention
Synthesis of (14) 5-Nitro-2-phenylbenzoxazol-7-ylmethylmethanesulfonate (10) (29.11 g, 83.6 mmol) was dissolved in THF (820 ml), and acetone (820 ml) was added. Add (S) -N-Boc-dichlorotyrosine methyl ester (13) (36.54 g, 100 mmol), sodium iodide (12.53 g, 83.6 mmol), potassium carbonate (17.37 g, 125 mmol) and bring to room temperature. And stirred for 21 hours. The reaction solution was filtered and the filtrate was concentrated to about ½. After ethyl acetate and water were added for liquid separation, the organic layer was washed with water, a 5% aqueous sodium thiosulfate solution and saturated brine, and the dry-bed dry solvent was distilled off. By a similar operation from 5-nitro-2-phenylbenzoxazol-7-ylmethylmethanesulfonate (10) (30.06 g), (S) -N-Boc-dichlorotyrosine methyl ester (13) (37.66 g) The obtained product was added, and a total of 116.2 g of the crude product was washed with diisopropyl ether and dried to obtain 98.43 g (94%) of a nitro compound (14) as a pale yellow solid.
(15) Synthetic nitro compound (14) (57.04 g, 92.5 mmol) was dissolved in THF (1340 ml), isopropanol (1340 ml) was added, and the internal temperature was heated to 60 ° C. 5M aqueous ammonium chloride solution (278 ml, 1.39 mol) was added, followed by iron powder (103.32 g, 1.85 mol), and the mixture was stirred at 60 ° C. for 3 hours. The reaction solution was filtered using celite, and the filtrate was concentrated to about 1/3. Unnecessary substances on Celite were washed three times with hot ethyl acetate, combined with the filtrate, washed with water and saturated brine, and then dried over dead glass. The solvent was distilled off, and the resulting solid was washed with diisopropyl ether and dried to obtain 53.03 g (98%) of the amino compound (15) as a yellow solid.
(16) Synthetic amino compound (15) (86.91 g, 0.148 mol) was dissolved in THF (1430 ml), and 0.5M lithium hydroxide was cooled in an ice bath so that the internal temperature was about 10 ° C. An aqueous solution (444 ml, 0.222 mol) was added dropwise, and the mixture was further stirred for 1 hour in an ice bath. The reaction mixture was concentrated to about ½, water (600 ml) was added, the pH was adjusted to about 4 with 10% aqueous citric acid solution, and the mixture was extracted with ethyl acetate-THF (10: 1). The extract was washed with water and saturated brine, dried over dry glass, and evaporated. The obtained residue was recrystallized from ethyl acetate-THF (10: 1) to obtain an almost colorless solid (72.94 g, crude yield 86%). This solid (10.03 g) was recrystallized from chloroform-methanol (10: 1) to obtain 8.63 g (recovery: 86%) of carboxylic acid (16) as an almost colorless solid.
(17) Synthesis of Compound Salts According to the Present Invention In a solution of carboxylic acid (16) (85.23 g, 0.149 mol) in THF (750 ml) under ice-cooling, 4M hydrogen chloride-dioxane (1117 ml, 4. 47 mol) was added dropwise over 2 hours. The reaction solution was stirred at the same temperature for 1 hour, then gradually warmed up and stirred at room temperature for 20 hours. Diisopropyl ether was added and the solid was collected by filtration and washed with diisopropyl ether. The obtained solid was divided into 3 lots and dissolved in ethanol (2300 ml in total), and insoluble matter was filtered off. The filtrate was concentrated to about 1000 ml under reduced pressure at room temperature, and the resulting solution was stirred at room temperature. The produced crystal was filtered and dried to obtain 66.11 g (81%) of a compound salt (17) according to the present invention as a pale yellow solid.
4) Synthesis of cyclodextrin inclusion bodies (1) Weigh 1000 g of Sulfobutyl ether-β-cyclodextrin (abbreviated as SBE-CD: trade name Captisol) and place it in a 5 L container in a sterile hood for cell culture. Then, 1800 ml of HPLC grade pure water was added and stirred overnight to obtain a clear solution (about 36% by weight).
(2) The next morning, warm to 32-36 ° C in a water bath and keep this temperature, 45.5 g of HCl salt of the compound according to the present invention; 35.5 g equivalent as a free base, 100 ml ethanol, 200 ml Of pure water and 300 ml of the above SBE-CD solution were added to obtain a compound solution. The pH of this solution was confirmed to be 3-4.
(3) 3.4 g of caustic soda was dissolved in 200 ml of pure water, and this sodium hydroxide water was added to the solution while stirring to adjust the pH to 6.5.
(4) In this warm state, the solution was sterile filtered with a 0.4 μm filter using a vacuum pump. The filtrate was placed in a sterile 5 L container and diluted with the SBE-CD solution prepared in (1) to adjust the final volume to 2800 ml.
(5) After further stirring for 5 minutes, the pH was measured and confirmed to be ~ 6.5. Using a sterile dispenser, each 15 ml was dispensed into 30 ml vials. The final compound concentration was 10 mg / ml. Each vial was processed in a lyophilizer (Lyostar3, FTS Systems SP Scientific) for 1 week.
(6) The vial after freeze-drying was sealed with a rubber stopper, metal creased, and stored in a refrigerator.
(7) For re-solutionization, 14.1 ml of distilled water for injection was added, and the solution was turned into a 15 ml light yellow solution by a voltex mixer for 5-10 minutes.

Test example 1
(Preparation of human T cells)
1) Obtaining written consent from a healthy person, blood is collected from the cubital vein, suspended in an equal amount of phosphate buffer, overlaid on the top of histpaque (Sigma), and centrifuged (2000 rpm, 10 Min, room temperature), and peripheral blood mononuclear cells (PBMCs) were isolated by collecting cells in the intermediate layer.
2) PBMCs were stained with fluorescently labeled anti-CD4 antibody and anti-CD25 antibody, and CD4-positive and CD25-negative human T cells were isolated using FACSAria (BD).
Test example 2
(Measurement of human T cell activation method and proliferation)
1) Anti-CD28 antibody (clone CD28.2, 2 μg / ml) is added to 1 × 10 ⁵ T cells, added to a petri dish coated with anti-CD3 antibody (clone OKT3, 5 μg / ml), and 10% fetal bovine serum Cultivation was continued in 1 ml of RPMI1640 medium containing 1 and activated for 3 days.
2) Cell proliferation was measured by the CFSE method. The purified T cells were mixed with carboxyfluorescein diacetate, succinimidyl ester (CFSE) to a final concentration of 0.7 μg / ml, and allowed to stand for 10 minutes to incorporate CFSE into the cells. After cell washing, the cells were activated for 3 days, and the fluorescence intensity decay of CFSE was measured by FACSCalibur (BD) to determine the number of divisions.
The principle and results are shown in FIGS.
Test example 3
(Preparation of test compound)
DMSO was added to each weighed test compound to prepare a 100 mM solution or suspension, which was used as a DMSO stock. At the time of assay, this stock was diluted with DMSO to prepare a solution having a concentration 1000 times the final concentration of each test compound, and each solution was diluted with a medium to adjust the final concentration of DMSO to 0.5%.
Test example 4
(Measurement method of cytokine release from activated human T cells)
1) The outline of the mechanism of cytokine release from human T cells is summarized in FIG.
2) Cytokine (interferon-gamma (INFγ), interleukin 2 (IL-2), interleukin 4) released in the medium after the T cell activation described in 1) of Test Example 2 is continued for 3 days. (IL-4) and interleukin 17 (IL-17)) were measured.
3) Each cytokine was measured by an antibody sandwich method using a cytometric bead array kit (BD). The beads to which the antibodies of each cytokine were bound and the cell culture solution were mixed and allowed to stand for 2 hours, and then the fluorescently labeled cytokine antibody was mixed and allowed to stand for 1 hour. After washing, the fluorescence intensity of the beads was measured with a FACSCalibur, and each cytokine was quantitatively analyzed.
4) The amount of each cytokine released in the absence of the compound and, as a specific example, the results in the presence of various concentrations of compound 6 are shown in FIG.

Claims (1)

  An immunosuppressant comprising O- (5-amino-2-phenylbenzoxazol-7-yl) methyl-3,5-dichloro-L-tyrosine or a pharmacologically acceptable salt thereof as an active ingredient.