JP2016193866A - Cyclooxygenase inhibitor, nadph-cytochrome p450 reductase inhibitor and tyrosinase inhibitor that contain mimosine or derivative thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide excellent inhibitors of cyclooxygenase, NADPH-cytochrome P450 reductase, or tyrosinase.SOLUTION: A mimosine derivative is represented by the general formula (1) in the figure, where X represents an amino acid residue selected from the group consisting of L or D form phenylalanine (Phe), alanine (Ala), proline (ProP), valine (Val) and tryptophan (Trp)SELECTED DRAWING: Figure 4

Description

  The present invention relates to mimosine or a derivative thereof, and more particularly, to mimosine or a derivative thereof having excellent cyclooxynase inhibitory activity, NADPH-cytochrome P450 reductase inhibitory activity, and tyrosinase inhibitory activity.

  Most chemical reactions in the body are catalyzed by enzymes, and enzymes are also involved in various diseases. For this reason, substances that inhibit the action of specific enzymes are used as drugs. For example, nonsteroidal anti-inflammatory drugs (NSAIDs) have therapeutic effects on various inflammatory diseases such as rheumatoid arthritis, but NSAIDs are primarily rate-limiting enzymes in the conversion of arachidonic acid to prostaglandins. It is based on the inhibition of a certain cyclooxynase (COX). COX has two isozymes called COX-1 and COX-2. COX-1 is mainly expressed in mammalian cells and tissues, and has a function of producing prostaglandins that maintain homeostasis. On the other hand, COX-2 is expressed in the brain, liver, ovary, etc., and is induced in response to inflammatory stimuli. It is considered that a COX-2 selective inhibitor can be an ideal anti-inflammatory agent with little gastrointestinal inhibitory action (Non-Patent Documents 1 to 5).

  Cytochrome P450 (CYP) plays an important role in the oxidative metabolism of drugs and is a target for cancer treatment because of its role in in vivo activation and tumor formation and development. NADPH-cytochrome P450 reductase (CPR), an electron transfer diflavoprotein, is essential for the catalysis of many CYP reactions and supplies electrons to many other proteins and small molecules. CPR has also been reported to produce reactive oxygen species (ROS) that cause oxidative stress and DNA damage. For this reason, CPR inhibitors are considered to be useful in the treatment of diseases involving CYP and the suppression of reactive oxygen species (Non-Patent Documents 6 to 11).

  On the other hand, the formation of melanin in the skin has an effect of protecting against cancer caused by ultraviolet rays. However, abnormal accumulation of melanin causes skin disorders such as merisma, blotches, post-inflammatory melanosis, and solar pigmented spots. Melanin production is controlled by other enzymes such as tyrosinase, a tyrosinase-related protein (TRP-1), and dopachrome tomerase (TRP-2), which are important enzymes in the melanin production pathway. Tyrosinase hydroxylates tyrosine by oxidation of 3,4-dihydroxyphenylalanine to o-dopaquinone by monophenolase and diphenolase reactions. Thus, since tyrosinase plays an important role in melanogenesis, the inhibitor may be used as a whitening agent (Non-Patent Documents 12 to 18).

  Although some substances are known as inhibitors of these enzymes (Patent Documents 1 and 2), some of them are not sufficiently active and safe, and are still powerful and safe inhibitors derived from natural products. Is required.

JP 56-7710 A JP-A 63-174910

Gautam, R .; Jachak, SM; Kumar, V .; Mohan, CG Synthesis, biological evaluation and molecular docking studies of stellatin derivatives as cyclooxygenase (COX-1, COX-2) inhibitors and anti-inflammatory agents.Bioorg. Med. Chem. Lett. 2011, 21, 1612-1616. Ulbrich, H .; Fiebich, B .; Dannhardt, G. Cyclooxygenase-1 / 2 (COX-1 / COX-2) and 5-lipoxygenase (5-LOX) inhibitors of the 6,7-diary-2,3- 1H-dihydropyrrolizine type. Eur. J. Med. Chem. 2002, 37, 953-959. Jain, H. K .; Mourya, V. K .; Agrawal, R. K. Inhibitory mode of 2-acetoxyphenyl alkyl sulfides against COX-1 and COX-2: QSAR analyzes. Bioorg. Med. Chem. Lett. 2006, 16, 5280-5284. Vitale, P .; Tacconelli, S .; Perrone, MG; Malerba, P .; Simone, L .; Scilimati, A .; Lavecchia, A .; Dovizio, M .; Marcantoni, E .; Bruno, A .; Patrignani , P. Synthesis, pharmacological characterization, and docking analysis of a novel family of diarylisoxazoles as highly selective cyclooxygenase-1 (COX-1) inhibitors. J. Med. Chem. 2013, 56, 4277-4299. Gierse, J .; Nickols, M .; Leahy, K .; Warner, J .; Zhang, Y .; Cortes-Burgos, L .; Carter, J .; Seibert, K .; Masferrer, J. Evaluation of COX- 1 / COX-2 selectivity and potency of a new class of COX-2 inhibitors. Eur. J. Pharmacol. 2008, 588, 93-98. Pandit, S .; Mukherjee, PK; Mukherjee, K .; Gajbhiye, R .; Venkatesh, M .; Ponnusankar, S .; Bhadra, S. Cytochrome P450 inhibitory potential of selected Indian spices-possible food drug interaction. Int. 2012, 45, 69-74. Sridhar, J .; Liu, J .; Foroozesh, M .; Stevens C. L. K. Inhibition of cytochrome P450 enzymes by quinones and anthraquinones. Chem. Res. Toxicol. 2012, 25, 357-365. Gan, L .; von Moltke, LL; Trepanier, LA; Harmatz, JS; Greenblatt, DJ; Court, MH Role of NADPH-cytochrome P450 reductase and cytochrome-b5 / NADH-b5 reductase in variability of CYP3A activity in human liver microsomes Drug Metab. Dispos. 2009, 37, 90-96. Gray, JP; Mishin, V .; Heck, DE; Laskin, DL Laskin, JD Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.Toxicol.Appl. Pharmacol. 2010, 247, 76-82. Pandey, A. V .; Fluck, C. E. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol. Ther. 2013, 138, 229-254. Pillai, V. C .; Mehvar, R. Inhibition of NADPH-cytochrome P450 reductase by tannic acid in rat liver microsomes and primary hepatocytes: methodological artifacts and application to ischemia-reperfusion injury.J. Pharm. Sci. 2011, 100, 3495-3505. Huh, S .; Jung, E .; Lee, J .; Roh, K .; Kim, J.-D .; Lee, J .; Park, D. Mechanisms of melanogenesis inhibition by propafenone. Arch Dermatol Res. 2010, 302, 561-565. Morikawa, T .; Nakanishi, Y .; Ninomiya, K .; Matsuda, H .; Nakashima, S .; Miki, H .; Miyashita, Y .; Yoshikawa, M .; Hayakawa, T .; Muraoka, O. Dimeric pyrrolidinoindoline-type alkaloids with melanogenesis inhibitory activity in flower buds of Chimonanthus praecox. J Nat Med. 2014. DOI 10.1007 / s11418-014-0832-1. Lee, J .; Jung, E .; Park, J .; Jung, K .; Park, E .; Kim, J .; Hong, S .; Park, J .; Park, S .; Lee, S .; Park, D. Glycyrrhizin induces melanogenesis by elevating a cAMP level in B16 melanoma cells.J Invest Dermatol. 2005, 124, 405-411. Liu, H .; Sui, X .; Li, X .; Li, Y. Lyoniresinol inhibits melanogenic activity through the induction of microphthalmia-associated transcription factor and extracellular receptor kinase activation. Mol Cell Biochem. 2013, 373, 211-216. Song, K.-K .; Huang, H .; Han, P .; Zhang, C.-L .; Shi, Y .; Chen, Q.-X. Inhibitory effects of cis- and trans-isomers of 3, 5-dihydroxystilbene on the activity of mushroom tyrosinase. Biochem. Biophys. Res. Commun. 2006, 342, 1147-1151. Delogu, G .; Podda, G .; Corda, M .; Fadda, MB; Fais, A .; Era, B. Synthesis and biological evaluation of a novel series of bis-salicylaldehydes as mushroom tyrosinase inhibitors. Bioorg. Med. Chem Lett. 2010, 20, 6138-6140. An, S. M .; Kim, H. J .; Kim, J.-E .; Boo, Y. C. Flavonoids, taxifolin and luteolin attenuate cellular melanogenesis despite increasing tyrosinase protein levels. Phytother. Res. 2008, 22, 1200-1207.

  Accordingly, an object of the present invention is to provide a highly safe inhibitor having excellent inhibitory activity against cyclooxygenase, NADPH-cytochrome P450 reductase (CPR) or tyrosinase.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that mimosine contained in tropical to subtropical plants such as ginnemu (Gingoukan) and mimosa, and mimosine dipeptide having a specific amino acid bound thereto, The present inventors have found that they exhibit excellent inhibitory activity against cyclooxygenase, CPR, and tyrosinase, and have completed the present invention.

（式中、XはL体又はD体のフェニルアラニン（Phe）、アラニン（Ala）、プロリン（Pro）
、バリン（Val）及びトリプトファン（Trp）よりなる群から選ばれるアミノ酸残基を示
す）
で表されるミモシン誘導体である。 That is, the present invention provides the following general formula (1);

(In the formula, X is L-form or D-form phenylalanine (Phe), alanine (Ala), proline (Pro)
, Amino acid residues selected from the group consisting of valine (Val) and tryptophan (Trp)
It is a mimosine derivative represented by

  The present invention is also a cyclooxygenase inhibitor, CPR inhibitor or tyrosinase inhibitor containing the mimosine derivative or mimosine as an active ingredient.

  The mimosine or derivative thereof used in the present invention has excellent cyclooxygenase inhibitory action, NADPH-cytochrome P450 reductase (CPR) inhibitory action, and tyrosinase inhibitory action, and has low cytotoxicity and excellent safety.

It is a figure which shows the reaction scheme of a mimosin dipeptide. It is a line weaver bark plot in the presence of 0-15 μM of each mimosine derivative. It is a graph which shows the influence of each mimosine derivative with respect to B16F10 cell viability. It is a graph which shows the inhibition rate of each mimosine derivative with respect to intracellular tyrosinase.

で表されるものである。 The mimosine derivative of the present invention has the following general formula (1):

It is represented by

  Mimosine (β- [N- (3-hydroxy-4-pyridone)]-α-aminopropionic acid) is a non-protein amino acid having an alanine side chain bonded to the nitrogen atom of the pyridine ring. The mimosine derivative of 1) is a dipeptide in which one amino acid is bound to this mimosine.

  In the general formula (1), X is not particularly limited as long as it is an amino acid residue. For example, alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D) ) Cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), Lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), valine (Val; V) and the like are exemplified. Of these, phenylalanine (Phe; F), alanine (Ala; A), proline (Pro; P), valine (Val; V), and tryptophan (Trp; Y) are preferred, and among them, cyclooxygenase inhibitory action, NADPH -Since it is excellent in cytochrome P450 reductase (CPR) inhibitory action, tyrosinase inhibitory action, etc., phenylalanine (Phe; F), alanine (Ala; A), proline (Pro; P), valine (Val; V), tryptophan (Trp) Y) is preferred. X has an amide bond with mimosine on the N-terminal side.

  When an optical isomer is present in the amino acid constituting the group X, it may be D-form or L-form, but D-form is particularly excellent in CPR inhibitory activity and tyrosinase inhibitory activity.

  As a preferred example of the mimosine derivative of the present invention, since it has excellent cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitory activity, as group X, D-Ala, L-Pro, D-Pro , L-Val, L-Trp and D-Trp are preferred. L-Pro, L-Val, L-Trp, and D-Trp are preferable because of their high cyclooxynase-2 (COX-2) inhibitory activity, and L-Val and L-Trp are more preferable. L-Val is suitable as an anti-inflammatory agent because of its high COX-2 selectivity. Moreover, since it is excellent in CPR inhibitory activity, D-Phe, D-Ala, D-Pro, D-Val, L-Trp, and D-Trp are preferable, and L-Trp and D-Trp are particularly preferable. On the other hand, D-Phe, D-Ala, L-Pro, D-Pro, L-Val, D-Val, L-Trp, and D-Trp are preferred because of their excellent tyrosinase inhibitory activity, and L-Pro, L-Pro Val and D-Val are more preferable, and D-Val is particularly preferable.

で表すこともできる。 A preferred mimosine derivative of the present invention is represented by the following general formula (2):

It can also be expressed as

  In the general formula (2), R represents a group selected from the group consisting of a benzyl group, a methyl group, a pyrrolidine group, an isopropyl group, and a 3-indolylmethyl group.

  In the above general formula (2), * is a symbol indicating the absolute configuration (S or R) of the asymmetric carbon. The mimosine derivatives of the present invention include mixtures thereof including enantiomers, diastereomers and racemates.

Examples of suitable mimosine derivatives of the present invention include the following (Mi represents a mimosine residue).
Mi-L-Phe
Mi-D-Phe
Mi-L-Ala
Mi-D-Ala
Mi-L-Pro
Mi-D-Pro
Mi-L-Val
Mi-D-Val
Mi-L-Trp
Mi-D-Trp

  The mimosine derivative of the present invention represented by the above general formula (1) or (2) can be produced, for example, by the following method.

  Mimosine is found in tropical and subtropical plants such as Ginnemu and Mimosa. This Ginnemu is an evergreen shrub belonging to the genus Ginkgoceae, distributed from the tropics to subtropical Asia, and in Japan from Okinawa Prefecture to the southern part of Kyushu.

  In order to obtain mimosine from the leaves of this gynem, first, the leaves of the gynem, preferably fresh young leaves, are preferably chopped or shredded to obtain a raw material for extraction.

  Next, an appropriate amount of water is heated with respect to the extraction raw material prepared as described above, and extracted with the obtained hot water. This hot water extraction can be performed with hot water at 70 ° C. or higher, preferably 75 ° C. to boiling. However, in order to deactivate the mimosine degrading enzyme and obtain highly pure mimosine, boiling water (100 ° C. It is particularly preferable to use hot water. The extraction time is about 5 to 30 minutes, and it is particularly preferable to perform boiling extraction for about 10 minutes. As an extraction solvent used for extraction, distilled water is preferable, and it is desirable to stir continuously or intermittently as necessary during extraction.

  A strong cation exchange resin is added to this ginnemu leaf extract to adsorb adsorbed components including mimosine. Next, the ion exchange resin is washed with water or a water-ethanol mixed solution and then immersed in ammonia water or the like to elute mimosine from the ion exchange resin. The eluate is treated with activated carbon as necessary, concentrated, and left at a low temperature to precipitate a mimosine salt. By collecting this, mimosine can be obtained. The obtained mimosine may be purified by means such as recrystallization as necessary.

  The mimosine derivative of the present invention can be obtained by binding an amino acid to the mimosine thus obtained using a known peptide synthesis method such as a peptide solid phase synthesis method.

  Mimosine and amino acids preferably protect amino groups with protecting groups such as 9-fluorenylmethyloxycarbonyl group (Fmoc) and t-butyloxycarbonyl group (Boc).

  Examples of the condensing agent for forming a peptide bond include diisopropylcarbodiimide (DIC), N, N-dicyclohexylcarbodiimide (DCC), and the like. Further, these condensing agents can be used by mixing with N-hydroxybenzotriazole (HOBt).

  Boc and Fmoc, which are protecting groups for the amino terminal amino group of a peptide or amino acid, can be removed with trifluoroacetic acid (TFA), piperidine and the like.

  Moreover, Wang resin etc. can be used as a peptide solid-phase synthetic resin. For releasing the peptide from the peptide solid phase synthetic resin, for example, TFA is used.

  A reaction scheme according to the Fmoc solid phase synthesis method for producing the mimosine derivative of the present invention is shown in FIG. In this scheme, Fmoc-mimosine is prepared by coupling the Fmoc group of N- (9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) to mimosine, which is linked to the amino acid bound to Wang resin. By binding, mimosine dipeptide is formed. More specific description will be given below.

(Preparation of Fmoc-mimosine)
Mimosine and sodium carbonate are dissolved in distilled water, and a solution of Fmoc-OSu in dioxane is added dropwise to this solution and incubated overnight at room temperature. Then, after adding sodium carbonate solution and stirring, the solution is filtered and then washed with ethyl acetate to remove unreacted Fmoc-OSu and by-products. Crystals of Fmoc-mimosine are precipitated by lowering the pH of the water fraction to about 4 using hydrochloric acid in an ice bath.

(Solid-phase synthesis of mimosine dipeptide)
Fmoc-amino acid (L-form or D-form), 1-hydroxy-1H-benzotriazole (HOBt) and N, N'-diisopropylcarbodiimide (DIC) added to N, N-dimethylformamide (DMF) solution in dichloromethane Add Wang resin swelled in, and stir. The resin is filtered, washed with DMF, dichloromethane and methanol and dried under vacuum conditions. After deprotection of Fmoc with 25% piperidine, a mixed solution of Fmoc-mimosine, HOBt, HBTU and N, N-diisopropylethylamine (DIEA) was added and stirred to bind Fmoc-mimosine to amino acids. To form a dipeptide. After shaking with 95% trifluoroacetic acid (TFA), the resin is filtered, washed with TFA, and the mimosine dipeptide is obtained from the resulting filtrate by precipitation with ice-cold diethyl ether.

  The mimosine derivative of the present invention obtained as described above can be used as a cyclooxygenase inhibitor, CPR inhibitor or tyrosinase inhibitor as it is or after purification by a known method such as liquid high-performance chromatography as necessary. Can do.

  On the other hand, not only mimosine derivatives but also mimosine itself has excellent cyclooxygenase inhibitory action and CPR inhibitory action, so that mimosine can be used as an active ingredient of the cyclooxygenase inhibitor or CPR inhibitor of the present invention.

  For example, the preparation of a cyclooxygenase inhibitor or CPR inhibitor containing mimosine or a mimosine derivative as an active ingredient can be carried out by treating a therapeutically effective amount of mimosine or a mimosine derivative with any pharmaceutically acceptable ingredient such as a conventional excipient, binder. , A lubricant, an aqueous solvent, an oily solvent, an emulsifier, a suspending agent, a preservative, a stabilizer, and the like.

  The cyclooxygenase inhibitor or CPR inhibitor of the present invention can be administered either orally or parenterally. In the case of oral administration, the cyclooxygenase inhibitor or CPR inhibitor of the present invention is a usual oral preparation, for example, solid preparations such as tablets, powders, granules, capsules, etc .; liquids; oily suspensions; or syrups or It can be used also as any dosage form of liquid agents, such as an elixir. In the case of parenteral administration, the cyclooxygenase inhibitor or CPR inhibitor of the present invention can be used as an aqueous or oily suspension injection or nasal solution.

  The cyclooxygenase inhibitor or CPR inhibitor of the present invention varies depending on the administration method, patient age, body weight, condition, and type of disease, but is usually about 10 to 200 mg per day for oral administration, preferably Is about 10 to 20 mg, and this may be divided into several doses as needed. In the case of parenteral administration, about 5 to 100 mg, preferably about 5 to 10 mg per day for an adult may be administered.

  The cyclooxygenase inhibitor of the present invention effectively inhibits COX-1 and / or COX-2. In particular, Mi-D-Ala, Mi-L-Pro, Mi-D-Pro, Mi-L-Val, Mi-L-Trp, Mi-D-Trp, and mimosine are suitable as COX-1 inhibitors. -1 inhibition can treat, prevent, or ameliorate a disease or condition that can be treated, prevented, or ameliorated. Examples of diseases or symptoms that can be treated, prevented or ameliorated by COX-1 inhibition include prevention of inflammation and thrombus formation. Mi-L-Pro, Mi-L-Val, Mi-L-Trp, Mi-D-Trp, mimosine are suitable as COX-2 inhibitors, especially Mi-L-Val, MI-L-Trp, mimosine Is preferred. A disease or condition that can be treated, prevented, or ameliorated by COX-2 inhibition can be treated, prevented, or ameliorated. Examples of the disease or symptom that can be treated, prevented or ameliorated by COX-2 inhibition include inflammation, arthritis, Alzheimer, pain, colon cancer and the like. Mi-L-Val and mimosine are suitable as anti-inflammatory agents with little gastrointestinal disorder because of high COX-2 selectivity. The CPR inhibitor of the present invention effectively inhibits CPR, which is the rate-limiting enzyme of cytochrome P450 catalytic reaction, and CPR serves as an electron donor for many proteins and low molecular weight compounds including heme oxygenase, squalene monooxygenase, and cytochrome b5. Since it works, it becomes a therapeutic, preventive or ameliorating agent for diseases and symptoms involving these in addition to active oxygen scavengers. As the CPR inhibitor, Mi-D-Phe, Mi-D-Ala, Mi-D-Pro, Mi-D-Val, Mi-L-Trp, Mi-D-Trp are suitable, and especially Mi-L- Trp and Mi-D-Trp are preferred.

  On the other hand, the tyrosinase inhibitor of the present invention contains the mimosine derivative as an active ingredient. Mimosin derivatives include Mi-D-Phe, Mi-D-Ala, Mi-L-Pro, Mi-D-Pro, Mi-L-Val, Mi-D-Val, Mi-L-Trp, Mi-D- Trp is preferable, Mi-L-Pro, Mi-L-Val, and Mi-D-Val are more preferable, and Mi-D-Val is particularly preferable. The tyrosinase inhibitor can be formulated as an oral or parenteral pharmaceutical for the treatment of skin abnormalities such as hyperpigmentation and hypopigmentation, in the same manner as the cyclooxygenase inhibitor or CPR inhibitor. It can also be used as an external preparation for skin, such as purified water, alcohols, water-soluble polymers, oils, surfactants, gelling agents, moisturizers, vitamins, antibacterial agents, fragrances, salts, pH adjusters, etc. It can be prepared by adding the above ingredients.

  The amount of the tyrosinase inhibitor of the present invention to be added varies depending on various conditions such as the type of addition target, administration route, dosage form, etc., for example, 2 mass% to 5 mass% is contained in the pharmaceutical preparation (total weight). Is preferred.

  The dose of the tyrosinase inhibitor of the present invention is not particularly limited and can be appropriately determined according to the patient's age, sex, body weight, degree of symptoms, administration method, etc. In the case of administration, it is about 10 to 200 mg per day for an adult, preferably about 10 to 20 mg. In the case of parenteral administration, about 5 to 100 mg, preferably about 5 to 10 mg per day may be administered.

  Furthermore, since the tyrosinase inhibitor of the present invention suppresses the production and deposition of melanin and has a whitening effect, it can be blended into cosmetics to obtain a whitening cosmetic. For example, about 3 to 5% by mass of a tyrosinase inhibitor is added to a known cosmetic base, and in the form of a solution, solubilized, emulsified, powdered, pasty, mousse or gel according to a conventional method And is provided as a lotion, emulsion, cream, pack, ointment and the like.

  Further, in the production of the above-mentioned whitening cosmetics, components that are usually used in cosmetics, that is, purified water, alcohols, water-soluble polymers, oils, as long as the effects of the present invention are not impaired as necessary. Surfactants, gelling agents, humectants, vitamins, antibacterial agents, fragrances, salts, pH adjusters and the like can be added.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Reference example 1
Preparation of mimosine:
Ginnemu leaves 1.5 kg collected around the Faculty of Agriculture, University of the Ryukyus were boiled in 5 L of water for 10 minutes. After cooling the extract, it was filtered by suction filtration (manufactured by AS ONE, Shaking Baths SB-20), and 2 kg of ion exchange resin (Amberlite IR120 plus (H)) was added to the filtrate. The extract / resin mixture was stirred for 30 minutes and then left overnight. The ion exchange resin was rinsed 5-6 times with distilled water, and 5 L of 80% ethanol was added dropwise to remove chlorophyll. This resin was eluted with 6 L of 2N ammonium hydroxide to obtain crude mimosine. The eluate was concentrated to 300 mL under reduced pressure at 40 ° C., pH was adjusted to 4.5-5.0 with 6N hydrochloric acid, and placed in a freezer overnight for crystallization. The obtained crystals were adjusted to pH 9.0 using 5N NaOH, and then recrystallized by adding 6N HCl to pH 4.5 to 5.0, and further left at 4 ° C. to obtain purified mimosine. . Mimosine was stored at -20 ° C.

Manufacturing example 1
Preparation of mimosine derivatives (Mi-L-Phe):
Dipeptides were synthesized by binding amino acids to mimosine (Mi) by Fmoc solid phase synthesis. Dipeptides were formed using Fmoc L-amino acids obtained from Hypep Laboratories. A more specific production method is shown below.

(Preparation of Fmoc-mimosine)
A solution prepared by dissolving 6.25 g of Fmoc-Osu in 37.5 mL of dioxane was added dropwise to an aqueous solution prepared by dissolving 2.5 g of mimosine and 2.75 g of sodium carbonate in 37.5 mL of distilled water. The mixture was stirred at room temperature for 20 hours and then 150 mL of sodium carbonate solution (0.1 M) was added. After stirring at 25 ° C. for 7 hours, the resulting solution was filtered and washed with 75 mL of ethyl acetate to remove Fmoc-OSu and by-products. In an ice bath, the pH of the water fraction was lowered to 4 with 6N hydrochloric acid, and left overnight at 4 ° C. The crystals were filtered, washed with distilled water, and dried under reduced pressure to obtain purified Fmoc-mimosine.

(Solid-phase synthesis of mimosine dipeptide)
Fmoc-L-Phe 2.35 mmol, 1-hydroxy-1H-benzotriazole (HOBt) 2.35 mmol (360 mg) and N, N′-diisopropylcarbodiimide (DIC) 2.35 mmol (365 μL) pre-activated in 5 mL DMF, To this solution was added 0.5 g of Wang resin expanded in 2 mL of dichloromethane for 30 minutes, and then a solution of 0.235 mmol (29 mg) N, N-dimethyl-4-aminopyridine (DMAP) in 500 μL DMF was added. added. The mixture was stirred at room temperature for 3 hours. This operation was repeated twice. This resin was washed with DMF, dichloromethane and methanol and dried under reduced pressure. Resin (5 mg) was used for analysis of Fmoc group content. After permeating at room temperature for 45 minutes and deprotecting Fmoc with 25% piperidine, Fmoc mimosine (4 equivalents to the amino acid of the resin), HOBt (4 equivalents), HBTU (3.6 equivalents): N, N- A DMF suspension of amino acid-resin was added to a mixture containing diisopropylethylamine (DIEA; 8 equivalents), and the mixture was stirred at room temperature for 1 hour.

  A Kaiser test was performed to examine the integrity of the binding. After final cleavage, the resin was shaken vigorously with 95% trifluoroacetic acid for 90 minutes, filtered and washed with TFA. The washing solution was precipitated with ice-cooled diethyl ether. The resulting precipitate was filtered, washed with diethyl ether, and then dried under reduced pressure to obtain the desired mimosine dipeptide (Mi-L-Phe). The obtained crude peptide was a white solid, and the yield was 132 mg. This crude peptide was further purified by liquid chromatography under the following conditions.

(HPLC conditions)
Column: Phenomenex column (150 x 14.6 mm; 4 μm)
Mobile phase: solvent A: water / 0.1% TFA, solvent B: acetonitrile / 0.1% TFA
Flow rate: 1mL / min Absorption wavelength: 210nm

The ¹ H and ¹³ C NMR spectra of the mimosine dipeptide (Mi-L-Phe) obtained are shown below. The ¹ H and ¹³ C spectra were recorded with a D ₂ O JEOL JNM-ECA400 (JEOL, Japan). Chemical shifts were expressed in ppm (δ) associated with TMS.

¹ H NMR (D ₂ O, 400 MHz) δ 7.44 (s, 1H, OH), 7.32-7.37 (m, 4H, aromatic), 6.60 (s, 1H, OH), 4.57 (s, 1H, NH), 4.43 (s, 2H, NH ₂ ), 3.99-4.19 (q, 1H, CH), 3.31 (d, 2H, CH ₂ ), 3.26 (d, 2H, CH ₂ ), 3.15 (d, 2H, CH ₂ ) , _{3.09 (d, 2H, CH 2} ). 13 C NMR (D 2 O, 400 MHz) δ 181.52, 173.89, 148.2, 140.2, 135.05, 129.32, 129.07, 127.65, 114.79, 97.49, 71.12, 55.99, 36.30. ESI- MS: m / z 345.13.

Manufacturing example 2
Preparation of mimosine derivatives (Mi-D-Phe):
Mimosine dipeptide (Mi-D-Phe) was obtained in the same manner as in Production Example 1 except that Fmoc-D-phenylalanine (Phe) was used as the Fmoc amino acid to be bound (yield 48.5 mg).

Manufacturing example 3
Preparation of mimosine derivatives (Mi-L-Ala):
A mimosine dipeptide (Mi-L-Ala) was obtained in the same manner as in Production Example 1 except that Fmoc-L-alanine (Ala) was used as the Fmoc amino acid to be bound (yield 45 mg). ¹ H and ¹³ C spectra of the obtained mimosine dipeptide (Mi-L-Phe) are shown.

δ 7.70 (s, 1H, OH), 6.61 (s, 1H, OH), 4.64 (s, 1H, NH), 4.45 (s, 2H, NH ₂ ), 4.20 (q, 1H, CH), 3.79-3.81 (m, 4H, aromatic), 3.75 (q, 1H, CH), 1.65 (d, 2H, CH ₂ ), 1.49 (d, 2H, CH ₂ ), 1.30 (d, 2H, CH ₃ ). ¹³ C NMR (D ₂ O, 400 MHz) δ 181.55, 175.75, 148.2, 140.2, 138.52, 130.70, 129.07, 127.65, 114.82, 97.79, 50.47, 42.80, 16.08. ESI- MS: m / z 269.10.

Manufacturing example 4
Preparation of mimosine derivatives (Mi-D-Ala):
Mimosine dipeptide (Mi-D-Ala) was obtained in the same manner as in Production Example 1 except that Fmoc-D-alanine (Ala) was used as the Fmoc amino acid to be bound (yield 79 mg).

Manufacturing example 5
Preparation of mimosine derivatives (Mi-L-Pro):
A mimosine dipeptide (Mi-L-Pro) was obtained in the same manner as in Production Example 1 except that Fmoc-L-proline (Pro) was used as the Fmoc amino acid to be bound (yield 105.5 mg).

Manufacturing Example 6
Preparation of mimosine derivatives (Mi-D-Pro):
Mimosine dipeptide (Mi-D-Pro) was obtained in the same manner as in Production Example 1 except that Fmoc-D-proline (Pro) was used as the Fmoc amino acid to be bound (yield 43 mg).

Manufacturing example 7
Preparation of mimosine derivatives (Mi-L-Val):
A mimosin dipeptide (Mi-L-Val) was obtained in the same manner as in Production Example 1 except that Fmoc-L-valine (Val) was used as the Fmoc amino acid to be bound (yield 17 mg).

Manufacturing Example 8
Preparation of mimosine derivatives (Mi-D-Val):
A mimosin dipeptide (Mi-D-Val) was obtained in the same manner as in Production Example 1 except that Fmoc-D-valine (Val) was used as the Fmoc amino acid to be bound (yield 25 mg).

Manufacturing example 9
Preparation of mimosine derivatives (Mi-L-Trp):
Mimosine dipeptide (Mi-L-Trp) was obtained in the same manner as in Production Example 1 except that Fmoc-L-tryptophan (Trp) was used as the Fmoc amino acid to be bound (yield 98 mg). ¹ H and ¹³ C spectra of the obtained mimosine dipeptide (Mi-L-Trp) are shown.

δ 7.52 (s, 1H, OH), 7.31 (s, 1H, NH), 7.28 (s, 1H, OH), 7.17-7.26 (s, 1H, CH), 6.58 (d, 1H, NH), 4.52 ( s, 2H, CH), 4.40 (s, 1H, CH), 4.16 (s, 1H, CH), 4.03 (m, 1H, CH), 3.51 (t, 2H, NH ₂ ), 3.49 (s, 1H, CH), 3.46 (m, 2H, CH ₂ ), _{3.27 (m, 2H, CH 2} ). 13 C NMR (D 2 O, 400 MHz) δ 181.52, 174.43, 148.2, 138.31, 136.34, 132.77, 124.97, 122.08, 119.41, 118.39, 111.89, 107.04, 98.99, 90.77, 68.44, 55.00, 48.07, 26.32. ESI-MS: m / z 384.14.

Manufacturing example 10
Preparation of mimosine derivatives (Mi-D-Trp):
Mimosine dipeptide (Mi-D-Trp) was obtained in the same manner as in Production Example 1 except that Fmoc-D-tryptophan (Trp) was used as the Fmoc amino acid to be bound (yield 71.5 mg).

Example 1
Cyclooxygenase (COX) inhibition test:
In the experiment, Colorimetric COX (ovine) Inhibitor Screening Assay (cat. 760111, Cayman) was used. To a 96-well microtiter plate coated with an anti-rabbit IgG antibody, 150 μL of Assay Buffer (0.1 M Tris-HCl, pH 8.0), 10 μL of hem and 10 μL of enzyme (COX-1 or COX-2) were added. Immediately, 10 μL of the mimosine obtained in Reference Example 1 or the mimosine dipeptide sample prepared in Production Example was added to the inhibitor well, and 10 μL of the solvent or Buffer was added to the 100% active well and the background well. After incubation at 25 ° C. for 5 minutes, 20 μL of colorimetric substrate was added to all wells, and 20 μL of 22 mM arachidonic acid was added. The mixture was shaken and then further incubated at 25 ° C. for 5 minutes. Absorbance at 590 nm was measured using a microplate reader (Benchmark Plus, Biorad, UK). Indomethacin was used as a positive control. The absorbance after correction of all samples and 100% active well was calculated by subtracting the absorbance of the back ground well, and the inhibition rate was calculated by the following formula. The results are shown in Table 1.
Inhibition (%) = (Ao-As) / Ao x 100
Ao: Absorbance of 100% activity after correction
As: Absorbance of the sample after correction

In all tests, statistical processing was performed using statistical analysis system (SAS) software Version 9.1.3 (SAS Institute Inc.). In significance analysis, data was analyzed by one-way analysis of variance ANOVA, and significant differences in mean values were determined by Duncan test (P ≦ 0.05). IC ₅₀ was determined from the graph as the concentration required for each sample to exhibit 50% inhibitory activity.

Mimosine showed strong inhibitory activity against COX-1 and COX-2, but IC ₅₀ was 28.86 μM and 20.93 μM, respectively, indicating that it is a stronger inhibitor against COX-2 than COX-1. Indicated. Mimosine has COX-2 inhibitory activity comparable to indomethacin with an IC ₅₀ of 27.70 μM. Therefore, mimosine is useful as a selective COX-2 inhibitor, an anti-inflammatory agent that reduces gastrointestinal disorders, or a therapeutic / preventive agent for inflammation-related diseases. All mimosine dipeptides showed COX-1 inhibitory action superior to mimosine, and their IC ₅₀ ranged from 17.91 to 26.18 μM, compared to 28.86 μM for mimosine. Mi-L-Val (IC ₅₀ 21.72 μM) and Mi-L-Trp (IC ₅₀ 19.31 μM) showed COX-2 inhibitory action superior to indomethacin. Mi-L-Trp showed the highest inhibitory activity against COX-1 and COX-2, and was higher than mimosine.

Example 2
NADPH-cytochrome P450 reductase inhibition test:
Inhibitory activity was measured using a cytochrome c reductase assay kit (Sigmaaldrich, Japan). The analytical principle is based on the measurement of cytochrome c by CPR in the presence of NADPH. Analysis was performed at 25 ° C. using cytochrome c as a substrate. Specifically, 950 μL of a diluted standard containing 0.3 M potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.45 mg / mL cytochrome c was transferred to a 1 mL cuvette, and 20 μL of CPR and the sample of mimosine of Reference Example 1 or the production example were used. 30 μL of mimosine dipeptide was added. Thereafter, 20 μL of 50 mM potassium cyanide aqueous solution was added to the reaction solution to stop the reaction. The reaction was initiated by the addition of 100 μL NADPH solution (0.85 mg / mL). Absorbance at 550 nm was measured with a spectrophotometer. The value given when no enzyme was added was used as a blank. Inhibitory activity was measured as a percentage relative to a blank using 1% water as 100%, and IC ₅₀ was determined from the result. The results are shown in Table 2.

Mimosine showed a high inhibitory effect on CPR with an IC _{50 of} 5.19 μM. All mimosine dipeptides showed strong CPR inhibitory activity in the IC ₅₀ range of 8.06 to 31.08 μM. For any amino acid, the CPR inhibitory activity of D-amino acid was higher than that of L-amino acid.
CPR, the rate-limiting enzyme for cytochrome P450 catalysis, acts as an electron donor for many proteins and small molecules including heme oxygenase, squalene monooxygenase, and cytochrome b5. For this reason, it is considered that mimosine may have a novel role in medicine, steroids, fatty acid metabolism, heme catabolism, and sterol biosynthesis.

Example 3
Tyrosinase inhibition test:
First, samples (20 μL) of various concentrations are taken into each well of a 96-well plate, and 120 μL of 20 mM sodium phosphate buffer (pH 6.8) and 20 μL of 500 U / mL mushroom tyrosinase enzyme dissolved in the buffer are added thereto. Mixed. The mixture was incubated at 25 ° C. for 15 minutes, then 20 μL of 0.85 mM L-tyrosine solution was added. Absorbance at 470 nm was recorded using a microplate reader (Benchmark Plus, Violad, UK). Kojic acid was used as a positive control.
Sample compounds that showed strong activity were reacted in a range of constant substrate concentrations (L-tyrosine) for several inhibitor concentrations for kinetic studies. The preincubation and measurement time were the same as described above. The inhibition mode of the enzyme was determined by the Lineweaver-Burk plot. The inhibition constant Ki was determined by replotting the Km / Vmax against the inhibitor concentration. The inhibition rate was calculated by the following formula, and IC ₅₀ was determined from this.

Inhibition (%) = [(C _E -C _O )-(S _E -S _O )] / (C _E -C _O ) × 100
C _E : Absorbance of control containing enzyme
C _O : Absorbance of control without enzyme
S _E : Absorbance of sample containing enzyme
S _O : Absorbance of sample without enzyme

All synthetic mimosine dipeptides showed better tyrosinase inhibitory activity than mimosine. Among amino acids, tryptophan, valine or proline dipeptides showed high tyrosinase inhibitory activity in both L and D forms. For all amino acids, the D-form tended to have higher inhibitory activity than the L-form. In particular, four mimosin dipeptides, Mi-L-Pro, Mi-D-Trp, Mi-L-Val and Mi-D-Val, showed excellent inhibitory activity, with IC ₅₀ of 13.11, 16.91, 11.52 and 10.39 μM, respectively. there were.
The kinetics of these four mimosine dipeptides were investigated. FIG. 2 shows a line weaver-Burk plot in which the horizontal axis represents the reciprocal 1 / [S] of the substrate concentration and the vertical axis represents the inverse 1 / v of the reaction rate at that time. The Km value increased with increasing inhibitor concentration, but Vmax did not change, so it was considered to be a competitive inhibitor. Table 4 shows the Ki values of these compounds. The inhibition constant of Mi-D-Val (0.021 mM) is lower than Mi-L-Val, Mi-L-Pro and Mi-D-Trp, indicating that Mi-D-Val has the most potent effect. It was done.

Example 4
Cytotoxicity test:
(B16F10 cell culture)
B16F10 cells are cultured in Dulbecco's Modified Minimum Essential Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in an environment with a temperature of 37 ° C, CO ₂ concentration of 5%, and saturated humidity. did.

(Cell viability analysis)
B16F10 cell viability is described in the literature (Tada, H .; Shiho, O .; Kuroshima, K.-I .; Koyama, M .; Tsukamoto, K. An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986, 93, 157-165.). This method is based on the fact that 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazolium compound, is cleaved by an enzyme and converted into purple formazan crystals. . B16F10 cells were seeded in each well at a density of 5 × 10 ³ cells / well. After culturing for 24 hours, the sample compound was added to DMEM containing 10% FBS and 1% penicillin / streptomycin. B16F10 cells were cultured for 40 hours in an environment of a temperature of 37 ° C., a CO ₂ concentration of 5%, and a saturated humidity. Thereafter, 20 μL of 0.5 mg / mL MTT solution was added to each well and cultured for 3 hours. The medium was removed and formazan was dissolved in 200 μL DMSO. The plate was shaken for 10 minutes and the absorbance at 590 nm was measured using a microplate reader (Benchmark Plus, Violad, UK). Mimosine (100 μM) and kojic acid (500 μM) were used as positive controls. Dimethyl sulfoxide (DMSO) was used as a blank. The absorbance of the blank was subtracted from the absorbance of the sample and control, the absorbance of the corrected sample was divided by the absorbance of the corrected control, and multiplied by 100 to determine the percentage of cell viability. The results are shown in Figure 3. Most mimosin dipeptides did not show cytotoxicity up to a concentration of 200 μM.

Example 5
Intracellular tyrosinase inhibition test:
B16F10 cells were seeded in a 96-well plate at a density of 5 × 10 ³ cells / well, cultured for 24 hours, and then a sample compound (10-500 μM) was added. After 48 hours of incubation, wash twice with 50 mM ice-cold phosphate buffer (pH 6.8), dissolve in 90 μL of 50 mM phosphate buffer (pH 6.8) containing 1% Triton-X, and at -80 ° C. Frozen for 30 minutes. After thawing and mixing, 20 μL of 5% L-DOPA was added to each well. This mixture was incubated at 37 ° C. for 2 hours, and the absorbance was measured at 490 nm. Mimosine (100 μM) and kojic acid (500 μM) served as positive controls. The results are shown in FIG.

  As shown in FIG. 4, all mimosine dipeptides suppressed intracellular tyrosinase activity by 56-72% at 200 μM.

  Since the mimosine derivative or mimosine of the present invention exhibits excellent cyclooxygenase inhibitory activity and NADPH-cytochrome P450 reductase inhibitory activity, it can be used as an anti-inflammatory agent, an active oxygen scavenger, or a medicine for treating or preventing a disease associated with them. It is a thing.

  In addition, since the mimosine derivative of the present invention has an excellent tyrosinase inhibitory activity, it can be used as a pharmaceutical or a whitening cosmetic for preventing or treating skin disorders.

Claims (5)

The following general formula (1);

(Wherein X is L or D form phenylalanine (Phe), alanine (Ala), proline (Pro)
, Amino acid residues selected from the group consisting of valine (Val) and tryptophan (Trp)
A mimosine derivative represented by:   A cyclooxygenase inhibitor comprising the mimosine derivative or mimosine according to claim 1 as an active ingredient.   A NADPH-cytochrome P450 reductase inhibitor comprising the mimosine derivative or mimosine according to claim 1 as an active ingredient.   A tyrosinase inhibitor comprising the mimosine derivative according to claim 1 as an active ingredient. A whitening agent comprising the tyrosinase inhibitor according to claim 4.

Images