JP5950390B2 - New mimosine derivatives - Google Patents
New mimosine derivatives Download PDFInfo
- Publication number
- JP5950390B2 JP5950390B2 JP2012068783A JP2012068783A JP5950390B2 JP 5950390 B2 JP5950390 B2 JP 5950390B2 JP 2012068783 A JP2012068783 A JP 2012068783A JP 2012068783 A JP2012068783 A JP 2012068783A JP 5950390 B2 JP5950390 B2 JP 5950390B2
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- Prior art keywords
- tyr
- mimosine
- phe
- fmoc
- group
- Prior art date
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Description
本発明は、ミモシン誘導体に関し、更に詳細には、優れたインフルエンザウイルスノイラミニダーゼ阻害活性およびチロシナーゼ阻害活性を有するミモシンテトラペプチドに関する。 The present invention relates to a mimosine derivative, and more particularly to a mimosine tetrapeptide having excellent influenza virus neuraminidase inhibitory activity and tyrosinase inhibitory activity.
インフルエンザウイルスによる感染には、宿主細胞に対する接着と脱離が重要であるが、この過程には、ヘマグルチニンとノイラミニダーゼ(シアリダーゼ)という2種類のタンパク質が関与している。特にノイラミニダーゼは、インフルエンザウイルスが感染した細胞から脱離する上で必須の酵素である。このため、ノイラミニダーゼの活性を有効に阻害することができれば、インフルエンザウイルスが、感染した細胞表面から遊離・拡散することを阻害することができる。従って、ノイラミニダーゼの阻害は、結果として、インフルエンザウイルスによる他の細胞への感染・増殖を抑制し、インフルエンザウイルス感染症の治療に応用することができる。 Adhesion and detachment from host cells are important for influenza virus infection, and this process involves two proteins, hemagglutinin and neuraminidase (sialidase). In particular, neuraminidase is an enzyme essential for detachment from cells infected with influenza virus. For this reason, if the activity of neuraminidase can be effectively inhibited, the influenza virus can be inhibited from being released and diffused from the infected cell surface. Therefore, inhibition of neuraminidase results in suppression of infection and proliferation of other cells by influenza virus, and can be applied to the treatment of influenza virus infection.
このようなノイラミニダーゼ阻害活性を有する化合物について、いくつか報告されており(特許文献1ないし4)、その作用を利用した医薬品も開発されている。例えば、シアリダーゼの阻害剤であるタミフル(商品名、ロシュ社)(一般名称:リン酸オセルタミビル)や、リレンザ(商品名、グラクソ・スミスクライン社)(一般名称:ザナミビル)が知られており、インフルエンザの特効薬として処方されている。
しかし、これら医薬品は、アナフィラキシー様症状等の副作用や耐性ウイルスの出現等の問題があり、特に、タミフルを服用した患者が異常行動を起こして死亡する事故も複数報告されており、タミフル服用による精神・神経症状(意識障害、異常行動、譫妄、幻覚、妄想、痙攣など)も問題となっている。
また、報告されている上記のインフルエンザウイルスノイラミニダーゼ阻害活性を有する物質も、その多くが複雑な構造を有するものであって、その生産には煩雑な工程が要求され、副作用の問題も懸念されるものであった。
Several compounds having such neuraminidase inhibitory activity have been reported (
However, these drugs have problems such as side effects such as anaphylactoid symptoms and the emergence of resistant viruses.In particular, there have been reports of multiple accidents in which patients taking Tamiflu die due to abnormal behavior.・ Nerve symptoms (impaired consciousness, abnormal behavior, delirium, hallucinations, delusions, convulsions, etc.) are also a problem.
In addition, many of the reported substances having the above-mentioned influenza virus neuraminidase inhibitory activity have a complicated structure, and their production requires complicated steps, and there are concerns about side effects. Met.
一方、銅含有酵素であるチロシナーゼは、モノフェノールのカテコールへのオルト−ヒドロキシル化を触媒し、次いで酸化によりオルトキノリンを生成する。哺乳類において、チロシナーゼは、髪および皮膚色素の褐変に関与しているだけでなく、低色素沈着あるいは色素沈着過剰などの皮膚異常を引き起こす。さらにチロシナーゼは、癌およびパーキンソン病のような神経変性疾患に関与している可能性も示唆されている。 On the other hand, tyrosinase, a copper-containing enzyme, catalyzes the ortho-hydroxylation of monophenol to catechol and then produces orthoquinoline by oxidation. In mammals, tyrosinase is not only involved in the browning of hair and skin pigments but also causes skin abnormalities such as hypopigmentation or hyperpigmentation. Furthermore, tyrosinase has been suggested to be involved in neurodegenerative diseases such as cancer and Parkinson's disease.
チロシナーゼ活性阻害物質の代表的なものとして、コウジ酸やアルブチン、アスコルビン酸などがよく知られており、メラニンの生成および沈着を抑制する美白剤として利用されている(特許文献5〜7)。しかし、これらの中には活性が十分でないものもあり、天然物由来で強力かつ安全性の高いチロシナーゼ阻害剤がなお強く求められている。
As typical tyrosinase activity inhibitors, kojic acid, arbutin, ascorbic acid and the like are well known, and are used as whitening agents that suppress the production and deposition of melanin (
従って、本発明の課題は、優れたノイラミニダーゼ阻害活性および/またはチロシナーゼ阻害活性を備えた安全性の高い新規物質を提供することである。 Accordingly, an object of the present invention is to provide a highly safe novel substance having an excellent neuraminidase inhibitory activity and / or tyrosinase inhibitory activity.
本発明者らは、上記課題を解決するために鋭意研究を行った結果、ギンネム(ギンゴウカン)やミモザなどの熱帯〜亜熱帯植物中に含まれるミモシンに、特定のトリペプチドを結合したミモシンテトラペプチドは、ミモシンよりも優れたノイラミニダーゼ阻害活性および/またはチロシナーゼ阻害活性を示すことを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that a mimosine tetrapeptide in which a specific tripeptide is bound to mimosine contained in tropical to subtropical plants such as ginnemu (Gingoukan) and mimosa, etc. The present inventors have found that it exhibits a neuraminidase inhibitory activity and / or tyrosinase inhibitory activity superior to mimosine, and has completed the present invention.
すなわち本発明は、下記一般式(1); That is, the present invention provides the following general formula (1);
で表されるミモシン誘導体である。
It is a mimosine derivative represented by
また本発明は、上記ミモシン誘導体を有効成分として含有するインフルエンザウイルスノイラミニダーゼ阻害剤またはチロシナーゼ阻害剤である。 The present invention is also an influenza virus neuraminidase inhibitor or tyrosinase inhibitor containing the mimosine derivative as an active ingredient.
本発明のミモシン誘導体は、優れたインフルエンザウイルスノイラミニダーゼ阻害作用および/またはチロシナーゼ阻害活性を有するものである。従って、これを有効成分として含有するインフルエンザウイルスノイラミニダーゼ阻害剤は、インフルエンザ治療用の医薬品またはその原料として利用できるものである。またこのミモシン誘導体を有効成分として含有するチロシナーゼ阻害剤は、医薬品のほか、美白用化粧料などとして利用することができる。 The mimosine derivative of the present invention has excellent influenza virus neuraminidase inhibitory activity and / or tyrosinase inhibitory activity. Therefore, an influenza virus neuraminidase inhibitor containing this as an active ingredient can be used as a medicine for treating influenza or a raw material thereof. The tyrosinase inhibitor containing this mimosine derivative as an active ingredient can be used as a whitening cosmetic in addition to pharmaceuticals.
本発明のミモシン誘導体は、下記一般式(1); The mimosine derivative of the present invention has the following general formula (1);
ミモシン(β−[N−(3−ヒドロキシ−4−ピリドン)]−α−アミノプロピオン酸)は、ピリジン環の窒素原子に結合したアラニン側鎖を有する非タンパク質アミノ酸であるが、上記一般式(1)のミモシン誘導体は、このミモシンにトリペプチドが結合したテトラペプチドである。 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 tetrapeptide in which a tripeptide is bound to this mimosine.
上記一般式(1)中、X1〜X3は、チロシン(Tyr;Y)、トリプトファン(Trp;Y)、フェニルアラニン(Phe;F)、グリシン(Gly;G)、グルタミン(Gln;Q)、バリン(Val;V)およびヒスチジン(His;H)よりなる群から選ばれるアミノ酸残基であり、これらは互いに独立して、同一であっても異なっていてもよい。 In the general formula (1), X 1 to X 3 are tyrosine (Tyr; Y), tryptophan (Trp; Y), phenylalanine (Phe; F), glycine (Gly; G), glutamine (Gln; Q), It is an amino acid residue selected from the group consisting of valine (Val; V) and histidine (His; H), and these may be the same or different independently of each other.
基X1〜X3を構成するアミノ酸に光学異性体が存在する場合は、D体であってもL体であってもよいが、L体であることが好ましい。X3はN末端側でミモシンとアミド結合している。 When optical isomers are present in the amino acids constituting the groups X 1 to X 3 , they may be D-forms or L-forms, but are preferably L-forms. X 3 has an amide bond with mimosine on the N-terminal side.
これらの中でも、基X3−X2−X1が、Phe−Phe−Tyr(FFY)、Phe−Tyr−Tyr(FYY)、Phe−Trp−Tyr(FWY)、Val−Gly−Tyr(VGY)、Gln−Gly−Tyr(QGY)、Trp−Gly−Tyr(WGY)、Phe−Gly−Tyr(FGY)およびHis−Gly−Tyr(HGY)よりなる群から選ばれるトリペプチド残基は、インフルエンザウイルスノイラミニダーゼ阻害剤および/またはチロシナーゼ阻害剤が優れるため好適である。 Among these, the group X 3 -X 2 -X 1 is Phe-Phe-Tyr (FFY), Phe-Tyr-Tyr (FYY), Phe-Trp-Tyr (FWY), Val-Gly-Tyr (VGY). , Gln-Gly-Tyr (QGY), Trp-Gly-Tyr (WGY), Phe-Gly-Tyr (FGY) and His-Gly-Tyr (HGY) are tripeptide residues selected from the group consisting of influenza virus Neuraminidase inhibitors and / or tyrosinase inhibitors are preferred because they are excellent.
この中でも、Phe−Phe−Tyr(FFY)、Phe−Tyr−Tyr(FYY)、Phe−Trp−Tyr(FWY)は、インフルエンザウイルスノイラミニダーゼ阻害剤に優れるため好ましく、特にPhe−Phe−Tyr(FFY)が好ましい。また、Phe−Phe−Tyr(FFY)、Phe−Tyr−Tyr(FYY)、Phe−Trp−Tyr(FWY)、Val−Gly−Tyr(VGY)、Gln−Gly−Tyr(QGY)、Trp−Gly−Tyr(WGY)、Phe−Gly−Tyr(FGY)は、チロシナーゼ阻害活性が高いため好ましく、Phe−Phe−Tyr(FFY)、Phe−Tyr−Tyr(FYY)、Phe−Trp−Tyr(FWY)がより好ましく、特にPhe−Phe−Tyr(FFY)が好ましい。 Among these, Phe-Phe-Tyr (FFY), Phe-Tyr-Tyr (FYY), and Phe-Trp-Tyr (FWY) are preferable because they are excellent influenza virus neuraminidase inhibitors, and in particular, Phe-Phe-Tyr (FFY). Is preferred. Also, Phe-Phe-Tyr (FFY), Phe-Tyr-Tyr (FYY), Phe-Trp-Tyr (FWY), Val-Gly-Tyr (VGY), Gln-Gly-Tyr (QGY), Trp-Gly -Tyr (WGY) and Phe-Gly-Tyr (FGY) are preferable because of their high tyrosinase inhibitory activity. Phe-Phe-Tyr (FFY), Phe-Tyr-Tyr (FYY), Phe-Trp-Tyr (FWY) Is more preferable, and Phe-Phe-Tyr (FFY) is particularly preferable.
また本発明のミモシン誘導体は下記一般式(2) The mimosine derivative of the present invention has the following general formula (2):
上記一般式(2)中、R1〜R3は、4−ヒドロキシベンジル基、3−インドリルメチル基、ベンジル基、水素原子、2−カルバモイルエチル基、イソプロピル基、イミダゾリルメチル基よりなる群から選ばれる基を示し、これらは互いに独立して、同一であっても異なっていてもよい。 In the general formula (2), R 1 to R 3 are selected from the group consisting of 4-hydroxybenzyl group, 3-indolylmethyl group, benzyl group, hydrogen atom, 2-carbamoylethyl group, isopropyl group, and imidazolylmethyl group. The groups to be selected are shown, and these may be the same or different independently of each other.
これらの中でもR1は4−ヒドロキシベンジル基が好ましい。また、R2は、4−ヒドロキシベンジル基、3−インドリルメチル基、ベンジル基および水素原子よりなる群から選ばれたものであることが好適である。R3は、3−インドリルメチル基、ベンジル基、2−カルバモイルエチル基、イソプロピル基およびイミダゾリルメチル基よりなる群から選ばれたものであることが好適である。 Among these, R 1 is preferably a 4-hydroxybenzyl group. R 2 is preferably selected from the group consisting of a 4-hydroxybenzyl group, a 3-indolylmethyl group, a benzyl group, and a hydrogen atom. R 3 is preferably selected from the group consisting of a 3-indolylmethyl group, a benzyl group, a 2-carbamoylethyl group, an isopropyl group, and an imidazolylmethyl group.
中でも、R1が4−ヒドロキシベンジル基、R3がベンジル基であって、R2がベンジル基、4−ヒドロキシベンジル基又は3−インドリルメチル基である組み合わせは、インフルエンザウイルスノイラミニダーゼ阻害活性およびチロシナーゼ阻害活性が優れるため好ましく、特にR1が4−ヒドロキシベンジル基で、R2とR3がともにベンジル基である組み合わせが好ましい。また、R1が4−ヒドロキシベンジル基、R2が水素原子であって、R3がイソプロピル基、2−カルバモイルエチル基、3−インドリルメチル基又はベンジル基である組み合わせは、チロシナーゼ阻害活性に優れるために好適である。 Among them, the combination in which R 1 is a 4-hydroxybenzyl group, R 3 is a benzyl group, and R 2 is a benzyl group, a 4-hydroxybenzyl group, or a 3-indolylmethyl group is an influenza virus neuraminidase inhibitory activity and tyrosinase The inhibitory activity is excellent, which is preferable. Particularly, a combination in which R 1 is a 4-hydroxybenzyl group and R 2 and R 3 are both benzyl groups is preferable. In addition, the combination in which R 1 is a 4-hydroxybenzyl group, R 2 is a hydrogen atom, and R 3 is an isopropyl group, a 2-carbamoylethyl group, a 3-indolylmethyl group, or a benzyl group is effective for tyrosinase inhibitory activity. It is suitable for superiority.
また上記一般式(2)中、R1、R2、R3が水素原子以外の場合には、これらが結合する炭素は不斉炭素となる。x、y、zは、不斉炭素となる場合のそれぞれの絶対配置(SまたはR)を示す符号を表わす。本発明のミモシン誘導体には、エナンチオマー、ジアステレオマーおよびラセミ体を含むこれらの混合物が包含されるが、x、y、zが全て(S)の立体配置であることが好ましい。 In the general formula (2), when R 1 , R 2 and R 3 are other than a hydrogen atom, the carbon to which they are bonded is an asymmetric carbon. x, y, and z each represent a sign indicating an absolute configuration (S or R) in the case of an asymmetric carbon. The mimosine derivatives of the present invention include mixtures thereof including enantiomers, diastereomers, and racemates, and it is preferable that x, y, and z are all in the (S) configuration.
上記一般式(1)または(2)で示される本発明のミモシン誘導体は、例えば以下の方法によって製造することができる。 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.
ミモシンはギンネムやミモザのような熱帯・亜熱帯植物に含まれる。このギンネムは、ネムノキ科ギンゴウカン属の常緑低木で、熱帯から亜熱帯アジアに分布し、日本では沖縄県から九州南部に分布する。 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.
このギンネムの葉からミモシンを得るには、まず、ギンネムの葉、好ましくは新鮮な若葉を、好ましくは細切ないし細断して抽出原料とする。 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.
次いで、上記のように準備した抽出原料に対し、適量の水を加熱し、得られた熱水で抽出する。この熱水抽出は、70℃以上、好ましくは75℃ないし沸騰状態の熱水で行うことができるが、ミモシン分解酵素を失活させ、純度の高いミモシンを得るためには、沸騰水(100℃程度)の熱水を用いることが特に好ましい。また、抽出時間は、5ないし30分程度であり、特に10分間程度煮沸抽出を行うことが好ましい。抽出に用いる抽出溶媒としては、蒸留水が好ましく、また、抽出中、必要により連続あるいは間欠的に攪拌することが望ましい。 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.
このギンネム葉抽出液中に、強陽イオン交換樹脂を加えて、ミモシンを含む被吸着成分を吸着させる。次いで、このイオン交換樹脂を、水や、水−エタノール混液で洗浄した後、アンモニア水中等に浸漬し、ミモシンをイオン交換樹脂から溶出させる。この溶出液を必要により活性炭処理した後、濃縮処理し、低温で放置することによりミモシン塩が析出してくるので、これを集めることでミモシンが得られる。得られたミモシンは必要に応じて再結晶等の手段により精製してもよい。 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.
このようにして得られたミモシンに、ペプチド固相合成法などの公知のペプチド合成法を用いてアミノ酸を結合させることにより本発明のミモシン誘導体を得ることができる。 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.
ミモシンおよびアミノ酸は、アミノ基を、9−フルオレニルメチルオキシカルボニル基(Fmoc)やt−ブチルオキシカルボニル基(Boc)などの保護基で保護することが好ましい。 Mimosine and amino acids preferably protect the amino group with a protecting group such as 9-fluorenylmethyloxycarbonyl group (Fmoc) or t-butyloxycarbonyl group (Boc).
ペプチド結合を形成するための縮合剤としては、例えば、ジイソプロピルカルボジイミド(DIC)、N,N−ジシクロヘキシルカルボジイミド(DCC)等が挙げられる。また、これらの縮合剤をN−ヒドロキシベンゾトリアゾール(HOBt)と混合して用いることもできる。 Examples of the condensing agent for forming a peptide bond include diisopropylcarbodiimide (DIC), N, N-dicyclohexylcarbodiimide (DCC), and the like. Moreover, these condensing agents can also be used by mixing with N-hydroxybenzotriazole (HOBt).
ペプチドまたはアミノ酸のアミノ末端アミノ基の保護基であるBocおよびFmocは、トリフルオロ酢酸(TFA)、ピペリジンなどにより除去することができる。 Boc and Fmoc, which are protecting groups for the amino terminal amino group of peptides or amino acids, can be removed with trifluoroacetic acid (TFA), piperidine and the like.
また、ペプチド固相合成樹脂としては、Wang樹脂などを用いることができる。ペプチドをペプチド固相合成樹脂より脱離させるにあたっては、例えば、TFAなどが用いられる。 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.
本発明のミモシン誘導体を製造するためのFmoc固相合成法による反応スキームを図1に示す。このスキームにおいては、N−(9−フルオレニルメトキシカルボニルオキシ)コハク酸イミド(Fmoc−OSu)のFmoc基をミモシンに結合してFmoc−ミモシンを調製し、これとFmoc−アミノ酸を用いて形成させたトリペプチドを結合させることによって、ミモシンテトラペプチドを形成させている。以下、より具体的に説明する。 A reaction scheme by the Fmoc solid phase synthesis method for producing the mimosine derivative of the present invention is shown in FIG. In this scheme, the Fmoc group of N- (9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) is coupled to mimosine to prepare Fmoc-mimosine, which is formed using Fmoc-amino acid. Mimosine tetrapeptides are formed by binding the tripeptides formed. More specific description will be given below.
(Fmoc−ミモシンの調製)
ミモシンおよび炭酸ナトリウムをジオキサンを含有する蒸留水に溶解し、この溶液にFmoc−OSuを添加し、室温で一晩インキュベートする。次いで、炭酸ナトリウム溶液を添加し、攪拌した後、この溶液をろ過し、次いで酢酸エチルで洗浄して、未反応のFmoc−OSu、副産物である9−フルオレニルメタノールおよび9−メチレンフルオレンを除去する。氷浴中で、塩酸を用いて水画分のpHを4程度にまで下げることによって、Fmoc−ミモシンの結晶が析出する。
(Preparation of Fmoc-mimosine)
Mimosine and sodium carbonate are dissolved in distilled water containing dioxane, Fmoc-OSu is added to this solution and incubated overnight at room temperature. Sodium carbonate solution is then added and stirred, then the solution is filtered and then washed with ethyl acetate to remove unreacted Fmoc-OSu, by-products 9-fluorenylmethanol and 9-methylenefluorene. To do. Crystals of Fmoc-mimosine are precipitated by lowering the pH of the water fraction to about 4 using hydrochloric acid in an ice bath.
(ミモシンテトラペプチドの固相合成)
Fmoc−アミノ酸(Fmoc−X1−OH)のジメチルアセトアミド溶液に、1−ヒドロキシ−1H−ベンゾトリアゾール(HOBt)およびN,N’−ジイソプロピルカルボジイミド(DIC)を添加し、攪拌する。この溶液にN,N−ジメチルホルムアミド(DMF)中で膨張させたWang樹脂を添加し、攪拌する(図1A参照)。この樹脂をろ過し、ジクロロメタン、イソプロピルアルコールおよびメタノールで洗浄し、真空条件下で乾燥する。DMF中にて25%ピぺリジン(試薬a)によりFmocの脱保護を行った後、次のアミノ酸(Fmoc−X2−OH)を、試薬b(Fmocアミノ酸、HOBt、HBTUおよびN,N−ジイソプロピルエチルアミン(DIEA)の混合物)に結合させ、さらに攪拌する(図1B参照)。このジペプチドに、同様にして、Fmocアミノ酸(Fmoc−X3−OH)を結合させてトリペプチドを形成する。さらに同様にして上記で調製したFmoc−ミモシンを加えて結合させた後、95%トリフルオロ酢酸(TFA;試薬k)で攪拌する(図1C参照)。この樹脂をろ過し、TFAで洗浄した後、得られたろ液から氷冷されたジエチルエーテルで沈殿を生じさせることによって、ミモシンテトラペプチドが得られる。
(Solid-phase synthesis of mimosine tetrapeptide)
1-hydroxy-1H-benzotriazole (HOBt) and N, N′-diisopropylcarbodiimide (DIC) are added to a dimethylacetamide solution of Fmoc-amino acid (Fmoc-X 1 -OH) and stirred. To this solution is added Wang resin swollen in N, N-dimethylformamide (DMF) and stirred (see FIG. 1A). The resin is filtered, washed with dichloromethane, isopropyl alcohol and methanol and dried under vacuum conditions. After deprotection of Fmoc with 25% piperidine (reagent a) in DMF, the next amino acid (Fmoc-X 2 -OH) was added to reagent b (Fmoc amino acid, HOBt, HBTU and N, N- (Mixture of diisopropylethylamine (DIEA)) and stirring (see FIG. 1B). Similarly, Fmoc amino acid (Fmoc-X 3 —OH) is bound to this dipeptide to form a tripeptide. Further, Fmoc-mimosine prepared as described above was added and bound in the same manner, followed by stirring with 95% trifluoroacetic acid (TFA; reagent k) (see FIG. 1C). The resin is filtered, washed with TFA, and then the mimosine tetrapeptide is obtained by causing precipitation from the obtained filtrate with ice-cooled diethyl ether.
以上のようにして得られた本発明のミモシン誘導体は、そのまま、あるいは必要に応じ、液体高速クロマトグラフィーなど公知の方法によって精製した後、インフルエンザウイルスノイラミニダーゼ阻害剤またはチロシナーゼ阻害剤として利用することができる。 The mimosine derivative of the present invention obtained as described above can be used as an influenza virus neuraminidase inhibitor or tyrosinase inhibitor as it is or after purification by a known method such as liquid high performance chromatography as necessary. .
例えば、ミモシン誘導体を有効成分とするインフルエンザウイルスノイラミニダーゼ阻害剤の調製は、治療有効量のミモシン誘導体を、製薬上許容される任意成分、例えば、慣用の賦形剤、結合剤、滑沢剤、水性溶剤、油性溶剤、乳化剤、懸濁化剤、保存剤、安定剤等と組み合わせ、混合することにより行うことができる。 For example, the preparation of an influenza virus neuraminidase inhibitor containing a mimosine derivative as an active ingredient can be carried out by treating a therapeutically effective amount of the mimosine derivative with any pharmaceutically acceptable ingredient such as conventional excipients, binders, lubricants, aqueous It can be carried out by combining and mixing a solvent, an oily solvent, an emulsifier, a suspending agent, a preservative, a stabilizer and the like.
本発明のインフルエンザウイルスノイラミニダーゼ阻害剤は、経口でも非経口でも投与することができる。経口投与による場合の本発明インフルエンザウイルスノイラミニダーゼ阻害剤は、通常の経口投与製剤、例えば、錠剤、散剤、顆粒剤、カプセル剤等の固形剤;水剤;油性懸濁剤;又はシロップ剤もしくはエリキシル剤等の液剤のいずれかの剤形としても用いることができる。非経口投与による場合には、本発明インフルエンザウイルスノイラミニダーゼ阻害剤は、水性又は油性懸濁注射剤、点鼻液として用いることができる。 The influenza virus neuraminidase inhibitor of the present invention can be administered either orally or parenterally. The influenza virus neuraminidase inhibitor of the present invention when orally administered is a usual oral preparation such as a solid preparation such as a tablet, powder, granule or capsule; a liquid preparation; an oil suspension; or a syrup or elixir. It can also be used as any dosage form of liquids such as In the case of parenteral administration, the influenza virus neuraminidase inhibitor of the present invention can be used as an aqueous or oily suspension injection or nasal solution.
本発明のインフルエンザウイルスノイラミニダーゼ阻害剤の投与量は、投与方法、患者の年齢、体重、状態および疾患の種類によっても異なるが、通常、経口投与の場合、成人1日あたり約10〜200mgであり、好ましくは、約10〜20mgであり、これを必要に応じて数回に分け投与すれば良い。また、非経口投与の場合は、成人1日あたり約5〜100mg、好ましくは、約5〜10mgを投与すれば良い。 The dose of the influenza virus neuraminidase inhibitor of the present invention varies depending on the administration method, the patient's age, body weight, condition, and type of disease, but in the case of oral administration, it is usually about 10 to 200 mg per adult day, Preferably, the dose is about 10 to 20 mg, which 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 may be administered.
なお、本発明のインフルエンザウイルスノイラミニダーゼ阻害剤を抗インフルエンザ薬として用いる場合は、特に経口剤が好ましい。 In addition, when the influenza virus neuraminidase inhibitor of the present invention is used as an anti-influenza drug, an oral preparation is particularly preferable.
一方、本発明のチロシナーゼ活性阻害剤も上記インフルエンザウイルスノイラミニダーゼ阻害剤と同様にして、色素沈着過剰、低色素沈着等の皮膚異常の治療用途のための経口あるいは非経口の医薬として製剤化することができる。また皮膚外用剤とすることもでき、例えば、精製水、アルコール類、水溶性高分子、油剤、界面活性剤、ゲル化剤、保湿剤、ビタミン類、抗菌剤、香料、塩類、pH調整剤等の成分を加えて調製することができる。 On the other hand, the tyrosinase activity inhibitor of the present invention can also be formulated as an oral or parenteral drug for the treatment of skin abnormalities such as hyperpigmentation and hypopigmentation, in the same manner as the influenza virus neuraminidase inhibitor. it can. It can also be used as an external preparation for skin, for example, 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.
本発明のチロシナーゼ活性阻害剤の添加量は、添加対象物の種類、投与経路、剤形等の諸条件によって異なるが、例えば、医薬製剤(全重量)中に2質量%〜5質量%含有させることが好適である。 The addition amount of the tyrosinase activity inhibitor of the present invention varies depending on various conditions such as the kind of the object to be added, the administration route, the dosage form, etc., for example, 2 mass% to 5 mass% is contained in the pharmaceutical preparation (total weight). Is preferred.
また本発明のチロシナーゼ活性阻害剤の投与量は、特に限定されるものではなく、患者の年齢、性別、体重、症状の程度、又は投与方法などに応じて適宜決定することができるが、例えば、経口投与の場合には、成人1日あたり約10〜200mgであり、好ましくは、約10〜20mgである。また非経口投与の場合は、成人1日あたり約5〜100mg、好ましくは、約5〜10mgを投与すれば良い。 The dosage of the tyrosinase activity inhibitor of the present invention is not particularly limited and can be appropriately determined according to the patient's age, sex, weight, degree of symptoms, administration method, etc. In the case of oral administration, it is about 10-200 mg per day for an adult, preferably about 10-20 mg. 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.
さらに、本発明のチロシナーゼ阻害剤は、メラニンの産生や沈着を抑制し、美白作用を有するため、化粧料に配合し美白化粧料とすることもできる。例えば、公知の化粧料基剤にチロシナーゼ阻害剤を、3〜5質量%程度配合し、常法に従って、溶液状、可溶化状、乳化状、粉末状、ペースト状、ムース状、ジェル状の形態とすることにより製造され、化粧水、乳液、クリーム、パック、軟膏等として提供される。 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.
また、上記美白化粧料の製造にあたっては、必要に応じて本発明の効果を損なわない範囲で、通常、化粧料に使用される成分、すなわち、精製水、アルコール類、水溶性高分子、油剤、界面活性剤、ゲル化剤、保湿剤、ビタミン類、抗菌剤、香料、塩類、pH調整剤等を加えることができる。 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.
次に実施例を挙げ、本発明を更に詳しく説明するが、本発明はこれら実施例に何ら制約されるものではない。 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.
参 考 例 1
ミモシンの調製:
ギンゴウカンの葉1kgを、5Lの水で10分間煮沸した。抽出液をろ過し、ろ液にカチオン交換樹脂(オルガノ株式会社、IR120BH)2kgを添加した。この抽出液・樹脂混合物を、振とう機(アズワン社製、 Shaking Baths SB−20)で室温にて一晩振とうした。ミモシン以外の不純物を取り除くために、このカチオン交換樹脂を、80%のエタノール5Lで洗浄し、さらに、数回程度水で蒸留でした。この樹脂を4N 水酸化アンモニウム6Lで溶出して粗ミモシンを得た。この溶出物を真空で濃縮し、さらに、粗ミモシンを水酸化ナトリウム水溶液に溶解した。この溶液のpHを6N塩酸で4.5〜5.0に調節し、冷凍庫に一晩置いて結晶化させた。得られた結晶をろ過し、さらに真空状態で乾燥することで、純度95%以上のミモシンを5.387g(0.54%)得た。
LC−MS(ESI−)m/z[M−H]+;197.2;[M+H]+;199.1
Reference example 1
Preparation of mimosine:
1 kg of ginkgo leaves was boiled with 5 L of water for 10 minutes. The extract was filtered, and 2 kg of a cation exchange resin (organo corporation, IR120BH) was added to the filtrate. The extract / resin mixture was shaken overnight at room temperature with a shaker (manufactured by AS ONE, Shaking Baths SB-20). In order to remove impurities other than mimosine, this cation exchange resin was washed with 5 L of 80% ethanol and further distilled several times with water. This resin was eluted with 6 L of 4N ammonium hydroxide to obtain crude mimosine. The eluate was concentrated in vacuo, and the crude mimosine was dissolved in an aqueous sodium hydroxide solution. The pH of this solution was adjusted to 4.5-5.0 with 6N hydrochloric acid and placed in a freezer overnight for crystallization. The obtained crystals were filtered and further dried under vacuum to obtain 5.387 g (0.54%) of mimosine with a purity of 95% or more.
LC-MS (ESI-) m / z [M-H] <+> ; 197.2; [M + H] < +>; 199.1
製 造 例 1
ミモシン誘導体の調製(M−FFY):
Fmoc固相合成法により、ミモシン(M)にトリペプチドを結合してテトラペプチドの合成を行った。ハイペップ研究所から入手したFmoc−アミノ酸を用いて、最初の結合は、チロシン(Y)で行い、次にフェニルアラニン(F)を結合してジペプチドを形成し、さらに、フェニルアラニン(F)を結合してトリペプチドを形成した。形成されたジペプチドを別途調製したFmoc−ミモシンと結合してミモシンテトラペプチドを得た。より具体的な製法を以下に示す。
Manufacturing example 1
Preparation of mimosine derivatives (M-FFY):
A tetrapeptide was synthesized by binding a tripeptide to mimosine (M) by Fmoc solid phase synthesis. Using Fmoc-amino acids obtained from Hypep Laboratories, the first coupling is performed with tyrosine (Y), then phenylalanine (F) is coupled to form a dipeptide, and further phenylalanine (F) is coupled. A tripeptide was formed. The formed dipeptide was combined with separately prepared Fmoc-mimosine to obtain a mimosine tetrapeptide. A more specific production method is shown below.
(Fmoc−ミモシンの調製)
5gのミモシンおよび5.5gの炭酸ナトリウムを75mLのジオキサンを含有する蒸留水75mLに溶解した。この溶液に12.5gのN−(9−フルオレニルメトキシカルボニルオキシ)コハク酸イミド(Fmoc−OSu)を添加し、この混合液を室温で一晩インキュベートした。次に、300mLの炭酸ナトリウム溶液(0.1M)を添加し、さらにマグネチックスターラー(300rpm)で5時間、25℃で攪拌した。得られた溶液(450mL)をろ過し、酢酸エチル(150mL)で洗浄して未反応のFmoc−OSu、副産物である9−フルオレニルメタノールおよび9−メチレンフルオレンを除去した。氷浴中で、6N 塩酸を用いて水画分のpHを4に下げ、Fmoc−ミモシンを結晶として得た。これをろ過し、真空条件下で乾燥した(収量7.108g)。
(Preparation of Fmoc-mimosine)
5 g mimosine and 5.5 g sodium carbonate were dissolved in 75 mL distilled water containing 75 mL dioxane. To this solution was added 12.5 g N- (9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) and the mixture was incubated overnight at room temperature. Next, 300 mL of sodium carbonate solution (0.1 M) was added, and the mixture was further stirred at 25 ° C. for 5 hours with a magnetic stirrer (300 rpm). The resulting solution (450 mL) was filtered and washed with ethyl acetate (150 mL) to remove unreacted Fmoc-OSu, by-products 9-fluorenylmethanol and 9-methylenefluorene. In an ice bath, the pH of the water fraction was lowered to 4 using 6N hydrochloric acid to obtain Fmoc-mimosine as crystals. This was filtered and dried under vacuum (yield 7.108 g).
(ミモシンテトラペプチドの固相合成)
Fmoc−L−チロシン1.6mmolのジメチルアセトアミド溶液5mLに、1−ヒドロキシ−1H−ベンゾトリアゾール(HOBt)1.6mmolおよびN,N’−ジイソプロピルカルボジイミド(DIC)1.6mmolを添加し、10分間攪拌した。この溶液にDMF中で膨張させたWang樹脂1gを添加し、反応混合物を17時間攪拌した(図1A参照)。この樹脂をろ過し、ジクロロメタン、イソプロピルアルコールおよびメタノールで洗浄し、真空条件下で乾燥した。DMF中にて30分間、25%ピぺリジン(試薬a)によりFmocの脱保護を行った後、次のアミノ酸Fmoc−L−フェニルアラニンを、樹脂混合溶液(Fmocアミノ酸:HOBt:HBTU:N,N−ジイソプロピルエチルアミン(DIEA)=4:3:3.6:8;試薬b)に結合させた。この反応混合物をさらに1時間攪拌した(図1B参照)。
(Solid-phase synthesis of mimosine tetrapeptide)
To 5 mL of a dimethylacetamide solution of 1.6 mmol of Fmoc-L-tyrosine, 1.6 mmol of 1-hydroxy-1H-benzotriazole (HOBt) and 1.6 mmol of N, N′-diisopropylcarbodiimide (DIC) were added and stirred for 10 minutes. did. To this solution was added 1 g of Wang resin swollen in DMF and the reaction mixture was stirred for 17 hours (see FIG. 1A). The resin was filtered, washed with dichloromethane, isopropyl alcohol and methanol and dried under vacuum conditions. After deprotection of Fmoc with 25% piperidine (reagent a) for 30 minutes in DMF, the next amino acid Fmoc-L-phenylalanine was added to a resin mixed solution (Fmoc amino acid: HOBt: HBTU: N, N -Diisopropylethylamine (DIEA) = 4: 3: 3.6: 8; reagent b). The reaction mixture was stirred for an additional hour (see FIG. 1B).
結合の完全性を調べるために、ニンヒドリン試験を行った。HOBt:酢酸:DIEA:DMF(0.8:19:9:400)混合液を樹脂1gあたり20mL用いて、未結合のFmoc−L−トリプトファンをアセチル基で保護した。
次に上記と同様にして、テトラペプチドを形成するために、Fmoc−L−フェニルアラニンをジペプチドに結合した。さらに上記で調製したFmoc−ミモシンを加え、同様にして結合させた後、この樹脂を、1時間、95%のトリフルオロ酢酸(TFA;試薬k)でゆっくり攪拌した(図1C参照)。この樹脂をろ過した後、TFAで洗浄し、得られたろ過液から、氷冷されたジエチルエーテルで沈殿を生じさせた。得られた沈殿をろ過し、ジエチルエーテルで3回洗浄した後、真空条件下で乾燥して目的のミモシンテトラペプチドを得た(M−FFY)。得られた粗ペプチドは白色固体であり、収量は80.2mgであった。この粗ペプチドをさらに下記条件の液体クロマトグラフィーによって精製した。
LC−MS(ESI−)m/z:693.2([M−H]+)
In order to examine the integrity of the binding, a ninhydrin test was performed. Unbound Fmoc-L-tryptophan was protected with acetyl groups using 20 mL of HOBt: acetic acid: DIEA: DMF (0.8: 19: 9: 400) mixture per gram of resin.
Next, in the same manner as described above, Fmoc-L-phenylalanine was bound to the dipeptide to form a tetrapeptide. Further, Fmoc-mimosine prepared above was added and bound in the same manner, and then the resin was slowly stirred with 95% trifluoroacetic acid (TFA; reagent k) for 1 hour (see FIG. 1C). The resin was filtered and then washed with TFA, and the resulting filtrate was precipitated with ice-cooled diethyl ether. The resulting precipitate was filtered, washed three times with diethyl ether, and then dried under vacuum conditions to obtain the desired mimosine tetrapeptide (M-FFY). The obtained crude peptide was a white solid, and the yield was 80.2 mg. This crude peptide was further purified by liquid chromatography under the following conditions.
LC-MS (ESI-) m / z: 693.2 ([M-H] <+> )
(HPLC条件)
カラム:Cadenza CD−C18 カラム(20×100mm;3μm)
移動相:0.1%トリフルオロ酢酸/CH3CN(1.5/8.5)
流量:5mL/分
(HPLC conditions)
Column: Cadenza CD-C18 column (20 × 100 mm; 3 μm)
Mobile phase: 0.1% trifluoroacetic acid / CH 3 CN (1.5 / 8.5)
Flow rate: 5 mL / min
製 造 例 2
ミモシン誘導体の調製(M−FYY):
2番目に結合させるFmocアミノ酸としてFmoc−L−チロシン(Y)、3番目に結合させるFmocアミノ酸としてFmoc−L−フェニルアラニン(F)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−FYY)を得た(収量65.7mg)。
LC−MS(ESI−)m/z:670.1([M−H]+)
Manufacturing example 2
Preparation of mimosine derivatives (M-FYY):
The mimotine tetrapeptide (Similar to Production Example 1 except that Fmoc-L-tyrosine (Y) was used as the Fmoc amino acid to be bound second, and Fmoc-L-phenylalanine (F) was used as the Fmoc amino acid to be bound third. M-FYY) was obtained (yield 65.7 mg).
LC-MS (ESI-) m / z: 670.1 ([M-H] <+> )
製 造 例 3
ミモシン誘導体の調製(M−FWY):
2番目に結合させるFmocアミノ酸としてFmoc−L−トリプトファン(W)、3番目に結合させるFmocアミノ酸としてFmoc−L−フェニルアラニン(F)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−FWY)を得た(収量71.5mg)。
LC−MS(ESI−)m/z:654.2([M−H]+)
Manufacturing example 3
Preparation of mimosine derivatives (M-FWY):
The mimotine tetrapeptide (Fmoc-L-tryptophan (W) was used as the Fmoc amino acid to be bound second, and Fmoc-L-phenylalanine (F) was used as the Fmoc amino acid to be bound third. M-FWY) was obtained (yield 71.5 mg).
LC-MS (ESI-) m / z: 654.2 ([M-H] <+> )
製 造 例 4
ミモシン誘導体の調製(M−VGY):
2番目に結合させるFmocアミノ酸としてFmoc−グリシン(G)、3番目に結合させるFmocアミノ酸としてFmoc−L−バリン(V)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−VGY)を得た(収量42.5mg)。
LC−MS(ESI−)m/z:603.2([M−H]+)
Manufacturing example 4
Preparation of mimosine derivatives (M-VGY):
The mimotine tetrapeptide (M-) was prepared in the same manner as in Production Example 1 except that Fmoc-glycine (G) was used as the second Fmoc amino acid to be bound, and Fmoc-L-valine (V) was used as the Fmoc amino acid to be bound third. VGY) was obtained (yield 42.5 mg).
LC-MS (ESI-) m / z: 603.2 ([M-H] <+> )
製 造 例 6
ミモシン誘導体の調製(M−QGY):
2番目に結合させるFmocアミノ酸としてFmoc−グリシン(G)、3番目に結合させるFmocアミノ酸としてFmoc−L−グルタミン(Q)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−QGY)を得た(収量85.6mg)。
LC−MS(ESI−)m/z:564.1([M−H]+)
Manufacturing Example 6
Preparation of mimosine derivatives (M-QGY):
The mimotine tetrapeptide (M-) was prepared in the same manner as in Production Example 1 except that Fmoc-glycine (G) was used as the second Fmoc amino acid to be bound, and Fmoc-L-glutamine (Q) was used as the Fmoc amino acid to be bound third. QGY) was obtained (yield 85.6 mg).
LC-MS (ESI-) m / z: 564.1 ([M-H] <+> )
製 造 例 7
ミモシン誘導体の調製(M−WGY):
2番目に結合させるFmocアミノ酸としてFmoc−グリシン(G)、3番目に結合させるFmocアミノ酸としてFmoc−L−トリプトファン(W)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−WGY)を得た(収量66.8mg)。
LC−MS(ESI−)m/z:545.1([M−H]+)
Manufacturing example 7
Preparation of mimosine derivatives (M-WGY):
The mimotine tetrapeptide (M-) was prepared in the same manner as in Production Example 1 except that Fmoc-glycine (G) was used as the second Fmoc amino acid to be bound, and Fmoc-L-tryptophan (W) was used as the Fmoc amino acid to be bound third. WGY) was obtained (yield 66.8 mg).
LC-MS (ESI-) m / z: 545.1 ([M-H] <+> )
製 造 例 7
ミモシン誘導体の調製(M−FGY):
2番目に結合させるFmocアミノ酸としてFmoc−グリシン(G)、3番目に結合させるFmocアミノ酸としてFmoc−L−フェニルアラニン(F)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−FGY)を得た(収量78.4mg)。
LC−MS(ESI−)m/z:554.2([M−H]+)
Manufacturing example 7
Preparation of mimosine derivatives (M-FGY):
The mimotine tetrapeptide (M-) was prepared in the same manner as in Production Example 1 except that Fmoc-glycine (G) was used as the second Fmoc amino acid to be bound, and Fmoc-L-phenylalanine (F) was used as the Fmoc amino acid to be bound third. FGY) was obtained (yield 78.4 mg).
LC-MS (ESI-) m / z: 554.2 ([M-H] <+> )
製 造 例 8
ミモシン誘導体の調製(M−HGY):
2番目に結合させるFmocアミノ酸としてFmoc−グリシン(G)、3番目に結合させるFmocアミノ酸としてFmoc−L−ヒスチジン(H)を用いた以外は製造例1と同様にしてミモチンテトラペプチド(M−HGY)を得た(収量78.4mg)。
LC−MS(ESI−)m/z:531.1([M−H]+)
Manufacturing Example 8
Preparation of mimosine derivatives (M-HGY):
The mimotine tetrapeptide (M-) was prepared in the same manner as in Production Example 1 except that Fmoc-glycine (G) was used as the second Fmoc amino acid to be bound, and Fmoc-L-histidine (H) was used as the Fmoc amino acid to be bound third. HGY) was obtained (yield 78.4 mg).
LC-MS (ESI-) m / z: 531.1 ([M-H] <+> )
実 施 例 1
インフルエンザウイルスノイラミニダーゼ阻害作用:
ノイラミニダーゼ阻害アッセイは、まず、酢酸ナトリウム緩衝液(pH 5.0)50mMに4−メチルウンベリフェリル−α−D−N−アセチルノイラミン酸ナトリウム水和物(No.M8639、シグマ社製)0.1mMを加え、これを基質として用いた。次に、酢酸緩衝液(pH 5.0)にウェルシュ菌(Clostridium perfringens)由来のノイラミニダ―ゼ(Sigma社)を加え、0.1U/mLになるようにし、これを酵素試薬として用いた。
Example 1
Influenza virus neuraminidase inhibitory action:
A neuraminidase inhibition assay was performed by first using sodium acetate buffer (pH 5.0) 50 mM in 4-methylumbelliferyl-α-DN-acetylneuraminate sodium hydrate (No. M8639, manufactured by Sigma) 0 .1 mM was added and used as a substrate. Next, Neuraminidase (Sigma) derived from Clostridium perfringens was added to the acetate buffer (pH 5.0) to adjust to 0.1 U / mL, which was used as an enzyme reagent.
次に、製造例1〜8で得られたミモシンテトラペプチドをメタノールに溶解し、0.5、1、2μMの濃度に希釈した。各サンプル20μLを酢酸緩衝液(pH6.8)80μLに加え、マイクロプレートのウェルに注入し、これに酵素試薬50μLを加えた。反応は、50μLの基質を加えることにより開始され、蛍光発光は、MTP−880蛍光量計(コロナ電気株式会社製)を用いて測定した。励起および発光波長は、それぞれ360および450nmに設定した。異なるサンプル濃度に対し、各基質濃度の範囲において記録された初期速度による時間駆動プロトコルを用いて反応速度試験を行った。結果を図2に示す。また、各ミモシンテトラペプチドの阻害様式およびKi値を表1に示す。 Next, the mimosine tetrapeptides obtained in Production Examples 1-8 were dissolved in methanol and diluted to concentrations of 0.5, 1, 2 μM. 20 μL of each sample was added to 80 μL of acetate buffer (pH 6.8) and injected into a well of a microplate, and 50 μL of enzyme reagent was added thereto. The reaction was started by adding 50 μL of substrate, and fluorescence was measured using an MTP-880 fluorometer (Corona Electric Co., Ltd.). Excitation and emission wavelengths were set at 360 and 450 nm, respectively. For different sample concentrations, kinetic tests were performed using a time-driven protocol with initial rates recorded in each substrate concentration range. The results are shown in FIG. Table 1 shows the inhibition mode and Ki value of each mimosine tetrapeptide.
また阻害率を、以下の式により計算しIC50を求めた。結果を表1に示す。
阻害率(%)=[1−(S−S0)/(C−C0)] x 100
(式中、SおよびCは反応後の試料および対照(メタノール)の相対蛍光単位(RFU
)をそれぞれ表わし、S0およびC0は、0時間での相対蛍光単位を表わす。)
ミモシンについても同様にして試験を行った。結果を表1に併せて示す。また、ミモシンテトラペプチドを構成する各アミノ酸単独による30μMでのノイラミニダーゼ阻害率(%)も上記と同様にして求めた。結果を表2に示す。
The addition inhibition rate was calculated and IC 50 was calculated by the following equation. The results are shown in Table 1.
Inhibition rate (%) = [1− (S−S 0 ) / (C−C 0 )] x 100
Where S and C are relative fluorescence units (RFU) of the sample after reaction and control (methanol).
) And S 0 and C 0 represent relative fluorescence units at 0 hours, respectively. )
Mimosine was similarly tested. The results are also shown in Table 1. Further, the neuraminidase inhibition rate (%) at 30 μM by each amino acid alone constituting the mimosine tetrapeptide was also determined in the same manner as described above. The results are shown in Table 2.
なお、全ての実験は3連で行い、2回繰り返した。データは、6回の結果の平均値±標準偏差(SD)を表す。IC50は各サンプルが50%の阻害活性を示すために必要とされる濃度としてグラフから求めた。反応速度試験における全ての計算は、マイクロソフト エクセル オフィス、2007を用いて行った。有意性分析においては、データを一元配置分散分析ANOVAで分析し、Tukey HSD法(P=0.01)を用いて平均値の有意差を算出した。 All experiments were performed in triplicate and repeated twice. Data represent the mean of 6 results ± standard deviation (SD). IC 50 was determined from the graph as the concentration required for each sample to exhibit 50% inhibitory activity. All calculations in the reaction rate test were performed using Microsoft Excel Office 2007. In significance analysis, data was analyzed by one-way analysis of variance ANOVA, and a significant difference in mean values was calculated using the Tukey HSD method (P = 0.01).
表1から明らかなように、合成されたミモシンテトラペプチドのうち、M−FFY、M−FYY、M−FWY、M−VGY、M−QGYはいずれもミモシンより優れた阻害活性を示した。特にM−FFYは、ミモシンの5倍の阻害活性を有するものであった。M−FFYの低いIC50値およびKi値は、ノイラミダ―ゼ阻害剤として有力な化合物であることを示すものである。ミモシンは競合阻害型であるが、ミモシンテトラペプチドはいずれも非競合阻害を示した(図2H参照)。個々のアミノ酸(30μM)によるノイラミダ―ゼ阻害活性は、非常に弱いものであった(表2参照)。ノイラミダ―ゼ阻害活性において、ミモシンテトラペプチドは個々のアミノ酸より優れた阻害作用を有することが確認された。
As is clear from Table 1, among the synthesized mimosine tetrapeptides, M-FFY, M-FYY, M-FWY, M-VGY, and M-QGY all showed inhibitory activity superior to mimosine. In particular, M-FFY had an
実 施 例 2
チロシナーゼ阻害作用:
製造例1〜8で得られた各ミモシンテトラペプチドのチロシナーゼ阻害作用を確認した。ミモシンテトラペプチドをメタノールで溶解し、いくつかの異なる濃度(μM)のサンプルを調製した。96ウェルプレートに、120μLのリン酸緩衝液(20mM,pH6.8)、20μLのサンプル、20μLのマッシュルームチロシナーゼ(20mM 緩衝液にて、500 units/mL)の順で添加した。25℃で15分間インキュベートした後、20μLのL−チロシナーゼ溶液(0.85mM)を各ウェルに添加して反応を開始した。マイクロリーダーを用いて470nmでの吸光度を測定することにより酵素活性を確認した。チロシナーゼ阻害率を以下の式により計算しIC50を求めた。結果を表3に示す。
Example 2
Tyrosinase inhibitory action:
The tyrosinase inhibitory action of each mimosine tetrapeptide obtained in Production Examples 1 to 8 was confirmed. Mimosine tetrapeptide was dissolved in methanol to prepare several different concentrations (μM) of samples. To a 96-well plate, 120 μL of phosphate buffer (20 mM, pH 6.8), 20 μL of sample, and 20 μL of mushroom tyrosinase (20 mM buffer, 500 units / mL) were added in this order. After incubation at 25 ° C. for 15 minutes, 20 μL of L-tyrosinase solution (0.85 mM) was added to each well to initiate the reaction. Enzyme activity was confirmed by measuring absorbance at 470 nm using a microreader. The tyrosinase inhibition rate was calculated according to the following formula to determine IC 50 . The results are shown in Table 3.
チロシナーゼ阻害率(%)=[(A−B)−(C−D)]/(A−B)× 100
(式中、AおよびBは、それぞれ、酵素存在下での対照(メタノール)および酵素存在
下でのサンプルのチロシナーゼ活性を示し、CおよびDは、それぞれ、酵素非存在下
での対照(メタノール)および酵素非存在下でのサンプルのチロシナーゼ活性を示す。)
Tyrosinase inhibition rate (%) = [(A−B) − (C−D)] / (A−B) × 100
(Wherein A and B indicate the tyrosinase activity of the sample in the presence of enzyme (methanol) and the sample in the presence of enzyme, respectively, and C and D are the controls in the absence of enzyme (methanol), respectively) And the tyrosinase activity of the sample in the absence of enzyme.)
実験は3連で行い、2回繰り返した。データは、6回の結果の平均値±標準偏差(SD)を表す。IC50は各サンプルが50%の阻害活性を示すために必要とされる濃度としてグラフから求めた。有意性分析においては、データを一元配置分散分析ANOVAで分析し、Tukey HSD法(P=0.01)を用いて平均値の有意差を算出した。 The experiment was performed in triplicate and repeated twice. Data represent the mean of 6 results ± standard deviation (SD). IC 50 was determined from the graph as the concentration required for each sample to exhibit 50% inhibitory activity. In significance analysis, data was analyzed by one-way analysis of variance ANOVA, and a significant difference in mean values was calculated using the Tukey HSD method (P = 0.01).
表3に示すとおり、ミモシンのチロシナーゼ阻害活性のIC50値は44.7±0.4μM)であるのに対し、合成されたミモシンテトラペプチドは、いずれもミモシンよりも優れたチロシナーゼ活性阻害を示した(5.6〜36.7μM)。
特にM−FFYは、合成されたミモシンテトラペプチドの中で、最も優れた阻害活性を有し、ミモシンの約8倍の活性を示した。
As shown in Table 3, the IC 50 value of tyrosinase inhibitory activity of mimosine is 44.7 ± 0.4 μM), whereas all of the synthesized mimosine tetrapeptides exhibited superior tyrosinase activity inhibition than mimosine. (5.6-36.7 μM).
In particular, M-FFY had the most excellent inhibitory activity among the synthesized mimosine tetrapeptides, and showed about 8 times the activity of mimosine.
本発明のミモシン誘導体は、優れたインフルエンザウイルスノイラミニダーゼ阻害活性を示すため、インフルエンザが関連する疾患に対し、治療効果が期待できるものである。 Since the mimosine derivative of the present invention exhibits excellent influenza virus neuraminidase inhibitory activity, it can be expected to have a therapeutic effect on diseases associated with influenza.
また、本発明のミモシン誘導体は、優れたチロシナーゼ阻害活性を有するため、皮膚障害を予防・治療するための医薬や美白化粧料等として利用できるものである。 Moreover, 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)
で表されるミモシン誘導体を有効成分とするインフルエンザウイルスノイラミニダーゼ阻害剤。 The following general formula (1);
An influenza virus neuraminidase inhibitor comprising a mimosine derivative represented by
で表されるミモシン誘導体を有効成分とするチロシナーゼ阻害剤。 The following general formula (1);
The tyrosinase inhibitor which uses the mimosine derivative represented by this as an active ingredient.
で表されるミモシン誘導体。
The following general formula (1);
A mimosine derivative represented by:
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JP6512546B2 (en) * | 2015-04-01 | 2019-05-15 | 有限会社バイオシステムコンサルティング | Cyclooxygenase inhibitor containing mimosine or its derivative, NADPH-cytochrome P450 reductase inhibitor and tyrosinase inhibitor |
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