JPWO2003010172A1 - Method for producing high-purity riboflavin-5'-phosphate sodium salt - Google Patents

Abstract

The present invention provides a method for producing high-purity riboflavin 5'-phosphate (5'-FMN) or a pharmaceutically acceptable salt thereof. That is, in a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, 1) converting crude 5'-FMN or a pharmacologically acceptable salt thereof to water Alternatively, an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4 to 8 dissolved in a buffer solution is prepared, and 2) the obtained aqueous solution is mechanically stirred. While adding an organic solvent soluble in water at a temperature of 0-60 ° C. to the previously obtained aqueous solution in an amount of 1-100% by weight, and adding 5′-FMN or its pharmacologically acceptable. 3) recrystallizing the salt, 3) dissolving the recrystallized 5′-FMN or a pharmaceutically acceptable salt thereof in water or a buffer to form an aqueous solution, 4) then performing reverse phase column chromatography. 5'-FMN purified using photographic treatment or activated carbon treatment or its pharmacology A method for producing high purity 5'-FMN or a pharmacologically acceptable salt thereof, characterized by obtaining an eluent of a pharmacologically acceptable salt.

Description

この粗製５’−ＦＭＮ又はその薬理学的に許容される塩は、ピリジン及びオキシ塩化リンの存在下で反応中間体リボフラビン・サイクリック４’−，５’−ホスホリデート（２）を黄色結晶として単離し、洗浄後乾燥して酸性条件で加水分解・異性化を行なうことにより製造される。加水分解・異性化の条件は、特に限定されないが、１−３５．５（ｗ／ｖ）％濃度の酸、望ましくは５−１８％濃度の酸を出発原料であるリボフラビン（１）に対し１−１０倍容量、望ましくは１−８倍容量、特に望ましくは２−４容量用いて、１０〜１００℃、望ましくは３０〜８０℃、特に望ましくは３０〜６０℃で攪拌反応により調製することが望ましい。酸の種類は、特に限定されないが、例えば塩酸、硝酸、硫酸等の鉱酸が挙げられ、望ましくは塩酸である。異性化の進行はＨＰＬＣにてチェックを行い５’−ＦＭＮの純度が９０％を超えた時点を仮の終点とし、これを確認後、得られた懸濁反応液中に酸又はアルカリを添加し溶解後に、先に溶解した液性とは逆のアルカリ又は酸でｐＨを４−８、望ましくはｐＨ５．５に調整する。この溶液を冷却後濾取し、アルコールで洗浄後乾燥して、５’−ＦＭＮの薬理学的に許容される塩、例えば５’−ＦＭＮ−Ｎａが得られるのである。
本発明においては、各種水溶液、反応液、反応工程、及びカラムクロマトグラフィーの溶離剤などに水を使用する。本発明において使用する水は特に限定されないが、望ましくは、蒸留水、イオン交換水及び／又は精製水である。
本発明における高純度５’−ＦＭＮ又はその薬理学的に許容される塩の製造法の最大の特徴は、粗製５’−ＦＭＮ又はその薬理学的に許容される塩の再結晶化による精製を行なうことである。
望ましくは、本発明における高純度５’−ＦＭＮ又はその薬理学的に許容される塩の製造法の最大の特徴は、その少なくとも２段階の精製工程にある。即ち、リボフラビンのホスホリル化工程により得られた粗製５’−ＦＭＮ又はその薬埋学的に許容される塩の再結晶化による精製を行なった後に、ＯＤＳカラム等の逆相カラムクロマトグラフィー処理によるカラム精製または活性炭処理による精製を行うのである。
本発明における高純度５’−ＦＭＮ又はその薬理学的に許容される塩、特に５’―ＦＭＮ−Ｎａの製造法においては、再結晶化工程で、粗製５’−ＦＭＮ−Ｎａ中の不純物のリボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）、リボフラビン−ジホスフェート等を除去し、次の逆相クロマトグラフィーによるカラム処理工程または活性炭処理工程により、粗製５’−ＦＭＮ−Ｎａ中の未反応のリボフラビン、ルミフラビン、ルミクロムを除去する。したがって、本発明に係る高純度５’−ＦＭＮ又はその薬理学的に許容される塩の製造法を使用することにより５’−ＦＭＮ又はその薬理学的に許容される塩中のほとんどの不純物を除去することが可能となるのである。
尚、従来、高純度５’−ＦＭＮ又はその薬理学的に許容される塩の製造法として、例えば、特許第２８５６７６０号公報には、吸着クロマトグラフィーによるカラム処理と逆相クロマトグラフィーによるカラム処理の２段階の精製工程が知られているが、両工程において主に未反応リボフラビン、ルミフラビン、ルミクロムを除去するのみであり、リボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）、リボフラビン−ジホスフェートは十分には除去できなかったのである。
本発明における高純度５’−ＦＭＮ又はその薬理学的に許容される塩、特に５’−ＦＭＮ−Ｎａの製造法においては、再結晶化工程で、粗製５’−ＦＭＮ−Ｎａ中の不純物のリボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）、リボフラビン−ジホスフェート等を除去し、次に活性炭処理工程により、粗製５’−ＦＭＮ−Ｎａ中の未反応のリボフラビン、ルミフラビン、ルミクロムを除去して高純度を確保した後、純度０．１％以下の微量不純物を更に除去する必要がある場合は、活性炭処理工程後に高分子量吸着樹脂で処理しても良い。
本発明においては、例えば、次の方法により、高純度５’−ＦＭＮ又はその薬理学的に許容される塩を製造することができる。
（１）粗製５’−ＦＭＮ−Ｎａの製造
コルベン中にアセトニトリル５００ｍｌ、ピリジン２５０ｇ、オキシ塩化リン５０７ｇを入れ１０℃にて攪拌する。次に、水３３ｍｌをアセトニトリル２００ｍｌに溶解した液を１時間かけて滴下してリン酸化試薬を調製し、リボフラビン１８０ｇ（０．４７８ｍｏｌ）を徐々に添加し１０℃にて１２時間攪拌する。析出した黄色の中間体リボフラビン・サイクリック−４’，５’−ホスホリデートの結晶を濾過し、アセトニトリル５００ｍｌにて洗浄後、風乾し、これを６％塩酸６００ｍｌ（１０℃）中に添加し、４０℃にて２７時間攪拌する。反応液を１０℃に冷却し、２５％水酸化ナトリウム水溶液５４０ｍｌを添加し生成物を完全に溶解後に、８℃に冷却しつつ濃塩酸４５ｍｌを滴下してｐＨを５．５に調製し、５’−ＦＭＮ−Ｎａの黄色結晶を析出させた。析出した結晶を濾過し、メタノール４００ｍｌにて洗浄後減圧乾燥して粗製５’−ＦＭＮ−Ｎａ１９５ｇを得る。
（２）粗製５’−ＦＭＮ−Ｎａの再結晶化精製
（１）で得た粗製５’−ＦＭＮ−Ｎａ５０ｇを１０００ｍｌコルベンに入れ、水４００ｍｌに懸濁下攪拌し、５０℃で加温攪拌することにより完全に溶解させる。この溶液にエタノール１００ｍｌを滴下し攪拌しつつ８℃に冷却し、５’−ＦＭＮ−Ｎａの黄色結晶を再結晶化により析出・濾過後、エタノール１５０ｍｌで洗浄し減圧乾燥して３８．３５ｇの精製５’−ＦＭＮ−Ｎａを得る。
（３）ＯＤＳカラムクロマトグラフィー処理による５’−ＦＭＮ−Ｎａのカラム精製
（２）で得た精製５’−ＦＭＮ−Ｎａ２０ｇを水４００ｍｌに溶解して、濃度５重量％のｐＨ５．５の５’−ＦＭＮ−Ｎａ水溶液を調製する。この５’−ＦＭＮ−Ｎａ水溶液をＯＤＳカラムにチャージした後、溶離剤として１０％アセトニトリルを用いて展開し、溶離液を捕集する。
（４）精製５’−ＦＭＮ−Ｎａの単離
（３）で得た５’−ＦＭＮ−Ｎａを含有する１０％アセトニトリル溶離液を、５０℃の水浴上にて約１５０ｍｌになるまで減圧濃縮し、濃縮液を攪拌しつつエタノール３０ｍｌを滴下し８℃に冷却して、黄色結晶を析出、濾過しエタノール３０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを１６．９ｇ得る。この精製５’−ＦＭＮ−Ｎａは、９８％以上の５’−ＦＭＮ−Ｎａ含量を有し、不純物のリボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）含有量は約１％であり、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）、リボフラビン−ジホスフェート、リボフラビンは検出されない。
また、本発明においては、例えば、次の方法によっても、高純度５’−ＦＭＮ又はその薬理学的に許容される塩を製造することができる。
（１）粗製５’−ＦＭＮ−Ｎａの製造、及び（２）粗製５’−ＦＭＮ−Ｎａの再結晶化精製の工程は、上記のとおりである。
（３）活性炭処理による５’−ＦＭＮ−Ｎａの精製
精製５’−ＦＭＮ−Ｎａ５ｇを水３０ｍｌに溶解して、１Ｎ−ＮａＯＨ水５．６ｍｌを加え、濃度１２重量％のｐＨ７．９の５’−ＦＭＮ−Ｎａ水溶液を調製する。この５’−ＦＭＮ−Ｎａ水溶液に活性炭（二村化学製の太閤Ｙ）０．７５ｇ（５’−ＦＭＮ−Ｎａに対して１５重量％）を添加し、３０℃で１時間懸濁後、活性炭をろ別する。ろ別した活性炭を水２５ｍｌで洗浄し、その洗浄水は先に得られたろ液と合わせる。
（４）精製５’−ＦＭＮ−Ｎａの単離
（３）で得た５’−ＦＭＮ−Ｎａを含有する水溶液に、１Ｎ−ＨＣｌ水４．３ｍｌを加え、ｐＨ６．０にして、攪拌しつつエタノール６０ｍｌを滴下し８℃に冷却して、黄色結晶を析出、濾過しエタノール１０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを３．６６ｇ得る。この精製５’−ＦＭＮ−Ｎａは、９７％以上の５’−ＦＭＮ−Ｎａ含量を有し、不純物のリボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）含有量は約２％であり、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）は約０．２％、リボフラビン−ジホスフェート０．１％以下、リボフラビンは約０．２％である。
さらに、本発明においては、例えば、次の方法によっても、高純度５’−ＦＭＮ又はその薬理学的に許容される塩を製造することができる。
（１）粗製５’−ＦＭＮ−Ｎａの製造、及び（２）粗製５’−ＦＭＮ−Ｎａの再結晶化精製の工程は、上記のとおりである。
（３）活性炭処理による５’−ＦＭＮ−Ｎａの精製
精製５’−ＦＭＮ−Ｎａ５ｇを水５０ｍｌに溶解して、濃度９重量％のｐＨ６の５’−ＦＭＮ−Ｎａ水溶液を調製する。この５’−ＦＭＮ−Ｎａ水溶液に活性炭（Ｎｏｒｉｔ製ＣＡＳＰ）０．６ｇ（５’−ＦＭＮ−Ｎａに対して１２重量％）を添加し、５０℃で１時間懸濁後、活性炭をろ別する。ろ別した活性炭を水１０ｍｌで洗浄し、その洗浄水は先に得られたろ液と合わせる。
（４）高分子量吸着樹脂による５’−ＦＭＮ−Ｎａの精製
（３）で得た５’−ＦＭＮ−Ｎａを含有する水溶液に、高分子量吸着樹脂（三菱化学製のＳＰ７００）５ｇを加え、３０℃で１時間懸濁後、高分子量吸着樹脂をろ別する。ろ別した高分子量吸着樹脂を水１５ｍｌで洗浄し、その水は先に得られたろ液と合わせる。
（５）精製５’−ＦＭＮ−Ｎａの単離
得られたろ液に攪拌しつつエタノール７５ｍｌを滴下し８℃に冷却して、黄色結晶を析出、濾過しエタノール１０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを３．２３ｇ得る。この精製５’−ＦＭＮ−Ｎａは、９７％以上の５’−ＦＭＮ−Ｎａ含量を有し、不純物のリボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）含有量は約２％であり、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）は約０．３％、リボフラビン−ジホスフェート０．１％以下、リボフラビンは約０．１％である。
本発明によるとリボフラビン５’−リン酸（５’−ＦＭＮ）又はその薬理学的に許容される塩、特に汎用されるリボフラビン−５’−リン酸ナトリウム（５’−ＦＭＮ−Ｎａ）の高純度製造法の提供が可能である。即ち、粗製リボフラビン５’−リン酸ナトリウム（５’−ＦＭＮ−Ｎａ）から、リボフラビン−４’−リン酸ナトリウム（４’−ＦＭＮ−Ｎａ）、リボフラビン−３’−リン酸ナトリウム（３’−ＦＭＮ−Ｎａ）、リボフラビン−ジホスフェート、リボフラビンポリホスフェート、リボフラビン、ルミフラビン、ルミクロム等の不純物を簡便な手段でほとんど除去することが可能である。その効果例を以下に示す。
実験例
（１）本発明における精製法（再結晶化精製及び逆相カラムクロマトグラフィーによるカラム精製）の５’−ＦＭＮ−Ｎａの純度に及ぼす効果
下記に示す組成の粗製５’−ＦＭＮ−Ｎａを用いて、（１）再結晶化精製を行い、次に、（２）逆相カラムクロマトグラフィー処理によるカラム精製を行ない、さらに引き続いて（３）精製５’−ＦＭＮ−Ｎａの単離を行なった。
使用した粗製５’−ＦＭＮ−Ｎａの粗成

即ち、最初に、上記の粗製５’−ＦＭＮ−Ｎａ５０ｇを水４００ｍｌに懸濁下攪拌しつつ、５０℃で加温攪拌して完全に溶解させ、この溶液にエタノール１００ｍｌを徐々に滴下し８℃で冷却攪拌して５’−ＦＭＮ−Ｎａの黄色結晶が析出させた。析出した結晶を濾過後、エタノール１５０ｍｌで洗浄し減圧乾燥して５’−ＦＭＮ−Ｎａ３８．５ｇを得た（（１）再結晶化精製）。この再結晶化精製した５’−ＦＭＮ−Ｎａの純度を下記に示す条件でＨＰＬＣによる評価を行なった。その結果を表１に示した。
ＨＰＬＣ分析条件
検出器：紫外吸光光度計（測定波長：２５４ｎｍ）
カラム：Ｎｕｃｌｅｏｓｉｌ−５Ｃ１８、４．６ｍｍｘ２５０ｍｍ
カラム温度：４０℃
移動相：２％リン酸二水素カリウム水溶液：メタノール：アセトニトリル＝４０：９：１
流量：１．０ｍｌ／ｍｉｎ
注入量：５ｕｌ
次に、上記で得た再結晶化された精製５’−ＦＭＮ−Ｎａ５ｇを水１００ｍｌに溶解し濃度５重量％のｐＨ５．５に調整した水溶液（原液）を、ポンプによりＹＭＣ社製のＯＤＳカラム（ＹＭＣ−ＡＱ−Ｃ１８、内径２０ｍｍ×長さ５０ｍｍ）にチヤージした後に、溶離剤として５％アセトニトリル水溶液（ｖ／ｖ）で展開し、溶離液はｕｖ２５４ｎｍで検出しフラクションごとに４画分に分割し、画分中の５’−ＦＭＮ−Ｎａ（（２）逆相カラムクロマトグラフィー処理によるカラム精製）の純度をＨＰＬＣで評価した。この評価結果を表２に示した。
さらに、次に、ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜４画分を合わせ、５０℃の水浴上にて約５０ｍｌになるまで減圧濃縮し、これにエタノール１０ｍｌを滴下し８℃に冷却攪拌して、５’−ＦＭＮ−Ｎａの結晶を析出させ、濾過後エタノール１０ｍｌにて洗浄し減圧乾燥して精製５’−ＦＭＮ−Ｎａ４．０ｇを得た（（３）精製５’−ＦＭＮ−Ｎａの単離）。この濃縮・結晶化精製した５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価し、その結果を表３に示した。

また、比較対照実験として、特許第２８５６７６０号公報に準じた方法により５’−ＦＭＮ−Ｎａの精製を行なった。即ち、「再結晶化精製」の代わりに（１）’高分子量吸着樹脂を充填した吸着カラムクロマトグラフィー処理によるカラム精製を行ない、次に、（２）逆相カラムクロマトグラフィー処理によるカラム精製を行ない、さらに引き続いて（３）精製５’−ＦＭＮ−Ｎａの単離を行なった。
即ち、最初に、先述した組成の粗製５’−ＦＭＮ−Ｎａ５ｇを水１００ｍｌに溶解し濃度５重量％のｐＨ５．５に調整した水溶液をポンプにより三菱化学製の吸着剤樹脂ＳＰ−８５０が充填された吸着カラム（内径２０ｍｍＸ長さ５００ｍｍ）にチヤージした後に、溶離剤として１０％メタノール水溶液（Ｖ／Ｖ）で展開した。溶離液はＵＶ吸収２５４ｎｍで検出し溶離液中の５’−ＦＭＮ−Ｎａ（（１）’高分子量吸着樹脂を充填した吸着カラムクロマトグラフィー処理によるカラム精製）の純度をＨＰＬＣで同様に評価し、その結果を表４に示した。
次に、上記の吸着カラム精製された精製５’−ＦＭＮ−Ｎａ５ｇを水１００ｍｌに溶解し濃度５重量％のｐＨ５．５に調整した水溶液を、ポンプによりＹＭＣ社製のＯＤＳカラム（ＹＭＣ−ＡＱ−Ｃ１８、内径２０ｍｍＸ長さ５００ｍｍ）にチャージした後に、溶離剤として１０％エタノール水溶液（ｖ／ｖ）で展開し、溶離液はＵＶ２５４ｎｍで検出しフラクションごとに４画分に分割し、画分中の５’−ＦＭＮ−Ｎａ（（２）逆相カラムクロマトグラフィー処理によるカラム精製）の純度をＨＰＬＣで評価した。この評価結果を表５に示した。
さらに、次に、ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜４画分を合わせ、５０℃で蒸発濃縮し、２０℃に冷却して５’−ＦＭＮ−Ｎａの結晶を析出させ、濾過後エタノール１０ｍｌにて洗浄し減圧乾燥して精製５’−ＦＭＮ−Ｎａを得た（（３）精製５’−ＦＭＮ−Ｎａの単離）。この濃縮・単離した５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価し、その結果を表６に示した。

本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法及び比較対照実験の対比の結果、本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法では、（１）再結晶化精製工程により、４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ及びリボフラビン・ジホスフェートがかなり除去され、（２）逆相カラム精製（溶離剤：５％アセトニトリル水溶液）によりリボフラビン及びリボフラビン・ジホスフェートが除去され、（３）結晶化による精製５’−ＦＭＮ−Ｎａの単離により、さらに４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａが除去され、最終の精製５’−ＦＭＮ−Ｎａの純度は９７％強の高純度であった。一方、比較対照実験（特許第２８５６７６０号公報に準じた精製方法）に係る５’−ＦＭＮ−Ｎａの製造・精製法では、（１）’高分子量吸着樹脂を充填した吸着カラム精製によりリボフラビンがかなり除去され、（２）逆相カラム精製（溶離剤：１０％メタノール水溶液）によりさらにリボフラビンが除去されたが、（３）精製５’−ＦＭＮ−Ｎａの単離では不純物量の変化は認められず、また、（１）〜（３）のいずれの精製工程においても、４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ、リボフラビン・ジホスフェートはほとんど除去されず、最終の精製５’−ＦＭＮ−Ｎａの純度は９０％強と低かった。また、逆相カラム精製では、溶離剤の種類が、リボフラビンの除去効率に影響を及ぼし、本発明に係るアセトニトリルの５〜１０％水溶液ではリボフラビンを完全に除去できたが、１０％メタノール水溶液ではリボフラビンのわずかな残存が認められた。尚、光分解物のルミフラビン、ルミクロムは、本発明及び比較対照実験のいずれにおいても検出されずほぼ完全に除去されていた。
本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法（粗製５’−ＦＭＮ−Ｎａを用いて、（１）再結晶化精製を行い、次に、（２）逆相カラムクロマトグラフィー処理によるカラム精製を行ない、さらに引き続いて（３）精製５’−ＦＭＮ−Ｎａの単離を行なう）は、従来法と比較して、主要な不純物（４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ、リボフラビン・ジホスフェート、リボフラビン）の除去効果が大きく、際立って優れた高純度５’−ＦＭＮ−Ｎａの製造法であることは明らかである。
（２）逆相カラムクロマトグラフィー処理によるカラム精製における溶離剤の種類の５’−ＦＭＮ−Ｎａの純度に及ぼす効果
逆相カラムクロマトグラフィー処理によるカラム精製において、溶離剤の種類の５’−ＦＭＮ−Ｎａの純度に及ぼす効果を評価するために、実施例１３（カラム：Ｗａｋｏｓｉｌ−４０−１８、溶離剤：１０％アセトニトリル水溶液）の対照実験として、実施例１３の溶離剤のみを１０％エタノールに代えた実験を行い、溶離液はＵＶ２５４ｎｍで検出しフラクションごとに５画分に分割し、画分中の５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価した。この評価結果を表７に示した。
次に、ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜５画分を合わせ、５０℃の水浴上にて約１５０ｍｌになるまで減圧濃縮し、濃縮液を攪拌しつつエタノール３０ｍｌを滴下し８℃に冷却して、黄色結晶を析出させた。この結晶を濾過しエタノール３０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａ１６．４ｇ（回収率８２．０％）を得て、この濃縮・精製した５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価し、その結果を表８に示した。また、旋光度は＋４１．８℃であった。

尚、また、旋光度は＋４１．８℃であった。
同様に、実施例１６（カラム：ＹＭＣ−ＡＱ−Ｃ１８、溶離剤：５％アセトニトリル水溶液）の対照実験として、実施例１６の溶離剤のみを１０％エタノールに代えた実験を行い、溶離液はＵＶ２５４ｎｍで検出しフラクションごとに６画分に分割し、画分中の５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価した。この評価結果を表９に示した。
次に、ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜６画分を合わせ、５０℃の水浴上にて約５０ｍｌになるまで減圧濃縮し、濃縮液を攪拌しつつエタノール１０ｍｌを滴下し８℃に冷却して、黄色結晶を析出させた。この結晶を濾過しエタノール１０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａ４．１ｇ（回収率８２．０％）を得た後に、この濃縮・精製した５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価し、その結果を表１０に示した。また、旋光度は＋４１．９度であった。

本発明に係る実施例１３及び１６の５’−ＦＭＮ−Ｎａの製造・精製法及び対照実験の対比の結果、本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法では、逆相カラム精製の溶離剤としてアセトニトリル水溶液の使用によりリボフラビンが完全に除去され５’−ＦＭＮ−Ｎａの純度も高かった。一方、逆相カラム精製の溶離剤としてエタノール水溶液を使用した対照実験では、不純物としてリボフラビンの残留が認められた。また、不純物除去に必要な溶離剤の量は、本発明（溶離剤：アセトニトリル水溶液）では、対照実験（溶離剤：エタノール水溶液）の１／２量で十分であった。
本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法において、逆相カラムクロマトグラフィー処理によるカラム精製に使用する溶離剤は、アセトニトリル水溶液がエタノール水溶液よりも優れた効果を有することは明らかである。
（３）本発明に係る精製法（再結晶化精製及び活性炭処理による精製）の５’−ＦＭＮ−Ｎａの純度に及ぼす効果
下記に示す組成の粗製５’−ＦＭＮ−Ｎａを用いて、（１）再結晶化精製を行い、次に、（２）活性炭処理による精製を行ない、さらに引き続いて（３）精製５’−ＦＭＮ−Ｎａの単離を行なった。
使用した粗製５’−ＦＭＮ−Ｎａの粗成

即ち、最初に、上記の粗製５’−ＦＭＮ−Ｎａ５．５ｇを水５５ｍｌに懸濁下攪拌しつつ、５０℃で加温攪拌して完全に溶解させ、この溶液にエタノール２８ｍｌを徐々に滴下し１℃で冷却攪拌して５’−ＦＭＮ−Ｎａの黄色結晶が析出させた。析出した結晶を濾過後、エタノール１０ｍｌで洗浄し減圧乾燥して５’−ＦＭＮ−Ｎａ４．８ｇを得た（（１）再結晶化精製）。この再結晶化精製した５’−ＦＭＮ−Ｎａの純度のＨＰＬＣによる評価結果を表Ａに示した。
次に、上記で得た再結晶化された精製５’−ＦＭＮ−Ｎａ４ｇを水５６ｍｌに溶解して、１Ｎ−ＮａＯＨ水４．７ｍｌを加え、濃度６重量％のｐＨ７．８の５’−ＦＭＮ−Ｎａ水溶液を調製した。この５’−ＦＭＮ−Ｎａ水溶液に活性炭（二村化学製の太閤Ｙ）３．２ｇ（５’−ＦＭＮ−Ｎａに対して８０重量％）を添加し、５０℃で２時間懸濁後、活性炭をろ別した。ろ別した活性炭を水１５ｍｌで洗浄し、その洗浄水は先に得られたろ液と合わせた（（２）活性炭処理による精製）。
さらに、この活性炭処理された精製５’−ＦＭＮ−Ｎａを含有する水溶液に、１Ｎ−ＨＣｌ水２．５ｍｌを加え、ｐＨ６．０にして、攪拌しつつエタノール４０ｍｌを滴下し４℃に冷却して、黄色結晶を析出、濾過しエタノール１０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを１．９８ｇ得た（（３）精製５’−ＦＭＮ−Ｎａの単離）。この結晶化精製した５’−ＦＭＮ−Ｎａの純度をＨＰＬＣで評価し、その結果を表Ｂに示した。

また、比較対照実験として、先に示した［（１）本発明における精製法（再結晶化精製及び逆相カラムクロマトグラフィーによるカラム精製）の５’−ＦＭＮ−Ｎａの純度に及ぼす効果］の評価の際に用いた［特許第２８５６７６０号公報に準じた方法による５’−ＦＭＮ−Ｎａの精製］を行なった。即ち、「再結晶化精製及び活性炭処理による精製」の代わりに（１）’高分子量吸着樹脂を充填した吸着カラムクロマトグラフィー処理によるカラム精製を行ない、次に、（２）逆相カラムクロマトグラフィー処理によるカラム精製を行ない、さらに引き続いて（３）精製５’−ＦＭＮ−Ｎａの単離を行なった。
本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法及び比較対照実験の対比の結果、本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法では、（１）再結晶化精製工程により、４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ及びリボフラビン・ジホスフェートがかなり除去され、（２）活性炭処理及びそれに続く精製５’−ＦＭＮ−Ｎａの単離によりリボフラビンが主に除去され、さらに４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａが除去され、最終の精製５’−ＦＭＮ−Ｎａの純度は９３％強の高純度であった。一方、比較対照実験（特許第２８５６７６０号公報に準じた精製方法）に係る５’−ＦＭＮ−Ｎａの製造・精製法では、（１）’高分子量吸着樹脂を充填した吸着カラム精製によりリボフラビンがかなり除去され、（２）逆相カラム精製（溶離剤：１０％メタノール水溶液）によりさらにリボフラビンが除去されたが、（３）精製５’−ＦＭＮ−Ｎａの単離では不純物量の変化は認められず、また、（１）〜（３）のいずれの精製工程においても、４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ、リボフラビン・ジホスフェートはほとんど除去されず、最終の精製５’−ＦＭＮ−Ｎａの純度は９０％強と低かった。尚、光分解物のルミフラビン、ルミクロムは、本発明及び比較対照実験のいずれにおいても検出されずほぼ完全に除去されていた。
本発明に係る５’−ＦＭＮ−Ｎａの製造・精製法（粗製５’−ＦＭＮ−Ｎａを用いて、（１）再結晶化精製を行い、次に、（２）活性炭処理による精製、さらに引き続いた（３）精製５’−ＦＭＮ−Ｎａの単離）は、従来法と比較して、主要な不純物（リボフラビン、４’−ＦＭＮ−Ｎａ、３’−ＦＭＮ−Ｎａ、リボフラビン・ジホスフェート）の除去効果が大きく、優れた高純度５’−ＦＭＮ−Ｎａの製造法であることは明らかである。
実施例
次に実施例を挙げて本発明を説明するが、本発明はこれらの実施例に限定される訳ではない。
実施例１ 粗製リボフラビン−５’−リン酸ナトリウム（５’−ＦＭＮ−Ｎａ）の再結晶化精製法
１）粗製５’−ＦＭＮ及び粗製５’−ＦＭＮ−Ｎａの製造
攪拌機、塩化カルシウム管、温度計、バッフルを付けた２Ｌ４頚コルベン中にアセトニトリル５００ｍｌ、ピリジン２５０ｇ（３．１４ｍｏｌ）、オキシ塩化リン５０７ｇ（３．３ｍｏｌ）を入れ１０℃にて攪拌した。次に、水３３ｍｌをアセトニトリル２００ｍｌに溶解した液を１時間かけて滴下してリン酸化試薬を調製し、リボフラビン１８０ｇ（０．４７８ｍｏｌ）を徐々に添加し１０℃にて１２時間攪拌した。析出した黄色の中間体リボフラビン・サイクリック−４’，５’−ホスホリデートの結晶を濾過し、アセトニトリル５００ｍｌにて洗浄後、風乾した（収量３６０ｇ）。
ついで２Ｌ４頚コルベンに６％塩酸（水）６００ｍｌを入れ、１０℃で攪拌しつつ上記に得た中間体３６０ｇを添加し、４０℃にて２７時間攪拌した。ＨＰＬＣにて原料中間体の消失を確認後、反応液を１０℃に冷却し、２５％水酸化ナトリウム（水）溶液５４０ｍｌを１時間かけて滴下し生成物を完全に溶解させた（ｐＨ＝９．２６）。
この反応液を８℃に冷却しつつ濃塩酸４５ｍｌを滴下してｐＨを５．５に調製し、５’−ＦＭＮ−Ｎａの黄色結晶を析出させた。析出した結晶を濾過し、メタノール４００ｍｌにて洗浄後減圧乾燥して５’−ＦＭＮ−Ｎａ１９５ｇを得た（収率８５．３％）。
ＨＰＬＣ評価の結果、この生成物は以下の組成であった。

２）粗製５’−ＦＭＮ−Ｎａの再結晶化精製
１）で得た５’−ＦＭＮ−Ｎａ５０ｇ（０．１０４５ｍｏｌ）を１０００ｍｌコルベンに入れ水４００ｍｌに懸濁下攪拌し、５０℃で加温攪拌することにより完全に溶解させた。この溶液にエタノール１００ｍｌを滴下し攪拌しつつ８℃に冷却し、５’−ＦＭＮ−Ｎａの黄色結晶が析出させた。
析出した結晶を濾過後、エタノール１５０ｍｌで洗浄し減圧乾燥して３８．３５ｇの精製５’−ＦＭＮ−Ｎａを得た（回収率７６．７％）。
ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の組成であった。また、旋光度は＋４１．５度であった。

実施例２−１２． 粗製５’−ＦＭＮ−Ｎａの再結晶化精製法
実施例１と同様に、実施例１の１）により得られた粗製５’−ＦＭＮ−Ｎａ５ｇ（０．０１０４ｍｏｌ）を１００ｍｌコルベンに入れ水４０ｍｌに懸濁下攪拌し、５０℃で加温攪拌することにより完全に溶解させた。この溶液に水に可溶な溶媒を滴下し攪拌しつつ８℃に冷却し、５’−ＦＭＮ−Ｎａの黄色結晶を得た。析出した結晶を濾過後、エタノール１５ｍｌで洗浄し減圧乾燥して精製５’−ＦＭＮ−Ｎａを得た（回収率７６．７％）。
ＨＰＬＣ評価により得られた各々の精製５’−ＦＭＮ−Ｎａの組成及び再結晶化精製法による回収率を、表１１に示した。

実施例２−１２においてリボフラビン−ジホスフェートの含有量は０．２１−０．２３％であった。
実施例１３． 再結晶化品５’−ＦＭＮ−ＮａのＯＤＳカラムクロマトグラフィー処理による高純度５’−ＦＭＮ−Ｎａの製造法
実施例１の２）で得られた再結晶化された精製５’−ＦＭＮ−Ｎａ２０ｇを水４００ｍｌに溶解し、濃度５重量％のｐＨ５．５の５’−ＦＭＮ−Ｎａ水溶液（原液）を調製した。和光純薬社製のＯＤＳカラムＷａｋｏｓｉｌ−４０−Ｃ１８（内径５０ｍｍｘ長さ５００ｍｍ）を１０％アセトニトリル水溶液であらかじめ置換しておき、上記の原液をポンプでＯＤＳカラムにチャージした後、溶離剤として１０％アセトニトリルを用いて展開し、溶離液をフラクションごとに４画分に分割した。
ＨＰＬＣ評価により得られた各々の画分の精製５’−ＦＭＮ−Ｎａの組成を、表１２に示した。

ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜４画分を合わせ、５０℃の水浴上にて約１５０ｍｌになるまで減圧濃縮し、濃縮液を攪拌しつつエタノール３０ｍｌを滴下し８℃に冷却して、黄色結晶を析出させた。この結晶を濾過しエタノール３０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを１６．９ｇ得た（回収率８４．５％）。ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の組成であった。また、旋光度は＋４２．２度であった。

実施例１４−１５． 再結晶化品５’−ＦＭＮ−ＮａのＯＤＳカラムクロマトグラフィー処理による高純度５’−ＦＭＮ−Ｎａの製造法
ＯＤＳカラムにチャージ後に展開に用いる溶離剤の種類だけを変え、それ以外は実施例１３と同様の方法で高純度５’−ＦＭＮ−Ｎａを製造した。
即ち、実施例１の２）で得られた再結晶化された精製５’−ＦＭＮ−Ｎａ２０ｇを水４００ｍｌに溶解し、濃度５重量％のｐＨ５．５の５’−ＦＭＮ−Ｎａ水溶液（原液）を調製した。和光純薬社製のＯＤＳカラムＷａｋｏｓｉｌ−４０−Ｃ１８（内径５０ｍｍｘ長さ５００ｍｍ）を用いて、上記の原液をポンプでＯＤＳカラムにチャージした後、溶離剤として１０％アセトン水溶液（実施例１４）又は１０％ＴＨＦ水溶液（実施例１５）を用いて展開し、溶離液の画分を捕集した。
ＨＰＬＣ評価により得られた各々の捕集画分の精製５’−ＦＭＮ−Ｎａの組成を、表１３に示した。

次に、ＯＤＳカラムクロマトグラフィー処理をし捕集した各々の溶離液の画分を、５０℃の水浴上にて約１５０ｍｌになるまで減圧濃縮し、濃縮液を攪拌しつつエタノール３０ｍｌを滴下し８℃に冷却して、黄色結晶を析出させた。この結晶を濾過しエタノール３０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを得た。
ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の表１４に示す組成であった。

実施例１６． 高純度５’−ＦＭＮ−Ｎａの製造法
実施例１の２）で得られた再結晶化された精製５’−ＦＭＮ−Ｎａ５ｇを水１００ｍｌに溶解し、濃度５重量％のｐＨ５．５の５’−ＦＭＮ−Ｎａ水溶液（原液）を調製した。ＹＭＣ社製のＯＤＳカラムＹＭＣ−ＡＱ−Ｃ１８（内径２０ｍｍＸ長さ５００ｍｍ）を５％アセトニトリル水溶液であらかじめ置換しておき、上記の原液をポンプでＯＤＳカラムにチャージした後、溶離剤として５％アセトニトリルを用いて展開し、溶離液をフラクションごとに４画分に分割した。
ＨＰＬＣ評価により得られた各々の画分の精製５’−ＦＭＮ−Ｎａの組成を、表１５に示した。

ＯＤＳカラムクロマトグラフィー処理をしたＮｏ．２〜４画分を合わせ、５０℃の水浴上にて約５０ｍｌになるまで減圧濃縮し、エタノール１０ｍｌを滴下して８℃に冷却攪拌して、高純度５’−ＦＭＮの結晶を析出させ、濾過後エタノール１０ｍｌにて洗浄し減圧乾燥して高純度５’−ＦＭＮ−Ｎａ４．２ｇを得た（回収率８４．０％）。
ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の組成であった。また、旋光度は＋４２．１度であった。

実施例１７． 再結晶化品５’−ＦＭＮ−Ｎａの活性炭処理による高純度５’−ＦＭＮ−Ｎａの製造法
下記に示す組成の再結晶化精製品５’−ＦＭＮ−Ｎａを用いて、活性炭処理を行ない、さらに引き続いて精製５’−ＦＭＮ−Ｎａの単離を行なった。
使用した粗製５’−ＦＭＮ−Ｎａの粗成

（１）活性炭処理による５’−ＦＭＮ−Ｎａの精製
上記組成の再結晶化精製品５’−ＦＭＮ−Ｎａ５ｇを水３０ｍｌに溶解して、１Ｎ−ＮａＯＨ水５．６ｍｌを加え、濃度１２重量％のｐＨ７．９の５’−ＦＭＮ−Ｎａ水溶液を調製した。この５’−ＦＭＮ−Ｎａ水溶液に活性炭（二村化学製の太閤Ｙ）０．７５ｇ（５’−ＦＭＮ−Ｎａに対して１５重量％）を添加し、３０℃で１時間懸濁後、活性炭をろ別した。ろ別した活性炭を水２５ｍｌで洗浄し、その洗浄水を先に得られたろ液と合わせ、活性炭処理による精製５’−ＦＭＮ−Ｎａを含有する水溶液を得た。
（２）精製５’−ＦＭＮ−Ｎａの単離
（１）で得た５’−ＦＭＮ−Ｎａを含有する水溶液に、１Ｎ−ＨＣｌ水４．３ｍｌを加え、ｐＨ６．０にして、攪拌しつつエタノール６０ｍｌを滴下し８℃に冷却して、黄色結晶を析出、濾過し、エタノール１０ｍｌにて洗浄後減圧乾燥して高純度５’−ＦＭＮ−Ｎａを３．６６ｇ得た。ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の組成であった。
精製後の５’−ＦＭＮ−Ｎａの粗成

実施例１８． 再結晶化品５’−ＦＭＮ−Ｎａの活性炭処理と高分子量吸着樹脂による高純度５’−ＦＭＮ−Ｎａの製造法
実施例１７で用いた再結晶化精製品５’−ＦＭＮ−Ｎａを用いて、活性炭処理を行ない、引き続いて高分子量吸着樹脂による精製を行い、さらに引き続いて精製５’−ＦＭＮ−Ｎａの単離を行なった。
（１）活性炭処理と高分子量吸着樹脂による５’−ＦＭＮ−Ｎａの精製
精製５’−ＦＭＮ−Ｎａ５ｇを水５０ｍｌに溶解して、濃度９重量％のｐＨ６の５’−ＦＭＮ−Ｎａ水溶液を調製した。この５’−ＦＭＮ−Ｎａ水溶液に活性炭（Ｎｏｒｉｔ製ＣＡＳＰ）０．６ｇ（５’−ＦＭＮ−Ｎａに対して１２重量％）を添加し、５０℃で１時間懸濁後、活性炭をろ別した。ろ別した活性炭を水１０ｍｌで洗浄し、その洗浄水は先に得られたろ液と合わせて、活性炭処理による精製５’−ＦＭＮ−Ｎａを含有する水溶液を得た。この活性炭処理による精製５’−ＦＭＮ−Ｎａを含有する水溶液に、高分子量吸着樹脂（三菱化学製のＳＰ７００）５ｇを加え、３０℃で１時間懸濁後、高分子量吸着樹脂をろした。ろ別した高分子量吸着樹脂を水１５ｍｌで洗浄し、その水は先に得られたろ液と合わせて、高分子量吸着樹脂処理による精製５’−ＦＭＮ−Ｎａを含有する水溶液を得た。
（２）精製５’−ＦＭＮ−Ｎａの単離
（１）で得た高分子量吸着樹脂処理による精製５’−ＦＭＮ−Ｎａを含有する水溶液を攪拌しつつエタノール７５ｍｌを滴下し、８℃に冷却して、黄色結晶を析出、濾過しエタノール１０ｍｌにて洗浄後、減圧乾燥して高純度の精製５’−ＦＭＮ−Ｎａを３．２３ｇ得た。ＨＰＬＣ評価の結果、この精製５’−ＦＭＮ−Ｎａは以下の組成であった。
精製５’−ＦＭＮ−Ｎａの単離

Technical field
The present invention relates to high-purity riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, particularly riboflavin 5'- useful as a pharmaceutical, a food additive, a feed, an industrial intermediate or the like. The present invention relates to a novel industrially excellent method for producing sodium phosphate (5'-FMN-Na).
Conventional technology
Riboflavin, riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof has an important role as a coenzyme in different enzymatic reactions in a living body, and particularly, a sodium salt (5'-FMN). FMN-Na) is often used as a pharmaceutical, food and feed additive. JP-A-5-201864 discloses riboflavin, riboflavin-5'-phosphate (5'-FMN) and pharmacologically acceptable salts thereof (riboflavin 5'-sodium phosphate (5'-FMN). -Na) and the like, a therapeutic agent for immunostimulation / infection protection using a riboflavin derivative is disclosed. Furthermore, Japanese Patent Publication No. Hei 6-506212 discloses that riboflavin is effective for the prevention and treatment of malaria diseases.
Conventionally, riboflavin is used to convert riboflavin 5′-phosphate (5′-FMN) or a pharmacologically acceptable salt thereof, particularly widely used sodium riboflavin-5′-phosphate (5′-FMN-Na). In the production method, it is extremely difficult to obtain a high-purity product, and as impurities, riboflavin 4′-sodium phosphate (4′-FMN-Na) and riboflavin-3 ′, which are inactive isomers in vivo, are contained as impurities. -Sodium phosphate (3'-FMN-Na), riboflavin-diphosphate, riboflavin polyphosphate, and a large amount of unreacted riboflavin.
As a method for producing riboflavin 5′-phosphate (5′-FMN) or a pharmacologically acceptable salt thereof, a phosphorylation method of riboflavin is generally used, and for example, Japanese Patent Application Laid-Open No. 48-54099. The reaction is carried out by adding riboflavin to a mixture of a polar solvent, a tertiary amine, phosphorus oxychloride and water, further decomposing excess phosphorus oxychloride by adding water, and hydrolyzing with hydrochloric acid generated there. Japanese Patent Application Laid-Open No. H11-49790 discloses a method of concentrating after pH adjustment, in which riboflavin is reacted with phosphorus oxychloride, and riboflavin cyclic 4′-, 5′-phosphoridate or a salt thereof is reacted outside the system. Disclosed is a method for producing the compound by taking it out, particularly by hydrolysis and isomerization under acidic conditions. Particularly in the latter production method, a high content of 5'-FMN-Na 87 to 90% is obtained. However, the purity of pharmaceuticals is not always at a sufficient level. Therefore, an additional purification step is required.
Riboflavin-5'-phosphate (5'-FMN) obtained by phosphorylation or the like or a pharmacologically acceptable salt thereof, particularly widely used sodium riboflavin-5'-phosphate (5'-FMN-Na) As a purification method, for example, an aqueous solution having a concentration of crude 5′-FMN or a pharmacologically acceptable salt thereof of 1 to 5% by weight and a pH of 4 to 8 is prepared as disclosed in Japanese Patent No. 2856760. A method of treating with a molecular weight adsorption resin, isolating from the resulting solution, further treating with RP (reverse phase) silica gel, eluting with water or an aqueous mixture having a lower aliphatic alcohol content of 0 to 80%, concentrating and crystallizing. It has been disclosed. However, the purity of 5′-FMN or a pharmacologically acceptable salt thereof obtained by this purification method, particularly, riboflavin 5′-sodium phosphate (5′-FMN-Na), which is widely used, is not yet sufficient, Impurities such as riboflavin-4'-sodium phosphate (4'-FMN-Na), riboflavin-3'-sodium phosphate (3'-FMN-Na), and riboflavin-diphosphate cannot be sufficiently removed. .
Therefore, high-purity production of riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, particularly, widely used sodium riboflavin-5'-phosphate (5'-FMN-Na). The law is very much anticipated.
In particular, riboflavin-4'-sodium phosphate (sodium riboflavin-4'-phosphate) is obtained from a mixture containing riboflavin-5'-phosphate (5'-FMN) obtained by a phosphorylation method or the like or a pharmaceutically acceptable salt thereof as a main component. Production capable of removing impurities such as 4'-FMN-Na), riboflavin-3'-sodium phosphate (3'-FMN-Na), riboflavin-diphosphate, riboflavin polyphosphate, riboflavin, lumiflavin, and lumichrome by simple means. Methods and / or purification methods are needed.
Disclosure of the invention
In view of the above situation, the present inventors have conducted intensive studies, and as a result, have found that the intended purpose can be achieved by the following configuration, and have completed the present invention.
The present invention relates to a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, comprising the steps of: 1) preparing crude 5'-FMN or a pharmacologically acceptable salt thereof; Dissolved in water or a buffer solution to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4 to 8; While stirring, an organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight based on the aqueous solution obtained above, and 5′-FMN or a pharmacologically acceptable salt thereof is added. And recrystallizing the resulting salt. 5) A method for producing high-purity 5′-FMN or a pharmaceutically acceptable salt thereof. The solution temperature at the time of dissolving the crude 5′-FMN or the pharmacologically acceptable salt thereof in 1) in water or a buffer is not particularly limited, but may be heated as necessary, and usually, The temperature is from 10 to 100 ° C, preferably from 10 to 90 ° C, particularly preferably from 30 to 80 ° C. The addition amount of the water-soluble organic solvent of 2) to the previously obtained aqueous solution is usually 1 to 100% by weight, preferably 1 to 50% by weight, more preferably 20 to 40% by weight. It is. Further, the temperature is usually 0-60 ° C, preferably 5-55 ° C, and more preferably 40-50 ° C.
The present invention also relates to a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof. 1) Crude 5'-FMN or a pharmacologically acceptable salt thereof. The salt is dissolved in water or a buffer to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. While mechanically stirring, an organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight based on the aqueous solution obtained above, and 5′-FMN or a pharmacological agent thereof is added. 3) The recrystallized 5′-FMN or a pharmacologically acceptable salt thereof is dissolved in water or a buffer to obtain a solution of pH 4 having a concentration of 1 to 20% by weight. −9) 4) Then, reverse the flow so that the liquid volume is at least 1-100% of the bed volume of the column. Obtaining an eluate of 5′-FMN or a pharmacologically acceptable salt thereof, which is subjected to column chromatography and then purified using a water-soluble organic solvent or a water-containing organic solvent as an eluent, The method for producing high purity 5'-FMN or a pharmacologically acceptable salt thereof characterized by the following.
Purified from the eluate of the purified 5′-FMN or the pharmaceutically acceptable salt thereof obtained in the steps 1) to 4), for example, by a method of concentration, evaporation to dryness, and crystallization. 5′-FMN or a pharmacologically acceptable salt thereof can be obtained, but purified 5′-FMN or a pharmacologically acceptable salt thereof can be obtained at a temperature of 10-60 ° C. by 5′-FMN. It is desirable to concentrate until the concentration of FMN becomes 5 to 20%, and to crystallize 5′-FMN by adding an organic solvent soluble in water to the concentrated solution at 0 to 60 ° C. at 1 to 150% by weight. Examples of the concentration method include, but are not particularly limited to, vacuum concentration and reverse osmosis membrane concentration.
The steps 1) and 2) are as described above. The column packing material used in the reversed phase column chromatography in step 4) is not particularly limited, but is usually ODS (C18, octadecylsilane), C8 (octylsilane) or C4 (butylsilane), preferably ODS (C18 Octadecylsilane) or C8 (octylsilane), particularly preferably ODS (C18, octadecylsilane). The eluent of 5′-FMN or a pharmaceutically acceptable salt thereof subjected to reverse phase column chromatography is not particularly limited as long as it is a water-soluble organic solvent or a water-containing organic solvent. With lower aliphatic alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, and isopropyl alcohol, acetonitrile, acetone, tetrahydrofuran (THF), DMF, DMSO, or an aqueous solution thereof, or a mixture of two or more thereof, or an aqueous solution thereof Yes, particularly preferably acetonitrile or an aqueous solution of acetonitrile. The content of the organic solvent in the aqueous organic solvent (aqueous organic solvent) used as an eluent for the reverse phase column chromatography is not particularly limited, but is preferably 0.1 to 50% by volume, more preferably 1 to 50% by volume. The content is 30% by volume, particularly preferably 3 to 20% by volume.
Further, the present invention relates to a method for purifying riboflavin 5-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, wherein 1) crude 5'-FMN or a pharmacologically acceptable salt thereof. Is dissolved in water or a buffer solution to prepare an aqueous solution of crude 5′-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. While mechanically stirring, an organic solvent soluble in water at a temperature of 0 to 60 ° C. is added in an amount of 1 to 100% by weight based on the aqueous solution obtained above to obtain 5′-FMN or its pharmacology. 3) recrystallized 5′-FMN or a pharmacologically acceptable salt thereof is dissolved in water or a buffer to give a concentration of 1-20% by weight. pH 4-9 aqueous solution 4) Next, about 1-10 for 5'-FMN or a pharmaceutically acceptable salt thereof After adding 0% by weight of activated carbon to the aqueous solution and adsorbing impurities in the aqueous solution to the activated carbon, the medium containing the activated carbon is filtered to obtain purified 5′-FMN or a pharmacologically acceptable salt thereof. A method for producing high-purity 5′-FMN or a pharmacologically acceptable salt thereof, characterized by obtaining an aqueous solution (filtrate). From the solution of the purified 5′-FMN or the pharmaceutically acceptable salt thereof obtained in the steps 1) to 4), for example, the solution was purified by a method of concentration, evaporation to dryness, and crystallization. 5′-FMN or a pharmacologically acceptable salt thereof can be obtained. That is, in a solution of the purified 5′-FMN or a pharmacologically acceptable salt thereof obtained in the steps 1) to 4), the water-soluble organic solvent in the solution (1) is usually reduced to 0. Add 1-150% by weight at −60 ° C., or (2) concentrate the solution at a temperature of 10−60 ° C. until the concentration of 5′-FMN becomes 5-20% and dissolve the water in the concentrated solution. The organic solvent can be added at 1-160% by weight at 0-60 ° C. to crystallize 5′-FMN, but in particular, (2) the solution is prepared by adding 5′-FMN at a temperature of 10-60 ° C. It is desirable that the 5'-FMN be crystallized by concentrating the solution to a concentration of 5 to 20% and adding a water-soluble organic solvent to the concentrated solution at 0 to 60 ° C at 1 to 150% by weight. Examples of the concentration method include, but are not particularly limited to, vacuum concentration and reverse osmosis membrane concentration.
The steps 1) to 3) are as described above.
In the activated carbon treatment of 4), impurities present in a dissolved state in an aqueous solution of 5′-FMN or a pharmacologically acceptable salt thereof are adsorbed. The amount of activated carbon added to the aqueous solution is usually about 1 to 100% by weight, preferably 5 to 50% by weight, more preferably 5 to 50% by weight, based on 5'-FMN or a pharmaceutically acceptable salt thereof. 15 to 30% by weight. Activated carbon according to the present invention, usually, in the case of activated carbon produced by the steam activation method, the raw material is peat, lignite, coal and the like, in the case of activated carbon produced by the chemical activation method, the raw material is wood and the like, However, it is not limited to these. The inner surface area of the activated carbon is not particularly limited, but is preferably 500-1500 m2 / g. The shape of the activated carbon is not particularly limited, but is preferably in the form of powder or granules, and more preferably in the form of powder. The particle size of the activated carbon powder is not particularly limited, is preferably 0.1 to 150 μm, and particularly preferably 0.1 to 150 μm of powdered activated carbon. Examples of commercially available activated carbon include, for example, activated carbon produced by the chemical activation method, such as Taiko Y (registered trademark) manufactured by Futmura Chemical Industry Co., Ltd., CASP (registered trademark) manufactured by Norit, etc., as activated carbon produced by the steam activation method. Include Shirasagi P (registered trademark) manufactured by Takeda Pharmaceutical Co., Ltd., but is not limited to these.
The present invention also relates to a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof. 1) Crude 5'-FMN or a pharmacologically acceptable salt thereof The salt is dissolved in water or a buffer to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4 to 8; While mechanically stirring, an organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight based on the aqueous solution obtained above, and 5′-FMN or a pharmacological agent thereof is added. 3) The recrystallized 5′-FMN or a pharmaceutically acceptable salt thereof is dissolved in water or a buffer solution to a concentration of 1 to 20% by weight of pH4. 4) Then, about 1-10 to 5'-FMN or a pharmacologically acceptable salt thereof. % By weight of activated carbon is added to the aqueous solution, and after the impurities in the aqueous solution are adsorbed on the activated carbon, the medium containing the activated carbon is filtered to obtain an aqueous solution of 5′-FMN or a pharmaceutically acceptable salt thereof, 5) Next, 10-200% by weight of a high molecular weight adsorption resin based on 5′-FMN or a pharmacologically acceptable salt thereof is added to the aqueous solution, and impurities in the aqueous solution are adsorbed on the high molecular weight adsorption resin. Thereafter, by filtering the medium containing the high molecular weight adsorption resin, to obtain a solution of purified 5'-FMN or a pharmacologically acceptable salt thereof, characterized in that high purity 5'-FMN or This is a method for producing a pharmacologically acceptable salt.
From the solution of the purified 5′-FMN or the pharmaceutically acceptable salt thereof obtained in the steps 1) to 5), for example, the solution was purified by a method of concentration, evaporation to dryness, and crystallization. 5′-FMN or a pharmacologically acceptable salt thereof can be obtained. That is, in a solution of the purified 5′-FMN or a pharmacologically acceptable salt thereof obtained in the steps 1) to 4), the water-soluble organic solvent in the solution (1) is usually reduced to 0. Add 1-150% by weight at −60 ° C., or (2) concentrate the solution at a temperature of 10−60 ° C. until the concentration of 5′-FMN becomes 5-20% and dissolve the water in the concentrated solution. The organic solvent can be added at 1-160% by weight at 0-60 ° C. to crystallize 5′-FMN, but in particular, (2) the solution is prepared by adding 5′-FMN at a temperature of 10-60 ° C. It is desirable that the 5'-FMN be crystallized by concentrating the solution to a concentration of 5 to 20% and adding a water-soluble organic solvent to the concentrated solution at 0 to 60 ° C at 1 to 150% by weight. Examples of the concentration method include, but are not particularly limited to, vacuum concentration and reverse osmosis membrane concentration.
The steps 1) to 4) are as described above.
In the high molecular weight adsorption resin treatment of 5), impurities existing in a dissolved state in an aqueous solution of 5′-FMN or a pharmacologically acceptable salt thereof are adsorbed. The amount of the high molecular weight adsorption resin to be added to the aqueous solution is usually 10 to 200% by weight, preferably 50 to 150% by weight, based on 5'-FMN or a pharmaceutically acceptable salt thereof. Preferably it is 80 to 110% by weight. The high molecular weight adsorption resin according to the present invention is not particularly limited, and is, for example, a styrene-divinylbenzene copolymer, a methacrylic acid polymer, or the like. The particle size of the high molecular weight adsorption resin is not particularly limited, and is preferably 3 to 1000 μm, and more preferably 50 to 250 μm. Examples of commercially available high molecular weight adsorption include DIAION SEPABEADS (registered trademark) manufactured by Mitsubishi Chemical Corporation, particularly SP207, SP700, SP850, HP20, HP2MG, and the like.
In the present invention, pharmacologically acceptable salts of riboflavin-5'-phosphate (5'-FMN) are not particularly limited, and examples thereof include inorganic salts such as sodium salt and potassium salt, and various organic salts. Preferably, it is sodium riboflavin-5'-phosphate (5'-FMN-Na).
In the present invention, the method for producing the crude 5′-FMN or a pharmacologically acceptable salt thereof used in 1) is not limited, but in particular, the following reaction between riboflavin (1) and phosphorus oxychloride, or Riboflavin (1) is reacted with phosphorus oxychloride and then produced in a sodium chloride reaction with sodium hydroxide. The riboflavin (1) is reacted with phosphorus oxychloride to produce crude 5'-FMN (3). In the reaction to be produced, riboflavin cyclic-4 ′, 5′-phosphoridate (2) or a salt thereof, which is a reaction intermediate, is taken out of the system and hydrolyzed / isomerized under acidic conditions. 5′-FMN (3) or a pharmacologically acceptable salt thereof, particularly 5′-FMN-Na (4) is desirable.

This crude 5′-FMN or a pharmacologically acceptable salt thereof is obtained by converting the reaction intermediate riboflavin cyclic 4 ′-, 5′-phosphoridate (2) into yellow crystals in the presence of pyridine and phosphorus oxychloride. It is produced by separating, washing, drying and hydrolyzing / isomerizing under acidic conditions. The conditions for the hydrolysis and isomerization are not particularly limited, but an acid having a concentration of 1-35.5 (w / v)%, preferably an acid having a concentration of 5-18% is added to riboflavin (1) as a starting material at a rate of 1 to 1%. It can be prepared by stirring at 10 to 100 ° C., preferably 30 to 80 ° C., particularly preferably 30 to 60 ° C., using -10 volumes, preferably 1 to 8 volumes, particularly preferably 2 to 4 volumes. desirable. The type of the acid is not particularly limited, and examples thereof include mineral acids such as hydrochloric acid, nitric acid, and sulfuric acid, and hydrochloric acid is preferable. The progress of isomerization is checked by HPLC, and the time when the purity of 5′-FMN exceeds 90% is set as a temporary end point. After confirming this, acid or alkali is added to the obtained suspension reaction solution. After dissolution, the pH is adjusted to 4-8, preferably 5.5, with an alkali or acid opposite to that of the solution previously dissolved. The solution is filtered after cooling, washed with alcohol and dried to obtain a pharmacologically acceptable salt of 5'-FMN, for example, 5'-FMN-Na.
In the present invention, water is used for various aqueous solutions, reaction solutions, reaction steps, eluents for column chromatography, and the like. The water used in the present invention is not particularly limited, but is preferably distilled water, ion-exchanged water and / or purified water.
The most significant feature of the method for producing high-purity 5'-FMN or a pharmaceutically acceptable salt thereof according to the present invention is that purification by recrystallization of crude 5'-FMN or a pharmaceutically acceptable salt thereof is carried out. It is to do.
Desirably, the most significant feature of the method for producing high-purity 5'-FMN or a pharmaceutically acceptable salt thereof according to the present invention resides in its at least two purification steps. That is, after purification by recrystallization of the crude 5′-FMN obtained by the phosphorylation step of riboflavin or its implantable salts, the column is subjected to reverse phase column chromatography such as an ODS column. Purification or purification by activated carbon treatment is performed.
In the method for producing high-purity 5'-FMN or a pharmacologically acceptable salt thereof, particularly 5'-FMN-Na in the present invention, in the recrystallization step, impurities in crude 5'-FMN-Na are removed. Riboflavin-4'-sodium phosphate (4'-FMN-Na), riboflavin-3'-sodium phosphate (3'-FMN-Na), riboflavin-diphosphate, etc. were removed and the following reverse phase chromatography was performed. Unreacted riboflavin, lumiflavin, and lumichrome in the crude 5'-FMN-Na are removed by a column treatment step or an activated carbon treatment step. Therefore, by using the method for producing high-purity 5′-FMN or a pharmaceutically acceptable salt thereof according to the present invention, most impurities in 5′-FMN or a pharmaceutically acceptable salt thereof can be reduced. It can be removed.
Conventionally, as a method for producing high-purity 5′-FMN or a pharmacologically acceptable salt thereof, for example, Japanese Patent No. 2856760 discloses a column treatment by adsorption chromatography and a column treatment by reverse phase chromatography. Although a two-step purification step is known, in both steps, only unreacted riboflavin, lumiflavine, and lumichrome are removed, and riboflavin-4'-sodium phosphate (4'-FMN-Na), riboflavin- 3'-Sodium phosphate (3'-FMN-Na) and riboflavin-diphosphate could not be sufficiently removed.
In the method for producing high-purity 5'-FMN or a pharmacologically acceptable salt thereof, particularly 5'-FMN-Na in the present invention, in the recrystallization step, impurities in crude 5'-FMN-Na are removed. Riboflavin-4'-sodium phosphate (4'-FMN-Na), riboflavin-3'-sodium phosphate (3'-FMN-Na), riboflavin-diphosphate, etc. are removed, and then activated carbon treatment step is performed. After removing unreacted riboflavin, lumiflavin and lumichrome in the crude 5'-FMN-Na to ensure high purity, if it is necessary to further remove trace impurities with a purity of 0.1% or less, an activated carbon treatment step It may be treated with a high molecular weight adsorption resin later.
In the present invention, for example, high-purity 5′-FMN or a pharmacologically acceptable salt thereof can be produced by the following method.
(1) Production of crude 5'-FMN-Na
500 ml of acetonitrile, 250 g of pyridine, and 507 g of phosphorus oxychloride are put in a kolben and stirred at 10 ° C. Next, a solution prepared by dissolving 33 ml of water in 200 ml of acetonitrile was added dropwise over 1 hour to prepare a phosphorylating reagent, and 180 g (0.478 mol) of riboflavin was gradually added thereto, followed by stirring at 10 ° C. for 12 hours. The crystals of the precipitated yellow intermediate riboflavin cyclic-4 ′, 5′-phosphoridate were filtered, washed with 500 ml of acetonitrile, air-dried, and added to 600 ml of 6% hydrochloric acid (10 ° C.). Stir at 27 ° C. for 27 hours. The reaction solution was cooled to 10 ° C., and 540 ml of a 25% aqueous sodium hydroxide solution was added to completely dissolve the product. After cooling to 8 ° C., 45 ml of concentrated hydrochloric acid was added dropwise to adjust the pH to 5.5. Yellow crystals of '-FMN-Na were precipitated. The precipitated crystals are filtered, washed with 400 ml of methanol, and dried under reduced pressure to obtain 195 g of crude 5'-FMN-Na.
(2) Recrystallization and purification of crude 5'-FMN-Na
50 g of the crude 5'-FMN-Na obtained in (1) is put in a 1000 ml kolben, stirred in 400 ml of water while being suspended, and heated and stirred at 50 ° C to completely dissolve. 100 ml of ethanol was added dropwise to this solution, and the mixture was cooled to 8 ° C. with stirring. Yellow crystals of 5′-FMN—Na were precipitated by recrystallization, filtered, washed with 150 ml of ethanol, and dried under reduced pressure to purify 38.35 g. 5'-FMN-Na is obtained.
(3) Column purification of 5'-FMN-Na by ODS column chromatography
20 g of the purified 5'-FMN-Na obtained in (2) is dissolved in 400 ml of water to prepare an aqueous 5'-FMN-Na solution having a concentration of 5% by weight and a pH of 5.5. After charging the 5'-FMN-Na aqueous solution to the ODS column, the solution is developed using 10% acetonitrile as an eluent, and the eluate is collected.
(4) Isolation of purified 5'-FMN-Na
The 10% acetonitrile eluate containing 5'-FMN-Na obtained in (3) was concentrated under reduced pressure on a water bath at 50 ° C until the volume became about 150 ml, and 30 ml of ethanol was added dropwise while stirring the concentrated solution. After cooling to 0 ° C, yellow crystals were precipitated, filtered, washed with 30 ml of ethanol, and dried under reduced pressure to obtain 16.9 g of high purity 5'-FMN-Na. This purified 5'-FMN-Na has a 5'-FMN-Na content of 98% or more, and the riboflavin-4'-sodium phosphate (4'-FMN-Na) content of impurities is about 1%. Yes, riboflavin-3'-sodium phosphate (3'-FMN-Na), riboflavin-diphosphate, and riboflavin are not detected.
In the present invention, for example, high-purity 5′-FMN or a pharmacologically acceptable salt thereof can also be produced by the following method.
The steps of (1) production of crude 5'-FMN-Na and (2) recrystallization and purification of crude 5'-FMN-Na are as described above.
(3) Purification of 5'-FMN-Na by activated carbon treatment
5 g of purified 5'-FMN-Na is dissolved in 30 ml of water, and 5.6 ml of 1N-NaOH water is added to prepare a 5'-FMN-Na aqueous solution having a concentration of 12% by weight and a pH of 7.9. 0.75 g (15% by weight based on 5'-FMN-Na) of activated carbon (Taiko Y manufactured by Nimura Chemical Co., Ltd.) was added to the 5'-FMN-Na aqueous solution, and suspended at 30 ° C for 1 hour. Filter off. The filtered activated carbon is washed with 25 ml of water, and the washing water is combined with the filtrate obtained earlier.
(4) Isolation of purified 5'-FMN-Na
To the aqueous solution containing 5′-FMN-Na obtained in (3), 4.3 ml of 1N HCl aqueous solution was added to adjust the pH to 6.0, 60 ml of ethanol was added dropwise with stirring, and the mixture was cooled to 8 ° C. Crystals are deposited, filtered, washed with 10 ml of ethanol and dried under reduced pressure to obtain 3.66 g of high purity 5'-FMN-Na. This purified 5'-FMN-Na has a 5'-FMN-Na content of 97% or more, and the riboflavin-4'-sodium phosphate (4'-FMN-Na) content of impurities is about 2%. Yes, riboflavin-3'-sodium phosphate (3'-FMN-Na) is about 0.2%, riboflavin-diphosphate 0.1% or less, and riboflavin is about 0.2%.
Further, in the present invention, for example, high-purity 5′-FMN or a pharmacologically acceptable salt thereof can also be produced by the following method.
The steps of (1) production of crude 5'-FMN-Na and (2) recrystallization and purification of crude 5'-FMN-Na are as described above.
(3) Purification of 5'-FMN-Na by activated carbon treatment
5 g of purified 5'-FMN-Na is dissolved in 50 ml of water to prepare an aqueous solution of 5'-FMN-Na having a concentration of 9% by weight and having a pH of 6. 0.6 g of activated carbon (CASP manufactured by Norit) (12% by weight based on 5'-FMN-Na) is added to the 5'-FMN-Na aqueous solution, and after suspending at 50 ° C for 1 hour, the activated carbon is filtered off. . The filtered activated carbon is washed with 10 ml of water, and the washing water is combined with the filtrate obtained earlier.
(4) Purification of 5'-FMN-Na by high molecular weight adsorption resin
To the aqueous solution containing 5'-FMN-Na obtained in (3), 5 g of a high molecular weight adsorption resin (SP700 manufactured by Mitsubishi Chemical) is added, and after suspending at 30 ° C. for 1 hour, the high molecular weight adsorption resin is filtered off. . The filtered high molecular weight adsorption resin is washed with 15 ml of water, and the water is combined with the filtrate obtained above.
(5) Isolation of purified 5'-FMN-Na
To the obtained filtrate, 75 ml of ethanol was added dropwise while stirring, and the mixture was cooled to 8 ° C. to precipitate yellow crystals, which was filtered, washed with 10 ml of ethanol, and dried under reduced pressure to obtain 3.23 g of high-purity 5′-FMN-Na. . This purified 5'-FMN-Na has a 5'-FMN-Na content of 97% or more, and the riboflavin-4'-sodium phosphate (4'-FMN-Na) content of impurities is about 2%. Yes, riboflavin-3'-sodium phosphate (3'-FMN-Na) is about 0.3%, riboflavin-diphosphate 0.1% or less, and riboflavin is about 0.1%.
According to the present invention, high purity of riboflavin 5′-phosphate (5′-FMN) or a pharmacologically acceptable salt thereof, particularly, widely used sodium riboflavin-5′-phosphate (5′-FMN-Na). It is possible to provide a manufacturing method. That is, from crude riboflavin 5′-sodium phosphate (5′-FMN-Na), riboflavin-4′-sodium phosphate (4′-FMN-Na), riboflavin-3′-sodium phosphate (3′-FMN) -Na), riboflavin-diphosphate, riboflavin polyphosphate, riboflavin, lumiflavin, lumichrome and the like can be almost completely removed by simple means. The effect example is shown below.
Experimental example
(1) Effect of the purification method (recrystallization purification and column purification by reverse phase column chromatography) on the purity of 5'-FMN-Na in the present invention
Using crude 5'-FMN-Na having the composition shown below, (1) recrystallization purification was performed, then (2) column purification was performed by reverse phase column chromatography, and subsequently (3) The purified 5'-FMN-Na was isolated.
Crude 5'-FMN-Na used

That is, first, 50 g of the above crude 5′-FMN-Na was suspended and stirred in 400 ml of water, and was heated and stirred at 50 ° C. to completely dissolve the solution. , And yellow crystals of 5'-FMN-Na were precipitated. The precipitated crystals were filtered, washed with 150 ml of ethanol and dried under reduced pressure to obtain 38.5 g of 5'-FMN-Na ((1) recrystallization and purification). The purity of the recrystallized and purified 5'-FMN-Na was evaluated by HPLC under the following conditions. The results are shown in Table 1.
HPLC analysis conditions
Detector: UV absorption photometer (measurement wavelength: 254 nm)
Column: Nucleosil-5C18, 4.6 mm x 250 mm
Column temperature: 40 ° C
Mobile phase: 2% potassium dihydrogen phosphate aqueous solution: methanol: acetonitrile = 40: 9: 1
Flow rate: 1.0 ml / min
Injection volume: 5ul
Next, an aqueous solution (stock solution) obtained by dissolving 5 g of the recrystallized purified 5'-FMN-Na obtained above in 100 ml of water and adjusting the pH to 5.5 at a concentration of 5% by weight was pumped by an ODS column manufactured by YMC. (YMC-AQ-C18, inner diameter 20 mm x length 50 mm), developed with 5% acetonitrile aqueous solution (v / v) as eluent, eluent was detected at uv 254 nm, and divided into 4 fractions for each fraction Then, the purity of 5'-FMN-Na ((2) column purification by reverse phase column chromatography) in the fraction was evaluated by HPLC. Table 2 shows the evaluation results.
Next, the ODS column chromatography No. The 2 to 4 fractions are combined, concentrated under reduced pressure on a 50 ° C. water bath until the volume becomes about 50 ml, 10 ml of ethanol is added dropwise, and the mixture is cooled and stirred at 8 ° C. to precipitate 5′-FMN-Na crystals. After filtration, the precipitate was washed with 10 ml of ethanol and dried under reduced pressure to obtain 4.0 g of purified 5'-FMN-Na ((3) Isolation of purified 5'-FMN-Na). The purity of the concentrated, crystallized and purified 5'-FMN-Na was evaluated by HPLC, and the results are shown in Table 3.

Further, as a comparative experiment, 5′-FMN-Na was purified by a method according to Japanese Patent No. 2856760. That is, instead of "recrystallization purification", (1) column purification by adsorption column chromatography filled with a high molecular weight adsorption resin is performed, and then (2) column purification by reverse phase column chromatography is performed. Subsequently, (3) isolation of purified 5'-FMN-Na was performed.
That is, first, 5 g of crude 5'-FMN-Na having the above-mentioned composition is dissolved in 100 ml of water, and an aqueous solution adjusted to pH 5.5 at a concentration of 5% by weight is filled with the adsorbent resin SP-850 manufactured by Mitsubishi Chemical using a pump. After charging to an adsorption column (inner diameter 20 mm × length 500 mm), the mixture was developed with a 10% aqueous methanol solution (V / V) as an eluent. The eluent was detected by UV absorption at 254 nm, and the purity of 5′-FMN-Na (column purification by adsorption column chromatography packed with (1) ′ high molecular weight adsorption resin) in the eluate was similarly evaluated by HPLC, Table 4 shows the results.
Next, 5 g of purified 5'-FMN-Na purified by the above adsorption column was dissolved in 100 ml of water and adjusted to pH 5.5 at a concentration of 5% by weight, and an ODS column manufactured by YMC (YMC-AQ- C18, inner diameter 20 mm × length 500 mm), developed with a 10% aqueous ethanol solution (v / v) as an eluent, the eluent was detected at UV 254 nm, divided into four fractions for each fraction, and The purity of 5'-FMN-Na ((2) column purification by reverse phase column chromatography) was evaluated by HPLC. Table 5 shows the evaluation results.
Next, the ODS column chromatography No. Fractions 2 to 4 were combined, concentrated by evaporation at 50 ° C., cooled to 20 ° C. to precipitate 5′-FMN-Na crystals, filtered, washed with 10 ml of ethanol, dried under reduced pressure, and purified 5′-FMN -Na was obtained ((3) isolation of purified 5'-FMN-Na). The purity of the concentrated and isolated 5'-FMN-Na was evaluated by HPLC, and the results are shown in Table 6.

As a result of comparison between the method for producing and purifying 5′-FMN-Na according to the present invention and the comparative experiment, the method for producing and purifying 5′-FMN-Na according to the present invention shows that (1) the recrystallization purification step , 4'-FMN-Na, 3'-FMN-Na and riboflavin diphosphate are considerably removed, and (2) riboflavin and riboflavin diphosphate are removed by reverse phase column purification (eluent: 5% acetonitrile aqueous solution). (3) Isolation of purified 5'-FMN-Na by crystallization further removed 4'-FMN-Na and 3'-FMN-Na, and the purity of the final purified 5'-FMN-Na was 97%. It was high purity of just over%. On the other hand, in the method for producing and purifying 5′-FMN-Na according to a comparative experiment (a purification method according to Japanese Patent No. 2856760), (1) riboflavin is considerably purified by an adsorption column purification packed with a high-molecular-weight adsorption resin. Riboflavin was further removed by (2) reverse phase column purification (eluent: 10% aqueous methanol solution), but (3) isolation of purified 5'-FMN-Na showed no change in the amount of impurities. Further, in any of the purification steps (1) to (3), 4′-FMN-Na, 3′-FMN-Na and riboflavin diphosphate were hardly removed, and the final purified 5′-FMN- The purity of Na was as low as over 90%. In reverse phase column purification, the type of eluent affected the riboflavin removal efficiency, and riboflavin could be completely removed with a 5 to 10% aqueous solution of acetonitrile according to the present invention, whereas riboflavin was removed with a 10% aqueous methanol solution. Slightly remained. Lumiflavin and Lumichrome as photodegradation products were not detected in any of the present invention and the comparative experiment, and were almost completely removed.
Production and purification method of 5'-FMN-Na according to the present invention ((1) recrystallization purification using crude 5'-FMN-Na, and then (2) reverse phase column chromatography The column purification is performed, and subsequently (3) the purified 5′-FMN-Na is isolated), the main impurities (4′-FMN-Na, 3′-FMN-Na) are compared with the conventional method. Riboflavin / diphosphate, riboflavin), and it is clear that this is a remarkably excellent method for producing high purity 5'-FMN-Na.
(2) Effect of type of eluent on purity of 5'-FMN-Na in column purification by reversed-phase column chromatography
In order to evaluate the effect of the type of eluent on the purity of 5'-FMN-Na in the column purification by reverse phase column chromatography, Example 13 (column: Wakosil-40-18, eluent: 10% As a control experiment for acetonitrile aqueous solution), an experiment was performed in which only the eluent of Example 13 was replaced with 10% ethanol. The eluent was detected at UV 254 nm, divided into 5 fractions for each fraction, and the 5′- The purity of FMN-Na was evaluated by HPLC. Table 7 shows the evaluation results.
Next, the ODS column chromatography was performed. Fractions 2 to 5 were combined, concentrated under reduced pressure on a water bath at 50 ° C until the volume became about 150 ml, and 30 ml of ethanol was added dropwise while stirring the concentrated solution, and the mixture was cooled to 8 ° C to precipitate yellow crystals. The crystals were filtered, washed with 30 ml of ethanol, and dried under reduced pressure to obtain 16.4 g of highly pure 5'-FMN-Na (recovery rate: 82.0%). The purity of the concentrated and purified 5'-FMN-Na was obtained. Was evaluated by HPLC, and the results are shown in Table 8. Further, the optical rotation was + 41.8 ° C.

In addition, the optical rotation was + 41.8 ° C.
Similarly, as a control experiment of Example 16 (column: YMC-AQ-C18, eluent: 5% acetonitrile aqueous solution), an experiment was performed in which only the eluent of Example 16 was replaced with 10% ethanol, and the eluent was UV 254 nm. The fraction was divided into 6 fractions for each fraction, and the purity of 5′-FMN-Na in the fractions was evaluated by HPLC. Table 9 shows the results of the evaluation.
Next, the ODS column chromatography was performed. Fractions 2 to 6 were combined, concentrated under reduced pressure on a 50 ° C. water bath until the volume became about 50 ml, and 10 ml of ethanol was added dropwise while stirring the concentrated solution, followed by cooling to 8 ° C. to precipitate yellow crystals. The crystals were filtered, washed with 10 ml of ethanol, and dried under reduced pressure to obtain 4.1 g of high-purity 5'-FMN-Na (recovery rate: 82.0%). The purity was evaluated by HPLC, and the results are shown in Table 10. Further, the optical rotation was +41.9 degrees.

As a result of comparison between the method for producing and purifying 5′-FMN-Na of Examples 13 and 16 according to the present invention and the control experiment, the method for producing and purifying 5′-FMN-Na according to the present invention showed that the reverse-phase column purification was performed. By using acetonitrile aqueous solution as eluent, riboflavin was completely removed and the purity of 5′-FMN-Na was high. On the other hand, in a control experiment using an ethanol aqueous solution as an eluent for reverse phase column purification, riboflavin remained as an impurity. Further, in the present invention (eluent: acetonitrile aqueous solution), the amount of the eluent required for removing impurities was １ ／ of the control experiment (eluent: ethanol aqueous solution).
In the method for producing and purifying 5'-FMN-Na according to the present invention, it is clear that the eluent used for column purification by reverse phase column chromatography has an effect of an aqueous solution of acetonitrile superior to that of an aqueous ethanol solution. .
(3) Effect of purification method (recrystallization purification and purification by activated carbon treatment) on the purity of 5'-FMN-Na according to the present invention
Using crude 5'-FMN-Na having the composition shown below, (1) recrystallization purification was performed, then (2) purification by activated carbon treatment, and (3) purified 5'-FMN -Na was isolated.
Crude 5'-FMN-Na used

That is, first, 5.5 g of the above crude 5'-FMN-Na was suspended and stirred in 55 ml of water while heating and stirring at 50 ° C. to dissolve completely, and 28 ml of ethanol was gradually added dropwise to this solution. After cooling and stirring at 1 ° C., yellow crystals of 5′-FMN—Na were deposited. The precipitated crystals were filtered, washed with 10 ml of ethanol and dried under reduced pressure to obtain 4.8 g of 5'-FMN-Na ((1) recrystallization and purification). Table A shows the results of HPLC evaluation of the purity of the recrystallized and purified 5'-FMN-Na.
Next, 4 g of the purified recrystallized 5'-FMN-Na obtained above was dissolved in 56 ml of water, and 4.7 ml of 1N-NaOH aqueous solution was added thereto. -Na aqueous solution was prepared. To this 5'-FMN-Na aqueous solution, 3.2 g (80% by weight based on 5'-FMN-Na) of activated carbon (Taiko Y manufactured by Nimura Chemical) was added, and suspended at 50 ° C for 2 hours. I filtered. The filtered activated carbon was washed with 15 ml of water, and the washing water was combined with the filtrate previously obtained ((2) purification by activated carbon treatment).
Further, to the aqueous solution containing the purified 5′-FMN-Na treated with activated carbon, 2.5 ml of 1N HCl was added to adjust the pH to 6.0, 40 ml of ethanol was added dropwise with stirring, and the mixture was cooled to 4 ° C. Yellow crystals were precipitated, filtered, washed with 10 ml of ethanol and dried under reduced pressure to obtain 1.98 g of high-purity 5'-FMN-Na ((3) isolation of purified 5'-FMN-Na). The purity of the crystallized and purified 5'-FMN-Na was evaluated by HPLC, and the results are shown in Table B.

In addition, as a comparative control experiment, the evaluation of [(1) Effect of the purification method (recrystallization purification and column purification by reverse phase column chromatography) on the purity of 5′-FMN-Na] shown above was performed. [Purification of 5'-FMN-Na by the method according to Japanese Patent No. 2856760]. That is, instead of "purification by recrystallization purification and activated carbon treatment", (1) column purification by adsorption column chromatography packed with a high molecular weight adsorption resin is performed, and then (2) reverse phase column chromatography treatment Was performed, followed by (3) isolation of purified 5'-FMN-Na.
As a result of comparison between the method for producing and purifying 5′-FMN-Na according to the present invention and the comparative experiment, the method for producing and purifying 5′-FMN-Na according to the present invention shows that (1) the recrystallization purification step , 4'-FMN-Na, 3'-FMN-Na and riboflavin diphosphate are considerably removed, (2) riboflavin is mainly removed by activated carbon treatment and subsequent isolation of purified 5'-FMN-Na, Further, 4'-FMN-Na and 3'-FMN-Na were removed, and the purity of the final purified 5'-FMN-Na was as high as 93% or more. On the other hand, in the method for producing and purifying 5′-FMN-Na according to a comparative experiment (a purification method according to Japanese Patent No. 2856760), (1) riboflavin is considerably purified by an adsorption column purification packed with a high-molecular-weight adsorption resin. Riboflavin was further removed by (2) reverse phase column purification (eluent: 10% aqueous methanol solution), but (3) isolation of purified 5'-FMN-Na showed no change in the amount of impurities. Further, in any of the purification steps (1) to (3), 4′-FMN-Na, 3′-FMN-Na and riboflavin diphosphate were hardly removed, and the final purified 5′-FMN- The purity of Na was as low as over 90%. Lumiflavin and Lumichrome as photodegradation products were not detected in any of the present invention and the comparative experiment, and were almost completely removed.
Production and purification method of 5′-FMN-Na according to the present invention (using crude 5′-FMN-Na, (1) recrystallization purification, then (2) purification by activated carbon treatment, and further (3) Isolation of purified 5'-FMN-Na) was compared with the conventional method, and the main impurities (riboflavin, 4'-FMN-Na, 3'-FMN-Na, riboflavin diphosphate) were removed. It is clear that this is a method for producing 5'-FMN-Na having a high removal effect and excellent high purity.
Example
Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
Example 1 Recrystallization and purification method of crude riboflavin-5'-sodium phosphate (5'-FMN-Na)
1) Production of crude 5'-FMN and crude 5'-FMN-Na
500 ml of acetonitrile, 250 g (3.14 mol) of pyridine, and 507 g (3.3 mol) of phosphorus oxychloride were placed in a 2 L 4-necked corbane equipped with a stirrer, calcium chloride tube, thermometer and baffle, and stirred at 10 ° C. Next, a solution prepared by dissolving 33 ml of water in 200 ml of acetonitrile was added dropwise over 1 hour to prepare a phosphorylation reagent, and 180 g (0.478 mol) of riboflavin was gradually added thereto, followed by stirring at 10 ° C. for 12 hours. The crystals of the precipitated yellow intermediate riboflavin cyclic-4 ', 5'-phosphoridate were filtered, washed with 500 ml of acetonitrile, and air-dried (yield 360 g).
Then, 600 ml of 6% hydrochloric acid (water) was added to a 2 L 4-necked kolben, 360 g of the intermediate obtained above was added while stirring at 10 ° C, and the mixture was stirred at 40 ° C for 27 hours. After confirming disappearance of the raw material intermediate by HPLC, the reaction solution was cooled to 10 ° C., and 540 ml of a 25% sodium hydroxide (water) solution was added dropwise over 1 hour to completely dissolve the product (pH = 9). .26).
While cooling the reaction solution to 8 ° C., 45 ml of concentrated hydrochloric acid was added dropwise to adjust the pH to 5.5, and yellow crystals of 5′-FMN—Na were precipitated. The precipitated crystals were filtered, washed with 400 ml of methanol, and dried under reduced pressure to obtain 195 g of 5'-FMN-Na (yield: 85.3%).
As a result of HPLC evaluation, this product had the following composition.

2) Recrystallization and purification of crude 5'-FMN-Na
50 g (0.1045 mol) of 5'-FMN-Na obtained in 1) was put into a 1000 ml kolben, stirred under suspension in 400 ml of water, and heated and stirred at 50 ° C. to completely dissolve it. 100 ml of ethanol was added dropwise to this solution, and the mixture was cooled to 8 ° C. with stirring to precipitate 5′-FMN-Na yellow crystals.
The precipitated crystals were collected by filtration, washed with 150 ml of ethanol, and dried under reduced pressure to obtain 38.35 g of purified 5'-FMN-Na (recovery rate: 76.7%).
As a result of HPLC evaluation, the purified 5′-FMN-Na had the following composition. In addition, the optical rotation was +41.5 degrees.

Embodiment 2-12. Recrystallization and purification method of crude 5'-FMN-Na
In the same manner as in Example 1, 5 g (0.0104 mol) of the crude 5'-FMN-Na obtained in 1) of Example 1 was placed in a 100 ml kolben, stirred under suspension in 40 ml of water, and heated and stirred at 50 ° C. Was completely dissolved. A water-soluble solvent was added dropwise to this solution, and the mixture was cooled to 8 ° C. with stirring to obtain 5′-FMN-Na yellow crystals. The precipitated crystals were filtered, washed with 15 ml of ethanol, and dried under reduced pressure to obtain purified 5'-FMN-Na (recovery rate: 76.7%).
Table 11 shows the composition of each purified 5'-FMN-Na obtained by HPLC evaluation and the recovery rate by the recrystallization purification method.

In Example 2-12, the content of riboflavin-diphosphate was 0.21-0.23%.
Embodiment 13 FIG. Method for producing high-purity 5'-FMN-Na by ODS column chromatography of recrystallized product 5'-FMN-Na
20 g of the recrystallized purified 5′-FMN-Na obtained in 2) of Example 1 was dissolved in 400 ml of water to prepare a 5′-FMN-Na aqueous solution (stock solution) having a concentration of 5% by weight and a pH of 5.5. did. An ODS column Wakosil-40-C18 (inner diameter 50 mm × length 500 mm) manufactured by Wako Pure Chemical Industries, Ltd. was previously substituted with a 10% aqueous acetonitrile solution, and the above stock solution was charged into the ODS column by a pump, and then 10% as an eluent. The eluate was developed with acetonitrile and the eluate was divided into 4 fractions per fraction.
The composition of purified 5'-FMN-Na of each fraction obtained by HPLC evaluation is shown in Table 12.

No. which was subjected to ODS column chromatography. Fractions 2 to 4 were combined, concentrated under reduced pressure on a water bath at 50 ° C. until the volume became about 150 ml, and 30 ml of ethanol was added dropwise while stirring the concentrated solution, followed by cooling to 8 ° C. to precipitate yellow crystals. The crystals were filtered, washed with 30 ml of ethanol, and dried under reduced pressure to obtain 16.9 g of high-purity 5'-FMN-Na (recovery rate: 84.5%). As a result of HPLC evaluation, the purified 5′-FMN-Na had the following composition. Further, the optical rotation was +42.2 degrees.

Embodiment 14-15. Method for producing high-purity 5'-FMN-Na by ODS column chromatography of recrystallized product 5'-FMN-Na
High purity 5'-FMN-Na was produced in the same manner as in Example 13 except that only the type of eluent used for developing after charging the ODS column was changed.
That is, 20 g of the purified and recrystallized 5'-FMN-Na obtained in 2) of Example 1 was dissolved in 400 ml of water, and a 5% by weight aqueous solution of 5'-FMN-Na having a pH of 5.5 (stock solution). Was prepared. Using an ODS column Wakosil-40-C18 (inner diameter 50 mm × length 500 mm) manufactured by Wako Pure Chemical Industries, the stock solution was charged into the ODS column by a pump, and then a 10% aqueous acetone solution (Example 14) or an eluent was used. It was developed using a 10% aqueous THF solution (Example 15), and the eluate fraction was collected.
Table 13 shows the composition of purified 5'-FMN-Na of each collected fraction obtained by HPLC evaluation.

Next, the fractions of each eluate collected by the ODS column chromatography were concentrated under reduced pressure on a 50 ° C. water bath to about 150 ml, and 30 ml of ethanol was added dropwise while stirring the concentrated solution. Upon cooling to ° C., yellow crystals precipitated. The crystals were filtered, washed with 30 ml of ethanol and dried under reduced pressure to obtain high purity 5'-FMN-Na.
As a result of HPLC evaluation, the purified 5′-FMN-Na had the composition shown in Table 14 below.

Embodiment 16 FIG. Method for producing high purity 5'-FMN-Na
5 g of the purified recrystallized 5'-FMN-Na obtained in 2) of Example 1 was dissolved in 100 ml of water to prepare a 5'-FMN-Na aqueous solution (stock solution) having a concentration of 5% by weight and a pH of 5.5. did. An ODS column YMC-AQ-C18 (20 mm ID × 500 mm length) manufactured by YMC was previously replaced with a 5% acetonitrile aqueous solution, and the stock solution was charged into the ODS column with a pump, and then 5% acetonitrile was used as an eluent. The eluate was divided into four fractions per fraction.
Table 15 shows the composition of purified 5'-FMN-Na of each fraction obtained by HPLC evaluation.

No. which was subjected to ODS column chromatography. The 2 to 4 fractions were combined, concentrated under reduced pressure on a 50 ° C. water bath to about 50 ml, 10 ml of ethanol was added dropwise, and the mixture was cooled and stirred at 8 ° C. to precipitate crystals of high purity 5′-FMN, After filtration, the precipitate was washed with 10 ml of ethanol and dried under reduced pressure to obtain 4.2 g of high-purity 5'-FMN-Na (recovery rate: 84.0%).
As a result of HPLC evaluation, the purified 5′-FMN-Na had the following composition. Further, the optical rotation was +42.1 degrees.

Embodiment 17 FIG. Method for producing high purity 5'-FMN-Na by activated carbon treatment of recrystallized product 5'-FMN-Na
Activated carbon treatment was performed using a recrystallized purified product 5'-FMN-Na having the composition shown below, and subsequently, purified 5'-FMN-Na was isolated.
Crude 5'-FMN-Na used

(1) Purification of 5'-FMN-Na by activated carbon treatment
5 g of recrystallized purified product 5'-FMN-Na having the above composition was dissolved in 30 ml of water, and 5.6 ml of 1N-NaOH water was added to prepare a 5% -FMN-Na aqueous solution having a concentration of 12 wt% and pH 7.9. did. 0.75 g (15% by weight based on 5'-FMN-Na) of activated carbon (Taiko Y manufactured by Nimura Chemical Co., Ltd.) was added to the 5'-FMN-Na aqueous solution, and suspended at 30 ° C for 1 hour. I filtered. The filtered activated carbon was washed with 25 ml of water, and the washing water was combined with the filtrate obtained previously to obtain an aqueous solution containing purified 5'-FMN-Na by activated carbon treatment.
(2) Isolation of purified 5'-FMN-Na
To the aqueous solution containing 5'-FMN-Na obtained in (1), 4.3 ml of 1N-HCl aqueous solution was added to adjust the pH to 6.0. Crystals were deposited, filtered, washed with 10 ml of ethanol, and dried under reduced pressure to obtain 3.66 g of high purity 5'-FMN-Na. As a result of HPLC evaluation, the purified 5′-FMN-Na had the following composition.
Crude 5'-FMN-Na after purification

Embodiment 18 FIG. Method for producing high purity 5'-FMN-Na by activated carbon treatment of recrystallized product 5'-FMN-Na and high molecular weight adsorption resin
Activated carbon treatment was performed using the recrystallized purified product 5′-FMN-Na used in Example 17, followed by purification with a high molecular weight adsorption resin, and further isolation of purified 5′-FMN-Na. Was performed.
(1) Purification of 5'-FMN-Na by activated carbon treatment and high molecular weight adsorption resin
5 g of purified 5'-FMN-Na was dissolved in 50 ml of water to prepare a 5'-FMN-Na aqueous solution having a concentration of 9% by weight and having a pH of 6. 0.6 g of activated carbon (CASP manufactured by Norit) (12% by weight based on 5'-FMN-Na) was added to the 5'-FMN-Na aqueous solution, suspended at 50 ° C for 1 hour, and the activated carbon was filtered off. . The filtered activated carbon was washed with 10 ml of water, and the washing water was combined with the filtrate previously obtained to obtain an aqueous solution containing purified 5'-FMN-Na by activated carbon treatment. To the aqueous solution containing purified 5'-FMN-Na by the activated carbon treatment, 5 g of a high molecular weight adsorption resin (SP700 manufactured by Mitsubishi Chemical Corporation) was added, and after suspending at 30 ° C for 1 hour, the high molecular weight adsorption resin was filtered. The filtered high molecular weight adsorption resin was washed with 15 ml of water, and the water was combined with the previously obtained filtrate to obtain an aqueous solution containing purified 5'-FMN-Na by treatment with the high molecular weight adsorption resin.
(2) Isolation of purified 5'-FMN-Na
75 ml of ethanol was added dropwise with stirring to the aqueous solution containing purified 5'-FMN-Na obtained by treatment with the high molecular weight adsorption resin obtained in (1), and the mixture was cooled to 8 ° C to precipitate yellow crystals. After washing and drying under reduced pressure, 3.23 g of high-purity purified 5'-FMN-Na was obtained. As a result of HPLC evaluation, the purified 5′-FMN-Na had the following composition.
Isolation of purified 5'-FMN-Na

Claims (15)

In a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, 1) dissolving crude 5'-FMN or a pharmacologically acceptable salt thereof in water or a buffer The resulting solution is dissolved in a liquid to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. 2) While mechanically stirring the obtained aqueous solution, An organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight based on the previously obtained aqueous solution to give 5′-FMN or a pharmaceutically acceptable salt thereof. A method for producing high-purity 5′-FMN or a pharmacologically acceptable salt thereof, characterized by recrystallizing. In a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, 1) dissolving crude 5'-FMN or a pharmacologically acceptable salt thereof in water or a buffer The resulting solution is dissolved in a liquid to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. 2) While mechanically stirring the obtained aqueous solution, An organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight based on the previously obtained aqueous solution to give 5′-FMN or a pharmaceutically acceptable salt thereof. 3) dissolving the recrystallized 5′-FMN or a pharmaceutically acceptable salt thereof in water or a buffer to form an aqueous solution having a concentration of 1 to 20% by weight and having a pH of 4 to 9; 4) Then, reverse the phase so that the liquid volume is at least 1 to 100% of the column bed volume. Treating with a column chromatography, followed by using a water-soluble organic solvent or a water-containing organic solvent as an eluent to obtain a solution of purified 5′-FMN or a pharmaceutically acceptable salt thereof, A method for producing high-purity 5′-FMN or a pharmacologically acceptable salt thereof, characterized by comprising: In a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, 1) dissolving crude 5'-FMN or a pharmacologically acceptable salt thereof in water or a buffer The resulting solution is dissolved in a liquid to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. 2) While mechanically stirring the obtained aqueous solution, An organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight to the previously obtained aqueous solution to give 5′-FMN or a pharmaceutically acceptable salt thereof. 3) dissolving the recrystallized 5′-FMN or a pharmaceutically acceptable salt thereof in water or a buffer to form an aqueous solution having a concentration of 1 to 20% by weight and having a pH of 4 to 9; 4) Next, about 1-100 weights with respect to 5'-FMN or a pharmacologically acceptable salt thereof. % Of the activated carbon is added to the aqueous solution, the impurities in the aqueous solution are adsorbed on the activated carbon, and the medium containing the activated carbon is filtered to obtain an aqueous solution of the purified 5′-FMN or a pharmacologically acceptable salt thereof. A method for producing high-purity 5′-FMN or a pharmaceutically acceptable salt thereof. In a method for purifying riboflavin-5'-phosphate (5'-FMN) or a pharmacologically acceptable salt thereof, 1) dissolving crude 5'-FMN or a pharmacologically acceptable salt thereof in water or a buffer The resulting solution is dissolved in a liquid to prepare an aqueous solution of crude 5'-FMN or a pharmacologically acceptable salt thereof having a concentration of 1 to 15% by weight and having a pH of 4-8. 2) While mechanically stirring the obtained aqueous solution, An organic solvent soluble in water at a temperature of 0-60 ° C. is added in an amount of 1-100% by weight to the previously obtained aqueous solution to give 5′-FMN or a pharmaceutically acceptable salt thereof. 3) dissolving the recrystallized 5′-FMN or a pharmaceutically acceptable salt thereof in water or a buffer to form an aqueous solution having a concentration of 1 to 20% by weight and having a pH of 4 to 9; 4) Next, about 1-100 weights with respect to 5'-FMN or a pharmacologically acceptable salt thereof. % Activated carbon is added to the aqueous solution, the impurities in the aqueous solution are adsorbed to the activated carbon, and the medium containing the activated carbon is filtered to obtain an aqueous solution of 5′-FMN or a pharmaceutically acceptable salt thereof. Next, 10-200% by weight of a high molecular weight adsorption resin based on 5′-FMN or a pharmacologically acceptable salt thereof is added to the aqueous solution, and the impurities in the aqueous solution are adsorbed on the high molecular weight adsorption resin. Obtaining a purified 5′-FMN or a solution of a pharmacologically acceptable salt thereof by filtering a medium containing a high molecular weight adsorption resin; A method for producing a pharmacologically acceptable salt. A solution of the purified 5′-FMN or the pharmacologically acceptable salt thereof obtained by the steps 1) to 4) of claim 2 is treated at a temperature of 10 to 60 ° C. to give a concentration of 5′-FMN. 5′-FMN is crystallized by adding water-soluble organic solvent to the concentrated solution at 0-60 ° C. at 1-160% by weight, and crystallizing 5′-FMN. '-A method for producing FMN or a pharmacologically acceptable salt thereof. In the solution of the purified 5′-FMN or the pharmaceutically acceptable salt thereof obtained by the steps of 1) to 4) of claim 3 or the steps of 1) to 5) of claim 4, 1) Add an organic solvent soluble in water to the solution at 0-60 ° C at 1-150% by weight, or 2) add the solution at a temperature of 10-60 ° C to a concentration of 5'-FMN of 5-20%. High purity 5′-FMN or a high-purity 5′-FMN characterized by crystallizing 5′-FMN by adding a water-soluble organic solvent to the concentrated solution at 0-60 ° C. at 1-160% by weight. A method for producing a pharmacologically acceptable salt. The high-purity 5'-FMN according to claim 2 or 5, or a pharmacologically acceptable salt thereof, wherein the column filler used for the reverse phase column chromatography is ODS (C18, octadecylsilane) and / or C8 (octylsilane). Method for producing salt. The organic solvent used as an eluent for reversed-phase column chromatography or the organic solvent in a water-containing organic solvent is a lower aliphatic alcohol, acetonitrile, acetone, tetrahydrofuran or a mixture of two or more thereof. A method for producing the high-purity 5′-FMN or the pharmaceutically acceptable salt thereof according to any one of the above. The high-purity 5'-FMN according to any one of claims 2, 5, 7, and 8, or an organic solvent content of the organic solvent in the water-containing organic solvent used as an eluent is 0.1 to 50% by volume. For the production of acceptable salts. The activated carbon according to claim 3, wherein the activated carbon is pulverized coal or granular coal, which is peat, lignite, coal produced by steam activation and / or wood produced by chemical activation. A method for producing 5'-FMN or a pharmacologically acceptable salt thereof. The method for producing high-purity 5'-FMN or a pharmaceutically acceptable salt thereof according to claim 4 or 6, wherein the high molecular weight adsorption resin is a styrene-divinylbenzene copolymer or a methacrylic acid polymer. The crude 5′-FMN or a pharmaceutically acceptable salt thereof is heated to 30-80 ° C. when dissolving the same in water or a buffer solution. A method for producing the high-purity 5′-FMN or the pharmaceutically acceptable salt thereof according to the above. Crude 5'-FMN or a pharmacologically acceptable salt thereof is reacted with riboflavin (1) and phosphorus oxychloride shown below, or reacted with riboflavin (1) and phosphorus oxychloride and then reacted with sodium hydroxide. In the reaction for producing crude 5'-FMN (3) by reacting riboflavin (1) with phosphorus oxychloride, which is produced in a sodium chloride reaction, riboflavin cyclic-4 ', which is a reaction intermediate, is produced. The method according to any one of claims 1 to 4, wherein the compound is produced by taking out 5,5'-phosphoridate (2) or a salt thereof out of the system, and hydrolyzing and isomerizing it under acidic conditions. A method for producing high-purity 5'-FMN or a pharmacologically acceptable salt thereof.

The high-purity 5 according to claim 13, wherein the acidic condition is a condition prepared by using an acid having a concentration of 1-35.5 (w / v)% with respect to riboflavin (1) as a starting material in a volume of 1-10 times. '-A method for producing FMN or a pharmacologically acceptable salt thereof. The pharmaceutically acceptable salt of riboflavin 5'-phosphate (5'-FMN) is riboflavin-5'-sodium phosphate (5'-FMN-Na). The method for producing high-purity 5′-FMN or a pharmacologically acceptable salt thereof according to the above item.