JP4913272B2 - Method for producing oligosaccharide, novel oligosaccharide and pharmaceutical composition containing the same - Google Patents

Description

（式中、Ｒ¹およびＲ²はそれぞれ独立してアラルキル基を示し、Ｒ³はアシル基又はシリル基を示し、Ｒ⁴はアミノ基保護基を示し、Ｒ⁵は脱離基を示す。）
【０００９】
【化１７】

（式中、Ｒ⁶およびＲ⁸はそれぞれ独立してアラルキル基を示し、Ｒ⁷はアシル基又はシリル基を示し、Ｒ⁹はアラルキル基、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。）
【００１０】
【化１８】

（式中、Ｒ¹⁰およびＲ¹¹はそれぞれ独立して水素原子または−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す。）を示し、Acはアセチル基を示す。また、Ｒ¹²は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。）
【００１１】
本発明製造方法において、Ｒ¹⁰およびＲ¹¹の少なくとも一方が−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す。）であり、上記一般式(1)で示される単糖と、上記一般式(2)で示される単糖とをグリコシド結合反応させる工程の後に、Ｒ³およびＲ⁷の少なくとも一方を水素原子に置換し、次いで当該水素原子を−ＳＯ₃Ｍに置換する工程が含まれることが好ましい。
【００１２】
本発明製造方法の好ましい態様においては、上記一般式(1)〜(3)が、それぞれ下記式(4)〜(6)で示される。
【００１３】
【化１９】

（式中、Bnはベンジル基を、Ｒ¹³はアセチル基又はレブリノイル基を、Phthはフタロイル基を、Ｘはハロゲン原子を示す。）
【００１４】
【化２０】

（式中、Bnはベンジル基を、Ｒ¹⁴はベンジル基、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。Pivはピバロイル基を示す。）
【００１５】
【化２１】

（式中、Acはアセチル基を、Ｍはプロトン又は１価のカチオンを示す。また、Ｒ¹⁵は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。）
【００１６】
本発明製造方法の別の好ましい態様においては、上記一般式(1)〜(3)が、それぞれ下記式(7)〜(9)で示される。
【００１７】
【化２２】

（式中、Bnはベンジル基を、Levはレブリノイル基を、Phthはフタロイル基を、Ｘはハロゲン原子を示す。）
【００１８】
【化２３】

（式中、Bn、Ｒ¹⁴及びＺは前記と同義である。また、Phはフェニル基を、Meはメチル基を示す。）
【００１９】
【化２４】

（式中、Ｒ¹⁵及びＺは前記と同義である。また、Acはアセチル基を、Ｍはプロトン又は１価のカチオンを示す。）
【００２０】
本発明製造方法のさらに別の好ましい態様においては、上記一般式(1)〜(3)が、それぞれ下記式(10)〜(12)で示される。
【００２１】
【化２５】

（式中、Bnはベンジル基を、Levはレブリノイル基を、Phthはフタロイル基を、Ｘはハロゲン原子を示す。）
【００２２】
【化２６】

（式中、Bn、Ｒ¹⁴及びＺは前記と同義である。また、Phはフェニル基を、Meはメチル基を示す。）
【００２３】
【化２７】

（式中、Ｒ¹⁵及びＺは前記と同義である。また、Acはアセチル基を、Ｍはプロトン又は１価のカチオンを示す。）
【００２４】
また、本発明は、下記一般式(13)で示されるオリゴ糖（以下、本発明オリゴ糖ともいう）を提供する。
【００２５】
【化２８】

（式中、Ｒ¹⁶およびＲ¹⁷はそれぞれ独立して水素原子又は−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）を示し、Acはアセチル基を示す。またＲ¹⁸は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。ただし、Ｒ¹⁶およびＲ¹⁷がいずれも水素原子であり、かつＺが酸素原子でＲ¹⁸が水素原子又はコレスタニル基であるもの、並びにＲ¹⁶が−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）でありかつＺが酸素原子でＲ¹⁷とＲ¹⁸がいずれも水素原子であるものを除く。）
【００２６】
本発明オリゴ糖の好ましい態様は、Ｒ¹⁶およびＲ¹⁷がいずれも−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）である。
【００２７】
また、本発明オリゴ糖の別の好ましい態様は、Ｒ¹⁶が水素原子であり、かつＲ¹⁷が−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）である。
【００２８】
本発明オリゴ糖において、好ましくは、Ｒ¹⁸が水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、Ｏ−アルキルグリセロール残基又はコレスタニル基であり、かつＺが酸素原子である。
【００２９】
本発明はさらに、下記一般式(13)で示される本発明オリゴ糖又はその薬学的に許容される塩を有効成分とする医薬（以下、本発明医薬ともいう）を提供する。
【００３０】
【化２９】

（式中、Ｒ¹⁶およびＲ¹⁷はそれぞれ独立して水素原子又は−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）を示し、Acはアセチル基を示す。またＲ¹⁸は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示す。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。ただし、Ｒ¹⁶およびＲ¹⁷がいずれも水素原子であり、かつＺが酸素原子でＲ¹⁸が水素原子又はコレスタニル基であるものを除く。）
【００３１】
特に、Ｒ¹⁶が−ＳＯ₃Ｍであり、かつＲ¹⁷が水素原子又は−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）を示す本発明オリゴ糖またはその薬学的に許容される塩は、抗アレルギー剤として、Ｒ¹⁶およびＲ¹⁷が−ＳＯ₃Ｍ（Ｍはプロトン又は１価のカチオンを示す）を示す本発明オリゴ糖またはその薬学的に許容される塩は、抗炎症剤として有用である。
【００３２】
【発明の実施の形態】
以下に、本発明の実施の形態を説明する。
まず、本明細書および図面において共通して用いた略号を、その意味（以下の略号に付した括弧内の記載）と共に以下に示す。
【００３３】
Ac（アセチル基）
Bn（ベンジル基）
Phth（フタロイル基）
Piv（ピバロイル基）
Lev（レブリノイル基）
Ph（フェニル基）
Me（メチル基）
All（アリル(allyl)基）
MP（パラメトキシフェニル基）
Ｍ（プロトン又は１価のカチオン）
Ｘ（ハロゲン原子）
【００３４】
＜１＞本発明製造方法
本発明製造方法は、一般式(3)で示されるオリゴ糖の製造方法であり、一般式(1)で示される単糖と一般式(2)で示される単糖とをグリコシド結合反応させる工程を少なくとも含むことを特徴とする。
【００３５】
一般式(1)〜(3)における置換基は以下のとおりである。
Ｒ¹、Ｒ²、Ｒ⁶およびＲ⁸は、それぞれ独立して、アラルキル基を示し、アラルキル基の例としては、ベンジル基、ｐ−メトキシベンジル基、フェネチル基、３−フェニルプロピル基、ｐ−ニトロベンジル基、ｏ−ニトロベンジル基、ｐ−ハロベンジル基、ｐ−シアノベンジル基、ジフェニルメチル基、トリフェニルメチル基（トリチル基）、αもしくはβ−ナフチルメチル基、α−ナフチルジフェニルメチル基などが挙げられる。Ｒ¹、Ｒ²、Ｒ⁶およびＲ⁸は、好ましくはベンジル基である。
【００３６】
Ｒ³およびＲ⁷は、それぞれ独立して、アシル基又はシリル基を示す。アシル基としては、アセチル基、ピバロイル基、レブリノイル基、ベンゾイル基、クロロアセチル基、ジクロロアセチル基、トリフルオロアセチル基、メトキシアセチル基、プロピオニル基、ｎ−ブチリル基、（Ｅ）−２−メチルブテノイル基、イソブチリル基、ペンタノイル基、ｏ−（ジブロモメチル）ベンゾイル基、ｏ−（メトキシカルボニル）ベンゾイル基、ｐ−フェニルベンゾイル基、２，４，６−トリメチルベンゾイル基、ｐ−トルオイル基、ｐ−アニソイル基、ｐ−クロロベンゾイル基、ｐ−ニトロベンゾイル基、α−ナフトイル基などが挙げられる。シリル基としては、トリメチルシリル基、トリエチルシリル基、ジメチルイソプロピリシリル基、イソプロピルジメチルシリル基、メチルジ−ｔ−ブチルシリル基、ｔ−ブチルジメチルシリル基、ｔ−ブチルジフェニルシリル基、トリイソプロピルシリル基、テトライソプロピルジシロキサニル基などが挙げられる。
【００３７】
Ｒ³およびＲ⁷は、好ましくは、それぞれアシル基であるか、又はＲ³がアシル基でありかつＲ⁷がシリル基である。アシル基としてはアセチル基、ピバロイル基、レブリノイル基が好ましく、シリル基としてはｔ−ブチルジフェニルシリル基が好ましい。
【００３８】
Ｒ⁴は、アミノ基保護基を示し、アミノ基保護基の例としては、フタロイル基、アセチル基、アリルオキシカルボニル基などが挙げられる。好ましくはフタロイル基である。
【００３９】
Ｒ⁵は、脱離基を示す。ここで脱離基とは、一般式(1)で示される単糖と一般式(2)で示される単糖とをグリコシド結合反応させる条件で脱離する基を意味する。脱離基の例としては、ハロゲン原子（フッ素原子、塩素原子、臭素原子等）、イミド基、メチルチオ基、フェニルチオ基などが挙げられるが、ハロゲン原子が好ましく、特にフッ素原子であることが好ましい。
【００４０】
なお式(4)、(7)および(10)中のＸは、脱離基のなかでも好ましいハロゲン原子である。このＸは、上記と同様にフッ素原子であることが好ましい。
【００４１】
Ｒ⁹は、アラルキル基、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基、又はペプチド残基を示す。ここで、残基とはその化合物の結合に関与する原子又は原子群をその化合物から除いた残りの部分を意味する。
【００４２】
アラルキル基の例、および好ましいアラルキル基については、前記と同様である。
【００４３】
６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基は、通常には、４位の水酸基が除かれた残基である。
【００４４】
アルキル基としては、炭素数１〜２３のものが例示され、炭素数８〜１４のものが好ましい。
【００４５】
グリセロール残基は、通常には、いずれかの水酸基が除かれた残基である。
【００４６】
Ｏ−アルキルグリセロール残基も特に限定されないが、通常にはいずれかの水酸基が除かれた残基であり、ジ−Ｏ−アルキルグリセロール残基が好ましく、２,３−ジ−Ｏ−アルキルグリセロール残基が好ましい。ここでいう「アルキル」としては、炭素数１〜２３のものが例示され、炭素数８〜１４のものが好ましい。
【００４７】
Ｏ−アシルグリセロール残基も特に限定されないが、通常にはいずれかの水酸基が除かれた残基であり、ジ−Ｏ−アシルグリセロール残基が好ましく、２,３−ジ−Ｏ−アシルグリセロール残基が好ましい。ここでいう「アシル」としては、炭素数１〜２３のものが例示され、炭素数８〜１４のものが好ましい。
【００４８】
コレステロール残基は、通常には、シクロペンタフェナントレン環のＣ−３の水酸基が除かれた残基である。
【００４９】
セラミド残基は、通常には、１位の水酸基が除かれた残基である。セラミド中のＮ−アシル基の炭素数は通常には１〜２８であり、炭素数１４〜２３のものが好ましい。
【００５０】
リン脂質残基としては、グリセロリン脂質（ホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルセリン、ホスファチジルイノシトール等）残基や、スフィンゴリン脂質（スフィンゴミエリン等）残基が挙げられる。
【００５１】
ビオチン残基は、通常には、カルボキシル基が除かれた残基である。
【００５２】
ペプチド残基は、通常には、アミノ基及びカルボキシル基のいずれかが除かれた残基である。
【００５３】
Ｚは酸素原子または−ＮＨＣＯ−を示す。−ＮＨＣＯ−の窒素原子及び酸素原子は、いずれがＲ⁹側となってもよい。
【００５４】
なお、Ｒ⁹がアラルキル基、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基又はリン脂質残基である場合には、Ｚは酸素原子であることが好ましい。
【００５５】
また、Ｒ⁹がビオチン残基またはペプチド残基である場合には、Ｚは−ＮＨＣＯ−であることが好ましい。
【００５６】
Ｒ¹⁰およびＲ¹¹は、それぞれ独立して、水素原子又は−ＳＯ₃Ｍを示す。
【００５７】
Ｒ¹²は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基、又はペプチド残基を示す。これらの基及び残基はＲ⁹について記載したのと同様である。
【００５８】
なお、Ｒ¹²が水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基又はリン脂質残基である場合には、Ｚは酸素原子であることが好ましい。
【００５９】
また、Ｒ¹²がビオチン残基またはペプチド残基である場合には、Ｚは−ＮＨＣＯ−であることが好ましい。
【００６０】
グリコシド結合反応させる条件は、用いる脱離基に合わせて適宜選択される。例えば、脱離基としてフッ素原子を選択した場合には、−７０〜６０℃で５分〜５０時間という条件が挙げられる。溶媒は、特に限定されず、１，２−ジクロロエタンなどを使用できる。
【００６１】
アラルキル基、アシル基又はシリル基およびアミノ基保護基は、脱離基が脱離する条件で、遊離しないものが選択される。これらの基は、同一の基となってもよい。
【００６２】
一般式(2)の単糖の製造方法としては、図２に概略を示したような方法が挙げられる。すなわち、化合物２から化合物９までの合成は、ガラクトース（化合物１）から伊藤らの報告している合成経路(Agric. Biol. Chem., 50, 3227(1986))に従って行うことができる。化合物１０から化合物１４までの合成は、以下の合成経路に従って行うことができる。トリクロロアセトイミド基からベンジル基への置き換え（化合物１０）、脱アセチル化（化合物１１）、ベンジル化（化合物１２）、脱アリル化（化合物１３）、ピバロイル化（化合物１４）。これらの工程の条件は、当業者が適宜設定することができる。
【００６３】
一般式(1)の単糖の製造方法としては、図３に概略を示したような方法が挙げられる。すなわち、化合物１６から化合物２０までの合成は、グルコサミン（化合物１５）から仲野らの報告している合成経路(Tetrahedron Lett., 31, 1597(1990))に従って行うことができる。化合物２１から化合物２４までの合成は、以下の合成経路に従って行うことができる。４位および６位の間のベンジリデン基の開環（化合物２１）、アセチル化（化合物２２）、脱メトキシフェニル化（化合物２３）、フッ素化（化合物２４）。これらの工程の条件は、当業者が適宜設定することができる。
【００６４】
一般式(1)の単糖と一般式(2)の単糖とをグリコシド結合反応させた後、アラルキル基およびアシル基又はシリル基を除去し、アミノ基保護基をアセチル基に置き換えることで、一般式(3)のオリゴ糖を得る。グリコシド結合反応、およびこのような基の除去および置換は公知の方法によって行うことができる（例えば、Synthesis, 384 (1989)等）。これらの具体例は、後述の実施例において詳述する。
【００６５】
一般式(3)において、Ｒ¹⁰およびＲ¹¹の少なくとも一方が−ＳＯ₃Ｍを示す場合には、上記一般式(1)で示される単糖と、上記一般式(2)で示される単糖とをグリコシド結合反応させる工程の後に、Ｒ³およびＲ⁷（アシル基又はシリル基）の少なくとも一方を選択的に除去して水素原子で置換し（これによりヒドロキシル基が生じる）、次いで当該水素原子を−ＳＯ₃Ｍに置換する（硫酸化）。
【００６６】
アシル基又はシリル基の選択的な除去（および水素原子への置換）は、アシル基又はシリル基を、アラルキル基およびアミノ基保護基に対して適切に選択することによって行うことができる。このような組み合わせとしては、アラルキル基としてベンジル基、アミノ基保護基としてフタロイル基、アシル基又はシリル基としてアセチル基又はピバロイル基の組み合わせ等が挙げられる。硫酸化の方法も特に限定されず、公知の方法を用いることができる。これらの具体例は、後述の実施例において詳述する。
【００６７】
具体的には、図４に概略を示したような方法によって一般式(3)のオリゴ糖を得ることができる。すなわち、アセチル基およびピバロイル基の除去（および水素原子への置換）ならびにフタロイル基のアセチル基への置換（化合物２６）、硫酸化（化合物２７）、脱ベンジル化（化合物２８）である。これらの工程の条件は、当業者が適宜設定することができる。
【００６８】
Ｒ¹⁰およびＲ¹¹のいずれか一方が−ＳＯ₃Ｍを示す場合には、Ｒ³およびＲ⁷のアシル基又はシリル基のいずれか一方を選択的に除去できるように選択する。例えば、Ｒ³としてレブリノイル基（式(7)又は(10)の単糖）、Ｒ⁷としてｔ−ブチルジフェニルシリル基（式(8)又は(11)の単糖）を選択する。レブリノイル基を選択的に除去（および水素原子に置換）してから硫酸化を行い、次いでその他のヒドロキシル基の保護基を除去することで、グルコサミン残基の６位のヒドロキシル基のみが硫酸化されたオリゴ糖（式(9)のオリゴ糖）を得ることができ、ｔ−ブチルジフェニルシリル基を選択的に除去（および水素原子に置換）してから硫酸化を行い、次いでその他のヒドロキシル基の保護基を除去することで、ガラクトース残基の６位のヒドロキシル基のみが硫酸化されたオリゴ糖（式(12)のオリゴ糖）を得ることができる。
【００６９】
アシル基又はシリル基の選択的な除去（および水素原子への置換）、これにより生じるヒドロキシル基の硫酸化、アラルキル基の除去、および、アミノ基保護基のアセチル基への置換は、公知の方法によって行うことができる。
【００７０】
一般式(1)の単糖と一般式(2)の単糖とをグリコシド結合反応等させて一般式(3)のオリゴ糖を得る方法の、他の例を図５〜図７に示す。
【００７１】
図５は、一般式(2)中のＲ⁹がＯ−アルキルグリセロール残基(2,3-ジ-O-テトラデシル-sn-グリセロール残基)の例であり、図６は、一般式(2)中のＲ⁹がアルキル基(オクチル基)の例であり、図７は、一般式(2)中のＲ⁹がコレスタニル基の例である。これらはいずれも上記と同様の方法で行うことができる。Ｒ⁹が他の基の場合も同様の方法で行うことができる。
【００７２】
得られる硫酸化されたオリゴ糖は塩となっていてもよく（すなわち該オリゴ糖中のＭが１価のカチオンであってもよく）、塩となっていなくてもよい（すなわち該オリゴ糖中のＭがプロトンであってもよい）。塩としては、後述の＜３＞本発明医薬の説明中に例示したものを挙げることができるが、アルカリ金属塩が好ましく、ナトリウム塩がより好ましい。また該オリゴ糖は電離した状態であってもよい。
【００７３】
本発明製造方法によれば、上記のオリゴ糖を収率よく、かつ少ない工程で製造することができる。
【００７４】
＜２＞本発明オリゴ糖
本発明オリゴ糖は、一般式(13)で表されるオリゴ糖である。
一般式(13)における置換基は以下のとおりである。
【００７５】
Ｒ¹⁶およびＲ¹⁷はそれぞれ独立して水素原子又は−ＳＯ₃Ｍを示す（ただし、Ｒ¹⁶およびＲ¹⁷がいずれも水素原子であり、かつＺが酸素原子でＲ¹⁸が水素原子又はコレスタニル基であるもの、並びにＲ¹⁶が−ＳＯ₃ＭでありかつＺが酸素原子でＲ¹⁷とＲ¹⁸がいずれも水素原子であるものを除く）。
【００７６】
またＲ¹⁸は水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を示すが、水素原子、６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基、アルキル基、Ｏ−アルキルグリセロール残基又はコレスタニル基が好ましい。これらの基及び残基はＲ⁹について記載したのと同様である。一般式(13)においてＲ¹⁸が水素原子の場合、Ｒ¹⁸により構成されるヒドロキシル基はβ位にあってもα位にあってもよい。またＲ¹⁸が６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基の場合、そのグリコシド結合はβ−グリコシド結合であることが好ましく、β−１,４グリコシド結合であることがより好ましい。Ｒ¹⁸が他の基又は残基の場合はβグリコシド結合であることが好ましい。また、Ｚは酸素原子又は−ＮＨＣＯ−を示す。Ｒ¹⁸がビオチン残基又はペプチド残基である場合には、Ｚは−ＮＨＣＯ−であることが好ましく、Ｒ¹⁸がそれ以外の基である場合には、Ｚは酸素原子であることが好ましい。
【００７７】
本発明オリゴ糖は塩となっていてもよく（すなわち該オリゴ糖中のＭが１価のカチオンであってもよく）、塩となっていなくてもよい（すなわち該オリゴ糖中のＭがプロトンであってもよい）。塩としては、後述の＜３＞本発明医薬の説明中に例示したものを挙げることができるが、アルカリ金属塩が好ましく、ナトリウム塩がより好ましい。また該オリゴ糖は電離した状態であってもよい。
【００７８】
Ｒ¹⁸が水素原子である本発明オリゴ糖は、上記本発明製造方法によって得ることができる。また、図５〜７に示すように、アルキル基、グリセロール残基、Ｏ−アルキルグリセロール残基、Ｏ−アシルグリセロール残基、コレステロール残基、コレスタニル基、セラミド残基、リン脂質残基、ビオチン残基又はペプチド残基を保持する単糖を他の単糖と結合させたり、Ｒ¹⁸が水素原子である本発明オリゴ糖と、グリセロール、コレステロール、セラミド、ビオチン又はペプチドとを公知のグリコシル化法により結合させたりすることによって、Ｒ¹⁸が水素原子以外の本発明オリゴ糖を得ることができる。
【００７９】
またＲ¹⁸が６−Ｏ−硫酸化Ｎ−アセチルグルコサミン残基である本発明オリゴ糖は、例えば公知のケラタン硫酸オリゴ糖に酸加水分解や酵素処理等の処理を施すことによって製造することができる。例えば公知のケラタン硫酸オリゴ糖であるNeuAc〜Galβ1-4GlcNAc(6S)β1-3Gal(6S)β1-4GlcNAc(6S)（式中、Galはガラクトース残基を、GlcNAcはＮ−アセチルグルコサミン残基を、NeuAcはＮ−アセチルノイラミン酸残基を、6Sは6-O-硫酸エステルをそれぞれ表す。また〜はα2,3結合又はα2,6結合を表す；ＷＯ９６／１６９７３参照）を、0.2Ｎ程度の強酸と共にインキュベートすることによりＮ−アセチルノイラミン酸(シアル酸)残基を除去し、次いでラクターゼ（β−ガラクトシダーゼ）と共にインキュベートすることによってガラクトース残基を除去することにより製造することができる。詳細は後述の実施例において詳述する。
【００８０】
本発明オリゴ糖又はその薬学的に許容される塩は、薬理作用を有し、医薬として使用できる。
【００８１】
＜３＞本発明医薬
本発明医薬は、本発明オリゴ糖又はその薬学的に許容される塩を有効成分とする。
【００８２】
薬学的に許容される塩とは、例えば、ナトリウム塩、カリウム塩、リチウム塩等のアルカリ金属塩、カルシウム塩等のアルカリ土類金属塩、アンモニウム塩等の無機塩基との塩、又はジエタノールアミン塩、シクロヘキシルアミン塩、アミノ酸塩等の有機塩基との塩のうち、薬学的に許容されるものであるが、これらに限定されるものではない。
【００８３】
本発明医薬は特に抗アレルギー剤として利用できる。抗アレルギー剤として利用する場合、本発明オリゴ糖のＮ−アセチルグルコサミン残基の６位のヒドロキシル基が硫酸化されている（すなわち一般式(13)におけるＲ¹⁶が−ＳＯ₃Ｍである）ことが好ましく、ガラクトース残基の６位のヒドロキシル基およびＮ−アセチルグルコサミン残基の６位のヒドロキシル基の両方が硫酸化されている（すなわち一般式(13)におけるＲ¹⁶およびＲ¹⁷がいずれも−ＳＯ₃Ｍである）ことがより好ましい。
【００８４】
本発明の抗アレルギー剤は、アレルギーが関与するあらゆる疾患に対して有効であり、具体的には、気管支喘息、アレルギ−性間質性肺炎、アレルギ−性鼻炎、アレルギ−性結膜炎、アトピ−性皮膚炎などの予防または治療を目的として適用することができる。
【００８５】
また、本発明オリゴ糖のガラクトース残基の６位のヒドロキシル基およびＮ−アセチルグルコサミン残基の６位のヒドロキシル基の両方が硫酸化されている場合、本発明医薬は特に抗炎症剤として利用できる。
【００８６】
本発明の抗炎症剤は、炎症が関与するあらゆる疾患に対して有効であり、具体的には、慢性関節リウマチ、全身性エリテマトーデス、変形性脊椎症、変形性関節症、腰痛症、手術後及び外傷後の炎症及び腫張の緩解、肩甲関節周囲炎、顎関節症、腱腱鞘炎、腱周囲炎、上腕骨顆炎（テニス肘）、筋肉痛、角結膜炎などの治療を目的として適用することができる。本発明の抗炎症剤は、その有効成分の作用により、これらの疾患に対して、鎮痛、消炎、解熱等の抗炎症作用を有する。
【００８７】
なお本発明医薬は、純然とした治療目的のみならず、疾患の予防、維持（悪化防止）、軽減（症状の改善）等を目的として適用することができる。
【００８８】
本発明においては、対象となる疾患の性質や進行状況、投与方法などに応じて、任意の剤形を適宜選択することができる。
【００８９】
すなわち、本発明医薬は注射（静脈内、筋肉内、皮下、皮内、腹腔内等）、経口、経皮、吸入などにより投与することができ、これらの投与方法に応じて適宜製剤化することができる。選択し得る剤形も特に限定されず、例えば注射剤（溶液、懸濁液、乳濁液、用時溶解用固形剤等）、錠剤、カプセル剤、顆粒剤、散剤、液剤、リポ化剤、軟膏剤、ゲル剤、外用散剤、スプレー剤、吸入散剤等から広く選択することができる。また、これらの製剤調製にあたり、慣用の賦形剤、安定化剤、結合剤、滑沢剤、乳化剤、浸透圧調整剤、pH調整剤、その他着色剤、崩壊剤等、通常医薬に用いられる成分を使用することができる。
【００９０】
本発明医薬中の有効成分であるケラタン硫酸オリゴ糖の配合量ならびに本発明医薬の投与量は、その製剤の投与方法、投与形態、使用目的、患者の具体的症状、患者の体重等に応じて個別的に決定されるべき事項であり、特に限定はされない。
【００９１】
アレルギ−性疾患では、気道や肺にあるＩｇＥ抗体をもった感作肥満細胞から抗原に誘導されて化学伝達物質が遊離される。化学伝達物質にはヒスタミンや好酸球走化性因子、ＳＲＳ−Ａなどがある。これらにより喘息では、呼吸困難、咳、発作が起き、また肺疾患では出血性肺炎、浮腫、間質性肺炎、血管炎などの症状がみられる。まれに肉芽腫もみられ、この疾患は後に肺線維症になることが多い。アレルギ−性疾患に対しては抗ヒスタミン剤やステロイド剤、抗アレルギ−剤（化学伝達物質遊離抑制薬）による治療が行われている。しかし、副作用として抗ヒスタミン剤では、脱力感、倦怠感、頭痛、嘔吐、頭重感、食欲不振等が報告されている。気管支喘息においては、抗コリン作用により気道分泌が抑制され、喀痰の喀出を困難とするため、軽症例を除いては使用されない。また緑内障、排尿困難の者にも禁忌である。一方、ステロイド剤の主たる作用は抗炎症作用と考えられ、通常アレルギ−疾患の治療には大量・連続投与が必要である。ステロイド剤は重篤な副作用があるため、一般的な治療方法でコントロ−ルできない場合に用いるのが原則とされている。また抗アレルギ−薬は、肝障害、出血性膀胱炎、胃腸障害を起こすこともあるので、定期的な検査を必要とする。
【００９２】
このように特にアレルギ−疾患の領域では、より副作用の少ない有効な治療法が求められているが、本発明医薬によれば、このような治療法を提供することができる。
【００９３】
なお、本発明医薬（本発明の抗アレルギー剤及び本発明の抗炎症剤を含む）において、一般式(13)における好ましいＲ¹⁸及びＺは前記と同様である。
【００９４】
【実施例】
以下に本発明を、実施例により具体的に説明する。しかしながら、これらにより本発明の技術的範囲が限定されるべきものではない。なお、実施例における溶媒の混合液の比率は、特記しない限り、容量比である。
【００９５】
【実施例１】
O-(2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル)-(1→3)-O-6-O-スルホ-β-D-ガラクトピラノース二ナトリウム塩の合成図２〜４に概略を示す手順によりO-(2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル)-(1→3)-O-6-O-スルホ-β-D-ガラクトピラノース二ナトリウム塩を合成した。なお、以下の実施例における各合成段階で共通して用いた方法は、以下の通りである。シリカゲルカラムクロマトグラフィーは、Kiesegel 60(MERCK)を用いて行った。薄層クロマトグラフィーは HPTLC-Fertigplatten Kieselgel 60 F₂₅₄ (MERCK)を使用した。¹H-NMR スペクトルおよび¹³C-NMR スペクトルは、JNM-EX-400（日本電子株式会社製）を用いて測定した。測定溶媒 CDCl₃, CD₃ODにおいてはテトラメチルシランを、またD₂Oにおいてはt-ブタノールを内部標準とした。
【００９６】
（１）化合物２から化合物１４の合成
ガラクトースシントン２−９は、ガラクトース（化合物１）から伊藤らの報告している合成経路(Agric. Biol. Chem., 50, 3227(1986))に従い合成を行った。化合物１０〜１４の合成は以下のようにして行った。
なお、以下、物質名の後の番号は、図２〜４における化合物の番号を示す。
【００９７】
(a) ベンジル2,4-ジ-O-アセチル-3,6-ジ-O-アリル-β-D-ガラクトピラノシド(benzyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside)１０
窒素ガス雰囲気下、事前に乾燥したモレキュラーシーブス４Ａ(30.0 g)の入った反応容器にベンジルアルコール(18.4 ml, 178.8 mmol)および化合物９(2,4-ジ-O-アセチル-3,6-ジ-O-アリル-D-ガラクトピラノシルトリクロロアセトイミデート(2,4-di-O-acetyl-3,6-di-O-allyl-D-galactopyranosyl trichloroacetimidate)；21.84 g, 44.67 mmol)を加えた後、氷冷下で15分間撹拌した。反応混合物に氷冷下でトリメチルシリルトリフルオロメタンスルホネート(1.7 ml, 8.93 mmol)を加えた後、同温で4時間撹拌した。反応液を酢酸エチルで希釈し、氷冷下、トリエチルアミンを加え中和後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝6:1)にて精製し、化合物１０(18.6
g, 96%)を得た。
【００９８】
Rf: 0.51 (トルエン：酢酸エチル=3:1)
C₂₃H₃₀O₈ MW: 434.47
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
2.037(s, 3H, OAc) 2.146(s, 3H, OAc) 4.445(d, 1H, J=7.8 Hz, H-1) 5.461(d, 1H, J=2.9 Hz, H-4) 5.715-5.914(m, 2H, CH₂=CH x2) 7.200-7.400(m, 5H, aromatic)
【００９９】
(b) ベンジル3,6-ジ-O-アリル-β-D-ガラクトピラノシド(benzyl 3,6-di-O-allyl-β-D-galactopyranoside)１１
化合物１０(10.84 g, 24.9 mmol)のメタノール溶液(30 ml)にナトリウムメトキシド(134 mg, 2.5 mmol)を加え窒素ガス雰囲気下室温で48時間撹拌した。反応混合物を酢酸にて中和後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝4:1)にて精製し、化合物１１(6.8 g, 78%)を得た。
【０１００】
Rf: 0.27 (トルエン：酢酸エチル=2:1)
C₁₉H₂₆O₆ MW: 350.40
【０１０１】
(c) ベンジル3,6-ジ-O-アリル-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(benzyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside)１２
窒素ガス雰囲気および氷冷下、60%水素化ナトリウム(3.8 g, 95.5 mmol)、化合物１１(6.7 g, 19.1 mmol)およびＤＭＦ20 mlの混合物にベンジルブロミド(11.4 ml, 95.5 mmol)を加え18時間撹拌した。反応混合物に氷冷下でメタノールを加え1時間撹拌後、減圧下溶媒を留去した。残渣をジエチルエーテルにて希釈後、水、飽和食塩水にて順次洗浄し、硫酸マグネシウムにて乾燥後、溶媒を減圧下留去した。得られた残渣をシリカゲルカラムクロマトグラフィー(n-ヘキサン：酢酸エチル＝10:1〜9:1)にて精製し、化合物１２(9.1 g, 90%)を得た。
【０１０２】
Rf: 0.27 (トルエン：酢酸エチル=10:1)
C₃₃H₃₈O₆ MW: 530.63
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
3.424(dd, 1H, J=2.9,9.8 Hz, H-3) 3.829(dd, 1H, J=7.8,9.8 Hz, H-2) 3.861(d, 1H, J=2.9 Hz, H-4) 4.453(d, 1H, J=7.8 Hz, H-1) 5.805-5.984(m, 2H, CH₂=CH x2) 7.200-7.450(m, 15H, aromatic)
【０１０３】
(d) ベンジル2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(benzyl 2,4-di-O-benzyl-β-D-galactopyranoside)１３
水素ガス雰囲気下、活性化されたイリジウムコンプレックス[Iｒ(CoD)(PMePh₂)₂PF₆(287 mg, 0.34 mmol)のテトラヒドロフラン溶液(60 ml)に室温で化合物１２(8.9 g, 16.7 mmol)のテトラヒドロフラン溶液(80 ml)を加え7時間撹拌した。次いで、水(100 ml)およびヨウ素(8.5 g, 67.1 mmol)を加え15時間撹拌した。反応混合物を酢酸エチルにて希釈後、飽和チオ硫酸ナトリウム溶液、飽和重曹水、飽和食塩水にて順次洗浄し、硫酸マグネシウムにて乾燥後、溶媒を減圧下留去した。得られた残渣を再結晶(エタノール−ジクロロメタン−ジエチルエーテル)し、化合物１３(7.4 g, 97%)を得た。
【０１０４】
Rf: 0.34 (n-ヘキサン:酢酸エチル=1:1)
C₂₇H₃₀O₆ MW: 450.51
【０１０５】
(e) ベンジル2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(benzyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)１４
窒素ガス雰囲気下、0℃で、化合物１３(7.3 g, 16.2 mmol)のピリジン溶液(50 ml)にピバロイルクロリド(4.2 ml, 35.7 mmol)を加え70分間撹拌した。反応液にメタノールを加え40分間撹拌した後に減圧下溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝6:1)にて精製し、化合物１４(7.81 g, 90%)を得た。
【０１０６】
Rf: 0.45 (トルエン：酢酸エチル=6:1)
C₃₂H₃₈O₇ MW: 534.62
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
1.202(s, 9H, OPiv) 2.326(bs, 1H, OH) 3.628-3.708(m, 3H, H-2, H-3 and H-5)3.786(d, 1H, J=3.9 Hz, H-4) 4.142(dd, 1H, J=6.4,10.7 Hz, H-6) 4.352(dd, 1H, J=6.8,11.2 Hz, H-6') 4.448(d, 1H, J=7.3 Hz, H-1) 6.650-7.150(m, 15H, aromatic)
【０１０７】
（２）化合物１６から化合物２４の合成
グルコサミンシントン１６−２０は、グルコサミン（化合物１５）から仲野らの報告している合成経路(Tetrahedron Lett., 31, 1597(1990))に従い合成を行った。化合物２１〜２４の合成は以下のようにして行った。
【０１０８】
(f) p-メトキシフェニル3,4-ジ-O-ベンジル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシド(p-methoxyphenyl 3,4-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside)２１
窒素ガス雰囲気下、事前に乾燥したモレキュラーシーブス４Ａ(60.0 g)の入った反応容器にボラン-トリメチルアミンコンプレックス(75.0 g, 1028 mmol)、化合物２０(21.0 g, 35.4 mmol)のジクロロメタン溶液(200 ml)、および、ジエチルエーテル(80 ml)を加え15分間撹拌した。反応容器を0℃に冷却し、無水塩化アルミニウム(20.0 g, 150 mmol)を少量ずつ1.5時間で加え、0℃で2.5時間撹拌した。反応混合物をセライトで濾過し、濾液を酢酸エチルで希釈後、1N硫酸水溶液、水、飽和重曹水、飽和食塩水にて順次洗浄し、硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。得られた残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝4:1)にて精製し、化合物２１(14.5 g, 69%)を得た。
【０１０９】
Rf: 0.40 (トルエン：酢酸エチル=3:1)
C₃₅H₃₃N₁O₈ MW: 595.62
400 MHz ¹H-NMR (CDCl₃+CD₃OD,TMS) δ：
3.620-3.662(m, 1H, H-5) 3.706(s, 3H, OMe) 3.783-3.849(m, 2H, H-4 and H-6) 3.939(dd, 1H, J=2.4,12.2 Hz, H-6') 4.351(dd, 1H, J=8.3,10.7 Hz, H-2) 4.435(dd, 1H, J=8.3,10.7 Hz, H-3) 5.693(d, 1H, J=8.3 Hz, H-1) 6.650-7.900(m, 18H, aromatic)
【０１１０】
(g) p-メトキシフェニル6-O-アセチル-3,4-ジ-O-ベンジル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシド(p-methoxyphenyl 6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside)２２
窒素ガス雰囲気下、化合物２１(10.5 g, 17.6 mmol)のピリジン溶液(200 ml)に無水酢酸(200 ml)およびDMAP(触媒量)を加え20時間撹拌した。反応液にエタノールを加え20分間撹拌した後に減圧下溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝4:1)にて精製し、化合物２２(9.6 g, 85%)を得た。
【０１１１】
Rf: 0.51 (トルエン：酢酸エチル=4:1)
C₃₇H₃₅N₁O₉ MW: 637.66
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
2.062(s, 3H, OAc) 3.680(s, 3H, OMe) 3.759-3.817(m, 2H, H-4 and H-5) 4.296(dd, 1H, J=4.4, 12.2 Hz, H-6) 5.631(d, 1H, J=7.8 Hz, H-1) 6.650-7.900(m, 18H, aromatic)
【０１１２】
(h) 6-O-アセチル-3,4-ジ-O-ベンジル-2-デオキシ-2-フタルイミド-D-グルコピラノース(6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimido-D-glucopyranose)２３
化合物２２(9.0 g, 14.1 mmol)をアセトニトリル：水 (4:1; 400 ml)に溶解し、硝酸第二セリウムアンモニウム(20.1 g, 36.7 mmol)を加え室温下、40分間激しく撹拌した。反応混合物を酢酸エチルにて希釈し、水、飽和重曹水、飽和食塩水にて順次洗浄後、硫酸マグネシウムにて乾燥し減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝2.5:1)にて精製し、化合物２３(6.1 g, 81%)を得た。
【０１１３】
Rf: 0.23 (トルエン：酢酸エチル=2:1)
C₃₀H₂₉N₁O₈ MW: 531.54
400 MHz ¹H-NMR (CDCl₃+D₂O,TMS) δ：
2.074(s, 3H, OAc) 3.680(t, 3H, J=9.3 Hz, H-4) 3.739-3.772(m, 1H, H-5) 4.100(dd, 1H, J=8.8,10.8 Hz, H-2) 4.240(dd, 1H, J=3.9,11.2 Hz, H-6) 5.386(d, 1H, J=8.3 Hz, H-1) 6.650-7.900(m, 14H, aromatic)
【０１１４】
(i) 6-O-アセチル-3,4-ジ-O-ベンジル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシルフルオライド(6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl fluoride)２４
窒素ガス雰囲気下、化合物２３(5.95 g, 11.2 mmol)の1,2−ジクロロエタン溶液(50 ml)に氷冷下で、ジエチルアミノサルファートリフルオリド(5.8 ml, 43.9 mmol)を加え2時間撹拌した。反応混合物を酢酸エチルにて希釈し、飽和重曹水、飽和食塩水にて順次洗浄後、硫酸マグネシウムにて乾燥し溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝4:1)にて精製し、化合物２４(5.9 g, 99%)を得た。
【０１１５】
Rf: 0.68 (トルエン：酢酸エチル=2:1)
C₃₀H₂₈N₁O₇F₁ MW: 533.53
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
2.097(s, 3H, OAc) 3.859(dd, 1H, J=8.3,9.8 Hz, H-4) 3.800-3.840(m, 1H, H-5) 5.810(d, 0.5H, J=7.8 Hz, H-1β) 5.943(d, 0.5H, J=7.8 Hz, H-1β) 6.800-7.800(m, 14H, aromatic)
【０１１６】
（３）化合物１４および化合物２４からの化合物２８の合成
化合物２５〜２８の合成は以下のように行った。
【０１１７】
(j) ベンジルO-(6-O-アセチル-3,4-ジ-O-ベンジル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→3)-O-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(benzyl O-(6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-O-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)２５
窒素ガス雰囲気下、事前に乾燥したモレキュラーシーブス４Ａ(20.0 g)の入った反応容器にシルバートリフレート(7.23 g, 28.2 mmol)、ハフノセンジクロリド(5.4 g, 14.1 mmol)および1,2−ジクロロエタン(20 ml)を加えた後、氷冷下20分間撹拌した。反応容器を-23℃に冷却し、化合物２４(5.8 g, 10.8 mmol)および化合物１４(5.4 g, 10.0 mmol)の1,2−ジクロロエタン溶液(45 ml)を加え、-23℃で1.5時間撹拌した。反応液を酢酸エチルで希釈し氷冷下、トリエチルアミンを加え20分間撹拌した後にセライトで濾過した。濾液を酢酸エチルで希釈し、飽和重曹水、飽和食塩水にて順次洗浄後、硫酸マグネシウムにて乾燥し減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン：酢酸エチル＝9:1)にて精製後、再結晶を行い化合物２５(9.3 g, 82%)を得た。
【０１１８】
Rf: 0.39 (トルエン：酢酸エチル=8:1)
C₆₂H₆₅N₁O₁₄ MW: 1048.15
400 MHz ¹H-NMR (CDCl₃,TMS) δ：
1.173(s, 9H, OPiv) 1.986(s, 3H, OAc) 3.859(bd, 1H, J=2.5 Hz, H-4) 4.063(dd, 1H, J=5.9,11.2 Hz) 5.454(d, 1H, J=8.3 Hz, H-1) 6.800-7.800(m, 24H, aromatic)
【０１１９】
(k) ベンジルO-(2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-β-D-グルコピラノシル)-(1→3)-O-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(benzyl O-(2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-2,4-di-O-benzyl-β-D-galactopyranoside)２６
化合物２５(8.0 g, 7.6 mmol)の1-ブタノール溶液(200 ml)に、エチレンジアミン(170 ml)を加え98℃にて46時間撹拌した。反応混合物の溶媒を減圧下留去し、残渣にトルエンおよびメタノールを加え減圧下溶媒を留去した。残渣をピリジン(200 ml)に溶解しＤＭＡＰ(触媒量)と無水酢酸(150 ml)を加え室温で2日間撹拌した。反応混合物の溶媒を留去し、トルエンおよびエタノールにて共沸を行った。得られた残渣をシリカゲルカラムクロマトグラフィー（トルエン：酢酸エチル＝4:1）にて精製し、2成分の混合物(6.84 g)を得た。さらにこの混合物のメタノール溶液 (100 ml)にナトリウムメトキシド(769 mg, 14.3 mmol)を加え窒素ガス雰囲気下室温で60時間撹拌した。アンバーリスト15で中和し濾過後、濾液を減圧下溶媒留去した。得られた残渣を再結晶(ジクロロメタン−イソプロピルエーテル）し化合物２６(6.0 g, 94%)を得た。
【０１２０】
Rf: 0.33 (トルエン：酢酸エチル=1:3)
C₄₉H₅₅N₁O₁₁ MW: 833.94
400 MHz ¹H-NMR (CDCl₃+CD₃OD,TMS) δ：
1.557(s, 3H, NAc) 4.438(d, 1H, J=7.3 Hz, H-1) 4.784(d, 1H, J=8.3 Hz, H-1) 7.200-7.450(m, 20H, aromatic)
100 MHz ¹³C-NMR (CDCl₃+CD₃OD,TMS) δ：22.92(Me-CO) 61.44,61.64(C-6 x2) 101.73(C-1),102.60(C-1) 170.29(Me-CO)
【０１２１】
(l) ベンジルO-(2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル)-(1→3)-O-2,4-ジ-O-ベンジル-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩(benzyl O-(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1→3)-O-2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt)２７
窒素ガス雰囲気下、化合物２６(212.5 mg, 0.255 mmol)とサルファートリオキシドトリエチルアミンコンプレックス(184.7 mg, 1.02 mmol)の混合物をDMF(1.0 ml)に溶解し、50℃で1時間撹拌した。反応液をそのままセファデックス LH-20(クロロホルム：メタノール＝1:1)にて精製し、糖画分を濃縮した。得られた残渣をメタノール(4 ml)に溶解後、Dowex 50(Na⁺, 4 g)を加え12時間撹拌し、対カチオンをナトリウムに変換した。更に得られた残渣をシリカゲルカラムクロマトグラフィー(クロロホルム：メタノール＝4:1)にて精製した後に、シリカゲルを除く目的でセファデックス LH-20(クロロホルム：メタノール＝1:1)にて精製し、化合物２７(252 mg, 95%)を得た。
【０１２２】
Rf: 0.53 (クロロホルム：メタノール=3:1)
C₄₉H₅₃N₁O₁₇S₂Na₂ MW: 1038.03
400 MHz ¹H-NMR (CDCl₃+CD₃OD,TMS) δ：
1.621(s, 3H, NAc) 7.200-7.450(m, 20H, aromatic)
100 MHz ¹³C-NMR (CDCl₃+CD₃OD,TMS) δ：
22.14(Me-CO) 66.25,66.61(C-6 x2) 102.04(C-1 x2) 170.98(Me-CO)
【０１２３】
(m) O-(2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル)-(1→3)-O-6-O-スルホ-β-D-ガラクトピラノース二ナトリウム塩(O-(2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1→3)-O-6-O-sulfo-β-D-galactopyranose disodium salt)２８
化合物２７(236.8 mg,0.228 mmol)のメタノール−水 (2:1, 6 ml)溶液に、20%水酸化パラジウム炭素(268 mg)を加え、反応系内を水素で置換し室温で17時間撹拌した。反応混合物をセライトで濾過し、残渣を水にて洗浄後、濾液と洗浄液を併せて減圧下溶媒を留去した。得られた残渣をセファデックスG−25(水)にて精製し、化合物２８(131 mg, 98%)を得た。
【０１２４】
Rf: 0.28 (1-ブタノール：エタノール：水=2:2:1)
C₁₄H₂₃N₁O₁₇S₂Na₂ MW: 587.44
400 MHz¹H-NMR (D₂O, t-BuOH, at 50℃) δ：
2.029(s, 3H, NAc) 4.580(d, 0.55H, J=8.3 Hz, H-1aβ) 4.727(d, 0.55H, J=8.3 Hz, H-1bβ) 4.742(d, 0.45H, J=8.3 Hz, H-1bα) 5.232(d, 0.45H, J=3.4 Hz, H-1aα)
100 MHz ¹³C-NMR (D₂O, t-BuOH, at 50℃) δ：
25.04(Me-CO) 69.81(C-6 b) 70.70(C-6 aβ) 70.94(C-6 aα) 95.21(C-1aα) 99.21(C-1aβ) 105.39(C-1b) 177.74(Me-CO)
【０１２５】
【実施例２】
O-(2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル)-(1→3)-O-(6-O-スルホ-β-D-ガラクトピラノシル)-(1→4)-O-2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノース三ナトリウム塩（以下、Ｇ４Ｌ４のナトリウム塩という）の製造
NeuAc〜Galβ1-4GlcNAc(6S)β1-3Gal(6S)β1-4GlcNAc(6S)（式中、Galはガラクトース残基を、GlcNAcはＮ−アセチルグルコサミン残基を、NeuAcはＮ−アセチルノイラミン酸残基を、6Sは6-O-硫酸エステルをそれぞれ表す。また〜はα2,3結合又はα2,6結合を表す；ＷＯ９６／１６９７３参照）１ｇを0.1 M 硫酸 10 mlに溶解させ、50℃で22時間保温することによりＮ−アセチルノイラミン酸残基(シアル酸残基)を切断した。反応後の溶液に1 M NaOHを少量加えてpH5に調整した後、0.5 M 酢酸ナトリウム緩衝液pH4.5を１ml、20％アジ化ナトリウムを25μl加えた。ラクターゼ(ケイアイ化成製) 5000 Uを加えて37℃で22時間保温することによりガラクトース残基を切断した。反応溶液を蒸留水で５倍希釈し、1 M NaClで平衡化したムロマックカラム(室町化学工業)(2.5×24 cm)にアプライした。1 M NaCl(500ml)から2.5 M NaCl(500ml)の塩濃度勾配をカラムに負荷し、溶出液を５mlづつ分取した。溶出画分をキャピラリー電気泳動で分析し、Ｇ４Ｌ４の溶出位置を確認した。Ｇ４Ｌ４を含む画分を集めてロータリーエバポレーターで約10 mlに濃縮した。濃縮溶液を蒸留水で平衡化したセルロファインGCL25sfカラム(生化学工業)(3×60 cm)にアプライし、蒸留水で溶出した。10 mlづつ分取した溶出画分をキャピラリー電気泳動で分析し、Ｇ４Ｌ４の溶出位置を確認した。Ｇ４Ｌ４を含む画分を集めてロータリーエバポレーターで約20 mlに濃縮した。分子量カット10000の限外ろ過膜でろ過してエンドトキシンを除去した後、凍結乾燥して最終サンプルとした。
【０１２６】
最終サンプルはキャピラリー電気泳動で単一ピークを示した。ヘキソース含量と硫酸含量の測定をおこなった結果、理論値１に対して各0.84、0.91の値を得た。
【０１２７】
また、最終サンプルを下記条件で高速液体クロマトグラフィーにかけた結果、保持時間１６．４分に単一ピークを示した。
【０１２８】
カラム：YMC-Pack PolyamineII (4.6×250 mm)((株)ワイエムシイ製)
カラム温度：３５℃
溶出液：150 mMリン酸二水素ナトリウム
流速：１ ml/分
測定波長：210 nm
サンプル：10 mg/mlＧ４Ｌ４(最終サンプル)
【０１２９】
また、最終サンプルのＮＭＲ測定の結果を以下に示す。
【０１３０】
400 MHz ¹H-NMR(D₂O, t-BuOH, at 22.9℃) δ：
2.024(s, 3H, NAc) 2.030(s, 3H, NAc) 4.526(d, 1H, J_1,2=7.8 Hz, H-1b) 4.699(d, 1H, J_1,2=8.8 Hz, H-1c) 4.729(d, 0.4H, J_1,2=7.8 Hz, H-1aβ) 5.211(d, 0.6H, J_1,2=2.5 Hz, H-1aα)
100 MHz ¹³C-NMR(D₂O, t-BuOH, at 26.0℃) δ：
24.74(NHCOCH₃), 25.05(NHCOCH₃), 69.62(C-6a or b or c), 69.77(C-6b or c or a), 70.57(C-6c or a or b), 93.31(C-1aα), 97.82(C-1aβ), 105.80(C-1b or c), 105.91(C-1c or b)
【０１３１】
【実施例３】
2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール二ナトリウム塩の合成
図５に概略を示す手順により、2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール二ナトリウム塩を合成した。
物質名の後の番号は、図５における化合物の番号を示す。
【０１３２】
(a) 2,4-ジ-O-アセチル-3,6-ジ-O-アリル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ- O-テトラデシル-sn-グリセロール(2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)３
2,3-ジ- O-テトラデシル-sn-グリセロール(500 mg, 1.03 mmol)、シクロペンタジエンハフノニウムダイクロライド(782 mg, 2.68 mmol)、シルバートリフレート(1.06 g, 5.36 mmol)、モレキュラーシーブ4A(1.8 g)を１,2-ジクロロエタン(3.0 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-15℃に冷却し 化合物１(536 mg, 1.55 mmol)を加え2.5時間攪拌した。反応液をトリエチルアミンを加え中和し、酢酸エチル(AcOEt)で希釈後、セライトでろ過し、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=10:1)にて精製し、化合物３(716.7 mg, 85.8%)を得た。
【０１３３】
Rf 0.58(トルエン:AcOEt=6:1)
C₄₇H₈₅O₁₀ MW : 810.179
¹H-NMR (CDCl₃) δ：
5.887 (m, 1H, Allyl), 5.816 (m, 1H, Allyl), 5.500 (d, 1H, J = 2.4Hz, H-4), 5.108 (dd, 1H, J = 8.3, 9.9Hz, H-2), 4.474 (d, 1H, J = 8.3Hz, H-1), 2.165, 2.015 (2s, 6H, 2Ac), 0.912 (t, 6H, J = 6.3Hz, 2CH₃)
【０１３４】
(b) 3,6-ジ-O-アリル-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール(3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)５
化合物３(430 mg, 0.531 mmol)をメタノールとテトラヒドロフランとの混合液(1:1、4 mL)に溶解し、1 N水酸化ナトリウム溶液(0.8 mL)を加え室温で1日間攪拌後、その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=5:2)にて精製し、化合物４(377.9 mg, 98.0%)を得た。
[Rf 0.43(トルエン:AcOEt=2:1)]
【０１３５】
続いて、化合物４(778 mg, 1.072 mmol)を、N,N-ジメチルホルムアミド(3 ml)に溶解し、アルゴンガス気流下-15℃で、水素化ナトリウム(308 mg, 6.99 mmol)を加え攪拌した。続いてベンジルブロマイド(0.84 ml, 6.99 mmol)を加え、徐々に室温にしながら3時間攪拌した。反応液にメタノールを加え中和し、酢酸エチルで希釈後、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:AcOEt=12:1)にて精製し、化合物５(864 mg, 89%)を得た。
【０１３６】
Rf 0.67(ヘキサン:AcOEt=6:1)
C₅₇H₉₃O₈ MW : 906.354
¹H-NMR (CDCl₃) δ：
5.845 (m, 1H, Allyl), 5.771 (m, 1H, Allyl), 4.859 (d, 1H, J = 11.7 Hz, Bn), 4.816 (d, 1H, J = 10.8 Hz, Bn), 4.665 (d, 1H, J = 10.7 Hz, Bn), 4.572 (d, 1H, J = 11.7 Hz, Bn), 4.272 (d, 1H, J = 7.3 Hz, H-1), 3.773 (d, 1H, J = 2.5 Hz, H-4), 3.660 (dd, 1H, J = 7.8, 9.8 Hz, H-2), 0.801 (t, 6H, J = 6.4 Hz, 2CH₃).
【０１３７】
(c) 2,4-ジ-O-ベンジル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール(2,4-di-O-benzyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)６
イリジウムコンプレックス (1,5-シクロオクタジエンビス(メチルジフェニルホスフィン)イリジウムヘキサフルオロホスフェート) (112 mg, 0.096 mmol)をテトラヒドロフラン(5 mL)に懸濁し、H₂気流下攪拌し活性化させた。その溶液に化合物５(864 mg, 0.95 mmol)をテトラヒドロフラン(5 mL)に溶解させて加え、アルゴンガス気流下室温で2時間攪拌後、ヨウ素(484 mg)、水(24.7 mL)、テトラヒドロフラン(15 mL)を加えさらに2時間室温で攪拌した。反応液をクロロホルムで希釈後、飽和チオ硫酸ナトリウム溶液、飽和重曹水、飽和食塩水で順次洗浄した。クロロホルム層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー(トルエン:EtOAc=5:2) にて精製し、化合物６(686.6 mg, 87.1%)を得た。
【０１３８】
Rf 0.32(トルエン:AcOEt=2:1)
C₅₁H₈₅O₈ MW: 826.225
¹H-NMR (CDCl₃) δ：
4.991 (d, 1H, J = 11.2 Hz, Bn), 4.834 (d, 1H, J = 11.7 Hz, Bn), 4.658 (d, 2H, J = 11.2 Hz, 2Bn), 4.385 (d, 1H, J = 7.3 Hz, H-1), 3.778 (d, 1H, J = 2.4 Hz, H-4), 0.881 (t, 6H, J = 6.8 Hz, 2CH₃)
【０１３９】
(d) 2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ- O-テトラデシル-sn-グリセロール(2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl-(1→1)-2,3-di- O-tetradecyl-sn-glycerol)７
化合物６(687 mg, 0.831 mmol)をピリジン(12 mL)を加え溶解させ、その溶液にピバロイルクロライド(130μL, 1.08 mmol)を加え-5℃にて1時間攪拌後、さらにピバロイルクロライド(130μL, 1.08 mmol)を加え-5℃にて1時間攪拌した。反応液を、酢酸エチルで希釈後、セライトでろ過し、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=10:1)にて精製し、化合物７(716.6 mg, 94.7%)を得た。
【０１４０】
Rf 0.57(トルエン:AcOEt=6:1)
C₅₆H₉₃O₉ MW : 910.342
¹H-NMR (CDCl₃) δ：
4.991 (d, 1H, J = 11.7 Hz, Bn), 4.855 (d, 1H, J = 11.7 Hz, Bn), 4.650 (d, 1H, J = 11.2 Hz, Bn), 4.646 (d, 1H, J = 11.7 Hz, Bn), 4.365 (d, 1H, J = 7.3 Hz, H-1), 4.302 (dd, 1H, J = 6.8, 11.8 Hz, H-6), 4.109 (dd, 1H, J = 6.4, 11.2 Hz, H-6'), 3.775 (d, 1H, J = 2.4 Hz, H-4), 1.179 (s, 9H, piv), 0.879 (t, 6H, J = 6.8 Hz, 2CH₃)
【０１４１】
(e) 3-O-アセチル-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール(3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)８
化合物７(10.8 mg, 11.9μmol)をピリジン(1 mL)を加え溶解させ、その溶液に無水酢酸(0.5 mL)を加え室温で1時間攪拌した。溶媒をトルエンで共沸後、残渣をセファデックスLH-20(CHCl₃:MeOH=1:2)にて精製し、化合物８(11.3 mg, qu.)を得た。
【０１４２】
Rf 0.46(トルエン:AcOEt=8:1)
C₅₈H₉₅O₁₀ MW : 952.379
¹H-NMR (CDCl₃) δ：
4.900 (dd, 1H, J = 3.4, 10.3 Hz, H-3), 4.882 (d, 1H, J = 11.7 Hz, Bn), 4.634 (d, 2H, J = 12.2 Hz, 2Bn), 4.543 (d, 1H, J = 11.2 Hz, Bn), 4.437 (d, 1H, J = 7.3 Hz, H-1), 4.299 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.087 (dd, 1H, J = 6.8, 11.2 Hz, H-6'), 3.850 (d, 1H, J = 2.9 Hz, H-4), 3.765 (dd, 1H, J = 7.8, 10.3 Hz, H-2), 1.926 (s, 3H, Ac), 1.188 (s, 9H, piv), 0.881 (t, 6H, J = 6.8 Hz, 2CH₃)
【０１４３】
(f) 3,4-ジ-O-ベンジル-2-デオキシ-6-O-レブロイル-2-フタルイミド-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール(3,4-di-O-benzyl-2-deoxy-6-O-levloyl-2-phtalimido-β-D-gulcopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)９
化合物７(685 mg, 0.752 mmol)、シクロペンタジエンハフノニウムダイクロライド(571 mg, 1.96 mmol)、シルバートリフレート(771 g, 3.91 mmol)、モレキュラーシーブ4A(2.5 g)を１,2-ジクロロエタン(10 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-15℃に冷却し化合物２(563 mg, 0.98 mmol)を加え1時間攪拌した。反応液をトリエチルアミンを加え中和し、酢酸エチルで希釈後、セライトでろ過し、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=9:1)にて精製し、化合物９(1.01 g, 91.5%)を得た。
【０１４４】
Rf 0.44(トルエン:AcOEt=6:1)
C₈₉H₁₂₄O₁₇N MW : 1479.949
¹H-NMR (CDCl₃) δ：
5.461 (d, 1H, J = 8.3 Hz, H-1b), 4.941 (d, 1H, J = 11.2 Hz, Bn), 4.874 (d, 1H, J = 10.7 Hz, Bn), 4.789 (d, 1H, J = 11.7 Hz, Bn), 4.648 (d, 1H, J = 11.2 Hz, Bn), 4.536 (d, 1H, J = 11.7 Hz, Bn), 4.460 (d, 1H, J = 11.7 Hz, Bn), 4.414 (d, 2H, J = 11.7 Hz, 2Bn), 4.223 (d, 1H, J = 7.8 Hz, H-1a), 3.897 (d, 1H, J = 3.2 Hz, H-4a), 2.130 (s, 3H, CH₃), 1.153 (s, 9H, piv), 0.881 (t, 6H, J = 6.6 Hz, 2CH₃)
【０１４５】
(g) 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール(2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-gulcopyranosyl-(1→3)-2,4-di-O-benzyl-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol)１０
化合物９(977.4 mg, 0.667 mmol)をエタノール(33.5 mL)に懸濁し、ヒドラジン水和物(3.35 mL)を加え、110℃で18時間攪拌した。溶媒を留去し、得られたアミノ体をピリジン (6.0 mL)に溶解し、無水酢酸(5.0 mL)を加え室温で17時間攪拌後、溶媒を留去した。残渣をメタノールとテトラヒドロフランとの混合液(1:1、20.0 mL)に溶解し、ナトリウムメトキシド(108 mg, 2.0 mmol)を加え、60℃で1時間攪拌した。その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:アセトン:CHCl₃=5:2:1)にて精製し、さらにセファデックスLH-20(CHCl₃:MeOH=2:3)による精製を行い、化合物１０(756 mg, 94.8%)を得た。
【０１４６】
Rf 0.18(トルエン:アセトン:CHCl₃=6:2:1)
C₇₃H₁₁₀O₁₃N MW : 1209.666
¹H-NMR (CDCl₃) δ：
4.823 (d, 1H, J = 8.3 Hz, H-1b), 4.354 (d, 1H, J = 6.8 Hz, H-1a), 1.497 (s, 3H, NHAc), 0.881 (t, 6H, J = 6.4 Hz, 2CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
173.06 (Me-CO), 105.51, 104.01 (C-1x2), 62.80, 62.51 (C-6x2)
【０１４７】
(h) 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-スルホ-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール二ナトリウム塩(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-gulcopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol disodium salt)１１
化合物１０(200 mg, 0.165 mmol)をN,N-ジメチルホルムアミド(1.5 mL)に溶解し、(C₂H₅)₃NSO₃(300 mg, 1.65 mmol)を加え、50℃で0.5時間攪拌した。その反応液を直接セファデックスLH-20(CHCl₃:MeOH=2:3)にて精製した。その溶媒をある程度留去し、残渣に水(2.0 mL)及びDowex-50 (Na⁺)タイプを加え、一昼夜攪拌し、セライトでろ過後、ろ液を留去した。残渣をDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=5:10:3)にて精製し、化合物１１(215 mg, 92.2%)を得た。
【０１４８】
Rf 0.38(CHCl₃:MeOH=6:1)
C₇₃H₁₀₈O₁₉NS₂Na₂ MW : 1413.74
¹H-NMR (CDCl₃+CD₃OD) δ：
4.403 (d, 1H, J = 7.8 Hz, H-1a), 1.593 (s, 3H, NHAc), 0.889 (t, 6H, J = 6.4 Hz, 2CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
172.93 (Me-CO), 105.24, 104.01 (C-1x2), 68.13, 68.00 (C-6x2)
【０１４９】
(i) 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシル-(1→1)-2,3-ジ-O-テトラデシル-sn-グリセロール二ナトリウム塩(2-acetamido-2-deoxy-6-O-sulfo-β-D-gulcopyranosyl-(1→3)-6-O-sulfo-β-D-galactopyranosyl-(1→1)-2,3-di-O-tetradecyl-sn-glycerol disodium salt)１２
化合物１１(200 mg, 0.141 mmol)をメタノールと水との混合液(3:1、15 mL)に溶解し、水酸化パラジウム-カーボン(200 mg)を加え、水素ガスで置換し室温で4時間接触還元を行った。反応液をセライトでろ過し、ろ液を留去した。残渣をセファデックスLH-20(CHCl₃:MeOH:H₂O=5:10:3)のカラムにて精製した。さらにDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=1:3:1)による精製を行い、最後にセファデックスLH-20 (CHCl₃:MeOH:H₂O=5:10:3)による再カラム精製を行い、化合物１２(119.6 mg, 80.5%)を得た。
【０１５０】
Rf 0.52(CHCl₃:MeOH:H₂O=12:8:1)
C₄₅H₈₄O₁₉NS₂Na₂ MW : 1053.24
¹H-NMR (DMSO+D₂O)δ：
4.666 (d, 1H, J = 8.3 Hz, H-1b), 4.162 (d, 1H, J = 7.3 Hz, H-1a), 4.067 (b.dd, 1H, H-6b), 4.006-3.924 (b.dd, 1H, H-6a), 1.882 (s, 3H, NHAc), 0.861 (t, 6H, J = 6.8 Hz, 2CH₃)
¹³C-NMR (DMSO+D₂O)δ：
171.32 (Me-CO), 103.49, 102.37 (C-1x2), 65.91, 65.85 (C-6x2)
【０１５１】
【実施例４】
オクチル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩の合成
図６に概略を示す手順により、オクチル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩を合成した。
物質名の後の番号は、図６における化合物の番号を示す。
【０１５２】
(a) オクチル 2,4-ジ-O-アセチル-3,6-ジ-O-アリル-β-D-ガラクトピラノシド(octyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside)１３
オクタノール(300 mg, 2.30 mmol)、シクロペンタジエンハフノニウムダイクロライド(1.75 g, 5.99 mmol)、シルバートリフレート(2.36 g, 12.0 mmol)、モレキュラーシーブ4A(2.3 g)を１,2-ジクロロエタン(5.0 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-15℃に冷却し化合物１(1.2 g, 3.47 mmol)を加え2.5時間攪拌した。反応液をトリエチルアミンを加えて中和し、酢酸エチルで希釈後、セライトでろ過し飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=10:1)にて精製し、化合物１３(0.8 g, 76%)を得た。
【０１５３】

C₂₄H₄₀O₈ MW : 456.572
¹H-NMR (CDCl₃) δ：
5.855 (m, 1H, Allyl), 5.784 (m, 1H, Allyl), 5.472 (d, 1H, J = 3.4 Hz, H-4), 5.086 (dd, 1H, J = 7.8, 9.8 Hz, H-2), 4.394 (d, 1H, J = 7.8 Hz, H-1), 2.125, 2.066 (2s, 6H, 2Ac), 0.879 (t, 3H, J = 6.8 Hz, CH₃)
【０１５４】
(b) オクチル 3,6-ジ-O-アリル-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(octyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside)１５
化合物１３ (0.8 g, 1.75 mmol)をメタノールとテトラヒドロフランとの混合液(1:1、5 mL)に溶解し、1N-水酸化ナトリウム溶液(1.0 mL)を加え室温で1日間攪拌後、その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=5:2)にて精製し、化合物１４(585 g, 89.7%)を得た。
[Rf 0.32(トルエン:AcOEt=2:1)]
【０１５５】
続いて、化合物１４(585 mg, 1.57 mmol)、N,N-ジメチルホルムアミド(3 ml)に溶解し、アルゴンガス気流下-15℃で、水素化ナトリウム(451 mg, 10.2 mmol)を加え攪拌した。続いてベンジルブロマイド(1.22 ml, 10.2 mmol)を加え、徐々に室温にしながら3時間攪拌した。反応液にメタノールを加え中和し、酢酸エチルで希釈後、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:AcOEt=15:1)にて精製し、化合物１５(811 mg, 93.5%)を得た。
【０１５６】
Rf 0.79(ヘキサン:AcOEt=6:1)
C₃₄H₄₈O₆ MW : 552.747
¹H-NMR (CDCl₃) δ：
5.947 (m, 1H, Allyl), 5.849 (m, 1H, Allyl), 4.959 (d, 1H, J = 11.7 Hz, Bn), 4.915 (d, 1H, J = 10.7 Hz, Bn), 4.765 (d, 1H, J = 10.7 Hz, Bn), 4.660 (d, 1H, J = 11.7 Hz, Bn), 4.346 (d, 1H, J = 7.6 Hz, H-1), 0.887 (t, 3H, J = 6.4 Hz, CH₃)
【０１５７】
(c) オクチル 2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(octyl 2,4-di-O-benzyl-β-D-galactopyranoside)１６
イリジウムコンプレックス (1,5-シクロオクタジエンビス(メチルジフェニルホスフィン)イリジウムヘキサフルオロホスフェート) (172 mg, 0.15 mmol) をテトラヒドロフラン(5 mL)に懸濁し、H₂気流下攪拌し活性化させた。その溶液に化合物１５(811 mg, 1.47 mmol)をテトラヒドロフラン(5 mL)に溶解させて加え、アルゴンガス気流下室温で1時間攪拌後、ヨウ素(745 mg)、水(38 mL)及びテトラヒドロフラン(15 mL)を加えさらに1時間室温で攪拌した。反応液をクロロホルムで希釈後、飽和チオ硫酸ナトリウム溶液、飽和重曹水、飽和食塩水で順次洗浄した。クロロホルム層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー(トルエン:EtOAc=5:2) にて精製し、化合物１６を(589 mg, 82.1%)得た。
【０１５８】
Rf 0.35(トルエン:AcOEt=2:1)
C₂₈H₄₀O₆ MW : 472.618
¹H-NMR (CDCl₃) δ：
5.000 (d, 1H, J = 11.7 Hz, Bn), 4.843 (d, 1H, J = 11.7 Hz, Bn), 4.674 (d, 1H, J = 11.7 Hz, Bn), 4.662 (d, 1H, J = 11.7 Hz, Bn), 4.360 (d, 1H, J = 7.3 Hz, H-1), 3.776 (d, 1H, J = 2.0 Hz, H-4), 0.868 (t, 3H, J = 6.8 Hz, CH₃)
【０１５９】
(d) オクチル 2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(octyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)１７
化合物１６(569 mg, 1.20 mmol)をピリジン(17 mL)を加えて溶解させ、その溶液にピバロイルクロライド(188μL, 1.57 mmol)を加え-5℃にて1時間攪拌後、さらにピバロイルクロライド(188μL, 1.57mmol)を加え-5℃にて1時間攪拌した。反応液を、酢酸エチルで希釈後、セライトでろ過し飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=10:1)にて精製し、化合物１７(648 mg, 96.7%)を得た。
【０１６０】
Rf 0.56(トルエン:AcOEt=6:1)
C₃₃H₄₈O₇ MW : 556.735
¹H-NMR (CDCl₃) δ：
4.986 (d, 1H, J = 11.2 Hz, Bn), 4.838 (d, 1H, J = 11.7 Hz, Bn), 4.664 (d, 1H, J = 11.7 Hz, Bn), 4.653 (d, 1H, J = 11.7 Hz, Bn), 4.332 (d, 1H, J = 7.3 Hz, H-1), 4.316 (dd, 1H, J = 6.8, 11.7 Hz, H-6), 4.113 (dd, 1H, J = 6.7, 11.0 Hz, H-6'), 3.772 (d, 1H, J = 2.4 Hz, H-4), 1.180 (s, 9H, piv), 0.867 (t, 3H, J = 6.8 Hz, CH₃)
【０１６１】
(e) オクチル 3-O-アセチル-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(octyl 3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)１８
化合物１７(10 mg, 18.0μmol)をピリジン(1 mL)を加えて溶解させ、その溶液に無水酢酸(0.5 mL)を加え室温で2時間攪拌した。溶媒をトルエンで共沸後、残渣をセファデックスLH-20(CHCl₃:MeOH=1:2)にて精製し、化合物１８(11 mg, qu.)を得た。
【０１６２】
Rf 0.58(トルエン:AcOEt=8:1)
C₃₅H₅₀O₈ MW : 598.772
¹H-NMR (CDCl₃) δ：
4.903 (dd, 1H, J = 3.2, 10.0 Hz, H-3), 4.883 (d, 1H, J = 11.7 Hz, Bn), 4.645 (d, 1H, J = 11.7 Hz, Bn), 4.638 (d, 1H, J = 11.7 Hz, Bn), 4.545 (d, 1H, J = 11.2 Hz, Bn), 4.405 (d, 1H, J = 7.3 Hz, H-1), 4.311 (dd, 1H, J = 6.8, 10.8 Hz, H-6), 4.087 (dd, 1H, J = 6.8, 10.7 Hz, H-6'), 3.847 (d, 1H, J = 2.9 Hz, H-4), 3.769 (dd, 1H, J = 7.8, 10.3 Hz, H-2), 1.937 (s, 3H, Ac), 1.188 (s, 9H, piv), 0.870 (t, 3H, J = 6.8 Hz, CH₃)
【０１６３】
(f) オクチル 3,4-ジ-O-ベンジル-2-デオキシ-6-O-レブロイル-2-フタルイミド-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(octyl 3,4-di-O-benzyl-2-deoxy-6-O-levloyl-2-phtalimido-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)１９
化合物１７(624 mg, 1.12 mmol)、シクロペンタジエンハフノニウムダイクロライド(850 mg, 2.91 mmol)、シルバートリフレート(1.15 g, 5.82 mmol)、モレキュラーシーブ4A(3.2 g)を１,2-ジクロロエタン(10 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-15℃に冷却し 化合物２(838 mg, 1.46 mmol)を加え1時間攪拌した。反応液をトリエチルアミンを加え中和し、酢酸エチルで希釈後、セライトでろ過し飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=10:1)にて精製し、化合物１９(1.03 g, 82.9%)を得た。
【０１６４】
Rf 0.47(トルエン:AcOEt=6:1)
C₆₆H₇₉O₁₅N MW : 1126.342
¹H-NMR (CDCl₃) δ：
5.475 (d, 1H, J = 8.3 Hz, H-1b), 4.189 (d, 1H, J = 7.8 Hz, H-1a), 3.874 (d, 1H, J = 2.4 Hz, H-4a), 2.133 (s, 3H, CH₃), 1.153 (s, 9H, piv), 0.826 (t, 3H, J = 7.1 Hz, CH₃)
【０１６５】
(g) オクチル 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-β-D-galactopyranoside)２０
化合物１９(1.0 g, 0.899 mmol)をエタノール(45 mL)に懸濁し、ヒドラジン水和物(4.5 mL)を加え、110℃で18時間攪拌した。溶媒を留去し、得られたアミノ体をピリジン (5.0 mL)に溶解し、無水酢酸(4.0 mL)を加え室温で17時間攪拌後、溶媒を留去した。残渣をメタノールとテトラヒドロフランとの混合液(1:1、20.0 mL)に溶解し、ナトリウムメトキシド(146 mg, 2.7 mmol)を加え、60℃で1時間攪拌した。その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:アセトン=2.3:1)にて精製し、さらにセファデックスLH-20(CHCl₃:MeOH=2:3)による精製を行い、化合物２０(739 mg, 96%)を得た。
【０１６６】
Rf 0.49(トルエン:アセトン=3:2)
C₅₀H₆₅O₁₁N MW : 856.06
¹H-NMR (CDCl₃) δ：
4.840 (d, 1H, J = 8.3 Hz, H-1b), 4.329 (d, 1H, J = 6.8 Hz, H-1a), 1.515 (s, 3H, NHAc), 0.848 (t, 3H, J = 6.8 Hz, CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
173.17 (Me-CO), 105.32, 104.01 (C-1x2), 62.82, 62.55 (C-6x2)
【０１６７】
(h) オクチル 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩(octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt)２１
化合物２０(150 mg, 0.175 mmol)をN,N-ジメチルホルムアミド(1.5 mL)に溶解し、(C₂H₅)₃NSO₃(319 mg, 1.75 mmol)を加え、50℃で0.5時間攪拌した。その反応液を直接セファデックスLH-20(CHCl₃:MeOH=2:3)にて精製した。溶出液の溶媒をある程度留去し、残渣に水(2.0 mL)、Dowex-50 (Na⁺)タイプを加え、一昼夜攪拌し、セライトでろ過後、ろ液を留去した。残渣をDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=5:10:3)にて精製し、化合物２１(185 mg, 99.6%)を得た。
【０１６８】
Rf 0.23(CHCl₃:MeOH=6:1)
C₅₀H₆₃O₁₇NS₂Na₂ MW : 1060.134
¹H-NMR (CDCl₃+CD₃OD) δ：
4.382 (d, 1H, J = 7.3 Hz, H-1a), 1.634 (s, 3H, NHAc), 0.854 (t, 3H, J = 6.8 Hz, CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
173.03 (Me-CO), 105.10, 103.99 (C-1x2), 68.18 (C-6x2)
【０１６９】
(i) オクチル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩(octyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl-(1→3)-6-O-sulfo-β-D-galactopyranoside disodium salt)２２
化合物２１(170 mg, 0.160 mmol)をメタノールと水との混合液(3:1、15 mL)に溶解し、水酸化パラジウム-カーボン(180 mg)を加え、水素ガスで置換し室温で4時間接触還元を行った。反応液をセライトでろ過し、ろ液を留去した。残渣をセファデックスLH-20 (CHCl₃:MeOH:H₂O=5:10:3)のカラムにて精製した。さらにDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=1:3:1)による精製を行い、最後にセファデックスLH-20 (CHCl₃:MeOH:H₂O=5:10:3)による再カラム精製を行い、化合物２２(101.4 mg, 90.6%)を得た。
【０１７０】
Rf 0.30(CHCl₃:MeOH:H₂O=12:6:1)
C₂₂H₃₉O₁₇NS₂Na₂ MW : 699.636
¹H-NMR (DMSO+D₂O)δ：
4.655 (d, 1H, J = 8.3 Hz, H-1b), 4.136 (d, 1H, J = 7.8 Hz, H-1a), 4.053 (dd, 1H, J = 2.0, 11.7 Hz, H-6b), 3.956-3.851 (b.dd, 1H, H-6a), 1.869 (s, 3H, NHAc), 0.861 (t, 3H, J = 7.1 Hz, CH₃)
¹³C-NMR (DMSO+D₂O)δ：
171.19 (Me-CO), 102.98, 102.30 (C-1x2), 66.09, 65.87 (C-6x2)
【０１７１】
【実施例５】
コレスタニル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩、およびコレスタニル 2-アセトアミド-2-デオキシ-β-D-グルコピラノシル-(1→3)-β-D-ガラクトピラノシドの合成
図７に概略を示す手順により、コレスタニル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩、およびコレスタニル 2-アセトアミド-2-デオキシ-β-D-グルコピラノシル-(1→3)-β-D-ガラクトピラノシドを合成した。
物質名の後の番号は、図７における化合物の番号を示す。
【０１７２】
(a) コレスタニル 2,4-ジ-O-アセチル-3,6-ジ-O-アリル-β-D-ガラクトピラノシド(cholestanyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside)２３
コレスタノール(700 mg, 1.80 mmol)、シクロペンタジエンハフノニウムダイクロライド(1.37 g, 4.68 mmol)、シルバートリフレート(1.85 g, 9.37 mmol)、モレキュラーシーブ4A(2.7 g)を１,2-ジクロロエタン(7.0 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-10℃に冷却し、化合物１(936 mg, 2.70 mmol)を加え2.5時間攪拌した。反応液を、トリエチルアミンを加えて中和し、酢酸エチルで希釈後、セライトでろ過し、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=10:1)にて精製し、化合物２３(970 mg, 75.3%)を得た。
【０１７３】
Rf 0.57(トルエン:AcOEt=6:1)
C₄₃H₇₀O₈ MW : 715.018
¹H-NMR (CDCl₃) δ：
5.880 (m, 1H, Allyl), 5.802 (m, 1H, Allyl), 5.480 (d, 1H, J = 3.4 Hz, H-4), 5.072 (dd, 1H, J = 8.3, 10.3 Hz, H-2), 4.510 (d, 1H, J = 8.3 Hz, H-1), 2.150, 2.095 (2s, 6H, 2Ac), 0.922 (d, 3H, J = 6.4 Hz, CH₃), 0.890 (d, 3H, J = 6.8 Hz, CH₃), 0.885 (d, 3H, J = 6.4 Hz, CH₃), 0.800, 0.667 (2s, 6H, 2CH₃)
【０１７４】
(b) コレスタニル 3,6-ジ-O-アリル-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(cholestanyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside)２５
化合物２３(0.98 g, 1.37 mmol)をメタノールとテトラヒドロフランとの混合液 (1:1、10 mL)に溶解し、1 N 水酸化ナトリウム溶液(1.0 mL)を加え室温で1日間攪拌後、さらにナトリウムメトキサイド (74 mg, 1.37 mmol) を加え室温で2時間攪拌した。その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=5:2)にて精製し、化合物２４(0.83 g, 96%)を得た。
[Rf 0.33(トルエン:AcOEt=2:1)]
【０１７５】
続いて、化合物２４(838 mg, 1.32 mmol)を、N,N-ジメチルホルムアミド(8 ml)に溶解し、アルゴンガス気流下0℃で、水素化ナトリウム(378 mg, 8.58 mmol)を加え攪拌した。続いてベンジルブロマイド(1.02 ml, 8.58 mmol)を加え、徐々に室温にしながら3時間攪拌した。さらに水素化ナトリウム(378 mg, 8.58 mmol)及びベンジルブロマイド(1.02 ml, 8.58 mmol)を加え、18時間攪拌した。反応液にメタノールを加え中和し、酢酸エチルで希釈後、飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:AcOEt=13:1)にて精製し、化合物２５(0.94 g, 88.1%)を得た。
【０１７６】
Rf 0.72(ヘキサン:AcOEt=6:1)
C₅₃H₇₈O₆ MW : 811.193
¹H-NMR (CDCl₃) δ：
5.935 (m, 1H, Allyl), 5.836 (m, 1H, Allyl), 4.931 (d, 1H, J = 11.7 Hz, Bn), 4.903 (d, 1H, J = 10.7 Hz, Bn), 4.739 (d, 1H, J = 10.7 Hz, Bn), 4.639 (d, 1H, J = 11.7 Hz, Bn), 4.438 (d, 1H, J = 7.8 Hz, H-1), 3.829 (d, 1H, J = 2.9 Hz, H-4), 3.719 (dd, 1H, J = 7.8, 9.8 Hz, H-2), 3.397 (dd, 1H, J = 2.9, 9.8 Hz, H-3), 0.893 (d, 3H, J = 6.4 Hz, CH₃), 0.862 (d, 3H, J = 6.8 Hz, CH₃), 0.858 (d, 3H, J = 6.4 Hz, CH₃), 0.799, 0.641 (2s, 6H, 2CH₃)
【０１７７】
(c) コレスタニル 2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(cholestanyl 2,4-di-O-benzyl-β-D-galactopyranoside)２６
イリジウムコンプレックス (1,5-シクロオクタジエンビス(メチルジフェニルホスフィン)イリジウムヘキサフルオロホスフェート) (136 mg, 0.12 mmol) をテトラヒドロフラン(5 mL)に懸濁し、H₂気流下攪拌し活性化させた。その溶液に、化合物２５(940 mg, 1.16 mmol)をテトラヒドロフラン(5 mL)に溶解させて加え、アルゴンガス気流下室温で1時間攪拌後、ヨウ素(588 mg)、水(30 mL)及びテトラヒドロフラン(15 mL)を加えさらに1.5時間室温で攪拌した。反応液をクロロホルムで希釈後、飽和チオ硫酸ナトリウム溶液、飽和重曹水、飽和食塩水で順次洗浄した。クロロホルム層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー(トルエン:アセトン=6:1)にて精製し、化合物２６を(748 mg, 88.2%)得た。
【０１７８】
Rf 0.49(トルエン:アセトン=4:1)
C₄₇H₇₀O₆ MW : 731.064
¹H-NMR (CDCl₃) δ：
5.006 (d, 1H, J = 11.2 Hz, Bn), 4.823 (d, 1H, J = 11.2 Hz, Bn), 4.678 (d, 1H, J = 10.7 Hz, Bn), 4.650 (d, 1H, J = 11.7 Hz, Bn), 4.476 (d, 1H, J = 6.8 Hz, H-1)
【０１７９】
(d) コレスタニル 2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(cholestanyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)２７
化合物２６(647 mg, 0.885 mmol)をピリジン(13 mL)に溶解させ、その溶液にピバロイルクロライド(138μL, 1.15 mmol)を加え-5℃から0℃にて0.5時間攪拌後、さらにピバロイルクロライド(138μL, 1.15 mmol)を加え-5℃から0℃にて1時間攪拌した。反応液を、酢酸エチルで希釈後、セライトでろ過し飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー (トルエン:AcOEt=10:1)にて精製し、化合物２７(663 mg, 91.9%)を得た。
【０１８０】
Rf 0.56(トルエン:AcOEt=6:1)
C₅₂H₇₈O₇ MW : 815.181
¹H-NMR (CDCl₃) δ：
4.993 (d, 1H, J = 11.7 Hz, Bn), 4.821 (d, 1H, J = 11.2 Hz, Bn), 4.667 (d, 1H, J = 11.2 Hz, Bn), 4.642 (d, 1H, J = 11.7 Hz, Bn), 4.450 (d, 1H, J = 7.3 Hz, H-1), 4.293 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.083 (dd, 1H, J = 6.3, 10.8 Hz, H-6'), 3.746 (d, 1H, J = 2.0 Hz, H-4), 1.176 (s, 9H, piv)
【０１８１】
(e) コレスタニル 3-O-アセチル-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(cholestanyl 3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)２８
化合物２７(10 mg, 12.3μmol)をピリジン(1 mL)に溶解させ、その溶液に無水酢酸(0.5 mL)を加え室温で1時間攪拌した。溶媒をトルエンで共沸後、残渣をセファデックスLH-20(CHCl₃:MeOH=1:2)にて精製し、化合物２８(9 mg, 85.4%)を得た。
【０１８２】
Rf 0.65(トルエン:AcOEt=8:1)
C₅₄H₈₀O₈ MW : 857.218
¹H-NMR (CDCl₃) δ：
4.889 (dd, 1H, J = 2.7, 10.5 Hz, H-3), 4.885 (d, 1H, J = 11.2 Hz, Bn), 4.647 (d, 1H, J = 11.7 Hz, Bn), 4.625 (d, 1H, J = 11.2 Hz, Bn), 4.533 (d, 1H, J = 11.7 Hz, Bn), 4.518 (d, 1H, J = 7.3 Hz, H-1), 4.289 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.061 (dd, 1H, J = 6.3, 11.2 Hz, H-6'), 3.766 (d, 1H, J = 2.4 Hz, H-4), 3.753 (dd, 1H, J = 7.3, 10.3 Hz, H-2), 1.924 (s, 3H, Ac), 1.185 (s, 9H, piv)
【０１８３】
(f) コレスタニル 3,4-ジ-O-ベンジル-2-デオキシ-6-O-レブロイル-2-フタルイミド-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-ピバロイル-β-D-ガラクトピラノシド(cholestanyl 3,4-di-O-benzyl-2-deoxy-6-O-levloyl-2-phtalimido-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside)２９
化合物２７(737 mg, 0.904 mmol)、シクロペンタジエンハフノニウムダイクロライド(686 mg, 2.35 mmol)、シルバートリフレート(927 mg, 4.70 mmol)及びモレキュラーシーブ4A(2.5 g)を１,2-ジクロロエタン(10 ml)に懸濁し、アルゴンガス気流下室温で攪拌後、-15℃に冷却し 化合物２(676.5 mg, 1.18 mmol)を加え1時間攪拌した。反応液を、トリエチルアミンを加えて中和し、酢酸エチルで希釈後、セライトでろ過し飽和重曹水、飽和食塩水で順次洗浄した。酢酸エチル層を硫酸マグネシウムにて乾燥後、減圧下溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:AcOEt=10:1)にて精製し、化合物２９(982.1 mg, 79.3%)を得た。
【０１８４】
Rf 0.58(トルエン:AcOEt=6:1)
0.07(ヘキサン:AcOEt=6:1)
C₈₅H₁₀₉O₁₅N MW : 1384.788
¹H-NMR (CDCl₃) δ：
5.475 (d, 1H, J = 8.3 Hz, H-1b), 4.453 (d, 1H, J = 7.8 Hz, H-1a), 3.852 (d, 1H, J = 2.9 Hz, H-4a), 2.133 (s, 3H, CH₃), 1.146 (s, 9H, piv)
【０１８５】
(g) コレスタニル 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-β-D-ガラクトピラノシド(cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-β-D-galactopyranoside)３０
化合物２９(668 mg, 0.488 mmol)をエタノール(24.5 mL)に懸濁し、ヒドラジン水和物(2.45 mL)を加え、110℃で18時間攪拌した。溶媒を留去し、得られたアミノ体をピリジン (5.0 mL)に溶解し、無水酢酸(4.0 mL)を加え室温で17時間攪拌後、溶媒を留去した。残渣をメタノールとテトラヒドロフランとの混合液(1:1、10.0 mL)に溶解し、ナトリウムメトキシド(78.8 mg, 1.46 mmol)を加え、60℃で1時間攪拌した。その反応液をアンバーリスト15E (H⁺)タイプで中和し、セライトでろ過後、ろ液を留去した。残渣をシリカゲルカラムクロマトグラフィー(トルエン:アセトン=3:1)にて精製し、さらにセファデックスLH-20(CHCl₃:MeOH=2:3)による精製を行い、化合物３０(534.2 mg, 99.6%)を得た。
【０１８６】
Rf 0.34(トルエン:アセトン=3:1)
C₆₉H₉₅O₁₁N MW : 1114.506
¹H-NMR (CDCl₃) δ：
5.044 (d, 1H, J = 12.2 Hz, NH), 4.875 (d, 1H, J = 11.2 Hz, Bn), 4.851 (d, 1H, J = 7.8 Hz, H-1b), 4.809 (d, 1H, J = 11.2 Hz, Bn), 4.740 (d, 1H, J = 11.7 Hz, Bn), 4.696 (d, 1H, J = 11.7 Hz, Bn), 4.672 (d, 1H, J = 12.2 Hz, Bn), 4.642 (d, 1H, J = 10.7 Hz, Bn), 4.571 (d, 1H, J = 11.2 Hz, Bn), 4.562 (d, 1H, J = 12.2 Hz, Bn), 4.439 (d, 1H, J = 6.4 Hz, H-1a), 1.524 (s, 3H, NHAc), 0.892 (d, 3H, J = 6.3 Hz, CH₃), 0.860 (d, 3H, J = 6.8 Hz, CH₃), 0.856 (d, 3H, J = 6.8 Hz, CH₃), 0.760, 0.635 (2s, 6H, 2CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
173.00 (Me-CO), 103.79, 103.06 (C-1x2), 62.77, 62.40 (C-6x2)
【０１８７】
(h) コレスタニル 2-アセトアミド-3,4-ジ-O-ベンジル-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-2,4-ジ-O-ベンジル-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩(cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt)３１
化合物３０(150 mg, 0.136 mmol)をN,N-ジメチルホルムアミド(1.5 mL)に溶解し、(C₂H₅)₃NSO₃(247 mg, 1.36 mmol)を加え、50℃で0.5時間攪拌した。その反応液を直接セファデックスLH-20(CHCl₃:MeOH=2:3)にて精製した。溶出液の溶媒をある程度留去し、残渣に水(2.0 mL)、Dowex-50 (Na⁺)タイプを加え、一昼夜攪拌し、セライトでろ過後、ろ液を留去した。残渣をDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=5:10:3)にて精製し、化合物３１(175 mg, 97.3%)を得た。
【０１８８】
Rf 0.22(CHCl₃:MeOH=8:1)
C₆₉H₉₃O₁₇NS₂Na₂ MW : 1318.58
¹H-NMR (CD₃OD) δ：
4.523 (d, 1H, J = 7.3 Hz, H-1a), 4.384 (dd, 1H, J = 2.0, 10.8 Hz, H-6b), 4.290 (dd, 1H, J = 4.4, 10.7 Hz, H-6a), 1.645 (s, 3H, NHAc), 0.910 (d, 3H, J = 6.8 Hz, CH₃), 0.873 (d, 3H, J = 6.4 Hz, CH₃), 0.868 (d, 3H, J = 6.8 Hz, CH₃), 0.730, 0.658 (2s, 6H, 2CH₃)
¹³C-NMR (CDCl₃+CD₃OD) δ：
102.97, 102.55 (C-1x2), 68.47, 67.22 (C-6x2)
【０１８９】
(i) コレスタニル 2-アセトアミド-2-デオキシ-6-O-スルホ-β-D-グルコピラノシル-(1→3)-6-O-スルホ-β-D-ガラクトピラノシド二ナトリウム塩(cholestanyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl-(1→3)-6-O-sulfo-β-D-galactopyranoside disodium salt)３２
化合物３１(170 mg, 0.129 mmol)をメタノールと水との混合液(3:1、15 mL)に溶解し、水酸化パラジウム-カーボン(180 mg)を加え、水素ガスで置換し室温で4時間接触還元を行った。反応液をセライトでろ過し、ろ液を留去した。残渣をセファデックスLH-20 (CHCl₃:MeOH:H₂O=5:10:3)のカラムにて精製した。さらにDowex-50 (Na⁺)タイプのカラム(CHCl₃:MeOH:H₂O=1:3:1)による精製を行い、最後にセファデックスLH-20 (CHCl₃:MeOH:H₂O=5:10:3)による再カラム精製を行い、化合物３２(109.4 mg, 88.6%)を得た。
【０１９０】
Rf 0.47(CHCl₃:MeOH:H₂O=12:8:1)
C₄₁H₆₉O₁₇NS₂Na₂ MW : 958.08
¹H-NMR (CDCl₃) δ：
4.727 (d, 1H, J = 7.8 Hz, H-1b), 4.310 (d, 1H, J = 7.8 Hz, H-1a), 4.093 (b.dd, 1H, H-6b), 4.010-3.924 (b.dd, 1H, H-6a), 1.926 (s, 3H, NHAc), 0.945 (d, 3H, J = 6.4 Hz, CH₃), 0.909 (d, 3H, J = 6.4 Hz, CH₃), 0.905 (d, 3H, J = 6.8 Hz, CH₃), 0.827, 0.689 (2s, 6H, 2CH₃)
¹³C-NMR (DMSO+D₂O)δ：
170.88 (Me-CO), 102.21, 100.73 (C-1x2), 65.85, 65.65 (C-6x2)
【０１９１】
(j) コレスタニル 2-アセトアミド-2-デオキシ-β-D-グルコピラノシル-(1→3)-β-D-ガラクトピラノシド(cholestanyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→3)-β-D-galactopyranoside)３３
化合物３０(230 mg, 0.209 mmol)をメタノールと水と酢酸エチルとの混合液(4:1:1、25 mL)に溶解し、水酸化パラジウム-カーボン(230 mg)を加え、水素ガスで置換し室温で20時間接触還元を行った。反応液をセライトでろ過し、ろ液を留去した。残渣をセファデックスLH-20 (CHCl₃:MeOH=1:1)のカラムにて精製し、化合物３３(126.1 mg, 81.5%)を得た。
【０１９２】
Rf 0.51(CHCl₃:MeOH=3:1)
C₄₁H₇₁O₁₁N MW : 754.008
¹H-NMR (C₅D₅N+D₂O)δ：
5.383 (d, 1H, J = 8.3 Hz, H-1b), 4.825 (d, 1H, J = 7.3 Hz, H-1a), 2.059 (s, 3H, NHAc)
【０１９３】
【実施例６】
安全性試験および薬効薬理試験
本実施例においてはケラタン硫酸オリゴ糖として、Ｌ４のナトリウム塩、２つのＬ４がβ１−３結合により結合したケラタン硫酸４糖（Ｌ４Ｌ４）のナトリウム塩、Ｋ４のナトリウム塩（実施例１の化合物２８）、Ｋ２のナトリウム塩、及びＧ４Ｌ４のナトリウム塩を使用した（略号により示すオリゴ糖の構造については図８参照）。Ｌ４およびＬ４Ｌ４は、国際公開パンフレット第ＷＯ９６／１６９７３号に記載の方法により得た。
【０１９４】
なおＫ２のナトリウム塩は、以下の方法により得た。
牛角膜由来のケラタン硫酸１０ｇを１２０ｍｌの０．１Ｍトリス塩酸緩衝液(pH 7.5)に溶解した。この液にシュードモナス(Pseudomonas sp.)由来ケラタナーゼ(生化学工業株式会社製)を１，０００ユニット加えて３７℃で５０時間分解を行った。反応終了後、１．３倍量のエタノールを加えて攪拌し、室温で一晩放置した。翌日、遠心分離（10,000rpm、２０分）により上清と沈殿を分離し、上清を減圧濃縮し、濃縮液を凍結乾燥して、乾燥物９ｇを得た。得られた凍結乾燥物を少量の蒸留水に溶解し、セルロファインＧＣＬ−９０ｍ(チッソ株式会社製)(4.5cmｘ125cm)を用い、食塩濃度０．２Ｍ液を溶出溶媒としてゲルクロマトグラフィーを行い、Ｋ２を含む画分を分取した。得られたＫ２画分を減圧濃縮し、セルロファインＧＣＬ−２５ｍ(チッソ株式会社製)(4.0cmｘ120cm)を用い蒸留水を溶出溶媒とし、ゲル濾過クロマトグラフィーにより脱塩し、凍結乾燥した。
【０１９５】
このＫ２を含む画分を少量の蒸留水に溶解し、予め蒸留水で平衡化したムロマック 1x4(200-400)(室町化学工業(株)製)(2.0cmｘ32cm)を用い、溶出溶媒に食塩を用い、食塩濃度を直線的に０から２Ｍに上昇させ、さらに精製したＫ２画分を分離溶出させた。得られたＫ２画分を減圧濃縮後、セルロファインＧＣＬ−２５ｍ(4.0cmｘ120cm)を用いたゲル濾過クロマトグラフィーにより脱塩し、凍結乾燥し、Ｋ２の乾燥物を１．９ｇを得た。
【０１９６】
（１）安全性試験
１．マウスを用いた単回投与毒性試験
正常マウス（１群５匹）にＫ４またはＧ４Ｌ４を２，０００ｍｇ／ｋｇの用量で単回静脈内投与し、１４日間一般症状を観察した。
【０１９７】
雌雄各群に死亡例は認められなかった。Ｋ４またはＧ４Ｌ４投与により雌雄の全例に投与直後から麻痺性歩行がみられたが、投与後約３分には全例が正常に回復した。体重では投与翌日に雄の投与群に軽微な体重減少がみられたが、その後は全例とも順調な体重増加を示した。剖検では全例ともＫ４またはＧ４Ｌ４投与に起因した異常は認められなかった。以上の結果から、Ｋ４およびＧ４Ｌ４の単回静脈内投与における最小死亡用量は２，０００ｍｇ／ｋｇ以上であると考えられる。従って、Ｋ４およびＧ４Ｌ４の単回静脈内投与におけるＬＤ₅₀値は、雌雄ともに２，０００ｍｇ／ｋｇを超えるものである。
【０１９８】
２．モルモットを用いた抗原性試験
モルモットの背部皮下にＫ４またはＧ４Ｌ４を単独、あるいは免疫補助剤であるフロイント完全アジュバント（ＦＣＡ）との乳化物を３回注射して感作した。最終感作から１２日後にＫ４またはＧ４Ｌ４を静脈内投与して能動的全身性アナフィラキシー反応を惹起した。また、陽性対照として卵白アルブミン（ＯＶＡ）についても同様に試験した。
【０１９９】
以上の結果を投与量とともに表１に示した。Ｋ４またはＧ４Ｌ４で惹起したモルモットにアナフィラキシー反応は認められなかった。また試験系の陽性対照である卵白アルブミンではアナフィラキシー反応が認められた。以上の結果より、Ｋ４およびＧ４Ｌ４はモルモットにおける能動的全身性アナフィラキシー反応を誘発しないと考えられる。
【０２００】
【表１】

【０２０１】
（２）薬効薬理試験
１．Ｃａ-イオノフォアーで誘発される血管透過性亢進に対する効果の検討
エーテル麻酔下でラットの背部を剃毛後、２％ＤＭＳＯに溶解したＣａ-イオノフォアー（A23187、和光純薬）溶液（１０μｇ／ｍｌ）０.１ｍｌを皮内の１カ所に投与した。陰性対照(control)として２％ＤＭＳＯ溶液０.１ｍｌを皮内の１カ所に投与した。被験物質は、Ｃａ-イオノフォアー溶液に溶解し、その０.１ｍｌを皮内の数カ所に投与した。直後に０.５％エバンスブルー１ｍｌを静脈内投与し、その３０分後にエーテル麻酔下で放血致死した。皮膚を剥離し、エバンスブルー漏出部位をトレパンで打ち抜き、これを色素漏出量の測定に供した。色素漏出量は Katayamaらの方法（Microbiol. Immunol., 22, 89-101 (1978)）に従って行った。すなわち採取した皮膚に１ＮＫＯＨを加えて一晩加水分解し、その後０.６ＮＨ₃ＰＯ₄含有アセトン溶液（５:１３の割合で混合）を加えて中和し遠心分離後、上清の吸光度（６２０ｎｍ）から色素漏出量を求めた。
【０２０２】
Ｌ４、Ｌ４Ｌ４、Ｋ４およびＫ２を被験物質としたときの結果を図９に示す。またＬ４およびＧ４Ｌ４を被験物質としたときの結果を図１０に示す。
【０２０３】
Ｌ４Ｌ４、Ｋ４およびＫ２は用量依存的に血管透過性の亢進を抑制し、それぞれ２.５〜１０ｍｇ／部位(site)、０.６３〜５ｍｇ／site、１.２５〜５ｍｇ／siteの濃度で有意な抑制効果を示した。抑制効果はＫ４が最も強く、次いでＫ２、Ｌ４Ｌ４の順であった。また、Ｇ４Ｌ４も血管透過性亢進の有意な抑制効果を示した。一方、Ｌ４は本モデルにおいて血管透過性の亢進をほとんど抑制しなかった。この結果から、Ｋ４、Ｋ２、Ｌ４Ｌ４およびＧ４Ｌ４は、血管透過性の亢進を抑制することにより抗アレルギー作用を発揮することが示唆される。特に、Ｋ４は優れた作用を発揮することが示唆される。
【０２０４】
本試験において、Ｌ４Ｌ４、Ｋ４、Ｋ２およびＧ４Ｌ４がＣａ-イオノフォアーによる血管透過性の亢進を有意に抑制したことから、その活性発現にはＫ２構造が重要で、さらにＫ４構造ではその作用が増すことが示唆された。またＬ４は抑制効果を示さなかったことから、本モデルではＬ４構造はあまり重要でないことが示唆された。一方、Ｌ４Ｌ４は２つのＬ４構造がβ-（１-３）結合したものだが、Ｋ４構造を有しているため本モデルにおいて抑制効果が認められたものと推察された。Ｌ４Ｌ４、Ｋ４、Ｋ２およびＧ４Ｌ４は膜安定化によるＣａ²⁺の細胞内への流入、もしくは脱顆粒化を阻害したことにより、または肥満細胞から分泌されたヒスタミン等を抑制したことにより、血管透過性の亢進を抑制することが示唆された。
【０２０５】
２．ＦＭＬＰ（N-ホルミル-Met-Leu-Phe）刺激によるモルモット好中球Ｏ₂ ^-産生に対する効果の検討
生理食塩液に溶解したグリコーゲンの０.２％水溶液を高圧蒸気滅菌後、ハートレー系雌性モルモットの腹腔内に２０ｍｌ投与した。１６時間後脱血死させ、ヘパリン１０Ｕ／ｍｌを含む生理食塩液２０ｍｌを腹腔内に注入し、腹腔浸出液を回収した。回収液を卓上遠心機を用いて１０００ｒｐｍで１０分間遠心分離し、沈査に精製水を加えて３０秒間溶血させ、２倍濃度のハンクス液で等張にもどし、１０００ｒｐｍで１０分間遠心分離した。沈査をハンクス液に再懸濁し、遠心分離の操作を２回繰り返し好中球を得た。採取したモルモット好中球をハンクス液に懸濁し、血球測定装置（Sysmex K-2000）を用いて白血球数を測定し、ハンクス液で２×１０⁶個／ｍｌに希釈したものを細胞浮遊液として実験に用いた。
【０２０６】
細胞浮遊液１ｍｌと既知濃度の被験物質の溶液１０μｌ（対照(control)としては、ケラタン硫酸オリゴ糖を加えないものを用いた）とを混和し、３７℃で１時間プレインキュベートした後、１.６ｍＭのチトクロームＣ液５０μｌおよび０.１ｍＭのＦＭＬＰ１００μｌを順次加え混和した。これを３７℃で１０分間インキュベートし、氷冷して反応を停止し、３０００ｒｐｍで５分間遠心分離後、上清の吸光度を波長５５０ｎｍで測定した。なお上記の操作はインキュベートを除いて全て氷冷下で行った。
【０２０７】
Ｌ４、Ｌ４Ｌ４、Ｋ４およびＫ２を被験物質としたときの結果を図１１に示す。またＬ４およびＧ４Ｌ４を被験物質としたときの結果を図１２に示す。
【０２０８】
Ｌ４Ｌ４、Ｋ４およびＧ４Ｌ４は、０.０１〜１ｍｇ／ｍｌの濃度で、ＦＭＬＰ刺激による好中球からのＯ₂ ^-産生(O₂ ^- generation)を顕著に抑制した。１ｍｇ／ｍｌでの抑制率は、それぞれ対照に対して５０.６％、４４.７％および５７.１％であった。Ｌ４とＫ２ではＯ₂ ^-産生抑制効果はほとんど認められなかった。この結果から、Ｌ４Ｌ４、Ｋ４およびＧ４Ｌ４は、好中球の活性酸素の産生を抑制することにより、抗炎症作用を発揮することが示唆される。
【０２０９】
本試験において、Ｌ４とＫ４は電荷、組成式および構成糖が等しいにもかかわらず、Ｋ４で抑制効果が認められ、さらにＬ４Ｌ４が抑制効果を示すのに対して構成糖であるＬ４では活性は認められなかった。Ｌ４Ｌ４、Ｋ４およびＧ４Ｌ４がＦＭＬＰ刺激によるモルモット好中球Ｏ₂ ^-産生を有意に抑制したことから、その活性発現にはＫ４構造が重要であることが示唆された。またＫ２は抑制効果を示さなかったことから、本モデルでは硫酸化の程度が重要であることが示唆された。Ｋ４構造を有するＬ４Ｌ４、Ｋ４およびＧ４Ｌ４はＧＴＰ結合蛋白質あるいはホスホリパーゼＣ等の刺激伝達系を阻害しているか、あるいはＦＭＬＰレセプターに直接拮抗して作用を発現している可能性が示唆された。
【０２１０】
【発明の効果】
本発明によれば、還元末端にガラクトース残基を有するケラタン硫酸オリゴ糖を効率よく製造することができる。特に、ガラクトース残基及び／又はＮ−アセチルグルコサミン残基の６位のヒドロキシル基が硫酸化されたケラタン硫酸オリゴ糖を提供できる。還元末端に、６位のヒドロキシル基が硫酸化されたガラクトース残基を有するケラタン硫酸オリゴ糖等は、優れた薬理効果を有し、安全で有効な新規医薬組成物を提供できる。
【図面の簡単な説明】
【図１】ケラタン硫酸の構造から予想されるケラタン硫酸二糖の構造を示す。
【図２】本発明のオリゴ糖の製造における出発材料の製造方法の一例の概略を示す。
【図３】本発明のオリゴ糖の製造における出発材料の製造方法の一例の概略を示す。
【図４】本発明のオリゴ糖の製造方法の一例の概略を示す。
【図５】本発明のオリゴ糖の製造方法の一例の概略を示す。
【図６】本発明のオリゴ糖の製造方法の一例の概略を示す。
【図７】本発明のオリゴ糖の製造方法の一例の概略を示す。
【図８】ケラタン硫酸オリゴ糖の構造を示す。
【図９】ケラタン硫酸オリゴ糖の血管透過性亢進に対する効果を示す。
【図１０】ケラタン硫酸オリゴ糖の血管透過性亢進に対する効果を示す。
【図１１】ケラタン硫酸オリゴ糖の好中球Ｏ₂ ^-産生に対する効果を示す。
【図１２】ケラタン硫酸オリゴ糖の好中球Ｏ₂ ^-産生に対する効果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an oligosaccharide. The present invention also relates to a novel oligosaccharide and a pharmaceutical composition containing the same.
[0002]
[Prior art]
Keratan sulfate is a glycosaminoglycan having a basic structure of N-acetyllactosamine in which the 6-position of an N-acetylglucosamine residue is O-sulfated. It has been reported that some of these keratan sulfate oligosaccharides, which are decomposed products, have a pharmacological action (see, for example, International Publication Pamphlet No. WO96 / 16973).
[0003]
The structure of the disaccharide derived from keratan sulfate is considered to be the one shown in FIG. 1, but the search for other keratin sulfate oligosaccharides having a pharmacological action is disaccharide (GlcNAcβ1) having a galactose residue at the reducing end. → 3Gal (where Gal is galactose, GlcN is glucosamine, and Ac is an acetyl group)), and it is difficult to obtain a keratan sulfate oligosaccharide having a sulfate group like keratan sulfate. Limited. For example, even when keratan sulfate was treated with a known endo-β-galactosidase, it was difficult to obtain a disaccharide having a galactose residue at the reducing end (GlcNAcβ1 → 3Gal) while retaining the sulfate group.
[0004]
[Problems to be solved by the invention]
The first object of the present invention is to provide a method capable of easily producing a keratan sulfate disaccharide having a galactose residue at the reducing end.
[0005]
The second object of the present invention is to provide a novel oligosaccharide having a pharmacological action and to provide it as a medicine.
[0006]
[Means for Solving the Problems]
The present inventors obtain a disaccharide having a galactose residue at the reducing end by glycoside bond reaction by protecting the hydroxyl group and amino group in glucosamine and the hydroxyl group in galactose in a specific manner. I found out. Moreover, it discovered that the disaccharide by which the hydroxyl group of the specific position was sulfated had the outstanding pharmacological activity. Based on these findings, the present invention has been completed.
[0007]
That is, the present invention is represented by the following general formula (3), which includes at least a step of subjecting a monosaccharide represented by the following general formula (1) and a monosaccharide represented by the following general formula (2) to glycoside bond reaction. An oligosaccharide production method (hereinafter also referred to as the production method of the present invention) is provided.
[0008]
Embedded image

(Wherein R¹And R²Each independently represents an aralkyl group, R^ThreeRepresents an acyl group or a silyl group, R^FourRepresents an amino protecting group, R^FiveRepresents a leaving group. )
[0009]
Embedded image

(Wherein R⁶And R⁸Each independently represents an aralkyl group, R⁷Represents an acyl group or a silyl group, R⁹Is an aralkyl group, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. )
[0010]
Embedded image

(Wherein R^TenAnd R¹¹Each independently represents a hydrogen atom or -SO_ThreeM represents M (M represents a proton or a monovalent cation), and Ac represents an acetyl group. R¹²Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. )
[0011]
In the production method of the present invention, R^TenAnd R¹¹At least one of -SO_ThreeM (M represents a proton or a monovalent cation), and after the step of reacting the monosaccharide represented by the general formula (1) and the monosaccharide represented by the general formula (2) with a glycosidic bond , R^ThreeAnd R⁷At least one of them is replaced by a hydrogen atom, and then the hydrogen atom is -SO_ThreeIt is preferable that the process of substituting for M is included.
[0012]
In a preferred embodiment of the production method of the present invention, the general formulas (1) to (3) are represented by the following formulas (4) to (6), respectively.
[0013]
Embedded image

(In the formula, Bn represents a benzyl group, R¹³Represents an acetyl group or a levulinoyl group, Phth represents a phthaloyl group, and X represents a halogen atom. )
[0014]
Embedded image

(In the formula, Bn represents a benzyl group, R¹⁴Are benzyl group, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. Piv represents a pivaloyl group. )
[0015]
Embedded image

(In the formula, Ac represents an acetyl group, M represents a proton or a monovalent cation.¹⁵Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. )
[0016]
In another preferred embodiment of the production method of the present invention, the general formulas (1) to (3) are represented by the following formulas (7) to (9), respectively.
[0017]
Embedded image

(In the formula, Bn represents a benzyl group, Lev represents a levulinoyl group, Phth represents a phthaloyl group, and X represents a halogen atom.)
[0018]
Embedded image

(Where Bn, R¹⁴And Z are as defined above. Ph represents a phenyl group, and Me represents a methyl group. )
[0019]
Embedded image

(Wherein R¹⁵And Z are as defined above. Ac represents an acetyl group, and M represents a proton or a monovalent cation. )
[0020]
In still another preferred embodiment of the production method of the present invention, the general formulas (1) to (3) are represented by the following formulas (10) to (12), respectively.
[0021]
Embedded image

(In the formula, Bn represents a benzyl group, Lev represents a levulinoyl group, Phth represents a phthaloyl group, and X represents a halogen atom.)
[0022]
Embedded image

(Where Bn, R¹⁴And Z are as defined above. Ph represents a phenyl group, and Me represents a methyl group. )
[0023]
Embedded image

(Wherein R¹⁵And Z are as defined above. Ac represents an acetyl group, and M represents a proton or a monovalent cation. )
[0024]
The present invention also provides an oligosaccharide represented by the following general formula (13) (hereinafter also referred to as the present oligosaccharide).
[0025]
Embedded image

(Wherein R¹⁶And R¹⁷Each independently represents a hydrogen atom or -SO_ThreeM represents M (M represents a proton or a monovalent cation), and Ac represents an acetyl group. Also R¹⁸Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. However, R¹⁶And R¹⁷Are both hydrogen atoms, Z is an oxygen atom and R¹⁸Is a hydrogen atom or a cholestanyl group, and R¹⁶-SO_ThreeM (M represents a proton or a monovalent cation) and Z is an oxygen atom and R¹⁷And R¹⁸Except where is a hydrogen atom. )
[0026]
A preferred embodiment of the oligosaccharide of the present invention is R¹⁶And R¹⁷Are all -SO_ThreeM (M represents a proton or a monovalent cation).
[0027]
Another preferred embodiment of the oligosaccharide of the present invention is R¹⁶Is a hydrogen atom and R¹⁷-SO_ThreeM (M represents a proton or a monovalent cation).
[0028]
In the oligosaccharide of the present invention, preferably R¹⁸Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, O-alkylglycerol residue or cholestanyl group, and Z is an oxygen atom.
[0029]
The present invention further provides a medicament (hereinafter also referred to as the present medicament) comprising the oligosaccharide of the present invention represented by the following general formula (13) or a pharmaceutically acceptable salt thereof as an active ingredient.
[0030]
Embedded image

(Wherein R¹⁶And R¹⁷Each independently represents a hydrogen atom or -SO_ThreeM represents M (M represents a proton or a monovalent cation), and Ac represents an acetyl group. Also R¹⁸Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, biotin residue or peptide residue is shown. Z represents an oxygen atom or -NHCO-. However, R¹⁶And R¹⁷Are both hydrogen atoms, Z is an oxygen atom and R¹⁸Except that is hydrogen atom or cholestanyl group. )
[0031]
In particular, R¹⁶-SO_ThreeM and R¹⁷Is a hydrogen atom or -SO_ThreeThe oligosaccharide of the present invention showing M (M represents a proton or a monovalent cation) or a pharmaceutically acceptable salt thereof is used as an antiallergic agent.¹⁶And R¹⁷-SO_ThreeThe oligosaccharide of the present invention or a pharmaceutically acceptable salt thereof showing M (M represents a proton or a monovalent cation) is useful as an anti-inflammatory agent.
[0032]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described.
First, abbreviations commonly used in this specification and the drawings are shown below together with their meanings (description in parentheses attached to the following abbreviations).
[0033]
Ac (acetyl group)
Bn (benzyl group)
Phth (phthaloyl group)
Piv (pivaloyl group)
Lev (levulinoyl group)
Ph (phenyl group)
Me (methyl group)
All (allyl group)
MP (paramethoxyphenyl group)
M (proton or monovalent cation)
X (halogen atom)
[0034]
<1> Production method of the present invention
The production method of the present invention is a method for producing an oligosaccharide represented by the general formula (3), wherein the monosaccharide represented by the general formula (1) and the monosaccharide represented by the general formula (2) are subjected to a glycosidic bond reaction. It is characterized by including at least.
[0035]
The substituents in the general formulas (1) to (3) are as follows.
R¹, R², R⁶And R⁸Each independently represents an aralkyl group, and examples of the aralkyl group include benzyl group, p-methoxybenzyl group, phenethyl group, 3-phenylpropyl group, p-nitrobenzyl group, o-nitrobenzyl group, p -Halobenzyl group, p-cyanobenzyl group, diphenylmethyl group, triphenylmethyl group (trityl group), α or β-naphthylmethyl group, α-naphthyldiphenylmethyl group and the like can be mentioned. R¹, R², R⁶And R⁸Is preferably a benzyl group.
[0036]
R^ThreeAnd R⁷Each independently represents an acyl group or a silyl group. As the acyl group, acetyl group, pivaloyl group, levulinoyl group, benzoyl group, chloroacetyl group, dichloroacetyl group, trifluoroacetyl group, methoxyacetyl group, propionyl group, n-butyryl group, (E) -2-methylbutenoyl group , Isobutyryl group, pentanoyl group, o- (dibromomethyl) benzoyl group, o- (methoxycarbonyl) benzoyl group, p-phenylbenzoyl group, 2,4,6-trimethylbenzoyl group, p-toluoyl group, p-anisoyl group , P-chlorobenzoyl group, p-nitrobenzoyl group, α-naphthoyl group and the like. Examples of the silyl group include trimethylsilyl group, triethylsilyl group, dimethylisopropylpropylsilyl group, isopropyldimethylsilyl group, methyldi-t-butylsilyl group, t-butyldimethylsilyl group, t-butyldiphenylsilyl group, triisopropylsilyl group, tetra Examples thereof include isopropyldisiloxanyl group.
[0037]
R^ThreeAnd R⁷Are preferably each an acyl group or R^ThreeIs an acyl group and R⁷Is a silyl group. The acyl group is preferably an acetyl group, a pivaloyl group, or a levulinoyl group, and the silyl group is preferably a t-butyldiphenylsilyl group.
[0038]
R^FourRepresents an amino group protecting group, and examples of the amino group protecting group include a phthaloyl group, an acetyl group, and an allyloxycarbonyl group. A phthaloyl group is preferable.
[0039]
R^FiveRepresents a leaving group. Here, the leaving group means a group capable of leaving under a condition in which a monosaccharide represented by the general formula (1) and a monosaccharide represented by the general formula (2) undergo a glycosidic bond reaction. Examples of the leaving group include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, etc.), an imide group, a methylthio group, a phenylthio group, and the like. A halogen atom is preferable, and a fluorine atom is particularly preferable.
[0040]
X in the formulas (4), (7) and (10) is a preferred halogen atom among the leaving groups. X is preferably a fluorine atom as described above.
[0041]
R⁹Are an aralkyl group, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, A phospholipid residue, a biotin residue, or a peptide residue is shown. Here, the residue means a remaining part obtained by removing an atom or a group of atoms involved in binding of the compound from the compound.
[0042]
Examples of the aralkyl group and preferred aralkyl groups are the same as described above.
[0043]
The 6-O-sulfated N-acetylglucosamine residue is usually a residue from which the hydroxyl group at position 4 has been removed.
[0044]
As an alkyl group, a C1-C23 thing is illustrated and a C8-C14 thing is preferable.
[0045]
The glycerol residue is usually a residue from which any hydroxyl group is removed.
[0046]
The O-alkylglycerol residue is not particularly limited, but is usually a residue from which any hydroxyl group is removed, and a di-O-alkylglycerol residue is preferred, and a 2,3-di-O-alkylglycerol residue is preferred. Groups are preferred. Examples of the “alkyl” herein include those having 1 to 23 carbon atoms, and those having 8 to 14 carbon atoms are preferable.
[0047]
The O-acylglycerol residue is not particularly limited, but is usually a residue from which any hydroxyl group is removed, and a di-O-acylglycerol residue is preferred, and a 2,3-di-O-acylglycerol residue is preferred. Groups are preferred. Examples of the “acyl” herein include those having 1 to 23 carbon atoms, and those having 8 to 14 carbon atoms are preferable.
[0048]
The cholesterol residue is usually a residue obtained by removing the C-3 hydroxyl group of the cyclopentaphenanthrene ring.
[0049]
The ceramide residue is usually a residue from which the hydroxyl group at position 1 has been removed. The N-acyl group in the ceramide usually has 1 to 28 carbon atoms, and preferably has 14 to 23 carbon atoms.
[0050]
Examples of phospholipid residues include glycerophospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, etc.) residues and sphingophospholipid (sphingomyelin, etc.) residues.
[0051]
The biotin residue is usually a residue from which a carboxyl group has been removed.
[0052]
Peptide residues are usually residues from which either an amino group or a carboxyl group has been removed.
[0053]
Z represents an oxygen atom or —NHCO—. Which of —NHCO— nitrogen atom and oxygen atom is R⁹It may be a side.
[0054]
R⁹Is an aralkyl group, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue or phosphorus In the case of a lipid residue, Z is preferably an oxygen atom.
[0055]
R⁹When is a biotin residue or a peptide residue, Z is preferably —NHCO—.
[0056]
R^TenAnd R¹¹Each independently represents a hydrogen atom or -SO_ThreeM is shown.
[0057]
R¹²Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus Indicates a lipid residue, biotin residue, or peptide residue. These groups and residues are R⁹Is the same as described above.
[0058]
R¹²Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue or phosphorus In the case of a lipid residue, Z is preferably an oxygen atom.
[0059]
R¹²When is a biotin residue or a peptide residue, Z is preferably —NHCO—.
[0060]
The conditions for the glycosidic bond reaction are appropriately selected according to the leaving group to be used. For example, when a fluorine atom is selected as the leaving group, the condition of −70 to 60 ° C. for 5 minutes to 50 hours can be mentioned. The solvent is not particularly limited, and 1,2-dichloroethane or the like can be used.
[0061]
As the aralkyl group, acyl group or silyl group, and amino group-protecting group, those that are not liberated under the condition that the leaving group is eliminated are selected. These groups may be the same group.
[0062]
Examples of the method for producing the monosaccharide of the general formula (2) include a method as schematically shown in FIG. That is, the synthesis from compound 2 to compound 9 can be performed according to the synthesis route reported by Ito et al. (Agric. Biol. Chem., 50, 3227 (1986)) from galactose (compound 1). Synthesis from Compound 10 to Compound 14 can be performed according to the following synthesis route. Replacement of trichloroacetimide group with benzyl group (compound 10), deacetylation (compound 11), benzylation (compound 12), deallylation (compound 13), pivaloylation (compound 14). Those skilled in the art can appropriately set the conditions for these steps.
[0063]
Examples of the method for producing the monosaccharide of the general formula (1) include a method as schematically shown in FIG. That is, the synthesis from compound 16 to compound 20 can be performed according to the synthesis route (Tetrahedron Lett., 31, 1597 (1990)) reported by Nakano et al. From glucosamine (compound 15). The synthesis from Compound 21 to Compound 24 can be performed according to the following synthesis route. Ring opening of the benzylidene group between the 4- and 6-positions (compound 21), acetylation (compound 22), demethoxyphenylation (compound 23), fluorination (compound 24). Those skilled in the art can appropriately set the conditions for these steps.
[0064]
After the glycoside bond reaction between the monosaccharide of the general formula (1) and the monosaccharide of the general formula (2), the aralkyl group and the acyl group or silyl group are removed, and the amino group protecting group is replaced with an acetyl group. An oligosaccharide of general formula (3) is obtained. Glycoside binding reactions, and removal and substitution of such groups can be performed by known methods (eg, Synthesis, 384 (1989), etc.). Specific examples thereof will be described in detail in Examples described later.
[0065]
In general formula (3), R^TenAnd R¹¹At least one of -SO_ThreeIn the case of indicating M, after the step of reacting the monosaccharide represented by the general formula (1) and the monosaccharide represented by the general formula (2) with a glycoside bond, R^ThreeAnd R⁷At least one of (acyl group or silyl group) is selectively removed and replaced with a hydrogen atom (this produces a hydroxyl group), and then the hydrogen atom is —SO 2_ThreeReplace with M (sulfation).
[0066]
The selective removal of the acyl group or silyl group (and substitution with a hydrogen atom) can be carried out by appropriately selecting the acyl group or silyl group with respect to the aralkyl group and the amino group protecting group. Examples of such a combination include a combination of a benzyl group as an aralkyl group, a phthaloyl group as an amino group protecting group, an acetyl group or a pivaloyl group as an acyl group or a silyl group. The method of sulfation is not particularly limited, and a known method can be used. Specific examples thereof will be described in detail in Examples described later.
[0067]
Specifically, the oligosaccharide of the general formula (3) can be obtained by the method as schematically shown in FIG. That is, removal of an acetyl group and a pivaloyl group (and substitution with a hydrogen atom), substitution of a phthaloyl group with an acetyl group (compound 26), sulfation (compound 27), and debenzylation (compound 28). Those skilled in the art can appropriately set the conditions for these steps.
[0068]
R^TenAnd R¹¹Either of them is -SO_ThreeWhen M is indicated, R^ThreeAnd R⁷The acyl group is selected so that either one of the acyl group and the silyl group can be selectively removed. For example, R^ThreeAs levulinoyl group (monosaccharide of formula (7) or (10)), R⁷T-butyldiphenylsilyl group (monosaccharide of formula (8) or (11)) is selected as By selectively removing (and replacing with a hydrogen atom) the levulinoyl group, sulfation is performed, and then the other hydroxyl group protecting groups are removed, so that only the hydroxyl group at the 6-position of the glucosamine residue is sulfated. The oligosaccharide (oligosaccharide of formula (9)) can be obtained, t-butyldiphenylsilyl group is selectively removed (and replaced with a hydrogen atom), sulfation is performed, and then other hydroxyl groups of By removing the protecting group, an oligosaccharide (oligosaccharide of formula (12)) in which only the hydroxyl group at the 6-position of the galactose residue is sulfated can be obtained.
[0069]
Selective removal of an acyl group or silyl group (and substitution with a hydrogen atom), sulfation of the resulting hydroxyl group, removal of an aralkyl group, and substitution of an amino group protecting group with an acetyl group are known methods. Can be done by.
[0070]
Other examples of the method for obtaining the oligosaccharide of the general formula (3) by subjecting the monosaccharide of the general formula (1) and the monosaccharide of the general formula (2) to a glycoside bond reaction and the like are shown in FIGS.
[0071]
FIG. 5 shows R in the general formula (2).⁹Is an example of an O-alkylglycerol residue (2,3-di-O-tetradecyl-sn-glycerol residue), and FIG. 6 shows R in general formula (2).⁹Is an example of an alkyl group (octyl group), and FIG. 7 shows R in general formula (2).⁹Is an example of a cholestanyl group. Any of these can be performed in the same manner as described above. R⁹In the case of other groups, the same method can be used.
[0072]
The resulting sulfated oligosaccharide may be a salt (that is, M in the oligosaccharide may be a monovalent cation) or may not be a salt (that is, in the oligosaccharide). M may be a proton). Examples of the salt include those exemplified in the description of <3> the pharmaceutical of the present invention described later, but an alkali metal salt is preferable, and a sodium salt is more preferable. The oligosaccharide may be in an ionized state.
[0073]
According to the production method of the present invention, the above-mentioned oligosaccharide can be produced in a high yield and with few steps.
[0074]
<2> The oligosaccharide of the present invention
The oligosaccharide of the present invention is an oligosaccharide represented by the general formula (13).
The substituents in the general formula (13) are as follows.
[0075]
R¹⁶And R¹⁷Each independently represents a hydrogen atom or -SO_ThreeM (however, R¹⁶And R¹⁷Are both hydrogen atoms, Z is an oxygen atom and R¹⁸Is a hydrogen atom or a cholestanyl group, and R¹⁶-SO_ThreeM and Z is an oxygen atom and R¹⁷And R¹⁸Except those in which all are hydrogen atoms).
[0076]
Also R¹⁸Is a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phosphorus A lipid residue, a biotin residue or a peptide residue is shown, and a hydrogen atom, 6-O-sulfated N-acetylglucosamine residue, alkyl group, O-alkylglycerol residue or cholestanyl group is preferable. These groups and residues are R⁹Is the same as described above. In general formula (13), R¹⁸R is a hydrogen atom, R¹⁸May be in the β-position or in the α-position. Also R¹⁸Is a 6-O-sulfated N-acetylglucosamine residue, the glycoside bond is preferably a β-glycoside bond, more preferably a β-1,4 glycoside bond. R¹⁸When is other group or residue, it is preferably a β glycoside bond. Z represents an oxygen atom or -NHCO-. R¹⁸Is preferably a biotin residue or a peptide residue, Z is preferably —NHCO— and R¹⁸When is a group other than that, Z is preferably an oxygen atom.
[0077]
The oligosaccharide of the present invention may be a salt (that is, M in the oligosaccharide may be a monovalent cation) or may not be a salt (that is, M in the oligosaccharide is a proton). May be). Examples of the salt include those exemplified in the description of <3> the pharmaceutical of the present invention described later, but an alkali metal salt is preferable, and a sodium salt is more preferable. The oligosaccharide may be in an ionized state.
[0078]
R¹⁸The oligosaccharide of the present invention in which is a hydrogen atom can be obtained by the production method of the present invention. In addition, as shown in FIGS. 5 to 7, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue A monosaccharide carrying a group or peptide residue can be combined with other monosaccharides, or R¹⁸By binding the oligosaccharide of the present invention in which is a hydrogen atom and glycerol, cholesterol, ceramide, biotin or a peptide by a known glycosylation method, R¹⁸Thus, the oligosaccharide of the present invention other than a hydrogen atom can be obtained.
[0079]
Also R¹⁸The oligosaccharide of the present invention in which is a 6-O-sulfated N-acetylglucosamine residue can be produced, for example, by subjecting a known keratan sulfate oligosaccharide to treatment such as acid hydrolysis or enzyme treatment. For example, the known keratan sulfate oligosaccharides NeuAc to Galβ1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) (wherein Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, NeuAc represents an N-acetylneuraminic acid residue, 6S represents a 6-O-sulfate, and ~ represents an α2,3 bond or α2,6 bond (see WO96 / 16973)) of about 0.2N. It can be prepared by removing N-acetylneuraminic acid (sialic acid) residues by incubating with a strong acid and then removing galactose residues by incubating with lactase (β-galactosidase). Details will be described in detail in Examples described later.
[0080]
The oligosaccharide of the present invention or a pharmaceutically acceptable salt thereof has a pharmacological action and can be used as a medicine.
[0081]
<3> Pharmaceutical of the present invention
The medicament of the present invention comprises the oligosaccharide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.
[0082]
Pharmaceutically acceptable salts include, for example, alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt, salts with inorganic bases such as ammonium salt, or diethanolamine salt, Among salts with organic bases such as cyclohexylamine salt and amino acid salt, pharmaceutically acceptable salts are not limited thereto.
[0083]
The medicament of the present invention can be used particularly as an antiallergic agent. When used as an antiallergic agent, the hydroxyl group at the 6-position of the N-acetylglucosamine residue of the oligosaccharide of the present invention is sulfated (ie, R in the general formula (13)).¹⁶-SO_ThreeIt is preferable that both the hydroxyl group at the 6-position of the galactose residue and the hydroxyl group at the 6-position of the N-acetylglucosamine residue are sulfated (ie, R in the general formula (13)).¹⁶And R¹⁷Are all -SO_ThreeM).
[0084]
The antiallergic agent of the present invention is effective for all diseases in which allergy is involved, specifically bronchial asthma, allergic interstitial pneumonia, allergic rhinitis, allergic conjunctivitis, atopic It can be applied for the purpose of preventing or treating dermatitis.
[0085]
In addition, when both the hydroxyl group at the 6-position of the galactose residue and the hydroxyl group at the 6-position of the N-acetylglucosamine residue of the oligosaccharide of the present invention are sulfated, the medicament of the present invention can be used as an anti-inflammatory agent. .
[0086]
The anti-inflammatory agent of the present invention is effective for all diseases related to inflammation, specifically rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, osteoarthritis, low back pain, postoperative and Applicable for the treatment of post-traumatic inflammation and swelling, scapuloarthritis, temporomandibular disorders, tendon tenosynovitis, peritonitis, condylar humerus (tennis elbow), muscle pain, keratoconjunctivitis, etc. Can do. The anti-inflammatory agent of the present invention has anti-inflammatory effects such as analgesic, anti-inflammatory, antipyretic and the like against these diseases by the action of its active ingredients.
[0087]
The pharmaceutical of the present invention can be applied not only for pure therapeutic purposes but also for the purpose of prevention, maintenance (prevention of deterioration), reduction (improvement of symptoms) and the like.
[0088]
In the present invention, any dosage form can be appropriately selected depending on the nature and progress of the target disease, the administration method, and the like.
[0089]
That is, the medicament of the present invention can be administered by injection (intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, etc.), oral, transdermal, inhalation, etc., and should be formulated appropriately according to these administration methods. Can do. The dosage form that can be selected is not particularly limited. For example, injections (solutions, suspensions, emulsions, solid preparations for use), tablets, capsules, granules, powders, liquids, lipolytic agents, It can be widely selected from ointments, gels, powders for external use, sprays, inhalation powders and the like. In addition, in preparing these preparations, conventional excipients, stabilizers, binders, lubricants, emulsifiers, osmotic pressure adjusting agents, pH adjusting agents, other colorants, disintegrating agents, etc. Can be used.
[0090]
The compounding amount of the keratan sulfate oligosaccharide, which is an active ingredient in the medicament of the present invention, and the dose of the medicament of the present invention depend on the administration method, dosage form, purpose of use, specific symptoms of the patient, patient weight, etc. These are matters to be determined individually and are not particularly limited.
[0091]
In allergic diseases, antigens are induced from sensitized mast cells with IgE antibodies in the airways and lungs to release chemical mediators. Chemical mediators include histamine, eosinophil chemotactic factor, and SRS-A. As a result, dyspnea, cough, and seizures occur in asthma, and symptoms such as hemorrhagic pneumonia, edema, interstitial pneumonia, and vasculitis are observed in lung diseases. Rarely, granulomas are present, and the disease often later becomes pulmonary fibrosis. Allergic diseases are treated with antihistamines, steroids, and antiallergic agents (chemical transmitter release inhibitors). However, as side effects, weakness, malaise, headache, vomiting, head feeling, loss of appetite, etc. have been reported with antihistamines. In bronchial asthma, airway secretion is suppressed by anticholinergic action, making it difficult to produce sputum, so it is not used except in light cases. It is also contraindicated for those with glaucoma and difficulty urinating. On the other hand, the main action of steroids is considered to be an anti-inflammatory action, and usually large doses and continuous administration are required for the treatment of allergic diseases. Since steroids have serious side effects, they are generally used when they cannot be controlled by general treatment methods. Antiallergic drugs can also cause liver damage, hemorrhagic cystitis, and gastrointestinal disorders, requiring regular testing.
[0092]
As described above, particularly in the area of allergic diseases, an effective treatment method with fewer side effects is required. According to the medicament of the present invention, such a treatment method can be provided.
[0093]
In the medicament of the present invention (including the antiallergic agent of the present invention and the antiinflammatory agent of the present invention), preferred R in the general formula (13)¹⁸And Z are the same as described above.
[0094]
【Example】
Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention should not be limited by these. The ratio of the solvent mixture in the examples is a volume ratio unless otherwise specified.
[0095]
[Example 1]
Synthesis diagram of O- (2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O-6-O-sulfo-β-D-galactopyranose disodium salt O- (2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O-6-O-sulfo-β-D by the procedure outlined in 2-4 -Galactopyranose disodium salt was synthesized. In addition, the method used in common at each synthesis step in the following examples is as follows. Silica gel column chromatography was performed using Kiesegel 60 (MERCK). Thin layer chromatography is performed using HPTLC-Fertigplatten Kieselgel 60 F₂₅₄(MERCK) was used.¹H-NMR spectrum and¹³The C-NMR spectrum was measured using JNM-EX-400 (manufactured by JEOL Ltd.). Measuring solvent CDCl_Three, CD_ThreeIn OD, tetramethylsilane and D₂In O, t-butanol was used as an internal standard.
[0096]
(1) Synthesis of Compound 14 from Compound 2
Galactose synthon 2-9 was synthesized from galactose (compound 1) according to the synthesis route reported by Ito et al. (Agric. Biol. Chem., 50, 3227 (1986)). The synthesis | combination of the compounds 10-14 was performed as follows.
Hereinafter, the number after the substance name indicates the compound number in FIGS.
[0097]
(a) benzyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside (benzyl 2,4-di-O-acetyl-3,6-di- O-allyl-β-D-galactopyranoside) 10
In a nitrogen gas atmosphere, a reaction vessel containing molecular sieves 4A (30.0 g) previously dried was charged with benzyl alcohol (18.4 ml, 178.8 mmol) and compound 9 (2,4-di-O-acetyl-3,6-di-). -O-allyl-D-galactopyranosyl trichloroacetimidate (21.84 g, 44.67 mmol) After that, the mixture was stirred for 15 minutes under ice cooling. Trimethylsilyl trifluoromethanesulfonate (1.7 ml, 8.93 mmol) was added to the reaction mixture under ice cooling, followed by stirring at the same temperature for 4 hours. The reaction mixture was diluted with ethyl acetate, neutralized by adding triethylamine under ice-cooling, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (toluene: ethyl acetate = 6: 1) to give compound 10 (18.6
g, 96%).
[0098]
Rf: 0.51 (toluene: ethyl acetate = 3: 1)
C_{twenty three}H₃₀O₈                 MW: 434.47
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
2.037 (s, 3H, OAc) 2.146 (s, 3H, OAc) 4.445 (d, 1H, J = 7.8 Hz, H-1) 5.461 (d, 1H, J = 2.9 Hz, H-4) 5.715-5.914 ( m, 2H, CH₂= CHx2) 7.200-7.400 (m, 5H, aromatic)
[0099]
(b) benzyl 3,6-di-O-allyl-β-D-galactopyranoside 11
Sodium methoxide (134 mg, 2.5 mmol) was added to a methanol solution (30 ml) of compound 10 (10.84 g, 24.9 mmol), and the mixture was stirred at room temperature for 48 hours under a nitrogen gas atmosphere. The reaction mixture was neutralized with acetic acid, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (toluene: ethyl acetate = 4: 1) to obtain Compound 11 (6.8 g, 78%).
[0100]
Rf: 0.27 (toluene: ethyl acetate = 2: 1)
C₁₉H₂₆O₆                 MW: 350.40
[0101]
(c) Benzyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside (benzyl 3,6-di-O-allyl-2,4-di- O-benzyl-β-D-galactopyranoside) 12
Under a nitrogen gas atmosphere and ice cooling, benzyl bromide (11.4 ml, 95.5 mmol) was added to a mixture of 60% sodium hydride (3.8 g, 95.5 mmol), compound 11 (6.7 g, 19.1 mmol) and DMF 20 ml and stirred for 18 hours. did. Methanol was added to the reaction mixture under ice-cooling and stirred for 1 hour, and then the solvent was distilled off under reduced pressure. The residue was diluted with diethyl ether, washed successively with water and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 10: 1 to 9: 1) to obtain Compound 12 (9.1 g, 90%).
[0102]
Rf: 0.27 (toluene: ethyl acetate = 10: 1)
C₃₃H₃₈O₆                 MW: 530.63
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
3.424 (dd, 1H, J = 2.9,9.8 Hz, H-3) 3.829 (dd, 1H, J = 7.8,9.8 Hz, H-2) 3.861 (d, 1H, J = 2.9 Hz, H-4) 4.453 (d, 1H, J = 7.8 Hz, H-1) 5.805-5.984 (m, 2H, CH₂= CHx2) 7.200-7.450 (m, 15H, aromatic)
[0103]
(d) benzyl 2,4-di-O-benzyl-β-D-galactopyranoside (benzyl 2,4-di-O-benzyl-β-D-galactopyranoside) 13
Activated iridium complex [Ir (CoD) (PMePh₂)₂PF₆To a tetrahydrofuran solution (60 ml) of (287 mg, 0.34 mmol), a tetrahydrofuran solution (80 ml) of compound 12 (8.9 g, 16.7 mmol) was added at room temperature and stirred for 7 hours. Then, water (100 ml) and iodine (8.5 g, 67.1 mmol) were added and stirred for 15 hours. The reaction mixture was diluted with ethyl acetate, washed successively with saturated sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate, and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was recrystallized (ethanol-dichloromethane-diethyl ether) to obtain Compound 13 (7.4 g, 97%).
[0104]
Rf: 0.34 (n-hexane: ethyl acetate = 1: 1)
C₂₇H₃₀O₆                 MW: 450.51
[0105]
(e) benzyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (benzyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D- galactopyranoside) 14
Under a nitrogen gas atmosphere, pivaloyl chloride (4.2 ml, 35.7 mmol) was added to a pyridine solution (50 ml) of compound 13 (7.3 g, 16.2 mmol) at 0 ° C. and stirred for 70 minutes. Methanol was added to the reaction mixture, and the mixture was stirred for 40 minutes. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (toluene: ethyl acetate = 6: 1) to obtain Compound 14 (7.81 g, 90%). Obtained.
[0106]
Rf: 0.45 (toluene: ethyl acetate = 6: 1)
C₃₂H₃₈O₇                 MW: 534.62
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
1.202 (s, 9H, OPiv) 2.326 (bs, 1H, OH) 3.628-3.708 (m, 3H, H-2, H-3 and H-5) 3.786 (d, 1H, J = 3.9 Hz, H-4 ) 4.142 (dd, 1H, J = 6.4, 10.7 Hz, H-6) 4.352 (dd, 1H, J = 6.8,11.2 Hz, H-6 ') 4.448 (d, 1H, J = 7.3 Hz, H-1) ) 6.650-7.150 (m, 15H, aromatic)
[0107]
(2) Synthesis of Compound 24 from Compound 16
Glucosamine synthon 16-20 was synthesized from glucosamine (compound 15) according to the synthesis route reported by Nakano et al. (Tetrahedron Lett., 31, 1597 (1990)). The synthesis | combination of the compounds 21-24 was performed as follows.
[0108]
(f) p-methoxyphenyl 3,4-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside (p-methoxyphenyl 3,4-di-O-benzyl-2-deoxy-2- phthalimido-β-D-glucopyranoside) 21
Under a nitrogen gas atmosphere, a borane-trimethylamine complex (75.0 g, 1028 mmol) and a solution of compound 20 (21.0 g, 35.4 mmol) in dichloromethane (200 ml) were placed in a reaction vessel containing molecular sieves 4A (60.0 g) previously dried. And diethyl ether (80 ml) was added and stirred for 15 minutes. The reaction vessel was cooled to 0 ° C., anhydrous aluminum chloride (20.0 g, 150 mmol) was added in small portions over 1.5 hours, and the mixture was stirred at 0 ° C. for 2.5 hours. The reaction mixture was filtered through celite, and the filtrate was diluted with ethyl acetate, washed successively with 1N aqueous sulfuric acid solution, water, saturated aqueous sodium hydrogen carbonate, and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 4: 1) to obtain Compound 21 (14.5 g, 69%).
[0109]
Rf: 0.40 (toluene: ethyl acetate = 3: 1)
C₃₅H₃₃N₁O₈             MW: 595.62
400 MHz¹H-NMR (CDCl_Three+ CD_ThreeOD, TMS) δ:
3.620-3.662 (m, 1H, H-5) 3.706 (s, 3H, OMe) 3.783-3.849 (m, 2H, H-4 and H-6) 3.939 (dd, 1H, J = 2.4, 12.2 Hz, H -6 ') 4.351 (dd, 1H, J = 8.3,10.7 Hz, H-2) 4.435 (dd, 1H, J = 8.3,10.7 Hz, H-3) 5.693 (d, 1H, J = 8.3 Hz, H -1) 6.650-7.900 (m, 18H, aromatic)
[0110]
(g) p-methoxyphenyl 6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside (p-methoxyphenyl 6-O-acetyl-3,4- di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside) 22
Under a nitrogen gas atmosphere, acetic anhydride (200 ml) and DMAP (catalytic amount) were added to a pyridine solution (200 ml) of compound 21 (10.5 g, 17.6 mmol), and the mixture was stirred for 20 hours. Ethanol was added to the reaction mixture, and the mixture was stirred for 20 minutes. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (toluene: ethyl acetate = 4: 1) to obtain Compound 22 (9.6 g, 85%). Obtained.
[0111]
Rf: 0.51 (toluene: ethyl acetate = 4: 1)
C₃₇H₃₅N₁O₉             MW: 637.66
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
2.062 (s, 3H, OAc) 3.680 (s, 3H, OMe) 3.759-3.817 (m, 2H, H-4 and H-5) 4.296 (dd, 1H, J = 4.4, 12.2 Hz, H-6) 5.631 (d, 1H, J = 7.8 Hz, H-1) 6.650-7.900 (m, 18H, aromatic)
[0112]
(h) 6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimide-D-glucopyranose (6-O-acetyl-3,4-di-O-benzyl-2- deoxy-2-phthalimido-D-glucopyranose) 23
Compound 22 (9.0 g, 14.1 mmol) was dissolved in acetonitrile: water (4: 1; 400 ml), ceric ammonium nitrate (20.1 g, 36.7 mmol) was added, and the mixture was vigorously stirred at room temperature for 40 minutes. The reaction mixture was diluted with ethyl acetate, washed successively with water, saturated aqueous sodium hydrogen carbonate, and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (toluene: ethyl acetate = 2.5: 1) to obtain Compound 23 (6.1 g, 81%).
[0113]
Rf: 0.23 (toluene: ethyl acetate = 2: 1)
C₃₀H₂₉N₁O₈             MW: 531.54
400 MHz¹H-NMR (CDCl_Three+ D₂O, TMS) δ:
2.074 (s, 3H, OAc) 3.680 (t, 3H, J = 9.3 Hz, H-4) 3.739-3.772 (m, 1H, H-5) 4.100 (dd, 1H, J = 8.8,10.8 Hz, H- 2) 4.240 (dd, 1H, J = 3.9,11.2 Hz, H-6) 5.386 (d, 1H, J = 8.3 Hz, H-1) 6.650-7.900 (m, 14H, aromatic)
[0114]
(i) 6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl fluoride (6-O-acetyl-3,4-di- O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl fluoride) 24
Under a nitrogen gas atmosphere, diethylaminosulfur trifluoride (5.8 ml, 43.9 mmol) was added to a 1,2-dichloroethane solution (50 ml) of compound 23 (5.95 g, 11.2 mmol) under ice cooling, and the mixture was stirred for 2 hours. The reaction mixture was diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (toluene: ethyl acetate = 4: 1) to obtain Compound 24 (5.9 g, 99%).
[0115]
Rf: 0.68 (toluene: ethyl acetate = 2: 1)
C₃₀H₂₈N₁O₇F₁             MW: 533.53
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
2.097 (s, 3H, OAc) 3.859 (dd, 1H, J = 8.3,9.8 Hz, H-4) 3.800-3.840 (m, 1H, H-5) 5.810 (d, 0.5H, J = 7.8 Hz, H -1β) 5.943 (d, 0.5H, J = 7.8 Hz, H-1β) 6.800-7.800 (m, 14H, aromatic)
[0116]
(3) Synthesis of Compound 28 from Compound 14 and Compound 24
The synthesis | combination of the compounds 25-28 was performed as follows.
[0117]
(j) Benzyl O- (6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 3) -O-2,4- Di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (benzyl O- (6-O-acetyl-3,4-di-O-benzyl-2-deoxy-2-phthalimido-β -D-glucopyranosyl)-(1 → 3) -O-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside) 25
Under a nitrogen gas atmosphere, silver triflate (7.23 g, 28.2 mmol), hafnocene dichloride (5.4 g, 14.1 mmol) and 1,2-dichloroethane were placed in a reaction vessel containing molecular sieves 4A (20.0 g) previously dried. (20 ml) was added, followed by stirring for 20 minutes under ice cooling. The reaction vessel was cooled to -23 ° C, and a 1,2-dichloroethane solution (45 ml) of compound 24 (5.8 g, 10.8 mmol) and compound 14 (5.4 g, 10.0 mmol) was added, followed by stirring at -23 ° C for 1.5 hours. did. The reaction mixture was diluted with ethyl acetate, triethylamine was added under ice-cooling, the mixture was stirred for 20 min, and filtered through celite. The filtrate was diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (toluene: ethyl acetate = 9: 1) and recrystallized to obtain Compound 25 (9.3 g, 82%).
[0118]
Rf: 0.39 (toluene: ethyl acetate = 8: 1)
C₆₂H₆₅N₁O₁₄              MW: 1048.15
400 MHz¹H-NMR (CDCl_Three, TMS) δ:
1.173 (s, 9H, OPiv) 1.986 (s, 3H, OAc) 3.859 (bd, 1H, J = 2.5 Hz, H-4) 4.063 (dd, 1H, J = 5.9, 11.2 Hz) 5.454 (d, 1H, J = 8.3 Hz, H-1) 6.800-7.800 (m, 24H, aromatic)
[0119]
(k) Benzyl O- (2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)-(1 → 3) -O-2,4-di-O-benzyl- β-D-galactopyranoside (benzyl O- (2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)-(1 → 3) -O-2,4- di-O-benzyl-β-D-galactopyranoside) 26
Ethylenediamine (170 ml) was added to a 1-butanol solution (200 ml) of compound 25 (8.0 g, 7.6 mmol), and the mixture was stirred at 98 ° C. for 46 hours. The solvent of the reaction mixture was distilled off under reduced pressure, toluene and methanol were added to the residue, and the solvent was distilled off under reduced pressure. The residue was dissolved in pyridine (200 ml), DMAP (catalytic amount) and acetic anhydride (150 ml) were added, and the mixture was stirred at room temperature for 2 days. The solvent of the reaction mixture was distilled off and azeotroped with toluene and ethanol. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 4: 1) to obtain a mixture of two components (6.84 g). Further, sodium methoxide (769 mg, 14.3 mmol) was added to a methanol solution (100 ml) of this mixture, followed by stirring at room temperature for 60 hours in a nitrogen gas atmosphere. After neutralization with Amberlyst 15 and filtration, the filtrate was evaporated under reduced pressure. The obtained residue was recrystallized (dichloromethane-isopropyl ether) to obtain Compound 26 (6.0 g, 94%).
[0120]
Rf: 0.33 (toluene: ethyl acetate = 1: 3)
C₄₉H₅₅N₁O₁₁              MW: 833.94
400 MHz¹H-NMR (CDCl_Three+ CD_ThreeOD, TMS) δ:
1.557 (s, 3H, NAc) 4.438 (d, 1H, J = 7.3 Hz, H-1) 4.784 (d, 1H, J = 8.3 Hz, H-1) 7.200-7.450 (m, 20H, aromatic)
100 MHz¹³C-NMR (CDCl_Three+ CD_ThreeOD, TMS) δ: 22.92 (Me-CO) 61.44, 61.64 (C-6 x2) 101.73 (C-1), 102.60 (C-1) 170.29 (Me-CO)
[0121]
(l) Benzyl O- (2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O-2,4- Di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt (benzyl O- (2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O- sulfo-β-D-glucopyranosyl)-(1 → 3) -O-2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt) 27
Under a nitrogen gas atmosphere, a mixture of compound 26 (212.5 mg, 0.255 mmol) and sulfur trioxide triethylamine complex (184.7 mg, 1.02 mmol) was dissolved in DMF (1.0 ml) and stirred at 50 ° C. for 1 hour. The reaction solution was directly purified by Sephadex LH-20 (chloroform: methanol = 1: 1), and the sugar fraction was concentrated. The obtained residue was dissolved in methanol (4 ml), and then Dowex 50 (Na⁺, 4 g) was added and stirred for 12 hours to convert the counter cation to sodium. Further, the obtained residue was purified by silica gel column chromatography (chloroform: methanol = 4: 1) and then purified by Sephadex LH-20 (chloroform: methanol = 1: 1) for the purpose of removing silica gel. 27 (252 mg, 95%) was obtained.
[0122]
Rf: 0.53 (chloroform: methanol = 3: 1)
C₄₉H₅₃N₁O₁₇S₂Na₂            MW: 1038.03
400 MHz¹H-NMR (CDCl_Three+ CD_ThreeOD, TMS) δ:
1.621 (s, 3H, NAc) 7.200-7.450 (m, 20H, aromatic)
100 MHz¹³C-NMR (CDCl_Three+ CD_ThreeOD, TMS) δ:
22.14 (Me-CO) 66.25, 66.61 (C-6 x2) 102.04 (C-1 x2) 170.98 (Me-CO)
[0123]
(m) O- (2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O-6-O-sulfo-β-D-galactopyranose disodium salt (O- (2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O-6-O-sulfo-β-D-galactopyranose disodium salt) 28
20% palladium hydroxide on carbon (268 mg) was added to a solution of compound 27 (236.8 mg, 0.228 mmol) in methanol-water (2: 1, 6 ml), the reaction system was replaced with hydrogen, and the mixture was stirred at room temperature for 17 hours. did. The reaction mixture was filtered through celite, the residue was washed with water, the filtrate and the washing solution were combined, and the solvent was evaporated under reduced pressure. The obtained residue was purified with Sephadex G-25 (water) to obtain Compound 28 (131 mg, 98%).
[0124]
Rf: 0.28 (1-butanol: ethanol: water = 2: 2: 1)
C₁₄H_{twenty three}N₁O₁₇S₂Na₂            MW: 587.44
400 MHz¹H-NMR (D₂O, t-BuOH, at 50 ° C) δ:
2.029 (s, 3H, NAc) 4.580 (d, 0.55H, J = 8.3 Hz, H-1aβ) 4.727 (d, 0.55H, J = 8.3 Hz, H-1bβ) 4.742 (d, 0.45H, J = 8.3 Hz, H-1bα) 5.232 (d, 0.45H, J = 3.4 Hz, H-1aα)
100 MHz¹³C-NMR (D₂O, t-BuOH, at 50 ° C) δ:
25.04 (Me-CO) 69.81 (C-6 b) 70.70 (C-6 aβ) 70.94 (C-6 aα) 95.21 (C-1aα) 99.21 (C-1aβ) 105.39 (C-1b) 177.74 (Me-CO)
[0125]
[Example 2]
O- (2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl)-(1 → 3) -O- (6-O-sulfo-β-D-galactopyranosyl)-( 1 → 4) Production of -O-2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranose trisodium salt (hereinafter referred to as G4L4 sodium salt)
NeuAc to Galβ1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) (where Gal is galactose residue, GlcNAc is N-acetylglucosamine residue, NeuAc is N-acetylneuraminic acid residue 6S represents 6-O-sulfate ester, and ~ represents an α2,3 bond or an α2,6 bond; see WO96 / 16973) 1 g is dissolved in 10 ml of 0.1 M sulfuric acid and 22 at 50 ° C. The N-acetylneuraminic acid residue (sialic acid residue) was cleaved by incubating for a period of time. After adjusting the pH to 5 by adding a small amount of 1 M NaOH to the solution after the reaction, 1 ml of 0.5 M sodium acetate buffer pH 4.5 and 25 μl of 20% sodium azide were added. Galactose residues were cleaved by adding lactase (manufactured by Keiai Kasei) 5000 U and incubating at 37 ° C. for 22 hours. The reaction solution was diluted 5-fold with distilled water and applied to a Muromac column (Muromachi Chemical Industries) (2.5 × 24 cm) equilibrated with 1 M NaCl. A salt concentration gradient from 1 M NaCl (500 ml) to 2.5 M NaCl (500 ml) was loaded onto the column, and the eluate was collected in 5 ml portions. The elution fraction was analyzed by capillary electrophoresis, and the elution position of G4L4 was confirmed. Fractions containing G4L4 were collected and concentrated to about 10 ml on a rotary evaporator. The concentrated solution was applied to a Cellulofine GCL25sf column (Seikagaku Corporation) (3 × 60 cm) equilibrated with distilled water and eluted with distilled water. The elution fractions collected every 10 ml were analyzed by capillary electrophoresis, and the elution position of G4L4 was confirmed. Fractions containing G4L4 were collected and concentrated to about 20 ml on a rotary evaporator. Endotoxin was removed by filtration through an ultrafiltration membrane with a molecular weight cut of 10,000, and then lyophilized to obtain the final sample.
[0126]
The final sample showed a single peak on capillary electrophoresis. As a result of measuring hexose content and sulfuric acid content, 0.84 and 0.91 were obtained for theoretical value 1, respectively.
[0127]
Further, as a result of subjecting the final sample to high performance liquid chromatography under the following conditions, a single peak was observed at a retention time of 16.4 minutes.
[0128]
Column: YMC-Pack PolyamineII (4.6 × 250 mm) (manufactured by YMC Co., Ltd.)
Column temperature: 35 ° C
Eluent: 150 mM sodium dihydrogen phosphate
Flow rate: 1 ml / min
Measurement wavelength: 210 nm
Sample: 10 mg / ml G4L4 (final sample)
[0129]
The results of NMR measurement of the final sample are shown below.
[0130]
400 MHz¹H-NMR (D₂O, t-BuOH, at 22.9 ° C) δ:
2.024 (s, 3H, NAc) 2.030 (s, 3H, NAc) 4.526 (d, 1H, J_1,2= 7.8 Hz, H-1b) 4.699 (d, 1H, J_1,2= 8.8 Hz, H-1c) 4.729 (d, 0.4H, J_1,2= 7.8 Hz, H-1aβ) 5.211 (d, 0.6H, J_1,2= 2.5 Hz, H-1aα)
100 MHz¹³C-NMR (D₂O, t-BuOH, at 26.0 ° C) δ:
24.74 (NHCOCH_Three), 25.05 (NHCOCH_Three), 69.62 (C-6a or b or c), 69.77 (C-6b or c or a), 70.57 (C-6c or a or b), 93.31 (C-1aα), 97.82 (C-1aβ), 105.80 (C-1b or c), 105.91 (C-1c or b)
[0131]
[Example 3]
2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranosyl- (1 → 1) -2,3 Of 2-di-O-tetradecyl-sn-glycerol disodium salt
According to the procedure outlined in FIG. 5, 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranosyl- (1 → 1) -2,3-Di-O-tetradecyl-sn-glycerol disodium salt was synthesized.
The number after the substance name indicates the number of the compound in FIG.
[0132]
(a) 2,4-Di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn- Glycerol (2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol) 3
2,3-di-O-tetradecyl-sn-glycerol (500 mg, 1.03 mmol), cyclopentadiene hafnonium dichloride (782 mg, 2.68 mmol), silver triflate (1.06 g, 5.36 mmol), molecular sieve 4A (1.8 g) was suspended in 1,2-dichloroethane (3.0 ml), stirred at room temperature under an argon gas stream, cooled to −15 ° C., compound 1 (536 mg, 1.55 mmol) was added, and the mixture was stirred for 2.5 hours. The reaction mixture was neutralized with triethylamine, diluted with ethyl acetate (AcOEt), filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 3 (716.7 mg, 85.8%).
[0133]
Rf 0.58 (Toluene: AcOEt = 6: 1)
C₄₇H₈₅O_Ten     MW: 810.179
¹H-NMR (CDCl_Three) δ:
5.887 (m, 1H, Allyl), 5.816 (m, 1H, Allyl), 5.500 (d, 1H, J = 2.4Hz, H-4), 5.108 (dd, 1H, J = 8.3, 9.9Hz, H-2 ), 4.474 (d, 1H, J = 8.3Hz, H-1), 2.165, 2.015 (2s, 6H, 2Ac), 0.912 (t, 6H, J = 6.3Hz, 2CH_Three)
[0134]
(b) 3,6-Di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn- Glycerol (3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol) 5
Compound 3 (430 mg, 0.531 mmol) was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 4 mL), 1 N sodium hydroxide solution (0.8 mL) was added, and the mixture was stirred at room temperature for 1 day. Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was purified by silica gel column chromatography (toluene: AcOEt = 5: 2) to obtain Compound 4 (377.9 mg, 98.0%).
[Rf 0.43 (toluene: AcOEt = 2: 1)]
[0135]
Subsequently, Compound 4 (778 mg, 1.072 mmol) was dissolved in N, N-dimethylformamide (3 ml), and sodium hydride (308 mg, 6.99 mmol) was added and stirred at −15 ° C. under an argon gas stream. did. Subsequently, benzyl bromide (0.84 ml, 6.99 mmol) was added, and the mixture was stirred for 3 hours while gradually warming to room temperature. The reaction solution was neutralized with methanol, diluted with ethyl acetate, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: AcOEt = 12: 1) to obtain Compound 5 (864 mg, 89%).
[0136]
Rf 0.67 (hexane: AcOEt = 6: 1)
C₅₇H₉₃O₈    MW: 906.354
¹H-NMR (CDCl_Three) δ:
5.845 (m, 1H, Allyl), 5.771 (m, 1H, Allyl), 4.859 (d, 1H, J = 11.7 Hz, Bn), 4.816 (d, 1H, J = 10.8 Hz, Bn), 4.665 (d, 1H, J = 10.7 Hz, Bn), 4.572 (d, 1H, J = 11.7 Hz, Bn), 4.272 (d, 1H, J = 7.3 Hz, H-1), 3.773 (d, 1H, J = 2.5 Hz , H-4), 3.660 (dd, 1H, J = 7.8, 9.8 Hz, H-2), 0.801 (t, 6H, J = 6.4 Hz, 2CH_Three).
[0137]
(c) 2,4-Di-O-benzyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol (2,4-di-O- benzyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol) 6
Iridium complex (1,5-cyclooctadienebis (methyldiphenylphosphine) iridium hexafluorophosphate) (112 mg, 0.096 mmol) was suspended in tetrahydrofuran (5 mL), and H₂The mixture was stirred and activated under a stream of air. Compound 5 (864 mg, 0.95 mmol) was dissolved in tetrahydrofuran (5 mL) and added to the solution. After stirring for 2 hours at room temperature under a stream of argon gas, iodine (484 mg), water (24.7 mL), tetrahydrofuran (15 mL) was added, and the mixture was further stirred at room temperature for 2 hours. The reaction solution was diluted with chloroform, and washed successively with saturated sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate, and saturated brine. The chloroform layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (toluene: EtOAc = 5: 2) to give compound 6 (686.6 mg, 87.1%). .
[0138]
Rf 0.32 (toluene: AcOEt = 2: 1)
C₅₁H₈₅O₈    MW: 826.225
¹H-NMR (CDCl_Three) δ:
4.991 (d, 1H, J = 11.2 Hz, Bn), 4.834 (d, 1H, J = 11.7 Hz, Bn), 4.658 (d, 2H, J = 11.2 Hz, 2Bn), 4.385 (d, 1H, J = 7.3 Hz, H-1), 3.778 (d, 1H, J = 2.4 Hz, H-4), 0.881 (t, 6H, J = 6.8 Hz, 2CH_Three)
[0139]
(d) 2,4-Di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol (2, 4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol) 7
Compound 6 (687 mg, 0.831 mmol) is dissolved by adding pyridine (12 mL), and pivaloyl chloride (130 μL, 1.08 mmol) is added to the solution, followed by stirring at -5 ° C. for 1 hour, and further pivaloyl chloride. (130 μL, 1.08 mmol) was added, and the mixture was stirred at −5 ° C. for 1 hour. The reaction mixture was diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 7 (716.6 mg, 94.7%).
[0140]
Rf 0.57 (Toluene: AcOEt = 6: 1)
C₅₆H₉₃O₉    MW: 910.342
¹H-NMR (CDCl_Three) δ:
4.991 (d, 1H, J = 11.7 Hz, Bn), 4.855 (d, 1H, J = 11.7 Hz, Bn), 4.650 (d, 1H, J = 11.2 Hz, Bn), 4.646 (d, 1H, J = 11.7 Hz, Bn), 4.365 (d, 1H, J = 7.3 Hz, H-1), 4.302 (dd, 1H, J = 6.8, 11.8 Hz, H-6), 4.109 (dd, 1H, J = 6.4, 11.2 Hz, H-6 '), 3.775 (d, 1H, J = 2.4 Hz, H-4), 1.179 (s, 9H, piv), 0.879 (t, 6H, J = 6.8 Hz, 2CH_Three)
[0141]
(e) 3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl- sn-glycerol (3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn- glycerol) 8
Compound 7 (10.8 mg, 11.9 μmol) was dissolved by adding pyridine (1 mL), acetic anhydride (0.5 mL) was added to the solution, and the mixture was stirred at room temperature for 1 hour. After azeotroping the solvent with toluene, the residue was separated by Sephadex LH-20 (CHCl_Three: MeOH = 1: 2) to obtain Compound 8 (11.3 mg, qu.).
[0142]
Rf 0.46 (Toluene: AcOEt = 8: 1)
C₅₈H₉₅O_Ten     MW: 952.379
¹H-NMR (CDCl_Three) δ:
4.900 (dd, 1H, J = 3.4, 10.3 Hz, H-3), 4.882 (d, 1H, J = 11.7 Hz, Bn), 4.634 (d, 2H, J = 12.2 Hz, 2Bn), 4.543 (d, 1H, J = 11.2 Hz, Bn), 4.437 (d, 1H, J = 7.3 Hz, H-1), 4.299 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.087 (dd, 1H, J = 6.8, 11.2 Hz, H-6 '), 3.850 (d, 1H, J = 2.9 Hz, H-4), 3.765 (dd, 1H, J = 7.8, 10.3 Hz, H-2), 1.926 (s , 3H, Ac), 1.188 (s, 9H, piv), 0.881 (t, 6H, J = 6.8 Hz, 2CH_Three)
[0143]
(f) 3,4-Di-O-benzyl-2-deoxy-6-O-levroyl-2-phthalimide-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-6 -O-Pivaloyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol (3,4-di-O-benzyl-2-deoxy-6- O-levloyl-2-phtalimido-β-D-gulcopyranosyl- (1 → 3) -2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranosyl- (1 → 1) -2, 3-di-O-tetradecyl-sn-glycerol) 9
Compound 7 (685 mg, 0.752 mmol), cyclopentadiene hafnonium dichloride (571 mg, 1.96 mmol), silver triflate (771 g, 3.91 mmol), molecular sieve 4A (2.5 g) in 1,2-dichloroethane ( 10 ml), stirred at room temperature under an argon gas stream, cooled to −15 ° C., added with compound 2 (563 mg, 0.98 mmol), and stirred for 1 hour. The reaction mixture was neutralized with triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 9: 1) to obtain Compound 9 (1.01 g, 91.5%).
[0144]
Rf 0.44 (Toluene: AcOEt = 6: 1)
C₈₉H₁₂₄O₁₇N MW: 1479.949
¹H-NMR (CDCl_Three) δ:
5.461 (d, 1H, J = 8.3 Hz, H-1b), 4.941 (d, 1H, J = 11.2 Hz, Bn), 4.874 (d, 1H, J = 10.7 Hz, Bn), 4.789 (d, 1H, J = 11.7 Hz, Bn), 4.648 (d, 1H, J = 11.2 Hz, Bn), 4.536 (d, 1H, J = 11.7 Hz, Bn), 4.460 (d, 1H, J = 11.7 Hz, Bn), 4.414 (d, 2H, J = 11.7 Hz, 2Bn), 4.223 (d, 1H, J = 7.8 Hz, H-1a), 3.897 (d, 1H, J = 3.2 Hz, H-4a), 2.130 (s, 3H, CH_Three), 1.153 (s, 9H, piv), 0.881 (t, 6H, J = 6.6 Hz, 2CH_Three)
[0145]
(g) 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-β-D-galactopyrano Syl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol (2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-gulcopyranosyl- (1 → 3 ) -2,4-di-O-benzyl-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol) 10
Compound 9 (977.4 mg, 0.667 mmol) was suspended in ethanol (33.5 mL), hydrazine hydrate (3.35 mL) was added, and the mixture was stirred at 110 ° C. for 18 hours. The solvent was distilled off, and the resulting amino compound was dissolved in pyridine (6.0 mL), acetic anhydride (5.0 mL) was added and the mixture was stirred at room temperature for 17 hours, and then the solvent was evaporated. The residue was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 20.0 mL), sodium methoxide (108 mg, 2.0 mmol) was added, and the mixture was stirred at 60 ° C. for 1 hr. The reaction solution was added to Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was subjected to silica gel column chromatography (toluene: acetone: CHCl_Three= 5: 2: 1), and Sephadex LH-20 (CHCl_Three: MeOH = 2: 3) to obtain Compound 10 (756 mg, 94.8%).
[0146]
Rf 0.18 (toluene: acetone: CHCl_Three= 6: 2: 1)
C₇₃H₁₁₀O₁₃N MW: 1209.666
¹H-NMR (CDCl_Three) δ:
4.823 (d, 1H, J = 8.3 Hz, H-1b), 4.354 (d, 1H, J = 6.8 Hz, H-1a), 1.497 (s, 3H, NHAc), 0.881 (t, 6H, J = 6.4 Hz, 2CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
173.06 (Me-CO), 105.51, 104.01 (C-1x2), 62.80, 62.51 (C-6x2)
[0147]
(h) 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-6 -O-sulfo-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol disodium salt (2-acetamido-3,4-di-O-benzyl -2-deoxy-6-O-sulfo-β-D-gulcopyranosyl- (1 → 3) -2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol disodium salt) 11
Compound 10 (200 mg, 0.165 mmol) was dissolved in N, N-dimethylformamide (1.5 mL) and (C₂H_Five)_ThreeNSO_Three(300 mg, 1.65 mmol) was added, and the mixture was stirred at 50 ° C. for 0.5 hr. The reaction solution was directly added to Sephadex LH-20 (CHCl_Three: MeOH = 2: 3). The solvent was distilled off to some extent, and water (2.0 mL) and Dowex-50 (Na⁺) Type was added, stirred for a whole day and night, filtered through Celite, and the filtrate was distilled off. The residue was added to Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂Purification at O = 5: 10: 3) gave Compound 11 (215 mg, 92.2%).
[0148]
Rf 0.38 (CHCl_Three: MeOH = 6: 1)
C₇₃H₁₀₈O₁₉NS₂Na₂    MW: 1413.74
¹H-NMR (CDCl_Three+ CD_ThreeOD) δ:
4.403 (d, 1H, J = 7.8 Hz, H-1a), 1.593 (s, 3H, NHAc), 0.889 (t, 6H, J = 6.4 Hz, 2CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
172.93 (Me-CO), 105.24, 104.01 (C-1x2), 68.13, 68.00 (C-6x2)
[0149]
(i) 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranosyl- (1 → 1)- 2,3-di-O-tetradecyl-sn-glycerol disodium salt (2-acetamido-2-deoxy-6-O-sulfo-β-D-gulcopyranosyl- (1 → 3) -6-O-sulfo-β -D-galactopyranosyl- (1 → 1) -2,3-di-O-tetradecyl-sn-glycerol disodium salt) 12
Compound 11 (200 mg, 0.141 mmol) is dissolved in a mixture of methanol and water (3: 1, 15 mL), palladium hydroxide-carbon (200 mg) is added, and the mixture is replaced with hydrogen gas for 4 hours at room temperature. Catalytic reduction was performed. The reaction solution was filtered through celite, and the filtrate was distilled off. The residue was separated by Sephadex LH-20 (CHCl_Three: MeOH: H₂It refine | purified in the column of O = 5: 10: 3). Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂O = 1: 3: 1) and finally Sephadex LH-20 (CHCl_Three: MeOH: H₂Re-column purification with O = 5: 10: 3) was performed to obtain Compound 12 (119.6 mg, 80.5%).
[0150]
Rf 0.52 (CHCl_Three: MeOH: H₂(O = 12: 8: 1)
C₄₅H₈₄O₁₉NS₂Na₂    MW: 1053.24
¹H-NMR (DMSO + D₂O) δ:
4.666 (d, 1H, J = 8.3 Hz, H-1b), 4.162 (d, 1H, J = 7.3 Hz, H-1a), 4.067 (b.dd, 1H, H-6b), 4.006-3.924 (b .dd, 1H, H-6a), 1.882 (s, 3H, NHAc), 0.861 (t, 6H, J = 6.8 Hz, 2CH_Three)
¹³C-NMR (DMSO + D₂O) δ:
171.32 (Me-CO), 103.49, 102.37 (C-1x2), 65.91, 65.85 (C-6x2)
[0151]
[Example 4]
Synthesis of octyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt
According to the procedure outlined in FIG. 6, octyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside The disodium salt was synthesized.
The number after the substance name indicates the compound number in FIG.
[0152]
(a) Octyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside (octyl 2,4-di-O-acetyl-3,6-di- O-allyl-β-D-galactopyranoside) 13
Octanol (300 mg, 2.30 mmol), cyclopentadiene hafnonium dichloride (1.75 g, 5.99 mmol), silver triflate (2.36 g, 12.0 mmol), molecular sieve 4A (2.3 g) in 1,2-dichloroethane (5.0 The mixture was suspended at room temperature under an argon gas stream, cooled to −15 ° C., compound 1 (1.2 g, 3.47 mmol) was added, and the mixture was stirred for 2.5 hours. The reaction mixture was neutralized with triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 13 (0.8 g, 76%).
[0153]

C_{twenty four}H₄₀O₈    MW: 456.572
¹H-NMR (CDCl_Three) δ:
5.855 (m, 1H, Allyl), 5.784 (m, 1H, Allyl), 5.472 (d, 1H, J = 3.4 Hz, H-4), 5.086 (dd, 1H, J = 7.8, 9.8 Hz, H-2 ), 4.394 (d, 1H, J = 7.8 Hz, H-1), 2.125, 2.066 (2s, 6H, 2Ac), 0.879 (t, 3H, J = 6.8 Hz, CH_Three)
[0154]
(b) Octyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside (octyl 3,6-di-O-allyl-2,4-di- O-benzyl-β-D-galactopyranoside) 15
Compound 13 (0.8 g, 1.75 mmol) was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 5 mL), 1N-sodium hydroxide solution (1.0 mL) was added, and the mixture was stirred at room temperature for 1 day. Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was purified by silica gel column chromatography (toluene: AcOEt = 5: 2) to obtain Compound 14 (585 g, 89.7%).
[Rf 0.32 (toluene: AcOEt = 2: 1)]
[0155]
Subsequently, compound 14 (585 mg, 1.57 mmol) and N, N-dimethylformamide (3 ml) were dissolved, and sodium hydride (451 mg, 10.2 mmol) was added and stirred at −15 ° C. under an argon gas stream. . Subsequently, benzyl bromide (1.22 ml, 10.2 mmol) was added and stirred for 3 hours while gradually warming to room temperature. The reaction solution was neutralized with methanol, diluted with ethyl acetate, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: AcOEt = 15: 1) to obtain Compound 15 (811 mg, 93.5%).
[0156]
Rf 0.79 (Hexane: AcOEt = 6: 1)
C₃₄H₄₈O₆    MW: 552.747
¹H-NMR (CDCl_Three) δ:
5.947 (m, 1H, Allyl), 5.849 (m, 1H, Allyl), 4.959 (d, 1H, J = 11.7 Hz, Bn), 4.915 (d, 1H, J = 10.7 Hz, Bn), 4.765 (d, 1H, J = 10.7 Hz, Bn), 4.660 (d, 1H, J = 11.7 Hz, Bn), 4.346 (d, 1H, J = 7.6 Hz, H-1), 0.887 (t, 3H, J = 6.4 Hz , CH_Three)
[0157]
(c) Octyl 2,4-di-O-benzyl-β-D-galactopyranoside 16
Iridium complex (1,5-cyclooctadienebis (methyldiphenylphosphine) iridium hexafluorophosphate) (172 mg, 0.15 mmol) was suspended in tetrahydrofuran (5 mL), and H₂The mixture was stirred and activated under a stream of air. Compound 15 (811 mg, 1.47 mmol) was dissolved in tetrahydrofuran (5 mL) and added to the solution. After stirring for 1 hour at room temperature under an argon gas stream, iodine (745 mg), water (38 mL), and tetrahydrofuran (15 mL) was added, and the mixture was further stirred at room temperature for 1 hour. The reaction solution was diluted with chloroform, and washed successively with saturated sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate, and saturated brine. The chloroform layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (toluene: EtOAc = 5: 2) to obtain Compound 16 (589 mg, 82.1%). .
[0158]
Rf 0.35 (toluene: AcOEt = 2: 1)
C₂₈H₄₀O₆    MW: 472.618
¹H-NMR (CDCl_Three) δ:
5.000 (d, 1H, J = 11.7 Hz, Bn), 4.843 (d, 1H, J = 11.7 Hz, Bn), 4.674 (d, 1H, J = 11.7 Hz, Bn), 4.662 (d, 1H, J = 11.7 Hz, Bn), 4.360 (d, 1H, J = 7.3 Hz, H-1), 3.776 (d, 1H, J = 2.0 Hz, H-4), 0.868 (t, 3H, J = 6.8 Hz, CH_Three)
[0159]
(d) Octyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (octyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D- galactopyranoside) 17
Compound 16 (569 mg, 1.20 mmol) was dissolved by adding pyridine (17 mL), and pivaloyl chloride (188 μL, 1.57 mmol) was added to the solution, followed by stirring at −5 ° C. for 1 hour, and further pivaloyl. Chloride (188 μL, 1.57 mmol) was added and stirred at −5 ° C. for 1 hour. The reaction mixture was diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 17 (648 mg, 96.7%).
[0160]
Rf 0.56 (Toluene: AcOEt = 6: 1)
C₃₃H₄₈O₇    MW: 556.735
¹H-NMR (CDCl_Three) δ:
4.986 (d, 1H, J = 11.2 Hz, Bn), 4.838 (d, 1H, J = 11.7 Hz, Bn), 4.664 (d, 1H, J = 11.7 Hz, Bn), 4.653 (d, 1H, J = 11.7 Hz, Bn), 4.332 (d, 1H, J = 7.3 Hz, H-1), 4.316 (dd, 1H, J = 6.8, 11.7 Hz, H-6), 4.113 (dd, 1H, J = 6.7, 11.0 Hz, H-6 '), 3.772 (d, 1H, J = 2.4 Hz, H-4), 1.180 (s, 9H, piv), 0.867 (t, 3H, J = 6.8 Hz, CH_Three)
[0161]
(e) Octyl 3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (octyl 3-O-acetyl-2,4-di-O- benzyl-6-O-pivaloyl-β-D-galactopyranoside) 18
Compound 17 (10 mg, 18.0 μmol) was dissolved by adding pyridine (1 mL), acetic anhydride (0.5 mL) was added to the solution, and the mixture was stirred at room temperature for 2 hours. After azeotroping the solvent with toluene, the residue was separated by Sephadex LH-20 (CHCl_Three: MeOH = 1: 2) to obtain Compound 18 (11 mg, qu.).
[0162]
Rf 0.58 (Toluene: AcOEt = 8: 1)
C₃₅H₅₀O₈    MW: 598.772
¹H-NMR (CDCl_Three) δ:
4.903 (dd, 1H, J = 3.2, 10.0 Hz, H-3), 4.883 (d, 1H, J = 11.7 Hz, Bn), 4.645 (d, 1H, J = 11.7 Hz, Bn), 4.638 (d, 1H, J = 11.7 Hz, Bn), 4.545 (d, 1H, J = 11.2 Hz, Bn), 4.405 (d, 1H, J = 7.3 Hz, H-1), 4.311 (dd, 1H, J = 6.8, 10.8 Hz, H-6), 4.087 (dd, 1H, J = 6.8, 10.7 Hz, H-6 '), 3.847 (d, 1H, J = 2.9 Hz, H-4), 3.769 (dd, 1H, J = 7.8, 10.3 Hz, H-2), 1.937 (s, 3H, Ac), 1.188 (s, 9H, piv), 0.870 (t, 3H, J = 6.8 Hz, CH_Three)
[0163]
(f) Octyl 3,4-di-O-benzyl-2-deoxy-6-O-levroyl-2-phthalimide-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl- 6-O-Pivaloyl-β-D-galactopyranoside (octyl 3,4-di-O-benzyl-2-deoxy-6-O-levloyl-2-phtalimido-β-D-glucopyranosyl- (1 → 3 ) -2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside) 19
Compound 17 (624 mg, 1.12 mmol), cyclopentadiene hafnonium dichloride (850 mg, 2.91 mmol), silver triflate (1.15 g, 5.82 mmol), molecular sieve 4A (3.2 g) in 1,2-dichloroethane ( 10 ml), stirred at room temperature under a stream of argon gas, cooled to −15 ° C., added with compound 2 (838 mg, 1.46 mmol), and stirred for 1 hour. The reaction mixture was neutralized with triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 19 (1.03 g, 82.9%).
[0164]
Rf 0.47 (Toluene: AcOEt = 6: 1)
C₆₆H₇₉O₁₅N MW: 1126.342
¹H-NMR (CDCl_Three) δ:
5.475 (d, 1H, J = 8.3 Hz, H-1b), 4.189 (d, 1H, J = 7.8 Hz, H-1a), 3.874 (d, 1H, J = 2.4 Hz, H-4a), 2.133 ( s, 3H, CH_Three), 1.153 (s, 9H, piv), 0.826 (t, 3H, J = 7.1 Hz, CH_Three)
[0165]
(g) Octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-β-D-galactopila Noside (octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-β-D-galactopyranoside) 20
Compound 19 (1.0 g, 0.899 mmol) was suspended in ethanol (45 mL), hydrazine hydrate (4.5 mL) was added, and the mixture was stirred at 110 ° C. for 18 hours. The solvent was distilled off, and the resulting amino compound was dissolved in pyridine (5.0 mL), acetic anhydride (4.0 mL) was added, and the mixture was stirred at room temperature for 17 hours. The residue was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 20.0 mL), sodium methoxide (146 mg, 2.7 mmol) was added, and the mixture was stirred at 60 ° C. for 1 hr. The reaction solution was added to Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was purified by silica gel column chromatography (toluene: acetone = 2.3: 1), and Sephadex LH-20 (CHCl_Three: MeOH = 2: 3) to obtain compound 20 (739 mg, 96%).
[0166]
Rf 0.49 (toluene: acetone = 3: 2)
C₅₀H₆₅O₁₁N MW: 856.06
¹H-NMR (CDCl_Three) δ:
4.840 (d, 1H, J = 8.3 Hz, H-1b), 4.329 (d, 1H, J = 6.8 Hz, H-1a), 1.515 (s, 3H, NHAc), 0.848 (t, 3H, J = 6.8 Hz, CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
173.17 (Me-CO), 105.32, 104.01 (C-1x2), 62.82, 62.55 (C-6x2)
[0167]
(h) Octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl- 6-O-sulfo-β-D-galactopyranoside disodium salt (octyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- ( 1 → 3) -2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt) 21
Compound 20 (150 mg, 0.175 mmol) was dissolved in N, N-dimethylformamide (1.5 mL) and (C₂H_Five)_ThreeNSO_Three(319 mg, 1.75 mmol) was added, and the mixture was stirred at 50 ° C. for 0.5 hr. The reaction solution was directly added to Sephadex LH-20 (CHCl_Three: MeOH = 2: 3). The solvent of the eluate is distilled off to some extent, and water (2.0 mL), Dowex-50 (Na⁺) Type was added, stirred for a whole day and night, filtered through Celite, and the filtrate was distilled off. The residue was added to Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂Purification at O = 5: 10: 3) gave Compound 21 (185 mg, 99.6%).
[0168]
Rf 0.23 (CHCl_Three: MeOH = 6: 1)
C₅₀H₆₃O₁₇NS₂Na₂    MW: 1060.134
¹H-NMR (CDCl_Three+ CD_ThreeOD) δ:
4.382 (d, 1H, J = 7.3 Hz, H-1a), 1.634 (s, 3H, NHAc), 0.854 (t, 3H, J = 6.8 Hz, CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
173.03 (Me-CO), 105.10, 103.99 (C-1x2), 68.18 (C-6x2)
[0169]
(i) Octyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt (octyl 2 -acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt) 22
Compound 21 (170 mg, 0.160 mmol) was dissolved in a mixture of methanol and water (3: 1, 15 mL), palladium hydroxide-carbon (180 mg) was added, and the mixture was replaced with hydrogen gas. Catalytic reduction was performed. The reaction solution was filtered through celite, and the filtrate was distilled off. The residue was separated by Sephadex LH-20 (CHCl_Three: MeOH: H₂It refine | purified in the column of O = 5: 10: 3). Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂O = 1: 3: 1) and finally Sephadex LH-20 (CHCl_Three: MeOH: H₂Re-column purification with O = 5: 10: 3) was performed to obtain Compound 22 (101.4 mg, 90.6%).
[0170]
Rf 0.30 (CHCl_Three: MeOH: H₂(O = 12: 6: 1)
C_{twenty two}H₃₉O₁₇NS₂Na₂    MW: 699.636
¹H-NMR (DMSO + D₂O) δ:
4.655 (d, 1H, J = 8.3 Hz, H-1b), 4.136 (d, 1H, J = 7.8 Hz, H-1a), 4.053 (dd, 1H, J = 2.0, 11.7 Hz, H-6b), 3.956-3.851 (b.dd, 1H, H-6a), 1.869 (s, 3H, NHAc), 0.861 (t, 3H, J = 7.1 Hz, CH_Three)
¹³C-NMR (DMSO + D₂O) δ:
171.19 (Me-CO), 102.98, 102.30 (C-1x2), 66.09, 65.87 (C-6x2)
[0171]
[Example 5]
Cholestanyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt, and cholestanyl 2-acetamide Synthesis of 2-deoxy-β-D-glucopyranosyl- (1 → 3) -β-D-galactopyranoside
According to the procedure outlined in FIG. 7, cholestanyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside The disodium salt and cholestanyl 2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 3) -β-D-galactopyranoside were synthesized.
The number after the substance name indicates the number of the compound in FIG.
[0172]
(a) Cholestanyl 2,4-di-O-acetyl-3,6-di-O-allyl-β-D-galactopyranoside (cholestanyl 2,4-di-O-acetyl-3,6-di- O-allyl-β-D-galactopyranoside) 23
Cholestanol (700 mg, 1.80 mmol), cyclopentadiene hafnonium dichloride (1.37 g, 4.68 mmol), silver triflate (1.85 g, 9.37 mmol), molecular sieve 4A (2.7 g) in 1,2-dichloroethane ( 7.0 ml) and stirred at room temperature under a stream of argon gas, cooled to −10 ° C., added with compound 1 (936 mg, 2.70 mmol), and stirred for 2.5 hours. The reaction solution was neutralized by adding triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 23 (970 mg, 75.3%).
[0173]
Rf 0.57 (Toluene: AcOEt = 6: 1)
C₄₃H₇₀O₈    MW: 715.018
¹H-NMR (CDCl_Three) δ:
5.880 (m, 1H, Allyl), 5.802 (m, 1H, Allyl), 5.480 (d, 1H, J = 3.4 Hz, H-4), 5.072 (dd, 1H, J = 8.3, 10.3 Hz, H-2 ), 4.510 (d, 1H, J = 8.3 Hz, H-1), 2.150, 2.095 (2s, 6H, 2Ac), 0.922 (d, 3H, J = 6.4 Hz, CH_Three), 0.890 (d, 3H, J = 6.8 Hz, CH_Three), 0.885 (d, 3H, J = 6.4 Hz, CH_Three), 0.800, 0.667 (2s, 6H, 2CH_Three)
[0174]
(b) Cholestanyl 3,6-di-O-allyl-2,4-di-O-benzyl-β-D-galactopyranoside (cholestanyl 3,6-di-O-allyl-2,4-di- O-benzyl-β-D-galactopyranoside) 25
Compound 23 (0.98 g, 1.37 mmol) was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 10 mL), 1 N sodium hydroxide solution (1.0 mL) was added, and the mixture was stirred at room temperature for 1 day. Metoxide (74 mg, 1.37 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was added to Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was purified by silica gel column chromatography (toluene: AcOEt = 5: 2) to obtain Compound 24 (0.83 g, 96%).
[Rf 0.33 (toluene: AcOEt = 2: 1)]
[0175]
Subsequently, Compound 24 (838 mg, 1.32 mmol) was dissolved in N, N-dimethylformamide (8 ml), and sodium hydride (378 mg, 8.58 mmol) was added and stirred at 0 ° C. under an argon gas stream. . Subsequently, benzyl bromide (1.02 ml, 8.58 mmol) was added, and the mixture was stirred for 3 hours while gradually warming to room temperature. Further, sodium hydride (378 mg, 8.58 mmol) and benzyl bromide (1.02 ml, 8.58 mmol) were added and stirred for 18 hours. The reaction solution was neutralized with methanol, diluted with ethyl acetate, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: AcOEt = 13: 1) to obtain Compound 25 (0.94 g, 88.1%).
[0176]
Rf 0.72 (Hexane: AcOEt = 6: 1)
C₅₃H₇₈O₆    MW: 811.193
¹H-NMR (CDCl_Three) δ:
5.935 (m, 1H, Allyl), 5.836 (m, 1H, Allyl), 4.931 (d, 1H, J = 11.7 Hz, Bn), 4.903 (d, 1H, J = 10.7 Hz, Bn), 4.739 (d, 1H, J = 10.7 Hz, Bn), 4.639 (d, 1H, J = 11.7 Hz, Bn), 4.438 (d, 1H, J = 7.8 Hz, H-1), 3.829 (d, 1H, J = 2.9 Hz , H-4), 3.719 (dd, 1H, J = 7.8, 9.8 Hz, H-2), 3.397 (dd, 1H, J = 2.9, 9.8 Hz, H-3), 0.893 (d, 3H, J = 6.4 Hz, CH_Three), 0.862 (d, 3H, J = 6.8 Hz, CH_Three), 0.858 (d, 3H, J = 6.4 Hz, CH_Three), 0.799, 0.641 (2s, 6H, 2CH_Three)
[0177]
(c) Cholestanyl 2,4-di-O-benzyl-β-D-galactopyranoside 26
Iridium complex (1,5-cyclooctadienebis (methyldiphenylphosphine) iridium hexafluorophosphate) (136 mg, 0.12 mmol) was suspended in tetrahydrofuran (5 mL), and H₂The mixture was stirred and activated under a stream of air. Compound 25 (940 mg, 1.16 mmol) dissolved in tetrahydrofuran (5 mL) was added to the solution, stirred for 1 hour at room temperature under a stream of argon gas, iodine (588 mg), water (30 mL) and tetrahydrofuran ( 15 mL) was added and the mixture was further stirred at room temperature for 1.5 hours. The reaction solution was diluted with chloroform, and washed successively with saturated sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate, and saturated brine. The chloroform layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (toluene: acetone = 6: 1) to obtain Compound 26 (748 mg, 88.2%). .
[0178]
Rf 0.49 (toluene: acetone = 4: 1)
C₄₇H₇₀O₆    MW: 731.064
¹H-NMR (CDCl_Three) δ:
5.006 (d, 1H, J = 11.2 Hz, Bn), 4.823 (d, 1H, J = 11.2 Hz, Bn), 4.678 (d, 1H, J = 10.7 Hz, Bn), 4.650 (d, 1H, J = 11.7 Hz, Bn), 4.476 (d, 1H, J = 6.8 Hz, H-1)
[0179]
(d) Cholestanyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (cholestanyl 2,4-di-O-benzyl-6-O-pivaloyl-β-D- galactopyranoside) 27
Compound 26 (647 mg, 0.885 mmol) was dissolved in pyridine (13 mL), pivaloyl chloride (138 μL, 1.15 mmol) was added to the solution, and the mixture was stirred at -5 ° C. to 0 ° C. for 0.5 hour, and further pivalo Ilchloride (138 µL, 1.15 mmol) was added and stirred at -5 ° C to 0 ° C for 1 hour. The reaction mixture was diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 27 (663 mg, 91.9%).
[0180]
Rf 0.56 (Toluene: AcOEt = 6: 1)
C₅₂H₇₈O₇    MW: 815.181
¹H-NMR (CDCl_Three) δ:
4.993 (d, 1H, J = 11.7 Hz, Bn), 4.821 (d, 1H, J = 11.2 Hz, Bn), 4.667 (d, 1H, J = 11.2 Hz, Bn), 4.642 (d, 1H, J = 11.7 Hz, Bn), 4.450 (d, 1H, J = 7.3 Hz, H-1), 4.293 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.083 (dd, 1H, J = 6.3, 10.8 Hz, H-6 '), 3.746 (d, 1H, J = 2.0 Hz, H-4), 1.176 (s, 9H, piv)
[0181]
(e) Cholestanyl 3-O-acetyl-2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside (cholestanyl 3-O-acetyl-2,4-di-O- benzyl-6-O-pivaloyl-β-D-galactopyranoside) 28
Compound 27 (10 mg, 12.3 μmol) was dissolved in pyridine (1 mL), and acetic anhydride (0.5 mL) was added to the solution, followed by stirring at room temperature for 1 hour. After azeotroping the solvent with toluene, the residue was separated by Sephadex LH-20 (CHCl_Three: MeOH = 1: 2) to obtain Compound 28 (9 mg, 85.4%).
[0182]
Rf 0.65 (Toluene: AcOEt = 8: 1)
C₅₄H₈₀O₈    MW: 857.218
¹H-NMR (CDCl_Three) δ:
4.889 (dd, 1H, J = 2.7, 10.5 Hz, H-3), 4.885 (d, 1H, J = 11.2 Hz, Bn), 4.647 (d, 1H, J = 11.7 Hz, Bn), 4.625 (d, 1H, J = 11.2 Hz, Bn), 4.533 (d, 1H, J = 11.7 Hz, Bn), 4.518 (d, 1H, J = 7.3 Hz, H-1), 4.289 (dd, 1H, J = 6.8, 11.2 Hz, H-6), 4.061 (dd, 1H, J = 6.3, 11.2 Hz, H-6 '), 3.766 (d, 1H, J = 2.4 Hz, H-4), 3.753 (dd, 1H, J = 7.3, 10.3 Hz, H-2), 1.924 (s, 3H, Ac), 1.185 (s, 9H, piv)
[0183]
(f) Cholestanyl 3,4-di-O-benzyl-2-deoxy-6-O-levroyl-2-phthalimide-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl- 6-O-Pivaloyl-β-D-galactopyranoside (cholestanyl 3,4-di-O-benzyl-2-deoxy-6-O-levloyl-2-phtalimido-β-D-glucopyranosyl- (1 → 3 ) -2,4-di-O-benzyl-6-O-pivaloyl-β-D-galactopyranoside) 29
Compound 27 (737 mg, 0.904 mmol), cyclopentadiene hafnonium dichloride (686 mg, 2.35 mmol), silver triflate (927 mg, 4.70 mmol) and molecular sieve 4A (2.5 g) were added to 1,2-dichloroethane ( 10 ml), stirred at room temperature under an argon gas stream, cooled to −15 ° C., added with compound 2 (676.5 mg, 1.18 mmol), and stirred for 1 hour. The reaction solution was neutralized by adding triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (toluene: AcOEt = 10: 1) to obtain Compound 29 (982.1 mg, 79.3%).
[0184]
Rf 0.58 (Toluene: AcOEt = 6: 1)
0.07 (hexane: AcOEt = 6: 1)
C₈₅H₁₀₉O₁₅N MW: 1384.788
¹H-NMR (CDCl_Three) δ:
5.475 (d, 1H, J = 8.3 Hz, H-1b), 4.453 (d, 1H, J = 7.8 Hz, H-1a), 3.852 (d, 1H, J = 2.9 Hz, H-4a), 2.133 ( s, 3H, CH_Three), 1.146 (s, 9H, piv)
[0185]
(g) Cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-β-D-galactopila Noside (cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl-β-D-galactopyranoside) 30
Compound 29 (668 mg, 0.488 mmol) was suspended in ethanol (24.5 mL), hydrazine hydrate (2.45 mL) was added, and the mixture was stirred at 110 ° C. for 18 hours. The solvent was distilled off, and the resulting amino compound was dissolved in pyridine (5.0 mL), acetic anhydride (4.0 mL) was added, and the mixture was stirred at room temperature for 17 hours. The residue was dissolved in a mixture of methanol and tetrahydrofuran (1: 1, 10.0 mL), sodium methoxide (78.8 mg, 1.46 mmol) was added, and the mixture was stirred at 60 ° C. for 1 hr. The reaction solution was added to Amberlyst 15E (H⁺) Type and neutralized with celite, and then the filtrate was distilled off. The residue was purified by silica gel column chromatography (toluene: acetone = 3: 1), and Sephadex LH-20 (CHCl_Three: MeOH = 2: 3) to obtain Compound 30 (534.2 mg, 99.6%).
[0186]
Rf 0.34 (toluene: acetone = 3: 1)
C₆₉H₉₅O₁₁N MW: 1114.506
¹H-NMR (CDCl_Three) δ:
5.044 (d, 1H, J = 12.2 Hz, NH), 4.875 (d, 1H, J = 11.2 Hz, Bn), 4.851 (d, 1H, J = 7.8 Hz, H-1b), 4.809 (d, 1H, J = 11.2 Hz, Bn), 4.740 (d, 1H, J = 11.7 Hz, Bn), 4.696 (d, 1H, J = 11.7 Hz, Bn), 4.672 (d, 1H, J = 12.2 Hz, Bn), 4.642 (d, 1H, J = 10.7 Hz, Bn), 4.571 (d, 1H, J = 11.2 Hz, Bn), 4.562 (d, 1H, J = 12.2 Hz, Bn), 4.439 (d, 1H, J = 6.4 Hz, H-1a), 1.524 (s, 3H, NHAc), 0.892 (d, 3H, J = 6.3 Hz, CH_Three), 0.860 (d, 3H, J = 6.8 Hz, CH_Three), 0.856 (d, 3H, J = 6.8 Hz, CH_Three), 0.760, 0.635 (2s, 6H, 2CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
173.00 (Me-CO), 103.79, 103.06 (C-1x2), 62.77, 62.40 (C-6x2)
[0187]
(h) Cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -2,4-di-O-benzyl- 6-O-sulfo-β-D-galactopyranoside disodium salt (cholestanyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- ( 1 → 3) -2,4-di-O-benzyl-6-O-sulfo-β-D-galactopyranoside disodium salt) 31
Compound 30 (150 mg, 0.136 mmol) was dissolved in N, N-dimethylformamide (1.5 mL) and (C₂H_Five)_ThreeNSO_Three(247 mg, 1.36 mmol) was added, and the mixture was stirred at 50 ° C. for 0.5 hr. The reaction solution was directly added to Sephadex LH-20 (CHCl_Three: MeOH = 2: 3). The solvent of the eluate is distilled off to some extent, and water (2.0 mL), Dowex-50 (Na⁺) Type was added, stirred for a whole day and night, filtered through Celite, and the filtrate was distilled off. The residue was added to Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂Purification at O = 5: 10: 3) gave Compound 31 (175 mg, 97.3%).
[0188]
Rf 0.22 (CHCl_Three: MeOH = 8: 1)
C₆₉H₉₃O₁₇NS₂Na₂    MW: 1318.58
¹H-NMR (CD_ThreeOD) δ:
4.523 (d, 1H, J = 7.3 Hz, H-1a), 4.384 (dd, 1H, J = 2.0, 10.8 Hz, H-6b), 4.290 (dd, 1H, J = 4.4, 10.7 Hz, H-6a ), 1.645 (s, 3H, NHAc), 0.910 (d, 3H, J = 6.8 Hz, CH_Three), 0.873 (d, 3H, J = 6.4 Hz, CH_Three), 0.868 (d, 3H, J = 6.8 Hz, CH_Three), 0.730, 0.658 (2s, 6H, 2CH_Three)
¹³C-NMR (CDCl_Three+ CD_ThreeOD) δ:
102.97, 102.55 (C-1x2), 68.47, 67.22 (C-6x2)
[0189]
(i) Cholestanyl 2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt (cholestanyl 2 -acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranosyl- (1 → 3) -6-O-sulfo-β-D-galactopyranoside disodium salt) 32
Compound 31 (170 mg, 0.129 mmol) was dissolved in a mixture of methanol and water (3: 1, 15 mL), palladium hydroxide-carbon (180 mg) was added, and the mixture was replaced with hydrogen gas. Catalytic reduction was performed. The reaction solution was filtered through celite, and the filtrate was distilled off. The residue was separated by Sephadex LH-20 (CHCl_Three: MeOH: H₂It refine | purified in the column of O = 5: 10: 3). Dowex-50 (Na⁺) Type column (CHCl_Three: MeOH: H₂O = 1: 3: 1) and finally Sephadex LH-20 (CHCl_Three: MeOH: H₂Re-column purification with O = 5: 10: 3) was performed to obtain Compound 32 (109.4 mg, 88.6%).
[0190]
Rf 0.47 (CHCl_Three: MeOH: H₂(O = 12: 8: 1)
C₄₁H₆₉O₁₇NS₂Na₂    MW: 958.08
¹H-NMR (CDCl_Three) δ:
4.727 (d, 1H, J = 7.8 Hz, H-1b), 4.310 (d, 1H, J = 7.8 Hz, H-1a), 4.093 (b.dd, 1H, H-6b), 4.010-3.924 (b .dd, 1H, H-6a), 1.926 (s, 3H, NHAc), 0.945 (d, 3H, J = 6.4 Hz, CH_Three), 0.909 (d, 3H, J = 6.4 Hz, CH_Three), 0.905 (d, 3H, J = 6.8 Hz, CH_Three), 0.827, 0.689 (2s, 6H, 2CH_Three)
¹³C-NMR (DMSO + D₂O) δ:
170.88 (Me-CO), 102.21, 100.73 (C-1x2), 65.85, 65.65 (C-6x2)
[0191]
(j) Cholestanyl 2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 3) -β-D-galactopyranoside → 3) -β-D-galactopyranoside) 33
Dissolve compound 30 (230 mg, 0.209 mmol) in a mixture of methanol, water and ethyl acetate (4: 1: 1, 25 mL), add palladium hydroxide-carbon (230 mg), and replace with hydrogen gas. Then, catalytic reduction was performed at room temperature for 20 hours. The reaction solution was filtered through celite, and the filtrate was distilled off. The residue was separated by Sephadex LH-20 (CHCl_Three: MeOH = 1: 1) to obtain compound 33 (126.1 mg, 81.5%).
[0192]
Rf 0.51 (CHCl_Three: MeOH = 3: 1)
C₄₁H₇₁O₁₁N MW: 754.008
¹H-NMR (C_FiveD_FiveN + D₂O) δ:
5.383 (d, 1H, J = 8.3 Hz, H-1b), 4.825 (d, 1H, J = 7.3 Hz, H-1a), 2.059 (s, 3H, NHAc)
[0193]
[Example 6]
Safety and pharmacological studies
In this example, as a keratan sulfate oligosaccharide, sodium salt of L4, sodium salt of keratan sulfate tetrasaccharide (L4L4) in which two L4s are linked by β1-3 bond, sodium salt of K4 (compound 28 of Example 1) , K2 sodium salt, and G4L4 sodium salt were used (see FIG. 8 for the oligosaccharide structures indicated by abbreviations). L4 and L4L4 were obtained by the method described in International Publication Pamphlet No. WO96 / 16973.
[0194]
The sodium salt of K2 was obtained by the following method.
10 g of keratan sulfate derived from bovine cornea was dissolved in 120 ml of 0.1 M Tris-HCl buffer (pH 7.5). To this solution, 1,000 units of Pseudomonas sp.-derived keratanase (manufactured by Seikagaku Corporation) was added and digested at 37 ° C. for 50 hours. After completion of the reaction, 1.3 times the amount of ethanol was added and stirred, and left at room temperature overnight. The next day, the supernatant and the precipitate were separated by centrifugation (10,000 rpm, 20 minutes), the supernatant was concentrated under reduced pressure, and the concentrated solution was lyophilized to obtain 9 g of a dried product. The obtained lyophilized product was dissolved in a small amount of distilled water, and gel chromatography was performed using Cellulofine GCL-90m (manufactured by Chisso Corporation) (4.5 cm x 125 cm) using a 0.2 M salt concentration solution as an elution solvent. Fractions containing were collected. The obtained K2 fraction was concentrated under reduced pressure, desalted by gel filtration chromatography using Cellulofine GCL-25m (manufactured by Chisso Corporation) (4.0 cm × 120 cm) with distilled water as an elution solvent, and freeze-dried.
[0195]
Dissolve the fraction containing K2 in a small amount of distilled water and equilibrate with distilled water in advance. Use Muromac 1x4 (200-400) (Muromachi Chemical Co., Ltd.) (2.0 cm x 32 cm), and add salt to the elution solvent. The salt concentration was linearly increased from 0 to 2M, and the further purified K2 fraction was separated and eluted. The obtained K2 fraction was concentrated under reduced pressure, desalted by gel filtration chromatography using Cellulofine GCL-25m (4.0 cm × 120 cm), and lyophilized to obtain 1.9 g of a dried product of K2.
[0196]
(1) Safety test
1. Single dose toxicity study in mice
Normal mice (5 mice per group) were administered a single intravenous dose of K4 or G4L4 at a dose of 2,000 mg / kg, and general symptoms were observed for 14 days.
[0197]
There were no deaths in either sex groups. Paralytic gait was observed in all male and female cases immediately after administration by K4 or G4L4 administration, but all cases recovered to normal approximately 3 minutes after administration. In terms of body weight, slight weight loss was observed in the male group on the day after administration, but thereafter all patients showed steady weight gain. At autopsy, no abnormalities due to K4 or G4L4 administration were observed in all cases. From the above results, it is considered that the minimum death dose in a single intravenous administration of K4 and G4L4 is 2,000 mg / kg or more. Thus, LD in a single intravenous dose of K4 and G4L4₅₀The value exceeds 2,000 mg / kg for both males and females.
[0198]
2. Antigenicity test using guinea pig
The guinea pig was sensitized by subcutaneous injection in the back of the guinea pig with three injections of K4 or G4L4 alone or an emulsion with Freund's complete adjuvant (FCA) as an immune adjuvant. 12 days after the final sensitization, K4 or G4L4 was intravenously administered to elicit an active systemic anaphylactic reaction. In addition, ovalbumin (OVA) was tested in the same manner as a positive control.
[0199]
The above results are shown in Table 1 together with the dose. No anaphylactic reaction was observed in guinea pigs induced with K4 or G4L4. An anaphylactic reaction was observed in ovalbumin, a positive control of the test system. From the above results, it is considered that K4 and G4L4 do not induce an active systemic anaphylactic reaction in guinea pigs.
[0200]
[Table 1]

[0201]
(2) Medicinal pharmacology test
1. Examination of the effect on Ca-ionophore-induced hypervascular permeability
After shaving the back of the rat under ether anesthesia, 0.1 ml of a Ca-ionophore (A23187, Wako Pure Chemicals) solution (10 μg / ml) dissolved in 2% DMSO was administered to one site in the skin. As a negative control, 0.1 ml of 2% DMSO solution was administered to one site in the skin. The test substance was dissolved in a Ca-ionophore solution, and 0.1 ml thereof was administered to several sites in the skin. Immediately thereafter, 1 ml of 0.5% Evans blue was intravenously administered, and 30 minutes later, the blood was lethal under ether anesthesia. The skin was peeled off, and the Evans blue leakage site was punched out with trepan, and this was used for measurement of the amount of pigment leakage. The amount of dye leakage was determined according to the method of Katayama et al. (Microbiol. Immunol., 22, 89-101 (1978)). That is, 1 NKOH is added to the collected skin and hydrolyzed overnight, then 0.6 NH_ThreePO_FourThe mixture was neutralized by adding an acetone solution (mixed at a ratio of 5:13), centrifuged, and the amount of dye leakage was determined from the absorbance (620 nm) of the supernatant.
[0202]
The results when L4, L4L4, K4 and K2 are used as test substances are shown in FIG. The results when L4 and G4L4 are used as test substances are shown in FIG.
[0203]
L4L4, K4, and K2 suppress vascular permeability enhancement in a dose-dependent manner, and are significant at concentrations of 2.5 to 10 mg / site, 0.63 to 5 mg / site, and 1.25 to 5 mg / site, respectively. The inhibitory effect was shown. The inhibitory effect was strongest for K4, followed by K2 and L4L4. G4L4 also showed a significant inhibitory effect on increased vascular permeability. On the other hand, L4 hardly suppressed the increase in vascular permeability in this model. From this result, it is suggested that K4, K2, L4L4 and G4L4 exert an antiallergic action by suppressing an increase in vascular permeability. In particular, it is suggested that K4 exhibits an excellent action.
[0204]
In this study, L4L4, K4, K2 and G4L4 significantly suppressed the increase in vascular permeability caused by Ca-ionophore, so that the K2 structure is important for the expression of its activity, and the action of the K4 structure may increase. It was suggested. In addition, L4 did not show an inhibitory effect, suggesting that the L4 structure is not very important in this model. On the other hand, although L4L4 is a β- (1-3) bond between two L4 structures, it was presumed that an inhibitory effect was observed in this model because it has a K4 structure. L4L4, K4, K2 and G4L4 are Ca stabilized by membrane stabilization²⁺It was suggested that the increase in vascular permeability was suppressed by inhibiting the inflow into cells or degranulation, or suppressing histamine secreted from mast cells.
[0205]
2. Guinea pig neutrophil O stimulated by FMLP (N-formyl-Met-Leu-Phe)₂ ^-Examination of effects on production
A 0.2% aqueous solution of glycogen dissolved in physiological saline was sterilized by autoclaving, and 20 ml was administered intraperitoneally to a Hartley female guinea pig. After 16 hours, blood was killed and 20 ml of physiological saline containing 10 U / ml of heparin was injected into the peritoneal cavity, and the peritoneal exudate was collected. The collected liquid was centrifuged at 1000 rpm for 10 minutes using a tabletop centrifuge, purified water was added to the precipitate, hemolyzed for 30 seconds, returned to isotonicity with a double concentration Hanks solution, and centrifuged at 1000 rpm for 10 minutes. The precipitate was resuspended in Hanks' solution and the centrifugation operation was repeated twice to obtain neutrophils. The collected guinea pig neutrophils are suspended in Hank's solution, the white blood cell count is measured using a blood cell measurement device (Sysmex K-2000), and 2 × 10 with Hank's solution.⁶Those diluted to cells / ml were used as cell suspension in the experiment.
[0206]
1 ml of cell suspension and 10 μl of a test substance solution having a known concentration (mixed with no keratan sulfate oligosaccharide as a control) were mixed and preincubated for 1 hour at 37 ° C. 50 μl of 6 mM cytochrome C solution and 100 μl of 0.1 mM FMLP were sequentially added and mixed. This was incubated at 37 ° C. for 10 minutes, cooled with ice to stop the reaction, centrifuged at 3000 rpm for 5 minutes, and the absorbance of the supernatant was measured at a wavelength of 550 nm. All the above operations were performed under ice-cooling except for incubation.
[0207]
The results when L4, L4L4, K4 and K2 are used as test substances are shown in FIG. The results when L4 and G4L4 are used as test substances are shown in FIG.
[0208]
L4L4, K4 and G4L4 are OLPs from neutrophils stimulated by FMLP at concentrations of 0.01-1 mg / ml.₂ ^-Production (O₂ ^-generation) was significantly suppressed. The inhibition rate at 1 mg / ml was 50.6%, 44.7% and 57.1%, respectively, with respect to the control. O for L4 and K2₂ ^-The production inhibitory effect was hardly recognized. From this result, it is suggested that L4L4, K4 and G4L4 exert an anti-inflammatory action by suppressing the production of active oxygen in neutrophils.
[0209]
In this test, although L4 and K4 have the same charge, composition formula and constituent sugar, K4 has an inhibitory effect, and L4L4 shows an inhibitory effect, whereas L4 which is a constituent sugar shows activity. I couldn't. L4L4, K4 and G4L4 are FMLP-stimulated guinea pig neutrophils O₂ ^-The production was significantly suppressed, suggesting that the K4 structure is important for the expression of its activity. Moreover, since K2 did not show an inhibitory effect, it was suggested that the degree of sulfation is important in this model. It was suggested that L4L4, K4 and G4L4 having the K4 structure may inhibit GTP-binding proteins or phospholipase C or other stimulus transmission systems, or may directly antagonize the FMLP receptor to express the action.
[0210]
【The invention's effect】
According to the present invention, a keratan sulfate oligosaccharide having a galactose residue at the reducing end can be efficiently produced. In particular, a keratan sulfate oligosaccharide in which the hydroxyl group at the 6-position of a galactose residue and / or an N-acetylglucosamine residue is sulfated can be provided. A keratan sulfate oligosaccharide having a galactose residue in which the hydroxyl group at the 6-position is sulfated at the reducing end has an excellent pharmacological effect and can provide a safe and effective novel pharmaceutical composition.
[Brief description of the drawings]
FIG. 1 shows the structure of keratan sulfate disaccharide predicted from the structure of keratan sulfate.
FIG. 2 shows an outline of an example of a method for producing a starting material in the production of the oligosaccharide of the present invention.
FIG. 3 shows an outline of an example of a method for producing a starting material in the production of the oligosaccharide of the present invention.
FIG. 4 shows an outline of an example of a method for producing an oligosaccharide of the present invention.
FIG. 5 shows an outline of an example of a method for producing an oligosaccharide of the present invention.
FIG. 6 shows an outline of an example of a method for producing an oligosaccharide of the present invention.
FIG. 7 shows an outline of an example of a method for producing an oligosaccharide of the present invention.
FIG. 8 shows the structure of keratan sulfate oligosaccharide.
FIG. 9 shows the effect of keratan sulfate oligosaccharide on enhanced vascular permeability.
FIG. 10 shows the effect of keratan sulfate oligosaccharide on enhanced vascular permeability.
FIG. 11. Keratan sulfate oligosaccharide neutrophil O₂ ^-The effect on production is shown.
FIG. 12: Neutrophil O of keratan sulfate oligosaccharide₂ ^-The effect on production is shown.

Claims (11)

A step of reacting a monosaccharide represented by the following general formula (1) and a monosaccharide represented by the following general formula (2) with a glycosidic bond, and substituting each of R ³ and R ⁷ with a hydrogen atom; Comprising the step of substituting atoms with —SO ₃ M, wherein the elimination of R ³ , R ⁴ and R ⁷ is carried out in one step by making the solvent alkaline after conversion of the protecting group of R ⁴ , A method for producing the oligosaccharide represented by (3).

(In the formula, R ¹ and R ² each independently represent an aralkyl group, R ³ represents an acyl group, R ⁴ represents an amino protecting group, and R ⁵ represents a leaving group.)

Wherein R ⁶ and R ⁸ each independently represent an aralkyl group, R ⁷ represents an acyl group, R ⁹ represents an aralkyl group, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, glycerol A residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue or a peptide residue, and Z is an oxygen atom or -NHCO-, where the aralkyl group is benzyl group, p-methoxybenzyl group, phenethyl group, 3-phenylpropyl group, p-nitrobenzyl group, o-nitrobenzyl group, p-halobenzyl group, p- Cyanobenzyl group, diphenylmethyl group, triphenylmethyl group (trityl group), α or β-naphthylmethyl group, or α-naphthyldiph An alkyl group having 1 to 23 carbon atoms, an O-alkylglycerol residue being a di-O-alkylglycerol residue, and an O-acylglycerol residue being a di-O-acylglycerol residue. The N-acyl group in the ceramide has 1 to 28 carbon atoms, and the phospholipid residue is a glycerophospholipid residue or a sphingophospholipid residue, wherein R ⁹ is an aralkyl group, 6 -O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue or phospholipid residue In some cases, Z is an oxygen atom, and when R ⁹ is a biotin residue or a peptide residue, Z is —NHCO—.)

(Wherein R ¹⁰ and R ¹¹ each independently represent —SO ₃ M (M represents a proton or a monovalent cation), Ac represents an acetyl group, R ¹² represents a hydrogen atom, 6 -O-sulfated N-acetylglucosamine residue, alkyl group, glycerol residue, O-alkylglycerol residue, O-acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue, phospholipid residue, biotin Z represents an oxygen atom or —NHCO—, wherein the alkyl group has 1 to 23 carbon atoms, and the O-alkylglycerol residue represents di-O-alkyl. Glycerol residue, O-acylglycerol residue is di-O-acylglycerol residue, N-acyl group in ceramide has 1 to 28 carbon atoms, phospholipid residue is Is a glycerophospholipid residue or sphingophospholipid residues. In addition, R ¹² is a hydrogen atom, 6-O-sulfated N- acetylglucosamine residue, an alkyl group, glycerol residue, O- alkyl glycerol residue, O- Z is an oxygen atom when it is an acylglycerol residue, cholesterol residue, cholestanyl group, ceramide residue or phospholipid residue, and when R ¹² is a biotin residue or a peptide residue, Z Is —NHCO—. The production method according to claim 1, wherein the general formulas (1) and (2) are represented by the following formulas (4) and (5), respectively.

(In the formula, Bn represents a benzyl group, R ¹³ represents an acetyl group or a levulinoyl group, Phth represents a phthaloyl group, and X represents a halogen atom.)

(Wherein, Bn is a benzyl group, R ¹⁴ is a benzyl group, 6-O-sulfated N- acetylglucosamine residue, an alkyl group, glycerol residue, O- alkyl glycerol residue, O- acylglycerol residue, A cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue or a peptide residue, Z represents an oxygen atom or —NHCO—, and Piv represents a pivaloyl group.) An oligosaccharide represented by the following general formula (13).

(Wherein R ¹⁶ and R ¹⁷ each independently represent —SO ₃ M (M represents a proton or a monovalent cation), Ac represents an acetyl group, R ¹⁸ represents a hydrogen atom, an alkyl group, A glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue or a peptide residue, and Z is an oxygen atom Or -NHCO-, wherein the alkyl group has 1 to 23 carbon atoms, the O-alkylglycerol residue is a di-O-alkylglycerol residue, and the O-acylglycerol residue is di- -O-acylglycerol residue, N-acyl group in ceramide has 1 to 28 carbon atoms, phospholipid residue is glycerophospholipid residue or sphingophospholipid residue It. Incidentally, R ¹⁸ is a hydrogen atom, an alkyl group, glycerol residue, O- alkyl glycerol residue, O- acylglycerol residue, a cholesterol residue, cholestanyl group, when ceramide residue, or a phospholipid residue In the above, Z is an oxygen atom, and when R ¹⁸ is a biotin residue or a peptide residue, Z is —NHCO—. The oligosaccharide according to claim 3, wherein Z is an oxygen atom. The oligosaccharide according to claim 3, wherein Z is an oxygen atom and R ¹⁸ is a hydrogen atom. The oligosaccharide according to claim 3, wherein Z is an oxygen atom, and R ¹⁸ is an alkyl group. The oligosaccharide according to claim 3, wherein Z is an oxygen atom and R ¹⁸ is an O-alkylglycerol residue. The oligosaccharide according to claim 3, wherein Z is an oxygen atom and R ¹⁸ is a cholestanyl group. A pharmaceutical comprising the oligosaccharide according to claim 5 or a pharmaceutically acceptable salt thereof as an active ingredient. An antiallergic agent comprising the oligosaccharide according to claim 5 or a pharmaceutically acceptable salt thereof as an active ingredient. An antiallergic agent comprising an oligosaccharide represented by the following general formula (13) or a pharmaceutically acceptable salt thereof as an active ingredient.

(Wherein R ¹⁶ and R ¹⁷ each independently represent —SO ₃ M (M represents a proton or a monovalent cation), Ac represents an acetyl group, and R ¹⁸ represents 6-O-sulfation.) N-acetylglucosamine residue, and Z represents an oxygen atom.

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