JP2009274955A

JP2009274955A - Osteoarthritis-treating medicine

Info

Publication number: JP2009274955A
Application number: JP2006227822A
Authority: JP
Inventors: Shinichiro Nishimura; 紳一郎西村
Original assignee: Hokkaido University NUC; Shionogi and Co Ltd
Current assignee: Hokkaido University NUC; Shionogi and Co Ltd
Priority date: 2006-08-24
Filing date: 2006-08-24
Publication date: 2009-11-26

Abstract

【課題】プロテオグリカン、特にコンドロイチン硫酸タイプの生成を選択的に促進する軟骨疾患の予防および／または治療薬を提供する。
【解決手段】下式

（式中、Ｘはハロゲン原子、水酸基またはアルコキシ基を表し、Ｒは水素原子、置換されていてもよいアルキル、置換されていてもよいアリール、置換されていてもよいヘテロアリールまたは天然若しくは非天然のアミノ酸からなるペプチド残基を表す。）
で表される化合物、その薬学上許容される塩、またはそれらの溶媒和物を有効成分とする医薬。
【選択図】なしDisclosed is a prophylactic and / or therapeutic agent for cartilage diseases that selectively promotes the production of proteoglycans, particularly chondroitin sulfate type.
[Solution] The following formula

(In the formula, X represents a halogen atom, a hydroxyl group or an alkoxy group, and R represents a hydrogen atom, an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or natural or non-natural. Represents a peptide residue comprising the amino acids of
Or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
[Selection figure] None

Description

本発明は軟骨疾患の予防または治療に有用な薬物に関する。より詳細には、プロテオグリカンの生成促進剤を有効成分とする、変形性関節症およびその他の軟骨疾患の予防または治療剤に関する。 The present invention relates to a drug useful for prevention or treatment of cartilage disease. More specifically, the present invention relates to a prophylactic or therapeutic agent for osteoarthritis and other cartilage diseases, which comprises a proteoglycan production promoter as an active ingredient.

軟骨は軟骨細胞とこれを取り囲む基質からなる結合組織であり、関節、脊柱の椎間板、肋軟骨、耳介、外耳道、恥骨結合、咽喉蓋などに存在する。軟骨は、軟骨細胞と、軟骨細胞が産生する軟骨基質とからなり、軟骨基質の主成分はプロテオグリカンおよびコラーゲン（II型、IX型など）である。
関節疾患は関節軟骨の変性を主たる病変とする疾患であって、種々の原因によりこれら軟骨組織からのプロテオグリカンの遊離が促進され、前後して該組織におけるプロテオグリカンの合成能が低下し始める。同時にＩ型コレゲナーゼ等の各種メタロプロテイナーゼの遊離と活性化が亢進し軟骨組織のコラーゲンが分解される。これら一連の反応により軟骨組織の破壊が進み、次いで、病変の進行により滑膜の増性、軟骨下骨の破壊、関節辺縁部の軟骨肥大あるいは骨新生がおこり、関節の変形を経て、重篤な場合には機能不全に至る。 Cartilage is a connective tissue composed of chondrocytes and the surrounding matrix, and is present in joints, spinal discs, costal cartilage, pinna, external auditory canal, pubic bone, pharyngeal palate and the like. Cartilage is composed of chondrocytes and cartilage matrix produced by chondrocytes, and the main components of cartilage matrix are proteoglycan and collagen (type II, type IX, etc.).
A joint disease is a disease whose main lesion is degeneration of articular cartilage, and the release of proteoglycan from these cartilage tissues is promoted by various causes, and the ability to synthesize proteoglycans in the tissue begins to decline. At the same time, the release and activation of various metalloproteinases such as type I collagenase are enhanced and the collagen in the cartilage tissue is degraded. These series of reactions lead to the destruction of the cartilage tissue, and then the progression of the lesion causes synovial growth, subchondral bone destruction, cartilage hypertrophy or osteogenesis at the joint margin, and after joint deformation, In severe cases it leads to dysfunction.

関節疾患の中でも最も患者数の多い疾患が変形性関節症である。本疾患では加齢が軟骨変性の原因の一つと考えられており、社会の高齢化に伴って今後さらに患者数が増えることが懸念されている。しかしながら、その治療法としては、軟骨変性・軟骨下骨破壊に伴う痛みを取る目的から鎮痛消炎剤やヒアルロン酸製剤が用いられているが、いずれも対症療法的に用いられているに過ぎず、十分な効果はあげていない。
これに対して、プロテオグリカンの生成促進剤が新しい治療剤として着目されている。プロテオグリカンとは、コアタンパク質と呼ばれるタンパク質に１本以上のグリコサミノグリカン（ＧＡＧ）鎖が結合している構造を有する分子であるが、西村等は、このプロテオグリカンの構造を模倣したＧＡＧ鎖伸張の開始剤（イニシエーター）となりうるポリ糖ペプチドを提案し、リウマチ性関節炎や変形性関節症治療剤としての有用性を示唆している（後記特許文献１参照）。 Among joint diseases, osteoarthritis is the most common disease. In this disease, aging is considered to be one of the causes of cartilage degeneration, and there is concern that the number of patients will increase further in the future as society ages. However, as its treatment method, analgesic anti-inflammatory agents and hyaluronic acid preparations are used for the purpose of taking pain associated with cartilage degeneration and subchondral bone destruction, but they are only used for symptomatic treatment, It is not effective enough.
In contrast, proteoglycan production promoters have attracted attention as new therapeutic agents. A proteoglycan is a molecule having a structure in which one or more glycosaminoglycan (GAG) chains are bound to a protein called a core protein. Nishimura et al. Have developed a GAG chain extension that mimics the structure of proteoglycans. Polypeptides that can serve as initiators have been proposed, suggesting their usefulness as therapeutic agents for rheumatoid arthritis and osteoarthritis (see Patent Document 1 below).

しかしながら、プロテオグリカンはそのＧＡＧ鎖を構成する糖の違いによりコンドロイチン硫酸プロテオグリカン（ＣＳＰＧ）およびヘパラン硫酸プロテオグリカン（ＨＳＰＧ）とに大きく分類され、ＣＳＰＧの多くは細胞外マトリクスとして存在し、例えばアグリカン（aggrecan）のように軟骨の主要構成成分としての役割を担う一方、ＨＳＰＧは細胞膜に挿入され例えば成長因子等のシグナル伝達に関与していることが知られている。変形性関節症等の軟骨疾患治療のためには、ＣＳＰＧの生成促進が重要であるところ、上記のポリ糖ペプチドよりなるイニシエーターはその選択性を確認するまでには至っていない。
ＷＯ２００４／０７６４７６号公報 However, proteoglycans are broadly classified into chondroitin sulfate proteoglycan (CSPG) and heparan sulfate proteoglycan (HSPG) depending on the difference in the sugars constituting the GAG chain. Thus, while playing a role as a major component of cartilage, it is known that HSPG is inserted into the cell membrane and is involved in signal transduction such as growth factors. In order to treat cartilage diseases such as osteoarthritis, it is important to promote the production of CSPG. However, the initiator composed of the above-mentioned polypeptide has not yet been confirmed to be selective.
WO2004 / 076476

本発明の目的は、プロテオグリカンの生成促進機能を有し、特に、ＣＳＰＧを選択的に生成促進させる、軟骨疾患の予防または治療剤を提供することである。 An object of the present invention is to provide a prophylactic or therapeutic agent for cartilage disease, which has a proteoglycan production promoting function and, in particular, selectively promotes the production of CSPG.

プロテオグリカンのＧＡＧ鎖はウロン酸とアミノ糖の２糖を繰り返し単位とする特徴的な構造を持ち、GlcA-Gal-Gal-Xylという共通四糖を介して、コアタンパク質中のセリン残基に結合している。

The proteoglycan GAG chain has a characteristic structure consisting of disaccharides of uronic acid and amino sugar, and binds to the serine residue in the core protein via a common tetrasaccharide called GlcA-Gal-Gal-Xyl. ing.

そして、ＧＡＧ鎖を構成する糖の違いによりＣＳＰＧおよびＨＳＰＧという二つのタイプに分類されることは上述の通りであるが、これら両タイプのＰＧの生合成分岐点にあたる共通四糖へのヘキソサミン転移メカニズムは未だ不明であった。
本発明者等は、共通四糖上の硫酸基の有無がＣＳＰＧ／ＨＳＰＧの仕分け制御に深く関与しているとの洞察から、当該硫酸エステル化が観測される水酸基をフッ素原子で置換した類縁体を合成した。そして、式（Ｉ）で表される化合物群が、ＣＳＰＧ伸張のイニシエーターとなることを見出し、更には軟骨細胞において実際にムコ多糖およびアグリカン量を増加させることを確認して本発明を完成した。 And, as described above, it is classified into two types, CSPG and HSPG, depending on the sugars constituting the GAG chain. The mechanism of hexosamine transfer to a common tetrasaccharide that is the biosynthetic branching point of both types of PGs. Was still unknown.
Based on the insight that the presence or absence of a sulfate group on a common tetrasaccharide is deeply involved in the sorting control of CSPG / HSPG, the present inventors have substituted the hydroxyl group in which the sulfate esterification is observed with a fluorine atom. Was synthesized. The inventors have found that the compound group represented by the formula (I) serves as an initiator of CSPG elongation, and further confirmed that the amount of mucopolysaccharide and aggrecan is actually increased in chondrocytes, thereby completing the present invention. .

本発明の化合物は、プロテオグリカンの生成促進機能を有し、特にＣＳＰＧの生成を選択的に促進するので、変形性関節症その他の軟骨疾患の予防または治療剤として有用である。 The compound of the present invention has a proteoglycan production-promoting function and, in particular, selectively promotes the production of CSPG. Therefore, it is useful as a prophylactic or therapeutic agent for osteoarthritis and other cartilage diseases.

本発明は以下のものを提供する。
（１）式（Ｉ）

The present invention provides the following.
(1) Formula (I)

（式中、Ｘはハロゲン素原子、水酸基またはアルコキシ基を表し、Ｒは水素原子、置換されていてもよいアルキル、置換されていてもよいアリール、置換されていてもよいヘテロアリールまたは天然若しくは非天然のアミノ酸からなるペプチド残基を表す。）
で表される化合物、その薬学上許容される塩、またはそれらの溶媒和物。
（２）Ｘがフッ素原子または水酸基である、式（Ｉ）の化合物、その薬学上許容される塩、またはそれらの溶媒和物。
（３）−ＯＲがセリンおよび／またはトレオニン含有オリゴペプチドであって、−ＯＲの酸素原子とセリンおよび／またはトレオニンが結合し、さらに該アミノ酸と三糖ユニットが結合した、式（Ｉ）の化合物、その薬学上許容される塩、またはそれらの溶媒和物。 (Wherein X represents a halogen atom, a hydroxyl group or an alkoxy group, R represents a hydrogen atom, an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, natural or non-substituted, Represents peptide residues consisting of natural amino acids.)
Or a pharmaceutically acceptable salt thereof, or a solvate thereof.
(2) A compound of formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein X is a fluorine atom or a hydroxyl group.
(3) A compound of formula (I), wherein -OR is a serine and / or threonine-containing oligopeptide, wherein the oxygen atom of -OR is bound to serine and / or threonine, and further, the amino acid and a trisaccharide unit are bound , A pharmaceutically acceptable salt thereof, or a solvate thereof.

（４）−ＯＲが下式

（式中、ＦＩＴＣはアミノ基がフルオレッセインイソチオシアネートで蛍光標識化されていることを表す。）
で表されるグリシルセリン誘導体である、式（Ｉ）の化合物、その薬学上許容される塩、またはそれらの溶媒和物。
（５）上記（１）から（４）のいずれかに記載された式（Ｉ）の化合物、その薬学上許容される塩、またはそれらの溶媒和物を有効成分とする医薬。
（６）上記（１）から（４）のいずれかに記載された式（Ｉ）の糖鎖誘導体、その薬学上許容される塩、またはそれらの溶媒和物を有効成分とする変形性関節症の予防および／または治療薬。 (4) -OR is the following formula

(In the formula, FITC represents that the amino group is fluorescently labeled with fluorescein isothiocyanate.)
A glycylserine derivative represented by the formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof:
(5) A medicament comprising as an active ingredient the compound of formula (I) described in any one of (1) to (4) above, a pharmaceutically acceptable salt thereof, or a solvate thereof.
(6) Osteoarthritis containing as an active ingredient the sugar chain derivative of the formula (I) described in any one of (1) to (4) above, a pharmaceutically acceptable salt thereof, or a solvate thereof. Preventive and / or therapeutic drug.

本発明のＰＧ生成促進剤は、下式（Ｉ）

（式中、Ｘはハロゲン原子、水酸基またはアルコキシ基を表し、Ｒは水素原子、置換されていてもよいアルキル、置換されていてもよいアリール、置換されていてもよいヘテロアリールまたは天然若しくは非天然のアミノ酸からなるペプチド残基を表す。）
で表される、化合物、その薬学上許容される塩、またはそれらの溶媒和物を有効成分とする。 The PG production promoter of the present invention has the following formula (I)

(In the formula, X represents a halogen atom, a hydroxyl group or an alkoxy group, and R represents a hydrogen atom, an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or natural or non-natural. Represents a peptide residue comprising the amino acids of
Or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

式（Ｉ）で表される化合物のすべての水酸基は、通常用いられる水酸基の保護基により保護されていても良い。好ましくは保護されていない水酸基である。
ここで、「アルキル」とは、炭素数１〜１０、好ましくは炭素数１〜６、さらに好ましくは炭素数１〜３までの直鎖状または分岐状のアルキルを包含し、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチル、ｎ−ペンチル、イソペンチル、ネオペンチル、ヘキシル、イソヘキシル、ｎ−へプチル、イソヘプチル、ｎ−オクチル、イソオクチル、ｎ−ノニルおよびｎ−デシル等が例示される。
「置換されていてもよいアルキル」の置換基としては、ハロゲン、ヒドロキシ、アルキル、アルコキシ、カルボキシ、アルコキシカルボニル、アシル等が挙げられる。
「ハロゲン」とはＦ、Ｃｌ、Ｂｒ、およびＩを包含する。 All the hydroxyl groups of the compound represented by the formula (I) may be protected by a commonly used hydroxyl-protecting group. Preferably, it is an unprotected hydroxyl group.
Here, “alkyl” includes linear or branched alkyl having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, and includes methyl, ethyl, n -Propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl and n- Examples are decyl and the like.
Examples of the substituent of “optionally substituted alkyl” include halogen, hydroxy, alkyl, alkoxy, carboxy, alkoxycarbonyl, acyl and the like.
“Halogen” includes F, Cl, Br, and I.

「アルコキシ」および「アルコキシカルボニル」のアルキル部分は前記「アルキル」と同意であって、いずれも、炭素数１〜６、さらに好ましくは炭素数１〜３までの直鎖状または分岐状のアルキルを包含し、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチル、ｎ−ペンチル、イソペンチル、ネオペンチル、ヘキシル、イソヘキシル等が例示される。特に好ましくはメチルまたはエチルである。
「アシル」とは、炭素数１〜７の脂肪族アシルおよびアロイルを包含する。具体的には、ホルミル、アセチル、プロピオニル、ブチリル、イソブチリル、バレリル、ピバロイル、ヘキサノイル、アクリロイル、プロピオロイル、メタクリロイル、クロトノイルおよびベンゾイル等が例示される。 The alkyl part of “alkoxy” and “alkoxycarbonyl” is the same as the above “alkyl”, and both are linear or branched alkyl having 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms. And methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl and the like. Particularly preferred is methyl or ethyl.
“Acyl” includes aliphatic acyl having 1 to 7 carbon atoms and aroyl. Specific examples include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, acryloyl, propioroyl, methacryloyl, crotonoyl and benzoyl.

「ヘテロアリール」とは、Ｏ、ＳおよびＮから任意に選択されるヘテロ原子を環内に１個以上有する芳香族複素環式基を包含し、具体的にはピロリル、イミダゾリル、ピラゾリル、ピリジル、ピリダジニル、ピリミジニル、ピラジニル、トリアゾリル、トリアジニル、テトラゾリル、イソオキサゾリル、オキサゾリル、オキサジアゾリル、イソチアゾリル、チアゾリル、チアジアゾリル、フリルおよびチエニル等の５〜６員環のヘテロアリール；インドリル、イソインドリル、インダゾリル、インドリジニル、インドリニル、イソインドリニル、キノリル、イソキノリル、シンノリニル、フタラジニル、キナゾリニル、ナフチリジニル、キノキサリニル、プリニル、プテリジニル、ベンゾピラニル、ベンズイミダゾリル、ベンゾトリアゾリル、ベンズイソオキサゾリル、ベンズオキサゾリル、ベンズオキサジアゾリル、ベンゾイソチアゾリル、ベンゾチアゾリル、ベンゾチアジアゾリル、ベンゾフリル、イソベンゾフリル、ベンゾチエニル、ベンゾトリアゾリル、イミダゾピリジル、ピラゾロピリジン、トリアゾロピリジル、イミダゾチアゾリル、ピラジノピリダジニル、キナゾリニル、キノリル、イソキノリル、ナフチリジニル、ジヒドロベンゾフリル、テトラヒドロキノリル、テトラヒドロイソキノリル、ジヒドロベンズオキサジン、テトラヒドロベンゾチエニル等の２環の縮合複素環式基；カルバゾリル、アクリジニル、キサンテニル、フェノチアジニル、フェノキサチイニル、フェノキサジニル、ジベンゾフリル、イミダゾキノリル等の３環の縮合複素環式基を包含する。好ましくは５〜６員環のヘテロアリールである。 “Heteroaryl” includes an aromatic heterocyclic group having one or more heteroatoms arbitrarily selected from O, S and N in the ring, specifically pyrrolyl, imidazolyl, pyrazolyl, pyridyl, 5-6 membered heteroaryl such as pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, furyl and thienyl; indolyl, isoindolyl, indazolyl, indolizinyl, indolinyl, isoindolinyl, Quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzopyranyl, benzimidazolyl, benzotriazolyl Benzisoxazolyl, Benzoxazolyl, Benzoxadiazolyl, Benzisothiazolyl, Benzthiazolyl, Benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, pyrazolopyridine, tria Bicyclic condensed heterocycles such as zolopyridyl, imidazothiazolyl, pyrazinopyridazinyl, quinazolinyl, quinolyl, isoquinolyl, naphthyridinyl, dihydrobenzofuryl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydrobenzoxazine, tetrahydrobenzothienyl Cyclic group; includes three-ring condensed heterocyclic groups such as carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, dibenzofuryl, imidazoquinolyl and the like. Preferably it is a 5-6 membered heteroaryl.

「アリール」とは、炭素数６から１６の単環性、二環性若しくは三環性の芳香族炭素環を示し、例えばフェニル、ナフチル、アントリルおよびフェナントリル等を包含し、特にフェニルが好ましい。
「置換されていてもよいアリール」および「置換されていてもよいヘテロアリール」の置換基は「置換されていてもよいアルキル」の場合と同様である。
「天然アミノ酸」は生物が普遍的に利用する２０種類のＬ−α−アミノ酸を意味し、また「非天然アミノ酸」は天然アミノ酸の光学異性体（Ｄ-体）の他、天然アミノ酸に存在しない側鎖を有するＬ−体若しくはＤ−体のアミノ酸誘導体を包含する。天然アミノ酸の光学異性体（Ｄ-体）が好ましい。
「ペプチド残基」はこれら天然および／または非天然アミノ酸が１個から１０個ペプチド結合でつながったオリゴペプチドを包含し、１個の天然または非天然アミノ酸を包含する。１個から６個のペプチドが好ましく、さらに１個から３個のペプチドが好ましい。また、このペプチド残基は、ペプチド合成のための保護基および／または当業者に一般的に使用される標識で適宜、修飾されていてもよい。 “Aryl” refers to a monocyclic, bicyclic or tricyclic aromatic carbocyclic ring having 6 to 16 carbon atoms, and includes, for example, phenyl, naphthyl, anthryl, phenanthryl and the like, and phenyl is particularly preferable.
The substituents of “optionally substituted aryl” and “optionally substituted heteroaryl” are the same as in the case of “optionally substituted alkyl”.
“Natural amino acid” means 20 kinds of L-α-amino acids universally used by organisms, and “unnatural amino acid” does not exist in natural amino acids other than optical isomers (D-form) of natural amino acids. It includes L- or D-amino acid derivatives having a side chain. The optical isomers (D-form) of natural amino acids are preferred.
“Peptide residues” include oligopeptides in which these natural and / or unnatural amino acids are connected by 1 to 10 peptide bonds, including one natural or non-natural amino acid. 1 to 6 peptides are preferred, and 1 to 3 peptides are more preferred. The peptide residue may be appropriately modified with a protecting group for peptide synthesis and / or a label generally used by those skilled in the art.

製薬上許容される塩としては、酸付加塩の場合、例えば塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、炭酸水素塩、過塩素酸塩等の無機酸塩；例えばシュウ酸塩、酢酸塩、プロピオン酸塩、乳酸塩、マレイン酸塩、フマール酸塩、酒石酸塩、リンゴ酸塩、クエン酸塩、アスコルビン酸塩等の有機酸塩；例えばメタンスルホン酸塩、イセチオン酸塩、ベンゼンスルホン酸塩、ｐ−トルエンスルホン酸塩等のスルホン酸塩；例えばアスパラギン酸塩、グルタミン酸塩等の酸性アミノ酸等を挙げることができる。また、塩基付加塩の場合、例えばリチウム、ナトリウム、カリウム等のアルカリ金属塩、カルシウム、マグネシウム等のアルカリ土類金属塩、アルギニン、リシン等の塩基性アミノ酸塩等を挙げることができる。
化合物（Ｉ）は、水、アセトニトリル、酢酸エチル、メタノール、エタノール等の溶媒和物であってもよい。又本発明化合物の溶媒和物の溶媒和数は通常、合成方法、精製方法または結晶化条件等によって変化し得るが、例えば、化合物１分子当り１〜５分子の範囲である。 Examples of pharmaceutically acceptable salts include, in the case of acid addition salts, inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, perchlorate; Organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate; for example, methanesulfonate, isethionate, benzenesulfone Examples thereof include sulfonates such as acid salts and p-toluenesulfonate; acidic amino acids such as aspartate and glutamate. Examples of the base addition salt include alkali metal salts such as lithium, sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and basic amino acid salts such as arginine and lysine.
Compound (I) may be a solvate such as water, acetonitrile, ethyl acetate, methanol, ethanol and the like. The solvation number of the solvate of the compound of the present invention can usually vary depending on the synthesis method, purification method, crystallization conditions, etc., but is, for example, in the range of 1 to 5 molecules per molecule.

本発明化合物は以下のようにして製造できる。
１）三糖ユニットの合成スキーム
フッ素置換されたグリコシルドナーは、その電子求引性のため反応性が悪く、アノマー位活性化基としてイミデートを用いた時のみ反応が進行した。また、グリコシルアクセプターとして４，６−ベンジリデン化Ｇａｌを用いた時には３位の水酸基の反応性が悪く反応が進行しなかったため、この水酸基の反応性を高める４，６−シリレン化Galをグリコシルアクセプターとして用いた時のみ反応が進行した。これらのことより、グリコシルドナーのアノマー位活性化基としてはイミデート、鍵フラグメントとなる真ん中のGalは４，６−シリレン化Galを用いる合成ルートを計画した。 The compound of the present invention can be produced as follows.
1) Synthetic scheme of trisaccharide unit The fluorine-substituted glycosyl donor was poor in reactivity due to its electron withdrawing property, and the reaction proceeded only when imidate was used as the anomeric position activating group. In addition, when 4,6-benzylidated Gal was used as the glycosyl acceptor, the reactivity of the hydroxyl group at position 3 was poor and the reaction did not proceed. Therefore, 4,6-silyleneated Gal, which increases the reactivity of the hydroxyl group, was converted to glycosyl acetate. The reaction proceeded only when used as a scepter. Based on these facts, a synthetic route was planned in which 4,6-silyleneated Gal was used as the gallate in the middle, which is an imidate as the anomeric position activating group of the glycosyl donor.

無置換体の三糖ユニット(41)は以下のスキームに従って合成される。

The unsubstituted trisaccharide unit (41) is synthesized according to the following scheme.

鍵化合物であるシリレン化ガラクトースアクセプター(17)に対し、フッ素置換されていない既知のガラクトシルドナー(18)を、それぞれグリコシル化して二糖ユニット(21)とした。シリレン基をＨＦで脱保護した後、そのままベンジル化して(24)とした。
二糖ユニット(24)のＭＰ（p-メトキシフェニル）基を、後述の通りイミデート体に変換し、これと、既知化合物であるキシロシルアクセプター(36)とグリコシル化して、三糖ユニット(41）とした。この際、アノマー位の選択比は約α：β（１：５）（ＮＭＲ比）となるが、β体のみフラッシュカラムクロマトグラフィーにより分離精製することができる。 The silanolated galactose acceptor (17), which is a key compound, was glycosylated with a known galactosyl donor (18) that was not fluorine-substituted to form a disaccharide unit (21). The silylene group was deprotected with HF and benzylated as it was to give (24).
The MP (p-methoxyphenyl) group of the disaccharide unit (24) is converted into an imidate form as described below, and this is glycosylated with a known compound, xylosyl acceptor (36), to give a trisaccharide unit (41 ). At this time, the selection ratio of the anomeric position is about α: β (1: 5) (NMR ratio), but only the β form can be separated and purified by flash column chromatography.

また、フッ素化三糖ユニット(40)の合成スキームは以下の通りである。

上記三糖ユニット(41)の合成に準じて、フッ素化三糖ユニット(40)を合成できる。 The synthesis scheme of the fluorinated trisaccharide unit (40) is as follows.

The fluorinated trisaccharide unit (40) can be synthesized according to the synthesis of the trisaccharide unit (41).

２）単糖誘導体の合成
4-Ｆフラグメント(13)の合成

2) Synthesis of monosaccharide derivatives
Synthesis of 4-F fragment (13)

反応条件；
ａ：p-メトキシフェノール, TMSOTf, MS4A, CH₂Cl₂、
ｂ：(1)NaOMe, MeOH, (2)PhCH(OMe)₂, CSA, DMF, (3)Ac₂O, ピリジン, 三工程で65%
ｃ：NaBH₃CN, 2N HCl/Et₂O, MS4A, THF, 79%
ｄ：(1)Tf₂O, ピリジン, CH₂Cl₂,(2)TBAF, THF, 二工程で81%
ｅ：(1)NaOMe, MeOH, (2)n-Bu₂SnO, トルエン, それからCACl, ピリジン,(3)BzCl, ピリジン, DMAP, CH₂Cl₂, (4)Et₃N, MeOH/CH₂Cl₂, 54%(1:1),
ｆ：(1)Pd/C, H₂, MeOH,(2)NaOMe, MeOH, (3)BzCl, ピリジン, DMAP, CH₂Cl₂,
ｇ：(1)CAN, CH₃CN:Tol: H₂O (1.5:1:1), 2, CCl₃CN, DBU Reaction conditions;
a: p-methoxyphenol, TMSOTf, MS4A, CH ₂ Cl ₂ ,
b: (1) NaOMe, MeOH, (2) PhCH (OMe) ₂ , CSA, DMF, (3) Ac ₂ O, pyridine, 65% in 3 steps
c: NaBH ₃ CN, 2N HCl / Et ₂ O, MS4A, THF, 79%
d: (1) Tf ₂ O, pyridine, CH ₂ Cl ₂ , (2) TBAF, THF, 81% in two steps
e: (1) NaOMe, MeOH, (2) n-Bu ₂ SnO, toluene, then CACl, pyridine, (3) BzCl, pyridine, DMAP, CH ₂ Cl ₂ , (4) Et ₃ N, MeOH / CH ₂ Cl ₂ , 54% (1: 1),
f: (1) Pd / C, H ₂ , MeOH, (2) NaOMe, MeOH, (3) BzCl, pyridine, DMAP, CH ₂ Cl ₂ ,
g: (1) CAN, CH ₃ CN: Tol: H ₂ O (1.5: 1: 1), 2, CCl ₃ CN, DBU

市販の(6)から既知化合物(7)へと導いた。この(7)を脱Ac化、ベンジリデンアセタールで選択的に4,6-位を保護して、未精製のまま2,3-位をアセチル化して(8)とした。ベンジリデンアセタールをNaBH₃CN、２N 塩酸で４位を選択的に開いて(9)とした。この４位水酸基に脱離基であるトリフレート（Tf）をかけた後、そのまま固体TBAFを加えてフッ素化し(10)とした。(10)のAc基を脱保護したものは、溶解性が悪く選択的保護基のかけわけが困難であったため、Snアセタールで2,3-位を保護した。この際、選択的に３位のみ活性化されるので、この部位をモノクロロアセチル基（CA）で保護した後、そのまま２位をBz基で保護した。その後モノクロロアセチル基を選択的に脱保護して(11)とした。
また、(10)を水素添加による６位ベンジル基を脱保護、NaOMeによる2,3-位Ac基の脱保護の後、そのままベンジル化して(12)とした。ｐ−メトキシフェニル基（ＭＰ）をＣＡＮで脱保護した後、イミデート化して(13)とした。 It was led from the commercially available (6) to the known compound (7). This (7) was deacylated, the 4,6-position was selectively protected with benzylidene acetal, and the 2,3-position was acetylated without purification to give (8). The benzylidene acetal was selectively opened at the 4-position with NaBH ₃ CN and 2N hydrochloric acid to give (9). This 4-hydroxyl group was subjected to triflate (Tf) as a leaving group, and solid TBAF was added as it was to obtain a fluorinated product (10). In the case where the Ac group of (10) was deprotected, the 2,3-position was protected with Sn acetal because of poor solubility and difficult application of the selective protecting group. At this time, since only the 3-position was selectively activated, this site was protected with a monochloroacetyl group (CA), and then the 2-position was protected with a Bz group. Thereafter, the monochloroacetyl group was selectively deprotected to give (11).
Further, (10) was deprotected from the 6-position benzyl group by hydrogenation, and after deprotection of the 2,3-position Ac group by NaOMe, it was benzylated as it was to obtain (12). The p-methoxyphenyl group (MP) was deprotected with CAN and then imidated to give (13).

4,6-シリレン化ガラクトース(17)の合成

反応条件
ａ：(1)NaOMe, MeOH, (2)n-Bu₂SnO, トルエン，還流，その後臭化アリル、臭化テトラブチルアンモニウム、
ｂ：(1)DTBP, 2, 6-ルチジン, (2)BzCl, ピリジン, DMAP、
ｃ：[Ir(COD)(PMePh₂)₂PF₆], H₂, その後TsOH H₂O Synthesis of 4,6-silyleneated galactose (17)

Reaction conditions a: (1) NaOMe, MeOH, (2) n-Bu ₂ SnO, toluene, reflux, then allyl bromide, tetrabutylammonium bromide,
b: (1) DTBP, 2, 6-lutidine, (2) BzCl, pyridine, DMAP,
c: [Ir (COD) (PMePh ₂ ) ₂ PF ₆ ], H ₂ , then TsOH H ₂ O

市販の(14)から既知化合物(15)へと導いた後、4,6-位を選択的にシリレン化した。これをそのまま２位をベンジル化して(16)とした。(16)のアリル基を脱保護して、シリレン化ガラクトースアクセプター(17)とした。
３）化合物（Ｉ）の合成
こうして得られた三糖ユニットはそのまま脱保護すれば、Ｒがp-メトキシフェニルである本発明化合物（I)となる。
また、常法に従ってイミデート体に変換した後、ＨＯＲとカップリングさせ、Ｂｚ基を脱保護すれば種々の化合物（Ｉ）に誘導することができる。
Ｒとしては、天然アミノ酸２個から４個のオリゴペプチドが好ましく、特にセリン若しくはスレオニンの側鎖ＯＨ基とカップリングさせたものが好ましい。また、そのＣＳＰＧ生成促進作用を損なわない範囲で、標識化した誘導体が好適に利用される。 After leading from commercially available (14) to known compound (15), the 4,6-position was selectively silyleneated. This was directly benzylated at the 2-position to give (16). The allyl group of (16) was deprotected to obtain a silylated galactose acceptor (17).
3) Synthesis of Compound (I) If the trisaccharide unit thus obtained is deprotected as it is, it will be the compound (I) of the present invention in which R is p-methoxyphenyl.
Moreover, after converting into an imidate body according to a conventional method, coupling with HOR and deprotecting the Bz group can lead to various compounds (I).
R is preferably an oligopeptide having 2 to 4 natural amino acids, particularly preferably one that is coupled to the side chain OH group of serine or threonine. In addition, a labeled derivative is preferably used as long as the CSPG production promoting action is not impaired.

本発明のＰＧ生成促進剤は、例えば慢性関節リウマチや変形性関節症等の軟骨疾患の治療または予防に有効である。好ましくは鎮痛剤として経口または非経口投与用に処方され得る。
経口投与による場合、本剤は通常の製剤、例えば錠剤、散剤、顆粒剤、カプセル剤等の固形剤；水剤；油性懸濁剤；またはシロップ剤もしくはエリキシル剤等の液剤のいずれかの剤形としても用いることができる。非経口投与による場合、水性または油性懸濁注射剤、点鼻液として用いることができる。その調製に際しては、慣用の賦形剤、結合剤、滑沢剤、水性溶剤、油性溶剤、乳化剤、懸濁化剤、保存剤、安定剤等を任意に用いることができる。 The PG production promoter of the present invention is effective for the treatment or prevention of cartilage diseases such as rheumatoid arthritis and osteoarthritis. Preferably it can be formulated for oral or parenteral administration as an analgesic.
In the case of oral administration, this drug is a normal formulation, for example, a solid preparation such as a tablet, powder, granule or capsule; a liquid preparation; an oil suspension; or a liquid preparation such as a syrup or elixir. Can also be used. In the case of parenteral administration, it can be used as an aqueous or oily suspension injection or nasal solution. In the preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be arbitrarily used.

本発明の製剤は、治療有効量の化合物を製薬上許容される担体または希釈剤とともに組み合わせる（例えば混合する）ことによって製造され、その場合、周知の、容易に入手できる成分を用いて既知の方法により製造される。
本発明の医薬組成物を製造する際、活性成分は担体と混合されるかまたは担体で希釈されるか、カプセル、サッシェー、紙、あるいは他の容器の形態をしている担体中に入れられる。担体が希釈剤として働く時、担体は媒体として働く固体、半固体、または液体の材料であり、それらは錠剤、丸剤、粉末剤、口中剤、エリキシル剤、懸濁剤、エマルジョン剤、溶液剤、シロップ剤、エアロゾル剤（液体媒質中の固体）、軟膏にすることができ、例えば、１０％までの活性化合物を含む。 The formulations of the present invention are prepared by combining (eg, mixing) a therapeutically effective amount of a compound with a pharmaceutically acceptable carrier or diluent, in which case, using known, readily available ingredients, known methods Manufactured by.
In preparing the pharmaceutical compositions of the invention, the active ingredient is mixed with or diluted with a carrier, or placed in a carrier that is in the form of a capsule, sachet, paper, or other container. When the carrier acts as a diluent, the carrier is a solid, semi-solid, or liquid material that acts as a medium, and they are tablets, pills, powders, mouthpieces, elixirs, suspensions, emulsions, solutions. Syrups, aerosols (solids in liquid media), ointments, eg containing up to 10% active compound.

当業者には公知の適当な担体はいずれもこの製剤のために使用できる。このような製剤では担体は、固体、液体、または固体と液体の混合物である。例えば、静脈注射のために有効成分の化合物を４％デキストロース／０．５％クエン酸ナトリウム水溶液中に溶解する。固形の製剤は粉末、錠剤およびカプセルを包含する。固形担体は、香料、滑沢剤、溶解剤、懸濁剤、結合剤、錠剤崩壊剤、カプセル剤にする材料としても役立つ１またはそれ以上の物質である。経口投与のための錠剤は、トウモロコシデンプン、アルギン酸などの崩壊剤、および／またはゼラチン、アカシアなどの結合剤、およびステアリン酸マグネシウム、ステアリン酸、滑石などの滑沢剤とともに炭酸カルシウム、炭酸ナトリウム、ラクトース、リン酸カルシウムなどの適当な賦形剤を含む。 Any suitable carrier known to those skilled in the art can be used for this formulation. In such formulations, the carrier is a solid, liquid, or a mixture of solid and liquid. For example, the compound of the active ingredient is dissolved in 4% dextrose / 0.5% aqueous sodium citrate solution for intravenous injection. Solid formulations include powders, tablets and capsules. A solid carrier is one or more substances that can also serve as a flavoring agent, lubricant, solubilizer, suspending agent, binder, tablet disintegrant, or capsule. Tablets for oral administration include calcium carbonate, sodium carbonate, lactose with disintegrants such as corn starch, alginic acid and / or binders such as gelatin, acacia, and lubricants such as magnesium stearate, stearic acid, talc A suitable excipient, such as calcium phosphate.

粉末剤では担体は細かく粉砕された活性成分と混合された、細かく粉砕された固体である。錠剤では活性成分は、適当な比率で、必要な結合性を持った担体と混合されており、所望の形と大きさに固められている。粉末剤および錠剤は約１〜約９９重量％の本発明の新規化合物である活性成分を含んでいる。適当な固形担体は、炭酸マグネシウム、ステアリン酸マグネシウム、滑石、砂糖、ラクトース、ペクチン、デキストリン、デンプン、ゼラチン、トラガカントゴム、メチルセルロース、ナトリウムカルボキシメチルセルロース、低融点ワックス、ココアバターである。 In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active ingredients are mixed in a suitable ratio with a carrier having the necessary binding properties and are consolidated into the desired shape and size. Powders and tablets contain from about 1 to about 99% by weight of the active ingredient which is the novel compound of the present invention. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter.

液体製剤は懸濁剤、エマルジョン剤、シロップ剤、およびエリキシル剤を含む。活性成分は、滅菌水、滅菌有機溶媒、または両者の混合物などの製薬上許容し得る担体中に溶解または懸濁することができる。活性成分はしばしば適切な有機溶媒、例えばプロピレングリコール水溶液中に溶解することができる。水性デンプン、ナトリウムカルボキシメチルセルロース溶液、または適切な油中に細かく砕いた活性成分を散布することによってその他の組成物を製造することもできる。
本発明における化合物の投与量は、投与方法、患者の年齢、体重、状態および疾患の種類によっても異なるが、通常、経口投与の場合、成人１日あたり約０．１ｍｇ〜７０００ｍｇ、好ましくは、約０．５ｍｇ〜２０００ｍｇを、要すれば分割して投与すればよい。また、非経口投与の場合、成人１日あたり約０．１ｍｇ〜１０００ｍｇ、好ましくは、約０．５ｍｇ〜５００ｍｇを投与する。 Liquid formulations include suspensions, emulsions, syrups, and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier such as sterile water, sterile organic solvent, or a mixture of both. The active ingredient can often be dissolved in a suitable organic solvent, for example an aqueous propylene glycol solution. Other compositions can be made by spraying the active ingredient finely ground in aqueous starch, sodium carboxymethylcellulose solution, or a suitable oil.
The dose of the compound in the present invention varies depending on the administration method, the patient's age, weight, condition, and type of the disease, but usually about 0.1 mg to 7000 mg per day for an adult when administered orally, preferably about 0.5 mg to 2000 mg may be divided and administered if necessary. In the case of parenteral administration, about 0.1 mg to 1000 mg, preferably about 0.5 mg to 500 mg is administered per day for an adult.

本発明化合物の効果を下記の方法により試験した。
試験例１ＣＳ／ＨＳ生成比の検証試験
アッセイはＣＨＯ−Ｋ１細胞で行った。まず、ＣＨＯ−Ｋ１細胞を２４ウェルプレート上にまき、これに合成したフッ素無置換体(Ia)（糖ペプチド(61)）およびフッ素置換体(Ib)（糖ペプチド(60))（それぞれ30ｍＭに調整）をそれぞれ１０μＬずつ加えた。これを３７℃で４８時間培養後、培養上清をフィルターろ過し、さらに上清をゲルろ過（Sephadex G-10）して脱塩等の簡易精製を行った。これを、ゲルろ過クロマトグラフィー（TSK-GEL SuperSW3000）により分析した。ＧＡＧ鎖が伸長したかどうかは、イニシエーターがゲルろ過カラムの排除体積領域から溶離するかどうかを調べることにより判断した。 The effect of the compound of the present invention was tested by the following method.
Test Example 1 Verification test of CS / HS production ratio The assay was performed on CHO-K1 cells. First, CHO-K1 cells were seeded on a 24-well plate, and the synthesized fluorine-unsubstituted product (Ia) (glycopeptide (61)) and fluorine-substituted product (Ib) (glycopeptide (60)) (each at 30 mM). 10 μL of each was added. This was cultured at 37 ° C. for 48 hours, the culture supernatant was filtered, and the supernatant was gel filtered (Sephadex G-10) for simple purification such as desalting. This was analyzed by gel filtration chromatography (TSK-GEL SuperSW3000). Whether the GAG chain was extended was determined by examining whether the initiator eluted from the excluded volume region of the gel filtration column.

続いて、この排除体積領域から溶出した分画をコンドロイチナーゼＡＢＣで酵素分解し、得られる生成物をＨＰＬＣで分析した。酵素分解の条件は次の通りである。
コンドロイチナーゼＡＢＣ酵素 10 mU
0.4M トリス塩酸緩衝液、pH 8.0
0.4M 酢酸ナトリウム
0.1% BSA、37℃、12h
コンドロイチナーゼＡＢＣにより消化されなかったピーク（ＨＳ）およびＣＳの酵素消化生成物のピークの比からＨＳ／ＣＳを推定した（図１および図２参照）。
フッ素無置換体(Ia)は、ＧＡＧ鎖が伸長し、ＣＳの割合が多くなっている。それでもかなりのＨＳ鎖が伸びているのは、Ｅｓｋｏらの報告によるとアグリコン芳香族の疎水性が増加するにつれＨＳ生成比が上昇することから、(Ia)は巨大芳香族環であるＦＩＴＣをラベル官能基として持っているためＨＳがある程度生成したものと推定される。尚、フッ素置換体（Ib）は伸長活性自体がそれほど高くなく、ＨＳの比率も多少高くなっている。 Subsequently, the fraction eluted from this excluded volume region was enzymatically degraded with chondroitinase ABC, and the resulting product was analyzed by HPLC. The conditions for enzymatic degradation are as follows.
Chondroitinase ABC enzyme 10 mU
0.4M Tris-HCl buffer, pH 8.0
0.4M sodium acetate
0.1% BSA, 37 ° C, 12h
HS / CS was estimated from the ratio of the peak that was not digested by chondroitinase ABC (HS) and the peak of the enzymatic digestion product of CS (see FIGS. 1 and 2).
In the fluorine non-substituted product (Ia), the GAG chain is elongated and the proportion of CS is increased. However, the substantial HS chain is extended because the HS production ratio increases as the hydrophobicity of the aglycone aromatic increases according to the report of Esko et al. (Ia) labels FITC, a macroaromatic ring. Since it has as a functional group, it is presumed that HS was generated to some extent. In addition, the fluorine substitution product (Ib) does not have a very high elongation activity itself, and the ratio of HS is somewhat high.

試験例２軟骨生成促進試験
ヒト正常軟骨細胞（NHAC-kn）を滅菌マイクロチューブ（1.5ml）内で細胞増殖用培地（ＣＧＭ培地）を用い３日間培養して細胞塊を形成させた後、超低接着プレート(コーニング社；24ウェル)に移して細胞分化用培地を用い培養した。各被験物質は終濃度1〜100 ng/mlになるように希釈して添加し、陰性対照としてはDMSO(基剤群)を、陽性対照としてはBMP-2（30, 100 ng/ml）を用いた。化合物を添加後37℃、0.5% CO₂条件下で14日間培養を行った後、各ウェルの培養上清と細胞塊を回収した。細胞塊は超音波処理で破砕した後、遠心分離して溶解液を回収した。細胞塊溶解液および細胞上清に含有するムコ多糖量とアグリカン量を、酸性ムコ多糖定量キット（ホクドー、プライマリーセル社）およびヒトアグリカンELISAキット（バイオソース社）で定量した。
ムコ多糖産生量およびアグリカン産生量をそれぞれ図３および図４に示す。
NHAC-kn細胞を用いた3D培養系において、糖ペプチド（Ia）および（Ib）はいずれも100 ng/mlでのみムコ多糖およびアグリカン産生を有意に亢進させた。プロテオグリカンのイニシエーターとして作用に加えて、アグリカン産生系も活性化することが示唆された。 Test Example 2 Chondrogenesis Promotion Test Human normal chondrocytes (NHAC-kn) were cultured in a sterile microtube (1.5 ml) for 3 days using a cell growth medium (CGM medium) to form a cell mass. The cells were transferred to a low adhesion plate (Corning; 24 wells) and cultured using a cell differentiation medium. Dilute each test substance to a final concentration of 1 to 100 ng / ml, add DMSO (base group) as a negative control, and BMP-2 (30, 100 ng / ml) as a positive control. Using. After adding the compound, the cells were cultured for 14 days under conditions of 37 ° C. and 0.5% CO ₂ , and then the culture supernatant and cell mass of each well were collected. The cell mass was crushed by sonication and then centrifuged to collect the lysate. The amount of mucopolysaccharide and aggrecan contained in the cell mass lysate and cell supernatant were quantified with an acidic mucopolysaccharide quantification kit (Hokudo, Primary Cell) and a human aggrecan ELISA kit (Biosource).
The production amount of mucopolysaccharide and the production amount of aggrecan are shown in FIGS. 3 and 4, respectively.
In the 3D culture system using NHAC-kn cells, both glycopeptides (Ia) and (Ib) significantly increased mucopolysaccharide and aggrecan production only at 100 ng / ml. In addition to acting as an initiator of proteoglycan, it was suggested that the aggrecan production system is also activated.

参考例１
4-Ｆフラグメント(13)の合成
アセタール(8)の合成

市販の(6)を既知化合物(7)へと導いた後、MeOHに懸濁させた。これにNaOMeを入れ１時間攪拌した後、DOWEX 50W×8[H⁺］を加えて反応を止めた。ろ過、濃縮後、これをDMFに溶かした。この溶液にベンズアルデヒドジメチルアセタール、ＣＳＡを加え８０℃で３時間攪拌した。トルエン共沸して溶媒を飛ばし、そのままピリジンに溶かした。０℃に冷却して無水酢酸をゆっくりと加え、室温で１２時間攪拌した。溶液を濃縮後EtOAcに溶かして、有機層を水、飽和重曹水、ブラインで洗浄後、MgSO₄で乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーで精製し、(8)を得た。
(8) : ¹H-NMR(600MHz, CDCl₃, δ) 7.47-7.10(5H, Ph), 6.97(2H, d, J=9.0Hz, MP), 6.85(2H, d, J=9.0Hz, MP), 5.55(1H, s, PhCH), 5.41(1H, t, J=7.8Hz, H3), 5.27(1H, t, J=7.8Hz, H2), 5.08(1H, d, J=7.8Hz, H1), 4.42(1H, dd, J=10.3, 5.0Hz, H6), 3.87(1H, t, J=10.3Hz, H6), 3.83(1H, dd, J=9.6, 7.8Hz, H4), 3.82(3H, s, OMe), 3.64(1H, ddd, J=10.3, 9.6, 5.0Hz, H5), 2.10(6H, Ac). Reference example 1
Synthesis of 4-F fragment (13) Synthesis of acetal (8)

The commercially available (6) was led to the known compound (7) and then suspended in MeOH. NaOMe was added to this and stirred for 1 hour, and then DOWEX 50W × 8 [H ⁺ ] was added to stop the reaction. After filtration and concentration, this was dissolved in DMF. Benzaldehyde dimethyl acetal and CSA were added to this solution and stirred at 80 ° C. for 3 hours. Toluene was azeotroped to remove the solvent and dissolved in pyridine as it was. After cooling to 0 ° C., acetic anhydride was slowly added and stirred at room temperature for 12 hours. The solution was concentrated and dissolved in EtOAc, and the organic layer was washed with water, saturated aqueous sodium hydrogen carbonate, and brine, and dried over MgSO ₄ . After filtration and concentration, purification by flash column chromatography gave (8).
(8): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 7.47-7.10 (5H, Ph), 6.97 (2H, d, J = 9.0 Hz, MP), 6.85 (2H, d, J = 9.0 Hz, MP), 5.55 (1H, s, PhCH), 5.41 (1H, t, J = 7.8Hz, H3), 5.27 (1H, t, J = 7.8Hz, H2), 5.08 (1H, d, J = 7.8Hz , H1), 4.42 (1H, dd, J = 10.3, 5.0Hz, H6), 3.87 (1H, t, J = 10.3Hz, H6), 3.83 (1H, dd, J = 9.6, 7.8Hz, H4), 3.82 (3H, s, OMe), 3.64 (1H, ddd, J = 10.3, 9.6, 5.0Hz, H5), 2.10 (6H, Ac).

フッ素体(10)の合成

(8)をTHFに溶かした後、この溶液を０℃に冷却し、MS4A、NaBH₃CNを加えて１０分間攪拌した。この溶液にpHチェックしながら酸性になるまで2HCl／Et₂O溶液を加え、室温に昇温し３時間攪拌した。セライトでMS4Aをろ過した後、有機層を飽和重曹水、ブラインで洗浄し、MgSO₄で乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーで精製し(9)を得た。
(9) : ¹H-NMR(600MHz, CDCl₃, δ) 7.35-7.26(5H, Ph), 6.95(2H, d, J=3.8Hz, MP), 6.78(2H, d, J=3.8Hz, MP), 5.16(1H, dd, J=9.6, 7.8Hz, H2), 5.11(1H, t, J=9.6Hz, H3), 4.93(1H, d, J=7.8Hz, H1), 4.60(1H, d, J=11.9Hz, Bn), 4.56(1H, d, J=11.9Hz, Bn), 3.83-3.77(3H, m, H4, H6, H6), 3.63(1H, m, H5), 2.10(3H, s, Ac), 2.06(3H, s, Ac).
次いで、(9)をCH₂Cl₂にとかした後、−１５℃に冷却しピリジン、Tf₂Oを加えた。１時間攪拌した後TBAFを加え室温に昇温後、さらに１２時間攪拌した。有機層を水、飽和重曹水、ブラインで洗浄後、MgSO₄で乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーにより精製し、(10)を得た。
(10) : ¹H-NMR(600MHz, CDCl₃, δ) 7.38-7.28(5H, Bn), 6.99(2H, d, J=9.6Hz, MP), 6.82(2H, d, J=9.6Hz, MP), 5.53(1H, dd, J=10.1, 8.3Hz, H2), 5.06(1H, ddd, J=26.4, 10.1, 2.2Hz, H3), 4.97(1H, dd, J=49.9, 2.2Hz, H4), 4.96(1H, d, J=8.3Hz, H1), 4.59(2H, s, Bn), 3.88(1H, td, J=26.4, 6.6Hz, H5), 3.81-3.74(5H, m, H6, H6, OMe), 2.07(6H, Ac). Synthesis of fluorine (10)

After (8) was dissolved in THF, this solution was cooled to 0 ° C., MS4A and NaBH ₃ CN were added, and the mixture was stirred for 10 minutes. To this solution, a 2HCl / Et ₂ O solution was added until acidity while checking the pH, and the mixture was warmed to room temperature and stirred for 3 hours. After filtration of the MS4A through celite, wash the organic layer saturated aqueous sodium bicarbonate solution, brine and dried over MgSO _4. After filtration and concentration, purification by flash column chromatography gave (9).
(9): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 7.35-7.26 (5H, Ph), 6.95 (2H, d, J = 3.8 Hz, MP), 6.78 (2H, d, J = 3.8 Hz, MP), 5.16 (1H, dd, J = 9.6, 7.8Hz, H2), 5.11 (1H, t, J = 9.6Hz, H3), 4.93 (1H, d, J = 7.8Hz, H1), 4.60 (1H , d, J = 11.9Hz, Bn), 4.56 (1H, d, J = 11.9Hz, Bn), 3.83-3.77 (3H, m, H4, H6, H6), 3.63 (1H, m, H5), 2.10 (3H, s, Ac), 2.06 (3H, s, Ac).
Next, (9) was dissolved in CH ₂ Cl ₂ , cooled to −15 ° C., and pyridine and Tf ₂ O were added. After stirring for 1 hour, TBAF was added, the temperature was raised to room temperature, and the mixture was further stirred for 12 hours. The organic layer was washed with water, saturated aqueous sodium hydrogen carbonate, and brine, and then dried over MgSO ₄ . After filtration and concentration, purification by flash column chromatography gave (10).
(10): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 7.38-7.28 (5H, Bn), 6.99 (2H, d, J = 9.6 Hz, MP), 6.82 (2H, d, J = 9.6 Hz, MP), 5.53 (1H, dd, J = 10.1, 8.3Hz, H2), 5.06 (1H, ddd, J = 26.4, 10.1, 2.2Hz, H3), 4.97 (1H, dd, J = 49.9, 2.2Hz, H4), 4.96 (1H, d, J = 8.3Hz, H1), 4.59 (2H, s, Bn), 3.88 (1H, td, J = 26.4, 6.6Hz, H5), 3.81-3.74 (5H, m, H6, H6, OMe), 2.07 (6H, Ac).

4-Ｆフラグメント(13)の合成

(10)をMeOHに溶かし、Pd炭素を加え水素雰囲気下で３時間攪拌した。セライトろ過、濃縮後MeOHに溶かしNaOMeを加え１時間攪拌した。DOWEX 50W×8［Ｈ⁺］を加えて反応をとめた後、ろ過、濃縮した。これをCH₂Cl₂に溶かし、ピリジン、BzCl、DMAPを加えて１２時間攪拌した。水を加えて反応をとめた後、１N HCl、飽和重曹水、ブラインで洗浄しMgSO₄乾燥した。ろ過、濃縮後フラッシュカラムクロマトグラフィーで精製し、(12)を得た。
(12) : ¹H-NMR(600MHz, CDCl₃, δ) 8.15-7.39(15H, Ph), 6.98(2H, d, J=9.0Hz, MP), 6.71(2H, d, J=9.0Hz, MP), 6.05(1H, dd, J=9.4, 8.0Hz, H2), 5.49(1H, dd, J=26.9, 9.4Hz, H3), 5.21(1H, d, J=52.0Hz, H4), 5.21(1H, d, J=8.0Hz, H1), 4.75(1H, dd, J=11.4, 7.4Hz, H6), 4.69(1H, dd, J=11.4, 5.8Hz, H6), 4.27(1H, ddd, J=25.8, 7.4, 5.8Hz, H5), 3.73(3H, s, OMe). Synthesis of 4-F fragment (13)

(10) was dissolved in MeOH, Pd carbon was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After Celite filtration and concentration, dissolved in MeOH, NaOMe was added and stirred for 1 hour. DOWEX 50W × 8 [H ⁺ ] was added to terminate the reaction, followed by filtration and concentration. This was dissolved in CH ₂ Cl ₂ and pyridine, BzCl and DMAP were added and stirred for 12 hours. Water was added to quench the reaction, and the mixture was washed with 1N HCl, saturated aqueous sodium bicarbonate, brine and dried over MgSO ₄ . After filtration and concentration, purification by flash column chromatography gave (12).
(12): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.15-7.39 (15H, Ph), 6.98 (2H, d, J = 9.0 Hz, MP), 6.71 (2H, d, J = 9.0 Hz, MP), 6.05 (1H, dd, J = 9.4, 8.0Hz, H2), 5.49 (1H, dd, J = 26.9, 9.4Hz, H3), 5.21 (1H, d, J = 52.0Hz, H4), 5.21 (1H, d, J = 8.0Hz, H1), 4.75 (1H, dd, J = 11.4, 7.4Hz, H6), 4.69 (1H, dd, J = 11.4, 5.8Hz, H6), 4.27 (1H, ddd , J = 25.8, 7.4, 5.8Hz, H5), 3.73 (3H, s, OMe).

次に、(12)をトルエン：CH₃CN：水＝1.5：１：１に溶かし、CANを加え１時間攪拌した。有機層を水、飽和重曹水、ブラインで洗浄し、MgSO₄で乾燥した。ろ過、濃縮後フラッシュカラムクロマトグラフィーで簡易精製した。不純物が混ざるため、この粗生成物をそのまま次の反応に用いた。粗生成物をCH₂Cl₂に溶かした後、０℃に冷却しCCl₃CN、DBUを加え１時間攪拌した。これをそのまま濃縮しフラッシュカラムクロマトグラフィーにより精製し、(13)を得た。
(13) : ¹H-NMR (600MHz, CDCl₃, δ) 8.65(1H, s, NH), 8.06-7.17(15H, Ph), 6.84(1H, d, J=3.5Hz, H1), 5.96(1H, dd, J=10.4, 3.8Hz, H2), 5.88(1H, ddd, J=28.6, 10.4, 2.3Hz, H3), 5.31(1H, dd, J=52.4, 2.3Hz, H4), 4.69-4.60(3H, m, H5, H6, H6). Next, (12) was dissolved in toluene: CH ₃ CN: water = 1.5: 1: 1, CAN was added, and the mixture was stirred for 1 hour. The organic layer was washed with water, saturated aqueous sodium hydrogen carbonate, brine and dried over MgSO ₄ . After filtration and concentration, it was simply purified by flash column chromatography. Since the impurities were mixed, this crude product was directly used in the next reaction. The crude product was dissolved in CH ₂ Cl ₂ , cooled to 0 ° C., CCl ₃ CN and DBU were added, and the mixture was stirred for 1 hour. This was concentrated as it was and purified by flash column chromatography to obtain (13).
(13): ¹ H-NMR (600MHz, CDCl ₃ , δ) 8.65 (1H, s, NH), 8.06-7.17 (15H, Ph), 6.84 (1H, d, J = 3.5Hz, H1), 5.96 ( 1H, dd, J = 10.4, 3.8Hz, H2), 5.88 (1H, ddd, J = 28.6, 10.4, 2.3Hz, H3), 5.31 (1H, dd, J = 52.4, 2.3Hz, H4), 4.69- 4.60 (3H, m, H5, H6, H6).

参考例２
シリレン化Gal(17)の合成

市販の(14)から既知化合物(15)へ文献に従い導いた。(15)をCH₂Cl₂に溶かし、t-Bu₂Si(OTf)₂、ピリジンを加え３時間攪拌した。有機層を飽和重曹水、ブラインで洗浄後、MgSO₄で乾燥した。ろ過、濃縮後、この粗生成物をCH₂Cl₂に溶かし、ピリジン、BzCl、DMAPを入れ、６０℃で５時間攪拌した。水を加えて反応をとめた後、有機層を飽和重曹水、ブラインで洗浄し、MgSO₄乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーで精製し、(16)を得た。
(16) : ¹H-NMR(600MHz, CDCl₃, δ) 8.19-7.46(5H, Ph), 6.94(2H, d, J=8.4Hz, MP), 6.76(2H, d, J=8.4Hz, MP), 5.85(1H, m, H8), 5.83(1H, dd, J=10.1, 8.0Hz, H2), 5.26(1H, dd, J=17.3, 1.5Hz, H9), 5.12(1H, dd, J=10.3, 1.5Hz, H9), 5.01(1H, d, J=8.0Hz, H1), 4.64(1H, d, J=2.7Hz, H4), 4.33(1H, m, H6, H6), 4.22(1H, dd, J=13.3, 5.2Hz, H7), 4.12(1H, dd, J=13.3, 5.9Hz, H7), 3.74(3H, s, OMe), 3.66(1H, dd, J=10.1, 2.7Hz, H3), 3.53(1H, m, H5), 1.11(9H, s, t-Bu), 1.03(9H, s, t-Bu). Reference example 2
Synthesis of silyleneated Gal (17)

The commercially available (14) was derived from the known compound (15) according to the literature. (15) was dissolved in CH ₂ Cl ₂ , t-Bu ₂ Si (OTf) ₂ and pyridine were added, and the mixture was stirred for 3 hours. The organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine, and dried over MgSO ₄ . After filtration and concentration, this crude product was dissolved in CH ₂ Cl ₂ , pyridine, BzCl and DMAP were added, and the mixture was stirred at 60 ° C. for 5 hours. After adding water to quench the reaction, the organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine and dried over MgSO ₄ . After filtration and concentration, purification by flash column chromatography gave (16).
(16): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.19-7.46 (5H, Ph), 6.94 (2H, d, J = 8.4 Hz, MP), 6.76 (2H, d, J = 8.4 Hz, MP), 5.85 (1H, m, H8), 5.83 (1H, dd, J = 10.1, 8.0Hz, H2), 5.26 (1H, dd, J = 17.3, 1.5Hz, H9), 5.12 (1H, dd, J = 10.3, 1.5Hz, H9), 5.01 (1H, d, J = 8.0Hz, H1), 4.64 (1H, d, J = 2.7Hz, H4), 4.33 (1H, m, H6, H6), 4.22 (1H, dd, J = 13.3, 5.2Hz, H7), 4.12 (1H, dd, J = 13.3, 5.9Hz, H7), 3.74 (3H, s, OMe), 3.66 (1H, dd, J = 10.1, 2.7Hz, H3), 3.53 (1H, m, H5), 1.11 (9H, s, t-Bu), 1.03 (9H, s, t-Bu).

次いで、Ir触媒をTHFに溶かし、水素雰囲気下で２時間攪拌した。フラスコ内を窒素置換した後、THFに溶かした(16)を加え室温で２時間攪拌した。TLCでアリル基の異性化を確かめた後、TsOH・H₂Oを加えて１時間攪拌した。有機層を飽和重曹水、ブラインで洗浄後、MgSO₄で乾燥した。ろ過、濃縮後、フラッシュクロマトグラフィーで精製し、(17)を得た。
(17) : ¹H-NMR(600MHz, CDCl₃, δ) 8.03-7.43(5H, Ph), 6.93(2H, d, J=9.0Hz, MP), 6.76(2H, d, J=9.0Hz, MP), 5.59(1H, dd, J=9.6, 8.4Hz, H2), 4.97(1H, d, J=8.4Hz, H1), 4.49(1H, d, J=3.0Hz, H4), 4.34(1H, dd, J=12.4, 2.0Hz, H6), 4.30(1H, dd, J=12.4, 1.2Hz, H6), 3.78(1H, td, J=9.6, 3.0Hz, H3), 3.74(3H, s, OMe), 3.58(1H, m, H5), 2.78(1H, d, J=9.6Hz, OH), 1.14(9H, s, t-Bu), 1.08(9H, s, t-Bu). Next, the Ir catalyst was dissolved in THF and stirred for 2 hours under a hydrogen atmosphere. The flask was purged with nitrogen, (16) dissolved in THF was added, and the mixture was stirred at room temperature for 2 hr. After confirming the isomerization of the allyl group by TLC, TsOH · H ₂ O was added and stirred for 1 hour. The organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine, and dried over MgSO ₄ . After filtration and concentration, purification by flash chromatography gave (17).
(17): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.03-7.43 (5H, Ph), 6.93 (2H, d, J = 9.0 Hz, MP), 6.76 (2H, d, J = 9.0 Hz, MP), 5.59 (1H, dd, J = 9.6, 8.4Hz, H2), 4.97 (1H, d, J = 8.4Hz, H1), 4.49 (1H, d, J = 3.0Hz, H4), 4.34 (1H , dd, J = 12.4, 2.0Hz, H6), 4.30 (1H, dd, J = 12.4, 1.2Hz, H6), 3.78 (1H, td, J = 9.6, 3.0Hz, H3), 3.74 (3H, s , OMe), 3.58 (1H, m, H5), 2.78 (1H, d, J = 9.6Hz, OH), 1.14 (9H, s, t-Bu), 1.08 (9H, s, t-Bu).

参考例３
二糖ユニットの合成

フッ素置換二糖ユニット(24)の合成
グリコシルドナー(13)、グリコシルアクセプター(17)、MSAW300をCH₂Cl₂/CH₃CN（１：１）に溶かし、窒素雰囲気下、室温で３０分攪拌した。０℃に冷却した後、CH₂Cl₂に溶かしたTMSOTfをゆっくりと加え１時間攪拌した。この溶液に飽和重曹水を加えて反応をとめた後、セライトろ過し、有機層をブラインで洗浄した。MgSO₄で乾燥後、ろ過、濃縮してフラッシュカラムクロマトグラフィーにより精製し、(20)を得た。
(20) : ¹H-NMR(600MHz, CDCl₃, δ) 8.06-7.07(20H, Ph), 6.83(2H, d, J=8.8Hz, MP), 6.71(2H, d, J=8.8Hz, MP), 5.81(1H, dd, J=10.5, 7.9Hz, H2’), 5.77(1H, dd, J=8.3, 7.9Hz, H2), 5.26(1H, dd, J=27.0, 10.5Hz, H3’), 5.09(1H, d, J=38.5Hz, H4’), 5.08(1H, d, J=7.9Hz, H1'), 4.87(1H, d, J=7.9Hz, H1), 4.78(1H, s, H4), 4.69(1H, dd, J=11.2, 7.1Hz, H6'), 4.51(1H, dd, J=11.2, 6.3Hz, H6'), 4.22(1H, d, J=12.1 Hz, H6), 4.16(1H, d, J=12.1Hz, H6), 4.09(1H, ddd, J=25.8, 7.1, 6.3Hz, H5'), 3.98(1H, d, J=8.3Hz, H3), 3.73(3H, s, OMe), 3.42(1H, s, H5). Reference example 3
Synthesis of disaccharide units

Synthesis of fluorinated disaccharide unit (24) Glycosyl donor (13), glycosyl acceptor (17) and MSAW300 are dissolved in CH ₂ Cl ₂ / CH ₃ CN (1: 1) and stirred at room temperature for 30 minutes under nitrogen atmosphere. did. After cooling to 0 ° C., TMSOTf dissolved in CH ₂ Cl ₂ was slowly added and stirred for 1 hour. Saturated aqueous sodium hydrogen carbonate was added to this solution to stop the reaction, followed by Celite filtration, and the organic layer was washed with brine. After drying over MgSO ₄ , filtration, concentration and purification by flash column chromatography gave (20).
(20): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.06-7.07 (20H, Ph), 6.83 (2H, d, J = 8.8Hz, MP), 6.71 (2H, d, J = 8.8Hz, MP), 5.81 (1H, dd, J = 10.5, 7.9Hz, H2 '), 5.77 (1H, dd, J = 8.3, 7.9Hz, H2), 5.26 (1H, dd, J = 27.0, 10.5Hz, H3 '), 5.09 (1H, d, J = 38.5Hz, H4'), 5.08 (1H, d, J = 7.9Hz, H1 '), 4.87 (1H, d, J = 7.9Hz, H1), 4.78 (1H , s, H4), 4.69 (1H, dd, J = 11.2, 7.1Hz, H6 '), 4.51 (1H, dd, J = 11.2, 6.3Hz, H6'), 4.22 (1H, d, J = 12.1 Hz , H6), 4.16 (1H, d, J = 12.1Hz, H6), 4.09 (1H, ddd, J = 25.8, 7.1, 6.3Hz, H5 '), 3.98 (1H, d, J = 8.3Hz, H3) , 3.73 (3H, s, OMe), 3.42 (1H, s, H5).

次に(20)をＴＨＦに溶かし０℃に冷却した後、(HF)x_・ピリジン(7.0ml, 9.0mmol)を加え６時間攪拌した。飽和重曹水を加えて反応をとめた後、EtOAcで２回抽出した。有機層をブラインで洗浄し、MgSO₄で乾燥後、ろ過、濃縮した。これをそのままCH₂Cl₂に溶かし、ピリジン（5ml, 2.95mmol）、BzCl(8.7ml, 1.0mmol)、DMAP(触媒量)を入れ、６０℃で５時間攪拌した。水を加えて反応をとめた後、有機層を飽和重曹水、ブラインで洗浄し、MgSO₄乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーで精製し、(23)を得た。
(23) : ¹H-NMR(600MHz, CDCl₃, δ) 5.25-7.16(30H, m, Ph), 6.88(2H, d, J=9.0Hz, MP), 6.56(2H, d, 9.0Hz, MP), 5.95(1H, d, J=3.2Hz, H4), 5.85(1H, dd, J=9.5, 8.0Hz, H2), 5.62(1H, dd, J=10.3, 8.0, H2’), 5.20(1H, ddd, J=27.0, 10.5, 2.4Hz, H3’), 5.04(1H, dd, J=50.3, 2.4Hz, H4'), 5.00(1H, d, J=8.0Hz, H1), 4.98(1H, d, J=8.0Hz, H1'), 4.71(1H, ddd, J=11.3, 6.4, H6'), 4.64(1H, dd, J=11.8, 4.2Hz, H6), 4.50(1H, dd, J=11.8, 8.3Hz, H6), 4.47(1H, dd, J=11.3, 7.0Hz, H6'), 4.38(1H, dd, J=9.5, 3.2Hz, H3), 4.21(1H, m, H5), 4.08(1H, m, H5'). Next, (20) was dissolved in THF and cooled to 0 ° C., (HF) x _· pyridine (7.0 ml, 9.0 mmol) was added, and the mixture was stirred for 6 hours. Saturated aqueous sodium bicarbonate was added to quench the reaction, and the mixture was extracted twice with EtOAc. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. This was dissolved in CH ₂ Cl _{2 as} it was, pyridine (5 ml, 2.95 mmol), BzCl (8.7 ml, 1.0 mmol) and DMAP (catalytic amount) were added, and the mixture was stirred at 60 ° C. for 5 hours. After adding water to quench the reaction, the organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine and dried over MgSO ₄ . After filtration and concentration, purification by flash column chromatography gave (23).
(23): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 5.25-7.16 (30H, m, Ph), 6.88 (2H, d, J = 9.0Hz, MP), 6.56 (2H, d, 9.0Hz, MP), 5.95 (1H, d, J = 3.2Hz, H4), 5.85 (1H, dd, J = 9.5, 8.0Hz, H2), 5.62 (1H, dd, J = 10.3, 8.0, H2 '), 5.20 (1H, ddd, J = 27.0, 10.5, 2.4Hz, H3 '), 5.04 (1H, dd, J = 50.3, 2.4Hz, H4'), 5.00 (1H, d, J = 8.0Hz, H1), 4.98 (1H, d, J = 8.0Hz, H1 '), 4.71 (1H, ddd, J = 11.3, 6.4, H6'), 4.64 (1H, dd, J = 11.8, 4.2Hz, H6), 4.50 (1H, dd, J = 11.8, 8.3Hz, H6), 4.47 (1H, dd, J = 11.3, 7.0Hz, H6 '), 4.38 (1H, dd, J = 9.5, 3.2Hz, H3), 4.21 (1H, m , H5), 4.08 (1H, m, H5 ').

無置換二糖ユニット(24)の合成
同様に既知化合物であるグリコシルドナー18、グリコシルアクセプター17、MSAW300をCH₂Cl₂/CH₃CN（１：１）に溶かし、窒素雰囲気下、室温で３０分攪拌した。０℃に冷却した後、CH₂Cl₂に溶かしたTMSOTfをゆっくりと加え１時間攪拌した。この溶液に飽和重曹水を加えて反応をとめた後、セライトろ過し、有機層をブラインで洗浄した。MgSO₄で乾燥後、ろ過、濃縮してフラッシュカラムクロマトグラフィーにより精製し、(21)を得た。
(21) : ¹H-NMR(600MHz, CDCl₃, δ) ¹H-NMR(600MHz, CDCl₃, δ) 8.09-7.01(25H, Ph), 6.83(2H, d, J=9.0Hz, MP), 6.71(2H, d, J=9.0Hz, MP), 5.95(1H, d, J=3.2Hz, H4’), 5.85(1H, dd, J=10.3, 7.9Hz, H2’), 5.78(1H, dd, J=9.7, 7.9Hz, H2), 5.51(1H, dd, J=10.3, 3.2Hz, H3’), 5.14(1H, d, J=7.9Hz, H1'), 4.88(1H, d, J=7.9 Hz, H1), 4.88(1H, s, H4), 4.68(1H, dd, J=11.1, 7.1Hz, H6'), 4.37(1H, dd, J=11.1, 5.9Hz, H6'), 4.33(1H, dd, J=7.1, 5.9Hz, H5'), 4.21(1H, d, J=12.1Hz, H6), 4.10(1H, d, J=12.1, H6), 3.95(1H, d, J=9.7Hz, H3), 3.40(1H, s, H5), 1.11(9H, t-Bu×2). Synthesis of unsubstituted disaccharide unit (24) Similarly, glycosyl donor 18, glycosyl acceptor 17, and MSAW300, which are known compounds, are dissolved in CH ₂ Cl ₂ / CH ₃ CN (1: 1), and 30 at room temperature in a nitrogen atmosphere. Stir for minutes. After cooling to 0 ° C., TMSOTf dissolved in CH ₂ Cl ₂ was slowly added and stirred for 1 hour. Saturated aqueous sodium hydrogen carbonate was added to this solution to stop the reaction, followed by Celite filtration, and the organic layer was washed with brine. After drying over MgSO ₄ , filtration, concentration and purification by flash column chromatography gave (21).
(21): ¹ H-NMR (600 MHz, CDCl ₃ , δ) ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.09-7.01 (25H, Ph), 6.83 (2H, d, J = 9.0 Hz, MP) , 6.71 (2H, d, J = 9.0Hz, MP), 5.95 (1H, d, J = 3.2Hz, H4 '), 5.85 (1H, dd, J = 10.3, 7.9Hz, H2'), 5.78 (1H , dd, J = 9.7, 7.9Hz, H2), 5.51 (1H, dd, J = 10.3, 3.2Hz, H3 '), 5.14 (1H, d, J = 7.9Hz, H1'), 4.88 (1H, d , J = 7.9 Hz, H1), 4.88 (1H, s, H4), 4.68 (1H, dd, J = 11.1, 7.1Hz, H6 '), 4.37 (1H, dd, J = 11.1, 5.9Hz, H6' ), 4.33 (1H, dd, J = 7.1, 5.9Hz, H5 '), 4.21 (1H, d, J = 12.1Hz, H6), 4.10 (1H, d, J = 12.1, H6), 3.95 (1H, d, J = 9.7Hz, H3), 3.40 (1H, s, H5), 1.11 (9H, t-Bu × 2).

(21)をＴＨＦに溶かし０℃に冷却した後、(HF)x・ピリジン(7.0ml, 9.0mmol)を加え６時間攪拌した。飽和重曹水を加えて反応をとめた後、EtOAcで２回抽出した。有機層をブラインで洗浄し、MgSO₄で乾燥後、ろ過、濃縮した。これをそのままCH₂Cl₂に溶かし、ピリジン（5ml, 2.95mmol）、BzCl(8.7ml, 1.0mmol)、DMAP(触媒量)を入れ、６０℃で５時間攪拌した。水を加えて反応をとめた後、有機層を飽和重曹水、ブラインで洗浄し、MgSO₄乾燥した。ろ過、濃縮後、フラッシュカラムクロマトグラフィーで精製し、(24)を得た。
(24) : ¹H-NMR(600MHz, CDCl₃, δ) 8.24-7.07(35H, Ph), 6.82(2H, d, J=9.0Hz, MP), 6.55(2H, d, J=9.0Hz, MP), 6.02(1H, d, J=3.6Hz, H4), 5.87(1H, d, J=3.4Hz, H4’), 5.84(1H, dd, J=9.6Hz, 7.9Hz, H2), 5.60(1H, dd, J=10.4, 7.8Hz, H2’), 5.40(1H, dd, J=10.4, 3.4Hz, H3'), 5.02(1H, d, J=7.8Hz, H1'), 4.98(1H, d, J=7.9Hz, H1), 4.71(1H, dd, J=11.2, 6.2Hz, H6'), 4.61(1H, dd, J=11.8, 4.1Hz, H6), 4.50(1H, dd, J=11.8, 8.3Hz, H6), 4.34-4.30(2H, m, H6', H3), 4.27(1H, t, J=6.2Hz, H5'), 4.18(1H, dd, J=8.3, 4.1Hz, H5), 3.66(1H, s, OMe). (21) was dissolved in THF and cooled to 0 ° C., (HF) x · pyridine (7.0 ml, 9.0 mmol) was added, and the mixture was stirred for 6 hours. Saturated aqueous sodium bicarbonate was added to quench the reaction, and the mixture was extracted twice with EtOAc. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. This was dissolved in CH ₂ Cl _{2 as} it was, pyridine (5 ml, 2.95 mmol), BzCl (8.7 ml, 1.0 mmol) and DMAP (catalytic amount) were added, and the mixture was stirred at 60 ° C. for 5 hours. After adding water to quench the reaction, the organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine and dried over MgSO ₄ . After filtration and concentration, the residue was purified by flash column chromatography to obtain (24).
(24): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.24-7.07 (35H, Ph), 6.82 (2H, d, J = 9.0 Hz, MP), 6.55 (2H, d, J = 9.0 Hz, MP), 6.02 (1H, d, J = 3.6Hz, H4), 5.87 (1H, d, J = 3.4Hz, H4 '), 5.84 (1H, dd, J = 9.6Hz, 7.9Hz, H2), 5.60 (1H, dd, J = 10.4, 7.8Hz, H2 '), 5.40 (1H, dd, J = 10.4, 3.4Hz, H3'), 5.02 (1H, d, J = 7.8Hz, H1 '), 4.98 ( 1H, d, J = 7.9Hz, H1), 4.71 (1H, dd, J = 11.2, 6.2Hz, H6 '), 4.61 (1H, dd, J = 11.8, 4.1Hz, H6), 4.50 (1H, dd , J = 11.8, 8.3Hz, H6), 4.34-4.30 (2H, m, H6 ', H3), 4.27 (1H, t, J = 6.2Hz, H5'), 4.18 (1H, dd, J = 8.3, 4.1Hz, H5), 3.66 (1H, s, OMe).

参考例４
ジペプチドの合成

市販のＺ−グリシン(28)とセリンベンジルエステル(29)をCH₂Cl₂に溶かし、DPPA、Et₃N、およびDMAPを加え、３時間攪拌した。有機層を５％クエン酸水溶液、飽和重曹水、ブラインで洗浄後、MgSO₄で乾燥した。ろ過、濃縮後、EtOAcから結晶化しZ-Gly-Ser(OH)-OBn (30)を得た。 Reference example 4
Dipeptide synthesis

Commercially available Z-glycine (28) and serine benzyl ester (29) were dissolved in CH ₂ Cl ₂ , DPPA, Et ₃ N and DMAP were added and stirred for 3 hours. The organic layer was washed with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate, and brine, and then dried over MgSO ₄ . After filtration and concentration, crystallization from EtOAc gave Z-Gly-Ser (OH) -OBn (30).

実施例１
ｐ-メトキシフェノール体の合成

(23)をトルエン：CH₃CN：水＝1.5：１：１に溶かし、CANを加え１時間攪拌した。有機層を水、飽和重曹水、ブラインで洗浄し、MgSO₄で乾燥した。ろ過、濃縮後フラッシュカラムクロマトグラフィーで簡易精製した。不純物が混ざるため、この粗生成物をそのまま次の反応に用いた。粗生成物をCH₂Cl₂に溶かした後、０℃に冷却しCCl₃CN、DBUを加え１時間攪拌した。これをそのまま濃縮しフラッシュカラムクロマトグラフィーにより精製し、(34)を得た。また、(24)も同様の条件で反応を行い、(35)を得た。 Example 1
Synthesis of p-methoxyphenol compounds

(23) was dissolved in toluene: CH ₃ CN: water = 1.5: 1: 1, CAN was added, and the mixture was stirred for 1 hour. The organic layer was washed with water, saturated aqueous sodium hydrogen carbonate, brine and dried over MgSO ₄ . After filtration and concentration, it was simply purified by flash column chromatography. Since the impurities were mixed, this crude product was directly used in the next reaction. The crude product was dissolved in CH ₂ Cl ₂ , cooled to 0 ° C., CCl ₃ CN and DBU were added, and the mixture was stirred for 1 hour. This was concentrated as it was and purified by flash column chromatography to obtain (34). In addition, (24) was reacted under the same conditions to obtain (35).

(34) : ¹H-NMR(600MHz, CDCl₃, δ) 8.50(1H, s, H1), 8.14-7.02(30H, Ph), 6.74(1H, d, J=3.6Hz, H1), 6.10(1H, d, J=3.2Hz, H4), 5.74(1H, dd, J=10.3, 3.6Hz, H2), 5.62(1H, dd, J=10.3, 7.8Hz, H2’), 5.26(1H, ddd, J=26.9, 10.3, 2.5Hz, H3’), 5.09(1H, d, J=7.8Hz, H1’), 5.06(1H, dd, J=50.0, 2.5Hz, H4'), 4.77(1H, dd, J=11.3, 6.6Hz, H6'), 4.65-4.62(2H, m, H3, H5'), 4.56(1H, dd, J=11.3, 6.6Hz, H6'), 4.48(1H, ddd, J=44.6, 11.8, 7.6Hz, H6), 4.48(1H, ddd, J=44.6, 11.8, 4.4Hz, H6), 4.20(1H, ddd, J=26.0, 7.6, 4.4Hz, H5).
(35) : ¹H-NMR(600MHz, CDCl₃, δ) 8.51(1H, s, H1), 8.16-7.07(35H, Ph), 6.75(1H, d, J=3.6 Hz, H1), 6.10(1H, d, J=3.2Hz, H4), 5.90(1H, d, J=3.4Hz, H4’), 5.74(1H, dd, J=10.3, 3.6Hz, H2), 5.62(1H, dd, J=10.3, 7.9Hz, H2’), 5.09(1H, d, J=7.9Hz, H1’), 5.55(1H, dd, J=10.3, 3.4Hz, H3'), 5.02(1H, d, J=7.8Hz, H1'), 4.71(1H, dd, J=11.2, 6.2Hz, H6'), 4.61(1H, dd, J=12.0, 4.0Hz, H6), 4.44(1H, dd, J=12.0, 8.3Hz, H6), 4.34-4.30(2H, m, H6', H3), 4.35(1H, t, J=6.2Hz, H5'), 4.20(1H, dd, J=8.3, 4.0Hz, H5). (34): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.50 (1H, s, H1), 8.14-7.02 (30H, Ph), 6.74 (1H, d, J = 3.6 Hz, H1), 6.10 ( 1H, d, J = 3.2Hz, H4), 5.74 (1H, dd, J = 10.3, 3.6Hz, H2), 5.62 (1H, dd, J = 10.3, 7.8Hz, H2 '), 5.26 (1H, ddd , J = 26.9, 10.3, 2.5Hz, H3 '), 5.09 (1H, d, J = 7.8Hz, H1'), 5.06 (1H, dd, J = 50.0, 2.5Hz, H4 '), 4.77 (1H, dd, J = 11.3, 6.6Hz, H6 '), 4.65-4.62 (2H, m, H3, H5'), 4.56 (1H, dd, J = 11.3, 6.6Hz, H6 '), 4.48 (1H, ddd, J = 44.6, 11.8, 7.6Hz, H6), 4.48 (1H, ddd, J = 44.6, 11.8, 4.4Hz, H6), 4.20 (1H, ddd, J = 26.0, 7.6, 4.4Hz, H5).
(35): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.51 (1H, s, H1), 8.16-7.07 (35H, Ph), 6.75 (1H, d, J = 3.6 Hz, H1), 6.10 ( 1H, d, J = 3.2Hz, H4), 5.90 (1H, d, J = 3.4Hz, H4 '), 5.74 (1H, dd, J = 10.3, 3.6Hz, H2), 5.62 (1H, dd, J = 10.3, 7.9Hz, H2 '), 5.09 (1H, d, J = 7.9Hz, H1'), 5.55 (1H, dd, J = 10.3, 3.4Hz, H3 '), 5.02 (1H, d, J = 7.8Hz, H1 '), 4.71 (1H, dd, J = 11.2, 6.2Hz, H6'), 4.61 (1H, dd, J = 12.0, 4.0Hz, H6), 4.44 (1H, dd, J = 12.0, 8.3Hz, H6), 4.34-4.30 (2H, m, H6 ', H3), 4.35 (1H, t, J = 6.2Hz, H5'), 4.20 (1H, dd, J = 8.3, 4.0Hz, H5) .

次に、グリコシルドナー(34)、既知化合物であるグリコシルアクセプター36、MSAW300をCH₂Cl₂/CH₃CN（１：１）に溶かし、窒素雰囲気下、室温で３０分攪拌した。０℃に冷却した後、CH₂Cl₂に溶かしたTMSOTfをゆっくりと加え１時間攪拌した。この溶液に飽和重曹水を加えて反応をとめた後、セライトろ過し、有機層をブラインで洗浄した。MgSO₄で乾燥後、ろ過、濃縮してフラッシュカラムクロマトグラフィーにより精製し、(40)を得た。また、(35)も同様の条件で反応を行い、(41)を得た。
(40) : ¹H-NMR(600MHz, CDCl₃, δ) 8.05-7.16(40H, Ph), 6.86(2H, d, J=9.0Hz, MP), 6.73(2H, d, J=9.0Hz, MP), 5.75(1H, d, J=3.2Hz, H4), 5.64(1H, t, J=6.2 Hz, H3), 5.55(2H, m, H2’, H2’’), 5.37(1H, dd, J=6.2, 4.7Hz, H1), 5.13(1H, ddd, J=27.0, 10.5, 2.2Hz, H4’’), 4.87(1H, d, J=7.9Hz, H1''), 4.77(1H, d, 8.0Hz, H1'), 4.63(1H, dd, J=11.3, 6.2Hz, H6''), 4.43(1H, dd, J=11.3, 7.0Hz, H6''), 4.25(1H, dd, J=10.5, 3.2Hz, H3'), 4.17(1H, dd, J=11.6, 4.7Hz, H6'), 4.12-3.97(3H, m, H5, H5'', H6'), 3.91(1H, dd, J=10.4, 6.4Hz, H4), 3.73(1H, dd, J=11.6, 7.8Hz, H5'), 3.71(3H, s, OMe), 3.36(1H, dd, J=12.5, 6.4Hz, H5).
(41) : ¹H-NMR(600MHz, CDCl₃, δ) 8.10-7.09(45H, Ph), 6.89(2H, d, J=9.1Hz, MP), 6.76(2H, d, J=9.1Hz, MP) 5.88(1H, d, J=3.2Hz, H4’), 5.87(1H, d, J=3.0Hz, H4), 5.68-5.56(2H, m, H2’’, H2’), 5.41-5.38(2H, m, H3’’, H2), 5.20(1H, d, J=4.9Hz, H1), 4.97(1H, d, J=7.7Hz, H1''), 4.81(1H, d, J=8.0Hz, H1'), 4.68(1H, dd, J=11.2, 6.3Hz, H6'), 4.32-4.24(3H, m, H3, H5', H6'), 4.20(1H, dd, J=11.6, 4.6Hz, H6''), 4.05(1H, m, H6''), 4.01(1H, dd, J=12.4, 3.9Hz, H5), 3.97(1H, m, H4), 3.77(1H, m, H5''), 3.76(3H, s, OMe), 3.40(1H, dd, J=12.4, 6.4Hz, H5). Next, the glycosyl donor (34), the known compound, glycosyl acceptor 36, and MSAW300 were dissolved in CH ₂ Cl ₂ / CH ₃ CN (1: 1) and stirred at room temperature for 30 minutes under a nitrogen atmosphere. After cooling to 0 ° C., TMSOTf dissolved in CH ₂ Cl ₂ was slowly added and stirred for 1 hour. Saturated aqueous sodium hydrogen carbonate was added to this solution to stop the reaction, followed by Celite filtration, and the organic layer was washed with brine. After drying over MgSO ₄ , filtration, concentration and purification by flash column chromatography gave (40). In addition, (35) was reacted under the same conditions to obtain (41).
(40): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.05-7.16 (40H, Ph), 6.86 (2H, d, J = 9.0 Hz, MP), 6.73 (2H, d, J = 9.0 Hz, MP), 5.75 (1H, d, J = 3.2Hz, H4), 5.64 (1H, t, J = 6.2 Hz, H3), 5.55 (2H, m, H2 ', H2``), 5.37 (1H, dd , J = 6.2, 4.7Hz, H1), 5.13 (1H, ddd, J = 27.0, 10.5, 2.2Hz, H4``), 4.87 (1H, d, J = 7.9Hz, H1 ''), 4.77 (1H , d, 8.0Hz, H1 '), 4.63 (1H, dd, J = 11.3, 6.2Hz, H6``), 4.43 (1H, dd, J = 11.3, 7.0Hz, H6''), 4.25 (1H, dd, J = 10.5, 3.2Hz, H3 '), 4.17 (1H, dd, J = 11.6, 4.7Hz, H6'), 4.12-3.97 (3H, m, H5, H5``, H6 '), 3.91 ( 1H, dd, J = 10.4, 6.4Hz, H4), 3.73 (1H, dd, J = 11.6, 7.8Hz, H5 '), 3.71 (3H, s, OMe), 3.36 (1H, dd, J = 12.5, 6.4Hz, H5).
(41): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.10-7.09 (45H, Ph), 6.89 (2H, d, J = 9.1 Hz, MP), 6.76 (2H, d, J = 9.1 Hz, MP) 5.88 (1H, d, J = 3.2Hz, H4 '), 5.87 (1H, d, J = 3.0Hz, H4), 5.68-5.56 (2H, m, H2``, H2'), 5.41-5.38 (2H, m, H3``, H2), 5.20 (1H, d, J = 4.9Hz, H1), 4.97 (1H, d, J = 7.7Hz, H1 ''), 4.81 (1H, d, J = 8.0Hz, H1 '), 4.68 (1H, dd, J = 11.2, 6.3Hz, H6'), 4.32-4.24 (3H, m, H3, H5 ', H6'), 4.20 (1H, dd, J = 11.6 , 4.6Hz, H6``), 4.05 (1H, m, H6 ''), 4.01 (1H, dd, J = 12.4, 3.9Hz, H5), 3.97 (1H, m, H4), 3.77 (1H, m , H5``), 3.76 (3H, s, OMe), 3.40 (1H, dd, J = 12.4, 6.4Hz, H5).

実施例２
糖ペプチドの合成（１）

(40)をトルエン：CH₃CN：水＝1.5：１：１に溶かし、CANを加え１時間攪拌した。有機層を水、飽和重曹水、ブラインで洗浄し、MgSO₄で乾燥した。ろ過、濃縮後フラッシュカラムクロマトグラフィーで簡易精製した。不純物が混ざるため、この粗生成物をそのまま次の反応に用いた。粗生成物をCH₂Cl₂に溶かした後、０℃に冷却しCCl₃CN、DBUを加え１時間攪拌した。これをそのまま濃縮しフラッシュカラムクロマトグラフィーにより精製し、(45)を得た。また、(41)も同様の条件で反応を行い、(46)を得た。 Example 2
Synthesis of glycopeptides (1)

(40) was dissolved in toluene: CH ₃ CN: water = 1.5: 1: 1, CAN was added, and the mixture was stirred for 1 hour. The organic layer was washed with water, saturated aqueous sodium hydrogen carbonate, brine and dried over MgSO ₄ . After filtration and concentration, it was simply purified by flash column chromatography. Since the impurities were mixed, this crude product was directly used in the next reaction. The crude product was dissolved in CH ₂ Cl ₂ , cooled to 0 ° C., CCl ₃ CN and DBU were added, and the mixture was stirred for 1 hour. This was concentrated as it was and purified by flash column chromatography to obtain (45). In addition, (41) was reacted under the same conditions to obtain (46).

(45) : ¹H-NMR(600MHz, CDCl₃, δ) 8.47(1H, s, NH), 8.05-7.09(40H, Ph), 6.52(1H, d, J=3.6Hz, H1), 5.88(1H, t, J=9.7 Hz, H3), 5.78(1H, d, J=3.1Hz, H4’), 5.53(1H, dd, J=10.4, 7.9Hz, H2’’), 5.44(1H, dd, J=9.9, 8.0Hz, H2’), 5.31(1H, dd, J=9.7, 3.6Hz, H2), 5.12(1H, ddd, J=27.1, 10.4, 2.6Hz, H3''), 4.97(1H, dd, J=50.1, 2.6Hz, H4''), 4.84(1H, d, J=7.9 Hz, H1''), 4.68(1H, d, J=8.0 Hz, H1'), 4.64(1H, dd, J=11.3, 6.2Hz, H6''), 4.45(1H, dd, J=11.3, 7.2Hz, H6''), 4.25(1H, dd, J=9.9, 3.1Hz, H3'), 4.13(1H, m, H4), 4.10-3.93(2H, m, H5'', H6'), 3.88(1H, t, J=6.2 Hz, H5'), 3.82(1H, dd, J=11.2, 6.2Hz, H6'), 3.71(1H, dd, J=11.3, 5.8Hz, H5), 3.62(1H, t, J=11.3Hz, H5).
グリコシルドナー(45)、ペプチドアクセプター30、MSAW300をCH₂Cl₂/CH₃CN（１：１）に溶かし、窒素雰囲気下、室温で３０分攪拌した。０℃に冷却した後、CH₂Cl₂に溶かしたTMSOTfをゆっくりと加え１時間攪拌した。この溶液に飽和重曹水を加えて反応をとめた後、セライトろ過し、有機層をブラインで洗浄した。MgSO₄で乾燥後、ろ過、濃縮してフラッシュカラムクロマトグラフィーにより精製し、(50)を得た。また(46)も同様の条件で反応を行い、(51)を得た。 (45): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.47 (1H, s, NH), 8.05-7.09 (40H, Ph), 6.52 (1H, d, J = 3.6 Hz, H1), 5.88 ( 1H, t, J = 9.7 Hz, H3), 5.78 (1H, d, J = 3.1Hz, H4 '), 5.53 (1H, dd, J = 10.4, 7.9Hz, H2``), 5.44 (1H, dd , J = 9.9, 8.0Hz, H2 '), 5.31 (1H, dd, J = 9.7, 3.6Hz, H2), 5.12 (1H, ddd, J = 27.1, 10.4, 2.6Hz, H3''), 4.97 ( 1H, dd, J = 50.1, 2.6Hz, H4``), 4.84 (1H, d, J = 7.9 Hz, H1 ''), 4.68 (1H, d, J = 8.0 Hz, H1 '), 4.64 (1H , dd, J = 11.3, 6.2Hz, H6 ''), 4.45 (1H, dd, J = 11.3, 7.2Hz, H6 ''), 4.25 (1H, dd, J = 9.9, 3.1Hz, H3 '), 4.13 (1H, m, H4), 4.10-3.93 (2H, m, H5``, H6 '), 3.88 (1H, t, J = 6.2 Hz, H5'), 3.82 (1H, dd, J = 11.2, 6.2Hz, H6 '), 3.71 (1H, dd, J = 11.3, 5.8Hz, H5), 3.62 (1H, t, J = 11.3Hz, H5).
Glycosyl donor (45), peptide acceptor 30, and MSAW300 were dissolved in CH ₂ Cl ₂ / CH ₃ CN (1: 1) and stirred at room temperature for 30 minutes under a nitrogen atmosphere. After cooling to 0 ° C., TMSOTf dissolved in CH ₂ Cl ₂ was slowly added and stirred for 1 hour. Saturated aqueous sodium hydrogen carbonate was added to this solution to stop the reaction, followed by Celite filtration, and the organic layer was washed with brine. After drying over MgSO ₄ , filtration, concentration and purification by flash column chromatography gave (50). Further, (46) was reacted under the same conditions to obtain (51).

(50) : ¹H-NMR(600MHz, CDCl₃, δ) 8.05-7.16(50H, Ph), 6.56(1H, d, J=7.4Hz, NH), 5.74(1H, d, J=3.6 Hz, H4’), 5.53(1H, dd, J=10.2, 7.8Hz, H2’), 5.53-5.49(2H, m, H3, H2’’), 5.24(1H, m, NH), 5.15(1H, ddd, J=26.9, 10.4, 2.7Hz, H3’’), 5.09(2H, d, J=12.6Hz, Bn×2), 5.04(1H, dd, J=6.0, 4.9Hz, H2), 4.98(2H, d, J=12.6 Hz, Bn×2), 4.97(1H, dd, J=50.4, 2.7Hz, H4''), 4.86(1H, d, J=7.8 Hz, H1''), 4.73(1H, d, J=7.8Hz, H1'), 4.71(1H, m, Ser-α-H), 4.63(1H, dd, J=11.4, 6.6Hz, H6''), 4.51(1H, m, H1), 4.43(1H, dd, J=11.4, 7.2Hz, H6''), 4.25(1H, dd, J=10.2, 3.6Hz, H3'), 4.13(1H, m, H6'), 4.01-3.96(2H, m, H6', H5''), 3.75-3.70(5H, m, H5', Gly-α-H×2, Ser-β-H×2), 3.65(1H, m, H4), 3.55(1H, dd, J=12.2, 3.4Hz, H5), 3.08(1H, m, H5).
(51) : ¹H-NMR(600MHz, CDCl₃, δ) 8.16-7.05(50H, Ph), 6.72(1H, m, NH), 5.88(2H, s, H4’, H4’’), 5.60-5.55(3H, m, H2’, H2’’, H3), 5.41(1H, dd, J=10.5, 3.2Hz, H3’’), 5.20-4.78(4H, Bn×4), 5.08(1H, m, H2), 4.79(1H, d, J=8.0Hz, H1’), 4.73(1H, m, Ser-α-H), 4.68(1H, dd, J=11.0, 6.1Hz, H6''), 4.57(1H, brs, H1), 4.32-4.25(4H, m, H3', H6', H5'', H6''), 4.15(1H, m, H6'), 4.06(1H, m, H5'), 3.77-3.74(4H, m, Gly-α-H×2, Ser-β-H×2), 3.69(1H, m, H4), 3.55(1H, m, H5), 3.09 (1H, m, H5). (50): ¹ H-NMR (600 MHz, CDCl ₃ , δ) 8.05-7.16 (50H, Ph), 6.56 (1H, d, J = 7.4 Hz, NH), 5.74 (1H, d, J = 3.6 Hz, H4 '), 5.53 (1H, dd, J = 10.2, 7.8Hz, H2'), 5.53-5.49 (2H, m, H3, H2 ''), 5.24 (1H, m, NH), 5.15 (1H, ddd , J = 26.9, 10.4, 2.7Hz, H3``), 5.09 (2H, d, J = 12.6Hz, Bn × 2), 5.04 (1H, dd, J = 6.0, 4.9Hz, H2), 4.98 (2H , d, J = 12.6 Hz, Bn × 2), 4.97 (1H, dd, J = 50.4, 2.7Hz, H4``), 4.86 (1H, d, J = 7.8 Hz, H1 ''), 4.73 (1H , d, J = 7.8Hz, H1 '), 4.71 (1H, m, Ser-α-H), 4.63 (1H, dd, J = 11.4, 6.6Hz, H6``), 4.51 (1H, m, H1 ), 4.43 (1H, dd, J = 11.4, 7.2Hz, H6 ''), 4.25 (1H, dd, J = 10.2, 3.6Hz, H3 '), 4.13 (1H, m, H6'), 4.01-3.96 (2H, m, H6 ', H5''), 3.75-3.70 (5H, m, H5', Gly-α-H × 2, Ser-β-H × 2), 3.65 (1H, m, H4), 3.55 (1H, dd, J = 12.2, 3.4Hz, H5), 3.08 (1H, m, H5).
(51): ¹ H-NMR (600MHz, CDCl ₃ , δ) 8.16-7.05 (50H, Ph), 6.72 (1H, m, NH), 5.88 (2H, s, H4 ', H4''), 5.60- 5.55 (3H, m, H2 ', H2``, H3), 5.41 (1H, dd, J = 10.5, 3.2Hz, H3''), 5.20-4.78 (4H, Bn × 4), 5.08 (1H, m , H2), 4.79 (1H, d, J = 8.0Hz, H1 '), 4.73 (1H, m, Ser-α-H), 4.68 (1H, dd, J = 11.0, 6.1Hz, H6''), 4.57 (1H, brs, H1), 4.32-4.25 (4H, m, H3 ', H6', H5 '', H6 ''), 4.15 (1H, m, H6 '), 4.06 (1H, m, H5' ), 3.77-3.74 (4H, m, Gly-α-H × 2, Ser-β-H × 2), 3.69 (1H, m, H4), 3.55 (1H, m, H5), 3.09 (1H, m , H5).

実施例３
糖ペプチドの合成（２）

(50)をMeOHに溶かし、Pdカーボンを加え水素雰囲気下で３時間攪拌した。セライトろ過、濃縮後MeOHに溶かしNaOMeを加え１時間攪拌した。DOWEX 50W×8［Ｈ⁺］を加えて反応をとめた後、ろ過してファルコンチューブにうつした。これにMeOHの１０倍量のEt₂Oを加えて生成物を析出させ、遠心分離（3500rpm, 10分間）し上澄みを取り除いた。沈殿物をゲルろ過クロマトグラフィー（Sephadex G-10）により精製し、(55)を得た。また、(51)も同様の条件で反応を行い、(56)を得た。 Example 3
Synthesis of glycopeptides (2)

(50) was dissolved in MeOH, Pd carbon was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After Celite filtration and concentration, dissolved in MeOH, NaOMe was added and stirred for 1 hour. After DOWEX 50W × 8 [H ⁺ ] was added to stop the reaction, the mixture was filtered and placed in a falcon tube. To this was added 10 times the amount of Et ₂ O of MeOH to precipitate the product, which was centrifuged (3500 rpm, 10 minutes) and the supernatant was removed. The precipitate was purified by gel filtration chromatography (Sephadex G-10) to obtain (55). In addition, (51) was reacted under the same conditions to obtain (56).

(55) : ¹H-NMR (600MHz, CDCl₃, δ) 4.68(1H, d, J=50.0Hz, H4’’), 4.57(1H, d, J=7.4 Hz, H1’’), 4.35(1H, d, J=7.4Hz, H1’), 4.29(1H, brs, Ser-α-H), 4.25(1H, d, J=7.3Hz, H1), 4.08(1H, m, Ser-β-H), 4.02(1H, s, H4’), 3.95(1H, m, H5), 3.78-3.41(15H, m), 3.28-3.15(2H, m, H2, H5).
(56) : ¹H-NMR (600MHz, CDCl₃, δ) 4.47(1H, d, J=7.4Hz, H1’’), 4.38(1H, d, J=7.3Hz, H1’), 4.32(1H, brs, Ser-α-H), 4.29(1H, d, J=7.2Hz, H1), 4.05(1H, d, J=3.2Hz, H4’), 4.04 (1H, m, Ser-β-H), 3.94(1H, dd, J=11.8, 5.3Hz, H5), 3.76(1H, d, J=3.2Hz, H4’’), 3.71-3.42(15H, m), 3.28-3.15(2H, m, H2, H5).
(55)をＤＭＦに縣濁させ、ＦＩＴＣ、飽和重曹水を加え室温で３時間攪拌した。この溶液をファルコンチューブに移してEtOH/Et₂O（１：１０）を加え、沈殿を析出させた。遠心分離（3500rpm, 10分間）し、上澄みを取り除いた後、沈殿物をゲルろ過クロマトグラフィー（Sephadex G-10）により精製し、(60)を得た。また、(56)も同様の条件で反応を行い、(61)を得た。生成物はMALDI-TOF MASSにより分子量を確認した。
質量分析の結果を図５に示す。 (55): ¹ H-NMR (600MHz, CDCl ₃ , δ) 4.68 (1H, d, J = 50.0Hz, H4``), 4.57 (1H, d, J = 7.4 Hz, H1 ''), 4.35 ( 1H, d, J = 7.4Hz, H1 '), 4.29 (1H, brs, Ser-α-H), 4.25 (1H, d, J = 7.3Hz, H1), 4.08 (1H, m, Ser-β- H), 4.02 (1H, s, H4 '), 3.95 (1H, m, H5), 3.78-3.41 (15H, m), 3.28-3.15 (2H, m, H2, H5).
(56): ¹ H-NMR (600MHz, CDCl ₃ , δ) 4.47 (1H, d, J = 7.4Hz, H1 ''), 4.38 (1H, d, J = 7.3Hz, H1 '), 4.32 (1H , brs, Ser-α-H), 4.29 (1H, d, J = 7.2Hz, H1), 4.05 (1H, d, J = 3.2Hz, H4 '), 4.04 (1H, m, Ser-β-H ), 3.94 (1H, dd, J = 11.8, 5.3Hz, H5), 3.76 (1H, d, J = 3.2Hz, H4 ''), 3.71-3.42 (15H, m), 3.28-3.15 (2H, m , H2, H5).
(55) was suspended in DMF, FITC and saturated aqueous sodium hydrogen carbonate were added, and the mixture was stirred at room temperature for 3 hours. This solution was transferred to a falcon tube and EtOH / Et ₂ O (1:10) was added to precipitate a precipitate. After centrifugation (3500 rpm, 10 minutes) and removal of the supernatant, the precipitate was purified by gel filtration chromatography (Sephadex G-10) to obtain (60). In addition, (56) was reacted under the same conditions to obtain (61). The molecular weight of the product was confirmed by MALDI-TOF MASS.
The results of mass spectrometry are shown in FIG.

本発明の化合物は、医薬、特に変形性関節症その他の軟骨疾患の予防または治療剤として有用である。 The compound of the present invention is useful as a pharmaceutical, particularly as an agent for preventing or treating osteoarthritis and other cartilage diseases.

糖ペプチドを添加してCHO-K1細胞の培養を行い、その上澄のゲル濾過クロマトグラフィーを示す。A1は、糖ペプチド(Ia)を添加した場合であり、溶出した分画をコンドロイチナーゼＡＢＣで消化した後のクロマトグラフィーがA2である。The CHO-K1 cells are cultured with the glycopeptide added, and the gel filtration chromatography of the supernatant is shown. A1 is the case where glycopeptide (Ia) is added, and the chromatography after digesting the eluted fraction with chondroitinase ABC is A2. 糖ペプチドを添加してCHO-K1細胞の培養を行い、その上澄のゲル濾過クロマトグラフィーを示す。B1は糖ペプチド(Ib)を添加した場合であり、溶出した分画をコンドロイチナーゼＡＢＣで消化した後のクロマトグラフィーがB2である。The CHO-K1 cells are cultured with the glycopeptide added, and the gel filtration chromatography of the supernatant is shown. B1 is the case where glycopeptide (Ib) is added, and the chromatography after digesting the eluted fraction with chondroitinase ABC is B2. 糖ペプチドを添加してヒト正常軟骨細胞を培養し、産生されたムコ多糖量を示すグラフである。It is a graph which shows the amount of mucopolysaccharides produced by culturing human normal chondrocytes with the addition of glycopeptides. 糖ペプチドを添加してヒト正常軟骨細胞を培養し、産生されたアグリカンを示すグラフである。It is a graph which shows the aggrecan produced by adding a glycopeptide and culturing human normal chondrocytes. 糖ペプチドIa（化合物６１）および糖ペプチドIb（化合物６０）の質量分析結果、および異なる水酸基にフッ素を有する糖ペプチドの質量分析スペクトルの比較結果である。It is a mass spectrometry result of glycopeptide Ia (compound 61) and glycopeptide Ib (compound 60), and a comparison result of mass spectrometry spectra of glycopeptides having fluorine at different hydroxyl groups.

Claims

Formula (I)

(In the formula, X represents a halogen atom, a hydroxyl group or an alkoxy group, and R represents a hydrogen atom, an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or natural or non-natural. Represents a peptide residue comprising the amino acids of
Or a pharmaceutically acceptable salt thereof, or a solvate thereof.

The compound according to claim 1, wherein X is a fluorine atom or a hydroxyl group in formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof.

In the formula (I), -OR is the following formula

(In the formula, FITC represents that the amino group is fluorescently labeled with fluorescein isothiocyanate.)
The compound of Claim 1 or 2 which is a glycylserine derivative represented by these, its pharmaceutically acceptable salt, or those solvates.

The pharmaceutical which uses the compound in any one of Claim 1 to 3, its pharmaceutically acceptable salt, or those solvates as an active ingredient.

A therapeutic agent for osteoarthritis comprising the compound according to any one of claims 1 to 3, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.