JP2009274955A - Osteoarthritis-treating medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chondropathy-preventing and/or treating medicine for selectively promoting the biosynthesis of proteoglycan, especially chondroitin sulfate type proteoglycan. <P>SOLUTION: The medicine contains a compound represented by formula (I) (wherein, X is a halogen atom, OH or an alkoxy group; R is H, an alkyl which may be substituted, an aryl which may be substituted, a heteroaryl which may be substituted, or a peptide residue comprising natural or non-natural amino acids). There is also provided a pharmaceutically acceptable salt thereof, or pharmaceutical product containing their solvates as active ingredients. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  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.

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.

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).

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

  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.

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.

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. .

  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.

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

(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.

（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.

（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.

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.

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.

  “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.

“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.

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.

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.

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

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.

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

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

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).

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

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.

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.

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.

  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.

  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.

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.

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.

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.

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.

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).

(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).

(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).

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).

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).

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).

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).

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 ').

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) 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).

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).

(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): 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) .

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).

(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): 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): 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): <sup>1</sup> H-NMR (600MHz, CDCl <sub>3</sub> , 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)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): 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.

  The compound of the present invention is useful as a pharmaceutical, particularly as an agent for preventing or treating osteoarthritis and other cartilage diseases.

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. 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. It is a graph which shows the amount of mucopolysaccharides produced by culturing human normal chondrocytes with the addition of glycopeptides. It is a graph which shows the aggrecan produced by adding a glycopeptide and culturing human normal chondrocytes. 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 (5)

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.

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