JP5481731B2 - Method for producing sugar chain dendrimer containing thiasialoside type oligosaccharide and use thereof - Google Patents

Description

  The present invention relates to a thialoolo-oligosaccharide-linked dendrimer compound and a medicament containing the dendrimer compound as an active ingredient. More specifically, the present invention relates to a dendrimer compound to which thiosialo-oligosaccharide is bound, a dendrimer compound having influenza neuraminidase inhibitory activity, and a medicament containing the dendrimer compound as an active ingredient.

Dendrimer is a general term for dendritic polymer compounds that are regularly branched from the Greek word “dendra” (tree). Since a spherical nanometer-scale space by dendrimers can be designed relatively freely by incorporating various functional groups, new dendrimers are being actively designed in the field of nanotechnology.
In particular, in recent years, the use of dendrimers in the field of biological functions has been remarkable, such as dendrimers that respond to external stimuli in biological systems, dendrimers that can be used for DDS (drug delivery systems), dendrimers that can function as molecular sensors, etc. Research is progressing to make use of sex.

Among them, the use of dendrimers is attracting attention as an effective defense tool against interference from the external environment with respect to living bodies, particularly infection with bacteria and viruses.
For example, development of dendrimers that can effectively protect the body from attack by verotoxin produced by enterohemorrhagic Escherichia coli O-157 has been carried out. Verotoxin produced by enterohemorrhagic E. coli O-157 is a protein belonging to the AB5 family of bacterial toxins similar to Shiga toxin derived from Shigella. These toxins are reported to be toxic by recognizing and adhering to the globotrisaccharide moiety in globotriosylceramide (Gb3, Galα1-4Galβ1-4Glcβ1-Cer) on kidney cells. Has been.

  The present inventors have already synthesized a cluster compound having a carbosilane dendrimer linked with the globotrisaccharide as a core skeleton, and reported that it has strong verotoxin inhibitory activity (Non-patent Documents 1 and 2). And Patent Documents 1 and 2).

In addition, the present inventors have specifically adhered hemagglutinin present on the surface of viruses such as influenza viruses based on the knowledge about various sugar chain-containing carbosilane dendrimer compounds (see Non-Patent Document 3), and virus infection to living bodies. A sialyl lactose-containing dendrimer has been disclosed as a substance capable of preventing (see Patent Document 2). Furthermore, the dendrimer which couple | bonds a sugar chain via the amide bond excellent in the compatibility and safety | security in the living body is also disclosed (refer patent document 3).
Adhesion and detachment from host cells are important for influenza virus infection, and this process involves two types of proteins, hemagglutinin and neuraminidase (sialidase), respectively. In particular, neuraminidase is an essential enzyme for influenza virus to detach from infected cells. Therefore, if the neuraminidase activity can be effectively inhibited, infection of other cells with influenza virus can be suppressed, and therefore, it can be applied to the treatment of influenza virus infection.
Influenza membrane protein ion channel inhibitors (Symmetrel ^R (amantadine)) and sialidase inhibitors (Tamiflu ^R (oseltamivir phosphate) and Relenza ^R (zanamivir)) have already been prescribed as specific medicines for influenza. . However, since the active ingredients of these specific drugs are not natural products, the emergence of resistant viruses is feared, and in recent years, there are reports that resistant viruses against simmetrel ^R and Tamiflu ^R have emerged.

Matsuoka et al., Tetrahedron Letters 40: 7839-7842 1999. Nishikawa et al., Proc. Natl. Acad. Sci. USA. 99: 7669-7664 2002 Matsuoka et al., Bull. Chem. Soc. Jpn. 71: 2709-2713 1998 JP 2004-107230 A International Publication WO02 / 02588 JP2003-212893

An object of the present invention is to provide a dendrimer compound that effectively inhibits the neuraminidase activity of influenza virus and becomes an active ingredient of a therapeutic agent for influenza virus infection.
Furthermore, this invention aims at provision of the pharmaceutical which contains this dendrimer compound as an active ingredient.

In view of the above circumstances, the present inventors have conducted extensive research for the purpose of developing a therapeutic agent for influenza virus infection that can retain a therapeutic effect against resistant viruses. The present inventors have found that a dendrimer conjugated with an oligosaccharide of sialic acid can effectively inhibit the neuraminidase activity of influenza virus, and completed the present invention.
As an influenza neuraminidase inhibitor, it is considered that a transition state analog of a substrate generated during hydrolysis is effective. It has been reported that sialic acid derivatives bind to the active center of influenza neuraminidase as a transition state analog and are effective in inhibiting the activity. Tamiflu ^R (oseltamivir phosphate), marketed as an anti-influenza drug, was developed with a focus on such a mechanism. However, since the sialic acid analog that is the effective site of Tamiflu ^R (oseltamivir phosphate) is not natural, a resistant virus may appear. As mentioned above, it is actually resistant to Tamiflu ^R (oseltamivir phosphate). There is also a report that a virus has appeared.
The inventors synthesized a dendrimer conjugated with a thioglycoside-linked sialic acid oligosaccharide for the purpose of avoiding the emergence of resistant viruses and creating a compound with high influenza neuraminidase inhibitory activity.

（式中、Ｅ^１及びＥ^２は、炭素、ケイ素、ゲルマニウムのいずれかであり、互いに同一でも異なっていてもよく、Ｒ^１、Ｒ^２は、同一又は異なった炭化水素基を示し、Ｒ^３、Ｒ^４及びＲ^５は酸素、窒素及び／又はカルボニル基を含んでもよい同一又は異なった炭化水素鎖を示し、Ｙはチオシアロオリゴ糖残基若しくは他の置換基であって少なくとも１つはチオシアロオリゴ糖残基を示し、ｌは０〜２の整数であり、ｍは０〜２の整数であり、ｋは０又は１の数を示し、ｋが０のときは３−ｍは１である）で表されるチオシアロオリゴ糖結合デンドリマー化合物若しくはその薬理学上許容される塩又はそれらの水和物、並びにこれらのチオシアロオリゴ糖結合デンドリマー化合物若しくはその薬理学上許容される塩又はそれらの水和物の他、薬理学上許容される担体を含む医薬である。 That is, the present invention provides the following formula (I)

(Wherein E ¹ and E ² are any of carbon, silicon and germanium, and may be the same or different from each other, R ¹ and R ² represent the same or different hydrocarbon groups, and R ³ , R ⁴ and R ⁵ represent the same or different hydrocarbon chains that may contain oxygen, nitrogen and / or carbonyl groups, Y is a thiosialogooligosaccharide residue or other substituent, at least one of which is a thiosialogooligosaccharide residue And 1 is an integer of 0 to 2, m is an integer of 0 to 2, k is a number of 0 or 1, and 3-k is 1 when k is 0). Thiosialogooligosaccharide-bonded dendrimer compounds or pharmacologically acceptable salts thereof or hydrates thereof, as well as these thiosialogooligosaccharide-bonded dendrimer compounds or pharmacologically acceptable salts thereof or hydrates thereof. A medicament containing a pharmacologically acceptable carrier.

  The thiosialo-oligosaccharide-linked dendrimer compound of the present invention effectively inhibits the activity of influenza neuraminidase. In addition, an effect of suppressing the emergence of resistant virus at a low rate can be expected.

In formula (I), E ¹ and E ² are any of carbon, silicon, and germanium, and may be the same or different from each other, but carbon or silicon is preferable, and silicon is most preferable.

R ¹ and R ² represent the same or different hydrocarbon groups, but any of an alkyl group having 3 to 6 carbon atoms, a phenyl group, a vinyl group, and an allyl group is preferable, and among them, the carbon number is 3 to 4 An alkyl group or a phenyl group is more preferable.

R ³ , R ⁴ and R ⁵ represent the same or different hydrocarbon group which may contain oxygen, nitrogen and / or carbonyl group, but an alkyl group, alkylene group, alkenylene group and alkoxylene having 3 to 12 carbon atoms. Any of the groups (oxyalkylene groups) is preferable, and among these, an alkylene group having 3 to 6 carbon atoms is more preferable.

また、本発明のデンドリマーにおいては、１分子中の全てのＹの位置が上記置換基のいずれかであることが望ましいが、必ずしも、全てのＹが上記置換基である必要はなく、例えば、水素、Ｃ＝Ｃ二重結合、水酸基などであってもよく、当該技術分野における通常の合成方法により、チオシアロオリゴ糖以外にＹの位置に結合し得ると当業者によって予測され得る置換基の如何なるものであってもよい。 At least one of Y represents a monosaccharide to a trisaccharide having a thioglycoside type sialic acid at a non-reducing end. As sialic acid, NeuAc (N-acetylneuraminic acid: Neu5Ac) or NeuGc (N-glycolylneuraminic acid: Neu5Gc) can be used, but NeuAc (NeuAcN -Acetylneuraminic acid) residue is preferred. Moreover, as sugars couple | bonded with sialic acid, 1-3 sugars are preferable, and what kind of sugar is well-known to those skilled in the art can be used, but it is not limited, For example, galactose, glucose, Lactose, cellobiose and the like can be suitably used. The bond between Y and the dendrimer skeleton is a glycosidic bond or a thioglycoside bond, and a thioglycoside bond is particularly preferable. Therefore, Y becomes the following substituents, for example.

Further, in the dendrimer of the present invention, it is desirable that all Y positions in one molecule are any of the above substituents, but not all Y need necessarily be the above substituents. , C = C double bond, hydroxyl group and the like, and any substituent that can be predicted by those skilled in the art to be able to bind to the Y position in addition to thiosialoglygosaccharides by a usual synthesis method in the art. There may be.

（式Ｉａ、Ｉｂ、Ｉｃ及びＩｄ中、Ｅ^１及びＥ^２は、炭素、ケイ素、ゲルマニウムのいずれかであり、互いに同一でも異なっていてもよく、Ｒ^１は、炭化水素基を示し、Ｒ^３、Ｒ^４及びＲ^５は酸素又は窒素あるいはカルボニル基を含んでもよい同一又は異なった炭化水素鎖を示し、Ｙはチオシアロオリゴ糖残基若しくは他の置換基であって少なくとも１つはチオシアロオリゴ糖残基を示す） The structure of the thiosialogooligosaccharide-bonded dendrimer compound of formula (I) can take various structures depending on the combination of k, l, and m, but typical chemical formulas are as follows.

(In Formulas Ia, Ib, Ic and Id, E ¹ and E ² are any of carbon, silicon and germanium, and may be the same or different from each other, R ¹ represents a hydrocarbon group, and R ³ , R ⁴ and R ⁵ represent the same or different hydrocarbon chain which may contain oxygen or nitrogen or a carbonyl group, Y is a thiosialogooligosaccharide residue or other substituent and at least one is a thiosialogooligosaccharide residue Show)

（上記式中、Ｘ^１はハロゲン原子、Ｘ^２は反応脱離性の保護基を示し、Ｙは、チオシアロオリゴ糖である）
本発明の式（Ｉ）の化合物は、式（ＩＩＩ）で表されるハロゲン化デンドリマーと式（ＩＶ）で表されるスルフィド化合物とを反応させ、必要に応じて、スルフィド化合物中のチオシアロオリゴ糖残基の保護基を脱離させることにより本発明の式（Ｉ）の化合物を製造できる。 The compound of the formula (I) of the present invention can be produced, for example, according to the following reaction formula.

(In the above formula, X ¹ represents a halogen atom, X ² represents a reaction-eliminating protecting group, and Y is a thiosialo-oligosaccharide)
The compound of the formula (I) of the present invention reacts the halogenated dendrimer represented by the formula (III) with the sulfide compound represented by the formula (IV), and if necessary, the thiosialo-oligosaccharide residue in the sulfide compound. The compound of formula (I) of the present invention can be produced by removing the protecting group.

As an example of the method for producing the compound of the formula (IV), the method for producing the compound (36) is shown in the Examples described later. In the case of producing thioacetate derivatives of other thiosialogooligosaccharides, they can be produced by the same method.
The halogenated dendrimer can be produced, for example, as follows.
Taking Fan (0) 3-Br as an example, it is prepared by introducing a mesyl group as a leaving group into a known compound triol (tris (3-hydroxypropyl) phenylsilane) and performing a nucleophilic substitution reaction with a bromine anion. (See, for example, Matsuoka et al., Biomacromolecules 7, pp. 2274-2283, 2006, etc. for this reaction). Moreover, it can manufacture similarly about the halogenated dendrimer which has another structure.
The reaction of the compound of formula (III) and the compound of formula (IV) can be carried out in the presence of a base such as sodium methoxide. The removal of the protecting group for Y can be carried out by hydrolysis using a base such as sodium methoxide.

    The obtained compound of the formula (I) of the present invention can be purified by washing, various chromatography, gel filtration and the like.

The compound of the present invention can be used as a medicament in the form of a pharmaceutical composition that does not adversely affect the living body.
The pharmaceutical composition may contain one or a plurality of types among the thiosialo-oligosaccharide-bound dendrimer compound represented by the general formula (I), a pharmacologically acceptable salt thereof, or a hydrate thereof.
Usually, the pharmaceutical composition includes a pharmacologically acceptable carrier in addition to the compound of the present invention.
“Pharmaceutically acceptable carrier” refers to those suitable for pharmaceutical administration, including solvents, dispersion media, coating agents, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like. . Examples of such carriers and those preferred for diluting the carriers include, but are not limited to, water, saline, finger solutions, dextrose solutions, and human serum albumin. Non-aqueous media such as liposomes and non-volatile oils are also used. In addition, certain compounds that protect or promote the activity of the compounds of the present invention may be included in the composition.

The medicament of the present invention includes intravenous, intradermal, subcutaneous, oral (including inhalation, etc.), transdermal and transmucosal administration, and is formulated to be suitable for a therapeutically appropriate route of administration. . Solutions or suspensions used for parenteral, intradermal, or subcutaneous application include, but are not limited to, sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents, benzyl alcohol or other preservatives such as methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, soothing agents such as benzalkonium chloride, procaine hydrochloride, ethylenediaminetetraacetic acid Chelating agents such as (EDTA), buffering agents such as acetate, citrate, or phosphate, and agents for osmotic pressure adjustment such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are contained in ampoules, glass or plastic disposable syringes or multiple dose vials.

  Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersion media and sterile powders for the preparation of sterile injectable solutions or dispersion media at the time of use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). When used as an injection, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes, corrosion, and the like during formulation and storage, and must prevent contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures. For example, using a coating agent such as lectin, maintaining a required particle size in the dispersion medium, and maintaining a proper fluidity by using a surfactant. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal can be used to prevent microbial contamination. In addition, polyalcohols such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition. Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by adding the required ingredients alone or in combination with other ingredients to the required amount of the active compound in a suitable solvent and sterilizing. Generally, a dispersion medium is prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the other necessary ingredients described above. Methods for preparing a sterile powder for preparing a sterile injectable solution include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired ingredients derived from the sterile solution. .

Oral compositions include inert diluents or carriers that are not harmful when incorporated into the body. Oral compositions are, for example, contained in gelatin capsules or compressed into tablets. For oral treatment, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a flowable carrier, and the composition in the flowable carrier is applied orally. In addition, pharmaceutically compatible binding agents, and / or adjuvant materials may be included.
Tablets, pills, capsules, troches and the like may contain any of the following components or compounds with similar properties: excipients such as microcrystalline cellulose, binding such as gum arabic, tragacanth or gelatin Agents; bulking agents such as alginic acid, PRIMOGEL, or corn starch; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl salicylic acid or orange flavor Perfume additive.

  The compounds of the present invention can be prepared as sustained release formulations such as delivery systems encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE), through a filter of appropriate pore size so as to be of a size suitable for use. Purified by evaporation.

In treating or preventing viral infections with the compounds of the present invention, the appropriate dosage level depends on the condition of the patient to be administered, the method of administration, etc., but can be easily optimized by those skilled in the art. It is.
In the case of injection administration, for example, it is preferable to administer about 0.1 μg / kg to about 500 mg / kg of the patient's body weight per day, and it will generally be possible to administer a single dose or divided into multiple doses. Preferably, the dosage level is about 0.1 μg / kg to about 250 mg / kg per day, more preferably about 0.5 to about 100 mg / kg per day.
For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 to 1000 mg of active ingredient, preferably 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750. 0, 800.0, 900.0 and 1000.0 mg. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily.

  A pharmaceutical composition or formulation must consist of uniform unit doses to ensure a constant dose. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. When determining the unit dosage of the present invention, physical and chemical characteristics of the compound to be formulated, expected therapeutic effects, considerations specific to the compound, and the like are considered.

  The pharmaceutical composition of the present invention can be included in the form of a kit in a container or pack together with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different constituents of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately in this way is to enable long-term storage without losing the function of the active component.

  Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.

  The kit also includes instructions for use. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

Furthermore, the present invention also includes a method for preventing or treating viral infections, particularly mammals infected with or at risk of infection with influenza viruses.
Here, “treatment” means to prevent or alleviate the progression of the pathology of the infectious disease in a mammal that is likely to be infected with a virus or is infected. It is used as a broad meaning including.
The “mammal” to be treated means any animal classified as a mammal, and is not particularly limited. For example, in addition to humans, pet animals such as dogs and cats, cows, pigs, sheep, horses It is a domestic animal. Particularly preferred “mammals” are humans.

  Examples are shown below, but the present invention is not limited thereto.

Synthesis Example 1 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Pyranosilonate)-(2 → 6) -2,3,4-tri-O-acetyl-6-thio-β-D-galactopyranoside (6) is synthesized as shown in the following scheme (6). Was synthesized.

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-Ot-butyldimethylsilyl-β-D-glucopyranoside (2) Under an argon atmosphere, the known pentenyl glucoside (1) (2.00 g , 8.06 mmol) was dissolved in pyridine (25 mL), TBDMSCl (2.19 g, 14.53 mmol) was added under ice cooling, and the mixture was stirred for 1 hour with ice cooling. Then, acetic anhydride (6.9 mL, 73.12 mmol) was added and stirred at room temperature for 2 hours. Methanol (10 mL) was added under ice cooling, and then the reaction solution was concentrated. The residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel 225 mL] to obtain the desired product (2) (3.63 g, 92.1%).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (3) Fully protected glucoside (2) (2.98 g, 6.10 mmol) was dissolved in acetic acid (24 mL) and water (6 mL) was added and it stirred at 50 degreeC for 2 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography [2: 1 (v / v) hexane-ethyl acetate, silica gel 180 mL] to obtain the desired product (3) (2.24 g, 98.2%). .

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-glucopyranoside (4) In an argon atmosphere, alcohol (3) (200 mg, 0.534 mmol) was pyridine. (3 mL), triphenylphosphine (210 mg, 0.801 mmol) and carbon tetrabromide (266 mg, 0.802 mmol) were sequentially added under ice-cooling, and the mixture was stirred at 45 ° C. for 15 minutes. Methanol (3 mL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was dissolved in a small amount of methanol, toluene was added thereto, and the by-product crystallized was removed by celite filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [5: 2 (v / v) hexane-ethyl acetate, silica gel 25 mL] to obtain the desired product (4) (224 mg, 95.7%).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- Synthesis of (2 → 6) -2,3,4-tri-O-acetyl-6-thio-β-D-galactopyranoside (6) Sialic acid thioacetate (5) (377 mg, 0 .686 mmol) and bromide (4) (200 mg, 0.457 mmol) were dissolved in DMF (1 mL), diethylamine (0.72 mL, 68.6 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with ethyl acetate, washed successively with 1M aqueous hydrochloric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was roughly purified by silica gel column chromatography [2: 3 (v / v) toluene-ethyl acetate, silica gel 45 mL], and then the desired product (6) (194 mg, 49.1%) was isolated by preparative GPC. .

Synthesis Example 2 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Pyranoshironate)-(2 → 6) -2,3,4-tri-O-acetyl-6-thio-β-D-galactopyranoside (11) is synthesized as shown in the following scheme. Was synthesized.

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-Ot-butyldimethylsilyl-β-D-galactopyranoside (8) Under an argon atmosphere, known pentenyl glucosides (eg, Matsuoka) And Nishimura, Macromolecules 28, pp. 2961-2968, 1995) (7) (2.87 g, 11.56 mmol) were dissolved in pyridine (25 mL) and TBDMSCl (3.48 g) was cooled with ice. , 23.09 mmol), and stirred for 5 hours while cooling with ice. Then, acetic anhydride (9.8 mL, 103.86 mmol) was added and stirred at room temperature for 6 hours. Methanol (15 mL) was added under ice cooling, and then the reaction solution was concentrated. The residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel 300 mL] to obtain the desired product (8) (4.45 g, 78.8%).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-β-D-galactopyranoside (9) Fully protected galactoside (8) (300 mg, 0.614 mmol) was dissolved in acetic acid (2.4 mL). Water (0.6 mL) was added and stirred at 50 ° C. for 2 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography [1: 1 (v / v) hexane-ethyl acetate, silica gel 180 mL] to obtain the desired product (9) (225 mg, 97.8%).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-galactopyranoside (10) Alcohol (9) (200 mg, 0.534 mmol) under argon atmosphere ) Was dissolved in pyridine (3 mL), triphenylphosphine (280 mg, 1.068 mmol) and carbon tetrabromide (266 mg, 0.802 mmol) were sequentially added under ice cooling, and the mixture was stirred at 50 ° C. for 1 hour. Under ice-cooling, methanol (3 mL) was added to stop the reaction, and the reaction solution was concentrated. The residue was dissolved in a small amount of methanol, toluene was added thereto, and the by-product crystallized was removed by celite filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel 25 mL] to obtain the desired product (10) (148 mg, 63.2%).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- Synthesis of (2 → 6) -2,3,4-tri-O-acetyl-6-thio-β-D-galactopyranoside (11) Under argon atmosphere, thioacetate (5) (364 mg, 0.662 mmol ) And bromide (10) (145 mg, 0.332 mmol) were dissolved in a DMF-methanol mixture (1: 1 (v / v), 1.4 mL), and potassium carbonate (91 mg, 0.662 mmol) was cooled with ice. ) And stirred at room temperature for 21 hours. Under ice-cooling, acetic acid (76 μL, 1.324 mmol) was added to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (4 mL), acetic anhydride (3 mL) was added, and the mixture was stirred overnight. The reaction solution was concentrated, and the residue was dissolved in chloroform, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. . The residue was roughly purified by silica gel column chromatography [15: 14: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 40 mL], and then the desired product (11) (122 mg, 42.5) by preparative GPC. %) Was isolated.

Synthesis Example 3 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Synthesis of pyranoshironate)-(2 → 6) -2,3,4-tri-O-acetyl-1,6-thio- [beta] -D-glucopyranoside (16) Compound (16) is Synthesis was performed as in the scheme.

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6- Ot -butyldimethylsilyl-1-thio-β-D-glucopyranoside (13) Under an argon atmosphere, a known pentenyl thioglucoside (Leuck And Kunz, J. prakt.Chem.339, pp.322-334, 1997, etc.) (12) (6.30 g, 23.83 mmol) was dissolved in pyridine (65 mL) under ice cooling. TBDMSCl (6.46 g, 42.86 mmol) was added, and the mixture was stirred for 1 hour with ice cooling. Then, acetic anhydride (13.5 mL, 143.07 mmol) was added and stirred at room temperature for 3 hours. Methanol (20 mL) was added under ice cooling, and then the reaction solution was concentrated. The residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel 0.8 L] to obtain the desired product (13) (11.39 g, 94.7%).

n-pentenyl 2,3,4-tri-O-acetyl-1-thio-β-D-glucopyranoside (14)
Fully protected thioglucoside (13) (5.90 g, 12.02 mmol) was dissolved in acetic acid (48 mL), water (12 mL) was added, and the mixture was stirred at 50 ° C. for 2 hr. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography [2: 1 (v / v) hexane-ethyl acetate, silica gel 250 mL] to obtain the desired product (14) (4.42 g, 97.6%). .

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-1-thio-β-D-glucopyranoside (15) Alcohol (14) (300 mg, 0. 768 mmol) was dissolved in pyridine (4.5 mL), triphenylphosphine (302 mg, 1.151 mmol) and carbon tetrabromide (382 mg, 1.151 mmol) were sequentially added under ice cooling, and the mixture was stirred at 45 ° C. for 15 minutes. . Methanol (3 mL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was purified by flash silica gel column chromatography [80: 1 (v / v) chloroform-methanol, silica gel 50 mL] to obtain the desired product (15) (318 mg, 91.4%).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- Synthesis of (2 → 6) -2,3,4-tri-O-acetyl-1,6-thio-β-D-glucopyranoside (16) Under argon atmosphere, thioacetate (3) (273 mg, 0.497 mmol ) And bromide (15) (150 mg, 0.331 mmol) were dissolved in DMF (2 mL), diethylamine (0.78 mL, 7.465 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 7 hours. The reaction mixture was concentrated, and the residue was roughly purified by silica gel column chromatography [2: 3 → 0: 1 (v / v) toluene-ethyl acetate, silica gel 40 mL], and then subjected to preparative GPC (16) (181 mg , 64.4%).

Synthesis Example 4 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Pyranosylonate)-(2 → 6) -2,3,4-tri-O-acetyl-1,6-thio-β-D-galactopyranoside (21) is synthesized by the following scheme (21). The synthesis was performed as follows.

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-Ot-butyldimethylsilyl-1-thio-β-D-galactopyranoside (18) Known pentenylthio under argon atmosphere Glucoside (see Leuck and Kunz, J. prakt. Chem. 339, pp. 322-334, 1997, etc.) (17) (5.30 g, 20.05 mmol) was dissolved in pyridine (55 mL) and iced. TBDMSCl (5.44 g, 36.09 mmol) was added under cooling, and the mixture was stirred for 5 hours while cooling with ice. Then, acetic anhydride (17.0 mL, 180.16 mmol) was added and stirred at room temperature for 1 hour. Methanol (30 mL) was added under ice cooling, and then the reaction solution was concentrated. The residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel 0.9 L] to obtain the desired product (18) (8.16 g, 80.7%).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-1-thio-β-D-galactopyranoside (19) Fully protected thiogalactoside (18) (550 mg, 1.090 mmol) was converted to acetic acid (4 8 mL), water (1.2 mL) was added, and the mixture was stirred at 50 ° C. for 3 hours. The reaction mixture was concentrated, and the residue was purified by flash silica gel column chromatography [2: 1 (v / v) hexane-ethyl acetate, silica gel 40 mL] to obtain the desired product (19) (388 mg, 91.3%).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-1-thio-β-D-galactopyranoside (20) Alcohol (19) (200 mg) under argon atmosphere , 0.512 mmol) was dissolved in pyridine (3 mL), triphenylphosphine (242 mg, 0.923 mmol) and carbon tetrabromide (255 mg, 0.769 mmol) were sequentially added under ice cooling, and the mixture was stirred at 45 ° C. for 45 minutes. did. Methanol (3 mL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was purified by flash silica gel column chromatography [80: 1 (v / v) chloroform-methanol, silica gel 40 mL] to obtain the desired product (20) (165 mg, 71.1%).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- Synthesis of (2 → 6) -2,3,4-tri-O-acetyl-1,6-thio-β-D-galactopyranoside (21) Thioacetate (5) (364 mg, 0 662 mmol) and bromide (20) (150 mg, 0.331 mmol) were dissolved in a DMF-methanol mixture (1: 1 (v / v), 1.5 mL), and potassium carbonate (92 mg 0.666 mmol) was cooled with ice. ) And stirred at room temperature for 32 hours. Under ice-cooling, acetic acid (76 μL, 1.328 mmol) was added to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (1 mL), acetic anhydride (1 mL) was added, and the mixture was stirred overnight. The reaction solution is concentrated, and the residue is dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through Celite, and the filtrate is concentrated. did. The residue was roughly purified by silica gel column chromatography [2: 3 (v / v) toluene-ethyl acetate, silica gel 30 mL], and then the desired product (21) (162 mg, 57.4%) was obtained by preparative GPC (chloroform). Isolated.

Synthesis Example 5 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Pyranoshironate)-(2 → 6) -O- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6- Synthesis of tri-O-acetyl- [beta] -D-glucopyranoside (26) Compound (26) was synthesized according to the following scheme.

n-pentenyl O- (2,3-di-O-acetyl-4,6-O-isopropylidene-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O -Synthesis of acetyl-β-D-glucopyranoside (23) Under an argon atmosphere, known pentenyl lactosides (see, for example, Matsuoka and Nishimura, Macromolecules 28, pp. 2961-2968, 1995) (22) ( 11.36 g (27.67 mmol) was dissolved in DMF (100 mL), and 2-methoxy-1-propene (5.2 mL, 55.53 mmol) and p-toluenesulfonic acid monohydrate (1.05 g) were cooled with ice. , 5.52 mmol) was added in order, and the mixture was stirred for 3 hours with ice cooling. Thereafter, pyridine (80 mL) and acetic anhydride (65.3 mL, 692.01 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated, the residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [4: 1 (v / v) toluene-ethyl acetate, silica gel 0.9 L] to obtain the desired product (23) (14.56 g, 79.6%).

n-pentenyl O- (2,3-di-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β-D-glucopyranoside ( Synthesis of 24) Completely protected lactoside (23) (200 mg, 0.303 mmol) was dissolved in acetic acid (1.6 mL), water (0.4 mL) was added, and the mixture was stirred at 50 ° C. for 2 hr. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography [1: 4 (v / v) toluene-ethyl acetate, silica gel 20 mL] to obtain the desired product (24) (178 mg, 94.7%).

n-pentenyl O- (2,3-di-O-acetyl-6-bromo-6-deoxy-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O— Synthesis of acetyl-β-D-glucopyranoside (25) Diol (24) (155 mg, 0.250 mmol) was dissolved in pyridine (2.5 mL) under an argon atmosphere, and triphenylphosphine (98 mg, 0. 374 mmol) and carbon tetrabromide (124 mg, 0.473 mmol) were sequentially added, and the mixture was stirred at 50 ° C. for 30 minutes and at 60 ° C. for 3 hours. Under ice cooling, triphenylphosphine (65 mg, 0.248 mmol) and carbon tetrabromide (83 mg, 0.250 mmol) were further added in this order, and the mixture was stirred at 60 ° C. for 30 minutes. Under ice cooling, methanol (1 mL) was added to stop the reaction, and the reaction solution was concentrated. The residue was dissolved in a small amount of methanol, toluene was added thereto, and the by-product crystallized was removed by celite filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [3: 1 (v / v) toluene-ethyl acetate, silica gel 25 mL] to obtain the desired product (25) (130 mg, 76.0%).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- (2 → 6) -O- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl Synthesis of β-D-glucopyranoside (26) Under argon atmosphere, thioacetate (5) (169 mg, 0.308 mmol) and bromide (25) (105 mg, 0.154 mmol) were mixed with DMF-methanol mixture (1: 1 ( v / v), 1 mL), potassium carbonate (43 mg, 0.311 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 36 hours. Under ice-cooling, acetic acid (35 μL, 0.611 mmol) was added to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (2 mL), acetic anhydride (1.5 mL) was added, and the mixture was stirred for 3 days. The reaction solution is concentrated, and the residue is dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through Celite, and the filtrate is concentrated. did. The residue was roughly purified by silica gel column chromatography [1: 1 → 2: 3 → 0: 1 (v / v) toluene-ethyl acetate, silica gel 25 mL], and then subjected to preparative GPC (26) (80 mg, 45 .2%) was isolated.

Synthesis Example 6 n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuro Pyranoshironate)-(2 → 6) -O- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O Synthesis of -acetyl- [beta] -D-glucopyranoside (35) Compound (35) was synthesized as shown in the following scheme.

O- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-α-D-glucopyranosyl Synthesis of bromide (28) Commercially available cellobiose α-acetate (PFANSTIHL LABORATORIES Inc., Waukegan, IL 60085-0439, USA) (27) (10.00 g, 14.47 mmol) acetic acid (20 mL) -acetic anhydride (2 mL) The mixture was dissolved in a mixed solution, 30% hydrogen bromide-acetic acid solution (5.9 mL, 29.48 mL) was added under ice-cooling, the mixture was sealed, and the mixture was stirred at room temperature for 3.5 hours. Further, 30% hydrogen bromide-acetic acid solution (5.9 mL, 29.48 mL) was added under ice cooling, and the mixture was stirred overnight. The reaction mixture was poured into ice water, extracted with ethyl acetate, washed 5 times with water, and washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine. After dehydration and drying with magnesium sulfate, the mixture was filtered through Celite, and the filtrate was concentrated. The residue was washed with diethyl ether to obtain the desired product (28) (10.20 g, 98.9%).

n-pentenyl O- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β-D-glucopyranoside (29) Under an argon atmosphere, cellobiose α-bromide (28) (28.10 g, 40.17 mmol) and 4-penten-1-ol (20.4 mL, 201.32 mmol), molecular sieves 4A (35 g) were added. Suspended in dichloromethane (180 mL) and stirred for 2 hours. Silver trifluoromethanesulfonate (12.39 g, 48.22 mmol) was added at −20 ° C., and the mixture was stirred for 40 minutes. The reaction solution was filtered through Celite, and the insoluble material was washed with chloroform. The filtrate and the washing solution were combined, washed successively with ice water, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by flash silica gel column chromatography [4: 1 (v / v) toluene-ethyl acetate, silica gel 0.9 L] to obtain the desired product (29) (15.23 g, 53.8%).

Synthesis of n-pentenyl O-β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranoside (30) Pentenyl cellobioside (29) (2.89 g, 4.10 mmol) in methanol (50 mL) -dichloromethane (5 mL) Dissolved in a mixed solution, 1M sodium methoxide / methanol solution (2 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was adjusted to pH˜4 by adding IR-120B (H + type) and filtered with a cotton plug, and the resin was washed with methanol. The filtrate and washings were combined and concentrated to obtain the desired product (30) (1.68 g, quantitative) as a residue.

n-pentenyl O- (2,3-di-O-acetyl-4,6-O-isopropylidene-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl- Synthesis of β-D-glucopyranoside (31) Cellobioside (30) (8.14 g, 19.83 mmol) was dissolved in DMF (80 mL) under an argon atmosphere, and 2-methoxy-1-propene (3. 7 mL, 39.51 mmol) and p-toluenesulfonic acid hydrate (0.75 g, 3.94 mmol) were added in order, and the mixture was stirred for 2 hours while cooling with ice and returning to room temperature for 4 hours. Thereafter, pyridi (50 mL) and acetic anhydride (47.0 mL, 498.08 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated, the residue was dissolved in ethyl acetate, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [5: 1 (v / v) toluene-ethyl acetate, silica gel 0.9 L] to obtain the desired product (31) (9.37 g, 71.5%).

n-pentenyl of O- (2,3-di-O-acetyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β-D-glucopyranoside (32) Synthetic fully protected cellobioside (31) (185 mg, 0.280 mmol) was dissolved in acetic acid (1.6 mL), water (0.4 mL) was added, and the mixture was stirred at 50 ° C. for 2 hr. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography [1: 2 (v / v) toluene-ethyl acetate, silica gel 20 mL] to obtain the desired product (32) (168 mg, 96.6%).

n-pentenyl O- (2,3-di-O-acetyl-6-bromo-6-deoxy-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β Synthesis of D-glucopyranoside (33) Diol (32) (1.71 g, 2.76 mmol) was dissolved in pyridine (25 mL) under an argon atmosphere, and triphenylphosphine (1.45 g, 5.53 mmol) was cooled with ice. ) And carbon tetrabromide (1.83 g, 5.52 mmol) were sequentially added, and the mixture was stirred at 50 ° C. for 1 hour. Under ice-cooling, methanol (5 mL) was added to stop the reaction, and the reaction solution was concentrated. The residue was dissolved in a small amount of methanol, toluene was added thereto, and the by-product crystallized was removed by celite filtration, and the filtrate was concentrated. The residue was purified by flash silica gel column chromatography [1: 1 (v / v) hexane-ethyl acetate, silica gel 25 mL] to obtain the desired product (33) (1.86 g, 98.9%).

n-pentenyl O- (2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl Synthesis of β-D-glucopyranoside (34) In an argon atmosphere, alcohol (33) (1.08 g, 1.58 mmol) was dissolved in pyridine (5 mL), and acetic anhydride (0.75 mL, 7. 95 mmol) was added and stirred at room temperature for 6 hours. The reaction mixture was concentrated, and the residue was purified by flash silica gel column chromatography [2: 1 (v / v) toluene-ethyl acetate, silica gel 80 mL] to obtain the desired product (34) (1.15 g, quantitative).

n-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)- (2 → 6) -O- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β- Synthesis of D-glucopyranoside (35) In an argon atmosphere, thioacetate (5) (227 mg, 0.413 mmol) and bromide (34) (200 mg, 0.276 mmol) were dissolved in DMF (0.8 mL), and ice-cooled. Diethylamine (0.43 mL, 4.11 mmol) was added and stirred at room temperature for 20 hours. The reaction mixture was concentrated, the residue was dissolved in chloroform, washed successively with 1M aqueous hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was roughly purified by silica gel column chromatography [100: 30: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 40 mL], and then subjected to preparative GPC (35) (249 mg, 78.3). %) Was isolated.

[Synthesis Example 7] Synthesis of thiosialogooligosaccharide-linked dendrimer A thiosialogooligosaccharide-linked dendrimer was synthesized as shown in the following scheme.

4-acetylthio-pentenyl S- (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate )-(2 → 6) -2,3,4-Tri-O-acetyl-1,6-thio-β-D-glucopyranoside (36s) Under an argon atmosphere, pentenyl glycoside (16) (216 mg, 0. 245 mmol) was dissolved in 1,4-dioxane (0.5 mL), thioacetic acid (1 mL, 13.99 mmol) was added, and the mixture was heated to 50 ° C. Therefore, AIBN (80 mg, 0.49 mmol) was added and stirred at 80 ° C. for 3 hours. Thereafter, cyclohexene (99 μL, 0.98 mmol) was added under ice cooling, and the mixture was returned to room temperature and stirred for several minutes. The reaction mixture was concentrated, and the residue was purified by flash silica gel column chromatography [toluene → ethyl acetate, silica gel 25 mL] to obtain the desired product (36s) (235 mg, quantitative).

Synthesis of Fan (0) 3-NeuSGlcS (OAc, OMe) (37s) Under an argon atmosphere, thioacetate (36s) (180 mg, 0.188 mmol) and Fan (0) 3-Br dendrimer (20 mg, 0.042 mmol) Was dissolved in a DMF (0.45 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (207 μL, 0.207 mmol) was added thereto and stirred at room temperature for 19 hours. Thereafter, acetic acid (12 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (0.5 mL) under an argon atmosphere, acetic anhydride (0.5 mL) was added, and the mixture was stirred at room temperature for 5 hours. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [15: 14: 1 → 10: 9: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 20 mL] to obtain the desired product (37s) (86 mg, 68.3). %). In addition, disaccharide dimer (41s) (31 mg) was obtained as a by-product.

Synthesis of Ball (0) 4-NeuSGlcS (OAc, OMe) (38s) Under argon atmosphere, thioacetate (36s) (180 mg, 0.188 mmol), and Ball (0) 4-Br dendrimer (17 mg, 0.033 mmol) Was dissolved in a DMF (0.45 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (207 μL, 0.207 mmol) was added thereto, and the mixture was stirred at room temperature for 24 hours. Thereafter, acetic acid (12 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (0.5 mL) under an argon atmosphere, acetic anhydride (0.5 mL) was added, and the mixture was stirred at room temperature for 22 hours. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed with water, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [15: 14: 1 → 7: 7: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 20 mL] to obtain the desired product (38s) (75 mg, 59.1%). ) Moreover, the disaccharide dimer (41s) (22 mg) was obtained as a by-product.

Synthesis of Dumbbell (1) 6-NeuSGlcS (OAc, OMe) (39s) Under argon atmosphere, thioacetate (36s) (190 mg, 0.199 mmol), and Dumbbell (1) 6-Br dendrimer (21 mg, 0.023 mmol) Was dissolved in a DMF (0.45 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (219 μL, 0.219 mmol) was added thereto, and the mixture was stirred at room temperature for 23 hours. Thereafter, acetic acid (13 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (0.5 mL) under an argon atmosphere, acetic anhydride (0.5 mL) was added, and the mixture was stirred at room temperature for 22 hours. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed with water, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [15: 14: 1 → 5: 4: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 20 mL] to obtain the desired product (39s) (87 mg, 64.9% ) In addition, disaccharide dimer (41s) (33 mg) was obtained as a by-product.

Synthesis of Ball (1) 12-NeuSGlcS (OAc, OMe) (40s) Under an argon atmosphere, thioacetate (36s) (180 mg, 0.188 mmol), and Ball (1) 12-Br dendrimer (20 mg, 0.011 mmol) Was dissolved in a DMF (0.45 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (207 μL, 0.207 mmol) was added thereto and stirred at room temperature for 8 hours. Thereafter, acetic acid (12 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (0.5 mL) under an argon atmosphere, acetic anhydride (0.5 mL) was added, and the mixture was stirred at room temperature for 24 hours. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed with water, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [15: 14: 1 → 15: 0: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 20 mL] to obtain the desired product (38s) (50 mg, 37.6%). )
The compound (36o) was synthesized in the same manner as the compound (36s).
Also, fan-type, ball-type, and dumbbell-type dendrimer compounds carrying the compound (36o) and the compound (35) were synthesized in the same manner as described above ([Synthesis Example 7]).
Further, fan-type, ball-type, and dumbbell-type dendrimer compounds carrying the compound (11) and the compound (26) were synthesized in the same manner as described above ([Synthesis Example 7]).

[Synthesis Example 8] Synthesis of ether and amide-extended thiosialo-oligosaccharide-linked dendrimer An ether and amide-extended thiosialo-oligosaccharide-linked dendrimer were synthesized as shown in the following scheme.

Synthesis of Fan (0) 3-ether-NeuSGlcO (OAc, OMe) (42o) Under argon atmosphere, thioacetate (36o) (249.6 mg, 265.5 μmol), and Fan (0) 3-ether-Br dendrimer ( 38.1 mg, 59.0 μmol) was dissolved in a DMF (0.25 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (266 μL, 266 μmol) was added thereto and stirred at room temperature for 24 hours. Thereafter, acetic acid (15 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (1.5 mL) under an argon atmosphere, acetic anhydride (1.5 mL) was added, and the mixture was stirred at 35 ° C. overnight. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [10: 9: 1 → 8: 6: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 30 mL] to obtain the desired product (42o) (144.7 mg, 79 .2%).
The ball type and dumbbell type were synthesized in the same manner.
Further, the ether-extended dendrimer carrying the compound (35), the compound (11) and the compound (26) was synthesized in the same manner as described above.

Synthesis of Fan (0) 3-amido-NeuSGlcO (OAc, OMe) (43o) Under argon atmosphere, thioacetate (36o) (250.0 mg, 266.0 μmol), and Fan (0) 3-amido-Br dendrimer ( 59.1 mg, 47.9 μmol) was dissolved in a DMF (0.25 mL) -methanol (0.25 mL) mixture. 1M sodium methoxide / methanol solution (266 μL, 266 μmol) was added thereto and stirred at room temperature for 24 hours. Thereafter, acetic acid (15 μL) was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue was suspended in pyridine (1.5 mL) under an argon atmosphere, acetic anhydride (1.5 mL) was added, and the mixture was stirred at 35 ° C. overnight. Thereafter, the reaction solution was concentrated, the residue was dissolved in chloroform, washed successively with 1M aqueous sulfuric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dehydrated and dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography [10: 9: 1 → 8: 6: 1 → 6: 5: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel 30 mL] to obtain the target compound (43o) ( 103.8 mg, 53.9%).
The ball type and dumbbell type were synthesized in the same manner.
Furthermore, the amide extension dendrimer carrying the compound (35), the compound (11) and the compound (26) was synthesized in the same manner as described above.

[Synthesis Example 9] Deprotection of Thiosialoside Compound The deprotection of the thiocyanoside compound containing dendrimer and monomer synthesized as described above was carried out according to the following scheme.

Synthesis of Fan (0) 3-NeuSGlcO (OH) (44o) Fan (0) 3-NeuSGlcO (OAc, OMe) (37o) (48.1 mg, 16.5 μmol) in methanol (0.5 mL) under an argon atmosphere Sodium methoxide (1M methanol solution, 8 μL, 8 μM) was added and stirred at room temperature for 1 hour. Thereafter, 0.5 M aqueous sodium hydroxide solution (2 mL) was added and stirred overnight, and after adding strong acidic cation exchange resin Dowex-50W-X8 (H ⁺ ) (appropriate amount) to pH ˜4, cotton plug Filtration was performed and the filtrate was concentrated. The residue was purified by gel filtration (Sephadex G-25: 250 mL, 5% AcOH aq.) And then freeze-dried to obtain the desired product (44o) (30.5 mg, 92.7%) as a white solid. .

アルゴン雰囲気下、ＮｅｕＳＧｌｃＯ(ＯＡｃ，ＯＭｅ)（６）（７９．１ｍｇ，９１．６μｍｏｌ）をメタノール（１．０ ｍＬ）に溶解させ、ナトリウムメトキシド（１Ｍ メタノール溶液， ５０μＬ， ５０μＭ）を加え、室温で３０分間撹拌した。その後、０．５Ｍ 水酸化ナトリウム水溶液（２ｍＬ）を加えて一晩撹拌し、強酸性陽イオン交換樹脂Ｄｏｗｅｘ−５０Ｗ−Ｘ８ (Ｈ^＋)を適量加えてｐＨ〜４とした後、綿栓ろ過を行った。ろ液を濃縮し、残渣をゲルろ過（Ｓｅｐｈａｄｅｘ Ｇ−１５：２２０ ｍＬ，５% ＡｃＯＨ ａｑ．）で精製した後、凍結乾燥を行い、白色固体として目的物（４５）（５４．３ｍｇ， ９９．３%）を得た。

n-pentenyl S- (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)-(2 → 6) -6-thio-β-D-galacto Synthesis of pyranoside (45)

Under an argon atmosphere, NeuSGlcO (OAc, OMe) (6) (79.1 mg, 91.6 μmol) was dissolved in methanol (1.0 mL), sodium methoxide (1M methanol solution, 50 μL, 50 μM) was added, and room temperature was added. For 30 minutes. Thereafter, 0.5 M aqueous sodium hydroxide solution (2 mL) was added and stirred overnight. After adding an appropriate amount of strong acidic cation exchange resin Dowex-50W-X8 (H ⁺ ) to pH ˜4, cotton plug filtration was performed. went. The filtrate was concentrated, and the residue was purified by gel filtration (Sephadex G-15: 220 mL, 5% AcOH aq.), And then lyophilized to give the target product (45) (54.3 mg, 99.99) as a white solid. 3%).

アルゴン雰囲気下、ＮｅｕＳＣｅｌ（ＯＡｃ，ＯＭｅ）（３５）（６１．１ｍｇ， ５３．０μｍｏｌ）をメタノール（０．８ｍＬ）に溶解させ、ナトリウムメトキシド（１Ｍ メタノール溶液， ４０μＬ， ４０μＭ）を加え、室温で３０分間撹拌した。その後、０．５Ｍ水酸化ナトリウム水溶液（２ｍＬ）を加えて一晩撹拌し、強酸性陽イオン交換樹脂Ｄｏｗｅｘ−５０Ｗ−Ｘ８（Ｈ^＋）を適量加えてｐＨ〜４とした後、綿栓ろ過を行った。ろ液を濃縮し、残渣をゲルろ過（Ｓｅｐｈａｄｅｘ Ｇ−１５: ２２０ｍＬ， ５%ＡｃＯＨ ａｑ．）で精製した後、凍結乾燥を行い、白色固体として目的物（４６）（３８．２ｍｇ， ｑｕａｎｔ．）を得た。

以下のシアリダーゼ阻害活性の測定には、脱保護した化合物を使用した。 n-pentenyl S- (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonopyranosilonate)-(2 → 6) -O- (6-thio-β- Synthesis of D-glucopyranosyl)-(1 → 4) -β-D-glucopyranoside (46)

Under an argon atmosphere, NeuSCel (OAc, OMe) (35) (61.1 mg, 53.0 μmol) was dissolved in methanol (0.8 mL), sodium methoxide (1 M methanol solution, 40 μL, 40 μM) was added, and at room temperature. Stir for 30 minutes. Thereafter, 0.5 M aqueous sodium hydroxide solution (2 mL) was added and stirred overnight. After adding an appropriate amount of strong acidic cation exchange resin Dowex-50W-X8 (H ⁺ ) to pH ˜4, cotton plug filtration was performed. went. The filtrate was concentrated, and the residue was purified by gel filtration (Sephadex G-15: 220 mL, 5% AcOH aq.) And then freeze-dried to obtain the target product (46) (38.2 mg, quant.) As a white solid. Got.

The deprotected compound was used for the following measurement of sialidase inhibitory activity.

９６穴プレートに種々の濃度の合成阻害剤（本発明に係る上記化合物及びポジティブコントロール（ザナミビルあるいはオセルタミビル））（４μＬ）とインフルエンザＡ型ウィルス（Ｈ１Ｎ１：Ａソ連型、Ｈ３Ｎ２：Ａ香港型）懸濁液（４μＬ）を加え、４℃、１時間インキュベートした。各ウェルにシアリダーゼの基質として０．４ｍＭ ４−Ｍｕ−Ｎｅｕ５Ａｃ（４−メチルウンベリフェリル−Ｎ−アセチル−α−Ｄ−ノイラミン酸）（２μＬ）を加え、３７℃、３０分インキュベートし、停止溶液（２００μＬ）を加えた。この混合物（１００μＬ）を新規の９６穴マイクロプレートに移し、励起波長 Ｅｘ．３５５ｎｍ、蛍光波長 Ｅｍ．４６０ｎｍの蛍光強度を測定し、インフルエンザウィルスシアリダーゼに対する各化合物のＩＣ_５０値を測定した。
測定結果を以下の表に示す。

[Experimental Example] Sialidase Inhibition Assay Method The sialidase inhibitory activity of the following compounds synthesized as shown in the synthesis examples was measured.

Suspension of influenza A virus (H1N1: A Soviet type, H3N2: A Hong Kong type) and suspension of various concentrations of synthesis inhibitor (the above compound according to the present invention and positive control (zanamivir or oseltamivir)) (4 μL) in 96-well plate The solution (4 μL) was added and incubated at 4 ° C. for 1 hour. To each well, 0.4 mM 4-Mu-Neu5Ac (4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid) (2 μL) was added as a sialidase substrate, incubated at 37 ° C. for 30 minutes, and stop solution (200 μL) was added. This mixture (100 μL) is transferred to a new 96-well microplate and the excitation wavelength Ex. 355 nm, fluorescence wavelength Em. The fluorescence intensity at 460 nm was measured, and the IC ₅₀ value of each compound against influenza virus sialidase was measured.
The measurement results are shown in the following table.

  Since the present invention effectively inhibits neuraminidase activity of influenza virus, it can be used for the development of anti-influenza drugs.

Claims (9)

Formula (I)

(In the formula, E ¹ and E ² are either carbon or silicon , and may be the same or different from each other, R ¹ and R ² represent the same or different hydrocarbon groups, and R ³ , R 2 ⁴ and R ⁵ is oxygen, nitrogen and / or optionally identical also contain a carbonyl group or a different alkyl group having 3 to 12 carbon atoms, an alkylene group, an alkynylene group and alkoxylene group (oxyalkylene group), Y is less shows a substituent, l is a number of 0 to 2, m is a number of 0 to 2, k represents a number of 0 or 1, 3-m when the k is 0, Ri 1 der In the case where k is 1, R ³ binds to E ² ), or a pharmacologically acceptable salt thereof or a hydrate thereof.

  R 1 and R 2 are the same or different alkyl groups having 1 to 6 carbon atoms, phenyl group, vinyl group, or alkenyl group, or the thiosialogooligosaccharide-bonded dendrimer compound according to claim 1, or a pharmacologically acceptable salt thereof, or a salt thereof Hydrate. The thiosialogooligosaccharide-bonded dendrimer compound according to claim 1 or ^{2, wherein} E ¹ and E ² are silicon, or a pharmacologically acceptable salt thereof, or a hydrate thereof. R ¹ is a phenyl group, R ³ is —C ₃ H ₆ —, —C ₃ H ₆ —O—C ₃ H ₆ — or —C ₃ H ₆ C ₅ H ₈ —NHCOC ₅ H ₁₀ —, and R ⁴ is — The thiosialogooligosaccharide-bonded dendrimer compound according to claim 3 , or a pharmacologically acceptable salt thereof, or a hydrate thereof, wherein C ₅ H _10- , k is 0, 1 is 1, and m is 2. R ³ is —C ₃ H ₆ —, —C ₃ H ₆ —O—C ₃ H ₆ — or —C ₃ H ₆ —NHCOC ₅ H ₁₀ —, R ⁴ is —C ₅ H ₁₀ , k is 0, l The thiosialogooligosaccharide-bonded dendrimer compound according to claim 3 , or a pharmacologically acceptable salt thereof, or a hydrate thereof. R ¹ is a methyl group, R ³ and R ⁵ are —C ₃ H ₆ —, —C ₃ H ₆ —O—C ₃ H ₆ — or —C ₃ H ₆ —NHCOC ₅ H ₁₀ —, and R ⁴ is —C. The thiosialogooligosaccharide-bonded dendrimer compound according to claim 3 or a pharmacologically acceptable salt thereof, or a hydrate thereof, wherein ₅ H ₁₀ , k is 1, 1 is 2, and m is 0. R ³ and R ⁵ are —C ₃ H ₆ —, —C ₃ H ₆ —O—C ₃ H ₆ — or —C ₃ H ₆ —NHCOC ₅ H ₁₀ —, R ⁴ is —C ₅ H ₁₀ , and k is The thialo-oligosaccharide-linked dendrimer compound according to claim 3 , wherein 1, 1 is 0, and m is 0, or a pharmacologically acceptable salt thereof, or a hydrate thereof. An infectious disease comprising the thiosialogooligosaccharide-bonded dendrimer compound according to any one of claims 1 to 7, a pharmaceutically acceptable salt and hydrate thereof, and a pharmaceutically acceptable carrier. Medicine for the treatment of The medicament according to claim 8 , wherein the infectious disease is an influenza virus infection.