JP2013063933A - Human neuraminidase inhibitor or chemical chaperone composition - Google Patents

Abstract

An object of the present invention is to provide an inhibitor for hNEU2 or a composition exhibiting a chemical chaperone effect on hNEU2.
Human neuraminidase 2 (hNEU2) comprising a thiosialogooligosaccharide-linked dendrimer compound represented by the following formula (I) or a pharmacologically acceptable salt thereof or a hydrate thereof, and a pharmacologically acceptable carrier: ) Inhibitors.

(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 Represents a group, l is an integer of 0 to 2, m is an integer of 0 to 2, k represents a number of 0 or 1, and 3-m is 1 when k is 0)
[Selection figure] None

Description

  The present invention relates to a human neuraminidase inhibitor comprising a thiosialogooligosaccharide-linked dendrimer compound, or a composition having a chemical chaperone effect of human neuraminidase.

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.

For example, the use of dendrimers has been considered so far as an effective defense tool against interference from the external environment on living bodies caused by infection with bacteria and viruses.
With regard to bacterial infections, development of dendrimers that can effectively prevent attack on the living body 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 have been reported to be toxic by recognizing and adhering to the globotrisaccharide moiety in globotriosylceramide (Gb3, Galα1-4Galβ1-4Glcβ1-Cer) on kidney cells. Has been.
Further, the present inventors based on the knowledge about various sugar chain-containing carbosilane dendrimer compounds (see Non-Patent Document 1), a thiasialoside oligosaccharide that inhibits neuraminidase (sialidase) activity present on the surface of viruses such as influenza virus. Have been reported and their effects have been reported (Patent Document 1, Patent Document 2).
Thus, with regard to the relationship between neuraminidase and sugar chains present in influenza viruses and the like, research has been advanced along with the development of infectious disease protection methods, and many findings are being revealed.

By the way, neuraminidase exists also in mammalian cells such as humans and plays an important role. Human neuraminidase (hNEU), also called exo-α-sialidase, hydrolyzes non-reducing sialic acid chains bound to glycoproteins, glycolipids, gangliosides, etc., thereby transporting molecules, antigen masking, proliferation and differentiation. It is involved in the regulation and maintenance of membrane function. Mammalian neuraminidase is derived from lysosome (NEU1), cytoplasm (NEU2), cell membrane (NEU3) and mitochondrial / lysosome (NEU4) based on its intracellular localization, pH optimum and substrate specificity. being classified.
When human neuraminidase does not function normally due to its functional abnormality, N-acetylneuraminic acid (NeuAc) bound to glycolipid or glycoprotein remains without being excised, and the carbohydrate with NeuAc added. Or accumulation of glycoprotein occurs and causes various diseases. For example, when a dysfunction occurs in hNEU that functions in lysosomes, an inborn error of metabolism that shows a serious symptom called “lysosomal disease” may occur. Among the diseases related to hNEU, for example, in the treatment of lysosomal disease, an enzyme replacement method or the like is currently used as an effective treatment method. Since the produced enzyme cannot cross the blood-brain barrier, it cannot reach the damaged site in the brain, and an effective therapeutic effect cannot be obtained. Therefore, among diseases caused by abnormalities in hNEU, particularly in diseases of the central nervous system, it is necessary to establish an effective treatment method other than the enzyme replacement method.

JP 2004-107230 A JP2009-242387

Matsuoka et al., Bull. Chem. Soc. Jpn. 71: 2709-2713 1998

As a method for treating hNEU-related diseases, the chemical chaperone method is expected to be effective even in central diseases in addition to the enzyme replacement method. A chemical chaperone stabilizes the structure of an enzyme by entering the active center of the enzyme. For example, some enzyme inhibitors function as a chemical chaperone at a low concentration.
Then, this invention aims at provision of the inhibitor with respect to hNEU2 among hNEU.
Furthermore, this invention aims at provision of the composition which shows the effect as a chemical chaperone with respect to hNEU2.
Another object of the present invention is to provide a therapeutic agent for diseases caused by dysfunction of hNEU2.

In view of the above circumstances, the present inventors searched for a substance having an inhibitory activity against hNEU2 and also functioning as a chemical chaperone. As a result, a dendrimer bound with a thioglycoside-linked sialic acid oligosaccharide exhibits hNEU2 activity. The present invention was completed by finding that it inhibits and also functions as a chemical chaperone.
The inventors have already found a compound capable of effectively inhibiting neuraminidase activity of influenza virus among dendrimers conjugated with a thioglycoside-linked sialic acid oligosaccharide (Patent Document 2). Was found to have an inhibitory effect on hNEU2 and function as a chemical chaperone.

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). An inhibitor of human neuraminidase 2 (hNEU2), comprising a thiosialogooligosaccharide-bonded dendrimer compound or a pharmacologically acceptable salt thereof or a hydrate thereof, and a pharmacologically acceptable carrier.

  Alternatively, the thialoolo-oligosaccharide-bound dendrimer compound represented by the above formula (I) (the description in the formula is the same as described above) or a pharmacologically acceptable salt thereof or a hydrate thereof, and a pharmacologically acceptable carrier It is a composition or pharmaceutical composition which shows an effect as a chemical chaperone with respect to hNEU2.

  The present invention provides an inhibitor that effectively inhibits the activity of hNEU2.

  The present invention provides a composition or pharmaceutical composition that exhibits an effect as a chemical chaperone of hNEU2. In particular, the compound of formula (I) contained in the chemical chaperone composition or pharmaceutical composition of the present invention is difficult to digest by hNEU2 because Y is composed of a thioglycoside bond. It is possible to fully exhibit the effect of.

It is a result which shows the effect as a chemical chaperone of the dendrimer compound of this invention. In (1), the activity was measured immediately after thawing hNEU2 which had been cryopreserved. Moreover, (2) performed the same process as another sample, without adding a dendrimer compound. The result of performing Native-PAGE after mixing by processing hNEU2 and various dendrimer compounds is shown.

In the compound of the formula (I) contained in the hNEU2 inhibitor or chemical chaperone composition of the present invention, in the formula (I), E ¹ and E ² are any of carbon, silicon and germanium and are the same or different from each other Although carbon or silicon is preferred, silicon is most preferred.
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.

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.

In addition, in the dendrimer contained in the inhibitor or composition of the present invention, it is desirable that all the Y positions in one molecule are any of the above-mentioned substituents. For example, hydrogen, C═C double bond, hydroxyl group and the like may be used, and it is predicted by a person skilled in the art that, in addition to thiosialo-oligosaccharide, it can be bonded to the Y position by a usual synthesis method in the art. Any of the substituents that can be made.

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)

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 (IV) reacts the halogenated dendrimer represented by the formula (III) with the sulfide compound represented by the formula (IV), and optionally protects the thiosialo-oligosaccharide residue in the sulfide compound. By removing the group, the compound of formula (I) of the present invention can be produced.

As an example of the method for producing the compound of formula (I), 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.

In addition, hNEU2 in the present invention is an enzyme generally referred to as “human neuraminidase 2”, and it may be not only natural hNEU2, but also hNEU2 whose activity is reduced by mutation or the like. hNEU2 is, for example, a protein containing the same or substantially the same amino acid sequence as a protein registered as NCBI Accession No: NM — 005383.2 (GI: 222352169, GeneID: 4759).
Here, the “protein containing substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, more preferably about 80% or 81%, with the amino acid sequence registered as NM_005383.2. , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, Most preferably a protein having an amino acid sequence having about 99% amino acid identity and having protein kinase activity.
Alternatively, as a protein having an amino acid sequence substantially identical to the amino acid sequence registered as NM_005383.2, one or several (preferably 1 to 30) amino acid sequences registered as NM_005383.2 About, more preferably about 1 to 10, more preferably 1 to 5 amino acids are deleted, substituted or added, and the protein has protein kinase activity.

  The hNEU2 inhibitor or chemical chaperone composition for hNEU2 of the present invention comprises a dendrimer compound of the general formula (I) or a pharmacologically acceptable salt thereof or a hydrate thereof. Furthermore, when the hNEU2 inhibitor of the present invention or the chemical chaperone composition for hNEU2 is used as “agent” or “composition”, it may contain a buffer, a salt, other additive substances, etc. depending on the purpose. Good. In particular, when the “inhibitor” or “composition” of the present invention is used as a therapeutic agent or a pharmaceutical composition for a disease caused by an abnormal function of hNEU2, it may contain a pharmacologically acceptable carrier. . Here, the “chemical chaperone” composition refers to a composition that has an effect of stabilizing hNEU2 (for example, by stabilizing the structure, etc.) and increasing or retaining activity, and can be used in vitro or in vivo. it can.

  “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.

When the inhibitor or chemical chaperone composition of the present invention is used as a pharmaceutical or pharmaceutical composition, it includes intravenous, intradermal, subcutaneous, oral (for example, including inhalation), transdermal and transmucosal administration, Formulated to suit 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, etc. Perfume additive.

  The pharmaceutical composition of the present invention can be prepared as a sustained-release preparation such as a delivery system 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-derivatized phosphatidylethanol (PEG-PE) through a filter of appropriate pore size to obtain a size suitable for use, and reverse phase. Purified by evaporation.

When the inhibitor or chemical chaperone of the present invention is used as a medicine or pharmaceutical composition, the appropriate dosage level depends on the disease of the subject, the condition of the patient to be administered, the administration method, etc. Can be easily optimized.
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.

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

  First, a method for synthesizing a dendrimer compound contained in the inhibitor or chemical chaperone composition of the present invention is described in detail in JP-A-2009-242387 (Patent Document 2). In the following, the synthesis route will be described.

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-6-thio-β-D-glucopyranoside (6) The target compound (6) can be synthesized as shown in the following scheme.

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-Ot-butyldimethylsilyl-β-D-glucopyranoside (2) Dissolve known pentenyl glucoside (1) in pyridine under argon atmosphere Add TBDMSCl under ice cooling and stir for 1 hour with ice cooling. Then acetic anhydride is added and stirred at room temperature for 2 hours. After adding methanol under ice cooling, the reaction mixture is 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, the filtrate was concentrated, and the residue was subjected to silica gel column chromatography [ 4: 1 (v / v) hexane-ethyl acetate, silica gel] to give compound (2).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (3) Dissolve the fully protected glucoside (2) in acetic acid, add water and stir at 50 ° C. for 2 hours. The reaction mixture is concentrated, and the residue is purified by silica gel column chromatography [2: 1 (v / v) hexane-ethyl acetate, silica gel] to give compound (3).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-glucopyranoside (4) In an argon atmosphere, alcohol (3) was dissolved in pyridine, and ice-cooled. Add triphenylphosphine and carbon tetrabromide in order and stir at 45 ° C. for 15 minutes. Methanol is added under ice cooling to stop the reaction, and the reaction mixture is concentrated. The residue is dissolved in a small amount of methanol, toluene is added thereto, and the by-product crystallized is removed by Celite filtration, and the filtrate is concentrated. The residue is purified by silica gel column chromatography [5: 2 (v / v) hexane-ethyl acetate, silica gel] to obtain compound (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-6-thio-β-D-galactopyranoside (6) In an argon atmosphere, sialic acid thioacetate (5) and bromide (4 ) Is dissolved in DMF (1 mL), diethylamine is added under ice cooling, and the mixture is stirred at room temperature for 6 hours. The reaction mixture is 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 is concentrated. The residue is roughly purified by silica gel column chromatography [2: 3 (v / v) toluene-ethyl acetate, silica gel], and then the target compound (6) can be isolated by preparative GPC.

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) The target compound (11) is synthesized as shown in the following scheme. Can do.

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, etc.) (7) is dissolved in pyridine, TBDMSCl is added under ice cooling, and the mixture is stirred for 5 hours with ice cooling. Then acetic anhydride is added and stirred at room temperature for 6 hours. After adding methanol under ice cooling, the reaction mixture is concentrated. 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. The residue is purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel] to obtain compound (8).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-β-D-galactopyranoside (9) Dissolve the fully protected galactoside (8) in acetic acid, add water and stir at 50 ° C. for 2 hours. To do. The reaction mixture is concentrated, and the residue is purified by silica gel column chromatography [1: 1 (v / v) hexane-ethyl acetate, silica gel] to give compound (9).

Synthesis of n-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-galactopyranoside (10) In an argon atmosphere, alcohol (9) was dissolved in pyridine. Triphenylphosphine and carbon tetrabromide are sequentially added under ice cooling, and the mixture is stirred at 50 ° C. for 1 hour. Under ice cooling, methanol is added to stop the reaction, and the reaction solution is concentrated. The residue is dissolved in a small amount of methanol, toluene is added thereto, and the by-product crystallized is removed by Celite filtration, and the filtrate is concentrated. The residue is purified by silica gel column chromatography [4: 1 (v / v) hexane-ethyl acetate, silica gel] to obtain compound (10).

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) and bromide (10) Dissolve in a DMF-methanol mixture (1: 1 (v / v)), add potassium carbonate under ice cooling, and stir at room temperature for 21 hours. Under ice-cooling, acetic acid was added to stop the reaction, and the reaction solution was concentrated. Suspend the residue in pyridine, add acetic anhydride and stir overnight. The reaction solution is concentrated, and the residue is 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 is concentrated. . The residue is roughly purified by silica gel column chromatography [15: 14: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel], and then the target compound (11) can be isolated by preparative GPC. .

3. 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) The target compound (26) can be synthesized as shown in 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 known argon pentenyl lactoside (see, for example, Matsuoka and Nishimura, Macromolecules 28, pp. 2961-2968, 1995, etc.) (22) Dissolve in DMF, add 2-methoxy-1-propene and p-toluenesulfonic acid monohydrate in order under ice cooling, and stir for 3 hours with ice cooling. Then add pyridine and acetic anhydride and stir overnight at room temperature. The reaction mixture is concentrated, 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. The residue is purified by silica gel column chromatography [4: 1 (v / v) toluene-ethyl acetate, silica gel] to obtain compound (23).

n-pentenyl O- (2,3-di-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-β-D-glucopyranoside ( 24) Synthesis Fully protected lactoside (23) is dissolved in acetic acid, water is added and stirred at 50 ° C. for 2 hours. The reaction mixture is concentrated, and the residue is purified by silica gel column chromatography [1: 4 (v / v) toluene-ethyl acetate, silica gel] to give compound (24).

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) In an argon atmosphere, diol (24) is dissolved in pyridine, and triphenylphosphine and carbon tetrabromide are added in that order under ice cooling, followed by 50 ° C for 30 minutes, 60 ° C. For 3 hours. Under ice cooling, triphenylphosphine and carbon tetrabromide are further added in this order, and the mixture is stirred at 60 ° C. for 30 minutes. Under ice cooling, methanol is added to stop the reaction, and the reaction solution is concentrated. The residue is dissolved in a small amount of methanol, toluene is added thereto, and the by-product crystallized is removed by Celite filtration, and the filtrate is concentrated. The residue is purified by silica gel column chromatography [3: 1 (v / v) toluene-ethyl acetate, silica gel] to obtain compound (25).

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) and bromide (25) were dissolved in a DMF-methanol mixture (1: 1 (v / v), 1 mL), and ice-cooled. Add potassium carbonate and stir at room temperature for 36 hours. Under ice-cooling, acetic acid was added to stop the reaction, and the reaction solution was concentrated. Suspend the residue in pyridine, add acetic anhydride and stir 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. To do. The residue is roughly purified by silica gel column chromatography [1: 1 → 2: 3 → 0: 1 (v / v) toluene-ethyl acetate, silica gel], and then the target compound (26) is isolated by preparative GPC. be able to.

4). 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 target compound (35) of D-glucopyranoside (35) can be 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) was dissolved in an acetic acid-acetic anhydride mixed solution, and 30% hydrogen bromide under ice-cooling. Add acetic acid solution, seal tightly and stir at room temperature for 3.5 hours. Further, a 30% hydrogen bromide-acetic acid solution is added under ice cooling, and the mixture is stirred overnight. The reaction mixture is 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 is washed with diethyl ether to obtain compound (28).

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) In an argon atmosphere, cellobiose α-bromide (28), 4-penten-1-ol, and molecular sieves 4A are suspended in dichloromethane and stirred for 2 hours. Silver trifluoromethanesulfonate is added at −20 ° C., and the mixture is stirred for 40 minutes. The reaction solution is filtered through Celite, and the insoluble matter is washed with chloroform. The filtrate and the washing solution are 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 is concentrated. The residue is purified by flash silica gel column chromatography [4: 1 (v / v) toluene-ethyl acetate, silica gel] to give compound (29).

Synthesis of n-pentenyl O-β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranoside (30) Pentenyl cellobioside (29) is dissolved in a methanol-dichloromethane mixed solution, and 1M sodium methoxide / methanol Add the solution and stir at room temperature for 2 hours. The reaction solution adjusted to pH˜4 by adding IR-120B (H + type) is filtered through a cotton plug, and the resin is washed with methanol. The filtrate and the washing solution are combined and concentrated to obtain the compound (30) 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) was dissolved in DMF under an argon atmosphere, and 2-methoxy-1-propene and p-toluenesulfonic acid-hydrate were sequentially added under ice-cooling. The mixture was allowed to cool for 2 hours and then returned to room temperature and stirred for 4 hours. Then, pyridine and acetic anhydride were added and stirred overnight at room temperature. The reaction mixture is concentrated, 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. The residue is purified by silica gel column chromatography [5: 1 (v / v) toluene-ethyl acetate, silica gel] to obtain compound (31).

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) is dissolved in acetic acid, water is added and stirred at 50 ° C. for 2 hours. The reaction mixture is concentrated, and the residue is purified by silica gel column chromatography [1: 2 (v / v) toluene-ethyl acetate, silica gel] to give compound (32).

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) The diol (32) is dissolved in pyridine under an argon atmosphere, triphenylphosphine and carbon tetrabromide are sequentially added under ice cooling, and the mixture is stirred at 50 ° C for 1 hour. Under ice cooling, methanol is added to stop the reaction, and the reaction solution is concentrated. The residue is dissolved in a small amount of methanol, toluene is added thereto, and the by-product crystallized is removed by Celite filtration, and the filtrate is concentrated. The residue is purified by flash silica gel column chromatography [1: 1 (v / v) hexane-ethyl acetate, silica gel] to give compound (33).

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) is dissolved in pyridine, acetic anhydride is added under ice cooling, and the mixture is stirred at room temperature for 6 hours. The reaction mixture is concentrated, and the residue is purified by flash silica gel column chromatography [2: 1 (v / v) toluene-ethyl acetate, silica gel] to give compound (34).

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) Thioacetate (5) and bromide (34) are dissolved in DMF under an argon atmosphere, diethylamine is added under ice cooling, and the mixture is 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 solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered through celite, and the filtrate was concentrated. The residue is roughly purified by silica gel column chromatography [100: 30: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel], and then the target compound (35) can be isolated by preparative GPC. .

5. Synthesis of Thiosialooligosaccharide-Linked Dendrimer Thiothialooligosaccharide-bound dendrimers can be synthesized as 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-6-thio-β-D-glucopyranoside (36) Synthesis of pentenyl glycoside (6) to 1,4-dioxane under argon atmosphere Dissolve, add thioacetic acid and heat to 50 ° C. Therefore, AIBN is added and stirred at 80 ° C. for 3 hours. Thereafter, cyclohexene is added under ice cooling, and the mixture is returned to room temperature and stirred for several minutes. The reaction mixture is concentrated, and the residue is purified by flash silica gel column chromatography [toluene → ethyl acetate, silica gel] to give compound (36).

Synthesis of Fan (0) 3-NeuSGlcS (OAc, OMe) (37) Thioacetate (36) and Fan (0) 3-Br dendrimer were dissolved in a DMF-methanol mixture under an argon atmosphere. 1M sodium methoxide / methanol solution is added thereto and stirred at room temperature for 19 hours. Then, acetic acid is added under ice cooling to stop the reaction, and the reaction solution is concentrated. The residue is suspended in pyridine under an argon atmosphere, acetic anhydride is added, and the mixture is stirred at room temperature for 5 hours. Thereafter, the reaction solution is concentrated, the residue is 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 is concentrated. The residue can be purified by silica gel column chromatography [15: 14: 1 → 10: 9: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel] to obtain the target compound (37).

Synthesis of Ball (0) 4-NeuSGlcS (OAc, OMe) (38) Thioacetate (36) and Ball (0) 4-Br dendrimer are dissolved in a DMF-methanol mixture under an argon atmosphere. 1M sodium methoxide / methanol solution is added thereto and stirred at room temperature for 24 hours. Then, acetic acid is added under ice cooling to stop the reaction, and the reaction solution is concentrated. The residue is suspended in pyridine under an argon atmosphere, acetic anhydride is added, and the mixture is stirred at room temperature for 22 hours. Thereafter, the reaction solution is concentrated, the residue is dissolved in chloroform, washed with water, dehydrated and dried over magnesium sulfate, filtered through Celite, and the filtrate is concentrated. The residue can be purified by silica gel column chromatography [15: 14: 1 → 7: 7: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel] to obtain the target compound (38).

Synthesis of Dumbbell (1) 6-NeuSGlcS (OAc, OMe) (39) Thioacetate (36) and Dumbbell (1) 6-Br dendrimer were dissolved in a DMF-methanol mixture under an argon atmosphere. 1M sodium methoxide / methanol solution is added thereto and stirred at room temperature for 23 hours. Thereafter, acetic acid was added under ice cooling to stop the reaction, and the reaction solution was concentrated. The residue is suspended in pyridine under an argon atmosphere, acetic anhydride is added, and the mixture is 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 can be purified by silica gel column chromatography [15: 14: 1 → 5: 4: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel] to obtain the target compound (39).

Synthesis of Ball (1) 12-NeuSGlcS (OAc, OMe) (40) In an argon atmosphere, thioacetate (36) and Ball (1) 12-Br dendrimer were dissolved in a DMF-methanol mixture. 1M sodium methoxide / methanol solution (added thereto) and stirred at room temperature for 8 hours. Then, acetic acid was added under ice cooling to stop the reaction, and the reaction mixture was concentrated. The residue was suspended in pyridine under an argon atmosphere. The reaction mixture was concentrated, and 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 can be purified by silica gel column chromatography [15: 14: 1 → 15: 0: 1 (v / v / v) chloroform-ethyl acetate-methanol, silica gel] to obtain the target compound (40).
Fan-type, ball-type and dumbbell-type dendrimer compounds carrying compound (11), compound (26) and compound (35) can also be synthesized in the same manner as in the above method 5.
In addition, synthesis of ether- or amide-extended thiosialo-oligosaccharide-linked dendrimers and deprotection of thiosialoside compounds are described in detail in JP-A-2009-242387 (Patent Document 2), and synthesized according to the method described therein. Went.

6). About the influence of various dendrimer compounds with respect to hNEU2 hNEU2 inhibitory activity was measured about the following compound synthesize | combined along the said synthesis example.

The purified recombinant hNEU2 (NCBI Accession No: NM — 005383.2) was measured for enzyme activity using 4MU-NeuAc, which is a synthetic substrate. The activity of hNEU2 was as follows: 0.2 mM sodium acetate buffer (pH 6.0), 2 μL of 50 mg / mL BSA, 5 μL of 2 mM 4MU-NeuAc, 20 μL of various dendrimer compounds at various concentrations, and 5 μL of recombinant hNEU2 (activity; 9.5 nmol) / 30 min / 5 μL) in a 40 μL reaction solution. After incubation of the reaction at 37 ° C. for 30 minutes, 770 μL of glycine-NaOH (pH 10.7) was added to stop the reaction. The fluorescence of the dissociated 4-methylumbelliferone (4-MU) was measured with a fluorescence spectrometer (excitation wavelength: 360 nm, fluorescence wavelength: 455 nm), and the concentration showing 50% inhibition (IC ₅₀ ) was determined.
The measurement results are shown in the following table.

From Table 1, the tendency of the inhibitory activity was recognized in the order of dumbbell type> fan type >> ball type as the dendrimer shape. Further, for the concentration (IC ₅₀ ) that inhibits 50%, a tendency of dumbbell (1) 6> fan (0) 3> ball (0) 4 was found. Furthermore, the linker between the sugar chain and the dendrimer was in the order of no linker> ether extension type> amide extension type.

7). Effects of various dendrimer compounds on hNEU2 as chemical chaperones Since the above-mentioned various dendrimer compounds were found to have inhibitory activity on hNEU2, next, the presence or absence of effects as chemical chaperones was examined.
To 5 μL of hNEU2 solution (activity: 9.5 nmol / 30 min / 5 μL), 5 μL of various dendrimer compounds were added so as to have respective concentrations of 0.05, 0.5, and 5 mM, and left on ice for 30 minutes. After 30 minutes, 10 μL of water was added to make the reaction solution 20 μL. Further, after being left on ice for 1 hour, 16 μL was taken out from the reaction solution and subjected to electrophoresis using Naive-PAGE (pH 8.0), and 2 μL was used for enzyme activity measurement.
As the dendrimer compound, dumbbell (1) 6-NeuSGal, dumbbell (1) 6-ether-NeuSGal, dumbbell (1) 6-NeuSLac, and dumbbell (1) 6-ether-NeuSLac were used (see below).

As a result of enzyme activity measurement, it was found that hNEU2 activity increased in any dendrimer compound depending on the concentration of the compound (FIG. 1). That is, it was revealed that the decrease in activity due to the denaturation of hNEU2 in vitro was suppressed by increasing the concentration of the dendrimer compound.
Further, as a result of Native-PAGE, a ladder-like band was observed at positions on both sides of the lower molecule than the hNEU2 band at an inhibitor concentration of 5 mM, and a dark band was observed near the upper end of the Running gel (FIG. 2). Thus, in the electrophoretic image, the observation of a dark band or a ladder-like band near the upper end of the Running gel indicates that hNEU2 and the dendrimer are bound in a plurality of association modes. Based on the binding of the dendrimer to hNEU2, it is considered that a stabilizing effect was observed in which inactivation due to denaturation of hNEU2 was suppressed.

  The present invention provides a composition comprising a dendrimer compound having hNEU2 inhibitory activity and chemical chaperone activity. The present invention can be used for development of therapeutic methods for hNEU2-related diseases.

Claims (7)

An inhibitor of human neuraminidase 2 (hNEU2), comprising a thialoolo-oligosaccharide-bound dendrimer compound represented by the following formula (I) or a pharmacologically acceptable salt thereof or a hydrate thereof, and a pharmacologically acceptable carrier: .

(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 Represents a group, l is an integer of 0 to 2, m is an integer of 0 to 2, k represents a number of 0 or 1, and 3-m is 1 when k is 0) The hNEU2 inhibitor according to claim 1, wherein 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. R ³ , R ⁴ and R ⁵ may be the same or different and may contain an amide bond, and are a linear alkyl group having 1 to 6 carbon atoms, an alkylene group, an alkenylene group, or an alkoxylene group (oxyalkylene group) The hNEU2 inhibitor according to claim 1 or 2. The hNEU2 inhibitor according to any one of claims 1 to 3, wherein E ¹ and E ² are silicon. R ¹ is a phenyl group or a methyl group, R ² 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 —, R ⁴ is —C ⁵ H 10 —, and Y is the following substituent.

The hNEU2 inhibitor according to claim 4, wherein   A composition showing a chemical chaperone effect on hNEU2, comprising the hNEU2 inhibitor according to any one of claims 1 to 5. A therapeutic agent for a disease caused by an abnormal function of hNEU2, comprising the hNEU2 inhibitor according to any one of claims 1 to 5.