Low-molecular polysulphated hyaluronic acid derivative and medicinal agent containing same

FIELD: chemistry.

SUBSTANCE: invention relates to an agent for preventing and/or treating an allergic disease selected from pollinosis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, which is a low-molecular polysulphated hyaluronic acid derivative.

EFFECT: obtaining a low-molecular polysulphated hyaluronic acid derivative.

15 cl, 103 dwg, 17 tbl, 55 ex

 

The technical field

The present invention relates to low molecular weight poliuretanovomu derived hyaluronic acid (perhaps), suitable for the prophylaxis and/or treatment of allergic diseases.

The level of technology

Hyaluronic acid is a linear polysaccharide polymer, which in turn are linked β-D-N-acetylglucosamine and β-D-glucuronic acid, it is relatively easily accessible mucopolysacharides and manifests certain physico-chemical properties and physiological properties, and thus, the very hyaluronic acid and its various derivatives are used as pharmaceuticals and cosmetics.

For example, it is known that poliolefinovoy hyaluronic acid which is a derivative of hyaluronic acid, has the activity of inhibiting kallickrein-kinin system (patent document 1) and the activity of inhibiting phospholipase A2 (patent document 2), can be used as medicines for allergic diseases (patent document 3), and it exhibits strong anti-inflammatory effect mediated by selectin, which is one of adhesion molecules (patent document 4).

In addition, it was described that polysulfonamide oligocarbonate with low molecular the popular mass, such as srednevozrastnoe molecular weight of 10000 or less, can be used as an active ingredient for cosmetics having excellent ability to penetrate the skin (patent document 5), and polysulfonamide hialuronowy oligosaccharides in the range from tetrasaccharide to eicosanoid have anticoagulant activity and antihyaluronidase activity and can be used as anticancer funds (non-patent document 1).

Patent documents

[Patent document 1] JP-A-1999-147901

[Patent document 2] JP-A-1999-269077

[Patent document 3] JP-A-1999-335288

[Patent document 4] JP-A-1996-277224

[Patent document 5] JP-A-1998-195107

Non-patent documents

[Non-patent document 1] Glycobiology, vol.11, No.1, pp. 57-64

The invention

However, the above poliuretonovaya hyaluronic acid and polysulfonyl hyaluronan oligomer themselves have a stimulating effect, such as increased vascular permeability, and thus, some of them are not suitable for clinical applications. The fact that only a small number of these compounds satisfy all the requirements, such as pharmacological activity and safety.

The present invention relates to the provision of low-molecular weight derivative of hyaluronic is islote, does not cause such problems and is suitable for the prophylaxis and/or treatment of allergic diseases.

The authors of the present invention conducted intensive studies to develop compounds that are suitable for the prophylaxis and/or treatment of allergic diseases, and as a result the inventors have discovered that low molecular weight polysulfonamide derivatives of hyaluronic acid corresponding to the General formulas (IA) and (IB), have anti-allergic and anti-inflammatory effect and does not have activity against the increase of vascular permeability, and thus suitable as pharmaceuticals.

Thus, the present invention relates to the following means (1) through (15):

(1) an agent for the prophylaxis and/or treatment of allergic disease selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, containing as an active ingredient of low molecular weight polysulfone derived hyaluronic acid corresponding to the following General formula (IA) or (IB), or its pharmaceutically acceptable salt:

where n is a number from 0 to 15; X corresponds to the following formula (a) or (b):

Y corresponds to the following fo the mule (c), (d) or (e):

each R independently represents a hydrogen atom or a group of SO3H (provided that the group of SO3H ranges from 80 to 100% of the total number of R); R1represents-OH, -OSO3H or-NZ1Z2(where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aracelio group, or optionally substituted heteroaryl group, or-NZ1Z2collectively represents an amino acid residue or a peptide residue; and each * represents the place of attachment to the oxygen atom);

where n is a number from 0 to 15; W corresponds to the following formula (f) or (g):

each R independently represents a hydrogen atom or a group of SO3H (provided that the group of SO3H ranges from 80 to 100% of the total number of R); R1represents-OH, -OSO3H or-NZ1Z2(where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aracelio g is the SCP or optionally substituted heteroaryl group, or-NZ1Z2collectively represents an amino acid residue or a peptide residue; and each * represents the place of attachment to the oxygen atom).

(2) the agent for the prophylaxis and/or treatment according to (1) above, where Y in the General formula (IA) corresponds to the formula (d) or (e);

(3) the agent for the prophylaxis and/or treatment according to (2) above, where X corresponds to the formula (a);

(4) the agent for the prophylaxis and/or treatment according to (3) above, where n is 3, 4 or 5;

(5) the agent for the prophylaxis and/or treatment according to (3) above, where n is 4 or 5;

(6) the agent for the prophylaxis and/or treatment according to (1) above, where a low-molecular polysulfone derivative of hyaluronic acid with the General formula (IB);

(7) the agent for the prophylaxis and/or treatment according to (6) above, where n is 3, 4 or 5;

(8) the agent for the prophylaxis and/or treatment according to (6) above, where n is 4 or 5;

(9) the use of low-molecular polysulfonamide derivative of hyaluronic acid or its pharmaceutically acceptable salt according to any one of (1)to(8) above, for the manufacture of products for the prevention and/or treatment of allergic disease selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma;

(10) a method of prevention and/or treatment allergies the CSOs disease, selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, including the introduction of a person or an animal an effective dose of low-molecular polysulfonamide derivative of hyaluronic acid or its pharmaceutically acceptable salt according to any one of (1)to(8) above;

(11) low-molecular polysulfone derived hyaluronic acid corresponding to General formula (IA') or (IB), or its pharmaceutically acceptable salt:

where n is a number from 0 to 15; X corresponds to the following formula (a) or (b):

Y' corresponds to the following formula (d) or (e);

each R independently represents a hydrogen atom or a group of SO3H (provided that the group of SO3H ranges from 80 to 100% of the total number of R); R1represents-OH, -OSO3H or-ΝΖ1Ζ2(where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aracelio group or optionally substituted heteroaryl group, or-ΝΖ1Ζ2collectively represents the amino acid OST the current or peptide residue); each * represents the place of attachment to the oxygen atom;

where n is a number from 0 to 15; W corresponds to the following formula (f) or (g):

each R independently represents a hydrogen atom or a group of SO3H (provided that the group of SO3H ranges from 80 to 100% of the total number of R); R1represents-OH, -OSO3H or-NZ1Z2(where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aracelio group or optionally substituted heteroaryl group, or-NZ1Z2collectively represents an amino acid residue or a peptide residue; and each * represents the place of attachment to the oxygen atom);

(12) low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to (11) above, where X in the General formula (IA') corresponds to the formula (a);

(13) low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to (11) above, which correspond to the General formula (IB);

(14) low-molecular polysulfate the TES-derived hyaluronic acid or its pharmaceutically acceptable salt according to (12) or (13) above, where n represents 3, 4 or 5; and

(15) a pharmaceutical composition comprising a low molecular weight polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to(11), (12), (13) or (14) above, and pharmaceutically acceptable excipient.

The effects of the invention

Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the present invention have excellent anti-allergic action and excellent anti-inflammatory effect and does not have activity against the increase of vascular permeability, and thus, they can be used as a tool for the prevention and/or treatment of allergic diseases such as pollinosis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, means fewer side effects and are excellent from the point of view of security. In addition, among the low-molecular polyzwitterionic derivatives of hyaluronic acid or pharmaceutically acceptable salts of the present invention, in particular, the group of compounds corresponding to General formula (IA), where Y corresponds to the formula (d) or (e), and groups of compounds corresponding to General formula (IB), have advantages in that connection E. the groups are highly stable in aqueous solutions and, thus, they easily take the form of drugs.

Brief description of drawings

Figure 1 presents chart1H-NMR for compound obtained according to example get 1.

Figure 2 presents chart1H-NMR for compound obtained according to example getting 2.

Figure 3 presents chart1H-NMR for compound obtained according to example receipt 3.

Figure 4 presents chart1H-NMR for compound obtained according to example 4.

Figure 5 presents chart1H-NMR for compound obtained according to example getting 5.

Figure 6 presents chart1H-NMR for compound obtained according to example receipt 6.

7 is a diagram of the1H-NMR for compound obtained according to example getting 7.

On FIG presents chart1H-NMR for compound obtained according to example receipt 8.

Figure 9 presents chart1H-NMR for compound obtained according to example receipt 9.

Figure 10 presents chart1H-NMR for compound obtained according to example 10.

Figure 11 presents chart1H-NMR for compound obtained according to example getting 11.

On FIG presents chart1H-NMR for travel is, obtained according to example getting 14.

On FIG presents chart1H-NMR for compound obtained according to example getting 15.

On FIG presents chart1H-NMR for compound obtained according to example 16.

On FIG presents chart1H-NMR for compound obtained according to example getting 17.

On FIG presents chart1H-NMR for compound obtained according to example getting 18.

On FIG presents chart1H-NMR for compound obtained according to example getting 19.

On FIG presents chart1H-NMR for compound obtained according to example getting 20.

On FIG presents chart1H-NMR for compound obtained according to example getting 21.

On FIG presents chart1H-NMR for compound obtained according to example getting 22.

On FIG presents chart1H-NMR for compound obtained according to example getting 23.

On FIG presents chart1H-NMR for compound obtained according to example getting 24.

On FIG presents chart1H-NMR for compound obtained according to example getting 25.

On FIG presents chart1H-NMR for compound obtained according to note the Roux get 26.

On FIG presents chart1H-NMR for compound obtained according to example getting 27.

On FIG presents chart1H-NMR for compound obtained according to example getting 28.

On FIG presents chart1H-NMR for compound obtained according to example getting 29.

On FIG presents chart1H-NMR for compound obtained according to example 30.

On FIG presents chart1H-NMR for compound obtained according to example getting 31.

On FIG presents chart1H-NMR for compound obtained according to example getting 32.

On FIG presents chart1H-NMR for compound obtained according to example receiving 33.

On FIG presents chart1H-NMR for compound obtained according to example getting 34.

On FIG presents chart1H-NMR for compound obtained according to example getting 35.

On FIG presents chart1H-NMR for compound obtained according to example getting 36.

On FIG presents chart1H-NMR for compound obtained according to example getting 37.

On FIG presents a chart of 1H-NMR for compound obtained according to example getting 38.

On FIG presents diagrams is and 1H-NMR for compound obtained according to example getting 39.

On FIG presents chart1H-NMR for compound obtained according to example getting 40.

On FIG presents chart1H-NMR for compound obtained according to example getting 41.

On FIG presents chart1H-NMR for compound obtained according to example getting 42.

On FIG presents chart1H-NMR for compound obtained according to example getting 43.

On FIG presents chart1H-NMR for compound obtained according to example getting 44.

On FIG presents chart1H-NMR for compound obtained according to example getting 45.

On FIG presents chart1H-NMR for compound obtained according to example getting 46.

On FIG presents chart1H-NMR for compound obtained according to example getting 47.

On FIG presents chart1H-NMR for compound obtained according to example getting 48.

On FIG presents chart1H-NMR for compound obtained according to example getting 49.

On FIG presents chart1H-NMR for compound obtained according to example getting 50.

On FIG presents chart1H-NMR for compound, p is obtained according to the example of obtaining 51.

On FIG presents chart1H-NMR for compound obtained according to example getting 52.

On FIG presents chart1H-NMR for compound obtained according to example get 53.

On FIG presents chart1H-NMR for compound obtained according to example getting 54.

On FIG presents chart1H-NMR for compound 1.

On FIG presents chart1H-NMR for compound 2.

On FIG presents chart1H-NMR for compound 3.

On FIG presents chart1H-NMR for compound 4.

On FIG presents chart1H-NMR for compound 5.

On FIG presents chart1H-NMR for compound 6.

On FIG presents chart1H-NMR for compound 7.

On FIG presents chart1H-NMR for compound 8.

On FIG presents chart1H-NMR for compound (9).

On FIG presents chart1H-NMR for compound 10.

On FIG presents chart1H-NMR for compound (11).

On FIG presents chart1H-NMR for compound 14.

On FIG presents chart1H-NMR for compound 15.

On FIG presents chart1H-NMR for compound 16.

On FIG presents chart1H-NMR for compound 17.

the and FIG presents chart 1H-NMR for compound 18.

On FIG presents chart1H-NMR for compound 19.

On FIG presents chart1H-NMR for compound 20.

On FIG presents chart1H-NMR for compound 21.

On FIG presents chart1H-NMR for compound 22.

On FIG presents chart1H-NMR for compound 23.

On FIG presents chart1H-NMR for compound 24.

On FIG presents chart1H-NMR for compound 25.

On FIG presents chart1H-NMR for compound 26.

On FIG presents chart1H-NMR for compound 27.

On FIG presents chart1H-NMR for compound 28.

On FIG presents chart1H-NMR for compound 29.

On FIG presents chart1H-NMR for compound 30.

On FIG presents chart1H-NMR for compound 31.

On FIG presents chart1H-NMR for compound 32.

On FIG presents chart1H-NMR for compound 33.

On FIG presents chart1H-NMR for compound 34.

On FIG presents chart1H-NMR for compound 35.

On FIG presents chart1H-NMR for compound 36.

On FIG presents chart1H-NMR for compound 37.

On FIG presents di is gram 1H-NMR for compound 38.

On FIG presents chart1H-NMR for compound 39.

On FIG presents chart1H-NMR for compound 40.

On FIG presents chart1H-NMR for compound 41.

On FIG presents chart1H-NMR for compound 42.

On FIG presents chart1H-NMR for compound 43.

On FIG presents chart1H-NMR for compound 44.

On FIG presents chart1H-NMR for compound 45.

On FIG presents chart1H-NMR for compound 46.

On FIG presents chart1H-NMR for compound 47.

On FIG presents chart1H-NMR for compound 48.

On FIG presents a graph which shows the inhibitory effect on allergic response, immediate type. ###: p<0,01, ##: p<0,01, †: p<0,05, *: p<0,05, **: P<0,01, N=8, mean +/- SE.

On FIG presents a graph illustrating the inhibitory effect on delayed allergic response. ###: p<0,01, ##: p<0,01, †: p<0,05, *: p<0,05, **: P<0,01, N=8, mean +/- SE.

On FIG presents a graph illustrating the activity in terms of increased vascular permeability. dex: the point of introduction of dextran. Control: the point of introduction of sulfated hyaluronic acid.

On FIG presents a graph illustrating the activity in respect to the NII increase vascular permeability. dex: the point of introduction of dextran. Control: the point of introduction of sulfated hyaluronic acid.

On FIG presents a graph illustrating the activity in terms of increased vascular permeability. dex: the point of introduction of dextran. Control: the point of introduction of sulfated hyaluronic acid.

Detailed description of the invention

Low-molecular polysulfone derived hyaluronic acid of the present invention is a derivative in which all the hydroxyl groups in the oligomer hyaluronan are excessively sulfated and sulfate crystallization degree (or a degree of substitution) is such that, for example, a group of SO3H ranges from 80 to 100% of the total number (all oligomer) R in the General formula (IA) and (IB) and the group of SO3H preferably is from 90 to 100%. Group SO3H in the oligomer may be unevenly distributed, however, as a rule, oligomer, in which groups of SO3H is uniformly distributed throughout the molecule is preferred from the point of view of its production and application.

The compound corresponding to General formula (IA), where Y corresponds to the formula (d) or (e) (i.e. a compound corresponding to General formula (IA')), and the compound corresponding to General formula (IB), are novel compounds not described in any document.

the reamers "lower alkyl" optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aranceles group and optionally substituted heteroaryl group, represented as Z1and Z2in the above General formulas (IA) and (IB)include linear or branched alkyl group having from 1 to 6 carbon atoms (hereinafter in this document abbreviated denoted as "C1-6"), such as methyl, ethyl, n-sawn, ISO-propyl, n-bucilina, isobutylene, second-bucilina, tert-bucilina, n-pentilla and n-exilda group. Among them, preferred is a group C1-4of alkyl, more preferred are methyl, ethyl, n-sawn, ISO-propyl, n-bucilina, isobutylene, second-bucilina and tert-bucilina group, and more preferred are methyl and ethyl group.

Examples of "aryl group" include C6-14from monocyclic to tricyclic aromatic hydrocarbon ring group such as phenyl, naftalina and antarctilyne group. Among them, preferred is a phenyl group.

Examples of "heteroaryl group" include saturated or unsaturated, monocyclic or polycyclic heterocyclic group having at least one heteroatom selected from a nitrogen atom, sulfur atom and oxygen atom.

Con the specific examples of the group include:

3-6-membered unsaturated monocyclic heterocyclic group having from 1 to 4 nitrogen atoms, such as pyrrolidine, pyrrolidinone, imidazolidine, pyrazolidine, perederina, pyrimidinyl, piratininga, pyridinoline, triazoline, tetrataenia and tetrahydropyridine group;

3-7-membered saturated monocyclic heterocyclic group having from 1 to 4 nitrogen atoms, such as pyrrolidinyl, imidazolidinyl, piperideine, piperazinyl and homopiperazine group;

unsaturated condensed heterocyclic group having from 1 to 5 nitrogen atoms, such as indayla, isoindoline, isoindolyl, benzimidazolyl, kinolinna, izochinolina, imidazopyridine, indazolinone, benzotriazolyl, tetrachloroethylene, khinoksalinona, pyridine, tetrahydropyridine, tetrahydroisoquinoline, indolenine and dihydropyrimidine group;

saturated condensed heterocyclic group having from 1 to 5 nitrogen atoms, such as pyrrolidineethanol, hinkleyville and pyrrolidinedione group;

3-6-membered unsaturated monocyclic heterocyclic group having an oxygen atom, such as Pernilla and furilla group;

3-6-membered saturated monocyclic heterocyclic gr is PPI, having an oxygen atom, such as 1H-tetrahydropyrimidine and tetrahydrofuranyl group;

3-6-membered unsaturated monocyclic heterocyclic group having one or two sulfur atom, such as thienyl group;

3-6-membered unsaturated monocyclic heterocyclic group having one or two oxygen atom and one to three nitrogen atoms, such as oxazolidine, isooxazolyl, oxadiazolyl and oxazolidinone group;

3-6-membered saturated monocyclic heterocyclic group having one or two oxygen atom and one to three nitrogen atoms, such as morpholinyl group;

saturated condensed heterocyclic group containing one or two oxygen atom and one to three nitrogen atoms, such as benzofurazanyl, benzoxazolinone and benzoxadiazole group;

3-6-membered unsaturated monocyclic heterocyclic group having one or two sulfur atom and one to three nitrogen atoms, such as thiazolidine and thiadiazolidine group;

3-6-membered saturated monocyclic heterocyclic group having one or two sulfur atom and one to three nitrogen atoms, such as thiazolidinedione group;

unsaturated condensed heterocyclic group containing one or two sulfur atom and one to three and the Ohm nitrogen, such as benzothiazoline, benzothiazolinone and triazolothiadiazepine group; and

unsaturated condensed heterocyclic group containing one or two oxygen atom, such as benzofuranyl, benzodioxolyl and chromadorina group.

The above-mentioned "lower alkyl group" may be substituted, for example, a halogen atom, carboxypropyl, aryl group, lower CNS group or acyl group, and each of the above-mentioned "aryl group" and "heteroaryl group" may be substituted, for example, a halogen atom, carboxypropyl, a lower alkyl group, lower CNS group or acyl group.

In this case, examples of aryl groups include phenyl and naftalina group, and examples of the lower alkyl group include the above-mentioned C1-6alkyl groups.

In addition, examples of the halogen atom include fluorine, chlorine, bromine and iodine.

In addition, examples of the lower CNS group include linear or branched C1-6CNS group, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentox and n-hexose. Among them, preferred is a C1-4CNS group, more preferred are methoxy, ethoxy-, n-propoxy, isopropoxy, h-butoxy, isobutoxy-, the tor-butoxy - and tert-butoxypropan and even more preferred are methoxy and ethoxypropan.

Examples of acyl groups include CHO, C1-6alkylcarboxylic, C1-6alkoxycarbonyl, arylcarbamoyl, aryl-C1-6alkilenkarbonatov, heteroarylboronic and heteroaryl-C1-6alkilenkarbonatov group.

In this case, examples of C1-6alkyl, C1-6alkoxy, aryl and heteroaryl groups are the same as described above. In addition, examples of C1-6alkilinity groups include linear or branched C1-6alkylene groups such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, propylene, methylmethyldopa, ethylethylene, 1,2-dimethylethylene and 1,1,2,2-tetramethylethylene group. Among them, preferred are methylene, ethylene and trimethylene group.

In addition, examples of "Uralkaliy groups include aryl-C1-6alkyl groups. In this case, examples of aryl and C1-6alkyl groups are the same as described above. However, suitable kalkilya group represents, for example, benzyl and fenetylline group.

Kalkilya group can be substituted, for example, any of the illustrative groups which may be substituted by the above aryl group or a lower alkyl group.

"Optionally substituted lower alkyl group", which represent the established with representation from each of Z 1and Z2preferably represents, for example, triptorelin, benzyl, 2-, 3 - or 4-methylbenzyl, 2-, 3 - or 4-methoxybenzyloxy, methoxymethyl or methoxycarbonylmethyl group,

"optionally substituted aryl group"represented by each of Z1and Z2preferably represents, for example, 2-, 3 - or 4-methylphenyl, 2-, 3 - or 4-metoksifenilny, 2-, 3 - or 4-florfenicol, 2-, 3 - or 4-triptorelin or 2-, 3 - or 4-carboxyphenyl group,

"optionally substituted kalkilya group, which is represented by each of Z1and Z2preferably represents, for example, benzyl, 3 - or 4-methylbenzyl or 2-, 3 - or 4-methoxybenzyloxy group, and

"optionally substituted heteroaryl group", which is represented by each of Z1and Z2preferably represents, for example, 2-, 3 - or 4-methylpyridine, 2-, 3 - or 4-methoxypyridine, 2-, 3 - or 4-perpillou, 1, 2-, 3 - or 4-cryptospiridium or 2-, 3 - or 4-carboxypropyl group.

Examples of amino acid residue or peptide residue when-ΝΖ1Ζ2collectively represents an amino acid residue or peptide residue include amino acid residues, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, Proline, serine, threonine, tryptophan, tyrosine, valine, β-alanine, sarcosine, phenylglycine, N-ethylglycine, N-n-papillitis, N-isopropylphenyl, N-n-butylglycol, N-tertbutylphenyl, N-n-pencillin and N-n-hexillion; and peptide residues, such as sarcozygium, glycylglycine, glycosamin, sarcocystosis, alanilglitsina, β-albertinelli, gliciltirosin, phenylalaninol, generalantilles, glycylglycylglycine, N-acylglycerols, N-n-propylpyrazine, sercotelhoteles, N-ethylpyrrolidin and phenylalanylglycyl. Amino acid residue or peptide residue can be aminirovanie on their terminal carboxyl group.

In the compounds of General formulas (IA) or (IB), n is a number from 0 to 15, preferably from 3 to 9, more preferably 3, 4 or 5 or even more preferably 4 or 5.

Compounds corresponding to the above General formulas (IA) or (IB)include various stereoisomers, optical isomers and a solvate such as a hydrate.

In addition, suitable salt of low molecular weight polysulfonamide derived hyaluronic acid of the present invention is a pharmaceutically acceptable salt. Examples of salts include metal salts, including salts of school is full of metals (such as sodium salt and potassium salt), and salts of alkaline earth metals (such as magnesium salt and calcium salt); salts with inorganic bases, such as ammonium salt, and the hydroxides, carbonates or bicarbonates of alkali metals (such as sodium and potassium) and alkaline earth metals (such as magnesium and calcium); and salts with organic bases such as organic amines such as trimethylamine and triethylamine), pyridine, quinoline, piperidine, imidazole, picoline, dimethylaminopyridine, dimethylaniline and N-methylmorpholin.

Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the present invention preferably has an average molecular weight of from 1500 to 13500, although the average molecular weight will vary depending on the type of salts.

The compound corresponding to General formula (IA) or (IB) of the present invention, can be obtained, for example, by sulfate crystallization of low molecular weight derivative of hyaluronic acid corresponding to the General formula (IIA) or (IIB), as shown in reaction 1 below. The above-mentioned suitable salts can be used as parent compounds or as the target compounds.

In addition, in the following formulae the substituents represented by Z3and Z4meet Z1and Z2respectively, and the values of the respective substituents are as described above.

[Reaction 1]

[In the formulas, X1match the following (a1) or (b1);

Y1follows (c1), (d1) or (e1);

W1follows (f1) or (g1);

R1'represents-OH or-NZ3Z4(where each of Z3and Z4independently represents a hydrogen atom, optionally substituted lower alkyl group, optionally substituted aryl group, optionally substituted aracelio group or optionally substituted heteroaryl group, or-NZ3Z4collectively represents an amino acid residue or a peptide residue; and each of n, X, Y, W, R * is as described above.]

The reaction can be performed using known reactions sulfate crystallization by dissolving the compound (IIA) or (IIB) and sulfatide agent in a suitable solvent and allowing them to react when heated.

Examples of the solvent used in the present invention include: N,N-dimethylformamide, dimethylsulfoxide, N-organic, N,N-dimethylacetamide, 1,1,3,3-tetramethylrhodamine, pyridine and N,N-dimethylacrylamide; ionic liquids, such as hexaphosphate 1-ethyl-3-methylimidazole, tetrafluoroborate 1-butyl-1-methylpyrrole the Shandong Jinlong and chloride 1-butylpyrazine, and solvents, representing a mixture.

Sulfatide agent is not specifically limited, and preferably use complexes anhydride of sulfuric acid with pyridine, picoline, 2,6-lutidine, trimethylamine, triethylamine, Ν,Ν-dimethylformamide, dioxane, etc. or sulfuric acid-dicyclohexylcarbodiimide, clorsulon etc. usually sulfatide agent is preferably used in an amount of from 1 to 100 equivalents relative to the amount of compounds of formula (IIA) or (IIB). In addition, in the reaction system, you can add an acid catalyst, such as triperoxonane acid, triftormetilfullerenov acid or similar.

The reaction temperature and reaction time are not specifically limited, and for example, they range from 0 to 120°C and from 30 minutes to 20 hours, respectively.

The compound corresponding to General formula (IIA), where Y1corresponds to the formula (d1) or (e1), and R1'represents-OH, or a compound corresponding to the General formula (IIB), where R1'represents-OH, can be obtained by restoring the below reactions-2. The original connection and end connections can represent the above-mentioned suitable salt.

[Reaction 2]

[In the formula, A1represents the next (h) or (i):

A2represents the following (j) or (k):

and n, Χ1and * are as described above.]

Thus, the compound (II-1) or (II-2) can be obtained by subjecting compound (III-1) or (III-2), for example, recovery in a suitable solvent in the presence of a reducing agent.

Examples of the solvent for use in the reaction include water; lower alcohols such as methanol, ethanol, isopropanol, butanol, tert-butanol and ethylene glycol; acetonitrile; fatty acids, such as formic acid and acetic acid; ethers such as diethyl ether, tetrahydrofuran, dioxane, monoglyme and diglyme; aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform and carbon tetrachloride; N,N-dimethylformamide; and their mixed solvents.

Examples of reducing agents include borohydride sodium, lithium borohydride, potassium borohydride, borohydride tetrabutylammonium, borohydride zinc, three(sec-butyl)borohydride lithium equivalents of borane, diisobutylaluminum and alumoweld lithium.

The reducing agent is generally used in a quantity approximately equal to the number of mol of compound (III-1) or (III-2), multiplied by 0.1 to 60.

In reaction C is theme you can add zinc chloride, chloride cobalt(II)chloride, samarium(III)chloride, cerium(III)chloride titanium(III)chloride iron(II)chloride iron(III)chloride Nickel(II) or similar in the presence of an amine, such as pyridine, trimethylamine, triethylamine or N-ethyldiethanolamine, inorganic bases such as sodium hydroxide, and/or ligand, such as dimethylglyoxime, 2,2'-bipyridyl or 1,10-phenanthroline.

Recovery can also be carried out by catalytic hydrogenation in the presence of a catalyst based on a transition metal such as palladium or platinum.

The reaction can be conducted usually at from -80 to 100°C, preferably at from about -80°to 70 ° C, and, as a rule, the reaction is completed within about 30 minutes to 60 hours.

The compound corresponding to General formula (IIA), where Y1corresponds to the formula (d1) or (e1), and R1'represents-NZ3Z4or a compound corresponding to the General formula (IIB), where R1'represents-NZ3Z4can be obtained by the reaction of reductive amination presented in reaction 3 below. The original connection and end connections can represent the above-mentioned suitable salt.

[Reaction 3]

[In the formula, each of the A1, A2, Z3and Z4 is the same as described above.]

The reaction is, for example, the reaction is called the reaction of reductive amination, in which the compound (III-1) or (III-2) allow to react with the amine (IV) in a suitable solvent in the presence of a reducing agent with the formation of Shipova base with subsequent recovery.

The amine (IV) is usually used in a quantity approximately equal to the number of mol of compound (III-1) or (III-2), multiplied by from 1 to 5.

Examples of the solvent for use in the reaction include water; lower alcohols such as methanol, ethanol, isopropanol, butanol, tert-butanol and ethylene glycol; acetonitrile; fatty acids, such as formic acid and acetic acid; ethers such as diethyl ether, tetrahydrofuran, dioxane, monoglyme and diglyme; aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform and carbon tetrachloride; Ν,N-dimethylformamide; and their mixed solvents.

Examples of reducing agents for use in the reaction include borohydride sodium, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, centralimediat sodium and createcachetable sodium. The reducing agent is usually used in a quantity approximately equal to the number of mol of compound (III), multiply the resultant by from 0.1 to 60.

The reaction can be conducted usually at from -80 to 100°C, preferably at from about -80°to 70 ° C, and, as a rule, the reaction is completed within about 30 minutes to 60 hours.

The reaction can be carried out in the presence of organic acids and their salts in a quantity equal to the number of moles multiplied by from 1 to 50, in accordance with necessity. Examples of organic acids or their salts include acetic acid, triperoxonane acid and alkali metal salts of these acids (such as sodium acetate).

In the reaction system can be added zinc chloride, cobalt chloride(II)chloride, samarium(III)chloride, cerium(III)chloride titanium(III)chloride iron(II)chloride iron(III)chloride Nickel(II) or similar in the presence of an amine, such as pyridine, trimethylamine, triethylamine or N-ethyldiethanolamine, inorganic bases such as sodium hydroxide, and/or ligand, such as dimethylglyoxime, 2,2'-bipyridyl or 1,10-phenanthroline.

In addition, in the reaction system, you can add appropriate amount of boric acid.

The transformation between the compounds with an even number of sugars, and compounds having an odd number of sugars, can be performed using reaction-4 or reaction of 5 with obtaining compounds in which one of the structural sugars removed from the original connection.

[Response 4]/p>

[In formulas (A2is the same as described above.]

The reaction is a reaction of elimination of N-acetylglucosamine with the processing of a weak alkali when heated.

The compound (VI) can be obtained by mixing the compound (V) by heating in a borate buffer having a pH 9,18 and then way Reissig et al. (Reissig. J. L., et al., J. Biol. Chem., 217, 959 (1955)).

The reaction can be conducted generally at about 50 to 120°C, preferably at from about 70 to 90°C, and, as a rule, the reaction is completed within about 30 minutes to 60 hours.

[Response 5]

[In formulas (D1meets the following (r) or (s), and

each of n and * is the same as described above.]

This reaction is a reaction elimination glucuronic acid using β-glucuronidase.

The compound (VIII) can be obtained by mixing the compound (VII) in the presence of β-glucuronidase in a suitable buffer.

The reaction typically can be performed at a temperature of from room temperature to 60°C, preferably at from about 30°to 40 ° C, and, as a rule, the reaction is completed within about 30 minutes to 60 hours.

Each of the final compounds obtained in the above reaction scheme, can be cleaned the ü purification methods, commonly used for a variety of modified sugars. Specific cleaning methods include desalting using gel filtration, neutralization and dialysis, the collection using precipitation by adding an organic solvent and collecting by freeze drying.

The compound of the present invention, as shown in the examples described below, demonstrates anti-allergic effect and anti-inflammatory effect and does not show any activity in relation to the increase of vascular permeability, and thus, it can be used in pharmaceuticals for the prevention and/or treatment of allergic diseases such as pollinosis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma.

These medicines are given as a General form of the medical staff with the use of the compounds of the present invention and is manufactured using a diluent, such as filler, extender, binder, humectant, dezintegriruetsja substance, a surfactant, or grease, or excipient.

These pharmaceutical agents can be selected from various shapes, depending upon therapeutic objectives, and typical examples of pharmaceuticals include tablet, pill, on osok, liquid, suspension, emulsion, granule, capsule, suppository, injectable form (liquid, suspension and the like), eye drops, ointment and inhalation form.

In the manufacture of tablets, you can use any well-known carrier. Their examples include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose; binders such as water, ethanol, propanol, simple syrup, liquid glucose, liquid starch, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone; dezintegriruetsja substances such as dry starch, ester of fatty acid and polyoxyethylene sorbitane, sodium lauryl sulfate, monoglyceride of stearic acid, starch and lactose; disintegration inhibitors such as sucrose, stearin, oil cocoa and hydrogenated oil; amplifying the suction means, such as Quaternary ammonium base and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; and lubricants such as purified talc, stearate, powdered boric acid and polyethylene glycol.

In addition, the tablet can be obtained, if necessary, as a coated tablet. Their PR is measures include covered sugar means, encapsulated in gelatin tablets, coated gastro-resistant shell tablets and film-coated tablets, or double-layer and multilayer tablets tablets.

In the manufacture of pills, you can use any well-known carrier. Their examples include excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc; binders such as powdered gum Arabic, powdered tragakant, gelatin and ethanol; and dezintegriruetsja substances, such as laminaran and agar.

In the manufacture of suppositories can be used any known media. Their examples include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic glycerides.

When the pharmaceutical agent are given as an injectable means, preferably a liquid, emulsion and suspension were sterilized and isotonic with blood. To obtain liquids, emulsions and suspensions can be applied widely used and known diluent. Examples of diluents include water, ethanol, propylene glycol, ethoxylated isostearoyl alcohol, polioksidony isostearoyl alcohol and an ester of fatty acids and polyoxyethylene sorbitan. In this case, the medical composition may contain sodium chloride, glucose or glycerin in number, sufficient to bring the liquid up to isotonic conditions, and alternative medical composition may contain, for example, conventional solubilizers substance, a buffer or a softening substance, and it may contain a dye, preservative, flavoring, giving the scent additive, sweetener or other pharmaceutical substance, depending on the need.

A number of compounds of the present invention contained in the pharmaceutical agent is not specifically limited, and can appropriately choose from a wide range. Generally, it is preferable that the compound of the present invention is contained in an amount of from 1 to 70 wt.% by weight pharmaceuticals.

Route of administration pharmaceutical agents in accordance with the present invention are not specifically limited, and the pharmaceutical agent is administered by any method, depending on the form of various drugs, age, sex and condition of the patient's medical condition and other conditions. For example, a tablet, pill, liquid, suspension, emulsion, granule and capsule administered orally. Alternative injection can be performed separately or in combination with conventional reducing agent liquid, such as liquid glucose or liquid with amino acids intravenously, and moreover, it can be done separately in utilisee, intradermally, subcutaneously or intraperitoneally, depending on the need. The suppository is administered rectally.

The dosage of the above pharmaceutical drug can be appropriately selected depending on the application, age, sex and stage of the disease the patient and other conditions, and pharmaceutical drug is administered at a daily dosage of from 0.001 to 100 mg, preferably from 0.001 to 50 mg per kg of body weight at once or in several portions a day. The above dosage varies depending on various conditions, in some cases, sufficient smaller dosage than the above ranges, and in other cases the dosage above these ranges.

EXAMPLES

Below the present invention is explained in more detail with illustrative examples.

Definition1H-NMR was performed using deuterated water (D2O) as solvent using an AVANCE III 400 (manufactured Burker) or AVANCE 500 (manufactured Burker).

Examples get from 1 to 54: Receiving source connections

The methods of examples obtain 1-54, presented below, was used to obtain the source compounds are presented in tables 1-7.

Mass spectrometry was performed using a Voyager DE-PRO (Applied Biosystems Japan Ltd.).

Example obtain 1

Hyaluronate on the rija (BIO Sodium Hyaluronate HA9, manufactured by Shiseido Co., Ltd.) subjected to the splitting obtained from bull testes hyaluronidase (Bovine Hyaluronidase Τ 100KU made Calbiochem Behring Corporation), by the method described in Glycobiology, vol. 12, No. 7, pp. 421 - 426, 2002, to obtain the 4-dimensional hyaluronan oligosaccharide. 4-dimensional hyaluronan oligosaccharide (20 mg) was dissolved in methanol (1 ml) and water (0.5 ml) and to the mixture was added sodium borohydride (10 mg) under ice cooling, followed by stirring. The temperature of the obtained product was raised to room temperature and the mixture was stirred over night. Completion of reaction was confirmed by mass spectrometry.

To the product was added 10% solution of acetic acid in methanol (0.5 ml) and water (1 ml) under ice cooling, and the mixture is then concentrated under reduced pressure. Added an additional 10% solution of acetic acid in methanol (0.5 ml) and the product was subjected to azeotropic distillation. After this was added methanol (2 ml) and the product twice subjected to azeotropic distillation.

The residue was dissolved in water (2 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). The filtrate was passed through a short column (Dowex (registered trademark) 50 Wx8, hydrogen form, manufactured by Sigma-Aldrich Corporation) to obtain the protonated compounds, and then concentrated under reduced pressure. For drawing concentrate dshell-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (12 mg, white powder).

MS [M+Na]+: 846,21

1H-NMR: chart illustrated in figure 1.

Example of getting 2

Carried out the same reaction process as in the example of obtaining 1, except that as the starting substances used 6-dimensional hyaluronan oligosaccharide (60 mg) to give the final product (50 mg, white powder).

MS [M-H]-: 1157,81

1H-NMR: chart illustrated in figure 2.

Example for the preparation of 3

8-dimensional hyaluronan oligosaccharide (60 mg) were subjected to reaction using the same process as in the example of obtaining 1, with the final product (51 mg, white powder).

MS [M-H]-: 1535,57

1H-NMR: chart illustrated in FIGURE 3.

Example 4

10-dimensional hyaluronan oligosaccharide (60 mg) were subjected to reaction using the same process as in the example of obtaining 1, with the final product (48 mg, white powder).

MS [M-H]-: 1915,72

1H-NMR: chart illustrated in FIGURE 4.

Example of getting 5

12-dimensional hyaluronan oligosaccharide (60 mg) were subjected to reaction using the same process as in the example of obtaining 1, with the doctrine of the final product (60 mg, white powder).

MS [M-H]-: 2294,98

1H-NMR: chart illustrated in FIGURE 5.

An example of obtaining 6

Carried out the same reaction process as in the example of obtaining 1, except that as the starting substances used 14-dimensional hyaluronan oligosaccharide (20 mg), to obtain the final product (20 mg, white powder).

1H-NMR: chart illustrated in FIG.6.

Example of getting 7

A 16-dimensional hyaluronan oligosaccharide (10 mg) was dissolved in methanol (0.6 ml) and water (0.3 ml) and to the mixture was added sodium borohydride (5 mg) under ice cooling, and then stirred. The temperature of the obtained product was raised to room temperature and the mixture was stirred over night. Completion of reaction was confirmed by mass spectrometry.

To the product was added 10% solution of acetic acid in methanol (0.1 ml) and water (0.2 ml) under ice cooling, and the mixture is then concentrated under reduced pressure.

The residue was dissolved in water (1 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). For the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (10 mg, white powder)

1H-NMR: chart illustrated in FIG.7.

Example obtain 8

Carried out the same reaction process as in the example of obtaining 1, except that as the starting substances used 18-dimensional hyaluronan oligosaccharide (20 mg), to obtain the final product (20 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example of getting 9

Carried out the same reaction process as in the example of obtaining 1, except that as the starting substances used 20-dimensional hyaluronan oligosaccharide (42 mg), to obtain the final product (40 mg, white powder).

1H-NMR: chart illustrated in FIG.9.

Example 10

10-dimensional hyaluronan oligosaccharide (20 mg) was dissolved in water (0.8 ml)and then cooled with ice. Anthranilic acid (30 mg), baronova acid (40 mg), sodium acetate (80 mg) and centralimediat sodium (5 mg) was dissolved in methanol (1 ml) and water (0.2 ml). The resulting solution was added to a solution of 10-dimensional hyaluronan oligosaccharide, and then stirred at 80°C for 5 hours. Completion of reaction was confirmed by mass spectrometry.

The obtained product was concentrated under reduced pressure and the residue was dissolved in methanol (1 ml) and water (1 ml). The mixture is then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 m is m). The filtrate was subjected to gel-filtration chromatography (LH-20, 18 mm × 500 mm, water:methanol = 1:1) for purification, and then the final fraction was subjected to freeze-drying to obtain the final product (24 mg, white powder).

1H-NMR: chart illustrated in FIGURE 10.

Example of getting 11

10-dimensional hyaluronan oligosaccharide (20 mg) was dissolved in water (0.8 ml)and then cooled with ice. Aniline (30 mg), baronova acid (40 mg), sodium acetate (80 mg) and centralimediat sodium (5 mg) was dissolved in methanol (1 ml) and water (0.2 ml). The resulting solution was added to a solution of 10-dimensional hyaluronan oligosaccharide, and then stirred at 80°C for 5 hours. Completion of reaction was confirmed by mass spectrometry.

The obtained product was concentrated under reduced pressure and the residue was dissolved in methanol (1 ml) and water (1 ml). The mixture is then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). The filtrate was subjected to gel-filtration chromatography (LH-20, 18 mm × 500 mm, water:methanol = 1:1) for purification, and then the final fraction was subjected to freeze-drying to obtain the final product (17 mg, white powder).

1H-NMR: chart illustrated in figure 11.

Example 12

Carried out the same reaction process as in the example of obtaining 1, except that as the starting material is the objects of study were 24-32-dimensional hyaluronan oligosaccharide (10 mg), to obtain the final product (10 mg, white powder).

Example of getting 13

Carried out the same reaction process as in the example of obtaining 1, except that as the starting substances used 34-46-dimensional hyaluronan oligosaccharide (10 mg), to obtain the final product (10 mg, white powder).

Structure of the final products of examples obtain 1 to 13 shown in table 1 below

Example of getting 14

Sodium hyaluronate (BIO Sodium Hyaluronate HA9, manufactured by Shiseido Co., Ltd.) subjected to the splitting obtained from bull testes hyaluronidase (Bovine Hyaluronidase Τ 100KU made Calbiochem Behring Corporation), by the method described in Glycobiology, vol. 12, No. 7, pp. 421 to 426, 2002, to obtain the 4-dimensional hyaluronan oligosaccharide. Thus obtained 4-dimensional hyaluronan oligosaccharide (40 mg) was dissolved in borate buffer (pH 9,18) (3 ml) and the mixture was stirred at 80°C for 1 hour. The temperature of the obtained product was raised to room temperature and to the mixture was added methanol (3 ml), and then it was concentrated under reduced pressure. The residue was dissolved in water (2 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). After that, for the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (izgotovlenie the GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was concentrated to obtain a white powder.

The resulting powder (25 mg) was dissolved in methanol (1 ml) and water (0.5 ml) and to the mixture was added sodium borohydride (10 mg) under ice cooling. The temperature of the obtained product was raised to room temperature and the mixture was stirred over night. Completion of reaction was confirmed by mass spectrometry. To the product was added 10% solution of acetic acid in methanol (0.2 ml) under ice cooling, and the mixture is then concentrated under reduced pressure. The residue was dissolved in water (2 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). After that, for the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (18 mg, white powder).

MS [M-H]-: 573,45

1H-NMR: chart illustrated in FIG.

Example get 15

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 6-dimensional hyaluronan oligosaccharide (60 mg), to obtain the final product (34 mg, white powder).

MS [M-H]-: 953,02

1H-NMR: chart proell is reported on FIG.

Example 16

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 8-dimensional hyaluronan oligosaccharide (10 mg), to obtain the final product (8 mg, white powder).

MS [M-H]-: 1331,54

1H-NMR: chart illustrated in FIG.

Example of getting 17

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 10-dimensional hyaluronan oligosaccharide (10 mg), to obtain the final product (8 mg, white powder).

MS [M-H]-: 1710,28

1H-NMR: chart illustrated in FIG.

Example of getting 18

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 12-dimensional hyaluronan oligosaccharide (20 mg), to obtain the final product (16 mg, white powder).

MS [M-H]-: 2090,01

1H-NMR: chart illustrated in FIG.

Example of getting 19

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 14-dimensional hyaluronan oligosaccharide (18 mg), to obtain the final product (11 mg, white powder).

MS [M-H]-: 2469,52

1H-NMR: chart PR is illustrated on FIG.

Example of getting 20

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 16-dimensional hyaluronan oligosaccharide (7 mg), to obtain the final product (5 mg, white powder).

MS [M-H]-: 2848,59

1H-NMR: chart illustrated in FIG.

Example of getting 21

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 18-dimensional hyaluronan oligosaccharide (10 mg), to obtain the final product (9 mg, white powder).

MS [M-H]-: 3225,70

1H-NMR: chart illustrated in FIG.

Example of getting 22

Carried out the same reaction process as in the example of a 14, except that as the starting substances used 20-dimensional hyaluronan oligosaccharide (15 mg), to obtain the final product (13 mg, white powder).

MS [M-H]-: 3604,16

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples obtain from 14 to 22 are presented in table 2 below.

An example of retrieving 23

The compound obtained according to example obtain 1 (17 mg), was dissolved in buffer (obtained by mixing an aqueous solution of sodium chloride (300 mm, 1 ml) and water races is the thief of sodium acetate (200 mm, 1 ml) and bringing the pH to 5.2 glacial acetic acid) (2 ml) and to the resulting product were added β-glucuronidase B-1 bovine liver (manufactured by Sigma-Aldrich Corporation) (8 mg), and then incubated at 37°C for 8 hours. The reaction solution was subjected to ultrafiltration (Amicon Ultra 4 ml 10K Nominal Molecular Weight Limit, manufactured by Millipore Corporation) for cleaning. After that, for the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (8 mg, white powder).

MS [M-H]-: 601,18

1H-NMR: chart illustrated in FIG.

Example of getting 24

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example getting 2 (11 mg), to obtain the final product (4 mg, white powder).

MS [M-H]-: 980,29

1H-NMR: chart illustrated in FIG.

Example get 25

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example for the preparation of 3 (10 mg), to obtain the final product (8 mg, Bel is th powder).

MS [M-H]-: 1359,37

1H-NMR: chart illustrated in FIG.

Example of getting 26

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example 4 (30 mg), to obtain the final product (15 mg, white powder).

MS [M-H]-: 1738,15

1H-NMR: chart illustrated in FIG.

Example of getting 27

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example getting 5 (14 mg), to obtain the final product (7 mg, white powder).

MS [M-H]-: 2117,50

1H-NMR: chart illustrated in FIG.

Example of getting 28

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example obtain 9 (10 mg), to obtain the final product (5 mg, white powder).

MS [M-H]-: 3633,84

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples received from 23 to 28 are presented in table 3 below.

An example of obtaining 29

Held the same of reaction the second process, as in the example of obtaining 23, except that as the starting substances used compound obtained according to example receiving 15 (15 mg), to obtain the final product (9 mg, white powder).

MS [M-H]-: 777,28

1H-NMR: chart illustrated in FIG.

Example 30

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example 16 (11 mg), to obtain the final product (7 mg, white powder).

MS [M-H]-: 1156,41

1H-NMR: chart illustrated in FIG.

An example of retrieving 31

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example getting 17 (23 mg), to obtain the final product (15 mg, white powder).

MS [M-H]-: 1535,07

1H-NMR: chart illustrated in FIG.

Example of getting 32

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example getting 18 (14 mg), to obtain the final product (8 mg, white powder).

MS [M-H]-: 1914,60

1H-NMR: )the MMA is illustrated in FIG.

An example of obtaining 33

Carried out the same reaction process as in the example of obtaining 23, except that as the starting substances used compound obtained according to example getting 22 (8 mg), to obtain the final product (5 mg, white powder).

MS [M-H]-: 3430,67

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples received from 29 to 33 are presented in table 4 below.

An example of retrieving 34

Sodium hyaluronate (Hyaluronic Acid FCH-SU made FOOD CHEMIFA CO., LTD.) subjected to cleavage hyaluronidase derived fromStreptomyces hyalurolyticus(Hyaluronidase "Amano" 1 made AmanoEnzyme Inc.) the method described in Glycobiology, vol. 11, No. 1, pp. 47 to 64, 2001, to obtain the 4-dimensional unsaturated hyaluronan oligosaccharide. 4-dimensional unsaturated oligosaccharide hyaluronan (8 mg) was dissolved in methanol (2 ml) and water (1 ml) and to the mixture was added borgert sodium (4 mg) under ice cooling, and then stirred. The temperature of the obtained product was raised to room temperature and the mixture was stirred over night. Completion of reaction was confirmed by mass spectrometry. To the product was added 10% solution of acetic acid in methanol (0.2 ml) under ice cooling, and the mixture is then concentrated under reduced pressure. Then for drawing concentrate on what I gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (3 mg, white powder).

MS [M-H]-: 759,61

1H-NMR: chart illustrated in FIG.

Example of getting 35

Carried out the same reaction process as in the example of obtaining 34, except that as the starting substances used 6-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (9 mg, white powder).

MS [M-H]-: 1139,15

1H-NMR: chart illustrated in FIG.

Example of getting 36

Carried out the same reaction process as in the example of obtaining 34, except that as the starting substances used 8-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (10 mg, white powder).

MS [M-H]-: 1518,49

1H-NMR: chart illustrated in FIG.

An example of retrieving 37

Carried out the same reaction process as in the example of obtaining 34, except that as the starting substances used 10-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (10 mg, white powder).

MS [M-H]-: 1897,57

1H-NMR: chart proell is reported on FIG.

An example of retrieving 38

Carried out the same reaction process as in the example of obtaining 34, except that as the starting substances used 12-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (8 mg, white powder).

MS [M-H]-: 2276,99

1H-NMR: chart illustrated in FIG.

An example of retrieving 39

Carried out the same reaction process as in the example of obtaining 34, except that as the starting substances used 20-dimensional unsaturated oligosaccharide hyaluronan (12 mg), to obtain the final product (10 mg, white powder).

MS [M-H]-: 3792,03

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples received from 34 to 39 are presented in table 5 below.

Example of getting 40

Sodium hyaluronate (Hyaluronic Acid FCH-SU made FOOD CHEMIFA CO., LTD.) subjected to cleavage hyaluronidase derived fromStreptomyces hyalurolyticus(Hyaluronidase "Amano" 1 made AmanoEnzyme Inc.), the method described in Glycobiology, vol. 11, No. 1, pp. 47 to 64, 2001, to obtain the 4-dimensional unsaturated hyaluronan oligosaccharide. Unsaturated 4-dimensional hyaluronan oligosaccharide (20 mg) was dissolved in borate buffer (pH 9,18) (1 ml) and the mixture was stirred at 80°C for 1 hour. The temperature of the obtained product stand the shawl to room temperature and to the mixture was added methanol (3 ml), and then it was concentrated under reduced pressure. The residue was dissolved in water (2 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). After that, for the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was concentrated to obtain a white powder.

The resulting powder (6 mg) was dissolved in methanol (1 ml) and water (0.5 ml) and to the mixture was added sodium borohydride (10 mg) under ice cooling. The temperature of the obtained product was raised to room temperature and the mixture was stirred over night. Completion of reaction was confirmed by mass spectrometry. To the product was added 10% solution of acetic acid in methanol (0.2 ml) under ice cooling, and the mixture is then concentrated under reduced pressure. The residue was dissolved in water (2 ml) and the product was filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). After that, for the application of the concentrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain the final product (4 mg, white powder).

MS [M-H]-: 556,77

1H-NMR: chart is proillyustrirovana on FIG.

An example of retrieving 41

Carried out the same reaction process as in the example of a 40, except that as the starting substances used 6-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (7 mg, white powder).

MS [M-H]-: 935,49

1H-NMR: chart illustrated in FIG.

An example of retrieving 42

Carried out the same reaction process as in the example of a 40, except that as the starting substances used 8-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (6 mg, white powder).

MS [M-H]-: 1314,21

1H-NMR: chart illustrated in FIG.

An example of retrieving 43

Carried out the same reaction process as in the example of a 40, except that as the starting substances used 10-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (7 mg, white powder).

MS [M-H]-: 1693,58

1H-NMR: chart illustrated in FIG.

An example of retrieving 44

Carried out the same reaction process as in the example of a 40, except that as the starting substances used 12-dimensional unsaturated oligosaccharide hyaluronan (10 mg), to obtain the final product (6 mg, white powder).

MS [M-H]-: 2073,41

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples obtain from 40 to 44 are presented in table 6 below.

An example of retrieving 45

To 4-dimensional hyaluronan oligosaccharide (10 mg) was added to the solution in which benzylamine (24 mg), baronova acid (20 mg), sodium acetate (40 mg) and centralimediat sodium (15 mg) were dissolved in methanol (0.5 ml) and water (0.5 ml)and then stirred at 50°C for 6 hours. Completion of reaction was confirmed by mass spectrometry.

The obtained product was concentrated under reduced pressure and the residue was dissolved in water (2 ml). The mixture is then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). The filtrate was subjected to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for cleaning, and then the final fraction was subjected to freeze-drying to obtain the final product (8 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 46

To 4-dimensional hyaluronan oligosaccharide (10 mg) was added to the solution in which phenylalanine (5 mg), acetic acid (10 ml), sodium acetate (10 mg) and centralimediat sodium (10 mg) were dissolved in methanol (0.2 ml) and water (0.2 ml)and then stirred at 60°C for 6 hours. Completion of reaction was confirmed by mass with what ectromelia.

The obtained product was concentrated under reduced pressure, and then to the precipitate was added dichloromethane (5 ml) in water (5 ml) for extraction. After concentration of the aqueous phase under reduced pressure, the resulting product was dissolved in water (2 ml). The mixture is then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). The filtrate was subjected to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for cleaning, and then the final fraction was subjected to freeze-drying to obtain the final product (11 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 47

Carried out the same reaction process as in the example of obtaining 46, except that instead of phenylalanine used Proline, with the final product (8 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example obtain 48

Carried out the same reaction process as in the example of obtaining 46, except that instead of phenylalanine used tryptophan, with the final product (7 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 49

Carried out the same reaction process as in the example of obtaining 46, except that instead of phenylalanine used amid pillpennsylvania, to receive the of the final product (4 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example of getting 50

Carried out the same reaction process as in the example of obtaining 46, except that instead of phenylalanine used phenylalaninol, with the final product (14 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 51

Carried out the same reaction process as in the example of a 45, except that as the starting material instead of 4-dimensional hyaluronan oligosaccharide (10 mg) used a 10-dimensional hyaluronan oligosaccharide (20 mg), and except that instead of benzylamine used 4-Chloroaniline, with the final product (10 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 52

Carried out the same reaction process as in the example of a 45, except that instead of 4-dimensional hyaluronan oligosaccharide used 10-dimensional hyaluronan oligosaccharide, and except that instead of benzylamine used 2-aminopyridine, with the final product (12 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example of getting 53

Carried out the same reaction process as in the example of obtaining 46, except that instead of the 4-m is REGO of hyaluronan oligosaccharide used 10-dimensional hyaluronan oligosaccharide, and except that instead of phenylalanine used phenylalanylglycyl, with the final product (5 mg, white powder).

1H-NMR: chart illustrated in FIG.

An example of retrieving 54

Carried out the same reaction process as in the example of obtaining 46, except that instead of 4-dimensional hyaluronan oligosaccharide used 10-dimensional hyaluronan oligosaccharide, with the final product (5 mg, white powder).

1H-NMR: chart illustrated in FIG.

Structure of the final products of examples received from 45 to 54 are presented in table 7 below.

Examples 1-48: obtaining the compounds of the present invention

The methods in examples 1-48, mentioned below, was used to obtain the compounds of the present invention, are presented in tables 8-14. Mass spectrometry was performed using a QSTAR pulsari (Applied Biosystems Japan Ltd.).

Example 1

The compound synthesized according to example extract 1 (12 mg), was dissolved in water (1 ml) and to the mixture was added tributylamine (100 μl)and then stirred. After that, the mixture was concentrated under reduced pressure. To the concentrate was added N,N-dimethylformamide (2 ml)and then twice conducted azeotropic distillation. The residue was dissolved in N,N-Dimethylol amide (1 ml) and the product was added pyridine-sulfur trioxide (150 mg), and then was stirred at 42°C for 3 hours in nitrogen atmosphere.

After cooling the resulting product to 4°C were added water (1 ml)and then was added a saturated solution of sodium acetate in ethanol (30 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Then the supernatant was removed, was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (20 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Again after removal of supernatant was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (20 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Then the supernatant was removed and the residue was dissolved in water (2 ml)and then concentrated under reduced pressure.

The residue was dissolved in water (2 ml), and then fil is listed through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). For applying filtrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain compound 1 (24 mg, white powder).

[M+2Na]2+: 994,75

1H-NMR: chart illustrated in FIG.

Example 2

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example getting 2 (47 mg), to obtain compound 2 (76 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 3

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example obtain 3 (51 mg), to obtain compound 3 (108 mg, white powder).

[M+3Na]3+: 1210,10

1H-NMR: chart illustrated in FIG.

Example 4

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example 4 (48 mg), to obtain compound 4 (92 mg, white powder).

[M+3Na]3+: 1479,73

1H-NMR: chart illustrated in FIG.

Example 5

Carried out the same process, is that in example 1, except that as starting substances used compound obtained according to example getting 5 (60 mg), to obtain compound 5 (112 mg, white powder).

[M+4Na]4+: 1317,74

1H-NMR: chart illustrated in FIG.

Example 6

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example obtain 6 (20 mg), to obtain compound 6 (22 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 7

The compound obtained according to example obtain 7 (10 mg), was dissolved in Ν,N-dimethylformamide (1 ml) and to the resulting product were added pyridine-sulfur trioxide (150 mg), and then the mixture was stirred at 42°C for 3 hours in nitrogen atmosphere.

The obtained product was cooled to 4°C, then to the obtained product was added water (1 ml) and a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. After that, the supernatant was removed, was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (20 ml) provided for the I deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Again, after removal of supernatant was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (20 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Then after removing the supernatant, the sediment was dissolved in water (2 ml), and then were concentrated under reduced pressure.

The residue was dissolved in water (2 ml)and then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). For applying filtrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain compound 7 (16 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 8

Carried out the same process as in example 7 except that as the starting substances used compound obtained according to example obtain 8 (20 mg), the obtaining of compound 8 (33 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 9

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example obtain 9 (39 mg), to obtain compound 9 (90 mg, white powder).

[M+5Na]5+: 1707,07

1H-NMR: chart illustrated in FIG.

Example 10

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example 10 (24 mg), to obtain compound 10 (48 mg, white powder).

[M+3Na]3+: 1493,42

1H-NMR: chart illustrated in FIG.

Example 11

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example obtain 11 (17 mg), to obtain compound (11) (34 mg, white powder).

[M+3Na]3+: 1505,11

1H-NMR: chart illustrated in FIG.

Example 12

Carried out the same process as in example 1 except that as the starting substances used compound obtained according to example 12 (10 mg), to obtain compound 12 (10 mg, white powder).

Example 13

Carried out the same process as in example 1, the drop is observed in as educt used the compound obtained according to example obtain 13 (10 mg), to obtain compound 13 (21 mg, white powder).

The structures of the compounds 1 to 13 shown in table 8 below.

Example 14

The compound obtained according to example obtain 14 (18 mg), was dissolved in N,N-dimethylformamide (2 ml) and to it was added pyridine-sulfur trioxide (300 mg)and then stirred at 42°C for 3 hours in nitrogen atmosphere.

After cooling the resulting product to 4°C to the resulting product were added water (1 ml)and then was added a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. After that, the supernatant was removed, was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (20 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Again after removal of supernatant was added water (1 ml) to dissolve the precipitate, and then obavljale a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Then after removing the supernatant, the sediment was dissolved in water (2 ml)and then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). For applying filtrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain compound 14 (30 mg, white powder).

[M+2Na]2+: 791,30

1H-NMR: chart illustrated in FIG.

Example 15

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example receiving 15 (34 mg), to obtain compound 15 (72 mg, white powder).

[M+3Na]3+: 804,83

1H-NMR: chart illustrated in FIG.

Example 16

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example 16 (8 mg), to obtain compound 16 (11 mg, white powder).

[M+3Na]3+: 1074,42

1H-NMR: chart illustrated in FIG.

17

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 17 (8 mg), to obtain compound 17 (14 mg, white powder).

[M+3Na]3+: 1344,06

1H-NMR: chart illustrated in FIG.

Example 18

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 18 (16 mg), to obtain compound 18 (23 mg, white powder).

[M+4Na]4+: 1217,00

1H-NMR: chart illustrated in FIG.

Example 19

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 19 (11 mg), to obtain compound 19 (9 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 20

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example receiving 20 (5 mg), to obtain compound 20 (8 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 21

Carried out the same process as in example 14, except that as the starting material COI is litovali connection, obtained according to example getting 21 (8 mg), to obtain compound 21 (11 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 22

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 22 (13 mg), to obtain compound 22 (14 mg, white powder).

1H-NMR: chart illustrated in FIG.

The structure of the compounds from 14 to 22 are presented in table 8 below.

Example 23

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example obtain 23 (8 mg), to obtain compound 23 (14 mg, white powder).

[M+2Na]2+: 793,81

1H-NMR: chart illustrated in FIG.

Example 24

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 24 (4 mg), to obtain compound 24 (7 mg, white powder).

[M+2Na]2+: 1176,25

1H-NMR: chart illustrated in FIG.

Example 25

Carried out the same process as in example 14, except that as the starting substances used connection polucen is E. according to the example of obtaining 25 (8 mg), getting connection 25 (11 mg, white powder).

[M+3Na]3+: 1076,10

1H-NMR: chart illustrated in FIG.

Example 26

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 26 (15 mg), to obtain compound 26 (26 mg, white powder).

[M+3Na]3+: 1345,72

1H-NMR: chart illustrated in FIG.

Example 27

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 27 (7 mg), to obtain compound 27 (11 mg, white powder).

[M+4Na]4+: 1616,67

1H-NMR: chart illustrated in FIG.

Example 28

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 28 (5 mg), to obtain compound 28 (7 mg, white powder).

1H-NMR: chart illustrated in FIG.

The structure of connections between 23 and 28 are presented in table 10 below.

Example 29

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example floor the treatment 29 (9 mg), getting connection 29 (16 mg, white powder).

[M+2Na]2+: 972,77

1H-NMR: chart illustrated in FIG.

Example 30

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example 30 (7 mg), to obtain compound 30 (8 mg, white powder).

[M+2Na]2+: 1377,20

1H-NMR: chart illustrated in FIG.

Example 31

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 31 (15 mg), to obtain compound 31 (21 mg, white powder).

[M+3Na]3+: 1210,07

1H-NMR: chart illustrated in FIG.

Example 32

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 32 (8 mg), to obtain compound 32 (9 mg, white powder).

[M+3H]3+: 1458,34

1H-NMR: chart illustrated in FIG.

Example 33

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 33 (5 mg), to obtain compound 33 (7 mg, white powder).

1H-NMR chart illustrated in FIG.

The structure of the compounds from 29 to 33 are presented in table 11 below.

Example 34

The compound synthesized according to example getting 36 (7 mg), was dissolved in N,N-dimethylformamide (0.7 ml) and to it was added triethylamine-sulfur trioxide (75 mg) and triftormetilfullerenov acid (12 μl)and then stirred at 0°C for 48 hours in nitrogen atmosphere.

After cooling the resulting product to 4°C to the resulting product were added water (1 ml)and then was added a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. After that, the supernatant was removed, was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution was stirred using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Again after removal of supernatant was added water (1 ml) to dissolve the precipitate, and then was added a saturated solution of sodium acetate in ethanol (25 ml) to provide deposition. The solution paramesh the Wali using a Voltex mixer, and then the obtained product was subjected to centrifugation at refrigeration (4°C at 3000 rpm for 15 minutes) to collect the precipitated substance. Then after removing the supernatant, the sediment was dissolved in water (2 ml)and then filtered through a disc filter (manufactured by Nihon Pall Ltd., 0.45 µm). For applying filtrate to gel-filtration chromatography (G-10, 16 mm × 600 mm, water) for desalting used the AKTA system (manufactured by GE Healthcare Bioscience Bioprocess Corp.), and then the final fraction was subjected to freeze-drying to obtain compound 34 (9 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 35

Carried out the same process as in example 34, except that as the starting substances used compound obtained according to example getting 37 (10 mg), to obtain compound 35 (14 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 36

Carried out the same process as in example 34, except that as the starting substances used compound obtained according to example getting 38 (10 mg), to obtain compound 36 (15 mg, white powder).

1H-NMR: chart illustrated in FIG.

The structure of the connections 34 to 36 are presented in table 12 below.

Example 37

rowdily the same process as in example 34, except that as the starting substances used compound obtained according to example getting 41 (9 mg), to obtain compound 37 (11 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 38

Carried out the same process as in example 34, except that as the starting substances used compound obtained according to example getting 44 (12 mg), to obtain compound 38 (20 mg, yellow powder).

1H-NMR: chart illustrated in FIG.

The structure of compounds 37 and 38 are presented in table 13 below.

Example 39

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 45 (8 mg), to obtain compound 39 (12 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 40

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 46 (10 mg), to obtain compound 40 (16 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 41

Carried out the same process as in example 14, except that the qualities of the original substance used connection obtained according to example getting 47 (8 mg), to obtain compound 41 (14 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 42

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example obtain 48 (7 mg), to obtain compound 42 (12 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 43

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 49 (4 mg), to obtain compound 43 (9 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 44

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 50 (10 mg), to obtain compound 44 (24 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 45

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 51 (10 mg), to obtain compound 45 (21 mg, white powder).

1H-NMR: chart illustrated in FIG

Example 46

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 52 (12 mg), to obtain compound 46 (15 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 47

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 53 (5 mg), with compound 47 (9 mg, white powder).

1H-NMR: chart illustrated in FIG.

Example 48

Carried out the same process as in example 14, except that as the starting substances used compound obtained according to example getting 54 (5 mg), with compound 48 (9 mg, white powder).

1H-NMR: chart illustrated in FIG.

The structure of compounds 37 and 38 are provided in table 14 below.

Reference examples 1 to 7

Compounds (known compounds) of the present invention, is presented in table 15, was obtained by a method described in the document, Glycobiology, vol. 11, No. 1, pp. 57 to 64, 2001.

Reference example 1

Sodium hyaluronate (BIO Sodium Hyaluronate HA9, manufactured by Shiseido Co., Ltd.) subjected to the splitting obtained from the testicles of a bull hyaluronan is dazai (Bovine Hyaluronidase Τ 100KU, manufactured by Calbiochem Behring Corporation), by the method described in Glycobiology, vol. 12, No. 7, pp. 421 to 426, 2002, to obtain the 4-dimensional hyaluronan oligosaccharide. 4-dimensional hyaluronan oligosaccharide (20 mg) was used as the starting material for obtaining according to the above document, Glycobiology, vol. 11, No. 1, pp. 57 to 64, 2001, to obtain the compound 49 (21 mg, white powder).

Reference example 2

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 10-dimensional hyaluronan oligosaccharide (43 mg), to obtain compound 50 (86 mg, white powder).

Reference example 3

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 16-dimensional hyaluronan oligosaccharide (75 mg), with compound 51 (80 mg, white powder).

Reference example 4

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 20-dimensional hyaluronan oligosaccharide (23 mg), to obtain the compound 52 (42 mg, white powder).

Reference example 5

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 22-dimensional hyaluronan oligosaccharide (17 mg),with compound 53 (25 mg, white powder).

Reference example 6

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 24-32-dimensional hyaluronan oligosaccharide (20 mg), with connections 54 (18 mg, white powder).

Reference example 6

Carried out the same reaction procedure as in reference example 1 except that as the starting substances used 34-46-dimensional hyaluronan oligosaccharide (24 mg), with connections 55 (39 mg, white powder).

The structure of the compounds 49 to 55 are presented in table 15 below.

Test example 1: anti-Allergic effect; model of allergic rhinitis in Guinea pigs (model nasal obstruction)

Were used in the experiments Hartley Guinea pigs (male, age 6 or 7 weeks after the first sensitization).

After receiving their animals previously kept for 7 days or more for quarantine and acclimatization before use in experiments.

As the first sensitization in the back of each animal was injected with physiological solution containing ovalbumin (OVA, 1 mg) and the gel of aluminum hydroxide (alum, 10 mg) in an amount of 1 ml per animal. Then in both the nasal cavity of an animal using a micropipette was administered physiological RA is creative, containing 10 mg/ml OVA in 20 µl of each, one time (i.e. one week after the first sensitization) or twice (i.e. one week and two weeks after the first sensitization)in order to conduct local sensitization.

The division of animals into groups was performed using the method of two-dimensional stratified random sampling, based on their body mass on the day of completion of sensitization and body weight changes since the initiation of sensitization before the expiry date of sensitization as indicators.

One week after the last sensitization in both the nasal cavity of an animal was injected with saline containing 20 mg/ml OVA in the amount of 10 μl, inducyruya reaction antigen-antibody. Similarly, in the control group (group without induction) were injected with saline.

30 before induction into both nasal cavities of the animal was injected 10 μl each of the test substances. Each of the test substances were dissolved in saline solution for use as a solution for injection, with a concentration of 500 µg/ml Similarly, in the control group (group without induction) and in the group of physiological solution (control group solvent) was injected with saline. As a control means (not containing compounds) used "Flunase" (from otoplenie GlaxoSmithKline K.K.).

Measure the resistance of the nasal airway was performed before induction, after 10 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours and 7 hours after the induction day induction. The resistance of the nasal passages (nRaw) for 100 breaths were measured once in each period of measurement and the average of the results was defined as nRaw in each measuring period. To calculate the speed increase nRaw for use as a measure of the resistance of the nasal Airways.

The rate of increase (%) resistance of the nasal passages (nRaw) was calculated based on the following equation.

The speed increase nRaw in each measuring period

R0: nRaw to induction

Rx: nRaw in each period of measurement (x hours later)

The evaluation was performed based on the speed increase nRaw 10 minutes after the induction (the resistance of the nasal Airways immediate type) and area under the curve speed increase nRaw from 3 to 7 hours after induction (AUC3-7 h, delayed resistance nasal airway)

I3-7 hthe rate of increase nRaw from 3 to 7 hours after induction

The results are presented on FIG and 100.

On FIG and 100 shows that the compounds of the present invention exhibit the effect of inhibiting the allergic response instant t is a and the effect of inhibition on delayed allergic response.

Test example 2: Effect of inhibition of passive cutaneous anaphylaxis (PCA)

Were used in the experiments Hartley Guinea pigs (male, 5 weeks or older).

Anticigarette against OVA obtained by immunization of Guinea pigs by OVA) was diluted 500 times with a saline solution (solution A).

Each of the test substances are given in table 16 below were diluted to 200 μg/ml of saline solution (solution B).

Solution B was mixed with a solution obtained by diluting the antisera Guinea pigs against OVA in 250 times in an equivalent amount, and the final concentration of the mixture was brought to 100 µg/ml for the test substance and up to 500-fold for antisera against OVA (solution C).

After ether anesthesia Guinea pig was intradermally injected with 100 μl of saline solution or A solution C to the area on the back, where he was to shave off his coat.

After about 3 hours Guinea pig was injected intravenously with saline with 0.5% Evans blue, containing from 0.2 to 0.4% OVA in an amount of from 0.8 to 1 ml/organism.

No later than 30 minutes was performed phlebotomy and Guinea pigs were skinned. Then determined the amount of pigment in each spot by means of image processing. When processing images, the amount of pigment on the spot where he entered anticigarette against OVA was determined ka is 100% and, based on the definition, assessed the degree of suppression caused by the test substance. The results are presented in table 16 (N=3 or 6).

The case where the leakage of pigment was 80% or more, indicating no inhibition effect on PCA, denoted as "D"; the case where the leakage of pigment ranges from 60 to 80%, designated as "C" (the percentage of suppression: from 20 to 40%); the case where the leakage of pigment ranges from 30% to 60% (the percentage of suppression: from 40 to 70%), designated as "B"; and the case where the leakage of the pigment is less than 30%, designated as "A".

Table 16 above shows that the compounds of the present invention exhibit the effect of suppressing the PCA.

Test example 3: Test the activity of increasing the permeability of blood vessels

Were used in the experiments Hartley Guinea pigs (male, 5 weeks or older).

The test substance was diluted in saline to 100 mcg/ml

Further, as control was used sulfated hyaluronic acid (a mixture of tetrasaccharide with almost heptanethiol, synthesized according to example (example obtain 1) JP-A-1999-147901, or similar).

After ether anesthesia Guinea pig was intradermally injected with 100 μl of saline or saline containing the test substance in the region on the back, where was shave off his coat.

A Guinea pig was injected intravenously with saline with 0.5% Evans blue in the amount of from 0.8 to 1 ml/organism.

No later than 30 minutes was performed phlebotomy and Guinea pigs were skinned. Then determined the amount of pigment in each spot by means of image processing.

The amount of pigment in the field, where he introduced extrasolar 10000, defined as 100%, and, based on this definition, the processing results are presented as the speed of vascular permeability test substance on FIG-103.

On FIG-103 shown that the compounds of the present invention, unlike the known polymer polysulfonamide hyaluronic acid, do not have activity against the increase of vascular permeability and do not show a stimulating action, which in itself is a side effect.

Test example 4: Long-term stability (in aqueous solution)

Aqueous solutions containing compound 4 and compound 50 in the amount of 1 mg/ml, were prepared for analysis HPLC.

Each of these solutions were stored in a cold place (2 to 8°C) and at room temperature and subjected dependent on time before HPLC analysis up to 4 months to check the changes of the pattern of peaks of each solution.

The HPLC conditions:

Column: Mightysil RP-18 GP (3 µm, 4.6 mm × 50 mm)

The column temperature: 40°C

The mobile phase A: 20 mm KH2PO4/MeCN (70:30)containing 2 mm TBAP

The mobile phase Β: 20 mm KH2PO4(80:20)containing MeCN/2 mm TBAP

The gradient conditions: Initial; A: 100%B: 0%, 0 to 20 minutes; A: 100 to 95%, B: from 0 to 5%, linear

Flow rate: 1 ml/min

Detection: UV (210 nm)

Injected amount: approximately 5 μg/5 μl (TBAP: tetrabutylammonium phosphate)

The results are presented in table 17.

Table 17
Storage conditions and storage periodConnection 4The connection 50
In the beginning of the testWatched one main peak and shoulder peaks, and the ratio of the areas of the main peak and shoulder peak was approximately 5 to 1Observed mainly three peaks, and the ratio of the areas of the second peak and the third peak was approximately 1 : 4
In a cool place for 4 monthsNo change in the pattern of the peaks compared to the pattern in the beginning of the testThe ratio of the areas of the second peak and the third is th peak was approximately 1 to 1
At room temperature for 10 days-*The ratio of the areas of the second peak and the third peak was approximately 3 : 2
At room temperature for 4 monthsNo change in the pattern of the peaks compared to the pattern in the beginning of the test-**
-: The test was not performed.
*: No change in the pattern of the peaks compared to the pattern in the beginning of the test, when the solution is kept at room temperature for 7 days.
**: When the solution is kept at room temperature for 10 days, it was found that the solution was obviously unstable, and thus, the test is immediately terminated.

The above table 17 shows that the observed changes in the pattern of peaks for compound 4 after storage at room temperature for 4 months, and thus, compound 4 is stable in aqueous solution, while the observed time-dependent increase in area of the second peak and time-dependent decrease in the square of the third peak among the three main peaks of the connection 50, despite the fact that it was stored in a cold place, and thus the om, the connection 50 is unstable in aqueous solution.

Among the low molecular weight polyzwitterionic derivatives of hyaluronic acid group of compounds corresponding to General formula (IA)and, where Y corresponds to the formula (d) or (e), or the group of compounds corresponding to General formula (IB), particularly suitable as compounds, which are stable in aqueous solution.

Industrial applicability

Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the present invention shows low activity in terms of increased vascular permeability (providing inflammatory side effects in a small degree), and, thus, it can be used as a largely safe means for the prevention and/or treatment of allergic diseases.

1. The remedy for the prevention and/or treatment of allergic disease selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, containing as an active ingredient of low molecular weight polysulfone derived hyaluronic acid corresponding to the following General formula (IA) or (IB), or its pharmaceutically acceptable salt:

where n made the focus of a number from 0 to 15; X corresponds to the following formula (a) or (b):

Y corresponds to the following formula (c), (d) or (e):

each R independently represents a hydrogen atom or a group of SO3H, provided that group SO3H ranges from 80 to 100% of the total number of R; R1represents-OH, -OSO3H or-NZ1Z2where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted phenyl group where the Deputy selected from a halogen atom, carboxypropyl, benzyl group or peredelnoj group, or-NZ1Z2collectively represents an amino acid residue selected from phenylalanine, Proline and tryptophan, or a peptide residue selected from pillpennsylvania, fenilalanina and phenylalanylglycyl, where amino acid residue or peptide residue can be aminirovanie on their terminal carboxyl group; and each * represents the place of attachment to the oxygen atom;
where n is a number from 0 to 15; W corresponds to the following formula (f) or (g):
each R independently represents a hydrogen atom or a group of SO3H, provided that group SO3H ranges from 80 to 100% of total Chi is La R; R1represents-OH, -OSO3H or-NZ1Z2where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted phenyl group where the Deputy selected from a halogen atom, carboxypropyl, benzyl group or peredelnoj group, or-NZ1Z2collectively represents an amino acid residue selected from phenylalanine, Proline and tryptophan, or a peptide residue selected from pillpennsylvania, fenilalanina and phenylalanylglycyl, where amino acid residue or peptide residue can be aminirovanie on their terminal carboxyl group; and each * represents the place of attachment to the oxygen atom.

2. The remedy for the prevention and/or treatment according to claim 1, where Y in the General formula (IA) corresponds to the formula (d) or (e).

3. The remedy for the prevention and/or treatment according to claim 2, where X corresponds to the formula (a).

4. The remedy for the prevention and/or treatment according to claim 3, where n is 3, 4 or 5.

5. The remedy for the prevention and/or treatment according to claim 3, where n is 4 or 5.

6. The remedy for the prevention and/or treatment according to claim 1, where the low molecular polysulfone derivative of hyaluronic acid with the General formula (IB) .

7. The remedy for the prevention and/or treatment according to claim 6, where n is 3, 4 or 5.

8. Means for PR is the prevention and/or treatment according to claim 6, where n is 4 or 5.

9. The use of low-molecular polysulfonamide derivative of hyaluronic acid or its pharmaceutically acceptable salt according to any one of claims 1 to 8 for the manufacture of products for the prevention and/or treatment of allergic disease selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma.

10. The method of prevention and/or treatment of allergic disease selected from hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, including the introduction of a person or an animal an effective dose of low-molecular polysulfonamide derivative of hyaluronic acid or its pharmaceutically acceptable salt according to any one of claims 1-8.

11. Low-molecular polysulfone derived hyaluronic acid corresponding to General formula (IA') or (IB), or its pharmaceutically acceptable salt:
where n is a number from 0 to 15; X corresponds to the following formula (a) or (b):

Y' corresponds to the following formula (d) or (e);
each R independently represents a hydrogen atom or a group of SO3H, provided that group SO3H ranges from 80 to 100% of the total number of R; R1the stand is made by a-OH, -OSO3H or-NZ1Z2where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted phenyl group where the Deputy selected from a halogen atom, carboxypropyl, benzyl group or peredelnoj group, or-NZ1Z2collectively represents an amino acid residue selected from phenylalanine, Proline and tryptophan, or a peptide residue selected from pillpennsylvania, fenilalanina and phenylalanylglycyl, where amino acid residue or peptide residue can be aminirovanie on their terminal carboxyl group; and each * represents the place of attachment to the oxygen atom;

where n is a number from 0 to 15; W corresponds to the following formula (f) or (g):

each R independently represents a hydrogen atom or a group of SO3H, provided that group SO3H ranges from 80 to 100% of the total number of R; R1represents-OH, -OSO3H or-NZ1Z2where each of Z1and Z2independently represents a hydrogen atom, -SO3H, optionally substituted phenyl group where the Deputy selected from a halogen atom, carboxypropyl, benzyl group or peredelnoj group, or-NZ1Z2in owls is kupeli represents an amino acid residue, selected from phenylalanine, Proline and tryptophan, or a peptide residue selected from pillpennsylvania, fenilalanina and phenylalanylglycyl, where amino acid residue or peptide residue can be aminirovanie on their terminal carboxyl group; and each * represents the place of attachment to the oxygen atom.

12. Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to claim 11, where X in the General formula (IA') corresponds to the formula (a).

13. Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to claim 11, which corresponds to General formula (IB).

14. Low-molecular polysulfone derivative of hyaluronic acid or its pharmaceutically acceptable salt according to item 12 or 13, where n represents 3, 4 or 5.

15. Pharmaceutical composition for prevention and/or treatment of allergic diseases such as pollinosis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and asthma, containing an effective amount of low molecular weight polysulfonamide derivative of hyaluronic acid or its pharmaceutically acceptable salt according to § § 11 to 14 and a pharmaceutically acceptable excipient.



 

Same patents:

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60 cl, 3 tbl, 65 ex

FIELD: food industry.

SUBSTANCE: this invention relates to a crystalline maltitol powder composition; its specificity consist in the fact that the average particle size across the bulk (according to laser diffraction results) is equal to 10 - 150 mcm; the content of maltitol in the composition is 80 - 99.9 wt %; at least 50 wt % of the particles pass through a sieve having retention threshold equal to 2000 mcm according to A1 test; at least 35 wt % of the particles pass through a sieve having retention threshold equal to 2000 mcm according to A2 test; the composition includes 0.1 - 20 wt % of at least one water-insoluble anti-clogging agent; the said anti-clogging agent has hydroscopic property (determined according to Test B) equal to 2.5 - 25%; the said anti-clogging agent is selected from the group including pyretogenous silicon dioxide, sodium aluminosilicate, anhydrous tricalcium phosphate and dehydrated potato starch (especially dehydrated potato starch containing less than 12% residual water, preferably containing less than 10% residual water, preferably containing less than 8% residual water, preferably containing less than 6% residual water) and their mixtures.

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13 cl, 5 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: method of producing modified alkyl polyglucoside by reacting glucose or water syrup of glucose with a C10-C16 alcohol in the presence of an acid catalyst, which is a strong organic or inorganic acid. Said catalyst is a mixture of alkyl polyalkoxy carboxylic acid and a strong acid in molar ratio of 4:1. The molar ratio glucose:alcohol is equal to 1:1.5; glucose:acid catalyst ranges from 1:0.025 to 1:0.03.

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1 cl, 5 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new compounds of general formula I [X]n-Y-ZR1R2, wherein the radicals are specified in the description, effective as heparan sulphate-binding protein inhibitors. The invention also refers to a pharmaceutical or veterinary composition having heparan sulphate-binding protein inhibitory activity for preventing or treating a disorder in a mammal, and to the use of these compounds and compositions for antiangiogenic, antimetastatic, anti-inflammatory, antimicrobial, anticoagulant and/or antithrombotic therapy in a mammal.

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10 cl, 31 ex, 11 tbl, 40 dwg

FIELD: medicine, pharmaceutics.

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26 cl, 8 ex, 26 dwg

FIELD: chemistry.

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25 cl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to group of compounds of general formula where R1, R2, R3, R4, R5 and R6 independently on each other represent C1-4 alkyl, -SO3H, polysulfated β-glycosyl or polysulfated diglycosyl group, on condition that, at least, one of R1-R6 represents polysulfated β-glycosyl or polysulfated diglycosyl group, or their pharmaceutically acceptable salts, where glycosyl group contains pentopyranose or hexopyranose molecule with configuration of choice, and diglycosyl group contains pentopyranose or hexopyranose molecule with configuration of choice, one hydroxyl group of which is glycosylated by other pentopyranose or hexopyranose molecule with configuration of choice. Invention also relates to pharmaceutical composition to be used in treatment of acute or chronic inflammatory diseases of respiratory ways in mammals on the basis of said compounds or their pharmaceutically acceptable salts.

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37 cl, 4 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to oligosaccharide, suitable for vaccine against meningitis A, which includes first mannose unit, which has spacer in alfa-configuration in C-1, where said spacer is able to conjugate with protein, and bound to second mannose unit by 1,6-bond, which binds C-6 of first unit with C-1 of second unit, 1,6-bond including phosphonate. Invention also relates to methods of obtaining oligosaccharide and improved methods of obtaining mannose derivative, suitable for obtaining immunogenic oligosaccharide. Invention also relates to pharmaceutical composition for induction of immune response, immunogenic composition, capable of inducing formation of protective antibodies against meningitis A and vaccine against meningitis A, which include oligosaccharide.

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51 cl, 4 dwg, 3 tbl, 16 ex

FIELD: chemistry.

SUBSTANCE: invention relates to carbohydrate-containing polycationic amphiphiles (1-3) which are trihydrochlorides of rac-N-[6-(β-D-glycopyranosyloxy)hexyl]-N-[2,3-di(tetradecyloxy)prop-1-yl]-4-[(12-amino-4,9-diazadodec-1-yl)amino-succinylamino]benzenesulfonamide of the given general formula , where A is a 1,2-di-O-tetradecyl-rac-glycerin residue, B is a galactose residue (for (1)), galactose (for (2)) and mannose (for (3)), C is a spermine residue, n= 6, m = 2.

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1 cl, 4 tbl, 2 dwg, 11 ex

FIELD: chemistry.

SUBSTANCE: in method of obtaining compound aminoalkyl glucosaminide 4-phosphate of formula , X represents , Y represents -O- or NH-; R1, R2 and R3, each is independently selected from hydrogen and saturated and unsaturated (C2-C24) aliphatic acyl groups; R8 represents -H or -PO3R11R11a, where R11a and R11a, each is independently -H or (C1-C4) aliphatic groups; R9 represents -H, -CH3 or -PO3R13aR14, where R13a and R14, each is independently selected from -H and (C1-C4) aliphatic groups, and where indices n, m, p, q each independently is a integer from 0 to 6 and r is independently integer from 2 to 10; R4 and R5 are independently selected from H and methyl; R6 and R7 are independently selected from H, OH, (C1-C4) oxyaliphatic groups -PO3H2, -OPO3H2, -SO3H, -OSO3H, -NR15R16, -SR15, -CN, -NO2, -CHO, -CO2R15, -CONR15R16, -PO3R15R16, -OPO3R15R16, -SO3R15 and -OSO3R15, where R15 and R16, each is independently selected from H and (C1-C4) aliphatic groups, where aliphatic groups are optionally substituted with aryl; and Z represents -O- or -S-; on condition that one of R8 and R9 represents phosphorus-containing group, but R8 and R9 cannot be simultaneously phosphorus-containing group, including: (a) selective 6-O- silylation of derivative of 2-amino-2-desoxy-β-D-glucopyranose of formula , where X represents O or S; and PG independently represent protecting group, which forms ester, ether or carbonate with oxygen atom of hydroxy group or which forms amide or carbamate with amino group nitrogen atom, respectively; by means of tri-substituted chlorosilane RaRbRcSi-Cl, where Ra, Rb and Rc are independently selected from group, consisting of C1-C6alkyl C3-C6cycloalkyl and optionally substituted phenyl, in presence of tertiary amin, which gives 6-silylated derivative; (b) selective acylation of 4-OH position of obtained 6-O-silylated derivative with 6-3-alkanoyloxyalcanoic acid or hydroxyl-protected (R)-3-hydroxyalkanoic acid presence of a carbodiimide reagent and catalytic 4-dimethylaminopyridine or 4-pyrrolidinopyridine to give a 4-O-acylated derivative; (c) selectively deprotecting the nitrogen protecting groups, sequentially or simultaneously and N,N-diacylating the resulting diamine with (R)-3-alkanoyloxyalkanoic acid or a hydroxy-protected (R)-3-hydroxyalkanoic acid in presence of peptide condensation reagent; (d) introducing a protecting phosphate group at 3-position with a chlorophosphate or phosphoramidite reagent to give a phosphotriester; and (e) simultaneous or sequential deprotecting phosphate, silyl, and remaining protecting groups.

EFFECT: method improvement.

11 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine and concerns preparing a fast-acting effective and safe agent for treating rhinitis. Solving the problem provides the agent for treating rhinitis, particularly allergi rhinitis containing C-type natriuretic peptide (CNP) and/or B-type natriuretic peptide (BNP) as an active ingredient.

EFFECT: invention provides the notable health improvement in rhinitis, particularly in allergic rhinitis, and besides, the therapeutic effect, ensures fast and prolonged action, and gives no local side effects.

21 cl, 7 ex, 7 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is using a composition containing galactooligosaccharide, fructooligosaccharide and uronic acid oligosaccharide in preparing a composition for oral administration into an infant for preventing the local administration of corticosteroids and/or preventing the administration of a calcineurin inhibitor into the above infant, wherein uronic acid oligosaccharide represents a pectin degradation product and/or an alginate degradation product, and wherein using the corticosteroids and/or administering the calcineurin inhibitor is applicable for treating eczema, infantile eczema, atopic dermatitis, dermatitis herpetiformis, contact dermatitis, seborrheic dermatitis, neurodermatitis, psoriasis and intertrigo. Particularly, the composition is a nutritional composition.

EFFECT: what is shown is reducing probability of the local administration of corticosteroids and dermatological preparations to be required for the purpose of preventing the above skin diseases, or reducing the length of using the corticosteroids.

5 cl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to naphthalene carboxamide derivatives of general formula I which possess the properties of protein kinase or histone deacetylase inhibitors. The compounds can find application for preparing a drug for treating inflammatory diseases, autoimmune diseases, oncological disease, diseases of the nervous system and neurodegenerative diseases, allergies, asthma, cardiovascular diseases and metabolic diseases or disease related to hormonal diseases. In general formula I: , Z represents CH or N; each of the groups R1, R2 and R3 represents hydrogen, halogen, alkyl, alkoxy or trifluoromethyl; R4 represents or X represents a benzene ring or a pyridine ring; R5 represents one or more substitutes specified in a group consisting of hydrogen, halogen, alkyl, alkoxy or trifluoromethyl. The invention also refers to a method for preparing the above compounds, a pharmaceutical preparation and using them.

EFFECT: preparing the compounds which possess the properties of protein kinase or histone deacetylase inhibitors.

13 cl, 10 tbl, 6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a complex of biologically active substance for treating allergic diseases of various genesis, characterised by the fact that it has been recovered from cod liver oil by gradual fractionation from ballast lipids by extraction in a two-phase oil and water extractant, centrifugation and ultrafiltration or diafiltration through a material with separation limit 25 kDa, and contains peptides 30-55%, amino acids 40-65%, carbohydrates 2-8%, micro and macroelements 2-13%.

EFFECT: invention provides the drug spectrum broadening.

8 ex, 3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel of 2,4-pyrimidine diamine compounds of formula I, which inhibit degranulation of immune cells and can be used in treating cell reactions mediated by FcεRI or FcγRl receptors. In formula (I) each R2 and R4 is independently phenyl substituted with one or more R8 groups or a heteroaryl selected from a group consisting of , where the heteroaryl is optionally substituted with one or more R8 groups and at least one of R2 and R4 is a heteroaryl; R5 is selected from a group consisting of (C1-C6)alkyl, optionally substituted with one or more identical or different R8 groups, -ORd, -SRd, fluorine, (C1-C3)halogenalkyloxy, (C1-C3)perhalogenalkyloxy, -NRcRc, (C1-C3)halogenalkyl, -CN, -NO2, -C(O)Rd, -C(O)ORd, -C(O)NRcRc, -C(NH)NRcRc, -OC(O)Rd, -OC(O)ORd, -OC(O)NRcRc; -OC(NH)NRcRc, - [NHC(O)]nORd, R35 is hydrogen or R8; each Y is independently selected from a group consisting of O, S and NH; each Y1 is independently selected from a group consisting of O, S and NH; each Y2 is independently selected from a group consisting of CH, CH2, S, N, NH and NR37. Other values of radicals are given in the claim.

EFFECT: improved efficiency.

19 cl, 6 tbl.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new 2-S-benzylpyrimidine derivatives having CRTH2 receptor antagonist activity. In formula 1: R1 means -CO2H; R4a and R4b mean hydrogen; W means -C(O)NR7-; R2 and R3, each independently mean F; Cl; Br;-NR10R11 or (C1-C6)alkoxy, optionally substituted by 1-3 halogen atoms; R5 means hydrogen; R6 means (C1-C6)alkyl; (C6-C19)aryl or (5-15)-member heteroaryl containing nitrogen, oxygen or sulphur atoms as heteroatoms, wherein above aryl and heteroaryl are optionally substituted by one or more substitutes specified in a group consisting of halogen; (C1-C6)alkyl optionally substituted by 1-3 halogen atoms; and (C1-C6)alkoxy optionally substituted by one, two or three halogen atoms; R7 means hydrogen; R10 and R11, each independently mean (C1-C6)alkyl; or R10 and R11, together with N, whereto attached form a 3-8- member saturated or unsaturated ring optionally containing one or more O or S atoms, or one or more additional N atoms in the ring; k is equal to 0; m is equal to 1.

EFFECT: invention also refers to using the above compounds for preparing a drug for treating allergic and inflammatory diseases mediated by CRTH2 receptor activity, such as asthma, atopic dermatitis, allergic conjunctivitis, Churg-Strauss disease, sinusitits, basophilic leukaemia, and recurrent urticaria.

27 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. The preparation contains an antibody, histidine and Polysorbate 80. Besides, a method of treating a subject with using the above preparation and a method of stabilising anti-human α-interferon antibody 13H5 are described. The invention can be used in medicine.

EFFECT: what is disclosed is a stable aqueous preparation containing the antibody or fragment thereof which are specifically bound to human α-interferon.

7 cl, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to paediatrics and may be used for preparing a drug or therapeutic nutritional composition for maturating an immune responses in a newborn infant. That is ensured by using an oligosaccharide specified in a group consisting of: lacto-N-tetrose, lacto-N-neotetrose, lacto-N-hexose, lacto-N-neohexose, para-lacto-N-hexose, para-lacto-N-neohexose, lacto-N-octose, lacto-N-neooctose, iso-lacto-N-octose, para-lacto-N-octose and lacto-N-decose. Also, the above oligosaccharide may be used for modulating the immune system of the newborn infant to ensure the developing beneficial intestinal microflora for the first weeks of life comparable to such found in breastfed infants.

EFFECT: group of inventions enables the developing beneficial intestinal microflora in the infant, and reduces the risk of a further allergy.

27 cl, 1 ex

FIELD: biotechnology.

SUBSTANCE: monocytes are isolated from venous blood MNCs using the Percoll cushion bi-gradient of density: 47.5% SIP and 15% SIP, respectively, at cooling to +4°C. The monocytes are placed in a completely nutritional culture medium with adding 20 ng/ml IL-4 and 20 ng/ml GM-CSF. The completely nutritional culture medium is replaced on the day 3 of cultivation. On the day 4 of cultivation the antigen of infectious origin Opisthorchis felineus is added at a dose of 40 mcg/ml and maturation of dendritic cells is simultaneously induced with lipopolysaccharide E.coli of serotype 055: B5 in a dose of 1 mcg/ml. On the day 6 of cultivation the dendritic cells are washed and analysed.

EFFECT: use of the method provides obtaining the mature, antigen-loaded dendritic cells.

2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to organic chemistry, namely to new 1,2-dihydroquinoline derivatives of general formula , or to a pharmaceutically acceptable salt thereof, wherein R1 represents a lower alkyl group; R2 represents a hydrogen atom; each of R3 and R4 represents a lower alkyl group; R5 represents a lower alkyl group; R6 represents a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group; X represents -CO-, -C(O)NR8 - or -S(O)2-; each of R7 and/or R8 may be identical or different, and represents a hydrogen atom, a lower alkyl group, a lower alkenyl group, a lower cycloalkyl group, a phenyl or naphthyl group, a saturated or unsaturated monocyclic 5- or 6-member heterocyclyl with one or two heteroatoms specified in nitrogen, oxygen and sulphur atoms, and 3-5 carbon atoms in a cycle, a lower alkoxy group, a phenoxy group; provided R7 and/or R8 represent a lower alkyl group, a lower alkoxy group, the mentioned lower alkyl group and lower alkoxy group may contain one or three groups specified in a halogen atom, a phenyl group, an unsubstituted monocyclic 6-member heterocyclyl with one heteroatom specified in a nitrogen atom, and 5 carbon atoms in a cycle, a lower alkoxy group, and -NRaRb as a substitute (substitutes); provided R7 and/or R8 represent a phenyl group, a saturated or unsaturated monocyclic 5- or 6-member heterocyclyl with one or two heteroatoms specified in nitrogen, oxygen and sulphur atoms, and 3-5 carbon atoms in a cycle, a phenoxy group, the mentioned phenyl group, saturated or unsaturated monocyclic 5- or 6-member heterocyclyl with one or two heteroatoms specified in nitrogen, oxygen and sulphur atoms, and 3-5 carbon atoms in a cycle, phenoxy group may contain one or two groups specified in a halogen atom, a lower alkyl group, a halogen-substituted lower alkyl group, a phenyl group, a hydroxyl group, a lower alkoxy group, a halogen-substituted lower alkoxy group, a lower alkylthio group, a lower alkylcarbonyl group, a lower alkoxycarbonyl group, a lower alkylcarbonyloxy group, -NRaRb, a nitro group and a cyano group as a substitute (substitutes); Ra and Rb may be identical or different, and each of them represents a hydrogen atom, a lower alkyl group, a lower alkoxycarbonyl group; Y represents a lower alkylene group; Z represents an oxygen atom; p is equal to 2, provided p is equal to 2, R6 may be identical or different. The invention also relates to a pharmaceutical composition and a glucocorticoid receptor modulator of the compound of formula (1).

EFFECT: there are produced new 1,2-dihydroquinoline derivatives possessing glucocorticoid receptor binding activity.

7 cl, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacy, medicine and veterinary science, particularly new drugs for inflammatory and allergic diseases. The assigned problem preparing a non-toxic, easy-to-use and storage-stable drug in the form of eye drops, intranasal drops and spray and causing anti-inflammatory and antiallergic action is solved by creating a composition containing a pigment-mineral complex of sea-urchin shell (0.05-2.0%) containing spinochromes B, D, dimer polyhydroxynaphthoquinone and a mineral ingredient (calcium, magnesium, phosphor, sodium, potassium) in the form of water-soluble salts, a co-solvent (0.01-10%), a preserving agent (0.01-0.2%), an antioxidant (0.01-0.2%), an acidity regulator (to pH 6.0-8.0) and water (to 100%).

EFFECT: what is described is the experimentally specified method for preparing the agent according to the invention eliminating the oxidation of the pigment-mineral complex as early as the stage of preparing.

3 cl, 3 tbl, 1 dwg, 6 ex

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