Functionalised beta 1, 6 glucosamine-disaccharides and method for preparing them

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a new method for the chemical synthesis of asymmetrically or symmetrically substituted β-(1→6)-bound glucosamine disaccharide of formula (1), as well as to a method for purifying it. The invention declared the intermediate compounds referred to the given method.

EFFECT: invention refers to a pharmaceutical composition comprising the mentioned compounds, and to the use of the compounds in treating the disorders affected by immune system activity modulation, including the inhibition or activation of the immune system, such as a disorder selected from immune disorders and/or cancer.

26 cl, 8 ex, 26 dwg

 

The technical FIELD

The present invention relates to a new method of chemical synthesis of β-(1→6)-linked glucosamine-disaccharides. Such compounds can be used as a derivative of lipid A. Example derivatives of lipid a isOM-174-DP®,first isolated firm OM Pharma1from partially decomposed lipopolysaccharidesEscherichia coli. The invention includes the design and chemical synthesis of new analogues of lipid A, which are deprived of both sugar-O-acyl substituents (O-3 and O-3') and thus contain only N-linked residues of fatty acids. Immunological activity of such compounds related to the biological activity of the parentOM-174-DP®.

BACKGROUND of the INVENTION

The lipopolysaccharides (LPS) are the major compounds, expressed in the outer membrane of almost all gram-negative bacteria. Data amphiphilic macromolecules have a common structure consisting of a hydrophilic polysaccharide (formed from the skeletal oligosaccharide and O-specific polysaccharide), covalently linked to a lipophilic fragment, called lipid And2, which serves as a membrane anchor LPS.

LPS, also known as endotoxins, are strong stimulants protection systems master as adjuvants DL the antigens 3vaccines and as inducers or activators nonspecific resistance to infection in animal models4. Data amphiphilic macromolecules are extremely strong immunostimulatory activity5. The biological activity of LPS is a consequence mainly of the lipid component And, although the toxicity of lipid And depends strictly on its primary structure.

In General, lipid And has a highly conserved structure. It usually consists of β-(1→6)-linked glucosamine-disaccharide glycosides of the skeleton, phosphorylated at positions O-1 and O-4', and six or more fatty acyl groups are associated in the form of esters and amides. Anomeric phosphate (1 position) repair parts glucosamine is exclusively the α configuration. For example, the full chemical structure of lipid A, isolated fromcells of E. coli(1)defined by the authors Imoto and other6contains β-(1→6)-linked glucosamine-disaccharide glycosides of the skeleton, phosphorylated at positions O-1 and O-4' and acylated 2.3 position (R)-3-hydroxymitragynine acid, 2' - position is (R)-3-dodecadodecahedron acid, and 3' position (R)-3-tetradecanoylphorbol acid.

Because of the wide spectrum of biological activity was shown a huge interest from both industry and academic the fir research laboratories. Much effort was devoted to chemical modifications of the structure of the lipid And to reduce natural endotoxicosis source connection and at the same time maintaining or improving its beneficial immunostimulatory properties in the eighties Ribi et al. studied the chemical process with the intention to separate the toxic effects of natural lipid And Salmonella Chlamydia RC595 from potentially useful immune modulating effects. This method is based on the selective hydrolysis of 1-phosphonopropyl7and (R)-3-hydroxymitragynine8residue attached to the 3-position of the sugar lipid And delivered a known immunostimulant monophosphoryl-lipid A (MPL®), which is an effective adjuvant for prophylactic and therapeutic vaccines9with significantly reduced toxicity compared to its original lipid A. However, MPL®as well as lipid And natural origin is a mixture of several components due to the inherent LPS heterogeneity and imperfect stages chemoselective hydrolysis or stages of purification. Consequently, adjuvant MPL®includes several less highly acylated compounds in addition to the main hexazinone connection.

In the early nineties the company OM PHARMA from partially decomposedLPS1E. coliwas allocated a new derivative of lipid A (OM-174-DP®,1). This derivative is devoid of both sugar-O-acyl substituents (O-3 and O-3') and, therefore, contains only N-linked residues of fatty acids lipid AndE. colinamely (R)-3-hydroxyacetanilide group in N-2 and (R)-3-dodecadodecahedron group in N-2', thus leaving that in the structure of only three acyl groups with long chain. Thorough pharmacological studies of this new connection has revealed that it has a strong antitumor activity in several in vivo models of tumor10and that it is an effective immunoadjuvant with very low toxicity.

Over the past two decades has been extensively studied the relationship between structure and activity of lipid A. Shiba with the staff sent a serious effort to study the relationship between structure and activity of synthetic lipid AndE. coliand on the development of chemical synthesis of such compounds. They first implemented the chemical synthesis monophosphoryl-lipid AndE. coli11and carefully confirmed the structure of the lipid AndE. coliusing total chemical synthesis based on the N-Troc-protected glucosamine derivative.12The same group reported on the many structural variations in the lipid AndE. coliin the sense acyl is of Rahmanov (types, number and position of the sugar skeleton)13and in the sense glycosylphosphatidyl fragment (phosphonacetyl similar to the α or β configuration at position 1).14

In 1997, they described the most effective synthesis of the precursor of lipid A.15By this time there were several non-natural analogs with modifications acyl chains16and modifications glycosylating fragment and the synthesis of the lipid A.17The group published the chemical synthesis of lipid A, isolated from Helicobacter pylori using a sophisticated method18. Publication includes triallylamine analogue of lipid a, which lacks both sugar-O-acyl substituents (O-3 and O-3'). However, in addition, in the connection there is no substitution at the 4'-O position.

Work Shiba was the starting point for the later synthesis of various lipids A. as evidence by the authors Kosma with employees recently synthesized a synthetic Tetra - and pentazocine analogues of lipid AndChlamydiato clarify the role of lipid And associated withChlamydiathe infections.19Group Biomira developed a framework for man-made synthetic lipid A, lipid containing new fragments that mimic naturally occurring originating fromE. coliandSalmonelladerivative structure of lipid A.20Khimicheskoi synthesis elipid AndP. gingivalis, triallylamine lipid containing onlyN-associated fatty acidremains and missing 4'-O-phosphate group, also reported by the authors Ogawa with employeesI.21

LPS and related compounds were studied mainly as LPS-agonists. In recent years related to lipid And compounds were studied as LPS antagonists, which may have potential as an immunosuppressive funds, and in autoimmune diseases and septicemia by deaktivirovana LPS-induced aggressive macrophages. For example, Qureshi with employees22allocated non-toxic lipid And how strong LPS antagonist ofRhodobacter sphaeroides(Rs-DPLA), and the Eisai group has developed a total synthesis of the proposed structure on their own methods23and related compounds, namely E, strong remedies against septicemia.24Existing previously described methods for the synthesis of lipid A, based on the final hydrogenolysis11-21could not be applied due to the presence of the proposed Rs-DPLA olefinic functionality. In recent years, were synthesized related Rs-DPLA and A connection.25

Molecules of lipopolysaccharides and lipid a are immunostimulating agents, because they activatetoll-likereceptor 4 (TLR4), one is about some LPS can activate TLT2, such as LPS from Porphyromonas gingivalis26. Usually TLR2 response induced only by such agents as muramylpeptide (MRM), bacterial lipopetides BLP, the composition (PGN) and lipoteichoic acid (LTA). Very interesting, the inventors of the present invention have found out currently that synthetic compounds of the invention (not justOM-174-DP,originating from naturalsources,as already described in the poster27or in a recent review28) preferably operate through TLR2 man, and not as in the case of cells of mice preferable expected TLR4 pathway. This noticeable interspecies difference (preferably TLR4 in murine cells and, rather, TLR2 in human cells) has not previously been detected.

The INVENTION

The prior art discussed above does not disclose synthetic analogues of lipid A, deprived of both sugar-O-acyl substituents (O-3 and O-3') and including 4'-O-phosphate group, or an alternative substitution at the 4'-O position. Such analogs of lipid And possess favorable properties and are useful in medicine (human). However, these analogues of lipid a can be obtained from natural sources only using complex ways, for example using process-specific hydrolysis. In addition, the data receive connections from prirodnykh sources of pharmaceutically acceptable purity is an additional problem, especially because the feedstock is mainly derived from potentially pathogenic organisms. In view of these problems, the present invention is the provision of such compounds in synthetic form. For this purpose, the present invention according to the first aspect provides a new method of chemical synthesis of β-(1→6)-linked glucosamine-disaccharides.

An additional aspect of the invention relates to a method suitable for processing products obtained using the synthetic method of the invention. Products that are processed using this method of processing, have altered the physico-chemical structure and in accordance with the preferred embodiment have an increased biological activity.

According to additional aspects of the present compound include the compounds obtained by the methods of the invention, intermediate compounds in the synthesis method, compositions comprising these compounds and to the use of these compounds in organic synthesis and/or medicine.

Is worth mentioning that the compounds of the invention preferably operate through TLR2 person.

DETAILED description of the INVENTION

An important stage in the method according to the invention is the glycosylation reaction between the compound of the formula10:

/p>

in which

R1represents a group selected from (C3-C6)alkenyl, such as C3or C4alkenyl, preferably 2-propenyl or 1-propenyl;

X represents hydrogen, a group selected from a benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl;

R0selected from R5or R2where R5chosen from:

(i) an acyl group derived from carboxylic acids with linear chain having from 2 to 24 carbon atoms, preferably hydroxyethylene group, such as 3-hydroxyethylene group, oxazoline group, such as 3-oxazoline group, aminoacyl group, such as 3-aminoaniline group;

(ii) acyloxyacyl groups, preferably 3-acyloxyacyl group, acylaminoalkyl groups, preferably 3-acylaminoacyl group, alltoall groups, preferably 3-alltoall group;

(iii) alkyloxyaryl, preferably (C2-C24)alkyloxyaryl group, alkenylacyl, preferably (C2-C24)alkenylamine group, alkyloxyalkyl, preferably (C2-C24)alkyloxyaryl group, acylaminoacyl, preferably (C2-C24)acylaminoacyl group, kanilirovannoy, preferably (C2-C24)alkenylbenzenes group, alkenylbenzenes, preferably (C2-C24)alkylamidoamines group; ancilliaries, preferably (C2-C24)alkylthiomethyl group, alkenylacyl, preferably (C2-C24)alkenylacyl group, alkenylacyl, preferably (C2-C24)alkenylacyl groups, acyl groups derived from carboxylic acids with branched chain, having from 2 to 48 carbon atoms, preferably carboxylic acids, branched at the 3-position;

where in groups (i), (ii), (iii) a hydrocarbon chain acyl may be saturated or unsaturated, hydrocarbon chain acyl, alkyl, alkenyl, quinil may be branched or linear, and optionally may be substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meaning as defined below; an amine or amine derivative-NHW, where W has the meanings given below; group-OZ, where Z is selected from (f), (g), (h), (i), (k), defined below;

and R2represents a group selected from (C1-C6)halogenated alkoxycarbonyl, such as 2,2,2-trichlorocyanuric (TROC) or 1,1-dimethyl-2,2,2-trichlorocyanuric (TCBOC);

<> with a compound of formula 7:

where R4selected from the

(a) acyl groups defined in (i), (ii) or (iii) for R5;

(b) a branched or linear alkyl, preferably branched or linear (C1-C24) alkyl group; a branched or linear alkenylphenol, preferably a branched or linear (C1-C24) alkenylphenol group, a branched or linear alkenylphenol, preferably a branched or linear (C1-C24) alkenylphenol group;

(c) a group -[(C1-C24)alkyl]-COOX, -[(C2-C24)alkenyl]-COOX or -[(C2-C24)quinil]-COOX, where X has the meanings given below;

(d) a group -[(C1-C24)alkyl]-NHW, -[(C2-C24)alkenyl]-NHW or -[(C2-C24)quinil]-NHW, where W has the meanings given below;

(e) formylalkyl group, preferably a formyl[(C1-C24)alkyl] group; formylalkyl group, preferably a formyl[(C1-C24)alkenylphenol] group; formylalkyl group, preferably a formyl[(C1-C24)alkenylphenol] group;

(f) dimethoxyphosphoryl group;

(g) group-P(O)(OY)2where Y has the meaning as defined below;

(h) the group-P(O)(OH)-O[(C1-C24)alkyl]NHW, -P(O)(OH)-O[(C2-C24)alkenyl]NHW or-P(O)(OH)-O[(C2-C24)quinil]NHW, where W has the meanings given below;

(i) group-P(O)(OH)-O[(C1-C24)alkyl], -P(O)(OH)-O[(C1-C24)alkenyl] or-P(O)(OH)-O[(C1-C24)quinil];

(j) group-P(O)(OH)-O[(C1-C24)alkyl]-COOH, -P(O)(OH)-O[(C1-C24)alkenyl]-COOH, -P(O)(OH)-O[(C1-C24)quinil]-COOH, where X has the values defined above;

(k) the group-S(O)(OH)2;

(l) a protective group selected from benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl; or (C3-C6)alkenyl, such as C3or C4alkenyl, preferably 2-propenyl or 1-propenyl;

where alkyl, Alchemilla, Alchemilla groups can be branched or linear and may be unsubstituted or optionally substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meaning as defined below; an amine or amine derivative-NHW, where W has the meaning as defined below; or a group OZ, where Z is selected from (f), (g), (h), (i), (j) (k);

and where Y is selected from hydrogen; (C3-C6)alkenyl, such as C2or C3alkenyl, preferably 2-propenyl or 1-propenyl; group selected from benzyl or substituted benzyl, the such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl;About-xylylenebis group;

andwhere W is selected from hydrogen; benzyloxycarbonyloxy group or 9-fluorenylmethoxycarbonyl;

and where R6represents a group selected from trichloroacetimidate, fluoride, chloride, bromide, and X and R2have the meanings given above.

The reaction may be carried out in accordance with the General method of glycosylation is known in the art, such as the method described in Angew. Chem., Int. Ed. Engl., (1986), 212. In this method using dichloromethane as solvent and a catalytic amount of acid, such as trimethylsilyltrifluoromethane. When used this way, receive only β-disaccharide according to the formula11h.

in which R1, R2, R4, R0and X have the meanings described above. The bond connection, consolidating OR1indicates that possible as α and β anomer.

R5can be selected from acyl group defined in (i), or alternative branched acyl group defined in (ii), (iii). The acyl group may be selected from the group comprising acyloxyacyl, acylaminoacyl, alltoallv, (C1-C24)alkyloxyalkyl, (C1-C24)acylaminoacyl and (C1-C4 )alkylthiomethyl group. (Cn-Cn), where n is an integer, such as (C1-C24) and (C2-C24used herein, means a saturated or unsaturated hydrocarbon chain to which this symbol may contain the number of carbon atoms in the range, such as, respectively 1-24 and 2-24 carbon atoms, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 carbon atom. Acyl, alkyl, Alchemilla and Alchemilla hydrocarbon chain acyl and acyl derivatives defined in (i), (ii) or (iii)may each, individually include a number from 1 to 50 carbon atoms, such as from 2 to 48 carbon atoms, including 1 to 24 carbon atoms, such as 2-24 carbon atoms, in particular 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 carbon atom. In (C2-C24) alkyloxyaryl group, for example, alkyl hydrocarbon may include from 2 to 24 carbon atoms, and the hydrocarbon chain acyl fragment may include from 2 to 24 carbon atoms.

Hydrocarbon chain acyl groups may be saturated or may contain one or more unsaturated carbon double or triple bonds. In addition, the hydrocarbon chain of the acyl, alkyl, alkenyl and quinil can be branched or linear and may be optionally substituted by one or more groups independently selected from halogen, such as fluorine,chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meanings given above; an amine or amine derivative-NHW, where W has the meanings as defined above; group-OZ, where Z is selected from (f), (g), (h), (i), (j),(k), defined above.

If acyloxyacyl group two acyl groups linked through an oxygen atom, in the case acylaminoacyl group through the NH group, and in the case alltoall group through a sulfur atom. (C1-C24)alkyloxyaryl, (C1-C24)acylaminoacyl and (C1-C24)allylthiourea group can be obtained from the corresponding hydroxy fatty acids.

Acyl group is preferably substituted at the 3-position and are such as 3-acyloxyacyl, 3-acylaminoacyl and 3-alltoall group. The same applies to the above-mentioned (C1-C24)alkyl equivalents.

Preferably the members of the group R5include one or two acyl fragment, preferably selected from fatty acid residues, of residues of hydroxy fatty acids and residues of hydroxy fatty acids. When acyloxyacyl group is a 3-acyloxyacyl group, data acyl fragments preferably comprise the remainder of the 3-hydroxy fatty acid or a related ester linkage group balance 3-oxo fatty acids. Typical examples of anilox the acyl groups are 3-hydroxy(C 4-C24)fatty acid-atilov, which are connected ester bond with the 3-hydroxy group (C1-C24)-carboxylic acid. Preferably acyloxyacyl group is a 3-hydroxy(C8-C18)-fatty acid-acyl, which is linked ester bond with the 3-hydroxy group (C10-C18)fatty acids. Such acyloxyacyl groups present in the lipid component And gram-negative bacteria, such as Escherichia coli, Haemophilus influenzae, Campylobacter jejuni, Rhodocyclus gelatinosus, Chromobacterium violaceum, Neisseria meningitides, Salmonella Minnesota.

In one group of preferred glucosamine-disaccharides according to the invention acyloxyacyl group selected for R5is 3-hydroxy-C14-fatty acid-acyl, ester linked connection with the 3-hydroxy group of the C12fatty acids, this acyloxyacyl group N2'-position. In yet another preferred glucosamine-disaccharide according to the invention acyloxyacyl group selected for R5is 3-hydroxy-C14-fatty acid-acyl, ester linked coupling with 3-hydroxy-group C14-fatty acids, and acyloxyacyl group is N-2' position.

In yet another preferred glucosamine-disaccharide according to the invention acyloxyacyl group selected for R5is 3-hydroxy-C14fatty acid is TA-acyl, associated ester coupling with 3-hydroxy-group C12fatty acids, this acyloxyacyl group in the N-2 position. In yet another preferred glucosamine-disaccharide according to the invention acyloxyacyl group selected for R5is 3-hydroxy-C14-fatty acid-acyl, ester linked coupling with 3-hydroxy-group C12fatty acids, with acyloxyacyl group and in the N2-position, and N2'-position.

When the compound of the invention includes a chiral center, the invention covers all R - and S-enantiomers and any racemic mixture.

Other choices for R5may be acyl group or acyloxyacyl group.

According to the second group of disaccharides according to the invention the acyl group is a 3-hydroxy (C4-C24)-fatty acid, preferably 3-hydroxy (C10-C18)-fatty acid. 3-hydroxy-group of such fatty acids can be protected by a group X, as defined previously. In the preferred disaccharides according to the invention the acyl group is 3-hydroxy, C14fatty acid, N2-position or N2'-position.

However, R5can also be acyloxyacyl group defined here above and including the number of 3-hydroxy-(C4-C24)fatty acid-acyl, which is related ester linkage is with the 3-hydroxy group (C 1-C20) carboxylic acid, preferably 3-hydroxy-(C8-C18)fatty acid-acyl, ester linked connection with the 3-hydroxy group (C10-C18) fatty acids. More preferred is a disaccharide in which R5in the N2-position is a 3-hydroxy-C14-fatty acid-acyl, ester linked connection with the 3-hydroxy group of the C12fatty acids or C16-fatty acids, and in which R5in N2'-position is 3-hydroxy-C14-fatty acid-acyl, ester linked connection with the 3-hydroxy group of the C12fatty acids, C12fatty acids or C14-fatty acids.

According to a preferred embodiment of the first group of R5selected from groups (i), as defined, and the second group of R5selected from groups (ii) or (iii)defined in claim 1 claims wherein the group R5in the N-2 position selected from (i). In alternative embodiments, the two groups R5selected identically or differently from the subgroup (i), or both selected identically or differently from the subgroups (ii) or (iii).

It is noted that in the group R5acyl groups and/or acyl and alkyl group may be linked.

In this description, the term “residue of a fatty acid” means essentially hydrophobic chain of the C2-C30atoms which can be the t to be linear, branched, saturated, mono - or polyunsaturated, having one or more heteroatoms, such as nitrogen, oxygen, sulfur, and which may be substituted by one or more substituents, such as hydroxyl, oxo, acyloxy, alkoxy, amino, nitro, cyano, halogen, sulfhydryl, provided that the biological activity is essentially not experience negative impacts. The sample residue substituted fatty acids (including amide-linked Deputy) described by the authors Onozuka, K. et al. in Int. J. Immunopharmac, Volume 15, pages 657-664 [1993]).

R4can be selected from (a)to(l)as defined above. Alkyl, Alchemilla, Alchemilla chain in these substituents for R4may be branched or linear and may be unsubstituted or optionally substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meanings given above; an amine or amine derivative-NHW, where W has the meanings given above. For groups (a), (b), (c), (d), (e) an optional substituents may further include a group-OZ, where Z is selected from (f), (g), (h), (i), (j), (k). Preferably R4selected from (f), (g), (h), (i) or (j), more preferably from (g). Preferably the group (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) include from 1 to 50 carbon atoms, for example the t 2 to 24 carbon atoms.

At a later stage a number (1-C6) halogenated alkoxycarbonyl protective groups R2hydrolytically removed from the compounds of formula11h. In this description, the number denotes one or more, if not specified otherwise. Preferably, all of the group R2compounds of the formula11hwere removed. If R5selected R0,then the compound of the formula11hwill include one group R2. If R0selected as R2then , the compound of the formula11hwill include two groups of R2and it would be preferable to delete both of these groups. Group R2can be removed by any appropriate means known in the art. Specialist it is known that (C1-C6) halogenated alkoxycarbonyl protective group, such as Troc, can be removed using zinc-copper couple in acetic acid and water.

If R5selected R0then you will obtain the connection formula12A.

in which R1, R4, R5and X have the meanings defined above. If R0selected as R2in the formula11hthen will preferably the compound of the formula12b.

where R1, R4and X have the meanings defined above.

To make the Noah amino group of compounds of the formula 12aor12bjoin the group R5. This can be achieved by reaction of compounds of formula12Aor12bwith (activated) carboxylic acid corresponding to the specified group R5. The reaction can be carried out by any method known in the art, such as using a condensing agent such as isobutylparaben or 1 isobutoxy-2-isobutylketone-1,2-dihydroquinoline or carbodiimide. In the reaction of the compound12A(activated) carboxylic acid corresponding to the specified group R5may include group R5identical or different groups R5compounds of the formula12A.

The reaction of the compound of the formula12Aor12bwith (activated) carboxylic acid corresponding to the specified group R5that results in the formation of compounds of the formula13:

in which R1, R4, R5and X have the meanings previously defined. Group R5may be the same or different. Whether the group R5connection13the same or different, may depend on whether the reaction of the compound12Aor connection12band from nature (activated) carboxylic acid used in the reaction. If the connection is used12byou can choose the group R 5(activated) carboxylic acid other than group R5connection12b. In this case the group R5connection13will be different. However, the group R5(activated) carboxylic acids may also be identical to the group R5connection12b. And clearly it is obvious that in this case the group R5connection13will be identical. If the connection12Asubjected to reaction with one (activated) carboxylic acid group, R5connection13will be identical. However, you can also use combinatorial chemistry, and subjecting the reaction of the compound12bwith a number of distinguished (activated) carboxylic acids. This will be a mixture of compounds according to General formula13in which groups R5are the same or different. The person skilled in the art clearly understands that the number of different compounds of General formula13and their ratio in the mixture will depend on the number of different (activated) carboxylic acids used in the reaction, and from their ratio. Preferably, at least one of R5was selected from branched acyl group defined in (ii), (iii). More preferably as branched acyl group is selected the group R5attached in N2'-position.

Palacete the ü formula 14:

in which R4, R5and X have the meanings defined above, is formed by removing groups of R1of the compounds of formula13. Unprotect (C3-C6)alkenylphenol group can be achieved by any method known to the expert. For example, (C3-C6)Alchemilla group can be removed by a two-step transformation. If (C3-C6)alkenylphenol group is, for example, 2-propenyl first allyl group13can be isomerized 1-propenyl by processing the hydrogen-activated iridium catalyst, such as industrial available hexaflurophosphate ([bis(methyldiphenylphosphine)]-(1, 5cyclooctadiene)iridium(I)) in a polar solvent such as tetrahydrofuran (Synthesis, (1981), 305-308). 1-protanilla group can then be chipped off the water source of iodine such as iodine orN-bromosuccinimide. (J. Chem. Soc., Chem. Commun., (1982), 1274). By analogy can be removed in different ways to group R1.

Connection13and Polyacetal formula14are important intermediate products in the synthesis method according to the invention. Depending on the reactions carried out with compounds13and14,and resulting from their intermediates can get a large number of different protected β-(1→6)-linked glucose is in disaccharides with different substituents R 8in O-1 status. Data β(→6)-linked glucosamine-disaccharides can be represented by the General formula15:

where R4, R5and X have the meanings previously defined, and R8selected from (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k), previously defined for R4.

In one of the embodiments of the method of synthesis of the invention the free hydroxyl group of compound14can be phosphorylated by any known specialist way. This can be used industrially available tetraethylpyrophosphate in the presence of a suitable base in a polar solvent. The base may be selected from bis(trimethylsilyl)amide lithium, and the solvent may be selected from tetrahydrofuran. Phosphorylation connection14results in the connection15A

Phosphorylation may be useful for producing compounds having O-1 position substituents selected from (g), (h), (i) or (j)defined for R4. If necessary, the phosphate group received in connection15A,may further be modified.

In yet another embodiment of a free hydroxyl group of compound14can sulfochromate any well-known specialist. The sulfation connection14results in a compound of the formula15b:

In yet another embodiment of the method according to the invention additionally provides for the reaction of the free hydroxyl group of compound14with (activated) carboxylic acid of formula R8OH, in which R8selected from (a), a previously defined for R4. The reaction may be carried out by any known professional way, such as in the presence of a condensing agent, such as isobutylparaben or 1 isobutoxy-2-isobutylketone-1,2-dihydroquinoline, or carbodiimide with the formation of compound15C:

where R4, R5and X have the meanings defined above, and R8selected from (a), a previously defined for R4and R8can be in α - or β-configuration, and preferably in the β-configuration.

In another embodiment the group, which can function in the subsequent reaction as a leaving group, such as trichloroacetimidate group, combined with the free hydroxyl group of the compound14. This can be done by any known specialist way, for example, through reaction of the compound14with trichloroacetonitrile in the presence of an inorganic base such as cesium carbonate or potassium carbonate, in a polar solvent, preferably an aprotic polar solvent such as dichloromethane. Dan is the first reaction of the compound 14results in the compound of the formula24:

Connection24may be subjected to reaction with an organic molecule R8OH replacing trichloroacetimidate group group R8. R8can be selected from (b), (c), (d), (e), defined for R4.

The reaction acetimidate group with organic alcohol. It can happen in a polar solvent, preferably an aprotic polar solvent, such as dichloromethane, in the presence of catalytic amount of acid, such as trimethylsilyltrifluoromethane, and can be carried out by analogy with the method described in Angew. Chem., Int. Ed. Engl., (1986), 212. The reaction of the compound24with a group R8results in a compound of the formula15d:

where R4, R5, R8and X have the meanings defined above, and where R8can be in α - or β-configuration, and preferably in the β-configuration.

Connection13,15A,15b,15Cand15dit may further be subjected to the reaction in order to remove any protective group selected from X, Y, W, different from N. Removing the protective groups can be achieved in accordance with known techniques methods.For example, the benzyl protective group can be removed by hydrogenolysis in the presence of the tvii noble metal, such as palladium-on-charcoal grill. Allyl group and similar groups can be removed, as discussed above for removal of the allyl group from a compound of13.Remove 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzidine, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxyaniline, or phenyl, or 4-metoksifenilny, or 3,4-dimethoxyaniline, or 2.5-dimethoxyaniline, or 2,3,4-trimethoxyphenyl, or 3,4,5-trimethoxyaniline groups can be carried out using oxidative cleavage, such as by using dichlorodicyanoquinone (DDQ) or ammoniumnitrate cerium (CAN).About-Killenemy group and benzyloxycarbonyl group can be removed by hydrogenolysis in the presence of a noble metal such as palladium-on-charcoal grill. 9-Fluorenylmethoxycarbonyl can be removed by a base, such as piperidine, morpholine. Obviously it is clear that the various protective groups can be removed independently. Therefore, before deleting X can be removed any protective group present in R8.

Reactive group originally present in the R8or after removal of the protective group, can be further subjected to reaction before deleting (optional) protective groups. If R8includes a number of free hydroxyl groups, can form complex EF the market, including phosphate and sulfate esters, and ethers using known methods. The free hydroxyl group can also be oxidized by known methods to obtain carboxylic acid or ketone. If R8includes a number of groups, carboxylic acid by known methods can form esters or amides. If R8includes a number of free amine groups, using known in the art methods can form amides. If R8includes a number of unsaturated carbon bonds, they are known methods can be subjected to reaction with osmium tetroxide with obtaining α,β-gidroksilirovanii group. Free hydroxyl groups, such as α,β-gidrauxilirovannaya group may optionally be subjected to reactions before removing the protective groups.

In addition, the phosphate group can metiliruetsa by known methods such as reaction with CH2N2. It should be noted that such methylation using CH2N2can occur before or after removal of the protective groups in the β-(1→6)-linked glucosamine-disaccharides, including a protective group selected from X, as defined above.

In an alternative embodiment of the method of the invention the protective group of the compound14removed by known methods, such as described above.

Additional al the alternative embodiment of the method of the invention unsaturated bond (C 3-C6)alkenylphenol group connection13such as3or2alkenyl, preferably 2-propenyl or 1-propenyl, hereroense in the appropriate alkyl.

In yet another alternative embodiment of the method of the invention (C3-C6)Alchemilla connection group13is in 2-propenyl, and unsaturated bond 2-propenyloxy group undergoes reaction with osmium tetroxide according to the method of obtaining α,β-gidroksilirovanii group. The free hydroxyl group such α,β-gidroksilirovanii group can be further subjected to reaction before removing the protective groups.

Obviously it is clear that by using the synthesis method according to the invention may produce a large number of β-(1→6)-linked glucosamine-disaccharides of formula 1:

where R4', R5' and R8' have the meanings according to the previously defined for R4, R5and R8where any Y or W are N, and where a value of R8' additionally includes N.

Connection7that is involved in the process according to the invention can be obtained by attaching the leaving group selected from trichloroacetimidate, fluoride, chloride, bromide, to the free hydroxyl group of compounds of formula6:

where R2R 4and X have the meanings previously defined. This can be implemented using any suitable technique known in the art. For example, treatment of compounds of formula6trichloroacetonitrile, preferably in the presence of a base, preferably an inorganic base such as cesium carbonate or potassium carbonate, in a polar solvent, preferably an aprotic polar solvent such as dichloromethane. Protection of chlorine and bromine may be carried out by the reaction of acetic anhydride in a solvent such as pyridine and subsequent reaction with gaseous respectively HCl or HBr in acetic acid. Protection of fluoride may be performed by reaction with acetic anhydride and subsequent reaction with the TRIFLUORIDE diarylamino (DAST).

The compound of the formula6can be obtained by removing the known methods group, R1of the compounds of formula5:

where R1, R2, R4and X have the meanings previously defined. For example, removing the protection of the allyl group can be achieved using a two-stage transformation. First, the allyl group can be isomerized 1-propenyl by processing the hydrogen-activated iridium catalyst, such as industrial available hexaflurophosphate ([bis(methyldiphenylphosphine)]-(1, 5cyclooctadiene)iridium(I) in polarn the m solvent, such as tetrahydrofuran, according to the method described inSynthesis, (1981), 305-308. Protanilla group can then be chipped off water source of iodine such as iodine or N-bromosuccinimide. (J. Chem. Soc., Chem. Commun., (1982), 1274).

The compound of the formula5can be obtained using a number of different reactions depending on the choice of the group R4. These reactions may proceed from compounds of the formula4:

where R1, R2and X have the meanings defined above. Based on the connection4to the free hydroxyl group of this compound as R4you can add a number of different substituents. These substituents can join by General methods known in the art.

If R4selected from (f),(g), (h), (i) or (j)the method according to the invention may include phosphorylation in suitable reaction conditions of the free hydroxyl group of the compounds of formula4:

where R1, R2and X have the meanings previously defined. This may be done, for example, by reaction with phosphoramidite reagent, such as diaryl-N,N-dialkylphosphinate or diallyl N,N-dialkylphosphinate, preferably diallyl N,N-diisopropylphosphoramidite, in the presence of a condensing agent, such as [1H]tetrazole, in a polar solvent, before occhialino aprotic polar solvent. In this reaction initially formed postit, which can subsequently be oxidized to the phosphate, for example, in the presence of aromatic nadarbazevi acid, such as m-chloroperoxybenzoic acid.

If R4selected from (k), the method according to the invention may include sulfation in suitable conditions, the reaction of the free hydroxyl group of the compounds of formula4

where R1, R2and X have the meanings defined above. This may be done, for example, by reaction with a complex of sulfur trioxide, for example trimethylamine complex with sulfur trioxide, in a polar solvent such as dimethylformamide (DMF).

If R4selected from (1), the method according to the invention may include the reaction of the free hydroxyl group of the compounds of formula4:

where R1, R2and X have the meanings defined above, with a compound suitable for attaching donor protective group to the specified free hydroxyl group of compounds of formula4. Such protective group of the compound of the donor may be preferably selected from benzyl-2,2,2-trichloroacetimidate or substituted benzyl-2,2,2-trichloroacetimidate, such as 4-methoxybenzyl-2,2,2-trichloroacetimidate, 3,4-dimethoxybenzyl-2,2,2-trichloroacetimidate, 2.5-dimethoxybenzyl-2,2,2-Tr is chloracetamide, 2,3,4-trimethoxybenzyl-2,2,2-trichloroacetimidate or 3,4,5-trimethoxybenzyl-2,2,2-trichloroacetimidate. Alternative protective group can be derived (C3-C6)alkenyl-2,2,2-trichloroacetimidate, such as3or4-2,2,2-trichloroacetamide, preferably 2-propenyl - 2,2,2-trichloroacetimidate or 1-propenyl-2,2,2-trichloroacetimidate. The reaction is preferably carried out in polar solventand/or in the presence of an acid catalyst, such as triftorbyenzola tin II or triftormetilfullerenov acid.

If R4selected from (a), the method according to the invention may include the reaction of the free hydroxyl group of the compounds of formula4:

where R1, R2and X have the meanings defined above, with carboxypropyl (activated) carboxylic acids of formula R4OH, in which R4selected from (a), defined above. The reaction is preferably conducted in the presence of a condensing agent, such as isobutylparaben or 1 isobutoxy 2-isobutylketone-1,2-dihydroquinoline or carbodiimide.

If R4selected from (b), (c), (d) or (e), the method according to the invention may include the reaction of the free hydroxyl group of the compounds of formula4:

where R1, R2and X have the value is, defined above, with a derivative 2,2,2-trichloroacetimidate-activated Olkiluoto alcohol corresponding to the specified group selection R4(b), (c), (d) or (e). The reaction is preferably carried out in polar solvent and/or in the presence of an acid catalyst, such as triftorbyenzola tin II or triftormetilfullerenov acid. Experts clearly understand that 2,2,2-trichloroacetimidate-activated alcohol derivative matching the specified selection (b), (c), (d) or (e) the group R4may be alkyl-2,2,2-trichloroacetimidate, such as, for example, propyl-2,2,2-trichloroacetimidate, when R4selected from (b) as the alkyl group. By analogy with this, you can choose other 2,2,2-trichloramine-activated alcohol derivative, corresponding to the choice (b), (c), (d) or (e), such as alkenyl-2,2,2-trichloroacetimidate, quinil-2,2,2-trichloroacetimidate.

Different substituents group R4can similar to the substituents of group R8to contain reactive groups such as hydroxyl group, amino group, carboxypropyl or carbon bonds, such as double bonds. Such reactive group in the compound5can be further modified, for example, by a reaction selected from the formation of ester, amidation, oxidation, hydrogenation or α,β-g is taxilane by osmium tetroxide.

Connection4can be obtained by using restorative disclosure ring benzylidene group of compounds of formula3:

where R1, R2and X have the meanings previously defined, and R3represents a group selected from an aromatic hydrocarbon, such as phenyl or 4-metoksifenilny, or 3,4-dimethoxyphenyl, or 2.5-dimethoxyaniline, or 2,3,4-trimethoxyphenyl, or 3,4,5-trimethoxyphenyl group. The reaction can be carried out by any method known in this field, such as using a hydride such as trimethylamine-bananowy complex and a Lewis acid such as aluminum chloride, in a polar solvent such as THF. This method is described in theCarbohydrate Research, (2003), 697-703 andTetrahedron Lett. (2000), 41, 6843-6847.

Connection10that in the method of the invention is introduced into the reaction together with the connection7with the formation of compound11can be obtained from a compound of9:

where R1and X have the meanings previously defined. For connection10the free amino group of the compound9acelerou by the reaction with (activated) carboxylic acid of formula R5OH, where R5has previously specified values. The method may be carried out under conditions known Spa is elistan, for example, a mixed anhydride, such as a mixed anhydride derived from (R)-3-benzyloxyethanol acid, described inBull. Chem. Soc. Jpn, (1987), 2197-2204, and alkylphosphonate, such as isobutylparaben.

Connection9can be formed by hydrolytic cleavage of known methods group, R2compounds of the formula8:

where R1, R2and X have the meanings defined above. For example, trichlorocarbanilide protective group (Troc) can be removed using zinc in acetic acid.

The compound of formula 8 can be restorative ring opening in a suitable reaction conditions benzylidene group of compounds of formula3:

where R1, R2, R3and X have the meanings defined above. This can be used any known method, such as using a hydride such as dimethylamine-bananowy complex as a reagent and a Lewis acid such as boron TRIFLUORIDE, in a polar solvent such as dichloromethane.

The compound of the formula3can be obtained by the reaction of compounds of the formula2:

where R1, R2, R3and X have the meanings defined above, with a compound suitable for introduction of protective the groupings based on the free carboxyl group of the compounds of formula 2. The connection, which is the donor of the protective group, preferably selected from benzyl-2,2,2-trichloroacetimidate, 4-methoxybenzyl-2,2,2-trichloroacetimidate, 3,4-dimethoxybenzyl-2,2,2-trichloroacetimidate, 2.5-dimethoxybenzyl-2,2,2-trichloroacetimidate, 2,3,4-trimethoxybenzyl-2,2,2-trichloroacetimidate or 3,4,5-trimethoxybenzyl-2,2,2-trichloroacetimidate. The reaction is preferably carried out in polar solvent and/or in the presence of an acid catalyst, such as triftorbyenzola tin II or triftormetilfullerenov acid. Suitable methods are described in theJ. Chem. Soc., Chem. Commun., (1981), 1240-1241). It is interesting to note that there wasn't any reaction when using the techniques described inTetrahedron Letters, (2001), 7613-7616 orTetrahedron Lett. (2000), 41, 6843-6847 for connection3and allocated only to the original substance2. Believe that these articles, as such, do not open the link3. Connection2the result was, as described inLiebigs Ann. (1996), 1599-1607.

According to an additional aspect of the invention relates to a method of processing glucosamine-disaccharides, preferably β-(1→6)-linked glucosamine-disaccharides. This method can be used for processing of compounds obtained using the synthesis method according to the invention. The method includes

(i) mixing a solution of compound f is rmula 1:

where R'4, R'5and R'8have the meanings defined above, with a solid resin with reversed phase under conditions suitable for binding at least part of the compounds of formula 1 with the solid phase;

(ii) removing the liquid phase and washing the solid phase of the drilling liquid comprising an aqueous phase, optionally superyoung at pH 6-9, preferably 7-8, and most preferably of 7.3 to 7.7, and the organic phase, which is mixed in a ratio of from 15:1 to 5:1, preferably 9:1 (vol./vol.);

(iii) removing drilling fluid and elution at least part of the connection 1 associated with the solid phase, eluting fluid comprising an aqueous phase and an organic phase, which is mixed in a ratio of between 1:15-1:5, preferably 1:9 (vol./vol.);

(iv) collecting the eluting liquid, including a certain number of connections1and optionally removing the organic phase from an eluting liquid.

In the preferred embodiment, the method further includes bringing the pH of the eluting liquid, including a certain number of compounds of the formula1to a pre-selected pH, preferably pH 6 to 9, more preferably 7-8 and most preferably a pH of 7.3 to 7.7. When the pH value products are the most stable.

It has been unexpectedly found that the processing of the connection forms of the crystals 1this method results in compounds with enhanced biological activity compared to the starting material.

Compounds of the formula1can contact the resin with reversed phase in a polar solvent such as2-C3organic alcohol, optionally mixed with water, such as a mixture of water and 2-propanol, mixed in a ratio of between 15:1 to 5:1, preferably 9:1 (vol./vol.). Resin with reversed phase can be resin VYDAC C18 or any other suitable resin with reversed phase.

The organic phase drilling fluids and/or eluting fluid may include an organic solvent such as a polar organic solvent, for example With2-C3organic alcohol.

The compound of formula 1 may be provided in a solvent, which is suitable for the reaction in which the protective groups are removed by hydrogenolysis. An example of such a solvent is tetrahydrofuran (THF). Compounds according to the invention can be processed in the processing method according to the present invention directly after their synthesis method of the invention. However, it is preferable to first purify compounds of the invention. Cleaning can be carried out by known methods such as the use of chromatography with reversed phase, preferably ion-pairs is Oh chromatography with reversed phase, such as using tetrabutylammonium phosphate.

Compounds obtained by the synthesis method according to the invention, represent a β-(1→6)-linked glucosamine-disaccharides formula1:

where R'4, R'5and R'8have the meanings given above. According to one aspect of the invention relates to these compounds. Preferred compounds of the invention represented in p of the claims and the attached figures. Qualified clearly understands that these compounds may exist in ionized form. The present invention relates also to a (pharmaceutically acceptable) salts such ionized species, such as salts of sodium, potassium or ammonium.

Many of the compounds according to the invention are new, with regard to their chemical structure. In addition, the compounds according to the invention differ from the compounds with known chemical structure, but originating from natural sources, the fact that they are free from any biological impurities such as traces of nucleic acids and/or peptides, and/or carbohydrates. Although they are present in small quantities, the presence of tracks of data of biological impurities is considered to be unacceptable for pharmaceutical products. The presence of b is ideological impurities may be determined by known methods, for example, selected from immunological methods or PCR methods. The aim of these methods, in particular, may be the detection of cellular components of gram-negative bacteria such asE. coli.

In another aspect the invention relates to certain new intermediate compounds of the method according to the invention. In particular, according to this aspect of the invention relates to compounds3, 7, 8, 10A, 11, 11b, 12b, 12A, 13, 14. The preferred embodiment of this aspect of the invention relates to compounds3b, 7b, 8b, 10b, 11a, 11C, 12C, 12d, 13b, 14b. These compounds can be used as intermediate compounds, including source materials, the method of synthesis of asymmetrically or symmetrically substituted β-(1→6)-linked glucosamine-disaccharides.

Compounds of the formula1useful medicine for the treatment of warm-blooded animals such as mammals, including humans. In particular, the compounds of the invention can be used in the treatment of immune disorders, such as immune-related disorders overproduction inflammatory cytokines or reduced production of inflammatory cytokines. Inflammatory cytokines can be produced activated T lymphocytes, monocytes, or cells present antigens and may belong to the group consisting of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IFN-γ, NF-α, or MCP-1. Painful condition that can be treated with compounds according to the invention, include including cancer, asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, diabetes, rheumatoid arthritis and others in which positive regulation of inflammatory cytokines increase and/or decrease. The fact that the compounds of the invention mainly operate through TLR2 person may be of clinical interest in the treatment of cancer (Garay et al., 2007). Cancers that can be treated by the compounds of the invention include colorectal cancer, breast cancer and melanoma.

Compounds of the invention may additionally reduce the secretion of histamine mast cells. They are useful in the treatment, including the improvement or relief of conditions under which involved excessive secretion of histamine mast cells. Such conditions may include allergic reactions, including hay fever (hay fever), allergic reactions caused by stinging or biting insects such as bees and wasps, or allergic reactions to food allergens.

Due to their stimulating effect on the immune system of the compounds of the invention useful as vaccine components.

Compounds of the invention can be administered to a subject in need of it, in the form of compositions, it is interesting in combination with a pharmaceutically acceptable carrier and/or other excipients oral, parenteral, intravenous, intratumoral, subcutaneous, rectal, local, or mucosal (mucous membranes) by. The introduction of peritoneal, subcutaneous, oral, intranasal, sublingual, intramuscular, or by aerosol. The selection of the suitable intervals dosages of the compounds of the invention will depend on the specific activity of selected compounds of the condition of the subject being treated disorders. A qualified technician will be able to choose the appropriate spacing of the doses given his General knowledge and his experience in this field. In conditions such as asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, diabetes or rheumatoid arthritis, appropriate intervals doses for men can be from 0.01 to 50 mg/sqm

Further aspects of the invention relate to methods of using and/or synthesized new and non-obvious (intermediate) compounds of the invention. Due to the usefulness and/or obtain new and unobvious compounds these methods are new and match the criterion of inventive step. These processes can be useful in the synthesis of asymmetrically or symmetrically substituted 1,6-β-disaccharides, including compounds of the invention.

Now the invention will be further illustrated by reference to the following the e following examples and accompanying figures, of which

Figure 1 shows the structure of lipid AndE. coliand OM-174-DP®;

Figure 2 gives a General overview of embodiments of the method of synthesis according to the invention;

Figure 3 provides an overview of the preferred embodiment of the method of synthesis according to the invention;

Figure 4-24 give an overview of the various alternative routes of synthesis for the preparation of compounds of formula 1 and/or their immediate predecessors;

Fig is a graph showing NO production by mouse macrophages in response to connection of the invention;

Fig presents experimental results illustrating the increased biological activity of β-(1→6)-linked glucosamine-disaccharides for the treatment by the method according to the invention.

In these figures the group R0, R1, R2, R4, R5, R6, R8, R4', R5', R8', X, Y and W have the meanings given in the claims and description for the various compounds. Bn denotes benzyl group, allyl denotes allyl group and Ipr is isopropyl group.

Molecular structure represented in figure 1, correspond to the lipid AndE. coliand OM-174-DP®as indicated. In figure 1, the designation 0-3 and 0-3'.

Figure 2 gives a General overview of embodiments of the method of synthesis according to the invention. From the above descriptions show is about clear, connection7you can enter into reaction with the compound10getting connection11hwhere R0is R5or an alternative connection8getting connection11hin which R0selected from R2. In the embodiment shown in figure 2, the connection7is introduced into the reaction with compound 10. This opens up the possibility of introducing various R5substituents in the molecule, which thus can asymmetrically replaced. Symmetrically substituted compounds can be obtained by the reaction of compounds7connection8and subsequent reaction of the obtained compound11hin which R0selected from R2for connection12b. Connection12bthe sequence of reactions may be carried out in a similar way, to obtain compounds which are symmetrically substituted N-2 and N-2' position.

Figure 3 gives a General overview of a preferred embodiment of the method of synthesis according to the invention. In this embodiment of the method according to the invention the final product is asymmetrically substituted OM-174-DP®.

Figure 4 shows the first possible reaction of phosphorylation of the free hydroxyl group of polyacetale formula14. In this reaction the connection14is introduced into the reaction tetraethylpyrophosphate in the presence of bis(trim ililil)lithium amide (LiHMDS). The reaction can take place in a polar solvent such as THF.

Figure 5 shows an alternative phosphorylation reaction of a free hydroxyl group polyacetale formula14. In this reaction the connection14is introduced into the reaction with dialkyl N,N-aminobutiramida phosphoramidite in the presence of a condensing agent, such as [1H] tetrazole. The reaction may proceed in a polar solvent, preferably an aprotic polar solvent. The reaction is first formed postit. This postit subsequently oxidized in a protected phosphate in the presence of aromatic nadarbazevi acid, such as m-chloroperoxybenzoic acid.

6 shows an exemplary formation of complex phosphodiester by the reaction of phosphonate-protected organic aminosterol formula HO-(C1-C24)-NHW. After the formation of the phosphodiester bond of the protective group W may be removed together or separately from the protective group X. When the group W is removed, while group X remain in the molecule, the free amino group can be further modified, for example, through the formation of amide with an organic acid.

Fig.7 shows an additional alternative reaction modification of the phosphate group. In this reaction, the phosphate group metiliruetsa using CH2N2. The reaction, shown in Fig.7, is on the forefront of the Le, in which none of the phosphate groups are not protected. Obviously it is clear that when a secure one of the phosphate groups, such as 1-O-phosphate group, or a 4'-O-phosphate group, such protected phosphate group will not metiliruetsa in the reaction. This opens up the possibility of choosing any modification or both phosphate groups.

Fig shows the reaction of sulfation connection14. In the reaction of the compound14is introduced into the reaction with a complex of sulfur trioxide.

In order to obtain compounds having a hydrocarbon chain attached directly to the 1-position, there are a number of possibilities. Some of them are shown in Fig.9. First, you can gidrirovanii (C3-C6)alkenyl attached to the 1-position in the compound of the formula13in the corresponding alkyl. 1-allyl group hereroense in various group. Secondly, you can attach a hydrocarbon chain first activating the hydroxyl function in the 1-position of compound 14 and the subsequent reaction of the activated groups with organic alcohol. Activating the free hydroxyl group of compound14can be achieved by the reaction of compounds14with trichloroacetonitrile in the presence of an inorganic base such as cesium carbonate or potassium carbonate. The reaction can occur in a polar solvent, preferably the aprotic polar solvent, such as dichloromethane. When the connection 14 is introduced into the reaction trichloroacetonitrile in such conditions, will produce the compound of the formula24.The reaction of the compound24with organic alcohol represented by the General formula ROH figure 9, will result in a compound having a hydrocarbon chain R attached in the 1-position.

Figure 10 shows an additional example of the reaction of the compound24with organic alcohol. Figure 10 connection24is introduced into the reaction with organic diola having 1-24 carbon atoms, in which one of the hydroxyl groups is protected by a group X, preferably PMB. Monogamistic organic diol represented by the General formula BUT-(C1-C24)-OH. Figure 10 additionally shows that after joining monoamino organic diol to About-1-position of the protective group X monoamino organic diol can be selectively removed, if it is different from group X in carbohydrate. After selective removal of the protective group X monoamino organic diol free hydroxyl group may be further modified, for example, by its phosphorylation by the methods described above. Obviously it is clear that the phosphate group can be further modified, as described above.

11 shows a reaction scheme similar to the scheme of Figure 10. However, in figure 1 after removal of the protective group X monoamino organic diol hydroxyl group is subjected to sulfation.

Alternatively, as shown in Fig, after removal of the protective group X monoamino organic diol hydroxyl group can be oxidized in carboxypropyl. It is clear that carboxypropyl may be further modified, for example, through education amide or ether complex.

Fig shows the reaction sequence, which makes possible the introduction of a hydrocarbon chain having α,β-dihydroxytoluene. This reaction scheme organic alcohol having an unsaturated carbon-carbon double bond, is introduced into the reaction with the compound24. The length of the hydrocarbon chain linking the hydroxyl group and an unsaturated bond shown organic alcohol is changeable and is composed of n carbon atoms, where n can vary between 1 and 24. Although shown unsaturated bond of the organic alcohol is present at the end of organic alcohol, obviously it is clear that it can also be present in any location in the hydrocarbon chain.After the accession of organic alcohol, 1-On-link location24unsaturated bond can undergo reaction with osmium tetroxide to attach the α,β-dihydroxy to the double bond. Hydroxyl group introduced by this method, may further be modified. For example, through education phosphate, as the show is but Fig, or alternatively through the formation of sulfate esters with organic acids or ethers. On Fig fosfauriliruetsa only one hydroxyl group. This can be achieved by reaction with a small amount of agent phosphorylation. Obviously it is clear that this reaction will also produce bisphosphate.

Fig shows the reaction sequence similar to the sequence shown in Fig. However, after joining of the α,β-dihydroxy to the double bond of the hydroxyl functions sulfatide.

Fig shows the reaction sequence similar to the reaction sequence shown in Fig. However, after joining of the α,β-dihydroxy to the double bond of the hydroxyl functions are subjected to reaction with an oxidizing agent such as NaIO4obtaining a carbonyl function.

To the reaction scheme shown in Fig, the connection24is introduced into the reaction with the protected organic aminosterol formula HO-(C1-C24)-NHW. After joining the protected organic amerosport protected aminophenol may be further processed, as discussed in connection with 6.

Fig shows part of the reaction sequence to obtain connections OM-174-MP (connection16from the connection14b. Details of the reaction follower of the spine are given in the examples of synthesis.

Fig shows part of the reaction sequence for obtaining compounds of OM-174-MP-PR (connection17from the connection14b. Details of the reaction sequence given in the examples of synthesis.

Fig shows part of the reaction sequence for obtaining compounds of OM-174-MP-PD (connection19from the connection13bthrough the connection18. Details of the reaction sequence given in the examples of synthesis.

Fig shows the reaction sequence for obtaining compounds of OM-174-MP-AC (connection26from the connection14b. Details of the reaction sequence shown in the examples of synthesis.

Fig shows the reaction sequence for obtaining compounds of OM-174-MP-ONES (compounds41pfrom the connection14b. Details of the reaction sequence shown in the examples of synthesis.

Fig shows the reaction sequence for obtaining compounds of OM-174-MP-SW (connection32from the connection18.Details of the reaction sequence shown in the examples of synthesis.

Fig shows the reaction sequence for obtaining compounds of OM-174-MP-EP (connection33from the connection32b. Details of the reaction sequence shown in the examples of synthesis.

Fig shows the reaction sequence of gaining the connections OM-174-MP-CM (connection 35Sfrom the connection32C. Details of the reaction sequence shown in the examples of synthesis.

Discussed above reaction can also be used to attach various substituents to O-4'-position of β-(1→6)-linked glucosamine-disaccharides of the invention. This can be achieved by using discussed above reactions the introduction of substituents in O-1 status. These reactions can be performed in a similar way to a free hydroxyl group of compounds of formula4.

From the foregoing it is clear that in O-1 and O - 4'-positions of β-(1→6)-linked glucosamine-disaccharides of the invention may join a huge variety of substitutions.

The following are experimental examples of synthesis of compounds of the invention and examples related to the biological activity of the compounds of the invention.

EXAMPLES SYNTHESIS

In the next section will discuss the synthesis of compounds of the present invention. Figure 3 shows the different synthesized compounds with their corresponding numbers indicate connections.

Allyl-3-About-benzyl-4,6-About-benzylidene-2-deoxy-2-(2,2,2-trichlorocarbanilide)-α-D-glucopyranosid (3b)

To a stirred suspension of allyl-4,6-About-benzylidene-2-deoxy-2-(2,2,2-trichlorocarbanilide)-α-D-glucopyranoside2b[Liebigs Ann. (1996), 1599-1607](5 g, 10,35 mmol) and mirceski available benzyl 2,2,2-trichloroacetimidate (2,9 ml, of 15.5 mmol) in a simple ether (200 ml) was added triftorbyenzola tin (863 mg, 2.1 mmol). The mixture was stirred for 17 hours at room temperature, neutralized with saturated solution of NaHCO3and concentrated. The residue was transferred into EtOAc, washed with water and the organic phase was separated and dried over MgSO4. The solvent is evaporated and the residue was recrystallized from EtOH, receiving the connection3b(4,43 g, 75%) as a white crystalline solid. TPL 168,7°C; [α]D+ 65 (c 0,24, CHCl3); vmaxcm-13301, 2915, 1709, 1546, 1075, 1013, 693;1H NMR (500 MHz, CDCl3): δ 7,54-7,26 (m, 10H, Ph), 5,90 (m, 1H, CH=CH2), 5,62 (s, 1H, PhCH), 5,32 with 5.22 (m, 2H, CH=CH2), 5,10 (d, 1H,J2,NHof 10.0 Hz, NH), 4.95 points (AB, 1H,J=to 11.9 Hz, CH2Ph)to 4.92 (d, 1H, J1,2the 3.7 Hz, H-1), to 4.81 (d, 1H,J12.0 Hz, CH2CCl3), 4,71 (AB q, 2H, CH2Ph, CH2CCl3), 4,30 (DD, 1H,J6,6the 10.1 Hz,J6,54,6 Hz, H-6), 4,19 (m, 1H, OCH2CH), 4,07-of 3.97 (m, 2H, H-2, OCH2CH), 3,88 (m, 1H, H-5), 3,83 of 3.75 (m, 3H, H-6', H-4, H-3);13C NMR (for 125.8 MHz, CDCl3): δ 154,3 (C=O), 138,2 (Cq), 137,2 (Cq), 133,2 (CH=CH2), 129,0, 128,7, 128,2, 126,0 (CH arene), 118,3 (CH=CH2), 101,2 (PhCH), of 97.3 (C-1), 95,4 (CH2CCl3), 82,7 (C-4), 76,2 (C-3), 74,8-74,4 (CH2CCl3CH2Ph), for 68.9 (C-6), 68,6 (OCH2CH), with 62.9 (C-5), 54,9 (C-2); MC-ES 596-594 [M+Na]+; Analysis Calculated for C26H28Cl3NO7: C, 54,51; H, is 4.93; N, 2.44 Percent, N is Geno: C, 54,51; H, 4,94; N, 2,34%.

Allyl-3,6-di-About-benzyl-2-deoxy-2-(2,2,2-trichlorocarbanilide)-α-D-glucopyranosid (4b)

To a stirred solution of compound3b(1.3 g, of 2.27 mmol) and borane trimethylamine complex (660 mg, remaining 9.08 mmol) in dry THF (45 ml) at room temperature was added aluminium chloride (1,81 g of 13.6 mmol). After dissolution of the reagents was added dropwise water (82 μl, of 4.54 mmol) and continued stirring at room temperature for 30 minutes. The reaction was stopped by adding water (20 ml) followed by addition of 1M HCl solution (20 ml) and dilution with EtOAc. The organic phase was separated, washed with saturated solution of NaCl, dried over MgSO4and the solvent was removed in vacuum. The residue was subjected to flash chromatography on silica gel (n-heptane/EtOAc, 3:1)to give compound4b(1.13 g, 87%) as a white solid. TPL 65°C; [α]D+ 64 (c 0,80, CHCl3); vmaxcm-13329, 2915, 1706, 1536, 1044, 730, 694;1H NMR (500 MHz, CDCl3): δ 7,40-7,26 (m, 10H, Ph), 5,90 (m, 1H, CH=CH2), 5,32-to 5.21 (m, 2H, CH=CH2), 5,14 (d, 1H,J2,NHof 10.0 Hz, NH), 4.92 in (d, 1H,J1,2the 3.7 Hz, H-1), 4,82 (d, 1H,J12.0 Hz, CH2CCl3), 4,80, and 4,77 (AB, 2H,Jthe 11.6 Hz, CH2Ph), of 4.67 (d, 1H,J12.0 Hz, CH2CCl3), with 4.64 and 4.57 (AB, 2H,J12.0 Hz, CH2Ph), 4,19 (m, 1H, OCH2CH), a 4.03-of 3.97 (m, 2H, H-2, OCH2CH), 3,82-3,68 (m, 4H, H-6, H-6', H-5, H-4), 3,63 (t, 1H, J2,3=J3,4of 10.2 Hz, H-3), 2,60 (s, 1H, OH);13C NMR (for 125.8 MHz, CDCl3): δ 154,2 (C=O), 138,2 (Cq), 137,7 (Cq), 133,4 (CH=CH2), 128,8, 128,5, 128,4, 127,8, 127,7, 127,6 (CH arene), to 118.0 (CH=CH2), to 96.8 (C-l), 95,4 (CH2CCl3), an 80.2 (C-3), 74,6, 74,5, 73,6 (CH2CCl3, 2×CH2Ph), 72,0, to 70.2 (C-4, C-5), was 69.7 (C-6), 68,3 (OCH2CH), 54,5 (C-2); MC-ES 598-596 [M+Na]+; Analysis Calculated for C26H30Cl3NO7: C, 54,32; H, 5,26; N, 2,44%, found: C, 54,55; H, of 5.39; N, 2,39%.

Allyl-3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-(2,2,2-trichlorocarbanilide)-α-D-glucopyranosid (5b)

To a stirred solution of compound4b(1.1 g, at 1.91 mmol) and commercially available solution of 1N-tetrazole in CH3CN (~0,45 M) (8.5 ml, 3.8 mmol) in CH2Cl2(33 ml) at room temperature was added dibenzyl diethylphosphoramidite (762 μl; 2,87 mmol). Stirring was continued at room temperature for 30 minutes and then the solution was cooled to -20°C. Then the solution was added mCPBA (57-86%, 1.22 g; 7,00 mmol) in CH2Cl2(20 ml) and the solution was stirred for 30 minutes at -20°C. was Added 10% aqueous sodium thiosulfate solution (50 ml) and the mixture was stirred for 10 minutes, then diluted with EtOAc and the organic phase was separated. The organic layer was sequentially washed with 10% aqueous solution of Na2S2O3(3×), saturated aqueous NaHCO3(2×), N HCl solution (1×) isolevel solution. The organic phase was dried over MgSO4and the solvent was removed in vacuum. The residue was subjected to flash chromatography on silica gel (n-heptane/EtOAc, 4:1)to give compound5b(1.25 g, 78%) as a colourless oil. [α]D+ 56 (c 1,32, CHCl3); vmaxcm-13301, 2920, 1728, 1542, 1453, 1264, 995, 731, 694;1H NMR (500 MHz, CDCl3): δ 7,40-7,10 (m, 20H, Ph), 5,90 (m, 1H, CH=CH2), 5,33-5,23 (m, 2H, CH=CH2), to 5.13 (d, 1H, J2,NHof 10.0 Hz, NH), is 4.93 (d, 1H,J1,23.5 Hz, H-1), 4,96-4,82 (m, 4H, 2×CH2Ph), a 4.86 (m, 1H, CH2CCl3), 4,76, and the 4.65 (AB, 2H,J12.0 Hz, CH2Ph), to 4.73 (m, 1H, CH2CCl3), 4,60 (t, 1H, J3,4=J4,59.5 Hz, H-4), 4,57 and 4,47 (AB, 2H,J12.0 Hz, CH2Ph), is 4.21 (m, 1H, OCH2CH), 4,10 (DDD, 1H, H-2), was 4.02 (m, 1H, OCH2CH), 3,92 (m, 1H, H-5), of 3.84 (DD, 1H, J2,39,3 Hz, H-3), of 3.80 (DD, 1H, J6,52.0 Hz, J6,6'and 11.0 Hz, H-6), 3,76 (DD, 1H, J6,5the 4.7 Hz, H-6');13C NMR (for 125.8 MHz, CDCl3): δ 154,0 (C=O), 138,1 (Cq), 137,8 (Cq), Br135.8 (Cq), 135,7 (Cq), 133,2 (CH=CH2), 128,6, 128,5, 128,4, 128,3, 128,2, 128,1, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5, 127,4 (CH arene), 118,1 (CH=CH2)that 96.4 (C-1), 95,3 (CH2CCl3), is 78.4 (C-3), 75,6 (C-4), 74,6, 73,7, 73,3 (CH2CCl3, 2×CH2Ph), 70,2 (C-5), 69,5, 69,4 (2×CH2Ph)and 68.4 (C-6, OCH2CH), 54,3 (C-2); MC-ES 858-856 [M+Na]+; Analysis Calculated for C40H43Cl3NO10P: C, 57,53; H, 5,19; N, 1,68%, Found: C, 57,41; H, 5,28; N, 1,74%.

3,6-Di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-(2,2,2-trichloro is oxycarbonyl)-D-glucopyranose (6b)

To a stirred solution of compound5b(659 mg; 0,79 mmol) in dry THF (10 ml) at room temperature was added hexaphosphate [bis(methyldiphenylphosphine)]-(1, 5cyclooctadiene)iridium(I) (67 mg). After activation of the iridium catalyst with hydrogen for 1 min (slightly red solution becomes colorless) and the mixture was stirred in nitrogen atmosphere for 1 hour. To the reaction mixture was added iodine (360 mg, of 1.42 mmol) and water (850 ml)and the mixture was stirred for an additional 30 minutes. To the mixture was added a 10% aqueous solution of Na2S2O3and the solution was extracted with EtOAc. The organic layer was sequentially washed with 10% aqueous solution of Na2S2O3(2×) and brine. The organic phase was dried over MgSO4the solvent was removed in vacuo and the residue was led from a mixture of n-heptane/EtOAc, receiving the connection6b(419 mg, 67%) as a pale yellow solid. vmaxcm-13361, 2920, 1716, 1522, 1452, 1216, 1006, 729, 693;1H NMR (500 MHz, CDCl3for α-anomer: δ 7,40 for 7.12 (m, 20H, Ph), 5,20 (d, 1H,J1,2=3,4 Hz, H-1), of 5.17 (d, 1H,J2,NH=9.8 Hz, NH), 4,96-and 4.40 (m, 10H, 4×CH2Ph, CH2CC13), of 4.45 (t, 1H,J3,4=J4,59.5 Hz, H-4), is 4.15 (m, 1H,J6,56,4 Hz,J4,59.5 Hz, H-5), of 4.00 (dt, 1H,J2,NH=J2,39.8 Hz, H-2), of 3.78 (DD, 1H, H-3), of 3.78 (DD, 1H,J6,5the 1.7 Hz, J6,6,=to 11.0 Hz, H-6'), 3,76(DD, 1H,J6,5=6,4 Hz, H-6);13C NMR (for 125.8 MHz, CDCl3for α-anomer: δ 154,1 (C=O), 137,8 (Cq), 137,7 (Cq), 135,7 (Cq), 135,6 (Cq), 128,6, 128,5, 128,4, 128,3, 128,2, 128,1, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5, 127,4 (CH arene), 95,3 (CH2CCl3), to 91.6 (C-1), up 77.9 (C-3), with 76.1 (C-4), 74,6, 73,7, 73,3 (CH2CCl3, 2×CH2Ph)70,6 (C-5), 69,5, 69,4 (2×CH2Ph), to 68.8 (C-6), 54,7 (C-2); MC-ES 818-816 [M+Na]+; Analysis Calculated for C37H39Cl3NO10P: C, 55,90; H, 4,94; N, 1,76%, Found: C, 55,67; H, 5,18; N, 1,62%.

3,6-Di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-(2,2,2-trichlorocarbanilide)-D-glyukopiranozil trichloroacetimidate (7b)

To a stirred solution of compound6b(419 mg of 0.53 mmol) in dry CH2Cl2(6.5 ml) at room temperature was added trichloroacetonitrile (528 μl; 5.3 mmol) and cesium carbonate (86 mg, 0.26 mmol). After stirring for 1 hour the reaction was suppressed saturated aqueous NaHCO3(5 ml) and the solution was extracted. The organic layer was washed with saline, dried over MgSO4and the solvent was removed in vacuum, obtaining the connection7b(400 mg) as a pale yellow oil, which was used in the next stage without further purification.

Allyl-3,4-di-About-benzyl-2-deoxy-2-(2,2,2-trichlorocarbanilide)-α-D-glucopyranosid (8b)

To a stirred solution of compound3b(937 mg; and 1.63 mmol) and berendililerin (482 mg, 8,18 mmol) in CH2Cl2/sub> (18 ml) at 0°C was slowly added BF3:Et2O (1 ml, 8,18 mmol). After stirring for 45 minutes the reaction was stopped by slowly adding saturated aqueous solution of NaHCO3. The organic phase was separated, washed with saturated solution of NaCl and dried over MgSO4. The solvent is evaporated and the residue was recrystallized from a mixture of EtOAc/n-heptane, receiving the connection8b(757 mg, 81%) as a white crystalline solid. TPL 119,9°C; [α]D+ 74 (c 0,59, CHCl3); vmaxcm-13312, 2916, 1702, 1538, 1023, 732, 692;1H NMR (500 MHz, CDCl3): δ 7,40-7,26 (m, 10H, Ph), 5,88 (m, 1H, CH=CH2), and 5.30-5,20 (m, 2H, CH=CH2), 5,07 (d, 1H,J2,NHof 10.0 Hz, NH), 4,89 (d, 1H,J1,23.5 Hz, H-l), to 4.87 (d, 1H,Jof 11.0 Hz, CH2CCl3), 4,87 and of 4.75 (AB, 2H,Jof 11.0 Hz, CH2Ph), 4,78 and to 4.68 (AB, 2H, J=12.0 Hz, CH2Ph), and 4.68 (d, 1H,Jof 11.0 Hz, CH2CCl3), to 4.16 (m, 1H, OCH2CH), was 4.02-of 3.94 (m, 2H, H-2, OCH2CH), 3,86-to 3.64 (m, 5H, H-6, H-6', H-5, H-4, H-3), of 1.78 (m, 1H, OH);13C NMR (for 125.8 MHz, CDCl3): δ 154,2 (C=O), 138,0 (Cq), 137,8 (Cq), 133,3 (CH=CH2), 128,5, 128,4, 128,1, 128,0, 127,8, 127,7 (CH arene), to 118.0 (CH=CH2), to 96.8 (C-1), 95,4 (CH2CCl3), an 80.2 (C-3), 78,0 (C-4), 75,2, 75,1, 74,6 (CH2CCl3, 2×CH2Ph), to 71.5 (C-5), 68,3 (OCH2CH), for 61.6 (C-6), 55,2 (C-2); MC-ES 598-596 [M+Na]+; Analysis Calculated for C26H30Cl3NO7: C, 54,32; H, 5,26; N, 2,44%, Found: C, 54,76; H, Of 5.53; N, 2,31%.

Allyl-amino-3,4-di- About-benzyl-2-deoxy-α-D-glucopyranosid (9b)

To a stirred solution of compound8b(245 mg; 0.43 mmol) in Asón (6 ml) at room temperature was added zinc powder (430 mg). After stirring overnight, the suspension was filtered through celite, the solvent was removed in vacuum and the residual solvent is evaporated simultaneously with toluene three times. The residue was transferred into EtOAc, washed with saturated aqueous NaHCO3and a salt solution. The organic phase was separated, dried over MgSO4and the solvent was removed in vacuum, obtaining the connection9b(157 mg) as a colourless oil, which was used in the next stage without further purification. A sample of the compound was purified using flash chromatography on silica gel (CH2Cl2/Acetone, 10:1→1:1), receiving the connection9bin the form of a white crystalline solid. TPL of 85.2°C; [α]D+ 98 (c 0,89, CHCl3); vmaxcm-13190, 2899, 1664, 1577, 1496, 1452, 1363, 1024, 737, 695;1H NMR (500 MHz, CDCl3): δ 7,50-7,26 (m, 10H, Ph), of 5.92 (m, 1H, CH=CH2), and 5.30-5,20 (m, 2H, CH=CH2), the 4.90 (d, 1H,J1,23.5 Hz, H-1), 5,01 and 4.73 (AB, 2H,Jof 11.0 Hz, CH2Ph), 4,88, and of 4.75 (AB, 2H, J 12.0 Hz, CH2Ph)to 4.16 (DD, 1H,J5.0 Hz,J12.9 Hz, OCH2CH), was 4.02 (DD, 1H,J6,0 Hz,J12.9 Hz, OCH2CH), 3,85-3,55 (m, 5H, H-6, H-6', H-5, H-4, H-3), 2,80 (DD, 1H, J2,39.4 Hz, H-2);13C NMR (for 125.8 MHz, CDCl ): δ 138,6 (Cq), 138,1 (Cq), 133,9 (CH=CH2), 128,6, 128,5, 127,9, 127,8, 127,7 (CH arene), 117,3 (CH=CH2), 98,8 (C-l), 83,8, 78,7, to 71.9 (C-3, C-4, C-5), 75,6, 74,8 (2×CH2Ph)68,3 (OCH2CH), to 61.4 (C-6), 56,0 (C-2); MC-ES 400 [M+H]+; Analysis Calculated for C23H29NO5: C, 69,15; H, To 7.32; N, 3,51%, Found: C, 69,21; H, Was 7.36; N, 3.25%Milkfat.

Allyl-2[(R)-3-benzyloxycarbonylamino]-3,4-di-About-benzyl-2-deoxy-α-D-glucopyranosid (10b)

To a cold solution (-15°C.) of (R)-3-benzyloxyethanol acid (145 mg; 0.43 mmol) [Bull. Chem. Soc. Jpn,60(1987), 2197-2204] in THF (5 ml) was addedN-methylmorpholine (47 μl; 0.43 mmol) and isobutylparaben (57 μl; 0.43 mmol). The reaction mixture was stirred at -15°C for 30 minutes. To the reaction mixture solution was added connection to9b(157 mg; 0,39 mmol) in THF (5 ml). Stirring was continued over night at room temperature. Then added water and EtOAc, the organic phase was separated and the aqueous phase again was extracted with EtOAc. The organic layers were combined, washed with water and brine and dried over MgSO4. The solvent is evaporated and the residue was recrystallized from MeOH, receiving the connection10b(176 mg, 63% over 2 stages) in the form of a white crystalline solid TPL 141,3°C; [α]D+ 61 (c 0,31, CHCl3); vmaxcm-13297, 2920, 2851, 1637, 1544, 1025, 732, 693;1H NMR (500 MHz, CDCl3): δ 7,40-7,26 (m, 15H, Ph), 6,32 (d, 1H, J2,NH9.0 HZ, NH), USD 5.76 (m, 1H, CH=CH2), 5,23-,10 (m, 2H, CH=CH2), 4,82 and the 4.65 (AB, 2H, J and 11.0 Hz, CH2Ph)4,80 (d, 1H,J1,24.0 Hz, H-1), 4,73 and 4.57 (AB, 2H,Jthe 11.5 Hz, CH2Ph), 4,54, and of 4.49 (AB, 2H,Jof 11.0 Hz, CH2Ph), 4,27 (m, 1H, J1,24.0 Hz, H-2), of 4.00 (m, 1H, OCH2CH), 3,85-3,62 (m, 7H, OCH2CH, H-6, H-6', H-5, H-4, H-3, H-3"), is 2.40 (DD, 1H, J 3,7, and 15.1 Hz, H-2"B), 2,28 (DD, 1H,J=7,6, and 15.1 Hz, H-2"A), was 1.58 (m, 1H, H-4"A), for 1.49 (m, 1H, H-4"B), 1,35 by 1.12 (m, 18H, 9 CH2), of 0.90 (t, 3H, CH3);13C NMR (for 125.8 MHz, CDCl3): δ of 171.2 (C=O), 138,3 (Cq), 138,2 (Cq), 137,9 (Cq), to 133.5 (CH=CH2), 128,5, 128,4, 128,3, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5 (CH arene), to 117.7 (CH=CH2), a 96.9 (C-1), an 80.2 (C-3), 78,0 (C-4), to 76.6 (C-3"), to 74.7 (CH2Ph)74,6 (CH2Ph), and 71.4 (C-5), from 71.3 (CH2Ph)68,3 (OCH2CH), to 61.8 (C-6), was 52.6 (C-2), of 41.5 (C-2'), and 33.8 (C-4"), 31,9, 29,6, 29,5, 29,4, 29,3, 25,1, 22,7 (CH2), 14,1 (CH3); MS-ES 739 [M+Na]+; Analysis Calculated for C44H61NO7: C, 73,81; H, 8,59; N 1,96%, Found: C, 73,62; H, To 8.57; N, Equal To 1.82%.

Allyl-3,4-di-About-benzyl-6-About-[3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-(2,2,2-trichlorocarbanilide)-β-D-glyukopiranozil]-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (11a)

To a stirred solution of compound10b(231 mg, 0.32 mmol) and imidate connection7b(400 mg, 0.42 mmol) in anhydrous CH2Cl2(9 ml) at -20°C was added 4 Å molecular sieves. After stirring for 30 minutes was added TMSOTf (12 μl, 64 mmol) and stirring was continued for additional 1 hour, the. The reaction mixture was filtered through celite, diluted with EtOAc and neutralized with a saturated aqueous solution of NaHCO3. The organic phase was separated, washed with saturated solution of NaCl, dried over MgSO4. The solvent is evaporated and the residue was recrystallized from MeOH, receiving the connection11a(335 mg, 70%) as a white crystalline solid TPL RUR 145.2°C; [α]D+ 37 (c 0,12, CHCl3); vmaxcm-13297, 2921, 1713, 1641, 1540, 1453, 1266, 997, 730, 693;1H NMR (500 MHz, CDCl3): δ 7,37-7,14 (m, 35H, Ph), 6,32 (d, 1H, J2,NH9.0 Hz, NH-2), USD 5.76 (m, 1H, CH=CH2), 5,23-5,10 (m, 3H, NH-2'CH=CH2), 4,95 was 4.42 (m, 15H, CH2CCl3, 7×CH2Ph), 4,79 (d, 1H,J1,23.5 Hz, H-1), 4,74 (d, 1H,J1',2'of 10.0 Hz, H-1'), to 4.46 (t, 1H,J3'4'=J4',5'that 8.5 Hz, H-4'), 4,32 (m, 1H,J1,24.0 Hz, H-2), 4,25 (AB, 1H, CH2Ph), of 4.13 (m, 1H, H-6A), 4,08 (m, 1H, H-3'), Android 4.04 (m, 1H, OCH2CH), 3,90-of 3.60 (m, 9H, OCH2CH, H-5', H-6'A, H-6'B, H-6B, H-5, H-4, H-3, H-3"), 3,42 (m, 1H, H-2'), is 2.40 (DD, 1H,J3,7, and 15.1 Hz, H-2"B), 2,28 (DD, 1H, J=7,6, and 15.1 Hz, H-2"A), was 1.58 (m, 1H, H-4"A), for 1.49 (m, 1H, H-4"B), 1,35 by 1.12 (m, 18H, 9 CH2), of 0.90 (t, 3H, CH3);13C NMR (for 125.8 MHz, CDCl3): δ 171,0 (C=O), 153,7 (C=O), 138,3 (Cq), 138,2 (Cq), 138,1 (Cq), 137,9 (Cq), 137,7 (Cq), 135,7 (Cq), 135,6 (Cq), 133,6 (CH=CH2), 128,5, 128,4, 128,3, 128,2, 128,1, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5, 127,4 (CH arene), to 117.7 (CH=CH2), and 99.8 (C-1'), a 96.9 (C-1), 95,1 (CH2CCl3), of 80.6 (C-3), of 78.6 (C-3'), 78,0 (C-4), to 76.6 (C-3"), 76,2 (C-4'), 74,4 (C-5'), 74,7, 74,2, 73,8, 73,3, 71, (CH 2CCl3, 5×CH2Ph), 70,2 (C-5), 69,6, 69,4 (2×P-OCH2Ph)69,0 (C-6 or C-6'), 68,1 (OCH2CH), is 67.7 (C-6 or C-6'), 57,3 (C-2'), for 52.6 (C-2), to 41.6 (C-2'), and 33.8 (C-4"), 31,9, 29,6, 29,5, 29,4, 29,3, 25,1, 22,7 (CH2), 14,1 (CH3); MC-ES 1515-1513 [M+Na]+; Analysis Calculated for C81H98Cl3N2O16P: C, 65,16; H, 6,62; N, 1,88%, Found: C, 65,31; H, 6,70; N, 1,77%.

Allyl-6-About-[2-amino-3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-β-D-glyukopiranozil]-2-[(R)-3-benzyloxycarbonylamino]-3,4-di-About-benzyl-2-deoxy-α-D-glucopyranosid (12d)

To a stirred solution of compound11a(310 mg, 0.21 mmol) in Asón/N2About 9:1 (5 ml) at room temperature was added zinc-copper couple (260 mg). After stirring for 1 hour was added to the zinc-copper couple (260 mg) and the operation repeated again. Stirring is continued for another 3 hours and the suspension was filtered through celite. The solvent was removed in vacuum and the residual solvent is evaporated simultaneously with toluene three times. The residue was transferred into EtOAc, washed with saturated aqueous NaHCO3(2×) and brine. The organic phase was separated, dried over MgSO4and the solvent was removed in vacuum, obtaining the connection12d(273 mg) as a colourless oil, which was used in the next stage without further purification. MC-ES 1317 [M+H]+, 1339 [M+Na]+.

Allyl-3,4-di-About-benzyl-6-About-3,6-di- About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (13b)

To a stirred solution of (R)-3-dodecadodecahedron acid [Bull. Chem. Soc. Jpn,60(1987), 2205-2214] (115 mg, 0.27 mmol) in THF (4 ml) at -15°C was added sequentiallyN-methylmorpholine (30 μl; 0.27 mmol) and isobutylparaben (35 μl; 0.27 mmol). Stirring is continued for 30 minutes at -15°C. Then the reaction mixture was added a solution of the crude compound12b(273 mg; 0.21 mmol) in THF (4 ml). After stirring over night at room temperature the solvent was removed in vacuo and to the residue was added water. Then the mixture was extracted with EtOAc, the organic phase is successively washed with saturated aqueous NaHCO3, brine and dried over MgSO4. The solvent is evaporated and the residue was led from MeOH, receiving the connection13b(259 mg, 72% over 2 stages) as a white solid TPL 173°C; [α]D+ 30 (c 0,90, CHCl3); vmaxcm-13302, 2919, 2850, 1726, 1636, 1544, 1453, 1357, 1266, 998, 730, 694;1H NMR (500 MHz, CDCl3): δ 7,40-7,10 (m, 35H, Ph), of 6.31 (d, 1H,J2,NH9.4 Hz, NH-2), 6,01 (d, 1H,J2',NH'7.5 HZ, NH-2'), USD 5.76 (m, 1H, CH=CH2), 5,23-5,08 (m, 2H, CH=CH2), to 5.08 (d, 1H,J1',2'8,2 Hz, H-1'), of 5.05 (m, 1H, H-3"'), 4,95-4,4 (m, 14H, 7×CH2Ph), 4,79 (d, 1H,J1,2of 3.4 Hz, H-1), to 4.46 (t, 1H,J3',4'=J4',5', to 8.7 Hz, H-4'), 4,32 (m, 2H, H-2, H-3'), 4.09 to (m, 1H, H-6A), a 4.03 (m, 1H, OCH2CH), 3,90-of 3.60 (m, 9H, OCH2CH, H-5', H-6'A, H-6'B, H-6B, H-5, H-4, H-3, H-3"), of 3.46 (m, 1H, H-2'), is 2.40 (DD, 1H,J3,3, 15.1 Hz, H-2"B), of 2.34 (DD, 1H,J7,2, 15,5 Hz, H-2"'B), 2,28 (DD, 1H, J=7,6, and 15.1 Hz, H-2"A), of 2.21 (DD, 1H, J 5,0, 15,5 Hz, H-2"'A), 2,11 (t, 2H, J of 7.5, H-2””), 1,58 (m, 1H, H-4"A), 1,55-of 1.36 (m, 3H, H-4"B, 2×H-4"'), 1,35-1,12 (m, 54H, 27 CH2), of 0.90 (m, 9H, 3×CH3);13C NMR (for 125.8 MHz, CDCl3): δ to 173.3 (C=O), 171,0 (C=O), is 170.0 (C=O), 138,4 (Cq), 138,3 (Cq), 138,2 (Cq), 138,1 (Cq), 137,8 (Cq), 135,7 (Cq), 135,6 (Cq), 133,6 (CH=CH2), 128,5, 128,4, 128,3, 128,2, 128,1, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5, 127,4 (CH arene), of 117.6 (CH=CH2), and 99.2 (C-1'), to 96.8 (C-1), 80,5 (C-3), to 78.3 (C-3', C-4), to 76.6 (C-3"), 76,0 (C-4'), 74,8, 74,7 (2×CH2Ph), 74,3 (C-5'), 73,2, 73,1 (2×CH2Ph), from 71.3 (CH2Ph), and 70.5 (C-3"'), 70,3 (C-5), 69,4, 69,3 (2×P-OCH2Ph), for 69.1 (C-6 or C-6'), 68,1 (OCH2CH), 67,6 (C-6 or C-6'), of 56.7 (C-2'), a 52.4 (C-2), 41,6 is 41.4 (C-2 ' C-2"'), 34,4, 34,1, 33,8 (C-4, C-4"'C-2""), 31,9, 29,9, 29,6, 29,5, 29,4, 29,3, 29,2, 29,0, 25,1, 25,0, 24,9, 22,7 (CH2), 14,1 (CH3); MC-ES 1747 [M+Na]+; Analysis Calculated for C104H145N2O17P: C, 72,36; H, Of 8.47; N, 1,62%, Found: C, 72,52; H, 8,43; N, 1,51%.

3,4-Di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-D-glucopyranose (14b)

To a stirred solution of compound13b(259 mg; 0.15 mmol) in dry the GF (13 ml) at room temperature was added hexaphosphate [bis(methyldiphenylphosphine)]-(1, 5cyclooctadiene)iridium(I) (13 mg). After activation of the iridium catalyst with hydrogen for 1 min (slightly red solution becomes colorless) and the mixture was stirred in nitrogen atmosphere for 1 hour. To the reaction mixture was added iodine (69 mg, 0.27 mmol) and water (13 ml) and the mixture was stirred for an additional 15 minutes. To the mixture was added a 10% aqueous solution of Na2S2O3and the solution was extracted with EtOAc. The organic layer was sequentially washed with 10% aqueous solution of Na2S2O3(2×) and brine. The organic phase was dried over MgSO4the solvent was removed in vacuo and the residue was led out of CH3CN, receiving the connection14b(165 mg, 65%) as a gray solid. vmaxcm-13388, 3276, 3062, 2919, 2850, 1726, 1641, 1544, 1453, 1358, 1263, 997, 731, 694;1H NMR (500 MHz, CDC13for α-anomer: δ 7,40-7,10 (m, 35H, Ph), to 6.39 (d, 1H,J2,NH9.4 Hz, NH-2), 6,18 (m, 1H, NH-2'), 5,38 (d, 1H,J1',2'7.7 Hz, H-1'), 5,12 (m, 1H,J1,2the 3.2 Hz, H-1), of 5.05 (m, 1H, H-3"'), 4,95-4,42 (m, 14H, 7×CH2Ph), 4,50 (m, 1H, H-4'), to 4.23 (dt, 1H,J1,2the 3.2 Hz,J2,NH=J2,39.4 Hz, H-2), 4,19 (t, 1H,J2',3'=J3',4'the 8.9 Hz, H-3'), 4,07 (m, 1H, H-5), of 3.96 (d, 1H,J6A,6Bof 11.4 Hz, H-6A), 3,89-of 3.80 (m, 2H, H-3", H-6'A), 3,80-the 3.65 (m, 4H, H-5', H-6'B, H-6B, H-3), the 3.35 (m, 1H, H-2'), 3,32 (t, 1H,J3,4=J4,59.5 Hz, H-4), 2,40-of 2.20 (m, 4H, 2×H-2", 2×H-2"'), of 2.16 (t, 2H,Jof 7.5, H-2""), 1,58 (m, 1H, H-4"A), 1,55-of 1.36 (m, 3H, H-4"B, 2×H-4"'), 1,35-1,12 (m, 54H, 27 CH2), of 0.90 (m, 9H, ×CH 3);13C NMR (for 125.8 MHz, CDCl3for α-anomer: δ 173,9 (C=O), 171,4 (C=O), 170,6 (C=O), 138,4 (Cq), 138,3 (Cq), 138,2 (Cq), 138,1 (Cq), 137,8 (Cq), 135,7 (Cq), 135,6 (Cq), 128,5, 128,4, 128,3, 128,2, 128,1, 128,0, 127,9, 127,8, 127,7, 127,6, 127,5, 127,4 (CH arene), 98,8 (C-1'), 91,7 (C-1), of 80.6 (C-3), of 78.8 (C-3', C-4), with 76.8 (C-3"), 76,2 (C-4'), 74,8 (CH2Ph)74,2 (C-5'), 73,6, 73,4, 73,3 (3×CH2Ph), total 71.8 (C-5), from 71.3 (CH2Ph), and 70.8 (C-3"'), 69,4, 69,3 (2×P-OCH2Ph)69,0 (C-6'), of 67.6 (C-6), 57,0 (C-2'), to 52.9 (C-2), and 41.7 (C-2 ' C-2"'), 34,4, 34,1, 33,8 (C-4, C-4"', C-2""), 31,9, 29,9, 29,6, 29,5, 29,4, 29,3, 29,2, 29,0, 25,1, 25,0, 24,9, 22,7 (CH2), 14,1 (CH3); MC-ES 1708 [M+Na]+; Analysis Calculated for C101H141N2O17P: C, 71,94; H, 8,43; N, 1,66%, Found: C, 71,68; H, 8,35; N, 1,61%.

3,4-Di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosyloxy (15b)

To a stirred solution of compound14b(1 g; of 0.60 mmol) in THF (90 ml) at -78°C was added a solution of lithium bis(trimethylsilyl)amide (1M in THF)(1.9 ml, 1.88 mmol). The mixture was stirred 5 minutes, then add tetraethylpyrophosphate (1.3 g, is 2.37 mmol). Stirring was continued at -78°C for 2 hours, then the solution was neutralized with a saturated aqueous solution of NaHCO3and diluted with EtOAc. The organic phase was separated, dried over MgSO4and the solvent was removed in vacuum, obtaining the connection15b(2 g) as a pale yellow oil which, which was used in the next stage without further purification.

2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glyukopiranozil dihydrophosphate (1b) (OM-174-DP)

The crude compound15b(2 g) in THF (100 ml) was first made for 17 hours in the presence of 5% Pd-C (1.5 g) at room temperature in hydrogen atmosphere (6 bar). The mixture is then neutralized Et3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified using HPLC according to the invention (Method D), receiving the connection1b(as sodium salt)(342 mg; 48% over 2 stages) as a white lyophilisate. [α]D+ 14 (c 0,6, H2O); vmaxcm-13305, 2918, 2849, 1713, 1648, 1553, 1465, 1376, 1174, 1039, 915, 719, 654;1H NMR (500 MHz, CDCl3/CD3OD/Pyridine-d5/DCl 37% 5/2/1/1): δ ceiling of 5.60 (DD, 1H,J1,27.5 Hz,J1,P2.0 Hz, H-1), at 5.27 (m, 1H, H-3"'), to 4.73 (d, 1H,J1',2'8.5 Hz, H-1'), 4.09 to (q, 1H,J3,4=J4,5=J4,P9.0 Hz, H-4'), 4,05-of 3.80 (m, 6H, 2×H-6, 2×H-6', H-4, H-3), of 4.00 (m, 1H, H-3"), 3,85 (m, 2H, H-2, H-3'), 3,85 (m, 1H, H-2'), 3,50 (m, 2H, H-5, H-5'), to 2.67 (m, 2H,,J6,4 Hz, 2×H-2"'), 2,43 (m, 2H,Jthe 6.1 Hz, 2×H-2"), to 2.29 (m, 2H,J7,3,J15 Hz, 2×H-2""), 1,60-1,36 (m, 6H, 2×H-4", 2×H-4"', 2×H-3""), 1,35-1,12 (m, 52H, 26 CH2), of 0.90 (m, 9H, 3×CH3);13C NMR (for 125.8 MHz, CDCl3/CD3OD/Pyridine-d /DCl 37% 5/2/1/1): δ 174,9 (C=O), 174,1 (C=O), 172,9 (C=O), Is 102.4 (C-1'), to 94.7 (C-1), 75,3 (C-5'), 73,8 (C-4'), 73,4, 70,4, 70,1, 69,6 (C-3', C-5, C-4, C-3), to 71.9 (C-3"'), to 69.6 (C-3"), is 69.4 (C-6), of 60.8 (C-6'), or 56.1 (C-2'), of 54.8 (C-2), a 44.2 (C-2'), and 41.7 (C-2"'), 37,7, 35,1, 34,6 (C-4, C-4"'C-2""), and 32.3 (C-12, C-12"', C-10""), 30,1-29,6 (C-6"->C-11, C-6"'->C-11"', C-4""->C-9""), 25,7, 25,6, 25,2 (C-5, C-5"', C-3""), 23,1 (C-13 C-13"'-11””), 14,5 (C-14 C-14"', C-12""); MC-ES 1155 [M+Na-2H]-, 1133 [M-H]-; Analysis Calculated for C52H97N2O20P2Na3+ H2O: C, 51,23; H, 8,18; N, 2,30%, Found: C, 51,11; H, 8,46; N, 2.20 Per Cent.

2-Deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranose (16) (OM-174-MP)

Connection14b(80 mg, 47 mmol) in THF (50 ml) was first made for 16 hours in the presence of 5% Pd-C (25 mg) at room temperature in hydrogen atmosphere (6 bar). The catalyst was removed by filtration and the filtrate was concentrated in vacuum. The residue was purified using HPLC according to the invention (Method B), receiving the connection16(as sodium salt)(25 mg; 50%) as a white lyophilisate. MS-ES 1053[M-H]-.

Propyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranoside (17) (OM-174-MP-PR)

Connection13b(100 mg, 58 mmol) in THF (100 ml) was first made for 17 hours in the presence of 5%Pd-C (50 mg) at room temperature in hydrogen atmosphere (6 bar). The mixture is then neutralized Et3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified using HPLC according to the invention (Method D), receiving the connection17(as sodium salt)(28 mg, 44%) as a white lyophilisate.1H NMR (500 MHz, CDCl3/CD3OD/Pyridine-d5/DCl 37% 5/2/1/1): δ a 5.25 (m, 1H, H-3"'), 4.72 in (d, 1H,J1,23.6 Hz, H-1), 4,70 (d, 1H,J1',2'9.9 Hz, H-1'), to 4.16 (q, 1H,J3,4=J4,5=J4,Pand 9.2 Hz, H-4'), to 4.01 (DD, 1H,J6A,6B9,3 Hz, H-6A), 3,95 (m, 1H, H-3"), 3,91 (t, 1H,J3',4'=J3',2'of 10.0 Hz, H-3'), with 3.89 (DDD, 1H,J1,23,6 Hz,J3,2the 10.5 Hz,JNH,28.0 Hz, H-2), 3,86-3,81 (m, 4H, 2×H-6', H-6B, H-2'), of 3.78 (DD, 1H,J3,49,0 Hz,J3,2the 10.5 Hz, H-3), to 3.64 (m, 1H, H-5), of 3.56 (t, 1H,J3,4=J4,58,8 Hz, H-4), of 3.54 (m, 1H, OCH2CH), 3,50 (m, 1H, H-5'), with 3.27 (m, 1H, OCH2CH), 2.63 in (m, 2H,J6.2 Hz, 2×H-2"'), 2,48 (DD, 1H,J2”,3”3,4 Hz,J2”,2”14,8 Hz, H-2"), 2,39 (DD, 1H,J2”,3”8,5 Hz,J2”,2”14,8 Hz, H-2"), 2,28 (m, 2H,J7,3,J15 Hz, 2×H-2""), 1,70-1,36 (m, 6H, 2×H-4", 2×H-4"', 2×H-3""), of 1.55 (m, 2H, OCH2CH2), 1,35-1,12 (m, 52H, 26 CH2), to 0.88 (m, 12H, 4×CH3);13C NMR (for 125.8 MHz, CDCl3/CD3OD/Pyridine-d5/DCl 37% 5/2/1/1): δ 174,7 (C=O), 174,0 (C=O), 172,5 (C=O), 101,9 (C-1'), a 97.5 (C-1), 75,3 (C-5'), 75,0 (C-4'), at 73.7 (C-3'), 71,7 (C-3"'), 71,6, 71,4, 70,6 (C-5, C-4, C-3), 70,1 (OCH2CH), 69,1 (C-3"), OR 69.0 (C-6), OF 60.8 (C-6'), 55,9 (C-2'), 54,4 (C-2), WHICH IS 43.4(C-2'), 41,5 (C-2"'), 37,4, 35,0, 34,5 (C-4, C-4"', C-2""), and 32.3 (C-12, C-12"', C-10""), 30,0-29,6 (C-6"->C-11, C-6"'->C-11"', C-4""->C-9""), 26,1, 25,6, 25,5 (C-5, C-5"', C-3""), 23,0, 22,9 (OCH2CH2CH3C-13 C-13"'-11””), 14,5 (C-14 C-14"', C-12""), 10,9 (OCH2CH2CH3); MS-ESI 1141 [M-H+2Na]+, HRMS-ESI calculated for C55H104N2O17Na2P [M-H+2Na]+1141,6868 found 1141,6879.

2,3-Dihydroxypropyl-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranoside (18)

To a stirred solution of compound13b(500 mg; 0.29 mmol) in a mixture of THF/tert-BuOH/H2O 10:10:1 (15 ml) at room temperature was sequentially added 4-methylmorpholine N-oxide (NMO)(156 mg; of 1.16 mmol) and OsO4in 2-propanol (2.5 percent; 580 μl; 58 mmol). After 4 hours was added saturated aqueous Na2S2O3and the mixture was extracted with EtOAc. The organic phase was washed with a saturated aqueous solution of Na2S2O3(2×), brine and dried over MgSO4. The solvent is evaporated and the residue was led from EtOH, receiving the connection18(300 mg, 59%) as a white solid. vmaxcm-13300, 2919, 2850, 1646, 1543, 1453, 1358, 1266, 998, 730, 694;1H NMR (500 MHz, CDCl3): δ 7,40-7,10 (m, 35H, Ph), is 6.54 (m, 1H, NH-2'), 6,38 (m, 1H, NH-2), to 5.08 (m, 0,5H, H-3"'), of 5.03 (m, 0,5H, H-3"'), 4,95-4,42 (�, 14H, 7×CH2Ph)4,88 (m, 1H, H-1'), 4,72 (m, 1H, H-1), to 4.52 (m, 1H, H-4'), 4,27 (m, 2H, H-2, H-3'), 4,18-3,30 (m, 15H, OCH2CH, CH(HE), CH2OH, H-2', H-5', 2×H-6', 2×H-6, H-5, H-4, H-3, H-3"), 2,50 at 2.45 (m, 1H, H-2"'), 2,45-2,35 (m, 1H, H-2"), 2,30-of 2.20 (m, 2H, H-2', H-2"'), of 2.15 (m, 1H, H-2""), 1,65-1,45 (m, 6H, 2×H-4", 2×H-4"', 2×H-3""), 1,35-1,12 (m, 52H, 26 CH2), of 0.90 (m, 9H, 3×CH3);13C NMR (for 125.8 MHz, CDCl3): δ 173,8 (C=O), 173,7 (C=O), RUB 171.1 (C=O), 170,6 (C=O), 170,4 (C=O), 138,2-138,02 (Cq), 137,7 (Cq), 137, 6mm (Cq), 135,7 (Cq), 135,6 (Cq), 128,4-127,4 (CH arene), and 99.4 (C-1'), And 99.2 (C-1'), to 98.5 (C-1), to 98.4 (C-1), of 80.6 (C-3), 80,4 (C-3), 78,7, is 78.4 (C-3', C-4), 76,8, 76,7 (C-3"), 75,6, 75,5, 75,4, 75,3 (C-4'), 74,8, 74,7 (2×CH2Ph), 74,1 (C-5'), 73,2, 72,6, 72,5, 72,0 (2×CH2Ph), 71,3, 71,2 (CH2Ph), 70,8-and 70.5 (C-3"', C-5, CH(OH)), 69,4, 69,3 (2×P-OCH2Ph), 68,8, 68,7, 68,6, 68,1 (C-6, (OCH2CH), C-6'), 63,7, 63,6 (CH2OH), 56,2, or 56.1 (C-2'), 52,6, 52,5 (C-2), compared with 41.8 41,4 (C-2 ' C-2"'), 34,4, 34,0, 33,7 (C-4, C-4"', C-2""), to 31.9 (C-12, C-12"', C-10""), 29,8-29,1 (C-6"->C-11, C-6"'->C-11”', C-4""->C-9""), 25,2, 25,1, 25,0, 24,9 (C-5, C-5"', C-3""), and 22.6 (C-13 C-13"', C-11""), a 14.1 (C-14 C-14"', C-12""); MC-ES 1782 [M+Na]+; Analysis Calculated for C104H147N2O19P: C, 70,96; H, 8,42; N, 1,59%, Found: C, 70,44; H, At 8.36; N, 1,47%.

2,3-Dihydroxypropyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranoside (19) (OM-174-MP-PD)

Connection18(113 mg, 64 mmol) in THF (100 ml) was first made for 19 hours in the presence of 5% Pd-C (50 mg) at room temperature in hydrogen atmosphere (6 bar). The mixture is then Nate who was alitwala Et 3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified using HPLC according to the invention (Method D), receiving the connection19(as sodium salt)(26 mg; 36%) as a white lyophilisate. MC-ESI 1173 [M-H+2Na]+, HRMS-ESI calculated for C55H104N2O19Na2P [M-H+2Na]+1173,6766 found 1173,6763.

Trichloroacetimidate 3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-D-glucopyranosyl (24b)

To a stirred solution of compound14b(260 mg; 150 mmol) in dry CH2Cl2(5 ml) at room temperature was added trichloroacetonitrile (155 μl; 1.54 mmol) and potassium carbonate (11 mg, 77 mmol). After stirring for 1 hour the reaction mixture is extinguished saturated aqueous NaHCO3(2 ml) and the solution was extracted. The organic layer was washed with saline, dried over MgSO4and the solvent was removed in vacuum, obtaining the connection24b(280 mg) as a pale yellow oil, which was used in the next stage without further purification.

(6-Benzyloxycarbonylamino)-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron the laminitis]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-β-D-glucopyranoside (25)

To a stirred solution of commercially available 6-benzyloxycarbonylamino-1-hexanol (43 mg, 0,17 mmol) and the crude of imidate connection24b(280 mg) in anhydrous CH2Cl2(5 ml) at -20°C was added 4 Å molecular sieves. After stirring for 30 minutes was added TMSOTf (6 μl, 31 mmol) and stirring was continued for additional 2 hours. The mixture was slowly heated to room temperature and was stirred overnight. The reaction mixture was filtered through celite, diluted with EtOAc and neutralized with a saturated aqueous solution of NaHCO3. The organic phase was separated, washed with saturated solution of NaCl, dried over MgSO4. The solvent is evaporated and the residue was recrystallized from MeOH, receiving the connection25(174 mg, 59% over 2 stages) in the form of a white crystalline solid. MS-ES 1942 [M+Na]+

6-Aminohexyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-β-D-glucopyranoside (26) (OM-174-MP-AC)

Connection25(102 mg, 53 mmol) in a mixture of Asón/2-propanol/CH2Cl23:3:1 (7 ml) was first made within 24 hours in the presence of 10% Pd-C (50 mg) at room temperature under atmospheric pressure. The catalyst was removed by filtration and the filtrate was concentrated in vacuum. The remainder of ciali using HPLC according to the invention (Method D), receiving the connection26(as sodium salt)(17 mg, 28%) as a white lyophilisate. MS-ES 1153 [M-N]-.

Tetradecyl-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-β-D-glucopyranoside (41b)

To a stirred solution of commercially available tetradecanol (14 mg, 65 mmol) and of untreated imidate connection24b(98 mg) in anhydrous CH2Cl2(2 ml) at -20°C was added 4 Å molecular sieves. After stirring for 30 minutes was added TMSOTf (2 μl, 12 mmol) and stirring was continued for additional 2 hours. The mixture was slowly heated to room temperature and was stirred overnight. The reaction mixture was filtered through celite, diluted with EtOAc and neutralized with a saturated aqueous solution of NaHCO3. The organic phase was separated, washed with saturated solution of NaCl, dried over MgSO4. The solvent is evaporated and the residue was recrystallized from MeOH, receiving the connection41b(58 mg, 52%) as a white crystalline solid.1H NMR (500 MHz, CDCl3): δ 7,37-7,11 (m, 35H, Ph), of 6.49 (d, 1H,JNH,28.0 Hz, NH-2), 6,21 (d, 1H,JNH,27.5 Hz, NH-2'), 5,11 (m, 1H, H-3"'), 5,02 (d, 1H,J1',2'8.0 Hz, H-1'), 4,95-and 4.40 (m, 14H, 7×CH2Ph), br4.61 (d, 1H, H-1), 4,51 (m, 1H, H-4'), or 4.31 (t, 1H,<> J3',4'=J3',2'9.5 Hz, H-3'), 4.09 to (m, 1H, H-6), of 3.96 (m, 1H, H-3), a-3.84 (m, 1H, H-6'), with 3.79 (m, 1H, OCH2CH), 3,79-the 3.65 (m, 5H, H-6', H-6, H-5', H-5, H-3"), 3,56-3,47 (m, 2H, H-4, H-2'), 3,44 (m, 1H, H-2), to 3.36 (m, 1H, OCH2CH), 2,42-of 2.23 (m, 4H, 2×H-2", 2×H-2"'), 2,12 (t, 2H,J7.2 Hz, 2×H-2""), 1,65-1,45 (m, 8H, 2×H-4", 2×H-4"', 2×H-3"", OCH2CH2), 1,35-1,12 (m, 74H, 37 CH2), of 0.90 (m, 12H, 4×CH3);13C NMR (for 125.8 MHz, CDCl3): δ 173,4 (C=O), is 171.3 (C=O), USD 170.1 (C=O), 138,2-138,0 (Cq), 135,7-137, 6mm (Cq), 128,5-127,4 (CH arene), to 99.9 (C-1), and 99.2 (C-1'), for 80.9 (C-3), was 78.5 (C-4), to 78.3 (C-3'), to 76.3 (C-3"), to 75.7 (C-4'), 74,6, 74,9 (2×CH2Ph), 74,2-74,1 (C-5', C-5), 73,2, 73,0 (2×CH2Ph)71,0 (CH2Ph)70,6 (C-3"'), 69,6 ((OCH2CH)), 69,4-69,0 (2×P-OCH2Ph, C-6'), To 67.3 (C-6), At 56.6 (C-2), 56,0 (C-2'), 41,4-41,2 (C-2 ' C-2"'), 34,4, 34,1, 33,6 (C-4, C-4"', C-2""), To 31.9 (C-12, C-12"', C-10", C-12""'), 29,7-29,1 (C-6"->C-11", C-6"'->C-11"', C-4""->C-9""C-3””'->C-11””'), 26,1, 25,2, 25,0 (C-5, C-5”', C-3""), an increase of 22.7 (C-13 C-13”'C-11""C-13””'), a 14.1 (C-14 C-14”', C-12""C-14””'); MC-ES 1905 [M+Na]+.

Tetradecyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-β-D-glucopyranoside (41p) (OM-174-MP-TE)

Connection41b(55 mg, 29 mmol) in THF (200 ml) was first made for 17 hours in the presence of 5% Pd-C (20 mg) at room temperature in hydrogen atmosphere (6 bar). The catalyst was removed by filtration and the filtrate was concentrated in vacuum. The residue was purified using HPLC according to the invention (Method B), receiving the connection41pin the form of sodium salt)(10 mg; 27%) as a white lyophilisate. MC-ES 1273 [M+Na]+, 1251 [M+H]+.

Formylmethyl-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (32C)

To a stirred solution of compound18(300 mg; 0,17 mmol) in THF/N2About 4:1 (5 ml) at room temperature was added periodate sodium (182 mg; 0.85 mmol). After stirring over night the reaction mixture was diluted with EtOAc and the mixture is washed with saturated aqueous NaHCO3. The organic layer was washed with saline, dried over MgSO4and the solvent was removed in vacuum. Flash chromatography of the residue on silica gel (Petroleum ether/EtOAc, 2:3) gave compound32C(250 mg; 85%) as a colourless oil. [α]D+ 26 (c 0,90, CHCl3);1H NMR (500 MHz, CDCl3): δ 9,42 (1H, s, CHO), 7,37-7,10 (m, 35H, Ph), 6,55 (d, 1H, JNH,2of 9.2 Hz, NH-2), 6,10 (d, 1H, JNH,2'7.7 Hz, NH-2'), 5,07 (m, 1H, H-3”'), of 4.95 (d, 1H, J1',2'and 7.6 Hz, H-1'), 4,94-and 4.40 (m, 15H, 7×CH2Ph, H-4'), 4,70 (d, 1H, J1,23.6 Hz, H-1), 4,35-of 4.25 (m, 2H, H-2, H-3'), of 4.05 (d, 1H, H-6), 3.95 to a 3.75 (m, 5H, H-5, OCH2CHO, H-6', H-3”), 3.75 to the 3.65 (m, 3H, H-6', H-5', H-3), of 3.60 (m, 1H, H-6), 3,55 is 3.40 (m, 2H, H-4, H-2'), 2,50-of 2.15 (m, 4H, 2×H-2", 2×H-2”'), 2,13 (t, 2H,J7.5 Hz, 2×H-2””), 1,70-1,45 (m, 6H, 2×H-4”, 2×H-4”', 2×H-3””), 1,35-1,12 (m, 52H, 26 CH2), of 0.90 (m, 9H, 3×CH3);13C NMR (for 125.8 MHz, CDCl3 ): δ 199,26 (CHO), 173,4 (C=O), is 171.3 (C=O)to 170.2 (C=O), 138,2-138,0 (Cq), 137, 6mm (Cq), 135,7 (Cq), 135,6 (Cq), 128,7-RUB 127.3 (CH arene), of 99.5 (C-1'), to 98.4 (C-1), an 80.2 (C-3), to 78.3 (C-4, C-3'), to 76.7 (C-3”), 75,9 (C-4'), 74,8, 74,7 (2×CH2Ph)74,2 (C-5'), 73,2, 73,1, 73,0 (2×CH2Ph-OCH2CHO), 71,2 (CH2Ph, C-5), 70,6 (C-3”'), 69,3, 69,2 (2×P-OCH2Ph)69,0 (C-6'), 68,1 (C-6), At 56.6 (C-2'), 52,3 (C-2), 41,4-41,2 (C-2 ' C-2”'), 34,4, 34,0, 33,8 (C-4, C-4”', C-2””), To 31.9 (C-12, C-12”', C-10””), 29,6-29,1 (C-6”->C-11, C-6”'->C-11”', C-4””->C-9””), 25,2, 25,1, 24,9 (C-5, C-5”', C-3””), an increase of 22.7 (C-13 C-13”'-11””), a 14.1 (C-14, C-14'”, C-12””); MS-ES 1750 [M+Na]+.

2-Hydroxyethyl-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (32b)

To a stirred solution of compound32c(119 mg; 69 mmol) in EtOH/CH2Cl24:1 (5 ml) at 0°C was added sodium borohydride (3 mg; 75 mmol). After stirring for 10 min the reaction mixture was extinguished saturated aqueous NH4Cl and diluted with CH2Cl2. The organic layer was extracted, washed with brine, dried over MgSO4. The solvent is evaporated and the residue was recrystallized from EtOH to obtain the connection32b(100 mg; 84%) as a white crystalline solid. TPL 175°C (EtOH); [α]D+ 27 (c 0,56, CHCl3); vmaxcm-13301, 2920, 2850, 1724, 1649, 1543, 1453, 1358, 1264, 1008, 731, 694;1 H NMR (500 MHz, CDC13): δ 7,40-7,10 (m, 35H, Ph), 6,44 (d, 1H,JNH,2'7,0 Hz, NH-2'), 6,40 (d, 1H,JNH,2the 10.1 Hz, NH-2), 5,14 (m, 1H, H-3”'), 5,00-4,42 (m, 14H, 7×CH2Ph)4,88 (m, 1H,J1',2'8,72 Hz, H-1'), to 4.73 (d, 1H,J1,23.6 Hz, H-1), a 4.53 (m, 1H, H-4'), 4,47 (m, 1H, H-3'), the 4.29 (DDD, 1H,JNH,2=J2,3the 10.1 Hz,Jl,23.6 Hz, H-2), is 4.15 (d, 1H, H-6), of 3.99 (m, 1H, H-5), 3,86 (m, 2H, H-6', H-3”), of 3.77-of 3.60 (m, 2H, H-6', H-5'), 3,66 (t, 1H,J3,4=J2,3the 10.1 Hz, H-3), 3,60-to 3.33 (m, 7H, H-6, H-4, H-2', OCH2CH2, OCH2CH2), 2,42 (m, 1H, H-2”'), of 2.28 (m, 3H, 2×H-2', H-2”'), of 2.15 (t, 2H,J7.5 Hz, 2×H-2””), 1,70-1,45 (m, 6H, 2×H-4”, 2×H-4”', 2×H-3””), 1,35-1,12 (m, 52H, 26 CH2), of 0.90 (m, 9H, 3×CH3);13C NMR (for 125.8 MHz, CDCl3): δ to 173.3 (C=O), 171,0 (C=O), 170,5 (C=O), 138,2-138,0 (Cq), 137, 6mm (Cq), 135,7 (Cq), 135,6 (Cq), 128,7-RUB 127.3 (CH arene), the 98.9 (C-1'), to 98.5 (C-1), of 80.3 (C-3), of 78.6 (C-4), of 78.1 (C-3'), to 76.7 (C-3”), 75,9 (C-4'), 74,8, 74,7 (2×CH2Ph), 74,0 (C-5'), 73,2, 73,1 (2×CH2Ph)72,3 (CH2OH), 71,2 (CH2Ph), and 70.8 (C-5), 70,6 (C-3”'), 69,3, 69,2 (2×P-OCH2Ph), to 68.8 (C-6'), of 68.2 (C-6), 61,8 (OCH2CH2), to 57.1 (C-2'), 52,5 (C-2), 41,4-41,2 (C-2 ' C-2”'), 34,4, 34,0, 33,7 (C-4, C-4”'C-2””), of 31.8 (C-12, C-12”', C-10””), 29,6-29,0 (C-6”->C-11, C-6”'->C-11”', C-4””->C-9””), 25,1, 25,0, 24,9 (C-5, C-5”'C-3””), and 22.6 (C-13 C-13”'C-11””), 14,0 (C-14 C-14”'C-12””); MC-ES 1753 [M+Na]+.

2-Hydroxyethyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranose is d (32) (OM-174-MP-SW)

Connection32b(160 mg, 92 mmol) in THF (100 ml) was first made for 17 hours in the presence of 5% Pd-C (50 mg) at room temperature in hydrogen atmosphere (6 bar). The mixture is then neutralized Et3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified using HPLC according to the invention (Method D), receiving the connection32(as sodium salt)(50 mg; 49%) as a white lyophilisate. MS-ESI 1143 [M-H+2Na]+, HRMS-ESI calculated for C54H102N2O18Na2P [M-H+2Na]+1143,6660 found 1143,6659.

2-(Dibenzalacetone)ethyl-3,4-di-About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (33b)

To a stirred solution of compound32b(125 mg, 72 mmol) and commercially available solution of 1N-tetrazole in CH3CN (~0,45 M) (321 μl, 0.14 mmol) in CH2Cl2(5 ml) at room temperature was added dibenzyl diethylphosphoramidite (29 μl; 0.11 mmol). Stirring was continued at room temperature for 10 minutes and then the solution was cooled to -20°C. Then the solution was added mCPBA (57-86%, 46 mg; 0.27 mmol) in CH2Cl2(3 ml) and the solution was stirred for 30 minutes at -20°C. was Added a 10% aqueous solution thiosul the ATA sodium (5 ml) and the mixture was stirred for 10 minutes, then was diluted with EtOAc and the organic phase was separated. The organic layer was sequentially washed with 10% aqueous solution of Na2S2O3(3×), saturated aqueous NaHCO3(2×), N HCl solution (1×) and brine. The organic phase was dried over MgSO4and the solvent was removed in vacuum. Flash chromatography of the residue on silica gel (Petroleum ether/EtOAc, 1:1-1:2) gave compound33b(130 mg, 90%) as a colourless oil.1H NMR (500 MHz, CDCl3): δ 7,40-7,10 (m, 45H, Ph), 7,07 (d, 1H,JNH,29.4 Hz, NH-2), 6,56 (d, 1H,JNH,2'7,4 Hz, NH-2'), 5,09 (m, 1H, H-3”'), of 5.05 (m, 1H,J1',2'and 7.8 Hz, H-1'), 5,00 is 4.36 (m, 18H, 9×CH2Ph), of 4.66 (d, 1H, J1,2the 3.2 Hz, H-1), 4,50 (m, 1H, H-4'), 4,43 (m, 1H, H-3'), to 4.33 (DDD, 1H,JNH,2=J2,39.4 Hz,Jl,2the 3.2 Hz, H-2), 4,10 (d, 1H, J6,6of 10.0 Hz, H-6), of 4.05 (m, 1H, OCH2CH2OP)3,93 (m, 1H, OCH2CH2OP), 3,88 (m, 1H, H-3”), 3,83 (d, 1H, J6',6'of 10.0 Hz, H-6'), 3,80 (m, 1H, H-5), of 3.73-3,63 (m, 3H, H-5', H-3, OCH2CH2OP)3,62 (DD, 1H, H-6), 3,51 (t, 1H,J3,4=J4,59.5 Hz, H-4), to 3.38 (DDD, 1H,JNH,2'7,4 Hz,J1',2'7,8 Hz,J2',3'8,8 Hz, H-2'), and 3.31 (m, 1H, OCH2CH2OP)of 2.44 (DD, 1H,J2”,3”7.5 Hz,J2”,2”of 14.7 Hz, H-2”), a 2.36 (m, 2H, H-2”', H-2”), of 2.23 (DD, 1H,J2”',3”'at 5.3 Hz,J2”',2”'15.2 Hz, H-2”'), 2,10 (t, 2H, J 7.5 Hz, 2×H-2””), 1,60-1,40 (m, 6H, 2×H-4”, 2×H-4”', 2×H-3””), 1,35-1,05 (m, 52H, 26 CH2), to 0.88 (m, 9H, 3×CH 3);13C NMR (for 125.8 MHz, CDCl3): δ to 173.3 (C=O), which is 171,5 (C=O), 170,3 (C=O), 138, 7mm (Cq), 138,5 (Cq), 138,3 (Cq), 138,2 (Cq), 138,0 (Cq), Br135.8 (Cq), 135,7 (Cq), 135,6 (Cq), 135,5 (Cq), 135,4 (Cq), of 128.6-127,8 (CH arene), and 99.2 (C-1'), by 98.7 (C-1), 80,8 (C-3), 78,1 (C-4, C-3'), with 76.8 (C-3”), 76,0 (C-4'), 74,9, 74,8 (2×CH2Ph)74,2 (C-5'), 73,2, 73,1 (2×CH2Ph), and 71.4 (CH2Ph), 70,7-70,6 (C-5, C-3”'), 69,5-69,3 (4×P-OCH2Ph), for 69.1 (C-6), to 68.0 (C-6'), 67,2 (OCH2CH2O-P)66,6 (OCH2CH2O-P)57,0 (C-2'), 52,5 (C-2), 41,6-41,3 (C-2 ' C-2”'), 34,4, 34,2, 34,0 (C-4, C-4”', C-2””), to 31.9 (C-12, C-12”', C-10””), 29,6-29,1 (C-6”->C-11, C-6”'->C-11”', C-4””->C-9””), 25,2, 25,0, 24,9 (C-5, C-5”', C-3””), an increase of 22.7 (C-13 C-13”'-11””), a 14.1 (C-14 C-14”'C-12””).

2-(Phosphonooxy)ethyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranoside (33) (OM-174-MP-EP)

Connection33b(107 mg, 54 mmol) in THF (80 ml) was first made for 17 hours in the presence of 5% Pd-C (70 mg) at room temperature in hydrogen atmosphere (6 bar). The mixture is then neutralized Et3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified using HPLC according to the invention (Method D), receiving the connection33(as sodium salt)(35 mg; 55%) as a white lyophilisate. MS-ESI 1245 [M-2H+3Na]+, HRMS-ESI calculated for C54H102N2O21Na3P2[M-2H+3Na]+1245,6143 found 1245,6136.

2 Carbox the methyl-3,4-di- About-benzyl-6-About-{3,6-di-About-benzyl-4-O-(dibenzalacetone)-2-deoxy-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil}-2-[(R)-3-benzyloxycarbonylamino]-2-deoxy-α-D-glucopyranosid (35d)

To a stirred solution of compound32C(100 mg; 58 mmol), NaH2PO4.H2O (8 mg, 58 mmol), 2-methyl-2-butene (28 μl, 260 mmol) in THF/N2About 4:1 (5 ml) at room temperature was added sodium chlorite (20 mg; 173 mmol). After stirring for 6 hours the reaction mixture was extinguished 1M HCl solution (1 ml) and was diluted in CH2Cl2. The organic layer was extracted, washed with brine, dried over MgSO4and the solvent was removed in vacuum. Flash chromatography of the residue on silica gel (CH2Cl2/Acetone, 5:1 + 1% Asón) gave compound35d(67 mg; 66%) as a white solid. MC-1766 ES [M+Na]+.

2-Carboxymethyl-2-deoxy-6-About-[2-deoxy-4-O-(dihydroxyfumaric)-2-[(R)-3-dodecadodecahedron]-β-D-glyukopiranozil]-2-[(R)-3-hydroxyacetylamino]-α-D-glucopyranosid (35c) (OM-174-MP-CM)

Connection35d(67 mg, 38 mmol) in THF (20 ml) was first made for 17 hours in the presence of 5% Pd-C (25 mg) at room temperature in hydrogen atmosphere (6 bar). The mixture is then neutralized Et3N (500 μl) and the catalyst was removed by filtration. The filtrate was concentrated in vacuum and the residue was purified using HPLC according to the invention (Method D), receiving the connection35S(as sodium salt)(25 mg, 58%) as a white lyophilisate. MS-ESI 1179 [M-2H+3Na]+, HRMS-ESI calculated for C54H99N2O19Na3P [M-2H+3Na]+1179,6273 found 1179,6275.

BIOLOGICAL ACTIVITY

1.metod processing And

The products were dissolved in a mixture of THF/water (1:1 vol./vol.). Processing using preparative reverse phase HPLC was performed under the following conditions:

Column:VYDAC C18, 22×250 mm, 10 μm, 300 Å

Mobile phase:

A: Acetonitrile - water (1:1 vol./vol.), 5 mm tetrabutylammonium monobasic

In: 2-propanol - water (9:1 vol./about.) 5 mm tetrabutylammonium monobasic

Flow rate:20 ml/min

Elution:

Time (min)% the mobile phase (In)
010
50100
5510
6010

Detection:UV, 210 nm (wavelength)

The fractions containing compounds in the form of tetrabutylammonium salt, was collected and concentrated using absorption on HPLC, VYDAC C18, 22×250 mm, 10 μm, 300 Å. Sodium salt compounds obtained by leaching 200 m the monobasic solution of sodium phosphate in water, pH 4.2 + 2-propanol (9:1, vol/about.)(5 volumes). After removal of excess monobasic phosphate by passing through 5 volumes of water + 2-propanol (9:1 vol./about.) the connection suirable a solution of water + 2-propanol (1:9 vol/vol.).

After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

2.metod processing

The products were dissolved in a mixture of THF/water (1:1 vol./vol.). Processing using preparative reverse phase HPLC was performed under the following conditions:

Column:VYDAC C18, 22×250 mm, 10 μm, 300 Å

Mobile phase:

A: Acetonitrile - water (1:1 vol./vol.), 5 mm tetrabutylammonium monobasic

In: 2-propanol - water (9:1 vol./about.) 5 mm tetrabutylammonium monobasic

Flow rate:20 ml/min

Elution:

Time (min)% the mobile phase (In)
010
50100
5510
6010

Detection:UV, 210 nm (wavelength)

The fractions containing compounds in the form of tetrabutylammonium salt, was collected and concentrated using absorb the AI on HPLC, VYDAC C18, 22×250 mm, 10 μm, 300 Å. Sodium salt compounds obtained by washing with 100 mm dibasic solution of sodium phosphate - monobasic solution of sodium phosphate in water, pH 7.5 + 2-propanol (9:1, vol/about.)(5 volumes) + 2-propanol (9:1, vol/about.)(5 volumes). After removal of excess of monobasic sodium phosphate - dibasic phosphate by passing through 5 volumes of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

Motod processing

The products were dissolved in a mixture of THF/water (1:1 vol./vol.). Processing using preparative reverse phase HPLC was performed under the following conditions:

Column:VYDAC C18, 22×250 mm, 10 μm, 300 Å

Mobile phase:

A: Acetonitrile - water (1:1 vol./vol.), 5 mm tetrabutylammonium monobasic

In: 2-propanol - water (9:1 vol./about.) 5 mm tetrabutylammonium monobasic

Flow rate:20 ml/min

Elution:

Time (min)% the mobile phase (In)
010
50100
5510
6010

Detection:UV, 210 nm (wavelength)

The fractions containing compounds in the form of tetrabutylammonium salt, was collected and concentrated using absorption on HPLC, VYDAC C18, 22×250 mm, 10 μm, 300 Å. Sodium salt compounds obtained by leaching of 200 mm dibasic solution of sodium phosphate in water, pH of 9.2 + 2-propanol (9:1, vol/about.)(5 volumes). After removal of excess dibasic phosphate by passing through 5 volumes of water + 2-propanol (9:1 vol./about.) the connection suirable a solution of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

Motod processing D

The products were dissolved in a mixture of THF/water (1:1 vol./vol.). Processing using preparative reverse phase HPLC was performed under the following conditions:

Column:VYDAC C18, 22×250 mm, 10 μm, 300 Å

Mobile phase:

A: Acetonitrile - water (1:1 vol./vol.), 5 mm tetrabutylammonium monobasic

In: 2-propanol - water (9:1 vol./about.) 5 mm tetrabutylammonium monobasic

Flow rate:20 ml/min

Elution:

Time (min)% the mobile phase (In)
010
50 100
5510
6010

Detection:UV, 210 nm (wavelength)

The fractions containing compounds in the form of tetrabutylammonium salt, was collected and concentrated using absorption on HPLC, VYDAC C18, 22×250 mm, 10 μm, 300 Å. Sodium salt compounds obtained by washing with 10 g/l solution of sodium chloride in water, pH 7.0 + 2-propanol (9:1, vol/about.)(5 volumes). After removal of excess sodium chloride by passing through 5 volumes of water + 2-propanol (9:1 vol./about.) the connection suirable a solution of water + 2-propanol (1:9 vol/vol.).

After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

5. Monitoring processing

After each stage of processing fractions were subjected to study by analytical HPLC chromatography reverse phase in accordance with the following conditions:

Column:Supelcosil C18, 3 ám and 4.6×150 mm, 100 Å, Supelco

Mobile phase:

A: water:acetonitrile (1:1 vol./vol.), 5 mm tetrabutylammonium monobasic

In: water - isopropanol (1:9 vol/about.) 5 mm tetrabutylammonium monobasic

Flow rate:1 ml/min

Elution:A:In the gradient (75:25-0:100) for 20 minutes.

Detection:UV at 210 and 254 nm (wavelength)

EXAMPLE 1:

The secretion of IL-6 and TNF-α managername cells in human peripheral blood (PBMC)

The effect on IL-6 and TNF-α synthetic compounds of the invention and comparison with the original biological molecule

The connection test on the secretion of IL-6:

The compounds of the invention that were tested here on the secretion of IL-6, were:

Connection 1b(OM-174-DP), the connection16(OM-174-MP); connection17(OM-174-MP-PR), the connection19(OM-174-MP-PD) and the connection26(OM-174-MP-AC).

In addition, for comparison was also tested the biological activity of the original molecule, OM-174-DP (P3).

Then in another series of experiments, the following compounds were tested for the secretion of TNF-α human RVMS:

Connection 1b(OM-174-DP), the connection16(OM-174-MP); connection17(OM-174-MP-PR), the connection19(OM-174-MP-PD), the connection26(OM-174-MP-AC), the connection41p(OM-174-MP-TE), the connection32(OM-174-MP-SW), the connection33(OM-174-MP-EP) and the connection35S(OM-174-MP-CM).

Introduction and explanation of:

The production of IL-6 managername cells in human peripheral blood (RVMS) is an important in vitro test for screening new compounds for the ability to stimulate the immune system. IL-6 is a multifunctional cytokine that plays an important role in protecting the host, acute-phase reactions and hematopoiesis.

F is a torus tumor necrosis (TNF-α) is pleiotrophin cytokine, produced a wide variety of cell types, mainly hematopoietic, but also not of hematopoietic origin. TNF-α is required to address the multiple infectious agents.

Therefore, activation of these cytokines, the compounds of the invention may represent important therapeutic value.

Ways:

Preparation of human RVMS and culture of cells:

Peripheral blood from healthy donors (Centre de transfusion, Hôpital Universitaire, Geneva) was centrifuged to obtain a yellow shell. Yellow shell mixed with a balanced physiological saline Hanks (HBSS, Sigma, Buchs, Switzerland), was layered on Ficoll Paque Plus (Amersham Pharmacia) to 1,077 g/ml and centrifuged (2800 rpm./min, 20°C, 25 min). Cells collected from the intermediate space, washed twice in HBSS at 800 rpm./min for 15 minutes at room temperature and pelletized cells re-suspended in HBSS. Counting cells produced using cells Neubauer. All cell culture was performed in medium RPMI-1640 with the addition of penicillin (100 units/ml), streptomycin (100 μg/ml), L-glutamine (2 mmol/l) and 10% fetal calf serum (FCS), all of which were obtained from Sigma. To stimulate in vitro the cells were cultured at a concentration of 1×106viable cells/well.

Stimulation and measurement of IL-6 and TNF-α in super nth culture:

RVMS incubated at 37°C and in an atmosphere of 5% CO2with controls (see below) and with the products of the invention at 1, 5 and 20 µg/ml for IL-6 and at 0.2, 2 and 20 μg/ml for the secretion of TNF-α. Environment: one RPMI.

Supernatant cultures were collected after 24 hours and measured the concentration of IL-6 and TNF-α using an enzyme-linked immunosorbent assay (ELISA) (set human IL-6 and TNF-α ELISA kit, BD OptEIA, San Diego, USA) according to manufacturer's instructions. The limits of detection were respectively 10 and 8 PG/ml

The results:

The results are shown below in tables:

A: For IL-6

Table 1.1
Negative (Wednesday) and positive (LPS) controls on the production of IL-6 ± STD. (PG/ml) managername cells in human peripheral blood
Products (number) + [download]+ concentration mcg/mlConc., mg/mlIL6 (PG/ml)Article off.
WednesdayNA6,50,2
WednesdayNA20,71,5
LPS [026:B6] of 0.01 mg/ml 0,00115105627,7
LPS [026:B6] of 0.01 mg/ml0,0113194632,9
LPS [026:B6] 0.1 mg/ml0,1160971004,7

Explanation of results:

LPS induced, as expected, very high levels of IL-6 from human RPMS even at the lowest tested doses.

Table 1.2
The effect of synthetic compounds (1b) of the invention (OM-174-DP) on the production of IL-6 human RVMS in comparison with the biological parent molecule (174-P3)
Products (number) + [download]+ concentration mcg/mlConc., mg/mlIL6 (PG/ml)Article off.
OM-174-DP [P3] 1 mg/ml19,81,9
OM-174-DP [P3] 5 mg/ml518,54,2
OM-174-DP [P3 20 mg/ml 2097,712,6

(1b) OM-174-DP [SMOR-189] 1 mg/ml120,22,0
(1b) OM-174-DP [SMOR-189] 5 mg/ml557,12,8
(1b) OM-174-DP [SMOR-189] 20 mg/ml20225,57,9

Explanation of results:

Both download OM-174 induce the secretion of IL-6 human RVMS, and the levels obtained with synthetic molecule, were slightly higher than the levels obtained with the biological molecule P3.

Table 1.3
Impact 5 examples of synthetic compounds of the invention on the production of IL-6 human RVMS
Products (number) + [download]+ concentration mcg/mlConc., mg/mlIL6 (PG/ml)Article off.
(1b) OM-174-DP [SMOR-189] 1 mg/ml120,2 2,0
(1b) OM-174-DP [SMOR-189] 5 mg/ml557,12,8
(1b) OM-174-DP [SMOR-189] 20 mg/ml20225,57,9
(16) OM-174-MP [SMORII-30] 1 mg/ml181,14,5
(16) OM-174-MP [SMORII-30] 5 mg/ml5473,332,0
(16) OM-174-MP [SMORII-30] 20 mg/ml201348,222,8
(17) OM-174-MP-PR [SMORII-24] 1 mg/ml1171,69,1
(17) OM-174-MP-PR [SMORII-24] 5 mg/ml5447,645,5
(17) OM-174-MP-PR [SMORII-24] 20 mg/ml20752,437,2
(19) OM-174-MP-PD [SMORII-32] 1 mg/ml144,51,3
(19) OM-14-MP-PD [SMORII-32] 5 mg/ml 595,91,9
(19) OM-174-MP-PD [SMORII-32] 20 mg/ml20199,16,4
(26) OM-174-MP-AC [F4] 1 mg/ml1269,010,2
(26) OM-174-MP-AC [F4] 5 mg/ml51923,269,3
(26) OM-174-MP-AC [F4] 20 mg/ml208349,3483,5

Explanation of results:

In comparison with the biological parent molecule OM-174-DP (download P3) synthetic molecule (1b) induces higher levels of IL-6 by human monocytes.

This is the same for OM-174-MP (16), because the biological product has not induced the production of IL-6 even at the highest tested dose (20 µg/ml, not shown), whereas the related synthetic molecule OM-174-MP (connection16) induces until 1348 PG/ml IL-6.

Basically all tested synthetic molecules (1b, 16, 17, 19and26)were able to induce the secretionIL-6.

C) For TNF-α

Table 1.4
Negative (Wednesday) and positive (LPS) controls on the production of TNF-α ± STD. (PG/ml) managername cells in human peripheral blood
Products (number) + [download]+ concentration mcg/mlConc., mg/mlTNF-α (PG/ml)Article off.
WednesdayNA246,8
WednesdayNA298,8
LPS [026:B6] of 0.2 μg/ml0,216112411
LPS [026:B6] 2 mg/ml214237494
LPS [026:B6] 20 mg/ml2013602472

Explanation of results:

LPS induced, as expected, very high levels of TNF-α human RPMS even at the three tested doses.

That the person 1.5
The effect of synthetic compounds (1b) of the invention (OM-174-DP) on the production of TNF-α human RVMS in comparison with the biological parent molecule (174-P3)
Products (number) + [download]+ concentration mcg/mlConc., mg/mlTNF-α (PG/ml)Article off.
OM-174-DP [P3] of 0.2 μg/ml0,22,52,1
OM-174-DP [P3] 2 mg/ml27,11,1
OM-174-DP [P3] 20 mg/ml2030031
(1b) OM-174-DP [SMORII-132] of 0.2 μg/ml0,237a 4.9
(1b) OM-174-DP [SMORII-132] 2 mg/ml211722
(1b) OM-174-DP [SMORII-132] 20 mg/ml20110569

Explanation of results:

Both download OM-174 induce the secretion of TNF-α human RVMS, aurone, obtained with a synthetic molecule, were slightly higher than the levels obtained with the biological molecule P3.

Table 1.6
The impact of 9 examples of synthetic compounds of the invention on the production of TNF-α human RVMS
Products (number) + [download]+ concentration mcg/mlConc., mg/mlTNF-α (PG/ml)Article off.
(1b) OM-174-DP [SMORII-132] of 0.2 μg/ml0,237a 4.9
(1b) OM-174-DP [SMORII-132] 2 mg/ml211722
(1b) OM-174-DP [SMORII-132] 20 mg/ml20110569
(16) OM-174-MP [SMORII-30] of 0.2 μg/ml0,20,690,1
(16) OM-174-MP [SMORII-30] 2 mg/ml24914
(16) OM-174-MP [SMORII-30] 20 mg/ml20 170516
(17) OM-174-MP-PR [KAS1-108] of 0.2 μg/ml0,2464,6
(17) OM-174-MP-PR [KAS1-108] 2 mg/ml247416
(17) OM-174-MP-PR [KAS1-108] 20 mg/ml20211435
(19) OM-174-MP-PD [SMORII-113] of 0.2 μg/ml0,29,24,1
(19) OM-174-MP-PD [SMORII-113] 2 mg/ml25,12,7
(19) OM-174-MP-PD [SMORII-113] 20 mg/ml2011,26,0
(26) OM-174-MP-AC [KAS1-103] of 0.2 μg/ml0,229,33,6
(26) OM-174-MP-AC [KAS1-103] 2 mg/ml2126433
(26) OM-174-MP-AC [KAS1-103] 20 mg/ml207016186
(41c) OM-174-MP-TE [KAS2-10] of 0.2 μg/ml0,257034
(41c) OM-174-MP-TE [KAS2-10] 2 mg/ml26036306
(41c) OM-174-MP-TE [KAS2-10] 20 mg/ml209769119
(32) OM-174-MP-EO [SMORII-74] of 0.2 μg/ml0,25,53,5
(32) OM-174-MP-EO [SMORII-74] 12 mcg/ml27,86,4
(32) OM-174-MP-EO [SMORII-74] 20 mg/ml2025120
(33) OM-174-MP-EP [SMORII-83] of 0.2 μg/ml0,28,01,4
(33) OM-174-MP-EP [SMORII-83] 2 mg/ml28,31,7
(33) OM-174-MP-EP [SMORII-83] 20 mg/ml2021the 4.7
(35c) OM-174-MP-CM [SMORII-135] of 0.2 μg/ml 0,2483,6
(35c) OM-174-MP-CM [SMORII-135] 2 mg/ml250211
(35c) OM-174-MP-CM [SMORII-135] 20 mg/ml2059111

Explanation of results:

With the exception of the synthesized molecules (19and (33) all the synthesized molecules (1b,16,17,26,41cand32) induced high levels of TNF-α by human monocytes, which suggests that they could act as immunostimulating drugs.

The results give reason to believe that it could be worthwhile to develop molecules (19i.e.OM-174-MP-PDand33i.e.OM-174-MP-EPas “anti-inflammatory” drugs. Very interestingly, both compounds (19and (33) showed“anti-inflammatory”properties.

Moreover, the connection (19) showed anti-inflammatory properties as “prophylactic”or “therapeutic” model LACK-induced asthma (see example 6), and inhibited induced release of compound 48/80 the secretion of histamine murine mast cells (see example 7). On this last models in example 7 was also active connection ( 33).

EXAMPLE 2:

Modification of the biological activity of a biological connection OM-174-DP:

The increased TNF-α-displayed secretion TNR-1 cells using the original method of cleaning the original biological molecules OM-174-DP

Compound:

The compounds of the invention tested here, are

The original biological loading (GMP004) molecule of the invention OM-174-DP was tested or from the original solution, or re-cleaned as described below, mainly by changing the pH of the HPLC mobile phase.

Introduction and explanation of:

The tumor necrosis factor (TNF-α) is pleiotrophin cytokine produced by a wide variety of cell types, mainly hematopoietic, but also not of hematopoietic origin. TNF-α is necessary for elimination of numerous infectious agents (Candida albicans, Listeria monocytogenes, Mycobacterium ...) and exhibits strong anti-inflammatory effects, for example, by inducing the expression of adhesion molecules such as VCAM-1, intercellular adhesion molecule 1 (ICAM-1), E-secretion in endothelial cells and other cell types.

Overproduction TNF, however, also involved in the pathogenesis of various diseases, such as rheumatoid arthritis, insulin-dependent diabetes and inflammatory bowel disease, particularly disease CZK is.

Therefore, TNF-α is required to run immunological responses; however, in order to avoid inflammatory pathologies, it is necessary to cope with this production.

One batch of the original biological product OM-174-DP (GMP004) reformulated in accordance with the two methods described below:

Methods

1. Method And

Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm. The fractions containing compounds in the form tetrabutylammonium salt, was collected and concentrated using adsorption on HPLC. The sodium salt of the compounds were obtained by flushing 200 mm monoammonium solution of sodium phosphate in water, pH to 4.23 + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess nonoonono phosphate passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

The resulting compounds were then tested with bringing pH (7.5) or without it on TNR-1 cells to analyze their capacity to induce the secretion of TNF-α (see below).

2. Method In

Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm. The fractions containing compounds in the form of t is tributylammonium salt, collected and concentrated using adsorption on HPLC. The sodium salt of the compounds were obtained by flushing 100 mm monoammonium solution of sodium phosphate dibasic phosphate in water,pH 7.5+ 2-propanol (9:1, vol/about.) (5 volumes) + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess nonoonono phosphate, sodium dibasic phosphate, sodium passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

The resulting compounds were then tested with bringing pH (7.5) or without it on TNR-1 cells to analyze their capacity to induce the secretion of TNF-α (see below).

Culture TNR-1 cell:

TNR-1, line, leukemia monocytic human cells, obtained from ATS (Manassas, USA)

Cells TNR-1 (106cells/ml, 200 l/well were cultured in 96-well flat-bottom plate with tissue culture (Costar) in RPMI medium with addition of 10% human serum (HS; Gibco-BRL)containing 10 mm HEPES buffer, 1 mm pyruvate, 0.1 M nonessential amino acids, 2 mm glutamine, 50 mm 2-mercaptoethanol, 100 u/ml penicillin and 10 mg/ml streptomycin (complete medium). Cells were stimulated by different concentrations of the compounds of the invention during various time periods at 37°C in wvla the applicable incubator with 5% CO 2. Supernatant culture was collected and stored at -20°C until determination of cytokines by ELISA method.

Stimulation and measurement of TNF-α in supernatant culture:

Cells were incubated at 37°C and in an atmosphere of 5% CO2with the products of the invention at 0.2, 2 and 20 ág/ml: environment: one RPMI.

Supernatant cultures were collected after 24 hours and measured the concentration of TNF-α using an enzyme-linked immunosorbent assay (ELISA) (BD OptEIA, San Diego, USA) according to manufacturer's instructions. The limit of detection was 8 PG/ml

Results:

The results are shown below in table 2.1:

Table I
The effect of the compounds of the invention on the production of TNF-α TNR-1 cell
Product[party]method/dose/LVTNF-α (PG/ml)Article off.
Wednesday0,510,33
Wednesday0,681,24
OM-174-DP [GMP004] (0.2 ág/ml)13,030,39
OM-174-DP [GMP004] (2 µg/ml)64,42OM-174-DP [GMP004] (20 µg/ml)193,0214,68
OM-174-DP [GMP004] according to the method A (0.2 ág/ml)10,853,59
OM-174-DP [GMP004] according to the method A (2 µg/ml)11,2012,56
OM-174-DP [GMP004] according to the method A (20 µg/ml)74,907,22
OM-174-DP [GMP004] according to the method A (0.2 ág/ml), then pH 7.51,220,46
OM-174-DP [GMP004] according to the method A (2 μg/ml), then pH 7.5a 9.091,20
OM-174-DP [GMP004] according to the method A (20 μg/ml), then pH 7.596,104,80
OM-174-DP [GMP004] according to the method B (0.2 ág/ml)205,9643,94
OM-174-DP [GMP004] according to the method B (2 µg/ml)447,366,00
OM-174-DP [GMP004] according to the method B (20 ág/ml)591,0923,14
OM-174-DP [GMP004] according to the method B (0.2 ág/ml), then pH 7.5 141,553,86
OM-174-DP [GMP004] according to the method B (2 μg/ml), then pH 7.5473,8918,56
OM-174-DP [GMP004] according to the method B (20 µg/ml), then pH 7.5636,7851,78

Explanation of results:

The amount of TNF-α, obtained with OM-174-DP biological products [GMP004] at 20 μg/ml was 193 PG/ml Here the authors demonstrate treatment method, which enhances the biological activity of the original biological product by a factor of 3.

In conclusion, the method of purification described here could therefore be applied to or adapted for the clinic to enhance therapeutic activity of the drug.

EXAMPLE 3:

The effect of biological activity of 3 synthetic monophosphoryl compounds of the invention: Production of nitric oxide by mouse macrophages stimulated three manufacturername synthetic compounds of the invention

Compound:

The compounds of the invention presented here, are

Connection16(OM-174-MP); connection17(OM-174-MP-PR) and the connection19(OM-174-MP-PD).

Introduction and explanation of:

The production of nitric oxide (NO) by macrophages is important in in vitro test for when rining new compounds for the ability to stimulate the immune system. It is an important signaling molecule in the body of mammals, including humans, one of the few known gaseous signaling molecules.

Molecule nitric oxide is a free radical, which makes it very reactive and unstable. In the body nitric oxide is synthesized from arginine and oxygen by various enzymes synthase nitric oxide (NOS) and the subsequent recovery of inorganic nitrate.

Macrophages produce nitric oxide to kill invading bacteria. In some conditions, this can backfire: fulminant infection (sepsis) causing excessive production of macrophage nitric oxide, which leads to vasodilation (dilation of blood vessels), probably one of the main causes of hypotension (low blood pressure) in sepsis.

Biological functions of nitric oxide were discovered in the 1980s, and nitric oxide was named “Molecule of the year” by Science magazine in 1992, it is Considered that published annually about 3,000 scientific articles about the biological role of nitric oxide.

Therefore, its activation by the compounds of the invention may represent important therapeutic value.

Materials and methods:

Experimental analysis of the production of nitric oxide by mouse macrophages: Male mice C57/BL6 six-week-old males six weeks of age, SPF quality, Charles Rivier, FR) killed with CO2inhalations. From the back of the removed hip, femur and tibia. Bone marrow was extracted from the lumen by injecting modified eagle medium of Dulbecco (DH) through the bone after cutting both end parts. After washing, the stem cells are re-suspended (40000 cells/ml) in the medium DH with the addition of 20% horse serum and 30% supernatant of L929 cells. Cell suspension was incubated for 8 days in an incubator at 37°C in an atmosphere of 8% CO2and plump. Macrophages were then separated ice PBS, washed and re-suspended in the medium DH with the addition of 5% fetal bovine serum (FCS), amino acids and antibiotics (Wednesday DHE). Cell density was adjusted to 700,000 cells/ml Directly in microtiter tablets diluted aqueous solutions of the products in the environment DHE. Products experience in three repetitions, and each microtiter tablet includes a negative control composed of environment. The final volume in each well is 100 μl. 100 μl of cell suspension is added to dilute the products and the cells incubated for 22 hours in an incubator at 37°C in an atmosphere of 8% CO2saturated with moisture. At the end of the incubation period, 100 μl of supernatant is transferred to another microtiter plate is through the reaction Griess determine the concentration of nitrite, produced in each supernatant. To each well was added 100 μl of Griess reagent (5 mg/ml sulfanilamide + 0.5 mg/ml of hydrochloride of N-(1-naphthyl)Ethylenediamine in 2.5% aqueous phosphoric acid. Microtiter tablets read by a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm against the reference at 690 nm. The nitrite concentration is proportional to the content of the resulting nitric oxide. The content of nitrite is determined on the basis of the standard curve. The results are given as average ± standard deviation and are drawn graphically as a curve of the response to the dose.

Results:

The results are shown in Fig. Three of the tested molecules were able to induce high levels of nitric oxide by mouse macrophages. Connection19(OM-174-MP-PD) was active at lower doses (0.01 mg/ml) in this test than the connection16(OM-174-MP) and17(OM-174-MP-PR).

EXAMPLE 4:

The effect of purification according To the method for the production of IL-6 managername cells in human peripheral blood previously inactive synthetic compounds of the invention obtained by the method D:

Tested compounds:

The compounds of the invention presented here, are

Party 14 synthetic product OM-174-DP (connection1bprocessed or unprocessed method D (see below).

Introduction and explanation of:

Party connection represented here (synthetic OM-174-DP), was initially inactive, because it was obtained according to method D, in which the final pH was handled improperly. In fact, the party had a pH of 14 4,88 before to be treated according To the method (see below). Very interestingly, the method of purification (see example 2) significantly increased the activity of the party 14.

Cm. also the example 1, what is the description of the biological effect on IL-6.

Cleaning method D

The method was described in detail above.

Synthetic product OM-174-DP(1b) (party 14) was dissolved in a mixture of THF-water (1:1 vol./vol.). Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm.

The fractions containing compounds in the form tetrabutylammonium salt, was collected and concentrated using adsorption HPLC, VYDAC C18, 22×250 mm, 10 µm, 300Å. Sodium salt compounds obtained by washing with 10 g/l solution of sodium chloride in water, pH 7.0 + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess sodium chloride passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.).

After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt. When and who use this method (D) final pH was not regulated properly. In fact, LV party 14 was 4,88.

Then the party was re-processed according To the method (see below).

Method of cleaning In

The method described in detail above. Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm. The fractions containing compounds in the form tetrabutylammonium salt, was collected and concentrated using adsorption on HPLC. Sodium salt compounds obtained by washing a 100 mm solution of dibasic sodium phosphate-nonoonono of sodium phosphate in water, pH 7.5 + 2-propanol (9:1, vol/about.) (5 volumes) + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess nonoonono of sodium phosphate - dibasic phosphate passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.).

After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

The resulting compounds were then tested with bringing pH (7.5) or without it on TNR-1 cells to analyze their capacity to induce the secretion of TNF-α (see below).

Preparation of human RVMS and cell culture:

Peripheral blood from healthy donors (Centre de transfusion, Hôpital Universitaire, Geneva) was centrifuged to obtain a yellow shell. Yellow shell mixed with a balanced Phi is biologicheskii salt Hanks solution (HBSS, Sigma, Buchs, Switzerland), was layered on Ficoll Paque Plus (Amersham Pharmacia) to 1,077 g/ml and centrifuged (2800 rpm./min, 20°C, 25 min). Cells collected from the intermediate space, washed twice in HBSS at 800 rpm./min for 15 minutes at room temperature and pelletized cells re-suspended in HBSS. Counting cells produced using cells Neubauer. All cell culture was performed in medium RPMI-1640 with the addition of penicillin (100 units/ml), streptomycin (100 μg/ml), L-glutamine (2 mmol/l) and 10% fetal calf serum (FCS), all of which were obtained from Sigma. To stimulate in vitro the cells were cultured at a concentration of 1×106viable cells/well.

Stimulation and measurement of IL-6 in supernatant culture:

RVMS incubated at 37°C and in an atmosphere of 5% CO2with the products of the invention.

Supernatant cultures were collected after 24 hours and measured the concentration of IL-6 using an enzyme-linked immunosorbent assay (ELISA) (set human IL-6 ELISA kit, BD OptEIA, San Diego, USA) according to manufacturer's instructions. The limit of detection was 10 PG/ml

The results:

The results are presented on Fig, which shows the application of the method (i.e. use during the cleanup procedure corresponding pH) to the compounds of the invention (here is the link1b), which converts inactive connection (n is RTE 14) in a fully effective activator of human RVMS (lot 39).

EXAMPLE 5:

Modification of the biological activity of compound 1b (OM-174-DP):

Strengthening induced TNF-α secretion TNR-1 cells, differentiated into macrophages by the original method of cleaning various parties molecules OM-174-DP

Tested compounds:

The compounds of the invention presented here, are

Two biological party (P3 and GMP004) connection1b(OM-174-DP) and the following synthetic party (14) was processed according to methods a or b (see below). LPS was used as positive control.

Introduction and explanation of:

TNF-α

The tumor necrosis factor (TNF-α) is pleiotrophin cytokine produced by a wide variety of cell types, primarily of hematopoietic, but also not of hematopoietic origin. TNF-α is necessary for elimination of numerous infectious agents (Candida albicans, Listeria monocytogenes, Mycobacterium ...) and exhibits strong anti-inflammatory effects, for example, by inducing the expression of adhesion molecules such as VCAM-1, intercellular adhesion molecule 1 (ICAM-1), or E-secretion in endothelial cells and other cell types.

Overproduction TNF, however, also involved in the pathogenesis of various diseases, such as rheumatoid arthritis, insulin-dependent diabetes and inflammatory bowel disease, in particular bol is June Crown.

Therefore, TNF-α is required to run immunological responses; however, in order to avoid inflammatory pathologies, it is necessary to manage this production. Inactive synthetic party (the party “14”, see example 4) was formulated in accordance with the two methods described below):

Methods

1. Method And

Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm. The fractions containing compounds in the form tetrabutylammonium salt, was collected and concentrated using adsorption on HPLC. Sodium salt compounds obtained by washing with 200 mm monoammonium solution of sodium phosphate in water, pH to 4.23 + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess nonoonono phosphate passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

The resulting compounds were then tested with bringing pH (7.5) or without it on TNR-1 cells to analyze their capacity to induce the secretion of TNF-α (see below).

2. Method In

Purification was performed using preparative HPLC with reversed phase. UV detection was performed at 210 nm. The fractions containing compounds in formaterrormessage salt, was collected and concentrated using adsorption on HPLC. Sodium salt compounds obtained by washing a 100 mm solution of dibasic sodium phosphate - nonoonono of sodium phosphate in water, pH 7.5 + 2-propanol (9:1, vol/about.) (5 volumes) + 2-propanol (9:1, vol/about.) (5 volumes). After removal of excess nonoonono of sodium phosphate - dibasic phosphate passing through the 5 volumes of water + 2-propanol (9:1 vol./about.) connection elute solution of water + 2-propanol (1:9 vol/vol.). After dilution with water and removal of the solvent by lyophilization get a connection in the form of sodium salt.

The resulting compounds were then tested with bringing pH (7.5) or without it on TNR-1 cells to analyze their capacity to induce the secretion of TNF-α (see below).

Culture TNR-1 cell:

TNR-1 cells (see method in example 1) were cultured (5×105cells/ml) in RPMI with 10% FCS + 100 ng/ml PMA (Sigma). After 3 days adherent cells were collected and brought to a concentration of 3×105cells per well and incubated with products at 37°C With 5% CO2within 6 hours.

Supernatant cultures were collected after 24 hours and measured the concentration of TNF-α using an enzyme-linked immunosorbent assay (ELISA) (BD OptEIA, San Diego, USA) according to manufacturer's instructions. The limit of detection was 8 PG/ml

Results:

The results are presented below in 3 RA the personal tables:

Table 5.1
The production of TNF-α TNR-1 cells, differentiated into macrophages, medium, LPS and biological parties GMP004 and P3 of the original product OM-174-DP
Product [party] dose mcg/mlmg/mlAverageStocks.
Wednesday03637±145
LPS 0,0010,0015400±1989
LPS 0,010,018770±687
LPS 0,10,153165±2536
OM-174-DP [GMP004] 225045±2275
OM-174-DP [GMP004] 20201050 ±3044
OM-174-DP [P3] 224628±206
OM-174-DP [P3] 20209579±2381

Explanation of results:

LPS induces, as expected, high levels of TNF-α. The production of TNF-α induced two biological parties (P3 and GMP004) OM-174 was much lower.

Table 5.2
Comparison of the production of TNF-α TNR-1 cells, differentiated into macrophages biological party GMP004, before and after treatment by the method a or the method of the invention (with the receiving parties 54)
Product [party], method, dose mcg/mlmg/mlAverageStocks.
Wednesday03637±145
OM-174-DP [GMP004] 22 5045±2275
OM-174-DP [GMP004] 202010150±3044
OM-174-DP [SMORII-54]A1 224208±308
OM-174-DP [SMORII-54]A1 202010223±2142

OM-174-DP [SMORII-54]A2 224328±421
OM-174-DP [SMORII-54]A2 202022802±5612
OM-174-DP [SMORII-54]B1 0,20,219039±5497
OM-174-DP [SMORII-54]B1 2222049±3442
OM-174-DP [SMORII-54]B1 20203301 ±3069
OM-174-DP [SMORII-54]B2 0,20,28079±1704
OM-174-DP [SMORII-54]B2 2211401±694
OM-174-DP [SMORII-54]B2 202022513±2584

Explanation of results:

The results clearly show that the application of the method In the party GMP004 greatly increases the biological activity of the original party GMP004.

Table 5.3
Comparison of the production of TNF-alpha THP-1 cells differentiated into macrophages initially inactive synthetic party (SMORII 14) connections OM-174-DP (see example 4) and a clear enhancement of its activity by using the method In the invention (generation “39”)
Product [party], dose mcg/mlmg/mlAverageStocks.
Wednesday03637±145
OM-174-DP [SMORII-14-150306] 2214603±1030
OM-174-DP [SMORII-14-150306] 20209582±2243
OM-174-DP [SMORII-14-So] 228853±2328
OM-174-DP [SMORII-14-So] 20209894±3319
OM-174-DP [SMORII-39-060509] 2223331±3019
OM-174-DP [SMORII-39-060509] 202037637±3945

Explanation of results:

The products encoded as a party “39”, obtained from synthetic compounds called party “14”, using the processes corresponding to method C. the Process was clearly actively reinforced the th of the original molecule. In contrast, the procedure of ultrasonic treatment was without effect (a number or series so).

EXAMPLE 6:

Effect of synthetic OM-174-DP and synthetic OM-174-MP-PDon the model LACK-induced asthma, “prophylactic” and “therapeutically”

Here is an example of in vivo biological activity of two representative compounds of the invention. Using the model of allergic asthma mice published previously (described in the work of Julia et al. Immunity. 2002 Feb; 16(2):271-83), the authors aimed to investigate whether I.P. Pavlova. the introduction of synthetic molecules OM-174-DP and OM-174-MP-PDto inhibit the inflammation of the Airways, caused from LACK-sensitized mice. For this purpose, mice were treated or along with the induction of asthma (preventive model) or therapeutically (i.e. three times after mice made sensitive to the allergen protein LACK). As the reading results were calculated eosinophils in bronchoalveolar lavah (BAL), and determined the number of well-known markers of allergic asthma, namely the Th2 cytokines IL-4, IL-5 and IL-13. In addition, the recorded levels of plasma IgE.

Protocol:

Material

As control was given saline solution in the form of an aerosol

Recombinant LACK protein was producyrovtsa inE. coliand the sight of what was little more than a Ni-NTA column for carrying out chromatography on affinity as described (Mougneau et al., 1995).

- Aluminum hydroxide (Alum) were purchased from the firm of Pierce

- Cytocentrifuge is Cytospin 4 (Thermo-Shandon, Cheschire, U.K.), Cytology purchased from Thermo-Shandon, and dyes Wright and Giemsa from Sigma.

The aerosols were made using an ultrasonic atomizer Ultramed (Medicalia, Forenze, Italy)

- Anti-IgE (R35-118) in combination with Biotin were purchased from BD Biosciences (Le Pont de Claix, France)

Animals

- Female mice BALB/c ByJ 6 weeks of age were purchased at The Centre d Elevage Janvier, France). Mice were kept in free from specific pathogens conditions and fed standard food supplied by the company Safe (Augy, France).

The experimental group

Experienced following 5 groups:

-A: NEGATIVE control:

Raw LACK-sensitized provoked saline mice (3 mice)

-In: POSITIVE Control:

Raw LACK-sensitized provoked mice (6 mice)

-With: 174-DP Preventive:

Processed OM-174-DP (I.P. Pavlova.) LACK-sensitized provoked mice (6 mice)

-D: 174-MP-PDPrevention:

Processed OM-174-MP-PD(I.P. Pavlova.) LACK-sensitized provoked mice (6 mice)

-E: 174-DP Therapeutic:

Processed OM-174-DP (I.P. Pavlova.) LACK-sensitized provoked mice (6 mice)

Treatment and schedule ispite is Oh or control groups

The experiment was started at day 0. In the days 0, 2, 3, 4, 7, 9, 10, 11 and 12 mice in groups C and D were injected I.P. Pavlova. synthetic OM-174-DP (connection1band OM-174-MP-PD(connection 19respectively at a dose of 1 mg/kg (20 μg per mouse).

Mice of group E were treated therapeutically I.P. Pavlova. on days 15, 17 and 19 had injected a drug in a dose of 1 mg/kg (20 μg per mouse).

In day 1 and day 8 mice were senzibilizirani I.P. Pavlova. LACK/Alum. From 16 to 20 day all groups of mice, with the exception of group a, provoked by the aerosol solution LACK (0,15%). Group And instead received saline (NaCl 0,9%) (group a) within 40 minutes.

Method

And: Bronchoalveolar lavage (BAL) and the counting of eosinophils.

All the animals did bronchoalveolar lavage BAL (reorganization abundant flow irrigation).

Two days after the last aerosol provocation mice were drawing blood and the trachea was inserted cannula. The lungs were washed 3 times with 1 ml of warm PBS. Cells were washed in PBS and red blood cells were literally using lisanova buffer for red blood cells. Cells were additionally washed in PBS and made calculations. For differential count BAL cells were prepared drugs cytospin and stained with Wright/Giemsa. For each slide was counting at least 400 cells with a microscope to determine the number of lymphocytes, neutrophils, eosinophils and macrophages/DC/pneum is Titov (the calculation was made as other managernew cells). It informs only about the eosinophilia.

In: Determination of pulmonary cytokines:

To analyze the number of pulmonary cytokines took the light, and for the preparation of protein extracts used left lungs. For each of the left lung was isolated 400 µl. Cytokines (IL-4 and IL-13 in the first series of tests, and then IL-5 and IFN-γ) was measured using multiplex analysis using FACSArray. The results, normalized for protein content, are presented in PG/ml.

From: Definition of LACK-specific IgE:

For the analysis of specific IgE groups of mice a, b and G let the blood via cardiac puncture two days after the last aerosol and to prepare serum. LACK-specific IgE was measured using ELISA.

Results:

A) Eosinophilia

Description the number of eosinophils in broncho-alveolar lavah:

The results for each individual mouse, and the average value of each of the tested groups are shown in the table below.

Eosino×106Mouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6Mean ±SEM
A: NEG CTRL 000NDNDND0
B: POS CTRL14454092189572864836646621831421895714890441 ± 641887*
C: OM-174-DP (1b) Preventive7535938561233412541581053524792109781 ± 139453*
D: OM-174-MP-PD (19) Preventive33286640104701825229482555123305100411 ± 80738*
E: OM-174-DP (1b) Therapeutic167443802741121739125002134313923 ± 7362*
*=p<0,05 (t student)

Compared to positive asthmatic group (C) animals were treated prophylactically connections1band19find about 8 times smaller BAL eosinophilia.

In addition, when animals were injected three times with therapeutic purpose, the compound1b(group E), the number of eosinophils they significantly decreased (by a factor of 64).

In: Characterization of pulmonary cytokines

In the first series of analyses, the results for each individual mouse, and the average value of each group is shown in the table below:

IL-4 andIL-13 (join1band19)

The number of IL-4 and IL-13 were first analyzed in the lungs of treated and untreated mice.

Whereas the levels of IL-4 was very low to undetectable in PBS-provoked animals, the number of IL-4 in LACK-provoking raw (not treated) control mice was increased 20-fold. After prophylactic treatment with OM-174-DP (1b) OM-174-MP-PD(19) amounts of IL-4 in the lungs was decreased in 6 and 4 times, respectively (p<0,001 Mann &Whitney) (see table below). It should be noted that a similar decrease in the number of IL-4 (4-fold compared with group b) was obtained with therapeutic treatment of a synthetic molecule1b(group E).

Groups is/mouse Mouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6The mean IL-4 (PG/ml)SEM of IL-4
A: raw/
PBS
0,490,440,39NANANA0,440,05
B: raw/
LACK
9,884,5110,47of 6.3111,279,548,662,65
C: (1b) OM-174-DP [SMORII-39] preventive1,13,330,712,431,230,171,50**1,17
D: (19) OM-174-MP-PD [SMORII-32] preventive1,011,47,91 of 5.681,791,662,09**1,79
E: (1b) OM-174-DP [SMORII-39] therapeutic2,511,583,481,943,482,33**1,02
**=p<0,01 (Mann &Whitney), compared to positive asthmatic group (In)

The PBS-provoking mice indicator of IL-13 was in the range of detection, but the asthmatic control mice was determined amount in the form of medium-50 PG/ml (see table below, group b). Compared with the data of asthmatic mice, the number of IL-13 was 4 times lower in mice treated prophylactically OM-174-DP (connection1)(p<0,001 Mann &Whitney), and 3 times lower in mice treated with OM-174-MP-PD(connection19)(p<0,001) (see table below).

It should be noted that a similar decrease in the number of IL-13 (3-fold compared with group b) was obtained with therapeutic treatment of a synthetic molecule1b(group E).

Group/mouse Mouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6Average IL-13 (PG/ml)SEM of IL-13
A: raw/
PBS
1,061,560,38NANANA1,000,59
B: raw/
LACK
50,8326,6255,0642,9162,2165,9850,6014,32
C: (1b) OM-174-DP [SMORII-39] preventive-cal8,3925,398,224,29to 12.521,5913,40*9,54
D: (19) OM-174-MP-PD [SMORII-32] preventive-cal12,0817,18,59 30,616,1415,2116,62*7,52
E: (1b) OM-174-DP [SMORII-39] therapeutic20,355,43to 9.9323,6821,99to 26.0217,90*compared to 8.26
*=p<0,05 (Mann &Whitney), compared to positive asthmatic group (In)

Additional cytokines measured after three therapeutic injections connection1b

Because after therapeutic treatment of subjects molecules eosinophilia airway sharply declined, the authors sought in another series of tests to check the content of the lungs of additional cytokines (IL-5 and IFN-γ) (only for connection1b). The results are shown in the table below.

In fact, when compared with light is not affected by the treatment of mice in the lungs of mice exposed to treatment, the number of Th2-cytokine: IL-5, strongly reduced. After treatment with OM-174-DP (connection1bp<0,01 Mann &Whitney) number of IL-5 decreased 2.8 times.

Group/mouse Mouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6The mean IL-5 (PG/ml)SEM of IL-5
A: raw/
PBS
0,250,250NANANA0,170,14
B: raw/
LACK
14,86to 7.6118,4211,7422,9923,916,596,40
C: (1b) OM-174-DP [SMORII-39] therapeutic7,411,062,159,23of 5.8110,336,00**3,75
Mann & Whitney, ** p<0,01

It is clear that, compared with the group In, therapeutic treatment of a synthetic molecule1b(GRU is PA (E) was reduced IL-5 cytokine, aware that he activates eosinophils. This decrease was not the result of a shift towards TH1 cytokine, because all mice compared with animals of the groups In the levels of IFN-γ did not increase (1.5-3 PG/ml) (for treatment of mice with compound1bthe levels of IFN-γ was even decreased in two times, see the table below):

Group/mouseMouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6The average IFN-γ (PG/ml)SEM IFN-g
A: raw/
PBS
0,510,560,45NANANA0,510,06
B: raw/
LACK
3,422,162,861,46a 3.92,252,680,90
C: (1b) OM-174-DP [SMORII-39] therapeutic 1,620,880,942,20,811,81,38*0,58
Mann & Whitney, ** p<0,05

From: Quantitative determination of serum allergen-specific IgE

In order to further characterize the immune status of mice after therapeutic treatment of OM-174-DP (compound 1b), the authors analyzed the LACK-specific IgE in the serum of treated mice and untreated mice by the method of ELISA. The results for each individual mouse and average values of each tested group shown in the table below (therapeutic experiment with the connection1b).

Group/mouseMouse 1Mouse 2Mouse 3Mouse 4Mouse 5Mouse 6The average IgE (PG/ml)SEM IgE
A: raw/
PBS
8432133NA NANA82,8250.25
B: raw/
LACK
679400996219559659585265.28
C: (1b) OM-174-DP [SMORII-39] therapeutic18815994382395129224,56*13064
Mann & Whitney, * p<0,05

While the levels of LACK-specific IgE was increased 7-fold after exposure to the action of the LACK of aerosols, the serum of mice treated with OM-174-DP contained 2.6 times less LACK-specific IgE compared with untreated LACK-provoking mice (p<0,05; Mann &Whitney).

Conclusion:

In the first part of this study compounds (1band (19) investigated on their action for preventive introduction. It is known that systemic injection of the two synthetic compounds had a significant negative effect on BAL eosinophilia and significantly reduced the number of IL-4 and IL-13 cytokines in the lungs.

In addition, it shows that synthetic compounds of the invention possess anti-asthma therapeutic potential, as illustrated by the results obtained with the connection1b(strongly and significantly reduces allergen-induced BAL eosinophilia, pulmonary IL-4, IL-5 and IL-13, and reduces the levels of IgE).

In sum, the results presented in example 6, to give a clear example of the fact that the compounds of the invention could be clinically tested preventively or therapeutically against asthma.

EXAMPLE 7:

The effect of OM-174-DP (compound 16), OM-174-MP-PD(compound 19), OM-174-MP-EP (compound 33), OM-174-MP-CM (connection 35C) and OM-174-MP-PR (compound 17) in vitro analysis on the model, stimulated by compound 48/80 secretion of histamine.

Using in vitro models degranulation of mast cells of the rat (proposed CRO CEREP, catalog number 2006: 771-(C) the intent of the authors was to study whether synthetic molecules OM-174-DP (connection1b), OM-174-MP-PD(connection19), OM-174-MP-ER (33), OM-174-MP-CM (35Sand OM-174-MP-PR(17) to inhibit the secretion of histamine stimulated by compound 48/80 mastocytoma. Protocol analysis is briefly summarized below:

Protocol

The General procedure

Origin
AnalysisReference connection.Bibliography
The secretion of histamine (stimulated Conn. 48/80)the fat cells of the ratSCG29Hakanson and other

Experimental conditions

TestStimulantIncubationReacts. productMethod detection
The secretion of histamine (soed/80-driven)Compound 48/80 (0.1 ág/ml)2 min/37°CHistamineFluorometry

Analysis and expression of results

The results are expressed as the percentage of the specific activity of control(measured spec. activity/spec. the activity of control)×100)obtained in the presence of the tested compounds.

The value of the IC50(the concentration that causes premaxillae inhibition of the specific activity of control) was determined using analysis of nonlinear regression curves of inhibition obtained with the average values of repeated experiments is the use of the compliance curve of the Hill equation (Y=D+[(A-D)/(1+(C/C50nH)], where Y = specific activity, D = minimum specific activity, A = maximum specific activity, C = concentration, C50 = IC50and nH = factor tilt). This analysis was performed using software developed by Cerep (software Hill) and was confirmed by comparison with data obtained using a commercial program SigmaPlot ® 4.0 Windows ® (© 1997, SPSS Inc.).

Compound

CompoundThe batch numberMolecular weight
OM-174-DP (1b)SMORII-69_1109061134
OM-174-MP-PD (19)SMORII-32_0509061128
OM-174-MP-EP (33)SMOR-II-831179
OM-174-MP-CM (35c)SMORII-1351113
OM-174-MP-PR (17)KAS1-1081097

Reference connection

In each experiment in parallel with the tested compounds were tested reference compounds in order to assess fit the TB evaluation. They were tested at several concentrations (IR50(IC50)), and data were compared with historical values defined CEREP. The assay was considered valid if the criteria of eligibility are met according to the appropriate standard operating procedure.

Results:

Value IR50defined for the tested compounds and the reference (5 different tests) are shown in the table below. Value IR50for the reference compounds are within the accepted limits of a historical average value obtained at CEREP.

CompoundIR50
OM-174-DP (1b)8,6E-08 M
OM-174-MP-PD (19)1,6E-07 M
OM-174-MP-EP (33)2,1E-07 M
OM-174-MP-CM (35c)3,2E-07 M
OM-174-MP-PR (17)1,6E-06 M
SCG (saloon. 1)4,7E-06 M
SGC (saloon. 2)1,1E-06 M
SGC (saloon. 3)1,3E-05 M
SCG (saloon. 4)2,3E-05 M
SCG (saloon. 5)9,5E-07 M
The average value of the reference8,6E-06 M

Conclusion:

Undoubtedly, the tested drugs are potent inhibitors of secretion of histamine induced stimulated by compound 48/80 fat cells. They are more active than the tested reference: SGS.

EXAMPLE 8:

The effect of OM-174-MP-PD(compound 19), OM-174-MP-EP (compound 33), OM-174-MP (compound 16) and OM-174-MP-PR (compound 17) on in vitro models of cells expressing toll-like receptors person (TLR)

Chemical structure and origin of products of the invention(OM-174-DPit was originally derived from decomposed LPS fromE. colican give us a reason to believe that drugs may be active through TLR receptors, more specifically through TLR4 and TLR2. TLR receptors are expressed mainly (but not exclusively) by immune cells such as monocytes, macrophages, dendritic cells, T cells, and others, and are key sensors of microbial products that can be interpreted by the host as danger signals. Even though they run cyclanilide innate immunity, activation of TLR initiates a full immunological cascade that will lead to the result, in the presence of antigens to the development of acquired immunity.

Cells that significantly Express due to functional TLR gene are valuable tools for many applications, such as the study of the mechanism involved in TLR recognition or signaling, and development of new potential drugs. Therefore, the aim of the experiment described below is the test of the 4 compounds of the invention on these key adapters immune response.

The response was tested in the following cell systems:

a) THP1-blue (lecture on optical density at 625 nm after 48 hours)

(b) HEK-TLR2 (IL-8 ELISA after 24 hours)

(c) HEK-TLR2-CD14 (IL-8 ELISA after 24 hours)

(d) HEK-MD2-TLR4-CD14 (IL-8 ELISA after 24 hours)

a) TNR-1 Blue

The first series of experiment was conducted on TNR-1 cells, which naturally Express and TLR2 and TLR4.

TNR-1cells are monocyte cells in human peripheral blood. Monocytes play a key role in natural immunity and Express the most TLR at different levels. With regard to primary cells, TNR-1 cells activate NF-κB and other transcription factors in response to TLR ligands. In contrast NECK cells that were engineering design is developed to answer specific TLR agonists (see below). TNR-1 cells naturally Express TLR genes and all genes involved in the signaling cascade.

To facilitate the analysis of TLR response in monocytes InvivoGen (Toulouse, France) presented TNR-1 clones stably transfection NF-κB-inducible reporter system, called THPI-Blue™. TNR-1 Blue cells steadily transactionalist reporter plasmid expressing the secretory embryonic gene alkaline phosphatase (SEAP) under the control of a promoter induced by several transcription factors such as NF-κB and AP-1. After stimulation of TLR cells TNR - Blue™ activate transcription factors and subsequently the secretion of SEAP, which is easily detected using QUANTI-Blueu2122 environment, which is becoming purple/blue in the presence of SEAP.

Cells stimulated controls and compounds of the invention according to the manufacturer's instructions.

Results: increased OD at 625 nm after 48 hours:

The results for the controls (negative = LPS K12CD25 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are given in the table below. The results (expressed as arbitrary OD units) show the average values of two measurements) optical density read at 625 nm, 48 hours after stimulation at 37°C controls (up to 1000 ng/ml):

Controls:

Controls ng/mlLPS averagePAM average
10001,001,35
3330,971,38
1110,691,30
370,461,22
120,271,11
40,160,85
10,130,48
00,100,11

Cell line responds clearly and TLR2 and TLR4 agonists PAM3CSK4 and LPS K12CD25 respectively ultrapure.

Then in this analysis tested the following four compounds of the invention OM-174-MP-PD (compound 19), OM-174-MP-EP(compound 33), OM-174-MP (compound 16)and OM-174-MP-PR (compound 17).

The results (the average of the OD values of the two repetitions at 625 nm after 48 hours) is shown below:

Connections:

Connection/
dose (ng/ml)
Average (compound 16)Average (compound 19)Average (compound 33)Average (compound 17)
100000,220,770,351,15
33330,180,580,251,02
11110,170,370,190,74
3700,160,240,160,36
1240,150,170,130,19
410,130,130,120,15
140,150,150,13 0,13
00,140,140,130,14

Connection(19)and(17)are good activators of TNR-1 cells, whereas the activity of compounds(16)and(33)weaker.

Since the compounds of the invention are active on the system, which expresses and TLR2, and TLR4, the authors had to check the activity of SOME cells expressing or only TLR2, or only TLR4, but not both TLR.

(b) HEK-TLR2

NECK cell line was chosen for her wishy-washy or a low basal expression of TLR genes. These cells make it possible to effectively control the activity of TLR using ELISA, such as IL-8 titration and systems-based reporter who control induced TLR NF-κB activation.

SOME TLR2 cells (Invivogen, Toulouse, France) are engineered NECK cells stably transfectional multiple genes of TLR2, which include TLR2 genes involved in the recognition or involved in the signaling cascade. These cells secrete IL-8 after stimulation of TLR2. The experiments were carried out according to the manufacturers ' instructions.

Briefly, 2×104cells/well (200 μl RPMI) incubated at 37°C for 3 days (5% CO2). The medium is removed, and the wells on billaut 90 μl RPMI+5%FCS. Then add agonists and controls (10 μl/well). The cells are returned to the incubator for 24 hours. Collect supernatant and perform the IL-8 ELISA according to the manufacturer's instructions.

Results: secretion of IL-8

The results for the controls (negative = LPS K12CD25 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are given in the table below. The results (expressed in PG/ml IL-8) show the average values of two measurements) the secretion of IL-8 after 24 hours of stimulation controls (up to 1000 ng/ml):

Controls:

Controls, ng/mlLPS averagePAM average
10000,9032,94
3330,4128,24
1110,3023,93
370,2318,33
120,199,12
40,233,89
10,29 1,84
00,280,38

The cell line is clearly only responds to a TLR2 agonist PAM3CSK4.

Then in this analysis had the following four compounds of the invention OM-174-MP-PD(compound 19), OM-174-MP-EP(compound 33), OM-174-MP(compound 16)and OM-174-MP-PR (compound 17).

The results (average of values of secreted IL-8 two repetitions in PG/ml after 24 hours) is shown below:

Connections:

Connection/
dose (ng/ml)
Average (compound 16)Average (compound 19)Average (compound 33)Average (compound 17)
100000,270,62of 3.642,02
33330,190,371,660,88
11110,150,270,680,39
3700,1 0,200,310,25
1240,150,160,230,24
410,090,150,180,20
140,070,060,120,19
00,180,120,170,23

Connection33and17clearly able to activate the secretion of IL-8 through TLR2.

C)SOME TLR2-CD14

Preliminary results were confirmed in another cell line expressing at the same time TLR2 and CD14. The procedure used is similar to the procedure described just above.

The results for the controls and the 4 tested compounds are shown in the 2 tables below:

Results secretion of IL-8

The results for the controls (negative = LPS K12CD25 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are given in the table below. The results (expressed in PG/ml IL-8) show sredneetazhnye two dimensions) the secretion of IL-8 after 24 hours of stimulation controls (up to 1000 ng/ml):

Controls:

Controls ng/mlLPS averagePAM average
10000,6739,04
3330,3233,30
1110,1922,64
370,1315,90
120,16of 6.78
40,112,55
10,111,33
00,220,32

As in the case of SOME TLR2 cell line, SOME TLR2-CD14 cells is clearly also responsible for TLR2 agonist PAM3CSK4.

Connections:

Then in this NECK TLR2-CD14 analysis tested the following four compounds of the invention:

OM-174-MP-PD (compound 19), OM-174-MP-EP(compound 33), OM-174-MP (compound 16)and OM-174-MP-PR (compound 17).

The results (average of two values p is Vtorov IL-8, secreted in PG/ml after 24 hours) is shown below:

Connection/
dose (ng/ml)
Average (compound 16)Average (compound 19)Average (compound 33)Average (compound 17)
100000,220,654,243,59
33330,130,261,491,06
11110,060,170,520,40
3700,010,100,190,22
1240,030,110,170,20
410,000,030,030,14
14 0,030,050,100,13
00,120,110,170,19

The results confirm the results obtained with the cell line SOME TLR2: Connection33and17able to activate the secretion of IL-8 through TLR2.

d)SOME of MD2-TLR4-CD14

TLR4 widely studied, as it is the main receptor involved in the recognition of lipopolysaccharide (LPS), responsible for septic shock.

SOME of MD2-TLR4-CD14 are highly sensitive to LPS. They got stable transfection NECK cells genes TLR4, MD2 and CD14 and NF-κB-inducible reporter system. They secrete IL-8.

The authors used the same experimental procedure as for SOME other cell lines described above.

The results for the controls and the 4 tested compounds is shown below in 2 tables:

Results: Secretion of IL-8

The results for the controls (positive = LPS K12CD25 ultrapure, TLR4 agonist; and PAM3CSK4 = negative, TLR2 agonist) are given in the table below. The results (expressed as PG/ml IL-8) show the average values of two measurements) the secretion of IL-8, and 24 hours after stimulation controls (up to 1000 ng/ml):

Controls:

<>
Controls, ng/mlLPS averagePAM average
100058,432,17
33356,581,87
11158,201,83
3750,241,72
1232,151,76
418,081,78
110,041,75
01,871,97

As expected, cell line SOME MD2-TLR4-CD14 clearly responds only to TLR4 positive control ultrapure LPS-K12, but not on the negative TLR2 in contrast to PAM3CSK4.

Connections:

Then in this analysis SOME of MD2-TLR4-CD14 tested the following four compounds of the invention:

OM-174-MP-PD (compound 19), OM-174-MP-EP(compound 33), OM-174-MP (compound 16)and OM-174-MP-PR (compound 1) .

The results (average of values of secreted IL-8 two repetitions in PG/ml after 24 hours) is shown below:

Connections:

Connection/
dose (ng/ml)
Average (compound 16)Average (compound 19)Average (compound 33)Average (compound 17)
100001,901,961,781,70
33331,631,881,621,89
11111,601,731,581,94
3701,661,671,641,85
1241,621,561,651,93
411,681,641,79 1,97
141,751,691,831,88
01,881,841,882,11

Clearly, in this TLR4 analysis none of the tested compounds is not active.

General conclusions from the TLR analysis:

Very interestingly, the results show that the compounds of the invention are active on human cells expressing mainly TLR2 receptor human.

References

1. The method of obtaining asymmetrically or symmetrically substituted β-(1→6)-linked glucosamine-disaccharide comprising the reaction of compounds of formula 10:

in which R1represents a group selected from (C3-C6)alkenyl, such as3or4alkenyl;
X represents hydrogen, a group selected from a benzyl or substituted benzyl, selected from the group comprising 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2.5-dimethoxybenzyl, 2,3,4-trimethoxybenzyl or 3,4,5-trimethoxybenzyl;
R0selected from R5or 2where R5chosen from:
(i) an acyl group derived from carboxylic acids with linear chain having from 2 to 24 carbon atoms;
(ii) acyloxyacyl group, acylaminoalkyl group, alltoall group;
(iii) alkyloxyaryl group, alkenylacyl group, alkyloxyalkyl group, acylaminoacyl group, alkenylbenzenes group, alkylamidoamines group, alkylthiomethyl group, alkenylacyl group, alkenylacyl groups, acyl groups derived from carboxylic acids with branched chain, having from 2 to 48 carbon atoms;
where in groups (i), (ii), (iii) a hydrocarbon chain acyl may be saturated or unsaturated, hydrocarbon chain acyl, alkyl, alkenyl, quinil may be branched or linear, and optionally may be substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meaning as defined below; an amine or amine derivative-NHW, where W has the meanings given below; group-OZ, where Z is selected from (f), (g), (h), (i), (j), (k), defined below;
and R2represents a group selected from (C1-C6)halogenated alkoxycarbonyl, such as 2,2,2-trichlorocyanuric (TROC) or 1,1-dimethyl-2,2,2-trichlorethene the Nile (TSOS);
with the compound of the formula 7

where R4selected from
(a) acyl groups defined in (i), (ii) or (iii) for R5;
(b) a branched or linear alkyl group; a branched or linear alkenylphenol group; a branched or linear alkenylphenol group;
(c) a group -[(C1-C24)alkyl]-COOH, -[(C2-C24)alkenyl] -COOH or - [(C2-C24)quinil]-COOH, where X has the meanings given below;
(d) a group -[(C1-C24) alkyl]-NHW, -[(C1-C24)alkenyl]-NHW or -[(C1-C24)quinil]-NHW, where W has the meanings given below;
(e) formylalkyl group; formylalkyl group; formylalkyl group;
(f) dimethoxyphosphoryl group;
(g) group-P(O)(OY)2where Y has the meaning as defined below;
(h) the group-P(O)(OH)-O[(C1-C24)alkyl]NHW, -P(O)(OH)-O[(C1-C24)alkenyl]NHW or-P(O)(OH)-O[(C1-C24)quinil]NHW, where W has the meanings given below;
(i) group-P(O)(OH)-O[(C1-C24)alkyl], -P(O)(OH)-O[(C1-C24)alkenyl] or-P(O)(OH)-O[(C1-C24)quinil];
(j) group-P(O)(OH)-O[(C1-C24)alkyl]-COOH, -P(O)(OH)-O[(C1-C24)alkenyl]-COOH, -P(O)(OH)-O[(C1-C24)quinil]-COOH, where X has the values defined above;
(k) the group-S(O)(OH)2;
(l) the protective group is, selected from benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl; or (C3-C6)alkenyl, such as3or4alkenyl; where alkyl, Alchemilla, Alchemilla groups can be branched or linear and may be unsubstituted or optionally substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine;
hydroxyl or hydroxyl derivative-OY, where Y has the meaning as defined below; an amine or amine derivative-NHW, where W has the meaning as defined below; or a group OZ, where Z is selected from (f), (g), (h), (i), (j), (k);
and where Y is selected from hydrogen; (C3-C6)alkenyl, such as2or3alkenyl; group selected from benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl,
or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl; xylylenebis group;
and where W is selected from hydrogen; benzyloxycarbonyloxy group or 9-fluorenylmethoxycarbonyl;
and where R6represents a group selected from trichloroacetimidate, fluoride, chloride, bromide, and X and R2have the meanings defined above, under the reaction conditions suitable for the formation of compounds of formula 1h:

in which R1, R2, R4, R0and X have the meanings given above, and the method optionally includes the addition of one or more of the following stages (2)to(11):
(2) the reaction of compounds of formula 11h under the reaction conditions suitable for hydrolytic removal of a number of groups R2the compounds of formula 11h, with the formation of the compounds of formula 12A:

in which R1, R4, R5and X have the meanings defined above, where R0selected as R5in the formula 11h, or the compounds of formula 12b:

in which R1, R4and X have the meanings defined above, when in the formula 11h as R2selected R0.
(3) reaction of the compound 12A or 12b under the reaction conditions suitable for the formation of amide bond between the free amino group of the compounds of formula 12A or 12b and carboxypropyl (activated) carboxylic acids of formula R5OH, in which R5has the values defined above, to form compounds of formula 13:

in which R1, R4, R5and X have the meanings defined above;
(4) education polyacetale formula 14:

in which R4, R5and X have the meanings given to enter the, by removing in a suitable reaction conditions group R1of the compounds of formula 13 defined above;
(5) phosphorylation of the free hydroxyl group of compound 14 in the reaction conditions suitable for the formation of compounds of formula 15A:

(6) the sulfation of the free hydroxyl group of compound 14 in the reaction conditions suitable for the formation of compounds of formula 15b:

(7) the reaction of the free hydroxyl group of compound 14 (activated) carboxylic acid of formula R8OH, in which R8selected from (a), a previously defined for R4with the formation of the compounds of formula 15C:

where R4, R5and X have the meanings defined above, and R8selected from (a), a previously defined for R4;
(8) attach the leaving group, such as trichloroacetimidate group to a free hydroxyl group of compound 14, under the reaction conditions suitable for the formation of compounds of formula 24:

in which R4, R5and X have the meanings previously defined;
(9) the reaction of compound 24 stage (8) with an organic molecule R8OH, in which R8selected from (b), (C), (d) or (e), previously defined for R4in the reaction conditions suitable for education what ia the compounds of formula 15d:

(10) the reaction in a suitable reaction conditions reactive groups such as hydroxyl group, amino group, carboxypropyl or carbon double bond in the compound of formula 15A, 15b, 15c, 15d or 13, for example by a reaction selected from the reaction of formation of ester, methylation, amidation, oxidation, hydrogenation or α,β-hydroxylation with osmium tetroxide, and in which the specified reaction of the reactive groups are not necessarily preceded by removal of protective groups, such as group Y, or W, for the release of these reactive groups; and
(11) remove some of the protective group X of the compounds 13, 14, 15A, 15b, 15C or 15d, under the reaction conditions suitable for the formation of compounds of formula 1:

in which R'4, R'5and R'7matter, according to the previously defined for R4, R5and R7and R'8selected from (a), (b), (C), (d), (e), (f), (g), (h), (i), (j) or (k), previously defined for R4or selected from N, and where Y and W are N.

2. The method according to claim 1, in which
R1represents a C3or4alkenyl selected from 2-propenyl or 1-propenyl;
X represents hydrogen, a group selected from a benzyl or substituted benzyl, selected from the group comprising 4-methoxybenzyl, 3,4-dimethoxybenzyl is, 2.5-dimethoxybenzyl, 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl;
R0selected from R5or R2where R5selected from
(i) hydroxyethylene group, such as 3-hydroxyethylene group, oxazoline group, such as 3-oxazoline group, aminoacyl group, such as 3-aminoaniline group;
(ii) 3-acyloxyacyl group, 3-acylaminoacyl group, 3-alltoall group;
(iii) (C2-C24)alkyloxyaryl group, (C2-C24)alkenylamine group, (C2-C24)alkyloxyaryl group, (C2-C24)acylaminoacyl group, (C2-C24)alkenylbenzenes group, (C2-C24)alkylamidoamines group; and (C2-C24)alkylthiomethyl group, (C2-C24)alkenylacyl group, (C2-C24)alkenylacyl groups, acyl groups derived from carboxylic acids with branched-chain, branched at the 3-position;
where in groups (i), (ii), (iii) a hydrocarbon chain acyl may be saturated or unsaturated, hydrocarbon chain acyl, alkyl, alkenyl, quinil may be branched or linear, and optionally may be substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y them the et values defined below; an amine or amine derivative-NHW, where W has the meanings given below; group-OZ, where Z is selected from (f), (g), (h), (i), (j), (k), defined below;
and R2represents a group selected from (C1-C6)halogenated alkoxycarbonyl, such as 2,2,2-trichlorocyanuric (TROC) or 1,1-dimethyl-2,2,2-trichlorocyanuric (TSOS);
R4selected from
(a) acyl groups defined in (i), (ii) or (iii) for R5;
(b) a branched or linear (C1-C24)alkyl group; a branched or linear (C1-C24)alkenylphenol group; a branched or linear (C1-C24)alkenylphenol group;
(c) a group -[(C1-C24)alkyl]-COOH, -[(C2-C24)alkenyl]-COOH or -[(C2-C24)quinil]-COOH, where X has the meanings given below;
(d) a group -[(C1-C24)alkyl]-NHW, -[(C1-C24) alkenyl]-NHW or -[(C1-C24)quinil]-NHW, where W has the meanings given below;
(e) a formyl[(C1-C24)alkyl]group; formyl [(C1-C24)alkenylphenol]group; formyl[(C1-C24)alkenylphenol]group;
(f) dimethoxyphosphoryl group;
(g) group-P(O)(OY)2where Y has the meaning as defined below;
(h) the group-P(O)(OH)-O[(C1-C24)alkyl]NHW, -P(O)(OH)-O[(C1-C24)alkenyl]NHW or-P(O)(OH)-O[(C1-C24)quinil]NHW, where W has the meanings given below;
(i) group-P(O)(OH)-O[(C1-C24)alkyl], -P(O)(OH)-O[(C1-C24)alkenyl] or-P(O)(OH)-O[(C1-C24)quinil];
(j) group-P(O)(OH)-O[(C1-C24)alkyl]-COOH, -P(O)(OH)-O[(C1-C24)alkenyl]-COOH, -P(O)(OH)-O[(C1-C24)quinil]-COOH, where X has the values defined above;
(k) the group-S(O)(OH)2;
(l) a protective group selected from benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, or 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl; or (C3or4alkenyl, such as 2-propenyl or 1-propenyl;
where alkyl, Alchemilla, Alchemilla groups can be branched or linear and may be unsubstituted or optionally substituted by one or more groups independently selected from halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl or hydroxyl derivative-OY, where Y has the meaning as defined below; an amine or amine derivative-NHW, where W has the meaning as defined below; or a group OZ, where Z is selected from (f), (g), (h), (i), (j), (k);
and where Y is selected from hydrogen; C2or3alkenyl, such as 2-propenyl or 1-propenyl; group selected from benzyl or substituted benzyl, such as 4-methoxybenzyl or 3,4-dimethoxybenzyl, or 2.5-dimethoxybenzyl, and 2,3,4-trimethoxybenzyl, or 3,4,5-trimethoxybenzyl; xylylenebis group;
and where W is selected from hydrogen; benzyloxycarbonyloxy group or 9-fluorenylmethoxycarbonyl.

3. The method according to claim 1, in which stage (3) is conducted in the presence of a condensing agent, such as isobutylparaben or 1 isobutoxy-2-isobutylketone-1,2-dihydroquinoline, or carbodiimide.

4. The method according to claim 1, wherein in stage (5) phosphorylation of the free hydroxyl group of compound 14, carried out using tetraethylpyrophosphate in the presence of a suitable base, such as bis(trimethylsilyl)amide lithium, in a polar solvent such as tetrahydrofuran.

5. The method according to claim 1, wherein in stage (6) the sulfation reaction is carried out with the help of a complex of sulfur trioxide, such as a complex of sulfur trioxide with trimethylamine, in a solvent such as DMF.

6. The method according to claim 1, in which stage (7) is carried out in the presence of a condensing agent, such as isobutylparaben or 1 isobutoxy-2-isobutylketone-1,2-dihydroquinoline, or carbodiimide.

7. The method according to claim 1, in which stage (8) is carried out using trichloroacetonitrile in the presence of an inorganic base such as cesium carbonate or potassium carbonate, in a polar solvent or in an aprotic polar solvent, or in dichloromethane.

8. The method according to claim 1, in which adieu (11) is carried out by hydrogenolysis of the protective groups in the presence of a noble metal, such as palladium-on-charcoal grill.

9. Synthetic asymmetrically or symmetrically substituted β-(1→6)-linked glucosamine-disaccharide of formula 1:

in which R'4represents-P(O)(OH)2,
R'5selected from
(i) an acyl group derived from carboxylic acids with linear chain having from 2 to 24 carbon atoms;
(ii) acyloxyacyl group;
where in groups (i) and (ii) a hydrocarbon chain acyl is saturated, and optionally may be substituted by a hydroxyl group;
and R'8selected from
(a) a branched or linear alkyl group, optionally substituted hydroxyl group(groups);
(b) a group -[(C1-C24)alkyl]-COOH;
(c) a group -[(C1-C24)alkyl]-NH2.

10. Disaccharide according to claim 9, which is selected from:





in which n represents 0 or an integer from 1 to 21

in which n represents an integer from 1 to 21, such as 13.

11. Purification method, including
(i) mixing a solution of the compounds of formula 1 obtained by the method according to claim 1 or 2:

where R4', R5' and R8' have the meanings previously defined, with the solid resin with reversed phase, under conditions suitable for binding at least part of the compounds of formula 1 with the solid phase;
(ii) removing the liquid phase and washing the solid phase of the drilling liquid comprising an aqueous phase buffered at pH 6-9, 7-8 or 7.3 to 7.7, and the organic phase and the aqueous phase and organic phase are mixed in a ratio of from 15:1 to 5:1 or 9:1 (vol./vol.);
(iii) removing drilling fluid and elution at least part of the connection 1 associated with the solid phase, eluting fluid comprising an aqueous phase and an organic phase, which is mixed in a ratio of from 1:15 to 1:5 or 1:9 (vol./vol.);
(iv) collecting the eluting liquid, including a certain number of compounds of formula 1;
(v) optionally removing the organic phase from an eluting liquid, comprising a compound of formula 1.

12. The method according to claim 11, further comprising bringing the pH of the eluting liquid, including a certain number of compounds of formula 1, to a pre-selected pH, or to a pH of 6-9, or to a pH of 7-8, or to a pH of 7.3 to 7.7.

13. Pharmaceutical composition comprising one or more compounds according to claim 9 or 10, optionally in combination with a suitable carrier or diluent, the composition exhibits immunostimulatory properties in respect of toll-like receptors person (TLR).

14. The connection according to claim 9 or 10 to obtain pharmaceutical compo is icii indicated in paragraph 13 for use in the treatment of disorders that affects the modulation of activity of the immune system, including the suppression or activation of the immune system, such as a disorder selected from immune disorders and/or cancers, or for use as a component of the vaccine, while the disorder is selected from
- immune disorders associated with overproduction inflammatory cytokines, such as overproduction inflammatory cytokines, activated T lymphocytes, monocytes, or antigen-providing cells with inflammatory cytokines or inflammatory markers preferably belong to the group consisting of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IFN-γ, TNF-α or MCP-1,
- immune disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, diabetes and rheumatoid arthritis
- immune disorders associated with reduced production of inflammatory cytokines,
disorders, the treatment for which benefits as a result of activation of the human immune system,
disorders, the treatment for which benefits by reducing the secretion of histamine fat cells.

15. The compound of formula 7:

in which R2, R4, R6and X have the meaning of the Oia, previously defined in claim 1.

16. The connection 15, which represents a compound of formula 7b:

where Bn is benzyl.

17. The compound of formula 10b:

where Bn is benzyl.

18. The compound of formula 11:

in which R1, R2, R4, R5and X have the meanings defined in claim 1.

19. Connection p, representing a compound 11a:

where Bn is benzyl.

20. The compound of formula 11b:

in which R1, R2, R4and X have the meanings defined in claim 1.

21. The compound of formula 12A:

in which R1, R2, R4, R5and X have the meanings defined in claim 1.

22. The compound of formula 12b:

in which R1, R4and X have the meanings defined in claim 1.

23. The compound of formula 13:

in which R1, R4, R5and X have the meanings defined in claim 1.

24. The compound of formula 14:

in which R4, R5and X have the meanings defined in claim 1.

25. A compound selected from the group including

where Bn is benzyl.

26. Application connection PP-25 as intermediate compounds, including as the starting material in the method of synthesis of asymmetrically or symmetrically substituted β-(1→6)-linked glucosamine-disaccharide.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to use of 2-mercaptobenzoyl hydrazones of monose of formula (where the name of monose and value of radicals are listed in the table) as antimocrobial and antifungal agents.

EFFECT: use of 2-mercaptobenzoyl hydrazones of monose of formula (I) as antimicrobial and antifungal agents.

2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: method involves preliminary acetylation of chitin with acetic anhydride, washing and drying the acetylated chitin in order to reduce degree of deacetylation thereof and, as a result, increase output of the desired product - D(+)-glucosamine hydrochloride when obtaining said product through hydrolysis of acetylated chitin with concentrated hydrochloric acid while heating, followed by evaporation, crystallisation, separation, washing and drying the desired product.

EFFECT: high output of the desired product while maintaining its high quality; method is more environmentally friendly since pre-treatment of chitin reduces the amount of processing wastes.

1 cl, 2 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to geranyl compounds represented by the following formulas (I-1) , (I-2) or (I-3) wherein R1 means compounds of the following formulas: or R2 means a group remaining after removing all carboxyl groups presenting in carboxylic acid chosen from group consisting of malic acid, citric acid, succinic acid, fumaric acid and others; m = 1, 2 or 3; n = 0, 1 or 2, and m + n represent a number of carboxylic groups presenting in indicated carboxylic acid; R3 means p-hydroxyphenyl or mercapto-group. Also, invention relates to derivatives of mevalonic acid represented by the following formula (I-4): wherein R means -CH2OH or CH3. Also, invention to an antitumor agent comprising as an active component geranyl compound of formulas (I-1), (I-2) or (I-3) or derivative of mevalonic acid of the formula (I-4), and optionally a pharmaceutically acceptable carrier or solvent. Also, invention relates to a method for treatment of liver cancer based on using geranyl compound of formulas (I-1), (I-2) or (I-3), or derivative of mevalonic acid of the formula (I-4) and using proposed compounds in manufacturing an antitumor agent. Invention provides using geranyl compounds or derivatives of mevalonic acid as antitumor agents.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

7 cl, 3 tbl, 17 ex

FIELD: organic chemistry, chemical technology, medicine.

SUBSTANCE: invention relates to a method for preparing 1-isonicotinyl-2-D-glucosyl hydrazone. In the glucosylation reaction of isonicotinic acid hydrazide the method involves using anionite AN 31 GS as a catalyst of the enhanced effectiveness, and using 95-96.5% ethanol or 85-90% isopropanol as the reaction medium. At the final stage the method involves sorption of contaminating impurities with activated carbon followed by drying the end product in nitrogen atmosphere.

EFFECT: improved preparing method.

2 cl, 1 ex

The invention relates to a method of joining azide function to organic connection

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25 cl, 9 ex

FIELD: medicine, pharmaceutics.

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EFFECT: application of said compounds or their pharmaceutically acceptable salts for obtaining medication for treatment of acute or chronic inflammatory diseases of respiratory ways in mammals.

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.

EFFECT: obtained glycoconjugates have C-phosphonate bond, which is much more stable than natural phosphodiester bonds, as well as higher immunologic activity.

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.

EFFECT: obtaining compounds which are capable of delivering nucleic acid into mammal cells.

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

The invention relates to an improved process for the preparation of agglomerates and molded products containing isomaltulose and/or gidrirovannoe isomaltulose

The invention relates to cationic charcterised surfactant with improved Biodegradability, which can be used as hydrotropes for non-ionic surface-active substances, thus increasing the cleaning effect of the latter

The invention relates to a carbohydrate derivative of the formula (I), where R1represents (1-4C)alkoxy; R2, R3and R4independently represent (1-4C)alkoxy or OSO3-; the total number of sulfate groups is 4-6; and their pharmaceutically acceptable salts

The invention relates to pentasaccharide in acid form or its pharmaceutically acceptable salts, anionic form which has the formula I, where R represents hydrogen or-SO3-, (C1-C3)alkyl or (C2-C3)acyl group; T represents hydrogen or ethyl group, n represents 1 or 2, and to a pharmaceutical composition based on them

The invention relates to a carbohydrate derivative of General formula I, where R1= H or CH2ОSO3-; R2and R3independently equal to N, (1-6C) alkyl or SO3-; R4= OSO3-; n= 0 or 1; p=1 or 2; or pharmaceutically acceptable salts

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely to allergens purified from apitoxin and derived allergoids for immunotherapy of individuals suffering specific allergic reactions. The allergens for immunotherapy are prepared by dialysis of native apitoxin with using a membrane with cut-off 10 kDa, and represent apitoxin substantially mellitin-free. The apitoxin allergoids for immunotherapy prepared by carbamylation or thiocarbamylation or treatment to form guanidine groups of primary amino groups of protein portions of mellitin-depleted apitoxin. The pharmaceutical composition for immunotherapy on the basis of apitoxin which contains an effective immunotherapeutic dose of the allergen or allergoid together with pharmaceutically acceptable excipients. The use of the allergens or allergoids for preparing a drug preparation for immunotherapy.

EFFECT: agents described above are effective for immunotherapy.

15 cl, 6 dwg, 1 tbl, 2 ex

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