JPH0565517B2 - - Google Patents

Description

[Detailed description of the invention] The present invention comprises fragments of acidic mucopolysaccharides,
or oligosaccharides consisting of it (oligosaccharides)
This relates to the organic synthesis method of (d). Sara
Furthermore, the present invention relates to the synthesis of derivatives of these oligosaccharides.
It is something that Furthermore, the present invention provides a drug that is of particular interest as a drug.
physical properties and/or experimental reagents, e.g.
of the above-mentioned types which have biological properties useful as
Concerning new oligosaccharides and their derivatives.
Ru. Furthermore, the present invention is applicable to those applications, particularly biological and biological applications.
and biochemical applications. ``Acidic mucopolysaccharide (acidic mucopolysaccharide)
Currently, the term glycosaminoglycan
It refers to a derivative also called lonoglycan. child
For example, heparin, heparan-sulfate
In the chain of biologically active derivatives such as derivatives of
oligosaccharides and polysaccharides especially found in
Ride). In natural products, the mucopolysaccharides are substantially
From mutual amino sugar-uronic acid units or vice versa
configured. In these units,Abelow
In more detail, the amino sugar shown is D-glucosamine structure.
It has a structure.UUronic acid, called uronic acid, is more
has D-glucuronic acid or L-iduronic acid structure
has. AThe basic structure for corresponds to the following formula a, and
U corresponds to the following formulas b and c: In the natural product, these various units are 1α → 4,1β → by 4 bonds are stereospecifically linked to each other. For example, in heparin 1α → Type 4 (between C and a, between a and b and between a and c) and 1β → Type 4 (between b and a) A combination of these can be seen. Furthermore, with reference to nature, the above units are specific
a substituent, i.e. some type of substitution at a given position
Note that it contains groups. For example, natural
The chain is -O-substituted unit, 2-O-sulfate-L
-iduronic acid, 3-O-sulfate-D-glue
Cosamine, 3,6-di-O-sulfate-D-
Glucosamine, 6-O-sulfate-D-glue
cosamine and non-O-substituted units, such as D-
Glucuronic acid, L-iduronic acid, D-glucosamine
Contains unit. Furthermore, the unit a is N-acetyl
and/or -N-sulfate group in the 2nd position.
N-substituted. Importance of therapeutic uses of the above acidic mucopolysaccharides
is particularly effective in preventing blood clots and damage to blood vessel walls and
known about treatment, especially thrombosis and
Prevention of teloma arteriosclerosis and arteriosclerosis
known about treatment. In addition, fragments with high affinity for AT and
To obtain biologically active fragments from heparin chains
Many studies by the applicant are known. this
The present invention, which was developed based on research by
The subject matter of the application, among which European patents are
Application No. 804014256 (October 6, 1980) and Fra.
Patent Application No. 8108604 (April 29, 1981)
be. European patent applications are of particular interest.
called ABCDEFGH which has certain anti-thrombotic properties
The octasaccharides (octasaccharides) are listed.
, which corresponds to the following structure: In this formula, R is SO₃ ^-group or hydrogen atom
shows. In the above French patent application of the present applicant, the structure
Uniform hexasaccharides (hexasaccharides) of C′DEFGH
compositions have been described, which also have high antithrombotic properties.
have sex. This structure has the formula: [In the formula, R is SO₃ ^-Indicates a group or hydrogen atom] corresponds to To date, no proposals have been made to obtain this type of product.
The methods used are from heparin or from heparin.
Extraction techniques or chemistry from the products obtained during production
of heparin chains in the action of drugs or enzymes.
Especially Affinitei chromatography following depolymerization technology
This is a technique of specific fractionation using a fee. As a result of the applicant's research in this field, this
provides a novel means of obtaining seed products and further
In detail, it is possible to pursue the possibility of obtaining them synthetically.
I made it into Noh. In this respect, the multiplicity generated by this type of synthesis
It is appropriate to address the following issues: fact,
On the other hand, there are several types of these products in the chain.
ofAandUunits, and on the other hand these
The bonds between the units correspond to a given stereochemistry and 1,
4 type, and its production is particularly difficult.
This is well known. Furthermore, each unit is
One or several specific locations depending on the type of product.
Contains substituents. In addition, glucosuria in natural products
The samine unit consists of two different nitrogen-containing groups (nitrogen
containing groups), i.e., N-acetyl group and -N-sa
The inclusion of sulfate groups should also be considered. As a result, this type of synthesis is particularly suitable for L-iduronic acid.
has not been practically described in the chemical literature to date.
Not yet. All of these elements are subject to common methods and synthetic methods.
Limitations that readily assess the difficulties involved in the development of the law
Emphasize the requirements. Oside suitable for developing this type of compound
By considering the synthesis conditions, the applicant has
selecting certain types of protection for substances used
We developed a method using Studies have shown that the conservation of this species
It is also possible to generate stereospecific chains using
and then insert the umbilical cord into the desired formed array.
A predetermined substituent can be introduced at a predetermined position of
Ta. An interesting aspect of its importance is that
The developed method has great flexibility. parable
especially regarding the specificity and purity of the synthetic method.
Advantageously, specific substituents or different substituents found in natural products are used.
have different substituents and/or different configurations.
Many oligosaccharide derivatives even contain units of similar structure.
can be reached. By this method, the applicant has achieved particularly high value
Certain medical properties, more specifically high antithrombotic activity
A oligosaccharide with Furthermore, the method of the present invention
for biological reagents and/or structural studies
Obtain a large number of oligosaccharides that are particularly useful as comparison compounds
be able to. Therefore, the object of the present invention is to prepare acidic mucopolysaccharides
oligosaccharides containing or corresponding to fragments of
We provide methods for synthetically producing such derivatives.
Is Rukoto. Furthermore, the purpose of the present invention is toAandUbetween the units of
Establish the glycosidic bond in the desired stereospecificity.
The goal is to provide a means by which people can Furthermore, it is an object of the present invention to
with certain functional groups, especially biologically active moieties, such as
Child chain, especially heparin, heparan-sulfei
Introducing specific substituents such as those found in
The goal is to provide a means to Furthermore, the object of the present invention is to provide substituents and/or
Chemical properties of sugars and/or interglycosidic bonds
position and configuration and/or monosaccharide
body positioning and/or (enquenchment,
enchainment) The order of the chain is different from that of the natural product.
This makes it possible to produce oligosaccharides such as those mentioned above.
It is to provide the means. According to another aspect, it is an object of the invention to
To provide new oligosaccharides that constitute intermediate products.
, where all the OH groups of the various units are protected
blocked by groups and possibly functional
Precursors of groups are present and these functional groups can be
You will also be protected. According to yet another aspect, the present invention provides the above-mentioned natural product.
Novel oligosaccharides with structure and their products
with the aim of providing oligosaccharides corresponding to fragments of
do. Furthermore, it is an object of the present invention to substitue specific substituents of natural products.
The object of the present invention is to provide a novel oligosaccharide having the following properties. Furthermore, the purpose of the present invention is to
The above natural product has a substituent and/or
By providing novel oligosaccharides containing different units
be. The invention particularly relates to active pharmaceutical substances, laboratory reagents or
especially for the study of compounds containing this type of structure.
Regarding the biological use of these oligosaccharides as reference substances.
It is something to do. The synthesis method of the present invention involves the reaction of the following two types of compounds:
It is characterized by: glucosamine structureAEach unit consists of
compounds that are terminated or terminated, especially D-glucosa
of min and glucuronic acid structures.UUnits, especially D-
Glucuronic acid or iduronic acid, especially L-iz
Ronic acid; unitAorUOne is alcohol;
The OH group of this alcohol is the unitAposition if
Occupies either 3, 4 or 6, and the unitU
occupies either 2, 3 or 4 in the case of
The other unit has an active anomeric carbon, i.e.
The desired glycosylation (glycosylation) together with the alcohol OH group
licosylation) -O- bond to the desired stereochemistry.
established in -A-U or -U-A sequence
Contains a reactive group (reactive group) that can form. AandUThe reactive groups in these units are
compatible with protecting groups and/or functional groups; other than those in which the anomeric carbon is activatedA
andUAll positions are OH group, amino group, or
Carboxyl groups or precursors of such groups
), and these groups, if present, may have one type.
or advantageously blocked by several protecting groups,
The various groups are compatible with each other and with respect to the precursors mentioned above.
These protecting groups and precursors are
is inert to the cosylation reaction and is reactive to the cosylation reaction.
together with the group at various positions during the course of subsequent operations.
Allows positioning of a given substituent and
This is then continued depending on the reaction to react the starting materials.
The conditions of use for these substances depend on the unit structure and
and the properties of the various substituents present.
However, the establishment of interglycosidic bonds is
[2-N-sulfate or (2-N-acetate)
(chill)-6-O-sulfate-D-glucosami
methyl-D-glucuronic acid] structure
Those that do not involve the production of
do. Due to the above considerations, the structureAandUcommon between units of
A bond can be formed, and this bond is
Entirement of species is already considered
The stereochemistry exhibited within biologically active molecules
It is formed by By means of the invention, the desired chain formation can be achieved in a predetermined manner.
in order and/or with a given stereospecificity.
can be formed. Therefore, the measures proposed by the invention are:
In particular, D-glucosamine units and D-glucuronic acid are also
or between L-iduronic acid 1α → type 4 bond, D-glucuronic acid unit and D-glucosamine unit
between 1β → type 4 bond and L-iduronic acid unit and D-glucosamine monomer
between 1α → type 4 bond enable establishment. The mono-intermediate or oligosaccharide in this synthesis is
semi-open or open
It is a product. The compound has a
Compounds that are activated or can potentially be activated
non-reducing end of a monosaccharide or oligosaccharide
If it can be moved (combined) to
Called open. "Compound half open on the left
The expression "object" refers to a single free or potentially
Contains free OH group, allowing specific glycosylation
refers to monosaccharides or oligosaccharides. as an example
The following formula 1 shows an example of a compound whose left side is half open.
An example of a compound whose right side is half open is shown in formula 2: Therefore, in the above definition, derivatives are defined as
If both the left side and the left side are half-open derivatives, open
This type of derivative is called a chain in both directions.
allows for the extension of This type of derivative is for example
Corresponds to formula 3 below: For closed derivatives, the nature of their substituents
It is a substance that cannot produce its unit or chain extension due to
Ru. A-U or U formed in the above step
- According to other features that can add units to the A array,
of the array formedAandUThe unit is temporary (temporary)
) must contain a protecting group, i.e. a new group
Intended to participate in lycosylation reactionAorUsingle
It must contain a group that can selectively block the position of
do not have. These groups are similar to other groups in the starting material unit.
Removal while generating alcohol in the presence of
This results in the glycosylation of the glucide skeleton.
Repeat the above process of elongation (stretching) by
can be done. Therefore, the present invention provides α or β stereospecificity.
and/or between a and b and/or c
Oligosaccharides with different linkages in terms of linkage order
, where the extension remains as desired.
Can be reproduced. According to further features of the method according to the invention,
The generated glucide chains are treated with one or several chemical reactions.
Depending on the type of functional group, a given type of functional group or several types in sequence
groups and then optionally modify these functional groups.
form a derivative of This functionalization step may contain some (some)
Protecting groups and/or certain types of amino derivatives
some) precursor groups only or protecting groups and/or
or remove the entire precursor group and place the desired species at that site.
Introducing similar substituents or sequentially different substituents
and then optionally remove the still blocked OH group.
This can be done by releasing some or all of the
Wear. Various groups present in the chain unit are introduced at each step.
is understood to be compatible with the substituents specified. one or more used during the functionalization process
The above chemical reactions are based on the chain structure and maintenance as required.
groups that are desirable to have and/or that have already been introduced.
This is done in a way that does not change the underlying group. To obtain oligosaccharides having the above-mentioned specific substituents
According to a preferred embodiment of the invention, advantageously several types of
Protecting groups, i.e. (1) one or more semi-permanent groups;
and (2) a starting material with one or more permanent groups.
substance is used. A semi-permanent group is a group in which the glucide skeleton is a large number of desired units.
After the glycosylation reaction, if present other
Removal of the group or removal without change
and then place the desired sensuality in those occupied positions.
It means a group into which a group can be introduced. A permanent group is a functional group introduced in place of a semi-permanent group.
This is a group that can continue to protect the OH group. These groups are introduced after removal of semi-permanent groups
Selected from those that are compatible with the functional group. Sara
These are performed to determine the position of these functional groups.
A group that is inert with respect to the reaction to be carried out and has a functional group.
It is a group that can be removed without being changed. Advantageously, the performance of these steps comprises:AandUsingle
Enables generation of glucide chains with selective substitutions at positions
Make it. Especially of the biologically active molecules mentioned above.Aand/also
teethUAdvantageous for producing oligosaccharides containing units
for example, an acyl group, which may be substituted
Relying on protecting groups such as alkyl or aryl groups
must be done. of the product usedAThe unit of
It contains a nitrogen group (nitrogen-containing group) at the 2nd position, and this nitrogen
The elementary functions of the operations carried out in this way by the elementary groups
(nitrogen function) can be maintained.
Wear. This nitrogen group is preferably for example -N₃if
Kuha-NHCOO-CH₂−C₆H_FiveGroups like , also
is an amine function (functional group) or an amine derivative.
Any other groups constituting the precursor, especially -
NHSO₃-or -NH-acyl, especially -NH
−COCH₃Composed by. UFor the carboxyl group of the unit, the protective group
It is inert with respect to the reaction used during substitution, and
The carboxylic acid is removed at the end of the synthesis for the purpose of salt formation.
The sil group is capped by a group capable of releasing it. Cal
These protecting groups for the boxyl function are preferably alkyl
or aryl group. Structures of compounds used during glycosylation reactions
is the desired gluside backbone unit as well as the desired position.
It is selected depending on the substituent. For example, producing a -U-A- type disaccharide
have a uronic acid structure and an amino sugar structure, respectively.
and further correspond to two types of compounds corresponding to the above definitions.
use. To extend the chain, generate the disaccharide.
These compounds used for
for the purpose of participating in (subsequent) glycosylation reactions.
Contains a temporary group (temporary group) at the position. U-A
- To extend the disaccharide to the left, this temporary
baseUTo make it exist in the unit and extend it to the right side
Exists in unit A. Thus, especially the chain U_wA_xU_yA_zYou can get
, where the sum of the subscripts is 2 to 12, and w and
y is not 0 at the same time. Normal chain is U (AU)_o,
(AU)_oA, (UA)_oOr (AU)_oand here
and n is 1 to 6. According to a variant of the method of the invention, the natural structure
There may be one type of A-U or U-A change.
There are several speciesAorUinstead of unitsAor
Sugars that constitute a structure equivalent to the U unit, e.g. neutral
sugars or desoxysugars or other uronic acids
Aminosugars with units or different configurationsUIf
teethAIt can be modified using . In a preferred embodiment of the method of the present invention, the above-mentioned
Alcohols, such as halides and imidates, are also
or react with reactive derivatives such as orthoesters.
make them respond. These condensations are carried out under anhydrous conditions.
Ru. Condensation reaction between halides and alcohols
is preferably of the Koenigs-Knorr type (Koenigs-Knorr type).
-Knorr type). Halides are
Preferably bromides or salts are used for reasons of ease of manufacture.
Composed of monsters. The operation is more preferably carried out in a solvent, especially an organic solvent.
is carried out in dichloromethane or dichloroethane.
be called. A catalyst is advantageously used, generally silver or mercury.
salts, such as silver trifluoromethanesulfonate
(commonly called silver triflate),
Silver carbonate, silver oxide, mercuric bromide or cyanide
Mercury is used. Additionally, for example sym coli
Possibly exists a gin-like proton receptor
to water and/or the generated hydrohalic acid.
This extractant is used in the same manner as the extractant for
For example, a 4 Å molecular sieve. Studies of reaction conditions indicate that room temperature or
At low temperatures that can reach 0℃ or below,
an inert gas such as nitrogen or argon
Suitable for operation in body atmosphere. These conditions apply to structures a and b or c
(or vice versa) in the desired stereochemistry.
can be condensed. In addition, these
Establishment of a covalent bond with a sexual or desoxy sugar
enable. Mercury derivatives, especially ferric cyanide, as catalysts
Variants using mercury and/or mercuric bromide
are alcohols of various structures and L-i of C structural unit.
Covalent bond between dose precursor (L-iduronic acid)
It was found to be suitable for forming composites. This variant
4Å molecular sheep was also used in
Ru. The organic solvent is selected depending on the reactivity of the alcohol.
be done. For example, it is advantageous if the condensation is higher than 100°C.
If higher temperatures are required, a nitrobenzene type solution may be used.
agent is used. For lower temperatures,
For example, benzene or dichloromethane
solvents are used. Solvent to carry out condensation reaction
Mixtures of are also suitable. UFor type units, especially C units, advantageously
Using an orthoester as a reactive group, then
and the reaction is preferably carried out at a temperature higher than 100°C.
be exposed. The solvent is chlorobenzene type or 100℃.
Any other solvent with a boiling point exceeding
It has a boiling point of 100 to 150°C. anti
To accelerate the reaction, e.g.
Using a catalyst such as lysinium perchlorate. An example of this condensation step is structure C (L-iduronic acid)
between the unit and the structure a (D-glucosamine) unit
of great interest in forming interglycosidic bonds in
It turns out that there is something. The use of orthoester groups has a particularly double advantage.
do. On the one hand, this means that the anomeric carbon of the C unit
Can provide the reactivity necessary for the cosylation reaction.
Wear. On the other hand, cleavage of this group occurs at the 2-position of the C unit.
It is possible to place a protecting group that can be selectively removed in
and thereby introduce a specific substituent at that position.
can be done. 1,2-O-methoxy-ethylidene group of C unit
By reacting with the OH group of the a unit, two types of
A compound capable of establishing an interglycosidic linkage between the compounds used.
Sometimes certain functional groups such as -SO₃ ^-Purpose of introducing
-OA can be selectively removed by_CGroup (A_Cis acetyl
(indicates a group) at the 2nd position of the C unit
be able to. This feature handles the 4 positions of the C unit.
can give you full opportunity to understand. These particularly advantageous advantages include, for example, heparin
2-O-sulfate-L as present in the chain
- It is capable of providing iduronic acid units. When imidoyl group is used as a reactive group, low
at temperatures, particularly at temperatures below or equal to about 0°C,
For example, 4 Å in a solvent such as dichloromethane.
In the presence of a molecular sieve and e.g.
Chemicals such as boron fluoride etherate
It has been found suitable to operate in the presence of a medium.
Ta. In the starting alcohol, free OH groups are
Occupying the desired position to participate in the cosylation bond
Melt. By appropriately selecting alcohol, 1-
2, 1-3, 1-4 or 1-6 type bonds
can be made. From the sequence formed at the end of the condensation reaction, the glyco
The desired number of units can be obtained by repeating the silation reaction step.
A chain containing the positions is generated. Units included in already configured glucide arraysA
orUone alcohol function, then that
Advantageously, it is freed from a temporary protecting group.
The selection of this group depends on the other groups present in the glucide chain.
It is easily determined by a person skilled in the art depending on the nature. Among the various groups that can be used, allyl group is mentioned.
For example, first Pd, Rh and Ir derivatives
isomerizing agents, especially Tris-triphenylpho
Suphine rhodium() or potassium t-chloride
treated with tooxides and then oxidized especially under acidic conditions
Treatment with a mixture of mercuric and mercuric chloride
and allows alcohol to easily occupy the position it occupies
can be played. Similarly, -O-acyl groups, especially -O-acetyl groups,
from the chloro or O-chloroacetyl group by saponification.
OH group can be obtained by These groups are preferably heated in a solvent at temperatures above 80°C.
thiourea at a temperature of about 100°C, preferably about 100°C.
The OH function is released by removing it by using
can be done. The above process can be performed selectively by A-U or U-A.
allows the production of glucide chains with units. This conventional method is suitable for glycosylation reactions.
It can be modified using quality. for exampleUtoo
For detailsAother units, especially neutral sugars or des
formation of irregular structures incorporating oxysugars.
You can also do it. Other types of irregular structures are
consecutiveAunit orUtwo unitsA−
Obtained by adding between U or U-A structural units
I can do it. AandUVarious processes of the present invention regarding units
For example, neutral sugars or desoxy sugars
It would be equally applicable to other units that may contain glucide chains.
It will be understood that As mentioned above,AandUeach present in the unit
Species groups are provided by imparting sufficient reactivity to these groups.
selected to generate the glycosidic bond.
Ru. Protecting groups for the OH group other than the temporary groups mentioned above are:
Generally acyl groups (particularly acetyl, alkyl, substituted
selected from the group consisting of alkyl, e.g. benzyl).
selected, and for two adjacent positions the aceter
group or ketal, such as benzyl
Den is selected. Other types of protection include epoxide
or two –OH groups in the form of a 1,6-anhydride bridge.
It has become known as a blockade.
Ru. Compounds advantageously used in glycosylation reactions
contains several protecting groups, and these groups can be functionalized
The sequential addition of one or several functional groups during the process
Introduction and optionally one or several OH groups
allows for the release of Generally, protecting groups are used in glycosylation reactions.
can preoccupy a predetermined position in a compound
Ru. Furthermore, these were composed of glucide skeletons.
Later, it can also be introduced from other groups. This variant
For example, OH groups at the 2nd and 3rd positions and
The OH groups at the 1st and 6th positions are 2 and 3, respectively.
- anhydrous forms of epoxides and 1,6-anhydros;
substances sequestered asAused for glycosylation
It is based on that. Due to this lockdown, Gurushi
During the generation of the skeleton, potentiallyAConfigure units
However, using elements that do not inhibit the reactions used in the synthesis
can be used. This process is based on other units.
When given wide freedom to react as desired,
It has the advantage of In addition, in this case epoxy with sodium acid
Cleavage of the oxide function is caused by the N₃Introduction of
, which constitutes a precursor for amine functionality.
Ru. Preferably, during the functionalization step one or more
Sequential introduction of several substituents, especially the specific substituents listed above
In order to obtain a glucide chain that can be
That is, semi-permanent groups and permanent groups defined above are
Use compounds that contain As described above,Aand the one in the two positions of the unit.
Apart from natural products, the substituents are substantially sulfate.
composed by the base. Complete appropriate sulfation conditions
According to the applicant's research to
The sulfation reaction can be carried out in the presence of benzyl groups.
It is possible and even advantageous. recognized in this field
In contrast to the theory that in the presence of -O-sulfate group
Removal of benzyl permanent groups can be carried out in
Ru. Preferably, the OH of the starting material to be sulfated
The group is then supported by an acyl group, especially an acetyl group.
The desired OH group is protected and released at the end of the synthesis.
protected by a permanent group, such as a benzyl group.
Ru. The high flexibility of the method of the present invention allows for the production of
All of the glucide chains are subjected to a predetermined chemical reaction.
A fixed variety of substituents can be introduced. This treatment can be carried out by e.g.
terification, or especially sulfation, and this
is carried out under conditions that do not change the ocide structure.
Ru. This sulfation is not specific, but if necessary
What to do with fully protected glycosides
I can do it. In a preferred embodiment of the invention, the functionalization step is
Several substituents are introduced into the chain sequentially, and then some
selectively to liberate the OH groups
Ru. Particularly favorable conditions allow the monkey to be placed in place on the unit.
Introducing a pheto group to replace OH groups at other positions
let me goAGenerates an amino derivative at the 2nd position of the unit
let, andUGenerates an acid derivative at the 6th position of the unit
However, the unit used in this case has the following characteristics:
Ru. The semi-permanent groups of these units are located at the desired position to be sulfated.
occupies the same position and is constituted by an -O-acetyl group.
Ru. At the position corresponding to the OH group to be released,
These are semi-permanent molecules composed of benzyl groups.
Occupied by Hisaki. AThe second place in the unit is, for example, N₃Or NH-
COO−CH₂−C₆H_Fivesubstituted by a group such as
The 6th position of the U unit is an alkyl group, especially a methyl group.
occupied by a protected carboxyl group
Ru. For example, this combination of conditions can be used to
process can be realized. After the -O-acetyl blocking group is first removed,
Selectively introduce sulfate groups. this reaction
is based on the benzyl groups and nitrogen-containing groups present.
This should be done in a way that does not affect the carboxyl groups.
It can be done. Advantageously in this respect, strong
A saponification reaction is carried out using a base. The reaction is preferably carried out at temperatures below room temperature, especially
It is carried out at a temperature close to 0°C. The products resulting from hydrolysis are treated with alkylating reagents.
It has been found that it is removed during hydrolysis.
Introducing a protective alkyl group to a carboxyl group
Ru. Released by hydrolysis by reaction with sulfating reagents.
and released after the action of the alkylating reagent.
A sulfate group is introduced at the position. Satisfactory reaction conditions for sulfation are e.g.
Trimethylamine/SO- Sulfation test like 3-complex
It is to use medicine. This reaction advantageously
in a solvent, especially dimethylformamide.
It is carried out in a similar solvent. Preferably, the operation is
It is carried out at a temperature higher than the normal temperature, generally around 50℃, and this
This corresponds to a reaction time of about 12 hours. After introducing the sulfate group for the alcohol function,
The OH group blocked by the ndyl group is liberated.
It can be done. Removal of the benzyl group is advantageously carried out by removing the sulfate group.
and the conversion of nitrogen-containing groups to amino groups.
It is carried out by catalytic hydrogenation under conditions of. Preferably, the operation is carried out under hydrogen pressure (hydrogen pressure).
pressure) in the presence of a Pd/c type catalyst.
be called. The reaction is carried out in an organic solvent, advantageously supplemented with water.
In particular, it is carried out in an alcoholic medium. Hydrogenation of precursor nitrogen-containing groups and protecting groups from OH groups
The reaction is carried out for about 3-4 days to obtain the removal of
is advantageous. As mentioned above, the amino functional group is biologically active.
N-acetyl or N-sulf in molecules such as
It is a derivative of the ate type. To generate N-acetyl group, hydrogenation reaction or
The resulting product is subjected to an acetylation reagent. this
In this respect, acetic anhydride is a particularly suitable reagent. may affect other substituents present on the unit.
To perform this selective acetylation reaction without
Especially when operating in an aqueous medium at a basic pH, especially a pH close to 8.
suitable for making. Furthermore, generating an N-sulfate group
is preferred, and this can be done using sulfating reagents of the type described above.
It can be done by For sulfation, higher than 9
A high pH, advantageously a pH of 9 to 10, is used. After the sulfation reaction, the carboxylic acid is removed by the addition of strong acid groups.
Liberation of syl groups can be carried out. The generated product is then converted into an appropriate form using an exchange resin.
can easily form salts in the on state. natural products
In particular, the cation is sodium. death
Exchange resin with sodium cations
is used advantageously. In addition, potassium, lithium, magnesium,
Calcium salts can also be formed. using a proton exchange resin and then produced
The acid is neutralized by the cationic base. Furthermore, the present invention provides for each step of the above synthesis method.
It is also directed toward oligosaccharides that constitute intermediates. In one type, these oligosaccharides are less
both contain one binary A-U and U-A unit
This is a fully protected and reducing end unit.
The anomeric carbon has a reactive group or is non-reduced.
It has a single free OH group on the original terminal unit, and this
The OH group isA3, 4 or 6 position for units
occupy,UOccupies 2nd, 3rd or 4th position in case of unit.
Melt. In other types, oligosaccharides are e.g.
Fully protected as obtained at the end of the silation process
It is composed of units. For other types, there may be one
or from products in which several OH groups have been liberated.
It's on. These various oligosaccharides have the structure (A-U)_oIf
is (U-A)_o[In the formula, n is a number from 1 to 6]
Consists of chains based on binary units. These oligosaccharide numbers correspond to a-b, a-c chains.
Ru. In one group of intermediate oligosaccharides of the present invention, glyco
A side chain is composed of a single type of these binary chains.
will be accomplished. In other groups, similar species of these types exist.
Ru. The corresponding oligosaccharides have a-b and
and a-c units. The above in one or several binary units
It is understood that the order of the chain can be reversed according to the present invention.
be understood. According to one variant of the method, even one of the intermediate oligosaccharides mentioned above is
or several consecutive a or b or c units
Contains. According to another variant, the intermediate oligosaccharides are
During the course, one or several units of neutral sugar
and/or contain several desoxy sugars. child
The various protecting groups for these sugars are as described above.AandUunit
This corresponds to the definition for . In these oligosaccharides, the structural units are 1-2,1
-3, 1-4 or 1-6 type bond
Depending on the nature of the alcohol used in the cosylation step,
are connected to each other. of heparin or heparan-sulfate fragments
Oligosaccharides with the structure are c1α → 4a, a1α → 4b, a1α → 4c and b1β → 4a Contains a combination of One group of preferred oligosaccharides is b1β → Has a structure of 4a containing at least one binary unit; That is, [D-glucuronic acid] 1β → 4 [D-glucosamine], which has the formula: [R in the formula₁may be the same or different, as required
Together with R, protecting groups, especially semi-permanent groups SP, can also be used.
or a permanent group P, and T is a temporary group t or
Represents a permanent group p or a hydrogen atom, N is a nitrogen-containing group,
amine or amine derivative precursor, R is a lipid
aliphatic or aromatic groups, especially 1 to 4 carbon atoms
is an alkyl group having
represents a reactive group such as a halide, and R
is an alkyl group, and M is a group that blocks the acid function.
and these various symbols have the meanings shown above.
do〕 corresponds to In a subgroup of the above kind, the radicals R,
R₁and T are all the same and p or sp
Indicates the group. In other groups, the group R₁are different from each other
Yes, at least one represents an sp-type group, and
together with R and one or more other
Group, R₁indicates a p group. The general meanings of the symbols in the formulas are as follows:
It is noted that it also applies to Eq. do the same
Each of these groups also has subgroups mentioned above.
(subgroup) is found. Suitable oligosaccharides correspond to formula (), () or ()
do: In the above formula, each symbol has the above meaning. Preferably, in formulas () to (), the indicated notation
The subtitles have the following meanings individually or in combination:
Has: M is a hydrogen atom or an alkyl group, especially methyl
show, sp indicates an acyl group, especially acetyl, p represents a substituted alkyl group, especially benzyl, R is an acyl group at α or β, especially an acetyl group.
thyl group, alkyl group, especially methyl or substituted alkyl group
kill, especially benzyl, or or halogen
especially a bromine group or an imidoyl group, N represents an azide group, T is an acyl group, especially acetyl, halogenated acyl
radicals, especially monochloro or trichloroacetyl groups.
a group t or a substituted alkyl group, especially ben
Zyl group, possibly paramethoxy or water
The group p represents an elementary atom. Other suitable groups of oligosaccharides include oligosaccharides of type a1→4b.
Both contain one type of unit, i.e. [D-gluco
Samin] 1 → 4 [D-glucuronic acid]
This corresponds to expression (): Suitable oligosaccharides correspond to the following formula () or ():
Ru: In these formulas () and (), the symbols M, N,
sp, p are preferably as defined above in relation to formulas () to ().
has a specific meaning, R is more preferably prope
Indicates nyl, allyl, imidoyl group or -H
, where N is more specifically -NH-acetyl
Indicates the group. It is thought that the order of chain formation of units can be reversed.
It will be served. In another preferred group, Oligosaccharide is C1α → 4a type containing at least one binary unit of That is, [L-iduronic acid] 1α → 4 [D-Gluco
Samin] and corresponds to the expression (): Suitable oligosaccharides correspond to formulas () and (): In preferred embodiments, these formulas () and
The symbols shown in parentheses have the following meanings: The various sp and p groups may be the same,
Indicates a sil group, especially an acetyl group, or a different
and an acyl group, especially acetyl or ben
Zoyl and aryl or substituted alkyl groups
selected from N is a precursor nitrogen-containing group, preferably of formulas () to ()
It is different from that present in compounds and has a special
NHCOO- (substituted alkyl group), especially -NH-
COO−CH₂−C₆H_Fivegroup, which refers to a nitrogen-containing group
allows for different processing, andAunit
form different amino derivatives at the 2-position of
It is possible, T is acetyl, halogenated acyl group, especially mono
Chlor or trichloroacetyl, p-methoxy
indicates cybenzoyl, symbols p, M and R are preferred
has the preferred meaning given above for expressions () to ().
have Other types of binary units of suitable oligosaccharides are a1α → It has a 4c structure, That is, [D-glucosamine] 1α → 4 [L-iduronic acid], which has the following formula (XI)
corresponds to: In particular, oligosaccharides correspond to formulas (XII) and (): [In the formulas, the preferred meanings of the symbols are for formulas () to ().
has the meaning set forth above]. Other suitable intermediate oligosaccharides falling within the scope of the invention
The protective group is partially removed during the synthesis process.
corresponds to the product that is In particular, products of this kind
contains an OH group instead of an sp group. Preferred intermediate products are the complete octasaccharides described above.
(ABCDEFGH) or hexasaccharide (C′DEFGH)
Compatible with oligosaccharides that have a sequence structure
do. Preferably these are disaccharides AB, BC, CD etc.
, trisaccharides ABC, BCD, etc., tetrasaccharides ABCD,
Hexasaccharides such as BCDE, pentasaccharides ABCDE, etc.
ABCDEF, etc., heptasaccharides ABCDEFG or
BCDEFGH or the octasaccharide itself. Among these oligosaccharides, especially structures BC, DE,
DEF, EF, GH, FGH, EFG, EFGH,
Mention may be made of DEFGH and CDEFGH. Preferred intermediate disaccharides are binary components of formulas ()-()
corresponds to the unit. A preferred group of intermediate trisaccharides has the structure DEF,
It corresponds to any one of formulas () to (XI). Preferably, N₁and N₂are the same or each other
different from azide or -NH-
Indicates an acyl group, especially an -NH-acetyl group. Other suitable trisaccharides have the formula: It has an FGH type structure, In the above formula, each symbol has the meaning shown above,
Two of the two glucosamine units of structure F and H
substituent N of₁and N₂are the same or advantageous
are different, as is the case with natural products.
and an azide or -NH-COO-acyl group,
especially -NH-COO-acetyl or -NH-
COO−CH₂−C₆H_Fiveselected from the group. Other suitable intermediate oligosaccharides are structured by tetrasaccharides.
will be accomplished. In more detail, the advantageous tetrasaccharide has a structure
EFGH, which corresponds to the following formula: In the formula, the preferred meanings of different symbols are shown in formula (XII).
correspond to what has been done. Other types of intermediate oligosaccharides are pentasaccharides, especially the following formula:
is constructed by the structure DEFGH. [wherein each symbol has the above preferred meaning,
N₁,N₂,N₃are the same or different from each other
It is selected from the above meanings. ] As mentioned above for binary units, the present invention
Furthermore, one, several, or in some cases all of the OH groups
Also related to the above oligosaccharides released during the synthesis process.
It is something. Furthermore, the present invention provides novel compounds, respectively.
Corresponding to each definition above, one or several official
Directed to oligosaccharides with functional groups, but disaccharides
[2-N-sulfate (or 2-N-acetate)
)-6-O-sulfate-D-glucosamine]
-Excluding methyl-D-glucuronic acid. These functional groups are preferably ester-based.
more specifically in the form of inorganic anions.
exist. Heparin or heparan-sulfate type organisms
Particularly suitable for esthetics as it exists in scientifically active molecules.
is composed of sulfuric acid ester. Other advantageous esters correspond to phosphoric esters
Ru. These functional groups contain one or similar types of primary atom.
alcohol and/or secondary alcohol and/or
or supported by a primary amine function. Suitable types of oligosaccharides according to the invention include, for example, those mentioned above.
anion at the 6 and/or 3 position
It includes an a unit with. Particularly preferred types are esters, especially sulfates.
Containing a units with groups in the 6- and 3-positions
Ru. This type of oligosaccharide favors the 2nd position of unit a.
is substituted by sulfate or by other substituents.
It has a converted primary amine functionality. The present invention contains at least two units a.
In sugars, the amine function at the 2-position is
Can be substituted by one or different groups
Wear. A preferred group of oligosaccharides of this type are secondary alcohols.
sulfates, especially on primary alcohol functional groups.
It includes a unit a containing a group. Preferred oligosaccharides of this group are
−NHSO₃ ^-It has a group. Other oligosaccharides are -NH-
It has a syl group, especially -NH-acetyl. Preferably, the esters listed below are inorganic or organic.
mechanical cations, especially metal cations, (especially alkalis)
cations) or nitrogen-containing organic bases.
Thiones such as salts with triethylammonium
It exists as such. The cations used are composed of sodium
be done. Other cations such as potassium, magne
sium or calcium, like a cation
is also suitable. Other suitable types of oligosaccharides according to the invention
Therefore, the carboxyl group of unit b or c is free.
or preferably as e.g.
It is in the form of salts with organic or inorganic cations. child
These may be protected as described above. Preferred compounds contain a sulfate group in the 2-position
Contains unit c. Other suitable compounds include a sulfate on the b unit.
contains. In these types of oligosaccharides, the hydrogen pyran ring
The roxyl group may be free or alkyl type.
protected by a permanent group of methyl groups. These various types of suitable compounds have the above properties.
Corresponding unitAandUContains a combination of. the biologically active molecules mentioned above, especially the octasaccharides;
presence in ABCDEFGH or hexasaccharide C′DEFGH.
In consideration of the present invention, suitable oligosaccharides have the above formulas () to (
) and () to () correspond to the compounds
but here the sp group is substituted by an anion.
Suitable compounds correspond to the salts of the above-mentioned compounds. Other suitable oligosaccharides include, in addition to the N group of the a unit, NH
-acyl group, especially -NHCOCH₃, −NHSO₃-
include. Suitable disaccharides of this type are BC, DE, EF or
has a GH-type structure, and each has the following formula ()
Corresponds to ~(): Other suitable oligosaccharides of the present invention have the following formula (XI):
and of structure DEF or FGH with ()
Contains or consists of a chain: Other suitable oligosaccharides correspond to the following formula (XI):
Contains or depends on the tetrasaccharide of EFGH with the structure
Consists of: Other particularly suitable oligosaccharides are the following oligosaccharides of formula (XII):
Contains or consists of sugars. Particularly suitable oligosaccharides of the present invention have the following formula (
) containing the hexasaccharide of the structure CDEFGH corresponding to
or is constituted by it. Other oligosaccharides are expressed by the above formulas () to ()
corresponds to either of
It has an OH group. These compounds are then completely
be “deprotected”. In other oligosaccharides, −OSO₃-The group is OH
can be substituted by groups. Preferably, the oligosaccharide of the present invention has the above anion and
It includes salts, preferably double salts, with the above cations. So
Due to the structure, the product of the present invention has polar
constitutes an interesting synthetic intermediate and is biologically active.
production of a given fragment or fragment derivative of a molecule
enable. These are especially suitable for comparison compounds for structural studies.
Configure. From the pharmacological study of oligosaccharides of the present invention, certain
These compounds play a role in certain steps in blood clotting.
biological activity that can be specifically inhibited.
It was found that Compounds of interest are
For example, the sulfated and deprotected formula ()
is composed of the trisaccharides of Example 13A.
It is a conductor. Of particular note is the sulfated and deprotected formula
The pentasaccharide of (), especially the derivative 50, becomes AT
It has a particularly high affinity for blood and
Activation of X-factor or extremely high degree of factor
It shows selective inhibitory activity. Therefore, the present invention is further developed in the laboratory.
Inhibition of Xa factor and measurement of antithrombin
Anticoagulant activity can be tested at defined levels.
as a comparative element to study other desirable substances.
and as a component of useful biological reagents.
It's about things. Structure DEF in which the D unit contains an N-sulfate group
For example, the trisaccharide of formula () having Yin−
Anti-Xa of around 7u/mg measured by Wessler test
Has activity. Pentasaccharide 50 of Example 9 is more obvious than heparin
It is characterized by a high (Yin-Wessler) titer. In particular, this pentasaccharide is equal to or near 2000μ/mg.
Greater anti-Xa activity (Yin-Wessler) and AT
has a high degree of affinity for In tests using chromogenic substrates, this activity
was 4000 anti-Xa units/mg (TEIEN A.M.
Method and LIE variant: Thrombosis Research
No. 10, 1977, pp. 388-410) This test was produced by STGMA Company, Inc.
Commercially available as an 8μ/ml solution in physical serum
It consists of using the Xa factor and the substrate
The concentration of is 1.33mM. To perform this test, do the following:
I can do it. 10 µl of the solution to be quantified and Trismaley
of human plasma diluted in phosphate buffer 0.02M.
Mix with 300 μl (PH5). Incubate it for 1 minute at 37°C. Add 100 μl of the above Xa factor (8 μ/ml),
Then, after 1 minute, inject the resulting solution into the substrate.
Ru. The total anticoagulant activity of this product was determined by the APTT test.
It is extremely low at 4μ/mg. This property makes certain steps in blood coagulation particularly
You can check. If this compound is considered, it has strong antithrombotic activity.
It shows that it can be achieved. Furthermore, according to the present invention
Derivatives that cause damage to blood vessel walls (atherosclerosis)
of extreme interest in overcoming arteriosclerosis (pressure and arteriosclerosis)
There is. Additionally, they have active effects on red blood cell aggregation
and does not cause thrombocytopenia.
It has the following advantages. Furthermore, this
It also has the advantage of virtually lacking the effect of
This eliminates the risk of bleeding. these two
properties are extremely important in medical applications. In addition, long-term pharmacology via the subcutaneous route
A positive reaction was observed, which further indicates that this compound
Show a marked interest in learning. The oligosaccharides of the present invention are further advantageous because they are non-toxic.
It is. Therefore, these compounds are particularly effective against thrombosis.
Developing drugs that are particularly useful in preventing and treating
It is useful for Therefore, the present invention provides even higher anti-Xa activity.
The oligosaccharides, especially the pentasaccharides mentioned above, have
It also relates to a pharmaceutical composition comprising: In particular, the present invention provides an effective amount of the active ingredient as a pharmaceutical adjuvant.
A pyrogen-free pharmaceutical composition containing
It is about things. Additionally, the present invention provides pharmaceutical vehicles suitable for oral administration.
The present invention relates to a composition comprising the following. For oral administration
Suitable dosage forms of the invention are advantageously gastric resistant capsules.
Can be made into cells, pellets or tablets, pills
Alternatively, it can be provided in liposome form.
Ru. Other pharmaceutical compositions include these oligosaccharides for enteral administration.
constituted in combination with suitable adjuvants. The corresponding throw
The dosage form is a suppository. Other dosage forms of the invention include aerosol or
It's pomade. Furthermore, the present invention provides intravenous, intramuscular or subcutaneous
Sterile or sterile injections suitable for administration in
It also relates to injectable pharmaceutical compositions. These solutions advantageously contain 1000
~100000μ (Yin−Wessler), preferably 5000~
50000, for example 25000μ/ml, in this case.
These solutions are intended for subcutaneous injection. Furthermore, this
If they are intended for intravenous injection or perfusion,
For example, 500 to 10,000μ per ml of oligosaccharide, especially 5,000μ
can contain. Advantageously, pharmaceutical compositions of this type are suitable for single-use injections.
It is provided in a form that can be used in a firearm. Furthermore, the present invention provides that the oligosaccharide is, for example, dihydro-
Vasoregulators like ergotamine, nicotinates
or blood clot-dissolving agents like urokinase.
Other activities particularly useful for the prevention and treatment of thrombosis
It also relates to pharmaceutical compositions containing the ingredients in combination.
It is. The pharmaceutical composition of the present invention can be used in humans and animals.
Inhibiting (preventing or preventing) certain stages of blood clotting
It is particularly suitable for patients undergoing surgery,
Process, tumor development and bacteria
Blood coagulation caused by chemical or enzymatically active substances, etc.
exposed to the risk of hypercoagulability, especially from disorders such as
It is especially suitable if To illustrate the present invention, the following is a description of the
An example of the pharmacology that can be used
the risk level of a patient's hypercoagulable or thrombotic state.
1000-25000μ (Yin−
Wessler) is administered subcutaneously to the patient.
Or 1,000 to 25,000μ/intravenously at regular intervals for 24 hours.
It consists of discontinuous administration or continuous administration by perfusion.
Consisting of continuous administration, or 1000 to 25000μ
Administer (3 times weekly) intramuscularly or subcutaneously.
(These titers are Yin−
(expressed as Wessler units). These doses are
Of course, the results obtained and the blood sample performed beforehand
analysis, the nature of the disorder the patient suffers from, and the general
may be adjusted for each patient depending on their health status.
I can do it. In addition to pharmaceutical compositions containing oligosaccharides as they are,
The invention further comprises at least one oligosaccharide as described above.
Covalently attached to soluble or insoluble supports
The body is advantageously bound by reducing terminal sugars.
A pharmaceutical composition comprising: Conjugate immobilized on a suitable soluble support
(conjugate) is an oligosaccharide combined with AT.
Ru. Conjugates of this type involving pentasaccharides 49 are particularly preferred.
It is. This type of product is a place of AT deficiency.
may be of particular interest in the prevention of thrombosis.
Ru. Other suitable conjugates with soluble supports include, for example
protein, especially polylysine or bovine serum albumen.
produced from oligosaccharides fixed in a vehicle such as
will be accomplished. These products are circulatory and produced in vivo.
Cloned in vitro using antibodies or appropriate techniques
Useful as an immunogen for monoclonal antibodies
be. In other preferred conjugates, oligosaccharides of the invention
is bound to an insoluble support. advantageously customary
A support is used. These conjugates have high specificity for e.g.
for the purification and estimation of
Novel thrombotic blood formation by fixation on synthetic polymers
As an immunoabsorbent for the development of liquid compatible polymers
Useful. Furthermore, the present invention provides the use of the oligosaccharide in nuclear medicine.
Items related to use as radiological medical products
It is. These products are currently used in this field
Tracers selected from those that have been
Marked by Cunetium 99m. For this purpose, techniques obtained from commercially available generators
Titanium-99m is converted into non-reactive heptavalent sodium pertechnetate.
Technetium reduced to 4 valence as thorium
, and this tetravalent one is in technetium
It is probably the most reactive. This conversion is
Certain tin salts (stanrous chloride), iron salts (ferrous sulfate)
and salts such as titanic acid (titanium trichloride).
This is done by a reduction system. In order to fix technetium to the molecule, a large amount of
This simple reduction of technetium is often
It is sufficient if it is carried out under PH conditions. On the one hand, the production of the invention constituting the support
The dosage is about 100 to 100μ (Yin-Wessler)
It can be used in To obtain these radiopharmaceuticals, P.V.
KULKARNI's ``The Journal of Nuclear
Medicine” Volume 21, No. 2, pp. 117-121
It can be operated in accordance with the law. Detection and long-term diagnosis of thrombosis and thrombotic conditions
Therefore, products labeled in this way cannot be tested in vivo.
It is advantageously used in experiments. Furthermore, the oligosaccharide of the present invention is a glycosaminoglucose.
Specificity of many enzymes involved in lonoglycan metabolism
It can also be used to measure gender. Other advantageous properties of the invention are that
See Figures 1-32 which illustrate the compounds used.
This will become clear from the following examples. Example 1 Derivative 13, i.e. (prop-1'-enyl
-2,3-di-O-benzyl-α-D glucopi
Synthesis of methyl uronate (ranoside) From glucose according to the following steps a) to j)
Perform this synthesis. a Production step of allyl derivative; b Benzylide at the 4- and 6-positions of the allyl derivative
protection step by a group; c step of introducing benzyl groups into the 2- and 3-positions; d 4- and 6-positions by removal of benzylidene group
removing the protecting group; e Introduction of a trityl group at the 6-position, followed by the addition of a trityl group at the 4-position.
Cetylation reaction stage; f Removal step of the trityl group at the 6-position; g oxidation stage of the primary alcohol group at position 6; h Methylation step of the carboxyl group at the 6-position; i step of introducing a propenyl group to the 1-position; j Removal step of acetyl group at position 4; These steps were carried out as follows (Section 1).
(See Figures 1 and 2). a Allyl α-D-glucopyranoside (compound
1) Manufacturing Gaseous hydrochloric acid (18g) to allyl alcohol
Heat the solution (600 ml) to 70 °C. next
Add anhydrous glucose (300g) and incubate for 3 hours.
Maintain temperature. The reaction was carried out using methanol/chloroform (1/4, v/
v) additionally by thin layer chromatography (t.l.c.) in solvent.
It can be traced. The brown solution obtained after 3 hours was placed in vacuo.
Dry and concentrate with
Mix and dry and concentrate again. to the resulting residue
Add acetone (500ml) and bring to a boil until the entire amount is dissolved.
Maintain this state until it is resolved. After cooling, this liquid
Decant the body. The same treatment is applied to the residue again.
I do. Residue of derivative 1 by extract T.L.C. analysis
Loss of material or very strong due to impurities
The above treatment is continued until extracts contaminated with
conduct. A portion (12 g) of the extracted first fraction was siluted.
Apply color chromatography. Acetone/Ethe
Derivative 1, which can crystallize in the mixture, is recovered.
(6.5g; M.P.95-99). The rest of the product is the same process
It can be purified with b Allyl 4,6-O-benzylidene-α-D-
Allyl derivative leading to glucopyranoside (compound 2)
Protection of conductor positions 4 and 6 Compound 1 (37g) was dissolved in dimethylformamide (200g).
ml). Then dimethoxytoluene (41
g) followed by hydrated paratoluene sulfonate.
Add phosphoric acid (130mg). After heating under vacuum and reflux for 2 hours (in a water bath), the reaction
ends (t.l.c. methanol/chloroform, 2/
25, v/v). Evaporate the solvent. Meal syrup
Dissolve in ethanol (minimum amount). Add this solution to
Drop into aqueous sodium carbonate solution (6.3 g in 320 ml of water)
Let it go down. The resulting precipitate was reconsolidated in ethanol.
Crystallize (21 g; M.P. 120-121°C). The mother liquor is further
Gives product 2. Overall yield (37g; 71.4%). c Allyl 2,3-di-O-benzyl-4,6-
O-benzylidene-α-D-glucopyranoside
Introduction of benzyl group leading to (compound 3) Compound 2 (45g) was dissolved in anhydrous DMF (500ml)
I let it happen. Sodium hydride (50% dispersion in oil 28
Add g). After 30 minutes, the mixture was cooled to 0°C and then treated with benzene bromide.
Zil (52ml) was added dropwise. The reaction is t.l.c.
(ether/hexane, 1/1, v/v).
Ru. Next, slowly add methanol (150ml),
Evaporate to dryness and remove again with chloroform.
vinegar. Wash the chloroform phase with water and dilute with sodium sulfate.
Dry on a dry surface. After evaporation of the solvent, the residue is
crystallize in a tel/hexane mixture (36.5
g; M.P. 83-84°C). The product is t.l.c. (ether/hexane, 1/1,
due to impurities migrating higher in v/v).
It is slightly contaminated. d Allyl 2,3-di-O-benzyl-α-D-
Benzyl leading to glucopyranoside (compound 4)
Removal of dene group Compound 3 (56g) in methanol (1) solution
Add water (450ml) and then add the hydrated paratolue.
Add sulfonic acid (17g). After 2 hours at 80°C, the mixture was allowed to cool and the solution
After evaporation of the medium, the residue was dissolved in chloroform (1
) and take it out again.The chloroform solution has a medium pH.
Wash with water until dry, then dry with sodium sulfate.
Let dry. In this way, pale yellow syrup (48g) was obtained.
Ru. This syrup is used in the next step (synthesis of compound 5).
used for. e Allyl 2,3-di-O-benzyl-6-O-
Trityl-α-D-glucopyranoside (compound
5) and its 4-O-acetyl analogue (compound
6a) Introduction of trityl group to the 6-position leading to continuous
followed by acetylation reaction at position 4. The obtained derivative 4 (48 g) was added to pyridine (250 ml).
and add trityl chloride (38.5g).
Ru. After 1 hour at 100°C, the reaction is terminated (t.l.c.
ether/hexane, 1/1, v/v). to the previous solution
Add acetic anhydride (200ml). Complete the reaction the next morning
(t.l.c., ether/hexane, 1/2, v/
v). Evaporate to dryness and dissolve the residue in chloroform (500
ml) and chloroform phase to 10% acidic
Wash with potassium sulfate solution, wash with water, then
Dry with sodium sulfate. Evaporate the chloroform. In this way
Compound 6a is obtained. Produce compound 7a as is.
Used in reaction. f Allyl 4-O-acetyl-2,3-di-O-
Benzyl-α-D-glucopyranoside (compound
7a) Removal of the trityl group leading to The obtained derivative 6a was added to chloroform (500ml).
Dissolve. Cool this solution to 0°C and
Add a methanol solution of boron trifluoride (20%,
120ml) dropwise while stirring. This anti
T.L.C. (toluene/acetone, 10/2, v/v)
Track with. Decant the reaction mixture into a separatory funnel. Black
The loform phase was washed with water (2 x 100 ml) and bicarbonate.
Wash with saturated solution of sodium and then to pH or neutrality
Wash with water until clean. After drying and evaporation, the obtained
The residue was equilibrated with silica gel in toluene.
(500 g) column. to pure toluene
After eluting most of the impurities, the product was evaporated into toluene.
Dissolved by a mixture of acetone/acetone (10/2, v/v).
Let go. 48 g of compound 7a are thus obtained.
This is used directly in the synthesis of compound 8a. A portion of compound 7a is obtained in pure form. [α]20 D = +11° (chloroform). This I.R. and
According to N.M.R. spectrum, elemental analysis
Similarly, check the structure. g (allyl-4-O-acetyl-2,3-di-
O-benzyl-α-D-glucopyranoside)
Primary alcohol at position 6 leading to rononic acid (compound 8a)
Oxidation of group Acetone (800ml) solution of compound 7a (48g)
Cool to -5°C. Next, add chromium trioxide (30g)
Add sulfuric acid (3.5M: 125ml) solution dropwise. Mixed
Leave the mixture to come to room temperature. The reactants are treated with T.L.C.
Test with alcohol/chloroform, 1/10, v/v).
Ru. At the end of the reaction, the reaction mixture was poured into water (500ml).
Open. Extract the product with chloroform (3
times, 250ml). Add the chloroform phase until the pH becomes neutral.
Wash with water and dry with sodium sulfate.
Concentrate. The obtained syrup (83g) is used as a compound as it is.
Used in the manufacture of 9a. h (allyl-4-O-acetyl-2,3-di-
O-benzyl-α-D-glucopyranoside)
Calculation at position 6 leading to methyl lonate (compound 9a)
Methylation of boxyl group The syrup obtained in the manufacturing stage of compound 8a was
(300ml). Diazomethane A
Add t.l. solution until compound 8a disappears (t.l.
c. ether/hexane, 1/1, v/v). With acetic acid
After acidification, the solvent is evaporated. Dissolve the resulting residue (53g) in hot ethanol.
let Derivative 9a crystallizes on cooling. recrystallization
After that, pure this compound 9a (18.4g) M.P.85-86
°C, [α]20 D = +12° (1,2 chloroform) obtained
Ru. This product was evaluated by its IR spectrum, N, M.R.
It is characterized by its spectrum and its elemental analysis. 7.6 g of compound 9a was also obtained from the recrystallization solution. The overall yield of compound 9a from compound 2 was 38%.
Ru. i (prop-1'-enyl-4-O-acetyl-
2,3-di-O-benzyl-α-D-glucopi
Ranoside) leads to methyl uronate (compound 10a)
Introduction of propenyl group to the 1st position Derivative 9a (4 g) was mixed with ethanol (119 ml).
Dissolved in a mixture of water (51ml) and water (17ml)
let Then diazabicyclooctane (170mg)
Add and reflux. Tris chloride (tris) in boiling solution
Phenylphosphine)-Rhodium() (550mg)
Add. Continue boiling for 4 hours (t.l.c., aete)
1/1, v/v). At the end of the reaction, filter the solution to remove the solvent.
Ru. The residue was dissolved in ethyl acetate/chloroform (1/50,
v/v) by silica gel (150 g) in a mixture
Perform chromatography. obtain compound 10a
(3.25g; 81%). This compound 10a is in ethanol
crystallizes out. [α]20 D = +12° (1, chloroform
). M.P.90℃. The structure was determined by elemental analysis and N.M.R.
and confirmed by IR spectrum. j (prop-1'-enyl-2,3-O-benzi
Methyl-α-D-glucopyranoside) uronic acid
Removal of the acetyl group at position 4 leading to (compound 13) Derivative 10a (350 mg) in methanol (5 ml)
Dissolve. Sodium methanol (0.2ml,
2M). After 1 hour at ambient temperature,
Kusu 50−H⁺Resin (dowex resin50−H⁺) added
to stop the reaction. After filtration, product 13 is obtained.
Product 13 is a small amount of product resulting from α-β elimination
contaminated by. Benzoylation instead of acetylation reaction
By modifying step e) in which the reaction is carried out, allyl-4-
O-benzoyl-2,3-di-O-benzyl-6
-O-trityl-α-D-glucopyranoid
Leading to compound 6b). Next, remove the trityl group,
Allyl-4-O-benzoyl-2,3-di-O-
Benzyl-α-D-glucopyranoside (compound
7b). These reactions are carried out as follows. Structure of compounds 6b and 7b: A solution of compound 5 in pyridine was then added to benzoic chloride.
(1.5 eq.) and the reaction was started with t.l.c. (ethyl acetate).
1/20, v/v). excess
benzoyl chloride, do not add excess methanol.
cause it to be destroyed by After evaporation to dryness, the residue is
Remove again with loloform and 10% KHSO_Fourmelting
Wash with liquid, wash with water, dry and concentrate to dryness.
let The resulting syrup was directly converted to compound 7b.
Use for synthesis. This syrup (105g, compound
(obtained using 30g of 3) in chloroform (300ml)
Dissolve in. Para-toluenesulfonic acid (methano
76 g of monohydrate in 100 ml of water). next morning,
Terminate the reaction (t.l.c., ethyl acetate/chlorophosate)
Lum, 1/20, v/v). The pH of the chloroform phase is medium.
Wash with water until dry, dry and concentrate
let The resulting syrup (98g) was mixed with silica gel.
(1.2Kg) column.
Elute with loloform (0.6) then ethyl acetate.
Dissolved in a mixture of chloroform/chloroform (1/20, v/v).
Let go. Thus pure derivative 7b (30g)
get. This derivative 7b was used directly to produce compound 8b.
Used during the construction stage. From compound 7b, − at position 6
CH₂oxidize the OH group, then the resulting carboxy
Introducing a methyl group into the allyl group and continuously (allyl-4
-O-benzoyl-2,3-di-O-benzyl-
α-D-glucopyranoside) uronic acid (compound
8b) and the corresponding methyl ester (compound 9b)
get. These derivatives are produced as follows. Production of compound 8b and its ester 9b: Compound 7b (27g) was added to the treatment of 7a in the production of 8a.
Process as explained above. at the end of processing
The syrup obtained was as described for compound 8a.
Contains compound 8b methylated with diazomethane
do. The residue obtained at the end of methylation was
(200g; ether/hexane, 1/1).
Ru. Compound 9b is thus obtained (21 g; 77.5
%). This IR and NMR spectrum reveals that the structure
Check the structure. From compounds 9b to 10a, depending on the handling method
The corresponding propenyl derivative 10b is prepared. Derivative 13 is produced by the same reaction as for 10a.
obtained from compound 10b. With other changes, (allyl-2,3-di-O-
benzyl-α-D-glucopyranoside) uronic acid
and (2,3-di-O-benzyl-α-D-glue
Copyranoside) methyl uronate (compound 11 and
12) Manufacture. Compound 8b (1.9 g) was dissolved in methanol (40 ml).
Let me understand. Next, add soda (5N) to a 1M concentration of soda.
Add only enough to give strength. The reaction is t,
l.c. (methanol/chloroform, 1/4, v/v)
Track with. When finishing the reaction, water (100ml)
Add. After washing with ether and acidifying
Extract the product with ether. acidic ether phase
Wash with water until the pH becomes neutral. Derivative 11
has not been isolated. Derivative 11 as diazometha
Compound 1 was methylated with an ether solution of
2 (900 mg; 56%). This compound 12 is silica
Gel column (ether/hexane, 1/1, v/
v). [α]20 D = +35.2° (1.3, chloro
Holm). Its IR and N.M.R. spectra and
Confirm the structure by elemental analysis. similar method
Then, derivative 11 is obtained from compounds 9a to 9b,
Furthermore, it is possible to obtain derivatives 12. Compound 13 was obtained from compound 12 by the following method.
It will be done. Derivative 12 was prepared as described for compound 9a.
It is then treated with a complex salt of rhodium. compound 13
obtained in 90% yield. This compound 13 has its IR and
Characterized by N.M.R. spectrum.
Furthermore, acetic anhydride (1 for 180 mg of compound 9a)
ml) to obtain compound 10a. Other changes make derivative 13 a compound 9a or 9b.
Compound 10a or
can be obtained from 10b. Example 2 Disaccharide 20, i.e. methyl (1-bromo-
3.6-di-O-acetyl-2-azido-4-O
-(2,3-di-O-benzyl-4-O-chloro
loacetyl-β-D-glucopyranosyl)uro
Synthesis of nate-β-D-glucopyranose This synthesis takes place in the following steps (see Figures 3 and 4).
(see). A Monosaccharide 16 from derivative 13 of Example 1, i.e.
(1-bromo-2,3-di-O-benzyl-4
-O-chloroacetyl-α-D-glucopyrano
sid) Production of methyl uronate; B Condensation of monosaccharide 17 and compound 16 leading to disaccharide 18; C Acetolysis of compound 18 leading to disaccharide 19; D Bromination to give disaccharide 20 A Production of monosaccharide 1b This synthesis involves monosaccharide 13, i.e. (prop-1′-e
Nyl-2,3-di-O-benzyl-α-D-glue
Copyranoside) from methyl uronate in the following three steps
Done: 1: Chloroacetylation of compound 13: 2: Unblocking anomeric carbon
(unblocking); 3: Bromination of anomeric carbon 1: Compound 14 i.e. (prop-1'-enyl
2,3-di-O-benzyl-4-O-chloroa
cetyl-α-D-glucopyranoside) uronic acid
Chloroacetylation of compound 13 leading to methyl Compound 13 (2.8g) in pyridine 30ml (6.56mmol)
dissolve in it. After cooling to 0°C, chloroacetyl chloride
Drop 10 ml of dichloromethane (20 ml) solution of 2 ml of chill
Remove and add. After 30 minutes, evaporate to dryness and clean the residue.
Remove with 200ml of loloform and KHSO_Four10% solution of
Wash with liquid and water in that order, dry, and concentrate. obtained
The syrup was soaked in silica gel (200g; eluent).
Clamatog of AcOEt/hexane; 1/3; v/v)
Spread roughly. In this way, 2.7g of pure
Compound 14 is obtained in the form of syrup (yield 80%);
[α]20 D=+2° (c=1.5; chloroform). elemental content
The desired structure is confirmed by analysis and N.M.R. spectrum.
recognized. 2: Compound 15, i.e. (2,3-di-O-be
D-gluco-4-O-chloroacetyl-D-gluco
Anomerits leading to methyl uronate (pyranoside)
Block carbon deblocking 2.7 g (5.3 mmol) of derivative 14 in acetone/water
Dissolve in 80 ml of (5/1; v/v) mixture. oxidation
Add mercuric (3.1 g) followed by mercuric chloride
(3.9 g) in acetone (27 ml) is added. 5 minutes
Afterwards, the salt is removed by filtration. Remains after concentration to dryness
Take things out again with chloroform. chloroform
Wash the phase with 10% KI solution and then water. After evaporation,
The product was crystallized in a methyl acetate/hexane mixture.
let M.P.105−107℃; [α]20 D = 4.7° (equivalent, 1
2 g of solid chloroform) are obtained. Elemental analysis and
NMR spectrum confirms the structure (yield
80%). 3: Compound 16 i.e. (1-bromo-2,3
-di-O-benzyl-4-O-chloroacetyl
-α-D-glucopyranoside) methyl uronate
Bromination of anomeric carbon leading to 2g (43.3mmol) of compound 15 was dissolved in dichloromethane.
Dissolve in 50ml of water. sym-collidine 4.8ml (34.4
m mol) at 0°C, HEPBURN D.R. and
HUDSON H.R., J. Chem. Soc. Perkin I (1976).
Bromomethylene dimer prepared according to 754-757
Add tylammonium bromide (17 mmol). After 4 hours of reaction, the mixture was diluted with 100 ml of dichloromethane.
Dilute and then pour into ice water. After washing with permanent water, remove the solvent.
evaporate. Silica gel (20g; eluent hexa
chromatograph of ethyl acetate, 2/1; v/v)
When poured, the compound is in the form of syrup weighing 2.06g.
16 is obtained (90% yield). [α]20 D = +82.5゜(c
= 1.5; chloroform). Elemental analysis and N.M.R. min.
Analysis confirms the structure. B disaccharide 18 or 3-O-acetyl-1,6-
anhydro-2-azido-4-O-(2,3-
di-O-benzyl-4-O-chloroacetyl-
β-D-glucopyranosyl) methyl uronate
Production of β-D-glucopyranose This synthesis consists of monosaccharides 16 and 870 mg (3.8 mmol) of 17
Based on the condensation of 870 mg (3.8 mmol) of compound 17 dichloromethane
Drierite (1g) and powder
4Å molecular sieve (0.5g) and new
Add freshly prepared silver carbonate (0.525 g). 2
After stirring for an hour, 670 mg (1.3 m
mol) added dropwise. After 6 days, filter the solids.
Remove. Transfer the syrup obtained after concentration to licagel.
(50g; eluent: chloroform/ethyl acetate; 4/
1, v/v). Disaccharide 18
obtained in the form of foam (421 mg; 50
%). [α]20 D=-17° (c=1, chloroform).
Elemental analysis confirms the structure. For NMR analysis
This confirms the configuration of the inter-sugar bonds. C Preparation of disaccharide of structure 19 by acetolysis of disaccharide 18
Construction Disaccharide 19 can be obtained by acetic acid decomposition of disaccharide 18 as shown below.
Manufactured. 300 mg of compound 18 was freshly distilled with 4 ml of acetic anhydride.
dissolved in a mixture with 0.5 ml of trifluoroacetic acid.
Let me understand. The reaction mixture was stirred at 18 °C for 10 h and then
Evaporate to dryness and co-evaporate with toluene.
Transfer the residue to a column chromatograph using silica gel (15 g).
Spread roughly. A mixture of dichloromethane and ethyl acetate
By elution with the compound (19:1, v/v), the structure
282 mg of a mixture of 19 anomeric acetates is a colorless
Obtained in syrup form (yield 86%). NMR
The type-to-type quantification determined by analysis is 4/1.
Ru. NMR spectrum confirms the desired structure.
It can be done. D Production of disaccharide 20 by bromination of disaccharide 19 TiBr to acetate mixture of structure 19_Fourto act
Ru. Mix 3 ml of dichloromethane and 0.3 ml of ethyl acetate.
140 mg of a mixture of 19 acetates in a combined solution at 17-18 °C.
140 mg of TiBr (2 equivalents) under dry Argo atmosphere.
Stir for 20 hours in the presence of Cool to 0℃,
After diluting with 30 ml of dichloromethane, the mixture was poured into ice water and then
Wash with 5% potassium bromide aqueous solution, water, and sulfuric acid.
Dry over sodium, filter and evaporate
Ru. The residue was purified using a silica gel column (10 g).
chromatograph. Ethyl acetate/dichloro
By elution of lomethane (19/1, v/v) mixture,
Collected in the following elution order: − Unstable in the form of colorless syrup (immediately
bromide 20 (74 mg; yield 50%).
The desired structure was confirmed by NMR spectrum.
Ru. − Fractions corresponding to unreacted starting material (28 mg;
rate 20%) - Corresponding to partially O-debenzylated compounds
fraction that hardly moves Example 3 Monosaccharide 22, i.e. benzyl-6-O-acetic acid of the formula
Chil-3-O-benzyl-2-benzyloxy
Carbonylamino-2-deoxy-α-D-g
Synthesis of lucopyranoside This derivative is benzyl-3-O-benzyl-
2-Benzyloxy-carbonylamino-2-deo
x-α-D-glucopyranoid (derivative 21)
It is manufactured by the following method (see FIG. 5). P.C.WYSS，J.KISS.Helv.Chim.Acta，58，
Compound 21 manufactured according to 1833-1847 (1975)
(987 mg, 2 mmol) was added to anhydrous 1,2-di
N-acetyl-imida in chloroethane (15 ml)
In the presence of sol (2.5 mmol freshly prepared)
Reflux for 30 hours. Cool and chloroform (50ml)
After diluting the organic phase with 1M chloric acid solution, water, carbonated water
Saturated aqueous solution of salt, washed with water and dried (sulfuric acid solution)
) and further filtered and distilled. residue
Chromatography using a column of silica gel (50 g)
Spread roughly. dichloromethane/acetone mixture
(15/1, v/v)
Derivative 22 is obtained in the form ethyl acetate/hexane
(759 mg; 71). M.
P.114-115℃; [α] D + 88° (c = 1, chlorophore
Lum). Example 4 Synthesis of monosaccharide 33 of the following formula Synthesis starts with compound 23 in the next step (6th step)
(see figure). 1. Introduction stage of benzoyl in 5th place 2 Methylation step of carboxyl group at position 6 3 Isomerization step of OH group at position 5 4 Formation stage of pyran ring 1 Benzoylation reaction 3-O-benzyl-1,2-O-isopropyly
Den-α-D-glucofuranoside (compound 23) 63
Dissolve g in 500 ml of anhydrous pyridine. Chloride
Add 85g of litil and heat to 80℃ for 1 hour. this
Obtain compound 24 in this way Optical rotation: [α] 20 D=-34.7°, chloroform. The structure of this compound is shown in infrared and N.M.R. spectra.
This was confirmed by elemental analysis. The mixture was then cooled to 0°C and benzoyl chloride 45
Add ml. The next morning, add 300ml of methanol and filter.
Allow excess reagent to decompose. Evaporate the resulting mixture
Dry and remove with chloroform. chloroform
Wash the phase, dry with sodium sulfate and concentrate.
Ru. Compound 25 is thus obtained. Dissolve the resulting syrup in 400ml of chloroform.
Let me understand. 5M para-toluenesulfonic acid methano
After adding 100ml of the solution, store the solution at 4°C until the next morning.
Leave it there. After washing the organic phase with water, mix 215 g.
get something This mixture was mixed with silica gel (ether).
chlorine/hexane, 2/1 (v/v) solvent)
Apply to matograph. In this way, compound 26 is
Get 36g. Optical rotation: [α] 20 D65.3゜, chloroform
Mu. The structure of compound 26 is infrared and N.M.R. spectra
Confirmed by . 2 Methylation of carboxyl group at position 6 Compound 26 (1.88g) was dissolved in acetone (20ml).
Let me understand. CrO₃(13g) of 3.5MH₂S.O._Four(29ml)
Add 3.5ml of the solution dropwise at 50℃. Leave to cool
and leave it in this state for 1 hour. Then anti
Pour the reaction mixture into a container, and remove the product with chloroform.
Extract. After washing with water, drying, and evaporating to dryness, the
Compound 27 is obtained. Dissolve the resulting mixture in methanol (20ml)
Then add 10ml of 1N soda and leave it in the room until the next morning.
Leave it warm. The reaction mixture was preliminarily diluted with methane.
H washed with water in a⁺Shape of Dowex 50 resin
Pass through rum (25 ml). The product is collected by concentrating the eluate.
I can get it. Compound 28 is thus obtained. This compound is dissolved in ether and
Methylated with diazomethane according to the method. evaporation
After that, compound 29 (1.08 g; 70.4%) is obtained. optical rotation
[α]20 D=-27°, chloroform. Based on elemental analysis, infrared and N.M.R. spectra
to confirm the structure of compound 29. 3 Isomerization of -OH group at position 5 triflic anhydride (0.8ml)
Cool the dichloromethane (16 ml) solution to -20°C.
Then, add pyridine (0.8ml) and dichloromethane to this.
(8 ml) solution dropwise. Then at -10℃
compound dissolved in dichloromethane (8 ml)
Add substance 29 (800 mg) dropwise. -50℃ for 1 hour
Afterwards, the reaction mixture was diluted with 160 mg of sodium bicarbonate.
Pour into the mixture (8 ml) of water and water. organic
Stir until the phase and aqueous phase separate. 3 organic phases
%HCl, H₂Wash with O and saturated NaCl solution.
Clean, dry and concentrate. In this way
Obtain compound 30. Remove the syrup again with DMF (10 ml). bird
Add sodium fluoroacetate (1.6g) and
Heat to 80 °C for an hour. In this way, compound 31 is
obtain. After evaporation, extract again with dichloromethane and water
Wash, dry and remove the residue with methanol.
and then evaporate the solvent after 1 hour. ether/
Column chromatograph using hexane (2/1) as a solvent
When applied roughly, compound 32 is obtained (450mg:
56.2%). Optical rotation: [α] 20 D=-33°, chloroform
Mu. Infrared and N.M.R. spectra of the structure of compound 32,
Confirm by elemental analysis. 4 Formation of pyran ring This synthesis is carried out from compound 32. Compound 32 (200
mg) in a mixture of trifluoroacetic acid/water (9/1)
Dissolve. After 15 minutes, the solvent is evaporated. residue
is crystallized in ethyl acetate/hexane. This way
In this manner, 110 mg of compound 33 was obtained. The characteristics of this derivative are as follows. Infrared spectrum: CHCl₃Medium cm^-1Unit γ: 3450
(OH), 3080, 3060, 3030 (CH₂: benzyl) and
and 1740 (COOCH₃) N.M.R. spectrum: ppm unit with TMS as standard
δ of place: 3.75 (S, 3H⁺, COOMe), 4.98 (1H⁺),
7.30 (S, 5H⁺,C₆H_Five) Optical rotation: [α] 20 D=+13°, methanol elemental analysis C₁₄H₁₈O₇ Theoretical value Actual value C…… 56.37 56.17 H……6.08 5.85 M.P.……125−126℃ Example 5 Derivative 38 of the following formula, i.e. 3-0-benzyl
-4-0-chloroacetyl-1,2-0-tertiary
Butoxyethylidene-β-L-methylidopyra
Synthesis of nuronate From the derivative 33 and the iduronic acid structure, the following step
It was synthesized according to the instructions (see Figure 7). acetylate α derivative 33; β A mixture of the obtained anomeric acetates 34 and 35
A brominating agent is created in the compound.
Introducing a bromine atom onto the anomeric carbon using
Ru; Forms an orthoester at γ 1- and 2-positions; δ Monochloroacetyl at position 4 of orthoester
implementation. α 1,2,4-tri-0-acetyl-3-0-
Benzyl-α or β-L-methylidopyranuro
Acetylation reaction to nates (derivatives 34 and 35) Compound 33 (3 g) with anhydrous pyridine (20 ml)
Add a mixed solution of water and acetic acid (10 ml) to
Stir at 0°C for 5 hours. Evaporate the reaction mixture to dryness
evaporate with toluene (4 x 20 ml) and dry in vacuo.
let Transfer the residue to a silica gel column (150g).
Apply to romatograph. Toluene: ethyl acetate
When eluted with a mixture of (volume ratio 4:1), the following
of substances are obtained sequentially. - a first fraction consisting of furan derivatives, - Compound 34 (alpha amanor), syrup (170mg,
4%), [α]_D=-43°;c.:1, Chlorophor
), N.M.R. (CDCl₃): δ: 6.23 (s, 1H, H
-1). - Compound 35 (β anomer), ether-hexane
Crystallized in the mixture, (2.688g, 63%), M.P.: 112−
113℃, [α]_D=+9゜(c.:1, chloroform),
N., M.R. (CDCl)₃): δ6.08(d, 1H, H-1,
J₁.₂:1.5Hz). α anomer 34 treated in the above synthesis process and
Does not separate from β anomer 35. leave the mixture as is
Used in syrup form for subsequent reactions. β Compound 36 i.e. 2,4-di-0-acetyl-3
-0-benzyl-α-L-methylidopyranuro
Bromination reaction to nil bromide Mixture of acetates 34 and 35 (212 mg,
0.5mM) with anhydrous dichloromethane (5ml)
Dissolve in ethyl acetate (0.5ml). Tetrabromide
Add Tan (250mg, 0.7mM) all at once and stir the reaction mixture.
The mixture is stirred at room temperature for 24 hours protected from moisture. 0
After cooling to °C and diluting with dichloromethane, the organic phase was placed on ice.
Washed with water (3 times) and dried (sodium sulfate)
After filtering and evaporating, a light colored syrupy derivative 36
(217 mg, 96%). N., M.R. (CDCl)₃);
δ: 6.41 (s, 1H, H-1). this compound
is extremely unstable and should not be used immediately for subsequent reactions.
do. γ 4-0-acetyl-3-0-benzyl-1,
2-0 tertiary butoxyethylidene-β-L-methy
Production of orthoester of luidopyranuronate (2.122g mixture of acetates 34 and 35, 5mM
anhydrous dichloromethane of bromide 36) freshly prepared from
(20 ml) solution at room temperature under a dry argon atmosphere.
Stir at warm temperature. sym-collidine (2.65ml,
20mM) and anhydrous tertiary butanol (3ml,
30mM) to the reaction mixture under the above conditions.
Stir for 15 hours. Dilute with dichloromethane (50ml)
After diluting, add saturated sodium bicarbonate aqueous solution and water in order.
Next wash the organic phase using (with sodium sulfate)
Dry, filter and evaporate. Remove residue from silica gel
Chromatograph lamb (120 g). (0.5
% triethylamine in a volume ratio of 2:1)
Elution with a xane:ethyl acetate mixture yields the compound
Compound 37 is pure syrup (1.542g, 70 of compound 34 + 35
%). [α)_D=-23゜(c.:1, chloroform), N, M.
R. (CDCl₃): δ: 5.48 (d, 1H, H-1, J_1.2:
2.5Hz). Monochloroacetylation of δ orthoester 37 Anhydrous solution of orthoester 37 (220mg, 0.5mM)
Stir the tanol (10ml) solution in a dry argon atmosphere.
Cool to −20° C. under stirring. Anhydrous potassium carbonate (40
mg) and stirred the reaction mixture for 5 hours under the above conditions.
Stir. Drain the solid, evaporate the liquid, and remove the residue.
Place in chloroform (50ml). Did you speed up the organic phase?
Wash with ice water (3 times) and dry (sodium sulfate).
Dry, filter and evaporate. Immediately wash the residue with anhydrous
gin (4 ml) and anhydrous dichloromethane (2 ml).
Dissolve. Cool to −20 °C under dry argon atmosphere.
After cooling, chloride (0.1 ml freshly distilled, 1.24 mM)
Dissolve chloroacetyl in anhydrous dichloromethane (1 ml)
Drop the liquid. The reaction mixture was incubated for 30 min under the above conditions.
Stir and then pour into water-ice mixture (100 ml). 15
After stirring for a minute, the mixture was dissolved in chloroform (3 x 20 ml).
Extract with Eishui, 2% sodium hydrogen carbonate solution
Wash the organic phase sequentially with solution and water (sulfuric acid solution).
(Thorium), filtered and evaporated. residue
Immediately transfer the chromatograph to a silica gel column (12 g).
Spread roughly. (Contains 0.2% triethylamine
in a hexane:ethyl acetate mixture (5:2 by volume)
Upon elution, the following substances are obtained in sequence. Unsaturated compound 39 (15 mg, 8%), Orthoester 38 Syrup (145mg, Compound 12
61%), [α]_D=+19゜(c.:1, chloroform)
，
N., M.R. (CDCl₃): δ: 5.45 (d, 1H, H-1,
J_1.2:2.5Hz), 5.24(d.d., 1H, H-4, J_3.4:2.5
Hz, J_4.5:1.5Hz), 4.00(s, 2H, Cl-CH₂−COO
-). Example 6 Disaccharide represented by the following formula 41, i.e. benzyl-6-0-
Acetyl-3-0-benzyl-2-benzyl
oxycarbonylamino-2-deoxy-4-0
-(2-0-acetyl-3-0-benzyl-α
-L-methylidopyranuronyl)-α-D-g
Synthesis of lucopyranoside by condensation of monosaccharides 38 and 22 according to step α).
40 is produced, and then in step β
Removal of the nochloroacetyl group yielded the desired disaccharide 41
(See Figure 8). Step α: disaccharide 40, i.e. benzyl-6-0-a
Cetyl-3-0-benzyl-2-benzyloxi
Cycarbonylamino-2-deoxy-4-0-
(2-0-acetyl-3-0-benzyl-4-
0-Chloroacetyl-α-L-methidopyranu
Production of α-D-glucopyranoside Orthoester 38 (284mg, 0.6mM) and alco
Anhydrous chlorobenze with alcohol 22 (214mg, 0.4mM)
(12 ml) solution in the presence of a slight stream of dry argon.
Heat to 140°C under stirring. 10ml of solvent
After slow distillation, (newly produced
0.006mM) 2,6-dimethylpyridinium peroxide
A solution of chlorate in chlorobenzene (4 ml) for 30 minutes.
Drip over a period of time. Solvent (4 ml) at the same time as the dropwise addition
Distill. Then a constant reaction volume equal to 4 ml is
Addition of fresh solvent (10 ml) and evaporation to maintain
Stir the reaction mixture for 1 hour while simultaneously distilling.
do. After cooling and diluting with chloroform, the organic phase is saturated.
Wash sequentially with sodium bicarbonate solution and water,
Dry (over sodium sulfate), filter and evaporate.
Chromatograph the residue on a silica gel column (40g).
Put it on the fu. Hexane:acetic acid (4:3 by volume)
When eluted with an ethyl mixture, the following substances appear in sequence:
can get. - product 22 (120 mg, 56%), - Disaccharide 40, crystallized in ether-hexane mixture
(112 mg, 30%), MP: 144-145℃, [α] 20 D=
+35° (c:1, chloroform), NMR
(CDCl₃): matches the desired structure. Step β: Removal of monochloroacetyl group Disaccharide 40 (56mg, 0.06mM) and thiourea (7
Pyridine (2.5 ml) containing
Stir the solution with tanol (0.5ml) at 100℃ for 30 minutes.
Stir. After cooling, the reaction mixture was evaporated to dryness and the residue
water-chloroform mixture (40 ml, volume ratio 1:
Put it in 1). The organic phase was washed with water and diluted with (sodium sulfate)
dry), filter and evaporate. Silica residue
Chromatograph on a gel column (2 g).
with ethyl acetate-hexane mixture (2:1 by volume)
Upon elution, disaccharide 41 is obtained. Crystal in the ether
(46mg, 30%), M.P.: 146-147℃, [α]_D=
44° (c:1, chloroform), NMR (CDCl₃): place
matches the desired structure Example 7 formula Synthesis of tetrasaccharide 43 shown as Tetrasaccharide 43 is produced in the following steps (see Figure 9).
(see). a Disaccharides 20 and 41 synthesized in Examples 2 and 6
condensation, b Selective -0 at position 4 of the formed tetrasaccharide 42
- Demonochloroacetylation. a Condensation reaction Newly prepared bromide 20 (64 mg, 80 μM), compound
Material 41 (51mg, 60μm) and molecular sieve 4Å
(powder, 80 mg) containing anhydrous dichloroethane (1.5
ml) solution for 30 min at room temperature under a dry argon atmosphere.
Stir for a while. The reaction mixture was cooled to −20 °C and the sym
- Collidine (20ml, 150μM) and silver trifler
Add (31 mg, 120 μM) sequentially. reaction mixture
The mixture was stirred at -20℃ for 1 hour and then warmed to room temperature in 15 hours.
let Dilute the solid with dichloromethane (50ml)
The dehydrated solution was diluted with cold 1M HCl solution and water (twice).
Washed, dried (with sodium sulfate), filtered and evaporated
let Transfer the residue to a silica gel column (8 g, 230-400 ml)
chromatograph (gel). Hexa
Elute with a mixture of ethyl acetate and ethyl acetate (volume ratio 4:3).
When washed, 42 tetrasaccharides were recovered in the form of colorless glass (39%).
It is possible. [α]20 D = +56° (c = 0.6; CHCl₃),
The NMR spectrum is consistent with the desired structure. The column was filled with an ethyl acetate-hexane mixture (by volume).
2:1), the starting material 41 was recovered.
(23 mg, 44%). b -0-Dechloroacetylation reaction 1.25 ml of pyridine containing 42 tetrasaccharides and absolute ethanol
Add 0.25ml of thiourea (7mg,
100 μM) for 20 min at 100 °C.
After cooling and evaporating to dryness, place the solid residue in 20 ml of water and evaporate.
Extract with loloform (5 x 5 ml). 10% organic phase
% sodium hydrogen sulfate aqueous solution and water,
Dry with sodium sulfate, filter and evaporate. Residue
Chromatograph the residue on a silica gel column (3 g)
Put it on. Ethyl acetate-hexane mixture (volume ratio
When eluted at a ratio of 3:2), derivative 43 was eluted with anhydrous glass.
(27 mg, 80%). [α]20 D = +61° (c
= 0.8, chloroform); NMR spectrum (third
(see figure) corresponds to the desired structure. Example 8 (see Figure 10) formula Synthesis of pentasaccharide 45 shown as A condensation reaction is carried out with tetrasaccharide 43 and monosaccharide 44 to form pentasaccharide
Get 45. H.PAULSEN and W.STENZEL, Chem.
Manufactured in accordance with Ber, 111 (1978) 2334-2347
Bromide 44 (27mg, 54μM), Tetrasaccharide 43 (26mg, 18μM)
and molecular sieve 4Å (powder, 50 mg).
Dry the chloroethane (0.8 ml) solution under argon atmosphere.
After stirring for 30 minutes at room temperature under air, cool to -20°C.
sym-collidine (16 ml, 120 μM) and silver trifle
(26 mg, 100 μM) was added sequentially. reaction mixture
The mixture was stirred for 18 hours and the temperature gradually rose to room temperature.
Warm up. Dilute the solid with dichloromethane (50ml)
The dehydrated solution was diluted with cold 1M HCl solution and water (twice).
Wash sequentially, dry (with sodium sulfate) and filter
Evaporate. Transfer the residue to a silica gel column (8 g, 230-400 ml)
chromatograph (gel). Hexa
Elute with a mixture of ethyl acetate and ethyl acetate (volume ratio 4:3).
When the pentasaccharide 45 is dissolved in the form of colorless glass (30mg, 90%)
It can be recovered as such. [α]20 D = +67° (c = 1;
CHCl₃); the NMR spectrum matches the desired structure.
Ru. In particular, the anomeric proton of the glucosamine unit
(anomeric Protons), 5.36 and 5.52ppm
Dislocations (δ, TMS) belong to H and F.D, respectively.
It turned out to be against Roton. Example 9 Production of pentasaccharide 50 (see Figures 10 and 11) This manufacturing takes place in the following steps. (a) Removal of acetyl group (pentasaccharide 46), (b) Sulfation of the −OH group thus liberated (pentasaccharide
47), (c) Hydrogenated and benzyl-protected −OH
liberate the group and -N₃-NH₂convert to base
(pentasaccharide 48), (d)NH₂group is sulfated (pentasaccharide 49), then the 6-position
-Saponify COOMe group (pentasaccharide 50) These steps are performed as follows. a Removal of acetyl group of derivative 45 Pentasaccharide 45 (28mg) of 1,2-dimethoxyethane
(2.5 ml) and methanol (0.8 ml) solutions were stirred.
Meanwhile, cool to 0°C. 1ml of 1M soda solution for 10 minutes
drip between. Stir the reaction mixture at 0°C for 1 hour.
and then stirred at room temperature for 12 hours. cooled to 0℃
After that, add 1M hydrochloric acid (3ml) and make a milky mixture.
Immediately extract with chloroform (5 x 5 ml).
The organic phase was washed with water, dried over sodium sulfate and filtered.
and evaporate. Put the residue in methanol (2 ml).
and treated with an ethereal solution of diazomethane for 30 minutes.
(yellow color persists due to the presence of excess diazomethane)
). After evaporation to dryness, the residue was filtered onto a silica gel column (2 g,
Chromatographed at 230-400 gels)
Ru. Dichloromethane-methanol mixture (volume ratio
15:1), the pentasaccharide 46 is a colorless gas.
Collected in the form of laths (18 mg, 72%). [α]20 D=+
57° (c=1; chloroform). The desired structure is confirmed in the NMR spectrum. b Sulfation of -OH group Compound 46 (22 mg) in diethylformamide (0.5
ml) solution, triethylamine/SO₃Complex (22
mg, 2.5 eq/OH). reaction mixture 50
Heat to ℃ for approximately 14 hours. Then triethylamine
N/SO₃Add the complex (10 mg) and carry out the reaction for 24 hours.
do. Add methanol (0.5 ml) and water to the reaction mixture.
Add loloform (0.5ml). CHCl₃/
CH₃Cefa equilibrated with OH (1:1 volume ratio)
Decks LH₂₀Introduce the solution at the top of the column.
Collect fractions containing sulfated products and evaporate the solvent.
let In this way a glassy substance is obtained
(30mg). Transfer this material to chroma on silica gel (10 g).
Put it on a tograph. As eluent, ethyl acetate/
Pyridine/acetic acid/water (volume ratio 6:2:0.6:1)
3 parts of a mixture of ethyl acetate/pyridine/acetic acid/water
(volume ratio 5:5:1:3)
Use a suitable solvent. Collect and concentrate fractions containing the desired product.
Ru. After evaporating the solvent, the resulting residue was dissolved in methane containing water.
Dissolve in methanol/water mixture (by volume)
Dowex 50W x 4 balanced with ratio 50:50),
Na⁺Pass through the column. Thus, the sodium of compound 47
Um salt is obtained. e Hydrogenation The product obtained above was added to a solution containing water (0.3 ml).
Dissolve in ethanol (3.7ml). A catalyst (Pd/C, 5%, 40 mg) was added to this solution.
and stirred under hydrogen atmosphere for 5 days. catalyst
After removal by filtration, the resulting solution was subjected to U.V. spectroscopy.
When analyzed, an absorption peak derived from the benzyl group
It is recognized that the has disappeared. Further evaporate the solvent.
Upon evaporation, a residue, compound 48, remains. d-NH₂After sulfation of the carboxyl group
transformation Compound 48 is dissolved in water (4 ml). pH 9.5
and add trimethylamine/SO to the solution.₃complex
(54 mg). Anti-oxidant by adding 0.1N soda
Maintain pH at 9.5 during treatment. After overnight, add more sulfating agent (27 mg).
Ru. The final addition is made 24 hours later. After 48 hours, soda (3M, 34ml) was formed
Add to compound. Stir the solution for 3 hours at room temperature
and the methyl ester of the uronic acid type unit is hydrolyzed
be done. The reaction mixture was then neutralized and approximately 2 ml of
Concentrate to soluble volume. The solution obtained in this way is
Introduce it to the top of the Adetskus G25 column (100ml),
Elute with water. The collected fractions were analyzed by UV absorption.
(206nm) and polarometry (265nm)
Do more. Collect the optically active fractions and add the solvent
Remove the residue, put it in about 2 ml of water, and freeze-dry it.
Ru. In this way, the derivative 50 is in the form of a white powder.
(5.6 mg, 25% of product 45). The desired structure is confirmed in the NMR spectrum.
In particular, for the anomeric proton of the glucosamine unit.
and dislocations (δ,
TMS) to protons belonging to H, F, and D, respectively.
It is recognized that it is aimed at Example 10 formula Disaccharide 51 represented by methyl 1-prop-1'-
enyl-2,3-di-O-benzyl-4-0-
[2-azido-3,4-di-0-benzyl-6-
0-acetyl-α-D-glucopyranoside
Synthesis of nate (see Figure 12) Monosaccharide 13 (0.215g, 0.5m mole) in dichloromethane
Monosaccharide 44 (0.49g, 1m mole)
in dichloromethane (3 ml) and molecular
Sequentially add 4 Å of sieve (powder). 0 mixture
After cooling to ℃, add sym-collidine (0.16 ml) and silver
Add reflate (0.3g). After 1 hour, mix
Dilute the mixture with dichloromethane (50ml). solid
was dehydrated and the solution was diluted with 5% sodium bicarbonate solution,
Wash sequentially with water, 10% potassium hydrogen sulfate, and water.
This gives 591 mg of residue after evaporation. Toluet
Syringe using a silica/acetone mixture (30:1 by volume).
Purification on Rica recovers pure disaccharide 51
(211 mg). This product is confirmed by elemental analysis. Example 11 formula Disaccharide 54 represented by methyl (1-trichloro
Acetimidyl-2,3-di-0-benzyl-4-
0-[2-acetylamide-2-deoxy-3,
4-di-0-benzyl-6-0-acetyl-α-
Synthesis of D-glucopyranoside] uronate (1st
(See Figure 2) Disaccharide 51 (180 mg) in acetone/water mixture (by volume)
5:1.6 ml) solution, mercury oxide (232 mg) and
Solution of mercuric chloride in acetone/water mixture (292mg/2
ml) are added sequentially. After filtration and evaporation, take up in chloroform and 10% iodide.
After sequential washing with potassium solution and water, disaccharide 52 was obtained.
(140 mg). Dissolve disaccharide 52 (100 mg) in 0.6 ml of methanol.
Ru. Add ammonium formate (160 mg) and
and 10% Pd/C catalyst (100 mg). 5 minutes
After that, remove the catalyst and add acetic anhydride (10 drops).
Ru. After evaporation, the product obtained was evaporated into toluene/acetic acid.
on silica using a mixture (volume ratio 4:1)
refine. Disaccharide 53 (61 mg) is thus obtained. Disaccharide 53 on silica plate using two types of solvents
Confirmed by Rf of (Merck, reference 5719)
Ta. Solvent: chloroform/ethyl acetate (volume ratio)
3:2) Rf=0.40 and toluene/acetone (volume ratio
4:1) Rf=0.20. Derivative 53 (60mg) in dichloromethane (1.5ml)
dissolve. Then trichloroacetonitrile
(75 μl) and sodium hydride (1.5 mg).
Ru. After 15 minutes, derivative 53 disappears and derivative 54 predominates
becomes. After filtration and evaporation 54 is obtained (67 mg).
Chloroform/ethyl acetate (volume ratio 2:1; Rf
= 0.59) using a silica plate (Merck,
Derivative 54 was confirmed by Rf of reference 5719).
(Rf of compound 53 = 0.37). Example 12 formula Derivative 57 represented by methyl 1,6-amphi
Dro-2,3-epoxy-4-0-[2,3-di
-0-benzyl-4-0-acetyluronate]
-Synthesis of α-D-glucopyranoside. This synthesis is carried out from derivatives 55 and 56 (13th
(see figure a). a Methyl (bromo-2,3-di-0-benzyl
-4-0-acetyl-D-glucopyranoside)
Preparation of uronate compound 55 (see Figure 13a)
(see) Synthesis of compound 1d Compound 1a (32g, 85.5m mole) of pyridine
(250ml) solution with trityl chloride (28.6g, 1.2eq)
and heat to 80℃. Furthermore, trityl chloride
(4.6 g, 0.2 eq) was added after 3 hours of reaction. 1b is
After complete formation (t.l.c. silica; methanol/
Chloroform, volume ratio 1:20), cool the solution to 0 °C.
Cool and add benzoyl chloride (15ml, 1.5eq).
Ru. After overnight, 1c is quantitatively formed. methanol
(1.5 ml) was added dropwise to the reaction mixture, then concentrated to dryness.
let The resulting residue was diluted with para-toluenesulfonic acid.
(95 g) in methanol (500 ml). reaction
After 2 hours, the reaction mixture was diluted with ice water (/2).
Transfer to liquid funnel. Extract product 1d with chloroform.
After exiting, use it as is for the following steps. A portion of this product was purified and analyzed by IR spectroscopy.
Confirm the structure by analysis. The product is colorless and gum-like.
Ru. [α]20 D = -61° (chloroform). Synthesis of compound 1k The product (95 g) obtained in the previous step was
(1) and cooled this solution to 0 °C.
After that, dissolve chromium oxide (52g) in 3.5M sulfuric acid (220ml).
Drop the liquid. After 2 hours of reaction, the reaction mixture was poured into ice water.
Pour into (1). Product 1e was dissolved in chloroform (5x
200ml). Change the chloroform phase to neutral pH.
Wash, dry, and concentrate to dryness. The resulting residue was dissolved in methanol (650 ml).
Add soda solution (20g/50ml) dropwise and mix.
Heat things to 50℃. After overnight, divide the resulting solution into
Concentrate fractionally and pour into water (1.5). Aqueous phase
After washing with hydrochloric acid and acidifying with hydrochloric acid, the product 1f was
Extract with tell. ether phase with sodium sulfate
When dried and concentrated to dryness, a yellow mass containing 1e
(50g) is obtained. This residue (50g) was mixed with acetic acid/trifluoroacetic acid.
(volume ratio 15:1, 615 ml). this
Stir the solution at 100°C and add water (160ml). After overnight, evaporate to dryness to remove trace amounts of acetic acid.
and evaporate the toluene. Unhydrolyzed 1f and
Partially formed residue from 1g in ether (400ml)
dissolve in This solution was diluted at 0°C until complete methylation.
Add ethereal solution of azomethane (t.l.c. silicon
ether/hexane, volume ratio 2:1). Next
Excess diazomethane was decomposed with acetic acid and the reaction mixture was
The mixture is concentrated to dryness. The residue is purified with a silica gel column (200 g).
The column was first filled with pure chloroform and then with chloroform.
Flow the roform/ether mixture (3:1 by volume).
vinegar. Thus 1k is obtained (8.6g, 22.2m
mole, (26% of 1a). Crystallize derivative 1k. m.p.122−123℃, original
Its structure was confirmed by elementary analysis and NMR spectrum.
be done. Synthesis of compound 1 1k (3.9g, 10m mole) dissolved in pyridine (50ml)
Add acetic anhydride (4 ml, 42 mmole) to the solution.
Ru. After 2 hours, the reaction mixture is evaporated to dryness. child
1 is obtained (4.62g, 98%). Synthesis of compound 55 1 (1.4 g) of dichloromethane (30 ml) and
titanium tetrabromide in ethyl acetate (3 ml) solution.
(1.5g). Stir the solution overnight at room temperature.
Ru. After dilution with dichloromethane, the reaction mixture was poured into ice water.
Pour into. The organic phase was dissolved in 5% aqueous sodium bicarbonate solution.
Wash, dry and concentrate. Silica the residue (50
g, ether/hexane, 1:1 volume ratio)
Apply to matograph. Compound 55 was thus obtained in the form of a colorless syrup.
(920 mg, 62%). [α]20 D = +97.5 (c = 1,
loloform). Elemental analysis and NMR spectra
The structure is confirmed. b Derivative 56 (432 mg, 3 mmole) of dichloromethane
Transfer the solution (10ml) to a 4Å molecular sieve.
(0.5g), drierite (1g) and
In the presence of freshly prepared silver carbonate (0.42 g), 0
Stir at °C. After cooling to 0°C, compound 55 (490
mg, 1 mmole) in dichloromethane (6 ml)
Drop the liquid. Continue the reaction for 2 hours. then anti
Filter the reaction mixture. Evaporate to dryness and silicate the residue.
When applied to a Kagel chromatograph (solvent: vinegar
ethyl acid/chloroform, volume ratio 1:6),
Conductor 57 is obtained (285 mg; 51%) The structure of derivative 57 was determined by elemental analysis and NMR spectroscopy.
Confirmed by Tor. [α]20 D = 39° (chlorophore
lum); MP=156-159℃. Example 13 formula Synthesis of derivative 59 shown in Trisaccharide 59 was manipulated as follows (example 12).
Obtained by removing the acetyl group at position 4 of compound 57)
Produced by reacting disaccharide 58 and monosaccharide 44 (No. 13)
(See figure a). Deacetylation reaction of compound 57: In a methanol (25 ml) solution of disaccharide 57 (260 mg),
Add 1N soda solution (25 ml) at 0°C. 1 o'clock
After a period of time, the mixture was acidified by the addition of 1N hydrochloric acid (30ml).
sexualize. Extract the product with chloroform. steaming
After evaporation, the residue was crystallized in an ethyl acetate/hexane mixture.
let Derivative 58 is obtained (167 mg, 70%). [α]20 D=-31° (chloroform), MP=169-
170℃. The structure of derivative 58 was further confirmed by NMR spectra.
be done. Condensation of disaccharide 58 and monosaccharide 44: Dichlorometh of compounds 44 (300mg) and 58 (155mg)
4Å sieves (powder, 500mg),
Add collidine (100 μl) and silver triflate sequentially.
Add. After 15 minutes, dissolve the solution in dichloromethane (50ml)
diluted with water, filtered, and dissolved in 10% potassium hydrogen sulfate.
Wash with liquid and water sequentially. After drying and concentration, sift the residue.
Apply silica gel column chromatography to the acetic acid solution.
Pass through a chill/chloroform mixture (1:10 by volume).
vinegar. Derivative 59 was thus obtained in the form of a white foam.
Ru. Elemental analysis and NMR spectrum of the structure of derivative 59
, optical rotation ([α]20 D=+25°; chloroform)
confirm. Example 13A formula Synthesis of the trisaccharide shown by The disaccharide 20 was synthesized under the same conditions as described above for the synthesis of the tetrasaccharide EFGH.
Reacted with methanol under conditions. Thus β-method
Tyl glycoside is obtained. MCA group conveniently
under the same conditions as the production of pentasaccharide to remove and then disaccharide.
Acts on 44 monosaccharides. The obtained trisaccharide is treated with conventional
Perform a reaction. Confirm the structure with NMR spectrum
Ru. Example 14 formula Synthesis of trisaccharide 62 shown as This trisaccharide 62 a N at position 2 of glucosamine unit₃-NHAc
Conversion to base, b 2,3-epoxy crosslinking of the unit at the reducing end
opening of, c Opening of the anhydro-1,6 bridge of the same unit, It is manufactured by steps (see Figure 14). a-N₃Conversion from -NHAc: Derivative 59 (10 mg) in DMF/ethanol mixture
(1/1, 1 ml) solution with catalyst Pd/CaCO₃(5%, 5
mg). Suspension under 1 atm hydrogen pressure
Stir for 96 hours. After passing through the catalyst and evaporating, the residue is dissolved in methanol.
and add 1 drop of acetic anhydride to acetylate.
Derivative 60 is obtained quantitatively. The structure of derivative 60 is shown by NMR spectrum and elemental content.
analysis, optical rotation ([α]20 D=+35.5°; chloroform)
will be confirmed. MP=147−149℃. b Cleavage of epoxy crosslink: Derivative 60 was saponified in the same manner as the synthesis of derivative 58.
and the acyl group at the 6-position of the non-reducing terminal unit and the middle
Removal of the methyl ester group at the 5-position of the intermediate unit
Ru. After extraction, the residue is dissolved in DMR and sodium
Heat to 120 °C for 48 hours in the presence of azide. evaporation
After that, extracted with chloroform, extracted with 0.1N-HCl and water.
After sequential washing, drying and evaporation of the solvent,
A residue is obtained. Treat this residue with diazomethane
and when acetylated with (pyridine-acetic anhydride),
Compound 61 is obtained. c Cleavage of anhydro-bridge: Compound 61 was prepared at −20°C under normal conditions (acetic anhydride,
Oxidized and decomposed with sulfuric acid). Induction after treatment of reaction mixture
62 bodies are obtained. Example 15 formula Synthesis of derivative 63 (see Figure 14) Derivative 62 obtained in Example 14 was converted into titanium tetrabromide.
of dichloromethane and ethyl acetate solution.
and halide 63 is obtained. Its structure is
Confirmed by NMR spectrum. Elemental analysis value
is in accordance with the theory. Example 16 formula Monosaccharide 68, i.e. methyl 2-acetamide
-3,6-di-0-benzyl-2-deoxy-α
-Synthesis of D-glucopyranoside This synthesis was published in the Journal of Chemical
Society1941, pages 50-51 A.
From the monosaccharide 64 produced by the method of Neuberger,
This is done in the following four steps (see Figure 15). 1 Benzylation of -OH group at position 3, 2 By removing the benzylidene group, - at the 4 and 6 positions
liberates OH group, 3 Tosylation of -OH group at position 6, 4 Substitution of -OTs group at position 6 with benzylate. Step 1: Benzylation reaction Compound 64 (6.5g, 20.10mM) in dimethylform
Barium hydroxide octahydrate in amide (120ml) solution
(3.6g) and barium oxide (166g) were added.
Ta. After 10 minutes, stir at room temperature and add benzyl bromide (4.5 ml).
drip. The reaction is continued overnight. chloroform
After diluting with (100 ml), the reaction mixture was
(Celite). Concentrate the liquid to dryness
let A white residue is thus obtained. This residue
Thin layer chromatography analysis reveals a single substance, i.e.
It is recognized that it contains conductor 65. derivative
65 will be used as is in the following steps. Step 2: Removal of benzylidene group The residue obtained in the previous step was dissolved in methanol (370
ml) and 130 ml of water). This melt
Add paratoluenesulfonic acid monohydrate (3g) to the liquid.
is added and the mixture is refluxed overnight. After cooling,
Evaporate most of the tanol and add water (250ml).
Add. After washing with a small amount of chloroform (100ml),
The aqueous phase is treated as follows. 1. Precipitation of barium salts with sulfuric acid, 2. The concentration of barium sulfate formed, 3 IRA resin 45 (OH^-) to remove excess acid. After removing and concentrating the resin, a pale yellow residue, i.e.
A conductor 66 is obtained (5.7 g). This derivative is
It is used in the production of compound 67. Step 3: Tosylation reaction This derivative 66 was mixed with dichloromethane (150ml) and
Dissolve in a mixture of DMF (10ml). This solution
Tosyl chloride (5.6g, 30mM), dimethylamine
Nopyridine (121 mg) and triethylamine (5
ml) are added sequentially. Keeps out moisture and allows dry nitrogen flow
Perform the reaction below. After 18 hours of reaction, add ice water and stir the mixture for about 14 hours.
Stir for a while. Dilute the reaction mixture with dichloromethane and dichloromethane.
The lomethane phase was mixed with 2M hydrochloric acid and saturated sodium bicarbonate.
Wash in this order, then wash with water until the pH becomes neutral.
Ru. After drying with sodium sulfate, evaporate the solvent.
let The obtained residue was applied to a silica gel column (200
g, ethyl acetate-hexane mixture (volume ratio 4:
1). Collect fractions containing derivative 67. remove solvent
A solid residue is then obtained (4.6 g). This residue
Used as is for synthesis of compound 68. Step 4: Benzylation reaction Derivative 67 obtained in the previous step was dissolved in anhydrous dimethyl
Dissolve in formamide (50ml). In this solution,
Benzyl alcohol of sodium benzylate (30
ml) 1 molar solution. Heat the mixture to 90℃ for 1 hour.
Heat for a while. After cooling to room temperature, the mixture was concentrated to dryness.
Ru. Take in chloroform (400ml) and add chloroform.
Wash the mucus phase with water and saturated sodium chloride and dry.
and concentrate to dryness. Transfer the residue to a silica gel column (200 g, chloroform).
Chromatogram of 1:1 volume/ethyl acetate
Put it on the fu. Derivative 68 is thus obtained (2.3 g). The yield is
It is 27.6% of compound 64. Compound 68 is crystalline. MP=149−150℃,
[α]20 D=87° (c=1, chloroform). infrared spec
Product 68 has the desired structure by vector and elemental analysis.
It is confirmed that the Example 17 Synthesis of disaccharide 73 (see Figure 15) This synthesis is 1 Step to obtain disaccharide 70 by condensing derivatives 68 and 69
Tsupu; 2 Steps to obtain derivative 71 by removing benzyl group
P; 3 Sulfate the -OH group of derivative 71 to create derivative 72
Furthermore, the anionic group is salted and the acetyl group is
step of removing; including. 1 Synthesis of disaccharide 70 This synthesis takes place from monosaccharides 68 and 69. Halides Journal of American
Chemical Society, 77 (1955), p.3312.
Manufactured by the method of G.N. Bollenback. Monosaccharide 68 (450mg, 1.1mM) in dichloromethane
(30ml) solution, add mercury bromide (400mg, 1.1mM).
Added. After diluting with about 10 ml of dichloroethane,
Add 4Å (powder) of recular sieves to the reaction mixture.
Add. Dichlorohalide 69 (1.1g, 2.75mM)
Add more ethane (10ml) solution. dichloroe
After diluting with 10 ml of chlorine, the reaction mixture was heated to 90-100°C.
Reflux at ℃ for about 14 hours. After cooling, the reaction mixture was
Dilute with chloromethane (100ml) and pleat the solid
Remove by passing through paper. 10% iodination of organic phase
Potassium solution (2 x 25ml), 5% sodium bicarbonate
Wash sequentially with um solution (2 x 25 ml) until the pH is neutral.
Rinse with water until dry. Dry with sodium sulfate,
After filtration and concentration, the residue was transferred to a silica gel column (150g).
Purify using. The column contains three types of acetate.
ton-ether mixture (volume ratio 1:5, 1:4,
1:2) in sequence. Disaccharide 70 is thus obtained in pure crystalline form.
(390mg). MP=189−190℃, [α]20 D = +60° (c
= 0.4, chloroform). Infrared spectrum, NMR
The desired structure was confirmed by spectra and elemental analysis.
Ru. 2 Synthesis of disaccharide 71 Derivative 70 (100mg) in methanol (20ml)
A catalyst (Pd/c, 5%, 100 mg) is added to the solution.
The suspension thus obtained was stirred for 3 days under a stream of hydrogen.
do. The catalyst is recovered by filtration. When evaporated, two
A residue consisting of sugar 71 is obtained (73 mg, 97%).
The NMR spectrum confirms the desired structure of the compound.
It can be done. Disaccharide 71 is the basic unit of heparan sulfate.
Note that it is a precursor. Said disaccharide
The following is used to produce derivative 73 from derivative 72:
Sufficiently deprotected to carry out the saponification reaction as described in
protected. 3 Synthesis of disaccharide 73 Compound 71 (70 mg) in dimethylformamide (2
ml) solution, add sulfating agent (trimethylamide-sulfur)
-trioxide complex, 75 mg) is added. One night
After this period, more complex (35 mg) is added. 6 hours
After that, the reaction was stopped, the mixture was evaporated to dryness, and the mixture
Take in loform, neutralize with triethylamine and evaporate.
let Silica gel column (20g, methanol/chloro
chromatographically with 1:2 volume ratio).
pure sulfated derivative 72 in the form of a white powder
be separated. This derivative is a synthesis of the deprotected disaccharide 73.
used directly for configuration. In a methanol (9 ml) solution of derivative 72 (71 mg)
Water (4 ml) was added followed by 1M soda solution (1
ml) attached. After stirring at room temperature for 4 hours, reaction mixture
Amberlite IR120H⁺Pass over the column. like this
The resulting solution was neutralized and the salt was diluted with water.
Remove by passing over a Sehadex G25 column. sulfuric acid
Collect the fractions containing disaccharides. When freeze-dried, derivative 73 is in the form of a white powder.
obtained (46 mg). [α]D 20=34.5° (C=1, water). As a result of the conductivity analysis method for this derivative, monkeys
Phate/carboxyl ratio=2. elemental content
Analysis, C¹³NMR analysis of the desired structure of this product
is confirmed. Example 18 Compounds 75, 76 and D-glucosamine structures
and 77 synthesis (see Figure 16) Compound 75: Methyl-3-0-benzyl-4,6-
0-Benzylidene-2-benzyloxyca
Rubonylamino-2-deoxy-α-D-
Glucopyranoside (ZU YONG KYI, Sci Sinica (Peking), 5
(1956) 461-467, CA52 (1958) 3694.
Compound 74 (415mg, 1mM) of anhydrous N,N
- Dimethylformamide (10 ml) solution was added to an anhydrous bag.
Ride (613 mg), barium hydroxide octahydrate (158
mg) and moisture in the presence of benzyl bromide (0.15 ml)
Shut off and stir at room temperature for 5 hours. Then the reaction mixture
Dilute the mixture with chloroform (50ml) and remove the organic phase.
Wash sequentially with 50% cold acetic acid and water (sulfuric acid solution).
Dry with thorium), filtrate, and evaporate. solid residue
Recrystallize with ethanol (461 mg, 91%);
Mp: 202-203℃; [α]_D= +46° (C:1, chloro
Holm) Compound 76: Methyl-3-O-benzyl-2-ben
Zyloxycarbonylamino-2-deoxy
C-α-D-glucopyranoside A suspension of compound 75 (300 mg) in 60% acetic acid (10 ml)
Stir at 100°C for 30 minutes. The solution is then cooled and
Evaporate to dryness, add water (4 x 10 ml) and evaporate.
Ru. The solid residue was dried under vacuum and dissolved in 2-propanol.
Compound 76 (220 mg, 89%) was obtained by recrystallization in a
Ru. MP: 151-152℃, [α]_D= +94° (C: 1, me
Tanol). Compound 77: Methyl 6-0-benzoyl-3-0-
Benzyl-2-benzyloxycarbocarbonate
Rubonylamino-2-deoxy-α-D-
Glucopyranoside Anhydrous pyridine (5 ml) and dichloromethane (12
Dissolve compound 76 (835mg, 2mM) in a mixture of
The solution is sealed from moisture and treated with benzoyl cyanide.
(400mg, 3mM) and stirred at room temperature for 5 hours.
Ru. Add methanol (5 ml) to excess reagent.
Disintegrate and stir for 30 minutes. Steam the reaction mixture.
Dry, add toluene and evaporate under vacuum.
dry. Mix solid residue with ethyl acetate and hexane
Compound 77 (935 mg, 90%) was recrystallized with
MP: 154-155℃, [α]_D= +74° (C: 1, chloro
holm). Example 19 Compounds 78 and 79 having L-iduronic acid structure
Synthesis (see Figure 16) Compound 78: 4-0-acetyl-3-0-benzyl
-1,2-0-methoxyethylidene-β-
L-methylide pyranuronate Bromide obtained by step β of Example 5
36 (from 0.425 g of mixture of acetate 34 and 35, 1mM)
(freshly prepared) in anhydrous dichloromethane (10 ml)
is stirred at room temperature under a dry argon atmosphere.
sym-collidine (0.66ml, 5mM) and anhydrous meth
Next, add Nord (0.40ml, 10mM) and react.
The mixture is stirred under the above conditions for 20 hours. Dichloro
Saturated sodium bicarbonate after dilution with methane (50 ml)
Wash the organic phase using aqueous solution and water sequentially.
dried (over sodium sulfate), filtered and evaporated.
let Collect the residue on a silica gel column (20g).
Hang it on the matograph. Hexane:ethyl acetate mixture
(volume ratio 3:2, containing 0.5% triethylamine)
Compound 78 in pure syrup form when eluted with
(302 mg, 76% of acetate 34 and 35) is obtained.
[α]_D=-21゜(c.:1, chloroform), NMR
(CDCl₃): δ: 5.52 (d, 1H, H-1, J_1.2:3
Hz). Compound 79: 3-0-benzyl 1,2-0-tert-
Butoxyethylidene-β-L-methylide
Pyranuronate Orthoester obtained in step γ of Example 5
37 (48mg, 1.1mM) in anhydrous methanol (15ml)
Cool the solution to −20 °C under stirring in a dry argon atmosphere.
Ru. Add anhydrous potassium carbonate (60 mg) and
The reaction mixture is stirred under conditions for 5 hours. dehydrate solids
Then, the liquid was evaporated and the residue was again dissolved in chloroform (50%
ml). Wash the organic phase with ice water (3 times)
and dried with (sodium sulfate), filtered and evaporated.
let Immediately transfer the residue to a silica gel column (25g)
chromatograph. Volume ratio 2 containing (0.5%) triethylamine:
1) Elute with hexane:ethyl acetate mixture
The following substances are obtained in sequence. Monounsaturated compound 39 (31 mg, 7%) syrup,
[α]_D=+103゜(c.:1, chloroform), NMR
(CDCl₃): δ: 6.27 (d.d., 1H, H-4, J_3.4:5
Hz, J_2.4: 1Hz), 5.67(d, 1H, H-1, J_1.2:
4Hz). One main fraction (271 mg, 62%). This fraction is hexane
-Crystallizes in ether mixture to form compound 79
(123mg, 28%), MP: 68-69℃ [α]_D=-19°
(c.:1, chloroform), NMR (CDCl₃):δ:
5.41 During chromatography using silica gel column
and during the crystallization process of compound 79, slightly more than compound 79.
New compounds with high Rf are produced. transformation
Silica gel chromatography of crystallized mother liquor of compound 79
By multiplying the pure fraction of this new compound 80
Some can be isolated (41 mg, 11%). syrup,
[α]_D=+21゜(c.:1, chloroform), NMR
(CDCl₃): δ: 5.83 (d, 1H, H-1, J_1.2:4.5
Hz). In the synthesis process of the present invention, the form of compound 80 is
The crude syrup 37 was chromatographed to prevent
Use directly for subsequent reactions without roughening. Example 20 Production of disaccharides 81, 82 and 83 (see Figure 17) Compound 81: Methyl-4-0-benzoyl-3-0
-benzyl-2-benzyloxycarbony
Ruamine-2-deoxy-4-0-(2,
4-di-0-acetyl-3-0-benzyl
-α-L-methylidopyranuronyl)-α
-D-glucopyranoside Orthoester 78 obtained in Example 19 (80 mg,
0.2mM) and alcohol 77 (52
mg, 0.1mM) in anhydrous chlorobenzene (8 ml)
The solution was stirred in the presence of a slight stream of dry argon.
Heat to 140℃ while stirring. Slowly add 6 ml of solvent.
After distillation (N.K.KOCHETKOV, A.F.
BOCHKOV, T.A.SOKOLOVSKAIA and V.J.
SNIATKOVA, Carbonhdr.Res., 16 (1971) 17
-0.002mM freshly prepared according to 27)2,
Chlorobe of 6-dimethylpyridinium perchlorate
2 ml solution was added dropwise over 15 minutes.
The solvent (2 ml) is distilled off simultaneously with the addition. next previous
Under the conditions described, a constant reaction volume equal to 2 ml is maintained.
Add fresh solvent (10 ml) and distill as
Stir the reaction mixture for 1 hour while simultaneously
Ru. After cooling and diluting with chloroform, the organic phase is saturated.
washed sequentially with sodium bicarbonate solution and water;
Dry (over sodium sulfate), filter and evaporate.
Chromatograph the residue on a silica gel column (15 g).
Put it on the fu. Hexane:acetic acid (4:3 by volume)
When eluted with an ethyl mixture, the following substances appear in sequence:
can get. Starting material 77 (20 mg, 38%), Homogeneous fraction (54 mg) by thin layer chromatography.
According to the NMR spectrum, this fraction contains several
with 0-methylgrunal (δ: 3.35-3.50)
These signals are associated with orthoester 78.
Derived from methyl glycosides generated by rearrangement
It is. This fraction crystallized in an ethanol-water mixture.
and recrystallized from ethyl acetate-hexane mixture to form
Generates compound 81 (44 mg, 50%). MP: 120−
121℃, [α]_D=+17゜(c.:1, chloroform),
NMR (CDCl)₃): matches the desired structure. Compound 82: Methyl 6-0-benzoyl-3-0-
Benzoyl-2-benzyloxycarboni
Ruamino-2-deoxy-4-0-(2-
0-acetyl-3-0-benzyl-4-0
-chloroacetyl-α-L-methylidopi
Ranulonil) α-D-glucopyranoside Orthoester 38 obtained in Example 5 (120 mg,
0.25mM) and alcohol 77 (66mg, 0.125mM)
of anhydrous chlorobenzene (8 ml) was added with a slightly dry solution.
Heat to 140° C. with stirring under a stream of dry argon.
After slowly distilling 6 ml of solvent, 2,6-dimethylene
Lupyridinium perchlorate (0.0025mM)
Simultaneously distill the lolobenzene solution and the solvent (2 ml)
Drip over 15 minutes. reaction mixture
After stirring for 1 hour, under the conditions described for the preparation of 81.
Process with. Transfer the residue to a silica gel column (15g)
Apply to chromatograph. Hexane: ethyl acetate
When eluted with a mixture of (7:4 elution ratio), the following
Substances are obtained sequentially. 1 Product 77 (40 mg, 60%) − Disaccharide 82, crystallized in ether-hexane mixture
(26mg, 30), MP: 143-144℃, [α]_D=
+(c.:1, chloroform), NMR (CDCl₃):
Match desired structure. Compound 83: 0-dechloroacetylation of disaccharide 82 and
Acetylation Disaccharide 82 (12mg) and thiourea (5mg)
in gin (1.2 ml) and absolute ethanol (0.3 ml).
The mixed mixture is stirred at 100° C. for 30 minutes. cold
After cooling, the reaction mixture was evaporated to dryness and the residue was purified by water-chromatography.
Take up again the loform mixture (1:1 by volume, 20 ml).
Ru. Wash the organic phase with water and dry (over sodium sulfate)
Strain, evaporate. Dab the residue with silica gel
(1 g). Ethyl acetate: hexane
When eluted with a mixture of (volume ratio 1:1), pure silica
Disaccharide 83 (3 mg) in the form of tup was obtained, and this disaccharide
was immediately acetylated without analysis (volume ratio 2:1).
of pyridine: acetic anhydride, 1.5 ml). Ambient temperature after 15 hours
Evaporate the reaction mixture to dryness at
Used in column (0.5g). Ethyl acetate: hexa
Disaccharide 81 when eluted with a mixture of
(7 mg) is obtained. ether-hexane mixture
MP: 120-120.5℃, mixture containing 81 crystallizes in
The MP of the compound is 120-121°C. Example 21 Synthesis of trisaccharide 85 (see Figure 18) Bromide 84 (H.PAULSEN & W.STENZEL.
Chem. Ber. , 111 (1978), 2334-2347, 110 mg, 0.25 mM) and alcohol 41 (Example 6).
A solution of 113 mg, 0.13 mM) in anhydrous dichloromethane (2.5 ml) is stirred for 30 min in the presence of molecular sieves 4 Å (100 mg powder), protected from light and under a dry argon atmosphere. After cooling to 20°C, sym-collidine (70μl, 0.55mM) and silver triflate (78mg, 0.30mM) were added sequentially.
Stirring is continued under the above conditions for 2 hours. The reaction mixture was then diluted with dichloromethane (50ml), the solid was dried and the liquid was diluted with 0.1M ice-cold hydrochloric acid solution, water,
Wash successively with saturated aqueous sodium bicarbonate solution, water, dry (sodium sulfate), filter and evaporate. The residue is chromatographed on a silica gel column (18 g). Elution with a mixture of hexane:ethyl acetate (4:3 by volume) gives trisaccharide 85 (139 mg, 88%) in the form of a colorless glass that cannot be crystallized [α] _D = +83° ( c : 1 , chloroform); NMR
Spectrum (90MHz, CDCl ₃ ): δ: 7.25 (m,
25H, 5Ph.); 5.44 (dd, 1H, H ₃ ″, J ₂ ″ ₃ ″: 10.5Hz, J ₃ ″, ₄ ″:
9
Hz); 5.26 (d, 1H, H ₁ ″, J ₁ ″, ₂ ″ ₃ ″: 3.5Hz); 3
.59
(s, 3H, COO Me ); 3.06 (dd, 1H, H ₂ ″, J ₁ ″,
₂ ″: 3.5Hz, J ₂ ″ ₃ ″: 10.5Hz); 2.12, 2.08, 2.01 and
1.97 (4s, 12H, 4CA c ). Example 22: Synthesis of trisaccharide 89 (see Figure 18) This synthesis involves the following four steps. a Removal of acetyl group, b Sulfation, c Hydrogenation, d Sulfation of amino functional groupa Production of trisaccharide 89 by removal of methyl group Trisaccharide 85 (122 mg) was dissolved in 1,2-dimethoxyethane (6
ml) and methanol (2 ml) is stirred at 0°C. A 1M aqueous solution of soda (2 ml) is added dropwise over 10 minutes and the reaction mixture is stirred at 0° C. for 6 hours. Next, add 1M hydrochloric acid,
Add dropwise until pH=0 (white precipitate appears). The mixture is poured into ice water (100 ml) and extracted with chloroform (5 x 10 ml). The organic phase was washed with ice water, dried (over sodium sulfate), filtered,
Evaporate. The syrupy residue was dissolved in methanol (2
ml) and treated with an ethereal solution of diazomethane until a persistent yellow color appears. 30 minutes later,
The reaction mixture is evaporated to dryness. The residue is chromatographed on a silica gel column (10 g). Trisaccharide in the form of a colorless foam that cannot be crystallized when eluted with a mixture of ethyl acetate: hexane (2:1 by volume)86
(85mg, 81%) is obtained. [α] _D = +77° (c: 1, chloroform), NMR spectrum (90 MHz, CDCl ₃ ): No C Ac signal (towards δ = 2 ppm). Elemental analysis: consistent with desired structure. b Production of trisaccharide 87 by sulfation Trimethylamine/sulfur trioxide complex (TMA/SO ₃ ;
60 mg; 2.5 equivalents per OH). Leave at 50°C overnight to complete the reaction. Methanol (0.5
ml), the solution is introduced into a Sephadex LH-20 column (1.5 x 25 cm) equilibrated in a chloroform/methanol mixture (1:1 by volume). Elution with the same mixture can separate the reaction product from the excess of reagent and reaction solvent. The residue obtained is chromatographed on a silica gel column (10 g) and eluted with a mixture of ethyl acetate/pyridine/acetic acid/water (98:56:13:32 by volume). After dissolving the obtained pure product in methanol,
Pass through Dowex 50W×4, Na ⁺ (5 ml) resin column. After evaporation and drying, derivative 87 (58 mg, 100
%) is obtained. This derivative has TLC (capacity ratio 5:
Homogeneity is confirmed with 5:1:3 ethyl acetate/pyridine/acetic acid/water and 7:3:0.1 ethyl acetate/methanol/acetic acid by volume. [α]
20 D = +55° (methanol). NMR spectrum matches the desired structure. c Production of trisaccharide 88 by hydrogenation A solution of compound 87 (20 mg) dissolved in a mixture of methanol (2 ml) and water (0.5 ml) was diluted with 5%
Stir in the presence of Pd/c (20 mg) at 0.2 bar hydrogen pressure for 96 hours. The catalyst is then removed by filtration. Ultraviolet analysis confirms the absence of aromatic rings. After evaporation, the product is used for the synthesis of trisaccharide 89. d Production of trisaccharide 89 by sulfation The derivative 88 obtained in the previous step was dissolved in water (2 ml).
dissolve in Adjust the pH of the solution to 9.5 and maintain this value using the PH-stat.
Add TMA/SO ₃ complex (14 mg; 5 eq. NH ₂ ).
After overnight, the same amount of complex is added. 48 hours later,
Adjust the pH to 12 using 2M soda and maintain this value for 2 hours. After neutralization with hydrochloric acid, the reaction mixture is chromatographed on a Sephadex G-25 column and eluted with water. Compound 89 is detected by a color reaction with carbazole, which is characteristic of uronic acids (Bitter & Muir, Anal.Biochem., 4).
(1962), 330-334). The fractions containing 89 are pooled together and passed through a Dowex resin column 50W x 4, Na ⁺ and eluted with water. After freeze-drying, 89 (4.5
mg) is obtained. According to colorimetric analysis of the carbohydrate components, there are 2.55 moles of uronic acid per 5.15 moles of glucose amine (ratio 1/2). The NMR spectrum of the product confirms the structure (chains, anomeric bonds, sulphate substitution). Example 23 Formula: Synthesis of disaccharide 92 (see Figure 19) Product dissolved in dichloromethane (50 ml)
91 (1 g) is stirred in the presence of dorierite (6 g) and freshly prepared silver carbonate (4.5 g) under an argon atmosphere for 1 hour. Halide 90 (2.8 g) dissolved in dichloromethane (10 ml) is then added and after 1.5 hours 2.8 g of halide 90 are added again. After overnight the solid is removed by filtration and the residue obtained after solvent evaporation is purified on a silica gel column using ethyl acetate/chloroform (volume ratio 1/30) as solvent. In this way, product 92 (866 mg, 42% yield)
get. The product crystallizes out in a hexane/ethyl acetate mixture. MP: 104-106℃; [α]20D
°C = 0 ° (c = 1; chloroform). Elemental analysis and NMR spectra are consistent with the desired structure. Example 24 Formula: Synthesis of derivative 94 (see Figures 19 and 20) Derivative 92 (1.5 g) is dissolved in a mixture of chloroform and methanol (volume ratio 1/1). Then add 2 ml of sodium methanolate (in methanol)
2M). After 20 minutes, evacuate the solution.
50 resin, thereby obtaining derivative 93, which is not isolated. After filtration and evaporation, the fraction of carboxylic acid that can be liberated can be re-esterified by conventional methylation with diazomethane in ether. After evaporation the residue is treated with a mixture of pyridine (20 ml) and acetic anhydride (2 ml) overnight. After evaporation, the residue is crystallized in ethyl acetate/hexane to give product 94 (1.125 g; yield 81.6%). MP: 103 ~ 105℃; [α] 20 D = +5.2° (c =
1; chloroform). Elemental analysis and NMR spectra are consistent with the desired structure. Also, derivative 94 is a derivative instead of derivative 91.
95 using the same procedure as described above. Example 25 Synthesis of derivative 97 (see Figure 20) The anhydride bridge is cleaved in the first step and the bromination reaction is carried out in the next step. a Cleavage of 1,6-anhydrobridge Compound 94 (1 g) was dissolved in acetic anhydride (10 ml),
Cool to −20 °C under argon. Add concentrated sulfuric acid (100 μl) to the cold solution. After 30 minutes the reaction mixture is diluted with chloroform (150 ml) and then poured into aqueous sodium bicarbonate solution (26.5 g in 400 ml). After gas evolution, the chloroform phase is washed twice with a saturated solution of NaCl, dried and concentrated. Chromatography on silica gel (50 g) using a mixture of ethyl acetate and chloroform (1/20 by volume) gives compound 96 (995 mg; yield 86.7%). This compound is a white foam. Spectral and elemental analysis confirm the production of the desired structure. b Bromination A solution of derivative 96 (0.2 g) in dichloromethane/ethyl acetate (volume ratio 9/1, 4 ml) is added to titanium tetrabromide (213 mg). After stirring overnight and subsequent dilution with dichloromethane, pour into a mixture of water and ice (50 ml) and wash twice with 50 ml of ice water. The slop obtained after drying and evaporation is chromatographed on silica gel in ethyl acetate/chloroform (1/20 by volume). Derivatives 97 are thus obtained in yields of 25-50%. NMR spectrum: (ppm, CDCl ₃ ): 2.04;
2.11: 3 broton 2-OAc singlet;
3.7:1, proton COO Me siglets; 6.33:
Doublet of proton H ₁ ; J _1.2 = 3.5Hz. Example 26 Synthesis of tetrasaccharide 98 (see Figure 20) A solution of bromide 97 (50 mg, 60 µM) and alcohol 41 (43 mg, 50 µm) prepared according to Example 6 in anhydrous dichloromethane (1 ml) was irradiated with light. Molecular sieve 4Å in a closed and dry argon atmosphere.
(powder, 100 mg) and stir for 15 min. −10
After cooling to <0>C, sym-collidine (11 [mu]l, 80 [mu]M) and silver trifluoromethanesulfonate (Ag triflate, 18 mg, 70 [mu]M) are successively added and stirring is continued under the above conditions for 3 hours. The reaction mixture was then diluted with dichloromethane (30 ml), the solid was dried, and the liquid was washed successively with 0.1 M ice-cold hydrochloric acid solution, water, saturated aqueous sodium bicarbonate solution, and water.
Dry (over sodium sulfate), filter and evaporate. The residue is chromatographed on a silica gel column (7 g). Elution with a mixture of hexane-ethyl acetate (4:3 by volume) gives the tetrasaccharide 98 in the form of a colorless glass (56 mg, 70% yield), which cannot be crystallized. Characteristics of NMR spectrum (270MHz, CDCl ₃ ):
S: 7.25 (m, 35H, 7Ph); 5.35 (dd, 1H,
H ₃ ″J ₂ ″, ₃ ″: Hz, J ₃ ″, ₄ ″OHz); 5.27 (d., 1H,
H ₁ ″J ₁ ″, ₂ ″: 3.5H); 5.31 (d., 1H, H ₁ J _{1 2} :
7.5Hz); 3.68 (s, 3H, COO Me IDO); 3.59 (s, 3H,
COO Me gluco); 3.37 (dd, 1H, H ₂ J ₁ , ₂ :
7.5Hz, J ₂ , ₃ : 9.5Hz); 3.18 (dd, 1H, H ₂ ″,
J ₁ ″, ₂ ″: 3.5Hz, J ₂ ″, ₃ ″: 11Hz, ) 2.06 and 1.97
(2s,
9 and 3H,4O Ac ). This spectrum is shown in FIG. Example 27 Synthesis of Tetrasaccharide 99 (See Figure 21) A solution of Tetrasaccharide 98 (28 mg) in anhydrous methanol (3 ml) is cooled to -15°C under a dry argon atmosphere.
Anhydrous potassium carbonate (12 mg) is added and the mixture is stirred under the above conditions for 6 hours. The solid is then dehydrated and
The liquid is evaporated and the residue is treated again with chloroform (15ml). The organic phase is washed successively with saturated aqueous sodium chloride solution and water, dried (over sodium sulphate), filtered and evaporated. The residue is chromatographed on a silica gel column (2 g). Elution with ethyl acetate/hexane (3:2 by volume) yields the tetrasaccharide in the form of a colorless glass.
99 (22 mg, 85%) is obtained. NMR spectrum (270MHz, CDCl ₃ ); δ:
7.30 (m, 35H, 7Ph); 5.37 (d, 1H, H″ ₁ , J ₁ ″,
₂ ″: 3.5Hz); 5.29(dd, 1H, H ₃ ″, J ₂ ″, ₃ ″: 10H
z、
J ₃ ″, ₄ ″: 8.5Hz); 5.09 (d, 1H, H ₁ , J ₁ , ₂ : 3.5
Hz); 3.57 (s, 3H, COO Me ido); 3.43 (s,
3H, COO Me gluco); 2.06 (s, 3H, OAc ). This compound 99 has 100 tetrasaccharides (3 of the second unit)
is an intermediate in the synthesis of analogues of this tetrasaccharide that would not be sulfated at the 3-position of the second unit. Example 28 Synthesis of Tetrasaccharide 103 (See Figure 21) This synthesis follows the following steps. a Removal of acetyl group, b Sulfation, c Hydrogenation, d Sulfation of amino group a Production of derivative 100 by removal of acetyl group Tetrasaccharide 98 (40 mg) was mixed with 1,2-dimethoxyethane (3 ml) and methanol (1 ml). ) is cooled to -15. A 1M aqueous solution of soda (1 ml) is added dropwise over 10 minutes and the reaction mixture is stirred at 0° C. for 5 hours. Next, 1M hydrochloric acid is added dropwise until PH=0 and the mixture is poured into ice water (50ml). Extract with chloroform (5 ml
After washing the organic phase with water (with sodium sulfate)
Dry, filter and evaporate. The residue is dissolved in methanol (1 ml) and treated with an ethereal solution of diazomethane until a persistent yellow color appears. After 30 minutes, the reaction mixture is evaporated to dryness.
The residue is chromatographed on a silica gel column (3 g). Ethyl acetate/hexane (volume ratio 2:
When eluted with the mixture of 1), the tetrasaccharide 100 (27 mg, 75
%) is obtained. MP: 126-127°C (ethanol); [α] _D = +55° (c:1, chloroform);
NMR spectrum (90MHz, CDCl ₃ ): Signal C A c
does not exist as a whole (as it approaches δ=2). Elemental analysis: consistent with desired structure. b Production of derivative 101 by sulfation In a solution of derivative 100 (2.4 mg) in DMF (1 ml),
Add TMA/SO ₃ complex (24 mg). The sulfation reaction is completed by standing at 50°C overnight. Methanol (0.5ml) was added to the reaction mixture;
This mixture was then placed in a cephadex equilibrated with chloroform-methanol (1:1 by volume).
Introduce into LH-20 column. Collect fractions containing 101. After evaporation to dryness, the residue was dissolved in ethyl acetate/
Pyridine/acetic acid/water (volume ratio 160:77:19:42)
Chromatograph the mixture on silica gel (10 g). Collect pure fractions. After concentration to dryness, the residue is passed through a Dowex 50W x 4, Na ⁺ column and eluted with water. The product obtained (30
mg) is homogeneous in thin layer chromatography using the above solvent. Its NMR spectrum confirms the structure. [α]20 D=+39° (c=1, methanol). c Production of derivative 102 by hydrogenation A solution of derivative 101 (10 mg) dissolved in a mixture of methanol (1.8 ml) and water (0.2 ml) was dissolved in 5%
Stir under 0.2 bar hydrogen pressure in the presence of Pd/C (10 mg). After 96 hours, the catalyst is removed by filtration. After evaporation, derivative 102 is used directly for the production of derivative 103. d Production of derivative 103 by sulfation Add derivative 102 obtained in the previous step to water (2 ml).
dissolve in Adjust the pH of this solution to 9.5 and maintain this value throughout the sulfation. Add TMA/SO ₃ complex (14 mg). Add again after 24 hours (14mg)
Do this. After 48 hours the pH is set to 12, then after another 2 hours it is set to 7. The reaction mixture was chromatographed on a Sephadex G-25 column (50 ml). The fractions containing derivative 103 (detected by color reaction of uronic acid) were collected and subjected to Dowex 50W×
4. Freeze-dry after passing through a Na ⁺ resin column. In this way, tetrasaccharide 103 (2 mg) is obtained. As a result of colorimetric analysis of the components of derivative 103, uronic acid
There are 1.84 moles of glucose amine in every 2.06 moles. The structure of derivative 103 (chain, anomeric form, position of sulfate group) was determined by NMR spectrum (270MHz,
This spectrum (δ) was confirmed by TMS)
are 4.72; 5.30; 5.50; and
It is 5.67. Example 29 Synthesis of Monosaccharide 115 (See Figure 22) This synthesis is carried out by the following steps 1-7. Step 1: Synthesis of monosaccharide 105 This compound was published in the Canadian Journal of Chemistry.
NL from Chemistry, 51 (1973), page 3357.
It is prepared from compound 104 obtained by the method of Holder and B. Fraser-Reid. Compound 104 (1g,
12.67mM) in dichloromethane (20ml),
Tosyl chloride (0.55 g), dimethylaminopyridine (16 mg) and triethylamine (0.7 ml) are added sequentially. After stirring for about 14 hours under nitrogen flow with exclusion of moisture, the reaction is stopped by adding ice and water. After diluting the reaction mixture with dichloromethane (50 ml), the dichloromethane phase is washed successively with 2M hydrochloric acid and saturated sodium bicarbonate solution, and finally with water until the pH becomes neutral. After drying and evaporation the residue i.e. derivative 105
(1.4g, 97%) is obtained. Derivative 106 is synthesized using this derivative as it is. Step 2: Synthesis of derivative 106 Monosaccharide 105 (31.8g) and sodium iodide (39g)
and is dissolved in acetonitrile (250 ml) and the solution is refluxed for 3 hours. After cooling the reaction mixture, filter the white precipitate formed. The liquid is concentrated, the residue is treated again with chloroform, then the chloroform phase is washed with water to neutral pH, dried over sodium sulfate and concentrated to dryness. Silica gel column (200g,
A syrup is obtained by chromatography with an ether-hexane volume ratio of 1/1. This gives the iodinated derivative (24.7 g, 71.5%). [α] 20 D = 24° (1, chloroform). infrared spectrum,
Compound 106 by NMR spectrum and elemental analysis
The structure of is confirmed. Step 3: Synthesis of derivative 107 To a solution of derivative 106 in anhydrous pyridine (200 ml) is added acetic anhydride (43 ml). The reaction is complete after about 14 hours of stirring. The reaction mixture is concentrated to dryness and the residue is purified on a silica gel column under pressure using ethyl acetate/hexane (volume ratio 1/6) as solvent. Collect multiple pure fractions together. This gives product 107 (16.4 g, 70%). The product is syrupy. [α]20 D=+4.5° (1.3.Chloroform). The structure is confirmed by elemental analysis and infrared spectral analysis. Step 4: Synthesis of derivative 108 Silver fluoride (AgF, 6.9 g) is added to a solution of derivative 107 (4 g) in pyridine (100 ml) cooled to 0°C. After 2 and a half hours, the reaction mixture is poured into a mixture containing chloroform and ether (volume ratio 1/4.1). The resulting suspension is passed through a pleated filter. The liquid was concentrated to dryness and the residue was treated again with chloroform (500 ml). 10 chloroform phase
% potassium sulfate aqueous solution and then water until neutral pH. The residue (2.7 g) obtained after drying over sodium sulfate and concentration to dryness is chromatographed on a silica gel column (200 g) (eluent: ethyl acetate-hexane in a volume ratio of 1:4). Fractions containing product 108 are combined and the solvent is evaporated to yield crystalline material (1.62 g, 54%). MP: 81~82℃, [α]D 25=-20゜(1,
chloroform). Compound 108 was determined by infrared spectrum analysis, elemental analysis and NMR spectrum analysis.
The structure of is confirmed. Step 5: Synthesis of derivative 109 Product 108 (2 g) is dissolved in methanol (20 ml) and chloroform (20 ml). Add sodium methanolate (2M, 2 ml) to this solution.
After 1.5 hours, the deacetylation reaction is complete.
Dilute the reaction mixture with chloroform. The chloroform phase is washed with water until neutral PH, dried and evaporated to dryness. This leaves compound 108 as a residue.
(1.8g, 100%) is obtained. This residue is immediately dissolved in tetrahydrofuran (50 ml) and then a solution of borohydride (BH ₃ , 1M) in tetrahydrofuran (10 ml) is added. After 1 hour of reaction, excess borohydride is decomposed by adding ethanol. After the end of gas generation, add tetrahydrofuran (100ml)
Dilute the reaction mixture by adding . Next, 3M sodium hydroxide (soda, 12 ml) and hydrogen peroxide (8 ml) (120 times the volume) are added sequentially. After heating at 50°C for 2 hours, the reaction is stopped. The solution was poured into chloroform (500 ml), the resulting organic phase was washed with water,
Next, wash with 2M hydrochloric acid and finally with water until the pH is neutral. This results in an extremely cloudy chloroform phase. This phase becomes transparent upon drying with sodium sulfate. After the filtration, chloroform was evaporated and the resulting residue was purified with silica gel (200 g).
Chromatograph (chloroform-methanol, 30:1 by volume). Idose derivative 109 (1.05g,
55%). The product is syrupy.
[α]D 20 = +85.5° (1, chloroform). Elemental and NMR analyzes confirm the desired structure. Step 6: Synthesis of derivative 112 This synthesis is carried out in one step from derivative 109 (without isolating intermediates 110 and 111).
Dimethylaminopyridine (60 mg,
0.24mM), triethylamine (1.7ml, 12mM)
and trityl chloride (2.5g, 9mM) are added sequentially. The reaction ends after about 14 hours. This results in derivative 110 in solution. Now add dimethylaminopyridine (150mg), triethylamine (1.7ml) and benzoyl chloride (1.05ml) to the reaction mixture.
Add. After 6 days, the dichloromethane is removed through a stream of nitrogen and replaced with dimethylformamide (40 ml). The reaction mixture is heated to 70°C and maintained overnight. Now benzoyl chloride (1 ml) and triethylamine (1.7 ml) are added again, heated to 70°C and maintained for 2 days. The dimethylformamide is then evaporated, the residue is treated again with chloroform, and the chloroform phase is washed successively with water, saturated sodium bicarbonate solution, 2M hydrochloric acid solution and finally with water until a neutral pH is reached. After drying, chloroform is evaporated to yield compound 111. Derivative 112 is obtained by immediate reaction to remove the trityl group from this compound. The residue containing derivative 111 was dissolved in 25 ml of chloroform, and para-toluenesulfonic acid hydrate in methanol (1M) was added to the solution.
Add 10ml of solution. The reaction is completed after 4 hours of reaction at room temperature. The reaction mixture is now diluted with chloroform, washed with water, dried and evaporated to dryness. The obtained residue was dissolved in silica gel (200 g, ether-hexane,
Chromatograph at a volume ratio of 3:1). Derivative 112 (1.5g; 52%) in pure state by this process
is obtained. This derivative is syrupy.
[α]D 20 = -8° (1, chloroform). Analysis of the infrared and NMR spectra confirms the structure of the desired product. Step 7: Synthesis of Compound 115 This synthesis is carried out directly from derivative 112 without isolation of intermediates 113 and 114. Compound 112 (1.2g)
After cooling an acetone (20 ml) solution to 0°C, a solution (2.9 ml) of chromium oxide (CrO ₃ ; 1.17 g) in 3.5 M sulfuric acid (5 ml) was added dropwise. After stirring at 0°C for 30 minutes, return to room temperature. The reaction lasts for 3 hours. Next, pour the reaction mixture into a separatory funnel containing ice water (100 ml). The formed product was dissolved in chloroform (3 x 50
ml). The chloroform phase is washed with water until neutral pH, dried over sodium sulfate, filtered and concentrated to dryness. The resulting residue (compound 113) is dissolved in methanol (130 ml). To this solution is added 3M sodium hydroxide (soda) solution (17 ml) and the mixture is kept under stirring for about 14 hours. Acidification with sulfuric acid and extraction of compound 114 with ether followed by immediate methylation with diazomethane provides compound 115 by conventional methods. After evaporation of the ether, the pure compound was chromatographed on silica gel (50 g, ether-hexane; 4/1 volume ratio).
Get 115. Pure fractions containing derivative 115 are combined and the solvent is removed. This process yielded iduronic acid derivative 115 (587 mg, 59% of derivative 112).
is obtained. The product is syrupy.
[α]D 25 = +98° (2.65, chloroform). The desired structure is confirmed by NMR analysis, infrared analysis and elemental analysis. Example 30 Synthesis of disaccharide 117 (see Figures 22 and 23) This synthesis consists of the monosaccharide 115 produced as described above and
The monosaccharide 44 produced by the method of H. Paulsen and W. Stenzel described in Chemische Berichte 111 (1978) 2234-2247. To a solution of Compound 115 (200 mg, 0.5 mM) in dichloromethane (10 ml) are sequentially added Compound 44 (0.450 g), sym-collidine (150 ml), and silver triflate (260 mg). The reaction mixture is kept under stirring at 0° C. for 3 hours, protected from moisture and light and under nitrogen flow. The reaction mixture is then diluted with dichloroethane (100 ml) and filtered through a pleated filter to remove solids. The resulting solution was washed sequentially with saturated sodium bicarbonate solution, water and 2M sulfuric acid, and finally neutralized.
Rinse again with water until pH is reached. The residue obtained after drying over sodium sulfate and evaporation of dichloromethane is chromatographed on silica gel (50 g, chloroform/ethyl acetate; 15/1 by volume). This process produced pure derivative 117 (327mg, 82%)
is obtained. The product is syrupy. [α]
D 20 = +57° (1, chloroform). NMR analysis and elemental analysis confirm the structure and anomeric form of disaccharide 117. Example 31 Synthesis of disaccharide 122 (see Figure 23) The synthesis follows the following steps. a Removal of acetyl groups, b Sulfation, c Hydrogenation, d Sulfation of primary amine groups. a Compound by removal of Me group from -COOH group
Preparation of 118 Dissolve disaccharide 117 (260 mg) in methanol (5 ml) and add 1M soda (1 ml) dropwise. After the reaction is complete, the reaction mixture is introduced to the top of a HT-type Dowex 50 resin column (5 ml). The effluent is concentrated to dryness, taken up again in methanol and the free acid product obtained after completion of the saponification of derivative 117 is methylated by addition of diazomethane. Derivative 18 was thus obtained, followed by a silica gel column (20 g;
Purification using ether/hexane (volume ratio 8/1). Derivative 118 is obtained. The yield of compound 118 is 92
mg. This product is used directly in the synthesis of derivative 119. b Preparation of disaccharide 119 by sulfation The product 118 (92 mg) obtained as above is dissolved in dimethylformamide (5 ml) and the trimethylamine/sulfur trioxide complex (25 mg) is added. Bring the solution to 50°C for approximately 14 hours. After evaporation to dryness,
The residue is taken up again in chloroform and the chloroform phase is then washed with water, dried and concentrated to dryness. The obtained solid is purified with a silica gel column (15 g; eluent: methanol/chloroform in a volume ratio of 1/4). After evaporation of the pure fraction, the sulfated disaccharide 119 (58
mg; 55.6%). c Production of disaccharide 120 by hydrogenation Disaccharide 119 (58 mg) was mixed with methanol-water (15 ml + 2
ml) mixture. Next, the catalyst (5%
Pd/C; 60 mg) is added and the suspension is stirred under hydrogen atmosphere for 48 hours. In this step, the benzyl group generated from derivative 119 completely disappears,
Similarly, the azide group of derivative 119 is reduced to an amino group. The catalyst is removed by filtration and the reaction mixture is then concentrated to dryness. Disaccharide 120 is thus obtained, and this disaccharide is the product
121 and 122 are immediately processed. Preparation of disaccharide 122 by sulfation of the d- _NH2 group Dissolve disaccharide 120 in water (6 ml). Trimethylamine/sulfur trioxide complex (25 mg) is added to this solution while the pH of the solution is maintained at 9.5 by addition of soda (0.1N). After 45 hours of reaction, 1N
Add soda to bring the pH to 12. 1 hour after that
Keep this value. After neutralizing the solution of 121 with 1N hydrochloric acid, apply it to a Na ⁺ type Dowex 50 column (5 ml).
Pass it through. Transfer the eluate from this column to column G1×
2 (16ml, 1.6 x 8cm). The column eluent is a sodium chloride gradient from 0 to 3M.
After collecting and concentrating the fractions containing the sodium salt derivative 122, the product is desalted by passing through a Sephadex G25 column (50 ml) with water as eluent. In this way disaccharide 122 (27
mg, 68%). Upon lyophilization, the product becomes a white powder. [α]D 20 = +95.5° (1.3; water). Carbon-13 NMR analysis confirms the desired structure of product 122. Example 32 Preparation of 2-0-(α-L-idopyranosyl)-D-galactose (compound 128) (see Figure 24) The synthesis is carried out in the following four steps a) to d). a 2,3,4,6-tetra-0-acetyl-α
-L-idopyranosyl bromide (compound 124)
Preparation of 5 g of penta-0-acetyl-α-L-idopyranose (compound 123; P.PERCHEMLIDES, T.
OSAWA, EADAVIDSON & R.W.JEANLOZ,
Manufactured according to Carbohydr.Res., 3 (1967), 463)
in anhydrous dichloromethane (100 ml) at 0°C.
Saturate with hydrogen bromide gas. After 2 hours at room temperature, the reaction medium is poured onto ice and extracted with chloroform. The organic phase is washed with water, dried (over calcium chloride) and evaporated. Crystallization of the residue in a dichloroethane-ether-pentane mixture yields the bromide.
124 (5 g, 95%) is obtained. MP: 126-127
°C, [α] _D = -120° (c; 0.75, chloroform). b Benzyl-3,4,6-tri-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-α-L-idopyranosyl)-β-D-
Production of galactopyranoside (compound 126) Benzyl-3,4,6-tri-O-benzyl-
β-D-galactopyranoside (Tetrahedron, 32
JC, published in Vol. 1963, 1976
Compound 125 prepared by the method of JACQUINET and P. SINAY A solution of 200 mg of anhydrous dichloromethane (10 ml) was added to
mg) and mercuric bromide (80 mg) at 90 °C under dry nitrogen until the volume was reduced by half. Bromide 124 (300mg) in dichloroethane (0ml)
The solution was added over a period of 3 hours, the volume of the reaction mixture being kept constant by continuous distillation of dichloroethane. Three hours after the end of this addition, the reaction medium was cooled to ambient temperature and dissolved in chloroform (100 ml).
The mixture was diluted with water, filtered, washed successively with 10% aqueous potassium iodide, dilute aqueous sodium bicarbonate, and water, dried (sodium sulfate), and evaporated. Ethyl acetate-hexane (1:1, V/V)
The mixture was purified by chromatography of the residue on a silica gel column (30 g) to give syrupy disaccharide 126 (290 mg, 90%;
[α]20D=-57° ( c : 1.1 chloroform)) was obtained. Theoretical value for C ₄₈ H ₅₄ O ₁₅ : C, 66.20; H,
6.25; O, 27.55, actual value: C, 65.98; H, 6.13; O, 27.35% c Benzyl-3,4,6-tri-O-benzyl-2-O-(α-L-idopyranosyl)-β- D
- Preparation of galactopyranoside (compound 127) Disaccharide 126 (200 mg) was dissolved in anhydrous methanol (100 ml), and a 1M solution of sodium methylate in anhydrous methanol (0.2 ml) was added. 1 hour later
Neutralize the reaction medium with Dowex50 (H ⁺ ) resin;
and evaporated. Dichloroethane-acetone (7:3, V/V)
The residue was purified by chromatography on a silica gel column (10 g) using the mixture to yield disaccharide 127 (154 mg, 95%) in pure form ([α]20 D = -43° ( c : 1,4-chloroform)). Theoretical value for C ₄₀ H ₄₆ O ₁₁ : C, 68.36; H,
6.60; O, 25.04; Actual value: C, 68.38; H, 6.64; O, 25.27%. d Preparation of 2-O-(α-L-idopyranosyl)-D-galactose (compound 128) Ethanol (10%) containing acetic acid (0.1 ml) in the presence of 10% palladium on charcoal (50 mg)
ml) for 48 hours.
The product was purified by chromatography on a silica gel column (15 g) using a methanol-chloroform (4:1, V/V) mixture to give the free disaccharide 128 as a hygroscopic white powder (97.5 mg,
100%; [α] 20 D = +28° (C: 1.2, methanol) +
15° (10 minutes) → +93° (2 hours) ( C : 1.2, Wednesday). Theoretical value for C ₁₂ H ₂₂ O ₁₁ : C, 42.10; H,
6.48, actual value: C, 41.71; H, 6.48%. Example 33: Synthesis of 1,2:3,4-di-O-isopropylidene-6-O-(α-L-idopyranosyl)-α-D-galactopyranose (compound 131) Initially 1,2:3 ,4-di-O-isopropylidene-6-O-(2,3,4,6-tetra-D-
Acetyl-α-L-idopyranosyl)-α-D-
Galactopyranose (compound 130) was produced.
J. Am. Chem, Soc., vol. 63, p. 2516, 1941 R.
C. HOCKETT, HGFLETCHER and JB
A solution of 300 mg of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose (compound 129) prepared according to AMES in anhydrous dichloroethane (20 ml) was added to a solution of
mg) and mercuric bromide (300 mg) at 90 °C.
Stir under dry nitrogen until the volume is reduced by half. Bromide 124 (550mg) in dichloroethane (10ml)
The solution was added and after 24 hours a further solution of bromide 124 (125 mg) in dichloroethane (2 ml) was added. 24 hours after the latter addition, the reaction medium was treated as already described for the preparation of disaccharide 126. Purification by chromatography on a silica gel column (50 g) using a dichloroethane-acetone (9:1, V/V) mixture gave disaccharide 130. The disaccharide was recrystallized in a dichloroethane/pentane mixture (650 mg, 95%, MP160-161
°C, [α]20D=-86° ( C : 1, chloroform)).
Theoretical value for C ₂₆ H ₃₈ O ₁₅ : C, 52.88; H,
6.48; O, 40.64; Actual value: C, 52.89; H, 6.41; O, 40.63%. Next, derivative 130 was added 1,2:
3,4-di-O-isopropylidene-6-O-
It was used in the production of (α-L-idopyranosyl)-α-D-galactopyranose (Compound 131). Disaccharides by the support mentioned above for the production of disaccharides
130 (300 mg) was deacetylated. Methanol-
Purification by chromatography on a silica gel column (15 mg) using a chloroform (4:1, V/V) mixture resulted in disaccharide 131 obtained in the form of an amorphous absorbent powder (204 mg, 95%). [α]20 D=-63° ( C : 0.7, methanol)). Theoretical value for C ₁₃ H ₃₀ O ₁₁ : C, 51.18; H,
7.16; O, 41.66, actual value: C, 50.86 (see Figures 24 and 25). Example 34: Preparation of benzyl-2,3,4-tri-O-benzyl-6-O-(α-L-idopyranosyl)-β-D-galactopyranoside (compound 134) First, benzyl-2 ,3,4-tri-O-benzyl-6-O-(2,3,4,6-tetra-O
-acetyl-α-L-idopyranosyl)-β-D
- Galactopyranoside (compound 133) was produced. Benzyl-2,3,4-tri-O-benzyl-β-D-galactopyranoside (K.MIYAT and
RWJEANLOZ's Carbohydr.Res., vol. 21, p. 45,
A solution of 200 mg of compound 132) prepared according to 1972 in anhydrous dichloroethane (15 ml) in the presence of 4 Å molekiller sieves (300 mg) and mercuric bromide (80 mg) at 90° C. under dry nitrogen. Stir until the volume reaches 5 ml. A solution of bromide 124 (160 mg) in dichloroethane (10 ml) was added and the reaction medium was heated at 90 °C.
Stirred for 24 hours. A similar treatment as described above for the preparation of disaccharide 126 was performed to obtain a residue. This residue was dissolved in dichloroethane-acetone (12:1,
V/V) mixture on a silica gel column (30
Purification by chromatography in g) gave disaccharide 133 (227 mg; 70%; [α] 20 D=-30° (C:1, chloroform)). Theoretical value for C ₄₈ H ₅₄ O ₁₅ : C, 66.20; H,
6.25; O, 27.55, actual value: C; 65.96; H, 6.23; O, 27.66%. Next, according to the procedure below, benzyl-2,
3,4-tri-O-benzyl-6-O-(α-L
Compound 133 was used in the preparation of -idopyranosyl)-β-D-galactopyranoside (compound 134).
Disaccharide 133 (200 mg) was deacetylated by the technique described above for the preparation of disaccharide 127. Disaccharide obtained in amorphous form by purification by chromatography on a silica gel (10 g) column using a chloroform-methanol (9:1, V/V) mixture
134 (147 mg, 90%; [α] 20 D = -88° (C: 0.8, chloroform)). Theoretical value for C ₄₀ H ₄₆ O ₁₁ : C, 68.36; H,
6.60; O, 25.04, Actual value: C, 68.74; H, 6.68; O, 25.37% (No.
(See Figure 25). Example 35: 2-acetamide-1,3,6-tri-
O-acetyl-4-O-(2,3,4,6
-Production of tetra-O-acetyl-α-L-idopyranosyl-2-desoxy-β-D-glucopyranose (compound 138) (No. 26)
Figure) The production of this compound follows the steps a) to
c) was carried out. a 2-acetamido-3-O-acetyl-1,
6-Anhydro-2-desoxy-4-O-
(2,3,4.6-tetra-O-acetyl-α-L
Production of -idopyranosyl-β-D-glucopyranose (compound 136) 2-acetamido-3-O-acetyl-1,6-
Anhydro-2-desoxy-β-D-glucopyranose (F.SCHMITT and P.SINAY)
Compound 135 prepared according to Carbohydr. Sheep (1g)
The mixture was stirred at 130°C for 2 hours in the presence of. Bromide 124
(1.43 g) in dichloroethane (10 ml),
The reaction medium was kept at 130°C for 10 hours. Thereafter, a solution of bromide 124 (0.7 g) in dichloroethane (5 ml) was further added, and the reaction was continued for 24 hours. disaccharide
Performing the same process as described for the manufacture of 126,
We reached 136 disaccharides. The compound was purified by chromatography on a silica gel column (200 g) using an ethyl acetate-ether (5:1, V/V) mixture to obtain the disaccharide 136
(1.8 g, 85%; [α] 20 D=70.6° (C:1, chloroform)) was obtained. Theoretical value for C ₂₄ H ₃₃ O ₁₄ N: C, 51.52; H,
5.94; N, 2.50; O, 40.03, Actual value: C51.35; H, 5.89; N, 2.51; O, 40.05
%. b 2-acetamido-1,6-anhydro-2-
Desoxy-4-O-(α-L-idopyranosyl)-β-D-glucopyranose (Compound 137)
Preparation of Disaccharide 136 (500 mg) was deacetylated by the technique already described for the preparation of Disaccharide 127.
Disaccharide 137 (300 mg, 90%; [α]20 D=-65.0゜(C:
1.6, methanol)) was obtained. Theoretical value for C ₁₄ H ₂₃ O ₁₀ N・0.5H ₂ O: C,
44.92; H, 6.46; N, 3.74, actual value: C, 44.95; H, 6.61; N, 4.27%. c 2-acetamido-1,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-
O-acetyl-α-L-idopyranosyl)-2
Production of -desoxy-β-D-glucopyranose (compound 138) Acetic anhydride, acetic acid and concentrated sulfuric acid (7:3:0.1,
V/V) in the presence of a mixture (5 ml) of disaccharide 136
(150 mg) was acetolyzed at ambient temperature for 12 hours.
The reaction medium was then poured into ice water, stirred for 4 hours and then extracted with chloroform (100 ml). The chloroform phase was washed with a dilute aqueous solution of sodium bicarbonate, water, dried (sodium sulfate) and evaporated. Ethyl acetate-ether mixture (5:1,
The residue was purified by chromatography on a silica gel column (10 g) using
Disaccharide 138 was obtained, which was recrystallized in an ethyl acetate-pentane mixture (120 mg, 64%; MP 120°C;
[α]20D=40°(C:1, chloroform)). Elemental analysis: Theoretical value for C ₂₈ H ₃₉ O ₁₈ N: C, 49.63; H,
5.80; O, 42.50; N, 2.07, Actual value: C, 49.68; H, 5.91; O, 42.16; N,
2.12%. Example 362-acetamido-2-desoxy-4-
Production of O-(α-L-idopyranosyl)-D-glucopyranose (compound 139) (Second
Figure 6) To prepare disaccharide 139, disaccharide 138 (100 mg) was deacetylated by the technique described above. The disaccharide 139 was purified by chromatography on a silica gel column (5 g) using a methanol-chloroform (1:2, V/V) mixture.
I got it. The disaccharide was recrystallized in aqueous ethanol (48 mg; 85%, MP 143-145 °C, [α] 20 D
= -20° ~ 31° [C: 0.8, water-methanol (19:1,
V/V), 14 hours later]. Elemental analysis: Theoretical values _{for C14H25NO11.0.5H2O} : C, 42.86; H, 6.68; _N , 3.57; Actual values: C, 42.83; H, 6.68; N, 3 _, 59%. Variation of the preparation of compound 138 according to Example 37 steps 1-6 (see Figure 27) 1: Benzyl 2-acetamide-3,6-di-O-
benzyl-2-desoxy-4-O-(6-O
-tosyl-β-D-glucopyranosyl)-α-
Production of D-glucopyranoside (compound 140) A solution of compound 139 (0.2 g) in pyridine (5 ml) was cooled to 0°C. Tosyl chloride (0.07g) dissolved in pyridine (2ml) was then added. The reaction continued for 24 hours at room temperature. After adding a few drops of water, the mixture was stirred for 30 minutes and poured onto ice. After extraction again with chloroform (0.2), the chloroform phase was washed successively with 10% aqueous _KHSO4 , water, saturated _NaHCO3 solution and water. After drying over sodium sulfate and concentration to dryness, the residue was dissolved in ethyl acetate/
It was chromatographed on silica gel (20 g) using a methanol mixture (15/1, V/V). In this way, pure compound 140 (150 mg;
60%, [α]20 D=+74° (C: 1.1, chloroform))
I got it. Elemental analysis: Theoretical value for C ₄₂ H ₄₉ O ₁₃ NS (807.912): C,
62.40; H, 6.11; N, 1.73; O, 25.74; S,
3.97, Actual value: C, 62.77; H, 6.13; N, 1.73; O,
24.98; S, 3.48, NMR spectrum was consistent with the expected structure. 2: Benzyl 2-acetamide-3,6-di-O-
Benzyl-2-desoxy-4-O-(2,3,
4-tri-3-acetyl-6-O-tosyl-β
-D-Glucopyranosyl)-α-D-glucopyranoside (Compound 141) Acetic anhydride (5 ml) was added to a solution of Compound 140 (200 mg) in pyridine (5 ml). After standing overnight at room temperature, the reaction mixture was concentrated to dryness. Ethyl acetate/
The residue was chromatographed on a silica gel column (25 g) using a hexane (3/1, V/V) mixture. In this way, syrupy compound 141 (208 mg, 90%; [α]20 D = +70° (C:
1, chloroform)) was obtained. Elemental analysis: Theoretical value for C ₄₈ H ₅₅ HSO ₁₆ (934.023): C,
61.78; H, 5.94; H, 1.5; O, 27.41; S, 3.43, Actual value: C, 61.58; H, 5.91; N, 1.27; S,
3.23, the NMR spectrum was consistent with the expected structure. 3: Benzyl 2-acetamide-3,6-di-O-
Benzyl-2-desoxy-4-O-(2,3,
4-tri-O-acetyl-6-desoxy-6
-iodo-β-D-glucopyranosyl)-α-
Production of D-glucopyranozide (Compound 142) 1 Production from Compound 141 Sodium iodide (150 mg) was added to a solution of Compound 141 (150 mg) in acetone (5 ml). This mixture was heated to 70° C. for 7 hours in a sealed tube. After evaporating to dryness, the residue was extracted again with water and chloroform. The chloroform phase was washed with water and dried over sodium sulfate. After evaporation to dryness, the residue was recrystallized in a chloroform/pentane mixture (102 mg, 70%; mp 173-174 °C, [α] 20 D=+
78.5° (C: 1.2, chloroform)). Elemental analysis: Theoretical value for C ₄₁ H ₄₈ O ₁₃ NI (889.733): C,,
55.34; H, 5.44; N, 1.57; O, 23.38; I,
14.28, Actual value: C, 54.98; H, 5.52; N, 1.45; O,
23.57; I, 14.10, NMR spectrum slope corresponds to the expected structure. 2. Preparation starting from compound 139 via compound 143 A solution of compound 139 (1 g) and N-iodo-succinimide (1 g) in DMF (50 ml) was stirred at 0°C for 30 minutes. Next, triphenylphosphine (1.2g)
was gradually added over the course of 1 hour. After heating at 50° C. for 1 hour, methanol (1 ml) was added and the reaction mixture was then concentrated to dryness. The product was extracted with chloroform. The chloroform phase was washed successively with water, sodium thiosulfate solution, and then again with water. After drying and evaporating the chloroform, the residue was placed on a silica gel column (50 g). Compound 143 contaminated with triphenylphosphine was eluted with an ethyl acetate-methanol mixture (15/1, V/V). After evaporating the chromatographic solvent to dryness, derivative 143 was dissolved in pyridine (10 ml) and
It was then acetylated using acetic anhydride (10 ml).
After working up in the usual manner, derivative 142 was recrystallized in a chloroform/pentane mixture. Compound
The yield for 139 was 85%. This compound is
It was similar in all respects to the compound obtained from compound 141. 4: Benzyl 2-acetamide-3,6-di-O-
Benzyl-2-desoxy-4-O-(2,3,
4-tri-O-acetyl-6-desoxy-β
-D-xylo-hexy-5-(enopyranosyl)
-α-D-glucopyranoside-2 (compound 144)
Preparation Silver fluoride (400 mg) was added to a solution of compound 142 (400 mg) in anhydrous pyridine (5 ml). The suspension was stirred in the dark for 48 hours. This mixture was then poured into ether (200ml) with stirring. After filtration, the ether phase was washed with 10% _NaHSO4 solution and then 10%
% _NaHCO3 solution and finally washed with water. dry,
After concentration to dryness, the residue was recrystallized in a chloroform/ether mixture (206 mg, 60
%; mp184-185; [α]20 D=+70° (C: 1.4; chloroform)). Elemental analysis: Theoretical value for C ₄₁ H ₄₇ NO ₁₃ (761821): C,
64.69; H, 6.22; N, 1.84; Actual value: C, 64.5; H, 5.96; N, 1.79; NMR spectrum was consistent with the expected structure. 5: Benzyl 2-acetamide-3,6-di-O-
benzyl-2-desoxy-4-O-(α-L
Preparation of -idopyranosyl-α-D-glucopyranoside (Compound 145) Compound 144 (380 mg) was dissolved in freshly distilled tetrahydrofuran (8 ml). After cooling to 0°C in a nitrogen atmosphere, boron hydride (BH ₃ , THF
(1M, 1 ml) was added and the temperature was then raised to room temperature again. After reacting for 1 hour, hydride (1 ml)
was further added. After 30 minutes, ethanol was added dropwise. When gas emission has stopped, add this mixture to
Diluted with THF (10ml). Soda (3M, 1.2ml)
was added followed by hydrogen peroxide (120 vol; 0.8 ml). After being kept at 50°C for 2 hours, the solution was poured into chloroform. The chloroform phase was washed with aqueous hydrochloric acid (0.1N) and then with water. After drying (Ba ₂ SO ₄ ) and concentration to dryness, the residue was chromatographed on a silica gel column (45 g) using an ethyl acetate/methanol mixture (15/4; V/V). Derivative 145 eluted first (63 mg; 15
%) and then derivative 139 (225 mg; 54%) eluted. Derivative 145 was recrystallized in an ethyl acetate/methanol mixture (mp 191°C; [α]20 D=+
64.4° (C:1, methanol)). Elemental analysis: Theoretical value for C ₃₅ H ₄₃ NO ₁₁・H ₂ O: C, 62.57;
H, 6.75; N, 2.08, actual value: C, 62.42; H, 6.55; N, 1.88, 6:2-acetamide-1,3,6-tri-O-acetyl-2-desoxy-4-O-( 2, 3,
4,6-tetra-O-acetyl-α-idopyranosyl)-D-glucopyranose (compound 138)
A solution of derivative 145 (35 mg) in methanol (10 ml) in the presence of a catalyst (Pd/c, 5%; 25 mg) was stirred in a hydrogen atmosphere for 48 hours. After filtration and evaporation, the residue (17 mg) was acetylated using a pyridine/acetic anhydride mixture (2 ml/1 ml). After the usual work-up, the residue was chromatographed on a silica gel column (10 g), eluting with ethyl acetate. After crystallization, compound 138 was obtained (14 mg, 32%; m, p. 191°C; [α]20 D=+8° (C:
0.6, chloroform)). Example 38 Synthesis of trisaccharide 149 having the following general formula This synthesis is done in three steps (28th
(see figure). First, the orthoester of the L-iduronic acid derivative is glucosylated. Then, selectively the monochloroacetyl group and then one of the alcohols formed is reacted with the disaccharide. 1 Orthoester with benzyl alcohol38
Glycosylation of orthoester 38 obtained according to Example 5
(118mg, 0.25mM) in benzyl alcohol (0.15
ml, 15mM, freshly distilled) in anhydrous chlorobenzene (10ml) was heated to 140°C with protection from moisture. After gradually distilling 8 ml of the solvent, a solution of 2,6-dimethylpyridinium perchlorate (2.5 μM) in chlorobenzene (2 ml) was added dropwise over a period of 30 minutes, and at the same time the solvent (2 ml) was distilled off. The reaction mixture was then stirred under these conditions for 30 minutes while the solvent was distilled off so that the reaction volume remained constant at about 2 ml while new solvent was added dropwise. After cooling, dilute with chloroform (50 ml), wash the organic phase with a 5% aqueous sodium bicarbonate solution and water, and dry (sodium sulfate).
filtered, filtered and evaporated. The residue was chromatographed on a silica gel column (8 g). Elution with hexane-ethyl acetate (2:1, V/V) gave a fraction containing 146 glycoside mixtures. The mixture was not separated at this stage [102 mg,
81%; N, MR (90MHz, CDCl ₃ ) δ: 7.30
(m.10H, 2ph), 3.98 (s, 2H, C1−CH ₂ −CO),
3.74 (s, 3H, COO Me ), 3.08 and 2.03 (2s, overall 3H, β and α forms of O Ac ; β:2:
1)]. 2 Selective O-demonochloroacetylation of the above mixture 146 (102 mg) in pyridine (5 ml)
Add thiourea (25 mg) to anhydrous ethanol (1 ml) solution.
and heated to 100°C for 20 minutes. After cooling,
The reaction mixture was evaporated to dryness and the residue was extracted again with a water-chloroform mixture (1:1, V/V, 50 ml). The organic phase was washed with water, dried (sodium sulphate), filtered and evaporated. The residue was chromatographed on a silica gel column (10 g). Glycosides were eluted with ethyl acetate-hexane mixture (4:3, V/V).
148 and glycoside 147, (in the elution order) were isolated. Glycoside 148 (26 mg, 25%): colorless syrup; [α]20 D = +79° ( C : 1, chloroform); N.
MR (90MHz, CDCl ₃ ): δ: 7.30 (m, 10H, 2
ph), 5.05 (m, 1H, H ₂ ), 4.90 (d, 1H, H ₁ ,
1.2J=2Hz), 3.78(s, 3H, COO Me ), 3.12
(1H, O H exchanged with D ₂ O), 2.05 (s, 3H, O
Ac). α-Glycoside 147 (54 mg, compounds 38-50
%): colorless syrup, [α]20 D=-65° ( C : 1, chloroform); NM
R. (90MHz, CDCl ₃ ): δ: 7.30 (m, 10H, 2
ph), 5.05 (2H, extremely weak coupling constant for H ₁ and H ₂ , J _1.2 ≦1Hz), 3.78 (s, 3H, COOMe),
2.80 (1H, OH exchanged with D ₂ O), 2.06 (s, 3H,
OAc). 3 Glycosylation of alcohol 147 with disaccharide 97 A solution of alcohol 147 (22 mg, 50 mM) and bromide 97 (57 mg, 70 mM) obtained in Example 6 in anhydrous dichloromethane (1.5 ml) was mixed with a 4 Å molecular sieve (powder). , 50 mg) and stirred protected from light and moisture. The reaction mixture was cooled to -20°C and sym-collidine (110ml) and silver triflate (26mg, 100mM) were added sequentially.
The reaction mixture was stirred under these conditions for 2 hours, diluted with dichloromethane (50 ml), the solid slowly dehydrated, and the liquid diluted with ice-cold 0.1 MHCl aqueous solution, water,
Washed sequentially with 5% aqueous sodium bicarbonate and water, dried (sodium sulfate), filtered and evaporated. Transfer the residue to a silica gel column (8 g, gel 230 ~
400 mesh) was subjected to chromatography.
Trisaccharide 149 (50 mg, 86%) was isolated as a colorless syrup by elution with toluene-ethyl acetate (5:1, V/V). The NMR spectrum (270MHz, CDCl ₃ ) was consistent with the expected structure. This spectrum is shown in FIG. Example 39: Immobilization of tetrasaccharides on BSA Bovine serum albumin (BSA: 7mg; 0.1μple)
Sodium cyanoborohydride (13 mg; 200 μM) was added to a phosphate buffer (0.15 M; PH7.0; 2.5 ml) solution of the tetrasaccharide (15 mg; 10 μM) and the solution was left at 37° C. for 5 days. The reaction mixture was then chromatographed on a Sephadex G-50 (1 x 100 cm) column and eluted with water to separate the pentasaccharides and salts that were not immobilized on the protein-oligosaccharide complex. Under these conditions, BSA1
It was possible to fix 12 moles of tetrasaccharide per mole. 2-aminoethyl-polyacrylamide or 2
- insoluble support such as aminoethyl-cellulose,
Or the same reaction could be performed on any other support containing primary amine functionality. Similarly, antithrombin (instead of BSA)
immobilize the oligosaccharides on antithrombin by operating under previously defined conditions in the presence of
Through this, permanently activated antithrombin was obtained (BJORK et al., FEBS Letters, vol. 143,
96-100, 1982). Synthesis of disaccharide 163 Compound 2 (5.6g) and mercuric cyanide (3.5g)
After about 1 ml of the solvent was distilled off, 4 Å molecular sieves (1 g) were added to a dichloromethane (40 ml) solution, and then Compound 1 (3.44 g; 8.82 mM) was added. After stirring overnight, the solids were removed by filtration and then washed with dichloromethane. The latter was combined with the solution and the organic phase obtained was then washed with saturated potassium iodide solution and then with water. After drying and concentrating to dryness, the resulting syrup (10 g)
was deacetylated in methanol (20 ml) in the presence of sodium methanolate (2M, 1 ml). Compound 163 obtained after chromatography on silica gel (50 g; chloroform/methanol; 20/1; V/V) is a syrup ([α]20 D=-
12° (C: 1.1, chloroform)). This syrup is used as is for the synthesis of compound 5. Synthesis of Compound 164 Disaccharide 163 (2.7 g) was dissolved in anhydrous DMF (27 ml), then trityl chloride (4.42 g), dimethylaminopyridine (135 mg) were added to this solution, and then triethylamine (2.7 ml) was added. added. After 2 days at room temperature, the reaction mixture was concentrated under vacuum. The residue was chromatographed on silica gel (50 g; hexane then hexane/ethyl acetate; 2/1 then 1/1; V/V). Compound 164 was thus obtained (2.6 g). Compound 164 is syrup ([α]20 D=-
The temperature was 16.3° (C: 1.3, chloroform)). Synthesis of disaccharide 166 The syrup obtained at the end of the preparation of compound 164 (2.4 g) was dissolved in DMF (40 ml). Next, barium hydroxide octahydrate (1.64 g), barium oxide (7.08 g), and finally benzyl bromide (2 ml) were added. After reacting for 4 hours, methanol was added followed by chloroform (100ml). The solids were drained and the chloroform phase was concentrated to dryness. The disaccharide 165 obtained at this step was directly converted to compound 166. For this, remove the residue in dichloromethane (20
ml) and then protected from moisture to 0.
A methanol solution of BF ₃ (2 ml) was added at °C. 4
After reacting for an hour, the reaction mixture was diluted with dichloromethane and then washed with aqueous sodium bicarbonate solution. After drying and concentration, the residue was transferred to a silica gel column (100 g; hexane/ethyl acetate; 4/1 then 1/1;
V/V) chromatography. In this way, compound 166 was obtained [α] 20 D=-2° (C: 0.7, chloroform)). Synthesis of Compound 168 Derivative 166 was dissolved in acetone (20ml).
Next, 3.5M sulfuric acid (3.5M) of chromium oxide () (670mg)
ml) solution was added at 0°C. After 1.5 hours, ice and water were added to the reaction mixture, and then the oxidation products were extracted with chloroform. The chloroform phase was washed with water, dried and concentrated to dryness. The residue dissolved in ether was methylated by adding diazomethitane to give compound 168. This compound was added to silica gel (hexane/ethyl acetate; 4/1,
It was then purified on 1/1; V/V). The purified compound was syrup ([α] 20 D=-8.5° (C:1, chloroform)). Elemental analysis and IR spectrum were consistent with the expected structure for compound 168. Note: Compound 168 can be converted to a halide by acetolysis using the method described in Example 25 (Compound 94 to Compound 97).

[Brief explanation of the drawing]

1 to 30 are process diagrams for producing the compounds of the present invention, and FIGS. 31 and 32 are diagrams showing NMR spectra of compounds 43 and 149 of the present invention, respectively. Ac...Acetyl group, Me...Methyl group, Bn...
...benzyl group, Bz...benzoyl group, MCAO...
...monochloroacetyl group, Tr...trityl group,
but...butyl group, S... _SO3- ^group .

Claims (1)

[Claims] 1 [D-glucosamine] 1α → 4 [D-glucuronic acid], [D-glucosamine] 1α → 4 [L-iduronic acid], [D-glucuronic acid] 1β → 4 [D-glucosamine] ], and [L-iduronic acid] 1α → 4[D-glucosamine] A method for organically synthesizing an oligosaccharide containing a chain selected from the following: A compound constituted by or terminated by an A unit is reacted with a compound constituted by or terminated by a U unit of a D-glucuronic acid or L-iduronic acid structure (herein, , one of the A and U units is an alcohol with an alcohol-functional -OH group occupying the 4-position, and the remaining units are reactive groups selected from halides, imidates and orthoesters. has an activated anomeric carbon substituted with a position of the activated anomeric carbon among the A and U positions and a - of the alcohol functionality.
All other positions except the position of the OH group carry amino or carboxyl groups or precursors of these groups or -OH groups; When the groups are present, they are blocked with amino- and carboxyl-functional protecting groups, respectively, and the -OH group is blocked by permanent and semi-permanent protecting groups, and in some cases by temporary protecting groups. Each type of protecting group can be removed under different conditions, and the permanent and semi-permanent protecting groups include alkyl groups, substituted alkyl groups, acyl groups,
an acetal group, an epoxide group between two adjacent OH groups, and a 1,6-anhydro bridge; the temporary protecting group is selected from an allyl group and an acyl group; (used to temporarily protect the position to be involved in the reaction), optionally repeating the glycosylation step to extend the oligosaccharide chain and remove the semi-permanent -OH protecting group; released
A method consisting of sulfation of the OH group followed by removal of the permanent protecting groups, amino protecting groups and carboxyl protecting groups (with the exception that by formation of interglycosidic bonds [2-N-sulfate or (2-N
-acetyl)-6-O-sulfate-D-glucosamine]-[methyl-D-glucuronic acid] structure). 2. It involves introducing one type of functional group or sequentially introducing multiple types of functional groups by subjecting the generated glucide chain to one or more chemical reactions, and these groups are used for heparin and heparan sulfate type 2. A method according to claim 1, characterized in that the group is selected from among the groups present in biologically active molecules. 3. In the functionalization step, only some or all of the temporary or semi-permanent protecting groups and/or precursor groups of the amino derivative are removed and one type of substituent is introduced in their place or multiple types of substituents are introduced. 3. The method according to claim 2, characterized in that the steps are sequentially introduced. 4. Any one of claims 1 to 3, characterized in that the A unit contains in its 2-position a nitrogen-containing group or any other group constituting an amine functional group or a precursor of an amine derivative. The method described in. 5 The carboxyl function of the U unit is a group which is inert to the reaction for substituting the protecting group and which can be removed at the end of the synthesis to liberate the carboxyl group, optionally for the purpose of salt formation. 5. A method according to claim 1, characterized in that the method is blocked by: 6 In order to extend the U unit side of the disaccharide I-A, a temporary protecting group is present in the U unit, and in order to extend the A unit side, a temporary protecting group is present in the A unit, and thereby,
Perform sequential glycosylation reactions to form a chain U _w A _x
U _y A _z [The sum of the subscripts is 2 to 12, each value of the subscripts is also included in this range, and w and y cannot be 0 at the same time]. The method according to any one of ranges 2 to 5. 7 The condensation reaction between the halide and the alcohol is carried out at room temperature in a solvent and using a proton acceptor and optionally present water and/or a scavenger for the hydrohalic acid formed. The method according to any one of claims 1 to 6, characterized in that the method is carried out in an inert gas atmosphere at a low temperature. 8. To form a covalent bond between the alcohol and the L-idose precursor of L-iduronic acid, a condensation reaction is carried out in an organic solvent using a mercury derivative as a catalyst and a molecular sieve. The method according to any one of claims 1 to 6, characterized in that: 9 Condensation with orthoester in the presence of a catalyst
7. Process according to any one of claims 1 to 6, characterized in that it is carried out at a temperature higher than 100°C in a chlorobenzene-type solvent or similar solvent with a boiling point higher than 100°C. 10. The method according to claim 1, wherein the condensation with imidate is carried out at low temperature in a solvent in the presence of a 4 Å molecular sieve and a catalyst. 11. The method according to any one of claims 2 to 10, characterized in that a temporary protecting group is released from one alcohol functional group of an A unit or a U unit contained in an already constituted glucide sequence. 12 Constructing the precursor of the amine function by ring opening of the epoxide function with sodium azide
12. The method according to any one of claims 1 to 11, characterized in that an _N3 group can be introduced at the 2-position. 13 The -OH group to be sulfated in the starting material is protected with an acyl group and the - to be liberated at the end of the synthesis.
13. A method according to any one of claims 1 to 12, characterized in that the OH group is protected with a permanent group. 14 A functionalization step is selectively carried out to sequentially introduce multiple substituents onto the chain, forming an amino derivative at the 2-position of the A unit and an acid derivative at the 6-position of the U unit; This functionalization step involves liberating the -OH group at the position of
The group occupying the position to be sulfated is composed of an -O-acetyl group, the position corresponding to the -OH group to be liberated is occupied by a benzyl group, and the 2nd position of the A unit is composed of N ₃ or 6-NH-COO-
is substituted with a group such as CH ₂ -C ₆ H ₅ , and U
14. A process according to claim 1, characterized in that a derivative is used in which the 6-position of the unit is occupied by a carboxyl group protected by an alkyl group. 15 The functionalization step - by a saponification reaction carried out using a strong base such as sodium hydroxide, preferably at a temperature below room temperature, in particular at a temperature close to 0°C -
After removing the O-acetyl group, the product obtained as a result of hydrolysis is treated with an alkylating agent to introduce the protected alkyl group removed during hydrolysis onto the carboxyl group, and after this alkylation, sulfuric acid The sulfate group is selectively introduced by carrying out a sulfate treatment to introduce a sulfate group into a position that is favored by hydrolysis and remains free even after the action of an alkylating agent (this sulfate treatment
(with a sulfating agent, in a solvent, corresponding to a reaction duration of about 12 hours), - preferably with retention of the sulfate group and conversion of the nitrogen-containing group into an amino function with water in an organic solvent. By removing the benzyl group by catalytic hydrogenation under conditions compatible with both, the -OH group blocked by the benzyl group is liberated and - N-acetyl groups are formed by the action of an acetylating agent, such as acetic anhydride, or N-sulfate groups are formed at a pH greater than 9 using sulfating agents similar to those described above, - by addition of a strong base. 15. The method according to claim 14, characterized in that it is carried out by liberating the carboxyl group and - salting the carboxyl group.