Synthetic polysaccharides, preparation method therefor and pharmaceutical compositions containing sa

专利摘要:
The present invention relates to a synthetic polysaccharide, a preparation method thereof and a pharmaceutical composition comprising the same. It comprises antithrombin III capable of binding to a region consisting of a series of five monosaccharides having a total of two carboxylic acid functional groups and at least four sulfo groups, wherein the region is independently a hydroxyl group (C 1 − C 6 ) by a thrombin-binding region comprising a series of 10 to 25 monosaccharide units selected from hexose, pentose or deoxy sugars which can be etherified with alkyl groups or esterified in the form of sulfo groups It is characterized by describing synthetic polysaccharides and their pharmaceutically acceptable salts which bind directly at their unreduced ends.
公开号:KR20000067909A
申请号:KR1019997000366
申请日:1997-07-18
公开日:2000-11-25
发明作者:드리게즈삐에르알렉상드르；뒤코쏘이필리뻬；에르베르쟝-마르크；쁘띠뚜모리스；반보에켈콘스탄트；그루텐후이스페터；바스텐죠한네스；드리이프-트롬프코르넬리아
申请人:디. 꼬쉬；사노피；샬크비즈크 피이터 코르넬리스; 페트귄터；아크조 노벨 엔.브이.；
IPC主号:

专利说明:

SYNTHETIC POLYSACCHARIDES, PREPARATION METHOD THEREFOR AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME
The present invention relates to synthetic polysaccharides having the anticoagulant and antithrombotic pharmacological activity of heparin.
Heparin belongs to the glycosaminoglycan (GAG) family, which is a heterologous natural sulfated polysaccharide.
Heparin preparations are chain mixtures consisting of a plurality of monosaccharides in the range of 10 to 100 and more. In addition to the size heterogeneity, structural heterogeneity exists in the nature of the constituent monosaccharides and the substituents they contain (L. Roden in: The Biochemistry of Glycoproteins and Glycosaminoglycans, Ed by Lennarz WJ, Plenum Press, New York and London, 267-371, 1980).
Each natural GAG family has a wide range of pharmacological activities. All are bound in preparations obtained from natural products. Thus, for example, heparin and heparan sulfate have antithrombotic activity associated with the simultaneous action of several coagulation elements.
Heparin catalyzes the inhibition of two enzymes, urea Xa and urea II (or thrombin), which are included in the coagulation cascade, in particular via antithrombin III (AT III). Low molecular weight heparin (LMWH) preparations contain chains composed of 4 to 30 monosaccharides and have a more selective action on urea Xa than thrombin.
Certain synthetic oligosaccharides, especially those described in EP 84,999, have no activity on thrombin and have the property to selectively inhibit urea Xa via antithrombin III.
Inhibition of urea Xa requires the binding of heparin to AT III via an antithrombin-binding region (ABR), and inhibition of urea IIa (thrombin) not only binds to AT (III) via ABR, but is not well known. It is known to require binding to thrombin via a binding region (TBR).
Synthetic oligosaccharides corresponding to the ABR region of heparin are known and demonstrate antithrombotic activity in venous thrombosis. Such compounds are described in EP 529,715 and EP 621,282 and Canadian patent 2,040,905.
The efficacy of the oligosaccharides in the prevention of arterial thrombosis is limited by the inability to inhibit thrombin.
The synthesis of heparin-type glycoaminoglycans that can inhibit thrombin through the AT (III) activator presents a number of difficulties and is not obtained in practice.
Rediscovering the activity of thrombin-inhibitor and urea Xa-inhibitor products, resulting in two identical oligosaccharides (ABR and TBR) by EP-A-0,649,854 that are not involved in biological activity ("spacers"). The connection is presented.
New polysaccharide derivatives have now been found to be relatively simple to synthesize and biologically active. These are especially anticoagulants and antithrombotic agents. In addition, the production of such polysaccharides by synthesis allows for selective modification of their structure and particularly the elimination of unwanted sulfate substituents involved in the reaction with specific proteins. Therefore, polysaccharides are obtained that are potent anticoagulants and antithrombotic agents and in vivo avoid the action of proteins, such as platelet element 4 (PF4), which neutralize the efficacy of heparin on thrombin.
It has therefore been found that sulfated and alkylated polysaccharides are potent antithrombotic and anticoagulant agents depending on the arrangement of alkyl and sulfate groups formed by carbohydrate backbones.
In general, it has been found that by preparing polysaccharide sequences, GAG-type activity can be precisely modified to obtain highly active products having the properties of heparin.
Therefore, according to one of its aspects, the present invention provides not only synthetic polysaccharides composed of antithrombin III-binding regions consisting of five monosaccharide sequences containing a total of two carboxylic acids and at least four sulfate groups, but also salts thereof, in particular pharmaceuticals thereof. With respect to the permissible salts, the region is selected from hexose, pentose or deox sugars in which all hydroxyl groups are each etherified with (C ₁ -C ₆ ) alkyl groups or esterified in the form of sulfate groups It is extended at its non-reducing end by a thrombin-binding region consisting of 10 to 25 monosaccharide unit sequences.
Preferably, the invention relates to the polysaccharides as defined above and to salts thereof, in particular to pharmaceutically acceptable salts thereof, wherein all of their hydroxyl groups are etherified with methyl or esterified in the form of sulfo groups. It is done.
The products of the present invention are in particular one of the polysaccharides represented by the following general formula (1) or salts thereof, in particular pharmaceutically acceptable salts:
(In Formula 1,
The wavy line represents a bond below or above the plane of the pyranose ring,
- Denotes a polysaccharide (Po) comprising the same or different monosaccharide units (n) bonded to Pe via its anomer carbon,
- Is a schematic representation of a monosaccharide unit of pyranose structure selected from hexose, pentose and the corresponding deoxy sugar, wherein the unit is bonded to another monosaccharide unit via its anomer carbon, The hydroxyl group is substituted with the same or different -X groups, the X group is selected from a (C ₁ -C ₆ ) alkyl group and a sulfo group,
n is an integer from 10 to 25,
Pe represents pentasaccharide of the general formula:
(In the above formula,
R ₁ represents a (C ₁ -C ₆ ) alkyl group and a sulfo group,
R ₁ a constitutes a C—CH ₂ —O group with R ₁ representing or attached to an oxygen atom and a carbon atom containing a carboxyl function on the same ring,
R represents (C ₁ -C ₆ ) alkyl,
W represents an oxygen atom or a methylene group))
It will be pointed out generally herein that the wavy line represents a bond below or above the plane of the pyranose ring.
Monosaccharides contained in Po are the same or different from each other, and the glycosidic linkage is α type or β type.
Said monosaccharide is selected from D hexose allose or L hexose allose, altrose, glucose, mannose, gallos, idose, galactose and talos (h = 3 in this case) or D pentose ribose or It is advantageously selected from L pentose ribose, arabinose, xylose and lyxos in this case h = 2. Other monosaccharides such as for example deoxy sugars may also be used (h = 1 and / or —CH ₂ OX═CH ₃ ).
When the unit W in the pentasaccharide (Pe) represents an oxygen atom and R ₁ a is defined as R ₁ , the pentasaccharide is described in particular in the literature as well as in EP 300,099, EP 529,715, EP 621,282 and EP 649,854. It constitutes a known compound. They are also described in C. van Boeckel and M. Petitou by Angew. Chem. Int. Ed. Obtained from synthons described in Engl., 1993, 32, 1671-1690.
If penta saccharide (Pe) R ₁ is a different R ₁ and / or W in a represent a carbon atom, wherein the penta-saccharides are produced by using the sinton form a further aspect of the present invention.
When a unit of the L-iduronic acid type in pentasaccharide (Pe) is replaced with a unit whose form is blocked by a bridge, the pentasaccharide is prepared using a syntone which forms another aspect of the present invention.
Therefore, according to another aspect thereof, the present invention relates to intermediates useful for preparing compound 1.
The polysaccharide moiety (Po) consists of 10 to 25 alkylated and disulfide or trisulfide monosaccharide units.
The polysaccharide moiety (Po) consists of 10 to 25 alkylated and monosulfated or disulfide monosaccharide units.
The polysaccharide moiety (Po) is composed of 10-25 uncharged and / or partially charged and / or fully charged alkylated monosaccharide units.
Charged or uncharged units are dispersed along the entire length of the chain or vice versa in groups within the charged or uncharged saccharide region.
Binding is 1,2; 1,3; 1,4; 1,5; 1,6; And α type or β type.
In the present specification, it is chosen to represent modification ¹ C ₄ for L-iduronic acid and modification ⁴ C ₁ for D-glucuronic acid, but in general it is well understood that the modification of monosaccharide units in solution is varied. Known. Therefore, L-iduronic acid has a ¹ C ₄ ² S ₀ or ⁴ C ₁ modification.
Preferred compounds according to the invention are compounds of the formula 1-A and salts thereof, in particular pharmaceutically acceptable salts thereof.
(In Chemical Formula 1-A,
- Denotes a specific polysaccharide (Po) system which is bonded to Pe via their anomer carbon as defined in Formula 1,
- Is as defined in Formula 1,
OX is as defined in formula 1 for the same polysaccharide, is the same or different,
monosaccharides contained within [] _m form disaccharides repeated m times, monosaccharides contained within [] _t form disaccharides repeated t times,
m is 1 to 8, t is 0 to 5, p is 0 to 1, and 5 ≦ m + t ≦ 12)
Advantageous compounds are salts in which the anion corresponds to formula (1-1) as well as the corresponding acid:
(In Formula 1-1, t is 5, 6 or 7, and the cation is a pharmaceutically acceptable monovalent cation.)
As well as the salts in which the anion corresponds to formula 1-2, the corresponding acids are also advantageous.
(In the above formula 1-2, t is 5, 6 or 7, the cation is a pharmaceutically acceptable monovalent cation)
Particularly advantageous are salts in which the anion has the structure of formula 1-3, as well as the corresponding acid.
(In Formula 1-3, m is 1, 2 or 3, t represents 2, 3, 4 or 5, and the cation is a pharmaceutically acceptable monovalent cation.)
Other preferred compounds according to the present invention are the compounds of formula 2-A and pharmaceutically acceptable salts thereof.
(In the above formula 2-A,
- Denotes a specific polysaccharide (Po) system which is bonded to Pe via their anomer carbon as defined in Formula 1,
- Is as defined in Formula 1,
-OX groups are as defined in formula 1 for the same monosaccharide, are the same or different,
monosaccharides contained within [] _{m '} are repeated m' times, monosaccharides contained within [] _{t '} are repeated t' times, and monosaccharides contained within [] _{p '} are p' times Repeated,
m 'is 1 to 5, t' is 0 to 24, p 'is 0 to 24, and 10≤m' + t '+ p'≤25)
Preferred salts of the present invention are those selected from alkali metal cations, more preferably the cations are Na ⁺ or K ⁺ .
Particular preference is given to the following polysaccharides:
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)] ₄ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)] ₅ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)] ₆ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-[O- (2,3-di- O-Methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-] _11- O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid )-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl -α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-[O- (2,3-di- O-Methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-] _13- O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid )-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl -α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-[O- (2,3-di- O-Methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-] _15- O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid )-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl -α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)] _2- [O- (2,3 , 6-Tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₂ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosyluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2, 3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)] _2- [O- (2,3 , 6-Tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosyluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2, 3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl)- (1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6- Tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) ₄ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D -Glucopyranosyluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2,3-di -O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl)- (1 → 4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6- Tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) ₃ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D -Glucopyranosyluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2,3-di -O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₄ -O-2,3-di-O-methyl-6-sulfo -α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1 → 4) -O- (2 , 3,6-Tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₅ -O-2,3-di-O-methyl-6-sulfo -α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1 → 4) -O- (2 , 3,6-Tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4)-(2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt
The present invention relates to a process for preparing a compound of formula (I), wherein in a first step a protected precursor of a Pe region (region shown in Scheme 1) extended by a sulfated polysaccharide (Po) at its non-reducing end The fully protected precursor of the desired polysaccharide (I) containing is synthesized and negatively charged groups are introduced and / or unshielded in the second step.
The structure of pentasaccharide (Pe) when W═O or C; The letter DEFGH is used in this scheme to indicate the relevant monosaccharide
In a first approach, a fully protected precursor of the tetrasaccharide portion of pentasaccharide (EFGH) is used. To obtain the total ABR recovered after binding, polysaccharide (Po) containing a loss unit (D) of Pe at its reduction-end is added.
In another approach, a fully protected precursor of the disaccharide portion (GH) of pentasaccharide is used. To obtain the total ABR recovered after binding, a polysaccharide (Po) precursor of TBR containing a loss unit (DEF) of Pe at its reduction-end is added.
The Pe precursor is synthesized as described above from the sintones described in the specification or part of the present invention.
The polysaccharide precursor portion of Po combines glycoside-linked-donor oligosaccharides with glycoside-linked-receptive oligosaccharides in order to induce other oligosaccharides whose size is the sum of the sizes of the two reactive species. Oligosaccharide or oligosaccharide when synthesized (GJ Boons, Tetrahedron. 1996, 52, 1095-1121), according to reactions known to those skilled in the art.
This sequence is repeated until the desired compound of formula (1) is obtained. The nature and profile of the desired final compound's charge determines the nature of the chemical species used in the various synthetic steps, according to rules known to those skilled in the art.
Preferred methods for preparing Po precursors according to the invention are shown in Scheme 2:
Synthesis of Protected Precursors of TBR
The term transient is understood to refer to a substituent that is conserved for a limited number of steps, the term semi-permanent is understood to refer to a substituent that is conserved for several steps, and the term permanent refers to a substituent that is conserved until the end of the synthesis. Is understood; Permanent substituents are removed during the final step. Certain permanent groups form part of the final molecule.
In Scheme 2, (a) is a glycoside wherein Z is a transient protecting group of hydroxyl functionality, Y is the same or the same, and is an anomer-carbon activator (Tn) that is a transient, semipermanent or permanent substituent of all other hydroxyl functionality. -Bond-donor monosaccharide.
Compound (b) having an unsubstituted hydroxyl group represents a glycoside-binding-receptor monosaccharide in which the same or different Tn is a temporary, semi-permanent or permanent substituent of the hydroxyl group. T ₁ is a temporary, semi-permanent or permanent protecting group at the anomer position. It is removed when desired to activate the anomer carbon.
To obtain a compound of the present invention, the glycoside-binding donor (a) and the glycoside-binding receptor (b) react together to give a disaccharide (c).
The obtained disaccharide (c) is specifically converted to glycoside-binding-donor disaccharide (d) by removing T ₁ and introducing Y and / or by removing Z, thereby removing the glycoside-binding-receptor ( e) is specifically converted.
Next, the glycoside-binding donor (d) and the glycoside-binding receptor (e) react together to give tetrasaccharide (f) in which t represents one.
When the reaction is repeated in the above order, an oligosaccharide or polysaccharide (f) having t of 1 or more is obtained.
Also, using the method shown in Scheme 2, various fully protected oligosaccharides such as (g) where oligosaccharides [] _m and [] _t are fully protected precursors of the otherwise charged regions of the compounds of the invention or Polysaccharides can be obtained.
In the following method steps, compounds such as (f) and (g) are converted into glycoside-linked donors and bound to the non-reducing end units of the fully protected precursor of Pe.
As mentioned above, when (g) is attached to the non-reducing end unit of a fully protected oligosaccharide that is a precursor of the residue of the structure of Pe, the non-reducing end of the glycoside-linked-donor polysaccharide (g) Oligosaccharides of the unit constitute part of Pe.
Compounds of the invention are obtained from their fully protected polysaccharide precursors using the following reaction sequence:
Alcoholic functionalities and carboxylic acids that need to be converted to sulfo groups are deprotected by removing the Tn groups used to protect them during skeletal development, and then
Sulfo groups continue to be induced.
Originally, compounds of the present invention are prepared using several methods known to those skilled in the art of oligosaccharide synthesis.
The method is a preferred method of the present invention. However, compounds of formula 1 are described, for example, in Monosaccharides, Their chemistry and their roles in natural products, P.M. Collins and R.J. Ferrier, J. Wiley & sons, 1995 and G.J. It can be prepared by other known methods of sugar chemistry described in Boons, Tetrahedron, 1996, 52, 1095-1121.
When W represents an oxygen atom and R ₁ a is R ₁ , some of the precursors of pentasaccharide (Pe) are oligosaccharide synthesis methods and in particular patents EP 84,999, EP 301,618, EP 454,220 and EP 529,715 and patent application EP 93204769 And the method described in EP 94202470. If full protection is given, free hydroxyl groups can be obtained on position 4 of the non-reducing terminal unit (D) using suitable protecting groups. The fully protected precursor of Pe is then bound to this position using known oligosaccharide synthesis methods.
Pentasaccharide (Pe) of formula (2) wherein W is a carbon atom and R ₁ a is R ₁ is obtained from a synthon of formula (2-1), and the resulting C-disaccharides are then obtained from C. van Boeckel and M. Convert to Sinton (Formula 2-1) according to the standard method according to Petitou.
(In Formula 2, R and R ₁ are as defined in Formula 1)
(In Formula 2-1, the same or different T ₁ and T _n represent a temporary, semi-permanent or permanent substituent, Z is a radical between a double bond containing monosaccharide and a free-radical-generating monosaccharide. Is a protecting group for hydroxyl functionality obtained by synthesis carried out by reaction)
Particularly useful to synthesize Compound 2, the sintone of Formula 2-1 has the structure:
(2-1) or compound 90
The sintone is prepared according to the following Scheme 22.
Penta-saccharide (Pe) of the constituting the L- biceps acid units of the block arrangement of the substituents R ₁ a to Formula 3 characterized is obtained from sinton of formula 3-1.
(In Formula 3, R and R ₁ are as defined in Formula 1, W represents an oxygen atom)
(In Formula 3-1, the same or different T ₁ and T _n represent temporary, semi-permanent or permanent substituents, Z denotes MK Gurjar et al., Tetrahedron letters, 1995, 36, 11, 1937-1940, 1933- 1936 and 1994, 35, 14, is a protecting group for hydroxyl functionality obtained by synthesis carried out according to the method described in 2241-2244).
Particularly useful to synthesize Compound 3, the sintone of Formula 3-1 has the structure of Formula 3-1:
(Formula 3-1)
The sintone is prepared according to Scheme 34 below.
Intermediates (2-1 and 3-1) are particularly useful intermediates for the preparation of compound (1) according to the invention.
Pentasaccharide (Pe) is the aforementioned C.A.A. van Boeckel and M. Petitou, Angew. Chem. Int. Ed. From the disaccharide synthons (2-1 or 3-1) in the method described in Engl.
The term semi-permanent group used above is first to be removed after glycosylation by introducing the desired functional groups into their occupied positions when the carbohydrate backbone contains the desired number of units without adversely affecting or eliminating other groups present. It is understood to refer to a group that can.
Permanent groups are those groups that can maintain protecting OH functionality while introducing functional groups in semi-permanent groups.
The group is selected from those compatible with the introduced functional group after removing the semi-permanent group. In addition, they are inert towards the reaction carried out to introduce the functional groups, which groups are removed without being adversely affected.
According to the invention, the permanent group is preferably a (C ₁ -C ₆ ) alkyl group.
Examples of semi-permanent and / or transient groups mentioned are benzyl and acetyl, levulinyl, p-methoxybenzyl groups and the like.
The substituent in position 3 of the uronic acid unit of the target compound is already present in the starting synthon together with the substituent R ₁ .
The protecting groups used in the process for preparing Compound 1 are those commonly used in sugar chemistry such as Protective Groups in Organic Synthesis, TW Greene, John Wiley & sons, New York, 1981.
The protecting group is, for example, an acetyl, halomethyl, benzoyl, levulinyl, benzyl, substituted benzyl, optionally substituted trityl, tetrahydropyranyl, allyl, pentenyl, tert-butyldimethylsilyl (tBDMS) or trimethylsilylethyl group It is advantageously selected from (etc.).
Active groups are sugar chemistry, for example G.J. It is conventionally used according to Boons, Tetrahedron, 1996, 52, 1095-1121. The active group is for example selected from imidate, thioglycoside, pentenylglycoside, xanthate, phosphite and halide.
By this method, the compound of the present invention can be obtained in the form of a salt. In order to obtain the corresponding acid, the compounds of the invention in salt form are placed in contact with the cation-exchange resin in acid form.
To obtain the desired salt, the compounds of the present invention in acid form are neutralized with salts.
To prepare salts of the compounds of formula (1), organic or inorganic bases are used together with the compounds of formula (1) to provide pharmaceutically acceptable salts.
Sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide are preferably used as the base. Sodium and calcium salts of the compound of formula 1 are preferred salts.
In step (a) of the method, the protecting group used is a sugar chemistry, for example those having ordinary skill in the art according to EP 84,999 or optionally Protective Groups in Organic Synthesis, TW Greene, J. Wiley & Sons, 1995. It is usually used by.
Compound 1 obtained is optionally chlorinated.
The compounds of formula 1 are also compounds in which one or more hydrogen or carbon atoms are substituted with their radioisotopes, for example tritium or carbon-14. The compounds indicated above are useful for pharmacokinetic metabolism or investigational studies and biochemical tests as ligands.
The compounds of the present invention formed as subjects of the biochemical and pharmacological studies shown have very advantageous properties.
Compounds of the invention that selectively bind AT III with an affinity equal to or greater than that of heparin have the anticoagulability and antithrombogenicity of heparin.
The total antithrombotic activity of the product of Formula 1 is not only a model of venous arrest and induction by thromboplastin according to the method described in Thrombosis Research, 1980, 18, 669-674 by J. Reyers et al. . As described in Haemost., 1978, 39, 74-83, a model of arterial thrombosis consisting of a shunt implanted between the carotid and jugular veins of the rat was measured intra-arterally or subcutaneously in the rat. In the two experimental models, the ED ₅₀ of the compounds of the present invention is in the order of less than or at least the same as other synthetic heparinoids already known (ED ₅₀ of 5 to 500 μg / kg). The compounds of the invention therefore have specificity of action and particularly advantageous anticoagulant and antithrombotic activity.
Due to their biochemical and pharmacological activity, the compounds of the present invention are very advantageous pharmaceutical products. Their toxicity is compatible with the use as a whole. They are also very stable and are particularly suitable for forming active ingredients of specialty pharmaceutical products.
In addition, the compounds of the present invention are not neutralized by high doses of cationic platelet proteins, such as platelet element 4 (PF4), released during activation of the protein during thrombosis. Therefore, the compounds of the present invention are particularly advantageous for treating and preventing thrombosis of arterial or venous sources.
They are persistent in modifying the hemostasis of the coagulation system, especially during cardiovascular and cerebrovascular diseases, such as unstable angina, brain shock, restenosis after angioplasty, arterial ablation and endovascular repair. Hemoembolic diseases associated with sclerosis and diabetes; Or several pathologies that appear selectively during rethrombosis, infarction, ischemia dementia, peripheral arterial disease, hemodialysis and hemoembolic diseases associated with atrial fibrillation or during coronary prosthesis of the aortic coronary bridge. Moreover, the product is used to treat or prevent hemoembolic pathologies of venous sources such as pulmonary embolism. They are used to prevent or treat thrombosis complications that occur during or with surgical intervention or with other pathologies such as cancer and bacterial or viral infections. When they are used during prosthetic installations, the compounds of the present invention coat the prostheses and make them blood compatible. In particular, they bind to endovascular repair (stents). In this case, they are chemically modified by introducing an appropriate arm onto the reducing or non-reducing end, optionally as described in EP 649,854.
The compounds of the present invention are also used as adjuvants during arterioplasty with small porous instruments.
The compounds of the present invention are very suitable and are particularly suitable for forming active ingredients of pharmaceuticals.
According to another aspect thereof, the subject of the present invention is a pharmaceutical composition containing a synthetic polysaccharide as an active ingredient as defined above.
The present invention preferably comprises as an active ingredient one of the compounds of the formula (1), (1-1), (1-2) or (1-3) or a pharmaceutically acceptable salt thereof, optionally in combination with one or more inert and suitable excipients It relates to a pharmaceutical composition containing.
In each dosage unit, the active ingredient is present in an amount suitable for the daily dose. In general, each dosage unit is a drop, subcutaneous or mucous form, such as tablets, gelatin capsules, small bags, ampoules, syrups, etc., so that one dosage unit contains 0.1 to 100 mg, preferably 0.5 to 50 mg of active ingredient. It is conveniently adjusted according to the dosage form and dosage as the patch.
The compounds according to the invention are useful for the desired therapy, for example as antithrombicides, anticoagulants or platelet-anticoagulants, such as, for example, antagonists of dipyridamole, aspirin, ticlopidine, clopidogrel or glycoprotein IIb / IIIa complexes. Used in combination with other active ingredients.
Pharmaceutical compositions are formulated for administration to mammals, including humans, for the treatment of such diseases.
The pharmaceutical compositions thus obtained are advantageously in various forms such as, for example, injectable or drinkable solutions, tablets, coated tablets or gelatin capsules. Injectable solutions are the preferred pharmaceutical form. The pharmaceutical composition of the present invention prevents or prevents medical conditions of blood vessel walls such as high coagulation states, atherosclerosis, observed after surgery, such as cancer development or deregulation of coagulation induced by bacterial, viral or enzymatically active agents. It is particularly useful for treating. Dosages vary widely depending on the age, weight and condition of the patient, the nature and severity of the disease and the route of administration. The administration comprises administration in one or more doses of 0.1 mg to 100 mg per day, preferably 0.5 mg to 50 mg per day, intramuscularly or subcutaneously, administered continuously or at regular intervals.
Subjects of the present invention are also pharmaceutical compositions containing, as an active ingredient, one of the compounds optionally combined with another active ingredient. The composition is prepared for administration by the digestive or parenteral route.
In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, subcutaneous, intramuscular, topical or rectal administration, the active ingredient is mixed with standard pharmaceutical excipients in animal and Administered to humans. Suitable unit forms of administration include oral forms such as tablets, gelatin capsules, powders, granules and oral suspensions or solutions, sublingual and oral dosage forms, subcutaneous, intramuscular, intravenous, intraocular or intraocular and rectal dosage forms. Include.
When the solid composition is prepared in tablet form, the main active ingredient is mixed with pharmaceutical excipients such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like. Tablets are coated with sucrose or other suitable material or are treated to selectively maintain or delay activity, and to continuously release a predetermined amount of active ingredient.
Preparation into gelatin capsules is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard gelatin capsules.
Powders or granules dispersed in water contain an active ingredient mixed with a dispersing or wetting agent or suspending agent such as polyvinylpyrrolidone as well as a sweetening or flavoring sensitizer.
For rectal administration, suppositories prepared with a binder that melts at the rectal temperature, such as cocoa butter or polyethylene glycol, are used.
For parenteral, nasal or eye administration, sterils, injectable solutions, isotonic saline solutions or aqueous suspensions containing pharmacologically compatible dispersants and / or wetting agents such as propylene glycol or butylene glycol are used.
For intramuscular administration, the active ingredient is for example hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectin, starch, gelatin, Formulation in the presence of hydrophilic polymers such as casein, acrylic acid, acrylic esters and copolymers thereof, vinyl polymers or copolymers, vinyl alcohols, alkoxypolymers, polyethylene oxide polymers and polyethers or mixtures thereof or in the presence of accelerators such as bile salts do.
The active ingredient is also optionally combined with one or more excipients or additives in the form of microcapsules.
The active ingredient is also in the form of a complex with a cyclodextrin such as α-, β- or γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin or methyl-β-cyclodextrin.
The active ingredient is also released by a vascular dilator introduced into the vessel or by a small device containing it. The pharmacological efficacy of the active ingredient is not adversely affected.
Subcutaneous administration is the preferred route.
The following methods, preparations and schemes illustrate the synthesis of various intermediates useful for obtaining the polysaccharides according to the invention.
The following examples also illustrate without limiting the invention.
The following abbreviations are used:
TBDMS: tert-butyldimethylsilyl; Lev: levulinyl; Bn: benzyl; Bz: benzoyl; TLC: thin layer chromatography; Olm: trichloroacetimidyl; LSIMS: liquid secondary ion gravimetry; ESIMS: electron spray ionization gravimetry; TMS: trimethylsilyl; TSP: sodium trimethylsilyltetradeutriopionate; Tf: triplicate; MS: molecular sieve; All: allyl; PMB: p-methoxybenzyl; SE: trimethylsilylethyl.
Dowex (trade name), Sephadex (trade name), Chelex (trade name), and Toyopearl (trade name) are registered trademarks.
In the above methods, preparations and examples below, catalysis of imidate, removal of levulinic acid esters, catalysis of thioglycosides, saponification, methylation and selective deprotection of p-methoxybenzyl groups, benzyl ethers Or the general procedure involving deprotection and sulfation of oligosaccharides and polysaccharides by hydrogenolysis of esters, saponification or antioxidant of esters is carried out in suitable intermediates by applying the following general methods.
Common way
Method 1. Binding with imidate catalyzed by tert-butyldimethylsilyl triflate
Tertiary-butyldimethylsilyl triflate solution (1M, imidate 0.2 mol / mol) in dichloromethane in -20 ° C argon atmosphere, in the presence of 4 Å molecular sieve, solution of imidate and glycosyl receptor in dichloromethane (17.5 ml / mmol). After 10-20 minutes (TLC), solid sodium bicarbonate is added. The solution is filtered, washed with water, dried and evaporated to dry.
Method 2. Removal of Levulinic Acid
The deprotected compound is dissolved in a 2/1 ethanol / toluene mixture (42 mL / mmol) and hydrazine acetate (5 mol / mol) is added. The mixture is stirred for 15-30 minutes (TLC) and concentrated.
Method 3. Binding with Thioglycosides Catalyzed by N-iosuccinimide / Sil Triplelate
Thioglycoside and glycosyl acceptor are dissolved in anhydrous toluene (18 mL / mmol) in the presence of a 4mm molecular sieve in a round bottom flask made of inert glass. The mixture is stirred at room temperature for 1 hour. It is cooled to 0 ° C., N-iodosuccinimide (thioglycoside 3 mol / mol) is added, followed by silver triflate (thioglycoside 0.28 mol / mol). After 10-15 minutes (TLC), solid sodium bicarbonate is added. After filtration, the solution is washed with an aqueous 1M sodium thiosulfate solution, water, dried and evaporated.
Method 4. Saponification, methylation and selective deprotection of p-methoxybenzyl groups
Saponification of esters. The saponified compound is dissolved in a 1/1 dichloromethane / methanol mixture (4 mL / mmol). Sodium methoxide is added and the mixture is stirred for 20 minutes and neutralized with 50 H ⁺ Dowex ™ resin. The solution is concentrated and the compound is used without purification.
Methylation. Sodium hydride is added dropwise at 0 ° C. and added to the mixture of the crude product and methyl iodide (7 mL / mmol) in N, N-dimethylformamide. After complete reaction, the mixture is poured into water and extracted with ethyl acetate. The organic phase is washed with water, dried and evaporated to dryness.
removal of p-methoxybenzyl. The crude compound is dissolved in a 9/1 acetonitrile / water mixture (20 mL / mmol). At 0 ° C., ammonium cerium nitrate (0.5 mol / mol) is added. The reaction mixture is stirred for 2 hours (measured by TLC), saturated sodium bicarbonate solution is added, the mixture is extracted with ethyl acetate, dried and evaporated.
Method 5. Deprotection and Sulfation of Oligosaccharides and Polysaccharides
Hydrolysis of Benzyl Ethers and Benzyl Esters. The compound solution in acetic acid is stirred for 6-12 hours (TLC) under hydrogen atmosphere (40 bar) in the presence of a 5% Pd / C catalyst (2 times the compound weight). After filtration, the product is used directly in the next step.
Saponification of esters. Aqueous 5M sodium hydroxide solution (in an amount such that the concentration of sodium hydroxide is 0.5M at the end of the addition) is added to the ester solution in methanol (150 mL / mmol). After 2-5 hours, water is added and the mixture is passed through a Sephadex ™ G-25 gel (1.6 × 115 cm) column eluted with water. The eluate is concentrated, passed through Dowex ™ 50H ⁺ column (2 mL) and lyophilized. In this step, the fact that all protecting groups are removed is confirmed by ¹ H NMR. If necessary, the product enters further hydrogenation and / or saponification.
Sulfated. Triethylamine / sulfur trioxide complex (hydroxyl functional 5 mol / mol) is added to the solution of the sulfated compound in dimethylformamide (5 mg / ml). After 1 day at 55 ° C., the solution is placed on top of a Sephadex ™ G-25 column (1.6 × 115 cm) and eluted with 0.2 M sodium chloride. The fraction containing the product is concentrated and desalted using the same column eluted with water. The final compound is obtained after lyophilization.
Synthesis of Monosaccharide 6 and Monosaccharide 9
Preparation Example 1
Ethyl 2,4,6-tri-O-methyl-3-O-methyl-1-thio-β-D-glucopyranoside (2)
1,2,4,6-tetra-O-acetyl-3-O-methyl-β-D-glucopyranose 1 (69 g, 0.19 mmol) (J. prakt. Chem., 132, 321 by B. Helferich et al. (1932)) is dissolved in toluene (580 mL). Ethanethiol (28 mL, 0.38 mmol) was added and a trifluoroborane diethyl etherate (1M in toluene, 190 mL) dropwise added. The mixture was stirred for 1.5 h (TLC) and solid sodium hydrogen carbonate was added and the mixture was filtered, washed with water, dried and concentrated. Chromatography on a silica (3/1 cyclohexane / ethyl acetate) column yields 2 (37 g, 54%). [a] _D -26 (c = 1. dichloromethane). ¹ H NMR (CDCl ₃ ). δ 5.05-4.96 (m, 2H, H-2, H-4), 4.39, (d, 1H, J = 9.5 Hz, H-1), 4.18-4.12 (m, 2H, H-6, H-6 '), 3.60 (m, 1H, H-5), 3.50 (dd, 1H, J = 9.3 Hz, H-3) 3.41 (s, 3H, OCH ₃ ), 2.65-2.53 (m, 2H, SCH ₂ CH ₃ ), 2.12, 2.11, 2.09, (3s, 9H, 3Ac), 1.25 (t, 1H, SCH ₂ CH ₃ ).
Preparation Example 2
Ethyl 4,6-O-benzylidene-3-O-methyl-1-thio-β-D-glucopyranoside (3)
Compound 2 (37 g, 0.1 mmol) is dissolved in a 1/2 mixture (1.5 L) of methanol and dichloromethane. 2M sodium methoxide solution (150 mL) is added. After 0.5 h at rt, the mixture is neutralized with Dowex ™ 50 (H ⁺ ) resin, filtered and concentrated.
The crude compound was dissolved in anhydrous acetonitrile (1 L), and α, α-dimethoxytoluene (30 mL, 0.2 mol) and camphorsulfonic acid (2.3 g, 10 mmol) were added. The mixture is stirred for 1.5 hours (TLC), triethylamine (1.4 mL) is added and the mixture is concentrated. The residue obtained is precipitated in ethyl ether and 3 is obtained (27 g, 81%). [α] _D -60 (c = 1.63, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.51-7.34 (m, 5H, Ph), 5.55 (s, 1H, C ₆ H ₅ CH), 4.56 (d, 1H, J = 9.2 Hz, H-1), 2.75 (m, 2H, SCH ₂ CH ₃ ), 1.32 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculation for C ₁₆ H ₂₂ O ₅ S (326.41): C, 58.58; H, 6.79; S, 9.82.
Found: C, 58.99; H, 6. 74; S, 9.75.
Preparation Example 3
Ethyl 2-O-benzyl-4,6-O-benzylidene-3-O-methyl-1-thio-β-D-glucopyranoside (4)
At 0 ° C., sodium hydride (2.00 g, 83.3 mmol) was added to a solution of benzyl bromide (11 mL, 93.0 mmol) in 3 (23 g, 71.0 mmol) and N, N-dimethylformamide (200 mL). The mixture is stirred for 2 hours (TLC), methanol is added and the reaction mixture is poured into water. It is extracted with ethyl acetate, washed with water, dried and concentrated. To afford 4 (18.8 g, 63%), the residue is precipitated in ethyl ether. mp 123 ° C. [α] _D -35 (c = 0.63, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.50-7.25 (m, 10H, 2Ph), 5.55 (s, 1H, C ₆ H ₅ CH), 4.54 (d, 1H, J = 9.7 Hz, H-1), 4.34 (m, 1H, H-6 ), 3.75 (t, 1H, J = 10.2 Hz, H-6 '), 3.65 (s, 3H, OCH ₃ ), 3.60-3.33 (m, 4H, H-5, H-4, H-3, H -2), 2.75 (m, 2H, SCH ₂ CH ₃ ), 1.32 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculation for C ₂₃ H ₂₈ O ₅ S (416.54): C, 66.32; H, 6. 78; S, 7.70.
Found: C, 66.25; H, 7. 28; S, 7.54.
Preparation Example 4
Ethyl 2,6-di-O-benzyl-3-O-methyl-1-thio-β-D-glucopyranoside (5)
Trifluoroacetic anhydride solution (0.65 mL, 4.50 mmol) in trifluoroacetic acid (16 mL, 0.21 mmol) was added to triethylsilane (33 in 4 (28.8 g, 69.0 mmol) and dichloromethane (120 mL) under an argon atmosphere. Ml, 0.21 mmol). The mixture is stirred for 2 hours, eluted with ethyl acetate, and added with aqueous 1M sodium hydroxide solution to pH 9. The mixture is extracted with ethyl acetate, washed with water, dried and evaporated to dryness. To obtain 5 (17.4 g, 60%), the residue is purified on a silica column (3/1 and then 2/1 cyclohexane / ethyl acetate). [α] _D -47 (c = 1, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.45-7.25 (m, 10H, 2Ph), 4.47 (d, 1H, J = 9.3 Hz, H-1), 3.66 (s, 3H, OCH ₃ ), 3.61-3.40 (m, 2H, H-4 and H-5), 3.36-3.19 (m, 2H, H-2 and H-3), 2.73 (m, 2H, SCH ₂ CH ₃ ), 1.31 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₂₃ H ₃₀ O ₅ S (418.55): C, 66.00; H, 7.22; S7.66. Found: C, 65.62; H, 7. 28; S, 7.21.
Preparation Example 5
Ethyl 2,6-O-di-benzyl-4-O-levulinyl-3-O-methyl-1-thio-β-D-glucopyranoside (6)
Compound 5 (17.3 g, 41.4 mmol) is dissolved in anhydrous dioxane (400 mL). Revulinic acid (9.60 g, 83.0 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (16 g, 86 mmol) and 4-dimethylaminopyridine (1 g, 8.3 mmol) are added. The mixture is stirred for 4 hours, extracted with ethyl acetate, washed successively with aqueous 5% potassium hydrogen sulfide solution, water, saturated aqueous sodium hydrogen carbonate solution, water, dried and concentrated. To obtain pure 6 (19,9 g, 93%), the residue is purified on a silica (6/1 toluene / ethyl acetate) column. [a] _D -5 (c = 1.46, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 539 (M + Na) ⁺ ; Thioglycerol + KF, 555 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.40-7.20 (m, 10H, 2Ph), 4.92 (m, 1H, H-4), 2.8-2.4 (m, 6H, SCH ₂ CH ₃ and O (C: O) CH ₂ CH ₂ (C: O ) CH ₃ ), 2.16 (s, 3H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.32 (t, 1H, J = 7.3 Hz, SCH ₂ CH ₃ ).
Analyzes by calculations for C ₂₈ H ₃₆ O ₇ S (516.65): C, 65.09; H, 7.02; S, 6.21. Found: C, 65.30; H, 7.03; S, 5.75.
Preparation Example 6
Allyl 4,6-O-benzylidene-3-O-methyl-β-D-glucopyranoside (7)
To a commercial suspension of 3-O-methylglucose (135 g, 0.7 mol) in allyl alcohol (1 L) is added trifluoromethanesulfonic acid (1.10 ml, 0.012 mol). The mixture is heated at 120 ° C. for 2 hours. Triethylamine (2 mL) was added to neutralize, evaporated to dryness.
To the crude compound dissolved in N, N-dimethylformamide (2 L) was added α, α-dimethoxytoluene (136 mL, 0.9 mol) and camphorsulfonic acid (25 g, 0.13 mmol). The mixture is heated in vacuo at 80 ° C. for 1 h. To obtain solid α / β mixture = 3/2 (144 g, 57%), triethylamine (21 mL) is added to neutralize, extracted with ethyl acetate, washed with water, dried and concentrated. The mixture is recrystallized from ethanol to obtain pure 7-α (60 g, 26%). A portion of the mother liquor is chromatographed on a silica column (3/1 cyclohexane / ethyl acetate) to give pure 7-β (7.6 g), 7-α / β (6.8 g) and pure 7-α (1.4 g).
Compound 7-β: [α] _D -43 (c = 1, dichloromethane). mp: 131 ° C. ¹ H NMR (CDCl ₃ ). δ 7.50-7.26 (m, 5H, Ph), 6.01-5.90 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.55 (s, 1H, C ₆ H ₅ CH), 5.38-5.32 (m, 2H , OCH ₂ (CH: CH ₂ )), 4.47 (d, 1H, J = 7.5 Hz, H-1), 4.42-4.32 (m, 2H, H-6 ′ and OCH ₂ (CH: CH ₂ )), 4.21 → 4.15 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.80 (dd, 1H, J = 10.2 Hz, H-6), 3.67 (s, 3H, OCH ₃ ).
Analysis by calculation for C ₁₇ H ₂₂ O ₆ (322.36): C, 63.34; H, 6.88. Found: C, 63.23; H, 7.12.
Preparation Example 7
Allyl-2-O-acetyl-4,6-O-benzylidene-3-O-methyl-β-glucopyranoside (8)
7 (11.5 g, 35.7 mmol) was dissolved in dichloromethane (100 mL), acetic anhydride (4.0 mL, 42.8 mmol), triethylamine (6.40 mL, 46.4 mmol) and 4-dimethylaminopyridine (440 mg, 3.60 mmol) is added. The mixture was stirred for 2 hours (TLC), washed successively with aqueous 5% potassium hydrogen sulfide solution, water, saturated aqueous sodium hydrogen carbonate solution, water, dried and evaporated until solid 8 (12.3 g, 95%) was obtained. Let's do it. mp 115 ° C. [α] _D -68 (c = 1, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 387 (M + Na) ⁺ ; Thioglycerol + KF, 403 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.51-7.34 (m, 5H, Ph), 5.98-5.78 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.56 (s, 1H, C ₆ H ₅ C ₆ H ₅ CH), 5.32-5.17 (m, 2H, OCH ₂ (CH: CH ₂ )), 4.99 (dd, J = 8 Hz, 1H, H-2), 4.55 (d, J = 7.9 Hz, 1H, H-1), 4.39-4.29 ( m, 2H, H-6 and OCH ₂ (CH: CH ₂ )), 4.14-4.04 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.82 (t, J = 10.2 Hz, 1H, H-6 '), 3.60 (s, 3H, OCH ₃ ), 2.12 (s, 3H, Ac).
Analysis by calculation for C ₁₉ H ₂₄ O ₇ (366.39): C, 62.63; H, 6.64. Found: C, 62.63; H, 6.64.
Preparation Example 8
Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranoside (9)
Trifluoroacetic anhydride solution (306 μl, 2.10 mmol) in trifluoroacetic acid (10 mL) was added with 8 (12.0 g, 33.3 mmol) and triethylsilane (21.3 mL, anhydrous dichloromethane (50 mL) at 0 ° C. 133 mmol). The mixture is stirred for 4 h (TLC), eluted with ethyl acetate and pH 9 by addition of aqueous 1 M sodium hydroxide solution. The mixture is extracted with ethyl acetate, washed with water, dried and concentrated. To obtain pure 9 (10 g, 82%), the residue is purified on a silica column (8/5 cyclohexane / acetone). [α] _D- 40 (c = 1.06, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.35-7.28 (m, 5H, 2Ph), 5.87-5.79 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.28-5.14 (m, 2H, OCH ₂ (CH: CH ₂ )), 4.43 ( d, 1H, J = 7.9 Hz, H-1), 4.41-4.28 (m, 1H, OCH ₂ (CH: CH ₂ )), 4.10-4.02 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.77-3.75 (m, 2H, H-6 and H-6 '), 3.51 (s, 3H, OCH ₃ ), 3.30 (dd, 1H, J = 8.9 Hz, H-3), 2.8 (d, 1H, OH).
Analysis by calculation for C ₁₉ H ₂₆ O ₇ (366.39): C, 62.28; H, 7.15. Found: C, 61.73; H, 7.19.
Synthesis of Disaccharide 13 and Disaccharide 14
Preparation Example 9
Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-4-O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D -Glucopyranosyl) -β-D-glucopyranoside (10)
Thioglycoside 6 (17.4 g, 33.7 mmol) and glycosyl receptor 9 (10.3 g, 28.1 mmol) are dissolved in dichloromethane (150 mL). 4 kM molecular sieve is added and the mixture is stirred for 1 hour. N-iodosuccinimide (8.30 g, 33.7 mmol) and trifluoromethanesulfonic acid (0.30 mL, 3.30) in a mixture of dichloroethane and ethyl ether (415 mL, 1: 1) at -20 ° C. under argon atmosphere. mmol) is added. The mixture is stirred for 10 minutes (TLC), sodium hydrogen carbonate is added, the mixture is filtered, washed successively with aqueous 1M sodium thiosulfate solution, water, saturated aqueous sodium hydrogen carbonate solution, water, dried and concentrated. . The residue is purified on silica column (11/1 dichloromethane / ethyl acetate) to obtain pure disaccharide 10-α (11.7 g, 52%). [α] _D +38 (c = 1.01, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.35-7.23 (m, 15H, 3Ph), 5.90-5.80 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.47 (d, 1H, J = 3.6 Hz, H-1 ′), 5.27-5.14 (m, 2H, OCH ₂ (CH: CH ₂ )), 5.05-4.90 (m, 2H, H-4 'and H-2), 4.42 (d, 1H, J = 7.6 Hz, H-1), 4.38 -4.32 (m, 1H, OCH ₂ (CH: CH ₂ )), 4.15-4.0 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.90 (dd, 1H, J = 8.8 Hz, H-4) , 3.54, 3.34 (2s, 6H, 2OCH ₃ ), 2.75-2.40 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.10 (2s, 6H, Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculation for C ₄₅ H ₅₆ O ₁₄ (820.94): C, 65.84; H, 6.88. Found: C, 65.74; H, 6.90.
Preparation Example 10
Prope-1'-Nyl-2-O-acetyl-6-O-benzyl-3-O-methyl-4-O- (2,6-di-O-benzyl-4-O-levulinyl-3- O-methyl-α-D-glucopyranosyl) -β-D-glucopyranoside (11)
1,5-cyclooctadienebis [methyldiphenylphosphine] iridium hexafluorophosphate (5.80 mg, 0.70 μmol) was added to 10 solutions (1.36 g, 1.66 mmol) in peroxide-free tetrahydrofuran (4.30 mL). Add. The solution is degassed, placed under argon atmosphere and injected with hydrogen. The mixture is stirred for 10 minutes (TLC) and evaporated. The residue is taken up with dichloromethane, washed with saturated aqueous sodium hydrogen carbonate, water, dried and concentrated. The residue is purified on silica column (3/1 toluene / ethyl acetate) to get pure 11 (1.04 g, 76%). [α] _D +47 (c = 1.1, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 951 (M + Na) ⁺ ; Thioglycerol + KF, 967 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.34-7.23 (m, 15H, 3Ph), 6.21-6.16 (m, 1H, O (CH: CH) CH ₃ ), 5.45 (d, 1H, J = 3.5 Hz,, H-1 ′), 5.13- 4.97 (m, 3H, H-4 ', H-2 and O (CH: CH) CH ₃ ), 4.6 (d, 1H, J = 7.55 Hz, H-1), 3.96 (dd, 1H, J = 8.9 Hz, H-4 '), 3.54, 3.34 (2s, 6H, 2CH ₃ ), 2.74-2.36 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.15, 2.08 , (2s, 6H, Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.56-1.51 (dd, 3H, O (CH: CH) CH ₃ ).
Analysis by calculation for C ₄₅ H ₅₆ O ₁₄ (820.94): C, 65.84; H, 6.88. Found: C, 66.21; H, 6.92.
Preparation Example 11
2-O-Acetyl-6-O-benzyl-3-O-methyl-4-O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D- Glucopyranosyl) -α, β-D-glucopyranose (12)
A solution of mercuric chloride (3.9 g, 14.3 mmol) in a mixture of acetone and water (26 mL, 5/1) was added dropwise to a solution of mercury oxide and 11 (7.8 g, 9.53 mmol) in the same solvent (80 mL). The mixture is stirred for 1 hour, filtered and concentrated. It is extracted with dichloromethane and the extract is washed with saturated aqueous potassium iodide solution, water, dried and concentrated. To obtain 12 (6.70 g, 90%), the residue is purified on silica column (10/1 and then 4/1 dichloromethane / acetone).
[α] _D +92 (c = 1.37, dichloromethane). TLC, R _F 0.31, 14/1 dichloromethane / acetone. ¹ H NMR (CDCl ₃ ). δ 7.37-7.24 (m, 15H, 3Ph), 5.46 (d, 1H J = 3.5 Hz, H-1 ′), 5.37 (d, J = 3.6 Hz, H-1α), 4.58 (d, J = 8 Hz, H-1β), 3.54 3.39, 3.36 (3s, 6H, 2OCH ₃ ), 2.75-2.4 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.15 (2s , 6H, Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.15 (2s, 6H, Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculation for C ₄₂ H ₅₂ O ₁₄ (780.83): C, 64.60; H, 6.71
Found: C, 65.09; H, 6.82.
Preparation Example 12
2-O-Acetyl-6-O-benzyl-3-O-methyl-4-O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D- Glucopyranosyl) -α, β-D-glucopyranose trichloroacetimidadate (13)
Compound 12 (5.11 g, 6.4 mmol) was dissolved in dichloromethane (50 mL) and trichloroacetonitrile (3.9 mL, 38.8 mmol) and potassium carbonate (1.6 g, 11.6 mmol) were added under argon. The mixture is stirred for 16 h (TLC) and filtered. To obtain a mixture of imidate 13 (5.22 g, 87%) (α / β = 60/40), the filtrate is purified on a silica column (8/1 and then 4/1 dichloromethane / acetone). TLC, R _F 0.66 and 0.51, 20/1 dichloromethane / acetone. ¹ H NMR (CDCl ₃ ). δ 8.62-8.59 (2s, 1H, N: H-α and β), 7.37-7.23 (m, 15H, 3Ph), 6.51 (d, 1H J = 3.7 Hz, H-1α), 5.81 (d, J = 7.1 Hz, H-1β), 5.50 (d, 1H, J = 3.5 Hz, H-1 '), 3.55, 3.41, 3.37 (3s, 9H, 3OCH ₃ ), 2.75-2.40 (m, 4H, O (C : O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.07, 2.04 (3s, 6H, Ac and (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculations for C ₄₄ H ₅₂ Cl ₃ NO ₁₄ (925.26): C, 57.12; H, 5. 66; N, 1.51. Found: C, 57.31; H, 5.87; N, 1.55.
Preparation Example 13
Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-4-O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl) -β -D-glucopyranoside (14)
To obtain 14 (2.70 g, 97%), Compound 10 (3.11 g, 3.80 mmol) was treated according to Method 2. [α] _D +25 (c = 1.7, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 745 (M + Na) ⁺ ; Thioglycerol + KF, 761 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.33-7.20 (m, 15H, 3Ph), 5.87-5.78 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.50 (d, 1H, J = 3.5 Hz,, H-1 ′), 5.30- 5.17 (m, 2H, OCH ₂ (CH: CH ₂ )), 5.02 (dd, 1H, H-2), 4.43 (d, 1H, J = 7.6 Hz, H-1), 4.34-4.28 (m, 1H , OCH ₂ (CH: CH ₂ )), 4.12-4.02 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.63, 3.36 (2s, 6H, 2OCH ₃ ), 2.10 (s, 3H, Ac).
Analyzes by calculations for C ₄₀ H ₅₀ O ₁₂ (722.84): C, 66.47; H, 6.97. Found: C, 66.31; H, 7.24.
Synthesis of Oligosaccharide 19
Preparation Example 14
Allyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D -Glucopyranosyl)-(1 → 4) -2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranoside (15)
A mixture of imidate 13 (4.22 g, 4.56 mmol) and glycosyl receptor 14 (2.63 g, 3.64 mmol) is treated according to Method 1. To obtain tetrasaccharide 15 (4.31 g, 80%), the product is purified on a silica column (3/2 and then 1/1 toluene / ethyl ether). [α] _D +52 (c = 0.66, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.35-7.23 (m, 30H, 6Ph), 5.83-5.79 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.47 (d, 2H J = 3.5 Hz, H-1 ′ '' and H-1 '), 5.25-5.14 (m, 2H, OCH ₂ (CH: CH ₂ )), 4.38 (d, 1H, J = 7.7 Hz, H-1 "), 4.30 (d, 1H, J = 8 Hz, H- 1), 4.32-4.25 (m, 1H, OCH ₂ (CH: CH ₂ )), 4.08-4.02 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.56, 3.53, 3.34, 3.27 (4s, 12H) , 4OCH ₃ ), 2.78-2.40 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.15, 2.09, 1.85 (3s, 9H, 2Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculation for C ₈₂ H ₁₀₀ O ₂₅ (1485.7): C, 66.29; H, 6.78. Found: C, 66.10; H, 6.79.
Preparation Example 15
O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2-O-acetyl- 6-O-Benzyl-3-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D- Glucopyranosyl)-(1 → 4) -2-O-acetyl-6-O-benzyl-3-O-methyl-α, β-D-glucopyranose (16)
Compound 15 (2.30 g, 1.54 mmol) was treated according to Preparation Example 10. After 10 minutes, a solution of N-bromosuccinimide (0.30 g, 1.70 mmol) and water (5.50 mmol) in dichloromethane (15 mL) was added to the reaction mixture. The mixture is stirred for 5 minutes (TLC). It is diluted with dichloromethane and washed with saturated aqueous sodium hydrogen sulfide solution, water, dried and concentrated. The residue is purified on silica column (3/2 toluene / ethyl acetate) to get pure 16 (1.57 g, 71% in two or more steps). [α] _D +69 (c = 0.87, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.38-7.20 (m, 30H, 6Ph), 5.47 (d, 1H J = 3.5 Hz, H-1 ′ '' and H-1 ′), 5.36 (d, 1H, J = 3.5 Hz, H-1α) , 4.55 (d, 1H, J = 8 Hz, H-1), 4.36 (d, 1H, J = 8 Hz, H-1 "), 3.56, 3.54, 3.39, 3.36, 3.28 (5s, 9H, 3OCH ₃ ), 2.75-2.35 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.13, 2.12, 1.86 (4s, 9H, 2Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Preparation Example 16
O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2-O-acetyl- 6-O-Benzyl-3-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D- Glucopyranosyl)-(1 → 4) -2-O-acetyl-6-O-benzyl-3-O-methyl-α, β-D-glucopyranos trichloroacetimidadate (17)
A mixture of 16 (1.5 g, 1.04 mmol), trichloroacetonitrile (0.63 mL, 6.22 mmol) and potassium carbonate (0.26 g, 1.87 mmol) in dichloromethane (15 mL) is stirred at room temperature for 16 hours. The solution is filtered and concentrated. Purification on silica column (triethylamine 4/1 toluene / acetone + 1 ‰) to obtain 17 (1.47 g, 89.6%). TLC, R _F 0.5 7/2 toluene / acetone.
Preparation Example 17
Allyl O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2-O-acetyl-6-O-benzyl- 3-O-Methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl)-( 1 → 4) -2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranoside (18)
To obtain 18 (1.05 g, 86%), delevulination of 15 (1.3 g, 0.87 mmol) is carried out according to Method 2. [α] _D +40 (c = 0.6, dichloromethane). ¹ H NMR (CDCl ₃ ). δ 7.36-7.23 (m, 30H, 6Ph), 5.83-5.78 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.50 (d, 1H, J = 3.5 Hz, H-1 ′ ''), 5.47 (d, 1H, J = 3.5 Hz, H-1 '), 5.25-5.21 (dd, 1H, J = 1.6 Hz, J = 17 Hz, OCH ₂ (CH: CH ₂ )), 5.16-5.13 (dd, 1H , J = 1.4 Hz, J = 10 Hz, OCH ₂ (CH: CH ₂ )), 4.38 (d, 1H, J = 6.5 Hz, H-1 "), 4.31 (d, 1H, J = 6.5 Hz, H- 1), 4.08-4.02 (m, 1H, OCH ₂ (CH: CH ₂ )), 3.59 (m, 1H, H-4 '''), 3.67, 3.53, 3.39, 3.29 (4s, 12H, 4OCH ₃ ) , 2.09, 1.86 (2s, 6H, 2Ac).
Preparation Example 18
Allyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1 → 4)-[O- (2-O- Acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,6-di-O-benzyl-3-O-methyl-α- D- glucopyranosyl) - (1 → 4)] 3 -2-O- acetyl -6-O- benzyl -3-O- methyl -β-D- glucopyranoside (19)
A mixture of 18 (842 mg, 0.53 mmol) and 17 (1.17 g, 0.74 mmol) is treated according to Method 1. To obtain 19 (1.44 g, 85%), the product is purified on a Toyopearl® HW-50 column (110 x 3.2 cm; 1/1 dichloromethane / ethanol). [α] _D +57 (c = 1.01, dichloromethane). ¹ H NMR (CDCl ₃ ). d 7.35-7.20 (m, 60H, 12Ph), 5.83-5.78 (m, 1H, OCH ₂ (CH: CH ₂ )), 5.24-5.21 (dd, 1H, OCH ₂ (CH: CH ₂ )), 5.16- 5.13 (dd, 1H, OCH ₂ (CH: CH ₂ )), 3.59, 3.56, 3.51, 3.47, 3.33, 3.26 (6s, 24H, 8OCH ₃ ), 2.75-2.35 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.15, 2.09, 1.85, 1.84 (4s, 15H, 4Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ );
Delta of the major anomer protons: 5.48; 4.37; 4.29; 4.23 ppm.
Analysis by calculations for C ₁₅₆ H ₄₇ O ₁₈₈ (2815.51): C, 66.56; H, 6.73. Found: C, 66.22; H, 6.75.
Synthesis of Polysaccharide 29
Preparation Example 19
O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D - _{glucopyranosyl) - (1-4)] 3 -2} -O- acetyl -6-O- benzyl -3-O- methyl -α, β-D- glucopyranose (20)
Compound 19 (720 mg, 0.25 mmol) was treated as in Preparation Example 15. The product is purified on silica column (3/2 and then 4/3 toluene / ethyl acetate) to give 20 (555 mg, 78%). [a] _D +70 (c = 0.94, dichloromethane). TLC, R _F 0.43, 1/1 toluene / ethyl acetate.
Preparation Example 20
O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4) -O- (2,6-di-O-benzyl-3-O-methyl-α-D - _{glucopyranosyl) - (1-4)] 3 -2} -O- acetyl -6-O- benzyl -3-O- methyl -α, β-D- gluconic nose acetamido imidate (21 to Lanos trichloromethyl)
Compound 20 (540 mg, 0.195 mmol) was treated as in Preparation Example 16. The product is purified on a silica column (3/2 toluene / ethyl acetate + triethylamine 1 ‰) to obtain a mixture of 21 (455 mg, 80%) (α / β = 27/73). TLC, R _F 0.48, 3/2 toluene / ethyl acetate. ¹ H NMR (CDCl ₃ ). δ 8.60, 8.59 (2s, 1H, N: Hα and β), 7.35-7.21 (m, 60H, 12Ph), 2.75-2.40 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.26, 2.06, 2.04, 1.85, 1.84 (5s, 15H, 4Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
¹ H NMR of the major anomer protons (CDCl ₃ ). delta: 6.50; 5.79; 5.51; 5.48, 4.29; 4.25 ppm.
Synthesis of Oligosaccharide 28
Preparation Example 21
Phenyl 2,4,6-tri-O-acetyl-3-O-methyl-1-thio-α-D-glucopyranoside (22)
1,2,4,6-Tetra-O-acetyl-3-O-methyl-β-D-glucopyranose 1 (5.23 g, 14.4 mmol) is dissolved in toluene (45 mL). Thiophenol (3.0 mL, 28.8 mmol) is added, trifluoroborane diethyl etherate (1.77 mL, 14.4 mmol) is added dropwise, and the mixture is heated at 50 ° C. for 0.5 hour. It is diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution, water, dried and concentrated. The residue is purified on silica column (5/2 cyclohexane / ethyl acetate) to yield 22-α (1.00 g, 17%) and 22-β (2.71 g, 46%).
22-α, R _F 0.44, 3/2 cyclohexane / ethyl acetate. [a] _D +230 (c = 1, dichloromethane). ESIMS, positive mode: m / z + NaCl, 435 (M + Na) ⁺ ; + KF, 451 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.46-7.27 (m, 5H, Ph), 5.89 (d, 1H, J = 5.6 Hz, H-1), 5.05-4.97 (m, 2H, H-2 and H-4), 4.49-4.42 (m , 1H, H-5), 4.25-4.18 (m, 1H, H-6), 4.05-4.00 (m, 1H, H-6 '), 3.66 (dd, 1H, J = 9.5 Hz, H-3) , 3.51 (s, 3H, OCH ₃ ), 2.16, 2.12, 2.00 (3s, 9H, 3Ac).
Analysis by calculation for C ₁₉ H ₂₄ O ₈ S (412.46): C, 55.33; H, 5.87; S, 7.77. Found: C, 55.25; H, 5. 90; S, 7.75.
Preparation Example 22
Phenyl 4,6-O-benzylidene-2,3-di-O-methyl-1-thio-α-D-glucopyranoside (23)
Compound 22 (970 mg, 2.35 mmol) was dissolved in a 2/1 mixture of methanol and dichloromethane (18 mL). 2M sodium methoxide solution (150 mL) is added. After 0.5 h at rt, neutralize with Dowex ™ 50 (H ⁺ ) resin, filter and concentrate.
α, α-dimethoxytoluene (0.7 mL, 40 mmol) and camphorsulfonic acid (51 mg, 0.22 mmol) are added to the crude reaction mixture in acetonitrile (22 mL). The mixture is stirred for 1 hour, neutralized by addition of triethylamine (0.50 mL) and concentrated.
Sodium anhydride (73.0 mg, 2.80 mmol) is added to a solution of methyl iodide (163 μl, 4.0 mmol) and the crude product in N, N-dimethylformamide (9 mL) at 0 ° C. The mixture is stirred for 1 hour and methanol is added. The mixture is extracted with ethyl acetate, washed with water, dried and concentrated to give 23 (840 mg, 94%) in solid form. mp: 178 ° C. [α] _D +330 (c = 1, dichloromethane). ESIMS, positive mode: m / z + NaCl, 411.4 (M + Na) ⁺ ; + KF, 427.4 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.50-7.24 (m, 10H, 2Ph), 5.71 (d, 1H, J = 3.4 Hz, H-1), 5.52 (s, 1H, C ₆ H ₅ CH), 3.62 (s, 3H, OCH ₃ ) , 3.55 (s, 3 H, OCH ₃ ).
Analysis by calculation for C ₂₁ H ₂₄ O ₅ S (388.48): C, 64.92; H, 6. 23; S, 8.25. Found: C, 64.87; H, 6. 17; S, 7.85.
Preparation Example 23
Phenyl 6-O-benzyl-2,3-di-O-methyl-1-thio-α-D-glucopyranoside (24)
Compound 23 (792 mg, 0.47 mmol) was treated as in Preparation Example 4. The product is purified on silica column (7/2 and then 2/1 cyclohexane / ethyl acetate) to give 24 (318 mg, 80%). [α] _D +243 (c = 1, dichloromethane). ESIMS, positive mode: m / z + NaCl, 413 (M + Na) ⁺ ; + KF, 429 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.52-7.22 (m, 10H, 2Ph), 5.71 (d, 1H, J = 5.3 Hz, H-1), 3.64 and 3.49 (2s, 6H, 20CH ₃ ), 3.36 (dd, 1H, H-3) .
Analyzes by calculations for C ₂₁ H ₂₆ O ₅ S (390.50): C, 64.59; H, 6.71; S, 8.21. Found: C, 64.05; H, 6.88; S, 7.74.
Preparation Example 24
Phenyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4) -6-O-benzyl-2,3-di-O-methyl-1-thio-α- D-glucopyranoside (25)
A mixture of 13 (436 mg, 0.47 mmol) and 24 (153 mg, 0.39 mmol) is treated according to Method 1. Purify the product on a column (Sephadex ™ LH20, 1/1 ethanol / dichloromethane) to give pure 25 (309 mg, 68%). [α] _D +144 (c = 1, dichloromethane). ESIMS, positive mode: m / z + NaCl, 1175 (M + Na) ⁺ ; + KF, 1191 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.51-7.21 (m, 25H, 5Ph), 5.73 (d, 1H, J = 5.2 Hz, H-1), 5.48 (d, 1H, J = 3.5 Hz, H-1 "), 4.46 (d, 1H , J = 8 Hz, H-1 '), 3.7, 3.54, 3.5, 3.31 (4s, 12H, 4OCH ₃ ), 2.70-2.41 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.16, 2.01 (2s, 6H, 1Ac, and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculations for C ₆₃ H ₇₆ O ₁₈ S: C, 65.61; H, 6.64, S, 2.78. Found: C, 65.02; H, 6. 60; S, 2.72.
Preparation Example 25
Methyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4) -O- (6-O-benzyl-2,3-di-O-methyl-α-D -Glucopyranosyl)-(1-4) -O- (benzyl 2,3-di-O-methyl-β-D-glucopyranosiluronate)-(1-4) -O- (3,6- Di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyl euro Nate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (27)
A solution of N-iosuccinimide (92 mg, 0.38 mmol) and triflic acid (37.5 μl, 0.38 mmol) in a 1/1 solution (22 mL) of 1,2-dichloroethane and ethyl ether A mixture of 25 (451 mg, 0.39 mmol) and 26 (434 mg, 0.31 mmol) in 1,2-dichloroethane (7.5 mL) in the presence of 4 cc molecular sieve (400 mg) at -25 ° C under argon (P. Westerduin). BioOrg, Med. Chem., 1994, 2, 1267). After 30 minutes, solid sodium bicarbonate was added. The solution is filtered, washed with sodium thiosulfate solution, water, dried and evaporated. Purify the residue on a Sephadex ™ LH-20 column (1/1 dichloromethane / ethanol) and purify on a silica column (1/1 and then 2/3 cyclohexane / ethyl acetate) to give pure 27 (487 mg, 64%). [α] _D +63 (c = 0.54, dichloromethane). TLC, R _F 0.28, 2/1 cyclohexane / ethyl acetate. ESIMS, positive mode: m / z + NaCl, 2454 (M + Na) ⁺ ; + KF, 2469 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.38-7.2 (m, 50H, 10Ph), 3.56, 3.52, 3.48, 3.46, 3.44, 3.42, 3.39, 3.30, 3.17 (9s, 27H, 9OCH ₃ ), 2.75-2.4 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.15, 1.98, 1.97, 1.87 (4s, 12H, 3Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ); Major anomer protons: 5.57; 5.47; 5.30; 5.18; 4.57; 4.29; 4.08.
Preparation Example 26
Methyl O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl-6-O-benzyl- 3-O-methyl-β-D-glucopyranosyl)-(1-4) -O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-( 1-4) -O- (benzyl 2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2 -O-benzyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyluronate)-(1-4 ) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (28)
Delevulinization of 27 (498 mg, 0.2 mmol) was carried out according to Method 2 to obtain 28 (402 mg, 84%). [α] _D +64 (c = 1, CH ₂ Cl ₂ ). ESIMS, positive mode: m / z 2352.9 (M + NH ₄ ) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.38-7.20 (m, 50H, 10Ph), 3.67, 3.52, 3.49, 3.46, 3.44, 3.41, 3.40, 3.28, 3.17 (9s, 27H, 9Ac), 2.65 (d, 1H, J = 2.14 Hz, OH) , 1.98, 1.96, 1.87 (3s, 9H, 3Ac); Major anomer protons: 5.55; 5.49; 5.30; 5.18; 4.56; 4.31; 4.08.
Analysis by calculation for C ₁₂₇ H ₁₅₂ O ₄₁ (2334.48): C, 65.34; H, 6.56. Found: C, 65.40; H, 6.62.
Preparation Example 27
Methyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4)-[O- (2,6-di-O-benzyl-3-O-methyl-α- D-glucopyranosyl)-(1-4) -O- (2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glycopyranosyl- (1-4)] _4- O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-β -D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4)- O- (benzyl 2,3-di-O-methyl-α-L-idopyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyrano Seed (29)
A mixture of 21 (340 mg, 1.16 mmol) and 28 (256 mg, 1.09 mmol) is treated according to Method 1. Purify the residue on a Toyopearl ™ HW-40 column (3.2 × 70 cm, 1/1 dichloromethane / ethanol) to afford pure 15-mer 29 (421 mg, 76%). [α] _D +65 (c = 1, dichloromethane). ESIMS, positive mode: m / z + KF, 2584.3 (M + 2K) ²⁺ ; 1736.5 (M + 3K) ³⁺ . ¹ H NMR (CDCl ₃ ). δ 7.35-7.18 (m, 105H, 21Ph), 2.75-2.4 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.15, 1.97, 1.95, 1.87, 1.84, 1.83 (6s, 24H, 7Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ); Major anomer protons: 5.55; 5.48; 5.30; 5.18; 4.56; 4.29; 4.22; 4.08.
Synthesis of Polysaccharide 91 (Example 1)
Preparation Example 28
Methyl O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl-6-O-benzyl- 3-O-methyl-β-D-glucopyranosyl)-(1-4)-[O- (2,6-di-O-benzyl-3-O-methyl-α-D-glucopyranosyl)- (1-4) -O- (2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glycopyranosyl- (1-4)] ₄ -O- (6-O- Benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-β-D-glucopyranosyl euro Nate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl 2,3 -Di-O-methyl-α-L-idopyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (30)
Compound 29 (342 mg, 0.067 mmol) was treated according to Method 2 to obtain 30 (253 mg, 75%). [α] _D +59 (c = 0.92, dichloromethane). ESIMS, positive mode: m / z + KF, 2535.6 (M + 2K) ²⁺ . ¹ H NMR of the major anomer protons (CDCl ₃ ). δ: 5.55; 5.50; 5.48; 5.30; 4.56; 4.30; 4.22; 4.08.
Analysis by calculations for C ₂₇₅ H ₃₂₈ O ₈₅ (4993.37): C, 65.57; H, 6.60. Found: C, 65.09; H, 6.57.
Preparation Example 29
Methyl O- (2,6-di-O-benzyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O-acetyl -6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4)-[O- (2,6-di-O-benzyl-3-O-methyl-α- D-glucopyranosyl)-(1-4) -O- (2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glycopyranosyl- (1-4)] _6- O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-β -D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4)- O- (benzyl 2,3-di-O-methyl-α-L-idopyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyrano Seed (31)
A mixture of 17 (32.7 mg, 20.6 mmol) and 30 (80.7 mg, 16.3 mmol) is treated according to Method 1. Purify the product on a Toyopearl ™ HW-40 column (1/1 dichloromethane / ethanol) to afford pure 19-mer 31 (60 mg, 59%). [α] _D +61 (c = 0.82, dichloromethane). ESIMS, positive mode: m / z + NaCl, 2162.4 (M + 3Na) ³⁺ ; + KF, 2178.5 (M + 3K) ³⁺ .
¹ H NMR of the major anomer protons (CDCl ₃ ). δ: 5.55; 5.48; 5.30; 5.17; 4.56; 4.22; 4.08.
Synthesis of Disaccharide 37
Preparation Example 30
Ethyl O- (4,6-O-benzylidene-α-D-glucopyranosyl)-(1-4) -6-O-trityl-1-thio-β-D-glucopyranoside (33)
Triethylamine in 32 (50.0 g, 0.105 mol) suspension (J. Westman and M. Nilsson, J. Carbohydr. Chem., 1995, 14 (7), 949-960) in dichloromethane (621 mL) under argon. 35 ml, 0.252 mol), trityl chloride (29.3 g, 0.105 mol) and 4-dimethylaminopyridine (1.28 g, 10 mmol) are added. The mixture is kept at reflux for 2 hours (TLC), cooled to room temperature, diluted with dichloromethane (500 mL) and washed successively with cold aqueous 10% potassium hydrogen sulfide, water and saturated sodium chloride solution. The solution is dried, concentrated and filtered over a silica column (65/35 and then 50/50 toluene / acetone) to obtain sufficiently pure crude product 33 used in the following steps. Analytical samples are chromatographed. [α] _D +53 (c = 0.74, dichloromethane). ESIMS, Voice mode: m / z 715 (MH) ^- . ¹ H NMR (CD ₂ Cl ₂ ). δ 7.52-7.25 (m, 20H, 4Ph), 5.42 (s, C ₆ H ₅ CH), 4.97 (d, J = 3.5Hz, H-1 '), 4.40 (d, J = 9.6Hz, H-1 ), 3.82 (t, J = 9.3 Hz, H-3 '), 3.70, 3.68 (m, 2H, H-3, H-4), 3.60 (dd, J 2.0, 11.0 Hz, H-6a), 3.55 (td, J = 5.2, 9.7, 9.7 Hz, H-5 '), 3.49-3.45 (m, 3H, H-2, H-2', H-5) , 3.38 (dd, J = 10.5 Hz, H-6'a), 3.33 (dd, H-6'b), 3.30-3.27 (m, 2H, H-4 ', H-6b), 2.90-2.77 ( m, 2H, SCH ₂ CH ₃ ), 1.40-1.37 (t, 3H, CH ₂ CH ₃ ).
Analysis by calculations for C ₄₀ H ₄₄ O ₁₀ S: C, 67.02; H, 6.19. S, 4.47. Found: C, 66.83; H, 6. 19; S, 4.19.
Preparation Example 31
Ethyl O- (4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -2,3-di-O-methyl-6- O-trityl-1-thio-β-D-glucopyranoside (34)
Under argon, methyl iodide (34 mL, 0.536 mol) is added dropwise to a solution of compound 33 (64.1 g) in N, N-dimethylformamide (600 mL). The solution is cooled to 0 ° C. and sodium hydride (13.5 g, 0.536 mol) is slowly added. The suspension was stirred at room temperature for 2 hours, cooled to 0 ° C. and then methanol (35 mL) was added dropwise and stirred for 2 hours before the mixture was diluted with ethyl acetate (500 mL) and water (600 mL). do. The aqueous phase is extracted with ethyl acetate and the organic phase is washed with water, dried and concentrated. Residue 34 is pure enough for the next step. Analytical samples are purified on silica columns (70/30 cyclohexane / acetone). [a] _D +45 (c = 0.83, dichloromethane). ESIMS, positive mode: m / z + NaCl, 795 (M + Na) ⁺ ; + KF, 811 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.52-7.19 (m, 20H, 4Ph), 5.51 (d, J = 3.3 Hz, H-1 '), 5.43 (s, C ₆ H ₅ CH), 4.45 (d, J = 9.8 Hz, H-1 ), 3.60, 3.59, 3.51, 3.49 (4s, 12H, 40CH ₃ ), 2.86 (q, 2H, J = 7.5, SCH ₂ CH ₃ ), 1.40 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₄₄ H ₅₂ O ₁₀ S: C, 68.37; H, 6.78. S, 4.15. Found: C, 68.28; H, 6.98; S, 4.09.
Preparation Example 32
Ethyl O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -2,3-di-O-methyl-1-thio-β-D-glucopyrano Seed (35)
The suspension of crude product 34 (67.4 g) is heated in aqueous 60% acetic acid solution (470 mL) for 2 hours. The reaction mixture is cooled, filtered and concentrated. The residue is treated with sodium methoxide (940 mg) in methanol (200 mL) for 1 hour. To obtain 35 (27.9 g, more than 60% in three steps), the solution was neutralized with Dowex ™ 50WX4 (H ⁺ ) resin, filtered and concentrated and purified on silica column (60/40 toluene / acetone) do. [α] _D +26 (c = 1.07, dichloromethane). ESIMS, positive mode: m / z + NaCl, 465 (M + Na) ⁺ ; + KF, 481 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.62 (d, J = 3.9 Hz, H-1 ′), 4.35 (d, J = 9.8 Hz, H-1), 3.64, 3.64, 3.59, 3.58 (4 s, 12H, 4OCH ₃ ), 1.29 (t, 3H, J = 7.4 Hz, SCH ₂ CH ₃ ).
Analysis by calculations for C ₁₈ H ₃₄ O ₁₀ S. H ₂ O: C, 46.94; H, 7.87. S, 6.96. Found: C, 47.19; H, 7.72; S, 6.70.
Preparation Example 33
Ethyl O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -6-O-acetyl-2,3-di-O-methyl -1-thio-β-D-glucopyranoside (36)
A solution of N-acetylimidazole (3.21 g, 29.1 mmol) and Triol 35 (5.86 g, 13.2 mmol) in 1,2-dichloroethane (120 mL) was refluxed for 16 h. Another portion of N-acetylimidazole (440 mg, 3.96 mmol) is added and the mixture is stirred for 4 hours. It is cooled to room temperature and then methanol (2 ml) is added. The mixture is further stirred for 1 h, diluted with dichloromethane (1 L), washed successively with cold aqueous 1M hydrochloric acid solution, cold water, saturated aqueous sodium hydrogen carbonate solution, water, dried and concentrated. The residue is chromatographed (3.5 / 1 toluene / acetone) to obtain diacetate 36 (3.97 g, 57%). [α] _D +33 (c = 1.90, dichloromethane). ESIMS, positive mode: m / z + NaCl, 549 (M + Na) ⁺ ; + KF, 565 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.51 (d, J = 3.9 Hz, H-1 ′), 4.34 (d, J = 9.8 Hz, H-1), 3.64, 3.63, 3.59, 3.56 (4s, 12H, 4OCH ₃ ), 2.11, 2.06 ( 2s, 6H, 2Ac), 1.31 (t, 3H, J = 7.4 Hz, SCH ₂ CH ₃ ).
Analysis by calculation for C ₂₂ H ₃₈ O ₁₂ : C, 50.17; H, 7.27. S, 6.09. Found: C, 50.15; H, 7. 49; S, 5.89.
Preparation Example 34
Ethyl O- (6-O-acetyl-4, O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -6-O-acetyl-2, 3-di-O-methyl-1-thio-β-D-glucopyranoside (37)
To a solution of diacetate 36 (19.4 g, 36.8 mmol) in dioxane (400 mL), levulinic acid (7.53 mL, 73.5 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (14.1 g) , 73.5 mmol) and 4-dimethylaminopyridine (900 mg, 7.35 mmol) were added under argon. The mixture is stirred for 3.5 hours, diluted with dichloromethane (1.5 L), washed successively with water, aqueous 10% potassium hydrogen sulfide solution, water, aqueous 2% sodium hydrogen carbonate solution, water, dried and concentrated. To obtain derivative 37 (21.8 g, 95%), the residue is chromatographed (97/3 and then 79/21 dichloromethane / acetone). [α] _D +40 (c = 0.72, dichloromethane). ESIMS, positive mode: m / z + NaCl, 647 (M + Na) ⁺ ; + KF, 663 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.56 (d, J = 3.9 Hz, H-1 ′), 4.35 (d, J = 9.8 Hz, H-1), 3.64, 3.60, 3.58, 3.55 (4s, 12H, 4OCH ₃ ), 2.76-2.71 ( m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.19, 2.08, 2.07 (3s, 9H, 2Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.31 (t, 3H, J = 7.4 Hz, SCH ₂ CH ₃ ).
Analysis by calculations for C ₂₇ H ₄₄ O ₁₄ S: C, 51.91; H, 7.10. S, 5.13. Found: C, 51.88; H, 7.05; S, 4.96.
Synthesis of Disaccharide 38 and Disaccharide 40
Preparation 35
O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -6-O-acetyl-2,3 -Di-O-methyl-α, β-D-glucopyranose trichloroacetimidadate (38)
To a solution of thioglycoside 37 (9.53 g, 15.3 mmol) in a 1/1 dichloromethane / ethyl ether mixture (180 mL), water (1.4 mL, 76.3 mmol), N-iodosuccinimide (6.84 g, 30.5 mmol) And silver triflate (0.51 g, 1.98 mmol). After 15 minutes (TLC), saturated aqueous sodium hydrogen carbonate solution (5 ml) is added and the reaction mixture is diluted with dichloromethane (1.5 L), water, aqueous 1M sodium thiosulfate solution and aqueous 2% sodium bicarbonate solution. Wash with After the solution is dried and concentrated, the residue is purified on a silica column (80/40 and then 100/0 ethyl acetate / cyclohexane) to give a solid that is used without characterization in the next step. The solution of the compound (7.88 g, 13.6 mmol) in dichloromethane (120 mL) under argon is treated with cesium carbonate (7.08 g, 21.7 mmol) and trichloroacetonitrile (6.81 mL, 67.9 mmol). After 40 minutes (TLC) to obtain imidate 38 (9.16 g, 83% in two or more steps), the mixture is filtered, concentrated and purified (85/15 toluene / acetone). [a] _D +118 (c = 1.00, dichloromethane). ESIMS, positive mode: m / z + NaCl, 746 (M + Na) ⁺ ; 741 (M + NH ₄ ) ⁺ . ¹ H NMR (CDCl ₃ ). δ 8.66, 8.65 (2s, 1H, α and β N: H), 6.52 (d, J = 3.6 Hz, H-1α), 5.70 (d, J = 7.5 Hz, H-1β), 5.58 (d, J = 3.7 Hz, H-1 '), 2.78-2.57 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18, 2.07, 2.06 (3s, 9H, 2Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculations for C ₂₇ H ₄₀ Cl ₃ NO ₁₅ . 0.5 H ₂ O: C, 44.18; H, 5.63. N, 1.98. Found: C, 44.14; H, 5.61; N, 1.97.
Preparation Example 36
2- (trimethylsilyl) ethyl O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -6- O-acetyl-2,3-di-O-methyl-α, β-D-glucopyranoside (39)
Thioglycoside 37 (10.6 g, 16.94 mmol) is treated with 2- (trimethylsilyl) ethanol (4.8 mL, 33.90 mmol) in a 1/2 dichloromethane / ethyl ether mixture (105 mL) according to Method 3. To obtain compound 39 (9.80 g, 85%) in the form of anomer mixture (α / β 65/35), the obtained residue is purified by chromatography (1/1 acetone / dichloromethane). ESIMS, positive mode: m / z + NaCl, 703 (M + Na) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.58 (d, J = 3.9 Hz, H-1 ′), 4.94 (d, J = 3.5 Hz, H-1α), 4.26 (d, J = 7.7 Hz, H-1β), 2.76-2.56 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.08, 2.05 (3s, 9H, 2Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.18-0.88 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.02 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₃₀ H ₅₂ O ₁₅ Si: C, 52.92; H, 7.69. Found: C, 53.29; H, 7.75.
Preparation Example 37
2- (trimethylsilyl) ethyl O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -6-O-acetyl-2,3 -Di-O-methyl-α, β-D-glucopyranoside (40)
Compound 39 (9.41 g, 13.82 mmol) was treated according to Method 2 with hydrazine acetate (10 mol / mol) in a 1/2 toluene / ethanol mixture (21 mL / mmol). The residue is chromatographed (60/40 acetone / toluene) to obtain 40α (4.81 g, 60%) as well as 40α / β mixture (3.06 g, 37%). 40α: [α] _D +132 (c = 0.61, dichloromethane). ESIMS, positive mode: m / z + NaCl, 605 (M + Na) ⁺ ; + KF, 621 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.55 (d, J = 3.8 Hz, H-1 ′), 4.95 (d, J = 3.6 Hz, H-1), 2.10, 2.08 (2s, 6H, 2Ac), 1.16-0.89 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.02 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₂₅ H ₄₆ O ₁₃ Si: C, 51.53; H, 7.96. Found: C, 51.37; H, 8.06.
Synthesis of Tetrasaccharide 41
Preparation Example 38
2- (trimethylsilyl) ethyl O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O -(6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₂ -6- (O-acetyl-2,3-di-O -Methyl-α-D-glucopyranoside (41)
Thioglycoside 37 (4.21 g, 6.74 mmol) and glycosyl acceptor 40 (3.57 g, 6.13 mmol) in a 1/2 dichloromethane / ethyl ether mixture (105 mL) were treated according to Method 3. The residue is purified by chromatography on silica (3/1 and then 9/1 acetone / cyclohexane) to afford 41 (4.81 g, 69%). [α] _D +143 (c = 0.56, dichloromethane). ESIMS, positive mode: m / z + NaCl, 1167 (M + Na) ⁺ ; + KF, 1183 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.57 (d, J = 3.9 Hz, H-1 ′ ''), 5.44, 5.41 (2d, J = 3.8 Hz, H-1 ″, H-1 ′), 4.96 (d, J = 3.6 Hz, H -1), 2.78-2.58 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18, 2.12, 2.12, 2.09, 2.06, (5s, 15H, 4Ac and O ( C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.21-0.97 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.03 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₅₀ H ₈₄ O ₂₇ Si: C, 52.44; H, 7.39. Found: C, 52.29; H, 7.46.
Synthesis of Tetrasaccharide 42
Preparation Example 39
Ethyl O- (6-O-Acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (6-O- Acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₂ -6- (O-acetyl-2,3-di-O-methyl-1-thio -β-D-glucopyranoside (42)
A solution of imidate 38 (1.10 g, 1.52 mmol) and glycosyl receptor 36 (806 mg, 1.38 mmol) is treated in a 1/2 dichloromethane / ethyl ether mixture (22 mL) according to Method 1. To afford 42 (1.12 g, 71%), the product is purified by chromatography on silica (2.5 / 1 and then 3/1 ethyl acetate / cyclohexane). [α] _D +95 (c = 1.00, dichloromethane). ESIMS, positive mode: m / z + NaCl, 1111 (M + Na) ⁺ ; + KF, 1127 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.55 (d, J = 3.9 Hz, H-1 ′ ''), 5.39, 5.37 (2d, J = 3.8 Hz, H-1 ″, H-1 ′), 4.34 (d, J = 9.7 Hz, H -1), 2.84-2.51 (m, 6H, SCH ₂ CH ₃ , O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.10, 2.09, 2.08, 2.04, (5s, 15H , 4Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.30 (t, 3H, J = 7.4 Hz, SCH ₂ CH ₃ ).
Analysis by calculations for C ₄₇ H ₇₆ O ₂₆ S: C, 51.83; H, 7.03; S, 2.94. Found: C, 51.66; H, 7.02; S, 2.94.
Synthesis of Polysaccharide 47
Preparation Example 40
2- (trimethylsilyl) ethyl- [O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₃ -6-O- Acetyl-2,3-di-O-methyl-α-D-glucopyranoside (43)
After column chromatography (3/2 cyclohexane / acetone), compound 41 (4.71 g, 4.11 mmol) was reacted according to Preparation Example 37, to obtain derivative 43 (4.11 g, 95%). [α] _D +154 (c = 0.63, dichloromethane). ESIMS, positive mode: m / z + NaCl, 1069 (M + Na) ⁺ ; + KF, 1085 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 5.46, 5.46 (2d, 3H, J = 3.9 Hz, H-1 ′, H-1 ″, H-1 ′ ''), 4.95 (d, J = 3.5 Hz, H-1), 2.81 (d, J = 4.4 Hz, OH), 2.11, 2.09, 2.08 (3s, 12H, 4Ac), 1.19-0.97 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.03 (s, 9H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₄₅ H ₇₈ O ₂₅ Si: C, 51.61; H, 7.51. Found: C, 51.39; H, 7.54.
Preparation Example 41
2- (trimethylsilyl) ethyl-O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[ O- (6-O- acetyl-2,3--O- methyl -α-D- _{glucopyranosyl) - (1-4) -] 6} -6-O- acetyl-2,3-di -O -Methyl-α-D-glucopyranoside (44)
Thioglycoside 42 (3.86 g, 3.54 mmol) and glycosyl receptor 43 (3.60 g, 3.44 mmol) were treated according to Preparation Example 38. The product is chromatographed (7/2 and then 2/1 dichloromethane / acetone) to give 44 (5.71 g, 80%). [α] _D +161 (c = 0.65, dichloromethane). ESIMS, positive mode: monoisotopic mass = 2072.8, chemical mass = 2074.2, experimental mass = 2074 ± 1 amu ¹ H NMR (CDCl ₃ ). δ 5.54, (d, J = 3.8 Hz, H-1, unit NR), 5.47-5.40 (m, 6H, H-1), 4.95 (d, J = 3.7 Hz, H-1 unit R), 2.84- 2.51 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.13, 2.12, 2.11, 2.11, 2.08, 2.05 (7s, 27H, 8Ac and O (C: O) ) CH ₂ CH ₂ (C: O) CH ₃ ), 1.18-0.97 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.03 (s, 9H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₉₀ H ₁₄₈ O ₅₁ Si: C, 52.12; H, 7.19. Found: C, 51.98; H, 7.25.
Preparation Example 42
2- (trimethylsilyl) ethyl- [O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₇ -6-O- Acetyl-2,3-di-O-methyl-α-D-glucopyranoside (45)
A solution of 1M hydrazine hydrate in 3/2 acetic acid / pyridine mixture (7.3 mL) at 0 ° C. is added to a solution of compound 44 (3.00 g, 1.45 mmol) in pyridine (5 mL). After stirring for 20 minutes, the reaction mixture is evaporated, diluted with dichloromethane (400 mL) and washed with aqueous 10% potassium hydrogen sulfide solution, water, aqueous 2% sodium bicarbonate solution and water. The solution is evaporated and concentrated and the residue is chromatographed to give 45 (2.43 g, 85%). [α] _D +167 (c = 0.57, dichloromethane). ESIMS, positive mode: monoisotopic mass = 1974.8, chemical mass = 1976.1, experimental mass = 1975.4 ± 2 amu. ¹ H NMR (CDCl ₃ ). δ 5.47-5.40 (m, 4H, H-1), 4.95 (d, J = 3.7 Hz, H-1, unit R), 2.80 (d, J = 4.4 Hz, OH), 2.13, 2.11, 2.10, 2.08 , 2.07 (5s, 24H, 8Ac), 1.18-0.97 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.03 (s, 9H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₈₅ H ₁₄₂ O ₄₉ Si: C, 51.66; H, 7.24. Found: C, 51.32; H, 7.26.
Preparation Example 43
2- (trimethylsilyl) ethyl-O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[ O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₁₀ -6-O-acetyl-2,3-di-O -Methyl-α-D-glucopyranoside (46)
Compound 42 (1.35 g, 1.24 mmol) and compound 45 (2.38 g, 1.20 mmol) were treated according to Preparation Example 38. The residue is chromatographed (4/3 cyclohexane / acetone) to give 46 (2.56 g, 71%). [α] _D +166 (c = 0.88, dichloromethane). ESIMS, positive mode: single isotope mass = 3001.3, chemical mass = 3003.2, experimental mass = 3004 ± 1 amu ¹ H NMR (CDCl ₃ ). δ 5.54, (d, J = 3.8 Hz, H-1 units NR), 5.47-5.40 (m, 10H, H-1), 4.95 (d, J = 3.7 Hz, H-1 units R), 2.81-2.51 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.13, 2.12, 2.11, 2.11, 2.08, 2.05 (7s, 39H, 12Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.17-0.96 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.03 (s, 9H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ) .
Analysis by calculations for C ₁₃₀ H ₂₁₂ O ₇₅ Si: C, 51.99; H, 7.12. Found: C, 51.63; H, 7.12.
Preparation Example 44
(6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (6-O-acetyl-2 , 3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₁₀ -6-O-acetyl-2,3-di-O-methyl-α, β-D-glu Copyranose trichloroacetimidadate (47)
(a) A solution of glycoside 46 (400 mg, 0.133 mmol) in a 2/1 trifluoroacetic acid / dichloromethane mixture (2 mL) is stirred for 1.5 h (TLC). The solution is diluted in 2/1 toluene / n-propyl acetate mixture (12 ml), concentrated and co-evaporated with toluene (5 x 10 ml). The residue is purified (4/3 acetone / cyclohexane) to give a solid (364 mg).
(b) The obtained solid is dissolved in dichloromethane (2.5 mL). Cesium carbonate (65 mg, 0.200 mmol) and trichloroacetonitrile (63 mL, 0.620 mmol) were added and the mixture was stirred for 2.5 h, filtered (celite), concentrated and imidate 47 (348 mg, 86%) is purified on a silica column (50/50 / 0.1 cyclohexane / acetone / triethylamine). [α] _D +185 (c = 0.91, dichloromethane). ESIMS, positive mode: single isotope mass = 3044.1, chemical mass = 3047.3, experimental mass = 3046.9 ± 0.2 amu ¹ H NMR (CD ₂ Cl ₂ ). δ 8.61, 8.58 (2s, 1H, α and β N: H), 6.35 (d, J = 3.7 Hz, H-1a units R), 5.59 (d, J = 7.5 Hz, H-1b units R), 5.38 (d, J = 3.8 Hz, NR in H-1), 5.32-5.25 (m, 10H, H-1), 2.64-2.40 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O ) CH ₃ ), 2.02, 1.96, 1.95, 1.94, 1.93, 1.89 (6s, 39H, 12Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculation for C ₁₂₇ H ₂₀₀ Cl ₃ NO ₂₅ : C, 50.06; H, 6. 61; N, 0.46. Found: C, 49.93; H, 6.52; N, 0.42.
Synthesis of Oligosaccharide 50
Preparation Example 45
(6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (6-O-acetyl-2 , 3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₂ -6-O-acetyl-2,3-di-O-methyl-α, β-D-glu Copyranose trichloroacetimidadate (48)
Compound 41 (200 mg, 0.174 mmol) was treated according to Preparation Example 44. To obtain imidate 48 (230 mg, 77%), the reaction mixture is purified on a silica column (3/2 toluene / acetone). ESIMS, positive mode: m / z, + NaCl, 1210 (M + Na) ⁺ ; + KF, 1226 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 8.66-8.64 (2s, 1H, α and β N: H), 6.51 (d, J = 3.6 Hz, H-1β), 5.71 (d, J = 7.5 Hz, H-1β), 2.90-2.52 (m , 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.11, 2.11, 2.09, 2.05 (5s, 4Ac and O (C: O) CH ₂ CH ₂ (C: O ) CH ₃ ).
Preparation Example 46
Methyl O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (6-O- Acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₂ -O- (benzyl-2,3-di-O-methyl-β-D-glucose Pyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl -2,3-di-O-methyl-α-L-iodo-pyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (49)
Imidate 48 (73 mg, 0.061 mmol) and glycosyl receptor 26 (82 mg, 0.059 mmol) were treated according to Preparation Example 39. The compound is purified on Sephadex ™ LH-20 chromatography column (1/1 dichloromethane / ethanol) and silica column (3/1 toluene / acetone) to obtain derivative 49 (98 mg, 69%). [α] _D +95 (c = 1.01, dichloromethane). ESIMS, positive mode: monoisotopic mass = 2414.97, chemical mass = 2416.97, experimental mass = 2416.2 ¹ H NMR (CDCl ₃ ). δ 7.43-7.20 (m, 30H, 6Ph), 5.55 (d, J = 3.9 Hz, NR in H-1), 5.50 (d, J = 3.9 Hz, NR-3 in H-1), 5.44, 5.38 ( 2d, J = 3.7 and 3.9 Hz, H-1 unit NR-1, unit NR-2), 5.29 (d, J = 6.8 Hz, H-1 unit R-1), 5.17 (d, J = 3.5 Hz, H-1 unit R-2), 4.56 (d, J = 3.7 Hz, H-1 unit R), 4.10 (d, J = 7.9 Hz, H-1 unit R-3), 2.81-2.50 (m, 4H , O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.15, 2.11, 2.09, 2.05, 2.00 (7s, 21H, 6Ac and O (C: O) CH ₂ CH ₂ (C : O) CH ₃ ).
Analysis by calculations for C ₁₂₀ H ₁₅₈ Cl ₃ O ₅₁ : C, 59.63; H, 6.59. Found: C, 59.23; H, 6.58.
Preparation 47
Methyl [O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₃ -O- (benzyl-2,3-di- O-methyl-β-D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-( 1-4) -O- (benzyl-2,3-di-O-methyl-α-L-iodo-pyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl -α-D-glucopyranoside (50)
Octacasaccharide 49 (120 mg, 0.050 mmol) was reacted according to Preparation Example 37. The residue is purified on silica column (3/1 toluene / acetone) to give compound 50 (95 mg, 83%). [α] _D +80 (c = 0.62, dichloromethane). ESIMS, positive mode: single isotope mass = 2316.9, chemical mass = 2318.4, experimental mass = 2318.2 ± 0.4 amu ¹ H NMR (CDCl ₃ ). δ 7.42-7.12 (m, 30H, 6Ph), 5.50, 5.46, 5.43, 5.40 (d, J = 3.9, 3.9, 3.7, 3.7 Hz, H-1 unit NR, unit NR-1, unit NR-2, unit NR-3), 2.14, 2.10, 2.09, 2.08, 2.00, 1.88 (6s, 18H, 6Ac).
Analysis by calculations for C ₁₁₅ H ₁₅₂ O ₄₉ : C, 59.57; H, 6.60.
Found: C, 59.49; H, 6.61.
Synthesis of Polysaccharide 97 (Example 4)
Preparation Example 48
Methyl O- (6-O-acetyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (6-O- Acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4)-] ₁₅ -O- (benzyl-2,3-di-O-methyl-β-D-glucose Pyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl -2,3-di-O-methyl-α-L-iodo-pyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (51)
Imidate 47 (84 mg, 0.027 mmol) was treated with glycosyl receptor (62 mg, 0.027 mmol) according to Preparation Example 39. To obtain derivative 51 (71 mg, 51% is not optimized), the residue is purified on Toyopearl ™ HW-40 column and then on silica column (1/1 cyclohexane / acetone). [α] _D +136 (c = 0.95, dichloromethane). ESIMS, positive mode: monoisotopic mass = 5200.11, chemical mass = 5203.39, experimental mass = 5203.5 ¹ H NMR (CDCl ₃ ). δ 7.42-7.18 (m, 30H, 6Ph), 5.54 (d, J = 3.8 Hz, H-1 unit NR), 5.51-5.40 (m, 15 H-1), 5.30 (d, J = 6.8 Hz, H -1 unit R-1), 5.17 (d, J = 3.5 Hz, H-1 unit R-2), 4.56 (d, J = 3.7 Hz, H-1 unit R), 4.09 (d, J = 7.9 Hz , H-1 unit R-3), 2.85-2.53 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.17, 2.16, 2.13, 2.11, 2.08, 2.05, 2.00 , 1.88 (8s, 57H, 18Ac and O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Analysis by calculation for C ₂₄₀ H ₃₅₀ O ₁₂₃ .4H ₂ O: C, 54.64; H, 6.84. Found: C, 54.51; H, 6.79.
Synthesis of Disaccharide 57
Preparation 49
Ethyl O- (4,6-O-p-methoxybenzylidene-α-D-glucopyranosyl)-(1-4) -1-thio-β-D-glucopyranoside (53)
At + 5 ° C. under argon, anisealdehyde dimethyl acetal (35.8 mL, 0.21 mol) and camphorsulfonic acid (4.44 g, 19.1 mmol) were added in a 3.5 / 1 acetonitrile / N, N-dimethylformamide mixture (990 mmol). Compound 52 (73.89 g, 0.19 mmol) solution (WE Dick Jr et JE Hodge, Methods in Carbohydrate Chemistry, 7, 1976, 15-18). After 1.5 hours of stirring at room temperature, triethylamine (2.96 ml, 21.0 eq.) Was added to the mixture and neutralized. The mixture is concentrated and the syrup is purified on a silica column (100/0 and then 50/50 dichloromethane / methanol) to give 53 (58.6 g, 61% not optimized). [α] _D + 47 (c = 1.03, methanol). ESIMS, positive mode: m / z, 503 (MH) ^- . ¹ H NMR (CD ₃ OD) δ 7.41, 6.89 (2d, 4H, CH ₃ OC ₆ H ₄ ), 5.51 (s, CHC ₆ H ₄ ), 5.20 (d, J = 3.6 Hz, H-1 ′), 4.39 (d, J = 7.1 Hz, H-1), 3.78 (s, 3H, CH ₃ OC ₆ H ₄ ), 2.75 (q, 2H, J = 7.0 Hz, SCH ₂ CH ₃ ), 1.29 (t, 3H , SCH ₂ CH ₃ ).
Analysis by calculations for C ₂₂ H ₃₂ O ₁₁ S: C, 52.37; H, 6.39; S, 6.35. Found: C, 52.15; H, 6. 61; S, 5.84.
Preparation 50
Ethyl O- (2,3-di-O-acetyl-4,6-p-methoxybenzylidene-α-D-glucopyranosyl)-(1-4) -2,3,6-tri-O- Acetyl-1-thio-β-D-glucopyranoside (54)
Triethylamine (65 mL, 0.47 mol) and acetic anhydride (89 mL, 0.94 mol) are added dropwise to a 53 (47.22 g, 93.6 mmol) suspension in dichloromethane (450 mL) at 0 ° C. 4-Dimethylaminopyridine (5.71 g, 46.8 mmol) is then added and the mixture is stirred at room temperature for 1.5 hours. Methanol (45 mL, 1.12 mol) is added to stop the reaction, which is then washed successively with cold aqueous 10% potassium hydrogen sulfide solution, water, saturated sodium bicarbonate solution and water. To obtain 54 (64.9 g, 97%), the solution is dried, concentrated and recrystallized (cyclohexane / ethyl acetate). [α] _D +21 (c = 1.00, dichloromethane). pf 213-215 ° C. ESIMS, positive mode: m / z + NaCl, 737 (M + Na) ⁺ ; + KF, 753 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.34, 6.86 (2d, 4H, CH ₃ OC ₆ H ₄ ), 5.43 (s, CHC ₆ H ₄ ), 5.34 (d, J = 4.1 Hz, H-1 ′), 4.54 (d, J = 9.9 Hz , H-1), 3.78 (s, 3H, CH ₃ OC ₆ H ₄ ), 2.74-2.60 (m, 2H, SCH ₂ CH ₃ ), 2.10, 2.06, 2.03, 2.01 (4s, 15H, 5Ac), 1.26 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₃₂ H ₄₂ O ₁₆ S: C, 53.78; H, 5.92; S, 4.49. Found: C, 53.74; H, 6.08; S, 4.40.
Preparation Example 51
Ethyl O- (2,3-di-O-acetyl-4-p-methoxybenzylidene-α-D-glucopyranosyl)-(1-4) -2,3,6-tri-O-acetyl- 1-thio-β-D-glucopyranoside (55) and ethyl O- (2,3-di-O-acetyl-4-p-methoxybenzylidene-α-D-glucopyranosyl)-(1 -4) -2,3-di-O-acetyl-1-thio-β-D-glucopyranoside (56)
A suspension of 54 (25.0 g, 35.0 mmol), borane / triethylamine complex (20.4 g, 0.28 mol) and molecular sieve (33 g, 4 cc) is stirred under argon in toluene (810 mL) for 1 h. The mixture is cooled to 0 ° C. and aluminum chloride (14.0 g, 0.11 mol) is added slowly. The reaction medium is stirred for 25 minutes (TLC), poured into cold aqueous 20% potassium hydrogen sulfide solution, stirred at 0 ° C. for 1 hour and filtered (celite). The organic phase is washed with water, aqueous 2% sodium bicarbonate solution, washed with water, dried and concentrated. The residue is purified on silica column to give 55 (7.37 g, 29%, not optimized) and 56 (1.36 g, 6%). An analytical sample of 55 is crystallized from a cyclohexane / ethyl acetate mixture. [α] _D +30 (c = 1.00, dichloromethane). mp 151-153 ° C. ESIMS, positive mode: m / z + NaCl, 739 (M + Na) ⁺ ; + KF, 755 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ). δ 7.19, 6.87 (2d, 4H, CH ₃ OC ₆ H ₄ ), 5.36 (d, J = 3.5 Hz, H-1 ′), 4.54 (s, 2H, C ₆ H ₄ CH ₂ ), 4.53 (d, J = 9.0 Hz, H-1), 2.70-2.65 (m, 2H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₃₂ H ₄₂ O ₁₆ S: C, 53.62; H, 6. 19; S, 4.47. Found: C, 53.57; H, 6. 21; S, 4.43.
Compound 56: [a] _D +19 (C = 1.11, dichloromethane). ESIMS, positive mode: m / z + NaCl, 697 (M + Na) ⁺ , + KF, 713 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.17, 6.84 (2d, 4H, CH ₃ OC ₆ H ₄ ), 5.36 (d, J = 4.5Hz, H-1 '), 4.54 (d, J = 10.1Hz, H- 1), 4.49 (s, 2H, C ₆ H ₄ CH ₂ ), 2.69-2.64 (m, 2H, SCH ₂ CH ₃ ), 2.02, 2.01, 2.00, 1.96 (4s, 12H, 4Ac), 1.25 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₃₀ H ₄₂ O ₁₅ S: C, 53.40; H, 6. 27; S, 4.75. Found: C, 53.29; H, 6.39; S, 4.53.
Preparation Example 52
Ethyl O- (2,3-tri-O-acetyl-4-p-methoxybenzyl-α-D-glucopyranosyl)-(1-4) -2,3,6-tri-O-acetyl-1 -Thio-β-D-glucopyranoside (57).
Acetic anhydride (1.47 ml, 15.5 mmol) was 55 (5.6 g, 7.77 mmol), triethylamine (1.19 g, 8.54 mmol) and 4-dimethylaminopyridine (190 mg, 1.55 mmol) in dichloromethane (40 ml) at 0 ° C. Is added to the mixture. After stirring for 40 minutes at room temperature (TLC), the mixture is diluted with dichloromethane (50 ml) and washed with cold aqueous 10% potassium hydrogen sulfate solution, water, saturated sodium bicarbonate solution and water. The solution is dried and concentrated, and the residue is purified on silica column (35/65 ethyl acetate / cyclohexane) to get 57 (5.66 g, 96%). [α] _D +44 (C = 1.03, dichloromethane). ESIMS, positive mode: m / z, + NaCl, 781 (M + Na) ⁺ , + KF, 797 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.15, 6.85 (2d, 4H, CH ₃ OC ₆ H ₄ ), 5.30 (d, J = 4.2 Hz, H-1 '), 4.53 (d, J = 10.0 Hz, H- 1), 2.69-2.64 (m, 2H, SCH ₂ CH ₃ ), 2.09, 2.07, 2.04, 2.01, 2.00, 1.98 (6s, 18H, 6Ac), 1.25 (t, 3H, SCH ₂ CH ₃ ).
Analysis by calculations for C ₃₄ H ₄₆ 0 ₁₇ S: C, 53.82; H, 6. 11; S, 4.22. Found: C, 53.77; H, 6. 24; S, 4.09.
Synthesis of Trisaccharide 62
Preparation Example 53
Ethyl 4,6-O-benzylidene-2,3-di-O-methyl-1-thio-β-D-glucopyranoside (59)
Sodium hydride (500 mg, 19.9 mmol) was methyl iodide (1.70 ml, 19.9 mmol) and compound 58 (2.59 g, 8.29 mmol) in N, N-dimethylformamide (25 ml) at 0 ° C. (Izv. Nauk SSSR, Ser.Khim. (1968), (1), 179) are added and the mixture is raised to a temperature of 20 ° C. The mixture is left to stir for 30 minutes (TLC) and methanol is added. The mixture is poured into water and extracted with ethyl acetate. The organic phase is successively washed with an aqueous 1 M sodium thiosulfate solution and water, dried and concentrated. The residue is triturated in ethyl ether to yield 59 (0.42 g, 15%); The mother liquor was purified in a column of silica (12/1 toluene / acetone) and 59 additional fractions obtained by crystallization were obtained (1.6 g, yield 71%). mp: 108 ° C. [α] _D -78 (C = 1.00, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 363 (M + Na) ⁺ , thioglycerol + KF, 379 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.51-7.33 (m, 5H, Ph), 5.52 (s, 1H, C ₆ H ₅ CH), 4.45 (d, 1H, J = 9.8 Hz, H-1), 3.64 ( s, 3H, OCH ₃ ), 3.62 (s, 3H, OCH ₃ ), 2.78-2.68 (m, 2H, SCH ₂ CH ₃ ), 1.31 (t, 3H, J = 2.7 Hz, SCH ₂ CH ₃ ).
Analysis by calculations for C ₁₇ H ₂₄ O ₅ S (340.44): C, 59.98; H, 7. 11; S, 9.42. Found: C, 59.91; H, 7. 15; S, 8.96.
Preparation Example 54
2- (trimethylsilyl) ethyl-4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranoside (60)
Compound 59 (23.0 g, 67.5 mmol) and 2- (trimethylsilyl) ethanol (19.4 ml, 135 mmol) are dissolved in a 2/1 mixture of ethyl ether and dichloromethane (345 ml), and 4 μg molecular sieves (11 g) are added. . The mixture is left to stir at 25 ° C. for 1 hour, N-iodosuccinimide (49.7 g, 220 mmol) is added, and at 0 ° C., silver triplate (2.20 g, 8.78 mmol) is added. The mixture is left to stir for 20 minutes (TLC) and solid sodium hydrogen carbonate is added. The mixture is diluted with dichloromethane, filtered through celite, washed successively with aqueous 1M sodium thiosulfate solution and water, dried and evaporated for dryness. The residue is purified on silica column (15/1, and 5/1 cyclohexane / ethyl acetate) to yield 60β (4.20g, 15%) and 60α (8.40g, 31%).
Compound 60α. [α] _D + 96 (C = 0.4, dichloromethane). ESIMS, positive mode: m / z, 419 (M + Na) ⁺ ; 435 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.52-7.35 (m, 5H, Ph), 5.54 (s, 1H, C ₆ H ₅ CH), 4.98 (d, 1H, J = 3.7 Hz, H-1), 3.64 ( s, 3H, OCH ₃ ), 3.62 (s, 3H, OCH ₃ ), 1.24-0.96 (m, 2H, OCH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, OCH ₂ CH ₂ Si ( CH ₃ ) ₃ ).
Analysis by calculations for C ₂₀ H ₃₂ O ₆ S (396.56): C, 60.58; H, 8.13. Found: C, 60.26; H, 8.39.
Preparation Example 55
2- (trimethylsilyl) ethyl 6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranoside (61)
Compound 60 (21.1 g, 53.3 mmol) is dissolved in dichloromethane (154 ml). Triethylsilane (34 ml, 213 mmol) is added at room temperature and is added dropwise to a mixture of trifluoroacetic acid (16.3 ml, 213 mmol) and trifluoroacetic anhydride (0.49 ml, 3.47 mmol). The mixture is stirred for 2 hours and an aqueous 1M sodium hydroxide solution is added until the pH is basic. After separation of the phases by precipitation, the aqueous phase is extracted with ethyl acetate, the organic phases are combined, dried and concentrated. The residue is purified on a column of silica (14/1, and 12/1 dichloromethane / acetone) to yield 61 (12.5 g, 59%). [a] _D + 100 (C = 1.45, dichloromethane). ¹ H NMR δ 7.40-7.20 (m, 5H, Ph), 4.98 (d, 1H, J = 3.5 Hz, H-1), 3.62 (s, 3H, OCH ₃ ), 3.49 (s, 3H, OCH ₃ ) , 1.14-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₂₀ H ₃₀ O ₆ Si (398.58): C, 60.27; H, 8.60. Found: C, 60.18; H, 8.81.
Preparation Example 56
2- (trimethylsilyl) ethyl O- (2,3,6-tri-O-acetyl-4-Op-methoxybenzyl-α-D-glucopyranosyl)-(1-4) -O- (2, 3,6-Tri-O-acetyl-β-D-glucopyranosyl- (1-4) -6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranoside (62 )
A mixture of thioglycoside 57 (19.1 g, 25.1 mmol) and glycosyl receptor 61 (7.5 g, 18.7 mmol) was treated according to Method 3, and subjected to silica column (20/1, and 10/1 dichloromethane / acetone). 62 (19.7 g, 95%) was obtained after purification. [a] _D + 90 (C = 1.15, dichloromethane).
Analysis by calculations for C ₅₂ H ₇₄ O ₂₃ Si (1095.24): C, 57.03; H, 6.81. Found: C, 57.38; H, 6.85.
Synthesis of Oligosaccharide 66
Preparation Example 57
2- (trimethylsilyl) ethyl O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O- Methyl-β-D-glucopyranosyl- (1-4) -6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranoside (63).
Compound 62 (19.7 g, 18.0 mmol) was treated according to Method 4, followed by purification with silica column (3/1, and 2/1 toluene / acetone) to give compound 63 (11.2 g, 79% over three steps). To obtain. [a] _D + 95 (C = 1.15, dichloromethane). ESIMS, positive mode: m / z + NaCl, 829 (M + Na) ⁺ ; + KF, 845 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.34-7.25 (m, 5H, Ph), 5.60 (d, 1H, J = 3.8 Hz, H-1 "), 4.96 (d, 1H, J = 3.8 Hz, H-1 ), 4.29 (d, 1H, J = 8.0 Hz, H-1 '), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Preparation 58
2- (trimethylsilyl) ethyl O- (2,3,6-tri-O-acetyl-4-Op-methoxybenzyl-α-D-glucopyranosyl)-(1-4) -O- (2, 3,6-Tri-O-acetyl-β-D-glucopyranosyl- (1-4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 -4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4) -6-O-benzyl-2,3-di-O-methyl- α-D-glucopyranoside (64).
A mixture of thioglycoside 57 (6.87 g, 9.10 mmol) and glycosyl receptor 63 (6.67 g, 8.30 mmol) was treated according to Method 3, and the residue was purified by silica column (2/5 toluene / acetone) 64 (10.7 g, 86%) is obtained. [a] _D + 90 (C = 0.83, dichloromethane). ESIMS, positive mode: m / z + NaCl, 1525 (M + Na) ⁺ ; + KF, 1541 (M + K) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.33-7.25 (m, 5H, Ph), 7.15-6.84 (m, 4H, C ₆ H ₄ OCH ₃ ), 5.56 (d, 1H, J = 3.9 Hz, H-1 " ), 5.29 (d, 1H, J = 4.0 Hz, H-1 ""), 4.97 (d, 1H, J = 8.1 Hz, H-1 "'), 4.27 (d, 1H, J = 7.9 Hz, H -1 '), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₇₀ H ₁₀₆ O ₃₃ Si (1503.69): C, 55.91; H, 7.11. Found: C, 56.05; H, 7.24.
Preparation Example 59
2- (trimethylsilyl) ethyl [O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O -methyl -β-D- glucopyranosyl _{- (1-4)] 2 -6-} O- methyl-benzyl-2,3-di -O- -α-D- glucopyranoside (65).
Compound 64 (10.7 g, 7.2 mmol) was treated according to Method 4, and the residue was purified by silica column (2/1, and 6/5 toluene / acetone) to give 65 (6.50 g, 74% over three steps). ). [α] _D + 102 (C = 0.68, dichloromethane). ¹ H NMR (CDCl ₃ ) δ 7.33-7.25 (m, 5H, Ph), 5.65 (d, 1H, J = 3.8 Hz, H-1 ""), 5.62 (d, 1H, J = 3.8 Hz, H- 1 "), 4.98 (d, 1H, J = 3.7 Hz, H-1), 4.31 (d, 1H, J = 8.1 Hz, H-1"'), 4.29 (d, 1H, J = 7.9 Hz, H -1 '), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₅₆ H ₉₈ O ₂₆ Si (1215.48): C, 55.34; H, 8.13. Found: C, 55.31; H, 8.19.
Preparation Example 60
2- (trimethylsilyl) ethyl O- (2,3,6-tri-O-acetyl-4-Op-methoxybenzyl-α-D-glucopyranosyl)-(1-4) -O- (2, 3,6-tri-O-acetyl-β-D-glucopyranosyl- (1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-( 1-4) -O- (2,3,6- tree -O- methyl -β-D- _{glucopyranosyl) - (1-4)] 2 -6} -O- -O-benzyl-2,3-di -Methyl-α-D-glucopyranoside (66).
A mixture of thioglycoside 57 (4.03 g, 5.31 mmol) and glycosyl receptor 65 (5.78 g, 4.75 mmol) was treated according to Method 3, and the residue was purified by silica column (2/5 toluene / acetone) 66 (8.63 g, 95%) is obtained. [α] _D + 94 (C = 0.74, dichloromethane). ESIMS, positive mode: monoisotopic mass = 1910.83, chemical mass = 1912.14, experimental mass = 1911.61 ± 0.12 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.25 (m, 5H, Ph), 7.15-6.83 (m, 4H , C ₆ H ₄ OCH ₃ ), 5.62 (d, 1H, J = 3.8 Hz, H-1 units R-2), 5.60 (d, 1H, J = 3.8 Hz, H-1 units NR-2), 5.30 (d, 1H, J = 4.0Hz, H-1 unit NR), 4.98 (d, 1H, J = 3.7Hz, H-1 unit R), 4.71 (d, 1H, J = 7.9Hz, H-1 unit NR-1), 4.30 (d, 1H, J = 8.1 Hz, H-1 unit C), 4.29 (d, 1H, J = 8.4 Hz, H-1 unit R-1), 2.08 (s, 3H, Ac ), 2.07 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₈₈ H ₁₃₈ O ₄₃ Si (1912.14): C, 55.28; H, 7.27. Found: C, 55.61; H, 7.35.
Synthesis of Oligosaccharide 72
Preparation Example 61
2- (trimethylsilyl) ethyl [O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O -methyl -β-D- glucopyranosyl _{- (1-4)] 3 -6-} O- methyl-benzyl-2,3-di -O- -α-D- glucopyranoside (67).
Compound 66 (8.63 g, 4.50 mmol) was treated according to Method 4 and the residue was purified by silica column (3/2 and 4/3 toluene / acetone) to give 67 (5.67 g, 77% over three steps). ). [α] _D + 102 (C = 0.70, dichloromethane). LSIMS, positive mode: m / z thioglycerol + NaCl, 1645.9 (M + Na) ⁺ . ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 5.65 (d, 2H, J = 3.8 Hz, H-1 units NR and H-1 units NR-2), 5.62 (d, 1H , J = 3.8Hz, H-1 unit R-2), 4.98 (d, 1H, J = 3.7Hz, H-1 unit R), 4.31 (d, 1H, J = 8.1Hz, H-1 unit NR- 1), 4.30 (d, 1H, J = 7.9 Hz, H-1 unit C), 4.29 (d, 1H, J = 7.9 Hz, H-1 unit R-1), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₇₄ H ₁₃₀ O ₃₆ Si (1912.14): C, 54.73; H, 8.07. Found: C, 54.61; H, 8.07.
Preparation Example 62
2- (trimethylsilyl) ethyl O- (2,3,6-tri-O-acetyl-4-Op-methoxybenzyl-α-D-glucopyranosyl)-(1-4) -O- (2, 3,6-tri-O-acetyl-β-D-glucopyranosyl- (1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-( 1-4) -O- (2,3,6- tree -O- methyl -β-D- _{glucopyranosyl) - (1-4)] 3 -6} -O- -O-benzyl-2,3-di -Methyl-α-D-glucopyranoside (68).
A mixture of thioglycoside 57 (1.47 g, 1.95 mmol) and glycosyl receptor 67 (2.89 g, 1.77 mmol) is treated according to Method 3. The residue was filtered through a silica column (3/2 cyclohexane / acetone) to give 68 (3.71 g, 92%). Analytical samples are purified on silica columns (3/2 toluene / acetone). [α] _D + 99 (C = 0.58, dichloromethane). ESIMS, positive mode: single isotope mass = 2319.03, chemical mass = 2320.59, experimental mass = 2319.03 ± 0.12 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 7.15-6.83 (m, 4H , C ₆ H ₄ OCH ₃ ), 5.62 (d, 1H, J = 3.8 Hz, H-1 units R-2), 5.60 (d, 2H, J = 3.8 Hz, H-1 units NR-2 and units C ), 5.29 (d, 1H, J = 4.0 Hz, H-1 units NR), 4.97 (d, 1H, J = 3.7 Hz, H-1 units R), 4.70 (d, 1H, J = 7.9 Hz, H -1 unit NR-1), 4.30 (d, 2H, J = 8.1 Hz, H-1 unit NR-3 and unit R-3), 4.29 (d, 1H, J = 7.9 Hz, H-1 unit R- 1), 2.08 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.96 ( s, 3H, Ac), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₁₀₆ H ₁₇₀ O ₅₃ Si (2320.59): C, 54.86; H, 7.38. Found: C, 54.76; H, 7.45.
Preparation Example 63
2- (trimethylsilyl) ethyl [O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl- (1-4)] _4-6 -O-benzyl-2,3-di-O-methyl-α-D-glucopyranoside (69).
Compound 68 (3.61 g, 1.55 mmol) was treated according to Method 4, and the residue was purified by silica column (4/5 toluene / acetone) to give 69 (2.22 g, 70% over three steps). [α] _D + 103 (C = 0.80, dichloromethane). ESIMS, positive mode: monoisotopic mass = 2031.01, chemical mass = 2032.38, experimental mass = 2032.38 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 5.65 (d, 1H, J = 3.8 Hz, H-1 units NR), 5.64 (d, 1H, J = 3.8 Hz, H-1 units NR-2), 5.62 (d, 2H, J = 3.8 Hz, H-1 units C and units R-2 ), 4.97 (d, 1H, J = 3.7 Hz, H-1 units R), 4.30 (d, 1H, J = 7.9 Hz, H-1 units NR-1), 4.29 (d, 3H, J = 7.9 Hz , H-1 unit R-1, unit R-3 and unit NR-3), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₉₂ H ₁₆₂ O ₄₆ Si (2032.38): C, 54.37; H, 8.03. Found: C, 54.71; H, 8.04.
Preparation Example 64
2- (trimethylsilyl) ethyl O- (4-O-levulinyl-2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2, 3,6-tri-O-methyl-β-D-glucopyranosyl- (1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-( 1-4) -O- (2,3,6- tree -O- methyl -β-D- _{glucopyranosyl) - (1-4)] 3 -6} -O- -O-benzyl-2,3-di -Methyl-α-D-glucopyranoside (70).
Compound 69 (855 mg, 0.42 mmol) was dissolved in dichloromethane (8 ml), triethylamine (83 μl, 0.59 mmol), 4-dimethylaminopyridine (5.14 mg, 0.04 mmol) and levulinic anhydride (117 mg, 0.55 mmol) ) Is added at 25 ° C. After stirring for 2 hours (TLC), dichloromethane is added and the mixture is washed with aqueous 10% potassium hydrogen sulfate and water, dried and evaporated to dryness. The compound will be used as a stock solution in the following step without purification. The analytical sample is purified by silica column (5/4 cyclohexane / acetone) to give pure 70. [α] _D + 101 (C = 0.89, dichloromethane). ESIMS, positive mode: single isotope mass = 2129.05, chemical mass = 2130.48, experimental mass = 2130.00 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 5.65 (d, 3H, J = 3.8 Hz, H-1 unit NR, unit NR-2 and unit C), 5.63 (d, 2H, J = 3.8 Hz, H-1 unit R-2), 5.02 (t, 1H, J = 10.1 Hz, H- 4 units NR), 4.98 (d, 1H, J = 3.7 Hz, H-1 units R), 4.32 (d, 1H, J = 7.9 Hz, H-1 units NR-1), 4.30 (d, 2H, J = 7.9 Hz, NR-3 in H-1 and R-3), 4.29 (d, 1H, J = 7.9 Hz, R-1 in H-1), 2.80-2.50 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18 (s, 3H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.08-0.91 (m, 2H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ), 0.00 (s, 9H, CH ₂ CH ₂ Si (CH ₃ ) ₃ ).
Analysis by calculations for C ₉₇ H ₁₆₈ O ₄₈ Si (2032.38): C, 54.69; H, 7.95. Found: C, 54.55; H, 8.07.
Preparation 65
O- (4-O-levulinyl-2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl- (1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tree -O- methyl -β-D- _{glucopyranosyl) - (1-4)] 3 -6} -O- methyl-benzyl-2,3-di -O- -α, β- D-glucopyranose (71).
Compound 70 (876 mg, 0.41 mmol) was treated as in Preparation 44 (a). The residue was purified by silica column to give 71 isomer mixture (a / b = 60/40) (600 mg, 50% over two steps). [a] _D + 89 (C = 0.74, dichloromethane). ESIMS, positive mode: monoisotopic mass = 2028.97, chemical mass = 2030.24, experimental mass = 2030.19 ± 0.09 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 5.65 (d, 3H, J = 3.8 Hz, H-1 unit NR, unit NR-2 and unit C), 5.63 (d, 1H, J = 3.8 Hz, H-1 unit R-2), 5.33 (d, 1H, J = 3.2 Hz, H-1α unit R), 5.02 (t, 1H, J = 10.1 Hz, H-4 unit NR), 4.59 (d, 1H, J = 5.3 Hz, H-1β unit R), 4.32 (d, 1H, J = 7.9 Hz, H-1 unit NR-1), 4.30 (d, 2H, J = 7.9 Hz, H-1 unit NR-3 and unit R-3), 4.29 (d, 1H, J = 7.9 Hz, H -1 unit R-1), 2.80-2.50 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18 (s, 3H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Preparation 66
O- (4-O-levulinyl-2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl- (1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tree -O- methyl -β-D- _{glucopyranosyl) - (1-4)] 3 -6} -O- methyl-benzyl-2,3-di -O- -α, β- D-glucopyranose trichloroacetimidadate (72).
To 71 solutions (593 mg, 0.29 mmol) in dichloromethane (7 ml) is added potassium carbonate (72.2 mg, 0.52 mmol) and trichloroacetonitrile (176 μl, 1.75 mmol). The mixture is left to stir for 16 hours, then filtered and evaporated. The residue is filtered through silica gel (5/4 cyclohexane / acetone + 1 ‰ triethylamine) to give a mixture of imidate 72 anomers (α / β = 47/53). [a] _D + 86 (C = 0.84, dichloromethane). ¹ H NMR (CDCl ₃ ) δ 7.33-7.32 (m, 5H, Ph), 6.50 (d, 1H, J = 3.5Hz, H-1α unit R), 5.65 (d, 1H, J = 8.2Hz, H- 1β, unit R), 5.65 (d, 1H, J = 3.8 Hz, H-1 unit NR), 5.64 (d, 2H, J = 3.8 Hz, H-1 unit R-2 and unit C), 5.61 (d , 1H, J = 3.8Hz, H-1 unit R-2), 5.02 (t, 1H, J = 10.1Hz, H-4 unit NR), 4.37 (d, 1H, J = 7.9Hz, H-1 unit R-1), 4.30 (d, 3H, J = 7.9 Hz, H-1 unit NR-1, unit NR-3 and unit R-3), 2.80-2.50 (m, 4H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18 (s, 3H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ).
Synthesis of Polysaccharide 73
Preparation Example 67
Methyl O- (4-O-levulinyl-2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4)-[O- (2,3,6-tri -O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4)] _3- O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl- (1-4) -O- (benzyl 2,3-di-O-methyl-β -D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1-4)- O- (benzyl 2,3-di-O-methyl-α-L-idopyranosyluronate)-(1-4) -2,3,6-tri-O-benzyl-α-D-glucopy Ranoside (73).
Glycosyl receptor 26 (220 mg, 0.16 mmol) and imidate 72 (344 mg, 0.16 mmol) are dissolved in a 1/2 dichloromethane / ethyl ether mixture (5 ml). 4 kPa molecular sieves (750 mg / mmol) are added and the mixture is left to stir at 25 ° C. for 1 hour. The mixture is cooled to -25 ° C and 1M of t-butyldimethylsilyl triflate (0.20 mol / mol imidate) in dichloromethane is added. After stirring for 15 minutes, solid sodium bicarbonate is added and the mixture is filtered and concentrated. The residue is placed on Toyopearl (tradename) HW-50 (1/1 dichloromethane / ethanol), and the glycosyl acceptor containing the fraction is returned to the reaction and treated as above. Purification was carried out continuously on silica columns (5/4 and 1/1 toluene / acetone) and fractions in 73α / β = 9/1 (201mg) and 73α / β = 7 / at a total yield of 57% (308 mg). A fraction in 3 (107 mg) is obtained. ¹ H NMR (CDCl ₃ ) δ 7.33-7.25 (m, 35H, 7Ph), 5.65 (d, 3H, J = 3.5 Hz, H-1 unit A, unit C and unit E), 5.57 (d, 1H, J = 3.9 Hz, H-1 unit G), 5.52 (d, 1H, J = 3.3 Hz, H-1 unit I), 5.29 (d, 1H, J = 6.8 Hz, H-1 unit L), 5.17 (d , 1H, J = 3.5Hz, H-1 unit K), 4.56 (d, 1H, J = 3.5Hz, H-1 unit M), 4.31 (d, 3H, J = 7.9Hz, H-1 unit B, Unit D and Unit F), 4.27 (d, 1H, J = 8.0 Hz, H-1 Unit H), 4.08 (d, 1H, J = 8.0 Hz, H-1 Unit J), 2.80-2.50 (m, 4H , O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 2.18 (s, 3H, O (C: O) CH ₂ CH ₂ (C: O) CH ₃ ), 1.97 (s, 3H , Ac), 1.81 (s, 3H, Ac).
Synthesis of Polysaccharides 74 and 75
Preparation Example 68
Methyl [O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D -Glucopyranosyl)-(1-4)] ₄ -O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (Benzyl 2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α- D-glucopyranosyl-uronate)-(1-4) -O- (benzyl 2,3-di-O-methyl-α-L-idopyranosyluronate)-(1-4) -2, 3,6-tri-O-benzyl-α-D-glucopyranoside (74).
Compound 73 (130 mg, 38.2 μmol) is treated according to Method 2, and the crude product is purified on silica to give 74 (118 mg, 93%). [α] _D + 78 (C = 0.48, dichloromethane); ESIMS, positive mode: single isotope mass = 3301.49; Chemical mass = 3303.65; Experimental mass = 3302.40 amu ¹ H NMR (CDCl ₃ ) δ 7.33-7.25 (m, 35H, 7Ph), 5.64 (d, 3H, J = 3.5 Hz, H-1 unit A, unit C and unit E), 5.57 ( d, 1H, J = 3.9Hz, H-1 unit G), 5.52 (d, 1H, J = 3.3Hz, H-1 unit I), 5.29 (d, 1H, J = 6.8Hz, H-1 unit L ), 5.17 (d, 1H, J = 3.5Hz, H-1 unit K), 4.56 (d, 1H, J = 3.5Hz, H-1 unit M), 4.31 (d, 3H, J = 7.9Hz, H -1 unit B, unit D and unit F), 4.27 (d, 1H, J = 8.0 Hz, H-1 unit H), 4.08 (d, 1H, J = 8.0 Hz, H-1 unit J), 1.97 ( s, 3H, Ac), 1.81 (s, 3H, Ac).
Preparation Example 69
Methyl O- (2,6-di-O-methyl-4-O-levulinyl-3-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2-O- Acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4) -O- (2,6-di-O-benzyl-3-O-methyl-α- D-glucopyranosyl)-(1-4) -O- (2-O-acetyl-6-O-benzyl-3-O-methyl-β-D-glucopyranosyl)-(1-4)-[ O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucose Pyranosyl)-(1-4)] ₄ -O- (6-O-benzyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (benzyl 2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D- Glucopyranosyl)-(1-4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyluronate- (1,4) -2,3,6,- Tri-O-benzyl-α-D-glucopyranoside (75).
Glycosyl receptor 74 (112 mg, 33.9 μmol) and imidate 17 (59.1 mg, 37.2 μmol) (see Preparation 16) are dissolved in toluene (2 ml). 4 cc molecular sieves (42 mg) are added and the mixture is left to stir at 25 ° C. for 1 hour. The mixture is cooled to −20 ° C. and 1 M t-butyldimethylsilyl triflate (0.20 mol / mol imidate) in toluene is added. After stirring for 15 minutes, solid sodium bicarbonate is added and the mixture is filtered and concentrated. The residue is placed in a column of TOYOPER ™ HW-50 (1/1 dichloromethane / ethanol), and the fraction containing the acceptor and 17-mer is returned to the reaction and treated as above. The product was continuously purified on a column of ToyoPharel® HW-50 and on a silica column to yield 75 (71 mg, 44%). [α] _D + 80 (C = 0.26, dichloromethane); ESIMS, positive mode: monoisotopic mass = 4728.10; Chemical mass = 4731.26; Experimental mass = 4731.27 ± 0.39 amu ¹ H NMR (CDCl ₃ ) δ of the primary anomeric protons: 5.64; 5.57; 5.52; 5.48; 5.47; 5.29; 5.17; 4.56; 4.50; 4.29; 4.27; 4.08 ppm.
Synthesis of Syntones Useful for the Synthesis of Pentasaccharide (Pe) Containing C-Internal Glycoside Bonds (Formula 2-1)
Preparation 70
Methyl 2,3-di-O-methyl-6-O-t-butyldimethylsilyl-α-D-glucopyranoside (77)
t-butyldimethylsilyl chloride (14.0 g, 92.9 mmol), triethylamine (15 ml, 108 mmol) and 4-dimethylaminopyridine (260 mg, 2.13 mmol) were solution of 76 in dichloromethane (300 ml) under argon (D. Trimmell , WM Doane, CR Russel, CE Rist, Carbohydr.Res., (1969) 11, 497) (15.84 g, 71.3 mmol). After stirring for 15 hours at room temperature, the solution is diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution, dried (with magnesium sulfate), filtered and concentrated. The residue was purified by flash chromatography (1.3 / 1 cyclohexane / ethyl acetate) to give 77 in the form of a colorless syrup (22.79 g, 95%). [α] _D + 87 (C = 1.2, chloroform)
Preparation Example 71
Methyl 2,3-di-O-methyl-6-O-t-butyldimethylsilyl-α-D-xyl-4-hexolopyranoside (78)
A solution of dimethyl sulfoxide in dichloromethane (20 ml) (8.7 ml, 123 mmol) is added to an oxalyl chloride solution (5.4 ml, 61.9 mmol) in dichloromethane (120 ml) at -70 ° C. under argon. After 15 minutes, a solution of 77 (18.78 g, 55.8 mmol) in dichloromethane is added dropwise. After magnetic stirring for 15 minutes, triethylamine (37 ml, 265 mmol) is added and after 15 minutes the mixture is returned to room temperature. Water (150 ml) is added and the aqueous phase is extracted with dichloromethane (150 ml). The organic phases are combined, washed with saturated aqueous sodium chloride solution, dried (with magnesium sulfate) and concentrated. The residue is purified by flash chromatography (9/1, and 4/1 cyclohexane / ethyl acetate) to give 78 in the form of a colorless oil. [α] _D + 98 (C = 1.0, Chloroform).
Preparation Example 72
Methyl 4-deoxy-2,3-di-O-methyl-4-C-methylene 6-O-t-butyldimethylsilyl-α-D-xyllo-hexapyranoside (79)
1.6 M n-butyllithium solution in n-hexane (75 ml) is added dropwise under argon to a suspension of methyltriphenylphosphonium bromide (43.3 g, 127 mmol) in tetrahydrofuran (250 ml). After 30 minutes at room temperature, the mixture is cooled to -70 ° C. 78 solutions (13.97 g, 41.8 mmol) in tetrahydrofuran (60 ml) were added. After 30 minutes at −70 ° C., the mixture is returned to room temperature. After 1 hour, saturated aqueous ammonium chloride solution (300 ml) is added and the aqueous phase is extracted with ether. The organic phase is dried (with magnesium sulfate), filtered and concentrated. The residue is purified by flash chromatography (7/1, and 4/1 cyclohexane / ethyl acetate) to give 79 (8.30 g, 60%) in the form of a colorless oil. [a] _D + 151 (C = 1.3, chloroform).
Preparation Example 73
Methyl 4-deoxy-2,3-di-O-methyl-4-C-methylene-α-D-xyllo-hexapyranoside (80)
Camphorsulfonic acid (pH 1) is added as a solution of 79 (8.50 g, 25.6 mmol) in a 5/1 dichloromethane / methanol mixture (250 ml). After the starting material disappears completely (TLC, 1/1 cyclohexane / ethyl acetate), the solution is neutralized by addition of triethylamine. After concentration, the residue is purified by flash chromatography (1/1, and 1/2 cyclohexane / ethyl acetate) to give 80 (5.32 g, 95%) in the form of a colorless syrup. [α] _D + 239 (C = 1.0, Chloroform).
Preparation Example 74
Phenyl 2,4,6-tri-O-acetyl-3-O-methyl-1-seleno-β-D-glucopyranoside (83).
Method 1: Selenophenol (5.7 ml, 53.7 mmol) in 81 solution of dichloromethane (120 ml) under argon (EL Hirst, E. Percival, Methods Carbohydrate Chem., (1963) 2, 145) (1/1 mixture) Anomer: 13.05 g, 36.0 mmol). The reaction medium is cooled to 0 ° C. and 48% of trifluoroborane diethyl etherate solution (8.8 ml, 71.9 mmol) is added dropwise. After 3 h at room temperature, the reaction mixture is diluted with dichloromethane (100 ml), washed with saturated aqueous sodium hydrogen carbonate solution, dried (with magnesium sulfate), filtered and concentrated. The crude product 83 (8.17 g, 68%) at 81 is used for the direct deacetylation reaction.
Method 2: Sodium borohydride (510 mg, 14.6 mmol) is added to a suspension of diphenyl diselenide (2.27 g, 7.27 mmol) in ethanol at 0 ° C. and under argon. If the reaction medium does not lose its original yellow color within 15 minutes, an additional portion of trifluoroborane diethyl etherate is added. The solution is transferred under argon to 82 solutions in dichloromethane (25 ml) (AK Sen, KK Sakar, N. Banerji, J. Carbohydr. Chem., (1988) 7, 645) (4.22 g, 11.0 mmol). After refluxing for 3 hours, the mixture is cooled to room temperature, sodium bromide is filtered off and the filtrate is concentrated. The residue is dissolved in dichloromethane (100 ml) and washed with aqueous 1 M sodium hydroxide solution (50 ml) and saturated aqueous ammonium chloride solution (50 ml). The aqueous phase is extracted with dichloromethane (20 ml) and the organic phase is dried (with magnesium sulfate), filtered and concentrated. The residue is purified by flash chromatography (1.7 / 1 cyclohexane / ethyl acetate) and crystallized in ethyl acetate to give 83 (4.35 g, 86%). mp 101-102 ° C. [α] _D -20 (C = 1.0, Chloroform)
Preparation 75
Phenyl 3-O-methyl-1-seleno-β-D-glucopyranoside (84).
The raw product 83 (32.3 g, 84.3 mmol) obtained in 82 is dissolved in methanol (500 ml) and sodium (1.2 g) is slowly added. After 1 hour, the solution is neutralized by addition of IR-120 (H ⁺ ) resin, filtered and concentrated. The residue is purified by flash chromatography (1.5 / 1/1 cyclohexane / ethylacetate / acetone) to give 84 in syrup form (22.7 g, 81 to 81%). [α] _D -58 (C = 1.0, methanol).
Preparation Example 76
Phenyl 4,6-O-benzylidene-3-O-methyl-1-seleno-β-D-glucopyranoside (85).
p-toluenesulfonic acid (45 mg) and benzaldehyde dimethyl acetal (5.4 ml, 36.0 mmol) are added to a solution of triol 84 (7.65 g, 23.0 mmol) in acetonitrile (150 ml) under argon. After stirring for 2 hours at room temperature, potassium carbonate (1.5 g) is added. After 30 minutes, the solution is filtered and concentrated. The residue is purified by flash chromatography (3.5 / 1 cyclohexane / ethyl acetate) to yield 85 (8.55 g, 88%) in the form of white crystals. mp 123-124 ° C. (cyclohexane / ethylacetate). [α] _D -38 (C = 1.0, Chloroform).
Preparation Example 77
Phenyl 4,6-O-benzylidene-3-O-methyl-2-O- (methyl-4-deoxy-6-O-dimethylsilyl-2,3-di-O-methyl-4-C-methylene -α-D-xyllo-hexopyranoside) -1-seleno-β-D-glucopyranoside (86).
1.6 M n-butyllithium solution (7.0 ml, 11.2 mmol) is added to 85 solutions (4.30 g, 10.2 mmol) in tetrahydrofuran (30 ml) placed in a Schlenk tube under argon and at −70 ° C. After 10 minutes, dichlorodimethylsilane (5.0 ml, 41.2 mmol) is added and the reaction medium is warmed to room temperature. After 3 hours, the mixture is concentrated and 80 solutions (2.10 g, 9.62 mmol) and imidazole (985 mg, 14.4 mmol) in tetrahydrofuran (20 ml) are added. After 30 minutes at room temperature, the solution is concentrated, water (50 ml) is added and the mixture is extracted with dichloromethane. The organic phase is dried (with magnesium sulfate), filtered and concentrated. 86 analytical samples are purified by flash chromatography (25/1 toluene / acetone with 0.5% triethylamine). Colorless syrup is obtained with a yield of 90%.
¹ H NMR (400 MHz, C ₆ D ₆ ) d 7.77-6.99 (m, 10H, aromatic), 5.51 (m, 1H, C: CH ₂ ), 5.24 (m, 1H, C: CH ₂ ), 5.16 (s , 1H, CHPh), 4.85 (d, 1H, J = 3.7 Hz, H-1), 4.81 (d, 1H, J = 9.8 Hz, H-1 '), 4.45 (m, 1H, H-5), 4.38 (dd, 1H, J = 10.8 Hz, 4.8 Hz, H-6a), 4.33 (dd, 1H, J = 6.2 Hz, H-6b), 4.20 (m, 1H, J = 9.2 Hz, H-3) , 4.05 (dd, 1H, J = 10.3 Hz, 4.9 Hz, H-6a '), 3.87 (dd, 1H, J = 8.1 Hz, H-2'), 3.52, 3.38, 3.31 and 3.27 (s, 3H, OCH ₃ ), 3.37 (t, 1H, J = 10.3 Hz, H-6b '), 3.35 (t, 1H, H-4'), 3.32 (dd, 1H, H-2), 3.22 (dd, 1H, J = 9.3 Hz, H-3 ′), 3.05 (ddd, 1H, J = 9.3 Hz, H-5 ′), 0.38 and 0.37 (s, 3H, Si (CH ₃ ) ₂ ). MS (m / z): 714 (M + NH ₄ ) ⁺ .
Preparation Example 78
Radical Cyclization Reaction (Formation of 87) and Degradation of Tether 88
A solution of 2,2'-azobisisobutyronitrile (200 mg, 1.22 mmol) and tributyltin hydride (6.1 ml, 22.7 mmol) in degassed toluene (14 ml) over 85 hours (10.2 mmol) and It is added as a solution of the stock solution 86 in toluene obtained at 80 (9.62 mmol).
After radical cyclization, the mixture is concentrated and the residue is dissolved in tetrahydrofuran. Excess (20 equivalents) of hydrofluoric acid (at a concentration of 40% in water) is added. After complete desilification (TLC, 4/1 toluene / acetone), the solution is neutralized by adding solid sodium bicarbonate, filtered and concentrated. The main compound 88 is purified by crystallization. mp 105 ° C. [a] _D + 119 (C = 1.1, chloroform). ¹³ C NMR (62.896 MHz, CDCl ₃ ) d 137.29 (quaternary aromatic C), 128.88-125.96 (aromatic C), 101.11 (CHPh), 97.64 (C-1), 83.52 (C-2), 82.76, 81.95, 80.84, 72.01, 71.92 and 64.31 (C-3, C-5, C-2 ', C-3', C-4 ', C-5'), 75.19 (C-1 '), 69.41 (C-6 '), 62.69 (C-6), 60.93, 60.70, 58.31 and 55.20 (OCH ₃ ), 38.80 (C-4), 25.58 (methylene C).
Analysis by calculation for C ₂₄ H ₃₆ O ₁₀ H ₂ O (502.558): C, 57.36; H, 7.62. Found: C, 57.31; H, 7.54.
Preparation Example 79
Methyl 6-O-acetyl-4-C- (2-O-acetyl-4,6-O-benzylidene-3-O-methyl-α-D-glucopyranosylmethyl) -4-deoxy-2, 3-di-O-methyl-α-D-glucopyranoside (89).
Compound 88 is quantitatively acetylated in a mixture of 1/1 acetic anhydride / pyridine in the presence of a catalytic amount of 4-dimethylaminopyridine. The product is obtained after concentration and chromatography. [a] _D + 87 (C = 1.0, Chloroform). ¹ H NMR (500MHz, CDCl ₃ ): see Table 1. ¹³ C NMR (62.896 MHz, CDCl ₃ ) d 170.81, 169.80 (C: O), 137.18 (quaternary aromatic C), 128.92-125.95 (aromatic C), 101.31 (CHPh), 97.51 (C-1), 83.22 ( C-2), 91.94, 69.25 and 63.83 (C-5, C-4 ', C-5'), 81.81 (C-3), 78.78 (C-3 '), 72.74 (C-2'), 71.96 (C-1 '), 69.49 (C-6'), 64.17 (C-6), 60.64, 59.82, 58.30 and 55.19 (OCH ₃ ), 39.00 (C-4), 26.47 (methylene C), 20.91, 20.76 (OCOCH ₃ ). Mass spectrum (m / z): 586 (M + NH ₄ ) ⁺ , 569 (M + H) ⁺ , 554 (M-OMe + NH ₃ ) ⁺ , 537 (M-OMe) ⁺ .
_{C 28 H 40 O 12 · H} 2 O Analysis by calculated for (586.632): C, 57.33; H, 7.22. Found: C, 57.28; H, 7.07.
The final part of the synthesis consists of converting 89 to imidate 90. As mentioned above, benzylidene is ring-opened using sodium cyanoborohydride and hydrochloric acid. The hydroxyl group thus liberated is temporarily protected in the form of p-methoxybenzyl ether. After deacetylation, the primary alcohol group is protected by the selective addition of t-butyldimethylsilyl ether, and the compound thus obtained is methylated. Under Jones conditions oxidation leads to euronic acid and benzylates. The p-methoxybenzyl ether is continuously degraded and the levulinic acid ester is added at this position. The system using a sulfuric acid / acetic acid / acetic anhydride mixture leads to acetolysis of not only benzylether as well as anomer methyl groups at the 6 ′ position, giving a mixture of two anomer acetates. Selective deacetylation at position 1 is carried out using hydrazine in dimethylformamide and the mixture of anomers dissolved in dichloromethane is present in the presence of 1,8-diazabicyclo [5.4.0] -unde-7-cene. Converted to 90 with trichloroacetonitrile.
Synthesis of Pentasaccharide 99
Preparation Example 80
Ethyl O- (2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-benzoyl- 1-thio-β-D-glucopyranose (92)
Benzoyl chloride (24.5 ml, 211 mmol) is added dropwise (16.7 g, 35.2 mmol) to a solution of cold (0 ° C.) compound 91 in pyridine (202 ml) over 20 minutes (J. Westman and M. Nilsson, J.). Carbohydr.Chem., 1995, 14 (7), 949-960). The reaction mixture is stirred at room temperature for 20 hours; TLC shows approximately 50% conversion. The mixture is diluted with water and dichloromethane. After extraction, the organic phase was washed with 10% sodium hydrogen carbonate solution, water, dried over magnesium sulfate and concentrated. The residue is treated again with benzoyl chloride according to the method described above. The original product is purified by chromatography on a column of silica gel to give 22 g of compound 92.
TLC: Rf = 0.80, silica gel, 9/1 v / v toluene / ethanol
Preparation Example 81
O- (2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Benzoyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranos (94).
Powdered 4 ′ molecular sieve (1.1 g) in toluene (18 ml), compound 93 (200 mg, 1.05 mmol) (Jeanioz et al., J. Org. Chem. 1961, 26, 3939-3944) and thioglycoside 92 (1.05) g, 1.05 mmol) is stirred under nitrogen atmosphere for 15 minutes. The mixture was then cooled to −20 ° C. and purified of trifluoromethanesulfonic acid (0.125 mmol) and N-iodosuccinimide (1.11 mmol) in 1/1 v / v dichloromethane / dioxane (6 ml). Freshly prepared solution is added above. After 10 minutes, the red reaction mixture is filtered, diluted with dichloromethane and extracted, washed successively with 10% sodium thiosulfate solution, 10% sodium bicarbonate solution and water, dried over magnesium sulfate and concentrated in vacuo. do. The residue is purified by chromatography on silica gel column to yield 1.25 g of compound 94.
TLC: Rf = 0.55, silica gel, 4/6 v / v heptane / ethyl acetate
Preparation Example 82
O- (4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (β-D-glucopyranosyl)-(1 → 4) -1,6-an Hydro-2,3-di-O-methyl-β-D-glucopyranose (95).
Potassium t-butoxide (about 50 mg) is added to a solution of compound 94 (1.24 g, 1.11 mmol) in 1/1 v / v methanol / dioxane (7 ml). The mixture is stirred for 1 hour, and additionally 50 mg of potassium t-butoxide is added; The mixture is stirred for an additional 60 minutes. The reaction mixture is neutralized with Doexx 50WX8 H ⁺ resin, filtered and concentrated in vacuo. After chromatography on a silica gel column, 665 mg of compound 95 is isolated in the form of an oil.
TLC: Rf = 0.50, silica gel, 85/15 v / v dichloromethane / methanol
Preparation Example 83
O- (4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranos (96).
Sodium hydride (387 mg, 9.65 mmol) is added to a cold (5 ° C.) 95 solution of compound 95 (660 mg, 1.1 mmol) in dry tetrahydrofuran (8 ml) under nitrogen atmosphere. Methyl iodide (0.51 ml, 8.22 mmol) is added dropwise and the mixture is stirred at room temperature for 20 hours. Excess sodium hydride is destroyed with methanol and the mixture is poured into 50 ml of cold water. After extraction with ethyl acetate (20 ml 3 times), the organic phase is washed with sodium chloride solution, dried over magnesium sulfate and concentrated to give 690 mg of pure compound 96.
TLC: Rf = 0.25, silica gel, 95/5 v / v dichloromethane / methanol
Preparation Example 84
O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O-2,3,6-tri-O-methyl-β-D-glucopyranosyl) -(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranose (97).
Pure Compound 96 (690 mg, 1.03 mmol) is dissolved in 80% acetic acid and stirred at 40 ° C. for 20 hours. The mixture is concentrated in vacuo and coevaporated with toluene. Chromatography of a silica gel column in 8/1/1 dichloromethane / ethylacetate / methanol afforded 569 mg of compound 97.
TLC: Rf = 0.40, silica gel, 9/1 v / v dichloromethane / methanol
Preparation Example 85
O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β -D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranose (98).
1-benzoyloxy-1H-benzotriazole (227 mg, 1.05 mmol) and triethylamine (1.15 mmol) were added to a solution of compound 97 (560 mg, 0.96 mmol) in dichloromethane, and the mixture was allowed to stand at room temperature for 20 hours. It is stirred. The reaction mixture is diluted with dichloromethane and washed with 10% sodium hydrogen carbonate solution and water. The organic phase is dried over magnesium sulfate, filtered and evaporated for dryness. The product was purified by chromatography on a silica gel column to yield 600 mg of compound 98.
TLC: Rf = 0.50, silica gel, 9/1 v / v dichloromethane / methanol
Preparation 86
O- (2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Benzoyl-β-D-glucopyranosyl)-(1 → 4) -O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl- (1 → 4)- O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D Glucopyranose (99).
Compound 98 is converted to compound 99 according to the method described in the preparation of compound 94. The coupling reaction is carried out at 5 ° C.
TLC: Rf = 0.50, silica gel, 2/8 v / v heptane / ethyl acetate.
Synthesis of Heptasaccharide 104
Preparation 87
O- (4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (β-D-glucopyranosyl)-(1 → 4) -O- (2, 3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-Anhydro-2,3-di-O-methyl-β-D-glucopyranose (100).
Compound 99 is converted to compound 100 according to the same method as described for the preparation of compound 95.
TLC: Rf = 0.35, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 88
O- (4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- ( 2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopy Lanos (101).
Compound 100 is converted to compound 101 according to the same method as described for the preparation of compound 96.
TLC: Rf = 0.50, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 89
O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl )-(1 → 4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranos (102).
Compound 101 is converted to compound 102 according to the same method as described for the preparation of compound 97.
TLC: Rf = 0.35, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation 90
O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β -D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3 , 6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranose (103 ).
Compound 102 is converted to compound 103 according to the same method as described for the preparation of compound 98.
TLC: Rf = 0.40, silica gel, 7.0 / 1.5 / 1.5 v / v / v toluene / ethylacetate / ethanol.
Preparation 91
O- (2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Benzoyl-β-D-glucopyranosyl)-(1 → 4) -O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-α-D- Glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2, 3-di-O-methyl-β-D-glucopyranose 104.
The pairing reaction of disaccharide 2 with compound 103 is carried out according to the method described in the preparation of compound 99.
TLC: Rf = 0.40, silica gel, 7.0 / 1.5 / 1.5 v / v / v toluene / ethylacetate / ethanol.
Synthesis of Oligosaccharide 109
Preparation Example 92
O- (4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (β-D-glucopyranosyl)-(1 → 4) -O- (2, 3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β- D-glucopyranosyl)-(1 → 4) -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranose (105).
Compound 104 is converted to compound 105 according to the same method as described for the preparation of compound 95.
TLC: Rf = 0.60, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 93
O- (4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₂ -1,6-anhydro-2,3-di-O-methyl-β-D Glucopyranose (106).
Compound 105 is converted to compound 106 following the same method as described for the preparation of compound 96.
TLC: Rf = 0.70, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 94
O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl )-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri- O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₂ -1,6-anhydro-2,3-di-O-methyl-β-D-glucopyranos (107).
A solution of Compound 106 (5.05 g, 2.0 mmol) in 80% acetic acid (50 ml) was stirred at 40 ° C. for 20 hours. The mixture is concentrated in vacuo and coevaporated with toluene. The residue is dissolved in ethyl acetate and extracted with water. The aqueous phase is extracted with dichloromethane and the organic phase is dried over magnesium sulfate, filtered and evaporated to dryness to give 2.68 g of compound 107.
TLC: Rf = 0.50, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 95
O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β -D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2, 3,6-Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₂ -1,6-anhydro-2,3-di-O-methyl-β-D-glucopy Lanos (108).
Compound 107 is converted to compound 108 following the same method as described for the preparation of compound 98.
TLC: Rf = 0.80, silica gel, 7.0 / 1.5 / 1.5 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 96
O- (2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Benzoyl-β-D-glucopyranosyl)-(1,4) -O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D -Glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₂ -1,6-anhydro -2,3-di-O-methyl-β-D-glucopyranose 109.
A mixture of powdered 4 ′ molecular sieve, toluene 108 (0.86 mg, 0.57 mmol) and thioglycoside 92 (1.97 g, 2.0 mmol, 3.0 eq) in toluene (22 ml) is stirred under nitrogen atmosphere for 15 minutes. Then, a newly prepared solution containing trifluoromethanesulfonic acid (0.808 mmol) and N-iodosuccinimide (496 mg, 2.2 mmol) in 1/1 v / v dichloromethane / dioxane (12 ml) was indoors. It is added dropwise at temperature. After 10 minutes, the reaction mixture is filtered, diluted with dichloromethane, extracted, washed with 10% sodium thiosulfate solution and 10% sodium bicarbonate solution, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by chromatography on a silica gel column to yield 1.09 g of compound 109.
TLC: Rf = 0.80, silica gel, 6/2/2 / v / v / v toluene / ethylacetate / ethanol.
Synthesis of Oligosaccharide 115
Preparation Example 97
O- (4,6-O-benzylidene-α-D-glucopyranosyl)-(1 → 4) -O- (β-D-glucopyranosyl)-(1 → 4) -O- (2, 3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-[(1 → 4) -O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β -D-glucopyranosyl)] _2- (1 → 4) -1,6-Anhydro-2,3-di-O-methyl-β-D-glucopyranos (110).
Compound 109 is converted to Compound 110 following the same method as described for the preparation of Compound 95.
TLC: Rf = 0.25, silica gel, 5.0 / 2.5 / 2.5 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 98
O- (4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O- Methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -1,6-anhydro-2,3-di-O-methyl-β-D Glucopyranose (111).
Compound 110 is converted to Compound 111 according to the same method as described for the preparation of Compound 96.
TLC: Rf = 0.50, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 99
O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl )-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri- O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -1,6-Anhydro-2,3-di-O-methyl-β-D-glucopyranos 112.
Compound 111 is converted to compound 112 according to the same method as described for the preparation of compound 97.
TLC: Rf = 0.20, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 100
O- (6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl- β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2 , 3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -1,6-anhydro-2,3-di-O-methyl-β-D-glu Copyranose (113).
Compound 112 is converted to compound 113 according to the same method as described for the preparation of compound 98.
TLC: Rf = 0.20, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 101
O- (6-O-benzoyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6 -Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -1,6-anhydro-2,3-di-O- Methyl-β-D-glucopyranose (114).
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (48mg, 0.25mmol), levulinic acid (29mg, 0.25mmol) and dimethylaminopyridine (4mg, 0.033mmol) dioxane (1ml) In solution 113 (320 mg, 0.167 mmol). The reaction mixture is stirred at room temperature under nitrogen atmosphere for 3 hours. Then dichloromethane and water are added and after extraction the organic phase is washed with water, dried over magnesium sulfate, filtered and concentrated. The crude product was purified by chromatography on a silica gel column to give 312 mg of compound 114.
TLC: Rf = 0.50, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 102
O- (6-O-benzoyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6 -Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -1,6-di-O-acetyl-2,3-di -O-methyl-α, β-D-glucopyranose (115).
A solution of compound 114 (312 mg, 0.155 mmol) in a mixture of acetic anhydride (2.25 ml), acetic acid (50 μl) and trifluoroacetic acid (0.14 ml) is stirred at room temperature for 4 hours. After addition of toluene (10 ml), the mixture is concentrated and co-evaporated with toluene (10 ml 3 times). After chromatography on a silica gel column, 324 mg of compound 115 are isolated.
TLC: Rf = 0.65, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Synthesis of Oligosaccharide 117
Preparation Example 103
O- (6-O-benzoyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6 -Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6- tree -O- methyl -β-D- glucopyranosyl) - (1 → 4)] 3 -6-O- acetyl-2,3--O- methyl α, β-D-glucopyranose 116.
A solution of morpholine (22.3 μl, 0.256 mmol) and compound 115 (324 mg, 0.153 mmol) in toluene (2 ml) is stirred at 35 ° C. for 4 hours. Then morpholine (22.3 μl) is added again and the reaction mixture is stirred at 35 ° C. for 20 hours. After the mixture is cooled rapidly with water and extracted with dichloromethane, the organic phase is washed successively with 0.1 N hydrochloric acid and water, dried and evaporated to dryness. After chromatography on a silica gel column, 280 mg of compound 116 are separated.
TLC: Rf = 0.45, silica gel, 6/2/2 v / v / v toluene / ethylacetate / ethanol.
Preparation Example 104
O- (6-O-benzoyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6 -Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6- tree -O- methyl -β-D- glucopyranosyl) - (1 → 4)] 3 -6-O- acetyl-2,3--O- methyl α, β-D-glucopyranose trichloroacetimidadate 117.
Trichloroacetonitrile (39 μl, 0.39 mmol) and cesium carbonate (4.7 mg) are added to a solution of compound 116 (138 mg, 0.066 mmol) in dichloromethane (1.5 ml). After stirring for 2 hours, the mixture is filtered, concentrated and the residue is chromatographed on a silica gel column to give 152 mg of imidate 117.
TLC: Rf = 0.35, silica gel, 8/1/1 v / v / v toluene / ethylacetate / ethanol.
Synthesis of Disaccharide 128
Preparation Example 105
Methyl 2-O-benzyl-4,6-O-benzylidene-α-D-glucopyranoside (119)
Compound 118 (60 g) (commercially available) is dissolved in dimethylformamide (858 ml) with benzyl bromide. After cooling to 10 ° C., an aqueous 20% sodium hydroxide solution is added dropwise. After stirring for 1 hour, the temperature is raised to 20 ° C. and the mixture is further stirred for 20 hours. The solution is then poured into a mixture of ice water and toluene and extracted. The organic phase is concentrated and the crude product is purified by crystallization to give 30.0 g of compound 119.
TLC: Rf = 0.60, silica gel, 7/3 v / v toluene / ethyl acetate.
Preparation Example 106
Methyl-2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxy-benzyl-α-D-glucopyranoside (120)
Compound 119 (26.4 g) is dissolved in dimethylformamide (211 ml) and cooled to 5 ° C. Sodium hydride (2.5 g) is added under nitrogen atmosphere. And 4-methoxybenzyl chloride (13.3 g) is added dropwise, and the mixture is stirred at room temperature for 1 hour. The mixture is washed with ethyl acetate, washed twice with water and concentrated to give 40.7 g of pure compound 120.
TLC: Rf = 0.80, silica gel, 7/3 v / v toluene / ethyl acetate.
Preparation Example 107
Methyl-2-O-benzyl-3-O-p-methoxybenzyl-α-D-glucopyranoside (121)
Compound 120 (34.9 g) is dissolved in aqueous 60% acetic acid and stirred at 60 ° C. for 4 hours. The mixture is diluted with toluene and concentrated. Purification by chromatography on a silica gel column yields 26.4 g of compound 121.
TLC: Rf = 0.07, silica gel, 7/3 v / v toluene / ethyl acetate.
Preparation Example 108
Methyl-2-O-benzyl-3-O-p-methoxybenzyl-6-O-methyl-α-D-glucopyranoside (122)
Compound 121 (26.4 g) is dissolved in dichloromethane (263 ml) under nitrogen atmosphere. Trimethyloxonium tetrafluoroborate (11.6 g) and 2,6-di-t-butyl-4-methylpyridine (17.4 g) are added at room temperature. After 4 hours, the mixture is poured into ice water and extracted with dichloromethane. The organic phase is washed with sodium hydrogen carbonate and concentrated. Purification of the stock solution by chromatography on a silica gel column yielded 18.5 g of compound 122.
TLC: Rf = 0.25, silica gel, 7/3 v / v toluene / ethyl acetate.
Preparation Example 109
Ethyl-2,4,6-tri-O-acetyl-3-O-methyl-1-thio-α-L-idopyranose (124)
Compound 123 (1,2,4,6-tetra-O-acetyl-3-O-methyl-α-L-idopyranose) (Jaurand et. Al. Bio. Med. Chem. Lett. 1992, 2, 897 -900) (48.4 g) is dissolved in toluene (175 ml). Boron trifluoride etherate and ethanethiol (20 ml) in toluene (134 ml) are added under nitrogen atmosphere. After stirring for 1 hour, aqueous sodium hydrogen carbonate (400 ml) is added and the mixture is stirred for an additional 1 hour. And the mixture is poured into ethyl acetate. The organic phase is washed twice with water and concentrated. Purification by chromatography on a silica gel column yields 29.6 g of Compound 124.
TLC: Rf = 0.45, silica gel, 6/4 v / v toluene / ethyl acetate.
Preparation 110
Methyl O- (2,4,6-tri-O-acetyl-3-O-methyl-α-L-idopyranosyl- (1 → 4) -2-O-benzyl-3-Op-methoxybenzyl -6-O-methyl-α-D-glucopyranoside (125)
Compound 122 (17.5 g) and Compound 124 (28.2 g) are dissolved in toluene (525 ml) under nitrogen atmosphere. After addition of 4 cc molecular sieve, the reaction is cooled to -20 ° C. A freshly prepared solution of trifluoromethanesulfonic acid (1.38 ml) and 0.1 M N-iodosuccinimide (17.4 g) in 1/1 v / v dioxane / dichloromethane was added dropwise under continuous flow of nitrogen. Is added. After 10 minutes, the red reaction mixture is filtered and washed successively with aqueous sodium thiosulfate and aqueous sodium hydrogen carbonate. The organic phase is concentrated in vacuo and 30.0 g of compound 125 is separated.
TLC: Rf = 0.45, silica gel, 8/2 v / v dichloromethane / ethyl acetate.
Preparation Example 111
Methyl O- (3-O-methyl-α-L-idopyranosyl- (1 → 4) -2-O-benzyl-3-Op-methoxybenzyl-6-O-methyl-α-D-glu Copyranoside (126)
Compound 125 (30.0 g) is dissolved in 460 ml of 1/1 v / v methanol / dioxane and potassium t-butoxide is added. After 15 minutes, the mixture is neutralized with Dowx® 50WX8H ⁺ resin and concentrated in vacuo. Purification by chromatography on a column of silica gel provided 17.4 g of compound 126.
TLC: Rf = 0.25, silica gel, 95/5 v / v dichloromethane / methanol.
Preparation Example 112
Methyl O- (4,6-O-isopropylidene-3-O-methyl-α-L-idopyranosyl)-(1 → 4) -2-O-benzyl-3-Op-methoxybenzyl- 6-O-methyl-α-D-glucopyranoside (127)
Compound 126 (17.4 g) is dissolved in dimethylformamide (77 ml) under nitrogen atmosphere. 2,2-dimethoxypropane (26 ml) and p-toluenesulfonic acid are added and the mixture is stirred for 30 minutes. The mixture was diluted with aqueous sodium hydrogen carbonate, extracted with ethyl acetate, and the solvent was evaporated to yield 19.7 g of compound 127.
TLC: Rf = 0.45, silica gel, 95/5 v / v dichloromethane / methanol.
Preparation Example 113
Methyl O- (4,6-O-isopropylidene-2,3-di-O-methyl-α-L-idopyranosyl)-(1 → 4) -2-O-benzyl-3-O- Methoxybenzyl-6-O-methyl-α-D-glucopyranoside (128)
Compound 127 (18.5 g) is dissolved in dimethylformamide (24.4 ml) and cooled to 0 ° C. Sodium hydride (1.47 g; 60% dispersion in oil) and iodomethane (2.36 ml) are added under nitrogen atmosphere. After 1 hour excess sodium hydride is decomposed into methanol and the mixture is extracted with dichloromethane and concentrated to give 20.0 g of compound 128.
TLC: Rf = 0.85, silica gel, 95/5 v / v dichloromethane / methanol.
Synthesis of Disaccharide 138
Preparation Example 114
Methyl O- (4,6-O-isopropylidene-2,3-di-O-methyl-α-L-idopyranosyl)-(1 → 4) -2-O-benzyl-6-O- Methyl-α-D-glucopyranoside (129)
Compound 128 (18.4 g) is dissolved in dichloromethane (838 ml) and water (168 ml). 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (7.1 g) is added and the mixture is stirred at 4 ° C. for 18 hours. The mixture is poured into aqueous sodium hydrogen carbonate and extracted with dichloromethane. The organic phase is concentrated to give 12.7 g of compound 129.
TLC: Rf = 0.40, silica gel, 95/5 v / v dichloromethane / methanol.
Preparation Example 115
Methyl O- (4,6-O-isopropylidene-2,3-di-O-methyl-α-L-idopyranosyl)-(1 → 4) -2,3-di-O-benzyl- 6-O-methyl-α-D-glucopyranoside (130)
Compound 129 (10.5 g) is dissolved in dry dimethylformamide (178 ml) and cooled to 0 ° C. under nitrogen atmosphere. Sodium hydride (1.91 g; 60% dispersion in oil) is added and benzyl bromide (3.3 ml) is added dropwise. After 30 minutes, the reaction is complete and excess sodium hydride is decomposed into methanol. Water is added and the mixture is extracted twice with ethyl acetate. Evaporating the solvent yields 13.6 g of compound 130.
TLC: Rf = 0.50, silica gel, 1/1 v / v toluene / ethyl acetate.
Preparation Example 116
Methyl O- (2,3-di-O-methyl-α-L-idopyranosyl)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D- Glucopyranoside (131)
Compound 130 is dissolved in 77/33 (v / v) acetic acid / water and stirred over night. The mixture is co-evaporated twice with toluene and purified by chromatography on a silica gel column to give 11.5 g of compound 131.
TLC: Rf = 0.09, silica gel, 1/1 v / v toluene / ethyl acetate.
Rf = 0.68, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 117
Methyl O- (2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D Glucopyranoside (132)
2,2,6,6-tetramethyl-1-piperidinyloxy [lacuna] (33 mg), sodium bicarbonate solution (40 ml), potassium bromide (218 mg) and tetrabutylammonium chloride (289 mg) were dichloromethane (60 ml In 131 solution (11.6 g). The mixture is cooled to 0 ° C. and a mixture of saturated sodium chloride solution (44 ml), saturated sodium bicarbonate solution (21.8 ml) and sodium hypochlorite (1.3 M, 50 ml) is added over 15 minutes. After stirring for 1 hour, the mixture is diluted with water and extracted with dichloromethane (3 times). The organic phase is washed with an aqueous sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to dryness to give 13.4 g of crude product 132.
TLC: Rf = 0.14, silica gel, 9/1 v / v dichloromethane / methanol.
Preparation Example 118
Methyl O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyluronate)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl- α-D-glucopyranoside (133)
Compound 132 is dissolved in dimethylformamide (110 ml) under nitrogen atmosphere. Potassium hydrogen carbonate (6.7 g) and benzyl bromide (10.7 ml) are added and the mixture is stirred for 90 minutes. Ethyl acetate and water are added and after extraction, the organic phase is concentrated. Purification by chromatography on a silica gel column yields 9.9 g of compound 133.
TLC: Rf = 0.43, silica gel, 4/6 v / v toluene / ethyl acetate.
Preparation Example 119
Methyl O- (benzyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α -D-glucopyranoside (134)
Compound 133 (9.9 g) is dissolved in 300 ml of methanol and heated while refluxing under nitrogen atmosphere. A solution of 1M sodium methoxide in methanol (65.2 ml) is added dropwise and the mixture is stirred and heated with reflux for 3 hours. The mixture is then cooled to room temperature, 1N sodium hydroxide (22.2 ml) is added and the reaction mixture is stirred for an additional 90 minutes. After neutralizing with Dowex 50WX8H ⁺ resin, it is filtered and the mixture is concentrated. Pure product is dissolved in dimethylformamide (192 ml) and molecular sieve is added under nitrogen atmosphere. Potassium hydrogen carbonate (3.2 g) and benzyl bromide (4.8 ml) are added and the mixture is stirred for 5 hours. After ethyl acetate and water are added, the two phases are extracted and separated and the organic phase is concentrated. The crude product is purified by chromatography on a silica gel column to provide 6.19 g of compound 134 and 1.88 g of starting compound 133.
TLC: Rf = 0.55, silica gel, 4/6 v / v toluene / ethyl acetate.
Preparation Example 120
Methyl O- (benzyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -2,3-di-O-benzyl -6-O-methyl-α-D-glucopyranoside (135)
Compound 134 (6.2 g) is dissolved in 40 ml of dioxane. Levulinic acid (2.1 g), dicyclohexylcarbodiimide (3.75 g) and 4-dimethylaminopyridine (0.2 g) are added and the mixture is stirred under nitrogen atmosphere for 2 hours. Diethyl ether (95 ml) is added and the precipitate is filtered off. The filtrate is washed with aqueous potassium hydrogen sulfate, dried over magnesium sulfate, filtered and concentrated. Crystallization in ether / heptane yields 6.2 g of compound 135.
TLC: Rf = 0.26, silica gel, 95/5 v / v dichloromethane / acetone.
Preparation Example 121
O- (benzyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -1,3-di-O-acetyl- 2-O-benzyl-6-O-methyl-α, β-D-glucopyranose (136)
Compound 135 (6.1 g) is dissolved in acetic anhydride (256 ml) under nitrogen atmosphere and cooled to -20 ° C. A mixture of sulfuric acid (4.9 ml) in acetic anhydride (49 ml) is added dropwise over 30 minutes. After 60 minutes, sodium acetate is added until a mixture with neutral pH is obtained. And ethyl acetate and water are added, and the organic phase is concentrated. Purification by chromatography on a column of silica gel affords 4.2 g of compound 136.
TLC: Rf = 0.24, silica gel, 8/2 v / v dichloromethane / ethyl acetate.
Preparation Example 122
O- (benzyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -3-O-acetyl-2-O- Benzyl-6-O-methyl-α, β-D-glucopyranose (137)
Compound 136 (4.2 g) is dissolved in tetrahydrofuran (42 ml) and piperidine (4.1 ml) is added. The mixture is stirred at room temperature overnight. Ethyl acetate is added and the mixture is washed with 0.5N hydrochloric acid. The organic phase is concentrated and the residue is purified by chromatography on silica gel column to give 3.2 g of compound 137.
TLC: Rf = 0.33, silica gel, 1/1 v / v dichloromethane / ethyl acetate.
Preparation Example 123
O- (benzyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyluronate)-(1 → 4) -3-O-acetyl-2-O- Benzyl-6-O-methyl-α-D-glucopyranose trichloroacetimidadate (138)
Compound 137 (1.59 g) is dissolved in dry dichloromethane under nitrogen atmosphere. Trichloroacetonitrile (1.1 ml) and cesium carbonate (72 mg) are added and the mixture is stirred for 1 hour. Cesium carbonate is filtered off and the filtrate is concentrated. Purification by chromatography on a silica gel column yields 1.57 g of compound 138.
TLC: Rf = 0.60, silica gel, 3/7 v / v toluene / ethyl acetate.
Synthesis of Tetrasaccharide 140
Preparation Example 124
Methyl O- (benzyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -O- (3-O-acetyl- 2-O-benzyl-6-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyl Uronate)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D-glucopyranoside (139)
A mixture of compound 133 (300 mg) and compound 138 (455.6 mg) is co-evaporated with toluene and dissolved in dichloromethane (6 ml) under nitrogen atmosphere. After addition of 4mm molecular sieve, the mixture is cooled to -20 ° C. After stirring for 20 minutes, trimethylsilyl trifluoromethanesulfonate (15 mol% relative to compound 138) is added. After 10 minutes, the mixture is cooled with aqueous sodium hydrogen carbonate. After filtration of the molecular sieve, the filtrate is diluted with dichloromethane, washed with water, concentrated and purified by chromatography on a silica gel column to give 560 mg of compound 139.
TLC: Rf = 0.50, silica gel, 3/7 v / v toluene / ethyl acetate.
Preparation Example 125
Methyl O- (benzyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -O- (3-O-acetyl-2-O-benzyl-6- O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyluronate)-(1 → 4 ) -2,3-di-O-benzyl-6-O-methyl-α-D-glucopyranoside (140)
Compound 139 (532.6 mg) is dissolved in pyridine (1.9 ml) and a mixture of hydrazine hydrate (0.3 ml) and acetic acid (2.4 ml) in pyridine (1.9 ml) is added at room temperature. After stirring for 9 minutes, dichloromethane and water are added. The organic phase is separated and washed successively with 0.1 N hydrochloric acid, aqueous sodium hydrogen carbonate and water. The organic phase is concentrated and purified by chromatography on silica gel column to give 451 mg of compound 140.
TLC: Rf = 0.45, silica gel, 3/7 v / v toluene / ethyl acetate.
Synthesis of Polysaccharide 142
Preparation Example 126
Methyl O- (6-O-benzoyl-4-O-levulinyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3, 6-Tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O- (6-O-acetyl-2,3-di -O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl 2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -O- (3-O-acetyl-2-O-benzyl-6-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O- Methyl-α-L-idopyranosyluronate)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D-glucopyranoside (141)
A mixture of compound 117 (144 mg, 0.064 mmol) and compound 140 (76 g, 0.058 mmol) is co-evaporated with toluene and dissolved in 1/2 v / v dichloromethane / diethyl ether (3.0 ml). 4 mm molecular sieve (140 mg) is added under nitrogen atmosphere and the mixture is cooled to 0 ° C. t-butyldimethylsilyl trifluoromethanesulfonate (128 μl of 0.1 mol solution in dichloromethane) is added and after 15 minutes the mixture is cooled with sodium hydrogen carbonate solution. After extraction with water and dichloromethane, the organic phase is dried and concentrated. The product is first purified by chromatography on Sephadex LH 20 (1/1 v / v dichloromethane / methanol) and chromatography on a silica gel column to give 124 mg of compound 141 at an α / β ratio of 8/2.
TLC: Rf = 0.60, silica gel, 1/1 v / v toluene / acetone.
Preparation Example 127
Methyl O- (6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl- β-D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2 , 3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O- (6-O-acetyl-2,3-di-O-methyl-α-D -Glucopyranosyl)-(1 → 4) -O- (benzyl 2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -O- (3-O- Acetyl-2-O-benzyl-6-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O-methyl-α-L-ido Pyranosyluronate)-(1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D-glucopyranoside (142)
Compound 141 is converted to compound 142 according to the method described in compound 140.
Compound 142 is isolated at an α / β ratio of 8/2.
TLC: Rf = 0.45, silica gel, 1/1 v / v toluene / acetone.
Synthesis of Trisaccharide 147
Preparation Example 128
O- (2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-acetyl-α-D -Glucopyranosyl)-(1 → 4) -1,2,3,6-tetra-O-acetyl-β-D-glucopyranose (144)
Maltotriose (7 g, 13.9 mmol) (commercially available) is added dropwise to a suspension of sodium acetate (7 g, 85 mmol) in acetic anhydride (70 ml) at 155 ° C. After 15 minutes, the clear solution is cooled and cooled with cold water (700 ml). After extraction with ethyl acetate, the organic phase is washed with water, dried over magnesium sulfate, filtered and concentrated to give 13.1 g of compound 144.
TLC: Rf = 0.53, silica gel, 7/3 v / v dichloromethane / ethyl acetate.
Preparation 129
Ethyl O- (2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-acetyl-α- D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (145)
Compound 144 (13 g, 13.5 mmol) is dissolved in toluene (80 ml). Ethane thiol (1.97 ml, 26.9 mmol) and boron trifluoride diethyl etherate (13.7 ml of 1 mol solution in toluene) are added under nitrogen atmosphere. After stirring for 60 hours, the mixture is diluted with water and dichloromethane. After extraction, the organic phase is washed with 10% sodium bicarbonate and water, dried, filtered and concentrated. The crude product is purified by chromatography on a silica gel column to give 8.6 g of compound 145.
TLC: Rf = 0.60, silica gel, 7/3 v / v dichloromethane / ethyl acetate.
Preparation Example 130
Ethyl O- (α-D-glucopyranosyl)-(1 → 4) -O- (α-D-glucopyranosyl)-(1 → 4) -1-thio-β-D-glucopyranoside ( 146)
Compound 145 is converted to compound 146 according to the method described in compound 95.
TLC: Rf = 0.80, silica gel, 13/7 / 1.6 / 4 v / v / v / v ethylacetate / pyridine / acetic acid / water.
Preparation Example 131
Ethyl O- (2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-benzoyl-α- D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (147)
Compound 146 is converted to compound 147 according to the method described in compound 92.
TLC: Rf = 0.50, silica gel, 9/1 v / v toluene / ethyl acetate.
Synthesis of Polysaccharide 150
Preparation Example 132
Methyl O- (2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-benzoyl-α- D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1 → 4) -O- (6-O- Benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl) -(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -O- (3-O-acetyl-2-O-benzyl -6-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl-2,3-di-O-methyl-α-L-idopyranosyluronate)-( 1 → 4) -2,3-di-O-benzyl-6-O-methyl-α-D-glucopyranoside (148)
Thioglycoside 147 (105 mg, 0.066 mmol) and receptor 142 (55 mg, 0.017 mmol), (α / β of 8/2) are coupled according to the method described for compound 109. The product was purified by chromatography on Sephadex LH 20 (1/1 dichloromethane / methanol) and chromatography on a column of silica gel to give 49 mg of compound 148.
TLC: Rf = 0.30, silica gel, 85 / 7.5 / 7.5 v / v / v diethyl ether / ethylacetate / ethanol.
Preparation Example 133
Methyl O- (2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-benzoyl-α- D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1 → 4) -O- (6-O- Benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl) -(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O- (6-O-acetyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1 → 4) -O- (3-O-acetyl-6-O-methyl-α -D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -6-O-methyl- α-D-glucopyranoside (149)
A solution of compound 148 (47 mg, 0.01 mmol) in ethyl acetate (10 ml) is stirred under nitrogen atmosphere in the presence of 10% palladium (90% w / w for compound 148) on charcoal for 3 hours and filtered. The filtrate was concentrated to give 42 mg of compound 149.
TLC: Rf = 0.35, silica gel, 20/7 / 1.6 / 4 v / v / v / v ethylacetate / pyridine / acetic acid / water.
Preparation Example 134
Methyl O- (α-D-glucopyranosyl)-(1 → 4) -O- (α-D-glucopyranosyl)-(1 → 4) -O- (β-D-glucopyranosyl)-( 1 → 4) -O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β- D-glucopyranosyl)-(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3 , 6-tri-O-methyl-β-D-glucopyranosyl)-(1 → 4)] ₃ -O- (2,3-di-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1 → 4) -O- (6-O-methyl-α-D-glucopyranosyl )-(1 → 4) -O- (2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -6-O-methyl-α-D-glucopy Ranozid (150)
A mixture of methanol (0.22 ml) and 0.66 N sodium hydroxide solution is added to compound 149 (41 mg, 0.01 mmol) and stirred at room temperature for 20 hours. The mixture is diluted with water and acidified with 0.5N of 0.5N hydrochloric acid solution to obtain a pH of 6.5. After concentration, the pure product is desalted on a Sephadex G-25 column using 9/1 v / v water / acetonitrile. Hexadecsaccharide fractions are combined and lyophilized to give 26 mg of compound 150 as an amorphous white powder.
TLC: Rf = 0.35, silica gel, 8/7 / 1.6 / 4 v / v / v / v ethylacetate / pyridine / acetic acid / water.
Preparation Example 135
6-O-t-butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl-α-D-glucofuranose (152)
Diol 151 (10 g, 42.7 mmol) was obtained in anhydrous dichloromethane (100 ml), and t-butyldimethylsilyl chloride (7.1 g, 47.3 mmol) and imidazole (5.8 g, 85.3 mmol) were added. The reaction mixture is stirred at room temperature. After 2 hours, the mixture is diluted with dichloromethane and washed with water. The organic phase is dried over magnesium sulfate and concentrated, and the residue is purified by chromatography on a silica gel column (1/9 v / v ethyl acetate / cyclohexane) to give the desired product 152 (11.9 g, 80%) in the form of a syrup. Obtained.
[α] _D -34 ° (c 1.9, CHCl ₃ )
Preparation Example 136
6-O-t-butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl-5-C-vinyl-α-D-glucofuranos (154)
Oxalyl chloride (3.2 ml, 36.8 mmol) and dimethyl sulfoxide (5.2 ml, 73.4 mmol) are added to anhydrous dichloromethane (40 ml) at −78 ° C. and the mixture is stirred for 30 minutes. Then compound 152 (6.4 g, 18.4 mmol) is added and the mixture is stirred for an additional 1 hour. Triethylamine (15.3 ml, 110.0 mmol) was added and after 30 minutes the reaction mixture was diluted with dichloromethane. The 5-urose compound 153 is obtained by standard methods and is used directly in the next reaction. The original ketone 153 is obtained in anhydrous tetrahydrofuran (100 ml), and 1 M of vinyl-magnesium bromide solution (28 ml, 27.6 mmol) in tetrahydrofuran is added at 0 ° C. After 1 hour, the reaction mixture is diluted with ammonium chloride and washed with water. The organic phase was dried over magnesium sulfate, concentrated and the residue was purified by chromatography on a silica gel column (1/9 v / v ethyl acetate / cyclohexane) to give the desired product 154 (4.8 g, 70%) in the form of syrup. Obtained.
[α] _D -40 ° (c 1.3, CHCl ₃ )
Analysis by calculation: C, 57.72, H, 9.15. Actual value: C, 57.77, H, 9.23
Preparation Example 137
1,2,4,6-tetra-O-acetyl-3-O-methyl-5-C-vinyl-β-D-glucopyranose (156)
Compound 154 (3.5 g, 9.4 mmol) was obtained in water (50 ml); IR-120 resin (1 g) is added above and the mixture is heated to a temperature of 80 ° C. for 6 hours. The resin is filtered off and the filtrate is concentrated. The crude product 155 is acetylated using acetic anhydride (12 ml) and pyridine (13 ml). Excess acetic anhydride is broken down into methanol and the solvent is concentrated. The residue is extracted with water and dichloromethane. The organic phase is dried over magnesium sulfate, concentrated and purified by chromatography on a silica gel column (3/2 v / v ethyl acetate / cyclohexane) to give tetraacetate compound 156 in solid form (75%, 2.7 g). m.p. 50 ° C.
[α] _D −84 ° (c 1.6, CHCl ₃ )
Analysis by calculation: C, 52.47, H, 6.19. Actual value: C, 52.51, H, 6.19
Cl-MS: 406 (M + NH ₄ ), 389 (M + l)
Preparation Example 138
Methyl 2,3,6-tri-O-benzyl-4-O- (2,4,6-tri-O-acetyl-3-O-methyl-5-C-vinyl-β-D-glucopyranosyl) -α-D-glucopyranoside (158)
Compound 156 (1.6 g, 4.1 mmol) and compound 157 (2.1 g, 4.5 mmol) (PJ Garegg and H. Hultberg, Carbohydr. Res. 1981, 93, C10) are dissolved in anhydrous dichloromethane (50 ml) and the molecular sieve (4.0 g) is added. The reaction mixture is stirred at room temperature for 1 hour and TMSOTf (0.95 ml, 5.2 mmol) is added at -78 ° C. The reaction mixture is then slowly raised to room temperature. After 2 hours, the reaction mixture is neutralized with triethylamine and filtered through celite; The filtrate is washed with water. The organic phase is dried over magnesium sulfate, concentrated and the residue is purified by chromatography on silica (4/1 v / v ethyl acetate / cyclohexane) to give the desired compound 158 (2.77 g, 85%) in solid form. To obtain. m.p. 47 ° C.
[α] _D -36 ° (c 0.6, CHCl ₃ )
Analysis by calculation: C, 65.14, H, 6.61. Actual value: C, 65.09, H, 6.70
Preparation Example 139
Methyl 2,3,6-O-tri-O-benzyl-4-O- (4,6-O-isopropylidene-3-O-methyl-5-C-vinyl-β-D-glucopyranosyl) -α-D-glucopyranoside (160)
Compound 158 (2.7 g, 3.4 mmol) is dissolved in methanol (40 ml). Sodium (catalyst) is added at 0 ° C. and the mixture is stirred at room temperature for 3 hours. The solvent is concentrated and the residue 159 is obtained in anhydrous acetone (40 ml) and 2,2-dimethoxypropane (2 ml) and p-toluenesulfonic acid (catalyst) are added. The reaction mixture is stirred at room temperature overnight. The solvent is evaporated and the residue is taken up with chloroform and washed with water. The organic phase is dried over magnesium sulfate, concentrated, and the residue is purified by chromatography on a column of silica (1/1 v / v ethyl acetate / cyclohexane) to give 4 ', 6'-isopropylidene in solid form. -O-derivative 160 (1.7 g, 70%) is obtained. m.p. 55 ° C.
[α] _D + 13 ° (c 0.8, CHCl ₃ )
Analytical calculation: C, 67.97, H, 7.13. Actual value: C, 67.87, H, 7.16
Cl-MS: 707 (M + 1), 724 (M + NH ₄ ).
Preparation 140
Methyl 2,3,6-tri-O-benzyl-4-O- (4,6-O-isopropylidene-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α -D-glucopyranoside (162)
Anhydrous DMSO (0.57 ml, 8.0 mmol) and oxalyl chloride (0.35 ml, 4.0 mmol) in anhydrous dichloromethane (10 ml) are stirred at −78 ° C. for 30 minutes. Compound 160 (1.4 g, 2.0 mmol) in anhydrous dichloromethane (10 ml) is added to the solution and the mixture is stirred for an additional 45 minutes. The reaction mixture is neutralized by adding anhydrous triethylamine (1.7 ml, 12.0 mmol) and diluted with dichloromethane. After washing with water, the organic phase is dried over magnesium sulfate, concentrated and the residue 161 is used directly in the next reaction without purification. Ketone 161 is obtained in anhydrous tetrahydrofuran (15 ml) and 1N super hydride solution in tetrahydrofuran (4 ml, 4.0 mmol) is added at -78 ° C. The reaction mixture is stirred for 1 h at room temperature and 5% sodium hydroxide (2 ml) and hydrogen peroxide (1 ml) are added. The solvent is evaporated and the residue is taken up in diethyl acetate and washed with water. The organic phase is dried over magnesium sulfate, concentrated and the residue is purified by chromatography (2/1 v / v ethyl acetate / cyclohexane) to give compound 162 (1.0 g, 70%).
[α] _D −11 ° (c 0.5, CHCl ₃ ).
Cl-MS: 724 (M + 18), 707 (M + l).
Preparation Example 141
Methyl 2,3,6-tri-O-benzyl-4-O- (2-O-acetyl-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α-D-glu Copyranoside (164)
Compound 162 (940 mg, 1.3 mmol) is dissolved in pyridine (3 ml) and acetic anhydride (0.3 ml) is added. The reaction mixture is stirred at room temperature for 3 hours. Excess pyridine and acetic anhydride are evaporated and the residue 163 is used directly for dehydrogenation of isopropylidene using 80% acetic acid (5 ml) at 60 ° C. for 2 hours. Excess acetic acid is evaporated and the residue is purified by chromatography on a silica gel column (4/1 v / v ethyl acetate / cyclohexane) to give diol 164 (660 mg, 70%) in solid form.
[α] _D −10 ° (c 0.8, CHCl ₃ ).
Cl-MS: 709 (M + 1), 726 (M + 18).
Preparation Example 142
Methyl 2,3,6-tri-O-benzyl-4- (2-O-acetyl-3-O-methyl-6-O-tosyl-5-C-vinyl-β-D-mannopyranosyl) -α -D-glucopyranose (165)
Compound 164 (600 mg, 0.9 mmol) is dissolved in pyridine (3 ml) and tosyl chloride (240 mg, 1.3 mmol) is added. The reaction mixture is stirred at room temperature for 3 hours. The solvent is evaporated and the residue is diluted with chloroform and washed with water. The organic phase was dried over magnesium sulfate and concentrated, and the residue was purified by chromatography on a silica gel column (1/1 v / v ethyl acetate / cyclohexane) to give syrup form tosyl compound 165 (297 mg, 80%). To obtain.
[α] _D −26 ° (c 0.8, CHCl ₃ ).
Preparation Example 143
Methyl 2,3,6-tri-O-benzyl-4- (2,6-anhydro-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α-D-glucopy Rano seed (166)
Compound 165 (550 mg, 0.6 mmol) was obtained in ethanol (3 ml), and 0.1 N ethanol sodium hydroxide (5 ml) was added. The reaction mixture is heated at 70 ° C. for 3 hours, neutralized with IR-120 resin (H ⁺ type) and filtered through celite. After concentration, the residue is purified by chromatography on a silica gel column (1/1 v / v ethyl acetate / cyclohexane) to give compound 166 (292 mg, 70%) in syrup form.
[α] _D + 13 ° (c 0.5, CHCl ₃ ).
Cl-MS: 666 (M + 18).
Preparation Example 144
Methyl 2,3,6-tri-O-benzyl-4- (benzyl 3-O-methyl-2-O-5-C-methylidene-α-L-idopyranouronate) -α-D- Glucopyranoside (167)
Compound 166 (260 mg, 0.4 mmol) is dissolved in dichloromethane (20 ml), the solution is stirred at −78 ° C. and ozone is bubbled for 30 seconds. The color of the solution is pale yellow. Dimethylsulfide is added into the solution and the reaction mixture is washed with water. The organic phase is dried over magnesium sulphate and concentrated to go directly to the next reaction without further purification. Raw aldehyde is obtained in t-butanol (16 ml) and 2-methyl-2-butene (5 ml) and water (16 ml) are added. NaH ₂ PO ₄ (700 mg) and NaClO ₂ (700 mg) are added successively in the mixture. The suspension is coarse stirred at room temperature for one night, diluted with water and extracted with ethyl acetate. The organic phase is dried over magnesium sulfate, concentrated and goes directly to the next reaction. Crude acid is obtained in dimethyl formamide (25 ml) and tetrabutyl ammonium iodide (0.7 g, 2.0 mmol), potassium bicarbonate (0.25 g, 2.5 mmol) and benzyl bromide (0.250 ml, 2.1 mmol) are added. The reaction mixture is stirred at room temperature for 5 hours. The reaction mixture is extracted with water and ether. The ether phase is dried over magnesium sulfate and concentrated, and the residue is purified by chromatography on a silica gel column (2/1 v / v ethyl acetate / cyclohexane) to give a derivative 167 (236 mg, 80%) in the form of a syrup. do.
Cl-MS: 774 (M + 18).
Synthesis of Sintones Useful for the Synthesis of Pentasaccharide Pe to Lock the Form of L-Iduronic Acid

Example 1
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6- Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α--D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] ₆ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idouronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt (168).
Compound 31 is treated according to Method 5, giving 168 (80% over three steps). [α] _D +41 (c = 0.8, water). ESIMS, negative mode: homoisomer mass = 7133.26; Chemical mass = 7138.90; Experimental mass = 7137.26 ± 0.0 amu ¹ H NMR (D ₂ O) δ of the primary anomeric protons: 5.71; 5.48; 5.46; 5.44; 5.17; 5.08; 4.81; 4.78; 4.67 ppm.
In the same manner, compounds 169 and 170 are obtained.
Example 4
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1-4)-[O- (2,3-di-O -Methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4)] _15- O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)- (1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-α -L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt (171).
Compound 51 (55 mg, 10.5 mmol) was treated according to Method 5 to give sulphate product 187 (50 mg, 77% over three steps) after lyophilization. [α] _D +10 7 (c = 0.52, water). ESIMS, positive mode: monoisomer mass = 6194.16; Chemical mass = 6198.83; Experimental mass = 6195.33 ± 1.79 ¹ H NMR (D ₂ O) δ: 5.71 of the primary anomer proton; 5.67; 5.48; 5.43; 5.17; 5.10; 4.68 ppm.
In the same manner, compounds 172 and 173 are obtained.
Example 7
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6- Di-O-sulfo-β-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl)-( 1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (2,3,6-tree -O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4)] ₄ -O- (2,3-di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β -D-glucopyranosyluronic acid)-(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2 , 3-di-O-methyl-α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt ( 174).
Compound 75 is treated according to Method 5 to yield 174 (84% over three steps). [α] _D + 62 (c = 0.46, water). ESIMS, positive mode: monoisomer mass = 4966.39; Chemical mass = 4970.04; Experimental mass = 4969.63 ± 0.78 amu ¹ H NMR (D ₂ O) δ of the primary anomeric protons: 5.69; 5.63; 5.57; 5.46; 5.44; 5.41; 5.15; 5.06; 4.79; 4.66; 4.62; 4.41 ppm.
Using suitable intermediates according to Example 7, Examples 8-12, described in Table 3 below, are prepared.
Example 13
Methyl O- (2,3,4,6-tetra-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α- D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-sulfo-β-D-glucopyranosyl)-(1 → 4) -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl) -(1 → 4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3,6-tri-O -Methyl-β-D-glucopyranosyl)-(1-4)] ₃ -O- (2,3-di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 -4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1 → 4) -O- (6-O-methyl-2,3-di-O Sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1-4) -6- O-methyl-2,3-di-O-sulfo-α-D-glucopyranoside 180.
Fully deprotected hexadecasaccharide 150 (26 mg, 0.0084 mmol) is dissolved in dimethylformamide (0.87 ml). Sulfur trioxide / triethylamine complex (125 mg, 0.67 mmol, 80 eq) is added under nitrogen atmosphere and the mixture is stirred at 50 ° C. for 16 hours. The mixture is cooled to 0 ° C. and aqueous sodium hydrogen carbonate (227 mg, 2.6 mmol) is added. The mixture is concentrated to a small volume and placed in a Sephadex G-25 column and eluted with 9/1 v / v water / acetonitrile. Appropriate fractions are separated, concentrated to a small volume, placed in a Dow's XW4 Na ⁺ ion exchange column in water, and the eluent is lyophilized to give 37 mg of compound 151 as a white powder. [a] ²⁰ _D = + 67.6 (c = 1, water). MS ESI: Molecular weight is 4370.6 (H ⁺ form) C ₁₂₈ H ₂₂₂ O ₁₁₃ S ₁₆ (theoretical mw = 4370.14).
NMR: Metastasis of Anomeric Protons (ppm):
Unit 1: 5.17; Unit 2: 5.03; Unit 3: 5.41; Unit 4: 4.42; Unit 5: 5.49; Unit 6: 4.66; Units 7, 9 and 11: 5.67; Units 8, 10 and 12: 4.46; Unit 13: 5.61; Unit 14: 4.94; Unit 15: 5.59; Unit 16: 5.69.
Structure of Polysaccharide 180 (Example 13)

权利要求:
Claims (17)
[1" claim-type="Currently amended] It has a region capable of binding to antithrombin III consisting of a series of five monosaccharides having a total of two carboxylic acid functional groups and at least four sulfo groups, wherein the hydroxyl groups are independently (C ₁ -C ₆ ) its reduction by a thrombin-binding region consisting of a series of 10 to 25 monosaccharide units selected from hexose, pentose or deoxy sugars which can be etherified with alkyl groups or esterified in the form of sulfo groups Synthetic polysaccharides and pharmaceutically acceptable salts thereof, characterized in that they are bonded directly at unterminated ends.
[2" claim-type="Currently amended] The method of claim 1,
Polysaccharide and a pharmaceutically acceptable salt thereof characterized by having a structure of formula (1).
(Formula 1)

(In Formula 1,
The wavy line represents a bond below or above the plane of the pyranose ring,
- Denotes a polysaccharide (Po) containing the same or different monosaccharide units (n) which is bonded to Pe via its anomer carbon,
- Is a schematic representation of a monosaccharide unit of pyranose structure selected from hexose, pentose and the corresponding deoxy sugar, wherein the unit is bonded to another monosaccharide unit via its anomer carbon, The hydroxyl group is substituted with the same or different -X groups, the X group is selected from a (C ₁ -C ₆ ) alkyl group and a sulfo group,
n is an integer from 10 to 25,
Pe represents pentasaccharide of the general formula:

(In the above formula,
R ₁ represents a (C ₁ -C ₆ ) alkyl group and a sulfo group,
R ₁ a constitutes C-CH ₂ -O together with an oxygen atom which represents or may be attached to R ₁ and a carbon atom containing a carboxyl functional group on the same ring,
R represents (C ₁ -C ₆ ) alkyl,
W represents an oxygen atom or a methylene group))
[3" claim-type="Currently amended] The method of claim 2,
The cation is a salt of polysaccharide, characterized in that selected from cations of alkali metals, in particular sodium and potassium.
[4" claim-type="Currently amended] The method of claim 2 or 3,
Polysaccharides and pharmaceutically acceptable salts thereof characterized by having the structure of Formula 1-A.
Formula 1-A

(In Chemical Formula 1-A,
- Denotes a particular mooring of polysaccharides (Po) that are bonded to Pe via their anomer carbon as defined in Formula 1,
- Is as defined in Formula 1,
OX is as defined in formula 1 for the same monosaccharide, is the same or different,
monosaccharides contained within [] _m form disaccharides repeated m times, monosaccharides contained within [] _t form disaccharides repeated t times,
m is 1 to 8, t is 0 to 5, p is 0 to 1, and 5 ≦ m + t ≦ 12)
[5" claim-type="Currently amended] The method of claim 2 or 3,
A polysaccharide having a structure of formula 2-A and a pharmaceutically acceptable salt thereof.
Formula 2-A

(In the above formula 2-A,
- Denotes a particular mooring of polysaccharides (Po) that are bonded to Pe via their anomer carbon as defined in Formula 1,
- Is as defined in Formula 1,
-OX groups are as defined in formula 1 for the same monosaccharide, are the same or different,
monosaccharides contained within [] _{m '} are repeated m' times, monosaccharides contained within [] _{t '} are repeated t' times, and monosaccharides contained within [] _{p '} are p' times Repeated,
m 'is 1 to 5, t' is 0 to 24, p 'is 0 to 24, and 10≤m' + t '+ p'≤25)
[6" claim-type="Currently amended] The method of claim 2 or 3,
A salt of a polysaccharide, characterized in that it has an anion of the formula (1-1).
(Formula 1-1)

(In Formula 1-1, t is 5, 6 or 7 and the cation is not only a pharmaceutically acceptable monovalent cation, but also a corresponding acid.)
[7" claim-type="Currently amended] The method of claim 2 or 3,
A salt of a polysaccharide characterized by having an anion of formula 1-2
(Formula 1-2)

(In the above formula 1-2, t is 5, 6 or 7, the cation is a pharmaceutically acceptable monovalent cation as well as the corresponding acid)
[8" claim-type="Currently amended] The method of claim 2 or 3,
A salt of a polysaccharide, characterized in that it has an anion of the formula 1-3.
(Formula 1-3)

(In Formula 1-3, m is 1, 2 or 3, t represents 2, 3, 4 or 5, and the cation is not only a pharmaceutically acceptable monovalent cation, but also a corresponding acid.)
[9" claim-type="Currently amended] Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] ₄ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] ₅ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] ₆ -O- (2,3- Di-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1-4)-[O- (2,3-di- O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4)] ₁₁ -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1-4)-[O- (2,3-di- O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4)] ₁₃ -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt (171),
Methyl O- (2,3-di-O-methyl-4,6-di-O-sulfo-α-D-glucopyranosyl)-(1-4)-[O- (2,3-di- O-Methyl-6-O-sulfo-α-D-glucopyranosyl)-(1-4)] _15- O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid) -(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] _2- [O- (2,3 , 6-Tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 -4)] ₂ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- β-D-glucopyranosyluronic acid)-(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4)-(2, 3-di-O-methyl-α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl) -(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)] _2- [O- (2,3 , 6-Tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1 -4)] ₃ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl- β-D-glucopyranosyluronic acid)-(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4)-(2, 3-di-O-methyl-α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl)- (1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (2,3,6- Tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4) ₄ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D -Glucopyranosyluronic acid)-(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4)-(2,3-di -O-methyl-α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6-di-O-sulfo-α-D-glucopyranosyl)- (1-4) -O- (3-O-methyl-2,6-di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (2,3,6- Tri-O-methyl-α-D-glucopyranosyl)-(1-4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4) ₃ -O-2,3-di-O-methyl-6-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D -Glucopyranosyluronic acid)-(1-4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4)-(2,3-di -O-methyl-α-L-idopyranosyluronic acid)-(1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1- 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4)] ₄ -O-2,3-di-O-methyl-6-sulfo -α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1-4) -O- (2 , 3,6-Tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-α-L-idopyranosyluronic acid)- (1-4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, sodium salt,
Methyl O- (3-O-methyl-2,4,6-tri-O-sulfo-α-D-glucopyranosyl)-(1-4) -O- (3-O-methyl-2,6 -Di-O-sulfo-β-D-glucopyranosyl)-(1-4)-[O- (2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1- 4) -O- (2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1-4)] ₅ -O-2,3-di-O-methyl-6-sulfo -α-D-glucopyranosyl)-(1-4) -O- (2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1-4) -O- (2 , 3,6-Tri-O-sulfo-α-D-glucopyranosyl)-(1-4)-(2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1 -4) -2,3,6-tri-O-sulfo-α-D-glucopyranoside, the polysaccharide selected from sodium salt.
[10" claim-type="Currently amended] In the first step, a fully protected precursor of the desired polysaccharide (I) comprising a protected precursor of the Pe region elongated at its unreduced end by a protected precursor of sulfated polysaccharide Po is synthesized and In the second step, the negatively charged group is added and / or demasking method of producing a compound of formula 1 characterized in that.
[11" claim-type="Currently amended] A compound having the structure of Formula 2-1.
(Formula 2-1)

(In Formula 2-1, the same or different T ₁ and T _n represent a temporary, semi-permanent or permanent substituent and Z is a protecting group for a hydroxyl functional group.)
[12" claim-type="Currently amended] A compound having the structure of Formula 3-1.
(Formula 3-1)

(In Formula 3-1, the same or different T ₁ and T _n represent a temporary, semi-permanent or permanent substituent and Z is a protecting group for a hydroxyl functional group.)
[13" claim-type="Currently amended] As an active ingredient, a mixture or a combination of a polysaccharide according to any one of claims 1 to 9 with a salt or an acid having a pharmaceutically acceptable base and an inert, nontoxic, pharmaceutically acceptable excipient Pharmaceutical composition comprising a.
[14" claim-type="Currently amended] The method of claim 13,
Pharmaceutical composition, characterized in that the active ingredient is mixed with at least one pharmaceutical excipient in the form of a dosage unit.
[15" claim-type="Currently amended] The method of claim 14,
Each dosage unit comprises 0.1 to 100 mg of the active ingredient.
[16" claim-type="Currently amended] The method of claim 15,
Each dosage unit comprises 0.5 to 50 mg of the active ingredient.
[17" claim-type="Currently amended] Use of a polysaccharide or a salt thereof, wherein the polysaccharide according to any one of claims 1 to 9 or a salt thereof is used to prepare a medicament useful for pathology depending on the dysfunction of coagulation. .

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-07-19|Priority to FR96/09116

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1996-07-19|Priority to FR9609116A

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1999-01-18|Application filed by 디. 꼬쉬, 사노피, 샬크비즈크 피이터 코르넬리스; 페트귄터, 아크조 노벨 엔.브이.

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2000-11-25|Publication of KR20000067909A

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2001-11-14|Application granted

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2001-11-14|Publication of KR100311857B1

优先权:

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申请号 | 申请日 | 专利标题

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FR96/09116|1996-07-19|

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FR9609116A|FR2751334B1|1996-07-19|1996-07-19|Synthetic polysaccharides, process for their preparation and pharmaceutical compositions containing them|