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WO1996016071A1 - PROCEDES POUR SYNTHETISER DE COMPOSES DE LEWISx SUBSTITUES EN 3' - Google Patents

PROCEDES POUR SYNTHETISER DE COMPOSES DE LEWISx SUBSTITUES EN 3' Download PDF

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Publication number
WO1996016071A1
WO1996016071A1 PCT/US1995/014573 US9514573W WO9616071A1 WO 1996016071 A1 WO1996016071 A1 WO 1996016071A1 US 9514573 W US9514573 W US 9514573W WO 9616071 A1 WO9616071 A1 WO 9616071A1
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compound
formula
group
contacting
produced
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PCT/US1995/014573
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English (en)
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Om Srivastava
Robert Ippolito
Geeta Srivastava
Roman Szweda
Toshio Ohuchi
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Glycomed, Inc.
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Priority to AU43641/96A priority Critical patent/AU4364196A/en
Publication of WO1996016071A1 publication Critical patent/WO1996016071A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical

Definitions

  • This invention is directed to processes for the chemical synthesis of 3'-substituted Lewis x -OR compounds where R is an aglycon of at least one carbon atom.
  • R is an aglycon of at least one carbon atom.
  • One particular compound prepared by processes of this invention is 8-methoxycarbonyloctyl-2-acetamido-3-O-( ⁇ -L-fucopyranosyl)-4-O-[3-O-sulfo- ⁇ -D-galactopyranosyl]-2-deoxy- ⁇ -D-glucopyranoside.
  • Lewis x trisaccharide glycosides modified at the 3-position of the galactose saccharide unit with sulfo or phosphoryl groups have been disclosed as possessing mammalian immunosuppressive and tolerogenic properties at least equivalent to sialyl Lewis x glycosides.
  • trisaccharide glycosides The complete chemical synthesis of trisaccharide glycosides is a difficult task involving the generation of differentially protected or blocked hydroxyl groups on at least some of the hydroxyl groups of each of the saccharide units so as to provide a means to selectively remove one or more of the blocking groups thereby permitting the necessary reactions to be conducted on the unblocked hydroxyl group(s) as required to generate the desired trisaccharide 2 . Additionally, the numerous reaction procedures required in blocking and deblocking different hydroxyl groups lead to a multistep chemical synthetic procedure and the generation of crystalline
  • glycosyltransferases to effect overall synthesis of the desired trisaccharide glycoside can be hindered by the lack of ready availability of the required glycosyltransferase, the difficulty in effecting large scale enzymatic reactions, the difficulty in coupling the desired saccharide to the nucleotide base required for coupling 3 , etc.
  • This invention is directed to novel processes for the overall chemical synthesis of derivatives of Lewis x glycosides modified at the
  • N-acetylglucosamine saccharide units The processes of this invention result in the 2 and 3 hydroxyl groups or the 3 hydroxyl group of the galactose unit in the Lewis x -OR compound being selectively exposed (not blocked) from the other hydroxyl groups of this trisaccharide thereby permitting the selective modification of the 3-hydroxyl group of the galactose unit.
  • saccharide units e.g., the appropriately protected and derivatized fucose, galactose, and N-acetylglucosamine units
  • these saccharide units are then assembled into fully hydroxyl protected Lewis x compounds in such a fashion that the number of manipulations at the disaccharide and trisaccharide levels is kept to a minimum and yield is improved.
  • the yield achieved by the processes of this invention for this conversion is approximately 10 to 20 percent.
  • this invention is directed to a process for the preparation of 3'-substituted Lewis x -OR compounds which process comprises:
  • R is an aglycon of at least 1 carbon atom
  • X 1 is selected from the group consisting of hydrogen, chloro, bromo, and OR 2 where R 2 is alkyl of from 1 to 4 carbon atoms
  • X 2 is selected from the group consisting of -NHB, -NB 2 and -NHC(O)R' where R 1 is alkyl of from 1 to 6 carbon atoms
  • each B is independently a removable blocking group
  • B' is a removable blocking group selected from the group consisting of benzyl and substituted benzyl
  • L is a leaving group
  • each B" is the same removable blocking group which group is different from any of the B and B' removable blocking groups and which group is separately (differentially) removable as compared to the B and the B' blocking groups, and L is a leaving group;
  • X 3 is hydrogen, bromo, Cl or -OR 3 where R 3 is alkyl of from 1 to 4 carbon atoms; and R 4 is alkyl of from 1 to about 4 carbon atoms;
  • R is as defined above
  • X is selected from the group consisting of HOSO 2 O- and (HO) 2 P(O)O-
  • X 4 is selected from the group consisting of -NH 2 and -NHC(O)R 1 wherein R 1 is as defined above
  • procedure (h) optionally converting the compound of formula X produced in (g) above to a pharmaceutically acceptable salt.
  • the reaction preferably employs from about 1 to about 2.0 and more preferably about 1.5 equivalents of the compound of formula II relative to the compound of formula I.
  • procedure (a) is conducted at a temperature of from about -20°C to about 35oC.
  • the hydride reducing agent is preferably selected from the group consisting of sodium cyanoborohydride/ethereal ⁇ HCl solution and aluminum trichloride/boron trialkylamine in trifluoroacetic acid wherein the alkyl groups of the boron trialkylamine are independently from 1 to 4 carbon atoms.
  • procedure (b) is conducted using from about 2 to about 20 equivalents preferably 5 to 10 equivalents of hydride reducing agent at a temperature of from about -15°C to about 20°C, preferably 0°C, when sodium cyanoborohydride is employed; or using from about 3 to about 10 and preferably 5 equivalents of the aluminum trichloride/borane trialkylamine complex at a temperature of from about -10°C to about +30°C when a borane trialkylamine complex is employed.
  • procedure (b) can be conducted using triethyl silane-trifluoroacetic acid as the reducing agent 18 .
  • reaction is preferably conducted at a temperature of from about -30°C to about 10°C preferably -10oC.
  • reaction is preferably conducted at a temperature of from about 0°C to about 35°C and more preferably from about 15oC to 25oC.
  • the reaction is preferably conducted at a temperature of from 0° to 30oC when the reagent employed is either the SO 3 -pyridine complex or the SO 3 (CH 3 ) 3 N complex in dimethylformamide; at a temperature of from about -50°C to -30°C when the reagent employed is SO 3 dimethyl formamide; or at a temperature of from -10°C to 30°C when the reagent employed is (BO) 2 P(O)L.
  • this invention is directed to a process for the preparation of 3'-substituted Lewis x -OR compounds which process comprises:
  • R is an aglycon of at least 1 carbon atom
  • X 1 is selected from the group consisting of hydrogen, chloro, bromo, and OR 2 where R 2 is alkyl of from 1 to 4 carbon atoms
  • X 2 is selected from the group consisting of -NHB, -NB 2 and -NHC(O)R' where R 1 is alkyl of from 1 to 6 carbon atoms
  • each B is independently a removable blocking group
  • B' is a removable blocking group selected from the group consisting of benzyl and substituted benzyl
  • L is a leaving group
  • B and B' are as defined above and each B" is the same removable blocking group which group is different from any of the B and B' removable blocking groups and which group is separately (differentially) removable as compared to the B and the B' blocking groups, and L is a leaving group;
  • R is as defined above
  • X is selected from the group consisting of HOSO 2 O-, and (HO) 2 P(O)O-
  • X 4 is selected from the group consisting of -NH 2 and -NHC(O)R 1 wherein R 1 is as defined above, and
  • procedure (a) of this embodiment the reaction preferably employs from about 1 to about 2.0 and more preferably about 1.5 equivalents of the compound of formula XII relative to the compound of formula XI.
  • procedure (a) is conducted at a temperature of from about -20°C to about 35°C.
  • the hydride reducing agent is preferably selected from the group consisting of sodium cyanoborohydride/ethereal ⁇ HCl solution and aluminum trichloride/boron trialkylamine in trifluoroacetic acid wherein the alkyl groups of the boron trialkylamine are independently from 1 to 4 carbon atoms.
  • procedure (b) of this embodiment is conducted using from about 2 to about 20 equivalents preferably 5 to 10 equivalents of hydride reducing agent at a temperature of from about -15°C to about 20°C, preferably 0°C, when sodium cyanoborohydride is employed; or using from about 3 to about 10 and preferably 5 equivalents of the aluminum trichloride/borane trialkylamine complex at a temperature of from about -10°C to about +30°C when a borane trialkylamine complex is employed.
  • procedure (b) can be conducted using triethyl silane-trifluoroacetic acid as the reducing agent 18 .
  • reaction is preferably conducted at a temperature of from about -30°C to about 10°C preferably -10°C.
  • the reaction is preferably conducted at a temperature of from 0° to 30°C when the reagent employed is either the SO 3 -pyridine complex or the SO 3 (CH 3 ) 3 N complex in
  • this invention is directed to a process for the preparation of 3'-sulfo-Lewis x -OR compounds which process comprises:
  • each Ac is as defined above; (d) selectively removing the acetyl blocking groups from the hydroxyl groups of the compound of formula XXIV produced in step (c) to provide for a compound of formula XXV:
  • R 4 is alkyl of from 1 to about 4 carbon atoms; (f) contacting the compound of formula XXVII produced in (e) above with from about 1.0 to about 1.5 equivalents of sulfur trioxide complex with pyridine wherein said contacting is conducted at a temperature of from about 0°C to about 30°C and further under conditions to provide for sulfo substitution at the 3 position of the galactose unit;
  • this invention is directed to a process for the preparation of S'-sulfo-Lewis x -OR compounds which process comprises:
  • Bz is benzoyl, and Ph and L are as defined above;
  • Figure 1 illustrates a reaction scheme which can be used to obtain a preferred fucose derivative used as a starting material in the preparation of the 3'-substituted Lewis x -OR compounds described herein.
  • Figures 2A and 2B illustrate reaction schemes which can be used to obtain a preferred N-acetylglucosamine derivative used as a starting material in the preparation of the 3 '-substituted Lewis x -OR compounds described herein.
  • Figures 3A and 3B illustrate reaction schemes which can be used to obtain preferred galactose derivatives used as starting materials in the preparation of the 3'-substituted Lewis x -OR compounds described herein.
  • Figures 4A and 4B illustrate preferred reaction schemes used to prepare the 3'-substituted Lewis x -OR compounds from the preferred fucose, N-acetylglucosamine, and galactose starting materials described in Figures 1; 2A and 2B; and 3A and 3B, respectively.
  • Figure 5 illustrates the increase in footpad swelling of
  • mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 ⁇ g of SuperCarrier (from Pierce, Rockford, IL 61105) antigen wherein some of the mice have been treated at 5 hours after the challenge with 100 ⁇ g of sialyl Lewis x -OR (SLeX) or with 3-O-sulfo-Lewis x -OR where R is -O(CH 2 ) 8 COOCH 3 in all cases.
  • SuperCarrier from Pierce, Rockford, IL 61105
  • Figure 6 illustrates the effect that 3'-sulfo-Lewis x -OR (LeX sulf) and sialyl Lewis x -OR (SleX) on lung injury arising from the intranasal administration of LPS to mice [R is -(CH 2 ) 8 COOCH 3 ].
  • Figure 7 demonstrates the effect that 3'-sulfo-Lewis x -OR has on the induction of an immune response to an antigen where R is -(CH 2 ) 8 COOCH 3 .
  • the term "aglycon of at least one carbon atom” refers to non-saccharide containing residues having at least one carbon atom.
  • the aglycon is selected from the group consisting of -(A)-Z wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the form -(WG) n - wherein n is an integer equal to 1 to 5;
  • W is a straight or branched chain alkylene group of from 2 to 10 carbon atoms optionally substituted with 1 to 3 substituents selected from the group consisting of aryl of 6 to 10 carbon atoms and aryl of from 6 to 10 carbon atoms substituted with from 1 to 3 substituents selected from the group consisting of amino, hydroxyl, halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms;
  • G is selected from the group consisting of the group consisting
  • each R 6 is independently alkyl of from 1 to 4 carbon atoms and R 7 is an alkenyl group of from 3 to 10 carbon atoms. Numerous aglycons are known in the art.
  • the nitro group is reduced to an amino group which can be protected as N- trifluoroacetamido.
  • the trifluoroacetamido group is removed thereby unmasking the amino group.
  • An aglycon containing sulfur is disclosed by Dahmen, et al.
  • the aglycon is derived from a 2-bromoe-hyl group which, in a substitution reaction with thionucleophiles, has been shown to lead to aglycons possessing a variety of terminal functional groups such as
  • exemplifications of known aglycons include the 7-methoxycarbonyl-3,6-dioxaheptyl aglycon 7 (-OCH 2 CH 2 ) 2 OCH 2 CO 2 CH 3 ; the 2-(4-methoxycarbonylbutancarboxamido)ethyl 8
  • allyl linking arms can be derivatized in the presence of 2-aminoethanethiol 11 to provide for the aglycon of the formula -OCH 2 CH 2 CH 2 SCH 2 CH 2 NH 2 . Still other aglycons are illustrated hereinbelow.
  • the R group can be an additional saccharide-OR' or an oligosaccharide-OR' at the reducing sugar terminus (where R' is an aglycon as defined above).
  • hydride reducing agents include, by way of example, sodium cyanoborohydride/ethereal ⁇ HCl solution; aluminum trichloride/boron trialkylamine in trifluoroacetic acid wherein the alkyl groups of the boron trialkylamine are independently from 1 to 4 carbon atoms; and triethyl silane-trifluoroacetic acid 18 .
  • the particular hydride reducing agent employed is not critical.
  • pharmaceutically acceptable salts include the pharmaceutically acceptable addition salts of 3'-substituted Lewis x -OR compounds derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like.
  • sulfate refers to the -OS(O) 2 OH substituent at the 3-position of the galactose unit, as well as pharmaceutically acceptable salts thereof.
  • phosphate refers to the -OP(O)(OH) 2 substituent at the 3-position of the galactose unit, as well as pharmaceutically acceptable salts thereof (e.g., -OP(O)(-ONa + ) 2 ).
  • removable blocking group refers to any group which when bound to one or more hydroxyl groups of the galactose, the N-acetylglucosamine, and/or the fucose units used to prepare the 3'-substituted Lewis x -OR derivatives described herein prevents reactions from occurring at these hydroxyl groups and which protecting groups can be removed by conventional chemical and/or enzymatic procedures to reestablish the hydroxyl group.
  • removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as benzyl, benzoyl, acetyl, chloroacetyl, benzylidene, t-butylbiphenylsilyl and any other group that can be introduced onto a hydroxyl functionality and later selectively removed by conventional methods in mild conditions compatible with the nature of the product.
  • the processes of this invention involve the synthesis of 3'-substituted Lewis x -OR derivatives from appropriately derivatized N-acetylglucosamine-OR, L-fucose, and galactose saccharide units.
  • the following discussion will first illustrate processes for the preparation of these individual saccharide starting materials and then how these starting materials are assembled into 3'-substituted Lewis x -OR derivatives. PREPARATION OF STARTING MATERIALS
  • FIG 1 the synthesis of preferred fucose starting material, compound 1, is illustrated.
  • compound 1 is prepared sequentially through intermediate compounds 1A, 1B, and 1C, the preparation of which are set forth in the examples hereinbelow.
  • the process to produce the highly crystalline fucose intermediate 1 from L-fucose as shown in Figure 1 maximizes the production of ⁇ -fucopyranose tetraacetate 1 A by adding acetic anhydride (AcOAc) dropwise to a slurry of fucose and about equimolar amounts (e.g., about 1.1 equivalents) of sodium acetate (NaOAc) maintained at about 50°C to about 55 °C in dichloroethane (DCE) and stirred at this temperature for a sufficient period of time to result in formation of compound 1A (e.g., for about 2-3 days).
  • AcOAc acetic anhydride
  • NaOAc sodium acetate
  • DCE dichloroethane
  • reaction mixture is then treated with water, quenched into ice water, extracted with dichloromethane and dried and partially concentrated to provide the peracylated compound 1A (about 4:1 ⁇ / ⁇ ratio of 1-acetate).
  • Compound 1A is then reacted with an approximately equivalent amount of p-chlorothiophenol (p-ClPhSH) and approximately 1 to 3
  • reaction conditions employed are not critical and temperatures of from about 15°C to about 30°C and reaction times of about 1 to about 10 hours can be used.
  • Compound 1C is, in turn, readily benzylated with benzyl chloride or benzyl bromide to yield p-chlorophenol-2,3,4-tri-O-benzyl- ⁇ -thiofucopyranoside, compound 1, in 45-50% overall yield from fucose.
  • the reaction conditions employed are not critical and temperatures of from about 15°C to about 30°C and reaction times of about 24 hours to about 48 hours can be used.
  • benzyl chloride or benzyl bromide are employed and the reaction is generally conducted in the presence of at least 4-5 equivalents of a suitable base (e.g., potassium hydroxide - KOH) in a suitable inert solvent (e.g., dimethoxysulfoxide ⁇ DMSO).
  • a suitable base e.g., potassium hydroxide - KOH
  • a suitable inert solvent e.g., dimethoxysulfoxide ⁇ DMSO
  • Figures 2A and 2B illustrate methods for synthesizing a preferred blocked derivative of N-acetylglucosamine, compound 2, which is useful as starting material in the herein described processes.
  • glucosamine hydrochloride is slurried in dichloroethane containing an equivalent of anhydrous sodium acetate and an equivalent of acetic acid to which the remaining acetic anhydride is added dropwise and, after addition is completed, the solution is refluxed for a period of from about 12 to about 36 hours to provide for the peracetylated compound 2 A (about 3:1 ratio of ⁇ / ⁇ ).
  • the aglycon is formed by conventional techniques.
  • compound 2 A is converted to 1- ⁇ -chloro compound 2B by well known chemistry which involves bubbling saturating amounts of hydrogen chloride directly into an inert diluent (e.g., the dichloroethane solution of compound 2A).
  • an inert diluent e.g., the dichloroethane solution of compound 2A
  • the solution used to prepare compound 2 A is preferably used in this reaction after that solution has been quenched into water to remove acetic anhydride and sodium acetate, dried and recovered.
  • the reaction generally proceeds over a period of about 4 to about 6 days at from about 0°C to about 20°C and hydrogen chloride is bubbled into the solution periodically (e.g., about once every 1 to 2 days).
  • the reaction is generally conducted at room temperature (about 15°C to about 30°C) for a period of from about 12 to about 24 hours.
  • reaction completion (as evidenced by thin layer chromatography)
  • the reaction solution is filtered through silica and the resulting solution is quenched by adding the reaction solution to cold water.
  • the organic layer is recovered and then washed twice with an aqueous potassium iodide solution (5 weight/vol percent) and then with a saturated aqueous sodium bicarbonate solution.
  • the resulting organic solution is then dried and the solvent removed by stripping to provide for compound 2C.
  • N-acetylglucosamine-OR compound 2D.
  • This compound is then reacted with C 6 H 5 CH(OCH 3 ) 2 in, for example, an acidic medium in an appropriate solvent at around 40°C to about 50°C for about 4 to about 6 hours to provide for the 4,6-O-protected benzylidene compound 2.
  • Figure 2A further illustrates a similar synthetic scheme for the preparation of compound 2H which contains a phthalimido amino blocking group which can subsequently be deblocked by conventional methods to provide for the 2-amino derivative.
  • compound 2 and similar aglycons can be prepared by the process illustrated in Figure 2B.
  • the 1- ⁇ -peracetylated compound 2A obtained from commercial sources is reacted with from about 1.1 to about 2 equivalents of HOR (e.g., HO(CH 2 ) 8 COOCH 3 ) in the presence of from about 0.5 to about 1.5 equivalents of trimethylsilyl trifluoromethanesulfonate and about twice the amount by weight of calcium sulfate as compared to the ⁇ -acetate of GlcNAc compound 2 A to provide compound 2C (shown in Figure 2A).
  • HOR e.g., HO(CH 2 ) 8 COOCH 3
  • This reaction is preferably conducted in an anhydrous inert organic solvent, such as dichloromethane.
  • reaction conditions are not critical and preferably the reaction is conducted at from about 0°C to about 20°C for about 5 to about 15 hours.
  • Compound 2C prepared by this reaction is generally not isolated. Instead, the reaction mixture is preferably further treated with sodium methoxide in methanol to deacetylate the 3, 4, and 6 hydroxyl groups of intermediate compound 2C to provide for N-acetylglucosamine-OR, compound 2D. This compound is then reacted with C 6 H 5 CH(OCH 3 ) 2 using the conditions described above to provide for the 4,6-O-protected benzylidene compound 2. Accordingly, in sequential form, the above described preferred methodology involves:
  • Figures 3 A and 3B illustrate the synthesis of preferred galactose derivatives (compounds 5 and 10) which are useful as starting materials in the herein described processes.
  • Figure 3A illustrates the synthesis of O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)trichloroacetimidate, compound 5, from ⁇ -D-galactose pentaacetate, compound 10A.
  • Compound 10A is produced by slurring D-galactose and about an equimolar amount (e.g., about 1.1 equivalents) of sodium acetate (NaOAc) in dichloroethane (DCE), heating to reflux and adding at least 5 equivalents of acetic anhydride (AcOAc) dropwise to the refluxing solution at about 80°C to about 85 °C and maintaining the reaction system at this temperature for a sufficient period of time (about 16 to about 32 hours) to result in formation of compound 10A.
  • This procedure optimizes the yield of ⁇ -D-galactose pentaacetate 10A and controls the exotherm of heretofore known procedures.
  • Compound 10A is then converted to 2,3,4,6-tetra-O-acetyl- ( ⁇ , ⁇ )-galactopyranoside 5A by reaction with hydrazine acetate (or
  • this reaction is conducted by contacting ⁇ -D-galactose pentaacetate 10A with from about 1.1 to about 1.5 equivalents of hydrazine acetate (prepared by known procedures from hydrazine and acetic anhydride) at a temperature of from about 0°C to about 20°C for about 5 to about 10 hours.
  • the reaction is conducted in an anhydrous solvent, such as dimethylformamide. Workup, preferably using the procedure described in Example 8, provides for compound 5A.
  • the reaction can be accomplished by using from about 1.5 to 2 equivalents of benzylamine based on compound 10A in tetrahydrofuran which reaction is conducted at from about 0°C to about 40°C for about 5 to about 10 hours.
  • Compound 5A is then converted into O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-trichloroacetimidate, compound 5, by treatment with trichloroacetonitrile in the presence of a base, such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
  • a base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
  • DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
  • This reaction is typically conducted in an anhydrous inert organic solvent, such as dichloromethane, using an excess of trichloroacetonitrile.
  • the reaction conditions are not critical and preferably the reaction is conducted at room temperature for from about 1 to about 5 hours. Workup, preferably using the procedures described in Example 9, provides for compound 5, which is a crystalline solid.
  • the above described preferred methodology involves: (a) contacting D-galactose with sodium acetate in
  • dichloroethane maintained at a temperature of from about 75°C to about 85°C while adding dropwise at least 5 equivalents of acetic anhydride;
  • FIG 3B illustrates the synthesis of another preferred galactose derivative (compound 10) which is useful as starting material in the processes described herein.
  • compound 10 another preferred galactose derivative
  • the synthesis of compound 10 parallels that of compound 1 as set forth above and illustrated in Figure 1.
  • benzyl 4,6-O-benzylidene- ⁇ -D-thiogalactopyranoside (compound 10D) has been produced without the necessity of chromatography. Compound 10D is then readily converted to compound 10.
  • ⁇ -D-galactose pentaacetate, compound 10A is produced as described above by reacting D-galactose with acetic anhydride.
  • the product is treated with approximately equimolar amounts of benzyl mercaptan (PhCH 2 SH) and from about 1 to about 3 (preferably 2) equivalents of boron trifluoride etherate (BF 3 ⁇ OEt 2 ) in dichloromethane.
  • the reaction conditions are not critical and the reaction is preferably conducted at from about 0°C to about 30°C for a period of from about 6 to about 16 hours to yield after crystallization from hot methanol or hot isopropanol 55 to 65% of benzyl 2,3,4,6-tetra-O-acetyl ⁇ -D-thiogalactopyranoside, compound 10B.
  • Deacetylation under Zemplen conditions sodium
  • reaction conditions are not critical and preferably the reaction is conducted at room temperature and is generally complete in about 12 to about 24 hours.
  • the benzyl 4,6-O-benzylidene ⁇ -D-thiogalactopyranoside, 10D is isolated and crystallized from hot isopropanol.
  • Compound 10 is then prepared by direct benzoylation of both the 2,3-hydroxyl groups of compound 10D. Specifically, compound 10 is benzoylated with at least 2 equivalents (and preferably from about 3 to about 5 equivalents) of benzoyl chloride in a suitable solvent containing a base (e.g., methylene chloride/pyridine) with from about 0.1 to about 1 weight percent of 4-(N,N-dimethylamino)pyridine (DMAP) as a catalyst.
  • a base e.g., methylene chloride/pyridine
  • DMAP 4-(N,N-dimethylamino)pyridine
  • reaction conditions are not critical and preferably the reaction is conducted at from about 0°C to about 30°C and for about 1 to about 4 hours (preferably room temperature for 2 hours) to give crystalline benzyl 4,6-O-benzylidene 2,3-di-O-benzoyl- ⁇ -D-thiogalactopyranoside, compound 10.
  • compound 10 can be converted to the 1-bromo derivative via known methodology (Norberg et al. 13 ) using bromine and tetraethylammonium bromide to provide for compound 10J.
  • the above procedure for producing compound 10 results in non-differentially blocked 2,3 hydroxyl groups on the galactose unit which, after deblocking, provide 2 sites for derivation. While selective derivation can be greatly controlled by careful use of reaction temperatures, Figure 3B further illustrates how to prepare 2,3 differentially blocked galactose units,
  • benzyl 4,6-O-benzylidene-3-O-chloroacetyl- ⁇ -D-thiogalactopyranoside, compound 10E is prepared from compound 10D by chloroacetylation using from about 1 to 3 (preferably 2) equivalents of chloroacetyl chloride which is added to a dimethylformamide (DMF) solution containing benzyl 4,6-O-benzylidene ⁇ -D-thiogalactopyranoside, compound 10D.
  • DMF dimethylformamide
  • the chloroacetyl chloride is added dropwise while maintaining the DMF solution at from about -40°C to about -15°C (preferably at -25°C).
  • Benzyl 4,6-O-benzylidene-3-O-chloroacetyl- ⁇ -D-thiogalactopyranoside (compound 10E) is benzoylated with at least 1 equivalent (and preferably about 2 equivalents) of benzoyl chloride in a suitable solvent containing a base (e.g., pyridine/methylene chloride) with from about 0.1 to about 1 weight percent of 4-(N,N-dimethylamino)pyridine (DMAP) as a catalyst.
  • a base e.g., pyridine/methylene chloride
  • DMAP 4-(N,N-dimethylamino)pyridine
  • the reaction conditions are not critical and preferably the reaction is conducted at from about 0°C to about 30°C and for about 1 to about 4 hours (preferably room temperature for 2 hours) to give crystalline benzyl 4,6-O-benzylidene 2-O-benzoyl-3-O-chloroacetyl- ⁇ -D-thiogalacto-pyranoside, compound 10F, in approximately 10 to 20% overall yield from galactose.
  • the advantage of this approach is that after subsequent assembly, the blocked intermediates will be simply deblocked and modified by sulfation, phosphorylation, and the like.
  • the material is crystalline and the process obviates the need for chromatography.
  • Compound 10F is converted to compound 10G via known methodology (Norberg, et al. 13 ) using bromine and tetraethylammonium bromide. Alternatively, compound 10F can be converted to compound
  • D-galactosyl pyranosides, and D-N-acetylglucosaminyl pyranosides suitable for use in the processes herein described can be achieved using methods known in the art including, by way of example, those described by Ippolito et al. 12 and International Patent Application Publication No. WO 92/22564 14 .
  • dimethylformamide, tetraethylammonium bromide and cupric bromide are combined into a suitable inert organic solvent such as dichloromethane and the solution is maintained in an anhydrous condition by the addition of 4A molecular sieves.
  • a suitable inert organic solvent such as dichloromethane
  • 4A molecular sieves At least molar equivalents and preferably about 1.2 molar equivalents of p-chlorophenyl 2,3,4-tri-O-benzyl- ⁇ -L-thiofucopyranoside, compound 1, and 8-methoxycarbonyloctyl-2-acetamido-4,6-O-benzylidene-2-deoxy- ⁇ -D-glucopyranoside, compound 2, are then combined into this solution.
  • the reaction is conducted using from about 1 to about 2
  • reaction conditions are not critical and the conditions are selected so that compound 2 reacts with compound 1 to provide for 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4,6-O-benzylidene-2-deoxy- ⁇ -D-glucopyranoside, compound 3.
  • the reaction is conducted at a temperature of from about -20°C to about 35°C for a period of from about 4 to about 15 hours. Workup, preferably as described in Example 5 hereinbelow, provides for compound 3, which is a crystalline solid.
  • Compound 3 is next converted to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-6-O-benzyl-2-deoxy D-glucopyranoside, compound 4, by reaction with a least a molar equivalent, and preferably an excess, of sodium cyanoborohydride or other hydride reducing agent.
  • the reaction is conducted in a suitable inert organic solvent such as tetrahydrofuran and is preferably maintained under anhydrous conditions which can be achieved, for example, by the inclusion 3A molecular sieves.
  • a pH indicator such as methyl orange is added to the reaction system and then a sufficient amount of acid, e.g., diethylether saturated with hydrogen chloride, is added until the pH of the system is about 3 or less and carbon dioxide evolution occurs. Achievement of this pH is indicated by the solution turning red.
  • the reaction conditions are not critical and the conditions are selected so as to produce compound 4.
  • about 2 to about 20, preferably 5 to 10 equivalents of sodium cyanoborohydride is employed at a reaction temperature of from about -15°C to about 20°C (preferably 0°C) for a period of from about 1 to about 7 hours.
  • the reaction is preferably conducted in an inert atmosphere and workup, preferably as described in Example 6 hereinbelow, provides for compound 4.
  • compound 3 can be converted to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside, compound 4, by reaction with from about 3 to about 10 equivalents (preferably 5) of an aluminum chloride borane trialkylamine complex, such as a complex with borane trimethylamine.
  • the reaction is conducted by adding from about 5 to about 10 equivalents of a Lewis acid, such as aluminum chloride, to the reaction system and then adding from about 1 to about 5 equivalents of an acid, such as trifluoroacetic acid.
  • the reaction is conducted in a suitable inert organic solvent, such as tetrahydrofuran, under an inert atmosphere.
  • a suitable inert organic solvent such as tetrahydrofuran
  • the temperature of the reaction system is preferably maintained in the range from about -10°C to about 30°C until the reaction is complete as determined by thin layer chromatography (about 1 to about 7 hours).
  • Compound 4 is next converted to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-( ⁇ -D-galactopyranosyl)-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside, compound 6, by reaction with from about 1.1 to about 2 equivalents of O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-trichloroacetimidate, compound 5, using conventional coupling conditions, followed by deacetylation of the resulting trisaccharide.
  • the coupling reaction is preferably conducted using an excess of boron trifluoride etherate relative to the galactose imidate and preferably from about 1.1 to about 2 equivalents.
  • the reaction is typically conducted at from about -30°C to about 10°C (preferably -10°C) in a suitable anhydrous organic solvent such as dichloromethane or a 1:2 mixture of
  • the product from the coupling reaction is then deacetylated using conventional reaction conditions, preferably using sodium methoxide in methanol. Workup, preferably as described in Example 10 hereinbelow, provides for compound 6, which is a crystalline solid.
  • this reaction can be conducted by using from about 2 to 3 equivalents of N-iodosuccinimide with about 1 equivalent of trifluoromethanesulfonic acid in an anhydrous mixture of diethyl ether and methylene chloride (1:1).
  • the reaction mixture is cooled to about -30°C and then compound 4 is added followed by addition of 2 equivalents of thiobenzyl-2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranoside.
  • the reaction is allowed to equilibrate for about 1 to 3 hours at -30°C.
  • the reaction solution is then quenched by the addition of triethylamine.
  • the solution is then diluted with methylene chloride and the organic layer washed with an aqueous solution of sodium thiosulfate (3 X), with an aqueous solution of sodium bicarbonate and then with water.
  • the resulting organic phase is filtered and dried and the resulting residue dissolved in 0.5 M solution of sodium methoxide in methanol to provide 8-methoxycarbonyloctyl-3-O-[2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl]-4-O-[ ⁇ -D-galactopyranosyl]- 6-O-benzyl-2-acetamido-2-deoxy- ⁇ -D-glucopyranoside.
  • Compound 6 is then converted to a 4,6-O-benzylidene derivative by reaction with from about 1 to about 2 equivalents of a substituted benzaldehyde dialkyl acetal, such as p-anisaldehyde dimethyl acetal or p-chlorobenzaldehyde dimethyl acetal.
  • a substituted benzaldehyde dialkyl acetal such as p-anisaldehyde dimethyl acetal or p-chlorobenzaldehyde dimethyl acetal.
  • the substituted benzaldehyde employed is p-anisaldehyde dimethyl acetal.
  • This reaction is preferably conducted in an inert organic solvent, such as acetonitrile, in the presence of an acidic catalyst, such as p-toluenesulfonic acid.
  • an acidic catalyst such as p-toluenesulfonic acid.
  • the reaction is conducted at a temperature of from about 0°C to about 35°C (preferably 15°C to 25°) for from about 1 to about 5 hours.
  • Workup preferably as described in Example 11, provides for the 4,6-O-benzylidene derivative.
  • the resulting 4,6-O-benzylidene derivative is 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[p-methoxy-4,6-O-benzylidene- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside, compound 7.
  • Compound 7 is then converted to the 3'-substituted derivative by reaction with a reagent selected from the group consisting of (BO) 2 P(O)L, sulfur trioxide, and sulfur trioxide in complexes with pyridine,
  • reaction typically employs an excess of such a reagent and preferably from about 1.2 to 2 molar equivalents of this reagent relative to compound 7.
  • reaction conditions are selected to favor substitution only at the 3' position of the galactose unit which, in the case of sulfur trioxide in DMF includes reaction at from about -30°C to about -50°C; whereas for sulfur trioxide complexes with pyridine or triethylamine includes reaction temperatures of 0°C to 30°C.
  • sulfur trioxide complexes with pyridine or triethylamine includes reaction temperatures of 0°C to 30°C.
  • such reaction conditions include reaction at from about -10°C to about 30°C.
  • the reaction is maintained at this temperature for a period of from about 1 to about 20 hours to provide for 3'-substitution of the blocked Lewis x -OR derivative.
  • the reagent is sulfur trioxide or a sulfur trioxide complex as described above, the resulting 3-O-sulfo derivative is optionally converted to a salt thereof, e.g. compound 8, by contact with a suitable cation exchange resin.
  • Compound 8 and other 3'-substituted blocked Lewis x -OR derivatives are then converted to the 3'-substituted Lewis x -OR derivatives by conventional deblocking methodology.
  • the particular methodology employed is selected relative to the blocking groups attached to the 3'-substituted blocked Lewis x -OR derivatives and it is well within the skill of the art to select suitable deblocking methodology.
  • deblocking is achieved by hydrogenation which provides for 8-methoxycarbonyloctyl-2-acetamido-3-O-( ⁇ -L-fucopyranosyl)-4-O-(3-O-sulfo- ⁇ -D-galactopyranosyl)-2-deoxy- ⁇ -D-glucopyranoside, which can be converted to a salt by contact with a cation exchange resin. Formation of the sodium salt, for example, provides for compound 9.
  • compound 9 and similar 3'-substituted Lewis x -OR derivatives can be prepared by the process illustrated in Figure 4B.
  • compound 4 prepared as described above, is converted to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside, compound 11, by reaction with from about 1.5 to about 2.5 equivalents of 4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-galactopyranosyl bromide, compound 10J, using conventional coupling conditions.
  • this reaction is conducted in a suitable anhydrous inert organic solvent such as a 1: 1 mixture of toluene:nitromethane.
  • a suitable anhydrous inert organic solvent such as a 1: 1 mixture of toluene:nitromethane.
  • Anhydrous conditions can be maintained by inclusion into the reaction medium of 4A molecular sieves.
  • the reaction is preferably conducted using an excess of silver trifluoromethanesulfonate relative to 4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-galactopyranosyl bromide and preferably from about 1.5 to about 3
  • the reaction is typically conducted at from about -30°C to about 10°C for a period of from about 5 to about 15 hours.
  • the reaction is preferably conducted in an inert atmosphere and workup, preferably as described in Example 16 hereinbelow, provides for compound 11.
  • This reaction can also be conducted by using about 2-3 equivalents of N-iodosuccinimide with about 1 equivalent of
  • trifluoromethanesulfonic acid in an anhydrous mixture of diethyl ether and methylene chloride (1:1).
  • the reaction mixture is cooled to about -30°C and then compound 4 is added followed by the addition of approximately 2 equivalents of thiobenzyl 4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-galactopyranoside.
  • the reaction is preferably cooled to -30°C prior to the addition of the trifluoromethanesulfonic acid.
  • the reaction is allowed to equilibrate at about -30°C for about 1-3 hours.
  • the reaction solution is then quenched at -30°C followed by the addition of triethylamine until neutral pH is reached.
  • Compound 11 can then be converted to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[4,6-O-benzylidene-3-O-sulfo- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside, compound 13, by first removing the benzoyl protecting groups on the 2,3-positions of the galactose unit under Zemplen conditions (NaOMe/MeOH) to provide for 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[4,6-O-benzylidene- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-
  • the reagent employed in this embodiment is sulfur trioxide or a sulfur trioxide complex as described above
  • the resulting 3-O-sulfo derivative is optionally converted to a salt thereof, e.g. compound 13, by contact with a suitable cation exchange resin.
  • Compound 13 and other 3'-substituted blocked Lewis x -OR derivatives prepared by this embodiment can then be converted to the 3'-substituted Lewis x -OR derivatives by conventional deblocking methodology.
  • the particular methodology employed is selected relative to the blocking groups attached to the 3'-substituted blocked Lewis x -OR derivatives and it is well within the skill of the art to select suitable deblocking methodology.
  • deblocking is achieved by hydrogenation which provides for 8-methoxycarbonyloctyl-2-acetamido-3-O-( ⁇ -L-fucopyranosyl)-4-O-(3-O-sulfo- ⁇ -D-galactopyranosyl)-2-deoxy- ⁇ -D-glucopyranoside, which can be converted to a salt by contact with a cation exchange resin. Formation of the sodium salt, for example, provides for compound 9.
  • the 3'-substituted Lewis x -OR compounds prepared by the processes described herein are useful in treating inflammation including the inflammatory component of an immune response to an antigen challenge in mammals which have been previously sensitized to that antigen as well as lung injury due to LPS.
  • the 3'-substituted Lewis x -OR compounds are administered within one-half the period required for maximal inflammatory response to the antigen challenge 1,12,14 .
  • the 3'-sulfate Lewis x -OR compounds described herein also induce tolerance to later challenges by the same antigen in the treated mammal. 1,12,14
  • the 3'-substituted Lewis x -OR compounds described herein are effective in modulating the inflammatory component of such immune responses as well as treating LPS lung injury when administered at a dosage range of from about 0.5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 to about 5 mg/kg of body weight.
  • the specific dose employed is regulated by the particular inflammatory component of the immune response being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like. Additionally, as is apparent, more active compounds will require less dosing than less active compounds.
  • the term "reducing sensitization” means that the 3'-sulfate Lewis x -OR compounds, when administered to a mammal in an effective amount along with a sufficient amount of antigen to induce an immune response, reduces the ability of the immune system of the mammal to become educated and thus sensitized to the antigen administered at the same time as the 3'-sulfate Lewis x -OR compounds.
  • an "effective amount" of this compound is that amount which will reduce sensitization (immunological education) of a mammal, including humans, to an antigen administered simultaneously as determined by a reduction in a cell-mediated response to the antigen such as a delayed type hypersensitivity (DTH) response as tested by the footpad challenge test set forth in the examples hereinbelow.
  • the reduction in sensitization will be at least about 20% and more preferably at least about 30% or more.
  • the 3'-sulfate Lewis x -OR compounds are effective in reducing sensitization when administered at a dosage range of from about 0.5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 mg to about 5 mg/kg of body weight.
  • the specific dose employed is regulated by the sensitization being treated as well as by the judgment of the attending clinician depending upon the age and general condition of the patient and the like.
  • “Simultaneous" administration of the compound with the antigen with regard to inhibiting sensitization means that the compound is administered once or continuously throughout a period of time within 3 hours of the administration of an antigen, more preferably the compound is administered within 1 hour of the antigen.
  • the 3'-substituted Lewis x -OR compounds are generally administered parenterally, such as intranasally, intrapulmonarily,
  • Examples 1-22 illustrate preferred synthetic processes of this invention whereas Examples 23-26 illustrate biological activity for the compounds of Examples 13, 19 and 20.
  • the organic layer was cooled with cold water. To the mixture of p-chlorothiophenol and fucose acetates was added 1.72 kg of boron trifluoride etherate in dropwise fashion. The mixture was then stirred for 6 hours (overnight is acceptable) allowing the reaction mixture to come to ambient temperature. A small aliquot was removed from the reaction mixture and quenched into sodium bicarbonate solution. Once carbon dioxide evolution ceases, the reaction was checked for completion by tic. When complete, the whole reaction mixture was quenched into 1 L of saturated sodium bicarbonate and the organic layer separated after carbon dioxide evolution had finished. The organic layer was separated and air bubbled through this layer for 1 hour.
  • Benzyl chloride (1.275 L) was then added dropwise to the stirring solution and the mixture stirred overnight at room temperature. Tic indicated incomplete reaction so an additional 300 g of powdered KOH was added to the reaction mixture followed 30 minutes later by 425 mL of benzyl chloride. The solution was stirred at room temperature until tic indicated the reaction was complete. (If the reaction was not complete after 24 hours, additional powdered KOH was added followed by 200 mL of benzyl chloride). The reaction was quenched into several volumes of water and the reaction product extracted from the aqueous solution with methylene chloride. The organic layer was backwashed twice with water, dried and the solvent evaporated.
  • Trimethylsilyl trifluoromethanesulfonate (2.5 mL, 12.8 mmol) in dichloromethane (5 mL) was added in portions over a period of 30 minutes to a cooled (0 to -5°C) suspension of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl- ⁇ -D-glucopyranoside (10 g, 25.7 mmol) (compound 2A), CaSO 4 (20 g pulverized) and 8-methoxycarbonyloctanol (5.8 g, 30.8 mmol) in
  • dichloromethane (5 mL) were added by syringe. After stirring for 15 hours, the reaction mixture was diluted with dichloromethane (500 mL), filtered through celite and washed with a 5% solution of EDTA (3 ⁇ 500 mL) until colorless. The solution was dried over anhydrous sodium sulfate and the solvent evaporated. The residue was passed through a silica gel column (70-230 mesh) using hexane:ethyl acetate (2:1) and (1:1) as the eluant. Fractions containing compound 3 were pooled and the solution evaporated. The resulting product was crystallized from hot isopropanol to provide 7 g (74.9% yield) of compound 3.
  • the product after work-up, can be crystallized directly from isopropanol in 75-80% yield without any column
  • the organic layer was transferred to a 2 L Erlenmeyer flask and the aqueous layer was extracted with 200 mL of dichloromethane.
  • the combined dichloromethane layer was washed with ice cold 6% sodium bicarbonate (200 g).
  • the solution was then filtered using a filtration flask (2 L) on a Buchner funnel (7.5 cm) and rinsed with dichloromethane (500 mL).
  • the filtrate was evaporated to dryness to provide a pale yellow solid.
  • the crude product was purified by chromatography using 70-230 mesh silica gel and a 3" HPLC column packed with dichloromethane.
  • 2,3,4,6-Tetra-O-acetyl-( ⁇ , ⁇ )-galactopyranoside (145 g, 0.42 mol) was dissolved in anhydrous dichloromethane (500 mL) and DBU (6.2 mL) was added. The reaction mixture was stirred for 10 minutes and then trichloroacetonitrile (73 mL, 73 mol) was added and the stirring continued for 1.5 hours by which time all the starting material had reacted to provide a new spot of higher Rf (hexane: ethyl acetate; 1: 1). The reaction mixture was then diluted with dichloromethane (1.5 L) and water (1.0 L).
  • the filtrate and washings were evaporated in a 5 L round bottom flask to obtain a thick syrup.
  • the syrup was dissolved in warm ethyl acetate (1.5 L) and transferred to an extractor.
  • the ethyl acetate layer was washed with water (2 ⁇ 500 mL) and the water layers were back extracted with warm ethyl acetate (300 mL).
  • the combined organic layers were dried over sodium sulfate (400 g) for 0.5 hours and then filtered and the sodium sulfate washed with warm ethyl acetate.
  • the solution was transferred to a 5 L round bottom flask and concentrated to about
  • Unreacted compound 4 was recovered by evaporating the mother liquor to a solid and recrystallizing the solid from isopropanol (400 mL) to provide 9.5 g of compound 4.
  • the yield of compound 6 based on consumed compound 4 was 80%.
  • the crude product was purified by chromatography on silica gel (70 - 230 mesh) using (dichloromethane:methanol:pyridine (95:5:0.1) as eluent to provide 110 g of compound 8 (75% yield) after conversion to the sodium salt by passage through Amberlite IR-120 (Na + ) ion exchange resin and crystallization with isobutyl alcohol (10 mL/g). Disulfated material was eluted in the later fractions of the column (12 g, 7%).
  • chromatography can also be conducted using dichloromethane:methanol:NH 1 OH (95:5:0.1) as the eluent in place of dichloromethane:methanol:pyridine (95:5:0.1).
  • the combined organic layers were returned to the 50 L reactor and extracted twice with 5 L portions of ice water for 10 minutes.
  • the organic layer was drained into a clean 20 L polyethylene pail and the aqueous layer was discarded as waste.
  • the organic layer was returned to the 50 L reactor, stirred and 1 kg of anhydrous sodium sulfate added. This mixture was stirred for 1-2 hours and then drained into a clean 20 L polyethylene pail and filtered using a 4 L vacuum filtration set.
  • the filtrate was concentrated to 8 L and then transferred into a clean 20 L reactor equipped with stirrer, 1 L addition funnel and cooling bath. Additional solvent was added if the level of the solution was below the thermowell.
  • the organic solution was cooled to 0°C using a cooling bath.
  • the reaction mixture was drained into a clean 20 L polyethylene pail.
  • the 50 L reactor was charged with 15 L of saturated sodium carbonate solution.
  • the reaction mixture in the 20 L polyethylene pail was then slowly transferred into the slowly stirring carbonate solution at such a rate that gas evolution was not overly vigorous.
  • the solution was stirred for 20 minutes then the rate of stirring was increased. When gas evolution ceased, air was bubbled through the entire solution for 24 to 36 hours.
  • the organic layer was drained into a clean 20 L polyethylene pail and stored in a hood.
  • the sodium carbonate solution was then extracted with 3 to 5 L of dichloromethane and drained into the same 20 L polyethylene pail.
  • the organic solution was filtered using a 4 L vacuum filtration set and the filtrate was evaporated under reduced pressure on the 20 L rotovap.
  • Methanol (7 L) was introduced into the rotavap flask and the residue heated with the rotavap bath until the residue dissolved in the warm methanol. The flask was rotated and allowed to cool. Ice water was added to the rotavap bath and the flask was slowly rotated for several hours. The flask was then removed from the rotovap and the white crystalline product, compound 10B, was filtered using a 4 L vacuum filtration set.
  • Benzyl 4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-thiogalactopyranoside, compound 5, was isolated by quenching the reaction mixture into saturated sodium bicarbonate solution and washing the organic extract with water, 5% copper sulfate solution, water, and then drying and evaporating the solvent. The residue was crystallized from isopropanol to give 10.7 g of benzyl-4,6-O-benzylidene-2,3-di-O-benzoyl- ⁇ -D-thiogalactopyranoside, compound 10.
  • tetraethylammonium bromide was added and the mixture was stirred for 2 to 3 hours at room temperature (the reaction was monitored by tlc on silica gel).
  • this product can be crystallized in the manner of the product obtained in Example 11 above.
  • reaction mixture was stirred for 20 hours at this temperature and 15 hours at -20°C, then quenched by adding methanol (10 mL).
  • methanol 10 mL
  • the reaction product was converted into its sodium salt form by passing the reaction mixture through a column of Amberlite IR-120 (Na + ) ion exchange resin. Fractions wore evaporated (after adding a trace of pyridine (5 mL)) and then co-evaporated with toluene (3 X 100 mL) to remove excess dimethylformamide to provide a white solid.
  • the solid was purified by chromatography on Iatrobeads using dichloromethane: methanol:pyridine (95:5:0.1) and (90:10:0.1) as eluant to provide 7.5 g of compound 13 (70.9% yield).
  • dichloromethane methanol:pyridine (95:5:0.1) and (90:10:0.1) as eluant to provide 7.5 g of compound 13 (70.9% yield).
  • the desired 3'-sulfo derivative can be crystallized from 2-methyl propanol.
  • compound 12 was prepared. This compound was converted to the title compound by first converting compound 12 to 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[3-O-diphenylphospho-4,6-O-benzylidene- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside by combining compound 12 (8.0 g, 6.97 mmol) in dry pyridine (50 mL) and then adding dimethylaminopyridine (1.6 g, 13.03 mmol).
  • reaction mixture was stirred at 0°C for 15 minutes and then diphenylphosphorochloridate (3.5g, 13.03 mmol) was added dropwise at 0°C and stirring was continued at this temperature for approximately 2 hours whereupon tic showed complete consumption of starting material.
  • diphenylphosphorochloridate 3.5g, 13.03 mmol
  • the reaction was worked up by addition of methanol, evaporation of the solvents and the solids taken in dichloromethane (250 mL) which was then washed with a cold aqueous 5% HCl solution (2 X 250 mL), cold saturated sodium bicarbonate solution (2 X 250 mL) and finally with cold water (2 X 250 mL).
  • the dichloromethane solution was filtered, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and subsequently purified via silica chromatography [hexane:ethyl acetate (2.5:1)] to provide for 8-methoxycarbonyloctyl-2-acetamido-3-O-(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-4-O-[3-O-diphenylphospho-4,6-O-benzylidene- ⁇ -D-galactopyranosyl]-6-O-benzyl-2-deoxy- ⁇ -D-glucopyranoside (7.81 g, 81% yield).
  • the title compound is prepared as follows.
  • Galactose pentaacetate (6 g, 15.4 mmol) was dissolved in 50 mL of tetrahydrofuran and 2.47 mL of benzylamine and the resulting solution stirred for 2 to 4 hours and checked by tic. After reaction completion, the tetrahydrofuran was removed under reduced pressure on the rotary evaporator and placed under high vacuum for 1 hour. The residue was taken up in dry dichloromethane, cooled to 0°C and DBU (460 mg) added to the reaction mixture followed by 3 mL of trichloroacetonitrile (2 equivalents).
  • reaction mixture was stirred at this temperature for 2 hours and the reaction progress monitored by tic. Upon reaction completion, the reaction mixture was poured into water, washed 2 or 3 times with water and the solvent removed under reduced pressure. If the color in the residue is too dark, silica can be added to the stirring reaction mixture just prior to work-up to remove some of the polar, dark impurities.
  • the residue was taken up in 60 mL of isopropanol, warmed to dissolve the residue, and allowed to cool first to room temperature and then to about 0°C in an ice bath for 1 to 2 hours whereupon the title compound precipitates from solution. The off-white imidate is filtered and dried under vacuum to give 3.6 grams (46%) of the title compound.
  • Examples 23 to 26 illustrate the immunomodulatory, antiinflammatory, and tolerogenic properties of compounds disclosed herein.
  • DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola 15 . Briefly, groups of Balb/c mice were immunized with SuperCarrier (SC) which have been shown to induce a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ⁇ g of SC. The resulting
  • mice were left untreated or received 100 ⁇ L of phosphate-buffered saline (PBS). The results of this test are set forth in Figure 5 which illustrate that the 3'-sulfo-Lewis x -OR provided roughly equivalent reduction in inflammation to sialyl Lewis x -OR.
  • Another group of mice (about 19-20 grams each) were immunized with 100 ⁇ g of the OVA antigen containing 20 ⁇ g of the adjuvant (DDA - - dimethyldioctadecylammonium bromide) which induces a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ⁇ g of the OVA antigen (without adjuvant). The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
  • DDA - - dimethyldioctadecylammonium bromide the adjuvant
  • LPS lipopolysaccharide
  • mice were anethesitized with Metofane (Pitman-Moore Ltd., Mississauga, Ontario, Canada) and a 50 ⁇ L drop of compound was placed on the nares of the mouse and was inhaled.
  • Metofane Pane-Moore Ltd., Mississauga, Ontario, Canada
  • the percent reduction was measured by subtracting from 100 the fraction derived by a numerator whose value is the weight of the treated lungs subtracted from the weight of normal lungs (lungs from mice not exposed to LPS, sialyl Lewis x -OR or 3'-sulfo-Lewis x -OR), and whose denominator value is the weight of the control lungs (mice that received only LPS) subtracted from the weight of normal lungs and multiplying the resulting fraction by 100.
  • the greater the percent reduction the better the compound was in alleviating lung damage.
  • Example 25 Effect of Administration of Sialyl Lewis x and 3'-Sulfo-Lewis x at the Time of Immunization on the Induction of an Immune Response to an Antigen
  • mice were immunized with 20 ⁇ g/mouse SC in 100 ⁇ l of PBS intramuscularly into the hind leg muscle which formulation also contained 100 ⁇ g/mouse of sialyl Lewis x -OR or
  • mice were footpad challenged with 20 ⁇ g/mouse of SC in 20 ⁇ L of PBS.
  • Control groups were either not immunized or received 20 ⁇ L of phosphate-buffered saline (PBS) in place of either sialyl Lewis x -OR or 3'-sulfo-Lewis x -OR.
  • PBS phosphate-buffered saline
  • Figure 7 shows that administering sialyl Lewis x -OR or 3'-sulfo-Lewis x -OR the mice at the time of immunization reduces the induction of an immune response to an antigen as compared to PBS control. This suggests that administration of a compound of this invention at the time of antigen immunization will reduce the ability of the mammal to become educated concerning this antigen.
  • mice treated with sialyl Lewis x -OR The identical groups of mice treated with sialyl Lewis x -OR and

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Abstract

L'invention concerne des procédés de synthèse chimique de composés de Lewisx-OR substitués en 3' dans lesquels R est un aglycone d'au moins un atome de carbone. Un composé particulier préparé par le procédé de cette invention est le 8-méthoxycarbonyl-octyl-2-acétamido-3-O-(α-L-fucopyranosyl)-4-O-[3-O-sulfo-β-D-galactopyranosyl]-2-désoxy-β-D-glucopyranoside.
PCT/US1995/014573 1994-11-21 1995-11-07 PROCEDES POUR SYNTHETISER DE COMPOSES DE LEWISx SUBSTITUES EN 3' WO1996016071A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002278A1 (fr) * 1995-06-30 1997-01-23 Nippon Shinyaku Co., Ltd. Derives formant une chaine saccharidique et procede pour les produire
CN102875610A (zh) * 2012-09-19 2013-01-16 淮海工学院 一种制备2-乙酰氨基-2-脱氧-3,4,6-三乙酰-β-D-氯代吡喃葡糖的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992022564A1 (fr) * 1991-06-10 1992-12-23 Alberta Research Council Composes modifies immunosuppresseurs et tolerogenes de lewisx et de lewis?a¿

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992022564A1 (fr) * 1991-06-10 1992-12-23 Alberta Research Council Composes modifies immunosuppresseurs et tolerogenes de lewisx et de lewis?a¿

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CARBOHYDRATE RESEARCH, Volume 185, issued 1989, SRIVASTAVA et al., "Synthesis of the 3''-Sulfate Ester of beta-D-GalpNAc-(1 4)-beta-D-GlcpNAc-(1 2)-alpha-D-Manp", pages 163-169. *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Volume 115, issued 1993, K.C. NICOLAOU et al., "Total Synthesis of Sulfated Lex and Lea-Type Oligosaccharide Selectin Ligands", pages 8843-8884. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, Number 33, issued 25 November 1992, E.V. CHANDRASEKARAN et al., "Ovarian Cancer alpha1,3-L-Fucosyltransferase", pages 23806-23814. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002278A1 (fr) * 1995-06-30 1997-01-23 Nippon Shinyaku Co., Ltd. Derives formant une chaine saccharidique et procede pour les produire
CN102875610A (zh) * 2012-09-19 2013-01-16 淮海工学院 一种制备2-乙酰氨基-2-脱氧-3,4,6-三乙酰-β-D-氯代吡喃葡糖的方法

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