Sialyl Le.sup.x analogues as inhibitors of cellular adhesion

FIELD OF THE INVENTION
The present invention relates to compounds that inhibit cellular adhesion, and more particularly relates to analogue compounds of sialyl Lewis.sup.x (sialyl Le.sup.x or SLe.sup.x) that inhibit selectin-mediated cellular adhesion, compositions containing and processes for using the same, and processes for preparing those analogues.
BACKGROUND OF THE INVENTION
Vascular endothelial cells and blood platelets play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the bloodstream. For example, endothelial cells preferentially bind monocytes and granulocytes prior to their migration through the blood vessel wall and into surrounding tissue in an inflammatory response.
Certain inflammation-triggering compounds are known to act directly on the vascular endothelium to promote the adhesion of leukocytes to vessel walls. Cells then move through the walls and into areas of injury or infection.
Cellular adhesion to vascular endothelium is also thought to be involved in tumor metastasis. Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.
Blood platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane fuses with the external plasma membrane. As a result, new cell surface proteins become expressed that confer on the activated platelet new functions, such as the ability to bind both other activated platelets and other cells. Activated platelets are recruited into growing thrombi, or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes that are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.
Recent work has revealed that specialized cell surface receptors on endothelial cells and platelets, designated E-selectin (endothelial leukocyte adhesion molecule-1; ELAM-1) and P-selectin (granule membrane protein-140; GMP-140), respectively, are involved in the recognition of various circulating cells by the endothelium and platelets. For example, E-selectin has been shown to mediate endothelial leukocyte adhesion, which is the first step in many inflammatory responses. Specifically, E-selectin binds human neutrophils, monocytes, eosinophils, certain T-lymphocytes �Graber et al., J. Immunol., 145:819 (1990)!, NK cells, and the promyelocytic cell line HL-60.
E-selectin is inducibly expressed on vascular endothelial cells �Bevilacqua et al., Science, 243:1160-1165 (1989) and Hession et al., Proc. Natl. Acad. Sci., 87:1673-1677 (1990)!. This receptor has been demonstrated to be induced by inflammatory cytokines such as interleukin I.beta. (IL-I.beta.) and tumor necrosis factor .alpha. TNF.alpha.), as well as bacterial endotoxin (lipopolysaccharide) �Bevilacqua et al., Proc. Natl. Acad. Sci., 84:9238-9242 (1987)!. These compounds augment polymorphonuclear leukocyte (neutrophil), and monocyte adhesion �Bevilacqua et al., Proc. Natl. Acad. Sci., 84:9238-9242 (1987)!.
P-selectin (also known as GMP-140 and PADGEM) is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions, �Geng et al., Nature, 343:757-760 (1990)!. Thus, for example, activated platelets that express P-selectin on their surface are known to bind to monocytes and neutrophils �Jungi et al., Blood, 67:629-636 (1986)!, and also to bind monocyte-like cell lines, e.g., HL-60 and U937 �Jungi et al., Blood, 67:629-636 (1986); Silverstein et al., J. Clin. Invest., 79:867-874 (1987)!.
P-selectin is an alpha granule membrane protein of molecular mass 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion �Hsu-Lin et al., J. Clin. Chem., 259:9121-9126 (1984); Stenberg et al., J. Cell Biol., 101:880-886 (1985); Berman et al., J. Clin. Invest., 78:130-137 (1986)!. It is also found in megakaryocytes �Beckstead et al., Blood, 67:285-293 (1986)!, and in endothelial cells �McEver et al., Blood, 70:335a (1987)! within the Weibel-Palade bodies �Bonfanti et al., Blood, 73:1109-1112 (1989)!. Furie et al., U.S. Pat. No. 4,783,330, describe monoclonal antibodies reactive with P-selectin.
A third receptor is the lymphocyte homing receptor, MEL-14 antigen or its human counterpart LAM-1 (L-selectin) �Gallatin et al., Nature, 304:30-34 (1983); Siegellman et al., Science, 243:1165-1172 (1989); Rosen, Cell Biology, 1:913-919 (1989); and Lasky et al., Cell, 56:1045-1055 (1989)!. In addition to lymphocyte homing, MEL-14 antigen/LAM-1 is believed to function early in neutrophil binding to the endothelium.
The term "selectin" has been suggested for a general class of receptors, which includes E-selectin (ELAM-1), P-selectin (GMP-140) and L-selectin (MEL-14), because of their lectin-like domain and the selective nature of their adhesive functions. The structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors �Bevilacqua et al., Science, 243:1160-1165 (1989), (ELAM-1); Geng et al., Nature, 343:757-760 (1990), (GMP-140); and Lasky et al., Cell, 56:1045-1055 (1989), (MEL-14 antigen)!.
The extracellular portion of selectins can be divided into three segments based on homologies to previously described proteins. The N-terminal region (about 120 amino acids) is related to the C-type mammalian lectin protein family as described by Drickamer, J. Biol. Chem., 263:9557-9560 (1988) that induces low affinity IgE receptor CD23. A polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif. Lastly, after the EGF domain are one or more tandem repetitive motifs of about 60 amino acids each, related to those found in a family of complement regulatory proteins.
U.S. Pat. No. 5,079,353 and its divisional U.S. Pat. No. 5,296,594 teach the synthesis and use of the sialyl Le.sup.x and sialyl Le.sup.a antigens that are present in cancerous tissues, and are ligands for the before-described selectin receptors. U.S. Pat. No. 5,143,712 teaches the binding interactions between various receptors such as ELAM-1 (E-selectin) and ligands such as sialyl Le.sup.x as well as ligands containing a plurality of N-acetyllactosamine (LacNAc) units along with a terminal sialyl group and one or more fucosyl groups that are bonded to the GlcNAc portion of a LacNAc unit.
Published International application WO 91/19501 and WO 91/19502 disclose that oligosaccharides containing the pentameric and hexameric structures shown below inhibited selective cellular binding between cells containing the ligand (below) and those containing a selectin receptor, and that the penta- and hexasaccharides assayed provided better inhibition than did SLe.sup.x.
NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc.beta.1,3Gal.beta.-;
NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc.beta.1,3Gal.beta.1,4Glc-; and
NeuAc.alpha.2-3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc=SLe.sup.x.
SUMMARY OF THE INVENTION
The present invention contemplates a sialyl Le.sup.x (SLe.sup.x) analogue compound that inhibits the adhesion of cells that express SLe.sup.x on their surfaces to a selectin receptor, intermediate compounds in the synthesis of an inhibitor, as well as a process for preparing such intermediates and a pharmaceutical composition containing an inhibitor.
In particular, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable diluent having dissolved or dispersed therein a cellular adhesion-inhibiting amount of a compound of the formula ##STR2## wherein: Z is selected from the group consisting of hydrogen, C.sub.1 -C.sub.6 acyl and ##STR3## Y is selected from the group consisting of C(O), SO.sub.2, HNC(O), OC(O) and SC(O); R.sup.1 is selected from the group consisting of a linking group for a lipid, a lipid, a linking group with attached lipid, an aryl, a substituted aryl and a phenyl C.sub.1 -C.sub.3 alkylene group, wherein an aryl group has one five-membered aromatic ring, one six-membered aromatic ring or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl, monooxahydrocarbyl, monothiahydrocarbyl, monoazahydrocarbyl and diazahydrocarbyl rings, and a substituted aryl group is said aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C.sub.1 -C.sub.12 alkyl, C.sub.1 -C.sub.12 alkoxy, amino, mono-C.sub.1 -C.sub.12 alkylamino, di-C.sub.1 -C.sub.12 alkylamino, benzylamino, C.sub.1 -C.sub.12 alkylbenzylamino, C.sub.1 -C.sub.12 thioalkyl and C.sub.1 -C.sub.12 alkyl carboxamido groups, or R.sup.1 Y is allyloxycarbonyl or chloroacetyl; R.sup.2 is selected from the group consisting of hydrogen, a linking group for a lipid, a lipid, a linking group with attached lipid, C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl, C.sub.1 -C.sub.6 alkyl C.sub.1 -C.sub.5 alkylene .omega.-carboxylate, .omega.-tri(C.sub.1 -C.sub.4 alkyl/phenyl)silyl C.sub.2 -C.sub.4 alkylene, monosaccharide and disaccharide, or OR.sup.2 together form a C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl carbamate; R.sup.4 is an alkyl group; R.sup.5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C.sub.1 -C.sub.6 acyl; R.sup.7 is methyl or hydroxymethyl; and X is selected from the group consisting of C.sub.1 -C.sub.6 acyloxy, C.sub.2 -C.sub.6 hydroxylacyloxy, hydroxy, halo and azido. In a presently preferred embodiment, R.sup.4 is a member selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, benzyl, pentyl and hexyl.
The present invention further provides intermediates which are useful in the preparation of the pharmaceutical compositions above. In particular, the intermediates are either lactones or esters, having the formulae (Va), (Vb) and (VI), respectively, ##STR4## in which Z, R.sup.4, R.sup.6, R.sup.7 and X are as defined above.
The invention further provides a process for the preparation of compounds of Formula IV, comprising:
(a) preparing a lactone intermediate of Formula (Va) or (Vb);
(b) treating the lactone intermediate with an alkoxide to form an ester intermediate of Formula (VI); and
(c) deprotecting, fucosylating and attaching R.sup.1 --Y to provide the pharmaceutical agents of Formula (IV).
The pharmaceutical compositions of the present invention are useful in methods of inhibiting intercellular adhesion in a patient for a disease process, such as inflammation. The selectin receptor, such as E-Selectin or P-Selectin, may be expressed on vascular endothelial cells or platelets. The inflammatory process may be, for example, septic shock, wound associated sepsis, rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), asthma, traumatic shock, nephritis, and acute and chronic inflammation, including atopic dermatitis, psoriasis, and inflammatory bowel disease. Various platelet-mediated pathologies such as atherosclerosis and clotting can also be treated. In addition, tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.

BRIEF DESCRIPTIONS OF THE DRAWINGS
FIG. 1 shows a reaction scheme for the preparation of compound 62.
FIG. 2 shows a reaction scheme for the preparation of compound 73.
FIG. 3 shows a reaction scheme for the preparation of compound 78.
FIG. 4 shows a reaction scheme for the preparation of compound 85.
FIG. 5 shows a reaction scheme for the preparation of compound 100.
FIG. 6 shows a reaction scheme for the preparation of compound 115.
FIG. 7 shows a reaction scheme for the preparation of compounds 116 to 120.

DESCRIPTION OF THE EMBODIMENTS
A. The Compounds
The nomenclature used to describe the oligosaccharide moieties of the present invention follows the conventional nomenclature. Standard abbreviations for individual monosaccharides are used. For instance, 2-N-acetylglucosamine is represented by GlcNAc, 2-N-acetylgalactosamine is GalNAc, fucose is Fuc, fructose is Fru, galactose in Gal, glucose is Glc, and mannose is Man. Unless otherwise indicated, all sugars except fucose (L-isomer) are D-isomers in the cyclic configuration (e.g., pyranose or furanose). The two anomers of the cyclic forms are represented by .alpha. and .beta..
The monosaccharides are generally linked by glycosidic bonds to form oligo- and polysaccharides. The orientation of the bond with respect to the plane of the rings is indicated by .alpha. and .beta.. The particular carbon atoms that form the bond between the two monosaccharides are also noted. Thus, a .beta. glycosidic bond between C-1 of galactose and C-4 of glucose is represented by Gal.beta.1.fwdarw.4Glc. For the D-sugars (e.g., D-GlcNAc, D-Gal, D-NeuAc and D-Man) the designation .alpha. means the hydroxyl attached to C-1 (C-2 in NeuAc) is below the plane of the ring and .beta. is above the ring. In the case of L-fucose, the .alpha. designation means the hydroxyl is above the ring and .beta. means it is below.
The present invention contemplates a SLe.sup.x analogue compound of structural Formula A, below, which structural formula encompasses a pentasaccharide compound of Formula I that is an analogue of sialyl Le.sup.x, as well as its penta- and tetrasaccharide precursors of Formulas II and III, respectively. A compound of structural Formula I inhibits cellular adhesion mediated by a selectin cell surface receptor. ##STR5##
In the above structural formulas,
Z is hydrogen (H) or C.sub.1 -C.sub.6 acyl, in which case a compound of Formula III is defined, or an .alpha.-L-fucosyl whose hydroxyl groups are free or blocked with a protecting group (benzyl or C.sub.1 -C.sub.6 acyl) thereby defining a compound of Formula I or II, depending upon the identities of R.sup.3, R.sup.4 and R.sup.5 (R.sup.3-5) groups;
Y is selected from the group consisting of C(O), SO.sub.2, HNC(O), OC(O) and SC(O);
R.sup.1 is selected from the group consisting of a linking group for a lipid, a lipid, a linking group with attached lipid, an aryl, a substituted aryl and a phenyl C.sub.1 -C.sub.3 alkylene group, wherein an aryl group has one five- or six-membered aromatic ring, fused five/six-membered aromatic rings, or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl, monooxahydrocarbyl, monothiahydrocarbyl, monoazahydrocarbyl and diazahydrocarbyl rings, and a substituted aryl group is a before-mentioned aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, amino, mono-C.sub.1 -C.sub.18 alkylamino, di-C.sub.1 -C.sub.18 alkylamino, benzylamino, C.sub.1 -C.sub.18 alkylbenzylamino, C.sub.1 -C.sub.18 thioalkyl and C.sub.1 -C.sub.18 alkyl carboxamido group, or
R.sup.1 Y is allyloxycarbonyl or chloroacetyl;
R.sup.2 is selected from the group consisting of hydrogen, a linking group for a lipid, a lipid, a linking group with attached lipid, C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl, C.sub.1 -C.sub.6 alkyl C.sub.1 -C.sub.5 alkylene .omega.-carboxylate, .omega.-tri(C.sub.1 -C.sub.4 alkyl/phenyl)silyl C.sub.2 -C.sub.4 alkylene, monosaccharide and disaccharide,
or OR.sup.2 together form a C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl carbamate;
R.sup.3 is hydrogen or C.sub.1 -C.sub.6 acyl;
R.sup.4 is hydrogen, C.sub.1 -C.sub.6 alkyl or benzyl;
R.sup.5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C.sub.1 -C.sub.6 acyl;
R.sup.7 is methyl (CH.sub.3) or hydroxymethyl (CH.sub.2 OH); and
X is selected from the group consisting of C.sub.1 -C.sub.6 acyloxy, C.sub.2 -C.sub.6 hydroxylacyloxy, hydroxy, halo and azido.
As noted above, Y can be one of a number of groups. When Y is C(O), R.sup.1 Y is an acyl substituent group so that an amide is formed with the saccharide amine nitrogen atom. When Y is SO.sub.2, R.sup.1 Y forms a sulfonyl substituent group so that a sulfonamide is formed with the saccharide amine nitrogen atom. When Y is HNC(O), R.sup.1 Y forms an aminocarbonyl substituent group so that a urea substituent is formed with that saccharide nitrogen atom. A urethane substituent is formed with the saccharide amine nitrogen where Y is oxycarbonyl, OC(O), whereas a thiourethane is formed where Y is thiocarbonyl, SC(O). A Y group is preferably a carbonyl group �C(O)!.
An R.sup.1 Y group can also be an allyloxycarbonyl or a chloroacetyl group. An allyloxycarbonyl R.sup.1 Y group is particularly preferred for a compound of Formula III as it provides a readily replaceable R.sup.1 group. An R.sup.1 Y allyloxycarbonyl or chloroacetyl group is present only in a compound of Formula III, and is not present in a compound of any of Formulas I, II, A, B or C (Formulas B and C are shown hereinafter).
As discussed before, an R.sup.1 group can be an aryl or substituted aryl group. Contemplated aryl groups are those that contain one aromatic five- or six-membered ring, fused five- and six- (five/six-) membered rings or two fused aromatic six-membered rings and include hydrocarbyl groups such as phenyl and naphthyl, as well as hydrocarbyl groups bearing an oxygen, a sulfur, or one or two nitrogen atoms that replace ring carbon atoms (mono- or diazahydrocarbyl). Exemplary aryl groups include furyl, thienyl, pyridyl, pyrazinyl, benzofuranyl (benzo�b!furyl), isobenzofuranyl (benzo�c!furyl), benzothienyl (benzo�b!thienyl), isobenzothienyl (benzo�c!thienyl), pyrimidinyl, pyridazinyl, quinolinyl, isoquinoyl, quinoxalinyl, naphthyridinyl, phthalazinyl and quinazolinyl. Each of those aryl groups can be unsubstituted, or each can have a substituent selected from the group consisting of halo, trifluoromethyl, nitro, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, amino, mono-C.sub.1 -C.sub.18 alkylamino, di-C.sub.1 -C.sub.18 alkylamino, C.sub.1 -C.sub.18 alkylbenzylamino and C.sub.1 -C.sub.18 alkyl carboxamido.
The above unsubstituted and substituted aryl R.sup.1 groups are well known in the art, and each can be bonded to the saccharide nitrogen atom using well known chemistry. The following discussion will therefore center upon aryl hydrocarbyl groups, phenyl and naphthyl, as being exemplary of the group, with the understanding that the other enumerated aryl and substituted aryl R.sup.1 groups can be utilized with substantially similar chemistry.
Where R.sup.1 is phenyl, benzoyl chloride or benzoic anhydride can be used to form a preferred amide bond. A benzenesulfonyl halide such as benzenesulfonyl chloride can similarly be used where Y is SO.sub.2. Phenyl isocyanate is used where Y is HNC(O). A phenyl chloroformate is used where Y is OC(O), whereas a phenyl chlorothioformate is used where Y is SC(O).
Specifically contemplated substituted phenyl R.sup.1 groups include those in which the substituent can be substituted at any position of the ring, with the meta and para positions being preferred. Mono-substituted R.sup.1 phenyl groups are preferred over di-substituted groups.
Contemplated halo substituents include fluoro, chloro, bromo and iodo groups, with p-fluorophenyl, m-chlorophenyl, m-iodophenyl, p-bromophenyl and o-fluorophenyl being exemplary. Dihalo-substituted phenyl R.sup.1 groups are also contemplated such as 3,4-dichlorophenyl, 3,5-dichlorophenyl, 2-chloro-4-fluorophenyl and 3-bromo-4-fluorophenyl.
Exemplary C.sub.1 -C.sub.18 alkyl groups present as substituent groups on a phenyl of R.sup.1 include straight and branched chain alkyl groups such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, t-butyl, pentyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl. C.sub.1 -C.sub.12 Alkyl groups are preferred, whereas C.sub.1 -C.sub.6 alkyl groups are particularly preferred, with methyl being most preferred. Exemplary, preferred R.sup.1 groups include o-, m- and p-tolyl (methylphenyl) and p-t-butylphenyl groups as well as 3,4-dimethylphenyl and 3,5-dimethylphenyl groups.
Exemplary C.sub.1 -C.sub.18 alkoxy groups are ethers containing a C.sub.1 -C.sub.18 alkyl group, or a particularly preferred C.sub.1 -C.sub.6 alkyl group. Methoxy is preferred here. Exemplary, preferred R.sup.1 groups include o, m- and p-anisyl (methoxyphenyl), as well as 3,4-dimethoxyphenyl and 3,5-dimethoxyphenyl.
A nitrophenyl R.sup.1 group is readily prepared by acylation using 3- or 4-nitrobenzoyl chloride. Acylation with 3,4- and 3,5-dinitrobenzoyl chloride provides the corresponding 3,4- and 3,5-dinitrophenyl R.sup.1 groups. Amide formation using 3- or 4-trifluoromethylbenzoyl chloride similarly provides 3- or 4-trifluoromethylphenyl R.sup.1 groups.
A substituted phenyl R.sup.1 group can also contain an amino, mono-C.sub.1 -C.sub.18 alkylamino, di-C.sub.1 -C.sub.18 alkylamino, benzylamino, C.sub.1 -C.sub.18 alkylbenzylamino or C.sub.1 -C.sub.18 alkyl carboxamido substituent, wherein C.sub.1 -C.sub.18 alkyl substituents are as discussed before.
Aminophenyl R.sup.1 groups are most readily prepared from corresponding nitrophenyl R.sup.1 groups discussed before by catalytic reduction of the nitro group after formation of the amide bond, as discussed before. Thus, for example, use of 3- or 4-nitrobenzoyl chloride to form the amide bond, upon reduction with palladium on carbon forms the corresponding 3- or 4-aminophenyl R.sup.1 group. A similar use of 3,4- or 3,5-dinitrobenzoyl chloride provides the corresponding 3,4- or 3,5-diaminophenyl R.sup.1 group after reduction.
Several di-C.sub.1 -C.sub.6 alkylaminobenzoic acids such as 4-diethylaminobenzoic acid and 3- and 4-dimethylaminobenzoic acids can be purchased commercially and used to form an appropriate benzoyl halide or anhydride for forming an R.sup.1 -containing amide. The remaining di-C.sub.1 -C.sub.18 alkylaminobenzoic acids and those compounds having two dialkylamino groups can be prepared using well known alkylation techniques from corresponding aminobenzoic acids or diaminobenzoic acids that are also commercially available.
A mono-C.sub.1 -C.sub.18 alkylaminophenyl R.sup.1 group can be prepared from the corresponding mono-C.sub.1 -C.sub.18 alkylaminobenzoyl halide, whose remaining nitrogen valence is blocked by a readily removable blocking group such as t-Boc that can be removed with acid or a benzyl group that can be removed by hydrogenation, if desired, using palladium on carbon. Thus, acylation can take place using N-benzyl-N-propylaminobenzoyl chloride, with the N-benzyl group being removed by catalytic hydrogenation to provide the mono-C.sub.1 -C.sub.18 alkylaminophenyl R.sup.1 group. Of course, the benzyl group need not be removed, thereby providing a C.sub.1 -C.sub.18 alkylbenzylamino group.
Each of the above-discussed phenyl or substituted phenyl substituents can be prepared by a well known amide-forming reaction. An exemplary reaction reacts an appropriate benzoyl halide or anhydride such as p-fluorobenzoyl chloride or benzoic anhydride with the unprotected amine group of an otherwise protected saccharide as is illustrated in detail hereinafter.
Both 1- and 2-naphthyl R.sup.1 groups are contemplated, with 2-naphthyl being particularly preferred. These compounds can also be prepared using standard amide-forming technology as above, such as by reacting 2-naphthoyl chloride with an amine of an appropriate saccharide as discussed above.
It is to be understood that similar substituents are present on the oxa-, thia-, aza- and diazahydrocarbyl aryl groups. For example, one can utilize any of the two furoic acid chlorides, the two thiophenecarboxyl chlorides, three pyridinecarboxyl chlorides, quinaldic acid chloride, 3-quinolinecarboxylic acid chloride, 2-quinoxaloyl chloride and the like to carry out an acylation reaction.
Similarly, where Y is SO.sub.2, a corresponding sulfonyl halide is used. For example, one may utilize benzenesulfonyl chloride, toluenesulfonyl chloride, 8-quinolinesulfonyl chloride, 1- or 2-naphthalenesulfonyl chloride, and the like to form the sulfonamide.
Where Y is HNC(O), the isocyanate corresponding to a before-described carboxylic acid is a convenient reactant. Such derivatives can be readily prepared from the acid halide by reaction with azide, to form the acyl azide, which undergoes the Curtius rearrangement to form the isocyanate upon heating.
Where Y is OC(O) or SC(O), a hydroxyl or mercapto substituted aryl R.sup.1 group is reacted with phosgene to form the chloroformate or chlorothioformate that can be reacted with the saccharide amine to form the urethane or thiourethane linkage to an R.sup.1.
A phenyl C.sub.1 -C.sub.3 alkylene R.sup.1 group is a C.sub.1 -C.sub.3 alkylene group that is itself substituted with a phenyl group, preferably at the terminal hydrocarbyl group carbon. This R.sup.1 C(O) group thus contains a phenyl ring linked to a chain of 2-4 carbon atoms. Exemplary C(O)R.sup.1 alkylene groups include 2-phenylacetoyl, 3-phenylpropionyl and 4-phenylbutanoyl �.phi.CH.sub.2 C(O), .phi.CH.sub.2 CH.sub.2 C(O) and .phi.(CH.sub.2).sub.3 C(O), respectively, where .phi.=phenyl!. These compounds can be prepared by reaction of an appropriate acid halide or anhydride with a saccharidal amine as above. Catalytic reduction using hydrogen and a palladium on carbon catalyst can be used to form saturated alkylene groups from the unsaturated hydrocarbyl chains; saturated hydrocarbyl chains being preferred.
An R.sup.2 group forms a .beta.-glycoside with the saccharide ring system. That glycoside bond can be formed from a simple C.sub.1 -C.sub.18 hydrocarbyl alcohol, from an .omega.-hydroxycarboxylic acid ester, from an .omega.-hydroxylated silylated alkyl group, or from a mono- or a disaccharide, or OR.sup.2 together form a C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl carbamate. A C.sub.1 -C.sub.6 hydrocarbyl group such as ethyl, a benzyl group or a monosaccharide such as 3-galactosyl is particularly preferred. R.sup.2 can also be hydrogen.
Exemplary R.sup.2 groups formed from simple precursor alcohol groups include C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl groups. Illustrative of such groups are the before-described C.sub.1 -C.sub.6 alkyl groups, which are preferred, as well as their unsaturated counterparts, such as allyl, 3-butenyl, 2-but-3-enyl, and but-3-ynyl, as well as longer hydrocarbyl groups such as benzyl, 4-methylcyclohexyl, decahydronaphthyl, nonyl, decyl (capryl), dodecyl (lauryl), dodec-7-enyl, myristyl, palmityl, stearyl, oleyl, linoleyl, linolenyl and ricinoleyl.
A C.sub.1 -C.sub.18 hydrocarbyl carbamate is prepared by reaction of an isocyanate corresponding to a before discussed C.sub.1 -C.sub.18 hydrocarbyl group with the hydroxyl group of the reducing end sugar. For example, the 1-hydroxyl group of a terminal glucosyl unit can be reacted with ethylisocyanate to form the corresponding ethyl carbamate (urethane). The carbonyl group of the carbamate is not included in the number of hydrocarbyl carbon atoms.
A C.sub.1 -C.sub.6 alkyl C.sub.1 -C.sub.5 -alkylene .omega.-carboxylate R.sup.2 group is a C.sub.1 -C.sub.6 alkyl ester of a C.sub.2 -C.sub.6 .omega.-carboxylic acid. Such esters are prepared from precursor .omega.-hydroxycarboxylic acid esters whose hydroxyl groups are used to form the glycosidic bond. Exemplary .omega.-hydroxycarboxylate esters include methyl 2-hydroxyacetate, ethyl 3-hydroxypropionate, t-butyl 4-hydroxybutyrate, hexyl 5-hydroxypentanoate and methyl 6-hydroxyhexanoate. Thus, the hydroxyl and carboxyl groups are at the termini of the chain and are separated by 1-5 methylene groups. Methyl 6-hydroxyhexanoate acid is preferred.
An .omega.-tri(C.sub.1 -C.sub.4 alkyl/phenyl)silyl C.sub.2 -C.sub.4 alkyl R.sup.2 group is formed from a corresponding precursor alcohol whose substituted silyl group is at the terminus (.omega.-position) of the chain opposite the hydroxyl group. As is well known in the art, substituted silyl groups can include many combinations of C.sub.1 -C.sub.4 alkyl and phenyl groups such as tri-C.sub.1 -C.sub.6 alkyl, di-C.sub.1 -C.sub.4 alkylphenyl, C.sub.1 -C.sub.4 alkyldiphenyl and triphenyl. Exemplary substituted silyl groups include trimethylsilyl, triphenylsilyl, di-t-butylmethylsilyl, dimethylphenylsilyl, t-butyldiphenylsilyl and the like.
Exemplary mono- and disaccharides include 3- and 4-glucosyl (3/4Glc), 3- and 4-galactosyl (3/4Gal), a 3-galactosyl group being particularly preferred, 3- and 4-N-acetylglucosyl (3/4GlcNAc), 2, 3-, 4- and 6-mannosyl (2/3/4/6Man), and 3- and 6-N-acetylgalactosyl (3/6 GalNAc) and Gal.beta.1.fwdarw.4GlcNAc. A monosaccharide can itself form a glycoside linkage with a group, R.sup.6, that includes all but a saccharide of an R.sup.2 group. Thus, R.sup.6 is R.sup.2 other than mono- or disaccharide.
A structural formula for a particularly preferred compound of Formula A having a reducing terminal 3Gal.beta.OR.sup.6 group is shown below in structural Formula B wherein X, Y, Z and R.sup.1-4, R.sup.6 and R.sup.7 are as defined before. ##STR6##
A particularly preferred compound of Formula B is an inhibitor of cellular adhesion having a structure of Formula C, below, wherein X, Y, R.sup.1, R.sup.6 and R.sup.7 are as before disclosed. ##STR7##
The .beta.-glycosyl bond formed with an R.sup.2 or R.sup.6 group can be prepared by well known organic chemical reactions with both the saccharides and other R.sup.2 (R.sup.6) group precursors, as by reaction of a 1-halo saccharide with a hydroxyl of a desired R.sup.2 (R.sup.6) group precursor alcohol in the presence of silver carbonate (Ag.sub.2 CO.sub.3) or silver triflate, as well as by enzymatic means as with a glycosyl transferase for the saccharides.
A contemplated R.sup.3 group can be hydrogen or C.sub.1 -C.sub.6 acyl, which is the acid portion of a C.sub.1 -C.sub.6 acyl carboxylic acid ester. A C.sub.1 -C.sub.6 acyl group is preferred for a compound of Formula II. Exemplary C.sub.1 -C.sub.6 acyl groups include formyl, acetyl, propionyl, butanoyl, isobutanoyl, pentanoyl and hexanoyl. An acetyl group is preferred. Acylation of saccharide hydroxyl groups is well known and can be carried out using an appropriate acid halide or anhydride.
A contemplated R.sup.4 group of Formula A can be hydrogen, a C.sub.1 -C.sub.6 alkyl, as was discussed before for such alkyl groups, or a benzyl group. An R.sup.4 group along with its bonded oxygen atom forms the alcohol portion of an ester. A methyl group is preferred. The R.sup.4 ester can be formed by standard means prior to the addition of the sialic acid group, after formation of the sialylated saccharide using a reagent such as diazomethane, or by reaction of a lactone with an appropriate alcohol as discussed in regard to Scheme 2, hereinafter.
The R.sup.4 group of a compound of Formula III can be either a proton, C.sub.1 -C.sub.6 alkyl or benzyl groups with C.sub.1 -C.sub.6 alkyl being preferred. When R.sup.4 is present as a proton, it is to be understood that that proton can be replaced by a pharmaceutically acceptable cation (M) such as ammonium, sodium, potassium, calcium, magnesium and the like. The R.sup.4 proton or other cation is typically not shown in the structures herein such as Formulas I and C because the sialyl carboxylic acid is usually ionized at physiological pH values of about 7.2-7.4 at which an inhibitor of Formulas I or C is utilized. Thus, the sialyl carboxyl group is often shown herein as a carboxylate.
An R.sup.5 group is a hydrogen, a benzyl, methoxybenzyl (3- or 4-methoxybenzyl being preferred), a dimethoxybenzyl such as 3,4- or 3,5-dimethoxybenzyl, or a C.sub.1 -C.sub.6 acyl group as discussed previously. A benzyl group is usually used where the fucosyl group is added by organic chemical synthesis.
R.sup.3, R.sup.4 and R.sup.5 groups other than hydrogen are protecting groups used during synthesis of intermediates such as a compound of Formulas B, II and III, above. When R.sup.3 .dbd.R.sup.4 .dbd.R.sup.5 .dbd.H (hydrogen) a compound of Formula II becomes a compound of Formula I, whereas a compound of Formula B becomes a compound of Formula C, when Z is fuco. Similarly, when Z is fuco and R.sup.3 .dbd.R.sup.4 .dbd.R.sup.5 =hydrogen, a compound of Formula A becomes a compound of Formula I.
An X substituent group can be a C.sub.1 -C.sub.6 acyloxy group; i.e., a C.sub.1 -C.sub.6 acyl ester of a precursor hydroxyl group at that position, a C.sub.2 -C.sub.6 hydroxylacyloxy group, a hydroxyl group, a halo group, as discussed previously, or an azido group. Exemplary C.sub.1 -C.sub.6 acyl groups have already been discussed, and a C.sub.1 -C.sub.6 acyloxy group is a C.sub.1 -C.sub.6 acyl group that further includes an additional oxygen atom bonded to the carbonyl carbon atom of an acyl group. A C.sub.2 -C.sub.6 hydroxylacyloxy group is an above-discussed C.sub.1 -C.sub.6 acyloxy group that further includes a substituent hydroxyl group. Exemplary C.sub.2 -C.sub.6 hydroxylacyloxy groups include hydroxyacetate, lactate, 3-hydroxybutyrate, 2-hydroxyisovalerate and 2-hydroxycaproate. An X substituent is usually other than C.sub.1 -C.sub.6 acyloxy or C.sub.2 -C.sub.6 hydroxylacyloxy unless both sialylation and fucosylation are carried out enzymatically, as is discussed hereinafter.
Syntheses of sialic acid derivatives containing an X substituent are disclosed in published international application WO 92/16640 that was published on Oct. 1, 1992. The use of those compounds for sialylating saccharides is also disclosed in that publication.
An R.sup.7 group is methyl or hydroxymethyl, so that along with the depicted carbonyl group �C(O)!R.sup.7 forms an N-acetyl or N-hydroxyacetyl group. Sialic acid derivatives containing either R.sup.7 group can be used in an enzymatic sialylation as described herein.
Particularly preferred inhibitor compounds of structural Formulas I and C are illustrated below, along with their compound numbers; i.e., Compounds 17, 30-38, and 43-51. ##STR8## B. Compound Syntheses
A before-described SLe.sup.x analogue compound can be prepared in numerous ways. Thus, completely enzymatic syntheses can be carried out, syntheses using only the techniques of organic chemistry can be used, and mixtures of both organic and enzymatic syntheses can be utilized, as is exemplified here.
One way to distinguish between organic and enzymatic syntheses is by the presence of one or more enzymes in a water-based reaction medium (enzymatic synthesis), versus the absence of any enzymes coupled with a reaction medium that is substantially free of water and utilizes an organic solvent such as acetonitrile, methanol, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), benzene, acetone, dichloromethane, tetrahydrofuran (THF) and the like (organic synthesis).
Regardless of which of those methods is utilized, the saccharides comprising lactosamine, galactose and glucosamine, must be joined together at some point in the syntheses. Somewhat surprisingly, the Gal.beta.1.fwdarw.4GlcN bond of lactosamine is also one of the more difficult bonds to form in the synthesis of a contemplated compound.
Lactosamine is a compound reported in the literature, but is not readily available. Nevertheless, lactosamine or a derivative of lactosamine provides a good starting material for synthesis of a contemplated compound.
Although lactosamine is not readily available, lactulose, a ketose that possesses no amine group but contains a Gal.beta.1.fwdarw.4Fru bond that is related to lactose and lactosamine, is commercially available. Lactulose, with its Gal.beta.1.fwdarw.4 bond already formed, provides a starting material for one contemplated synthesis of lactosamine. A synthesis of lactosamine (Compound 3) as an acid addition salt is illustrated generally and specifically below in Schemes 1 and 1A, respectively, as are the syntheses of peracetyl N-phthalimidolactosamine (Compound 5) and peracetyl N-phthalimidolactosamine .beta. chloride (Compound 6). Numbered compounds in both schemes are the same compounds. ##STR9##
Thus, lactulose was reacted neat with a primary amine that is an ammonia derivative whose nitrogen atom is bonded to a reductively removable blocking group (benzylamine) as both reactant and solvent to form the corresponding N-glycoside, lactulose N-benzyl glycoside, (Bn=benzyl; Compound 1). Reaction of Compound 1 in methanol with about a stoichiometric amount of an organic carboxylic acid having a pK.sub.a value of about 2.5 to about 5.0 (glacial acetic acid) provided N-benzyl lactosammonium acetate (Compound 2) in 50-55 percent yield. Lactosammonium acetate (Compound 3) was prepared by hydrogenolysis of the above methanolic solution using palladium on carbon (Pd/C).
It is noted that other reductively removable blocked amines can be used in place of benzylamine. For example, mono- and dimethoxybenzylamines can be viewed as reductively removable blocked ammonia derivatives in that after reaction with the saccharide, the mono- and dimethoxybenzyl groups can also be removed by hydrogenolysis. Allylamine can similarly be used, with the allyl blocking group being removed by reaction with polymethylhydrosiloxane (PMSH) and palladium-tetrakistriphenylphosphine �Pd(PPh.sub.3).sub.4 ! in THF as solvent.
Thus, although a benzyl group (Scheme 1A) is used as R.sup.A in Scheme 1, it is to be understood that a monomethoxybenzyl, dimethoxybenzyl or allyl group can be used as R.sup.A.
The discussion above and reactions illustrated in Schemes 1 and 1A illustrate a process for preparing lactosamine or a lactosammonium salt from lactulose. In accordance with this process, lactulose is admixed with a primary amine that is a monosubstituted ammonia derivative whose nitrogen atom is bonded to a reductively removable blocking group to form a reaction mixture. The blocked ammonia derivative serves both as the reaction and solvent in this process.
The blocked ammonia derivative (or primary amine) is present in a 2- to about 10-fold molar excess over the moles of lactulose utilized. The primary amine is preferably present in about a 4- to about 8-fold molar excess.
As noted before, primary amines containing other reductively removable blocking groups are contemplated. Thus, allylamine and p-methoxybenzylamine have been successfully used to form the lactulose N-glycoside, and rearranged to the corresponding N-substituted lactosamine.
The reaction mixture so formed is maintained at a temperature of about 10.degree. C. to about 60.degree. C. for a time period sufficient for the corresponding lactulose N-glycoside to form; i.e., for the primary amine to replace the lactulose 2-hydroxyl group. Temperatures from ambient room temperature (about 20.degree. C.) to about 50.degree. C. are preferred.
The maintenance time is a function of several variables such as the molar excess of primary amine, maintenance temperature, and the amount of lactulose N-glycoside desired, and can range from about 8 hours, where little of the product is desired, to as much as two weeks, using low temperatures and amounts of primary amine. For example, when 4-7.5 molar excesses of primary amine (here, benzylamine) were used, the reaction was complete after a maintenance time of seven days at room temperature, but less than 50 percent complete over the same time when a 2-fold excess of benzylamine was used under the same conditions. When the maintenance temperature was raised to 50.degree. C., the reaction using a 4-fold excess of amine was complete after two days (48 hours), whereas a 70.degree. C. reaction temperature caused decomposition.
The presence of a Lewis acid catalyst such as zinc chloride, zinc trifluoromethanesulfonate or magnesium trifluoromethanesulfonate in the reaction medium increased the reaction rate so that reactions using a 7.5-fold excess of benzylamine that were complete after seven days at room temperature without catalyst were completed in two days (48 hours). A similar result was obtained using trifluoroacetic acid as catalyst, which is preferred.
Lactulose is insoluble in alcohol solvents, including methanol. Lactulose can be dissolved in hot DMF and remain in solution after cooling. Both methanol and DMF can be used as cosolvents with the primary amine when an above-discussed catalyst is also present. For example, when methanol was used as a cosolvent, no reaction was had at either room temperature or 50.degree. C. However, when a zinc chloride catalyst was used with a 4-fold excess of benzylamine and methanol as cosolvent, the reaction was complete after 48 hours at room temperature.
The lactulose N-glycoside prepared as discussed above is hygroscopic, and is therefore used quickly after its preparation. That N-glycoside is reacted with about 0.1 equivalents up to an equivalent amount (for best yield) of a carboxylic acid having a pK.sub.a value of about 2.5 to about 5.0 in a C.sub.1 -C.sub.3 alcohol solvent at a temperature of about 10.degree. C. to about 30.degree. C. to rearrange the lactulose N-glycoside into a lactosammonium salt whose amine group is blocked with an above reductively removable blocking group; i.e., an amine-blocked lactosammonium salt having a reductively removable blocking group bonded to the amine nitrogen atom.
The carboxylic acid utilized can be any of a number of such acids as are well known in the art such as acetic (pK.sub.a =4.76), propionic (pK.sub.a =4.88), butyric (pK.sub.1 =4.82), chloroacetic (pK.sub.a =2.80), methoxyacetic (pK.sub.1 =3.52), and the like. Glacial acetic acid is preferred. Exemplary C.sub.1 -C.sub.3 alcohols include methanol, which is preferred, ethanol, propanol and iso-propanol. A reaction temperature of ambient room temperature is preferred.
The concentration of lactulose N-glycoside can range from about 0.1M to substantial saturation. Typically utilized concentrations are about 0.5 to about 1.5M in the solvent.
The reductively removable blocking group is then removed. Hydrogenolysis using a palladium catalyst is a preferred process for that removal, particularly where benzylamine or a methoxybenzylamine is used. PMHS and Pd(PPh.sub.3).sub.4 are used where allylamine is the primary amine.
The above reduction can take place in any appropriate solvent for the lactosammonium derivative. For example, hydrogenolysis can be carried out in acidic water or C.sub.1 -C.sub.3 alcohol as above. PMHS and Pd(PPh.sub.3).sub.4 are typically utilized in THF or a similar solvent.
A thus produced lactosammonium salt is generally recovered after preparation, although, depending upon the solvent used and the use to be put to the compound, recovery is not necessary. Where it is desired to recover the lactosammonium salt, whose anion is the anion form of the acid used in the reduction, can be obtained by well known methods such as chromatography or precipitation. Free lactosamine can be prepared from the salt by ion exchange chromatography or by neutralization, followed by extraction of the free base into an appropriate organic solvent.
The Compound 3-containing methanolic solution resulting from the hydrogenolysis reaction, or another appropriate solution, was then reacted with phthalic anhydride (PhthO) in the presence of a basic catalyst such as Na.sub.2 CO.sub.3 to form the N-phthalamide half-acid, Compound 4. After a suitable amide half-acid, e.g. Compound 4, was formed, any reactive solvent such as methanol was removed. The hydroxyls of the disaccharide were then peracylated and the phthalimide ring closed to provide peracetylated (Ac) phthalimido Compound 5 in over 10 percent yield from starting material.
An additional synthesis of a lactosammonium salt from lactulose is also contemplated.
Here, lactulose is reacted in a stainless steel autoclave with an equimolar amount of ammonium acetate and liquid ammonia as solvent, the liquid ammonia being added to the autoclave cooled to -78.degree. C. The resulting reaction mixture is warmed to a temperature from zero degrees C. to about 80.degree. C., and maintained for a period of about five hours to about five days, depending upon the temperature used and desired conversion. This reaction forms lactulose aminoglycoside.
After removing the ammonia and ammonium acetate, the latter being typically removed under vacuum, the resulting ammonia-free material is treated with a carboxylic acid as before to form the lactosammonium salt, e.g. Compound 3. The lactosammonium salt is also treated as discussed before to form Compound 5. The .beta.-anomer of Compound 5 was recovered in 3.8 percent overall yield in the first crystallization, where a reaction temperature of 35.degree. C. and reaction time of 24 hours was utilized in the first reaction step.
Although the yield of Compound 5 was less using this procedure than the previously discussed process, this process obviates the need for reductive removal of the amine blocking group used in that process. The palladium-containing catalyst used in that reduction is the most expensive reagent utilized in these syntheses. It is also noted that methanolic ammonia can be used as solvent rather than liquid ammonia, thereby obviating the need for use of an autoclave.
The amine of Compound 5 in Scheme 1 is shown bonded to R.sup.B and R.sup.B groups that together with the depicted nitrogen atom form a C.sub.4 -C.sub.8 cyclic imide such as an exemplary phthalimide (Phth) in Compound 5. It is noted that succinic anhydride, maleic anhydride, mono- and dimethylsuccinic anhydrides and citraconic anhydride can also be used to form similar imides, so that R.sup.B and R.sup.B together with the nitrogen atom form a corresponding imide. A cyclic imide formed by the --NR.sup.B R.sup.B group provides an amine protecting group that is stable under conditions in which O-acyl groups such as acetate are removed, but can be readily removed with hydrazine. It is also noted that an anhydride need not be used, but can be replaced by a C.sub.1 -C.sub.6 alkyl half ester halide such as methyl phthaloyl chloride.
Compound 5 is shown as the .beta.-anomer. The .alpha.-acetate is also formed and the yield of the desired .beta.-acetate can be almost doubled by concentrating the mother liquor from which Compound 5 was obtained to a foam followed by redissolution in DMF and then reaction with hydrazinium acetate, which cleaved the acetate group and caused formation of the .beta.-OH anomer. After isolation of the reaction product by usual extraction techniques and drying, dissolution of the dried material in pyridine, treatment of the pyridine solution with excess acetic anhydride, reaction, and a further extraction, an additional 8.3 percent overall percent yield of Compound 5 was obtained. The final yield of Compound 5 of 18.7 percent was obtained, based on starting materials.
Reaction of Compound 5 with AlCl.sub.3 in dichloromethane at room temperature provided a substantially quantitative yield of Compound 6.
Scheme 2, hereinafter, illustrates the transformation of Compound 6, peracetyl N-phthalimidolactosamine .beta.-chloride, into the fully protected sialylated tetrasaccharide, Compound 13. Thus, Compound 6 was reacted at ambient temperature for two hours in step a with Compound 9, whose synthesis is discussed in the examples, in the presence of molecular sieves, collidine and silver trifluoromethanesulfonate (triflate) using dichloromethane as solvent to prepare the corresponding trisaccharide. That fully protected trisaccharide was first treated in step b with 80 percent aqueous acetic acid for two hours at 80.degree. C. to remove the benzylidene protecting group at the 4- and 6-positions of the terminal Gal unit. Hydrazine hydrate was then reacted at reflux for 17 hours with the recovered, partially deprotected trisaccharide in step c to remove the phthalimido and acetyl groups, and form the completely deprotected trisaccharide. Reaction of the deprotected trisaccharide in methanol:water (5:1) with diallylpyrocarbonate in step d provided Compound 10, where AL is allylcarbamoyl.
Where R.sup.2 is not a glycoside as described in the syntheses of Scheme 2, and is rather a preferred C.sub.1 -C.sub.18 hydrocarbyl group such as benzyl, the glycosylation steps a and b are omitted, providing a tetrasaccharide of Formulas A, I or II, where R.sup.2 is other than mono- or disaccharide.
Compound 10 was then sialylated enzymatically in step e in an aqueous buffer using .alpha.-(2,3)-sialyltransferase (EC 2.4.99.6) and a number of other enzymes. The reaction was followed by TLC for 10-12 days at ambient temperature, at which time more than 95 percent of Compound 10 had been consumed, and Compound 11 was prepared.
Compound 11 was recovered as a thick syrup that was coevaporated twice with pyridine and then kept under vacuum for 20 hours. The thus dewatered material was redissolved in pyridine to which a catalytic amount of 4-dimethylaminopyridine (DMAP) was added as was acetic anhydride. Two more additions of acetic anhydride over the ensuing 44 hours completed the acetylation reaction and formation of a lactone with the sialyl carboxyl and a saccharide hydroxyl in step f. Methanol was thereafter added to the recovered material to form the sialyl methyl ester and thereafter, another addition of acetic anhydride was made to acetylate the freed hydroxyl to form completely protected Compound 12 in step g.
It should be apparent that Compounds 11 and 12 are compounds of structural Formulas A and III. Using Compound 12 as exemplary, Z is C.sub.1 -C.sub.6 acyl (acetyl), X is C.sub.1 -C.sub.6 acyloxy (acetoxy), R.sup.2 is 3Gal.beta.O-ethyl, R.sup.3 is acetyl, R.sup.4 is methyl and R.sup.1 is allyloxy. It should be equally apparent that the before-mentioned other X groups for a compound of any of the structural formulas are conveniently introduced at the sialylation step. If it is desired that sialyl unit X substituents that are C.sub.1 -C.sub.6 acyloxy or C.sub.1 -C.sub.6 hydroxylacyloxy be present in an inhibitor of structural Formulas I or C, it is preferred that Compound 10 (or a disaccharide without the 3Gal.beta.OR.sup.2 group) be peracetylated, the allyloxy carbamoyl group (AL) of Compound 10 be removed as in step h, and replaced by one of the phenyl ring-containing R.sup.1 acyl groups as in step c of Scheme 3. The molecule is then deprotected and enzymatically sialylated and fucosylated as is discussed hereinafter. For other of the R.sup.1-4 groups or a similar compound of structural Formulas A, B or III, one can substitute the 3Gal.beta. glycoside R.sup.2 of Compound 9, the acylating agent of steps f and g, and the esterifying alcohol of step f.
Treatment of recovered, dried Compound 12 with polymethylhydrosiloxane (PMHS) in anhydrous THF at room temperature followed by palladium tetrakistriphenylphosphine �Pd(PPh.sub.3).sub.4 ! for 18 hours provided Compound 13 in 87 percent yield in step h. ##STR10##
Scheme 3, hereinafter, outlines one remaining synthesis to illustrative inhibitor Compound 17 of Formulas I and C. Thus, reaction of Compound 13 with one equivalent of glacial acetic acid in aqueous methanol for 48 hours at 50.degree. C. provided selective deacylation of the Glc 3-hydroxyl and gave Compound 14 in 65 percent yield in step a.
Compound 14 was then selectively benzoylated in step b in 83 percent yield by reaction with benzoyl chloride in dichloromethane with solid sodium bicarbonate at room temperature for 24 hours to form Compound 15. The alterative R.sup.1 groups of a compound of structural Formulas A, I, II and III are added at this step or at an analogous step where R.sup.2 is not a saccharide unit.
An organic chemical fucosylation was carried out in step c of Scheme 3 by mixing Compound 15 with tri-O-benzyl fucosyl fluoride, molecular sieves and tetramethylurea in dichloroethane, followed by cooling to -20.degree. C. and addition of stannous chloride and silver perchlorate. After warming slowly to room temperature and stirring for 24 hours, Compound 16 was prepared in 77 percent yield.
Compound 16 is thus a compound of structural Formulas A and B, where Z is a blocked fucosyl group, as well as a compound of Formula II. Use of alternative R.sup.5 groups provide the remaining compounds of those structural formulas when combined with the before-discussed X and R.sup.1-4 groups.
The O-benzyl blocking groups, R.sup.5, of the fucosyl saccharide unit were removed in step d by hydrogenation using palladium hydroxide on carbon �Pd(OH).sub.2 /C! in methanol as solvent. Reaction for one hour at room temperature provided complete removal of the O-benzyl groups. Filtration and concentration of the debenzylated compound provided an oil that was redissolved in methanol:water (4:1) to which was added sodium methoxide powder in step e. After 16 hours of reaction at room temperature, a 72 percent yield of inhibitor Compound 17 was obtained.
Where R.sup.5 is a C.sub.1 -C.sub.6 acyl group, the hydrogenation step is not used and the R.sup.5 C.sub.1 -C.sub.6 acyl group is removed along with the R.sup.3 and R.sup.4 groups. Use of an R.sup.5 C.sub.1 -C.sub.6 acyl group and the avoidance of a hydrogenation step, also provides a route for synthesis of nitro group-containing R.sup.1 groups. ##STR11##
Where the R.sup.2 group is a mono- or disaccharide, an appropriately blocked mono- or disaccharide is used such as Compound 9 of Scheme 2. For example, lactose, a lactose C.sub.1 -C.sub.18 glycoside or melibiose can be made into protected (blocked) benzylidine derivatives similar to that of Compound 9 and then used in the coupling step a of Scheme 2, and the resulting product used in subsequent steps of Schemes 2 and 3.
It is to be understood that lactosamine and its derivatives can be prepared by other methods well known to skilled workers. It is to be further understood that the trisaccharide Compound 10 can be prepared enzymatically by reaction of ethyl 3-O-(2-N-allyloxycarbonyl-2-amino-2-deoxy-.beta.-D-glucopyranosyl)-.beta.-D-galactoside using uridine-5'-diphosphate-galactosyl transferase with UDP-Gal, and other appropriate enzymes following known procedures. Similarly, Compound 11 can be fucosylated enzymatically using a fucosyl transferase (FT), such as fucosyl transferase V, as well as the nucleotide sugar donor GDP-fucose, and other enzymes useful in the regeneration of GDP-fucose, using known procedures. Of course, slight changes in the reaction schemes shown are necessitated by those synthetic changes, but those changes are well within the skill of an ordinary worker.
Still another useful synthetic procedure is shown in Scheme 4, below. Here, the starting material is the free base, Compound 14a, of Compound 14 of Scheme 3. ##STR12##
Thus, Compound 14a was reacted in step a with a slight excess of carbobenzoxy chloride (CBZ-Cl) in dichloromethane, in the presence of sodium bicarbonate followed by another equal amount of CBZ-Cl about eighteen hours later to form the amine-protected Compound 39 in 65 percent yield. Step b of Scheme 4 is substantially the same glycosylation step shown as step c of Scheme 3, with Compound 40 being formed in 73 percent yield, plus recovery of 17 percent starting Compound 39.
The fucosylated free amine, Compound 41, was thereafter formed in 96 percent yield in step c by reaction with ten percent Pd--C in ammonium formate in ethanol at reflux. The free amine of Compound 41 was thereafter reacted in step d with an acyl (YR.sup.1) chloride in dichloromethane in the presence of sodium bicarbonate to provide the corresponding hydroxy-blocked N-acylated compound, here, the 3,5-dichlorbenzamide derivative, Compound 42, in high yield. The hydroxyl groups were de-blocked by reaction in 28 percent sodium methoxide-methanol in substantially quantitative yield.
The structures of several particularly preferred inhibitors, Compounds 17, 30-38 and 43-51 have already been shown. Compounds 30-33 were prepared from their respective precursor Compounds 26-29 as described for conversion of Compound 16 into Compound 17 in Scheme 3. Compounds 34-38 and 51 were prepared in manners analogous to those of Scheme 3. Compounds 43-49 were prepared similarly, using the general approach shown in Scheme 4. Compound 50 was prepared following Scheme 3, using the reduction of Scheme 4, step c. These are compounds of structural Formula I, as well as Formula A.
D. Pharmaceutical Compositions
A pharmaceutical composition containing a contemplated SLe.sup.x analogue compound dissolved or dispersed in a pharmaceutically acceptable carrier or diluent is also contemplated. Such a composition contains a cell adhesion-inhibiting amount of a before-discussed, contemplated SLe.sup.x analogue compound. As is discussed in detail below, the present invention provides pharmaceutical compositions which are SLe.sup.x analogue compounds, liposomal formulations of a lipid-SLe.sup.x conjugate (e.g., liposomal formulations of compounds of formula IV in which either R.sup.1 or R.sup.2 is a lipid or a linking group with an attached lipid), and liposomal formulations of a lipid-SLe.sup.x conjugate in which the liposome portion comprises additional therapeutic agent(s). For the latter group of pharmaceutical compositions, the SLe.sup.x portion of the liposome formulation can serve as a pharmaceutical agent in combination with an additional encapsulated drug, or it can serve as a targeting agent for the liposome formulation, or it can provide dual advantage of both a pharmaceutical agent and a targeting agent. For the description below, any reference to the SLe.sup.x portion as a targeting agent is not meant to be limiting on its actual function in vivo. Each of the pharmaceutical compositions has utility as a pharmaceutical preparation which will be understood by those of skill in the art, in the context of the disclosure below.
A cellular adhesion-inhibiting amount can vary widely. That amount is, however, sufficient to inhibit binding of cells that express sialyl Le.sup.x on their cell surfaces to selectin, particularly E-selectin (ELAM-1) preferably by about one-half or more. An exemplary cellular adhesion-inhibiting amount is about 5 to about 60 mg/kg.
A contemplated pharmaceutical composition can be used to block or inhibit cellular adhesion associated with a number of disorders. For instance, a number of inflammatory disorders are associated with selectins expressed on vascular endothelial cells and platelets. The term "inflammation" is used here to refer to reactions of both the specific and non-specific defense systems. A specific defense system reaction is a specific immune system reaction to an antigen. Exemplary of specific defense system reactions include antibody response to antigens, such as viruses, and delayed-type hypersensitivity. A non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include macrophages, eosinophils and neutrophils. Examples of non-specific reactions include the immediate swelling after a bee sting, and the collection of peripheral mononuclear (PMN) leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonia and pus formation in abscesses).
Other treatable disorders include, e.g., rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation, including atopic dermatitis, psoriasis, and inflammatory bowel disease. Various platelet-mediated pathologies such as atherosclerosis and clotting can also be treated. In addition, tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
By way of example, reperfusion injury is particularly amenable to treatment by a contemplated pharmaceutical composition. A composition that inhibits a P-selectin-ligand interaction can be particularly useful for treating or preventing reperfusion injury. A contemplated pharmaceutical composition can be used prophylactically prior to heart surgery to enhance postsurgical recovery.
Because P-selectin is stored in Weibel-Palade bodies of platelets and endothelial cells and is released upon activation by thrombin to mediate adhesion of neutrophils and monocytes, inhibitors of the P-selectin-ligand interaction can be especially useful in minimizing tissue damage that often accompanies thrombotic disorders. For instance, such inhibitors can be of therapeutic value in patients who have recently experienced stroke, myocardial infarctions, deep vein thrombosis, pulmonary embolism, etc. The compounds are especially useful in pre-thrombolytic therapy.
A contemplated composition finds particular use in treating the secondary effects of septic shock or disseminated intravascular coagulation (DIC). Leukocyte emigration into tissues during septic shock or DIC often results in pathological tissue destruction. Furthermore, these patients can have widespread microcirculatory thrombi and diffuse inflammation. A therapeutic composition provided herein inhibits leukocyte emigration at these sites and mitigates tissue damage.
An inhibitor of a selectin-cellular SLe.sup.x ligand interaction is also useful in treating traumatic shock and acute tissue injury associated therewith. Because the selectins play a role in recruitment of leukocytes to the sites of injury, particularly E-selectin (ELAM-1) in cases of acute injury and inflammation, inhibitors thereof can be administered locally or systemically to control tissue damage associated with such injuries. Moreover, because of the specificity of such inhibitors for sites of inflammation, e.g., where ELAM-1 receptors are expressed, these compositions can be more effective and less likely to cause complications when compared to traditional anti-inflammatory agents.
Thus, the present invention also provides a pharmaceutical composition that can be used in treating the aforementioned conditions. A contemplated pharmaceutical composition is comprised of a before-described SLe.sup.x analogue compound that inhibits the interaction between a cellular SLe.sup.x ligand and a selectin receptor, which compound is dissolved or dispersed in a pharmaceutically acceptable diluent. A contemplated pharmaceutical composition is suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see, Langer, Science, 249:1527-1533 (1990).
In light of the complexity of the inflammatory response in mammals, one of skill will readily recognize that a contemplated pharmaceutical composition can further include other compounds known to interfere with the function of other cellular adhesion molecules. For instance, members of the integrin family of adhesion molecules are thought to play a role in the extravasation of leukocytes at points of infection. For a review of intercellular adhesion receptors, including selectin receptors, and their role immune function, see Springer, Nature, 346:425-434 (1990). In addition, successful treatment using a contemplated pharmaceutical composition can also be determined by the state of development of the condition to be treated. Because different adhesion molecules can be up or down regulated in response to a variety of factors during the course of the disease or condition, one of skill will recognize that different pharmaceutical compositions can be required for treatment of different inflammatory states.
In another embodiment, a before-described SLe.sup.x analogue compound of the pharmaceutical composition can be used to target conventional anti-inflammatory drugs or other agents to specific sites of tissue injury. By using such a compound to target a drug to a selectin receptor on, e.g., a vascular endothelial cell, such drugs can achieve higher concentrations at sites of injury. Side effects from the conventional anti-inflammatory chemotherapeutic agents can be substantially alleviated by the lower dosages, the localization of the agent at the injury sites and/or the encapsulation of the agent prior to delivery.
The targeting component, i.e., the SLe.sup.x analogue compound that binds to a selectin, can be directly or indirectly coupled to the chemotherapeutic agent. The coupling, which can be performed by means, generally known in the art, should not substantially inhibit the ability of the ligand to bind the receptor nor should it substantially reduce the activity of the chemotherapeutic agent. A variety of chemotherapeutics can be coupled for targeting. For example, anti-inflammatory agents that can be coupled include immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, and leukotriene antagonists. Some preferred moieties include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, etc. Similarly, anti-oxidants, e.g., superoxide dismutase, are useful in treating reperfusion injury when targeted by a contemplated saccharide compound. Likewise, anticancer agents can be targeted by coupling the SLe.sup.x analogue compound to the chemotherapeutic agent. Examples of agents that can be coupled include daunomycin, doxorubicin, vinblastine, bleomycin, etc. Here, again, a C.sub.1 -C.sub.6 alkyl C.sub.1 -C.sub.5 alkylene .omega.-carboxylate R.sup.1 group can be used for coupling.
The selectin receptor targeting can also be accomplished via amphipaths, or dual character molecules (polar:nonpolar) that exist as aggregates in aqueous solution. Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are generically referred to herein as liposomes. In these preparations the drug to be delivered is incorporated as part of a liposome in conjunction with a SLe.sup.x analogue compound that binds to the selectin receptor.
A contemplated SLe.sup.x analogue compound whose R.sup.2 group is a C.sub.12 -C.sub.18 hydrocarbyl group is particularly useful in such liposome preparations. Still other contemplated SLe.sup.x analogue compounds are those in which either R.sup.1 or R.sup.2 is a lipid or a linking group with an attached lipid. Methods of attaching lipids to oligosaccharides as well as suitable lipids for use in such conjugates are described in PCT/US94/03103 (WO 94/21235), incorporated herein by reference. Thus, liposomes filled with a desired chemotherapeutic agent can be directed to a site of tissue injury by the selectin-SLe.sup.x analogue compound interaction. When the liposomes are brought into proximity of the affected cells, they deliver the selected therapeutic compositions.
The liposomes of the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream.
Typically, the major lipid component in the liposomes is phosphatidylcholine, phosphatidylcholines having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In general, less saturated phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below about 0.3 microns, for purposes of filter sterilization. Methods used in sizing and filter-sterilizing liposomes are discussed below. The acyl chain composition of phospholipid can also affect the stability of liposomes in the blood. One preferred phosphatidylcholine is partially hydrogenated egg phosphatidylcholine.
Targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in such a manner that the target agents are available for interaction with the selectin receptor. In one group of embodiments, the liposome is typically fashioned in such a way that a connector portion is first incorporated into the membrane at the time of forming the membrane. The connector portion has a lipophilic portion that is firmly embedded and anchored in the membrane. It also has a hydrophilic portion that is chemically available on the aqueous surface of the liposome. The hydrophilic portion is selected so that it is chemically suitable to form a stable chemical bond with the targeting agent (e.g., SLe.sup.x analog) which is added later. Therefore, the connector molecule has both a lipophilic anchor and a hydrophilic reactive group suitable for reacting with the SLe.sup.x analog and holding the SLe.sup.x analog in its correct position, extended out from the liposome's surface. In some cases one can attach the target agent to the connector molecule directly, but in most instances it is more suitable to use a third molecule to act as a chemical bridge, thus linking the connector molecule which is in the membrane with the target agent which is extended, three dimensionally, off the vesicle surface. In another group of embodiments, the targeting agent (SLe.sup.x analog) is conjugated to a lipid, typically a phospholipid, and then brought into contact with a liposome. Examples of SLe.sup.x analogs which are covalently attached to a phospholipid are provided in Examples 30 and 31, below. One of skill in the art will understand that this method, and others, are widely applicable to other SLe.sup.x analogs described herein as the overall chemical manipulations employed are similar. For example, a phosphatidyl thioethanol was coupled (in Example 30) to a pentasaccharide bearing a reactive .alpha.-bromoacetamide (see FIG. 6). In a similar fashion, compounds 62 (see FIG. 1), 73 (see FIG. 2), 78 (see FIG. 3), 85 (see FIG. 4), and 100 (see FIG. 5) have also been coupled to a phospholipid (see Example 31). Alternatively, other phospholipids bearing suitable nucleophilic functionality could also be coupled with the saccharides bearing a reactive .alpha.-bromoacetamide groups. Still further, other methods of coupling a phospholipid to a SLe.sup.x analog could be employed. For example, diacylphosphatidylethanolamine can be coupled to a SLe.sup.x analog bearing a suitable carboxylic acid or ester in an amide-forming reaction. Similarly, other suitable bond-forming reactions could be employed to link a phospholipid to a SLe.sup.x analog via a new ester, ether, disulfide, thioether, or hydrazone. The phospholipids or other lipid species which are conjugated to a SLe.sup.x analog can have fatty acid side chains of from about 8 to about 24 carbons in length which are saturated or unsaturated. The position of the SLe.sup.x analogue on the surface of the liposome can be adjusted and controlled by the length of the spacer between the analogue and the phospholipid. For example, compound 114 (see FIG. 6) has a propylene group which connects the pentasaccharide portion and the .alpha.-bromoacetamide portion. Alternative groups such as longer alkylene chains (e.g., --(CH.sub.2).sub.5 --, --(CH.sub.2).sub.10 --, --(CH.sub.2).sub.14 -- and --(CH.sub.2).sub.20 --) could also be used. In this manner, the SLe.sup.x analogue can be positioned on the surface of the liposome and outside the area occupied by any circulation enhancing agents (e.g., polyethylene glycols or polyethylene glycol-lipid conjugates).
Liposome charge is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the reticuloendothelial system (see Juliano, Biochem. Biophys. Res. Commun., 63:651 (1975)) and thus having shorter half-lives in the bloodstream. Liposomes with prolonged circulation half-lives are typically desirable for therapeutic and diagnostic uses.
Typically, the liposomes are prepared with about 5-15 mole percent negatively charged phospholipids, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol. The added negatively charged phospholipids, such as phosphatidylglycerol, serve to prevent spontaneous liposome aggregating, and thus minimize the risk of undersized liposomal aggregate formation. Membrane-rigidifying agents, such as sphingomyelin or a saturated neutral phospholipid, at a concentration of at least about 50 mole percent, and 5-15 mole percent of monosialylganglioside, can provide increased circulation of the liposome preparation in the bloodstream, as generally described in U.S. Pat. No. 4,837,028, incorporated herein by reference.
Additionally, the liposome suspension can include lipid-protective agents that protect lipids and drug components against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.
Several methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, incorporated herein by reference. One method produces multilamellar vesicles of heterogeneous sizes. In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film. If desired, the film can be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture that is in a more easily hydrated powder-like form. This film is covered with an aqueous solution of the targeted drug and the targeting component and allowed to hydrate, typically over a 15-60 minute period with agitation. The size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
The hydration medium contains the targeted drug at a concentration that is desired in the interior volume of the liposomes-in the final liposome suspension. Typically the drug solution contains between 10-100 mg/mL in a buffered saline. The concentration of the targeting SLe .sup.X analogue compound which binds a selectin is generally between about 0.05-20 mg/mL.
Following liposome preparation, the liposomes can be sized to achieve a desired size range and relatively narrow distribution of liposome sizes. One preferred size range is about 0.1-0.2 microns, which allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 micron filter. The filter sterilization method can be carried out on a high through-put basis if the liposomes have been sized down to about 0.1-0.2 microns.
Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 0.05 microns in size. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination.
Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes can be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
Even under the most efficient encapsulation methods and methods for incorporating a lipid-SLe.sup.x conjugate, the initial sized liposome suspension can contain up to 50 percent or more drug or targeting agent (lipid-SLe.sup.x conjugate) in free form (e.g., non-encapsulated or unincorporated). Therefore, to maximize the advantages of liposomal-targeted drug, it is important to remove free drug and targeting agent from the final injectable suspension.
Several methods are available for removing non-entrapped or unincorporated compounds from a liposome suspension. In one method, the liposomes in the suspension are pelleted by high-speed centrifugation leaving free compound and very small liposomes in the supernatant. Another method involves concentrating the suspension by ultrafiltration, then resuspending the concentrated liposomes in a drug-free replacement medium. Alternatively, gel filtration can be used to separate large liposome particles from solute molecules.
Following treatment to remove free drug and/or targeting agent, the liposome suspension is brought to a desired concentration for use in intravenous administration. This can involve resuspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultrafiltration, or concentrating the suspension, where the drug removal step has increased total suspension volume. The suspension is then sterilized by filtration as described above. The liposome-ligand preparation may be administered parenterally or locally in a dose which varies according to, e.g., the manner of administration, the drug being delivered, the particular disease being treated, etc.
For a pharmaceutical composition that comprises a SLe.sup.x analogue compound that binds to selectin receptors and inhibits binding thereto by SLe.sup.x ligand-containing cells, the dose of the compound varies according to, e.g., the particular compound, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician. For example, for the treatment of reperfusion injury, the dose of a contemplated SLe.sup.x analogue compound is in the range of about 50 .mu.g to 10,000 mg/day for a 70 kg patient. Ideally, therapeutic administration should begin as soon as possible after the myocardial infarction or other injury. A pharmaceutical composition is intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. A pharmaceutical composition can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and dragees.
Preferably, a pharmaceutical composition is administered intravenously. Thus, this invention provides a composition for intravenous administration that comprises a solution of a contemplated SLe.sup.x analogue compound dissolved or dispersed in a pharmaceutically acceptable diluent (carrier), preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4 percent saline, and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. A composition can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concentration of SLe.sup.x analogue compound utilized is usually about 0.1 to about 10-30 mol % and is selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. As described above, the composition components can be delivered via liposome preparations.
Thus, a typical pharmaceutical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 25 mg of the SLe.sup.x analogue compound. Actual methods for preparing parenterally administrable compounds are known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.
For solid compositions, conventional nontoxic solid diluents (carriers) may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95 percent of active ingredient, that is, a before-described SLe.sup.x analogue compound, preferably about 20 percent (see, Remington's, supra).
For aerosol administration, a contemplated SLe.sup.x analogue compound is preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of a SLe.sup.x analogue compound are about 0.5 to about 30 percent by weight, and preferably about 1 to about 10 percent. The surfactant must of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides can be employed. The surfactant can constitute about 0.1 to about 20 percent by weight of the composition, and preferably about 0.25 to about 5 percent. The balance of the composition is ordinarily propellant. Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above can also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
A pharmaceutical composition containing a SLe.sup.x analogue compound can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, a composition is administered to a patient already suffering from a disease, as described above, in an amount sufficient to inhibit binding between cells expressing a selectin and neutrophils or HL-60 cells; i.e., cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose" or "a cell adhesion-inhibiting amount". Amounts effective for this use depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.5 mg to about 10,000 mg of SLe.sup.x analogue compound per day for a 70 kg patient, with dosages of from about 5 mg to about 2,000 mg of a compound per day being more commonly used.
In prophylactic applications, a composition containing a contemplated compound is administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective dose" and is also an amount sufficient to inhibit adhesion (binding) of SLe.sup.x -containing cells to selectin. In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.5 mg to about 5,000 mg per 70 kilogram patient, more commonly from about 5 mg to about 2,000 mg per 70 kg of body weight.
Another way to assess an adhesion-inhibiting amount of a contemplated SLe.sup.x analogue compound is to compare binding inhibition exhibited by the SLe.sup.x analogue compound to that provided by SLe.sup.x itself. One convenient way to make that comparison is by use of IC.sub.50 (the concentration needed to inhibit binding by one-half) of the two compared materials, and base the amount used on the amount of SLe.sup.x and an amount of the SLe.sup.x analogue compound that is a multiple of the IC.sub.50 value for that compound.
Typically, a compound whose IC.sub.50 value is about one-tenth that of SLe.sup.x itself, when used at ten times the molar amount of SLe.sup.x is a useful cell adhesion-inhibiting amount. More preferably, the amount is about four times the amount of SLe.sup.x. More preferably still, the amount is equal to that of SLe.sup.x. Most preferably, as is the case with most of the SLe.sup.x analogue compounds described herein, the amount used is less than the amount of SLe.sup.x used such as about one-half to about one-tenth the molar amount of SLe.sup.x itself.
Single or multiple administrations of a composition can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of a SLe.sup.x analogue compound sufficient to effectively treat the patient.
The compounds can also find use as diagnostic reagents. For example, labeled compounds can be used to locate areas of inflammation or tumor metastasis in a patient suspected of having an inflammation. For this use, the compounds can be labeled with .sup.125 I, .sup.14 C, or tritium.
EXAMPLES
Example 1
This example provides a synthesis of lactulose N-benzyl glycoside (Compound 1).
A 500 mL 3-neck round bottom flask was immersed in an ice bath and charged with lactulose (23.9 gm, 69.8 mmol) and benzylamine (109 mL, 526 mmol, 7.5 equivalent). The flask was then capped and stirred using a magnetic stirbar. The ice bath was permitted to melt and the reaction was permitted to slowly warm to room temperature. Dissolution of the solid material occurred over several hours and the reaction became yellow in color. TLC in 60:50:15 CHCl.sub.3 :MeOH:15 mM CaCl.sub.2 can be used to monitor the progress of the reaction (lactulose R.sub.f =0.45, product R.sub.f =0.75, orcinol visualization).
The reaction was quite slow and appeared to reach completion in 5-7 days. At the time the reaction was judged to be complete, the stirbar was removed from the reaction, the flask was fitted with an overhead mechanical stirrer, and the apparatus was immersed in an ice bath. Hexane (250 mL) was then added to the flask and the mixture was stirred vigorously for approximately 60 seconds. Stirring was then discontinued and the mixture was permitted to separate into two distinct layers (this separation takes from 15 minutes to one hour). At this time, the upper hexane/benzylamine layer was removed through a tube by suction. Extraction of benzylamine was repeated twice more using hexane (250 mL portions) and then was done three more times using 250 mL portions of diethyl ether (all extractions were done on ice).
After these extractions were performed a viscous pale yellow residue was left. This material was dissolved in ethanol (300 mL) and was transferred to a 2 liter single neck round bottom flask. The yellow solution was concentrated by rotary evaporation to a thick syrup. Reagent grade acetone (1000 mL) was then rapidly stirred with a magnetic stirbar at zero degrees C., and the solution was then slowly treated with the ethanolic syrup. As the syrup was slowly added, a milky white precipitate began to form. After addition was complete, the flask was capped and stored in a -20.degree. C. freezer overnight (about 18 hours). After removal from the freezer, a white solid cake was apparent at the bottom of the flask and the supernatant was clear yellow. The solution was then decanted off and the crude solid Compound 1 was pulled under high vacuum to remove residual acetone. The product (Compound 1) is a very unstable solid and was used immediately in the next reaction.
Example 2
This example provides a synthesis of N-benzyl lactosamine acetate salt (Compound 2).
The crude product (Compound 1) from above (30.1 gm, 69.8 mmol, theoretical) was dissolved in 1000 mL of reagent grade methanol and was stirred at room temperature. Glacial acetic acid (4 mL, 70 mmol) was then added and the flask was capped. The pale yellow reaction mixture was permitted to stir at room temperature and was monitored by TLC in the same solvent system as described above. The product Compound 2 appeared at R.sub.f =0.65 (residual lactulose is noticeable by TLC from the beginning of this reaction but its amount does not seem to increase substantially as the reaction progresses). When Compound 1 appeared to have been completely consumed by TLC (24-48 hours), 100 .mu.L was withdrawn from the reaction mixture and was evaporated under a stream of argon. The yellow residue was then dissolved in CD.sub.3 OD and evaporated again to a yellow residue. This material was then dissolved in D.sub.2 O and was analyzed by .sup.1 H-NMR.
This crude solution of Compound 2 was then used in the next reaction. For yield calculation purposes, a small aliquot of known volume can be removed from the reaction mixture, concentrated to dryness, dissolved in H.sub.2 O, brought to pH>10, and chromatographed using reverse phase silica gel flash chromatography first eluting with H.sub.2 O and then with 2:1H.sub.2 O:MeOH. Typical yields from lactulose were 50-55 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to HOD) 7.44 (m, 5H), 5.49 (d, J=3 Hz, 1H), 5.05 (d, J=8 Hz, 1H), 4.39 (d, J=7 Hz, 1H), 4.38 (d, J=8 Hz, 2H), 4.35 (d, J=7 Hz, 1H), 4.10-3.5 (m, 11H), 3.24 (dd, J=3 Hz, J=10 Hz, 1H), 3.00 (dd, J=8 Hz, J=10 Hz, 1H), 2.87 (s, 3H).
Example 3
This example provides the preparation of lactosamine acetate salt (Compound 3).
The 2 liter flask containing the crude acidic methanolic solution of Compound 2 from the previous reaction was equipped with a three-way stopcock and was put through an argon/vacuum/purge cycle three times using a balloon of argon and a house vacuum line. The flask was opened and 10 percent palladium on carbon was added (7.4 gm, 6.98 mmol). The flask was then re-equipped with a three-way stopcock and put through a vacuum/purge cycle three times using hydrogen gas. The reaction was then held under a hydrogen atmosphere using a balloon.
The reaction was monitored closely by TLC (product R.sub.f =0.2). When starting material was consumed, a 100 .mu.L aliquot was withdrawn, placed in an eppendorf tube, spun in a microfuge, and the clear supernatant was removed and was used to prepare an NMR sample as in the previous reaction. Once the NMR showed complete loss of Compound 2, the slurry was filtered through a plug of celite on a medium porosity sintered glass funnel using methanol. The clear yellow solution was then concentrated by rotary evaporation to 140 mL in a 500 mL round bottom flask and used crude in the following reaction. Compound 3: .sup.1 H-NMR (300 MHz, .delta. in ppm relative to HOD) 5.40 (d, J=3 Hz, 1H), 4.90 (d, J=8 Hz, 1H), 4.41 (d, J=8 Hz, 1H), 4.00-3.5 (m, 11H), 3.28 (dd, J=3 Hz, J=8 Hz, 1H), 2.98 (dd, J=7 Hz, J=8 Hz, 1H).
Example 4
This example illustrates the preparation of 2-Deoxy-2-(2'-carboxy)-benzamido-4-O-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside (Compound 4) and 1,3,6-Tri-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside (Compound 5)
The crude acidic methanolic solution of Compound 3 was diluted with 14 mL of H.sub.2 O and treated with sodium carbonate (29.7 gm, 280 mmol) followed by phthalic anhydride (20.7 gm, 140 mmol). The reaction was watched carefully because some foaming occurs initially. After four hours, the reaction was complete, and the slurry was filtered through a sintered glass funnel to remove residual sodium carbonate and phthalate-based material. The filtrate was then concentrated to a paste first by rotary evaporation and then under high vacuum to provide Compound 4. Removing as much of the trace methanol and H.sub.2 O left in the material is essential to avoid side reaction with acetic anhydride in the following acetylation.
When the material was judged to be dry enough, pyridine (212 mL) was added followed by acetic anhydride (106 mL, 1.12 mol). The mixture was shaken manually at first to promote dissolution, but once an initial exotherm began to occur, dissolution proceeded and magnetic stirring was then used. After stirring overnight (about 18 hours), TLC in 20:1 CHCl.sub.3 :MeOH indicated preponderance of one major UV active spot which cospotted with authentic Compound 5. The solution was cooled to zero degrees C., treated with 32 mL of H.sub.2 O, and stirred for 15 minutes to hydrolyze excess acetic anhydride. The solution was then diluted to 1000 mL with dichloromethane and washed (3.times.1000 mL) with 2N HCl, (3.times.1000 mL) with saturated NaHCO.sub.3, and (.times.1000 mL) with saturated NaCl. The organic solution was then dried (MgSO.sub.4), filtered, and concentrated to a crude product. .sup.1 H-NMR was then run in CDCl.sub.3 and indicated an approximately 1:1 mixture of .alpha.- and .beta.-anomers. This crude product was dissolved in a minimum amount of methanol (about 30 mL) and crystallization ensued within a matter of minutes. After remaining at room temperature for several hours, the solid was collected by filtration and rinsed with ice cold methanol. After air drying the product, pure Compound 5 was collected (5.6 gm, 10.4 percent) as a white powder. .sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.90-7.70 (m, 4H), 6.50 (d, J=8 Hz, 1H), 5.83 (dd, J=10.5 Hz, J=8 Hz, 1H), 5.36 (d, J=3.5 Hz, 1H), 5.15 (dd, J=8 Hz, J=10.5 Hz, 1H), 4.97 (dd, J=10 Hz, J=3.5 Hz, 1H), 4.56-3.83 (m, 9H), 2.20-190 (7s, 21H).
Example 5
The example provides the conversion of 1,3,6-tri-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside to Compound 5.
The .alpha.-acetate-containing mother liquor from the crystallization of Compound 5 discussed above was concentrated to a foam and dissolved in DMF (110 mL). This solution was stirred under argon at 55.degree. C. Hydrazinium acetate (9.5 gm, 104 mmol) was then added. After 15 minutes, TLC in 20:1 CHCl.sub.3 :MeOH indicated complete loss of starting material and appearance of a slightly lower R.sub.f spot. The reaction was cooled to room temperature and diluted to 1000 mL with ethyl acetate. The solution was then washed (2.times.1000 mL) with H.sub.2 O and (1.times.1000 mL) with saturated NaCl. The organics were dried (MgSO.sub.4), filtered and concentrated.
The crude concentrated product was dissolved in pyridine 50 mL and treated with acetic anhydride (25 mL). After stirring overnight (about 18 hours), TLC in 20:1 CHCl.sub.3 :MeOH indicated preponderance of one major UV active spot that cospotted with authentic Compound 5. The solution was cooled to zero degrees C., treated with 7.5 mL of H.sub.2 O, and stirred for 15 minutes to hydrolyze excess acetic anhydride. The solution was diluted to 250 mL with dichloromethane and washed (3.times.250 mL) with 2N HCl, (3.times.250 mL) with saturated NaHCO.sub.3, and (1.times.250 mL) with saturated NaCl. The organic solution was dried (MgSO.sub.4), filtered, and concentrated to a crude product. The crude product was then dissolved in a minimum of methanol and once again crystallization occurred. After several hours, the solid Compound 5 was isolated as before to provide another crop of product (4.4 gm, 8.3 percent) as a white powder. Overall yield of Compound 5 for two crops, 18.7 percent, 10 gm.
Example 5A
This example provides an alternative preparation of Compound 5 from Lactulose.
A. Lactulose aminoglycoside (Compound 1A)
A 300 mL stainless steel autoclave containing a stirbar, lactulose (17.1 g, 50 mmol), and ammonium acetate (3.85 g, 50 mmol) was cooled to -78.degree. C. and charged with 80 mL of liquid ammonia. The autoclave was sealed and allowed to warm to room temperature with stirring. Once the autoclave had reached room temperature, it was placed in an oil bath and heated to 35.degree. C. for 24 hours. The autoclave was then cooled to room temperature and carefully vented to the atmosphere. Once all of the ammonia had dissipated, approximately two hours, the entire autoclave was placed in a vacuum desiccator containing phosphorous pentoxide and carefully put under high vacuum. After being held under high vacuum overnight, the contents of the autoclave had become a pale yellow foam. The compound was quite hygroscopic and was quickly removed from the autoclave and placed in a sealed jar. This material was used crude in the following reaction.
B. Lactosamine acetate (Compound 2A)
Lactulose aminoglycoside (Compound 10) (3.41 gm, 10 mmol) was dissolved in 100 mL of anhydrous methanol and stirred at room temperature under argon. Glacial acetic acid (572 uL, 10 mmol) was then added. After 24 hours, the yellow solution was concentrated to a foam that appeared to contain lactosamine acetate salt as a 1:1 mixture of .alpha. and .beta. anomers. Two other products were apparent which are thought to be the .alpha. and .beta. anomers of galactopyranosyl mannosamine. This product was used crude in the following reaction.
Compound 5 was then prepared from Compound 2A by using the crude material obtained in step B., above, with the procedures of Example 4 at about 1/7-1/10 scale. Acetone constituted about one-third of the solvent utilized to form the phthalamide half-acid. The ultimately produced peracetyl phthalimide (Compound 5) was prepared in 3.8 percent yield based on lactulose, with no second crop of crystals being sought.
Example 6
This example provides a synthesis of 1-Chloro-3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-.beta.-D-glycopyranoside (Compound 6).
The anomeric acetate (Compound 5) (3.3 gm, 4.3 mmol) was stirred in 43 mL of dry CH.sub.2 Cl.sub.2 under argon at room temperature. Aluminum trichloride (2.9 gm, 21.5 mmol) was then added as a solid. After 40 minutes, the mixture was rinsed into a separatory funnel to a volume of 400 mL in 1:1 CH.sub.2 Cl.sub.2 :H.sub.2 O. The mixture was shaken, the aqueous phase removed, and the organic solution was washed 2.times.200 mL with H.sub.2 O and 3.times.200 mL with saturated NaHCO.sub.3 solution. The clear pale yellow solution was then dried (MgSO.sub.4), filtered and concentrated to a pale yellow powder (3.2 gm, 10096). This material was then used for the condensation in Example 7.
Example 7
This example provides a synthesis of Ethyl .beta.-D galactopyranoside (Compound 7).
A solution of 2,3,4,6,-tetra-O-acetyl-galactosyl bromide (2.5 kg) in dichloromethane (4 L) was added at a rate of 20-25 mL/minute to a reactor charged with silver carbonate (3.13 kg, 11.4 mol), 4 .ANG. molecular sieves (2.37 kg), dichloromethane (16 L), and anhydrous ethanol (4.0 L). Agitation was maintained to provide vigorous mixing of the reagents. Two hours after complete addition of the bromide solution was achieved, TLC on silica gel developed with hexane:ethyl acetate 1:1 showed no bromide present. At that time the reaction mixture was filtered through a celite pad (1 kg), and the filtrate was evaporated at 30.degree.-35.degree. C. under vacuum to give a brown oil (1.95 kg). This oil was dried under vacuum for 17 hours. .sup.1 H-NMR (CDCL.sub.3) .delta.: 5.36(1H, d, J.sub.3,4 =3.7 Hz, H-4), 5.17(1H, dd, J.sub.2,3 =11.0 Hz, H-2), 4.99(1H, dd,H-3), 4.46)1H, d, J.sub.1,2 =8.3 Hz, H-1), 2.15, 2.05, 2.04, 1.95(12H, 4s, OAc), 1.21(3H, t, OCH.sub.2 CH.sub.3).
The crude ethyl tetraacetyl galactopyranoside (1.95 kg) was dissolved in anhydrous methanol (11.7 L) and a 25 percent sodium methoxide in methanol solution (90 mL) was added dropwise. The solution was stirred for one hour at which time TLC on silica gel developed with ethyl acetate:methanol 2:1 showed no starting material to be present. The product had an R.sub.f =0.6. The solution was neutralized by the addition of Amberlite IR-120(H.sup.+) resin (0.6 kg) and stirring. When the solution pH=6-7, the resin was removed by filtration and the filtrate was evaporated under vacuum to afford a pale yellow solid. This solid was dissolved in boiling ethanol (11 L). The resulting solution was permitted to cool to 25.degree. C. and then cooled to zero degrees C. to give a white precipitate. Filtration of this solid gave ethyl .beta.-D-galactopyranoside, Compound 7, (0.851 kg). .sup.1 H-NMR (D.sub.2 O) .delta.: 4.38(1H, d, J.sub.1,2 =8.0 Hz, H-1), 3.89(1H, bd, J.sub.3,4 =3.7 Hz, H-4), 1.2(3H, t, OCH.sub.2 CH.sub.3).
Example 8
This example provides the synthesis of Ethyl 4,6-O-benzylidene-.beta.-D-galactopyranoside (Compound 8).
Ethyl .beta.-D-galactopyranoside, Compound 7, (0.851 kg, 4.09 mol) was charged into a 20 L rotovap flask with toluene sulfonic acid (1.5 g, 7.9 mmol). The evaporator flask was fixed to the evaporator and benzaldehyde dimethyl acetal (1.23 L, 8.18 mol) was added by aspiration. The mixture was tumbled for four hours. Between thirty and forty minutes after addition of the acetal, near complete solution was obtained followed rapidly by the appearance of a heavy precipitate. Rotation was continued for four hours at which time triethylamine (1.5 mL) was added to neutralize the reaction mixture. A vacuum was applied and the solvent was removed to give a solid mass. Hexane (6 L) was charged into the flask and the mixture tumbled for 0.5 hours. The resulting solid was filtered and washed on the filter with hexane:ethyl ether 1:1 (2 L). The white solid so obtained was dried under vacuum for 17 hours to give pure ethyl 4,6-O-benzylidene-.beta.-D-galactopyranoside Compound 8, (1.0 kg,3.38 mol) in 83 percent yield. .sup.1 H-NMR (CDCl.sub.3) .delta.: 7.53(2H, m, aromatics), 7.37(3H, m, aromatics), 5.57(1H, s, CHPh), 4.29(1H, d, J.sub.1,2 =7.0 Hz, H-1), 4.21(1H, d, J.sub.3,4 =3.27 Hz, H-4), 1.29(3H, t, OCH.sub.2 CH.sub.3).
Example 9
This example provides the synthesis of Ethyl 2-O-benzoyl-4,6-O-benzylidene-.beta.-D-galactopyranoside (Compound 9).
Ethyl 4,6-O-benzylidene-.beta.-D-galactopyranoside, Compound 8, (0.924 kg, 3.12 mol) was put into a 20 liter reactor equipped with an air drive, a pressure equalizing addition funnel with gas inlet, cooling bath, and a gas outlet. Before sealing the flask, dichloromethane (9.3 L) and pyridine (2 L) were added, which gave a homogeneous solution. The addition funnel was charged with chloroacetyl chloride (0.388 kg, 3.43 mol, 273 mL) as a 60 percent solution in dichloromethane. The flask was sealed and a low flow of dry nitrogen was begun. The bath was cooled to -65.degree..+-.5.degree. C. and the reaction mixture was stirred for 30 minutes. At that time dropwise addition of the acyl chloride solution was begun at a rate of 3-4 mL per minute. After complete addition of this solution, the reaction mixture was maintained at -65.degree..+-.5.degree. C. for an additional one hour. At that time benzoyl chloride (0.614 kg, 4.37 mol, 0.507 L) was added to the reaction mixture at a rate of 8-12 mL per minute. The reaction mixture was permitted to warm to room temperature and left for 17 hours. The reaction mixture was filtered to remove precipitated salts, and the filtrate was concentrated in vacuo to remove most of the dichloromethane. A small sample was set aside for .sup.1 H-NMR. .sup.1 H-NMR (CDCl.sub.3) .delta.: 5.75(1H, dd, J.sub.2,3 =10.6 Hz, H-2), 5.56(1H, s, CHPh), 5.25(1H, dd, J.sub.3,4 =3.44 Hz, H-3), 4.69(1H, d, J.sub.1,2 =8.48 Hz, H-1), 4.48(1H, bd, H-4), 1.15(3H, t, OCH.sub.2 H.sub.3).
Water (180 mL) was added to the concentrate and the resulting mixture was agitated for two hours at 40.degree. C. At that time, the reaction mixture was further concentrated to give a yellow residue that was dissolved in dichloromethane (11 L) and transferred to a 50 liter extractor. The organic solution was successively extracted with ice cold aqueous 0.5N HCl (11 L), aqueous saturated sodium hydrogen carbonate (11 L), and cold water (11 L). The organic layer was dried over anhydrous sodium sulfate (1.0 kg), filtered, and the filtrate was evaporated to give a yellow solid that was dried under high vacuum. This reaction was monitored by TLC on silica gel developed with hexane:ethyl acetate 1:1. This solid was dissolved in hot ethanol (9.5 L) that, after cooling and filtration, gave ethyl 2-O-benzoyl-4,6-O-benzylidene-.beta.-D-galactopyranoside, Compound 9, (0.737 kg, 1.85 mol) in 59 percent yield. .sup.1 H-NMR (CDCl.sub.3) .delta.: 5.59(1H, s, CHPh), 5.36(1H, dd, J.sub.2,3 =10.07 Hz, H-2), 4.64(1H, d, J.sub.1,2 =8.21 Hz, H-1), 1.15(3H, t, OCH.sub.2 CH.sub.3).
To confirm that the benzoate was at the C-2 and that C-3 carried a free hydroxyl group, a drop of trichloroacetyl isocyanate was added to the nmr sample and the spectrum was reacquired. This spectrum contained a low field doublet of doublets at .delta.=5.27 typical of H-3 of galactose which is esterified at C-3. The original filtrate obtained from the reaction mixture contained additional quantities of product.
Example 10
This example provides the preparation of Ethyl (.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside Compound 10).
To a mixture of ethyl 2-O-benzoyl-4,6-O-benzylidene-.beta.-D-galactopyranoside, Compound 9, (0.76 g, 1.9 mmol), 4 .ANG. molecular sieves (2 g), dichloromethane (10 mL), collidine (0.278 mL, 2.1 mmol), and silver trifluoromethanesulfonate (0.522 g, 2 mmol) cooled to -25.degree. C. was added dropwise a solution of 4-O-(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-.beta.-D-glucopyranosyl chloride (Compound 6; 1.484 g, 2 mmol) dissolved in dichloromethane (5 mL). The resulting mixture was stirred and warmed to ambient temperature after complete addition of the chloride. After two hours, the mixture was diluted with dichloromethane and filtered. The filtrate was washed successively with aqueous sodium bisulfite, aqueous hydrochloric acid, aqueous sodium hydrogen carbonate, and finally water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to give a solid mass that was recrystallized from dichloromethane:hexane.
The resulting fully blocked trisaccharide (0:66 g) was treated with 80 percent aqueous acetic acid (5 mL) at 80.degree. C. for two hours at which time the solvent was removed by evaporation. The residue was coevaporated with toluene-ethyl acetate two times to give a residue that was dissolved in ethanol (10 mL). Hydrazine hydrate (0.3 mL) was added and the resulting mixture was refluxed for 17 hours to give a precipitate that was filtered to give a solid (0.45 g) after drying. This solid was dissolved in methanol:water 5:1 and treated with diallylpyrocarbonate (0.166 mL) for one hour. The resulting mixture was evaporated and partitioned between dichloromethane and water. The aqueous layer was separated and concentrated to provide Compound 10 as a residue that solidified upon trituration with ethyl acetate:acetone 2:1.
This provided the title trisaccharide (Compound 10) which was enzymatically sialylated to give ethyl �sodium (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-nonulopyranosylonate)!-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (Compound 11) which was identical to that produced in the following procedure.
Example 11
This example provides the preparation of Ethyl �sodium (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-nonulopyranosylonate)!-2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (Compound 11).
The following describes the enzymatic conversion of a disaccharide (Compound 9) to produce the title compound (Compound 11) using galactosyl transferase and sialyl transferase.
To water (12 L), N-�2-hydroxyethyl!piperazine-N'-�2-ethanesulfonic acid! (0.410 Kg) was added and the pH of the resulting solution was adjusted to 7.5. Bovine serum albumin (17 g) was added and the mixture stirred until a complete solution was obtained. Ethyl 3-O-(2-N-allyloxycarbonyl-2-amino-2-deoxy-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (Compound 9) (0.3 kg), glucose-1-phosphate (0.271 kg), phosphoenolpyruvate (0.177 kg), potassium chloride (0.087 kg), sodium azide (8.4 g), and uridine-5'-diphosphate (8.76 g) were added and the resulting mixture stirred until all of the solids are dissolved. Manganese chloride (1M, 506 mL) and magnesium chloride (1M, 168 mL) were then added. Pyruvate kinase (42,000 U), uridine-5'-diphosphate-glycose pyrophosphorylase (2,000 U), inorganic pyrophosphatase (8,400 U), uridine-5'-diphosphate-galactose epimerase (91,000 U), and uridine-5'-diphosphate-galactosyl transferase (8,850 U) were then added. The final volume of the reaction mixture was adjusted to 17 L with water. After 48 hours magnesium chloride (1M, 340 mL) was added. The reaction was monitored by TLC on silica gel developed with isopropanol:1M ammonium acetate 4:1. After 8-9 days TLC indicated that the reaction had proceeded to >95 percent at which time the following solution was prepared.
A solution of N-�2-hydroxyethyl!piperazine-N'-�2-ethanesulfonic acid! (0.528 kg) was prepared in water (15 L) and the pH of the resulting solution was adjusted to 7.5. Bovine serum albumin (22 g), sodium azide (11.5 g), sialic acid (0.242 kg), phosphoenolpyruvate (0.395 kg), cytidine-5'-monophosphate (25 g), adenosine-5'-triphosphate (4.7 g), manganese chloride (1M, 780 mL) are added. To this solution was added pyruvate kinase (207,000 U), myokinase (125,000 U), cytidine-5'-monophosphate-N-acetylneuraminic acid synthetase (3245 U), inorganic pyrophosphatase (9400 U), and .alpha.-2,3-sialyltransferase (1640 U). The volume of this mixture was adjusted to 22 L and this solution was added to the galactosyl transferase reaction. The reaction was monitored by TLC on silica gel developed with isopropanol:1M ammonium acetate 4:1. After 10-12 days, TLC indicated that the reaction had proceeded to give >95 percent of the title compound, Compound 11.
Example 12
This example provides the synthesis of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-nonulopyranosylonate)!-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-(3,6-di-O-acetyl-2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 12).
A solution (40 L) of ethyl �sodium (5-acetamido-3,5-dideoxy-.alpha.-glycero-D-galacto-nonulopyranosylonate)!-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (Compound 11) was filtered through paper. The filtrate was evaporated to a thick syrup in a 50 L rotavapor. The syrup was coevaporated twice with pyridine (2.times.2 L), then kept under vacuum for 20 hours. The evaporation flask was charged with a solution of N,N-dimethylaminopyridine (20 g) in pyridine (12 L). The rotavapor bath was charged with ice-water mixture, and rotation was continued while acetic anhydride (6 L) was added during a period of one hour. Two hours after complete addition, more acetic anhydride (2 L) was added and the resulting mixture was left for 20 hours rotating slowly at room temperature. To ensure compete acetylation, more acetic anhydride (1 L) was added and the mixture was rotated for an additional 24 hours. The reaction was checked by TLC (ethyl acetate:hexane:ethanol, 10:10:3).
Upon complete reaction vacuum was applied and 14 L of distillate were collected. To the resulting residue, methanol (15 L) was added over a period of one hour and the mixture was rotated at room temperature for 20 hours. At this time, TLC on silica gel (ethyl acetate:hexane:ethanol, 10:10:3 and dichloromethane:acetone 3:2) showed complete conversion of the lactone to a slower-moving spot that was the methyl ester monohydroxy compound. The mixture was then concentrated (18 L evaporated) and the mixture was cooled in ice water while acetic anhydride (3 L) was added over a period of 30 minutes. The mixture was left for 20 hours. TLC on silica gel (dichloromethane:acetone 3:2) showed complete acetylation with the product running slightly faster.
Methanol (1 L) was added to destroy excess acetic anhydride during which a slight exotherm was noticed. After one hour, the mixture was concentrated to a syrup, which was transferred to a 50 L extractor with the aid of ethyl acetate-water mixture (13/13 L). The mixture was agitated vigorously. After phase separation, the lower aqueous layer was drawn off, and the remaining organic layer was filtered through paper. The filtrate was washed with 5 percent aqueous hydrochloric acid (15 L, the aqueous layer should still be strongly acidic to pH-paper after washing), and aqueous 1M sodium bicarbonate (15 L, the aqueous layer should still be alkaline to pH paper after washing). The organic layer was then transferred to a 20 L container, dried over anhydrous sodium sulfate and filtered.
The filtrate was concentrated to a semi-solid residue. This residue was dissolved in dichloromethane (3 L), and applied to a silica gel column (10 kg), packed in dichloromethane. Elution first with dichloromethane (25 L), then with 3:1 dichloromethane:acetone (25 L), and finally with 1:1 dichloromethane:acetone (50 L) gave fractions containing product. Base-line separation was achieved from the disaccharide material, but very little separation was achieved from the traces of slightly faster moving material. The fractions containing product were evaporated, and redissolved in dichloromethane (1.5 L). This solution was slowly added to a vigorously stirred mixture of ethyl ether (7.5 L) and hexane (10 L). The resulting precipitate was filtered and washed with 2:1 ether:hexane, air-dried overnight, then dried in high vacuum for 48 hours. The precipitate was shown to be the title Compound 12 by .sup.1 H-NMR, and contained a small amount of residual solvent (1-5 percent, weight/weight). .sup.1 H-NMR (CDCl.sub.3) .delta.: 4.67(d, 1H, H-1"), 4.49(d, 1H, H-1'), 4.33(d, 1H, H-1).
Example 13
This example provides the synthesis of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-amino-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 13).
To a stirred solution of ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-allyloxycarbonylamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 12) (5.10 gm, 3.80 mmol) in anhydrous THF under argon at room temperature was added polymethylhydrosiloxane (420 .mu.L). The reaction mixture was put through a vacuum/purge cycle three times with argon to degas the solution. The flask was wrapped in aluminum foil to protect the solution from light, and the solution was treated with palladium tetrakistriphenylphosphine �Pd(PPh.sub.3).sub.4 ; 158 mg, 0.14 mmol!. After stirring for 18 hours at room temperature, TLC in 10:1 CHCl.sub.3 :MeOH indicated complete consumption of Compound 12 and the presence of a single lower rf product. The reaction mixture was diluted with 600 mL of EtOAc and washed 1.times.200 mL with H.sub.2 O and 1.times.200 mL with saturated NaCl solution. The organic solution was dried (MgSO.sub.4), filtered, concentrated by rotary evaporation, and flash chromatographed on a 65mm.times.10" column of silica gel using 3:1 EtOAc:acetone as eluant. The product-containing fractions (as judged by TLC) were pooled and concentrated to provide Compound 13 (4.42 gm, 87 percent) as a tan solid. .sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 5.50 (m, 1H), 5.44 (dd, J=6 Hz, J=2 Hz, 1H), 5.35-5.01 (m), 4.89 (m, 2H), 4.63 (d, J=6 Hz, 1H), 4.59-4.35 (m), 4.22-3.38 (m), 3.81 (s, 3H), 2.69 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.21 (t, J=5 Hz, 3H).
Example 14
This example provides the synthesis of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-amino-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 14).
To a stirred solution of Compound 13 (4.42 gm, 3.29 mmol) in 366 mL of 4:1 MeOH:H.sub.2 O at room temperature in a capped flask was added glacial acetic acid (188 .mu.L, 3.29 mmol). The pale yellow solution was then heated to 50.degree. C. After 48 hours, TLC in 10:1 CHCl.sub.3 :MeOH indicated nearly complete disappearance of Compound 13 and appearance of a predominant, slightly higher R.sub.f product. The reaction was cooled to room temperature, concentrated by rotary evaporation to an oil, and flash chromatographed on a 65mm.times.10" column of silica gel using 10:10:4 EtOAc:hexane:MeOH as eluant. The product-containing fractions (as judged by TLC) were pooled and concentrated to give Compound 14 (2.78 gm, 65 percent) as a foam.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 5.50 (m, 1H), 5.40 (d, J=2 Hz, 1H), 5.25 (d, J=7 Hz, 1H), 5.17 (dd, J=6 Hz, J=7 Hz, 1H), 5.04 (dd, J=6 Hz, J=7 Hz, 1H), 4.89 (d, J=3 Hz, 1H), 4.63 (d, J=6 Hz, 1H), 4.59 (dd, J=3 Hz, J=7 Hz, 1H), 4.42-3.40 (m), 3.81 (s, 3H), 2.69 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.21 (t, J=5 Hz, 3H).
Example 15
This example provides the synthesis of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-benzamido-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 15).
To a stirred solution of Compound 14 (150 mg, 0.12 mmol) in 2 mL of dichloromethane at room temperature under an argon atmosphere was added anhydrous NaHCO.sub.3 (40 mg, 0.48 mmol), and benzoyl chloride (34 mg, 0.24 mmol, 28 .mu.L). After stirring for 24 hours, TLC in 80:20 EtOAc:acetone indicated complete consumption of starting material and the appearance of a slightly higher R.sub.f material. The reaction mixture was diluted with 150 mL of ethyl acetate and washed 1.times.50 mL with H.sub.2 O. The organic solution was dried (MgSO.sub.4), filtered, concentrated, and flash chromatographed on a column of silica gel using 90:10 EtOAc:acetone as eluant. The product-containing fractions (as judged by TLC) were pooled and concentrated by rotary evaporation and then by high vacuum to a cream waxy solid, Compound 15: (140 mg, 83 percent). .sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.75 (d, J=7 Hz, 2H), 7.45 (d, J=7 Hz, 1H), 7.39 (dd, J=7 Hz, J=7 Hz, 2H), 6.45 (d, J=5 Hz, 1H), 5.50 (m, 1H), 5.40 (d, J=2 Hz, 1H), 5.37 (d, J=2 Hz, 1H), 5.27 (m, 1H), 5.09 (m,1H), 4.82 (d, J=3 Hz, 1H), 4.63 (d, J=6 Hz, 1H), 4.59 (dd, J=3 Hz, J=7 Hz, 1H), 4.39-3.40 (m), 3.81 (s, 3H), 3.19 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.15 (t, J=5 Hz, 3H).
Example 16
This example provides the synthesis of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-benzamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 16).
To a stirred solution of Compound 15 (140 mg, 0.1 mmol) in 1 mL of dichloroethane at room temperature under an argon atmosphere were added powdered, flame-dried 4 .ANG. molecular sieves (100 mg), tetramethylurea (120 uL, 1 mmol), and tri-O-benzyl fucosyl fluoride (218 mg, 0.5 mmol). After stirring for one hour at room temperature, the reaction was cooled to -20.degree. C. and treated with SnCl.sub.2 (95 mg, 0.5 mmol) and AgClO.sub.4 (126 mg, 0.5 mmol). The reaction was then allowed to slowly warm to room temperature. After stirring for 24 hours, TLC in 10:1 CHCl.sub.3 :MeOH indicated near complete consumption of starting material and the appearance of a slightly lower R.sub.f material.
The reaction mixture was filtered through a plug of celite with 50 mL of dichloromethane, and the filtrate was washed 2.times.50 mL with H.sub.2 O. The organic solution was dried (MgSO.sub.4), filtered, concentrated, and flash chromatographed on a 20mm.times.6" column of silica gel using 10:10:3 EtOAc:hexane:MeOH as eluant. The product-containing fractions (as judged by TLC) were pooled and concentrated by rotary evaporation and then by high vacuum to a white film, Compound 16 (140 mg, 77 percent). .sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.46 (d, J=7 Hz, 2H), 7.35-7.12 (m, 18H), 6.45 (d, J=6 Hz, 1H), 3.82 (s, 3H), 3.20 (m, 1H), 2.55 (dd, J=4 Hz, J=12 Hz, 1H), 1.18 (d, J=6 Hz, 3H), 1.10 (t, J=6 Hz, 3H).
Example 17
This example provides the synthesis of Ethyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-benzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 17).
To a stirred solution of Compound 16 (140 mg, 77 .mu.mol) in 4 mL of methanol was added palladium hydroxide on carbon (140 mg, 20 percent by weight palladium). The slurry was then put through a vacuum/purge cycle three times with hydrogen gas and then held under hydrogen at one atmosphere pressure at room temperature. After one hour, TLC in 5:1 EtOAc:MeOH indicated complete disappearance of Compound 16 and the appearance of a single lower R.sub.f material. The slurry was filtered through a plug of celite with 50 mL of methanol and concentrated by rotary evaporation to an oil.
This oil was dissolved in 5 mL of 4:1 MeOH:H.sub.2 O and stirred at room temperature in a capped flask. Sodium methoxide powder (140 mg, 2.6 mmol) was added to the stirred solution. After 16 hours, TLC in 60:50:15 CHCl.sub.3 :MeOH:15 mM CaCl.sub.2 indicated complete disappearance of starting material and the appearance of a single lower R.sub.f product.
The mixture was treated with 1 gram of Dowex 50.times.8-400 cation exchange resin (hydrogen form, freshly methanol washed) and stirred for one minute. The mixture was filtered through a fritted funnel and the filtrate concentrated by rotary evaporation to an oil. This material was chromatographed on a 40 mm.times.8" column of Bio-Rad Bio-Gel P2 gel filtration media (mesh size: fine) using 0.1M ammonium bicarbonate as eluant. The product-containing fractions (as judged by TLC) were pooled and lyophilized to a white powder for Compound 17 (60 mg, 72 percent). .sup.1 H-NMR (300 MHz, .delta. in ppm relative to HOD) 7.70 (d, J=7 Hz, 2H), 7.55 (d, J=7 Hz, 1H), 7.47 (dd, J=7 Hz, J=7 Hz, 2H), 5.08 (d, J=4 Hz, 1H), 4.50 (d, J=8 Hz, 1H), 4.27 (d, J=8 Hz, 1H), 4.10 (d, J=3 Hz, 1H), 4.05-3.40 (m), 2.70 (dd, J=4.6 Hz, J=12.4 Hz, 1H), 1.97 (s, 3H), 1.74 (dd, J=12.4 Hz, J=12.4 Hz, 1H), 1.10 (t, J=7 Hz, 3H), 1.07 (d, J=7 Hz, 3H).
Example 18
This example provides the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-2'-napthamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 29).
To a stirred solution of Compound 25 (prepared analogously to Compound 16; 90 mg, 48 .mu.mol) in 5 mL of methanol was added palladium hydroxide on carbon (40 mg, 40 percent by weight palladium). The slurry was put through a vacuum/purge cycle three times with hydrogen gas and held under hydrogen at one atmosphere pressure at room temperature. After 24 hours, TLC in 90:10 CH.sub.2 Cl.sub.2 :MeOH indicated complete disappearance of Compound 25 and the appearance of a single lower R.sub.f material. The slurry was filtered through a plug of celite with 50 mL of methanol and concentrated by rotary evaporation to a cream waxy solid. The product was treated by flash column chromatography on a column of silica gel using 90:10 CH.sub.2 Cl.sub.2 :MeOH as eluant. The product containing fractions (as judged by TLC) were then pooled and concentrated to give Compound 29 (55 mg, 72%) as a white waxy solid. .sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 8.39 (s, 1H), 7.94 (d, J=7 Hz, 1H), 7.82 (m, 2H), 7.57 (m, 2H), 7.37 (m,1H), 5.57-5.41 (m, 3H), 5.22 (d, J=7 Hz, 1H), 5.15 (m,1H), 4.97-4.39 (m), 4.35 (d, J=4 Hz, 2H), 4.19-3.42 (m), 3.81 (s, 3H), 3.23 (m, 1H), 2.75 (bs,1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.23 (d, J=5 Hz, 3H), 1.05 (t, J=5 Hz, 3H).
Following procedures substantially similar to those discussed above and as to Scheme 3 for the conversion of Compound 14 into Compounds 15, 16 and 17, Compounds of 18-38 were also prepared. Tables 1, 2 and 3, below show the generalized structures for groups of compounds corresponding to Compounds 15, 16 or 17, and provides other pertinent data for each of those compounds. Table 1 shows the acylating agent used to prepare each R.sup.1 group. Tables 1-3 are followed by NMR and added data for several of those compounds, and inhibitor Compounds 30-38, including last step yields.
TABLE 1__________________________________________________________________________ ##STR13## R.sup.1 GroupCompound # Acylating agent Yield R.sub.f (solvent)__________________________________________________________________________18 ##STR14## 148 mg, 87% 0:4(90:10 EtOAc:acetone)19 ##STR15## 136 mg, 78% 0.43(90:10 EtOAc:acetone)20 ##STR16## 133 mg, 78% 0.40(90:10 EtOAc:acetone)21 ##STR17## 143 mg, 82% 0.45(90:10 EtOAc:acetone)__________________________________________________________________________
TABLE 2__________________________________________________________________________ ##STR18##Compound # Glycosyl acceptor Yield R.sub.f (solvent)__________________________________________________________________________22 18 135 mg, 74% 0.35(92:8 EtOAc:acetone)23 19 100 mg, 58% 0.39(92:8 EtOAc:acetone)24 20 105 mg, 65% 0.37(92:8 ETOAc:acetone)25 21 100 mg, 58% 0.37(92:8 ETOac:acetone)__________________________________________________________________________
TABLE 3__________________________________________________________________________ ##STR19##Compound # Benzylate pentasaccharide Yield R.sub.f (solvent)__________________________________________________________________________26 22 62 mg, 60% 0.32(90:10 CH.sub.2 Cl.sub.2 :MeOH)27 23 35 mg, 50% 0.39(90:10 CH.sub.2 Cl.sub.2 :MeOH)28 24 73 mg, 65% 0.31(90:10 CH.sub.2 Cl.sub.2 :MeOH)__________________________________________________________________________
Example 19
This example provides the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-benzyloxycarbonylamido-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 39).
A solution of benzyloxycarbonyl chloride (CBZ-Cl) (1.2 ml, 8.4 mmol) in CH.sub.2 Cl.sub.2 (2.0 ml) was added dropwise to a mixture of Compound 14a (10.8 g, 8.3 mmol) and NaHCO.sub.3 (1.4 g, 16.6 mmol) in CH.sub.2 Cl.sub.2 (100 ml), and the reaction mixture was stirred overnight (about 18 hours). To this mixture were added NaHCO.sub.3 (1.4 g, 16.6 mmol) and CBZ-Cl (1.2 ml, 8.4 mmol), and the resulting mixture was stirred an additional four hours. The resulting mixture was diluted with AcOEt, washed with H.sub.2 O, dried over MgSO.sub.4, filtered, and concentrated. The residue was chromatographed on silica gel to provide Compound 39 (7.75 g, 65 percent yield) as a white solid.
Example 20
This example provides the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-benzyloxycarbonylamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 40).
To a stirred solution of Compound 39 (3.90 g, 2.72 mmol) in 100 ml of ClCH.sub.2 CH.sub.2 Cl were added powdered molecular sieves (MS4A) (12 g), tetramethyl urea (TMU) (3.25 ml, 27.2 mmol) and 2,3,4-tri-O-benzyl-L-fucosyl fluoride (CMH-048, 5.94 g, 13.6 mmol). After stirring for 90 minutes at room temperature, the mixture was shielded from light, cooled to -20.degree. C. and treated with SnCl.sub.2 (2.59 g, 13.6 mmol) and AgClO.sub.4 (98 percent, 2.88 g, 13.6 mmol). The reaction mixture was permitted to warm to room temperature over a 90 minute time period, and stirred for 24 hours. In order to complete the reaction, TMU (1.95 ml, 16.3 mmol), CMH-048 (3.56 g, 8.16 mmol), SnCl.sub.2 (1.55 g, 8.17 mmol) and AgClO.sub.4 (1.73 g, 8.17 mmol) were added again to the mixture at zero degrees C., which was then permitted to slowly warm to room temperature. After 48 hours, the resulting mixture was filtered through a pad of Celite and the filtrate was washed with H.sub.2 O. The organic phase was dried over Na.sub.2 SO.sub.4, filtered, concentrated, and chromatographed on silica gel (Hexane/AcOEt/MeOH=10/10/2) to provide Compound 40 (3.65 g, 73 percent yield) and recovered starting material, Compound 39, (672 mg, 17 percent yield).
Example 21
This example provides the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-amino-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 41).
The mixture of Compound 40 (3.06 g, 1.66 mmol), HCOONH.sub.4 (1.05 g, 16.6 mmol) and 10 percent Pd--C (wet, 3.0 g) in EtOH (80 ml) was refluxed with stirring for 9.5 hours. To this mixture were added more HCOONH.sub.4 (1.05 g, 16.6 mmol) and 10 percent Pd--C (3.0 g), and the resulting mixture was refluxed an additional 11 hours. That resulting mixture was filtered through a pad of Celite and concentrated to provide Compound 41 (2.30 g, 96 percent yield) as a white solid.
Example 22
This example illustrates the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(3,5-dichlorobenzoylamido)-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl!-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 42).
To a stirred solution of Compound 41 (40 mg, 0.028 mmol) in CH.sub.2 Cl.sub.2 (8.9 ml) were added NaHCO.sub.3 (46 mg, 0.54 mmol) and 3,5-dichlorobenzoyl chloride (58.6 mg, 0.28 mmol). After 12 hours at room temperature, the reaction mixture was diluted with EtOAc and washed with H.sub.2 O. The organic phase was dried over MgSO.sub.4, filtered, and evaporated to afford crude Compound 42 (98.4 mg) as a pale yellow oil.
Example 23
This example illustrates the preparation of Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(3,5-dichlorobenzamido)-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl!-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 43).
To a stirred solution of crude Compound 42 (98.4 mg) in MeOH (8.9 ml) was added 28 percent NaOMe--MeOH (300 .mu.l). After 48 hours at room temperature, the mixture was neutralized with DOWEX 50W-X8 (H.sup.+ -form) and filtered. The filtrate was concentrated, diluted with EtOAc, and extracted with H.sub.2 O. The aqueous phase was evaporated to give the corresponding ester. The ester was treated with 1N--NaOH (200 .mu.l) in H.sub.2 O (5.0 ml). The mixture was stirred for 12 hours at room temperature, neutralized with DOWEX 50W-X8 (H.sup.+ -form) and filtered. The filtrate was concentrated, purified by Gel (p-2) filtration (H.sub.2 O as eluent), and lyophilized to afford Compound 43 (31.6 mg, quantitative yield) as a white powder.
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.61 (s, 3H), 5.00 (d, J=3.96 Hz, 1H), 4.47 (d, J=7.59 Hz, 1H), 4.26 (d, J=7.92 Hz, 1H), 4.09 (d, J=2.97 Hz, 1H), 4.04-3.30 (m), 2.67 (m, 1H), 1.94 (s, 3H), 1.72 (t, J=11.88 Hz, 1H), 1.09 (m, 6H).
Example 24
This example illustrates the preparation of esterified forms of compound 17 beginning with Ethyl (sodium (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-nonulopyranosylonate))-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (Compound 11)
An aqueous solution (40 L) of ethyl (sodium (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-nonulopyranosylonate))-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (Compound 11) was filtered through paper. The filtrate was evaporated to a thick syrup in a 50 L rotavapor. The syrup was coevaporated twice with pyridine (2.times.2 L), then kept under vacuum for 20 hours. The evaporation flask was charged with a solution of N,N-dimethylaminopyridine (20 g) in pyridine (12 L). The rotavapor bath was charged with ice-water mixture, and rotation was continued while acetic anhydride (6 L) was added during a period of one hour. Two hours after complete addition, more acetic anhydride (2 L) was added and the resulting mixture was left for 20 hours rotating slowly at room temperature. To ensure compete acetylation, more acetic anhydride (1 L) was added and the mixture was rotated for an additional 24 hours. The reaction was checked by TLC (ethyl acetate:hexane:ethanol, 10:10:3). Upon complete reaction, vacuum was applied and 14 L of distillate were collected.
To the resulting residue, an alcohol (15 L) is added over a period of one hour and the mixture is rotated at room temperature for 20 hours, or until TLC on silica gel (ethyl acetate:hexane:ethanol, 10:10:3 and dichloromethane:acetone 3:2) shows complete conversion of the lactone to a slower-moving spot which is the ester monohydroxy compound. The mixture is then concentrated (18 L evaporated) and cooled in ice water while acetic anhydride (3 L) is added over a period of 30 minutes. The mixture is left for 20 hours, or until TLC on silica gel (dichloromethane:acetone 3:2) shows complete acetylation with the product running slightly faster.
Alcohol (1 L) is added to destroy excess acetic anhydride during which a slight exotherm is typically noticed. After 1 hour, the mixture is concentrated to a syrup, and transferred to a 50 L extractor with the aid of ethyl acetate-water mixture (13/13 L). The mixture is agitated vigorously. After phase separation, the lower aqueous layer is drawn off, and the remaining organic layer is filtered through paper. The filtrate is washed with 5 percent aqueous hydrochloric acid (15 L, the aqueous layer should still be strongly acidic to pH-paper after washing), and aqueous 1M sodium bicarbonate (15 L, the aqueous layer should still be alkaline to pH paper after washing). The organic layer is then transferred to a 20 L container, dried over anhydrous sodium sulfate and filtered.
The filtrate is concentrated to a semi-solid residue. This residue is dissolved in dichloromethane (3 L), and applied to a silica gel column (10 kg), packed in dichloromethane and eluted first with dichloromethane (25 L), then with 3:1 dichloromethane:acetone (25 L), and finally with 1:1 dichloromethane:acetone (50 L) to give fractions containing product. The fractions containing product are evaporated, and redissolved in dichloromethane (1.5 L). This solution is slowly added to a vigorously stirred mixture of ethyl ether (7.5 L) and hexane (10 L). The resulting precipitate is filtered and washed with 2:1 ether:hexane, air-dried overnight, then dried in high vacuum for 48 hours to provide a purified product of the ester-form of the blocked tetrasaccharide.
To a stirred solution of blocked tetrasaccharide (5.10 g, 3.80 mmol) in anhydrous THF (8 L) under argon at room temperature is added polymethylhydrosiloxane (PMSH, 420 .mu.L). The reaction mixture is put through a vacuum/purge cycle three times with argon to degas the solution. The flask is wrapped in aluminum foil to protect the solution from light, and the solution is treated with palladium tetrakistriphenylphosphine (Pd(PPh.sub.3).sub.4 ; 158 mg, 0.14 mmol). The resulting reaction mixture is then stirred at room temperature for 18 hours, or until TLC (10:1 CHCl.sub.3 :MeOH) shows completion of the reaction. The resulting reaction mixture is diluted with acetic acid (600 mL) and washed 1.times.200 mL with H.sub.2 O and 1.times.200 mL with saturated NaCl solution. The organic solution is dried (MgSO.sub.4), filtered, concentrated by rotary evaporation, and flash chromatographed on a 65 mm.times.10" column of silica gel using 3:1 EtOAc:acetone as eluant. The product-containing fractions (as judged by TLC) are pooled and concentrated.
To a portion of the resulting product in 4:1 alcohol:H.sub.2 O (366 mL) at room temperature in a capped flask is added glacial acetic acid (188 .mu.L, 3.29 mmol). The solution is stirred and heated to 50.degree. C. for 48 hours, or until TLC (10:1 CHCl.sub.3 :MeOH) shows a complete reaction. The reaction is cooled to room temperature, concentrated by rotary evaporation to an oil, and flash chromatographed on a 65 mm.times.10" column of silica gel using 10:10:4 EtOAc:hexane:MeOH as eluant. The product-containing fractions (as judged by TLC) are pooled and concentrated.
To a stirred solution of the resulting product in 2 mL of dichloromethane at room temperature under an argon atmosphere is added anhydrous NaHCO.sub.3 (40 mg, 0.48 mmol), and benzoyl chloride (34 mg, 0.24 mmol, 28 .mu.L) and stirred for 24 hours, or until TLC (80:20 EtOAc:acetone) shows the reaction is completed. The reaction mixture is diluted with 150 mL of ethyl acetate and washed 1.times.50 mL with H.sub.2 O. The organic solution is dried (MgSO.sub.4), filtered, concentrated, and flash chromatographed on a column of silica gel using 90:10 EtOAc:acetone as eluant. The product-containing fractions (as judged by TLC) are pooled and concentrated by rotary evaporation and high vacuum.
To a stirred solution of the resulting product in 1 mL of dichloroethane at room temperature under an argon atmosphere are added powdered, flame-dried 4 .ANG. molecular sieves (100 mg), tetramethylurea (120 .mu.L, 1 mmol), and tri-O-benzyl fucosyl fluoride (218 mg, 0.5 mmol). After stirring for one hour at room temperature, the reaction is cooled to -20.degree. C. and treated with SnCl.sub.2 (95 mg, 0.5 mmol) and AgClO.sub.4 (126 mg, 0.5 mmol). The reaction is then allowed to slowly warm to room temperature while stirring for 24 hours, or until TLC (10:1 CHCl.sub.3 :MeOH) shows the reaction is complete.
The reaction mixture is filtered through a plug of celite with 50 mL of dichloromethane, and the filtrate is washed 2.times.50 mL with H.sub.2 O. The organic solution is dried (MgSO.sub.4), filtered, concentrated, and flash chromatographed on a 20 mm.times.6" column of silica gel using 10:10:3 EtOAc:hexane:MeOH as eluant. The product-containing fractions (as judged by TLC) are pooled and concentrated by rotary evaporation and high vacuum.
To a stirred solution of the resulting product in 4 mL of an alcohol is added palladium hydroxide on carbon (140 mg, 20 percent by weight palladium). The slurry is put through a vacuum/purge cycle three times with hydrogen gas and then held under hydrogen at one atmosphere pressure at room temperature for 1 hour, or until TLC (5:1 EtOAc:MeOH) shows completion of the reaction. The slurry is filtered through a plug of celite with 50 mL of ethyl acetate and concentrated by rotary evaporation to an oil.
This oil is dissolved in 5 mL of an alcohol and stirred at room temperature in a capped flask. An alkoxide powder or pyridine is added to the stirred solution. The reaction mixture is stirred for 16 hours, or until TLC (60:50:15 CHCl.sub.3 :MeOH:15 mM CaCl.sub.2) shows completion of the reaction. The mixture is treated with 1 gram of Dowex 50.times.8-400 cation exchange resin (hydrogen form, freshly alcohol washed) and stirred for one minute. The mixture is filtered through a fritted funnel and the filtrate concentrated by rotary evaporation to an oil. This material is chromatographed on a 40 mm.times.8" column of Bio-Rad Bio-Gel P2 gel filtration media (mesh size: fine) using 0.1M ammonium bicarbonate as eluant. The product-containing fractions (as judged by TLC) are pooled and lyophilized to give the prodrug pentasaccharide esters having the formula: ##STR20## wherein: Z is selected from the group consisting of hydrogen, C.sub.1 -C.sub.6 acyl and ##STR21## Y is selected from the group consisting of C(O), SO.sub.2, HNC(O), OC(O) and SC(O); R.sup.1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C.sub.1 -C.sub.3 alkylene group, wherein an aryl group has one five-membered aromatic ring, one six-membered aromatic ring or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl, monooxahydrocarbyl, monothiahydrocarbyl, monoazahydrocarbyl and diazahydrocarbyl rings, and a substituted aryl group is said aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C.sub.1 -C.sub.12 alkyl, C.sub.1 -C.sub.12 alkoxy, amino, mono-C.sub.1 -C.sub.12 alkylamino, di-C.sub.1 -C.sub.12 alkylamino, benzylamino, C.sub.1 -C.sub.12 alkylbenzylamino, C.sub.1 -C.sub.12 thioalkyl and C.sub.1 -C.sub.12 alkyl carboxamido groups, or R.sup.1 Y is allyloxycarbonyl or chloroacetyl; R.sup.2 is selected from the group consisting of hydrogen, C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl, C.sub.1 -C.sub.6 alkyl C.sub.1 -C.sub.5 alkylene .omega.-carboxylate, .omega.-tri(C.sub.1 -C.sub.4 allyl/phenyl)silyl C.sub.2 -C.sub.4 alkylene, monosaccharide and disaccharide, or OR.sup.2 together form a C.sub.1 -C.sub.18 straight chain, branched chain or cyclic hydrocarbyl carbamate; R.sup.4 is an alkyl group; R.sup.5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C.sub.1 -C.sub.6 acyl; R.sup.7 is methyl or hydroxymethyl; and X is selected from the group consisting of C.sub.1 -C.sub.6 acyloxy, C.sub.2 -C.sub.6 hydroxylacyloxy, hydroxy, halo and azido. In a presently preferred embodiment, R.sup.4 is a member selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl. benzyl, pentyl and hexyl.
NMR Data of Compounds 18-28
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-p-fluorobenzamido-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 18)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.79 (m, 2H), 7.15 (m, 2H), 6.41 (d, J=5 Hz, 1H), 5.53 (m, 1H), 5.42 (m,1H), 5.23 (d, J=7 Hz, 1H), 5.17 (m,2H), 4.89 (d, J=3 Hz, 1H), 4.63 (d, J=6 Hz, 1H), 4.59 (dd, J=3 Hz, J=3 Hz, J=7 Hz, 1H), 4.42-3.40 (m), 3.81 (s, 3H), 3.19 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-p-nitrobenzamido-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 19)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 8.22 (d, J=8 Hz, 2H), 7.95 (d, J=8 Hz, 2H), 6.81 (d, J=5 Hz, 1H), 5.59-5.37 (m, 2H), 5.21 (d, J=4 Hz, 1H), 5.11 (m, 2H), 4.89 (d, J=2 Hz, 1H), 4.63 (d, J=5 Hz, 1H), 4.59 (dd, J=1 Hz, J=7 Hz, 1H), 4.42-3.40 (m), 3.79 (s, 3H), 3.19 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galacto-pyranosyl)-(1,4)-O-�2-(E-1-oxo-3-phenylprop-2-ene)amino-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl!-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 20)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.57 (d, J=11 Hz, 1H), 7.45-7.25 (m, 5H), 6.39 (d, J=11 Hz, 1H), 5.87 (d, J=4 Hz, 1H), 5.45 (m, 1H), 5.39 (m, 2H), 5.21 (t, J=7 Hz, 1H), 5.17-4.97 (m,2H), 4.89 (d, J=2 Hz, 1H), 4.63 (d, J=5 Hz, 1H), 4.59 (dd, J=1 Hz, J=7 Hz, 1H), 4.42-3.40 (m), 3.79 (s, 3H), 3.05 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-(2-2'-napthamido-2-deoxy-6-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 21)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 8.25 (s, 1H), 7.95-7.42 (m, 6H), 6.58 (d, J=5 Hz, 1H), 5.53 (m, 1H), 5.44 (d, J=2 Hz, 1H), 5.41-5.23 (m), 5.17-5.01 (m), 4.89 (d, J=3 Hz, 1H), 4.63 (d, J=6 Hz, 1H), 4.59 (dd, J=3 Hz, J=7 Hz, 1H), 4.42-3.40 (m), 3.81 (s, 3H), 3.27 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-fluorobenzamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-b-D-galactopyranoside (Compound 22)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.42 (m, 2H), 7.39-7.17 (m, 15H), 6.95 (t, J=7 Hz, 2H), 6.45 (d, J=5 Hz, 1H), 5.57-5.37 (m, 3H), 5.27 (d, J=7 Hz,1H), 5.17-4.45 (m), 4.39 (d, J=7 Hz, 1H), 4.25-3.41 (m), 3.81 (s, 3H), 3.21 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.19 (d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-nitrobenzamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 23)
.sup.1 H-NMR (300 MHz, .DELTA. in ppm relative to CHCl.sub.3) 8.15 (d, J=8 Hz, 2H), 7.55 (d, J=8 Hz, 2H), 7.41-7.15 (m, 15H), 6.63 (d, J=5 Hz, 1H), 5.48 (m, 1H), 5.43 (dd, J=6 Hz, J=2 Hz, 1H), 5.37 (d, J=6 Hz,1H), 5.19 (d, J=8 Hz,1H), 5.15-4.45 (m), 4.42 (t, J=4 Hz, 1H), 4.25 (m, 2H), 4.18-3.40 (m), 3.82 (s, 3H), 3.25 (m, 1H), 2.59 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.19 (d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(E-1-oxo-3-phenylprop-2-ene)amino-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl!-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 24)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.42 (d, J=11 Hz, 1H), 7.39-7.15 (m, 20H), 5.94 (d, J=11 Hz, 1H), 5.85 (d, J=4 Hz, 1H), 5.55-5.29 (m, 4H), 5.17-4.42 (m), 4.25 (m, 2H), 4.17-3.40 (m), 3.79 (s, 3H), 3.05 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.19 (d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-2'-napthamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 25)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 8.13 (s, 1H), 7.84 (d, J=7 Hz, 1H), 7.78 (d, J=7 Hz, 1H), 7.57 (m, 2H), 7.37-7.11 (m, 16H), 6.98 (d, J=7 Hz, 1H), 6.65 (d, J=5 Hz, 1H), 5.57-5.35 (m, 2H), 5.22 (d, J=7 Hz, 1H), 5.15-5.01 (m, 3H), 4.97-4.45 (m), 4.25 (m, 2H), 4.19-3.42 (m), 3.81 (s, 3H), 3.23 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.19 (d, J=5 Hz, 3H), 1.05 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-fluorobenzamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 26)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.83 (m, 2H), 7.17 (m, 2H), 5.45 (m,1H), 6.40 (m, 2H), 5.23 (d, J=5 Hz, 1H), 5.17-4.75 (m,3H), 4.77-4.45 (m, 4H), 4.36 (m, 2H), 4.19-3.41 (m), 3.81 (s, 3H), 3.09 (bs, 1H), 2.62 (m, 1H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.24(d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-aminobenzamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 27)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.61 (d, J=8 Hz, 2H), 2H), 6.75 (d, J=5 Hz, 1H), 6.57 (d, J=8 Hz, 2H), 5.57 (m, 1H), 5.43 (dd, J=6 Hz, J=2 Hz, 1H), 5.27 (d, J=2 Hz,1H), 5.19 (d, J=8 Hz,1H), 5.09 (m, 1H), 4.95 (m, 2H), 4.77-4.63 (m), 4.55 (dd, J=7 Hz, J=1 Hz, 1H), 4.42 (t, J=4 Hz, 1H), 4.35 (m, 2H), 4.21-3.38 (m), 3.82 (s, 3H), 3.17 (m, 1H), 2.95 (bs, 1H), 2.59 (dd, J=3 Hz, J=10 Hz, 1H), 2.42 (bs, 1H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.22 (d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Ethyl �methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(3'-phenyl)-propionamido-2-deoxy-3,6-di-O-acetyl-.beta.-D-glucopyranosyl!-(1,3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (Compound 28)
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to CHCl.sub.3) 7.29(m, 5H), 6.39 (d, J=2 Hz, 1H), 5.85 (d, J=4 Hz, 1H), 5.55-5.19 (m, 5H), 5.11 (t, J=5 Hz,1H), 4.95 (m, 4H), 4.71-4.35 (m), 4.17-3.22 (m), 3.79 (s, 3H), 2.95 (t, J=3H, 2H), 2.57 (dd, J=3 Hz, J=10 Hz, 1H), 2.47 (t, J=3 Hz, 2H), 2.27-1.85 (12s, 36H), 1.77 (dd, J=10 Hz, J=10 Hz, 1H), 1.24 (d, J=5 Hz, 3H), 1.15 (t, J=5 Hz, 3H).
Data for Compounds 30-38
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-fluorobenzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 30)
R.sub.f =0.62 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 41 mg, 96 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.83 (m, 2H, aromatic), 7.25 (m, 2H, aromatic), 5.18 (d, J=5 Hz, 1H), 4.95 (m), 4.56 (d, J=8 Hz, 1H), 4.37 (d, J=8 Hz, 1H), 4.19 (d, J=3.5 Hz, 1H), 4.15-3.42 (m), 2.77 (dd, J=3 Hz, J=10 Hz, 1H), 2.05 (s, 3H), 1.79 (dd, J=10 Hz, J=10 Hz, 1H), 1.19 (m, 3H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-aminobenzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 31)
R.sub.f =0.52 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 26 mg, 96 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.65 (d, J=9 Hz, 2H, aromatic), 6.82 (d, J=9 Hz, 2H), 5.19 (d, J=3 Hz, 1H), 4.95 (m), 4.59 (d, J=8 Hz, 1H), 4.38 (d, J=8 Hz, 1H), 4.19 (d, J=2 Hz, 1H), 4.15-3.42 (m), 3.19 (q, J=6 Hz, 2H), 2.79 (dd, J=3, J=11 Hz, 1H), 2.05 (s, 3H), 1.77 (dd, J=10, J=10 Hz, 1H), 1.19 (d, J=6 Hz, 3H), 1.17 (t, J=6 Hz, 3H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�(2-(3'-phenyl)-propionamido-2-deoxy-.beta.-D-glucopyranosyl!-(1,3)-O-.beta.-D-galactopyranoside (Compound 32)
R.sub.f =0.62 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 47 mg, 98 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.42-7.25(m, 5H), 5.19 (d, J=4 Hz, 1H), 4.95 (m), 4.57 (d, J=8 Hz, 1H), 4.38 (d, J=8 Hz, 1H), 4.13 (d, J=2 Hz, 1H), 4.11-3.42 (m), 2.95 (t, J=5 Hz, 2H), 2.75 (dd, J=3, J=10 Hz, 1H), 2.63 (t, J=5 Hz, 2H), 2.05 (s, 3H), 1.80 (dd, J=10, J=10 Hz, 1H), 1.24 (t, J=5 Hz, 3H), 1.18 (d, J=5 Hz, 3H).
Ethyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2,2'-napthamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 33)
R.sub.f =0.52 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 35 mg, 96 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 8.39 (s, aromatic,1H), 8.02 (m, aromatic, 2H), 7.82 (d, J=7 Hz, aromatic,1H), 7.63 (m, aromatic, 3H),5.19 (d, J=4 Hz, 1H), 4.95 (m), 4.57 (d, J=8 Hz, 1H), 4.35 (d, J=8 Hz, 1H), 4.19 (d, J=2 Hz, 1H), 4.15-3.42 (m), 2.77 (dd, J=3 Hz, J=11 Hz, 1H), 2.05 (s, 3H), 1.77 (dd, J=10, J=10 Hz, 1H), 1.19 (d, J=6 Hz, 3H), 1.05 (t, J=6 Hz, 3H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)!-(1,3)-O-(2,2'-phenylacetamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 34)
R.sub.f =0.62 (4.5:1 i-PrOH:NH.sub.4 OAc), white solid, 24 mg, 68 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.45-7.27(m, 5H), 4.85 (d, J=3 Hz, 1H), 4.75 (m), 4.55 (d, J=8 Hz, 1H), 4.38 (d, J=8 Hz, 1H), 4.13 (d, J=2 Hz, 1H), 4.09-3.42 (m), 2.78 (dd, J=3, J=10 Hz, 1H), 2.05 (2s, 5H), 1.80 (dd, J=10, J=10 Hz, 1H), 1.24 (t, J=5 Hz, 3H), 1.18 (d, J=5 Hz, 3H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-methoxybenzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 35)
R.sub.f =0.52 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 46 mg, 90 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.75 (d, J=9 Hz, 2H, aromatic), 7.05 (d, J=9 Hz, 2H), 5.11 (d, J=3 Hz, 1H), 4.95 (m), 4.52 (d, J=8 Hz, 1H), 4.25 (d, J=8 Hz, 1H), 4.19 (d, J=2 Hz, 1H), 4.15-3.39 (m), 3.82 (s, 3H), 2.75 (dd, J=3, J=11 Hz, 1H), 1.99 (s, 3H), 1.77 (dd, J=10, J=10 Hz, 1H), 1.17 (m, 5H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-p-tert-butylbenzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 36)
R.sub.f =0.52 (3:1 i-PrOH:NH.sub.4 OAc), white solid, 46 mg, 90 percent.
.sup.1 H-NMR (300 MHz, .delta. in ppm relative to H.sub.2 O) 7.65 (d, J=9 Hz, 2H, aromatic), 7.58 (d, J=9 Hz, 2H), 5.19 (d, J=4 Hz, 1H), 4.95 (m), 4.57 (d, J=8 Hz, 1H), 4.38 (d, J=8 Hz, 1H), 4.19 (d, J=2 Hz, 1H), 4.15-3.39 (m), 2.73 (dd, J=3, 11 Hz, 1H), 2.05 (s, 3H), 1.77 (dd, J=10, 10 Hz, 1H), 1.24 (s, 9H), 1.17 (m, 5H).
(5-Acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-benzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 37)
.sup.1 H NMR, (300 MHz, .delta. in ppm relative to H.sub.2 O) 1.15 (3H, d, J=6.5 Hz), 1.81 (1H, t, J=10.4 Hz), 2.02 (3H, s), 2.78 (1H, dd, J=10.4, 3.2 Hz), 3.5-4.2 (m), 4.4-4.8 (m), 5.09, 5.16 (d, d, 1H, .alpha.,.beta.), 5.2 (d, J=3.4 Hz, H-1 .alpha.), 7.5-7.8 (5H, Aromatic).
Benzyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�.alpha.-L-fucopyranosyl-(1,3)!-O-(2-benzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 38)
.sup.1 H NMR, (300 MHz, .delta. in ppm relative to H.sub.2 O) 1.08 (3H, d, J=6.4 Hz), 1.76 (1H, t, J=10.4 Hz), 1.97 (3H, s), 2.7 (1H, dd, J=10.4, 3.2 Hz), 3.4-4.2 (m), 4.5 (1H, d, J=7.7 Hz), 4.6 (1H, d, J=8.0 Hz), 5.02 (1H, d, J=3.8 Hz), 7.1-7.8 (10H, Aromatic).
NMR Data of Compounds 44-49
Ethyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(3,4-dichlorobenzamido)-2-deoxy-.beta.-D-glucopyranosyl!-(1,3)-O-.beta.-D-galactopyranoside (Compound 44)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.82 (s, 1H), 7.56 (m, 2H), 5.99 (d, J=3.96 Hz, 1H), 4.47 (d, J=7.59 Hz, 1H), 4.25 (d, J=7.91 Hz, 1H), 4.15-3.22 (m), 2.66 (dd, J=12.54 Hz, J=3.96 Hz, 1H), 1.94 (s, 3H), 1.76 (t, J=12.54 Hz, 1H), 1.10 (m, 6H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)!-(1,3)-O-(2-furanamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 45)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.59 (d, J=1.98 Hz, 1H), 7.10 (d, J=3.63 Hz, 1H), 6.54 (dd, J=3.36 Hz, J=1.98 Hz, 1H), 5.05 (d, J=4.29 Hz, 1H), 4.46 (d, J=7.59 Hz, 1H), 4.23 (d, J=7.92 Hz, 1H), 4.06 (d, J=2.97 Hz, 1H), 4.02-3.32 (m), 2.68 (dd, J=12.87 Hz, J=3.96 Hz, 1H), 1.95 (s, 3H), 1.77 (t, J=12.87 Hz, 1H), 1.08 (m, 6H).
Ethyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-(2-thiophenamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 46)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.63 (m, 2H), 7.10 (m, 1H), 5.06 (d, J=3.63 Hz, 1H), 4.46 (d, J=7.92 Hz, 1H), 4.23 (d, J=7.92 Hz, 1H), 4.06 (d, J=3.30 Hz, 1H), 4.04-3.30 (m), 2.67 (dd, J=12.21 Hz, J=3.96 Hz, 1H), 1.94 (s, 3H), 1.73 (t, J=12.21 Hz, 1H), 1.07 (m, 6H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)!-(1,3)-O-�2-(2-thiomethyl)nicotinamido-2-deoxy-.beta.-D-glucopyranosyl!-(1,3)-O-.beta.-D-galactopyranoside (Compound 48)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.62 (m, 2H), 7.06 (m, 1H), 5.04 (d, J=3.96 Hz, 1H), 4.43 (d, J=7.59 Hz, 1H), 4.23 (d, J=7.92 Hz, 1H), 4.10-3.20 (m), 2.68 (m, 1H), 2.14 (s, 3H), 2.09 (s, 3H), 1.70 (m, 1H), 1.05 (m, 6H).
Ethyl (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)-(1,3)!-O-�2-(6-dodecyloxy-2-naphthamido)-2-deoxy-.beta.-D-glucopyranosyl!-(1,3)-O-.beta.-D-galactopyranoside (Compound 47)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to CH.sub.3 OH) 8.32 (s, 1H), 7.90-7.78 (m, 3H), 7.26-7.16 (m, 2H), 5.17-5.13 (m, 1H), 4.48-4.40 (m, 1H), 4.22-3.32 (m), 2.88-2.82 (m, 1H), 2.01 (s, 3H), 1.85-1.19 (m), 0.91-0.85 (m, 3H).
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)!-(1,3)-O-(2-m-butyloxybenzamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 49)
.sup.1 H-NMR (270 MHz, .delta. in ppm relative to H.sub.2 O) 7.39-7.22 (m, 3H), 7.13-7.09 (m, 1H), 5.03 (d, J=3.96 Hz, 1H), 4.46 (d, J=7.92 Hz, 1H), 4.23 (d, J=7.92 Hz, 1H), 4.07-3.34 (m), 2.68-2.64 (m, 1H), 1.93 (s, 3H), 1.74-1.62 (m, 3H), 1.30-1.44 (m, 2H), 1.07 (t, J=7.25 Hz, 3H), 1.06 (d, J=5.60 Hz, 3H), 0.84 (t, J=7.58 Hz, 3H).
Data for Compounds 50 and 51
Ethyl �(5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�(.alpha.-L-fucopyranosyl)!-(1,3)-O-(2-nicotinamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (Compound 50)
R.sub.f =0.22 (silica, iso-propanol/1M NH.sub.4 OAc); .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.1.13 (d, 3H, J=6.6 Hz), 1.15 (t, 3H, J=6.7 Hz), 1.78 (t, 1H, J=11.9 Hz), 2.01 (s, 3H), 2.74 (dd, 1H, J=4.4, 11.9 Hz), 3.41-4.33 (multiple peaks, 34H), 4.31 (d, 1H, J=8.1 Hz), 4.53 (d, 1H, J=8.0 Hz), 5.10 (d, 1H, J=3.7 Hz), 7.56 (m, 1H), 8.16 (dd, 1H, J=1.3, 8.1 Hz), 8.68 (m, 1H), 8.85 (s, 1H).
Ethyl �Sodium (5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)!-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�.alpha.-L-fucopyranosyl-(1,3)-O-!-(2-benzenesulfonamido-2-deoxy-.beta.-D-glucopyranoside)-.beta.-D-galactopyranoside (Compound 51)
R.sub.f =0.28 (silica, 20 percent 1 m NH.sub.4 OAc/iso-propanol). .sup.1 H NMR (300 MHz, D.sub.2 O, ppm relative to H.sub.2 O): .delta.7.92 (d, J=7.4 Hz, 2H), 7.69 (d, J=7.2 Hz, 1H), 7.60 (t, J=7.2 Hz, 2H), 5.47 (d, J=4.0 Hz, 1H), 4.62 (d, J=8.1 Hz, 1H), 4.51 (d, J=7.6 Hz, 1H), 4.24 (d, J=8.0 Hz, 1H), 4.07 (dd, J=3.1, 9.6 Hz, 1H), 3.99 (d, J=3.1 Hz, 1H), 3.96=3.46 (m, 29H), 2.75 (dd, J=4.6, 12.5 Hz, 1H), 2.68 (dd, J=8.1, 8.1 Hz, 1H), 2.02 (s, 3H), 1.78 (t, J=12.1 Hz, 1H), 1.20 (t, 3H), 1.16 (d, J=6.5 Hz, 3H).
Example 25
This example describes the synthesis of preferred compound 62 according to the reaction scheme depicted in FIG. 1.
Ethyl 3,4,6tri-O-acetyl-2-deoxy-2-allyloxycarbonylamino-.beta.-D-gluco-pyranoside (52)
To a mixture cooled at -35.degree. C. (CH.sub.3 CN/dry ice) of .beta.-1,3,4,6-tetra-O-acetyl-2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside (2.70 g, 6.26 mmol), ethanol (386 .mu.L, 6.58 mmol), 1.0 g of powdered 5 .ANG. molecular sieves and 125 mL of dry dichloromethane was added TMSOTf (1.21 mL, 6.26 mmol). After being stirred for 10 min, the mixture was warmed to 0.degree. C. and stirred for 1.5 h at which time TLC showed the reaction was complete. After quenching with sat'd NaHCO.sub.3 (aq), the organic layer was separated, washed with water, dried with Na.sub.2 SO.sub.4, filtered and concentrated. The residue was purified by flash chromatography on silica gel column to provide the pure compound as a white solid (2.5 g, 96%). .sup.1 H NMR (300 mHz, CDCl.sub.3): .delta.5.83-5.94 (m, 1H), 5.18-5.31 (m, 3H), 5.05 (t, 1H, J=9.6 Hz), 4.62 (br d, 1H, J=7.6 Hz), 4.56 (br d, 2H, J=4.8 Hz), 4.26 (dd, 1H, J=4.8, 12.2 Hz), 4.12 (dd, 1H, J=2.3, 12.2 Hz), 3.87-3.97 (m, 1H), 3.61-3.71 (m, 1H), 3.42-3.59 (m, 2H), 2.08 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.21 (t, 3H, J=7.1 Hz).
Ethyl 2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside (53)
To a solution of 2.4 g (5.76 mmol) of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside in 8 mL of MeOH was added 40 .mu.L of 25% NaOMe in MeOH solution. The resulting mixture was stirred at room temperature for 1 h, and an additional portion of NaOMe solution (40 .mu.L) was added. The mixture was stirred for another 1 h at which time TLC showed the starting material had been consumed. Neutralization using cation-exchange resin (AG 50WX8) followed by filtration and concentration afforded the desired product as a white solid in quantitative yield. .sup.1 H NMR (300 mHz, CD.sub.3 OD): .delta.5.85-5.96 (m, 1H), 5.29 (br d, 1H, J=16.5 Hz), 5.14 (dd, 1H, J=1.3, 10.3 Hz), 4.51 (br s, 2H), 4.33 (d, 1H, J=7.6 Hz), 3.82-3.92 (m, 2H), 3.64 (dd, 1H, J=5.3, 11.8 Hz), 3.50-3.56 (m, 1H), 1.15 (t, 3H, J=6.9 Hz).
Ethyl 4-(.beta.-D-galactopyranosyl)-2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside (54)
Galactosyltransferase (240 u, 10 u/mL) and uridine 5'-diphosphogalactose 4'-epimerase (330 u, 10 u/.mu.L) were added to a solution containing sodium cacodylate (pH 7.5, 1M, 5.5 mL), water (45 mL), MnCl.sub.2 (1M, 1.4 mL), alkaline phosphatase (1000 u, 1 u/.mu.L), uridine diphosphoglucose disodium salt (3.86 g, 6.81 mmol), and ethyl 2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside (1.32 g, 4.54 mmol). The reaction mixture was inverted several times and then allowed to sit at room temperature. After 2 days, the pH value was adjusted to 7.4 by adding 1M NaOH (aq), and then additional galactosyltransferase (50 u, 10 u/mL) and uridine 5'-diphosphogalactose 4'-epimerase (50 u, 10 u/.mu.L) were added. The mixture was allowed to stand for another 2 days. Filtration over celite followed by concentration and purification by chromatography on reverse phase silica (Biosil C-18) using 5% to 100% MeOH in H.sub.2 O provided 1.98 g of disaccharide in 97% yield as a white solid. .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.5.85-5.97 (m, 1H), 5.29 (br d, 1H, J=17.1 Hz), 5.14 (br d, 1H, J=10.3 Hz), 4.51 (br s, 2H), 3.37-4.36 (m, 16H), 1.15 (t, 3H, J=6.8 Hz).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-3,6-di-O-acetyl-2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranoside (56)
The pH value of a solution containing 1.96 g (4.33 mmol) of disaccharide 54, 3.85 g (7.28 mmol) of cytidine triphosphate, 2.25 g (7.28 mmol) of sialic acid, 55 mL of water, 4.4 mL of 1M MnCl.sub.2, 440 mg of MgCl.sub.2.6H.sub.2 O and 30 mL of sodium cacodylate was adjusted to 7.5 with 1N NaOH, and then N-type .alpha.(2-3)-sialyl transferase (220 u), CMP-NeuAc synthetase (450 u) and inorganic pyrophosphatase (300 u) were added. The resulting mixture was allowed to stand for 2 days (the pH value was readjusted to 7.5 every day). The mixture was filtered over celite with washing with water. Concentration and drying overnight in vacuo gave a syrup which was treated with 150 mL of dry pyridine and 110 mL of Ac.sub.2 O at 0.degree. C., and then at room temperature for 2 days at which time TLC indicated the acetylation was complete.
Concentration of this mixture provided a syrup which was dissolved in 50 mL of pyridine and 120 mL of MeOH. After being stirred at room temperature for 1 day, the mixture was concentrated and ice-water/EtOAc were added. The organic layer was separated, washed with sat. NaHCO.sub.3 (aq) and brine. Drying and concentration gave a residue which was treated with 100 mL of pyridine and 10 mL of Ac.sub.2 O. The mixture was stirred at room temperature for 1 day, then poured into cold sat'd NaHCO.sub.3 and extracted with EtOAc. After washing with water once, the organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column using MeOH/CH.sub.2 Cl.sub.2 /EtOAc (1:8:8 to 1:4:4) to give 4.4 g of peracetylated trisaccharide 56 as a white solid in 98% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.84-5.93 (m, 1H), 5.51-5.55 (m, 1H), 5.39 (dd, 1H, J=2.6, 9.2 Hz), 5.27 (br d, 1H, J=17.5 Hz), 5.15-5.20 (m, 2H), 5.09 (t, 1H, J=9.6 Hz), 4.92 (dd, 1H, J=8.0, 10.2 Hz), 4.87 (d, 1H, J=3.2 Hz), 4.82 (d, 1H, J=9.6 Hz), 4.67 (d, 1H, J=8.0 Hz), 3.83 (s, 3H), 3.52-4.56 (m 15H), 2.58 (dd, 1H, J=4.4, 12.6 Hz), 2.24, 2.15, 2.10, 2.08, 2.07, 2.05, 2.00, 1.85 (8s, 30H), 1.19(t, 3H, J=7.0 Hz). MS(FAB.sup.+) Calcd for C.sub.48 H.sub.68 N.sub.2 O.sub.29 1052; found 1075(M+Na.sup.+), 1185(M+Cs.sup.+).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-3,6-di-O-acetyl-2-deoxy-2-amino-.beta.-D-glucopyranoside (57)
To a solution of trisaccharide 56 (2.0 g, 1.76 mmol), polymethyl hydrosiloxane (235 .mu.L) and 20 mL of dry THF was added Pd(PPh.sub.3).sub.4 (102 mg). The resulting mixture was stirred under argon at room temperature for 10 h. After dilution with EtOAc, the mixture was washed with ice-water and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel using MeOH/CH.sub.2 Cl.sub.2 /EtOAc (1:8:8 to 1:4:4) as the eluant provided the amine as a white solid (1.4 g, 76%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.50 (m, 1H), 5.40 (dd, 1H, J=2.3 Hz, 9.3 Hz), 5.07 (d, 1H, J=10.1 Hz), 4.85-4.98 (m, 3H), 4.63 (d, 1H, J=7.9 Hz), 4.51 (dd, 1H, J=3.2, 10.2 Hz), 3.84 (s, 3H), 3.56-4.44 (m 14H), 2.77 (dd, 1H, J=8.1, 10.1 Hz), 2.56 (dd, 1H, J=4.5, 12.6 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.10 (s, 3H), 2.09 (s, 9H), 2.06 (s, 6H), 2.00 (s, 3H), 1.85 (s, 3H), 1.67 (dd, 1H, J=12.5, 12.5 Hz), 1.24 (t, J=7.0 Hz, 3H).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)-.beta.-D-glucopyranoside (58)
A mixture of 1.35 g (1.28 mmol) of trisaccharide 57, 73 .mu.L (1.28 mmol) of HOAc, 6.5 mL of H.sub.2 O and 20 mL of MeOH was heated at 50.degree. C. for 1.5 days. After being cooled to room temperature, the mixture was concentrated and dried in vacuo overnight to give a crude deacetylated product which was used for the next step without further purification.
The residue was dissolved in 50 mL of CH.sub.2 Cl.sub.2, and 430 mg (5.12 mmol) of NaHCO.sub.3 and 475 mg (2.56 mmol) of 4-nitrobenzoyl chloride were added at 0.degree. C. The suspension was stirred at 0.degree. C. for 10 min, and at room temperature for 2 h. After additional NaHCO.sub.3 (215 mg) and 4-nitrobenzoyl chloride (238 mg) were added, the mixture was continued to be stirred for another 5 h. After dilution with EtOAc and washing with ice-water and brine, the organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel column using EtOAc/CH.sub.2 Cl.sub.2 /MeOH (8:8:1 to 4:4:1) provided 1.32 g of the desired product as a white solid in 89% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.8.29 (d, 2H, J=8.6 Hz), 7.93 (d, 2H, J=8.6 Hz), 6.31 (br d, 1H, J=6.8 Hz), 5.55 (m, 1H), 5.40 (dd, 1H, J=6.8 Hz), 5.55 (m, 1H), 5.40 (dd, 1H, J=2.5, 9.1 Hz), 5.09 (d, 1H, J=10.0 Hz), 5.04 (dd, 1H, J=8.0, 10.2 Hz), 4.98 (m, 1H), 4.90 (d, 1H, J=3.5 Hz), 4.68 (d, 1H, J=8.0 Hz), 4.59 (dd, 1H, J=3.4, 10.1 Hz), 3.84 (s, 3H), 3.52-4.42 (m, 14H), 2.58 (dd, 1H, J=4.7, 12.8 Hz), 2.17 (s, 3H), 2.11 (s, 3H), 2.10 (s, 6H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.86 (s, 3H), 1.64 (dd, 1H, J=12.8, 12.8 Hz), 1.27 (t, 3H, J=7.2 Hz). MS(LSIMS.sup.+) Calcd for C.sub.49 H.sub.65 N.sub.3 O.sub.29 1159; found 1292(M+Cs.sup.+).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galacto-pyranosyl)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)-.beta.-D-gluco-pyranoside (59)
A mixture of 464 mg (0.4 mmol) of glycosyl acceptor 58, 84 mg of tetraethylammonium bromide, 0.26 mL of DMF, 1.5 mL of dry CH.sub.2 Cl.sub.2 and 0.8 g of activated 4 .ANG. molecular sieves was stirred under argon at room temperature for 6 h. A solution of freshly prepared 2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl bromide (approx. 2 mmol) in 1 mL of CH.sub.2 CH.sub.2 was added. Stirring was then continued at room temperature for 3 days. The reaction mixture was filtered through celite and the filtrate was concentrated. The residue was chromatographed on silica gel (EtOAc/hexane/MeOH, 10:8:3) and afforded 400 mg (64%) of 59 as a white solid. .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.8.12 (d, 2H, J=8.7 Hz), 7.70 (d, 2H, J=8.7 Hz), 7.28 (m, 15H), 6.83 (d, 1H, J=7.7 Hz), 5.52-5.56 (m, 1H), 5.42 (dd, 1H, J=2.5, 9.4 Hz), 5.16 (d, 1H, J=3.4 Hz), 5.08 (d, 1H, J=10.2 Hz), 3.84 (s, 3H), 3.47-4.97 (m, 31H), 2.58 (dd, 1H, J=4.6, 12.6 Hz), 2.24 (s, 3H), 2.16 (s, 3H), 2.08 (s, 6H), 2.06 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.86 (s, 3H), 1.70 (dd, 1H, J=12.6, 12.6 Hz), 1.17 (d, 3H, J=6.4 Hz), 1.08 (t, 3H, J=7.0 Hz). MS(FAB.sup.+) Calcd for C.sub.76 H.sub.93 N.sub.3 O.sub.33 1575; found 1708 (M+Cs.sup.+).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-(4-nitrobenzamido)-.beta.-D-glucopyranoside (60)
A mixture of 370 mg of tetrasaccharide 59, 400 .mu.L of 25% methanolic solution of NaOMe and 4 mL of MeOH was stirred at room temperature for 24 h. After addition of 0.5 mL of H.sub.2 O, stirring was continued for another 24 h. Two drops of HOAc was added to adjust the pH to 8.5. The mixture was concentrated and purified by flash chromatography on reverse phase silica (Biosil C-18; 75% MeOH in H.sub.2 O) to afford 250 mg of 60 in 85% yield. .sup.1 H NMR (300 MHz, CD.sub.3 OD) .delta.8.28 (d, 2H, J=8.7 Hz), 8.00 (d, 2H, J=8.7 Hz), 7.04-7.33 (m, 15H), 5.33 (d, 1H, J=3.6 Hz), 3.46-4.87 (m, 33H), 2.85 (br dd, 1H, J=3, 10 Hz), 2.00 (s, 3H), 1.75 (dd, 1H, J=10, 10 Hz), 1.15 (d, 3H, J=6.3 Hz), 1.07 (t, 3H, J=7.1 Hz). MS (negative ion spray) Calcd for C.sub.59 H.sub.74 N.sub.3 O.sub.25 Na 1247; found 1224 (M-Na.sup.+).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-(4-amino-benzamido)-.beta.-D-glucopyranoside (61)
Palladium hydroxide on carbon (70 mg, 20%) was added to a solution of tetrasaccharide 60 (200 mg, 0.127 mmol) in 16 mL of H.sub.2 O. The mixture was purged three times with hydrogen gas and then stirred under an atmosphere of hydrogen for 14 h. The mixture was then degassed, filtered through celite, and concentrated. The residue was chromatographed on reverse phase silica (Biosil C-18; 5% MeOH in H.sub.2 O) to afford 130 mg of product as a white solid in 91% yield. .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.57 (d, 2H, J=8.8 Hz), 6.80 (d, 2H, J=8.8 Hz), 5.06 (d, 1H, J=3.9 Hz), 4.6 (br d, 1H, J=8.1 Hz), 4.50 (d, 1H, J=7.8 Hz), 3.45-4.07 (m, 25H), 2.72 (dd, 1H, J=2.4, 12.3 Hz), 1.98 (s, 3H), 1.75 (dd, 1H, J=12.3, 12.3 Hz), 1.12 (d, 3H, J=6.5 Hz), 1.03 (t, 3H, J=7.0 Hz).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-(4-bromoacetamido-benzamido)-.beta.-D-glucopyranoside (62)
Bromoacetic anhydride (428 mg, 1.64 mmol) was added to a mixture of 130 mg (0.137 mmol) of tetrasaccharide 61, 9.5 mL of sat. NaHCO.sub.3 (aq), 6 mL of EtOH and 6 mL of H.sub.2 O at 0.degree. C. After being stirred for 10 min, the mixture was warmed to room temperature and stirring was continued for 2 h. Additional bromoacetic anhydride (214 mg) and NaHCO.sub.3 (120 mg) were added, and stirring was continued for another 2 h. Concentration followed by column chromatography on reverse phase silica (Biosil C-18; 5% MeOH in H.sub.2 O) afforded 130 mg of the desired product as a white solid after lyophilization (89% yield). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.75 (d, 2H, J=8.6 Hz), 7.58 (d, 2H, J=8.6 Hz), 5.08 (d, 1H, J=3.9 Hz), 4.51 (d, 1H, J=7.9 Hz), 4.05 (s, 2H), 3.46-4.01 (m, 25H), 2.72 (dd, 1H, J=4.8, 12.8 Hz), 1.99 (s, 3H), 1.75 (dd, 1H, J=12.8, 12.8 Hz), 1.11 (d, 3H, J=6.8 Hz), 1.05 (t, 3H, J=7.1 Hz). MS(negative ion spray) Calcd for C.sub.40 H.sub.59 BrN.sub.3 O.sub.24 Na 1067/1069; found 1066/1068 (M-H.sup.+), 1044/1046 (M-Na.sup.+).
Example 26
This example describes the synthesis of preferred compound 73 according to the reaction scheme depicted in FIG. 2.
3'-azidopropyl 3,4,6-tri-O-acetyl-O-2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranoside (63)
To a mixture cooled at -35.degree. C. (CH.sub.3 CN/dry ice) of 3,4,6-tri-O-acetyl-2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranosyl acetate (2.20 g, 5.1 mmol), 3-bromopropanol (485 .mu.L, mmol), 1.0 g of powdered 5 .ANG. molecular sieves and 100 mL of dry dichloromethane was added TMSOTf (1.0 mL, 5.2 mmol). After being stirred for 10 min, the mixture was warmed to 0.degree. C. and stirred for 2 h at which time TLC showed the reaction was complete. After quenching with sat. NaHCO.sub.3 (aq), the separated organic layer was washed with water, dried with Na.sub.2 SO.sub.4 and filtered. Concentration and drying in vacuo gave a crude glycoside which was used for the next step without purification.
The crude product was dissolved in 15 mL of dry DMF, 500 mg (7.69 mmol) of sodium azide and 821 mg (2.55 mmol) of tetrabutylammonium bromide were added. The resulting mixture was heated at 100.degree. C. for 10 min. After being cooled to room temperature, the mixture was poured into ice-water, and extracted with CHCl.sub.3. The organic layer was washed with brine, dried over Na.sub.2 SO.sub.4 and filtered. Concentration followed by chromatography (silica gel, EtOAc/hexane: 1:1-2:1) afforded the desired product (2.22 g, 92%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.85-5.95 (m, 1H), 5.16-5.33 (m, 3H), 5.06 (t, 1H, J=9.6 Hz), 4.82 (br s, 1H), 4.56 (br d, 3H, J=5.0 Hz), 4.27 (dd, 1H, J=4.8, 12.2 Hz), 4.13 (dd, 1H, J=1.5, 12.2 Hz), 3.93-4.00 (m, 1H), 3.54-3.71 (m, 3H), 3.37 (t, 2H, J=6.5 Hz), 2.09, 2.04, 2.02 (3s, 9H), 1.82-1.88 (m, 2H).
3'-Azidopropyl 2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranoside (64)
The title compound was prepared in a manner similar to compound 53. Yield 1.5 g of 64 as a white solid (100%). .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.5.86-5.97 (m, 1H), 5.30 (br d, 1H, J=17.0 Hz), 5.15 (dd, 1H, J=1.2, 17.0 Hz), 4.52 (br d, 2H), 4.32 (d, 1H, J=7.8 Hz), 3.90-3.97 (m, 1H), 3.85 (dd, 1H, J=2.0, 12.0 Hz), 3.65 (dd, 1H, J=5.5, 12.0 Hz), 3.50-3.57 (m, 1H), 3.20-3.38 (m, 6H), 1.74-1.84 (m, 2H).
3'-Azidopropyl 4(.beta.-D-galactopyranosyl)-2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranoside (65)
The title compound was prepared in a manner similar to compound 54. Yield 2.0 g of 65 as a white solid (100%). .sup.1 H NMR (300 MHz, CD3OD): .delta.5.82-5.92 (m, 1H), 5.24 (br d, 1H, J=17.4 Hz), 5.10 (br d, 1H, J=10.3 Hz), 4.46 (br s, 2H), 4.27-4.32 (m, 2H), 3.23-3.91 (m, 16H), 1.69-1.77 (m, 2H).
3'-azidopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranyl)-(1-4)-O-2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranoside (66)
The pH value of a solution containing 1.95 g (3.84 mmol) of disaccharide 65, 3.0 g (5.76 mmol) of cytidine triphosphate, 1.78 g (5.76 mmol) of sialic acid, 25 mL of water, 4.4 mL of 1M MnCl.sub.2, 440 mg of MgCl.sub.2.6H.sub.2 O and 33 mL of sodium cacodylate was adjusted to 7.5 with 1N NaOH, and then N-type .alpha.(2-3)-sialyl transferase (200 u), CMP-NeuAc synthetase (400 u) and inorganic pyrophosphatase (300 u) were added. The resulting mixture was allowed to stand for 36 h (the pH value was readjusted to 7.5 after 12 h). The mixture was filtered over celite. The filtrate was concentrated, and the residue was chromatographed on reverse phase silica gel (Biosil C-18; using 5%-10% MeOH in H.sub.2 O) to afford trisaccharide 66 as a white solid (2.8 g, 89%). .sup.1 H NMR(300 MHz, D.sub.2 O): .delta.5.87-5.93 (m, 1H), 5.30 (br d, 1H, J=16.6 Hz), 5.26 (br d, 1H, J=2.3, 9.8 Hz), 3.29-4.59 (m, 25H), 2.70 (dd, 1H, J=4.5, 12.4 Hz), 1.98 (s, 3H), 1.79-1.90 (m, 2H), 1.75 (dd, 1H, J=12.4, 12.4 Hz).
3'-Azidopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyrany)-(1-4)-O-3,6-di-O-acetyl-2-deoxy-2-allyloxycarbonylamino-.beta.-D-glucopyranoside (67)
A mixture of trisaccharide 66 (1.18 g, 1.44 mmol), 100 mg of DMAP, 5.0 mL of Ac.sub.2 O and 50 mL of pyridine was stirred at room temperature overnight. After being cooled to 0.degree. C., 10 mL of MeOH was added. The resulting mixture continued to be stirred at room temperature until TLC showed near complete conversion of the intermediate lactone to a slower-moving spot which is the methyl ester. Concentration of this mixture and drying in vacuo gave a residue which was treated with 50 mL of pyridine and 8 mL of Ac.sub.2 O for 48 h. After dilution with EtOAc, the mixture washed with cold water, sat. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel (acetone:dichloromethane, 1:1) afforded 1.63 g of product 67 in 95% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.80-5.90 (m, 1H), 5.51-5.51 (m, 1H), 5.39 (dd, 1H, J=2.6, 9.2 Hz), 5.28 (br d, 1H, J=16.6 Hz), 5.20 (br d, J=10.3 Hz), 4.79-5.10 (m, 6H), 4.68 (d, 1H, J=7.9 Hz), 3.84 (s, 3H), 3.52-4.56 (m, 14H), 3.36 (t, 2H, J=6.6 Hz), 2.58 (dd, 1H, J=4.6, 12.5 Hz), 2.24 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H), 2.08 (s, 6H), 2.07 (s, 6H), 2.06 (s, 3H), 2.01 (s, 3H), 1.85 (s, 3H), 1.79-1.89 (m, 2H), 1.64 (dd, 1H, J=12.5, 12.5 Hz). MS(LSIMS.sup.+) Calcd for C.sub.49 H.sub.69 N.sub.5 O.sub.29 1191; found 1192 (M+H.sup.+) and 1214 (M+Na.sup.+).
3'-Azidopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-3,6-di-O-acetyl-2-deoxy-2-amino-.beta.-D-glucopyranoside (68)
The title compound was prepared in a manner similar to compound 57. Yield 0.99 g of 68 as a white solid (86%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.47-5.51 (m, 1H), 5.38 (dd, 1H, J=2.7, 9.3 Hz), 5.16 (d, 1H, J=10.2 Hz), 4.83-4.98 (m, 3H), 4.62 (d, 1H, J=7.9 Hz), 3.82 (s, 3H), 3.55-4.52 (m, 15H), 3.39 (t, 2H, J=6.7 Hz), 2.78 (dd, 1H, J=8.3, 10.0 Hz), 2.56 (dd, 1H, J=4.6, 12.6 Hz), 2.21, 2.14, 2.10, 2.07, 2.06, 2.05, 1.99, 1.84 (8s, 30H), 1.66 (dd, 1H, J=12.6, 12.6 Hz).
3'-Azidopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyrany)-(1-4)-O-6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranoside (69)
To a solution of 0.95 g (0.858 mmol) of 68 in 25 mL of THF was introduced 74 .mu.L (1.29 mmol) of acetic acid and 144 .mu.L (1.46 mmol) of piperidine. The mixture was stirred at room temperature for 8 h, then diluted with EtOAc and washed with ice-water once. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel (MeOH/EtOAc/hexane, 3:10:8) provided 0.93 g of 69 as a white solid in 98% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.58 (d, 1H, J=8.2 Hz), 5.47-5.52 (m, 1H), 5.38 (dd, 1H, J=2.4, 9.0 Hz), 5.20 (d, 1H, J=10.2 Hz), 5.02 (dd, 1H, J=8.1, 10.0 Hz), 4.89 (m, 2H), 4.64 (d, 2H, J=7.7 Hz), 5.58 (dd, 1H, J=3.2, 10.0 Hz), 3.50-4.41 (m, 15H), 3.83 (s, 3H), 3.36 (t, 2H, J=6.1 Hz), 2.56 (dd, 1H, J=4.5, 12.6 Hz), 2.26 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H), 2.08 (s, 6H), 2.06 (s, 6H), 1.99 (s, 6H), 1.84 (s, 3H), 1.67 (dd, 1H, J=12.6 Hz). MS(positive ion spray) Calcd for C.sub.45 H.sub.65 N.sub.2 O.sub.27 1107; found 1108 (M+H.sup.+) and 1130 (M+Na.sup.+).
3'-Azidopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranoside (70)
The title compound was prepared in a manner similar to compound 59. Yield 540 mg of 70 as a white solid (88%). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.22-7.42 (m, 15H), 5.98 (d, 1H, J=8.1 Hz, NH), 5.50-5.54 (m, 1H), 5.42 (dd, 1H, J=2.5, 9.4 Hz), 5.08 (m, 2H), 3.84 (s, 3H), 3.44-4.98 (m, 31H), 3.26 (t, 2H, J=6.5 Hz), 2.57 (dd, 1H, J=4.6, 12.6 Hz), 2.21 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.01 (s, 6H), 1.92 (s, 3H), 1.86 (s, 3H), 1.68 (m, 3H), 1.16 (d, 3H, J=6.4 Hz).
3'-Azidopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-acetamido-.beta.-D-glucopyranoside (71)
The title compound was prepared in a manner similar to compound 60. Yield 220 mg of 71 as a white solid (96%). .sup.1 H NMR (300 MHz, CD.sub.3 OD) .delta.7.37 (br s, 5H), 7.33 (br s, 5H), 7.31 (br s, 5H), 5.38 (d, 1H, J=3.5 Hz), 4.40-4.74 (m, 8H), 3.44-4.04 (m, 25H), 3.31 (t, 2H, J=6.3 Hz), 2.73 (dd, 1H, J=4.5, 12.4 Hz), 1.99 (s, 3H), 1.97 (s, 3H), 1.72-1.80 (m, 3H), 1.09 (d, 1H, J=6.5 Hz).
3'-Aminopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-acetamido-.beta.-D-glucopyranoside (72)
The title compound was prepared in a manner similar to compound 61. Yield 160 mg of 72 as a white solid (90%). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.5.06 (d, 1H, J=3.8 Hz), 4.47 (br d, 2H, J=7.9 Hz), 3.45-4.05 (m, 25H), 3.02 (t, 2H, J=6.8 Hz), 2.72 (dd, 1H, J=4.0, 12.5 Hz), 1.98 (s, 6H), 1.86 (m, 2H), 1.74 (t, J=12.5 Hz), 1.12 (d, 3H, J=6.4 Hz), MS(negative ion spray) Calcd for C.sub.34 H.sub.58 N.sub.3 O.sub.23 Na 899; found 876 (M-Na.sup.+).
3'-(Bromoacetamido)propyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-acetamido-.beta.-D-glucopyranoside (73)
The title compound was prepared in a manner similar to compound 62. Yield 130 mg of 73 as a white solid after lyophilization (88%). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.5.06 (d, 1H, J=3.9 Hz), 4.48 (d, 2H, J=8.2 Hz), 4.04 (dd, 1H, J=3.1, 9.9 Hz), 3.85 (br s, 2H), 3.47-3.99 (m, 24H), 3.17-3.31 (m, 1H), 2.72 (dd, 1H, J=4.5, 12.5 Hz), 1.99 (s, 6H), 1.75 (m, 3H), 1.12 (d, 3H, J=6.5 Hz). MS(positive ion spray) Calcd for C.sub.36 H.sub.59 BrN.sub.3 O.sub.24 Na 1019/1021; found 1020/1022 (M+H.sup.+), 1042/1044 (M+Na.sup.+).
Example 27
This example describes the synthesis of preferred compound 78 according to the reaction scheme depicted in FIG. 3.
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-(3,6-di-O-acetyl-2-deoxy-2-amino-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (13)
The title compound was prepared in a manner similar to compound 57. Yield 6.2 g of 13 as a white solid (82%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.5.45-5.50 (m, 1H), 5.36-5.39 (m, 1H), 5.20 (d, 1H, J=10.2 Hz), 5.14 (dd, 1H, J=8.0, 10.0 Hz), 4.81-4.90 (m, 3H), 4.65 (d, 1H, J=8.0 Hz), 4.47-4.56 (m, 2H), 4.39 (d, 1H, J=7.9 Hz), 4.37 (dd, 1H, J=2.2, 10.0 Hz), 4.20 (d, 1H, J=7.8 Hz), 3.46-4.15 (m, 17H), 3.81 (s, 3H), 2.68 (dd, 1H, J=7.8, 10.2 Hz), 2.54 (dd, 1H, J=4.6, 12.6 Hz), 2.20 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 9H, 3Ac), 2.04 (s, 6H), 2.02 (s, 3H), 1.98 (s, 3H), 1.82 (s, 3H), 1.64 (dd, 1H, J=12.6, 12.6 Hz), 1.23 (t, 3H, J=7.1 Hz).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-(6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-.beta.-D-galactopyranoside (74)
The title compound was prepared in a manner analogous to compound 58 and used directly in the following step. MS(FAB) Calcd for C.sub.61 H.sub.81 N.sub.3 O.sub.37 1447; found 1580 (M+Cs.sup.+).
Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6O-acetyl-2-deoxy-2-(4-nitrobenzamido)-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-.beta.-D-galactopyranoside (75)
The title compound was prepared in a manner similar to compound 59. Yield 2.4 g of 75 as a white solid (67%). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.8.08 (d, 2H, J=8.6 Hz), 7.52 (d, 2H, J=8.6 Hz), 7.16-7.33 (m, 15H), 6.60 (d, 1H, J=6.8 Hz), 5.50-5.54 (m, 1H), 5.43 (dd, 1H, J=2.5, 8.4 Hz), 5.33 (d, 1H, J=7.9 Hz), 5.09 (d, 1H, J=10.2 Hz), 5.03 (dd, 1H, J=8.3, 10.2 Hz), 4.55 (dd, 1H, J=3.3, 10.0 Hz), 4.43 (br t, J=8.9 Hz), 4.61-4.94 and 3.30-4.40 (m, 35H), 3.84 (s, 3H), 2.55 (dd, 1H, J=4.4, 12.5 Hz), 2.23, 2.17, 2.12, 2.11, 2.08, 2.07, 2.06, 2.04, 2.03, 2.01, 1.86 and 1.81 (12s, 36H), 1.68 (dd, 1H, J=12.5, 12.5 Hz), 1.23 (t, 3H, J=7.1 Hz), 1.18 (d, 3H, J=6.4 Hz).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-�2-deoxy-2-(4-nitrobenzamido)-.beta.-D-glucopyranosyl!-(1-3)-O-.beta.-D-galactopyranoside (76)
The title compound was prepared in a manner similar to compound 60. Yield 1.4 g of 76 as a white solid (100%). .sup.1 H NMR (300 MHz, CD.sub.3 OD) .delta.8.26 (d, 2H, J=8.6 Hz), 8.04 (d, 2H, J=8.6 Hz), 7.04-7.33 (m, 15H), 5.33 (d, 1H, J=3.7 Hz), 4.30-4.88 (m, 9H), 3.25-4.21 (m, 31H), 2.87 (br d, J=4.3 Hz, 12.0 Hz), 1.99 (s, 3H), 1.72 (br t, 1H, J=12.0 Hz), 1.15 (m, 6H).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-�2-deoxy-2-(4-amino-benzamido)-.beta.-D-glucopyranosyl!-(1-3)-O-.beta.-D-galactopyranoside (77)
The title compound was prepared in a manner similar to compound 61. Yield 230 mg 77 as a white solid. .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.58 (d, 2H, J=8.3 Hz), 6.81 (d, 2H, J=8.3 Hz), 5.08 (d, 1H, J=3.5 Hz), 4.51 (d, 1H, J=7.7 Hz), 4.28 (d, 1H), J=8.0 Hz), 4.10 (d, 1H, J=2.6 Hz), 3.39-4.09 (m, 30H), 2.72 (dd, 1H, J=4.4, 12.5 Hz), 1.99 (s, 3H), 1.75 (dd, 1H, J=12.5, 12.5 Hz), 1.13 (m, 6H).
Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-�2-deoxy-2-(4-bromoacetamido-benzamido)-.beta.-D-glucopyranosyl!-(1-3)-O-.beta.-D-galactopyranoside (78)
The title compound was prepared in a manner similar to compound 62. Yield 220 mg of 78 as a white solid after lyophilization (84%, two steps). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.75 (d, 2H, J=8.6 Hz), 7.57 (d, 2H, J=8.6 Hz), 5.09 (d, 1H, J=3.6 Hz), 4.81 (d, 1H, J=7.5 Hz), 4.51 (d, 1H, J=8.0 Hz), 4.29 (d, 1H, J=7.9 Hz), 4.12 (d, 1H, J=3.0 Hz), 4.04 (s, 2H), 3.40-4.04 (m, 30H), 2.72 (dd, 1H, J=4.5, 12.6 Hz), 1.98 (s, 3H), 1.75 (dd, 1H, J=12.5, 12.5 Hz), 1.12 (m, 6H). MS(negative ion spray) Calcd for C.sub.46 H.sub.69 BrN.sub.3 O.sub.29 Na 1229/1231; found 1206/1208 (M-Na.sup.+).
Example 28
This example describes the synthesis of preferred compound 85 according to the reaction scheme depicted in FIG. 4.
(methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-acetyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-1,2,4,6-tetra-O-acetyl-.alpha./.beta.-D-galactopyranoside (80)
Palladium on carbon (1.0 g of 10%) was added to a solution of 1.0 g (0.55 mmol) of benzyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyrany)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fuco-pyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-D-galactopyranoside79 in 5 mL of EtOH. The mixture was purged three times with hydrogen gas and then stirred under an atmosphere of hydrogen for 3 days. After being degassed, the mixture was then filtered through celite with washing with MeOH. The filtrate was concentrated and dried in vacuo. The residue was dissolved in 10 mL of pyridine, and the solution was cooled to 0.degree. C. Three mL of Ac.sub.2 O was added and stirring was continued at 0.degree. C. for 30 min, and then at room temperature for 2 days at which time TLC showed acetylation was complete. The reaction mixture was diluted with EtOAc, and washed with ice-water, sat. NaHCO.sub.3 (aq) and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel using MeOH/hexane/EtOAc (3:8:10) as the eluant provided 0.80 g of 80 in 90% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.6.27 (d, 1H, J=3.8 Hz), 5.60 (d, 1H, J=8.2 Hz), 5.08 (d, 1H, J=10.3 Hz), 4.51 (dd, 1H, J=3.2, 10.0 Hz), 4.38 (dd, 1H, J=6.5, 11.5 Hz), 5.00-5.52, 4.74-5.16 and 3.82-4.27 (m, 29H), 3.85 (s, 3H), 3.62 (dd, 1H, J=2.6, 10.8 Hz), 3.50 (m, 1H), 3.25 (m, 1H), 2.58 (dd, 1H, J=4.5, 12.6 Hz), 1.68 (dd, 1H, J=12.6, 12.6 Hz), 1.17 (d, 3H, J=6.4 Hz).
(methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4tri-O-acetyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-D-galactopyranose (81)
To a solution of 690 mg of 80 in 15 mL of DMF was added 47 mg of hydrazine acetate. The resulting mixture was stirred at room temperature for 24 h at which time TLC indicated the starting material had been consumed. After dilution with EtOAc and washing with ice-water, the organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel using MeOH/EtOAc/hexane (3:8:10 to 1:2:2) as the eluant and provided 625 mg of the desired product 81 as a white solid in 93% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.4.52 (dd, 1H, J=2.9, 9.8 Hz), 4.67-5.52 and 3.85-4.40 (m, 311), 3.85 (s, 3H), 3.62 (dd, 1H, J=2.0, 10.8 Hz), 3.48 (m, 1H), 3.32 (m, 1H), 2.57 (dd, 1H, J=4.3, 12.4 Hz), 1.67 (dd, 1H, J=12.4, 12.4 Hz), 1.17 (d, 3H, J=6.3 Hz).
Trichloroacetimido (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4tri-O-acetyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.alpha.-D-galactopyranoside (82)
To a solution cooled at -40.degree. C. containing 500 mg of 81, 1.25 mL of trichloroacetonitrile and 12.5 mL of dichloromethane was added 30 mg of DBU. The resulting solution was stirred at -40.degree. C. for 1 h. Concentration followed by chromatography on silica gel (EtOAc/hexane/MeOH, 8:8:1 to 4:4:1) afforded the product 82 as a mixture of .alpha.-isomer (408 mg, 73%) and .beta.-isomer (100 mg, 18%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .alpha.-anomer .delta.8.63 (s, 1H), 6.50 (d, 1H, J=3.6 Hz), 5.18 (dd, 1H, J=2.6, 10.8 Hz), 5.07 (d, 1H, J=10.1 Hz), 5.27-5.53, 4.71-5.00 and 3.86-4.38 (m, 28H), 4.53 (dd, 1H, J=3.0, 10.2 Hz), 3.86 (s, 3H), 3.63 (dd, 1H, J=2.2, 11 Hz), 3.50-3.57 (m, 1H), 3.36 (m, 11H), 2.58 (dd, 1H, J=4.6, 12.4 Hz), 2.22 (s, 3H), 2.16 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.10 (s, 6H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 6H), 2.05 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H), 1.91 (s, 3H), 1.85 (s, 3H), 1.68 (dd, 1H, J=12.4, 12.4 Hz), 1.17 (d, 3H, J=6.4 Hz).
3'-Benzyloxycarbonylaminopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyrany)-(1-4)-O-�2,3,4-tri-O-acetyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6tri-O-acetyl-D-galactopyranoside (83)
A mixture of pentasaccharide imidate (82) (400 mg, 0.231 mmol), 3-benzyloxy-carbonylamino-propanol (97 mg, 0.462 mmol), activated powdered 4 .ANG. molecular sieves (5.5 g) and CH.sub.2 Cl.sub.2 (5.5 mL) was stirred at room temperature for 3 h, and then cooled to -20.degree. C. After BF.sub.3.Et.sub.2 O (108 .mu.L) was added, the resulting suspension was stirred at -20.degree. C. for 1 h and at room temperature for 3 h. After addition of 0.2 mL of Et.sub.3 N, the mixture was filtered through celite and washed with EtOAc. The filtrate was washed with sat. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. The residue was chromatographed on silica gel (MeOH/EtOAc/hexane: 1:8:8 to 3:10:10) to afford the desired product 83 as a white solid (250 mg, 62%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.7.34 (m, 5H), 5.09 (br s, 2H), 5.18 (dd, 1H, J=3.3, 10.8 Hz), 4.51 (dd, 1H, J=2.3, 9.9 Hz), 5.30-5.52, 4.70-5.09 and 3.71-4.40 (m, 32H), 3.85 (s, 3H), 3.62 (dd, 1H, J=2.0, 11.0 Hz), 3.45-3.57 (m, 2H), 3.18-3.31 (m, 3H), 2.57 (dd, 1H, J=4.5, 12.6 Hz), 2.20, 2.19, 2.15, 2.13, 2.11, 2.10, 2.09, 2.07, 2.06, 2.05, 2.04, 2.03, 1.99, 1.95, 1.91 and 1.84 (16s, 48H), 1.78 (m, 2H), 1.67 (dd, 1H, J=12.6, 12.6 Hz), 1.17 (d, 3H, J=6.4 Hz).
3'-Benzyloxycarbonylaminopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-D-galactopyranoside (84)
A mixture of 240 mg (0.135 mmol) of pentasaccharide 83, 380 .mu.L of 25% methanolic NaOMe solution and 6 mL of MeOH was stirred at room temperature for 16 h, and then 2 mL of water was added. Stirring was continued for one day. After the addition of one drop of HOAc, the reaction mixture was concentrated to provide a residue which was chromatographed on reverse phase silica (Biosil C-18; 5%. MeOH in H.sub.2 O) to afford 84 as a white solid (145 mg, 90%). .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.7.37 (br s, 5H), 5.06 (br s, 3H), 4.47 (d, 1H, J=7.8 Hz), 4.26 (d, 1H, J=7.8 Hz), 4.08 (d, 1H, J=2.7, 10.0 Hz), 4.03 (dd, 1H, J=2.8, 10.0 Hz), 3.44-3.95 (m, 29H), 3.18 (m, 2H), 2.71 (dd, 1H, J=4.6, 12.6 Hz), 1.98 (s, 3H), 1.95 (s, 3H), 1.74 (m, 3H), 1.12 (d, 3H). MS(ion spray) Calcd for C.sub.48 H.sub.74 N.sub.3 O.sub.30 Na 1195; found 1196 (M+H.sup.+) and 1218 (M+Na.sup.+).
3'-Bromoacetamidopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-D-galactopyranoside (85)
Palladium hydroxide on carbon (100 mg of 20%) was added to a solution of 140 mg (0.117 mmol) of 84 in 5 mL of EtOH and 1.5 mL of water. The mixture was purged three times with hydrogen gas and then stirred under an atmosphere of hydrogen for 3 h. The mixture was degassed and filtered through celite with washing with 50% MeOH in water. The filtrate was concentrated and dried in vacuo overnight to provide amine which was used for the next reaction.
The residue was dissolved in a mixture of 8 mL of sat. NaHCO.sub.3, 5 mL of water and 5 mL of EtOH, and 353 mg (1.36 mmol) of bromoacetic anhydride was then added. The resulting suspension was stirred at room temperature for 1 h. After additional bromoacetic anhydride (100 mg) and NaHCO.sub.3 (56 mg) were added, stirring was continued for another hour. The mixture was concentrated and purified on reverse phase silica (Biosil C-18; 0-5% MeOH in H.sub.2 O) to afford 85 as a white solid after lyophilization (130 mg, 94%). .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.5.07 (d, 1H, J=3.8 Hz), 4.66 (d, 1H, J=8.2 Hz), 4.47 (d, 1H, J=7.7 Hz), 4.31 (d, 1H, J=7.7 Hz), 4.09 (d, 1H, J=2.9 Hz), 4.03 (dd, 1H, J=2.9, 9.8 Hz), 3.44-3.97 (m, 29H), 3.84 (br s, 2H), 3.29 (m, 2H), 2.71 (dd, 1H, J=4.7, 12.4 Hz), 1.97 (s, 3H), 1.96 (s, 3H), 1.80 (m, 2H), 1.74 (dd, 1H, J=12.4, 12.4 Hz), 1.11 (d, 3H, J=6.4 Hz). MS(negative ion spray) Calcd for C.sub.42 H.sub.69 BrN.sub.3 O.sub.29 Na 1181/1183; found 1158/1160 (M-Na.sup.+).
Example 29
This example describes the synthesis of preferred compound 100 according to the reaction scheme depicted in FIG. 5.
Benzyl 2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranoside (86)
A suspension of benzyl alcohol (12.6 g, 116.8 mmol, 12.1 mL), activated 4 .ANG. molecular sieves (12 g), silver carbonate (58 g, 0.214 mmoles) and dichloromethane (400 mL) was stirred under an argon atmosphere for 4 h. A solution of 2,3,4,6-tetra-O-acetyl-.alpha.-D-galactosyl bromide (Sigma Chemical Co., 40.0 g, 97.3 mmol) in anhydrous dichloromethane (400 mL) was then added and the reaction stirred for 1 h. The mixture was filtered through Celite, concentrated, and chromatographed (silica, EtOAc/hexane, 35:65) to afford 33.0 g (77%) of yellow oil; R.sub.f =0.4 (hexane/EtOAc, 7:3). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.38-7.26 (m, 5H), 5.38 (d, J=3.3 Hz, 1H), 5.28 (dd, J=8.0, 10.4 Hz, 1H), 4.98 (dd, J=3.4, 10.5 Hz, 1H), 4.90 (d, J=12.3 Hz, 1H), 4.63 (d, J=12.3 Hz, 1H), 4.52 (d, J=8.0 Hz, 1H), 4.25-4.11 (m, 2H), 3.91-3.86 (m, 1H), 2.16 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H).
Benzyl .beta.-D-galactopyranoside (87)
Sodium methoxide (25% in methanol, 1.25 g, 23 mmol, 1 mL) was added to a solution of 86 (33.0 g, 75.3 mmol) in anhydrous methanol (200 mL) and stirred for 1.5 h. The mixture was neutralized with acidic cationic exchange resin (AG 50W-X8 resin, 100-200 mesh) and concentrated to afford 20.0 g (99%) of white solid; R.sub.f =0.22 (MeOH/CH.sub.2 Cl.sub.2, 1:9). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.46-7.39 (m, 5H), 4.92 (d, J=11.6 Hz, 1H), 4.45 (d, J=7.5 Hz, 1H), 3.90 (d, J=2.8 Hz, 1H), 3.83-3.34 (m, 5H).
Benzyl 4,6benzylidene-.beta.-D-galactopyranoside (88)
To a suspension of pulverized starting 87 (18.9 g 70.0 mmol) in acetonitrile (800 mL) was added benzaldehyde dimethylacetal (16.0 g, 105 mmol, 15.8 mL) and para-toluene sulfonic acid monohydrate (0.945 g). After 6 h, the mixture was cooled to -15.degree. C. for 18 h, and then filtered. The resulting solid was washed with diethyl ether and dried under high vacuum for 18 h to afford 21.5 g (86%) of shiny white solid; R.sub.f =0.35 (EtOAc, 100%). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.72-7.26 (m, 10H), 5.67 (s, 1H), 5.00 (d, J=11.7 Hz, 1H), 4.64 (d, J=11.7 Hz, 1H), 4.48 (d, J=8.0 Hz, 1H), 4.37 (d, J=3.6 Hz, 1H), 4.22 (d, J=3.7 Hz, 1H), 3.83 (dd, J=7.5, 9.2 Hz, 1H), 3.68 (dd, J=3.7, 9.5 Hz, 1H), 3.49 (s, 1H), 2.51-2.37 (br s, 2H).
Benzyl 2-benzoyl-4,6-benzylidene-.beta.-D-galactopyranoside (89)
Choroacetyl chloride (8.51 g, 75.4 mmol, 6.0 mL) was added over 30 min to a suspension of 88 (19.3 g, 53.9 mmol) in pyridine (40.7 g, 65.9 mmol, 39.8 mL) at -65.degree. C. The reaction mixture was stirred at -65.degree. C. under argon atmosphere for 6 h. Benzoyl chloride (9.09 g, 64.7 mmol, 7.51 mL) was then added dropwise at -65.degree. over 10 min. The stirring was continued for 2 h at -65.degree. C. and then at room temperature for 18 h. The reaction mixture was then washed with 1N HCl (400 mL), water (250 mL.times.2), saturated NaHCO.sub.3 solution (500 mL). The aqueous layers were extracted with EtOAc/EtOH (8:2) and the combined organic layers were dried (MgSO.sub.4) and concentrated. The resulting orange solid was suspended in methanol (500 mL) and cooled to -30.degree. C. A 2M methanolic ammonia solution (33 mL) was added to the suspension and stirring was continued for 5 h. The reaction mixture was diluted with dichloromethane (500 mL), washed with water (2.times.500 mL), and saturated NaCl solution (500 mL), and dried (Na.sub.2 SO.sub.4). Chromatography (silica, CH.sub.2 Cl.sub.2) afforded 13.8 g (66%) of off-white solid; R.sub.f =0.41 (EtOAc/hexance, 1:1); .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.8.03-8.01 (m, 2H), 7.61-7.19 (m, 13H), 5.60 (s, 1H), 5.45 (dd, J=8.0, 9.8 Hz, 1H), 4.93 (d, J=12.6 Hz, 1H), 4.71 (d, J=12.6 Hz, 1H), 4.63 (d, J=8.0 Hz, 1H), 4.43 (dd, J=1.1, 12.5 Hz, 1H), 4.26 (d, J=3.7 Hz, 1H), 4.13 (dd, J=1.4, 12.5 Hz, 1H), 3.85 (dt, J=3.8, 10.5 Hz, 1H), 3.53 (s, 1H), 2.65 (d, J=11.1 Hz, 1H).
Benzyl 2-benzoyl-4,6-benzylidene-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (90)
A suspension of 89 (12.2 g, 26.4 mmol), anhydrous dichloromethane (350 mL), collidine (4.31 g, 35.6 mmol, 4.7 mL) and activated 4 .ANG. molecular sieves (25 g) were stirred for 2.5 h under argon atmosphere. The mixture was cooled to 0.degree. C. and silver trifluoromethane sulfonate (8.81 g, 34.3 mmol) and a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido chloride (18.0 g, 39.6 mmol) in anhydrous dichloromethane (100 mL) were added. After stirring for 18 h at room temperature, the suspension was filtered, washed with 1M citric acid solution, water, saturated NaHCO.sub.3, water, saturated NaCl, and dried (Na.sub.2 SO.sub.4). Concentration and chromatography (silica, EtOAc/hexane, 1:1) afforded 20.4 g (88%) of white solid; R.sub.f =0.30 (EtOAc/hexane, 1:1). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.63-7.01 (m, 19H), 5.67-5.56 (m, 2H), 5.58 (s, 1H), 5.48 (dd, J=8.6, 9.2 Hz, 1H), 5.15 (dd, J=9.5, 9.9 Hz, 1H), 4.75 (d, J=12.7 Hz, 1H), 4.57 (d, J=12.9 Hz, 1H), 4.49 (d, J=8.2 Hz, 1H), 4.41-4.33 (m, 4H), 4.17-4.11 (m, 2H), 3.90 (dd, J=3.0, 9.8 Hz, 1H), 3.81 (dd, J=2.4, 7.7 Hz, 1H), 3.44 (s, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H).
Benzyl 2-benzoyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (91)
A suspension of 90 (20 g) in 80% acetic acid/water was heated to 80.degree. C. and after 30 min was diluted with water (300 mL) and cooled at 5.degree. C. for 18 h. The filtered solid was washed with cold water and chromatographed (silica, EtOAc/CH.sub.2 Cl.sub.2, 6:4) to afford 14.0 (76%) of shiny white solid; R.sub.f =0.26 (EtOAc/hexane, 7:3); .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.55-7.01 (m, 19H), 5.64 (dd, J=9.1, 10.3 Hz, 1H), 5.55 (d, J=8.3 Hz, 1H), 5.39 (dd, J=8.3, 9.6 Hz, 1H), 5.10 (dd, J=9.2, 10.0 Hz, 1H), 4.73 (d, J=12.8 Hz, 1H), 4.57 (d, J=12.7, 1H), 4.44 (d, J=8.1 Hz, 1H), 4.37-4.32 (m, 1H), 4.32 (dt, J=2.1, 11.9 Hz, 1H), 4.18 (d, J=4.3 Hz, 1H), 4.14 (d, J=5.3 Hz, 1H), 4.01 (dd, J=6.7, 11.8 Hz, 1H), 3.90-3.85 (m, 2H), 3.79 (dd, J=3.2, 9.7 Hz, 1H), 3.5 (dd, J=6.1, 5.6 Hz, 1H), 2.10 (s, 3H), 2.01 (s, 3H), 1.77 (s, 3H).
Benzyl-3-O-(2-deoxy-2-N-allyloxycarboxamido-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (92)
A suspension of 91 (15.5 g, 19.6 mmol) was heated in ethanol and hydrazine monohydrate at 80.degree. C. for 22 h. After cooling, acetone (160 mL) was added and the mixture was stirred for a few minutes. The solid was filtered, and dried under high vacuum at 50.degree. C. for 18 h. The solid was then suspended in MeOH (424 mL) and water (23 mL), and NaHCO.sub.3 (24.1 g, 0.287 mol) was added. After 30 min, allyl chloroformate (5.28 g, 43.8 mmol, 4.67 mL) was added and stirring was continued for 1 h. The reaction mixture was filtered and the precipitate was washed with methanol. The resulting white solid (8.59 g, 85% crude yield) was used in the subsequent step; R.sub.f =0.59 (EtOAc/MeOH/AcOH/H.sub.2 O, 90:15:15:10). .sup.1 H NMR (300 MHz, MeOD) .delta.7.41-7.38 (m, 2H), 7.33-7.34 (m, 3H), 6.00-5.87 (m, 1H), 5.28 (dd, J=1.3, 17.0 Hz, 1H), 5.16 (dd, J=1.2, 10.4 Hz, 1H), 4.90 (d, J=11.9 Hz, 1H), 4.77-4.58 (m, 1H), 4.65 (d, J=11.6 Hz, 1H), 4.52 (d, J=5.2 Hz, 2H), 4.29 (d, J=7.7 Hz, 1H), 4.01 (d, J=2.8 Hz, 1H), 3.83 (dd, J=2.0, 11.9 Hz, 1H1), 3.79-3.63 (m, 3H), 3.57 (dd, J=3.1, 9.7 Hz, 1H), 3.50 (dd, J=6.1, 5.6 Hz, 1H), 3.39 (dd, J=6.5, 7.2 Hz, 1H), 3.34-3.21 (m, 3H).
Benzyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-Dgalactopyranosyl)-(1-4)-O-(2-deoxy-2-N-allyloxycarboxamido-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (93)
Reaction conditions similar to compound 55 were used. Concentration and chromatography (reverse phase C-18, MeOH/H.sub.2 O, 5-10%) afforded 13.3 g (81%) of white solid; R.sub.f =0.26 (1M NH.sub.4 OAc/i-PrOH, 15:85). .sup.1 H NMR (300 MHz, D.sub.2 O) .delta.7.48-7.38 (m, 5H), 5.99-5.90 (m, 1H), 5.26 (d, J=17.2 Hz, 1H), 5.22 (d, J=10.4 Hz, 1H), 4.93 (d, J=11.8 Hz, 1H), 4.70 (d, J=8.3 Hz, 1H), 4.59-4.53 (m, 1H), 4.54 (d, J=7.9 Hz, 11H), 4.45 (d, J=7.7 Hz, 1H), 4.13 (dd, J=2.9, 10.0 Hz, 2H), 3.97-3.34 (m, 26H), 2.75 (dd, J=4.4, 12.3 Hz, 1H), 2.02 (s, 3H), 1.79 (dd, J=12.0 Hz, 1H).
Benzyl (sodium 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-(3,6-O-diacetyl-2-deoxy-2-N-allyloxycarboxamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (94)
A suspension of 93 (12.0 g) was suspended in pyridine (50 mL) and concentrated to dryness. The resulting residue was suspended in anhydrous pyridine (300 mL) and cooled to 0.degree. C. Acetic anhydride (250 mL) was then added dropwise over 20 min and the solution was stirred under argon for 48 h. The suspension was concentrated and methanol (410 mL) was added. After 5 days of stirring, the suspension was concentrated to 150-200 mL and pyridine (10 mL) and acetic anhydride (110 mL) were added. After stirring for 18 h at room temperature, the reaction mixture was concentrated, diluted with EtOAc (300 mL), washed with saturated NaHCO.sub.3 (3.times.300 mL), and dried (Na.sub.2 SO.sub.4). Concentration and chromatography (silica, CH.sub.2 Cl.sub.2 /acetone, 25:10) afforded 15.9 g of white solid (80%); R.sub.f =0.59 (hexane/EtOAc/EtOH, 10:10:3).
.sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.36-7.26 (m, 5H), 5.93-5.84 (m, 1H), 5.54-5.50 (br m, 1H), 5.49-5.37 (br m, 1H), 5.26 (d, J=16.4 Hz, 1H), 5.21-5.05 (m, 4H), 4.95-4.84 (m, 4H), 4.71 (d, J=8.0 Hz, 1H), 4.65-4.49 (m, 6H), 4.44-4.37 (m, 2H), 4.17-4.05 (m, 3H), 4.00-3.96 (m, 4H), 3.84 (s, 3H), 3.82-3.69 (m, 5H), 3.63 (dd, J=2.6, 10.8 Hz, 1H), 3.51-3.39 (m, 2H), 3.57 (dd, J=4.5, 12.6 Hz, 1H), 2.23-2.00 (s, 39H), 1.85 (s, 3H), 1.67 (dd, J =12.4 Hz, 1H).
Benzyl (sodium 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-D-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (95)
Compound 94 (15.9 g, 10.7 mmol) was dissolved in tetrahydrofuran (100 mL) and polymethylsiloxane (1.28 mL) and tetrakis(triphenylphosphine)palladium (0.255 g, 0.21 mmol) was added. The suspension was stirred under argon for 2 h. Acetic acid (1.02 mL) and piperidine (1.3 mL) were then added and stirring was continued for 23 h. Additional acetic acid (1.6 mL) was then added and the solution concentrated. The resulting residue was chromatographed (silica, acetone/CH.sub.2 Cl.sub.2, 4:6) to afford 14.6 g (97%) of slightly yellow solid; R.sub.f =0.3 (CH.sub.2 Cl.sub.2 /acetone, 4:6-developed 2.times.). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.37-7.26 (m, 5H), 5.66-5.63 (br m, 1H), 5.53-5.50 (br m, 1H), 5.40 (dd, J=2.3, 7.6 Hz, 2H), 5.17 (dd, J=9.1, 8.8 Hz, 1H), 5.06-5.02 (m, 2H), 5.03 (dd, J=4.2, 10.2 Hz, 1H), 4.99-4.84 (m, 2H), 4.88 (d, J=3.8 Hz, 1H), 4.68-4.57 (m, 3H), 4.50-4.35 (m, 3H), 4.18-3.86 (m, 10H), 3.84 (s, 3H), 3.79-3.75 (m, 3H), 3.63 (dd, J=2.3, 10.7 Hz, 1H), 3.55-3.41 (m, 2H), 3.00-2.9 (m, 1H), 2.57 (dd, J=4.6, 12.7 Hz, 1H), 2.25, 2.18, 2.17, 2.16, 2.13, 2.10, 2.08, 2.07, 2.06, 2.05, 2.01, 1.95, 1.86 (13s, 39H), 1.68 (dd, J=6.8 Hz, 1H).
Benzyl (sodium 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-benzyl-.alpha.-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-acetamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (96)
A solution of compound 95 (14.6 g, 10.4 mmol) in CH.sub.2 Cl.sub.2 (100 mL), DMF (6 mL), tetraethylammonium bromide (2.0 g, 10.4 mmol) and activated 4 .ANG. molecular sieves (25 g) was stirred under argon for 6 h. Tri-O-benzyl fucose (61.6 g, 131 mmol) was dissolved in CH.sub.2 Cl.sub.2 (135 mL), DMF (22 mL) and 2N oxalyl bromide solution in dichloromethane (170 mmol, 85 mL) and added at 0.degree. C. over 40 min. After stirring for an additional 20 min, the solution was washed with cold water (150 mL) and the aqueous solution was extracted with dichloromethane (70 mL). The combined organic layers were washed with cold water (150 mL.times.2), dried (MgSO.sub.4), and filtered. The solution was added to the tetrasaccharide reaction mixture above. After strung for 2 weeks, diisopropylethylamine (11 mL) and methanol (11 mL) were added and the reaction was stirred for 30 min. The mixture was diluted with dichloromethane. (110 mL) and filtered through Celite, concentrated, and chromatographed (silica, CH.sub.2 Cl.sub.2, 100% to EtOAC/hexane/EtOH, 5:5:1) to afford 10.7 g (79%) of yellow foam; R.sub.f =0.38 (hexane/EtOAc/EtOH, 50:50:15). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.7.43-7.23 (m, 5H), 5.53-5.47 (m, 3H), 5.42 (dd, J=2.2, 9.7 Hz, 1H), 5.34 (d, J=3.1 Hz, 1H), 5.23 (d, J=7.8 Hz, 1H), 5.15 (dd, J=8.4, 9.8 Hz, 1H), 5.04-4.50 (m, 17H), 4.38-4.25 (m, 2H), 4.16-3.91 (m, 8H), 3.83 (s, 3H), 3.81-3.66 (m, 6H), 3.61 (dd, J=1.9, 10.5 Hz, 1H), 3.48-3.45 (m, 1H), 2.55 (dd, J=4.2, 12.2 Hz, 1H), 2.18, 2.15, 2.08, 2.07, 2.06, 2.05, 2.03, 2.00, 1.97, 1.85, 1.79, 1.65, 1.59 (13s, 39H).
Benzyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl-(1,3)-O!-(2-acetamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (97)
Compound 96 (1.5 g, 1.1 mmoles) was dissolved in methanol (14 mL) and 25% NaOMe in MeOH (1.05 mL) was added under argon. After stirring overnight, water (0.58 mL) was than added and the reaction stirred for 24 h. The pH was then adjusted to 7.0 with 0.25M HCl and the solution concentrated. Chromatography (silica, ethyl acetate/ethanol/water (8/2/1)) afforded 1.35 g (90%) of a water solid, R.sub.f =0.50 (silica, 90% iso-propanol/1M NH.sub.4 OAc); .sup.1 H NMR (CD.sub.3 OD) .delta.7.41-7.18 (m, 20H), 5.32 (d, J=3.8 Hz, 1H), 4.83 (d, J =12.0 Hz, 1H), 4.57 (d, J =8.1 Hz, 1H), 4.53 (d, J=12.0 Hz, 1H), 4.51 (d, J=7.8 Hz, 1H), 4.28 (d, J=7.7 Hz, 1H), 4.11-3.3 (m, 28H), 2.86 (dd, J=12.1, 4.1 Hz, 1H), 1.98 (s, 6H), 1.72 (dd, J=12.1, 12.1 Hz, 1H), 1.12 (d, J=6.3 Hz, 3H).
(sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-acetamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranose (98)
A solution of Compound 97 (1.35 g, 0.99 mmoles) and 10% Pd/C (1.47 g) in ethanol (50 mL) and water (2.5 mL) was placed under a hydrogen atmosphere. After stirring for 24 h, the catalyst was filtered and the filtrate concentrated. The residue was chromatographed (C-18 silica, water) to afford 0.634 g (86%) of 98 as a white solid after lyophilization, R.sub.f =0.46 (silica, 70% iso-propanol/1M NH.sub.4 OAc). .sup.1 H NMR (D.sub.2 O) .delta.5.21 (d, J=3.4 Hz, 0.3H), 5.11 (d, J=3.9 Hz, 0.7H), 5.08 (d, J=3.9 Hz, 0.3H), 4.53 (m, 2.7H), 4.20-3.47 (m, 28H), 2.75 (dd, J=4.5, 12.1 Hz, 1H), 2.01 (s, 3H), 1.90 (s, 3H), 1.78 (dd, J=12.1, 12.1 Hz, 1H), 1.15 (d, J=6.6 Hz, 3H).
Amino (ammonium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1,4)-O-�.alpha.-L-fucopyranosyl-(1,3)-O!-(2-acetamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (99)
A saturated NH.sub.4 HCO.sub.3 solution containing 98 (0.4 g, 0.398 mmol) was stirred for 3 days adding NH.sub.4 HCO.sub.3 to maintain a saturated solution. The solution was lyophilized and the residue dissolved in water (20 mL) and relyophilized (3.times.) to afford 0.202 g (50%) of 99 as a white solid, R.sub.f =0.31 (silica, 70% iso-propanol/1M NH.sub.4 OAc).
2-Bromoacetamido (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1,3)-O!-(2-acetamido-2-deoxy-.beta.-D-glucopyranosyl)-(1,3)-O-.beta.-D-galactopyranoside (100)
Bromoacetic anhydride (0.31 g, 1.195 mmoles) was added to a solution of 99 (0.1 g, 0.0996 mmol) containing ethanol (2 mL), water (2 mL) and sat. NaHCO.sub.3 (2 mL). After 30 min, the reaction mixture was chromatographed (Biogel P-2, 20% ethanol/water) to afford 86 mg (76%) of a white solid after lyophilization, R.sub.f =0.63 (silica, 70% iso-propanol/1M NH.sub.4 OAc); .sup.1 H NMR (D.sub.2 O) .delta.5.11 (d, J=3.9 Hz, 1H), 4.92 (d, J=9.7 Hz, 1H), 4.55 (d, J=7.9 Hz, 1H), 4.21 (d, J=2.7. Hz, 1H), 4.08 (dd, J=2.9, 10.1 Hz, 1H), 4.03-3.49 (m, 28H), 2.76 (dd, J=4.4, 12.2 Hz, 1H), 2.02 (s, 3H), 2.00 (s, 3H), 1.78 (dd, J=12.2, 12.2 Hz, 1H), 1.16 (d, J=6.5 Hz, 3H). MS (FAB) C.sub.39 H.sub.63 BrN.sub.3 O.sub.28 Na, calc. 1123.268; found 1100 �M-Na.sup.+ !.sup.-.
Example 30
This example describes the synthesis of preferred compound 115 according to the reaction scheme depicted in FIG. 6.
3'-Benzyloxycarbonylaminopropyl 4,6-benzylidene-.beta.-galactopyranoside (103)
To a mixture of 20 g (48.7 mmol) of 2,3,4,6-tetra-O-acetyl-.alpha.-D-galactosyl bromide, 15.3 g (73 mmol) of 3-benzyloxyaminopropyl alcohol, 20 g of activated 4 .ANG. molecular sieves and 250 mL of CH.sub.2 Cl.sub.2 was added 40 g (145.8 mmol) of Ag.sub.2 CO.sub.3. The resulting suspension was stirred at 0.degree. C. for 30 min, and then at room temperature for 24 h. After filtration through celite, the filtrate was washed with sat. NaHCO.sub.3 and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated. The residue was dried in vacuo to dryness, and then dissolved in 50 mL of dry MeOH, 1 mL of 25% NaOMe in MeOH was added. Stirring continued for 2 h at which time the reaction was completed. Neutralization with ion-exchange resin, filtration and concentration afforded the crude compound 102 which was used for the next step.
To a suspension of the crude galactoside 102, 10 mL of benzaldehyde dimethyl acetal and 150 mL of dry acetonitrile was added 400 mg of p-toluenesulfonic acid monohydrate. The mixture was stirred at room temperature for 2 h at which time the starting material had been consumed. Concentration of the solution gave a residue which was taken up in CH.sub.2 Cl.sub.2 and washed with sat. NaHCO.sub.3 and brine. Drying over MgSO.sub.4, filtration and concentration followed by chromatography on silica gel column MeOH/EtOAc/hexanes, 3:20:20 to 3:10:10) afforded the desired product 103 as a white solid (13.5 g, 60.5% yield). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.7.26-7.51 (m, 10H), 5.54 (s, 1H), 5.28 (br s, 1H), 5.08 (s, 2H), 3.24-4.37 (m, 11H), 2.68 (br s, 2H), 1.81 (m, 2H).
3'-Benzyloxycarbonylaminopropyl 2-benzoyl-4,6-benzylidene-.beta.-D-galactopyranoside (104)
To a solution cooled at -65.degree. C. (dry ice/CHCl.sub.3) containing (13 g, 28.3 mmol) of 3'-benzyloxycarbonylaminopropyl 4,6-benzylidene-.beta.-D-galactopyranoside 103 in pyridine (20 mL) and CH.sub.2 Cl.sub.2 (100 mL) was added dropwise 2.6 mL (32.6 mmol) of chloroacetyl chloride over 1 h. After stirring for another hour, 4.7 mL (40.5 mmol) of benzoyl chloride was added dropwise over 5 min. The mixture continued to be stirred at this temperature for 30 min, and then at room temperature overnight. The mixture was filtered through celite with washing with small portion of CH.sub.2 Cl.sub.2. The filtrate was concentrated to approximately 40 mL, and 50 mL of water was added. The mixture was heated with vigorous stirring for 3.5 h. After being cooled to room temperature, it was treated with cold water, and extracted with CH.sub.2 Cl.sub.2. The organic layer was washed with sat. NaHCO.sub.3, brine and dried (MgSO.sub.4). Purification by flash chromatography on silica gel column using EtOAc/hexanes (1:2-2:1) afforded the product (8.0 g, 50.3%).
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.8.04 (d, 2H, J=7.6 Hz), 7.27-7.57 (m, 8H), 5.59 (s, 1H), 5.37 (dd, 1H, J=8.0, 9.8 Hz), 5.07 (br s, 1H), 5.02 (s, 2H), 4.60 (d, 1H, J=8.0 Hz), 4.38 (d, 1H, J=12.4 Hz), 4.28 (d, 1H, J=3.7 Hz), 4.10 (dd, 1H, J=12.4, 1.0 Hz), 3.85-4.50 (m, 2H), 3.62 (m, 1H), 3.56 (br s, 1H), 3.15-3.23 (m, 4H), 2.60 (d, 1H, J=11.1 Hz), 1.74 (m, 2H).
3'-Benzyloxycarbonylaminopropyl 3-(2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-.beta.-D-glucopyranosyl)-(2-benzoyl-4,6benzylidene-.beta.-D-galactopyranoside (105)
A mixture of 7.8 g (13.85 mmol) of 104, 2.56 mL (19.4 mmol) of collidine, 50 mL of CH.sub.2 Cl.sub.2, 5.4 g (20.8 mmol) of AgOTf and 10 g of 4 .ANG. molecular sieves was stirred at room temperature for 1 h, and then cooled to -20.degree. C. 2-Phthalimido-3,4,6-tri-O-acetyl-.beta.-D-glucopyranoside (7.53 g, 16.6 mmol) was added. The resulting mixture continued to be stirred for 2 h, warmed to room temperature and stirred overnight. Filtration over celite was followed by washing with sat. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. The residue was chromatographed on silica gel column (EtOAc/hexanes, 1:1-2:1) to afford the desired product (13.6 g, 85%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.7.10-7.65 (m, 14H), 5.66 (t, 1H, J=9.8 Hz), 5.60 (d, 1H, J=8.5 Hz), 5.57 (s, 1H), 5.39 (dd, 1H, J=8.1, 9.9 Hz), 5.16 (t, 1H, J=9.8 Hz), 4.98 (s, 2H), 4.92 (m, 1H), 4.49 (d, 1H, J=8.0 Hz), 3.80-4.44 (m, 10H), 2.97 (m, 4H), 2.06 (s, 3H), 2.01 (s, 3H), 1.77 (s, 3H).
3'-Benzyloxycarbonylaminopropyl 3-(2-deoxy-2-allyloxycarboxamido-.beta.-D-glucopyranosyl)-.beta.-D-galactopyranoside (106)
A mixture of 9.5 g (9.70 mmol) of 105 and 95 mL of 80% acetic acid was heated at 70.degree. C. for 2 hr at which time TLC showed the reaction was completed. The mixture was concentrated, and the residue was taken up in EtOAc with washing with sat. NaHCO.sub.3 and brine. The organic layer was dried over MgSO.sub.4 and filtered. The filtrate was concentrated and dried in vacuo to dryness. The residue obtained was dissolved in 135 mL of EtOH. The mixture was refluxed with 4.2 mL of hydrazine monohydrate for 16 h. After being cooled to room temperature, the suspension was concentrated to provide a solid which was dried in vacuo overnight after washing with EtOAc several times.
The solid was added to a mixture containing 150 mL of MeOH, 15 mL of water and 9.7 g of NaHCO.sub.3. The suspension was stirred for 15 min, and then allyl chloroformate (1.44 mL) was added dropwise over 10 min. The resulting mixture was stirred for 1 h. After additional 1.44 mL of allyl chloroformate was added, the mixture was stirred for another hour. The mixture was filtered and the filtrate was concentrated to give a residue which was purified by flash chromatography on silica gel column (MeOH/CHCl.sub.3 /H.sub.2 O, 3:7:0 to 4:6:0.1) to give 4.78 g of product 106 (80%). .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.7.38 (br s, 5H), 5.86-5.96 (m, 1H), 5.28 (br d, 1H, J=18.6 Hz), 5.20 (br d, 1H, J=10.6 Hz), 5.07 (s, 2H), 4.53 (br d, 2H, J=2.5 Hz), 4.28 (d, 1H, J=7.4 Hz), 4.08 (d, 1H, J=2.7 Hz), 3.42-3.91 (m, 11H), 3.20 (m, 2H), 1.76 (m, 2H).
The enzymatic glycosylation of the compound 106 and the acetylation: The preparation of the compound 108
Galactosyltransferase (600 u), pyruvate kinase (762 u), UDP-glucose pyrophosphorylase (1000 u), UDP-galactose 4'-epimerase (1000 u) were added to a solution (pH, 7.4) containing 106 (4.0 g, 6.5 mmol), glucose-1-phosphate (4.6 g, 15.1 mmol), 2-phosphoenol pyruvate (2Na.sup.+) (8.4 g), UDP(3Na.sup.+) (460 mg), BSA (800 mg), water (1100 mL), MeOH (20 mL), MnCl.sub.2 (40 mL) and sodium cacodylate (100 mL). The mixture was inverted several times, and then allowed to sit at room temperature for 2 days. The pH was adjusted to 7.4 by addition of 1M NaOH solution, and then additional enzymes (50% amounts of first addition) were added. The mixture was allowed to stand for 5 days at which time the starting material had been consumed.
To the mixture were added potassium 2-phosphoenol pyruvate (3.75 g), cytidine monophosphate (289 mg), ATP (100 mg), sialic acid (4.1 g), sodium cacodylate (pH 7.4, 1M, 40 mL). After the pH value was adjusted to 7.4, pyruvate kinase (762 u), myokinase (500 u), CMP-NeuAc synthetase (200 u), and sialyl transferase (100 u) were added. The resulting mixture was allowed to stand for 2 days. Additional enzymes (the same amount of the first addition) were added. The mixture was left for 5 days (the pH value was re-adjusted every day).
Concentration of this mixture provided a syrup which was dried in vacuo for 24 h, and it was then treated with 1.5 L of dry pyridine and 1.1 L of Ac.sub.2 O in the presence of 4 g of DMAP at 0.degree. C., and at room temperature for 3 days. The mixture was concentrated, 500 mL of MeOH and 500 mL of pyridine were added. After being stirred at room temperature for 2 days, the mixture was concentrated. The residue was taken up in EtOAc and washed with sat. NaHCO.sub.3 and brine. Drying and concentration gave a residue which was treated with 1 L of pyridine and 300 mL of Ac.sub.2 O for 2 days.
The mixture was poured into cold sat. NaHCO.sub.3, extracted with EtOAc. After washing with brine, the organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. The residue was purified by flash chromatography on silica gel column (MeOH/EtOAc/hexanes, 1:3:3) to afford 6.7 g of fully protected tetrasaccharide 108 in 65% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.7.31 (m, 5H), 5.80-5.89 (m, 1H), 5.47 (m, 1H), 5.36 (m, 2H), 5.21 (d, 1H, J=16.6 Hz), 4.67 (d, 1H, J=7.9 Hz), 4.80-5.15 and 3.40-4.53 (m, 31H), 3.80 (s, 3H), 3.13-3.36 (m, 3H), 2.53 (dd, 1H, J=4.5, 12.6 Hz), 2.20 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 6H), 2.03 (s, 6H), 2.00 (s, 6H), 1.99 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.81 (s, 3H), 1.74 (m, 2H), 1.62 (dd, 1H, J=12.6, 12.6 Hz). MS(positive ion spray) Calcd for C.sub.69 H.sub.93 N.sub.3 O.sub.39 1588; found 1589 (M+H.sup.+).
3'-Benzyloxycarbonylaminopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-(3,6-di-O-acetyl-2-deoxy-2-amino-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (109)
To a solution of tetrasaccharide 108 (4.5 g, 2.84 mmol), polymethylhydrosiloxane (1.15 mL) and 65 mL of dry THF was added Pd(PPh.sub.3).sub.4 (384 mg). The resulting mixture was stirred under argon at room temperature for 1.5 h at which time TLC indicated the starting material had been consumed. After dilution with EtOAc, the mixture was washed with ice-water and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel column using MeOH/EtOAc (0:1 to 1:10) as the irrigant provided amine as a yellow solid (3.8 g, 89%). .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.7.32 (m, 5H), 5.47 (m, 1H), 5.38 (dd, 1H, J=2.6, 9.0 Hz), 5.21 (d, 1H, J=10.2 Hz), 5.13 (dd, 1H, J=8.2, 10.0 Hz), 5.11 (br s, 1H), 5.06 (s, 2H), 4.85-4.99 (m, 3H), 4.65 (d, 1H, J=8.0 Hz), 3.48-4.56 (m, 21H), 3.81 (s, 3H), 3.19-3.30 (m, 3H), 2.67 (dd, 1H, J=8.0, 10.0 Hz), 2.55 (dd, 1H, J=4.6, 12.6 Hz), 2.20 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.06 (s, 6H), 2.05 (s, 6H), 2.03 (s, 6H), 2.01 (s, 3H), 1.98 (s, 3H), 1.82 (s, 3H), 1.75 (m, 2H), 1.65 (dd, 1H, J=12.6, 12.6 Hz).
3'-Benzyloxycarbonylaminopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranyl)-(1-4)-O-(3,6-di-O-acetyl-2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl-.beta.-D-galactopyranoside (110)
A mixture of 706 mg (0.47 mmol) of tetrasaccharide 109, 27 .mu.L (0.47 mmol) of HOAc, 3 mL of H.sub.2 O and 9 mL of MeOH was heated at 50.degree. C. for 1.5 days. After being cooled to room temperature, the mixture was concentrated and dried in vacuo overnight to give the crude deacetylated product which was used for the next step without further purification.
The residue was dissolved in 12 mL of CH.sub.2 Cl.sub.2, and 159 mg (1.88 mmol) of NaHCO.sub.3 and 180 mg (0.94 mmol) of naphthoyl chloride were added at 0.degree. C. The suspension was stirred at 0.degree. C. for 10 min, and then at room temperature for 2 h. After additional NaHCO.sub.3 (80 mg) and naphthoyl chloride (90 mg) were added, the mixture was continued to be stirred for 12 h. After dilution with EtOAc and washing with ice-water and brine, the organic layer was dried over Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash chromatography on silica gel column using EtOAc/CH.sub.2 Cl.sub.2 /MeOH (8:8:1 to 3:3:1) provided 420 mg of the desired product as a white solid in 56% yield.
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.8.27 (s, 1H), 7.78-7.93 (m, 4H), 7.55 (m, 2H), 7.29 (m, 5H), 6.53 (d, 1H, J=7.7 Hz), 5.54 (m, 1H), 5.35-5.47 (m, 3H), 5.03 (br s, 2H), 4.70 (d, 1H, J=8.0 Hz), 4.60 (dd, 1H, J=3.4, 10.0 Hz), 4.88-5.15 and 3.50-4.42 (m, 25H), 3.83 (s, 3H), 3.16-3.26 (m, 3H), 2.58 (dd, 1H, J=4.5, 12.6 Hz), 2.28 (s, 3H), 2.16 (s, 3H), 2.13 (s, 3H), 2.10 (s, 6H), 2.08 (s, 9H), 2.01 (s, 3H), 2.00 (s, 3H), 1.93 (s, 3H), 1.86 (s, 3H). MS(positive ion spray) Calcd for C.sub.74 H.sub.93 N.sub.3 O.sub.37 1616; found 1639 (M+Na.sup.+).
3'-Benzyloxycarbonylaminopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�2,3,4-tri-O-para-methoxybenzyl-.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-.beta.-D-galactopyranoside (111)
A suspension of CuBr.sub.2 (138 mg, 0.62 mmol), Et.sub.4 NBr (138 mg, 0.64 mmol), DMF (1.0 mL), CH.sub.2 Cl.sub.2 (6.0 mL) and 4 .ANG. molecular sieves (1.6 g) was stirred at room temperature for 5 h, tetrasaccharide 110 (400 mg, 0.248 mmol) and thiomethyl 2,3,4-tri-O-(4-methoxybenzyl)-.beta.-L-fucopyranoside (274 mg, 0.496 mmol) were added. The mixture was stirred at room temperature under argon for 48 h. After dilution with EtOAc, it was filtered through celite, and the filtrate was then washed with sat. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2 SO.sub.4, filtered. Concentration followed by chromatography on silica gel column using MeOH/EtOAc/hexanes (8:8:1 to 3:3:1) afforded 330 mg of pentasaccharide 111 in 64% yield. .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.8.11 (s, 1H), 7.84 (m, 3H), 7.53-7.62 (m, 3H), 7.21-7.34 (m, 7H), 7.15 (d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.4 Hz), 6.81 (d, 2H, J=8.6 Hz)), 6.74 (d, 2H, J=8.5 Hz), 6.64 (d, 1H), 6.53 (d, 2H, J=8.5 Hz), 3.10-5.58 (m, 47H), 3.83 (s, 3H), 3.77 (s, 3H), 3.74 (s, 3H), 3.66 (s, 3H), 2.55 (dd, 1H, J=4.5, 12.6 Hz), 2.22 (s, 3H), 2.16 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 (s, 6H), 2.00 (s, 3H), 1.85 (s, 3H), 1.74 (s, 3H), 1.67 (m, 3H), 1.14 (d, 3H, J=6.4 Hz). MS(positive ion spray) Calcd for C.sub.104 H.sub.127 N.sub.3 O.sub.44 2122; found 2146 (M+Na.sup.+ +1C.sup.13).
3'-Benzyloxycarbonylaminopropyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl-.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(6-O-acetyl-2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-.beta.-D-galactopyranoside (112)
To a solution of 300 mg (0.141 mmol) of pentasaccharide 111 in 2 mL of acetonitrile and 0.2 mL of water was added 600 mg (1.095 mmol) of ceric ammonium nitrate. The mixture was vigorously stirred at room temperature for 15 min at which time the reaction was completed. After being diluted with EtOAc, it was washed with sat. NaHCO.sub.3 /water (1:1, v/v) and brine. The organic layer was dried over Na.sub.2 SO.sub.4 and filtered. Concentration followed by purification by flash chromatography on silica gel column (CH.sub.2 Cl.sub.2 /MeOH, 20:1 to 10:1) afforded 225 mg of product 112 in 90% yield.
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.8.34 (s, 1H), 7.82-7.96 (m, 4H), 7.55 (m, 2H), 7.30 (br s, 5H), 6.81 (br d, 1H, J=6.3 Hz), 3.20-5.55 (m, 21H), 3.85 (s, 3H), 2.59 (dd, 1H, J=4.0, 12.5 Hz), 2.25 (s, 3H), 2.17 (s, 3H), 2.14 (s, 3H, 2.12 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 6H), 2.01 (s, 6H), 1.86 (s, 3H), 1.67 (m, 3H), 1.29 (d, 3H, J=6.4 Hz). MS(positive ion spray) Calcd for C.sub.80 H.sub.103 N.sub.3 O.sub.41 1762; found 1785(M+Na.sup.+), 1895(M+Cs.sup.+).
3'-Benzyloxycarbonylaminopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-D-galactopyranoside (113)
A mixture of 225 mg (0.128 mmol) of pentasaccharide 112, 300 .mu.L of 25% methanolic NaOMe solution and 4 mL of MeOH was stirred at room temperature for 24 h, and then 1 mL of water was added. The resulting solution was continued to be stirred for another 24 h. After addition of one drop of HOAc to adjust pH about 8, the reaction mixture was concentrated to give a residue which was chromatographed on reverse phase silica (Biosil C-18; 10-80% MeOH in H.sub.2 O) to afford 113 as a white solid (155 mg, 93%). .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.8.30 (s, 1H), 7.89-8.00 (m, 3H), 7.78 (dd, 1H, J=1.5, 8.6 Hz), 7.53-7.61 (m, 2H), 7.30 (br s, 3H), 7.20 (m, 2H), 5.15 (d, 1H, J=4.0 Hz), 4.53 (d, 1H, J=7.6 Hz), 4.22 (d, 1H, J=7.7 Hz), 4.13 (d, 1H, 3.1 Hz), 3.40-4.11 (m, 31H), 3.06 (m, 2H), 2.73 (dd, 1H, J=4.6, 12.5 Hz), 1.99 (s, 3H), 1.76 (dd, 1H, J=12.5, 12.5 Hz), 1.66 (m, 2H), 1.13 (d, 3H, J=6.6 Hz).
3'-Bromoacetamidopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (114) 70 mg of 5% Pd/BaSO.sub.4 was added to a solution of 145 mg (0.11 mmol) of 3'-Benzyloxycarbonylaminopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside 113 in 4 mL of water. The mixture was purged three times with hydrogen gas and then stirred under hydrogen atmosphere (1 atm) for 1 h. The mixture was degassed and concentrated. 10 mL of MeOH was added, and the mixture was filtered through celite with washing with MeOH. The filtrate was concentrated and dried in vacuo overnight to provide amine which was used for the next reaction.
The residue was dissolved in a mixture of 8.5 mL of sat. NaHCO.sub.3, 5.6 mL of water and 5.6 mL of EtOH, and 372 mg (1.44 mmol) of bromoacetic anhydride was then added. The resulting suspension was stirred at room temperature for 1 h. After additional bromoacetic anhydride (150 mg) and NaHCO.sub.3 (120 mg) were added, stirring was continued for another hour. Concentration of the mixture followed by purification on reverse phase silica (Biosil C-18; 0-5% MeOH in H.sub.2 O) afforded 114 as a white solid after lyophilization (130 mg, 91%). .sup.1 H NMR (300 MHz, D.sub.2 O): .delta.8.32 (s, 1H), 7.96-8.04 (m, 3H), 7.80 (br d, J=8.4 Hz), 7.62 (m, 2H), 5.16 (d, 1H, J=3.7 Hz), 4.53 (d, 1H, J=8.0 Hz), 4.26 (d, 1H, J=8.0 Hz), 4.14 (d, 1H, J=3.3 Hz), 3.42-4.12 (m, 33H), 3.63 (s, 2H), 3.15 (m, 2H), 2.72 (dd, 1H, J=4.3, 12.6 Hz), 1.99 (s, 3H), 1.76 (dd, 1H, J=12.6, 12.6 Hz), 1.70 (m, 2H), 1.12 (d, 3H, J=6.5 Hz). MS(negative ion spray) Calcd for C.sub.51 H.sub.73 N.sub.3 O.sub.29 BrNa 1293/1295; found 1270/1272 (M-Na.sup.+).
Preparation of compound 115
A mixture of 5 mg (3.86 .mu.mol) of 3'-Bromoacetamidopropyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-(2-deoxy-2-naphthamido-.beta.-D-glucopyranosyl)-(1-3)-O-.beta.-D-galactopyranoside (114), 4.0 mg (5.8 .mu.mol) of dimyristoylphosphatidyl thioethanol, 2.0 mg (6.13 .mu.mol) of Cs.sub.2 CO.sub.3 and 0.6 mL of DMF was stirred under argon at room temperature for 2.5 h. The solvent was removed by co-evaporation with water under the reduced pressure. The residue obtained was purified by chromatography on reverse phase silica (Biosil C-18; 50-85% MeOH in H.sub.2 O) to afford the liposomal pentasaccharide 115 as a white solid after lyophilization (4.0 mg, 55%). .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.8.42 (s, 1H), 7.88-8.01 (m, 4H), 7.52-7.56 (m, 2H), 5.20 (m, 1H), 5.13 (d, 1H, J=4.0 Hz), 4.54 (d, 1H, J=7.8 Hz), 4.41 (dd, 1H, J=3.3, 12.0 Hz), 3.20-4.24 (m, 41H), 2.86 (dd, 1H, J=2.6, 12.0 Hz), 2.74 (t, 2H, J=6.6 Hz), 2.26-2.342 (m, 4H), 1.99 (s, 3H), 1.76 (m, 3H), 1.57 (m, 4H), 1.27 (br s, 44H), 1.14 (d, 3H, J=6.6 Hz), 0.88 (br t, 6H, J=6.4 Hz). MS(positive ion spray) Calcd for C.sub.84 H.sub.136 N.sub.3 O.sub.37 PSNa 1888; found 1890 (M+H.sup.+ +1C.sup.13).
Example 31
This example provides a general procedure for coupling of a lipid to compounds 62, 73, 78, 85 and 100 as provided in FIG. 7.
(a) Preparation of Compound 116
A mixture of 10 mg (9.36 .mu.mol) of Ethyl (sodium 5-acetamido-3,5-dideoxy-.alpha.-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(g.beta.-D-galactopyranosyl)-(1-4)-O-�.alpha.-L-fucopyranosyl-(1-3)-O!-2-deoxy-2-(4-bromoacetamido-benzamido)-.beta.-D-glucopyranoside, 62, 4.2 mg (6.24 .mu.mol) of dimyristoylphosphatidyl thioethanol, 3.0 mg (35.8 .mu.mol) of NaHCO.sub.3 and 1.0 ml of DMF was stirred under argon at room temperature for 5 hr. The solvent was removed by co-evaporation with water under the reduced pressure. The residue obtained was purified by chromatography on reverse phase silica (Biosil C-18; 65% MeOH in H.sub.2 O) to afford the liposomal tetrasaccharide as a white solid after lyophilization (6.8 mg, 65%). .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.7.77 (d, 2H, J=8.7 Hz), 7.70 (d, 2H, J=8.7 Hz), 5.19 (m, 1H), 5.03 (d, 1H, J=3.9 Hz), 3.45-4.62 (m, 35H), 2.87 (m, 3H), 2.28-2.31 (m, 4H), 1.98 (s, 3H), 1.88 (dd, 1H, J=13, 13 Hz), 1.57 (m, 4H), 1.26 (br s, 44H), 1.12 (d, 3H, J=6.5 Hz), 1.10 (t, 3H, J=7.0 Hz), 0.88 (br t, 6H). MS(ion spray) Calcd for C.sub.73 H.sub.122 N.sub.3 O.sub.32 PNa.sub.2 S 1662; found 1617(M-2Na.sup.+ +H.sup.+).
(b) Compound 117: yield 26 mg of a white solid after lyophilization (38%); .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.5.20 (m, 2H), 5.02 (d, 1H, J=3.8 Hz), 4.48 (d, 1H, J=7.8 Hz), 4.41-4.45 (m, 2H), 4.16 (dd, 1H, J=6.6, 12.0 Hz), 3.18-4.03 (m, 32H), 2.78-2.87 (m, 3H), 2.28-2.35 (m, 4H), 1.99 (s, 3H), 1.96 (s, 3H), 1.58-1.87 (m, 7H), 1.27 (br s, 44H), 1.14 (d, 3H, J=6.5 Hz), 0.88 (br t, 6H).
(c) Compound 118: yield 9 mg of a white solid after lyophilization (50%); .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.5.19-5.22 (m, 1H), 5.04 (d, 1H, J=3.8 Hz), 4.67 (d, 1H, J=8.0 Hz), 4.49 (d, 1H, J=7.8 Hz), 4.43 (dd, 1H, J=3.1, 12.1 Hz), 4.13-4.23 (m, 2H), 3.24-4.04 (m, 38H), 2.77-2.87 (m, 3H), 2.28-2.35 (m, 4H), 1.99 (s, 3H), 1.80 (m, 2H), 1.70 (dd, 1H, J=12.4, 12.4 Hz), 1.59 (m, 4H), 1.27 (br s, 44H), 1.13 (d, 3H, J=6.5 Hz), 0.88 (br t, 6H, J=6.4 Hz).
(d) Compound 119: yield 40 mg of a white solid after lyophilization (81%); .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.7.81 (d, 2H, J=8.7 Hz), 7.69 (d, 2H, J=8.7 Hz), 5.20 (m, 1H), 5.05 (d, 1H, J=3.9 Hz), 4.52 (d, 1H, J=7.7 Hz), 4.41 (dd, 1H, J=3.4, 12.0 Hz), 3.42-4.20 (m, 38H), 2.85 (m, 3H), 2.26-2.34 (m, 4H), 1.99 (s, 3H), 1.70 (dd, 1H, J=12.6, 12.6 Hz), 1.59 (m, 4H), 1.27 (br s, 44H), 1.17 (t, 3H, J=7.1 Hz), 1.13 (d, 3H, J=6.6 Hz), 0.88 (br t, 6H, J=6.4 Hz). MS(negative ion spray) Calcd for C.sub.79 H.sub.132 N.sub.3 O.sub.37 PNa.sub.2 S 1824; found 1801(M-Na.sup.+), 1779(M-2Na.sup.+ +H.sup.+).
(e) Compound 120: yield 6 mg of a white solid after lyophilization (20%); .sup.1 H NMR (300 MHz, CD.sub.3 OD): .delta.5.19-5.25 (m, 1H), 5.05 (d, 1H, J=3.9 Hz), 4.74 (d, 1H, J=8.0 Hz), 4.48 (d, 1H, J=7.8 Hz), 4.43 (dd, 1H, J=12.1, 3.1 Hz), 4.16 (dd, 1H, J=6.6, 12.1 Hz), 3.30-4.10 (m, 35H), 2.84 (m, 3H), 2.28-2.35 (m, 4H), 1.99 (s, 3H), 1.70 (dd, 1H, J=12.5, 12.5 Hz), 1.58 (m, 4H), 1.27 (s, 44H), 1.14 (d, 3H, J=6.5 Hz), 0.88 (br t, 6H).

Number	Name	Date
4324663	Lowe	Apr 1982
5079353	Ratcliffe et al.	Jan 1992
5143712	Brandley et al.	Sep 1992
5211936	Brandley et al.	May 1993
5211937	Brandley et al.	May 1993
5296594	Ratcliffe et al.	Mar 1994
5352670	Venot et al.	Oct 1994
5576305	Ratcliffe	Nov 1996

Number	Date	Country
WO 9119502	Dec 1991	WOX
WO 9119501	Dec 1991	WOX
WO 9207572	May 1992	WOX
WO 9216440	Oct 1992	WOX
WO 9222563	Dec 1992	WOX
WO 9426760	Nov 1994	WOX

	Number	Date
Parent	485453	Jun 1995
Parent	345072	Nov 1994
Parent	241645	May 1994
Parent	62120	May 1993

Sialyl Le.sup.x analogues as inhibitors of cellular adhesion

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Entry
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