WO2003041639A2

WO2003041639A2 - Methods and compositions for treating rotavirus-mediated disease

Info

Publication number: WO2003041639A2
Application number: PCT/US2002/034664
Authority: WO
Inventors: Kazuo Takahashi; Shiro Shigeta
Original assignee: Microbiotix, Inc.
Priority date: 2001-10-30
Filing date: 2002-10-29
Publication date: 2003-05-22
Also published as: AU2002353915A1; WO2003041639A3

Abstract

Methods and medicaments comprising a sulfated sialyl lipid compound are described for treating rotavirus-mediated gastroenteritis in an individual.

Description

METHODS AND COMPOSITIONS FOR TREATING ROTAVIRUS-MEDIATED DISEASE

FIELD OF THE INVENTION This invention is generally in the field of treating virus-mediated diseases in animals.

In particular, this invention relates to compositions and methods comprising sulfated sialyl lipid compounds for the treatment of rotavirus-mediated gastroenteritis.

BACKGROUND OF THE INVENTION Human rota virus ("HRV") is the leading etiological agent of severe dehydrating gastroenteritis in children worldwide, causing more than 850,000 deaths per year. Although most commonly seen in children in the age range of 3 months to 2 years, HRV-mediated gastroenteritis may also occur in infants of less than 2 months as well as adults ("Rotaviruses," in Encyclopedia of Virology. Vol. 3. pp. 1274-1281 (Webster and Granoff, eds.) (Academic Press, New York, 1994)). The predommant clinical symptoms of HRV- mediated gastroenteritis are, foremost, diarrhea, and vomiting, both of which can be severe, as well as fever and abdominal pain. As the result of the diarrhea and vomiting, patients may experience serious volume depletion giving rise to a range of additional symptoms such as irritability, confusion, and eventual vascular collapse leading to death. In recent years, a number of compounds have been identified that inhibit various aspects of animal viral replication or infection and, thus, are candidate compounds that may be used to treat one or more virus-mediated diseases. For example, azidothymidine ("AZT") and dideoxy inosine ("DDI"), which inhibit the reverse transcriptase of human immunodeficiency virus ("HIV"), and various compounds, which inhibit a protease of HlV, have been used in regimens to treat acquired immune deficiency syndrome ("AIDS"). However, use of such compounds to treat various viral diseases have been marked by a number of undesirable problems in a patient, which may counter-indicate repeated administration, as well as result in generation of virus strains that are resistant to such drugs. Although rotaviruses have been known to be diarrheal pathogens in animals for more than 40 years and in humans for more than 20 years, little progress has been made in developing effective therapies to prevent or treat rotavirus-mediated gastroenteritis.

SUMMARY OF THE INVENTION

This invention provides compositions, such as medicaments, and methods for treating rotavirus-mediated disease, particularly rotavirus-mediated gastroenteritis in mammals, including humans. Such methods comprise administering to a mammal in need of treatment thereof, one or more sulfated sialyl lipid compounds, such as those previously described (see, e.g., EP 0 957 107 Al , incorporated herein by reference) and described herein, which have been found to unexpectedly prevent replication (propagation) of rotavirus particles that infect and cause a viral gastroenteritis in mammals, including humans and especially children. According to the invention, sulfated sialyl lipid compounds may be administered to a mammal for prophylactic or therapeutic treatment of rotavirus-mediated gastroenteritis. In one embodiment, the invention provides methods of prophylactically or therapeutically treating rotavirus-mediated gastroenteritis in a mammal comprising administering to the mammal a sulfated sialyl lipid compound, or salt thereof, comprising a monosaccharide moiety linked to a lipid moiety, wherein the monosaccharide moiety is a sialic acid or a 2-keto-3-deoxy-D-glycero-2-nonuloic aci (KDN), wherein the lipid moiety is linked to an anomeric carbon of the sialic acid or KDN, and wherein all hydroxyl groups of the sialic acid or KDN are sulfated.

In a preferred embodiment, methods of treating rotavirus-mediated gastroenteritis in a mammal comprise administering a sulfated sialyl lipid compound comprising a monosaccharide moiety linked to a lipid moiety wherein the monosaccharide moiety of the compound comprises a sialic acid or a KDN, and wherein the monosaccharide moiety and lipid moiety of the compound are linked by an O-glycosidic linkage or an S-glycosidic linkage at position 2 of said sialic acid or KDN, or an amide linkage at position 1 of said sialic acid or KDN.

More preferably, the methods of the invention for prophylactically or therapeutically treating rotavirus-mediated gastroenteritis in a mammal comprise administering to the mammal a sulfated sialyl lipid compound as described above and further wherein the lipid moiety of the compound is a linear lipid comprising a branched chain structure at a site of branching on said linear lipid. Particularly preferred, the site of branching on the linear lipid is located at position 2, and even more preferably the branched chain structure is a two- branched chain structure. The two chains at the branching site of the linear may be identical or different structures.

In yet another embodiment, the invention provides methods of prophylactically or therapeutically treating a rotavirus-mediated disease, such as gastroenteritis, in a mammal comprising administering to the mammal a sulfated sialyl lipid compound, which comprises a monosaccharide moiety and a lipid moiety, wherein the lipid moiety of the compound is a linear lipid comprising 2 to 8 skeleton-forming atoms substituted by a branched chain structure as described herein or an aromatic lipid substituted with two chain structures on the aromatic ring. Particularly preferred are methods of treating rotavirus-mediated gastroenteritis in a mammal, including humans, comprising administering to the mammal a sulfated sialyl lipid compound that is sodium [2,2-bis(docosyloxymethyl)propyl-5-acetamido-3,5-dideoxy- 4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate. In another aspect of the invention, a sulfated sialyl lipid compound may be administered to a mammal to treat rotavirus-gastroenteritis by parenteral or non-parenteral routes. A preferred route for administering a sulfated sialyl lipid compound according to the invention is a parenteral route, and more preferably, the compound is administered orally. However, non-parenteral routes of administration may also be used in the methods described herein, including by not limited to intravenous, intramuscular, intraperitoneal, intra-arterial, subcutaneous, percutaneous, sublingual, by inhalation, and rectal routes.

Medicaments of the invention comprise one or more of the compounds mentioned above. Such medicaments may be prepared for administration to a mammal to treat a rotavirus-mediated disease orally as well as any of the other various routes mentioned above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of COMPOUND 29 of Example 29 on binding of the HRV Wa strain to MA 104 cells. Bar graphs show binding of radiolabeled HRV strain Wa (2 x 10⁴ cpm/lOO μl/well, 48 FFU) to MA104 cells in wells of a 96-well microplate in the presence of varying concentrations of COMPOUND 29 (0, 4, 20, and 100 μg/ml). y axis: HRV binding (% control); x axis: concentration of COMPOUND 29.

Figures 2A-2E show protective effect of varying amounts of COMPOUND 29 of Example 29 on HRV-induced diarrhea in suckling mice (mouse model) in graphs of Diarrhea Index (DI) as a function of days after viral infection. Litters of 6 or 7-day-old mice were orally administered water (Figure 2A) or various amounts of COMPOUND 29 of Example 29 (i.e., 50 μg COMPOUND 29 (Figure 2B), 10 μg COMPOUND 29 (Figure 2C), 2 μg compound (Figure 2D), or 0.4 μg COMPOUND 29 (Figure 2E)), three times a day, for four days, beginning 30 minutes before oral inoculation (infection) with HRV strain MO (3 x 10⁶ pfu of HRV strain MO/mouse). A six point scoring system (Diarrhea Index, DI) was used to characterize development of diarrhea in mice: 1, no stool; 2, brown formed stool; 3, soft brown stool; 4, soft mucous brown-yellow stool; 5, muddy mucous yellow stool; 6, liquid yellow stool, y axis: Diarrhea Index (DI); x axis: days after infection.

Figures 3A-3E show the influence of starting time on the protective effect of compound 29 against HRV-mediated diarrhea in suckling mice in graphs of Diarrhea Index (DI) as a function of days after viral infection. Litters of 6 or 7-day-old mice were orally administered either water (Figure 3E) or 10 μg COMPOUND 29 beginning 30 minutes prior to virus inoculation (Figure 3 A), or 6 hours (Figure 3B), 12 hours (Figure 3C), or 18 hours (Figure 3D) after virus inoculation (3 x 10⁶ pfu of MO strain/mouse). Development of diarrhea in mice assessed and scored as described for Figures 2A-2E. y axis: Diarrhea Index (DI); x axis: days after infection.

DETAILED DESCRIPTION Sulfated sialyl lipid compounds have previously been described as anti-viral compounds effective at preventing or inhibiting replication of certain unrelated animal viruses (see, e.g., EP 0 957 107 Al, incorporated herein by reference). Such compounds are glycosides comprising a monosaccharide moiety linked to a lipid moiety, wherein the monosaccharide moiety is a sialic acid or 2-keto-3-deoxy-D-glycero-2-nonuloic acid (KDN), wherein the lipid moiety is linked to an anomeric carbon of the sialic acid or KDN, and further wherein all hydroxyl groups of the sialic acid or KDN are sulfated. For example, one such representative sulfated sialyl lipid compound, sodium [2,2-bis (docosyloxymethyl) propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D- galacto-2-nonulopyranosid]onate ("COMPOUND 29" of Example 29), and has previously been shown to inhibit replication of several diverse types of viruses, such as, human immunodeficiency virus (HIV), feline immunodeficiency virus, respiratory syncytial virus, herpes simplex II virus, and adenovirus. However, none of these previous findings suggested that sulfated sialyl lipid compounds, such as COMPOUND 29, would also be effective at inhibiting replication of rota virus particles and at prophylactically or therapeutically treating rotavirus-mediated gastroenteritis. Furthermore, the level of anti-rotavirus activity of sulfated sialyl lipid compounds used in the methods of the invention is unexpectedly high. Unless otherwise noted, a sulfated sialyl lipid compound useful in the compositions and methods of the invention is understood to also include its various salt forms as well. In order that the invention may be more fully understood and described, the following definitions of additional terms are provided below.

The term "anti-viral activity", and similar terms, means the property of a compound or method described herein to prevent or inhibit replication (i.e., propagation) of virus particles either in a mammal, including humans, or in a cell culture, and/or to prophylactically or therapeutically treat a virus-mediated disease, without limitation to any particular mechanism or mode of action. In addition, any compound that has an anti-viral activity may be referred to as an "anti-viral", "anti-viral compound", "anti-viral agent", and similar terms. These latter terms may be applied to sulfated sialyl lipid compounds described herein as well as other compounds, such as any type of antibody, or fragment thereof, which binds viruses or cell receptors to viruses to prevent or inhibit viral replication and/or treat a virus-mediated disease.

As used in this specification, the term "nonuloic acid" is meant the same nonuloic acid as used and understood in the field of organic chemistry generally, which has an acidic carbohydrate having a carboxyl group at its position 1 and 9 carbon atoms. "Nonuloic acid derivatives" used in this specification include neuraminic acid (5-amino-3,5-dideoxy-D- glycero-D-galacto-nonuloic acid) and neuraminic acid derivatives, including "sialic acids". "Sialic acid" comprises a series of N-acyl derivatives of neuraminic acid, particularly N- acetylneuraminic acid and N-glycolylneuraminic acid (Yasuo Inoue, Seitai Bunsi no Kagaku, Tositu no Kagaku (Chemistry of Biomolecules 1, Chemistry of Carbohydrates), pp. 80-81 , Baifukan; Lehninger, Biochemistry, second edition, (Worth Publishers, Inc., New York, 1975), p. 261). Since "sialic acids" are acyl derivatives of neuraminic acid, they are also included in "nonuloic acid derivatives".

By "KDN" used in this specification is meant 2-keto-3-deoxy-D-glycero-2-nonuloic acid. By "two branched chains", "two chains at a site of branching", and similar terms is meant a structure comprising two chains of atoms, each chain having 7 or more skeleton- forming (backbone) atoms. Accordingly, alkyl groups, wherein the total number of skeleton- forming atoms is less than 7, are not included in "chain" in these terms. By "skeleton-forming atoms" is meant atoms constructing the skeleton or backbone of a chain, including carbon atom, oxygen atom, nitrogen atom, sulfur atom, etc. However, a monovalent atom, such as hydrogen atom, is not included in the "skeleton-forming atoms", because it cannot form the skeleton portion of the chain. By "salt" is meant the cationic salt, such as sodium and potassium salts, of a compound described herein. Such a salt is formed by neutralizing one or more, preferably all, carboxylic acid and/or sulfonic acid groups of a compound with a base containing one or more counter cations, which are attracted to the resulting anionic group(s) (e.g., carboxylate or sulfonate) without decreasing a desired biological activity (e.g., anti-viral activity) of the compound. According to the invention, any cation that does not lower a desired biological activity of a compound described herein may be useful in a salt form of a compound described herein. Preferred cations of salts used in the compositions and methods of the invention include sodium and potassium cations.

In principle, a compound useful in this invention is a glycosidic compound, or salt thereof, comprising a saccharide (i.e., a sugar) moiety and a lipid moiety, wherein the lipid moiety is linked, preferably covalently, to the anomeric carbon of the saccharide moiety, and wherein all hydroxyl groups of the saccharide moiety are sulfated. When the saccharide moiety of the glycoside is sialic acid or KDN, the glycoside not only has a strong anti-viral activity, but also a lower cytotoxicity, and can preferably be used in a method of treating a mammal, including humans, for rotavirus-mediated gastroenteritits. In this case, the hydroxyl groups of the saccharide moiety are all sulfated when the saccharide is a nonuloic acid such as N-acetylneuraminic acid, wherein sulfate groups replace the hydroxyl groups at positions 4, 7, 8 and 9 as well as the glycolyl hydroxyl group at position 5 when present. In the case wherein the nonuloic acid is KDN, all the hydroxyl groups at positions 4, 5, 7, 8 and 9 are sulfated.

The link or bond between the monosaccharide moiety and lipid moiety of a compound related to the present invention can be of any type. Preferably, the link is a covalent bond, such as an O-glycosidic linkage or an S-glycosidic linkage. Furthermore, in the case where the saccharide moiety of a compound used in the methods described herein is a nonuloic acid, the amide linkage and ester linkage may be formed using the carboxyl group at position 1 , in addition to a glycosidic linkage with the carbon atom at position 2. The bond between saccharide and lipid moieties may be such amide linkage and ester linkage. Therefore, although, by the term "glycoside" is generally meant a compound wherein the saccharide and lipid moieties are linked by a glycosidic linkage, as used herein, the term "glycoside" comprises not only compounds having a monosaccharide moiety and a lipid moiety linked by a glycosidic linkage, but also compounds having a monosaccharide moiety and lipid moiety linked by an amide or by an ester linkage, e.g., with the carboxyl group at position 1 of a nonuloic acid derivative. Preferred compounds useful in the methods described herein are glycosides with an O-glycosidic, S-glycosidic, or amide linkage.

By "lipid moiety" in a compound related to this invention is meant a lipid in a broad sense including steroid, carotinoid, terpenoid, etc., and other lipid compounds, such as cholesterol. However, the lipid moiety related to this invention is preferably a linear lipid having 2 to 8 skeleton-forming atoms or an aromatic lipid and further preferably is substituted by a branched chain structure. The branched chain structure may be two- branched or three-branched, located at position 2 of the linear lipid chain or may be a substituted aromatic lipid in which two chain structures are substituted on the ring of the aromatic ring (see, e.g., aromatic compounds in Examples 33-35 below). The lipid is preferably two-branched at the β position (i.e., the β position with respect to the sugar moiety) of said lipid moiety. For example, the compound of Example 29 (COMPOUND 29) has a three-carbon atom linear lipid substituted at position 2 with a two-branched chain structure, each branched chain having 24 skeleton-forming atoms.

The above-described two-branched chain structure may be a hydrocarbon chain, wherein one or more individual carbon atoms may be substituted with one or more heteroatoms such as oxygen, nitrogen, sulfur. Furthermore, regardless of the species of component atoms, the total number of the skeleton-forming atoms of all branched chain structures is preferably 18 to 60. In addition, the above-described two-branched chain structures may have unsaturated bonds between carbon atoms of the chain. Also, although the above-described two-branched chain can be further branched, they are preferably linear. In the case where the above-described two-branched chain contains heteroatoms as the component atom, each branched chain preferably contains an ester bond or ether bond, and furthermore, said ester bond or ether bond is preferably localized at position 1 or 2 of said branched chain.

Herein, when the ester bond or ether bond is present at position 1 of the linear lipid, the compound related to this invention will become the sulfated derivative of a sialoglycerolipid with anti-viral activity. In this connection, when an ether bond is present, said compound will be an alkyl glycerol wherein a long-chain alcohol is linked to the glycerol residue, and when an ester bond is present, said compound will be amyl glycerol. Furthermore, in the case where the ester bond or ether bond is located at position 2 of the branched chain, the glycerol residue in the glycerol area is suitably modified to become pseudo-glycerol.

As to the length of branched chains, the number of skeleton-forming atom is preferably 10 to 28, more preferably 18 to 26, most preferably 24. In addition, branched chains can be of different lengths, but preferably of the same length, most preferably of the same structure comprised of the same component atoms.

Methods of Treatment, Pharmaceutical Compositions, Modes of Administration

The invention provides methods of treating rotavirus infections, especially rotavirus- mediate gastroenteritis in a mammal, including humans, which comprise administering to the mammal a sulfated sialyl lipid compound to inhibit or prevent replication (propagation) of rotavirus in the mammal. Such methods may be therapeutic prophylactic (preventative or protective). Although a sulfated sialyl lipid compound described herein may be administered to a mammal alone, preferably the sulfated sialyl compound is present in a pharmaceutical composition. Such compositions are useful in the methods of the invention which comprise administering to a mammal one or more sulfated sialyl lipid compounds in an amount effective to prevent or inhibit replication of rotavirus as evidence by prevention of gastroenteritis or diminution of symptoms of gastroenteritis, most notably, diarrhea, but also other known clinical symptoms, such as vomiting, fever, abdominal pain, blood in stool, volume depletion, lethargy, irritability, and confusion.

A pharmaceutical composition (medicament) comprising sulfated sialyl lipid compound may be in any of a variety of forms particularly suited for the intended mode of administration, including solid, semi-solid or liquid dosage forms, for example, tablets, lozenges, pills, capsules, powders, suppositories, liquids, powders, aqueous or oily suspensions, syrups, elixirs, and aqueous solutions. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or multiple of a dose which is calculated to produce the desired affect on gut motility. A composition used in the methods of the invention will preferably include, an effective amount of one or more sulfated sialyl lipid compounds in combination with a pharmaceutically acceptable carrier and/or buffer, and, in addition, may further include other medicinal agents or pharmaceutical agents, carriers, diluents, fillers and formulation adjuvants, or combinations thereof, which are non-toxic, inert, and pharmaceutically acceptable. In liquid mixtures or preparations, a pharmaceutically acceptable buffer, such as a phosphate buffered saline may be used. By "pharmaceutically acceptable" is meant a material that is not biologically, chemically, or in any other way, incompatible with mammalian body chemistry and metabolism and also does not adversely affect any other component that may be present in the pharmaceutical composition.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Pharmaceutically acceptable liquid compositions can, for example, be prepared by dissolving or dispersing an active compound that regulates gut motility as described herein and optimal pharmaceutical adjuvants in an excipient, such as, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, triethanolamine oleate.

Standard methods of preparing dosage forms are known, or will be apparent, to those skilled in this art (see, for example, Remington's Pharmaceutical Sciences (Martin, E.W. (ed.) latest edition Mack Publishing Co., Easton, PA).

The primary active ingredient of a composition used in the methods of this invention is a sulfated sialyl lipid compound, such as COMPOUND 29, which inhibits or prevents replication of rotavirus in a mammal. A preferred method of treating rotavirus-mediated gastroenteritis in a mammal comprises orally administering a sulfated sialyl lipid to the mammal. Accordingly, a preferred composition for use in the methods of this invention is formulated for oral administration. However, according to methods of the invention a sulfated sialyl lipid may be administered by any of a variety of other routes of administration, including, by not limited to parenteral or non-parenteral routes, including but not limited to, intravenously, intramuscularly, intraperitoneally, intra-arterially, subcutaneously, percutaneously, sublingually, inhalationally, or rectally.

For oral administration, which is preferred, compositions of the invention may be formulated as fine powders or granules containing of the compound that affects gut motility and may also contain diluting, dispersing, and/or surface active agents. Compositions for oral administration may also be presented in water or in a syrup as a solution or suspension, in pills, tablets, capsules or sachets in the dry state, or in a non-aqueous solution or suspension wherein suspending agents may be included. Binders and lubricants may also be used in compositions for oral administration. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

Parenteral administration, if used, is generally a method of injection. Injectable preparations can be prepared in conventional forms, either liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. For most purposes, a compound useful in regulating gut motility may be injected intravenously in a pharmaceutically acceptable buffer. However, it is within the scope of this invention that such a compound may alternatively be prepared as a bolus, which may contain a mordant for gradual release from an injection site. One approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained (see, for example, U.S. Patent No. 3,710,795).

The exact, effective amount of a sulfated sialyl lipid compound useful in treating rotavirus mediated gastroenteritis in the compositions and methods described herein will vary from subject to subject, depending on the age, weight and general condition of the subject, the advancement of the rotavirus infection, the particular compound used, its mode of administration, and the like. Prophylactically, the goal is to prevent development of clinical symptoms of rotavirus infection. Therapeutically, the goal is to reduce or eliminate one or more known symptoms of rotavirus-mediated gastroenteritis, such as diarrhea, vomiting, fever, and/or other known symptoms. For commercial pharmaceutical compositions, it is understood that a pharmaceutically effective and suitable amount of sulfated sialyl lipid compound will be determined, in the case of human use, by the healthcare professional in studies acceptable to the standards of the United States Food and Drug Administration (or comparable agency). For use in other mammals, an appropriate composition will be determined and formulated according to the standards and practices for veterinary medicine.

Additional embodiments and features of the invention will be apparent from the following non-limiting examples.

EXAMPLES Example 1 : Sodium [methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosid] onate .

Methyl 5-acetamido-3,5-dideoxy-D-glycero- (α)-D-galacto-2-nonulopyranosidonate (Chem. Ber., 99, 611(1966)) (200 mg, 0.58 mmol) and sulfur trioxide-trimethylamine complex (1600 mg, 11.5 mmol) were stirred in anhydrous dimefhylformamide (5.8 ml) under the argon atmosphere at 80-85°C for 2 h. The reaction mixture was purified directly by silica gel column chromatography (gel 209 g, chloroform/methanol/water, 5:4: 1), and further treated with Dowex 50W-X8 (Na form) resin. The product was further purified by gel chromatography (Sephadex G-25, 400 ml, water) to obtain the title compound (359 mg, 82%) as white solid.

Η-NMR (D₂O) δ: 3.37 (s, 3H, OCH₃), 2.88 (dd, 1H, J =4.0, 11.1 Hz, H-3eq), 1.97 (s, 3H, NAc), 1.77 (t, 1H,J =12.1 Hz, H-3ax).

Example 2: Sodium [methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-

D-glycero-β-D-galacto-2-nonulopyranosid] onate.

Methyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonate

(Chem. Ber., 99, 611(1966)) (200 mg) was reacted by the general procedure according to

Example 1 to obtain the title compound (218 mg, 50%) as white solid. ¹H-NMR (D₂O) δ: 3.33 (s, 3H, OCH₃), 2.57 (dd, 1H, J =5.1, 13.2 Hz, H-3eq), 1.96 (s,

3H, NAc), 1.90 (t, 1H, J =13.2 Hz, H-3ax).

Example 3: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl) - D-glycero-α -D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-hexyl-Sn-glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-hexyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Hei 1- 125394) (19 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (15 mg, 46%) as white solid.

Η-NMR (CD₃OD) δ: 3.02 (dd, 1H, J =4.9, 12.6 Hz, H-3eq), 1.92 (s, 3H, NAc), 1.83 (t, 1H, J =12.1 Hz, H-3ax), 1.39-1.26 (m, 12H, 6CH₂), 0.90 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 4: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero-β-D-galacto-2-nonulopyranosyl} onate]- 1,2-di-O-hexyl-Sn-glycerol.

3-O- [Sodium (5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-hexyl-Sn-glycerol (Japanese Patent Laid-Open Publication No.Heil-125394) (26 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (22 mg, 48%) as white solid. Η-NMR (CD₃OD) δ: 2.83 (dd, IH, J =5.1, 12.8 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.87 (t, IH, J =12.3 Hz, H-3ax), 1.37-1.34 (m, 12H, 6CH₂), 0.90 (t, 3H, J =6.8 Hz, CH₂CH₃), 0.90 (t, 3H, J =7.0 Hz, CH₂CH₃).

Example 5: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-decyl-Sn-glycerol.

3-O- [Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-decyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Hei 1 - 125394) (190 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (268 mg, 88%) as white solid.

Η-NMR (D₂O) δ: 2.94 (dd, IH, J =5.0, 13.0 Hz, H-3eq), 1.99 (s, 3H, NAc), 1 .96 (t, IH, J =12.5 Hz, H-3ax), 1.44-121 (m, 28H, 14CH₂), 0.89 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 6: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl) - D-glycero-β-D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-decyl-Sn-glycerol.

3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl) onate]- 1,2-di-O-decyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Hei l -125394)

(197 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (240 mg, 76%) as white solid. Η-NMR (D₂O) δ: 2.67 (dd, IH, J =5.0, 13.0 Hz, H-3eq), 2.01 (s, 3H, NAc), 1.93 (t,

IH, J =12.2 Hz, H-3ax), 1.42-1.20 (m, 28H, 14CH₂), 0.88 (t, 6H, J =6.8 Hz; 2CH₂CH₃).

Example 7: 3-O- [Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-tetradecyl-Sn-glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate]-l ,2-di-O-tetradecyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Sho59- 164798) (406 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (479 mg, 78%) as white solid.

Η-NMR (CD3OD) δ: 3.01 (dd, IH, J =4.9, 1 1.9 Hz, H-3eq), 1.93 (s, 3H, NAc), 1.74 (t, IH, J =12.1 Hz, H-3ax), 1.38-1.25 (m, 44H, 22CH₂), 0.90 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 8: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D- glycerol-β-D-galacto-2-nonulopyranosyl} ornate]-! ,2-di-O-tetradecyl-Sn- glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-β α-D-galacto-2- nonulopyranosyl) ornate) - 1 ,2-di-O-tetradecyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Sho59- 164798) (87 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (120 mg, 91%) as white solid. Η-MR (CD₃OD) δ: 2.83 (DD, IH, J =5.5, 12.8 Hz, H-3eq), 2.03 (s, 3H, ANC), 1.85

(t, IH, J =12.0 Hz, H-ax), 1.45-1.20 (m, 44H, 22CH₂), 0.90 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 9: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D- glycerol-α-D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-octadecyl-Sn-glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-octadecyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Hei 1- 125394) (45 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (44 mg, 66%) as white solid.

Η-NMR (CD₃OD) δ: 3.02 (dd, IH, J =5.1, 13.2 Hz, H-3eq), 1.92 (s, 3H, NAc), 1.82 (t, IH, J =12.5 Hz, H-3ax), 1.38-1.22 (m, 60H, 30CH2, 0.90 (t, 6H, J =6.9 Hz, 2CH₂CH₃).

Example 10: 3-O- [Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiurn oxysulfonyl)- D-glycero- -D-galacto-2-nonulopyranosyl} onate]-2-O- (sodium oxysulfonyl)- l-O- octadecyl-Sn-glycerol . 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate]- 1-O-octadecyl-Sn-glycerol (Japanese Patent Publication No. Heil-125394) (132 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (125 mg, 53%) as white solid.

Η-NMR (CD₃OD) δ: 3.07 (dd, IH, J =5.1, 12.1 Hz, H-3eq), 1.95 (s, 3H, NAc), 1.68 (t, IH, J =12.1 Hz, H-3ax), 1.36-1.22 (m, 30H, 15CH₂), 0.90 (t, 3H, J =7.0 Hz, CH₂CH₃).

Example 1 1 : 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-β-D-galacto-2 -nonulopyranosyl} onate]-2-O-(sodium oxysulfonyl)- 1-O-octadecyl- Sn-glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2 -nonulopyranosyl) onate]- 1-O-octadecyl-Sn-glycerol (Japanese Patent Publication No. Heil-125394) (66 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (31 mg, 49%) as white solid. Η-NMR (CD₃OD) δ: 2.81 (dd, IH, J= 4.4, 12.1 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.88 (t, IH, J =11.9 Hz, H-3ax), 1.38-1.17 (m, 30H, 15CH₂), 0.90 (t, 3H, J =7.0 Hz, CH₂CH₃).

Example 12: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiurn oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-l,2-di-O-docosyl-Sn-glycerol.

3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate] -1,2-di-O-docosyl-Sn-glycerol (Japanese Patent Publication No. Heil-125394) (502 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (447 mg, 54%) as white solid. Η-NMR (CD₃OD-D₂O, 1 :1, 50°C) δ: 2.96 (br. dd, IH, H-3eq), 1.97 (s, 3H, NAc),

1.83 (br. t, IH, H-3ax), 1.40-1.21 (m, 76H, 38CH₂), 0.89 (t, 6H, J =6.4 Hz, 2CH₂CH₃).

Example 13: 3-O-[Sodium {5- acetamido-3,5-dideoxy- 4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-β-D-galacto-2 -nonulopyranosyl} onate]- 1,2-di-O-docosyl-Sn-glycerol. 3-O-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl) onate]- 1,2-di-O-docosyl-Sn-glycerol (Japanese Patent Publication No. Heil-125394) (44 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (25 mg, 40%) as white solid.

Η-NMR (CDCl₃-CD₃OD-D₂O, 3:4:2) δ: 2.75 (br. dd, IH, H-3eq), 2.02 (s, 3H, NAc), 1.89 (br. t, IH, H-3ax), 1.40-1.18 (m, 76H, 38CH₂), 0.89 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 14: 3-S-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-l,2-di-O-tetradecyl-Sn-thioglycerol.

3-S-[Sodium (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl) onate]- 1,2-di-O-tetradecyl-Sn-thioglycerol ( Japanese Patent Laid-Open Publication No. Sho64-52794) (41 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (47 mg, 77%) as white solid.

Η-NMR (CD₃OD) δ: 3.17 (dd, IH, J =5.1, 12.1 Hz, H-3eq),1.92 (s, 3H, NAc), 1.77

(t, IH, J =11.7 Hz, H-3ax), 1.41-1.23 (m, 44H, 22CH₂), 0.90 (t, 6H, J =7.1 Hz, 2CH₂CH₃).

Example 15: 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-eicosyl-Sn-glycerol. A. 3-O-benzyl-l ,2-di-O-eicosyl-Sn-glycerol: 3-O-Benzyl-Sn-glycerol (Agric. Biol. Chem., 46, 255 (1982)) (300 mg, 1.65 mmol), 1-bromo-eicosane (2.38 g, 6.58 mmol) and pulverized sodium hydroxide (293 mg, 7.33 mmol) were azeotropically heated at reflux in benzene (10 ml) for 2 days to remove water from the mixture. After the reaction solution was diluted with ether and washed with water, the organic layer was dried over anhydrous magnesium sulfate and then condensed in vacuo. The residue was purified by silica gel column chromatography (100 g of gel, hexane: toluene = 3:2) to obtain the title compound (929 mg, 76%) as white powder.

Η-NMR (CDC1₃) δ: 7.34-7.26 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m, 68H, 34H2), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

B . 1 ,2-Di-O-eicosyl-Sn-glycerol :

3-O-benzyl-l,2-di-O-eicosyl-Sn-glycerol (1.47 g, 1.98 mmol) and 10% palladium- charcoal (200 mg) were stirred in ethyl acetate (30 ml) under the hydrogen atmosphere at room temperature for 18 h. The reaction mixture was filtered through celite, and the filtrate was concentrated in vacuo to obtain the title compound (934 mg, 72%) as white powder. Η-NMR (CDCI3) δ: 1.25 (m, 68H, 34CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

C. 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-eisosyl-Sn-glycerol. A mixture comprising 1,2-di-O-eicosyl-Sn-glycerol (497 mg, 0.76 mmol), methyl 5- acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-tn deoxy-D-glycero-β-D-galacto-2- nonulopyranosonate (Chem. Ber., 99, 611(1966) ) (422 mg, 0.83 mmol), mercury (II) cyanide (336 mg, 1.33 mmol), mercury (II) bromide (478 mg, 1.33 mmol) and dried molecular sieve 4A (1.0 g) in anhydrous chloroform (5.0 ml) was stirred under the nitrogen atmosphere at room temperature overnight. After the reaction mixture was filtered, the filtrate was concentrated in vacuo, and purified by intermediary pressure silica gel column chromatography (125 g of gel, toluene:ethyl acetate = 3:2) to obtain the title compound (390 mg, 46%) as white power.

Η-NMR (CDCI₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, IH, J=4.8, 12.8 Hz, H-3 eq.), 2.14, 2.13, 2.06, 2.04, 1.88 (5s, 15H, 5Ac), 1.98 (t, IH, J =12.8 Hz, H-3ax.), 1.25 (m, 68H, 34CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃). Example 16: 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2-yl]-l,2-di-O-eicosyl-Sn-glycerol.

3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-eicosyl-Sn-glycerol (455 mg, 0.40 mmol) and sodium methoxide was stirred in a mixture (13 ml) of THF:methanol (1 : 1) overnight. To this reaction mixture was added 3N sodium hydroxide (380 μl), and the mixture was stirred at 60 °C for 1 h. After the reaction mixture was concentrated in vacuo, the residue was dissolved in ethanol and water, and the solution was adjusted to pH 2 with formic acid. Precipitates were collected by filtration, washed successively with water, methanol and ether, and dried in vacuo to obtain the title compound (365 mg, 96%) as white powder.

Η-NMR (CDCI₃-CD₃OD, 1 : 1) δ: 2.79 (dd, IH, J =3.9, 12.3 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.69 (t, IH, J =11.5 Hz, H-3ax.), 1.43-1.20 (m, 68H, 34CH₂), 0.89 (t, 6H, J =7.0 Hz,

Example 17: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- -D-galacto-2-nonulopyranosyl} onate]- 1,2-di-O-eicosyl-Sn-glycerol:

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid) -2- yl] -1,2-di-O-eicosyl-Sn-glycerol (241 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (236 mg, 67%) as white solid. Η-NMR (CD₃OD) δ: 3.02 (dd, IH, J =4.4, 12.4 Hz, H-3eq.), 1.92 (s, 3H, NAc), 1.80

(t, IH, J =11.0 Hz, H-3ax.), 1.44-1.13 (m, 68H, 34CH₂), 0.909 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 18: 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-tetracosyl-Sn-glycerol. A. 3-O-Benzyl-l,2-di-O-tetracosyl-Sn-glycerol:

3-O-Benzyl-Sn-glycerol (Agric. Biol. Chem., 46, 255 (1982)) and 1 -bromotetracosane (J. Am. Chem. Soc, 115, 3840 (1993)) were reacted by the general procedure according to Example 1 to obtain the title compound (236 mg, 67%) as white solid.

Η-NMR (CDCI₃) δ: 7.34-7.26 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m, 84H, 42CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃). B. 1 ,2-Di-O-tetracosyl-Sn-glycerol:

3-O-Benzyl-l,2-di-O-tetracosyl-Sn-glycerol (1.47 g) was reacted in a mixture (30 ml) of toluene-ethyl acetate (1 : 1) by the general procedure according to 15-B to obtain the title compound (0.80 g, 66%) as white solid. Η-NMR (CDC1₃) δ: 1.25 (m, 84H, 42CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

C. 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-tetracosyl-Sn-glycerol:

1 ,2-Di-O-tetracosyl-Sn-glycerol (497 mg, 0.76 mmol) and methyl-5-acetamido- 4,7,8,9-tetra-O-acetyl-2-chloro-2, 3,5 -trideoxy-D-glycero-β-D-galacto-2- nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (422 mg, 0.83 mmol) were reacted by the general procedure according to 15-C to obtain the title compound (224 mg, 14%) as white solid.

Η-NMR (CDCI₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, IH, J=4.8, 12.8 Hz, H-3eq), 2.14, 2.13, 2.04, 2.03, 1.88 (5s, 15H, 5Ac), 1.98(t, IH, J =12.8 Hz, H-3ax), 1.25 (m, 84H, 42CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 19: 3-O- [(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2-yl]-l,2-di-O-tetracosyl-Sn-glycerol. 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate] -1,2-di-O-tetracosyl-Sn-glycerol (240 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (160 mg, 79%) as white solid.

1H-NMR (CDCI₃-CD₃OD, 1 :1) δ: 2.81 (dd, IH, J =3.7, 12.1 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.66 (t, IH, J =1 1.3 Hz, H-3ax),1.38-1.16 (m, 84H, 42CH₂), 0.89 (t, 6H, J =6.8 Hz,

Example 20: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- -D-galacto-2 -nonulopyranosyl} onate]- 1,2-di-O-tetracosyl-Sn-glycerol. 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid) reacted by the general procedure according to Example 1 to obtain the title compound (21 mg, 19%) as white solid. 1H-NMR (CD₃OD-D₂O, 1 : 1, 50°C) (δ): 2.96 (br. dd, IH, H-3eq), 2.01 (s, 3H, NAc), 1.81 (br. t, IH, H-3ax), 1.36-1.10 (m, 84H, 42CH2), 0.89 (t, 6H, J =6.4 Hz, 2CH₂CH₃).

Example 21 : 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1,2-di-O-hexacosyl-Sn-glycerol.

A. 3-O-Benzyl-l ,2-di-O-hexacosyl-Sn-glycerol:

3-O-Benzyl-Sn-glycerol (Agric. Biol. Chem., 40, 391 (1976)) (242 mg) and 1- bromohexacosane (Agric. Biol. Chem., 46, 255 (1982)) (1.30 g) were reacted by the general procedure according to Example 15-A. Then, the compound thus obtained was, without purification, subjected to the debenzylation by the general procedure according to Example 15-B to obtain the title compound (615 mg, 57%) as white solid.

1H-NMR (CDCI₃) δ: 1.25 (m, 92H, 46CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

B. 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-hexacosyl-Sn-glycerol:

1,2-Di-O-hexacosyl-Sn-glycerol (610 mg, 0.74 mmol) and methyl 5-acetamido-4, 7, 8, 9-tetra-O-acetyl -2-chloro-2, 3, 5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (454 mg, 0.89 mmol) were reacted by the general procedure according to Example 15-C to obtain the title compound (76 mg, 8%) as white solid. 1H-NMR (CDCI₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, IH, J=4.8, 12.8 Hz, H-3eq), 2.14,

213, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.97 (t, IH, J =12.8 Hz, H-3ax), 1.25 (m, 92H, 46CH₂), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 22: 3-O-(5-Acetamido-3,5-dideoxy-D-glycero-(α)-D-galacto-nonulopyranosonic acid) -2-yl)-l,2-di-O-hexacosyl-Sn-glycerol.

3-O- ( Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-(α)-D- galacto-2-nonulopyranosyl) onate) -1,2-di-O-hexacosyl-Sn-glycerol (76 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (51 mg, 78%) as white solid. Η-NMR (CDCI₃-CD₃OD, 1:1, 60°C) δ: 2.76 (dd, IH, J=4.4, 12.8 Hz, H-3eq), 2.02 (s,

3H, NAc), 1.75 (t, IH, J =11.7 Hz, H-3ax), 1.42-1.14 (m, 92H, 46CH₂), 0.89 (t, 6H, J =6.8

Hz, 2CH₂CH₃). Example 23: 3-O- [Sodium {5- acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-l,2-di-O-hexacosyl-Sn-glycerol.

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid) -2- yl]-l,2-di-O-hexacosyl-Sn-glycerol (47 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (29 mg, 45%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 :1, 60°C) δ: 2.96 (br. dd, IH, H-3eq), 1.96 (s, 3H, NAc), 1.79 (br. t, IH, H-3ax), 1.42-1.14 (m, 92H, 46CH2), 0.88 (br. t, 6H, J=6.2 Hz, CH₂CH₃).

Example 24: 3β-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero- -D-galacto-2-nonulopyranosyl} onate]-5-cholestene.

3 β-[(5-Acetamido-3 ,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonic acid)-2- yl]-5-cholestene (Japanese Patent Laid-Open Publication No, Sho 61-243096) (387 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (232 mg, 37%) as white solid. Η-NMR (CD₃OD-D₂O, 1 :1) (δ) : 2.92 (dd, IH, J =4.5, 11.5 Hz, H-3eq), 1.96 (s, 3H,

NAc), 0.99 (s, 3H, CH₃-19 chole), 0.68 (s, 3H, CH₃-I8 chole).

Example 25: Sodium [oleyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-

D-glycero-α-D-galacto-2-nonulopyranosid] onate. A. Sodium (oleyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid) onate:

To a solution of methyl (oleyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero- α-D-galacto-2-nonulopyranosid) onate (Japanese Patent Laid-Open Publication No.

Sho 63-264493) (115 mg, 0.154 mmol) dissolved in methanol (1.5 ml) was added 3N sodium hydroxide (0.30 ml), and the mixture was stirred at room temperature for 16 h. The reaction solution was evaporated to dryness in vcuo, and the residue was purified by gel chromatography (Sephadex LH-20, 180 ml, methanol) to obtain the title compound (78 mg,

87%) as white solid.

Η-NMR (CD₃OD) δ: 5.34 (m, 2H, CH = CH), 2.81 (dd, IH, J=4.0, 12.1 Hz, H-3 eq), 2.00 (s, 3H, NAc), 1.59 (t, IH, J =11.7 Hz, H-3ax), 1.42-1.23 (m, 20H, 10CH₂), 0.90 (t, 3H, J

B. Sodium [oleyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- α-D-galacto-2-nonulopyranosid] onate :

Sodium (oleyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulo-pyranosid) onate (62 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (86 mg, 82%) as white solid.

1H-NMR (CD₃OD) (δ): 3.03 (dd, IH, J=3.7, 12.3 Hz, H-3eq), 2.19 (m, 4H, CH2CH= CHCH2), 1.92 (s, 3H, NAc), 1.73 (t, IH, J=12.1 Hz, H-3ax), 1.42-1.22 (m, 20H, 10CH2), 0.90 (t, 3H, J =6.8 Hz, CH₂CH₃).

Example 26: Sodium [octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid] onate.

A. Methyl (octadecyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D- galacto-2-nonulopyranosid)onate:

To a solution of methyl (oleyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero- α-D-galacto-2-nonulopyranosid)onate (Japanese Patent Laid-Open Publication No. Sho 63-264493) (111 mg, 0.149 mmol) dissolved in ethanol (1.5 ml) was added 10 % palladium-charcoal (16 mg) was added, and the mixture was stirred under the hydrogen atmosphere at room temperature for 18 h. The reaction solution was filtered through celite to obtain the title compound (107 mg, 96%) as white solid. Η-NMR (CDCI₃) δ: 3.79 (s, 3H, COOCH₃), 2.58 (dd, IH, J=4.8, 12.8 Hz, H-3 eq),

2.15, 2 14, 2 04, 2 03, 1.88 (5s, 15H, 5Ac), 1.95 (t, IH, J =12.6 Hz, H-3ax), 1.38-1.19 (m, 30H, 15CH₂), 0.88 (t, 3H, J=7.0 Hz, CH₂CH₃).

B. Sodium (octadecyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid) onate:

Methyl (octadecyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosid) onate (107 mg) was reacted by the general procedure according to Example 25-A to obtain the titled compound (70 mg, 83%) as white solid.

1H-NMR (CD₃OD) δ: 2.81 (dd, IH, J =3.8, 12.3 Hz, H-3eq), 2.00 (s, 3H, NAc), 1.59 (t, IH, J =11.9 Hz, H-3ax), 1.38-1.22 (m, 30H, 15CH₂), 0.90 (t, 3H, J =6.8 Hz, CH₂CH₃).

C. Sodium [octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D- glycero-α-D-galacto-2-nonulopyranosid] onate : Sodium (octadecyl 5-acetamido-3 ,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosid) onate (72 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (109 mg, 92%) as white solid.

¹H-NMR (CD₃OD) δ: 3.02 (dd, 1H, J =3.7, 11.4 Hz, H-3eq), 1.92 (s, 3H, NAc), 1.73 (t, IH, J =11.7 Hz, H-3ax), 1.40-1.31 (m, 30H, 15CH₂), 0.90 (t, 3H, J =7.0 Hz, CH₂CH₃).

Example 27: Methyl [2,2-bis (docosyloxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero- -D-galacto-2-nonulopyranosid] onate.

A. 2,2- bis (docosyloxymethyl) propanol: 1,1,1 -Tris(hydroxymethyl) ethane (1.0 g, 8.32 mmol) and sodium hydride (732 mg,

18.3 mmol) were stirred in dehydrated dimethylformamide (30 ml) at room temperature for 15 min. Then, the reaction solution was cooled in ice, and docosyl bromide (7.1 g, 18.3 mmol) and benzene (10 ml) were added thereto, and the resulting mixture was stirred at room temperature for 16 h. The reaction solution was concentrated in vacuo, and the residue was suspended in chloroform, and washed with 2N HC1. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (130 g of gel, toluene/ethyl acetate, 19: 1) to obtain the title compound (2.32 g, 38%) as white powder.

'H-NMR (CDC1₃) δ: 3.56 (d, 2H, J =5.9 Hz, CH₂OH), 1.26 (m, 76H, 38CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃), 0.85 (s, 3H, CCH₃).

B. Methyl [2,2-bis (docosyloxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-α-D-galacto-2-nonulopyτanosid] onate :

2,2-Bis (docosyloxymethyl) propanol (1.20 g, 1.63 mmol) and methyl 5-acetamido- 4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-(β)-D-galacto-2-nonulo pyranosonate (Chem. Ber., 99, 611 (1966)) (913 mg, 1.79 mmol) were reacted by the general procedure according to Example 15-C to obtain the title compound (786 mg, 40%) as white solid.

Η-NMR (CDCI₃) δ: 3.78 (s, 3H, COOCH₃), 2.57 (dd, IH, J=4.4, 12.8 Hz, H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.95 (t, IH, J =12.5 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃). Example 28: 2,2-Bis (docosyloxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosidonic acid:

Methyl [2,2-bis (docosyloxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid] onate (786 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (599 mg, 90%) as white solid.

Η-NMR (CDCI₃-CD₃OD, 1 :1, 60°C) δ: 2.79 (dd, IH, J=4.8, 12.8 Hz, H-3eq), 2.02 (s, 3H, NAc), 1.75 (t, IH, J =11.9 Hz, H-3ax), 1.36-1.22 (m, 76H, 38CH₂), 0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 29: Sodium [2,2-bis(docosyloxymethyl)propyl-5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulo pyranosid] onate

("COMPOUND 29").

2,2-Bis (docosyloxymethyl)propyl-5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto- 2-nonulopyranosidonic acid (94 mg) was reacted by the general procedure according to

Example 1 to obtain the title compound (63 mg, 47%) as white solid. (599 mg, 90%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 :1, 60°C) δ: 2.93 (br. dd, IH, H-3eq), 1.96 (s, 3H, NAc),

1.80 (br. t, IH, H-3ax), 1.43-1.16 (m, 76H, 38CH₂), 0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J =6.6 Hz, 2CH2CH3).

Example 30: Methyl [2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid] onate.

A. 2,2-Bis (eicosyl oxymethyl) propanol: 1,1,1-Tris (hydroxymethyl) ethane (0.50 g, 4.16 mmol) and eicosyl bromide (3.30 g,

9.15 mmol) were reacted by the general procedure according to Example 27-A to obtain the title compound (1.43 g, 51%) as white solid.

Η-NMR (CDCI₃) δ: 3.57 (d, 2H, J =5.9 Hz, CH₂OH), 1.26 (m, 68H, 34CH₂), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃), 0.85 (s, 3H, CCH₃).

B. Methyl [2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid] onate: 2,2-bis (eicosyl oxymethyl) propanol (267 mg, 0.392 mmol) and methyl 5-acetamido- 4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (200 mg, 0.392 mmol) were reacted by the general procedure according to Example 15-C to obtain the title compound (175 mg, 39%) as white solid. Η-NMR (CDC1₃) δ: 3.78 (s, 3H, COOCH₃), 2.58 (dd, IH, J=4.8, 12.8 Hz, H-3eq.),

2.13, 2.13, 2.04, 2.02, 1.89 (5s, 15H, 5Ac), 1.95 (dd, IH, J =12.5, 12.8 Hz, H-3ax.), 1.25 (m, 68H, 34CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 31: 2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosidonic acid.

Methyl (2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-0-acetyl-3,5- dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid) onate (174 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (127 mg, 87%) as white solid. Η-NMR (CDCI₃-CD₃OD, 1 : 1) (δ): 2.75 (dd, IH, J =4.6, 12.6 Hz, H-3eq), 2.03 (s,

3H, NAc), 1.75 (t, IH, J =11.7 Hz, H-3ax), 1.43-1.19 (m, 68H, 34CH₂), 0.92 (s, 3H, CCH₃),

0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 32: Sodium [2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulo pyranosid] onate.

2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto- 2-nonulopyranosidonic acid (103 mg) acid was reacted by the general procedure according to Example 1 to obtain the title compound (96 mg, 64%) as white solid.

Η-NMR (CD₃OD-D₂O, 1: 1, 60°C) δ: 2.96 (br. dd, IH, H-3eq), 1.98 (s, 3H, NAc), 1.78 (t, IH, J =12.6 Hz, H-3ax), 1.37-1.17 (m, 68H, 34CH₂), 0.94 (s, 3H, CCH₃), 0.90 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 33: Methyl [3,5-didocosyl oxyphenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-α-D-galacto-2-nonulo pyranosid] onate. A. 3,5-didocosyl oxyphenol:

After phloroglucinol (2.0 g, 15.9 mmol) and sodium hydride (1.59 g, 39.7 mmol) were stirred in dehydrated dimethylformamide (30 ml) at room temperature for 15 min, docosyl bromide (13.0 mg, 33.3 mmol) and benzene (30 ml) were added thereto, and the resulting mixture was stirred at 40°C for 2 days. The reaction solution was diluted with chloroform, washed with 2 N hydrochloric acid, and the organic layer was dried over anhydrous magnesium sulfate. The solvent was concentrated in vacuo, and the residue was purified by silica gel column chromatography (90 g of the gel, hexane/ethyl acetate, 6: 1) to obtain the title compound (2.40 g, 20%) as light yellow powder.

Η-NMR (CDC1₃) δ: 6.06 (d, IH, J =2.2 Hz, H-4 phenyl), 5.99 (2d, 2H, J =2.2 Hz, H- 2,6 phenyl), 3.89 (t, 4H, J =6.8 Hz, 2OCH₂), 1.39-1.17 (m, 72H, 36CH₂), 0.88 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

B. Methyl (3,5-didocosyloxyphenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero-α-D-galacto-2-nonulopyranosid) onate :

3,5-Didocosyloxyphenol (1.20 g, 1.61 mmol) and sodium hydride (77.5 g, 1.94 mmol) were stirred in dimethylformamide:benzene (1 : 1, 40 ml) at room temperature for 20 min.

Then, to this mixture was added methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5- trideoxy-D-glycero-(β) -D-galacto-2-nonulopyranosonate (Chem. Ber., 99, 61 1 (1966)) (823 mg, 1.61 mmol), and the resulting mixture was stirred at room temperature for 3 h. After the reaction solution was diluted with chloroform, and washed with 2N-hydrochloric acid and, the organic layer was dried over anhydrous magnesium sulfate. The solvent was condensed in vacuo, and the residue was purified by silica gel column chromatography (90 g of the gel, toluene/ethyl acetate, 2:3) to obtain the title compound (386 mg, 20%) light yellow powder.

Η-NMR (CDC1₃) δ: 6.28 (2d, 2H, J =1.8 Hz, H-2,6 phenyl), 6.22 (d, IH, J =1.8 Hz,

H-4 phenyl), 3.78 (s, 3H, COOCH₃), 2.58 (dd, IH, J = 4.8, 12.8 Hz, H-3eq.), 2.14, 2.10, 2.03,

2.01, 1.90 (5s, 15H, 5Ac), 1.26 (m, 76H, 38CH₂), 0.88 (t, 6H, J = 6.6 Hz, 2CH₂CH₃).

Example 34: 3,5-Didocosyloxyphenyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyrasidonic acid.

Methyl (3,5-didocosyloxyphenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero-α-D-galacto-2-nonulopyranosid) onate (385 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (163 mg, 50%) as light yellow solid.

Η-NMR (CDCI₃-CD₃OD, 1 :1, 60°C) δ: 6.42 (2d, 2H, J=2.2 Hz, H-2,6 phenyl), 6.20

(d, IH, J =2.2 Hz, H-4 phenyl), 2.86 (dd, IH, J =4.2, 12.6 Hz, H-3eq), 2.02 (s, 3H, NAc), 1.95 (t, IH, J =11.9 Hz, H-3ax), 1.40-1.23 (m, 72H, 36CH₂), 0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 35: Sodium [3,5-Didocosyloxyphenyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)-D-glycero-β-D-galacto-2-nonulo pyranosid] onate.

3,5-Didocosyloxyphenyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulo pyranosidonic acid (16 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (11 mg, 47%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 : 1, 50°C) δ: 6.42 (br. s, 2H, H-2,6 phenyl), 6.17 (br. s, IH, H-4 phenyl), 2.93 (br. dd, IH, H-3eq), 2.00 (s, 3H, NAc), 1.38-1.18 (m, 72H, 36CH₂), 0.90 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 36: 3-O- [Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1,2-di-O-docosanoyl-Sn-glycerol. A. 3-O-Benzyl-l,2-di-O-docosanoyl-Sn-glycerol:

3-O-Benzyl-Sn-glycerol (Agric. Biol. Chem., 40, 391 (1976)) (0.67 g, 3.68 mmol), behenic acid (3.76 g, 11.04 mmol), dicyclohexylcarbodiimide (2.28 g, 11.04 mmol) and 4- dimethylaminopyridine (0.13 g, 1.06 mmol) were stirred in pyridine (37 ml) at room temperature for 22 h. The reaction solution was filtered, and evaporated to dryness in vacuo. The residue was purified by silica gel column chromatography (using 90 g of gel, hexane/ethyl acetate, 9: 1) to obtain the title compound (1.55 g, 51%) as light yellow powder. 'H-NMR (CDC1₃) δ: 7.30 (m, 5H, C₆H₅), 5.24 (m, IH, H-2), 4.56 (d, IH, J =12.1 Hz,

C₆H₅CH), 4.52 (d, IH, J =12.1 Hz, C₆H₅CH), 1.25 (m, 72H, 36CH₂), 0.88 (t, 6H, J =70 Hz,

2CH₂CH₃).

B. 1 ,2-Di-O-docosanoyl-Sn-glycerol:

3-O-Benzyl-l,2-di-O-docosanoyl-Sn-glycerol (1.25 g) was reacted by the general procedure according to Example 15-B to obtain the title compound (0.98 g, 88%) as white solid. Η-NMR (CDCI₃) δ: 5.08 (m, IH, H-2), 4.32 (dd, IH, J =4.6, 11.9 Hz, H-3), 4.24 (dd,

IH, J =5.7, 11.9 Hz, H-3'), 3.73 (m, 2H, CH₂-1), 1.25 (m, 72H, 36CH₂), 0.88 (t, 6H, J =7.0

C. 3-O- (Methyl (5-acetamido-4,7,8,9-tetra-O-levulinoyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate) - 1 ,2-di-O-docosanoyl-Sn-glycerol :

Methyl 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (3.23 g, 10.0 mmol), 4-dimethylaminopyridine (0.61 g, 0.50 mmol), levulinic acid (12.3 ml, 120 mmol) and dicyclohexylcarbodiimide (24.76 g, 120 mmol) were stirred in pyridine (30 ml) at room temperature for two days. The reaction solution was filtered through celite, and the filtrate was evaporated to dryness in vacuo. The residue was purified by silica gel column chromatography (with 170 g of gel, chloroform/methanol, 24: 1) to obtain the penta-levulinoyl derivative (4.62 g, 65%) of the title compound. Then the penta- levulinoyl derivative (2.50 g, 3.07 mmol) was dissolved in acetyl chloride (30 ml), saturated with hydrogen chloride gas at 0°C, left at standing at 2°C for five days. The reaction solution was evaporated to dryness in vacuo to obtain the chloride derivative (2.15 g, 95%) of the title compound. Then, the chloride derivative (1.22 g, 1.66 mmol) and 1 ,2-di-O-docosanoyl-Sn- glycerol (0.89 g, 1.21 mmol) were reacted by the general procedure according to Example 15-C to obtain the title compound (64 mg, 4%) as white solid.

Η-NMR (CDC1₃) δ: 5.06 (dt, IH, J =4.6, 11.7 Hz, H-4), 3.81 (s, 3H, COOCH₃), 2.19 (6H), 2.18, 2.17 (3s, 12H, 3CH₃CO), 1.89 (s, 3H, NAc), 1.85 (t, IH, J =12.5 Hz, H-3ax), 1.25 (m, 72H, 36CH₂), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 37: 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonic acid-2-yl]-l,2-di-O-docosanoyl-Sn-glycerol:

3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-levulinoyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-docosanoyl-Sn-glycerol (52 mg, 0.036 mmol) and anhydrous lithium iodide (49 mg, 0.366 mmol) were stirred in dehydrated pyridine (1.8 ml) at 80°C for 6 h. The reaction solution was directly purified by gel chromatography (using Sephadex LH-20, 100 ml, chloroform/methanol, 1: 1) to obtain lithium salt of title compound (34.6 mg, 67%) as white solid. The lithium salt (34.6 mg, 0.0242 mmol) was dissolved in methanol (0.5 ml) and chloroform (0.5 ml), added with hydrazine acetate (48 mg, 0.524 mmol), and the mixture was stirred at room temperature for 10 min. The reaction solution was adjusted to pH 4 with 0. lN-hydrochloric acid, and purified by gel chromatography (Sephadex LH-20, 75 ml, chloroform/methanol, 1 : 1) to obtain the title compound (23 mg, 92%) as white solid. Η-NMR (CDCI₃-CD₃OD, 1 : 1, 40°C) δ: 2.76 (dd, IH, J=4.2, 12.6 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.74 (t, IH, J =11.9 Hz, H-3ax), 1.28 (m, 72H, 36CH₂), 0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 38: 3-O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tretra-O-(sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulopyranosyl} onate]-l ,2-di-O-docosanoyl-Sn-glycerol:

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonic acid)- 2-yd]-l ,2-di-O-docosanoyl-Sn-glycerol (12 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (9 mg, 53%) as white solid. Η-MR (CD₃OD-D₂O, 1 : 1, 50°C) δ: 2.94 (BR. DD, IH, H-3eq), 1.95 (s, 3H, ANC),

1.71 (t, IH, J =1 1.0 Hz, H-ax), 1.26 (m, 72H, 36CH₂), 0.87 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 39: 3-O-[Methyl (4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero- -D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-docosyl-Sn-glycerol. 1 ,2-Di-O-docosyl-Sn-glycerol (Japanese Patent Laid-Open Publication No. Hei 1 -

125394) (467 mg) and methyl 4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideoxy-D-glycero-(β) -D-galacto-2-nonulopyranosonate (Chem. Pharm. Bull., 39, 3140 (1991)) (281 mg) were reacted by the general procedure according to Example 15-C to obtain the title compound

(313 mg, 48%) as white solid. Η-NMR (CDC1₃) δ: 3.80 (s, 3H, OCH₃, 2.68 (dd, IH, J=4.0,12.8Hz, H-3eq), 2.15,

2.09, 2.04, 2.00, 2.00 (5s, 15H, 5Ac), 1.25 (m, 76H, 38CH₂), 0.88(t, 6H, J=6.6Hz, 2CH₂CH₃).

Example 40: 3-O-[(3-Deoxy-D-glycero-( )-D-galacto-2-nonulopyranosonic acid) -2-yl]-l ,2- di-O-docosyl-Sn-glycerol. 3-O-[Methyl (4,5,7, 8,9-penta-O-acetyl-3-deoxy-D-glycero-α-D-galacto-2- nonulopyranosyl) onate]- 1 ,2-di-O-docosyl-Sn-glycerol (313 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (186 mg, 75%) as white solid.

Η-NMR (CDCI₃-CD₃OD, 1 : 1 , 60°C) δ: 2.68 (dd, IH, J=4.6, 12.6 Hz, H-3eq), 1.73 (t, IH, J =12.1 Hz, H-3ax), 1.29 ( , 76H, 38CH₂), 0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 41 : 3-O-[Sodium {3-deoxy-4,5,7,8,9-penta-O-(sodium oxysulfonyl)-D-glycero-α- D-galacto-2-nonulopyranosyl} onate]- 1 ,2-di-O-docosyl-Sn-glycerol. 3-O-[(3-Deoxy-D-glycero-α-D-galacto-2-nonulopyranosonic acid)-2-yl]- 1 ,2-di-O- docosyl-Sn-glycerol (97 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (15 mg, 10%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 :1, 50°C) δ: 2.89 (br. dd, IH, H-3eq), 1.81 (br. t, I H, H- 3ax), 1.26 (m, 76H, 38CH₂), 0.86 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 42: l-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl)onate]-L-2,3-di-O-docosyl-Sn-glycerol.

A. L-l-O-Benyl-2,3.-di-O-isopropylidene-Sn-glycerol: After L-2,3-di-O-isopropylidene-Sn-glycerol (5.00 g, 37.8 mmol) and 60% sodium hydride (3.03 g, 75.8 mmol) were stirred in dehydrated dimethylformamide (110 ml) at room temperature for 10 min, benzyl bromide (6.7 ml, 56.7 mmol) was added, and the resulting mixture was stirred at room temperature for 3 h. The reaction solution was diluted with ether, washed successively with water and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was distilled under reduced pressure (110°C/3 torr) to obtain the benzyl derivative (6.65 g, 80%). Then, to a solution of this product (6.65 g, 29.9 mmol) dissolved in dichloromethane (30 ml), methanol (9 ml) and water (3 ml) was added trifluoroacetic acid (3 ml), and the mixture was stirred at room temperature for 3 h. After the reaction solution was neutralized with sodium hydroxide and condensed in vacuo, the residue was purified by distillation under reduced pressure to obtain the title compound (4.71 g, 86%).

Η-NMR (CDCI₃) δ: 7.32 (m, 5H, C₆H₅), 4.56 (s, 2H, CH₂Ph).

B. L-l-O-Benzyl-2,3-di-O-docosyl-Sn-glycerol: L-1-O-Benzyl-Sn-glycerol (1.90 g) and 1-bromo docosane (9.70 g) were reacted by the general procedure according to Example 15-A to obtain the title compound (7.0 g, 85%). Η-NMR (CDCI₃) δ: 7.30 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

C. L-2,3-Di-O-docosyl-Sn-glycerol:

L-l-O-Benzyl-2,3-di-O-docosyl-Sn-glycerol (1.40 g) was reacted by the general procedure according to Example 15-B to obtain the title compound (0.99 g, 79%) as white solid. Η-NMR (CDC1₃) δ: 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

D. 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl)-onate]-L-2,3-di-O-docosyl-Sn-glycerol: L-2,3-Di-O-docosyl-Sn-glycerol (990 mg) and methyl 5-acetamido-4,7,8,9-tetra-O- acetyl-2-chloro-2,3,5-trideoxy-D-glycero-(β) -D-galacto-2- nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (850 mg) were reacted by the general procedure according to Example 15-C to obtain the title compound (662 mg, 40%) as white solid.

Η-NMR (CDC1₃) δ: 3,79 (s, 3H, OCH₃), 2.60 (dd, IH, J=4.8, 12,8 Hz, H-3eq.), 2.15, 2.09, 2.04, 2.00, 2.00 (5s, 15H, 5Ac), 1.96 (t, IH, J =12.8 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 43: l-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2-yl]-L-2,3-di-O-docosyl-Sn-glycerol: l-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosyl)onate]-L-2,3-di-O-docosyl-Sn-glycerol (632 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (500 mg, 92%) as white solid.

Η-NMR (CDCl₃-CD₃OD, 1 : 1, 40°C) δ: 2.77 (dd, IH, J=4.6, 13.4 Hz, H-3eq.), 2.02 (s, 3H, NAc), 1.74 (t, IH, J =11.9 Hz, H-3ax.), 1.28 (m, 76H, 38CH₂), 0.89 (t, 6H, J =6.8 Hz,

2CH2CH₃).

Example 44: l -O-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D- glycero- -D-galacto-2-nonulopyranosyl}onate]-L-2,3-di-O-doosyl-Sn-glycerol: l-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2- yl]-L-2,3-di-O-docosyl-Sn-glycerol (200 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (237 mg, 83%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 : 1, 40°C) δ: 2.93 (br.dd, lH,H-3eq), 1.96 (s, 3H, NAc), 1.77

(br.t, IH, H-3ax), 1.28 (m, 76H, 38CH₂), 0.87 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 45: Methyl [2,2-bis (oleyl oxymethyl)propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-

3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate.

A. 2,2-Bis(Oleyl oxymethyl) propanol: 1,1,1-Tris (hydroxymethyl) ethane (1.0 g) and oleyl chloride (5.25 g) were reacted by the general procedure according to Example 15-A to obtain the title compound (2.91 g, 57%) as white solid.

Η-NMR (CDC1₃) δ: 5.36 (m, 4H, 2CH=CH), 3.56 (d, 2H, J =5.9 Hz, CH₂OH), 1.27 (m, 40H, 20CH₂), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃), 0.85 (s, 3H, CCH₃).

B. Methyl [2,2-bis(oleyl oxymethyl)propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- D-glycero-α-D-galacto-2-nonulopyranosid]onate:

2,2-Bis(oleyl oxymethyl) propanol (400 mg) and methyl 5-acetamido-4,7,8,9-tetra-O- acetyl-2-hloro-2,3,5-trideoxy-D-glycero-(β) -D-galacto-2-nonulopyranosonate (Chem. Ber., 99, 611(1966)) (328 mg) were reacted by the general procedure according to Example 15-C to obtain the title compound (564 mg, 80%) as white solid.

Η-NMR (CDCI₃) δ: 5.35 (m, 4H, 2CH=CH), 3.78 (s, 3H, COOCH3), 2.58 (dd,l H, J =5.0, 12.8 Hz, H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.27 (m, 40H, 20CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J =6.9 Hz, 2CH₂CH₃).

Example 46: 2,2-Bis(oleyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosidonic acid.

Methyl [2,2-bis(oleyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero- -D-galacto-2-nonulopyranosid]onate (250 mg) was reacted by the general procedure according to Example 16 to obtain the title compound ( 190 mg, 89%) as white solid.

Η-NMR (CDCI₃-CD₃OD, 1 :1) δ: 5.35 (m, 4H, 2CH=CH), 2.76 (dd, IH, J =4.2, 13.4 Hz, H-3eq), 2.05 (s, 3H, NAc), 1.70 (t, IH, J =11.9 Hz, H-3ax), 1.30 (m, 40H, 20CH₂), 0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 47: Sodium [2,2-bis(oleyl oxymethyl)propyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyτanosid] onate .

2,2-Bis(oleyl oxymethyl)propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosidonic acid (913 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (63 mg, 47%) as white solid. Η-NMR (CD₃OD-D₂O, 1:1, 50°C) δ: 5.37 (m, 4H, 2CH=CH), 2.95 (dd, IH, J =5.3, 12.6 Hz, H-3eq), 1.97 (s, 3H, NAc), 1.74 (t, IH, J =12.8 Hz, H-3ax), 1.28 (m, 40H, 20CH₂), 0.94 (s, 3H, CCH₃), 0.89 (t, 6H, J =6.8 Hz, 2CH₂CH₃).

Example 48: Methyl [2,2-bis(docosyl oxymethyl)butyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate.

A. 2,2-Bis(docosyl oxymethyl)butanol: l,l,l-Tris(hydroxymethyl)propane (1.0 g) and 1-bromo docosane (7.3 g) were reacted by the general procedure according to Example 15-A to obtain the title compound (3.12 g, 56%) as white solid.

Η-NMR (CDC1₃) δ: 3.59 (d, 2H, J =5.9 Hz, CH₂OH), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J =6.7 Hz, 2CH₂CH₃), 0.84 (t, 3H, J =7.5 Hz, CCH₂CH₃).

B. Methyl [2,2-bis(docosyloxymethyl)butyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- D-glycero-α-D-galacto-2-nonulopyranosid]onate:

2,2-Bis(docosyloxymethyl)butanol (2.26 g) and methyl 5-acetamido-4,7,8,9-tetra-O- acetyl-2-chloro-2,3,5-trideoxy-D-glycero-(β) -D-galacto-2- nonulopyranosonate (Chem. Ber., 99, 611 (1966)) (1.02 g) were reacted by the general procedure according to Example 15-C to obtain the title compound (539 mg, 22%) as white solid. Η-NMR (CDCI₃) δ: 3.78 (s, 3H, COOCH₃), 2.58 (dd, IH, J=4.8, 12.8 Hz, H-3eq.),

2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.95 (t, IH, J =12.5 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃), 0.84 (t, 3H, J =7.5 Hz, CCH₂CH₃).

Example 49: 2,2-Bis(docosyloxymethyl)butyl 5-acetamido-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosidonic acid.

Methyl (2,2-bis(docosyloxymethyl) butyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-(α)-D-galacto-2-nonulopyranosid)onate (520 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (393 mg, 90%) as white solid. Η-NMR (CDCI₃-CD₃OD, 1 : 1, 60°C) δ: 2.72 (dd, IH, J=5.1, 12.1 Hz, H-3eq), 2.02 (s,

3H, NAc), 1.80 (t, IH, J =11.9 Hz, H-3ax), 1.28 (m, 76H, 38CH₂), 0.89 (t, 6H, J =7.0 Hz,

2CH₂CH₃), 0.86 (t, 3H, J =7.7 Hz, CCH₂CH₃). Example 50: Sodium [2,2-bis(docosyloxymethyl)butyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate:

2,2-Bis(docosyloxymethyl)butyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosidonic acid (388 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (332 mg, 61%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 : 1, 60°C) δ: 2.95 (br.dd, IH, H-3eq), 1.96 (s, 3H, NAc), 1.78 (br.t, IH, H-3ax), 1.27 (m, 76H, 38CH₂), 0.87 (t, 9H, J =7.0 Hz, 3CH₂CH₃).

Example 51 : 3-O-[Methyl {5-N-(O-acetylglycolyl)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero- -D-galacto-2-nonulopyranosyl}onate]-l ,2-di-O-docosyl-Sn-glycerol.

1,2-Di-O-docosyl-Sn-glycerol (400 mg) and methyl 5-N-(O-acetylglycolyl)-4,7,8,9- tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate (Carbohydr. Res., 174, 73 (1988)) (300 mg) were reacted by the general procedure according to Example 15-C to obtain the title compound (66 mg, 10%) as white solid. Η-NMR (CDC1₃) δ: 4.60, 4.29 (2d, 2H, J =5.0 Hz, NHCOCH₂), 3.81 (s, 3H, OCH₃),

2.62 (dd, 1H, J =4.8, 12.8 Hz, H-3eq), 2.20, 2.15, 2.13, 2.04, 2.01 (5s, 15H, 50Ac), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 52: 3-O-[(5-N-glycolyl-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2-yl]- 1,2-di-O-docosyl-Sn-glycerol.

3-0-[Methyl {5-N-(O-acetylglycolyl)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- α-D-galacto-2 -nonulopyranosyl} onate]- 1 ,2-di-O-docosyl-Sn-glycerol (60 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (43 mg, 88%) as white solid. Η-NMR (CDCI₃-CD3OD, 1 : 1, 60°C) δ: 2.78 (dd, IH, J=4,0, 12.0 Hz, H-3eq), 1.79 (t,

I H, J =12.0 Hz, H-3ax), 1.28 (m, 76H, 38CH₂), 0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 53: 3-O-[Sodium {5-N-(O-sodium oxysulfonylglycolyl)-3,5-dideoxy-4,7,8,9-tetra- O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-l ,2-di-O- docosyl-Sn-glycerol.

3-0-[(5-N-glycolyl-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2- yl]- 1 ,2-di-O-docosyl-Sn-glycerol (41 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (16 mg, 26%) as white solid. Η-NMR (CD₃OD-D₂O, 1 : 1, 60°C) δ: 2.99 (br.dd, IH, H-3eq), 1.92 (br.t, IH, H-3ax), 1.30 (m, 76H, 38CH₂), 0.90 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 54: 3-S-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α -D-galacto-2-nonulopyranosyl)onate]-l,2-di-O-docosyl-Sn-thioglycerol.

A. 3-Bromo-3-deoxy-l ,2-di-O-docosyl-Sn-glycerol:

1,2-Di-O-docosyl-Sn-glycerol (200 mg, 0.28 mmol), N-bromosuccinimide (90 mg, 0.51 mmol) and triphenylphosphine (170 mg, 0.65 mmol) were stirred in toluene (12 ml) at room temperature for 3 days. The reaction solution was condensed in vacuo, and the residue was purified by silica gel column chromatography (with 20 g of gel, hexane/toluene, 3:2) to obtain the title compound (168 mg, 77%) as white powder.

Η-NMR (CDC1₃) δ: 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B. 3-S-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosyl)onate]-l,2-di-O-doosyl-Sn-thioglycerol:

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thio-D-glycero- ( )-D-galacto-2-nonulopyranosonate (J. Carbohydr. Chem., 5,11 (1986)) (114 mg, 0.21 mmol) and sodium methoxide (11 mg, 0.20 mmol) were stirred in anhydrous methanol (0.5 ml) at -10°C for 1 h, and the reaction solution was evaporated to dryness in vacuo. To the residue were added a solution of 3-bromo-3-deoxy-l,2-di-O-docosyl-Sn-glycerol (160 mg, 0.21 mmol) in dehydrated dimethylformamide (1.0 ml) and toluene (1.0 ml), and the mixture was stirred at room temperature for two days. The reaction solution was diluted with chloroform, washed with a saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. After the solvent was distilled off in vacuo, the residue were purified by silica gel column chromatography (27 g of gel, toluene-acetone, 5: 1) to obtain the title compound (85 mg, 34%) as white powder.

Η-NMR (CDC1₃) δ: 3.79 (s, 3H, COOCH₃), 2.91 (dd, IH, J=4.8, 13.0 Hz, H-3eq.), 2.72 (m, 2H, SCH₂), 2.15, 2.13, 2.04, 2.03, 1.88 (5s, 15H, 5Ac), 2.00 (t, IH, J =12.8 Hz, H- 3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J =7.1 Hz, 2CH₂CH₃).

Example 55: 3-S-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2-yl]- 1 ,2-di-O-doosyl-Sn-thioglycerol. 3-S-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto- 2-nonulopyτanosyl)onate]-l,2-di-O-docosyl-Sn-thioglycerol (154 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (115 mg, 86%) as white solid. Η-NMR (CDCl₃-CD₃OD, 1 :1, 60°C) δ: 2.94 (dd, IH, J=4.6, 12.8 Hz, H-3eq), 2.84

(m, 2H, SCH2), 2.02 (s, 3H, NAc), 1.84 (t, IH, J =11.6 Hz, H-3ax), 1.29 (m, 76H, 38CH₂), 0.89 (t, 6H, J =6.9 Hz, 2CH₂CH₃).

Example 56: 3-S-[Sodium {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D- glycero-α-D-galacto-2 -nonulopyranosyl} onate]- 1,2-di-O-docosyl-Sn-thioglycerol.

3-S-[(5-Acetamido-3 ,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonic acid)-2- yl]-l,2-di-O-doosyl-Sn-thioglycerol (422 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (29 mg, 45%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 : 1, 50°C) δ: 3.06 (br.dd, IH, H-3eq), 2.90 (m, 2H, SCH₂), 1.90 (s, 3H, NAc), 1.30 (m, 76H, 38CH₂), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 57: l-[N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β- D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane.

A. 2,2-Bis(docosyloxymethyl)propyl methanesulfonate: After a mixture of 2,2-bis(docosyloxymethyl) propanol (7.37 g; 10 mmol), methanesulfonyl chloride (1.3 ml; 16.8 mmol) and pyridine (150 ml) was stirred at 70°C for 1 h, the reaction mixture was poured into water. Precipitates thus obtained were collected by suction, washed successively with water and acetone, and dried to obtain the title compound (6.66 g, 82%). Η-NMR (CDC1 ) δ: 4.13 (s, 2H, CH₂OMs), 2.98 (s, 3H, CH₃SO₂), 1.26 (m, 76H,

38CH₂), 1.00 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B. 1 - Azido-2,2- bis(docosyloxymefhyl) propane:

After a mixture of 2,2-bis(docosyloxymethyl)propyl methanesulonate(22.7 g; 27.8 mmol), sodium azide (0.27 g; 83.5 mmol), and dimethylformamide (150 ml) was stirred at 110°C for 20 h, the reaction mixture was poured into water. Precipitates thus obtained were collected by suction, washed successively with water and acetone, and dried to obtain the title compound (20.8 g; 98%). Η-NMR (CDCI₃) δ: 1.25 (m, 76H, 38CH₂), 0.89 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

C. l-Amino-2,2- bis(docosyloxymethyl) propane: l-Azido-2,2-bis(docosyloxymethyl) propane (12.8 g; 16.8 mmol) and palladium hydroxide on carbon (6.0 g) in tetrahydrofuran (100 ml) was stirred under the hydrogen atmosphere at 50°C for 3 h. The reaction solution was filtered through celite, and the filtrate was evaporated to dryness in vacuo. The residue was purified by silica gel column chromatography (60 g of gel, chloroform/methanol, 10: 1) to obtain the title compound (8 g, 65%) as white powder.

D. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosidonic acid.

After a mixture of methyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosidonic acid (Chem. Ber., 99, 61 1 (1966)) (150 mg; 0.464 mmol), acetic anhydride (2.3 ml) and pyridine (2.3 ml) was stirred at room temperature for 7 h, the reaction solution was evaporated to dryness in vacuo. The residue was dissolved in chloroform, and treated with Amberist-15 (H type) to obtain the title compound (218 mg, 96%) as white powder. Η-NMR (CDCI₃) δ: 3.33 (s, 3H, OCH₃), 2.56 (dd, IH, J=4.8, 13.2 Hz, H-3eq.), 2.16,

2.1 1 , 2.06, 2.04, 1.91 (5s, 15H, 5Ac).

E. l- N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane: After a mixture of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-

(β)-D-galacto-2 -nonulopyranosidonic acid (260 mg; 0.53 mmol), l-amino-2,2- bis(docosyloxymethyl) propane (390 mg, 0.53 mmol), dicyclohexylcarbodiimide (165 mg; 0.80 mmol), 1-hydroxybenztriazole (95 mg; 0.70 mmol) and chloroform (1 1 ml) was stirred at room temperature for 17 h, the reaction solution was filtered through celite, an the solvent was distilled off in vacuo. The residue was purified by silica gel column chromatography (with 60 g of gel, chloroform-acetone, 17:3) to obtain the title compound (620 mg, 97%) as white powder. Η-NMR (CDC1 ) δ: 3.18 (s, 3H, OCH₃), 2.51 (dd, IH, J=4.8, 13.2 Hz, H-3eq), 2.1 4, 2.08, 2.03, 2.01 , 1.90 (5s, 15H, 5Ac), 1.77 (t, IH, J =1 1.7 Hz, H-3ax), 1.25 (m, 76H, 38CH₂), 0.91 (s, 3H, CCH₃), 0.88 (t, 6H, J =6.6 Hz, 2CH₂CH₃).

Example 58: l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane.

After a mixture of l-[N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-0-methyl- D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane (620 mg; 0.51 mmol), sodium methoxide (28 mg; 0.52 mmol), methanol (5 ml) and tetrahydrofuran (5 ml) was stirred at room temperature for 7 h, the reaction solution was neutralized with Amberlist-15 (H type), and evaporated to dryness in vacuo. The residue was purified by silica gel column chromatography (50 g of gel, chloroform/methanol, 10: 1) to obtain the title compound (480 mg, 90%) as white powder.

Η-NMR (CDCI₃-CD3OD, 1 : 1) δ: 3.24 (s, 3H, OCH₃), 2.40 (dd, IH, J =4.9, 13.0 Hz, H-3eq), 2.05 (s, 3H, NAc), 1.27 (m, 76H, 38CH₂), 0.91 (s, 3H, CCH₃), 0.89 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 59: l-[N- {5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2- bis(docosyloxymethyl) propane. l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane (831 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (810 mg, 70%) as white solid. Η-NMR (CD₃OD-D₂O, 1 :1 , 40°C) δ: 2.66 (br.dd, IH, J=4.4, 12.5 Hz, H-3eq), 2.03

(s, 3H, NAc), 1.79 (br.t, IH, H-3ax), 1.31 (m, 76H, 38CH₂), 0.91 (s, 3H, CCH₃), 0.91 (t, 6H,

J =6.8 Hz, 2CH₂CH₃).

Example 60: l-[N-(5-Aetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β- D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane. A. 2,2-Bis(eicosyloxymethyl)propyl methanesulfonate:

2,2-Bis(eicosyloxymethyl) propanol (1.19 g) was reacted by the general procedure according to Example 57-A to obtain the title compound (1.08 g, 82%) as white solid. 1)H- NMR (CDCI₃) (δ): 4.13 (s, 2H, CH₂OMs), 2.98 (s, 3H, CH₃SO₂), 1.25 (m, 68H, 34CH₂), 1.00 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

B. l-Azido-2,2-bis(eicosyloxymethyl) propane: 2,2-Bis(eicosyloxymethyl)propyl methanesulfonate (1.05 g) was reacted by the general procedure according to Example 57-B to obtain the title compound (0.94 g, 96%) as white solid.

Η-NMR (CDCI₃) δ: 1.25 (m, 68H, 34CH₂), 0.94 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH3).

C. l-Amino-2,2-bis(eicosyloxymethyl) propane: l-Azido-2,2-bis(eicosyloxymethyl) propane (360 mg) was reacted by the general procedure according to Example 57-C to obtain the title compound (206 mg, 59%) as white solid. Η-NMR (CDCI₃) δ: 2.74 (s, 2H, CH₂), 1.25 (m, 68H, 34CH₂), 0.90 (s, 3H, CCH₃),

0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

D. l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane: Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-(β)-D-galacto-2- nonulopyranosidonic acid (179 mg) and l-amino-2,2-bis(eicosyloxymethyl) propane (206 mg) were reacted by the general procedure according to Example 57-E to obtain the title compound (327 mg, 94%) as white solid.

Η-NMR (CDCI₃) δ: 3.18 (s, 3H, OCH₃), 2.51 (dd, IH, J=5.0, 13.0 Hz, H-3eq), 2.14, 2.09, 2.04, 2.01, 1.91 (5s, 15H, 5Ac), 1.77 (dd, IH, J =11.7, 12.8 Hz, H-3ax), 1.25 (m, 68H, 34CH2), 0.91 (s, 3H, CCH₃), 0,88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 61 : 1 -[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane. l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane (216 mg) was reacted by the general procedure according to Example 58 to obtain the title compound (202 mg, 74%) as white solid. Η-NMR (CDCI₃-CD₃OD, 10:1) δ: 3.22 (s, 3H, OCH₃), 2.36 (dd, IH, J =4.8, 13.2 Hz, H-3eq), 2.04 (s, 3H, NAc), 1.26 (m, 68H, 34CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.1 Hz, 2CH₂CH₃).

Example 62: l-[N-{5-Aetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2- bis(eicosyloxymethyl) propane. l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane (422 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (49 mg, 36%) as white solid.

Η-NMR (CD₃OD-D₂O, 1 : 1, 40°C) δ: 2.66 (br.dd, IH, H-3eq), 2.02 (s, 3H, NAc), 1.78 (br.t, IH, J =12.3 Hz, H-3ax), 1.30 (m, 68H, 34CH₂), 0.90 (s, 3H, CCH₃), 0.90 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 63: l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-β- D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane. A. Methyl (ethyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosid)onate: A mixture of N-acetylneuraminic acid (1.00 g; 3.23 mmol), Dowex-50 (H form) (15 g) and dehydrated ethanol (100 ml) was heated at reflux for 18 h. The reaction mixture was packed into a column, and eluted with 2N-HCl-methanol (100 ml). After the solvent was distilled off in vacuo. The residue thus obtained was stirred with acetic anhydride (20 ml) and pyridine (20 ml) at room temperature for 17 h, and then the reaction solution was evaporated to dryness in vacuo. The residue was dissolved in chloroform, washed successively with 0. IN HC1, water and saturated NaCl solution, dried over anhydrous magnesium sulfate, and then the solvent was distilled off in vacuo. After the residue thus obtained was stirred with sodium methoxide (31 mg; 0.575 mmol) in methanol (23 ml) at room temperature for 17 h, the reaction mixture was neutralized with Dowex-50 (H form), and then the solvent was distilled off. The residue was purified by silica gel column chromatography (40 g of gel, chloroform methanol, 4: 1) to obtain the title compound (142 mg, 12%)) as white powder.

Η-NMR (CD₃OD) δ: 3.78 (s, 3H, OCH₃), 2.36 (dd, IH, J=4.9, 13.0 Hz, H-3eq.), 2.00 (s, 3H, NAc), 1.62 (dd, IH, J =11.4, 12.8 Hz, H-3ax.), 1.16 (t, 3H, J =7.0 Hz, CH₂CH₃). B. Methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosid)onate :

Methyl (ethyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosid)onate (142 mg) was reacted by the general procedure according to Example 57-D to obtain the title compound (209 mg, 99%) as white solid.

Η-NMR (CDC1₃) δ: 3.80 (s, 3H, COOCH₃), 2.45 (dd, IH, J=5.1, 12.8 Hz, H-3eq.), 2.15, 2.08, 2.03, 2.02, 1.89 (5s, 15H, 5 Ac), 1.87 (dd, 1H, J =11.4, 12.8 Hz, H-3ax.), 1.22 (t, 3H, J =7.1 Hz, CH₂CH₃).

C. Ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2- nonulopyranosidonic acid:

A mixture of methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero-β-D-galacto-2-nonulopyranosid)onate (203 mg, 0.39 mmol), anhydrous lithium iodide (523 mg, 3.9 mmol) and pyridine (8.0 ml) was stirred at 90°C for 14 h. The reaction solution was subjected to gel filtration (LH-20, 150 ml, methanol), and then purified by silica gel column chromatography (20 g of gel, chloroform/methanol, 7:3) to obtain the title compound (92 mg, 47%) as white solid.

Η-NMR (CDCI₃) δ: 3.52 (m, 2H, CH₂CH₃), 2.56 (dd, IH, J=4.5, 12.2 Hz, H-3eq.), 2.15, 2.11, 2.06, 2.04, 1.91 (5s, 15H, 5Ac), 1.92 (t, IH, J =11.8 Hz, H-3ax.), 1.24 (t, 3H, J =7.0 Hz, CH₂CH3).

D. l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-(β)-D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane: Ethyl 5-acetamido-4,7,8,9-tetτa-O-acetyl-3,5-dideoxy-D-glycero-(β)-D-galacto-2- nonulopyranosodonic acid (92 mg) and l-amino-2,2-bis(docosyloxymethyl) propane (268 mg) were reacted by the general procedure according to Example 57-E to obtain the title compound (191 mg, 86%) as white solid.

Η-NMR (CDCI₃) δ: 2.52 (dd, IH, J =4.8, 13.2 Hz, H-3eq), 2.14, 2.08, 2.03, 2.01 , 1.91 (5s, 15H, 5Ac), 1.76 (t, IH, J = 12.4 Hz, H-3ax), 1.25 (m, 76H, 38CH₂), 1.19 (t, 3H, J = 7.0 Hz, OCH₂CH₃), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J = 7.0 Hz, 2CH₂CH₃). Example 64: 1 -[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2- nonulopyranosonyl) amino]-2,2-bis(docosyloxymethyl) propane. l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-(β) -D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane (185 mg) was reacted by the general procedure according to Example 58 to obtain the title compound (1 14 mg, 71%) as white solid.

Η-NMR (CDCI₃-CD₃OD, 10:1) δ: 2.38 (dd, IH, J =4.8, 13.2 Hz, H-3eq), 2.04 (s, 3H, NAc), 1.26 (m, 76H, 38CH₂), 1.17 (t, 3H, J=7.0 Hz, OCH₂CH₃), 0.89 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.3 Hz, 2CH₂CH₃).

Example 65: l-[N-{5-Acetamido-3,5-dideoxy-2-O-ethyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2- bis(docosyloxymethyl) propane. l-[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane (422 mg) was reacted by the general procedure accordng to Example 1 to obtain the title compound (140 mg, 93%) as white solid.

Η-NMR (CD₃OD-D₂O, 1:1, 40°C) δ: 2.67 (br.dd, IH, H-3eq), 2.02 (s, 3H, NAc), 1.77 (br.t, IH, H-3ax), 1.30 (m, 76H, 38CH₂), 1.25 (t, 3H, J =7.0 Hz, OCH₂CH₃), 0.91 (t, 6H, J =6.8 Hz, 2CH₂CH3), 0.90 (s, 3H, CCH₃).

Example 66: l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero- α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane:

A. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosidonic acid:

Methyl 5-acetamido-3,5-dideoxy-D-glycero-(α)-D-galacto-2 -nonulopyranosidonic acid (Chem. Ber., 99, 611 (1966)) (150 mg) was reacted by the general procedure according to Example 57-D to obtain the title compound (220 mg, 96%>) as white solid.

Η-NMR (CDCl₃-CD3OD, 1 :1) δ: 3.20 (s, 3H, OCH₃), 2.42 (dd, IH, J =4.4, 12.5 Hz, H-3eq.), 1.96, 1.95, 1.87, 1.83, 1.70 (5s, 15H, 5Ac), 1.56 (t, IH, J =12.5 Hz, H3ax.).

B. l-[N-(5-Acetamido-4,7,8,9-tetτa-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-α-D- galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane: Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-(α)-D-galacto-2- nonulopyranosidonic acid (191 mg) and l-amino-2,2-bis(docosyloxymethyl) propane (396 mg) were reacted by the general procedure according to Example 57-E to obtain the title compound (345 mg, 73%) as white solid. Η-NMR (CDC1₃) δ: 3.36 (s, 3H, OCH₃), 2.13, 2.08, 2.03, 2.01, 1.89 (5s, 15H, 5Ac), 1.98 (t, IH, J = 11.7 Hz, H3ax.) 1.25 (m, 76H, 38CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 67: l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis (docosyloxymethyl) propane. l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-α-D- galacto-2-nonulopyranosonyl)amino)-2,2-bis(doosyloxymethyl) propane (340 mg) was reacted by the general procedure according to Example 58 to obtain the title compound (216 mg, 74%>) as white solid. Η-NMR (CDCI₃-CD3OD, 10: 1) δ: 3.36 (s, 3H, OCH₃), 2.55 (dd, IH, J =4.8, 13.2 Hz,

H-3eq), 2.03 (s, 3H, NAc), 1.82 (dd, IH, J=11.2, 13.0 Hz, H3ax.), 1.26 (m, 76H, 38CH₂),

0.91 (s, 3H, CCH₃), 0.88 (t, 6H, J =7.0 Hz, 2CH₂CH₃).

Example 68: l-[N-{5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosonyl}amino]-2,2- bis(docosyloxymethyl) propane. l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane (117 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (98 mg, 60%) as white solid.

Η-NMR (CD₃OD-D₂O, l :l, 40°C) δ: 2.55 (br.dd, IH, J=5.1, 13.6 Hz, H-3eq), 1.99 (s, 3H, NAc), 1.30 (m, 76H, 38CH₂), 0.90 (br.t, 9H, J =6.6 Hz, CCH₃) and 2CH₂CH₃).

Example 69: In vitro and in vivo inhibition of human rotavirus (HRV) by sodium [2,2-bis (docosyloxymethyl) propyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero-α-D-galacto-2-nonulo pyranosid] onate ("COMPOUND 29"). Materials and Methods

Cells and viruses. MA 104 cells (African rhesus monkey kidney cells) were cultured in MEM supplemented with 10% fetal calf serum (FCS). Wa (Gl), S2, DS-1 (G2), MO (G3), and Hochi (G4) strains of human rotavirus (HRV) were propagated in MA 104 cells, and harvested after two freeze-thaw cycles. HRV titer was determined by plaque assay. For experiments of mouse diarrhea model, MO strain was purified by ultracentrifugation at 100,000 x g for 3 hours (h). The pellet was suspended in PBS containing 1 mM each of CaCl₂ and MgCl₂ and stored until use in aliquots at -80° C. Chemicals. COMPOUND 29 (sodium (2,2-bis (docosyloxymethyl)propyl 5-acetamido-3,5- dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulo pyranosid)onate), a sulfated sialyl lipid described in Example 29, above, was synthesized and purified at more than 98% at the Central Research Institute of Nissin Food Products Co., Ltd. (Kusatu, Shiga, Japan). Antiviral assays. The inhibitory effect of COMPOUND 29 on the replication of rotavirus was determined by the inhibition of virus-induced cytotoxicity in MA 104 cells. In brief, a confluent monolayer culture of MA 104 cells was infected with Wa strain of HRV (100 pfu/well) with various concentrations of COMPOUND 29 in a 96-well microplate and cultured for 3 days. The rate of viable cells was determined colorimetrically by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Focal forming unit (FFU) assay. Wa strain of HRV was also determined by focus forming unit (FFU) assay. MA104 cells in a 96-well plate were infected with 10-fold serial dilutions of virus stock and cultured for 24 h. The cells were fixed with methanol, washed with PBS, and reacted with rabbit anti-rotavirus (KU strain) serum. After washing, the cells were reacted with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase. HRV- infected cells were visualized by diaminobenzidine (DAB) and the number of infected cells (focus) was counted under a microscope to determine the FFU titer of the virus stock. Time of addition experiments. MAI 04 cells in a 96-well microplate were pretreated with various concentrations of COMPOUND 29 before virus adsorption (100 FFU/well) for 1 h or treated with COMPOUND 29 during or after virus adsorption. The cells were cultured for 24 h and virus-infected cells were visualized and counted as described above. The 50% effective concentration (EC50) was defined as the concentration of COMPOUND 29 required for reducing the number of focuses by 50%. Virus binding assay. Virus binding assay was performed with radiolabeled purified Wa strain of HRV. HRV Wa strain-infected MA104 cells were radiolabeled with 100 mCi ³⁵S- methionine for 12 h until massive CPE was observed. The radiolabeled virus was purified with sucrose gradient by ultracentrifugation. Fractions of 100 μl aliquots were tested for virus titer by a FFU assay and for radioactivity. For a virus binding assay, a confluent monolayer of MA 104 cells in a 96-well microplate was incubated with a radiolabeled virus (48 FFU = 2 x 10⁴ counts per minute (cpm)/100 μl of MEM) in the presence or absence of COMPOUND 29 for 1 h on ice, after which cells were washed free of medium and excess labeled virus. The cells were lysed with lysing solution (1% Triton X-100, 0.15 M NaCl, 10 mM Tris/HCl), and bound virus was counted in a liquid scintillation counter (Aloka). The percentage of bound viruses was calculated as:

(cpm of membrane-bound virus/cpm of total input Wa strain of HRV in medium) x 100% Animals. ICR suckling mice were maintained in a laminar flow hood in the Animal Experiment Facility of the Fukushima Medical University. Mouse diarrhea model studies. A mouse model for gastroenteritis (Ebina et al., Microbiol. Immunol, 35(7): 583-588 (1991)) was used to study aspects of the protective effect of COMPOUND 29 on development of diarrhea. In the basic protection study, litters of 6 or 7- day old mice were orally inoculated (50 μl) with 3 x 10⁶ pfu of MO strain of HRV by a gavage. Stools were examined daily for characteristics of diarrhea by gentle abdominal palpitation beginning at one day after inoculation until 5 days. A six-point rating system (diarrhea index, DI) was used to characterize diarrhea: 1 , no stool; 2, normal brown formed stool; 3, soft brown stool; 4, soft mucous brown-yellow stool; 5, muddy mucous yellow stool; 6, liquid mucous yellow stool.

To determine the effective amount of COMPOUND 29 required to prevent rotavirus- induced diarrhea, COMPOUND 29 was given orally by gavage (50 μl) three times per day (9 AM, 3 PM, 9 PM) for 4 days starting 30 minutes before virus inoculation. To evaluate initiation time of treatment, treatment with COMPOUND 29 started 6, 9, 12, or 18 h after inoculation after virus inoculation. In the both experiments stools were examined and scored daily and the efficacy of COMPOUND 29 was evaluated by the prevention of development of diarrhea. Body weight of mice was measured every morning during experiments. Immunofluorescence. Antibody titer of mice to MO strain HRV was determined by immunofluorescence. Mice were anesthetized and blood collected 14 days after HRV inoculation. MA104 cells were cultured on 24-well multi-spotted glass slides and infected with MO strain of HRV at a multiplicity of infection (m.o.i.) of 0.5 for 24 h. Slides were washed with PBS, dried, and fixed with cold acetone. The infected cells were reacted with serial dilutions of the blood serum for 30 minutes at room temperature. After two washes with PBS, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antibody (1 :200, DAKO JAPAN, Kyoto, Japan). After washings, antibody titer was determined by fluorescent microscopy. The antibody titer was expressed as the highest serum dilution showing a detectable signal.

Results: Inhibitory effect of COMPOUND 29 on the HRV replication in vitro and in vivo. The inhibitory effect of COMPOUND 29 on HRV strain Wa (Gl) in infected cell was examined by the MTT assay (see above). The values for the effective concentration (EC50), cytotoxicity concentration (CC₅₀), and selective index (SI) are shown in Table 1 below. EC50 values shown in Table 1 were determined by two independent experiments. To determine whether COMPOUND 29 would retain HRV inhibitory activity under the acidic conditions of the gut, the assay included a test for HRV inhibition by COMPOUND 29 previously exposed to HCl (pH 2) for 30 minutes. As shown in Table 1, no significant reduction of the HRV inhibitory activity of COMPOUND 29 was detected by prior exposure of the compound to HCl (pH 2).

Table 1. HRV Inhibitory Activity of COMPOUND 29 in MTT assay

Results: Inhibitory effect of COMPOUND 29 on human rotavirus adsorption. To explore the mechanism of the inhibitory activity of COMPOUND 29 toward HRV, time-of-addition experiments were performed by FFU assays in which only one cycle of HRV replication occurred. Various concentrations of COMPOUND 29 were added to MA 104 cells before (pretreatment, -1 - 0 hours), during (0 - 1.5 hours), and after (1.5 - 24 hours) HRV adsorption, and the inhibitory effects were evaluated on the basis of EC₅₀ values. The data are shown in Table 2 below. The EC₅₀ values shown in Table 2 were determined by two independent experiments. The data in Table 2 clearly show that the greatest inhibition of HRV replication occurred when COMPOUND 29 was added during virus adsorption (0-1.5 hours). Pretreatment of MAI 04 cells with COMPOUND 29 resulted in little inhibitory activity. These data indicate that the predominant HRV inhibitory activity of COMPOUND 29 resides in the ability of the compound to inhibit virus adsorption.

Table 2. Time-Of- Addition Study of COMPOUND 29 Inhibition of HRV

Results: Inhibitory effect of COMPOUND 29 on growth of four serotypes of HRV. The HRV inhibitory activity of COMPOUND 29 toward representatives of the four major serotypes of HRV responsible for gastroenteritis in humans throughout the world was tested in a plaque reduction assay. A further test was made for the ability of COMPOUND 29 to the Rhesus RV serotype of simian rotavirus strain SAl 1. The data, expressed as EC50 values, are shown in Table 3 below. Each of the EC₅₀ values shown in Table 3 was determined by two independent experiments.

Table 3. COMPOUND 29 Inhibition of Adsorption of Selected Rotavirus Serotypes

The data in Table 3, above, show that COMPOUND 29 effectively inhibited adsorption each of the four major serotypes of HRV with EC₅₀ values ranging from 1.5 to 4.7 μg/ml.

Results: Effect of COMPOUND 29 on the binding of Wa strain to MAI 04 cells. COMPOUND 29 was examined for inhibition of binding of radiolabeled, purified HRV Wa strain to MA104 cells (2 x 10⁴ cpm/well, 48 FFU). The data as shown in the bar graphs of Figure 1 indicate that COMPOUND 29 blocked binding of HRV to cells in a dose-dependent manner. For example, as shown in Figure 1, COMPOUND 29 inhibited approximately 66% of the control binding at 20 μg/ml.

Results: Protective efficacy of COMPOUND 29 against rotavirus-induced diarrhea in a mouse model. Groups of 4 to 5 pups were orally inoculated with 3 x 10° pfu of MO strain rotavirus by gavage. COMPOUND 29 was also orally administered three times per day, beginning 30 minutes before inoculation with virus, as described above. The results are shown in Figures 2A-2E. All mice treated with water alone as a control developed diarrhea (i.e., Diarrhea index (DI) values of 4, 5, or 6) 2 to 3 days after inoculation (see, Figure 2A). To determine the effective amount of COMPOUND 29 to prevent rotavirus-induced diarrhea, mice were orally give 50, 10, 2, or 0.4 μg of COMPOUND 29, three times per day for four days. All mice given 10 μg of COMPOUND 29 did not develop diarrhea (see, Figure 2C). Only one of the five mice that received 10 μg of COMPOUND 29 showed soft brown stool, and the other showed no stool or brown formed stool (Figure 2C), indicating that COMPOUND 29 prevented diarrhea in these mice. Mice give 50 μg of COMPOUND 29 showed partial protection (Figure 2B). Two of the five mice that received 50 μg of COMPOUND 29 developed soft mucous diarrhea (DI = 4), but the others showed brown formed to brown soft stool during the course of the study. In contrast, administering 2 or 0.4 μg of COMPOUND 29 to mice inoculated with the rotavirus MO strain had little effect on the development of diarrhea (see Figures 2D and 2E). The mice in these latter groups all developed soft mucous to muddy mucous diarrhea (DI = 4 or 5). All mice recovered by the 5th day after inoculation without mortality.

Results: Determination of effective initiation time of treatment of rotavirus-induced diarrhea in mouse model. To further determine the effective initiation time of treatment with COMPOUND 29 after virus inoculation, mice were treated with 10 μg COMPOUND 29 beginning 30 minutes before infection, and 6, 12, or 18 hours after virus inoculation (infection) until the 4th day of infection. Mice of the control group were inoculated with virus, but received water instead of COMPOUND 29. All control mice (treated with water) developed diarrhea (Figure 3E). All mice treated with COMPOUND 29 beginning 30 minutes before (Figure 3A) and 6 hours after (Figure 3B) virus inoculation were protected from diarrhea. One of 5 mice in both of these groups showed soft brown stool (DI=3) during the course and the others showed no stool or brown formed stool. In contrast, treatment beginning 12 hours (Figure 3 C) or 18 hours (Figure 3D) after virus inoculation had little effect on the development of diarrhea. All mice of these two groups developed diarrhea with DI greater than 4, although none of the mice developed liquid, mucous yellow stool (i.e., no DI = 6).

Results: Effect of COMPOUND 29 on serum IgG responses of mice to HRV MO strain in a mouse diarrhea model. Serum IgG titers to MO strain 14 days after inoculation with HRV MO strain were determined by immunofluorescence to estimate the extent of suppression of viral replication in mice treated with COMPOUND 29 starting before virus inoculation, as shown in Figures 2A-2E. Titers of IgG antibody in mice infected with the MO strain are shown in Table 4 below.

Table 4. Effect of COMPOUND 29 treatment of serum anti-HRV IgG response to HRV in mice infected with MO strain of HRV

^* p < 0.05 compared to untreated group

As can be seen in Table 4, the mean titer of anti-HRV (MO strain) IgG in the MO strain- infected, untreated, control mice was significantly higher than those in mice treated three times per day with 10 μg and 50 μg COMPOUND 29 (3 times daily), but not significantly different from the group of mice treated three times per with 2 μg and 0.4 μg of COMPOUND 29. The above studies indicate that COMPOUND 29 of Example 29 inhibits replication of rotavirus. COMPOUND 29 can be administered orally to treat HRV-mediated gastroenteritis. COMPOUND 29 inhibits the four major serotypes of HRV that are known to cause the vast majority of cases of infantile gastroenteritis. Although not wishing to be bound by any theory or mechanism, COMPOUND 29 may inhibit HRV replication by binding to HRV particles, but not to cellular receptors for HRV, and may prevent virus attachment to receptor sites on cells.

Other variations and embodiments of the invention described herein will now be apparent to those of ordinary skill in the art without departing from the spirit of the invention or the scope of the claims below.

All patents, applications, and publications cited in the above text are incorporated herein by reference.

Claims

What is claimed is:

1. A method of treating rotavirus-mediated gastroenteritis in a mammal comprising administering to said mammal a sulfated sialyl lipid compound, or the salt thereof, comprising: a monosaccharide moiety linked to a lipid moiety, wherein said monosaccharide moiety is a sialic acid or a 2-keto-3-deoxy-D-glycero-2 -nonuloic acid (KDN), and said lipid moiety is linked to the anomeric carbon of said sialic acid or of said KDN, and all hydroxyl groups of said sialic acid or said KDN are sulfated.

2. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 1, wherein the link between said sialic acid or said KDN and said lipid moiety is an O- glycosidic linkage or an S-glycosidic linkage at position 2 of said sialic acid or said KDN, or an amide linkage at position 1 of said sialic acid or said KDN.

3. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 2, wherein said lipid moiety is a linear lipid comprising a branched chain structure.

4. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 3, wherein said lipid moiety comprises a branched chain structure, wherein said branched chain structure is located at position 2 of said linear lipid.

5. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 4, wherein said branched chain structure of said linear lipid comprises two chains.

6. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 5, wherein said linear lipid has 2 to 8 skeleton-forming atoms.

7. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 6, wherein the total number of skeleton-forming atoms of all branched chains is 18 to 60.

8. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 7, wherein said branched chains comprise an unsaturated bond between carbon atoms.

9. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 7, wherein said branched chains of said lipid moiety are linear.

10. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 7, wherein said branched chains comprise an ester linkage or an ether linkage.

11. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 10, wherein said ester linkage or said ether linkage is localized at position 1 or 2 of said branched chain structure.

12. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 7, wherein the number of skeleton-forming atoms per each branched chain is 10 to 28.

13. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 12, wherein the number of skeleton-forming atoms per each branched chain is 18 to 26.

14. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 13, wherein the number of skeleton-forming atoms per each branched chain is 24.

15. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 14, wherein said branched chains have the same length.

16. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 15, wherein said branched chains have the same structure.

17. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 16, wherein said branched chains have an ester linkage or an ether linkage at position 1 or 2 of said branched chains.

18. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 17, wherein the sulfated sialyl lipid compound is sodium [2,2-bis (docosyloxymethyl) propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D- galacto-2-nonulopyranosid]onate, or the acid thereof.

19. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 18, wherein one or more sodium atoms is replaced by a different cation.

20. The method of treating rotavirus-mediated gastroenteritis in a mammal according to claim 19, wherein said different cation is potassium.

21. The method according to any one of claims 1-20, wherein said sulfated sialyl lipid compound is administered to said mammal orally.

22. The method according to any one of claims 1-20, wherein said sulfated sialyl lipid compound is administered through a route selected from the group consisting of an intravenous route, an intramuscular route, an intraperitoneal route, an intra-arterial route, a subcutaneous route, a percutaneous route, a sublingual route, an inhalation route, and a rectal route.

23. A medicament to treat rotavirus-mediated gastroenteritis in a mammal comprising a sulfated sialyl lipid compound, or the salt thereof, comprising: a monosaccharide moiety linked to a lipid moiety, wherein said monosaccharide moiety is a sialic acid or a 2-keto-3-deoxy-D-glycero-2-nonuloic acid (KDN), and said lipid moiety is linked to the anomeric carbon of said sialic acid or of said KDN, and all hydroxyl groups of said sialic acid or said KDN are sulfated.

24. The medicament according to claim 23, wherein the link between said sialic acid or said KDN and said lipid moiety is an O-glycosidic linkage or an S-glycosidic linkage at position 2 of said sialic acid or said KDN, or an amide linkage at position 1 of said sialic acid or said KDN.

25. The medicament according to claim 24, wherein said lipid moiety is a linear lipid comprising a branched chain structure.

26. The medicament according to claim 25, wherein said lipid moiety comprises a branched chain structure, wherein said branched chain structure is located at position 2 of said linear lipid.

27. The medicament according to claim 26, wherein said branched chain structure of said linear lipid comprises two chains.

28. The medicament according to claim 27, wherein said linear lipid has 2 to 8 skeleton- forming atoms.

29. The medicament according to claim 28, wherein the total number of skeleton- forming atoms of all branched chains is 18 to 60.

30. The medicament according to claim 29, wherein said branched chains comprise an unsaturated bond between carbon atoms.

31. The medicament according to claim 29, wherein said branched chains of said lipid moiety are linear.

32. The medicament according to claim 29, wherein said branched chains comprise an ester linkage or an ether linkage.

33. The medicament according to claim 32, wherein said ester linkage or said ether linkage is localized at position 1 or 2 of said branched chain structure.

34. The medicament according to claim 29, wherein the number of skeleton- forming atoms per each branched chain is 10 to 28.

35. The medicament according to claim 34, wherein the number of skeleton- forming atoms per each branched chain is 18 to 26.

36. The medicament according to claim 35, wherein the number of skeleton-forming atoms per each branched chain is 24.

37. The medicament according to claim 36, wherein said branched chains have the same length.

38. The medicament according to claim 37, wherein said branched chains have the same structure.

39. The medicament according to claim 38, wherein said branched chains have an ester linkage or an ether linkage at position 1 or 2 of said branched chains.

40. The medicament according to claim 23, wherein the sulfated sialyl lipid compound is sodium [2,2-bis(docosyloxymethyl) propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyτanosid]onate, or the acid thereof.

41. The medicament according to claim 40, wherein one or more sodium atoms is replaced by a different cation.

42. The medicament according to claim 41, wherein said different cation is potassium.

43. The method according to any one of claims 23-42, prepared for administration to a mammal through a route selected from the group consisting of an oral route, intravenous route, an intramuscular route, an intraperitoneal route, an intra-arterial route, a subcutaneous route, a percutaneous route, a sublingual route, an inhalation route, and a rectal route.