Nothing Special   »   [go: up one dir, main page]

WO2003049672A2 - Methods and compositions for treating flavivirus-mediated disease - Google Patents

Methods and compositions for treating flavivirus-mediated disease Download PDF

Info

Publication number
WO2003049672A2
WO2003049672A2 PCT/US2002/034659 US0234659W WO03049672A2 WO 2003049672 A2 WO2003049672 A2 WO 2003049672A2 US 0234659 W US0234659 W US 0234659W WO 03049672 A2 WO03049672 A2 WO 03049672A2
Authority
WO
WIPO (PCT)
Prior art keywords
flavivirus
glycero
treating
mediated disease
galacto
Prior art date
Application number
PCT/US2002/034659
Other languages
French (fr)
Other versions
WO2003049672A3 (en
Inventor
Kazuo Takahashi
Shiro Shigeta
Kirk A. Field
Original Assignee
Microbiotix, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Microbiotix, Inc. filed Critical Microbiotix, Inc.
Priority to AU2002342188A priority Critical patent/AU2002342188A1/en
Publication of WO2003049672A2 publication Critical patent/WO2003049672A2/en
Publication of WO2003049672A3 publication Critical patent/WO2003049672A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is generally in the field of treating flavivirus-mediated diseases in animals.
  • this invention relates to methods and compositions comprising sulfated sialyl lipid compounds for the treatment of flavivirus-mediated diseases, such as dengue fever and hepatitis C.
  • Flaviviridaes Animal viruses of the family Flaviviridae, commonly known as flaviviruses, include a number of human viral pathogens, which cause diseases that continue to erupt annually in widespread, debilitating, and life threatening epidemics. Such notable pathogenic flaviviruses include dengue virus and hepatitis C virus (HCV). The dengue flavivirus continues to infect people, in vast areas of the tropics, such as Asia and Africa, and in recent years has also advanced into the Americas, thereby placing over 2 billion people at risk, with millions of cases of infection reported yearly.
  • HCV hepatitis C virus
  • dengue fever (or simply "dengue") is now primarily an urban disease following the rise in tropical urban centers where large and crowded human populations live in intimate contact with the principal mosquito vector, Aedes aegypti, although a number of other species of mosquitoes are known to be effective vectors as well (see, “Dengue Viruses,” in Encyclopedia of Virology, Vol. 1, pp. 324-331 (Webster and Granoff, eds.) (Academic Press, New York, 1994)).
  • dengue viruses The only known natural hosts for dengue viruses are mosquitoes, humans, and lower primates. Dengue viruses can be propagated in baby mice and some vertebrate cell lines.
  • Mosquitos as the disease vectors, do not appear to be adversely affected by lifelong infection by dengue. Although some dengue infections in humans may result in only a mild illness, the most widely recognized forms of the dengue-mediated disease is "dengue fever" and classical dengue hemorrhagic fever or dengue shock syndrome, which can be fatal. Patients infected with dengue virus that do not present classical dengue hemorrhagic fever or dengue shock syndrome may still experience any of a variety of disease symptoms, such as severe, uncontrollable bleeding, particularly, from the upper gastrointestinal tract; skin hemorrhages; fever; liver damage; encephalopathy; rash; joint pain; nausea; vomitting; headache; myalgias (Id. at p. 329). Absent any current effective pharmaceutical regimen, therapeutic treatment of dengue fever is largely symptomatic, such as early fluid replacement and shock control for the patient. Preventive measures are predominantly centered mainly on mosquito control in tropical urban centers.
  • HCV infection can cause both acute as well as chronic hepatitis in humans and certain other primates, with up to 20% of the disease state in human patients progressing to liver cirrhosis (see, "Hepatitis C Virus,” in Encyclopedia of Virology, Vol. 2, pp. 569-574 (Webster and Granoff, eds.) (Academic Press, New York, 1994)).
  • a predominant mode of transmission HCV to individuals appears to be parenteral transfer of contaminate blood, body fluids, and blood products.
  • HCV was originally indicated by the discovery that most cases of transfusion-associated hepatitis were in fact caused by neither the hepatitis A virus nor the hepatitis B virus (Id. at p. 569).
  • HCV human immunodeficiency virus
  • compositions and methods to treat flavivirus-mediated diseases SUMMARY OF THE INVENTION
  • This invention provides compositions, such as medicaments, and methods to prophylactically or therapeutically treat an individual (e.g., a human or other primate) for a disease caused by a virus of the family Flaviviridae.
  • viruses are also referred to as flaviviruses, which include dengue viruses and the hepatitis C virus (HCV).
  • Methods of the invention comprise administering to an individual 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) and described herein, which have been discovered to unexpectedly prevent replication (propagation) of strains of flaviviruses and, therefore, useful for treating certain flavivirus- mediated diseases, such as dengue fever and hepatitis C.
  • one or more sulfated sialyl lipid compounds such as those previously described (see, e.g., EP 0 957 107 Al) and described herein, which have been discovered to unexpectedly prevent replication (propagation) of strains of flaviviruses and, therefore, useful for treating certain flavivirus- mediated diseases, such as dengue fever and hepatitis C.
  • a method of treating, prophylactically or therapeutically, a flavivirus-mediated in an individual comprising administering to the individual 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 2-keto- 3-deoxy-D-glycero-2-nonuloic (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.
  • 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 2-keto- 3-deoxy-D-glycero-2-nonuloic (KDN), wherein the lipid moiety is linked to an
  • methods of treating flavivirus-mediated disease 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.
  • the methods of the invention for prophylactically or therapeutically treating flavivirus-mediated disease 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.
  • the site of branching on the linear lipid is located a 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 lipid may be identical or different structures.
  • the invention provides methods of prophylactically or therapeutically treating a flavivirus-mediated disease 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.
  • sulfated sialyl lipid compound that is sodium [2,2-bis(docosyloxymethyl)propyl-5-acetamido-3,5-dideoxyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- -D-galacto-2-nonulopyranosid]onate.
  • a sulfated sialyl lipid compound may be administered to an individual to treat a flavivirus-mediated disease by parenteral or non- parenteral routes.
  • a particularly preferred route for administering a sulfated sialyl lipid compound according to the invention is a parenteral route, most preferably, orally.
  • 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 flavivirus-mediated disease orally as well as any of the other various routes mentioned above.
  • the methods and medicaments of the invention are directed to a flavivirus-mediated disease that is a dengue virus-mediated disease, including, but not limited to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome.
  • the methods and medicaments of the invention are directed to a flavivirus-mediated disease that is a HCV-mediated disease, particularly hepatitis C.
  • 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 a 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.
  • KDN 2-keto-3-deoxy-D-glycero-2-nonuloic acid
  • 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- ⁇ -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.
  • HAV human immunodeficiency virus
  • feline immunodeficiency virus feline immunodeficiency virus
  • respiratory syncytial virus herpes simplex II virus
  • herpes simplex II virus herpes simplex II virus
  • sulfated sialyl lipid compounds such as COMPOUND 29 would also be effective at inhibiting replication of a flavivirus, such as dengue virus or hepatitis C virus (HCV), and therefore useful for prophylactically or therapeutically treating certain flavivirus-mediated diseases.
  • a flavivirus such as dengue virus or hepatitis C virus (HCV)
  • HCV hepatitis C virus
  • the level of a particular anti-flavivirus activity of sialyl lipid compounds used in the methods described herein is unexpectedly high.
  • a sulfated sialyl lipid compound useful in the methods and compositions of the invention is understood to also include its various salt forms as well.
  • anti-viral activity 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.
  • 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.
  • virus refers to one or more viruses of the family Flaviviridae, also known as a flaviviruses, and includes strains of dengue virus and HCV.
  • nucleic 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”.
  • KDN used in this specification is meant 2-keto-3-deoxy-D-glycero-2-nonuloic acid.
  • two branched chains 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.
  • skeleton-forming atoms is meant atoms constructing the skeleton or backbone of a chain, including carbon atom, oxygen atom, nitrogen atom, sulfur atom, etc.
  • 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.
  • salt is meant the cationic salt, such as sodium and potassium salts, of a compound described herein.
  • 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.
  • 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.
  • 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.
  • the saccharide moiety of the glycoside is sialic acid or KDN
  • the glycoside not only has a strong anti-viral activity, but also lower cytotoxicity, and can preferably be used in a method of treating a mammal, including humans, for a flavivirus-mediated disease.
  • 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.
  • 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.
  • the link is a covalent bond, such as an O-glycosidic linkage or an S-glycosidic linkage.
  • 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.
  • glycoside is generally meant a compound wherein the saccharide and lipid moieties are linked by a glycosidic linkage
  • 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.
  • 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.
  • 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 ⁇ position (i.e., the ⁇ position with respect to the sugar moiety) of said lipid moiety.
  • 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, or sulfur.
  • the total number of the skeleton-forming atoms of all branched chain structures is preferably 18 to 60.
  • 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.
  • the compound related to this invention 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.
  • said compound 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.
  • the glycerol residue in the glycerol area is suitably modified to become pseudo-glycerol.
  • the number of skeleton-forming atom is preferably 10 to 28, more preferably 18 to 26, most preferably 24.
  • 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.
  • the invention provides methods of treating a flavivirus-mediated disease in a mammal (including humans and other primates) which comprise administering to the mammal a sulfated sialyl lipid compound to inhibit or prevent replication (propagation) of a flavivirus in the mammal.
  • 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.
  • compositions are useful in the methods of the invention which comprise administering to a mammal one or more sulfated sialyl lipid compounds to prevent or inhibit replication of a flavivirus as evidence by prevention or diminution of one or symptoms of a particular disease.
  • symptoms of dengue virus infections include varying degrees of fevers, from mild to classical dengue hemorrhagic fever or dengue shock syndrome, which can be fatal.
  • Other symptoms include severe, uncontrollable bleeding, particularly, from the upper gastrointestinal tract; skin hemorrhages; fever; liver damage; encephalopathy; rash; joint pain; nausea; vomitting; headache; myalgias.
  • symptoms may include both acute as well as chronic hepatitis in humans and certain other primates, including progression to liver cirrhosis.
  • 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.
  • 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.
  • a pharmaceutically acceptable buffer such as a phosphate buffered saline may be used.
  • 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.
  • 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.
  • 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.
  • 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 a flavivirus in an infected mammal.
  • a preferred method of treating flavivirus- mediated disease 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.
  • 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, mhalationally, or rectally.
  • 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 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.
  • a compound useful in regulating gut motility may be injected intravenously in a pharmaceutically acceptable buffer.
  • a compound useful in regulating gut motility may be injected intravenously in a pharmaceutically acceptable buffer.
  • 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).
  • a sulfated sialyl lipid compound useful in treating a flavivirus-mediated disease 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 particular flavivirus infection, the particular compound used, its mode of administration, and the like.
  • the goal is to prevent development of clinical symptoms of a flavivirus infection, such as dengue virus or HCV infection.
  • the goal is to reduce or eliminate one or more known symptoms of a flavivirus-mediated disease, such as one or more symptoms of dengue virus or HCV-mediated disease.
  • compositions 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 primates or other mammals, an appropriate composition will be determined and formulated according to the standards and practices for veterinary medicine.
  • Example 1 Sodium [methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosid] onate.
  • Example 2 Sodium [methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-
  • Example 3 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl) - D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-hexyl-Sn-glycerol.
  • Example 4 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-hexyl-Sn-glycerol.
  • Example 5 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-decyl-Sn-glycerol.
  • Example 6 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl) - D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l ,2-di-O-decyl-Sn-glycerol.
  • Example 7 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-tetradecyl-Sn-glycerol.
  • Example 8 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D- glycerol- ⁇ -D-galacto-2-nonulopyranosyl ⁇ ornate]- 1,2-di-O-tetradecyl-Sn- glycerol.
  • Example 9 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D- glycerol- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]- 1 ,2-di-O-octadecyl-Sn-glycerol.
  • Example 10 3-O- [Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-2-O- (sodium oxysulfonyl)- l-O- octadecyl-Sn-glycerol.
  • Example 11 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-2-O-(sodium oxysulfonyl)- 1 -O-octadecyl- Sn-glycerol.
  • Example 12 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-docosyl-Sn-glycerol.
  • Example 13 3-O-[Sodium ⁇ 5- acetamido-3,5-dideoxy- 4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-docosyl-Sn-glycerol.
  • Example 14 3-S-[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-tetradecyl-Sn-thioglycerol.
  • Example 15 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D- galacto-2-nonulopyranosyl) onate]- 1 ,2-di-O-eicosyl-Sn-glycerol.
  • Example 16 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero- ⁇ -D-galacto-nonulopyranosonic acid)-2-yl]- 1 ,2-di-O-eicosyl-Sn-glycerol.
  • 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]-l,2-di-O-eicosyl-Sn-glycerol: 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero- -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.
  • Example 18 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D- galacto-2-nonulopyranosyl) onate]-l,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- ⁇ -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.
  • Example 19 3-O- [(5-Acetamido-3,5-dideoxy-D-glycero- ⁇ -D-galacto-nonulopyranosonic acid)-2-yl]-l,2-di-O-tetracosyl-Sn-glycerol.
  • 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]-l,2-di-O-tetracosyl-Sn-glycerol.
  • Example 21 3-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D- galacto-2-nonulopyranosyl) onate]-l ,2-di-O-hexacosyl-Sn-glycerol.
  • Example 22 3-O-(5-Acetamido-3,5-dideoxy-D-glycero-( ⁇ )-D-galacto-nonulopyranosonic acid) -2-yl)-l ,2-di-O-hexacosyl-Sn-glycerol.
  • Example 23 3-O- [Sodium ⁇ 5- acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-hexacosyl-Sn-glycerol.
  • Example 24 3 ⁇ -[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O- (sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-5-cholestene.
  • Example 25 Sodium [oleyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosid] onate.
  • Example 26 Sodium [octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- -D-galacto-2-nonulopyranosid] onate.
  • Example 27 Methyl [2,2-bis (docosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosid] onate.
  • A. 2,2- bis (docosyl oxymethyl) propanol: l,l,l-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.
  • 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.
  • Example 28 2,2-Bis (docosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero- ⁇ -D- galacto-2-nonulopyranosidonic acid:
  • Example 29 Sodium [2,2-bis (docosyloxymethyl)propyl-5-acetamido-3,5-dideoxy-4,7,8,9- tetra-0-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulo pyranosid] onate ("COMPOUND 29").
  • 2,2-Bis (docosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero- -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.
  • Example 30 Methyl [2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3 ,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosid] onate.
  • Example 31 2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero- -D- galacto-2-nonulopyranosidonic acid.
  • Example 32 Sodium [2,2-bis (eicosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulo pyranosid] onate.
  • Example 33 Methyl [3,5-didocosyl oxyphenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero- ⁇ -D-galacto-2-nonulo pyranosid] onate.
  • Example 34 3,5-Didocosyloxyphenyl 5-acetamido-3,5-dideoxy-D-glycero- ⁇ -D-galacto-2- nonulopyrasidonic acid.
  • Methyl (3,5-didocosyloxyphenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D- glycero- ⁇ -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.
  • Example 35 Sodium [3,5-Didocosyloxyphenyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-
  • Example 36 3-O- [Methyl (5-acetamido-4,7,8,9-tet ⁇ a-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -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-dimethylamino ⁇ yridine (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.
  • 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 O.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.
  • Example 38 3-O-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tretra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]- 1 ,2-di-O-docosanoyl-Sn-glycerol: 3-0-[(5-Acetamido-3,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosonic acid)-
  • 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.
  • Example 40 3-O-[(3-Deoxy-D-glycero-( )-D-galacto-2-nonulopyranosonic acid) -2-yl]-l,2- di-O-docosyl-Sn-glycerol.
  • Example 41 3-O-[Sodium ⁇ 3-deoxy-4,5,7,8,9-penta-O-(sodium oxysulfonyl)-D-glycero- ⁇ - D-galacto-2-nonulopyranosyl ⁇ onate]- 1 ,2-di-O-docosyl-Sn-glycerol. 3-0-[(3-Deoxy-D-glycero- ⁇ -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.
  • Example 42 l-O-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D- galacto-2-nonulopyranosyl)onate]-L-2,3-di-O-docosyl-Sn-glycerol.
  • Example 43 1 -O-[(5-Acetamido-3,5-dideoxy-D-glycero- -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- ⁇ -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.
  • 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- ⁇ -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.
  • 1H-NMR CD 3 OD-D 2 0, 1:1, 40°C) ⁇ : 2.93 (br.dd, lH,H-3
  • Example 45 Methyl [2,2-bis (oleyl oxymethyl)propyl 5-acetamido-4,7,8,9-tetra-0-acetyl- 3,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosid]onate.
  • 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.
  • 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.
  • Example 47 Sodium [2,2-bis(oleyl oxymethyl)propyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosid] onate .
  • Example 48 Methyl [2,2-bis(docosyl oxymethyl)butyl 5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosid]onate.
  • Example 49 2,2-Bis(docosyloxymethyl)butyl 5-acetamido-3,5-dideoxy-D-glycero- ⁇ -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-( ⁇ )-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.
  • Example 50 Sodium [2,2-bis(docosyloxymethyl)butyl 5-acetamido-3,5-dideoxy-4,7,8,9- tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosid]onate:
  • Example 51 3-O-[Methyl ⁇ 5-N-(O-acetylglycolyl)-4,7,8,9-tetra-0-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
  • methyl 5-N-(O-acetylglycolyl)-4,7,8,9- tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero- ⁇ -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.
  • Example 52 3-O-[(5-N-glycolyl-3,5-dideoxy-D-glycero- ⁇ -D-galacto-nonulopyranosonic acid)-2-yl]- 1 ,2-di-O-docosyl-Sn-glycerol.
  • Example 53 3-O-[Sodium ⁇ 5-N-(O-sodium oxysulfonylglycolyl)-3,5-dideoxy-4,7,8,9-tetra- O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]- 1 ,2-di-O- docosyl-Sn-glycerol.
  • Example 54 3-S-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D-galacto-2-nonulopyranosyl)onate]-l,2-di-O-docosyl-Sn-thioglycerol.
  • 1,2-Di-O-docosyl-Sn-glycerol 200 mg, 0.28 mmol
  • N-bromosuccinimide 90 mg, 0.51 mmol
  • triphenylphosphine 170 mg, 0.65 mmol
  • 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.
  • Example 55 3-S-[(5-Acetamido-3,5-dideoxy-D-glycero- ⁇ -D-galacto-nonulopyranosonic acid)-2-yl]-l ,2-di-O-doosyl-Sn-thioglycerol.
  • Example 56 3-S-[Sodium ⁇ 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-0-(sodium oxysulfonyi)- D- glycero- ⁇ -D-galacto-2-nonulopyranosyl ⁇ onate]-l,2-di-O-docosyl-Sn-thioglycerol.
  • Example 57 l-[N-(5-acetamido-4,7,8,9-tet ⁇ a-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero- ⁇ - D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane.
  • Example 58 l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero- ⁇ -D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane.
  • Example 59 l-[N- ⁇ 5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosonyl ⁇ amino]-2,2- bis(docosyloxymethyl) propane.
  • Example 60 l-[N-(5-Aetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero- ⁇ - 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-( ⁇ )-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.
  • Example 61 1 -[N-(5-Acetamido-3,5-dideoxy-2-0-methyl-D-glycero- ⁇ -D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane.
  • Example 62 l-[N- ⁇ 5-Aetamido-3,5-dideoxy-2-0-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosonyl ⁇ amino]-2,2- bis(eicosyloxymethyl) propane.
  • Example 63 l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero- ⁇ - D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl) propane.
  • Methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- ⁇ -D-galacto-2- nonulopyranosid)onate Methyl (ethyl 5-acetamido-3,5-dideoxy-D-glycero- ⁇ -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.
  • Example 64 l-[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero- ⁇ -D-galacto-2- nonulopyranosonyl) ammo]-2,2-bis(docosyloxymethy ⁇ ) propane.
  • Example 65 l-[N- ⁇ 5-Acetamido-3,5-dideoxy-2-O-ethyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosonyl ⁇ amino]-2,2- bis(docosyloxymethyl) propane.
  • Example 66 l-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-0-methyl-D-glycero- ⁇ -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- ⁇ -D-galacto-2- nonulopyranosidonic acid:
  • Methyl 5-acetamido-3,5-dideoxy-D-glycero-( )-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.
  • Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-( ⁇ )-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.
  • Example 67 l-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero- ⁇ -D-galacto-2- nonulopyranosonyl)amino]-2,2-bis (docosyloxymethyl) propane.
  • Example 68 l-[N- ⁇ 5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulopyranosonyl ⁇ amino]-2,2- bis(docosyloxymethyl) propane.
  • Example 69 In vitro inhibition of human dengue virus (HDV) by sodium [2,2-bis (docosyl oxymethyl)propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- ⁇ -D-galacto-2-nonulo pyranosidjonate ("COMPOUND 29" of Example 29).
  • HDV human dengue virus
  • Vero cells are cultured in MEM supplemented with 10% fetal calf serum (FCS).
  • FCS fetal calf serum
  • DEN type 2 strain of human dengue virus (HDV) are propagated in Vero cells, and harvested after two freeze-thaw cycles. HDV titer is determined by plaque assay. Chemicals.
  • COMPOUND 29 of Example 29 above sodium (2,2-bis (docosyloxymethyl)propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)- D-glycero- ⁇ -D-galacto-2-nonulo pyranosid] onate
  • Antiviral assays The inhibitory effect of COMPOUND 29 on the replication of dengue virus is determined by the inhibition of vims-induced cytotoxicity in Vero cells.
  • a confluent monolayer culture of Vero cells is infected with DEN type 2 strain of HDV (100 pfu/well) with various concentratons of COMPOUND 29 in a 96-well microplate and cultured for 3 days.
  • the rate of viable cells is determined colorimetrically by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
  • DEN type 2 strain of HDV is also determined by focus forming unit (FFU) assay. Vero cells in a 96-well plate are infected with 10-fold serial dilutions of virus stock and cultured for 24 hours.
  • the cells are fixed with methanol, washed with PBS, and reacted with mouse anti-dengue virus (DEN type 2 strain) serum. After washing, the cells are reacted with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase. HDV-infected cells are visualized by diaminobenzidine (DAB) and the number of infected cells (focus) is counted under a microscope to determine the FFU titer of the virus stock. Time of addition experiments. Vero cells in a 96-well microplate are pretreated with various concentrations of COMPOUND 29 before virus adsorption (100 FFU/well) for 1 hour or treated with COMPOUND 29 during or after virus adsorption. The cells are cultured for 24 hours and virus-infected cells are visualized and counted as described above. The 50% effective concentration (EC 50 ) is defined as the concentration of COMPOUND 29 required for reducing the number of focuses by 50%.
  • EC 50 50% effective concentration
  • Virus binding assay is performed with radiolabeled purified DEN type 2 strain of HDV.
  • HDV strain-infected Vero cells are radiolabeled with 100 mCi 35 S- methionine for 12 hours until massive CPE is observed.
  • the radiolabeled virus is then purified with sucrose gradient by ultracentrifugation. Fractions of 100 ⁇ l aliquots are tested for virus titer by a FFU assay and for radioactivity.
  • the cells are lysed with lysing solution (1% Triton X-100, 0.15 M NaCl, 10 mM Tris/HCl), and bound virus is counted in a liquid scintillation counter (Aloka). The percentage of bound viruses is calculated as:
  • Example 70 Efficacy of Sulfated Sialyl Lipid Compounds against Hepatitis C Virus (HCV) Pseudosystems and Surrogates.
  • HCV does not replicate efficiently in mammalian cells and the difficulty of obtaining cell-free infectious virus has hindered efforts to study the entry of HCV into cells.
  • pseudotype virus systems also called “pseudovirus” systems
  • surrogate models have provided useful alternatives for tests and studies on viruses, such as HCV, that do not replicate efficiently in cell cultures.
  • VSV vesicular stomatitis virus
  • GFP green fluorescent protein
  • the inhibitory effect of COMPOUND 29 on the chimeric proteins El and E2 HCV proteins in an HCV-VSV pseudotype virus system is tested by examining the reduction in viral infectivity in the presence of COMPOUND 29 or other sulfated sialyl lipid compound.
  • the HCV-VSV pseudotype virus system is propagated in Hep2 cells.
  • Bovine Diarrhea Virus Surrogate Model. Although at present there is no known direct in vitro system for the analysis of compounds against HCV, a bovine diarrhea virus (BVDV) is an accepted surrogate model for the analysis of compound efficacy against HCV. Below is outlined the procedure for determining compound efficacy against BVDV. Stocks of BVDV were prepared by infecting bovine kidney (BK) cells at a multiplicity of infection (m.o.i.) of ⁇ 0.01 plaque-forming units (pfu.) per cell. Cell growth medium was changed every four days until cytopathology was evident in all cells (approximately 21 days). Supernatant fluids were retained as the virus stock (see, Chapter 4, In Virus Culture.
  • BK bovine kidney
  • pfu. plaque-forming units
  • Virus titers were determined using monolayer cultures of BK cells (ibid.). Briefly, BK cells were plated as described above in 96-well cluster dishes and incubated overnight at 37°C in a humidified 3% C0 2 - 97% air atmosphere. The next day cultures were inoculated with BVDV and serially diluted 1:3 across the remaining eleven columns of the 96-well plate. Cultures were incubated at 37°C for 2 hours to permit virus adsorption and then virus inoculums were replaced with 0.2 ml of fresh medium. Cultures were incubated for seven days.
  • BK cells were plated into 24-well cluster dishes and infected with
  • COMPOUND 29 of Example 29
  • COMPOUND 29 and other sulfated sialyl compounds described herein can be administered orally as well as by other routes to treat a flavivirus-mediated disease. While not desiring to be bound by any particular mechanism or theory, COMPOUND 29 appears to inhibit viral replication by binding to the surface of virus particles, but not to cellular receptors, and may prevent virus attachment to receptor sites on cells.
  • COMPOUND 29 appears to inhibit viral replication by binding to the surface of virus particles, but not to cellular receptors, and may prevent virus attachment to receptor sites on cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Compositions and methods comprising a sulfated sialyl lipid compound are described for treating a flavivirus-mediated disease, such as dengue fever or hepatitis C, in an individual.

Description

METHODS AND COMPOSITIONS FOR TREATING FLAVIVIRUS-MEDIATED DISEASE
FIELD OF THE INVENTION This invention is generally in the field of treating flavivirus-mediated diseases in animals. In particular, this invention relates to methods and compositions comprising sulfated sialyl lipid compounds for the treatment of flavivirus-mediated diseases, such as dengue fever and hepatitis C.
BACKGROUND OF THE INVENTION
Animal viruses of the family Flaviviridae, commonly known as flaviviruses, include a number of human viral pathogens, which cause diseases that continue to erupt annually in widespread, debilitating, and life threatening epidemics. Such notable pathogenic flaviviruses include dengue virus and hepatitis C virus (HCV). The dengue flavivirus continues to infect people, in vast areas of the tropics, such as Asia and Africa, and in recent years has also advanced into the Americas, thereby placing over 2 billion people at risk, with millions of cases of infection reported yearly. With increased migration of populations to cities and travel between cities, dengue fever (or simply "dengue") is now primarily an urban disease following the rise in tropical urban centers where large and crowded human populations live in intimate contact with the principal mosquito vector, Aedes aegypti, although a number of other species of mosquitoes are known to be effective vectors as well (see, "Dengue Viruses," in Encyclopedia of Virology, Vol. 1, pp. 324-331 (Webster and Granoff, eds.) (Academic Press, New York, 1994)).
The only known natural hosts for dengue viruses are mosquitoes, humans, and lower primates. Dengue viruses can be propagated in baby mice and some vertebrate cell lines.
Mosquitos, as the disease vectors, do not appear to be adversely affected by lifelong infection by dengue. Although some dengue infections in humans may result in only a mild illness, the most widely recognized forms of the dengue-mediated disease is "dengue fever" and classical dengue hemorrhagic fever or dengue shock syndrome, which can be fatal. Patients infected with dengue virus that do not present classical dengue hemorrhagic fever or dengue shock syndrome may still experience any of a variety of disease symptoms, such as severe, uncontrollable bleeding, particularly, from the upper gastrointestinal tract; skin hemorrhages; fever; liver damage; encephalopathy; rash; joint pain; nausea; vomitting; headache; myalgias (Id. at p. 329). Absent any current effective pharmaceutical regimen, therapeutic treatment of dengue fever is largely symptomatic, such as early fluid replacement and shock control for the patient. Preventive measures are predominantly centered mainly on mosquito control in tropical urban centers.
HCV infection can cause both acute as well as chronic hepatitis in humans and certain other primates, with up to 20% of the disease state in human patients progressing to liver cirrhosis (see, "Hepatitis C Virus," in Encyclopedia of Virology, Vol. 2, pp. 569-574 (Webster and Granoff, eds.) (Academic Press, New York, 1994)). A predominant mode of transmission HCV to individuals appears to be parenteral transfer of contaminate blood, body fluids, and blood products. In fact, HCV was originally indicated by the discovery that most cases of transfusion-associated hepatitis were in fact caused by neither the hepatitis A virus nor the hepatitis B virus (Id. at p. 569). Accordingly, identifiable populations of people at high-risk for HCV infection have in the past included hemophiliacs, illicit parenteral drug users, and health care professionals who come in contact with blood and tissues of HCV patients. Screening for carriers of HCV and improved manufacturing methods to inactivate contamination by human immunodeficiency virus (HIV) have reduced incidences of transfusion-acquired hepatitis. However, the mode of transmission of many community- acquired HCV infections remains to be fully elucidated. No effective prophylactic or therapeutic treatment is currently available for HCV-mediated hepatitis.
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 HIV, 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.
Clearly, needs remain for compositions and methods to treat flavivirus-mediated diseases. SUMMARY OF THE INVENTION This invention provides compositions, such as medicaments, and methods to prophylactically or therapeutically treat an individual (e.g., a human or other primate) for a disease caused by a virus of the family Flaviviridae. Such viruses are also referred to as flaviviruses, which include dengue viruses and the hepatitis C virus (HCV). Methods of the invention comprise administering to an individual 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) and described herein, which have been discovered to unexpectedly prevent replication (propagation) of strains of flaviviruses and, therefore, useful for treating certain flavivirus- mediated diseases, such as dengue fever and hepatitis C.
In one embodiment of the invention is provided a method of treating, prophylactically or therapeutically, a flavivirus-mediated in an individual comprising administering to the individual 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 2-keto- 3-deoxy-D-glycero-2-nonuloic (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 flavivirus-mediated disease 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 flavivirus-mediated disease 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 a 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 lipid may be identical or different structures. In yet another embodiment, the invention provides methods of prophylactically or therapeutically treating a flavivirus-mediated disease 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 a flavivirus-mediated disease in an individual comprising administering to the mammal a sulfated sialyl lipid compound that is sodium [2,2-bis(docosyloxymethyl)propyl-5-acetamido-3,5-dideoxyl-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- -D-galacto-2-nonulopyranosid]onate.
In another aspect of the invention, a sulfated sialyl lipid compound may be administered to an individual to treat a flavivirus-mediated disease by parenteral or non- parenteral routes. A particularly preferred route for administering a sulfated sialyl lipid compound according to the invention is a parenteral route, most preferably, 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 flavivirus-mediated disease orally as well as any of the other various routes mentioned above.
In a preferred embodiment, the methods and medicaments of the invention are directed to a flavivirus-mediated disease that is a dengue virus-mediated disease, including, but not limited to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome.
In yet another embodiment, the methods and medicaments of the invention are directed to a flavivirus-mediated disease that is a HCV-mediated disease, particularly hepatitis C.
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 a 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 a flavivirus, such as dengue virus or hepatitis C virus (HCV), and therefore useful for prophylactically or therapeutically treating certain flavivirus-mediated diseases. Furthermore, the level of a particular anti-flavivirus activity of sialyl lipid compounds used in the methods described herein is unexpectedly high. Unless otherwise noted, a sulfated sialyl lipid compound useful in the methods and compositions 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. Unless stated otherwise, the terms "virus", "viral", "viral-mediated", "anti-viral activity", and similar terms refer to one or more viruses of the family Flaviviridae, also known as a flaviviruses, and includes strains of dengue virus and HCV. 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 lower cytotoxicity, and can preferably be used in a method of treating a mammal, including humans, for a flavivirus-mediated disease. 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, or 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 a flavivirus-mediated disease in a mammal (including humans and other primates) which comprise administering to the mammal a sulfated sialyl lipid compound to inhibit or prevent replication (propagation) of a flavivirus in the mammal. Such methods may be either therapeutic or 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 to prevent or inhibit replication of a flavivirus as evidence by prevention or diminution of one or symptoms of a particular disease. For example, symptoms of dengue virus infections include varying degrees of fevers, from mild to classical dengue hemorrhagic fever or dengue shock syndrome, which can be fatal. Other symptoms include severe, uncontrollable bleeding, particularly, from the upper gastrointestinal tract; skin hemorrhages; fever; liver damage; encephalopathy; rash; joint pain; nausea; vomitting; headache; myalgias. In the case of HCV infection, symptoms may include both acute as well as chronic hepatitis in humans and certain other primates, including progression to liver cirrhosis.
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 a flavivirus in an infected mammal. A preferred method of treating flavivirus- mediated disease 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, mhalationally, 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 a flavivirus-mediated disease 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 particular flavivirus infection, the particular compound used, its mode of administration, and the like. Prophylactically, the goal is to prevent development of clinical symptoms of a flavivirus infection, such as dengue virus or HCV infection. Therapeutically, the goal is to reduce or eliminate one or more known symptoms of a flavivirus-mediated disease, such as one or more symptoms of dengue virus or HCV-mediated disease. 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 primates or 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 dimethylformamide (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.
1H-NMR (D2O) δ: 3.37 (s, 3H, OCH3), 2.88 (dd, IH, J =4.0, 11.7 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. Η-NMR (D2O) δ: 3.33 (s, 3H, OCH3), 2.57 (dd, IH, J =5.1, 13.2 Hz, H-3eq), 1.96 (s,
3H, NAc), 1.90 (t, IH, 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]-l,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.
!H-NMR (CD3OD) δ: 3.02 (dd, IH, J =4.9, 12.6 Hz, H-3eq), 1.92 (s, 3H, NAc), 1.83 (t, IH, 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]-l,2-di-O-hexyl-Sn-glycerol.
3-O- [Sodium (5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl) onate]-l,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.
1H-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.
!H-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]-l ,2-di-O-decyl-Sn-glycerol.
3-0-[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. Heil-125394) (197 mg) was reacted by the general procedure according to Example 1 to obtain the title compound (240 mg, 76%) as white solid. tø-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), 0.8.8 (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]- 1 ,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, 11.9 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]- 1,2-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.
!H-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-α-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. -NMR CDsOD) 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-(sodium oxysulfonyl)- D-glycero-α-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]-l-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.
1H-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 11 : 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]-l-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.
1H-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-(sodium 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.
1H-NMR (CD3OD-D20, 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]-l,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%o) as white solid.
5H-NMR (CDCi3-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- -D-galacto-2-nonulopyranosyl} onate]- 1 ,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. -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. 3-0-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. !H-NMR (CDCI3) 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.
!H-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-tetτa-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- nonulopyranosyl) onate]-l ,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 (CDC13) 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]- 1 ,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, 2CH2CH3).
Example 17: 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-eicosyl-Sn-glycerol: 3-O-[(5-Acetamido-3,5-dideoxy-D-glycero- -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. H-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]-l,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-0-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. 1H-NMR (CDCI3) 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.
!H-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- -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 =11.3 Hz, H-3ax),1.38-1.16 (m, 84H, 42CH2), 0.89 (t, 6H, J =6.8 Hz, 2CH2CH3).
Example 20: 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-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 (CD30D-D20, 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]-l ,2-di-O-hexacosyl-Sn-glycerol.
A. 3-0-Benzyl-l,2-di-0-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. 3-0-[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 (CDC13) 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.
!H-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- -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-α-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.
!H-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, CH3-18 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.
!H-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 =7.0 Hz, CH2CH3).
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- -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- -D-galacto-2-nonulopyranosid] onate.
A. Methyl (octadecyl 5-acetamido-4,7,8,9-tetra-O-acetyl-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) (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.
!H-NMR (CDC13) 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. !H-NMR (CD3OD) δ: 3.02 (dd, IH, 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 (docosyl 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 (docosyl oxymethyl) propanol: l,l,l-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 ( , 76H, 38CH2), 0.88 (t, 6H, J =6.6 Hz, 2CH2CH3), 0.85 (s, 3H, CCH3).
B. Methyl [2,2-bis (docosyl 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 ( docosyl oxymethyl) 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.
!H-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 (docosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero-α-D- galacto-2-nonulopyranosidonic acid:
Methyl [2,2-bis (docosyl oxymethyl) propyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero- -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.
'H-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-0-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulo pyranosid] onate ("COMPOUND 29"). 2,2-Bis (docosyl oxymethyl) propyl 5-acetamido-3,5-dideoxy-D-glycero- -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. !H-NMR (CD3OD-D20, 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: l,l,l-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. H-NMR (CDC13) 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. 'H-NMR (CDCI3) 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- -D- galacto-2-nonulopyranosidonic acid.
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 (174 mg) was reacted by the general procedure according to Example 16 to obtain the title compound (127 mg, 87%) as a white solid. !H-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 2N 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.
1H-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, 611 (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-2,6phenyl), 6.22 (d, IH, J =1.8 Hz, H-4phenyl), 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.
1H-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.
1H-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-tetτa-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.
>H-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
Figure imgf000027_0001
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-dimethylaminoρyridine (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. !H-NMR (CDCI3) 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-nonuloρyranosonic acid-2-yl]- 1 ,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]-l ,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 O.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.
1H-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]- 1 ,2-di-O-docosanoyl-Sn-glycerol: 3-0-[(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. H-MR (CD3OD-D20, 1:1, 50°C) δ: 2.94 (BR. DD, IH, H-3eq), 1.95 (s, 3H, ANC),
1.71 (t, IH, J =11.0 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-α-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-ρenta-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. !H-NMR (CDCI3) 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.
3-0-[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. ^-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 (m, 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. 3-0-[(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.
*H-NMR (CD3OD-DA 1:1, 50°C) δ: 2.89 (br. dd, IH, H-3eq), 1.81 (br. t, IH, 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-0-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%).
'H-NMR (CDC13) δ: 7.32 (m, 5H, C6H5), 4.56 (s, 2H, CH2Ph).
B . L- 1 -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%). tø-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. !H-NMR (CDCI3) 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 (CDCI3) 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 : 1 -O-[(5-Acetamido-3,5-dideoxy-D-glycero- -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. 'H-NMR (CDCI3-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-α-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. 1H-NMR (CD3OD-D20, 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-0-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.
1H-NMR (CDCI3) 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-0-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,lH, J =5.0, 12.8 Hz, H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.27 ( , 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-α-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.
1H-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-nonulopyranosid] 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.
1H-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.
!H-NMR (CDCI3) 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.
'H- MR CCDC ) 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.
!H-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-0-acetyl-3,5-dideoxy-D- glycero-α-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, IH, 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-O-[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.
1H-NMR (CDCI3-CD3OD, 1:1, 60°C) δ: 2.78 (dd, IH, J=4,0, 12.0 Hz, H-3eq), 1.79 (t, IH, 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]- 1 ,2-di-O- docosyl-Sn-glycerol.
3-O-[(5-N-glycolyl-3,5-dideoxy-D-glycero- -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.
1H-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.
1H-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-0-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. 1H-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]-l ,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-nonulopyranosyl)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. 1H-NMR (CDCI3-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-0-(sodium oxysulfonyi)- D- glycero-α-D-galacto-2-nonulopyranosyl}onate]-l,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-0-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.
1H-NMR (CD3OD-DA 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-tetτa-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%).
1H-NMR (CDC13) δ: 4.13 (s, 2H, CH2OMs), 2.98 (s, 3H, CH3S02), 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(docosyloxymethyl) 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%).
1H-NMR (CDC13) 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-0-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, 611 (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.
!H-NMR (CDC13) δ: 3.33 (s, 3H, OCH3), 2.56 (dd, IH, J=4.8, 13.2 Hz, H-3eq.), 2.16, 2.11, 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 (11 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 (CDCI3) 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 =11.7 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-O-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.
'H-NMR (CDCl3-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.
!H-NMR (CD3OD-D20, 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.
1H-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.
1H-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. 1 -[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.
1H-NMR (CDC13) 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-2-0-methyl-D-glycero-β-D-galacto-2- nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl) propane. l-[N-(5-Acetamido-4,7,8,9-tetτa-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.
1H-NMR (CDC13-CD30D, 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-2-0-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.
1H-NMR (CD3OD-D20, 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. tø-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.
!H-NMR (CDCI3) 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, 5Ac), 1.87 (dd, IH, 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.
1H-NMR (CDC13) 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-tetra-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.
1H-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: l-[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2- nonulopyranosonyl) ammo]-2,2-bis(docosyloxymethyι) propane. l-[N-(5-Acetamido-4,7,8,9-tetτa-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 (114 mg, 71%) as white solid.
1H-NMR (CDCl3-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. !H-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-2-0-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-( )-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 CDCh-CDSOD, 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-tetra-O-acetyl-3,5-dideoxy-2-0-methyl-D-glycero- -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.
!H-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%o) as white solid.
]H-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.
'H-NMR (CD3OD-D20, 1:1, 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 inhibition of human dengue virus (HDV) by sodium [2,2-bis (docosyl oxymethyl)propyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodium oxysulfonyl)-D-glycero- α-D-galacto-2-nonulo pyranosidjonate ("COMPOUND 29" of Example 29). Materials and Methods
Cells and viruses. Vero cells are cultured in MEM supplemented with 10% fetal calf serum (FCS). DEN type 2 strain of human dengue virus (HDV) are propagated in Vero cells, and harvested after two freeze-thaw cycles. HDV titer is determined by plaque assay. Chemicals. COMPOUND 29 of Example 29 above (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) is synthesized and purified to more than 98%o purity 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 dengue virus is determined by the inhibition of vims-induced cytotoxicity in Vero cells. In brief, a confluent monolayer culture of Vero cells is infected with DEN type 2 strain of HDV (100 pfu/well) with various concentratons of COMPOUND 29 in a 96-well microplate and cultured for 3 days. The rate of viable cells is determined colorimetrically by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Focal forming unit ("FFU") assay. DEN type 2 strain of HDV is also determined by focus forming unit (FFU) assay. Vero cells in a 96-well plate are infected with 10-fold serial dilutions of virus stock and cultured for 24 hours. The cells are fixed with methanol, washed with PBS, and reacted with mouse anti-dengue virus (DEN type 2 strain) serum. After washing, the cells are reacted with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase. HDV-infected cells are visualized by diaminobenzidine (DAB) and the number of infected cells (focus) is counted under a microscope to determine the FFU titer of the virus stock. Time of addition experiments. Vero cells in a 96-well microplate are pretreated with various concentrations of COMPOUND 29 before virus adsorption (100 FFU/well) for 1 hour or treated with COMPOUND 29 during or after virus adsorption. The cells are cultured for 24 hours and virus-infected cells are visualized and counted as described above. The 50% effective concentration (EC50) is defined as the concentration of COMPOUND 29 required for reducing the number of focuses by 50%.
Virus binding assay. Virus binding assay is performed with radiolabeled purified DEN type 2 strain of HDV. HDV strain-infected Vero cells are radiolabeled with 100 mCi 35S- methionine for 12 hours until massive CPE is observed. The radiolabeled virus is then purified with sucrose gradient by ultracentrifugation. Fractions of 100 μl aliquots are tested for virus titer by a FFU assay and for radioactivity. For a virus-binding assay, a confluent monolayer of MA104 cells in a 96-well microplate is 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 hour on ice, after which cells are washed free of medium and excess labeled virus. The cells are lysed with lysing solution (1% Triton X-100, 0.15 M NaCl, 10 mM Tris/HCl), and bound virus is counted in a liquid scintillation counter (Aloka). The percentage of bound viruses is calculated as:
(cpm of membrane-bound virus/cpm of total input DEN type 2 strain of HDV in medium) x 100%. Results Protocols as described above have provided data that indicate that COMPOUND 29 inhibits DEN type 2 strain of HDV at an EC50 of approximately 1 μg/ml.
Example 70: Efficacy of Sulfated Sialyl Lipid Compounds against Hepatitis C Virus (HCV) Pseudosystems and Surrogates. HCV does not replicate efficiently in mammalian cells and the difficulty of obtaining cell-free infectious virus has hindered efforts to study the entry of HCV into cells. However, pseudotype virus systems (also called "pseudovirus" systems) and surrogate models have provided useful alternatives for tests and studies on viruses, such as HCV, that do not replicate efficiently in cell cultures.
Pseudovirus Virus Systems (Vesicular Stomatitis Virus). For example, whether a sulfated sialyl lipid compound (such as COMPOUND 29 of Example 29 above) is effective against HCV can be tested in the pseudotype virus model system reported by Matsuura et al (Virology, 286: 263-275 (2001)). In order to investigate the cell surface determinants important for HCV infection, this system uses a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene is replaced with a reporter gene encoding green fluorescent protein (GFP) to produce HCV-VSV pseudotypes possessing chimeric HCV El or E2 glycoproteins either individually or together. The inhibitory effect of COMPOUND 29 on the chimeric proteins El and E2 HCV proteins in an HCV-VSV pseudotype virus system is tested by examining the reduction in viral infectivity in the presence of COMPOUND 29 or other sulfated sialyl lipid compound. The HCV-VSV pseudotype virus system is propagated in Hep2 cells.
A similar pseudovirus system has been described by Meyer et al. (Virology, 276: 214- 226 (2000), incorporated herein by reference). The EC50 for COMPOUND 29 in this system is shown in the first row of the Table shown below.
Bovine Diarrhea Virus ( VDV) Surrogate Model. Although at present there is no known direct in vitro system for the analysis of compounds against HCV, a bovine diarrhea virus (BVDV) is an accepted surrogate model for the analysis of compound efficacy against HCV. Below is outlined the procedure for determining compound efficacy against BVDV. Stocks of BVDV were prepared by infecting bovine kidney (BK) cells at a multiplicity of infection (m.o.i.) of <0.01 plaque-forming units (pfu.) per cell. Cell growth medium was changed every four days until cytopathology was evident in all cells (approximately 21 days). Supernatant fluids were retained as the virus stock (see, Chapter 4, In Virus Culture. A Practical Approach, pp. 81-109 (A.J. Cann, ed.) (Oxford University Press, 1999)). Virus titers were determined using monolayer cultures of BK cells (ibid.). Briefly, BK cells were plated as described above in 96-well cluster dishes and incubated overnight at 37°C in a humidified 3% C02 - 97% air atmosphere. The next day cultures were inoculated with BVDV and serially diluted 1:3 across the remaining eleven columns of the 96-well plate. Cultures were incubated at 37°C for 2 hours to permit virus adsorption and then virus inoculums were replaced with 0.2 ml of fresh medium. Cultures were incubated for seven days. The medium was removed, and the cell sheets were stained with 0.1% crystal violet in 20% methanol. Plaques were enumerated under 20-fold magnification in wells having the dilution that gives 5 to 20 plaques per well. Virus titers were calculated according to the following formula: Titer (pfu/ml) = number of plaques x 5 x 3n; wherein "n" represents the nth dilution of the virus used to infect the well in which plaques were enumerated.
The effect of a compound on the replication of BVDV was measured using a plaque reduction assay. BK cells were plated into 24-well cluster dishes and infected with
2 approximately 100 pfu of HCMV per cm cell sheet using the procedures detailed above. Following virus adsorption, compounds dissolved in growth medium were added to duplicate wells in three to six selected concentrations. Following incubation at 37°C for 7 to 10 days, cell sheets were fixed, stained with crystal violet and microscopic plaques enumerated as described above. The effect of a compound was calculated as a percentage of reduction in number of plaques in the presence of each compound concentration compared to the number observed in the absence of drug. Table
Figure imgf000046_0001
The above systems and assays demonstrate that sulfated sialyl lipid compounds, such as COMPOUND 29 of Example 29, inhibited propagation of strains of disease-causing flavivimses. COMPOUND 29 and other sulfated sialyl compounds described herein can be administered orally as well as by other routes to treat a flavivirus-mediated disease. While not desiring to be bound by any particular mechanism or theory, COMPOUND 29 appears to inhibit viral replication by binding to the surface of virus particles, but not to cellular receptors, 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease in a mammal according to claim
4, wherein said branched chain structure of said linear lipid comprises two chains.
6. The method of treating a flavivirus-mediated disease in a mammal according to claim
5, wherein said linear lipid has 2 to 8 skeleton-forming atoms.
7. The method of treating a flavivirus-mediated disease 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 a flavivirus-mediated disease in a mammal according to claim
7, wherein said branched chains comprise an unsaturated bond between carbon atoms.
9. The method of treating a flavivirus-mediated disease in a mammal according to claim 7, wherein said branched chains of said lipid moiety are linear.
10. The method of treating a flavivirus-mediated disease in a mammal according to claim 7, wherein said branched chains comprise an ester linkage or an ether linkage.
11. The method of treating a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease 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 a flavivirus-mediated disease in a mammal according to claim
14, wherein said branched chains have the same length.
16. The method of treating a flavivirus-mediated disease in a mammal according to claim
15, wherein said branched chains have the same structure.
17. The method of treating a flavivirus-mediated disease 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 a flavivirus-mediated disease in a mammal according to claim 1, wherein the sulfated sialyl lipid compound is sodium [2,2-bis (docosyloxymethyl) propyl- 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-0-(sodium oxysulfonyl)-D-glycero-α-D-galacto-2- nonulopyranosid]onate, or the acid thereof.
19. The method of treating a flavivirus-mediated disease in a mammal according to claim
18, wherein one or more sodium atoms is replaced by a different cation.
20. The method of treating a flavivirus-mediated disease in a mammal according to claim
19, wherein said different cation is potassium.
21. The method of treating a flavivirus-mediated disease in a mammal according to claim 1, wherein the flavivirus is a dengue virus or a hepatitis C virus.
22. The method of treating a flavivirus-mediated disease in a mammal according to any one of claims 1-21, wherein said sulfated sialyl lipid compound is administered to said mammal through a route selected from the group consisting of an oral route, 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 for treating a flavivirus-mediated disease 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-0- (sodium oxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]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 medicament according to claim 23, wherein the flavivirus is a dengue virus or a hepatitis C virus.
44. The medicament according to any one of claims 23-42, prepared for administration to said mammal through a route selected from the group consisting of an oral route, 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.
PCT/US2002/034659 2001-10-30 2002-10-29 Methods and compositions for treating flavivirus-mediated disease WO2003049672A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002342188A AU2002342188A1 (en) 2001-10-30 2002-10-29 Methods and compositions for treating flavivirus-mediated disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34073501P 2001-10-30 2001-10-30
US60/340,735 2001-10-30

Publications (2)

Publication Number Publication Date
WO2003049672A2 true WO2003049672A2 (en) 2003-06-19
WO2003049672A3 WO2003049672A3 (en) 2003-07-17

Family

ID=23334710

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/034659 WO2003049672A2 (en) 2001-10-30 2002-10-29 Methods and compositions for treating flavivirus-mediated disease

Country Status (2)

Country Link
AU (1) AU2002342188A1 (en)
WO (1) WO2003049672A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008074720A (en) * 2006-09-19 2008-04-03 Noguchi Inst GM3 sugar chain probe
WO2009152589A1 (en) 2008-06-17 2009-12-23 Universidade Federal De Minas Gerais-Ufmg Use of paf receptor for treating infections caused by flaviviridae
WO2023062386A1 (en) * 2021-10-14 2023-04-20 Owlstone Medical Limited Method for the synthesis of evoc probes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0957107A1 (en) * 1996-05-16 1999-11-17 Nissin Food Products Co., Ltd. Novel compounds having antiviral activity
WO2000038697A1 (en) * 1998-12-29 2000-07-06 Glukor, Inc. Treatment and prevention of hiv and other viral infections

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0957107A1 (en) * 1996-05-16 1999-11-17 Nissin Food Products Co., Ltd. Novel compounds having antiviral activity
WO2000038697A1 (en) * 1998-12-29 2000-07-06 Glukor, Inc. Treatment and prevention of hiv and other viral infections

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008074720A (en) * 2006-09-19 2008-04-03 Noguchi Inst GM3 sugar chain probe
WO2009152589A1 (en) 2008-06-17 2009-12-23 Universidade Federal De Minas Gerais-Ufmg Use of paf receptor for treating infections caused by flaviviridae
WO2023062386A1 (en) * 2021-10-14 2023-04-20 Owlstone Medical Limited Method for the synthesis of evoc probes

Also Published As

Publication number Publication date
AU2002342188A1 (en) 2003-06-23
AU2002342188A8 (en) 2003-06-23
WO2003049672A3 (en) 2003-07-17

Similar Documents

Publication Publication Date Title
JP3571061B2 (en) Pharmaceutical preparations that suppress the replication of hepatitis B virus (HBV)
JP3049258B2 (en) New antiviral compounds
US6835720B2 (en) Compounds having antiviral activity
CA2182007A1 (en) Substituted lactose derivatives as cell adhesion inhibitors
EP2555779A1 (en) Glycomimetic compounds and methods to inhibit infection by hiv
Oka et al. Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl α (2→ 3) lactose moieties as inhibitors for human influenza viruses
JP2873313B2 (en) Compound
Yarchoan et al. Clinical and basic advances in the antiretroviral therapy of human immunodeficiency virus infection
CN101810600A (en) Methods and compositions for prevention and treatment of inflammatory disease, autoimmune disease, and transplant rejection
US20070225254A1 (en) Prophylactic and therapeutic treatment of infectious and other diseases with mono- and disaccharide- based compounds
AU8230091A (en) Hexa-n-(o-hydroxybenzyl) neomycin b for inhibiting human retroviruses and for the treatment of aids
US6518302B1 (en) Remedies
WO2003049672A2 (en) Methods and compositions for treating flavivirus-mediated disease
CN102639520B (en) Sialic acid simulation compound
US4868157A (en) Dipeptidyl 2-amino-1,2-dideoxy-D-glucose derivatives as host resistance enhancers in AIDS-immunocompromised hosts and methods of use
US4866035A (en) Dipeptidyl saccharides as host resistance enhancers in AIDS-immuno-compromised hosts and methods of use
WO2003041639A2 (en) Methods and compositions for treating rotavirus-mediated disease
US5571796A (en) Administration of valienamine-related disaccharide compounds in reducing inflammation in a sensitized mammal arising from exposure to an antigen
See et al. WIN 54954 treatment of mice infected with a diabetogenic strain of group B coxsackievirus
JPS63179885A (en) Novel glucopyranose derivative, production thereof and medicine containing said derivative as active ingredient
See et al. Occurrence of Coxsackievirus Hepatitis in Baby Rabbits and Protection by a Formalin-lnactivated Polyvalent Vaccine
EP1095653A9 (en) Glucopyranose derivatives and preventives and/or remedies for hiv infection containing the same as the active ingredient
JP5777200B2 (en) Anti-dengue virus agent
Huffman et al. Lack of suppression of resistance factors to influenxa infections in mice pretreated with virazole
JPH06206893A (en) New disaccharide derivative

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP