Aminoalkylaminoglucoside 4-phosphate derivatives

Abstract

The present application provides novel compounds or pharmaceutically acceptable salts thereof having TLR4 activating effects, and useful as immunostimulants or adjuvants in vaccine or allergen immunotherapy. The present application provides a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof. In the formula (I), X, Y, Z and n are each as defined in the specification.[Formula 1]

Description

Technical Field

[0001] This invention relates to novel compounds with immunostimulatory effects. Specifically, this invention relates to novel aminoalkyl aminoglucosinolate 4-phosphate derivatives that enhance the efficacy of vaccines or allergen immunotherapy. Background Technology

[0002] Attenuated or inactivated viruses have traditionally been used in vaccines for infectious diseases. In terms of specifications and safety, increasing the purity of the antigen in the vaccine formulation has led to the development of vaccine formulations with fewer safety issues. However, this has introduced new problems related to reduced immunogenicity, resulting in the addition of adjuvants to enhance vaccine efficacy (Non-Patent Literature 1).

[0003] The endotoxin in the outer membrane of the cell wall of Gram-negative bacteria, discovered in 1892, is now considered to be lipopolysaccharide (LPS) and is known to induce shock symptoms or systemic inflammatory responses. On the other hand, small amounts of endotoxin are also known to have therapeutic effects on certain diseases. In vaccine research, the possibility that endotoxins can enhance vaccine efficacy has been known since the 1800s (Non-Patent Literature 2).

[0004] In 1989, the existence of receptors that recognize pathogen-associated molecular patterns (PAMPs) was proposed (Non-Patent Document 3). This concept was supported by the discovery of mammalian Toll-like receptors (TLRs), and the LPS receptor TLR4 was discovered in 1998 (Non-Patent Document 4).

[0005] Lipid A has been isolated as a component of LPS, and it has been found to be important for TLR4-mediated activation of intracellular signaling (Non-Patent Literature 5 and 6). In 1982, monophospholipid A (MPLA) was screened from LPS fractions of Salmonella Typhimurium (Non-Patent Literature 7) and developed as an adjuvant for injectable vaccines, including prophylactic vaccines for cervical cancer and hepatitis B vaccines. In these vaccine formulations, MPLA exhibits immunostimulatory effects and enhances the production of antiviral antigen-specific IgG in the blood, thereby improving efficacy. The usefulness of MPLA in allergen immunotherapy (AIT) has also been demonstrated, and MPLA shows the ability to induce allergen-specific IgG within a short time, thereby suppressing allergy symptoms (Non-Patent Literature 8).

[0006] Infection via mucosa is a known route for many human pathogens, and secretory IgA present on the mucosal surface is important for mucosal protection against viruses or bacteria (Non-Patent Literature 9). However, conventional injectable vaccines for infections do not effectively induce mucosal IgA. On the other hand, mucosal vaccines have been extensively developed because mucosal-mediated immunity effectively induces mucosal IgA (Non-Patent Literature 10). However, there are technical hurdles to the clinical application of such vaccines, and only a small number are currently available. The importance of adjuvants as one of the technologies for clinical application is mentioned (Non-Patent Literature 11).

[0007] AIT has been identified as a therapy for allergy symptoms caused by subcutaneous administration (subcutaneous immunotherapy: SCIT) of allergens inducing allergic rhinitis (Non-Patent Literature 12). Under the proposed mechanism of AIT, induced allergen-specific IgG, particularly IgG4, captures the allergen, leading to competitive inhibition of allergen binding to IgE on effector cells such as mast cells (Non-Patent Literature 13-15). Specifically, clinical results have shown that administration of an allergen-specific IgG4 formulation against cat antigen improved symptoms of allergic rhinitis induced by cat antigen (Non-Patent Literature 16), strongly suggesting that allergen-specific IgG4 induced in AIT is also a major mechanism of action for AIT.

[0008] SCIT presents challenges in terms of its universality, as the necessity of weekly subcutaneous administration, typically lasting 3-5 years, or the risk of inducing severe systemic allergic responses has been noted (Non-Patent Literature 17). Therefore, sublingual immunotherapy (SLIT), pointing towards a safer AIT, has been investigated, and the Food and Drug Administration (FDA) approved sublingual formulations in 2011. SLIT targeting various allergens has then rapidly gained popularity due to its safety and convenience. However, the treatment period for SLIT is also long, lasting 3-5 years. Therefore, there is an unmet need for treatment with higher efficacy and shorter treatment duration.

[0009] Although food allergy-targeting immunotherapy (AIT) is not performed by SCIT, especially due to high safety concerns, oral immunotherapy (OIT) has been investigated as an alternative. Several comparative trials of OIT and SLIT have been conducted using peanut antigen. OIT is generally considered superior in efficacy, while SLIT is characterized by excellent safety. However, there is an unmet need for even greater safety and higher efficacy. It is hoped that mucosal immunostimulants (adjuvants) for SLIT formulations will be developed as one approach (Non-Patent Literature 18).

[0010] CRX-527 is considered to be a similar compound to lipid A (Patent Document 1).

[0011] Citation List

[0012] Patent documents

[0013] Patent Document 1: WO1998 / 50399

[0014] Non-patent literature

[0015] Non-patent literature 1: McKee AS et al., BMC biology. 2010; 8:37

[0016] Non-patent literature 2: Arakawa T. Expert review of vaccines. 2011; 10(1):1-5

[0017] Non-patent literature 3: Janeway CA, Jr. Cold Spring Harbor symposia on quantitative biology. 1989; 54Pt 1:1-13

[0018] Non-patent literature 4: Poltorak A, et al., Science. 1998; 282(5396): 2085-8

[0019] Non-patent literature 5: Luderitz O, Galanos C, Lehmann V, Nurminen M, Rietschel ET, Rosenfelder G, et al., The Journal of Infectious Diseases. 1973; 128:Suppl:17-29

[0020] Non-patent literature 6: Mata-Haro V, et al., Science. 2007; 316(5831):1628-32

[0021] Non-patent literature 7: Qureshi N, et al., The Journal of biological chemistry. 1982; 257(19):11808-15

[0022] Non-patent literature 8: Drachenberg KJ, et al., Allergy. 2001; 56(6):498-505 Non-patent literature 9: McGhee JR, Fujihashi K. PLoS biology. 2012; 10(9):e1001397

[0023] Non-patent literature 10: Lycke N. Nature reviews Immunology. 2012; 12(8):592-605

[0024] Non-patent literature 11: Holmgren J, Czerkinsky C. Nature medicine. 2005; 11(4Suppl): S45-53

[0025] Non-patent literature 12: Noon L. Lancet. 1911; 177(4580): 1572-3

[0026] Non-patent literature 13: Shamji MH, Durham SR. The Journal of allergy and clinical immunology. 2017; 140(6):1485-98

[0027] Non-patent literature 14: Larsen JN, et al., Drug discovery today. 2016; 21(1):26-37

[0028] Non-patent literature 15: Akdis M, Akdis CA. The Journal of allergy and clinical immunology. 2014; 133(3):621-31

[0029] Non-patent literature 16: Orengo JM, et al., Nature Communications. 2018; 9(1):1421

[0030] Non-patent literature 17: CSM update: desensitizing vaccines. Committee on the safety of medicines. Br Med J. 1986; 293:948

[0031] Non-patent literature 18: Nowak-Wegrzyn A, et al., Current opinion in allergy and clinical immunology. 2019; 19(6):606-13 Summary of the Invention

[0032] Technical issues

[0033] This invention provides novel compounds or pharmaceutically acceptable salts thereof that have TLR4 activating activity and can be used as immunostimulants or adjuvants in vaccine or allergen immunotherapy.

[0034] Solution to the problem

[0035] The present invention relates to (1) to (15) below.

[0036] (1) A compound of general formula (I) or a pharmaceutically acceptable salt thereof

[0037] [Formula 1]

[0038]

[0039] in,

[0040] X represents an oxygen atom or CH2.

[0041] Y represents CH2 or C=O.

[0042] Z represents a halogen atom or OR 1 ,

[0043] R 1 Represents a hydrogen atom or the following formula (II):

[0044] [Equation 2]

[0045]

[0046] in,

[0047] R 2 Represents a hydrogen atom or a carboxyl group.

[0048] R 3 Represents a hydrogen atom, hydroxyl group, or acetamino group.

[0049] R 4 Represents a hydrogen atom or the following formula (III):

[0050] [Formula 3]

[0051]

[0052] R 5 Represents a hydrogen atom or a phosphate group, and

[0053] R 6 Indicates hydroxymethyl, methyl phosphate group, carboxyl group or (1S)-1,2-dihydroxyethyl, and

[0054] n represents 0 or 1,

[0055] The condition is to exclude compounds, where X represents oxygen atom, n represents 0, and Z represents OR.1 , and R 1 It represents a hydrogen atom.

[0056] (2) The compound or a pharmaceutically acceptable salt thereof according to (1), wherein,

[0057] In equation (I),

[0058] X represents an oxygen atom or CH2.

[0059] Y represents C=O,

[0060] Z represents the following expression (IV):

[0061] [Formula 4]

[0062]

[0063] in,

[0064] R 7 Represents a hydrogen atom or formula (III), and

[0065] n represents 0 or 1,

[0066] The condition is to exclude compounds, where X represents CH2, n represents 1, and R... 7 Expression (III).

[0067] (3) The compound or a pharmaceutically acceptable salt thereof according to (1), wherein,

[0068] In equation (I),

[0069] X represents an oxygen atom.

[0070] Y represents C=O,

[0071] Z represents the following expression (V),

[0072] [Formula 5]

[0073]

[0074] in,

[0075] R 8 Indicates hydroxyl or acetamino.

[0076] R 9 Represents a hydrogen atom or a phosphate group, and

[0077] R 10 Indicates hydroxymethyl, methyl phosphate or carboxyl groups, and

[0078] n represents 0.

[0079] (4) Any compound selected from the following groups:

[0080] (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid,

[0081] (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-{[3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→4)-3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}propionic acid,

[0082] (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-OD-glucuronic acid-4-O-phosphono-β-D-glucuronic acid}oxy)propionic acid,

[0083] 6,10-Dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-11-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-9-O-phosphono-D-erythro-L-galactose-undecanoic acid, and

[0084] 5,9-Dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-10-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-8-O-phosphono-D-erythro-L-galactose-decanoic acid,

[0085] Or its pharmaceutically acceptable salt.

[0086] (5) (3R)-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid or a pharmaceutically acceptable salt thereof.

[0087] (6) A pharmaceutical composition comprising any one of (1) to (5) or a pharmaceutically acceptable salt thereof.

[0088] (7) A pharmaceutical composition comprising any one of (1) to (5) a compound or a pharmaceutically acceptable salt thereof and an antigen.

[0089] (8) A pharmaceutical composition wherein the compound of any one of (1) to (5) or a pharmaceutically acceptable salt thereof and an antigen are administered simultaneously or separately.

[0090] (9) The pharmaceutical composition according to (7) or (8), wherein the antigen is selected from one or more of the following: influenza virus, adenovirus, rubella virus, mumps virus, RS virus, enterovirus, rotavirus, norovirus or coronavirus attenuated virus, inactivated virus and recombinant protein of viral structural protein, Japanese cedar pollen, cypress pollen, birch pollen, ragweed pollen, sage pollen, Japanese hop pollen, orchardgrass pollen, spinach pollen, black pine pollen, cattail pollen, red pine pollen, chrysanthemum pollen, artemisia pollen, timothy pollen, bermudagrass pollen, Kentucky grass pollen, meadow fescue pollen, red-capped grass pollen, ryegrass pollen, yellow clover pollen, white quinoa pollen, mites, cat hair, eggs, milk, peanuts, wheat and buckwheat.

[0091] (10) The pharmaceutical composition according to any one of (6) to (9) is used for the prevention or treatment of viral infections, allergic diseases, bacterial infections and bacterial-derived toxins, cancer or intracellular parasites.

[0092] (11) The pharmaceutical composition according to any one of (6) to (9) is used for the prevention or treatment of influenza virus, coronavirus, RS virus, norovirus or rotavirus infection.

[0093] (12) The pharmaceutical composition according to any one of (6) to (9) is used for the prevention or treatment of allergic diseases caused by pollen of Japanese cedar, cypress, birch, ragweed, sage, Japanese hops, orchardgrass, spinach, black pine, cattail pollen, red pine pollen, chrysanthemum, artemisia pollen, timothy pollen, bermudagrass pollen, Kentucky grass pollen, meadow fescue pollen, red cap grass pollen, ryegrass pollen, yellow clover pollen, white quinoa pollen (quinoa [lamb's purslane or lamb's leg quinoa]), mites, cat hair, eggs, milk, peanuts, wheat or buckwheat.

[0094] (13) A TLR4 activator comprising any one of (1) to (5) or a pharmaceutically acceptable salt thereof.

[0095] (14) An immunostimulant comprising any one of (1) to (5) or a pharmaceutically acceptable salt thereof.

[0096] (15) The immunostimulant according to (14), wherein the immunostimulant is a vaccine adjuvant.

[0097] Beneficial effects of the invention

[0098] The aminoalkyl glucosinolate 4-phosphate derivatives of the present invention, or pharmaceutically acceptable salts thereof, have TLR4 activating activity and are effective in preventing or treating viral infections, allergic diseases, bacterial infections and bacterial-derived toxins, cancer, or diseases caused by intracellular protozoa. Brief description of the attached diagram

[0100] [ Figure 1 ] Figure 1 The time-dependent changes in blood ovalbumin-specific IgG concentrations after simultaneous sublingual administration of the compound described in Example 2 (2e) and ovalbumin are shown.

[0101] [ Figure 2 ] Figure 2 The time-dependent changes in blood ovalbumin-specific IgA concentrations after simultaneous sublingual administration of the compound described in Example 2 (2e) and ovalbumin are shown.

[0102] [ Figure 3 ] Figure 3 The time-dependent changes in the titer of Japanese cedar pollen antigen-specific IgG in the blood after simultaneous sublingual administration of the compound described in Example 2 (2e) and the Japanese cedar pollen antigen extract are shown.

[0103] [ Figure 4 ] Figure 4The time-dependent changes in the blood mite antigen extract-specific IgG titer after simultaneous sublingual administration of the compound described in Example 2 (2e) and the mite antigen extract are shown.

[0104] [ Figure 5 ] Figure 5 The time-dependent changes in the blood titer of ragweed pollen antigen extract-specific IgG after simultaneous sublingual administration of the compound described in Example 2 (2e) and the ragweed pollen antigen extract are shown.

[0105] [ Figure 6 ] Figure 6 The time-dependent changes in the blood titer of timothy pollen antigen extract-specific IgG after simultaneous sublingual administration of the compound described in Example 2 (2e) and timothy pollen antigen extract are shown.

[0106] [ Figure 7 ] Figure 7 The time-dependent changes in the blood peanut antigen extract-specific IgG titer after simultaneous sublingual administration of the compound described in Example 2 (2e) and the peanut antigen extract are shown.

[0107] [ Figure 8 ] Figure 8 The time-dependent changes in blood milk antigen-specific IgG titers after simultaneous sublingual administration of the compound described in Example 2 (2e) and milk antigen are shown.

[0108] [ Figure 9 ] Figure 9 The concentration of anti-Cryj 1 IgG in the blood is shown after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen.

[0109] [ Figure 10 ] Figure 10 The concentration of anti-Cryj 1IgA in the blood is shown after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen.

[0110] [ Figure 11 ] Figure 11 The concentration of anti-Cryj 1IgA in nasal wash solution after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen is shown.

[0111] [ Figure 12 ] Figure 12 The amount of IL-10 produced by cervical lymph node immune cells via Cry j 1 stimulation after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen is shown.

[0112] [ Figure 13 ] Figure 13The amount of IFN-γ produced by cervical lymph node immune cells via Cry j 1 stimulation after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen is shown.

[0113] [ Figure 14 ] Figure 14 The amount of IL-4 produced by cervical lymph node immune cells via Cry j 1 stimulation after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen is shown.

[0114] [ Figure 15 ] Figure 15 This shows the amount of mast cell degranulation via anti-Cryj 1 IgE stimulation after Cryj 1 stimulation.

[0115] [ Figure 16 ] Figure 16 The amount of mast cell degranulation is shown in a concentration-dependent manner by Cryj 1.

[0116] [ Figure 17 ] Figure 17 This demonstrates the inhibitory effect of serum IgG on Cryj 1-induced mast cell degranulation after simultaneous sublingual administration of the compound described in Example 24 and Japanese cedar pollen antigen.

[0117] [ Figure 18 ] Figure 18 The concentration of anti-RBDIgG in the blood is shown after simultaneous sublingual administration of the compound described in Example 2 (2e) with a recombinant protein of the novel coronavirus receptor-binding domain (RBD).

[0118] [ Figure 19 ] Figure 19 The concentration of anti-RBD IgA in the blood is shown after simultaneous sublingual administration of the compound described in Example 2 (2e) and recombinant RBD protein.

[0119] [ Figure 20 ] Figure 20 The anti-RBD IgA (OD) in nasal wash solution after simultaneous sublingual administration of the compound described in Example 2 (2e) and recombinant RBD protein is shown.

[0120] [ Figure 21 ] Figure 21 The nasal wash solution, after simultaneous sublingual administration of the compound described in Example 2 (2e) and the recombinant novel coronavirus RBD protein, demonstrates the inhibitory activity of the nasal wash solution on the binding of the recombinant RBD protein to the recombinant hACE2 protein. Detailed Implementation

[0121] In this invention, "*" represents a bonding site with a carbon or oxygen atom.

[0122] In formula (II) of the present invention, the “wavy line” indicates that the substituent is located in an axial or flat position.

[0123] In this invention, a "halogen atom" is, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. A fluorine atom is preferred.

[0124] Next, the preferred substituents in general formula (I) will be explained.

[0125] X is preferably an oxygen atom.

[0126] Y is preferably C=O.

[0127] Z is preferably one of the following formulas (VI) or (VII):

[0128] [Formula 6]

[0129]

[0130] n is preferably 1.

[0131] For the preferred combination of X, Y, Z and n, X is an oxygen atom, Y is C=O, Z is formula (VI), and n is 1.

[0132] For another preferred combination of X, Y, Z and n, X is an oxygen atom, Y is C=O, Z is formula (VII), and n is 0.

[0133] The preferred compound of the present invention is (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid or a pharmaceutically acceptable salt thereof.

[0134] The preferred compound of the present invention is (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-{[3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→4)-3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}propionic acid or a pharmaceutically acceptable salt thereof.

[0135] The preferred compound of the present invention is (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-OD-glucuronide-4-O-phosphono-β-D-glucuronide}oxy)propionic acid or a pharmaceutically acceptable salt thereof.

[0136] The preferred compound of the present invention is 6,10-dehydro-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-11-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-9-O-phosphono-D-erythro-L-galactose-undecanoic acid or a pharmaceutically acceptable salt thereof.

[0137] The preferred compound of the present invention is 5,9-dehydro-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-10-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-8-O-phosphono-D-erythro-L-galactose-decanoic acid or a pharmaceutically acceptable salt thereof.

[0138] A more preferred compound of the present invention is (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid meglumine.

[0139] A more preferred compound of the present invention is sodium (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyrate.

[0140] The compounds represented by general formula (I) of this invention, or pharmaceutically acceptable salts thereof, may be used as active ingredients or additives in pharmaceuticals. Whether they are treated as active ingredients or additives depends on the laws of each country.

[0141] The compounds of general formula (I) of the present invention can be prepared as pharmaceutically acceptable salts as needed. Pharmaceutically acceptable salts are those that are non-toxic and can be used as medicines. The compounds of general formula (I) of the present invention can react with a base to form salts.

[0142] Examples of such salts may include: alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminum and iron salts; inorganic salts such as ammonium salts; and amine salts including organic salts such as tert-butylamine salts, tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, alkyl phenylglycine ester salts, ethylenediamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, triethanolamine salts, N-benzylphenylethylamine salts, piperazine salts, tetramethylammonium salts, tris(hydroxymethyl)aminomethane salts, and meglumine salts. Pharmaceutically acceptable salts are preferably meglumine salts or sodium salts, more preferably meglumine salts.

[0143] The compounds of general formula (I) of the present invention, or their pharmaceutically acceptable salts, can form hydrates by incorporating water molecules when exposed to air or recrystallized. These hydrates are also covered by the compounds or salts of the present invention.

[0144] The compounds of general formula (I) of the present invention, or their pharmaceutically acceptable salts, can form solvates by absorbing a solvent when placed in a solvent or recrystallized. Such solvates are also covered by the compounds or salts of the present invention.

[0145] Within the scope of this invention, "pharmaceutically acceptable prodrug compounds" include compounds of general formula (I) that are converted into active ingredients in pharmaceutical compositions of this invention by reactions such as acid under physiological conditions in vivo, i.e., by enzyme oxidation, reduction, hydrolysis, etc., or by hydrolysis caused by gastric acid, etc.

[0146] When the compound of general formula (I) has an amino group, examples of the prodrug may include: compounds formed by amidation, alkylation, or phosphorylation of the amino group (e.g., compounds formed by eicosanoylation, alanylation, pentylaminocarbonylation, (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methoxycarbonylation, tetrahydrofuranylation, pyrrolidinylmethylation, neopentyloxymethylation, or tert-butylation). When the compound of general formula (I) has a hydroxyl group, examples include compounds formed by acylation, alkylation, phosphorylation, or borateation of the hydroxyl group (e.g., compounds formed by acetylation, palmitoylation, propionylation, neopentylation, succinylation, fumarylation, alanylation, or dimethylaminomethylcarbonylation of the hydroxyl group). When the compound represented by general formula (I) has a carboxyl group, examples include compounds formed by esterification or amidation of the carboxyl group (e.g., compounds formed by ethyl esterification, phenyl esterification, carboxymethyl esterification, dimethylaminomethyl esterification, neopentyloxymethyl esterification, ethoxycarbonyloxyethyl esterification, or methylamidation of the carboxyl group).

[0147] The prodrugs described in this invention can be manufactured from compounds of general formula (I) by methods known in the art. The prodrugs described in this invention also include compounds that, under physiological conditions, are converted into compounds of general formula (I), as described in "Iyakuhin No Kaihatsu" Volume 7, Bunshi Sekkei, and Hirokawa-Shoten Ltd., 1990, pp. 163-198.

[0148] The compounds of general formula (I) of the present invention, or their pharmaceutically acceptable salts, cover all stereoisomers.

[0149] For compounds represented by general formula (I) of the present invention, or pharmaceutically acceptable salts thereof, their isomers and mixtures of such isomers are all represented by a single formula, namely general formula (I). Therefore, the present invention includes all such isomers, and even mixtures of such isomers in any proportion.

[0150] The compounds of general formula (I) of the present invention, or pharmaceutically acceptable salts thereof, may also contain atomic isotopes in non-natural proportions at more than one atom constituting the compound. Examples of atomic isotopes include deuterium (…). 2 H), tritium ( 3 H), Iodine-125 ( 125 I) and carbon-14 ( 14 C). Compounds can be made using radioactive isotopes such as tritium ( 3 H), Iodine-125 ( 125 I) or carbon-14 ( 14C) Radiolabeling. Radiolabeled compounds can be used as therapeutic or preventative agents, research reagents (e.g., assay reagents), and diagnostic agents (e.g., in vivo diagnostic imaging agents). All isotopic variants of the compounds of this invention are included within the scope of this invention, regardless of whether they are radioactive.

[0151] In this invention, "antigen" refers to a general term for substances that induce an immune response. Specifically, substances containing antigens that cause allergic reactions are also called "allergens." For example, attenuated viruses, inactivated viruses, recombinant proteins of viral structural proteins, various pollens, insects, organisms, and foods are known as antigens or allergens. Examples include: influenza virus, adenovirus, rubella virus, mumps virus, RS virus, enterovirus, rotavirus, norovirus, or attenuated, inactivated, and recombinant proteins of viral structural proteins; pollen from Japanese cedar, cypress, birch, ragweed, sage, Japanese hops, orchardgrass, spinach, black pine, cattail pollen, red pine pollen, chrysanthemum, artemisia pollen, timothy grass pollen, bermudagrass pollen, Kentucky grass pollen, meadow fescue pollen, red-capped grass pollen, ryegrass pollen, yellow clover pollen, white quinoa pollen (quinoa [lamb's quarters or lamb's leg quinoa]), mites, cat hair, eggs, milk, peanuts, wheat, and buckwheat.

[0152] More specifically, for example, Japanese cedar pollen (Cryj 1, Cryj 2, and Cryj 3), cypress pollen (Chao 1, Chao 2, and Chao 3), birch pollen (Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v 7, and Bet v 8), ragweed pollen (short ragweed pollen, Amb a 1, Amb a 2, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 7, Amb a 8, Amb a 9, Amb a 10, Amb a 11, and Amb a 12), sage pollen, Japanese hop pollen, spinach pollen, black pine pollen, cattail pollen, red pine pollen, chrysanthemum pollen, artemisia pollen, and timothy pollen (Timothy grass, Phl p 1, Phl p). 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12 and Phl p 13), Bermuda grass pollen (Cyn d 1), Kentucky grass pollen (Poa p 1, Poa p 5 and Poa p 9), meadow fescue pollen (Fes e 1, Fes e 3, Fes e 4 and Fes e 5), orchardgrass pollen (Dac g 1, Dac g 2 and Dac g 5), red-capped grass pollen (Agr a 1), ryegrass pollen (Lolp 1, Lolp 2, Lolp 3, Lolp 5 and Lolp 9), yellow clover pollen (Anto 1), white quinoa pollen (quinoa [lamb's quarters or lamb's leg quinoa], Che a 1, Che a 2 and Che a 3), mites (Der f 1. Derf2, Derf3 to 39, Derp1, Derp2 and Derp3-38), cat hair, eggs (Gal d1, Gal d2, Gal d3, Gal d4 and Gal d5), milk (Bos d4, Bos d5, Bos d6, Bos d7, Bos d8, Bos d9, Bos d10, Bos d11 and Bos d12), peanuts (Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16 and Ara h 17), wheat (Tri a 14, Tri a 15, Tri a 19, Tri a 20, Tri a 21, Tri a 26, Tri a 28, Tri a29. Tri a 30 and Tri a 36) or buckwheat, or allergen extracts obtained from them, as antigens.

[0153] In this invention, "viral infection" refers to the state of being infected by a virus through ingestion or inhalation, insect bites, trauma, or sexual contact, and also includes the state of disease development caused by viral infection.

[0154] In this invention, "allergic disease" refers to a systemic or localized pathological condition in vivo based on an immune response triggered by the entry of an allergen into the body. Examples of allergic diseases include allergic rhinitis, food allergies, and atopic dermatitis.

[0155] Allergic reactions can be classified into immediate and non-immediate allergic reactions. Immediate allergic reactions occur when mast cells (found in the skin, intestinal mucosa, bronchial mucosa, nasal mucosa, conjunctiva, etc.) encounter an antigen while bound to IgE antibodies, triggering the release of chemical neurotransmitters such as histamine or leukotrienes. Non-immediate allergic reactions are independent of IgE antibodies, and the possibility of T cell involvement has been proposed.

[0156] "Ig" is an abbreviation for immunoglobulin, and specifically refers to antibodies. Antibodies are proteins produced and released by B cells that bind to foreign substances, such as pathogens, that have entered the body.

[0157] IgE is a human serum immunoglobulin, and it is particularly involved in allergic reactions.

[0158] As methods for dealing with and treating allergic diseases, known approaches include pharmacological therapy such as avoiding or removing triggering antigens and using anti-allergy medications, as well as allergen immunotherapy (AIT).

[0159] In this invention, "TLR4" refers to Toll-like receptor 4, which is a receptor that recognizes characteristic molecules of pathogens. It is known that activation of TLR4 promotes the induction of antigen-specific IgG and IgA.

[0160] "IgG" is a human serum immunoglobulin that is involved in the detoxification of risk factors and the recognition of antigen-antibody complexes by leukocytes or macrophages.

[0161] "IgA" is human serum immunoglobulin, which is found in large quantities in serum, nasal discharge, saliva, breast milk, intestinal fluid, etc., and participates in mucosal immunity.

[0162] In this invention, "adjuvant" refers to a substance that is administered simultaneously or sequentially with an antigen or allergen and is used to enhance the immune response to the antigen or allergen.

[0163] In this invention, "treatment" means the recovery, relief, reduction, and / or delay of the deterioration of the clinical symptoms of a patient suffering from a viral infection, allergic disease, bacterial infection and bacterial-derived toxins, cancer, or a disease caused by intracellular protozoa.

[0164] In this invention, "prevention" means reducing the incidence of viral infections, allergic diseases, bacterial infections and bacterial-derived toxins, cancer, or diseases caused by intracellular protozoa. Prevention includes reducing the risk of progression of viral infections, allergic diseases, bacterial infections and bacterial-derived toxins, cancer, or diseases caused by intracellular protozoa, or reducing the deterioration of diseases. This invention induces a protective immune response in the human body, and therefore can effectively prevent said diseases.

[0165] The compounds of general formula (I) of the present invention, or pharmaceutically acceptable salts thereof, can be administered in various forms. Examples of dosage forms include tablets, capsules, granules, emulsions, pills, powders, and syrups (solutions) for oral administration, and injections (intravenous, intramuscular, subcutaneous, or intraperitoneal), drops, and suppositories (rectal administration) for parenteral administration. These various formulations can be formulated using conventional methods, in addition to the active ingredient, using excipients commonly used in the field of pharmaceutical formulation, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, and coating agents.

[0166] Examples of carriers that can be used for tablets include: excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silica; binders such as water, ethanol, propanol, monosaccharide syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants such as dry starch, sodium alginate, agar powder, kelp polysaccharide powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, starch, and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; absorption enhancers such as quaternary ammonium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silica; and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol. Optionally, tablets can be prepared in a conventional manner as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, bilayer tablets, and multilayer tablets.

[0167] Examples of carriers that can be used for use in pills include: excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; binders such as gum arabic powder, powdered tragacanth, gelatin, and ethanol; and disintegrants such as kelp polysaccharide and agar.

[0168] For use as suppositories, a wide range of conventional carriers known in the art can be used. Examples include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0169] For use as injections or sublingual solutions, solutions, emulsions, or suspensions can be used. These solutions, emulsions, or suspensions are preferably sterilized and adjusted to be isotonic with blood. In the preparation of these solutions, emulsions, or suspensions, any solvent suitable for use as a medical diluent can be used without limitation. Examples include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, each formulation may contain an amount sufficient to prepare an isotonic solution of sodium chloride, glucose, or glycerol. Additionally, each formulation may contain conventional solubilizers, buffers, soothing agents, etc.

[0170] These preparations may also contain colorants, preservatives, flavorings, flavorings, sweeteners, and other pharmaceuticals as needed.

[0171] There are no particular limitations on the amount of compounds contained in each of these formulations, and a suitable range can be selected. The composition typically contains 0.5-70% by weight, preferably 1-30% by weight, of the compound based on the total weight.

[0172] The amount of compound used varies depending on the patient's (warm-blooded animal, especially human) symptoms, age, etc. The daily oral dose for adults is 10 mg (preferably 1 mg) as the upper limit and 0.001 mg as the lower limit, and it is recommended to administer it 0-3 times a day depending on the symptoms.

[0173] Next, a representative method for manufacturing the compound represented by general formula (I) will be described. The compounds of the present invention can be manufactured by various methods. The manufacturing methods shown below are given for illustrative purposes. It should be understood that the present invention is not limited to these examples.

[0174] The compounds of general formula (I) of the present invention, or pharmaceutically acceptable salts thereof, can be manufactured by means of characteristics based on their skeleton or substituent type using various manufacturing methods known in the art. Methods known in the art include, for example, those described in "Organic Functional Group Preparations," 2nd edition, Academic Press, Inc., 1989, and "Comprehensive Organic Transformations," VCH Publishers Inc., 1989.

[0175] Depending on the type of functional groups present in the compound, the functional groups in the starting materials or intermediates can be protected with appropriate protecting groups, or replaced with groups that can be readily converted into those functional groups. This method may be effective for production techniques.

[0176] Examples of such functional groups include amino, hydroxyl, and carboxyl groups. Examples of their protecting groups include those described in TW Greene and PGWuts, “Protective Groups in Organic Synthesis (4th Edition, John Wiley & Sons, Inc., 2006)”.

[0177] Protecting groups or groups that are easily converted into functional groups can be appropriately selected and used according to the reaction conditions of each manufacturing method for the compound.

[0178] According to this method, the reaction can be carried out after the introduction of the group, and then the protecting group can be removed or converted into the desired group as needed to obtain the desired compound.

[0179] The prodrug of the compound can be manufactured by introducing specific groups into the starting material or intermediate in the same manner as the protecting group described above, or by reacting the resulting compound. The reaction to manufacture the prodrug can be carried out using methods commonly known to those skilled in the art, such as conventional esterification, amidation, dehydration, or hydrogenation.

[0180] Hereinafter, for the purpose of referring to compounds, the compound numbers shown in the respective reaction formulas will be used. Specifically, compounds will be referred to as "(1)", etc. This also applies to compounds with other numbers.

[0181] In the method AC given below, X, Y, and n in each formula are as defined above.

[0182] The abbreviations used in this paragraph, examples, and tables have the following meanings.

[0183] Ac: Acetyl, Bn: Benzyl, Boc: tert-butoxycarbonyl, Cbz: benzyloxycarbonyl, CDCl3: deuterated chloroform, CD3OD: deuterated methanol, D2O: heavy water, DMSO: dimethyl sulfoxide, TBDPS: tert-butyldiphenylsilyl, TES: tetraethylsilyl, R: (3R)-3-(decyloxy)tetradecanoyl, and R': (3R)-3-(decyloxy)tetradecanoyl.

[0184] Method A

[0185] The compound shown in (1) of the present invention or a pharmaceutically acceptable salt thereof can be manufactured according to method A below.

[0186] [Formula 7]

[0187]

[0188] [Formula 8]

[0189]

[0190] (Step A-1)

[0191] This step involves glycosylation of (1a) with (2a) under ice-cooled conditions using a Lewis acid to obtain (7a), where X is an oxygen atom. The preferred starting material for the synthesis of (1a) is allyl 2-deoxy-4,6-O-(1-methylethylidene)-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside, which can be prepared from glucosamine hydrochloride using the method described in the report by Imoto et al. (Tetrahedron Lett. 1985, 26, 1545-1548).

[0192] (Step A-1')

[0193] This step involves the production of (7a) from (3a) via a carbon-carbon bond formation reaction with alkyne (5a) or (6a) when X is a carbon atom. C-glycosylyne is synthesized by coupling lithium-ionized alkyne with (3a) at low temperature, followed by reduction of the nitro group, protection with a primary amine, and reduction of the alkyne under heating conditions. In the case of alkyne (5a), the protecting group on the hydroxyl group is deprotected, and the resulting hydroxyl group is converted to an aldehyde by Dess-Martin oxidation, followed by conversion to a carboxylic acid by Pinnick oxidation. In the case of alkyne (6a), the carboxylic acid is prepared by simultaneously deprotecting and oxidizing the acetal group using Jones' reagent. Next, the benzyl protecting group is deprotected by hydrogenation, and the carboxylic acid is then protected with benzyl under heating conditions, followed by acetal protection of the 4- and 6-hydroxyl groups of glucosamine, and acylation of the 3-hydroxyl group using (4a) to synthesize (7a).

[0194] (Step A-2)

[0195] This step involves the preparation of (8a) from (7a) via deprotection, acylation with (4a), and phosphorylation at the 4-position. Under heating conditions, the acetal group of (7a) is deprotected with a mixed solvent of acetic acid and water. The Boc protecting group on the primary amine is removed by acid treatment, and then the resulting primary amine is amidated with (4a). Next, the Troc protecting group is removed by reduction, followed by amidation with (4a). The hydroxyl group at the 6-position of the glucosamine is temporarily protected with a silyl protecting group, and then the hydroxyl group at the 4-position is phosphorylated. Subsequently, the silyl protecting group is deprotected to synthesize (8a).

[0196] (Step A-3)

[0197] This step involves deprotecting the benzyl protecting group of (8a) by hydrogenation to produce (1).

[0198] Method B

[0199] The compound shown in (2) of the present invention or a pharmaceutically acceptable salt thereof can be manufactured according to method B below.

[0200] [Formula 9]

[0201]

[0202] The steps of manufacturing (2) from (2a) can be performed in the same manner as A-3 of method A.

[0203] [Formula 10]

[0204]

[0205] (Step B-1)

[0206] This step involves the glycosylation of (8a) obtained by method A and the KDO (2-keto-3-deoxyoctyl ketone acid) unit (2b). The 6-hydroxyl group of the glucosamine in (8a) is protected with TES, and (2b) is coupled with a Lewis acid under ice-cooling conditions. The acetal protecting group is then deprotected by acid treatment to synthesize (2a).

[0207] Method C

[0208] The compound shown in (3) of the present invention or its pharmaceutically acceptable salt can be manufactured according to method C below.

[0209] [Equation 11]

[0210]

[0211] The step of manufacturing (3) from (3a) can be performed in the same manner as A-3 of method A.

[0212] [Equation 12]

[0213]

[0214] (Step C-1)

[0215] This step involves the glycosylation of (2a) and the KDO unit (2b) obtained by method B to produce (3a). The 4-hydroxyl group of the KDO unit of (2a) is protected with TES, and (2b) is coupled with a Lewis acid under ice-cooling conditions. The acetal protecting group is then deprotected by acid treatment to synthesize (3a).

[0216] In the above method AC, the acetal protecting group is preferably dimethylacetal, and may be benzyleneacetal, etc. The acetal protecting groups of the two hydroxyl groups can be independent protecting groups. The protecting group on the hydroxyl group is preferably benzyl or tert-butyldiphenylsilyl, and may be tert-butyldimethylsilyl, allyl, benzyloxycarbonyl, etc. As the protecting group of the primary amine, 2,2,2-trichloroethylcarbonyl or tert-butoxycarbonyl is preferred, and may be allyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, etc. The protecting group on the phosphoric acid or carboxylic acid is preferably benzyl, and may be allyl, tert-butyl, phenyl, etc. The low temperature condition is -100 to -20°C, preferably -80 to -50°C. Ice cooling is -20 to 10°C, preferably -10 to 5°C. The heating condition is 35 to 130°C, preferably 50 to 100°C. The unspecified temperature conditions are -10 to 100°C, preferably 15°C to 35°C.

[0217] Example

[0218] The present invention will now be described in more detail with reference to the embodiments. However, the scope of the invention is not limited to these embodiments, and these embodiments are not to be construed as limiting in any way. In this specification, unless otherwise stated, reagents, solvents, and starting materials are readily available from commercial suppliers.

[0219] Proton nuclear magnetic resonance spectroscopy ( 1 H-NMR was measured using a 400 MHz NMR system manufactured by JEOL Ltd., a 400 MHz NMR system manufactured by Varian Ltd., or a 500 MHz NMR system manufactured by Varian Ltd. Spectral data were expressed as chemical shifts (expressed as relative ppm(δ) using tetramethylsilane as a standard), proton number, peak splitting multiplicity (expressed as follows: s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br: broad peak, etc.), and the J value (in Hz) as the spin coupling constant at the explicit time.

[0220] Mass spectrometry (MS m / z) was performed by electrospray ionization (ESI).

[0221] Silica gel column chromatography was performed using commercially available packed columns and automated preparative separation and purification systems (Isorela One manufactured by Biotage Japan Co., Ltd., EPCLC-W-Prep2XY manufactured by Yamazen Co., Ltd., Purif-α2 manufactured by Shoko Science Co., Ltd., etc.), and only the various solvents used as the mobile phase are described. Elution was performed under observation using thin-layer chromatography (TLC) on Merck KGaA silica gel 60F. 254 or 60NH2F 254 NH2 silica gel 60F manufactured by Wako Pure Chemical Industries Co., Ltd. 254 The plate, or the CHROMATOREX NH TLC plate manufactured by Fuji Silysia Chemical Co., Ltd., was used as the TLC plate, the mobile phase used in column chromatography was used as the developing solvent, and a UV detector or colorimetric reagent was used as the detection method.

[0222] (Example 1) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl}oxy)ammonium butyrate

[0223] [Equation 13]

[0224]

[0225] (1A) Allyl 3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-4,6-O-(1-methylethylidene)-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside

[0226] At room temperature, a solution of allyl 2-deoxy-4,6-O-(1-methylethylidene)-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside (7.70 g) (Tetrahedron Letters 1985, 26(12), 1545-1548) in dichloromethane (80 mL) was added, along with (3R)-3-(decanoyloxy)tetradecanoic acid (6.0 g) (Tetrahedron Letters 2006, 47(13), 2087-2092), 4-dimethylaminopyridine (55.5 mg), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.49 g). The mixture was stirred overnight at the same temperature. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to obtain the title compound (9.51 g).

[0227] (1b) 3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-4,6-O-(1-methylethylidene)-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glucopyranose

[0228] At room temperature, 1,5-cyclooctadiene bis(methyldiphenylphosphine)iridium(I)hexafluorophosphate (284 mg) was added to a tetrahydrofuran (50 mL) solution of the compound (5.47 g) obtained in Example 1 (1a), and the mixture was stirred for 1 minute at the same temperature under a hydrogen atmosphere. After further stirring for 2 hours under a nitrogen atmosphere, water (10 mL), pyridine (1.6 mL), and iodine (3.41 g) were added at the same temperature, and the mixture was stirred for 2 hours. The reaction was terminated by adding a 5% aqueous sodium thiosulfate solution to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (1.89 g).

[0229] (1c)(3R)-3-[(tert-butoxycarbonyl)amino]-4-[(3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-4,6-O-(1-methylethylidene)-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)oxy]benzyl butyrate

[0230] At 0°C, trichloroacetonitrile (1.2 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.036 mL) were added to a 10 mL solution of the compound (930 mg) obtained in Example 1 (1b) in dichloromethane, and the mixture was stirred for 1 hour at the same temperature. After concentrating the reaction mixture, the residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the product (1.07 g). At room temperature, (3R)-3-[(tert-butoxycarbonyl)amino]-4-hydroxybutyrate benzyl ester (500 mg) and molecular sieve 4A,1 / 16 (300 mg) were added to a 10 mL solution of the product in dichloromethane, and the mixture was stirred for 20 minutes at the same temperature. Then, trimethylsilyl trifluoromethanesulfonate (0.021 mL) was added at 0°C, and the mixture was stirred for 3 hours at the same temperature. The reaction was terminated by adding triethylamine. The molecular sieve was then filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (370 mg).

[0231] (1d)(3R)-3-amino-4-[(3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)oxy]benzyl butyrate

[0232] At room temperature, water (1 mL) was added to a 10 mL solution of the compound (370 mg) obtained in Example 1 (1c) in acetic acid, and the mixture was stirred at 60 °C for 2 hours. The reaction mixture was concentrated to give a product (350 mg). This product was combined with a product (310 mg) obtained in the same manner as above, and the resulting product (660 mg) was dissolved in dichloromethane (20 mL). Trifluoroacetic acid (4 mL) was added to the solution at room temperature, and the mixture was stirred at the same temperature for 1 hour. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / methanol] to give the title compound (470 mg).

[0233] (1e)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-[(3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)oxy]benzyl butyrate

[0234] At room temperature, (3R)-3-(decanoyloxy)tetradecanoic acid (400 mg) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (420 mg) were added to a tetrahydrofuran-methanol (1:1, 8 mL) solution of the compound obtained in Example 1 (1d), and the mixture was stirred overnight at the same temperature. The reaction was terminated by adding 0.5 N hydrochloric acid to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate / dichloromethane] to give the title compound (520 mg).

[0235] (1f)(3R)-4-({2-amino-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-deoxy-β-D-glucopyranoyl}oxy)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}benzyl butyrate

[0236] At room temperature, acetic acid (10 mL) and zinc powder (520 mg) were added to a tetrahydrofuran (5 mL) solution of the compound (520 mg) obtained in Example 1 (1e), and the mixture was stirred for 1 hour at the same temperature. The zinc was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / methanol] to give the title compound (450 mg).

[0237] (1g)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-[(3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]benzyl butyrate

[0238] At room temperature, (3R)-3-(decanoyloxy)tetradecanoic acid (317 mg) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (330 mg) were added to a tetrahydrofuran-methanol (1:1, 8 mL) solution of the compound obtained in Example 1 (1f) (450 mg), and the mixture was stirred overnight at the same temperature. The reaction was terminated by adding 0.5 N hydrochloric acid to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (476 mg).

[0239] (1h)(3R)-4-[(6-O-[tert-butyl(diphenyl)silyl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl]-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}benzyl butyrate

[0240] At room temperature, tert-butyldiphenylchlorosilane (410 mg) and imidazole (21 mg) were added to a solution of the compound (1.90 g) obtained in Example 1 (1 g) in dichloromethane (20 mL), and the mixture was stirred at the same temperature for 2 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (2.08 g).

[0241] (1i)(3R)-4-[(4-O-[bis(benzyloxy)phosphoryl]-6-O-[tert-butyl(diphenyl)silyl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl]oxy]-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0242] At room temperature, dibenzyl N,N-diisopropylphosphonamide (0.626 mL) and 1H-tetrazole (166 mg) were added to a solution of the compound (2.08 g) obtained in Example 1 (1 h) in dichloromethane (25 mL), and the mixture was stirred for 1 h at the same temperature. 3-chloroperbenzoic acid (400 mg) was added at 0 °C, and the mixture was stirred for 15 min at the same temperature. The reaction was terminated by adding 5% aqueous sodium thiosulfate and saturated sodium bicarbonate solution to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (2.39 g).

[0243] (1j)(3R)-4-[(4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0244] At room temperature, a solution of acetic acid (0.34 mL) and a solution of 1 M tetrabutylammonium fluoride in tetrahydrofuran (5.94 mL) were added to a 20 mL solution of the compound (2.39 g) obtained in Example 1 (1i), and the mixture was stirred overnight at the same temperature. The reaction was terminated by adding water to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (1.90 g).

[0245] (1k)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl}oxy)diammonium butyrate

[0246] At room temperature, 10% carbon-supported palladium (60.0 mg) was added to a tetrahydrofuran (3 mL) solution of the compound (88.9 mg) obtained in Example 1 (1j), and the mixture was stirred at the same temperature under a hydrogen atmosphere for 8 hours. The palladium catalyst was filtered off, and the filtrate was concentrated under reduced pressure. At -78 °C, a tetrahydrofuran (10 mL) solution of the residue was added to a methanol (0.25 mL) solution of 4% ammonia, and the mixture was concentrated under reduced pressure at room temperature. The residue was washed with acetonitrile, filtered, and collected to give the title compound (57.1 mg).

[0247] (Example 2) (3R)-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)ammonium butyrate

[0248] [Formula 14]

[0249]

[0250] (2a) (3R)-4-({4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(triethylsilyl)-β-D-glucopyranoyl}oxy)-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0251] At room temperature, triethylamine (0.12 mL), 4-dimethylaminopyridine (27.2 mg), and trichloroethylsilane (0.049 mL) were added to a 5 mL solution of the compound (394 mg) obtained in Example 1 (1j) in dichloromethane, and the mixture was stirred for 1 hour at the same temperature. The reaction mixture was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (384 mg).

[0252] (2b)(3R)-4-[(6-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl]-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl)oxy]-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0253] To a solution of the compound (384 mg) obtained in Example 2 (2a) in dichloromethane (4 mL), (3aR,4R,6S,7aR)-4-[(1R)-1,2-bis(benzyloxy)ethyl]-6-fluoro-2,2-dimethyltetrahydro-2H,4H-[1,3]dioxacyclopenteno[4,5-c]pyran-6-carboxylate (336 mg) (Angewandte Chemie, International Edition 2001, 40, 1475-1480) and molecular sieve 5A, 1 / 16 (1.0 g) were added, and the mixture was stirred at the temperature for 15 minutes. At 0 °C, a boron trifluoride-diethyl ether complex (0.255 mL) was added to the reaction mixture, and the mixture was stirred at the same temperature for 20 minutes. The reaction was terminated by adding triethylamine. The molecular sieve was then filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (267 mg).

[0254] (2c)(3R)-4-{[6-O-(1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0255] At room temperature, water (0.48 mL) and trifluoroacetic acid (0.72 mL) were added to a 10 mL solution of the compound (267 mg) obtained in Example 2 (2b), and the mixture was stirred at the same temperature for 30 minutes. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (237 mg).

[0256] (2d)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid

[0257] At room temperature, 10% carbon-supported palladium (360 mg) was added to a tetrahydrofuran (12 mL) solution of the compound (600 mg) obtained in Example 2 (2c), and the mixture was stirred at the same temperature under a hydrogen atmosphere for 7 hours. The palladium catalyst was filtered off, and the filtrate was then concentrated under reduced pressure to give the title compound (420 mg).

[0258] (2e)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)ammonium butyrate

[0259] At -78°C, a solution of 4% ammonia in methanol (1.2 mL) was added to a 20 mL solution of the compound (420 mg) obtained in Example 2 (2d), and the mixture was concentrated under reduced pressure at room temperature. The residue was washed with acetonitrile, filtered, and collected to give the title compound (421 mg).

[0260] (Example 3) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-{[3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}ammonium butyrate

[0261] [Formula 15]

[0262]

[0263] (3a)(3R)-4-[(6-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4-O-(triethylsilyl)-α-D-mannooct-2-onepyranosyl]-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl)oxy]-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0264] At room temperature, triethylamine (0.38 mL), 4-dimethylaminopyridine (33.5 mg), and trichloroethylsilane (0.232 mL) were added to a 7 mL solution of the compound (620 mg) obtained in Example 2 (2c) in dichloromethane, and the mixture was stirred for 1 hour at the same temperature. The reaction mixture was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (606 mg).

[0265] (3b)(3R)-4-{[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0266] At room temperature, (3aR,4R,6S,7aR)-4-[(1R)-1,2-bis(benzyloxy)ethyl]-6-fluoro-2,2-dimethyltetrahydro-2H,4H-[1,3]dioxacyclopenteno[4,5-c]pyran-6-carboxylate (655 mg) and molecular sieve 5A,1 / 16 (2.0 g) were added to a dichloromethane solution (606 mg) obtained in Example 3 (3a), and the mixture was stirred at the same temperature for 20 minutes. At 0 °C, boron trifluoride-ethyl ether complex (0.0415 mL) was added to the reaction mixture, and the mixture was stirred at the same temperature for 1 hour. The reaction was terminated by adding triethylamine. The molecular sieve was then filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (383 mg).

[0267] (3c)(3R)-4-{[1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl butyrate

[0268] At room temperature, water (0.24 mL) and trifluoroacetic acid (0.36 mL) were added to a 12 mL solution of the compound (484 mg) obtained in Example 3 (3b), and the mixture was stirred at the same temperature for 3 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (269 mg).

[0269] (3d)(3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-{[3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→4)-3-deoxy-α-D-manno-oct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}ammonium butyrate

[0270] The title compound (151 mg) was obtained by reacting the compound (236 mg) obtained in Example 3 (3c) in the same manner as in Example 1 (1k).

[0271] (Example 4) (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)ammonium propionate

[0272] [Formula 16]

[0273]

[0274] (4a)(2S)-3-({4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(triethylsilyl)-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0275] The title compound (1.49 g) was obtained by reacting (2S)-3-[(4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate (1.40 g) (Bioorganic & Medicinal Chemistry Letters 2008, 18, 5350-5354) in the same manner as in Example 2 (2a).

[0276] (4b)(2S)-3-[(6-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl]-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl)oxy]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0277] The title compound (1.39 g) was obtained by reacting the compound (1.49 g) obtained in Example 4 (4a) in the same manner as in Example 2 (2b).

[0278] (4c)(2S)-3-{[6-O-(1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0279] The title compound (1.25 g) was obtained by reacting the compound (1.39 g) obtained in Example 4 (4b) in the same manner as in Example 2 (2c).

[0280] (4d)(2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)ammonium propionate

[0281] The title compound (213 mg) was obtained by reacting the compound (309 mg) obtained in Example 4 (4c) in the same manner as in Example 1 (1k).

[0282] (Example 5) (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-{[3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}ammonium propionate

[0283] [Equation 17]

[0284]

[0285] (5a)(2S)-3-[(6-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4-O-(triethylsilyl)-α-D-mannooct-2-onepyranosyl]-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl)oxy]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0286] The title compound (614 mg) was obtained by reacting the compound (619 mg) obtained in Example 4 (4c) in the same manner as in Example 3 (3a).

[0287] (5b)(2S)-3-{[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0288] The title compound (527 mg) was obtained by reacting the compound (614 mg) obtained in Example 5 (5a) in the same manner as in Example 3 (3b).

[0289] (5c)(2S)-3-{[1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranosyl]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0290] The title compound (319 mg) was obtained by reacting the compound (484 mg) obtained in Example 5 (5b) in the same manner as in Example 3 (3c).

[0291] (5d)(2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-{[3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→6)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranosyl]oxy}ammonium propionate

[0292] The title compound (167 mg) was obtained by reacting the compound (264 mg) obtained in Example 5 (5c) in the same manner as in Example 1 (1k).

[0293] (Example 6) (2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-OD-glucuronic acid-4-O-phosphono-β-D-glucuronic acid}oxy)ammonium propionate

[0294] [Formula 18]

[0295]

[0296] (6a)(2S)-3-{[6-O-(6-benzyl-2,3,4-tri-O-benzyl-D-glucuronic acid)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucuronic acid]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0297] At 0 °C, trichloroacetonitrile (0.31 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.009 mL) were added to a dichloromethane (3 mL) solution of 170 mg of benzyl pyranose (tri-O-benzyl-D-glucuronide) and stirred at the same temperature for 1 hour. After concentrating the reaction mixture, the residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the product (201 mg). At room temperature, (2S)-3-[(4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}benzyl propionate (150 mg) and molecular sieve 4A, 1 / 16 (100 mg) were added to a dichloromethane (3 mL) solution of the product, and the mixture was stirred at the same temperature for 20 minutes. Then, trimethylsilyl trifluoromethanesulfonate (0.003 mL) was added at 0 °C, and the mixture was stirred at the same temperature for 30 minutes. The reaction was terminated by adding triethylamine. The molecular sieve was then filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the lower polar diastereomer (6a-1, 48.7 mg) and the higher polar diastereomer (6a-2, 59.7 mg) of the title compound.

[0298] (6b)(2S)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-OD-glucuronic acid-4-O-phosphono-β-D-glucuronic acid}oxy)ammonium propionate

[0299] The title compound (29.4 mg) was obtained by reacting the highly polar diastereomer (6a-2, 59.7 mg) obtained in Example 6 (6a) in the same manner as in Example 1 (1k).

[0300] (Example 7) (2S)-3-{[6-O-(2-acetamido-2-deoxy-β-D-glucopyranoyl)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl]oxy}-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0301] [Formula 19]

[0302]

[0303] (7a)(2S)-3-({4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3,4,6-tri-O-benzyl-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0304] The title compound (187 mg) was obtained by reacting 3,4,6-tri-O-benzyl-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glucopyranose (250 mg) (Peptide Science (2009), 45th, 179-182) in the same manner as in Example 6 (6a).

[0305] (7b)(2S)-3-{[6-O-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranoyl)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0306] At room temperature, zinc powder (200 mg) was added to a 4 mL solution of the compound (187 mg) obtained in Example 7 (7a) in acetic acid, and the mixture was stirred for 1 hour at the same temperature. The zinc was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane / methanol] to give the product (90.6 mg). At room temperature, triethylamine (0.1 mL) and acetic anhydride (0.5 mL) were added to a 3 mL solution of the product in dichloromethane-methanol (1:2), and the mixture was stirred for 15 minutes at the same temperature. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (57.2 mg).

[0307] (7c)(2S)-3-{[6-O-(2-acetamido-2-deoxy-β-D-glucopyranoyl)-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl]oxy}-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0308] The title compound (28.5 mg) was obtained by reacting the compound (57.2 mg) obtained in Example 7 (7b) in the same manner as in Example 1 (1k).

[0309] (Example 8) (2S)-3-({6-O-[2-acetamido-2-deoxy-4-O-(hydroxyphosphono)-β-D-glucopyranoyl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0310] [Formula 20]

[0311]

[0312] (8a) Allyl 3-O-benzyl-6-O-[(benzyloxy)carbonyl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside

[0313] At room temperature, pyridine (1.26 mL) and benzyl chloroformate (1.67 mL) were added to a tetrahydrofuran (30 mL) solution of allyl 3-O-benzyl-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside (3.78 g) (Journal of Endotoxin Research 1994, 1(3), 149-163), and the mixture was stirred at the same temperature for 2 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate / dichloromethane] to give the title compound (3.88 g).

[0314] (8b) Allyl 3-O-benzyl-6-O-[(benzyloxy)carbonyl]-4-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside

[0315] The title compound (5.51 g) was obtained by reacting the compound (3.88 g) obtained in Example 8 (8a) in the same manner as in Example 1 (1i).

[0316] (8c)3-O-benzyl-6-O-[(benzyloxy)carbonyl]-4-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glucopyranose

[0317] The title compound (4.11 g) was obtained by reacting the compound (5.51 g) obtained in Example 8 (8b) in the same manner as in Example 1 (1b).

[0318] (8D)(2S)-3-{[6-O-(3-O-benzyl-6-O-[(benzyloxy)carbonyl]-4-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl]oxy}-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0319] The title compound (261 mg) was obtained by reacting the compound (380 mg) obtained in Example 8 (8c) in the same manner as in Example 6 (6a).

[0320] (8e)(2S)-3-[(6-O-{2-acetamido-3-O-benzyl-6-O-[(benzyloxy)carbonyl]-4-O-[bis(benzyloxy)phosphoryl]-2-deoxy-β-D-glucopyranoyl}-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0321] At room temperature, zinc powder (520 mg) was added to a 4 mL solution of the compound (261 mg) obtained in Example 8 (8d) in acetic acid, and the mixture was stirred for 1 hour at the same temperature. The zinc was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane / methanol] to give the product (131 mg). At room temperature, acetic anhydride (1 mL) was added to a 1 mL solution of the product in pyridine, and the mixture was stirred for 30 minutes at the same temperature. The reaction mixture was concentrated under reduced pressure. The residue was then purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (102 mg).

[0322] (8f)(2S)-3-({6-O-[2-acetamido-2-deoxy-4-O-(hydroxyphosphono)-β-D-glucopyranoyl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0323] The title compound (62.1 mg) was obtained by reacting the compound (102 mg) obtained in Example 8 (8e) in the same manner as in Example 1 (1k).

[0324] (Example 9) (2S)-3-({6-O-[2-acetamido-2-deoxy-6-O-(hydroxyphosphono)-β-D-glucopyranoyl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0325] [Equation 21]

[0326]

[0327] (9a) Allyl 3,4-di-O-benzyl-6-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside

[0328] The title compound (4.52 g) was obtained by reacting allyl 3,4-di-O-benzyl-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-α-D-glucopyranoside (3.11 g) (Synlett 2007, 1, 164-166) in the same manner as in Example 1 (1i).

[0329] (9b) 3,4-Di-O-benzyl-6-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glucopyranose

[0330] The title compound (3.34 g) was obtained by reacting the compound (4.52 g) obtained in Example 9 (9a) in the same manner as in Example 1 (1b).

[0331] (9c)(2S)-3-({4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3,4-di-O-benzyl-6-O-[bis(benzyloxy)phosphoryl]-2-deoxy-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-β-D-glucopyranoyl)-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0332] The title compound (215 mg) was obtained by reacting the compound (360 mg) obtained in Example 9 (9b) in the same manner as in Example 6 (6a).

[0333] (9d)(2S)-3-[(6-O-{2-acetamido-3,4-di-O-benzyl-6-O-[bis(benzyloxy)phosphoryl]-2-deoxy-β-D-glucopyranoyl}-4-O-[bis(benzyloxy)phosphoryl]-3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-β-D-glucopyranoyl)oxy]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}benzyl propionate

[0334] The title compound (108 mg) was obtained by reacting the compound (169 mg) obtained in Example 9 (9c) in the same manner as in Example 8 (8e).

[0335] (9e)(2S)-3-({6-O-[2-acetamido-2-deoxy-6-O-(hydroxyphosphonoester)-β-D-glucopyranoyl]-3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-4-O-phosphono-β-D-glucopyranoyl}oxy)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}ammonium propionate

[0336] The title compound (43.3 mg) was obtained by reacting the compound (70.9 mg) obtained in Example 9 (9d) in the same manner as in Example 1 (1k).

[0337] (Example 10) 6,10-dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-9-O-phosphono-D-erythro-L-galactosyl-undecanoic acid ammonium

[0338] [Equation 22]

[0339]

[0340] (10a)[(3R)-5-{[tert-butyl(diphenyl)silyl]oxy}pent-1-yn-3-yl]tert-butyl carbamate

[0341] At 0 °C, tert-butyl carbamate (27.7 g) (Tetrahedron Letters 2007, 48, 7279-7282) in methanol (270 mL) was mixed with dimethyl (1-diazo-2-oxopropyl)phosphonate (14.1 mL) and potassium carbonate (17.4 g), and the mixture was stirred overnight at room temperature. The reaction was terminated by adding saturated aqueous ammonium chloride solution to the reaction mixture, followed by extraction with n-hexane. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (23.9 g).

[0342] (10b) 6,10-dehydr-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-1-O-[tert-butyl(diphenyl)silyl]-2,3,4,5,7-pentadeoxy-7-nitro-D-erythro-L-galactose-undec-4-ynyl alcohol

[0343] At -78°C, a solution of 1.6 M n-butyllithium in n-hexane (75 mL) was added to a tetrahydrofuran (200 mL) solution of the compound (23.9 g) obtained in Example 10 (10a), and the mixture was stirred for 1 hour at the same temperature. At -78°C, a tetrahydrofuran solution of the organolithium compound prepared above was added dropwise to a tetrahydrofuran (200 mL) solution of 1,5-dehydr-3,4,6-tris-O-benzyl-2-deoxy-2-nitro-D-arabino-hex-1-enol (25.2 g) (European Journal of Organic Chemistry 1998, 8, 1609-1613). The mixture was stirred for 1 hour at the same temperature, and the reaction was terminated by adding a saturated aqueous solution of ammonium chloride to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to obtain the title compound (18.4 g).

[0344] (10c)7-Amino-6,10-dehydr-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-1-O-[tert-butyl(diphenyl)silyl]-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undec-4-ynyl alcohol

[0345] At room temperature, acetic acid (25 mL) and zinc powder (3.30 g) were added to a tetrahydrofuran (25 mL) solution of the compound (4.47 g) obtained in Example 10 (10b), and the mixture was stirred at the same temperature for 7 hours. The zinc was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (2.78 g).

[0346] (10d)6,10-dehydration-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-1-O-[tert-butyl(diphenyl)silyl]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-undec-4-ynyl alcohol

[0347] At room temperature, N,N-diisopropylethylamine (1.11 mL) and trichloroethyl 2,2,2-chloroformate (0.652 mL) were added to dichloromethane (30 mL) containing 2.78 g of the compound obtained in Example 10 (10c), and the mixture was stirred at the same temperature for 2 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (2.87 g).

[0348] (10e)6,10-dehydration-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-1-O-[tert-butyl(diphenyl)silyl]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-undec-4-ynyl alcohol

[0349] At room temperature, p-toluenesulfonyl hydrazine (4.09 g) was added to 30 mL of dimethoxyethane containing 2.87 g of the compound obtained in Example 10 (10 d), and 1 M sodium acetate aqueous solution (15 mL) was added in six portions every 30 minutes at 80 °C. The mixture was stirred at the same temperature for 2 hours, and then extracted with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (2.57 g).

[0350] (10f)6,10-dehydr-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-undecanol

[0351] At room temperature, a solution of acetic acid (0.421 mL) and 1 M tetrabutylammonium fluoride in tetrahydrofuran (7.35 mL) was added to 20 mL of tetrahydrofuran containing 2.57 g of the compound obtained in Example 10 (10e), and the mixture was stirred overnight at the same temperature. The reaction was terminated by adding water to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (1.99 g).

[0352] (10g) 6,10-dehydro-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-undecanose

[0353] At room temperature, Dess-Martin periodane (1.25 g) was added to a 30 mL solution of the compound (1.99 g) obtained in Example 10 (10f) in dichloromethane, and the mixture was stirred at the same temperature for 3 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (1.85 g).

[0354] (10h)6,10-dehydration-8,9,11-tri-O-benzyl-3-[(tert-butoxycarbonyl)amino]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-undecanoic acid

[0355] At room temperature, to a solution of the compound (1.85 g) obtained in Example 10 (10 g) in tert-butyl alcohol (20 mL), water (4 mL), 80% sodium chlorite (311 mg), 2-methyl-2-butene (1.5 mL), and sodium dihydrogen phosphate dihydrate (536 mg) were added, and the mixture was stirred at the same temperature for 2 hours. Ethyl acetate was added to the reaction mixture for extraction. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (1.64 g).

[0356] (10i)6,10-Dehydr-3-[(tert-butoxycarbonyl)amino]-2,3,4,5,7-pentadeoxy-9,11-O-(1-methylethylidene)-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-undecanoate benzyl ester

[0357] At room temperature, 10% carbon-supported palladium (1.6 g) was added to a tetrahydrofuran (20 mL) solution of the compound (1.64 g) obtained in Example 10 (10 h), and the mixture was stirred at the same temperature under a hydrogen atmosphere for 8 h. The palladium catalyst was filtered off, and the filtrate was concentrated under reduced pressure to give the product. At room temperature, sodium bicarbonate (983 mg) and benzyl bromide (1.18 mL) were added to a N,N-dimethylformamide (10 mL) solution of the residue, and the mixture was stirred at 50 °C for 4 h. The sodium bicarbonate was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane / methanol] to give the product. At room temperature, 2,2-dimethoxypropane (8 mL) and p-toluenesulfonic acid monohydrate (23.6 mg) were added to an N,N-dimethylformamide (8 mL) solution of the product, and the mixture was stirred overnight at the same temperature. The reaction was terminated by adding triethylamine, and the reaction mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (610 mg).

[0358] (10j)6,10-Dehydration-3-[(tert-butoxycarbonyl)amino]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,3,4,5,7-pentadeoxy-9,11-O-(1-methylethylidene)-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-undecanoate benzyl ester

[0359] The title compound (380 mg) was obtained by reacting the compound (300 mg) obtained in Example 10 (10i) in the same manner as in Example 1 (1a).

[0360] (10k)3-amino-6,10-dehydration-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-undecanoate benzyl ester

[0361] The title compound (270 mg) was obtained by reacting the compound (380 mg) obtained in Example 10 (10j) in the same manner as in Example 1 (1d).

[0362] (10l)6,10-Dehydration-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-7-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-undecanoate benzyl ester

[0363] The title compound (320 mg) was obtained by reacting the compound (270 mg) obtained in Example 10 (10k) in the same manner as in Example 1 (1e).

[0364] (10m)7-amino-6,10-dehydration-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactosyl-undecanoate benzyl ester

[0365] The title compound (212 mg) was obtained by reacting the compound (320 mg) obtained in Example 10 (10 l) in the same manner as in Example 1 (1 f).

[0366] (10n)6,10-dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactosyl-undecanoate benzyl ester

[0367] The title compound (270 mg) was obtained by reacting the compound (212 mg) obtained in Example 10 (10 m) in the same manner as in Example 1 (1 g).

[0368] (10o)6,10-dehydrated-11-O-[tert-butyl(diphenyl)silyl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactosyl-undecanoate benzyl ester

[0369] The title compound (1.93 g) was obtained by reacting the compound (1.66 g) obtained in Example 10 (10n) in the same manner as in Example 1 (1h).

[0370] (10p)6,10-dehydrated-9-O-[bis(benzyloxy)phosphoryl]-11-O-[tert-butyl(diphenyl)silyl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactosyl-undecanoate benzyl ester

[0371] The title compound (2.15 g) was obtained by reacting the compound (1.93 g) obtained in Example 10 (10o) in the same manner as in Example 1 (1i).

[0372] (10q)6,10-dehydrated-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactosyl-undecanoate benzyl ester

[0373] The title compound (1.54 g) was obtained by reacting the compound (2.15 g) obtained in Example 10 (10p) in the same manner as in Example 1 (1j).

[0374] (10r)6,10-dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-9-O-phosphono-D-erythro-L-galactosyl-undecanoic acid ammonium

[0375] The title compound (69.0 mg) was obtained by reacting the compound (106 mg) obtained in Example 10 (10q) in the same manner as in Example 1 (1k).

[0376] (Example 11) 6,10-dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-11-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-9-O-phosphono-D-erythro-L-galactose-undecanoic acid ammonium

[0377] [Equation 23]

[0378]

[0379] (11a) 6,10-dehydrated-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-11-O-(triethylsilyl)-D-erythro-L-galactose-undecanoate benzyl ester

[0380] The title compound (1.49 g) was obtained by reacting the compound (1.40 g) obtained in Example 10 (10q) in the same manner as in Example 2 (2a).

[0381] (11b) 6,10-dehydration-11-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl]-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undecanoate benzyl ester

[0382] The title compound (1.28 g) was obtained by reacting the compound (1.49 g) obtained in Example 11 (11a) in the same manner as in Example 2 (2b).

[0383] (11c)6,10-dehydration-11-O-(1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl)-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undecanoate benzyl ester

[0384] The title compound (1.13 g) was obtained by reacting the compound (1.24 g) obtained in Example 11 (11b) in the same manner as in Example 2 (2c).

[0385] (11d)6,10-dehydrated-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-11-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-9-O-phosphono-D-erythro-L-galactose-undecanoic acid ammonium

[0386] The title compound (192 mg) was obtained by reacting the compound (274 mg) obtained in Example 11 (11c) in the same manner as in Example 1 (1k).

[0387] (Example 12) 3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→4)-3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→11)-6,10-dehydration-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-9-O-phosphono-D-erythro-L-galactose-undecanoic acid ammonium

[0388] [Equation 24]

[0389]

[0390] (12a) 6,10-dehydration-11-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4-O-(triethylsilyl)-α-D-mannooct-2-onepyranosyl]-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undecanoate benzyl ester

[0391] The title compound (580 mg) was obtained by reacting the compound (562 mg) obtained in Example 11 (11c) in the same manner as in Example 3 (3a).

[0392] (12b) 1-Benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→11)-6,10-dehydration-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undecanoate benzyl ester

[0393] The title compound (420 mg) was obtained by reacting the compound (580 mg) obtained in Example 12 (12a) in the same manner as in Example 3 (3b).

[0394] (12c) 1-Benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→11)-6,10-dehydr-9-O-[bis(benzyloxy)phosphoryl]-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-D-erythro-L-galactose-undecanoate benzyl ester

[0395] The title compound (223 mg) was obtained by reacting the compound (420 mg) obtained in Example 12 (12b) in the same manner as in Example 3 (3c).

[0396] (12d)3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→4)-3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→11)-6,10-dehydration-8-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-3,7-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,5,7-pentadeoxy-9-O-phosphono-D-erythro-L-galactose-undecanoic acid ammonium

[0397] The title compound (141 mg) was obtained by reacting the compound (223 mg) obtained in Example 12 (12c) in the same manner as in Example 1 (1k).

[0398] (Example 13) 5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0399] [Equation 25]

[0400]

[0401] (13a) 3,7-dehydro-5,6,8-tris-O-benzyl-1-[(4S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4-yl]-1,2,4-trideoxy-4-nitro-D-glycerol-D-gulolo-octyl-1-ynyl alcohol

[0402] The title compound (9.34 g) was obtained by reacting (4S)-4-ethynyl-2,2-dimethyl-1,3-oxazolidine-3-carboxylic acid tert-butyl ester (5.50 g) (Tetrahedron 2007,63,8499-8513) in the same manner as in Example 10 (10b).

[0403] (13b) 4-Amino-3,7-dehydro-5,6,8-tri-O-benzyl-1-[(4S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4-yl]-1,2,4-trideoxy-D-glycerol-D-gulolo-octyl-1-ynyl alcohol

[0404] The title compound (6.40 g) was obtained by reacting the compound (9.30 g) obtained in Example 13 (13a) in the same manner as in Example 10 (10c).

[0405] (13c)3,7-dehydro-5,6,8-tri-O-benzyl-1-[(4S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4-yl]-1,2,4-trideoxy-4-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glycerol-D-gulolo-octyl-1-ynyl alcohol

[0406] The title compound (7.10 g) was obtained by reacting the compound (6.40 g) obtained in Example 13 (13b) in the same manner as in Example 10 (10d).

[0407] (13d)3,7-dehydro-5,6,8-tri-O-benzyl-1-[(4S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4-yl]-1,2,4-trideoxy-4-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-glycerol-D-gulol-octitol

[0408] The title compound (7.10 g) was obtained by reacting the compound (7.10 g) obtained in Example 13 (13c) in the same manner as in Example 10 (10e).

[0409] (13e)5,9-Dehydration-7,8,10-Tri-O-benzyl-2-[(tert-butoxycarbonyl)amino]-2,3,4,6-Tetradeoxy-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-taro-decanoic acid

[0410] Jones' reagent (24 mL) was added to a solution of the compound (6.70 g) obtained in Example 13 (13d) in acetone (120 mL) at 0 °C, and the mixture was stirred at room temperature for 1 hour. The reaction was terminated by adding 2-propanol at 0 °C, followed by extraction with dichloromethane. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [n-hexane / ethyl acetate] to give the title compound (4.71 g).

[0411] (13f) 5,9-dehydro-2-[(tert-butoxycarbonyl)amino]-2,3,4,6-tetradeoxy-8,10-O-(1-methylethylidene)-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-decanoate benzyl ester

[0412] The title compound (930 mg) was obtained by reacting the compound (1.95 g) obtained in Example 13 (13e) in the same manner as in Example 10 (10i).

[0413] (13g) 5,9-dehydrated-2-[(tert-butoxycarbonyl)amino]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,3,4,6-tetradeoxy-8,10-O-(1-methylethylidene)-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-benzyl decanoate

[0414] The title compound (1.23 g) was obtained by reacting the compound (930 mg) obtained in Example 13 (13f) in the same manner as in Example 1 (1a).

[0415] (13h)2-Amino-5,9-dehydration-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,3,4,6-tetradeoxy-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-benzyl decanoate

[0416] The title compound (928 mg) was obtained by reacting the compound (1.23 g) obtained in Example 13 (13 g) in the same manner as in Example 1 (1 d).

[0417] (13i)5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactosyl-decanoate benzyl ester

[0418] The title compound (1.24 g) was obtained by reacting the compound (928 mg) obtained in Example 13 (13h) in the same manner as in Example 1 (1e).

[0419] (13j)6-Amino-5,9-dehydration-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0420] The title compound (960 mg) was obtained by reacting the compound (1.24 g) obtained in Example 13 (13i) in the same manner as in Example 1 (1f).

[0421] (13k)5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0422] The title compound (752 mg) was obtained by reacting the compound (960 mg) obtained in Example 13 (13j) in the same manner as in Example 1 (1 g).

[0423] (13l)5,9-dehydrated-10-O-[tert-butyl(diphenyl)silyl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0424] The title compound (2.28 g) was obtained by reacting the compound (2.19 g) obtained in Example 13 (13k) in the same manner as in Example 1 (1h).

[0425] (13m)5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-10-O-[tert-butyl(diphenyl)silyl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0426] The title compound (2.62 g) was obtained by reacting the compound (2.28 g) obtained in Example 13 (13 l) in the same manner as in Example 1 (1 i).

[0427] (13n)5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0428] The title compound (1.94 g) was obtained by reacting the compound (2.62 g) obtained in Example 13 (13m) in the same manner as in Example 1 (1j).

[0429] (13o)5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0430] The title compound (73.1 mg) was obtained by reacting the compound (103 mg) obtained in Example 13 (13n) in the same manner as in Example 1 (1k).

[0431] (Example 14) 5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-10-O-(3-deoxy-α-D-mannooct-2-onepyranosyl)-8-O-phosphono-D-erythro-L-galactose-decanoic acid ammonium

[0432] [Equation 26]

[0433]

[0434] (14a) 5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-10-O-(triethylsilyl)-D-erythro-L-galactose-benzyl decanoate

[0435] The title compound (1.49 g) was obtained by reacting the compound (1.40 g) obtained in Example 13 (13n) in the same manner as in Example 2 (2a).

[0436] (14b) 5,9-dehydr-10-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl]-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0437] The title compound (1.19 g) was obtained by reacting the compound (1.49 g) obtained in Example 14 (14a) in the same manner as in Example 2 (2b).

[0438] (14c) 5,9-dehydro-10-O-(1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl)-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0439] The title compound (1.10 g) was obtained by reacting the compound (1.19 g) obtained in Example 14 (14b) in the same manner as in Example 2 (2c).

[0440] (14d)5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-10-O-(3-deoxy-α-D-mannooct-2-onepyranosyl)-8-O-phosphono-D-erythro-L-galactose-decanoic acid ammonium

[0441] The title compound (196 mg) was obtained by reacting the compound (275 mg) obtained in Example 14 (14c) in the same manner as in Example 1 (1k).

[0442] (Example 15) 3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→4)-3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→10)-5,9-Dehydration-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactose-decanoic acid ammonium

[0443] [Equation 27]

[0444]

[0445] (15a) 5,9-dehydr-10-O-[1-benzyl-7,8-di-O-benzyl-3-deoxy-4-O-(triethylsilyl)-α-D-mannooct-2-onepyranosyl]-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0446] The title compound (554 mg) was obtained by reacting the compound (541 mg) obtained in Example 14 (14c) in the same manner as in Example 3 (3a).

[0447] (15b) 1-Benzyl-7,8-di-O-benzyl-3-deoxy-4,5-O-(1-methylethylidene)-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→10)-5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0448] The title compound (422 mg) was obtained by reacting the compound (554 mg) obtained in Example 15 (15a) in the same manner as in Example 3 (3b).

[0449] (15c) 1-Benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→4)-1-benzyl-7,8-di-O-benzyl-3-deoxy-α-D-mannooct-2-onepyranosyl-(2→10)-5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0450] The title compound (246 mg) was obtained by reacting the compound (422 mg) obtained in Example 15 (15b) in the same manner as in Example 3 (3c).

[0451] (15d)3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→4)-3-Deoxy-α-D-Mannooct-2-onepyranosyl-(2→10)-5,9-Dehydration-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactose-decanoic acid ammonium

[0452] The title compound (145 mg) was obtained by reacting the compound (225 mg) obtained in Example 15 (15c) in the same manner as in Example 1 (1k).

[0453] (Example 16) 5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6,10-pentadeoxy-10-fluoro-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0454] [Equation 28]

[0455]

[0456] (16a) 5,9-dehydrated-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6,10-pentadeoxy-10-fluoro-D-erythro-L-galactose-benzyl decanoate

[0457] At 0°C, bis(2-methoxyethyl)aminosulfur trifluoride (0.05 mL) was added to a 2 mL solution of the compound (142 mg) obtained in Example 13 (13n) in dichloromethane, and the mixture was stirred at the same temperature for 8 hours. The reaction was terminated by adding a saturated aqueous solution of sodium bicarbonate to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / methanol] to give the title compound (90.0 mg).

[0458] (16b) 5,9-dehydrated-7-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2,3,4,6,10-pentadeoxy-10-fluoro-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0459] The title compound (56.8 mg) was obtained by reacting the compound (90.0 mg) obtained in Example 16 (16a) in the same manner as in Example 1 (1k).

[0460] (Example 17) 5,9-dehydrated-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0461] [Equation 29]

[0462]

[0463] (17a) 5,9-dehydro-2-[(tert-butoxycarbonyl)amino]-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,3,4,6-tetradeoxy-8,10-O-(1-methylethylidene)-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-decanoic acid benzyl ester

[0464] The title compound (2.51 g) was obtained by reacting the compound (1.47 g) obtained in Example 13 (13f) and (3R)-3-(decyloxy)tetradecanoic acid (928 mg) (Bioorganic & Medicinal Chemistry Letters 2008, 18, 5350-5354) in the same manner as in Example 1 (1a).

[0465] (17b) 2-Amino-5,9-dehydr-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,3,4,6-tetradeoxy-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-benzyl decanoate

[0466] The title compound (444 mg) was obtained by reacting the compound (2.05 g) obtained in Example 17 (17a) in the same manner as in Example 1 (1d).

[0467] (17c)5,9-dehydrated-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-6-{[(2,2,2-trichloroethoxy)carbonyl]amino}-D-erythro-L-galactose-benzyl decanoate

[0468] The title compound (590 mg) was obtained by reacting the compound (444 mg) obtained in Example 17 (17b) and (3R)-3-(decyloxy)tetradecanoic acid (381 mg) in the same manner as in Example 1 (1e).

[0469] (17d)6-Amino-5,9-dehydration-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoate benzyl ester

[0470] The title compound (470 mg) was obtained by reacting the compound (590 mg) obtained in Example 17 (17c) in the same manner as in Example 1 (1f).

[0471] (17e)5,9-dehydrated-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoate benzyl ester

[0472] The title compound (570 mg) was obtained by reacting the compound (470 mg) obtained in Example 17 (17d) and (3R)-3-(decyloxy)tetradecanoic acid (332 mg) in the same manner as in Example 1 (1 g).

[0473] (17f) 5,9-dehydrated-10-O-[(benzyloxy)carbonyl]-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-benzyl decanoate

[0474] The title compound (560 mg) was obtained by reacting the compound (570 mg) obtained in Example 17 (17e) in the same manner as in Example 8 (8a).

[0475] (17g) 5,9-dehydrated-10-O-[(benzyloxy)carbonyl]-8-O-[bis(benzyloxy)phosphoryl]-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-D-erythro-L-galactose-decanoic acid benzyl ester

[0476] The title compound (221 mg) was obtained by reacting the compound (190 mg) obtained in Example 17 (17f) in the same manner as in Example 1 (1i).

[0477] (17h)5,9-Dehydrated-7-O-[(3R)-3-(decyloxy)tetradecanoyl]-2,6-bis{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2,3,4,6-tetradeoxy-8-O-phosphono-D-erythro-L-galactosyl-decanoic acid ammonium

[0478] The title compound (144 mg) was obtained by reacting the compound (221 mg) obtained in Example 17 (17 g) in the same manner as in Example 1 (1 k).

[0479] (Example 18) Sodium (3R)-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyrate

[0480] At room temperature, triethylamine (0.033 mL) was added dropwise to a tetrahydrofuran (10 mL) solution of the compound (104 mg) obtained in Example 2 (2 d), and the mixture was then concentrated under reduced pressure. DOWEX-50W (1.0 g) was converted to Na salt with a 1N aqueous sodium hydroxide solution (10 mL), washed with water (10 mL), injected with an aqueous solution of the compound (10 mL), and eluted with water (10 mL). The resulting fraction was concentrated under reduced pressure, the above ion exchange operation was repeated twice, and then diluted in water. The solution was lyophilized to give the title compound (66.5 mg).

[0481] (Example 19) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)potassium butyrate

[0482] The title compound (52.1 mg) was obtained by reacting the compound (104 mg) obtained in Example 2 (2d) and 1N potassium hydroxide aqueous solution (10 mL) in the same manner as in Example 18.

[0483] (Example 20) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)tetra(triethanolamine)butyrate

[0484] At room temperature, a solution of triethanolamine (0.04 mL) in tetrahydrofuran (0.4 mL) was added dropwise to a solution of the compound (96 mg) obtained in Example 2 (2d) in tetrahydrofuran (3 mL), and the mixture was then concentrated under reduced pressure. The resulting residue was washed with acetonitrile and dissolved in water. The solution was then lyophilized to give the title compound (116 mg).

[0485] (Example 21) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid monoglucamine

[0486] At room temperature, a methanol solution of meglumine (36 mg) was added dropwise to a tetrahydrofuran (5 mL) solution of the compound (320 mg) obtained in Example 2 (2 d), and the mixture was then concentrated under reduced pressure. The resulting residue was washed successively with acetonitrile and isopropanol, and then dissolved in water. The solution was then lyophilized to give the title compound (260 mg).

[0487] (Example 22) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy) butyrate diglucamine

[0488] The title compound (336 mg) was obtained by reacting the compound (300 mg) obtained in Example 2 (2d) and meglumine (68 mg) in the same manner as in Example 21.

[0489] (Example 23) (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)triglucamine butyrate

[0490] The title compound (352 mg) was obtained by reacting the compound (279 mg) obtained in Example 2 (2d) and meglumine (95 mg) in the same manner as in Example 21.

[0491] (Example 24) (3R)-3-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decyloxy)tetradecanoyl]-2-{[(3R)-3-(decyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)tetraglucamine butyrate

[0492] The title compound (1.08 g) was obtained by reacting the compound (780 mg) obtained in Example 2 (2d) and meglumine (354 mg) in the same manner as in Example 21.

[0493] [Table 1-1]

[0494]

[0495] [Table 1-2]

[0496]

[0497] [Table 1-3]

[0498]

[0499] [Table 1-4]

[0500]

[0501] [Table 1-5]

[0502]

[0503] [Table 1-6]

[0504]

[0505] [Table 1-7]

[0506]

[0507] [Table 1-8]

[0508]

[0509] [Table 1-9]

[0510]

[0511] [Table 1-10]

[0512]

[0513] [Table 1-11]

[0514]

[0515] [Table 1-12]

[0516]

[0517] [Table 1-13]

[0518]

[0519] [Table 1-14]

[0520]

[0521] [Table 1-15]

[0522]

[0523] [Table 1-16]

[0524]

[0525] [Table 2-1]

[0526]

[0527] [Table 2-2]

[0528]

[0529] [Table 2-3]

[0530]

[0531] [Table 2-4]

[0532]

[0533] [Table 2-5]

[0534]

[0535] [Table 2-6]

[0536]

[0537] [Table 2-7]

[0538]

[0539] [Table 2-8]

[0540]

[0541] [Table 2-9]

[0542]

[0543] [Table 2-10]

[0544]

[0545] [Table 2-11]

[0546]

[0547] [Table 2-12]

[0548]

[0549] (Experimental Example 1) Activation of human TLR4

[0550] Human TLR4 activation was studied using the compounds described in Examples 1-17 and monophospholipid A as Comparative Example A.

[0551] Triethanolamine was dissolved in distilled water for injection at 0.5% (v / v) to prepare an aqueous solution. 1 mg of each test drug was dissolved in 0.98 mL of the aqueous triethanolamine solution, and the pH was adjusted to 7.2–7.4 by adding 20 μL of 1M HCl to prepare a 1 mg / mL solution. A series of dilutions of the test drugs were prepared using culture media [DMEM (Nacalai Tesque, Inc.), 10% FBS (Sigma-Aldrich Co. LLC), 50 U / mL penicillin-50 μg / mL streptomycin (Thermo Fisher Scientific Inc.), 1×HEK-Blue Selection (InvivoGen), and 100 μg / mL Normocin (InvivoGen)]. 2×10⁶ wells were seeded into each well of a 96-well microplate for tissue culture (Iwaki). 5Cells / mL HEK-Blue™ human TLR4 cells (InvivoGen) were then treated with the test drug and cultured at 37°C and 5% CO2 for 24 hours. The cultured cells were then centrifuged at 400×g for 3 minutes at 4°C. Recombinant SEAP protein (InvivoGen) was used for the standard curve. 180 μL of QUANTI-Blue (InvivoGen) was added to each well of a 96-well TC-treated microplate (Corning, Inc.), along with 20 μL of cell culture supernatant and standard dilution solution. The plate was incubated at 37°C for 4 hours. After the reaction, the absorbance (wavelength: 655 nm) was measured using a microplate reader (PerkinElmer, Inc., EnSpire). The SEAP protein concentration was calculated from the standard curve and used as an indicator of TLR4 agonist activity. The EC50 (ng / mL) corresponding to the 50% induced response of the maximum response was calculated from the linear expression and measurements of the two drug concentrations. In each experiment, the drug concentration corresponding to the 50% induced response of the maximum response was between the two drug concentrations. The results are shown in Table 3.

[0552] [Table 3]

[0553]

[0554]

[0555] (Experimental Example 2) Immunostimulatory effect of sublingual administration of ovalbumin (OVA) antigen

[0556] (Test drug preparation)

[0557] 5 μg of ovalbumin (OVA, Hyglos GmbH, endotoxin-free) and 0.01, 0.1 or 1 μg of the compound described in Example 2 (2e) were dissolved in 2 μL of distilled water. The solution was used as a vaccine formulation to study its immunostimulatory effect on ovalbumin (OVA) antigen when administered sublingually.

[0558] (Sublingual administration test)

[0559] Mice (BALB / c mice, female, 6 weeks old, Charles River Laboratories Japan, Inc.) were deprived of food and water for 1 hour prior to sublingual administration. The mice were then anesthetized with 1-4% vaporized isoflurane (Pfizer Inc.). 2 μL of the test drug was administered sublingually, followed by a 10-minute maintenance sublingual anesthesia. Upon awakening, a second administration was given 1 hour after the initial administration. After sublingual administration, the mice were kept deprived of food and water for another 1 hour. This administration was repeated for 4 weeks at 1-week intervals. Starting 2 weeks after the initial administration, blood was continuously collected from the tail vein at 1-week intervals, and the serum was cryopreserved (-20°C).

[0560] (Measurement of anti-OVA IgG and IgA in blood)

[0561] Add 50 μL of OVA (Sigma-Aldrich Co. LLC, 1 μg OVA / mL, PBS) to each well of a 96-well semi-volume transparent flat-bottom polystyrene high-binding microplate (Corning, Inc.), then incubate overnight at 4°C, followed by washing three times with washing buffer (0.05% Tween 20 and PBS). Add 120 μL of ELISA solution (1% BSA, 0.05% Tween 20 and PBS) to each well, then incubate the plate at room temperature for 1 hour, followed by washing three times. Dilute serum samples with ELISA solution. Use anti-OVA mouse IgG (Chondrex, Inc.) and anti-OVA mouse IgA (Chondrex, Inc.) as samples for the standard curve. Add 50 μL of each sample and each standard curve sample to each well, incubate the plate at room temperature for 1 hour, and then wash three times. Add 50 μL of HRP-labeled anti-mouse IgG (Southern Biotech, 1 / 4000 dilution) or IgA (Southern Biotech, 1 / 4000 dilution) to each well, and then incubate the plate at room temperature for 1 hour. After washing three times with washing buffer, add 50 μL of TMB substrate (SERACARE Life Sciences Inc.) to each well and incubate the plate for 10 minutes. Terminate the reaction by adding 50 μL / well of TMB stop solution (SERACARE Life Sciences Inc.). Measure the absorbance at 450 nm using a microplate reader (PerkinElmer, Inc., EnSpire). Calculate the amounts of anti-OVA IgG and IgA in the blood using the standard curve. The limits of detection for anti-OVA IgG and IgA are 0.1 and 0.01 μg / mL, respectively. Values ​​below the detection limits are used for samples. Results are shown below. Figure 1 and 2 .

[0562] (Experimental Example 3) Immunostimulatory effects of sublingual administration on various allergens

[0563] The compounds described in Example 2 (2e) were used to study their immunostimulatory effects on various allergens when administered sublingually.

[0564] Japanese cedar pollen antigen extract was prepared by treating Japanese cedar pollen (YamizoPollen Study Group) with a solution containing 0.125M NaHCO3 and 0.5M NaCl for 24 hours (4°C), followed by filtration to remove insoluble matter. The concentration of Cry j 1 in the extract was measured, and the extract was diluted to 12.5 μg / mL (10000 JAU / mL). Allergen scratch extracts (Torii Pharmaceutical Co., Ltd.) were used for mites, ragweed, timothy hay, peanuts, and milk. Allergen sensitization was performed by intramuscular administration of the allergen (20 μL) into the femoral region of mice one week before the start of sublingual administration. Allergen scratch extracts of mites, ragweed, timothy hay, peanuts, or milk were diluted 10-fold with PBS and used for allergen sensitization. Sublingual administration began one week after sensitization. Prototype vaccine formulations were prepared by mixing each allergen with distilled water or the compound described in Example 2 (2e) dissolved in distilled water at 1 mg / mL before administration. 2 μL of the prototype vaccine formulation was administered sublingually once daily and 3-5 times per week for 12 weeks. Blood was collected from the tail vein at weeks 4, 8, and 12 after the start of sublingual administration, and the separated serum was stored at -20°C.

[0565] (Measurement of allergen-specific IgG)

[0566] Add 25 μL / well of each allergen (Japanese cedar pollen, 1 μg Cryj 1 / mL; mites, ragweed, timothy hay, peanuts, and milk, 1:1000 dilution) to an ELISA plate and incubate overnight at 4°C. After 16–24 hours, wash the plate three times with washing buffer. Then, add 100 μL of ELISA solution to each well, incubate the plate at room temperature for 1 hour, and wash three times. Using the ELISA solution, prepare an 8-series dilution series of serum samples with a 1 / 128 dilution as the highest concentration and a 1 / 2 dilution ratio. Add 25 μL of the sample to each well of the plate, incubate the plate at room temperature for 1 hour, and wash three times. Dilute HRP-labeled anti-mouse IgG 1:10000 with ELISA solution and add 25 μL / well, incubate the plate at room temperature for 1 hour, and wash three times. Add 30 μL of TMB substrate to each well and let the plate stand for 10 minutes. Then, add 30 μL of TMB stop solution to each well and measure the absorbance at 450 nm. Appropriate OD values ​​were set for each allergen, and the dilution ratio of serum reaching that OD value was expressed as the allergen-specific IgG titer. Results are shown in... Figure 3-8 .

[0567] In allergen-sensitized mice, in the control group (without sublingual administration), no significant increase in allergen-specific IgG titers was observed from the start of sublingual administration up to 12 weeks. For all allergens, the groups receiving sublingual administration of the compounds described in Example 2 (2e) induced significantly higher allergen-specific IgG titers compared to sublingual administration of the allergen alone.

[0568] (Experimental Example 4) Immunostimulatory effect of sublingual administration on Japanese cedar pollen antigen

[0569] The compound described in Example 24 was used to study its immunostimulatory effect on Japanese cedar pollen antigen when administered sublingually.

[0570] (Allergen Immunotherapy Model)

[0571] Three weeks and one week prior to the start of sublingual administration, mice were sensitized to allergens by subcutaneous administration of 50 μL of Japanese cedar pollen antigen extract (allergen scratch extract "Torii" Japanese cedar pollen, Torii Pharmaceutical Co., Ltd.) to the base of their tails. Blood samples were collected four days after the second sensitization, and serum anti-Cryj 1 IgG levels were measured as per subsequent sections. Mice were grouped to ensure even distribution of measurements between groups. Sublingual administration (sublingual allergen immunotherapy model) began three days later.

[0572] The antigen used was Japanese cedar pollen antigen (5000 JAU CEDARCURE[R], Torii Pharmaceutical Co., Ltd.) dissolved in 100 μL PBS. The compound from Example 24 was dissolved in water for injection (Otsuka Pharmaceutical Co., Ltd.) to prepare a 5 mg / mL solution. The antigen and the compound from Example 24 were mixed in equal volumes immediately before administration.

[0573] One hour prior to sublingual administration, mice were deprived of food and water. Under isoflurane anesthesia, 2 μL of the original vaccine formulation was administered sublingually at 5-minute intervals, followed by 5-minute awakening. This sublingual administration was repeated twice at 1-hour intervals, with food and water being resumed one hour later. This administration was repeated three times per week.

[0574] Blood was drawn from the tail vein 3 and 4 weeks after the start of sublingual administration. Blood, nasal wash, and cervical lymph nodes were collected 5 weeks after the start of sublingual administration.

[0575] (Measurement of anti-Cryj 1 IgG or IgA)

[0576] Add 25 μL of 10 μg / mL ImmunoPure streptavidin (Thermo Fisher Scientific Inc., Cat No. 21125) to each well of a 96-well microplate (Corning, Inc., Cat No. 3690) and incubate overnight at 4°C. After washing three times with washing buffer, add 100 μL of ELISA solution to each well and incubate the plate at room temperature for 1 hour. After washing three times, add 25 μL of 1 μg / mL biotin-labeled Cry j 1 (BioDynamics Laboratory Inc., Cat No. HBL-BC-1) to each well and incubate the plate at room temperature for 1 hour. After washing three times, add 25 μL of sample to each well and incubate the plate at room temperature for 1 hour. After washing three times, add 25 μL of HRP-labeled anti-mouse IgG (1 / 8000) or HRP-labeled anti-mouse IgA (1 / 4000) to each well and incubate the plate at room temperature for 1 hour. After washing three times, add 30 μL of the TMB microwell peroxidase substrate system to each well. Incubate the plate at room temperature for 10 minutes, then add 30 μL of TMB stop solution to each well, and measure the absorbance at 450 nm.

[0577] Positive serum was used as the serum standard sample, and the anti-Cryj 1 IgG or IgA content was set at 1000 units / mL. Six serial dilutions were prepared by 4-fold dilution from the 100-fold dilution of this standard sample, and a standard curve was constructed. Evaluation samples were diluted 1000-fold with ELISA solution and measured. The standard curve was used to determine the unit value of anti-Cryj 1 IgG or IgA in the evaluation samples. The results are shown below. Figure 9 and 10 .

[0578] Nasal washes collected from four individuals in the positive group were mixed in equal volumes and used as a standard sample for nasal washes. The anti-Cryj 1 IgA content was set at 1000 units / mL. Twelve serial dilutions were prepared by diluting the undiluted solution of this standard sample by 2-fold, and a standard curve was constructed. The evaluation samples were diluted 8-fold with ELISA solution and measured, and the unit value of anti-Cryj 1 IgA in the evaluation samples was determined using the standard curve. The results are shown in... Figure 11 .

[0579] Compared with mice that did not receive sublingual administration (no SLIT group) and mice that received only sublingual administration of Japanese cedar pollen antigen (Japanese cedar pollen SLIT group), mice that received sublingual administration of Japanese cedar pollen antigen and the compound of Example 24 (Japanese cedar pollen + compound 24 SLIT group) had significantly higher serum levels of anti-Cry j 1 IgG. Figure 9 ) and anti-Cry j 1IgA ( Figure 10 Anti-Cryj 1 IgA in nasal wash was induced at higher levels. In the Japanese cedar pollen + compound 24SLIT group, anti-Cryj 1 IgA was induced at higher levels. Figure 11 ).

[0580] (Cryj 1-specific cell-mediated immunoassay)

[0581] Neck lymph nodes were collected, combined, and homogenized in PBS (1% BSA). The mixture was passed through a 70 μm filter. After adding 5 mL of RPMI / FBS / ps (RPMI 1640, 10% FBS, and 100 units / mL penicillin and streptomycin), the mixture was centrifuged at 400 g for 3 min. Cells were recovered using RPMI / FBS / ps, and the cell count was prepared to 2 × 5 × 10⁻⁶ cells / mL. 6Cells / mL. 40 μL / well was seeded into 96-well U-plates, followed by the addition of 40 μL of RPMI or 40 μL of RPMI / FBS / ps containing 2 × 5 μg / mL Cryj 1 to each well. Cells were incubated at 37°C for 44 hours. The levels of IL-10, IFN-γ, and IL-4 in the culture supernatant were measured using a mouse Th1 / Th2 / Th17 cytokine kit (BDCytometric Bead Array). Results are shown in... Figure 12-14 .

[0582] In the Japanese cedar pollen + compound 24SLIT group, Cry j 1 stimulation strongly induced IL-10 ( Figure 12 On the other hand, IFN-γ ( Figure 13 ) and IL-4 Figure 14 All values ​​were at or below the detection limit (9.77 pg / mL).

[0583] (Measurement of mast cell degranulation using Cryj 1 and inhibition of degranulation using IgG from immune serum)

[0584] Mice (C57BL / 6J, 8 months old, male, Charles River Laboratories Japan, Inc.) were euthanized by exsanguination under isoflurane anesthesia. 10 mL of ice-cold RPMI / BSA / hep (RPMI 1640, 1 mg / mL BSA and 10 units / mL heparin) was injected intraperitoneally, followed by gentle massage for approximately 60 minutes. The peritoneal lavage fluid was then collected. The lavage fluids from four individuals were combined and centrifuged at 400g for 3 minutes. The supernatant was then discarded, and the peritoneal cells were resuspended in 1 mL of RPMI / BSA / hep. The RPMI / BSA / hep was heated to 37°C, and 0.235 g / mL Histodenz was dissolved in it to prepare Histodenz solution. 2 mL of Histodenz solution was added to a 15 mL test tube, and then further coated with 1 mL of the peritoneal cell suspension. The tube was then centrifuged at 400g for 15 minutes. Discard the cell population contained in the intermediate layer and recover the peritoneal mast cells at the bottom of the tube using 15 mL of RPMI / FBS / ps (RPMI 1640, 10% FBS, and 100 units / mL penicillin-streptomycin). Centrifuge the cells at 400g for 3 minutes, then resuspend them in 5 mL of RPMI / FBS / ps and seed them into 24-well plates at 0.5–1 mL / well. Immediately add 5 μg / mL Cryj 1-01-F11 mouse monoclonal IgE (mIgE) or 5 μg / mL Cryj 1-02-F02 mIgE, or both, to each well. Incubate the cells at 37°C for 24 hours.

[0585] 10 mL of Tyrode solution (Sigma Aldrich Co. LLC, T2145-10X1L) was added to mast cells, and the mixture was centrifuged at 400 g for 3 minutes. The cells were then centrifuged at approximately 10... 6 Cells were suspended in Tyrode solution at a concentration of 1 / mL. 10 μL of the suspension was added to a 1.5 mL test tube (Eppendorf) and allowed to stand at room temperature until the test substance was processed. The test substance (Cry j 1 [Hayashibara Co., Ltd., HBL-C-1]) was prepared using Tyrode solution to twice the final concentration. Separately, a 2×1% Triton-X100 solution was prepared using Tyrode solution. 10 μL of the test substance or Triton-X100 was added to the cells, and the tube was immediately incubated at 37°C. After 10 minutes, the reaction was terminated by immediate ice cooling. After centrifugation at 400 g for 3 minutes, 10 μL of the supernatant was added to 50 μL of 4-nitrophenyl N-acetyl-bD-aminoglycoside (Sigma Aldrich Co. LLC, β-N-acetylglycylase Assay Kit). The background sample used was 4-nitrophenyl N-acetyl-bD-aminoglucosinolate with only 10 μL of Tyrode solution. The result was incubated at 37°C for 1 hour, and the reaction was terminated by adding 100 μL of sodium carbonate. The absorbance was then measured at 405 nm. An EnSpire microplate reader (PerkinElmer, Inc.) was used for the measurements.

[0586] The percentage of mast cell degranulation is calculated according to the following expression.

[0587] Departicle amount (%) = [OD(sample) - OD(background)] / OD(Triton-X100) - OD(background)]

[0588] Mast cells that were not sensitized by IgE or were sensitized by only one of two monoclonal IgEs (Cryj1-01-F11 mIgE or Cryj1-02-F02 mIgE) were not degranulated by Cryj1. Figure 15 This is presumably because a cross-linking between IgE and FcεRI mediated by Cryj 1 was not formed. On the other hand, mast cells sensitized with both clones were stimulated with Cryj 1 in a concentration-dependent manner to degranulate. Figure 15 and 16 ).

[0589] Following the protocol of Protein G HP spin trap (Cytiva, 28-9031-34), IgG was purified from 200 μL of serum from each mouse in an allergen immunotherapy model. Using a Vivaspin 500, 10 kDa MWCO polyethersulfone (Cytiva, 28-9322-25), the solvent in the purified IgG was replaced with 50 μL of Tyrode solution. Cryj 1 (2 × 10 × 200 ng / mL, 1.5 μL, Tyrode solution) was added to the purified IgG (13.5 μL, Tyrode solution), and the mixture was incubated at room temperature in the dark for 1 hour. Mast cells sensitized with anti-Cryj 1 IgE were treated with 10 μL of this sample as the test substance, and the amount of mast cell degranulation was measured (final concentration: 200 ng / mL, Cryj 1 stimulation).

[0590] Purified IgG from mouse serum obtained from the Japanese cedar pollen + compound 24SLIT group inhibited anti-Cryj1 IgE-mediated mast cell degranulation at significantly lower levels compared to the SLIT-free group and the Japanese cedar pollen SLIT group. Figure 17 ).

[0591] (Experimental Example 5) Immunostimulatory effect of sublingual administration on SARS-CoV-2 RBD

[0592] The antigen used was a recombinant SARS-CoV-2 RBD (receptor-binding domain) protein (recombinant 2019-nCoV RBD protein, Sino Biological Inc., Cat. No. 40592-V08H) dissolved in 1 mg / mL of the novel coronavirus (SARS-CoV-2). A 1 mg / mL solution was prepared by dissolving the compound described in Example 2 (2e) in 0.5% triethanolamine aqueous solution and neutralizing with HCl. Equal volumes of the antigen and the compound from Example 2 (2e) were mixed immediately before use. The solution was administered sublingually (2 μL) twice daily per week. Blood and nasal washes were collected 6 weeks after the initial administration. Nasal washes were performed with 200 μL of PBS.

[0593] (Measurement of anti-RBD IgG and IgA in blood)

[0594] Add 25 μL of 10 μg / mL streptavidin (Thermo Fisher Scientific Inc., Cat#21125, dissolved in PBS) to each well of an ELISA plate, then incubate overnight at 4°C, followed by three washes. Add 100 μL of ELISA solution to each well, incubate at room temperature for 1 hour, and then wash three times. Add 25 μL of 0.2 μg / mL recombinant RBD protein (Acro Biosystems, Cat. No. SPD-C82E9) prepared with ELISA solution to each well, incubate at room temperature for 1 hour, and then wash three times. Add 25 μL of serum diluted with ELISA solution to each well, incubate at room temperature for 1 hour, and then wash three times. Add 25 μL of HRP-labeled anti-mouse IgG (1 / 10000 dilution) or HRP-labeled anti-mouse IgA (1 / 4000 dilution) diluted with ELISA solution to each well. Incubate the plate at room temperature for 1 hour, then wash three times. Add 30 μL of TMB substrate to each well and incubate the plate for 10 minutes. Then, terminate the reaction by adding 30 μL of TMB stop solution. Measure the absorbance at 450 nm. Use the serum of mice treated with RBD and the compound of Example 2 (2e) as standard samples, and set the anti-RBD IgG and IgA content in the standard serum to 1000 units / mL to prepare a standard curve. Calculate the anti-RBD IgG and IgA in each independent sample. Sample values ​​equal to or below the lower limit of the standard curve are considered as the lower limit of the standard curve: 0.78 units / mL (anti-RBD IgG) and 15.6 units / mL (anti-RBD IgA).

[0595] (Measurement of anti-RBD IgA in nasal wash solution)

[0596] 0.2 μg / mL recombinant RBD protein (Acro Biosystems, SPD-C82E9) was immobilized on a solid phase in the same manner as the previous step, and 25 μL of nasal wash solution diluted 1 / 2 with ELISA solution was added. Subsequent procedures were performed in the same manner as the previous step. The absorbance at 450 nm is shown in the results.

[0597] (RBD-hACE2 binding inhibitory activity in nasal wash)

[0598] Add 25 μL of 10 μg / mL streptavidin (Thermo Fisher Scientific Inc., Cat#21125, dissolved in PBS) to each well of an ELISA plate, then incubate overnight at 4°C, followed by three washes. Add 100 μL of ELISA solution to each well, incubate the plate at room temperature for 1 hour, and then wash three times. Immobilize 0.04 μg / mL recombinant RBD protein (Acro Biosystems, SPD-C82E9) onto the solid phase, and wash the plate three times. Add 25 μL of nasal wash buffer to each well, incubate the plate at room temperature for 1 hour, and then wash three times. Add 25 μL of 0.1 μg / mL recombinant hACE2 protein (Acro Biosystems, Cat.No.AC2-H5257) diluted with ELISA solution to each well, incubate the plate at room temperature for 1 hour, and then wash three times. Add 25 μL of HRP-labeled anti-human IgG1 (Cygnus Technologies Inc., Cat. No. Im50) diluted 500-fold with ELISA solution to each well. Incubate the plate at room temperature for 1 hour, then wash three times. Add 30 μL of TMB substrate to each well and incubate the plate for 10 minutes. Then, stop the reaction by adding 30 μL of TMB stop solution. The absorbance at 450 nm is shown in the results.

[0599] (result)

[0600] The test results are shown in Figure 18-21 When recombinant RBD protein was administered sublingually only, serum anti-RBD IgG or IgA levels were at or below the detection limit. Conversely, in mice that were simultaneously administered sublingually with the compound described in Example 2 (2e), serum anti-RBD IgG levels were significantly higher. Figure 18 ) and IgA( Figure 19 The amount of ) was significantly higher. Furthermore, in the nasal wash of mice simultaneously administered sublingually the compound described in Example 2 (2e) and recombinant RBD protein, anti-RBD IgA was induced at high levels ( Figure 20 The interaction between the surface RBD of the novel coronavirus and hACE2 on host cells is important for viral infection. Simultaneously, sublingual administration of the compound described in Example 2 (2e) and nasal wash of mice containing recombinant RBD protein inhibited the binding of RBD to hACE2. Figure 21 These results suggest the possibility that simultaneous sublingual administration of the compounds described in Example 2 (2e) with recombinant RBD protein into the nasal cavity induces RBD-specific IgA and effectively inhibits the binding of RBD and hACE2 in the nasal cavity, which serves as a gateway for viral entry.

Claims

1. A compound, which is (3R)-3-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-4-({3-O-[(3R)-3-(decanoyloxy)tetradecanoyl]-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-6-O-(3-deoxy-α-D-manno-oct-2-onepyranosyl)-4-O-phosphono-β-D-glucopyranosyl}oxy)butyric acid or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and an antigen.

4. A pharmaceutical composition, wherein, The compound of claim 1 or its pharmaceutically acceptable salt and antigen are used for simultaneous or non-simultaneous administration in combination.

5. The pharmaceutical composition according to claim 3, wherein, The antigen is selected from one or more of the following: influenza virus, adenovirus, rubella virus, mumps virus, RS virus, enterovirus, rotavirus, norovirus, or attenuated virus, inactivated virus, recombinant protein of viral structural protein, and Japanese cedar pollen, cypress pollen, birch pollen, ragweed pollen, sage pollen, Japanese hop pollen, orchardgrass pollen, spinach pollen, black pine pollen, cattail pollen, red pine pollen, chrysanthemum pollen, artemisia pollen, timothy pollen, bermudagrass pollen, Kentucky grass pollen, meadow fescue pollen, red-capped grass pollen, ryegrass pollen, yellow clover pollen, white quinoa pollen, mites, cat hair, eggs, milk, peanuts, wheat, or buckwheat extracts.

6. The pharmaceutical composition according to any one of claims 2 to 5, for the prevention or treatment of viral infections, allergic diseases, bacterial infections and bacterial-derived toxins, cancer or intracellular protozoa.

7. The pharmaceutical composition according to any one of claims 2 to 5, for the prevention or treatment of influenza virus, coronavirus, RS virus, norovirus or rotavirus infection.

8. The pharmaceutical composition according to any one of claims 2 to 5, for the prevention or treatment of allergic diseases caused by pollen of Japanese cedar, cypress, birch, ragweed, sage, Japanese hops, orchardgrass, spinach, black pine, cattail pollen, red pine, chrysanthemum, artemisia pollen, timothy pollen, bermudagrass pollen, Kentucky grass pollen, meadow fescue pollen, red-capped grass pollen, ryegrass pollen, yellow clover pollen, white quinoa pollen, mites, cat hair, eggs, milk, peanuts, wheat, or buckwheat.

9. A TLR4 activator comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 2 to 5.

10. An immunostimulant comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 2 to 5.

11. The immunostimulant according to claim 10, wherein, The immunostimulant is a vaccine adjuvant.

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