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PHYTOSPHINGOSINE DERIVATIVES AS ADJUVANTS IN IMMUNE STIMULATION

20230129118 · 2023-04-27

    Inventors

    Cpc classification

    International classification

    Abstract

    Phytosphingosine derivatives suitable as adjuvants in immune stimulation, pharmaceutical compositions comprising such compounds and the medical use of the compounds and/or compositions in therapeutic or prophylactic methods of immune stimulation in a subject, and for use in the treatment of a disease, for which stimulation of an immune response in a subject produces a therapeutic benefit. The phytosphingosine derivative may also be used as adjuvants in vaccinating a subject. The phytosphingosine derivative may also be used in stimulating antibody production, stimulating an immune response against infection, stimulating an immune response against a cancer, or preventing and/or treating septic shock. Methods for the manufacture of the derivatives comprising an Ugi-4-component reaction (Ugi-4CR) is also disclosed.

    Claims

    1. A compound according to formula (I): ##STR00066## wherein R1 is a linear or branched alkyl, alkenyl, or alkoxy group, wherein R1 is optionally substituted with -halogen, —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, or wherein R1 is absent, wherein R10, R11 and R12 are, independently, a linear or branched alkyl or alkenyl group, cycloalkyl or an aromatic or heteroaromatic group, or protecting groups, and R13 is H or a linear or branched alkyl or alkenyl group; A is H, cycloalkyl, an aromatic or heteroaromatic group, a linear or branched alkyl, alkenyl, or alkoxy, wherein A is optionally substituted with: —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, wherein preferably R1 is absent when A is a cycloalkyl group, or wherein A is an amino acid or a polypeptide; R2 is a linear or branched alkyl, alkenyl, or alkoxy group, wherein R2 is optionally substituted with -halogen, —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, or wherein R2 is absent, D is H, cycloalkyl, an aromatic or heteroaromatic group, a linear or branched alkyl, alkenyl, or alkoxy, wherein D is optionally substituted with: —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; ═O, NR10.sub.2, acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, or wherein D is an amino acid or a polypeptide; E is, independently, —H, alkyl, -halogen, —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, or —OPO.sub.3R13; G is a saccharide, wherein the saccharide is optionally substituted with -halogen, —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, alkyl, or ester-, alkyl- or amide-aromatic or heteroaromatic substituents; wherein optionally two compounds according to formula (I) are covalently bonded to each other at their respective R2 groups, thereby forming R15, wherein R15 is a linear or branched alkyl, alkenyl or alkoxy group, and wherein D is absent, or wherein optionally two compounds according to formula (I) are covalently bonded to each other at their respective R1 groups, thereby forming R18, wherein R18 is a linear or branched alkyl, alkenyl or alkoxy group, and wherein A is absent.

    2. The compound according to claim 1, of a structure according to formula (II): ##STR00067## wherein A, D, R1, R2 and E are as defined above for formula (I), and R3 is —OH, OC.sub.1-C.sub.12 alkyl, —CO.sub.2H, or R19, wherein R19 is —NHCONH—R20, —OCONH—R20, —OCOC.sub.1-C.sub.12 alkyl- or —NHCOC.sub.1-C.sub.12 alkyl-, optionally bound to R20, wherein R20 is an aromatic group, wherein the aromatic group comprises or consists of 1-2 aromatic or heteroaromatic 5- or 6-membered ring structures; R4 and R5 are either: R4 is —H and R5 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R5 is —H and R4 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R6 and R7 are either: R6 is —H and R7 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R7 is —H and R6 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R8 and R9 are either: R8 is —H and R9 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R9 is —H and R8 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19.

    3. The compound according to claim 2, wherein R3 to R9 are selected to form galactosyl or glucosyl, GalNAc, or a deoxy sugar group.

    4. The compound according to claim 1, wherein at least one of R1 and/or R2 is a C.sub.6-C.sub.30 linear or branched alkyl or alkenyl group, or an oligomeric- or polymeric-ethylene glycol chain, optionally substituted with —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO3R13.

    5. The compound according claim 1, comprising at least 3 C.sub.6-C.sub.30 linear or branched alkyl, alkenyl, or alkoxy groups, optionally substituted as for R1 or R2 according to claim 1.

    6. The compound according to claim 1, of a structure according to formula (III): ##STR00068## wherein A, E and R1 are as defined in claim 1, R3 is —OH, OC.sub.1-C.sub.12 alkyl, —CO.sub.2H, or R19, wherein R19 is —NHCONH—R20, —OCONH—R20, —OCOC.sub.1-C.sub.12 alkyl- or —NHCOC.sub.1-C.sub.12 alkyl-, optionally bound to R20, wherein R20 is an aromatic group, wherein the aromatic group comprises or consists of 1-2 aromatic or heteroaromatic 5- or 6-membered ring structures; R4 and R5 are either: R4 is —H and R5 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R5 is —H and R4 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R6 and R7 are either: R6 is —H and R7 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R7 is —H and R6 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R8 and R9 are either: R8 is —H and R9 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R9 is —H and R8 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, and R15 is a linear or branched alkyl, alkenyl or alkoxy group, optionally substituted with —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, or R15 is —R16PhR17-, wherein Ph is phenyl, or a para/meta di-substituted phenyl ring and R16 and R17 are, independently, a linear or branched alkyl or alkenyl group.

    7. A compound according to claim 1, of a structure according to formula (IV): ##STR00069## wherein D, E and R2 are as defined in claim 1, R3 is —OH, OC.sub.1-C.sub.12 alkyl, —CO.sub.2H, or R19, wherein R19 is —NHCONH—R20, —OCONH—R20, —OCOC.sub.1-C.sub.12 alkyl- or —NHCOC.sub.1-C.sub.12 alkyl-, optionally bound to R20, wherein R20 is an aromatic group, wherein the aromatic group comprises or consists of 1-2 aromatic or heteroaromatic 5- or 6-membered ring structures; R4 and R5 are either: R4 is —H and R5 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R5 is —H and R4 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R6 and R7 are either: R6 is —H and R7 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R7 is —H and R6 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19; R8 and R9 are either: R8 is —H and R9 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, or R9 is —H and R8 is —H, —OH, —OC.sub.1-C.sub.12 alkyl, —NH.sub.2, or R19, and R18 is a linear or branched alkyl, alkenyl or alkoxy group, optionally substituted with —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13, or R18 is —R16PhR17-, wherein Ph is phenyl, or a para/meta di-substituted phenyl ring and R16 and R17 are, independently, a linear or branched alkyl or alkenyl group.

    8. The compound according to claim 1, wherein at least at least two lipophilic groups are present at R1 and/or R2, wherein said lipophilic groups are C.sub.6-C.sub.30 linear or branched alkyl or alkenyl groups, optionally substituted with —OH, —NH.sub.2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO3R13.

    9. The compound according to claim 1, wherein at least at least two hydrophilic groups are present at R1 and/or R2, wherein said hydrophilic groups are an oligomeric- or polymeric-ethylene glycol chain, optionally substituted with —OH, —NH2, —NHR10, —N.sub.3, —C═O; acetal, —CO.sub.2H, —CO.sub.2R11, —SO.sub.3H, —SO.sub.3R11, —SH; —SR12, maleimide, —OPO.sub.3R13.

    10. A compound according to claim 1, according to: ##STR00070## ##STR00071## ##STR00072## ##STR00073##

    11. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.

    12. A method for immune stimulation in a subject that produces a therapeutic benefit, comprising administering a compound according to claim 1 to a subject in need thereof.

    13. The method according to claim 12, wherein the compound is administered as an adjuvant in a method of vaccinating a subject.

    14. The method according to claim 12, for stimulating dendritic cell (DC), natural killer (NK) cell, B cell, T cell or macrophage activity, stimulating antibody production, stimulating an immune response against infection, or treating septic shock.

    15. A method for the manufacture of a compound according to claim 1, comprising an Ugi-4-component reaction (Ugi-4CR) followed by deprotection, said reaction comprising: ##STR00074## wherein A, D, G, R1, R2, E are as defined in claim 1 for formula (I), and reactive groups of A, D, G, R1, R2 are protected prior to and during the Ugi-4CR, and X is a protecting group.

    16. The method according to claim 14, wherein the method stimulates an immune response against a cancer.

    17. The compound according to claim 1, wherein A is an amino acid or a polypeptide, wherein A is a residue with the following formula: ##STR00075## wherein R14 is a side chain of a naturally occurring amino acid.

    18. The compound according to claim 1, wherein R2 is absent when D is a cycloalkyl group.

    19. The compound according to claim 1, wherein D is an amino acid or a polypeptide, wherein D is a residue with the following formula: ##STR00076## wherein R14 is a side chain of a naturally occurring amino acid.

    20. The compound according to claim 1, wherein E is H.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0205] FIG. 1: Potent activators of iNKTs.

    [0206] FIG. 2: Analysis of unspecific proliferative responses of murine cells stimulated without or with enhanced concentrations of the (B/C/E/F) Phytosphingosine-compounds or (A/D) controls shown by thymidine uptake (count per minute, cpm) and Stimulation index (SI).

    [0207] FIG. 3A: Cell proliferation tracked by CFSE dilution of CD19+ B cells after interaction with matured Antigen Presenting Cells (APC).

    [0208] FIG. 3B: Cell proliferation tracked by CFSE dilution of CD4+ T cells after interaction with matured Antigen Presenting Cells (APC).

    [0209] FIG. 3C: Cell proliferation tracked by CFSE dilution of CD8+ T cells after interaction with matured Antigen Presenting Cells (APC).

    [0210] FIG. 4: Effect after treatment with IPB 0964-1917—Cell proliferation tracked by CFSE loss in A/B) CD4+ and C/D) CD8+ T cells after interaction with matured Antigen Presenting Cells (APC).

    [0211] FIG. 5: Immunization protocol—Administration (intramuscular route) of Ovalbumin with adjuvants.

    [0212] FIG. 6: Development of the weight of mice vaccinated with Ovalbumin-containing control formulations and ovalbumin combined with adjuvants via the intramuscular route.

    [0213] FIG. 7: Systemic humoral immune responses induced in mice.

    [0214] FIG. 8: Antigen-specific multifunctional CD4+ T cells.

    [0215] FIG. 9: Immunization protocol—Administration (intranasal route) of Ovalbumin with adjuvants.

    [0216] FIG. 10: Development of the weight of mice vaccinated with Ovalbumin-containing control formulations and ovalbumin combined with adjuvants via the intranasal route.

    [0217] FIG. 11: Systemic humoral immune responses induced in mice.

    DETAILED DESCRIPTION OF THE FIGURES

    [0218] FIG. 1: Potent activators of iNKTs. Presented is a selection of prior art compounds including KRN7000 and α-GalCer derivatives.

    [0219] FIG. 2: Analysis of unspecific proliferative responses of murine cells stimulated without or with enhanced concentrations of the Phytosphingosine-compounds. Splenocytes from mice were re-stimulated for 96 h with different concentrations of adjuvant (1, 10 and 20 μg/ml).

    [0220] The results are presented by are expressed (A-C) as counts per minute (cpm) and (D-F) stimulation index (SI) being the ratio of [.sup.3H]-thymidine uptake of stimulated versus non-stimulated samples. (A) and (D) show data for α-GalCer controls, (B) and (E) show data for IPB1901-1910, and (C) and (F) show data for IPB1911-1917.

    [0221] FIG. 3A: Cell proliferation tracked by CFSE dilution of B cells after interaction with matured Antigen Presenting Cells (APC). With every cell division, the CFSE signal strength is approximately halved. The observed cell numbers indicate the ratio of proliferating viable CD19+ B cells.

    [0222] FIG. 3B: Cell proliferation tracked by CFSE dilution of CD4+ T cells after interaction with matured Antigen Presenting Cells (APC). With every cell division, the CFSE signal strength is approximately halved. The shown cell numbers indicate the ratio of proliferated live CD4+ T cells.

    [0223] FIG. 3C: Cell proliferation tracked by CFSE dilution of CD8+ T cells after interaction with matured Antigen Presenting Cells (APC). With every cell division, the CFSE signal strength is approximately halved. The shown cell numbers indicate the ratio of proliferated live CD8+ T cells

    [0224] FIG. 4: Effect after treatment with IPB 0964-1917—Cell proliferation tracked by CFSE loss of T cells after interaction with matured Antigen Presenting Cells (APC). With every cell division, the CFSE signal strength is reduced. The shown cell numbers indicate the ratio of proliferating T and B cells (7 steps are shown). (A) Numbers of CD4+ T cells are shown at different proliferation steps up to 7 days measuring the loss of CFSE. Naïve cells were treated with one of IPB1901-1912 together with DCs activated with Ova prior to the CFSE assay. (B) Numbers of CD4+ T cells are shown at different proliferation steps up to 7 days measuring the loss of CFSE. Naïve cells were treated with one of IPB2033-2044 together with DCs activated with Ova prior to the CFSE assay. (C) Numbers of CD8+ T cells are shown at different proliferation steps up to 7 days measuring the loss of CFSE. Naïve cells were treated with one of IPB1901-1912 together with DCs activated with Ova prior to the CFSE assay. (D) Numbers of CD8+ T cells are shown at different proliferation steps up to 7 days measuring the loss of CFSE. Naïve cells were treated with one of IPB2033-2044 together with DCs activated with Ova prior to the CFSE assay.

    [0225] FIG. 5: Immunization protocol—Administration (intramuscular route) of Ovalbumin with adjuvants. Ovalbumin (30 μg) was co-administered (intramuscular) without or with different adjuvants, such as IPB 2033-2044, c-di-AMP or αβGalCerMPEG (15 μg), on days 0, 14 and 28.

    [0226] FIG. 6: Development of the weight of mice vaccinated with Ovalbumin-containing control formulations and ovalbumin combined with adjuvants via the intramuscular route. (A) Mice weight after administration of controls ovalbumin, owa+c-di-AMP or ova+alpha-beta-GalCerMPEG. Animal body weight was monitored throughout the whole experimental setting. (B) Development of the weight of mice vaccinated with different Ovalbumin-containing IPB 2033-2044 formulations. Animal body weight was monitored throughout the whole experimental setting. No signs of acute toxicity were observed in animals receiving Ovalbumin-containing IPB 2033-2044 formulations by i.m. route.

    [0227] FIG. 7: Systemic humoral immune responses induced in mice. Shown is the IgG titer as a readout of systemic humoral immune responses induced in mice after three immunizations with Ovalbumin co-administered with different adjuvants via intramuscular route. Ova-specific IgG titers were measured via ELISA in sera 14 days after the last immunization.

    [0228] FIG. 8: Antigen-specific multifunctional CD4+ T cells. (A) Groups of 3 BALB/c mice were immunized intramuscularly with PBS (control) or with three doses (14 days apart) of Ovalbumin (30 μg), alone or adjuvanted with c-di-AMP or αβGalCerMPEG or IPB 2033-2044 (15 μg). At 14 days after the second immunization, spleen cells were harvested, restimulated with Ovalbumin, intracellularly stained for double positive Th cytokines (IFN-γ, IL-2, TNF-α, IL-4 and IL-17), and analyzed by flow cytometry. (B) Groups of 3 BALB/c mice were immunized intramuscularly with PBS (control) or with three doses (14 days apart) of Ovalbumin (30 μg), alone or adjuvanted with c-di-AMP or αβGalCerMPEG or IPB 2033-2044 (15 μg). At 14 days after the second immunization, spleen cells were harvested, restimulated with Ovalbumin, intracellularly stained for triple positive Th cytokines (IFN-γ, IL-2, TNF-α, IL-4 and IL-17), and analyzed by flow cytometry.

    [0229] FIG. 9: Immunization protocol—Administration (intranasal route) of Ovalbumin with adjuvants. Immunization protocol: Mucosal administration (intranasal (i.n.) route) of Ovalbumin (30 μg) co-administered without or with different adjuvants, such as IPB 2033-2044, c-di-AMP or αβGalCerMPEG (15 μg), on days 0, 14 and 28.

    [0230] FIG. 10: Development of the weight of mice vaccinated with Ovalbumin-containing control formulations and ovalbumin combined with adjuvants via the intranasal route. (A) Development of the weight of mice vaccinated with different Ovalbumin-containing control formulations by i.n. route. Animal body weight was monitored throughout the whole experimental setting. (B) Development of the weight of mice vaccinated with different Ovalbumin-containing IPB 2033-2044 formulations by i.n. route. Animal body weight was monitored throughout the whole experimental setting. No signs of acute toxicity were observed in animals receiving Ovalbumin-containing IPB 2033-2044 formulations.

    [0231] FIG. 11: Systemic humoral immune responses induced in mice. Shown is the IgG titer as a readout of systemic humoral immune responses induced in mice after three immunizations with Ovalbumin co-administered with different adjuvants via intranasal route. Ova-specific IgG titers in sera 14 days after the last immunization.

    EXAMPLES

    [0232] The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration.

    Example 1: Unspecific T and B Cell Stimulation

    [0233] In order to assess the properties of the inventive compounds in antigen-unspecific immune response stimulation the compounds were administered to splenocytes in an established proliferation assay.

    [0234] For the proliferation assay, splenocytes (5×10.sup.6 cells/ml) are seeded at 100 μl per well in a U-bottomed 96-well microtiter plate (Sarstedt Inc., Newton, N.C.) and cultured in quadruplicates 4 days in the presence of enhanced concentrations of adjuvant (1, 10 and 20 μg/ml)), 5 μg/ml of Concanavalin A (max); or medium alone. Eighteen hours before harvesting, 1 μCi of [3H]thymidine (Amersham International, Freiburg, Germany) is added to each well. Cells are harvested on paper filters (Filtermat A; Wallac, Freiburg, Germany) using a cell harvester (Inotech, Wohlen, Switzerland) and the incorporation of [3H] thymidine into the DNA of proliferating cells is determined using a scintillation counter (Wallac 1450, Micro-Trilux).

    [0235] The novel phytosphingosine derivatives, IPB 1901 (SI 4), IPB 1902 (SI>3), IPB 1903 (SI>10), IPB 1904 (SI>3), IPB 1910 (SI ca. 2) in FIG. 2E and IPB 1911, IPB 1913 (SI>1.5), IPB 1912 (SI>2.5), amongst others in FIG. 2F, showed induction of unspecific proliferation after restimulation of splenocytes with different concentrations of the Phytosphingosine-compounds (1, 10 and 20 μg/ml) for 96 h. Refer to FIG. 2.

    Example 2: Antigen-Specific T and B Cell Stimulation by Maturated Bone Marrow Derived DCs

    [0236] In order to assess the properties of the inventive compounds in antigen-specific T and B cell stimulation, the compounds were administered to cultured bone marrow cells in a CFSE assay.

    [0237] Briefly, femurs and Tibiae of female 4-12 weeks old C57Bl6 (or BALB/c for TCR HA) were removed and purified from the surrounding muscle tissue (Kleenex tissues). Intact Bones were left in 70% ethanol for 2-5 min for disinfection. Both ends were cut with scissors and the bone marrow were flushed with PBS using a syringe with a 0.45 mm diameter needle, clusters were disintegrated by vigorous pipetting.

    [0238] In order to obtain activated antigen presenting dendritic cells (DCs), bone marrow cells were cultured with GM-CSF. On Day 0, cells were seeded in a concentration of 2×10.sup.6 cells per 100 mm dish (bacteriological petri dishes) in 10 ml RPMI-10 (100 U Pen/100 μg Strept/2 mM Glutamin/50 μm β-Mercaptoethanol/10% FCS heat-inactivated and filtered 0.22 μm Filter) containing 200 Units GM-CSF. On Day 3 another 10 ml RPMI-10 containing 200 U GM-CSF were added to the plates. On Day 6 half of the culture supernatants (10 ml) were collected, centrifuged and the cell pellet resuspended in 10 ml fresh RPMI-10/200 U GM-CSF and given back into the original plates.

    [0239] Subsequently, the DCs were incubated with Ovalbumin (as antigen) alone or co-administered with known adjuvants, such as α-GalCerMPEG, αβGalCerMPEG or TLR2/6 ligand (BPPcysMPEG), or the novel phytosphingosine derivatives of the present invention. The activation of DCs was controlled by FACS analysis of different CD markers, such as CD40, CD54, CD80, CD86, CD83, MHCI and MHCII.

    [0240] The effect of the compounds of the present invention was then ascertained via co-incubation of naïve T or B cells, obtained from mice (OTI or OTII mice) with previous exposure to the Ova antigen, with DCs that had been treated with the compounds of the invention.

    [0241] Naïve T cells were derived from LN/spleen from OTI or OTII mice. The MagniSort™ Mouse CD4 naïve or CD8 naïve T cell Enrichment Kit is designed for the magnetic separation of CD4 or CD8 naïve T cells by negative selection from mouse spleens or lymph nodes utilizing a biotinylated antibody cocktail and streptavidin-coated magnetic beads. Undesired cells are bound by antibody (Anti-Mouse CD8 or CD4/CD11b CD19/CD24/CD44/CD45R(B220)/CD49b(Integrin α 2)/Ly-6G (Gr-1)/γδ TCR/TER-119) and then magnetic beads that, when placed in a magnetic field, leave CD4 or CD8 naïve T cells untouched and free in solution.

    [0242] CFSE staining of the CD4+ or CD8+ naïve T cells is then carried out according to standard procedures. Subsequently, 2-5×10.sup.4 DCs (as mature antigen presenting cells APCs) are incubated with 4×10.sup.5 CFSE+ T cells (OTI or OTII) per well (ratio DC/T cells is 1:4 or 1:8). A CFSE proliferation assay is then conducted, using on Day +5 or on Day +7 a FACS analysis (CFSE/LD/CD4/CD8/CD19/Thy1.1), providing a cell count for the desired cell type based on CFSE. With every cell division, the CFSE signal strength is approximately halved. The cells marked with CFSE can therefore be used to determine the proliferation of CD19+ B cells, CD4+ or CD8+ T cells in response to treatment with activated DCs, that were treated with or without the compounds of the invention.

    Example 3A: Antigen-Specific CD19+ B Cell Proliferation

    [0243] As is shown in FIG. 3A, cell proliferation of B cells is tracked by CFSE dilution after interaction with APCs. With every cell division, the CFSE signal strength is approximately halved. Therefore, the compounds of the invention show an effect on inducing antigen specific CD19+ cell proliferation.

    [0244] On day −1, mature DCs were restimulated with Ovalbumin (antigen) co-administered with 10 μg of the different novel phytosphingosine derivatives (0964-1917) or with positive controls (α-GalCerMPEG, αβGalCerMPEG, CDA or BPPcysPEGdef) for 24 h. The novel phytosphingosine derivatives showed comparable or higher capacity to stimulate B cells via DC interaction.

    [0245] The novel phytosphingosine derivatives, IPB 1909, IPB 1905, IPB 1906, IPB 1904 and IPB 1912 in FIG. 3A showed a particularly high capacity to induce antigen specific CD19+ B cell proliferation after interaction with matured Antigen Presenting Cells (APC) for 5 days. These compounds showed a greater effect than known adjuvants as controls. Additionally, essentially all compounds showed comparable effects to the adjuvant controls, with the exception of IPB 1916, IPB 1901, IPB 1902, IPB 1903, which may show some toxicity against B cells.

    Example 3B: Antigen-Specific CD4+ T Cell Proliferation

    [0246] As is shown in FIG. 3B, cell proliferation is of CD4+ T cells tracked by CFSE dilution after interaction with matured Antigen Presenting Cells (APC).

    [0247] On day −1, mature DCs were restimulated with Ovalbumin (antigen) co-administered with 10 μg of the different novel phytosphingosine derivatives (IPB 0964-1917) or with positive controls (α-GalCerMPEG, αβGalCerMPEG, CDA or BPPcysPEGdef) for 24 h. The novel Phytosphingosine-compounds showed higher capacity to stimulate CD4+ T cells via DC interaction.

    [0248] The novel phytosphingosine derivatives IPB 1909, IPB 0964, IPB 1903, IPB 1911 and IPB 1910 in FIG. 3B showed high capacity to induce antigen specific CD4+ T cell proliferation after interaction with matured Antigen Presenting Cells (APC) for 5 days. These compounds showed the greatest effect, although essentially all compounds showed improved effects over the adjuvant controls, with the exception of IPB 1902, which may show some toxicity against CD4+ T cells.

    Example 3C: Antigen-Specific CD8+ T Cell Proliferation

    [0249] As is shown in FIG. 3C, cell proliferation of CD8+ T cells is tracked by CFSE dilution after interaction with matured Antigen Presenting Cells (APC).

    [0250] On day −1, mature DCs were restimulated with Ovalbumin (antigen) co-administered with 10 μg of the different novel phytosphingosine derivatives (IPB 1901-0964) or with positive controls (α-GalCerMPEG, αβGalCerMPEG, CDA or BPPcysPEGdef) for 24 h. The novel phytosphingosine derivatives showed comparable or higher capacity to stimulate CD8+ T cells via DC interaction. A strong capacity was seen to stimulate more efficiently antigen specific CD8+ T cells in comparison to the positive controls.

    [0251] The novel phytosphingosine derivatives IPB 1909, IPB 0964, IPB 1911, IPB 1912 and IPB 1910 in FIG. 3C also showed a strong effect to induce antigen specific CD8+ T cell proliferation after interaction with matured Antigen Presenting Cells (APC) for 5 days.

    Example 4: Cell Proliferation of CD4+ and CD8+ T Cells

    [0252] As is shown in FIG. 4, cell proliferation of T and B cells is tracked by CFSE loss after interaction with matured Antigen Presenting Cells (APC). On day −1, mature DCs were restimulated with Ovalbumin (antigen) and co-administered with 10 μg of the different novel phytosphingosine derivatives (IPB 1901-0964 and IPB 2033-2044) for 24 h. The novel phytosphingosine derivatives showed comparable or higher capacity to stimulate CD4+ T cells and 8+ T cells via DC interaction.

    [0253] In FIG. 4A the novel phytosphingosine derivatives IPB 1901, IPB 1902, IPB 1903 and IPB 1912 showed a stimulating effect of CD4+ T cells beyond controls in which only the antigen Ova was used to activate the antigen presenting DCs. In FIG. 4B the novel phytosphingosine derivatives IPB 2036, IPB 2038, IPB 2039 showed a stimulating effect of CD4+ T cells beyond controls in which only the antigen Ova was used to activate the antigen presenting DCs.

    [0254] In FIG. 4C the novel phytosphingosine derivatives IPB 1901, IPB 1902, IPB 1903 and IPB 1912 showed a stimulating effect of CD8+ T cells beyond controls in which only the antigen Ova was used to activate the antigen presenting DCs. In FIG. 4D the novel phytosphingosine derivatives IPB 2036, IPB 2039 showed a stimulating effect of CD8+ T cells beyond controls in which only the antigen Ova was used to activate the antigen presenting DCs.

    [0255] The inventive compounds therefore showed high capacity to induce antigen-specific B or T cell proliferation after interaction with matured Antigen Presenting Cells (APC) for 7 days.

    Further Examples

    [0256] Additional experimentation was undertaken and is ongoing to investigate the properties of the compounds of the present invention with respect to their immune-stimulating action and potential as adjuvants. In particular, the compounds IPB 2033-2044 have been subjected to further analysis.

    [0257] Many adjuvants from preclinical studies interact with receptors of the innate immune system, e.g. toll-like receptors (TLR) on antigen-presenting immune cells, thereby triggering an activation cascade. In contrast, the novel IPB compounds 2033-2044 interact with the surface molecule CD1d on antigen-presenting cells (e.g. dendritic cells). The contact of IPB 2033-2044 with CD1d mediates the bond between natural killer cells (NK cells) or iNKT cells and antigen-presenting cells, thus enhancing adjuvant activity.

    [0258] The following experimental protocols are of relevance:

    [0259] IPB 2033-2044 Adoptive Transfer Experiment (In Vitro):

    [0260] Adoptive Transfer Models OTI and OTII:

    [0261] OTII mice transgenic for αβTCR specific for 323-339 OVA-peptide in the context of H-2 I-Ab, and OTI mice transgenic for αβTCR specific for SIINFEKL OVA-peptide in the context of H-2Kb were crossed to Thy1.1 C57BL/6J congenic mice. For adoptive transfer, CD4 T cells from LN and spleen of OTII mice were purified using Affymetrix enrichment kit for naïve CD4+ T cells; CD8 T cells from LN and spleen of OTI mice were purified using Affymetrix enrichment kit for naïve CD8+ T cells. In all experiments, OTI or OTII cells were stained with CFSE (Cambridge Bioscience) before injection into Thy1.2+ recipient mice.

    [0262] Where indicated, small naïve Thy1.1+CD4+CD62L+CD44− OT-II cells were sorted by flow cytometry (MoFlo, DakoCytomation, UK). To ensure high purity, and the exclusion of memory (CD4+CD62L-CD44+) cells from the naïve CD4+CD62L+CD44-OT-II cell population, samples were sorted. Sorted cell purity was assessed on a MoFlo or based on a BD FACSsort. Before transfer Thy1.1+OT-II T cells were labeled with CFSE (Cambridge Bioscience, Cambridge, UK), as follows: [0263] Prepare suspension of cells in PBS at about 0.5-3×10.sup.7/ml [0264] Add equal volume of 2 μM CFDA in PBS (stock 10 mM in DMSO) (=>1:5000; 5 ml PBS+1 μl CFDA stock) [0265] Incubate the cells for 5 minutes at RT in the dark. [0266] Add equal volume of FCS (bind to unbound CFSE) [0267] Incubate the cells for further 5 minutes. [0268] Centrifuge the cells and wash with complete medium one time. [0269] Suspend the cells in medium [0270] 1 to 3×10.sup.6 cells per congenic Thy1.2+ recipient mouse were injected i.v., unless otherwise stated. Mice were immunized the following day.

    [0271] Where indicated, small naïve Thy1.1+CD8+CD62L+CD44− OT-1 cells were sorted by flow cytometry (MoFlo, DakoCytomation, UK). To ensure high purity, and the exclusion of memory (CD8+CD62L-CD44+) cells from the naïve CD8+CD62L+CD44− OT-1 cell population, samples were sorted. Sorted cell purity was assessed on a MoFlo or based on a BD FACSsort. Before transfer Thy1.1+OT-1 T cells were labeled with CFSE (Cambridge Bioscience, Cambridge, UK) and were injected iv. at 1 to 3×10.sup.6 cells per congenic Thy1.2+ recipient mouse, unless otherwise stated. Mice were immunized the following day.

    [0272] The chimeras were immunized by different routes (e.g. i.n. or i.m.) with different volumes and concentrations (Table below). As antigen, endotoxin-free OVA protein (Hyglos) was used, formulated or conjugated to nanoparticles, and co-administered with different adjuvants (e.g. IPB 2033-2044, c-di-AMP, etc.).

    TABLE-US-00001 TABLE Routes of immunization Route Volume Ova Concentration i.n. 20 μl 20 μg Oral 100 μl 75 μg Rectal 50 μl 75 μg i.p. 100 μl 75 μg Pulmonal 75 μl 20 μg i.v. 100 μl 1 to 3 × 10.sup.6 cells s.c. 50-100 μl 20 μg

    [0273] Flow Cytometry Analysis and FACS Cell Sort (CFSE) was carried out as follows: [0274] Draining LNs and spleen were removed [0275] single-cell suspensions were prepared in RPMI medium containing 5% FCS. [0276] The antibodies used for surface staining are listed in Table 2. [0277] Thy1.1+ cells were analysed for the CFSE loss of LN or spleen cell suspensions by flow cytometry using a FACS Fortessa (Becton Dickinson). [0278] Final analysis and graphical output were performed using FlowJo software (Treestar). [0279] The number of Thy1.1 positive cells in combination with the loss of CFSE staining per sample (LN, spleen, etc.) and the numbers of proliferating CFSE-cells in response to the vaccine versus non-proliferating CFSE+ cells was calculated.

    TABLE-US-00002 TABLE Antibody list Flourochrom Marker Action 1 FITC CFSE Proliferation 2 UV Live/dead 3 PE CD3+ T cells 4 APC CD4+ CD4+ T cells 5 APC-Cy7 CD8+ CD8+ T cells (6) PE-Cy7 Thy1.1+ (CD90)− Sorting (7) PE CD69+/or CD19+ Activation

    [0280] Immunization Protocol (In Vivo)

    [0281] Groups of mice (3-5 animals) were immunized either intranasal (i.n.) or intramuscular (i.m.) on days 0, 14 and 281 with PBS, or with Ovalbumin (30 μg); the latter were administered alone or with different adjuvants—c-di-AMP, αβGalCerMPEG or IPB 2033 to 2044—made up to a maximal volume of 20 μl (i.n.) or 50 μl (i.m.) in PBS. Vaccinated animals showed no adverse effects when vaccinated with Ova in combinations of IPB 2033-2044.

    [0282] Blood samples were collected on days 1, 14, 28 and 42 via retro-bulbar bleeding. Spleens of vaccinated mice were aseptically removed. For the subsequent methods, cell suspensions of spleens (n=5) of each immunized groups were prepared and erythrocytes were lysed. These splenocyte pools of each group were cultured in the presence of different concentrations of Ovalbumin; controls received 5 μg/mL concanavalin A. The incorporation of [.sup.3H] thymidine into the DNA of proliferating cells was determined using a scintillation counter (Wallac 1450, Micro-Trilux).

    [0283] Immunization Using IPB 2033-2044 Effectively Stimulates T-Cell-Mediated Proliferation Responses when Co-Administered with a Soluble Model Antigen (Proliferation)

    [0284] The spleens were removed from sacrificed animals and combined for the analysis of the cell immune reactions. The cells were then resolved in RPMI 1640, supplemented by 10% fetal calf serum, 10 U/ml penicillin, 50 μg/ml streptomycin, 5×10.sup.5 M p-mercaptoethanol and 1 mM L-glutamine (GIBCO BRL, Karlsruhe, Germany) and stored at 37° C. in a humid atmosphere with 5% CO.sub.2. The spleen cell suspensions were adjusted to 5×10.sup.6 cells/ml in the complete medium, placed in a flat-bottomed microtiter plate with 96 wells (Nunc) at 100 μl/well, and the plates were incubated for four days in the presence of different concentrations of soluble IPB 2033-2044. Each concentration was tested in groups of three. During the last 18 hours of incubation, 1 μCi of .sup.3H thymidine (Amersham International, Freiburg, Germany) was added to each well. The cells were then harvested on filter paper (Filtermat A; Wallac, Freiburg, Germany) using a cell harvester (Inotech, Wohlen, Switzerland) and the amount of .sup.3H thymidine embedded in the DNA of the multiplied cells was determined using a γ-scintillation counter (Wallac 1450, Trilux). The results were presented as the arithmetic mean of the .sup.3H thymidine uptake in cpm.

    [0285] Detection of Antigen-Specific IgG and IgA in Serum (Humoral Response)

    [0286] The Ova-specific antibodies were determined in serum samples by ELISA. Endpoint titers were expressed as reciprocal values of the last dilution, which gave an optical density at 405 nm of two times above the values of the negative controls. For calculation purposes, negative samples were assigned an arbitrary titer of the lowest dilution measured.

    [0287] Immunization Using IPB 2033-2044 Effectively Stimulates Cytokines Secretion when Co-Administered with a Soluble Model Antigen (Elispot)

    [0288] The number of Ova-specific cytokine-producing cells was determined using an ELISpot assay. 96-well plates (BD Pharmingen) were coated with anti-IFN-γ, anti-IL2, anti-IL4, anti-IL-10 or anti-IL17 antibodies overnight at 4° C. Then plates were washed one time with culture medium (RPMI, 10% fetal calf serum (FCS), PenStrep, L-glutamine, and p-mercaptoethanol) and cells were seeded in culture medium with or without Ova (5 μg/ml). Plates were incubated 24 h for IFN-γ and 48 h for the other cytokines. Then, cells were removed and the plates processed according to manufacturer's instructions. Colored spots were counted with an ELISpot reader (CTL-Europe GmbH) and analyzed using the ImmunoSpot image analyzer software v3.2.

    [0289] CDA-Adjuvanted Dose Elicits Multiple-Cytokine Producers Among Antigen-Specific T Cells (Multifunctional T Cells)

    [0290] In order to characterize more accurately the cellular immune response, the production of intracellular cytokines was measured in CD4+ and CD8+ T cells. Multifunctional CD4+ and CD8+ cells were stimulated when mice received OVA+IPB 2033-2044. Vaccinated animals showed enhanced secretions of double or triple positive cytokines. The above-mentioned in vitro approach is very useful as an initial screening for the evaluation of cytokine profiles promoted by candidate adjuvants after stimulation of different types of cells. However, it does not allow accurate prediction of important effector functions of an adjuvant, such as CTL stimulation capabilities, target cell subpopulations and capacity to confer protective immunity.

    [0291] Therefore, mice were immunized to assess the in vivo performance of CDA in an active vaccination setting. To assess vaccination effectivity, the frequencies of cytokine producers among the CD4+ and CD8+ T cell populations were evaluated by intracellular cytokine staining and flow cytometry. The production of multiple cytokines (especially IL-2, IFN-γ, IL-17, IL-10 and TNF-α) by T cells has been described to correlate with vaccine protective efficacy (Darrah, P. A., et. al., 2007, Nat Med 13 (7):843-50. doi: 10.1038/nm1592). Thus, the frequency of T cells producing single cytokines or combinations (positive events/million) was analyzed by flow cytometry. In contrast, the use of CDA and by IPB 2033-2044 adjuvanted Ovalbumin increased the frequency of triple, double and single cytokine producers.

    [0292] NK Cells Combined with IPB 2033-2044 (FACS)

    [0293] PBMCs were thawed and 1×106 to 4×106 cells/sample were re-stimulated for 16 h in complete RPMI 1640 (Gibco, supplemented with 10% FCS, 5% Penicillin/Streptomycin and 5% Glutamine) containing the vaccine formulation with a final concentration of 5 μg Ovalbumin/mL model antigen combined with IPB 2033-2044. Unstimulated samples were incubated for the same time in complete RPMI without the vaccine formulation. Brefeldin A and monensin were added to all samples after 5 h of incubation. Cells were collected and stained for flow cytometric analysis. Surface marker staining was performed for 20 min at 4° C. The following antibodies were used diluted in PBS: CD56 (PE-Cy7, clone B159, BD, Franklin Lakes, N.J., USA), CD3 (V450, clone UCHT1, BD), CD14 (Pacific Blue, clone M5E2, BD), CD19 (V450, clone HIB19, BD Horizon), CD16 (APC-H7, clone 3G8, BD Pharmingen), NKG2C (PE, clone 134591, R&D Systems, Minneapolis, Minn., USA), CD57 (APC, clone HCD57, BioLegend, San Diego, Calif., USA), Live/Dead (Fixable Blue, Invitrogen, Carlsbad, Calif., USA). The expression of CD107a was used as a correlate of degranulation. To this end, the anti-CD107a antibody (PE-Cy5, clone eBioH4A3, eBioscience, San Diego, Calif., USA) was added to the culture. The secretion of IFNγ (Alexa Fluor 700, clone B27, BioLegend) was detected by intracellular staining using Cytofix/Cytoperm solution (BD Biosciences). Samples were acquired at a BD Fortessa flow cytometer and analyzed using FlowJo (FlowJo, LLC, Ashland, Oreg., USA). Unstained, single stained (one antibody/sample) as well as fluorescence-minus-one (FMO) samples were used as controls for the acquisition as well as the subsequent analysis. Statistical differences were determined by the GraphPad Prism software.

    Example 5: Immunization Using IPB 2033-2044 Effectively Stimulates Cytokines Secretion when Co-Administered with a Soluble Model Antigen

    [0294] As described in the protocols above, groups of mice (3-5 animals) were immunized either intranasal (i.n.) or intramuscular (i.m.) on days 0, 14 and 281 with PBS, or with Ovalbumin (30 μg); the latter were administered alone or with different adjuvants—c-di-AMP, αβGalCerMPEG or IPB 2033 to 2044—is shown in FIG. 5-11, intramuscular.

    [0295] The data presented in FIG. 5 to 11 shows the immunization protocols, measurements of animal weight, measurements of cellular immune response via multifunctional CD4+ T cells and measurements of the systemic humoral immune responses via IgG titer.

    [0296] As is shown in FIGS. 6 and 10, both intramuscular and intranasal administration had no detrimental effect on mice weight development during the immunization and sampling protocol, indicating no or negligible toxicity to the mice in this experimental setting.

    [0297] As shown in FIGS. 7 and 11, both intramuscular and intranasal administration of the inventive adjuvants IPB 2033 to 2044 lead to enhanced antigen-specific IgG titer in sera of immunized mice. Of note is that the inventive adjuvants provide at least equivalent (IPB 2038, 2041, 2044 in i.m.; 2034, 2035, 2037, 2044 in i.n.), or in many cases improved stimulation (IPB 2033, 2034, 2035, 2036, 2037, 2039, 2040, 2042, 2043 in i.m.; 2033, 2036, 2038, 2039, 2040, 2041, 2042, 2043 in i.n.) of antigen specific IgG production compared to the structurally-related relevant control (αβGalCerMPEG+ova).

    [0298] As shown in FIG. 8, the production of multifunctional CD4+ T cells is enhanced when using the inventive adjuvants IPB 2033 to 2044, using a readout of antigen-specific CD4+ T cells that express particular immune-stimulatory cytokines. All inventive adjuvants IPB 2033 to 2044 provide enhanced levels of these CD4+ T cells compared to the structurally-related relevant control (αβGalCerMPEG+ova), as is evident from staining and sorting cells according to e.g. IL-2 and TNFa, or any 2 of IL-2, TNFa or IFNg.

    Synthesis and Analytical Chemistry

    1. General Procedure for the Synthesis of Phytosphingosine Derivatives (Also Described as α-GalCer Analogues) by Ugi-4CR

    [0299] ##STR00019## ##STR00020##

    [0300] A suspension containing compound 1 (1 mmol) and paraformaldehyde (1 mmol) in MeOH/THF 2:1 (v/v) (3 mL) is stirred overnight at room temperature. Then, the acid component—carboxylic acid—(1 mmol) and the isocyanide (1 mmol) are added and the reaction mixture is protected from light and stirred at room temperature for 72 h. The product formation is checked by TLC, the volatiles are removed under high vacuum, and the obtained crude is purified by column chromatography (n-hexane/EtOAc) to obtain the protected α-GalCer analogue.

    [0301] In other embodiments, the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR may be as follows:

    ##STR00021##

    [0302] A suspension containing compound 2 (1 mmol) and paraformaldehyde (1 mmol) in MeOH/THF 2:1 (3 mL) is stirred overnight at room temperature. Then, the acid component—(1 mmol) and the isocyanide (1 mmol) are added and the reaction mixture is protected from light and stirred at room temperature for 72 h. The product formation is checked by TLC, the volatiles are removed under high vacuum, and the obtained crude is purified by column chromatography (n-hexane/EtOAc) to obtain the protected α-GalCer analogue.

    2. General Procedure for the Benzyl Ether/Azide/4-Phenyl-1,3-Dioxolane Removal

    [0303] To a three necked flask containing a suspension of Pd/C 10% (5 g per g of the α-GalCer analogue) in THF (1 mL) under nitrogen atmosphere, the Ugi-GalCer analogue—dissolved in THF (2 mL)—and formic acid (300 μL) are added and the reaction mixture is stirred for 4 h. The product formation is checked by ESI-MS, and finally the reaction mixture is filtered over celite and washed thoroughly with THF. All volatiles are removed under reduced pressure to afford the deprotected α-GalCer analogue.

    3. General Procedure for the p-Methoxybenzyl Ether Removal

    [0304] To a solution of the per-PMB-protected Ugi-GalCer analogue (0.01 mmol) in 1,4-dioxane (1 mL) are added sequentially anisole (0.1 mmol) and HCl (1 mL, 4M in 1,4-dioxane) at rt. The progress of the reaction is followed by ESI-MS. The volatiles are removed under reduced pressure to afford the deprotected α-GalCer analogue.

    4. Synthesis of Isocyanides

    [0305] The amine (1 mmol) is dissolved in ethyl formate, in the presence of base (Et.sub.3N or DIPEA) when necessary, and the solution is refluxed overnight at 70-80° C. The solvent is removed under reduced pressure and the corresponding formamide (checked by TLC) is purified when necessary and dissolved in dry DCM or THF. Et.sub.3N (5 mmol) and POCl.sub.3 (1 mmol)—drop wise and over 15 min—are then added under nitrogen atmosphere at 0° C. and the reaction mixture is allowed to reach room temperature and stirred for 2 to 3 additional hours until completeness. A saturated solution of NaHCO.sub.3 is added drop wise to neutralize and quench the reaction and the organic phase is separated and washed twice with brine. The volatiles are removed under reduced pressure and the product is immediately purified by column chromatography and stored under nitrogen atmosphere at −20° C.

    4.1. Synthesis and Characterization of Isocyanide 31

    [0306] ##STR00022##

    4.2. Synthesis and Characterization of Isocyanide 4

    [0307] ##STR00023##

    [0308] Tetradecylamine (3.0 g, 14.1 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 4 (2.7 g, 87%) as a light-yellow oil over two steps and a final column chromatography purification (n-hexane/EtOAc 2:1); R.sub.f=0.90 (n-hexane/EtOAc 2:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 3H, J=6.6 Hz, CH.sub.3); 1.22-1.34 (m, 20H); 1.38-1.47 (m, 2H); 1.63-1.72 (m, 2H); 3.34-3.41 (m, 2H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 22.8, 26.5, 28.8, 29.3, 29.5, 29.6, 29.7, 29.8, 32.1 (CH.sub.2); 43.7 (t, J=6.0 Hz, CH.sub.2); 155.7 (t, J=6.0 Hz, C≡N).

    4.3. Synthesis and Characterization of Isocyanide 5.SUP.1

    [0309] ##STR00024##

    4.4. Synthesis and Characterization of s Isocyanide 6.SUP.1

    [0310] ##STR00025##

    4.5. Synthesis and Characterization of Isocyanide 7

    [0311] 3-Phenylpropan-1-amine (5.0 g, 31 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 and the resulting product purified by column chromatography (n-hexane/EtOAc 4:1) to afford isocyanide 7 (3.6 g, 80%). R.sub.f=0.85 (n-hexane/EtOAc 2:1). .sup.1H NMR (400 MHz, CDCl.sub.3): 1.93-2.05 (m, 2H, CH.sub.2); 2.78 (t, 2H, J=7.4 Hz, CH.sub.2); 3.32-3.39 (m, 2H, CH.sub.2); 7.10-7.29 (m, 5H, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=30.6, 32.3 (CH.sub.2); 40.8 (t, J=6.5 Hz, CH.sub.2); 126.5, 128.6, 128.7 (CH); 139.9 (C); 156.4 (t, J=5.7 Hz, C≡N).

    4.6. Synthesis and Characterization of Isocyanide 8

    [0312] ##STR00026##

    [0313] 2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethan-1-amine (1.0 g, 4.6 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 8 (0.62 g, 59%) as a light yellow liquid over two steps and two column chromatography purifications: for the formamide (DCM/MeOH 20:1; R.sub.f=0.86 (DCM/MeOH 10:1)); for the isocyanide (EtOAc/n-hexane 1:1; R.sub.f=0.75 (EtOAc/n-hexane 1:2)). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=3.39 (t, 2H, J=5.3 Hz, CH.sub.2); 3.57 (t, 2H, J=5.3 Hz, CH.sub.2); 3.64-3.74 (m, 12H, 6×CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=41.9 (t, J=7.1 Hz, CH.sub.2); 50.8, 68.8, 70.2, 70.8, 70.9, 71.0 (CH.sub.2); 157.4 (t, J=5.5 Hz, C≡N).

    4.7. Synthesis and Characterization of Isocyanide 9

    [0314] ##STR00027##

    [0315] 6-Azidohexan-1-amine (TFA salt) (1.17 g, 4.9 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 9 (0.49 g, 66%) as a light yellow liquid over two steps and two column chromatography purifications: for the formamide (EtOAc/n-hexane 2:1-3:1; R.sub.f=0.44 (EtOAc/n-hexane 1:1)); for the isocyanide (EtOAc/n-hexane 1:2; R.sub.f=0.60 (EtOAc/n-hexane 1:4)). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.36-1.54 (m, 4H, 2×CH.sub.2); 1.57-1.75 (m, 4H, 2×CH.sub.2); 3.28 (t, 2H, J=6.8 Hz, CH.sub.2); 3.35-3.44 (m, 2H, CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=26.0, 28.8, 29.9 (CH.sub.2); 41.5 (t, J=6.4 Hz, CH.sub.2); 51.8, (CH.sub.2); 156.1 (t, J=5.8 Hz, C≡N).

    4.8 Synthesis and Characterization of Isocyanide 10

    [0316] ##STR00028##

    [0317] 2,5,8,11-Tetraoxatridecan-13-amine (1.0 g, 4.8 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 and the resulting product purified by column chromatography (DCM/methanol 40:1) to afford isocyanide 10 (1.0 g, 96%) as a light yellow liquid; R.sub.f=0.35 (DCM/methanol 30:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=3.36 (s, 3H, CH.sub.3); 3.51-3.58 (m, 4H, 2×CH.sub.2); 3.61-3.71 (m, 12H, 6×CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=41.8 (t, J=7.1 Hz, CH.sub.2); 53.5 (CH.sub.2); 59.1 (CH.sub.3); 68.8, 70.6, 70.7, 70.9, 72.0 (CH.sub.2); 157.3 (t, J=5.4 Hz, C≡N).

    4.9. Synthesis and Characterization of Isocyanide 11

    [0318] ##STR00029##

    [0319] 2,5,8,11,14-Pentaoxahexadecan-16-amine (1.0 g, 4.0 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 and the resulting product purified by column chromatography (DCM/methanol 40:1) to afford isocyanide 11 (0.40 g, 38%) as a light yellow liquid; R.sub.f=0.37 (DCM/methanol 30:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=3.35 (s, 3H, CH.sub.3); 3.50-3.59 (m, 4H, 2×CH.sub.2); 3.60-3.72 (m, 16H, 8×CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=41.8 (t, J=7.1 Hz, CH.sub.2); 59.1 (CH.sub.3); 68.8, 70.6, 70.7, 70.9, 72.0 (CH.sub.2); 157.3 (t, J=5.6 Hz, C≡N).

    4.10. Synthesis and Characterization of Isocyanide 12

    [0320] ##STR00030##

    [0321] 2,2′-(Ethane-1,2-diylbis(oxy))bis(ethan-1-amine) (2.0 g, 13.5 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 12 (1.7 g, 77%) as a light yellow liquid over two steps and two column chromatography purifications: for the formamide (DCM/MeOH 20:1; R.sub.f=0.42 (DCM/MeOH 10:1)); for the isocyanide (EtOAc/n-hexane 2:1; R.sub.f=0.25 (EtOAc/n-hexane 1:1)). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=3.56-3.61 (m, 4H, 2×CH.sub.2); 3.69-3.75 (m, 8H, 4×CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=42.0 (t, J=7.0 Hz, CH.sub.2); 68.8, 71.0 (CH.sub.2); 157.5 (t, J=5.7 Hz, C≡N).

    4.11. Synthesis and Characterization of Isocyanide 13

    [0322] ##STR00031##

    [0323] 2,2-Dimethoxyethylamine (2.0 g, 19.0 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 13 (1.5 g, 66%) as a light-yellow oil over two steps and a final column chromatography purification (n-hexane/EtOAc 1:1); R.sub.f=0.85 (n-hexane/EtOAc 2:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=3.43 (s, 6H, 2×OCH.sub.3); 3.50 (d, 2H, J=5.2 Hz, CH.sub.2); 4.60 (d, 1H, J=5.2 Hz, CH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=43.7 (t, J=7.5 Hz, CH.sub.2); 54.5 (OCH.sub.3); 101.1 (CH); 158.6 (t, J=5.2 Hz, C≡N).

    4.12. Synthesis and Characterization of Isocyanide 14

    [0324] ##STR00032##

    [0325] L-Alanine tert-butyl ester hydrochloride (4.0 g, 22.0 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 to afford isocyanide 14 (2.5 g, 74%) as a light yellow liquid over two steps and two column chromatography purifications: for the formamide (EtOAc/n-hexane 1:1-5:1; R.sub.f=0.35 (EtOAc/n-hexane 1:1)); for the isocyanide (EtOAc/n-hexane 1:1; R.sub.f=0.75 (EtOAc/n-hexane 1:2)). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.46 (s, 9H, 3×CH.sub.3); 2.62 (tt, 2H, J=6.9/2.1 Hz, CH.sub.2); 3.63 (tt, 2H, J=6.9/1.9 Hz, CH.sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=28.1 (CH.sub.3); 35.4, 37.6 (CH.sub.2); 82.1 (C); 157.3 (t, J=5.4 Hz, C≡N); 168.7 (C═O).

    4.13. Synthesis and Characterization of Isocyanide 15

    [0326] ##STR00033##

    [0327] A solution containing NaN.sub.3 (11.5 g, 177.2 mmol) in DCM/H.sub.2O 1:1 (40 mL) was cooled to 0° C., and Tf.sub.2O (10.0 g, 35.4 mmol) was added dropwise. After 2 h of stirring at 0° C., the organic phase was separated, the aqueous layer was extracted with DCM and the combined organic layers were washed with H.sub.2O. This freshly prepared TfN.sub.3 solution in DCM (20 mL) was added to a suspension of 6-amino-1-hexanol (2.0 g, 17.1 mmol), K.sub.2CO.sub.3 (4.3 g, 31.4 mmol), CuSO.sub.4 (0.04 g, 0.2 mmol), in MeOH/H.sub.2O 3:1 (v:v) (160 mL). The reaction mixture was stirred overnight, filtered and the organic solvents were evaporated under reduced pressure. The remaining aqueous solution was extracted with EtOAc (5×30 mL) and the combined organic layer was washed with H.sub.2O, NH.sub.4OH (12%) and brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to afford 6-azido-1-hexanol (15a) (2.2 g, 89%) as a colorless oil. R.sub.f=0.46 (n-hexane/EtOAc 2.1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.26 (s, 1H, OH); 1.41 (p, 3H, J=3.6 Hz); 1.53-1.67 (m, 5H); 3.27 (t, 2H, J=6.9 Hz, CH.sub.2); 3.65 (t, 2H, J=6.5 Hz, CH.sub.2). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=25.5, 26.7, 29.0, 32.7, 51.5, 63.0 (CH.sub.2). A solution of DMSO (4.3 mL, 60.9 mmol) in DCM (5 mL) was added over 30 min to a stirred solution of oxalyl chloride (2.6 mL, 30.5 mmol) in DCM (20 mL) at −78° C. Upon completion of the addition, the mixture was stirred at −78° C. for 5 min, followed by addition of a solution of 15a (2.2 g, 15.2 mmol) in DCM (5 mL) over 30 min at −78° C. and the resulting mixture was stirred for 40 min. Then Et.sub.3N (13 mL, 91.4 mmol) was added dropwise over 10 min. The resulting mixture was allowed to warm to 0° C. and stirred at 0° C. for 1 h. H.sub.2O (30 mL) was added to quench the reaction and the organic layer was then separated and further washed with H.sub.2O (2×20 mL) and brine (20 mL), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure, and the residue purified by column chromatography (n-hexane/EtOAc 4:1) to afford 6-azidohexanal (15b) (1.4 g, 65%) as a colorless oil. R.sub.f=0.65 (n-hexane/EtOAc 4:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.35-1.48 (m, 2H); 1.56-1.71 (m, 4H); 2.46 (td, 2H, J=7.3/1.6 Hz, CH.sub.2 α); 3.28 (t, 2H, J=6.8 Hz, CH.sub.2); 9.77 (t, 1H; J=1.6 Hz, CHO). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=21.7, 26.4, 28.8, 43.8, 51.3 (CH.sub.2); 202.3 (C═O). 15b (0.8 g, 5.7 mmol) was dissolved in toluene (200 mL) and then placed in a two necked flask equipped with a Dean Stark apparatus, a reflux condenser and a thermometer. Then, p-TSA (32 mg, 0.17 mmol) and 1-phenyl-1,2-ethanediol (1.96 g, 14.2 mmol) were added and the mixture was heated up until toluene distillation. The reaction was carried out for five hours and then the reaction mixture was allowed to cool down, diluted with diethyl ether and washed with a saturated NaHCO.sub.3 solution (30 mL). The organic layer was further washed with H.sub.2O (2×30 mL) and brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/EtOAc 20:1) to afford the cyclic acetal 2-(5-azidopentyl)-4-phenyl-1,3-dioxolane (15c) (1.3 g, 85%) as a pale-yellow liquid. R.sub.f=0.84 (n-hexane/EtOAc 4:1). For the major diastereomer: .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.43-1.57 (m, 3H); 1.64 (p, 3H, J=7.1 Hz); 1.80-1.86 (m, 1H); 3.28 (t, 2H, J=6.9 Hz, CH.sub.2); 3.75 (dd, 1H; J=7.7/6.3 Hz); 4.20 (t, 1H, J=7.7 Hz); 5.01 (t, 1H, J=6.3 Hz); 5.09 (t, 1H, J=4.7 Hz); 7.27-7.40 (m, 5H, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=23.8, 26.8, 28.9, 33.9, 51.5, 72.1 (CH.sub.2); 78.5, 105.4, 126.5, 128.3, 128.7 (CH); 139.7 (C). A solution of trimethylphosphine in THF (24 mL, 1 M) was added dropwise to a solution of 15c (1.26 g, 4.8 mmol) in THF (10 mL) at room temperature. After stirring for 2 h at room temperature, a solution of NaOH (24 mL, 1 M) was added and the mixture was allowed to stir for additional 2 h. The reaction mixture was diluted with EtOAc (50 mL), washed with H.sub.2O (3×20 mL) and brine (20 mL), dried over MgSO.sub.4, filtered, and evaporated to dryness to afford the corresponding amine.sup.2 5-(4-phenyl-1,3-dioxolan-2-yl)pentan-1-amine (15d) (quantitative) as a colorless oil, that was employed in the next step without further purification. R.sub.f=0.11 (DCM/MeOH 20:1). For the major diastereomer: .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.36-1.58 (m, 6H, 3×CH.sub.2); 1.71-1.79 (m, 1H); 1.79-1.86 (m, 1H); 2.71 (t, 2H, J=6.8 Hz, CH.sub.2); 3.74 (dd, 1H; J=7.7/6.8 Hz, CH.sub.2); 4.19 (t, 1H, J=7.7 Hz, CH.sub.2); 5.09 (t, 1H, J=6.8 Hz, CH); 5.09 (t, 1H, J=4.8 Hz, CH); 7.25-7.43 (m, 5H, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=24.1, 27.0, 33.7, 34.5, 42.2, 72.1 (CH.sub.2); 78.4, 105.8, 126.5, 128.1, 128.7 (CH); 139.8 (C). 15d (0.89 g, 3.79 mmol) was subjected to the standard procedure for the isocyanide synthesis described in section 4 and the resulting product purified by column chromatography (n-hexane/EtOAc 4:1) to afford isocyanide 15 (0.35 g, 38%). R.sub.f=0.79 (n-hexane/EtOAc 2:1). For the major diasteromer: .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.48-1.61 (m, 5H); 1.66-1.89 (m, 3H); 3.37-3.43 (m, 2H, CH.sub.2); 3.75 (dd, 1H; J=8.1/6.5 Hz, CH.sub.2); 4.41 (dd, 1H, J=8.1/6.5 Hz, CH.sub.2); 5.10 (t, 1H, J=6.5 Hz, CH); 5.26 (t, 1H, J=4.7 Hz, CH); 7.27-7.40 (m, 5H, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=23.3, 26.4, 29.2, 33.8, 41.5, 72.0 (CH.sub.2); 78.5, 105.2, 126.5, 128.3, 128.7 (CH); 193.6 (C); 155.9 (t, J=5.7 Hz, C≡N).

    4.14. Synthesis and Characterization of Isocyanide 16.SUP.3

    [0328] ##STR00034##

    4.15. Synthesis and Characterization of Isocyanide 17

    [0329] ##STR00035##

    [0330] Formic acid (39.1 mL, 1164.1 mmol) and acetic anhydride (73.3 mL, 776.1 mmol) were mixed and reacted for 3 h at 60° C. To the cooled mixture diluted with THF, methyl α-aminoisobutyrate (9.1 g, 77.6 mmol) was added. After stirring for 12 h at room temperature, the crude mixture was co-evaporated with toluene under reduced pressure and then diluted with EtOAc (200 mL), washed with H.sub.2O (50 mL) and brine (50 mL) and the organic phase dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure to afford 17a as a yellowish liquid (10.3 g, 92%). R.sub.f=0.23 (n-hexane/EtOAc 1:1). 17a (10.3 g, 71.0 mmol) was dissolved in DCM (100 mL) and Et.sub.3N (49.5 mL, 355.1 mmol) and POCl.sub.3 (7.9 mL, 85.2 mmol)—drop wise and over 15 min—were subsequently added under nitrogen atmosphere and the reaction mixture was stirred for 2 to 3 h until completeness. A saturated solution of NaHCO.sub.3 (10 mL) was added drop wise and the organic phase was separated and washed with brine (2×20 mL). All volatiles were removed under reduced pressure and the product was purified by column chromatography (n-hexane/EtOAc 10:1-6:1) to afford 17b as a yellowish liquid (9.0 g, >99%). Rf=0.81 (n-hexane/EtOAc 1:1). 17b (2 g, 15.6 mmol) was mixed with a solution of KOH (3 mL, 1 M in MeOH) and the mixture was stirred for 2 h at room temperature. The product formation was checked by TLC and ESI-MS and after completeness, the volatiles were removed under reduced pressure and the product (17c) stored without further purification step. Triethylamine (2.2 mL, 15.8 mmol) was added dropwise to a mixture of 17c (1.2 g, 9.8 mmol) and L-Tyr(Bn)-OBzl-HCl (3.0 g, 7.5 mmol) in DMF (5 mL). After stirring for 20 min at room temperature, the reaction mixture was cooled to −10° C. (ice-salt bath; internal thermometer). HBTU (4.3 g, 11.3 mmol) was added and the mixture was stirred for 12 h until reaction completion (monitored by TLC). The reaction mixture was then diluted with EtOAc (100 mL), transferred to a separatory funnel and washed with brine (50 mL). The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to dryness. The crude product was purified by column chromatography (n-hexane/EtOAc 6:1) to afford isocyanide 17 (1.5 g, 44%). R.sub.f=0.24 (n-hexane/EtOAc 6:1). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=1.55 (s, 3H, CH.sub.3); 1.58 (s, 3H, CH.sub.3); 3.03-3.19 (m, 2H, CH.sub.2); 4.79 (dt, 1H, J=7.9/6.0 Hz, CH); 5.02 (s, 2H, CH.sub.2); 5.13 (d, 1H, J=12.1 Hz, CH.sub.2); 5.23 (d, 1H, J=12.1 Hz, CH.sub.2); 6.86 (d, 2H, J=8.6 Hz, 2×CH, Ar); 6.95 (d, 2H, J=8.6 Hz, 2×CH, Ar); 7.30-7.45 (m, 10H, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=27.7, 27.8 (CH.sub.3); 36.9 (CH.sub.2); 53.7 (CH); 67.6, 70.1 (CH.sub.2); 115.3, 127.4, 127.6, 128.1, 128.8, 130.4 (CH); 135.1, 137.0, 158.2 (C); 160.6 (t, J=3.6 Hz, C≡N); 168.8 (C); 170.7 (C═O).

    5.1. Synthesis of α-GalCer Analogue 19 (IPB001901

    [0331] ##STR00036##

    [0332] 1 (50 mg, 0.05 mmol), paraformaldehyde (1.5 mg, 0.05 mmol), myristic acid (11 mg, 0.05 mmol) and lauric isocyanide 3 (9.6 μL, 0.05 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 5:1) afforded the protected Ugi product 18 (60 mg, 84%). R.sub.f=0.24 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 19 (33 mg, 88%) as a colorless oil. [α].sub.D.sup.23.5=23.9 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 9H, J=6.7 Hz, 3×CH.sub.3); 0.98-1.36 (m, 61H); 1.36-1.67 (m, 9H), 2.17 (s, 1H); 2.26 (d, 1H, J=8.8 Hz); 2.32 (t, 1H, J=7.5 Hz); 3.08-3.26 (m, 2H); 3.47 (s, 2H); 3.55-4.31 (m); 7.43 (t, 1H, J=7.5 Hz, NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 22.8, 25.0, 29.3, 29.4, 29.5, 29.6, 29.8, 29.9, 30.0, 32.1, 34.1, 40.0 (CH.sub.2); 50.8 (CH); 63.5 (CH.sub.2), 70.3, 77.4 (CH); 178.1 (C═O). HRMS: m/z=937.7428 [M+Na].sup.+ (calculated for C.sub.52H.sub.102N.sub.2NaO.sub.10: 937.7432).

    5.2. Synthesis of α-GalCer Analogue 21 (IPB002040

    [0333] ##STR00037##

    [0334] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), lignoceric acid (54 mg, 0.15 mmol) and stearic isocyanide 6 (41 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1) afforded the protected Ugi product 20 (160 mg, 65%). R.sub.f=0.33 (n-hexane/EtOAc 10:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 21 (68 mg, 63%) as a colorless oil. [α].sub.D.sup.23.5=ND. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 5=0.85 (t, 9H, J=6.8 Hz, 3×CH.sub.3); 1.13-1.38 (m, 70H); 1.38-1.68 (m, 30H); 2.31-2.34 (m); 2.39-2.45 (m, 2H), 3.36-4.27 (m). .sup.13C NMR* (100 MHz, CDCl.sub.3): δ=13.8 (CH.sub.3); 22.0, 24.3, 25.4, 28.8, 29.0, 31.2, 68.8 (CH.sub.2); 69.7 (CH); 70.3 (CH.sub.2), 70.2, 70.8, 71.6 (CH). HRMS: m/z=1140.0106 [M+H].sup.+ (calculated for C.sub.68H.sub.135N.sub.2O.sub.10: 1140.0117).

    5.3. Synthesis of α-GalCer Analogue 23 (IPB002033

    [0335] ##STR00038##

    [0336] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and stearic isocyanide 6 (41 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1) afforded the protected Ugi product 22 (140 mg, 56%). R.sub.f=0.33 (n-hexane/EtOAc 10:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 23 (47 mg, 49%) as a colorless oil. [α].sub.D.sup.23.5=ND. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.80-0.89 (m, 9H, 3×CH.sub.3); 1.14-59 (m, 104H); 1.95-2.10 (m); 2.30-2.33 (m, 2H); 3.13-4.66 (m); .sup.13C NMR* (100 MHz, DMSO-d.sub.6): δ=13.8 (CH.sub.3); 21.9, 23.0, 25.2, 25.5, 28.6, 28.8, 31.2 39.6 (CH.sub.2); 48.5 (CH); 61.9 (CH.sub.2). HRMS: m/z=1168.0422 [M+H].sup.+ (calculated for C.sub.70H.sub.139N.sub.2O.sub.10: 1168.0430).

    5.4. Synthesis of α-GalCer Analogue 25 (IPB001902

    [0337] ##STR00039##

    [0338] 1 (70 mg, 0.07 mmol), paraformaldehyde (2.1 mg, 0.07 mmol), octanoic acid (11 μL, 0.07 mmol) and benzyl isocyanide (8 μL, 0.07 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 5:1) afforded the protected Ugi product 24 (36 mg, 40%). R.sub.f=0.31 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 25 (20 mg, 95%) as a colorless oil. [α].sub.D.sup.24.2=52.9 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.07-1.39 (m, 34H); 1.61 (p, 2H, J=6.6 Hz); 2.31 (t, 2H, J=7.5 Hz); 3.35-3.44 (m, 2H); 3.25 (s, 2H); 3.47-3.78 (m); 7.11-7.37 (m, 5H, Ar); 7.43 (t, 1H, J=7.6 Hz, NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 22.7, 22.8, 24.9, 29.0, 29.2, 29.5, 29.8, 31.8, 32.0, 34.1, 45.9 (CH.sub.2); 50.8 (CH); 63.7, 68.6, 70.6 (CH.sub.2), 75.1, 75.2, 77.4, 128.5, 128.7, 130.2 (CH); 178.7 (C═O). HRMS: m/z=775.5078 [M+Na].sup.+ (calculated for C.sub.41H.sub.72N.sub.2NaO.sub.10: 775.5085).

    5.5. Synthesis of α-GalCer Analogue 27 (IPB002042

    [0339] ##STR00040##

    [0340] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and benzyl isocyanide (18 μL, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section). Column chromatography (n-hexane/EtOAc 7:1) afforded the protected Ugi product 26 (85 mg, 37%). R.sub.f=0.35 (n-hexane/EtOAc 6:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 27 (53 mg, 96%) as a colorless oil. [α].sub.D.sup.24.2=20.0 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.82 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.10-1.30 (m, 64H); 1.32-1.59 (m, 10H); 2.32-2.39 (m, 2H); 3.24-3.28 (m, 2H); 3.34-3.37 (m, 2H); 3.37-3.40 (m, 1H); 3.40-4.60 (m); 7.18-7.31 (m, 5H, Ar); 7.95 (t, 1H, J=6.3 Hz, NH). .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ=13.9 (CH.sub.3); 22.0, 24.6, 24.8, 25.4, 28.7, 29.0, 29.1, 30.7, 31.3, 33.1 (CH.sub.2), 39.4 (CH); 42.0; 46.4, 60.4 (CH.sub.2), 68.2, 68.5, 69.7, 71.3, 98.9, 126.3, 126.9, 128.0 (CH); 162.8, 165.9 (C═O). HRMS: m/z=1005.8065 [M+H].sup.+ (calculated for C.sub.59H.sub.109N.sub.2O.sub.10: 1005.8082).

    5.6. Synthesis of α-GalCer Analogue 29 (IPB002037

    [0341] ##STR00041##

    [0342] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and 3-phenylpropyl isocyanide 7 (21 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 7:1) afforded the protected Ugi product 27 (110 mg, 46%). R.sub.f=0.34 (n-hexane/EtOAc 6:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 29 (70 mg, 97%) as a colorless oil. [α].sub.D.sup.24.2=34.6 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.84 (t, 6H, J=6.3 Hz, 2×CH.sub.3); 1.10-1.33 (m, 61H); 1.34-1.57 (m, 8H); 1.58-1.74 (m, 4H); 2.30-2.40 (m, 2H); 2.98-3.07 (m, 2H); 3.08-3.19 (m, 3H); 3.40-4.90 (m); 7.10-7.26 (m, 5H, Ar); 7.56 (t, 1H, J=6.6 Hz, NH). .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ=13.7 (CH.sub.3); 20.9, 22.2, 24.5, 24.9, 26.5, 27.3, 28.0, 29.0, 30.0, 31.3, 31.4, 32.3, 32.4, 33.1, 38.2 (CH.sub.2), 39.7 (CH); 46.4 (CH.sub.2), 57.1 (CH), 60.3, 64.3 (CH.sub.2), 68.6, 68.7, 69.7, 71.2, 75.7, 99.0, 125.4, 126.3, 128.1 (CH); 166.0 (C═O). HRMS: m/z=1033.8365 [M+H].sup.+ (calculated for C.sub.61H.sub.113N.sub.2O.sub.10: 1033.8395).

    5.7. Synthesis of α-GalCer Analogue 31 (IPB001903

    [0343] ##STR00042##

    [0344] 1 (50 mg, 0.05 mmol), paraformaldehyde (1.5 mg, 0.05 mmol), lauric acid (9.8 mg, 0.05 mmol) and cyclohexyl isocyanide (6.0 μL, 0.05 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 5:1) afforded the protected Ugi product 30 (36 mg, 54%), R.sub.f=0.34 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 31 (20 mg, 93%) as a colorless oil. [α].sub.D.sup.24.7=30.8 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.07-1.40 (m, 44H); 1.49-1.89 (m, 10H); 1.53 (s, 1H); 1.57 (s, 1H); 2.04 (s, 1H); 2.26 (s, 1H); 2.28-2.33 (m, 2H); 3.35-3.44 (m, 2H); 3.43-3.56 (m, 2H); 3.55-4.30 (m); 7.42 (t, 1H, J=7.5 Hz, NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 22.8, 25.0, 29.5, 29.9, 30.0, 32.1, 34.5, 68.6 (CH.sub.2), 70.2 (CH); 70.7 (CH.sub.2), 75.1, 75.3, 77.4, 110.0 (CH); 174.7, 175.8 (C═O).

    5.8. Synthesis of α-GalCer Analogue 33 (IPB002039

    [0345] ##STR00043##

    [0346] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and cyclohexyl isocyanide (18 μL, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 8:1) afforded the protected Ugi product 32 (108 mg, 48%), R.sub.f=0.24 (n-hexane/EtOAc 8:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 33 (68 mg, 97%) as a colorless oil. [α].sub.D.sup.24.7=36.1 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 1.11-1.38 (m, 70H), 1.46-2.03 (m, 15H); 2.13-2.48 (m, 5H); 3.44-4.96 (m). .sup.13C NMR* (100 MHz, CDCl.sub.3) δ=14.1 (CH.sub.3); 22.7, 24.7, 25.3, 25.6, 29.7, 31.9, 32.6, 33.6 (CH.sub.2), 48.8, 70.4 (CH). HRMS: m/z=997.8374 [M+H].sup.+ (calculated for C.sub.58H.sub.113N.sub.2O.sub.10: 997.8395).

    5.9. Synthesis of α-GalCer Analogue 35 (IPB001904

    [0347] ##STR00044##

    [0348] 1 (80 mg, 0.08 mmol), paraformaldehyde (2.4 mg, 0.08 mmol), L-Phe (Ac) (16 mg, 0.08 mmol) and myristic isocyanide 4 (18 mg, 0.08 mmol) in MeOH/THF reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 3:1) afforded the protected Ugi product 34 (34 mg, 30%), R.sub.f=0.27 (n-hexane/EtOAc 4:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 35 (21 mg, 98%) as a colorless oil. [α].sub.D.sup.23.2=25.1 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.10-1.34 (m, 50H); 1.38 (s, 1H); 2.05 (s, 1H); 2.17 (s, 1H); 2.24 (s, 1H); 3.35-3.44 (m, 2H); 3.46-3.52 (m, 2H); 3.55-3.76 (m); 7.10-7.25 (m, 5H, Ar); 7.44 (t, 1H, J=7.7 Hz, NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3, 17.3, 18.5 (CH.sub.3); 22.8, 27.3, 29.5, 29.6, 29.9, 30.0, 30.1, 32.1, 40.4, 58.6, 68.6, 70.7 (CH.sub.2); 75.1, 77.4, 110.1, 117.4, 121.1, 127.0, 128.6, 129.2 (CH); 136.9 (C). HRMS: m/z=944.6534 [M+Na].sup.+ (calculated for C.sub.51H.sub.91N.sub.3NaO.sub.11: 944.6551).

    5.10. Synthesis of α-GalCer Analogue 37 (IPB001905

    [0349] ##STR00045##

    [0350] 1 (57 mg, 0.056 mmol), paraformaldehyde (1.7 mg, 0.056 mmol), lauric acid (11 mg, 0.056 mmol) and isocyanide 10 (12 mg, 0.057 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (5). Column chromatography (n-hexane/EtOAc 5:1) afforded the protected Ugi product 36 (43 mg, 52%). R.sub.f=0.34 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 37 (25 mg, 94%) as a colorless oil. [α].sub.D.sup.23.6=16.4 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CD.sub.3OD): δ=0.91 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.12-1.18 (m, 14H); 1.25-1.40 (m, 31H); 2.00 (s, 2H); 3.37 (s, 1H); 3.38-3.73 (m); 7.46 (t, 1H, J=7.7 Hz, NH). .sup.13C NMR (100 MHz, CD.sub.3OD): δ=14.4, 17.5 (CH.sub.3); 23.7, 26.3, 29.6, 29.8, 29.9, 30.5, 30.8, 33.1, 40.4 (CH.sub.2); 59.2 (CH.sub.3); 62.0, 62.2, 64.3, 69.6, 71.1, 71.3, 71.5. (CH.sub.2); 73.0, 73.6 (CH); 76.1 (CH.sub.2); 78.0, 100.8 (CH); 172.8 (C═O). HRMS: m/z=931.6423 [M+Na].sup.+ (calculated for C.sub.47H.sub.92N.sub.2NaO.sub.14: 931.6446).

    5.11. Synthesis of α-GalCer Analogue 39 (IPB001906

    [0351] ##STR00046##

    [0352] 1 (62 mg, 0.061 mmol), paraformaldehyde (1.8 mg, 0.061 mmol), palmitic acid (16 mg, 0.061 mmol) and isocyanide 10 (13.2 mg, 0.061 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 1:2) afforded the protected Ugi product 38 (46.7 mg, 51%). R.sub.f=0.31 (n-hexane/EtOAc 1:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 39 (18 mg, 60%) as a colorless oil. [α].sub.D.sup.23.4=12.9 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 6H, J=7.0 Hz, 2×CH.sub.3); 1.08-1.61 (m, 54H); 1.96-2.41 (m); 3.03 (q, 2H; J=7.3 Hz); 3.34-4.36 (m); 7.45 (t, 1H; J=7.8 Hz, NH); 8.05-8.10 (m, 1H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3, 17.4 (CH.sub.3); 22.8, 29.5, 29.8, 29.9, 32.1, 42.3 (CH.sub.2); 59.1 (CH.sub.3); 68.6, 70.7, 70.9, 71.0, 71.1 (CH.sub.2); 75.1 (CH); 75.3 (CH.sub.2); 77.4, (CH); 169.3 (C═O). HRMS: m/z=987.7045 [M+Na].sup.+ (calculated for C.sub.51H.sub.100N.sub.2NaO.sub.14: 987.7072).

    5.12. Synthesis of α-GalCer Analogue 41 (IPB002036

    [0353] ##STR00047##

    [0354] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), lignoceric acid (54 mg, 0.15 mmol) and isocyanide 10 (31.9 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 2:1) afforded the protected Ugi product 40 (139 mg, 58%). R.sub.f=0.15 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 41 (91 mg, 98%) as a colorless oil. [α].sub.D.sup.23.4=28.5 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.84 (t, 6H, J=6.5 Hz, 2×CH.sub.3); 1.12-1.32 (m, 60H); 1.33-1.56 (m, 8H), 1.73-1.79 (m, 3H), 3.22-3.26 (m, 4H); 3.39-3.45 (m, 4H); 3.47-3.53 (m); 3.56-3.63 (m); 3.66-4.66 (m); 7.55 (t, 1H, J=5.8 Hz, NH); .sup.13C NMR* (100 MHz, DMSO-d.sub.6) δ=13.8, (CH.sub.3); 22.1, 23.3, 24.2, 24.6, 24.9, 26.5, 27.3, 28.0, 29.1, 30.1, 31.4, 32.4, 32.6, 33.0, 33.7, 38.4 (CH.sub.2); 46.3 (CH.sub.2); 58.0 (CH.sub.3); 60.3, 64.2, 64.3, 67.0 (CH.sub.2); 68.2, 68.6 (CH); 68.9, 69.7, 71.3 (CH.sub.2); 71.3, 73.7, 75.8, 99.0 (CH). HRMS: m/z=1077.8487 [M+H].sup.+ (calculated for C.sub.59H.sub.117N.sub.2O.sub.14: 1077.8505).

    5.13. Synthesis of α-GalCer Analogue 43 (IPB002034

    [0355] ##STR00048##

    [0356] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and isocyanide 10 (31.9 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 2:1) afforded the protected Ugi product 42 (126 mg, 52%). R.sub.f=0.20 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 43 (71.7 mg, 85%) as a colorless oil. [α].sub.D.sup.23.4=25.1 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.83 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 0.96-1.35 (m, 63H); 1.36-1.56 (m, 7H), 2.09-2.43 (m, 4H); 3.14-4.31 (m); 4.56-4.66 (m, 2H); 5.27-5.38 (m, 2H); 7.55 (bs, 1H, NH); .sup.13C NMR* (100 MHz, DMSO-d.sub.6) δ=13.7 (CH.sub.3); 22.0, 23.1, 24.5, 25.0, 27.3, 29.0, 30.1, 31.3, 32.6, 33.0, 34.8, 38.4, 46.2 (CH.sub.2); 57.0 (CH); 58.0 (CH.sub.3); 60.2, 60.7, 64.2, 65.6, 66.9, 68.2 (CH.sub.2); 68.2, 68.5 (CH); 68.8, 69.6, 71.2 (CH.sub.2); 71.2, 73.6, 75.7, 98.9 (CH). HRMS: m/z=1105.8961 [M+H].sup.+ (calculated for C.sub.61H.sub.121N.sub.2O.sub.14: 1105.8818).

    5.14. Synthesis of α-GalCer Analogue 45 (IPB002044

    [0357] ##STR00049##

    [0358] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and isocyanide 11 (38.4 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 1:1) afforded the protected Ugi product 44 (110 mg, 44%). R.sub.f=0.17 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 45 (52.9 mg, 71%) as a colorless oil. [α].sub.D.sup.23.4=25.8 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.83 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.12-1.34 (m, 58H); 1.35-1.66 (m, 14H), 2.10-2.18 (m, 2H); 2.39-2.46 (m, 2H); 3.18-4.29 (m); 4.56-4.66 (m, 2H), 7.55 (t, 1H, J=6.0 Hz, NH); .sup.13C NMR* (100 MHz, DMSO-d.sub.6) δ=13.8 (CH.sub.3); 22.0, 21.6, 23.2, 24.5, 25.0, 27.3, 29.0, 31.4, 32.4, 33.0, 35.7, 38.3, 40.1, 46.2, 57.0 (CH.sub.2); 57.0 (CH); 57.9 (CH.sub.2); 57.9 (CH.sub.3); 60.2, 60.7, 64.3, 65.6, 66.9 (CH.sub.2); 68.1 (CH); 68.2 (CH.sub.2); 68.6 (CH); 68.8, 69.7, 71.2 (CH.sub.2); 71.3, 73.5, 75.7, 99.1 (CH). HRMS: m/z=1149.9048 [M+H].sup.+ (calculated for C.sub.63H.sub.125N.sub.2O.sub.15: 1149.9080).

    5.15. Synthesis of α-GalCer Analogue 47 (IPB002035

    [0359] ##STR00050##

    [0360] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.15 mmol) and isocyanide 9 (22 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 6:1) afforded the protected Ugi product 46 (119 mg, 51%), R.sub.f=0.28 (n-hexane/EtOAc 6:1) that was further deprotected following the general procedure for the benzyl ether/azide protecting groups removal (section 2) to afford the α-GalCer analogue 47 (59 mg, 78%) as a colorless oil [α].sub.D.sup.23.4=28.1 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.83 (t, 6H, J=5.9 Hz, 2×CH.sub.3); 0.95-1.85 (m, 80H); 2.10-2.20 (m, 3H); 2.31-2.38 (m, 2H); 2.42 (t, 2H, J=8.1 Hz, CH.sub.2); 2.95-4.71 (m); 7.46-7.54 (m, 1H). .sup.13C NMR* (100 MHz, DMSO-d.sub.6): δ=13.7 (CH.sub.3); 21.9, 25.7, 29.0, 31.3, 34.0, 38.4, 46.2 (CH.sub.2), 57.0 (CH); 60.0, 63.1 (CH.sub.2); 68.0, 68.5, 71.2, 99.0 (CH). HRMS: m/z=1014.8637 [M+H].sup.+ (calculated for C.sub.58H.sub.116N.sub.3O.sub.10: 1014.8661).

    5.16. Synthesis of α-GalCer Analogues 49 and 50 (IPB001910 and IPB000964

    [0361] ##STR00051##

    [0362] 1 (800 mg, 0.785 mmol), paraformaldehyde (23.6 mg, 0.785 mmol), lignoceric acid (289 mg, 0.785 mmol) and isocyanide 14 (122 mg, 0.785 mmol) in MeOH/THF (9 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1-5:1) afforded the protected Ugi product 48 (428 mg, 42%). R.sub.f=0.47 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford α-GalCer analogue 49. In order to remove the tert-butyl group, the product was dissolved in a solution containing 25% TFA in DCM (3 mL) and the reaction mixture was stirred for 3 h. Then, the solvent was evaporated under reduced pressure and the remaining TFA co-evaporated with DCM to afford the final deprotected α-GalCer analogue 50 (167 mg, 63% over two steps) as a colorless oil. [Ca]°=28.8 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=6.6 Hz, 2×CH.sub.3); 1.02-1.70 (m); 2.04-2.79 (m); 2.93-4.53 (m). ESI-MS: m/z=959.8 [M+H].sup.+ (calculated for C.sub.53H.sub.103N.sub.2O.sub.12: 959.8); 997.7 [M+K].sup.+ (calculated for C.sub.53H.sub.102KN.sub.2O.sub.12: 997.7). HRMS: m/z=995.7487 [M(OMe)+Na].sup.+ (calculated for C.sub.54H.sub.104N.sub.2NaO.sub.12: 995.7466).

    5.17. Synthesis of α-GalCer Analogue 52 (IPB001909

    [0363] ##STR00052##

    [0364] 1 (100 mg, 0.098 mmol), paraformaldehyde (2.9 mg, 0.098 mmol), stearic acid (28 mg, 0.098 mmol) and isocyanide 14 (15 mg, 0.098 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 8:1-1:1) afforded the protected Ugi product 51 (51 mg, 35%). R.sub.f=0.73 (n-hexane/EtOAc 1:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2). to afford the final deprotected α-GalCer analogue 52 (32 mg, 99%) as a colorless oil. [α].sub.D.sup.24.0=17.9 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CD.sub.3OD): δ=0.89 (t, 6H, J=6.9 Hz, 2×CH.sub.3); 1.24-1.38 (m, 54H); 1.43-1.48 (m, 9H, 3×CH.sub.3 (tBu)); 2.23-2.30 (m, 2H); 2.43-2.50 (m, 2H); 3.22-4.38 (m). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 18.2, 22.8, 24.9 (CH.sub.2); 28.2 (CH.sub.3); 29.2, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 32.0, 34.0 (CH.sub.2); 53.5 (CH); 58.5 (CH.sub.2); 77.4 (CH); 81.6 (C); 163.9, 175.6, 178.2 (C═O); HRMS: m/z=953.8387 [M+Na].sup.+ (calculated for C.sub.51H.sub.98N.sub.2NaO.sub.12: 953.7017).

    5.18. Synthesis of α-GalCer Analogue 54 (IPB002043

    [0365] ##STR00053##

    [0366] 1 (225 mg, 0.22 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.098 mmol) and isocyanide 14 (23 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 6:1) afforded the protected Ugi product 53 (190 mg, 54%). R.sub.f=0.28 (n-hexane/EtOAc 6:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2). to afford the final deprotected α-GalCer analogue 54 (125 mg, 99%) as a colorless oil. [α].sub.D.sup.24.0=32.2 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.84 (t, 6H, J=7.2 Hz, 2×CH.sub.3); 1.04-1.35 (m, 67H); 1.36-1.42 (m, 9H, 3×CH.sub.3 (tBu)); 1.42-1.56 (m, 5H); 2.25-2.42 (m, 4H); 3.15-4.93 (m); 7.52 (t, 1H, J=6.0 Hz, NH). .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ=13.8 (CH.sub.3); 22.2, 24.6 (CH.sub.2); 27.7 (CH.sub.3); 28.9, 29.2, 31.4, 32.5, 34.6, 35.0 (CH.sub.2); 54.9 (CH); 60.3, 64.1 (CH.sub.2); 68.2, 68.6, 69.7, 71.3, 75.7 (CH); 79.7 (C); 98.9 (CH); 166.1, 169.9, 170.6 (C═O); HRMS: m/z=1043.8430 [M+H].sup.+ (calculated for C.sub.59H.sub.115N.sub.2O.sub.12: 1043.8450).

    5.19. Synthesis of α-GalCer Analogue 56 (IPB001911

    [0367] ##STR00054##

    [0368] 1 (400 mg, 0.392 mmol), paraformaldehyde (11.8 mg, 0.392 mmol), lignoceric acid (144.5 mg, 0.392 mmol) and isocyanide 13 (45.1 mg, 0.392 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1-5:1) afforded the protected Ugi product 55 (200 mg, 34%). R.sub.f=0.16 (n-hexane/EtOAc 4:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 1) to afford the α-GalCer analogue 56 (114 mg, 89%) as a colorless oil. [α].sub.D.sup.24.1=28.1 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=7.0 Hz, 2×CH.sub.3); 1.11-1.42 (m, 66H); 1.52-1.70 (m, 4H); 2.17 (s, 1H); 2.19-2.22 (m, 2H); 2.34 (t, 1H; J=7.6 Hz); 3.33-3.41 (2×s, 6H; 2×OCH.sub.3); 3.47-4.44 (m); 8.27 (s, 1H; NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.9, 29.5, 29.6, 29.8, 29.9, 30.0, 32.1 (CH.sub.2), 79.8 (CH). HRMS: m/z=997.7627 [M+Na].sup.+ (calculated for C.sub.54H.sub.106N.sub.2NaO.sub.12: 997.7643).

    5.20. Synthesis of α-GalCer Analogue 58 (IPB002038

    [0369] ##STR00055##

    [0370] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58.3 mg, 0.15 mmol) and isocyanide 13 (17 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 6:1) afforded the protected Ugi product 57 (125 mg, 55%). R.sub.f=0.25 (n-hexane/EtOAc 6:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 1) to afford the α-GalCer analogue 58 (81 mg, 99%) as a colorless oil. [α].sub.D.sup.24.1=32.6 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6/CDCl.sub.3 6.5:1 (v:v)) 6=0.85 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 1.15-1.31 (m, 69H), 1.34-1.59 (m, 7H), 2.10-2.16 (m, 1H); 2.31-2.33 (m, 1H); 2.35 (t, 1H; J=7.3 Hz); 3.15 (t, 2H, J=5.8 Hz); 3.22-3.27 (2×s, 6H; 2×OCH.sub.3); 3.38-4.79 (m); 8.24 (s, 1H; NH). .sup.13C NMR* (100 MHz, DMSO-d.sub.6/CDCl.sub.3 6.5:1 (v:v)) 6=12.8 (CH.sub.3); 21.8, 24.4, 24.6, 28.8, 31.0, 32.3, 32.4, 39.9, 40.3 (CH.sub.2), 52.9 (CH.sub.3); 56.7, 68.0, 68.5, 69.6, 70.9, 71.1, 98.8 (CH); 170.3 (C═O). HRMS: m/z=1003.8111 [M+H]+(calculated for C.sub.56H.sub.111N.sub.2O.sub.12: 1003.8137).

    5.21. Synthesis of α-GalCer Analogue 60 (IPB000970

    [0371] ##STR00056##

    [0372] 1 (400 mg, 0.392 mmol), paraformaldehyde (11.8 mg, 0.392 mmol), lignoceric acid (144.5 mg, 0.392 mmol) and isocyanide 15 (96.2 mg, 0.392 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1-5:1) afforded the protected Ugi product 59 (469 mg, 72%). R.sub.f=0.74 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether/4-phenyl-1,3-dioxolane protecting groups removal (section 2) to afford the α-GalCer analogue 60 (160 mg, 57%) as a colorless oil. [α].sub.D.sup.24.0=30.6 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.78-0.95 (m, 7H); 0.88 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 0.96-1.74 (m, 73H); 2.12-2.50 (m, 4H); 3.10-4.44 (m); 4.89 (s, 1H); 8.09 (s, 1H); 9.73 (s, 1H; CHO); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.8, 25.3, 26.4, 29.9, 32.1, 34.0, 43.8 (CH.sub.2); 69.0, 70.2, 110.2 (CH); 202.9 (CHO). ESI-MS: m/z=986.0 [M+H].sup.+ (calculated for C.sub.56H.sub.109N.sub.2O.sub.11: 985.8). HRMS: m/z=1053.8332 [M(dimethyl acetal)+Na]+(calculated for C.sub.58H.sub.114N.sub.2NaO.sub.12: 1053.8269).

    5.22. Synthesis of α-GalCer Analogue 62 (IPB002041

    [0373] ##STR00057##

    [0374] 1 (150 mg, 0.15 mmol), paraformaldehyde (4.4 mg, 0.15 mmol), cerotic acid (58 mg, 0.392 mmol) and isocyanide 16 (41 mg, 0.15 mmol) in MeOH/THF (6 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 10:1-5:1) afforded the protected Ugi product 61 (129 mg, 82%). R.sub.f=0.74 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether/4-phenyl-1,3-dioxolane protecting groups removal (section 2) to afford the α-GalCer analogue 62 (72 mg, 82%) as a colorless oil. [α].sub.D.sup.24.0=26.8 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=0.77-0.85 (m, 6H); 1.01-1.51 (m, 72H); 2.40-2.45 (m, 2H); 3.03 (s, 3H, CH.sub.3); 3.06 (s, 3H, CH.sub.3); 3.09-4.86 (m); 7.07-7.33 (m, 3H, Ar); 8.17-8.49 (m, 2H, Ar). HRMS: m/z=1181.8628 [M+H].sup.+ (calculated for C.sub.68H.sub.117N.sub.4O.sub.12: 1181.8668).

    5.23. Synthesis of α-GalCer Analogue 64 (IPB001912

    [0375] ##STR00058##

    [0376] 1 (100 mg, 0.098 mmol), paraformaldehyde (2.9 mg, 0.098 mmol), stearic acid (27.9 mg, 0.098 mmol) and isocyanide 12 (8.2 mg, 0.049 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 6:1) afforded the protected Ugi product 63 (15.1 mg, 5%). R.sub.f=0.44 (n-hexane/EtOAc 5:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 64 (9.0 mg, 98%) as a colorless oil. [α].sub.D.sup.24.8=26.8 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.77-0.94 (m, 14H); 0.88 (t, 12H, J=6.7 Hz, 4×CH.sub.3); 1.07-1.46 (m, 102H); 1.55-1.70 (m, 8H); 2.20-2.30 (m, 4H); 2.35 (t, 5H, J=7.5 Hz); 2.50 (t, 3H, J=8.2 Hz); 3.58-5.24 (m), 4.35 (t, J=7.0 Hz); 8.00-8.18 (m, 2H; 2×NH); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.3, 22.8, 24.8, 25.1, 28.0, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 32.1, 34.0, 51.6, 68.7 (CH.sub.2); 77.4, 110.0 (CH); 179.0 (C═O).

    5.24. Synthesis of α-GalCer Analogue 66 (IPB001915

    [0377] ##STR00059##

    [0378] 1 (100 mg, 0.098 mmol), paraformaldehyde (2.9 mg, 0.098 mmol), adipic acid (7.2 mg, 0.049 mmol) and isocyanide 8 (22.4 mg, 0.098 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 2:1-1:2) afforded the protected Ugi product 65 (59.1 mg, 45%). R.sub.f=0.21 (n-hexane/EtOAc 1:1) that was further deprotected following the general procedure for the benzyl ether/azide protecting groups removal (section 2) to afford the α-GalCer analogue 66 (18.3 mg, 54%) as a colorless oil. [α].sub.D.sup.24.7=14.3 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, DMSOd.sub.6): δ=0.85 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 1.04 (d, 2H, J=6.2 Hz); 1.05-1.29 (m, 48H); 1.36-1.59 (m, 11H); 2.07-2.22 (m, 4H); 2.36-2.45 (m, 2H); 2.89-3.00 (m, 4H); 3.17-4.68 (m); 8.26 (s, 2H, 2×NH). .sup.13C NMR (100 MHz, DMSOd.sub.6): δ=13.9 (CH.sub.3); 22.1, 28.7, 29.1, 29.2, 29.4, 31.3, 38.5, 45.5 (CH.sub.2); 57.1 (CH); 58.0, 60.6, 66.6 (CH.sub.2); 68.3, 68.9 (CH); 69.6, 69.7, 69.8, 60.9 (CH.sub.2), 71.3, 107.0 (CH). HRMS: m/z=767.5122 [M+2H].sup.2+ (calculated for C.sub.74H.sub.144N.sub.6O.sub.26/2: 767.5144).

    5.25. Synthesis of α-GalCer Analogue 68 (IPB001917

    [0379] ##STR00060##

    [0380] 1 (100 mg, 0.098 mmol), paraformaldehyde (2.9 mg, 0.098 mmol), adipic acid (7.2 mg, 0.049 mmol) and isocyanide 14 (15.2 mg, 0.098 mmol) in MeOH/THF (3 mL) reacted following the general procedure for the synthesis of α-GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 3:1-2:1) afforded the protected Ugi product 67 (23.1 mg, 9%). R.sub.f=0.77 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the benzyl ether protecting group removal (section 2) to afford the α-GalCer analogue 68 (13 mg, 99%) as a colorless oil. [α].sub.D.sup.24.6=36.7 (c 1.0 CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.87 (t, 6H, J=6.8 Hz, 2×CH.sub.3); 1.14-1.36 (m, 24×CH.sub.2; 6×CH.sub.3); 1.35-1.50 (m); 1.74-2.10 (m); 2.21-2.53 (m); 3.36-4.71 (m); 8.07 (s, 2H, NH). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.8, 24.0 (CH.sub.2); 28.2 (CH.sub.3); 29.3, 29.5, 29.8, 32.1 (CH.sub.2); 53.9 (CH); 67.6, 67.8, 67.9, 68.7 (CH.sub.2), 77.4 (CH); 82.6 (C); 106.5, 107.8 (CH); 171.0, 172.1 (C═O). HRMS: m/z=1439.9477 [M+H].sup.+ (calculated for C.sub.72H.sub.135N.sub.4O.sub.24: 1439.9466).

    5.26. Synthesis of GalCer Analogue 70 (IPB002611

    [0381] ##STR00061##

    [0382] 2 (120 mg, 0.100 mmol), paraformaldehyde (3.0 mg, 0.100 mmol), biotin (24.4 mg, 0.100 mmol) and isocyanide 6 (27.9 mg, 0.100 mmol) in MeOH/DCM (3 mL) reacted following the general procedure for the synthesis of GalCer analogues by Ugi-4CR (section 1). Column chromatography (DCM/MeOH 80:1-20:1) afforded the protected Ugi product 69 (54 mg, 31%). R.sub.f=0.35 (DCM/MeOH 20:1) that was further deprotected following the general procedure for the p-methoxybenzyl ether protecting group removal (section 3) to afford the GalCer analogue 70 (31 mg, 99%) as a light-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=6.7 Hz, 2×CH.sub.3); 0.94-2.00 (m); 3.32-5.00 (m). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.8, 27.2, 29.5, 29.8, 29.9, 32.1, 35.0, 37.2, 46.6 (CH.sub.2); 55.4, 55.6 (CH); 70.9 (CH.sub.2); 110.6 (CH). HRMS: m/z=1015.7391 [M+H].sup.+ (calculated for C.sub.54H.sub.103N.sub.4O.sub.11S: 1015.7344).

    5.27. Synthesis of GalCer Analogue 72 (IPB002613

    [0383] ##STR00062##

    [0384] 2 (120 mg, 0.100 mmol), paraformaldehyde (3.0 mg, 0.100 mmol), 4-(trifluoromethyl) benzoic acid (19.0 mg, 0.100 mmol) and isocyanide 5 (25.1 mg, 0.100 mmol) in MeOH/DCM (3 mL) reacted following the general procedure for the synthesis of GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 4:1-2:1) afforded the protected Ugi product 71 (20 mg, 12%). R.sub.f=0.25 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the p-methoxybenzyl ether protecting group removal (section 3) to afford the GalCer analogue 72 (11 mg, 99%) as a light-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (bs, 6H, 2×CH.sub.3); 1.02-1.48 (m); 3.46-5.20 (m), 7.00-7.60 (m, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2, 14.3 (CH.sub.3); 22.8, 27.2, 29.5, 29.8, 30.0, 32.0, 32.1, 37.2 (CH.sub.2); 55.4, 55.6, 113.7, 128.7, 128.8 (CH). HRMS: m/z=933.6357 [M+H].sup.+ (calculated for C.sub.50H.sub.88F.sub.3N.sub.2O.sub.10: 933.6391).

    5.28. Synthesis of GalCer Analogue 74 (IPB002614

    [0385] ##STR00063##

    [0386] 2 (120 mg, 0.100 mmol), paraformaldehyde (3.0 mg, 0.100 mmol), Ac-L-Lys(Z)—OH (32.2 mg, 0.100 mmol) and isocyanide 6 (27.9 mg, 0.100 mmol) in MeOH/DCM (3 mL) reacted following the the general procedure for the synthesis of GalCer analogues by Ugi-4CR (section 1). Column chromatography (DCM/MeOH 99:1) afforded the protected Ugi product 73 (20 mg, 11%). R.sub.f=0.30 (DCM/MeOH 20:1) that was further deprotected following the general procedure for the p-methoxybenzyl ether protecting group removal (section 3) to afford the GalCer analogue 74 (12 mg, 99%) as a light-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=6.4 Hz, 2×CH.sub.3); 0.94-1.65 (m); 2.93-5.52 (m), 7.15-7.61 (m, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2, 14.3 (CH.sub.3); 22.8, 29.5, 29.8, 30.1, 32.0, 32.1 (CH.sub.2). HRMS: m/z=1093.7944 [M+H].sup.+ (calculated for C.sub.60H.sub.109N.sub.4O.sub.13: 1093.7991).

    5.29. Synthesis of GalCer Analogue 76 (IPB002615

    [0387] ##STR00064##

    [0388] 2 (120 mg, 0.100 mmol), paraformaldehyde (3.0 mg, 0.100 mmol), linoleic acid (31.0 mL, 0.100 mmol) and isocyanide 10 (21.7 mg, 0.100 mmol) in MeOH/DCM (3 mL) reacted following the general procedure for the synthesis of GalCer analogues by Ugi-4CR (section 1). Column chromatography (DCM/EtOAc 5:1-1:2) afforded the protected Ugi product 75 (70 mg, 41%). R.sub.f=0.28 (DCM/EtOAc 1:1) that was further deprotected following the general procedure for the p-methoxybenzyl ether protecting group removal (section 3) to afford the GalCer analogue 76 (40 mg, 99%) as a light-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.85-0.90 (m, 6H, 2×CH.sub.3); 1.15-1.44 (m); 1.46-1.68 (m), 1.74-1.90 (m); 1.99-2.08 (m); 2.71-2.78 (m); 3.31-4.50 (m); 5.27-5.41 (m). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.2 (CH.sub.3); 22.7, 22.8, 25.7, 27.3, 29.4, 29.5, 29.8, 29.9, 31.6, 32.0, 37.9, 46.6 (CH.sub.2); 55.3, 55.6 (CH); 59.1 (CH.sub.3), 59.3, 64.9 (CH); 67.4, 69.7, 70.2, 70.4, 70.6, 70.7, 72.0 (CH.sub.2); 72.9, 82.7, 110.5, 113.8, 128.0, 128.2, 130.1, 130.3 (CH); 165.6, 173.4 (C═O). HRMS: m/z=989.7216 [M+H].sup.+ (calculated for C.sub.53H.sub.101N.sub.2O.sub.14: 989.7253).

    5.30. Synthesis of GalCer Analogue 78 (IPB002612

    [0389] ##STR00065##

    [0390] 2 (120 mg, 0.100 mmol), paraformaldehyde (3.0 mg, 0.100 mmol), lignoceric acid (36.9 mg, 0.100 mmol) and isocyanide 17 (45.6 mg, 0.100 mmol) in MeOH/DCM (3 mL) reacted following the general procedure for the synthesis of GalCer analogues by Ugi-4CR (section 1). Column chromatography (n-hexane/EtOAc 5:1-2:1) afforded the protected Ugi product 77 (30 mg, 15%). R.sub.f=0.30 (n-hexane/EtOAc 2:1) that was further deprotected following the general procedure for the p-methoxybenzyl ether protecting group removal (section 3) to afford the GalCer analogue 78 (19 mg, 99%) as a light-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=0.88 (t, 6H, J=7.2 Hz, 2×CH.sub.3); 1.05-1.60 (m); 3.34-5.33 (m); 6.71-7.14 (m, Ar); 7.19-7.54 (m, Ar). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=14.3 (CH.sub.3); 22.8 (CH.sub.2); 24.9 (CH.sub.3); 29.5, 29.9, 32.1, 33.2, 46.4 (CH.sub.2); 55.3, 55.5 (CH); 67.8, 70.1 (CH.sub.2); 114.3, 115.0, 127.6, 128.7, 130.2, 130.3 (CH); 135.3, 136.8 (C); 168.6, 176.5 (C═O). ESI-MS: m/z=1317.3 [M+H].sup.+ (calculated for C.sub.77H.sub.126N.sub.3O.sub.14: 1316.9). [0391] (1) Brouard, I.; Rivera, D. G. Multicomponent Synthesis of Ugi-Type Ceramide Analogues and Neoglycolipids from Lipidic Isocyanides. 2012. https://doi.org/10.1021/jo300462m. [0392] (2) Trappeniers, M.; Goormans, S.; Van Beneden, K.; Decruy, T.; Linclau, B.; Al-Shamkhani, A.; Elliott, T.; Ottensmeier, C.; Werner, J. M.; Elewaut, D.; et al. Synthesis and in Vitro Evaluation of α-GalCer Epimers. ChemMedChem 2008, 3, 1061-1070. https://doi.org/10.1002/cmdc.200800021. [0393] (3) Rotstein, B. H.; Mourtada, R.; Kelley, S. O.; Yudin, A. K. Solvatochromic Reagents for Multicomponent Reactions and Their Utility in the Development of Cell-Permeable Macrocyclic Peptide Vectors. 2011, No. Scheme 2, 12257-12261. https://doi.org/10.1002/chem.201102096.