Sphingoglycolipid analogues

Abstract

The invention relates to sphingoglycolipid analogues which are useful in treating or preventing diseases and conditions such as those relating to infection, atopic disorders, autoimmune diseases or cancer.

Claims

1. A compound of formula (I): ##STR00077## wherein Z is S, S—S, SO or SO.sub.2; D is selected from the group consisting of: hydrogen, halogen, hydroxyl, cyano, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aralkyl group, an optionally substituted aryl group, a radical of formula D1, a radical of formula D2 and a radical of formula D3; ##STR00078## wherein R.sup.32 is selected from the group consisting of: an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group and an optionally substituted aryl group; R.sup.33 is halogen; m is an integer from 2 to 10, and wherein in D2 denotes an optional double bond; provided that if D is halogen then Z is not S or S—S and provided that if D is cyano then Z is not S—S, SO or SO.sub.2 and provided that if D is hydroxyl then Z is not S, S—S or SO; R.sup.1 is H or glycosyl, provided that if R.sup.1 is glycosyl then R.sup.2 and R.sup.3 are both OH; R.sup.2 is selected from the group consisting of H, OH, F and OR.sup.10; provided that if R.sup.2 is H, F or OR.sup.10, then R.sup.1 is H, R.sup.3 is OH; R.sup.3 is selected from the group consisting of H, OH, F and OR.sup.10; provided that if R.sup.3 is H, F or OR.sup.10, then R.sup.1 is H, R.sup.2 is OH; R.sup.6 is OH or H; R.sup.7 is OH or H; wherein when R.sup.7 is H, R.sup.8 is C.sub.1-C.sub.15 alkyl and X is O, denotes an optional double bond linking the carbon adjacent to R.sup.7 with the carbon adjacent to R.sup.8; R.sup.8 is H or C.sub.1-C.sub.15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group; R.sup.10 is glycosyl; R.sup.12 is C.sub.6-C.sub.30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C.sub.1-C.sub.6 amide, nitro, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 acyloxy and C.sub.1-C.sub.6 thioalkyl; X is O, CH.sub.2 or S; wherein, when X is CH.sub.2 then the following must all be true: the stereochemistry of the 6-membered sugar ring in formula (I) is α-D-galacto; R.sup.1 is H; R.sup.2 and R.sup.3 are both OH; and: either R.sup.6 is OH and R.sup.7 is OH and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S); or R.sup.6 is OH and R.sup.7 is H, and R.sup.8 is C.sub.13H.sub.27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); or when X is S then the following must all be true: the stereochemistry of the 6-membered sugar ring in formula (I) is α-D-galacto; R.sup.1 is H; R.sup.2 and R.sup.3 are both OH; and: either R.sup.6 is OH and R.sup.7 is OH and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R); or R.sup.6 is OH and R.sup.7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2S, 3S); n is 1 when X is O or S; or n is 0 or 1 when X is CH.sub.2; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 which is a compound of formula (Ia) ##STR00079## wherein X, Z, D, R.sup.1, R.sup.2, R.sup.3, R.sup.6, R.sup.7, R.sup.8, R.sup.10, R.sup.12, R.sup.32 and n are all as defined in claim 1.

3. The compound of claim 1 wherein X is O.

4. The compound of claim 1 wherein n in formula (I) is 1, the stereochemistry of the 6-membered sugar ring of formula (I) is α-D-galacto, R.sup.6 is OH, R.sup.7 is OH and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R).

5. The compound of claim 1 wherein n in formula (I) is 0, the stereochemistry of the 6-membered sugar ring of formula (I) is α-D-galacto, R.sup.6 is OH, R.sup.7 is OH and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R).

6. The compound of claim 1 wherein X is O, R.sup.6 is OH, R.sup.7 is H, R.sup.8 is C.sub.1-C.sub.15 alkyl and is a double bond linking the carbon adjacent to R.sup.7 with the carbon adjacent to R.sup.8 and the stereochemistry at the carbon atoms 2, 3 is (2S, 3S).

7. The compound of claim 1 wherein D is an alkyl group.

8. The compound of claim 1 wherein D is hydrogen.

9. The compound of claim 1 which is a compound of formula (I.2), (I.3) or (I.4) ##STR00080## wherein X, Z, R.sup.1, R.sup.2, R.sup.3, R.sup.6, R.sup.7, R.sup.8, R.sup.10, R.sup.12, R.sup.32, R.sup.33 and n are all as defined in claim 1.

10. The compound of claim 1 wherein R.sup.12 is a C.sub.26-acyl group.

11. A compound of formula (IV) ##STR00081## wherein X, Z, R.sup.1, R.sup.2, R.sup.3, R.sup.6, R.sup.7, R.sup.8, R.sup.10, R.sup.12, R.sup.32, R.sup.33 and n are all as defined in claim 1; and wherein denotes an optional double bond; or a pharmaceutically acceptable salt thereof.

12. A compound of formula (V) ##STR00082## wherein X, Z, R.sup.1, R.sup.2, R.sup.3, R.sup.6, R.sup.7, R.sup.8, R.sup.10, R.sup.12 and n are all as defined in claim 1; or a pharmaceutically acceptable salt thereof.

13. The compound of claim 11 wherein R.sup.12 is a C.sub.26 acyl group.

14. The compound of claim 12 wherein R.sup.12 is a C.sub.26-acyl group.

15. The compound of claim 1, selected from the group consisting of: ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##

16. The compound of claim 1 wherein R.sup.8 is C.sub.1-C.sub.15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group.

17. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of claim 1, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17 which is a vaccine optionally comprising an antigen.

19. A method of modifying an immune response in a patient, comprising administering to the patient either (i) the compound of claim 1, or (ii) the compound of claim 1 and an antigen.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows CD86 expression on dendritic cells. The data show that injection of compounds of the invention induces activation of iNKT cells and subsequent maturation of dendritic cells, as indicated by up-regulation of expression of the activation marker CD86. Groups of C57BL/6 mice (n=3) are injected intravenously with 0.23 nmol of the indicated compounds and then the spleens removed 20 h later for the analysis of CD86 expression on CD11c.sup.+dendritic cells by antibody labelling and flow cytometry. Mean fluorescence index (MFI)±SEM are presented. *P<0.05, *P<0.01, ***P<0.001

(2) FIG. 2 shows CD86 expression on dendritic cells. The data show that injection of compounds of the invention induces activation of iNKT cells and subsequent maturation of dendritic cells, as indicated by up-regulation of expression of the activation marker CD86. Groups of C57BL/6 mice (n=3) are injected intravenously with 0.23 nmol of the indicated compounds and then the spleens removed 20 h later for the analysis of CD86 expression on CD11c.sup.+ dendritic cells by antibody labelling and flow cytometry. Mean fluorescence index (MFI)±SEM are presented. *P<0.05, *P<0.01, ***P<0.001

(3) FIG. 3 shows the ratio of cytokines IFN-γ/IL-4 as measured in blood at 18 h (IFN-γ) and 3 h (IL-4) respectively. The data show that injection of some compounds of the invention induces higher ratios of IFN-γ/IL-4 as compared to α-GalCer. Groups of C57BL/6 mice (n=3) are injected intravenously with 0.23 nmol of the indicated compounds.

(4) FIG. 4 shows progression of B16.OVA melanoma over 37 days after challenge. Groups of C57BL/6 mice (n=5) are injected with 1×10.sup.5 live B16.OVA tumour cells subcutaneously on day 0. On day 6 0.23 nmol of the indicated compounds (α-GalCer or CN161) mixed with OVA protein (200 μg) are administered intraveneously. The tumour size is monitored regularly with calipers until the first animal in each group reached 200 mm.sup.2, at which point the whole group is culled.

(5) FIG. 5 shows the cytotoxic capacity of T cells with specificity for the H-2K.sup.b-restricted peptide SVYDFFVWL from the tumour-associated antigen TRP-2 following intravenous administration of vaccines comprising compounds of the invention (0.23 nmol) and the long peptide sequence SVYDFFVWLKFFHRTCKCTGNFA from the TRP-2 protein (0.57 nmol). Control animals are injected with the long peptide alone, or α-GalCer alone. Flow cytometry is used to assess the killing of target cells comprised of syngeneic splenocytes loaded ex vivo with 5 μM SVYDFFVWL injected intravenously 7 days after vaccination. To discriminate the targets from host tissue, the injected cells are labelled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE). A cohort of syngeneic splenocytes (without peptide) labelled with the fluorescent dye cell tracker orange are also injected to serve as controls. Killing is defined as the percentage of peptide-loaded targets killed relative to control cells. Each treatment group contained 5 animals with mean percentage of killing per group±SEM shown. The data show that injection of CN161 with the long TRP2 peptide induces a greater cytotoxic response against SVYDFFVWL-loaded targets than co-injection with α-GalCer or injection of peptide alone.

(6) FIG. 6 shows the antitumour activity against B16.OVA melanoma following intravenous administration of vaccines comprising compounds of the invention (0.23 nmol) and the long peptide sequence SVYDFFVWLKFFHRTCKCTGNFA from the TRP2 protein (0.57 nmol). Control animals are injected with the long peptide alone, α-GalCer alone, or left untreated. The mean tumour sizes per group (n=5)±SEM are shown. These data show that vaccination with CN161 and long TRP2 peptide results in anti-tumour activity.

(7) FIG. 7 shows the antitumour activity against B16.OVA melanoma following intravenous administration of vaccines comprising compounds of the invention and the long peptide sequence KISQAVHAAHAEINEAGRESIINFEKLTEWT from chicken ovalbumin (OVA) protein, a “model” tumour antigen encoded by the melanoma cells. These data show that vaccination with CN161 and long OVA peptide results in equivalent anti-tumour activity compared to injection of the peptide with α-GalCer.

(8) FIG. 8 shows the anti-leukemia activity of intravenously administered cell-based vaccines used to prevent challenge with C1498 acute leukemia cells. The prophylactic vaccines consist of irradiated C1498 cells that are incubated with compounds of the invention for 24 h. Control animals are injected with irradiated C1498 cells alone, compound alone, or left untreated. The time to onset of leukemia-associated symptoms are shown. (A) Analysis of vaccines incorporating CN161. (B) Analysis of vaccines incorporating α-GalCer. These data show that cell-based vaccines incorporating CN161 or α-GalCer provide significant protection against leukemia development. Also, CN161 alone, but not α-GalCer alone, can provide protection in this model.

(9) FIG. 9 shows the effect of compounds of the invention on proliferation of human NKT cells. Peripheral blood mononuclear cells from one donor are cultured for 7 days with different doses of the indicated compounds in the presence of IL-2, and then the percentages of NKT cells in the final cultures determined by flow cytometry with fluorescent α-GalCer-loaded CD1d tetramers and anti-CD3. Data are expressed as percentage of NKT cells (α-GalCer/CD1d tetramer and anti-CD3-binding cells) of total T cells (all anti-CD3-binding cells).

ABBREVIATIONS

(10) NKT cells Natural killer T-cells iNKT cells Invariant natural killer T-cells CD1 Cluster of differentiation 1 DC Dendritic cells MHC Major histocompatibility complex PBMC Peripheral blood mononuclear cell TLR2 Toll-like receptor 2 MUC-1 Mucin 1, cell surface associated Th1 T helper cells, type 1 Th2 T helper cells, type 2 MFI Mean fluorescence index SEM Structural equation modelling IFN-γ Interferon-gamma IL-4 Interleukin 4 TMS Trimethylsilyl TBAI Tetrabutylammonium iodide DIPEA Diisopropylethylamine NMR Nuclear magnetic resonance spectrometry HRMS High resolution mass spectrometry ESI Electrospray ionisation Q-Tof Quadrupole time-of-flight mass spectrometer RT Room temperature TLC Thin layer chromatography THF Tetrahydrofuran m-CPBA meta-chloroperoxybenzoic acid DAST Diethylaminosulfur trifluoride DCM Dichloromethane DMF Dimethylformamide DMSO Dimethylsulfoxide DMAP Dimethylaminopyridine DME Dimethoxyethane DTT Dithiothreitol TCEP-HCl Tris(2-carboxyethyl)phosphine hydrochloride NHS N-hydroxy succinimide NCS N-chloro succinimide OTs O-Tosyl (O-p-toluenesulfonyl) OMs O-Mesyl (O-methane sulfonyl) OTf O-Triflate (O-trifluoromethanesulfonyl) EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide IgE Immunoglobulin E FACS Fluorescence-activated cell sorting EDTA Ethylenediaminetetraacetic acid Ab Antibody RBC Red blood cell cDNA Complementary deoxyribonucleic acid PBS Phosphate-buffered saline FCS Fetal calf serum OVA Chicken ovalbumin

EXAMPLES

(11) The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples. The examples described herein are for the purposes of illustrating embodiments of the invention. Other embodiments, methods, and types of analyses are within the capabilities of persons of ordinary skill in the art and need not be described in detail herein. Other embodiments within the scope of the art are considered to be part of this invention.

(12) Anhydrous solvents are obtained commercially. Air sensitive reactions are carried out under Ar. Thin layer chromatography (TLC) is performed on aluminium sheets coated with 60 F.sub.254 silica. Flash column chromatography is performed on Merck or SiliCycle silica gel (40-63 μm) or SiliCycle reversed phase (C18) silica gel (40-63 μm). NMR spectra are recorded on a Bruker 500 MHz spectrometer. .sup.1H NMR spectra are referenced to tetramethylsilane at 0 Ppm (internal standard) or to residual solvent peak (CHCl.sub.3 7.26 ppm, CHD.sub.2OD 3.31 ppm). .sup.13C NMR spectra are referenced to tetramethylsilane at 0 ppm (internal standard) or to the deuterated solvent peak (CDCl.sub.3 77.0 ppm, CD.sub.3OD 49.0 ppm). CDCl.sub.3-CD.sub.3OD solvent mixtures are always referenced to the methanol peak. High resolution electrospray ionization mass spectra are recorded on a Q-Tof Premier mass spectrometer.

Example 1

Synthesis of (2S,3S,4R)-1-(6-Deoxy-64-butylthio-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN153)

(13) ##STR00048##

Example 1.1

(2S,3S,4R)-1-(2,3-Di-O-benzyl-6-O-(4-toluenesulfonyl)-α-D-galactopyranosyloxy)-3,4-di(benzyloxy)-2-hexacosanoylamino-octadeca-6-ene

(14) ##STR00049##

(15) Tosyl chloride (0.400 g, 2.10 mmol) is added to diol 1 (0.180 g, 0.154 mmol) (which is prepared as described in Lee, A., K. J. Farrand, et al. (2006) “Novel synthesis of alpha-galactosyl-ceramides and confirmation of their powerful NKT cell agonist activity.” Carbohydr Res 341(17): 2785-2798.) stirring in pyridine (8 mL) at RT. After 5 h the reaction mixture is diluted with CH.sub.2Cl.sub.2 (50 mL) and MeOH (10 mL) and stirred at RT for 18 h. The solvent is removed in vacuo. Purification of the resulting residue by silica gel chromatography (100% toluene changing to 20% EtOAc/toluene) gave the mono-tosylated material 2 (0.470 g, 0.343 mmol, 93%) as a white foam [α].sub.D.sup.20=+16.4 (c 0.005, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.88 (t, J=6.8 Hz, 6H), 1.22-1.32 (m, 62H), 1.47-1.51 (m, 2H), 1.86-1.95 (m, 2H), 2.02-2.06 (m, 2H), 2.40 (s, 3H), 2.43-2.53 (m, 2H), 3.60-3.63 (m, 1H), 3.74-3.77 (m, 4H), 3.80 (dd, J=9.7, 3.2 Hz, 1H), 3.93-3.94 (m, 1H), 3.97-3.99 (m, 1H), 4.09-4.17 (m, 2H), 4.33-4.38 (m, 1H), 4.51-4.54 (m, 2H), 4.58 (d, J=11.7 Hz, 1H), 4.60 (d, J=11.6 Hz, 1H), 4.66 (d, J=11.6 Hz, 1H), 4.70 (d, J=11.7 Hz, 2H), 4.74 (d, J=11.5 Hz, 1H), 4.77 (d, J=3.4 Hz, 1H), 5.43-5.52 (m, 2H), 5.68-5.72 (m, 1H), 7.22-7.32 (m, 22H), 7.74 (d, J=8.3 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1, 22.7, 25.6, 27.6, 27.8, 29.3, 29.4, 29.6, 29.7, 31.9), 36.7, 49.8, 67.1, 67.9, 68.5, 68.9, 71.5, 72.7, 73.3, 75.7, 77.0, 79.29, 79.31, 98.5, 125.4, 127.5, 127.6, 127.68, 127.72, 127.76, 127.8, 128.0, 128.3, 128.4, 128.5, 129.8, 132.1, 137.8, 138.2, 138.5, 138.6, 144.8, 172.8; HRMS (ESI): m/z calcd for C.sub.85H.sub.127NO.sub.11SNa [M+Na].sup.+ 1392.9028, found 1392.9031.

Example 1.2

(2S,3S,4R)-1-(2,3-Di-O-benzyl-6-deoxy-6-t-butylthio-α-D-galactopyranosyloxy)-3,4-di(benzyloxy)-2-hexacosanoylamino-octadeca-6-ene

(16) ##STR00050##

(17) 2-Methyl-2-propanethiol (0.016 ml, 0.146 mmol) and NaH (5.0 mg, 0.125 mmol) are added to tosylate 2 (0.100 g, 0.073 mmol) stirring in anhydrous THF (2 mL) at RT. After 18 h, the reaction is warmed to 30° C. and additional 2-Methyl-2-propanethiol (0.016 ml, 0.146 mmol) and NaH (5.0 mg, 0.208 mmol) are added. After a further 18 h the reaction is quenched by the addition of H.sub.2O and stirred for 15 mins. The layers are then separated, and the organic phase is washed with H.sub.2O, brine, dried (MgSO.sub.4) and concentrated in vacuo. Purification by silica gel chromatography (10% EtOAc/petroleum ether changing to 18% EtOAc/petroleum ether) gives product 3 (61 mg, 0.047 mmol, 65%). [α].sub.D.sup.20=+24.0 (c 0.007, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.86-0.87 (m, 6H), 1.21-1.33 (m, 62H), 1.30 (s, 9H), 1.48-1.51 (m, 2H), 1.90-1.99 (m, 2H), 2.01-2.05 (m, 2H), 2.41-2.51 (m, 2H), 2.81 (d, J=7.0 Hz, 2H), 3.58-3.61 (m, 1H), 3.79-3.85 (m, 6H), 4.08 (br s, 1H), 4.29-4.34 (m, 1H), 4.52-4.62 (m, 4H), 4.70-4.77 (m, 4H), 4.817-4.822 (m, 1H), 5.42-5.51 (m, 2H), 5.85-5.89 (m, 1H), 7.23-7.35 (m, 20H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1, 22.7, 25.7, 27.6, 27.9, 28.7, 29.36, 29.44, 29.7, 30.9, 31.9, 36.8, 42.3, 50.0, 68.1, 68.4, 69.8, 71.8, 72.5, 73.3, 73.5, 75.8, 77.7, 79.3, 79.7, 98.7, 125.7, 127.5, 127.6, 127.8, 127.9, 128.31, 128.36, 128.4, 128.5, 132.0, 138.0, 138.3, 138.64, 138.67, 172.7; HRMS (ESI): m/z calcd for C.sub.82H.sub.129NO.sub.8SNa [M+Na].sup.+ 1310.9337, found 1310.9340.

Example 1.3

(2S,3S,4R)-1-(6-Deoxy-6-t-butylthio-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN153)

(18) ##STR00051##

(19) Protected 6-S.sup.tBu 3 (0.060 g, 0.047 mmol) is dissolved in MeOH (5 mL) and anhydrous THF (3 mL) before the addition of formic acid (0.3 mL) and Pd(OH).sub.2/C (10% Pd; 104 mg). The reaction vessel is evacuated, flushed with hydrogen and stirred at 30° C. for 5 h followed by 20° C. for 68 h. Once cooled, the product mixture is filtered through celite, washing repeatedly with CHCl.sub.3:MeOH (3:1), and is then concentrated. The crude material is then purified by silica gel chromatography (100% CH.sub.2Cl.sub.2 changing to 5% MeOH/CH.sub.2Cl.sub.2) to isolate the target material CN153 as the major fraction alongside some partially hydrogenated material. This partially hydrogenated material is resubmitted to the same hydrogenation conditions as detailed above and purified by silica gel chromatography (100% CH.sub.2Cl.sub.2 changing to 5% MeOH/CH.sub.2Cl.sub.2) to give the product CN153 as a white solid, which is combined with that obtained earlier (30 mg, 0.032 mmol, 68%). [α].sub.D.sup.20=+44.7 [c 0.003, CHCl.sub.3:MeOH (3:1)]; .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=7.0 Hz, 6H), 1.24-1.41 (m, 68H), 1.33 (s, 9H), 1.52-1.68 (m, 4H), 2.19-2.23 (m, 2H), 2.79 (d, J=7.0 Hz, 2H), 3.35-3.37 (m, 1H), 3.54-3.59 (m, 2H), 3.70-3.74 (m, 2H), 3.78 (dd, J=10.0, 3.8 Hz, 1H), 3.83 (dd, J=7.4, 7.1 Hz, 1H), 3.90 (dd, J=10.6, 4.3 Hz, 1H), 3.94 (d, J=2.7 Hz, 1H), 4.18-4.21 (m, 1H), 4.87 (d, J=3.8 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.1, 22.9, 26.1, 28.8, 29.55, 29.60, 29.64, 29.8, 29.9, 30.0, 31.0, 32.1, 32.7, 36.8, 49.9, 50.3, 68.1, 69.0, 70.2, 70.7, 70.9, 72.4, 74.9, 100.0, 174.4; HRMS (ESI): m/z calcd for C.sub.54H.sub.107NO.sub.8SNa [M+Na].sup.+ 952.7615, found 952.7623.

Example 2

(2S,3S,4R)-1-(6-Deoxy-6-mercapto-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN237)

(20) ##STR00052##

Example 2.1

(2S,3S,4R)-2-Hexacosanoylamino-1-(6-O-(4-toluenesulfonyl)-α-D-galactopyranosyloxy)-3,4-octadecandiol

(21) ##STR00053##

(22) Pd(OH).sub.2/C (20% Pd; ˜5 mg) is added to protected tosylate 2 (0.040 g, 0.029 mmol) stirring in anhydrous CH.sub.2Cl.sub.2:MeOH (4 mL; 1:1). The reaction vessel is evacuated and flushed with hydrogen and stirred at RT for 24 h. The product mixture is filtered through celite, washed repeatedly with CHCl.sub.3:MeOH (3:1) and then concentrated. Purification by silica gel chromatography (100% CHCl.sub.3 changing to 10% MeOH/CHCl.sub.3) gives the target 4 (23 mg, 0.023 mmol, 79%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=6.9 Hz, 6H), 1.23-1.42 (m, 68H), 1.52-1.68 (m, 4H), 2.16-2.26 (m, 2H), 2.46 (s, 3H), 3.35-3.36 (m, 1H), 3.52-3.58 (m, 2H), 3.64 (dd, J=10.7, 4.0 Hz, 1H), 3.70-3.76 (m, 2H), 3.83-3.87 (m, 2H), 4.03-4.06 (m, 1H), 4.13-4.23 (m, 2H), 4.85 (d, J=3.4 Hz, 1H), 7.58 (d, J=8.1 Hz, 2H), 7.79 (d, J=8.1 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.2, 21.7, 22.9, 26.1, 29.59, 29.63, 29.7, 29.8, 29.92, 29.95, 30.04, 32.2, 32.8, 36.8, 50.4, 68.1, 68.9, 69.2, 69.6, 70.1, 72.4, 74.9, 77.8, 99.9, 128.2, 130.2, 132.8, 145.5, 174.6; HRMS (ESI): m/z calcd for C.sub.57H.sub.105NO.sub.11SNa [M+Na].sup.+ 1034.7306, found 1034.7317.

Example 2.2

(2S,3S,4R)-2-Hexacosanoylamino-1-(2,3,4-tri-O-acetyl-6-O-(4-toluenesulfonyl)-α-D-galactopyranosyloxy)-3,4-di(acetyloxy)octadecane

(23) ##STR00054##

(24) Tosylate 4 (10 mg, 9.9 μmol) is dissolved in pyridine (0.10 mL, 1.2 mmol) and cooled to 0° C. Acetic anhydride (0.10 mL, 1.0 mmol) and 4-(dimethylamino)pyridine (1.0 mg, 8.1 μmol) are then added and stirred at RT for 5 h. The product mixture is diluted with CH.sub.2Cl.sub.2, and is washed with 1M HCl, saturated NaHCO.sub.3, brine, dried (MgSO.sub.4) and the solvent removed in vacuo. Purification by silica gel chromatography (20% EtOAc/petroleum ether changing to 30% EtOAc/petroleum ether) affords the acetylated compound 5 (10 mg, 8.2 μmol, 83%) as a colourless oil. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.88 (t, J=6.9 Hz, 6H), 1.22-1.33 (m, 68H), 1.62-1.75 (m, 4H), 1.97 (s, 3H), 1.99 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.23-2.29 (m, 2H), 2.45 (s, 3H), 3.37 (dd, J=10.8, 2.7 Hz, 1H), 3.62 (dd, J=10.8, 2.9 Hz, 1H), 3.98 (dd, J=10.3, J=5.9 Hz, 1H), 4.04 (dd, J=10.2, J=6.7 Hz, 1H), 4.16 (t, J=6.9 Hz, 1H), 4.36 (tt, J=9.7, 2.7 Hz, 1H), 4.87-4.90 (m, 2H), 5.10 (dd, J=10.9, 3.6 Hz, 1H), 5.23 (dd, J=9.8, 2.5 Hz, 1H), 5.29 (dd, J=10.9, 3.4 Hz, 1H), 5.41 (br d, J=2.8 Hz, 1H), 6.24 (d, J=9.7 Hz, 1H), 7.34 (d, J=8.2 Hz, 2H), 7.75 (d, J=8.2 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1, 20.4, 20.5, 20.6, 20.7, 20.9, 21.6, 22.6, 25.6, 25.7, 27.4, 29.27, 29.33, 29.4, 29.5, 29.6, 31.9, 36.7, 47.8, 66.6, 66.7, 67.2, 67.4, 67.6, 67.7, 70.9, 73.4, 97.2, 128.0, 129.9, 132.5, 145.1, 169.8, 169.88, 169.94, 170.5, 171.0, 172.8; HRMS (ESI): m/z calcd for C.sub.67H.sub.115NO.sub.16SNa [M+Na].sup.+ 1244.7834, found 1244.7844.

Example 2.3

(2S,3S,4R)-2-Hexacosanoylamino-1-(2,3,4-tri-O-acetyl-6-deoxy-6-acetylthio-α-D-galactopyranosyloxy)-3,4-di(acetyloxy)octadecane

(25) ##STR00055##

(26) Potassium thioacetate (0.010 g, 8.2 μmol) is added to a solution of acetylated tosylate 5 (0.010 g, 8.2 μmol) stirring in anhydrous DMF (0.3 mL, 4 mmol) under Ar. The reaction mixture is heated to 80° C. and potassium thioacetate is added in aliquots until the reaction is complete by TLC (30% EtOAc/petroleum ether). Once cool, Et.sub.2O and H.sub.2O are added, the layers are separated and the aqueous layer is extracted with Et.sub.2O several times. The combined organics are washed with H.sub.2O, brine, dried (MgSO.sub.4) and then concentrated. Purification of the crude residue by silica gel chromatography (20% EtOAc/petroleum ether changing to 25% EtOAc/petroleum ether) affords thioacetate 6 (5 mg, 4.4 μmol, 54%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.88 (t, J=6.9 Hz, 6H), 1.21-1.35 (m, 68H), 1.55-1.66 (m, 4H), 1.986 (s, 3H), 1.989 (s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.15 (s, 3H), 2.24-2.28 (m, 2H), 2.32 (s, 3H), 2.85 (dd, J=13.9, 8.0 Hz, 1H), 3.08 (dd, J=13.8, 5.9 Hz, 1H), 3.38 (dd, J=10.7, 2.3 Hz, 1H), 3.67 (dd, J=10.6, 2.8 Hz, 1H), 3.89 (t, J=7.0 Hz, 1H), 4.36 (tt, J=10.0, 2.4 Hz, 1H), 4.86-4.89 (m, 2H), 5.10 (dd, J=10.9, 3.7 Hz, 1H), 5.26 (dd, J=10.2, 2.3 Hz, 1H), 5.30 (dd, J=10.9, 3.3 Hz, 1H), 5.46 (br d, J=2.6 Hz, 1H), 6.30 (d, J=9.8 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1, 20.5, 20.6, 20.7, 20.9, 21.6, 22.6, 25.6, 25.7, 27.2, 28.8, 29.28, 29.34, 29.4, 29.7, 29.6, 30.4, 31.9, 36.7, 47.7, 67.2, 67.6, 67.7, 67.9, 69.0, 70.5, 73.5, 97.0, 169.6, 170.1, 170.2, 170.7, 171.1, 172.9, 194.5; HRMS (ESI): m/z calcd for C.sub.62H.sub.111NO.sub.14SNa [M+Na].sup.+ 1148.7623, found 1148.7627.

Example 2.4

6,6′-Disulfanediylbis[6-deoxy-1-((2S,3S,4R)-3,4-dihydroxy-2-(hexacosanoylamino)octadecyl)-α-D-galactopyranose]

(27) ##STR00056##

(28) Sodium methoxide (30% in MeOH; 5.0 μL, 0.027 mmol) is added to compound 6 (4.00 mg, 3.55 μmol) stirring in anhydrous CH.sub.2Cl.sub.2:MeOH (0.5 mL; 1:1) under Ar at RT for 24 h. TLC analysis (12% MeOH/CHCl.sub.3) during this time showed the presence of the target material, although this is not always reproducible. The product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 20% MeOH/CHCl.sub.3) to yield target thiol CN161 as the disulfide (2 mg, 2.3 μmol, 64%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.89 (t, J=7.0 Hz, 6H), 1.23-1.41 (m, 68H), 1.51-1.69 (m, 4H), 2.20-2.23 (m, 2H), 3.00 (d, J=6.7 Hz, 2H), 3.54-3.59 (m, 2H), 3.65-3.68 (m, 1H), 3.74-3.79 (m, 3H), 3.90 (br d, J=2.5 Hz, 1H), 3.93 (dd, J=10.7, 4.7 Hz, 1H), 3.97 (dd, J=6.9, 6.8 Hz, 1H), 4.20-4.23 (m, 1H), 4.89 (d, J=3.4 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) 14.2, 22.9, 26.1, 26.2, 29.6, 29.67, 29.72, 29.9, 30.0, 32.2, 32.8, 36.8, 40.22, 50.5, 67.9, 69.0, 69.7, 70.4, 70.6, 72.4, 75.0, 99.9, 174.6; HRMS (ESI): m/z calcd for C.sub.50H.sub.99NO.sub.8SNa [M+Na].sup.+ 896.6989, found 896.7007. MS on CN161 is obtained in the presence of reducing agent DTT.

Example 2.5

(2S,3S,4R)-1-(6-Deoxy-6-mercapto-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN237)

(29) ##STR00057##

(30) Sodium methoxide (0.5 M in MeOH; 500 μL, 0.25 mmol) is added to compound 6 (130 mg, 0.115 mmol) stirring in anhydrous CH.sub.3Cl:MeOH (2:1, 6 mL) under Ar at RT for 1 h. Formic acid (100 μL) is added, the product mixture concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 20% MeOH/CHCl.sub.3) to yield target thiol CN237 (74 mg, 0.083 mmol, 72%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=7.1 Hz, 6H), 1.23-1.33 (m, 68H), 1.50-1.69 (m, 4H), 2.21 (td, J=8.0, 2.3 Hz, 2H), 2.66 (dd, J=13.7, 6.5 Hz, 1H), 2.79 (dd, J=13.7, 7.6 Hz, 1H), 3.52-3.59 (m, 4H), 3.68-3.79 (m, 5H), 3.94 (dd, J=10.5, 4.5 Hz, 1H), 3.99-4.01 (m, 1H), 4.18-4.23 (m, 1H), 4.89 (d, J=3.8 Hz, 1H) 7.17 (d, J=8.5 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.8, 23.4, 25.3, 26.7, 30.1, 30.2, 30.4, 30.5, 32.7, 33.4, 37.4, 50.9, 68.5, 69.6, 70.2, 71.2, 73.0, 73.4, 75.5, 100.4, 175.0; HRMS (ESI): m/z calcd for C.sub.50H.sub.99NO.sub.8SNa [M+Na].sup.+ 896.6989, found 896.6997.

Example 3

Synthesis of (2S,3S,4R)-1-(4,6-Anhydro-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN154)

(31) ##STR00058##

Example 3.1

(2S,3S,4R)-1-(4,6-Anhydro-2,3-di-O-benzyl-α-D-galactopyranosyloxy)-3,4-di(benzyloxy)-2-(hexacosanoylamino)octadeca-6-ene

(32) ##STR00059##

(33) Tosylated galactose 2 (0.029 g, 0.021 mmol) is dissolved in anhydrous Et.sub.2O (0.3 mL) under an Ar atmosphere and cooled to 0° C. Sodium hydride (0.002 g, 0.042 mmol; 60% dispersion in mineral oil) is then added and the reaction mixture left to warm to RT over 18 h. After this time, the mixture is warmed to 25° C. and NaH added in aliquots to push the reaction to completion. Once all the starting material is consumed (TLC), the mixture is diluted with EtOAc and H.sub.2O added. The layers are separated and the aqueous layer re-extracted with EtOAc. The combined organics were washed with H.sub.2O, brine, dried (MgSO.sub.4) and the solvent removed in vacuo. Purification of the resulting residue by silica gel chromatography (23% EtOAc/petroleum ether changing to 30% EtOAc/petroleum ether) afforded the target material 7 as a colourless oil (0.019 g, 0.016 mmol, 54%).

(34) [α].sub.D.sup.20=+27.1 (c 0.0095, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.86-0.89 (m, 6H), 1.21-1.33 (m, 62H), 1.42-1.48 (m, 2H), 1.83-1.86 (m, 2H), 2.00-2.04 (m, 2H), 2.38-2.50 (m, 2H), 3.56 (dt, J=6.4, 4.8 Hz, 1H), 3.66-3.70 (m, 2H), 3.81 (dd, J=9.4, 5.1 Hz, 1H, H-3), 3.85 (dd, J=10.4, 4.6 Hz, 1H), 4.03 (dd, J=9.5, 2.7 Hz, 1H), 4.08 (d, J=7.3 Hz, 1H), 4.30-4.34 (m, 1H), 4.36 (td, J=3.9, 1.5 Hz, 1H), 4.47 (d, J=11.6 Hz, 2H), 4.58 (d, J=11.6 Hz, 1H), 4.63-4.67 (m, 3H), 4.69 (d, J=11.8 Hz, 1H), 4.70 (d, J=11.8 Hz, 1H), 4.84 (d, J=11.8 Hz, 1H), 4.88 (d, J=2.6 Hz, 1H), 5.09 (t, J=4.5 Hz, 1H), 5.42-5.51 (m, 2H), 5.56 (d, J=8.6 Hz), 7.23-7.36 (m, 20H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1 (CH.sub.3), 22.7, 23.3, 23.8, 24.7, 25.7, 27.6, 28.1, 29.3, 29.4, 29.7, 31.9, 36.6, 36.7, 50.1, 67.8, 69.4, 71.7, 72.0, 73.4, 73.7, 74.1, 74.8, 77.7, 78.1, 79.2, 80.0, 98.9, 125.1, 127.6, 127.72, 127.77, 127.82, 128.3, 128.36, 128.43, 132.3, 138.3, 138.4, 138.5, 172.7; HRMS (ESI): m/z calcd for C.sub.78H.sub.120NO.sub.8 [M+H].sup.+ 1198.9014, found 1198.9014; m/z calcd for C.sub.78H.sub.119NO.sub.8Na [M+Na].sup.+ 1220.8833, found 1220.8818.

Example 3.2

(2S,3S,4R)-1-(4,6-Anhydro-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN154)

(35) ##STR00060##

(36) Pd(OH).sub.2 on carbon (20% Pd; ˜5 mg) is added to protected oxetane 7 (19 mg, 16 μmol) stirring in anhydrous CH.sub.2Cl.sub.2:MeOH (4 mL; 1:1). The reaction vessel is evacuated and flushed with hydrogen (×4) and stirred at 25° C. for 20 hrs. Once cooled, the product mixture is filtered through celite, washed repeatedly with CHCl.sub.3:MeOH (3:1), and then concentrated. Purification by silica gel chromatography (100% CHCl.sub.3 changing to 10% MeOH/CHCl.sub.3) yielded the target oxetane CN154 as a white solid (6 mg, 7 μmol, 44%).

(37) [α].sub.D.sup.20=+66.0 [c 0.0025, CHCl.sub.3:MeOH (3:1)]; .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=7.0 Hz, 6H), 1.52-1.68 (m, 76H), 2.18-2.21 (m, 2H), 3.50-3.56 (m, 2H), 3.68 (dd, J=10.7, 4.0 Hz, 1H), 3.71 (br s, 1H), 3.77 (dd, J=9.6, 5.2 Hz, 1H), 3.87 (dd, J=10.6, 5.0 Hz, 1H), 3.98 (dd, J=9.6, 2.8 Hz, 1H), 4.14-4.18 (m, 2H), 4.48 (td, J=3.9, 1.5 Hz, 1H), 4.78 (dd, J=7.3, 3.8 Hz, 1H), 4.99 (d, J=2.8 Hz, 1H), 5.08 (t, J=4.6 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.1, 22.9 26.1, 29.5, 29.6, 29.9, 32.1, 32.7, 36.7, 50.4, 67.36, 67.43, 69.5, 70.7, 72.2, 74.7, 75.4, 80.5, 100.2, 174.5; HRMS (ESI): m/z calcd for C.sub.50H.sub.97NO.sub.8Na [M+Na].sup.+ 862.74112, found 862.7109.

Example 4

(2S,3S,4R)-1-(6-Deoxy-6-carboxymethylthio-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN224)

(38) ##STR00061##

(39) Iodoacetic acid (2.0 mg, 0.011 mmol) is added to CN237 (5.2 mg, 0.0059 mmol) and triethylamine (10 μL, 0.072 mmol) stirring in anhydrous DMF (5 mL) under Ar. The solution was heated to 70 C for 2 h. TLC analysis (10% MeOH/CHCl.sub.3) during this time showed the presence of the target material. The product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 20% MeOH/CHCl.sub.3 with 1% formic acid) to yield target acid CN224 (3 mg, 0.0032 mmol, 56%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=7.0 Hz, 6H), 1.22-1.42 (m, 68H), 1.49-1.71 (m, 4H), 2.18-2.25 (m, 2H), 2.80-2-95 (m, 2H), 3.20-3.30 (m, 2H), 3.56-3.63 (m, 2H), 3.65-3.71 (m, 1H), 3.72-3.80 (m, 2H) 3.90-4.03 (m, 3H), 4.20-4.24 (m, 1H), 4.85 (d, J=2.6 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.52H.sub.101NO.sub.10SNa [M+Na].sup.+ 954.7044, found 954.7039.

Example 5

2S,3S,4R)-1-(6-Deoxy-6-((4-iodo-N-phenylmaleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN170)

(40) ##STR00062##

(41) Triethylamine (30 μL, 0.22 mmol) is added to 3,4-diiodo-N-phenylmaleimide (50 mg, 0.12 mmol) and CN237 (10 mg, 0.011 mmol) stirring in anhydrous CHCl.sub.3/MeOH (1:1, 5 mL) under Ar at RT. After 60 min. the product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 30% MeOH/CHCl.sub.3) to yield the target compound CN170 (10 mg, 0.0085 mmol, 77%) as a yellow solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=6.8 Hz, 6H), 1.20-1.38 (m, 68H), 1.48-1.68 (m, 4H), 2.16 (td, J=7.7, 2.6 Hz, 2H), 3.38 (bt, J=1.5 Hz, 1H), 3.40-3.55 (m, 2H), 3.85 (dd, J=10.7, 3.9 Hz, 2H), 3.67-3.74 (m, 2H), 3.79 (dd, J=10.0, 3.9 Hz, 1H), 3.93 (dd, J=10.8, 5.3 Hz, 1H), 3.96-3.95 (m, 1H), 4.01 (t, J=6.5 Hz, 1H), 4.20 (q, J=4.5 Hz, 1H), 4.91 (d, J=3.8 Hz, 1H), 7.30-7.40 (m, 3H), 7.44-7.49 (m, 3H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.2 (CH.sub.3), 22.8 (CH.sub.2), 26.0 (CH.sub.2), 29.5 (CH.sub.2), 29.6 (CH.sub.2), 29.9 (CH.sub.2), 32.1 (CH.sub.2), 32.3 (CH.sub.2), 33.0 (CH.sub.2) 36.7 (CH.sub.2), 50.1, 67.9, 68.8, 70.1, 70.3, 70.9, 72.4, 75.1, 95.0, 99.9, 126.4, 128.4, 129.3, 131.6, 150.8, 164.4, 165.6, 174.5 (CO) HRMS (ESI): m/z calcd for C.sub.60H.sub.103N.sub.2O.sub.10SINa [M+Na].sup.+ 1193.6276, found 1193.6282.

Example 6

(2S,3S,4R)-1-(6-Deoxy-6-((4-iodo-N-propargylmaleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN225)

(42) ##STR00063##

(43) A solution of CN237 (5.0 mg, 0.0057 mmol) in CHCl.sub.3/MeOH (1:1, 400 μL) is added to a stirred mixture of 3,4-diiodo-N-propargylmaleimide (16 mg, 0.041 mmol) and potassium acetate (0.60 mg, 00.61 mmol) in CHCl.sub.3 (400 μL) at RT. After 60 min. the product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 30% MeOH/CHCl.sub.3) to yield the target compound CN225 (3.0 mg, 0.0026 mmol, 46%) as a yellow solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.89 (t, J=7.0 Hz, 6H), 1.18-1.40 (m, 68H), 1.50-1.70 (m, 4H), 2.20 (bt, J=7.7 Hz, 2H), 2.43-2.45 (m, 1H), 2.53-3.56 (m, 2H), 3.60 (dd, J=13.8, 5.1 Hz, 1H), 3.65 (dd, J=10.5, 4.2 Hz, 1H), 3.72-3.81 (m, 3H), 3.91 (dd, J=10.8, 5.1 Hz, 1H), 3.95-4.01 (m, 2H), 4.07-4.24 (m, 3H), 4.28-4.38 (m, 2H), 4.90 (d, J=3.7 Hz, 1H), 7.38 (bt, J=8.0 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 15.1, 23.9, 27.17, 27.21, 29.4, 30.6, 30.7, 30.8, 30.9, 31.1, 33.2, 33.7, 37.8, 51.5, 68.6, 70.0, 71.4, 71.5, 72.2, 73.3, 73.4, 75.9, 78.1, 95.0, 101.0, 152.3, 165.6, 166.5, 175.6; HRMS (ESI): m/z calcd for C.sub.57H.sub.101N.sub.2O.sub.10NaSI [M+Na]+ 1155.6119, found 1155.6108.

Example 7

(2S,3S,4R)-1-(6-Deoxy-6-((N-propargylmaleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN223)

(44) ##STR00064##

(45) A solution of CN237 (5.0 mg, 0.0057 mmol) in CHCl.sub.3/MeOH (1:1, 200 μL) is added to a stirred mixture of 3-bromo-N-propargymaleimide (3.4 mg, 0.016 mmol) and potassium acetate (0.60 mg, 0.0061 mmol) in MeOH (200 μL) at RT. After 10 hrs. the product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 20% MeOH/CHCl.sub.3) to yield the target compound CN223 (3.0 mg, 0.0030 mmol, 52%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.89 (t, J=7.2 Hz, 6H), 1.19-1.42 (m, 68H), 1.50-1.70 (m, 4H), 2.20 (td, J=7.7, 2.0 Hz, 2H), 2.37 (t, J=2.5 Hz, 1H), 3.18 (dd, J=13.3, 5.2 Hz, 1H), 3.30 (dd, J=13.3, 8.4 Hz, 1H), 3.51-3.57 (m, 2H), 3.62 (dd, J=10.7, 4.3 Hz, 1H), 3.74 (dd, J=10.0, 3.2 Hz, 1H), 3.80 (dd, J=10.1, 3.8 Hz, 1H), 3.88-3.92 (m, 2H), 4.00-4.05 (m, 3H), 4.21 (q, J=4.7, Hz, 1H), 4.28-4.38 (m, 2H), 4.91 (d, J=3.7 Hz), 6.31 (s, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 13.7, 22.5, 25.7, 25.8, 26.9, 29.2, 29.3, 29.5, 29.6, 31.8, 32.4, 50.0, 67.2, 68.5, 69.9, 70.0, 70.2, 71.5, 71.9, 74.6, 99.6, 118.2, 151.6, 166.8, 168.4, 174.3; HRMS (ESI): m/z calcd for C.sub.57H.sub.103N.sub.2O.sub.10S [M+H]+ 1007.7333, found 1007.7337.

Example 8

(2S,3S,4R)-1-(6-Deoxy-6-ethylthio-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN227)

(46) ##STR00065##

(47) Sodium hydride (60% dispersion in mineral oil, 9.4 mg, 0.24 mmol) is added to a stirred solution of 5 (12.0 mg, 0.00981 mmol) and ethane thiol (21 μL, 0.27 mmol) in dry DMF (96 μL) under argon. After 3 days at RT the product mixture is concentrated and purified by silica gel chromatography (4% MeOH/CH.sub.2Cl.sub.2) changing to 10% MeOH/CH.sub.2Cl.sub.2) to yield the target compound CN227 (2.1 mg, 24%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:3) 0.86 (t, J=7.1 Hz, 6H), 1.23 (t, J=7.5 Hz, 3H), 1.22-1.39 (m, 68H), 1.50-1.65 (m, 4H), 2.20 (t, J=7.4, Hz, 2H), 2.58 (q, J=7.5 Hz, 2H), 2.68-2.78 (m, 2H), 2.68-2.78 (m, 2H), 3.52-3.58 (m, 2H), 3.66 (dd, J=10.6, 4.0 Hz, 1H), 3.72 (dd, J=10.0, 3.1 Hz, 1H), 3.76 (dd, J=10.0, 3.6 Hz, 1H), 3.84 (t, J=7.0 Hz, 1H), 3.88 (dd, J=10.4, 4.5 Hz, 1H), 3.90-3.92 (m, 1H), 4.20-4.16 (m, 1H), 4.84 (d, J=3.6 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 2:3), d 14.3, 15.1, 23.3, 26.6, 26.7, 27.3, 30.0, 30.1, 30.2, 30.3, 30.4, 32.5, 32.6, 32.8, 37.1, 51.0, 67.9, 69.6, 70.9, 71.2, 71.7, 72.7, 75.0, 100.4, 175.1; HRMS (ESI): m/z calcd for C.sub.52H.sub.103NO.sub.8SNa [M+Na]+ 924.7302, found 924.7299.

Example 9

(2S,3S,4R)-1-(6-Deoxy-6-ethylsulfinyl)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN228)

(48) ##STR00066##

(49) A solution of ˜55% mCPBA in CH.sub.2Cl.sub.2 (10 mg/mL, 150 μL, 0.0048 mmol) is added to a stirred solution of CN227 (4.2 mg, 0.0047 mmol) in CH.sub.2Cl.sub.2/MeOH (10:1, 220 μL) cooled to −50° C. The mixture is warmed to RT over 4 hrs and diluted with CH.sub.2Cl.sub.2/MeOH (85:15, 20 mL) and sat aq sodium bicarbonate (20 mL). The phases are separated and the organic phase re-washed with sodium bicarbonate (2×20 mL). The organic phase is dried (MgSO.sub.4) and the solvent removed in vacuo. Purification of the resulting residue by silica gel chromatography (4% MeOH/CH.sub.2Cl.sub.2) changing to 10% MeOH/CH.sub.2Cl.sub.2) afforded CN228 (0.34 mg, 8%) as a thin film. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 1:1) δ 0.89 (t, J=7.0 Hz, 6H) 1.22-1.39 (m, 68H), 1.37 (t, J=7.3 Hz, 3H), 1.50-1.65 (m, 4H), 2.22 (t, J=7.5 Hz, 2H), 2.75-2.82 (m, 2H), 2.86-2.92 (m, 1H), 3.16 (dd, J=13.2, 11.2 Hz, 1H), 3.54-3.58 (m, 2H), 3.63 (dd, J=10.5, 5.2 Hz, 1H), 3.79-3.82 (m, 3H), 3.95 (dd, J=10.5, 5.4 Hz, 1H), 4.27 (dd, J=10.0, 4.9 Hz, 1H), 4.32 (bd, J=10.9 Hz, 1H), 4.90 (bs, 1H); HRMS (ESI): m/z calcd for C.sub.52H.sub.103NO.sub.9SNa [M+Na]+ 940.7251, found 940.7244.

Example 10

(2S,3S,4R)-1-(3,6-Anhydro-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN230)

(50) ##STR00067##

(51) Sodium hydride (60% dispersion in mineral oil, 9.4 mg, 0.24 mmol) is added to a stirred solution of 5 (12.0 mg, 0.00981 mmol) and ethanethiol (21 μL, 0.27 mmol) in dry DMF (96 μL) under argon. After 3 days at RT the product mixture is concentrated and purified by silica gel chromatography (4% MeOH/CH.sub.2Cl.sub.2 changing to 10% MeOH/CH.sub.2Cl.sub.2) to yield a fraction containing a ˜3:7 mixture of title compound CN230 and CN227. The mixture is treated with mCPBA to oxidise the thioether CN227 and repurified by silica gel chromatography to give CN230 as a white solid (1.1 mg, 13%). .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:3) δ 0.89 (t, J=7.1 Hz, 6H), 1.22-1.45 (m, 68H), 1.50-1.65 (m, 4H), 2.21 (t, J=7.5 Hz, 2H), 3.52-3.57 (m, 1H), 3.58-3.62 (m, 1H), 3.84 (dd, J=10.0, 4.3 Hz, 1H), 3.90 (dd, J=5.4, 2.4 Hz, 1H), 3.93 (dd, J=10.2, 3.7 Hz, 1H), 4.02 (dd, J=10.0, 2.4 Hz, 1H), 4.06 (d, J=10.0 Hz, 1H), 4.13-4.19 (m, 1H), 4.22-4.26 (m, 2H), 4.47-4.48 (m, 1H), 4.80 (d, J=2.5 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3:CD.sub.3OD 2:3) δ 14.3, 23.3, 26.6, 30.0, 30.1, 30.2, 30.3, 30.4, 32.5, 32.6, 37.1, 51.0, 69.6, 70.0, 70.6, 70.7, 73.0, 74.9, 78.4, 82.1, 97.9, 175.3; HRMS (ESI): m/z calcd for C.sub.50H.sub.97NO.sub.8Na [M+Na]+ 862.7112, found 862.7114.

Example 11

(2S,3S,4R)-1-(6-Deoxy-6-phenylthio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN231)

(52) ##STR00068##

(53) Sodium hydride (60% dispersion in mineral oil, 1.5 mg, 0.037 mmol) is added to a stirred solution of 5 (6.2 mg, 0.0051 mmol) and thiophenol (5.0 μL, 0.048 mmol) in dry DMF (50 μL) under argon. After 1 h at 65° C. the cooled reaction mixture is partitioned between ethyl acetate (1 mL) and sat aq sodium bicarbonate (1 mL). The aqueous phase is thoroughly extracted with ethyl acetate and the combined organic extracts are dried (MgSO.sub.4) and concentrated at reduced pressure to afford a solid (9.2 mg). The crude material is dissolved in 2:3 CH.sub.2Cl.sub.2/MeOH (0.25 mL), treated with NaOMe (0.5 M in MeOH, 20 μL, 0.01 mmol) and stirred at RT for 1 h. The reaction mixture is quenched with the addition of formic acid (2 μL, 0.053 mmol), and purified by silica gel chromatography (2% MeOH/CH.sub.2Cl.sub.2 changing to 6% MeOH/CH.sub.2Cl.sub.2) to yield the target compound CN231 (3.0 mg, 62%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:3) δ 0.89 (t, J=7.1 Hz, 6H), 1.22-1.45 (m, 68H), 1.50-1.68 (m, 4H), 2.16 (t, J=7.7 Hz, 2H), 3.14-3.24 (m, 2H), 3.52-3.61 (m, 2H), 3.69 (dd, J=10.6, 3.8 Hz, 1H), 3.72 (dd, J=10.0, 3.4 Hz, 1H), 3.79 (dd, J=10.0, 3.9 Hz, 1H), 3.84 (dd, J=10.7, 4.4 Hz, 1H), 3.89-3.92 (m, 1H), 3.95-3.96 (m, 1H), 4.16-4.19 (m, 1H), 4.88 (d, J=3.9 Hz) 7.15-7.18 (m, 1H), 7.26-7.30 (m, 2H), 7.33-7.36 (m, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 2:3) δ 14.2, 23.0, 26.3, 29.7, 29.8, 30.0, 30.1, 30.2, 32.3, 32.7, 34.2, 36.9, 50.5, 68.1, 69.2, 70.27, 70.33, 70.8, 72.5, 74.9, 100.2, 126.4, 129.1, 129.4, 136.7, 174.6; HRMS (ESI): m/z calcd for C.sub.56H.sub.103NO.sub.8SNa [M+Na]+ 972.7302, found 972.7294.

Example 12

(2S,3S,4R)-1-(6-Deoxy-6-napthalen-2-ylthio-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN236)

(54) ##STR00069##

(55) Sodium hydride (60% dispersion in mineral oil, 1.5 mg, 0.037 mmol) is added to a stirred solution of 5 (6.8 mg, 0.0056 mmol) and 2-napthalenethiol (8.6 mg, 0.053 mmol) in dry DMF (56 μL) under argon. After 1 h at 65° C. the cooled reaction mixture is partitioned between ethyl acetate (1 mL) and sat aq sodium bicarbonate (1 mL). The aqueous phase is thoroughly extracted with ethyl acetate and the combined organic extracts are dried (MgSO.sub.4) and concentrated at reduced pressure to afford a solid (13.8 mg). The crude material is dissolved in 2:3 CH.sub.2Cl.sub.2/MeOH (0.28 mL), treated with NaOMe (0.5 M in MeOH, 22 μL, 0.011 mmol) and stirred at RT for 1 h. The reaction mixture is quenched with the addition of formic acid (2 μL, 0.053 mmol), and purified by silica gel chromatography (2% MeOH/CH.sub.2Cl.sub.2 changing to 6% MeOH/CH.sub.2Cl.sub.2) to yield the target compound CN236 (3.7 mg, 66%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:3) δ 0.89 (t, J=7.1 Hz, 6H), 1.20-1.34 (m, 68H), 1.47-1.65 (m, 4H), 2.04-2.07 (m, 2H), 3.26 (dd, J=13.6, 6.5 Hz, 1H), 3.33-3.37 (m, 1H), 3.52-3.59 (m, 2H), 3.73 (dd, J=10.2, 3.4 Hz, 1H), 3.81 (dd, J=9.9, 3.9 Hz, 1H), 3.89 (dd, J=10.5, 4.4 Hz, 1H), 3.97-4.00 (m, 2H), 4.17-4.20 (m, 1H), 4.91 (d, J=3.9 Hz, 1H), 7.40-7.49 (m, 3H), 7.73-7.80 (m, 4H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 2:3) δ 14.3, 23.3, 26.5, 29.9, 30.00, 30.02, 30.1, 30.31, 30.34, 30.37, 30.41, 32.6, 32.7, 34.4, 37.0, 50.9, 68.2, 69.5, 70.8, 70.9, 71.1, 72.7, 75.0, 100.5, 126.3, 126.9, 127.3, 127.5, 127.7, 128.3, 129.2, 132.6, 134.7, 134.8, 175.0; HRMS (ESI): m/z calcd for C.sub.60H.sub.105NO.sub.8SNa [M+Na]+ 1022.7459, found 1022.7456.

Example 13

(2S,3S,4R)-1-O-[6-Deoxy-6-(2-pyridyl)disulfanyl]-α-D-galactopyranosyl)-2-hexacosanoylamino-3,4-octadecandiol (CN208)

Example 13.1

(2S,3S,4R)-1-(2,3,4-Tri-O-acetyl-[6-deoxy-6-(2-pyridyl)disulfanyl]-α-D-galactopyranosyloxy)-3,4-di(acetyloxy)-2-hexacosanoylamino-octadecaene

(56) ##STR00070##

(57) A degassed solution of hydrazine acetate (1.00 mL, 5 mg/mL, DMF/MeOH, 7:3, 0.054 mmol) is added over 2 hrs to a degassed solution of 6 (12 mg, 0.011 mmol) and 2,2′-dithiodipyridine (36 mg, 0.16 mmol) in DMF (5.5 mL). After 14 hrs at RT the mixture is diluted with CH.sub.2Cl.sub.2 (20 mL) and brine (sat. 20 mL). The layers are separated and the aqueous is re-extracted with EtOAc (20 mL) and the combined organic layers are dried (MgSO.sub.4) and the solvent removed in vacuo. Purification of the resulting residue by silica gel chromatography (100% PE changing to 80% PE/EtOAc) afforded CN500 (4.0 mg, 31%) as a yellow solid. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 0.88 (t, J=7.2 Hz, 6H), 1.22-1.32 (m, 68H), 1.60-1.72 (m, 4H), 1.98 (s, 3H), 1.99 (s, 3H), 2.06 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H), 2.19-2.30 (m, 2H), 2.81 (dd, J=13.7, 5.2 Hz, 1H), 2.93 (dd, J=13.7, 5.2 Hz, 1H), 3.41 (dd, J=10.7, 2.6 Hz, 1H), 3.75 (dd, J=10.7, 2.8 Hz, 1H), 4.29 (bt, J=7.0 Hz, 1H), 4.42 (tt, J=10.0, 2.8 Hz, 1H), 4.87-4.92 (m, 2H), 5.13 (dd, J=10.5, 3.7 Hz, 1H), 5.28 (dd, J=9.8, 2.2 Hz, 1H), 5.32 (dd, J=10.9, 3.5 Hz, 1H), 5.49 (br d, J=2.8 Hz, 1H), 6.40 (d, J=9.6 Hz, 1H), 7.11-7.15 (m, 1H), 7.54-7.57 (m, 1H), 7.61-7.65 (m, 1H), 8.52-8.55 (m, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 14.1, 20.58, 20.63 (2×), 20.7, 21.0, 22.7, 25.7, 25.8, 27.3, 29.3, 29.4, 29.7, 31.9, 36.7, 39.1, 47.7, 67.5, 67.8, 69.4, 70.9, 73.5, 97.2, 121.0, 121.4, 137.1, 150.1, 158.7, 169.7, 170.0, 170.2, 170.7, 171.1, 172.8; HRMS (ESI): m/z calcd for C.sub.65H.sub.112N.sub.2O.sub.13S.sub.2Na [M+Na].sup.+ 1215.7504, found 12.15.7491.

Example 13.2

(2S,3S,4R)-1-O-[6-Deoxy-6-(2-pyridyl)disulfanyl]-α-D-galactopyranosyl)-2-hexacosanoylamino-3,4-octadecandiol (CN208

(58) ##STR00071##

(59) A solution of NaOMe (0.5 M, in MeOH, 40 μL, 0.020 mmol) is added to a stirred solution of CN500 (10.0 mg, 0.0083 mmol) in CHCl.sub.3/MeOH (3:2, 2 mL). After 2 hrs formic acid (50 μL) was added and the mixture concentrated. Purification of the resulting residue by silica gel chromatography (100% CHCl.sub.3 changing to 90% CHCl.sub.3/MeOH) afforded CN208 (7.0 mg, 0.0071 mmol, 85%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.88 (t, J=7.2 Hz, 6H), 1.20-1.42 (m, 68H), 1.50-1.72 (m, 4H), 2.22 (td, J=7.5, 3.3 Hz, 2H), 3.02 (dd, J=13.7, 6.1 Hz, 1H), 3.11 (dd, J=13.7, 7.5 Hz, 1H), 3.54-3.62 (m, 2H), 3.67-3.72 (m, 2H), 3.75-3.80 (m, 2H), 3.94 (dd, J=10.2, 5.0 Hz, 1H), 4.07 (t, J=6.7 Hz, 1H), 4.23 (q, J=4.9 Hz, 1H), 4.90 (d, J=3.9 Hz, 1H), 7.16-7.19 (m, 1H), 7.72-7.80 (m, 2H) 8.41-8.43 (m, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 15.2, 24.0, 27.2, 30.7, 31.0, 33.2, 34.0, 37.9, 40.8, 51.5, 69.2, 70.1, 70.5, 70.9, 71.2, 71.6, 73.4, 76.2, 101.1, 122.2, 122.6, 139.1, 150.7, 161.1, 175.6; HRMS (ESI): m/z calcd for C.sub.55H.sub.103N.sub.2O.sub.8S.sub.2Na [M+Na]+ 983.7156, found 983.7156.

Example 14

(2S,3S,4R)-1-(6-Deoxy-6-((4-iodo-N-(5-azidopentyl)-maleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN235)

Example 14.1

N-(5-azidopentyl)-3,4-diiodomaleimide

(60) ##STR00072##

(61) A solution of 5-azidopentan-1-amine (10 mg, 0.078 mmol) in CH.sub.2Cl.sub.2 (100 μL) is added to a stirred solution of N-methoxycarbonyl-3,4-diiodomaleimide (29 mg, 0.071 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. After 20 min. the solvent removed in vacuo. Purification of the resulting residue by silica gel chromatography (100% PE changing to 70% PE/EtOAc) afforded the title compound (30 mg, 92%) as a yellow solid. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.35-1.41 (m, 2H), 1.59-1.67 (m, 4H), 3.27 (t, J=6.9 Hz, 2H), 3.64 (t, J=7.2 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 23.8, 28.0, 28.3, 39.9, 51.4, 117.2, 166.3; HRMS (ESI): m/z calcd for C.sub.9H.sub.10N.sub.4O.sub.2I.sub.2Na [M+Na]+ 482.8791, found 482.8785.

Example 14.2

2S,3S,4R)-1-(6-Deoxy-6-((4-iodo-N-(5-azidopentyl)-maleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN235)

(62) ##STR00073##

(63) A solution of CN237 (5.0 mg, 0.0057 mmol) in CHCl.sub.3 (1.2 mL) is added to a stirred mixture of N-(5-azidopentyl)-3,4-diiodomaleimide (26 mg, 0.057 mmol) and potassium acetate (0.60 mg, 0.0061 mmol) in CHCl.sub.3 (800 μL). After 60 min. at RT the product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 30% MeOH/CHCl.sub.3) to yield the target compound CN235 (6.0 mg, 0.0026 mmol, 87%) as a yellow solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 0.89 (t, J=6.9 Hz, 6H), 1.18-1.40 (m, 70H), 1.50-1.70 (m, 8H), 2.20 (bt, J=7.7, 2H), 3.30 (t, J=6.8 Hz, 2H), 3.53-3.61 (m, 4H), 3.62-3.66 (m, 2H), 3.70-3.81 (m, 3H), 3.90-4.00 (3H), 4.19-4.22 (m, 1H), 4.90 (d, J=3.7 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 3:1) δ 14.3, 23.2, 24.4, 26.5, 28.6, 28.8, 29.87, 29.92, 29.98, 30.18, 30.22, 30.35, 32.4, 33.0, 37.0, 39.6, 50.7, 51.7, 63.8, 67.8, 69.3, 69.5, 70.7, 70.8, 71.4, 72.5, 75.1, 94.6, 100.3, 150.9, 166.3, 167.1, 174.8; HRMS (ESI): m/z calcd for C.sub.59H.sub.109N.sub.5O.sub.10SI [M+H]+ 1206.6940, found 1206.6946.

Example 15

(2S,3S,4R)-1-(6-Deoxy-6-(N-ethylmaleimid-3-yl)thio)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecandiol (CN234)

(64) ##STR00074##

(65) A solution of CN237 (5.0 mg, 0.0057 mmol) in CHCl.sub.3 (1 mL) is added to a stirred mixture of N-ethylmaleimide (30 mg, 0.240 mmol) and triethylamine (10 μL). After 60 min. at RT the product mixture is concentrated and purified by silica gel chromatography (100% CHCl.sub.3 changing to 25% MeOH/CHCl.sub.3) to yield a diastereomeric mixture of the target compound CN234 (5.0 mg, 0.005 mmol, 87%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 1:1) δ 0.89 (t, J=6.9 Hz, 12H), 1.18 (t, J=7.2 Hz, 6H), 1.125-1.37 (m, 140H), 1.50-1.70 (m, 8H), 2.21 (bt, J=7.7, 4H), 2.50 (dd, J=4.0, 18.1 Hz, 1H), 2.56 (dd, J=3.6, 18.1 Hz, 1H), 2.89 (dd, J=5.5, 13.8 Hz, 1H), 2.98 (dd, J=8.3, 14.0 Hz, 1H), 3.06 (dd, J=5.5, 14.1 Hz, 1H), 3.18 (dd, J=9.1, 18.2 Hz, 1H), 3.22 (dd, J=9.1, 18.2 Hz, 1H), 3.30 (dd, J=8.2, 13.8 Hz, 1H), 3.53-3.59 (m, 8H), 3.64-3.70 (m, 2H), 3.73-3.80 (m, 4H), 3.91-4.01 (m, 8H), 4.20-4.24 (m, 2H), 4.89 (d, J=4.4 Hz, 1H), 4.90 (d, J=4.4 Hz, 1H), .sup.13C NMR (125 MHz, CDCl.sub.3/CD.sub.3OD 1:1) δ 13.0, 14.3, 23.2, 26.4, 26.5, 29.8, 29.9, 30.1, 30.18, 30.22, 30.3, 32.4, 32.6, 32.7, 32.9, 33.0, 34.5, 36.5, 36.6, 36.8, 36.9, 37.01, 37.03, 40.3, 40.4, 50.86, 50.92, 67.6, 67.7, 69.3, 70.6, 70.79, 70.84, 71.8, 72.5, 75.1, 75.2, 100.1, 100.2, 174.98, 175.04, 176.0, 176.1, 177.6, 178.1; HRMS (ESI): m/z calcd for C.sub.56H.sub.107N.sub.2O.sub.10S [M+H]+ 999.7646, found 999.7657.

Example 16

Methyl 1-(((2S,3S,4R)-1-(6-Deoxy-)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-dihydroxyoctadecandyl)-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-tetracosaoxa-1,2-dithiaoctaheptacontan-77-oate (CN238)

Example 16.1

Methyl 1-(pyridin-2-yldisulfanyl)-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oate

(66) ##STR00075##

(67) A solution of NaOMe (0.5 M, in MeOH, 100 μL, 0.05 mmol) is added to a solution of S-acetyl-dPEG.sub.24-NHS ester (Quanta Biodesign, product #10188) (11.7 mg, 0.00898 mmol) and 2,2′-dipyridyl disulphide (25 mg, 0.113 mmol) in DMF (0.5 mL). After 2 h at RT MeOH (5 mL) is added and the solvents removed in vacuo. Purification of the resulting residue by silica gel chromatography (100% CHCl.sub.3 changing to 85% CHCl.sub.3/MeOH) afforded CN510 (7 mg, 0.0054 mmol, 60%) as a yellow oil. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 1:1) δ 2.61 (t, J=6.3 Hz, 2H), 3.02 (t, J=6.1 Hz, 2H), 3.57-3.60 (m, 2H), 3.61-3.69 (m, 90H), 3.70 (s, 3H), 3.73 (t, J=6.1 Hz, 2H), 3.77 (t, J=6.3 Hz, 2H), 7.20 (bdd, J=4.8, 7.6 Hz, 1H), 7.80 (ddd, J=1.8, 7.6, 8.0 Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 8.39, (bd, J=4.8 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 1:1) δ 35.4, 39.4, 52.1, 67.2, 69.4, 70.8, 70.98, 71.01, 71.05, 71.13, 71.2, 120.7, 121.7, 138.7, 149.7, 161.2, 173.3; HRMS (ESI): m/z calcd for C.sub.57H.sub.107NO.sub.26S.sub.2Na [M+Na]+ 1308.6420, found 1308.6423.

Example 16.2

Methyl 1-(((2S,3S,4R)-1-(6-Deoxy-)-α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-dihydroxyoctadecandyl)-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-tetracosaoxa-1,2-dithiaoctaheptacontan-77-oate (CN238)

(68) ##STR00076##

(69) An aqueous solution of NaHCO.sub.3 (1M, 30 μL) is added to a solution of CN237 (4 mg, 0.0046 mmol) and disulphide CN510 (6 mg, 0.0042 mmol) in CHCl.sub.3/MeOH (1;1, 2 mL). After 1 h the reaction mixture is diluted with CHCl.sub.3/MeOH (1;1, 10 mL) and the solvents removed in vacuo. Purification of the resulting residue by silica gel chromatography (100% CHCl.sub.3 changing to 70% CHCl.sub.3/MeOH) afforded CN238 (4 mg, 0.0020 mmol, 43%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 9:1) δ 0.88 (t, J=6.8 Hz, 6H), 1.20-1.44 (m, 70H), 1.47-1.70 (m, 4H), 2.21 (bt, J=7.7, 2H), 2.61 (t, J=6.4 Hz, 2H), 2.87-2.92 (m, 2H), 2.95-3.04 (m, 2H), 3.51-3.56 (m, 2H), 3.62-3.69 (m, 93H), 3.70 (s, 3H), 3.73-3.80 (m, 4H), 3.89-3.99 (m, 3H), 4.19-4.23 (m, 1H), 4.88 (d, J=3.8 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.102H.sub.201NO.sub.34S.sub.2Na [M+Na]+ 2071.3369, found 2071.3381.

Example 17

Formulating Compounds of the Invention for Intravenous Injection

(70) Compounds of the invention are formulated analogously to reported methods for α-GalCer. Briefly, solubilisation is based on excipient proportions described by Giaccone et al (Giaccone, Punt et al. 2002). Thus, 100 μL of a 10 mg/mL solution of α-GalCer or a compound of the invention in 9:1 THF/MeOH is added to 1.78 mL of an aqueous solution of Tween 20 (15.9 mg), sucrose (177 mg) and L-histidine (23.8 mg). This homogeneous mixture is freeze dried and the resulting foam is stored under Ar at −18° C. This material is reconstituted with 1.0 mL of phosphate-buffered saline (PBS) or water prior to serial dilutions in PBS to achieve final injectable solutions of α-GalCer or compounds of the invention.

Example 18

Biological Studies

(71) Mice.

(72) C57BL/6 are from breeding pairs originally obtained from Jackson Laboratories, Bar Harbor, Me., and used according to institutional guidelines with approval from the Victoria University of Wellington Animal Ethics Committee.

(73) Media and Reagents

(74) The tumour cells used (B16.OVA, C1498) are cultured in complete media consisting of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 5% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, 50 M 2-mercaptoethanol.

(75) Administration of Compounds of the Invention.

(76) Each compound of the invention is supplied as formulated product (see example 3), and diluted in water for delivery (0.23 nmol/mouse) by intravenous injection into the lateral tail vein. In humans the expected therapeutic dose lies in the 50-4800 (μg/m.sup.2) range (Giaccone, Punt et al. 2002). Note, 0.23 nmol in a mouse is a human equivalent dose of 30 μg/m.sup.2 for α-GalCer.

(77) All antibody labelling is performed on ice in FACS buffer (PBS supplemented with 1% FCS, 0.05% sodium azide, and 2 mM EDTA). Non-specific FcR-mediated antibody staining is blocked by incubation for 10 min with anti-CD16/32 Ab (24G2, prepared in-house from hybridoma supernatant). Flow cytometry is performed on a BD Biosciences FACSCalibur or BD LSRII SORP flow cytometer with data analysis using FlowJo software (Tree Star, Inc., OR, USA).

(78) Phenotyping DC from Spleen.

(79) Antibody staining and flow cytometry are used to examine the expression of maturation markers on dendritic cells in the spleen following injection of compounds of the invention. Splenocyte preparations are prepared by gentle teasing of splenic tissue through gauze in Iscove's Modified Dulbecco's Medium with 2 mM glutamine, 1% penicillin—streptomycin, 5×10.sup.−5 M 2-mercapto-ethanol and 5% fetal bovine serum (all Invitrogen, Auckland, New Zealand), followed by lysis of red blood cells with RBC lysis buffer (Puregene, Gentra Systems, Minneapolis, Minn., USA). Antibody staining is performed in PBS 2% fetal bovine serum and 0.01% sodium azide. The anti-FcgRII monoclonal antibody 2.4G2 is used at 10 mg/mL to inhibit non-specific staining. Monoclonal antibodies (all BD Biosciences Pharmingen, San Jose, Calif., USA) are used to examine expression of the maturation markers CD40, CD80 and CD86 on CD11c+ dendritic cells.

(80) Analysis of Cytokine Release into Serum.

(81) Blood is collected from the lateral tail vein at different time intervals after glycolipid administration. Serum is collected after blood has clotted, and levels of cytokines IL-12p70, IL-4 and IFN-γ are assessed by cytokine bead array technology (Biolpex, Biorad), according to the manufacturer's instructions.

(82) Analysis of Anti-Tumour Activity.

(83) Groups of C57BL/6 mice (n=5) receive a subcutaneous injection into the flank of 1×10.sup.5 B16.OVA melanoma cells, which express a cDNA encoding the chicken ovalbumin (OVA) sequence. The different groups are treated 7 days later, when tumours are fully engrafted, by intravenous injection of one of the following; vaccines as indicated in text and figure legends. Mice are monitored for tumour growth every 3-4 days, and tumour size for each group is calculated as the mean of the products of bisecting diameters (+SEM). Measurements are terminated for each group when the first animal develops a tumour exceeding 200 mm.

(84) Analysis of Anti-Leukemia Activity.

(85) To generate cell-based vaccines, C1498 acute leukemia cells are cultured for 24 h in complete IMDM supplemented with 200 ng/ml of α-GalCer or 200 ng/ml CN161, washed three times with PBS, and γ-irradiated (150 Gy). Vaccines comprising of 7.5×10.sup.5 cells are administered intravenously via the lateral tail vein. Mice are monitored for onset of leukemia-associated symptoms, such as weight loss, hunching or reduced grooming. All experiments are conducted with five animals per treatment group, with controls including a leukemia-only group.

(86) Analysis of Reactivity of Human NKT Cells to Compounds of the Invention.

(87) Peripheral blood is drawn into heparinized tubes, diluted 1:1 in PBS, and layered over a sodium diatrizoate and polysaccharide solution (Lymphoprep; Axis-Shield, Oslo, Norway) before centrifugation at 800×g for 25 minutes at room temperature to collect the peripheral blood mononuclear cell (PBMC) fraction, which contains NKT cells. To assess proliferation of NKT cells, PBMC (2×10.sup.5 per well) are cultured at 37° C. in Iscove's Modified Dulbecco's Medium with 5% human AB serum and the indicated concentrations of α-GalCer, or CN161, with recombinant human IL-2 50 U/mL (Chiron Corporation, Emeryville, Calif.) added after 24 hours. After 7 days of culture, the cells are analysed by flow cytometry, using fluorescent soluble CD1d tetramers that have been loaded with α-GalCer to identify the NKT cells. Data are presented as percentage of NKT cells (CD1d/α-GalCer tetramer-binding cells) of total T cells (identified by binding of antibody specific for CD3) in the final cultures.

(88) Where the foregoing description reference has been made to integers having known equivalents thereof, those equivalents are herein incorporated as if individually set forth.

(89) Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

(90) It is appreciated that further modifications may be made to the invention as described herein without departing from the spirit and scope of the invention.

INDUSTRIAL APPLICABILITY

(91) The invention relates to sphingoglycolipid analogues which are useful in treating or preventing diseases and conditions such as those relating to infection, atopic disorders, autoimmune diseases or cancer.

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