Potent sialyltransferase inhibitors

Abstract

The present invention relates to a new class of sialyltransferase inhibitors. The class features a carbamate or similar moiety on the amine of a neuraminic acid derivative. Such inhibitors are suitable for use as a medicament, for example for treating, preventing, or delaying bacterial infection, viral infection, cancer, a disorder of sialic acid metabolism, or an autoimmune disease.

Claims

1. A compound of general formula (I): ##STR00082## wherein X is in each instance independently chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 acyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety, and wherein the acyl chain is optionally unsaturated; Z is chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety; Q and Q′ are each independently chosen from the group consisting of O, S, and NH; c′ is C; L is either —CH.sub.2— or is absent; and R is a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety; wherein the compound is not of general formula (I) wherein Z is methyl, Q is O, Q′ is O, L is absent, R is tert-butyl, X at the anomeric position is axial and is H, and each other X is acetyl.

2. The compound according to claim 1, wherein it is of general formula (II-ax) or (II-eq): ##STR00083## wherein each of X, Z, Q, Q′, c′, L, and R are as defined in claim 1.

3. The compound according to claim 1, wherein it is of general formula (III): ##STR00084## wherein each of X, Z, Q, Q′, c′, and R are as defined in claim 1.

4. The compound according to claim 1, wherein X is in each instance chosen from the group consisting of acetyl, propionyl, and butyryl; and/or Z is chosen from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, allyl, propargyl, n-butyl, tert-butyl, sec-butyl, isobutyl, and cyclobutyl; and/or Q is chosen from the group consisting of O and S; and/or Q′ is chosen from the group consisting of O, S, and NH; and/or L is absent; and/or R is a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety.

5. The compound according to claim 1, wherein X is acetyl; and/or Z is methyl; and/or Q is O; and/or Q′ is O; and/or L is absent; and/or R is chosen from the group consisting of methyl, ethyl, isobutyl, tert-butyl, n-butyl, allyl, propargyl, acetyl, 2-methoxyethyl, 2,2,2-trichloroethyl, and 2-fluoroethyl.

6. A method of treating, preventing, or delaying a bacterial infection, viral infection, cancer, a disorder of sialic acid metabolism, or an autoimmune disease in a subject in need thereof, the method comprising administrating to the subject a compound of general formula (I): ##STR00085## wherein X is in each instance independently chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 acyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety, and wherein the acyl chain is optionally unsaturated; Z is chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety; Q and Q′ are each independently chosen from the group consisting of O, S, and NH; c′ is C; L is either —CH.sub.2— or is absent; and R is a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety.

7. A compound of general formula (I): ##STR00086## wherein c′ is S(═O); X is in each instance independently chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 acyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety, and wherein the acyl chain is optionally unsaturated; Z is chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety; Q is O and Q′ is absent or is chosen from the group consisting of O, S, and NH; L is either —CH.sub.2— or is absent; and R is a linear, branched, or cyclic C.sub.1-6 hydrocarbon moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety, or optionally R is H when L is absent and Q′ is not absent.

8. The compound according to claim 7, wherein it is of general formula (II-ax) or (II-eq): ##STR00087## wherein each of X, Z, Q, Q′, c′, L, and R are as defined in claim 7.

9. The compound according to claim 7, wherein it is of general formula (III): ##STR00088## wherein each of X, Z, Q, Q′, c′, and R are as defined in claim 7.

10. The compound according to claim 7, wherein X is in each instance chosen from the group consisting of acetyl, propionyl, and butyryl; and/or Z is chosen from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, allyl, propargyl, n-butyl, tert-butyl, sec-butyl, isobutyl, and cyclobutyl; and/or Q is chosen from the group consisting of O and S; and/or Q′ is chosen from the group consisting of O, S, and NH; and/or L is absent; and/or R is a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety.

11. The compound according to claim 7, wherein X is acetyl; and/or Z is methyl; and/or Q is O; and/or Q′ is O; and/or L is absent; and/or R is chosen from the group consisting of methyl, ethyl, isobutyl, tert-butyl, n-butyl, allyl, propargyl, acetyl, 2-methoxyethyl, 2,2,2-trichloroethyl, and 2-fluoroethyl.

12. A method of treating or delaying a bacterial infection, viral infection, cancer, a disorder of sialic acid metabolism, or an autoimmune disease in a subject in need thereof, the method comprising administrating to the subject a compound of general formula (I): ##STR00089## wherein c′ is S(═O); X is in each instance independently chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 acyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety, and wherein the acyl chain is optionally unsaturated; Z is chosen from the group consisting of hydrogen and a linear, branched, or cyclic C.sub.1-6 alkyl, alkenyl, or alkynyl moiety wherein each carbon atom is optionally substituted by a halogen, an alkoxy, or a haloalkoxy moiety; Q is O and Q′ is absent or is chosen from the group consisting of O, S, and NH; L is either —CH.sub.2— or is absent; and R is a linear, branched, or cyclic C.sub.1-6 hydrocarbon moiety, wherein each carbon atom is optionally substituted by a halogen, an alkoxy, a haloalkoxy, a hydroxyl, or an oxo moiety, or optionally R is H when L is absent and Q′ is not absent.

Description

SHORT DESCRIPTION OF DRAWINGS

(1) FIG. 1: A) Working model of metabolic sialyltransferase inhibitors. The metabolic precursor is taken-up via passive diffusion and deacetylated by intracellular esterases. CMP activation in the nucleus by CMAS produces the active inhibitor Sia-3Fax-CMP which competitively blocks sialyltransferases and induces feedback inhibition of the de novo sialic acid synthesis pathway. B) Structure of the metabolic sialyltransferase inhibitors that were prepared containing various C-5 amide- or carbamate modifications.

(2) FIG. 2A: strategy for synthesis of various compounds according to the invention, or of reference compounds. i) Dowex H+, MeOH, r.t., 24 hrs ii) Ac.sub.2O, Pyr, r.t., 24 hrs iii) HSTol, BF.sub.3.Et.sub.2O, r.t., 21 hrs, 95% (three steps), iv) Boc.sub.2O, DMAP, THF, 70° C., 1 hr, 88% v) K.sub.2CO.sub.3, MeOH, r.t., 24 hrs, 83% vi) Ac.sub.2O, Pyr, r.t., 7 hrs, 91% vii) Br.sub.2, DCM, r.t., 2.5 hrs viii) TEA, DCM, r.t., 24 hrs, 78% (two steps) ix) Select-Fluor, H.sub.2O, DMF, 60° C., 3 hrs, 72% (based on recovery of 7) x) Ac.sub.2O, Pyr, r.t., 24 hrs, 95%, xi) TfOH or TFA, DCM, r.t., 5 min-4 hrs xii) R—Cl or R—OSu, DCM, TEA or Pyr, 16-24 hrs, 6-60%.

(3) FIG. 2B: depictions of R groups.

(4) FIG. 3: synthesis of C-5 modified metabolic sialic acid biosynthesis inhibitors. Reagents and Conditions: i) Ac.sub.2O, Pyridine, r.t., 48 hrs, 95%; ii) DCM, water, TFA, r.t., 2 hrs; iii) Activated acyl substituents (AAS), TEA, DCM, r.t.; 19: AAS=chloroacetyl chloride, overnight, 60%; 11: AAS=acetoxyacetyl chloride, overnight, 43%; 12: AAS=Azidoacetic acid NHS ester, 23 hrs, 16%; 13: AAS=4-Pentynoic acid NHS ester, overnight, 7%; 14: AAS=N-(propargyloxycarbonyloxy)-succinimide, 15 hrs, 40%; 15: AAS=Allyl chloroformate, 21.5 hrs, 16%; 16: AAS=methyl chloroformate, overnight, 33%; 17: AAS=ethyl chloroformate, overnight, 16%; 18: AAS=isobutyl chloroformate, overnight, 33%; 20: AAS=benzyl chloroformate, 21.5 hrs, 6%; iv) Benzyl azide, TBTA, CuI, Cu, DMF, water, t-butanol, r.t., overnight, 46%.

(5) FIG. 4: Amide and carbamate fluorine sialic acids inhibit sialylation in B16-F10 cells. B16-F10 cells were treated for three days with 0.1-204.8 μM amide or carbamate fluorine sialic acids or DMSO control and stained with the biotinylated lectins MALII or SNA-1 that recognize α2,3-linked or α2,6-linked sialic acids, respectively and streptavidin-PE. Binding of the lectins was determined by flow cytometry and data of three independent experiments are presented as mean percentage lectin binding±SEM normalized to control cells. Graphs show MALII and SNA-1 binding to cells treated with amide fluorine sialic acids (A, C) and carbamate fluorine sialic acids (B, D) shown in FIG. 1B.

(6) FIG. 4E: MALII data for additional fluorine sialic acids, shown in example 3.

(7) FIG. 4F: SNA-1 data for additional fluorine sialic acids, shown in example 3.

(8) FIG. 5: Effect of fluorine sialic acids on cell metabolic activity/viability. B16-F10 cells were cultured for three days with increasing concentrations of fluorine sialic acids or DMSO and subjected to an MTT assay. Representative graphs show absorbance at 595 nm for cells treated with amide (A) or carbamate (B) fluorine sialic acids.

(9) FIG. 6: Carbamate fluorine sialic acids inhibit sialylation in 9464D and EL4 cells. 9464D cells or EL4 cells were treated for three days with DMSO control or 102.4 μM amide or carbamate fluorine sialic acids. Cells were stained with MALII or SNA-1 lectin and streptavidin-PE and lectin binding was determined by flow cytometry. Bar diagrams show mean binding percentages±SEM of MALII and SNA-1 to 9464D cells (A, B) and EL4 cells (C, D) of three independent experiments.

(10) FIG. 7: Recovery of sialylation after amide or carbamate fluorine sialic acid treatment. B16-F10 cells were incubated for three days with 51.2 μM amide or carbamate fluorine sialic acids or DMSO control. Fluorine sialic acids were removed from the culture and the cells were reseeded. During a period of six days, sialylation was assessed daily with flow cytometry by MALII or SNA-1 lectins. Graphs show recovery of α2,3-sialylation (A, B) or α2,6-sialylation (C, D) in time presented as mean percentage lectin binding±SEM normalized to control (n=3).

(11) FIG. 8: urea, sulphonamide, and thiocarbamate fluorine sialic acids inhibit sialylation in B16-F10 cells. B16-F10 cells were treated for three days with 0.1-204.8 μM amide, carbamate, urea, or thiocarbamate fluorine sialic acids or DMSO control and stained with the biotinylated lectins MALII or SNA-1 that recognize α2,3-linked or α2,6-linked sialic acids, respectively and streptavidin-PE. Binding of the lectins was determined by flow cytometry and data of at least 4 independent experiments are presented as mean percentage lectin binding±SEM normalized to control cells. Graphs show MALII binding to cells (A) and SNA-1 binding to cells (B).

EXAMPLES

Example 1—Synthesis

(12) General Synthetic Procedures

(13) .sup.1H and .sup.13C NMR spectra were recorded on a Varian Inova 400 MHz or Bruker Avance III 500 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal standard. NMR data is presented as follows: Chemical shift, multiplicity (s=singlet, bs=broad singlet, d=doublet, t=triplet, dd=doublet of doublet, dt=doublet of triplet, m=multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz). All NMR signals were assigned on the basis of .sup.1H, .sup.13C, .sup.19F NMR, COSY and HSQC experiments. Mass spectra were recorded on a JEOL JMS-T1000S AccuTOF mass spectrometer. Automatic column chromatography was performed on Biotage Isolera Spektra One, using SNAP cartridges 10-50 g filled with normal silica (Biotage, 30-100 μm, 60 Å) or water resistant iatro beads. Microwave reactions were performed on a Biotage Initiator 4.1.3. TLC analysis was conducted on TLC Silicagel, 60, F254, Merck, with detection by UV absorption (254 nm) where applicable, and by spraying with 20% H.sub.2SO.sub.4 in methanol followed by charring at −150° C. or by spraying with a solution of (NH.sub.4).sub.6Mo.sub.7O.sub.24.H.sub.2O (25 g l.sup.−1) in 10% H.sub.2SO.sub.4 in methanol followed by charring at −300° C. DCM, ACN and Tol were freshly distilled. Reactions were carried out under an argon atmosphere.

Commonly Used Abbreviations

(14) Ac.sub.2O—Acetic anhydride; Acet—Acetone; ACN—Acetonitrile; AcOH—Acetic acid; Alloc—Allyloxycarbonyl; Az—Azidoacetic acid; BF.sub.3.Et.sub.2O—Boron trifluoride etherate; Boc.sub.2O—Di-tert-butyl dicarbonate; Br.sub.2—Bromine; Cbz—Carboxybenzyl; CD.sub.3OD—Deuterated methanol; CDCl.sub.3—Deuterated chloroform; ClAcCl—Chloroacetyl chloride; D.sub.2O—Deuterium oxide; DCM—Dichloromethane; DMAP—Dimethylaminopyridine; DMF—N,N-Dimethylformamide; EtOAc—Ethyl acetate; EtOAc—Ethyl acetate; Hept—Heptane; iBu—Iso-butyl; Me—Methyl; n-Bu—Butyl; Poc—Propargylcarboxycarbonyl; Pyr—Pyridine; ROSu—Hydroxysuccinimide ester; SAda—Adamantyl-thiol; STol—4-methylthiophenol; TBTA—Tris(benzyltriazolyl methyl)amine; tBu—Tert-butyl; TEA—Triethylamine; TFA—Trifluoroacetic acid; TFAA—Trifluoroacetic acid anhydride; TfOH—Trifluoromethanesulfonic acid; TMSOTf—Trimethylsilyl trifluoromethanesulfonate; Tol—Toluene; Troc—2,2,2 trichloroethoxycarbonyl.

(15) General Synthetic Strategy

(16) FIG. 2 shows the strategy for synthesis of various compounds according to the invention, or of reference compounds. The P-3F.sub.ax-NeuNAc derivatives were prepared from a common C-5 Boc protected precursor 9 (FIG. 3) to allow for rapid derivatization of the C-5 amine after Boc deprotection in a divergent manner. Thioglycoside 6 (Büll et al., 2015, DOI: 10.1021/acschembio.5b00501) was reacted with bromine to afford a mixture of the corresponding glycosyl bromide and glycal 7. Reaction of the mixture under basic conditions then afforded glycal 7 in a good overall yield (78%). Electrophilic fluorination using Selectfluor in a mixture of water/DMF afforded axial-fluorine 8 as the major product (Burkart et al., 1997, DOI: 10.1021/ja9723904). Finally, 8 was acetylated to afford 9 in high yield (95%).

(17) Next, inhibitor precursor 9 was modified in a two-step sequence of Boc deprotection followed by acylation. Including precursor 9, inhibitor derivatives (11-21, FIG. 3) were prepared, carrying a variety of acyl groups in low to moderate yields (6-60%). Derivatives 12, 13 and 14 contain an alkyne or azide group which is amendable for modification using the copper catalyzed azide alkyne cycloaddition (CuAAC) reaction. To explore whether modifications introduced in this manner are tolerated by the sialic acid biosynthesis, derivative 21 was prepared using 14, benzylazide, and CuI/TBTA.

Intermediate: Methyl (5-acetamido-3,5-dideoxy-5-D-glycero-D-galacto)onate (1)

(18) ##STR00049##

(19) To a solution of N-acetylneuraminic acid (20 g; 64.7 mmol) in MeOH (600 ml; 0.1 M), Dowex (10 g) was added. After stirring at r.t. for 24 hrs, the mixture was filtered. The residue was washed with an excess of MeOH. The filtrate was concentrated in vacuo affording OH 1 (20.9 g; 64.7 mmol; quant.) TLC: (H.sub.2O:ACN, 20:80 v/v) R.sub.f=0.39. .sup.1H NMR (500 MHz, CD.sub.3OD) δ 4.07-3.98 (m, 2H, H-4; H-6), 3.85-3.79 (m, 2H, H-5; H-9.sub.a), 3.78 (s, 3H, OMe), 3.70 (ddd, J=8.8, 5.7, 2.8 Hz, 1H, H-8), 3.62 (dd, J=11.3, 5.7 Hz, 1H, H-9.sub.b), 3.48 (dd, J=9.2, 1.1 Hz, 1H, H-7), 2.22 (dd, J=12.9, 4.9 Hz, 1H, H-3.sub.eq), 2.02 (s, 3H, Me, Ac), 1.89 (dd, J=12.8, 11.5 Hz, 1H, H-3.sub.ax). .sup.13C NMR (126 MHz, CD.sub.3OD) δ 175.10 (C-1), 171.78 (CONH), 96.65 (C-2), 72.06 (C-6), 71.62 (C-8), 70.16 (C-7), 67.83 (C-4), 64.82 (C-9), 54.29 (C-5), 53.71 (OMe), 40.68 (C-3), 22.68 (Me, Ac). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.12H.sub.21NNaO.sub.9, 346.11140; found, 346.11286.

Intermediate: Methyl (5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-5-D-glycero-D-galacto)onate (2)

(20) ##STR00050##

(21) To a solution of 1 (20.9 g; 64.6 mmol) in Pyr (250 ml; 3.09 mol; 47.8 eq.), Ac.sub.2O (125 ml; 1.59 mol; 24.6 eq.) was slowly added. After stirring at r.t. for 24 hrs, the mixture was concentrated in vacuo using Tol for co-evaporation. The residue was dissolved in EtOAc and washed successively with HCl (0.1 M) and sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4 and filtered. The filtrate was concentrated in vacuo affording 2 (34.5 g; 64.6 mmol; quant.) as a white foam. TLC: (Ace:DCM, 40:60 v/v) R.sub.f=0.51. .sup.1H NMR (500 MHz, CDCl.sub.3, major anomer) δ 5.38-5.37 (m, 1H, H-7), 5.26 (ddt, J=10.1, 7.5, 4.9 Hz, 1H, H-4), 5.07 (ddd, J=6.8, 5.1, 2.5 Hz, 1H, H-8), 4.50 (dd, J=12.5, 2.6 Hz, 1H, H-9.sub.a), 4.15-4.10 (m, 3H, H-9.sub.b; H-6; H-5), 3.80 (s, 3H, OMe), 2.55 (dd, J=13.5, 5.0 Hz, 1H, H-3.sub.eq), 2.15 (s, 3H, Me, OAc), 2.14 (s, 3H, Me, OAc), 2.10-2.09 (m, 1H, H-3.sub.ax), 2.07 (s, 3H, Me, OAc), 2.05-2.03 (m, 6H, 2×Me, OAc), 1.90 (s, 3H, Me, NHAc). .sup.13C NMR (126 MHz, CDC.sub.3, major anomer) δ 171.15 (CO), 170.75 (CO), 170.41 (2×CO), 170.38 (CO), 168.37 (CO), 166.46 (CO), 97.65 (C-2), 73.00 (C6), 71.52 (C-8), 68.44 (C-4), 67.98 (C-7), 62.27 (C-9), 53.35 (OMe), 49.49 (C-5), 36.05 (C-3), 23.33 (Me, Ac), 21.05 (Me, Ac), 20.99 (Me, Ac), 20.93 (2×Me, Ac) 20.91 (Me, Ac). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.31NNaO.sub.14, 556.16422; found, 556.16487.

Intermediate: Methyl 5-acetamido-4,7,8,9-penta-O-acetyl-2,3,5-dideoxy-2-para-methylthiophenol-D-glycero-β-galacto-non-2-ulopyranosonate (3)

(22) ##STR00051##

(23) Similar to the previously described procedure (Chao et al., 2008, DOI: 10.1016/j.carres.2008.01.014), 2 (33.87 g; 63.5 mmol) was dissolved in DCM (400 ml; 0.16 M). HSTol (9.26 ml; 76 mmol; 1.2 eq.) was added to form a slightly yellow solution. BF.sub.3.Et.sub.2O (15.7 ml; 127 mmol; 2 eq.) was added and the reaction stirred for 21 hrs, washed with 10% Na.sub.2S.sub.2O.sub.3 aq. and sat. NaHCO.sub.3 aq. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was crystallized using DCM and Hept. The crystals were filtered, washed with Hept and collected with Ace. Evaporation of solvents in vacuo afforded 3 (35.92 g; 60.1 mmol; 95%) as a white foam. TLC: (Acet/DCM, 30/70): R.sub.f=0.4; .sup.1H NMR (500 MHz, CDCl.sub.3, major anomer) δ 7.30 (dd, J=8.1, 1.8 Hz, 2H, 2×CH, meta STol), 7.12 (d, J=7.2 Hz, 2H, 2×CH, ortho STol), 5.68 (d, J=10.3 Hz, 1H, NH), 5.46 (t, J=2.4 Hz, 1H, H-7), 5.37 (td, J=11.1, 4.8 Hz, 1H, H-4), 4.95 (d, J=8.5 Hz, 1H, H-8), 4.61 (dd, J=10.5, 2.5 Hz, 1H, H-6), 4.49 (dd, J=12.3, 2.2 Hz, 1H, H-9.sub.a), 4.12 (q, J=10.4 Hz, 1H, H-5), 4.02 (dd, J=12.2, 8.6 Hz, 1H, H-9.sub.b), 3.58 (s, 3H, OMe), 2.64 (dd, J=13.8, 4.7 Hz, 1H, H-3.sub.eq), 2.31 (s, 3H, Me, STol), 2.11-2.08 (m, 4H, H-3.sub.ax; Me, Ac), 2.07 (s, 3H, Me, Ac), 2.03 (s, 3H, Me, Ac), 1.95 (s, 3H, Me, Ac). .sup.13C NMR (126 MHz, CDCl.sub.3, major anomer) δ 171.13 (CO, Ac), 170.90 (CO, Ac), 170.23 (CO, Ac), 170.19 (CO, Ac), 170.16 (CO, Ac), 168.21 (C-1), 140.08 (CCH.sub.3, STol), 136.14 (2×CH, meta STol), 129.80 (2×CH, ortho STol), 125.17 (C-5, STol), 88.82 (C-2), 73.10 (C-6), 73.01 (C-8), 69.05 (C-4), 68.78 (C-7), 62.64 (C-9), 52.51 (OMe), 49.36 (C-5), 37.33 (C-3), 23.10 (Me, Ac), 21.25 (Me, STol), 21.04 (Me, Ac), 20.83 (Me, Ac), 20.68 (Me, Ac), 20.64 (Me, Ac). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.27H.sub.35NNaO.sub.12S, 620.17776; found, 620.17694.

Intermediate: Methyl 5-[(tert-butoxycarbonyl)amino]-4,7,8,9-penta-O-acetyl-2,3,5-dideoxy-2-para-methylthiophenol-D-glycero-β-galacto-non-2-ulopyranosonate (4)

(24) ##STR00052##

(25) Similar to the previously described procedure (Büll et al., 2015, DOI: 10.1021/acschembio.5b00501), 3 (30.93 g; 51.7 mmol) was dissolved in THE (500 ml; 0.1 M). Successively, Boc.sub.2O (24.0 ml; 103 mmol; 2 eq.) and 4-dimethylaminopyridine (3.16 g; 25.9 mmol; 0.5 eq.) were added. After stirring at 70° C. for 2 hrs, the 4 mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with sat. aq. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.10% Ace in DCM) afforded 4 (31.66 g; 45.4 mmol; 88%) as a slightly yellow foam. TLC: (Acet:DCM, 5:95 v/v) R.sub.f=0.47 .sup.1H NMR (500 MHz, CDCl.sub.3, major anomer) δ 7.34 (d, J=8.2 Hz, 2H, 2×CH, meta STol), 7.14 (d, J=8.0 Hz, 3H, 2×CH, ortho STol), 5.80 (td, J=11.0, 4.9 Hz, 1H, H-4), 5.44 (dd, J=10.1, 2.1 Hz, 1H, H-6), 5.35 (t, J=2.5 Hz, 1H, H-7), 5.12 (dt, J=8.2, 2.6 Hz, 1H, H-8), 4.85 (t, J=10.5 Hz, 1H, H-5), 4.49 (dd, J=12.4, 2.3 Hz, 1H, H-9.sub.b), 4.06 (dd, J=12.4, 8.2 Hz, 1H, H-9.sub.a), 3.58 (s, 3H, OMe), 2.74 (dd, J=13.8, 4.9 Hz, 1H, H-3.sub.eq), 2.36 (s, 3H, Me, NAc), 2.34 (s, 3H, Me, STol), 2.13 (dd, J=13.8, 11.1 Hz, 1H, H-3.sub.ax), 2.07 (s, 3H, Me, OAc), 2.06 (s, 3H, Me, OAc), 1.97 (s, 3H, Me, OAc), 1.97 (s, 3H, Me, OAc), 1.72 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3, major anomer) δ 173.93 (CO, Ac), 170.64 (CO, Ac), 170.50 (CO, Ac), 170.33 (CO, Ac), 169.94 (CO, Ac), 168.28 (C-1), 152.10 (CO, Boc), 140.21 (CCH.sub.3, STol), 136.40 (2×CH meta STol), 129.95 (2×CH ortho STol), 125.76 (C-5, STol), 89.50 (C-2), 85.42 (C(CH.sub.3).sub.3, Boc), 72.91, (C-8), 72.29 (C-6), 68.68 (C-7), 66.54 (C-4), 62.53 (C-9), 52.98 (C-5), 52.57 (OMe), 38.88 (C-3), 28.29 (tBu, Boc), 26.73 (Me, NHAc), 21.44 (Me, STol), 21.13 (Me, OAc), 20.93 (Me, OAc), 20.81 (2×Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.32H.sub.43NNaO.sub.14S, 720.23019; found, 720.23055.

Intermediate: Methyl 5-(tert-butoxycarbamado)-2,3,5-dideoxy-2-para-methylthiophenol-D-glycero-β-galacto-non-2-ulopyranosonate (5)

(26) ##STR00053##

(27) As described previously (Büll et al., 2015, DOI: 10.1021/acschembio.5b00501), 4 (31.65 g; 45.4 mmol) was dissolved in MeOH (200 ml; 0.23 M) and K.sub.2CO.sub.3 (3.13 g; 22.7 mmol; 0.5 eq.) was added. After stirring at r.t. for 24 hrs the reaction was quenched with AcOH to pH 6 and filtered. The filtrate was concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.10% MeOH in DCM) afforded 5 (18.39 g; 37.7 mmol; 83%). TLC: (MeOH:DCM, 10:90 v/v) R.sub.f=0.56 .sup.1H NMR (500 MHz, CDCl.sub.3, major anomer) δ 7.35 (d, J=8.1 Hz, 2H, 2×CH, meta STol), 7.11 (d, J=8.0 Hz, 2H, 2×CH, ortho STol), 5.27 (d, J=8.7 Hz, 1H, NH), 4.30 (d, J=10.4 Hz, 1H, H-6), 4.08 (td, J=10.9, 4.6 Hz, 1H, H-5), 3.80-3.65 (m, 5H, H-9.sub.a; H-9.sub.b; H-8; H-7; H-5), 3.57 (s, 3H, OMe), 2.71 (dd, J=13.8, 4.7 Hz, 1H, H-3.sub.eq), 2.32 (s, 3H, Me, STol), 2.05-1.99 (m, 1H, H-3.sub.ax), 1.45 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3, major anomer) δ 169.49 (C-1), 157.82 (CO, Boc), 139.98 (C-Me STol), 135.73 (2×CH, meta STol), 129.87 (2×CH, ortho STol), 126.24 (C-5 STol), 89.59 (C-2), 81.13 (C(CH.sub.3).sub.3, Boc) 72.85 (C-6), 70.56 (C-7), 70.23 (C-8), 68.20 (C-4), 64.55 (C-9), 54.12 (C-5), 52.78 (OMe), 40.67 (C-3), 28.47 (tBu, Boc), 21.39 (Me, STol). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.33NNaO.sub.9S, 510.17737; found, 510.17732.

Intermediate: Methyl 5-(tert-butoxycarbamado)-4,7,8,9-penta-O-acetyl-2,3,5-dideoxy-2-para-methylthiophenol-D-glycero-β-galacto-non-2-ulopyranosonate (6)

(28) ##STR00054##

(29) As described previously (Büll et al., 2015, DOI: 10.1021/acschembio.5.sub.b00501), 5 (8.39 g; 17.2 mmol) was dissolved in Pyr (62 ml; 764 mmol; 44.4 eq.). Ac.sub.2O (36 ml; 382 mmol; 22.2 eq.) was slowly added. After stirring at r.t. for 7 hrs, the mixture was concentrated in vacuo using Tol for co-evaporation. The residue was dissolved in EtOAc and washed successively with HCl (0.1 M) and sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4 and filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 6 (10.26 g; 15.6 mmol; 91%) as a white foam. TLC: (EtOAc:Hept, 50:50 v/v) R.sub.f=0.47 .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.32 (d, J=7.7 Hz, 2H, 2×CH, meta STol), 7.14 (d, J=7.7 Hz, 2H, 2×CH, ortho STol), 5.55 (t, J=2.6 Hz, 1H, H-7), 5.33 (td, J=11.1, 4.8 Hz, 1H, H-4), 5.01 (dt, J=8.5, 2.5 Hz, 1H, H-8), 4.55 (dd, J=10.5, 2.6 Hz, 1H, H-6), 4.51 (dd, J=12.2, 2.3 Hz, 1H, H-9.sub.a), 4.45 (d, J=10.7 Hz, 1H, NH), 4.04 (dd, J=12.2, 8.5 Hz, 1H, H-9.sub.b), 3.79 (q, J=10.6 Hz, 1H, H-5), 3.61 (s, 3H, OMe), 2.67 (dd, J=13.8, 4.8 Hz, 1H, H-3.sub.eq), 2.34 (s, 3H, Me, STol), 2.10 (s, 3H, Me, OAc), 2.08 (s, 3H, Me, OAc), 2.04 (s, 4H, H-3.sub.ax; Me, OAc), 1.97 (s, 3H, Me, OAc), 1.40 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.98 (CO, OAc), 170.61 (CO, OAc), 170.43 (CO, OAc), 169.93 (CO, OAc), 168.48 (C-1), 155.35 (CO, Boc), 140.22 (C-Me, STol) 136.38 (2×CH, meta STol), 129.99 (2×CH ortho STol), 125.41 (C-5, STol), 88.87 (C-2), 80.29 (C(CH.sub.3).sub.3, Boc), 73.34 (C-6), 72.90 (C-8), 69.51 (C-4), 69.12 (C-7), 62.92 (C-9), 52.68 (OMe), 50.96 (C-5), 37.59 (C-3), 28.28 (tBu, Boc), 21.44 (Me, STol), 21.21 (Me, OAc), 20.94 (Me, OAc), 20.83 (Me, OAc), 20.82 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.30H.sub.41NNaO.sub.13S, 678.21963; found, 678.21799.

Intermediate: Methyl 5-(tert-butoxycarbamado]-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonate (7)

(30) ##STR00055##

(31) To a solution of 6 (2.757 g; 4.22 mmol) in DCM (42.2 ml; 0.1 M), slowly Br.sub.2 (0.239 ml; 4.64 mmol; 1.1 eq.) was added. After 2.5 hrs stirring at r.t., the reaction was diluted with DCM and washed with 10% Na.sub.2S.sub.2O.sub.3. The milky organic layer was dried over MgSO.sub.4, filtered and the clear filtrate was extracted once more with 10% Na.sub.2S.sub.2O.sub.3 aq. before drying over MgSO.sub.4, filtering. The filtrate was concentrated in vacuo, redissolved in DCM (42.0 ml; 0.1 M) and TEA (1.699 g; 16.79 mmol; 4 eq.) was added. The reaction was stirred overnight at r.t. and concentrated in vacuo. The residue was dissolved in EtOAc and washed successively with HCl (0.1 M) and sat. aq. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and again concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.45% EtOAc in Hept) afforded 7 (1.734 g; 3.26 mmol; 78% two steps) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.62 .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.99 (d, J=3.1 Hz, 1H, H-3), 5.55 (t, J=4.3 Hz, 1H, H-7), 5.47 (dd, J=7.5, 3.1 Hz, 1H, H-4), 5.37 (ddd, J=6.8, 4.9, 3.4 Hz, 1H, H-8), 4.65 (d, J=9.9 Hz, 1H, NH), 4.60 (dd, J=12.3, 3.4 Hz, 1H, H-9.sub.a), 4.33 (dd, J=9.0, 3.8 Hz, 1H, H-6), 4.19 (dd, J=12.2, 6.8 Hz, 1H, H-9.sub.b), 4.09 (q, J=8.9 Hz, 1H, H-5), 3.80 (s, 3H, OMe), 2.13 (s, 3H, Me, OAc), 2.08 (s, 3H, Me, OAc), 2.06 (s, 3H, Me, OAc), 1.41 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.73 (CO, Ac), 170.68 (CO, Ac), 170.05 (CO, Ac), 169.91 (CO, Ac), 161.78 (C-1), 154.98 (CO, Boc), 145.15 (C-2), 108.11 (C-3), 80.57 (C(CH.sub.3).sub.3, Boc), 76.94 (C-6), 70.58 (C-8), 68.60 (C-4), 67.92 (C-7), 62.10 (C-9), 52.66 (OMe), 48.01 (C-5), 28.27 (tBu, Boc), 20.96 (Me, OAc), 20.91 (Me, OAc), 20.85 (Me, OAc), 20.80 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.23H.sub.33NNaO.sub.13, 554.18496; found, 554.18611.

Methyl 5-(tert-butoxycarbamado)-4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (8)

(32) ##STR00056##

(33) To a solution of 7 (1.724 g; 3.24 mmol) in a 1:3 mixture of H.sub.2O and DMF (32 ml; 0.1 M), Selectfluor (3.45 g; 9.73 mmol; 3 eq.) was added. The reaction was stirred at 60° C. for 3 hrs. The mixture was quenched with sat. aq. NaHCO.sub.3 and concentrated in vacuo—even though conversion was incomplete. The residue was dissolved in EtOAc and washed successively with HCl (0.1M) and sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 8 (789 mg; 1.39 mmol; 72% based on recovery of starting material) as a white foam. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.47 .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.52 (s, 1H, OH), 5.47 (dd, J=4.4, 2.3 Hz, 1H, H-7), 5.37-5.23 (m, 2H, H-8; H.sub.4), 4.94 (d, J=10.3 Hz, 1H, NH), 4.92 (dd, J=49.8, 2.1 Hz, 1H, H-3), 4.81 (dd, J=12.3, 2.5 Hz, 1H, H-9.sub.a), 4.26 (dd, J=10.6, 2.3 Hz, 1H, H-6), 4.18-4.08 (m, 2H, H-5; H-9.sub.b), 3.85 (s, 3H, OMe), 2.16 (s, 3H, Me, OAc), 2.09 (s, 3H, Me, OAc), 2.09 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 1.40 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.68 (CO, Ac), 171.48 (CO, Ac), 170.60 (CO, Ac), 170.19 (CO, Ac), 167.70 (C-1), 155.22 (CO, Boc), 94.45 (d, J=25.5 Hz, C-2), 87.06 (d, J=185.1 Hz, C-3), 80.09 (C(CH.sub.3).sub.3, Boc), 71.91 (C-8), 71.46 (C-6), 69.96 (d, J=17.3 Hz, C-4), 68.63 (C-7), 63.01 (C-9), 53.50 (OMe), 46.36 (C-5), 28.31 (tBu, Boc), 21.13 (Me, OAc), 20.99 (Me, OAc), 20.85 (Me, OAc), 20.78 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.23H.sub.34FNNaO.sub.14, 590.18610; found, 590.18498.

Methyl 5-(tert-butoxycarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (9)

(34) ##STR00057##

(35) To a solution of 8 (789 mg; 1.39 mmol) in Pyr (12 ml; 148 mmol; 107 eq.), Ac.sub.2O (6 ml; 63.6 mmol; 45.7 eq.) was slowly added. After stirring at r.t. for 24 hrs, the mixture was concentrated in vacuo using Tol for co-evaporation. The residue was dissolved in EtOAc and washed successively with HCl (0.1M) and sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 9 (717 mg; 1.176 mmol; 95%) as a white foam. TLC: (EtOAc:Hept, 50:50 v/v) R.sub.f=0.42 .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.46-5.34 (m, 2H, H-7; H-4), 5.18-5.11 (m, 1H, H-8), 4.92 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.60-4.54 (m, 1H, H-9.sub.a), 4.48 (d, J=9.5 Hz, 1H, NH), 4.19 (dd, J=12.4, 6.6 Hz, 1H, H9.sub.b), 4.12-3.97 (m, 2H, H-6; H-5), 3.84 (s, 3H, OMe), 2.17 (s, 3H, Me, OAc), 2.16 (s, 3H, Me, OAc), 2.12 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 1.40 (s, 9H, tBu, Boc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.73 (CO, Ac), 170.48 (CO, Ac), 170.36 (CO, Ac), 170.25 (CO, Ac), 167.24 (CO, Ac), 165.28 (C-1), 154.88 (CO, Boc), 95.33 (d, J=29.0 Hz, C-2), 87.21 (d, J=185.1 Hz, C-3), 80.51 (C(CH.sub.3).sub.3, Boc), 72.67 (C-6), 71.39 (C-8), 68.97 (d, J=17.2 Hz, C-4), 68.09 (C-7), 62.32 (C-9), 53.60 (OMe), 46.32 (C5), 28.27 (tBu, Boc), 21.00 (Me, OAc), 20.90 (Me, OAc), 20.86 (Me, OAc), 20.82 (Me, OAc), 20.74 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.25H.sub.36FNNaO.sub.15, 632.19667; found, 632.19540.

Reference Compound: Methyl 5-(trifluoroacetamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (10)

(36) ##STR00058##

(37) TFA protected Sialic acid 10 was a common side product after TFA deprotection. Further investigation discovered that TFA, if not removed carefully, forms a mixed anhydride with chloroformates resulting in a sometimes quantitative coupling of TFA to the relatively unreactive amine. Later deprotection reactions were therefore done with TfOH which avoids this problem. Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:2 mixture of TFA and DCM (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then concentrated in vacuo. The residue was dissolved in DCM (0.83 ml; 0.1 M) and additional TFA (188 μl, 2.5 mmol, 30 eq) and TEA (690 μl; 4.95 mmol; 60 eq.) were added. Isobutyl chloroformate (also possible with other chloroformates) (76 μl; 589 μmol; 20 eq.) was added and the reaction was stirred for 16 hrs. The mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 10 (44 mg; 74 μmol; 90%) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.50 .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.02 (d, J=9.1 Hz, 1H, NH), 5.61 (ddd, J=27.4, 11.0, 2.5 Hz, 1H, H-4), 5.29 (dd, J=5.3, 2.0 Hz, 1H, H-7), 5.13 (ddd, J=6.1, 5.2, 2.4 Hz, 1H, H-8), 4.97 (dd, J=48.9, 2.5 Hz, 1H, H-3), 4.57 (dd, J=12.5, 2.5 Hz, 1H, H-9.sub.a), 4.38 (ddd, J=10.7, 2.0, 0.8 Hz, 1H, H-6), 4.26-4.17 (m, 2H, H-5; H-9.sub.b), 3.85 (s, 3H, OMe), 2.19 (s, 3H, Me, OAc), 2.18 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.70 (CO, Ac), 170.78 (CO, Ac), 170.76 (CO, Ac), 170.51 (CO, Ac), 167.22 (C-1), 164.94 (CO, Ac), 157.68 (q, J=37.8 Hz, CO, TFA), 115.50 (q, J=288.4 Hz, CF.sub.3), 95.16 (d, J=29.0 Hz, C-2), 86.90 (d, J=186.1 Hz, C-3), 71.27 (C-8), 71.00 (C-6), 68.00 (d, J=16.9 Hz, C-4), 67.91 (C-7), 62.06 (C-9), 53.75 (OMe), 46.43 (d, J=2.4 Hz, C-5), 20.99 (Me, OAc), 20.82 (Me, OAc), 20.80 (Me, OAc), 20.60 (Me, OAc), 20.56 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.27F.sub.4NNaO.sub.14, 628.12654; found, 628.12591.

Methyl 5-(acetoxyamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (11)

(38) ##STR00059##

(39) Boc inhibitor 9 (40 mg; 66 μmol) was dissolved in a 1:1:2 mixture of DCM, H.sub.2O and TFA (0.7 ml; 0.1 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The residue was dissolved in DCM (0.7 ml; 0.1 M) and successively acetoxyacetyl chloride (11 μl; 97 μmol; 1.5 eq.) and TEA (45 μl; 324 mol; 5 eq.) were added. After stirring at r.t. overnight the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.60% EtOAc in Hept) afforded 11 (17 mg; 28 μmol; 43%) as a white solid. TLC: (EtOAc:Hept, 80:20 v/v) R.sub.f=0.19. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 6.18 (d, J=9.0 Hz, 1H, NH), 5.64 (ddd, J=27.9, 11.0, 2.5 Hz, 1H, H-4), 5.28 (dd, J=5.3, 2.0 Hz, 1H, H-7), 5.13 (ddd, J=6.3, 5.2, 2.4 Hz, 1H, H-8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.61 (d, J=15.3 Hz, 1H, CHH Glc), 4.56 (dd, J=12.5, 2.4 Hz, 1H, H-9.sub.a), 4.32 (d, J=15.3 Hz, 1H, CHH Gc), 4.30-4.26 (m, 1H, H-6), 4.22-4.15 (m, 2H, H-9.sub.b; H-5), 3.84 (s, 3H, OMe), 2.20 (s, 3H, Me, OAc), 2.19 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.05 (CO, Ac), 170.88 (CO, Ac), 170.83 (CO, Ac), 170.53 (CO, Ac), 169.98 (CO, Ac), 168.03 (CO, Ac), 167.35 (CO, C-1), 165.23 (CO, NHGc), 95.38 (d, J=29.0 Hz, C-2), 87.28 (d, J=185.5 Hz, C-3), 71.91 (C-6), 71.41 (C-8), 68.27 (C-7), 67.98 (d, J=17.1 Hz, C-4), 63.01 (CH.sub.2, Gc), 62.20 (C-9), 53.76 (OMe), 45.88 (d, J=2.6 Hz, C-5), 21.11 (Me, OAc), 21.07 (Me, OAc), 20.95 (Me, OAc), 20.85 (2×Me, OAc), 20.80 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.24H.sub.32FNNaO.sub.16, 632.16028; found, 632.15804.

Reference Compound: Methyl 5-(azidoacetamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (12)

(40) ##STR00060##
Boc inhibitor 9 (20 mg; 33 μmol) was dissolved in a 1:1:2 mixture of respectively DCM, H.sub.2O and TFA (0.65 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with water and concentrated in vacuo. The residue was dissolved in DCM (0.33 ml; 0.1 M) and successively AzOSu (42 mg; 167 μmol; 5 eq.), Pyr (27 μl; 334 μmol; 10 eq.) and DMAP (2 mg, 17 μmol; 0.5 eq.) were added. After stirring at r.t. for 23 hrs the mixture was diluted with DCM and washed successively with 0.1 M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 12 (3.1 mg; 5.2 μmol; 16%). TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.20. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 6.31 (d, J=8.6 Hz, 1H, NH), 5.54 (ddd, J=27.5, 10.6, 2.5 Hz, 1H, H-4), 5.34 (dd, J=5.3, 1.8 Hz, 1H, H-7), 5.13 (td, J=5.7, 2.4 Hz, 1H, H-8), 4.95 (dd, J=49.0, 2.5 Hz, 1H, H-3), 4.55 (dd, J=12.5, 2.4 Hz, 1H, H-9.sub.a), 4.30-4.22 (m, 2H, H-5; H-6), 4.19 (dd, J=12.6, 6.3 Hz, 1H, H-9.sub.b), 3.94-3.83 (m, 5H, CH.sub.2 NAz; OMe), 2.20 (s, 3H, Me, OAc), 2.16 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.94 (CO, Ac), 170.84 (CO, Ac), 170.66 (CO, Ac), 170.47 (CO, Ac), 167.52 (CO, Ac), 167.31 (C-1), 165.20 (CO, Az), 95.64 (d, J=185.7 Hz, C-2), 87.16 (d, J=185.7 Hz, C-3), 71.95 (C-6), 71.43 (C-8), 68.49 (d, J=17.2 Hz, C-4), 68.02 (C-7), 62.16 (C-9), 53.82 (OMe), 52.89 (CH.sub.2, Az), 49.50 (C-5), 21.13 (Me, OAc), 21.06 (Me, OAc), 21.00 (Me, OAc), 20.91 (Me, OAc), 20.81 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.29FN.sub.4NaO.sub.14, 615.15620; found, 615.15758.

Reference Compound: Methyl 5-(4-pentynacetamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (13)

(41) ##STR00061##

(42) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:2 mixture of respectively TFA, H.sub.2O and DCM (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with water and concentrated in vacuo. The residue was dissolved in DCM (0.83 ml; 0.1 M) and successively 4-Pentynoic acid-OSu (226 mg; 1.158 mmol; 14 eq.) and TEA (69 μl; 495 μmol; 6 eq.) were added. After stirring at r.t. overnight the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.55% EtOAc in Hept) afforded 13 (3.3 mg; 5.60 μmol; 7%) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.60 (ddd, J=27.9, 11.0, 2.6 Hz, 1H, H-4), 5.54 (d, J=9.0 Hz, 1H, NH), 5.37 (dd, J=5.5, 2.0 Hz, 1H, H-7), 5.15 (ddd, J=6.3, 5.4, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.53 (dd, J=12.5, 2.4 Hz, 1H, H-9.sub.a), 4.30-4.26 (m, 1H, H-6), 4.21 (dd, J=12.5, 6.3 Hz, 1H, H-9.sub.b), 4.13 (q, J=10.3 Hz, 1H, H-5), 3.84 (s, 3H, OMe), 2.56-2.44 (m, 2H, CH.sub.2C≡CH), 2.40-2.27 (m, 2H, CH.sub.2—CONH), 2.19 (s, 3H, Me, OAc), 2.16 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 2.02 (t, J=2.6 Hz, 1H, HC≡C). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.38 (CO, Ac), 170.85 (CO, Ac), 170.69 (CO, AC), 170.53 (CO, Ac), 170.31 (CO, Ac), 167.28 (C-1), 165.21 (CONH), 95.29 (d, J=29.0 Hz, C-2), 87.12 (d, J=185.1 Hz, C-3), 82.88 (HC≡C), 71.75 (C-6), 71.21 (C-8), 69.79 (HC≡C), 68.33-68.14 (m, C-4; C-7), 62.22 (C-9), 53.65 (OMe), 46.01 (C-5), 35.67 (CH.sub.2CONH), 21.02 (Me, OAc), 20.98 (Me, OAc), 20.93 (Me, OAc), 20.92 (Me, OAc), 20.74 (Me, OAc), 14.80 (CH.sub.2C≡CH). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.25H.sub.32FNNaO.sub.14, 612.17045; found, 612.16924.

Methyl 5-(propargylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (14)

(43) ##STR00062##

(44) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:2 mixture of respectively DCM, H.sub.2O and TFA (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The residue was dissolved in DCM (0.83 ml; 0.1 M) and successively PocOSu (98 mg; 497 μmol; 6 eq.) and TEA (35 μl; 248 μmol; 3 eq.) were added. After stirring at r.t. for 15 hrs the mixture was diluted with DCM and washed successively with 0.1 M HCl and sat. aq. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.60% EtOAc in Hept) afforded 14 (20 mg; 83 μmol; 40%) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.36. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.53 (dd, J=26.9, 10.8 Hz, 1H, H-4), 5.39-5.36 (m, 1H, H-7), 5.18 (td, J=5.9, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.87 (d, J=9.5 Hz, 1H, NH), 4.73 (ddd, J=15.5, 5.4, 2.5 Hz, 1H, CHH. Poc), 4.57-4.49 (m, 2H, CHH, Poc; H-9.sub.a), 4.23-4.17 (m, 2H, H9.sub.b; H-6), 3.95-3.86 (m, 1H, H-5), 3.84 (s, 3H, OMe), 2.47 (t, J=2.4 Hz, 1H, C≡CH, Poc), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.13 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.75 (CO, Ac), 170.69 (CO, Ac), 170.46 (CO, Ac), 170.24 (CO, Ac), 167.27 (C-1), 165.17 (CO, Ac), 154.75 (CO, Poc), 95.20 (d, J=28.8 Hz, C-2), 87.18 (d, J=185.1 Hz, C-3), 77.95 (C≡CH, Poc), 75.00 (C≡CH, Poc), 71.89 (C-6), 71.00 (C-8), 68.32-67.98 (m, C-4; C-7), 62.20 (C-9), 53.66 (OMe), 53.16 (CH.sub.2, Poc), 47.37 (C-5), 21.00 (Me, OAc), 20.94 (Me, OAc), 20.92 (Me, OAc), 20.82 (Me, OAc), 20.72 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.24H.sub.30FNNaO.sub.15, 614.14972; found, 614.15007.

5-(alloxycarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (15)

(45) ##STR00063##

(46) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:2 mixture of respectively TFA, H.sub.2O and DCM (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with water and concentrated in vacuo. The residue was dissolved in DCM (0.83 ml; 0.1 M) and Alloc-Cl (11 μl; 99 μmol; 1.2 eq.) and TEA (69 μl; 495 μmol; 6 eq.) were added. After stirring at r.t. for 1 hr additional Alloc-Cl (18 μl; 165 μmol; 2 eq.) was added. The reaction was stirred for 15 hrs after which the reaction was still not finished, so additional Alloc-Cl (90 μl; 844 μmol; 10.2 eq.) and TEA (35 μl; 252 μmol; 3 eq.) were added. After stirring at r.t. for 5.5 hrs the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 15 (7.7 mg; 13 μmol; 16%) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.32. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.89 (tdd, J=16.1, 8.3, 3.3 Hz, 1H, CHH═CH, Alloc), 5.54 (dd, J=27.7, 10.9 Hz, 1H, H-4), 5.39 (dd, J=5.5, 2.0 Hz, 1H, H-7), 5.28 (dd, J=16.1, 2.4 Hz, 1H, CHH═CH, Alloc), 5.21 (d, J=11.2 Hz, 1H, CHH═CH, Alloc), 5.18 (td, J=6.0, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.79 (d, J=9.3 Hz, 1H, NH), 4.60-4.45 (m, 3H, H-9.sub.a; OCH.sub.2 Alloc), 4.24-4.17 (m, 2H, H-6; H-9.sub.b), 3.94-3.87 (m, 1H, H-5), 3.84 (s, 3H, OMe), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.75 (CO, Ac), 170.66 (CO, Ac), 170.45 (CO, Ac), 170.27 (CO, Ac), 167.30 (C-1), 165.22 (CO, Ac), 155.41 (CO, Alloc), 132.65 (CH.sub.2═CH, Alloc), 117.84 (CH.sub.2═CH, Alloc), 95.21 (d, J=28.8 Hz, C-2), 87.20 (d, J=184.9 Hz, C-3), 71.98 (C-6), 71.07 (C-8), 68.17-67.92 (C-4; C-7), 66.09 (OCH.sub.2, Alloc), 62.24 (C-9), 53.64 (OMe), 47.22 (C-5), 20.99 (Me, OAc), 20.93 (Me, OAc), 20.91 (Me, OAc), 20.80 (Me, OAc), 20.71 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.24H.sub.32FNNaO.sub.15, 616.16537; found, 616.16544.

Methyl 5-(methylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (16)

(47) ##STR00064##

(48) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:3 mixture of respectively TFA, water and DCM (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The residue was dissolved in DCM (0.8 ml; 0.1 M) and Me-chloroformate (183 μl; 1.649 mmol; 20 eq.) and TEA (229 μl; 1.649 mmol; 20 eq.) were added. The reaction was stirred at r.t. overnight after which the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 16 (16 mg; 27 μmol; 33%). TLC: (EtOAc:Hept:MeOH, 45:45:10 v/v) R.sub.f=0.27. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.59-5.47 (m, 1H, H-4), 5.41-5.38 (m, 1H, H-7), 5.17 (ddd, J=6.3, 5.3, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.1, 2.6 Hz, 1H, H-3), 4.77 (d, J=9.2 Hz, 1H, NH), 4.54 (dd, J=12.5, 2.6 Hz, 1H, H-9.sub.a), 4.25-4.17 (m, 2H, H-9.sub.b; H-6), 3.88-3.85 (m, 1H, H-5), 3.84 (s, 3H, MeO—C-1), 3.64 (s, 3H, MeO—CONH), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.12 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.73 (2×CO, Ac), 170.45 (CO, Ac), 170.30 (CO, Ac), 167.30 (C-1), 165.21 (CO, Ac), 156.17 (NHCO), 95.20 (d, J=28.7 Hz, C-2), 87.18 (d, J=184.8 Hz, C-3), 72.04 (C.sub.6), 71.20 (C-8), 68.42-68.16 (m, C-4; C-7), 62.26 (C-9), 53.62 (MeO—C1), 52.76 (MeO—CONH), 47.26 (C-5), 20.99 (Me, OAc), 20.91 (Me, OAc), 20.90 (Me, OAc), 20.78 (Me, OAc), 20.68 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.30FNNaO.sub.15, 590.14972; found, 590.14874.

Methyl 5-(ethylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (17)

(49) ##STR00065##

(50) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:3 mixture of respectively TFA, water and DCM (1.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with water and concentrated in vacuo. The 1 residue was dissolved in DCM (0.8 ml; 0.1 M) and Et-chloroformate (197 μl; 1.649 mmol; 20 eq.) and TEA (229 μl; 1.649 mmol; 20 eq.) were added. The reaction was stirred at r.t. overnight after which the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 17 (8 mg; 13 μmol; 16%). TLC: (EtOAc:Hept:MeOH, 45:45:10 v/v) R.sub.f=0.28. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.58-5.46 (m, 1H, H-4), 5.40 (dt, J=5.0, 2.4 Hz, 1H, H-7), 5.18 (td, J=5.8, 2.7 Hz, 1H, H-8), 4.95 (dd, J=49.2, 2.5 Hz, 1H, H-3), 4.65 (d, J=9.3 Hz, 1H, NH), 4.57-4.51 (m, 1H, H-9.sub.a), 4.24-4.16 (m, 2H, H-9.sub.b; H6), 4.11-4.03 (m, 2H, CH.sub.2, Et), 3.90 (d, J=10.3 Hz, 1H, H-5), 3.84 (s, 3H, OMe), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.12 (s, 3H, Me, OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 1.25-1.20 (m, 3H, Me, Et). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.73 (CO, Ac), 170.66 (CO, Ac), 170.42 (CO, Ac), 170.29 (CO, Ac), 167.27 (C-1), 165.42 (CO, Ac), 155.74 (CONH), 95.24 (d, J=28.6 Hz, C-2), 87.23 (d, J=184.8 Hz, C-3), 72.15 (C-6), 71.16 (C-8), 68.20 (C-4), 67.89 (C-7), 62.27 (C-9), 61.63 (CH.sub.2, Et), 53.62 (OMe), 47.13 (C-5), 20.99 (Me, OAc), 20.93 (Me, OAc), 20.91 (Me, OAc), 20.78 (Me, OAc), 20.71 (Me, OAc), 14.62 (Me, Et). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.23H.sub.32FNNaO.sub.15, 604.16537; found, 604.16438.

Methyl 5-(isobutylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (18)

(51) ##STR00066##

(52) Boc inhibitor 9 (18 mg; 30 μmol) was dissolved in a 1:1:3 mixture of respectively TFA, H.sub.2O and DCM (0.6 ml; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f 0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The mixture was redissolved in Tol and concentrated in vacuo three times. The residue was then dissolved in DCM (0.3 ml; 0.1 M) and .sup.iBu chloroformate (76 μl; 589 μmol; 20 eq.) and TEA (82 μl; 589 μmol; 20 eq.) were added. The reaction was stirred at r.t. overnight after which the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 9 (6 mg; 9.8 μmol; 33%). TLC: (EtOAc:Hept:MeOH, 45:45:10 v/v) R.sub.f=0.40. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.64-5.49 (m, 1H, H-4), 5.42-5.37 (m, 1H, H-7), 5.18 (td, J=6.0, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.2, 2.5 Hz, 1H, H-3), 4.73 (d, J=9.1 Hz, 1H, NH), 4.53 (dd, J=12.5, 2.5 Hz, 1H, H-9.sub.a), 4.25-4.17 (m, 2H, H-9.sub.b; H-6), 3.92-3.75 (m, 6H, H-5; OMe; CH.sub.2, .sup.iBu), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.11 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 1.95-1.84 (m, 1H, CH, .sup.iBu), 0.90 (d, J=6.8 Hz, 6H, 2×Me, .sup.iBu). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.72 (CO, Ac), 170.39 (CO, Ac), 170.21 (CO, Ac), 170.00 (CO, Ac), 167.28 (C-1), 165.24 (CO, Ac), 155.87 (CONH), 95.25 (d, J=29.0 Hz, C-2), 87.24 (d, J=184.8 Hz, C-3), 72.00 (C-6), 71.56 (CH.sub.2, .sup.iBu), 71.06 (C-8), 68.16-68.01 (m, C-7; C-4), 62.21 (C-9), 53.62 (OMe), 47.18 (C-5), 28.05 (CH.sub.2, .sup.iBu), 20.98 (Me, OAc), 20.92 (Me, OAc), 20.89 (Me, OAc), 20.75 (Me, OAc), 20.70 (Me, OAc), 19.02 (2×Me, .sup.iBu) HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.25H.sub.36FNNaO.sub.15, 632.19667; found, 632.19698.

Reference Compound: Methyl 5-(chloroacetamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (19)

(53) ##STR00067##

(54) Boc inhibitor 9 (200 mg; 329 μmol) was dissolved in a 1:1:2 mixture of DCM, H.sub.2O and TFA (3.3 mL; 0.1 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The residue was dissolved in DCM (3.3 mL; 0.1 M) and successively ClAcCl (39 μL; 492 μmol; 1.5 eq.) and TEA (273 μL; 1.97 mmol; 6 eq.) were added. After stirring at r.t. overnight the mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with sat. aq. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 19 (116 mg; 198 μmol; 60%) as a white solid. TLC: (EtOAc:Hept, 80:20 v/v) R.sub.f=0.48).sup.1H NMR (500 MHz, CDCl.sub.3) δ 6.63 (d, J=8.6 Hz, 1H, NH), 5.58 (ddd, J=27.5, 10.6, 2.5 Hz, 1H, H-4), 5.34 (dd, J=5.2, 1.8 Hz, 1H, H-7), 5.12 (ddd, J=6.4, 5.1, 2.4 Hz, 1H, H-8), 4.95 (dd, J=49.0, 2.5 Hz, 1H, H-3), 4.59 (dd, J=12.5, 2.5 Hz, 1H, H-9.sub.a), 4.33-4.23 (m, 2H, H-6; H-5), 4.21 (dd, J=12.5, 6.5 Hz, 1H, H-9.sub.b), 4.02-3.93 (m, 2H, CH.sub.2, ClAc), 3.85 (s, 3H, OMe), 2.19 (s, 3H, Me, OAc), 2.16 (s, 3H, Me, OAc), 2.12 (s, 3H, Me, OAc), 2.06 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.77 (CO, Ac), 170.66 (CO, Ac), 170.61 (CO, Ac), 170.47 (CO, Ac), 167.23 (C-1), 166.73 (CO, Ac), 165.08 (CO, ClAc), 95.28 (d, J=29.0 Hz, C-2). 87.01 (d, J=185.7 Hz, C-3), 71.79 (C-6), 71.50 (C-8), 68.14 (d, J=17.2 Hz, C-4), 67.88 (C-7), 62.15 (C-9), 53.68 (OMe), 45.91 (d, J=2.6 Hz, C-5), 42.56 (CH.sub.2, ClAc), 21.03 (Me, Ac), 20.91 (Me, Ac), 20.86 (Me, Ac), 20.76 (Me, Ac), 20.66 (Me, Ac). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.29ClFNNaO.sub.14, 608.11583; found, 608.11438.

Reference Compound: Methyl 5-(benzylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (20)

(55) ##STR00068##

(56) Boc inhibitor 9 (50 mg; 82 μmol) was dissolved in a 1:1:2 mixture of respectively TFA, H.sub.2O and DCM (1.6 mL; 0.05 M). The mixture was stirred for 2 hrs at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09). The mixture was then diluted with H.sub.2O and concentrated in vacuo. The residue was dissolved in DCM (0.83 mL; 0.1 M) and Cbz-Cl (14 μl; 99 μmol; 1.2 eq.) and TEA (69 μl; 495 μmol; 6 eq.) were added. After stirring at r.t. for 1 hr additional Cbz-Cl (24 μl; 165 μmol; 2 eq.) was added. The reaction was stirred for 15 hrs after which the reaction was still not finished, so additional Cbz-Cl (125 μl; 874 μmol; 10.5 eq.) and TEA (35 μl; 252 μmol; 3 eq.) were added. After stirring at r.t. for 5.5 hrs the mixture was diluted with DCM and washed successively with 0.1M HCl and sat. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 20 (3.3 mg; 5.1 μmol; 6%) as a white solid. TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.43. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.37-7.30 (m, 5H, 5×CH, Cbz), 5.52 (dd, J=27.7, 11.0 Hz, 1H, H-4), 5.41 (dd, J=5.8, 2.0 Hz, 1H, H-7), 5.18 (td, J=5.9, 2.6 Hz, 1H, H-8), 5.15 (d, J=12.4 Hz, 1H, CHH, Cbz), 5.02-4.88 (m, 2H, CHH, Cbz; H-3), 4.78 (d, J=9.5 Hz, 1H, NH), 4.56-4.49 (m, 1H, H-9.sub.a), 4.24-4.17 (m, 2H, H9.sub.b; H-6), 3.91 (q, J=10.5 Hz, 1H, H-5), 3.83 (s, 3H, OMe), 2.16 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.03 (s, 3H, Me, OAc), 2.02 (s, 3H, Me, OAc), 1.97 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.74 (CO, Ac), 170.64 (CO, Ac), 170.43 (CO, Ac), 170.20 (CO, Ac), 167.26 (C-1), 165.21 (CO, Ac), 155.54 (CO, Cbz), 136.39 (C, Cbz), 128.68 (2×CH, ortho Cbz), 128.36 (CH, para Cbz), 128.15 (2×CH, meta Cbz), 95.22 (d, J=29.0 Hz, C-2), 87.21 (d, J=184.9 Hz, C-3), 71.99 (C-6), 70.98 (C-8), 68.11-67.91 (m, C-4; C-7), 67.18 (CH.sub.2, Cbz), 62.21 (C-9), 53.64 (OMe), 47.27 (C-5), 20.97 (Me, OAc), 20.94 (Me, OAc), 20.92 (Me, OAc), 20.72 (Me, OAc), 20.62 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.28H.sub.34FNNaO.sub.15, 666.18102; found, 666.18010.

Reference Compound: Methyl 5-[(1-benzyl-1H-1,2,3-triazol-4-yl)methylcarbamado]-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (21)

(57) ##STR00069##

(58) To a mixture of 14 (10 mg; 17 μmol) in a 1:9 mixture of H.sub.2O and .sup.tBuOH (0.19 ml; 0.09 M), Bn-N.sub.3 (4.5 mg; 34 μmol; 2 eq.) was added. A premixture of TBTA (29 mg), DMF (750 μL) and CuI (5.1 mg) was agitated until a homogenous solution was obtained. The TBTA mixture (95 μl) was added to the H.sub.2O/.sup.tBuOH mixture and 10 mg of copper flakes were added. The reaction was stirred at r.t. overnight, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.80% EtOAc in Hept) afforded 21 (5.7 mg; 17 μmol; 46%) as a white solid. TLC: (EtOAc:Hept, 80:20 v/v) R.sub.f=0.37. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.64 (s, 1H, CH, triazole), 7.37 (d, J=2.2 Hz, 2H, 2×CH, ortho Bn), 7.32-7.29 (m, 2H, 2×CH, meta Bn), 6.91 (dd, J=10.3, 6.8 Hz, 1H, CH, para Bn), 5.58-5.41 (m, 2H, CH.sub.2, Bn), 5.34-5.28 (m, 1H, H-7), 5.23-5.05 (m, 2H, CH.sub.2, Poc), 5.00 (d, J=8.9 Hz, 1H, NH), 4.93 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.50 (dd, J=12.6, 2.6 Hz, 1H, H-9.sub.a), 4.20-4.14 (m, 2H, H-9.sub.b; H-6), 3.92 (q, J=10.4 Hz, 1H, H-5), 3.83 (s, 3H, OMe), 2.16 (s, 3H, Me, OAc), 2.14 (s, 3H, Me, OAc), 2.02 (s, 3H, Me, OAc), 2.02 (s, 3H, Me, OAc), 1.97 (s, 3H, Me, OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.88 (CO, Ac), 170.72 (CO, Ac), 170.52 (CO, Ac), 170.34 (CO, Ac), 167.39 (C-1), 165.27 (CO, Ac), 155.63 (CO, Poc), 134.64 (C, Bn), 129.38 (2×CH, meta Bn), 129.09 (CH, para Bn), 128.44 (2×CH, ortho Bn), 125.00 (CH, triazole), 95.25 (d, J=28.9 Hz, C-2), 87.21 (d, J=185.0 Hz, C-3), 72.15 (C-6), 71.15 (C-8), 68.47 (d, J=17.0 Hz, C-4), 68.30 (C-7), 62.30 (C-9), 58.65 (CH.sub.2, Poc), 54.52 (CH.sub.2, Bn), 53.74 (OMe), 47.22 (C-5), 21.07 (Me, OAc), 21.05 (Me, OAc), 21.01 (Me, OAc), 20.78 (Me, OAc), 20.74 (Me, OAc). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.31H.sub.37FN.sub.4NaO.sub.15, 747.21371; found, 747.21371.

Methyl 5-(butylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (22)

(59) ##STR00070##

(60) Boc inhibitor 9 (25 mg; 41 μmol) was dissolved in DCM (1 ml; 0.041 M) and TfOH (14 μl; 164 μm; 4 eq.)) was added and stirred for 5 min at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09) then n-Bu chloroformate (27 μl; 205 μm; 5 eq.) and TEA (57 μl; 410 μm; 10 eq.) were added. The reaction was left stirring for 16 hrs at r.t., diluted with an excess of DCM washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 22 (8 mg; 41 μmol; 32%) as a white solid. TLC: (EtOAc) R.sub.f=0.90. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.54 (dd, J=27.9, 11.3 Hz, 1H, H-4), 5.39 (d, J=5.9 Hz, 1H, H-7), 5.18 (td, J=6.0, 2.5 Hz, 1H, H-8), 4.95 (dd, J=49.2, 2.5 Hz, 1H, H-3), 4.73-4.66 (m, 1H, NH), 4.53 (dd, J=12.4, 2.6 Hz, 1H, H-9.sub.a), 4.24-4.17 (m, 2H, H-9.sub.b; H-6), 4.08-3.96 (m, 2H, (CO)CH.sub.2, n-Bu), 3.93-3.84 (m, 1H, H-5), 3.84 (s, 3H, OMe), 2.18 (s, 3H, Me, OAc), 2.17 (s, 3H, Me, OAc), 2.11 (s, 3H, Me OAc), 2.05 (s, 3H, Me, OAc), 2.04 (s, 3H, Me, OAc), 1.61-1.54 (m, 2H, CH.sub.2CH.sub.2CH.sub.2, n-Bu), 1.39-1.32 (m, 2H, CH.sub.2CH.sub.3, n-Bu), 0.93 (t, J=7.4 Hz, 3H, CH.sub.2CH.sub.3, n-Bu). .sup.13C NMR (126 MHz, Chloroform-d) δ 170.59 (CO, Ac), 170.45 (CO, Ac), 170.25 (CO, Ac), 170.09 (CO, Ac), 167.14 (C-1), 165.10 (CO, Ac), 155.70 (CONH), 95.09 (d, J=28.8 Hz, C-2), 87.08 (d, J=184.7 Hz, C-3), 71.92 (C-6), 70.96 (C-8), 69.30 (d, J=13.5 Hz, C-4), 68.00 (C-7), 65.31 (C(O)CH.sub.2, n-Bu), 62.09 (C-9), 53.48 (OMe), 46.99 (C-5), 30.85 (CH.sub.2CH.sub.2CH.sub.2, n-Bu), 20.84 (Me, OAc), 20.78 (Me, OAc), 20.75 (Me, OAc), 20.62 (Me, OAc), 20.56 (Me, OAc), 18.93 (CH.sub.2CH.sub.3, n-Bu), 13.68 (CH.sub.2CH.sub.3, n-Bu). .sup.19F NMR (470 MHz, CDCl.sub.3) δ −209.10 (dd, J=49.1, 27.9 Hz). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.25H.sub.36FN.sub.4NaO.sub.15, 632.19667; found, 632.19640.

Methyl 5-(2-methoxy-ethylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-R-galacto-non-2-ulopyranosonate (23)

(61) ##STR00071##

(62) Boc inhibitor 9 (40 mg; 66 μmol) was dissolved in DCM (1 ml; 0.066 M) and TfOH (23 μl; 262 μm; 4 eq.)) was added and stirred for 5 min at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09) F then 2-methoxyethyl chloroformate (38 μl; 328 μm; 5 eq.) and TEA (91 μl; 656 μm; 10 eq.) were added. The reaction was left stirring for 16 hrs at r.t., diluted with an excess of DCM washed successively with 0.1M HCl and sat. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 23 (36 mg; 66 μmol; 90%) as a white solid. TLC: (EtOAc) R.sub.f=0.80. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.51 (ddd, J=27.9, 11.1, 3.1 Hz, 1H, H-4), 5.39 (dd, J=5.7, 1.9 Hz, 1H, H.sub.7), 5.21-5.14 (m, 1H, H.sub.8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H.sub.3), 4.86 (d, J=9.1 Hz, 1H, NH), 4.52 (dd, J=12.5, 2.6 Hz, 1H, H9.sub.a), 4.23-4.15 (m, 4H, H9.sub.b, H.sub.6, CH.sub.2CH.sub.2C(O)), 3.92-3.86 (m, 1H, H.sub.5), 3.83 (s, 3H, COOMe), 3.55 (ddd, J=6.6, 5.2, 2.7 Hz, 2H, MeOCH.sub.2CH.sub.2), 3.37 (s, 3H, OMe), 2.17 (s, 3H, Me OAc), 2.15 (s, 3H, Me OAc), 2.11 (s, 3H, Me OAc), 2.04 (s, 3H, Me OAc), 2.03 (s, 3H, Me OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.61 (CO), 170.44 (CO), 170.26 (CO), 170.04 (CO), 167.17 (CO), 165.07 (CO), 155.43 (CONH), 95.05 (d, J=28.9 Hz, C.sub.2), 87.01 (d, J=184.8 Hz, C.sub.3), 71.82 (C.sub.6), 70.66 (C.sub.8), 70.61 CH.sub.2CH.sub.2C(O), 68.16 (d, J=17.1 Hz, C.sub.4), 67.98 (C.sub.7), 64.46 (MeOCH.sub.2CH.sub.2), 62.06 (C.sub.9), 58.87 (OMe), 53.47 (COOCH.sub.3), 46.99 (C.sub.5), 20.83 (Me OAc), 20.78 (Me OAc), 20.76 (Me OAc), 20.63 (Me OAc), 20.56 (Me OAc). .sup.19F NMR (470 MHz, CDCl.sub.3) δ −209.21 (dd, J=49.2, 27.9 Hz). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.24H.sub.34FNNaO.sub.16, 634.17593; found, 634.17656.

Methyl 5-(2,2,2-trichloroethylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (24)

(63) ##STR00072##

(64) Boc inhibitor 9 (27 mg; 44 μmol) was dissolved in DCM (1 ml; 0.044 M) and TfOH (16 μl; 262 μm; 4 eq.)) was added and stirred for 5 min at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09) Troc-Cl (31 μl; 221 μm; 5 eq.) and TEA (62 μl; 443 μm; 10 eq.) were added. The reaction was left stirring for 16 hrs at r.t., diluted with an excess of DCM washed successively with 0.1M HCl and sat. NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 24 (5.2 mg; 66 μmol; 17%) as a white solid. TLC: (EtOAc) R.sub.f=0.85. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.67 (ddd, J=27.9, 11.1, 2.5 Hz, 1H, H.sub.4), 5.37 (dd, J=6.3, 1.7 Hz, 1H, H7), 5.26-5.16 (m, 2H, NH, H8), 4.97 (dd, J=49.1, 2.5 Hz, 1H, H3), 4.91 (d, 1H, CHH Troc), 4.53 (d, J=12.1 Hz, 1H, CHH Troc), 4.49 (dd, J=12.6, 2.5 Hz, 1H, H9.sub.a), 4.29 (d, J=10.4 Hz, 1H, H6), 4.23 (dd, J=12.6, 5.6 Hz, 1H, H9.sub.b), 3.84 (s, 3H, OMe), 3.83-3.78 (m, 1H, H5), 2.18-2.17 (m, 6H, 2×Me OAc), 2.17 (s, 3H, Me OAc), 2.04 (s, 3H, Me OAc), 2.04 (s, 3H, Me OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.75 (CO), 170.56 (CO), 170.12 (CO), 169.87 (CO), 167.04 (CO), 164.93 (CO), 153.75 (CONH), 95.05 (d, J=28.9 Hz, C2), 86.97 (d, J=184.9 Hz, C3), 95.11 (CCl.sub.3 Troc), 74.33 (CH.sub.2 Troc), 70.99 (C6), 70.50 (C8), 67.85 (C7), 67.62 (d, J=17.5 Hz, C4), 61.84 (C9), 53.50 (OMe), 47.43 (C5), 30.93 (Me OAc), 20.79 (Me OAc), 20.73 (Me OAc), 20.65 (Me OAc), 20.52 (Me OAc). .sup.19F NMR (470 MHz, CDCl.sub.3) δ−209.54 (dd, J=49.1, 27.7 Hz). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.23H.sub.29Cl.sub.3FNNaO.sub.15, 706.04845; found, 706.04999.

Methyl 5-(2-fluoroethylcarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (25)

(65) ##STR00073##

(66) Boc inhibitor 9 (27 mg; 44 μmol) was dissolved in DCM (1 ml; 0.044 M) and TfOH (16 μl; 262 μm; 4 eq.)) was added and stirred for 5 min at r.t. (TLC: (EtOAc:Hept, 60:40 v/v) R.sub.f=0.09) 2-fluoroethyl chloroformate (21 μl; 221 μm; 5 eq.) and TEA (62 μl; 443 μm; 10 eq.) were added. The reaction was left stirring for 16 hrs at r.t., diluted with an excess of DCM washed successively with 0.1M HCl and sat. NaHCO.sub.03. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. Silicagel flash column chromatography (0%.fwdarw.50% EtOAc in Hept) afforded 25 (16 mg; 66 μmol; 60%) as a white solid. TLC: (EtOAc) R.sub.f=0.85. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.51 (ddd, J=27.9, 11.0, 2.5 Hz, 1H, H4), 5.40 (dd, J=5.4, 2.0 Hz, 1H, H7), 5.17 (td, J=5.9, 2.5 Hz, 1H, H8), 4.95 (dd, J=49.1, 2.5 Hz, 1H, H3), 4.94 (d, J=9.4 Hz, 1H, NH), 4.67-4.46 (m, 3H, FCH.sub.2CH.sub.2, H9.sub.a), 4.41-4.24 (m, 2H FCH.sub.2CH.sub.2), 4.25-4.16 (m, 2H, H9.sub.b, H6), 4.00-3.89 (m, 1H, H5), 3.84 (s, 3H, OMe), 2.18 (s, 3H, Me OAc), 2.16 (s, 3H, Me OAc), 2.12 (s, 3H, Me OAc), 2.05 (s, 3H, Me OAc), 2.04 (s, 3H, Me OAc). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.63 (CO), 170.48 (CO), 170.37 (CO), 170.12 (CO), 167.13 (CO), 165.04 (CO), 155.24 (CONH), 95.07 (d, J=28.7 Hz, C2), 87.03 (d, J=185.2 Hz, C3), 81.60 (d, J=170.2 Hz, FCH.sub.2CH.sub.2), 71.91 (C6), 71.00 (C8), 68.21 (d, J=17.3 Hz, C4), 67.96 (C7) 64.29 (d, J=19.9 Hz, FCH.sub.2CH.sub.2), 62.02 (C9), 53.50 (OMe), 47.05 (C5), 20.83 (Me OAc), 20.77 (Me OAc), 20.75 (Me OAc), 20.57 (Me OAc), 20.54 (Me OAc). .sup.19F NMR (470 MHz, CDCl.sub.3) δ −209.07 (dd, J=49.1, 27.9 Hz). HR-ESI-TOF/MS (m/z): [M+Na].sup.+ calcd. for C.sub.23H.sub.31F.sub.2NNaO.sub.15, 622.15594; found, 622.15531.

Intermediate: Methyl 5-(acetamido)-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonate (26)

(67) ##STR00074##

(68) Peracetylated sialic acid 2 (1.02 g, 1.912 mmol) was dissolved in ACN (4 ml; 0.478 M) and cooled to 0° C. TMSOTf (1.03 ml; 5.736 mmol; 3 eq.) was added dropwise to the solution which turned slightly yellow. After 4 hours, the reaction was diluted with an excess EtOAc and washed three times with sat. aq. NaHCO.sub.3 (30 ml). The organic layer was concentrated in vacuo and the product was purified on silicagel flash column chromatography (0.fwdarw.30% ACE in DCM), and 26 (0.552 g, 1.166 mmol, 61% yield) was obtained as a white foam. TLC: (Acet:DCM, 30/70) R.sub.f=0.50. H NMR (400 MHz, CDCl.sub.3) δ 6.05 (d, J=8.9 Hz, 1H, NH), 5.99 (d, J=3.1 Hz, 1H, H-3), 5.54-5.48 (m, 2H, H4; H7), 5.35 (ddd, J=7.5, 4.4, 3.1 Hz, 1H, H-8), 4.65 (dd, J=12.3, 3.1 Hz, 1H, H-9.sub.a), 4.40 (dd, J=7.8, 5.1 Hz, 2H, H-6; H-5), 4.20 (dd, J=12.3, 7.3 Hz, 1H, H-9.sub.b), 3.81 (s, 3H, OMe), 2.13 (s, 3H, Me, Ac), 2.08 (s, 3H, Me, Ac), 2.07 (s, 3H, Me, Ac), 2.06 (s, 3H, Me, Ac), 1.93 (s, 3H, Me, Ac). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.82 (CO, Ac), 170.61 (CO, Ac), 170.19 (CO, Ac), 170.17 (CO, Ac), 161.63 (C-1), 145.06 (C-2), 107.98 (C-3), 76.64 (C-6), 70.85 (C-8), 68.03 (C-7), 67.64 (C-4), 61.97 (C-9), 52.57 (OMe), 46.46 (C-5), 23.09 (Me, Ac), 20.85 (Me, Ac), 20.76 (Me, Ac), 20.72 (Me, Ac), 20.70 (Me, Ac). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.20H.sub.27NO.sub.12, 496.1431; found, 496.1430.

Reference Compound: Methyl 5-(acetamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (27)

(69) ##STR00075##

(70) Sialic acid glycal 26 (1.5 g, 3.17 mmol) was solved in DMF (23.8 ml) and H.sub.2O (7.9 ml; 0.1 M). Selectfluor (3.37 g, 9.51 mmol; 3 eq.) was added and the reaction was heated to 60° C. for 16 hours, then concentrated in vacuo. Note: Longer reaction times did in our hands not increase the conversion but led to the formation of side-products. The compound was redissolved in EtOAc washed with sat. aq. NaHCO.sub.03, dried with MgSO.sub.4, filtered, concentrated in vacuo. TLC: (Acet:DCM, 30:70 v/v) R.sub.f=0.40. HRMS (m/z): [M+Na].sup.+ calcd for C.sub.20H.sub.28FNO.sub.13, 532.1443; found, 532.1442. The crude fluorinated alcohol was dissolved in Pyr (14.5 ml) and Ac.sub.20 (7.3 ml), stirred for 16 hours at r.t. and was evaporated in vacuo. The resulting solid dissolved in EtOAc and sat. aq. NaHCO.sub.03. The organic phase was separated and the solvent evaporated in vacuo and purified on silica flash-column chromatography (0.fwdarw.30% ACE in DCM) affording 27 (1.46 g, 2.54 mmol, 80% yield two steps) as a slightly yellow foam. TLC: (Acet:DCM, 30:70 v/v) R.sub.f=0.55. .sup.1H-NMR (500 MHz, CD.sub.3OD, major anomer) δ 5.58 (d, J=8.9 Hz, 1H, NH), 5.46 (dd, J=27.9, 10.7 Hz, 1H, H-4), 5.29 (dd, J=5.0, 1.8 Hz, 1H, H-7), 5.05 (ddd, J=6.7, 5.2, 2.5 Hz, 1H, H-8), 4.87 (dd, J=49.1, 2.5 Hz, 1H, H-3), 4.51 (dd, J=12.5, 2.5 Hz, 1H, H-9.sub.a), 4.21-4.10 (m, 3H, H-9.sub.b; H-5; H-6), 3.77 (s, 3H, OMe), 2.12-2.09 (m, 6H, 2×Me, OAc), 2.04 (s, 3H, Me, OAc), 1.98 (s, 3H, Me, OAc), 1.97 (s, 3H, Me, OAc), 1.85 (s, 3H, Me, NHAc); .sup.13C-NMR (126 MHz, CD.sub.3OD) δ 170.58 (CO, Ac), 170.56 (CO, Ac), 170.50 (CO, Ac), 170.34 (CO, Ac), 167.11 (C-1), 95.16 (d, J=28.8 Hz, C-2), 86.95 (d, J=185.3 Hz, C-3), 71.91 (C-6), 71.37 (C-8), 68.40 (d, J=17.2 Hz, C-4), 67.93 (C-7), 62.09 (C-9), 53.49 (OMe), 45.53 (C-5), 29.27 (Me, Ac), 20.88 (Me, Ac), 20.79 (Me, Ac), 20.74 (Me, Ac), 20.65 (Me, Ac), 20.51 (Me, Ac); HRMS (m/z): [M+Na].sup.+ calcd for C.sub.22H.sub.3FNO.sub.14, 574.1548; found, 574.1548.

Methyl 5-(S-ethylthiocarbamado)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (28)

(71) ##STR00076##

(72) Boc inhibitor 9 (0.1 mmol) was dissolved in acetonitrile (1 ml; 0.1M) and trimethysilyl iodide (0.15 mmol; 1.5 eq.) was added. The mixture was stirred at rt. for 2 hrs. The mixture was concentrated in vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 (1 ml; 0.1M) and DIPEA (0.4 mmol; 4 eq.), followed by S-ethylchlorothioformate (0.4 mmol; 4 eq.), were added. The reaction was stirred for 16 h at rt after which the mixture was diluted with CH.sub.2Cl.sub.2 and washed with 0.1M HCl and sat. NaHCO.sub.3. The water layer was extracted twice with CH.sub.2Cl.sub.2. The combined organic layers were dried over MgSO.sub.4 and concentrated in vacuo. Silicagel flash column chromatography (0% to 60% EtOAc in heptanes) afforded compound 28 (10 mg; 23%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.54 (dd, J=27.8, 10.6 Hz, 1H), 5.41 (d, J=8.9 Hz, 1H), 5.34 (dd, J=5.5, 1.9 Hz, 1H), 5.17 (td, J=6.0, 2.5 Hz, 1H), 4.94 (dd, J=49.1, 2.5 Hz, 1H), 4.53 (dd, J=12.4, 2.6 Hz, 1H), 4.28-4.06 (m, 3H), 3.84 (s, 3H), 2.86 (q, J=7.3 Hz, 2H), 2.18 (s, 3H), 2.17 (s, 2H), 2.12 (s, 2H), 2.05 (s, 3H), 2.04 (s, 3H), 1.27 (t, J=7.3 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.60, 170.33, 170.19, 167.11, 165.03, 95.22, 94.99, 87.86, 86.38, 71.76, 70.95, 68.11, 67.09, 53.49, 47.24, 24.58, 20.84, 20.75, 20.64, 20.55, 15.53. .sup.19F NMR (470 MHz, CDCl.sub.3) δ −209.00 (dd, J=49.0, 28.2 Hz). HR-ESI-TOF/MS (m/z): [M+Na.sup.+] calcd. for C.sub.23H.sub.32FNO.sub.14S, 620.14252; found, 620.14154.

Methyl 5-N-ethylamidnocarbonyl)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (29)

(73) ##STR00077##

(74) Synthesized as described for compound 28, using ethyl isocyanate instead of S-ethylchlorothioformate. Silicagel flash column chromatography (0% to 60% EtOAc in heptanes) afforded compound 29 (14 mg; 35%). H NMR (500 MHz, CDCl.sub.3) δ 5.54 (ddd, J=28.4, 11.0, 2.4 Hz, 1H), 5.42 (dd, J=5.0, 2.0 Hz, 1H), 5.14 (ddd, J=7.3, 5.0, 2.5 Hz, 1H), 4.94 (dd, J=49.2, 2.5 Hz, 1H), 4.61-4.57 (m, 2H), 4.51 (d, J=9.1 Hz, 1H), 4.28-4.18 (m, 2H), 4.03 (q, J=10.3 Hz, 1H), 3.84 (s, 3H), 3.13 (dq, J=12.8, 7.2 Hz, 2H), 2.18 (s, 6H), 2.11 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.10 (t, J=7.2 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 170.77, 170.67, 170.61, 170.46, 167.30, 165.27, 156.82, 95.33, 95.10, 88.00, 86.53, 72.51, 71.51, 69.06, 68.92, 68.24, 62.25, 53.45, 46.45, 35.39, 20.89, 20.83, 20.75, 20.71, 20.56, 15.31. .sup.19F NMR (470 MHz, CDCl.sub.3) δ −208.73 (dd, J=48.9, 28.4 Hz). HR-ESI-TOF/MS (m/z): [M+Na.sup.+] calcd. for C.sub.23H.sub.33FN.sub.2O.sub.14, 603.18135; found, 603.18059.

Methyl 5-(methylsulfonamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (30)

(75) ##STR00078##

(76) Synthesized as described for compound 28, using mesyl chloride instead of S-ethylchlorothioformate. Silicagel flash column chromatography (0% to 60% EtOAc in heptanes) afforded compound 30 (6.6 mg; 24%). .sup.1H NMR (500 MHz, CDCl.sub.3 δ 5.53 (dd, J=4.7, 1.2 Hz, 1H), 5.47-5.33 (m, 1H), 5.11 (ddd, J=7.1, 4.7, 2.6 Hz, 1H), 4.92 (dd, J=49.0, 2.4 Hz, 1H), 4.62-4.55 (m, 2H), 4.18 (dd, J=12.5, 6.9 Hz, 1H), 4.06-4.01 (m, 2H), 3.85 (s, 3H), 3.02 (s, 3H), 2.21 (s, 3H), 2.18 (s, 3H), 2.16 (s, 3H), 2.06 (s, 3H), 2.05 (s, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.56, 170.88, 170.58, 170.48, 167.02, 164.93, 95.03, 94.80, 87.96, 86.48, 73.05, 71.78, 69.06, 68.92, 67.96, 62.05, 53.57, 48.68, 48.66, 42.54, 21.03, 20.89, 20.79, 20.77, 20.50. .sup.19F NMR (470 MHz, CDCl.sub.3) δ− 208.29 (dd, J=49.1, 27.8 Hz).

Methyl 5-(phenylsulfonamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (31)

(77) ##STR00079##

(78) Synthesized as described for compound 28, using benzenesulfonyl chloride instead of S-ethylchlorothioformate. Silicagel flash column chromatography (0% to 60% EtOAc in heptanes) afforded compound 31 (5.5 mg; 34%). .sup.1H NMR (500 MHz, CDCl.sub.3 δ 7.92-7.82 (m, 1H), 7.66-7.56 (m, 1H), 7.52 (dd, J=8.4, 6.9 Hz, 1H), 5.47 (dd, J=6.0, 1.5 Hz, 1H), 5.38 (ddd, J=26.9, 11.3, 2.5 Hz, 1H), 5.23 (td, J=5.9, 2.6 Hz, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.95 (dd, J=49.0, 2.5 Hz, 0H), 4.49 (dd, J=12.5, 2.6 Hz, 1H), 4.26-4.19 (m, 1H), 3.84-3.71 (m, 2H), 2.21 (s, 2H), 2.19 (s, 2H), 2.09 (s, 2H), 2.06 (s, 2H), 1.68 (s, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.30, 170.65, 170.46, 170.15, 167.05, 164.90, 141.32, 132.92, 129.18, 127.01, 94.78, 94.55, 87.44, 85.97, 72.61, 70.57, 68.70, 68.07, 67.93, 62.04, 53.46, 49.68, 49.67, 20.87, 20.85, 20.76, 20.50, 20.30. .sup.19F NMR (470 MHz, CDCl.sub.3) δ −210.10 (dd, J=49.1, 26.9 Hz). HR-ESI-TOF/MS (m/z): [M+Na.sup.+] calcd. for C.sub.26H.sub.32FNO.sub.15S, 672.13744; found, 672.13539.

Methyl 5-(propylsulfonamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-glycero-β-galacto-non-2-ulopyranosonate (32)

(79) ##STR00080##

(80) Synthesized as described for compound 28, using 1-propanesulfonyl chloride (1.0 mmol; 10 eq.) instead of S-ethylchlorothioformate (0.4 mmol; 4 eq.). Silicagel flash column chromatography (0% to 60% EtOAc in heptanes) afforded compound 32 (7 mg; 11%). HR-ESI-TOF/MS (m/z): [M+Na.sup.+] calcd. for C.sub.23H.sub.34FNO.sub.15S, 638.15309; found, 638.15182.

Example 2—Assay Conditions and Methods

(81) Cell Culture

(82) Mouse B16-F10 melanoma cells (ATCC CRL-6475) were cultured in Minimum Essential Medium (MEM) (Gibco, Invitrogen, Carlsbad, Calif.) containing 5% fetal bovine serum (FBS) (Greiner Bio-one, Frickenhausen, Germany), 1% MEM non-essential amino acids (Gibco), 0.15% sodium bicarbonate (Gibco), 1 mM sodium pyruvate (Gibco), 1.5% MEM vitamins (Gibco), 1% antibiotic-antimycotic solution (50 U/ml penicillin, 50 μg/ml streptomycin and 125 ng/ml amphotericin B) (Gibco). The murine 9464D neuroblastoma cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Glutamax, Gibco) with 10% FBS, 1% non-essential amino acids, 50 μM 2-mercaptoethanol (Sigma-Aldrich) and 1% antibiotic-antimycotic solution (Kroesen et al., 2014, DOI: 10.1002/ijc.28463, Stauffer et al., 2012, DOI: 10.3109/07357907.2012.664670). The murine EL4 T lymphocyte cell line (ATCC TIB-39) was cultured in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 5% FBS and 1% antibiotic-antimycotic solution. Human HEK293 (ATCC CRL-1573) kidney cells and human HeLa (ATCC CCL-2) cervical cancer cells were cultured in DMEM containing 10% FBS, 2 mM glutamine (Lonza, Walkersville, Md.) and 1% antibiotic-antimycotic solution. Human monocytic THP-1 cells (TIB-202, ATCC) were cultured in RRMI-1640 medium (Gibco) supplemented with 10% FBS, 2 mM glutamine and 1% antibiotic-antimycotic solution. All cell lines were cultured in a humidified 5% CO.sub.2 incubator at 37° C.

(83) Titration and Long-Term Effect of Fluorine Sialic Acids

(84) To assess the effect of the synthesized compounds on cell surface sialylation, different murine and human cell lines were cultured for three days in the presence of 0-204.8 μM amide and carbamate fluorine sialic acids. Cells treated with an equal final percentage of DMSO were used as control and all cells were subjected to lectin staining and flow cytometry as described below. To investigate the longevity of the effect of the fluorine sialic acids on cell surface sialylation, B16-F10 cells treated with 25.6 μM or 51.2 μM were thoroughly washed with medium to remove fluorine sialic acids from the culture and reseeded. During a period of six days, cells were harvested every day and sialylation was assessed by flow cytometry.

(85) Lectin Staining and Flow Cytometry

(86) For the lectin staining, all cells were harvested and washed with 1× carbo-free blocking solution (Vector Laboratories, Inc., Burlingame, Calif., USA) and stained for 45 minutes at 4° C. in 1× carbo-free blocking solution containing 1 mM CaCl.sub.2), 1 mM MgCl.sub.2 and biotinylated MALII (5 μg/ml), SNA-1 (1 μg/ml) or PNA (5 μg/ml) all obtained from Vector Laboratories Inc. MALII recognizes α2,3-linked sialic acids, SNA-1 recognizes α2,6-linked sialic acids and PNA binds to terminal 3-galactose. In addition, B16-F10 cells were stained with 2 μg/ml biotinylated AAL (α-linked Fucose), LCA (α-linked Mannose), PHA-L (complex glycans), GSL-I (α-linked Galactose and N-Acetylgalactosamine), WGA (chitobiose), SJA (β-linked Galactose and N-Acetylgalactosamine) and PSA (Glucose and Mannose) purchased from Vector Laboratories Inc. Next, the cells were washed thrice in PBA (1×PBS, 1% BSA, and 0.02% sodium azide) and stained for 10 minutes at 4° C. with 2 μg/ml streptavidin-phycoerythrin (PE) (BD Pharmingen, Franklin Lakes, N.J., USA). The cells were washed thrice again with PBA and fluorescence was assessed using a CyAn ADP flow cytometer (BD Biosciences, San Jose, Calif.) followed by analysis with FlowJo software (Tree Star Inc., Ashland, Oreg.). Untreated cells stained with streptavidin-PE only served as background fluorescence control. The percentages lectin binding was calculated by normalizing the mean fluorescence intensity values from the treated cells with the DMSO controls.

(87) MTT Assay

(88) B16-F10 cells treated for three days with 0.1-204.8 μM fluorine sialic acids or DMSO control were washed with PBS. Next, 60 μl growth medium and 10 μl PBS containing 4 mg/ml Thiazolyl Blue Tetrazolium Blue (MTT) (Sigma-Aldrich, St. Louis, Mo., USA) were added to the cells. The cells were incubated for 30 minutes at 37° C. until blue crystals were clearly detectable inside the cells. Medium was removed and the cells were lysed in 100 μl lysis buffer (isopropanol containing 0.5% SDS, 4% 1N HCl and 3.5% MQ) for 30 minutes at room temperature. Absorbance was measured at 595 nm using an iMark absorbance reader (BioRad, Hercules, Calif., USA).

(89) Statistical Analysis

(90) IC.sub.50 values and statistics were calculated using Prism 5.03 (GraphPad Software, Inc., La Jolla, Calif.). Statistical significance between the different groups was calculated using one-way ANOVA followed by Bonferroni post-hoc testing and P-values<0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***).

Example 3—C-5 Carbamate Sialic Acid Inhibitor are More Potent than the Corresponding C-5 Amide Derivatives

(91) The inhibitory potency of 2-14 on the sialic acid biosynthesis was assayed. To this end, B16-F10 cells were fed sialic acid precursors for three day at various concentrations (0.1-204.8 μM). The decrease in cell surface sialylation was measured using biotinylated lectins that are specific for binding α2,3-linked (Maackia amurensis Lectin II, MALII) or α2,6-linked (Sambucus nigra Lectin, SNA-I) sialic acid residues, subsequently visualized using streptavidin-phycoerythrin. Conversely, binding of the underlying galactose residue uncovered by desialylation was measured using a galactose specific lectin (Peanut, Agglutinin Lectin, PNA). No inhibition was observed without a P-3F.sub.ax-Neu derivative present. From the concentration dependent inhibition curves, EC.sub.50 values were extrapolated (FIG. 4). The EC.sub.50 is defined as the concentration where a 50% decrease in lectin binding is measured. The 5-carbamate derivatives with small substituents proved to be much more potent than the amide derivatives, including the known inhibitor based on natural sialic acid (Ac). The most potent inhibitor showed a 26 fold decrease in EC.sub.50 for α2,3-inked sialic acid inhibition compared to P-3F.sub.ax-Neu5Ac. Extended carbamates (—NH—C(═O)—CH.sub.2—O— motif) also showed improved potency relative to amide analogues. In addition to the analogues shown in FIG. 1B, the analogues shown below were tested on the same SiaFR scaffold as shown in FIG. 1A.

(92) This scaffold is of compounds according to the invention of general formula (I-ax) wherein each X is acetyl, Z is methyl, L is absent, Q and Q′ are both O, and R is variable:

(93) ##STR00081##

(94) The inhibitors were tested for toxicity (FIG. 5) using the MTT assay. From the results it is clear that larger substituents are worse inhibitors in terms of inhibition and toxicity. Carbamates with smaller hydrophobic alkyl substituents were most potent. In this screen, linear alkyl substituents of α2-3 carbons (7, 8, 10) were best, as including sidechains (11, 12) or 1-carbon substituents (9) gave slightly higher EC.sub.50 values. The importance of the carbamate was confirmed by an amide homolog (6) of a potent carbamate inhibitor (7), which gave respectively a 44 (MALII) and 22 (SNA-I) times higher EC.sub.50. Overall, we observed an increased inhibition towards α2,3-linked over α2,6-linked sialic acids. Importantly, the compounds also induce inhibition of sialic acid biosynthesis in cell lines resistant to Ac.sub.5SiaNAc-3Fax, namely 9464D cells and EL4 cells (FIG. 6). Tables 1 through 6 show IC50 values of various analogues in different cell lines.

(95) TABLE-US-00003 TABLE 1 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in B16-F10 cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc 26.79 ± 5.72 32.80 ± 3.08 SiaFTFA 14.32 ± 0.77 33.33 ± 2.63 SiaFAz 19.94 ± 2.08 32.82 ± 3.55 SiaFPen 40.56 ± 5.67 52.49 ± 4.31 SiaFAcC 17.98 ± 2.12 ND Extended carbamates SiaFGc 04.98 ± 0.33 11.78 ± 2.27 Carbamates SiaFPoc 00.72 ± 0.20 02.59 ± 0.07 SiaFAII 01.39 ± 0.21 02.87 ± 0.80 SiaFMe 04.89 ± 0.25 07.54 ± 1.34 SiaFEt 01.78 ± 0.16 03.87 ± 1.14 SiaFiBu 03.33 ± 0.12 15.07 ± 3.70 SiaFBoc 03.48 ± 0.56 08.21 ± 0.67 SiaFCbz 14.46 ± 3.51 ND SiaFnBu  7.45 ± 0.92  7.91 ± 1.95 SiaFMox 25.49 ± 2.53 32.38 ± 1.38 SiaFTro  3.83 ± 1.06  7.85 ± 2.07 SiaFFEt  1.25 ± 1.07  2.05 ± 1.29

(96) TABLE-US-00004 TABLE 2 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in THP-1 cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc 08.50 ± 1.54 10.87 ± 2.47 SiaFTFA 03.30 ± 1.26 07.19 ± 3.60 SiaFAz 10.93 ± 1.98 13.23 ± 3.02 SiaFPen 10.39 ± 2.14 12.58 ± 3.51 Extended carbamates SiaFGc 04.24 ± 2.00 05.94 ± 2.18 Carbamates SiaFPoc 00.42 ± 0.90 00.44 ± 1.59 SiaFAII 00.41 ± 0.73 00.38 ± 1.67 SiaFMe 01.98 ± 0.91 02.65 ± 1.60 SiaFEt 00.45 ± 0.74 00.37 ± 1.76 SiaFiBu 00.50 ± 1.34 00.62 ± 1.77 SiaFBoc 01.28 ± 1.36 01.72 ± 2.30

(97) TABLE-US-00005 TABLE 3 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in HEK293 cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc 45.22 ± 8.30 36.69 ± 3.59  SiaFTFA 42.75 ± 2.38 41.36 ± 10.44 SiaFAz 45.55 ± 5.14 47.21 ± 2.89  SiaFPen 26.24 ± 2.37 44.71 ± 4.33  Extended Carbamates SiaFGc 53.60 ± 5.25 54.06 ± 15.34 Carbamates SiaFPoc 01.19 ± 1.47 05.42 ± 1.40  SiaFAII 01.83 ± 1.19 01.68 ± 3.20  SiaFMe 03.31 ± 1.24 04.77 ± 2.80  SiaFEt 02.02 ± 2.72 01.57 ± 4.23  SiaFiBu 11.42 ± 4.73 09.61 ± 4.83  SiaFBoc 10.44 ± 4.76 28.03 ± 2.57 

(98) TABLE-US-00006 TABLE 4 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in HeLa cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc 52.95 ± 3.61 34.50 ± 2.18 SiaFTFA 25.54 ± 2.61 33.38 ± 4.38 SiaFAz 80.61 ± 3.26 42.19 ± 8.42 SiaFPen 62.31 ± 3.91 51.23 ± 6.06 Extended carbamates SiaFGc 16.61 ± 3.91 12.50 ± 2.52 Carbamates SiaFPoc 01.65 ± 2.27 01.37 ± 0.81 SiaFAII 01.77 ± 1.78 02.04 ± 1.40 SiaFMe 06.33 ± 3.26 05.23 ± 1.88 SiaFEt 02.02 ± 1.52 01.67 ± 1.31 SiaFiBu 05.15 ± 2.40 04.32 ± 1.82 SiaFBoc 12.83 ± 1.61 20.55 ± 1.95

(99) TABLE-US-00007 TABLE 5 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in 9464D cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc >102.4 >102.4 SiaFTFA 46.53 ± 4.41 >102.4 SiaFAz >102.4 >102.4 SiaFPen >102.4 >102.4 Extended carbamates SiaFGc >102.4 >102.4 Carbamates SiaFPoc 10.70 ± 2.85 40.19 ± 6.56 SiaFAII 06.91 ± 3.56 09.64 ± 3.93 SiaFMe 18.31 ± 2.67 29.60 ± 4.67 SiaFEt 08.91 ± 2.38 28.13 ± 5.51 SiaFiBu 24.45 ± 2.95 36.15 ± 6.40 SiaFBoc 36.48 ± 3.81 >102.4

(100) TABLE-US-00008 TABLE 6 IC.sub.50 values (μM) of amide and carbamate fluorine sialic acids in EL4 cells. Group Compound α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Amides SiaFAc >102.4 >102.4 SiaFTFA >102.4 >102.4 SiaFAz >102.4 >102.4 SiaFPen >102.4 >102.4 Extended carbamate SiaFGc >102.4 >102.4 Carbamates SiaFPoc 51.00 ± 8.62  51.65 ± 2.62 SiaFAII 39.92 ± 4.95   50.9 ± 6.44 SiaFMe 59.18 ± 4.17  52.68 ± 5.45 SiaFEt 42.03 ± 11.04 50.58 ± 8.84 SiaFiBu 51.82 ± 18.70 51.07 ± 8.97 SiaFBoc 54.58 ± 3.22  50.31 ± 3.99

(101) The effect on total cell surface glycosylation was also assessed. For this, B16-F.sub.10 cells were treated for three days with 102.4 μM fluorine sialic acids or DMSO control and were stained with a panel of biotinylated lectins and streptavidin-PE. Lectin binding was determined in two independent experiments by flow cytometry. The following lectins were used: MALII (for α2,3Neu5Ac), PNA (for βGal), AAL (for αFuc), LCA (for αMan), PHA-L (for complex glycans), GSL-I (for αGal, αGalNAc), WGA (for chitobiose), SJA (for βGal, βGalNAc), and PSA (for Glc, Man). Only MaLII showed decreased signal (data not shown).

Example 4—C-5 Carbamate Sialic Acid Inhibitors Induce Prolonged Inhibition of the Sialic Acid Biosynthesis

(102) The same compounds as in Example 3 were used to assess the duration of the inhibitory effect. Recovery of sialylation was assessed after treatment of cells with amide and carbamate fluorine sialic acid. For this, B16-F10 cells were incubated for three days with 51.2 μM amide or carbamate fluorine sialic acids or DMSO control. Fluorine sialic acids were removed from the culture and the cells were reseeded. During a period of six days, sialylation was assessed daily with flow cytometry by MALII or SNA-I lectins. FIG. 7 shows recovery of α2,3-sialylation or α2,6-sialylation in time presented as mean percentage lectin binding±SEM normalized to control (n=3). Treatment at 25.6 μM showed similar results (data not shown).

Example 5—C-5 Urea, Thiocarbamate, and Sulphonamide Sialic Acid Inhibitors Performance

(103) Analogous to example 3, the inhibitory potency of thiocarbamate 28, urea 29, and sulphonamides 30 and 31 on the sialic acid biosynthesis was assayed. To this end, B16-F10 cells were fed sialic acid precursors for three day at various concentrations (0.1-204.8 μM). The decrease in cell surface sialylation was measured using biotinylated lectins that are specific for binding α2,3-linked (MALII) or α2,6-linked (SNA-1) sialic acid residues, subsequently visualized using streptavidin-phycoerythrin. Conversely, binding of the underlying galactose residue uncovered by desialylation was measured using a galactose specific lectin (Peanut, Agglutinin Lectin, PNA). No inhibition was observed without a P-3F.sub.ax-Neu derivative present (DMSO vehicle control). From the concentration dependent inhibition curves, EC.sub.50 values were extrapolated (FIG. 8). The EC.sub.50 is defined as the concentration where a 50% decrease in lectin binding is measured. The urea and thiocarbamate derivatives proved to be comparable to the carbamate, each being superior to the known inhibitor based on natural sialic acid (Ac). Sulphonamides also proved to be viable inhibitors, with methylsulphonamide 30 outperforming the acetate at various concentrations. IC.sub.50 values are shown in table 7.

(104) TABLE-US-00009 TABLE 7 EC.sub.50 values (μM) of urea, thiocarbamate, and sulphonamide analogues. α2,3-Sia IC.sub.50 α2,6-Sia IC.sub.50 Compound (MAL) (SNA) Acetate 2.165 20.88 Carbamate 17 0.9291 3.915 Thiocarbamate 28 1.675 4.825 Urea 29 4.201 4.892 Sulphonamide 30 9.899 — Phenylsulphonamide Could not be — 31 determined

REFERENCES

(105) Adams and Gahl (GeneReviews, 13 Jun. 2003, PMID: 20301643) Almaraz et al., 2012, DOI 10.1002/bit.24363 Angata and Varki, 2002, DOI: 10.1021/cr000407m Bode et al., 2011, doi: 10.1586/erv.10.174 Büll et al., 2013, DOI: 10.1158/1535-7163.MCT-13-0279 Büll et al., 2014, DOI: 10.1016/j.bbcan.2014.07.005 Büll et al., 2014, DOI: 10.1158/0008-5472.CAN-14-0728 Büll et al., 2015, DOI: 10.1021/nn5061964 Büll et al., 2015, DOI: 10.1021/acschembio.5b 00501 Büll et al., 2016, doi: 10.1016/j.tibs.2016.03.007 Burkart et al., 1997, DOI: 10.1021/ja9723904 Burkart et al., 1999, DOI: 10.1039/A9033621 Chao et al., 2008, DOI: 10.1016/j.carres.2008.01.014 Kroesen et al., 2014, DOI: 10.1002/ijc.28463 Ley, 2003, DOI: 10.1016/S1471-4914(03)00071-6 Macauley et al., 2014, doi: 10.1038/nri3737 Pagan et al., 2018, doi: 10.1016/j.cell.2017.11.041 Rillahan et al., 2012, DOI: 10.1038/nchembio.999 Sedlacek and Seiler, 1978, DOI: 10.1007/BF00199623 Stauffer et al., 2012, DOI: 10.3109/07357907.2012.664670 Stencel-Baerenwald 2014, doi:10.1038/nrmicro3346 Varki and Schauer, 2009, PMID: 20301246 Volkers et al., 2015, DOI: 10.1038/nsmb.3060 Yin et al., 2017, DOI: 10.1002/bit.26291 WO2008/068638/EP2591787A1/WO2016071431/WO2015148915