Sialic acid (A-(2-6))-D-aminopyranose derivatives, synthesis methods and uses thereof
09695208 ยท 2017-07-04
Assignee
Inventors
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
C07H15/10
CHEMISTRY; METALLURGY
A61K47/549
HUMAN NECESSITIES
A61K47/643
HUMAN NECESSITIES
C07H13/00
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07H15/10
CHEMISTRY; METALLURGY
C07H15/04
CHEMISTRY; METALLURGY
C07H13/00
CHEMISTRY; METALLURGY
C07K1/107
CHEMISTRY; METALLURGY
C07K2/00
CHEMISTRY; METALLURGY
Abstract
N-acyl modified sialic acid (-(2.fwdarw.6))-D-aminopyranose derivatives, their synthesis methods and uses are disclosed. Sialic acid (-(2.fwdarw.6))-D-aminopyranose derivatives represented by formula 1 are synthesized by using D-aminogalactose (glucose) and sialic acid as raw materials, which are coupled with carrier proteins or polypeptides to obtain glycoprotein (glycopeptide) conjugates. Acetyl is replaced by derivative acyl in the structures of said compounds, therefore the compounds show good activity in antitumor vaccines.
Claims
1. A sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt of the following Formula (1): ##STR00002## wherein R.sub.1 is independently selected from the group consisting of NHC(O)CH.sub.xCl.sub.y, NHC(O)CH.sub.xF.sub.y, NHC(O)CH.sub.xBr.sub.y, NHC(O)H, NHC(O)C.sub.aH.sub.2a+1, NHC(O)C.sub.aH.sub.2aOH, NHC(O)C.sub.bH.sub.2b1, and NHC(O)C.sub.bH.sub.2b3, NH.sub.2, N.sub.3, and OH, where x and y is 0, 1, 2, or 3, and the sum of x and y is 3; a is an integer between 1 and 20; and b is an integer between 2 and 20; R.sub.2 is independently selected from the group consisting of NHC(O)CH.sub.pCl.sub.q, NHC(O)CH.sub.pF.sub.q, NHC(O)CH.sub.pBr.sub.q, NHC(O)H, NHC(O)C.sub.aH.sub.2a+1, NHC(O)C.sub.aH.sub.2aOH, NHC(O)C.sub.bH.sub.2b1, NHC(O)C.sub.bH.sub.2b3, acylamino, NH.sub.2, N.sub.3, and OH, where p and q is 0, 1, 2 or 3, and the sum of p and q is 3; a is an integer between 1 and 20; and b is an integer between 2 and 20; R.sub.1 and R.sub.2 are not NHC(O)CH.sub.3 simultaneously; R.sub.3 is independently selected from the group consisting of CO.sub.2H, CH.sub.2OH, CO.sub.2M, C(O)NHOH and hydrogen, where M is alkyl, aryl or heteroaryl, and the orientation of the R.sub.3 is an equatorial bond or an axial bond; R.sub.4 is allyloxy; R.sub.5 is OH, and the orientation of the R.sub.5 is an equatorial bond or an axial bond; and R.sub.6 is Hydrogen or -galactosyl-.
2. A glycoconjugate characterized by conjugating the sialic acid--(2.fwdarw.6)-D-aminopyranose derivative or salt of claim 1 to a protein or a polypeptide.
3. The sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt claim 1, wherein the sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt is characterized in that the salt is formed with a base.
4. A process of preparing the sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt of the following Formula (1), the process comprising: an acylation reaction in a solvent with carboxylic acid anhydrides, carboxylic acids or carboxylic esters in the presence of a reaction promoter, and using a sialic acid with 5-naked amino- and/or aminogalactose (aminoglucose) with 2-naked amino- as raw materials; ##STR00003## wherein R.sub.1 is independently selected from the group consisting of NHC(O)CH.sub.xCl.sub.y, NHC(O)CH.sub.xF.sub.y, NHC(O)CH.sub.xBr.sub.y, NHC(O)H, NHC(O)C.sub.aH.sub.2a+1, NHC(O)C.sub.aH.sub.2aOH, NHC(O)C.sub.bH.sub.2b1, and NHC(O)C.sub.bH.sub.2b3, NH.sub.2, N.sub.3, and OH; where x and y is 0, 1, 2 or 3, and the sum of x and y is 3; a is an integer between 1 and 20; and b is an integer between 2 and 20; R.sub.2 is independently selected from the group consisting of NHC(O)CH.sub.pCl.sub.q, NHC(O)CH.sub.pF.sub.q, NHC(O)CH.sub.pBr.sub.q, NHC(O)H , NHC(O)C.sub.aH.sub.2a+1, NHC(O)C.sub.aH.sub.2aOH , NHC(O)C.sub.bH.sub.2b1, NHC(O)C.sub.bH.sub.2b3, NH.sub.2, N.sub.3, and OH; where p and q is -0, 1, 2 or 3, and the sum of p and q is 3; a is an integer between 1 and 20; and b is an integer between 2 and 20; R.sub.1and R.sub.2 are not NHC(O)CH.sub.3 simultaneously; R.sub.3 is independently selected from the group consisting of CO.sub.2H, CH.sub.2OH, CO.sub.2M, C(O)NHOH and hydrogen, where M is alkyl, aryl or heteroaryl, and the orientation of the R.sub.3 is an equatorial bond or an axial bond; R.sub.4 is allyloxy; R.sub.5 is OH, and the orientation of the R.sub.5 is an equatorial bond or an axial bond; and R.sub.6 is Hydrogen or -galactosyl-.
5. The Process of claim 4, wherein the preparation process is characterized in that the raw materials include allyl 4-O-(5-amino-3,5-dideoxy--neuraminopyranosyl)-2-acetylamino-2-deoxy--D-galactopyranoside, allyl 4-O-(5-acetylamino-3,5-dideoxy--neuraminopyranosyl)-2-amino-2-deoxy--D-galactopyranoside or allyl 4-O-(5-amino-3,5-dideoxy--neuraminopyranosyl)-2-amino-2-deoxy--D-galactopyranoside; the carboxylic acid anhydrides include aliphatic carboxylic acid anhydrides, with/without fluoro, chloro or bromo substituents; that the carboxylic acids include aliphatic acids, with/without fluoro, chloro or bromo substituents; that the carboxylic esters include aliphatic esters, with/without fluoro, chloro or bromo substituents; the promoter include organic or inorganic bases; and that the solvents include water or organic solvents.
6. The Process of claim 5, wherein the preparation process is characterized in that the carboxylic acid anhydrides include acetic anhydride, propionic anhydride, n-butyric anhydride, iso-butyric anhydride and caproic anhydride; the carboxylic acids include mono-, di- and trifluoroacetic acid and mono-, dichloroacetic acid; and that the esters include methyl mono-, di- and trifluoroacetate and methyl dichloroacetate.
7. A process of preparing a glycoconjugate, including (1) ozonization of the sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt of claim 1 to give the corresponding disaccharide with aldehyde ; and (2) reductamination of the production obtained in step (1) with the carrier protein or polypeptide to give the conjugate.
8. A method of producing an anti-tumor drug, the method comprising preparing the sialic acid (-(2.fwdarw.6))-D-aminopyranose derivative or salt of claim 1 to produce the anti-tumor drug.
9. A method of producing an anti-tumor drug, the method comprising preparing the glycoconjugate of claim 2 to produce the anti-tumor drug.
10. A method of treating a tumor, comprising: administering to a subject in need thereof a composition comprising an effective dose the glycoconjugate of claim 2 and a pharmaceutically acceptable carrier or adjuvant.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION
(7) Below is the detailed description of the embodiments revealing the advantages of the present invention. The scope of protection of the present invention is not constrained by the representative embodiments described herein. And it should be noted that modificative and alternative technical solutions are included in this invention as well.
(8) Embodiment 1 Synthesis of Compound 2
(9) Compound 48 (100 mg, 0.119 mmol) (For its synthesis, see Embodiment 42.) was dissolved in 10 ml methanol, NaOMe/MeOH (30%, 0.02 g, 0.11 mmol) was added. The mixture was stirred at R.T. for 0.5 h. The reaction mixture was neutralized with 1N HCl/MeOH to pH=3. The mixture was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent. Fractions containing the expected product were collected to afford 2 (yield=95%).
(10) .sup.1H-NMR (400 MHz, D.sub.2O) 5.90-5.77 (m, 1H), 5.22 (dd, 1H, J.sub.1=1.6 Hz, J.sub.2=17.6 Hz), 5.14 (d, 1H, J=10.4 Hz), 4.75 (d, 1H, J=3.6 Hz, anomeric H of GalNAc), 4.15 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=13.6 Hz), 4.05 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.8 Hz), 3.96-3.87 (m, 3H), 3.82-3.74 (m, 5H), 3.60-3.48 (m, 4H), 3.46 (dd, 1H, J.sub.1=1.6 Hz, J.sub.2=8.8 Hz), 2.59 (dd, 1H, J.sub.1=4.4 Hz, J.sub.2=12.4 Hz, siaH-3 eq), 1.91 (s, 6H), 1.56 (t, 1H, J=12.4 Hz, siaH-3ax); .sup.13C-NMR (75 MHz, D.sub.2O) 175.59, 175.16, 173.99, 134.221, 118.64, 100.94, 96.79, 73.14, 72.33, 70.09, 69.28, 69.05, 68.80, 68.12, 64.32, 63.17, 52.42, 50.42, 40.83, 22.59, 22.47; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.36N.sub.2NaO.sub.14].sup.+, 575.2059. found, 575.2050
(11) Embodiment 2 Synthesis of Compound 3
(12) 10 mg Compound 14 [allyl 4-O-(5-amino-3,5-dideoxy--D-neuraminopyranosyl)-2-acetylamino-2-deoxy--D-galactopyranoside, (10 mg) See Embodiment 13] used as raw material was dissolved in 1 ml methanol. For its synthesis, 2-3 mg NaHCO3 was added. On ice bath, one drop of propionic anhydride (5 L) was added. The reaction mixture was stirred for 1 h. Another drop of propionic anhydride was added. After TLC showed the reaction is largely completed. The reaction mixture was then stirred overnight. The temperature was raised to R.T. After TLC showed the reaction is completed, the reaction mixture was neutralized with strong acidic resin and filtered The filtrate was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent and was further purified by C18 reverse phase column chromatography with water-water/MeOH as eluent, affording 3 (8-9 mg, yield=75-85%).
(13) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.96 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 16.5 Hz), 5.29-5.24 (m, 1H), 4.93 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.21 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.5 Hz, J.sub.4=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=8.0 Hz), 4.04-4.00 (m, 2H), 3.93-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.28 (q, 2H, J=12.5 Hz), 1.69 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.12 (t, 3H, J=8.0 Hz); .sup.13C-NMR(75 MHz, D.sub.2O) 179.58, 175.13, 173.98, 134.19, 118.63, 100.91, 96.76, 73.15, 72.31, 70.08, 69.26, 69.03, 68.80, 68.68, 68.09, 64.29, 63.13, 52.27, 50.40, 40.88, 29.80, 22.46, 10.08; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.23H.sub.38N.sub.2NaO.sub.14].sup.+, 589.2215. found, 589.2223
(14) Embodiment 3 Synthesis of Compound 4
(15) This compound was prepared from Compound 14 and n-butyric anhydride, affording 4 (yield=75-85%). The synthetic procedure was the same as that of Compound 3.
(16) .sup.1HNMR (500 MHz, D.sub.2O) 6.02-5.96 (m, 1H), 5.35 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.0 Hz), 5.26 (dd, 1H, J.sub.1=1.0 Hz, J.sub.2=3.0 Hz, J.sub.3=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.22 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.5 Hz, J.sub.4=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.06 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.04-4.00 (m, 2H), 3.93-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.26 (t, 2H, J=7.5 Hz), 2.03 (s, 3H), 1.68 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.60 (hexad, 2H, J=7.5 Hz), 0.91 (t, 3H, J=7.5 Hz); .sup.13C-NMR(75 MHz, D.sub.2O) 178.88, 175.31, 174.08, 134.40, 118.81, 101.08, 96.98, 73.36, 72.47, 70.26, 69.45, 69.22, 69.10, 68.80, 68.29, 64.46, 63.35, 52.50, 50.59, 41.10, 38.59, 22.66, 19.74, 13.56; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.24H.sub.40N.sub.2NaO.sub.14].sup.+, 603.2372. found, 603.2379
(17) Embodiment 4 Synthesis of Compound 5
(18) This compound was prepared from Compound 14 and iso-butyric anhydride, affording 5 (yield=75-85%). The synthetic procedure was the same as that of Compound 3.
(19) .sup.1H-NMR (500 MHz, D.sub.2O) 5.95-6.02 (m, 1H), 5.36 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.5 Hz), 5.26 (m, 1H), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.22 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.5 Hz, J.sub.4=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.06 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.04-3.98 (m, 2H), 3.94-3.78 (m, 5H), 3.74-3.60 (m, 4H), 3.52 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.75 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.54 (heptad, 1H, J=7.0 Hz), 2.02 (s, 3H), 1.68 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.12 (d, 3H, J=7.0 Hz), 1.11 (d, 3H, J=7.0 Hz); .sup.13C-NMR(125 MHz, D.sub.2O) 182.92, 174.64, 134.01, 119.50, 103.39, 101.79, 100.52, 78.99, 76.22, 75.91, 75.50, 75.14, 73.66, 73.54, 72.50, 71.40, 70.10, 68.95, 68.81, 68.19, 63.26, 61.76, 60.80, 52.19, 40.55, 35.95, 19.69, 19.16; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.24H.sub.40N.sub.2NaO.sub.14].sup.+, 603.2372. found, 603.2379
(20) Embodiment 5 Synthesis of Compound 6
(21) This compound was prepared from Compound 14 and caproic anhydride, affording 6 (yield=100%). The synthetic procedure was the same as that of Compound 3.
(22) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.96 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.1=3.0 Hz, J.sub.2=17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=10.5 Hz), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalNAc), 4.22 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08-4.00 (m, 3H), 3.93-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.28 (t, 2H, J=7.5 Hz), 2.03 (s, 3H), 1.68 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.60 (m, 2H), 1.29 (m, 4H), 0.86 (t, 3H, J=7.0 Hz); .sup.13C-NMR(75 MHz, D.sub.2O) 179.14, 175.31, 174.12, 134.40, 118.82, 101.11, 96.98, 73.38, 72.51, 70.27, 69.45, 69.22, 69.16, 68.79, 68.29, 64.46, 63.41, 52.49, 50.59, 41.14, 36.62, 31.25, 25.82, 22.66, 22.39, 13.95; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.26H.sub.44N.sub.2NaO.sub.14].sup.+, 631.2685. found, 631.2684
(23) Embodiment 6 Synthesis of Compound 7
(24) 10 mg Compound 14 used as raw material was dissolved in 1 ml methanol, under N.sub.2 atmosphere, triethylamine (TEA) (0.4 mL) was added. On ice bath, methyl fluoroacetate (0.2 mL) was added. The temperature was raised to R.T. The reaction mixture can be heated when necessary. When the reaction was near completion (by TLC monitoring), the mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as eluent and then on a Biogel P-2 column with water as the eluent. The product was still further purified by C18 reverse-phase column chromatography with water-water/MeOH as eluent, affording 7 (yield=75-85%).
(25) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.92 (m, 1H), 5.38-5.30 (m, 1H), 5.28-5.22 (m, 1H), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalNAc), 4.92 (d, 2H, J=46.0 Hz), 4.23-4.17 (m, 1H), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.06 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.04-3.80 (m, 9H), 3.71 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=10.0 Hz), 3.64 (dd, 1H, J.sub.1=6.0 Hz, J.sub.2=12.5 Hz), 3.59-3.55 (m, 1H), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.79 (t, 1H, J=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.34, 172.48, 172 (d, 1C, J=18.25 Hz), 134.37, 118.80, 100.02, 97.02, 80 (d, 1C, J=180.25 Hz), 73.15, 71.89, 70.20, 69.50, 69.26, 68.94, 68.29, 68.15, 64.56, 63.60, 52.10, 50.56, 40.20, 22.64; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.35FN.sub.2NaO.sub.14].sup.+, 593.1965. found, 593.1977
(26) Embodiment 7 Synthesis of Compound 8
(27) This compound was prepared from Compound 14 and methyl difluoroacetate, affording 8 (yield=69%). The synthetic procedure was the same as that of Compound 7.
(28) .sup.1H-NMR (500 MHz, D.sub.2O) 6.17 (t, J=54.0 Hz), 6.02-5.92 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.1=3.0 Hz, J.sub.2=17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=10.5 Hz), 4.92 (d, 1H, J=3.5, anomeric H of GalNAc), 4.22 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08-3.89 (m, 9H), 3.75 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.5 Hz, J.sub.3=12.0 Hz), 3.65-3.61 (m, 2H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.74 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.71 (t, 1H, J=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.32, 174.05, 166.30 (m, 1C, CF.sub.2HCO), 134.41, 118.81, 109.03 (t, 1C, J=247.0 Hz, CF.sub.2HCO), 101.13, 96.98, 72.70, 72.60, 70.28, 69.46, 69.24, 68.86, 68.73, 68.28, 64.55, 63.33, 52.62, 50.59, 40.95, 22.65; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.22H.sub.35F.sub.2N.sub.2O.sub.14].sup.+, 589.2051. found, 589.2053
(29) Embodiment 8 Synthesis of Compound 9
(30) This compound was prepared from Compound 14 and methyl trifluoroacetate, affording 9 (yield=85%). The synthetic procedure was the same as that of Compound 7.
(31) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.91 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=10.5 Hz) 4.93 (d, 1H, J=4.0 Hz, anomeric H of GalNAc), 4.20 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.07 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.0 Hz), 4.04-3.80 (m, 9H), 3.70 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.0 Hz), 3.66-3.60 (m, 1H) 3.53 (dd, 1H, J.sub.1=1 Hz, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (m, 3H), 1.79 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.34, 172.69, 160.19 (q, J=37.2 Hz), 134.39, 118.80, 116.49 (q, J=284.5 Hz), 100.23, 97.02, 72.72, 72.01, 70.21, 69.50, 69.26, 69.00, 64.61, 63.54, 53.07, 50.57, 49.60, 40.36, 22.65; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.22H.sub.34F.sub.3N.sub.2O.sub.14].sup.+, 607.1957. found, 607.1936
(32) Embodiment 9 Synthesis of Compound 10
(33) Under N.sub.2 atmosphere, Compound 14 (10-15 mg, 0.02-0.03 mmol), HBTU [O-(benzotriazol-1-yl)-tetramethyluronium hexafluorophosphate] (1.3 eq. per amino group) and fluoroacetic acid were dissolved in 2 ml DMF at R.T., DIPEA (N,N-diisopropylethylamine) (13 eq. to Compound 14) was added. After 1-4 h, the reaction was near completion (by TLC monitoring). The mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as eluent and then on a on a Biogel P-2 column with water as the eluent. The product was still further purified by C18 reverse-phase column chromatography with water-water/MeOH as eluent, affording 10 (yield=56%).
(34) .sup.1H-NMR (300 MHz, D.sub.2O) 5.84-5.65 (m, 1H), 5.35 (d, 1H, J=17.5 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.72 (d, 1H, J=3.6 Hz, anomeric H of GalNAc), 4.04-3.59 (m, 15H), 3.49-3.35 (m, 3H), 2.52 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.83 (m, 3H), 1.56 (t, 1H, J.sub.1=J.sub.2=12.0 Hz, siaH-3ax); .sup.13C-NMR(75 MHz, D.sub.2O) 175.13, 172.86, 170.99, 134.13, 118.60, 100.08, 96.78, 72.91, 71.86, 70.01, 69.28, 69.04, 68.75, 68.07, 64.37, 63.32, 52.72, 50.35, 42.82, 40.26, 22.42; HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.35ClN.sub.2NaO.sub.14, 609.1669. found, 609.1675
(35) Embodiment 10 Synthesis of Compound 11
(36) This compound was prepared from Compound 14 and methyl dichloroacetate, affording 11 (yield=46%). The synthetic procedure was the same as that of Compound 7.
(37) .sup.1H-NMR (300 MHz, D.sub.2O) 6.14 (s, 1H), 5.87-5.72 (m, 1H), 5.17 (d, 1H, J=17.1 Hz), 5.08 (d, 1H, J=10.2 Hz), 4.77 (d, 1H, J=3.6 Hz, anomeric H of GalNAc), 4.06-3.56 (m, 18H), 2.56 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=12.6 Hz, siaH-3 eq), 1.86 (s, 3H), 1.62 (t, 1H, J.sub.1=J.sub.2=12.0 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.34, 172.45, 168.22, 134.39, 118.81, 100.03, 97.03, 73.09, 71.95, 70.20, 69.51, 69.27, 69.15, 68.30, 68.00, 66.93, 64.58, 63.61, 53.25, 50.56, 40.32, 22.65; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.34Cl.sub.2N.sub.2NaO.sub.14].sup.+, 643.1279. found, 643.1304
(38) Embodiment 11 Synthesis of Compound 12
(39) This compound was prepared from Compound 14 and methyl trichloroacetate, affording 12 (yield=64%). The synthetic procedure was the same as that of Compound 7.
(40) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.92 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.0 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.93 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.21 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.5 Hz), 4.15 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=12.5 Hz), 4.09-3.83 (m, 9H), 3.67-3.57 (m, 3H) 3.53 (dd, 1H, J.sub.1=1 Hz, J.sub.2=9.0 Hz), 2.75 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.74 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.34, 173.76, 165.54, 134.42, 118.82, 100.90, 97.00, 72.67, 72.59, 70.28, 69.48, 69.25, 69.22, 68.29, 64.58, 63.38, 54.58, 50.59, 41.13, 22.67; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.33Cl.sub.3N.sub.2NaO.sub.14].sup.+, 677.0890. found, 677.0902
(41) Embodiment 12 Synthesis of Compound 13
(42) Compound 14 (15.0 mg, 0.029 mmol) was dissolved in 2.0 ml methanol, CuSO.sub.4 (0.5 mg, 0.003 mmol) was added. TfN.sub.3/pyridine (8.0 mL, 0.56 mmol See Tetrahedron Lett. 2005, 46, 8993-8995) was then added to the mixture. The reaction went to completion after 1 h (by TLC monitoring). The mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as eluent and then on a Biogel P-2 column with water as the eluent. The product was still further purified by C18 reverse-phase column chromatography with water-water/MeOH as eluent, affording 13 (yield=96%).
(43) .sup.1H-NMR (500 MHz, D.sub.2O) 5.93-6.00 (m, 1H), 5.35 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.5 Hz), 5.26 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=2.5 Hz, J.sub.3=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.21 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08-3.99 (m, 3H), 3.93-3.88 (m, 4H), 3.80 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=9.0 Hz), 3.72-3.64 (m, 2H), 3.64-3.58 (m, 2H), 3.47 (dd, 1H, J.sub.1=J.sub.2=10.0 Hz), 2.72 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, siaH-3 eq), 2.03 (s, 3H), 1.68 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.33, 173.99, 134.42, 118.80, 101.11, 96.98, 73.27, 72.67, 70.28, 70.13, 69.46, 69.29, 69.25, 68.28, 64.58, 63.49, 63.33, 50.60, 40.80, 22.66; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.20H.sub.32N.sub.4NaO.sub.13].sup.+, 559.1858. found, 559.1847
(44) Embodiment 13 Synthesis of Compound 14
(45) Compound 52 (For its synthesis, see Embodiment 44) (380 mg, 0.426 mmol) was dissolved in 20 ml methanol, NaOMe/MeOH (30%, 0.02 g, 011 mmol) was added. The mixture was stirred at R.T. for 1 h. The mixture was concentrated in vacuum. The solution of NaOH in water (2N, 10 mL) was added. The mixture was stirred at R.T. for 10 h. The reaction mixture was neutralized with 1N HCl/MeOH to pH=3. The mixture was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent. Fractions containing the expected product were collected to afford 2 (yield=86%). The crude product may also be directly used in the next step.
(46) .sup.1H-NMR (500 MHz, D.sub.2O) 5.97 (m, 1H), 5.35 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.5 Hz), 5.26 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=10.5 Hz), 4.93 (d, 1H, J=4.0 Hz, anomeric H of GalNAc), 4.20 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=12.0 Hz), 4.07 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.0 Hz), 4.04-3.89 (m, 7H), 3.80-3.71 (m, 3H), 3.62 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.5 Hz), 3.22 (t, 1H, J.sub.1=J.sub.2=10.0 Hz), 2.77 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (m, 3H), 1.71 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (75 MHz, D.sub.2O) 175.06, 173.57, 134.14, 118.57, 100.80, 96.71, 72.25, 72.15, 70.04, 69.20, 69.04, 68.37, 67.98, 67.72, 64.51, 62.77, 52.79, 50.34, 40.82, 22.45; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.20H.sub.34N.sub.2NaO.sub.13].sup.+, 533.1953. found, 533.1953
(47) Embodiment 14 Synthesis of Compound 15
(48) This compound was prepared from Compound 14 and bromoacetic acid, affording 15 (yield=37%). The synthetic procedure was the same as that of Compound 10.
(49) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dq, 1H, J=1.5 Hz, 10.5 Hz), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalNAc), 4.22 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=4.0 Hz, J.sub.3=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=6.0 Hz), 4.07 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.0 Hz), 4.05-4.00 (m, 2H), 3.96-3.85 (m, 7H), 3.80 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 3.71 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.5 Hz, J.sub.3=12.0 Hz), 3.66-3.59 (m, 3H), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.04 (s, 3H), 1.69 (dd, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.34, 174.12, 171.51, 134.42, 118.82, 101.15, 96.99, 73.13, 72.63, 70.29, 69.47, 69.24, 69.02, 68.30, 64.52, 63.38, 53.03, 50.61, 41.04, 28.73, 22.67; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.22H.sub.36BrN.sub.2O.sub.14].sup.+, 631.1344. found, 631.1324
(50) Embodiment 15 Synthesis of Compound 16
(51) This compound was prepared from Compound 26 [allyl 4-O-(5-acetylamino-3,5-dideoxy--D-neuraminopyranosyl)-2-amino-2-deoxy--D-galactopyranoside. See Embodiment 25] and propionic anhydride, affording 16 (yield=56%). The synthetic procedure was the same as that of Compound 3.
(52) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.91 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.5 Hz), 5.25 (d, 1H, J=10.5 Hz), 4.92 (d, J=3.5 Hz, anomeric H of GalN), 4.21 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.5 Hz), 4.07 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=7.5 Hz), 4.04-3.99 (m, 2H), 3.94-3.81 (m, 5H), 3.73-3.62 (m, 4H), 3.57 (1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (t, 1H, J.sub.1=J.sub.2=8.0 Hz), 2.29 (q, 2H, J=7.5 Hz), 2.03 (s, 3H), 1.70 (t, 1H, J.sub.1=J.sub.2=12.0 Hz, siaH-3ax), 1.11 (t, 3H, J.sub.1=J.sub.2=7.5 Hz); .sup.13C-NMR (125 MHz, D.sub.2O) 179.33, 175.76, 174.14, 134.36, 118.89, 101.13, 96.96, 73.33, 72.50, 70.28, 69.48, 69.25, 68.98, 68.22, 64.49, 63.36, 52.59, 50.49, 41.00, 29.84, 22.76, 10.29; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.23H.sub.38N.sub.2NaO.sub.14].sup.+, 589.2215. found, 589.2225
(53) Embodiment 16 Synthesis of Compound 17
(54) This compound was prepared from Compound 26 and n-butyric anhydride, affording 17 (yield=53%). The synthetic procedure was the same as that of Compound 3.
(55) .sup.1H-NMR (500 MHz, D.sub.2O) 6.00-5.90 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.5 Hz), 5.26 (d, 1H, J.sub.1=1.0 Hz, J.sub.2=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.20 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.5 Hz), 4.06 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=7.5 Hz), 4.03-3.99 (m, 2H), 3.94-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.78 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=5.0, J.sub.2=12.5 Hz, siaH-3 eq), 2.28 (t, 2H, J=7.0 Hz), 2.03 (s, 1H), 1.68 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.65-1.56 (m, 2H), 0.93 (t, 3H, J=7.5 Hz); .sup.13C-NMR(125 MHz, D.sub.2O) 178.26, 175.59, 173.89, 134.14, 118.82, 100.88, 96.8473.16, 72.29, 70.10, 69.36, 69.10, 68.78, 67.96, 64.33, 63.19, 52.41, 50.36, 40.79, 38.19, 22.59, 19.60, 13.26; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.24H.sub.40N.sub.2NaO.sub.14].sup.+, 603.2372. found, 603.2374
(56) Embodiment 17 Synthesis of Compound 18
(57) This compound was prepared from Compound 26 and iso-butyric anhydride, affording 18 (yield=76%). The synthetic procedure was the same as that of Compound 3.
(58) .sup.1H-NMR (500 MHz, D.sub.2O) 6.00-5.90 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.0 Hz), 5.26 (d, 1H, J.sub.1=10.0 Hz), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.20 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz), 4.14 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.5 Hz), 4.06 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.0 Hz), 4.03-3.99 (m, 2H), 3.94-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.78 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=8.5 Hz), 2.73 (dd, 1H, J.sub.1=4.5, J.sub.2=12.5 Hz, siaH-3 eq), 2.56 (septenary, 1H, J=7.0 Hz), 2.03 (s, 3H), 1.83 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax), 1.10 (d, 3H, J=7.0 Hz), 1.09 (d, 3H, J=7.0 Hz); .sup.13C-NMR(125 MHz, D.sub.2O) 182.45, 175.76, 174.12, 134.31, 119.00, 101.11, 96.91, 73.32, 72.49, 70.29, 69.50, 69.28, 68.97, 6810, 64.49, 63.36, 52.58, 50.43, 40.98, 35.71, 22.76, 19.61, 19.19; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.24H.sub.40N.sub.2NaO.sub.14].sup.+, 603.2372. found, 603.2384
(59) Embodiment 18 Synthesis of Compound 19
(60) This compound was prepared from Compound 26 and caproic anhydride, affording 19 (yield=87%). The synthetic procedure was the same as that of Compound 3.
(61) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.93 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.0 Hz, J.sub.2=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.22 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz), 4.15 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.0 Hz), 4.07 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.08-4.00 (m, 2H), 3.93-3.80 (m, 5H), 3.71-3.61 (m, 4H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.28 (m, 2H), 2.03 (s, 3H), 1.68 (t, 1H, J=12.5 Hz, siaH-3ax), 1.60 (m, 2H), 1.29 (m, 4H), 0.86 (t, 3H, J=7.0 Hz); .sup.13C-NMR(125 MHz, D.sub.2O) 178.62, 175.76, 174.12, 134.32, 118.98, 101.13, 97.02, 73.33, 72.50, 70.28, 69.54, 69.26, 68.98, 68.13, 64.48, 63.36, 52.59, 50.52, 40.99, 36.44, 31.14, 25.83, 22.76, 22.41, 13.95; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.26H.sub.44N.sub.2NaO.sub.14].sup.+, 631.2685. found, 631.2678
(62) Embodiment 19 Synthesis of Compound 20
(63) This compound was prepared from Compound 26 and methyl fluoroacetate, affording 20 (yield=77%). The synthetic procedure was the same as that of Compound 7.
(64) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.92 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.0 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.97 (d, J=4.0 Hz, anomeric H of GalN), 4.97&4.88 (d, 2H, J.sub.F,H=46.5 Hz), 4.26 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.0 Hz), 4.22 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz), 4.08 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.0 Hz), 4.05-3.99 (m, 3H), 3.94-3.81 (m, 4H), 3.71-3.62 (m, 4H), 3.58 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.72 (dd, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.68 (t, 1H J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.77, 174.14, 171.89, 171.74, 134.35, 118.94, 101.13, 96.88, 81.18, 79.74, 73.33, 72.50, 70.33, 69.50, 69.22, 69.00, 68.10, 64.45, 63.36, 52.59, 50.28, 41.00, 22.76; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.35FN.sub.2NaO.sub.14].sup.+, 593.1965. found, 593.1975
(65) Embodiment 20 Synthesis of Compound 21
(66) This compound was prepared from Compound 26 and methyl difluoroacetate, affording 21 (yield=78%). The synthetic procedure was the same as that of Compound 7.
(67) .sup.1H-NMR (500 MHz, D.sub.2O) 6.19 (t, 1H, J.sub.F,H=53.6 Hz), 6.01-5.91 (m, 1H), 5.34 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.25 (dq, 1H, J=1.5 Hz, 10.5 Hz), 4.99 (d, J=4.0 Hz, anomeric H of GalN), 4.26-4.19 (m, 2H), 4.10 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.06-3.99 (m, 3H), 3.99-3.93 (m, 1H), 3.90-3.84 (m, 3H), 3.80-3.69 (m, 3H), 3.65 (dd, 1H, J.sub.1=6.5 Hz, J.sub.2=12.5 Hz), 3.57 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=8.5 Hz), 2.71 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, siaH-3 eq), 2.03 (s, 3H), 1.76 (t, J.sub.1=J.sub.2=12.0 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.73, 172.88, 166.05 (t, 1C), 134.29, 118.97, 118.79, 108.88 (t, 1C, J.sub.F-C=246.1 Hz), 100.27, 96.61, 73.50, 71.99, 70.28, 69.55, 69.20, 69.02, 68.50, 67.93, 64.46, 63.57, 52.51, 50.90, 50.82, 40.36, 22.79; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.34F.sub.2N.sub.2NaO.sub.14].sup.+, 611.1870. found, 611.1861
(68) Embodiment 21 Synthesis of Compound 22
(69) This compound was prepared from Compound 26 and methyl trifluoroacetate, affording 22 (yield=76%). The synthetic procedure was the same as that of Compound 7.
(70) .sup.1H-NMR (500 MHz, D.sub.2O) 5.92-5.82 (m, 1H), 5.35 (d, 1H, J=17.0 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.20-4.11 (m, 2H), 4.01 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=7.0 Hz), 4.00-3.91 (m, 3H), 3.86-3.72 (m, 5H), 3.65-3.51 (m, 4H), 3.50 (d, 1H, J=9.0 Hz), 2.65 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.95 (s, 3H), 1.60 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.07, 173.34, 159.38 (q, J.sub.F-C=37.5 Hz), 133.59, 118.30, 116 (q, J.sub.F-C=284.1 Hz), 100.40, 95.65, 72.63, 71.77, 69.62, 68.82 68.46, 68.29, 68.23, 66.97, 63.69, 62.68, 51.89, 50.75, 40.25 HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.33F.sub.3N.sub.2NaO.sub.14].sup.+, 629.1776. found, 629.1787
(71) Embodiment 22 Synthesis of Compound 23
(72) This compound was prepared from Compound 26 and chloroacetic acid, affording 23 (yield=54%). The synthetic procedure was the same as that of Compound 10.
(73) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.27 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 4.95 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.24-4.18 (m, 3H), 4.16 (d, 1H, J=0.5 Hz), 4.08 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=8.0 Hz), 4.06-4.00 (m, 2H), 3.96 (dd, 1H, J.sub.1=3.0 Hz, J.sub.2=11.0 Hz), 3.92-3.80 (m, 4H), 3.70 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=8.5 Hz), 3.68-3.60 (m, 3H), 3.58 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=9.0 Hz), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.83 (s, 3H), 1.56 (t, 1H, J=12.0 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.77, 174.12, 170.85, 134.33, 118.98, 101.12, 96.75, 73.33, 72.50, 70.34, 69.53, 69.21, 68.99, 68.21, 64.46, 63.36, 52.59, 51.11, 42.97, 40.99, 22.76; HRMS (m/z): [M+NH.sub.4].sup.+ calcd. for [C.sub.22H.sub.39ClN.sub.3O.sub.14].sup.+, 604.2115. found, 604.2112
(74) Embodiment 23 Synthesis of Compound 24
(75) This compound was prepared from Compound 26 and methyl dichloroacetate, affording 24 (yield=54%). The synthetic procedure was the same as that of Compound 7.
(76) .sup.1H-NMR (500 MHz, D.sub.2O) 6.35 (s, 1H), 6.01-5.92 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.5 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.22 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.17 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=6.0 Hz), 4.08 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.0 Hz), 4.05-4.00 (m, 2H), 3.98 (dd, 1H, J.sub.1=3.0 Hz, J.sub.2=11.0 Hz), 3.94-3.80 (m, 4H), 3.71 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=10.5 Hz), 3.69-3.62 (m, 3H), 3.58 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=8.5 Hz), 2.73 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.69 (t, 1H, J=12.0 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 175.77, 174.06, 168.02, 134.28, 119.15, 101.09, 96.40, 73.34, 72.48, 70.36, 69.60, 69.24, 69.00, 68.96, 68.03, 66.77, 64.44, 63.38, 52.59, 51.64, 40.96, 22.77; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.22H.sub.35Cl.sub.2N.sub.2O.sub.14].sup.+, 621.1460. found, 621.1477
(77) Embodiment 24 Synthesis of Compound 25
(78) Compound 54 (For its synthesis, see Embodiment 45) (145 mg, 0.176 mmol) was dissolved in 8.0 ml methanol, NaOMe/MeOH (30%, 0.02 g, 0.11 mmol) was added. The mixture was stirred at R.T. for 2 h. The mixture was concentrated in vacuum. The solution of NaOH in water (1N, 3 mL) was added. The mixture was stirred at R.T. for 24 h. The reaction mixture was neutralized with 1N HCl/MeOH to pH=3. The mixture was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent. Fractions containing the expected product were collected to afford 25 (yield=100%).
(79) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.94 (m, 1H), 5.35 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.5 Hz), 5.26 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=2.5 Hz, J.sub.3=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN.sub.3), 4.21 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08-3.99 (m, 3H), 3.93-3.88 (m, 4H), 3.80 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=9.0 Hz), 3.72-3.64 (m, 2H), 3.64-3.58 (m, 2H), 3.47 (dd, 1H, J.sub.1=J.sub.2=10.0 Hz), 2.72 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, siaH-3 eq), 2.03 (s, 3H), 1.68 (t, 1H, J=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.33, 173.99, 134.42, 118.80, 101.11, 96.98, 73.27, 72.67, 70.28, 70.13, 69.46, 69.29, 69.25, 68.28, 64.58, 63.49, 63.33, 50.60, 40.80, 22.66; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.20H.sub.32N.sub.4NaO.sub.13].sup.+, 559.1858. found, 559.1851
(80) Embodiment 25 Synthesis of Compound 26
(81) Compound 25 (94 mg, 0.176 mmol. For its synthesis, see Embodiment 24) was dissolved in solution comprising of 15 ml pyridine, 10 ml TEA and 5 ml water, H.sub.2S was introduced. The reaction mixture was stirred for 10 h. The mixture was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent. Fractions containing the expected product were collected to afford 26 (yield=67%).
(82) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.97 (m, 1H), 5.38 (d, 1H, J=17.0 Hz), 5.28 (d, J=10.0 Hz), 5.17 (d, 1H, J=3.5 Hz, anomeric H of GalNH.sub.2), 4.28 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.5 Hz), 4.14-4.00 (m, 4H), 3.93-3.80 (m, 4H), 3.71-3.56 (m, 5H), 3.48 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.03 (s, 3H), 1.68 (t, 1H, J=12.0 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.61, 173.94, 133.74, 119.33, 100.95, 94.97, 73.16, 72.34, 70.29, 69.56, 68.74, 68.56, 67.10, 64.04, 63.16, 52.40, 51.36, 40.78, 22.58; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.20H.sub.34N.sub.2NaO.sub.13].sup.+, 533.1953. found, 533.1957
(83) Embodiment 26 Synthesis of Compound 27
(84) This compound was prepared from Compound 50 [allyl 4-O-(5-amino-3,5-dideoxy--D-neuraminopyranosyl)-2-amino-2-deoxy--D-galactopyranoside. For its synthesis, see embodiment 43] and propionic anhydride, affording 27 (yield=55%). The synthetic procedure was the same as that of Compound 3.
(85) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=10.5 Hz), 4.91 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.22 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.07 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=8.0 Hz), 4.03-4.00 (m, 2H), 3.96-3.81 (m, 5H), 3.75-3.62 (m, 4H), 3.56 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.73 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.30 (q, 4H, J=7.5 Hz), 1.68 (t, 1H, J=12.5 Hz, siaH-3ax), 1.17 (t, 6H, J=7.5 Hz); .sup.13C-NMR (75 MHz, D.sub.2O) 134.13, 118.69, 100.91, 96.74, 73.14, 72.30, 70.09, 69.26, 69.05, 68.79, 68.68, 68.00, 64.32, 63.11, 52.25, 50.29, 40.89, 29.79, 29.64, 10.10, 10.07; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.24H.sub.40N.sub.2NaO.sub.14].sup.+, 603.2372. found, 603.2378.
(86) Embodiment 27 Synthesis of Compound 28
(87) This compound was prepared from Compound 50 and n-butyric anhydride, affording 28 (yield=56%). The synthetic procedure was the same as that of Compound 3.
(88) .sup.1H-NMR (300 MHz, D.sub.2O) 5.83-5.68 (m, 1H), 5.35 (d, 1H, J=17.5 Hz), 5.26 (d, 1H, J=10.5 Hz), 4.72 (d, 1H, J=3.6 Hz, anomeric H of GalN), 4.05-3.36 (m, 15H), 2.53 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.07 (t, 3H, J=7.2 Hz), 1.49 (t, 1H, J=12.3 Hz, siaH-3ax), 1.42 (hexad peaks, 4H, J=7.2 Hz), 0.71 (t, 6H, J=7.2 Hz); .sup.13C-NMR(125 MHz, D.sub.2O) 178.89, 178.41, 174.141, 134.32, 118.98, 101.12, 97.01, 73.35, 72.50, 70.28, 69.53, 69.26, 69.10, 68.83, 68.14, 64.48, 63.33, 52.50, 50.53, 41.13, 38.59, 38.36, 19.74, 13.54, 13.42; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.26H.sub.44N.sub.2NaO.sub.14].sup.+, 631.2685. found, 631.2675
(89) Embodiment 28 Synthesis of Compound 29
(90) This compound was prepared from Compound 50 and i-butyric anhydride, affording 29 (yield=60%). The synthetic procedure was the same as that of Compound 3.
(91) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.91 (m, 1H), 5.35 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.0 Hz, J.sub.3=10.5 Hz) 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.22 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.14 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.07 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=7.5 Hz), 4.04-4.00 (m, 2H), 3.94-3.86 (m, 5H), 3.73-3.62 (m, 4H), 3.52 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.74 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 2.56 (heptad peaks, 1H, J=7.0 Hz), 2.46 (heptad peaks, 1H, J=7.0 Hz), 1.68 (t, 1H, J=12.5 Hz, siaH-3ax), 1.13-1.09 (m, 12H); .sup.13C-NMR (125 MHz, D.sub.2O) 182.93, 182.46, 174.17, 134.32, 119.01, 101.13, 96.91, 73.37, 72.49, 70.30, 69.51, 69.28, 69.08, 68.72, 68.11, 64.49, 63.31, 52.36, 50.43, 41.17, 35.96, 35.71, 19.71, 19.61, 19.18 (2C); HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.26H.sub.44N.sub.2NaO.sub.14].sup.+, 631.2685. found, 631.2673
(92) Embodiment 29 Synthesis of Compound 30
(93) This compound was prepared from Compound 50 and caproic anhydride, affording 30 (yield=77%). The synthetic procedure was the same as that of Compound 3.
(94) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.91 (m, 1H), 5.35 (dd, 1H, J.sub.1=17.0 Hz, J.sub.2=1.0 Hz), 5.26 (d, 1H, J.sub.1=10.5 Hz), 4.91 (d, 1H, J=3.5 Hz), 4.21 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.16 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.0 Hz), 4.07 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.02-3.99 (m, 2H), 3.93-3.79 (m, 5H), 3.71-3.61 (m, 4H), 3.55 (d, 1H, J=7.0 Hz), 2.72 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, siaH-3 eq), 2.31-2.25 (m, 4H), 1.68 (t, 1H, J=12.5 Hz siaH-3ax), 1.62-1.57 (m, 4H), 1.36-1.24 (m, 8H), 0.86 (m, 6H); .sup.13C-NMR(125 MHz, D.sub.2O) 178.94, 178.48, 173.36, 134.11, 118.81, 100.51, 96.85, 73.28, 72.10, 70.08, 69.38, 69.10, 68.98, 68.40, 67.96, 64.35, 63.31, 52.27, 50.34, 40.68, 36.46, 36.26, 31.08, 30.96, 25.66, 22.25, 13.78; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.30H.sub.53N.sub.2O.sub.14].sup.+, 665.3491. found, 665.3495
(95) Embodiment 30 Synthesis of Compound 31
(96) This compound was prepared from Compound 50 and methyl fluoroacetate, affording 31 (yield=67%). The synthetic procedure was the same as that of Compound 7.
(97) .sup.1H-NMR (500 MHz, D.sub.2O) 5.90-5.85 (m, 1H), 5.27 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=17.0 Hz), 5.27 (d, 1H, J=10.5 Hz), 4.87 (d, 1H, J=3.0 Hz, anomeric H of GalN), 4.8 (d, 2H, J.sub.F-H=46.0 Hz), 4.7 (d, 2H, J.sub.F-H=46.0 Hz), 4.17 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.5 Hz), 4.13 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz), 4.00 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 3.96-3.72 (m, 8H), 3.65 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=10.0 Hz, J.sub.3=12.0 Hz), 3.57-3.49 (m, 2H), 3.50 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.79 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.36, 171.5 (d, 1C, J.sub.F-C=18.12 Hz), 171.1 (d, 1C, J.sub.F-C=18.62 Hz), 133.65, 118.22, 100.44, 96.19, 80 (d, 1C, J.sub.F-C=189.53 Hz), 79 (d, 1C, J.sub.F-C=179.56 Hz), 72.21, 71.87, 69.62, 68.79, 68.51, 68.149, 68.08, 67.40, 63.77, 62.66, 51.50, 49.58, 40.27; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.32F.sub.4N.sub.2NaO.sub.14].sup.+, 611.1870. found, 611.1872
(98) Embodiment 31 Synthesis of Compound 32
(99) This compound was prepared from Compound 50 and methyl difluoroacetate, affording 32 (yield=72%). The synthetic procedure was the same as that of Compound 7.
(100) .sup.1H-NMR (500 MHz, D.sub.2O) 6.10 (t, 1H, J.sub.F-H=53.6 Hz), 6.09 (dd, 1H, J.sub.F-H=53.6 Hz), 5.92-5.86 (m, 1H), 5.30 (d, 1H, J=17.5 Hz), 5.18 (d, 1H, J=10.5 Hz), 4.91 (d, 1H, J=3.5 Hz, anomeric H of Gal), 4.20-4.13 (m, 2H), 4.02 (dd, 1H, J.sub.1=4.5 Hz, J.sub.3=8.0 Hz), 4.00-3.78 (m, 9H), 3.71-3.65 (m, 1H), 3.58-3.54 (m, 1H), 3.49 (d, 1H, J=9.0 Hz), 2.67 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.69 (dd, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 173.33, 165.3 (m, 2C), 133.61, 118.26, 108.29 (t, 1C, J.sub.F-C=46.5 Hz), 108.17 (t, 1C, J.sub.F-C=5.6 Hz), 100.45, 95.86, 71.99, 71.91, 69.63, 68.81, 68.48, 68.16, 68.02, 67.21, 63.78, 51.91, 50.14, 40.24; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.32F.sub.4N.sub.2NaO.sub.14].sup.+, 647.1682. found, 647.1669
(101) Embodiment 32 Synthesis of Compound 33
(102) This compound was prepared from Compound 50 and methyl trifluoroacetate, affording 33 (yield=49%). The synthetic procedure was the same as that of Compound 7.
(103) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=10.5 Hz), 5.01 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.21-4.27 (m, 2H), 4.11 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.07-3.86 (m, 8H), 3.76 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.5 Hz, J.sub.3=12.0 Hz), 3.67-3.61 (m, 2H), 3.56 (dd, 1H, J.sub.1=1.5, J.sub.2=9.0 Hz), 2.76 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.72 (t, 1H, J=12.5 Hz, siaH-3ax); .sup.13C-NMR(125 MHz, D.sub.2O) 174.04, 160.1 (m, 2C), 134.28, 119.01, 116.51 (dd, 2C, J.sub.F-H=285.12 Hz), 101.18, 96.36, 72.66, 72.51, 70.35, 69.54, 69.19, 68.91, 68.65, 67.68, 64.53, 63.32, 53.13, 51.46, 40.99; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.30F.sub.6N.sub.2NaO.sub.14].sup.+, 683.1493. found, 683.1501
(104) Embodiment 33 Synthesis of Compound 34
(105) This compound was prepared from Compound 50 and chloroacetic acid, affording 34 (yield=60%). The synthetic procedure was the same as that of Compound 10.
(106) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.93 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.27 (dq, 1H, J=1.5 Hz, 10.5 Hz), 4.97 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.21-4.18 (m, 2H), 4.19-4.17 (m, 3H), 4.09 (dd, 1H, J.sub.1=4.0 Hz, J.sub.3=8.5 Hz), 4.06-4.01 (m, 2H), 3.93-3.86 (m, 5H), 3.78 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.5 Hz, J.sub.3=12.0 Hz), 3.68 (dd, 1H, J.sub.1=4.5 Hz, J.sub.3=10.5 Hz), 3.65 (dd, 1H, J.sub.1=6.5 Hz, J.sub.3=12.0 Hz), 3.58 (dd, 1H, J.sub.1=1.5 Hz, J.sub.3=9.0 Hz), 2.71 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.75 (t, J=12.0 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 173.29, 171.20, 170.86, 134.33, 118.98, 100.58, 96.79, 73.10, 72.24, 70.31, 69.57, 69.24, 68.98, 68.46, 68.21, 64.52, 63.49, 52.97, 51.09, 43.05, 42.97, 40.62; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.34Cl.sub.2N.sub.2NaO.sub.14].sup.+, 643.1279. found, 643.1306
(107) Embodiment 34 Synthesis of Compound 35
(108) This compound was prepared from Compound 50 and methyl dichloroacetate, affording 35 (yield=43%). The synthetic procedure was the same as that of Compound 7.
(109) .sup.1H-NMR (500 MHz, D.sub.2O) 6.35 (s, 1H), 6.32 (s, 1H), 6.01-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dq, 1H, J=1.5 Hz, 10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.22 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.5 Hz, J.sub.4=13.0 Hz), 4.18 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.10 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.0 Hz), 4.08-3.84 (m, 8H), 3.75 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.0 Hz, J.sub.3=12.0 Hz), 3.66-3.61 (m, 2H), 3.57 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.5 Hz), 2.74 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.69 (t, 1H, J=12.0 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 173.99, 168.21, 168.04, 134.29, 119.14, 101.10, 96.41, 72.89, 72.65, 70.37, 69.60, 69.25, 69.08, 68.67, 68.03, 66.96, 66.76, 64.52, 63.36, 53.32, 51.64, 41.04; HRMS (m/z): [M+H].sup.+ calcd. for [C.sub.22H.sub.33Cl.sub.4N.sub.2O.sub.14].sup.+, 689.0680. found, 689.0671
(110) Embodiment 35 Synthesis of Compound 36
(111) This compound was prepared from Compound 50 and methyl trichloroacetate, affording 36 (yield=64%). The synthetic procedure was the same as that of Compound 7.
(112) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.91 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.26 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 5.01 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.25 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.0 Hz, J.sub.4=13.0 Hz), 4.20 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.14-3.80 (m, 10H), 3.66 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=11.0 Hz), 3.63 (dd, 1H, J.sub.1=6.5 Hz, J.sub.2=11.5 Hz), 3.59 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.5 Hz), 2.76 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, siaH-3 eq), 1.72 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR(75 MHz, D.sub.2O) 173.96, 165.38, 134.06, 119.05, 100.98, 96.14, 72.60, 72.46, 70.26, 69.47, 69.18, 69.18, 69.01, 68.27, 67.56, 64.41, 63.14, 54.42, 52.84, 41.14; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.30Cl.sub.6N.sub.2NaO.sub.14].sup.+, 778.9720. found, 778.9698
(113) Embodiment 36 Synthesis of Compound 37
(114) Compound 39 (For its synthesis, see Embodiment 38) (18.0 mg, 0.032 mmol) was dissolved in 5 ml methanol, 50% NH.sub.2OH/water (0.5 mL, 7.57 mmol) and KCN (1.0 mg, 0.016 mmol) were added. The mixture was stirred at R.T. for 20 h. The mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as eluent and then on a on a Biogel P-2 column with water as the eluent, affording 37 (yield=50%).
(115) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.93 (m, 1H), 5.36 (d, 1H, J.sub.1=17.0 Hz), 5.26 (d, 1H, J.sub.1=10.5 Hz), 4.92 (d, 1H, J=4.0 Hz, anomeric H of GalN), 4.20 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.15 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.06 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=8.0 Hz), 4.04-3.98 (m, 2H), 3.96-3.82 (m, 5H), 3.79-3.72 (m, 2H), 3.71-3.62 (m, 3H), 2.73 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=13.5 Hz, siaH-3 eq), 2.03 (s, 6H), 1.83 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.64, 175.22, 166.20, 134.26, 118.61, 100.03, 96.91, 74.23, 71.78, 70.02, 69.34, 69.08, 68.28, 68.14, 67.62, 63.76, 63.44, 52.16, 50.41, 39.13, 22.64, 22.52; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.37N.sub.3NaO.sub.14].sup.+, 590.2168. found, 590.2163
(116) Embodiment 37 Synthesis of Compound 38
(117) Compound 39 (For its synthesis, see Embodiment 38) (10.0 mg, 0.017 mmol) was dissolved in 2 ml methanol, NaBH.sub.4 (6.0 mg, 0.158 mmol) was added in three batches. The mixture was stirred at R.T. for 1 h. The mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as eluent and then on a on a Biogel P-2 column with water as the eluent, affording 38 (yield=94%).
(118) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.92 (m, 1H), 5.34 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=12.5 Hz), 5.25 (d, 1H, J=10.5 Hz), 4.94 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.21 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.16 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.08-4.00 (m, 3H), 3.95-3.78 (m, 8H), 3.74-3.68 (m, 2H), 3.62 (dd, 1H, J.sub.1=6.0 Hz, J.sub.2=12.0 Hz), 3.52 (d, 1H, J=9.0 Hz), 2.30 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=13.5 Hz, siaH-3 eq), 2.04 (s, 3H), 2.03 (s, 3H), 1.75 (t, 1H, J=13.0 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 177.44, 177.09, 136.14, 120.44, 102.79, 98.77, 74.09, 73.07, 72.25, 71.21, 71.04, 70.80, 70.25, 70.08, 65.59, 63.56, 62.79, 54.69, 52.33, 38.67, 24.54, 24.38. HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.38N.sub.2NaO.sub.13].sup.+, 561.2266. found, 561.2267
(119) Embodiment 38 Synthesis of Compound 39
(120) Compound 48 (For its synthesis, see Embodiment 42) (100 mg, 0.119 mmol) was dissolved in 10 ml methanol, NaOMe/MeOH (30%, 0.02 g, 011 mmol) was added. The mixture was stirred at R.T. for 4 h. The reaction mixture was neutralized with 1N HCl/MeOH to pH=6. The mixture was concentrated in vacuum. The residue was purified by C18 reverse-phase column chromatography with MeOH/water as the eluent. Fractions containing the expected product were collected to afford 39 (yield=95%).
(121) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.27 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 4.92 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.21-4.14 (m, 2H), 4.06-3.96 (m, 4H), 3.93-3.83 (m, 8H), 3.7 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=10.0 Hz, J.sub.3=12.0 Hz), 3.71-3.64 (m, 2H), 3.56 (d, 1H, J=9.5 Hz), 2.71 (dd, 1H, J.sub.1=4.5 Hz, J.sub.3=13.0 Hz, siaH-3 eq), 2.04 (s, 6H), 1.83 (t, J.sub.1=J.sub.2=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.70, 175.36, 170.58, 134.36, 118.79, 99.64, 97.06, 73.62, 71.30, 70.10, 69.51, 69.25, 69.01, 68.31, 67.89, 64.55, 63.85, 54.16, 52.44, 50.53, 39.83, 22.82, 22.64; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.23H.sub.38N.sub.2NaO.sub.14].sup.+, 589.2215. found, 589.2223
(122) Embodiment 39 Synthesis of Compound 40
(123) This compound was prepared from Compound 61 (For its synthesis, see Embodiment 47), affording 40 (yield=95%). The synthetic procedure was the same as that of Compound 3.
(124) .sup.1H-NMR (500 MHz, D.sub.2O) 6.04-5.95 (m, 1H), 5.38 (dq, 1H, J=1.5 Hz, 17.5 Hz), 5.28 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 4.97 (d, 1H, J=3.5 Hz, anomeric H of Gal), 4.21 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=5.5 Hz, J.sub.3=13.0 Hz), 4.16 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 4.10-4.02 (m, 2H), 3.98 (d, 1H, J=2.5 Hz), 3.96-3.60 (m, 9H), 3.58 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=8.5 Hz), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.3=13.0 Hz, siaH-3 eq), 2.03 (s, 3H), 1.74 (t, J=12.5 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.76, 173.21, 134.35, 119.10, 100.47, 98.31, 73.45, 72.13, 70.07, 70.00, 69.54, 69.02, 68.85, 68.63, 64.45, 63.51, 52.55, 40.56, 22.80; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.20H.sub.33NNaO.sub.14].sup.+, 534.1793. found, 534.1789
(125) Embodiment 40 Synthesis of Compound 41
(126) This compound was prepared from Compound 63 (For its synthesis, see Embodiment 48), affording 41 (yield=95%). The synthetic procedure was the same as that of Compound 3.
(127) .sup.1H-NMR (500 MHz, D.sub.2O) 6.01-5.92 (m, 1H), 5.35 (dq, 1H, J=1.5 Hz, 17.0 Hz), 5.28 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 4.97 (d, 1H, J=3.5 Hz, anomeric H of GlcNAc), 4.21 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=5.0 Hz, J.sub.3=13.0 Hz), 4.02 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=6.0 Hz, J.sub.3=13.0 Hz), 3.98 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=10.5 Hz), 3.93 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.5 Hz), 3.91-3.79 (m, 4H), 3.78-3.61 (m, 5H), 3.59 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=9.0 Hz), 3.53 (dd, 1H, J.sub.1=9.5 Hz, J.sub.2=10.0 Hz), 2.75 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.0 Hz, siaH-3 eq), 2.04 (s, 3H), 1.71 (t, J=12.0 Hz, siaH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 175.80, 175.16, 174.15, 134.32, 118.80, 100.89, 96.77, 73.24, 72.51, 71.75, 71.32, 70.68, 69.38, 69.00, 63.56, 63.36, 54.31, 52.63, 40.87, 22.76, 22.58; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.22H.sub.36N.sub.2NaO.sub.14].sup.+, 575.2059. found, 575.2067
(128) Embodiment 41 Synthesis of Compound 42
(129) This compound was prepared from Compound 49 (For its synthesis, see Embodiment 42), affording 42 (yield=95%). The synthetic procedure was the same as that of Compound 3.
(130) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.94 (m, 1H), 5.35 (dd, 1H, J.sub.1=17.0 Hz,), 5.26 (d, 1H, J.sub.1=10.5 Hz), 4.95 (d, 1H, J=3.5 Hz, anomeric Hon GalNAc), 4.21 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz), 4.18 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=8.5 Hz), 4.11-4.02 (m, 4H), 3.93-3.83 (m, 5H), 2.75 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=10.0 Hz), 3.76 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.0 Hz), 3.56 (d, 1H, J=9.0 Hz), 3.47 (dd, 1H, J.sub.1=8.5 Hz, J.sub.2=10.0 Hz), 2.39 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.05 (s, 3H), 2.04 (s, 3H), 1.68 (dd, 1H, J.sub.1=12.5 Hz, J.sub.2=12.0 Hz, sialH-3ax); .sup.13C-NMR (125 MHz, D.sub.2O) 177.45, 177.20, 174.52, 136.43, 120.45, 101.42, 99.08, 73.28, 72.59, 72.01, 71.44, 71.27, 70.52, 70.24, 69.11, 65.92, 65.00, 54.50, 52.45, 41.92, 24.70, 24.53; HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.36N.sub.2NaO.sub.14, 575.2059. found, 575.2050
(131) Embodiment 42 Synthesis of Compound 48 and 49
(132) Compound 46 (1.07 g, 2.93 mmol, 1.00 eq. For its synthesis, see U.S. Pat. No. 6,013,779.) and Compound 47 (2.06 g, 3.38 mmol, 1.15 eq. For its synthesis, see J. Org. Chem. 2000, 65, 144-151.) were added into the 100 mL double-necked flask together with activated molecular sieves. Under argon atmosphere, 40 mL THF was added. The mixture was stirred at R.T. for 0.5 h and then was cooled to 72 C. To the mixture, TMSOTf (70 L, 0.36 mmol, 0.11 eq.) was added in three batches during 15 min. The reaction went to completion in 4 h (by TLC monitoring). TEA (1.0 mL) was added. The mixture was filtered through diatomite. The filtrate was concentrated in vacuum. The residue was purified on silica gel, affording 48 and 49 (48:49=1:1.2, yield=84%).
(133) Compound 48: .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.08-8.05 (m, 2H), 7.60-7.50 (m, 1H), 7.50-7.40 (m, 2H), 5.97-5.87 (m, 1H), 5.75 (d, 1H, J=10.0 Hz, NHAc), 5.42 (d, 1H, J=10.0 Hz, NHAc), 5.40-5.31 (m, 3H), 5.29 (dd, 1H, J.sub.1=3.0 Hz, J.sub.2=11.0 Hz), 5.23 (ddd, 1H, J.sub.1=1.0 Hz, J.sub.2=3.0 Hz, J.sub.3=10.5 Hz), 4.96 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.92-4.85 (m, 2H), 4.36 (dd, 1H, J.sub.1=2.5 Hz, J.sub.2=12.5 Hz), 4.25-4.20 (m, 2H), 4.18-4.00 (m, 5H), 3.88 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=10.0 Hz), 3.80 (s, 3H), 3.75 (dd, 1H, J.sub.1=7.0 Hz, J.sub.2=10.0 Hz), 3.02 (d, 1H, J=3.0 Hz, OH), 2.59 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.14 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.98 (t, 1H, J=13.0 Hz, sialH-3ax), 1.97 (s, 3H, OAc), 1.88 (s, 3H, NAc), 1.87 (s, 3H, NAc); .sup.13C-NMR (125 MHz, CDCl.sub.3) 170.93, 170.86, 170.25, 170.18, 170.15, 169.92, 168.03, 166.67, 133.53, 133.31, 129.92, 129.52, 128.46, 117.90, 98.70, 96.96, 72.78, 72.19, 69.10, 69.03, 68.68, 68.36, 67.40, 66.89, 62.96, 62.54, 23.30, 23.13, 21.03, 20.82, 20.77, 20.63. HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.38H.sub.50N.sub.2NaO.sub.19].sup.+, 861.2900. found, 861.2903
(134) Compound 49: .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.12-8.08 (m, 2H), 7.60-7.52 (m, 1H), 7.47-7.40 (m, 2H), 5.96-5.84 (m, 2H), 5.62 (d, 1H, J=10.0 Hz, NHAc), 5.44-5.28 (m, 5H), 5.23 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 4.94 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.85 (ddd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.0 Hz, J.sub.3=11.0 Hz), 4.78 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=12.5 Hz), 4.40-4.32 (m, 2H), 4.21 (ddt, 1H, J.sub.1=J.sub.2=1.0 Hz, J.sub.3=5.0 Hz, J.sub.4=12.5 Hz), 4.16 (dd, 1H, J.sub.1=8.0 Hz, J.sub.2=12.5 Hz), 4.11 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=8.5 Hz), 4.02-3.96 (dd, 1H, J.sub.1=6.0 Hz, J.sub.2=12.0 Hz), 3.88 (t, 1H, J.sub.1=J.sub.2=9.0 Hz), 3.82 (s, 3H), 3.82-3.72 (m, 1H), 3.57 (d, 1H, J=4.0 Hz, OH), 3.50 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=9.0 Hz), 2.51 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.14 (s, 3H, OAc), 2.16-2.10 (1H, overlapped, sialH-3ax), 2.11 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.85 (s, 3H, NAc), 1.80 (s, 3H, NAc); .sup.13C-NMR (125 MHz, CDCl.sub.3) 171.43, 170.77, 170.47, 170.00, 167.13, 166.73, 133.54, 133.31, 129.97, 129.44, 128.40, 117.63, 98.29, 97.08, 72.07, 71.59, 70.95, 68.88, 68.38, 68.23, 68.11, 66.46, 62.45, 61.57, 52.77, 49.90, 47.83, 37.41, 23.19, 23.14, 21.09, 20.85, 20.81, 20.75. HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.38H.sub.50N.sub.2NaO.sub.19].sup.+, 861.2900. found, 861.2882
(135) Embodiment 43 Synthesis of Compound 50
(136) Compound 48 (100 mg, 0.119 mmol) was dissolved in 10 ml methanol (10 mg/mL), NaOMe/MeOH (30%, 0.02 g, 011 mmol) was added. The mixture was stirred at R.T. for 4 h. The mixture was heated to 90 C. and stirred for 8 h. The reaction mixture was neutralized with 1N HCl/MeOH to pH=4-7. The mixture was concentrated in vacuum. The residue was purified on a Biogel P-2 column with water as the eluent. Fractions containing the expected product were collected to afford 50 (yield=88%).
(137) .sup.1H-NMR (500 MHz, D.sub.2O) 6.02-5.96 (m, 1H), 5.37 (ddd, 1H, J.sub.1=1.5 Hz, J.sub.2=3.0 Hz, J.sub.3=17.5 Hz), 5.29 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 5.11 (d, 1H, J=3.5 Hz, anomeric H of GalN), 4.26 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.5 Hz), 4.09-4.05 (m, 2H), 3.99-3.96 (m, 2H), 3.93-3.88 (m, 3H), 3.80 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=9.0 Hz), 3.73-3.68 (m, 2H), 3.61 (dd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.5 Hz), 3.57-3.54 (m, 1H), 3.38-3.35 (m, 1H), 2.95-2.91 (m, 1H), 2.72 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=12.5 Hz, sialH-3 eq), 1.64 (t, 1H, J=12.5 Hz, sialH-3ax); .sup.13C-NMR (75 MHz, D.sub.2O) 176.08, 135.91, 121.21, 102.96, 8.07, 76.19, 74.57, 72.38, 71.83, 71.58, 70.82, 70.60, 70.18, 66.26, 65.07, 54.99, 53.35, 42.87; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.18H.sub.32N.sub.2NaO.sub.12].sup.+, 491.1847. found, 491.1852
(138) Embodiment 44 Synthesis of Compound 52 and 55
(139) These compounds were prepared from Compound 51 (For its synthesis, see U.S. Pat. No. 6,013,779) and Compound 46 (For its synthesis, see U.S. Pat. No. 6,013,779), affording 52 and 55 (52:55=2.6:1, yield=81%). The synthetic procedure was the same as that of Compound 48 and 49.
(140) Compound 52: .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.08-8.04 (m, 2H), 7.60-7.52 (m, 1H), 7.50-7.39 (m, 2H), 7.10 (d, 1H, J=10.0 Hz, NHTFA), 5.98-5.87 (m, 1H), 5.78 (d, 1H, J=10.0 Hz, NHAc), 5.38-5.27 (m, 4H), 5.25 (dd, 1H, J.sub.1=1.0 Hz, J.sub.2=10.0 Hz), 5.23 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=10.5 Hz, J.sub.3=12.0 Hz), 4.96 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.88 (ddd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.0 Hz, J.sub.3=11.0 Hz), 4.38 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=12.5 Hz), 4.30 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=12.5 Hz), 4.25-4.20 (m, 2H), 4.08 (dd, 1H, J.sub.1=6.0 Hz, J.sub.2=12.5 Hz), 4.06-4.00 (m, 3H), 3.88 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=9.5 Hz), 3.80 (s, 3H, COOMe), 3.78 (dd, 1H, J.sub.1=7.0 Hz, J.sub.2=9.5 Hz), 3.08 (d, 1H, J=3.0 Hz, OH), 2.63 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.14 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.12 (s, 3H, OAc), 1.97 (t, 1H, J.sub.1=J.sub.2=12.5 Hz sialH-3ax), 1.99 (s, 3H, OAc), 1.96 (s, 3H, OAc), 1.88 (s, 3H, NAc); .sup.13C-NMR (125 MHz, CDCl.sub.3) 170.90, 170.86, 170.42, 170.13, 169.94, 167.86, 166.86, 157.59 (q, 1C, J=37.5 Hz, carbonyl in TFA) 133.51, 133.37, 129.89, 129.46, 128.46, 117.92, 115.05 (q, 1C, J=286.2 Hz, CF.sub.3), 98.70, 96.96, 72.21, 71.99, 69.31, 68.71, 68.47, 68.38, 67.26, 66.84, 63.06, 62.38, 53.10, 49.91, 47.32, 37.10, 23.24, 21.00, 20.59, 20.57, 20.54; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.38H.sub.47F.sub.3N.sub.2NaO.sub.19].sup.+, 915.2617. found, 915.2615.
(141) Compound 55: .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.10-8.04 (m, 2H), 7.56 (t, 1H, J=7.5 Hz), 7.43 (q, 2H, J=8.0 Hz), 5.98-5.87 (m, 1H), 5.88 (d, 1H, J=10.0 Hz, NHTFA), 5.42-5.28 (m, 4H), 5.25 (dd, 1H, J.sub.1=1.0 Hz, J.sub.2=10.0 Hz), 4.96 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.84 (ddd, 1H, J.sub.1=4.0 Hz, J.sub.2=10.0 Hz, J.sub.3=11.0 Hz), 4.78 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=12.0 Hz), 4.46 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=10.5 Hz), 4.35 (br, 1H), 4.21 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=12.5 Hz), 4.14 (dd, 1H, J.sub.1=8.0 Hz, J.sub.2=12.0 Hz), 4.10 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=8.0 Hz), 4.00 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=12.5 Hz), 3.96-3.88 (m, 2H), 3.83 (s, 3H, COOMe), 3.58-3.50 (m, 2H), 3.45-3.87 (br, 1H), 2.54 (dd, 1H, J.sub.1=5.0 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.26 (br, 1H) 2.13 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.96 (s, 3H, OAc), 1.86 (s, 3H, NAc), 1.86 (t, 1H, J.sub.1=J.sub.2=12.5 Hz, overlapped, sialH-3ax); .sup.13C-NMR (75 MHz, CDCl.sub.3) 171.56, 170.84, 170.64, 170.27, 170.20, 166.89, 157.76 (q, 1C, J=37.7 Hz, carbonyl in TFA), 133.48, 129.95, 129.29, 128.51, 117.87, 115.40 (q, 1C, J=285.7 Hz, CF.sub.3) 98.30, 97.05, 72.09, 71.81, 70.57, 68.67, 68.48, 68.03, 67.88, 66.53, 62.31, 61.69, 52.92, 50.13, 47.70, 37.35, 23.22, 21.02, 20.79, 20.58; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.38H.sub.47F.sub.3N.sub.2NaO.sub.19].sup.+, 915.2617. found, 915.2626
(142) Embodiment 45 Synthesis of Compound 54
(143) This compound was prepared from Compound 47 (See Tetrahedron Lett. 1992, 33, 6123-6126) and Compound 53 (For its synthesis, see Embodiment 46), affording 54 (yield=58%). The synthetic procedure was the same as that of Compound 48.
(144) .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.14-8.10 (m, 2H), 7.62-7.56 (m, 1H), 7.50-7.39 (t, 2H, J=8.0 Hz), 6.00-5.91 (m, 1H), 5.49 (dd, 1H, J.sub.1=3.5 Hz, J.sub.2=11.0 Hz), 5.42-4.80 (m, 3H), 5.25 (dd, 1H, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz), 5.22 (d, 1H, J=10.0 Hz, NHAc), 5.11 (d, 1H, J=3.5 Hz, anomeric H of GalNAc), 4.88 (ddd, 1H, J.sub.1=4.5 Hz, J.sub.2=9.5 Hz, J.sub.3=12.0 Hz), 4.38 (dd, 1H, J.sub.1=2.0 Hz, J.sub.2=12.5 Hz), 4.36-4.33 (m, 1H), 4.28 (ddt, 1H, J.sub.1=J.sub.2=1.5 Hz, J.sub.3=5.0 Hz, J.sub.4=13.0 Hz), 4.13-3.97 (m, 6H), 3.87 (dd, 1H, J.sub.1=5.5 Hz, J.sub.2=9.5 Hz), 3.81 (s, 3H, COOMe), 3.78 (dd, 1H, J.sub.1=7.0 Hz, J.sub.2=9.5 Hz), 3.20 (d, 1H, J=4.5 Hz, OH), 2.57 (dd, 1H, J.sub.1=4.5 Hz, J.sub.2=13.0 Hz, sialH-3 eq), 2.12 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (t, 1H, J.sub.1=J.sub.2=12.5 Hz sialH-3ax), 1.91 (s, 3H, OAc), 1.87 (s, 3H, NAc); .sup.13C-NMR (75 MHz, CDCl.sub.3) 171.00, 170.86, 170.45, 170.29, 170.11, 167.96, 165.66, 133.29, 133.15, 129.89, 129.46, 128.35, 117.85, 98.54, 97.13, 72.76, 71.55, 69.36, 68.91, 68.55, 68.45, 67.32, 66.78, 62.77, 62.54, 57.60, 52.98, 49.07, 36.70, 23.00, 20.97, 20.73, 20.66, 20.48; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.36H.sub.46N.sub.4NaO.sub.18].sup.+, 845.2699. found, 845.2690
(145) Embodiment 46 Synthesis of Compound 60 and 53
(146) Compound 56 (For its synthesis, see Tetrahedron Lett. 2005, 46, 8993-8995)(7.91 g, 21.2 mmol) was dissolved in CH.sub.2Cl.sub.2 (66 mL), and AllylOH (7.0 mL, 102 mmol) and BF.sub.3.Et.sub.2O (25 mL, 197.5 mmol) was added. The mixture was heated under reflux for 3 h. BF.sub.3.Et.sub.2O (10 mL, 79 mmol) was added and the mixture was refluxed for another 3 h. The solvent was evaporated under reduced pressure, and the residue was neutralized with saturated KHCO.sub.3 aq. The mixture was extracted with ethyl acetate (150 mL3). The organic layer were collected, dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuum. The residue was purified by column chromatography on silica gel (acetone:petroleum ether=1:6) to afford 57 (5.68 g). The anomeric isomers could not be separated, and were employed for the following reaction without further purification.
(147) To a stirred solution of the above product 57 (4.82 g, 13.0 mmol) in methanol (70 mL) was added a sodium methoxide solution in methanol (30%, 0.5 g, 2.78 mmol). The mixture was stirred at R.T. for 0.5 h. The reaction mixture was neutralized with strongly acidic resin, which was filtered off later. The solvent was removed under reduced pressure to afford 58 (2.88 g, yield=91%).
(148) To a solution of the above product 58 (2.88 g, 11.75 mmol) in MeCN (10 mL) was added PhCH(OMe).sub.2 (2.15 g, 14.13 mmol) and CSA (203 mg, 0.88 mmol). The mixture was stirred at R.T. for 18 h. After triethylamine (0.2 g, 2.0 mmol) was added, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to afford 59 (2.78 g). The unstable a isomer was separated in this stage. The product employed for the following reaction was without complete purification.
(149) The above product 59 (2.78 g, 8.35 mmol) and DMAP (70 mg, 0.6 mmol) were dissolved in pyridine (10 mL), and the solution was cooled on ice bath. BzCl (1.8 mL, 15 mmol) was added dropwise. The mixture was stirred at R.T. for 24 h. The solvent was evaporated under reduced pressure. The residue was neutralized with saturated KHCO.sub.3 aq., and extracted with ethyl acetate (150 mL3). The organic layer were collected, dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone:petroleum ether=1:5) to afford 60 (3.21 g, yield=88%).
(150) Compound 60: .sup.1H-NMR (300 MHz, CDCl3) 8.14-8.07 (m, 2H), 7.63-7.55 (m, 1H), 7.48-7.43 (m, 4H), 7.37-7.33 (m. 3H), 6.02-5.95 (m, 1H), 5.60 (dd, 1H, J1=3.6 Hz, J2=11.1 Hz, H-3), 5.55 (s, 1H), 5.37 (dq, 1H, J1=1.5 Hz, 17.1 Hz, terminal H of vinyl in allyl), 5.26 (dd, 1H, J1=1.5 Hz, J2=10.2 Hz, terminal H of vinyl in allyl), 5.19 (d, 1H, J=3.0 Hz, anomeric H), 4.63 (d, 1H, J=2.7 Hz), 4.29 (dd, 1H, J1=2.5 Hz, J2=12.3 Hz, H-6a), 4.28 (ddt, 1H, J1=J2=1.5 Hz, J3=5.4 Hz, J4=13.0 Hz, OCH2 in allyl), 4.16-4.08 (m, 3H), 3.90-3.86 (br, 1H), 13C-NMR (75 MHz, CDCl3) 171.89, 165.95, 137.44, 133.68, 133.44, 133.04, 130.13, 129.90, 129.29, 128.89, 128.74, 128.44, 128.08, 125.96, 118.20, 100.48, 97.50, 73.51, 70.05, 69.07, 68.83, 62.67, 57.53; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.23H.sub.23N.sub.3NaO.sub.6].sup.+, 460.1479. found, 460.1488.
(151) To a solution of Compound 60 (688 mg, 1.57 mmol, For its synthesis, see the foregoing two paragraphs) in MeOH (10 mL) was added strongly acidic resin (668 mg). The mixture was stirred at 40 C. for 4 h. The resin was filtered off, and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (acetone:petroleum ether=1:3) to afford 53 (402 mg, yield=73%):
(152) Compound 53: .sup.1H-NMR (500 MHz, CDCl.sub.3) 8.09-8.05 (m, 2H), 7.60-7.55 (m, 1H), 7.46-7.42 (m, 2H), 5.97-5.88 (m, 1H), 5.45 (dd, 1H, J1=3.0 Hz, J2=11.0 Hz, H-3), 5.34 (dq, 1H, J1=1.5 Hz, J2=17.5 Hz, terminal H of vinyl in allyl), 5.24 (dq, 1H, J=1.5 Hz, J2=10.0 Hz, terminal H of vinyl in allyl), 5.08 (d, 1H, J=3.5 Hz, anomeric H), 4.39 (d, 1H, J=2.5 Hz, H-4), 4.22 (ddt, 1H, J1=J2=1.5 Hz, J3=6.5 Hz, J4=13.0 Hz, OCH2 in allyl), 4.05 (ddt, 1H, J1=J2=1.5 Hz, J3=6.5 Hz, J4=13.0 Hz, OCH2 in allyl), 4.00-3.94 (m, 2H), 3.91-3.81 (m, 2H), 3.55 (br, 1H, OH), 2.93 (br, 1H, OH); 13C-NMR (75 MHz, CDCl3) 165.74, 133.55, 133.04, 129.81, 129.10, 128.49, 118.03, 97.28, 71.61, 69.14, 68.73, 62.85, 57.45; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.16H.sub.19N.sub.3NaO.sub.6].sup.+, 372.1166. found, 372.1158
(153) Embodiment 47 Synthesis of Compound 61
(154) This compound was prepared from Compound 47 and 62 (See Carbohydr. Res. 1994, 253, 167-183), affording 61 (yield=51%). The synthetic procedure was the same as that of Compound 48.
(155) Compound 61: .sup.1H-NMR (500 MHz, D.sub.2O) 8.05-7.98 (m, 4H), 7.53-7.48 (m, 2H), 7.40-7.35 (m, 4H), 5.90-5.81 (m, 1H), 5.73-5.67 (m, 2H), 5.40-5.26 (m, 5H), 5.16-5.12 (m, 1H), 4.90 (ddd, 1H, J1=4.5 Hz, J2=9.5 Hz, J3=11.5 Hz), 4.41-4.37 (m, 2H), 4.25 (ddt, 1H, J1=J2=1.5 Hz, J3=5.5 Hz, J4=13.5 Hz), 4.18 (t, 1H, J1=J2=6.0 Hz), 4.16-4.02 (m, 4H), 3.92 (dd, 1H, J1=5.5 Hz, J2=9.5 Hz), 3.82 (s, 3H, COOMe, overlapped), 3.84-3.80 (m, 1H, overlapped), 3.12 (b, 1H, OH), 2.59 (dd, 1H, J1=5.0 Hz, J2=13.0 Hz, sialH-3 eq), 2.14 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (t, 1H, J1=J2=12.5 Hz sialH-3ax), 1.94 (s, 3H, OAc), 1.88 (s, 3H, NAc); 13C-NMR (125 MHz, CDCl3) 170.93, 170.27, 170.23, 170.19, 168.05, 165.99, 165.92, 133.57, 133.12, 129.79, 129.77, 129.66, 129.52, 128.33, 117.38, 98.70, 95.73, (77.25, 77.00, 76.74 CDCl3), 72.85, 71.13, 69.24, 68.96, 68.87, 68.51, 68.22, 67.65, 67.40, 62.93, 62.57, 53.03, 49.35, 36.98, 23.14, 21.04, 20.81, 20.76, 20.54; HRMS (m/z): [M+Na].sup.+ calcd. for [C.sub.43H.sub.51NNaO.sub.20].sup.+, 924.2897. found, 924.2905
(156) Embodiment 48 Synthesis of Compound 63
(157) This compound was prepared from Compound 47 and 64 (See Monatshefte fuer Chemie. 2002, 133, 531-540), affording 63 (yield=51%). The synthetic procedure was the same as that of Compound 48.
(158) Compound 63: .sup.1H-NMR (500 MHz, D.sub.2O) 8.06-8.03 (m, 2H), 7.58-7.53 (m, 1H), 7.45-7.41 (m, 2H), 5.94-5.85 (m, 2H), 5.49-5.41 (m, 2H), 5.35-5.21 (m, 4H), 5.05-4.97 (m, 1H), 4.92 (d, 1H, J=4.0 Hz, anomeric H on GlcNAc), 4.45 (ddd, 1H, J1=4.0 Hz, J2=10.0 Hz, J3=11.0 Hz), 4.29-4.22 (m, 2H), 4.25 (ddt, 1H, J1=J2=1.5 Hz, J3=5.5 Hz, J4=12.5 Hz), 4.14 (dd, 1H, J1=1.5 Hz, J2=10.5 Hz), 4.10-3.96 (m, 4H), 3.81 (s, 3H, COOMe), 3.83-3.78 (1H, overlapped), 3.76 (dd, 1H, J1=1.5 Hz, J2=11.2 Hz), 3.44 (br., 1H, OH), 2.66 (dd, 1H, J1=5.0 Hz, J2=13.5 Hz, sialH-3 eq), 2.11 (s, 3H, OAc), 1.90-1.80 (1H, sial H-3ax, overlapped), 2.06 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.90 (s, 3H, OAc), 1.86 (s, 3H, NAc), 1.84 (s, 3H, NAc); 13C-NMR (125 MHz, CDCl3) 170.99, 170.60, 170.34, 170.05, 169.98, 169.56, 168.24, 167.34, 133.34, 133.22, 129.80, 129.58, 128.36, 118.00, 97.86, 96.74, 73.80, 72.22, 71.16, 68.85, 68.49, 68.37, 68.01, 67.11, 62.76, 62.18, 52.87, 51.86, 49.59, 37.77, 23.13, 23.09, 21.02, 20.87, 20.65, 20.27; HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.38H.sub.50N.sub.2NaO.sub.19, 861.2900. found, 861.2872
(159) Embodiment 49 Synthesis of Glycoconjugates by Conjugation Reaction between Hapten 2-14, 16-24, 29-34, 42 and Carrier Protein/Peptide
(160) The hapten (Compound 2, 10 mg) was dissolved in anhydrous methanol (2 mL). At 78 C., ozone was introduced into the mixture. When the color of the mixture turned blue (about 1030 min), the ozone was cut out. The color stayed blue for about 10 min. N.sub.2 was then introduced into the flask to extrude the ozone in excess. Dimethylsulfide (0.5 mL) was added dropwise. The temperature was allowed to R.T. spontaneously. After 2 h, the solvent was removed in vacuum, affording the aldehyde derivative. The aldehyde derivative of the carbohydrate hapten and the protein (or polypeptide), such as BSA or KLH (10 mg), were dissolved in pH 7.2 buffer. NaBH.sub.3CN (3 mg) was added. The reaction flask was placed on a shaking table and the mixture was shaken for 24 h. The mixture was dialyzed to afford the desired glycoconjugate STn-KLH.
(161) According to aforementioned procedure, the compounds of Embodiment 3-47 (2-14, 16-24, 29-34, 42) are conjugated to the protein carrier to afford the corresponding glycoconjugates 3-KLH, 4-KLH, 5-KLH, 6-KLH, 7-KLH, 8-KLH, 9-KLH, 10-KLH, 11-KLH, 12-KLH, 14-KLH, 16-KLH, 17-KLH, 18-KLH, 19-KLH, 20-KLH, 21-KLH, 22-KLH, 23-KLH, 24-KLH, 29-KLH, 30-KLH, 31-KLH, 32-KLH, 33-KLH, 34-KLH, 42-KLH.
(162) It should be noted that although the structurally typical R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 have been employed in the syntheses of STn derivatives and their conjugates with proteins (or polypeptides), the art described in the present invention can be applied to syntheses of structurally diverse R substituents, with R.sub.1 being any acylamino, NH.sub.2, N.sub.3 or OH group; R.sub.2 any acylamino, NH.sub.2, N.sub.3, OH group; R.sub.3 equatorial or axial CO.sub.2H, CH.sub.2OH, CO.sub.2M, C(O)NHOH, H wherein M may be alkyl, aryl, heterocycle or cation); R.sub.4 different linkers; R.sub.5 equatorial or axial OH; and R6 H or -galactosyl. These synthetic tasks can be accomplished without any creative endeavor by the person in the art. It is obvious that the scope of the patent protection is not constrained with the limited compounds of the presented Embodiments.
(163) Characterizing the Immune Response
(164) 1. Materials and resources: Test antigens: glycoproteins (or -polypeptides) prepared in Embodiment 49 of the present invention. 2. Test method: (1) Immunization of mice
(165) Groups of six mice (female pathogen-free BALB/c, age 6-8 weeks, Number: SCXKjing2007-0001, SPF/VAF, from Department of Laboratory Animal Science, Peking University Health Science Center) were immunized four times at 2-week intervals with STn-KLH or modified STn-KLH glycoconjugates (each containing 2 g of carbohydrate in PBS). The vaccines were administered intraperitoneally. Mice were bled prior to the initial vaccination, 13 days after the second and the third vaccinations, and 14 days after the fourth vaccination. Blood was clotted to obtain sera, which were stored at 80 C. (2) Serological assays. The total antigen-specific antibody titers of the pooled sera were assessed by means of ELISA. 1 ELISA plate was coated with 100 L of STn-BSA (including 0.02 g of STn) overnight at 4 C. (0.1 M bicarbonate buffer, pH=9.6). 2 After being washed three times (200 L per time for per microwell) with PBS-Tween20 (0.05%), microwells were blocked with 3% BSA-PBS (200 L per microwell) and incubated for 1 h at 37 C. 3 After the plate was washed for three times, serially diluted sera (1% BSA-PBS, double diluted) were added to microwells (100 L/well) and incubated for 1 h at 37 C. 4 The plate was washed for three times and incubated with 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (-chain specific) or IgM (-chain specific) (Southern Biotechnology Associates, Inc., Buckingham, Ala.) for 1 h at 37 C. 5 The plate was washed for three times, developed with o-phenylenediamine (OPD) substrate in the dark for 15 min terminated by addition of 2M H.sub.2SO.sub.4. 6 The OD value is read at 490 nm on the Microplate reader. The antibody titer was defined as the highest dilution showing an absorbance of 0.1, after subtracting background. (3) Result 1)
(166) TABLE-US-00001 TABLE 1 Table 1. ELISA titers anti-STn 13 days after the second vaccination Group IgG IgM immunized with STn-KLH 910 <1000 immunized with 3-KLH <1000 <1000 immunized with 4-KLH <1000 <1000 immunized with 5-KLH <1000 <1000 immunized with 6-KLH <1000 <1000 immunized with 7-KLH <1000 <1000 immunized with 8-KLH 7734 <1000 immunized with 9-KLH <1000 <1000 immunized with 10-KLH 7933 <1000 immunized with 11-KLH <1000 <1000 immunized with 12-KLH <1000 <1000 immunized with 13-KLH <1000 <1000 immunized with 14-KLH <1000 <1000 immunized with 16-KLH <1000 <1000 immunized with 17-KLH <1000 <1000 immunized with 18-KLH 1381 <1000 immunized with 19-KLH <1000 <1000 immunized with 20-KLH 11000 <1000 immunized with 21-KLH 1266 <1000 immunized with 22-KLH <1000 <1000 immunized with 23-KLH <1000 <1000 immunized with 24-KLH 6618 <1000 immunized with 29-KLH <1000 <1000 immunized with 30-KLH <1000 <1000 immunized with 31-KLH 20000 <1000 immunized with 32-KLH <1000 <1000 immunized with 33-KLH <1000 <1000 immunized with 34-KLH <1000 <1000 immunized with 42-KLH <1000 <1000 2)
(167) TABLE-US-00002 TABLE 2 ELISA titers anti-STn 13 days after the third vaccination Group IgG IgM immunized with STn-KLH 50,144 5763 immunized with 3-KLH <2500 <1000 immunized with 4-KLH 17,040 1604 immunized with 5-KLH 38,878 1872 immunized with 6-KLH <2500 <1000 immunized with 7-KLH 23,279 4706 immunized with 8-KLH 43,201 4963 immunized with 9-KLH 4714 2897 immunized with 10-KLH 55,004 <1000 immunized with 11-KLH 4054 <1000 immunized with 12-KLH 11,783 <1000 immunized with 13-KLH 24,512 <1000 immunized with 14-KLH <2500 <1000 immunized with 16-KLH <2500 2571 immunized with 17-KLH 11,329 2047 immunized with 18-KLH 56,276 1842 immunized with 19-KLH <2500 <1000 immunized with 20-KLH 185,354 2581 immunized with 21-KLH 150,504 9236 immunized with 22-KLH 24,154 1586 immunized with 23-KLH 38,173 2880 immunized with 24-KLH 93,488 <1000 immunized with 29-KLH <2500 1979 immunized with 30-KLH <2500 <1000 immunized with 31-KLH 276,162 1804 immunized with 32-KLH <2500 <1000 immunized with 33-KLH <2500 <1000 immunized with 34-KLH 6108 <1000 immunized with 42-KLH <2500 <1000 3)
(168) TABLE-US-00003 TABLE 3 IgG antibody titer anti-STn of individual mouse immunized with 20-KLH, 21-KLH, 31-KLH and 2-KLH after the 3rd vaccination STn-KLH 20-KLH 31-KLH 21-KLH Mouse 1 5,170 296,647 179,666 24,235 Mouse 2 6,385 79,799 356,282 182,259 Mouse 3 65,524 283,403 291,807 71,044 Mouse 4 96,812 63,561 83,396 85,645 Mouse 5 6,187 69,508 104,519 89,172 Mouse 6 18,663 68,401 351,977 472,834 4) The combination between the mice sera and the tumor cells 13 days after the 3.sup.rd vaccination The result is shown in
(169) In vivo test of the effect of the synthetic glycoconjugates on killing tumor cells was not conducted. However, it is reported that (See British J. Cancer 2009, 100, 1746-1754) the anti-STn antibody (obtained after immunizing the mice) indeed represses tumor growth. Since higher titers of antibody were obtained in the present invention, it is expected that the repressive activity of the synthetic glycoconjugates surpasses that of the unmodified STn-KLH.