GB2401107A

GB2401107A - Preparation of a galactopyranose derivative

Info

Publication number: GB2401107A
Application number: GB0415573A
Authority: GB
Inventors: Hiroshi Tomiyama; Naoto Ueyama; Masahiro Yanagiya; Yasufumi Ohkura
Original assignee: Kotobuki Seiyaku Co Ltd
Current assignee: Kotobuki Seiyaku Co Ltd
Priority date: 2000-08-11
Filing date: 2001-08-13
Publication date: 2004-11-03
Anticipated expiration: 2021-08-13
Also published as: GB2401107B; GB0415573D0

Abstract

Use of a galactosamide derivative of general formula (V) <EMI ID=1.1 HE=38 WI=71 LX=518 LY=813 TI=CF> <PC>wherein: <DL TSIZE=3> <DT>R1<DD>is H or a hydroxyl protecting group; <DT>R2<DD>is a leaving group; and <DT>G<DD>is allyl or a protected hydroxyl group, </DL> in the preparation of a galactopyranose derivative of general formula (VI) <EMI ID=1.2 HE=37 WI=71 LX=610 LY=1737 TI=CF> <PC>wherein R1 and G are as defined above.

Description

2401 1 07

SPECIFICATION

TITLE OF INVENTION

PREPARATION OF A GALACTOPYRANOSE DERIVATIVE

BACKGROUND OF THE INVENTION

l.The Subject of the Invention The present invention concerns non-mucin type synthetic compounds which were linked to carrier, that is, non-mucin type synthetic compounds or it's carrier conjugated compounds. The present invention further concerns the use of non-mucin type synthetic compounds or it's carrier conjugated compounds for preparation of monoclonal antibodies, human immunodeficiency virus (HIV) agents, antitumor agents and immunostimulants.

2.Current Technology Mucin type antigens such as Tn (GalNAcol O-Ser/Thr), TF (Galp13GalNAcol O-Ser/Thr), STn (NeuAc c2,6GalNAclO-Ser/Thr) as shown in following below figure, are highly expressed in tumor tissues, and appearance in the nomal tissues are restricted.(G.F.Springer, J. Natl, Cancer Inst., 1975, 54, 335., S.Hakomori, Advanced in Cancer Research, 1989, 52, 257) OH Qua OH CH2OH CH2OH CH2OH J:, NH2 - NHz HO iCO2H to NHAc OH NHAc 2 HO

HO

Tn TF STn Recentry, Tn and STn epitopes, were found on the gpl20 as human immunodeficiency virus (HIV) specific glycoprotein.(Hanse, J. E., J. Viol. ; 1990, 64, 2833., J. Viol., 1991, 65, 6416.; Arch. Viol., 1992, 126, 11.) And it was reported that the monoclonal antibodies for the O-linked oligosaccharide block HIV infections.(Hanse, J. E., J. Viol.; 1990, 64, 2833.; Kumar A., Virology, 2000, 274, 149.) The administration of mucin type tumor antigens and/or attached to pharmaceutically acceptable carriers are expected as a specific immunotherapy for cancer and HIV.

Wherein these carriers are pharmaceutically acceptable proteins such as albumine (ALB), Keyhole limpet hemocyanin (KLH), BCG, or synthetic compounds such as palmitoyl derivatives, aromatic compounds, aliphatic compounds, alkyl, aminoalkyl, peptide and peptoid, which can obtain induction of immune response. (S. J. Danishefsky, J. Am. Chem. Soc. 1998, 120, 12474.; G. Ragupathi, Glycoconjugate J., 1998, 15, 217.; B. M. Sandmeier, J. Immunotherapy, 1999, 22(1), 54.; A. Singhal, Cancer Res., 1991, 51, 1406.; T. Shimizu, 1987, 55, 2287-2289.) However, above mentioned mucine type antigens-carrier have O-glycoside linkage between sugar and carrier moiety. Therefore, considering about their metabolic stability and immunogenicity, these O-glycoside linkage are susceptible to hydrolyze by glycosidase such as N-acetyl galactosaminidase (EC 3.2.1.49) (Eq A), or hydrolyze of peptide bond by peptidase,as shown in following equation (Eq B), and their activities are anticipated to decrease or attenuate.

ICH2OA glycosdaseCH2OA 0: '0 f-N-acetyl garactosamndaseHQg BO 0-43 (A) BO1J., HO 3 NHAc NHAc CH2OA CH2OA HQJ O peptidase 0: 0 NHAc I NHAc BO À O4e) (B) BO \/ o - OH+ H NHAc NHAc On the other hand, Beau et al have been synthesized the C- glycosides (GalNAca 1 CH2-Ser) that have the carbon atom instead of the oxygen atom which connect with serine and N-acetyl galactosamine as a metabolically stable Tn antigen, shown in following equation (A). This Tn antigen is stable against glycosidase such as N-acetyl galactosaminidase (Beau et al, J. C. S. Chem. Commun., 1998, 955.). But, when these C- glycosides are attached to peptides, these compounds could be hydrolyzed by peptidases, and their stabilities are not satisfactory in a living body.

CH2OH CH2OH Hero Hero NHAc NH2 NHAc NH2 H (A) (B) And Roy et al have been synthesized the glycopeptoids as the metabolic stable mimic of carbohydrate antigen, which was attached to peptoid that is metabolically stable against hydrolysis by peptidase, as shown in following equation (Tetrahedron Lett., 1997, 38, 3487.). But, his reported compounds are thought to be unstable, because it is also expected to be hydrolyzed by glycosidases such as N- acetyl galactosaminidase.

Beau and Roy have not reported to pharmacological activities of Tn antigen linked to carrier proteins. CH2OH HOg O

CH2OH HOJ -: "o TO Ac O AcHN O HO: "I o N N: OH NHAc o HOH2C - 'O."0 HO: 'NHAc

OH

As mentioned above, the compound, prepared from a coupling of the precedented naturally occuring mucin type antigen and the carrier, is hydrolyzed at it's glycoside bond by glycosidase which exists widely in a living body and at it's peptide bond by peptidase. Therefore it is expected to obtain an insufficient effect.

3.The Subject of Invention Under these situations, this invention was attained to solve these problems. The purposes of this invention are to prepare the non-mucin type synthetic compounds- carrier conjugated compounds which are stable to hydrolyze against both enzymes, glycosidase and peptidase.

and to prepare non-mucin type synthetic compounds-carrier conjugated compounds which have the ability of specific reactivity to induce immune response for cancer and HIV and have excellent active immunization activities.

And to prepare non-mucin type synthetic compounds-carrier conjugated compounds which are able to obtain selective monoclonal antibodies for cancer and HIV.

And to prepare anti-tumor agents, anti-HIV agents and immunostimulants which contain this non-mucin type synthetic compounds-carrier conjugated compounds as active ingredients.

And the method to prepare N-acetyl galactosamine in cost effectivly, starting material of non-mucin type synthetic compounds-carrier conjugated compounds, is the another object of present invention.

4. A Solution of the Problem Under these backgrounds, we maked effort to consider to prepare the metabolic stable compounds, having C-glycoside and peptoid, against to glycosidase and peptidase and the non-mucin compound as shown in following figure, were synthesized for the first time. These compounds are, so to speak, C-glycopeptoid and new compounds.

OH

CH2OH CH2OH CH2OH OH N^O H 'g'o H(io H: o HO, , O9,, ,, o \ r HO "' N OH HO ', N rOH OH TV O NHAc OH NHAc OH H: / NHAc C-Tn C-TF C-STn These C-glycopeptoids are more metabolically stable against glycosidase and peptidase than known vaccines. Furthermore, they are able to show their effects for longer time and to store for long term at room temperature. When these novel C-glycopeptoids which are attached to pharmaceutically acceptable carrier proteins, these compounds are more metabolically stable in a living body than known vaccines against glycosidase and peptidase and also expected to show the excellent active immunization activities. It is expected that these novel C-glycopeptoids have potent passive immunogenicities for cancer and HIV. Monoclonal antibodies which were prepared using these compounds are expected to have activities for cancer therapy as a passive immune response. And these compounds have antitumor, anti HIV activity and immunopotentiation.

As a result of investigation of novel compounds as anti HIV agents and immunostimulant, the antibodies which was prepared using novel compound have excelent antitumor, anti HIV activity and immunostimulaion as shown in general formura (l).

DETAILED DESCRIPYTION OF THE INVENTION

A compound ofthe general formula (l), CH2A NO 1 1 1 t B; ,''(CH)m (XX-WE C-M (l) NHAc wherein A represents OH or sialic acid and/or it's derivatives, and B represents OH or galactose and/ or it's derivatives; T represents H or protecting groups of amine; M represents H or OH; X represents oxygen atom, -NH- or S(O)z (where z is 0,l or 2); Q is H or oxygen atom; V represents lower alkyl or H; W is straight or branched alkylene groups from 0 to 5; Z is straight or branched alkylene groups from l to 5; i, m, and t is 0 or l; non-mucin type synthetic compounds or it's carrier conjugated compounds, which have above mentioned compounds as a core structure of antigen.

In the explanation of T. protecting groups of amin are alkyl, acetyl, tbutyloxycarbonyl, benzyloxycarbonyl group,and others. W is straight or branched alkylene groups from 0 . to 5. And general formura (1) is called non-mucin compound in this invention.

Compound (1) have immunopotentiation and non-mucin type synthetic compounds or it's carrier conjugated compounds can be prepared from compound (1) or 2-5 clustered of compound(1) linked with the synthetic compounds such as palmitoyl derivatives which can obtain inductions of immune response.

A compound of the general formula (2), CH2A : (CH)m (X)i-WAN-Z: l-(F)'-E (2) NHAc wherein A, B. T. X, Q. V, W. Z. i, m,and t have above-mentioned meanings; E represents pharmaceutically acceptable carrier compounds; 1 is O or 1; F is showed followings, -N-Jp-(CH2)2 tin- INS} ) Y -N-Jp-(CH2)2 CH2-S; Nit By -N-J - CH N -N-J - CH C -N-Jp CH2 I P ( 2)2 At, P ( 2)2 tl 1 3: d I wherein J is -CH2CH2X- or -N(L)-CH2CO- (where X have above-mentioned meanings; L is H or lower alkyl); G is H or lower alkyl; p is O to 3; y is O or 1; non-mucin type synthetic compounds or it's carrier conjugated compounds, which have above compounds as a core structure of antigen. CH2A to IV

CH2A Boy: ','(CH)(x),-W (CH) (X)-W-N-Z: CO-N Z:CO-lZ)iC-(F)l-E at' \ (CH)rn-(X)l-W I: CH2A wherein A, B. T. X, Q. V, W. Z. i, m, t, E,, and 1 have above-mentioned meanings; r is from 1 to 4; non-mucin type synthetic compounds or it's carrier conjugated compounds, which have above compounds as a core structure of antigen.

A compound of the general formula (4), CH2A A: 1 lV H2N-C-CH CH2A BRA-"'(cHm(x)- O IV T NHAc Q i) '''(CH)m#X\-W-N-Z( CO-N-Zen co Liz, j c-JP-N- CH (3) NHAc NHAc | r U CH2 B: (CIH)m (Xh W H: CH2A wherein A, B. T. X, Q. V, W. Z. J. i, m, t, p, and r, have above-mentioned meanings; U represents H or lower alkyl; w is O to 50; y is 1 or 50.

In the explanation of E, pharmaceutically acceptable proteins are such as albumine (ALB), Keyhole limpet hemocyanin (KLH), BCG, or synthetic compounds such as palmitoyl derivatives, aromatic compounds, aliphatic compounds, alkyl, aminoalkyl, peptide and peptoid, which can obtain induction of immune response.

Non-mucin type synthetic compounds or it's carrier conjugated compounds contain general formula (1) as a core structure. These compounds are able to apply to mammal such as human, and these are used as anti-tumor agents and/or anti-HIV agents having immunostimulate activities. These compounds are also used for the preparation of monoclonal antibodies. These novel compounds are able to elongate of effective time, to decrease of dosage, and to reduce of side effects, further the compounds of the invention are expected to have the potent immunogenicities for cancer and HIV than known vaccines. It is expected that monoclonal antibodies, prepared from this invention, have excellent antitumor and anti HIV activity. Futhermore, when neuraminidase inhibitors such as Zanamivir or Oseltamivir are co-administrated with sialic acid contained compounds in this invention, these sialic acid contained compounds are expected to be more stable in a living body.

N-ac etyl galactopyrano se moi e ty in m uc in type ( O -Tn, O- S Tn. O TF) or non-mucin type (C-Tn, C-STn, C-TF) antigens was synthesized from Nacetyl galactosamine that is very expensive as a starting material. On the other hand, N-acetyl glucosamine, isomer of N-acetyl galactosamine at C-4 hydroxy group, is cheaper and readily available. So it is hoped to use cheaper N-acetyl glucosamine as starting material.

Following this invention, N-acetyl galactosamine derivatives can be synthesized from N-acetyl glucosamine via inversion of C-4 hydroxy group.

That is to say the process for the preparation of N-acetyl galactosamine derivatives, general formula (6) can be prepared from the invention of OR: group to OR group at C-4 position in N-acetyl glucosamine derivatives, general formula (5).

CH2OR, R2O,,, R4O (s) NHAc wherein OR is H or a protecting group of a hydroxy group such as acetyl group; R: is a leaving group such as tosylate, trifluoromesylate or methansulfonate; G is allyl or protected hydroxyl groups.

CH2OR, RIO - t RIO G (6)

NHAC

Herein we descrived the method for preparing the key intermediate, galactose derivatives, (la-11), and also general formula (1).

1) Synthesis of intermediate 1 a- 1 1 (i) Route 1-a CH2OH CH2OTr CH2OH CH2OAc HO,, l TCI AcO, AcO,, l HO,, l Tf2O 1 Pyndlne | AcOH 'A 0 Pyndme HO OH Ac25 AcO OAc AcO MePh OCAHc2c12 NHAc NHAc NHAc NHAc la-l la-2 la-3 lam CH2OAc CH2OAc CH2OH CH2OAc TfO,, l AcO_ HOW AcO l 1 CsOAc at, O OH- 0 1)AcCI it, O AcO DMSO OAc HO 2)C-allylabon,., ''> NHAc NHAc NHAc NHAc la-S lam la-7 lam CH2OAc CH2OAc CH2OAc N-acetylabon C:: oxidation: reduction AcQ Ac i''b Ac 'REECHO AcO - OH NAc2 NAc2 NHAc la-9 la-10 la-11 The intermediate 1 a- 11 is synthesized starting from readily available N acetylgulucosamine as shown in route 1-a via inversion of C-4 hydroxy group.

N-acetylglucosamine is selectively protected by trityl ether at C-6 position (B.

Helferich et al, Ann., 1920, 450, 219.), followed by acetylation at C-3, 4 and treated with formic acid aford compound 1 a-3 (M.Bessodes, Tetrahedron Lett., 1986, 27, 579).

The 4-hydroxyl intermediate la-4 is obtained via acetyl migration of the compound la- 3by heating with a acetic acid in toluene (D.Chaplin et al, J. Chem. Soc. Perkin Trans. 1, 1992, 235.).

The preparation of 4-hydroxyl derivative is selectively protected as benzoyl or pivaloyl ester at position C-3 and 6 by only one step procedure.

4-hydroxyl group is transformed to triflate la-5 and the inversion step is carried out using cesium acetate to give N-acetyl-1,3,4,6-tetra-Oacetyl-D-galactosamine la-6.

Methanesulfonyl chloride or p-toluenesulfonyl chloride can be used instead of trifluoromethanesulfonyl chloride. Then compound I a-6 is deacetylated to N-acetyl-D- galactosamine. And this epimerization at C-4 position was carried out by the procedure of Cipolla et al (Tetrahedron Asymmetry, 2000, 295-303). Allyl group is introduced into compound 1 a-7 by Horton's procedure (Carbohydr. Res., 1996, 309, 319-330).

Compound la-7 is reacted with acetyl chloride, followed by allylation using allyltributyltin and 2, 2'-azobis isobutylonitril (AIBN) to obtain compound I a-8. But, the method of allylation is not restricted by this allylation method.

2-Acetamide group is protected as N. N-diacetyl using isopropenyl acetate in the presence of catalytic amount of acid to afford compound 1 a-9 (J. Oui. Horton et al, Carbohydr. Res, 1996, 309, 319-330.). Compound la-9 is reacted with osumium oxide and NaIO4 to obtain aldehyde compound I a- 10. Compound 1 a- 10 is subjected to reduction using sodium brohydride to give compound la-11.

(ii) Route 1-b CH2OH ICH2OAc CH2OAc CH2OH Oc AcC, Àb c ailylation AcO, OH Ac a Ac HO

_ _ _

NHAc NHAc NHAc NHAc la-l Ib-1 Ib-2 Ib-3 CH2OTBDMS CH2OTBDMS CH2OH TBDMSCI HO,, 1 AcO, 1 AcO 1 mldazó, 1 Ac2O r 1 HF ' 1 AcOH CH2CI2 HO, AcO AcO PhMe

_ _ _

NHAc NHAc NHAc Ib Ib-S Ib-6 CH2OAc Tf2O CH2OAc CH2OAc CH2OAc H., O Pyndne TfO,, O CsOAc AcC O N-acetyiabon C O AcO "' 2 2 A O^J'"/ AcO^; AcO NHAc NHAc NHAc NAc2 1 b-7 1 b-8 1 a-8 1 a-9 CH2OAc CH2OAc oxydation Ac::o reduction oX.J Ac ''/'CHO Ac -OH NAc2 NHAc I a-10 la-11 The intermediate la-11 is also synthesized starting from N-acetyl-D-glucosamineas shown in route 1-b. The compound la-1 is inverted at C-4 hydroxyl group after induction of allyl group (B.A.Roe et al, J. Org. Chem, 1996, 61, 64426445.). N-acetyl- D-glucosamine is treated with acetyl chloride followed by C-allylation with allyltributyltin afford compound lb-2. Compound lb-2 is deacetylated with NaOMe to afford compound lb-3. Then compound lb-3 is selectively protected as t- butyldimethylsilyl (TBS) ether at C-6 position, followed by acetylation with acetic anhydride under basic conditions to give compound lb-5. Compound lb-5 is desilylated by acids and rearrangement to compound 1 b-7 by heating with a catalytic amount of acetic acid in toluene.

Synthesis of compound la-11 from 4-hydroxyl compound Ib-7 is obtained by simillar method as described in route l-a.

2) Synthesis of compound 2-5: Route 2 ACO41 CH2OAc AcO, CH2OAc ACQ CH2OAc reduction of Br(CH2)nCOOtBu \ azidation \ azide / \ base AGO- O ACO- o AcC- ., ., m=1-5

ACHN - ACHN CH ACHN

1a-11 (CH2)n 2 ( 2)n (CH2)n n=lor2 OH N3 NH2 AcO:CH2OAc AcO:<CH2OAc ACON CH2OAc Ac(:o Ncetylation 0_( H+ AcO-CO ACHN (CH2)n AcHN (CH2)n ACHN (CH2)n 2-3 HN-(CH2) 24 ACN-(CH2)N 2-s AcN(CH2)m CO OtBu CO OtBu CO OH Compound la-11 is converted to 2-1 by Mitsunobu reaction (O.Mitsunobu, Synthesis, 1, 1981.). Then azide group of compound 2-1 is reduced to primary amine; for example hydrogenation using Pd-C.

Alkylation of compound 2-2 with haloester, for example butyl bromo acetate, give compound 2-3.

Compound 2-3 is protected as acetamide using acetic anhydride or acetyl chloride.

Compound 2-5 was obtained by deprotection of compound 2-4.

3) Synthesis of compound 2-3: Route 3 AcO' CH2OAc AcO. CH2OAc AcO, CH2OAc ) tBuOOC-(CH 2)m-NH / \ halogenabon / \ AcO: O Ac - :O ba3S-2 AcO-. O nl-5 AdHN (CH2)n n=lor2 OH HN-(CH2) X=CI, Br, I COOtBu The hydroxyl group in compound la-11 is converted to leaving group such as halogen, followed by coupling with compound 3-2 in the presence of base give compound 2-3.

But, the leaving group is not restricted to halogens.

AcO CHzOAc AcO. CH2OAc AcO CH2OAc protection of oxydative cleavage AcO: O N atom Ac: of olefine AcO::O AdHN Ac 3 3 q B-N q=Oor2 AcO CH 20Ac AcO CH2OAc tBuOOC-(CHz)mNH2 X deprotection of AcC - :O AcO: O \ reductve aminabon. À, ; Bn-N (CH2) AcHN (CH2)\ Ac 3-6 HN(CH2) mcOot 2-3 HN(CH2)m Acetamide group in compound 3-2 is protected as suitable protecting group, for example benzylamide, followed by oxdative- cleveage of olefine to provide aldehyde 3-4.

Reductive amination with compound 3-5, followed by deprotection give compound 2-3.

4) Synthesis of compound 4-5: Route 4 AcO:CH2OAc AcO - CH2OAc AcO:CH2OAc / Bu3P / mCPBA base AcO- O AcO-( O AcO- O \ pyndine \ CH2CI2 AcHN AcHN AcHN la-ll OH I SPh '2 SOPh AcO. CH2OAc AcO, CH2OAc AcO" CH2OAc oxydabve cleavage of olefine /\ reduction \ AcO- O > AcO::O > AcO-\:O AcHN = AcHN CHO AcHN -OH 4-3 4 4 s Compound la-11 is reacted with diphenyldisulfide to give compound 4-1, followed by oxdation using m-chloroperbenzoic acid to give compound 4-2. Then, heating in the presence of amine give olefin compound 4-3. The compound 4-3 is oxidized, followed by reduction give compound 5- 8 as described route 1.

5) Synthesis of compound 5-8: Route 5 ACO CH2OAC o ACO CH2OAc ACON_ oxydative cleavage Ac(:O AcO Pd(OACk <_:O of olefine \ pyrdne benzene ACHN (CH2)n ACHN (CH2)\n O ACHN (CH2)n n=lor2 OH 0:, = 0\= AcO CH2OAc ACO CH2OAc AcO, CH2OAc H2 Pd-C ( reductive amination:( Br(CH2)mCOOtB u <4 \ AcOH ACO-\:O AcC:O \ - MeOH ACHN (CH2)n AcHN (CH2)n ACHN (cH2)n \ 0: s s 0: CHO NHBn (CH2)mCOOtBu AcO CH2OAC ACON CH2OAC AcO - CH2OAc N-acetylat on O Ac:O AcO Ac: : ', CH) AcHN (CH2)n ACHN (CH2n ACHN ( 2 \ s-6 NH NA C 5-8 NA c (CH2)mCOOtBu (CH2)mCootBu (CH2)mCOOH Allyl group is introduced into compound 1 a-11 by the method of Curibe et al (Tetrahedron Lett., 1981, 22, 3591-94). Ozonolysis or oxidative cleveage of compound 5-2 afford compound 5-3 by OsO*. The reductive amination of compound 5-3 using amine, for example benzylamine, give compound 5-4. The compound 5-4 is coupled with haloester ( for example butyl bromoacetate) provide compound 5-5. Then amino group of the compound 5-5 is deprotected by hydrogenation, followed by acetylation and debutylation give compound 5-o.

6) Synthesis of sialyl acid derivatives: Route 6 (i) Route 6-a Ac:CH2OAc HO, CH2OH AcOocO'Me / 1) Nrotecton AcO AcO- O HO O 2) OH- \ Lews acd A cH N -(CH 2)m A cH N -(CH 2)m 6-1 (X)(CH2)n 6-2 (x)r(cH2)n rn-lorO R6=O:Bu HN-(CH2)l-CO-R6 R2N-(CH2h-CO- R6 O. S. NH NH-(CH2CH2O)U-CH2CH=CH2 R2=Ac or Benzyl =0 or I OAc COOMe OAc COOMe AcQ: OAc | AcQ: OAc l AcN 1\ 0 I JNdeprotection A cN O, AcO HQlo AcO H: O 6-3 AcHN (CH2)m AcHN (CH2)m (X)(cH2)n (X)(cH2)n R2N-(CH2h-CO-R6 HN-(CH2\-CO-R6 The protection of amino group, followed by deacetylation give compound 6-2. The compound 6- 2 is glycosilated with sialic acid derivatives by Danishefsky's procedure (J.

Am. Chem. Soc., 1999, 121, 2662-2673.). And, leaving group in this reaction is not restricted to halogens. Obtained compound 6-3 can be converted to compound 6-4 as a intermediate of cluster.

(ii) Route 6-b AcO:OMe AcO( OAc COOMe AcNH O. C-allylabon AcNH\O route 15 AcO AcO>-O AcO AcO O 6-3 6-S AcO OAc 6-6 AcO4 R8 R8 R8=-N-Rg, Na R,=Ac, benzyl, or H R10 R'o=Ac, benzyl, or H The a-C-glycoside 6-6 is obtained by C-allylation of compound 6-5. Synthesis of compound 6-3 from compound 6-6 is simillar to described route 1-5. And this reaction also can be proceeded using sialyl transferase and sialic acid derivatives (C.Pauison et al. J. Am. Chem. Soc., 1990, 112, 9308-9309).

7) Synthesis of galactose derivatives: Route 7 (i) Route 7-a h\ AcO ACED HO:CH2OH 0\-;? Ac4Br7-2 / PhCH(OMe)2/ H< O PTS HO-DO Lewis acid CH3NO2 ' ACHN -(CH24m AcHN (CH2(m 6-2 (X)r(CH27n (X)r(CH27n R2 N-(CH2)i-COR6 R2 N-(CH2)'-COMERS m=lorO n=1- 5 Rs=OtBu X=O, S. NH NH-(CH2CH2O)U-CH2CH=CH2 =0 or I R2=Ac or Benzyl ACE N-deprotechon ACT A CH N -(CH\2)m (x)r(CH In (X)r(CH In R2N-(CH2)l-CO-Rr' HN-(CH2)l-CO-R6 Compound 7-1 is obtained by hydroxyl group protection of compound 6-2. The compound 7-1 is glycosilated with acetobromo galactose to give compound 7-3. And, the leaving groups in this reaction is not restricted to halogen. Compound 73 can converted to compound 7-4 as a intermediate of cluster.

(ii) Route 7-b pi pi AcO ACQUIT O AcO ACO\ ) O - ? C-aDylabonAcO_ O: route 1-5 AcO o<0 AcO o::O 7 5 R8 OAc 7-6 R8,< R8=-Rg, N3 R9=Ac, benzyl, or H Rio R'Ac, beryl, or H The o-C-glycoside 7-6 is obtained by C-allylation of compound 7-5. Synthesis of compound 7-3 from compound 7-6 is simillar to described route 1-5. And this reaction also can be proceeded using sialyl transferase and sialic acid derivatives (C. Paulson et al. J. Am. Chem. Soc., 1990, 112, 9308-9309).

8) Synthesis of culster derivatives: Route 8 CH2A H:'o CH2A gJJ. (CH2)m () r (CH2)n (CH2);E. (X), (CH2)n-N- (CH2)q-C(>iN- (CH2)q coil (CH2 - GT OH B) " (cH2) ii: (x)r - (cH2)n HC:O 8-la (r=0, P0) CH:A A=salc acd denvatves orAcO- =o or I, m=0-2 8- lb (r= 1-3, t=0-2) B=galactose denvatves or AcOn=0-5, q=1-5 T--Ac or Benzyl r=0-6, t =0-1 X=OorNHorS CH2A HWo B; J'(cH2)m (X3(CH2)H-(CH2) COOtBu NHAC 8-2 couplmg reagents CH2A HWo CH2A B>; '''(CH2)i(X)r-(cH2)n X) )'(cH2)m-(r (CH2)n-N-(CH2) colN-(cH2)r cofl (CHzl;Ot Bu B:" (CH2)iiF(X) r(cH2)n

H O

8-3a (r=1, P0) 8-3b (r= 1-3, t-0-2) CH2A CH2A H:-o CH2A B; ''(CH2)m(x)l (CH2)n '(CH2(1 (CH2)n I (CH2)-C]N(CH2)q CO<L(CH2= 8i OH NHAc r NHAc B:'' (CH2)(l (CH2)n 8-lb(r=1-3, t=0-2) o 8-4 (r=3, t=1) H CH2A Compound 8-3 is obtained by coupling compound 8-la with compound 8-2 by P.Roy's l procedure (Tetrahedron Lett., 1997, 38, 13478-13490.). Compound 8-lb is synthesized by deprotection of compound 8- 3, followed by coupling with compound 8-2 afford compound 8-3b. Compound 8-lb, 4 is obtained by deprotection of ester in compound 8 3a,b.

9) Coupling linker with haptens: Route 9 CH2A

O

CHzA F:>','(CH2)ij:(X)i (CH2)n AcO I NHAc I 11 H2(CH2CH2X),, R7 B:), /(CH2)n-(X),-(CH2)n-N(CH2)q e(:(CH2)rCOiN-(CHz - CiOH couplng reagents 15 NHAc lHAc | r R7=CHH2CCHH CH2SAc (9-3) ' (CH2)ii(X),-(CH2)n A 0: 8-la (r=0, t=0) c I A=salc acd denvadves orAcO- =o or I m=0-2 8-lb (r=1-3, t=0-2) CH2A B=galactose denvauves or AcO- n=0 5 q=1 5 T=Ac or Benzyl r=0-6, t =0-1 X=OorNHorS CH2A CHzA B)\; ,''(CH2)(X)(CH2)n Ac IT NHAc IO' X)') '(CHz)m (X)l (CHz)n-N- (CHz) COi N- (CH2)q eoil (CHz'C: NH- (CH 2C H2X)= R7 NHAc NHAc | r :' (CH2)m- (X)l (CH2)n R,=CH2CH=CH2 (94) AcO: CH2CH2CH2SAc (9-5) CHzA CH2A Hgo CH2A I^J-'z (CH2)iir (X)(CH2)n depntecbon HO NHAc | O. B), (CHz)m (X)' (CH2)n-N- (CH2)q-C] N- (CH2)q-COiN- (C12+ C: NH-(CH 2C H2X>R7 NHAc tIHAc | r P: (CH2)m ()I (CH2)n HO:O CH2A Compound 9-4 and 9-5 are prepared from coupling linker 9-2 or 9-3 with compound 9-1 respectively (P. Roy et al, Tetrahedron Lett., 1997, 38, 3478-3490). And this coupling can be proceeded used to other reagents such as N. N-dicyclocarbodiimide, Danishefsky's procedure (J. Am. Chem. Soc., 1998, 120, 12474-12485) or 2- isobutyl-1isobutoxycarbonyl-1,2-dihydroquinoline. Compound 9-7 is obtained by deprotection of hydroxy group.

10) Coupling carrier with hapten: Route 10 CO NH-Pa I Hapten H2N-: 1 0-6 /amde coupimg

COOH

Hapten J H2N-E 10-1 CO NH-E amdecoupimg Hapten J 10-7 HOOC-E NHCO-E oxydahon mduchon old NH2 Smarten J amp Hapten amdecoupimg 10-13 oxydahon i= CHO HaptenJ Hapten J -E 2HN-N:N,;3 reductive ammah Hapten J 10-3 O reductive amnabon 10-9 AcSH N) HS-E N> MN-NH MN-NH sfE HaptenJ 10-4 o' HaptenJ 0 Ha ten,SAc OH- SH O 1 11 P 95 Hasten Hapten/ HE 10-5 10-12 Compound 9-4 is oxidized to aldehyde 10-2, followed by reductive amination with protein using sodium cyanoborohydride in phosphate buffer (pH7.2) affod compound 10-9 by Livingston's method (Glycoconjugate Journal, 1998, 115, 217-221.). Compound 9-4 is also coupled with protein by Slovin's method (Proc. Nat. Acad. Sci. USA, 1999, 96, 5710.). Compound 9-4 is converted to maleimide compound 10-4, followed by coupling with thiol group in protein or compound 9-4 is converted to thioacetate compound 9-5. Compound 9-5 is coupled with maleimidated protein by Khono's method (J. Clin. Lab. Anal., 1999, 10, 91. ) afford compound 10-12.

Compound 10-7 is obtained by coupling carboxylic group in compound 10-1 with amino groups. Compound 10-13 is obtained by coupling aminogroup in compound 10-8 with carboxylic groups in protein. And Coupling compound 101 with palmitoyl derivatives by Danishefsky's method (J. Am. Chem. Soc., 1999, 121, 2662-2673,.) give compound 10-6.

11) Synthesis of polymer derivatives: Route 11-a tCH2A H 'U'o CH2A E^J ', (CH2= (art (CH2)n (:O T NHAc | H2N-(CH2CH2X)U-N3 L: (C}l2)(x)r(cH2)n N-(CH2)rC N-(CH2 CO(N-(CH2 oc i oH - at' (CH2)i(' (ClI2)n 12-1 H: CE12A Aslalic acid derivatives orAcO- I=0 or I, m=0-2 B=galactose derivatives or AcO- r=0-S, q-1-5 T--Ac or Berzyl r=0- 6, t =0-1 X=0 or NH or S CH2A Hgo H2A J,'(CH2);j:('-(CH2)n H(g F NHAc | Reduction of:'de B;,,'(CH2 (r(cH2)n N- (CH2)q CO {N- (Ct2) COIN- (CHAT OC: N - CHzC H2X)U N B:-' (CH2);;r()(CH2)n 12-3 H( - O CH2A ICH2A CH2A W, '(CH2)ii7('-(CH2)n :(CH2)(X),-(CH2)n N(CH2)q-CCr[N-(CH2) Co-((CH2oC3N - CH2CH2X)u NH2 W(CH2)(X)i-(CH2)n 1' 12 - CH2A CH2A HI lo CH2A B>: ','(CH2) (X)r (cH2)n :,'(CH2)m- (X)r (CH2)n N- (CH2)q-C]N- (CH2 cowl (chid oc: NtCH2C H2X)U-HI B: (CH2)m-(X)r-(Ci2)n 12-5 polymenzation CH2A CH2A HQJ Q 1 - 1 NH2-C-CH CH2A BE -(cH2);iF(x)r-(cH2)n l H2 X), (CH2)m- (X),- (CH2)n-N- (CH2)q-CLAN (CHASE CO\I(CH2 OC:NtCH2CH2X N-C- CH B:" (CH2)m (X)i-(CH2)n _ c: 1-50 1 Y CH2A 12-6 To couple a linker 12-2 with carboxylic acid compound 12-1 afford compound 12-3.

Compound 12-3 is reduced to compound 12-4, followed by condensation with acroyl chloride and afforded compound 12-5. Compound 12-5 is converted to compound 12-6 by the method of K.Eklind et al (J.Carbohydrate Chem., 1996, 15, 1161) or J.Domb et al (J.Med.Chem., 2000, 43, 2591). The method of polymerization is not restricted to this method.

(ii) Route 11-b CH2OH,l_ (: N H2-C-CH CH2OH Ho^; 'l'(CH2)m-(x) (CH2)n CH2 H: NHAc | R t H:Q; \ (CH2(X)' (CH2)o y=1 50:Y H 12-6 CH2OH CMP-Neu-5Ac 2,6-slalyltransferase CH2A 1 0,0, CH2A HO^;'.(CH2)m (X)' (CH2)o CH2 O I NHAc | H IO' w Ni; ', (CH2)(X)r(CH2)o-h'L (CH2)q Co5N(CH2 CO-(N-(CH OCiN CH2CH2X) H-C- ICH NHAc NHAc | r CH2 H(g-- (cH2)nr(x)l-(CH2)o H>:O I Y CH2A 13 1 A=sialic acid derivatives orHO Synthesis of sialic acid derivative 13-1, having a polymer, can be achieved using CMP Neu-5Ac and sialyltransferase from compound 12-6 (C. Pauison, J. Am. Chem. Soc., 1990, 1 12, 9308-9309).

(iii) Route I l-c :20H NH2-"OC- CH2OHH "/ (CH2)m (X)r- (CH2)n CH 2 H NHAc I O W H: (CH2) (X)- (CH2)n NL (CH2)q-CO{N- (CH2 CoiN- (CH2= oc: N - CH 2C H2X); N C- CH H( > (CH2)m(X),- (CH2)n ty 1 o w=1-50 HO- y=1-50 CH2OH 12-6 PNP-Gal Galactosldase tCH2OH q:O NH2-C-CH H20H BtJ '/ (CH2)m (X)-(CH2)n CH 2 H:(cH2(r(cH2)n W(cH2)q-C] N-(CH2 Coil(CH2ociN - CH2CH2X - H-C- ICH NHAc NHAc | r CH 2 B" (CH2)m(X) r(clI2)n M_ 1 l w=1-50 1 Y HO y=1-50 CH2OH 14-l B=galactose derivatives or O Synthesis of galactose derivative 14-1 can beachieved from compound 12-6 using galactosiase and galactose derivatives PNG-Gal after polymerization (C. Pauison, J. Am. Chem. Soc., 1990, 112, 9308-9309).

The Biological stability for glycosidase such as N-acetyl galactosaminidase was tested using allyl derivatives (11-1, 11-2) by Mark von Itzstein's method(Org. Leyy., 1999, 443-446) CH 20H H:o f&-N-acetyl galactosamtnldse I I > No Reaction i"' NHAc 11-1 CH2OH CH2OH Id''' ,-Nacetylgalactosamintdse;i) /OH NHAc NHAc 11-2 enzyme; a-N-acetyl galactosaminidase 0.32unit (1.69 unit/ml 0.1% BSA containing 0.5M sodium citrate buffer) solvent; citric acid buffer (pD=3) 0.6ml temperature; 35 C procedure; Substrate (2mg) was disolved in citric acid buffer (0.6ml) and o-N-acetyl galactosaminidase (0.32 unit) was added. NMR spectrum was determined in every constant time.

Results of this test, Substrate persistence, were shown in table 29.

Table 29

substrate 2 4 6 8 10 12 18 24 11-2 89 79 68 57 50 45 40 22 I l_l 100 100 100 100 100 100 100 100 From above results, evidently, 78% of O-glycoside linkage allyl ether (11-2) that have was hydrolyzed after 24 h. As it is expected, compound 11-1, replaced ether bond to C C bond, was unaffected by enzyme, degradation was not observed after 24h.

This result show the C-glycoside is metabolic and catabolic more stable than O- glycoside.

One or more than two medicinal compounds can be contained compounds that of described general formula (1) in the present invention as active ingredients. And the general formula (1) can be administered to human. And monoclonal antibodies of general formula (1) can be administered to human. The compounds with the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic propaties. And the compound itself and/or it can be formed injections, powders, granules, tablets, capsules, troches, dry-syrups, lipozome preparations and others. The appropriate dose and dosage times, that of the compound of the present invention, must be determined by the conditions of patient, age, body weight etc. The compounds are exemplified as follow, but the invention is not limited to these compounds.

(A (1-111), B (1-111), or C (1-111)) (A (1-11), B (1-111), Or C (11I)(CH2 qtC(CH2 q<Cl(CH2)q$C-(Ft (A (1-111), B (1-111), or C (1-111)) (wherein T. E, F. Q. t, 1, and r have above-mentioned meaning; q represents O to 5; u is O or 1.) A (I), A (II), A (m), B (I), B (II),B (m) , C (I), C (II), and C (m) in above equation and table 1-28 were shown in bellow equation HOH2C HOH2C HOH2C HC Hi o HOW o HOWL CH2CH2OCH2CH2 HOW; CH2CH2 HOW: ''CH2NHAc NHAc NHAC A(l) A(II) A(111) OH Hi OH Hi H OH Hi HO HO, - H: Hi HO Of O HO o: O HO Of LO AcHN CH2CH2OCH2CH2 ACHN " AcHN CH2- B(l) B(II) B(111) O 4,iCHzCH2OCH2CHm HooH H ''NHA HO OH NHAC C(l) C(ll) C(TII)

RH NKLH

Number | R | A(0 2 B(I) 3 C(I) Number | 4 HOH2[

HO

HO: '-O Nit N'-O-NKLH NHAc HOH2[ o Nit NO-N-KLH NHAc Rip NO New o H Number Number Number R 6 A(9 B(9 12 C(I) 7A() 10 B(In 13 C(Ip 8A(III) 11 B(III) 1 4 C( R'N- Nan Or N-BSA

H H

Number Number R Number Am 18 B(I) 21 C(9 16 A(II) 19 Bow) 22 C(I 17 A(In) 20 B() 23 C(III) R' N oO O NKLH

R O

Number Number Number A(I) 18 B(I) 21 C(I) 16 A() 19 B() 22 C() 17 A(m) 20 B(m) 23 C(m) Ac O H R' N oOO BSA

R O

Number Number R Number 24 A(I) 27 B(I) 30 C(0 A(n) 28 B(II) 31 C(II) 26 A(HI) 29 B(m) 32 C(In) R t N KLH R4 o H H Number | R3 | R4 R5 Number R3 R5 33 A(0 A(I) A(9 41 C (HI) C (m) C (HI) 34 A() A(r A() 42 A() B() A() A(HI) A(HI) A(m) 43 B(II) A(r B(II) 36 B(I) B(I) B(I) 44 A(II) c(r A() 37 B(II) B() B() 45 C(II) A(r C(II) 38 B(m) B(m) B(III) 46 B(II) C() B(II) 39 C(I) C(0 C(I) 47 C() B(II) C() C(II) C(Ib C() 48 A(II) B(r C(II) R4 o H H Number R3 R4 R5 Number R3 | R4 1 5 49 A(I) A(I) A(b 57 C(III) 1 C( A(II) A(II) A(II) 58 A(II) B(II) A() 51 A(m) A( A(III) 59 B(I1) A(II) B(II) 52 B(I) B(I) B(I) 60 A(II) C(II) A(II) 53 B(Ip B(II) B(II) 61 C(II) A( C(II) 54 B(III) B(III) B(III) 62 B(II) C(II) B(II) C(I) C(I) C(I) 63 C(II) B(1I) C(In 56 C(II) C(II) C(II) 64 A(II) B( C(II) A o HNKLH

R HN- O

umber Number R | Number R A(I) 68 B(I) 71 c(r) 66 A() 69 B( 72 C(Ib 67 A(m) 70 B(m) 73 C(III) RhN'--O)-NBSA umber. Number Number 74 A(1) 77 B(I) 80 C(I) A() 78 B() 81 C(II) 76 A(m) 79 B(m) 82 C(III) R'aN_ o O-o '\ KLH Number Number R Number 83 A(I) 86 B(0 89 C(I) 84 A(In 87 B(II) 90 C(Ip A(In) 88 B(m) 91 C(nI) R' mN O o' BSA

O O

Number Number R Number 92 A(I) 9S B(I) 98 C(I) 93 A() 96 B(II) 99 C(I 94 A(III) 97 B(m) 100 C(III) s) +KLH Number R3 R4 R5 Number R3 R4 R5 1 O 1 A(0 A(I) A(I) 109 C(m) C(III) C(III) 102 A(II) A(II) A(II) 110 A(II) B(II) A() 103 A(III) A(m) A(m) 111 B(II) A(II) B(II) 104 B(I) B(0 B(I) 112 A(II) C(II) A() B(II) B( B(II) 113 C(II) A(II) C(II) 106 B(III) B(III) B(IIl) 114 B(II) C(II) B(II) 107 C(I) C(I) C(1) 115 C(II) B(II) C(II) 108 C(II) C(ll) C(II) 116 A(II) B( C(II) //0 H

N--N-BSA

R3'ap: H Number R3 R4 R5 Number R3 R4 R5 117 A(I) A(I) A(I) 125 C(III) C(m) C(0 1 1 i A(II) A( A() 126 A(II) B(II) A() 119 A(m) A(III) A(m) 127 B(II) A( B(II) B(I) B(I) B(I) 128 A(II) C( A(II) 121 B(II) B( B(II) 129 C(II) A( C(II) 122 B(III) B(m) B(III) 130 B(II) C(II) B(Ib 123 C(I) C(I) C(I) 131 C(II) B( C() 124 C(II) C(II) C(II) 132 A(II) B(II) C(II)

Table 15

R'N(NO-oO N-N - N: H H H 9-KLH o Number Numbe R Numbe R 133 A(b 136 B(9 139 C(I) 134 A( 137 B() 140 C(II) A(III) 138 B(nI) 141 C(HI)

Table 16

R'N) N0 o H}BSA o - C(l) _ C() c(m

Table 17

Rota - N 0OO H} - KLH

Table 18

R'a:Na'Ot) H HNJ:4S-BSA Numbe R Numbe R Numbe R A(I) 163 B(I) 166 C(I) 161 A(In 164 B(II) 167 C( 162 A(m) 165 B(HI) 168 C(m)

Table 19

R3' N--O oO N-N-ILLS-KLH Numbe R3 R4 | R5 Numbe R3 R4 R5 169 A(0 A(0 A(I) 177 C(HI) c(m) C(0 A(II) A() A() 178 A(n) Bun) A() 171 A(Ul) At A(m) 179 B(I Am B(II) 172 B(I) B(I) B(I) 180 A() C() A() 173 B(r B() B() 181 C(II) A() C(II) 174 B(m) B(m) B(0 182 B(II) C() B(II) C(I) C(0 C(I) 183 C(I B(n) C(II) 176 C() C() c(n) 184 A() Bun) C(Ib

Table 20

R,'6 H IS-BSA

Table 21

Rat NO opera? N'- S AC H Hats,,, Numbe Numbe R Numbe 201 A(I) 204 B(0 207 C(I) 202 A(n) 205 B(ro 208 C(Ip 203 A(m) 206 B(m) 209 C(HI)

Table 22 S S 0

R N'N o O4t N Ny1 N: 50

AC O H S H H

Numbe R Numbe R Numbe R 210 A(I) 213 B(0 216 C(I) 211 A() 214 B() 217 C(II) 212 A(m) 215 B(m) 218 C(nI)

Table 23

H O H,, O H-_ R. N. o oJI N N. N-54 14

AC O H O H H o

Numbe R Numbe R Numbe R 219 A(I) 222 B(I) 225 C(I) 220 A() 223 B() 226 C(II) 221 A(m) 224 B(m) 227 C(nI)

Table 24 HO n

R. N', N o Q_7 NNg NgS4 AC O H 0 H H- o Numbe R Numbe R Numbe R 228 A(0 231 B(I) 234 C(I) 229 A(II) 232 B(II) 235 C(II) 230 A(m) 233 B(ID) 236 C(III)

Table 25

HO of _ tic H O H O Q R' NO Nat N. oOJL N N1> N S-l-(r- R o H O H Hl o Numbe R Numbe R Numbe R 237 A(0 240 B(0 243 C(I) 238 A() 241 B(10 244 C(Ip 239 A(11) 242 B(m) 245 C(rl)

Table 26 row

94 P H O HI O r o Rats ON LION oN N SILO Ac O Rs O H 0 H Hi Numbo R3 R4 R5 | Numbe R3 R4 R5 246 A(0 A(0 A(9 254 C( C(m) C(0 247 A(II) A() A(n) 255 A() B() A() 248 A(m) A(m) A(m) 256 B(II) A(Ic B(II) 249 B(I) B(I) B(I) 257 A() C(I1) A() 250 B(II) B(n) B(IT) 258 C(I A() C(II) 251 B(n[0 B(n[I) B(HI) 259 B(n] C(I10 B(II) 252 C(I) C(h C(I) 260 C(I B(n) C(I 253 C(I1[) C(I1[) C(n[) 261 A(n[) B(n) C(II)

Table 27

R 'aJ(N'O-o' NKLH Numbe R3 R4 R5 Numbe R3 R4 R5 262 A(0 A(I) A(0 270 C(m) C(m) C(I1[I) 263 A(n[) A(n[) A(n[) 271 A(II) B(n[) A(n[) 264 A(IL[) A(III) A(IL[) 272 B(II) A(II) B(II) 265 B(I) B(0 B(I) 273 A(n[) C(II) A(n[) 266 B(II) B(n[) B(II) 274 C(I A(II) C(II) 267 B(n[I) B(m) B(III) 275 B(I C(I10 B(II) 268 C(I) C(0 C(I) 276 C(I B(n) C(II) 269 C(II) C(II) C(I1[) 277 A(I1[) B(I1[) C(I

Table 28

R3 - 0 H H Numbe | R3 | R4 | R5 I Numbe | R3 | R4 | R5 278 T A(I) T A(I) | A(I) | 286 1 C() | C(m) | C(III) 279 1 A(II) T A() 1 A(II) T 287 1 A(II) 1 B(II) 1 A(II) 280 A(III j A( A(m) 288 B (II) A(II) B (II) 281 B(I) B(I) B(I) 289 A(II) C(II) A() 282 B(II) B(II) B(II) 290 C(II) A(II) C(II) 283 B(III) B(III) B(III) 291 B(II) C(II) B(II) 284 C(I) C(I) C(I) 292 C(II) B(II) C(II) 285 C(II) C(II) C(11) 293 A(II) B( C(II)

PHARMACOLOGICAL EXPERIMENT

Immunization and antiserum preparation Vaccine used to immunize were prepared as below. Glycoproteinic antigen (ox. lmg) suspended in phosphate buffered saline (ex. lmg) were mixed with equivalent volume of adjuvant (ex. freund complete adjuvant, and BCG etc.). Female BALB/c mice (6weeks of age) were subcutaneously immunized with 200,u1/mouse of vaccine. Mice were injected on days 0, 14, 28, and bled 1 week after the 3rd immunization. Antiserum (-) was obtained from the blood centrifuged at 1,200Xg for 20min.

Measurement of antibody titer.

Microtiter 96-well plate were coated with Tn antigen. IgG and IgM antibody titers were measured by ELISA with horse anti-mouse IgG antibody and anti-mouse IgM antibody, respectively, as second antibody. Human colon carcinoma cell line LS-174T cells cultured in microtiter 96-well plates, and were imorbilized with methanol. As described, IgG and IgM antibody titers were measured by ELISA. Effects of each compounds described below on antibody titer were evaluated by this assay.

_ H2OH _ H OOH I P H 3 HO H H

H H NH

_ _

pH2OH H2OH 4 H: ''^O LH 5 NHAc -^O H H2OH H H tOOH 6 H it, O-- H 12 O NHAC H H -^o^_ H

_ _ ACHN OH

l H H H (ad H H HOH2 HOH2C,O HO 'NHAC.1 1

OH HO DUNHAM

_

_ AcHN^OH _ AcHNAOH H Ho2 H io\CH2OH tH2OH oCH2OH (3b3) H _O-oONKL H (3c3) H- _o H NHAc HO NHAc

_ OH _ OH

IgG and IgM antibody titer (against Tn antigen) in mouse serum after vaccination were shown in table 30 and 31 respectively. IgG and IgM antibody titer (against LS-174T cell) after vaccination were shown in table 32.

Table 30

1st immunization 2nd immunization 3rd immunization Number dose Adjuvant IgG IgG IgG l lO0g BCG Not tested Not Tested 2667+ 3233 l Og BCG 800+0 2160+ 1431 4960+4879 lO,ug BCG Not tested Not tested 2640+1252 10pg BCG 200+200 1600+980 7680+ 10207 12 1g BCG 600+283 1920+ 1213 7680+ 10207 lOg BCG 560+358 2080+1073 6400+ 5879 33(a) 1 g BCG 200+346 1760+ 1315 6880+ 10516 33(b) 10pg BCG 240+220 1920+720 7700+3320 33(c) 10pg BCG 1040+780 1760+ 1320 4840+5010

Table 31

l |lst immunization Number dose Adjuvant IgM 1 lO,ug BCG Not tested OUg BCG 40i89 lOg BCG Not tested l Og BCG 0+0 12 1 kg BCG 160+358.

l Og BCG 100+ 173 33 1 Ng BCG 0+0 33(b) lOg BCG 100+100 33(c) 10,ug BCG 10400+5400

Table 32

Number dose Adjuvant IgG IgM 10,ug BCG 2400+1131 15201073 1 OL'g BCG 800+0 2160+ 1431 10pg BCG 6401590 11201438 10pg BCG 1120+ 1242 1200+560 33(a) hug BCG 1520ilO73 1440+358 Antibody depended cell mediated cytotoxic response (ADCC) LS-174T cells used as target cells and mononuclear cells from peripheral blood in human were used as effecter cells. The target cells were seeded into microtiter 96-well plate (1 x 103 cells/ weld 50Lrl), and were added 0.5pCi/ well of 5'CrCk. the cells supernatants were harvested and counted in a gamma counter. The cytotoxicity was calculated as the percentage of releasable counts subtracting the spontaneous release.

The results were shown in Table 33.

Table 33 HOHz; , H

HO'O N- KLH NHAc Dilution cpm/ 1000cell 913 400 685 800 318 1600 281 3200 103 6400 46 Purified carrier protein Keyhole limpet hemocyanin (KLH, CHEMICONINTERNATIONAL INC.) was purified by the previously published method. KLH (SOOmg) was suspended in 50ml phosphate buffered saline (PBS()) and centrifuged at I,200xg for 20 min. Resulted supernatant was centrifuged at 43,000xg for 15min. Resulted sediment was suspended in PBS(-) and further centrifuged at 43,000xg for I Smin and resulted sediment was used as carrier protein.

Immunization C-linked Tn-KLH conjugate or C-linked sTn-KLH conjugate (1 to thug) were immunized subcutaneously to female BALB/c mice with BCG(50, ug) 3 times at two weeks interval. One week after the last immunization, mice were anesthetized and bloods were collected from abdominal vein. Antisera were separated by centrifugation, and IgG or IgM antibody titers against gpl20 were assayed by ELISA. The titer was defined as the highest dilution yielding an absorbance of greater over that of normal sera.

The results were shown in Table 34.

Table 34

Number IgG IgM 3 12,800 6,400 12,800 12,800 12,800 6,400 6,400 12,800 33 6,400 6,400 Our results also shows the potent immunogenicity of metabolic and catabolic stable "C-glycopeptoid" with or even without carrier protein. On other hand, Danishefsky's team reported the O-Tn, O-STn, O-TF antigens dose not have less potent immunogenicity themselves, but attached to carrier proteins such as KLH.

(S.J.Danishefsky et al, 1998, 120, 1427-14285.) We firstely showed the concept and efficacy of using "C-glycopeptoid" for the promising immunotherapy of cancer and HIV.

EXAMPLE

The following Examples are provide only for the purpose of the preparation of the compounds and not restrict to the disclosed invention.

Referencial Example 1 The preparation of 2-Acetylamino-1,3,4-tri-O-acetyl-6-O-triphenylmethyl-2- deoxy-O-Dglucopyranose (compound la-2) CH2OTr AcO., 1 Act OAc NHAc A suspension of N- acetylglucosamine (200g, 0.9mol) and trityl chloride (250g, O.9mol) in pyridine (363ml) was heated to 85 C. After the suspension was dissolved, acetic anhydride (280ml, 2.97mol) was added and stirred for 23h at room temperature. The reaction mixture was slowly poured into ice water acetic acid. The mixture was stirred for 3h and the resulting precipitate was collected, followed by washing with water. 400g (75%) of the objective compound was obtained.

MS (m/ z): 590, 531, 452, 243, 165.

IR (cm') neat: 3364, 1749, 1656, 1218.

H-NMR (CDC13) b: 3.02 (1H, dd,]=10.8, 3.9Hz), 3.27 (1H, dd, ]=10.3, 2.0Hz) , 3.87 (1H, ddd, J=9.3, 2.0, 2.0Hz), 4.54 (1H, ddd, J=11.2, 9.3, 3.9Hz), 5.17 (1H, dd, J=11.2, 9.8Hz), 5.35 (1H, dd, ]=9.8, 9.8Hz), 5.53 (1H, d, ]=8.8Hz), 6.29 ((1H, d, ]=3.4Hz), 7.31-7.17 (9H, m), 7.41-7.43 (6H, m).

Referencial Example 2 The preparation of 2-acetylamino-1,3,4-tri-0-acetyl2-deoxy-a-D-glucopyranose (compound la-3) HOH29 Act ACO'V 'OAc NHAc The trityl compound (168g) obtained from the above mentioned Referencial Example 1 was dissolved in diethylether (420ml), then formic acid (420ml) was added at room temperature and the mixture was stirred for 7h. After the reaction was finished, the reaction mixture was poured into ice cold water and neutralized by NaHCO3, followed by removal of diethylether, and the resulting precipitate was filtrated. The filtrate was extracted with chloroform. After drying (Na2SO4), the solvent was removed under reduced pressure. 46g (46%) of the objective alcohol was obtained.

MS (m/ z): 347, 304, 228, 114.

IR (cm') neat: 3280, 3076, 1749, 1665, 1221.

H-NMR (CDC13) b: 1.91 (3H, s), 2.04 (3H, s), 2.16 (3H, s), 3.55 (1H, dd, 1=12.8, 4.4Hz), 3.66 (1H, dd, ]=12.8, 2.2Hz), 3.78 (1H, ddd, 1=10.1, 4.3, 2.2Hz), 4.43 (1H, ddd, J=10.9, 9.0, 3.6Hz), 5.14 (1H, t, J=9.7Hz), 5.25 (1H, dd, J=10.8, 9.6Hz), 5.76 (1H, d, J=9.OHz), 6.15 (1H, d, J=3.6Hz).

Referencial Example 3 The preparation of 2-acetylamino- 1,3,6-tri-0acetyl-2-deoxy--D-glucopyranose (compound la-4) AcOH2 AcO: 'OAc NHAc To a solution of the primary alcohol compound (81 g, 0.23mol) obtained from the above mentioned Referencial Example 2 in toluene (1600ml) was added acetic acid (16ml) and the mixture was stirred at 80 C for 15h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The redsidue was purified by silicagel column chromatography (AcOEt). 99g (58%) of the objective compound was obtained as a colorless oil.

MS (m/ z): 347, 304, 262, 228, 114.

IR (cm') neat: 3370, 3010, 1737, 1659, 1230.

H-NMR (CDC13) b: 1.93 (3H, s), 2.13 (3H, s), 2.17 (3H, s), 3.70 (1H, dd, J=9.8, 3.5Hz), 3.84 (2H, d, ]=3.5Hz), 3.90 (1H, dd, ]=9.8, 9.2Hz), 4.30 (1H, ddd, ]=11.1, 9.0, 3.7Hz), 5.12 (1H, dd, ]=11.1, 9.2Hz), 5.71 (1H, d, 1=9.OHz), 6.11 (1H, d, ]=3.6Hz).

Referencial Example 4 The preparation of 2-acetylamino-1,3,6-tri-0-acetyl-4- trifluoromethanesulfonyl-2- deoxy-o -D- glucopyrano se (compound la-5) AcOH27 AcO: 'OAc NHAc The alcohol compound (5.0g, 14. 3mmol) obtained from the above mentioned Referencial Example 3 was dissolved in dichloromethane (50ml) and added pyridine (5ml). The solution was cooled to -40 C, then triflic anhydride (3.1ml 18.7mmol) was added dropwise to the mixture. After stirring for 2h, the reaction mixture was poured into ice cold water and extracted with dichloromethane. The organic layer was washed with 10% HCI and dried (Na2SO4). The solvent was removed under reduced pressure.

7.83g of the objective compound was obtained as a colorless oil.

Referencial Example 5 The preparation of 3-(2-acetylamino-3,4,6-tetra-0-acetyl-2-deoxy-a-D- galactopyranosyl)- 1 -propene (compound 1 a-6) AcOH29 AcOJ Act NHAc To 2. 0g (9.Ommol) of N-acetylgalactosamine was slowly added acetyl chloride (4. 0ml) at 0 C. The mixture was stirred for 14h at room temperature. After the reaction, the mixture was poured into ice cold water and extracted with chloroform. The organic layer was neutralized by satd. NaHCO3, and washed with water and brine. After drying (Na2SO4), the solvent was removed under reduced pressure. 3.3g of N- acetylamino-l Chrolo-tri-O-acetyl-2-deoxy-galactosamine was obtained as a colorless oil. To a solution of the obtained compound (3.3g, 9.0mmol) in toluene, was added allyltributyltin (8.5ml) and 2, 2'- azobisisobutyronitrile (AIBN) (0.25g) under argon atmosphere. The reaction mixture was heated to 80 C and stirred for 6h. After the reaction was completed, the mixture was cooled to room temperature. The solvent was removed under reduced pressure. The resulting redsidue was purified by silicagel column chromatography (BW-200, AcOEt: n-hexane=4: 1) . 0.85g (25.4%) of the oily objective compound was obtained as a colorless oil.

Mass (m/e): 371, 330, 210, 150, 101, 59.

IR (cm) KBr: 3290, 3071, 1746, 1658, 1020.

H-NMR(C6D6) b: 1.47 (3H, s), 1.63 (3H, s), 1.66 (3H, s), 1.67 (3H, s), 1. 99 (1H, m), 2.19 (1H, m), 3.94 (IH, m), 4.26 (1H, d,d, J=3, SHz), 4.37 (2H, m), 4.83 (1H, m), 5.00 (2H, m), 5.17 (1H, d, J=7Hz), 5.43 (1H, t, J=3Hz), 5.68 (1H, m), 6.19 (1H, S).

Referencial Example 6 The preparation of 3-(2-diacetylamino-3,4,6-tetra-O-acetyl-2-deoxy-a-D- galactopyranosyl)- 1 -propene (compound la-9) AcOH2 ,0 Act NAc2 To a solution of the compound (1.5g, 4. 0mmol) obtained from the above mentioned Referencial Example S in isopropenyl acetate (lSml) was added p-toluensulfonic acid (20mg). The reaction mixture was stirred at 55 C for 42h. After the mixture was cooled to room temperature, triethylamine was added and stirred for 1 Smin. The mixture was concentrated. The residue was purified by silicagel column chromatography (BW-200, AcOEt: n-hexane=1:1). l.Og (66%) ofthe objective diacetate compound was obtained as a colorless oil.

Mass (m/e): 413, 372, 330, 270, 210, 179, 150, 126, 101, 59.

IR (calf) KBr: 3050, 1749, 1668, 1233, 780 H-NMR(CDC13) 5: 1.95 (3H,s), 2. 03 (3H, S), 2.16 (3H, s), 2.17 (1H, m), 2.39 (3H, s), 2.75 (1H, m), 4.05 (2H, m), 4.15 (2H, m), 4.61 (1 H. d,d, J=4,8Hz), 5.11 (2H, m), S.SO(1H, d, d=3Hz), 5.75 (1H, m), 5.95 (1H, dd, J=3,1 lHz).

Referencial Example 7 The preparation of 3-(2-acetylamino-3,4,6-tetra-O-acetyl-2-deoxy-a-D- galactopyranosyl)- 1 -acetaldehyde (compound la-10) AcOH29 AcOJ art. CHO NAc2 To a solution of the compound (0.74g, 1.78mmol) obtained from the above mentioned Referencial Example 6 in tetrahydrofrane (lOml) was added water (lOml), NaIO4 (1.9g, 8.91mmol) and 4% OsO4 solution under an atmosphere of argon. The mixture was stirried for 4h at room temperature. After the reaction was completed, the reaction mixture was extracted with ethyl acetate and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure. 0.77g (98%) of the objective aldehyde compound was obtained as a colorless oil.

IR (emu) KBr: 1746, 1371, 1230, 1054, 665.

H-NMR(CDC13) is: 1.95 (3H, s), 2.04 (3H, s), 2.16 (3H, s), 2.37 (6H, s), 2.85 (1H, m), 3.17 (1H, dd, J=2,8Hz), 4.11 (3H, m), 4.75 (2H, m), 5.54 (1H, m), 5.81 (1H, d,d, r=3.5,1 lHz), 9.67 (lH,s).

Referencial Example 8 The preparation of 3-(2-acetylamino-3,4,6-tetra-0-acetyl-2-deoxy--Dgalactopyranosyl)-l-ethanol (compound la-l l) AcOH2 AcOJ: Act '-OH NHAc To a solution of the compound (0.77g, 1.85mmol) obtained from the above mentioned Referencial Example 7 in methanol (lOml) was added sodium borohydride (O.lg, 2.78mmol) at 0 C and the mixture was stirred for lOmin. The reaction mixture was poured into satd.NH4C1 and extracted with dichloromethane. The organic layer was washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt: MeOHe=10: 1). 0.25g (36%) of the oily objective compound was obtained as a colorless oil.

Mass (m/e): 357(M+), 316, 238, 183, 141, 101, 59.

IR (emu) KBr: 1743, 1680, 1398, 1236.

IH-NMR(CDCl3) 6: 1.60 (1H, m), 1.95 (1H, m), 2.00 (3H, s), 2.09 (3H, m), 2.10 (3H, s), 2.12 (3H, s), 3.17 (1H, dd, J=3, 8Hz) 3.76 (2H, m), 4.05-4. 18 (3H, m), 4.42 (3H, m), 5.32 (1 H. t, ]=3Hz), 5.73 (1H, d, ]=8Hz).

Referencial Example 9 The preparation of 3-(2-acetylamino-3,4,6-tetra-O-acetyl-2-deoxy-a-Dgluctopyranosyl)-1-propene (compound lb-2) AcOH2i AcO Act NHAc To Nacetylglucosamine 100g (0.45mol) was added acetyl chloride (200ml) at 0 C and stirred for 23h. After the reaction, the mixture was extracted with chloroform and the mixture was poured into ice cold water and stirred for 10min. The organic layer was neutralized by satd. NaHCO3 and dried (Na2S04). The solvent was removed under reduced pressure. Diethyl ether was added to the residue and the resulting precipitate was collected. 117g (71%) of 2-acetylamino-1-chloro-3,4,6-tetra-O-acetyl-2-deoxy-cI-D gluctose was obtained as a colorless solid. To a solution of the obtained compound (78g, 0.21mol) in tetrahydrofuran (400ml) was added allyltributyltin (198ml, 0. 64mol) and 2,2'-azobisisobutyronitrile (AIBN) (3.4g, 0.02mol). The reaction mixture was heated to 80 C and stirred for 16h under argon atmosphere. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt: n-hexane=4: 1). The mixture of allyl compound (1.62g) was obtained. To a solution of the obtained mixture in aceton (lOml) was added I % HCI (6ml) and stirred for 2h. The mixture was concentrated under reduced pressure and the residue was extracted with chloroform (30ml). The organic layer was neutralized by satd. NaHCO3 and dried (Na2S04). The solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt: n-hexane=4: 1). 73g (92%) of the objective compound was obtained as a colorless solid.

MS (m/ z): 371, 330, 312, 210, 126.

IR (cm') neat: 3290, 3071, 1746, 1658, 1020.

H-NMR (CDCI3) 6: 1.47 (3H, s), 1.63 (3H, s), 1.66 (3H, s), 1.67 (3H, s), 1.99 (1H, m), 2.19 (1H, m), 3.94 (1H, m), 4.26 (1H, dd, J=3, 5Hz), 4.37 (2H, m), 4.38 (1H, m), 5.01 (2H, m), 5.17 (1H, d, J=7Hz), 5.43 (1H, t, J=3Hz), 5.68 (1H, m), 6. 19 (1H, s).

Referencial Example 10 The preparation of 3-(2-acetylamino-3,4-di-0-acetyl-2-deoxy-a-D- glucopyranosyl)-l- prpene (compound lb-4)

TBDMSOH

AcO,,, AcO'V NHAc To a solution of the acetate compound (73g, 0.2mol) obtained from the above mentioned Referencial Example 9 in methanol (400ml) was added sodium methoxide (5g, 0.95mmol) at 0 C and stirred for 90min. After the reaction was completed, the reaction mixture was neutralized by IR-120 resin, filtrated and concentrated. 54.8g of the triol compound was obtained as a colorless solid. To a solution of the obtained triol compound (54.8g, 224mmol) in N,N-dimethylformamide (224ml) was added imidazole (30.8g, 448mmol), tert-butyldimethylsilyl chloride (40.5g, 268mmol) and dimethylaminopyridine (2.7g, 22.4mmol) and the mixture was stirred for 70h at 35 C.

The reaction mixture poured into water and extracted with chloroform. The organic layer was neutralized by said. NaHCO3 and dried (Na2SO4). The solvent was removed under reduced pressure and 120g of the silyl compound was obtained. To the obtained silyl compound was added pyridine (108ml, 1. 34mol), acetic anhydride (84.7ml, 0.89mol) and dimethyaminopyridine (13.7g, 0.1 lmol). The reaction mixture was stirred for 1 h. Afire the reaction was finished, the mixture was poured into water and extracted with ethyl acetate. After drying (Na2SO4), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt: n-hexane=2: 1). 33.4g (35%) of the objective alcohol compound was obtained as a colorless oil.

MS(m/z): 428, 386, 326, 117.

H-NMR (CDC13) b: 0.84 (9H, s), 1.92 (3H, s), 2.00 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.18-2.25 (1H, m), 2.33-2.39 (1H, m), 3.69 (2H, s), 4.04-4.20 (3H, m), 4. 93-5.11 (4H, m), 5.71-5.86 (2H, m).

Referencial Example 11 The preparation of 3-(2-acetylamino-3,4-di-0-acetyl-2-deoxy--D- glucopyranosyl)-l- propene (compound lb-6) HOH2i AcO,,, o Act / NHAc A solution of the silyl compound (leg, 23. lmmol) obtained from the above mentioned Referencial Example 10 in a mixture of tetrahydroLrane (1 Oml), acetic acid (30ml) and water (lOml) was stirred for 62h at 30 C. The reaction mixture was poured into water and extracted with chloroform, the organic extract was neutralized by satd.NaHCO3, and dried over Na2SO4. The solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt). 7.5g (100%) of the f ( objective alcohol compound was obtained as a colorless solid.

MS (m/ z): 330, 288, 268, 228, 126, 101.

IR (cm') KBr: 3352, 2926, 1734, 1656, 1233.

H-NMR (CDC13) 6: 1.96 (3H, s), 2.05 (3H, s), 2.08 (3H, s), 2.28-2.35 (1H, m), 2.43 2.49 (1H, m), 3.57-3.69 (3H, m), 4.26-4.32 (2H, m), 4.97 (1H, dd, ]=8.3, 8.3Hz), 5.10 5.19 (3H, m), 5.78-5.86 (2H, m).

Referencial Example 12 The preparation of 3-(2-acetylamino-3,6-di-O-acetyl-2-deoxy-a-D- glucopyranosyl)-1- propene (compound lb-7) AcOH HO,, AcO NHAc A mixture ofthe primary alcohol compound (7.5g, 23.1mmol) obtained from the above mentioned Referencial Example 11 and acetic acid (0.75ml) in toluene (75ml) was stirred for 18h at 80 C. The mixture was concentrated under reduced pressure and the resulting residue was purified by silicagel column chromatography (AcOEt). 5.24g (70%) of the objective compound was obtained as a colorless oil.

MS (m/ z): 330, 228, 209, 168, 126, 101, 83.

IR (cm') KBr: 3352, 1734, 1656, 1233.

H-NMR (CDC13) :1.95 (3H, s), 2.13 (3H, s), 2.14 (3H, s), 2.30-2.36 (1H, m) , 2.40 2.49 (1H, m), 3.55-3.59 (1H, m), 3.66-3.70 (1H, m), 4.18 (1H, dd, J=12.2, 2.9Hz), 4.22 4.29 (2H, m), 4.51 (IH, dd, J=12.2, 4.9Hz), 4.99 (IH, dd, J=8.3, 9.7Hz), 5.10-5.16 (2H, m), 5.72-5.82 (1H, m), 5.90 (1H, d, J=8.3Hz).

Example 1

The preparation of 2-acetylamino-1,3,4,6-tetra-O-acetyl-2-deoxy-a-Dgalactopyranose (compound la-7) AcOH2i AcO: OAc NHAc To a solution of cesium acetate (13. 7g, 71.5mmol) in dimethylsulfoxide (lSml) was added a solution of the triLrate compound (7.83g) obtained from the above mentioned Referencial Example 4 in dimethylsulfoxide (15ml). After the mixture was stirred for 3h, the mixture was concentrated under reduced pressure. The residue was poured into water and extracted with dichloromethane, then dried over Na2SO4. The solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt). 3.4g (61 %) of the objective compound was obtained as a colorless oil.

MS (m/ z): 389, 330, 287, 241, 114.

IR(cm')neat: 1746, 1656, 1218, 1128.

lH-NMR (CDC13) 6: 1.88 (3H, s), 1.96 (3H, s), 1.97 (3H, s), 2.10 (3H, s), 3.99 (1H, dd, J=11.2, 6.6Hz), 4.04 (1 H. dd, J=11.2, 6.8Hz), 4.20 (1H,ddd, J=6.8, 6.6, O.9Hz), 4.63 (1H, ddd, J=11.6, 9.0, 3.6Hz), 5.14 (1H, dd, J=11.7, 3.2Hz), 5.36 (1H, dd, J=3.1, 0.7Hz), 5.82 (1H, d, ]=9.OHz), 6.15(1H, d, 1=3.6Hz).

Example 2

The preparation of 3-(2-acetylamino-3,4,6-tetra-0-acetyl-2-deoxy--Dgalactopyranosyl)- 1 -propene (compound 1 a-8) AcOH2 AcO^ o AcO NHAc The alcohol compound (13.2g, 40.1mmol) obtained from the above mentioned / Referencial Example 12 was disolved in a mixture of dichloromethane (130ml) and pyridine(l3ml). Then triflic anhydride (8.1ml, 48.1mmol) was added dropwise at 40 C and stirred for 4h. The mixture was poured into ice cold water and extracted with dichloromethane and the organic extract was washed with 10%HCI and dried over Na2SO4. The solvent was removed under reduced pressure and 16. lg of trifrate compound (16.1g) was obtained. A solution ofthe obtained triflate compound (16.1g) in dimethylsulfoxide (60ml) was added to a solution of cesium acetate (20.0g, 104mmol) in dimethylsulfoxide (lOOml). After the mixture was stirred for 3h, the mixture was concentrated under reduced pressure. The residue was poured into water and extracted with dichloromethane, then dried over Na2SO4. The solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt). l O.9g (84%) of the objective compound was obtained as a colorless solid.

Example 3

The preparation of 2-(2-acetylamino-3,4,6-tri-0-acetyl-2-deoxy-cI-Dgalactopyranosyl) 1-ethylazide (compound 2-la) AcO H2C AcCl;p Ac, N3 AcH To a solution of the alcohol compound (2.33g, 6.22mmol) obtained from the above mentioned Referencial Example 8 in tetrahydrofran (62ml) was added diphenylphosphoryl azide (2.68ml, 12.4mmol) and triphenylphosphine (3.25g, 12.4mmol). The solution was cooled to 0 C, diisopropyl azodicarboxylate (2.44ml, 12.4mmol) was added slowly to the solution and the mixture was stirred for lh. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silicagel column chromatography (AcOEt:benzene=1: l). 1.92g (77%) of the objective compound was obtained as a colorless oil. (

MS (m/e): 401, 357, 313, 277, 166, 101.

IR (cm') neat: 3244, 3046, 2092, 1737, 1656.

H-NMR(CDC13) :1.66-1.72 (1H, m), 1.83-1.89 (1H, m), 2.00 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 2.12 (3H, s), 3.35-3.39 (2H, m), 4.02-4.12 (2H, m), 4.31-4. 35 (2H, m), 4.45 (1H, ddd, ]=8.3, 8.3, 4.9Hz), 5.14 (1H, dd, J=8.8, 3.4Hz), 5.33 (1H, dd, ]=3.4, 3.4Hz), 6.23 (1H, d, ]=8.3Hz).

Example 4

The preparation of 2-(2-acetylamino-3,4,6-tri-O-acetyl-2-deoxy-a-Dgalactopyranosyl) 1-ethylamine (compound 2-2a) AcOH29

I

* AcO;- , NH2 Achy The azide compound (982mg, 2.46mmol) obtained from the above mentioned Example 3 was disolved in methanol (lOml), acetic acid (0.lml) and 10% Pd-C (98mg) were added to the solution. The reaction mixture was stirred for 88h under an atmosphere of Ha. The suspension was filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silicagel column chromatography (CHC13:MeOH:H:O=8:2:0.2). 662mg (72%) of the objective compound was obtained as a colorless oil.

MS (m/e): 374, 317, 256, 166, 115.

IR (cm') neat: 3280, 2932, 1740, 1656.

H-NMR (CD30D) 5: 1.75-1.79 (1H, m), 1.97-2.01 (1H, m), 1.99 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.09(3H, s), 3.02-3.04 (2H, m), 4.07 (1H, dd, J=11.7, 4.4Hz) , 4.18 4.19(1H, m), 4.31-4.45 (3H, m), 5.12 (1H, dd, J=9.3, 3.4Hz), 5.42 (1 H. dd, J=3.4, 3.4Hz).

Example 5 lK

The preparation of t-butyl-2-({2-[2-acetylamino-3,4,6-tri-0-acetyl-2deoxy-a-D galactopyranosyl] ethyl}amino) acetate (compound 2-3a) AcOH29 Acp A o:>'-'' NH-'' AcHN To a solution of the amine compound (59Omg, 1. 58mmol) obtained from the above mentioned Example 4 in dichloromethane (15.8ml) was added triethylamine (0.33ml, 2.73mmol) and tert-butyl bromoacetic acid (0.35ml, 2.37mmol). After the mixture was stirred for 2h at 60 C, the mixture was concentrated under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt:MeOH = 10: 1). 225mg (27%) of the objective amide compound was obtained as a colorless oil.

MS (m/e): 489, 414, 387, 224, 164, 88.

IR (cm') neat: 3328, 1740, 1656, 1233.

H-NMR (CD30D) 6: 1.54 (9H, s), 1.61-1.65 (1H, m), 1.96-1.98 (1H, m), 1.96 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.10 (3H, s), 2.62-2.77 (2H, m), 3.28-3.37 (2H, m), 4.10 (1H, dd, J=10.7, 4.9Hz), 4.16 (1H, ddd, J=8.3, 8.3, 2.4Hz), 4.22 (1H, ddd, J=8.3, 8.3, 3.4Hz), 4.24-4.32 (1H, m), 4.40 (1H, dd, J=9.8, 4.9Hz), 5.12 (1H, dd, ]=9.8, 2. 9Hz), 5.40 (1H, dd, J=2.9, 2.9Hz).

Example 6

The preparation of t-butyl 2-(N-{2-[2-acetylamino-3,4,6-tri-0-acetyl-2deoxy-o-D galactopyranosyl] ethyl} acetamino) acetate (compound 2-4a) AcOH29 AcO' p AcO:J'. -COOtBu Ac O AcHN The amine compound (lOOmg, 0. 205mmol) obtained from the above mentioned Example 5 was disolved in pyridine (lml), acetic anhydride (0.039ml, 0.41mmol) and dimethylaminopyridine (12mg, 0.103mmol) were added to the solution. After the solution was stirred for lh, the mixture was poured into water and extracted with ethyl acetate, the organic layer was washed with said. CuSO4 and brine, and dred over Na2SO4. The solvent was removed under reduced pressure, then the resulting residue was purified by silicagel column chromatography (AcOEt:MeOH=20: 1). 100mg (92%) of the objective compound was obtained as a colorless oil.

MS (m/e): 530, 487, 429, 387, 222, 57.

IR (cm') neat: 2968, 1740, 1650, 1230.

H-NMR (CD30D) 6: 1.45 (9H, s), 1.73-1.77 (IH, m), 1.92-1.97 (1H, m), 1.97 (3H, s), 2.00 (3H, s), 2.03 (3H, s), 2.10 (3H, s), 2.16 (3H, s), 3.40-3.60 (2H, m), 3.89-4.30 (6H, m), 4.40-4.44 (IH, m), 5.07-5.14 (1H, m), 5.38-5.40 (1H, m).

Example 7

The preparation of 2-(N-{2-[2-acetylamino-3,4,6-tri-O-acetyl-2-deoxy--Dgalactopyranosyl] ethyl} acetamino) acetic acid (compound 2-Sa) AcOH2,C AcO9 oats,,,, ,COOH AcHh A mixture of the ester compound (9Omg, 0.17mmol) obtained from the above mentioned Example 6 and trifluoroacetic acid (0. 2ml) in dichloromethane (lml) was stirred for 3h. The mixture was concentraed under reduced pressure and the resulting residue was purified by silicagel column chromatography (CHCI3:MeOH:AcOH=18:2:1). 70mg (87%) ofthe objective compound was obtained as a colorless oil.

MS (m/e): 474, 429, 314, 222, 69.

IR (cm') neat: 1740, 1370, 1230.

H-NMR (CD30D) b: 1.76-1.82 (1H, m), 1.92-1.97 (1H, m), 1.99 (3H, s), 2.03 (3H, s), 2.11 (3H, s), 2.14 (3H, s), 2.17 (3H, s), 3.70-3.52(2H, m), 4.00-4.30 (6H, m), 4.43 4.46(1H, m), 5.08-5.14 (1H, m), 5.38-5.40 (1H, m), 4.43-4.46 (1H, m), 5. 08-5.14 (IH, ! m), 5.38-5.40 (1H, m).

Example 8

The preparation of 3-(2-acetylamino-3,4-tri-O-acetyl-2-deoxy-a-Dgalactopyranosyl)-l- phenylthioethane (compound 4-1) CH2OAc AcO: AcO^.; ASPS NHAc The compound (0.25g, 0.67mmol) obtained from the above mentioned Example 7 was disolved in pyridine (3ml), tributylphosphine (0.42ml) and diphenyldisulfide (0.32g) were added to the solution. The mixture was stirred for 3h at 60 C under argon atmosphere. The reaction mixture was extracted with ethyl acetate and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW- 200, AcOEt:n-Hexane=10:1). 0.18g (56%) of the objective thiophenyl compound was obtained as a colorless oil.

Mass (m/e): 467 (M).

IH-NMR(CDCl3) b: 1.63 (lH, m), 1.94 (3H, s), 1.96 (1H, m), 2.03 (3H, s), 2.06 (3H, s), 2.56 (3H, s), 2.91 (1H, m), 3.24 (1H, m), 3.98 (lH, m), 4. 51 (2H, m), 4.32 (1H, m), 4.42 (2H, m), 5.07 (1H, dd, ]= 4, 9Hz), 5.29 (1H, t, J=3Hz), 5.55 (1H, d J=7Hz), 7.21 7.38 (5H, m).

Example 9

The preparation of 3-(2-acetylamino-3,4-tri-O-acetyl-2-deoxy-a-Dgalactopyranosyl)-l- phenylsulutenylethane l (compound 4-2) CH2OAc Act AcO:; SOPh NHAc To a solution ofthe compound (0. 14g, 0.29mmol) obtained from the above mentioned Example 8 in dichloromethane (2ml) was slowly added a solution of 3- chloroperoxybenzoic acid in dichloromethane (l.Oml) at -78 C. After stirring for 30min, diethylether (lOml) and 10% NaOH (lml) was added to the reaction mixture and the mixture was stirred for 15min. The organic layer was separated and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure.

O.15g (99%) of the objective compound was obtained as a colorless oil.

Mass (m/e): 483(M).

H-NMR(CDCI3) 5: 1.89 (IH, m), 1.91 (3H, s), 1.95 (3H, s), 2.05 (3H, s), 2. 09 (1H, m), 1.96 (1H, m), 2.58 (1H, m), 2.80 (1H, t, J=8Hz), 3.01( 1H, m), 3.80 (1H, m), 3.95 4.10 ( 2H, m), 4.20 (1H, m), 4.35 (1H, m), 4.56 (2H, m), 5.10( 1H, dd, J= 4, 9Hz), 5.27 (1H, t, ]=3Hz), 6.50 (1H, d, ]=8Hz), 7.4-7.60 (5H, m).

Example 10

The preparation of 3-(2-acetylamino-3,4-tri-0-acetyl-2-deoxy-a-Dgalactopyranosyl) 1-vinylene (compound 4-3) CH2OAc AcO> AcO'J '' NHAc A mixture of the compound (0.14g, 0.29mmol) obtained from the above mentioned Example 9 and diisopropylethylamine (0.09ml) in toluene (2ml) was refluxed for 18h.

After the reaction mixture was cooled to room temperature, the mixture was extracted l with ethyl acetate and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt). 0.07g (70%) of the oily objective compound was obtained as a colorless oil.

Mass (m/e): 357 (M+), 298, 255, 165, lOl(BP), 59.

H-NMR(CDCI3) 5: 1.96 (3H, s), 2.05 (3H, s), 2.06 3H, s), 2.16 (3H, s), 4. 14 (3H, m), 4.62 (1H, m), 4.76 (1H, m), 5.03 (1H, dd, J=4, lOHz), 5.35 (1H, d, J=2Hz), 5.45 (3H, m), 5.95 (1H, m).

Example 11

The preparation of 3-(2-acetylamino-3,4-tri-0-acetyl-2-deoxy-a-Dgalactopyranosyl)-1- carbaldehyde (compound 4-4) CH2OAc AcO: AcO CHO NHAc To a mixture of the compound (0. 07g, 0.20mmol) obtained from the above mentioned Example 10, in tetrahydroDran (2ml) and water was added NaIO4 (0.16g, 0.78mmol) and 4% OsO4 solution (O.Olml). After the mixture was stirred for 4h, the reaction mixture was extracted with ethyl acetate and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure. 0.705g (69.6%) of the objective aldehyde compound was obtained as a colorless oil.

Mass (m/e): 360(M+'), 330, 300, 199, 139, 97(BP), 59.

IH-NMR(CDCl3) b: 1.98 (3H, s), 2.06 (3H, s), 2.02 (3H, S), 2.17 (3H, s), 3.92 (1H, t, J=7Hz), 4.20 (2H, m), 4.59 (1H, d, J=Hz), 4.80 (1H, m), 5.09 (1H, dd, J=3, 9Hz), 5.38 (1H, d, J=3Hz), 6.22 (1H, d, J=9Hz), 9.83 (1H, S). i

Example 12

The preparation of 3-(2-acetylamino-3,4-tri-0-acetyl-2-deoxy-a-Dgalactopyranosyl)-l- methanol (compound 4-5) CHzOAc Act

OH NHAc

A mixture of the compound (0.77g, 1.85mmol) obtained from the above mentioned Example 11 and sodium borohydride (O.lg, 2.78mmol) in methanol (lOml) was stirred for lOmin at 0 C. The reaction mixture was poured into said. NHCI and the mixture was extracted with dichloromethane, the organic layer was washed with water and brine.

After drying (MgS04), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt). 0.25g (36%) of the objective alcohol compound was obtained as a colorless oil.

Mass (m/e): 362(M+'), 330, 300, 199, 139, 97(BP), 59.

H-NMR(CDC13) 5: 1.98 (3H, s), 2.06 (3H, s), 2.02 (3H, s), 2.17 (3H, s), 3. 92 (1H, t, J=7Hz), 4.20 (2H, m), 4.59 (1H, d, J=3Hz), 4.80 (1H, m), 5.09 (1H, dd, J=3, 9Hz), 5.38 (1H, d, J=3Hz), 6.22 (1H, d, J=9Hz), 9.83 (1H, s).

Example 13

The preparation of 3-(2-acetamino-3,4,6-tetra-0-acetyl-2-deoxy-a-Dgalactopyranosyl) 1 -ethylvinyloxyformate (compound 5-la) i' AcO\ &H2OAC Ace-CO : : ACHN too - O The compound (0. 09g, 2.27mmol) obtained from the above mentioned Example 8 was disolved in tetrahydrofuran (5ml), allyl chloroformate (0.026ml, 2.5mmol) was added to the solution in the presence of pyridine (lml). After the solution was stirred for 30min, the reaction mixture was extracted with ethyl acetate and the organic layer was washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure and the resulting residue was purified by silicagel column chromatography (BW-200, AcOEt:n-hexane=4: 1). 0.08g (70%) of the objective compound was obtained as a colorless oil.

IR(cm-)KBr: 1743, 1392, 1245, 1020.

tH-NMR(CDC13) 6: 1.58 (1H, m), 1.99, (3H, s), 2.07 (3H, s), 2.08 (3H, s), 2.12 (3H, s), 4.12 (2H, m), 4.19-4.35 (4H, m), 4.38 (1H, m), 4.48 (1H, m), 4.62 (2H, d, J=6Hz), 5.13 (1H, dd, 1=3, 8Hz), 5.27-5.39 (3H, m), 5.64 (1H, d, ]=8Hz).

Example 14

The preparation of 3-(2-acetamino-3,4,6-tetra-O-acetyl-2-deoxy-a-Dglactopyranosyl) 1 -ethoxyprop-2 -one (compound 5-2a) Act CH2OAc

A

AcO: p

ACHN

The compound (0.07g, 0.15mmol) obtained from the above mentioned Example 13 was disolved in benzene (2ml), Pd(OAC): (0.7mg) and triphenylphosphine (4mg) were / added to the solution under argon atmosphere. After the mixture was stirred for 2h at 70 C, the mixture was concentrated. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt:hexane =4: 1). 0.045g (72. 3%) of the objective compound was obtained as a colorless oil.

Mass (m/e): 415(M), 358, 314, 277, 181, 152, 101, 59.

IH-NMR(CDCl3) 6: 1.82 (1H, m), 1.91 (1H, m), 1.97 (3H, s), 2.05 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 3.50 (2H, m), 3.97 (2H, d, J=3Hz), 4.06 (2H, m), 4.22 (1H, m), 4.28 (lH, m), 4.50 (1H, m), 5.12 (1H, dd, J=3,5Hz), 5.15 (1H, dd, J=2,7Hz), 5. 30 (IH, m), 5.76 (1H, d, I=8Hz), 5.90 (1H, m).

Example 15

The preparation of 2-(2-acetylamino-3, 4,6-tri O-acetyl-2-deoxy--Dglactopyranosyl) 1-[2-(benzylamino) ethoxy] ethane (compound 5-3a) AcO CH 20Ac AcC::o : AcHN 0W NHBn A solution of the compound (0.69g, 1.65mmol) obtained from the above mentioned Example 14 in methanol (Sml) and dichlorometane (Sml) was ozonized at -78 C. After the reaction was completed, dimethylsulfide was added to the mixture and the solution was stirred at room temperature. The mixture was concentrated and 0.69g (99%) of the aldehyde was obtained. To a solution of the obtained aldehyde in dichloromethane (Sml) was added benzylamine (0.22ml). After stirring for 1 S min. sodium triacetoxyborohydride (0.5g) was added to the mixture and the mixture was stirred for 12h. The reaction mixture was extracted with chloroform and the organic layer was washed with water and brine. After drying (Na2SO*), the solvent was removed under reduced pressure and the resulting residue was purified by silicagel column chromatography (BW-200, chloroform:methanol=20:1). O. 51g (64.4%) ofthe objective compound was obtained as a colorless oil.

Mass (m/e): 449 (M-NHAc), 383, 192, 120, 91.

IR Acme) KBr: 3290, 2950, 1740, 1660, 1378, 1230, 1000.

H-NMR(CDC13) 5: 1.87 (1H, m), 1.95 (3H, s), 1.97 (1H, m), 2.05 (6H, m), 2. 19 (3H, s), 2.81 (2H, t, J=5.OHz), 3.51 (2H, m), 3.57 (2H, t, J=5.OHz), 3. 84 (1H, s), 4.03 (1H, m), 4.08 (1H, m), 4.38 (1H, m), 4.50 (1H, m), 5.13 (IH, dd, ]=8.3, 2.1Hz), 5.31 (1H, t, ]=3Hz), 5.85 (1H, d,l=8.0Hz), 7.32 (5H, m).

Example 16

The preparation of t-butyl 2-[(2-{2-[2-acetylamino-3, 4, 6-tri-0-acetyl-2deoxy-0-D glactopyranosyl] ethoxy} ethyl) benzylamino] acetate (compound 5-4a) AcO CH 20Ac Ac(f AcHN 0W hIBn CO of Bu A mixture of the compound (0. 5 Ig, 1.03mmol) obtained from the above mentioned Example 15 and tertbutyl bromoacetic acid (0.3ml) in dichloromethane (5ml) was stirred for 16h at 60 C. After the reaction was completed, triethylamine was added to the mixture and stirred for 15min. The mixture was extracted with ethyl acetate and washed with water and brine. After drying (MgS04), the solvent was removed under reduced pressure and the resulting residue was purified by silicagel column chromatography (ABW-200, CHC13:MeOH=10: 1). 0.23g (36.9%) of the objective compound was obtained as a colorless oil.

IH-NMR(CDC13) b: 1.45 (9H, s), 1.75 (1H, m), 1.84 (1H, m), 1.97 (3H, s), 2.05 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 2.86 (2H, t, J=6.0Hz), 3.29 (2H, s), 3.40-3.60 (4H, m), 3.83 (2H, s), 4.02 (1H, m), 4.11 (1H, m), 4.20 (1H, m), 4.32 (1H, m), 4. 50 (1H, m), 5.11 (1H, dd, ]=6.0,2.0Hz), 5.31 (1H, t, ]=3Hz), 5.70 (1H, d, ]=6.0Hz), 7.33 (5H, m).

Example 17

The preparation of t-butyl 2-[(2- {2-[2-acetylamino-3, 4, 6-tri-0-acetyl2-deoxy-a-D glactopyranosyl] ethoxy} ethyl) amino] acetate (compound 5-5a) AcO,wCH 20Ac Act: P AcHN 0W HAc COOtBu The compound (0.21g, 0.34mmol) obtained from the above mentioned Example 16 disolved in methanol (lOml), acetic acid (0.5ml) and 10% Pd-C (20mg) were added to the solution. The reaction mixture was stirred for 3h under an atmosphere of Ha, then the suspension was filtered through celite and the filtrate was concentrated. 0.18g (99%) of the objective compound was obtained as a colorless oil.

Mass(m/e) :431,373,314,91,78.

H-NMR(CDC13) 5: 1.45 (9H, s), 1.91 (1H, m), 1.95 (1H, m), 2.01 (3H, s), 2. 05( 6H, s), 2.05 (3H, s), 2.50 (1H, s), 2.87 (2H,t,J=6.0Hz), 3.40-3.70 (4H, m), 4.10 (2H, m), 4.204.41 (3H, m), 4.50 (2H, m), 5.20 (1H, dd, I=6.0, 2.0Hz), 5.33 (1H, t, ]=3Hz), 6.05 (1 H. d, J=6.0Hz).

Example 18

The preparation of t-butyl 2-[(2-{2-[2-acetylamino-3, 4, 6-tri-0-acetyl-2deoxy-a-D glactopyranosyl] ethoxy} ethyl) acetylamino] acetate (compound 5-6a) ACO.:CH2OAc Act AcHN CO OtBu To a solution of the compound (O.18g, 0.34mmol) obtained from the above mentioned Example 17 in dichloromethane (5ml) was slowly added acetyl chloride (0.36ml) in the presence of diisopropylethylamine (O. Iml). After the solution was stirred for 2h, the mixture was concentrated under reduced pressure and the resulting residue was purified by silicagel column chromatography (BW-200, AcOEt). 0.13g (66.5%) of the objective compound was obtained as a colorless oil.

Mass (m/e): 517 (M-Bu), 501, 431, 358, 314, 199, 144, 99, 72.

IH-NMR(CDCl3) 6: 1.47 9H, s), 1.83 (IH, m), 1.93 (1H, m), 1.86 (3H, s), 1. 91-2.20 (15H, m), 3.40-3.60 (1OH, m), 3.96-4.40 (9H, m), 4.50 (1H, m), 5.18 (1H, dd, ]=6.0, 3.0), 5.30 (1H, t, ]=3.0Hz), 5.75 (1H, m).

Example 19

The preparation of 2-[N-(2- {2-[2-acetylamino-3, 4, 6-tri-0-acetyl-2deoxy-a-D glactopyranosyl] ethoxy} ethyl) benzylamino] acetic acid (compound 5-7a) Ac:CH2OAc AcC:_:O AcHN NAc \COOH A mixture of the compound (0.15g, 0.26mmol) obtained from the above mentioned Example 18 and trifluoroacetic acid (0.4ml) in dichloromethane (2ml) was added to the mixture and stirred for 3h. The reaction mixture was concentrated and 0.13g (66.8%) of the objective compound was obtained as a colorless oil.

Mass (m/e): 517 (M-Bu), 501, 431, 358, 314, 199, 144, 99, 72.

IH-NMR(CDCI3) 6: 0.89 (1H, m), 0.97 (1H, m), 2.00 (3H, s), 2.03 (3H, s), 2.04 (3H, s), 2.15 (3H, s), 2.19 (3H, s), 3.40-3.65 (6H, m), 3.95-4.18 (3H, m), 4.30 (2H, m), 4.46 (1H, m), 5.10 (1H, d, J=4.0Hz), 5.18 (1H, m).

Example 20

The preparation of O-(methyl 5-acetylamino- 4,8,9-tetra-O- acetyl-3,5dideoxy--D- glycero-D-galacto-2-nuno pyranosynate)-(2 6)-2-(2-acetylamino-3,4-di-O- acetyl-2deoxy-a-D-galctopyranosyl)-1-(prop-2-enyloxy) ethane (compound 6-2a) OAC coOMe Act 1 OAc l AcNH\-O\ H:'o HOW; 'I/ O AcHN A mixture ofthe alcohol compound (173mg, 0.66mmol) obtained from the above mentioned Example 14 and MS4A in tetrahydrofuran (lOml) was added di-tert- butylpyridine (0.29ml) and AgOTf (337mg) and the mixture was stirred for 30min.

After cooling to -78 C, a solution of the sialyl chloride (670mg, 0. 66mmol) in tetrahydrofurane (8ml) was added dropwise to the mixture and the mixture was stirred for 28h. The suspension was filtered through Celite and the filtrate was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (CHCI3:MeOH= 10: 1). 8 lmg (18%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e): 785(M).

IR (cm'): 3340, 2944, 1744, 1656.

H-NMR (CDC13) 6: 1.88 (3H, s), 1.99 (3H, s), 2.03 (3H, s), 2.04 (3H, s), 2.13 (3H, s), 2.14 (3H, s), 2.58 (1H, dd, ]=17.4, 4.4Hz), 2.97 (1H, d, ]=3.9Hz), 3.99-4. 10 (4H, m), 4.14-4.27 (2H, m), 4.34 (1H, dd, ]=12.2, 2.5Hz), 4.40-4.43 (1H, m), 4.84- 4.91(1H, m), 5.21-5.39 (4H, m), 5.87-5.96 (1H, m), 6.74 (1H, d, ]=5.9Hz).

Example 21

The preparation of O-(methyl 5-acetylamino-3,5-dideoxy--D-glycero-Dgalacto-2- nuno pyranosinate)-(2 6)-2-(2-acetylamino-2-deoxy-a-D-galactopyranosyl)- I -(prop-2- enyloxy) ethane (compound 6-3a)

OH COOH

ACNE-OX

HO Hat HOE, 'I/ 0'- AcHN A mixture of the compound (21 ma, 0.027mmol) obtained from the above mentioned Example 20 and 2% K2CO3 (3ml) in methanol (9ml) was stirred for 20h. The reaction mixture was neutralized by 1% HCI, then the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silicagel column chromatography (PR-18, H:O:AcOH=100:1). 13mg (81%) ofthe objective compound was obtained as a colorless oil.

MS (ESI, m/e): 579 (M-H)+ IR (cm' ) neat: 3268, 1638, 1566.

H-NMR (CD30D) 6: 1.60-1.80 (2H, m), 2.01-2.05 (1H, m), 2.01 (3H, s), 2.05 (3H, s), 2.85-2.88 (1H, m), 3.50-3.60 (3H, m), 3.65-3.76 (5H, m), 3.81-3.95 (5H, m) , 4.02 (2H, d, J=5.4Hz), 4.21-4.33 (2H, m), 5.18-5.21(1H, m), 5.29-5.34 (IH, m), 5.91-6.00 (1H, m).

Example 22

The preparation of O-(methyl 5-acetylamino-3,5-dideoxy--D-glycero-Dgalacto-2nonuropysanosinate)-(2)6)-2-[N-(2-{2-[2-acetylamino-3,4,6-tri-0-acetyl-2deoxy-cI- D-galactopyranosyl]ethoxy} ethyl)acetylamino]-N-(2- {2-[2-(2oxyoethoxy)ethoxy] ethoxy} ethyl)acetamide 0 6-3b L1 HO | OH COOH

ACNE-O

HO o AC HOE '-O H O Of

ACHN

To use of the compound obtained from the following mentioned Example 32, the objective compound was obtained according to the method described in Example 20-21.

MS (ESI, m/e): 877 (M+Na)+ IR (cm') KBr: 3400, 2950, 1650, 1400, 1125.

lH-NMR(CD30D) 6: 1.62 (2H, m), 2.01 (3H, s), 2.04 (3H, s), 2.20 (3H, s), 2.81 (1H, dd, ]=2.8Hz), 3.40-3.98 (30H, m), 4.18 (4H, m), 4.21 (4H, m), 5. 20 (1H, d, ]=12Hz), 5.19 (1H, d, ]=12Hz), 6.98 (1H, m).

Example 23

The preparation of the following compound.

HO: '-O

NHAC

To a solution of the compound (O.12g, 0.41 mmol) obtained from the above mentioned Example 14 in acetonitrile (lOml) was added benzaldehyddimethylacetal (0.12ml) and p-toluensulfonate (3.8mg) and the mixture was stirred for 6h at 60 C under argon atmosphere. After cooling to room temperature, the mixture was extracted with ethyl acetate and the organic layer was washed with water and brine. After drying (MgSO4), the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt). 0.10g (64.4%) of the objective acetal compound was obtained as a colorless oil.

MS (ESI, m/e), 377 (M)+ H-NMR (CDC13) 6: 1.96 (2H, m), 1.99 (3H, s), 3.433.60 (3H, m), 3.75 (1H, d, J=3Hz), 4.01 (3H, m), 4.11 (2H, m), 4.42 (2H, m), 5.20 (2H,, dd, J=3, 8Hz), 5.60 (1H, s), 5.90 (1H, m), 6.20 (1H, d, J=3Hz), 7.30-7.56 (5H, m).

Example 24

The preparation of the following compound. in Act

AC0 4 0'- AcO NHAc A mixture of the compound (100mg, 0.4 lmmol) obtained from the above mentioned Example 23 and MS4A in dichloromethane (lOml) was added di tert-butylpyridine (0.12ml) and AgOTf (0.14g) and the mixture was stirred for 30min. After cooling to 78 C, a solution of the galactose derivatives (0.22g 0.41 mmol) in dichloromethane was added dropwise to the mixture. After the reaction was completed, the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (BW-200, AcOEt). 0.10g (64.4%) of the objective compound was obtained as a colorless oil.

MS (ESI, mte): 707 (M)+ H-NMR (CDC13) b: 1.96 (2H, m), 1.99 (3H, s), 3.433.60 (3H, m), 3.75 (1H, d, J=3Hz), 4.01 (3H, m), 4.11 (2H, m), 4.42 (2H, m), 5.20 (2H,, dd, J=3, 8Hz), 5.60 (IH, s), 5.90 (1H, m), 6.20 (1H, d, J=3Hz), 7.30-7.56 (5H, m).

Example 25

The preparation of the following compound.

HO

HO>: Her,' HO 4 "-O

HO NHAC

A solution of the compound (0.11 g, 0.16mmol) obtained from the above mentioned Example 24 in 80% acetic acid was heated to 70 C and stirred for 2h. The solvent was removed under reduced pressuremixture and the obtained dial compound was dissolved in methanol (5ml). Sodium methoxide (2mg) was added to the solution and the mixture was stirred for 2h at room temperature. The reaction mixture was neutralized by Amberlite IR- 120 and filtrated and the filtrate was concentrated under reduced pressure.

0. I g (64.4%) of the objective compound was obtained.

MS (ESI, m/e): 451 (M)+ H-NMR (CDC13) 5: 1.75 (1H, m), 2.00 (IH, m), 2.13 (3H, s), 3.16 (1H, m), 3.55 (2H, m), 3.60 (1H, m), 3.69-3.82 (3H, m) 3.98 (2H, m), 4.01-4.10 (5H, m), 4.30 (1H, m), 4.80 (3H, m), 5.11 (1H, m), 5.18 (2H, m), 5.23 (1H, m), 5.40 (1H, m), 5. 90 (1H, m).

Example 26

The preparation of t-butyl 2-[N-(2-{2-[2-acetylamino-3, 4, 6-tri-O-acetyl2-deoxy-a-D glactopyranosyl] ethyl) -2-[N-(2-{2-[2-acetylamino-3, 4, 6tri-O-acetyl-2-deoxy-a-D glactopyranosyl] ethoxy} ethyl) acetylamino] acetate (compound 8-3a) HrCH2O H H; AcH HaN_COOtBu nHAc Ac A mixture of carboxylic acid (67mg, 0.14mmol) and amine (69mg, 0.14mmol) obtained from above mentioned Example 7 and 5 were disolved in acetonitorile (1.4ml), diisopropylethylamine (0.027ml) and O-(benzotriazol-1-yl) N. N. N', N'tetramethylhydroniumtetrafluoroborate (TBTU) (SOmg) were added to the mixture.

After the reaction mixture was stirred for 24h, the mixture was poured into brine and extracted with chloroform, the organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt:MeOH=10: 1). 72mg (54%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e): 944 (M).

H-NMR (CD30D) 0: 1.46 (9H, s), 1.77-1.83 (1H, m), 1.92-1.97 (1H, m), 1.90 (3H, s), 1.91 (3H, s), 1.94 (3H, s), 1.95 (3H, s), 2.00 (3H, s), 2.01 (3H, s), 2. 04 (3H, s), 2.05 (3H, s), 2.11 (3H, s), 3.41-3.68 (4H, m), 3.90-4.59 (14H, m), 5.09-5.16 (2H, m), 5.40-5.42 (2H, m)

Example 27

The preparation of 2-[N-(2-{2-[2-acetylamino-3, 4, 6-tri-O-acetyl-2-deoxya-D glactopyranosyl] ethyl) -2-[N-(2- {2-[2-acetylamino-3, 4, 6-tri-oacetyl-2-deoxy-a-D glactopyranosyl] ethoxy} ethyl) acetylamino] acetic acid (compound 8-lb)

H

HVCH2OH HC; AcH Ha:4COOH HAc Ac A solution of the ester compound (62mg, 65.7'umol) obtained from the above mentioned Example 26 and trifluoroacetic acid (0.2ml) in dichloromethane (lml) was stirred for 4h.

The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silicagel column chromatography (CHC13:MeOH:AcOH=18:2: 1). 50mg (86%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e): 888 (M+).

H-NMR (CD30D) 6: 1.77-1.82 (2H, m), 1.94 (3H, s), 1.95 (3H, s), 1.97 (3H, s), 1.99 (3H, s), 2.00 (3H, s), 2.02 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.11 (3H, s), 3.34-3.77(4H, m), 4.06-4.87 (14H, m), 5.10-5.15 (2H, m), 5.33-5.41 (2H, m).

Example 28

The preparation of t-butyl N- {2-[2-acetylamino-3, 4, 6-tri-o-acetyl-2deoxy-a-D glactopyranosyl] ethyl] -2-(N-{2-acetylamino-3, 4, 6-tri-Oacetyl-2-deoxy-a-D glactopyranosyl] ethyl} acetylamino)-N-( {N- {2-[2acetylamino-3, 4, 6-tri-O-acetyl-2 deoxy-a-D-glactopyranosyl] ethyl} -N[(N-carbamoyl)methyl]carbamoyl} methyl) acetate (compound 8-3b)

AH

H('CH2O H HC) Achy H(I - COOBu

SHAD C

p - ,..NHAc HOH2COH

OH

To a solution of carboxylic acid (48mg, 54.1 remold and amine (26.3mg, 54. 1 Wool) obtained from above mentioned Example 27 and 17 in acetonitorile (Iml) was added diisopropylethylamine (101, 59.5pmol) and O-(benzotriazol-1-yl) N. N. N', N'- tetramethylhydroniumtetrafluoroborate (TBTU) (19mg, 59.5pmol). After stirring for 38h, the mixture was poured into brine and extracted with chloroform, the organic layer was dried overNa2SO4 and the solvent was removed under reduced pressure. The resulting residue was purified by silicagel column chromatography (AcOEt:MeOH=5:1).

40mg (54%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e): 1382 (M+Na)+.

H-NMR (CD30D) b: 1.46 (9H, s), 1.73-1.83 (3H, m), 1.94-2.18 (42H, m), 3. 41-3.78 (8H, m), 4.03-4.55 (21H, m), 5.10-5.15 (3H, m), 5.40-5.42 (3H, m).

Example 29

The preparation of N-{2-[2-acetylamino-3, 4, 6-tri-O-acetyl-2-deoxy-a-Dglactopyranosyl] ethyl] -2-(N-{2-acetylamino-3, 4, 6-tri-O-acetyl-2-deoxya-D- glactopyranosyl] ethyl} acetylamino)-N-( {N- {2-[2-acetylamino-3, 4, 6- tri-O-acetyl-2- deoxy-a-D-glactopyranosyl] ethyl}-N-[(N-carbamoyl)methyl]carbamoyl}methyl) acetic acid (compound 8-lc) iAc AcO CHzOAc C)20Ac

AC_OH

o+lHAc ACO H2C: OAc OAc A solution of the ester compound (40mg, 29.5,umol) obtained from the above mentioned Example 28 and trifluoroacetic acid (0. 2ml) in dichloromethane (lml) was stirred for 16h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silicagel column chromatography (CHCl3:MeOH:AcOH=18:2:1). 18mg (47%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e) 1302 (M+).

H-NMR (CD30D) b: 1.65-1.75 (3H, m), 2.00-2.14 (42H, m), 3.31-3.60 (6H, m), 4.05-4.50 (21H, m), 5.05-5.10 (3H, m), 5.30-5.39 (3H, m).

Example30

The preparation of the following compound. AcO

CO2Ak Act OVA Sac ACE AcHN j0 AcOH (60 2: To use of the compound obtained from the above mentioned Example 20, the objective compound was obtained according to the method described in Example 26-28.

H-NMR (CD30D) 5: 1.80-2.25 (69H, m), 3.01-3.68 (50H, m), 3.90-4.58[26H, m) .

Example 31

The preparation of 2-(2-Acetylamino-3,4,6-tri-O-acetyl-2-deoxy-a-Dgalactopyranosyl)- 1 -(2- tN-[(N- {2-[2-(2-prop 2enyloxyethoxy)ethoxy]ethyl} carbamoyl)methyl]acetylamino} ethoxy)ethane (compound 8-4) AcOH2 Act AcOJ) ''-O N NQ of NHAc To a solution of carboxylic acid (23mg, 44.4pmol) obtained from above mentioned Example 19 and amine (17mg, 88.8,umol) in acetonitorile (lml) was added diisopropylethylamine (9'u1, 48.8,umol), O-(benzotriazol-1-yl) N. N. N', N' tetramethylhydroniumtetrafluoroborate (TBTU) (16mg, 48.8pmol). After the mixture was stirred for 4h, the mixture was poured into brine and extracted with chloroform, the organic layer was washed with 10% HCI and said. NaHCO3. After drying (Na2SO4), the solvent was removed under reduced pressure and the resulting residue was purified by silicagel column chromatography (AcOEt:MeOH=8: 1). 20mg (65%) of the objective compound was obtained as a colorless oil.

MS (ESI, m/e): 688 (M).

IR (cm') neat: 3286, 2860, 1743, 1650.

H-NMR (CDC13) 6: 1.80-1.86 (1H, m), 2.00 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 2.13 (3H, s), 2.18 (3H, s), 3.42-3.67 (18H, m), 4.40-4.11 (6H, m), 4.204.30 (1H, m), 4.34 4.41 (1H, m), 4.42-4.47 (1H, m), 5.10-5.14 (1H, m), 5.17-5.20 (1H, m), 5. 25-5.30 (1H, m), 5.31-5.32 (1H, m), 5.86-5.98 (1H, m).

Example 32

The preparation of 2-[N-(2-{2-[2-Acetylamino-2-deoxy--D-galactopyranosyl] ethoxy} ethyl) acetylamino]-N-}2-[2-(2-prop-2-oxyethoxy) ethoxy] ethyl} acetamide (compound 8-6) HOH27 HO: ''-O Not N'-O-0

H NHAc

A mixture of the acetate compound (19.5mg, 29.0,umol) obtained from the above mentioned Example 31 and sodium methoxide (3mg, 58.0pmol) in methanol (lml) was stirred for 1.5h at 0 C. The reaction mixture was neutralized by IR-120, filtered and the filtrate was removed under reduced pressure. 15.7mg (99%) of the objective trial compound was obtained as a colorless oil.

MS (ESI, m/e): 562(M+).

IR (cm') neat: 3272, 2932, 1636.

H-NMR (CD30D) 5: 1.68-1.78 (1H, m), 1.92-2.00 (1H, m), 1.92 (3H, s), 2.21 (3H, s), 3.64-3.78 (24H, m), 3.83-3.88 (1H, m), 4.05-4.10 (2H, m), 4.21-4.29 (2H, m), 5.19-5.22 (1H, m), 5.30-5.53 (1H, m), 5.91-6.01 (1H, m).

Example 33

The preparation of the following compound. H2OH

Hi IO H S HO- N o' o) NHAc AC O H o H He- o To a solution ofthe carboxylic acid (20mg, 57.5,umol) obtained from the above mentioned Example 7 and amine (136mg, 115,umol) in dimethylformamide (Iml) was added diisopropylethylamine (42,u1, 230pmol), HATU (87mg, 230pmol) and HOAt (16mg, 115pmol). After the mixture was stirred for 24h, the mixture was poured into brine and extracted with chloroform, the organic layer was washed with 10% HCI and said. NaHCO3. After drying (Na2SO4), the solvent was removed under reduced pressure and the resulting residue was purified by silicagel column chromatography (CHCl3:MeOH:AcOH=18:2: 1). 5mg (6%) of the objective compound was obtained as a colorless oil.

MS(ESI,m/e): 1150 H-NMR (CDC13, ppm): 51.70-2.12 (45H, m), 3.41-3.79 (15H, m), 4.09-4.58 (26H, m), 5.08-5.36 (1H, m), 5.40-5.48 (3H, m), 5.90-6.05 (1H, m).

Example 34

The preparation of the following compound. [H2OH HO)

HO: "-ONN'-O-0-NH-KLH NHAc A solution of the compound obtained from the above in methanol and dichlorometane was ozonized at -78 C. The reaction mixture was treated with dimethylsulfide and concentrated to obtain the aldehydeTo the mixture of this aldehyde and KLH in phosphate buffer was added sodium cyanoborohydride and stirred for 30h. After purified by dialysis using PBS(-), the objective glycoprotein antigen was obtained.

Example 35

The preparation of the following compound. HOH2[

4., oNN'-O-0-N- - KLH

NHAC O

The aldehyde obtained from the above mentioned Example 34 reacted with 4(4-N- maleimidomethyl)cyclohexyl-1-carbonylhydrzine to obtin a maleimide derivative. To the mixture of this compound and KLH in phosphate buffer was added sodium cyanoborohydride. After purified by dialysis using PBS(-) , the objective glycoprotein antigen was obtained.

Example 36

The preparation of 2-(2-acetylamino-3, 4, 6-tri-O-acetyl-2-deoxy-a-D galactopyranosyl)-l -(2- {N-[(N- {2-[2-(3 acetylthiopropoxy)ethoxy]ethyl} carbamoyl)methyl]acetylamino}ethoxy)ethane [H2OAc

ACE

ACOW -oNNo-OO-SAC NHAc To a solution of the olefine (22mg, 0.032mmol) obtained from the above mentioned Example 31 in dioxane (2ml) was added thioacetic acid (0.02ml) and the mixture was heated at 80 C for 6h. The solvent was removed under reduced pressure. The redsidue was purified by silicagel column chromatography (AcOEt:MeOH=9: 1). 0.02g (82%) of the objective compound was obtained.

Example 37

The preparation of 2 (2-{2-[2-acetylamino-2-deoxy--D-galactopyranosyl] ethoxy} ethyl) acetylamino] -N- } 2- [2 -(2-sulfenylpropoxy)ethoxt] ethyl} acetamide [H2OH

HI

HO '-oN (No -oO-SH

NHAC

To a solution of the compound (20mg, 0.029mmol) obtained from the above mentioned Example 36 in methanol (lml) was added sodium methoxide (2mg, 0. 058mmol) and stirred for 12h. The reaction mixture neutralized by IR-120, filtered using celite, and the solvent was removed under reduced pressure. 8mg (51%) of the objective compound was obtained.

Example 38

The preparation of the following compound. [H2OH

HOW. "-ON; N-TO-00-/KLH : H

NHAC

The compound obtained from the above mentioned Example 37 was added to maleimidated KLH with stirring and allowed to stand over 2h at 4 C. The reaction mixture was dialyzed with phosphate buffered saline (pH7.4) for 48h and with distilled water for 48h, followed by byophilization and obtained the objective compound.

Claims

1. Use of a galactosamide derivative of general formula (V) CH2ORi R2O'lle.o 1 1 (V)

RIO-G NHAc

wherein: Rat is H or a hydroxyl protecting group; R2 is a leaving group; and G is allyl or a protected hydroxyl group, in the preparation of a galactopyranose derivative of general formula (VI) CH2OR RIO,,, 0 l I (VI) RO. G NHAc wherein Rat and G are as defined above.

2. Use according to claim 1 wherein Rat is acetyl.

3. Use according to claim 1 or claim 2 wherein R2 is tosylate, trifluoromesylate or methanesulfate.