CA2110707A1 - Immunosuppressive and tolerogenic oligosaccharide modified lewis x and lewis a compounds - Google Patents

Abstract

Disclosed are novel Lewisx and Lewisa analogues, pharmaceutical compositions containing such analogues, methods for their preparation and methods for their use.

Description

WO 92/22564 PCl /CA92/00245

2 1 1 0 ~

I1~08~ 8I~5 aND ~rO~æROG2~IC
~ODIFI~D I~I~3X I~ID I.EWI8~ C0~5POV~

BAC~CGR~9~D OF ~ ~ ~IO~
' , ~

S T~ presenlt invent:ion is directed t~ novel ~` Lewi~;X and Lewis8 analogues, pharmaceutical c:os~po it~or~s ~ont;aining such an~loguel~:, an~thods for their pr~para l:ion and m~khods ~or their use .

2. ~e~en~es 1~ Th~ following referenc:es are Gited in this application a~; supersc:rip~ numbers a~ ~he relevant po~ion of the appl ication:
::
Horowitz, the Glycoconj uga~:es I Vols . I-V, Pigman, Editor, New York Ac:ad2mic Pres~; (1977, is lg78, l9B2, 1983~.
: 2 Ippolito et al., U.S. Patent ~pplication 5erial No. 07~714,161, filed June 10~ l99I for "Tmmunosuppresive ~nd Tolerogenic Oligosaccharide Glycosides.
~o 3 Sialic Acids in "Cell Biology Monographsl' Schauer, ~ditor, Vol. 10 ~1982~.
4 Lowe et al., Cell, 63:47~-485 (1990).

W092/22564 Pr/CA92/00245 ~ 0 6 __ 2 --Phillips et al., Science, 250:1130-1132 (1~90).
6 Walz et alO, Science 250:1132 et seq. (1990).
7 Larsen et a}., Cell, 63:467-47~ (19903.
8 Ratcli~e et al., U.S. Patent No. 5,07~,353, i~sued January 7, 1992, for "Sialic Acid Glycosides, Antigens, Immunoadsorbents, and Methods for their Preparation".
9 Ratcliffe et al~, U.S. Patent Application Serial No. 07/278,106, filed November 30, 1988, ~10 for "Sialic Acid ~lyc~sides, ~n~igens, Immunoadsorbents t and Methods for their Preparation".
Venot et al., ~.SO Patent Applica~ion S~rial No. 07/771,007, "~ethods for the Enzymatic ~5 Synthe~is o Alpha-sialylated Oligosaccharide Glycosidess~, iled October 2, 1991.
11 Wein~tein et al~, J~ Biol. Che~., 257:13845-13~53 (lg8~).
12 Reuter et al., Glycoconjugate J. 5:133-135 ~19~8).
13 Palcic et al., Caxbohydx~ ~e~
:~ (19893.
14 Prieels et al., J. Biol. ~hem., 56:10456 10463 (19~
Eppenberger-C~stori et alO, Glycocon;. J9 6:101~ (1989).
16 Go~hale et al., ~an. J. Chem., 68:10~3-1071 ( 1990~ .
17 Jiang et al., U.S. Patent Application Serial No. 07/848,223 filed March 9, 19929 for "Chemical Synthesis of GDP Fucosel'.
18 Ekberg et al., Carbohydr. Res. 110:55-67 (19~2~.
19 Dahmen et al~, Carbohydr~ Res. 1~8:~92-301 t1983)~
Rana et al., Carbohydr. Res. ~1:149-157 ~1981).

W092/225~ 2 1 1 ~ 7 0 7 PC~tCA92/~45 21 Amvam-Zollo et al., Car~ohydr. Res. 150:~99-212 (1986).
22 Paulsen et al., Carbohydr. Res. 104:19S 219 (1982).
23 Chernyak et al., Carbohydr. Res. 128:269-282 (1984).
24 Fernandez-Santana et al., J. Carbohydr. Chem.
8:531-537 (19~9).
Lee ~t al., Carbohydr. Res., 37:193 et seq.
~1~ (1974~
2S Norberg et al., Carbohydr. Res. 183-71 ~t seq.
~: (1988).
27 ~atta et alO, Carbohydro. Res. 208:51-58 (1980).
~: 28 ~handras~kar~n et al., Ab~tracts of ~he 11th I~ternational Symposium on Glycoconjugates, ~une 30, 1991.
29 ~kowska-Latallo et al., Genes and Development, 0 ~-1288-1303 ~199~).
Duma et al., Bioorg. Med. Letters, ~:425-428 (1991).
31 Okamoto et al.,:Tetrahedron, Vol. 46~ No. 17, pp. 5835-5837 (1990)~
32 Abbas et al., Proc. JapanesQ-G~nman Symp.
Berlin, pp. ~0-21 (l9&B~.
33 Paulsen, ~gne~. Chem. Int. Ed. Eng., ~1:155-173 (1982).
34 Schmidt, Agnew. Chem. Int. Ed. Eng., 25:~12-23S

3~ (lg86).
~jedi et al., Glycoconj. J., 4:97-108 (1987~.
: 36 Kameyama et al., Carbohydr. R~s., ~2 Cl-C4 .. ~1991).
37 Inazu et al., Bull. Soc. Chim~ Jap., 611:44S7 3~ (lg88).
38 Bernotas et al., ~iochem. J., 2?0:539-540 (1990).

~ Q~ ~7 4 __ 39 Wollenberg et al., U.S. Patent NoO 4,612,132, for Issued September 21, l9B6 for "Modified Succinimides".
Greig et al., J. Chem. Soc., p. 879 ~1961).
~1 Piekarska-Bartowzewicz et al., Carbohydr. Res., 2.03:302-307 (1990~.
42 Petitou et a~., Carbohydr. Res., 1472210~36 (198~).
43 Trumtez et al., Carbohydr. Res., 191:29_52 ~10 (19893.
~ 44 ~emieux et al., J. Amer. Chem~ Soc., g7:4076-:` 40~3 (1975).
Ogaw~ et al., Tet. Letters, ~2:4759-4761 (198~).
~ 5 46 Lemieux et al., canO J. ~hem. ~Q:63-67 (1982).
: 47 ~icolaou et al., JACS, 112:3693-3695 (1990).
48 Hindsgaul e al O ~ J. Org. Chem~ 2:2869-2875 : (1975) 49 Richard~on et al., Carbohydr. Reæ. ~ 271-287 ~1991~
~; 50 Smith et al., Immunology, 58:245 (1986).
~: ~ 51 Sleytr et al~, ~rch. Microbiol., ~ 19 (1986).
: 52 Ziola et al., J. Neuroimm~nol., 7:315--330 (I985).
53 ~okhale et al., Can. J. Chem., 68:1063-1071 ~1990) 54 Schmidt, et al., Liebigs Ann. Ch~mO, 121-124 ( 1991~
Nu~e~ , et al O, Can. J . Chem., ~9 : 2086-2095 (1981) 5Ç Veeneman, et al., Tetrahedron Lett., 32 : 6175-6178 (1991) W~92/22564 2 ~ i PCT/CA92/00245 __ 5 __ All publications and patent applications mentioned in this specification are indicative of the level of skill of tho e skilled in the art to which this invention pertains. ~11 publications and S patent applications are herein incorporat~d by reference in their entir~ty to ~he same exten as if each individual publication or patent application was specifically and individually indicated to be incorporated by r~ference in its entirety.

3. ~ z~JYh~
Carbobydratas and/or oligosac harides are present on a variety of natural and pathological glycoconjugates1. Of particular inter~t are : carbohydrates and oligosaccharides containing sialyl ~: 15 and/or fucosyl residues3. Such sialyl and/or : fuco~yl ~arbohydrates and oligosaccharides are :~ pre~en in a n~mber of products which ha~e been ~: implicated in a wid~ range of biological phen~mena ~: bAsed, in part, on the conc~pt of recognition sign~ls carried by the car~ohydrate structures and by their bi~ding to ~pecific ligands.
8pecifically, a numb~r of sialylat~d and : ~ialylated/fucosylated oligosaccharide glycoside :~ ha~e be~n proposed as mediators of cell adhesion in : 25 that they are ligands ~or selectins tor LEC-~AM's)4,5,6~7 Sialylat~d, fucosylated, and sialylated and fucosylated oligosaccharide structures relating to blood group determinants, including LewisX, Lewisa, sialyl LewisX and sialyl Lewis~, have also been shown by Ippolito et al.2 to po~sess in ivo im~unomodulating and tolerogenic properties in mammals including anti-inflammatory immunomodulating properties. In this regard, the DTH anti-inflammatory immunomodulating properties of C~`
LewisX and sialyl LewisX reported by Ippolito et al. 2 demon~trate that the presence of the sialyl residue on sialyl LewisX re~ults in enhanced anti-in~lammatory acti~ity as compared to LewisX.
Contrarily, sialyl LewisX, and sialyl Lewis~ and related compounds are dif f icult to chemically synthesize in high yield with anomeric specificity for the a~2~3) linkag~ of NeuSAc to galactose.
~nown chemical methodologies include a multistep `10 synthesis which fir~t generatas a blocked ~Neu5Ac(2~3)Gal disaccharide having a suitable leaving ~roup at the reducing sugar tsrminus of the galactose8~9. This disaccharide is then reacted with a suitably protected GlcNAc-O~ saccharida glycoside lS and then ~ suitably protected L-fucose d~rivative which, after d~protection, pr~ides for the ~ialyl ~: L~wisX g}yco~ide ~Neu5Ac(2~3~Gal(~4)t~uc~1~3)]
~GlcNAc-OR~ or the ~ialyl L~wis~ glyaoside ti~e.~
~N~u5Ac(2~3)0Gal~1-3)~uc(1~4)]~1eN~c-OR] where R
is an aglycon of at lea~t one carbon ~to~
Additionally, sialyl LewisX, sialyl lewisa and relat~d co~pound~ can al80 be synthesi2ed via ~hemical/enzy~atic synthesi~2~9~l0. In ~neral, the ~Gal(1~4~GlcNAc-OR, the derivatiz~d ~al(1~4)~GlcN~c-OR, the ~al(1~33~GlcN~c-OR, or the deri~tized ~Gal(1~3~GlcNAc-QR backbone is f irst ~ynthesized chemically and then the ~ialic acid r~sidue (e.g., Neu5Ac) is attached to th~ galactose to form the aNeuSAc(2~3)~Gal(1~3)~GlcNA~OR or the ~Neu5Ac(2~3~Gal~1~4~GlcNAc-OR structures by use of a compatible sialyltransferase and the fuco~e residue is then attached to the 4-hydroxyl position of the N-acetylglucosamine residue by use of a compatib~e fucosyltransferase.

W092/22564 21 1 0 7 ~ 7 PCT/CA92/00245 However, the enzymatic synthesis of sialyl LewisX and related compounds is restricted by the availabilîty of compatibl~ sialyltransferases. For example, the ~Gal(1~3J4~GlcNAc a(2~3)sialyltrans-ferase disclosed in the art for sialylating the ~al(1~4)~GlcNAc backbone is currently recovered from ra~ livers11.
In any event, the inclusion of a sialyl residue on LewisX, Lewis~ and related compounds so as to `~0 pro~ide for sialyl LewisX, ~ialyl Lewis~ and relat~d compounds results in a more c~mplex and costly synthesis.

8~a~Y OF T~ INVE~TIO~
The pre~ent inv~n~ion is directed, in part, t~
: 15 the discov2ry that modified L~wisX and modified ; ~ Lewi~a compounds having a sulfate, a phosphate or a ~: carboxylate containing group posse~s enhanc~d mun~uppressive and tolerogenic propert~e as ;~` ; oompared to imilar co~pounds lacking such 8 ~ ~titution. ~oreover, the 3-sulfa~e LewisX
compound posse~se8 at least eg~iva~ent immunosuppressive and tolerogenic properti~s as :~ :
compared to sialyl LewisX. This result is ~:~ particularly surprising insofar as unmodif i~d LewisX
25 po~;~esse inferior immunosuppressive and tol ~rogenic prcperties as co~pared to sialyl LewisX.
Additionally, bec:ause these modif ied LewisX and : ~ ~ modified Lewis~ compolmds do not contain a sialyl residue at thç~ 3-position of the g~lac~ose so as to 30 ~brm an ~ (2~3) linkage, the problems inherent with f orming such a 1 inkag~ are avoided .

~t~ 8 --Accordingly, in one of its composition aspects, the present invention is directed to compounds of Formula I:
XQ
IOH
C~3 ~ OH
HO ~ OXl ¦ ~
X2O ~ O ~ Y-R
R3 Rl : S where R is ~elected from the group eonsi~ting ~: of hydrogen, a ~accharide ORl4, an oligosaccharide-ORl4, or an aglycon having at le~st 1 carbon atom ~: where R14 is hydrogen or an aglycon of at least one : carbon ato~; :~
O Y i~ sel~cted;from ~he group consisting of o~ygen, ~ulfur, and -NH-:
R1 is selected from the group con~i~tin,g of hydrogen, -NH2, -N3, -NHS03H, -N~C (O j R~, -NzC(R~)2, -NHC~ NHR6, N(R~)2, OH,- OR~, 3, -S(O)R~ -S(O)~and sulfate, whereîn R4 is~selected from the group : consistin,g of ~: :hydrogen, : ~ :
: alkyl of from 1 to 4 carbon atom~, : -OR7 whereîn R7 i5 alkyl of from 1 to 4 ~: : carbon ato~s, or alkyl of from 2 to 4 carbon atoms substîtuted with a hydroxyl group, and -NR~R~ wherein R8 and Rg are îndependently selected from the group consîstîng of hydrogen and al~yl of from 1 to 4 carbon atoms, each R~ is selected from the group consi~ting : of hydrogen and alkyl of from 1 to 4 carbon atoms, each R6 îs alkyl of from 1 to 4 carbon at~ms, W(~ ~2/22564 PCr/C~92/0024S
21 ~ D 7 (3 i R2 is selected fxom the group consisting of hydrogen, -N3, -NH2, NHS03H, -NRl~C (O) R10 ~
-N=C (R71 ) 2 ~ (Rl1 ) 21 ~ 2 ~ -N (R12) 2~ OH and --ORl 2 ~
wherein R~o is selected from the group con~;isting of hydrogen, alkyl o~ f rom 1 to 4 carbon atoms, -O~R13 wherein R13 is alkyl of from 1 to 4 car!bon atoms, or alkyl of from 2 to 4 car~on atoms substitut~d with a hydroxyl group, and 4Rls wherein R14 and R15 are independç~ntly seleated îrom t:he group consistinS~ of hydrogen and alkyl of frs~ 1 to 4 carbon atc~ms, ~: ~ 15 ~ each R11 is select~d from the group consisting of hydrogen and alkyl of from 1 to 4 carbon ~toms;
~ach R1;! is alkyl oP îrom 1 to 4 carbon a~o~as, R3 is ~elec:ted from the group c:onsisting of hydrogen, fluoro, sulfate and ~ydroxy;
~: 20 X is s~lected from the group consisting of hydrogen, sulfate, ansl phosphate;
X1 is select~d i~rom ~ group consistirlg of ~: hydrogen, sulfat~, phosphate, and CHR18COOH wh~re R18 is selected rom the group con~;isting of hydrcgen, alkyl of from 1 to 7 carbon atoms and ~COOH;
X2 is selected from the group con~isting of ~, hydrogen, sul~a~e, phosphate, and -CHRl3COOH where R~8 is ~:elect~d ~rom the group consisting of hydrogen, alkyl of from 1 to 7 c:arbon atoms ~nd -COO~I: and pharmaceutically ac~eptable salts thereof;
and with the proviso that either at least one of X, X1 0 or X2 is sulfate or phosphate or at least or~e of X1 or X2 is -CHR1~,COOH.

4 PC~/CA92/00245 r~ 10 --The present invention is also directed to compounds of Formula II:
X~
IOH
CH~ ~ OH , R1 II

where R is selected from the group consisting s of hydrogen, a saccharide-OR1~, an oligo-: ~ saccharide-OR14, or an aglycon having at least l carbon ~tom where R14 is hydrogen or an aglycon ~f at : least one carbon atom;
Y is selected from the group consisting of oxygen, sulfur, and -NH-;
R1 is selected from the group consisting of hydrog~n, -NH2, -N3~ -NHS~H, -NR~C(O)R~, N=C~R~2~ -N~C~(R~)2, -NHR6, -N(R~32~ -OH~ OR~
: S(O~R~, -S(0~2R~ and sulfate, ; wherein h~is selected from the group consisting of :~ ~
: hydrogen, alkyl of from 1 to 4 carbon atoms;
OR7 wh~rein R7 is alkyl of from l to 4 carbon ato~s, or alkyl of from 2 to 4 carbon at~ms ~: substitut~d with a hydroxyl group, and :: N~bR~ wherein Rb and R9 ar~ independently selected from the group consisting of hydrogen and alkyl of from l to 4 carbon atoms, : 25 each ~ is selected from the group consisting :: ~ of hydrogen and alkyl of from l to 4 carb~n atoms, each R6 is alkyl of from l to 4 carbon atoms, R2 i5 select~d from the group consisting of hydrogen, -N3, -NH2, -NHS03H, -NR~1C(O)R10, ~092/22564 PCT/CA92/00245 ~11 1) 7 1,' ~

-N=C(R11)2, -NHCH(R1~)2, -NHR12, -N (R12) 2, -OH and -R12 ~
wherein R1~ is selected from the grsup cvnsisting of hydrogen, alkyl of from 1 to 4 carbon atoms, -OR13 wherein Rl3 is alkyl of from 1 to 4 ~ carbon a~oms, or alkyl of from 2 to 4 carbon atoms ; ~ubstituted with a hydroxyl group, and -N~14~5 wh~rein R14 and R15 are ind~pendently sele~ted from th~ group consisting of hydrogQn and alkyl of fro~ l to 4 carbon atoms, ea~h R~ s~lected from the sroup consisting o~ hydrogen and alkyl of from 1 to 4 carbon atoms;
~ch R12 is al ~ l Qf fr~m 1 to 4 carbon atom~, :~ R3 i. elect~d from th~ group consi~ting of hydrog~n, fluoro, sulfa~e and hydroxy;
X i~ ~elect~d from the grou~ consisting of h~ rogsn, ~ulfate, and ph~sphate;
20:: X1 is selected ~rom the ~roup consisting of : hydrogen, sulfa~e,:phoæphate~ and -CHRl8COOH where R18 is seleated from the group aonsiæting of hydrogen,:al ~ l of ~r~ 1 to 7 caxb~n a~oms a~d :-COO~
~ X2 i~ ~elected from the group consis~ing o~
hydrogen, sulfate, phosphate, and -CHR18COQH where . elected ~frs~m the group consisting o~
hydrogen, alkyl of fxom 1 to 7 carbon atoDI~; and -COOH; and 3~ pharmaceutically acc~ptabla salts thereof;
and with the proviso that sithQr at least one of X, X1, or X2 is sulfate or phospha e or at least one of Xl or X2 is -CHR,8COOH.
The compounds of Formula I and II are particularly useful in modulating a cell-mediated W092J22564 PCT/CA92fO0245 ~ 12 --immune response to an antigen and in particular a cell-mediated immune inflammatory response to an antigen. In this regardl when the compounds of : Formula I and II are administered to a mammal in response to an antigen challenge, such administration induces tolerance to later challenges from this same antigen.
In another of its composition aspects, the present invention is directed to a pharmaceutical ~10 composition suitable for administration to a mammal ; (e.g., human) which comprises a pharmaceutically inert carrier~and an effective amount of the compound of Formula I or Formula II to modulate a cell-mediated immune re~ponse in said ~ammal.
~5 In still another of its composition aspects, the present i m ention is directed to novel intermediates~useful in preparing the compounds of Formula I and~Formula II. In this regard, so~e of the:intermediate~mono accharides disclosed herein 20: are:highly:crystalline and can be produced in large quantities in~high~purity without the n~ed for chromatography~;to separa~e these compounds.
In~:one of its method aspects, the present invention is directed to a method for ~odulating a :cell-mediated~immune response to an antigen in a : : mammal:which~method comprises administering to said mammal an amount of a compound of Formula I or Formula II effective in modulating said i~mune response.
~:: 30 In another:of its method aspects, the present ::
~ : invention is directed to a me~hod for preparing the :~: c~mpounds o~ Formula I and II above and to the : ~ preparation of intermediates useful in preparin~ the compoumds of Formula I and II.

~' W092~22564 PCT/CA92J00245 -- 13 -- 2 ~ 1 0 7 ~ ~

BRI~F D~gcRIpTIsN OF T~ DRA~I~G8 Figure 1 illustrates reaction scheme~ for the synthesis of partially blocked N-ace~yl glucosamine d~ri~atives which are then used to prepare either S modified Lewis~ compounds or modified Lewisa co~pounds.

Figure 2 illustrates reaction schemes for the synthesis of blocked fucose derivative~ which are ~hen used to prepare either modified LewisX or 10 ~odified L~wis compounds.

Figure 3 illustrates r~action schemes for the ~ynthesis of partially blocked galactose derivatives : which are then u~ed to prepare either modi~ied : L~ X co~pounds or modified Lewi5a co~pound~.

: 15 Figure 4 illustrates the synthesi~ of modifi~d ~ewisX ~ompound~ having a sulga~e substi~uent in the 3 po~ition of the galactose unit. In thi~ scheme, ~he 2,3 positions of galaotose are diffexentially : blocked ~o that th~ 3-po~i~ion c~n be ~electi~ely 20: deblock~d and ~hen s~lec~i~ely con~e~t~d to the sulfate æ~bstituent.
::
Figure 5 illustrates the ~ynthesis o~ modified LQwisX compounds having a sul~ate substituent in the 3 po ition of the galactose unit~ In this scheme, the 2,3 positions of galactose are not di~ferentially blocked. Accordingly, deprotection of the 3-position of the galactose unit also results in deprQtection of the 2-position and subsequent reaction to form the sulfate at the 3-position does not proceed with 100% yield but rather some of the product has a sulfate substituent at the 2-position WO 92/?2~i64 PCI'/CAg2/00245 of the galactose which is then separated by chromatoqraphy.

Fi~ure 6 illustrates the synthesis of modified LewisX derivatives bearing a sulfate ~ubstituent at S the 3-position of the galactose and which utiliæe a 6-benzyl and 2-N-ph~haloyl blocked glucosa~ine which can be later deblocked to provide for a glucosamine derivative .
In this figure, because the 3,4-dihydroxyl :~ 10 groups of th~ 6-benzyl and 2-N-phthaloyl blocked glucosamine 15 are not blocked, reaction with 1-bromo-2j3 9 4 ~ 6-tetraacetyl galactose will result in fo~mation of both the blocked ~Gal(1~4)~GlcNH2 d~rivative 48 and the block~d ~Gal(1~3)~GlcNH2 ~5 derivatîve ~n~t sho~n).
In turn, these materials can be further :~ derivatîzQd ~t an appropriate point in t~e æynthesis so as to provide for N-functionalized derivatives of L2WisX and Lewîs~O

~O Figure 7 illustrates a second synthe is of m~difigd Lewi~X co~pounds bearing a sulfat~
sub~tituent at the 3-position of the galactose and which utilize a different N-phthaloyl blocked glucosamine intermediate that allow~ for th~
selecti~e preparation of 2-amino or N-functionalized ~, LewisX derivatives. In this figure, only the 4-pocition of the glucosamine is not ~locked ~o that only the blocked ~al(1~4)~GlcNH2 derivativ~ 68 is formed.
:
Figure 8 illustrates the preparation of modified Lewis~ analogues having a sulfate substituent in the 3 position of the galactose unit.

211~7i37 In this scheme, the 2, 3 positis:~ns of galactose are differentially blocked so that the 3-position c:an be sel . ctively deblocked and then selectively converted to the sul f ate substituent .

Figure 9 illustrates the synthesis o~E the 6 azido derivative of GlcNAc-OR.

Figure 10 illustrates the synthesi~; of the 6-alkoxy derivatives and the 6-deoxy derivatives of Glc:NAc: .

Figure 11 illustrates the increase in footpad 2~w~ of immunized mic~ arising from a DTH
infla~atory resporlse mea~ursd ~4 hours ~fter chall¢Iage with 10 ~g of th~ L~ll S-Layer protein a~ti~en wh~2rein ~;ome: of the mice ha~e been treated at 5 hours after ~he challenge with 100 ~g of l~ewi~;X-OR and ~ialyl LewisX-OR (R= -O (CR2) 8COOC~I3) .

Fis~ure 12 illustrates the increa~e in f ootpad swelling of immunized mice arising from a DTH
inflasamato~y r~ ponse measure~ 24 houxs after challenge with 20 ,ug o~ Super~:arrier gfro~ Pierc~, ~ckford, IL 61105) antigen wher~in some o~ the mice have been treat~d at 5 hours after the challenge with lQ0 ~g of sialyl LewisX-OR (S}.eX) and with 3-O-sulphate (on the gal~ctose moiety) of the ~wisX OR -(SULF9NATE:-LeX~ whexe R is -o (CH2) 8COOCH3 in both cas~s .

Figure 13 demonstrates the ef~Ee~t that sulfated LewisX-OR (SULFONATE-LeX3 has on the induction of an immune response t~ an antigen where R i5 ;IS de~ined 3 0 in Figure 12 .

WO 92/22564 PCr/C~92/n0245 Figure 14 illustrates the effect that sulfated LewisX-OR (LeXsulf~ and sialyl I.ewisX-OR (SleX) on lung injury arising f rom the intranasal administration of LP5 to mice ~R is as def ined in S Figure 12 ) .

Figure 15 demonstrates the long term tolerogenic effec:t of sulfated IRwisX-OR tSULEONATE
LeX~ and sialyl IA~wisX-OR (SLeX) on an i~rmune re~ponse to an antigen w~ere R is as def ined in Figure 12 ) .

I:U3 g!~II.13D D~8C~Pq!IO~ OF ~E P~ D ~l~ODTNE5~!8 P.s noted above, the present in~rention is direeted, in pa~, to t:he dise:ov~ry oi~ novel Lewi~;
and novel Lewi~i" a~alogues whiah, in mammals, inc1udirlg hu~a~s, ~ are useful for ~ v .ro modulaltion (eSg. ~ suppression) of a c:ell mediated immune response inc1uding cell-mediated arld immune directed inf1amma~ory respons~ to an antigen in a m ~al ~e.g., a DTH response).
- 20 Additiona11y, th~ present in rent~on is direated, in part, to nc~vel methods for the ~ynthesis of L~wi~;X and for the synthesi~ of L@wis~
ana1Ogue~; and to novel inter~edial:es useful in these syntheæes .
However, prior to discussin~ t~is in~ ntion in further detail, the following ~erms will f ~ rst be def ined .

Def initions As used herein, the f ol1Owing terms have the de~initions given be1Ow:

W092/22~64 2 t 1 ~ 7 ~ 7 PCT/CAg2/00245 The term "cell-mediated immune response to an antigen in a mammal" refers to ~hose mammalian immune responses which are mediated by cell-cell interactions~ Included within this term are cell mediated inflammatory responses to an antigen such ~s DTH responses as well as cell-mediated inflammatory responses arising from myocardial infarction, virus-induced pneumonia, shock and :~ s~quelae (e.g., multiple organ failure), adult `10 respirato~y distress syndrome, psoriasis, arthritis, ~: and the like. Preferably, the cell-medi~ted immune : response is a leucocyte-mediated response.
The term "LacNAc" refers to the disaccharide ~Gal(1~4)~GlcNAc which is repr~sented by the lS ~or~ula:

~: HO OH ~Hac ~ ~ OH
OH oH

: ~
he term "LacNH2" refers to the LacNAc der~vative wherein the N-acetyl group of LacNAc has been repl~ced wi~h an amine (-NH2).
The te~m "LacN3~l refers to the LacNAc d~riYative wh rein the N-acetyl group of LacNAc has ~: been replaced with an azido (-N3~.
The term "~ewisX" (sometimes referred to "LeX") refers to *he trisaccharide having the following : structure:

W092/225~ PCT/CA92/00245 OH

H~OH
OH
0~

Because of its relation~hip to blood group de~e ~inants, th~ core ~Gal(1-43~GlcNAc struckure of Lewi5X i5 often referred a~ a "type II structure~' or "LacNAc structure".
Th~ term ~'Le~i~n" (sometimes ref~rred to IlLe~
re~rs to the tri~accharide hav.ing the followi~g ~: struc~ure: :

HO

C H3 S~0H ~ OH

H~\~l ~ OH

Becauss of its relationship to blood group dete~minants, the core ~Gal(1~3)~GlcNAc structure of 10 Lewis3 is often referrad as a "type I structure'~.
The term ~modified LRwisX glycosides and deri~ati~es thereof" refer to deri~atives of the LeX
modified in one or more of the fucose, galactose and N-acetylglucosamine saccharid~ units of ~ewisX and which have an -YR substituent as defi~ed above.
When the R subs~ituent is an aglycon yroup, this group has at least one carbon a~om, but neverthel2ss are different from glycoconjugates because such aglycon moieties are neither a protein nor a lipid capable ~f forming a micelle or other large aggregate stru~ture.
The term "modified Lewis~ glycosides and derivatives thereof" refer to derivatives of the Le~
modified in one or more of the fucose, galaatose and N-acetylglucosamine saccharide units of Lewi8~ and which have an -YR substituent as defined abov~.
When t~e R ~ub~tituent is an aglycon group, this group has at least one carbon atom, but ne~ertheless are different ~rom glycoconjugat~s because such aglyc~n moieties are neither a pro~ein ~or a lipid aapable of forming a micelle or o~her large aggr~gate structure.
The term "aglycon of at leas~ one carbon a~om~' xefers to non-saccharide co~taining residues having ~: a~ lea~t one carbon atsm. Preferably, the aglycon 20 i8 ~elected fro~ th~ gro~p con~isting of ~ Z
wherein A represents a bond, an ~lkylene group of f~om 2 to lO carbon atoms, and a moiety of the form . - (~H~-~5G~ n~ wher~in n i~ an integer equa~ tv 1 to ~ 5~: Rl5 is ~elocted ~rom th~ group aonsisti~g o~
hydrogen, methyl, or ethyl; and G i5 s~lected from the group consisting of hydrogen, haloge~, oxygen, sulphur, nitrogen, phenyl and phenyl s~bstituted with 1 to 3 substituents selected from t~e group consisting o~ amine, hydroxyl, halo, alk~l of from 1 to 4 carbon atom~ and alkoxy of ~r~m 1 to 4 carbon at~ms. and Z is s~lected from the group consisting of hydrogen, methyl, phenyl, nitrop~enyl and, when G
is not oxygen, sulphur or nitrogen and ~ is not a bond, the~ Z is also selected ~rom the group consi5ting of -OH, -SH, -~H2, -NHR16, -N(R16) 2 W O 92/22564 PC~r/CA92/00245 ~rl Q~ - 20 ---C(O)OH, -C(O~OR21, -C(O)~nH-NH2, C(O)NnH2, ( ) 16~ C()N(R16)2~ and -OR17 wherein each R16 is independently alkyl of from 1 to 4 carbon atoms and R17 is an alkenyl group of from 3 to 10 carbon atoms.
Nu~nerous aglycons are hno~nn in the a ~ . For example, an aglycon comprising a para-nitrophenyl group (i.e., -YR = -OC6H4pN02) has been disclosed by Ekberg et al-18 At the appropriat~ time during ~10 synthesist the nitro ~roup is reduced to an amino group which can ~e protected as N-~ri~luoroacet-amido. When desir~d, the trifluoroace amido group i~ remov~d thereby unmasking the amino group.
~n aglycon containing sulfur is di~closed by ~ ~5 D~h~en et al.19. Sp~ci~ically, the aglycon i~
: deriv~d fro~ a 2-bromoe~hyl group which, in a ubstitution reaction with thionucleophiles, has b~en h~wn to lead to aglycons pos~es~ing a variety ten~inal functional groups ~uch as ~: 20 O~H2cH2s~H2co2c~3 and -OCH2c~2sc6H~-pNH2 ana et al.~ di d o es a 6-trifluoroacetamido-h~xyl aglycon ~-0-(CH2)6-N~COCF3~ i~ which th~
~ri~luoroacet~mido protecting group can b~ removed unmasking the pri~ary amino group.
~5 Oth~r exemplification of kn~wn aglycon~ include the 7~methoxycarbonyl-3,6,dioxaheptyl a~lycon~l ( OCH2-~H2~ zOCH2CO2C~3; the 2-~4-methoxyc~rbonyl-butancarboxamido)ethyl22 ~-O~H2CH~NHC (O) (C~2~ bC2~H3);
and th@ allyl aglycon23 ~O~H2CH~CH2) which, by r~dical co-polymerization with an appropriate monomer,leads to co-polymers; other allyl linking aglycons24 are known [e~g., -O(CH2~H20)2CH2CH=CH2].
Additionally, allyl linkin~ arms can be derivati~ed in the presence of 2-aminoethanekhiol25 to provide W092/22~64 PCT/CAg2/0024~
i O ~r~7 ~ t ri for aglycons -OCH2CHzCH2SCH2CH2NH2~ Still other aglycons are illustrated hereinbelow.
Additionally, as shown by Ratcli~fe et al. 9, the R group can be an additional saccharide OR14 or

5 an oligosaccharide-OR14 at the reducing sugar term,inus (where R14 is as defined above).
Preferably, the aglycon moiety is a hydrophobic group and most preferably, ~he aglycon moiety is a hyd~ophobic group selected from the group consi~ting f -(CH2)8COOCH3, -(CH2)sOCH2CHzC~2 and ~(CH2)sCH
The t~rm, "oligosaccharide" re~ers to a carbohydrate ~t~ucture having from 2 to about lO
saccharide units. The particular saccharide units : ~mployed are not critical and include, by way of ~5 exam~le, ~ll natural and ~ynthetic deriv~t.ives o~
glucoæe, galactose t N-acetylgluco~amine, N-acetyl-~galactosamin~, fucos~, sialic acid, 3-deoxy-D,L-oatulo onic acid, and the like.
In addition ~o b~ing in ~heir pyranose ~orm~
all saccharide units described herein are in their D
fo ~ except for fuc~se which is in its L fonmO
The term 3'sialic acid~' or ~'sialyl" me~ns all naturally oc~urring structures of sialic acid and analo~ue~ of sialic acid. Naturally occuxring ~tructures o~ sialic acid include, by way o~
exa~ple, 5-acetamido-3,5-dideoxy-D-glycero-D-galactononulo-pyranosylonic acid ("N~uSAc3a), N-glycoyl neuraminic acid (NeuSGc~ and 9-O acetyl neuraminic acid ~Neu5,9Ac~)O Analogues of sialic acid r~fers to analo~ues of naturally ocGurring struc~ur~s of sialic acid including those wherein ~ t~e sial ic acid unit has been ch~mically modified so as to introduce and/or remove one or more functionalities from such st~uctures~ For example, such modi~ication can result in ths removal of an -OH functionality, the introduction of an amine functionality, the introduction of a halo functionality, and the like.
Certain analo ~ es of sialic acid are known in S the art and include, by way of example, S-azido-NeuSAc, 9-amino-NeuSAc, 9-deoxy-NeuSAc, 9-fluoro-NeuSAc, 9-bromo-Neu5Ac, 7-deoxy-NeuSAc, ~ epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Nsu5Ac, 4-N-acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac, ~-oxo-Neu5Ac, as `10 well as the 6-thio analogues of NeuSAc. The : nomenclature employed herein in describing analogues of sialic acid is as set forth by Reuter et al.12 The term "fucose" or "fucosyl" refers to ~-fucose and~analogues thereof. The ~ucose : substituent ca~:be:attached to the derivatized Gal~ 3)GlcNAc~-disaccharide (type I) or the -^
derivatized;Ga1~ 4)GlcNAcO-disaccharide (type II) so as to f~orm the Lewis' or LewisX molety by eithar chemical ox:;enzymatic means. While che~ical means ~are illustrsted:~in the figures, enzymatic means for attaching L-fucose to~th~ 3-position of the GlcNAc ~; unit~of ~Gal(1-4)~GlcNAcO-Iipid containing a sulfate : on the 3-po~ition;:~of the galactose unit has been : repo ~ ed~. In~uch~ca~es, ~he fucose e~ployed,~- as its GDP derivative,~is one which is compatib~e with the fucosyltransferase (e.g., ~Gal(l~3j4)~GlcNAc a(l-3/4)fucosyltransferase). The ~Gal(l~3/4~GlcNAc 4(1 ~3~4~fucosyltransferase is readily isolated from human milkl3~l4~15. : M ditionally, it is contemplated that these fucose or fucosyl compounds will also be :~ compatible with other furosyltransferases of ::: appropriate specificity such as cloned fucosyltransferases~30.
: In regard to the absve, any fucose compound ~: 35 which, as its GDP-derivative, is recognized by the :::

W092/225~ PCTJCA92/00245 2~ 7 ,~ ~

~Gal~l~3/4)-~GlcNAc ~ 3/4)fucosyltransferase so as to bind to the enzyme and is then available for transfer to the compound of Formula III or IV:

Y-R III

R~
S s:~
I10 0~
X2 ~ ~ Y-R IV

R~ Rl where R, R1, R2, R3, Xl and X~ are as defined abov~, is said to be compatible with this fucos~1~ransf~rase.
An2logues of ~ucose refer to naturally occurring and synthetic analogues of fucose : including those where the Pucose unit has b~en che~ically modified 80 as to introduce and~or remove ~: o~e or ~ore ~unctionalities from this structure.
lS For example, such modification ~an result in the r~mo~al of an -OH functionality, the introduction of ~an ami~e functionality, ~he introduc~ion of a halQ
~unc~ionality, a~d ~h~ like.
: Certain compatible analogues o~ fucose are known in the art and include, by way of example, 3-deoxy-ucose, arabinose, and the like .16 The GDP--derivative of fucose refer ~o guanosine 5'~ L fucopyranosyl)diphospha~e and any and ~11 compatible salts thereof which has the - 25 formula:

W092/22564 PCT/CA92/0024~

O.
~Z~ o~ O~

~ethods ~or preparing GDP-fucose are kn~wn in the art. However, GDP-fucose is preferably prepared by the m~thod described by.Jiang et al.l7 in U.S~ Patent Application Serial No. 07/848,223 which is ~; 5 incorpor~t~d herein ~y re~erence in i~s entirety.
he texm "co~patlbl~ salts" as it îs u~ed in rela~ion to guanosine 5'~ L-fucopyranosyl~-: ~ ~ip~osphate refers to those salts of guanosine 5'-L-~ucopyranosyl)diphosphate which readily form 10~ count~r ions (i-eO ~ cations) and which are co~pa$ibl~ with~the i~tended reactions and/or pu~ifications. ~Suitable co~pati~le alts include :~ tho~e prepared fr~m counter ion~ ~uch as ~odiu~, pota~ium~ lithium,~:calcium, magnesium, ammonium, mono-, di-, tri- or te~ra-alkylammonium, iro~, zinc, and t~e lik~
: The term "pharmaceutically acceptable salts3' ; includes the pharmaceutically acceptable addition salts of the co~pounds of F~rmula I derived from a variety of organic and inorganic counter salts well :: Xnown in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, The term "sulfate" such as used to define the : 25 substituents -OX, -OX~, and -OX2 refers to WQ92/22564 PCT/CA92~00245 -- 25 -- 2~ 7 substituents which, with the oxygen of a hydroxyl group of the galactose unit and/or fucose group, form a sulfate group (i.e., -0-S(0)2-OH). Thus, when X, X1 or X2 is a sulfa~el the resulting -OX, -OX1 and/or -OX2 group is -0-S(Q)2-OH, which readily forms pharmaceutically acceptable salts thereof (e-g-, -0-S(0)2-O~Na~, The term "phosphate" such as used to define the substituents -OX, -X1, and -OX2 refers to substituents which, with the oxygen of a hydroxyl group of the gal~ctose unit and/or fucose group, form a phosphate group (i.e., -O-P(0)2-OH). Thu., when X, X1 or X2 i~ a phosphate, t~e resulting -OX, -X1 and/or -OX2 group is -0-P(0)2-OH~ which readily ~5 form~ pharmaceutiGally acceptable salts ther~of (e.g~ -0-P~0)2-0-N~
The term '~re~ovable blocking group'~ refers to any group which when bound to one or more hydroxyl groups of the galactosa 9 the N-acetylglucosamine, and/or the fucose units of L~wi X and ~ewis moieties : ~ prevents reactions ~rom occurring at ~hese hydroxyl groups and which protecting group ~an ba removed ~y con~entional ~ iaal or e~zym~tic ~eps to xeestabli h the hydroxyl group. The particular r~movable ~locking group employed is not critical and pr~ferred re~ovable hydroxyl bloc~ing groups include conventq nal substituants such as benzyl, benzoyl acPtyl, chloroacetyl, benzylldine, t-butyldiphenylsilyl and any o~her group that can be introduced either enzymatically or chemically onto a hydroxyl functionality and later selectively removed either by e~zymatic or chemical methods in mild conditions compa~ible with the nature of the product. One such additional contemplated blocking WO 92/22564 P~/CA92/00245 group is a ~-galactose which can be remo~ed enzymatically with an ~-galactosidase.

2. ~hQ~
The modif ied LewisX and modif ied Lewis~
co~pounds disc10s2d herein are readily prepared either by complete chemic:al syntheses or by chemical/enzymatic: syntheses wherein glycosyltransferase~ are employed to effect the æaquent~al addition of one or more of sugar units onto a GlcN~c OR saccharide strurture, a deriv~tized Glc:NAc~-C)R saccharide structure, a Lac~Ac OR
disac:charide structure, a derivatized L~acNAc-OR
saccharide ~ tru~::ture, a ~Gal ( 1~3 ) ~GlcNAc-OR
dis;lccharide struct;ure, or a derivatized ~Gal ( 1~3 ) ~GlcNAc-OR disaccharide struoture .
Th~ f i~uras a~ached hereto elabc~rate on a variety o~ complete che~aical synthetic schemes which re~ult in ~he :pr~paration of modified LewisX an~
modi~i~d Lewi~8 co~pounds.
Enzyma~ic means to prepare modified LswisX
compounds and modi~ied Lewis~ compounds can be used at di~fer@nt~;teps. For ~xample~ L-fucos~ c:an be n~ymatically transferr~d on~o ~h~ d~bloak~d ~ul~ate, p~osph~t~, or carboxylat~ containing : 25 derivatives of:
a LacNAc-OR structure or a derivatized LacNAc OR structure (modified ~ewisX compounds); or a ~Gal(~33~GlcNAc-OR structura or a der vatized ~Gal(1-33~GlcNAc-OR ~tructure (modifiad Lewisa compound ) by an appropriate fucosyltran ferase such as : the ~Gal(1~3/4)~GlcNAc ~(1~3/4)fucosyltransferase which is readily obtained from human milkt3~l4~5.

7 ~ 7 The LacNAc-OR disaccharide can be made enzymatically from an N-acetyl glucosamine glycoside and the known ~-galactose~1~4)transferase. The ~Gal(1~3)~GlcNAc-OR disaccharide glycoside can be made chemically.
Additio~ally, it is con~emplated that sulfotransferases may be used to effect sulfation at the 3-position of galactose on either the type I or type II structures~ As is apparent, this can be followed by transfer of fucose using an appropriate fucosyl~ransferase as de cribed above.
Alte~natively, chemical and enz~matic means can be coupled wherein, for example, the sulfated, phospho ~ lated, or ~ 1~COOH substituted Laa~c-OR
: 15 ~ructure or ~ulfated, phosphorylated, or -CHR1aCOOH
: substituted ~&al(1~3)~GlcNAc-OR structure is made chemically and the fucosyl group transferred enzyma~ically.
Chemic~l syn~hesis is a convenient method for : 20 ~ preparing either the complete oligosaccharide ~lycoside; for chemically modifying a saccharide unit which can then be ch~ically or enzymatically coupled to an oligosaccharide glycoside; or ~or chemically preparing an olig~saccharide glycosi~e ~o which can be enzy~a~lcally coupled one or more : saccharide unit Several chemical syntheses of blocked intermediates exist46~47~48. These intermediat~s are ~uitable for the modifications described herein.
30 Syntheses of the~;e int rmediates or similar ones utilizing methods known in the art allow the synthesis of the modified L~wisX and modifie~ I.ewisa compounds contained herein.
Chemical modif ications include introduc:tion of s the sulphate or phosphate group or a -oCHR18COOH at WO ~2/22564 P~/CA92/00245 c~ 28 --the 3 and/or 6 position of the terminal galactc~se.
Additionally the 3 or 4 position of the fucose can be sulphated or phosphorylated if the fucose intermediate 25 of Figure 2 is employed rather than S fucose ~ntermediate 20.

2Ao CHE~fICAL SYNTHESIS OF SACCHARIDE MONOMERS
Chemical methods f or the synthesis of LewisX
and Lewis~ and some analogues thereof are known in the art. The~e materials are generally assembled .0 using suitably protected individual monosa ::charides including the desirable glu~osamine, fucose and galacto~e, lactose or ~Gal(1 ~3),8GlcNAc:
intermediate~. The modifications to the final structures are acromplished using kno~n methods or 15 ~;ul~atic~n or pho~;phorylation after ~ppropriate s~lec:tive d~blocking of the to-be functionalized ;: hydroxyl group (s) of the fully blocked I,ewisX or I~di8a o The s~ec:if i c method~ employed are genérally 20 adapl:ed and opti~Qized for each individual structure to be synth~sized. In general, the chemical synthesis of all or part of the oligo~acc~aride glycosides first involves formation of a glycosidic linkage on the anomeric carbon atom of the reducing sugar. Specifically, an appropriately protected form of a naturally occurring or of a chemically modified saccharide structure (the glycosyl donor) is selectiYely modified at the an~meric center of the reducing unit so as to introduce a leaving group comprising halides, trichloroacetimidate, acetyl, thioglycoside, etc. The donor is then reacted under catalytic conditivns well known in the art with an aglycon or an appropriate form of a car~ohydrate acceptor which possess one free hydroxyl group at WO 92f22564 PCr/CA92/00245 -- 29 -- ~11 1)70 ~

the position whexe the glycosidic linkage is to be established. A large variety of aglycon moieties are known in the art and can be attached with the proper conf iguratio~ to the anomeric center of the S reducing unit. Appropriate use of compatible blocking groups, well knowrl in the art of carbohydra~te synthesis, will allow selective modif icatîon of the ~;ynthesized structures or the further attachment of additional sugar units or 10 ugar blocks to t:he acceptor s~ructures.
After formatiorl of the glyc:c~sidic linkage, the sacchaL~ide glycoside can be used to effect coupling of additional saccharide unit ~s) or chemically Dlodif i~d at sel~cted positions or, a~ter 15 conventional deprotection, used in an enzymatic `~ ~ ynthesis. In g~neral, chemical coupling of a naturally occurrins~ or chemically modi~ied sac: charide unit to ~e saccharide glycoside is acc~mpli~hed by employing established c:hemistry well 2 0 doaw~l~nt~d in the l ~ tQrature . See, f or ~xample, Oka~oto ~t al.3l, Ratcliffe et al.8, Abbas et al.32, Paulson33, Schmidt34, Fugedi et al . 35, and Kameyama al.36 With referenc:e to the figures, Figure 1 illus . rates the synthesis of numerous blocked derivatives of glucosamine and N-acetylgluc:osamine whic:h are then useful in the preparation of blocked LacNH2-OR, LacN~c-OR, ,BGal(l ~33,~GlcNAc:-OR;
,~lGal ( 1 ~ 3 ) ~Gls:NH2-OR, etc . structures . Spec:if ically, ~: 30 in Figure 1, glucosa~mine hydrochloride is slurried in dichloroethane containing an equivalent of a~hydrous sodium acetate to whic:h acetic anhydride is added dropwise and, after addition is completa~d, the solution is refluxed for a period of from about W092/22~64 PCT/CA92/0024~

~ 91~ ~ 30 __ 12-16 hours to provide for the peracylated compound 10 (about 3:1 ratio of a/~).
Alternatively, the glucosamine hydrochloride is first taken up in methanol and then treated with 1 eguivalent of metallic fiodium to neutralize the HClo Phthali~ anhydride is then quickly to the reaction mix~ure followed shortly thereafter ~y triethylamine to provide for the phthalimido derivative. This compound is then isolated and acetylated with acetic ~10 a~hydride/pyridine using conventional techni~ues to provide for peracylated compound 1 having a phthalimide blocking group protecting ~he amine.
Afterwards, th~ aglycon is formed by conventional techni~ue~. For example, compound 10 lS is converted to l-a-chloro compound ~ by w~ll known chemiætry which involves~ bubbling ~aturating a~un~s :of~hydrogen chloride directly into a di~hloroethane solution of ~co~pound 10. In thi~ rega~, the solution used to~prepare c:ompound 10 ~an be used in : 20 ~his reaction after that solution has been guenc:~ed ~into water to ;remove ac:etic anhydride and sodium acetate, dried and r~covered. Th~ reaction genera~lly proceeds~ ov~r a~period ~of about 4-6 day~
arld::hydrogen chloride is bu}~bled ~nto the ~;olution 2S perios~ic~lly ~.g., about o~ce: ever 1-2 days).
Aft~r reaction ~coBpl~tiont the solution is quenched in aqueous :sodiumi :bicar~onate at abo~lt .
0-5 c C and the product is recoveredi after drying the organic layer and tripping the solution to providl3 0 for compound 2 (one spot on t~I~co) Compound 2 is then converted ts: the (CHz~8COOCH3 açllycon by well known ch~imiætry which involves reaction of compound 2 with H0(~2)8C:OOCH3 in anhiydrous dichIoromethane - 35 containirlg molecular sieves in the presence of an ~092f~2564 PCT/CA92/00245 -- 31 __ ~I10~7 equivalent amount of mercuric cyanide. The reaction is generally conducted at room temperature for a period of about 12 to 24 h~urs. Upon reaction completion (as evidenced by t.l.c.), the reaction solu~ion is filter through silica and the resulting solution i5 quenched by adding the reaction solution to cold wat~r. Th~ organic layer is recovered and the washed twice with an aqueous potassium iodide (5 weight/vol percent) solution and then with a `10 saturat~d aqueous sodium bicarbonate solution. The re8ulting organic ~olution is then dried and the solv~nt removed by stripping to pro~ide for co~pou~d 3.
The 3, 4, and 6 hydroxyl groups of compound 3 ar then deprotected by reaction with sodium methoxide in met~anol to prc~vide for N-ace~yl-gluco~;amine-0~, compound 4. This compouIld s::an r~acted with Cs~5CH:(OCH3)2 in, for example, an acidic ~: me~ium in arl appropriate solvent ~t around ~10-50C
for about 4-6 hours to provide for the 4, 6-0-dipxotected b~nzylidine co~pQund 5. In turn, compound S can be reacted wi~h p-metho~ybenzyl :: ~ : t~ichlo~oacetiiaidate in an appropriate olvent e.g. ,- DMF, dich~oromethane) in the pre~;enc:e oi~ a catalytic amount:of aal acid (e.g., p-tolu~nesul~onic acid) to provide for the p-methoxybenzyl protec:~ed 3-hydroxy compound 6. Trea~ment of compound 6 with sodium cyanoborohydride in acetonitrile followed by he dropwise additio~ of HCl saturated ether at about 0C leads to~compound 7.
: Alternatively~ compound S can be blocked at the 3-hydroxyl group by reaction with, for example, allyl bro~ide and base (e.g., barium hydroxide/barium oxide) to provide for comp~und ~.
3~ Treatment of compound 8 with sodium cyanoborohydride W092/22564 PCT/CA92/002~45 c~ 32 --in tetrahydrofuran followed by the dropwise addition of HCl saturated ether at about 0C lead~ to compound ~.
Because compoundæ 7 and ~ contain only a freo hydroxyl ~roup at the 4-position of the blocked GlcNAc-OR saccharide, subseguent reaction with an appropriately blocked galactose will result in for~ation of a type II LacNAc-OR structure t~Ga~ 4)~GlCNAc O~3.
~ cause compound 5 contains a free hydroxyl group only at the 3-po~ition of the hlocked GlcNAc-0~ saccharide, subsequent reaction with an appropriately blocked galactose will resu}t in formation of ~ type I structure t,BGal ( ~--3 ~ ,BGlcNAc-15 OR].
:~ ~lternatively, compound 1 can be converted to ^
co~pourld 11 by reaction of compound 1 with an eguivalent of p-c:hlorothiophenol in dichloromethane at room te~perature in the presen ::e of 2 equi~ral~nts 20 o~ boron trifluoride etherate BF3-~therate ~ to provide for co~apound 1~.
In yet another embodiment, compound 1 is ao~rt~d to c~mpound 12 (or th~ bromo analogu~ by follow~ng simi~ar procedures s~t forth above ~or compound 2.
Compound I2 is con~erted to compound 13 (R =
~C~H3~ by reac~ion wi~h ethanol in manner similar to that of compound 3 wi~h the exception that ethanol replaces HO(~H2) 8COOCH3. Compound 13 is then conYerted to compound 1~ with sodium methoxide/me~hanol and is then con~erted to c~mpound lS by reaction with bis~tributyltin] oxide in refluxing toluene containing tetraethylammonium bromide followed by reaction with benzyl bromide.

3 7 ~3 ,' Because compound 15 contains free hydroxyl groups at the 3- and 4-positions of the blocked Glc~A~-OR saccharide, subsequent reaction with an appropriately blockad galactose will result in S formation of both a type I structure ~Gal(1~3)~GlcNAc-OR] and type II structure ~Gal(1~4~GlcNAc-OR~ which are readily separated by : co~entional technique~ including chromatography.
Com~ound a6 is prepared by treating p-chloro-thiophenol with 0.95 equivalents of potassiumhydroxide in ethanol followed by heating the solution to abou~ 4OA-5O-C and then adding about 0-5 equivalents of c~mpound 2 to the reaction solution.
The reaction is ~aintained at 40-50-C for about 1-2 : 15 hours and the product 16 precipitates upon cooling the ~olution and is reco~ered by filtration.
In Figure 2, the synthesis o~ c~poun~s 17 - 20 are set ~o ~ h in the ex~m~les hereinbelow. ~he : process to prod~ce ~he highly crystalline fucose intermediate 20~ from L-fucose as shown in Fisure 2 i o~el. This procedure opti~izes the production : of ~-~ucopyra~ose tetraacetate 17 by adding acetic anhydride (AcOAc~dropwi~e to ~ slurry of fucose and about ~quLmolar ~mounts ~e~g., abo~t 1.1 2S ~ui~alents) of ~odium acetate tNaO~c) maintain~d at bout 50-55 C in~dichloroethane (DCE3 and stirred at thi te~perature for a suffici~nt period of ti~e to result in ~ormation of compound 17 ~e.g., for about .
2-3 days)O The reaction mixture is treated with :: 30 water, quenched into ice water, extracted with additional dichloromethane and dried and partially concentrated to provide ~he peracylated compound 17 (about 4~ ratio of l-acetate~.
Compound 17 is then r~acted with an approximately equivalent amount of p-chl~rothio-W092/22~ PCT/CA92/0024 ~ - 34 --~,~' phenol (p-Cl-Ph-SH) and approximately 1 to 3 (preferably 2) equivalents of boron trifluoride etherate (BF3-OEt2) in a suitable solvent (e.g., dichloromethane) to provide the p-chlorophenyl 2,3,4-tri-O-acetyl-B-thiofucopyranoside, compound 18. The reaction conditions employed are not critical and temperatures of from about O~ to about :~: 25-C (preferably at room temperature) and reaction times of about 3 to about 16 hours can be used.
`10 Compound 18 is quickly deacetylated under Zemplen conditions:(NaOMe, MeOH) to yield p-chlorophenyl ~-thiofucopyranoside 19 as a crystalline product in S5-65% overall yield from fucose after recrstal}ization from an appropriate l5~ solvent (e~g., isobutanol). Again, the reaction : conditions employed are not critical and : ~emperatures of from about 15- to about 30-C and reaction times of about 1 to about 10 hours can be :; used.
Compound~a9 is, in turn, readily benzylated with benzyl chloride:or benzyl bromide to yield p-chlorophenyl~2,3,4-tri-O-benzyl-B-thiofuco-pyranoside, compound 2Q, in 45-50% overall yield ~:~ from fucose. ~As before, the reaction conditions ~: ~ 25 employed are not~critical and temperatures of from : about 15- to about 3 a ~ c and reaction times of about ~ 4 to about 48~hours can be used. In general, at ~:
least 3 equivalents of benzyl chloride or bromide are employed and the reaction is generally conducted in the presence~of at least about 4-5 equivalents of a ~uitable base (e.g., potassium hydroxide -- K~H) in a suitable inert solvent (e.g., dimethoxy-sulfoxide -- DMSO).
In a preferred embodiment, about 3 equivalent 3s of base are added to the reaction system prior to W092/22564 PCT/CA92/00~4 __ 35 __ h 1, ~ ri ~j~ r~

addition of about 3 equivalents benzyl chloride or benzyl bromide. ~fter about 18 hours, an additional 1.5 equivalents of b~se and an additional equivalent of benzyl chloride are addad.
The simple reagents, easy processing and highly crystalline products eliminate the chromatography that frequently has been required using heretofore described methodology.
Thus, in this aspect, the present invention `10 relates to ~ method for the preparation of p-chlorophenyl 2,3,4-tri-O-benzyl-B-thiofucopyranoside which co~prises ~he ~teps:
(a) contacting L-fucose wi~h sodium ac~tate in dichloroethane maintained at a temperature of from about 50-55CC while adding dropwise at least 4 equivalents of acetic anhydride;
(b) ~aintaining the ~olutio~ pro~uced in ~tep (a) above at about 50-55-C for a suf~icient p~riod of time o as to produce the 1,2,3,4 tetraacetylated ~0 deriv~tive of L-~ucose;
(c) re~cting the product produced in step ~b) : abo~e wi~h at lea t one equivale~t of p- hlorothio~
phenol and Pxom about 1 ~o about 3 equivalents of boron trifluoride eth~ra~e under conditions suf~icient to prsvide the p chlorophenyl 2,3,4-tri-O ac~tyl-B-~hiofucopyranosid~;
~ d) remuving ~he acs yl blocking groupC by contact~ng the p-chloropheny} 2,3,4-tri-O-acetyl-B
thiofucopyranoside wi~h sodium methoxide and m~thanol under conditions sufficient to provide for p-chlorophenyl B-thiofucopyranoside; and (e) contacting the p-chlorophenyl B~thiofuco-pyranoside produced in step (d~ with benzyl c~loride or benzyl bromide in the presence of a bace and 9 ~ 36 --under conditions sufficient to provide for p-chloro-phanyl 2,3,4-tri-O-benzyl-B-thiofucopyranoside.
The synthesis of compounds 21-24 are conducted by following known techniques, for example those S described by Matta et al. 27 In the procedure of Matta et al., compound 23 can be con~erted to either a 3-acetyl (co~pound 24~ or the 4-acetyl blocking group (not shown). In turn, both of theee compounds are then blocked at the remaining hydroxyl group ` 10 with a chloroacetyl blocking group by acetylation with chloraacetylchloride in pyridine/dichloro-methane at about O-C a This resul~s in compounds which have di~ferentially protected 3,4-hydroxy groups. The : 15 chloroaaetyl blooking group in either co~pound can be lectively re~ov~ at ~he appropria~e point in^
ths æynth~ by tr~atment with thiourea in pyridine/ethanol (6:1) anà then reac~ed to form the sul~ate or phosphate in the manner describad }: elow.
l~e ~synthe~is of compounds 26 - 31 are depicted ~ in Fi~ure 3 a~d are 5~t forth in the exa~ples :~ ~ herei~elou n In ~is f igure, ~he synthesis o~
ao~pou~ds ~6, 27, 28, and 3C parallels that of ce~pounds ~7,: ~8:, 19, and 20 as se~ ~orth above and illustrated in Figure 20 In this regard, benzyl 4, 6-0-be~azyliderle-2 0-benzoyl-3 0-chloroacetyl-B D-thiogalactopyranoside ~compound 31) ha~: be~n produced wilthout the necessity of chromatography.
D Galac~ose pentaacetate ~6 is produs::ed by slurring D-galartose and about an equimolar amount (e.g., abou~ 1.1 equivalents) of sodium acetate tNa0~c:~ in dichloroethane ~DC~:), heating to reflux and addirlg at least 5 equivalents of acetic anhydride ~cOAc~
dropwise to the refluxing solution (about ~0-85 C) 3s and then maintaining the reaction system at this 2 1 ~` O r~ (?

temperature for a sufficient period of time (about 16-32 hour~) to result in formation of compound 26.
This procedure optimizes the yield of ~-D-galactose pentaacetate 2~ and controls the exotherm of heretofore known procedures.
After workup of the solution in a similar ~anner to that described above for c~mpound 17, the product is treated with approximately e~uimolar amounts of benzyl mercaptan (Ph-CH2-SH) and from `10 about 1-3 ~preferably ~wo) eguivalent sf boron trifluorida eth~rate (BF30Et2) in dichloromethane.
The reaction conditi ~ ~ are not critical and the reaction is preferably conducted at from about O-C
to about 30-C for a period a~ouut 6 to 16 hou~s to yieId after crystallization from hot m~thanol or hot : isopropanol 55-65% of benzyl 2,3,4,6-tetra-O-acetyl^
~-D-t~iogalaato-pryanoside, compound Z7O
Deacetyla~ion under Z~mplen conditions (sodium ~ethoxide/meth~nol) leads to compound 28~
Deacetylation reaction conditions are not critical and ~he reaction is generally conduct~d at roo~
temp~rature for a period of ~rom about 2 to ab~ut 15 hours. ~fter th~ deacetylation reac~ion is complete : (a. judged by t.l.c~), the solution is neutralized with an acid ion exchange resin7 filtered and e~ap~rated to dryness to provide for compound 28.
The r~sidue is crystallized from hot acetone ~nd the product is taken up in dim~thylformamide or acetonitrile and treated with from 1 to 2 equi~alents (preferably 1.4 equivalents) of benzaldehyde dimethyl acetal and about 0.25 to 3 weight percent of p-toluenesulphonic acid (based on compound 28). The reaction conditions are not critical and preferably the reaction is conducted at ~5 room temperature and is generally complete in about W0~2/2~564 PCT/C~9 /00245 t 12 to 24 hours. After neutralization, the benzyl 4,6-O-benzylidene B-D-thiogalactopyranoside, 29, is isolated and crystallized from hot isopropanol.
Benzyl 4,6-O benzylidene-3-o-chloroac~tyl-B-D-thiogalactopyranoside 30 is prepared by chloroacetylation using from about 1 to 3 ~preferably 2) equivalents of chloroacetylchloride which is added to a dimethylformamide (D~F) solution containing benzyl 4,6-O-benzylidene B-D-thiogalacto-~0 pyranoside 29. The chloroacetylchlorid~ is add~ddropwi~e while maintaining the DMF solution at from about 40- to about -lS-C (preferably at -25C~.
Under these conditions, it is unexpectedly been found that the use of D~F permits selective chloroacetylation of compound 29 without the need for additional basè. The reaction is generally complete in about 10-2~ hour~.
Benz~l 4 r ~-O-benzylidene-3-o-chloroacetyl-B-D-thiogalactopyranosi~e (compound 30) i5 ben~ylated ~ 20 with at least 1 equivalent (and preferably~about 2 : : e~uivalent ) of benzoyl chloride in a suitable ~olvent co~taining a ba~e (e.g., pyridine/methyl ne chloride) wi~ fr~m about 0~1 to about 1 weight p~rcent of dimethylaminopyridine ~DM~P~ as a cataly~t. Th~reaction conditions are not critical and preferably th~ reaction is conducted at from about O~C to about 30C and for.about 1 to about 4 hours tpreferably room temperature for 2 hours) to give cryst~lline benzyl 4,6-0-benzylidene 2-O-benzoyl-3-O-chloroacetyl-B-D-thio-galacto-pyranoside, compound 31, in approximately 10-20%
overall yield from galactose.
The advantag of this approach is that after subsequent assenbly the block d intermedia~es will 35 be simply deblocked and modified by sulfation or W09~/22564 2 1 1 ~ 7 ~J r/ PCT/CAg2/00245 phosphorylation. The mat rial is crystalline and the process obviates the need for chromatography. The sulfates and phosphates of the galactose moiety of blocked Lewisa and LewisX can also be made using compound 32 in the synthesis of these compound.
This compound is made by direct benzoylation of both the 2,3-hydroxyl groups of compound 29. Howe~er, after deblocking, both the 2 and 3 hydroxyl groups of galactose are then available for sulfation and phosphorylation and the selectivity is not as e~fici~n~0 Compound 29 can be converted to the 2,3-dibenzoyl prot@cted compound 32 in a manner similar to that described above for the preparation of compound 3~. In this case, 3-5 equiv~lents of benzoyl chloride are generally 2mployed.
~: Co~pounds 3~ and 32 are convert~d to compounds ~: 33 and:32~ (shown in Figure 5~ via known methodology Norberg et al~ 26) U ing bromine tetra~thylammonium ~: bromide.
Alternatively, compound 3~ can be converted to ~;~ compound 3~ by contacting compound 31 with 80%
:~ acetic acid~water at approxLm~tely 50~C for about 1-hours. Compound~34 is th~n conver~ed to compound 35 ~y *rea ~ en~ with acetic anhydride/pyridine in di~hloro~ thane. ~
In ano~h~r embodiment, ;ompound 32 is treat~d with sodium cyanoborohydride and ceric chloride to ~, provide for the benzyl-2,3-0-dibenzoyl-4-o-ben2yl-~-D-thiogalactopyranoside (not shown~. In turn, this compound is chloroacetylated at the 6-hydroxyl group. After formation of the LewisX or ~ewis~
: struc~ures, the ~hloroacetyl group can be electively removed (as described above) and then : either phosphorylated or sulphonated so as to provide for the 6-phosphate or 6 sulfate deriYative.

W092/22564~ P~T/C~92/~0245 Thus, in ano~her of i~s me~hod aspects, the pr~sent in~ention relates to a method for the preparation of benzyl 4,6-di-0-benzylidene-2-0-benzoy~-3-0-chloroacetyl-B-D-thiogalactopyranoside which comprises the step~:
(a) contacting D-galactose wi~h sodium ac~tate in dichloroethane maintained at a temperature of from about 75-~5-C while adding dropwise at least 5 ~uivalents of acetic anhydride;
(b) maintaining the solution produced in step ~) a~ove at about 7~-85-C for a suffici~nt period of time so as to produce the 1,2,3,4,6-penta-acetylated derivative of D-galactose;
(c~ reacting the product produced in step (b) lS ab~ve with at least one ~uivalent of benzyl ~; ~ercaptan and about 1-3 equivalents of boron -^
trifluorid~ etherate under conditions suf~icient to pro~ide the benzyl 2,3,4,~-tetra 0-acetyl-B-thiogalactopyranoside;
(d~ remo~ing ~he ace~yl blocking groups by contacting the b~nzyl 2,3~4,6-t~tra--O-acetyl-~
thiogalactopyranoside with sodium methoxide and methanol under conditions sufficien* to provide ~or phenyl B-thiogalactopyxanoside;
~e) contacting ~he benzyl ~-thiogalaG~o-pyranoside produced in step (d) aboYe with :: benzylaldehyde dimethyl acetal and p-toluene~u}fonic acid under conditions sufficisnt to provide the benzyl 4,6-di-O-benzylidene-B~D-thiogalacto ~0 pyrano~ide;
(f) adding chloroacetylchloride to a dime~hylformamide (DMF) solution containing the benzyl 4,6-di-O-benzylidene-B-D-thiogalacto-pyranoside produced in step (e) above maintain~d at a temperature of from about -40C to about -15C for W092/22~64 PCT/CA92/00245 -- 41 __ ~Jl 1 ~ 7~ 1~

a sufficient period of time so as to provide benzyl 4,6-di-O benzylidene 3-O-chloroacetyl-B-D-thiogalactopyranoside; and (g) adding benzoyl chloride or other suitable ~enzoylating agent to a solution of benzyl 4,6-di-O-benzylidene-3-Ochloroacetyl-B-D-thiogalacto-pyranoside in a suitable solvent containing a base and dimethylaminopyridine under conditions sufficient to provide for benzyl 4,6-di-O-~enzylidene-2-O-benzoyl-3-O-chloroacetyl-~-D-thiogalactopyranoside.

2B. S~NTHESIS OF TYPE LEWISX STRUC~URES
[~Gal(1-4)~Fuc(1~3)]~GlcNAc-OR]
-~ Fiyure 4 illustrates one m~thod for lS synthesizlng blocked type II b~ckbones and conv~rsion to blocked LewisX struatu~esO In ~hi8 ~igureO the 2,3 hydroxyl groups of the galactose are di~ferentially blocked ~o tha . at the appropriate point in the synthesis of LewisX and L2wisa derivative~, the chloroacetyl protecting group at ~he 3-position of galactose is selectively removed and then co~verted to the sulfate, pho~phate or OoH~8~OO~ group. ~1BO~ as noted above, ~he chloroac~tyl protQcting ~roup can be selectiv~ly placed a~ the 6-position o~ the galactose and then .elQctively removed ~o as to allow for the formation of the sulfate, phosphate or -OCHRl8COOH group at th~
~-position of galactose~
Specifically, in Figure 4, ¢ompound 7 and co~pound 33 are co~bined to form compound 37. ~his is acc~mplished by dissolving co~pound 7 and approximately 1.5 equivalents of compound 33 in dichloromethane containing molecular sieves to which is added about 1 equivalent (based on compound 7~ of W092/72~64 PCT/CA92/00245 c; ~

2,6-di-t-butyl-4-methylpyridine. The reaction is stirred for 30 minutes at room temperature and then cooled to -50~C. An anhydrous toluene solution containing approximately a slight excess (e.g., S about l.~ equivalents) of silver trifluoromethane sulfonate is then ~dded to the solution and the reaction is allowed to warm to -15C o~er 2 hours and maintained at that temperature fDr an additional 5 hours.
At thi~ time, the molecular sieves are removed by ~iltration by passing t~rough celite a~d the recovered solution is guenched by addition to a saturated sodium bicarbonate solution. The organic extract is then wash~d with water, with aqueous O.SN
:~ 15 ~Cl, and then wi~h water~ The organic solution is ~ ~hen dri~d and conce~trated in vacuo to provide a ;~ ~ c~uds product of compound 37. This is then purified : by conven~ional techniqu~s such as colu~n ~:~: chr~matography using silica gel and hexane~ethyl acetate (1:1) as~the eluant.
To a dichloromethane solution containing compound 37 is added an excess of dichlorodicyano ~ inone ~DDQ) which selectively :: r~moves the p-methoxybenzyl protecting group to ~ 25 ~provide compo~nd 38. This co~pound is fucosylated ; ~ wi*h an ~xc~ss of ~ompound 20 ~about 1. 3-1r 5 e~ui~alents) in dichloromethane containing mercuric bromide or cupric bromide and about 1-1.5 volume perce~t DMF to give blocked LewisX compound 39.
After work-up and chromatography compound 39 is treat~d with thiourea to removP the chloroacetyl group and the compound is sulphonated with sulphur : trioxide/pyridine complex in DMF at 0C for 2 hours to provide compound ~1. The blocking groups on 35 compound 41 are then removed by conventional techniques to provide for the LewisX analogue having a sulfate group at the 3-position of the galactose unit.
Alternatively, compound 25 (or the 3-chloroacetyl analo~ue of compound 25 describ~dabove--not shown3 can be used in place o~ compound 20 in the above synthesis. Removal of the chloroacetyl blocking groups on the 3-hydroxyl of the galactose and the 4-hydroxyl of the fucose pr~vides an facile route to the preparation of a ~isulfated, diphosphQrylated ~wisX derivatives.
In another embodiment, compound 40 can then be alkylated by ~irst adding an appropriated ba~e (e.g., silv~r oxide, barium hydroxide, ~odium hydrid~) and then adding benzyl bromide acetate (BrCH2COO8n) or other ~imilar acetate~ (e.g., :~ BrCHR18,COOBn - where R18, is alkyl of from 1 to 7 ¢arbon ato~s or -COOBn) to the reaction m~dium in an appropria e solvent such a~ DMF. After reaction co~ple~ion, the be~zyl e~ter(s~ ic (ar~) readily :: r~moved by conven~ional hydrogenation techniques which additionally removes the other be~zyl prot~cting groups and the benzylidine protecting grsupO Trea~menk with sodium methoxide/methanol pr~vides for a OCH2COOH (or -OC~R1~COOH wh~re R18 i~
alkyl of fr5m 1 to 7 carbon atoms or -CQO~) substîtuted to the 3-position of galactose. Similar type chemistry can be performed at the &-hydro~yl group of the galactos~ or at the 4-hydroxyl group of the ~ucose by use o appropriate blocking groups.
In another embodiment, compound 40 can be treated by known methods48 t~ pro~ide for the 3-phosphate compound~ Specifically, compound 40 can be treated with diphenylphosphorochloridate and ~-dimethylaminopyridine (1:1) in pyridin~ at O~C. The W092/22564 PCT/CA9~/00245 ?~ 44 --solution is allowed to warm to room t~mpera~ure over 0.5 hours and stirred for 15 hours. The resulting compound is then hydrogenated under conv~ntional conditions (first with H2 in EtOH with Pd on carbon for 15 hours and then with H2 in EtO~ with PtO2 for 3 hours~ to provide for the phosphate derivative at the 3-position of galactose. Further deprotection leads to the modified LewisX compound having a pho~phate substituent at the 3-position of ~0 galacto e) which is purified and converted to its di~odium salt by contac*ing a solution of ~his compound with a sodium form of Dowex 50 x 8.
As is apparent, t~e procedures set forth above can also be used to introduce a phosphate or a -OCH~8COOH group at the 6-position of galactose or a phosphate group on the fucose. -~

Figure 5 illustrates another method foryn~hesizing bl~cked type II ~ackbones and~
con~ersion ko blocked Lewis~ structur~s. In this fi ~ e, th~ 2,3 hydroxyl gr~ups of the galactose ~re not di~f~rentïally blocked and, accordinglyd while the resulting com~ound ~5 tand ~he ~yp~ ~ ~nalogu~) i~ u~ul ~or preparing the 3-sul~ate (as p rt of a :~ mixture with the ~-sulfate which can be purifi~d by chromatography) t is not as ver~akile as the synthetic scheme set forth in Figure 4.
In any eYent, in Figure 5, compound 7 and approximately 1O6 1.7 equivalents of compound 32 are dissolved in dichloromethane containing molecular sieves to which is added about 1 eguivalant (based on compound 7~ of 2,6 di-t-butyl-4-methylpyridine. The reaction is stirred for 30 minutes at room temperature and then cooled to -~092/225~ PCT/CA92/00245 -- 4S -- 2 11 0 7~l~

50C. An anhydrous toluene solution containing approximately a slight excess (e.g., about 1.2 equivalents) of silver trifluoromethane sulfonate is then added to the solution and the reaction was S allowed to warm to -15-C over 2 hours and maintained at that temperature ~or an addi~ional 5 hours.
After reaction completion, the reaction system was worked up to provide a crude product of compound : ~2. This is then purified by conventional :~ 10 te¢hnigues such as column chromatography using ~ ~ silica gel and toluene-ethyl acetate (1:1) as the : eluant. : ~
To a dichlorame~hane solution containing :~ ~ compound ~2 i ~added an exce s of dichlorodicyano-15: guinone ~DDQ) which celectively ramoves the p-methoxybenzyl protectins group to pro~ide compoun~
3. This compound is;fucosylat~d with an excess of compound 20 (about 1-3 equivalents and pre~erably about 1.3-1.5~equivalents) in dichlor~methane : 20~ con~aining mercuric bromide vr cupric bromid~ and :abo~t 1~2 volume percent DNF to give blocked LewisX
co~pound A4. ; After work-up and chromatography compound ~: is~treated with sodium :: ~
~ :~ethoxide/methanol:to remove the benzoyl blocking : : : 25 ~group~ a~ the 2,3-positions of the galactose o as to provide for co~pound 45. This compound is th~n sulphonated with~sulphur trioxide/pyridine complex in DMF at 0C for Z hours to pro~ide compound 46.
Compound ~6 is produced as a mixture o~ the 3-: 30 sulfate and the 2-sulfate (or 2,3-disulfate) which :: : is separated by chromatography (e.g., column : chromatography on silica). Conventional : : deprotection of the removable protecting groups provides for the sulfate derivative at the 3~
position of galactose for LewisX, compound 47, which W092/22564 ~, PCT/CA92/0024S

i ~-' -- ~6 --can be passed onto an anion exchange resin (sodium form) to generate the sodium salt.
Additionally, lactose can be used in the methods of this invention in place LacNAc by merely a suitable b~ocking group at the 2-hydroxy of the glucos moiety of the lactose structure49.
Dif~erential blocking of the lactose provides for a compo~ition having a electively removable blocking group at the 3 a~d/or 6 position of the galactose.
~10 Thi8 compound is th~n selecti~ely deblocked at the 3 and/or 6 po~ition and then derivatized to the 3 and/or 6 sul~ate, phosphate or -OCHR~8COOH.
Afterwards, the remaining blocking groups are removed and the fucosyl unit added enzymatically s ( ~ee below) .

2C. S~NTHESIS OF LEWISA STRUCTURES
~Gal(1~3j[~uc(1~4)]~GlcNAc-OR]
While Figures 4 and 5 illustrzte ~he synthesis of LewisX struc~ures, ~ewisa structures are readily prep~red in a ~imilar manner, a~ illustrated in Figure 8, using appropriately blocked GlcN~c-OR
structures. The ~Gal(1~3)~Glc~c-~R structures can be prepar~d, for example, ~rom c3mpounds 5 and 35.
Specifically, compound 3~ is first conver~ed to the 2S l-~-bro~o derivative via known methodology (Norberg et al.26) using bromine (Br2~ and ~etraethylammonium bromide (Et4~Br ) at about O~C. About 1.5 equivalent~ of this compound and compound 5 are dissolved in dichloromethane (Cl2CH2~ containing molecular sieves to which is added about 1 equivalent (based on compound 5) of 2,6-di-t-butyl-~-methylpyridin~. The reaction is stirred for 30 minutes at room temperature and then cooled ko ~092/22564 2 ¦ PCT/CA92/0024 47 __ -50C. An anhydrous toluene solution containing approximately a slight excess (e.g., about 1~2 equivalents) of sil~er trifluoromethane sulfonate ~silver triflate) is then added to the solution and the reaction is allowed to warm to -~5-C over 2 hours and maintained at that temperature f or an additional 5 hours. Afterwards, the solution is allowed t~ come to room temperatur~ and ~îrred o~ernight.
; ~10 At this time, pyridine and dichloromethane are :: ad~ed and the molecular sieves are remo~ed by filtration by pa~sing through celite and the recovered solution is quenched by addition to a saturated sodium bicarbonate solutio~. The organic 15 extract is then washed with water, with asIueous 0.5N
HCl, and then~ with water~, The organic ~olution is ^
then dried and concentrat~d in vacuo ~o provide a ; arude product which; is then purified by conventional techni~ues ~uGh a~; column chromatography usirlg 20: ~ silica gel and hexane-ethyl ac~at. (1~ as the eluant to pro~ride :for compQund 100. The benzylidine protecting ~ro~p of c~mpound 100 is then selecti~ely remo~red by tre~ nt;~with 80% ac:etic acid ~AcOH~ in water ~3~2) t~ proYide for aompound ~01. Compound 25 :01 . is selectively acetylated at th~ 6-hydroxy group of the Glc~Ac unit by trea~ment with acetic , ~
anhydride (AcOAc~ in pyridine at about -20-C to provide for co~pound 102. (i.e., 8-methoxy-~, ;
carbonyloctyl-2-~cetamido-3(2-O-benzoyl-3-~: 30 chloroacetyl-4,6-di-O-acetyI-~-D galactopyranosyl)-

6-O-acetyl-2 deoxy-~-D-glucopyranoside~ This compound is th~n fucosylated in the manner similar to compound 38 as described above to provide for compound 103 and ~hen deblocked and sulfa~ed in the W092/22~64 PCT/CA92/00245 manner described above ~or compounds 40, 41, and ~7 to provide for compounds 104, 105, and 106.
Alternatively, compound 32 is converted to the ~ bromo derivatiYe via known methodology ~Norberg S et al. 26) as described above and the resulting compound is then treated with sodium cyanoborohydride and ceric chloride to provide for the benzyl-2~3-O-dibenzoyl-4-O-benzyl-~-D-thiogalactopyranoside (not shown). In turn, this compound is chloroacetylated at the 6-hydroxyl group and then rsacted with compound 5 in the manner : d~cribed above to provide for the 8 metho~y-carbonyloctyl-2-acetamido-3(4-O benzoyl-6-chloroacetyl 2,3-di O-benzoyl-~-D-galactopyranosyl)-~5 6-O-ac~tyl-2-deoxy-~-D-glucopyranoside. T~is compound is th~n tr~ated in the manner de~cribed above for compound 102 so as to provide for a Lewi~
derivative having a sulfate at ~he 6-position of the galacto~.
In yet another embodiment, both type I and t~pe II st ~ ctures ~an be made simultan@ou~ly by combining compound 15 and compound 33 under appropriate condition~ well known in the art~ For example, compound 15 and approximately 1.5 equivalents of compound 33 ar~ added to dichloromethane containing molecular sieves to whiah is added about 1 equivalent ~based on compound 15) of 2,6-diot-butyl-4-methylpyridine~ The reactlon is .
stirr d for 30 minutes at room temperature and then cooled to -50C. An anhydrous toluene solution contai~ing approximately a slight excess (e.g., a~out 1~2 equivalent~) of silver trifluoromethane sulfonate is then added ~o the solution and the reaction is allowed to warm to -15C over 2 hours and maintained at that temperature for an additional 2 ~ 1 0 7 ~ J
-- 4g --5 hours. Afterwards, the solution is allowed to come to room temperature and stirred overnight.
At this time, pyxidine and dichloromethane are added and the molacular sieves are removed by filtration by passing through celite and the r~covered solution is quenched by addition to a saturated sodium bicarbonate solution. The or~anic : extract is then ~ashed with water, with aqueous 0.5N
~Cl, and then with water. The organic solution is then dried and concentrated in vacuo to provide a crude pro~uct which contains both the type I and type II structures which are separated and purîfied by conv~ntional techniques such as column chro~atography using ~ilica gel and hexane-ethyl lS acetate (1:1) as th~ ~luant.
The ratio o~ type I structure to type II
:~ ~ structur~ rssulting from this rea~tion can be imprQv~d by using the 2-NAc derivati~e of ~lcNH2 co~pound 5. This c~mpound can be readily prepared 20 by reac~ing co~ound 15 with hydrazine, ac~tylating the resulting product with asetic anhydrid~/pyridine :an~ ~ ~n deacetylating the 3,4-hydroxyl groups by treatment with ~odium methoxid~/m~thanol.

2D. ENZY~ATIC RE~CTIONS
In addition to th~ chemical ynth~ses of the Lewis~ and LewisX analogues described above, the appropriately blocked type I t~al(l-3)~Gl~NAc-OR]
and type II t~Gal(1~4)~GlcN~c-ORJ structures (e~g., : compound 37) can be selectively deblocked ~o proYide 3~ for a hydroxyl group at the 3-position of galactose (or at the 6-position) and then sulfonated, phosphorylated, or converted to OCHR~8COOH (each of which are described above). Th~ resulting compou~d is then totally deblocked and fucosylated by using, W092/22~64 PCT/CA92/00245 for example, ~Gal(1-3/~3~GlcNAc ~ 3/4) fucosyltransferase~. The enzymatic transfer of fucose onto the 4-position of &lcNAc to form Lewisa and to the 3-position of GlcNAc to form LewisX
structur~s requires the prior synthesis of its nucleotide (GDP) derivatives. Synthesis of G~P-fucose is preferably accomplished in the manner recited by Jiang et al.17 and which is exemplified in the exampl~ hereunder.
GDP-fucos2 (GDP-Fuc) is then combined with the d~rivatiz~d ~Galtl~4)~1cNAc-oR compound or the derivatized ~Gal(1~3)~GlcNAc-OR compound in the pr~ence of a ~uitable ~ucosyltra~s~erase ~e.g., ~Gal(1~3/4)~GlcNAc ~ 3/4)~ucosyltrans~rase~ under ~5 conditlons wherein fucose is transferred to ~he 3 po5ition of GieN~c of ~he deri~atiz~d ~Gal(1~4~0GlcN~c-OR or the 4-posi~ion of the deri~atized ~Gal(1~3)~GlcNAc-OR compound to form a Lewi~X or Lewis~ ~txuctures respectively.
: 20 Suitable fucosylations conditions, ~nown in the : art, include the addition of ~he fucosyltran~erase to a mix~ure of the derivatized ~Gal(1~4~GlcN~c-OR
(or al~ernatively~the d~rivatized ~Galtl~3)~GlcNAc-OR compound) and the GDP-fucos~ in a appropria~e ~S buffer s~c~ as 50 mM sodium cacodylate in appropriate conditions of pH and temperature such as at a pH of 6.5 and a temperatuxe between 30 and 45C, preferably 35 to 40-C while incubating for about 12 hours to 4 days. The re~ulting ~ucosylated produ t can be isolated and purified using conventional methodology comprising HPLC, ion exchange-, gel-, reverse-phase- or adsorption : chro~atography.

2 1 ~ ~ 7 ~ ~
~- 51 --It is also contemplated that the deblocked t~peI and II structures can be sulfated by u e of an appropriate sulfotransferase.
i 2E, M~DIFICATION ON THE 2 ~ND/OR 6 POSITIONS OF
GlcNAc Figure~ 6 and 7 illustrate two different synthese~ for the retention of the 2-amino s~b~tituent on the glucosamine (i.e., a derivati~e :~ o~ ~Gal(1~3)~GlcN~c-OR or ~Gal(1~4)~Glc~Ac-OR where ~:~ 10 ~he N~c group of GlaNAc has been converted to an amine). As shown b~low, the retention of ~he amino ~roup on the glucosamine unit allows ~or the ~acile preparation of di~fere~t 2-substituted derivati~esu In F~gure 6~ compounds 1, 13, 14, and lS are ~pr~pared in ~he manner described above and : illustrated~in Figure 1~ Likewi~e, l-~bromo-2~3,4,6~tetracetyl-galactose i prepared by ~ir~t ~fonmi~g th~ perace~ylated derisrati~re o~ galactose, compourld ~6. Co~pou~d 26 i~ then convert~d to the 20 1; a-b~omo deri~ative via known methodology IBr/~c2tic acid --: at about 0C ~o 20C) ~o as to :provide for l-~-bromo-2, 3, 4, 6~e . rac:etylated ~: : galactos~.
The l-a-bro~ao-2,3,4,6-~etracetylated galactose 2S (about 1. 2 to about 1. 5 equi~Jalent~;) is add~d dropwise to a solution of c:ompound 15 in dichloro~nethane at abou~ 50 ' C in the pr~sencs of ~: excess calc::ium su}fate, about 4 equivalents of silver carbonate and about o . 5 equivalents of sil~er 30 triflate. The~ rea~::tion is then a}lowed to warm to -30-C and maintained there for-about 1-3 days. The reaction is then quenched by the addition of methanol, warmed to room temperature, and f iltered W092t22564 PCT/CA92/00245 through celite. The filtrate is washed with aqueous sodi~m bicarbonate and aqueous ethylene diamine t~traacetic acid (EDTA). The recovered solution is dried and then stripped in vacuo to provide a crude product containing both the type I structure (not shown~ and the type II structure (compound 48). The residue is chromatographed on a silica gel column eluted with toluene:acetone:methanol (20:3:1) to give compound ~8 as well as the type I analogue (not `10 sho~n).
For convenience sake, further reactions are ~hown on compound ~8, it being understood however, that the same reactions could be conducted on the type I analogue to provide m~dified Lewisa compounds.
Compound 20 is then reacted with one equivalent of bromine in dichloromethane at -20-C for about 1 hour to pro~ide for the 1 ~-bromo d~rivative of : compound 20. The solution is then quenched with a : 20 cold agueous sodium bicarbonate solution. -The ~: : organic solution is dried and concentrated to : approximately half the original volume in ~acuo at room t~mperature. About 2 equivalen~s of this compound are then add to a dichloromethane solution of compound ~8 ~hat contains about 2 egui~al~nt~ of ~:~ mercuric bromide (HgBr2), molecular si~ves and tetraethylammonium bromide. The reaction iæ stirred at room temp~rature for approximately 48 hour~ and the solution is filtered through celite and the 3D ~iltrate washed with water, a 5% EDTA solution, ~aturated a~ueous sodium bicarbonate, and then water. The organic layer is then dried and the solve~t removed in vacuo to provide for compound ~9 which is purified by chromatography on silica gel.

wo g2,22564 2 1 1 ~ 7 0 PCT/CA9210024$

Compound 49 is converted to compound 5Q by conventional Z~mplen condi~ions and compound S0 is then con~erted to compound 5l by conventional methodology (e.g., benzaldehyde dimethylacetal, D~F, S pTSA). In turn, compound 51 is treated with hydrazine acetate in methanol at room temperature for about l - 5 hours to provide for ccmpound 52 which is co~erted to compound 53 by contacting with trifluoroacetic anhydride in methanol.
Alternatively, compound 52 serves as a convenient point in the synthesis to convert this amine to an amide, a carbamate, an urea, a -NHS03H ~roup, etc.
in the manner described below.
Compound S3 can then be sulfated in the same ~anner as described above *or compound ~S.
Alt~rnatiYely, c~pound 53 can ~e differentially blocked at the 2,3 hydroxyl groups of the galacto~e in ~h~ same set forth above for co~pound~ 29 - 31 so as to pro~ide for compounds 54 and 55. In ~urn, compound 55 is ~electively deblock~d with thiourea to provide for c~mpound 5S in the s~e manner escribed above for compound 39 (to provide compound ~ 40). ~ompound 56 is then ~electiYely sulfated in ;~ the mann~r descri~ed above to pro~id~ for compound 57. Alternativ~ly, compound 56 can be converted to the 3~phosphate group on the galactose by reaction with diphenylphosphorochloridate an~ 4-dim~thyl-aminopyridine (l:l) in pyridine at O-C~ The solution is allowed to warm to ro~m temperature ~ver O~S hours and stirred for lS hours. The resulting compound is then hydrogenated under conventional conditions (first with H2 in EtO~ with Pd on carbon for 15 hours and then with H2 in EtOH with P*02 for 3 hours) to provide for the phosphate derivative at the 3-position of galactose. Further deprotection WO g2/22564 PCI/CA92/lD024~i r l leads to the modified LewisX compound having a phosphate substituent at the 3-position of galactose) which is purified and converted to its disodium salt by contacting a solution of this compound with a sodium form of Dowex S0 x 8.
Compound S6 can also be converted to the -CHR~8COOH
in the manner described above.
Lastly, co~pound 57 is deblocked ~y conventional techniques to provide for co~pound 60 which is a LewisX analogue having a 2-amino glucose ~accharide unit in~tead of a GlcNAc saccharide and further having a sulfate at the 3-position of the galactose saccharide unit.
Figure 7 parallels somewhat the chemistry ; ~ 15 depicted in Figure :6 but, because the 3-hydroxyl group of the GlcN~2 derivati~e is blocked (compound^
69), this synthe~ic results only in type II
:stru~tures. ~n particular, in Figure 7, compound 13 prepared by th~ ~sthods described above~ Thi~
a~mpound is then deacety~ated by conven~io~al techni ~ es (sodiu~ ~ethoxide/methanol) to provide for c~mpound l~ which i~ then benzylidenated under co~ventional technigues to provide co~pound 66~
Compound 6C is~th~n treat~d with benzyl chlorids and ~: ~5 ~odium ~ydride in di~ethylformamide at about -20~C
to 20-C to pr~vide fo~ co~pound 670 ~e benzylidine ~:~ group of compo~nd 67 is then rem~ved with 80~
aqueous acetic acid at about 80C for about 1~4 , ~
hours to provide for compound 68. This co~pound is : ~ 30 then selectively acetylated at the 6-position by use of approximately e~uimolar amounts of acetyl chloride/pyridine in dichloromethan at about -10C
~o provide for compound 69. Approximately 1.2 - 1.5 equivalents of the l-a-bromo-2,3,4,6-tetraacetylated galactose (described above) is added dropwise ~o a W092~22564 2 ~ I PCr/CA92/00245 solution of compound 69 in dichloromethane maintained at about -30-C in the presence of about 1.3 equivalents of 2,6,-di-t-butyl-4-methyl-pyridine and about 1.3 eguivalents of silver triflate. The S reaction i~ then maintained at -30-C for 1 hour and then allowed to warm to S-C and maintained there for about 2 hours. The reaction is then quenched by the addition of nethanol, warmed to room temperature, and filtered through celite. The filtrate is washed ~0 with aqueous sodium bicarbonate. The recovered solution is dried and then stripped in vacuo to provide a csude product containing compound 70 which is purified by chromatography on a 8iliC8 gel column eluted with ethyl acetate:hexanes (~:2) to give lS compound 70.
$he b nzyl protecting group is then renoved by^
hydrog nolysi~ (H~/Pd on C) to provide fos compound

7~. Co~pound 71, in turn, is fucosylated in the ~ane manner as described above for compound ~8 (to provide for compound ~9 as illustrated in Figure 6) so as to provide for co~pound 72. Compound 72 is deacetylated by conve~tional techniques described above to provide for compound 73. Compound 73 is then converted to compound 7~ by conventional methodology (e.g., benzaldehyde dimethylacetal, DMF, pTSA), followed by selective acetylation at the 6-position of the partially protected GlcNH2 derivative by an approximately equivalent amount of acetyl chloride/pyridine in dichloromethane maintained at about -50 to about -20-C). Compound 7~ is then converted to compound 79 in the same manner (described above) as compound 53 was treated to provide for compound 60.
Alternatively, the free hydroxyl groups of compound 7~ can be acetylated with acetyl chloride~pyridine in the manner described above and the benzylidine group selectively opened by sodium cyanoborohydride and ceric or aluminum chloride to give the 2,3-diacetyl-4-benzyl-6-hydroxy deri~ative on the galactose moiety (not shown). This compound is then functionalized at the 6-position of the galactose so as to contain a sulfate, phosphate or -CHR~8COOH group at this position.

` In addition to the above, th~ 2,6 positions of the GlcNAc unit can be modified prior ~o coupling so as to provide for type I and type II structures ~odified at these positions.which are then further modifiad in th~ manner described above to prepare :the sul~at~d, phosphorylated or -CHR18COOH
subctitut~d ~wi~ and LewisX structures. As shown ~- by Venot et al., U.S. Patent Application Serial NoO
a7/~ filed ~ay 22, 1992 as Attorney Docket No. 000475-011 and~entitled l'MODIFIE~ SIALYL LEWISA
COMPOUNDS" and by Venot et al., U.S. Patent~
Appl cation Serial No~ 07/ , , filed ~ay 22, ~ ~; 1992 as ~ttorney~Docket No. 900475-029 and entitled : ~ "M~DIFIED SIALYL ~EWISX CONP~UNDS'I, modification at ~; : ~ e 2 and/or 6-positions of ~he GlcN~ moi~ty of typ~ I structures ~ ,~Gàl ( 1~3 ) ~GlcNAc-OR~ and on type 25 II stxuctures ~:,BGal ( ~--3 ) ~GlcNAc OR -- LacNAc-OR3 s~ill permit the use of the ~BGal(1~3/4)~GlcNAc 3/4~fu~osyltransferase on the deblocked compound. The disclos~res of both of these applications are incorporated herein by reference in their en~irety.

i. Modification at the 2-position of GlcNAc W092/22564 P~T/CA92/00245 ? l ~ 3, ~ odification at the 2-position of GlcNAc can be accomplished by a variety of ways. For example, the known8 2-azido-2-deoxy-glucose-OR compound (prepared, for example, by azi~onitration of 4,5,6-triacetylglucal) can be protected at the 6 positionwith a removable protecting group (i.e., Si(C6H5)2tBu) by conventional techniques8 and then combined with an appropriate blocked galactos~
compound in the manner descri~ed above to provide a mixtur@ of blocked ~Gal(1-3)GlcN3-OR and ~Gal(1~4~GlcN3-OR derivatives which are readily separated by conventional techniques.
At the appropriat~ tim~ during synthesis of the Lewis~ or Lewi~ structures, the azido group i~
reduced to ~n a~ino group which can be protected as N-tri~luoxoacztamido~ In turn, the trifluoroacetamido group is remov~d a~ the ~ppropriate point in the ~ynthesis thereby unmasking th~ a~ino group.
The amino group can also be derivatized by con~entional m~thods to provide f~r -NR1~C(O~R~or ( 11 ) 2 ~ ~HCH((~l)zi -NHR12~ a~d ~N(R~)2 groups by co~entio~al me~hods. :For example, ~he -N~2 group can be re~ated, using con~entional t~hniques, with:
a carboxylic acid, anhydride or chloride to provide for amides. Alternatively/ the de~ired acid can be actiYatad, as reported ~y In~zu et al37 and then reac~ed with the amino group~ The carbo~ylic 39 acid, anhydride, chloride, or activated acid is selected so as to provide for an Rto group (i~eO ~ as part of the ~NRl1C(O~R1~ substituent) which is hydrogen or alkyl of from 1 to 4 carbon atoms, with an aldehyde or ketone (of from 1 to 4 3S carbon atoms~ at controlled pH to form an imine W092/22564 PCr/CA92/00245 [-N=C(Rl1)2] which upon reduction (e.g., with sodium cyanoborohydride) provides for an alkylamine substituent ~i.e., -NHCH(R~,~2~ as reported by Bernotas et al.~, with a cyclic car~onate such as ethylene carbonate or propylene carbonate which ring opens upon reaction with the amine to form a carbamate group having an HO-alkylene-OC(O~NH- substi~uent where alkyl~ne is from 2 to 4 carbon atoms as reported by Wollenberg et al.39, U.S. Patent No.
4,612,13Z, with a chloroformate ti.e., ClC(O)OR~3~ in the manner di~clo ed by Greig et al.~~ In thi5 case, the chloroformate has an Rl3 group which i~ alkyl of lS from 1 t~ 4 carbon ato~s, Wit~ O=C ~-C~ pN2) 2 which le ds to an -~
activated intermediate which is then re~cted with an a~ine (HNR14R15) to provide for ur~as r-N~C(O)NR,4R15 as de~cribed by Piekarska-Bartosz~wicz et al.~l, wikh trime~hylamine, sulfur trioxide~(SO3) so as to form the -NHSO3H ~roup as d~scribed by Petitou42, and with derivatized formic acid or other materials to form a fonmamîde (-NH-CHO)43 which can be further 25 functionalized to the isocyano (-NzC=O~ and reduced to the deoxy derivati~e by tributyltin hydride (Bu3SnH) 43 .
Alternatively, the 2-deoxy (R2 = H) and 2-alkoxy glucose derivatiYes [ i ~ e., derivatives of 30 GlcNAc where the N~c has been replac:ed by -H (deoxy or by an -~12 (alkoxy~ ] are prepared using a synthetic scheme similar to that rec:i~ed by Trumtez et al . 43 Specif ically, the known 3, 4, 6-triacylated 1, 2-ortho ester of glucose is deacylated under 3S conventional conditions to give the 1, 2-ortho ester W092/225~ PCT/CA92/00245 7 ~

of glucose. This compound is then converted to the 3,4,6-tribenzyl 1,2-ortho ester of glucose using conventional techniques. The 1,2-ortho ester of the resulting compound is then opened by conventional techniques to provide a protected gl~cosyl donor such as the 1 ~-bromo-2-acetyl-3,4,6-tribenzyl deriYati~e of glucose. This 1 ~-bromo derivative is the~ converted to the glycoside (-OR) by conventional techniques and the 2-acetyl group is `10 then removed. ~he 2-position is now ready for formation of the 2-deoxy by conventional methods such as firs~ treating with carbon disulfide an~
methyl iodide in the presence of one equivalent of a base to form the -C~S)SCH3 deri~ative, followed by 75 reaction with ~ributyltin hydride~ or for the preparation of the 2-~lkoxy. The remaining ~; ~ proteating s~roups are removed æo as to prc~vide ~or 2-deoxy-glucose glycoside or a 2-alkoxygluco~;e : glycoside which c:an then be deriva~ized in ~he 20 nlanne:r described above and illustrated in~- Figure 1 ~:~ without t~e need to form the aglycon.
: :
~: ii. ~odific~tion at the 6-Po~ition of GlcN~c m~ 6-deoxy deri~atiYe of ~lcNAc-OR is : ~ynthe~ized from a known benzylidene ring blocked sa~charid~ (~-methoxycarbonyloctyl-2-acetamido-4,6-O-benzylidene-2-deoxy-~-D-glucopyranoside)~ which is protected at the 3-hydroxy position with a removable benzoyl blocking group (Bz) by reaction with be~zoic : anhydride in pyridine. Further conversion of this aompound by reaction with N-bro~osuccinimide and barium car~onate in car~on tetrachloride ( CC14) at 65C leads to the 3,4-dibenzoyl-6-bromo-GlcNAc-OR
compound. This compound is, in turn, converted to the 3,4-dibenzyl-6-d~oxy-GlcNAc-OR by reaction with W092/22564 PCT/C~9~/00245 (c4Hg)3snH in the presence of AIBN ~azo bis-isobutyronitrile~ a~ 110C followed by treatment with me~hanol/sodium methoxide. This compound can then be d protected by conventional techniques to pro~ide for the 6-deoxy~lcNAc-OR glycoside which can then be derivatized in the manner described a~ove and illustrated in Figure 1 withou~ the n~ed to form the aglycon.
The 6-azido derivatives of GlcNAc-OR can be prepared in the manner described in Figure 9.
Specifically, GlcN~c-OR, compound 1~0, is converted o the pmethoxybenzylidine blocked compound 141 by reaCtiQn wi~h ( CH30) 2CH-C6H4-p-OCH3. This compound is th~n protected at the 3-hydroxyl position by reaction with 4-~H3O-C6H~CH2Br to pro~ide for compound 1~2 where X' i~ 4-CH3O-C6H4-CH2-. Compound~
14~ is partially deprotecte~ at the 4 and 6 positions by reaction with acetic acid ~AcOH) in water at about 45C to provide for co~pound 1~3.
:~:2~ The 6-mecylate, c~mpound 144, is prepared by ~:~; reacting compound 1~3 with mesyl chIoride in pyridine (~sCl/py). The 6-azido derivative, c~mpo~ d 1~5, is then formed ~y reaction with sodium agide in dime~hylformamide (DMF) and removal of th~
3-blocking group with dichlorodicyanoquinone (DDQ) yields comp~und 1~6.
The 6-mesyl compound ~ can also be derivatized to any o~ a number of 6-sub tituents including alkoxy s~bsti~uent~, and the like by well known chemistry.
The 6-azido compound 1~5 can be derivatized to the 6-amino at an appropriate point in the synthesis of the ~ewisA or LewisX analogues in the manner described above. The 6-amino deriva~ive can then be W092/22~64 PCT/CA92/00245 2l~ 07~ ;i further functionalized by conventional me~hods to provide for -NF~C~O)R4, -NHSO3H, -N=C(R~)2, NHCH(R~)2, -NHR6 and -N(R6) 2. For example, the -NH2 group can be reacted, using conventional techniques, with:
a carboxylic acid, a~hydride or chloride to provide ~or amides. Alternatively, the de~ired acid can be acti~ated, as report~d by Inazu et al37 and then reacted with ~he amino group. The carboxylic acid, anhydride, ch~oride, or activ2ted acid i~
: s~lected ~o as to provide or an R4 group ti.e. f a~
part of the ~NR~C(O)R~ substituent) which is hydrogen or alkyl of from 1 to 4 car~on atoms, with an aldehyde or ketone (of from 1 to 4 car~Dn ato~s) at co~troll~d p~ to form an imine t~ (F~)23 which upon reduction (e.g.~ with sodium cyanoborohydride) pr~vides for an alkylamine 5ub8tituent ~i.a., NHCH~R~)2] as reported by rno~as et alO~
with a cyclic carbonate such as ethylene carbonat~ or propylene carbonat@ which ring opens upon reaction with the amine ~o form a ¢arbamate group having a~ HO-alkylene-OC(O)N~ su~sti~u~nt ~ where alkylene i~ fr~m 2 to 4 car~on ato~s 2S
: 25 reported by Woll~berg e~ al.39, U.S. Pat~n~ No.
: 4,612,132, with a chloro~onmate ~i.e., ClC(O)OR7] in the manner disclosed by Greig et al. 40 . In this case, the hloroPormate has an R7 group which is alkyl of from 1 to 4 car~on atoms, with O=C(O-C6H4-pNO2)2 which laads to an activated intermediate which is then reacted with an amine (HNE~F~) to provîde for ureas ~-NHC(O~NR~R~) as described by Piekarska-Bartoszewic2 et al . 41, W092/22564 PCT/CA92/~0245 with trimethylamine, sul~ur trioxide (S03~ at pH 9.5 so as to form the -NHS03H group as described by Petitou42~ and with derivatized formic acid or other materials S to form a formamide (NH-CHO) 43 which can b~ further functionalized to the isocyano (-N=C=O) and reduced to the deoxy d~rivative by tributyltin hydride (Bu3Sn~)43.
The 6-alkoxy derivat~ves of GlcNAc c~n be prepared in the manner descri~ed in Figure 10.
Sp~cificallyf GlcNAc-OR, compound 1~0 t is reacted with C6H5CH(OCH3~2 in an acidic medium in acetonitrile to provide for the 4,6-diprotected benzylidine compound 1~7. Ih turn, compound 147 can I5 be reacted with benzy~ (Bn) bromide and sodium hydride in ~he presence of dLme~hyl~ormamide at around 0C to provide for a benzyl prokec~ing group at ~he 3 po ition, i.e., compound 148. Deprotection at ~ e 4,6 positions by contacting co~pound 1~8 with ac~tic acid a~d water at about 80-~O-C provides for compound 14~. R~action of compound 1~9 with dibutyltin oxide t~Bu~2SnO3 and R~Br pro~ide~ for the : 6-alkoxy com~ound 150. Conven~ional deprotection of ~h~ benæyl group with hydrogan in palladium/carbon yield~ compound 151.
In anoth@r embodiment, compound ~47 can ~P
react~d with ~C6H~C(0)320 in pyridine to provide for a benzoyl protecting group (Bz~ at the 3-poaition, i~e., compound 152. ~eaction of compound ~52 wi~h N-bromosuccinimide in carbon tetrachlorid~ yields ~he 6-bromo compound 153. Compound 153 can be reacted with tributyltin hydride ~(Buj3SnH] in toluene to provide for the 6-deoxy compound 155 which after conventional deprotection of the benzoy W092/22564 21 1 0 7 O ~ PCT/CA92/00245 groups with sodium methoxide in methanol gives the 6-deoxy compound 156.
Th~ 6-SR6 compounds are prepared from the 6-mesyl derivative, compound 1~4, by reaction with potassium thioacetate, CH3C(O)S ~, to give the thioace~ate derivative at the 6-position. This deri~ative is then treated with mild base to produce the 6-SH derivativ~ The 6-SH can be reacted with an alkyl halide (e.g., CH3Br) to provide the -SR~
derivatives which, in turn, can be partially or fully oxidized to the 6-sulfone or the 6 sulfoxide d~rivatives, -S(O)R6 and -S(0)~ where R6 is alkyl of from 1 to 4 carbon atoms.

c. ~L~Y
Without being limited to any theory, it is believed that the modified L~wisX and Lewi5a glycoside~ disclo~ed herein affect the c~ll mediated mune response in a number of ways. Specifically, ~he~Q compounds can inhibit the ability o~ the immune response to become edu~ated about a specific aN~igen when the compound is adminis~ered simultaneously with ~he first exposure of the immun~
~ystem to the antigen. Also, the modified LewisX
and Lewi glyco ide~ disclosed herein can inhibit ~he ef~ector phase of a cell-mediated i~mun~
response (eg., the inflammatory component of a DTH
re~ponse~ when administered after second or later exposures of the iDune system to the same antigen.
A, dditionally, the modif ied LewisX and Lewis~
30 glycosides disclosed herein ran induce tolerance to antigens when administered at the time of sacond or later exposures of the immune syst m to the antigen.

W092/~2564 PCT/CA92/00245 The suppression of the inflammatory component of the immune response by the modified ~ewisX and ~ewisa glycosides disclosed herein is ~elieved to require the initiation of a secondary immune response (i.e., a response to a se ond exposure to antigen~. The modified LewisX and Lewis~ glycoside is generally administered to the patient at least about O.S hours after an inflammatory episode, preferably, at least ab~ut l hour after, and most ~: 10 preferably, ~t least about 5 hours after an inflammatory episode or exacerbation.
The modified LewisX and Lewis~ glyco~ides disclosed h~rein are ef~ective in suppressing cell-: mediated immune responses to an antigen (eg. the inflammatory co~ponent of a DTH response) when~ad~inistered at a dosage range of from about O.5 to about SO mg/kg of body weight, and preferably from about 0.5 tc ~bou~ 5 mgJkg of body weight. The : : : speoif ic dose e~ployed is regulated 3~y the 20 particular cell-mediated im~une response ~eing reated as well as :by the judgment of the attending : cIinician depending upon factors such as the seYerity of the: adverse i~amune response, the age and g eneral condition of the patient, and ~he like. ~he 25 dified I.swis~ or I,ewi nalogue is gen2rally dministered par~nterally, such as intranasally, intrapulmonarily, transdermally and intravenously, alths)ugh other forms of administration are contemplated . Pref erably, the suppression of a 30 cell-mediated immune response t eg . the inflammatory component of a DTH response, is reduced by at least abou~ IO% as oppc~sed to control measurç~d 24 hours after administration of the challenge to the mammal and 19 hours after administration of the modif ied 35 LewisX or Lewisa glycoside as per this invention.

W092/22~64 PCT/CA92/00245 2~ 7 (~ r~;

-- 6~ --In addition to providing supp ession of the inflammatory component of the cell-mediated immune response to an antigen, administration of the modified LewisX and Lewis~ glycosides disclosed s herein also impa ~ s a tolerance to additional challenges from the same antigen. In this regard, re-challenge by the ame antigen weeks after administration of the modified LewisX or Lewisa ~lycoside results in a significantly reduced immune response.
Administration of the modified LewisX and Lewisa glycosides disclosed herein simultaneously with f irst exposure to an antigen imparts suppression of a cell-mediated immune response to the antigen and tolera~ce to future challenges with that antigen.
In this regard the term "reducing censi~iz~tio~"
means that the modified LewisX or Lewisa glycoside, when administered to a mammal in an ef~ect~ve amount along with a suffieient amount of antigen to in~uce an immune response, reduces the ability ofYth~
mune system~of the mammal to become educated and thus sensitized~to:the antigen administered at the same~time as the~:~odified LewisX or Lewis~ glycoside compound. An :"effective amount" of ~his co~pound i~
:: : 25 ~ at amount which~will reduce sensitiza~ion (immunological~education) of a mammal, including humans, to an antigen admînistered simultaneously as determined by a reduction in a cell-mediated response to the antigen such as DTH responses as ~ested by the footpad challenge test. Pr~er~bly the reduction in sensitization will be at least a~out 20% and more preferably at least about 30% or more. Generally, modified LewisX and Lewis~
glycoside compounds related to blood group ~5 determinants are effective in reducing sensitization when administPred at a dosage range of from about O. 5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 mg to about S mg/kg of body weight. The specific dose employed is regulated by th~ sen~itization being treated as well as the judgement of thOE attending clinician depending upon the age and general condition of the patient and the like. "Simultaneous" administration of the compound with the antigen with regard to i~hibiting sensitization means that the compound is administered once or continuously throughout a period o~ time within 3 hours of the administration of an antigen, ~ore preferably the compound is administered within 1 hour of the antigen.
~ The methods of thiæ invention are generally :; achi~ved by use of a pbarmaceutical composition uitable for use in the parenteral administration of : an:e~fective amount of an oligosaccharide glycoside : : related to a blood group determinant. These compositions çomprise a pharmaceutically inert ~-: carrier such as water, buffered saline, ~tco and an effective amount of a modified LewisX or Lewi~
~ :glycoside co~pound 50 as to provide the abo~e-noted : ~ do~age of ~he oligosaccharide glycoside when administered~to a patient. It i~ contemplated that : :suitable pha~maceutical compositions can additionally contain optional components such as an adjuvant, a preservative, etc.
It is also contemplated that other suitable pharmaceutical compositions can include oral compositions, transdermal compositions or bandages etc., which are well known in the art~
The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this W092/22564 P~CT/CA92/#0245 B7 jf~

inventionO Unlfess otherwise stated, all temperatures are in degrees Celsius. Also, in these example~, unless otherwise defined bfelow, the abbreviations femployed have their generally accepted meaning:

s A = ~ngstroms AB - AB pattern ax = axial bs = broad singlet BSA = ~fofvine serum albumin ~10 13C-n.m.r = C'''3 nuclear magnetic resonance ~ = doublet dd = doublet of doubflets ddd = doublet of doublets of doublffPts DDQ = dichlorodicyano~uinone 15 DTH = fdelayed-typfe hypfersensitivity eq = efquatorial : f~ = gram r~-n.m.r. = proton nufclear magnetic resonance i.r. f- infra red : 20 kg = kilogram L = liter m - muItiplet mL - milliliter ~ a ~uartet : 25 s = singlet t - triplet t.l.c. = thin layer chromatofgraphy U = Unitfc fum = microns :
AG 1 ~ 8 ~ormate form) = ion exchanffge resin AG 1 x

8 (formate form) available from Bio-Rad Lahoratoriffes, Richmond, '{~A
Dowex SOW x B (H~ form~ = ion exchange resin Dowex f 50 x 8 (H~ form) available from Df~W
Chemical, Midland, MI
IR-120 resin (H~ form) - amberlite resin a~fallable from Rohm f~ Haas, Philadelphia, PA
IR-C50 resin (H~ form3 = ion exchange resin IR-C50 (H~ form) available from Rohm & Haas, Philadelphia, PA

WO 92/22~i64 r. ~ PCI'/CA92/00~45 ~8 --Commercial~y availa~le components are listed by manufacturer and~where appropriate, the order number. Some of the recited manufacturers are as follow~:

S Merck = E. ~erck AG, Darmstadt, Germany Millipore 3 Millipore Corp., Bedford, MA.
Waters = Waters Associates, Inc., Milford, MA.

The following examples are divided into two parts. The first par* (part I) relates to the ~ynthetic proceduras to make the recited compounds whereas the second part (part II) relates to the biologica} results.

Part I -- Synthetic Procedures ~ Examples 1-24 illustrate syntheses of the :: lS described compounds.

EXAMPLE 1 - Syn~hesis of Benzyl-2-0-benzoyl-4,6-0-benzylidene-3-0-chloroacetyl-~-D-thiogalactopyranoside (compound 31~
:: Dry a 20 L stirred reactor equipped with reflux condenser, heating mantle and 1 L addition funnel.
Charge to thi. reactor 10 L of dichloroethane. Begin to stir the reactor then charge 1 kg D-g~lactose and 500 g anhydrous sodium acetate to the d1chloroethane. Heat this slurry to reflux. Add ~5 dropwise 4 L of acetic anhydride to he rea~tion mixture using the 1 L addition funnel on the reactor. Reflux is to ~e maintained during the 2-4 W092/22~64 PCT/CA92tO0245 -- 69 _~ 7~ j hour addition period. Continue to stir and heat the mixture at reflux overnight.
When the reac~ion is complete as determined by t.l.c., t~rn off the heat to the reactor a~d add 250 mL of water in slow dropwise fashion using the addition funnel. ThiC ~eaction is quite vigorous but is controlled by slowing the addition of the water. Stir the reaction for 1-2 hours. Charge 30 L of cold water ~o a 50 L stirred reactor and begin ~0 stirxing. Drain the co~tents of the 20 L reactor into a 20 ~ polyethylene pail and pour into the ~tirring ice water in the 50 ~ react~r. Stir this mixture for twenty minutes. Drain the lower organic layer into a 20 L polyethylene pail. Extract the agu~ous layer in the 50 L r~actor with an additional 5 L of dichloro~ethane. ~ombine the dichlorome*hane extraet with the first organic layer. Drain th~
agueous la~er to polyethylene pails and discard as a~ueou~ waste.
Return the combined organic layers to the 50 L
reactor and extract t~ice with S L portions of ic~
water for 10 ~inutes. Drain th~ organic layer to a clean 20 L polyethylene pail. Drain the a~u~ous to wa~tP, return the organic layer to the 50 L reactor, stir and add 1 kg of anhydrous sodium sul~ate. Stir for 1-2 hours ~nd then drain the ~olution into a clean 20 L polyethylene pail and ~ilter the solution using a 4 L ~acuum filtration ~et ~or large Buchner attached to a collector~.
Concentrate the filtrate to 8 L then ~ransfer into a cl~an 20 L reactor equipped with stirrer, 1 L
addition funnel and cooling bath. Additional solvent can be added if the level of the solutiDn is below the thermowell. Cool the organic solution to 0 G C using a cooling bath. Charge to this cool W092/22~64 ~ PCT/CA92/00245 solution 724 g of benzyl mercaptan. Add a total of 1.1 L of colorless boron trifluoride etherate in slow dropwise fashion over 2 hsurs using the 1 L
addition funnel. Stir the reaction 3-4 hours after S the addition is complete maintaining the temperature at 0C. The reaction is checked for comp}etion by t.l~c. on silica gel. ~The reaction can be left to ~it ov8rnight].
The reaction mixture is drained into a clean 20 L polyethylene pail. The 50 L reac~or is charged wikh lS L of fiaturated sodium carbonate solution.
T~e 20 L polyethylene pail is slowly transferred into the slowly stirring ~arbonate solution at such a rate that the gas evolution is not ~verly lS vigorous. Stir the ~olu~ion for 20 minutss then increase the rate of stirring. When ga~ evolution -~C . S88 bubble air through the entire ~olution for ~-36 hours.~ Drain ~he organic layer into a clean 20 L polyethylene pail and store in a hood. Extract the sodium carbonate solution with 3-5 L of dichloromethane and :drain this solution into the same 20 L polyethylene pail.
Once th~ ~mell has be~n reduced the organic ~olution can be filt~red u ing a 4 L vacuum ~ltration set and the filtrate evaporated under : r~duced pressure on the 20 L rotovap. 7 L of methanol is introduced into the rotavap flask and the residue heated with the rotavap bath till the re~idue dissolves in the warm methanol. The flask is rotated and allowed to cool. Cool ice water is added to the ro avap bath and t~ flask slowly rotated for several hours. The flask is removed from the rotovap and ~he white crystalline product filtered using a 4 L vacuum filtration set.

W092/225~4 PCT/CA92fO0245 -- 7 1 -- 2 f ~ O ^.~

The benzyl 2,3,4,6-tetra-0-acetyl-B-D-thiogalactopyranoside (-1.3 kg~ is charged into a clean dry 20 L reactor with stirring motor and 7 L
of dry methanol is added to dissolve the material.
The solution is treated with 3 g of freshly surfaced sodium and stirred for two hours. The reaction is checked by t.l.c~ on silica gel using a retained ~ample of the benzyl 2,3,4,6-tetra-0-acetyl-B-D-thiogalactopyranoside with 80:20 ethyl acetate:
m~thanol (v/v) the eluant. The absence of starting material indicates the reac~ion is compl~te.
50 g of fresh methanol washed H~ ion exchange resin is added, the reac on stirred for 15 minutes.
The pH is ch~cked using pH paper to ensure a ne~tral ~: 15 .olution. The r~sin is filtered off under r~duced prassure and the methanol is remov~d under reduced ^
pr~s~ure using the 20L roto~ap. To the r~sidue, 5 L
o~ aceton~ is added to the 20 ~ flask and the solution warmed to reflux. The residue dis~ol~es and is allowed to cool to room temperatur~at which ::~ tim~ ice is added to the bath, the solution ro~at~d with cooling ov@rnight. 800-9OOg o~ benzyl B-D-thiogalactopyr noside cry~tallizes and is filt~red and dried under va~uum.
To 8 L of dry acetonitrile is added 800 g of benzyl B-D-galactopyrano~e, 600 g of ~enzaldehyde : dimethyl acetal and 2-5 g of p-toluenesulphonic acid. The solution is stirred at room temperature ove ~ ight. The reaction pro~ress is checked by t.l.c. When complete, the reaction is brought to pH
7 by ~he addition of triethy}amine. The volume of acetonitrile is reduced to a minimum, 7 L of isopropanol is added and the mixture is heated to near reflux. Most of the product gQeS into the hot isopropanol after ~arming for several hours. The W092/22564 PCTlCA92/00245 mixture is cooled and ice added to the bath and cooling continued overnight to give a precipitate.
After filtering and drying the precipitate, 760 g of benzyl-4,6-O-benzylidene-B-D-thiogalactopyranoside S is obtained.
: 180 g of benzyl-4,6-O-benzylidene-~-D-thiogalactopyranoside was dissol~ed in dry DMF and placed in a jacketed reactor. The reactor was : cooled using a recirculating cooling bath maintained ~10 at a temperature of -25-C and treated dropwise with 108 g of chloroacetyl chloride over 3 hours while stirring:the:reaction mixture. Stirring was continued 24 hours at this temperature then the reaction was quenched into se~eral vo}umes of cold bicarbonate solution. The product was extracted into methylene chloride~ water washed several times, dried over sodium~:sulphate and evaporat d to : dryness. The product was crystallized from isopropanol. Yield: lZ5 g of benzyl 4,6-O-20~ enzyl:idene-3-O-chloroacetyl-B-~-thiogalac~o-pyranoside. ~
S g Benzyl~4,6-O-benzylide~e-3-O-chloroacetyl-,~:
:B-D-thiogalac~opyranoside was benzoylated at room temperature~in mothylene chloride/pyridine using 3 e~ui~alents ~benzoyl chloride and a catalytic amount of dimethylaminopyridine. The solution is quenched into cold sodium bicarbonate solution, the organic layer is washed with saturated copper sulphate solution to remoYe the pyridine the organic layer dried and evaporated. Th~ residue is taken up in hot isopropanol~ and benzyl 4,6-O-benzylidene-2 O-bénzoyl-3-O-chloroacetyl-B-D-thiogalactopyranoside crystallizes from solution. 1H-n.m.r. (CDCl3): ~ =
7.96, 7.4 (2m, lSH, aromatic, 5.79 (t~ lHr H 2~1 5.5 (s, lH, CH), 5.2 (q, lH, H-4, J23 9 9 HZr J3,4 3-3 W092/225~ PCT/CA92/00245 __ 73 __ 2 ~

Hz), 4.5 ~m, 2H), 4.4 (d, lH), 3.99 (m, 5H), 3.~5 ~s, lH).

Example 2 -- Synthesis of 4,6-O-benzylidene-2,3-di-O-benzoyl-B-D~galactopyranosyl bromide (compound 32A) Benzyl-4,6-O-benzylidene ~-D-thiogalacto-pyranoside (10 g) was dissolved in 100 mL
dichloromethane and 6.35 g of pyridine was added.
To the solution was added 9 g o~ benzoyl chloride in dropwi~e fashion and after 1 hour, 50 mg of dimethylaminopyridine was added to the solution and the mix~ure was stirred for an addition 2 to 4 hours. The progress of the reaction was checked by ; to l-c- on silica gel. Benzyl-~,6-O benzylidene-2,3-di-O-benzoyl-~-D-thiogalactopyranoside (compound 3O
waæ i~olat~d by ~uenching the reaction mixture into ~ ~, satura~ed sodium bicar~onate solution and wa~hing the organic extract with water, 5% copper sulfate solution, water, drying and e~apoxating the solvent.
: ~ 20 The residue was crystallized from isopropanol ~o ~ ~ give 10.7 g of compound 32.
: Compound 32, benzyl-4,6-O-b~nzylidene-2,3-di-O
::~ benzoyl-~-D-thiogalactopyranoside (~.89 g), was di$solved i:n lOO ~L of dichloromethane, cooled to O~C, and treated wi ~ a solution of bromine (2.85 g) in 10 mL of dichloromethan After 15 minutes, 1.8 grams of tetraethylammonium bromide was added to the mixtur~ and the mix~ure stirred for 2-3 hours at room t~mperature (~ollowed by t.l.c. on silica gel).
30 A smal l quantity o~ cyclohexane was added to ~uench excess bromine and the reaction mixture wa~ quenched into cold saturate sodium bicarbona~e solution, washed with water, dried and volume of the solution reduced to 30 mL. This dichloromethane solution of 3~ compound 32a was used directly in the synthesis of W092/22~64 PCT/CA92/OQ24~

?~ G1 74 compound 42 without further isolation and/or purification.

Examp}e 3 -- Synthesis of p-Chlorophenyl 2,3,4-tri-O-benzyl-~-L thiofucopyranoside S (compound 20) Dry a 2 L thr~e neck round bottomed flask, r~flux condenser and 500 mL addition funnel. Then cool under a fIow of nitrogen. Charge to the flask 1000 g of L-fucose, 500 g of anhydrous sodium acatate and 800 mL of dxy dichloroethane. ~eat the mixture with stirring to 50-C. Charge to the addition fu~nel 400 mL of acetic anhydride. Add t~e ace~ic anhydrid~ to the stirring, warm ~ 50 o _55 o C) : ~lu~ry in dropwi e fa.hion at a rate that does not : 15 cool the reaction appreciably. Upon completion of ; the additiv~ stir the mixture for 72 hours at thi~
t~mp~rature, removi~g aliquots from the reaction mixture ev~ry 24 hours to check the progr~s of the r~action by t.l.c~
: :~ 20 Wh~n the reaction appears to be complete add 200 m~ of water to he warm stirring mixture dropwi~ over 30 min. and stir for 1 hour at this : temper~ure. This converts the remaining acetic an~ydride to acetic acid. The reaction mixture is quenched int~ 3-4 volumes of water. T~e organic layer is removed and the aqueous layer is extr~cted with 4 L dichloromethane. The combined orga~ic : layers are backwashed three times with 2 L portions of wat~r~ The organic layers ara dried o~er sodium sulphate and concentrated under reduced pressure to approximately 5 L.
To the organic layer is added 925 g of p-chlorothiophenol. The organic layer is cooled with cold water. To the mixture of p-chloro-W092/22~64 PCT/CA92/00245 2 ~ 7 (~

thiophenol and fucose acetates is added 1.72 kg of boxon trifluoride etherate in dropwise fashion. The mixture is then stirred for 6 hours (overnight is ~cceptable~ allowing the reaction mixture to come to S ambient temperature. A small aliquot is rem~ved from the rea~tion mixture and quenched into sodium bicarbonate solution. Once CO2 evolution has ceased, the reaction is checked for completion by t.l.c. If complete, the whole reac~ion mixture is quench2d into 1 L of satura~ed sodium bicarbonate and the organic layer separated after CO2 evolution has fini~hed. The organic layer is separated and air bubbled through this layer for 1 hour.
me separated organic layer is then dried over sodium sulphate and eYaporated to dryness. The ; re~i~ue is;taken up in 1 L of dry methanol in a 2 L^
round ~ottom flask and treated with 1 g of freshly ~ su~faced sodium. The reaction i~ kept under :~ nitrogen for se~eral hours then checked by t.l.c.
2~ for r moval of the acetate groups. The reaction is ne~tralized with H~ ion exchange r~sin and filtered and evaporated under reduced pr~ssure. The re~idue is taken up in a minim ~ of hot isobuta~ol and the : p-chloroph~nyl-8-~-thiofucopyranQside crystallizes from solution aft~r cooling overnight at O-C.
~: Yield: 1060 g.
: p-Chlorophenyl-B-L-~hiofucopyranoside is dissolved in 7 L of dry dimethylsulphoxide. To the .
solution is added 600 g of powdered KOH and the reaction mixture tirred for 30 minutes. Benzyl chloride (1.275 L) is added dropwise to the stirring solution and the mixture stirred o~ernight at room temperature. T.l.c. indicates incomplete reaction so an additional 300 g of powdared KOH is added to the reaction mixture followed 30 minutes later by ~ 91 -- 76 --425 mL of benzyl chloride. The solution is stirred at room temperature until t.l.c. indicates the reaction is complete. If the reaction is not c~mplete after 24 hours, powdered KOH is added followed by 200 mL of benzyl chloride. The reaction is quenched in~o several volumes of water, extracted with methylene chloride, backwashed ~wice with wat~r, dried and evaporated. The residue is taken up in hot hexanes. p-Chlorophenyl-2,3,4-tri-O-~enzyl-~-L-thiofu o-pyranoside crystallizes and is filtered and dried under vacuum. Yield: 1.3 kg.
H-n.m.r. (CDCl3): ~ = 7.57 (m, l9H, aromatic), 4.99 (d, lH), 4.65 (m, SH), 4.55 (d, lH, J12 9 5 Hz)~
3.9B ~t, 1~), 3.55 ~m, 3H), 1.26 (d, 3~ Js6 6-2 ~ 6).

Example 4 -- Synthesi~ of 8-methoxycarbonyloctyl-2-acetamido-4,6-di-O-benzylidene 2-deoxy-B-D-glucopyranoside Compound 5) A 20L glass reactor was charged with 8 L of dichloroethane, 1 L of acetic anhydride and 1 kg of anhydrous ~odium acetate. To the skirring mixture Was added 1 kg of glucosamine hydrochloride and the ~ixture was brought to reflux. ~ further 3.5 L of acetic anhydride~was added dropwise to the refluxing solution over 3-4 hours and the solution maintained at reflux for 36 hours. During the last hour of re~lux 200 mL of water was added dropwise to the solution. The reaction was ~hen cooled and added to 35 L o~ ice water in a 50 L stirr d reactor. The organic layer was removed and then water washed a second time with an additional 20 L of water. The organic layer was dried over sodium sulphate, filtered, and saturated with anhydrous ~aseous HCl WO 92/22S64 ~ 1 L 0 7 o r;

~~ 7 7 ~~

for 2 hours. The reaction was allowed to sit for 6 days being saturated with ~Cl for 1 hour every second day. 2-acetamido-2-deoxy-3,4,6-tri-0-acetyl-B-D-glucopyranosyl chloridc was isolated by S quenching into ice cold sodium bicarbonate solution.
The organic layer was dried over sodium sulphate and evaporated to a brown solid.
: Four hundred grams of 2-acetamido-2-deoxy-3,4,6-tri-O~acetyl-B-D-glucopyranosyl chloride was dissolved in 2 L of anhydrous dichloromethane ~ containing 200 g of activated molecular sieves. 266 : g of 8-methoxycarbonyloctanol was charged to the reaction mixture along with 317 g of mercuric cyanide. The solution was stirred rapidly at room ~: 15 temperature ~or 24 hours. A ter checking for ~: rea~tion completion by t.l.c. ~he reaction mixture ^
was filtered through a buchner funnel of silica and the organic lay~r~washed twic~ with water, twice wi~h a 5% ~olution of potassium iodide and twice 20:~: with a satura~ed solution of sodium bica~bonate.
The soIutio~ was dried over sodium sulphate and ~: ~ :ev~porated to dryne s.~ The residue was taken up in anhydrous methanol and treated with 1 g of freshly cut sodium t~n stirred at roo~ temperature ~ 25 o~ernight. The solution of 8-methoxycarbonyloc~yl ;;~ 2-acetamido-2-deoxy-B-D-gl~copyranoside was neutralized with~acid ion exchange resin and , . filtered and evaporated to yield 218 ~ o~ product : after crystallization from isopropanol /diisopropyl ether.
Two hundred grams of 8-me~oxy arbonyloctyl 2-acetamido-2-deoxy-~-D-glucopyranoside was dissolved in 1.2 L of anhydrous dimethylformamide and treated with 169 mL of dimethoxytoluene (benzylaldehyde dimethyl acetal) and 1-2 g of p toluenesulphonic ~r`~ ~~ 7 B ~~

acid. The reaction was stirred and heated to 40C
for 5 hours, then chec~ed for completion by t.l.c.
When the reaction appears complete the mixture was neutralized with triethylamine and quenched into S several volume~ of ice water, extracted into dichlorome~hane and backwashed several times with water. The organic layer was dried over sadium ~ulphate, evaporated to dryness and taken up in hot isopropanol. After cooling 8-me~hoxycar~onyloctyl-2-acetamido~4,6-O benzyliden~-2-deoxy B-D-glucopyranoside precipitates. It is filtered and dried to yi~ld 106 g of product. 1H-n.m.r. (CDCl3):
~ = 7.41 ~m,SH, aromatic), 6.11 (d, lH, NH), 5.5 (s, lH, C~), 4.63 (d~ lH, H l, J-2 7.4 Hz), 2.29 (t, ~5 2H~, 1.99 ~s, 3H, Ac), 1.58 Im, 4H), 1~29 (bs, 8H).
..

: ~xample 5 -- Synthesis of 8-Methoxycarbonyloctyl-2 acetamido-3-O-p-methoxyb~nzyl~4,6-O benzylidene-B-D~glucopyranoside ~co~pound ~).
To a stirred solution of compound 5 (17.5 g, -3 mmol) in dry dichloromethane (100 mL~ and catalytic amount of p-toluen sulfonic acid (0.2~ to 3 weight perc~nt ba5e~ on compound S~ was added dropwi~e a solution of p-~ethoxybenzyl trichloroacetimide (10 g Z5 in 25 mL CH2C12). The reaction ~ixture was stirred at room temperature overnight. Triethylamine was added to quench ~he reaction, the organic layer was washed with sodium bicarbonate solution and the organic layer dried and evaporated to dryness;
Crystallization in hot ethanol gave 20 g of the desired product. 1H-n.m.r. (CDCl3): ~ 7~56-6.90 (m, 9H, aromatic~, 5.60 (d, lH, NH), 5.3~ (s, lH, PhCH), 4.94 (d~ lH~ J12 8.0Hz, H-1), 3.80 (s, 3H, CH3), WO 9~/22564 n PCr/CA92/00245 ~1~o~

3.60 (s, 3H, CH3Ph), 2.30 (t, 2H, CH2C0), 1.90 (s, 3H, AcNH), 1.80-1.10 (m, 12H, (CH2)b).

Example 6 -- Syn~hesis of 8-~ethoxycarbonyloctyl-2-acetamido-2-deoxy-3-0-p methoxybenzyl-Ç-0-benzyl-2-B-D-glucopyranoside (compound 7~
To a stirred solution of compound 6 (15.0 g, -3 mmol) in 200 m~ of dry T~F were added, 11.0 g of sodium cyanoborohydride, 10 g of molecular sieves 4A
and 5 mg of methyl ora~ge~ The solution w~s cooled to -lO^C and then ethereal hydrochloric acid was added dropwîse un~il the solution remain~d acidic.
on completion of the r~action, i~ was diluted with :; dichlorome ~ ane ~200 mL), filtered through celite lS~ and washed successive~y with aqueous sodium bicarbonate (2 x 100 mL) and water ~2 x 100 mL) and t~en the solvent dried and evaporated to give a yrup. Purification of the mixture on column chroma~ography using silica gel as adsorbent and eluting with hexane:ethyl acetate:ethanol ~20:10:1) ~ave 7~ in 70% yield. 1H-n.m.r. (CDCl3~: ~ 7.40-6.90(m, 9H~ aromaticj, 5.70(d, lH, NH), 4.64(d, 1~, J12 8.0Hz, H-l), 3.86(s, 3H, CH30~, 3.68~s, 3Hp C~30Ph), 2.30(t, 2~, CH2CO), 1~90~s, 3H, N~Ac), 1.80-l~lO(m, 12~, t~H2)s)-pl~ 7 -- Synthesis of 8-Methoxycarbonyloctyl 2-acetamido-2-deoxy-3-0-p-methoxybenzyl-4-0-~4,6-o-benzylidene-2~3-0-dibenzoyl-B-D-galactopyranosyl)~6-0-benzyl-2-deoxy-~-D-glucopyranoside (compound 42) A solution of compound 7 (10.61 g, 19.7 mmol) and compound 32A (1.6-1.7 equivalents based on W092/225~ PCT/CA92/00245 compound 7~ and 2,6-di-t-butyl-4-methyl pyridine (3.11 g, 15~2 mmol) in 250 L of dichloromethane and 40 g of molecular sieves (4A) was stirred at room temperature for 30 minutes, and then coole~ to -50~C
under nitrogen. A dry so$ution of silver triflate (4.47 g, 17.3 mmol) in toluene (40 mL) was added to the stirred mixture. Th~ mixture was warmed ~o -15-C during two hours and kept at -15-C for an : additional 5 hours. At the end of which the mixture ~ ~o w~s warmed to room temperature and stirred : : overnight. 3 mL of pyridine and 250 mL of dichloromethane~were added to the mixture and was filtered over celite, filtrate was washed with saturated aqueouC sodium hydrogen carbonate (200 mL) and then with water (200 m~), aqueous hydrogen chloride (0.5N, 200 mL) and water (200 m~
` concentrated in vacuo. 6.0 g of compound ~ was crystallized as white crystals from ethyl acetate-diethyl ether-hexane. ;The moth~r liquor was ; :20 concentrated, purified over chromatography-(300 g silica gel, toluene:ethyl acetate, (1:1) to gi~e 4.5 ; g pure compound~:~2.~ Total yield was 10.5 g (68%).
Rf~0.~48 (methanol~:dichloromethane, 4:96). ~H-n.m.r.
(CDCl3]: ~ 5.80(t,~1H,~J2 3 ll.O~z, H-2 ), 5.S2(~, ; 25 lN~, CNPhj, 5.25(dd,:1H, J3~ 4.0Hz, ~-3'), 4.88(d, ~ : : lH, Jl 2 ll.OEz,~H-l ), 4.70(d, lH, J12 9 OHz~ ~~
:5~ ~ 1), 3.78~s, 2~,~CH30j, 3.64(s, 3H, CH30Ph).
~: ,.

Example 8 -- Synthesis of 8-Methoxycarbonyloctyl :- 2-ace~amido-4-0-~4,6-0-benzylidene-2 3 -di-O-benzoyl-B-D-yalacto-: pyranosyl)-6-0-benzyl-2-deoxy-B-D-glucopyranoside (compound 43) DDQ (126 mg, 0.5 mmol) was added to a stirred ~ solution of compound ~2 (350 mg, 185 ~mol~ in :; 35 dichloromethane (10 mL) saturated with water. After W092/22564 2 l 1 ~ 7 o ~ PCT/CA92~00245 2 hours at room temperature, the reaction was complete, and organic layer was successi~ely washed with aqu~ous sodium bicarbonate and water, dried and concentrated. Col~mn chromatography gave the desired compound ~3 in 85~ yield. 1~-n.m.r. (CDCl3~:
~ 5.65(dd, lH, J2 3- 10.8Hæ, H-2 ), 5.61(d, lH, 33 $ 4.0Hz, H-3 ), 4.68~d, lH, Jl 2- ll.OHz, H~
4.62(d, lH, Jl2 lOHz, H-l), 3.60(s, 3H, COOCH3).

Example 9 - Synthesis of 8-methoxycarbonyloctyl 2-ac~tamido-3-0-~2,3,4-tri-o-benzyl--fucopyranosyl)-4-0-(4~6-0-benzylidene-2,3-di-o-benzoyl-B-D-galactopyranosyl)-6-o-benzyl-2-deoxy-B-D-glucopyr~no~ide~cQmpound 44) ~5 ~o a mixture o~ copper (cupric) bromide (40 g,^
17.~ ~mol) and 5 g of molecular sieves 4A in lo mL
sf dry dichloromethane were added 1.2 mL of dry DMF
and tetraethylammonium bromide (1.85 g, 8.8 mmol).
The ~ixture was stirred at room temperature for 1 hour and th~n a sclution of compound 43 (5.0 g, 5.7S
mmol) and the thiofucoside 20 (7.5 g, 11.8 mmol) in 30 ~L dry dichloromethane was adde~ dropwise at 0-C
fox 30 minutes. The mixture was stixred at room te~p~rature for 48 hours, at t~e end of which time 5 mL o~ methanol wa~ added and tirred for 30 minutes.
Further, 3 mL of pyridine, 100 mL of ethyl acetate and 100 mL of toluene were zdded to the reaction mixture. The mixture was filtered over celite pad and the ~olvent evaporated to give a brown syrup.
Purification over column chromatography with silica gél and eluted with toluene:ethyl acetate (2:1) gave the compound 4~ in 86% yield. 1H-n.m.r. (CDCl3):
~ 5.BO(dd, lH, J2 3- ll.OHz, H-2 ~, 5.60(s, lH, W092~22564 PCT/CA92/00245 ` 9 ~ 82 --CHPh), 5.50~d, lH, NH), 5.10(dd, lH, 33 4. 4.0Hz, H-3 ), 3.60~s, 3H, OCH3~ 1.20(d, 3H, CH3, ucose)O

Example 10 -- Synthesis of 8-methoxycarbonyloctyl 2-acetamido-3~0-(2,3,4,-tri-D-benzyl-~-L-fucopyranosyl~-4-0-(4,6-O~
benzylidene-B-D-galactopyranosyl)-6-O-benzyl-2-deoxy-B-D-glucopyranoside (compou~d 45~
Compound 44 (200mg) was treated with 20 mL of sodium methoxide in methanol. After 3 hours, t.l.cO
(tolu~ne-ethyl acetate, 1:1) indicated the disappaarance of the star ing material and the : appearance of a slower movin~ spot. The solution was neutralized with ~Qrli~e re~;in IR-120 H~ and th~
:; 15 solvent evaporated under reduced pressure to gi~e a uantitative yield of crude compound 45. ~he product wa~ purif ied on silica gel using toluene-et~yl acetate (2 : 1) as ~luant. ~H-n.m. r. (CDCl3): ~
7.15-7.55 (aro~atic, 25H), 5.62 ~d, lH,N~I), 5.58 (s, lH, ~I benzylidene), 5.06(d, lH, J1.. ;2u 7.0Hz9 H-1"3, 4O95 ~d, l~t Jl~,2~: 3.8Hz, ~ ) 4.85 ~d, 1~, Jl 2=9.0~1z, H ) 3.62 ~s, 3~, COO~I3) 1.O(d, 2H, Fu~-C~3) ~:

:E5xample 11 -- Synthesis of B-methoxycarbonyloctyl 2-acetamido-3-0- ~oL-fucopyrano~yl) -4-0- ( 3-O-slllphate-B-D-galacto pyranosyl ) 2-deoxy-B-D-gluc:o-pyranoside ( compound 47 ) Diol (100 mg -- compound 45~ was dissolved in 39 5mL dry dimethylformamid~. Pyridine: sulfur trioxide complex ( 120 mg) was addPd to the solution and the reaction mixture stirred ar room temperature for 1 hour. The reaction was followed by t.l.c. to monitor the disappearance of the diol (Rf =0.28 in W092/22564 ~ ~ O PCT/CA92/0024 EtO~c: MeOH 80:20). Solvent was evaporated to dryness and taken up in 50mL of methanol then treated with Na' resin to convert it to the sodium ~alt. Purification by column chromatography on silica gel gave 65 mg of compound ~6 which was immediately hydrogenated with 10% Pd(OH)z on carbon to give 35 mg of compound 47. 13C-n.m.r. (DzO): ~
103.94(C-l, Gal), 103.4~(Cl, GlcNAc), 101.07(C-1, Fuc~, 82.7(C-3, Gal), 63.83~C-6, Gal), 62.~(C~, GlcNAc), 54.55(C-N, Glc~Ac), 17.75(C6-Fuc).

Example 12 -- Synthesis of 2-0-benzoyl-4 9 6-0-b~nzylidene-3-0-chloroacetyl-~-D
galactopyranosyl bromide (compound ~ 3) : 15 C~mpo~nd 32, benzyl 4,6-0-benzylid~ne-2-0-benzQyl-3-chloroacet~ D-thiogalactopyranoside : (8.87 g) wa di~olvad in 100 mL of dichloromethane, ~ ~ co~led to O-C and ~reated with a ~olution.of br~mine : ~ t 2 . 7 g j in 10 mL of dichlorome~hane. A~ter 15 minutes, 1.7 g o~ tetraethy~ammonium bromide was add~d to the mixture and th mixture ~tirred for 2 to 3 h~urs at room temperature (follo~ed by t.l.c.
on silica gel). A small quantity of cyclohexene was added to ~uench excess bromine and the reaction : 25 ~ixture was quenched into cDld saturate sodium bicarbonate solution, washed with water, dried, and the volume of the solution reduced to 30 mL so as to provide a dichloromethane solution of compound 330 This solution was used directly in the synthesis of com~ound 38.

W09~/22564 PCT~CA92/00245 Example 13 -- Synthesis o* 8-methoxycarbonyloctyl 2-acetamido-4-0-(2'-o-benzoyl-4',6'-O~benzylidena-3'-0-chloroacetyl-~-D-galactopyranosyl)-6-o-benzyl-2-deoxy-3 0-p-m~thoxybenzyl-B-D-gluco-pyranosid~ ~compound 37) A solution of the compound 7 (5.0 g, 0.9 mmol~
and compound 33 (1.4 to 1.5 equivalents -- from example 12) and 2,6-di t~butyl-4-methyl pyridine (1.78 g, 1.0 ~mol~ in 50 mL of dichloromethane and 20 g of molecular sieves ~4A) was stirred at room tQmperature for 30 minutes, and then cooled to -50'C
und~r nitrogen. A dry solution of silver triflate : (3.3 g~ 1.5 mLj in toluena (10 mL) was added to the stirred ml~ture. The miXture was warmed to -15-C
over two hour~ and kept at -15~C for an additional 5 hour~, then allo~ed ~o warm to roo~ temperature and stirred overni~ht. 1 mL of pyridine and 100 mL
of dichlorc)~thane ~were ~dd~d to the mixture and it 2 0 w~æ filtered o~er c:elite, the filtrate wa~ washed wlt~ a~eous sodium bicarbonate ( 100 mL) and then with water ( 10~0 mL), aqueous hydrogen chloride ( 0 O 5 N , 100 mI-) and water ( 100 ~L~, then cor~centrated in vac:uo~ Purii~lcation of the crude mixture on colu~
25 chromatography WIth silica gel as adso~ent eluted with h~xane:~yl :ac~tate (1:1) gave 5.2 g of pure ompound 37. lH ~mOr. ~C~DCl3) ~ 85(d, lh, NH), 5.62(t, lH, 32 3 10.8Hz, H-2 3, 5~52ts, lH-C:H
benzylidene~, 5.0B ~dd, lH, J3. ,~ 4H2:r H-3 ) ~ 4~85 3~ (d, lH, J1 2 11.0Hz, H-l ), 4.Ç8 (d, lH, Jl 2 9.0Hz, :: H 1), 3~72 and 3.64 (2s, 6~, OCH3 and C:OOCH3); 13c-n .m~ r~: 15g ~ 0 (aromatic c-p-methoxyl ) 165 . 15 ~c:~0 , chloroacetyl) ~ 167 . 12 (c-o, acetyl), 174 . 2 ~c=0, COQC~13), 99.64(c 1), 100.26(c-1 ), 101.0(PhCH).

21-i~7~

Compound 37 was then treated with DDQ in th~
same manner as Example 8 to give co~pound 38 in near quantitative yields.

Example 14 -- Synthesi~ of 8~methoxycarbonyloctyl S 2-acetamido-3-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl-4-0-(2-0-benz~yl-4,6-0-benzylidene-3-0-chloroacetyl-B-D-galactopyranosyl)-6-0-benzyl-2 deoxy-B-D glucopyranoside (compound ~10 39) Thiofucoside 20 (4 g) was stirred in dry dichloromethane ~50mL) and bromine (0.60g) wa~
added. The mixture was cooled to -~0-C~ The conversion to the bromide was complete in 1 hour and th~ reaction mixtur~ was washed with cold a~ueous sodiu~ bicaEbonate, dried and concentrated to lOmh and syringed i~to a fla~k cDntaining the alcohol 3 : (2.97 g, 3~56 mmol), CuBr2 (2.39 g)~ tetraethyl ammonium bromide (2.24 g), molecular ~ieves 4 A (4g) :in dimethylfonmamide (1 mL) in dry dichloxomethane (75 mL). The mixture wa stirred at room te~p~rature for 48 hours after which the t~l.c~
: .ho~ed the disappearanc~ of ~h~ alcohol 38 and a ~s~er ~oving ~pot ~f 0.5S -- toluene:e~yl acetate 2:1). ~fter the usual work up, th~ crude mixture was purified ~y colu~n chromatography to gi~e compo~nd 39 (4~2 g, about 80% yield). 1H-n.m.r.
(CDC13): ~ 7.1-8.0 ~m,aromatic-30H), 5.53, 5.61 (m~
2~, NH and ~H-benzylidene, overlapping), 5~56 (d, 1~, J1u2" 7.0 Hz, H~ , 4.98 (d, lH, J12 8.0 Hz, H-1), 4.95 (d, lH, J~2~ 3~ Hz, H-1'), 3.6S (s, 3H, COOCH3~ a~d ~ d, 3H, C~3-Fuc).

Compound 39 i5 then dechloroacetyla~ed by treatment with thiourea and the compund is sulphated ~ - 86 with sulfur trioxide/pyridine complex in dimethylformamide at 0C for 2 hours to provide for compound 41. The blocking groups on compound 41 are then remcved by conventional techniques to pr~vid~
for co~pound ~7.

Example 15 -- Synthesis of 2-deoxy-2-phthalimido-1,3,4,6 tetra-0-acetyl-~-D-glucopyranoside (compound 1) (D+) Glucosamine hydrochlorid~ (100 g, 0.46 mol) was added to a solution of sodium methoxide in methanol which was prepar~d from equimolar amount of sodium metal i~ methanol (O.5 L). The resultant mixture was treated with equimolar equivalent of phthalic a~hydrid2 and triethylamine (80 mL). The mi~ture was then s~irred for 2 hours, filtered and :~ the ~olid was dried in ~acuum for 12 hours. The dry - solid wa~ dissolved in pyridine (300 ~h) and treated ~` ~ with acetic a~hydride ~200 mL, 2.1 mol). m e : mixture was then stirred at room temperatùre for 48 20 ~hours. The reaction mixture was then treated with ~:` an ice-water mixture, and the resultant precipitate was filtered, con~ntrated and crys~allized fro~
di~thylether to 98.3 g (4~%3 of the title compou~d.
~ : 7H-n.m~r. (CDC13): ~ 7.75 ~m, 4h, aromatic)l 6.45 (d, :: 25 lH, H-1~ J12 9.0H2), 5.85 (t, lH), 5.15 ~t, lH), 4~4 ~t, lH), 4.3 ~q, lH), 4.1 ~q,. lH), 4~00 (m, lH), 2.05, 2.00, 1.95, 1.80-(4s, 12H, 4Ac). 13C-n.m.r.
~CDCl3) ~ 89.7 (C-1), 72.6, 70.5, 68.3 (3C, C-3, C-4, C-5), 61.45 (~-6~, 53.42 ~C-2).
: .

W092/22S64 PCT/~92/00245 2l~7~

Example 16 -- Synthesis o~ 2-deoxy-2-phthalamido-3,4,6-tri-0-acetyl-~-D-glucQpyranosyl bromide (c~mpound 12) 2-deoxy-2 phthalamido-1,3,~,6-tetra-0-acetyl-~-D~glucopyranoside 1 (20g, 41.9mmol) was treated with hydrogen bromide solution in acetic acid ~30%, 200mL) and stirred at room temperature for 2 hrs. The mixture was then poured into an ice water mixture and extracted with dicloro~ethane.
`10 The ~xtract was washed with ~aHCO3 solution and water followed by ~gS04 drying. The mixture is :~ ~iltered, dried and concentrated in vacuo to give compound 12 as a dry syrup (compound 12) ~xample ~7 - Synthesi~ of E~hyl 2-deoxy-2-phthalimido-3,4,6-tri 0-acetyl-B-D-glucopyranoside (compound 13) 2-Deoxy-o2-phthalamido-3,4,6-tri-0-acetyl-~-D~
glucopyranosyl bromide (co~pound 12) fro~ example 16 was taken up in dry ethanol and treat~d ~irectly with dry etha~ol (200 mL), mercuric ~yanide (1307 g~
~ :5~ ~mol) and stirred at room ~emperat~re for 48 hr.
: i The mixture was then filtered and concen~ra~ed~ ~he ~: residu~ was takèn up in 200 mL of dichloromethane and wa~hed with a ~olution of 10~ potas~ium iodide, 5% sodium bicarbonate, water, dried over MgSC~ and concentrated to a syxupO

Example 18 -- Synthesis of Ethyl 2-deoxy 2-phthalimido-B-D-glucopyranoside . ~Compound 143 Ethyl 2-deoxy-2-phthalamido-3,4,~-tri-o-acetyl-~-D-glucopyra~osyide (compound 133 from example 17 was taken up in 100 mL of dry methanol and treated W0~2/22564 PCT/CA92J00245 with 100 mg of sodium metal. The solution was stirred at room temperature for 24 hours and then neutralized with Am~erlite ER-120(H+)] resin, filter~d, and evaporated to dryness in vacuo. This compound was used in the preparation of compound 15 and compound 66.

Example 19 -- Synthesis of Ethyl 2-deoxy-2-: ~ phthalimido-6-O-benzyl-B-D-glucopyranoside (Compound 15) Compound 1~ (2.:1 g, 6.23 mmol~ was taken up in 100 mL of toluene. To it waC ~dded bis(tributyl tin) oxide (2.22 mL,-4.35 mmol) and tetrabutylammonium bromide (0.983 g, 3.05 mmol~.
The mixture was~heated at 150C for 4 hours and the~
toluene ~50 m$): was~distilled off from the mixture.
The:reaction mixture was eooled to:room temperature and benzyl bro~ de ~2.17 mL, 18.27 mmol) was added and the reaction heated to llO-C for 36 hours.
Toluene was ev~porated and the residue taken up ino ~: ~ 20 ethyl acetate ~:22 mL),:washed successively with aqueous sodium bicarbona~e, saturated sodium : chloride solution~and water. The organic layer wa~
dried and e~aporated~to dryness to giv~ a crude solid. Purification~by column chroma~ography on :: 25 silica gel gave a:crystalline solid 15 gl.4 g, 70%).
H-n.m.r.(CDCl3) ~ 7.~3-8.1 (9H, aromatic), 4.5 (dd, i 2H, Ca2Ph), 5.18 (d, lH, J12 lO.OHz, H-l), 4.36 ~dd, ; lH, H-3), 4.25 (dd, H, J21 lO.OHz, J23 8.0Hz, H-2) and l.O(t, 3H,~ CH~).

:

~ ~ :
:

3 7 ~ !

Example 20 -- Synthesis of Ethyl 6-O-benzyl-2-deoxy-2-phthalimido-3 0-(2,3,~,-tri-O benzyl-~-L-fucopyranosyl)-4-Oo (2,3,4,6-tetra O-ac tyl-B-D-galacto-pyranosyl)-B-D-glucopyranoside (Compound 49~
To a stirred solution of compound 15 (2.49, 5.71 mmol) in dry dichloromethane (50 mL~ was added dry CaSO4 (7.5 g), sllver triflate (0.73 g, 2.8 m~ol) and silver carbonate (7.0 g, 2S.7 ~mol) and the reaction mixture cooled to 50-C. 2,3,4,6~
t~traacetyl~ bromogalactose (3OS g, 8~5 ~mol) in dry dichloromethane (15 mL) was added dropwise througk a dropping funnel. The reaction mixt~re was lS warmed to -3~-C and stirred .for 48 hours and then m~thanol (S m~) was adde~ to cease the reaction and the mixture allowed to warm to room e~perature.
Aft~-r filtration th~ough a celite p~d and the filtrat~ was wa~h~d with agueous ~i~ar~o~ate and S%
: 20 ~DT~ ~olution. Evaporation of the solvent in vacuo gave a r*ddish bro ~ syrup which was chromatographed on:silica with toluene: acetone:~eOH (20:3:1~ as elu~nt o give compound 48 (Rf O.S28) as the major co~pound~ I
Thiofuco~ide 20 ~1.5 g, 2.8 mmol3 was stirred in dry dichloro~ethane (S0 mL) cooled to -20c and br~mine (0.40 y) was added. The co~version to br~mide w~ c~m~lete in 1 hour a~d the reaction mixture was washed with cold aqueous bicaxbonate, dried a~d concentrated to 50 ml and syring~d into a ~lask containing compound ~8 (1 g, 1.4 ~mol), H~r2 ~1~08 g, 3 mmol), molecular sieves 4A ~2g) and tetraethylammonium bromide (1 g~ in dry dichloromethane (50 mL). The mixture was stirred at room temperature for 48 hours. T.l.c. showed a ~as~er moving spot. The reaction mixture was filtered through celite, and the filtrate washed with water, 5% EDTA, saturated aqueous sodium bicarbonate, water, then dried over sodium sulphate, filtered and evaporated to dryness in vacuo.
Purification of the crude product by silica gel chromatography gave the title compound 4Q (1.2 g, 70%, Rf 0.669 in toluene; acetone; MeOH 20:3:1).
1~ n.m.r.(CD~13): ~ 7.00 - 7.8 (aromatic 24 H) 5.35(d,1H, J-2 9.0Hz, H-l), 5.15 (d, lH, J-2 3.8Hz, ~ Fuc~, 4.35(dd, lH, J2 ,3 10.0Hz, H-3 ) 2.1(s, 3H, ace~yl CH3~ 1.95(s, 6H, acetyl CH3), l.9O(S, 3H, acetyl CH3), l.l(t, 3H, C 3~, and 0.S ~d, 3H, CH3-Fuc). 13C-n.m.r.: ~ 168, 170 (C=O, ph~halimido and aee~yl), 101.0 (C-l, Gal), 100.0(C-1, GlcNPhth) 97.7(C-l-Fuc), 2O.6(CH2~ ) and 15.98(C-6-Fuc).
~; :

:Example 2~ -- Synthesis of Ethyl 2-acetamido-6-O-acetyl 3-O-benzyl-2-deoxy-B-D-: glucopyranoside ~ A ~olution of compound 1 (2 g, 4.68 mmol3 in :~ agueous acetic a~id (80%r 150 mL) was heated at 80-C:~ 20 for 2 hour~. The:mixture then was evaporated and the r~ulta~t solid was dried over P205 in high : ~ ~ vacuu~. The dry solid was selectively acetylated with acetyl chloride (0.33 mL, 4O7 mmol) and :~ pyridine ~10 mh) in dichloromethane (~00 mL) at ~
:~ 25 ~:0C to SC. The mixture was then diluted with diehloromethane (50 m~), washed with a~ueous NaHCO3, dried ~ver MgSQ4 and evaporated. The residue was chro~tographed on a silica gel colu~n using ET~Ac: hexanes, 3:1 (v:v) as eluant to give 0.82 g (46%) of the title compound: 1H-n.m.r. (300 MHz, CDC13): ~7.3(m, 5H, aromatic), 5.67(bs, lH, NH~, 4.86(d, lH, H-l), 4.75(m, 2H), ~.48(q, lH), ~.27(d, lH), 4.1(t, lHj, 3.85(m, lH), 3.5(m, 3H), 3.16(m, lH), 2.70(bs, lH, OH), 2.1(s, 3H, Ac), l.9(s, 3H, SIJBST~TI~TE SI~E~T

~V0~2/22564 PCT/C~92/~0245 ~~ g 1 ~~ ~ 1 Ac), 1.18(t, 3H, CH3), 13C-n.m.r. (CDC13): ~
99.45(C-1), 79.85, 74.5(CH2ph~, 73.7, 71.09, 65.25 (C-6), 63.36(CH2-), 57.7(C-2~, 23.6(Ac), 20.86(Ac), 15.06(CH3)~

Exa~ple 22 -- Synthesis of Ethyl 6-O-acetyl-3-O-: benzyl-2-deoxy-2-phthalimido-B-D-glucopyranoside (compound 69) A solution of ethyl ~-deoxy-2-phthalimido-~-D-` glucopyranoside (compound 14) from Example 18 was taken up in dry acetonitrile (100 mL) and ~reated ~:~ with benzyaldehyde dimethylacetal (9.6 g) and a catalyt~c amount of p-toluenesulphonic acid tlO0 mg). The mixture was stirred for 17 hours at room temperature and then neutralized to pH 7 with ~15 triethyla~ine. The mixture was evaporated and :~: crystalli~ed ~rom hot hexanes to give 12.7 grams of ethyl 4~6-0-benzylidene-2-deoxy-2-phthalimido-B-D-glucopyranoside compound 66.
Compound 66 ( 10 g) was dissolved in d~y : ~ ~ 20 ~dimethylformamide (D~qF) at -5 - C and treated with 1.1 :~ : g ~:46. 6 mol) ~odium hydride arld benzoyl bro~ide (5.46 mL, 22 mmol).~ The mixture was stirred a~ 0C
or 2 hours and then treated slowly with 20 mL
methanol then slowly brought to room temperature and trea~ed with ~ICl (lN) to pH 7 and then extracted three times with dichlor~me~ane. The organic layer was dried ~rer anhydrous magnesium sulfate then filtered, concentrated to dryness and taken up in : hot ethanol to give 7.2 g of compound 67~ Compound 67 (5.43 g, 10.50 mmol) in aqueous acetic acid ~80%, 200 mL) w~s heated at 80C for 2 hours. The mixture was evaporated and the resultant solid was dried over P205 in high vacuum. The dry solid was selectively acetylated with acetyl chloride (0.8 mL, 3S 11.0 mmol) and pyridine (lo mL) in dichloromethane WO 92/22564 ~r~, PCI`/CA92/00245 Grl (200 mL) at -10C to 0C. The mixture was then diluted with dichloromethane (1 0 m~), washed with aqueous NaHCO3, dried over MgSO4 and evaporated. The residue was chromatographed on a silica gel column using EtOAc:hexane, 1:2 (v:v) as eluant to give 3.5 g (71%~ of the compound 69: lH-n.m.r. (300 MHz, CDCl3): ~ 7.7(ml 4H, aromatic), 7.0(m, 5H, aromatic), 5.16(d~ lH, H~ .7(d, lH), 4.5(m, 2H), 4.2(m, 3H), 3.8(m, lH), 3.6(m, 2H), 3.45~m, lH), 1~0 2.9(bs, lH, OH), 2.1(s, 3H, Ac), 1.95(t, 3H, CH3).
3C-n.m.r. (CDCl3): ~ g8.09(C-1), 78.45, 7~.5, 73.9, 71.7, 65.1, 63.1, 55.5, 20.87 (Ac), 14.92 (CH3~.

Example 23 -- Sy~thesis of Ethyl 6-0-acety'-3-benzy}-2-deoxy-2-phthalimido-~0 ~2,3,4,6-tetra-0-acetyl-B-D-galactosyl)-B-D-glucopyranoside o~pound 70) : To a stirred solution of c~mpound 9 (80 mg, :: O.17 mmol) in dichloromethane (10 m~) containing ; 20 molecular sie~es (3A, 1 g), 2,6-di-tert-butyl-4-: methyl-pyridine ~45 ~g, 0.22 mmol~ and silver triflate (57 mg, 0.22 mmol) was added, at -30-C
under nitrogen, 2,3,4~6-tetra O-acetyl-~-D-ga~actosyl bromide in dichloromethane (5 mL). The mixture wa~ stirrcd at this temperature for 1 h and : then warmed up to 5~C over 2h. The mixture was then diluted with d~hloromethane (10 m~), filtered and the insoluble material was washed with dichloromethane (5 mL). The combined filtrates were washed with saturate~ aqueous sodium hydrogen carbonate and water, dried over MgSO4, and concentrated. The residue was chromatographed on a silica gel column using ethyl acetate: hexanes, 1:2 ~v:v) as eluant to give 120 mg (80~) of the title W092/22~64 PCT/CA92/00245 21t ~Brl -- ~3 --compound: ~H-n.m.r. (300 MHz, CDC13): ~ 7.68, 6.96(2m, 9H, aromatic), 5.3(m, 2H), 5.13(d, lH, H-1~, J1 z 8.0Hz), 4.99(q, lH), 4.82(d, lH), 4.62~d~
lH, H-l, J12 7.7Hz), 4~54(d, lH), 4.42(d, lH), 4.3~q, 1~), 4.15(m ,2H), 3.99(m, 2H), 3.87(m, 2H), 3.72(m, 2H), 3.46~m, lh), 2.15, 2.12, 2.09, 2.00, 1.98(5s, 15H, 5X~c), l.OO(t, 3H, C~3).
3C-n.m.r. (CDCl3): ~ 101.2, 97.8(C-1, C-l ), 14.85(~H3)-The 2-amine of Compound 67 above can be r~generated by contacting this compound with hydrazine acetate and then acetylated with acetic anhydride pyridine or other acetylating agents to provide for a disaccharide (90) 5 Example 24 ~- Synthesis of Ethyl 2-acetamido-Ç-O-acetyl-2-deoxy-4-0-(2,3,4,6-tetra-0-acetyl-B-D-galacto~yl)-3-0-(2,3,4-tri-O-benzyl ~-h-fuco yl)-B-D-glucopyranoside ?o To a stirred solution o,f the disaccharidQ 90 (80 mg, 0.129 mmol) in dichloromethane (2 mL) conSaîning molecular sîev~s (3A-, 1 g), tetra~thylammonî~m bromîde (41 mg, 0.195 mmol), dimethylformamîde (0.1 mL) and diisopropylethylamine (0.087 mL, 0.5 mmol~ was added, at room t~mperature under nitrogen~ a solution of 2,3,4-tri-0-~enzyl fucosyl bromîde (130 mg, 0.26 mmol -- as per Example

9) în dichloromethane (2 mL~. The mixture was stirred at room temperature under nitrogen for 72 h and then filtered, and the insoluble material was washed with dichloromethane (10 mL). The combined filtratPs were washed with saturated aqueous sodium hydrogen carbonate and water, dried over M~SO4, and concentrated~ The residue was chromatographed on a WO 92/22~64 . PCr/CA92/00245 J I
~7,~

silica gel column using ethyl acetate:hexanes, 3: 1 (v:~r) as eluant to give 115 mg (90%) of the title trisaccharide 6: ~H-n.m.r. ~300 M~Iz, CDCl3): S
7 . 30 (m, 15H, aroma~ic), 6 ~ 00 (d, lH, NH, J 8 . 0Hz), 5.38(d, lH, H-l Fuc, J1 2 3.3Hz), 5.14(d, lH, H 1 Glc, J1 2 7.8Hz~, 5.1(m, lH), 4.98(m, 2H), 4.80(m, 6~), 4 . 40 (m, 2H), 4 . 33 ~q, lH), 4 . 1 (m, 5H), 3 . 77 (m, 7~I), 3.48(m, 1~). 2.09, ~.07, 2.01, 2.00, 1.97(5XAc, lSH), 1.80(s, 3H, NAc), 1.18(d, 3H, H--6 EUC, J5 6 6.~Hz), 10087(t,, 3H, CH30fEt) . 13C-n.m.r.
(C:~C13): ~ 99 0 ~ (C-l ~;al), 99.19 (C-l Glc~, 97.,18(C-l Fuc~, 16.67(C--6 Fuc:), 14.79(CH30fEt).

1 :xample 2 5 -- Synthe~;is of GDP-Fucose As not~d above, fucosylation of the sulfated 15 LewisX and L~wi~ ;tructure~ c:an ba ac:~ie~red by use o:l~ an approp:riate fucosyltransf eras~ which ar~ well known in the art. Enzymatic fucosylation requires the use of GDP-fucose. Accordingly, the purpose of l:his e~am~le i8 to illustrate the synthesi~: of GDP-20 fucos~. This is achieved in a 3 step pros~ess asillustrated below:

A. Preparation of Bis (tetra n-butylammoniu~) ~5 Tetra-n-butyla~nonium hydroxide (40% aq. w/w, abo~t 150g) was added dropwise to a ~;olution of phosphoric acid (85% aq, w/w, 18g~ 0. lS5 mmol) in water ( 15 0 mL) until the pH reached 7 . Water was t~ien evaporated in vacllo to give a syrup which was 30 s::o-evaporated with dry aceto-nitrile (2 x ~Q0 mL~
followed by dry toluene (2 x 400 mL). The resulting `~092/22564 PCT/CA92/00245 211~

white solid (75g) was dried in ~acuo and stored over phosphoru~ pentoxide under vacuum until used.

B. Preearation of_~-L-Fuco~ranosyl-l-~hQsPhate A solution of bis(tetra-n-bu~ylammonium) hydrogen phosphate (58g, 127.8 mmol) in dry acetonitrile (300 mL) was stirred at room temperature under nitrogen in the presence of mole ular sieves (4~, 20g) for about one hour. A
solution of tri-0-acetyl fucosyl-l-bromide (freshly : 10 prepared ~r~m 31g, 93 mmol of L-fucose tetraacetate in the man~er of Nunez et al.53) in dry toluene (lO0 mL3 was added dropwise in about 0.5 hour to the a~oYe solution, cooled at 0-C. After one more hour at 0-C~ the ~ixtur~ was brought to ro~m temperature and stirred for 3 hour. Tlc ~l:l toluene:~thyl acet~te) indicated a main spot on the base line and sev~ral faster moving smaller spots.
The mix~ure was filt~red o~er a pad of Celite : (which was further washed with acetonitrilè) and the ~olvent~ e~aporated in vacuo to give a red syrup.
~his material was dissolved in water (400 mL) a~d ; ~ ~ racted with eth~l acetate (250 mL, twi~e). The aqueous layer was then evaporated in vacuo leaving a yellowish syrup to which ~ solution of a~monium 2S hydroxide ~2~% aqO, 200 mL) was added. The mix~ure w s stirred at room temperakure for 3 hours after which tlc (6~:35:8 chloroform:methanol:water) indicated a baseline spot. The solve~t was evaporat~d in vacuo to give a yellowish syrup which was diluted with water ~400 mL). The pH of this solution was checked and brought to 7, if necessary, by addition of a small amount of hydrochloric acid.
The solution was slowly absorbed onto a column of ion exchange resin Dowex 2 X 8 ~200-40Q mesh, 5 x 45 W092/~2564 PCT/CA9~/00245 ~ - g6 --cm, bicarbonate form which had been prepared by sequential washing of the resin with methanol (800 mL~, water (1200 mL), ammonium bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water (1000 mL) was then run through the column followed by a solution of ammonium bicarbonate (O.5 M; 2.3 mL/minute, overnight). The eluate was collected in fractions (15 m~ and the product detected by charring after spotting on a tlc plate. Fractions 20 to 57 were pooled and vaporated in vacuo leaving a white solid which was further co-evaporated with water (3 x 300 mL) and freeze drying of the last 50 mL and then dryin~ of the residue with a vacuum pump to give ~-LDfucopyransyll-phocphate (9O5g, 40%) as a 12:1 mixkure of ~ and ~ ano~ers containing some ammonium a¢et~te identified by a singlet at ~=1.940 in the 1H-n.m.~. spectrum. This product was slowly run through a col~mn o~ Dow~x S X 8 resin tlOO-200 mesh, tri~thylammonium fo ~) and eluted with water to provide the bis triethylammonium salt of ~-L-: fucopyransyl-l-phosphate as a sticky gum after reeze d ~ ing of the eluate. lHDn,m~r~
~ 4-84g ~dd, J12 ~ J1P = 7~5 Hz, H-l), 3.82 (q, lH, J56 6.5 Nz, H-5~, 3.750 (dd, lH, J34 3.5, J45 1.0 Hz, H-4), 3.679 (dd, lH, J23 10.0 Hz, H-3), 3.S20 ~ddt lH, H-2), 1.940 (s, acetate), 1.26 (d, ~ 6).
Int~gral of th~ sign~ls at 3.20 (q, J 7~4 Hz, NCH2) and 1.280 and 1.260 ~NCH2CH3 and H-6) indicates that the product is the bis-triethyl-ammonium salt which may loose some triethylamine upon extensive drying.
13C-n.m.r. ~:98.3 (d, J lP 3.4 Hz, C-l), 72.8 (d, JC 2P 7-5 HZ, C_2), 16.4(C-6); 31P_nmr ~ +2.6~S) .
~ -L-fucopyransyl-1-phosphate appears to slowly degrade upon prolong~d storage (l+ days) in water at 22C and, accordingly, the material should not be ~092/22564 PCT/CA92/00245 -- g7 - ~ 7 ~

left, handled or stored as an aqueous solution at 22C or higher temperatures. In the present case, this material was kept at -18C and dried in vacuo o~er phs~phoru~ pentoxide prior to being used in the next step.

C. Preparation of Guanosine 5'~ fucopy-ranosyl)-diphosphate : Guanosine S'~ fucopyranosyl)-diphosphate was prepared from ~-L-fucopyranosyl-l-phosphate

10 using two different a ~ recognized procedures as set forth below:
, PROCBD~RB #
~ ~ L-fuc~pyranosyl-1-phosphate and guanosiné 5'-:~ mono~pho~phomorpholidate (4-msrpholine-N,N'-di-cy d ~hexyl~carboxamidine salt, available from Si~ma, St. Louis, ~issouri, t'G~P-~orpholidate") were reacted a~ described in a recent modification54~56 of Nunez's original procedureS5. Accordingly, tri~n : o~tylamine (0.800g, available from Aldrich Chemical Company, ~ilwaukee, Wisconsin) was added to a mixture of ~ -~ucopyranosyl-1-p~osphate (triethyl-ammonium salt, 1.00~, about 2~20 mmol) in dry pyridine (10 ~) under nitrogen the sol~ent removed in vacuo. The process was repeat~d three times with care to allow only dry air to enter the flaskO GMP
morpholidate (2O4g, about 3.30 mmol) was dissolved in a 1:1 mixture of dry dimethylformamide and pyridine (10 mL). The solvents wexe evaporated in vacuo and the proce~ure repeated three times as abo~e. The re idue was dissolved in the same mixture of solvents ~20 mL) and the solu~ion added to the reaction flask accompanied by crushed molecular sieves (2g, 4A). The mixture was stirred q~ 98 --at room temperature under nitrogen. Tlc (3:5:2 25%
aq. ammonium hydroxide, isopropanol and water) showed spots corresponding to the starting GMP-morpholidate (Rf-0.8, U.V.), guanosine S'~
fucopyranosyl)-diphosphate (Rf-0.5, U.V. and charring), followed by the tailing spot of the starting fucose-1-phosphate (Rf-0.44, charring).
Additional ~.V. active minor spots were also present. After stirring for 4 days at room io temperature, the yellowish mixture was co-evaporated in vacuo with toluene and the yellowish residue further dried overnight at the vacuum pump leaving a thick residue (2.43g). Water (10 mL) was then added into the flask to give a yelIow cloudy solution which was added on top o~ a column of AG 50W-X12 ~:: (fro~ Biorad) resin~100-200 mesh, 25 x 1.5 cm, Na~ ^
: form). ~The product eluted with water after the void :volume. The ~ractions which were active, both by U.V. and charring:after ~potting on a tlc plate, 20 ~ were recovered and the solution freeze-dried overnight in vacuo providing a crude material ~:
(1.96g). ~ : :
This residue was dissolved in water (10 mL
- ~ :
overall) and slowly absorbed on*o a column of hydrophobic C18 :~silica gel (Wateræ, 2.5 x 30 cm~
: which had been conditioned by washing with water, ~: methanol and water (250 mL each). Water was then run through ihe column tO.4 mL~min) and the eluate collected in fractions (0.8 m~) which wer~ checked : : 30 by klc (3:~:2 25%~aq. ammonium hydroxide, isopropanol and water). ~ fucopyranosyl 1-p~o~phate, (Rf-0.54, charring) was eluted in fractions 29 to 45. A product showing a strongly ~ U.V. active spot (Rf~0.51) eluted mainly in : 35 fractions 46 to 65. Other minor U.V. active spots ~92/22564 PCT/C~92/00245 ~.llG~o~
__ 99 __ of higher or lower Rf were observed. Fractions 59 to 86, which contained guanosine 5'~
fucopyranosyl)-diphosphate ~Rf~0.~2), also showed a narrow U.V. active spot (Rf-0.57~. Fractions 59 to 86 were pooled and freeze-dried overnight providing O.353g of material enriched in guanosine 5'-(~
fucopyranosyl)-diphosphate. 1H-n.m.r. indicated that this material was contaminated by a small amount of impurities giving signals at ~ = 4.12 and ~ = 5.05.
Fractions 29 to 45 and 47 to 57 were separately pooled and freeze-dried pro~iding recovered ~ L-~uco-pyranosyl-l-phosphate (0.264g and 0.223g, re~pectively, in which the seco~d fraction contains so~e impuriti~s). Qcca~ionally, pooling of appropriate fractions provid@d some amou~t of guanosine 5'o(~-1 fucopyranosyl)-diph~sphate in good purity (1H-n.m.r.). Generally, all the material enriched in guanosine 5'~ l fuco-pyranosyl)-~: ~0 diphosphate wa~ dissol~ed i~ a mini~um a~ount of water and run on the same column which had been regenerated ~y washing with large amounts of methanol follcwed by water. The fractions containing the purified guanosine S'~
fucopyranosyl~-diphosphate (tlc) were pooled and freezed dried in vacuo leaving a white fluf*y material (187 mg, 16%). ~-n.m.r~ was iden$ical to the previously reported data53.

PROCED~R$ #2 . ~-L-fucopyranosyl-l-phosphat and ~uanosine 5/-monophosphomorpholidate (4-morpholine-N,N'-di cyclohexyl-carboxamidine salt -- ''GMP-morpholidate~) were reacted in dry pyridine as indica ed in the original procedure55. Accordingly, the ~

W092J22~64 PCT/CA92/00245 ~i fucopyranosyl~l-phosphate (triethyl-ammonium salt, o.528g, about 1~18 mmol) was dissolved in dry pyridine (20 mL) and the solvent removed in vacuo.
The process was repeated three times with care ~o allow only dry air to enter the flask. GMP-morpholidate (1.2g, 1.65 mmol3 and pyridine (20 mL) were added into the reaction flask, the solvent : evaporated in vacuo and the process repeated three times as abo~e. Pyridine ~20 mL) was added to the final residue and the heterogeneous mixture was stirred for 3 to 4 days at room temperature under nitrogen. An insoluble mass was formed which had to be occasionally broken down by sonication~
The reaction was followed by tlc and worked up as indicated in the first procedure to provide the GDP-fucose (120~mg, 16%).
~:
:, .
II. Biological Results Examples:26-29~illustrate the immunomodulatory, antii~flammatoryt ahd tolerogenic properties of 20 ~ c~mpounds disclosed herein.

: ~ Examp~e 26 - Inhibition of ~ Inflammatory Response : DTH infl~matory~responses were measured using :~ the mouse footpad:swélling assay as described by , Smith and Ziola55. Briefly, groups of Balb/c mice were immunized with S-layer protein, a bacterial surface protein5l from Clostridium hermohvdrosulfur um Ll11-69 ~Llll] or with ~ SuperCarrier (SC) which have been shown to induce a : strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 10 ~g of L-lll S-Layer protein or with 20 ~g of SC. The resulting inflammatory footpad swelling was m~asured :: ~

WO9~/22564 ~ 7~ 7 PCT/~A9~/00245 with a Mitutoyo Engine~ring micrometer 24 hours after challenge.
To assess the effect on the DTH inflammatory response by sialyl LewisX-OR and sulfated IRwisX-OR
(compound ~7), groups of mice challenged with 10 ~g of the same anti~en 7 d~ys after immunization with the antigen received 100 ~g of sialyl L2wisx-oR or sulfated LewisX-OR tR = -(CH2)8COOCH3] injected into the tail vein, 5 hours after challenge. Control `~0 groups were left untreated. The results of this experiment are shown in FIG. 11 which demonstrates that while mice injected with either sialyl LewisX-OR or sulfat~d LRwisX OR exhibited reduced inflammation, those injected with sialyl LewisX
~xhibited the greater reduc~ion in in~lammation.
- To ass~ss the e~fect on the DTH infl~mmatory : : ~r~æponse by the modified LewisX-OR c~pound containing a sulfate substituent at the 3-position o~ galactose (c~m~ound ~7), group; of mice challenged with either 10 ~g of the L111 antigen ~Figure 11) or with 20 ~g of the SC antigen (Figure 12~ 7 days after i ~ unization with the antigen received 100 ~g of sialyl LewisX or sul ated LewisX
i~ject d into the tail vein, S hours after ~5 challenge. Control groups were l~ft untrea~ed or ~: received 100 ~L of phosphate-buffered saline (PBS).
The results of this part of this example are set ~orth in Figure 12 which illustrates that the sulfated LewisX provided roughly e~uivalent reduction in inflammation. This result is particularly surprising when compared to Figure 11 and dem~nstrates that substitution of the sulfat group at the 3-position of the galactose of LewisX
significantly enhances the anti-inflammatory 3S properties of these compounds.

WO 92/22S64 PCI/CA92/01)245 ~?~r~
/~ 3 ---- 102 ~--Example 27 -- Effect of Oligosaccharide G-ycosides on LPS Caused Lung Injury LPS (lipopolysaccharide) caused lung injury is measured by weighing the lungs of sacrificed mice 24 S hours after mice are given LPS intranasally.
Briefly, groups of 8-10 weeX old Balb/c mice were s~nsitized with S ~g /mouse of ~PS in 50 ~1 of PBS
intranasally under light anesthesia.
The method of administering compound ~0 intranasally ic descr~bed in Smith et al., Infection and Immunity, 31: 129 (1980), which is incorporated by reference. Briefly, mice are anethesitized with Metofane (Pitman-~oore Ltd., Mississauga, Ontario, Canada) and a 50 ~1 drop of compound is placed on ~5 the nares o~ the mouse and is inhaled.
Five hours later, 100 ~g/mouse of sialyl LewisX
or ~ul~ated LewisX in 200 ~1 o~ PBS are given to the : mouse in~ravenously. After 24 hours, the mice are sacrificed and ~ e lungs removed and weighed. The weight of the lungs of mice treated with either sialyl L~wisX or ~ulfated Lswis~ are c:ompared against control ( i . ~ ., mice treaked with LPS but to which nt3ither sialyl LewisX or I,ewisX has been administered~. Th~ percent reduction is measured by 25 s~btracting f rom 100 the following:
The fraction derived by a num~rator whose value is the weight of the treated lungs subtracted f rom the weight of normal lungs ~lungs from mice not exposed to LPS, sialyl LewisX or sulfated LRwisX), and whose denominator whose value is the weight of the control lungs (mice that received only LPS) subtracted from he weight of normal lun~s and multiplying the resulting fraction by 100.
The greater the percent reduction, the better the compound is in alle~ating lung damage.

~092/22564 PCT/CA92/00245 ~s ~ 7 ~ 7 The results of this test are set forth in Figure 13 which illustrates that sulfated LewisX
provid~s about 50~ reduction whereas sialyl Lewisa provides only about a 30% reducti~n in the DTH
inflammatory response in lungs. This suggests that not only do both sialyl LewisX and sulfated LewisX
can b~ use~ul in reducing inflamation in lungs exposed to antigen, for example Acute Respiratory Distress Syndrome but that sulfated LewisX actually provides significantly enhanced results.

Exa~le 28 -- ~ffect of Adminis~ration of Sialyl Lewis~ and Sulfated LewisX at the Time of Immunization on the Induction of an Immune Response to an ~ntigen Groups of Balb/c female mice were immunized : with 20 ~g/mouse SC in 100 ~1 of PBS in~ramuscularl~
i ~ o the hind leg muscle which formulation also contained 100 ~g/mouse of sialyl LewisX or ~ulfated ~ewisX. SeYen day later ~he mice were ~ootpad challenged with 20 ~g/mouse of 5C in 20 ~L of PBS.
Control groups were ei~her not immunized or received 20 ~ of phosphat~-buffered salin~ (PBS) in place of either sialyl LewisX or sulfated LewisX. The footpad swelling was measured 24 hours later with a 2S Mitutoyo Engineering micrometer.
FIG. 14 shows that admini~tering sialyl LewisX
or sulfated LewisX to the mice at the ti~e of immunization reduces ths induction of an immune response to an antigen as compared to PBS control.
This suggests that administration of a compound of thi invention a~ the tim~ of antigen immunization will reduce the ability sf the mammal to become educated concerning this antigen.

W092/22564 PCT/CA92/00~45 Example 29 -- Persistence of Suppression of the DTH
Inflammatory Response at 6 Weeks After Challenge i. The identical groUps of mice treated with S sialyl LewisX and sul~ated LRwisx in Example 26 were re-challenged 6 weeks after primayr immmuniZation with 20 ~g/mouse with SC. Untreated controls r~spond~d with the usual degree of footpad swelling wherea~ all other groups showed reduced footpad swelling as shown in Figure 15.
In addition to providing an anti-inflammatory effect as well as modulation of a cell-mediated immune response, the abo~e data demonstrate that treatment with sul~ated LewisX as per this invention ; ~5 also imparts tolerance to additional challenges from t~e same antigen.

~ ditionally, o ~ er compounds disclosed herein can be used in place of the sulfated LewisX
: including the following ~alartose substitu~ed ~0 materials: 6-sulfated ~RWiSX, 3- or 6-sulfated Lewisa, 3- or 6-pho phated LewisX, 3-6-phosphated i. 3t and th~ following fuco~ed . ubstituted ma~ rials: 3 :3r 4- sulfated or phospha~ed ~uc:o e groups on Le~isX or L~wis~ and the 1 ike .

Claims (18)

WHAT IS CLAIMED IS:

1. A compound of Formula I:

I
or Formula II:
II
where R is selected from the group consisting of hydrogen, a saccaride-OR14, or an aglycon having at least 1 carbon atom where R14 is hydrogen or an aglycon of at least one carbon atom;
Y is selected from the group consisting of oxygen, sulfur, and -NH-;
R1 is selected from the group consisting of hydrogen, -NH2, -N3, -NHSO3H, -NR5C(O)R4, -N=C(R5)2, -NHCH(R5)2, -NHR6, -N(R6)2, -OH, -OR6, -S(O)R6, -S(O)2R6 and sulfate, wherein R4 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms;

-OR7 wherein R7 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group, and -NR8R9 wherein R8 and R9 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R5 is selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R6 is alkyl of from 1 to 4 carbon atoms, R2 is selected from the group consisting of hydrogen, -N3, -NH2, -NHSO3H, -NH11C(O)R10, -N=C(R11)2, -NHCH(R11)2, -NHR12, -N(R12)2, -OH and -OR12, wherein R10 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, -OR13 wherein R13 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group, and -NR14R15 wherein R14 and R15 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R11 is selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms;
each R12 is alkyl of from 1 to 4 carbon atoms, R3 is selected from the group consisting of hydrogen, fluoro, sulfate and hydroxy;
X is selected from the group consisting of hydrogen, sulfate, and phosphate;
X1 is selected from the group consisting of hydrogen, sulfate, and phosphate;
X2 is selected from the group consisting of hydrogen, sulfate, and phosphate; and pharmaceutically acceptable salts thereof;

and with the proviso that either at least one of X, X1, or X2 is sulfate or phosphate.

2. A compound of Claim 1 wherein said compound is of Formula I.

3. A compound of Claim 1 wherein said compound is of Formula II.

4. A compound of Claim 1 wherein R1 is selected from the group consisting of hydroxyl, hydrogen or alkoxy of from 1 to 4 carbon atoms.

5. A compound of Claim 1 wherein R2 is selected from the group consisting of hydroxyl, alkoxy of from 1 to 4 carbon atoms, -NH2, -N3 and -NHC(O)R10.

6. A compound of Claim 1 wherein R3 is -OH.

7. A compound of Claim 1 wherein X and X1 are hydrogen.

8. A compound of Claim 1 wherein X and X2 are hydrogen.

9. A compound of Claim 2 wherein X is hydrogen, X1 is hydrogen, X2 is sulfate, R1 is hydroxyl, R2 is selected from the group consisting of -NH2 and -NHC(O)CH3 and R3 is hydroxyl.

10. A compound of Claim 3 wherein X is hydrogen, X1 is hydrogen, X2 is sulfate, R1 is hydroxyl, R2 is selected from the group consisting of -NH2 and -NHC(O)CH3 and R3 is hydroxyl.

11. A pharmaceutical composition suitable for administration to a mammal which comprises a pharmaceutically inert carrier and an effective amount of the compound of Claim 1 to modulate a cell-mediated immune response in said mammal.

12. A method for modulating a cell-mediated immune response in a mammal which method comprises administering to said mammal an amount of a compound of Claim 1 effective in modulating said immune response.

13. A method for the preparation of p-chlorophenyl 2,3,4-tri-O-benzyl-.beta.-thiofucopyranoside which comprises the steps:
(a) contacting L-fucose with sodium acetate in dichloroethane maintained at a temperature of from about 50-55°C while adding dropwise at least 4 equivalents of acetic anhydride;
(b) maintaining the solution produced in step (a) above at about 50-55°C for a sufficient period of time so as to produce the 1,2,3,4-tetraacetylated derivative of L-fucose;
(c) reacting the product produced in step (b) above with at least one equivalent of p-chlorothio-phenol and at least two equivalents of boron trifluoride etherate under conditions sufficient to provide the p-chlorophenyl 2,3,4-tri-O-acetyl-.beta.-thiofucopyranoside;
(d) removing the acetyl blocking groups by contacting the p-chlorophenyl 2,3,4-tri-O-acetyl-.beta.-thiofucopyranoside with sodium methoxide and methanol under conditions sufficient to provide for p-chlorophenyl .beta.-thiofucopyranoside; and (e) contacting the p-chlorophenyl .beta.-thiofuco-pyranoside produced in step (d) with benzyl chloride or benzyl bromide in the presence of a base and under conditions sufficient to provide for p-chloro-phenyl 2,3,4-tri-0-benzyl-.beta.-thiofucopyranoside.

14. A method for the preparation of benzyl 4,6-di-0-benzylidene-2-0-benzoyl-3-0-chloroacetyl-.beta.-D-thiogalactopyranoside which comprises the steps:
(a) contacting D-galactose with sodium acetate in dichloroethane maintained at a temperature of from about 80-85°C while adding dropwise at least 5 equivalents of acetic anhydride;
(b) maintaining the solution produced in step (a) above at about 75-85°C for a sufficient period of time so as to produce the 1,2,3,4,6-penta-acetylated derivative of D-galactose;
(c) reacting the product produced in step (b) above with at least one equivalent of benzyl mercaptane and about 1-3 equivalents of boron trifluoride etherate under conditions sufficient to provide the benzyl 2,3,4,6-tetra-0-acetyl-.beta.-thiogalactopyranoside;
(d) removing the acetyl blocking groups by contacting the benzyl 2,3,4,6-tetra-0-acetyl-.beta.-thiogalactopyranoside with sodium methoxide and methanol under conditions sufficient to provide for phenyl .beta.-thiogalactopyranoside;
(e) contacting the benzyl .beta.-thiogalacto-pyranoside produced in step (d) above with benzylaldehyde dimthyl acetal and p-toluenesulfonic acid under conditions sufficient to provide the benzyl 4,6-di-0-benzylidene-.beta.-D-thiogalacto-pyranoside;

(f) adding chloroacetylchloride to a dimethylformamide (DMF) solution containing the benzyl 4,6-di-O-benzylidene-.beta.-D-thiogalacto-pyranoside produced in step (e) above maintained at a temperature of from about -40°C to about -15°C for a sufficient period of time so as to provide benzyl 4,6-di-O-benzylidene-3-O-chloroacetyl-.beta.-D-thiogalactopyranoside; and (g) adding benzoyl chloride or other suitable benzoylating agent to a solution of benzyl 4,6-di-O-benzylidene-3-O-chloroacetyl-.beta.-D-thiogalacto-pyranoside in a suitable solvent containing a base and dimethylaminopyridine under conditions sufficient to provide for benzyl 4,6-di-O-benzylidene-2-O-benzoyl-3-O-chloroacetyl-.beta.-D-thiogalactopyranoside.

15. A method for reducing sensitization of a mammal to an antigen which comprises administration of an effective amount to the mammal of a compound of Claim 1.

16. A method for reducing sensitization of a mammal to an antigen which comprises administration of an effective amount to the mammal of a compound of Claim 2.

17. A method for reducing sensitization of a mammal to antigen which comprises administration of an effective amount to the mammal of a compound of Claim 3.

18. A method according to Claim 12 wherein said suppression of an immune response comprises suppression of a delayed type hypersensitivity (DTH) response and induction of tolerance to an antigen.